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CHEMISTRY RESEARCH AND APPLICATIONS
CLICK CHEMISTRY
APPROACHES, APPLICATIONS
AND CHALLENGES
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CHEMISTRY RESEARCH
AND APPLICATIONS
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CHEMISTRY RESEARCH AND APPLICATIONS
CLICK CHEMISTRY
APPROACHES, APPLICATIONS AND
CHALLENGES
YU CHEN
AND
ZONG-RUI TONG
EDITORS
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Library of Congress Cataloging-in-Publication Data

Names: Chen, Yu (Chemist), editor. | Tong, Zong-Rui, editor.
Title: Click chemistry : approaches, applications, and challenges / editors,
Yu Chen and Zong-Rui Tong (School of Material Science and
Engineering, Beijing Institute of Technology, Beijing, China, and others).
Description: Hauppauge, New York : Nova Science Publishers, Inc., [2017] |
Series: Chemistry research and applications | Includes index.
Identifiers: LCCN 2017017342 (print) | LCCN 2017018840 (ebook) | ISBN
9781536119237H%RRN | ISBN 9781536119039 (hardcover)
Subjects: LCSH: Chemical equilibrium. | Chemical reactions. |
Biocompatibility.
Classification: LCC QD503 (ebook) | LCC QD503 .C565 2017 (print) | DDC
547/.2--dc23
LC record available at https://lccn.loc.gov/2017017342
Published by Nova Science Publishers, Inc. † New York

CONTENTS
Chapter 1 The Introduction of Click Chemistry Reaction 1

Yu Chen
Chapter 2 Nitrile N-Oxide-Based Click Reactions
Accompanying C-C Bond Formation 21
Yasuhito Koyama and Toshikazu Takata
Chapter 3 Bi-1,2,3-Triazoles: Synthesis and Perspectives 51
Ivette Santana-Martinez and Erick Cuevas-Yañez
Chapter 4 Click Chemistry of Natural Polymers 69
Yu-Tong Zhang, Zhu-Yun Li and Yu Chen
Chapter 5 Synthesis and Functionalization of Hydrogel through
“Click-Chemistry” 107
Mi-Heng Dong and Yu Chen
Chapter 6 Using of Click Chemistry for Elastomer 139
Ya-Lun Wang and Yu Chen
Chapter 7 Surface Engineering of Porous Monoliths via
Click Chemistry: Towards Functional Materials for
Flow Chemistry Applications 157
Seydina Ibrahima Kebe, Hela Kammoun,
Mohamed Guerrouache, Samia Mahouche-Chergui,
Sabrina Belbekhouche, Benjamin Le Droumaguet,
Yosra Dridi-Zrelli and Benjamin Carbonnier
vi Contents
Chapter 8 Click Chemistry for Membrane Preparation and

Surface Modification 211
Zhu-Fang Hu, Jin Zhou and Hai-Yin Yu
Chapter 9 Copper-Mediated Click Chemistry Applications to
Assemble Polyaromatic Structures 235
Celedonio M. Álvarez, Héctor Barbero and
Sergio Ferrero
Chapter 10 Application of Click Chemistry in Biomedical Fields 281
Yu Chen, Ying Zhang, Xiaoyu Sun and Jingjing Yuan
Chapter 11 Click Chemistry: Optical Sensing in
Biological Analysis 313
Suyan Qiu, Fang Luo, Linguang Luo, Longhua Guo,
Bin Qiu, Zhenyu Lin and Guonan Chen
Chapter 12 Telechilic Polybutadiene Solid Propellant Binders
Based on ‘Çlick’ Chemistry Approach 347
S. Reshmi, E. Arunan and C. P. Reghunadhan Nair
Editor Contact Information 367
Index 369
In: Click Chemistry ISBN: 978-1-53611-903-9
Editors: Y. Chen and Z. R. Tong ©2017 Nova Science Publishers, Inc.
Chapter 1
THE INTRODUCTION OF
CLICK CHEMISTRY REACTION
Yu Chen*
School of Materials Science and Engineering,
Beijing Institute of Technology, Beijing, China
ABSTRACT
Click chemistry, which is also called as linkage chemistry, dynamic,
combinatorial chemistry or quick linking combinatorial chemistry,
describes the reaction that joins molecular fragments as simple, efficient
and versatile as clicking a mouse. The two units with specific click
structures can be linked by click reaction, no matter what is attached to
the structure and only the specific click structures can be joined. It
emphasizes the development of new combinatorial chemistries on the
basis of the synthesis of efficient and highly selective carbon-heteroatom
bond (C-X-C) and effectively prepares molecules with high diversity by
these simple reactions. It significantly simplified and promoted the
development of synthesis chemistry. Click chemistry has become one of
the most useful and attractive synthetic strategy in many fields. In the
current chapter, the definition of the click chemistry was explained, the
characteristics and types of the click chemistry was introduces, some
specific reaction types were focused. Application of click chemistry in
different fields and its development were briefly introduced. It is helpful
*
Corresponding Author Email: cylsy@163.com.
2 Yu Chen
for the whole comprehension of the readers to the characteristics and

development of this type reaction.
1. DEVELOPMENT OF CLICK CHEMISTRY

With the development of modern science and technology, the
conventional chemical synthesis cannot meet the demands on the rapid
synthesis a large number of target compounds. To overcome this challenge,
the “click chemistry” was proposed based on the synthesis of natural products
and biosynthesis pathways [1]. The nature can generate complicated biological
molecules, such as proteins and polysaccharides, by joining the modular
units of choice with specific biomolecules, such as amino acids and
monosaccharides. This process is characterized with obvious “modular”
features and can form carbon-heteroatom bonds between specific functional
groups, which connect amino acids and monosaccharides into various
biological molecules.
With the growth of the new drug demand and the development of high-
throughput screening since the late 20th century, the rapid synthesis of a large
number of new molecules has become an urgent task for organic synthesis
[2, 3]. Studies have been focused on constructing molecular libraries [4, 5],
developing molecular diversity [6] and meet the needs for synthesis of new
drugs [7]. The modern technologies and tools, such as chiral techniques, high-
throughput techniques, have significantly improved the quality of drug
chemical synthesis and promoted drug development.
A pharmaceutically-acceptable compound should consist of at least 30
non-hydrogen atoms with a molecular weight of no less than 500 Da [8]. It
should be composed of hydrogen, carbon, nitrogen, oxygen, phosphorous,
sulfur, chlorine and bromine and highly tolerant to oxygen and water at room
temperature [9]. In 1996, Guida et al. found by computer simulation that there
were as many as 1063 compounds meeting these criteria, which is one million
times of total atoms in the sun [10]. However, only 106 -107 known
compounds fulfill these criteria, indicating that only a small portion of drugs
have been developed largely due to the low efficiency of current synthesis
methods. Therefore, the development of reliable, high efficiency and highly
selective click reaction that follows the “modular” synthesis in nature would
have a revolutionary effect on the drug synthesis, as well as the development
and synthesis of other new materials [11].
The Introduction of Click Chemistry Reaction 3
In the past 150 years of modern chemistry history, a variety of synthesis

techniques have been developed to join molecular fragments. Many of them,
such as the “combinatorial chemistry” invented in the 1990s, are very delicate
and require careful manipulations of highly reactive reactants under very
restricted conditions. However, combinatorial chemistry relies on the reactions
between monomers more than traditional synthetic chemistry, which limits its
application in the synthesis of compounds of diverse structures.
In 1893, Michael et al. [12]. reported for the first time the azide-acetylenic
cycloaddition reaction under non-catalytic conditions. In the mid-late 20th
century, Huisgen et al. [13] established a new type of reaction, e.g., 1, 3-
dipolar cycloaddition via thorough mechanistic investigation and synthetic
application. However, such reaction under non-catalytic conditions requires
high reaction temperature (80-120℃) and long reaction time (12-24h). In 2001,
Meldal et al. [14] in Denmark and Sharpless et al. of Scripps Research
Institute in the United States independently discovered the copper catalyzed
azide-alkyne cycloaddition that could be completed in 8h at room temperature.
The reaction is easy to perform and produce target products quickly with high
yields and little or no by-products. It can be conducted under a variety
conditions, especially in water, without affected by the neighbor functional
groups. The reaction under the optimal conditions was named by the
researchers as “click chemistry” that could rapidly join small units to yield
various compounds. The core of this reaction is to generate heteroatom-
containing compounds by a series of reliable modular reactions. Click
chemistry fulfilled the requirements to the chemical synthesis of molecular
diversity. In 2001, Sharpless et al. was awarded the Nobel Prize in chemistry
for their pilot work in “click chemistry.”
Click chemistry can also be called as Linkage Chemistry, Dynamic,
Combinatorial Chemistry or Quick Linking Combinatorial Chemistry.
Click chemistry, also known as tagging, describes the reaction that joins
molecular fragments as simple, efficient and versatile as clicking a mouse. The
two units with specific click structures can be linked by click reaction, no
matter what is attached to the structure and only the specific click structures
can be joined. It emphasizes the development of new combinatorial
chemistries on the basis of the synthesis of efficient and highly selective
carbon-heteroatom bond (C-X-C) and effectively prepares molecules with high
diversity by these simple reactions [15, 16]. It significantly simplified and
promoted the development of synthesis chemistry. Click chemistry has
become one of the most useful and attractive synthetic strategy in many fields,
4 Yu Chen
such as drug development and the synthesis and preparations of biomedical

materials and polymers.
2. CHARACTERISTICS OF CLICK CHEMISTRY REACTION

“Click chemistry” is a new concept of chemical synthesis rather than a
new chemical synthesis technology, which opens up new ways for chemists to
develop chemical synthesis pathways. It is a powerful and practical method for
synthesizing a large number of new compounds at low cost by linking carbon
and heteroatoms (C-X-C) via rapid, reliable and selective chemical reactions
using readily available raw materials. By the way, click chemistry reactions
have the following common characteristics [17-22].
1) Click chemistry reactions use readily available raw materials. Many

of the reactants for the click chemistry reactions are the olefins and
alkynes that are the products of petroleum cracking. Their carbon-
carbon multibonds can be used as the active components of click
chemistry reactions.
2) The majority of click chemistry reactions involve the formation of
carbon-heteroatom (mainly nitrogen, oxygen, sulfur) bonds, which is
different from focus of organic chemistry on the carbon-carbon bond
formation in recent years.
3) Click chemistry reactions are simple to perform under mild conditions
and are rarely affected by water and oxygen. The presence of water
can even accelerate the reaction.
4) Click chemistry reactions are highly stereoselective, highly yielding
and produce by-products that are non-toxic.
5) Click chemistry reactions are usually exothermic, which is achieved
by high-energy reactants and stable products.
6) In general, click chemistry reaction is a fusion process that produces
no by-products or a condensation process that produce water as the
by-product.
7) Click chemistry reactions is characterized by high thermodynamic
driving force (>84kJ/mol).
8) Click chemistry reactions produce the products that can be purified
simply by crystallization and distillation without complex
chromatographic separation needed.
9) Click chemical reaction is also characterized with high yield and low-
cost.
These characteristics of click chemistry reaction indicate that its chemical

energy minimizes the damage of chemical reaction to the environment and
reduces environmental pollution. It is an important way to implement green
chemistry. Synthesizing known or new compounds with click chemistry
reaction has positive effects on the environment.
3. TYPES OF CLICK CHEMISTRY REACTION

So far, four major types of click chemistry reactions have been developed,
e.g., cycloaddition reaction that includes heterocyclic Diels-Alder reactions,
especially 1, 3-dipolar cycloaddition reactions; nucleophilic ring opening
reaction, especially the ring opening of the electrophilic group of high tension
heterocycles; mild condensation reaction of non-alcohol aldehyde carbonyl
compounds; and addition reaction of carbon-carbon multibond.
1) Cycloaddition reaction
Cycloaddition reaction usually joins unsaturated reactants to form a

variety of five-membered and six-membered heterocycles, which fully realizes
the idea of click chemistry and covers a wide range of reactions, such as the
Diels-Alder reaction [23-25]. The reactive groups for the cycloaddition
reaction are usually non-polar. Both azide and alkynyl groups can be easily
introduced into the desired compound structure. Their 1, 3-dipolar
cycloaddition has become the most widely used click chemistry reaction
because it is simple to perform and stereospecific, can be conducted in water
or organic solvents under mild conditions without significant interferences and
create only the by-products that are easily removed [26, 27]. It is thus called as
“Cream of the Crop” [28].
Cycloaddition of azide compounds and acetylene was reported in as early
as the early 20th century. However, the reaction produced a mixture of 1, 4-
and 1, 5-disubstituted triazoles. Later, Cu (I) catalyzed cycloaddition was
proposed [29], which promoted the selectivity of cycloaddition to produce
only 1, 4-triazole with yields up to 91% and reduced the reaction time from 18
h to 8 h. Currently, the most commonly used Cu (I) catalysts are CuI, Cu (I),
6 Yu Chen
(PPh3)3Br, sodium ascorbate reduced CuSO4, Cu and Cu reduced CuSO4 [30,

31]. The reaction proceeds as follows.
2) Nucleophilic ring-opening reaction
The nucleophilic ring-opening reaction is realized mainly by the

nucleophilic ring-opening of ternary heteroatom rings, such as epoxy
derivatives, aziridines, cyclic sulfates, cyclic sulfamides, aziridinium ions,
cyclosulfonium ions and so on [32]. Their internal tension energy is released
during the ring-opening reaction. Among these ternary heterocyclic
compounds, epoxy derivatives and aziridinium ions are the most commonly
used substrates for click chemical reactions [33, 34]. Their ring-opening
produces various highly selective compounds.
Figure 1. Mechanism of Cu (I) catalysis for terminal azide-alkyne coupling.

Epoxy compounds contain a three-membered ring with great tension,

which is very favorable to their ring-opening reaction. The nucleophile can
only attack one of the carbon atoms along the axial direction of the CO bond
under certain conditions [35]. The orbital arrangement is unfavorable to the
elimination reaction that compete with the ring-opening reaction, avoiding the
production of by-products and promoting the yield. In addition, although the
epoxides have low reactivity with water, the hydrogen-bonding ability and
polar property of water are favorable to the ring-opening reaction of epoxides
with other nucleophiles. The ring-opening reaction of epoxides by amine
attack can be expressed as the following formula:
Such reactions can be carried out in an alcohol/water or solvent free
media. For example, the ring-opening reactions of dioxirane with benzylamine
produce 1, 4-diol with a 90% yield in the presence of protic methanol and
produce 1, 3-diol with a yield of 94% in the absence of solvent. The reactions
are expressed as follows.
3) Carbonyl condensation reaction
Figure 2. The ring-opening reaction of epoxides by amine attack.
Figure 3. Regioselectivity of oxirane opening.

8 Yu Chen
The reactions of aldehydes or ketones with 1, 3-diols to produce 1, 3-

dioxolane [36], the reaction of aldehydes with hydrazines or hydroxylamine
ethers to produce hydrazones or oximes [37], and the reaction of α-/β-carbonyl
aldehydes and ketones with esters to form heterocyclic compounds [38], etc.
are the most widely used carbonyl condensation reactions.
For example, the acetals of D-isoascorbic acid containing 1, 3-dioxolane
can be obtained by the carbonyl condensation reaction of linear saturated
aliphatic aldehyde with D- isoascorbic acid in N, N-dimethylacetamide using
p-toluenesulfonic acid as the catalyst and cyclohexane as the water-carrying
agent [39]. The reaction is shown below.
4) Addition reaction of carbon-carbon multibonds
The click reaction of thiol–ene/yne is a typical addition reaction of carbon-

carbon multibonds discovered after Huisgen reaction. Such reaction is usually
carried out under UV irradiation that initiate the click reaction of unsaturated
bonds with thiol using a photoinitiator. It is characterized with wide
application scope [40-41]. The click reaction of thiol-ene is a simple metal-
free-catalyzed reaction and has become an efficient tool for curing reaction
and polymer modification. Alkynyl is also an ideal material for click chemistry
reaction because it can form a variety of structures and is stable under normal
conditions [42-47].
Because the reaction mechanisms of the thiol-ene and thiol-yne are very
similar, only the click reaction of thiol-ne is discussed here to elaborate the
addition reaction of carbon-carbon multibonds.
Figure 4. Acetals of D-isoascorbic acid containing 1, 3-dioxolane via the carbonyl

condensation reaction.
Figure 5. Mechanism of thiol-ene click chemistry.
The thiol-ene click reaction was first discovered by Posner in 1905 [48].
Later in 1938, Kharasch et al. [49] proposed photochemical reaction
mechanism of the thioltor absorbs photons to produce free radicals that seize a
hydrogen from the thiol compounds nearby to convert thiol into thiyl radicals.
The thiol radicals attack the e-ene as shown in Figure 5. The reaction is subject
to three stages: photoinitiation, free radical addition, and free radical
termination. During the first stage of photoinitiation, the initialectron-rich
double bonds to undergo the free radical addition reaction. The free radicals
complete the thiol-ene photochemical reaction with the participation of thiols.
The unreacted thiol radicals combine with each other to terminate the reaction.
In the early 1990s, Jacobine [50] systematically reviewed the different
types of thiol-ene reaction and their reaction mechanisms, and pointed out the
great potential of the thiol-ene click chemistry reaction in the fields of UV-
curable adhesives and coatings. At the early 21st century, Hoyle et al. [51]
summarized the research status of thiol-ene photochemical reaction in the past
ten years and concluded that the photocrosslinking of thiol-ene had a
promising application prospect in the preparation of polymers with tunable
mechanical properties and physicochemical properties. In recent years, Holye
and Bowman [52-53] pointed out that the free radicals produced by the
photocleavage of the photoinitiator functioned as the catalyst to catalyze the
highly selective reaction between the specific regions with functional group.
The thiol-ene photocrosslinking reaction combines the advantages of
photoinitiation and click chemistry. The thiol-ene/yne click chemistry,
especially the combination of thiol-ene/yne click chemistry and controllable
polymerization, provides a new strategy for the synthesis of organic functional
materials.
10 Yu Chen
4. APPLICATION OF CLICK CHEMISTRY REACTION

AND ITS DEVELOPMENT
Click chemistry has been rapidly developed and applied in a variety of

fields, such as DNA [54, 55], self-assembly [56], surface modification
[57, 58], supramolecular chemistry [59, 60], dendritic molecules [61, 62],
functional polymers [63, 64], combinatorial chemistry [65], proteomics [66],
biocomposites [67], biomedicine [68, 69], and so on. The following chapters
of this book will introduce the application of click chemistry in different fields
in detail.
Studies have shown that click chemistry reaction can effectively overcome
the shortcomings of traditional synthesis methods and is a novel technique for
the synthesis and surface modifications in many fields. Click chemistry
reaction is usually conducted under very mild conditions, even at room
temperature, which make it very conducive to the biological applications, such
as protein modification, synthesis of proteinase inhibitor, DNA modification,
nucleotide grafting and so on. The reaction does not have significant effects on
the activity of these biomolecules, and even can greatly increase their activity
[70, 71].
Click chemistry reaction is stereospecific and high yielding. It is not
disturbed by water and oxygen. Therefore, click chemistry provides a power
tool for the design and development of new materials, especially for the
development of novel biomaterials, such as biodetection [72], biosensors [73],
bioseparation [74, 75], drug release [76, 77], and so on. However, there are
still some issues of click chemistry to be solved in the future work.
First, although click chemistry reactions and Diels-Alder reactions of
acetylenes and azides by ring tension have been reported, most works adopt
the Cu(I) catalyzed click reaction reported by Sharpless et al. Developing new
click chemistry reactions is still one of the major research directions.
Second, the introduction of Cu (I) catalyst can produce a certain degree of
cytotoxicity and also cause the aggregation of nano-dispersed systems,
limiting its in vivo and nano-level applications. In addition, Cu (I) catalysts are
easily oxidized. The oxygen in the reaction system needs to be removed.
Therefore, developing novel catalytic systems is also a research direction.
Third, most works have been focused on the preparation of functional
polymers by the combination of click chemistry reaction and ATRP [78, 79].
The combinations of click chemistry reaction with NMP and RAFT [80, 81],
especially with RAFT, have been rarely studied. All ATRP, NMP and RATP
free radical polymerization methods have their advantages and disadvantages.

ATRP is the simplest method and NMP and RAFT are more suitable to the
preparation of polymers containing functional groups, such as amide and
carboxyl. Therefore, the combination of click chemistry reaction with the
NMP and RAFT free radical polymerization methods will provide a possible
means for the preparation of novel polymers.
Fourth, nanomaterials are involved in many fields as an interdisciplinary
field. However, the application of click chemistry reaction to the modification
of nanomaterials still needs further studies in detail.
So far, click chemistry has not been fully utilized. Expanding the scope of
its application is still a challenge. Click chemistry is still limited to the 1, 3-
dipolar cycloaddition reaction of azides and terminal alkyne compounds.
Future studies should be focused on exploring more novel click reactions. In
addition, most reactions use Cu (I) as the catalyst, which requires the oxygen
removal from the reaction system and thus limits the development of click
chemistry.
The application of click chemistry is still underexplored in many fields
and the mechanisms of some phenomena are still unclear. For example, the
electron transfer rate of the triazole ring linked compounds formed with
acetylene double-cyclopentadienyl groups is slightly higher than that of the
triazole ring linked compounds formed by the longer alkynyl chain
cyclopentadienyl, which, theoretically, should be much higher due to the steric
hindrance effect of the latter. Therefore, the deep study of click chemistry has
become more and more important.
REFERENCES
[1] Hur, GH; Meier, JL; Baskin, J; Codelli, JA; Bertozzi, CR; Marahiel,
MA; Burkart, MD. Crosslinking studies of protein-protein interactions in
nonribosomal peptide biosynthesis. Chemistry & Biology, 2009, 16(4),
372-381.
[2] Cleary, JD; Walker, LA; Hawke, RL. Antimycotic drug discovery in the
age of genomics. American Journal of Pharmacogenomics, 2005, 5(6),
365-386.
[3] Smith, AB; Pitram, SM; Boldi, AM; Gaunt, MJ; Sfouggatakis, C;
Moser, WH. Multicomponent linchpin couplings, Reaction of dithiane
anions with terminal epoxides, epichlorohydrin, and vinyl epoxides:
Efficient, rapid, and stereocontrolled assembly of advanced fragments
12 Yu Chen
for complex molecule synthesis. Journal of the American Chemical

Society, 2003, 125(47), 14435-14445.
[4] Austin, CP; Brady, LS; Insel, TR; Collins, FS. NIH molecular libraries
initiative. Science, 2004, 306(5699), 1138-1139.
[5] Maddry, JA; Ananthan, S; Goldman, RC; Hobrath, JV; Kwong, CD;
Maddox, C; Rasmussen, L; Reynolds, RC; Secrist, JA; Sosa, MI.
Antituberculosis activity of the molecular libraries screening center
network library. Tuberculosis, 2009, 89(5), 354-363.
[6] Patterson, DE; Cramer, RD; Ferguson, AM; Clark, RD; Weinberger, LE.
Neighborhood behavior: A useful concept for validation of “molecular
diversity” descriptors. Journal of Medicinal Chemistry, 1996, 39(16),
3049-3059.
[7] Martin, YC; Willett, P; Lajiness, M; Johnson, M; Maggiora, G; Martin,
E; Bures, MG; Gasteiger, J; Cramer, RD; Pearlman, RS. Diverse
viewpoints on computational aspects of molecular diversity. Journal of
Combinatorial Chemistry, 2001, 3(3), 231-250.
[8] Shakeel, F; AlAjmi, MF; Haq, N; Siddiqui, NA; Alam, P; Al-Rehaily,
AJ. Solubility and thermodynamic function of a bioactive compound
bergenin in various pharmaceutically acceptable neat solvents at
different temperatures. Journal of Chemical Thermodynamics, 2016,
101, 19-24.
[9] Berry, DJ; Seaton, CC; Clegg, W; Harrington, RW; Coles, SJ; Horton,
PN; Hursthouse, MB; Storey, R; Jones, W; Friscic, T. Applying hot-
stage microscopy to co-crystal screening: A study of nicotinamide with
seven active pharmaceutical ingredients. Crystal Growth & Design,
2008, 8(5), 1697-1712.
[10] Bohacek, RS; McMartin, C; Guida, WC. The art and practice of
structure-based drug design: A molecular modeling perspective.
Medicinal Research Reviews, 1996, 16(1), 3-50.
[11] Malkoch, M; Thibault, RJ; Drockenmuller, E; Messerschmidt, M; Voit,
B; Russell, TP; Hawker, CJ. Orthogonal approaches to the simultaneous
and cascade functionalization of macromolecules using click chemistry.
Journal of the American Chemical Society, 2005, 127(42), 14942-14949.
[12] Michael, A. Journal fur Praktische Chemie-Practical Applications and
Applied Chemistry, 1893, 48, 94-95.
[13] Huisgen, R; Bayer, HO; Schaefer, FC; Gotthardt, H. New type of
mesoionic aromatic compound +ITS 1,3-dipolar cycloaddition reactins
with acetylene dervatives, Angewandte Chemie – International Edition,
1964, 3(2), 136-137.
[14] Tornoe, CW; Christensen, C; Meldal, M. Peptidotriazoles on solid

phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar
cycloadditions of terminal alkynes to azides. Journal of Organic
Chemsitry, 2002, 67(9), 3057-3064.
[15] Moses, JE; Moorhouse, AD. The growing applications of click
chemistry. Chemical Society Reviews, 2007, 36(8), 1249-1262.
[16] Mandhare, A; Banerjee, P; Bhutkar, S; Hirwani, R. ‘Click chemistry’ for
diagnosis: a patent review on exploitation of its emerging trends. Expert
Opinion on Therapeutic Patents, 2014, 24(12), 1287-1310.
[17] Han, YM; Yuan, L; Li, GY; Huang, LH; Qin, TF; Chu, FX; Tang, CB.
Renewable polymers from lignin via copper-free thermal click
chemistry. Polymer, 2016, 83, 92-100.
[18] Oria, L; Aguado, R; Pomposo, JA; Colmenero, J. A versatile “click”
chemistry precursor of functional polystyrene nanoparticles. Advanced
Materials, 2010, 22(28), 3038-3041.
[19] Wang, C; Wang, N; Zhou, W; Shen, YM; Zhang, L. Application of
“click chemistry” in synthesis of radiopharmaceuticals. Progress in
Chemistry, 2010, 22(8), 1591-1602.
[20] Bayraktar, A; Saracoglu, B; Golgelioglu, C; Tuncel, A. Click-chemistry
for surface modification of monodisperse-macroporous particles.
Journal of Colloid and Interface Science, 2012, 365(1), 63-71.
[21] Gao, Y; Zhang, ZW; Chen, LL; Gu, WW; Li, YP. Synthesis of 6-N,N,N-
trimethyltriazole chitosan via “click chemistry” and evaluation for gene
delivery. Biomacromolecules, 2009, 10(8), 2175-2182.
[22] Prim, D; Rebeaud, F; Cosandey, V; Marti, R; Passeraub, P; Pfeifer, ME.
ADIBO-based “click” chemistry for diagnostic peptide micro-array
fabrication: physicochemical and assay characteristics. Molecules, 2013,
18(8), 9833-9849.
[23] Arumugam, S; Orski, SV; Mbua, NE; McNitt, C; Boons, GJ; Locklin, J;
Popik, VV. Photo-click chemistry strategies for spatiotemporal control
of metal-free ligation, labeling, and surface derivatization. Pure and
Applied Chemistry, 2013, 85(7), 1499-1513.
[24] Ramachary, DB; Barbas, CF. Towards organo-click chemistry:
Development of organocatalytic multicomponent reactions through
combinations of Aldol, Wittig, Knoevenagel, Michael, Diels-Alder and
Huisgen cycloaddition reactions. Chemistry – A European Journal,
2004, 10(21), 5323-5331.
14 Yu Chen
[25] Martinek, M; Filipova, L; Galeta, J; Ludvikova, L; Klan, P.

Photochemical formation of dibenzosilacyclohept-4-yne for Cu-free
click chemistry with azides and 1,2,4,5-tetrazines. Organic Letters,
2016, 18(19), 4892-4895.
[26] Vieyres, A; Lam, T; Gillet, R; Franc, G; Castonguay, A; Kakkar, A.
Combined Cu-I-catalysed alkyne-azide cycloaddition and furan-
maleimide Diels-Alder “click” chemistry approach to thermoresponsive
dendrimers. Chemical Communications, 2010, 46(11), 1875-1877.
[27] Hansell, CF; Espeel, P; Stamenovic, MM; Barker, IA; Dove, A; Du
Prez, FE; O’Reilly, RK. Additive-free clicking for polymer
functionalization and coupling by tetrazine-norbornene chemistry.
Journal of the American Chemical Society, 2011, 133(35), 13828-13831.
[28] Wang, Q; Chittaboina, S; Barnhill, HN. Advances in 1,3-dipolar
cycloaddition reaction of azides and alkynes - A prototype of “click”
chemistry. Letters in Organic Chemistry, 2005, 2(4), 293-301.
[29] Zheng, ZJ; Wang, D; Xu, Z; Xu, LW. Synthesis of bi- and bis-1,2,3-
triazoles by copper-catalyzed Huisgen cycloaddition: A family of
valuable products by click chemistry. Beilstein Journal of Organic
Chemistry, 2015, 11, 2557-2576.
[30] Liu, Y; Diaz, DD; Accurso, AA; Sharpless, KB; Fokin, VV; Finn, MG.
Click chemistry in materials synthesis. III. Metal-adhesive polymers
from Cu(I)-catalyzed azide-alkyne cycloaddition. Journal of Polymer
Science Part A-Polymer Chemistry, 2007, 45(22), 5182-5189.
[31] Tale, RH; Gopula, VB; Toradmal, GK. ‘Click’ ligand for ‘click’
chemistry: (1-(4-methoxybenzyl)-1-H-1,2,3-triazol-4-yl)methanol
(MBHTM) accelerated copper-catalyzed [3+2] azide-alkyne
cycloaddition (CuAAC) at low catalyst loading. Tetrahedron Letters,
2015, 56(43), 5864-5869.
[32] Mespouille, L; Coulembier, O; Paneva, D; Degee, P; Rashkov, I;
Dubois, P. Synthesis of adaptative and amphiphilic polymer model
conetworks by versatile combination of ATRP, ROP, and “Click
chemistry.” Journal of Polymer Science Part A-Polymer Chemistry,
2008, 46(15), 4997-5013.
[33] Scheunemann, M; Hennig, L; Funke, U; Steinbach, J. High regiocontrol
in the nucleophilic ring opening of 1-aralkyl-3,4-epoxypiperidines with
amines-a short-step synthesis of 4-fluorobenzyltrozamicol and novel
anilidopiperidines. Tetrahedron, 2011, 67(19), 3448-3456.
[34] Markiewicz, KH; Wilczewska, AZ; Chernyaeva, O; Winkler, K. Ring-

opening reactions of epoxidized SWCNT with nucleophilic agents: a
convenient way for sidewall functionalization. New Journal of
Chemistry, 2014, 38(6), 2670-2678.
[35] Ganguly, B. A semi-empirical and density functional study on the origin
of regioselective epoxy-ring opening of 2’ 3’-lyxoanhydrothymidine.
Journal of Molecular Structure-Themchem, 2010, 673(1-3), 127-132.
[36] Sax, M; Frohlich, R; Schepmann, D; Wunsch, B. Synthesis and NMDA
receptor affinity of ring and side chain homologues of dexoxadrol.
European Journal of Organic Chemistry, 2008, 35, 6015-6028.
[37] Hong, ZY; Lv, C; Liu, AA; Liu, SL; Sun, EZ; Zhang, ZL; Lei, AW;
Pang, DW. Clicking hydrazine and aldehyde: the way to labeling of
viruses with quantum dots. ACS Nano, 2015, 9(12), 11750-11760.
[38] Medina, FG; Marrero, JG; Macias-Alonso, M; Gonzalez, MC; Cordova-
Guerrero, I; Garcia, AGT; Osegueda-Roblesa, S. Coumarin heterocyclic
derivatives: chemical synthesis and biological activity. Natural Product
Reports, 2015, 32(10), 1472-1507.
[39] Zheng, DG; Yu, YW. Synthesis of n-alkyl aldehyde acetals of D-
isoascorbic acid. Chinese Journal of Synthetic Chemistry, 2008, 16(4),
456-459.
[40] Pounder, RJ; Stanford, MJ; Brooks, P; Richards, SP; Dove, AP. Metal
free tlliol-maleimide click reaction as a mild functionalisation strategy
for degradable polymers. Chemical Communications, 2008, 41, 5158-
5160.
[41] Stanford, MJ; Dove AP. One-pot synthesis of alpha, omega-chain end
functional，stereoregular, star-shaped poly(lactide)．Macromoleeules,
2009, 42(1), 141-147.
[42] Lowe, AB; Hoyle, CE; Bowman CN. Thiol-yne click chemistry: a
powerful and versatile methodology for materials synthesis．Journal of
Materials Chemistry, 2010, 20(23), 4745-4750.
[43] Wang, ZC; Xu, XD; Chen, CS; Yun, L; Song, JC; Zhang, XZ; Zhuo,
RX. In situ formation of thermosensitive PNIPAAm-based hydrogels by
michael-type addition reaction. Applied Materials & Interfaces, 2010,
2(4), 1009-1018.
[44] Ruthergle, BG; McBath, RA; Huang, YL; Shipp, DA．Polyanhydride
networks from thiol-ene polymerizations. Macromolecules, 2010,
43(24), 10297-10303.
16 Yu Chen
[45] Chan, JW; Shin, J; Hoyle, CE; Bowman, CN; Lowe AB. Synthesis,
thiol-yne “click” photo-polymerization, and physical properties of
networks derived from novel multifunctional alkynes. Macromolecules,
2010, 43(11), 4937-4942.
[46] Fairbanks, BD; Scott, TF; Kloxin, CJ; Anseth, KS; Bowman, CN.
Thiolt-Yne photopolymerizations: novel mechanism, kinetics, and step-
growth formation of highly cross-linked networks. Macromolecules,
2009, 42(1), 211-217.
[47] Park, HY; Kloxin, CJ; Scott, TF; Bowman, CN. Stress relaxation by
addition-fragmentation chain transfer in highly cross-linked Thiol-yne
networks. Macromolecules, 2010, 43(24), 10188-10190.
[48] Posner T. Information on unsaturated compounds II The addition of
mercaptan to unsaturated hydrocarbon. Berichte der deutschen
chemischen Gesellschaft, 1905, 38: 646-657.
[49] Kharasch, MS; May, EM; Mayo, FR. The peroxide effect in the addition
of reagents to unsaturated compounds. Xviii. The addition and
substitution of bisulfite. The Journal of Organic Chemistry, 1938, 3(2),
175-192.
[50] Jacobine, AF; Fouassier, JD; Rabek, JF. Radiation curing in polymer
science and technology III. Polymerisation Mechanisms. Elsevier
Applied Science, 1993, 219 Chapter.
[51] Hoyle, CE; Lee, TY; Roper, T. Thiol–enes: Chemistry of the past with
promise for the future. Journal of Polymer Science Part A: Polymer
Chemistry, 2004, 42(21), 5301-5338.
[52] Hoyle, CE; Bowman, CN. Thiol–ene click chemistry. Angewandte
Chemie International Edition, 2010, 49(9), 1540-1573.
[53] Hoyle, CE; Lowe, AB; Bowman, CN. Thiol-click chemistry: a
multifaceted toolbox for small molecule and polymer synthesis.
Chemical Society Reviews, 2010, 39(4), 1355-1387.
[54] El-Sagheer, AH; Brown, T. Click chemistry with DNA. Chemical
Society Reviews, 2010, 39(4), 1388-1405.
[55] Kumar, R; El-Sagheer, A; Tumpane, J; Lincoln, P; Wilhelmsson, LM;
Brown, T. Template-directed oligonucleotide strand ligation, covalent
intramolecular DNA circularization and catenation using click
chemistry. Journal of the American Chemical Society, 2007, 129(21),
6859-5864.
[56] Zhou, QH; Zheng, JK; Shen, ZH; Fan, XH; Chen, XF; Zhou, QF.
Synthesis and hierarchical self-assembly of rod rod block copolymers
via click chemistry between mesogen-jacketed liquid crystalline
polymers and helical polypeptides. Macromolecules, 2010, 43(13),
5637-5646.
[57] Goldmann, AS; Walther, A; Nebhani, L; Joso, R; Ernst, D; Loos, K;
Barner-Kowollik, C; Barner, L; Muller, AHE. Surface modification of
poly(divinylbenzene) microspheres via thiol-ene chemistry and alkyne-
azide click reactions. Macromolecules, 2009, 42(11), 3707-3714.
[58] Wu, XM; Wang, LL; Wang, Y; Gu, JS; Yu, HY. Surface modification of
polypropylene macroporous membrane by marrying RAFT
polymerization with click chemistry. Journal of Membrane Science,
2012, 421, 60-68.
[59] Wong, CH; Zimmerman, SC. Orthogonality in organic, polymer, and
supramolecular chemistry: from Merrifield to click chemistry. Chemical
Communications, 2013, 49(17), 1679-1695.
[60] Binder, WH; Sachsenhofer, R; Straif, CJ; Zirbs, R. Surface-modified
nanoparticles via thermal and Cu(I)-mediated “click” chemistry:
Generation of luminescent CdSe nanoparticles with polar ligands
guiding supramolecular recognition. Journal of Materials Chemistry,
2007, 17(20), 2125-2132.
[61] Whittaker, MR; Urbani, CN; Monteiro, MJ. Synthesis of 3-miktoarm
stars and 1st generation mikto dendritic copolymers by “living” radical
polymerization and “click” chemistry. Journal of the American
Chemical Society, 2006, 128(35), 11360-11361.
[62] Gopin, A; Ebner, S; Attali, B; Shabat, D. Enzymatic activation of
second-generation dendritic prodrugs: Conjugation of self-immolative
dendrimers with poly(ethylene glycol) via click chemistry. Bioconjugate
Chemistry, 2006, 17(6), 1432-1440.
[63] Lutz, JF; Borner, HG; Weichenhan, K. Combining atom transfer radical
polymerization and click chemistry: A versatile method for the
preparation of end-functional polymers. Macromolecular Rapid
Communications, 2005, 26(7), 514-518.
[64] Wang, YP; Chen, JC; Xiang, JM; Li, HJ; Shen, YQ; Gao, XH; Liang, Y.
Synthesis and characterization of end-functional polymers on silica
nanoparticles via a combination of atom transfer radical polymerization
and click chemistry. Reactive & Functional Polymers, 2009, 69(6), 393-
399.
18 Yu Chen
[65] Ghosh, KK; Ha, HH; Kang, NY; Chandran, Y; Chang, YT. Solid phase
combinatorial synthesis of a xanthone library using click chemistry and
its application to an embryonic stem cell probe. Chemical
Communications, 2011, 47(26), 7488-7490.
[66] Ismail, HM; Barton, VE; Panchana, M; Charoensutthivarakul, S;
Biagini, GA; Ward, SA; O’Neill, PM. A click chemistry-based
proteomic approach reveals that 1,2,4-trioxolane and artemisinin
antimalarials share a common protein alkylation profile. Angewandte
Chemie-International Edition, 2016, 55(22), 6401-6405.
[67] Zhang, XH; He, XW; Chen, LX; Zhang, YH. A combination of
distillation-precipitation polymerization and click chemistry: fabrication
of boronic acid functionalized Fe3O4 hybrid composites for enrichment
of glycoproteins. Journal of Materials Chemistry B, 2014, 2(21), 3254-
3262.
[68] Crow, JM. New tools for biomedicine just a click away. Chemistry
World, 2006, 3(5), 26.
[69] Astruc, D; Liang, LY; Rapakousiou, A; Ruiz, J. Click dendrimers and
triazole-related sspects: catalysts, mechanism, synthesis, and functions.
A bridge between dendritic architectures and nanomaterials. Accounts of
Chemical Research, 2012, 45(4), 630-640.
[70] Tron, GC; Pirali, T; Billington, RA; Canonico, PL; Sorba, G; Genazzani,
AA. Click chemistry reactions in medicinal chemistry: Applications of
the 1,3-dipolar cycloaddition between azides and alkynes. Medicinal
Research Reviews, 2008, 28(2), 278-308.
[71] Mamidyala, SK; Finn, MG. In situ click chemistry: probing the binding
landscapes of biological molecules. Chemical Society Reviews, 2010,
39(4), 1252-1261.
[72] Cernat, A; Tertis, M; Cristea, C; Sandulescu, R. Applications of click
chemistry in the development of electrochemical sensors. International
Journal of Electrochemical Science, 2015, 10(8), 6324-6447.
[73] Liu, Y; Yu, Y; Lam, JWY; Hong, YN; Faisal, M; Yuan, WZ; Tang, BZ.
Simple biosensor with high selectivity and sensitivity: thiol-specific
biomolecular probing and intracellular imaging by AIE fluorogen on a
TLC plate through a thiol-ene click mechanism. Chemistry- A European
Journal, 2010, 16(28), 8433-8438.
[74] Chu, CH; Liu, RH. Application of click chemistry on preparation of
separation materials for liquid chromatography. Chemical Society
Reviews, 2011, 40(5), 2177-2188.
[75] Fugier, E; Dumont, A; Malleron, A; Poquet, E; Pons, JM; Baron, A;

Vauzeilles, B; Dukan, S. Rapid and specific enrichment of culturable
gram negative bacteria using non-lethal copper-free click chemistry
coupled with magnetic beads separation. PLOS ONE, 2015, 10(6),
e0127700.
[76] N’Guyen, TTT; Duong, HTT; Basuki, J; Montembault, V; Pascual, S;
Guibert, C; Fresnais, J; Boyer, C; Whittaker, MR; Davis, TP. Functional
iron oxide magnetic nanoparticles with hyperthermia-induced drug
release ability by using a combination of orthogonal click reactions.
Angewandte Chemie-International Edition, 2013, 52(52), 14152-14156.
[77] Wu, XJ; Zhou, LZ; Su, Y; Dong, CM. Comb-like poly(L-cysteine)
derivatives with different side groups: synthesis via photochemistry and
click chemistry, multi-responsive nanostructures, triggered drug release
and cytotoxicity. Polymer Chemistry, 2015, 6(38), 6857-6869.
[78] Lutz, JF; Borner, HG; Weichenhan, K. Combining ATRP and “click”
chemistry: a promising platform toward functional biocompatible
polymers and polymer bioconjugates. Macromolecules, 2006, 39(19),
6376-6383.
[79] Gao, HF; Louche, G; Sumerlin, BS; Jahed, N; Golas, P; Matyjaszewski,
K. Gradient polymer elution chromatographic analysis of alpha,omega-
dihydroxypolystyrene synthesized via ATRP and click chemistry.
Macromolecules, 2005, 38(22), 8979-8982.
[80] Gozgen, A; Dag, A; Durmaz, H; Sirkecioglu, O; Hizal, G; Tunca, U.
ROMP-NMP-ATRP combination for the preparation of 3-miktoarm star
terpolymer via click chemistry. Journal of Polymer Science Part A-
Polymer Chemistry, 2009, 47(2), 497-504.
[81] Lutz, JF; Borner, HG; Weichenhan, K. Combining ATRP and “click”
chemistry: a promising platform toward functional biocompatible
polymers and polymer bioconjugates. Macromolecules, 2006, 39(19),
6376-6383.
Chapter 2
NITRILE N-OXIDE-BASED
CLICK REACTIONS ACCOMPANYING
C-C BOND FORMATION
Yasuhito Koyama1, and Toshikazu Takata2,†

1
Department of Biotechnology, Faculty of Engineering,
Toyama Prefectural University, Toyama, Japan
2
Department of Chemical Science and Engineering,
Tokyo Institute of Technology, Tokyo, Japan
ABSTRACT
This article provides a review on the recent progress concerning
nitrile N-oxide-based click reactions, which allow to fabricate the multi-
component organic architectures. Chapter 1 deals with a brief summary
of the preparation method of nitrile N-oxide. Eight types of precursors are
reported to be available for the preparation of nitrile N-oxide, whose
characteristics are discussed on the basis of the historical survey. Chapter
2 focuses on the reactions of nitrile N-oxide, in which we first describe
the feasibility and problems of the 1,3-dipolar cycloaddition reaction of
nitrile N-oxide related to the click reaction. The problem comes mainly
from the instability or high reactivity in its use. Subsequently, we discuss

Corresponding author: Y. Koyama: Tel: +81-766-56-7500 (ext. 782), E-mail: ykoyama@pu-
toyama.ac.jp
†
Corresponding author: T. Takata: Tel: +81-3-5734-2898, Fax: +81-3-5734-2888, E-mail:
ttakata@ polymer.titech.ac.jp.
22 Yasuhito Koyama and Toshikazu Takata
the reported self-reactions including (i) dimerization to furoxan, (ii)

polymerization, and (iii) thermal isomerization to isocyanate, along with
their suppression methods. Fourth degradation pathway, i.e., the
intramolecular cycloaddition of nitrile N-oxide moiety to simple benzene
ring, is also introduced in this chapter. In Chapter 3, we discuss the
structure of nitrile N-oxide in solution as determined by 13C NMR and
UV spectroscopic studies of 13C-labelled nitrile N-oxides in addition to
the crystalline structure. The results indicate the temperature-dependent
dynamic structural change of nitrile N-oxide in solution. Chapter 4
describes the usefulness of nitrile N-oxide-based click agents we
developed, such as homoditopic nitrile N-oxides, orthogonal agents
possessing both nitrile N-oxide and electrophile functions, and nitrile N-
oxide-terminated polymers. These agents undergo catalyst- and solvent-
free cycloaddition reactions with unsaturated bond-containing polymers
and material surfaces, resulting in cross-linking of polymers, introduction
of functional parts to polymers and surfaces, and grafting of vinyl
polymers onto common polymers. In conclusion, we emphasize the
advantages of the nitrile N-oxide agents in click chemistry along with the
future prospects of nitrile N-oxide agents.
INTRODUCTION
Development of new sophisticated systems is always required in the broad
areas of scientific and technological fields. The term “system” refers to an
assembly of several units that exhibit some specific interactions. For instance,
a personal computer can be referred to an information system, comprising a
rational combination of hardware, software, and the internet. The situation is
the case for chemistry and related areas such as chemical biology, polymer
chemistry, and supramolecular chemistry. Even in organic chemistry, an
assembly of modified molecules can produce various functional systems
including molecular probes and sensors containing sites for molecular
recognition and emission, high performance polymers with versatile functions,
and supramolecular systems that link a function to the response of stimuli.
Regarding the organic synthesis of such systems, we must carefully bind
functionalized units in consideration of each molecular skeleton. The first
problem an organic chemist faces is to decide what bond-forming reactions are
most suitable for connecting different units. We should select an efficient
reaction based on ideal conditions, such as high reactivity, chemo-selectivity,
free of byproduct and catalyst, and easy purification, that take into account the
Nitrile N-Oxide-Based Click Reactions Accompanying C-C Bond … 23
chemical reactivity and stability of each unit and the intended functions of the
product.
Click reactions are categorized as one of the covalent bond-forming
reactions useful for synthesizing organic systems [1]. If the click reaction is
applied to the connection of a few units, new architectures can be easily
obtained without optimizing the reaction conditions. The most frequently used
click reaction is a copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition
reaction, i.e., Huisgen reaction [2]. In contrast to it, the authors have
extensively studied the synthesis of stable nitrile N-oxide agents and their use
for the 1,3-dipolar cycloaddition as the click reaction.
Nitrile N-oxide is recognized as a highly reactive species since the 1930s
[3]. The dipole comprises sp hybridized orbitals similar to that of the azide
functionality. To date, the structure [4] and physical property [5] of nitrile N-
oxide have been considerably investigated. The reaction of nitrile N-oxide
often appears in multi-step synthesis of bioactive natural products, which is
used for introducing -ketol or 1,3-diketone framework to the molecular
skeleton [6]. According to Sustman’s classification of 1,3-dipoles based on its
reactivity [7], nitrile N-oxide is categorized as having an ambiphilic dipole
(type II) with a high reactivity with both electron-rich and -deficient
unsaturated bonds. However, it is hardly utilized as a tool for chemical
ligation, probably due to the complexity of handling nitrile N-oxide. The high
reactivity of nitrile N-oxide is accompanied by chemical instability, leading to
spontaneous self-decomposition reactions. Despite its disadvantageous
instability, we are still interested in its use for click reaction. Because the
reaction has several advantages compared with those of the Huisgen reaction,
such as (i) the 1,3-dipolar cycloaddition of nitrile N-oxide with dipolarophile
does not require catalyst, (ii) the reaction can avoid the use of azide which is
usually explosive, (iii) the reaction is applicable to various unsaturated bonds
as reaction points, and (iv) the reaction with alkene and alkyne proceeds with
C-C bond-formation. Considering these merits, we have developed chemically
stable nitrile N-oxide-based click agents as a new ligation tool.
This review comprises of the following four chapters: in Chapter 1, we
describe the preparation methods of nitrile N-oxide. In Chapter 2, we describe
the 1,3-dipolar cycloaddition reactions and the self-decomposition reactions of
nitrile N-oxide, where the intramolecular cycloaddition of nitrile N-oxide to a
benzene ring is also introduced. In Chapter 3, we discusses the precise
structure of nitrile N-oxide in solution as determined by 13C NMR and UV
spectroscopic studies of 13C-labelled nitrile N-oxides, in addition to the
crystalline structure. In Chapter 4, we introduce the development and
application of nitrile N-oxide-based click agents, such as homoditopic nitrile

N-oxide, orthogonal agents possessing both nitrile N-oxide and electrophile
moieties, and nitrile N-oxide-terminated polymers. In conclusion, the
advantages of nitrile N-oxide in click chemistry is described along with the
future prospects of nitrile N-oxide.
1. PREPARATION METHODS
OF NITRILE N-OXIDE
Nitrile N-oxide can be generated from various precursors. Scheme 1

shows the representative preparation methods of nitrile N-oxide. The oldest
and most investigated method is the dehydrochlorination reaction of
hydroxamoyl chloride with base (Scheme 1, I) [8]. Although a variety of bases
including Et3N are available, the use of molecular sieve 4A (MS 4A) or KF
with weak basicity results in the highly efficient yield of nitrile N-oxide [9].
The diastereo-selective cycloaddition of nitrile N-oxide can be achieved using
a Grignard agent as a result of chelation control [10].
Nitrile N-oxide can also be produced by the dehydration of nitroalkanes
(Scheme 1, II). Although phenylisocyanate is commonly used as a dehydrator
of nitroalkane [11], acetic anhydride [12], Burgess agent (MeO2CNSO2NEt3)
[12], DAST (Et2NSF3) [12], and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholium chloride [13] are also available as dehydrators.
Scheme 1. Preparation of nitrile N-oxide.

The other preparation methods of nitrile N-oxide include the dehydration

of O-silylated hydroxamic acid with Ts2O (Scheme 1, III) [14], oxidation of
oxime using dimethyldioxyrane or Pb(OAc)4 as an oxidant (IV) [15],
oxidative decarboxylation with ceric ammonium nitrate (CAN) (V) [16],
thermal degradation of ethylnitroacetate (VI) [17], and stepwise dehydration
of nitro group including nucleophilic addition of nucleophile to nitroethene
derivative and subsequent dehydration with H2SO4 (VII) [18]. Compared with
I-VI, pathway VII was the most viable method, because it is expected to be
useful for the prompt construction of a nitrile N-oxide library via the addition
of various nucleophiles to the commercially-available nitroethene precursor. In
fact, we developed several nitrile N-oxide agents via pathway VII.
In regards to generation of vic-nitrile N-oxide, retro-1,3-dipolar
cycloaddition of furoxan is an excellent method. Upon heating, strained
furoxan frameworks generate the corresponding vic-nitrile N-oxides as an
equilibrium mixture (Scheme 2) [19]. The vic-nitrile N-oxides were utilized in
cross-linking reactions of linear polymers to give network polymers.
Scheme 2. Retro-1,3-dipolar cycloaddition of strained furoxan to give vic-nitrile N-

oxide.
2. REACTION OF NITRILE N-OXIDE

Nitrile N-oxide serves as either HOMO and LUMO agent in a 1,3-dipolar
cycloaddition reaction, and usually exhibits high reactivity to various
unsaturated bonds, e.g., C = C, C  C, C  N, C = N, C = O, C = S, C = Se, N
= N, C = P(V), C = P(III), C = As, C  P, and N = P(V) [3]. The cycloaddition
reactions of nitrile N-oxide also occur with highly reactive aromatic
compounds such as strained aromatics and multiply fused aromatics.
Benzocyclopropane [20], fullerene [21], anthracene [22], phenanthrene [22],
pyrene [22], perirene [22], and related nitrogen-containing aromatics [22] are
known to be reactive with nitrile N-oxide under harsh conditions. Scheme 3
shows the representative 1,3-dipolar cycloaddition reactions of nitrile N-oxide

to a few dipolarophiles.
Scheme 3. 1,3-Dipolar cycloaddition reactions of nitrile N-oxide to unsaturated bonds.
Despite its high reactivity as described above, nitrile N-oxide has hardly
been utilized as a chemical ligation tool, possibly due to its instability. Nitrile
N-oxide is not ordinarily isolable and spontaneously self-decomposes (Scheme
4). Although the dimerization to furoxan has long been considered the primary
reason for self-decomposition (Scheme 4, I) [8a, 11, 23], degradation via
polymerization (II) [24] and thermal and photochemical isomerization to
isocyanate (III) have also been reported [25].
The self-decompositions (I) and (II) can be suppressed by two methods.
One method is the use of nitrile N-oxide precursor in the presence of an excess
dipolarophile as the reactant. Because the slow generation of nitrile N-oxide
leads to lower concentration of nitrile N-oxide than that of the dipolarophile,
the cycloaddition reaction of nitrile N-oxide with the dipolarophile is
preferential over the self-reactions between the generated nitrile N-oxides. The
second method is the introduction of bulky substituents around nitrile N-oxide
moiety, resulting in the isolation of kinetically-stabilized nitrile N-oxides
(Scheme 4, IV) [26]. When nitrile N-oxide has a quaternary carbon center or
an ortho-disubstituted aromatic ring at the neighboring position of the CNO
moiety, the compounds often become isolable. The substituents suppress the
self-reactions of nitrile N-oxides increase by increasing bulkiness. Some
aromatic nitrile N-oxides with high crystallinity can be isolated as a crystal
even if there is no bulky substituent for kinetic stabilization [27]. However, the
suppression method of self-decomposition III has not been reported. Our
research has demonstrated that the isomerization rate is strongly dependent on

the migration capability of the substituent from a carbon to nitrogen atom [28],
implying that pathway III might be suppressed by the control of electron
demand of the substituent.
Based on this background, we began to develop nitrile N-oxide-based
click agents as useful tools in various research fields, including the
development of homoditopic nitrile N-oxide for homo-junction (Figure 1, A)
[29], an orthogonal agent possessing nitrile N-oxide and an electrophilic
functionality for hetero-junction (B) [28a, 30], and one-pot introduction
method of nitrile N-oxide to prepare versatile molecular framework (C) [28b,
28c], In cases (A) and (B), we first selected an appropriate aromatic skeleton
for yielding the nitrile N-oxide with high crystallinity.
Scheme 4. Possible self-decompositions (I-III) of nitrile N-oxide and the structures of

kinetically stabilized alkyl and aryl nitrile N-oxides (IV).
Figure 1. Homoditopic nitrile N-oxide (A), orthogonal agent (B), and one-pot
introduction of nitrile N-oxide moiety to compound R (C).
Scheme 5. Intramolecular cycloaddition of nitrile N-oxide to non-strained, monocyclic

benzene ring.
In the structural optimization of the aromatic nitrile N-oxide agents, we

fortuitously found the progress of the cycloaddition of nitrile N-oxide to the
non-strained, monocyclic benzene ring located at the neighboring position
(Scheme 5) [31]. The reaction was accompanied by the dearomatization of the
benzene ring, leading to the formation of tetracyclic, partially reduced
skeletons in excellent yield and high regioselectivity. Coustard et al. reported a
related study that a nitrile N-oxide generated in situ in the presence of an
excess amount of trifluoromethane sulfonic acid (TfOH) reacted with the
neighboring benzene ring to give tricyclic isoxazolines in 41-75% yields [32].
To determine the generality of the reaction and the substituent effects of
the ortho-phenoxy group on the stabilization of nitrile N-oxide, we prepared
several 2-phenoxybenzonitrile N-oxide derivatives 2a-f from the
corresponding oximes 1a-f and performed the intramolecular cycloaddition of
2a-f to 3a-f by simple heating (Table 1).
We first studied with 6-methoxy-2-(p-tolyloxy)benzonitrile N-oxide 2a
(R1 = R2 = H, R3 = Me) for the intramolecular cycloaddition. Oxidation of
precursor oxime 1a with N-chlorosuccinimide (NCS) and Et3N in CHCl3 at
0°C gave the corresponding nitrile N-oxide 2a in 91% NMR yield. Nitrile N-
oxide 2a was stably isolated initially but gradually isomerized to 3a even at
room temperature. Therefore, the reaction mixture was directly refluxed for 3
h after confirming the generation of 2a using TLC analysis, to eventually yield
71% of tetracyclic 3a. The structure of 3a was examined by various
spectroscopic analyses and finally determined by the X-ray single crystal
structure analysis. The ORTEP drawing of 3a clearly suggests that the nitrile
N-oxide was added to the benzene ring in a cis-manner. Because the two
carbons at the annulated positions adopt sp [3] hybrid orbitals, the four rings
are not in coplanar. The strain-induced stereo electronic effect can
significantly decrease the acidity at the  position of C = N bond, which could
suppress the re-aromatization of the cyclohexadiene ring via the E1cB reaction
of the oxygen atom. It is expected that the strain of the dihedral angle is
attributed to the steric repulsion between the methoxy group at the peri
position and the C = N bond on the isoxazoline ring.
As shown in Table 1, the reaction of meta-substituted substrate (2b)
proceeded most smoothly to give the cyclization product 3b (entry 2), whereas
only a trace amount of 3c was obtained from ortho-substituted one 2c. These
results suggest that the steric hindrance of o-methyl group toward the nitrile N-
oxide group (2c) may destabilize the transition state of the [3 +
2]cycloaddition reaction, being much different from that of the meta-positions
(2b).
Table 1. Intramolecular cycloaddition of 2-phenoxynitrile N-oxides
entry 1 R1 R2 R3 2 yield/% time/h 3 yield/%

1 1a H H Me 2a 95 3 3a 71
2 1b H Me H 2b 96 3 3b 81
3 1c Me H H 2c quant. 3 3c trace
4 1d H H H 2d 96 4.5 3d 52
5 1e H H OMe 2e 95 3 3e 93
6 1f H H COOEt 2f 96 72 3f 4f: 75
Reprinted with permission from ref [31]. Copyright 2012 American Chemical Society.
We next investigated the electronic effect of the benzene ring on the

reaction (entries 4-6). The reaction of 2d required slightly longer time for the
completion, possibly due to the decrease in the lower electron density of the
benzene ring. The yield of 3d was lower (52%) than those of the other
substrates (71-93%), probably because the further cycloaddition occurred
between 3d and 2d. Conversely, the methoxy-substitution at the para-position
(2e) resulted in the highest yield (3e, 93%). The electron-withdrawing
ethoxycarbonyl group at the para-position (2f) decreased the reaction rate (for
3 d). In addition, the product was not isoxazoline 3f but iminophenol 4f.
Although the detailed reaction mechanism is not clear at present time, it can be
explained by the occurrence of the stepwise reaction consisting of initial
intramolecular cycloaddition of nitrile N-oxide moiety and the subsequent
intramolecular redox reaction between the isoxazoline and cyclohexadiene
rings [6e], which eventually accompany the cleavage of the isoxazoline ring.
Based on these results, the reactivity of the aromatic ring to the nitrile N-
oxide group is concluded to strongly depend on both electronic and steric
factors. The benzene skeleton directly attached by the nitrile N-oxide
functionality is not reactive, whereas the benzene ring at the ortho-
functionality of aromatic nitrile N-oxide is highly reactive. Note that the
substituent pattern of 2c efficiently suppressed the intramolecular self-
decomposition by unabling the aromatization.
3. STRUCTURE OF NITRILE N-OXIDE

The structure of nitrile N-oxide in crystalline state is studied by X-ray
single crystal structural analyses of some stable nitrile N-oxides [4]. Nitrile N-
oxide takes a hetero-cumulene-type linear structure in crystalline state. When
nitrile N-oxide group is directly attached at aromatic ring, the nitrile N-oxide
functionality occupies the same plane as the aromatic ring.
However, several issues remain unclear in terms of the spectroscopic
analysis of nitrile N-oxides in solution. Despite the importance of NMR data
for the structural analysis, 13C NMR chemical shift value of nitrile N-oxide sp
carbon remained a matter of controversy. The reported 13C signals of nitrile N-
oxides (35.6 ppm [33b] and 34.7-42.2 ppm [33c]) are barely detectable as they
are very weak and broad signals [33]. The weak signal intensity of the nitrile
N-oxide sp carbon can be explained by the quadrupolar relaxation mechanism
of the adjacent 14N nucleus [33c].
Scheme 6. Synthesis of 13C-labeled nitrile N-oxide 7 and 8.
Thus, we investigated the synthesis of 13C-labeled nitrile N-oxide and the

re-analysis of the 13C NMR measurement [34] for the purpose of obtaining
reference data for future analyses of nitrile N-oxide-based click agents. The
dynamic character of nitrile N-oxide in solution was also evaluated by 13C
NMR and UV-vis spectra at varying temperature mode (VT-13C NMR and
VT-UV).
Formylation of 1,3-dimethoxybenzene with 13C-labeled DMF (99 atom%)
yielded aldehyde 5 (Scheme 6). The oximation of 5 and the successive
treatment of 6 with NCS afforded the corresponding nitrile N-oxide 7. The
reaction of 6 to nitrile N-oxide 7 proceeded in a quantitative yield without any
self-decomposition to isolate 7 as a stable material after purification using
silica gel column chromatography. 13C-Labeled nitrile N-oxide 8 was also
prepared in a similar manner.
Figure 2 shows 13C NMR spectra of 13C-labeled compounds (5, 6, 7, and
8). In the spectrum of 5 (a), the intense singlet signal at 190 ppm is assigned to
the carbon of the aldehyde. The carbon (Ca) adjacent to the 13C-labeled carbon
appears at 118 ppm as a doublet signal based on the scalar coupling of a C-C
bond. In the spectrum of 6 (b), the labeled signal is shifted to 144 ppm by the
conversion to the oxime. In the nitrile N-oxide 7 spectrum (c), the labeled
signal appears at 35 ppm as a triplet signal in good agreement with the
chemical shift observed by Paton [34b]. The labeled signal of the other nitrile
N-oxide 8 was also observed at a similar chemical shift (38 ppm). The results
could provide clear proof for the chemical shift of the nitrile N-oxide carbon in
the 13C NMR spectrum. The nitrile N-oxide carbon signals were remarkably
upfield-shifted compared with those expected from unsaturated carbons such
as carbonyl and nitrile carbons, clearly indicating the strong shielding effect of
the nitrile N-oxide group.
The reason for the triplet splitting of the signal is caused by the scalar
coupling to the adjacent 14N nucleus (I = 1), and the coupling constants (1JCN)
are 53.5 Hz for 7 and 43.2 Hz for 8. The magnitudes are significantly larger
than those of typical nitrogen-containing compounds [35].
We performed VT-13C NMR measurements of 7 and 8 to evaluate the
temperature dependence on the electronic and/or structural characteristics of
them. Figure 3 shows the VT-13C NMR spectrum of 7. The labeled signal was
split to a triplet signal in the temperature region higher than room temperature,
whereas the signal fused to a singlet at low temperature. The signal pattern
change was reversible, indicating the reversible electronic and/or structural
change of the nitrile N-oxide moiety in the NMR time scale. The similar
reversible pattern change was also observed in the VT-13C NMR spectra of 8.
Figure 2. 13C NMR spectra (100 MHz, 293 K, CDCl3) of (a) 5, (b) 6 (in DMSO-d6), (c)
7, and (d) 8. Reprinted with permission from ref [34]. Copyright 2015 Elsevier.
Figure 3. VT-13C NMR spectra of 7 (100 MHz, CDCl3) at (a) 313 K, (b) 293 K, (c) 273
K, (d) 253 K, (e) 243 K, (f) 233 K, and (g) 223 K. Reprinted with permission from ref
[34]. Copyright 2015 Elsevier.
Figure 4 displays the plots of 1JCN, the chemical shift of nitrile N-oxide
carbon, 1JCC, and the chemical shift of Ca, as a function of temperature. Both
plots of 1JCN and 13C NMR chemical shift in (A) monotonously changed with
inflections at 260 and 300 K, respectively, implying the electronic and/or
structural change of 7 at the temperature around the inflections. The plots of
1J
CC and the chemical shift of Ca in (B) clearly contrast those in (A), which had
no inflection point. These results indicate that the chemical environment
around the C-N bond of the nitrile N-oxide moiety could be parted on the
inflection temperature, whereas that of the C-C bond between the nitrile N-
oxide moiety and the aromatic ring was hardly influenced by temperature. The
temperature dependencies of the coupling constants and chemical shifts of 8
were almost similar to those of 7.
Figure 4. (A) Effects of temperature on 1JCN of 7 (circle) and 13C chemical shift of
nitrile N-oxide (square) and (B) effects of temperature on 1JCC between nitrile N-oxide
and Ca of 7 (square) and 13C chemical shift of Ca (circle).
The VT-UV-vis spectra of 7 and 8 provided clear information concerning

the mutation of the nitrile N-oxide moiety triggered by heating (Figure 5). The
spectra exhibited the reversible thermochromism. Upon heating,
hyperchromism was observed around 300 nm, indicating an increase in
effective conjugation length along with an increase in temperature. We also
plotted the molar absorption of 7 and 8 as a function of temperature, in which
the plots had the inflections around 280-290 K. The inflection temperature
seems to meet with those in Figure 5 (A).
Figure 5. VT-UV-vis spectra of 7 (THF, 100 M). Reprinted with permission from ref
[34]. Copyright 2015 Elsevier.
Figure 6. Possible temperature-dependent structural change of nitrile N-oxide in

solution.
These phenomena observed in the VT-13C NMR and VT-UV-vis spectra

indicate the dynamic structural change of the nitrile N-oxide moiety in
solution. One of the plausible changes is shown in Figure 6. Heating an
aromatic nitrile N-oxide having ortho-substituents modulates its structure from
a bent conformation to a coplanar conformation toward the benzene ring. The
bent conformation at low temperature is induced by the steric repulsion
between the nitrile N-oxide moiety and the ortho-substituents, which seems to
be in clear contrast to the coplanar structure of the nitrile N-oxides in a solid
state. Recently, Hosoya et al. have proposed a twisted structure of an aromatic
azide with ortho-disubstituents, being similar to our results [36].
4. SYNTHESIS AND APPLICATIONS OF

NITRILE N-OXIDE-BASED CLICK AGENTS
Based on the results described in Chapters 1-3, we developed several
nitrile N-oxide-based click agents. We first synthesized a homoditopic nitrile
N-oxide 9 derived from a stable nitrile N-oxide skeleton 3c as shown in
chapter 2 [29]. Scheme 7 shows the synthetic scheme for 9 [29a]. We selected
commercially available 4,4’-isopropylidene-bis(2,6-dimethylphenol) as a
spacer between two nitrile N-oxide groups, by considering both the utility of
the 3c skeleton and the good solubility of the product 9. Aryl etherification of
2-fluoro-6-methoxybenzaldehyde with a bisphenol yielded 91% of a
bisaldehyde. Subsequent oximation and oxidation with NCS yielded the stable
homoditopic nitrile N-oxide 9 as a solid (overall yield: 88%). The reaction
protocol was scalable up to 20 g. We also synthesized another homoditopic
nitrile N-oxide 10 from 2-hydroxy-1-naphthaldehyde [37]. The chemical
stability of 9 and 10 was sufficiently high that it could be stored for 1 year in a
refrigerator without any decomposition.
Scheme 7. Synthesis of homoditopic nitrile N-oxide 9 and the structure of 10.
Both 9 and 10 served as a catalyst-free chemical ligation tool between

unsaturated bonds. We demonstrated the catalyst-free polycycloaddition
reaction of 9 and 10 to diene, diyne, and dinitrile to give the corresponding
heterocycle-containing polymers in excellent yields [29a, 37].
Polyisoxazole prepared by polycycloaddition of homoditopic nitrile N-
oxide to diyne is available for the reductive transformation of the isoxazole
moiety to other groups without the cleavage of C-C bond in the main chain
(Scheme 9). The isoxazole moiety can be transformed to -aminoalcohol and
iminoenol (or enaminoketone) moiety [38]. Poly(-aminoalcohol) thus formed
was highly reactive to various electrophiles and easily cross-linked by ditopic

electrophiles [38]. The enaminoketone skeleton could be used as a ligand and
derived to fluorescent poly(boron enaminoketonate)s [37].
Scheme 8. Catalyst-free click polymerization using homoditopic nitrile N-oxide.
Scheme 9. Reductive polymer transformation of polyisoxazole to polymers having -

aminoalcohol and iminoenol (or enaminoketone) moieties.
The homoditopic nitrile N-oxides are also useful as a cross-linker for

polymers possessing unsaturated bonds. Various common polymers such as
polyacrylonitrile (PAN), nitrile-butadiene rubber (NBR), natural rubber (NR),
and ethylene-propylene-diene terpolymer (EPDM) were efficiently cross-
linked by a simple mixing with 9 at a low temperature around 60°C (Scheme
10) [39]. Rubbers and elastomers underwent the solvent-free cross-linking
reaction, which was also sufficiently facilitated by a hot press fabrication to
give a big film or sheet.
The utility of homoditopic nitrile N-oxides is seen in not only
polymerization and cross-linking but also chemical ligation between highly
sophisticated elements. We have reported on the simple construction of
interlocked molecules such as rotaxane [40], polyrotaxane [41], and catenane
[42], and the development of a fluorescence switch system exploiting the
rotaxane skeleton [43].
Scheme 10. Catalyst-free click cross-linking of common polymers using homoditopic

nitrile N-oxide.
We next examined the development of a different type of click agent to

provide the hetero-junction of building units [28a, 30]. In particular, we
designed and synthesized orthogonal agents possessing a stable nitrile N-oxide
moiety and an electrophilic functionality including epoxide (11), ester (12),
oxetane (13), and Meldrum’s acid (ketene equivalent, 14) as shown in Figure
7.
Figure 7. Structures of orthogonal agents.
Scheme 11 illustrates a typical example of the one-pot modification of

polyacrylonitrile (PAN) using 14 [30a]. A mixture of PAN, 14, and
polyethylene glycol monomethylether (MeO-PEG-OH) in DMF was heated at
60°C in the presence of CaSO4, which resulted in the introduction of 14
skeleton into PAN via the catalyst-free 1,3-dipolar cycloaddition of the nitrile
N-oxide moiety to the nitrile group. After stirring for 1 d at the same
temperature, the reaction temperature was increased up to 160°C, leading to
the thermal decomposition of the Meldrum’s acid moiety to generate a reactive
ketene. The following nucleophilic addition of the terminal alcohol of PEG to
the ketene efficiently proceeded to yield PEG-grafted PAN in one-pot. As a
result of the sequential processes, the orthogonal agent 14 was found to act as
a molecular glue between the nitrile groups on PAN and the alcohol on PEG
because the 1,3-dipolar cycloaddition and the nucleophilic addition proceeded
stepwise without competing reactions.
Scheme 11. Catalyst-free connection between PAN and PEG mediated by the
orthogonal agent 14.
We also investigated the development of a method for introducing a nitrile

N-oxide functionality to versatile molecular frameworks [28b, 28c]. One-pot
introducing method of nitrile N-oxide exploiting 1,1-diphenylnitroethene [28b]
or trans--nitrostyrene [28c] as a key compound was developed. As shown in

Scheme 12, the treatment of 1,1-diphenynitroethene with BuLi as a
nucleophile afforded a Michael adduct, which was followed by the
dehydration with H2SO4 to yield a stable aliphatic nitrile N-oxide having a
quaternary carbon center at the adjacent position to the nitrile N-oxide moiety.
This reaction was applied to the propagation end modification of living
polymer anions to form the corresponding nitrile N-oxide-terminated vinyl
polymers (Scheme 12).
Scheme 12. General synthetic pathways of polymer nitrile N-oxide.
A bulky initiator for anionic polymerization was first prepared from sec-
BuLi and 1,1-diphenylethene in THF at -78C. The living anionic
polymerization started by the addition of a vinyl monomer to the initiator.
After the polymerization, 1,1-diphenylnitroethene or trans--nitrostyrene as a
terminator was added to the polymerization mixture to lead to the
functionalization of the propagation end by the terminator. After the
confirmation of the consumption of the terminator by TLC, conc. H2SO4 (10
eq.) was added in one portion to the reaction mixture. The resulting mixture
was warmed up to 0°C and stirred for 30 min to complete the dehydration
reaction. Typical work-up and purification by reprecipitation afforded the
corresponding polymer nitrile N-oxide in an excellent yield. This method is
applicable to various vinyl polymers except for polystyrene homopolymer.
Polymer nitrile N-oxide prepared from styrene was not stable and
spontaneously self-decomposed. In contrast, when different polymer segment

except for polystyrene was introduced at the polystyrene terminus,
polystyrene-based nitrile N-oxide having block copolymer structure showing
significant stability was obtained, although it contained a polystyrene segment.
Intramolecular cycloaddition of the nitrile N-oxide moiety to the neighboring
benzene ring of the polystyrene terminal at the adjacent position as shown in
Chapter 2 would be the most plausible mechanism [31], although the precise
decomposition pathway is not yet clear.
Various grafting reactions of the PMMA-based polymer nitrile N-oxide
(P1, Mn 2,200, Mw/Mn 1.2) were investigated (Scheme 13). Press grinding of
polymer containing allyl group (poly-1) with 1.0 eq. of P1 in a mortar at
200°C under air afforded a quantitative yield of P1-grafted Poly-1 (Scheme
13a).
Scheme 13. Grafting reactions of polymer nitrile N-oxide onto unsaturated bond-
containing polymers.
It is noteworthy that the covalent bond connection between two

macromolecules proceeded with the complete conversion despite the use of a
stoichiometric amount of P1 to the olefin moiety. The grafting reaction of
natural rubber (NR) with P1 took place with low efficiency, likely due to the
instability of NR to oxygen and the low reactivity of the sterically hindered
trisubstituted olefin moiety of NR (Scheme 13b). P2 with a less hindered
nitrile N-oxide (Mn 1600, Mw/Mn 1.24) exhibited relatively higher reactivity
than P1. Meanwhile, the grafting reactions of P1 onto PAN and polyacetylene
P3 with internal acetylenes quantitatively yielded the corresponding graft
copolymers (Scheme 13c and d). These results emphasize the potential usage
of this direct grafting method via the cycloadditions of polymer nitrile N-oxide
to various dipolarophiles including common polymers and materials.
Nitrile N-oxide-type click agents can be used for surface modifications,
because the catalyst-free reactions of them are available in solid state, such as
the modifications of the surfaces of allyl-functionalized glass, unsaturated
bond-containing resins, and rubbers [44]. The surface modification can be
achieved just by coating the nitrile N-oxide agents onto such surfaces and
successive heating at an arbitrary temperature. Both hydrophobic and
hydrophilic surfaces were prepared from perfluoroalkyl group-containing
nitrile N-oxide (RF-CNO) [45] and poly(methacrylic acid)-based polymer
nitrile N-oxide (RPMA-CNO), respectively (Scheme 14) [28c].
Scheme 14. Surface modification using nitrile N-oxide-based click agents.
In addition, surface modification using orthogonal agent (RMDA-CNO)

enables flexible integration of a variety of materials via the covalent bond
formation [30b]. We successfully fabricated several surfaces by the
modification with PEG and perfluoroalkyl groups in addition to functional
moieties such as photochromic spiropyran group which leads to a photo-

responsive surface, via stepwise reactions comprising of initial formation of
electrophilic surface and subsequent reaction of it with nucleophilic modifier
[30]. The results can suggest that combining the orthogonal agent-modified
surface and appropriate supramolecules can easily provide a more unique
surface possessing supramolecular property.
CONCLUSION
This review article deals with the development and application of nitrile
N-oxide-type click agents. Kinetically stabilized nitrile N-oxides displayed
sufficiently high reactivity to various unsaturated bond-containing molecules,
macromolecules, and materials involving surfaces under catalyst- and solvent-
free conditions. Because the internal olefins and nitrile groups in the polymer
chain are available as the click reaction points for nitrile N-oxides, the polymer
modifications by the click chemistry using nitrile N-oxides mentioned above
might develop a new polymer chemistry exploiting common polymers as a
scaffold for molecular integration. Although the reactions of nitrile N-oxides
have been utilized in the creation of limited organic systems [46] at present
time, the use of nitrile N-oxide agent will be widely spread by recognizing
their advantageous merits such as C-C bond forming catalyst-free reaction
besides the their stability and safely in comparison with azides. Some of these
nitrile N-oxide-type click agents will be commercially available soon. We
hope that the present review article contributes to the creation of versatile
sophisticated and useful organic systems in the near future.
ACKNOWLEDGMENTS
The authors thank all co-workers and collaborators for their great
contributions to the studies covered in this review. Moreover, the authors are
grateful to the financial support from JSPS KAKENHI (Grant Numbers:
22750101, 21106508, 24685023, and 25102510), the Mizuho Foundation for
the Promotion of Science, and the Eno Foundation for the Promotion of
Science.
REFERENCES
[1] (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click chemistry: diverse
chemical function from a few good reactions, Angew. Chem., Int. Ed.,
2001, 40, 2004-2021; (b) Tornøe, C. W.; Christensen, C.; Meldal, M.,
Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific
Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to
Azides, J. Org. Chem., 2002, 67, 3057-3064.
[2] Takata, T.; Koyama, Y.; Fukase, K., In: Click Chemistry: Fundamentals
and Practical Technologies, CMC Publishing Co. Ltd., Tokyo, 2014.
[3] Belen’Kii, L., I. In: Feuer, H. Nitrile Oxides, Nitrones, and Nitronates in
Organic Synthesis (2nd ed.), Wiley, New York, 2008, p 1-128.
[4] (a) Shiro, M.; Yamakawa, M.; Kubota, T.; Koyama, H., Molecular
structure of 4-methoxy-2,6-dimethylbenzonitrile N-oxide, Chem.
Commun., 1968, 1409-1410; (b) Shiro, M.; Yamakawa, M.; Kubota, T.,
The structure of 4-methoxy-2,6-dimethylbenzonitrile N-oxide, 4-bromo-
2,6-dimethylbenzonitrile N-oxide, and 2,4,6-trimethylbenzonitrile N-
oxide, Acta Cryst., 1979, B35, 712-716; (c) Stoyanovich, F. M.;
Krayushkin, M. M.; Mamaeva, O. O., Stable O-sulfamoylbenzonitrile
oxides, Gazz. Chim. Ital., 1993, 123, 39-44.
[5] (a) Miyazaki, H.; Nishikida, K.; Kubota, T., Anion radicals produced by
the nonaqueous polarographic reduction of several benzonitrile N-oxide,
Bull. Chem. Soc. Jpn., 1971, 44, 277-278; (b) Gilbert, B. C.; Malatesta,
V.; Norman, R. O. C., Reactions of aromatic nitrile N-oxide with organic
radicals. New type of spin-trapping reagent, J. Am. Chem. Soc., 1971,
93, 3290-3291; (c) Sosonkin, I. M.; Domarev, A. N.; Kuznetzova, A. L.;
Niyazymbetov, M. E.; Petrosyan, V. A., Electrochemical behavior of
nitrile N-oxides, Izv. Akad. Nauk. Ser. Khim., 1989, 281-286; (d)
Morozov, V. I.; Buzykin, B. I.; Il’yasov, A. V., Phosphorylformonitrile
oxides as spin traps for carbon-centered free radicals: Co-formation with
radicals in the generation of nitrile oxides from C-
phosphorylformohydroximoyl halides in the presence of alcohols or
ethers, Russ. Chem. Bull., 2005, 54, 342-347.
[6] (a) Yamashita, Y.; Hirano, Y.; Takada, A.; Takikawa, H.; Suzuki, K.,
Total synthesis of the Antibiotic BE-43472B, Angew. Chem., Int. Ed.,
2013, 52, 6658-6661; (b) Maimone, T. J.; Shi, J.; Ashida, S.; Baran, P.
S., Total synthesis of Vinigrol, J. Am. Chem. Soc., 2009, 131, 17066-
17067; (c) Takada, A.; Hashimoto, Y.; Takikawa, H.; Hikita, K.; Suzuki,
K., Total Synthesis and Absolute Stereochemistry of Seragakinone A,
Angew. Chem., Int. Ed., 2011, 50, 2297-2301; (d) Wright, B. J. D.;
Hartung, J.; Peng, F.; de Water, R. V.; Liu, H.; Tan, Q.-H.; Chou, T.-C.;
Danishefsky, S. J., Synthesis of Pluraflavin A “Aglycon,” J. Am. Chem.
Soc., 2008, 130, 16786-16790; (e) Koyama, Y.; Yamaguchi, R.; Suzuki,
K., Total synthesis and structure assignment of the anthrone C-glycoside
cassialoin, Angew. Chem., Int. Ed., 2007, 47, 1084-1087.
[7] Sustman, R.; Trill, H., Substituent effects in 1,3-dipolar cycloadditions
of phenyl azide, Angew. Chem., Int. Ed., 1972, 11, 838-840.
[8] Quilico, A.; Fusco, R., New researches in the isoxazole group. IV. The
action of benzohydroxamic chlorides on -ketonic esters, Gazz. Chim.
Ital., 1937, 67, 589-603.
[9] (a) Kim, J. N.; Ryu, E. K., 1,3-Dipolar cycloaddition: molecular sieve
assisted generation of nitrile oxides from hydroximoyl chlorides,
Heterocycles, 1990, 31, 1693-1697; (b) Kim, J. N.; Chung, K. H.; Ryu,
E. K., Alkali metal fluoride promoted generation of hydroximoyl
chlorides, Heterocycles, 1991, 32, 477-480.
[10] Kanemasa, S.; Nishiuchi, M.; Kamimura, A.; Hori, K., First Successful
Metal Coordination Control in 1,3-Dipolar Cycloadditions. High-Rate
Acceleration and Regio- and Stereocontrol of Nitrile Oxide
Cycloadditions to the Magnesium Alkoxides of Alkylic and Homoallylic
Alcohols, J. Am. Chem. Soc., 1994, 116, 2324-2339.
[11] Mukaiyama, T.; Hoshino, T., The reactions of primary nitro paraffins
with isocyanates, J. Am. Chem. Soc., 1960, 82, 5339-5342.
[12] Maugein, N.; Wagner, A.; Mioskowski, C., New conditions for the
generation of nitrile oxides from primarynitroalkanes, Tetrahedron Lett.,
1997, 38, 1547-1550.
[13] Giacomelli, G.; Luca, L. D., Porcheddu, A., A method for generating
nitrile oxides from nitroalkanes: a microwave assisted route for
isoxazoles, Tetrahedron, 2003, 59, 5437-5440.
[14] Muri, D.; Bode, J. W.; Carreira, E. M., A novel, general method for the
synthesis of nitrile oxides: dehydration of O-silylated hydroxamic acids,
Org. Lett., 2000, 2, 539-543.
[15] (a) Tanaka, S.; Sawamoto, S.; Yamamoto, T.; Yamada, K., A mild and
convenient method of generation of nitrile oxides by dimethyldioxirane
oxidation, Nippon Kagaku Kaishi, 1992, 420-422; (b) Barrow, S. J.;
Easton, C. J., Exploiting the 1,3-dithiane of 2-oxopropanenitrile oxide to
limit competing dimerization in 1,3-dipolar cycloaddition reactions,
Tetrahedron Lett., 1997, 38, 2175-2178.
[16] Arai, N.; Iwakoshi, M.; Tanaka, K.; Narasaka, K., Generation of nitrile
oxides from oxime derivatives by the oxidation with ammonium
hexanitratocerate(IV), Bull. Chem. Soc. Jpn., 1999, 72, 2277-2285.
[17] Huffman, B. S.; Schultz, R. A.; Schlom, P. J., Novel reagents for heat-
active polymer cross-linking, Polym. Bull., 2001, 47, 159-166.
[18] Yao, C.-F.; Kao, K.-H.; Liu, J.-T.; Chu, C.-M.; Wang, Y.; Chen, W.-C.;
Lin, Y.-M.; Lin, W.-W.; Yan, M.-C.; Liu, J.-Y.; Chuang, M.-C.; Shiue,
J.-L., Generation of nitroalkanes, hydroximoyl halides and nitrile oxides
from the reactions of -nitrostyrenes with Grignard or organolithium
reagents, Tetrahedron, 1998, 54, 791-822.
[19] Crosby, J.; Rennie, R. A. C.; Tanner, J.; Paton, R. M., US, US 3931106
(1976).
[20] Nitta, M.; Sogo, S.; Nakayama, T., Cycloaddition reaction of
benzocyclopropene with aromatic nitrile oxides: a synthetic entry into a
bridged oxazonine, Chem. Lett., 1979, 8, 1431-1434.
[21] (a) Meier, M. S.; Poplawska, M., Addition of nitrile oxides to C60:
formation of isoxazoline derivatives of fullerenes, J. Org. Chem., 1993,
58, 4524-4525; (b) Meier, M. S.; Poplawska, M.; Compton, A. L.; Shaw,
J. P.; Selegue, J. P.; Guarr, T. F., Preparation and Isolation of Three
Isomeric C70 Isoxazolines: Strong Deshielding in the Polar Region of
C70, J. Am. Chem. Soc., 1994, 116, 7044-7048; (c) Irngartinger, H.;
Weber, A., Twofold cycloaddition of [60]fullerene to a bifunctional
nitrile oxide, Tetrahedron Lett., 1996, 37, 4137-4140; (d) Irngartinger,
H.; Weber, H.; Escher, T.; Fettel, P. W.; Gassner, F., Synthesis of
isoxazolo[60]fullerenes with dumb-bell-type structure and atropisomeric
properties, Eur. J. Org. Chem., 1999, 2087-2092.
[22] (a) Corsaro, A.; Librando, V.; Chiacchio, U.; Pistarà, V., 1,3-Dipolar
cycloaddition reaction of polycyclic aromatic hydrocarbons with 3,5-
dichloro-2,4,6-trimethyl- and 2,4,6-trimethylbenzonitrile oxide,
Tetrahedron, 1996, 52, 13027-13034; (b) Corsaro, A.; Librando, V.;
Chiacchio, U.; Fisichella, S.; Pistarà, V., 1,3-Dipolar cycloadditions of
polycyclic aromatic hydrocarbons with nitrile oxides under microwave
irradiation in the absence of solvent, Heterocycles, 1997, 45, 1567-1572;
(c) Corsaro, A.; Librando, V.; Chiacchio, U.; Pistarà, V.; Rescifina, A.,
Cycloaddition of nitrile oxides to aza-analogs of phenanthrene,
Tetrahedron, 1998, 54, 9187-9194; (d) Corsaro, A.; Pistarà, V.;
Rescifina, A.; Piperno, A.; Chiacchio, M. A.; Romeo, G., A DFT
rationalization for the observed regiochemistry in the nitrile oxide
cycloaddition with anthracene and acridine, Tetrahedron, 2004, 60,

6443-6451.
[23] (a) Quilico, A., Advances in nitrile oxide chemistry, Experientia, 1970,
26, 1169-1183; (b) Barbaro, G.; Battalia, A.; Dondoni, A., Kinetics and
mechanism of dimerization of benzonitrile N-oxide to furazan N-oxide,
J. Chem. Soc. (B), 1970, 4, 588-592.
[24] De Sarlo, F.; Guarna, A.; Brandi, A.; Mascagni, P., Behavior of nitrile
oxides towards nucleophiles. VI. Synthesis and polymerization of
aliphatic nitrile oxides, Gazz. Chim. Ital., 1980, 110, 341-344.
[25] (a) Grundmann, C.; Kochs, P., Nitrile oxide. 14. Investigation of the
nitrile oxide-isocyanate rearrangement, Angew. Chem., 1970, 82, 635;
(b) Grundmann, C.; Kochs, P.; Boal, J. R., Nitrile oxides. XV. Thermal
isomerization of nitrile oxides to isocyanates, Liebigs Ann. Chem., 1972,
761, 162-181.
[26] (a) Beltrame, P.; Veglio, C.; Simonetta, M., Kinetic and mechanism of
1,3-cycloaddition of a substituted benzonitrile oxide to a series of
arylacetylenes, J. Chem. Soc. B, 1967, 867-873; (b) Zinner, G.;
Guenther, H., Hydroxylamine derivatives. XVII. Aliphatic nitrile oxides,
Angew. Chem., 1964, 76, 440; (c) Matsuura, T., Bode, J. W.; Hachisu,
Y.; Suzuki, K., Molecular sieve (MS 4A) promoted cyclocondensation
of hindered, aromatic nitrile oxides and cyclic diketones under mild
conditions, Synlett, 2003, 1746-1748.
[27] Hayashi, J.; Furukawa, J.; Yamashita, S., Vulcanization of rubbers by
1,3-dipolar addition reactions. V. Vulcanization of EPDM
[ethylene/propylene/diene terpolymer] with several dinitrile oxide,
Nippon Gomu Kyokaishi, 1970, 43, 807-817.
[28] (a) Koyama, Y.; Miura, K.; Cheawchan, S.; Seo, A.; Takata, T., Cascade
functionalization of unsaturated bond-containing polymers using
ambident agents possessing both nitrile N-oxide and electrophilic
functions, Chem. Commun., 2012, 48, 10304-10306; (b) Wang, C.-G.;
Koyama, Y.; Yonekawa, M.; Uchida, S.; Takata, T., Polymer nitrile N-
oxides directed toward catalyst- and solvent-free click grafting, Chem.
Commun., 2013, 49, 7723-7725; (c) Wang, C.-G.; Koyama, Y.; Uchida,
S.; Takata, T., Synthesis of Highly Reactive Polymer Nitrile N-Oxides
for Effective Solvent-Free Grafting, ACS Macro Lett., 2014, 3, 286-290.
[29] (a) Lee, Y.-G.; Yonekawa, M.; Koyama, Y.; Takata, T., Synthesis of a
kinetically stabilized homoditopic nitrile N-oxide directed toward
catalyst-free click polymerization, Chem. Lett., 2010, 39, 420-421; (b)
Lee, Y.-G.; Koyama, Y.; Yonekawa, M.; Takata, T., Synthesis of Main-
Chain-Type Polyrotaxanes by New Click Polymerization Using

Homoditopic Nitrile N-Oxides via Rotaxanation-Polymerization
Protocol, Macromolecules, 2010, 43, 4070-4080; (c) Koyama, Y.;
Yonekawa, M.; Takata, T., New click chemistry: click polymerization
via 1,3-dipolar addition of homo-ditopic aromatic nitrile oxides formed
in situ, Chem. Lett., 2008, 37, 918-919.
[30] (a) Cheawchan, S.; Koyama, Y.; Uchida, S.; Takata, T., Catalyst-free
click cascade functionalization of unsaturated-bond-containing polymers
using masked-ketene-tethering nitrile N-oxide, Polymer, 2013, 54, 4501-
4510; (b) Cheawchan, S.; Uchida, S.; Sogawa, H.; Koyama, Y.; Takata,
T., Thermotriggered Catalyst-Free Modification of a Glass Surface with
an Orthogonal Agent Possessing Nitrile N-Oxide and Masked Ketene,
Langmuir, 2016, 32, 309-315.
[31] Yonekawa, M.; Koyama, Y.; Kuwata, S.; Takata, T., Intramolecular 1,3-
dipolar cycloaddition of nitrile N-oxide accompanied by
dearomatization, Org. Lett., 2012, 14, 1164-1167.
[32] Soro, Y.; Bamba, F.; Siaka, S.; Coustard, J. M., One-step synthesis of
diazadihydroacenaphthylene derivatives with an isoxazoline ring,
starting from 1-benzylamino-1-methylsulfanyl-2-nitroethenes,
Tetrahedron Lett., 2006, 47, 3315-3319.
[33] (a) Christl, M.; Warren, J. P.; Hawkins, B. L.; Roberts, J. D., Carbon-13
and nitrogen-15 nuclear magnetic resonance spectroscopy of nitrile
oxides and related reaction products. Unexpected carbon-13 and
nitrogen-15 nuclear magnetic resonance parameters of 2,4,6-
trimethylbenzonitrile oxide, J. Am. Chem. Soc., 1973, 95, 4392-4397; (b)
Mitchell, W. R.; Paton, R. M., Isolation of nitrile oxides from the
thermal fragmentation of furazan N-oxides, Tetrahedron Lett., 1979, 20,
2443-2446; (c) De Sarlo, F.; Brandi, A.; Guarna, A., A multinuclear
magnetic resonance study of nitrile oxides, J. Magn. Reson., 1982, 50,
64-70.
[34] Koyama, Y.; Lee, Y.-G.; Kuroki, S.; Takata, T., Synthesis, 13C NMR,
and UV spectroscopic study of 13C-labeled nitrile N-oxide, Tetrahedron
Lett., 2015, 56, 7038-7042.
[35] Levy, G. C.; Lichter, R. L., In: Nitrogen-15 Nuclear Magnetic
Resonance Spectroscopy, John Wiley and Sons, Inc., New York, 1979.
[36] Yoshida, S.; Shiraishi, A.; Kanno, K.; Matsushita, T.; Johmoto, K.;
Uekusa, H.; Hosoya, T., Enhanced clickability of doubly sterically-
hindered aryl azides, Sci. Rep., 2011, 1, 82, 4 pp.
[37] Matsumura, T.; Koyama, Y.; Uchida, S.; Yonekawa, M.; Yui, T.;
Ishitani, O.; Takata, T., Fluorescent poly(boron enaminoketonate)s:
synthesis via the direct modification of polyisoxazoles obtained from the
click polymerization of a homoditopic nitrile N-oxide and diynes,
Polym. J., 2014, 46, 609-616.
[38] Lee, Y.-G.; Koyama, Y.; Yonekawa, M.; Takata, T., New Click
Chemistry: Polymerization Based on 1,3-Dipolar Cycloaddition of a
Homo Ditopic Nitrile N-Oxide and Transformation of the Resulting
Polymers into Reactive Polymers, Macromolecules, 2009, 42, 7709-
7717.
[39] Koyama, Y.; Takata, T., In: Kadooka, M.; Shirai, M. Crosslinking and
Degradation of Polymers III, CMC Publishing Co. Ltd., Tokyo, 2012, p
82-92.
[40] Matsumura, T.; Ishiwari, F.; Koyama, Y.; Takata, T., C-C Bond-
Forming Click Synthesis of Rotaxanes Exploiting Nitrile N-Oxide, Org.
Lett., 2010, 12, 3828-3831.
[41] (a) Jang, K.; Miura, K.; Koyama, Y.; Takata, T., Catalyst- and Solvent-
Free Click Synthesis of Cyclodextrin-Based Polyrotaxanes Exploiting a
Nitrile N-Oxide, Org. Lett., 2012, 14, 3088-3091; (b) Koyama, Y.;
Suzuki, Y.; Asakawa, T.; Kihara, N.; Nakazono, K.; Takata, T., Polymer
architectures assisted by dynamic covalent bonds: synthesis and
properties of boronate-functionalized polyrotaxane and graft
polyrotaxane, Polym. J., 2012, 44, 30-37; (c) Iguchi, H.; Uchida, S.;
Koyama, Y.; Takata, T., Polyester-Containing -Cyclodextrin-Based
Polyrotaxane: Synthesis by Living Ring-Opening Polymerization,
Polypseudorotaxanation, and End Capping Using Nitrile N-Oxide, ACS
Macro Lett., 2013, 2, 527-530.
[42] Yuki, T.; Koyama, Y.; Matsumura, T.; Takata, T., Click Annulation of
Pseudo[2]rotaxane to [2]Catenane Exploiting Homoditopic Nitrile N-
Oxide, Org. Lett., 2013, 15, 4438-4441.
[43] Koyama, Y.; Matsumura, T.; Yui, T.; Ishitani, O.; Takata, T.,
Fluorescence Control of Boron Enaminoketonate Using a Rotaxane
Shuttle, Org. Lett., 2013, 15, 4686-4689.
[44] (a) Koyama, Y.; Seo, A.; Takata, T., Polymer modification exploiting
stable nitrile N-oxide, Nippon Gomu Kyokaishi, 2011, 84, 111-116; (b)
Koyama, Y.; Wang, C.-G.; Miura, K.; Cheawchan, S.; Seo, A.; Takata,
T., Development of nitrile N-oxide agents directed toward catalyst-free
solid-state modification of common polymers, Hyomen, 2012, 50, 228-
238; (c) Koyama, Y.; Takata, T., Efficient Grafting and Cross-Linking
Reaction Exploiting Nitrile N-Oxide Agents via Click Protocol, Nippon

Gomu Kyokaishi, 2014, 87, 96-102.
[45] Wang, C.-G.; Cheawchan, S.; Qiagedeer, A.; Monjiyama, S.; Uchida, S.;
Koyama, Y.; Takata, T., Synthesis of Perfluorocarbon-containing Nitrile
N-Oxide for Catalyst- and Solvent-Free Direct Surface Modification,
manuscript in preparation.
[46] (a) Koyama, Y.; Takata, T., Polymer synthesis exploiting nitrile N-
oxide, Kobunshi Ronbunshu, 2011, 68, 147-159; (b) Altintas, O.;
Glassner, M.; Rodriguez-Emmenegger. C.; Welle, A.; Trouillet, V.;
Barner-Kowollik, C., Macromolecular Surface Design: Photopatterning
of Functional Stable Nitrile Oxides, Angew. Chem., Int. Ed., 2015, 54,
5777-5783; (c) Singh, I.; Zarafshani, Z.; Lutz, J.-F.; Heaney, F., Metal-
Free “Click” Chemistry: Efficient Polymer Modification via 1,3-Dipolar
Cycloaddition of Nitrile Oxides and Alkynes, Macromolecules, 2009,
42, 5411-5413; (d) Grecian, S.; Fokin, V. V., Ruthenium-Catalyzed
Cycloaddition of Nitrile Oxides and Alkynes: Practical Synthesis of
Isoxazoles, Angew. Chem., Int. Ed., 2008, 120, 8409-8411.
Chapter 3
BI-1,2,3-TRIAZOLES: SYNTHESIS
AND PERSPECTIVES
Ivette Santana-Martinez and Erick Cuevas-Yañez*

Centro Conjunto de Investigación en Química Sustentable UAEM-UNAM,
Toluca, México
Universidad Autónoma del Estado de México, Toluca, Mexico
ABSTRACT
Bi-1,2,3-triazoles are interesting molecules derived from copper-
catalyzed alkyne-azide cycloaddition (CuAAC) under oxidative
conditions. Other methods to synthesize bi-1,2,3-triazoles involve
condensation reactions or palladium catalyzed couplings. On the other
hand, bi-1,2,3-triazoles have attracted to many research groups due to
their promising properties as ligands for catalysis as well as the design of
compounds with photochemical properties.
1. INTRODUCTION
4,4’-(1,2,3-triazolyl)-1,2,3-triazoles, namely, bi-1,2,3-triazoles (1, Scheme
1), are interesting molecules derived in most cases from copper-catalyzed
alkyne-azide cycloaddition (CuAAC) under oxidative conditions. Since their
*
Corresponding Author Email: ecuevasy@uaemex.mx
52 Ivette Santana-Martinez and Erick Cuevas-Yañez
discovery, bi-1,2,3-triazoles have attracted considerable attention due to their

promising properties as ligands for catalysis and other applications.
Bi-1,2,3-triazoles present a similar structure to other biaryl compounds
such as binaphthyls like BINOL [1] 2 and derivatives which are widely used
as chiral ligands and reagents as well as bipyridines 3 [2], the most extended
kind of heterocyclic biaryl compounds that are also often used in asymmetric
catalysis as chiral ligands. Other less common heterocyclic biaryl systems are
biimidazoles 4 and 5 [3], bipyrazoles [4] 6 and 4,4’-bi-1,2,4-triazoles 7 [5]. All
these compounds have been used in catalysis as ligands using their ability to
coordinate to metal centers.
In contrast, the chemistry of bi-1,2,3-triazoles have been less studied. This
is due in part to there being few synthetic methods to obtain these kinds of
compounds. However, in recent years reports that describe the synthesis of bi-
1,2,3-triazoles have increased considerably. In this revision, synthetic
protocols reported for bi-1,2,3-triazoles will be analyzed from a future
application perspective.
Scheme 1. Important biaryl compounds.
Scheme 2. Formation of bi-1,2,3-triazole 9 through cycloaddition of compound 8.

Bi-1,2,3-Triazoles 53
2. SYNTHESIS OF BI-1,2,3-TRIAZOLES
2.1. Condensations and Non-Catalyzed Cycloadditions
Before the arrival of the Click Chemistry approach, the synthesis of bi-
1,2,3-triazoles was limited to some condensation/cycloaddition reactions.
Initial reports by Dornow and Rombusch showed that bisacetylenes, such as
molecule 8, in the presence of phenyl azide gave bi-1,2,3-triazole 9 under
thermal conditions (Scheme 2) [6]. A similar behavior was observed by
Tikhonova et al. using benzyl azide with uncoupled and conjugated
diacetylenes [7].

Other sources of bi-1,2,3-triazoles proceed from oxidative processes.

Through a destructive nitration, triazolyl acetone 10 produced bi-1,2,3-triazole
11 [8], whereas bi-1,2,3-triazole 13 was obtained from the oxidation of
tricyclic compound 12 (Scheme 3) [9].
Moreover, p-toluenesulfonyl azide and α,β-Unsaturated Imine 14
underwent a condensation to yield bi-1,2,3-triazole 18 through successive
formation of intermediate 16 and subsequent cyclization of diazo derivative 17
(Scheme 4) [10].
2.2. Copper-Catalyzed Alkyne-Azide Cycloaddition
Although Copper-Catalyzed Alkyne-Azide Cycloaddition (CuAAC), the

most used click reaction, is not the only chemical process that allows the
synthesis of bi-1,2,3-triazoles, it is the most important synthetic procedure to
prepare this kind of compounds, with the highest number of reports about
synthesis displaying a wide range of variations [11].
In its original context, the CuAAC reaction has been used for the selective
synthesis of 1,2,3-triazoles. However, the discovery of bi-1,2,3-triazoles as
additional products, obtained initially in trace amounts, motivated the
development of a new approach to the study of this reaction.
In 2007, Angell and Burgess documented the first report about the
formation of bi-1,2,3-triazoles from CuAAC reaction in the presence of
inorganic bases, optimizing the process from trace amounts to moderate yields
[12]. In this study, the use of the base proved decisive because inorganic bases,
such as Na2CO3, generated mixtures of triazoles bi-1,2,3-triazoles in contrast
to the traditional organic bases used in CuAAC like DIPEA which provide
exclusively 1,2,3-triazoles (Scheme 5). However, the sensitivity to steric
hindrance represents an important limitation in this process.
This pioneering work inspired several research groups. They have
investigated and studied this process thoroughly in order to improve yields and
develop new synthetic protocols for bi-1,2,3-triazoles by changing the reaction
conditions.
One of the first questions that concerned chemists is related to the reaction
mechanism. In this regard, the Straub group supplied some insights based on
mechanistic studies by Fokin and Finn, and proposed the formation of a
copper triazolide as an intermediate in the catalytic cycle in CuAAC reactions
[13]. They demonstrated this fact by synthesizing the copper triazolide 20
derived from sterically hindered copper acetylide 19 (Scheme 6) and noted
that the rate-determining step in the intermediate conversion to triazole

depends on protonation of the triazolide copper complex and therefore the rate
of conversion is determined by the acidity. In addition, the isolation of
triazolide 20 suggests that this molecule can participate as an intermediate in
the catalytic cycle which would produce triazoles and bi-1,2,3-triazoles under
the same conditions.
Scheme 5. Formation of bi-1,2,3-triazole 9 from CuAAC reaction.
Scheme 6. Formation of triazolide 20 from copper acetylide 19.
Scheme 7. Synthesis of alkynyl triazole 24.
Other groups have focused on studying the cycloaddition of bisacetylenes

and derivatives under the idea that a bisacetylene intermediate is produced in a
former step during the formation of bi-1,2,3-triazole. A fact that supports this
hypothesis is that alkynyl triazole 24 is obtained from the CuAAC reaction
between benzyl azide 21 and pentyne 22 catalyzed by a mixture of CuI, N-
methylmorpholine N-oxide, DIPEA and a nucleophilic amine as 23 (Scheme

7) [14].
Fiandanesse and his coworkers designed a methodology for the synthesis
of bi-1,2,3-triazoles from consecutive CuAAC reactions on buta-1,3-diynyl-
trimethyl-silane 26; in an earlier reaction, bis acetylene 26 experimented a
cycloaddition in the presence of an organic azide and a catalytic Cu(OAc)2 to
afford alkynyl triazole 27 which in turn reacted with diverse azides using CuI
as catalyst and 1,1,4,7,7-pentamethyldiethylenetriamine as an additive
(Scheme 8) [15]. This methodology proved useful in the preparation of
unsymmetrical bi-1,2,3-triazoles. A variation of this protocol was described by
the Simpson group using both TMS- and TIPS-bisacetylenes and
CuSO4/sodium ascorbate catalytic system [16].
Scheme 8. Synthesis of bi-1,2,3-triazole 28.
Scheme 9. Synthesis of 1,1’-disubsituted bi-1,2,3-triazole 32.
Other efforts to prepare alkynyl triazoles and bi-1,2,3-triazoles are shown

in Scheme 9 and consist of converting 4-hydroxymethyl-1,2,3-triazole 29 or
bis-(trimethylsilylacetylene) 30 into 4-alkynyl-1,2,3-triazole 31 which is then
transformed into the target compound 32 using similar reaction conditions
described in previous reports [17]. Thus, unsymmetrical 1,1’-disubsituted bi-

1,2,3-triazoles are synthesized through this “double click” strategy.
The formation of alkynyl triazoles can be modulated and directed to the
synthesis of bi-1,2,3-triazoles without an intermediate isolation step. The
Zhang group established a protocol for controllable synthesis of bi-1,2,3-
triazoles and alkynyl triazoles using copper bromide as a catalyst,
demonstrating that the use of a strong base like sodium ethoxide promotes the
efficient bi-1,2,3-triazole generation at 0°C in ca. 90% yield. However, this
effect is negligible at higher reaction temperatures. When the reaction
temperature is increased to 60°C, bi-1,2,3-triazoles were not detected.
Therefore, the reaction temperature plays an important role in regulating the
distribution of reaction products derived from the oxidative CuAAC reaction
(Scheme 10) [18].
The conditions and catalysts in which the bi-1,2,3-triazole formation
occurs are diverse. The simplest catalytic system involves the use of a copper
(I) salt and a preferably inorganic base. On this premise, Cuevas-Yañez and
their coworkers studied the bi-1,2,3-triazole formation process using copper (I)
iodide and sodium hydroxide and they observed a similar temperature effect
which enhances the bi-1,2,3-triazole ratio, obtaining the best yields at -35°C
(Scheme 11) [19]. Another important factor was the sodium hydroxide
concentration. The best bi-1,2,3-triazole yields are related to high
concentrations of sodium hydroxide (10 molar equivalents). These results
suggest that bi-1,2,3-triazoles would probably be kinetically controlled
products under oxidative conditions. This pattern was also observed using
CuSO4-glucose as catalytic source instead of a copper (I) salt [20], as well as
solventless conditions through direct treatment of alkynes and azides with
NaOH and catalytic CuI [21]. Despite the triazole/bi-1,2,3-triazole formation
is still unclear, the presence of a parent intermediate precursor as a putative
copper triazolide, could explain the observations made in these studies.
Scheme 10. Synthesis of bi-1,2,3-triazole 1 and alkynyl triazole 33.

Scheme 11. Synthesis of bi-1,2,3-triazole 1 and triazole 34.
The group of Jeon reported an evaluation of the catalyst/base effect on

CuAAC between acetylenic amides 35 and alkyl azides, demonstrating that
CuI/DIPEA/DMF system catalyzes the bi-1,2,3-triazole 37 formation as major
product, whereas the use of a CuSO4/Na ascorbate/tBuOH:H2O system is
selective for triazole formation (Scheme 12) [22].
The use of a simple copper (I) salt in combination with a base provides a
versatile method for the preparation of bi-1,2,3-triazoles with a high functional
group tolerance. For example, the group of Tajima verified a CuAAC reaction
on terminal alkyne 38 containing a fullerene moiety with hexyl azide to afford

bi-1,2,3-triazole 39 using pentamethyl diethylenetriamine as basic ligand and
copper bromide as a catalyst (Scheme 13) [23]. This group noted that the
presence of oxygen is a determining factor in the reaction which allows bi-
1,2,3-triazole obtaining as opposed to the use of inert atmospheric conditions
which promotes only triazole formation.
Xu and coworkers reported the oxidative CuAAC reaction catalyzed by
copper (I) chloride and mediated by a polysiloxane supported secondary amine
42 at 0°C with the objective of preparing bi-1,2,3-triazoles 43 from benzyl
azides 40 and alkynes 41 (Scheme 14) [24]. The authors suggest that
compound 42 helps the oxidative process because a mononuclear bistriazole-
copper complex is stabilized and dispersed by the secondary amine-functional
polysiloxane allowing bi-1,2,3-triazole formation.
A particularly challenging group of molecules to functionalize are
carbohydrates. Through oxidative CuAAC, β-D-glucosyl azide 44 is converted
to bi-1,2,3-triazole 46 in 56% yield [25]. A noteworthy feature is that the use
of CuCl gave the expected product while other copper (I) salts as CuBr or CuI
yielded only 1-glucosyl-1,2,3-triazoles 45 (Scheme 15), indicating a
counterion influence in this process.
Scheme 14. Synthesis of bi-1,2,3-triazole 39 mediated by amine 42.
Scheme 15. Synthesis of bi-1,2,3-triazole 46 from glucosyl azide 44.

However, the role played by the counterion or ligand is not clear. For
instance, when a CuI/Et3N mixture was used to synthesize bi-1,2,3-triazoles 49
and 54. The compound 49 was obtained as the only reaction product in 40%
yield from diazidoferrocene 47 and tetrahydro-[5]-helicenequinone 48
(Scheme 16), contrary to the use of CuSO4/sodium ascorbate which generates
exclusively 1,2,3-triazole [26]. In this case, a cyclic copper triazolide is
proposed in order to rationalize the formation of a chiral compound and the
stability required to favor the bi-1,2,3-triazole formation.
A similar behavior was observed when bisazide 50 was treated with the
corresponding alkynes in the presence of catalytic amounts of CuI/DIPEA to
yield bistriazoles 52 and cyclic bi-1,2,3-triazoles 51 as major products
(Scheme 17) [27]. Moreover, the best bi-1,2,3-triazole yields were detected
using bulkier alkynes which can be an additional factor for oxidative CuAAC
dimerization.
On the other hand, compound 54 was obtained in 23% yield as a mixture
together with the corresponding triazole 55 (Scheme 18) [28]. Therefore, a
definitive conclusion is not possible to determine from these results.
Other catalytic systems designed for the synthesis of bi-1,2,3-triazoles
involve the use of copper (I) complexes. N-heterocyclic Carbene copper (I)
complex 58 catalyzed the last step of a multicomponent synthesis of triazoles
in water at room temperature through the generation of a micellar medium
which promotes the in situ formation of organic azides followed by oxidative
CuAAC to produce compounds 59 and 60 (Scheme 19) [29].
In addition, the effect of phenylboronic acid/CuSO4 catalyst on the
CuAAC reaction was studied by López-Ruiz et al. which found that when
benzyl azide 21 and 4-Ethynylbenzonitrile 61 were combined at room
temperature, a mixture of triazole 62 and bi-1,2,3-triazole 69 was obtained
(Scheme 20) [30]. The oxidative CuAAC process was possible due to the
phenylboronic acid’s reducing power at room temperature, which produces
copper (I) species for the synthesis of the bi-1,2,3-triazole product.
According to Zhu and coworkers, copper (II) acetate catalyzes the
formation of bi-1,2,3-triazoles from organic azides and terminal alkynes using
K2CO3 and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) as
additives under an oxygen atmosphere (Scheme 21) [31].

Scheme 21. Synthesis of bi-1,2,3-triazoles catalyzed by Cu(OAc)2 under an oxygen

atmosphere.
2.3. Homocoupling Reactions
The Rowan’s group developed a synthesis for cyclic bi-1,2,3-triazole 65

from 1,2-Bis(1’-Benzyl-5’-iodotriazolyl)benzene 64 using a palladacycle
catalyst (Scheme 22) [32]. This cross coupling reaction proved to be a very
efficient method for preparing compound 65 instead of direct oxidative
CuAAC reaction on 1,2-Diethynylbenzene which gave only traces of bi-1,2,3-
triazole product.
Scheme 23. Synthesis of metal complexes 68 and 71 from bi-1,2,3-triazoles 66 and 69.
3. REACTIONS OF BI-1,2,3-TRIAZOLES
In spite of bi-1,2,3-triazole chemistry having been recently developed, the
applications found for this class of compounds are quite promising. Two
particular properties should be taken into account to understand the chemistry
of bi-1,2,3-triazoles, the presence of a series of consecutive lone pair electrons
around nitrogen atoms, and the high aromaticity/low reactivity from the
heterocyclic moiety. These properties contribute to the role of bi-1,2,3-
triazoles as nucleophiles or ligands.
The nucleophilic properties of bi-1,2,3-triazoles were observed and used
almost simultaneously by the groups of Bertrand [33] and Aizpurua [34, 35]
on bi-1,2,3-triazoles 66 and 69 which were methylated to give bitriazolium
salts 67 and 70 respectively (Scheme 23). These bitriazolium salts were
departure materials to synthesize metal complexes 68 and 71 through the
formation of abnormal N-heterocyclic carbene ligands which were bound to
metal centers as both monodentate or bidentate ligands.
The tendency of carbenes derived from bitriazolium salts to react as
bidentate ligands is displayed in bitriazolium salt 72 that was functionalized
and coordinated to ruthenium and iridium salts to generate complexes 73 and
74 (Scheme 24). These compounds were used as catalysts in the conversion of
nitrobenzene to various products through a hydrogenation transfer process
[36], as well as catalytic oxygenations [37].
Scheme 24. Synthesis of metal complexes 73 and 74 from bi-1,2,3-triazolium salt 72.
Scheme 25. Synthesis of metal complexes 77 and 78.
On the other hand, bi-1,2,3-triazoles can bind directly to a metal through

nitrogen atoms. For example, dibenzyl bi-1,2,3-triazole 76 was combined with
ruthenium complex 75 affording a new bi-1,2,3-triazolyl complex 77 which
exhibits metal-to-ligand charge transfer absorption bands at 425 and 446 nm

respectively showing a progressive blue-shift in the absorption on increasing
the bi-1,2,3-triazolyl ligand content when compared to compound 75 (Scheme
25) [38]. In Addition, these kinds of complexes can undergo a photochemical
ejection of the bi-1,2,3-triazole ligand in the absence of any promotion through
steric congestion to generate cis-bis(solvent) complexes such as molecule 78
where bi-1,2,3-triazolyl ligand is exchanged by acetonitrile using a domestic
fluorescent lamp [39].
Similar iridium complexes are luminescent and show structured emission
bands with vibronic progressions at 532 and 568 nm [40]. Unlike ruthenium
systems, osmium (II) bi-1,2,3-triazole complexes display intense emission in
the far-red/near-infrared and one of these complexes exhibits highly intense
blue emission. Thus, light-emitting electrochemical cell (LEC) devices were
fabricated from these complexes [41].
These examples demonstrate that bi-1,2,3-triazoles represent an emerging
group of compounds which are useful points of departure en route to catalysts
or compounds with novel photochemical properties. Moreover, the synthesis
of bi-1,2,3-triazoles is effected under mild conditions, and is broad in scope.
These characteristics suggest that bi-1,2,3-triazoles will enjoy widespread
application.
REFERENCES
[1] Parmar, D.; Sugiono, E.; Raja, S.; Rueping. M. Chem. Rev. 2014, 114,
9047. (b) Brunel, J. M. Chem. Rev. 2005, 105, 857. (c) Chen, Y.; Yekta,
S.; K. Yudin, A. K. Chem. Rev. 2003, 103, 3155.
[2] Kaes, C.; A.; Katz, A.; Hosseini, M. W. Chem. Rev. 2000, 100, 3553.
[3] Murata, T.; Yakiyama, Y.; Nakasuji, K.; Morita, Y. Cryst. Growth Des.
2010, 10, 4898. (b) Kennedy, D. C.; and Brian R. James, B. R. Can. J.
Chem. 2010, 88, 886. (c) Murata,T.; Morita, Y.; Yakiyama, Y.;
Yamamoto, Y.; Yamada, S.; Nishimura, Y.; Nakasuji, K. Cryst. Growth
Des. 2008, 8, 3058. (d) Zhang, W.; Landee, C. P.; Willett, R. D.; M.
Turnbull, M. M. Tetrahedron 2003, 59, 6027. (e) Morita, Y.; Murata, T.;
Yamada, S.; Tadokoro, M.; Ichimura, A.; Nakasuji, K. J. Chem. Soc.
Perk. T. 1 2002, 2598. (f) Cliff, M. D.; Pyne, S. G. Synthesis 1994, 681.
[4] Baig, R. B. N.; Varma, R. S. Green Chem. 2013, 15, 398. (b) Luque, R.;
Baruwati, B.; Varma, R. S. Green Chem. 2010, 12, 1540.
[5] Liu, X.; Gao, W.; Sun, P.; Su, Z.; Sanping Chen, S.; Wei, Q.; Xie, G.;
Gao, S. Green Chem. 2015, 17, 331. (b) Wang, X. L.; Cao, J. J.; Liu, G.
C.; Tian, A. X.; Luan, J.; Lin, H. Y.; Zhang, J. W.; Li, N. Cryst. Eng.
Comm. 2014, 16, 5732.
[6] Dornow, A.; Rombusch, K. Chem. Ber. 1958, 91, 1841.
[7] Tikhonova, L. G.; Serebryakova, E. S.; Vereshchagin, L. I. Zh. Org.
Khim. 1982, 18, 1619.
[8] Semenov, V. V.; Shevelev, S. A.; Bruskin, A. B.; Kanishchev, M. I.;
Baryshnikov, A. T. Russ. Chem. Bull. Int. Ed. 2009, 58, 2077.
[9] Sainsonov, V. A.; Volodarskii, L. B.; Korolev, V. L.; Khisamutdinov, G.
K. Chem. Heterocycl. Compd. 1993, 29, 1169.
[10] Bátori, S.; Bokotey, S.; Messmer, A. ARKIVOC 2012, (v), 146.
[11] Zheng, Z. J.; Wang, D.; Xu, Z.; Xu, L. W. Beilstein J. Org. Chem. 2015,
11, 2557.
[12] Angell, Y.; Burgess, K. Angew. Chem. Int. Ed. 2007, 46, 3649.
[13] Nolte, C.; Mayer, P.; Straub, B. F. Angew. Chem. Int. Ed. 2007, 46,
2101.
[14] Gerard, B.; Ryan, J.; Beeler, A. B.; Porco, J. A. Tetrahedron 2006, 62,
6405.
[15] Fiandanese, V.; Bottalico, D.; Marchese, G.; Punzi, A.; Francesca
Capuzzolo, F. Tetrahedron 2009, 65, 10573.
[16] Doak, B. C.; Scanlon, M. J.; Simpson, J. S. Org. Lett. 2011, 13, 537.
[17] Aizpurua, J. M.; Azcune, I.; Fratila, R. M.; Balentova, E.; Sagartzazu-
Aizpurua, M.; Miranda, J. I. Org. Lett. 2010, 12, 1584.
[18] Li, L.; Fan, X.; Zhang, Y.; Zhu, A.; Zhang, G. Tetrahedron 2013, 69,
9939.
[19] González, J.; Pérez, V. M.; Jiménez, D. O.; Lopez-Valdez, G.; Corona,
D.; Cuevas-Yañez, E. Tetrahedron Lett. 2011, 52, 3514.
[20] García, M. A.; Ríos, Z. G.; González, J.; Pérez, V. M.; Lara, N.; Fuentes,
A.; González, C.; Corona, D.; Cuevas-Yañez, E. Lett. Org. Chem. 2011,
8, 701.
[21] Pateraki, M.; Morales-Ortiz, G. K.; López-Guzmán, A.; Fuentes-Benites,
A.; Cuevas-Yañez, E. Bulg. Chem. Commun. 2016, 48, 253.
[22] Kwon, M.; Jang, Y.; Yoon, S.; Yang, D.; Jeon, H. B. Tetrahedron Lett.
2012, 53, 1606.
[23] Miyanishi, S.; Zhang, Y.; Hashimoto, K.; Tajima, K. Macromolecules
2012, 45, 6424.
[24] Zheng, Z. J.; Ye, F.; Zheng, L. S.; Yang, K. F.; Lai, G. Q.; Wen Xu, L.
W. Chem. Eur. J. 2012, 18, 14094.
[25] Goyard, D.; Chajistamatiou, A. S.; Sotiropoulou, A. I.; Chrysina, E. D.;
Praly, J. P.; Vidal, S. Chem. Eur. J. 2014, 20, 5423. (b) Goyard, D.;
Praly, J. P.; Vidal, S. Carbohyd. Res. 2012, 362, 79.
[26] del Hoyo, A. M.; Latorre, A.; Diaz, R.; Antonio Urbano, A.; Carreño, M.
C. Adv. Synth. Catal. 2015, 357, 1154.
[27] Oladeinde, O. A.; Hong, S. Y.; Holland, R. J.; Maciag, A. E.; Keefer, L.
K.; Saavedra, J. E.; Nandurdikar, R. S. Org. Lett. 2010, 12, 4256.
[28] Key, J. A.; Cairo, C. W.; Ferguson, M. J.; Acta Crystallogr. E 2008,
E64, o1910.
[29] Elena Tasca, E.; La Sorella, G.; Sperni, L.; Strukul, G.; Scarso, A. Green
Chem. 2015, 17, 1414.
[30] de la Cerda-Pedro, J. E.; Rojas-Lima, S.; Santillan, R.; López-Ruiz, H. J.
Mex. Chem. Soc. 2015, 59, 130.
[31] Brassard, C. J.; Zhang, X.; Brewer, C. R.; Liu, P.; Clark, R. J.; Lei Zhu,
L. J. Org. Chem. 2016, 81, 12091.
[32] Jurícek, M.; Stout, K.; Kouwer, P. H. J.; Rowan, A. E. J. Porphy.
Phthalocya. 2011, 15, 898.
[33] Guisado-Barrios, G.; Jean Bouffard, J.; Donnadieu, B.; Bertrand, G.
Organometallics 2011, 30, 6017.
[34] Aizpurua, J. M.; Sagartzazu-Aizpurua, M.; Azcune, I.; Miranda, J. I.;
Monasterio, Z.; García-Lecina, E.; Fratila, R. M. Synthesis 2011, 2737.
[35] Aizpurua, J. M.; Sagartzazu-Aizpurua, M.; Monasterio, Z.; Azcune, I.;
Mendicute, C.; Miranda, J. I.; García-Lecina, E.; Altube, A.; Fratila, R.
M. Org. Lett. 2012, 14, 1866.
[36] Hohloch, S.; Suntrup, L.; Sarkar, B. Organometallics 2013, 32, 7376.
[37] Hohloch, S.; Kaiser, S.; Duecker, F. L.; Bolje, A.; Maity, R.; Košmrlj, J.;
Sarkar, B. Dalton T. 2015, 44, 686.
[38] Welby, C. E.; Grkinic, S.; Adam Zahid, A.; Uppal, B. S.; Gibson, E. A.;
Rice, C. R.; Elliott, P. I. P. Dalton T. 2012, 41, 7637.
[39] Welby, C. E.; Armitage, G. K.; Bartley, H.; Sinopoli, A.; Uppal, B. S.;
Elliott, P. I. P. Photochem. Photobiol. Sci. 2014, 13, 735.
[40] Welby, C. E.; Gilmartin, L.; Marriott, R. R.; Zahid, A.; Rice, C. R.;
Gibson, E. A.; Elliott, P. I. P. Dalton T. 2013, 42, 13527.
[41] Ross, D. A. W.; Scattergood, P. A.; Babaei, A.; Pertegás, A.; Bolink, H.
J.; Elliott, P. I. P. Dalton T. 2016, 45, 7748.
Chapter 4
CLICK CHEMISTRY OF NATURAL POLYMERS
Yu-Tong Zhang, Zhu-Yun Li and Yu Chen*

Beijing Institute of Techology, Beijing, China
ABSTRACT
In recent years, natural polymers, such as chitosan, cellulose, starch,
alginate, collagen, gelatine, chondroitin sulphate, hyaluronic acid,
heparin, and so on, have been attracted more and more attentions in
different fields for their versatile properties, such as biocompatibility,
biodegradability, nontoxicity, econony and environmental friendly. The
above natural polymers are also easy to be modified by chemical grafting
of new functional groups to promote their properties.
Nowadays, click chemistry has become a powerful tool for materials
modification by material chemists for their number of advantages such as
readily available starting material, high reaction rate, high reliability, mild
reaction conditions, high oxygen and moisture stability, good
stereoselectivity, high yield, simple workup and easy purification. Click
chemistry not only has broad applications in synthetic organic chemistry,
but also has been employed in immobilizing small molecules, linear
polymers, dendrimers and biological macromolecules into the skeleton of
natural polymers. Modification of natural polymers by click chemistry
will help to overcome their shortages such as complicated reaction
*
70 Yu-Tong Zhang, Zhu-Yun Li and Yu Chen
conditions, low selectivity, various side reactions and low yields, and
remarkably improve their immobilizing efficiency.
In the current chapter, we want to introduce the progress in
modification of the natural polymers through the click reactions, and the
application of the above composite in different fields.
Keywords: natural polymers, click chemistry, modification, application
INTRODUCTION
Proposed by Sharpless and coworkers in 2001, click chemistry is the most
excellent chemical reactions to connect a diversity of structures. There are
about 4 kinds of “click” chemistry: ① The Cu (I) catalyzed azide-alkyne 1, 3 -
dipolar cycloaddition, including Diels-Alder reactions [1]. Among these
reactions, Cu (I) catalyzed Huisgen 1, 3-dipolar cycloaddition of azide and
terminal alkyne functionalities to form 1, 2, 3-triazoles, which called “cream
of the crop” in the field of “click chemistry.” But it is difficult in the
modification of polysaccharides introducing the azide or alkyne functionalities
necessary for the subsequent CuAAc reaction. ②Nucleophilic ring opening
reaction, especially the tension of heterocyclic electrophilic reagents open
loop. ③Non-alcoholic aldehyde carbonyl reactions. ④Addition reactions of
carbon-carbon bonds, especially the oxidation of epoxidation reaction.
The substance of “click chemistry” is to choosing the raw material which
is easy to get, achieving the linking of C-X-C by reliable, efficient and
selective chemistry reactions. It is a low cost, efficient way to synthesizing lots
of new compounds. The core of “click” chemistry is to using a series of
reliable and modular reactions to produce heteroatom-containing
compounds [2].
These days, ‘click’ chemistry explores some new approach in organic and
polymer synthesis, involving a number of functional groups, reactions. It has a
lot of good features, such as wide range of applications, mild reaction
conditions, high selectivity and high purity. For these outstanding
characteristics, “click” chemistry reactions have been widely applied in many
research areas.
This chapter mainly introduces the way for application of different nature
polymers including application of natural polymers derivatives, natural
polymers hydrogels, natural polymers nanomaterials and the linked
fluorophore in the natural polymers via “click” chemistry.
Click Chemistry of Natural Polymers 71
1. CLICK CHEMISTRY APPLICATION

Click chemistry has been being used in natural polymers’ field from 2001
when it was invented. For example, click chemistry can have some assistance
in the natural polymers derivatives, natural polymers hydrogels, natural
polymers nanomaterials, fluorescent natural polymers and so on. However,
click chemistry still has vast potential for future development and more and
more scientists devote themselves to the click chemistry of natural polymers.
1.1. Click Chemistry Application of Natural

Polymers Derivatives
Many scientists bend themselves to the research and preparation of

chitosan and cellulose such as C6 quaternary ammonium chitosan derivative,
Side-Chain type benzoxazine-functional cellulose and so on. However,
traditional preparation ways are inefficiency and reaction conditions are also
complex. Therefore, efficiency and easy preparation methods which synthetic
chemists search for all the time are the important research field. Click
chemistry has the good prospect because of mild reaction conditions, simple
ways, less outgrowth and other advantages.
1.2. C6 Quaternary Ammonium Chitosan Derivative
Recently, we explored a new method to prepare C6 quaternary ammonium

chitosan (CTS) derivatives via click chemistry. The C2-NH2 of CTS was
protected by benzaldehyde firstly. The C6-OH of CTS was then transformed
into a sulfonyl ester, and then sulfonyl ester was reacted with NaN3 through
nucleophilic substitution to introduce the -N3 group at the CTS C6 position.
This intermediate was reacted with a terminal alkynyl quaternary ammonium
salt in a click chemistry reaction; then it was followed by the deprotection of
C2-NH2 with acid to furnish the C6 quaternary ammonium CTS derivative
(CTS-6-DMPOAB). The structures and properties of synthesized products
were characterized by all kinds of characterization methods in the reactions.
By using the largest inhibition zone test and the MIC test, we compared the
inhibitory effects of CTS and CTS-6-DMPOAB against S. aureus and E. coli.
We found that the prepared CTS-6-DMPOAB had obviously improved
antibacterial activity toward S. aureus and E. coli compared to CTS [3].
Figure 1. Reaction routes for the preparation of a C6 quaternary ammonium CTS

derivative through the CTS Schiff base with click chemistry.
1.3. Hollow Tubes [4]-A Novel Cellulose-Click-Chitosan Polymer
Chitosan and cellulose are both reproducible and abundant polymers on

earth. The high density of free hydroxyl groups on cellulose polymer chains
and the reactive amino groups on the structural units of chitosan make
cellulose and chitosan suitable to be chemically modified for advanced and
high-valued biopolymers. New concepts of molecular design require perfect
tailoring and allow extensive freedom to manipulate by chemical and/or
biochemical coupling. Therefore, attempts to introduce functional groups into
macromolecules followed by development of uniform structure via chemical
reaction have attracted a great deal of interest. Functional biopolymers were
triumphantly prepared by the introduction of terminal alkyne and azide groups
onto the cellulose and chitosan chains, respectively. The cellulose-click-
chitosan polymer was made via click reaction, that is, the Cu (I)-catalyzed
Huisgen 1, 3-dipolar cycloaddition reaction, between the terminal alkyne
groups of cellulose and the azide groups on the chitosan backbone at room
temperature. It was found that cellulose-click-chitosan polymer had a high
thermal stability, which indicate its superb potential in the applications of
heating-tolerable materials. What’s more, some hollow tubes with near
millimeter length were also found.
Figure 2. Synthesis of Pg-CE, CH-N3, and cellulose-click-chitosan.
1.4. Chitosan-Oxanorbornadiene [5]
New chitosan derivatives, chitosan-oxanorbornadienes, were synthesized

enebling metal free click chemisty. The hydroxyl and the amine groups at the
polymeric chain acted as nucleophiles to show the reaction with the NHS-
oxanorbornadiene. The high degree of oxanorbornadiene substitution on
chitosan (80%) could be achieved when oxanorbonadiene was used with a
certain spacer chain length in the form of a NHS-active ester. A series of
water-based model reactions at room temperature between chitosan-

oxanorbonadiene (CS-3) and azido-carboxylic acid derivative, disulfide
derivative, and silane derivative proved a successful triazole linkage.
Therefore, the chitosan oxanorbornadiene derivative, combined with metal-
free click chemistry, is an expedient derivative to provide simple ligation to
other functional molecules like polymers or inorganic particles (e.g.,
magnetite, gold, silica particles) and makes chitosan useful in advanced
applications, such as biomedical field.
Figure 3. Synthesis of Chitosan-Oxanorbornadiene.
1.5. Locating Substitution Derivatives
There is a novel method [6] which prepares the macrocyclic compound

locating substitution derivatives of chitosan, by using cyclodextrin as the
model of macrocyclic compound. This method combines the goodness of
activated 6-OH of chitin and high efficiency of click reaction. Chitin C6-OH p-
toluenesulfonate (CTN-6-OTs) was generated and subsequently transferred to
chitin C6-N3 via nucleophilic substitution. Next, β-cyclodextrin was
immobilized at 6-OH of chitin via click reaction to afford CTN-6-CD.
Eventually, CTS-6-CD was obtained by removing the acetyl group of chitin
unit. It was found that CTN-6-CD synthesized at the optimum conditions has a
stable loading of 1.6126 × 10−4mol/g and that of the corresponding CTS-6-CD,
generated by removal of the acetyl group, was 1.6891 × 10−4 mol/g. The
prepared 6-OH substituted macrocyclic compound derivatives of chitosan with
high loading capacity have superb application prospect in the field of chemical
biosensor, slow release drug carrier, chromatographic support, and so on.
Figure 4. Synthesis of CTS-6-CD via click reaction.
1.6. Regioselective Sequential Modification of Chitosan
Recently, the attention of researchers has been drawn to the synthesis of

chitosan derivatives [7] and their nanoparticles with improved antimicrobial
activities. In this study, chitosan derivatives with different azides and alkyne
groups were synthesized using click chemistry, and these were further
transformed into nanoparticles by using the ionotropic gelation method.
Nanoparticles of synthesized derivatives were made by ionic gelation to form
complexes of polyanionic penta-sodium tripolyphosphate and cationic

chitosan derivatives. The derivatives and corresponding nanoparticles were
assessed in vitro for antibacterial and antifungal activities against three gram-
positive and gram-negative bacteria and three fungal strains, respectively. The
hemolytic assay on erythrocytes and cell viability test on two disparate cell
lines (Chinese hamster lung fibroblast cells V79 and Human hepatic cell line
WRL68) demonstrated the safety; suggesting that these derivatives could be
utilized in future medical applications. Chitosan derivatives with triazole
functionality, synthesized by Huisgen 1, 3-dipolar cycloaddition, and their
nanoparticles indicated important enhancement in antibacterial and antifungal
activities in comparison to those attached with native, non-altered chitosan.
Figure 5 indicates 4 chitosan derivatives. Synthetic scheme of chitosan azide
functional derivatives.
Figure 5. Chitosan derivatives. (i) phthalic anhydride,DMF 5%(v/v), 8h,N2

atmosphere,120℃, (ii) THF,CDI, 5h,N2 atmosphere, 40℃, propargylamine, THF,24h,
25℃, (iii) sodium ascorbate, copper (II) acetate, (1-azidoadamantane/ azidobenzene/1-
azidomethyl-2-methyl benzene/ azidomethyl phenyl sulphide/ 2-azidomethy-l-1-boc
pyrolidine), tertbutanol/ water, 24h, 25℃, (iv) NH2NH2.H2O,water, 18h, 100℃.
1.7. Side-Chain Type Benzoxazine-Functional Cellulose [8]
A side-chain type benzoxazine-functional cellulose which using click

chemistry has been developed via the reaction of the alkynyl of the ethynyl-
monofunctional benzoxazine monomer and azide-functional cellulose. The
crosslinking reaction of the benzoxazine side-chain unusually takes place at
low-temperatures in comparison to an ordinary benzoxazine resins. Upon
crosslinking, the polymer exhibits high char yield of 40%, which is a marked
improvement from a just 4% of the unfunctionalized cellulose. Combining
cellulose with benzoxazine could possibly make a more environmental-
friendly material than benzoxazine synthesized by conventional method using
petroleum-derived raw materials only. It also produces more flame resistant
material than pure cellulose fiber.
Figure 6. Preparation of 3-ethynyl monofunctional benzoxazine monomer.

And benzoxazine-functional cellulose.
1.8. Surface Functionalization of Cellulose Membrane
Cellulose membranes (CMs) [9] which contains zwitterionic sulfobetaine

groups were prepared from cellulose membranes with azide groups and N, N-
Diethyl-N-Propargyl-N-(3-sulfopropyl) ammonium via click chemistry in a
one pot two-step grafting reaction. The reactions process was shown in Figure
7. The study results showed that zwitterionic monomers were triumphantly
grafted from CM surfaces and the surface roughness of zwitterionic
sulfobetaine functionalized cellulose membranes were more superb than the
beginning cellulose membranes which was found by scanning electron
microscope and atomic forced microscope. This new member has potential in
some fields such as biomedical applications.
Figure 7. Zwitterionic sulfobetaine functionalization of cellulose membrane.
1.8. Linking Cellulose and Clay
There is an efficient method reported for covalently linking of cellulose

and clay using a click chemistry based strategy. [10]. Azide and alkynyl
derivatives of silane were synthesized and used for silanization of cellulose
and clay respectively. Functionalized cellulose and clay were then coupled
utilizing Cu(I) catalyzed azide–alkyne cycloaddition reaction, leading to a
covalent linkage between them. Triumphant synthesis of the silane derivates
was set up using Fourier transform infrared (FTIR) and nuclear magnetic
resonance. Silanization of cellulose and clay with azide and alkynyl
derivatives and the formation of a triazole linkage were confirmed by FTIR.
Figure 8 shows this method scheme.
Figure 8. Linking of cellulose and clay via click reaction.
1.9. Novel 1, 2, 3-Triazole-Linked Starch Derivatives [11]
An unfamiliar amphiprotic starch that derived from “click reaction” of

antioxidant activity reveals a singular improvement over starch. The first
synthesized reaction by selection between the C6OH of starch and N-
bromosuccinimide (NBS), N-dimethylformamide (DMF)/LiBr, resulted in the
6-bromo-6-deoxy starch. It was considered to be time-saving as well as
efficient thus to be selected as the reaction medium. Then, the 6-azido-6-deoxy
starch was obtained by reaction between the 6-bromo-6-deoxy starch and
NaN3.Along with alkyne components were introduced into 6-azido-6-deoxy
starch through the Huisgen 1,3-dipolar cycloaddition reaction. Programmed in
this way the starch derivatives could possibly meet other’s expectation as it
tends to have high antioxidant activity and good water solubility as its
advantageous characteristics [12].
Intrinsically unbefitting the native starch with advanced industrial
applications. There’s an effectual resolution is often suitable through
chemically towards worthy functional properties. Moreover, the increasing
attention has been paid to structure–activity relationship of polysaccharides,
what’s more, it’s been noticed that the biological activities of polysaccharide
are associated to its molecular structure. The Cu(I) catalyzing the azide-alkyne
[3+2] cycloaddition (CuAAC) known as‘click chemistry’, for whom the
Sharpless et al. Intiated, has coming forth as a formidable strategy as to
creating intricate biomaterials with high levels of precision and control.
1.10. Side-Chain Modification of Dextran derivatives
Dextran’s excellent solubility in water, wide availability, biocompatibility

and nonfouling properties make it outstanding and reliable in biomaterial field.
But dextran only has hydroxyl groups, which to the disadvantage of
widespread applications. Therefore, Introduce other functional groups may be

helpful to improve Dextran’s performance [13].
By esterification, etherification and reductive amination methods, we can
achieve side chain modifications of Dextran, and obtain a new member or
improved properties, but it has a lot of limitation. For example, it needs very
high reaction temperature and long reaction time, which will demand high-
ranking equipments. Besides, some side reactions are inevitable.
These days, a new approach has been proposed that combining base-
catalyzed epoxide ring-opening and thiol-ene click chemistry contributes to
the side-chain modification of dextran. In 0.1mol/L NaOH, a basic epoxide
ring opening reaction happens, followed by thiol-addition click reaction of
three model sulfhydryl compounds using water-soluble Irgacure 2959 as the
photoinitiator. It can modify side-chain functionalized dextran with bidentate
dicarboxyl, carboxyl or other groups [14]
In aqueous media, this is the first example of combining epoxide ring-
opening and thiol-ene click chemistry for side-chain modification of dextran.
Figure 9. Modification of Dextran via Epoxide Ring-opening and Thiol-ene Click

Chemistry.
1.11. Facile synthesis of β-Cyclodextrin-Dextran

Derivatives Polymers [15]
Cyclodextrin (CD) polymers are of high interest in biomedical science for

their numbers of applications, which are known to be used widely in
medication and food industry, applied for poorly water-soluble drugs,
masking odors and several other intent. In many cases the inclusion of “guest”
drugs into the hydrophobic interior of the CD cavities improving its apparent
solubility, chemical stability and bioavailability, so CDs become very
convenient for achieving a number of drug delivery goals [16].
In these ten years, many types of CD polymers have been synthesized. For
example, three series of novel water-β-cyclodextrin-dextran polymers’
preparation. Grafted by a copper (I)-catalyzed azide-alkyne cycloaddition

(CuAAC), the polymers were prepared from alkyne-modified dextrans(AMDs)
onto which mono-6-O-deoxy-monoazido-β CD (N3β CD).
Figure10. Reaction scheme for the synthesis of 6-monodeoxy-6-moncazido-βCD (N3β

CD) Made by ‘click’ chemistry, these are the first βCD polymers based on native
alkyne grafted dextran. βCD polymers have the excellent properties such as high water
solubility and the great binding skill, which makes it very competitive in biomedical
and supramolecular studies.
1.12. Synthesis of Dextran-Graft-PHBHV Amphiphilic

Copolymer Derivatives [17]
When limiting an essential nutrient, poly (3-hydroxyalkanoates) (PHAs)

are a class of natural polyesters accumulated by many bacteria as a carbon and
energy supply. A series of PHAs can be synthesized through various
substrates.
For example, preparing Dextran-grafted-PHBHV copolymers by using
click-chemistry. Functional dextran which contains azide groups has been put
forward by tosylation and subsequent nucleophilic displacement reaction with
sodium azide (DSN3 = 1). Prepared in one step reaction by direct alcoholysis
from natural polyesters, well defined PHBHV oligomers contain an alkyne end
group using propargyl alcohol with dibutyltin dilaurate as catalyst [18].
1.13. Grafting of Oligocaprolactones onto Starch Backbone
Diverse PCL chain lengths that the Polycaprolactone-grafted starch

copolymers with,it can be obtained by click chemistry from a propargylated
starch and tailor-made azido-polycaprolactones in a breeze [19].
Despite for the common chemical processes including etherification or

esterification, 1,3 dipolar cycloaddition (click chemistry CuAAC) comprising
another versatile tool, now applied broadly in chemically modify
polysaccharides by organic moieties, and to obtain graft copolymers based on
various polysaccharides as well [20]. We kept our eyes on the reaction
between propargylated starch and azido-PCL with various controlled
oligocaprolactone chain lengths. Previously synthesized by functional ring
opening polymerization of -CL in the presence of 11-bromo-1-undecanol as a
transfer agent and the subsequent substitution by sodium azide, these
oligocaprolactone chains are.
2. CLICK CHEMISTRY APPLICATION OF

NATURAL POLYMERS HYDROGELS
Natural polymers hydrogels play the important role in the drug delivery
and extracellular matrix materials Scientists pay attention to their fabrication
and modifications all the time. However, it is really difficult because of its
complex structure, low yield and unnecessary outgrowth. Therefore, synthetic
chemists started to develop the potential click chemistry which is used in the
chitosan hydrogels, hyaluronic acid hydrogels, gelatin-based hydrogels and
alginate hydrogels to solve these problems.
2.1. Chitosan/Hyaluronan Hydrogels [21]
Injectable hydrogels are significant cell scaffolding materials for tissue

engineering and regenerative medicine. Also, scientists report a new kind of
biocompatible and biodegradable polysaccharide hydrogels derived from
chitosan and hyaluronan via a metal-free click chemistry, without the
adjunction of copper catalyst. As for the metal-free click reaction, chitosan and
hyaluronan were altered with oxanorbor-nadiene and 11-azido-3, 6, 9-
trioxaundecan-1-amine (AA), respectively. The gelation is attributed to the
triazole ring formation between oxanorbor-nadiene (OB) and azido groups of
polysaccharide derivatives. The potential of the metal-free hydrogel as a cell
scaffold was decided by encapsulation of human adipose-derived stem cells
(ASCs) within the gel matrix in vitro. Cell culture showed that this metal-free
hydrogel could support survival and multiplication of ASCs. A preliminary in
vivo study demonstrated the effectiveness of the hydrogel as an injectable
scaffold for adipose tissue engineering. These characteristics provide a
potential opportunity to utilize the metal-free click chemistry to prepare the
biocompatible hydrogels for soft tissue engineering applications.
Figure 11. Chemical structures of CS–OB (a), HA–AA (b) and formed hydrogel (c).
The hydrogel is crosslinked via a metal-free click chemistry.
2.2. Furan Chitosan Hydrogels [22]
In this research, furan groups were combined with chitosan chain via
reaction of 6-azido-6-deoxy chitosan and furfuryl propargyl ether. Via this
method, 6-azido-6-deoxy chitosan was synthesized by bromination and the
subsequent nucleophilic substitution with sodium azide on the C6 hydroxyl
groups of a previously amino-protected N-phthaloyl chitosan. Next,6-azido-6-
deoxy chitosan was reacted with furfuryl propargyl ether by the Cu(I)-
catalyzed Huisgen 1,3-dipolar cycloaddition reaction, resulting in an o-
substituted furan–chitosan derivative with adegree of substitution of 10%. The

structure was cross-linked with a bismaleimide to creat a polymer network via
Diels–Alder reaction. The system presented a solgel transition with some
syneresis. The generated chitosan–furan–maleimide polymer network
indicated the typical pattern of a soft polymer hydrogel, in which both moduli
were almost frequency independent with values lower than 10 Pa. These feeble
mechanical properties were explained as a result of the polymer degradation,
which happened during the N-phthaloyl deprotection procedure.
2.3. Hyaluronic Acid Hydrogels for Tissue Engineering
Hyaluronic acid (HA) is a naturally occurring polymer that holds

important promise for tissue engineering applications [23]. Present cross-
linking chemistries often request a coupling agent, catalyst, or photoinitiator,
which may be cytotoxic, or involve a multistep synthesis of functionalized-
HA, increasing the intricacy of the system. With the target of creating a
simpler one-step, aqueous-based cross-linking system, Nimmo’s team
synthesized HA hydrogels via Diels-Alder “click” chemistry. Furan-modified
HA derivatives were synthesized and cross-linked via dimaleimide poly
(ethylene glycol). By controlling the furan tomaleimide molar ratio, both the
mechanical and degradation features of the resulting Diels-Alder cross-linked
hydrogels can be tuned. Rheological and degradation studies demonstrate that
the Diels-Alder click reaction is a compatible cross-linking method for HA.
These HA cross-linked hydrogels were exhibited to be cytocompatible and
may represent a potential material for soft tissue engineering. Figure 12 shows
that Diels Alder “click” reaction.
Figure 12. Diels Alder”click” reaction.

2.4. Biocompatible Hydrogels
Hyaluronan (HA) based hydrogels [24] have been synthesized combining

chemical modification of the polysaccharide by partial oxidation, reductive
amination and click “chemistry.” HA was oxidized by 4-acetamido-TEMPO-
mediated reaction, using sodium hypochlorite as primary oxidant and NaBr in
buffered pH, in order to produce the aldehyde moieties (hemiacetals) which
was trapped in situ by subjoining primary amines containing azide or alkyne-
terminal groups. In addition, azido- and alkynyl derivatives underwent cross-
linking by click chemistry into hydrogels. The above material shows potential
application as scaffold for tissue engineering. Next research orientation is that
optimizing the properties of scaffolds such as organized porosity and superb
mechanical integrity.
2.5. Core−Shell Poly (Vinyl Alcohol)-Hyaluronic Acid Microgels
There is one of the main synthetic ways which is chemoselective

chemistry for the project of bioactive constructs [25]. In this contribution
scientists have some new finding about the fabrication of core−shell microgel
particles, obtained by “click chemistry” and “inverse emulsion droplets”
techniques. Azido and alkyne derivatives of poly (vinyl alcohol) (PVA) were
crosslinked by click chemistry method. The microgel particles were spherical
in shape with a mean diameter of about 2μm and with a narrow size
distribution. Residual unreacted alkyne groups present on the particle surface
were “clicked” with an azido-grafted hyaluronic acid. These microgel particles
with a PVA core and a hyaluronic acid shell were measured for
bioorthogonality, that is, for the lack of cytotoxicity in the presence of
unreacted clickable functionalities and showed a notable ability to target
adenocarcinoma colon cells (HT- 29) as well as to liberate locally the
antitumor drug, doxorubicin. This is a concept device based on chemoselective
chemistry, which may devote to the progect of micro- and nanoplatforms
having controlled and multifunctional structures.
2.6. Emulating the Extracellular Matrix by hyaluronic acid
Hydrogels are used to design 3D microenvironments with features of

direct cell function.[26] The Present study shows the versatility of hyaluronic
acid (HA)-based hydrogels with independent control over hydrogel properties

such as mechanics, architecture, and the spatial distribution of biological
factors. Hydrogels were prepared by reacting furan-modified HA with bis-
maleimide-poly (ethylene glycol) in a Diels−Alder click reaction.
Biomolecules were photopatterned into the hydrogel by two-photon laser
processing, leading to spatially defined growth factor gradients. The Young’s
modulus was controlled by either altering the hydrogel concentration or the
furan substitution on the HA backbone, thereby decoupling the hydrogel
concentration from mechanical properties. Porosity was controlled by
cryogelation, and the pore size distribution, by the thaw temperature. The
additament of galactose further impacted the porosity, pore size, and Young’s
modulus of the cryogels. These HA-based hydrogels provide a tunable
platform with a variety of properties for directing cell function, with potential
applications in tissue engineering and renewable medicine.
Figure 13. Schematic representation of the crosslinked network of PVA microgels.

2.7. Biodegradable Hyaluronic Acid Hydrogels for Adipose

Tissue Engineering
There is a robust synthetic strategy [27] of biopolymer-based hydrogels

which develops where hyaluronic acid derivatives reacted through aqueous
Diels–Alder chemistry without the containment of chemical catalysts,
allowing for control and sustain release of dexamethasone. To combine the
hydrogel, furan and maleimide functionalized hyaluronic acid were
synthesized, respectively, as well as furan functionalized dexamethasone, for
the covalent fixation. The consequences showed that the aqueous Diels–Alder
chemistry offers an excessive selective reaction and proceeds with high
efficiency for hydrogel conjugation and covalent immobilization of
dexamethasone. Cell culture consequences exhibited that the dexamethasone
fixed hydrogel was noncytotoxic and preserved proliferation of entrapped
human adipose-derived stem cells. This synthetic method uniquely allows for
the direct synthesis of biologically functionalized gel scaffolds with ideal
structures for adipose tissue engineering, which offers a competitive
alternative to traditional conjugation techniques such as copper mediated click
chemistry.
2.8. Biological Hydrogel
In order to imitate the natural cartilage extracellular matrix [28], which

consists of core proteins and glycosaminoglycans, a biological hydrogel was
synthesized from the biopolymers hyaluronic acid (HA),chondroitin sulfate
(CS) and gelatin via click chemistry. HA and CS were modified with 11-
azido-3,6,9-tri-oxaundecan-1-amine (AA) and gelatin was modified with
propiolic acid (PA). Giving substitution degrees of 29%, 89% and 44% for
HA–AA, CS–AA and gelatin–PA (G–PA), respectively. The N3 groups of
HA–AA and CS–AA were reacted with the acetylene groups of G–PA,
catalyzed by Cu (I) to form triazole rings, therefore forming a cross-linked
hydrogel. The gelation time was decreased monotonically with increasing Cu
(I) concentration up to 0.95 mg ml-1. The hydrogel achieved was in a highly
swollen state and exhibited the characteristics of elastomer. In vitro cell
culture indicated that the hydrogel could support the adhesion and proliferation
of chondrocytes.
2.9. Photodegradable Gelatin-Based Hydrogels
Truong’s team presented an approach for the fabrication of in situ forming

gelatin and poly(ethylene glycol)-based hydrogels [29] utilizing bioorthogonal,
strain-promoted alkyne−azide cycloaddition as the cross-linking reaction. By
incorporating nitrobenzyl moieties within the network structure, these
hydrogels can be designed to be degradable upon irradiation with low intensity
UV light, allowing precise photopatterning. Fibroblast cells encapsulated
within these hydrogels were viable at 14 days and could be readily harvested
using a light trigger. Potential applications of this new class of injectable
hydrogel include the 3D culturing platform that allows the capture and release
of cells, as well as light-triggered cell delivery in renewable medicine.
Figure 14. Mechanisms of poly(ethylene glycol)-based hydrogels.
2.10. Hydrogel Tissue Engineering Scaffolds
The integration of biological extracellular matrix (ECM) components and

synthetic materials is a hopeful pathway to creat the next generation of
hydrogel-based tissue scaffolds [30] that more accurately emulate the
microscale heterogeneity of natural ECM. Daniele’s team reported the
development of a bio/synthetic interpenetrating network (BioSINx), containing
gelatin methacrylamide (GelMA) polymerized within a poly (ethylene glycol)
(PEG) framework to form a mechanically robust network capable of
supporting both internal cell encapsulation and surface cell adherence. The
covalently crosslinked PEG networkwas formed by thiol-yne coupling, while
the bioactive GelMAwas integrated using a concurrent thiol-ene coupling
reaction. BioSINx displayed superior physical properties and significantly
lower gelatin dissolution. These benefits led to promote cytocompatibility for
both cell adhesion and encapsulation; furthermore, the increased physical
strength provided for the generation of a micro-engineered tissue scaffold.
Endothelial cells showed extensive cytoplasmic spreading and the formation

of cellular adhesion sites when cultured onto BioSINx; what’s more, both
encapsulated and adherent cells showed sustained viability and proliferation.
Daniele’s team intends to incorporate additional ECM proteins and
macromolecules, e.g., heparin and hyaluronan respectively, to generate a more
biologically accurate and complex system for targeting the biofabrication of
specific tissue types.
2.11. Alginate Hydrogel Capsules
Ionically crosslinked alginate hydrogels [31] have been extensively

developed for encapsulation and immunoisolation of living cells/tissues to
explore implantable cell therapies, such as islet encapsulation for bioartificial
pancreas. Chemical instability of these hydrogels during long-term
implantation hinders the development of viable cell therapy. The exchange
between divalent crosslinking ions (e.g., Ca+2) with monovalent ions from
physiological environment causes alginate hydrogels to degrade, leading to the
exposure of the donor tissue to the host’s immune system and graft failure.
Covalent “click” crosslinking can promote stability of alginate hydrogels
while preserving other biomedically viable hydrogel properties. Alginate was
first functionalized to contain either pendant alkyne or azide functionalities,
and subsequently reacted by “click” chemistry to form “click” gel capsules.
When compared with Ca12 capsules, “click” capsules showed superior
stability in ionic media, while exhibiting higher permeability to small size
diffusants and the same molecular weight cut-off and water swelling.
Physicochemical properties of “click” alginate hydrogels show their potential
utility for therapeutic cell encapsulation and other biomedical applications.
2.12. Alginate Hydrogels [32]
Alginate hydrogels are well-characterized, biologically inert materials that

are utilized in many biomedical applications for the delivery of drugs,
proteins, and cells. Unfortunately, canonical covalently crosslinked alginate
hydrogels are formed utilizing chemical strategies that can be biologically
detrimental because of their lack of chemoselectivity. Desai’s team introduced
tetrazine and norbornene groups to alginate polymer chains and subsequently
form covalently crosslinked click alginate hydrogels capable of encapsulating
cells without damaging them. They created the click alginate hydrogels by the
reaction of AIg-N and AIg-T. Figure 15 shows the reaction. The click alginate
hydrogel can be altered after gelation to display cell adhesion peptides for 2D
cell culture utilizing thiol-ene chemistry. In addition, click alginate hydrogels
combine the numerous benefits of alginate hydrogels with powerful
bioorthogonal click chemistry for utilizing in tissue engineering applications
such as stable encapsulation, delivery of cells and bioactive molecules.
3. CLICK CHEMISTRY APPLICATION OF NATURAL

POLYMERS NANOMATERIALS
Natural polymers nanomaterials are the significant field of material
science. On the one hand, scientists need to utilize all kinds of technology to
keep the nanomaterials dispersity in its application. On the other hand,
scientists also need to do different functionalized modifications in the specific
applications. However, functionalization of nanomaterials has been the mutual
problem for all science researchers because of the strict conditions. Click
chemistry offers us one suitable and simple way to solve this problem.
Figure 15. Fabrication of click alginate hydrogels.

3.1. Radiolabeling Nanoparticles
There is an efficient and facile method urgently needed to understand the

in vivo biodistribution of nanoparticles [33] for radiolabeling nanoparticles. In
this respect, Lee’s team, investigated a straightforward and highly efficient
way to prepare radiolabeled glycol chitosan nanoparticles with 64Cu via a
strain-promoted azide−alkyne cycloaddition strategy, which is often referred
to as click chemistry. Firstly, the azide (N3) group, which allows for the
preparation of radiolabeled nanoparticles by copper-free click chemistry, was
contained to glycol chitosan nanoparticles (CNPs). Secondly, the strained
cyclooctyne derivative, dibenzyl cyclooctyne (DBCO) conjugated with a 1, 4,
7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) chelator, was
synthesized for preparing the preradiolabeled alkyne intricate with 64Cu
radionuclide. Following incubation with the 64Cu-radiolabeled DBCO
complex, the azide-functionalized CNPs were radiolabeled successfully with
64Cu, with a high radiolabeling efficiency and a high radiolabeling yield.
Importantly, the radiolabeling of CNPs by copper-free click chemistry was

achieved in 30 minutes, with great efficiency in aqueous conditions. It
demonstrated that the goodness of copper-free click chemistry as a facile,
preradiolabeling way to conveniently radiolabel nanoparticles for evulating the
real-time in vivo biodistribution of nanoparticles.
Figure 16. Synthesis of radiolabeled glycol chitosan nanoparticles with 64Cu via click
reaction.
3.2. Grafting Chitosan on the Surface

of Hydroxyapatite Nanoparticles [34]
In order to modify the surface properties of hydroxyapatite (HA)

nanoparticles and prevent them from aggregation, there was an effective way
proposed to graft chitosan (CS) molecules on the surface of HA via “click”

reaction. This mild method is versatile for the covalent immobilization of other
functional biomacromolecules on the surface of HA. “Clickable”
Hydroxyapatite (AHA) was dispersed in the deionized water, and then CS-N3
and CuSO4·5H2O were added in the AHA suspension system. The mixture
was bubbled with N2 for several minutes, and sodium ascorbic (NaAsc) was
added to the mixture under nitrogen atmosphere. Thermal gravimetric analysis
(TGA) shows that CS was triumphantly grafted on the surface of HA
nanoparticles and the grafting quantity was about 8.9 g of CS on per hundred
grams of HA. The grafted chitosan chains can prevent HA nanoparticles from
aggregation and extradinatory improve the colloidal stability of HA in water.
The cytotoxicity of CS modified HA (HA-CS) is negligible and thus HA-CS
may have possible applications in biomedical fields.
3.3. Cellulose Nanocrystals
In comparison to the typical nanocomposite approach, many of the

mechanically excellent biological nanocomposites involve self-assembled and
space-filled structures of hard reinforcing and soft toughening domains, with
high weight fraction of reinforcements [35]. Katas’ team find a new concept
toward mimicking such structures by studying whether percolation of
intercalated domains comprises the alternating rigid and strengthening, and
soft rubbery domains could allow a transition to a reinforced state or not.
Toward that, they showed the functionalization of rigid native cellulose
nanocrystals (CNCs) by esterification with a dense hydrocarbon chain brush
containing cross-linkable double bonds. Such modified CNCs (mCNCs)
within a poly (butadiene) (PBD) rubber matrix were prepared via cross-linking
by UV-light initiated thiol−ene click reaction. Transmission electron
microscopy exhibited structures at two length scales, where the mCNCs and
PBD form domains having internal aligned self-assemblies of alternating hard
mCNCs and soft PBD, and where additional PBD connects such domains. It
was found that exceptional insensitivity to air humidity from the mechanical
properties of the composites. The shown simple conception of percolative
intercalated nanocomposites propels searching for more general biomimetic
compositions involving some deformation mechanisms for improved
mechanical properties.
3.4. Cellulose Acetate Nanofibers
Beta-cyclodextrin (β-CD) functionalized cellulose acetate (CA) nanofibers

have been triumphantly prepared by combining electrospinning and “click”
reaction [36]. Firstly, β-CD and electrospun CA nanofibers were modified so
as to be azide-β-CD and propargyl-terminated CA nanofibers, respectively.
Then “click” reaction was performed between modified CD molecules and CA
nanofibers to obtain permanent grafting of CDs onto nanofibers surface. It was
observed from the SEM image that, while CA nanofibers have smooth surface,
there were some anomaly and roughness at nanofibers morphology after the
modification. However, the fibrous structure was still protected. ATR-FTIR
and XPS revealed that, CD molecules were triumphantly grafted onto surface
of CA nanofibers. The adsorption capacity of β-CD-functionalized CA (CA-
CD) nanofibers was also decided by removing phenanthrene (polycyclic
aromatic hydrocarbons, PAH) from its aqueous solution. It was found that CA-
CD nanofibers have potential to be used as the molecular filters for the
purpose of water purification and sewage treatment by integrating the high
surface area of nanofibers with inclusion complexation property of CD
molecules.
3.5. Functionalized Cellulose Nanocrystals [37]
Natural rubber/cellulose nanocrystals (NR/CNCs) form true

biocomposites from reproducible resources and are demonstrated to exhibit
significantly improved thermomechanical performance and reduced stress-
softening. The nanocomposites were prepared from chemically functionalized
CNCs bearing thiols. CNCs were prepared from cotton, and the crosslinkable
mercapto-groups were introduced onto the surface of CNCs by esterification.
Nanocomposite films were prepared by scattering the modified CNCs (m-
CNCs) in NR matrix by solution casting. The cross-links at the filler−matrix
(m-CNCs−NR) interface were created by photochemically initiated thiol−ene
reactions. In comparison to biocomposites from NR with unmodified CNCs,
the NR/m-CNCs nanocomposites exhibited increase in tensile strength, strain-
to-failure, and work-of-fracture.
As the present concept is common in material synthesis and design, we
can predict the potential application of modified cellulose nanocrystals bearing
thiols in the tire industry and general elastomeric composites. Note that the
present experiments were designed to prove the effect of thiol-functional
CNCs for covalent cross-linking at the NR/CNCs interface. If the NR network

had been also cross-linked by conventional vulcanization mechanisms, the
intension effects would have been even stronger.
3.6. Nano- and Microfibrillar Cellulose
The modification of cellulose as a reproducible resource has received

much attention in research and industry [38]. A major issue regarding
chemical modification, including heating and drying, is related to hornification
that causes pore-system collapse and leads to decreased reactivity and alters in
the 3D structure of the material. Amild and green approach for the
modification of distinct never-dried and thus wet cellulose substrates (pulp,
nanostructured celluloses, and viscose fibers) by an alkoxysilane-azide in
water is showed. Figure 17 shows the reation scheme. A kinetic research of the
silanization reaction demonstrates that alkoxy-trans-sila-nization of the
cellulose surface is achieved in water as a suspension medium within a few
hours at room temperature.
Figure 17. General procedure for the introduction of azide functionalities onto
cellulose substrates using (3-azidopropyl)triethoxysilane and subsequent click reaction
with propargylated Rhodamine B. Conditions: a)NaOH, water; b)L-ascorbic acid,
CuSO4·5H2O, water.
The resulting, azido-equipped celluloses are generally applicable

precursor materials for subsequent functionalization by so-called click
chemistry, for example, with a fluorescent Rhodamine derivative as a typical
reagent. Triumphant covalent bonding was exhibited by GPC and a model
reaction. The 3D structure of the materials remained intact, as was inter alia
visualized by optical and fluorescence microscopy.
3.7. Thiol-yne Click on Nano-Starch [39]
Processing the natural starch particles to starch nanocrystals as a

reinforcing agent of polymer matrix filler, has attracted wide interest. The
surface chemical modification of starch nanocrystals may constitute a
promising way to expand its application, supporting other functions of
anchoring, depend on existence of a large number of hydroxyl groups. In the
other hand, grafting of homogeneous metal complexes with the uses of
chemically modified starch nanocrystals, are being explored these days. For
grafting of homogeneous oxo-vanadium Schiff base catalyst to the chemically
modified starch nanocrystalline support, an efficient radical mediated thiol-yne
click reaction is the new trend. For the oxidation of kinds of alcohols by t-
BuOOH to the corresponding carbonyl compounds, the prepared catalyst has
been used [40].
It is known as an efficient, mild and quality approach that the The metal-
free stoichiometric click reaction between yne-functional oxo-vanadium Schiff
and thiol-functionalized nanocrystalline starch, to develop heterogenized
homogeneous complex via covalent bonding. The oxidation of various
alcohols to corresponding carbonyl compounds with t-BuOOH under mild
reaction conditions has made the developed catalyst recyclable and realiable. It
is important in the preparation of immobilized catalysts for its simplicity in
use, multi-functionality, green nature and high efficiency.
Figure 18. Click reaction between yne-functional oxo-vanadium Schiff and thiol-
functionalized nanocrystalline starch.
4. CLICK CHEMISTRY APPLICATION OF THE

LINKED FLUOROPHORE IN THE NATURAL POLYMERS
Click chemistry has entered into the practical field from the theoretical
field. Click chemistry reaction conditions are usually mild and under the room
temperature. It is found that click chemistry can be used to modify natural
polymers for specific applications from lots of experiments such as fluorescent
polymeric nanoparticle, multicolor fluorescent labeling of cellulose nanofibril
and so on.
4.1. Multicolor Fluorescent Labeling of Cellulose Nanofibril
There are chemically modified cellulose nanofibrils (CNF) with furan and
maleimide groups, and selectively labeled the modified CNF with fluorescent
probes [41] which are 7-mercapto-4-methylcoumarin and fluorescein diacetate
5-maleimide through two specific click chemistry reactions: Diels−Alder
cycloaddition and the thiol-Michael reaction. Figure 19 shows that
Diels−Alder cycloaddition’s reaction scheme. Feature by solid-state 13C NMR
and infrared spectroscopy was utilized to follow the surface modification and
estimate the substitution degrees. Navarro’s team found that the two
luminescent dyes could be selectively labeled onto CNF, yielding a multicolor
CNF that was marked by UV/visible and fluorescence spectroscopies. It was
demonstrated that the multicolor CNF could be imaged that using a confocal
laser scanning microscope. It was found through the study of fourier transform
infrared spectroscopy and solid-state that the first step in the surface
modification of CNF interest for biological application such as multimodality
molecular imaging.
Figure 19. Grafting Furan Groups for Diels-Alder Cycloaddition onto Cellulose
Nanofibrils.
4.2. New Fluorescent Polymeric Nanoparticle [42]
There is a novel kind of biopolymeric nanoparticles prepared which

comprises cellulose nanowhisker (CNW) as support system and polyglycerol
(PG) as surface modifying agent. PG was linked to the surface of CNW by
click chemistry reaction. CdSe quantum dots then interact with the prepared
system by noncovalent interaction. These new synthesized biopolymeric
nanoparticles were characterized by spectroscopic measurement ways. Due to
the presence of hydrophilic polymer at the surface of CNW, synthesized
nanomaterials were water soluble, and have a large number of functional
group for future modification. Also the presence of fluorescence quantum dots
(QDS) caused fluorescence feature of synthesized system. These novel
synthesized system has many potential applications to be utilized in widely
filed such as drug delivery, biomedical imaging etc.
4.3. Fluorescent Hyperbranched Polymeric Sensors [43]
A new hyperbranched fluorescent polymer called P1000-NAPHT have

been already synthesized through click chemistry by introducing 1, 8-
naphthalimide chromophore into the side-chain of an advantageous
hyperbranched polymer.
The reactive 4-(N, N-dimethylaminoethylenamino-N-propargyl-l, 8-
naphthali-mide (NAPHTyne) was synthesized in two steps, by using
microwave irradiation as the energy source [44].
Used as the starting hyperbranched polymer, P1000 is obtained through
chain reaction, getting along a lot of distinct structures. The perfect structure
and lots of reactive groups which provided by the company has been used by
us. Recently, some experts report that the hydroxyl groups of this kind of
molecules could be successfully changed into azide groups. For this reason, a
active azido-functionalized hyprebranched polymer (HBP-N3) reacts with
acetylenic-functionalized compounds through the Huisgen 1,3-dipolar
cycloaddition, through a mild click reaction. After these three steps, the
starting –OH groups of P1000 have been transformed to azides opportunely by
using mesylates as intermediates.
Figure 20. Two step synthesis of NAPHTyne.
In several of polymeric medias, the fluorescent hyperbranched polymer is

suitable to large scale preparation and for a pragmatic use as sensors because
of its availability of reagents, the optimization of synthesis and purification of
the products, and the loss of extraction from a polymer matrix.
4.4. A Turn-on Fluorescent Probe for Specific Detection

of Cysteine
Based on the thiol-chromene click chemistry, an opening fluorescent

probe for distinctive detection of cysteine (Cys) was developed quickly. The
comparatively strong nucleophilic reactivity of SH of Cys in comparison with
Hcy and GSH brings great selective ability to Cys [45].
The thiol-chromene “click” chemistry is a suitable way for developing
chromene derivative as fluorescent probe for Cys detection. The extraordinary
click ring open reaction between Cys and chromene makes it possible that
probe 1 display high sensitivity and selective ability opening fluorescent
detection of Cys and for bioimaging. It is known that in thiol-contained amino
acids detection, the chromene moiety shows excellent selectivity. And some
optical probes for thiols have been developed, which based on the thiol-
chromene nucleophilic addition reaction.
Some great properties of fluorescent detection make it play an important
role in biological application, such as its low price, great sensitiveness and
operability. It is a big challenge that Cys, Hcy and GSH have similar
molecular structure that makes it difficult to measure them correctly [46].
Having been successfully applied in living cells, this probe occupies an
important position in biological applications.
5. CLICK CHEMISTRY APPLICATION OF

SILK FIBROIN PREPARATION [47]
Silk is a natural protein fiber, some forms of which can be woven into
textiles. The protein fiber of silk is composed mainly of fibroin and is
produced by certain insect larvae to form cocoons. The best-known silk is
obtained from the cocoons of the larvae of the mulberry silkworm Bombyx
mori reared in captivity (sericulture). The shimmering appearance of silk is
due to the triangular prism-like structure of the silk fibre, which allows silk
cloth to refract incoming light at different angles, thus producing different
colors.
5.1. Surface Modification of Silk Fibroin Films
Biocompatible the silk fibroin is, being widely studied as well as applied
as biomaterial for diverse applications. And full-bodied protein polymer that
can be made optically trans-parent mechanically [48]. Its chemical alteration is
off absorbing a way for tuning the properties and broaden its adhibition
aspects. Herein, PEG grafting on the surface of regenerated silk fibroin films is
incurred by direct linking via a click reaction between the azido activated silk
surface and an alkyne terminated PEG. Thus the surface properties have been
improved through the so obtained PEGylated films, compares to the
unmodified films.
Figure 21. Reaction scheme for PEG grafting onto silk fibroin films.
Chemical modification of SF is a fascinating way for tun-ing the

properties of this natural material. In this study, PEG grafting on the surface of
regenerated SF films is obtained by direct linking via a click reaction between
the azido activated SF surface and an alkyne ter-urinated PEG [49]. Differ
from other existing methods, the exact method that we mentioned above is off
more reliable to chemical modification of the only external surface of silk,
making the bulk of the material more consummate. The supporting evidence
that meet our demonstration is that: SF surface properties have been modified
in terms of both morphology and hydrophilicity at the end of this process.
5.2. Silk Fibroin-Polyethylene Glycol Conjugate Films
Primarily the silk from Bombyx mori silk fibres are composed of two
types of protein: the antigenic gum-like protein surrounding the fibres called
sericin, and fibroin, the core fibres. Silk fibroin belongs to a group of high
molecular weight organic polymers characterized by repetitive hydrophobic
and hydrophilic peptide sequences [50].
Synthesized the Azide silk fibroin (azido SF) and alkyne terminal
polyethylene glycol) (PEG) 2000 (acetylene-terminal PEG 2000) are, Azido
SF would have reaction with acetylene-terminal PEG 2000 results in
producing films through a copper-mediated 7,3-cycloaddition ('click'
chemistry) generating a triazole linkage along with the networking forming
reaction. Novel silk-based films with various weight ratios were prepared and
investigated through click chemistry.
With diazonium coupling chemistry, an aromatic azide functional moiety
was successfully introduced into SF. Similarly, an alkyne functional group was
successfully introduced into PEG 2000 via esterification.
6. PROSPECT
Click chemistry has been noticed by more and more scientists since the
click chemistry is put forward, for its several advantages that simple materials,
mild reaction conditions, high yield, good selectivity and easy separation and
purification and so on [51]. Click chemistry have been entered into the
practical application from the original research in drug and polymer
development. However, the development and application of click chemistry
are still in the initial stage. Next target of click chemistry is that scientists are
able to find more reaction which is efficient, reliable, rapid and high selectivity
and expending its application from the original drug exploitation to the
synthesis of polymer, nanotechnology, supra-momolecular science, surface
modification and other fields. In terms of nature polymers, they will be easier
to be modified by chemical grafting of new functional groups to promote their
properties via click chemistry. This kind of method will be the main
technology in all kinds of fields about nature polymers in the future.
REFERENCES
[1] Solimana S M A, Colombeaua L, Nouvela C, et al. Amphiphilic
photosensitive dextran-g-poly (o-nitrobenzyl acrylate)glycopolymers.
Carbohydrate Polymers 136 (2016) 598-608.
[2] Liu Z, Wei Z, Zhu X, et al. Dextran- based hydrogel formed by thiol-
Michael addition reaction for 3D cell encapsulation. Colloids and
Surfaces B: Biointerfaces 128 (2015) 140-148.
[3] Chen Y, Wang F, Yun D, et al. Preparation of a C6 Quaternary
Ammonium Chitosan Derivative Through a Chitosan Schiff Base with
Click Chemistry [J]. 2013.
[4] Peng P, Cao X, Peng F, et al. Binding cellulose and chitosan via click
chemistry: Synthesis, characterization, and formation of some hollow
tubes [J]. Journal of Polymer Science Part A: Polymer Chemistry, 2012,
50(24): 5201-10.
[5] Jirawutthiwongchai J, Krause A, Draeger G, et al. Chitosan-
Oxanorbornadiene: A Convenient Chitosan Derivative for Click
Chemistry without Metal Catalyst Problem [J]. ACS Macro Letters,
2013, 2(3): 177-80.
[6] Chen Y, Ye Y, Jing Y, et al. The Synthesis of the Locating Substitution
Derivatives of Chitosan by Click Reaction at the 6-Position of Chitin [J].
International Journal of Polymer Science, 2015, 2015:1-9.
[7] Agag T, Vietmeier K, Chernykh A, et al. Side-chain type benzoxazine-
functional cellulose via click chemistry [J]. Journal of Applied Polymer
Science, 2012, 125(2): 1346-51.
[8] Lee D E, Na J H, Lee S, et al. Facile method to radiolabel glycol
chitosan nanoparticles with (64)Cu via copper-free click chemistry for
MicroPET imaging [J]. Molecular pharmaceutics, 2013, 10(6): 2190-8.
[9] Huang W, Huang J, Xu C, et al. Surface functionalization of cellulose

membrane via heterogeneous “click” grafting of zwitterionic
sulfobetaine [J]. Polymer Bulletin, 2014, 71(10): 2559-69.
[10] Yadav P, Chacko S, Kumar G, et al. Click chemistry route to covalently
link cellulose and clay [J]. Cellulose, 2015, 22(3): 1615-24.
[11] Fan M, Ma Y, Mao J, et al. Cytocompatible in situ forming
chitosan/hyaluronan hydrogels via a metal-free click chemistry for soft
tissue engineering [J]. Acta biomaterialia, 2015, 20:60-8.
[12] Tan, W, Li, Q, Li W, et al. Synthesis and antioxidant property of novel
1, 2, 3-triazole-linked starch derivatives via 'click chemistry'.
International Journal of Biological Macromolecules 82 (2016) 404–410.
[13] Tan W, Li Q, Wang H, et al. Synthesis, characterization, and
antibacterial property of novel starch derivatives with 1,2,3-triazole.
[14] Li M, Tanga Z, Wang C, et al. Efficient Side-chain Modification of
Dextran via Base-catalyzed Epoxide Ring-opening and Thiol-ene Click
Chemistry in Aqueous Mediax. Chinese Journal of Polymer Science
Vo1. 32, No. 8, (2014), 969 974.
[15] Lia F, Peib D, Huang N, et al. Synthesis and properties of novel
biomimetic and thermo-responsive Dextran-based biohvbrids.
[16] Nielsen T T, Wintgens V, Amiel C, et al. Facile Synthesis of
β-Cyclodextrin-Dextran Polymers by 'Click' Chemistry. Department of
Biofechnolo_qy, Chemistry, and Environmental Engineering, Aalborq
University.2010, 11, 1710-1715.
[17] Mai K, Zhang S, Liang B ， et al. Water soluble cationic dextran
derivatives containing poly (amidoamine) dendrons for efficient gene
delivery. Carbohydrate Polymers 123 (2015) 237-245.
[18] Lemechlco P, Renard E, Guezennec J, et al. Synthesis of dextran-graft-
PHBHV amphiphilic copolymer using click chemistry approach.
Reactive & Functional Polymers 72 (2012) 487-494.
[19] Antoniuk I,, Volet G, Wintgens V. Synthesis of a new dextran-PEG-β-
cyclodextrin host polymer using “Click” chemistry. Catherine Amiel. J
Incl Phenom Macrocycl Chem (2014) 80:93-100 DOI 10.1007/s 10847-
014-0401-y.
[20] Uliniuc A, Popab M, Drockenmullera E, et al. Thierry Hamaide Toward
tunable amphiphilic copolymers via CuAAC click chemistry of
oligocaprolactones onto starch backbone. Carbohydrate Polymers 96
(2013) 259–269.
[21] Besset C, Binauld S, Ibert M, et al. Copper-Catalyzed vs Thermal Step

Growth Polymerization of Starch-Derived a-Azide-ω –Alkyne
Dianhydrohexitol Stereoisomers: To Click or Not To Click?
Macromolecules (2010) 43(1): 17-19.
[22] Montiel-Herrera M, Gandini A, Goycoolea F M, et al. Furan–chitosan
hydrogels based on click chemistry [J]. Iranian Polymer Journal, 2015,
24(5): 349-57.
[23] Nimmo C M, Owen S C, Shoichet M S. Diels-Alder Click cross-linked
hyaluronic acid hydrogels for tissue engineering [J].
Biomacromolecules, 2011, 12(3): 824-30.
[24] Huerta-Angeles G, Nemcova M, Prikopova E, et al. Reductive alkylation
of hyaluronic acid for the synthesis of biocompatible hydrogels by click
chemistry [J]. Carbohydr Polym, 2012, 90(4): 1704-11.
[25] Owen S C, Fisher S A, Tam R Y, et al. Hyaluronic acid click hydrogels
emulate the extracellular matrix [J]. Langmuir: the ACS journal of
surfaces and colloids, 2013, 29(24): 7393-400.
[26] Kupal S G, Cerroni B, Ghugare S V, et al. Biointerface properties of
core-shell poly(vinyl alcohol)-hyaluronic acid microgels based on
chemoselective chemistry [J]. Biomacromolecules, 2012, 13(11): 3592-
601.
[27] Fan M, Ma Y, Zhang Z, et al. Biodegradable hyaluronic acid hydrogels
to control release of dexamethasone through aqueous Diels-Alder
chemistry for adipose tissue engineering [J]. Materials science &
engineering C, Materials for biological applications, 2015, 56:311-7.
[28] Truong V X, Tsang K M, Simon G P, et al. Photodegradable Gelatin-
Based Hydrogels Prepared by Bioorthogonal Click Chemistry for Cell
Encapsulation and Release [J]. Biomacromolecules, 2015, 16(7): 2246-
53.
[29] Kupal S G, Cerroni B, Ghugare S V, et al. Biointerface properties of
core-shell poly(vinyl alcohol)-hyaluronic acid microgels based on
chemoselective chemistry [J]. Biomacromolecules, 2012, 13(11): 3592-
601.
[30] Lee D E, Na J H, Lee S, et al. Facile method to radiolabel glycol
chitosan nanoparticles with (64)Cu via copper-free click chemistry for
MicroPET imaging [J]. Molecular pharmaceutics, 2013, 10(6): 2190-8.
[31] Daniele M A, Adams A A, Naciri J, et al. Interpenetrating networks
based on gelatin methacrylamide and PEG formed using concurrent thiol
click chemistries for hydrogel tissue engineering scaffolds [J].
Biomaterials, 2014, 35(6): 1845-56.
[32] Breger J C, Fisher B, Samy R, et al. Synthesis of "click" alginate

hydrogel capsules and comparison of their stability, water swelling, and
diffusion properties with that of Ca(+2) crosslinked alginate capsules [J].
Journal of biomedical materials research Part B, Applied biomaterials,
2015, 103(5): 1120-32.
[33] Desai R M, Koshy S T, Hilderbrand S A, et al. Versatile click alginate
hydrogels crosslinked via tetrazine-norbornene chemistry [J].
Biomaterials, 2015, 50:30-7.
[34] Wei J, Wang P, Cui L, et al. Novel method to graft chitosan on the
surface of hydroxyapatite nanoparticles via “click” reaction [J].
Chemical Research in Chinese Universities, 2014, 30(6): 1063-5.
[35] Parambath Kanoth B, Claudino M, Johansson M, et al. Biocomposites
from Natural Rubber: Synergistic Effects of Functionalized Cellulose
Nanocrystals as Both Reinforcing and Cross-Linking Agents via Free-
Radical Thiol-ene Chemistry [J]. ACS applied materials & interfaces,
2015, 7(30): 16303-10.
[36] Parambath Kanoth B, Claudino M, Johansson M, et al. Biocomposites
from Natural Rubber: Synergistic Effects of Functionalized Cellulose
Nanocrystals as Both Reinforcing and Cross-Linking Agents via Free-
Radical Thiol-ene Chemistry [J]. ACS applied materials & interfaces,
2015, 7(30): 16303-10.
[37] Celebioglu A, Demirci S, Uyar T. Cyclodextrin-grafted electrospun
cellulose acetate nanofibers via “Click” reaction for removal of
phenanthrene [J]. Applied Surface Science, 2014, 305:581-8.
[38] Navarro J R, Conzatti G, Yu Y, et al. Multicolor fluorescent labeling of
cellulose nanofibrils by click chemistry [J]. Biomacromolecules, 2015,
16(4): 1293-300.
[39] Verma S, Brass J L, Jain S, et al. Thiol-yne click on nano-starch:An
expedient approach for grafting of oxo-vanadium Schiff base catalyst
and its use in the oxidation of alcohols. Applied Catalysis A: General
4G8 (2013) 334-340.
[40] Tankam P F, Miiller R, Mischnicka P, et al. Alkynyl polysaccharides:
synthesis of propargyl potato starch followed by subsequent
derivatizations. Carbohydrate Research 342 (2007) 2049-2060.
[41] Parsamanesh M, Dadkhah Tehrani A. Synthesize of new fluorescent
polymeric nanoparticle using modified cellulose nanowhisker through
click reaction [J]. Carbohydrate Polymers, 2016, 136:1323-31.
[42] Medel S, Bosch P, Grabchev N, et al. Click chemistry to fluorescent

hyperbranched polymeric sensors. 2. Synthesis, spectroscopic and
cation-sensing properties of new green fluorescent 1,8- naphthalimides.
European Polymer Journal 74 (2076) 247-255.
[43] Xie Q, Weng X, Lu L, et al. A sensitive fluorescent sensor for
quantification of alpha-fetoprotein based on immunosorbent assay and
click chemistry. Biosensors and Bioelectronics 77 (2016) 46-50.
[44] Xu L, Li N, Zhang B, et al. PEGylated Fluorescent Nanoparticles from
One-Pot Atom Transfer Radical Polymerization and "Click Chemistry".
Polymers (2015) 7(10): 2119-2130.
[45] Zhang S, Yang H, Ma Y, et al. A fluorescent bis-NBD derivative of
calixarene: Switchable response to Ag+ and HCHO in solution phase.
Sensors and Actuators B 227 (2016) 271-276.
[46] Galeotti F, Andicsova A, Bertini F, et al. A versatile click-grafting
approach to surface modification of silk fibroin films. Journal of
Material Science (2013) 48:7004-7010.
[47] Sampaio S, Miranda T M R, Santos J G, et al. Soares Preparation of silk
fibroin 一 polyethylene glycol) conjugate films through click chemistry.
Wiley Online Library.
[48] Yue Y, Yin C, Huo F, et al. Thiol-chromene click chemistry: A turn-on
fluorescent probe for specific detection of cysteine and its application in
bioimaging. Sensors and Actuators B 223 (2016) 496-500.
[49] Parsamanesh M, Tehrani A D. Synthesize of new fluorescent polymeric
nanoparticle using modified cellulose nanowhislcer through click
reaction. Carbohydrate Polymers 136 (2016) 1323-1331.
[50] Pahimanolis N, Vesterinen A, Rich J, et al. Modification of dextran
using click-chemistry approach in aqueous media. Carbohydrate
Polymers 82 (2010) 78-82.
[51] Caprioglio D, Torretta S, Ferrari M, et al. Triazole-curcuminoids: A new
class of derivatives for `tuning’ curcumin bioactivities? Bioorganic &
Medicinal Chemistry 24 (2076) 740-752.
Chapter 5
SYNTHESIS AND FUNCTIONALIZATION

OF HYDROGEL THROUGH
“CLICK-CHEMISTRY”
Mi-Heng Dong1,2 and Chen Yu1,*

1
2
College of Engineering and Computer Science,
Australian National University, Canberra, Australia
ABSTRACT
Hydrogels are polymer cross-linked networks with ability to retain
large amount water yet remain insoluble. With this special property, quite
a lot of studies have been done on biomedical and tissue-engineering
applications of hydrogel. In recent years, hydrogel synthesized through
“Click-Chemistry” method has developed in large range. Because the
“Click-Chemistry” reactions could happen in relatively mild environment
(temperature and PH similar to organism's internal environment) with
highly selectivity and controllable gelation-time. Meanwhile, “Click-
Chemistry” also allows further functionalization of hydrogels which
greatly enlarges the applications of hydrogels. In this chapter, we will
cover some of the popular synthesis methods of hydrogels through
“Click-Chemistry” in recent years. The properties of different hydrogels
*
Corresponding Author email: cylsy@163.com
108 Mi-Heng Dong and Yu Chen
will be analyzed, including physical and chemical properties, the

degradation of “click” hydrogels. Further functionalization and potential
application of them on biomedical engineering will also be introduced to
give the readers a general idea of the current biomedical research interest
in this specialization.
Keywords: Click-chemistry, Hydrogel, Functionalization, Application.
1. HYDROGELS PREPARED FROM AZIDE-ALKYNE

CYCLOADDITION CATALYZED BY CU (I)
Since the copper (I)-catalyzed azide-alkyne (CuAAC) click reaction was
found in 2002 by Sharpless and Meldal, it has been widely used as a fast
synthesis method for bio-compatible hydrogels [1]. It has proven effective for
the synthesis of numerous different types of rotaxanes, catenanes and
molecular shuttles by passive as well as active template strategies [2].
Stimuli-responsive hydrogel is a hot topic in recent year. Its ability to
change volume or shape has drawn close interests in potential applications
such as drug delivery or protein separation. poly (N-isopropylacrylamide)
(PNIPAAm) hydrogels could exhibit volume phase transition (VPT) behavior
along with temperature variation. However, due to the slow rate of water
molecules diffusion in this hydrogel, the swelling and deswelling rate were not
ideal. To improve its property, in 2011, Wang et al. reported a synthesizing
method of POSS-capped PNIPAAm telechelics with a “POSS-spreading”
strategy based on CuAAC reaction. POSS microdomains works as cross-
linking sites to enable the PNIPAAm physical hydrogels. As cross-linking
density of the hydrogel network was determined by temperature, its swelling
and deswelling ratio could be altered. Applying the POSS-capping method
improves the PNIPAAm response to temperature. Below the temperature of
volume phase transition, the swelling ratio increased with decreasing the
percentage of POSS (or with increasing the length of PNIPAAm chains). The
relationship could be interpreted to the density change of physically cross-
linked network [3].
Apart from the thermos-responsive hydrogels, electroconductive
hydrogels are highly focused because of its fast response to electric stimuli. In
2015, Choi et al. introduced synthesizing of CNT-wrapping alkyne-
functionalized PVA chains. 1mg CNT was firstly dispersed into a solution of
a-PVA prepared from a-PVA powder (30 mg) in 1 mL of 0.1 M KCl aqueous
Synthesis and Functionalization of Hydrogel … 109
solution. Then, the mixture was sonicated with a probe-tip sonicator (26 W)
for 30 min. The mixture was centrifuged (13 000 rpm) for 90 min, and then
decants were collected. The 30 mg mL−1 of a-PVA, 6 mM of bis-azide and 10
mM CuSO4·5H2O in aqueous solution were prepared with 0.1 M KCl as the
supporting electrolyte. Next, the hydrogel was deposited on the ITO-coated
glass electrode by chronoamperometry and the reduction potential of Cu (II)
was applied. In this approach, the apparent electrochemically active surface
area in the pre-gelation solution with CNTs was 2.27 times larger than that
without CNTs, leading to a faster deposition of hydrogels with increased Cu
(I) catalysts concentration. At pH=8, the release rate of model drug
tetracycline is faster than pH=5, indicating that this might be a new method for
in situ electro-stimulated drug delivery system [4].
Figure 1. The synthesis of CNT enhanced hydrogel (a) and the drug release
model (b) [4].
However, due to the use of toxic Cu (I) as catalyst, the application of this
method in biomedical is limited. Azide-Alkyne reaction of same strategy that
doesn’t require catalyst was found in recent years. In 2014, Truong et al.
reported click reaction between PEG-alkyne and CS azide. The hydrogel
formed within 5–60 min at 37°C. It was prepared in 10 mL vials previously
silanized with Sigmacote® to prevent gels adhering to the glassware. In a
typical procedure for a hydrogel with [azide]; [alkyne] of 1; 1, 25 μL of PEG-
alkyne solution (80 wt%) was added to 200 μL of a 2.25 wt% solution of CS-
azide and then mixed for 5 s using a vortex mixer before transferring into a
water bath at 37°C. The gelation time was determined by vial tilt method. The
human mesenchymal stem cells (MSCs) was encapsulated in vitro and cell
viability was assessed after 24 hours. MSCs demonstrated high viability
(>95%). MCSs seeded to formed hydrogel and cultured for 7days also showed
adhesion to hydrogel with no cytotoxic response. The hydrogels were also
shown to display a rupture compressive stress of 81 ± 11, 160 ± 15 and 192 ±
20 kPa. During this process density of hydrogel are also found to be enhanced
with higher polymer concentration of precursor [5].
Figure 2. The reaction between PEG-alkyne and CS-azide [5].
To introduce further properties for better functionality, the hydrogels are

usually required to take additional modification steps. If the hydrogel could
contain clickable reactive sites, further functionalization could be faster and
easier. Yilmaz et al. discovered a hydrogel synthesis method combining three
monomers in one step with –N3 enabling further functionalization through
SPAAC. The hydrogel was firstly synthesized through AAm, BAAm, PAm by
photoinitiated free radical polymerization. To imitate the functionalization of
hydrogel through Cu (I) catalyzed click reaction, 2-azidomethyl pyrene (Py-
N3) was used. The fluorescence spectra showed successful functional reaction
[6].
Figure 3. The reaction among AAm, BAAm and PAm [6].
Figure 4. The Pyrene functionalization of hydrogel [6].

The azide groups could not only be applied to enable further functionality
as mentioned in the previous example, it could also be utilized as a part of the
reaction cycle to realize auto-adjust temperature control of hydrogels. He et al.
synthesized a self-regulated hydrogel to realize the automatic temperature
control through the several exothermic catalytic reactions containing a click
reaction. The thermal-response of poly (N-isopropylacrylamide) is enabled by
4 reactions including the click reaction between ‘click’ reaction between
octylazide and phenylacetylene catalyzed by Cu (PPh3)2NO3 as illustrated in
the Figure 5. The shape change of this temperature responsive hydrogel is also
shown in Figure 6. Although the hydrogel synthesis is not directly related to
the click reaction in this example, we can still get a broad idea of the potential
application of hydrogels with click reactions.
Figure 5. The ‘click’ reaction between octylazide and phenylacetylene catalyzed by Cu

(PPh3)2NO3[7].
Figure 6. The process of temperature regulation process [7].

2. HYDROGELS PREPARED FROM

THIOL-ENE PHOTO COUPLING
The thiol-ene is a click reaction involving a radical-mediated addition of a
thiol to a double bond under light irradiation [1]. It is in use for the formation of
networks or for the purpose of controlling molecular weight in radical
polymerization [8]. Thiol-ene reaction could provide a promising replacement
to the chain-polymerization synthesis of hydrogel. With shorter gelation time,
the free radicals generated by photoinitiator cause less damage to cells. Lin et
al. developed a thiol-ene hydrogel system composed of a PEG4NB macromer
and a simple bis-cysteine-terminated and chymotrypsin-sensitive peptide
sequence (CGGYYC, arrow indicates enzyme cleavage site) for the
encapsulation of MIN6 b-cells. Chain-growth PEG hydrogels were
photopolymerized from desired concentrations of PEGDA (10 kDa) and in the
presence of 1 mM LAP (3 min UV at 365 nm, 5 mW/cm2). Step-growth thiol-
ene hydrogels were formed from PEG4NB (20 kDa) and a chymotrypsin-
sensitive peptide crosslinker (CGGYC). Gels were formed in 1 mL syringes
with open tips for gel removal. Compared to PEGDA hydrogels, the β -cells
encapsulated in this systems were more viable and formed functional cell
spheroids which was later retrieved through hydrogel degradation. The results
showed MIN6 β -cells remained 93±1.4% alive in PEG4NB hydrogel while
had only 45 ± 2.2% viability in PEGDA [9].
Another approach to speed up synthesizing progress is using Eosin-Y
(EY) as the Sole Photoinitiator. With the increase of EY concentration, the
initiation rate of both thick and thin hydrogels is accelerated. Shih et al.
introduced using Eosin-Y (EY) as the sole photoinitiator initialized through
visible light. Photoinitiator Eosin-Y (concentration: 0.1−2.0 mM) was added to
the precursor solution and the gelation was accomplished after exposing the
solution to a halogen cold light lamp (AmScope, Inc.) for 4 min at 70 000 Lux.
Wide thickness ranging (from tens of micrometers to a few millimeters)
multilayered hydrogels could be synthesized through this step-growth method.
An obvious decrease of gel fraction in thick hydrogel is found yet the thin
hydrogels, the gel fraction remains stable in the high level. As the
concentration of EY increases, it is more likely to form pendant in polymer by
the quenching and termination of EY leading to a decrease of network-
crossing linking density. In that case, the swelling ratios of both thick and thin
hydrogels increase and the mechanical properties are reduced [10].
Figure 7. Schematics of non-gelling photopolymerizations [9].
Figure 8. Formation of Gel Particles by Cross-Linking of Hyperbranched Polyglycerol

(hPG) and Polyethyleneglycol (PEG) [11].
Although the utilizing step growth polymerization could reduce the

damage of free radicals to cell viability, the synthesizing method could be
further improved. In 2012, Rossow et al. utilized droplet microfluidic to
prepare the hydrogel to reduce free radicals harmful to cell viability. The
gelation of these microgels is achieved via the nucleophilic Michael addition
of dithiolated PEG macro-cross-linkers to acrylated hPG building blocks and
does not require any initiator. The first precursor contained hPGs with a
weight-average molecular weight (Mw) of 16.5 kDa functionalized with 10
acrylate groups (hPG16.5Dea), and the other contained PEG-diamines with
different molecular weights. The PEG-diamines are then converted with 2-
iminothiolane hydrochloride to yield thiol terminated PEG (PEG-dithiol) in
situ. Both two solution and cells solution (yeast cells as model) was mixed in
Droplet-microfluidic device and subsequently gelled into yeast-cell-laden
particles. The highest yeast cell viability was obtained with the PEG6.0 kDa
gels with high precursor concentration [11].
Figure 9. The gelation and cells encapsulation [16].
Conventionally, the effects of receptor inhibitors are tested on 2D surfaces

before they were tested using animal models. However, the 2D surface is not
an ideal platform for test because more and more researches are indicating that
extracellular niches could influence tumor metastasis [12-14]. In order to test
the matrix properties on antitumor drugs, Ki et al. discovered a new approach
by using thiol-ene to control the stiffness of hydrogels through the alternation
cross-linking degree. A 3D hydrogel matrix was synthesized using MMP-
sensitive peptide as crosslinker for PEG-hydrogel. The gelation process was
illustrated in Figure 9. This new system serves as a promising platform for
pancreatic ductal adenocarcinoma cell (PANC-1) culture with adaptable and
well-defined biophysical and biochemical properties. It could mimic the
aspects of tumor niches for studying cancer cell fate processes under the
influence of various environmental stimuli. In their research, this hydrogel
proved that EGFR peptide inhibitor could only lead to significant cell death in
stiff hydrogel (G’~12kPa) indicating the non-negligible importance of matrix

properties in antitumor medication [15].
Similarly, McKinnon et al. created a system thiol-ene cross-linked and
functionalized with ECM-mimic peptides, a cationic peptide, and bFGF as
crosslinker. 8 equivalents of 5-norbornene-2-carboxylic acid were dissolved in
anhydrous DMF and activated with 7.5 equivalents of HBTU (Sigma). 16
equivalents of N-methylmorpholine (Sigma) were added as base. After
activating for 5 minutes, this mixture was added to 1 equivalent of 20 kDa 4-
arm PEG-amine (JenKem) dissolved in anhydrous DMF and reacted overnight
at room temperature. PEG-norbornene was precipitated in diethyl ether,
redissolved in DI water, and dialyzed against DI water for 24 h (2000
MWCO). The study results indicated that this new system could support the
survival of motor neurons and a robust extension of motor axons. This in vivo
study system could serve as a tool providing insights for neurons study and
improve medical treatments for cellular delivery vehicles for treatment of
neurodegenerative disease. Besides, this useful platform could be utilized to
study the fundamental aspects of neuronal development and provides insight
into the requirement of in vivo cell delivery vehicles [17].
Figure 10. The precursors of hydrogel (a) and the polymerization of hydrogel [17].
Besides, the medical application of hydrogel could be expanded to

antibacterial materials. In 2014, Cleophas et al. discovered a method to
synthesis of bactericidal hydrogel surface. PEGDA 700 and PTMP were
crosslinked via thiol-ene reaction in the presence of Inverso-CysHHC10. The
biological activity of AMP remains the same after the immobilization. The
resulting AMP-hydrogels showed potent bactericidal activity against Gram-
positive S. aureus and S. epidermidis and Gram-negative E. coli in vitro. It
killed >99.9% of inocula of S. aureus ATCC 49230, S. epidermidis ATCC
35984, and E. coli ATCC 8739. Besides, in cytotoxicity testing, all tested
peptides showed less than 2% lysis as compared to the control (1% Triton X-
100). It could be generalized that this new hydrogel has a high antibacterial
property with low hazard to red blood cells [18].
Figure 11. Initiation and Polymerization Mechanisms for Visible-Light-Mediated

Thiol-ene Photopolymerization, Using Eosin-Y (EY) as the Sole Photoinitiator [10].
Figure 12. Single step immobilization/polymerization of bactericidal hydrogel

surface [18].
3. STRAIN-PROMOTED AZIDE-ALKYNE
CYCLOADDITION (SPAAC)
SPAAC is a method that could enable cyclooctyne molecules with azides
to react quickly without copper catalyst as a result of ring strain and electron-
withdrawing fluorine substituents [1].
As mentioned in the previous chapters, the thiol-ene reaction has been
used to control the stiffness of hydrogel. To further enable more diversified
and more precise control of hydrogel in its functionality and architecture,

DeForest et al. introduced a novel method to prepare the hydrogel network.
PEG-tetraDIFO3 and bis (azide)-functionalized polypeptide was reacted with
four-arm poly (ethylene glycol) (PEG) tetracyclooctyne. The system contains
functional precursors for gel formation, chemical patterning-thiol ene
functionalization and mechanical patterning-photodegradation. Terminal
difluorinated cyclooctyne (DIFO3) and azide (2N3) moieties with 1:1
stoichiometry at 10 wt% total macromere concentration experienced a SPAAC
reaction to form hydrogels in about 2mins, as the illustrated in Figure 13. This
hydrogel follows first order degradation and the cleavage could be control by
focused multiphoton laser light (=740 nm) to form precise 3D shape. After
the functionalization of RGD, a cell-adhesive fibronectin motif in degraded
channels, the hydrogel could support the migration of human mesenchymal
stem cells (hMSCs) and could enable hMSCs to form complex structure with
the presence of 3T3 fibroblasts [19].
Figure 13. The various functional components of hydrogel [19].

Microfluidic polymerization has high equipment cost and lack of

scalability [20, 21]. To overcome these problems, replica molding was
invented to create non-spherical micro particles [20-22]. Jung et al. tested the
conjugation of chitosan-PEG particles with single-stranded (ss) DNAs via
SPAAC reaction. The chitosan−PEG microparticles were incubated in 5 ×
SSC buffer solution containing 0.05% (v/v) TW20 with 500 μM DBCO-sulfo-
NHS ester for 1 h on a rotator at room temperature. The unreacted DBCO-
sulfo-NHS ester molecules were rinsed 5 times using the rinsing procedure.
The DBCO-activated microparticles were then reacted with 10 μM of azide-
terminated ssDNAs (i.e., F-DNAazide or capture DNA) for 1 day at room
temperature. The unconjugated DNAs were then rinsed 5 times using the
rinsing procedure. In this paper, tobacco mosaic virus (TMV) is used as an
example of supramolecular targets. After the Hybridization-Based Assembly
of TMV, the results indicated that this could be a promising method to enable
robust biomolecular conjugation or assembly platform. The observed TMV is
related to the DNA surface density of the DNA-conjugated particles. This new
method provides higher DNA density than the hydrogels functionalized via
PDMSbased microfluidic procedures. Through the click reaction, there is no
legible decrease of DNA fluorescence intensity upon hydrogel for three
months which confirms this Cu-free fabrication- conjugation scheme [23].
Figure 14. The reaction between Chitosan-PEG and DBCO-Sulfo-NHS Ester with the
fluorescein [23].
Figure 15. Azide-functionalized RAFT-copolymer (PEG-N) 1 crosslinks with PEG-

DBCO crosslinker 2 [26].
Figure 16. Fluorescent signal of Gremilin1in hydrogel and PEG-N3 on 0 to 14 days

[26].
Figure 17. Synthesis of photocleavable caged ciprofloxacin (PC-CIP) [27].

Figure 18. The synthesis of hydrogel with conjugated PC-CIP [27].
Figure 19. UV-induced cleavage reaction of caged PC-CIP [27].
Figure 20. Synthesis of aminooxy-8-arm PEG [24].

Figure 21. Synthesis of 8-arm alkyne PEG [24].
More complicated conjugation of proteins could be realized on multi

layered hydrogels. Broyer et al. reported reactions containing Oxime reaction
and SPAAC reaction in synthesizing protein microarrays on the modified
surfaces of hydrogels. In this approach, proteins were immobilized either side-
by-side or in multilayer constructs. Two 8-arm PEG were firstly synthesized
through Figure 20 & Figure 21. Then the Aminooxy and alkyne PEGs were
patterned to Silicon wafer by e-beam lithography. The attaching of ubiquitin
and myoglobin is illustrated in the Figure 22. The proteins were subsequently
visualized by fluorescent antibody staining [24].
Previous several introductions have been focusing on the gelation of more
than one precursor. To create a dendron−polymer conjugate based single-
component system that enables fabrication of photopatternable “clickable”
hydrogels, Kaga et al. introduced a dendron−polymer conjugate- based single-
component system that enables fabrication of photopatternable “clickable”
hydrogels is synthesized for gel formation through only one precursor. alkene
and alkyne reactive groups attached onto the surface of these
dendron−polymer conjugates provided the desired multifunctionality [25].
Delivery of therapeutic factors is closely related to the degradation of
hydrogels releasing therapeutic factors. In order to sustain stable therapeutic
concentration, a hydrogel with constant and controllable degradation is
expected. SPAAC click reaction also provides a possible solution for drug
encapsulation and controlled release solution for hydrogel. Hermann et al.
reported a hydrogel formed of PEG and dibenzylcyclooctyne (DBCO)-
crosslinker via SPAAC, encapsulating Alexa Fluor tagged glutathione s-
transferase (GST-647) to mimic the target medicine Gremlin1. Gel could be
formed in 2 minutes to eliminate influence on other tissues. Azide-
functionalized RAFT-copolymer (PEG-N3) rapidly cross links with PEG-
DBCO (weight percent 12.5%) cross-linker, forming a hydrogel in 2 minutes

with nearly linear releasing simulated drug within 14 days meanwhile
maintaining the stable mechanical property or 7 days. Meanwhile, the control
group showed no fluorescent signal after 5 days (Figure 16) [26].
More than the control release of drug in vivo, Shi et al. synthesized the
drugs into hydrogels with a click reaction for two monomers (4-arm PEG–
dibenzocyclooctyl (DBCO) and 4-arm PEG-N3) and photocleavable caged
ciprofloxacin (PC-CIP). The PC-CIP was first reacted with DCBO, and then
the product was polymerized with PEG-N3 with a total polymer concentration
of 10wt%. Hydrogel formation with 20mol% of conjugated ciprofloxacin
could form good mechanical property within 5 minutes, suitable for ‘‘spray-
on’’ wound dressing materials. Via the results for the analysis of
photodegradation, it was found that the total amount release of drugs is related
to the total irradiation of UV light. The degradation process was shown in the
Figure 19 [27].
Figure 22. The successful myoglobin (green) and ubiquitin (red) fluorescence images
in multilayer structures [24].
Figure 23. Fabrication and Functionalization of Photo-Cross-Linked “Clickable”

Hydrogels from Dendron−Polymer Conjugates [25].
Figure 24. Structures and Naming of Macromers and the Orthogonally Cross-Linked
ClickGel Networks [29].
As shown above, the degradable hydrogels have huge application for

either drug release in or out of organisms. Several experiments have proved
the degradation of hydrogels is first order reaction [19, 28-31]. The placement
of labile ester linkage near cross-linking site with adjustments of macromer
ratio, enables broad tuning of disintegration rate to precisely match the
theoretical predictions based on first-order linkage cleavage kinetics. Xu et al.
created this type of hydrogel synthesized from 4 types of precursors in order to
alter the degradation rates prior to reaching the network disintegration in a
wide range (from 2 to >250 days). The click SPAAC and degradation data are
shown in the following Figure 24 and Figure 25 [29].
Figure 25. The degradation time from ClickGel-A to ClickGel-D in PBS

and α-MEM [29].
4. DIELS-ALDER REACTION
4.1. Common Diels-Alder Reaction
As a natural material with broad application in tissue engineering and

wound-healing [32], Hyaluronic acid (HA) is biocompatible and
biodegradable and elicits low levels of immune response [33]. Nimmo et al.
synthesized Furan-modified HA (diene) using 4- (4,6-dimethoxy-1,3,5-triazin-
2-yl)-4-methylmorpholinium chloride (DMTMM) and then formed hydrogels
with dimaleimide poly (ethylene glycol) (dienophile) as crosslinker. With the
increase of dimaleimide poly (ethylene glycol), the hydrogel formed by Furan-
modified HA presented a plateau of G’, which demonstrating that Diels-Alder

click reaction is a promising way in synthesis of HA hydrogels [34].
Original enzymatic crosslinked hydrogels have limited application due to
the poor mechanical and rapid degradation properties. To overcome these
defects, Yu et al. combined the HA Diels-Alder click reaction with the
enzymatic crosslinking method to improve mechanical properties of hydrogel.
This new hydrogel was synthesized with strain-stress curves similar to the
stress–strain curve of PAC (the porcine articular cartilage tissue) through
Diels-Alder reaction enabling its ability (breakage strength of 109.4 kPa) to
support cell attachment and growth. The TA groups (the red triangle showed
in the picture) were firstly crosslinked by enzymes and then the DA click
reaction happened between furan (the blue three quarters of fan shown in the
picture) and MAL–PEG–MAL groups. The live dead assay and the CCK-8
cytotoxicity assay proved that this hydrogel provided high cell viability with
high cell proliferation [35].
Figure 26. The reaction between PEG-dienophile and DMTMM [34].
Figure 27. The linkage of hydrogel [35].

HA is not the only materials that could be utilized to enhance the property
of hydrogel. Cellulose nanocrystals (CNCs) are nanoentities obtained from the
partial hydrolysis of a variety of cellulosic materials with length of 100–
500nm and width of 5-30nm [36, 37]. Utilizing Maleimide-grafted cellulose
nanocrystals (CNC-Mal) as cross-linkers in Diels-Alder Click reaction,
García-Astrain et al. synthesized new hydrogel of which G’ and G” stays in
high level during Dynamic modulus tests in all frequencies. This fact proved
the enhancement provided by nanocrystals as cross-linkers [38].
Figure 28. The Diels-Alder click reaction between GF and CNC-Mal [38].
4.2. Special Diels-Alder Reactions
There is also a special type named as inverse electron demand Diels-Alder

reaction. It happens between tetrazine and an appropriate dienophile
(norbornene, trans-cyclooctene). This method has been proved useful for
conformationally strained reactions for in vitro and in vivo cell labelling and
imaging [39].
Alge et al. functionalized hydrogel via thiol-ene click reaction. The PEG-
Tz reacted with norbornene-functionalized peptide to encapsulate hMSCs
model cells. The swelling ratio decreases and the G’ & G” increase with
higher weight percent of PEG-Tz as initial monomer. The result showed
cytocompatibility: 92 ± 3% viability after 24hr encapsulation and 79 ± 6%
viability after 72hr encapsulation [16].
Moreover, Hetero-Diels-Alder reaction could be a new addition to the

existing bioorthogonal chemistry. Li et al. found a click Hetero-Diels−Alder
cycloaddition reaction between o-quinolinone quinone methide (oQQM) and
vinyl thioether (VT). o-Quinone methides (oQMs) are highly reactive and
versatile synthetic intermediates that can undergo rapid and selective Hetero-
Diels−Alder (HDA) cycloadditions with electron-rich dienophiles to generate
a multitude of benzannulated tetrahydropyran derivatives [41, 42]. This
ligation has high efficiency under physiological condition and is compatible
with other SPAAC reactions [41].
Figure 29. Tetrazine cycloaddition with trans-cyclooctene forming

a dihydropyrazine [40].
Figure 30. The precursors A, B, C, the click reaction D, and the encapsulation
of cells [16].
Figure 31. The synthesis of GelMA [44].

4.3. Thiol-yne Reaction
Since thiol-ene is capable of bonding to only a single sulfur in thiol-ene

coupling reaction, the maximum density of the crosslinked network is limited
by the monomer functionality. In reports from 1940-1960s, scientists have
provided speculation of how alkynes could be utilized for network formation
and polymer functionalization [43]. Nowadays, thiol-yne reaction has been
extended to hydrogel formation. Thiol-yne could be combined with thiol-ene
to reached mechanical integrity and cytocompatibility. Daniele et al.
synthesized hydrogels covalently crosslinked gelatin methacrylamide
(BioSINx) in which the gelatin methacrylamide reacts with both itself and the
PEG network has synthetic network. The gelatin methacrylamide (GelMA)
was firstly synthesized. Then, two types of PEG (PEG tetra-thiol and PEG
tetra-alkyne) were synthesized. The reaction of three precursors formed the
interpenetrating network as shown in Figure 34.
Figure 32. Development of Bioorthogonal Ligations Using oQMs.
Hydrogel remained short-term structure reliability enabling cell growth

(Endothelial cells (EA.hy926) for 1 week with high level of cell viability
(>90%). The hydrogel gains compressive stress 15.56 ± 0.51 MPa at ca. 98%
compression without fracture, stronger than the hydrogel is only synthesized
by either one of the methods [25]. In Daniele’s report, the interpenetrating

network synthesized by Thiol-ene and Thiol-yne exhibited modulus from 10.8
to 327.7 kPa. Meanwhile the physically-incorporated gelatin and PEG-co-
GelMA hydrogels only exhibited modulus between 8.2-66.3 kPa and 9.5-247.2
kPa, showing a stronger mechanical property [44].
Figure 33. The synthesis of PEG tetra-alkyne [44].
Figure 34. The polymerization of precursors and the formation of interpenetrating

network [44].
4.4. Thiol-Michael Reaction
Thiol-ene reaction is not limited to radical-mediated process and can also

proceed via ionic mechanism and typically anionic reactive center. Thiol-
Michael reaction is an example of base-catalyzed addition of thiols [45]. The

Michael addition is facile reaction between nucleophiles and activated olefins
and alkynes in which the nucleophile adds across a carbon–carbon multiple
bond [46]. Kharkar et al. discovered a new method for employ different
degradable links via Thiol-Michael enabling precise control of degradation
time for different application. The hydrogel was synthesized by functionalized
PEG. The D1E and D2ER functional group has one or two degradable group
respectively. The D1E hydrogels can only undergo degradation by ester
hydrolysis. D2ER hydrogels can undergo degradation by ester hydrolysis and
by thiol exchange reactions. The result showed release of model protein
(bovine serum albumin (BSA-488)) in the Control (~33%), D1E (~36%), and
D2ER (~90%). It indicated GSH-responsive hydrogels as a drug carrier for
controlled cargo release applications. To accurately adjust the degradation of
hydrogels, D2ER was synthesized through click reaction with property to
undergo degradation by both ester hydrolysis and thiol exchange reactions.
With medium concentration of GSH, the degradation could become a second-
order reaction that not only controlled by the number of crosslinks, but also by
the concentration of GSH, making it capable to be complex degradation
hydrogels [28].
Figure 35. The different properties of each hydrogel composition [28].
Tibbit et al. compared the chain polymerized hydrogel and step

polymerized hydrogel in 2013. Chain-polymerized hydrogels were fabricated
through the copolymerization of PEGdiPDA with PEGA via free-radical
polymerization. The Step-polymerized hydrogels were fabricated through the

copolymerization of PEGdiDPA with PEG4SH via Michael-addition
polymerization. As illustrated in Figure 37. Compared to the chain-
polymerized gels, step-polymerized hydrogels showed increased ductility,
tensile toughness, and shear strain. While rheometry results indicate no
relationship between the photodegradation rate and network structure of
hydrogel, the erosion rate of chain-polymerized is much faster than step-
polymerized gels, on account of the lower network connectivity [47].
Figure 36. The degradation of hydrogel of D1E and D2ER [28].
Figure 37. The chain polymerization and step polymerization [47].

CONCLUSION
Hydrogels have broad application in medical treatment and industry.
Through various types click chemistry, it is expected to get versatile new
hydrogel products with versatile properties. In this chapter, we have covered 6
main hydrogel synthesis methodology (CuAAC, thiol-ene, SPAAC, Diels
Alder, thiol-yne, Thiol-Michael reaction), especially two uncommon Diels-
Alder reactions (Hetero-Diels-Alder reaction and inverse electron demand
Diels-Alder reaction). The mechanical properties, chemical properties and
their influences on application has been introduced. The corresponding
relationship with click chemistry is also stated to provide readers a broad view
on how to combine new materials, use various click chemistry process for one
hydrogel to improve the property of hydrogels via click chemistry.
Functionalization of hydrogels such as electro-control and thermos-response
via click reactions are also described. Moreover, this chapter also shed lights
on potential new applications of hydrogels. Hydrogel could have further
application with the progress of researches.
REFERENCES
[1] Jiang Y, Chen J, Deng C, Suuronen EJ, Zhong Z. Click hydrogels,
microgels and nanogels: Emerging platforms for drug delivery and tissue
engineering. Biomaterials. 2014;35 (18):4969-85.
[2] Hanni KD, Leigh DA. The application of CuAAC 'click' chemistry to
catenane and rotaxane synthesis. Chemical Society Reviews. 2010;39
(4):1240-51.
[3] Wang L, Zeng K, Zheng S. Hepta (3,3,3-trifluoropropyl) Polyhedral
Oligomeric Silsesquioxane-capped Poly (N-isopropylacrylamide)
Telechelics: Synthesis and Behavior of Physical Hydrogels. ACS Appl
Mater Interfaces. 2011;3 (3):898-909.
[4] Choi EJ, Shin J, Khaleel ZH, Cha I, Yun S-H, Cho S-W, et al. Synthesis
of electroconductive hydrogel films by an electro-controlled click
reaction and their application to drug delivery systems. Polym Chem.
2015;6 (24):4473-8.
[5] Truong VX, Ablett MP, Gilbert HTJ, Bowen J, Richardson SM,
Hoyland JA, et al. In situ-forming robust chitosan-poly (ethylene glycol)
hydrogels prepared by copper-free azide-alkyne click reaction for tissue
engineering. Biomater Sci. 2014;2 (2):167-75.
[6] Yilmaz G, Kahveci MU, Yagci Y. A One Pot, One Step Method for the
Preparation of Clickable Hydrogels by Photoinitiated Polymerization.
Macromolecular Rapid Communications. 2011;32 (23):1906-9.
[7] He X, Aizenberg M, Kuksenok O, Zarzar LD, Shastri A, Balazs AC, et
al. Synthetic homeostatic materials with chemo-mechano-chemical self-
regulation. Nature. 2012;487 (7406):214-8.
[8] Koo SPS, Stamenović MM, Prasath RA, Inglis AJ, Du Prez FE, Barner-
Kowollik C, et al. Limitations of radical thiol-ene reactions for polymer–
polymer conjugation. Journal of Polymer Science Part A: Polymer
Chemistry. 2010;48 (8):1699-713.
[9] Lin C-C, Raza A, Shih H. PEG hydrogels formed by thiol-ene photo-
click chemistry and their effect on the formation and recovery of insulin-
secreting cell spheroids. Biomaterials. 2011;32 (36):9685-95.
[10] Shih H, Fraser AK, Lin C-C. Interfacial Thiol-ene Photoclick Reactions
for Forming Multilayer Hydrogels. ACS Appl Mater Interfaces. 2013;5
(5):1673-80.
[11] Rossow T, Heyman JA, Ehrlicher AJ, Langhoff A, Weitz DA, Haag R,
et al. Controlled Synthesis of Cell-Laden Microgels by Radical-Free
Gelation in Droplet Microfluidics. J Am Chem Soc. 2012;134 (10):
4983-9.
[12] Miroshnikova YA, Jorgens DM, Spirio L, Auer M, Sarang-Sieminski
AL, Weaver VM. Engineering strategies to recapitulate epithelial
morphogenesis within synthetic three-dimensional extracellular matrix
with tunable mechanical properties. Physical Biology. 2011;8
(2):026013.
[13] Leight JL, Wozniak Ma Fau - Chen S, Chen S Fau - Lynch ML, Lynch
Ml Fau - Chen CS, Chen CS. Matrix rigidity regulates a switch between
TGF-beta1-induced apoptosis and epithelial-mesenchymal transition.
2012 (1939-4586 (Electronic)).
[14] Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, et al.
Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin
Signaling. Cell.139 (5):891-906.
[15] Ki CS, Shih H, Lin C-C. Effect of 3D Matrix Compositions on the
Efficacy of EGFR Inhibition in Pancreatic Ductal Adenocarcinoma
Cells. Biomacromolecules. 2013;14 (9):3017-26.
[16] Alge DL, Azagarsamy MA, Donohue DF, Anseth KS. Synthetically
Tractable Click Hydrogels for Three-Dimensional Cell Culture Formed
Using Tetrazine-Norbornene Chemistry. Biomacromolecules. 2013;14
(4):949-53.
[17] McKinnon DD, Kloxin AM, Anseth KS. Synthetic hydrogel platform for
three-dimensional culture of embryonic stem cell-derived motor
neurons. Biomater Sci. 2013;1 (5):460-9.
[18] Cleophas RTC, Riool M, Quarles van Ufford HC, Zaat SAJ, Kruijtzer
JAW, Liskamp RMJ. Convenient Preparation of Bactericidal Hydrogels
by Covalent Attachment of Stabilized Antimicrobial Peptides Using
Thiol–ene Click Chemistry. ACS Macro Letters. 2014;3 (5):477-80.
[19] DeForest CA, Anseth KS. Cytocompatible click-based hydrogels with
dynamically tunable properties through orthogonal photoconjugation and
photocleavage reactions. Nat Chem. 2011;3 (12):925-31.
[20] Lewis CL, Choi C-H, Lin Y, Lee C-S, Yi H. Fabrication of Uniform
DNA-Conjugated Hydrogel Microparticles via Replica Molding for
Facile Nucleic Acid Hybridization Assays. Analytical Chemistry.
2010;82 (13):5851-8.
[21] Merkel TJ, Herlihy KP, Nunes J, Orgel RM, Rolland JP, DeSimone JM.
Scalable, Shape-Specific, Top-Down Fabrication Methods for the
Synthesis of Engineered Colloidal Particles. Langmuir. 2010;26
(16):13086-96.
[22] Zhao X-M, Xia Y, Whitesides GM. Soft lithographic methods for nano-
fabrication. Journal of Materials Chemistry. 1997;7 (7):1069-74.
[23] Jung S, Yi H. Fabrication of Chitosan-Poly (ethylene glycol) Hybrid
Hydrogel Microparticles via Replica Molding and Its Application toward
Facile Conjugation of Biomolecules. Langmuir. 2012;28 (49):17061-70.
[24] Broyer RM, Schopf E, Kolodziej CM, Chen Y, Maynard HD. Dual Click
reactions to micropattern proteins. Soft Matter. 2011;7 (21):9972-7.
[25] Kaga S, Yapar S, Gecici EM, Sanyal R. Photopatternable “Clickable”
Hydrogels: “Orthogonal” Control over Fabrication and
Functionalization. Macromolecules. 2015;48 (15):5106-15.
[26] Hermann CD, Wilson DS, Lawrence KA, Ning X, Olivares-Navarrete R,
Williams JK, et al. Rapidly Polymerizing Injectable Click Hydrogel
Therapy to Delay Bone Growth in a Murine Re-synostosis Model.
Biomaterials. 2014;35 (36):9698-708.
[27] Shi Y, Truong VX, Kulkarni K, Qu Y, Simon GP, Boyd RL, et al. Light-
triggered release of ciprofloxacin from an in situ forming click hydrogel
for antibacterial wound dressings. J Mater Chem B. 2015;3 (45):8771-4.
[28] Kharkar PM, Kloxin AM, Kiick KL. Dually degradable click hydrogels
for controlled degradation and protein release. J Mater Chem B. 2014;2
(34):5511-21.
[29] Xu J, Feng E, Song J. Bioorthogonally Cross-Linked Hydrogel Network
with Precisely Controlled Disintegration Time over a Broad Range. J
Am Chem Soc. 2014;136 (11):4105-8.
[30] DeForest CA, Tirrell DA. A photoreversible protein-patterning approach
for guiding stem cell fate in three-dimensional gels. Nat Mater. 2015;14
(5):523-31.
[31] Santi DV, Schneider EL, Reid R, Robinson L, Ashley GW. Predictable
and tunable half-life extension of therapeutic agents by controlled
chemical release from macromolecular conjugates. Proceedings of the
National Academy of Sciences. 2012;109 (16):6211-6.
[32] Allison DD, Grande-Allen KJ. Review. Hyaluronan: a powerful tissue
engineering tool. Tissue engineering. 2006;12 (8):2131-40.
[33] Termeer C, Sleeman JP, Simon JC. Hyaluronan–magic glue for the
regulation of the immune response? Trends in immunology. 2003;24
(3):112-4.
[34] Nimmo CM, Owen SC, Shoichet MS. Diels-Alder Click Cross-Linked
Hyaluronic Acid Hydrogels for Tissue Engineering. Biomacromolecules.
2011;12 (3):824-30.
[35] Yu F, Cao X, Li Y, Zeng L, Yuan B, Chen X. An injectable hyaluronic
acid/PEG hydrogel for cartilage tissue engineering formed by integrating
enzymatic crosslinking and Diels-Alder "click chemistry". Polym Chem.
2014;5 (3):1082-90.
[36] Lin N, Dufresne A. Nanocellulose in biomedicine: current status and
future prospect. European Polymer Journal. 2014;59:302-25.
[37] Yang D, Peng X, Zhong L, Cao X, Chen W, Wang S, et al. Fabrication
of a highly elastic nanocomposite hydrogel by surface modification of
cellulose nanocrystals. RSC Advances. 2015;5 (18):13878-85.
[38] García-Astrain C, González K, Gurrea T, Guaresti O, Algar I, Eceiza A,
et al. Maleimide-grafted cellulose nanocrystals as cross-linkers for
bionanocomposite hydrogels. Carbohydrate Polymers. 2016;149:94-
101.
[39] Seitchik JL, Peeler JC, Taylor MT, Blackman ML, Rhoads TW, Cooley
RB, et al. Genetically Encoded Tetrazine Amino Acid Directs Rapid
Site-Specific in Vivo Bioorthogonal Ligation with trans-Cyclooctenes.
Journal of the American Chemical Society. 2012;134 (6):2898-901.
[40] Devaraj NK, Thurber GM, Keliher EJ, Marinelli B, Weissleder R.

Reactive polymer enables efficient in vivo bioorthogonal chemistry.
Proceedings of the National Academy of Sciences. 2012;109 (13):4762-
7.
[41] Li Q, Dong T, Liu X, Lei X. A Bioorthogonal Ligation Enabled by Click
Cycloaddition of o-Quinolinone Quinone Methide and Vinyl Thioether.
J Am Chem Soc. 2013;135 (13):4996-9.
[42] Willis NJ, Bray CD. ortho-Quinone Methides in Natural Product
Synthesis. Chemistry – A European Journal. 2012;18 (30):9160-73.
[43] Lowe AB, Hoyle CE, Bowman CN. Thiol-yne click chemistry: A
powerful and versatile methodology for materials synthesis. Journal of
Materials Chemistry. 2010;20 (23):4745-50.
[44] Daniele MA, Adams AA, Naciri J, North SH, Ligler FS. Interpenetrating
networks based on gelatin methacrylamide and PEG formed using
concurrent thiol click chemistries for hydrogel tissue engineering
scaffolds. Biomaterials. 2014;35 (6):1845-56.
[45] Chan JW, Hoyle CE, Lowe AB, Bowman M. Nucleophile-Initiated
Thiol-Michael Reactions: Effect of Organocatalyst, Thiol, and Ene.
Macromolecules. 2010;43 (15):6381-8.
[46] Mather BD, Viswanathan K, Miller KM, Long TE. Michael addition
reactions in macromolecular design for emerging technologies. Progress
in Polymer Science. 2006;31 (5):487-531.
[47] Tibbitt MW, Kloxin AM, Sawicki LA, Anseth KS. Mechanical
Properties and Degradation of Chain and Step-Polymerized
Photodegradable Hydrogels. Macromolecules. 2013;46 (7):2785-92.
Chapter 6
USING OF CLICK CHEMISTRY

FOR ELASTOMER
Ya-Lun Wang and Yu Chen*

School of Materials Science and Engineering
Beijing Institute of Technology
Beijing, China
ABSTRACT
Since the “click” reaction has been reported, it has become a useful
and powerful tool for chemistry. Using the “click” reaction, elastomer or
rubber can be conveniently prepared and modified. Different researchers
have reported a lot of new materials and surface modification methods. In
this review, a series of works were introduced. The main progresses of
the research on various “click” reactions applied to elastomer and rubber
were pointed out.
Keywords: Click Chemistry; Elastomer; Preparation and Modification;

CuAAC; Thiol-ene Reaction
*
140 Ya-Lun Wang and Yu Chen
INTRODUCTION
Elastomer is a kind of polymer that has a crosslinked network and shows
rapid and large reversible strain in response to stress.1 So it shows a high
elasticity and resilience than ordinary liner polymer. The crosslinked network
within an elastomer can be formed through chemistry or physical methods,
such as linked through sulfur bridge, a typical chemistry method, or and
through hydrogen bond, a physical method.
Different methods can obviously change the properties of final product.
For example, if we combine a polymer through building hydrogen bond, we
can change the situation to connect or disconnect this bond. In this way,
crosslinked network may be reconstructable and the material may be reusable
or self healing after broken. Or crosslink degree will affect to the glass
transform temperature (Tg) and modulus of an elastomer, i.e., a higher
crosslink degree usually means a higher Tg and modulus. Thus, controlling the
crosslink process is an effect way to gain an ideal material with great
properties.
In the other hand, single material, in many cases, usually need to be
conjuncted with other materials for better properties. So it is necessary to
combine variety materials together, such as form a silicone rubber coating on
natural rubber (NR) to anti-ozone. Thus, we need to think that, how to
combine two or more material stably.
“Click” chemistry gave us a new enlightenment to reach these purposes.
Since the term, “click” reaction was proposed in 2001, more and more
new processes and materials have been developed. These reactions have
various advantages such as rapid reaction rate, high yields, minimal oxygen or
water sensitive and developed tremendously over the past decade. 2 Therefore,
“click” reactions are used between monomers or crosslink reagents to obtain
various elastomers, to introduce different functional groups to change
properties of products, or just for increasing reaction rate or reducing reaction
temperature. In the other words, the using of “click” reaction enriched our
method to improve both of these performance and efficiency of the materials.
1. THE PREPARATION OF ELASTOMERS

AND RUBBER WITH “CLICK” REACTION
Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction and Thiol-

ene reaction are two famous “click” reaction. These two methods have been
Using of Click Chemistry for Elastomer 141
widely used for the preparation, modification and recycling of the elastomers
and rubbers. Besides, more reactions were used to aim to various purposes and
applications.
1.1 Using of Alkyne-azide Reaction
Alkyne-azide reaction could be easily catalyzed by Cu(I) salt, or in some

case it can occurs under heating without the need of catalyzer.3-4 These
reaction results 1,2,3-triazole ring into the elastomer and the it would offer a
stable covalently link between two parts. So the material containing triazole
rings structure can exhibit good chemical and physical stability. These
properties cause it become a powerful tool.
Zhai et al. found that using tripropargylamine as crosslink reagent,
different functional molar ratios elastomers can be prepared from azide
terminated ethylene oxide-tetrahydrofuran copolymer (ATPET) through the
bulk CuAAC reaction. Firstly, ethylene oxide-tetrahydrofuran copolymer
(PET) and SOCl2 was mixed together, then slowly heated to 75℃ and kept 20
hours, to reserve chlorine terminated ethylene oxide-tetrahydrofuran
copolymer (ClTPET). Next, the ClTPET were mixed with sodium azide,
heated to 80℃ for 24 hours and reserved ATPET. Then it was cured with
tripropargylamine at 50℃ for 7 days and formed hexagon lattices. This process
showed in Figure 1. And they reported that the mechanical properties of the
elastomers parabolically depend on the functional molar ratios: at first the
modulus increased and then decrease while the functional molar ratios are
increasing. The maximum modulus and stress and minimum strain
simultaneously occur near the stoichiometric ratio. At the stoichiometric ratio,
the elastomer network is comprised of hexagon lattices and a smaller mesh
size. This structure leads to lower equilibrium volume-swelling ratio.When
R<1.0 there were more ATPET dangling strands and when R>1.0 was more
free alkynyl groups, form larger-size meshes. This property leaded to that the
modulus of the elastomers first increase, and then decrease. This elastomer has
a glass transition temperature at about -66℃, and the R values hardly affect it.
Comparing with PET, the APTET elastomer showed a low viscosity because
there were less hydrogen bonding with other oxygen atoms. Thermal stability
showed that the thermal degradation of polyether strands more than the end-
crosslinking point triazole groups and it was limited by polyether but not
triazole. 5
Figure 1. Bulk CuAAC reaction between ATPET and tripropargylamine
With triazide terminated organic small molecules, Yang et al. reported

a new synthesized perfluoropolyether-based material via CuAAC
reaction. PFPE-dialkyne ether and 1,3,5-tris((3-azidopropoxy)methyl)-2,4,6-
trimethylbenzene (triazide) was prepared. As shown in Figure 2, PFPE-
dialkyne ether and triazide was mixed, to used to form the perfluoropolyether
gel, catalyzed by added copper wire pieces or copper turnings.This gel can be
cured into a tough, highly durable elastomer and had high chemical resistance
and low swell ability. The 1,2,3-triazole linkages were very stable that it's
possible to against harsh acidic or basic conditions. Also, the thermal stability
test showed that the material was stable after heat at 50℃ for 5 hours, without
weight or color change. The modulus of it was 3.5MPa and Tg was -10℃,
below the room temperature. Due to these properties, synthesized gel showed
a well mechanical properties. In addition, this fluoro-elastomer showed strong
adhesion to glass and itself, so it can be used to fabricate a multi-layer
microfluidic device. All of these properties let the fluoro-elastomer can be
applied for organic based microfluidic devices.6
Figure 2. PFPE-dialkyne ether and triazide formed the gel, finally got a three-
dimensional body structure.
But CuAAC reaction may cause copper-ion contamination inside

elastomer, so that the product will not suit to be used on biochemistry or
medicine field. This problem limits the application of obtained elastomers. So
it’s necessary to develop metal-free method to extend the application fields of
CuAAC reaction. Fortunately, alkyne-azide reaction can be easily carried out
without the using of copper-ion.
In the work of Gonzaga et al., thermally initiated Huisgen 1,3-dipolar
cycloaddition was studied. The work showed that in some case, reaction was
carried out without metal catalysts.3-4 Then, based on these these works,
Rambarran et al. used the thermally initiated Huisgen 1,3-dipolar
cycloaddition to form and functionalize a silicone elastomers without the using
of catalyst.7 Silicone elastomers were prepared by linking different reactivities
of di- or multi alkyne functionalized polysiloxanes with poly(azide)-containing
siloxanes. Different structure and molecular weight of an alkyne-modified
PEGs was used to obtain different properties. The results of studies showed,
the metal-free “click” reaction can be used to high-throughput preparation of
potential biomaterials surfaces.
In one word, CuAAC reaction and derived ractions showed a great
application prospect from biological materials to chemical equipment.
1.2 Using of thiol-ene reaction
Another widely used “click” reaction, thiol-ene “click” reaction, is a kind

of reaction using thiol group and carbon double bond to form the carbon-sulfur
bond. This reaction is a radical reaction, so that it can be easily initiated by

UV-light and don’t need any catalysts. This property let the reaction can be
used on biomaterials without the trepidation of heavy metal ion contamination.
Using thiol-ene reaction, polysiloxane-based luminescent elastomers were
prepared by Zuo et al. At first N-acetyl-L-cysteine functionalized polysiloxane
(PNL) was prepared via “click” N-acetyl-cysteine and prepared vinyl-
functional polysiloxanes. Then europium-ions were coordinated to the side-
chains of PNL in a THF solution, followed by the step that thiol-ene reaction
was used to produce silicone luminescent elastomers (Figure 3). The obtained
silicone rubber via UV-curing has fine mechanical properties and excellent
thermal stabilities, at the same time the above process can avoid the using of
heavy metal catalysts. On the other hand, they grafted the carbonyl-containing
monomers (MP, MPE and MS) onto vinyl-functionalized polysiloxane by
using thiol–ene reaction and obtained a series of side-chain carbonyl-modified
polysiloxanes (PMP, PMPE, and PMS). The functionalization ratio can be
controlled by adjusting the molar ratio of vinyl moietiescarbonyl-containing
monomers. The luminescence color of elastomers was pure and could be
changed by choosing different lanthanide ions.8-9
Figure 3. The Preparation of the Polysiloxane-based Luminescent Elastomers10

In others works, Zuo et al. obtained polysiloxane-based autonomic self-

healing elastomers via thiol-ene reaction.11 Because of the reversibility of the
borate hydrolysis process, they choose 4-[(allyloxy)methyl]-2-(4-vinylphenyl)-
1,3,2-dioxaborolane (VPD) as a crosslink reagent. VPD was synthesized and
then incorporated into a polysiloxane matrix. It was used to crosslink
poly[(mercaptopropyl)methylsiloxane] (PMMS) or both PMMS and vinyl-
functional polysiloxanes (VP) to obtain dual crosslinked network. The reaction
was initiated by a photoinitiator 2,2-dimethoxy-2-phenylacetophenone
(DMPA) and mercury lamp. The product could heal by itself after cut, via the
reversibility of the boronic ester crosslinkages to restore the original silicone
sample within 30 min. When the sample was cut, the polysiloxane would be
destroyed and exposed under the air. At that time the hydrolysis of boric acid
ester will finish. Then the dehydration will happen to form a new chemical
bond bonded with two cut polymers and become a new network, to finish the
self-healing process. The work showed that the dynamic boronic ester linkage
can be incorporated into polysiloxanes via thiol–ene “click” method.
And more others crosslink reagent can enhance the stability and
mechanical properties. Thus, by constructing a suitable crosslink reagent, a
functional structure can be introduced into the elastomer. Alzahrani et al. tried
to use two sequential click reactions, thiol-ene reaction and CuAAC reaction,
to prepared an elastomer and generate persistent wrinkles on surface.
Compared with traditional lithographic fabrication, the new method was a
simple and low cost way fitting to be used in smart adhesives, biological
material, anti-fouling coating, etc.12 Using two different reaction made it was
easy to control the progress. The first step, thiol-ene reaction, was used to
form a crosslinked polymer matrix and remain reactive alkyne sites
throughout the network. Pentaerythritol tetramercaptopropionate (PETMP),
trimethylolpropane triacrylate (TMPTA) and propargyl acrylate (PA) were
used to form elastomer network, catalyzed by triethylamine. ATR-FT IR
spectra was used to confirm and evaluate the functional group and characterize
the resulting crosslinked polymer networks. This work was shown in Figure 4.
The second step using the CuAAC reaction formed the wrinkles pattern. This
section will be described later.
Combined with microfluidics technique, Fleischmann et al. prepared
micrometer-sized liquid crystalline elastomer (LCE) particles via thiol-ene
reaction under UV light.13 Using 1(4-(4-pentenyloxy)phenyl-4-(6-
mercaptohexyloxy)-benzoate) LC1 as monomer, pentaerythritol tetrakis(3-
mercaptopropionate) (PETMP) and glyoxal bis(diallylacetal) (GBDA) as
crosslink reagents, and Lucirin TPO as photoinitiator, the mixture flow out the
micro tube to form droplets and “fly in” the co-flowing silicone oil. That will
form small droplets. Then let the droplets exposed under ultraviolet irradiation
about 2s to crosslink to form an elastomer network (Figure 5). The network
shows a fully reversible contraction upon heating. This work showed us a new
method for preparing particles.
Figure 4. Process of Wrinkle Formation via a Dual Click Reaction Approacha 12
Figure 5. Monomers and the Preparation Process via Thiol-ene Reaction 13
Rosilo et al. used 10-undecenoyl chloride to modify a native cellulose

nanocrystals (CNCs) surface and formed molecular brush with thiol end on the
surface of CNCs. Then they mixed the modified CNCs(mCNCs),
poly(butadiene) (PBD) and a bifunctional dithiol crosslink reagent to get a
composite film via UV-light initiated thiol-ene reaction. The crosslink process
will give the product a well and stable mechanical properties. By changing the
content ratio of mCNCs from 0 wt% to 80 wt%, they studied the effect of
weight fractions on the composite structure with TEM, mechanical and
thermal properties. They found that the increasing of the mCNCs weight
fraction will cause an uncommon abrupt transition from soft PBD-dominated
to reinforced mCNCs-dominated structure. All of the composite shows
stability to air humidity. The work found a way to improve the mechanical
properties by using biomimetic compositions.14
Figure 6. Reaction Process of the Preparation of the Recyclable Rubber System 15
Combining with a reversible crosslink network, the obtained elastomer

was reusable and it can be used as an environment friendly materials. Bai and
Li et al. used thiol−ene reaction and Diels−Alder “click” reaction to obtain a
thermally recyclable polybutadiene elastomer. The polybutadiene reacted with
modified furan via thiol−ene reaction: the thiol on the furan reacted with
polybutadiene to obtain a modified branched polymer. The obtained polymer

was then crosslinked by bismaleimide via Diels−Alder reaction: bismaleimide
react with furan on different polymers to form a crosslinked network. For the
second step, the D-A reaction was reversible under a high temperature, i.e., the
D-A reaction will aim forward under low temperature to form an elastomer
and aim backward to return liquid state when heat it for reforming. This
process was shown in Figure 6.This property caused that the elastomer can
easily remold to manufacture new product without obvious negative effects on
mechanical properties and shows a self-healing behaviors. The work illustrated
a new way to prepare thermoplastic elastomer. Obtained elastomer had well
solvent resistance at ambient temperature and tunable mechanical properties
depending on different crosslinking degree.15 This work provided us with a
new way to introduce a reversible structure into elastomer conveniently, let the
elastomer can be reusable and environment friendly.
1.3 Others reactions and applications
Tillet et al. used a cycloaddition reaction between azide and nitrile to

simplify the crosslink process of perfluoro elastomer. This azide–nitrile
cycloaddition reaction is similar with CuAAC reaction but in the absence of
any catalyst. They used this reaction to crosslink DuPont™ KalrezV®
perfluoroelastomer and changed the curing process from at 300℃ for two days
to at 150℃ for 24 h while pressurized. As it is shown in Figure 7, the pendant
of DuPont™ KalrezV® perfluoroelastomer (2) was cyano groups, and reacted
with prepared bis-azido fluorinated curing agent (1), formed a linkage between
two perfluoroelastomers. This change was simple to implement and reduced
the used energy than that currently used in the fluoro elastomers industry. The
results of thermogravimetric analyzes showed that resulting press cured fluoro
elastomer revealed an improvement by about 20℃ of the thermal degradation
profile in air compared with uncured fluoro elastomer.16
Electrospinning has used widely in polymer fibers production. But the low
glass transition temperature will cause the rubber fiber too unstable to produce.
Thielke et al. found an efficient way to fabricate stable electrospun fiber mats
of polybutadiene rubber by crosslinking via UV irradiation.
Trimethylolpropane trimercaptoacetate (TMPMA) was added as crosslink
reagent. After a electrospinning process under UV, the fibers would not melt
together or stick to the collector. The result related to the consent of the
TMPMA. It showed that the mass fraction of 2 wt%, and 3 wt% TMPMA
form individual fibers.17 But they found that the hydrophilicity of fiber mat
was decreased. So they modified the electrospun fiber mats via photo
initialized thiol-ene reaction and this stage will be explained in next part.
Figure 7. Preparation of Perfluoro Elastomer
According to the results of above studies, we can find that kinds of “click”
reactions offer us a powerful tool to prepare more high performance
elastomers and rubbers. The above reactions have become a powerful tool for
formation of the elastomers and rubbers.
2. USING OF “CLICK” REACTION ON SURFACE

MODIFICATION OF ELASTOMER
At some conditions, the ordinary prepared elastomer could not be used

directly due to the poor surface properties. These problems limited the
application of elastomer. Besides, traditional modification methods may be
poor efficient or difficult to be applied. But if there are some groups like
alkynyl or thiol within elastomer matrix, they can react with other
corresponding groups and then, using a high effect “click” reaction to
introduce expected parts onto or into the elastomer. Comparing with the
traditional processes, the above method shows high efficiency and it is simple.
It provides us a new idea to obtain the elastomer that can be used for its ideal
properties.
Numerous elastomer and rubber is hydrophobic material. This property
sometimes usually limits their using. Introduce hydrophilic groups onto the
surface of the elastomer via “click” reaction is a high effect way to change the
hydrophilicity.
Thielke et al. formed the fibers mat via electrospinning firstly. And they
found that the hydrophilicity of the polybutadiene fiber mat was decreased
than spincoat film. The rest carbon double bond in the polybutadiene fiber
offered a reactive point to react with thiol. Using hydrophilic mercaptoethanol
or thioglycolic acid to modify it, the contact angles of polymer fibers mat were
obviously changed. The contact angles were dependent on the specific thiol
used and it could be reduced to 0°, which showed super hydrophilic.17
Traditional silicone elastomers show the poor hydrophilicity, which
causing it cannot be used as a biomaterials surface. In order to get a
hydrophilic silicone gel trimethylolpropane trimercaptoacetate elastomers,
Rambarran et al. used a thermally induced metal-free reaction between azido
and alkyne to graft PEGs onto both functionalized and crosslinked silicone
elastomers and enhanced hydrophilic of silicone elastomers. They introduced
the alkynyl-modified PEGs or silicone to azidoalkyl-modified silicone first,
followed by crosslinking the obtained polymers by reacting the unreacted
azido groups and α,ω-alkynyl-PDMS to elastomers. The weight proportion of
PEG controlled the crosslink density and others properties such as viscoelastic,
wetting and protein adsorption ability. The rest of azide groups can be used for
further modification process.18
Natural rubber (NR) is a useful and renewable material. But many
properties of NR are not satisfactory, such as easy to oxidation, too smooth or
rough, etc. Exploiting the carbon double bond inside NR via “click” reaction,
certain groups can be introduced efficiently to modify the surface of NR.
The carbon double bond in the natural rubber causes it has a low oxidation
resistance. This property limits the application of NR. Shown in Figure 8,
Zheng et al. reported a polymethylvinylsiloxane (PMVS) coating layer
combined on natural rubber and given the elastomers a well ozone-resistance.
Using PMVS, trimethylolpropane tris(3-mercaptopropionate) (TMPMP) as a
crosslink reagent, 2-methyl-4-(methylthio)-2-morpholinopropiophenone as
photoinitiator, they prepared the coating layer via UV initiated thiol-ene
reaction. TMPMP crosslinked the PMVS to form the coating layer and offered
covalent crosslinking between natural rubber layer and PMVS layer. The
PMVS layer showed a great anti-ozone properties that, only microcracks can
be observed on the surface of coated elastomer, while pure natural rubber has
wider cracks at the same ozone concentration.19
Figure 8. PMVS Layer Coated NR Layer19
To obtain a surface modified natural rubber with tunable tailored friction

properties and surface pattern, two step thiol-ene reaction was used by
Manhart et al. and a new photochemical modification route was developed.
Trimethylolpropane tris(3-mercaptopropionate) (TriThiol) was grafted onto
natural rubber to offer an immobilization point for vinyl-functionalized silica
particles, which can increase the surface roughness. Using different patterns,
they precisely controlled the coefficient of friction value by changing the
width of silica particles modified banded regions. The patterns lead to a
difference between two orthogonal directions.20
Not only the surface pattern of the NR can be changed to improve the
tailored friction properties, synthetic elastomer surface also can form special
pattern. As previously described, Alzahrani et al. formed an elastomer via

thiol-ene reaction. The elastomer contained BPADA and photoinitiator I819.
After preparation, the rest alkynyl groups can continue the next step of
CuAAC reaction. The elastomer was immersed into a solution of copper(II)
sulfate, let copper(II) sulfate diffused into the film surface. After being
exposed to irradiation, the photoinitiator changed copper(II) to copper(I) and
initiated the CuAAC reaction. The diffusion-limited presence of copper(I)
limits the second polymerization to a thin skin layer on the surface. Finally,
wrinkles were formed on the surface of the elastomer. After CuAAC reaction,
the modulus and glass transition temperature of the elastomer was raised from
1.6 MPa and 2 °C to 4.4 MPa and 22 °C. Wrinkles on the surface of the
elastomer had wavelength and amplitude of 8.50 ± 1.60 and 1.41 μm. They
studied the wrinkles by SEM and AFM and control the generation of wrinkles
by changing the Cu-catalyst concentration, crosslink density, light intensity,
and monomer types offer many opportunities and this approach further enables
spatial selectivity of wrinkle formation by photopatterning.12
“Click” reaction is a good tool to introduce various structures or parts into
an elastomer. These structures or parts can have a unique performance to reach
the objective that once difficult to achieve.
Non-covalent crosslinking system has an advantage that the crosslinking
is reversible, so the elastomers is recyclable and repurposable. Rambarran et
al. obtained a hydrogen bond interactions a,x-(b-cyclodextrin terminated)
polydimethylsiloxane telechelic copolymers via metal-free “click” chemistry.
The thermal Huisgen 1,3-dipolar cycloaddition was used to create linkage
between mono-6-deoxy-6-azido-b-cyclodextrin (CD) and 1,3-Bis-
(propiolatobutyl)-capped polydimethylsiloxanes. The reaction was taken at at
80℃ for 5 days. Then, physical association between CDs which were grafted
onto different chain was created and soft viscoelastic elastomer was obtained
final. This physical association and elastomer were stable even up to 100℃.
Using a chaotropic agent, the physical association can be destructed and let
elastomers became small patches releasing active principle. In this way,
obtained elastomer can be formed again.21
Another application of reversible linkage is to prepared a rewritable
surface. Roling et al. developed a rewritable polymer brush micropattern on
glass or Si via thiol-ene reaction and triazolinedione (TAD) click reactions.
TAD reaction could be proceeded at room temperature and show a high
reaction rate without catalyst, irradiation, or other external stimuli. And the
used of indole will cause the reaction temperature rise to 120℃ or higher. An
indole functionalized substrate was prepared via thiol-ene reaction between
reactive self-assembled monolayers (SAMs) and thiol functionalized indole
derivative. Then the film was contacted with a PDMS stamp and was soaked
with ATRP-TAD solution in acetonitrile to get the micropatterns. After the
printing step, the substrates were immersed into 2,4-hexadien-1-ol solution in
DMF or α-phellandrene, to erase the micropattern by transclick reaction. This
step ATRP-TAD would detach to regain indole groups on the surface. At that
time, it can be printed again.22
The works showed above described high versatility for the preparation of
reversible chemical structure. The introduction of functional groups was more
easy to occur with kinds of “click” reactions.
CONCLUSION
In summary, various “click” reactions can rapidly combine two parts
together with high selectivity. Therefore, it will be more simple to develop
kinds of elastomer. Thus, introduction of functional group will be more
convenient, and then obtained elastomer will get ideal physical and chemical
properties, even unimaginable new features. For preparing processes, “click”
reaction is a powerful tool to create a stable crosslinked network, to give
obtained elastomer a suitable modulus or Tg. Or by using a pair of groups
likes diene and dienophile, a reversible network will be obtained. That mains,
the elastomer is an environment friendly renewable elastomer. For
modification processes, a special group or structure which is hard to combine
with a matrix will be easily linked with “click” reaction and change properties
of elastomer. Or for this process, by using “click” reaction, reaction time or
energy cost can be reduced obversely. So, we can focus on the design of
polymer, crosslink reagent and modified components but not the reaction that
how to combine them together. Compared with traditional chemical synthesis
methods, this process has great advantage, such as diversified, controllable,
efficient, low-consumption and low-pollution. Nowaday, elastomer industry
focus on not only just economic benefits of producing a elastomer, but also
impact on the environment and energy saving. And “click” reaction will
promote the rapid development of rubber and elastomer industry.
REFERENCES
[1] Mark, J. E.; Erman, B. In Networks, elastomeric, John Wiley & Sons,
Inc.: 2014; pp 1-46.
[2] Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click chemistry: diverse
chemical function from a few good reactions. Angew. Chem., Int. Ed.
2001, 40 (11), 2004-2021.
[3] Gonzaga, F.; Yu, G.; Brook, M. A., Polysiloxane Elastomers via Room
Temperature, Metal-Free Click Chemistry. Macromolecules
(Washington, DC, U. S.) 2009, 42 (23), 9220-9224.
[4] Gonzaga, F.; Yu, G.; Brook, M. A., Versatile, efficient derivatization of
polysiloxanes via click technology. Chem. Commun. (Cambridge, U. K.)
2009, (13), 1730-1732.
[5] Zhai, J.; Zhang, N.; Guo, X.; He, J.; Li, D.; Yang, R., Study on bulk
preparation and properties of click chemistry end-crosslinked copolyether
elastomers. Eur. Polym. J. 2016, 78, 72-81.
[6] Yang, Y.-W.; Hentschel, J.; Chen, Y.-C.; Lazari, M.; Zeng, H.; van Dam,
R. M.; Guan, Z., "Clicked" fluoropolymer elastomers as robust materials
for potential microfluidic device applications. J. Mater. Chem. 2012, 22
(3), 1100-1106.
[7] Rambarran, T.; Gonzaga, F.; Brook, M. A., Generic, Metal-Free Cross-
Linking and Modification of Silicone Elastomers Using Click Ligation.
Macromolecules (Washington, DC, U. S.) 2012, 45 (5), 2276-2285.
[8] Xue, L.; Zhang, Y.; Zuo, Y.; Diao, S.; Zhang, J.; Feng, S., Preparation
and characterization of novel UV-curing silicone rubber via thiol-ene
reaction. Materials Letters 2013, 106, 425-427.
[9] Zuo, Y.; Lu, H.; Xue, L.; Wang, X.; Wu, L.; Feng, S., Polysiloxane-
Based Luminescent Elastomers Prepared by Thiol-ene "Click"
Chemistry. Chem. - Eur. J. 2014, 20 (40), 12924-12932.
[10] Zuo, Y.; Lu, H.; Xue, L.; Wang, X.; Ning, L.; Feng, S., Preparation and
characterization of luminescent silicone elastomer by thiol-ene "click"
chemistry. J. Mater. Chem. C 2014, 2 (15), 2724-2734.
[11] Zuo, Y.; Gou, Z.; Zhang, C.; Feng, S., Polysiloxane-Based Autonomic
Self-Healing Elastomers Obtained Through Dynamic Boronic Ester
Bonds Prepared by Thiol-Ene “Click” Chemistry. Macromolecular Rapid
Communications 2016, 37 (15), 1300-1300.
[12] Alzahrani, A. A.; Nair, D. P.; Smits, D. J.; Saed, M.; Yakacki, C. M.;
Bowman, C. N., Photo-CuAAC Induced Wrinkle Formation in a Thiol-
Acrylate Elastomer via Sequential Click Reactions. Chem. Mater. 2014,
26 (18), 5303-5309.
[13] Fleischmann, E.-K.; Forst, F. R.; Koeder, K.; Kapernaum, N.; Zentel, R.,
Microactuators from a main-chain liquid crystalline elastomer via thiol-
ene "click" chemistry. J. Mater. Chem. C 2013, 1 (37), 5885-5891.
[14] Rosilo, H.; Kontturi, E.; Seitsonen, J.; Kolehmainen, E.; Ikkala, O.,
Transition to Reinforced State by Percolating Domains of Intercalated
Brush-Modified Cellulose Nanocrystals and Poly(butadiene) in Cross-
Linked Composites Based on Thiol-ene Click Chemistry.
Biomacromolecules 2013, 14 (5), 1547-1554.
[15] Bai, J.; Li, H.; Shi, Z.; Yin, J., An Eco-Friendly Scheme for the Cross-
Linked Polybutadiene Elastomer via Thiol-Ene and Diels-Alder Click
Chemistry. Macromolecules (Washington, DC, U. S.) 2015, 48 (11),
3539-3546.
[16] Tillet, G.; Lopez, G.; Hung, M.-H.; Ameduri, B., Crosslinking of
fluoroelastomers by "click" azide-nitrile cycloaddition. J. Polym. Sci.,
Part A: Polym. Chem. 2015, 53 (10), 1171-1173.
[17] Thielke, M. W.; Bruckner, E. P.; Wong, D. L.; Theato, P., Thiol-ene
modification of electrospun polybutadiene fibers crosslinked by UV
irradiation. Polymer 2014, 55 (22), 5596-5599.
[18] Rambarran, T.; Gonzaga, F.; Brook, M. A.; Lasowski, F.; Sheardown, H.,
Amphiphilic thermoset elastomers from metal-free, click crosslinking of
PEG-grafted silicone surfactants. J. Polym. Sci., Part A: Polym. Chem.
2015, 53 (9), 1082-1093.
[19] Ning, N. Y.; Zheng, Z. P.; Zhang, L. Q.; Tian, M., An excellent ozone-
resistant polymethylvinylsiloxane coating on natural rubber by thiol-ene
click chemistry. eXPRESS Polym. Lett. 2015, 9 (6), 490-495.
[20] Manhart, J.; Lenko, D.; Muehlbacher, I.; Hausberger, A.; Schaller, R.;
Holzner, A.; Kern, W.; Schloegl, S., Photo-patterned natural rubber
surfaces with tunable tribological properties. Eur. Polym. J. 2015, 66,
236-246.
[21] Rambarran, T.; Bertrand, A.; Gonzaga, F.; Boisson, F.; Bernard, J.;
Fleury, E.; Ganachaud, F.; Brook, M. A., Sweet supramolecular
elastomers from a,?-(ß-cyclodextrin terminated) PDMS. Chem.
Commun. (Cambridge, U. K.) 2016, 52 (40), 6681-6684.
[22] Roling, O.; De Bruycker, K.; Vonhoeren, B.; Stricker, L.; Koersgen, M.;
Arlinghaus, H. F.; Ravoo, B. J.; Du Prez, F. E., Rewritable Polymer
Brush Micropatterns Grafted by Triazolinedione Click Chemistry.
Angew. Chem., Int. Ed. 2015, 54 (44), 13126-13129.
Chapter 7
SURFACE ENGINEERING OF POROUS

MONOLITHS VIA CLICK CHEMISTRY:
TOWARDS FUNCTIONAL MATERIALS FOR
FLOW CHEMISTRY APPLICATIONS
Seydina Ibrahima Kebe1, Hela Kammoun1,2,

Mohamed Guerrouache1, Samia Mahouche-Chergui1,
Sabrina Belbekhouche1, Benjamin Le Droumaguet1,
Yosra Dridi-Zrelli2 and Benjamin Carbonnier1,*
1
Université Paris-Est, ICMPE (UMR7182), CNRS, UPEC, Thiais, France
2
Département de Génie Chimique-Université Libre de Tunis,
Tunis, Tunisie
ABSTRACT
Herein, we review methods to prepare and/or functionalize monoliths
through a plethora of click chemistries in view of developing materials
meant for flow-through applications. The latter range from separation
science (electrochromatography, chromatography, preceoncentration) and
catalysis technology (microreactor). The emphasis is on copper-catalyzed
azide-alkyne Huisgen dipolar cycloaddition, thiol-ene and thiol-yne
coupling, thiol-(meth)acrylate and thiol-epoxy reactions and Diels-Alder
* Corresponding Author address Email: carbonnier@icmpe.cnrs.fr.

158 S. Ibrahima Kebe, H. Kammoun, M. Guerrouache et al.
[4+2] cycloaddition and both organic, inorganic and hybrid variants of

the monolithic materials are covered. These chemical strategies are
proved efficient to provide materials with well-defined surface chemistry.
As such monoliths with hydrophobic, hydrophilic, ionic or chiral surface
functionalities are discussed and examples of their applications in
reversed-phase, hydrophilic interaction and enantioselective (electro)
chromatographies are provided and discussed with respect to the
chemical nature of the via click chemistry incorporated molecular units. It
is also highlighted that monoliths prepared via Click chemistry provide
excellent platforms for supporting metal nanoparticles (gold, copper) for
high catalytic performances (reduction of nitrophenol). We thus
anticipate that click chemistry strategies will increasingly be exploited for
designing monolithic materials with unique properties.
Keywords: capillary column, chromatography, click chemistry, micro-reactor,

monolith, surface functionalization
1. INTRODUCTION
Control at the molecular-leveled of the chemical nature of pores surface is
of utmost importance for predicting the overall behavior of porous materials
with potential flow-through uses [1]. This is notably true for monoliths which
are defined as single continuous pieces of highly cross-linked and porous
materials. Monoliths of organic, inorganic and hybrid nature, have
continuously gained interest over the last quarter of century as stationary
phases for capillary chromatographic and electrochromatographic applications,
as solid sorbents for extraction and preconcentration purposes [2, 3].
Monoliths exhibit unmatched intrinsic properties as they offer, for instance,
highly efficient mass transfer kinetics in contrast to their particulate
counterparts. Monoliths can be prepared via different synthetic strategies as
the traditional free radical polymerization and vinyl-like monomers and the
inorganic polymerization, i.e., sol-gel process, of silane precursors [4, 5]. With
the aim to combine both properties of organic and inorganic materials, hybrid
monoliths have also met great success.
In this regard, many research efforts have been devoted to implement easy
elaboration methods with a special focus over the last 15 years on using click
chemistry. The copper I-catalyzed Huisgen and Diels-Alder implementations
are cycloaddition reactions involving 6π (4+2) electrons ended up with the
formation of 5- and 6-membered cycles, respectively. In contrast, the addition
Surface Engineering of Porous Monoliths via Click Chemistry 159
of thiols to unsatured carbon-carbon double and triple bonds is mediated by

thiyl radicals, formed in the presence or not of a hydrogen abstractor and
leading to the formation of thioether bridge. Alkaline-catalyzed thiol-epoxy
click reaction is a well-established polymerization method to prepare linear
polymer chains substituted with free hydroxyl groups [6]. Indeed, beside its
high efficiency, this synthetic method leads to the formation of a reactive OH
upon ring opening. Such reactive polymers can be further functionalized
through simple one-step chemistry. Commercial availability, at low cost, of a
large range of di-epoxide and bis-sulfur reactants that can serve as
comonomers, are additional attributes of this strategy which proved powerful
for industrial and biomedical applications.
As the reader will discover, or simply remember, by browsing the
hereafter sections, these reactions have been implemented either as unique
synthesis step, or in combination with other chemical process such as sol-gel,
or even as functionalization method of clickable surface for the design of
functional monoliths.
Much has been said about click reaction in the general domain of organic
synthesis and materials science, however their applications to separation and
catalysis sciences is incipient, hence the motivation for this contribution. We
are presenting a detailed overview of the click chemistry strategies recently
implemented for the both aspects of the monolithic matrices elaboration and
surface modification of monoliths is discussed with respect to the type of the
click reaction, nature of the surface grafts and the resulting flow-through
applications in the context of analytical sciences for separation, extraction and
preconcentration purposes and catalysis technology for the stabilization of
nanometals [7, 8, 9, 10, 11, 12].
Considering the monolith and click chemistry pair, it will be shown that
while the copper(I)-catalyzed Huisgen 1,3-dipolar cyclo-addition and the thiol-
ene addition reaction have been mainly applied, successful achievement of
thiol/yne, Diels-Alder 4+2 cycloaddition and thiol-maleimide Michael
Addition remains challenging.
This work conclusively highlights the synergy of surface click chemistry
and porous monolithic materials for the design of highly permeable materials
with specific and selective surface interaction ability. Both fundamental and
applied issues are of utmost importance to our ongoing research aiming at
developing monolith-based microsystems allowing on-line catalytic reactions
and monitoring of the said chemical processes.
2. SCOPE AND AIM

In this contribution, general overview about monolithic supports with a
specific focus on synthetic routes exploiting the multifaceted toolbox of click
chemistry for either the synthesis or surface functionalization of the monolithic
skeleton is proposed. Application of the as-prepared monoliths for flow-
through processes, namely (electro)chromatographic separation, on line solid
phase extraction or preconcentration, as well as enzymatic and heterogeneous
catalysis is also discussed and rationalized on the basis of the chemical nature
of the used click-compatible partners. The discussion encompasses the field of
organic, inorganic and hybrid monolithic materials and includes also a rapid
discussion about major characteristics of monolithic materials and click
reactions.
This chapter is mainly intended to provide a condensed vision of the
potential of the monolith & click chemistry tandem to providing functional
materials with high potential in flow-chemistry. Hence, the deliberate choice
from the authors to discuss well-selected studies independently of
chronological considerations but mainly guided by relevance and benefits
allowing scientific breakthrough in monolithic materials-related technological
fields. This contribution is meant to provide the necessary scientific
background to postgraduate students in chemistry and materials science as
well as to illustrate very recent developments in the area of monoliths for
senior scientists.
3. KEY FEATURES OF MONOLITHS

A monolith, also referred to as a “continuous rod,” can be described as a
single large solid particle that does not contain interparticular voids [13]. In
their original variants, in the early 1960s, polymer monoliths were proposed in
the form of hydrogel-like materials with loose crosslinking density featuring
high compressibility and limited flow rate resistance [14]. The studies reported
studies by the group of Hjertén [15, 16, 17], further complemented by the
impressive contribution of Svec [18, 19, 20] and his collaborators marked
definitively the enshrinement of monoliths as a new generation of stationary
phases for liquid-phase separation applications. Tanaka's group [21, 22] has
played a major role in the development and reduction to practice of silica-
based monolithic columns for separation science. Such columns were proved
to provide high-speed, high-efficiency and operation methods allowing

significant enhancement of chromatographic performances. Success of
monolithic materials in separation science can be rationalized by the presence
of large separation pores network providing high permeability and enhanced
convective flow-driven mass transfer characteristics [23]. Figure 1 presents
scanning electron microscopy images of polymeric monolithic structures. In
contrast to particles filled chromatographic media, for which mass transfer is
primarily controlled by diffusion effects and speed is detrimental to resolution,
monoliths offer high-resolution separations and no peaks broadening even at
high-flow velocity. To date, organic and inorganic monoliths have been
applied to reversed-phase [24], charge-transfer [25, 26], hydrophilic
interaction [27, 28], ion exchange [29, 30] chromatographic modes
demonstrating their potential for the separation of amino acids, peptides,
proteins, nucleic acids, nitrogenous bases, enantiomers, polar and apolar
species with a large panel of molar mass [3, 31, 32].
Figure 1. Scanning electron microscopy images showing typical morphology of

polymer-based monolith. The two monoliths were prepared within fused-silica
capillaries though UV-initiated free radical polymerization with different
monomer/crosslinker ratios. The white bar in the figure represents size of 10 µm.
Reproduced from ref. 31 by permission of Wiley.
Low backpressures, outstanding reproducibility, large loading capacity,

stability over a broad range of pH and adjustable surface chemistry are other
positive features of monolithic materials. The latter is particularly true for
polymeric monoliths whose synthesis can be easily performed through thermal
or photochemical free radical polymerization in/on varied substrates such as
glass slides, fused silica capillaries as well as PDMS and COC chips [33, 34].
The large variety of readily available (meth)acrylate/styrenic monomers and
crosslinkers allows the preparation of monolithic structures with nearly
endless possibilities in terms of surface functionality. Through judicious
choice of the synthesis conditions, pore size and surface area can be easily
tuned. Although the in-microchannel synthesis of silica monoliths is a more
complex operation, inorganic-organic monoliths can be easily obtained
through various preparation methods [35].
All these reasons explain why application features of monoliths are today
far beyond separation science and have been generalized to the domain of flow
chemistry.
4. KEY FEATURES OF CLICK CHEMISTRY

“Click” chemistry including radical addition, cycloaddition, ring opening
addition has emerged as an efficient and versatile tool for the synthesis of
functional materials [36, 37]. Among the numerous relevant scientific and
technological fields, one may cite (i) environmental sciences with chelating
magnetic copolymer composites for the removal of metal ions [38, 39], (ii) life
sciences with boronic acid functionalized nanomagnets for glycoproteins
enrichment [40] and enzyme immobilized on magnetic beads for proteomics
[41], (iii) heterogeneous catalysis with magnetic nanoparticle-supported
palladium catalysts for Suzuki-Miyaura coupling reactions [42] bifunctional
gold nanoparticles and monolith-immobilized copper nanoparticles for
nitrophenol reduction [12], (iv) sensing with electro-active PEDOT electrodes
for label-free electrochemical DNA sensing [43], molecularly imprinted
polymers grafted onto electrodes for the detection of antioxidant [44] and
bifunctional gold nanoparticles for warfare agent analogues detection [45], to
mention but a few…
Click chemistry, term originally employed by Sharpless and coworkers to
describe the copper-catalyzed azide-alkyne Huisgen dipolar cycloaddition
reaction (also shortened as CuAAC), is now generally used to refer to a
restricted pool of particular organic reactions that shared together a certain
number of synthetic features. Indeed, according to Sharpless, such click

reactions should be regio- (and even stereo-) selective, carried out in mild
conditions and afford thermodynamically stable products with high yields
without the need for tedious purification procedures [46]. Additionally, they
should only produce very few inoffensive byproducts and be wide in scope. In
this context, different reactions have been classified within this conceptual
click chemistry toolbox. Thus, in the last 15 years, a particular attention has
been paid by the scientific community to a restricted pool of chemical
reactions that notably encompass the CuAAC, the thiol-ene and thiol-yne, the
thiol-(meth)acrylate Michael addition, the thiol-epoxyde and the Diels-Alder
reactions, as shown on Figure 2.
Figure 2. General scheme of the most commonly implemented reactions of the ‘‘click’’
type so far developed for the synthesis and functionalization of monoliths.
Implementation of these click reactions in organic, but more importantly

in polymer, materials and surface chemistry has flourished and even exploded
in the literature, especially for the design and synthesis of macromolecular
architectures (Figure 3). Numerous reviews in the field already gave a rather
exhaustive overview of all possible applications of this click chemistry toolbox
in such research area [47, 48, 49, 50, 51]. It is worth mentioning that most of
these click reactions have been discovered a long time ago but so far neglected
by scientists that only found them a limited synthetic interest.
Figure 3. Histograms obtained from Web of ScienceTM displaying the number of

published items in each year and the number of citations for each year when searching
for “click chemistry” and “polymer” topics on the 2016, April 12th. Figure reproduced
from Web of ScienceTM.
This section will emphasize on the particular features of these reactions

that make them appealing for material scientists regarding the conception and
functionalization of inorganic, hybrid and polymer-based monolithic materials.
First of all, considering the topic of this book chapter, it is worth mentioning
that chemical functions involved in click chemistry reactions can be easily
installed both on organic/inorganic compounds, to be grafted, and onto
surfaces, to be functionalized, regardless to the reaction yield. Additionally,
some of these starting compounds, and notably a large variety of thiols,
alkynes, (meth)acrylates or even alkenes and dienes are rather cheap and
generally easy to purchase from chemical providers. On the other hand, such
installed clickable functions are chemically inert in various environmental
conditions, which is a crucial point regarding the stability in time of such
starting materials. More importantly, they are inert under a large variety of
reaction conditions, allowing for sequential click reactions to be easily
envisioned in both one-pot and orthogonal fashion for instance [52, 53, 54, 55,
56, 57]. All these click reactions also give really better yields than more
classical coupling ligation strategies, i.e., amidation, esterification, etc. Last
but not least, monitoring of such click chemistry reactions, realized either in
solution or at the surface of engineered materials is quite easy to investigate as
the consumption of most of the starting materials but also the formation of the
resulting products can be easily followed using diverse characterization
techniques such as NMR, FT-IR, XPS and Raman spectroscopy for instance.
Indeed, clickable functions generally possess particular chemical functions,
e.g., azides, alkynes, alkenes, thiols, etc. that have very specific signature as
examined using such physico-chemical techniques.
All these above-mentioned click reactions have been extensively
implemented during the last 15 years in macromolecular engineering and for
the conception of smart advanced materials and nanomaterials [58] and
especially for the design and synthesis of chromatographic supports based on
inorganic, hybrid or polymeric monoliths.
4.1. Copper-Catalyzed Azide-Alkyne Huisgen Dipolar

Cycloaddition Reaction (CuAAC)
First discovered by Rolf Huisgen [59] in 1963 and considered in the early
2000’s as the cream of the crop of the click reactions, CuAAC reaction has
since been fallen into disuse when compared to other click reactions. Its
reaction mechanism has not been elucidated for a long time. Recent studies
notably bring to light two distinct possible routes to this mechanism, thanks to
the isolation of CuAAC reaction intermediates [60, 61]. In a general manner, it
involves the regioselective copper(I)-catalyzed cycloaddition between an azide
and a terminal alkyne, as shown on Figure 2a. More precisely, two
concomitant pathways can lead to the formation of the triazole ring (Figure 4).
While the first one, slow, involves mononuclear catalytic species, the second
one, underpinned by the isolation of bis(copper) key intermediates, relies on a
fast and thus kinetically favoured synthetic route.
However, this reaction is more and more abandoned because of the
potential toxicity of the required copper catalyst, even though it is generally
removed from the products in a nearly quantitative fashion. Nevertheless, a
strain promoted version of the CuAAC reaction [62] has been since developed
especially by Bertozzi and collaborators [63] that implemented the strain-
promoted azide-alkyne cycloaddition (SPAAC) reaction that is a copper-free
version of the CuAAC, involving in this case cyclooctyne-based compounds.
The main advantage in that particular version relies in the absence of the
copper catalyst that notably allows for using such click reaction for
biofunctionalization and modification of biomolecules in living systems
notably. Nevertheless, it has been extensively implemented for the
functionalization of monoliths with selectors of interest for applications in
separation sciences notably, as reported by different reviews in the field [64,

65].
Figure 4. Two synthetic pathways, underpined by the isolation of copper-based key

intermediate species, proposed for the CuAAC click reaction.
4.2. Thiol-Ene (TEC) and Thiol-Yne (TYC)

Coupling Chemistries
Thiol-based chemistries and especially radical-based version of this

reaction class has re-emerged in the late 2000’s [66]. Since, they have been
applied to various research area and notably in the chemical, biological,
physical, materials and engineering fields [67]. Originally discovered by
Posner in 1905 [68], the thiol-ene click reaction is a step-growth radical-
mediated organic reaction coupling between a thiol and an alkene to form the
corresponding thioether (Figure 2b). As showed on Figure 5, it involves the
formation of a thiyl radical that can be achieved via either photo-triggered,
thermal or redox activation pathway in the presence of a suitable radical
initiator (benzophenone (BP) and dimethoxyphenylacetophenone (DMPA) or
azobisisobutyronitrile (AIBN) as photoinitiators or thermal initiator,
respectively) during the initiation step. The photochemical initiation strategy
generally affords, in such surface chemistry reactions, for spatially controlling
the coupling reaction as photomasks can be in this particular case easily
implemented [8]. Further, the thiyl radical undergoes a radical addition onto
the alkene function in an anti-Markovnikov fashion during the propagation
step. The regioselectivity of this thiol-ene reaction can be easily explained by
the enhanced stability of the intermediate carbon-centered radical upon

addition to the less substituted alkene carbon [69]. The radical-mediated
formation of such thioethers from unsaturated alkenes can also be achieved
from disulfide-containing compounds. Termination reactions can occur rapidly
with a bimolecular reaction between any of the radical species generated
during the reaction. Electron rich and strained alkenes, like vinyl ethers, react
more rapidly.
Figure 5. Commonly accepted mechanism of the radical thiol-ene addition reaction

between a thiol and an olefin.
The alkyne-based version of the thiol-ene reaction, i.e., the thiol-yne click
reaction, operates in the same conditions but allows in that particular case for
the grafting of two thiol-containing compounds per alkyne moiety (Figure 2c)
[70, 71]. In surface chemistry area, this is actually an important parameter to
take into consideration when one wants to obtain a denser surface
functionalization with the thiol-containing compound, provided that steric
hindrance is limited. It is worth mentioning that in such an attempt of denser
surface functionalization, the supports should bear alkyne functions.
Figure 6. Reaction mechanism for addition of thiols to alkynes.
The mechanism is similar to that of the thiol-ene reaction, as depicted on

Figure 6. Upon addition of the thiyl radical to the triple bond and abstraction
of a hydrogen from another thiol molecule, the as-obtained vinyl thioether
undergoes in the same way a radical addition of another thiyl radical to form
the resulting carbon-centered radical that abstracts a hydrogen from another
thiol of the reaction medium to finally generate a bisthioether. Similarly to the
thiol-ene reaction, the first thiyl addition is regioselectively achieved on the
less substituted carbon of the alkyne function so as to produce the more stable
radical. The second addition of the thiyl radical occurs to the other carbon of
the unsaturation as the obtained carbon-centered radical is stabilized by
electron-donating mesomer effect of the thioether moiety.
4.3. Thiol-(Meth)Acrylate Reaction
Oppositely to the thiol-ene and thiol-yne reaction, the thiol-(meth)acrylate

click coupling relies on a chemical process involving ionic species. It is
generally catalyzed by Lewis bases such as amines and phosphines, for
instance (Figure 2d). The mechanism notably involves the addition of the
catalyst to the less substituted carbon of the activated C-C double bond via a
1,4-Michael type addition. The zwiterrionic intermediate then abstracts a
hydrogen from a thiol molecule in the medium. Finally the as-formed thiolate
substitutes the catalyst, thus generating the corresponding thioether (Figure 7).
In order for this reaction to proceed, the alkene-containing substrate requires
an electron-withdrawing group (generally an ester, a ketone or an aldehyde)
directly conjugated to the C-C double bond to proceed.
Figure 7. Mechanism of the Lewis acid-catalyzed thiol-(meth)acrylate click reaction.
4.4. Thiol-Epoxy Click Coupling
This reaction operates via ring opening of epoxide moieties in the

presence of rather strong nucleophiles. In the case of the addition of thiols to
the epoxy group, it requires the presence of a base in the medium as thiols are
not enough nucleophiles to undergo such addition reaction (Figure 2e) [72,
73]. Bases can be directly added to the reaction medium or formed in situ by
using photobase generators, depending whether the reaction is realized under
its ionic or photo-initiated version, respectively. In such conditions the thiolate
active species is generated by proton abstraction in the presence of the base
and can then achieve the nucleophilic addition, as shown on Figure. The last
step of the reaction involves hydrogen abstraction from another thiol molecule
in the medium to afford the final -mercaptoalcohol.
Figure 8. Generally accepted mechanism for the thiol-epoxy click coupling.
4.5. Diels-Alder Click Coupling
Diels-Alder click coupling generally occurs upon thermal process and

has the advantage to be reversible. The mechanism, discovered by Diels and
Alder [74] and awarded from the chemistry Nobel Prize in 1950, is based on
the [4π + 2 π] cycloaddition between diene and dienophile (i.e., alkene). It
generally involves alkene-based maleimide derivatives and diene-based
cyclopentadiene-, furan- or anthracene-functionalized compounds to afford
cycloadduct products with high yield via photochemical or thermal pathways
[75, 76, 77].
5. CLICK CHEMISTRY APPLIED TO

MONOLITH TECHNOLOGY
Monoliths can be fully regarded as complex polymeric systems in the
sense that their overall properties result from a delicate balance between
interface and bulk characteristics. As discussed above, monolithic structures
made of a network of together-fused solid nodules with in-between tortuous
pore channels accounts for the flow-through properties and efficient fluid
transport through monolithic elements, while interaction ability originates
from the chemical functionality on the monolith surface. Such an interaction
can be based on partitioning or adsorption for chromatographic processes,
biomolecular recognition for separation, extraction and purification purposes,
electrostatic forces for nanoparticular elements immobilization
Here below, we discuss specific cases where click chemistry was
implemented towards either the synthesis or surface chemical modification of
monoliths. Both organic, inorganic and organic-inorganic hybrid monolithic
skeletons are considered. The following section is segmented in three parts
describing successively click chemistry for the preparation of monoliths, the
synthesis of clickable monoliths and click chemistry for the surface
functionalization of monoliths.
5.1. Click Chemistry for the Preparation of Monoliths
Radical-mediated addition of enes and thiols is a well-established method

in the field of polymer science and engineering to design crosslinked materials
[78, 79]. In the context of monolith, thiol-ene click reactions have found their
way for the preparation of organic and hybrid structures [80, 81].
Photo-triggered (λ = 365 nm, 120 mJ/cm2) thiol-ene click polymerization
of 1,2,4-trivinylcyclohexane (TVCH) and pentaerythriol tetra(3-mercapto-
propionate) (4SH) was implemented for the fast (6 min) preparation of
monoliths [82]. The monolith was synthesized within UV-transparent capillary
using 2,2-dimethoxy-2-phenylacetophone (DMPA) as initiator and a mixture
of diethyl ether (DEGDE) and polyethylene glycol 200 (PEG200) as binary
porogenic solvent. Commonly used porogens (toluene/dodecanol and
cyclohexanol/decanol) did not provide monolith-filled and permeable capillary
column. Interestingly, the authors did not discuss only the effect of porogen
mixture composition on permeability but they also clearly highlighted effects
of photoinitiator content and UV irradiation time. Permeability was found to

decrease from 5.3 to 2.8 x 10-14 m2 when the DMPA content was increased
from 0.19 to 1.87% while it decreased from 6.1 to 4.2 x 10-14 m2 while the UV
irradiation time increased from 2 to 15 min.
The group of Feng described an efficient method for the one-step
preparation of organic-inorganic hybrid monoliths though a synthetic method
based on polycondensation and thiol-ene click reaction (Figure 9) [83]. To this
end, homogeneous mixture made of vinyl-end organic monomers,
azobisisobutyronitrile (AIBN), hydrolyzed tetramethoxysilane (TMOS) and 3-
mercaptopropyltrimethoxysilane (MPTMS) was maintained at 40°C during
12h within fused-silica capillary providing monolithic materials. Depending
on the chemical nature of the organic monomer, namely [2-
(methacryloyloxy)ethyl]trimethylammonium (META) and acrylamide (AA),
monoliths with anion exchange and hydrophilic interaction ability were
obtained, respectively. Although polycondensation and thiol-ene occurred
simultaneously, the latter reaction can be considered as mainly contributing to
the surface functionalization of the monolith as, in some way, it performed
end-capping of the SH groups arising from silane-like monomers. In other
words, surface functionality of monoliths was dictated by the chemical nature
of the vinyl monomers in the course of thiol-ene reaction.
Figure 9. Schematic illustration of the synthetic path developed to synthesize hybrid

monolith versatile surface functionality through thiol-ene click chemistry. Reproduced
from ref 83 by permission of Elsevier.
In 2014, the group of Zou reported on the successful synthesis of

macroporous hybrid monoliths via thiol-ene polymerization meant for cLC
application (Figure 10) [84]. The polymerization of vinyl monomers – 2,4,6,8-
tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane (TMTVS) or tetravinylsilane
(TVS) – with thiol – 1,6-hexanedithiol (dithiol) or pentaerythritol tetrakis(3-
mercapto-propionate) (tetrathiol) – was initiated by UV light irradiation at
365 nm using 2,2-dimethoxy-2-phenylacetophenone (DMPA) and (diethylene

glycol diethyl ether (DEGDE) as a photoinitiator and good solvent,
respectively. Fourier-transform infrared spectroscopy (FT-IR) revealed the
almost disappearance of signals assigned to the vinyl (1592 cm-1) and also
thiol (2555 cm-1) groups confirming occurrence of the thiol-ene reaction in
high yields.
Figure 10. Scanning electron microscopy images showing the morphology of hybrid
monolith prepared by through photodriven thiol-ene click chemistry using different
tetra-vinyl monomers and muli-thiols. Reproduced from ref 84 by permission of Royal
Society of Chemistry.
The same group applied thiol-yne click chemistry type in the area of
monolithic materials [85].
Dual-process for the preparation of organic-inorganic monolith, involving
simultaneous polycondensation and thiol-mediated radical addition, has also
been exploited by the group of Huanghao Yang from the Fuzhou University
[86]. Glutathione, a molecular combination of three proteins building blocks,
namely cysteine, glycine and glutamine, was used in mixture with 2,2-
azobisisobutyronitrile (AIBN), hydrolyzed tetramethyloxysilane (TMOS) and
γ-methacryloxypropyltrimethoxysilane (γ-MAPS) to provide multifunctional
monoliths (Figure 11). A two-step thermal treatment involving, initial heating
at 40°C for 12 h and additional treatment at 70°C for another 12 h was applied
with success. Such gradual temperature treatment can be easily rationalized by

the intrinsic nature of the reaction mechanisms. Indeed radicals-forming
decomposition of AIBN is known to occur efficiently in a temperature range
60°C-85°C. As such, we may assume that thiol-ene reaction occurs mainly in
the second half, i.e., in the higher temperature regime, of the synthesis process.
This is of course beneficial to the overall design of monolith with surface-
controlled properties as the thiol-ene, in the present case, leads to the
incorporation of glutathione that should occur on the surface of the inorganic
skeleton to provide its chemical functions, –COOH, –NH2, –CO–NH2–,
available for interacting with molecular targets. Silica network of GSH–hybrid
monolith exhibited remarkable mechanical stability under pressure up to 18.6
MPa, the permeability and surface area were quantitatively determined to be
1.47 × 10−14 m2 and 273.5 m2/g, respectively. Pore size analysis revealed a
narrow mesoporous distribution centered at about 3.6 nm.
monolith zwitterionic surface functionality through thiol-ene click chemistry.
Reproduced from ref 86 by permission of elsevier.
Although vinyl-POSS derivatives have gained great interest over the last
years for the preparation of organic polymer-based materials they suffer from
low reactivity of the tightly tethered vinyl group in free radical polymerization.
Radical-mediated step-growth process in the presence of thiol and initiator has
been exploited to the chemical cross-linking of POSS precursors in an efficient
and controlled way [87].
Thiol-methacrylate Michael addition click reaction between multi-
methacrylate polyhedral oligomeric silsesquioxane and multi-thiols was
reported as a facile strategy for the direct preparation of hybrid monolithic
capillary columns [88]. Methacrylate-polyhedral oligomeric silsesquioxane
(POSS-MA) (cage mixture, n = 8, 10, 12, POSS-MA) was allowed to
react with different multi-thiol crosslinkers – 1,6-hexanedithiol (HDT),
trimethylolpropane tris(3-mercaptopropionate) (TPTM) and pentaerythritol
tetrakis(3-mercaptopropionate) (PTM) – in a two component (n-propanol and
PEG 200) porogenic system. The Michael addition was efficiently catalyzed
by adding 2, 4 or 6 wt% of dimethylphenylphosphine (DMPP) as catalyst as

suggested by spectroscopic evidences such as increase in peak intensity of
carbon sulfur bonds (697 cm−1) and almost disappearance of peaks typical for
stretching vibrations of –C= C– (1637 cm−1) and –S–H (2569 cm−1) groups.
Such an increase in the DMPP content was accompanied by an increase in the
permeability (by nearly one order of magnitude) leading to value of 3.12 ×
10−14 m2). Such a dependence is related to the fact that large feeding amount
of catalyst induces faster rate of polymerization and the resulting formation of
oligomers promotes early phase separation and large macropores.
In 2014, a contribution from Shufen Shen et coll. described the
elaboration of novel organic-inorganic hybrid monoliths based on POSS via
thiol-ene click reaction between polyhedral oligomeric silsesquioxane
methacryl substituted (POSS-MA) and 1,4-bis(mercaptoacetoxy) butane
(BMAB) (Figure 12) [89]. Influence of monomers ratio (POSS-MA–BMAB
from 1:1.0 to 1:3 mol:mol), composition (toluene vs. dodecanol) of porogenic
solvents and temperature (50, 55 and 60°C) on monoliths morphology and
permeability were thoroughly investigated. Differences in morphology, as
observed with different BMAB crosslinker content, were assumed to result
from change in local degree of polymerization. For high crosslinker density,
i.e., high degree of polymerization, crosslinked nanoglobular polymers are
formed and separate from the solvent phase.
These hybrid materials are very appealing alternatives to organic and
silica monoliths as they combine advantages of both partners without the
inherent drawbacks. Hybrid monoliths were also synthesized though initial
alkaline-catalyzed thiol–epoxy click polymerization. A multi-epoxy monomer,
octaglycidyldimethylsilyl polyhedral oligomeric silsesquioxane (POSS-epoxy)
was copolymerized with either trimethylolpropanetris(3-mercaptopropionate)
(TPTM) or pentaerythritoltetrakis(3-mercaptopropionate) (PTM), as multi-
thiols precursors (Figure 13) [90].
Assuming high efficiency of thiol–epoxy click reaction, i.e., almost equal
consumption of epoxy and thiol groups, the mole ratio of the two functional
groups was kept equal to 1. This assumption was confirmed to a large extent
through FT-IR characterization as the authors observed significant decrease in
the characteristic absorption peaks of epoxy group (in the range from 725 to
910 cm−1) together with an almost disappearance of the absorption peak of the
thiol group at 2570 cm−1. Typical 3D skeleton with well-controlled
microstructures and high thermal, mechanical and chemical stabilities were
obtained for both, POSS-epoxy–TPTM and POSS-epoxy–PTM, hybrid
monoliths. Permeability values as calculated according to Darcy’s law were
found in the range 0.19 – 60.51 x 10-14 m2 for the POSS-epoxy–PTM hybrid
monoliths depending on the composition of the binary porogenic mixture
(EtOH/PEG 10,000) and catalyst (KOH) content. The same research team
extended successfully the thiol-epoxy approach to other multi-epoxy monomer
– tetraphenylolethane glycidyl ether – and multi-thiol monomer –
trimethylolpropane tris(3-mercaptopropionate) and pentaerythritol tetrakis(3-
mercaptopropionate) – using ternary porogenic system – DMSO/PEG200/H2O
– as ascertained by systematic characterizations of the obtained monoliths by
complementary methods including SEM, FT-IR spectroscopy, pore size
measurement, thermal gravimetric analysis and nitrogen adsorption/desorption
measurement [91].
In another implementation, methacrylate-silica monolithic columns of
hybrid nature and bearing sulfonic acid surface groups were newly synthesized
within capillary columns through a one pot approach [92]. To establish
the method, organic monomer (3-sulfopropyl methacrylate potassium) and
initiator (2,2’-azobis (2-methyl propionamidine) dihydrochloride) were
simply added to the hydrolysis solution of TMOS and mercaptopropyl-
trimethoxysilane (MPTS) so that polycondensation and thiol-ene click reaction
occurred simultaneously between the precondensed siloxanes and organic
monomers. The reaction was conducted in presence of urea, PEG and acetic
acid at low temperature (35 –55°C) for 12h. Monolithic materials with both
homogeneous structure and good permeability, and referred to as ideal
monoliths by the authors, were obtained only using an intermediate
temperature of 45°C. The morphology was found to be highly dependent on
the amount of added porogen. Low and high PEG amounts led to monoliths
with poor permeability and weak attachment onto the silica wall, respectively.
A multi-step and elegant preparation approach was implemented by
Cuicui Liu with the aim to design two-dimensional monoliths within capillary
columns for biomolecules analysis [93, 94]. The 1st dimension acted as pre-
concentration segment. The corresponding monolithic structure was in situ
prepared in a single step via Michael addition reaction and radical
polymerization. Such mechanism combination involved thiol graphene (500
m2/g), 4-vinylphenylboronic acid (VPBA), ethylene dimethacrylate (EDMA),
1-vinyl-3-octylimidazolium chloride (ViOcIm+Cl−) and was initiated at 70°C
in presence of 2, 2-azobis(isobutyronitrile) (AIBN). Extensive ultra-sonication
was required to dissolve the graphene derivative in a N,N-dimethylformamide
and 1,4-butanediol solvent mixture.
Figure 12. Schematic illustration of the synthetic path developed to synthesize POSS-
based hybrid monolith through thiol-ene click chemistry. Reproduced from ref 89 by
permission of Royal Society of Chemistry.
Figure 13. Schematic illustration of the synthetic path developed to synthesize POSS-
based hybrid monolith through thiol-epoxy click chemistry. Reproduced from ref 90 by
permission of Elesevier.
5.2. Synthesis of Clickable Monoliths
In contrast to the historical one-step synthetic pathway applied to produce

permeable polymeric monoliths, post-polymerization chemical modification of
monoliths prevents tedious and time-consuming steps required for the
concomitant optimization of both surface chemistry and flow-through
properties [95]. Indeed, change in the nature of the functional monomer, i.e.,
monomer bearing as a side chain a selector/ligand for further flow-chemistry
uses, requires tedious re-optimization of the polymerization conditions.
Porogen or porogenic mixture, a crucial parameter in the design of monolithic
structures, has to be well-fitted to solubility parameters of both monomers and
polymers. Post-polymerization approach, enables tuning of these two
characteristics independently. For generic monoliths, mechanical stability and
porous structure, resulting from the polymerization kinetics and related phase
separation process, can be initially optimized in the course of the free radical
polymerization step independently of the ultimate desired surface chemical
nature [96]. In a further step, monoliths with very different surface
characteristics can be prepared through simple grafting of functional
molecules without troubles inherent to the reactivity or solubility of the
corresponding functional monomer.
Two strategies can be envisioned for each type of click reaction.
Restricting the discussion to the case of copper(I)-catalyzed (3 + 2) azide-
alkyne cycloaddition, either azide or alkyne units can be surface-grafted on the
solid supports [97, 98]. Then, the surface with surface yne groups can
subsequently react with a selector/ligand bearing an azide unit and vice versa.
This allows a variety of combination taking advantage of the numerous and
commercially available functional thiols, alkenes, alkynes, azides, epoxy,
methacrylates… Extended this introductive section to silica-based monoliths,
alkynyl- or azido-hybrid monoliths can be prepared through condensation of
the hydrolyzed silane precursors tetramethoxysilane and (3-iodopropyl)-
trimethoxysilane in the presence of neutralized functional amine,
propargylamine or 11-azido-3,6,9-trioxaundecan-1-amine, respectively [98].
Using ethylene dimethacrylate (EDMA), as a crosslinker, and glycidyl
methacrylate, as monomer, P(GMA-EDMA) monoliths carrying epoxy surface
groups have proved very useful to serve as generic platforms for the
preparation of monolithic materials with a variety of functionality
(hydrophobic, hydrophilic, anion/cation exchange, chiral, affinity) [5]
including, more recently, clickable groups.
P(GMA-EDMA) can be prepared by free radical polymerization using
heavy alcohol (cyclohexanol/dodecanol mixtures) as porogen. Svec et al.
reported on the synthesis of thiol monoliths by one-[99, 100] and two-step
[101, 102] surface modification of GMA-based monoliths. Surface thiols were
obtained by direct aminolysis of epoxy groups with 2-aminoethanethiol while
the grafting of 2,2′-dithiobis(ethylamine) required additional treatment under
reducing conditions to provide disruption of the S-S bonds. Considering the
surface modification of generic P(GMA-co-EDMA) with cystamine, the same
group of authors showed that repeating the grafting twice (1.0 mol/L
cystamine dihydrochloride in 2.0 mol/L aqueous sodium hydroxide at RT for 1
h and subsequent heating at 50°C for 1 h) enables increasing the sulfur content
from 2.6% to 3.7%. Such monolith with enriched sulfur, i.e., thiol, content can
serve as versatile clickable platform for efficient grafting of methacrylate
monomers.
Lämmerhofer proposed the bonding of thiolated polymers instead of their
molecular counterparts to design tentacle-like surface-structured monoliths
with the aim to enhance both surface coverage of functional groups and
sample loading abilities [103]. The proof of principle was proposed for
polythiol-grafted monoliths following a three-step derivatization protocol.
Poly(GMA-co-EDMA) was subjected to (i) amination – ammonia – (ii)
vinylation – allyl glycidyl ether – and (iii) coating – poly-3-mercaptopropyl
methylsiloxane (PMPMS*) – in combination with crosslinking by thermally-
driven radical addition click reaction. Such an approach allowed more than a
2-fold increase in thiol coverage as compared to a control monolith obtained
through direct functionalization by nucleophilic substitution with PMPMS.
Thiolated silica monolith was also designed via the polycondensation of

TMOS and 3- mercaptopropyltrimethoxysilane (MPTS*). Usual conditions
were applied consisting of acetic acid solution of PEG, acting as porogen, and
thermal activation at 55°C for 12 h to complete the condensation reaction.
Svec et al. illustrated the suitability of the CuAAC for producing brush-
type monoliths [104]. Azide functionalities were first introduced on the pore
surface of monolithic silica by reaction with 3-(azidopropyl)trimethoxysilane.
The click step was achieved in dimethylformamide (DMF) in the presence of
catalytic amounts of copper (I) iodide providing high yield grafting of a
proline derivative containing an alkyne moiety. F. Tang et al. discussed a
facile and efficient “single-step” procedure to design azide-functionalized
silica monoliths [105]. To a homogeneous precondensation mixture, made of
tetramethoxysilane, 3-(chloropropyl)trimethoxysilane, water, methanol and
PEG, aqueous solution of sodium azide and potassium iodide was added and
the reactive solution was sonicated prior to gelation. Such an approach was
compared to the traditional sol–gel process using TMOS and CTMS as
precursors, and involving an in-column-functionalization step for the grafting
of azide units. The authors notably concluded that their new approach afforded
larger amount of accessible azide, prone to further click grafting, on the
monolith surface.The presence of azide moieties as surface functionalities was
evidenced by the characteristic peak at 2,100 cm-1 observed in the FTIR
spectrocopic spectra. The average pore diameter of the monolith was
calculated as 173 nm by mercury intrusion porosimetry, and the total surface
area was 17.69 m2·g-1. Permeability values were calculated to be 6.05×10-13 m2
and 3.72×10-13 m2 for water and methanol, respectively.
A one-step approach for the in situ preparation of click-functionalizable
monolithic stationary phase with alkyne surface functionality was presented by
the group of Delépée [106]. Poly(propargyl acrylate–pentaerythritol
triacrylate–trimethylolpropane trimethacrylate), poly(PA–PETRA–TRIM),
monolith was obtained through a photochemically initiated copolymerization
process using a mixture of methanol, acetonitrile and sodium phosphate buffer
(5 mM, pH 6.8) (2/6/2, v/v/v) as porogen. The authors provided systematic
investigation of the effects of molar ratios of both crosslinkers (PETRA vs.
TRIM) on the durability and permeability of the resulting polymers.
Schachtschneider and his co-workers discussed the design of periodically
ordered mesoporous organosilicas and aerogel-like monolithic bodies
exhibiting an optimized density of azide as clickable surface groups [107]. The
1,3-dipolar Huisgen cycloaddition was judiciously selected because of its high
versatility, enormous tolerance towards a large panel of functional groups and
its general feasibility in pores [108]. The use of bridging phenylazide moieties
offer a variety of unique possibilities for the grafting of nearly any desired
chemical functionality via click chemistry. Surface functionalization was
achieved through click grafting of an alkyne-modified fluorescein onto azide-
containing aerogel-like monoliths. Fluorescence imaging in combination to
spatially resolved IR spectroscopy measurements highlighted undoubtedly the
possibility to generate chemical gradient through this elegant approach.
Although the authors did not report applications they claimed that materials
exhibiting property gradients – chemical, optical and structural – may be
potential candidates for uses requiring directionality as it is the case for
chromatography.
Beside the more conventional methacrylate- and styrenic-based monoliths,
polycarbonate monoliths were also considered as functional platforms with
click reactivity [109]. Bisphenol A (BPA), 4,4′-(1-methylethylidene)bis(2-
allylphenol) (MBP) and 4-nitrophenyl chloroformate (NPC) were initially
dissolved in acetonitrile and the polymerization was further conducted under
argon at 70°C during 20 h after addition of 4-dimethylaminopyridine (DMAP)
and triethylamine. Polycarbonate with allyl side units was thus obtained and
further processed in a monolithic structure through a phase separation
approach. Cyclohexane was used as precipitation solvent providing 3-D
porous network with specific surface area of 145 m2/g as determined by
adsorption/desorption measurements.
Thiol-sensitive monoliths were also prepared in their hybrid variant
through sol-gel process [110, 80]. Inorganic polymerization of
tetramethoxysilane (TMOS) and γ-methyl methacrylate trimethoxysilane (γ-
MAPS) in presence of PEG (6000, 10000 and 20000 g/mol) urea and acetic
acid provided hybrid skeleton with the required flow-through properties and
carrying clickable C=C units [110].
Other implementation of the “generic monolith” concept with easy surface
functionalization features and thus allowing fast preparation of supports with
versatile surface properties was proposed by some of us nearly 10 years ago
[24]. Organic polymer monoliths were prepared in UV-transparent fused-silica
capillaries by photo-triggered free radical copolymerization of N-
acryloxysuccinimide as reactive monomer, ethylene dimethacrylate as
crosslinker, azobisisobutyronitrile as initiator and toluene as porogen.
Chemical and porous structures of such N-hydroxysuccinimide (NHS)
decorated monoliths can be simply tuned through judicious choice of synthesis
conditions. Addition of a limited amount of polar solvent (DMF or DMSO) to
toluene allows controlling the pore size in the range 0.4 – 2.2 µm, whereas
increasing the monomer/crosslinker ratio enables enriching the monolith

surface with NHS units [31]. Activated esters with good leaving group
properties have been widely applied to chemically modify surface of organic
or inorganic substrates providing nanostructuration, chemical functionality or
biological activity [111]. Carboxylates (–COOH) can be reacted to NHS or
sulfo-NHS in the presence of a carbodiimide such as EDC leading to the
formation of a semi-stable NHS of sulfo-NHS ester, which may, in a further
step, be reacted with primary amines to form amide anchoring units. Another
path to design materials with amine-reactive esters of carboxylate groups relies
on the use of polymerizable NHS-derivatives such as N-acryloxysuccinimide
and N-methacryloxysuccinimide.
NHS-ester can be advantageously used through a two-step reactional
mechanism path for initial functionalization of NHS-containing materials
surface via prompt reaction with functional amines in organic media
(providing solubility of the NHS leaving group) and further hydrolysis of the
remaining NHS units providing –COO- or –COOH groups depending on the
pH of the surrounding medium (Figure 14).
Figure 14. Schematic illustration of the strategy developed by our group to design
functional monoliths via the post-functionalization of generic monolithic skeleton with
N-hydroxysuccinimide surface functionality.
In the context of separation science, the proof of concept was established

through investigation of the electro-osmotic flow generation ability of
Poly(NAS-co-EDMA) monoliths initially functionalized with alkylamine in
ACN and further hydrolysed at pH [31]. Indeed, NHS esters are known to
exhibit a half-life of 4-5 hours at pH 7, 1 hour at pH 8 and only 10 minutes at
pH 8.6 [112, 113, 114]. We have shown that using background buffer with pH
value of about 6 that is near the pKa of –COOH groups for polyelectrolyte-like
polymeric surfaces, no electrodriven-flow was possible through the monolithic
material.
Considering the generic synthetic route presented in Figure2 clickable
monolith with alkene or alkyne surface functionalities were prepared through
reaction with allylamine [10, 11] and propargylamine [7, 8, 9, 10],
respectively. Success of the reactions was ascertained on the basis of in situ
micro Raman spectroscopy analysis showing the disappearance of the peaks at
about 1730 cm-1 (imide asymmetric stretching), 1780 cm-1 (imide symmetric
stretching) and 1810 cm-1 (activated ester stretching) together with clear
appearance of Raman signals at 2125 cm-1 and 1640 cm-1 supporting the
presence of –C≡C– and –C=C–units, respectively, on the monolith surface.
Thiol groups, known as complementary to enes and ynes for radical
addition click reactions, can also be surface-attached onto poly(NAS-co-
EDMA) [115]. The said generic monolith is then reacted with 2-
aminoethanethiol, cysteamine, via nucleophilic substitution of the NHS units.
The reaction is conducted in ethanolic solution for 2 hours at room
temperature. Through such an approach, free thiols are present on the monolith
surface as demonstrated by the appearance of a peak at 2574 cm-1 typical for –
SH units. Moreover, the Raman profile did not indicate the presence of a
signal in the 700 cm-1 Raman shift range that would suggest the formation of a
carbon–sulfur bond. All these data confirm the grafting of cysteamine through
amide coupling reaction. It is interesting mentioning that thiolated-monoliths
can be designed via the grafting of disulphide-intermediate [101].
Nucleophilic-sensitive monoliths can be reacted with 2,2′-
dithiobis(ethylamine), cystamine, followed by the cleavage of the disulphide
links. The approach was implemented for epoxy- and succinimide ester-based
generic monolith providing thiol-rich monolith after treatment of their surface
with tris(2-carboxylethyl)phosphine and threo-1,4-dimercapto-2,3-butanediol,
respectively, to reveal thiol functions.
In a recent report, the unprecedented use of glycerol carbonate
methacrylate as functional monomers was presented for the preparation of
generic porous polymer monoliths (Figure 15) [116]. GCMA was
copolymerized with EDMA under UV irradiation at 365 nm for 2h. To

optimize the porous properties of the resulting monoliths, different porogens
combination (toluene/dodecanol or nonane) and monomer/crosslinker ratios
were used. Binary porogen mixtures with different solubility parameters
enables producing monoliths with bimodal pore size distribution as evidenced
by mercury intrusion porosimetry measurements. Micrometer-sized pores
provided flow-through properties while mesopores enhanced the surface area
of the monolithic structures. Permeability values were calculated to be in the
range 0.9 – 4.3x10-14 m2. Cyclic carbonates such as glycerol carbonate
methacrylate can undergo ring opening when reacted with primary amines-
containing compounds and this reaction scheme was applied to graft ene-
functionalities through reaction with cysteamine.
Figure 15. Schematic illustration of the strategy developed by our group to prepare
clickable monoliths based on glycerol carbonate methacrylate. Surface
functionalization of the monolith through thiol-ene click chemistry is also presented.
Reproduced from ref 116 by permission of Royal Society of Chemistry.
To best of our knowledge, unique example of surface functionalizable

monolith via Diels-Alder click reaction was reported by our group [10].
Monolith with diene surface group was prepared through nucleophilic
substitution reaction of the N-hydroxysuccinimide units with 1,3-
cyclopentadiene-1-ethanamine (1M in ACN). The reaction was conducted at
room temperature for 2h.
5.3. Click Chemistry for the Surface Functionalization

of Monoliths
Simplicity, rapidness and high efficiency of “thiol-ene” click reaction

account for the broad interest by materials scientist to implement it as unique
preparation method, or as a part of a multi-step mechanism process, for
designing new monoliths with smart surface properties.
Organic polymer-based monolithic skeletons with surface-grafted alkene
groups have proved to be versatile platforms for thiol-ene surface click
chemistry as hydrophobic, hydrophilic, oligomeric moieties could be grafted
with high yields [11, 116]. Thiol-ene click grafting implies the presence of
either alkene or thiol groups onto the pore surface. Post-grafting was
developed by Lämmerhofer and co-workers about 10 years ago to design
chiral monoliths [117, 118]. After transformation of the epoxide groups of
poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) into 3-
mercapto-2-hydroxy-propyl residues, the thiol surface groups were allowed to
react with quinine or phosphonic derivatives via thermal radical addition and
the columns were used for enantioseparations. As a representative indications
of experimental conditions, O-9-tert-butylcarbamoylquinine (6 mg.mL-1 in
MeOH) was allowed to react with the mercapto-functionalized monolithic
matrix at 60°C, during 24h in the presence of AIBN.
Thiol–maleimide Michael addition click reaction was implemented for the
first as an efficient and versatile surface modification method of thiol-
containing monoliths (Figure 16) [115]. The surface characteristics of clicked
monoliths were found to be mainly governed by the chemical nature of the
surface-grafted maleimide-ligand providing easily monoliths with very
different surface interaction ability. As such hydrophobic and hydropholic
monolith can be obtained from the same generic matrix through click
attachment of aliphatic or carboxylic acid units, respectively.
Figure 16. Schematic illustration of the strategy developed by our group to

functionalize the surface of monoliths via thiol-maleimide Michael addition click
chemistry. Reproduced from ref 115 by permission of Elsevier.
Photochemical-initiation process (365 nm) of thiol-ene click grafting from

monolith surface was reported for the first in 2012 by Tijunelyte et al. and
grafting efficiencies were comparatively studied with the results obtained
under thermal (65°C) conditions [11]. Pore surface of generic poly(NAS-co-
EDMA) with pendant allyl moieties was functionalized via a two-step thiol-
ene click reaction with thiol-containing oligo(ethylene glycol) and
mercaptoethanol, successively. In both cases, the click reaction was carried out
in the presence of AIBN at different molar concentrations (0.1-0.5 M) while
the concentration of thiol derivatives was kept at 1M in toluene. Conversion of
double bonds was considered as a direct indication for the success of the
grafting and was evaluated as a function of reaction time through in situ
Raman spectroscopy monitoring of the (C=C) signal at 1647 cm-1. For
thermal operating conditions, no noticeable changes were observed in the
Raman profile after 3h of reaction whereas decrease in the (C=C) band
intensity became obvious when the reaction time was extended to 24h.
Fivefold increase in the initiator concentration (from 0.1 M to 0.5 M) did not
improve the grafting yield that remained limited and did not exceed 50%. To
provide reliable comparison of the effect of photochemically-driven radical
formation, photo-thiol-ene was conducted using AIBN. Interestingly,
comparable grafting yields of about 50% were obtained after 3h or UV-
irradiation as compared to the 24h or thermal activation. We also mentioned
the use of 2,2-dimethoxy-2-phenylacetophenone, a true photoinitiator,
however the Raman characterization did not show significant differences
within the reaction time range 0-4h. To illustrate the versatility of the
approach, the authors synthesized monoliths with surface-grafted octadecyl
groups, i.e., hydrophobic monolith, through the grafting of 1-octadecanethiol.
The reaction was performed on the same generic poly(NAS-co-EDMA) and

did not require any optimization step. Only the reaction solvent was changed
to ethanol to fit with solvation properties of 1-octadecanethiol.
Figure 17. Schematic illustration of the synthetic path developed by our group to
functionalize the surface of ene-monolith through thiol-ene click chemistry.
Reproduced from ref 11 by permission of Elsevier.
Although less commonly applied, thiol-yne chemistry was also

investigated in the context on monolithic materials. Our first report mentioned
the hydrophilization of monolith surface through the radical addition of
cysteamine onto alkyne containing porous monolith [8]. The reaction was
preformed through UV-irradiation at 313 nm for 4 h in the presence of 2-
hydroxy-2-methyl-1-phenyl-propanone (20 wt% with respect to cysteamine)
as photoinitiator.
Of particular interest we have shown that the grafting reaction, although
initiated through UV-process, occurred homogeneously along the in-capillary
monolith section but also over its cross-section. Indeed, Raman mapping with
a resolution step of 2 µm afforded 2D-plots indicative for the Raman intensity
in the 2125 cm-1 region, typical for the –C≡C– moieties, as a function of the
measurement location on the sample. No functionalization gradients were
observed after UV-irradiation of the 75 µm thick monolith and photo thiol-yne

click reaction was proved to proceed up to completion as ascertained by the
disappearance of the alkyne Raman signature. Raman spectroscopy proved
also to be a powerful technique to investigate the click reaction mechanism.
Although evidence for the radical-mediated mechanism is not accessible
through Raman measurements, it was possible to monitor the successive
grafting of two equivalent of 1-adamantanethiol per triple bond unit of
alkynylated monolith [9]. Indeed, unique observation of the consumption of
the C≡C is not a proof for a fully thiol-yne addition mechanism as presented in
Figure 6. Time dependence investigation (over a period of 180 min) of the
Raman profiles in the 2100–2160 cm-1 and 1500–1800 cm-1 regions revealed
the decrease in the signal intensity at 2125cm-1 and the appearance within an
intermediate reaction time window (30 – 120 min) of a band at 1610 cm-1. The
former indicates consumption of the alkyne group that occurs from the early
stage of the reaction and is total after 2h of reaction. The latter is considered as
indicative of the temporary presence of –C=C–R groups which are produced
within the course of the thiol-yne reaction after addition of one equivalent of
thiol derivatives per –C≡C– unit and are further consumed through addition of
the second equivalent. These spectroscopic features indicate that the bis
addition of 1-adamantanethiol is the major reaction mechanism, in accordance
with the thiol-yne mechanism presented in Figure 6.

functionalize the surface of yne-monolith through thiol-yne click chemistry.
Reproduced from ref 9 by permission of Wiley.
Indisputably, alkyne-functionalized monoliths have been mainly meant for

the Huisgen 1,3-dipolar cyclo-addition. Here again our group reported the first
implementation of the CuAA of for the covalent attachment of β-cyclodextrin
onto organic polymer monolith bearing alkyne units [7]. Mono-(6-azido-6-
deoxy)-β-CD was synthesized from β-CD using p-(toluene-sulfonyl)imidazole
as tosylation agent as reported elsewhere [119]. Azido-reactive monolith
surface was further grafted with β-cyclodextrin via a triazole ring (Figure 19).
The reaction was performed in presence of copper iodide and 1,1,4,7,7-
pentamethyldiethylenetriamine for 16 h at a temperature of 60°C. Raman
spectroscopy confirmed the consumption of the alkyne bond.

functionalize the surface of yne-monolith through CuAAC click chemistry. Two
examples of enantioseparation of flavanone obtained by nan-LC and CEC are also
shown. Reproduced from ref 7 by permission of Wiley.
Besides the surface-grafting of molecular segments acting as

chromatographic selectors, some researchers have also considered the design
of biofunctional monoliths through the well-controlled attachment of
biological macromolecules on clickable monoliths. This can be achieved in a
straightforward way, while still compatible with biological environment. To
this end, thiol groups can be purposely introduced as end-functionality of the
considered biomolecule or in situ generated through disruption of the –S–S–
bridges. Aptamer with 5′-end modified by a hexyl spacer arm containing end
thiol was immobilized on organic-silica hybrid monolith bearing methacrylate

surface groups [110]. The aptamer was first mixed with 2,2'-azobis (2-
methylpropionamidine) dihydrochloride as a thermal initiator and reacted with
the ene hybrid monolith at 55°C for 5 h. Coverage density of modified aptamer
was calculated from sulfur content to reach value of 420 pmol μL-1. This
coverage density is about two fold larger than the ones reported for other
systems proving the superiority of the click chemistry approach for monolith
biofunctionalization purposes [120, 121].
To date, our group published the unique example in the literature of
implementation of Diels-Alder click reaction for the surface functionalization
of monolithic stationary phases [10]. Diene-decorated monolithic matrix was
allowed to react with N-phenylmaleimide (1M in ACN) for 4h at room
temperature. The efficiency of the click grafting was evidenced by Raman
spectroscopic investigations with the presence of a signal characteristic for the
aromatic ring.
6. APPLICATION OF “CLICKED” MONOLITHS

TO FLOW CHEMISTRY
To date, the panel of applications of polymer monoliths encompasses

microreactors for chemical and enzymatic processes [122, 123], reversed-
phase, chiral and affinity chromatographic stationary phases [124], solid
supports for surface-enhanced laser desorption/ionization time-of-flight mass
spectrometry of small molecules [125] surface-enhanced Raman spectroscopy
sensors for detection of proteins [126] sorbents for preconcentration and solid
phase extraction [127], to mention but a few. Here-after we discuss
successively applications of clicked monoliths to liquid phase separation,
sample treatment and enzymatic and heterogeneous catalysis.
6.1. Clicked Monoliths for (Electro)Chromatographic

Separation Science
Herein, rather than presenting an long list of applications, we have

rationalized our discussion about chromatographic and electrochromatographic
applications as a function of the chemical nature of the monolith surface.
Indeed, monoliths involving a click step in their preparation process have been
designed to fulfill specifications for the different chromatography modes.
Reversed phase capillary (electro)chromatography requires the use of
stationary phases with apolar surface features in combination with aqueous-
based mobile phase containing usually methanol or acetonitrile as organic
modifiers. As such monolith with aliphatic-graft have been prepared as
analogous to the famous C4, C8, C18 particulate stationary phases [102]. The
said monoliths with dodecyl or octadecyl grafts provided efficient solutions for
the separation of low (alkylbenzenes) and large (protein) molar mass analytes.
Chromatographic properties of POSS-containing monoliths have been
thoroughly investigated under reversed phase separation mode [88, 89]. POSS-
epoxy–pentaerythritoltetrakis(3-mercaptopropionate) (PTM) and POSS-
epoxy–trimethylolpropanetris (3-mercaptopropionate) (TPTM) provided the
baseline separation of five alkylbenzenes according to their hydrophobicity,
from low to high (Figure 20) [90]. Using ACN/H2O mixture (50/50, v/v) as
mobile phase, the former provided lower retention time and lower plate height
(of about 6 µm). Both columns were further applied for the separation of five
polycyclic aromatic hydrocarbons (PAHs), six phenols, six anilines, five
benzoic acids, five pesticides, three dipeptides and four intact proteins. These
results demonstrated the potentiality of this type of monoliths for the analysis
of both small molecules and (bio)macromolecules.
Additionally, POSS-epoxy–PTM monolith was implemented for cLC–
MS/MS analysis of BSA digest. Fifty three unique peptides were positively
identified with protein sequence coverage of 58.65%, while C18-particle-
packed column, used for comparison purpose, provided lower analytical
performances where 51 unique peptides were positively identified with protein
sequence coverage of 56.01%.
Monolithic matrices, easily prepared through thiol ene, with carbon rich
skeleton exhibit hydrophic surface properties as suggested by contact angle
measurements indicating values well above 90°C [82]. This behavior can be
used for the LC separation of apolar solutes such as alkylbenzenes. For such
homologous series, selectivity values were extracted from the plots of log k =
f(n) with n being the number of carbon atom in the aliphatic side chain and
were found to increase from 1.27 to 1.43 when the ACN content was changed
from 80 to 65%. The separation properties were further investigated towards
five basic compunds, four pesticides and EPA610, consisting of sixteen
priority PAHs pollutants with potential health hazards (Figure 21). The authors
claimed for very high columns efficiency with values up to 1130,000
plates/meter although phenanthrene and anthracene were not baseline

resolved.
Figure 20. Separation of alkylbenzenes obtained under reversed-phase

chromatographic mode using POSS-based hybrid monoliths prepared by thiol-ene
click chemistry. Efficiencies are shown vs flow velocity. Reproduced from ref 90 by
permission of Elsevier.
Figure 21. Separation of (A) basic compounds, (B) pesticides (C) EPA610 obtained
under reversed phase chromatographic mode using organic monoliths prepared by
photothiol-ene click chemistry. Reproduced from ref 82 by permission of Elsevier.
Incorporation within the polymeric matrix of monomer such as

tetraphenylolethane glycidyl, i.e., containing phenyl units, provided monoliths
with π-π conjugate interaction ability besides the expected RP retention
mechanism [91]. This was proved to a large extent for the separation of
analytes with π electron, whose retention was assigned to the large number of
benzene rings in their structure. Interestingly enough, the authors showed that
for these monoliths obtained though thiol-ene reaction between polyglycidyl
and polythiol partners, the monolith may also exhibit some retention
mechanism correlated to hydrophilic interaction chromatography (HILIC).
Such behavior was assigned to the fact that through ring opening of glycidyl
units, hydroxyls are formed and act as hydrophilic interaction sites.
Cyclodextrin derivatives became increasingly important as chiral selectors
for enantioseparations in capillary electrophoresis [128], gas [129] and liquid
[130] chromatography, capillary electrochromatography [131]. Our group
reported the first enantioseparation obtained with a monolithic stationary
phase with surface-immobilized β-Cyclodextrin via Huisgen click reaction [7].
The same column was successfully applied to both capillary chromatography
and electrochromatography for the separation of flavanone. Other
cyclodextrin-functionalized monoliths obtained by alkyne–azide 1,3-dipolar
cycloaddition enabled the separation of three flavonols and eleven PAHs
[106]. Other type of clickable chiral selector includes a quinine carbamate
derivative, namely O-9-tert-butylcarbamoylquinine, allowing the separation of
dinitrobenzoyl-(R,S)-leucine in CEC mode [79].
Organic-silica hybrid monolithic columns incorporating [2-
(methacryloyloxy)ethyl]trimethylammonium or acrylamide served for anion-
exchange/hydrophilic interaction liquid chromatography [83]. Different types
of analytes, including benzoic acids, inorganic ions, nucleosides, and
nucleotides, were well separated with column efficiency in the range 80,000–
130,000 plates/m (Figure 22). The columns were found to provide highly
reproducible separation properties with batch-to batch, column-to-column and
run-to-run relative standard deviation (RSD) of 7.7%, 2.3% and 0.9%,
respectively, for the retention time of five nucleosides. Increase in the
retention time with the increase in ACN content suggested HILIC retention
mechanism. The low RSD values were considered as indicative of both
column homogeneity and uniform distribution of functional groups onto the
monolith surface.
Figure 22. Separation of (A) nucleosides, (B) and (C) nucleotides obtained under
hydrophilic interaction chromatographic mode using organic-silica hybrid monoliths
prepared by thiol-ene click chemistry. Reproduced from ref 83 by permission of
Elsevier.
Monolithic materials with multifunctional surface made of unique –

positive or negative –or combined – zwitterionic or amphoteric – charges,
aliphatic segment – methylene-based spacer arm – dipolar units – amide
anchoring site – can provide mixed separation mode allowing, with the use of
unique column though simple adjustment of the composition of the mobile
phase, the separation of polar and apolar solutes according to their intrinsic
hydrophobic/hydrophilic character [31]. Silica hybrid monoliths with click
grafted gluthathione were used as chromatographic stationary phases for the
separation small molecules and protein tryptic digests [86]. Separation abilities
were tested in terms of hydrophobic, hydrophilic, and cation-exchange
interactions using binary mobile phases with varied ratios of ACN/H2O with
or without different pH and concentration of phosphate buffered sodium. The
results suggested that hybrid monoliths with grafts of tripeptidic nature may
afford hydrophobic interaction, hydrophilic interaction and cation-
exchange/hydrophobic interaction modes for the chromatographic separation
of alkylbenzene, nucleotides and peptides. Phase mobile-dependent
electrochromatographic separation mode was also observed for oligoethylene
glycol-clicked poly(acrylate) monolith [11]. Polyethylene glycol-containing
monoliths are often applied to reversed phase chromatography providing
stationary phases with mild hydrophobic character [132]. Our results showed
that the elution order of acrylamide and toluene was reversed when changing
the ACN–phosphate buffer (5 mM, pH 8) mobile phase composition from
60/40 (v/v) to 80/20. Toluene was eluted prior to acrylamide for rich ACN
content.
Phenol-like solutes – 2,3-dimethylphenol, p-chlorophenol, catechol and
resorcinol – were injected on the same column and the separation mechanism
was found to governed by hydrophilic interaction as hydroxy-substituted

phenols exhibited larger retention (Figure 23). All these results illustrated
typical HILIC behaviour for the oligoethylene glycol click grafted monolith
[133].
Figure 23. Separation of phenol obtained under (a) hydrophilic interaction and (b)
reversed phase electrochromatographic modes using (a) oligoethylene-clicked and (b)
C6-grafted monoliths having the same generic skeleton. Reproduced from ref 11 by
permission of Elsevier.
Moreover, we showed that opposite separation behaviour was observed

when using a C6-monolithic column prepared though functionalization of the
same generic monolith.
Monoliths with a highly hydrophilic surface chemistry were also obtained
following the general scheme of thiol-mediated click chemistry for the surface
grafting of amine [8] or 3-sulfopropyl ammonium betaines [102] moieties.
Examples of successfully performed separation concern phenols, peptides and
nucleotides.
Classical reversed-phase and ion-exchange chromatographic retention
mechanism was reported by Huihui Yang et al. using (3-sulfopropyl
methacrylate potassium)-silica hybrid monolithic column [92]. The former
was proved from the retention of alkylbenzene, i.e., the retention increased
with the number of methyl groups on the benzene ring, while anilines were
used to establish the ion-exchange mechanism. Anilines were less retained

when the pH rose from 4.0 to 6.0 (Figure 24). Anilines exhibited strong
retention at low pH, due to their positive charge and the resulting cation-
exchange interaction with the sulfonated monolith. Increasing the pH led to
anilines deprotonation and lower retention. Above pH 6.0, the four anilines –
aniline, N-methylaniline, p-toluidine, N,N’-dimethylaniline – are neutral and
their apolar character accounted for their retention.
Figure 24. Separation of anilines obtained under ion-exchange electrochromatographic

mode using hybrid monoliths with sulfopropyl surface functionality prepared by thiol-
ene click chemistry. Reproduced from ref 75 92 by permission of Wiley.
6.2. Clicked Monoliths for Sample Pretreatment Technology
Sample treatment is a crucial step in the successful implementation of

analytical methods. This is even more challenging when considering the area
of bio-related samples analysis as the target (macro)molecules can be present
at ultra-trace level and often, if not always, in mixture with interferent of
complex chemical nature and topology that may generates false results. As
such bioanalysis requires combining advanced technology relevant to
microfluidics, surface science, analytical chemistry…
One area of great interest relies on the utilization of monolithic supports,
especially the one made of polymers, as building elements of bioanalytical
setups [134].
Recent literature reported about the click chemistry-mediated covalent
attachment of bio-recognition elements on monolithic materials. Aptamers
which are the artificial single-stranded DNA or RNA sequences have the
ability to bind to complementary targets with high selectivity and affinity
[135]. Hence, aptamer technology offers a powerful approach for the one step
selective extraction and concentration of a target analyte from liquid matrices
as well as sample purification from solid matrices extracts. In a recent study,
affinity matrix made of organic-silica hybrid monolithic skeleton surface-
decorated with aptamer against human α-thrombin linked through thioether
bond was applied to the enrichment of trace proteins [110]. Thrombin was
extracted from human serum with recovery of 91.8%. All the results were
compared with those obtained with a monolith grafted with a DNA oligo
control.
Incorporation of nanoparticles with high surface/volume ratios within
monolithic materials greatly improved their specific surface area, especially in
the case of organic polymer monoliths (Figure 25) [93]. Such effect is
beneficial to preconcentration ability even for low abundance peptides.
Monoliths incorporating crosslinked and functionalized graphene – through
Michael addition between thiolated graphene and EDMA and 4-
vinylphenylboronic acid, respectively – exhibited large specific surface area of
about 130 m2/g and enhanced binding capacity towards glycoproteins
including horseradish peroxidase, ovalbumin, transferrin, alpha fetoprotein and
ribonudease A. The binding values were calculated as 10.16 and 10.47 mg/g
(at pH 9.0) for disease markers transferrin and alpha fetoprotein, respectively,
and were well-larger than the ones calculated for boronic acid-functionalized
monolithic materials [105, 136, 137]. Effect of the preconcentration segment
length was optimized in a further step. Indeed, short preconcentration monolith
led to poor enrichment factor while too-long preconcentration monolith
resulted in high back pressure. Reasonable compromise was obtained for 14
cm long monoliths.
Figure 25. Schematic illustration of the synthetic path developed to prepare 2D

monolith with a graphene-contained preconcentration segment incorporated through
thiol-ene click chemistry. Reproduced from ref 93 by permission of American
Chemical Society.
Specificity of boronate affinity hybrid monolith was evaluated toward

nucleosides (adenoside) and glycoproteins (horseradish peroxidase,
ovalbumin, transferrin), selected as cis-diol-containing model compounds
under neutral conditions [105]. pH of the phosphate buffer (0.1 M) was set at
7.5 and sodium chloride (0.3 M) was additionally used to prevent non-specific
electrostatic interactions. Under these conditions, non-glycoproteins from an
egg white sample were eluted at the dead volume while glycoproteins were
specifically eluted under acidic (0.2M acetic acid) conditions.
6.3. Clicked Monoliths for Micro-Reactor Science

and Technology
From the above, it is obvious that the click chemistry and monolith pair
has been to date mainly applied to separation science. However, one should
mention the utilization of clicked monolith for catalytic reaction both of

enzymatic and chemical nature.
Some of us have shown that click chemistry in its thiol-maleimide
implementation can afford efficient strategy to graft spacer arms on monolithic
surface with enhanced reactivity towards lysine-containing biomacromolecules
(Figure 26) [115].
functionalize the surface of thiol-monolith through thiol-maleimide click chemistry.
Reproduced from ref 115 by permission of Wiley.
A pepsin micro-reactor was prepared by conjugating pepsin onto azido–

silica monolith via the CuAAC, with the aim to prove the feasibility of
clickable monoliths for versatile design enzyme-based micro-reactors [98].
Efficiency of the as-designed micro-reactor was tested towards the digestion
of immunoglobulin G. The monolith digest was analyzed by means of
SDS-PAGE gel electrophoresis and the results were compared with
immunoglobulin G digest obtained by from in-solution digestion. The authors
illustrated that the pepsin micro-reactor efficiency is directly dependent on the
flow rate, complete digestion of immunoglobulin G was obtained at 1µL.min-1
at room temperature, corresponding to a reaction time of 2.8 min. In contrast,
solution digestion required 40 min of incubation at 37°C. Moreover, the
alkynyl modification of pepsin, a prerequisite prior to grafting on azido-
monolith, did not affect significantly the enzyme activity.
Silica hybrid monoliths functionalized with vinyl groups were
biofunctionalized with trypsin taking advantage of the reactivity of free thiols
produced through reduction of trypsin disulfide bonds (Figure 27) [138].
Cysteamine labeled by fluorescein isothiocyanate (FITC) was used as model
coupling ligand to evidence the free-radical reaction of ene groups on surface

of the vinyl monolith. Then the trypsin containing free thiol groups was
attached on the vinyl hybrid monolithic column via thiol-ene click chemistry
in mild conditions (25°C, 5 h). Tris(2-carboxyethyl) phosphine hydrochloride
was used as reducing agent. BSA and BHb with molecular weight of 67,000
Da and 64,500 Da, respectively were chosen as standard proteins for testing
the enzymatic activity of the trypsin-based monolithic microreactor. The
results showed that hydrolytic digestion of high molecular weight proteins can
be achieved in short time (less than 30 s). Stability of the monolithic
microreactors was evaluated over 100 times showing reminiscence of the
initial enzyme activity in the range 45.3 – 85.7%. Finally, lifetime of the
enzymatic microreactors was tested by assaying five times per day over 15
days. Although activity of the surface grafted enzyme was found to decrease
slowly with time, up to 88.2% of the initial activity could be preserved.
monolith-based enzymatic microreactors through thiol-ene click chemistry.
Our group has reported the use of clicked monoliths for heterogeneous
catalysis applications using monolith-immobilized nano-metals as catalysts.
The proof of concept was reported in an initial paper published in 2012,
describing the versatility of thiol-yne surface click chemistry for the well-
controlled functionalization of porous polymer-based monoliths (Figure 28)
[8].
functionalize the surface of ene-monolith through thiol-ene click chemistry.
Reproduced from ref 8 by permission of Royal Society of Chemistry.
Site specific immobilization of gold nanoparticles was successfully

achieved through molecular-scale structuration of the monolithic column with
amine groups, acting as gold nanoparticle ligands, using photo-initiated click
reactions. The commercial availability of a large panel of functional thiols
allows an easy change in the monolith surface functionality so that it can be
judiciously adjusted with respect to the nature of the immobilized metal
nanoparticles. Using the same generic monolithic matrix bearing N-
hydroxysuccinimide surface reactive groups, we developed successively
alkenyl- and carboxylated monolith via grafting of allylamine and thiol-ene-

assisted addition of 4-mercaptobutyric acid, respectively (Figure 29) [12]. The
monolith surface with carboxylic acid functionality enabled the robust
anchoring of copper nanoparticles. Two pathways were envisioned for the
nanoparticles immobilization through either percolation of a suspension of
pre-formed copper nanoparticles through the capillary or by in situ reduction
with sodium borohydride of Cu(II) Br2 salt solution preliminary flown through
the monolith. The so-called ex-situ and in-situ approaches afforded efficient
catalytic microreactors for the reduction of nitroarenes.
Figure 29. Schematic illustration of the synthetic path developed by our group to apply
COOH-monolith obtained thiol-ene click chemistry as catalytic microreactor.
We reported further the design of platinum nanoparticles-decorated

monoliths applied as catalytic micro-reactor for the reduction of para-
nitrophenol, as a model compound, into the corresponding amino-phenol
[116].
CONCLUSION
For about 25 years now, monolithic materials have been synthesized using
a plethora of synthetic routes enabling the fine control of both skeleton and
surface chemistry. The former affords mechanical resistance in combination
with transport properties for applications under flow conditions. The latter
ensures the functionality of the monolithic surface providing specific
interaction with analytes or reactants. Organic (methacrylate, acrylate,
carbonate) inorganic (silica, silsesquioxanes) and hybrid monolithic matrices
have all be subjects of investigation by the chemists interested in spreading
click reactions in all range of materials science. Although thiol-ene radical
addition is the prominent click method in the context of monolithic materials,
we attempted, in this contribution, to summarize the synthetic strategies,
involving a click chemistry step, developed recently for synthesizing or
functionalizing monoliths. Undoubtedly, click chemistry has allowed
developing fast, one-step, if not one-pot, preparation methods of highly
functional monoliths providing advanced solutions in analytical chemistry as
exemplified with versatile stationary phases for separation science and smart
sorbents for pretreatment of complex sample matrices preparation as well as in
micro-reactors technology.
REFERENCES
[1] M. S. Silverstein, Special Issue of Polymer on porous polymers. Polymer
2014, 55(1), 302.
[2] F. Svec, T.B. Tennikova, Z. Deyl, Monolithic materials: preparation,
properties and applications. 2003: Elsevier.
[3] M. Guerrouache, A.M. Khalil, S. Kebe, B. Le Droumaguet, S.
Mahouche-Chergui, B. Carbonnier, Surface Innovations, 2015, 3(2), 84.
[4] M. R. Buchmeiser, Polymer 2007, 48, 2187.
[5] F. Svec, J. Chromatogr. A 2010, 1217(6), 902.
[6] Andreas Brändle, Anzar Khan, Polym. Chem., 2012, 3, 3224-3227.
[7] M. Guerrouache, M-C. Millot, B. Carbonnier, Macromol Rapid
Commun, 2009, 30(2), 109.
[8] M. Guerrouache, S. Mahouche-Chergui, M. M. Chehimi, B. Carbonnier,
Chem. Commun. 2012, 48(60), 7486.
[9] T. T. H. Dao, M. Guerrouache, B. Carbonnier, Chinese J. Chin. Chem.

2012, 30(10) 2281.
[10] M. Guerrouache, S. Mahouche-Chergui, T. Mekhalif, T.T.H. Dao, M.M.
Chehimi, B. Carbonnier, Surf. Interface Anal. 2014, 46(10-11) 1009-
1013.
[11] I. Tijunelyte, J. Babinot, M. Guerrouache, G. Valincius, B. Carbonnier,
Polymer 2012, 53(1), 29.
[12] R. Poupart, B. Le Droumaguet, M. Guerrouache, B. Carbonnier, Mater.
Chem. Phys. 2015, 163, 446.
[13] Svec F., Frechet J.M.J. Anal. Chem. 1992, 64(7), 820.
[14] Kubin, M, Spacek P, Chromecek R, Collect Czech Chem Commun,
1967, 32, 3881.
[15] Hjertén S, Liao JL, Zhang R, J. Chromatogr. 1989, 473, 273.
[16] Hjertén S, Li YM, Liao JL, Mohammad J, Nakazato K, Pettersson G,
Nature 1992 356(6372): 810-811.
[17] Nakazato K, Mohammad J, Hjerten S, Chromatographia, 1994, 39(11-
12), 655.
[18] Svec F., Frechet, JMJ, Science 1996, 273, 205.
[19] Tennikova T B, Belenkii BG, Svec F, J. Liq. Chromatogr. 1990, 13, 63.
[20] Tennikova TB, Bleha M, Svec F, Almazova TV, Belenkii BG, J.
Chromatogr. A 1991, 555(1/2), 97.
[21] Minakuchi, H., Nakanishi, K., Soga, N., Ishizuka, N., Tanaka, N., Anal.
Chem. 1996, 68, 3498 – 3501.
[22] Ishizuka, N., Minakuchi, H., Nakanishi, K., Soga, N., Nagayama, H.,
Hosoya, K., Tanaka, N., Anal. Chem. 2000, 72, 1275.
[23] Siouffi AM, J. Chromatogr. A, 2006, 1126, 86.
[24] Guerrouache, M Carbonnier, B Vidal-Madjar, C Millot, MC. J.
Chromatogr. A, 2007, 1149(2) 368.
[25] Guerrouache, Mohamed; Millot, Marie Claude; Carbonnier, Benjamin,
J. Sep. Sci. 2011, 34(16-17), 2271.
[26] Meinusch, R Hormann, K Hakim, R Tallarek, U Smarsly, BM, RSC
Advances 2015, 5(26) 20283-20294.
[27] Guerrouache, M; Pantazaki, A; Millot, MC; Carbonnier, J. Sep. Sci.
2010, 33 (6-7), 787.
[28] Gunasena, Dilani N.; El Rassi, Ziad, J. Chromatogr. A 2013, 1317, 77.
[29] Lin, XC; Feng, SH; Jia, WC; Ding, K; Xie, ZH, J. Chromatogr. A 2013,
1316, 104.
[30] Yang, Gengliang, Bai, Ligai, Yan, Cuihong, Gu, Yanzhao, Ma, Junjie,
Talanta, 2011, 85(5) 2666.
[31] Carbonnier, B, Guerrouache, M, Denoyel, R, Millot, MC, J Sep Sci,

2007, 30(17), 3000.
[32] Dinh, NP Cam, QM Nguyen, AM Shchukarev, A Irgum, K, J. Sep. Sci.
2009, 32(15-16), 2556.
[33] Araya-Farias, M Taverna, M Woytasik, M Bayle, F Guerrouache, M
Ayed, I Cao, HH Carbonnier, B Tran, NT, Polymer 2015, 66, 249.
[34] Ladner, Y; Bruchet, A; Cretier, G; Dugas, V; Randon, J; Faure, K, Lab
Chip 2012, 12(9) 1680.
[35] Zhu, Tao; Row, Kyung Ho, J. Sep. Sci. 2012, 35(10-11) 1294.
[36] Lowe, Andrew B, Polym. Chem. 2014, 5(17) 4820.
[37] Lowe, Andrew B, Polymer 2014, 55(22) 5517.
[38] Lapwanit, S., Trakulsujaritchok, T., Nongkhai, P.N., Chem. Eng. J.
2016, 289, 286.
[39] Ghaemy, M, Shabzendedar, S, Taghavi, M, J. Polym. Res. 2014, 21(6)
464.
[40] Zhang, ST He, XW Chen, LX Zhang, YK, N. J. Chem. 2014, 38(9)
4212.
[41] Bayramoglu, G Celikbicak, O Arica, MY Salih, B, Ind. Eng. Chem. Res.
2014, 53(12) 4554.
[42] Zhang, Q, Su, H, Luo, J, Wei, Y, Catal. Sci. Technol. 2013, 3(1), 235.
[43] Galan, T, Prieto-Simon, B, Alvira, M, Eritja, R, Gotz, G, Bauerle, P,
Samitier, J, Biosens. Bioelectron. 2015, 74, 751.
[44] Wang, TY, Shannon, C, Anal. Chim. Acta 2011, 708(1-2) 37-43.
[45] Li, N, Zhao, PX, Liu, N, Echeverria, M, Moya, S, Salmon, L, Ruiz, J,
Astruc, D, Chem. Eur. J. 2014, 20 (27), 8363-8369.
[46] Kolb, H. C.; Finn, Finn, M. G.; Sharpless, K.B.; Angew. Chem. Int. Ed.
2001, 40 (11), 2004-2021.
[47] Lutz, J.-F.; Angew. Chem. Int. Ed. 2007, 46 (7), 1018-1025.
[48] Binder, W. H.; Sachsenhofer, R.; Macromol. Rapid Commun. 2007, 28
(1), 15-54.
[49] Iha, R. K.; Wooley, K. L.; Nystrom, A. M.; Burke, D. J.; Kade, M. J.;
Hawker, C. J.; Chem. Rev. 2009, 109 (11), 5620-5686.
[50] Moses, J. E.; Moorhouse, A. D.; Chem. Soc. Rev. 2007, 36 (8), 1249-
1262.
[51] Binder, W. H.; Sachsenhofer, R.; Macromol. Rapid Commun. 2008, 29
(12-13), 952-981.
[52] Yu, B.; Chan, J. W.; Hoyle, C. E.; Lowe, A. B.; J. Polym. Sci. A Polym.
Chem. 2009, 47 (14), 3544-3557.
[53] Chan, J. W.; Hoyle, C. E.; Lowe, A. B.; J. Am. Chem. Soc. 2009, 131
(16), 5751-5753.
[54] Aimetti, A. A.; Feaver, K. R.; Anseth, K. S.; Chem. Commun. 2010, 46
(31), 5781-5783.
[55] Tunca, U.; J. Polym. Sci. A Polym. Chem. 2014, 52 (22), 3147-3165.
[56] Peng, H. Y.; Wang, C.; Xi, W. X.; Kowalski, B. A.; Gong, T.; Xie, X.
L.; Wang, W. T.; Nair, D. P.; McLeod, R. R.; Bowman, C. N.; Chem.
Mater. 2014, 26 (23), 6819-6826.
[57] Sumerlin, B. S.; Vogt, A. P.; Macromolecules 2010, 43 (1), 1-13.
[58] Mahouche Chergui, S, Ledebt, A. Mammeri, F. Herbst, F. Carbonnier,
B. Ben Romdhane, H. Delamar, M. Chehimi, M. M. Langmuir 2010,
26(20) 16115-21.
[59] Huisgen, R.; Angew. Chem. 1963, 75 (13), 604-37.
[60] Jin, L.; Tolentino, D. R.; Melaimi, M.; Bertrand, G.; Science Advances
2015, 1(5), e1500304, DOI: 10.1126/sciadv.1500304.
[61] Makarem, A.; Berg, R.; Rominger, F.; Straub, B. F.; Angew. Chemie Int.
Ed. 2015, 54 (25), 7431.
[62] Becer, C.R.; Hoogenboom, R.; Schubert, U. S.; Angew. Chemie Int. Ed.
2009, 48 (27), 4900.
[63] Agard, N. J.; Prescher, J. A.; Bertozzi, C. R.; J. Am. Chem. Soc. 2004;
126 (46), 15046.
[64] Marechal, A.; El-Debs, R.; Dugas, V.; Demesmay, C.; J. Sep. Science
2013, 36 (13), 2049.
[65] Chu, C.; Liua, R.; Chem. Soc. Rev. 2011, 40, 2177.
[66] Hoyle, C. E.; Bowman, C. N., Angew. Chemie Int. Ed. 2010; 49 (9),
1540.
[67] Hoyle, C. E.; Lowe, A. B.; Bowman, C. N.; Chem. Soc. Rev. 2010, 39
(4), 1355.
[68] Posner, T. Ber. Dtsch. Chem. Ges. 1905, 38, 646.
[69] Griesbaum, K. Angew. Chem. Int. Ed. 1970, 9, 273.
[70] Fairbanks, B. D.; Scott, T. F.; Kloxin, C. J.; Anseth, K. S.; Bowman, C.
N.; Macromolecules 2009, 42 (1), 211.
[71] Hoogenboom, R.; Angew. Chemie Int. Ed. 2010; 49 (20), 3415.
[72] Grazu, V.; Abian, O.; Mateo, C.; Batista-Viera, F.; Fernandez-Lafuente,
R.; Guisan, J. M.; Biotechnol. Bioeng. 2005, 90 (5), 597.
[73] Carioscia, J. A.; Stansbury, J. W.; Bowman, C. N.; Polymer 2007, 48
(6), 1526.
[74] Diels, O.; Alder, K.; Liebigs Ann. Chem. 1928, 460 (1), 98.
[75] Sun, X. L.; Stabler, C. L.; Cazalis, C. S.; Chaikof, E. L.; Bioconjugate
Chem. 2006, 17 (1), 52.
[76] Goodall, G. W.; Hayes, W.; Chem. Soc. Rev. 2006, 35 (3), 280.
[77] Gacal, B.; Durmaz, H.; Tasdelen, M. A.; Hizal, G.; Tunca, U.; Yagci,
Y.; Demirel, A. L.; Macromolecules 2006, 39 (16), 5330.
[78] Dai, J, Jiang, Y. Liu, X, Wang, J, Zhu, J, RSC Advances 2016, 6(22)
17857.
[79] Zhang, D, Hu, P, Xu, Z, Chen, S, Zhang, J, Zhang, A, Miao, M, J. Appl.
Polym. Sci., 2015, 132(30) 42316.
[80] El-Debs R, Cadoux F, Bois L, Bonhommé A, Randon J, Dugas V,
Demesmay C, Langmuir 2015, 31, 11649.
[81] R. Goebel, P. Hesemann, A. Friedrich, R. Rothe, H. Schlaad and A.
Taubert, Chem. Eur J., 2014, 20(52), 17579.
[82] L. Chen, J. Ou, Z. Liu, H. Lin, H. Wang, J. Dong and H. Zou, J.
Chromatogr. A, 2015, 1394, 103.
[83] M-L. Chen, J. Zhang, Z. Zhang, B-F. Yuan, Q.-W. Yu and Y-Q. Feng, J.
Chrom. A, 1284 (2013) 118.
[84] Z. Liu, J. Ou, H. Lin, Z. Liu, H. Wang, J. Dong, H. Zou, Chem.
Commun., 2014, 50, 9288.
[85] Z. Liu, J. Ou, H. Lin, H. Wang, Z. Liu, J. Dong, H. Zou, Anal. Chem. 86
(2014) 12334.
[86] Z. Lin, X. Tan, R. Yu, J. Lin, X. Yin, L. Zhang, H. Yang, J. Chrom. A,
2014, 1355, 228.
[87] F. Alves, I. Nischang, Chem.-Eur. J. 2013, 19, 17310.
[88] Lin H, Ou J, Liu Z, Wanga H, Dong J, Zou H, J. Chrom. A, 2015, 1379,
34.
[89] Shen S, Ye F, Zhang C, Xiong Y, Su L, Zhao S, Analyst 2015, 140(1),
265.
[90] Lin H, Chen L, Ou J, Liu Z, Wanga H, Donga J, Zou H, J. Chrom. A,
2015, 1416, 74.
[91] Lin H, Ou J, Liu Z, Wang H, Dong J, Zou H, Anal. Chem. 2015, 87 (6),
3476.
[92] Yang H, Chen Y, Liu Y, Nie L, Yao S, Electrophoresis 2013, 34, 510–
517.
[93] Liu C, Deng Q, Fang G, Huang X, Wang S, He J, ACS Appl. Mater.
Interfaces 2015, 7(36), 20430.
[94] Liu C, Deng, Q, Fang, G, Huang, X, Wang S, J. Mater. Chem. B 2014,
2, 5229−5237.
[95] Currivan S, Jandera P, Chromatography 2014, 1(1), 24.
[96] Viklund C, Ponten E, Glad B, Irgum K, Horstedt P, Svec F, Chem Mater

1997, 9(2) 463.
[97] Slater M, Snauko M, Svec F, Frechet JMJ, Anal. Chem. 2006 78(14)
4969.
[98] Wu M, Zhang H, Wang Z, Shen S, Le XC, Li XF, Chem. Commun.
2013, 49, 1407.
[99] Cao Q, Xu Y, Liu F, Svec F, Fréchet JMJ, Anal. Chem. 2010, 82(17),
7416.
[100] Xu Y, Cao Q, Svec F, Fréchet JMJ, Anal. Chem. 2010, 82(8), 3352.
[101] Lv Y, Alejandro FM, Fréchet JMJ, Svec F, J. Chromatogr. A 2012,
1261, 121.
[102] Lv Y, Lin Z, Svec F, Analyst 2012, 137, 4114-4118.
[103] Carrasco-Correa EJ, Ramis-Ramos G, Herrero-Martínez JM,
Lämmerhofer M, J. Chrom. A, 2014, 1367, 123.
[104] Slater MD, Fréchet JMJ, Svec F, J. Sep. Sci. 2009, 32, 21.
[105] Yang F, Mao J, He XW, Chen LX, Zhang YK, Anal. Bioanal. Chem.
2013, 405, 5321.
[106] Salwiński A, Roy V, Agrofoglio LA, Delépée R, Macromol. Chem.
Phys. 2011, 212, 2700.
[107] Schachtschneider A, Wessig M, Spitzbarth M, Donner A, Fischer C,
Drescher M, Polarz S, Angew. Chem. Int. Ed. 2015, 54, 10465.
[108] Malvi B, Sarkar BR, Pati D, Mathew R, Ajithkumar TG, Sen Gupta S, J.
Mater. Chem. 2009, 19, 1409.
[109] Xin Y, Sakamoto J, van der Vlies AJ, Hasegawa U, Uyama H, Polymer
2015, 66, 52.
[110] Wang Z, Zhao JC, Lian HZ, Chen HY, Talanta 2015, 138, 52.
[111] Maguis S, Laffont G, Ferdinand P, Carbonnier B, Kham K, Mekhalif T,
Millot MC, Opt. Express 2008, 16(23) 19049.
[112] Lomant AJ, Fairbanks G, J. Mol. Biol. 1976, 104, 243-61.
[113] Cuatrecaseas P, Parikh I, Biochemistry 1972, 11, 291.
[114] Staros JV, Wright RW, Swingle DM, Anal. Biochem. 1986, 156, 220.
[115] Belbekhouche S, Guerrouache M, Carbonnier B, Macromol. Chem.
Phys. 2016, 217, 997.
[116] Poupart, R; El Houda, DN.; Chellapermal, D; Guerrouache, M;
Carbonnier, B; Le Droumaguet, B; RSC ADVANCES, 2016 (17) 13614.
[117] Preinerstorfer B, Bicker W, Lindner W, Lämmerhofer M. J.
Chromatogr. A 2004, 1044, 187.
[118] Preinerstorfer B, Lindner W, Laemmerhofer M, Electrophoresis, 2005,
26, 2005.
[119] Tang W, Ng SC, Nature Protocols 2008, 3, 691.

[120] Zhao Q, Li XF, Shao Y, Le XC, Anal. Chem., 2008, 80, 3915-3920.
[121] B. Han, C. Zhao, J. Yin, H. Wang, J. Chromatogr. B, 2012, 903, 112-
117.
[122] Gömann A, Deverell JA, Munting KF, Jones RC, Rodemann T, Canty
AJ, Smith JA, Guijt RM, Tetrahedron, 2009, 65(7), 1450.
[123] Krenkova J, Lacher NA, Svec F, Anal. Chem. 2009, 81(5), 2004.
[124] Pfaunmiller EL, Paulemond ML, Dupper CM, Hage DS, Anal. Bioanal.
Chem. 2013, 405, 2133.
[125] Peterson DS, Luo Q, Hilder EF, Svec F, Frechet JMJ, Rapid Commun.
Mass Spectrom. 2004, 18(13), 1504.
[126] Liu J, White I, DeVoe DL, Anal. Chem. 2011, 83, 2119.
[127] Wang H, Zhang H, Lv Y, Svec F, Tan T, J. Chromatogr. A 2014, 1343,
128.
[128] Wang Y, Zhang S, Breitbach ZS, Petersen H, Ellegaard P, Armstrong
DW, Electrophoresis 2015, 37(5-6) 841.
[129] Plotka JM, Simeonov V, Morrison C, Biziuk M, Namiesnik J, J.
Chromatogr. A 2014, 1347, 146.
[130] Janus, L, Carbonnier, B, Deratani, A, Bacquet, M, Crini, G, Laureyns, J,
Morcellet, M New J. Chem. 2003, 27(2), 307.
[131] Pedehontaa-Hiaa G, Guerrouache M, Carbonnier B, Le Derf F, Morin
CJ, Chromatographia 2015, 78(7-8) 533.
[132] Li Y, Tolley HD. Lee ML. J. Chromatogr. A 2010;1217:4934-4945.
[133] Alpert AJ, J. Chromatogr. 1990, 499, 177.
[134] Saunders KC, Ghanem A, Hon WB, Hilder EF. Haddad PR. Anal. Chim.
Acta 2009, 652(1-2), 22.
[135] Pichon V, Brothier F, Combes A, Anal. Bioanal. Chem. 2015, 407(3)
681.
[136] Li Q, Lu C, Li H, Liu Y, Wang H, Wang X, Liu Z, J. Chromatogr. A
2012, 1256, 114.
[137] Lin, Z.; Wang, J.; Tan, X.; Sun, L.; Yu, R.; Yang, H.; Chen, G. J.
Chromatogr. A 2013, 1319, 141−147.
[138] Chen Y, Wu M, Wang K, Chen B, Yao S, Zou H, Nie L, J. Chromatogr.
A, 2011, 1218, 7982.
Chapter 8
CLICK CHEMISTRY FOR

MEMBRANE PREPARATION
AND SURFACE MODIFICATION
Zhu-Fang Hu1, Jin Zhou1,2 and Hai-Yin Yu1*

1
College of Chemistry and Materials Science,
Anhui Normal University, Wuhu, China
2
Department of Material and Chemical Engineering,
Chizhou University, Chizhou, China
ABSTRACT
Polymeric membranes exhibit high potentials for comprehensive
applications. However, the low surface energy and relatively high
hydrophobicity restrict their wide usage. Modulation of their bulk
and surface characteristics is especially important practically and
theoretically. The CuI-catalyzed triazo-alkynyl cycloaddition enjoys high
efficiency, less side reaction and high selectivity, and is vastly used in
polymer synthesis and preparation currently. Through this method the
polymers with low polydispersity, preset molecular weight, controlled
composition, and functionality could be introduced to membrane surface
or bulk materials. This chapter is focused on the utilization of click
chemistry for the membrane preparation and surface modification.
*
Corresponding Author Email: yhy456@ahnu.edu.cn
212 Zhu-Fang Hu, Jin Zhou and Hai-Yin Yu
Keywords click chemistry, membrane surface modification, membrane

preparation
INTRODUCTION
Click Chemistry
Click chemistry, a new molecular approach proposed by Sharpless and co-

workers in 2001, is designed through a small unit of stitching, quick to
complete a variety of molecular chemical synthesis (EJ Corey, 1989; Kolb H
C, 2001a; Nicolaou K C, 1996). The most practical and reliable chemical
reactions to connect a diversity of structures has been proved effective in
modification of polymeric materials (L. A. B. Hoyle C E, Bowman C N, 2010;
Kolb H C, 2001a). Click reaction with modular stereoselectivity and wide
range of applications, such as high yield characteristics, can accelerate the
reaction rate, and to get a single product. In recent years, click reactions have
converged into a powerful tool set for materials, and it has been widely applied
in syntheses of functional polymers and biomacromolecules, surfaces
modification (L. A. B. Hoyle C E, Bowman C N, 2010; Kolb H C, 2001b;
Tamao K, 1976).
Among the main click reactions, CuI-catalyzed Huisgen cycloaddition
between azide and alkyne is the largely applied one (Kolb H C, 2001b),
(Tamao K, 1976) for the mild reaction conditions, this click reaction is
essentially inert to most biological molecules, oxygen, water, and tolerant of a
wide range of solvents, pH values and temperatures, easy purification. It also
has excellent selectivity in chemical synthesis, and allows mild reaction
conditions. The azide and alkyne groups are facile introduced into the structure
of molecules and form stable 1,2,3-trizoles via click reaction; it is widely used
for modification of polymer. There are many important research results,
progress and advances of click chemistry are prospected in recent years (Kolb
H C, 2001a), (Pearson H A, 2014).
Click chemistry (S. R. Binder W H, 2007; Kolb H C, 2001a) is one of the
highly efficient (>95%) synthetic routes for achieving high reaction yields in a
relatively short time under mild conditions. For example, Heather A. et al.
combined highly efficient microwave plasma reactions and click chemistry to
create antimicrobial PE and PP surfaces. While the first step generates acid
groups, the second step is not limited to “click” chemistry. It may also include
Huisgen 1,3-dipolar cycloaddition (Hensarling R M, 2009).
Click Chemistry for Membrane Preparation and Surface Modification 213
Notably, surface reactions utilizing cycloaddition click reactions with high

efficiencies on Cu (S. R. Binder W H, 2007), Au (Zhu K, 2012), Si (X.-L. Sun,
2006), and carbon nanotube14 surfaces have shown promising results. Thus,
the attachment of bioactive molecules can be particularly important in creating
synthetic-biological interfaces. Other unique attributes of click chemistry also
broke new grounds for selective reactions with complex dendrimers (Sumerlin
B S, 2009; Wu P, 2004) and nanoparticles (O’Reilly R K, 2005).
As we know that free radical grafting polymerization is difficult to control
the chain structure. But delightedly, click chemistry may be the best solution
to the problem; it could be a perfect grafting-to route, capable of providing
superior site selectivity and almost complete reaction under benign conditions
nearly without side reactions or byproducts. It is also a modular synthetic
approach to linking small blocks together (K. C. Binder W H, 2006; K. C. J. C.
O. C. Binder W H, 2006). Particularly, click chemistry yields a well-controlled
product without formatting an inter-intramolecular crosslink, which would
jeopardize the correlation between grafting chain structure and membrane
performance. Besides, the resulting aromatic 1,2,3-triazoles linkages are rather
stable and thus make the functional grafting chain covalently linked to the
substrate membrane (S. R. Binder W H, 2007). The azide-alkyne click reaction
promisingly offers a more cost-friendly and sustainable approach to modifying
membrane surface.
Click Chemistry Reaction Types
Click chemistry can be divided into two classes: those in which protons
must be shuffled about (epoxide ring opening, for example) and those in which
no -bond connections are severed (cycloaddition reactions, the most useful
and reliable being the Huisgen dipolar cycloadditions). The former tends to
benefit dramatically from an aqueous environment, while the latter reveals
little solvent dependence and are better overall in their adherence to click
chemistry ideals. Indeed, the azide acetylene triazole version of Huisgens [2 +
3] cycloaddition family of processes is about as good as a reaction can get.
Nevertheless, it is the “less ideal” epoxide and aziridine opening processes
which are the workhorses for installing, often in the penultimate step of a
block synthesis, the azide or alkyne moieties (Kolb H C, 2001a).
The following four kinds of click reactions possess the merits of click
chemistry, namely the cycloaddition reaction, nucleophilic ring-opening
reaction, aldol carbonylation reaction, carbon carbon addition reaction
(Gandini, Coelho, & Silvestre, 2008). Nucleophilic openings of three-

membered rings, such as epoxides and aziridines, are important and reliable
methods for making carbon-heteroatom bonds because the competing
elimination processes are stereoelectronically disfavored. As a result, ring-
opened products are commonly obtained in high yield (Chanda A, 2009). The
nucleophilic opening of epoxides with azide anion proceeds efficiently on
water to generate a variety of useful azide-containing products. Hydroxylation
reaction, aziridine is the typical reaction of carbon carbon multiple bond
addition reaction. Thiol-ene reaction was worth mentioning, this click
chemistry without metal catalyst, the reaction can be under solvent-free
conditions and be able to pass through the light trigger and control, has
become a synthesis and modification of functional materials, is a typical click
reaction (Chanda A, 2009). The condensation reaction of carbonyl compounds
include: (1) aldehydes (ketones) and glycol under the condition of acid
condensation formation of acetal (ketal), reaction of reversibility in organic
chemistry. They can be used for protection of carbonyl and hydroxyl groups;
(2) the derivative reaction of aldehydes (ketones) and ammonia hydrazone,
oxime and urea, such reactions used in the identification of aldehydes
(ketones); (3) α,β-unsaturated aldehydes (ketones) formed heterocyclic
compound, such reactions are commonly used in the construction of a
molecular compound (Chanda A, 2009; Guthrie J P.[J], 1978; Kolb H C,
2001a).
CuI-catalyzed 1,3-dipolar cycloaddition reactions between organo-azides
and acetylenes (CuAAC) have played a dominant role in the synthesis of
functional polymers, this coupling chemistry has been rapidly adopted by
polymer scientists in the synthesis and post-polymerization modification of
polymers (R, 2014).
The Diels-Alder reaction is a [4 + 2] cycloaddition involving a diene and a
dienophile as precursors. Alkenes and alkynes with electron-withdrawing
substituents, which make the unsaturated groups more electron-poor, are
suitable dienophiles to react with a diene to perform the DA reaction. Like
other addition reactions, the DA reaction is useful for polymer preparation
with multifunctional diene and dienophile compounds as monomers (F, 2007;
Inglis A J, 2010). The “click” characteristics of the DA reaction provide some
convenience for polymer design and synthesis. Moreover, the DA reaction is
thermally reversible. The group formed by the DA reaction (DA adducts) is
thermally unstable and could undergo a reverse reaction (retro-DA reaction) at
higher temperatures to regenerate the diene and dienophile involved in DA
reaction (Figure 1). DA chemistry has been applied for the preparation of
thermo-responsive optical polymers38, novel materials (Dag A, 2008), and

smart coatings (Liu Y L, 2013).
Figure 1. General mechanism of Diels-Alder/retro Diels-Alder reactions of dienophile

and diene.
Figure 2. General mechanism of CuI catalysis for terminal azide-alkyne coupling.
Figure 3. General mechanism of thiol-Michael addition reaction.
Click chemistry ideas in cycloaddition of heteroatomic participation are

fully reflected, the modular reaction process combine two unsaturated
reactants, generated a lot of interesting five-membered heterocyclic and six-
membered heterocyclic. Currently, it is reported that the most common form
of this kind of reaction is the 1,3-dipolar cycloaddition reaction. The click
reaction essence known as the pile of nitrogen compounds and acetylene
cycloaddition was first reported by Huisgen. Rostovtsev respectively reported
CuI-catalytic azide-acetylene cycloaddition, highly selectivite to generate 1,4-
triazole, the production rate is as high as 91%, the reaction time is reduced
from the original 18 h for 8 h (Rostovtsev V V, 2002).
Over the past years, the thiol-Michael addition reaction has been tailored
to progress under mild, solventless reaction conditions using mild catalysts
to yield a highly efficient, modular click reaction. In fact, numerous thiol-
X reactions have been broadly classified as click reactions in which the
thiol reacts via pathways as diverse as radical-mediated thiol-ene reactions,
amine-catalyzed thiol-epoxy reactions, thiourethane-forming thiol-isocyanate
reactions, and thiol-halide reactions (L. A. B. Hoyle C E, Bowman C N.[J]. , , ,
2010; Hoyle, Lowe, & Bowman, 2010a).
New methods to synthesize and functionalize polymers are of constant
interest to the polymer scientist. In polymer science, a clear transition occurred
from simple plastic production to the generation of diverse functional
materials targeted for use in applications such as electronic devices,
nanomaterials, and medical treatments (Chu C, 2011; Fournier D, 2007; K. A.
K. C. m. e. Franc G, simple and greener routes to design dendrimers[J], &
MLA, 2010; K. A. K. J. Franc G, & MLA, 2010; Golas P L, 2010; L. A. B.
Hoyle C E, Bowman C N.[J]. 2010; P. Theato, and Harm-Anton Klok 2013).
Highly efficient linking reactions have played an indispensable role in polymer
science.
This chapter will provide the context of Click Chemistry Reaction used
for membrane preparation and surface modification, particularly in
functionalization, modification, orthogonally functionalizing polymers, and
its integration with Reversible Addition-Fragmentation Chain Transfer
Polymerization (RAFT). The utility of the click reaction has been
demonstrated in living radical polymerization. Among controlled free radical
polymerizations, RAFT has arguably played an important role in membrane
modification because it works with the greatest range of vinyl monomers
(Ranjan R, 2007).
Membrane Technology
Polymeric membranes currently are widely applied in many fields,

including sea water desalination, sewage treatment, energy production, and
food processing. However, there are still several problems with practical
application of these membranes, which are related to their fouling, chemical
mechanical and thermal stability. With the rapid development of membrane
science and technology, the functional membrane occupies a more and more
important role in modern life and industry status. On the membrane surface,
the surface wettability and hydrophilicity is poor, this leads not only to low
water flux but also to serious membrane fouling. Thus, membrane materials
restrict the potential applications of these membranes in biomedical systems
and the separation of aqueous solutions. The advanced membranes should be
designed to meet specific water treatment applications by tuning their
structural and physicochemical characteristics, including hydrophilicity,
porosity, membrane charge, and thermal and mechanical stability as well as
introducing additional functionalities such as antibacterial, photocatalytic or
adsorption capabilities (Kochkodan V, 2015). Due to their exceptional
mechanical, chemical and thermal stability and conductive and antibacterial
properties carbon-based nanomaterials are among the most promising
candidates to tackle this challenge (Daer S, 2015; El-Saied H, 2003; Tian M,
2015).
Advantages of Membrane Surface Modification
Membrane surface modification plays important roles both in applied and

basic research (Azari & Zou, 2012; Bernstein, Belfer, & Freger, 2012;
Wandera, Himstedt, Marroquin, Wickramasinghe, & Husson, 2012). This
technique is widely used to modify the surface properties of a wide range of
polymeric substrate membranes via a number of approaches, such as physical
adsorption of surfactant (Nasrul, Bastian, Sri, Yoshikage, & Hideto, 2011),
plasma treatment (Wei et al., 2012), chemical grafting (M. Zhang et al., 2012).
Among these techniques, chemical grafting is of particular significance,
because it can lead to introducing specific functional moieties onto the
polymer surface through covalent bond, thus to retain the functionalities
permanently. Traditionally there are two main approaches for chemical
grafting, grafting-to and grafting-from (Golas & Matyjaszewski, 2010; Ranjan
& Brittain, 2007; Zhao & Brittain, 2000). The former typically proceeds via
coupling a preformed, end functionalized polymer to an activated surface,
while the latter via the propagation from a surface immobilized initiator. The
grafting-to route enables the individual synthesis and the characterization of
the backbone and side chains, but often runs into low grafting densities due to
steric congestion. The grafting-from route has been extensively studied due to
its capability of preparing brushes of high grafting density from a backbone
with a predetermined number of initiation sites (X. M. Wu, L. L. Wang, Y.
Wang, J. S. Gu, & H. Y. Yu, 2012).
SH + CS2 + CHCl3 + CH3COCH3
Substrate Membrane H NaOH

UV / Br2
S
OH
S S
O
Carboxyl terminated RAFT CTA
Br
Esterification
OH
NaN3
S
O
S S
O
Alkyne terminated RAFT CTA
N3
N-vinylpyrrolidone RAFT Polymerization
Cu(I) Click Coupling
O
N
S
O C12H25
N
nS S
N N O
Figure 4. Surface modiﬁcation of polypropylene macroporous membrane via a one-pot

RAFT polymerization and click chemistry.
Membrane Modification with Click Chemistry
Several research papers have been recently published on using click

chemistry, for example, Jin Zhou’s work. In this study, to obtain a hydrophilic
membrane, poly(N-vinyl pyrrolidone) (PNVP) was grafted onto the
macroporous polypropylene membrane (MPPM) surface via reversible-
addition fragmentation chain transfer (RAFT) polymerization combined with
click chemistry reaction in one-pot (Figure 4). Various characterization
techniques were used to verify the successful grafting of PNVP onto the
membranes. The antifouling performances of the membranes before and after
PNVP grafting were examined by filtrating bovine serum albumin (BSA)
dispersion. By contrast to the nascent membrane, the grafted membrane
efficiently obstructed protein molecules because of the compactly grafted
polymer chains. The hydrophilicity and antifouling properties of MPPM were
greatly ameliorated after modification (Zhou & Hu, 2015).
Figure 5. Schematic representation for the layer-by-layer assembly of GO to MPPM by

click chemistry (first layer).
In order to tether grapheme oxide (GO) to the MPPM by azide-alkyne

click reaction, alkynyl-terminated GO was synthesized by the esterification
between carboxyl and amino, azido-terminated GO was prepared via the ring-
opening of epoxy groups with sodium azide. Meanwhile azido-MPPM was
prepared according to Hai-Yin Yu’s previous work (X.-M. Wu, L.-L. Wang,
Y. Wang, J.-S. Gu, & H.-Y. Yu, 2012). Further, GO was assembled to MPPM
layer-by-layer by click chemistry. For the first layer, alkynyl-GO was coupled
to azido-MPPM with a click reaction, the detailed reaction mechanism
between alkynyl-GO and azido-MPPM is illustrated in Figure 5. Because of
the steric effect, some of the alkyne groups on the GO would remain after the
reaction in the first step and the remained alkyne groups were able to react
with the azide groups on the azido-GO in the following step. For the second
layer, the azido-GO was coupled to MPPM-GO1 via a click reaction, where
some of the azide groups still remained on the azido-GO. Each of the process
was repeated for several times, thus MPPM was coupled with GO by the layer-
by-layer assembly to the membrane using click chemistry. Highly permeable
polypropylene membrane with remarkable protein fouling resistant and better
antibacterial was prepared via the layer-by-layer assembly of graphene oxide
(GO) nanosheets through azide-alkyne click reaction (Z. B. Zhang et al.,
2015).
The number of colonies in the bacterial culture plates gradually decrease
with the increase of layers of GO. The number of the bacterial colonies on the
5-layered GO modified MPPM decreases almost two-thirds due to that the
cutting edge can produce high membrane pressure; kill the bacteria (Figure 6).
Figure 6. Photograph showing the bacterial culture plates of E. coli for the unmodified
and modified MPPMs. (a) nascent MPPM; (b) MPPM-GO2; (c) MPPM-GO4 and (d)
MPPM-GO5, respectively.
The grafting-to approach is experimentally simple and can provide better

control over the grafted polymer, but it usually suffers from a lower grafting
density. A novel three-step method for polyacrylamide grafting-to the
polypropylene macroporous membrane was carried out by marrying click
chemistry with reversible addition-fragmentation chain transfer radical
polymerization (Figure 7). First, the membrane was brominated via a gas
phase free radical photochemical pathway, followed by SN2 nucleophilic
exchange of bromine atoms in the brominated membrane with azide groups in
NaN3; second, alkyne-terminated polyacrylamide with determined structure
was synthesized by using reversible addition-fragmentation transfer radical
polymerization method; third, alkyne-terminated polyacrylamide was coupled
onto the azide-functionalized membrane surface by the CuI-catalyzed azide-
alkyne cycloaddition click reaction. The permeation performances of the
modified membranes were tested by the filtration of protein dispersion. The
protein filtration experiments show that, in comparison with the unmodified
membrane, the modified membrane can effectively reject proteins due to the
densely grafted polymer chains. A novel and distinctive strategy has been
developed for the polypropylene macroporous membrane surface modification
by marrying click chemistry with controlled radical polymerization, which can
be readily extended to other polymeric membranes. Hai-Yin Yu group report
the attachment of a RAFT polymer via the grafting-to approach. The grafted
polymer with determined structure can individually be prepared controllably
and characterized elaborately. This approach opens a new avenue to the
fabrication of polymer membrane surfaces with different functional polymers.
More research is needed to improve the reaction efficiencies: the SN2
nucleophilic exchange of bromine groups on the substrate membrane surface
with azide group; the click coupling of functional polymers with long chain
onto the membrane surfaces (X. M. Wu et al., 2012).
Xiao-Jun Huang et al. presented a novel approach to constructing
glycosylated surface for microporous membrane. Carbohydrate derivative can
be facilely bound onto the alkyne-modified membrane surface via thiol-yne
click chemistry. The glycosylated membrane surface shows an excellent
affinity adsorption to lectin on the basis of carbohydrate-protein recognition.
Click chemistry between azide and alkyne is one of the methods to
construct a “glycoside cluster effect” on the surface of microporous
polypropylene membrane (MPPM) for lectin recognition and affinity
adsorption (C. Wang, Wu, & Xu, 2010). However, the triazole moieties
derived from this azide/alkyne click reaction seems to engage in hydrogen
bonding and stacking interactions with the amino acid residues of proteins
(Manetsch et al., 2004). These unexpected interactions unavoidably cause the
nonspecific adsorption of proteins, which decrease the recognition capability
between the glycosylated membrane surface and its protein receptor.
1st step
2nd step
3rd step
Figure 7. Schematic representation of the membrane surface modification by three-step

click coupling. First, surface UV bromination and subsequently exchange Br atoms
with azide groups. Second, synthesis of polyacrylamide with propargyl terminated
groups by the RAFT technique. Third, click coupling between alkyne-PAAm and
azide-PPMM membrane.
Recently, radical-mediated thiol-ene reaction has attracted much attention

after having been almost forgotten for a few decades (Chan, Hoyle, & Lowe,
2009; Hoyle & Bowman, 2010; Hoyle, Lowe, & Bowman, 2010b; Kade,
Burke, & Hawker, 2010; Lee, Cramer, Hoyle, Stansbury, & Bowman, 2009).
A series of studies have shown the high efficiency of this reaction meets the
“click” criteria (Hoyle, Lee, & Roper, 2004). In 2009, reaction between thiol
and alkyne, termed as thiol-yne click chemistry, was firstly introduced for the
synthesis of cross-linked polymer networks by Bowman and co-workers
(Fairbanks, Scott, Kloxin, Anseth, & Bowman, 2009). As with the thiol-ene
reaction, the thiol-yne click chemistry proceeds rapidly under a variety of
experimental conditions and yields selectively mono- or bis- addition products
(Hoogenboom, 2010). It employs the most promising metal-free reaction and
can be catalyzed photochemically or by nucleophiles with near-quantitative
yields in a period of seconds at ambient atmosphere (Lowe, Hoyle, &
Bowman, 2010). While this robust reaction has been well-documented in the
synthesis of polymers including dendrimers (Chan, Shin, Hoyle, Bowman, &
Lowe, 2010), hyperbranched polymers (Chan, Zhou, Hoyle, & Lowe, 2009)
and polymeric networks (Konkolewicz, Gray-Weale, & Perrier, 2009), it has
been essentially overlooked in the surface modification of biomaterials
(Hensarling, Doughty, Chan, & Patton, 2009) such as membranes for
bioseparation. Herein, thiol-yne click chemistry has been firstly used to
construct a glycosylated surface on MPPM for the affinity adsorption of lectin
(C. Wang, Ren, Huang, Wu, & Xu, 2011).
Layer-by-layer (LbL) assembly is a versatile technique for fabricating
tailored thin films of diverse composition (Decher, 1997; Hon, 1991). The
majority of work has focused on the assembly of polyelectrolyte (PE) films by
either electrostatic (Hon, 1991; Losche, Schmitt, Decher, Bouwman, & Kjaer,
1998) or hydrogen bonding (Stockton & Rubner, 1997; L. Y. Wang et al.,
1997) interactions. More recent work has reported polymer multilayer
assembly facilitated by covalent bonding (Kohli & Blanchard, 2000; Serizawa,
Nanameki, Yamamoto, & Akashi, 2002). Covalently bound films offer the
advantage of higher stability due to the cross-linked polymer networks and are
not susceptible to disassembly under varying solution conditions (e.g., salt,
pH), as is typically observed for a range of electrostatically coupled and H-
bonded films (Cho & Caruso, 2003; Sukhishvili & Granick, 2000). Frank
Caruso’s group report a highly efficient and generalizable method based on
click chemistry to construct LbL polymer films.
In the work of Frank Caruso report the LbL assembly of poly (acrylic
acid) multilayer films using click chemistry. They demonstrate that this
technique provides a simple and general method for the assembly of PE films
of controlled thickness and that the click moiety provides stable cross-links
within the films. Poly (acrylic acid) with either azide (PAA-Az) or alkyne
functionality (PAA-Alk) was synthesized using living radical polymerization.
LbL assembly was performed by sequentially exposing the substrates (quartz,
silicon, or gold) to PAA-Az and PAA-Alk solutions containing copper sulfate
and sodium ascorbate for 20 min, with water rinsing after deposition of each
layer (Such, Quinn, Quinn, Tjipto, & Caruso, 2006).
In Zhi-Kang Xu’s group, identification of an effective reaction is the

critical determinant for the fabrication of glycosylated membrane surfaces. It
can be expected that, therefore, click chemistry with its high yield and
specificity makes this strategy one of the most reliable glycosylation methods
for fabricating the desirable glycosyl density on MPPMs. Following this idea,
carboxyl groups were covalently bound on the membrane surfaces by UV-
induced grafting of acrylic acid (AA) (Yu, Xu, Yang, Hu, & Wang, 2006). The
AA-grafted membrane surfaces were then rendered to react with
propargylamine to give terminal alkyne-modified MPPMs. Subsequently,
azide containing glucose pendants were linked to the membrane surfaces by
click chemistry. Recognition and adsorption of lectins on the glycosylated
membrane surfaces were studied to evaluate the ‘glycoside cluste’ effect. A
straightforward strategy has been developed for the fabrication of glycosylated
surfaces on MPPMs by click chemistry. Considerable advances have been
made with respect to the fabrication of functional surfaces by the CuI-
catalyzed 1,3-dipolar cycloaddition of azides and alkynes. Advantages of high
yield and specificity for click chemistry have allowed a quantitative
introduction of glycosyl groups onto various surfaces, such as polymeric resin
beads (Chen et al., 2007), metal (Y. Zhang et al., 2006), silicon/silica(Sun,
Stabler, Cazalis, & Chaikof, 2006), and microtiter plate(Fazio, Bryan, Blixt,
Paulson, & Wong, 2002). To date, however, this method has not been applied
to fabricate a glycosylated surface for a microporous polymer membrane. In
comparison to the materials mentioned above, microporous polymer
membranes have great merits of high porosity, large surface area-to-volume
ratio, and good mechanical properties, which are facile to be used in the field
of bioseparation (C. Wang et al., 2010).
Controlling and improving the surface property of membrane materials is
an indispensable prerequisite for marketing applications. Several approaches
for the modification of membrane materials have been developed to confer
excellent properties. Numerous materials have also been employed for these
purposes. Many scientist studies have demonstrated that click chemistry show
great promise in the realm of surface-active compounds.
ACKNOWLEDGMENTS
This material is financially supported by National Natural Science
Foundation of China under Grant No. 21371008. This support is gratefully
acknowledged.
SUMMARY AND OUTLOOK

CuI-catalyzed click chemistry corresponds to an efficient and selective
reaction between alkynes and azides to form heteroatom links. These reactions
employ mild reaction conditions and simple work up procedures, but still
proceed in high yields. Click functionalized polymers can be used for
functionalization and modification of a variety of substrates. Controlled radical
polymerization has received increasing attention in recent years. It permits
synthesis of polymers with predetermined molecular weight, low
polydispersity, controlled composition and functionality. Combining the
chain-end functionality control of living free radical polymerization and the
efficiency and diversity of click chemistry is desirable.
Although the use of click reactions in polymer chemistry is undoubtedly
effective for material scientists, well-established click reactions show some
inevitable drawbacks: (1) the installation of a clickable reactive unit within the
desired functional compounds is mandatory, (2) only one functional unit per
clickable unit can be installed, and (3) required reagents are often not available
commercially. Therefore, the direct use of readily available functional
compounds without any organic transformation is highly desirable in order to
accelerate inter disciplinary application of such polymers (P. Theato, 2015).
In summary, many chemists have demonstrated a new approach to prepare
LbL films by using click chemistry. Using this technique, layers can be
sequentially deposited based on covalent interactions under mild aqueous
conditions. It is particularly well suited to biological systems. Currently, they
are extending this approach to prepare a tailored click multilayer membrane
and capsules of various materials (Such et al., 2006).
REFERENCES
Azari, S., and Zou, L. (2012). Using zwitterionic amino acid l-DOPA to
modify the surface of thin film composite polyamide reverse osmosis
membranes to increase their fouling resistance. Journal of Membrane
Science, 401-402, 68-75. doi: http://dx.doi.org/10.1016/j.memsci.2012.01.
041.
Bernstein, R., Belfer, S., and Freger, V. (2012). Improving performance of

spiral wound RO elements by in situ concentration polarization-enhanced
radical graft polymerization. Journal of Membrane Science, 405-406, 79-
84. doi: http://dx.doi.org/10.1016/j.memsci.2012.02.046.
Binder W H, K. C. (2006). Azide/alkyne-“click” reactions: applications in
material science and organic synthesis. Current Organic Chemistry,
10(14).
Binder W H, K. C. J. C. O. C. (2006). Azide/alkyne-“click” reactions:
applications in material science and organic synthesis. 10(14), 1791.
Binder W H, S. R. (2007). ‘Click’chemistry in polymer and materials science.
Macromolecular Rapid Communications, 28(1).
Chan, J. W., Hoyle, C. E., and Lowe, A. B. (2009). Sequential Phosphine-
Catalyzed, Nucleophilic Thiol-Ene/Radical-Mediated Thiol-Yne
Reactions and the Facile Orthogonal Synthesis of Polyfunctional
Materials. Journal of the American Chemical Society, 131(16), 5751-+.
doi: 10.1021/ja8099135.
Chan, J. W., Shin, J., Hoyle, C. E., Bowman, C. N., and Lowe, A. B. (2010).
Synthesis, Thiol-Yne “Click” Photopolymerization, and Physical
Properties of Networks Derived from Novel Multifunctional Alkynes.
Macromolecules, 43(11), 4937-4942. doi: 10.1021/ma1004452.
Chan, J. W., Zhou, H., Hoyle, C. E., and Lowe, A. B. (2009).
Photopolymerization of Thiol-Alkynes: Polysulfide Networks. Chemistry
of Materials, 21(8), 1579-1585. doi: 10.1021/cm803262p.
Chanda A, F. V. V. (2009). Organic synthesis “on water.” Chemical reviews,
109(2).
Chen, G. J., Tao, L., Mantovani, G., Geng, J., Nystrom, D., and Haddleton, D.
M. (2007). A modular click approach to glycosylated polymeric beads:
Design, synthesis and preliminary lectin, recognition studies.
Macromolecules, 40(21), 7513-7520. doi: 10.1021/ma071362v.
Cho, J., and Caruso, F. (2003). Polymeric multilayer films comprising
deconstructible hydrogen-bonded stacks confined between
electrostatically assembled layers. Macromolecules, 36(8), 2845-2851.
doi: 10.1021/ma021049n.
Chu C, L. R. (2011). Application of click chemistry on preparation of
Reviews, 40(5).
Daer S, K. J., Giwa A et al. [J], (2015). Recent applications of nanomaterials
in water desalination: a critical review and future opportunities. VCH,
Weinheim, 367.
Dag A, D. H., Demir E et al. [J], 2008, 46(20): 6969-6977. (2008). Heterograft
copolymers via double click reactions using one‐pot technique. Journal of
Polymer Science Part A: Polymer Chemistry, 46(20).
Decher, G. (1997). Fuzzy nanoassemblies: Toward layered polymeric
multicomposites. Science, 277(5330), 1232-1237. doi: 10.1126/science.27
7.5330.1232.
EJ Corey, X.-M. C. (1989). The Logic of Chemical Synthesis.
El-Saied H, B. A. H., Barsoum B N et al. (2003). Cellulose membranes for
reverse osmosis Part I. RO cellulose acetate membranes including a
composite with polypropylene. Desalination, 159(2).
F, L. J. (2007). 1, 3‐Dipolar cycloadditions of azides and alkynes: a universal
ligation tool in polymer and materials science. Angewandte Chemie
International Edition, 46(7).
Fairbanks, B. D., Scott, T. F., Kloxin, C. J., Anseth, K. S., and Bowman, C. N.
(2009). Thiol-Yne Photopolymerizations: Novel Mechanism, Kinetics,
and Step-Growth Formation of Highly Cross-Linked Networks.
Macromolecules, 42(1), 211-217. doi: 10.1021/ma801903w.
Fazio, F., Bryan, M. C., Blixt, O., Paulson, J. C., and Wong, C. H. (2002).
Synthesis of sugar arrays in microtiter plate. Journal of the American
Chemical Society, 124(48), 14397-14402. doi: 10.1021/ja020887u.
Fournier D, H. R., Schubert U S. [J]. (2007). Clicking polymers: a
straightforward approach to novel macromolecular architectures.
Chemical Society Reviews, 36(8).
Franc G, K. A. K. C. m. e., simple and greener routes to design dendrimers [J],
and MLA. (2010). “Click” methodologies: efficient, simple and greener
routes to design dendrimers. Chemical Society Reviews, 39(5).
Franc G, K. A. K. J., and MLA. (2010). “Click” methodologies: efficient,
simple and greener routes to design dendrimers. Chemical Society
Reviews, 39(5).
Gandini, A., Coelho, D., and Silvestre, A. J. D. (2008). Reversible click
chemistry at the service of macromolecular materials. Part 1: Kinetics of
the Diels–Alder reaction applied to furan–maleimide model compounds
and linear polymerizations. European Polymer Journal, 44(12), 4029-
4036. doi: 10.1016/j.eurpolymj.2008.09.026.
Golas P L, M. K. J., (2010). Marrying click chemistry with polymerization:
expanding the scope of polymeric materials. Chemical Society Reviews,
39(4).
Golas, P. L., and Matyjaszewski, K. (2010). Marrying click chemistry with

polymerization: expanding the scope of polymeric materials. Chemical
Society Reviews, 39(4), 1338-1354. doi: 10.1039/b901978m.
Guthrie J P.[J]. (1978). Equilibrium constants for a series of simple aldol
condensations, and linear free energy relations with other carbonyl
addition reactions. Canadian Journal of Chemistry, 56(7).
Hensarling R M, D. V. A., Chan J W et al. (2009). “Clicking” polymer brushes
with thiol-yne chemistry: indoors and out. Journal of the American
Chemical Society, 131(41).
Hensarling, R. M., Doughty, V. A., Chan, J. W., and Patton, D. L. (2009).
“Clicking” Polymer Brushes with Thiol-yne Chemistry: Indoors and Out.
Journal of the American Chemical Society, 131(41), 14673-+. doi: 10.102
1/ja9071157.
Hon, G. D. a. J.-D. (1991). <Buildup Of Ultrathin Multilayer Films By A Self-
Assembly Process, 1 Consecutive Adsorption Of Anionic And Cationic
Bipolar Amphiphiles On Charged Surfaces.pdf>. Makromol. Chem.,
Macromol. Symp., 46, 321-327.
Hoogenboom, R. (2010). Thiol-Yne Chemistry: A Powerful Tool for Creating
Highly Functional Materials. Angewandte Chemie-International Edition,
49(20), 3415-3417. doi: 10.1002/anie.201000401.
Hoyle C E, L. A. B., Bowman C N. (2010). Thiol-click chemistry: a
multifaceted toolbox for small molecule and polymer synthesis. Chemical
Society Reviews, 39(4).
Hoyle C E, L. A. B., Bowman C N. [J] (2010). Thiol-click chemistry: a
multifaceted toolbox for small molecule and polymer synthesis. Chemical
Society Reviews, 39(4).
Hoyle, C. E., and Bowman, C. N. (2010). Thiol-Ene Click Chemistry.
Angewandte Chemie-International Edition, 49(9), 1540-1573. doi: 10.100
2/anie.200903924.
Hoyle, C. E., Lee, T. Y., and Roper, T. (2004). Thiol-enes: Chemistry of the
past with promise for the future. Journal of Polymer Science Part a-
Polymer Chemistry, 42(21), 5301-5338. doi: 10.1002/pola.20366.
Hoyle, C. E., Lowe, A. B., and Bowman, C. N. (2010a). Thiol-click chemistry:
a multifaceted toolbox for small molecule and polymer synthesis. Chem.
Soc. Rev., 39(4), 1355-1387. doi: 10.1039/b901979k.
Hoyle, C. E., Lowe, A. B., and Bowman, C. N. (2010b). Thiol-click chemistry:
a multifaceted toolbox for small molecule and polymer synthesis.
Chemical Society Reviews, 39(4), 1355-1387. doi: 10.1039/b901979k.
Inglis A J, B. K. C. J. M. r. c., 2010, 31(14): 1247-1266. (2010). Ultra rapid

approaches to mild macromolecular conjugation. Macromolecular Rapid
Communications, 31(14).
Kade, M. J., Burke, D. J., and Hawker, C. J. (2010). The Power of Thiol-ene
Chemistry. Journal of Polymer Science Part a-Polymer Chemistry, 48(4),
743-750. doi: 10.1002/pola.23824.
Kochkodan V, H. N. (2015). A comprehensive review on surface modified
polymer membranes for biofouling mitigation. Desalination, 356.
Kohli, P., and Blanchard, G. J. (2000). Applying polymer chemistry to
interfaces: Layer-by-layer and spontaneous growth of covalently bound
multilayers. Langmuir, 16(10), 4655-4661. doi: 10.1021/la000120k.
Kolb H C, F. M. G., Sharpless K B. (2001a). Click Chemistry: Diverse
Chemical Function from a Few Good Reactions. Angewandte Chemie
International Edition, 40(11).
Kolb H C, F. M. G., Sharpless K B. (2001b). Click‐Chemie: diverse
chemische Funktionalität mit einer Handvoll guter Reaktionen [Click
chemistry: diverse chemical functionality with a handful of good
reactions]. Angewandte Chemie, 113(11).
Konkolewicz, D., Gray-Weale, A., and Perrier, S. (2009). Hyperbranched
Polymers by Thiol-Yne Chemistry: From Small Molecules to Functional
Polymers. Journal of the American Chemical Society, 131(50), 18075-+.
doi: 10.1021/ja908206a.
Lee, T. Y., Cramer, N. B., Hoyle, C. E., Stansbury, J. W., and Bowman, C. N.
(2009). (Meth)Acrylate Vinyl Ester Hybrid Polymerizations. Journal of
Polymer Science Part a-Polymer Chemistry, 47(10), 2509-2517. doi: 10.
1002/pola.23327.
Liu Y L, C. T. W. (2013). Self-healing polymers based on thermally reversible
Diels-Alder chemistry. Polymer Chemistry, 4(7).
Losche, M., Schmitt, J., Decher, G., Bouwman, W. G., and Kjaer, K. (1998).
Detailed structure of molecularly thin polyelectrolyte multilayer films on
solid substrates as revealed by neutron reflectometry. Macromolecules,
31(25), 8893-8906. doi: 10.1021/ma980910p.
Lowe, A. B., Hoyle, C. E., and Bowman, C. N. (2010). Thiol-yne click
chemistry: A powerful and versatile methodology for materials synthesis.
Journal of Materials Chemistry, 20(23), 4745-4750. doi: 10.1039/b91710
2a.
Manetsch, R., Krasinski, A., Radic, Z., Raushel, J., Taylor, P., Sharpless, K.
B., and Kolb, H. C. (2004). In situ click chemistry: Enzyme inhibitors
made to their own specifications. Journal of the American Chemical
Society, 126(40), 12809-12818. doi: 10.1021/ja046382g.
Nasrul, A., Bastian, A., Sri, M., Yoshikage, O., and Hideto, M. (2011).
Improved fouling reduction of PES hollow fiber membranes by
incorporation with non-ionic surfactant. Research Journal of Chemistry
and Environment, 15(2), 212-216.
Nicolaou K C, S. E. J. (1996). Classics in Total Synthesis: Targets, Strategies,
Methods. VCH, Weinheim, 789.
O’Reilly R K, J. M. J., Wooley K L et al. Functionalization of micelles and
shell cross-linked nanoparticles using click chemistry [J] (2005).
Functionalization of micelles and shell crosslinked nanoparticles using
click chemistry. Chemistry of materials, 17(24).
Pearson H A, U. M. W. (2014). Simple click reactions on polymer surfaces
leading to antimicrobial behavior. Journal of Materials Chemistry B,
2(15).
R, K. (2014). Metal-catalyzed multicomponent reactions for the synthesis of
polymers. Multi-Component and Sequential Reactions in Polymer
Synthesis.
Ranjan, R., and Brittain, W. J. (2007). Tandem RAFT polymerization and
click chemistry: An efficient approach to surface modification.
Macromolecular Rapid Communications, 28(21), 2084-2089. doi: 10.100
2/marc.200700428.
Ranjan R, B. W. J. J., 2007, 28(21): 2084-2089. (2007). Tandem RAFT
polymerization and click chemistry: an efficient approach to surface
modification. Macromolecular Rapid Communications, 28(21).
Rostovtsev V V, G. L. G., Fokin V V et al. (2002). A stepwise huisgen
cycloaddition process: copper (I)‐catalyzed regioselective “ligation” of
azides and terminal alkynes. Angewandte Chemie, 114(14).
Serizawa, T., Nanameki, K., Yamamoto, K., and Akashi, M. (2002).
Thermoresponsive ultrathin hydrogels prepared by sequential chemical
reactions. Macromolecules, 35(6), 2184-2189. doi: 10.1021/ma011465s.
Stockton, W. B., and Rubner, M. F. (1997). Molecular-level processing of
conjugated polymers 4. Layer-by-layer manipulation of polyaniline via
hydrogen-bonding interactions. Macromolecules, 30(9), 2717-2725. doi:
10.1021/ma9700486.
Such, G. K., Quinn, J. F., Quinn, A., Tjipto, E., and Caruso, F. (2006).
Assembly of ultrathin polymer multilayer films by click chemistry.
Journal of the American Chemical Society, 128(29), 9318-9319. doi: 10.
1021/ja063043+.
Sukhishvili, S. A., and Granick, S. (2000). Layered, erasable, ultrathin
polymer films. Journal of the American Chemical Society, 122(39), 9550-
9551. doi: 10.1021/ja002410t.
Sumerlin B S, V. A. P. (2009). Macromolecular engineering through click
chemistry and other efficient transformations. Macromolecules, 43(1).
Sun, X. L., Stabler, C. L., Cazalis, C. S., and Chaikof, E. L. (2006).
Carbohydrate and protein immobilization onto solid surfaces by sequential
Diels-Alder and azide-alkyne cycloadditions. Bioconjugate Chemistry,
17(1), 52-57. doi: 10.1021/bc0502311.
Tamao K, S. K., Kiso Y. (1976). Nickel-phosphine complex-catalyzed
Grignard coupling. I. Cross-coupling of alkyl, aryl, and alkenyl Grignard
reagents with aryl and alkenyl halides: General scope and limitations.
Bulletin of the Chemical Society of Japan, 49(7).
Theato, P. (2015). Multi-Component and Sequential Reactions in Polymer
Synthesis Advances in Polymer Science, 1-209.
Theato, P., and Harm-Anton Klok (2013). Functional polymers by post-
polymerization modification: concepts, guidelines and applications. [M]
John Wiley and Sons.
Tian M, W. R., Goh K et al. [J] (2015). Synthesis and characterization of high-
performance novel thin film nanocomposite PRO membranes with tiered
nanofiber support reinforced by functionalized carbon nanotubes. Journal
of Membrane Science, 486.
Wandera, D., Himstedt, H. H., Marroquin, M., Wickramasinghe, S. R., and
Husson, S. M. (2012). Modification of ultrafiltration membranes with
block copolymer nanolayers for produced water treatment: The roles of
polymer chain density and polymerization time on performance. Journal
of Membrane Science, 403-404, 250-260. doi: http://dx.doi.org/10.1016/j.
memsci.2012.02.061.
Wang, C., Ren, P. F., Huang, X. J., Wu, J. A., and Xu, Z. K. (2011). Surface
glycosylation of polymer membrane by thiol-yne click chemistry for
affinity adsorption of lectin. Chemical Communications, 47(13), 3930-
3932. doi: 10.1039/c1cc10634a.
Wang, C., Wu, J., and Xu, Z. K. (2010). High-density glycosylation of

polymer membrane surfaces by click chemistry for carbohydrate-protein
recognition. Macromol Rapid Commun, 31(12), 1078-1082. doi: 10.1002/
marc.200900866.
Wang, L. Y., Wang, Z. Q., Zhang, X., Shen, J. C., Chi, L. F., and Fuchs, H.
(1997). A new approach for the fabrication of an alternating multilayer
film of poly(4-vinylpyridine) and poly(acrylic acid) based on hydrogen
bonding. Macromolecular Rapid Communications, 18(6), 509-514. doi:
10.1002/marc.1997.030180609.
Wei, X., Zhao, B., Li, X.-M., Wang, Z., He, B.-Q., He, T., and Jiang, B.
(2012). CF4 plasma surface modification of asymmetric hydrophilic
polyethersulfone membranes for direct contact membrane distillation.
Journal of Membrane Science, 407-408, 164-175. doi: http://dx.doi.org/
10.1016/j.memsci.2012.03.031.
Wu P, F. A. K., Nugent A K et al. (2004). Efficiency and fidelity in a
click‐chemistry route to triazole dendrimers by the copper (I)‐catalyzed
ligation of azides and alkynes. Angewandte Chemie, 116(30).
Wu, X.-M., Wang, L.-L., Wang, Y., Gu, J.-S., and Yu, H.-Y. (2012). Surface
modification of polypropylene macroporous membrane by marrying
RAFT polymerization with click chemistry. Journal of Membrane
Scienceo, 421-422, 60-68. doi: 10.1016/j.memsci.2012.06.033.
Wu, X. M., Wang, L. L., Wang, Y., Gu, J. S., and Yu, H. Y. (2012). Surface
modification of polypropylene macroporous membrane by marrying
RAFT polymerization with click chemistry. Journal of Membrane
Science, 421, 60-68. doi: 10.1016/j.memsci.2012.06.033.
X.-L. Sun, C. L. S., C. S. Cazalis and E. L. Chaikof, (2006). Carbohydrate and
protein immobilization onto solid surfaces by sequential Diels-Alder and
azide–alkyne cycloadditions. Bioconjugate chemistry, 17(1).
Yu, H. Y., Xu, Z. K., Yang, Q., Hu, M. X., and Wang, S. Y. (2006).
Improvement of the antifouling characteristics for polypropylene
microporous membranes by the sequential photoinduced graft
polymerization of acrylic acid. Journal of Membrane Science, 281(1-2),
658-665. doi: 10.1016/j.memsci.2006.04.036.
Zhang, M., Zhang, L., Cheng, L.-H., Xu, K., Xu, Q.-P., Chen, H.-L, ... Tung,
K.-L. (2012). Extracorporeal endotoxin removal by novel l-serine grafted
PVDF membrane modules. Journal of Membrane Science, 405-406, 104-
112. doi: http://dx.doi.org/10.1016/j.memsci.2012.02.057.
Zhang, Y., Luo, S. Z., Tang, Y. J., Yu, L., Hou, K. Y., Cheng, J. P, ... Wang,
P. G. (2006). Carbohydrate-protein interactions by “clicked” carbohydrate
self-assembled monolayers. Analytical Chemistry, 78(6), 2001-2008. doi:
10.1021/ac051919+.
Zhang, Z. B., Wu, J. J., Su, Y., Zhou, J., Gao, Y., Yu, H. Y., and Gu, J. S.
(2015). Layer-by-layer assembly of graphene oxide on polypropylene
macroporous membranes via click chemistry to improve antibacterial and
antifouling performance. Applied Surface Science, 332, 300-307. doi: 10.
1016/j.apsusc.2015.01.193.
Zhao, B., and Brittain, W. J. (2000). Polymer brushes: surface-immobilized
macromolecules. Progress in Polymer Science, 25(5), 677-710. doi: http:
//dx.doi.org/10.1016/S0079-6700(00)00012-5.
Zhou, J., and Hu, B. (2015). Fabrication of a poly(N-vinyl-2-pyrrolidone)
modified macroporous polypropylene membrane via one-pot reversible-
addition fragmentation chain-transfer polymerization and click chemistry.
Journal of Applied Polymer Science, 132(42). doi: 10.1002/app.42649.
Zhu K, Z. Y., He S et al. (2012). Quantification of Proteins by Functionalized
Gold Nanoparticles Using Click Chemistry. Analytical chemistry, 84(10).
Chapter 9
COPPER-MEDIATED CLICK CHEMISTRY

APPLICATIONS TO ASSEMBLE
POLYAROMATIC STRUCTURES
Celedonio M. Álvarez*, Héctor Barbero

and Sergio Ferrero
GIR MIOMeT, IU CINQUIMA/Química Inorgánica, Facultad de Ciencias,
Universidad de Valladolid, Valladolid, Spain
ABSTRACT
Carbon nanostructures and Polycyclic Aromatic Hydrocarbons
(PAHs) have been widely studied over the last few decades due to their
great ability to establish supramolecular associations among them. This
gives rise to a new generation of hybrid nanomaterials with very
interesting properties. Furthermore, these species can be conjugated in
order to create electron donor-acceptor systems with potential
applications in innovative devices. Additionally, the presence of a
polyaromatic compound in the structure of a biomolecule can be used as a
marker, due to its luminescent properties, or as a scaffold for other
complex macrostructures. On the other hand, Copper catalyzed
Azide−Alkyne Cycloaddition (CuAAC), known as “Click Chemistry”, is
a simple and practical route to prepare a huge variety of new materials.
This regioselective procedure, in contrast to the uncatalyzed Huisgen 1,3-
*
Corresponding author Email: celedonio.alvarez@uva.es.
236 Celedonio M. Álvarez, Héctor Barbero and Sergio Ferrero
dipolar cycloaddition, is being used nowadays in many different fields of

science. The scope of “Click” reaction has been thoroughly studied to
include aromatic moieties to all kind of materials. However, when a
polyaromatic entity is considered to be assembled, especially when it is
not planar, all those studies must be revised because they do not follow
the usual trend observed for non-conjugated aromatic compounds. In this
chapter we highlight the use of this reaction as a synthetic method to add,
as building blocks, a big range of polyaromatic species into other
structures or into other polyaromatic species.
Keywords: carbon nanostructures, fullerenes, carbon nanotubes, graphene,

polyaromatic, pyrene, perylene, corannulene
INTRODUCTION
Click chemistry serves as a powerful synthetic approach opening a new
area towards the assembly of original molecular entities based on 1,2,3-
triazole scaffold becoming a straightforward way to create heterocyclic
systems [1].
Since its discovery in the last century, the information about this topic is
overwhelming. In fact, a considerable number of periodic revisions attending
these chemical approaches are wealth and extensive. As a result, it is very
difficult to find specific information without getting lost in it. In order to shed
light, the reader can discover some general and interest reviews on “click
chemistry” in these references [2].
The term of click chemistry was introduced by Sharpless [3] as a general
concept for organic synthesis and became very popular. This notion is
summarized in one sentence: “all searches must be restricted to molecules that
are easy to make”.
Among the various click reactions responding to the requirements of this
concept, nowadays the most popular click reaction is based on the well-known
Huisgen 1,3-dipolar cycloaddition [4], known currently as CuAAC reaction
(copper-catalyzed azide-alkyne cycloaddition) when it is catalyzed by this
metal. It consists of the transformation of an acetylene group into a five-
membered heteroaromatic 1,2,3-triazole ring and have numerous applications
in synthetic organic chemistry [5], drug discovery [6], sugar derivatization [2g,
7], biochemistry [8], polymer chemistry [9] and materials science [10].
The main limitation of the azide-alkyne cycloaddition was associated with
the formation of two triazolinic regioisomers (1,4 and 1,5) when performed at
Copper-Mediated Click Chemistry Applications to Assemble … 237
high temperatures, being very difficult to separate using classical

chromatographic techniques. This drawback was successfully overcome when
the groups of Sharpless [11] and Meldal [12] described a new method using
copper(I) catalysts for the 1,3-dipolar cycloaddition of organic azides and
alkynes, getting a breakthrough due to the total regioselectivity towards the
1,4-substituted 1,2,3-triazole (over 1,5 isomer) and a tremendous increase in
reaction rates [13]. This allowed room-temperature cycloadditions (Figure 1).
Other general benefits can be summarized as follows: the obtained yields are
high, simple workup and no purification is generally required or purification
procedures are easy to execute. The mechanism aspects of this reaction are
examined in this review [13] and deeply studied by Finn et al. [14].
There are different procedures on the literature aiming to introduce alkyne
[15] and azide [16] functionalities in a strainforward way. Thanks to their
kinetic stability, tolerance to numerous functional groups and their little
sensitivity to steric factors, make these complementary coupling partners
particularly attractive. For this reason, it has led to its rapid evolution into a
common tool in various research areas, as commented previously.
Two very useful and easy to perform systems to obtain the desired
copper(I) catalyst, among others, are as follows: generation in situ by using
copper(II) sulfate and sodium ascorbate as reducing agent, or the use of a
copper(I) halide together with a stabilizing N-donor ligand. Additionally, it
can be carried out particularly well in aqueous media with the help of an
organic solvent. In this regard, it fulfills the requirements of “green
chemistry”.
Figure 1. Schematic comparison between Huisgen reaction (above) and copper-

catalyzed azide-alkyne cycloaddition, CuAAC (down).
There has been an explosive growth in the use of microwave-assisted

organic synthesis [17] due to great advantages over traditional methodologies.
In this field, click chemistry has not been left behind. Although Cu(I)-
catalyzed alkyne–azide coupling often requires no additional heating,
microwave radiation can dramatically reduce reaction times in many cases

from over twelve hours to less than one hour [18].
Finally, click reactions are not limited only to Cu(I). Alternatively, there
are several examples with catalysts based on various metals [19], being
ruthenium-based catalysts as the most interesting because they can specifically
furnish 1,4 regioisomer. However, only copper-mediated cycloadditions will
be addresed here.
CLICK ON CARBON NANOSTRUCTURES

In the last decade, nanoscience and nanotechnology has impacted on
different areas of science. Inside this topic, carbon has shown great potential
thanks to its different nano-allotropic forms [20]. As examples of these forms,
fullerenes, carbon nanotubes (CNTs) and graphene are, by far, the most known
[21].
The solubility of this kind of compounds is limited, which makes their
manipulation and application difficult, being this fact an important drawback.
In response to this situation, it is fair to note the efforts of scientists to create
new approaches to solve this limitation via functionalization. This can be done
through covalent bonding, which this book chapter is mostly focused, or via
the non-covalent association based on supramolecular interaction.
Among all fullerenes [22], the most known is buckminsterfullerene, or
C60. It consists of 20 six-membered rings (hexagons) and 12 five-membered
rings (pentagons). Five-membered rings provide the curvature while strain is
minimized by sharing their sides with five hexagons preventing two pentagons
to be adjacent. It has been studied for a broad range of novel applications, like
liquid crystals [23], superconductors [24], solar cells [25], biological systems
[26] and biosensors [27], among others.
Carbon nanotubes (CNTs) [28] are made of graphene sheets which are
rolled up to become hollow cylinder-shaped macromolecules and they can be
classified as Multi-Walled nanotubes (MWCNTs) [29], composed by a
concentric arrangement of many cylinders, or Single-Walled nanotubes
(SWCNTs) [30] which possess the simplest geometry. They have been used in
the development of composite materials [31], nanoelectronic [32], energy
storage [33], biosensors [34], biomedical [35] and nanobioelectronics [36] and
many others.
Graphene [37] is defined as a single layer of sp2 bonded carbon atoms
arranged in a honeycomb pattern, although they may also contain some sp3
carbon atoms considered defect sites. It has a very wide range of potential
applications in photovoltaic devices [38], transparent conductive films [39],
photosensitive transistors [40], batteries [41], supercapacitors [42], cancer
therapy [43], biosensing [44] and many more.
Fullerenes
The compatibility of 1,3 dipolar cycloadditions between terminal alkynes

and azides with fullerene derivatives is not obvious because organic azides
may undergo [3+2] cycloadditions to the double bonds of fullerenes. However,
CuAAC reaction allowed to decrease the temperature of these procedures,
giving rise to a lack of competition between both processes, dominating the
one leading to the desired 1,2,3-triazole derivatives [45].
The first attempt was performed by Nakamura and coworkers [46]
achieving great results when linking five equal moieties to a functionalized C60
bearing five terminal alkynes (Figure 2). It was reacted with an excess of the
organic azide in DMSO or toluene with CuBr·SMe2 and DIPEA at room
temperature or up to 50ºC for oligosaccharide derivatives, which were
revealed to be the most difficult to link among all the molecules tested.
Interestingly, great yields, easy purification procedure and high recovery of
starting material were obtained.
Figure 2. First set of fullerenes functionalized by click chemistry by Nakamura et al.

Ten examples were reported.
Figure 3. First modified Buckminsterfullerenes with CuAAC by Cheng and coworkers (5) and Nierengarten and
coworkers (2, 3, 4 and 6).
Figure 4. First triazole-linked dendro [60] fullerenes. 5 examples were reported.
On the other hand, almost simultaneously, Cheng's [47] and

Nierengarten’s group [48] developed other strategies by linking
buckminsterfullerene molecule to a polymer scaffold or by having terminal
azide in C60 moiety, respectively (Figure 3). In the case of Cheng group’s
work, a monoalkyne fullerene (called Fulleryne) was prepared in 3 steps and
reacted with a polystyrene having terminal azides, CuBr and PMDETA in
toluene at room temperature. These conditions were very similar to those
reported previously by Nakamura et al. and, not surprisingly, good yields were
obtained due, mainly, to the inexistence of reaction between the polymer and
C60 core.
At the same time, Nierengarten’s group experimented with
methanofullerenes functionalized both with terminal alkyne or azide groups by
a previous Bingel reaction [49]. The best results were observed when using the
well-known biphasic system CH2Cl2/H2O having CuSO4 and sodium ascorbate
dissolved in the aqueous phase and reactants in the organic phase, avoiding the
use of thermodynamically unstable copper (I) species. All yields obtained
ranged from moderate to very good depending on the solubility of fullerene
derivatives in the organic solvent. Otherwise, the reactions could take longer,
favoring undesired side reactions. Interestingly, monofunctionalized fullerenes
(3 and 4) gave worse yields if compared to their bifunctionalized counterparts
(2 and 6) pointing out that C60 core was still reactive under such conditions. It
must be noteworthy that azide-functionalized buckminsterfullerene was very
hard to handle as it underwent intramolecular cycloadditions giving rise to
intractable material.
Further investigation with alkynyl or azide-functionalized C60 [50]

confirmed the trends observed initially and it was established that alkynyl
derivatives were better building blocks to be prepared easily and stored for
longer times than azide derivatives, whose inherent unstability did not allow to
be synthesized in great amounts and stored for further uses. Furthermore, a
compound with potential optical properties [51], consisting of a bis-adduct
between a porphyrin and two fullerene subunits, could be obtained.
At the same time, the group of Zhao approached to this chemistry by
exploring the formation of buckminsterfullerene-based dendrons via CuAAC
click reaction (Figure 4) [52]. Yields increased with dendron generations (up
to G3). This depended on the number of attached groups because they
increased the solubility of the compound due to the long alkyl chains.
Problems associated with azide-functionalized C60 were partially solved
after a hexakis-adduct based on Bingel reaction was published later [53]. The
compound comprised up to six cyclopropanes bearing two azide tails per
group, ready for cycloaddition (Figure 5). The solution was accomplished in
two ways: on one hand, multiple functionalization of the fullerene core
decreased the reactivity towards intramolecular reactions; on the other hand,
the presence of a large number of appending groups prevented intramolecular
cyclyzation by means of steric hindrance. However, the compound could not
be stored upon purification for longer than half a day in solution. Nevertheless,
dodecatriazoles could be prepared, by using the biphasic system disclosed
above, from good to excellent yields. Interesting groups could be linked, such
as ferrocene or porphyrin units, which are useful for photosynthetic models
[54].
The complementary approach (terminal triple bond on C60 moiety),
including dendron functionalization, was also explored [55], leading to better
yields. This could permit a well-designed strategy to [5:1] hexa-aducts.
All these findings led immediately to the decoration of
buckminsterfullerene with sugars for future applications as inhibitors of
biomolecules [56] (Figure 6). Due to the nature of these new functional
groups, the water/DCM biphasic system could not be applied and the solvent
was changed to DMSO, but CuSO4·5H2O and sodium ascorbate were still used
in catalytic amounts. Moderate to excellent yields were obtained.
From that point, two interrelated branches of C60 decoration by click
reaction were developed, being one of them, the search of functionalization
with photoactive units, and the other one, the functionalization with
saccharides for new treatments by inhibition of biomolecules.
Figure 5. Hexakis-adduct fullerene bearing up to twelve terminal azides.
Figure 6. Two sets of fullerene sugar balls prepared by CuAAC click chemistry
in both approaches.
Excellent examples of the first branch are the work from Schuster, Guldi
and coworkers [57], in which several connections between porphyrin and
buckminsterfullerene moieties were studied. In all cases, cycloaddition of
phenyl alkyne and a phenyl azide were obtained by using copper iodide and
sodium ascorbate in a mixture of H2O and DMSO at 80ºC with MW
irradiation under inert atmosphere, but the attachment to C60 was performed by
Prato reaction [58] and the link between both building blocks was not as
excellent as expected. Other approach was developed by Campidelli et al. [59]
in which they attached up to two porphyrins by CuAAC Huisgen reaction in a
dendron-like fashion so that a host-guest complexation effect was observed. In
this case, the salt [Cu(NCMe)4)]PF6 was used in near stoichiometric amounts
along with 2,6-lutidine in a mixture of degassed THF and water at room
temperature to give the expected product in moderate yields, mainly due to the
low solubility of the fullerene derivative. Other interesting approach was
applied by Jiang et al. [60] in an exceptional organic framework built by
phthalocyanine subunits. The cavities were filled by fullerenes through
copper-mediated cycloaddition to give donor-acceptor heterojunctions in the
form of [C60]y-ZnPc-COFs; y = 0.3, 0.4 and 0.5. This was achieved by stirring
all species in degassed dimethyl acetamide at 50ºC with copper (I) iodide salt
for 24h. Porphyrins were not the only photoactive groups tested in a C60
scaffold. Boron dipyrromethenes (bodipys) could be linked to this molecule in
a [5:1] hexakis-adduct by click chemistry [61] thanks to the development
previously carried out [55]. The classical method of CuSO4 and sodium
ascorbate in a biphasic mixture of DCM and water by using an azide-
functionalized fullerene was applied giving great results. It is noteworthy that
protected triple bonds did not react at all under these conditions, allowing
further synthesis to get the [5:1] model, which became an artificial light-
harvesting antenna (Figure 7).
Figure 7. Artificial light-harvesting antenna developed by Nierengarten and

Ziessel groups.
Recently, Fukuzumi, Sastres-Santos and collaborators reported the

functionalization of C60 with perylene moieties [62] by using the system
CuI/DIPEA in toluene obtaining moderate yields.
On the other hand, several authors have addressed the preparation of
multivalent saccharide-functionalized fullerenes (kindly called sweet or sugar
balls) finding excellent results in order to bind proteins like Concavalin A,
bacterial adhesins, bacterial lectins and almost any biomolecule whose
recognition motif depends on carbohydrates [63]. Both hexaalkyne and
hexaazide fullerenes were tested and the conditions did not differ substantially
from previous optimized conditions. Copper source came from CuSO4·5H2O
and CuBr·SMe2 salts, and chosen solvents were DCM/H2O, DMSO or a
combination of all three. Martín, Delgado, Rojo and collaborators tested a C60
scaffold with 36 attached sugars in a cellular infection model [64]. The
strategy followed known conditions: CuBr·SMe2 in degassed DMSO at room
temperature, but, this time, they added a little piece of Cu(0) to the mixture.
The most impressive example was developed later as a set of ‘super balls’ [65]
consisting on a core of 13 fullerenes with 120 peripheral carbohydrate groups
(Figure 8), all linked by CuAAC chemistry under similar conditions stated
above, adding MW irradiation to increase reactions rates.
Figure 8. Set of sugar balls capable of inhibit a model of Ebola virus infection.
We would like to account for a closely related family of nanostructures,

called carbon nano-onions or multi shell fullerenes [66] which share properties
between fullerenes and carbon nanotubes, and their functionalization by
copper-mediated cycloaddition (Figure 9) [67]. Their low solubility forced
researchers to carry out the reaction in a dispersed system in DMF or a mixture
of TMP/DMF by using the couple CuSO4/sodium ascorbate or
[Cu(NCMe)4]PF6 salt helped by coligands such as 2,6-lutidine or THPTA.
Figure 9. Model of the first CNO functionalized with click chemistry by Giordani
and coworkers.
Carbon Nanotubes
Unlike the previously addressed group of carbon nanostructures, this

family does not suffer cycloadditions with azides on the π surface, so it can be
handled more easily. Despite this advantage, CNTs show a major drawback:
their low solubility might complicate their functionalization. The general
strategy towards click-chemistry-mediated connection of molecules
commented above can be applied with this system [68], as seen in Figure 10. It
is possible to attach a phenylene group with an azide or a triple bond and the
choice will heavily depend on the availability of the counterpart which will be
linked.
The pioneer work from Adronov et al. [69] disclosed the possibility of
performing click chemistry in a carbon nanotube by first appending a
propargyl aniline by known diazotization and coupling procedures in CNTs
[70] and a subsequent CuAAC cycloaddition to an azide-
functionalized polystyrene (Figure 11). An organosoluble [CuBr(PPh3)3] salt
was utilized as catalyst in DMF at temperatures from 20 to 90ºC with a large
excess of polymer. The purification was not too difficult as only an ultra-
filtration and prolonged washing of THF and aqueous ammonia were
necessary.
Figure 10. Easiest complementary approaches to CNTs functionalization towards [3+2]

azide-alkyne cycloadditions.
Figure 11. First set of polymer/CNT hybrid developed by Adronov and coworkers.
Other approach was further investigated by the same group [71] with
hidroxyl functionalized MWNTs. Two isocyanates bearing terminal triple
bonds were firstly attached. Then, PDMA-PNIPAM diblock copolymer
micelles of different molecular weights and polydispersity with azide groups
on the outer shell were clicked in water by using the couple CuSO4/sodium
ascorbate. Purification was similar to that discussed earlier by these authors.
The observed increase in grafting efficiency for the copolymer micelles as the
temperature increased allowed to conclude that preorganization of the reactive
polymer chains deeply influenced such efficiency. Parallel to this work, Gao’s
group reported the synthesis of amphiphilic polymer brushes on carbon
nanotubes [72] in a Gemini-grafting strategy clicking a macroinitiator that
underwent atom transfer radical polymerization (ATRP). An alkyne-
terminated carbon nanotube was used and cycloaddition occurred in degassed
DMF with CuBr and PMDETA at room temperature. Further investigations by
the same group and other groups were developed to attach nanoparticles
through a soft polymer interlayer [73], to get a sequential layer-by-layer
grafting [74], to combine Pd nanoparticles [75] and to link polymers based on
caprolactames [76] by applying previously optimized conditions.
Figure 12. MWNT/MSN nanohybrids attached with CuAAC Huisgen reaction by Qu’s
group. Carbon nanotube was depicted as SWNT for clarity.
All addressed examples regarding the covalent union of carbon nanotubes

and nanoparticles needed a “soft layer” to work efficiently. Nonetheless, Qu
and coworkers were able to join magnetic silica nanoparticles (MSN) into
MWNT [77] by directly functionalizing nanotubes with just a terminal alkyne,
with no spacer groups and carrying out a CuAAC cycloaddition in very simple
conditions (Figure 12). They just mixed MSNs and nanotubes in water with
dissolved copper sulfate and sodium ascorbate and stirred for 24h; following a
magnetic separation and washing as purification method.
Zheng et al. could synthesize a hybrid between a carbon nanotube and a
cyclodextrin [78] by using the same alkyne-terminated SWNT used by
Adronov and coworkers before, in the paper initially addressed in this section.
However, CuI in combination with DBU were utilized instead of an
organosoluble salt.
Other functionalizations were explored almost simultaneously by Swager
et al. [79] with zwitterion-mediated transformations on fullerenes and
nanotubes surfaces to give two triple bonds per anchoring group. Click
reactions were carried out with CuI and DBU in DMF at 65ºC. The group of
Hamer [80] achieved direct-azide functionalized carbon nanofibers
(complementary to Qu’s method disclosed above) by drawing upon the
procedures developed before [81] for further CuAAC-mediated cycloaddition
with ferrocene (Figure 13) and, later on, these findings were used to click
pyrene derivatives [82] or ionic liquids [83] by the same method.
Giambastiani and collaborators attained other expected azide-terminated
alternative [84] by appending p-phenylene azides to single walled carbon
nanotubes and, then, clicking them to a variety of alkynyl derivatives (Figure
14) either with CuSO4·H2O/sodium ascorbate or organosoluble [CuI(P(OEt)3)]
salt in degassed DMF at 85ºC for 48h with periodic sonication cycles.
In other related research field, carbon nanotubes have been widely
transformed to attach photoactive molecules by click chemistry for future
applications as devices. Campidelli, Torres, Guldi et al. reported the first
nanohybrid between azide-functionalized Zn phthalocyanines and alkyne-
terminated SWNT by this reaction [85]. They used the couple
CuSO4·5H2O/sodium ascorbate in NMP at 70ºC for 2 days. Shortly, a second
related contribution was published [86]. In this case, SWNT was clicked with
one or two Zn porphyrins per appending group, but they chose to follow a
different method. [Cu(NCMe)4]PF6/2,6-lutidine couple was used, along with

THPTA coligand in NMP at room temperature. This dendron-like approach
was further explored by Campidelli and coworkers [87]. A heterohybrid
consisting on an SWNT, Zn phthalocyanine and Zn porphyrin was obtained by
similar procedures. Finally, these investigations culminated in a porphyryn
polymer grafted to a single walled carbon nanotube (Figure 15) by the same
group [88]. Multiple CuAAC Huisgen reactions occurred under the same
conditions stated already and, interestingly, the polymer was observed to be
formed in solution before grafting to the nanotube, which was the key step.
Along the same line covered in the previous section regarding fullerenes
derivative, porphyrins and phthalocyanines were not the only family of
photoactive molecules to be considered for carbon nanotubes
functionalization, as other families, like bodipys [89] and exTTFs [90], have
been studied. With respect to the first family, [CuI(P(OEt)3)] was used in
degassed DMF at 70ºC for 48h with periodic additions of more catalyst
batches; and, in relation to the second one, copper sulfate and sodium
ascorbate dissolved in DMP with the addition of some wires of Cu(0) were
used at 70 degrees for 24h.
Figure 13. Carbon nanofiber functionalized with azides by Hamers’ group and
conditions for further cyclyzation with an alkyne ferrocene.
Figure 14. Giambastiani’s group method towards azide-SWNT and subsequent

click reactions.
Figure 15. Hyperbranched porphyrin polymers on carbon nanotubes via a CuAAC

“grafting from” approach.
Recently, D’Souza, Sastre-Santos, Langa and coworkers described the

functionalization of double-walled CNTs with perylene [91] in a similar
fashion stated before [85].
Graphene
Unlike fullerenes or carbon nanotubes, this promising carbon

nanostructure have sp2 (in the surface) and sp3 (along the edge of the sheet)
carbons and reactions might be carried out in one of those sections, or in both
[92]. sp3 carbons are considered defects in the overall structure and the
aromaticity near the edge is widely decreased, concomitantly increasing the
reactivity of this material. Moreover, functionalization can be done in pristine
graphene (by anchoring groups resembling the chemistry of CNTs) or in
graphene oxide (by performing reactions in functional groups containing
oxygen) [93]. Those groups can be situated on the surface and/or along the
edges (Figure 16).
After the previously mentioned work regarding a graphene functionalized
by azides along the edges [81], Wu, Feng et al. reported the conversion of
terminal carboxyl groups in graphene oxide (GO) [94] with propargyl alcohol
into an esther bearing a triple bond. This function was utilized in a subsequent
copper-mediated Huisgen cycloaddition with a polystyrene derivative to give a
graphene/polymer nanohybrid [95]. The system CuBr/PMDETA was used in
degassed DMF, along with an excess of polymer, and the mixture was stirred
at room temperature for 48h. Similar conditions were further applied in other
studies by using the same propargyl esther or a propargyl amide [96].
The opposite approach was later performed by Peng, Huang and
collaborators by obtaining an azide-terminated amide, ready to be clicked with
a group bearing a terminal alkyne [97]. In this case, modified DNA was used
and the new composite was tested to establish interactions with gold
nanoparticles. The conditions were simple: CuSO4, an excess of sodium
ascorbate in water and stirring at room temperature for 1 day. Interestingly,
purification was easy, as only a sonication followed by PBS washing
procedures were carried out. Namazi’s group studied the possibility of
anchoring saccharides to get hydrophilic graphene nanosheets [98] for future
applications in biological recognition by developing two approaches. On the
one hand, GO was directly functionalized with azide groups on the surface,
and, on the other hand, carboxyl groups were transformed into an esther
bearing terminal azides. CuAAC click reaction was carried out in H2O/DMF
with the well-known CuSO4/sodium ascorbate system. Simultaneously,
Somwangthanaroj and coworkers applied similar conditions to those disclosed
in this paragraph to achieve a link between GO and a PEDOT:PSS ensemble
along the edge of the sheets [99]. The most interesting recent work from Al-
Jamal et al. addressed the possibility of double and orthogonal
functionalization of graphene oxide by anchoring azides and terminal triple
bonds protected by TMS in a sequential multi-step procedure [100]. Then,
click reaction occured with an alkyne-terminated building block and, after
TMS removal, a second click was performed with a different azide-terminated
moiety (Figure 17).
Regarding surface functionalization of graphene, one initial contribution
belongs to van der Wiel and coworkers [101]. In such publication, epoxide
groups on GO were attacked by sodium azide to give a graphene comprised of
OH and N3 groups. These nanosheets suffered reduction to get the amine
derivative, restoring sp2 network partially and becoming a conductor material
again. It was utilized to perform other transformations, but the previous azide-
functionalized graphene was linked to long-chain alkyne-terminated
hydrocarbons with already commented procedures. Other groups approached
in the same way [102]. Later on, the group of Strano et al. [103] added the
same alkynyl-terminated anchoring group discussed earlier [69] on the surface
of a CVD-synthesized graphene for ulterior attachment to a PEG building
block with terminal azides. Click conditions were simple: CuSO4 in
combination with sodium ascorbate were used in water with the help of
THPTA as coligand and the suspension was vigorously stirred for 18h. This
methodology was later covered again [104]. In parallel with these studies, Tu
and collaborators proposed another approach by using OH groups on the
surface of GO [105]. An esther was formed by nucleophilic attack to an aryl
bromide and further functionalization to an azide derivative was carried out
and, finally, clicked to a polystyrene derivative. This approach
(functionalization of surface OH groups) was revisited later [106], and the
work published by Binder and coworkers is particularly noticeable [106b]. In
that contribution, copper (I) nanoparticles were immobilized on GO surface by
click chemistry to use the resulting nanocomposite as a renewable catalyst for
CuAAC cycloadditions (Figure 18).
Figure 16. Sketch of a pristine graphene sheet (left) and graphene oxide (right).
Figure 17. Sequential functionalization of GO using two CuAAC click reactions by Al-
Jamal’s group.
Figure 18. Immobilized copper (I) nanoparticles on graphene surface by Binder

and collaborators.
Figure 19. Photoresponsive GO-C60 nanostructure obtaind by CuAAC

Huisgen cycloaddition.
Following the methods from Campidelli et al. in carbon nanotubes [85],

Zhang and coworkers were able to attach photoactive molecules over the
surface of pristine graphene [107] by clicking azide-functionalized porphyrins
and bipy-based Ru complexes in similar conditions. Later, Salavagione and

coworkers explored the same functionalization linking a poly(fluorene) to
graphene [108]. They also clicked this polymer by the edge of the carbon
nanostructure. However, the conditions were different, as CuBr and PDMTA
were used in degassed DMF and the suspension was stirred at 60ºC for 150
minutes. García, Langa and collaborators prepared a photoresponsive GO-C60
hybrid by click chemistry (Figure 19) [109] with the same conditions stated
before [85]. Recently, other photoactive molecules could be attached by
similar procedures [90].
Other current interesting tendencies found in literature deal with silicon-
derived graphene oxide for further cyclization with other structures by click
chemistry [110], or simultaneous exploration of surface and edge
functionalization [111, 107].
CLICK ON SMALL POLYCYCLIC

AROMATIC HYDROCARBON
Polycyclic Aromatic Hydrocarbons (PAHs) are considered, from a
simplistic point of view, as little fragments of those carbon nanostructures
revised in the first part of this chapter. Their molecular nature makes them to
be soluble in all cases, facilitating handling and characterization. Among the
vast variety known so far [112], just a few of them have been employed,
mainly due to a commitment between ease of preparation and excellent
photophysical properties [113]. In fact, pyrene and perylene are the most used
for sensing purposes [114] by quenching or enhancing fluorescence depending
upon the molecule to be detected. Furthermore, both molecules are totally
planar, permiting supramolecular association with carbon nanostructures such
as CNTs and graphene thanks to a moderately strong π-π stacking effect [20,
68f] giving rise to new composites with interesting potential applications.
Biomolecules, especially carbohydrates, have been connected extensively
with these molecules to give new families of hybrid conjugates [2g].
On the other hand, corannulene is a special PAH because of the
arrangement of its atoms. It consists of a five-membered ring sharing all sides
with six-membered rings. This connection results in a bowl shape and this is
why it is commonly known as a buckybowl inside the family of geodesic
polyarenes [115]. This structure resembles that from C60 and is considered one
third of the fullerene. This special fact, combined with a net dipole moment
arising from different electronic densities in both faces, give corannulene a

special ability to associate fullerenes thanks to an excellent concave-convex
complementary between both surfaces [116].
Pyrene
The first click reaction involving pyrene was reported by Anslyn et al.
[117]. They studied the possibility of monitoring the reaction with the system
CuSO4/ascorbate in the presence of EDTA (inhibitor by chelating copper ion)
along with different additions of other exogenous metals (effectors due to a
better coordination with EDTA releasing copper species). An observed Förster
resonance energy transfer (FRET) phenomenon occured when pyrene was
connected by triazole linkage, meaning that the reaction worked.
From that point, the chemistry of pyrene by CuAAC cycloaddition has
been widely explored and the most relevant works will be covered here.
Kim, Matthews, Vicens and collaborators developed the first calix [4]
arene with triazole-linked pyrenes for detection of Cd2+ and Zn2+ (Figure 20)
[118]. The structure of this receptor was substantially modified upon cation
coordination, affecting its emission spectra. An azidomethyl pyrene was
coupled to an alkynyl-terminated calixarene in DMF at 90ºC for 2h by using
CuI catalyst, furnishing moderate yields only. Parallel to this paper, Yang and
coworkers reported similar structures [119] and, aftwerwads, the same group
published more homologues [120].
Figure 20. First calix [4] arenes with triazole-linked pyrenes for cation detection.
The group of Yamato and collaborators added a third pyrene in an

oxacalix [3]arene scaffold for lead and zinc detection and the formation of
INHIBIT and OR logic gates [121]. Reaction conditions were very similar to
those described previously, but modifying the solvent by a mixture of THF
and water achieving moderately better conversions. A similar approach was
explored by Chung et al. using 1-azidopyrene, with no methylene spacers, in
the same calix [4] arene [122]. They used almost equal conditions and,
interestingly, this azide gave improved yields, probably due to longer reaction
times (1 day) or to the special nature of the azide derivative. Similar other
approaches have been recently explored by mentioned groups [123], but a
great different example came from Li et al. [124]. The reported calixarene was
prepared with the opposite model; this means that the terminal triple bond was
located in the pyrene derivative and the azide group in the calixarene. CuSO4
and sodium ascorbate were used instead of CuI and the solvent utilized was
DMF at 90ºC giving moderate yields. The resulting compound was employed
to construct a nanocomposite with GO to be tested as a pesticide sensor.
Some other different approaches for the construction of pincer-like pyrene
derivatives have been attained. For instance, Chung’s group prepared a set of
triazole-chained pyrenes by polyalkyl or polyoxyethylene tethers for ions
recognition by formation/disruption of an excimer as a consequence of the
absence/existence of a metal (Figure 21) [125]. The clicking strategies carried
out did not differ substantially from conditions addressed before [120] and,
consequently, yields were moderate. This approach was later revised [126].
Figure 21. Set of triazole-chained pyrenes developed by Chung et al.

Other kind of tether was explored by Zhu, Cheng and collaborators [127].
In such work, a BINOL-pyrene derivative was synthesized by copper-
catalyzed azide-alkyne cycloaddition between an azidomethyl pyrene and a
dipropargyl (S)-BINOL as a sensor for Ag+ and Hg2+. Regarding click
conditions, copper (I) iodide salt was used too, along with an excess of DIPEA
for metal stabilization. All reagents were mixed in THF under N2 atmosphere
at room temperature for 12h obtaining, again, moderate yields.
Later on, Seela and coworker prepared a tripodal pyrene derivative based
upon a propargyl amine for detection of a wide variety of cations, especially
for Zn2+ [128]. This time, [Cu(NCMe)]PF6/2,6-lutidine system was employed
in acetonitrile leaving the reaction for 3 days at room temperature, having
slightly better conversions.
The best results, in terms of isolated products yields, were obtained by
Zhao and coworkers and, then, by Tárraga and Molina et al. in a TTFV
tweezer derivative and a three-armed triazole-linked compound, respectively
[129]. In the first case, CuI was also used, but in the presence of iPr2EtN, in
THF at 60ºC overnight. In the second case, [Cu(NCMe)4]PF6 was utilized
along with TBTA, sodium ascorbate and DIPEA in THF at room temperature,
getting the best yields. It is noteworthy that pyrene derivative involved in the
last examined publication is an alkynyl-terminated compound, instead of the
azidomethyl pyrene discussed in the other examples.
According to the general trends regarding functionalization of
polyaromatic structures, polymer appendage with pyrene moieties has been
explored by several chemists. Thus, Yagci and collaborators firstly reported
several polymers with pending azido groups which were linked to propargyl
pyrene [130] by using an aqueous solution of copper sulfate and sodium
ascorbate in DMSO, achieving near-quantitative conversion. Later, a
polysulfonate was functionalized with the same pyrene derivative by the that
group [131]. The most interesting contribution has been recently developed by
Qiao and coworkers [132]. An alkynyl-terminated pyrene butyrate suffered
cycloaddition with a PEG-N3 polymer by using CuBr2 and a photocatalyst
designed by them for the conversion of copper (II) into copper (I) with a light
input (Figure 22).
A different approach, hardly addressed for other polyaromatic structures,
was firstly covered by Tárraga, Molina et al. in a ferrocene-pyrene derivative
[133]. This molecule had dual-mode recognition due to electrochemical
properties from ferrocene moiety and photophysical issues from pyrene
subunit. The compound was able to bind pyrophosphate anion; it was prepared
with azidoferrocene and 1-ethynylpyrene in THF along with the couple
CuSO4/ascorbate in water at room temperature, but only moderate yield was

obtained, probably due to short reaction time. This procedure was utilized later
in a similar system [134]. Gaubicher and coworkers explored the opposite
version with ethynylferrocene and an azide-terminated pyrene derivative
[135]. CuI salt with DIPEA in acetonitrile gave the expected compound in
good yield. The group of Ghosh synthesized two multi-channel probes for
mercury detection based upon unsimmetrically substituted ferrocene, being
one of them a pyrene-derivative [136]. An azidomethylferrocene was reacted
with the same propargyl pyrene covered before [129] with CuI/DBU system in
DMF at 65ºC for 6 hours furnishing good yields. Further derivatives were
investigated by Tárraga, Molina et al., [137] but the same CuSO4/ascorbate
system was applied giving, consequently, moderate to good yields.
Functionalization of biomolecules with pyrene groups to introduce
fluorescent properties by CuAAC Huisgen reaction has been also covered.
Thus, Fujimoto, Inouye and coworker prepared a pyrene-derived pair of
complementary-sequenced oligonucleotides linked with a triazole to a
cyclodextrin for detection of unsaturated fatty acids by the switch from
monomer emission to excimer emission after binding the guest molecule
[138]. CuBr and TBTA were used in a complex mixture of
H2O/DMSO/tBuOH at room temperature followed by HPLC separation.
Although a click reaction was reported here, pyrene was not covalently linked
to the oligomer by this reaction until Nielsen and collaborators published their
work regarding pyrene-functionalized oligonucleotides [139]. In such study,
both approaches were covered, as 1-ethynylpyrene or 1-azidopyrene were
coupled to azide-terminated or alkynyl-derivative, respectively. Copper (II)
sulfate and sodium ascorbate were widely used along with complex mixtures
of solvents (THF/H2O/tBuOH or THF/H2O/pyridine) and, sometimes, TBTA
as coligand for copper (I) stabilization, giving rise to a range of yields from
good to excellent. Thereafter, Seela et al. developed a stepwise
functionalization of DNA in terms of click chemistry helped by the use of a
bifunctional 2,5-bis(azidomethyl)pyridine [140]. The elegant strategy relied
upon a previous use of a copper (II) salt without a reducing agent furnishing a
monotriazole at the carbon 2 in mentioned tether and leaving the other azide
group unreacted. Then, a second click could be performed with the same
system, this time with a reducing agent, to get a second triazole at carbon 5.
This protocol could permit cross-linked and pyrene-linked oligonucleotides.
The success of this method is based on a chelating effect from azide group and
pyridine nitrogen that took place only with one of the substituents but not with
the other. The same group contributed with nitroindole oligonucleotides
bearing alkynyl side chains to be coupled with azidomethylpyrene (Figure 23)

by using well-known CuSO4/ascorbate system in THF/H2O obtaining
moderate yields [141].
Figure 22. Synthesis of PEG-pyrene by CuAAC with a solid state photocatalyst

developed by Qiao and collaborators.
Figure 23. Molecular models of two sets of complementary oligonucleotides

functionalized by copper-mediated Huisgen cycloaddition with pyrene group
(in green).
There have been other studies reported by several researchers in which a

pyrene is involved in a click reaction and they are worth to be consulted by the
reader [142].
Perylene
This molecule has been utilized in three main groups. The simplest one is
pristine perylene, in which funcionalization might be performed at peri or bay
positions. However, the most widely used are those whose peri positions are
already occupied by one imide (PMI) or by two imides (PDI). In such cases,
functionalization can be performed in imide pending group, too. All stated
possibilities are gathered in Figure 24.
The first click reaction was reported by Koshun et al.[143] for
bioconjugation between oligonucleotides and PDI. In that work, perylene
moiety had azido groups at the end of the chain supported by nitrogen atoms
and oligonucleotides had a modified phosphate bearing a terminal alkyne.
CuSO4 and sodium ascorbate, along with TBTA as stabilizing reagent, were
used in DMSO at 95ºC overnight. A separable mixture of monoadduct and
diadduct was obtained in moderate yields. PDI derivative was not quite
soluble, as high temperatures had to be used. The same group explored other
approaches with subtle modifications and yields were slightly enhanced [144].
Simultaneously, Langhals and coworker explored more deeply the chemistry
of PDI in terms of copper-mediated cycloadditions by synthesizing a set of
monoazide(or monoalkynyl)-terminated PDI derivatives, with several spacer
groups, for further click reactions [145]. In such study, all reactions were
performed with copper (I) iodide, N-ethylbis(isopropyl)amine (for stabilizing
purposes) in THF at room temperature getting a range of yields from moderate
to good. Next, Nielsen and coworkers used one alkynyl-terminated PMI
already developed by Langhal’s group to conjugate with a TTF derivative for
optical and electrochemical studies [146]. The system CuSO4/ascorbate in
DMF was used and moderate conversion was obtained. One of the greatest
advances in the study of this reaction involving perylenes came from
Finlayson and Kouwer’s groups in a publication where eight click reactions
were simultaneously carried out between an unsymmetrical PDI and a
phthalocynanine bearing eight alkynyl groups (Figure 25) [147]. Copper (I)
iodide with PMDTA in the presence of TBAF in THF as a solvent were

reacted for 24h achieving excellent yields of isolated product. The in situ
deprotection of triple bonds were immediately captured by copper species and
coupled to azide groups giving the expected triazole.
In a parallel work, Thelakkat et al. studied click reaction of PDI in
semiconducting polymers by using similar azide-terminated perylene diimides
[148]. Other brilliant example of multiple copper-mediated Huisgen
cycloaddition was published by Rybtchinski and coworkers [149]. A tri and
hexa adduct were prepared with amphiphilic PDI in a hexasubstitued benzene
as a tether (Figure 26). It must be noticed that this is the first example in which
a PDI functionalized in bay position was employed for this type of reaction.
The system utilized for this synthesis was a mixture of CuI in THF and sodium
ascorbate in water. The reaction was stirred at 80ºC for 4 days and yields were
moderate.
Figure 24. Three most common perylenes addressed in this text.

Figure 25. Phthalocynanines-PDI octads obtained by in situ deprotection followed by

CuAAC reaction by Finlayson, Kouwer and collaborators.
Figure 26. Amphiphilic tri and hexa adducts of PDI prepared by click chemistry.
Simultaneously, Zimmerman and collaborators reported dendrimeric PDIs

attached to several groups (including biomolecules) for biolabeling purposes
[150]. Excellent yields were attained, most likely because the only CuAAC
occured far away from perylene core. The same group prepared later
hyperbranched polyglycerols with perylene as a core [151]. In that work, a
bay-functionalized PDI was reacted with the alkynyl dendrimer to get the
expected product. Four click reactions occurred with CuI as the catalyst and
DIPEA as the coligand giving rise to moderate conversion.
Astakhova’s group contributed with studies regarding pristine perylene
monofunctionalized in peri position with an azide group. A set of
oligonucleotides coupled to fluorescent molecules were prepared for nucleic
acids, autoimmune antibodies and natural RNA sensing [152]. A Cu(II)-TBTA
complex was used in the presence of ascorbic acid and an aqueous phosphate
buffer. The mixture was stirred in DMSO and heated by conventional methods
or by using MW radiation, obtaining the best results with the second system. A
different approach involving click chemistry of perylene and biomolecule was
reported by a collaboration between Astakhova and Wengel’s groups. In that
study a doubly functionalized PDI was used as a branching unit in DNA
nanostructures to form FRET pairs with pyrene moieties linked to the same
material [153]. The same protocol stated before was applied furnishing very
good yields.
Later, Bhattacharjya, Yeow, Xing and collaborators reported a magnetic
nanoparticle bearing a PDI attached by copper-assisted Huisgen reaction with
an appending peptide for detection of bacterial lypopolysaccharides by
fluorescence self-quenching or self-activation [154]. Copper sulfate and
ascorbic acid were used in a mixture of water and tert-butanol. The dispersion
was shaked for 48h achieving good conversions.
A current contribution came from Bozdemir et al. applying their approach
by using a pristine perylene tetrafunctionalized in peri positions and a peri-
disubstituted PMI [155]. In all cases, perylene moiety had propargyl groups
and were reacted with azide-terminated molecules with the well-known
CuSO4/ascorbate system in a mixture of CHCl3/EtOH/H2O. The reaction was
heated at 65ºC assisted by MW irradiation getting excellent yields of isolated
products. Simultaneously with that work, Wang, Li and collaborators
described two perylenes attached to carbohydrates in bay and imide positions
[156].
Recently, McGrath et al. has reported asymetric phthalocyanines [157]

with terminal triple bonds for further linking with PDIs and other structures in
a similar way to that addressed before [146].
Corannulene
The first reported preparation of corannulene via CuAAC click chemistry

was performed by Stuparu [158]. In this study alkynyl, azide or azidomethyl-
functionalized corannulene were used to obtain dimers and a trimer, as seen in
Figure 27. In all cases, the system CuSO4·5H2O/sodium ascorbate was used in
a mixture of water and tert-butanol at 80ºC achieving very good yields.
Simultaneously, Siegel’s group could transform a sym-pentachloro
corannulene into several derivatives by using the appropriate Grignard’s
reagent [159]. In such work, a sym-pentalakynyl derivative was clicked to an
azide-terminated sugar by utilizing a singular method: they reacted the
building blocks in DMF at 60ºC inside a microwave reactor in the presence of
copper nanoparticles. Further approaches of corannulene pentafunction-
alization were investigated by the same group afterwards [160]. Along a
different line, the same group, in colaboration with Sukwattanasinitt’s group,
developed a set of triskelion-shaped fluorophores containing a triphenylamine
coupled to pyrene (in a smiliar way to what Seela et al. disclosed in a previous
work [127]) or corannulene by copper-mediated Huisgen cycloaddition,
among others [161]. The conditions were simpler and very similar to the initial
work addressed above, being the only difference the use of THF instead of
tBuOH.
Figure 27. First triazole-linked corannulene dimers and trimer reported.

Figure 28. Set of triazole-linked corannulene pincers for supramolecular interactions

with C60 developed by Álvarez et al.
Later on, Álvarez and coworkers reported the synthesis of two

tripodal molecules based upon a tris(triazomethyl)benzene or a tris
(triazomethylethane) tethers and corannulene for supramolecular assembly
with Buckminsterfullerene along with the only example of biscorannulene-
helicene assembly known so far (Figure 28) [162]. In all cases, terminal triple
bond was located in corannulene units and click reaction was performed with
CuSO4/ascorbate system in a mixture of THF and water at 60ºC for a few days
furnishing excellent yields.
CONCLUSION
In the last few years since CuAAC reaction was born, it has dramatically
influenced many research fields in science as inferred from the emerging
number of contributions; but development of this reaction is yet far to be
completed. Regarding the main topic of this chapter, many approaches have
been addressed in the search for an efficient way to link a diverse variety of
building blocks containing polyaromatic species. All procedures were aimed to
overcome some difficulties found in this kind of compounds, such as low
solubilities or high reactivity of the π electronic density, for instance.
The most popular protocol covered here is the use of copper sulfate along
with a reductor, but this system has had a huge number of variations in order
to obtain reasonable yields, being oftenly the selection of a solvents mixture or
the addition of a nitrogen-donor ligand for stabilizing copper catalyst.
A different proposal has consisted on the way polyaromatic structures are
functionalized. The place where the terminal azide (or alkyne) is anchored
prior to the click reaction is crucial in terms of stability, solubility or ease of
handling. In addition, the proximity of those groups to the polycyclic aromatic

scaffold is another decisive fact, giving rise to different behaviors.
Finally, we must say that the versatility of Cu(I)-catalyzed Huisgen
cycloaddition has been demonstrated in this chapter and seems endless;
however, as a perspective, there is an important problem to solve. Nowadays,
catalyst elimination is considered a secondary step during purification.
Reaction conditions should be improved in such a way that copper removal
becomes completely effective. This issue must be addressed in future
approaches.
REFERENCES
[1] Jurícek, M; Kouwer, PHJ; Rowan, AE. Chem. Commun., 2011, 47,
8740-8749.
[2] a) Lutz, JF. Angew. Chem. Int. Ed., 2007, 46, 1018-1025. b) Moses, JE;
Moorhouse, AD. Chem. Soc. Rev., 2007, 36, 1249−1262. c) Meldal, M;
Tornoe, CW. Chem. Rev., 2008, 108, 2952−3015. d) Hein, JE; Fokin,
VV. Chem. Soc. Rev., 2010, 39, 1302-1315. e) Liang, L; Astruc, D.
Coord. Chem. Rev., 2011, 255, 2933−2945. f) Haldón, E; Nicasio, MC;
Pérez, PJ. Org. Biomol. Chem., 2015, 13, 9528-9550. g) Tiwari, VK;
Mishra, BB; Mishra, KB; Mishra, N; Singh, AS; Chen, X. Chem. Rev.,
2016, 116, 3086-3240.
[3] Kolb, HC; Finn. MG; Sharpless, KB. Angew. Chem. Int. Ed., 2001, 40,
2004-2021.
[4] Huisgen, R. Angew. Chem. Int. Ed., 1963, 2, 565−598.
[5] Singh, MS; Chowdhury, S; Koley, S. Tetrahedron, 2016, 72, 1603-1644.
[6] a) Kolb, HC; Sharpless, KB. Drug Discov. Today, 2003, 8, 1128-1137.
b) Tron, GC; Pirali, T; Billington, RA; Canonico, PL; Sorba, G;
Genazzani, AA. Med. Res. Rev., 2008, 28, 278-308. c) Thirumurugan, P;
Matosiuk, D; Jozwiak, K. Chem. Rev., 2013, 113, 4905-4979.
[7] a) Fazio, F; Bryan, MC; Blixt, O; Paulson, JC; Wong, CH. J. Am. Chem.
Soc., 2002, 124, 14397-14402. b) Guo, Z; Lei, A; Zhang, Y; Xu, Q; Xue,
X; Zhanga, F; Liang, X. Chem. Commun., 2007, 2491-2493.
[8] a) Lutz, JF; Zarafshani, Z. Adv. Drug Delivery Rev., 2008, 60, 958-970.
b) Sagheer, AHE; Brown, T. Chem. Soc. Rev., 2010, 39, 1388-1405. c)
Mamidyala, SK; Finn, MG. Chem. Soc. Rev., 2010, 39, 1252-1261. d)
Glassford, I; Teijaro, CN; Daher, SS; Weil, A; Small, MC; Redhu, SK;
Colussi, DJ; Jacobson, MA; Childers, WE; Buttaro, B; Nicholson, AW;
MacKerell, Jr. AD; Cooperman, BS; Andrade, RB. J. Am. Chem. Soc.,
2016, 138, 3136-3144.
[9] a) Fournier, D; Hoogenboom, R; Schubert, US. Chem. Soc. Rev., 2007,
36, 1369-1380. b) Kempe, K; Krieg, A; Becer, CR; Schubert, US. Chem.
Soc. Rev., 2012, 41, 176-191.
[10] a) Hanni, KD; Leigh, DA. Chem. Soc. Rev., 2010, 39, 1240-1251. b) Xi,
W; Scott, TF; Kloxin, CJ; Bowman, CN. Adv. Funct. Mater., 2014, 24,
2572-2590.
[11] Rostovtsev, VV; Freen, LG; Fokin, VV; Sharpless, KB. Angew. Chem.
Int. Ed., 2002, 41, 2596-2599.
[12] Tornoe, CW; Christensen, C; Meldal, M. J. Org. Chem., 2002, 67, 3057-
3064.
[13] Bock, VD; Hiemstra, H; van Maarseveen, JH. Eur. J. Org. Chem., 2006,
51-68.
[14] Rodionov, VO; Fokin, VV; Finn, MG. Angew. Chem. Int. Ed., 2005, 44,
2210-2215.
[15] Chinchilla, R; Nájera, C. Chem. Soc. Rev., 2011, 40, 5084-5121.
[16] Bräse, S; Gil, C; Knepper, K; Zimmermann, V. Angew. Chem. Int. Ed.,
2005, 44, 5188-5240.
[17] Kappe, CO. Angew. Chem. Int. Ed., 2004, 43, 6250-6284.
[18] a) Appukkuttan, P; Dehaen, W; Fokin, VV; Van der Eycken, E. Org.
Lett., 2004, 6, 4223−4225. b) Cintas, P; Martina, K; Robaldo, B;
Garella, D; Boffa, L; Cravotto, G. Collect. Czech. Chem. Commun.
2007, 72, 1014−1024. c) Kappe, CO; Van der Eycken, E. Chem. Soc.
Rev., 2010, 39, 1280-1290.
[19] a) McNulty, J; Keskar, K. Eur. J. Org. Chem., 2012, 2012, 5462−5470.
b) Wang, C; Ikhlef, D; Kahlal, S; Saillard, JY; Astruc, D. Coord. Chem.
Rev., doi:10.1016/j.ccr.2016.02.010.
[20] Georgakilas, V; Perman, JA; Tucek, J; Zboril, R. Chem. Rev., 2015, 115,
4744-4822.
[21] Li, Z; Liu, Z; Sun, H; Gao, C. Chem. Rev., 2015, 115, 7046-7117.
[22] a) Kroto, HW; Heath, JR; O’Brien, SC; Curl, RF; Smalley, RE. Nature,
1985, 318, 162-163. b) Kratschmer, W; Lamb, LD; Fostiropoulos, K;
Huffman, DR. Ibid. Nature, 1990, 347, 354-358. c) Martín, N;
Nierengarten, JF. Supramolecular Chemistry of Fullerenes and Carbon
Nanotubes, 2012, Wiley-VCH Verlag GmbH & Co. KGaA.
[23] Zhang, X; Hsu, CH; Ren, X; Gu, Y; Song, B; Sun, HJ; Yang, S; Chen,
E; Tu, Y; Li, X; Yang, X; Li, Y; Zhu, X. Angew. Chem. Int. Ed., 2015,
54, 114-117.
[24] Jerome, D. Science, 1991, 252, 1509-1514.
[25] Lai, YY; Cheng, YJ; Hsu, CS. Energy Environ. Sci., 2014, 7, 1866-
1883.
[26] Gharbi, N; Pressac, M; Hadchouel, M; Szwarc, H; Wilson, SR; Moussa,
F. Nano Lett., 2005, 5, 2578-2585.
[27] Afreen, S; Muthoosamy, K; Manickam, S; Hashim, U. Biosens.
Bioelectron., 2015, 63, 354-364.
[28] a) Balasubramanian, K; Burghard, M. Small, 2005, 2, 180-192. b) Tasis,
D; Tagmatarchis, N; Bianco, A; Prato, M. Chem. Rev., 2006, 106, 1105-
1136.
[29] Iijima, S. Nature, 1991, 354, 56-58.
[30] Iijima, S; Ichihashi, T. Nature, 1993, 363, 603-605.
[31] Coleman, JN; Khan, U; Blau, WJ; Gun’ko, YK. Carbon, 2006, 44,
1624-1652.
[32] Avouris, P; Chen, Z; Perebeinos, V. Nat. Nanotechnol., 2007, 2, 605-
615.
[33] Che, GL; Lakshmi, BB; Fisher, ER; Martin, CR. Nature, 1998, 393,
346-349.
[34] Allen, BL; Kichambare, PD; Star, A. Adv. Mater., 2007, 19, 1439-1451.
[35] Bianco, A; Kostarelos, K; Partidos, CD; Prato, M. Chem. Commun.,
2005, 571-577.
[36] Katz, E; Willner, I. ChemPhysChem, 2004, 5, 1084-1104.
[37] a) Novoselov, KS; Geim, AK; Morozov, SV; Jiang, D; Zhang, Y;
Dubonos, SV; Grigorieva, IV; Firsov, AA. Science, 2004, 306, 666-669.
b) Huang, X; Yin, Z; Wu, S; Qi, X; He, Q; Zhang, Q; Yan, Q; Boey, F;
Zhang, H. Small, 2011, 7, 1876-1902. c) Choi, W; Lee, Jw. Graphene:
Synthesis and Applications, 2011, CRC Press, Taylor & Francis Group.
[38] Liu, Z; Lau, SP; Yan, F. Chem. Soc. Rev., 2015, 44, 5638-5679.
[39] Zheng, Q; Li, Z; Yang, J; Kim, JK. Prog. Mater. Sci., 2014, 64, 200-
247.
[40] Sun, ZH; Chang, HX. ACS Nano, 2014, 8, 4133-4156.
[41] a) Wang, C; Li, D; Too, CO; Wallace, GG. Chem. Mater., 2009, 21,
2604-2606. b) Zhao, X; Hayner, CM; Kung, MC; Kung, HH. ACS Nano,
2011, 5, 8739-8749.
[42] Huang, T; Zheng, B; Kou, L; Gopalsamy, K; Xu, Z; Gao, C; Meng, Y;
Wei, Z. RSC Adv., 2013, 3, 23957-23962.
[43] Zhang, LM; Xia, JG; Zhao, QH; Liu, LW; Zhang, ZJ. Small, 2010, 6,
537-544.
[44] Lu, CH; Yang, HH; Zhu, CL; Chen, X; Chen, GN. Angew. Chem. Int.
Ed., 2009, 48, 4785-4787.
[45] a) Nierengarten, JF. Pure Appl. Chem., 2012, 84, 1027-1037. b)
Nierengarten, I; Nierengarten, JF. Chem. Rec., 2015, 15, 31-51.
[46] Isobe, H; Cho, K; Solin, N; Werz, DB; Seeberger, PH; Nakamura, E.
Org. Lett., 2007, 9, 4611-4614.
[47] Zhang, WB; Tu, Y; Ranjan, R; Van Horn, RM; Leng, S; Wang, J; Polce,
MJ; Wesdemiotis, C; Quirk, RP; Newkome, GR; Cheng, SZD.
Macromolecules, 2008, 41, 515-517.
[48] Iehl, J; de Freitas, RP; Nierengarten, JF. Tetrahedron Lett., 2008, 49,
4063-4066.
[49] Bingel, C. Chem. Ber., 1993, 126, 1957–1959.
[50] a) Pereira de Freitas, R; Iehl, J; Delavaux-Nicot, B; Nierengarten, JF.
Tetrahedron, 2008, 64, 11409-11419. b) Iehl, J; Osinska, I; Louis, R;
Holler, M; Nierengarten, JF. Tetrahedron Lett., 2009, 50, 2245-2248.
[51] Iehl, J; Vartanian, M; Holler, M; Nierengarten, JF; Delavaux-Nicot, B;
Strub, JM; Van Dorsselaer, A; Wu, Y; Mohanraj, J; Yoosaf, K;
Armaroli, N. J. Mat. Chem., 2011, 21, 1562-1573.
[52] Mahmud, IM; Zhou, N; Wang, L; Zhao, Y. Tetrahedron, 2008, 64,
11420-11432.
[53] Iehl, J; Pereira de Freitas, R; Delavaux-Nicot, B; Nierengarten, JF.
Chem. Commun., 2008, 2450-2452.
[54] Yoosaf, K; Iehl, J; Nierengarten, I; Hmadeh, M; Albrecht-Gary, AM;
Nierengarten, JF; Armaroli, N. Chem. Eur. J., 2014, 20, 223-231.
[55] a) Iehl, J; Nierengarten, JF. Chem. Eur. J., 2009, 15, 7306-7309. b)
Guerra, S; Iehl, J; Holler, M; Peterca, M; Wilson, DA; Partridge, BE;
Zhang, S; Deschenaux, R; Nierengarten, JF; Percec, V. Chem. Sci.,
2015, 6, 3393-3401.
[56] a) Pereira, GR; Santos, LJ; Luduvico, I; Alves, RB; de Freitas, RP.
Tetrahedron Lett., 2010, 51, 1022-1025. b) Nierengarten, JF; Iehl, J;
Oerthel, V; Holler, M; Illescas, BM; Munoz, A; Martin, N; Rojo, J;
Sanchez-Navarro, M; Cecioni, S; Vidal, S; Buffet, K; Durka, M;
Vincent, SP. Chem. Commun., 2010, 46, 3860-3862.
[57] de Miguel, G; Wielopolski, M; Schuster, DI; Fazio, MA; Lee, OP;
Haley, CK; Ortiz, AL; Echegoyen, L; Clark, T; Guldi, DM. J. Am.
Chem. Soc., 2011, 133, 13036-13054.
[58] Prato, M; Maggini, M. Acc. Chem. Res., 1998, 31, 519-526.
[59] Ho, KHL; Hijazi, I; Rivier, L; Gautier, C; Jousselme, B; de Miguel, G;
Romero-Nieto, C; Guldi, DM; Heinrich, B; Donnio, B; Campidelli, S.
Chem. Eur. J., 2013, 19, 11374-11381.
[60] Chen, L; Furukawa, K; Gao, J; Nagai, A; Nakamura, T; Dong, Y; Jiang,
D. J. Am. Chem. Soc., 2014, 136, 9806-9809.
[61] Iehl, J; Nierengarten, JF; Harriman, A; Bura, T; Ziessel, R. J. Am. Chem.
Soc., 2012, 134, 988-998.
[62] Pla, S; Martín-Gomis, L; Ohkubo, K; Fukuzumi, S; Fernández-Lázaro,
F; Sastre-Santos, Á. Asian J. Org. Chem., 2014, 3, 185-197.
[63] a) Compain, P; Decroocq, C; Iehl, J; Holler, M; Hazelard, D; Mena
Barragán, T; Ortiz Mellet, C; Nierengarten, JF. Angew. Chem. Int. Ed.,
2010, 49, 5753-5756. b) Sánchez-Navarro, M; Muñoz, A; Illescas, BM;
Rojo, J; Martín, N. Chem. Eur. J., 2011, 17, 766-769. c) Durka, M;
Buffet, K; Iehl, J; Holler, M; Nierengarten, JF; Taganna, J; Bouckaert, J;
Vincent, SP. Chem. Commun., 2011, 47, 1321-1323. d) Cecioni, S;
Oerthel, V; Iehl, J; Holler, M; Goyard, D; Praly, JP; Imberty, A;
Nierengarten, JF; Vidal, S. Chem. Eur. J., 2011, 17, 3252-3261. e)
Durka, M; Buffet, K; Iehl, J; Holler, M; Nierengarten, JF; Vincent, SP.
Chem. Eur. J., 2012, 18, 641-651. f) Rísquez-Cuadro, R; García
Fernández, JM; Nierengarten, JF; Ortiz Mellet, C. Chem. Eur. J., 2013,
19, 16791-16803.
[64] Luczkowiak, J; Muñoz, A; Sánchez-Navarro, M; Ribeiro-Viana, R;

Ginieis, A; Illescas, BM; Martín, N; Delgado, R; Rojo, J.
Biomacromolecules, 2013, 14, 431-437.
[65] Muñoz, A; Sigwalt, D; Illescas, BM; Luczkowiak, J; Rodríguez-Pérez,
L; Nierengarten, I; Holler, M; Remy, JS; Buffet, K; Vincent, SP; Rojo, J;
Delgado, R; Nierengarten, JF; Martín, N. Nat. Chem., 2016, 8, 50-57.
[66] a) Ugarte, D. Nature, 1992, 359, 707-709. b) Kroto, HW. Nature, 1992,
359, 670-671.
[67] a) Flavin, K; Chaur, MN; Echegoyen, L; Giordani, S. Org. Lett., 2010,
12, 840-843. b) Frasconi, M; Marotta, R; Markey, L; Flavin, K;
Spampinato, V; Ceccone, G; Echegoyen, L; Scanlan, EM; Giordani, S.
Chem. Eur. J., 2015, 21, 19071-19080. c) Bartelmess, J; Frasconi, M;
Balakrishnan, PB; Signorelli, A; Echegoyen, L; Pellegrino, T; Giordani,
S. RSC Adv., 2015, 5, 50253-50258.
[68] a) Han, J; Gao, C. Nano-Micro Letters, 2010, 2, 213-226. b) Rana, S;
Cho, JW. Nanoscale, 2010, 2, 2550-2556. c) Clave, G; Campidelli, S.
Chem. Sci., 2011, 2, 1887-1896. d) Clave, G; Campidelli, S. Chem. Sci.,
2011, 2, 1887-1896. e) Grennberg, H. Organic Synthesis and Molecular
Engineering, 2013, 76-127. f) Dirian, K; Herranz, MA; Katsukis, G;
Malig, J; Rodriguez-Perez, L; Romero-Nieto, C; Strauss, V; Martin, N;
Guldi, DM. Chem. Sci., 2013, 4, 4335-4353. g) Ménard-Moyon, C.
Chemistry of Organo-Hybrids, 2014, 1-35.
[69] Li, H; Cheng, F; Duft, AM; Adronov, A. J. Am. Chem. Soc., 2005, 127,
14518-14524.
[70] Dyke, CA; Tour, JM. J. Am. Chem. Soc., 2003, 125, 1156-1157.
[71] Liu, J; Nie, Z; Gao, Y; Adronov, A; Li, H. J.Polym. Sci. Part A: Polym.
Chem., 2008, 46, 7187-7199.
[72] Zhang, Y; He, H; Gao, C. Macromolecules, 2008, 41, 9581-9594.
[73] He, H; Zhang, Y; Gao, C; Wu, J. Chem. Commun., 2009, 1655-1657.
[74] Zhang, Y; He, H; Gao, C; Wu, J. Langmuir, 2009, 25, 5814-5824.
[75] Mahouche Chergui, S; Ledebt, A; Mammeri, F; Herbst, F; Carbonnier,
B; Ben Romdhane, H; Delamar, M; Chehimi, MM. Langmuir, 2010, 26,
16115-16121.
[76] Rana, S; Yoo, HJ; Cho, JW; Chun, BC; Park, JS. J. Appl. Polym. Sci.,
2011, 119, 31-37.
[77] Song, Y; Qu, K; Xu, C; Ren, J; Qu, X. Chem. Commun., 2010, 46, 6572-
6574.
[78] Guo, Z; Liang, L; Liang, JJ; Ma, YF; Yang, XY; Ren, DM; Chen, YS;
Zheng, JY. J. Nanopart. Res., 2007, 10, 1077-1083.
[79] Zhang, W; Sprafke, JK; Ma, M; Tsui, EY; Sydlik, SA; Rutledge, GC;
Swager, TM. J. Am. Chem. Soc., 2009, 131, 8446-8454.
[80] Landis, EC; Hamers, RJ. Chem. Mater., 2009, 21, 724-730.
[81] Devadoss, A; Chidsey, CED. J. Am. Chem. Soc., 2007, 129, 5370-5371.
[82] Jing, L; Liang, C; Shi, X; Ye, S; Xian, Y. Analyst, 2012, 137, 1718-
1722.
[83] Zhao, L; Zeng, B; Zhao, F. Electrochimica Acta, 2014, 146, 611-617.
[84] Tuci, G; Vinattieri, C; Luconi, L; Ceppatelli, M; Cicchi, S; Brandi, A;
Filippi, J; Melucci, M; Giambastiani, G. Chem. Eur. J., 2012, 18, 8454-
8463.
[85] Campidelli, S; Ballesteros, B; Filoramo, A; Díaz, DD; de la Torre, G;
Torres, T; Rahman, GMA; Ehli, C; Kiessling, D; Werner, F; Sgobba, V;
Guldi, DM; Cioffi, C; Prato, M; Bourgoin, JP. J. Am. Chem. Soc., 2008,
130, 11503-11509.
[86] Palacin, T; Khanh, HL; Jousselme, B; Jegou, P; Filoramo, A; Ehli, C;
Guldi, DM; Campidelli, S. J. Am. Chem. Soc., 2009, 131, 15394-15402.
[87] Le Ho, KH; Rivier, L; Jousselme, B; Jegou, P; Filoramo, A; Campidelli,
S. Chem. Commun., 2010, 46, 8731-8733.
[88] Hijazi, I; Jousselme, B; Jegou, P; Filoramo, A; Campidelli, S. J. Mat.
Chem., 2012, 22, 20936-20942.
[89] a) Fedeli, S; Paoli, P; Brandi, A; Venturini, L; Giambastiani, G; Tuci, G;
Cicchi, S. Chem. Eur. J., 2015, 21, 15349-15353. b) Tuci, G; Luconi, L;
Rossin, A; Baldini, F; Cicchi, S; Tombelli, S; Trono, C; Giannetti, A;
Manet, I; Fedeli, S; Brandi, A; Giambastiani, G. ChemPlusChem, 2015,
80, 704-714.
[90] Mateos-Gil, J; Rodriguez-Perez, L; Moreno Oliva, M; Katsukis, G;
Romero-Nieto, C; Herranz, MA; Guldi, DM; Martin, N. Nanoscale,
2015, 7, 1193-1200.
[91] Barrejon, M; Pla, S; Berlanga, I; Gomez-Escalonilla, MJ; Martin-Gomis,
L; Fierro, JLG; Zhang, M; Yudasaka, M; Iijima, S; Gobeze, HB;
D’Souza, F; Sastre-Santos, A; Langa, F. J. Mater. Chem. C, 2015, 3,
4960-4969.
[92] a) Georgakilas, V. Covalent Attachment of Organic Functional Groups

on Pristine Graphene. Functionalization of Graphene, 2014, 21-58,
Wiley-VCH Verlag GmbH & Co. KGaA. b) Paulus, GLC; Wang, QH;
Strano, MS. Acc. Chem. Res., 2013, 46, 160-170. c) Umeyama, T;
Imahori, H. J. Phys. Chem. C, 2013, 117, 3195-3209.
[93] a) Choi, W; Lahiri, I; Seelaboyina, R; Kang, YS. Crit. Rev. Solid State,
2010, 35, 52-71. b) Rao, CNR; Sood, AK. Graphene: Synthesis,
Properties, and Phenomena, 2012, Wiley-VCH Verlag GmbH & Co.
KGaA. c) Chang, H; Wu, H. Energy Environ. Sci., 2013, 6, 3483-3507.
d) Rodriguez-Perez, L; Herranz, MaA; Martin, N. Chem. Commun.,
2013, 49, 3721-3735.
[94] a) He, HY; Riedl, T; Lerf, A; Klinowski, J. J. Phys. Chem., 1996, 100,
19954 - 19958. b) Lerf, A; He, HY; Riedl, T; Forster, M; Klinowski, J.
Solid State Ionics, 1997, 101, 857 - 862. c) Lerf, A; He, HY; Forster, M;
Klinowski, J. J. Phys. Chem. B, 1998, 102, 4477 - 4482. d) He, HY;
Klinowski, J; Forster, M; Lerf, A. Chem. Phys. Lett., 1998, 287, 53 - 56.
e) Cai, WW; Piner, RD; Stadermann, FJ; Park, S; Shaibat, MA; Ishii, Y;
Yang, DX; Velamakanni, A; An, SJ; Stoller, M; An, JH; Chen, DM;
Ruoff, RS. Science, 2008, 321, 1815 - 1817.
[95] Sun, S; Cao, Y; Feng, J; Wu, P. J. Mater. Chem., 2010, 20, 5605-5607.
[96] a) Cao, Y; Lai, Z; Feng, J; Wu, P. J. Mater. Chem., 2011, 21, 9271-
9278. b) Pan, Y; Bao, H; Sahoo, NG; Wu, T; Li, L. Adv. Funct. Mat.,
2011, 21, 2754-2763. c) Yang, H; Kwon, Y; Kwon, T; Lee, H; Kim, BJ.
Small, 2012, 8, 3161-3168. d) Yadav, SK; Yoo, HJ; Cho, JW. J. Polym.
Sci. Part B: Polym. Phys., 2013, 51, 39-47. e) Zhang, W; Shi, X; Zhang,
Y; Gu, W; Li, B; Xian, Y. J. Mater. Chem. A, 2013, 1, 1745-1753.
[97] Wang, Z; Ge, Z; Zheng, X; Chen, N; Peng, C; Fan, C; Huang, Q.
Nanoscale, 2012, 4, 394-399.
[98] Namvari, M; Namazi, H. Carbohyd. Res., 2014, 396, 1-8.
[99] Deetuam, C; Samthong, C; Thongyai, S; Praserthdam, P;
Somwangthanaroj, A. Compos. Sci. Technol., 2014, 93, 1-8.
[100] Mei, KC; Rubio, N; Costa, PM; Kafa, H; Abbate, V; Festy, F; Bansal,
SS; Hider, RC; Al-Jamal, KT. Chem. Commun., 2015, 51, 14981-14984.
[101] Salvio, R; Krabbenborg, S; Naber, WJM; Velders, AH; Reinhoudt, DN;
van der Wiel, WG. Chem. Eur. J., 2009, 15, 8235-8240.
[102] Huang, W; Wang, S; Guo, C; Yang, X; Li, Y; Tu, Y. Polymer, 2014, 55,
4619-4626.
[103] Jin, Z; McNicholas, TP; Shih, CJ; Wang, QH; Paulus, GL. C; Hilmer,
AJ; Shimizu, S; Strano, MS. Chem. Mater. 2011, 23, 3362-3370.
[104] Ye, YS; Chen, YN; Wang, JS; Rick, J; Huang, YJ; Chang, FC; Hwang,
BJ. Chem. Mater., 2012, 24, 2987-2997.
[105] Yang, X; Ma, L; Wang, S; Li, Y; Tu, Y; Zhu, X. Polymer, 2011, 52,
3046-3052.
[106] a) Campos, JM; Ferraria, AM; Botelho do Rego, AM; Ribeiro, MR;
Barros-Timmons, A. Mater. Chem. Phys., 2015, 166, 122-132. b)
Shaygan Nia, A; Rana, S; Döhler, D; Jirsa, F; Meister, A.; Guadagno, L;
Koslowski, E; Bron, M; Binder, WH. Chem. Eur. J., 2015, 21, 10763-
10770.
[107] Wang, HX; Zhou, KG; Xie, YL; Zeng, J; Chai, NN; Li, J; Zhang, HL.
Chem. Commun., 2011, 47, 5747-5749.
[108] Castelaín, M; Martínez, G; Merino, P; Martín-Gago, JÁ; Segura, JL;
Ellis, G; Salavagione, HJ. Chem. Eur. J., 2012, 18, 4965-4973.
[109] Barrejon, M; Vizuete, M; Gomez-Escalonilla, MJ; Fierro, JLG;
Berlanga, I; Zamora, F; Abellan, G; Atienzar, P; Nierengarten, JF;
Garcia, H; Langa, F. Chem. Commun., 2014, 50, 9053-9055.
[110] a) Meng, D; Sun, J; Jiang, S; Zeng, Y; Li, Y; Yan, S; Geng, J; Huang, Y.
J. Mater. Chem. 2012, 22, 21583-21591. b) Yenchalwar, SG; Reddy
Devarapalli, R; Deshmukh, AB; Shelke, MV. Chem. Eur. J., 2014, 20,
7402-7409.
[111] a) Namvari, M; Namazi, H. J. Mater. Sci., 2015, 50, 5348-5361. b)
Zhang, ZB; Wu, JJ; Su, Y; Zhou, J; Gao, Y; Yu, HY; Gu, JS. Appl. Surf.
Sci., 2015, 332, 300-307.
[112] Moss, GP. Pure App. Chem., 1998, 70, 143-216.
[113] Birks, JB. Photophysics of Aromatic Molecules, 1970, Wiley-
Interscience, London. b) Winnik, FM. Chem. Rev., 1993, 93, 587-614. c)
Lakowicz, JR. Principles of Fluorescence Specroscopy, 2006 Springer,
New York.
[114] a) Lau, YH; Rutledge, PJ; Watkinson, M; Todd, MH. Chem. Soc. Rev.,
2011, 40, 2848-2866. b) Manandhar, E; Wallace, KJ. Inorg. Chim. Acta,
2012, 381, 15-43.
[115] a) Wu, YT; Siegel, JS. Chem. Rev., 2006, 106, 4843-4867. b) Wu, YT;
Siegel, JS. Top. Curr. Chem., 2014, 349, 63-120.
[116] a) Sygula, A; Fronczek, FR; Sygula, R; Rabideau, PW; Olmstead, M. M.
J. Am. Chem. Soc., 2007, 129, 3842-3843. b) Yanney, M; Sygula, A.
Tetrahedron Lett., 2013, 54, 2604-2607. c) Sygula, A; Yanney, M;
Henry, WP; Fronczek, FR; Zabula, AV; Petrukhina, MA. Cryst. Growth
Des., 2014, 14, 2633-2639. d) Álvarez, CM; García-Escudero, LA;
García-Rodriguez, R; Martín-Álvarez, JM; Miguel, D; Rayón, VM.
Dalton Trans., 2014, 43, 15693-15696. e) Yanney, M; Fronczek, FR;
Sygula, A. Angew. Chem. Int. Ed., 2015, 54, 11153-11156. f) Abeyratne
Kuragama, PL; Fronczek, FR; Sygula, A. Org. Lett., 2015, 17, 5292-
5295.
[117] Zhu, L; Lynch, VM; Anslyn, EV. Tetrahedron, 2004, 60, 7267-7275.
[118] Park, SY; Yoon, JH; Hong, CS; Souane, R; Kim, JS; Matthews, SE;
Vicens, J. J. Org. Chem., 2008, 73, 8212-8218.
[119] Zhu, LN; Gong, SL; Gong, SL; Yang, CL; Qin, JG. Chin. J. Chem.,
2008, 26, 1424-1430.
[120] Kim, JS; Park, SY; Kim, SH; Thuéry, P; Souane, R; Matthews, SE;
Vicens, J. Bull. Korean Chem. Soc., 2010, 31, 624-628.
[121] a) Ni, XL; Wang, S; Zeng, X; Tao, Z; Yamato, T. Org. Lett., 2011, 13,
552-555. b) Ni, Xl; Zeng, X; Redshaw, C; Yamato, T. J. Org. Chem.,
2011, 76, 5696-5702.
[122] Wang, NJ; Sun, CM; Chung, WS. Sensors Actuat. B-Chem., 2012, 171–
172, 984-993.
[123] a) Hung, HC; Chang, YY; Luo, L; Hung, CH; Diau, EWG; Chung, WS.
Photochem. Photobiol. Sci., 2014, 13, 370-379. b) Tomiyasu, H; Shigyo,
N; Ni, XL; Zeng, X; Redshaw, C; Yamato, T. Tetrahedron, 2014, 70,
7893-7899.
[124] Sun, Y; Mao, X; Luo, L; Tian, D; Li, H. Org. Biomol. Chem., 2015, 13,
9294-9299.
[125] a) Hung, HC; Cheng, CW; Ho, IT; Chung, WS. Tetrahedron Lett., 2009,
50, 302-305. b) Hung, HC; Cheng, CW; Wang, YY; Chen, YJ; Chung,
WS. Eur. J. Org. Chem., 2009, 2009, 6360-6366.
[126] Wang, HF; Wu, SP. Tetrahedron, 2013, 69, 1965-1969.
[127] Liu, X; Yang, X; Fu, Y; Zhu, C; Cheng, Y. Tetrahedron, 2011, 67,
3181-3186.
[128] Ingale, SA; Seela, F. J. Org. Chem., 2012, 77, 9352-9356.

[129] a) Mulla, K; Shaik, H; Thompson, DW; Zhao, Y. Org. Lett. 2013, 15,
4532-4535. b) Gonzalez, MdC; Oton, F; Espinosa, A; Tarraga, A;
Molina, P. Org. Biomol. Chem., 2015, 13, 1429-1438.
[130] a) Gacal, BN; Koz, B; Gacal, B; Kiskan, B; Erdogan, M; Yagci, Y. J.
Polym. Sci., Part A: Polym. Chem., 2009, 47, 1317-1326. b) Odaci, D;
Gacal, BN; Gacal, B; Timur, S; Yagci, Y. Biomacromolecules, 2009, 10,
2928-2934. c) Karagoz, B; Durmaz, YY; Gacal, BN; Bicak, N; Yagci,
Y. Des. Monomers Polym., 2009, 12, 511-522.
[131] Toiserkani, H; Yilmaz, G; Yagci, Y; Torun, L. Macromol. Chem. Phys.,
2010, 211, 2389-2395.
[132] Fu, Q; McKenzie, TG; Ren, JM; Tan, S; Nam, E; Qiao, GG. Sci. Rep.,
2016, 6, 20779.
[133] Romero, T; Caballero, A; Tárraga, A; Molina, P. Org. Lett., 2009, 11,
3466-3469.
[134] Otón, F; González, MdC; Espinosa, A; Ramírez de Arellano, C; Tárraga,
A; Molina, P. J. Org. Chem., 2012, 77, 10083-10092.
[135] Madec, L; Bouvree, A; Blanchard, P; Cougnon, C; Brousse, T; Lestriez,
B; Guyomard, D; Gaubicher, J. Energy Environ. Sci., 2012, 5, 5379-
5386.
[136] Mandal, D; Deb, P; Mondal, B; Thakur, A; Ponniah, SJ; Ghosh, S. RSC
Adv., 2013, 3, 18614-18625.
[137] a) Romero, T; Orenes, RA; Tárraga, A; Molina, P. Organometallics,
2013, 32, 5740-5753. b) González, MadC; Otón, F; Orenes, RA;
Espinosa, A; Tárraga, A; Molina, P. Organometallics, 2014, 33, 2837-
2852.
[138] Fujimoto, K; Yamada, S; Inouye, M. Chem. Commun., 2009, 7164-
7166.
[139] Kumar, P; Shaikh, KI; Jørgensen, AS; Kumar, S; Nielsen, P. J. Org.
Chem., 2012, 77, 9562-9573.
[140] Ingale, SA; Seela, F. J. Org. Chem., 2013, 78, 3394-3399.
[141] Ingale, SA; Leonard, P; Yang, H; Seela, F. Org. Biomol. Chem., 2014,
12, 8519-8532.
[142] a) Manandhar, E; Broome, JH; Myrick, J; Lagrone, W; Cragg, PJ;

Wallace, KJ. Chem. Commun., 2011, 47, 8796-8798. b) Chakrabarty, R;
Stang, PJ. J. Am. Chem. Soc., 2012, 134, 14738-14741. c) Yang, Y; Yao,
Z; Tang, B; Yu, J; Bi, X; Zhao, Y; Wu, HC. Anal. Methods, 2014, 6,
4977-4981. d) Vega, B; Wondraczek, H; Bretschneider, L; Näreoja, T;
Fardim, P; Heinze, T. Carbohydr. Polym., 2015, 132, 261-273.
[143] Ustinov, AV; Dubnyakova, VV; Korshun, VA. Nucleos. Nucleot. Nucl.,
2007, 26, 751-754.
[144] Ustinov, AV; Dubnyakova, VV; Korshun, VA. Tetrahedron, 2008, 64,
1467-1473.
[145] Langhals, H; Obermeier, A. Eur. J. Org. Chem., 2008, 2008, 6144-6151.
[146] Qvortrup, K; Petersen, MÅ; Hassenkam, T; Nielsen, MB. Tetrahedron
Lett., 2009, 50, 5613-5616.
[147] Albert-Seifried, S; Finlayson, CE; Laquai, F; Friend, RH; Swager, TM;
Kouwer, PHJ; Juríček, M; Kitto, HJ; Valster, S; Nolte, RJM; Rowan,
AE. Chem. Eur. J., 2010, 16, 10021-10029.
[148] a) Lang, AS; Neubig, A; Sommer, M; Thelakkat, M. Macromolecules
2010, 43, 7001-7010. b) Lang, AS; Thelakkat, M. Polym. Chem., 2011,
2, 2213-2221.
[149] Ustinov, A; Weissman, H; Shirman, E; Pinkas, I; Zuo, X; Rybtchinski,
B. J. Am. Chem. Soc., 2011, 133, 16201-16211.
[150] Yang, SK; Shi, X; Park, S; Doganay, S; Ha, T; Zimmerman, SC. J. Am.
Chem. Soc., 2011, 133, 9964-9967.
[151] Zill, AT; Licha, K; Haag, R; Zimmerman, SC. New J. Chem., 2012, 36,
419-427.
[152] Jorgensen, AS; Gupta, P; Wengel, J; Astakhova, IK. Chem. Commun.,
2013, 49, 10751-10753. b) Okholm, A; Kjems, J; Astakhova, K. RSC
Adv., 2014, 4, 45653-45656.
[153] Astakhova, IK; Santhosh Kumar, T; Campbell, MA; Ustinov, AV;
Korshun, VA; Wengel, J. Chem. Commun., 2013, 49, 511-513.
[154] Liu, F; Mu, J; Wu, X; Bhattacharjya, S; Yeow, EKL; Xing, B. Chem.
Commun., 2014, 50, 6200-6203.
[155] Aydin, E; Nisanci, B; Acar, M; Dastan, A; Bozdemir, OA. New J.
Chem., 2015, 39, 548-554.
[156] a) Wang, KR; An, HW; Wang, YQ; Zhang, JC; Li, XL. Org. Biomol.
Chem., 2013, 11, 1007-1012. b) Wang, KR; Han, D; Cao, GJ; Li, XL.
Chem. As. J., 2015, 10, 1204-1214. c) Wang, KR; Han, D; Cao, GJ; Li,
XL. RSC Adv., 2015, 5, 47728-47731.
[157] Chen, X; Lu, CW; Huang, Y; McGrath, DV. Tetrahedron, 2015, 71,
9154-9160.
[158] Stuparu, MC. Tetrahedron, 2012, 68, 3527-3531.
[159] Mattarella, M; Siegel, JS. Org. Biomol. Chem., 2012, 10, 5799-5802.
[160] a) Mattarella, M; Haberl, JM; Ruokolainen, J; Landau, EM; Mezzenga,
R; Siegel, JS. Chem. Commun., 2013, 49, 7204-7206. b) Mattarella, M;
Berstis, L; Baldridge, KK; Siegel, JS. Bioconj. Chem., 2014, 25, 115-
128.
[161] Niamnont, N; Kimpitak, N; Wongravee, K; Rashatasakhon, P;
Baldridge, KK; Siegel, JS; Sukwattanasinitt, M. Chem. Commun., 2013,
49, 780-782.
[162] Álvarez, CM; Aullón, G; Barbero, H; García-Escudero, LA; Martínez-
Pérez, C; Martín-Álvarez, JM; Miguel, D. Org. Lett., 2015, 17, 2578-
2581.
Chapter 10
APPLICATION OF CLICK CHEMISTRY

IN BIOMEDICAL FIELDS
Yu Chen*, Ying Zhang, Xiaoyu Sun and Jingjing Yuan

ABSTRACT
During the last ten years, click chemistry has become a powerful tool
for materials modification and application for their number of advantages
such as readily available starting material, mild reaction conditions, high
reliability, high reaction rate, high yield, good stereoselectivity, high
oxygen and moisture stability, simple workup and easy purification. A
large number of recent original publications and specialized reviews have
focused on applications of the click reaction technique in the biomedical
fields. In the current chapter, the progress on preparation via click
reaction and application of the multifunctional and intelligent materials
for chemical sensor in biomedical field, pharmaceutical science and gene
transfer carriers is summarized. The limitations and prospective
applications of the click chemistry technique in these fields are also
discussed.
*
Corresponding author Email: cylsy@163.com.
282 Yu Chen, Ying Zhang, Xiaoyu Sun et al.
INTRODUCTION
In 2001, Sharpless and co-workers coined the concept of “click chemistry”
[1] to classify a particular set of nearly perfect reactions. It is undeniably one
of the most important trends in contemporary chemistry [2]. Click chemistry
has attracted great attentions, because the click chemistry reaction can be
carried out under mild and simple reaction conditions [3, 4], it can be
proceeded well in aqueous medium and therefore may be efficiently performed
under physiological conditions [5-7], resulting in high reaction efficiency with
easy post-treatment for the obtained products [9-11]. Moreover, it holds great
chemoselectivity and can therefore be used for modifying highly functional
biomolecules such as poly-peptides, nucleic acids or polysaccharides [12, 13].
Since a large number of recent original publications and specialized
reviews have been published during the past years on applications of the click
reaction technique in the biomedical fields [14-16]. In the current chapter, the
progress on preparation via click reaction and application of the
multifunctional and intelligent materials for chemical sensor in biomedical
field, pharmaceutical science and gene transfer carriers is summarized. The
limitations and prospective applications of the click chemistry technique in
these fields are also discussed.
1. THE APPLICATION OF CLICK CHEMISTRY

IN BIOLOGICAL PROBE AND SENSOR’S DETECTION
Chemical sensor is a kind of chemical device which can be converted to

the target analysis signal device. Chemical sensors have a wide range, and the
principle of each have differences, the target parameter related to the target
detection is endless also and same. After years of rapid development of
chemical sensors, it has become an important part of the detection of the
medical science, foodstuff, environmental protection and so on.
1.1. The Role of Click Chemistry in the Research

of Chemical Sensor
Click chemistry as a rapid, simple, high selectivity and effective synthesis

method, it can give full play to its advantages in the process of preparing
Application of Click Chemistry in Biomedical Fields 283
sensors. Click chemistry can be used in the bonding by covalent to fix

sensitive membrane or tag active matter. In the study of chemical sensors, the
function of click chemistry mainly reflects in the following aspects, namely
fast connection fixed sensitive content or tag material, synthetic sensitive
element, and the use of click reaction directly or indirectly detect click
reactants.
Lau et al. [17] reported a way to use click chemistry to prepare
fluorophore sensors by using selective anion induction metal process to
identify the copper ion and mercury ion. They first synthesized a kind of
structure based on a new type of triazole ring pull amine pendant arm as
fluorescence sensors, sensor through the pendant arm structure material and
the copper ion and mercury ions in neutral aqueous solution to form
complexes and produce high sensitive response, and in about 50 times more
likely to interfere with the presence of metal ions can still achieve high
selective determination. When added anions such as I- Or S2O32-, fluorescence
intensity restores to original numerical. This method can simply and efficiently
identify Cu2+ or Hg2+ (Figure 1).
Su et al. [18] reported the way indirectly determine Cu2+ by using click
chemistry and business blood glucose meter. They first use sodium
ascorbicum to reduce Cu2+ to Cu+. Then, the restored Cu+ catalysis acetylene
group DNA which is fixed on the one-off srceen print carbon electrode, to
initiate click reaction with azide DNA attached to the yoke of the compound
enzyme/magnetic beads. This reaction let sucrose marked on the magnetic
beads transform into glucose. And the glucose meter was used to monitor
glucose levels so as to indirectly detect the Cu2+.
1.2. The Application of Chemical Sensor Based

on Click Chemistry
For most of the organic compound, because of their groups are more
easily functional, some of them even own groups that can click on the
reaction. So for the detection of this kind of material, we can use the reaction
of the click directly to the object under test, and effectively improve the
selectivity of sensors. The click reaction can also be used to achieve the
enhancement purpose to the amplified sensor signal. Therefore, this method
can significantly improve the sensitivity of the sensor [19, 20].
Figure 1. A click fluorophore sensor that can distinguish Cu2+ and Hg2+ via selective
anion - induced demetallation.
Lu et al. [21] prepared a kind of sensors based on click on the chemical

which used in the detection of propiolic fluorine grass amine. They make
through the catalysis of Cu+, propiolic fluorine grass amine on the object under
test alkynyl and weak fluorescence 3-azide group-7-hydroxyl coumarinazide
groups on the click reaction, generate a strong fluorescence components
(1,2,3-benzene triazole), which can be treated for high selectivity, high
sensitive detection measurement.
Zeng et al. [22] used thiol click chemistry to prepare electrochemical
luminescence (ECL) fixed sensors based on Ru (bpy)32+, and then used it to
SanZhengpropylamine detection. They first synthesized pentenyl
functionalization of Ru (bpy)3Cl2. Then they used thiol-click reaction to fix it
on thiol propyl silane pretreatment of top oxygen radicals of conductive glass.
The above fixed method is rapid, simple and efficient. The stability of the Ru
(bpy)32+ film in organic solvents is better than previously reported similar
membrane (as shown in Figure 2). The sensor is used for propylamine
detection. It was found that the response range is wide (5 x 10-6 ~ 5 x 10-3
mol/L), and the detection limit could reach 1 x 10-6 mol/L.
For ionic detection, there are also some reports in the field of
electrochemical sensor [23-26]. Ion under test can be generated form
complexes with the product of click on the reaction to produce electrical or
optical signal detection in order to achieve the purpose, thus improve the
selectivity and sensitivity of the sensor. And for the detection of some metal
ions, such as Cu2+, the methods of electrochemistry and electrochemical

luminescence can also be used directly or indirectly.
Zhang [27] reported a high sensitive method that using Cu+ as catalyst for
clicking reaction used for visual detection of Cu2+ in aqueous solution. They
used water soluble azide functionalization of AuNPs with alkynyl on glass
slide in the click reaction under the catalysis of copper ions. After reaction,
AuNPs are fixed on the slide. Due to the effect of increase of the Ag, the dark
degree of AuNPs was increased. The detection limit was lower than previously
reported methods for 2~3 orders of magnitude, up to 62 nmol/L.
Ge [28] also prepared fluorescent sensors for determination of Cu2+. By
+
Cu catalytic via azide and alkynyl modified, short rich G respectively
sequence click on the reaction in aqueous solution. Then, after elution steps,
the G-four couplet body structure can be formed, including Cu+ from sodium
ascorbate reduction of Cu2+. Chlorine in high iron red element and the
presence of K+, self-assembly of chlorine high iron red element/G - four
conjoined structure has a catalytic colorless tetramethylbenzidine the activity
of color. So the colorimetric analysis can be carried out on the concentration of
Cu2+.
Figure 2. Preparation of the electrochemical luminescence fixed sensors based on Ru

(bpy)32+ via thiol click chemistry.
Different from general organic matter detection, some biology have large
biological molecules. And some are even aggregated level. So the participate
of them in the click on the response of sensor is less reported. The report are
mostly about fixing enzymes or other active substances on the sensor by
clicking on the reaction to get detection signal which is being catalytic reaction
between enzyme and the object under test, so as to realize the detection of
object treatment test [29, 30].
Trilling [31] used click reaction to study the influence of biological
receptor of directional on the sensitivity of biosensor. For example, they
recognize FMDV viruses by VHH protein biosensor. Putting VHH protein
azide into functionalization firstly and click on the reaction with the exposed
plasma resonance chip ring sheen acetylene, by comparing VHH modified
biosensor which was many times azide functional marked or the single azide
functional marked. They found the former’s sensitivity raised 800 times than
the latter’s.
Li et al. [32] used a click reaction based on fixed glucose oxidase
biosensors for the determination of glucose. They first used azidebifunctional
molecules to get end azide carbon nanotubes (CNTs-N-3). By the way, alkynyl
modified glucose oxidase was synthesized at the same time. And then they
proceeded the click reaction under catalytic of Cu+, mixing with the help of
perfluorinated sulfonic acid soak to the surface of glassy carbon electrode, to
prepare the sensor. At the same time they studied its electrochemical
performance by cyclic voltammetry and chronoamperometry. Results showed
that the linear range of glucose was 6. 0 × 10-7 ~ 1. 4 × 10-3 mol/L, the
detection limit was 2. 0 × 10-7 mol/L.
According to the results in the relevant report, chemical sensors based on
the click chemistry have great stability and reproducibility [33, 34]. As a rapid,
simple, highly selective synthesis method, whether for quick connection fixed
sensitive content, tag material, synthesis of sensitive element, or directly or
indirectly detect click reactants, click chemistry can combine its advantages
with chemical sensor and application, greatly improve the response
performance of the sensor.
Applications of the products prepared from click chemistry in chemical
sensors is still in its initial stage, therefore, there are still some problems for
application of it in chemical sensors. Click chemistry requires specific groups,
it is preferred to the reactant functionalization. Some of the reactants to target
functionalization condition is too harsh, which limits the application range of
the click chemistry. Besides, currently the type of click reaction is less. The
reported type including azide and end group, the 1,3-azo addition reaction of
acetylene. In recent years, more mercapto-ene addition reaction has been

reported [35]. On the other hand, the researchers also need to apply more click
chemical groups selectively to prepare the sensor sensitive membrane, explore
more simple and efficient method for preparation of electrochemical sensor
based on click chemistry.
2. THE APPLICATION OF CLICK CHEMISTRY

IN GENE TRANSFER CARRIER
The completion of the work of the human genome project will enable
people to better understand the relationship between genes and diseases, so as
a new treatment gene therapy method has very broad application prospects.
However, the field of genetics has identified a number of genes having the
potential to treat and prevent disease, but the progress of gene therapy is very
slow. The main reason is the current lack of the appropriate gene carrier that
can deliver effectively gene drug safety to the location of the lesion and realize
nuclear expression, and gene delivery to the target cell and expression of the
corresponding protein has multiple barrier function. Therefore, the researchers
need to compress plasmid DNA into nano particles and overcome various
barriers of the carrier system, which cannot be directly used in the DNA.
Gene therapy holds a great promise for the treatment of diseases with a
genetic origin that are currently incurable. The success of gene therapy largely
depends on the availability of suitable delivery vehicles. Gene vectors include
viral vectors and non-viral vectors. Viral vectors have high gene delivery
efficiency. But the virus vector is not suitable for mass production; at the same
time, the DNA size that they can carry is limited; and the viral vectors may
cause the body’s immune response and other safety problems. So the
development of the virus vector has been restricted. More and more studies
have focused on the non-viral vectors, which including the cationic polymer
carrier, such as polyethylene imine (PEI), chitosan, poly lysine (PLL), and
methacrylic acid ethyl ester two methylamine (PDMAEMA) and its
derivatives [36]. PEI has become the most effective cationic polymer carrier
due to its strong DNA load capacity and high transfection efficiency, and it
can be a measure of cationic polymer vector of common reference [37, 38].
But the cytotoxicity of PEI is also an unavoidable problem [38]. Researchers
hope to design a new carrier that possesses high transfection efficiency and
low cytotoxicity. As the high selectivity and reaction efficiency of click
chemistry, it can be a valuable tool to synthesize these new polymers for

nonviral drug delivery systems.
Labeling DNA Oligonucleotides have recently been found many
applications and are considered to be the most important tools to many areas
of research [39]. They have been used for gene therapy [40], as antisense
agents to treat diseases like leukemia [41], as molecular probes [42], etc.
Adding different functional groups further increases their versatility,
especially when one considers that functionalization can be introduced at
either the 3’-end, 5’-end, or an internal position. The newly added functional
groups can serve as handles for bioconjugation with a wide variety of
biological molecules. However, current methods for DNA bioconjugation are
inefficient. The procedure must be able to tolerate aqueous conditions, give
high yields, and the resulting linkage must be stable in biological conditions
[43]. This is the perfect situation for click chemistry.
Besides, Seo et al. tagged a fluorogen to the 5’-end of single-stranded
DNA [43]. The oligonucleotide was modified, through several reactions, to
display a terminal alkyne at its 5’-end and the fluorogen contained an azido
functional group. A 91% product yield was obtained, but no catalyst was used
in the reaction, leading to a mixture of 1, 4-substituted and 1,5-substituted
1,2,3-triazole products. If a copper catalyst were used, it is highly likely that
only 1,4-substituted triazoles would be produced and that the reaction would
proceed much faster. Seela et al. took DNA labeling one step further and
synthesized nucleosides that each contained a single terminal alkyne on their
aromatic nucleobase [44]. Modified deoxyadenosine (dA), deoxyguanosine
(dG), deoxycytidine (dC), and deoxythymidine (dT) were all included. Using
solid phase synthesis, several oligonucleotides were subsequently synthesized,
which either contained one modified nucleoside, two, or none at all (to serve
as controls). The properties of the oligonucleotides and their duplexes were not
significantly affected by the modified nucleosides, as evidenced by the similar
melting temperatures when compared to the controls. Reporter molecules
containing azido functional groups were then conjugated to the modified bases
through click chemistry. Upon conjugation the reporter molecules began to
fluoresce, indicating that the oligonucleotides had been successfully labeled. If
made commercially available, Seela’s modified nucleosides could make
oligonucleotide labeling trivial.
The polymer with the highest secondary amine density gave polyplexes
with low toxicity and high cellular delivery. The transfection efficiency of the
trehalose polymers was an order of magnitude higher than Jet-PEI, one of the
most efficient in vitro gene delivery polymers, in serum-free conditions. In the
study [45], the same group investigated the influence of the molecular weight
of the trehalose click polymer on polyplex stability and pDNA cellular
delivery efficiency, It was shown that a higher degree of polymerization
resulted in a higher polyplex stability, although no effect was observed in
pDNA binding affinity, cellular uptake, and DNase protection in relation to the
Mw.
Ideally, suitable polymeric transfectants should be nontoxic,
nonimmunogenic, and preferably biodegradable in a controlled manner.
Furthermore, biodegradable polymers should yield degradation products with
a molecular weight lower than 30 kDa, because these degradation products can
be excreted by the kidneys [46, 47]. To reduce the cytotoxicity of cationic
polymers, Hennink and co-workers [48] grafted a low-molecular-weight
cationic poly(2-dimetylamino) ethyl methacrylate (pDMAEMA) onto the
polymer backbone of uncharged hydrophilic polymer, poly(hydroxyethyl
methacrylate) (pHEMA), via biodegradable linkages. Both pDMAEMA and
pHEMA were synthesized by atom transfer radical polymerization (ATRP)
(Figure 3). For this goal, pDMAEMA was end-functionalized with an azide
[49], while pHEMA was randomly functionalized with acetylene moieties
[50]. The polymers were “clicked” together via the CuAAC in DMF at 50℃
with CuBr as catalyst. The molecular weight of the polymer as well as the
number of grafts could easily be varied. Upon incubation at physiological
conditions (pH 7.4, 37C), the carbonate ester bonds were readily hydrolyzed
(t1/2: 96 h). The molecular weight of the final main degradation product was
very close to that of the starting pDMAEMA, indicating that the carbonate
esters were quantitatively hydrolyzed. Furthermore, the synthesized polymers
were able to condense DNA into small particles, which were able to transfect
cells efficiently in the presence of endosome-disruptive INF/7 peptide. Finally,
the polymers had a lower toxicity compared to high molecular weight
pDMAEMA, making this an effective approach to reduce the toxicity of high-
molecular-weight cationic polymers [48].
Dervan et al. showed that macromolecules that containing various
heterocyclic residues, such as derivatives of pyrrole and imidazole, are able to
bind nucleic acids [51]. Reineke and his colleagues were inspired by them.
They used copper catalyzed azide-alkyne cycloaddition click reaction to
synthesis a series oligomeric amine modified polymers based on sugars
(trehalose/cyclodextrin) gene carriers [52-54]. Firstly, through the hydroxyl
group of sugar unit, they connected two groups of nitrogen with every
molecule, and then the molecule was polymerised with different double
acetylene based oligomers, getting the cationic polymers with different degree
of polymerization. Hydrophobic ring supplement by click chemistry can

increase the Van Der Waals force and hydrogen bonding interactions between
polymers and nucleic acids. They found that changing the length of the
oligomer can regulate the bounding ability of cationic polymer to DNA. By
optimizing the reaction temperature, reactant and catalyst concentration,
reaction time and so on, the cyclodextrin can be introduced into the main chain
of polymer. Reineke and co-workers [55] exploited to use CuAAC to
synthesize a family of trehalose-based glycopolymers (Figure 4). Three
polymers with different amine stoichiometry were synthesized from diazide
functionalized trehalose monomers and dialkyne-amide comonomers in the
presence of CuSO4/Na-ascorbate. The polymers contained a trehalose unit, an
oligoamineunit for electrostatic interactions with DNA, and a triazole
functionality for hydrophobic, van der Waals, and hydrogenbonding
interactions with nucleic acids.
Méndez-Ardoy and his colleagues explored two affinity beta
cyclodextrincluster like system via the CuAAC click chemistry to use as the
gene vector. Different spacer groups can be introduced to regulate the
flexibility, charge density and hydrophilic hydrophobic property of polymer by
CuAAC reaction. And then the ability to bind and protect the plasmid DNA
was optimized, so that the transfection efficiency of the gene vector system
was improved.
Figure 3. Synthesis of degradable-brushed pHEMA-pDMAEMA.

Figure 4. Structure of trehalose polymers synthesized by Reineke and co-workers.
Note: M: main-chain (backbone) unit, DL: degradable linkage, CP: side-chain cationic
polymer.
Figure 5. Schematic structure of the degradable-brushed cationic polymers synthesized

by click chemistry.
In general, with the increase of the molecular weight of the cationic

polymer, the gene transfection efficiency and toxicity could be improved.
Jiang and his colleagues [56] prepared polymer whose backbone is poly 2-
hydroxyethyl methacrylate (PHEMA) and side chain is poly methyl
methacrylate two methylamino ethyl (PDMAEMA) comb change polymer by
the atom transfer radical polymerization (ATRP) method combined with click
chemistry method, besides both the backbone and the side chain of the
polymer polymerized narrow distribution polymers by ATRP.
Yang and his colleagues came up with more systemic ways. They used the
click chemistry to link low molecular weight cationic polymers to a high
molecular weight cationic polymer via a readily degradable covalent bond
(Figure 5). They used the click chemistry to synthesize the biodegradable
micro - crosslinked polyethylene (PEI-SS-CL) - based and the reduced

degradation of PEI and its application as gene carrier [57].
Li et al. designed a gene vector based on chitosan. Chitosan has good
biodegradability and biocompatibility, and it also has low cytotoxicity. So it
can be an important non-viral gene vector. However, the solubility of chitosan
in water is not good, so it needs to be modified. After protecting C2 bit amino
on the chitosan, they made the hydroxyl group on the C6 siteconnected with
the group of nitrogen and then, together with C and C, connected with
modified quaternary ammonium group on the chitosan side chain. The results
showed that the modified chitosan has good water solubility, and the better
ability to bind and protect DNA than unmodified chitosan.
It can be seen from the results of the above study that click chemistry has
important applications in construction of multifunctional cationic polymer
gene vector and achieving a higher gene transfection efficiency and low cell
toxicity.
3. APPLICATION OF CLICK CHEMISTRY

IN PHARMACEUTICAL SCIENCE
Chemistry, traditionally being the science of synthesis and structural

manipulations of molecules, has gradually undertaken the more challenging
task of biology-oriented synthesis [58]. The generation of molecules/molecular
assemblies possessing well-defined biological functions remains an extremely
challenging task; immediate refinements in conventional synthetic tactics are
necessary. New and more efficient chemical reactions and methodologies,
which may override the laborious protection/deprotection and purification
steps in conventional total synthesis, could revolutionize the next-generation
chemical and biological research [59]. A set of chemical reactions, known as
bioorthogonal reactions, that are orthogonal to most functional groups in
biological systems has so far promising applications in biological research
[60]. Click chemistry has recently emerged to become one of the most
powerful tools in drug discovery, chemical biology, and proteomic
applications. In recent years, the design and synthesis of pharmacologically
relevant heterocyclic molecules by combinatorial techniques have proven to be
a promising strategy in the search for new pharmaceutical lead structures.
Click chemistry is one of the powerful reactions for making carbon–
heteroatom–carbon bonds in aqueous environment with a wide variety of

chemical and biological applications in various fields [61].
Despite many successes, drug discovery approaches that are based on
nature’s secondary metabolites are often hampered by slow and complex
syntheses. Through the use of only the most facile and selective chemical
transformations, click chemistry simplifies compound synthesis, providing the
faster lead discovery and optimization. The click reaction must be modular,
wide in scope, give very high yields, generate only inoffensive byproducts that
can be removed by nonchromatographic methods, and be stereospecific (but
not necessarily enantioselective). The required process characteristics include
simple reaction conditions (ideally, the process should be insensitive to
oxygen and water), readily available starting materials and reagents, the use of
no solvent or a solvent that is benign (such as water) or easily removed, and
simple product isolation [62]. Purification, if required, must be by
nonchromatographic methods, such as crystallization or distillation, and the
product must be stable under physiological conditions. The traditional process
of drug discovery based on natural secondary metabolites has often been slow,
costly, and labor-intensive. Even with the advent of combinatorial chemistry
and high-throughput screening in the past two decades, the generation of leads
is dependent on the reliability of the individual reactions to construct the new
molecular framework [63].
Click chemistry is a newer approach to the synthesis of drug-like
molecules that can accelerate the drug discovery process by utilizing a few
practical and reliable reactions. Sharpless and co-workers defined the click
reaction as wide in scope and easy to perform, uses only readily available
reagents, and is insensitive to oxygen and water. In fact, in several instances,
water is the ideal reaction solvent, providing the best yields of the product with
the highest rates. For reaction workup and purification, eco-friendly solvents
are used to avoid purification techniques like chromatography [64].
Current drug discovery relies on massive screening of chemical libraries
against various extracellular and intracellular molecular targets to find novel
chemotypes with the desired mode of action. In recent years, high-throughput
technologies for combinatorial and multiparallel chemical synthesis,
automation technologies for the isolation of natural products, and also
availability of large compound collections from commercial sources have
substantially increased the size and diversity of compound collections among
most pharma and biotech companies, in some cases exceeding one million
distinct chemical entities [65]. At the same time, sequencing of the human
genome as well as sequencing the genomes of various pathogens, such as
microbes, bacteria, and viruses, have delivered hundreds to thousands of

potentially novel biological targets that have poor or no clearly precedented
chemical starting point for lead optimization [65, 66]. Click chemistry-based
drug discovery mainly falls into three types: (1) high-throughput screening, (2)
fragment-based drug discovery, and (3) dynamic template-assisted strategies
in fragment-based drug discovery.
3.1. Click Chemistry and Drug Discovery
3.1.1. High-Throughput Screening

Click chemistry combined with high-throughput enzyme assay
technologies such as microarrays revolutionized lead-finding and lead-
optimization steps in drug discovery. Small molecule libraries assembled using
click chemistry has been successfully employed in generating unique inhibitor
and activity-based fingerprints of important enzymes. Such fingerprinting
strategies may lead to the identification and characterization of new enzyme
subclasses in the future and even address the issue of functional convergence
of enzymes at a broader scale. Rapid developments in high-throughput
screening will continue to be fueled by click chemistry and its novel variants
in combination with powerful technologies like microarrays and other
ingenious characterization techniques [61].
3.1.2. Fragment-Based Drug Discovery

Fragment-based drug discovery (FBDD) has become a main-stream
alternative to high-throughput screening in the past few years. There are an
increasing number of compounds in clinical development, which can trace
their origins back to fragment screening, and a number of reviews have been
published recently highlighting the progress that has been made [67]. A recent
review highlights in FBDD the following concepts: (1) Inappropriate physical
properties are a major source of attrition for small molecule drugs. (2)
Although weak in potency, fragments actually form high-quality interactions.
(3) Ligand efficiency is a way to judge the relative optimizability of differently
sized molecules. (4) Relatively small libraries of fragments are required to
sample chemical space (Figure 1). It also highlights the following challenges:
(1) Specialized methods are needed to detect fragment binding. (2) Efficient
optimization of fragment hits is required.
Figure 6. Workflow of the process of hit-to-lead optimization from click chemistry and
drug candidate selection. FBDD, SAR, and QSAR studies are essential elements of this
complex paradigm. FBDD, fragment-based drug design; QSAR, quantitative SAR;
SAR, structure–activity relationship.
Fragment-based drug discovery is based on the consideration that the free

binding energy of a protein ligand results from the contributions of its
molecular components. Therefore, small contributions from molecular
fragments can add up to yield a high-affinity protein ligand. First, a small
molecule fragment that binds to the protein pocket of interest is identified. The
starting fragment is then chemically modified to generate a binder of higher
affinity, which is subsequently further optimized to a lead structure. The
concept has become very popular for two main reasons. First, initial screening
of fragment libraries is expected to sample the chemical space much more
efficiently than traditional approaches ever could [68]. The second reason is
that fragment-derived lead structures have significantly higher ligand
efficiency (free binding energy per non-hydrogen atom of the ligand) than
molecules discovered by screening of large compound libraries. An
investigation of 150 known natural and synthetic ligands revealed that the free
binding energy increased in proportion to ligand size up to a maximum of 15

atoms. The maximum average free-energy contribution per heavy atom was -
1.5 kcal/mol. For molecules larger than this, no further increase in ligand
efficiency was observed. These observations confirm how crucial limiting the
molecular size is for the efficiency of protein ligands, thereby supporting the
preference of fragment hits (<12 heavy atoms per molecule) over typical HTS
hits. These results also indicate easier optimization and hit-to-lead
development of fragment hits relative to that of HTS hits [69, 70a].
Many enzymes are known to possess multiple binding pockets; yet
conventional inhibitor developments generally focus more on only the active
site. However, in many cases, the secondary/allosteric binding sites confer
selectivity as well as potency. Within this context, click chemistry, due to its
highly modular and efficient reaction nature, has been identified as one of the
most practical methods. Because of the efficiency and water compatible nature
of the click reaction, in most cases, the assembled products could be directly
screened for inhibition without the need for any purification [70b].
3.1.3. Dynamic Template-Assisted Strategies in Fragment-Based

Drug Discovery
The major challenges of fragment-based drug discovery are the
identification of low-affinity fragments and efficient biologically active
linkage of the fragments identified. Weakly binding ligands are difficult to
detect. To address this problem, alternative so-called dynamic and template-
assisted strategies have been proposed for fragment-based drug discovery by
researchers. All of these methods use a target protein as a template for
selection and/or assembly of optimal fragment combinations. All dynamic
template-assisted approaches covered here have in common a chemical
reaction (reversible or irreversible, enzymatic or nonenzymatic), which is
exploited for detection of the best fragment combination [71].
For example, the azide and alkyne reagents are structurally selected to
partially interact with the enzymatic binding site. Thus, fragments that interact
favorably with the enzyme are selected, and the 1, 3-dipolar cycloaddition
“click” reaction connects the fragments to form the final inhibitor. FBDD and
TGS both have in common the fact that they capitalize on molecular
recognition patterns imprinted on molecular targets to select suitable
molecular fragments “ex vivo,” which are then tethered through synthetic
optimization (FBDD) or in situ self-assembling to form the final ligands.
Figure 7. Concepts in lead discovery. (a) High-throughput screening (HTS). A diverse

library of chemical compounds is collected and tested against the drug target. (b)
Fragment-based lead discovery. The binding of small molecular fragments to the
protein is detected. Low-affinity fragments can be linked to provide high-affinity
ligands. The binding constant KAB is an exponential function of the binding energy.
(c) Dynamic strategies in fragment-based drug discovery. Reactive fragments are
incubated with the protein and form specific combinations of fragments on the protein
template, which facilitates fragment detection and linkage to a new ligand.
These ligands can then be resynthesized and scaled up for subsequent

biological evaluations through the drug development stages (i.e., in vivo
animal models and clinical development). However, when considering
Rideout’s ideas and recent developments in TGS together, one could shift this
paradigm of fragment-based approaches a step foreword toward the use of
molecular fragments for direct clinical use. The approach would allow for the
in vivo - in situ synthesis of the final ligand already in close proximity to its
biological target (protein or DNA) in the host. In the context of cancer,
formation of the final drug only at close proximity of its biological targeted
can lead to amplification of selectivity between neoplastic and normal cells.
Thus, it is foreseeable that in the future, purposely prepared pharmaceutical
compositions of closely related but different fragmented prodrugs could be
designed with the help of the click chemistry precursor to synergistically
interact with specific molecular targets that have different isoforms (i.e.,
proteins) or polymorphism (i.e., genes) within patients, paving the way for
personalized prodrugs.
3.2. Using of Click Chemistry in the Development of

Enzyme Inhibitors
3.2.1. Protein Tyrosine Phosphatase Inhibitors

Protein tyrosine phosphatases (PTPs, protein tyrosine phosphatase class
enzyme) contain an important class of signaling enzymes that can catalyze the
dephosphorylation of phosphotyrosine residues in a protein substrate. Among
them, PTP1B (protein tyrosine phosphatase 1B, nonreceptor phosphor-trosine
PTP) has been identified as the major regulator of both insulin and leptin
signaling pathways. Malfunctioning of PTP1B leads to various human diseases
like cancer, diabetes, obesity, and inflammation [72].
Zhang and co-workers prepared a highly potent and selective mPTPB
inhibitor using a novel, double click chemistry strategy. The most potent
mPTPB inhibitor (compound 1, Figure 3) from this approach possesses a Ki
value of 160 nM and a >25-fold selectivity for mPTPB over 19 other protein
tyrosine phosphatase inhibitors, and molecular docking study of the enzyme–
inhibitor complex provides a rationale for the high potency and selectivity of
the lead compound and reveals an unusual binding mode (IC50 = 4.9 μM
against PTP1B and 0.27 μM against mPTPB) [73].
Xie et al. [74] reported new PTP inhibitor entities by simply “clicking”
alkynyl amino acids onto diverse azido sugar templates. Triazolylglucosyl,
galactosyl, and mannosyl serine and threonine derivatives were efficiently
synthesized via click reaction, one of which (compound 2, Figure 3) was
identified as a potent and selective PTP1B inhibitor against a panel of
homologous PTPs with IC50 = 5.9 ± 0.4 μM for R1 = H against PTP1B and
IC50 = 7.1 ± 1.0 μM for R1 = Me against PTP1B.
Tang and co-workers [75] recently reported a library of benzyl 6-
triazolo(hydroxy)benzoic glucosides via the Cu(I)-catalyzed azide–alkyne 1,3-
dipolar cycloaddition. These glycoconjugates bearing alkyl chain, sugar, and
(hydroxy)-benzoic derivatives (compound 3, Figure 3) (IC50 = 8.7 ± 1.4 μM
for n = 2 and IC50 = 6.7 ± 0.5 μM for n = 3) were identified as new PTP1B
inhibitors with selectivity over T-Cell PTP (TCPTP), SH2-containing PTP-1
(SHP-1), SHP-2, and leukocyte antigen-related tyrosine phosphatase (LAR).
Zhou and co-workers [76] recently reported a potent and selective mPTPB
inhibitor (compound 4, Figure 8) with highly efficacious cellular activity, from
a combinatorial library of bidentatebenzofuran salicylic acid derivatives
assembled by click chemistry. The inhibition of mPTPB with compound (4) in
macrophages reversed the altered host immune responses induced by the
bacterial phosphatase and prevents TB growth in host cells with IC50 = 19 ±
1.5 μM against PTP1B, 1.6 ± 0.22 μM against mPTPB, and 77.3 ± 1.5 μM
against mPTPA.
Figure 8. Chemical structures of protein tyrosine phosphatase inhibitors synthesized

via click chemistry.
Yao et al. [77] reported a solid-phase reaction strategy for high-throughput

synthesis of a 96-member azide library. A 384-member PTP inhibitor l was
synthesized by clicking the azide library with an alkyne-modified isoxazole
warhead. The entire operation was performed in 96/384-well plates without
any purification. From a homologous series of bidentate inhibitors (compound
5, Figure 3) was identified a most potent inhibitor of PTP1B with an IC50 of
11.1 μM.
3.2.2. Protein Kinase Inhibitors

Protein kinase phosphorylating enzymes play a pivotal role in cellular
signal transduction, and many diseases are characterized by abnormalities in a
kinase or its expression level. Protein kinases (PK) are a family of enzymes
that are involved in controlling the function of other proteins through the
phosphorylation of hydroxyl groups of serine and threonine amino acid
residues on these proteins. PK enzymes in turn are activated by signals such as
increases in the concentration of diacylglycerol or Ca2+. PK enzymes play
important roles in several signal transduction cascades. Hence, a significant
portion of drug discovery efforts has made protein kinases as primary targets
Liskamp and co-workers [78] synthesized bisubstrate-based kinase
inhibitors that target the more selective peptide-binding site in addition to the
ATP-binding site. Dynamic peptide microarrays were used to find peptide
binding site PKa inhibitors. These active binding peptides were linked with
chemo-selective click chemistry to an ATP-binding site kinase inhibitor, and
this led to novel bisubstrate structures. The most promising potent inhibitors
(compounds 6 and 7, Figure 9) had nanomolar affinity and selectivity toward
PKCalpha and PKCteta among three isozymes. Compound 23 showed IC50 =
1.0 ± 0.2 μM against PKCalpha and IC50 = 0.6 ± 0.1 μM against PKCteta. On
the other hand, compound 24 showed IC50 = 0.43 ± 0.03 μM against PKCteta.
Liskamp et al. [79] employed click chemistry for the synthesis of
bisubstrate-based kinase inhibitors using arginine residues featuring acetylene
or azide moieties in their side chain. Developed bisubstrate-based kinase
inhibitor was tested for affinity and selectivity toward three highly
homologous PKC isozymes. The resulting inhibitor (compound 8, Figure 9)
showed improved affinity and a highly interesting shift in selectivity toward
PKCteta with IC50 = 0.17 ± 0.029 μM.
Kumar et al. [80] recently synthesized two classes of 1, 4-disubstituted
1,2,3-triazoles using one-pot reaction of α-tosyloxy ketones/α-halo ketones,
sodium azide, and terminal alkynes in the presence of aqueous PEG (1:1, v/v)
using click chemistry. 1,4-Disubstituted 1,2,3-triazoles (compounds 9 and 10,
Figure 9) exhibited modest Src kinase inhibitory activity among the

synthesized 1,2,3-triazoles with IC50 values in the range of 32.5 and 33.9 μM.
Merrer and co-workers [81] reported a library of pyrido[2,3-d]
pyrimidines as inhibitors of FGFR3 (fibroblast growth factor receptor 3)
tyrosine kinase allowing possible interactions with an unexploited region of
the ATP binding-site. This library was built-up with an efficient step of click
chemistry giving easy access to triazole-based compounds bearing a large
panel of substituents. Among the 27 analogues synthesized, more than one-
half exhibited 55-89% inhibition of in vitro FGFR3 kinase activity at 2 μM,
and one of the pyrido[2,3-d]pyrimidine derivatives (compound 11, Figure 9)
was able to inhibit autophosphorylation of mutant FGFR3-K650 M in
transfected HEK (human embryonic kidney) cells.
Figure 9. Chemical structures of protein kinases inhibitors synthesized via click

chemistry.
3.3. Click Chemistry in Drug Development Using Fragment-

Based Drug Discovery
The main target in medicinal chemistry is to synthesize compounds or

libraries of compounds during the process of drug discovery or lead
optimization, and for this reason, this field is particularly attracted to synthetic
methodologies that allow rapid construction of molecules. The identification
of such rapid synthetic strategies should allow the medicinal chemist to
assemble a large number of biologically active compounds in a very short
period of time, speeding up the process of discovery and lead optimization.
Click chemistry is one of the powerful tools to synthesize many drugs using
fragment-based drug screening methods. This click-FBDD-based screening
allows for more efficient lead identification and lead optimization procedures
in medicinal chemistry. In this context, it is easy to predict the usefulness of
this reaction in fragment-based ligand design. Triazolepeptidomimetic
fragments are generated, and these could act as building blocks to be used in
the fragment-based approach to drug discovery, thereby rendering this
technique feasible in drug discovery. Hence, we are describing some potential
applications of the click chemistry reaction found in the literature toward
novel drug development for many incurable diseases such as anticancer, anti-
TB, etc. [82-83].
Blagg and his co-workers [84] employed click chemistry for the synthesis
of a series of triazole-containing novobiocin analogues. These compounds
contain a triazole ring in lieu of the amide moiety present in the natural
product. The antiproliferative effects of these compounds were evaluated
against two breast cancer cell lines (SKBr-3 and MCF-7), and manifested
activities similar to those of their amide-containing counterparts. In addition,
Hsp90-dependent client protein degradation was observed via Western blot
analyses, supporting a common mode of Hsp90 inhibition for both structural
classes. Compounds 12 and 13 showed most potent inhibition against SKBr-3
and MCF-7 among homologous series with IC50 = 13.16 ± 3.85 μM against
MCF-7 and IC50 = 21.22 ± 5.99 μM against SKBr-3 for compound 12.
Compound 13 exhibited a IC50 value of 18.33 ± 4.67 μM against MCF-7 and
IC50 = 8.17 ± 0.11 μM against SKBr-3.
A series of benzo-macrolactonedervatives were prepared by click
chemistry reaction and evaluated as inhibitors of heat shock protein 90
(Hsp90), an emerging attractive target for novel cancer therapeutic agents. A
new synthesis of these resorcylic acid macrolactone analogues of the natural
product radicicol is described in which the key steps are the acylation and
ring-opening of a homophthalic anhydride to give an isocoumarin, followed by
a ring-closing metathesis to form the macrocycle. The novel triazole-
macrocyclic lactones were evaluated for Hsp90 inhibition in two Hsp90
binding assays: the fluorescence polarization (FP) assay and the TR-Fret
assay. Their growth inhibitory potency in HCT116 human colon cancer cell
line, as measured by the SRB assay, was also determined. Compound 14
(Figure 10) showed good inhibition of Hsp90 in the FP assay in the series with
IC50 = 2.7 ± 1.3 μM [85].
1, 2, 3-triazole-based molecules are useful pharmacophores for several
DNA-alkylating and cross-linking agents. A series of A/C8, C/C2, and A/C8-
C/C2-linked 1,2,3-triazole-pyrrolo[2,1-c] [1,4] benzodiazepines (PBD)
conjugates was synthesized by employing “click” chemistry. These molecules
exhibited promising DNA-binding affinity and were evaluated for their in vitro
anticancer activity in selected human cancer cell lines of breast (Zr-75-1,
MCF7), oral (KB, DWD, Gurav), ovary (A2780), colon (Colo205), lung
(A549), prostate (PC3), and cervix (SiHa) by using the sulforhodamine B
(SRB) method. Especially, compound 15 (Figure 5) showed good inhibition
(GI50 = 0.15 μM against DWD, GI50 = 0.16 μM against A2780, GI50 = 0.17
μM against PC3, and GI50 = 0.12 μM against SiHa) against various cancer
cells such as oral, ovary, colon, lung, prostate, and cervix [86].
Figure 10. Chemical structures of anticancer drugs constructed via click chemistry.
In the development of enzyme inhibitors, click chemistry plays an

important active role. It has been identified as a convenient strategy toward
fragment-based inhibitor assembly, where large libraries of potential bidentate
inhibitors were generated with minimum synthetic efforts by several research
groups. Many research groups developed a nanomolar potent ligand for
specific enzyme inhibition using click chemistry. Ingenious strategies, such as
in situ click chemistry, have so far shed some light onto new ways of
generating extremely potent inhibitors against certain enzymes. Nevertheless,
click chemistry is used for the development of agonists, antagonist, and
selective ligand in receptor-ligand binding studies for drug development in the
field of medicinal chemistry.
Bioorthogonal-click chemistry reactions are paving the way for new
innovations in biology. These reactions possess extreme selectivity and
biocompatibility, such that their participating reagents can form covalent
bonds within richly functionalized biological systems. The bioorthogonal
reactions described in this Review and related transformations have proven
powerful tools for bioconjugation. Thus, the click chemistry reaction was
successfully used for site-specific protein, glycans, lipids, and cell surfaces,
inside living animals under physiological conditions. Click-ABPP platforms
enable both the discovery of various disease relevant enzymes and the
selective pharmacological probes to perturb and characterize these proteins in
cells. Activity-based proteomics can provide insight into the metabolic and
signaling pathways that support illuminate new strategies for disease diagnosis
and treatment.
The portability of azide labeling and Cu-free click chemistry enables
applications in many areas of chemical biology. Cu-free click chemistry has
been used to monitor azidosugars, proteins bearing azido amino acids, lipids,
and site-specifically labeled proteins, DNA, and RNA in live cells.
In the short period since click chemistry was conceived, it has had a
dramatic and diverse impact in many areas of modern chemistry.
REFERENCES
[1] Kolb, HC; Finn, MG; Sharpless, KB. Click chemistry: diverse chemical
function from a few good reactions. Angewandte Chemie-International
Edition, 2001, 40, 2004-2021.
[2] Nwe, K; Brechbiel, MW. Growing applications of “click chemistry” for

bioconjugation in contemporary biomedical research. Cancer Biotherapy
and Radiopharmaceuticals, 2009, 24(3), 289-302.
[3] Moses, JE; Moorhouse, AD. The growing applications of click
chemistry.Chemical Society Reviews, 2007, 36(8), 1249-1262.
[4] Hoyle, CE; Lowe, AB; Bowman, CN. Thiol-click chemistry: a
multifaceted toolbox for small molecule and polymer synthesis.
Chemical Society Reviews, 2010, 39(4), 1355-1387.
[5] Chu CH, Liu RH. Application of click chemistry on preparation of
Reviews, 2011, 40(5), 2177-2188.
[6] Lecomte, P; Riva, R; Jerome, C; Jerome, R. Macromolecular
engineering of biodegradable polyesters by ring-opening polymerization
and ‘Click’ chemistry. Macromolecular Rapid Communications, 2008,
29(12-13), 982-997.
[7] Wangler, C; Schirrmacher, R; Bartenstein, P; Wangler, B. Click-
chemistry reactions in radiopharmaceutical chemistry: fast and easy
introduction of radiolabels into biomolecules for in vivo imaging.
Current Medicinal Chemistry, 2010, 17(11), 1092-1116.
[8] Dirks, AJ; Cornelissen, JJLM; van Delft, FL; van Hest, JCM; Nolte,
RJM; Rowan, AE; Rutjes, FPJT. From (bio)molecules to biohybrid
materials with the click chemistry approach. QSAR and Combinatorials
Science, 2007, 26(11-12), 1200-1210.
[9] Lowe, AB. Thiol-yne ‘click’/coupling chemistry and recent applications
in polymer and materials synthesis and modification. Polymer, 2014,
55(22), 5517-5549.
[10] Zeng, DX; Zeglis, BM; Lewis, JS; Anderson, CJ. The growing impact of
bioorthogonal click chemistry on the development of
radiopharmaceuticals. Journal of Nuclear Medicine, 2013, 54(6), 829-
832.
[11] Akeroyd, N; Klumperman, B. The combination of living radical
polymerization and click chemistry for the synthesis of advanced
macromolecular architectures. European Polymer Journal, 2011, 47(6),
1207-1231.
[12] Arnold, RM; Huddleston, NE; Locklin, J. Utilizing click chemistry to
design functional interfaces through post-polymerization modification.
Journal of Materials Chemistry, 2012, 22(37), 19357-19365.
[13] Application of “click” chemistry to the construction of supramolecular

functional systems. Asian Journal of Organic Chemistry, 2014, 3(5),
582-602.
[14] van Dijk, M; Rijkers, DTS; Liskamp, RMJ; van Nostrum, CF; Hennink,
WE. Synthesis and applications of biomedical and pharmaceutical
polymers via click chemistry methodologies. Bioconjugate Chemistry,
2009, 20(11), 2001-2016.
[15] Yang, QZ; Liu, J; Jiang, XL. Application of click chemistry in
biomedical polymers. Progress in Chemistry, 2010, 22(12), 2377-2387.
[16] Zhao, ZD; Yuan, WZ; Gu, SY; Ren, TB; Ren, J. “Click Chemistry” and
its growing applications in biomedical field. Progress in Chemistry,
2010, 22(2-3), 417-426.
[17] Lau, YH; Price, JR; Todd, MH; Rutledge, PJ. Chemical sensors that
incorporate click-derived triazoles. Chemical Society Reviews, 2011,
17(10), 2850-2858.
[18] Su, J; Xu, J; Chen, Y; Xiang, Y; Yuan, R; Chai, YQ. Sensitive detection
of copper(II) by a commercial glucometer using click chemistry.
Biosensors and Bioelectronics, 2013, 45, 219-222.
[19] Zhang, GF; Zhu, XL; Miao, FJ; Tian, DM; Li, HB. Design of switchable
wettability sensor for paraquat based on clicking calixarene. Organic
and Biomolecular Chemistry, 2012, 10(16), 3185-3188.
[20] Fujimoto, K; Yamada, S; Inouye, M. Synthesis of versatile fluorescent
sensors based on Click Chemistry: detection of unsaturated fatty acids
by their pyrene-emission switching. Chemical Communications, 2009,
46, 7164-7166.
[21] Lu, LJ; Yang, LL; Cai, HJ; Zhang, L; Lin, ZY; Guo, LH; Qiu, B; Chen,
GN. Determination of flumioxazin residue in food samples through a
sensitive fluorescent sensor based on click chemistry. Food Chemistry,
2014, 162, 242-246.
[22] Zeng, K; Guo, ML; Zhang, YJ; Qing, M; Liu, A; Nie, Z; Huang, Y; Pan,
YL; Yao, SZ. Thiol-eneclick chemistry for the fabrication of
Ru(bpy)32+-based solid-state electrochemiluminescence sensor.
Electrochemistry Communications, 2011, 13(12), 1353-1356.
[23] Maity, D; Govindaraju, T. Conformationally constrained (coumarin-
triazolyl-bipyridyl) click fluoroionophore as a selective Al3+ sensor.
Inorganic Chemistry, 2010, 49(16), 7229-7231.
[24] Maity, D; Govindaraju, T. Pyrrolidine constrained bipyridyl-dansyl click
fluoroionophore as selective Al(III) sensor. Chemical Communications,
2010, 46(25), 4499-4501.
[25] Li, HB; Zheng, QL; Han, CP. Click synthesis of podand triazole-linked
gold nanoparticles as highly selective and sensitive colorimetric probes
for lead(II) ions. Analyst, 2010, 135(6), 1360-1364.
[26] Ruan, YB; Maisonneuve, S; Xie, JA. Highly selective fluorescent and
colorimetric sensor for Hg2+ based on triazole-linked NBD. Dyes and
Pigments, 2011, 90(3), 239-244.
[27] Zhang, Z; Li, WQ; Zhao, QL; Cheng, M; Xu, L; Fang, XH. Highly
sensitive visual detection of copper (II) using water-soluble azide-
functionalized gold nanoparticles and silver enhancement. Biosensors
and Bioelectronics, 2014, 59, 40-44.
[28] Ge, CC; Luo, Q; Wang, D; Zhao, SM; Liang, XL; Yu, LX; Xing, XR;
Zeng, LW. Colorimetric detection of copper(II) ion using click
chemistry and hemin/G-quadruplex horseradish peroxidase-mimicking
DNAzyme. Analytical Chemistry, 2014, 86(13), 6387-6392.
[29] Szunerits, S; Niedziolka-Jonsson, J; Boukherroub, R; Woisel, P;
Baumann, JS; Siriwadena, A. Label-free detection of lectins on
carbohydrate-modified boron-doped diamond surfaces. Analytical
Chemistry, 2010, 82(19), 8203-8210.
[30] Odaci, D; Gacal, BN; Gacal, B; Timur, S; Yagci, Y. Fluorescence
sensing of glucose using glucose oxidase modified by PVA-pyrene
prepared via “click” chemistry. Biomacromolecules, 2009, 10(10), 2928-
2934.
[31] Trilling, AK; Hesselink, T; Van Houwelingen, A; Cordewener, JHG;
Jongsma, MA; Schoffelen, S; Van Hest, JCM; Zuilhof, H; Beekwilder, J.
Orientation of llama antibodies strongly increases sensitivity of
biosensors. Biosensors and Bioelectronics, 2014, 60, 130-136.
[32] Li, WJ; Yu C; Wang, YX; Yang Z. Chinese Journal of Analytical
Chemistry, 2012, 40(11), 1642-1647.
[33] Bhalla, V; Gupta, A; Kumar, M; Rao, DSS; Prasad, SK. Self-assembled
pentacenequinone derivative for trace detection of picric acid. ACS
Applied Materials and Interfaces, 2013, 5(3), 672-679.
[34] Sui, BL; Kim, B; Zhang, YW; Frazer, A; Belfield, KD. Highly selective
fluorescence turn-on sensor for fluoride detection. ACS Applied
Materials and Interfaces, 2013, 5(8), 2920-2923.
[35] Norberg, O; Lee, IH; Aastrup, T; Yan, MD; Ramstrom, O.
Photogenerated lectin sensors produced by thiol-ene/yne photo-click
chemistry in aqueous solution. Biosensors and Bioelectronics, 2012,
34(1), 51-56.
[36] Haag, R; Kratz, F. Polymer therapeutics: concepts and applications.

Angewandte Chemie-International Edition, 2006, 45, 1198-1215.
[37] Parhamifar L; Larsen AK; Hunter AC; Andresen TL; Moghimi SM;
Moghimi M. Polycation cytotoxicity: a delicate matter for nucleic acid
therapy-focus on polyethylenimine. Soft Matter, 2010, 6 (17), 4001-
4009.
[38] Deng R; Yue Y; Jin F; Chen Y; Kung H; Lin MCM; Wu CJ. Revisit the
complexation of PEI and DNA-How to make low cytotoxic and highly
efficient PEI gene transfection non-viral vectors with a controllable
chain length and structure. Control Release, 2009, 140, 40-46.
[39] Caruthers MH. Gene synthesis machines: DNA chemistry and its uses.
Science, 1985, 230, 281-285.
[40] Tewary, HK; Iversen PL. Qualitative and quantitative measurements of
oligonucleotides in gene therapy: part II in vivo models. Journal of
Pharmaceutical and Biomedical Analysis, 1997, 15, 1127-1135.
[41] Gewirtz AM. Antisense oligonucleotide therapeutics forhuman
leukemia. Current Opinion in Hematology, 1998, 5, 59-71.
[42] Schena, M; Shalon D; Davis RW; Brown PO. Quantitative monitoring of
gene expression patterns with a complementary DNA microarray.
Science, 1995, 270, 467-470.
[43] Seo, TS; Li, Z; Ruparel, H; Ju, J. Click chemistry to construct
fluorescent oligonucleotides for DNA sequencing. Journal of Organic
Chemistry, 2003, 68, 609-612.
[44] Seela, F; Sirivolu, VR; Chittepu P. Modification of DNA with
octadiynyl side chains: synthesis, base pairing, and formation of
fluorescent coumarin dye conjugates of four nucleobases by the alkyne-
azide “click reaction.” Bioconjugate Chemistry, 2008, 19, 211-224.
[45] Srinivasachari, S; Liu, YM; Prevette, LE; Reineke, TM. Effects of
trehalose click polymer length on pDNA complex stability and delivery
efficacy. Biomaterials, 2007, 28, 2885-2898.
[46] Petersen H; Merdan T; Kunath K; Fischer D; Kissel T.
Poly(ethylenimine-co-L-lactamide-co-succinamide): a biodegradable
polyethylenimine derivative with an advantageous pH-dependent
hydrolytic degradation for gene delivery. Bioconjugate Chemistry, 2002,
13, 812-821.
[47] Luten, J; van Nostrum, CF; De Smedt, SC; Hennink, WE. Biodegradable
polymers as non-viral carriers for plasmid DNA delivery. Journal of
Controlled Release, 2008, 126, 97-110.
[48] Jiang, X.; Lok, MC; Hennink, WE. Degradable brushed pHEMA-
pDMAEMA synthesized via ATRP and click chemistry for gene
delivery. Bioconjugate Chemistry, 2007, 18, 2077-2084.
[49] Dirks, AJ; Cornelissen, JJLM; van Delft, FL; van Hest, JCM; Nolte,
RJM; Rowan, AE; Rutjes FPJT. From (bio) molecules to biohybrid
materials with the click chemistry approach. QSAR and Combinatorial
Science, 2007, 26, 1200-1210.
[50] Le Droumaguet, B; Velonia, K. Click chemistry: A powerful tool to
create polymer-based macromolecular chimeras. Macromolecular Rapid
Communications, 2008, 29, 1073-1089.
[51] White, S; Szewczyk, JW; Turner, JM; Baird, EE; Dervan, PB.
Recognition of the four Watson-Crick base pairs in the DNA minor
groove by synthetic ligands. Nature, 1998, 391, 468-471.
[52] Srinivasachari, S; Liu, YM; Zhang, GD; Prevette, L, Reineke, TM.
Trehalose click polymers inhibit nanoparticle aggregation and promote
pDNA delivery in serum. Journal of the American Chemical Society,
2006, 128, 8176-8184.
[53] Srinivasachari, S; Liu, YM; Prevette, LE; Reineke, TM. Effects of
trehalose click polymer length on pDNA complex stability and delivery
efficacy. Biomaterials, 2007, 28, 2885-2898.
[54] Srinivasachari, S; Reineke, TM. Versatile supramolecular pDNA
vehicles via “click polymerization” of β-cyclodextrin with
oligoethyleneamines. Biomaterials, 2009, 30(5), 928-938.
[55] Srinivasachari, S; Liu, YM; Zhang, GD; Prevette, L, Reineke, TM.
Trehalose click polymers inhibit nanoparticle aggregation and promote
pDNA delivery in serum. Journal of the American Chemical Society,
2006, 128, 8176-8184.
[56] Jiang, X; Lok, MC; Hennink, WE. Degradable brushed pHEMA-
pDMAEMA synthesized via ATRP and click chemistry for gene
delivery. Bioconjugate Chemistry, 2007, 18, 2077-2084.
[57] Jiang, XL; Liu, J; Xu, L; Zhuo, RX. Disulfide-containing hyperbranched
polyethylenimine derivatives via click chemistry for nonviral gene
delivery. Macromolecular Chemistry and Physics, 2011, 212, 64-71.
[58] (a) Boyce, M; Bertozzi, CR. Bringing chemistry to life. Nature Methods,
2011, 8(8), 638-642. (b) Noren-Muller, A. Discovery of protein
phosphatase inhibitor classes by biology-oriented synthesis. Proceedings
of the National Academy of Sciences of the United States of America,
2006, 103, 10606-10611.
[59] Baran, PS; Maimone, TJ; Richter, JM. Total synthesis of marine natural
products without using protecting groups. Nature (London, United
Kingdom), 2007, 446(7134), 404-408.
[60] Baran, PS; Maimone, TJ; Richter, JM. Total synthesis of marine natural
products without using protecting groups. Nature (London, United
Kingdom), 2007, 446(7134), 404-408.
[61] (a) Kolb, HC; Sharpless, KB. The growing impact of click chemistry on
drug discovery. Drug Discovery Today, 2003, 8(24), 1128-1137. (b)
Moses, JE; Moorhouse, AD. The growing Applications of Click
Chemistry, 2007, 36(8), 1249-1262.
[62] Vsevolod, V; Rostovtsev, D; Luke, G; Green, D; Valery, V; Fokin. K;
Barry S. Stepwise huisgen cycloaddition process: Copper(I)-catalyzed
regioselective “ligation” of azides and terminal alkynes. Angewandte
Chemie-International Edition, 2002,114, 2708-2711.
[63] (a) Appukkuttan, P; Dehaen, W; Fokin, VV; Van der Eycken, E. A
microwave-assisted click chemistry synthesis of 1,4-disubstituted 1,2,3-
triazoles via a copper(I)-catalyzed three-component reaction. Organic
Letters, 2004, 6(23), 4223-4225. (b) Binder, WH, Sachsenhofer, R.
‘Click’ chemistry in polymer and materials science. Macromolecular
Rapid Communications, 2007, 28(1), 15-54.
[64] Bouillon, C; Meyer, A; Vidal, S; Jochum, A; Chevolot, Y; Cloarec, JP;
Praly, JP; Vasseur, Jean-Jacques; Morvan, Francois. Microwave assisted
“click” chemistry for the synthesis of multiple labeled-carbohydrate
oligonucleotides on solid support. Journal of Organic Chemistry, 2006,
71(12), 4700-4702.
[65] Colombo, M; Peretto, I. Chemistry strategies in early drug discovery: an
overview of recent trends. Drug Discovery Today, 2008, 13(15-16), 677-
684.
[66] (a) Borshell, Nigel; Congreve, Miles. Deal watch Valuation benefits of
structure-enabled drug discovery. Nature Reviews Drug Discovery,
2011, 10(3), 166. (b) Welsch, ME.; Snyder, SA; Stockwell, BR.
Privileged scaffolds for library design and drug discovery. Current
Opinion in Chemical Biology, 2010, 14(3), 347-361.
[67] Hajduk, PJ; Greer, J. A decade of fragment-based drug design: strategic
advances and lessons learned. Nature Reviews Drug Discovery, 2007,
6(3), 211-219.
[68] De Kloe, GE. Transforming fragments into candidates: small becomes
big in medicinal chemistry. Drug discovery today, 2009, 14, 630-646.
[69] Salum, LB; Andricopulo, AD. Fragment-based QSAR strategies in drug

design. Expert Opinion on Drug Discovery, 2010, 5(5), 405-412.
[70] (a) Murray, CW; Blundell, TL. Structural biology in fragment-based
drug design. Current Opinion in Structural Biology, 2010, 20(4), 497-
507. (b) Brik, A; Wu, CY; Wong, CH. Microtiter plate based chemistry
and in situ screening: a useful approach for rapid inhibitor discovery.
Organic and Biomolecular Chemistry, 2006, 4(8), 1446-1457.
[71] Schmidt, MF; Rademann, J. Dynamic template-assisted strategies in
fragment-based drug discovery. Trends in Biotechnology, 2009, 27(9),
512-521.
[72] Lu LP. Metal-based inhibitors of protein tyrosine phosphatases. Anti-
Cancer Agents in Medicinal Chemistry, 2011, 11,164-171.
[73] He, RJ; Yu, ZH; He, YT; Zeng, LF; Xu, J; Wu, L; Gunawan, AM;
Wang, LN; Jiang, ZX; Zhang, ZY. Double Click Reaction for the
Acquisition of a Highly Potent and Selective mPTPB Inhibitor. Chem.
Med. Chem., 2010, 5(12), 2051-2056.
[74] He, XP; Deng, Q; Gao, LX; Li, CZ; Wei Z, Yu, B; Tang, Y; Shi, XX;
Xie, J; Li, J; Cuo-R; Chen, Kaixian. Facile fabrication of promising
protein tyrosine phosphatase (PTP) inhibitor entities based on clicked’
serine/threonine-monosaccharide hybrids. Bioorganic and Medicinal
Chemistry, 2011, 19(13), 3892-3900.
[75] Li, C; He, XP; Zhang, YJ; Li, Z; Gao, L-X; Shi, XX; Xie, J; Li, J, Chen,
GR, Click to a focused library of benzyl 6-triazolo(hydroxy)benzoic
glucosides: Novel construction of PTP1B inhibitors on a sugar scaffold.
European Journal of Medicinal Chemistry, 2011, 46(9), 4212-4218.
[76] Zhou, B; He, YT; Zhang, X; Xu, J; Luo, Y; Wang, YH; Franzblau, SG;
Yang, ZY; Chan, RJ; Liu, Y; Zheng, JY; Zhang, ZY. Targeting
mycobacterium protein tyrosine phosphatase B for antituberculosis
agents. Proceedings of the National Academy of Sciences of the United
States of America, 2010, 107(10), 4573-4578.
[77] Srinivasan, R; Tan, LP; Wu, H; Yang, PY; Kalesh, KA.; Yao, SQ. High-
throughput synthesis of azide libraries suitable for direct “click”
chemistry and in situ screening. Organic and Biomolecular Chemistry,
2009, 7(9), 1821-1828.
[78] Mohapatra, DK; Maity, PK.; Shabab, M; Khan, MI. Click chemistry
based rapid one-pot synthesis and evaluation for protease inhibition of
new tetracyclic triazole fused benzodiazepine derivatives. Bioorganic
and Medicinal Chemistry Letters, 2009, 19(17), 5241-5245.
[79] Van Ameijde, J; Poot, AJ; van Wandelen, LTM; Wammes, AEM;
Ruijtenbeek, R; Rijkers, DTS; Liskamp, RMJ. Preparation of novel
alkylated arginine derivatives suitable for click-cycloaddition chemistry
and their incorporation into pseudosubstrate- and bisubstrate-based
kinase inhibitors. Organic and Biomolecular Chemistry, 2010, 8(7),
1629-1639.
[80] Kumar, D; Reddy, VB; Kumar, A; Mandal, D; Tiwari, R; Parang, K.
Click chemistry inspired one-pot synthesis of 1,4-disubstituted 1,2,3-
triazoles and their Src kinase inhibitory activity. Bioorganic and
Medicinal Chemistry Letters, 2011, 21(1), 449-452.
[81] Le Corre, L; Girard, AL; Aubertin, J; Radvanyi, F; Benoist-Lasselin, C;
Jonquoy, A; Mugniery, E; Legeai-Mallet, L; Busca, P; Le Merrer, Y.
Synthesis and biological evaluation of a triazole-based library of
pyrido[2,3-d] pyrimidines as FGFR3 tyrosine kinase inhibitors. Organic
and Biomolecular Chemistry, 2010, 8(9), 2164-2173.
[82] Jorgensen, WL. Efficient Drug Lead Discovery and Optimization.
Accounts of Chemical Research, 2009, 42(6), 724-733.
[83] Dwards, AM; Bountra, C; Kerr, DJ; Willson, TM. Open access chemical
and clinical probes to support drug discovery. Nature Chemical Biology,
2009, 5(7), 436-440.
[84] Peterson, LB.; Blagg, BSJ. Click chemistry to probe Hsp90: Synthesis
and evaluation of a series of triazole-containing novobiocin analogs.
Bioorganic and Medicinal Chemistry Letters, 2010, 20(13), 3957-3960.
[85] Day, JEH.; Sharp, SY; Rowlands, MG; Aherne, W; Workman, P;
Moody, CJ. Targeting the Hsp90 chaperone: synthesis of novel
resorcylic acid macrolactone inhibitors of Hsp90. Chemistry - A
European Journal, 2010, 16(9), 2758-2763.
[86] Kamal, A; Shankaraiah, N; Devaiah, V; Reddy, KL; Juvekar, A; Sen, S;
Kurian, N; Zingde, S. Synthesis of 1,2,3-triazole-linked
pyrrolobenzodiazepine conjugates employing ‘click’ chemistry: DNA-
binding affinity and anticancer activity. Bioorganic and Medicinal
Chemistry Letters, 2008, 18(4), 1468-1473.
Chapter 11
CLICK CHEMISTRY: OPTICAL SENSING

IN BIOLOGICAL ANALYSIS
Suyan Qiu1, Fang Luo2, Linguang Luo1, Longhua Guo2,

Bin Qiu2, Zhenyu Lin2,* and Guonan Chen2
1
Institute for Quality & Safety and Standards of Agricultural Products
Research, Jiangxi Academy of Agricultural Sciences,
Nanchang, Jiangxi, China
2
MOE Key Laboratory of Analysis and Detection for Food Safety,
Fujian Provincial Key Laboratory of Analysis and Detection Technology
for Food Safety, Department of Chemistry, Fuzhou University,
Fuzhou, China
ABSTRACT
The common limitations of optical sensing are caused by the
complicated functional process, the deficiency of reporter with a robust
signal, and the poor specificity. Therefore, it is still full of challenges to
develop the rapid and simple optical sensing systems with high sensitivity
and selectivity. Click chemistry, the simple and robust connecting
approach with versatile superiorities containing easy to perform,
insensitive to oxygen and water, high specificity and high rate, becomes a
good candidate to relieve these problems of optical sensing. In this
review, we focus on several classic click reactions that used to design
*
Corresponding author: E-mail: zylin@fzu.edu.cn; Fax/Tel: 86-591-22866135.
314 Suyan Qiu, Fang Luo, Linguang Luo, Longhua Guo et al.
optical sensing systems and highlight recent examples to illustrate the

contribution of click chemistry in the biological analysis fields including
biomolecules sensing, pathogens detection, and living cells detection.
Keywords: click chemistry, optical sensing, biological analysis, CuAAC

reaction, SPAAC reaction, thiol-ene reaction
1. INTRODUCTION
Optical sensing has drawn great attention in biomedical [1, 2] diagnostic,
and environmental sciences [3-5] as well as industrial applications [6], because
it has significant advantages compared with conventional sensor: (i) great
sensitivity and wide dynamic range; (ii) electrical passiveness and freedom
from electromagnetic interference; (iii) easy operation and low cost; (iv) can
be carried out on site. Generally, optical sensor consists of two functional
components: an identification element to provide the selective binding with
target analytes and an optical transducer component to output the detectable
signal [7]. Several different types of optical sensings have been frequently
reported depending on the different output signals of transducer, such as
fluorescence sensors, colorimetric sensors, electrochemiluminescence sensors,
photo-luminescence sensors and surface-enhanced Raman scattering sensors.
Among these sensors, fluorescence sensors are one of the most widely
applications in diverse fields with their unique prerequisites except for the
common features of optical sensing platform [8, 9], such as low damage to
targets and easy to handle. Most of fluorescence sensors are designed
depending on the special fluorescent materials or the chemical reaction to
change the property of the fluorescence emission, such as dyes, quantum dots
(QDs) [10, 11] and nano-materials [12, 13] including nanoparticles,
nanowires, carbon nanotubes and graphene. Colorimetric sensors have drawn
high attention in biological analysis by taking into account advantages of low
cost, quick feedback, no need of any advanced instrument, and only relying on
the human eyes conveniently [14, 15]. Currently, nanoparticle-based
colorimetric sensor has attracted much concern in biological molecules
detection and metal ions recognition [16, 17].
The common difficulties of optical sensings are interferences from
multiple effects, such as complicated functional process, deficiency of reporter
with a robust signal, and poor specificity. Therefore, it is still full of challenges
to develop the rapid and simple optical sensing systems with high sensitivity
Click Chemistry: Optical Sensing in Biological Analysis 315
and selectivity. A great deal of work has been studied to improve these
characteristics, such as promoting the binding ability between targets and other
ligands, accelerating the reaction rate, increasing the yield and amplifying the
response signal by nanotechnology.
Click chemistry is termed by Sharpless as the reaction that generates
substances with high selectivity and high efficiency by joining small units
together with heteroatom links (C-X-C) [18]. It is a promising candidate for
the service in the above mentioned missions with various superiorities, such as
high yield, excellent purity and high regioselectivity, good biocompatibility,
mild conditions and high tolerance with versatile solvents [19]. Several
click chemistry reactions have been reported to date, such as copper(I)
catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne
cycloaddition (SPAAC), thiol-ene reaction, oxime ligation and Diels-
Alder reaction (See Fig.1). Table 1 displays the unique advantages and
disadvantages of click chemistry among the reaction rate, yield, stability and
biocompatibility. These “click” reactions have the potential to breakthrough in
the field of labeling and functionalization with high efficiency and specificity,
whereupon becoming an ideal choice to serve as the sensing platform [20, 22].
Figure 1. Summary of classic click chemistry reactions.

Table 1. The advantages and disadvantages of several different click

chemistry reactions
Type Reaction Yield Advantages Disadvantages Reference

rate
CuAAC 10-103 >90% High purity Metal toxicity [23, 24]
M−1s−1 High rate Poor
High stability biocompatibility
Mild condition
SPAAC 2.0-2.9 >89% High purity Low rate [25, 26]
M−1s−1 High stability
Good
biocompatibility
Mild condition
Thiol- 105-107 73%- High purity Need photo [27, 28]
ene M−1s−1 99% High stability initiation
High rate
Good
biocompatibility
Type Reaction Yield Advantages Disadvantages Reference
rate
[
Oxime 10- 45%- High rate Poor stability 29, 30]
ligation 103M−1s−1 72% Good Poor purity
biocompatibility pH-sensitive
Mild condition
Diels- >2000 >87% High rate Poor stability [31, 32]
Alder M−1s−1 Good Poor purity
biocompatibility
Mild condition
In this review, we highlight the properties and routes of different types of

“click” reactions in the optical sensing strategies. Diverse optical sensings that
utilized click chemistry in the biological analysis in recent decades are
summarized including cells, proteins, nucleic acids and small biomolecules
detections, as well as the advantages of different “click” reactions in
biosensing systems are demonstrated. Through this review, we aim to provide
a summary on how to employ a suitable “click” reaction to design the
outstanding sensing platform.
2. CLICK CHEMISTRY FOR OPTICAL SENSING

IN BIOLOGICAL ANALYSIS
2.1.1. Copper(I) Catalyzed Azide-Alkyne Cycloaddition

(CuAAC) Reaction
The CuAAC reaction is a typical Huisgen 1,3-dipolar cycloaddition.

Rostovtsev et al. have claimed CuAAC reaction is initiated by the formation of
an acetylide preassociation complex through the coordination of the terminal
azide group to the copper(I), and then regioselectively generates a 1,4-
disubstituted 1,2,3-triazole by a stepwise mechanism [33]. The participation of
dinuclear copper clusters in the catalytic process is verified by Fokin group
through real-timely monitoring of a representative cycloaddition process. They
demonstrated that monomeric copper acetylide complexes were reactive
toward terminal azides under the participation of an exogenous copper catalyst
[34, 35]. The product of 1,2,3-triazole is high stable against water and ambient
oxygen, shows a good biocompatibility to many other biomolecules.
Chidsey demonstrated the 1,2,3-triazole compounds can be mediated
quantitatively by the copper(I) ions concentration [36, 37], suggesting that the
copper(I) ions can be used as a bridge to connect the sensing [38, 39]. The
CuAAC reaction is a convenient synthetic approach for conjugating the two
components, making it to be a good choice to unite the reporter and the
binding domain of target molecules in the design of optical sensing platforms
[40, 41]. Hulme and coworkers firstly employed the catalytic effect on the
CuAAC reaction to detect the copper(I) concentration by monitoring the
formation of the 1,2,3-triazole compounds in biology (complex 1 in Figure
2A) [42]. They found that the triazole ring of the complex 1 could be acted as
a spacer to prevent dansyl from coordinating with the europium, inducing a
great enhancement of the europium luminescence emission. While lacking a
triazole ring in the similar structure to the complex 1, the luminescence
intensity was weak owing to the quenching from the coordination with the
dansyl sulfonamide. Subsequently, Jiang and coworkers reported an approach
for visual detection of copper(II) upon the CuAAC reaction [43]. As shown in
Figure 2B, in the presence of copper(II) and sodium ascorbate, the previous
well-dispersed azide- and terminal alkyne- functionalized AuNPs were
aggregated together under the catalysis of copper(I) species, leading to the
solution color change from red to colorless. Another colorimetric sensor for
copper(II) ions detection was demonstrated using the terminal azide- and
terminal alkyne-DNA modified AuNPs by Mirkin and coworkers [44]. These

studies further confirmed the CuAAC reaction can be quantitatively triggered
to produce 1,2,3-triazole ligands by the copper(I) catalyst. Because the
copper(I) catalyst can be derived from the oxidation of copper(II) by
ascorbate, a CuAAC reaction-based visual method was further expanded to
detect ascorbic acid [45]. The limit of detection (LOD) was as low as 3 nM,
which was caused by the high efficiency of CuAAC reaction. In addition, the
fluorescence sensing systems based on the CuAAC reaction have been
developed for copper ions determination as well. For example, Huang and
coworkers reported a “turn-on” fluorescent probe for copper ions detection
based on the DNA-templated CuAAC reaction [46]. A novel near-infrared
fluorescence sensing platform was introduced to detect copper ions by
combining the CuAAC ligation and energy transfer [47].
Figure 2. (A) Sensitized Eu(III) chelate 1 generated through the CuAAC reaction in the
presence of a copper(I) glutathione complex with micromolar concentrations.
Reprinted with permission from J. Am. Chem. Soc. (ref 42). Copyright 2006 American
Chemical Society. (B) The copper(II) ions detection based on CuAAC reaction
between the alkyne (1) and the azide (2) terminated gold nanoparticles. Reprinted with
permission from Angew. Chem. Int. Ed. (ref 43). Copyright 2008 Wiley-VCH Verlag
& Co. KGaA, Weinheim, Germany.
2.1.2. CuAAC Reaction in DNAs and Proteins Detection
Most of the above stated sensors are limited in the detection of copper(II)
ions and ascorbic acid, other targets are rarely involved based on the catalytic
effect. In this context, Jiang and coworkers proposed a colorimetric
immunoassays by the copper-mediated amplification, which breaks through

the constraint in the copper(II) ions determination [48], in which a traditional
sandwich structure consisting of target antigen, primary antibody and
secondary antibody was designed. It is noted that the secondary antibody was
previously labeled with copper monoxide nanoparticle (CuO), which could
release copper(II) ions by HCl, therefore catalyzing the CuAAC reaction
between azide- and terminal alkyne-DNA modified AuNPs and causing the
aggregation of AuNPs. Thus, the solution color would change from red to
purple by triggering the CuAAC reaction once the antibody captured target
antigen. 150 ng/mL of target antigen concentration could be detected by the
naked eyes, which was the same level as that measured by the UV/vis
spectrometry, suggesting the method shows high sensitivity. Besides,
compared with previous published method based on the CuAAC reaction, the
detection time was greatly shorten (from 24 hours to 10 minutes), further
indicating the CuAAC reaction is high rate and high efficiency. Such method
provides a way to expand the CuAAC reaction to other targets detection with
high sensitivity in short time, not just the catalysts such as copper(II) ions and
ascorbic acid.
Lin and coworkers have reported several optical sensors to other macro-
biomolecules targets by exploring the effects of other species on the catalytic
system, such as histidine [49], DNAs [50, 51], and enzymes [52], which
further confirms that the CuAAC reaction can be used to identify more other
targets except for the antigen. For example, as shown in Figure 3,
pyrophosphate can capture copper(II) ions to form a new complex of
Cu(II)/pyrophosphate, which inhibits the reduction of copper(II) into copper(I)
catalyst by ascorbate, resulting in the fact that CuAAC reaction cannot be
triggered effectively. While the Cu(II)/pyrophosphate complex is destroyed in
the presence of pyrophosphatase that can catalyze the hydrolysis of
pyrophosphate into inorganic phosphate, and releases copper(II) ions, which in
turn initiates the CuAAC reaction to produce a highly fluorescent triazole
complex. Besides, a bioluminescent nanosensor for protease detection based
on AuNPs-luciferase conjugates with site-specifically labeling through the
CuAAC reaction was demonstrated [53]. Dash and coworkers employed the
double CuAAC reactions to synthesize a guanosine-based fluorescent probe to
discriminate the c-myc G-quadruplex DNA sequence with high selectivity
[54]. Moreover, the probe exhibited a specific cytotoxicity to the human
melanoma A375 cells over normal human keratinocyte cells, promoting the
cell death.
Figure 3. The structure and sensing mechanism of fluorescent sensor for

pyrophosphatase detection. Reprinted with permission from Anal. Chem. (ref 52).
Copyright 2015 American Chemical Society.
2.1.3. CuAAC Reaction in Small Biomolecules Sensing
Several other optical sensing platforms were developed to identify small

molecules depending on the 1,2,3-triazole as a ligand rather than the above
mentioned that using copper(I) catalyst as a linking bridge [55-57]. For
instance, Dash and coworkers synthesized a water soluble fluorescent
molecular probe to recognize heavy and transition metal ions in living cells,
such as Zn2+ ions that can greatly increase the fluorescence intensity, and Fe2+
ions and Cu2+ ions which can quench the fluorescence effectively [58]. Bi and
coworkers prepared a series of acidic fluorescent pH probes using the rapid
CuAAC reaction, in which all probes were non-fluorescent under basic
conditions, whereas the strong fluorescence intensities were achieved when
these probes were shifted to acidic conditions. The best performance of these
probes could increase over 800 folds as the pH value decreased from 8.0 to
4.1. Furthermore, the probe was not affected by the cell mediums when
applied to functionally determine pH alterations in cancer cells, revealing the
outstanding capacity of the probe for monitoring the intracellular [H+] levels
(Figure 4A) [59]. Except for the above mentioned cations determination [60,
61], the triazole and triazolium groups have been explored to identify anions
as well. For example, Belfield and coworkers developed a highly selective

fluorescence turn-on sensor to determine fluoride by the formation of the
hydrogen-bonding complex between the triazolium ring and fluoride [62]. As
shown in Figure 4B, two types of interactions were involved in the turn-on
sensing process: the first was the formation of the hydrogen-bonding complex
with initial addition of fluoride; the second one was deprotonation of the
triazolium group when added more fluoride, resulting in the formation of FHF-
and two deprotonated species, igniting the fluorescence. A high specificity was
validated for the fluoride detection through investigating the interference
effect from other anion ions.
Figure 4. A: (a) Fabrication of fluorescent pH probes 1-3; (b) Absorbance and emission
spectra of probe 3 with different pH values at 480 nm excitation wavelength. Reprinted
with permission from Bioorg. Med. Chem. Lett. (ref 59). Copyright 2012 Elsevier
B.V.. B: (a) Schematic illustration of the fluorescence turn-on sensing for fluoride
detection; (b) Fluorescence spectra and intensity with the addition of various other
anions (λex = 385 nm). Reprinted with permission from ACS Appl. Mater. Interfaces
(ref 62). Copyright 2013 American Chemical Society.
Besides, several other CuAAC reactions were demonstrated in the optical

platforms relying on the unique structure and property of alkynyl/azido group
[63]. For instance, Chen and coworkers reported a novel colorimetric
molecular switch for sensing flumioxazin based on the activity of the G-
quadruplex-hemin DNAzyme [64]. As shown in Figure 5, azido group can

coordinate with hemin to form hemin-N3 complex, hence the G-quadruplex
cannot combine with hemin to form the G-quadruplex-hemin DNAzyme
which is a mimic horseradish peroxidase. In the presence of propargylamine,
azido group reacted with propargylamine rapidly through the CuAAC reaction,
inducing that the hemin was free in the solution, and bound with the G-
quadruplex to yield G-quadruplex-hemin DNAzyme, and then catalyzed the
substrate effectively. In the study, flumioxazin, one of the pesticides with
alkynyl group, was chosen as an example to demonstrate the extensive
application of the proposed switch. The high sensitivity as well as good
specificity was confirmed, which attributed to the high efficiency and high
selectivity of the CuAAC reaction.
Figure 5. A: Scheme of the colorimetric switch based on the CuAAC reaction. B:

Photograph images of the different reaction tubes: (a) hemin + CatG4; (b) hemin +
CatG4 + NaN3; (c) hemin + CatG4 + NaN3 + propargylamine + Cu(II) +sodium L-
ascorbate; (d) hemin + CatG4 + NaN3 + Cu(II) + sodium L-ascorbate. Reprinted with
permission from Analyst (ref 64). Copyright 2013 Royal Society of Chemistry.
2.1.4. CuAAC Reaction In Vivo Sensing
Owing to the copper toxicity to proteins and cells, the CuAAC reaction is
considered to be not appropriate for vivo detection. However, Pezacki group
found the Cu(II)-L-histidine complex (Cu(his)2) showed no significant toxicity
in human cell lines and lipid metabolism after 72 h at micromolar

concentration and became an effective catalyst for the CuAAC reaction in
living cells [65]. The result reveals the CuAAC reaction is possible to be
exploited in living system. Subsequently, Rubino group presented a chemo-
selective modification of viral surface using the CuAAC reaction [66], which
broadens a way to explore the implementation of the CuAAC reaction in
pathogen assays. Meanwhile, a high through-put approach for pathogen-host
AMPylation (adenylylation) detection was described by Yu and coworkers
using self-assembled protein microarrays [67]. However, these studies cannot
satisfy the vivo assay because of metal toxicity. It still remains a challenge to
enlarge the application for vivo sensing using the CuAAC reaction.
2.2. STRAIN-PROMOTED AZIDE-ALKYNE

CYCLOADDITION (SPAAC) REACTION
Strained cyclooctyne exhibits a massive bond angle deformation which

can induce the destabilization of the ground state and provides a dramatic rate
acceleration to react with azides without auxiliary reagents, which is known as
the SPAAC reaction [68]. Thus the SPAAC reaction avoids the problem from
the metal catalyst, such as cell toxicity and protein toxicity [69]. In other
words, the SPAAC reaction can be used to sensing in living system with its
particular merits. Chirivi and coworkers compared two “click” reactions in
enzyme-linked immunosorbent assay (ELISA) through the reactions between
the azide functionalized human anti-citrulline antibody and the coating with
terminal alkyne or with bicyclooctyne [70]. They found the coating with
terminal alkyne showed a high background level due to the coordination of
copper catalyst with the amide bonds of the immobilized peptides, which
readily introduced a false positive signal. The coating with cyclononyne was
successfully utilized in the ELISA assay with a much lower background
signal. Hence, the excellent optical properties of the product via the SPAAC
reaction maybe more appropriate for the utilization in optical sensing system
for the bioanalysis, such as DNAs analysis, proteins and pathogens detection,
and cells detection [71-74].
Figure 6. A: (a) Structure of PA with a cyclooctyne group. (b) Scheme of the process
of fabricating PA-decorated surfaces using the SPAAC reaction. (c) Light microscopy
image of water condensation experiment on PA-decorated glass surface (1 μM, 60
min). Reprinted with permission from J. Am. Chem. Soc. (ref 75). Copyright 2013
American Chemical Society. B: Schematic illustration of the principle for DNA (RNA)
detection based on the SPAAC reaction. Reprinted with permission from ACS Nano.
(ref 76). Copyright 2014 American Chemical Society.
2.2.1. SPAAC Reaction in DNAs Analysis
A polyamide (PA) microarray for recognizing the double strand DNA

(dsDNA) sequence was designed by Ravoo and coworkers [75]. As shown in
Figure 6A, the SPAAC reaction was utilized to immobilize PA on the glass
substrate, inducing a significant decrease of water contact angle because of the
higher hydrophilic nature of PA to the azide surface (Figure 6Ac), and then
selectively coupled with Cy3-dsDNA. The development of the sensing
platform not only provides an initial high-throughput screening tool to
interrogate the dsDNA binding profile, but also offers a new opportunity to
facilitate the immobilization of DNA nanostructures in predefined locations.

Oishi group reported an ultrasensitive colorimetric DNA and RNA detection
using an enzyme-free SPAAC reaction between N3-DNA-modified AuNPs
and a probe-dibenzocyclooctyne (probe-DBCO) (Figure 6B) [76]. During the
SPAAC reaction, a thermal cycling process of hybridization between probe
DNA and target DNA (RNA) was adopted to amplify the sensitivity. The
combination of the SPAAC reaction with high efficiency and thermal cycling
leads a 50 zM of both target DNA and target RNA to be detected. More
importantly, after magnetic separation, the supernatant was selected to be
detected by the UV/vis measurement rather than the precipitate that consisted
of more complicated sandwich structure among AuNPs/dsDNA/magnetic
beads, making the method operate easily. The proposed method greatly
improve the sensitivity and specificity using the SPAAC reaction, further
indicating the SPAAC reaction may provide an excellent optical sensing for
the DNA analysis.
2.2.2. SPAAC Reaction in Proteins and Pathogens Detection
Hu and coworkers developed a colorimetric and plasmonic method for

lectins determination based on the SPAAC reaction, which breaks through the
restriction in DNAs analysis [77]. In the report, an amphiphilic glycolipid
produced from the SPAAC reaction between azido galactoside and a lipid
cyclooctyne can be embedded into the polyethylene glycol-coated AuNPs,
therefore forming core-shell Au glycoNPs. The core-shell Au glycoNPs were
dispersed well in the solution and displayed a red color. While aggregated Au
glycoNPs were produced with the addition of lectins because of their specific
interaction with glycolipids, the solution color changed from red to purple and
the plasmonic resonance scattering enhanced significantly (Figure 7A).
In addition, several SPAAC reactions were exploited to detect pathogens.
Chen group developed an ultrasensitive sensing for pathogen detection with
the naked eyes depending on the stable triazole ligation through the SPAAC
reaction [78]. Vauzeilles and coworkers proposed a rapid and specific
enrichment way for the Gram negative bacteria by combining the SPAAC
reaction with magnetic beads separation, which may greatly improve the
sensitivity for the pathogenic bacteria identification [79]. They immobilized
the azido group onto the surface of bacteria, and the cyclooctyne was coupled
with magnetic beads through the biotin-streptavidin interaction. The bacteria
could be trapped after the magnetic separation when the azido group
functionalized bacterial bound with cyclooctyne functionalized magnetic
beads. So the high specific enrichment is attributed to the high efficiency and
high selectivity of the SPAAC reaction.
Figure 7. A: Scheme of the simple fabrication for core−shell glycol AuNPs promoted
by the SPACC reaction. Reprinted with permission from ACS Appl. Mater. Interfaces
(ref 77). Copyright 2015 American Chemical Society. B: (a) Schematic depiction for
the reaction of azide terminated EpCAM with DIBO-AF594 to produce a fluorescently
labeled aptamer through the SPACC reaction (Right); (b) Dark field-fluorescent
microscopic imaging of cancer cells with EpDNA-DIBO-AF594. Left panel of each
cell line corresponds to: dark field + DAPI and on the right panel: DAPI + AF594
merged. Scale bar indicates 50 µm. Reprinted with permission from Chem. Commun.
(ref 80). Copyright 2014 Royal Society of Chemistry.
2.2.3. SPAAC Reaction in Cells Detection
The SPAAC reaction was used to image cells as well due to its excellent
biocompatibility. In the Krishnakumar group, the SPAAC reaction was
adopted to synthesize a novelfluorescent aptamer probe to image cancer cells

[80]. An alexa-fluor 594 fluorescent dye conjugated dibenzocyclooctynol
(DIBO-AF594) reacted with the azide terminated epithelial cell adhesion
molecule (EpCAM) aptamer. Four different cell lines (breast cancer cell line:
MCF7 and MDAMB453; retinoblastoma cell line: Weri-Rb1; prostate cancer
cell line: PC3 and low EpCAM expressing-Muller glial cell line: MIO-M1.)
were selected to study the percentage of the EpCAM expression by using the
previously prepared aptamer-fluorescent conjugation. Over 80 to 90% was
expressed in MCF7 and MDAMB453 cells, and 35 to 45% was detected in
PC3 and Weri-RB1 cells, while EpCAM expression in MIO-M1 cells was less
than 5%. They also investigated the internalization of the EpDNA-DIBO-
AF594 in the above four different cells. As displayed in Figure 7Bb, because
of the internalized EpDNA-DIBO-AF594 in MCF7, MDAMB453, Weri-Rb1
and PC3 cells, the cytoplasmic and nuclear straining indicated by white arrows
could be observed clearly. Moreover, the SPAAC reaction has also been
expanded to the live cells analysis. The implication of the SPAAC reaction in
intracellular imaging was evaluated through comparing with Halo Tag protein
technology by Johnson group [81]. Lemke group described a protocol for the
rapid labeling of cell-surface proteins in living mammalian cells based on the
SPAAC reaction [82], in which noncanonical amino acids functionalized by
the ring-strained alkynes firstly and then rapidly reacted with dyes bearing
azide groups. These results further confirm that the SPAAC reaction may have
great potential applications in vivo detection, which breaks through the metal
toxicity from the CuAAC reaction. However, most of strained- alkynes are
expensive and not readily available, limiting the application of SPAAC
reaction. It is an essential issue to develop techniques to reduce the cost and
simplify the synthetic process of strained-alkynes.
2.3. THIOL-ENE REACTION

Generally, the thiol-ene radical reaction (termed as the thiol-ene reaction)
contains two steps: (1) the hydrogen radical from the thiol group is abstracted
to form the thiyl radical by the carbon-centered radical; (2) the cycle process is
triggered between the thiyl radical propagation across the ene group and the
chain-transfer reaction [83]. Owing to the light-mediated thiol-ene reaction
combines the advantages of both CuAAC reaction and SPAAC reaction, such
as triggering in aqueous solution with high efficiency and high reaction speed
and without toxic metal catalyst [84-88], it becomes one of the most prevalent
tools in chemical synthesis and biological conjugation. More importantly, the

reaction can be activated at specific time and location by the ultra-violate (UV)
irradiation. On the basis of these facts, Hawker and coworkers fabricated
multifunctional high-throughput microarrays on the surface of a hydrogel
substrate successfully, which provides a perfect platform to collect assorted
chemical signals [89]. Lafleur and coworkers presented a rapid and simple
method to prepare emulsion-templated monoliths in microfluidic channels
depending on the thiol-ene reaction. They claimed that the method exhibited
monolith synthesis and anchoring inside the micro-channels in a single
photoinitiated step. Moreover, the enzymes can be readily immobilized on the
prepared monoliths via the formation of disulfides with a good reversibility
and still remained a high enzymatic activity in enzyme assay, which offers a
promising stationary phase for the on-chip separation [90]. These studies
suggest the possibility of the thiol-ene reaction applied in biomolecules
sensing by taking advantage of the excellent biocompatibility and cost-
effectiveness.
2.3.1. Thiol-ene Reaction in DNAs and Proteins Detection
Several optical sensing systems were developed using the thiol-ene

“click” reaction as a labeling process [91-96]. For instance, an aptamer
decorated organic-silica hybrid monolithic column was prepared utilizing the
thiol-ene reaction [97]. The high surface area and uniform distribution of
active groups on the hybrid silica monolith offered a high aptamer coverage
density (which was up to 420 pmol/µL), greatly increasing the binding sites
with the target (Figure 8A). Meanwhile, the strategy showed a high specificity
to capture thrombin. 91.8% of the extraction recovery in human serum was
achieved. Simon and coworkers firstly immobilized the thiolated fluorescein-
substituted lysine on the surface of porous cellulose nanocrystal-poly(vinyl
alcohol) substrates and then used it as a fluorescent biosensing platform to
identify the protease activity by binding a Förster-type resonance energy
transfer chromophore pair that can quench the fluorescence [98]. The
originally quenched fluorescence was switched on because protease can
separate the chromophores through degrading the protein linker. In the
meanwhile, a novel two-step method started from bulk silicon wafers to
oligonucleotide (ODN) conjugated silicon nanoparticles (SiNPs) was
illustrated by Fink and coworkers [99]. Firstly, they employed reactive high-
energy ball milling (HEBM) to produce alkene grafted SiNPs. And then the
thiol-labeled ODN with fluorescein reporter was added to the above solution,
which reacted with the alkene moieties to yield ODN-SiNPs conjugates via the
thiol-ene reaction. A further application of these ODN-SiNPs conjugates was
explored to detect cancer-associated miR-21 through introducing a quencher
strand (Figure 8B), suggesting that the labeling ODN-SiNPs based on the
thiol-ene reaction is carried out readily and remains highly stable in the
biological environment.
Figure 8. A: Scheme for preparation of aptamer-based hybrid monolithic column via

“thiol-ene” reaction and the implementation in capturing thrombin with chromogenic
assay. Reprinted with permission from Talanta (ref 97). Copyright 2015 Elsevier B.V..
B: Schematic depiction for the ODN-SiNPs conjugates fabrication based on the thiol-
ene reaction and the application for miR-21 detection. Reprinted with permission from
Bioconjug. Chem. (ref 99). Copyright 2014 American Chemical Society.
2.3.2. Thiol-ene Reaction in Small Biomolecules Detection
Several optical small biomolecules sensing platforms were designed

depending on the thiol-ene reaction directly rather than that used as a labeling
approach. For example, Carlson and coworkers described a simple method for
the chemical differentiation of thiols and thiophosphorylated groups, such as
thiophosphorylated kinase substrates [100]. In the Lou’s group, a series of
non-emissive dye tetraphenylethene (TPE) derivatives were synthesized to
detect biological thiols including glutathione (GSH) and its dimeric form
(GSSH) based on the aggregation-induced emission (AIE) [101, 102]. The
fluorescence of these dyes can be switched on/off when the analytes alter the
aggregation behavior or change the intramolecular motions of the fluorescent
species. In the study, the GSH and GSSH can react with TPE derivatives to
form poorly soluble aggregates through the thiol-ene reaction, which turns on
the fluorescence. Thus the approach can serve as a label-free sensor for
enzymatic activity assay of glutathione reductase. Also, it is further explored
to detect the intracellular GSH in living cells, suggesting the thiol-ene reaction
shows a high specificity for vivo sensing.
2.4. OTHER CLICK REACTIONS

2.4.1. Oxime Ligation
The oxime ligation is a reaction between the aminooxy group and

aldehyde group or ketones group, producing an oxime-conjugate and one
molecule of H2O, which shows fast and low cost, without metal catalysts.
However, aldehyde groups may be oxidized and react with themselves or other
nucleophiles, so the aminooxy groups can bind with electrophiles and the
products of oxime-conjugates are sensitive to pH [103], leading to less
attention paid to oxime ligation. However, the specificity and the rate of
exchange can be further improved by the catalysis including heat, acids and
nucleophiles [104]. Therefore, the applications in biological analysis were
investigated [105, 106], Meijler and coworkers firstly exploited an aniline-
catalyzed oxime formation to immobilize the fluorescent ligands to the
proteins, and then it was used to visualize the specific native proteins in living
cells [107]. To avoid the disturbance from the endogenous carbonyl moieties
including pyruvic acid and glucose that may react with aminooxy group, the
aniline-catalyzed oxime reaction was optimized, and followed by
appropriately washing the cells to remove unreacted ligands. Wu and
coworkers reported an intramolecular three-color fluorescence resonance
energy transfer (FRET) probe to study the protein structure, folding and
interactions by the site-specific labelin [108]. In the study, three different
fluorophores were labeled on the protein: (1) N-terminal fused with the
enhanced green fluorescent protein (EGFP, donor); (2) C-terminal bonded
with the keto-rhodamine (Acceptor 1) through oxime ligation; (3) a cysteine
side chain within the protein conjugated with the Dy630-maleimide (Acceptor
2) (Figure 9A). The Förster distances (R0s) for the EGFP/rhodamine pair and
rhodamine/Dy630 pair were estimated to be 5.8 nm and 6.8 nm, respectively,
which suggesting that the distance among three-color probes were sufficient to
confer efficient FRET. Furthermore, a novel chemical conjugation through
biorthogonal oxime ligation was developed to target cancer cells in vivo by
Cheng and coworkers [109]. As shown in Figure 9Ba, an oxyamine (Oa) group
was decorated on 4T1 murine breast cancer cells through liposome delivery
and fusion firstly, and then reacted with a fluorescent rhodamine with
aldehyde groups functionalized poly(ethylene glycol)-polylactide (PEG-PLA)
nanoparticles (Ald-NPs) to form the fluorescent Ald-NPs immobilized cells,
which can be embedded into cells depending on the liposome fusion process,
inducing a intracellular fluorescence. In the study, Native 4T1 cells and o4T1
cells were treated with Cy5 labeled NPs or Ald-NPs. It was noticed from
Figure 9Bb that only a slightly higher level of NP binding or uptake were
found in native 4T1 cells treated with Ald-NPs and o4T1 cells treated with
NPs or without Ald on the surface (Figure 9Bb). The cellular binding and
internalization were significantly enhanced when the o4T1 cells were treated
with Ald-NPs, because of the specific and efficient oxime ligation between the
Oa group of cell surface and Ald group on Ald-NPs. This outcome suggests
that the cancer targeting can be greatly improved by the Oa-Ald oxime
ligation, which is important to intracellular delivery of anticancer drugs and
the efficacy against cancer cells.
2.4.2. Diels-Alder Reaction
Another common type of “click” reaction is the Diels-Alder reaction,

which refers to a [4+2] cycloaddition that occurs between the diene group and
the dienophile, yielding cyclohexene derivative. Owing to the high kinetic of
the Diels-Alder reaction with high quantification, it is exploited to establish
several new methods for analysis, such as cholesterol and cholecalciferol [110,
111]. Moreover, the Diels-Alder reaction is accepted by cellular enzymes
because of its sufficiently small molecules. Wittmann and coworkers used a
small strained cycloproene to monitor the glycosylation of both cell-surface
glycoconjugates and isolated glycoproteins [112]. The N-acylgalactosamine
and its derivatives were labeled by cyclopropene tags, leading to a significant
fluorescence straining of cell-surface glycoconjugates through the Diels-Alder
reaction.
Figure 9. A: (a) Schematic illustration for the preparation of the triple FRET probe. (b)
Structural model for the calculated and experimentally determined distances of triple-
labeled protein probes. Structures of Rab1b (PDB: 3NKV) and EGFP (PDB: 2Y0G)
were used. Reprinted with permission from J. Pept. Sci. (ref 108). Copyright 2014
European Peptide Society and John Wiley & Sons, Ltd. B: (a) Scheme for cancer
targeting of aldehyde groups modified PEG-PLA nanoparticles surface depending on
the bioorthogonal oxime ligation. Cancer cells were coupled with Oxyamine (Oa)
groups by membrane fusion of liposomes bearing Oa groups (Oa-Lip). (b) Confocal
microscopic images of native 4T1 cells (4T1) and 4T1 cells with surface expressed Oa
groups (o4T1) by treating with Cy5 labeled PEG-PLA NPs (red) in the absence or
presence of Ald groups (denoted as NP and Ald-NP respectively). The nuclei were
stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 5 mm. Reprinted
with permission from Chem. Sci. (ref 109). Copyright 2015 Royal Society of
Chemistry.
CONCLUSION AND OUTLOOKS

Click chemistry with high efficiency, mild condition, and excellent
specificity becomes a good candidate for optical sensing platforms in the
biological analysis. Easy functionalization routes for surfaces of various
material and simple synthetic paths for diverse molecules are provided by
click chemistry, which can be utilized to carry out the transduction of the
binding event toward analytes with adaptable affinity and specificity.
Particularly, the product can be generated quantitatively with high efficiency
and high selectivity through the clickable reaction, which offers a powerful
tool to identify targets. The sensing sensitivity can be further improved by
taking advantage of unique materials, such as nanoparticles, films and
microarrays, which enables the sensing platform to expand over a broad range
of targets in the biological analysis, even high throughput detection.
Although some restrictions of clickable reactions are discovered by taking
into account the sensing conditions. For instance, owing to the toxicity of
copper(I), the implementations of the CuAAC reaction in several living beings
readily are subject to the copper toxicity. However, the SPAAC reaction
overcomes the interference of metal toxicity, and the reaction rate and
conversion can be greatly improved by the chemical structure of cyclooctyne,
leading to a wild application in many living systems. Therefore, as described
in this review, numerous click chemistry-based optical sensing platforms have
been fashioned to illustrate the importance of biological analysis. Given the
particular advantages, click chemistry will continue to emerge as the high
efficient and robust synthetic method for the design of the optical sensors, and
continue to breakthrough for monitoring biological process in living systems.
ACKNOWLEDGMENTS
This work was financially supported NSFC for Excellent Youth Scholars
of China (21222506), NSFC (21175024, 21275031 and 21605063), Nature
Sciences Funding of Fujian Province (2014J06005), program for New Century
Excellent Talents in University (NCET-12-0619), and the innovation fund of
Jiangxi Academy of Agricultural Sciences (No. 2013CBS001).
Conflict of Interest: The authors declare that they have no conflict of interest.
REFERENCES
[1] Stanca, S. E.; Matthäus, C.; Neugebauer, U.; Nietzsche, S.; Fritzsche,
W.; Dellith, J.; Heintzmann, R.; Weber, K.; Deckert, V.; Krafft, C.;
Popp, J. (2015) Chemo-spectroscopic sensor for carboxyl terminus
overexpressed in carcinoma cell membrane. Nanomedicine, 11, 1831-
1839.
[2] Tran, T. M.; Alan, Y.; Glass, T. E. (2015) A highly selective fluorescent
sensor for glucosamine. Chem. Commun., 51, 7915-7918.
[3] Yadav, A. K.; Shen, D. L.; Shan, X.; He, X.; Kermode, A. R.; Vocadlo,
D. J. (2015) Fluorescence-quenched substrates for live cell imaging of
human glucocerebrosidase activity. J. Am. Chem. Soc., 137, 1181-1189.
[4] Sletten, E. M.; Swager, T. M. (2014) Fluorofluorophores: fluorescent
fluorous chemical tools spanning the visible spectrum. J. Am. Chem.
Soc., 136, 13574-13577.
[5] Payra, S.; Soni, M.; Kumar, A.; Prakash, D.; Verma, S. (2015)
Intercomparison of aerosol optical thickness derived from MODIS and
in situ ground datasets over Jaipur, a semi-arid zone in India. Environ.
Sci. Technol., 49, 9237-9246.
[6] Kouroussis, G.; Caucheteur, C.; Kinet, D.; Alexandrou, G.; Verlinden,
O.; Moeyaert, V. (2015) Review of trackside monitoring solutions: from
strain gages to optical fibre sensors. Sensors, 15, 20115-20139.
[7] Wang, Y.; Yan, B.; Chen, L. (2013) SERS tags: novel optical
nanoprobes for bioanalysis. Chem. Rev., 113, 1391-1428.
[8] Tan, C.; Yu, P.; Hu, Y.; Chen, J.; Huang, Y.; Cai, Y.; Luo, Z.; Li, B.;
Lu, Q.; Wang, L.; Liu, Z.; Zhang, H. (2015) High-yield exfoliation of
ultrathin two-dimensional ternary chalcogenide nanosheets for highly
sensitive and selective fluorescence DNA sensors. J. Am. Chem. Soc.,
137, 10430-10436.
[9] Jin, Z.; Geißler, D.; Qiu, X.; Wegner, K. D.; Hildebrandt, N. (2015) A
rapid, amplification-free, and sensitive diagnostic assay for single-step
multiplexed fluorescence detection of microRNA. Angew. Chem. Int.
Ed., 54, 10024-10029.
[10] Wu, Q.; Chen, L.; Huang, L.; Wang, J.; Liu, J.; Hu, C.; Han, H. (2015)
Quantum dots decorated gold nanorod as fluorescent-plasmonic dual-
modal contrasts agent for cancer imaging. Biosens. Bioelectron., 74, 16-
23.
[11] Yan, X.; Li, H.; Han, X.; Su, X. (2015) A ratiometric fluorescent
quantum dots based biosensor for organophosphorus pesticides
detection by inner-filter effect. Biosens. Bioelectron., 74, 277-283.
[12] Nair, L. V.; Nazeer, S. S.; Jayasree, R. S.; Ajayaghosh, A. (2015)
Fluorescence imaging assisted photodynamic therapy using
photosensitizer-linked gold quantum clusters. ACS Nano, 9, 5825-5832.
[13] Jeong, H. Y.; Baek, S. H.; Chang, S. J.; Cheon, S. A.; Park, T. J. (2015)
Robust fluorescence sensing platform for detection of CD44 cells based
on graphene oxide/gold nanoparticles. Colloids Surf. B: Biointerfaces,
135, 309-315.
[14] Xianyu, Y.; Xie, Y.; Wang, N.; Wang, Z.; Jiang, X. (2015) A
dispersion-dominated chromogenic strategy for colorimetric sensing of
glutathione at the nanomolar level using gold nanoparticles. Small, 11,
5510-5514.
[15] Lu, L.; Xia, Y. (2015) Enzymatic reaction modulated gold nanorod end-
to-end self-assembly for ultrahigh sensitively colorimetric sensing of
cholinesterase and organophosphate pesticides in human blood. Anal.
Chem., 87, 8584-8591.
[16] Saha, K.; Agasti, S. S.; Kim, C.; Li, X.; Rotello, V. M. (2012) Gold
nanoparticles in chemical and biological sensing. Chem. Rev., 112,
2739-2779.
[17] Alsager, O. A.; Kumar, S.; Zhu, B.; Travas-Sejdic, J.; McNatty, K. P.;
Hodgkiss, J. M. (2015) Ultrasensitive colorimetric detection of 17β-
estradiol: The effect of shortening DNA aptamer sequences. Anal.
Chem., 87, 4201-4209.
[18] Kolb, H. C.; Finn, M. G.; Sharpless, K. B. (2001) Click chemistry:
diverse chemical function from a few good reactions. Angew. Chem. Int.
Ed., 40, 2004-2021.
[19] McKay, C. S.; Finn, M. G. (2014) Click chemistry in complex mixtures:
bioorthogonal bioconjugation. Chem. Biol., 21, 1075-1101.
[20] Horisawa, K. (2014) Specific and quantitative labeling of biomolecules
using click chemistry. Front Physiol., 5, 457.
[21] Tobimatsu, Y.; Van de Wouwer, D.; Allen E.; Kumpf, R.; Vanholme,
B.; Boerjan, W.; Ralph, J. (2014) A click chemistry strategy for
visualization of plant cell wall lignification. Chem, Commun., 50,
12262-12265.
[22] Lau, Y. H.; Rutledge, P. J.; Watkinson, M.; Todd, M. H. (2011)
Chemical sensors that incorporate click-derived triazoles. Chem. Soc.
Rev., 40, 2848-2866.
[23] Uttamapinant, C.; Tangpeerachaikul, A.; Grecian, S.; Clarke, S.; Singh,
U.; Slade, P.; Gee, K. R.; Ting, A. Y. (2012) Fast, cell-compatible click
chemistry with copper-chelating azides for biomolecular labeling.
Angew. Chem. Int. Ed., 51, 5852-5856.
[24] Hein, J. E.; Fokin, V. V. (2010) Copper-catalyzed azide-alkyne
cycloaddition (CuAAC) and beyond: new reactivity of copper(I)
acetylides. Chem. Soc. Rev., 39, 1302-1315.
[25] Dommerholt, J.; van Rooijen, O.; Borrmann, A.; Guerra, C. F.;
Bickelhaupt, F. M.; van Delft, F. L. (2014) Highly accelerated inverse
electron-demand cycloaddition of electron-deficient azides with
aliphatic cyclooctynes. Nat. Commun., 5, 5378.
[26] Ornelas, C.; Broichhagen, J.; Weck, M. (2010) Strain-promoted alkyne
azide cycloaddition for the functionalization of poly(amide)-based
dendrons and dendrimers. J. Am. Chem. Soc., 132, 3923-3931.
[27] Northrop, B. H.; Coffey, R. N. (2012) Thiol-ene click chemistry:
computational and kinetic analysis of the influence of alkene
functionality. J. Am. Chem. Soc., 134, 13804-13817.
[28] Tyson, E. L.; Ament, M. S.; Yoon, T. P. (2013) Transition metal
photoredox catalysis of radical thiol-ene reactions. J. Org. Chem., 78,
2046-2050.
[29] Dirksen, A.; Dawson, P. E. (2008) Rapid oxime and hydrazone ligations
with aromatic aldehydes for biomolecular labeling. Bioconjug. Chem.,
19, 2543-2548.
[30] Ulrich, S.; Boturyn, D.; Marra, A.; Renaudet, O.; Dumy, P. (2014)
Oxime ligation: a chemoselective click-type reaction for accessing
multifunctional biomolecular constructs. Chemistry, 20, 34-41.
[31] Blackman, M. L.; Royzen, M.; Fox, J. M. (2008) The tetrazine ligation:
fast bioconjugation based on inverse-electron-demand Diels-Alder
reactivity. J. Am. Chem. Soc., 130, 13518-13519.
[32] Thayumanavan, R.; Dhevalapally, B.; Sakthivel, K.; Tanaka, F.; Barbas,
C. F. (2002) Amine-catalyzed direct Diels–Alder reactions of α,β-
unsaturated ketones with nitro olefins. Tetrahedron Lett., 43, 3817-
3820.
[33] Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. (2002)
A stepwise huisgen cycloaddition process: copper(I)-catalyzed
regioselective “ligation” of azides and terminal alkynes. Angew. Chem.
Int. Ed., 41, 2596-2599.
[34] Worrell, B. T.; Malik, J. A.; Fokin, V. V. (2013) Direct evidence of a
dinuclear copper intermediate in Cu(I)-catalyzed azide-alkyne
cycloadditions. Science, 340, 457-460.
[35] Calvo-Losada, S.; Pino-González, M. S.; Quirante, J. J. (2015)
Rationalizing the catalytic activity of copper in the cycloaddition of
azide and alkynes (CuAAC) with the topology of ∇(2)ρ(r) and
∇∇(2)ρ(r). J. Phys. Chem. B, 119, 1243-1248.
[36] Devadoss, A.; Chidsey, C. E. (2007) Azide-modified graphitic surfaces
for covalent attachment of alkyne-terminated molecules by “click”
chemistry. J. Am. Chem. Soc., 129, 5370-5371.
[37] Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. (2002)
A stepwise huisgen cycloaddition process: copper(I)-catalyzed
regioselective “ligation” of azides and terminal alkynes. Angew. Chem.
Int. Ed., 41, 2596-2599.
[38] Zhou, L.; Shen, Q.; Zhao, P.; Xiang, B.; Nie, Z.; Huang, Y.; Yao, S.
(2013) Fluorescent detection of copper(II) based on DNA-templated
click chemistry and graphene oxide. Methods, 64, 299-304.
[39] Su, J.; Xu, J.; Chen, Y.; Xiang, Y.; Yuan, R.; Chai, Y. (2013) Sensitive
detection of copper(II) by a commercial glucometer using click
chemistry. Biosens. Bioelectron., 45, 219-222.
[40] Lau, Y. H.; Price, J. R.; Todd, M. H.; Rutledge, P. J. (2011) A click
fluorophore sensor that can distinguish Cu(II) and Hg(II) via selective
anion-induced demetallation. Chemistry, 17, 2850-2858.
[41] Ingale, S. A.; Seela, F. (2012) A ratiometric fluorescent on-off Zn2+
chemosensor based on a tripropargylamine pyrene azide click adduct. J.
Org. Chem., 77, 9352-9356.
[42] Viguier, R. F. H.; Hulme, A. (2006) A sensitized europium complex

generated by micromolar concentrations of copper(I): toward the
detection of copper(I) in biology. J. Am. Chem. Soc., 128, 11370-11371.
[43] Zhou, Y.; Wang, S.; Zhang, K.; Jiang, X. (2008) Visual detection of
copper(II) by azide- and alkyne-functionalized gold nanoparticles using
click chemistry. Angew. Chem. Int. Ed., 47, 7454-7456.
[44] Xu, X. Y.; Daniel, W. L.; Wei, W.; Mirkin, C. A. (2010) Colorimetric
Cu(2+) detection using DNA-modified gold-nanoparticle aggregates as
probes and click chemistry. Small, 6, 623-626.
[45] Zhang, Y. F.; Li, B. X.; Xu, C. L. (2010) Visual detection of ascorbic
acid via alkyne-azide click reaction using gold nanoparticles as a
colorimetric probe. Analyst, 135, 1579-1584.
[46] Shen, Q.; Zhou, L.; Yuan, Y.; Huang, Y.; Xiang, B.; Chen, C.; Nie, Z.;
Yao, S. (2014) Intra-molecular G-quadruplex structure generated by
DNA-templated click chemistry: “turn-on” fluorescent probe for copper
ions. Biosens. Bioelectron., 55, 187-194.
[47] Lee, A.; Chin, J.; Park, O. K.; Chung, H.; Kim, J. W.; Yoon, S. Y.; Park,
K. (2013) A novel near-infrared fluorescence chemosensor for copper
ion detection using click ligation and energy transfer. Chem. Commun.,
49, 5659-5671.
[48] Qu, W.; Liu, Y.; Liu, D.; Wang, Z.; Jiang, X. (2011) Copper-mediated
amplification allows readout of immunoassays by the naked eye.
Angew. Chem. Int. Ed., 50, 3442-3445.
[49] Qiu, S.; Miao, M.; Wang, T.; Lin, Z.; Guo, L.; Qiu, B.; Chen, G. (2013)
A fluorescent probe for detection of histidine in cellular homogenate
and ovalbumin based on the strategy of click chemistry. Biosens.
Bioelectron., 42, 332-336.
[50] Qiu, S.; Li, X.; Xiong, W.; Xie, L.; Guo, L.; Lin, Z.; Qiu, B.; Chen, G.
(2013) A novel fluorescent sensor for mutational p53 DNA sequence
detection based on click chemistry. Biosens. Bioelectron., 41, 403-408.
[51] Yue, G.; Ye, H.; Huang, X.; Ye, W.; Qiu, S.; Qiu, B.; Lin, Z.; Chen, G.
(2014) Quantification of DNA through a fluorescence biosensor based
on click chemistry. Analyst, 139, 5669-5673.
[52] Xu, K.; Chen, Z.; Zhou, L.; Zheng, O.; Wu, X.; Guo, L.; Qiu, B.; Lin,
Z.; Chen, G. (2015) Fluorometric method for inorganic pyrophosphatase
activity detection and inhibitor screening based on click chemistry.
Anal. Chem., 87, 816-820.
[53] Kim, Y. P.; Daniel, W. L.; Xia, Z.; Xie, H.; Mirkin, C. A.; Rao, J.
(2010) Bioluminescent nanosensors for protease detection based upon
gold nanoparticle-luciferase conjugates. Chem. Commun., 46, 76-78.
[54] Kumar, Y. P.; Bhowmik, S.; Das, R. N.; Bessi, I.; Paladhi, S.; Ghosh,
R.; Schwalbe, H.; Dash, J. (2013) A fluorescent guanosine dinucleoside
as a selective switch-on sensor for c-myc G-quadruplex DNA with
potent anticancer activities. Chem. Eur. J., 19, 11502 -11506.
[55] Yao, Y.; Tian, D.; Li, H. (2010) Cooperative binding of bifunctionalized
and click-synthesized silver nanoparticles for colorimetric Co(2+)
sensing. ACS Appl. Mater. Interfaces, 2, 684-690.
[56] Tamanini, E.; Flavin, K.; Motevalli, M.; Piperno, S.; Gheber, L. A.;
Todd, M. H.; Watkinson, M. (2010) Cyclam-based “clickates”:
homogeneous and heterogeneous fluorescent sensors for Zn(II). Inorg.
Chem., 49, 3789-3780.
[57] Jing, L.; Liang, C.; Shi, X.; Ye, S.; Xian, Y. (2012) Fluorescent probe
for Fe(III) based on pyrene grafted multiwalled carbon nanotubes by
click reaction. Analyst, 137, 1718-1722.
[58] Midya, G. C.; Paladhi, S.; Bhowmik, S.; Saha, S.; Dash, J. (2013)
Design and synthesis of an on-off “click” fluorophore that executes a
logic operation and detects heavy and transition metal ions in water and
living cells. Org. Biomol. Chem., 11, 3057-3063.
[59] Yapici, N. B.; Mandalapu, S. R.; Chew, T. L.; Khuon, S.; Bi, L. (2012)
Determination of intracellular pH using sensitive, clickable fluorescent
probes. Bioorg. Med. Chem. Lett., 22, 2440-2443.
[60] Yang, Y.; Yu, K.; Yang, L.; Liu, J.; Li, K.; Luo, S. (2015) One single
molecule as a multifunctional fluorescent probe for ratiometric sensing
of Fe3+, Cr3+ and colorimetric sensing of Cu2+. Sensor, 15, 49-58.
[61] Yao, Y.; Sun, Z.; Zou, Z.; Li, H. (2011) Quinolino-triazole linked gold
nanoparticles as sensitive ‘turn-on’ fluorescent Cd(2+) probes.
Nanotechnology, 22, 435502.
[62] Sui, B.; Kim, B.; Zhang, Y.; Frazer, A.; Belfield, K. D. (2013) Highly
selective fluorescence turn-on sensor for fluoride detection. ACS Appl.
Mater. Interfaces, 5, 2920-2923.
[63] Lu, L.; Yang, L.; Cai, H.; Zhang, L.; Lin, Z.; Guo, L.; Qiu, B.; Chen, G.
(2014) Determination of flumioxazin residue in food samples through a
sensitive fluorescent sensor based on click chemistry. Food Chem., 162,
242-246.
[64] Xie L, Zheng H, Ye W, Qiu S, Lin Z, Guo L, Qiu B, Chen G (2013)
Novel colorimetric molecular switch based on copper(I)-catalyzed
azide-alkyne cycloaddition reaction and its application for flumioxazin
detection. Analyst 138: 688-692.
[65] Kennedy, D. C.; McKay, C. S.; Legault, M. C.; Danielson, D. C.; Blake,
J. A.; Pegoraro, A. F.; Stolow, A.; Mester, Z.; Pezacki, J. P. (2011)
Cellular consequences of copper complexes used to catalyze
bioorthogonal click reactions. J. Am. Chem. Soc., 133, 17993-18001.
[66] Rubino, F. A.; Oum, Y. H.; Rajaram, L.; Chu, Y.; Carrico, I. S. (2012)
Chemoselective modification of viral surfaces via bioorthogonal click
chemistry. J. Vis. Exp., 66, 4246.
[67] Yu, X.; Woolery, A. R.; Luong, P.; Hao, Y. H.; Grammel, M.; Westcott,
N.; Park, J.; Wang, J.; Bian, X.; Demirkan, G.; Hang, H. C.; Orth, K.;
LaBaer, J. (2014) Copper-catalyzed azide-alkyne cycloaddition (click
chemistry)-based detection of global pathogen-host AMPylation on self-
assembled human protein microarrays. Mol. Cell Proteomics, 13, 3164-
3176.
[68] Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. (2004) A strain-promoted
[3 + 2] azide-alkyne cycloaddition for covalent modification of
biomolecules in living systems. J. Am. Chem. Soc., 126, 15046-15047.
[69] Tummatorn, J.; Batsomboon, P.; Clark, R. J.; Alabugin, I. V.; Dudley,
G. B. (2012) Strain-promoted azide-alkyne cycloadditions of
benzocyclononynes. J. Org. Chem., 77, 2093-2097.
[70] Canalle, L. A.; Vong, T.; Adams, P. H.; van Delft, F. L.; Raats, J. M.;
Chirivi, R. G.; van Hest, J. C. (2011) Clickable enzyme-linked
immunosorbent assay. Biomacromolecules, 12, 3692-3697.
[71] Jung, S.; Yi, H. (2014) An integrated approach for enhanced protein
conjugation and capture with viral nanotemplates and hydrogel
microparticle platforms via rapid bioorthogonal reactions. Langmuir, 30,
7762-7770.
[72] Teo, C. F.; Wells, L. (2014) Monitoring protein O-linked b-N-
acetylglucosamine status via metabolic labeling and copper-free click
chemistry. Anal. Biochem., 464, 70-72.
[73] Ledin, P. A.; Kolishetti, N.; Boons, G. J. (2013) Multi-Functionalization
of Polymers by Strain-Promoted Cycloadditions. Macromolecules, 46,
7759-7768.
[74] Kim, E. J.; Kang, D. W.; Leucke, H. F.; Bond, M. R.; Ghosh, S.; Love,
D. C.; Ahn, J. S.; Kang, D. O.; Hanover, J. A. (2013) Optimizing the
selectivity of DIFO-based reagents for intracellular bioorthogonal
applications. Carbohydr. Res., 377, 18-27.
[75] Singh, I.; Wendeln, C.; Clark, A. W.; Cooper, J. M.; Ravoo, B. J.;
Burley, G. A. (2013) Sequence-selective detection of double-stranded
DNA sequences using pyrrole-imidazole polyamide microarrays. J. Am.
Chem. Soc., 135, 3449-3457.
[76] Kato, D.; Oishi, M. (2014) Ultrasensitive Detection of DNA and RNA
based on enzyme-free click chemical ligation chain reaction on
dispersed gold nanoparticles. ACS Nano, 8, 9988-9997.
[77] Hu, X. L.; Jin, H. Y.; He, X. P.; James, T. D.; Chen, G. R.; Long, Y. T.
(2015) Colorimetric and plasmonic detection of lectins using core-shell
gold glyconanoparticles prepared by copper-free click chemistry. ACS
Appl. Mater. Interfaces, 7, 1874-1878.
[78] Liu, D.; Wang, Z.; Jin, A.; Huang, X.; Sun, X.; Wang, F.; Yan, Q.; Ge,
S.; Xia, N.; Niu, G.; Liu, G.; Hight Walker, A. R.; Chen, X. (2013)
Acetylcholinesterase-catalyzed hydrolysis allows ultrasensitive
detection of pathogens with the naked eye. Angew. Chem. Int. Ed., 52,
14065-14069.
[79] Fugier, E.; Dumont, A.; Malleron, A.; Poquet, E.; Mas Pons, J.; Baron,
A.; Vauzeilles, B.; Dukan, S. (2015) Rapid and specific enrichment of
culturable gram negative bacteria using non-lethal copper-free click
chemistry coupled with magnetic beads separation. PLoS One, 10,
0127700.
[80] Subramanian, N.; Sreemanthula, J. B.; Balaji, B.; Kanwar, J. R.; Biswas,
J.; Krishnakumar, S. (2014) A strain-promoted alkyne-azide
cycloaddition (SPAAC) reaction of a novel EpCAM aptamer-
fluorescent conjugate for imaging of cancer cells. Chem. Commun., 50,
11810-11813.
[81] Murrey, H. E.; Judkins, J. C.; Am Ende, C. W.; Ballard, T. E.; Fang, Y.;
Riccardi, K.; Di, L.; Guilmette, E. R.; Schwartz, J. W.; Fox, J. M.;
Johnson, D. S. (2015) Systematic evaluation of bioorthogonal reactions
in live cells with clickable HaloTag ligands: Implications for
intracellular imaging. J. Am. Chem. Soc., 137, 11461-11475.
[82] Nikić, I.; Kang, J. H.; Girona, G. E.; Aramburu, I. V.; Lemke, E. A.
(2015) Labeling proteins on live mammalian cells using click chemistry.
Nat. Protoc., 10, 780-791.
[83] Hoyle, C. E.; Bowman, C. N. (2010) Thiol–ene click chemistry. Angew.
Chem. Int. Ed., 49, 1540-1573.
[84] Dübner, M.; Gevrek, T. N.; Sanyal, A.; Spencer, N. D.; Padeste, C.
(2015) Fabrication of thiol-ene “clickable” copolymer-brush
nanostructures on polymeric substrates via extreme ultraviolet
interference lithography. ACS Appl. Mater. Interfaces, 7, 11337-11345.
[85] Pahimanolis, N.; Kilpeläinen, P.; Master, E.; Ilvesniemi, H.; Seppälä, J.
(2015) Novel thiol- amine- and amino acid functional xylan derivatives
synthesized by thiol-ene reaction. Carbohydr. Polym., 131, 392-398.
[86] Buhl, M.; Vonhören, B.; Ravoo, B. J. (2015) Immobilization of
enzymes via microcontact printing and thiol-ene click chemistry.
Bioconjug. Chem., 26, 1017-1020.
[87] Arseneault, M.; Wafer, C.; Morin, J. F. (2015) Recent advances in click
chemistry applied to dendrimer synthesis. Molecules, 20, 9263-9264.
[88] Kumar, R.; Saima Shard A, Andhare, N. H.; Richa Sinha, A. K. (2015)
Thiol-ene “click” reaction triggered by neutral ionic liquid: the
“ambiphilic” character of [hmim]Br in the regioselective nucleophilic
hydrothiolation. Angew. Chem. Int. Ed., 54, 828-832.
[89] Gupta, N.; Lin, B. F.; Campos, L. M.; Dimitriou, M. D.; Hikita, S. T.;
Treat, N. D.; Tirrell, M. V.; Clegg, D. O.; Kramer, E. J.; Hawker, C. J.
(2010) A versatile approach to high-throughput microarrays using thiol-
ene chemistry. Nat. Chem., 2, 138-145.
[90] Lafleur, J. P.; Senkbeil, S.; Novotny, J.; Nys, G.; Bøgelund, N.; Rand,
K. D.; Foret, F.; Kutter, J. P. (2015) Rapid and simple preparation of
thiol-ene emulsion-templated monoliths and their application as
enzymatic microreactors. Lab Chip, 15, 2162-2172.
[91] Sulistio, A.; Lowenthal, J.; Blencowe, A.; Bongiovanni, M. N.; Ong, L.;
Gras, S. L.; Zhang, X.; Qiao, G. G. (2011) Folic acid conjugated amino
acid-based star polymers for active targeting of cancer cells.
Biomacromolecules, 12, 3469-3477.
[92] Melnik, E.; Muellner, P.; Bethge, O.; Bertagnolli, E.; Hainberger, R.;
Laemmerhofer, M. (2014) Streptavidin binding as a model to
characterize thiol-ene chemistry-based polyamine surfaces for reversible
photonic protein biosensing. Chem. Commun., 50, 2424-2427.
[93] Zhao, L.; Xiao, C.; Ding, J.; He, P.; Tang, Z.; Pang, X.; Zhuang, X.;
Chen, X. (2013) Facile one-pot synthesis of glucose-sensitive nanogel
via thiol-ene click chemistry for self-regulated drug delivery. Acta
Biomater., 9, 6535-6543.
[94] Seto, H.; Yamashita, C.; Kamba, S.; Kondo, T.; Hasegawa, M.;
Matsuno, M.; Ogawa, Y.; Hoshino, Y.; Miura, Y. (2013) Biotinylation
of silicon and nickel surfaces and detection of streptavidin as biosensor.
Langmuir, 29, 9457-9463.
[95] Marechal, A.; Jarrosson, F.; Randon, J.; Dugas, V.; Demesmay, C.
(2015) In-line coupling of an aptamer based miniaturized monolithic
affinity preconcentration unit with capillary electrophoresis and Laser
Induced Fluorescence detection. J. Chromatogr. A, 1406, 109-117.
[96] Escorihuela, J.; Bañuls, M. J.; Grijalvo, S.; Eritja, R.; Puchades, R.;
Maquieira, A. (2014) Direct covalent attachment of DNA microarrays
by rapid thiol-ene “click” chemistry. Bioconjug, Chem., 25, 618-627.
[97] Wang, Z.; Zhao, J. C.; Lian, H. Z.; Chen, H. Y. (2015) Aptamer-based
organic-silica hybrid affinity monolith prepared via “thiol-ene” click
reaction for extraction of thrombin. Talanta, 138, 52-58.
[98] Schyrr, B.; Pasche, S.; Voirin, G.; Weder, C.; Simon, Y. C.; Foster, E. J.
(2014) Biosensors based on porous cellulose nanocrystal-poly(vinyl
alcohol) scaffolds. ACS Appl. Mater. Interfaces, 6, 12674-12683.
[99] Su, X.; Kuang, L.; Battle, C.; Shaner, T.; Mitchell, B. S.; Fink, M. J.;
Jayawickramarajah, J. (2014) Mild two-step method to construct DNA-
conjugated silicon nanoparticles: scaffolds for the detection of
microRNA-21. Bioconjug. Chem., 25, 1739-1743.
[100] Garber, K. C.; Carlson, E. E. (2013) Thiol-ene enabled detection of
thiophosphorylated kinase substrates. ACS Chem. Biol., 8, 1671-1676.
[101] Lou, X.; Hong, Y.; Chen, S.; Leung, C. W.; Zhao, N.; Situ, B.; Lam, J.
W.; Tang, B. Z. (2014) A selective glutathione probe based on AIE
fluorogen and its application in enzymatic activity assay. Sci. Rep., 4,
4272.
[102] Liu, Y.; Yu, Y.; Lam, J. W.; Hong, Y.; Faisal, M.; Yuan, W. Z.; Tang,
B. Z. (2010) Simple biosensor with high selectivity and sensitivity:
thiol-specific biomolecular probing and intracellular imaging by AIE
fluorogen on a TLC plate through a thiol-ene click mechanism. Chem.
Eur. J., 16, 8433-8488.
[103] Ulrich, S.; Boturyn, D.; Marra, A.; Renaudet, O.; Dumy, P. (2014)
Oxime ligation: a chemoselective click-type reaction for accessing
multifunctional biomolecular constructs. Chem. Eur. J., 20, 34-41.
[104] Rashidian, M.; Mahmoodi, M. M.; Shah, R.; Dozier, J. K.; Wagner, C.
R.; Distefano, M. D. (2013) A highly efficient catalyst for oxime
ligation and hydrazone-oxime exchange suitable for bioconjugation.
Bioconjug. Chem., 24, 333-342.
[105] Uth, C.; Zielonka, S.; Hörner, S.; Rasche, N.; Plog, A.; Orelma, H.;
Avrutina, O.; Zhang, K.; Kolmar, H. (2014) A chemoenzymatic
approach to protein immobilization onto crystalline cellulose
nanoscaffolds. Angew. Chem. Int. Ed., 53, 12618-12623.
[106] Křenek, K.; Gažák, R.; Daskhan, G. C.; Garcia, J.; Fiore, M.; Dumy, P.;
Sulc, M.; Křen, V.; Renaudet, O. (2014) Access to bifunctionalized
biomolecular platforms using oxime ligation. Carbohydr. Res., 393, 9-
14.
[107] Rayo, J.; Amara, N.; Krief, P.; Meijler, M. M. (2011) Live cell labeling
of native intracellular bacterial receptors using aniline-catalyzed oxime
ligation. J. Am. Chem. Soc., 133, 7469-7475.
[108] Voss, S.; Zhao, L.; Chen, X.; Gerhard, F.; Wu, Y. W. (2014) Generation
of an intramolecular three-color fluorescence resonance energy transfer
probe by site-specific protein labeling. J. Pept. Sci., 20, 115-120.
[109] Tang, L.; Yin, Q.; Xu, Y.; Zhou, Q.; Cai, K.; Yen, J.; Dobrucki, L. W.;
Cheng, J. (2015) Bioorthogonal oxime ligation mediated in vivo cancer
targeting. Chem. Sci., 6, 2182-2186.
[110] Abernethy, G. A. (2012) A rapid analytical method for cholecalciferol
(vitamin D3) in fortified infant formula, milk and milk powder using
Diels–Alder derivatisation and liquid chromatography–tandem mass
spectrometric detection. Anal. Bioanal. Chem., 403, 1433-1440.
[111] Liu, W.; Xu, L.; Lamberson, C.; Haas, D.; Korade, Z.; Porter, N. A.
(2014) A highly sensitive method for analysis of 7-dehydrocholesterol
for the study of Smith-Lemli-Opitz syndrome. J. Lipid Res., 55, 329-
337.
[112] Späte, A. K.; Schart, V. F.; Häfner, J.; Niederwieser, A.; Mayer, T. U.;
Wittmann, V. (2014) Expanding the scope of cyclopropene reporters for
the detection of metabolically engineered glycoproteins by Diels-Alder
reactions. Beilstein. J. Org. Chem., 10, 2235-2242.
Chapter 12
TELECHILIC POLYBUTADIENE SOLID

PROPELLANT BINDERS BASED ON
‘ÇLICK’ CHEMISTRY APPROACH
S. Reshmi1,*, PhD, E. Arunan2, PhD

and C. P. Reghunadhan Nair1, PhD
1
Polymers and Special Chemicals Group, Vikram Sarabhai Space Centre,
Thiruvananthapuram, Kerala, India
2
Department of Inorganic and Physical Chemistry,
Indian Institute of Science, Bengaluru, Karnataka, India
ABSTRACT
Solid propellants are widely used for launch vehicle and missile
applications. The polymeric fuel binder is a critical ingredient of a
composite solid propellant. It acts as the matrix for holding together the
oxidiser, metallic fuel and other additives and also imparts structural
integrity, mechanical properties to the propellant and contribute to the
combustion phenomena. In recent years, the impetus has been to improve
the energetics by the use of binders with energetic functional groups. The
introduction of ‘triazole’ groups in the polymer network via ‘Click
chemistry’ is one such approach. This imparts superior processability,
mechanical properties and ballistics to the propellant. Amongst the
different types of polymeric binders used in composite solid propellants,
*
Corresponding Author Email: reshmiskurup@gmail.com.
348 S. Reshmi, E. Arunan and C. P. Reghunadhan Nair
hydroxyl terminated polybutadiene (HTPB) is considered as the most

versatile. In the present approach, hydroxyl terminated polybutadiene
(HTPB) was chemically modified to derive propargyl end capped
polymers. The polymers were then cured by ‘Click Chemistry’ approach
to form triazole network using an azide bearing polymer viz.
Glycidylazide polymer (GAP) referred to as PTPB triazole. The curing
parameters were studied using Differential Scanning Calorimetry (DSC).
Curing occurs only for an alkyne:azide molar equivalence of 1:0.1 and
beyond this stoichiometry, phase separation occurs. Rheological studies
of PTPB triazole was carried out and the properties were compared with
HTPB-tolylenediisocyanate (TDI) based urethane system (based on
hydroxyl: isocyanate). The studies revealed that the gel time for curing
through the 1, 3 dipolar addition is higher for triazole curing route than
urethanes based systems indicating a higher ’pot life’. The mechanical
properties of the triazole mediated networks were evaluated and
compared to those of HTPB-urethanes. Thermo gravimetric analysis (TG)
of the triazoles derived from the polymers were investigated and the
mechanism of decomposition of PTPB triazole with AP as oxidiser was
elucidated by pyrolysis GC-MS.The propellant level properties of PTPB
triazolewere evaluated and compared with propellants based on HTPB-
urethane system. The studies reveal that propellant based on PTPB
triazolehavesuperior processabilitywith acceptable mechanical properties,
than HTPB-urethanes It also provide improved ballistic properties in
terms of higher gas generation during combustion.
INTRODUCTION
Hydroxyl terminated polybutadiene (HTPB) is the most popular binder
used in solid propellants both for boosters, pyrogenigniters, gas generators and
upper stage motors [1-5].The mode of crosslinking in HTPB based systems is
through the reaction of hydroxyl groups with polyisocyanates, resulting in
polyurethanes [5]. The major limitation of this cure methodology is the low
pot life and intervention of extraneous side reactions causing microvoids in the
cured propellant matrix [6].Hence it is always desirable to have end groups
that can give crosslinked matrices wherein side reactions can be avoided [6-9].
It is also desirable if the addition product adds to the energy and ballistics of
the propellant. Several reports exist on the modification of HTPB [10-18] such
as grafting of energetic groups such as poly(glycidylazide) [14],anchoring of
iron pentacarbonyl [15], grafting of 2-(ferrocenylpropyl) dimethylsilane
(FPDS) etc. Most of these are aimed at improving the ballistic performances of
HTPB based propellants [19-20]. A comprehensive approach of achieving
Telechilic Polybutadiene Solid Propellant Binders … 349
improved processability and superior mechanical properties for the propellant

without compromising its ballistics is essential to meet the future
requirements. However, these have never addressed the issue related to
processability or compatibility with chlorine free high energy oxidiser like
ammonium dinitramide (ADN), hydraziniumnitroformate (HNF) etc. ‘Click’
reaction between certain azide-alkyne groups is one reaction which can be
extensively exploited for replacing urethanes due to high yields and absence of
side reactions [21-23]. There have been few reports [24-29] on alkyne-azide
‘click reaction’ through a 1,3-dipolar cycloaddition to form 1,2,3-triazole
networks for crosslinking polymers as well as propellant binders including a
recent article [29] on synthesis and characterisation of PTPB. However, in all
these cases, the aspects of processability, mechanical properties and propellant
energetics have not been addressed.
The present chapter reports modification of the hydroxyl groups of HTPB
to ‘clickable alkyne groups which can be crosslinked using azides to yield
triazoles. The chapter details the synthesis and characterisation of
propargyloxy terminated polybutadiene (PTPB) polymeric binder. This binder
was cured with azide containing polymer viz. glycidylazide polymer (GAP) to
give triazoles. The curing, thermal decomposition mechanism, mechanical
characterisation and dynamic mechanical characteristics of the cured triazole
network in neat polymer, the processability aspects, mechanical properties of
the cured polymer and propellant, energetics, burn rate and thermal
decomposition of the propellant with ammonium perchlorate (AP) as oxidiser
have been investigated.
2. EXPERIMENTAL
2.1. Materials
HTPB, sodium hydride,propargyl bromide, ammonium perchlorate (AP)

and aluminium powder were used for the studies. The solvents namely
methanol, toluene, pentane and tetrahydrofuran (THF) of high purity (AR
grade) were used.
2.2. Instrumental
FTIR spectra were recorded on a Perkin Elmer Spectra GXA FTIR

spectrometer in the range of 4000-650 cm-1 using KBr plates. 1H NMR
analysis was done using a Bruker Avance (300 MHz) Spectrometer. Curing
was monitored using differential scanning calorimeter, (DSC, TA Instruments
Q 20) at heating rate of 5oC/min respectively. Mechanical properties of the
polymer and propellant viz. tensile strength, elongation and modulus were
evaluated using Universal Testing Machine (INSTRON Model 4469) at a
cross head speed of 50 mm/min.The heat of combustion data as generated in a
bomb calorimeter (Parr Instrument 600) in oxygen atmosphere at 3 MPa and
the heat of formation was computed. Dynamic mechanical analysis (DMA)
was done using 01 dbMetravib Viscoanalyser (Model VA 2000) using
rectangular specimens of dimension (25x15x5 mm).Pyrolysis GC-MS studies
were conducted using a Thermo Electron Trace Ultra GC directly coupled to a
Thermo Electron Polaris Q (Quadruple ion trap) mass spectrometer and SGE
pyrolyser. TG-MS studies were conducted using TGA attached with
Quadruple mass spectrometer at heating rate of 5oC/min for cured polymer and
at 2oC/min for the propellant samples.Heat of combustion were measured
using bomb calorimeter model Parr Instrument 6200 in oxygen atmosphere.
2.3.Curing
The binder PTPB was cured with GAP at an alkyne: azide equivalent ratio
of 1:0.1. The mixtures were then cast in aluminiummoulds and the curing
reaction was carried out at 60oC for a period of 5 days. For comparison,
HTPB-TDI urethanes (stoichiometric ratio of 1:1 with respect to isocyanate
and hydroxyl, NCO: OH) were also prepared and evaluated.
2.4. Propellant Processing
The thermochemical performance evaluation of the propellant based on

PTPB was carried out using a thermo chemical code NASA CEAgui [30]..
Propellant batches were processed in a 1 kg scale in a Guitard horizontal
mixing system at 40oC and an average mixing time of three hours. The typical
solid propellant formulation consisted of PTPB as binder, aluminium as

metallic fuel (2% by weight) and ammonium perchlorate as oxidiser (77% by
weight) and GAP as curing agent. The samples were cured at 60oC for 5 days
without a catalyst. For comparison, propellant based on HTPB-urethane was
also processed in the same manner. The burn rates of the cured propellant
samples were measured using acoustic emission technique at an operating
pressure of 6.93MPa using cured propellant strands (size: 80x6x6mm).
2.5. Synthesis
2.5.1. Synthesis of Propargyloxy Terminated Polybutadiene (PTPB)

Propargyloxy terminated polybutadiene (PTPB) was synthesised from
HTPB by treating with propargyl bromide in presence of sodium hydride
(NaH) (Scheme 1). In a typical reaction, 15g (0.006 mol) of moisture free
HTPB was dissolved in THF and reacted with 1.25g (0.052 mol) of NaH at
40oC for 3 hours with stirring under nitrogen blanket. To the mixture, 5 ml
(0.03 mol) of propargyl bromide was added and reaction was continued for 24
hrs. Following the reaction, 30 ml of methanol was added to remove excess
NaH. The product was washed with hot water, followed by methanol. The
product was dried at 60oC to remove methanol and water. The product was
extracted using pentane and dried under reduced pressure at 80oC for 6hrs.
Yield: ~89%.
The presence of propargyl group in PTPB was confirmed by FTIR (Figure
1) by the characteristic absorption at 2130 cm-1 corresponding to -CC-H,
absorption at 3307 cm-1 due to alkenyl C-H stretch and absence of broad peak
at 3400-3600 cm-1 corresponding to hydroxyl groups. The spectrum of PTPB
is given in Figure 1. The double bond and microstructures of the butadiene
[32-34] remains unaltered even after the modification of the polymer
backbone.
IH NMR of PTPB (Figure 2) showed all the chemical shifts as that of
HTPB [33] and the microstructure of PTPB was found to be identical to that of
HTPB. In addition, the chemical shifts at 2.5ppm due to ─C≡C─H and the one
at 4.2 ppm due to O─CH2─ bonded to the propargyl group confirms the
anchoring of propargyl oxy groups to HTPB and this matches with reported
literature [34].
Scheme 1. Typical Synthesis Scheme for PTPB.
Figure 1. FTIR spectrum of PTPB.
1.0 28a.001.1r.esp
0.9
k g
j f e
d O CH2 CH
HC CH2O c h
i a b n
0.8
0.7
Normalized Intensity
0.6
0.5
0.4
b,c,d
0.3
0.2
f,g
0.1 h a
,I,j
16 14 12 10 8 6 4 2 0 -2 -4
Chemical Shift (ppm)
Figure 2. 1H NMR spectrum of PTPB (in CDCl3)

Figure 3.GPC chromatogramofHTPB, PTPB.

IH NMR of PTPB (Figure 2) showed all the chemical shifts as that of
HTPB [32] and the microstructure of PTPB was found to be identical to that of
HTPB. In addition, the chemical shifts at 2.5ppm due to ─C≡C─H and the one
at 4.2 ppm due to O─CH2─ bonded to the propargyl group confirms the
anchoring of propargyl oxy groups to HTPB and this matches with reported
literature [33].
GPC traces of the PTPB and HTPB corrected for hydrodynamic volume is
given in Figure 3. The calculated number average molecular weight for PTPB
is 3627 and weight average molecular weight (Mw) is 15551. Polydispersity
index (PDI) is 2.4. The end functionalisation, does not add to any change in
molecular weight.
3. RESULTS AND DISCUSSION

3.1. Curing Characteristics of PTPB
The curing of PTPB with GAP results in the formation of triazole. To

study the curing, non-isothermal differential scanning calorimetry (DSC)
analysis was done at a heating rate of 5oC/min. Initially DSC study was carried
out for an azide to alkyne molar equivalence of 1:1. It was observed that at this
composition completion of cure does not occur. Instead, azide decomposition
is more predominant (Figure 4a). Curing is found to occur only for an alkyne-
azide molar equivalence of 1:0.1 and beyond this, phase separation occurs.
This can be attributed to the difference in the solubility parameters of the
polymers. HTPB [35] has a solubility parameter of 17.6 MPa1/2and that of
GAP [35] is 22.8 MPa1/2 which causes miscibility issues beyond a certain
concentration. The poor compatibility between GAP and HTPB due to polar
nature of azide groups and non-polar nature of HTPB backbone has been
reported by Ding et al. [29] also. The cure reaction of PTPB with GAP
resulting in the formation of triazolesfor alkyne-azide equivalence of 1:0.1
occurs in the temperature range of 110-185oC with an associated enthalpy of
50 ±2 J/g. This is followed by decomposition of the residual azide at ~186oC
(Figure.4b) as the reaction is not complete in a DSC cell. The azide groups can
also react with double bonds in polybutadiene backbone [30] which gives rises
to triazoline networks. Thus, azide-alkyne curing in PTPB with GAP results in
triazole-triazoline networks.
Figure 4.DSC Traces of Curing of PTPB with GAP a) Azide-alkyne equivalence 1:1;
b) Azide-alkyne equivalence (1:0.1).
The rheological behaviour of the curing reaction of PTPB and GAP (molar
stoichiometry of 1:0.1) was investigated at 80oC. The isothermal evolution of
storage modulus (G’) and loss modulus (G”) vs. reaction time for the curing
reaction for PTPB triazole is given in Figure 5.Both moduli (storage and loss)
increase as a result of the increase in crosslinking as observed in the rheogram.
The cross over point of loss modulus with storage modulus is considered as
the gel point. The gel point for PTPB triazole-triazoline system occurs after
190 minutes. A higher modulus build up may be attributed to triazole and
triazoline formation [30-35]. The gel point is higher than for HTPB-urethane
system which is 120 minutes. This indicates a higher ‘pot-life’ for the cure
reaction involving PTPB-GAP.
Figure 5.Rheogram of PTPB with GAP at 80oC.
3.2. Mechanical Properties
The mechanical properties viz. tensile strength (T.S), elongation and

modulus of the cured polymers (PTPB-GAP) was determined. The tensile
strength of the PTPB- triazole-triazoline for an azide –alkyne molar
equivalence of 1:0.1 is 1.18 MPa, elongation at break is 21% and the modulus
is 0.88 MPa.
Ding et al. [29] have reported the mechanical properties of PTPB-GAP

system for various stoichiometries of alkyne: azide and a tensile strength of
2.7 MPa, elongation break ~47% and modulus of 5.36 MPa. However, they
have not discussed the problems related to miscibility and phase separation
between PTPB and GAP during the preparation of composite.
Table 1. Mechanical Properties of cured PTPB and PTMP polymer
Mechanical Properties PTPB-GAP HTPB-urethane

(-C≡CH:N3=1:0.1) (-NCO:OH=1:1)
Tensile strength, (MPa) 1.18 0.86
Elongation at break (%) 21 240
Modulus (MPa) 0.88 0.52
The mechanical properties of HTPB urethanes cured using different

isocyanates like isophoronediisocyanate, hexamethylenediisocyanate are
reported [36-38]. The tensile strength is reported in to be in the range 0.3-
0.7MPa, elongation at break is in the range 170-400% and Young’s modulus
in the range 0.3-1.04 MPa. For triisocyanate based HTPB-urethane, the
reported values of 0.4-1.18 MPa and elongation at break is 90-140%. The
decrease in elongation may be due the increase in the rigidity of the networks
formed from the triisocyantes. Similar is the present observation wherein the
rigid triazole groups are decreasing the elongation characteristics of the cured
polymer.
3.3. Dynamic Mechanical Characterisation
Dynamic mechanical analysis (DMA) of cured networks of PTPB was

undertaken. The cured PTPB networks, exhibits a biphasic transition with two
glass transitions (Tg). The first transition occurs at -40.5oC which may be
attributed to the butadiene backbone and the second one occurring at 18.4oC,
islikely due to the triazole-triazoline network (Figure.6).
3.4. Thermal Decomposition Studies
The thermal decomposition characteristics of triazole-triazoline of PTPB

triazole was studied using thermogravimetric analysis (TGA). TGA was done
at a heating rate of 5oC/min in nitrogen atmosphere. The cured PTPB triazole

undergoes a single-stage decomposition (Figure 7a).The decomposition occurs
in the temperature range of 250-460oC with a weight loss of 94%.
Figure. 6. Tan δ and Storage modulus of Cured PTPB triazole
The peak reaction temperature is 452oC. The residue left over at 600oC is
6%. This is different from HTPB-urethane system where two-stage
decomposition is reported [39].
The mechanism of HTPB urethane has been studied by flash pyrolysis
[40] and it is reported that initially the cleavage of urethane bond occurs
liberating the curing agent which vaporises. This is followed by decomposition
of polymer back bone. The mechanism of the decomposition reaction was
investigated using pyrolysis GC-MS and TG-MS. The pyrolysis studies at
300oC gave butylated hydroxyl toluene (BHT) which is the antioxidantused in
HTPB.
Unlike in HTPB-urethane, in cured PTPB, the cleavage of triazole-
triazoline group occurs along with degradation of polymer backbone which is
supported by the pyrolysis data.Further, the pyrolysis characteristics were
studied at a higher temperature of 500oC. This revealed that at 500oC, cleavage
of the triazole group occurs (Figure 7b) liberating N2 (retention time, RT 1.74)
in addition to the degradation of polybutadiene back bone giving rise to
butadiene(RT 1.83), cyclohexadiene(RT 2.03), 4-vinyl cyclohexene (RT 4.99),
xylene (RT 5.93), methylidene(RT 10.05) and BHT (RT 14.90) as reported in
literature [39-40].
Figure7a. TGA-DTGtrace of PTPB triazoles (Heating rate 5oC/min).
Figure 7b. Pyrogram of of cured PTPB-GAP at500°C.

3.5. Propellant Studies
Propellant studies were done using PTPB as binder, ammonium

perchlorate as oxidiser and aluminium as metallic fuel (2%) for oxidiser
loading of 77% by weight. For this, thermochemical perfromance evaluation
of the propellant was carried out and the results are compared with
conventional HTPB propellant of the same formulation. In order to compute
the thermochemical performance, the heat of formation of the polymers were
computed for the polymer from the heat of combustion data.
3.5.1. Thermochemical Measurements

The heat of combustion of PTPB was measured using bomb caloriemter.
The theoretical empirical formula can be calculated from the molecular weight
of the polymer. From the heat of combustion data and molecular weight of the
polymer, the heat of formation of the polymer was obtained to be -290.5
kJ/mol.
Theoretical performance evaluation using NASA CEA programme of low
aluminised (with 2% aluminium) propellant was completed at an operating
pressure of 6.93MPa and area ratio of 10. Analysis was conducted for a typical
AP content of 77% AP, the performance of PTPB -AP was computed and
compared with HTPB urethane-AP propellant (Table2). It is observed that
PTPB-AP propellant releases higher N2, CO2 and H2O content than
conventional HTPB urethane–AP propellant.
Table 2. Thermochemical Performance Parameters of PTPB Propellant

(Aluminium content 2%)
Parameters HTPB-AP(urethane) PTPB-AP(triazole)

Isp (s) 241.4 235.6
V.Isp (s) 224.0 220.0
Flame temperature (Chamber) K 2421 2293
Combustion products,
(mass %)
CO 42.5 7.8
CO2 0.8 16.2
HCl 15.5 15.5
H2 5.1 1.3
H2O 1.6 12.9
N2 6.3 15.6
Al2O3 8.2 8.2
3.5.2. Propellant Processabiity, Mechanical Properties, Thermal

Decomposition and Burn Rate
The viscosity build up of PTPB -GAP was evaluated and compared with
HTPB propellant. The ‘end of mix viscosity’ of PTPB-GAP propellant is 173
Pa.s as against HTPB-TDI propellant which is 480 Pa.s. The build up rate is
lower (Table 3) which brings out the obvious advantage of the azide-alkyne
curing reaction with respect to processability over the conventional reaction
involving diisocyanate-hydroxyl groups of HTPB propellant.
Table 3. Viscosity build up of PTPB propellant
Propellant Viscosity in Pa.s at 400C

Time (h) HTPB-urethane PTPB-triazole
(NCO:OH=0.85:1) (-C≡CH:N3=1:0.1)
0 480 173
2 540 368
3 900 235
Table 4. Mechanical Properties of PTPB propellant
Mechanical Properties HTPB-urethane PTPB-triazole

(NCO:OH=0.85:1) (-C≡CH:N3=1:0.1)
Tensile strength, (MPa) 0.49 0.80
Elongation at break (%) 35 10
Modulus (MPa) 3.92 2.94
The mechanical properties of propellant based on PTPB-triazole was

evaluated and compared with HTPB- urethanes (Table 4). It is observed that
propellant based on PTPB has a higher tensile strength and lower elongation
than HTPB-urethane based propellant. This could be due to the rigid
characteristics of the triazole groups in comparison to urethanes. The void
related problems can be totally overcome by curing through the new triazole
route.
The thermal decomposition of PTPB-AP propellantwas studied by TG-
DSC at a heating rate of 1oC/min (Figure.8). The propellant undergoes two-
stage decomposition which is similar to HTPB--AP propellant. The first stage
decomposition occurs in the temperature range of 205-265oC corresponding to
the decomposition of AP with a weight loss of 23%.The second stage
decomposition occurs in the temperature range of 267-312oC which is due to
the decomposition of binder and second stage decomposition of AP with a

weight loss of 65% and residue obtained at 356oC is 12%. The termal stability
of both the propellants are comparable.
The burn rate of the PTPB-triazole propellant was evaluated (Table 6) and
compared with HTPB urethane based propellants at 6.93MPa. The present
formulation has been designed for gas generator/igniter application and Isp is
not of concern. The burn rate values are found to be comparable for all the
propellants at the present solid loading that were used.
100
80
Weight (%)
60
40 HTPB-AP Propellant
PTPB-AP Propelant
20
0
0 50 100 150 200 250 300 350
o
Temperature ( C)
Figure 8. TGA of PTPB-AP and HTPB-AP propellant.
Table 6. Burn rate of PTPB propellant
Burn rate (mm/s) at HTPB-urethane PTPB-triazole

6.93 MPa 16.09±0.08 16.10±0.12
From the study, it can be concluded that the triazole formation has no
adverse effects on the ballistic properties of the propellant and is advantageous
with respect to the combustion products.
CONCLUSIONS
PTPB was obtained by the end functionalisation of HTPB by alkyne
functional groups through a direct method. Curing of the polymer was effected
through ‘click mechanism’ by reaction of the alkyne groups with an azide
containing polymer namely GAP to form triazole network in the presence of
cuprous iodide as cure catalyst. The curing reaction was monitored by DSC.
While, curing of PTPB with GAP occurred at higher alkyne-azide molar
equivalence, phase separation occurs due to difference in solubility parameters
of PTPB and GAP. For this system, curing occurs only at an alkyne-azide ratio
of 1:0.1. Rheological studies reveals that gel point for PTPB-GAP system
occurs after 190 minutes (at 80oC) in comparison to 120 minutes for HTPB-
TDI system, which is advantageous for processing. The mechanical properties
of the triazoles based on PTPB were evaluated and compared with HTPB-TDI
urethanes. DMA studies indicate a biphasic transition for PTPB-GAP with two
glass transitions (Tg) occurring at -40.5oC which may be due to the butadiene
backbone and the second one at 18.4oC may be due to the triazole network.
The thermal decomposition studies indicate that the thermal stability of
the neat polymers is improved by triazole-triazoline formation. The
mechanism of decomposition was elucidated by pyrolysis GC-MS. studies. It
is observed that the degradation of the polymer does not occur at lower
temperature of 250oC. At higher temperatures, the decomposition is complete
and proceeds with cleavage of triazole-triazoline groups and the polymer back
bone. The decomposition of the polymers in the presence of AP was evaluated
as oxidiser. The decomposition pattern of PTPB- propellant system is similar
to HTPB-urethane decomposition. The mechanical properties and burn rates of
the propellant based on PTPB- and are comparable to conventional HTPB-
urethane propellants. Thus, propellants based on PTPB yield more gaseous
products which are conducive for specialised applications such as gas
generator or as pyrogen igniter propellant.
ACKNOWLEDGMENT
The authors thank Director, Vikram Sarabhai Space Centre for permission
to publish this work. Support from Analytical and Spectroscopy Division,
VSSC for characterisation is acknowledged.
REFERENCES
[1] DeLuca, L.; Shimada, T.; Sinditskii, V.P.; Valery, P.; Calabro, M. (Eds);
Chemical Rocket propulsion-a Comprehensive survey of energetic
materials; Springer International Publishing, Switzerland, p.127-138.
[2] DeLuca, L.T.; Galfetti, L.; Maggi, F.; Colomb, G.; Merotto, L.; Boiocchi,
M.; Paravan, C.; Reina, A.; Tadini, P.; Fanton, L. ActaAstronautica,
2013, 92, 150-162.
[3] Guery, J. F.; Chang, I.S.; Shimada. T., Glick. M.; Boury, D.; Robert, E.
ActaAstronautica, 2010, 66, 201-219.
[4] BrunoC., AccetturaA.G. (Eds); advanced propulsion systems and
Technology Today to 2020, AIAA, Reston, VA, 2008.
[5] Moore, T.L.; Polybutadienes Dominate for 40 Years, Chemical
Propulsion Information Agency Bulletin, vol. 24, No. 2, March 1988.
[6] Woods,G.; The ICI Polyurethanes Book, John Wiley and Sons, New
York, 1990.
[7] Celina, M.; Graham, A.C.; Gillen, K.T.; Assink, R.A.; Minier, L.M.
Rubber Chem. Techn., 2000, 73, 678-693.
[8] Hailu, K.; Guthausen, G.; Becker, W.; König, A.; Bendfeld, A.; Geissler,
E. Polymer Testing, 2010, 29, 513-519.
[9] Daesilets, S.; Villeneuve, S.; Laviolette, M.; Auger M. J. Polymer
Science, Part A: Polymer Chemistry, 1997, 35, 2991-2998.
[10] Gopala Krishnan, P.S.; Ayyaswamy, K.; Nayak, S.K. J. Macromolecular
Science, Part A: Pure and Applied Chemistry, 2012, 50, 128-138.
[11] Fabio, L.B.; Marcio, A.A.; Bluma, G.S. J. Appl.Polym. Sci., 2002, 83,
838-849.
[12] Ji, H.; Sato, N.; William, K.N.; Mays, J.W. Polymer, 2002, 43, 7119-
7123.
[13] Wang, Y.; Hillmyer, M.A. Macromolecules, 2000, 33, 7395-7403.
[14] Eroglu M.S.; Hazer B.; Guven O. Polym. Bull., 1996, 36, 695-701.
[15] Subramanian, K.; Sastri, K.S. J. Appl.Polym. Sci., 2003, 90, 2813-2823.
[16] Cho, B.S.; Noh, S.T. J. Appl.Polym. Sci., 2011, 121, 3560-3568.
[17] Saravanakumar, D.; Sengottuvelan, N.; Narayanan, V.; Kandaswamy,
M.; Varghese T.L.J. Appl.Polym. Sci., 2011, 119, 2517-2524.
[18] Barcia, F.L.; Thiago, P.A.; Bluma, G.S. Polymer, 2003, 44, 5811-5819.
[19] Murali, M.Y.; Raju, M.K. Designed Monomers and Polymers, 2005, 8,
159-175.
[20] Shankar, R. M.; Roy, T. K.; Jana, T.J.Appl.Polym. Sci., 2009, 114, 732-
741.
[21] Huisgen. R. Angew. Chem. Int., Ed.,1963,2, 633–645.

[22] Fireston,R. J. Organic Chemistry, 1968,33, 2285–2290.
[23] Rostovtsev, V.V.; Green.L.G., Fokin. V.V., Sharples, K.B. Angew.
Chem. Int., Ed,2002,41, 2596-2599.
[24] Binder, W.H., Sachsenhoer, R.Macromol. Rapid Commun., 2007, 28,
15-54.
[25] Binauld. S., Damiron. D., Hamaide;Pascault, J.P.;Fleury,
E.;Drockenmuller E.Chem. Commun., 2008, 35, 4138-4140.
[26] Rahm. M., Green Propellants. Stockholm, Sweden: Royal Institute of
Technology; 2010.
[27] Jung, J.H.,Lee, K.H., Koo, B.T.Tetrahedron Letters, 2007, 48, 6442-
6448.
[28] Wang. L., Song, Y., Gyanda, R.; Sakhuja, R.; Nabin, K.; Hanci, C.;
Gyanda, K.; Mathai, S.; Sabri, F.; Ciaramitaro, D.A.; Bedford, C.D.;
Katritzky, A.R.; Duran, R.S.J.Appl.Polym. Sci., 2010, 117, 2612-2621.
[29] Song, Y.; Wang, L.; Gyanda, R.; Sakhuja, R.; Cavallaro, M.; Jackson, D.
C.; Meher, N. K.; Ciaramitaro, D. A.; Bedford, C. D.; Katritzky, A. R.;
Duran, R. S. J.Appl.Polym. Sci.,2010, 117, 473-478.
[30] Lee, D.H.; Kim, K.T.; Jang, Y.; Lee, S.; Jeon, H.B.; Paik, H.; Min, B.S.;
Kim, W. J.Appl.Polym. Sci., 2014, doi: 10.1002/APP.40594.
[31] Ding, Y.; Hu, C.; Guo, X.; Che, C.; Huang, J. J.Appl.Polym. Sci., 2014,
doi: 10.1002/app.40007.
[32] Gordon. S., McBride B.J. Computer programme for calculation of
complex chemical equilibrium compositions and applications II, NASA
reference publication, NASA RP-1311-P2. Lewis research Centre.
Cleveland. Ohio. USA; 1994.
[33] Frankland, J. A.; Edwards, H. G. M.; Johnson, A. F.; Lewis, I. R.,
Poshyachinda, S.Spectrochimica Acta, 1991,47A,1511-1524.
[34] Pham, Q. T. Proton and carbon NMR spectra of Polymers, Florida, 1991.
[35] Sreelatha, S.P.; Ninan, K.N. J. Appl. Polym. Sci., 1995, 56, 1797-1804.
[36] Huang, S.; Lai J.-Y.J.Membrane Science, 1995, 105, 137-145.
[37] Bräse, S.; Gil, C.; Knepper, K.; Zimmerman,V. Angew. Chem. Int., Edn,
2005, 44 5188-5240.
[38] Sekkar, V.; J. Appl. Polym. Sci. 2010, 117, 920-925.
[39] Jain, S.R.; Sekkar, V.; Krishanmurthy, V.N.J.Appl.Polym. Sci., 1993, 48,
1515-1523.
[40] Wingborg, N. Polymer Testing, 2002, 21, 283-287.
[41] Arisawa, H.; Brill, T.B.Combustion and Flame, 1996, 106, 131-143.
[42] Yang, V.; Brill, T.B.; Ren, -Z. Solid Propellant Chemistry, Combustion
and Motor Interior Ballistics, Vol. 185, Progress in Astronautics and
Aeronautics, AIAA.Inc, Virginina, 2000.
EDITOR CONTACT INFORMATION
Dr. Yu Chen
Associate Professor
School of Material Science and Engineering
Beijing, PR China
Email: cylsy@163.com
Zong-Rui Tong
Beijing, PR China
INDEX
bitriazolium salts, 63
# bi-1,2,3-triazolyl complex formation, 64
from Condensations and Non-Catalyzed
1,3-dipolar cycloaddition, 12, 13, 14, 18, 21, Cycloadditions, 53
23, 25, 39, 45, 48, 79, 83, 97, 143, 152, from Copper-Catalyzed Alkyne-Azide
193, 212, 214, 215, 224, 236, 237, 299, Cycloaddition, 54
317, 349 from Homocoupling Reactions, 62
13C-labeled nitrile N-oxide, 31, 48
C
A
capillary, 158, 171, 174, 176
addition reaction of carbon-carbon capillary column, 158, 171, 174, 176
multibonds, 8 carbon, 8, 235, 236, 246, 255
ambiphilic dipole, 23 carbon nanostructures, 235, 236, 246, 255
application, 1, 3, 8, 9, 10, 11, 13, 18, 24, 43, carbon nanotubes, 231, 236, 238, 246, 248,
52, 65, 70, 71, 75, 82, 85, 90, 93, 95, 96, 249, 250, 251, 254, 286, 314, 339
98, 99, 100, 105, 108, 109, 112, 125, carbonyl condensation reaction, 7, 8
126, 131, 133, 135, 143, 150, 151, 152, catalytic microreactor, 202
160, 162, 172, 190, 216, 225, 226, 238, catenane, 37, 49, 133
281, 282, 283, 286, 287, 292, 305, 306, characteristics, 1, 4, 5, 13, 21, 32, 65, 70,
322, 323, 327, 329, 333, 340, 343, 344, 79, 83, 87, 160, 161, 171, 178, 185, 211,
361 212, 214, 217, 232, 293, 315, 349, 353,
356, 357, 360
B chemical, 23, 26, 30, 31, 33, 34, 36, 37, 80,
341, 351, 353
biological analysis, 314, 316, 330, 332, 333 chemical ligation, 23, 26, 36, 37, 341
biomedical engineering, 108 chemical shift, 30, 31, 33, 34, 351, 353
biomedical fields, 92, 281, 282 chemical stability, 36, 80
biomolecules, 314, 328, 329 chromatography, 18, 31, 157, 158, 181, 191,
biomolecules sensing, 314, 328, 329 193, 194, 207, 226, 293, 305, 345
370 Index
clickable surface, 159, 180

colorimetric sensors, 314
E
condensation, 7, 8
Elastomer, 139, 140, 149, 155
corannulene, 236, 255, 265, 266
enaminoketone, 36, 37
cross-linker, 37, 115, 123, 127, 136
enzyme inhibitors, 230, 304
cross-linking, 22, 25, 37, 38, 46, 84, 85, 88,
92, 94, 108, 115, 125, 174, 303
CuAAC, 14, 51, 54, 55, 56, 57, 58, 59, 61, F
62, 79, 81, 82, 102, 108, 133, 139, 140,
141, 142, 143, 145, 148, 152, 155, 162, flow chemistry, 162
165, 166, 180, 189, 199, 214, 235, 236, fluorescence sensors, 283, 314
237, 239, 240, 242, 243, 244, 245, 247, fullerenes, 46, 236, 238, 239, 241, 244, 245,
248, 249, 250, 251, 252, 253, 254, 256, 246, 249, 250, 251, 256, 269
259, 260, 263, 264, 265, 266, 289, 290, functionalization, 12, 14, 15, 40, 47, 48, 78,
314, 315, 316, 317, 318, 319, 320, 321, 90, 92, 95, 102, 107, 108, 110, 111, 118,
322, 327, 333, 336, 337 124, 129, 133, 135, 144, 159, 163, 164,
CuAAC reaction, 54, 55, 56, 57, 58, 59, 61, 165, 167, 179, 180, 181, 182, 184, 185,
62, 108, 141, 142, 143, 145, 148, 152, 187, 195, 201, 216, 225, 230, 238, 242,
165, 236, 239, 263, 266, 290, 314, 317, 245, 246, 247, 250, 251, 252, 253, 255,
318, 319, 320, 321, 322, 327, 333 258, 259, 261, 274, 284, 285, 286, 288,
cycloaddition reaction, 3, 5, 11, 13, 22, 25, 315, 332, 336, 341
26, 29, 46, 53, 72, 78, 128, 148, 158, furoxan, 22, 25, 26
162, 213, 340
G
D
gene transfer carrier, 281, 282
dearomatization, 28, 48 grafting reaction, 41, 42, 78, 187
degradation, 22, 25, 26, 49, 84, 108, 113, graphene, 176, 197, 198, 220, 233, 236,
118, 122, 123, 125, 126, 131, 132, 136, 238, 251, 252, 253, 254, 255, 269, 274,
137, 141, 148, 289, 292, 302, 308, 357, 314, 335, 337
362
development, 1, 2, 3, 10, 11, 13, 18, 22, 23,
27, 37, 38, 39, 43, 49, 54, 71, 72, 88, 89, H
100, 116, 129, 153, 160, 216, 238, 244,
266, 282, 287, 294, 296, 297, 298, 302, homoditopic nitrile N-oxide, 22, 24, 27, 36,
304, 305, 324 37, 38, 47, 49
Diels-Alder click coupling, 170 Huisgen reaction, 8, 23, 237, 244, 248, 250,
dimerization, 22, 26, 45, 47, 61 259, 264
dipolarophile, 23, 26 Hydrogel, 87, 88, 89, 107, 108, 123, 129,
133, 135, 136
hydroxamoyl chloride, 24
Index 371
I N
iminoenol, 36, 37 natural polymers, 69, 70, 71, 82, 90, 96

intramolecular cycloaddition, 22, 23, 28, 29, nitrile N-oxide, 21, 23, 24, 25, 26, 27, 28,
30, 41, 241 29, 30, 31, 32, 33, 34, 35, 36, 39, 40, 42,
isomerization, 22, 26, 27, 47 43, 44, 47, 48, 49, 50
isoxazole, 36, 45, 300 nitrile N-oxide-terminated polymer, 22, 24
nitroalkane, 24
nucleophilic ring-opening reaction, 6, 213
K
ketene, 38, 39, 48 O
optical sensing, 313, 314, 316, 317, 320,

L 323, 325, 328, 332, 333
orthogonal agent, 22, 24, 27, 38, 39, 42
living cells detection, 314
P
M
pathogens, 314, 323
medical, 76, 116
pathogens detection, 314, 323
medical application, 76, 116
perylene, 236, 245, 251, 255, 261, 262, 264
membrane preparation, 211, 216
pharmaceutical science, 281, 282
membrane surface modification, 211, 217,
poly(boron enaminoketonate), 37, 49
221, 222
Poly(-aminoalcohol), 36
micro-reactor, 158, 199, 202, 203
polyaromatic, 235, 236, 258, 266
modification, 8, 10, 11, 17, 39, 40, 42, 48,
polycycloaddition, 36
49, 50, 69, 70, 75, 79, 80, 85, 93, 94, 95,
polyisoxazole, 36, 37
96, 97, 99, 100, 102, 105, 110, 139, 141,
polymer nitrile N-oxide, 40, 41, 42, 47
150, 151, 153, 154, 155, 165, 171, 178,
polyrotaxane, 37, 49
179, 199, 211, 212, 214, 216, 217, 218,
porous materials, 158
224, 225, 231, 232, 281, 305, 308, 323,
pyrene, 25, 111, 236, 249, 255, 256, 257,
340, 348, 349, 351
258, 259, 260, 261, 264, 265, 306, 307,
monolith, 158, 159, 160, 161, 162, 171, 172,
337, 339
173, 174, 177, 178, 179, 180, 181, 183,
184, 185, 186, 187, 188, 189, 190, 191,
193, 194, 195, 196, 197, 198, 199, 200, Q
201, 202, 328, 343
quadrupolar relaxation, 30
372 Index
R T
rotaxane, 37, 49, 133 thiol-ene reaction, 9, 113, 116, 117, 130,
134, 140, 143, 144, 145, 147, 149, 151,
152, 154, 166, 167, 168, 172, 173, 174,
S 193, 214, 216, 222, 314, 315, 327, 328,
329, 336, 342
self-decomposition, 23, 26, 27, 30, 31
thiol-ene/yne, 8, 9, 15, 17, 18, 80, 88, 90,
SPAAC reaction, 118, 119, 122, 128, 314,
102, 104, 113, 115, 116, 117, 127, 129,
323, 324, 325, 326, 327, 333
130, 133, 134, 139, 140, 143, 144, 145,
stable nitrile N-oxide, 23, 30, 36, 38, 49
146, 147, 149, 151, 152, 154, 155, 157,
stationary phase, 158, 160, 180, 190, 191,
159, 163, 166, 167, 168, 171, 172, 173,
193, 194, 203, 328
174, 175, 176, 177, 184, 185, 186, 187,
stimuli-responsive hydrogel, 108
192, 193, 194, 196, 198,200, 201, 202,
surface functionalization, 102, 158, 160,
203, 214, 216, 222, 228, 229, 306, 307,
167, 171, 172, 181, 184, 190, 252
314, 315, 316, 327, 328, 329, 336, 342,
surface modification, 10, 13, 17, 42, 96,
343, 344
101, 105, 136, 139, 150, 159, 179, 185,
thiol-epoxy click coupling, 170
211, 216, 217, 223, 230, 232
types, 1, 5, 9, 21, 80, 89, 100, 108, 125, 129,
133, 152, 193, 213, 294, 314, 316, 321,
347

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CHEMISTRY RESEARCH AND APPLICATIONS

Additional books in this series can be found on Nova’s website

Additional e-books in this series can be found on Nova’s website

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Published by Nova Science Publishers, Inc. † New York

Chapter 1 The Introduction of Click Chemistry Reaction 1

Chapter 8 Click Chemistry for Membrane Preparation and

for the whole comprehension of the readers to the characteristics and

1. DEVELOPMENT OF CLICK CHEMISTRY

In the past 150 years of modern chemistry history, a variety of synthesis

such as drug development and the synthesis and preparations of biomedical

2. CHARACTERISTICS OF CLICK CHEMISTRY REACTION

1) Click chemistry reactions use readily available raw materials. Many

These characteristics of click chemistry reaction indicate that its chemical

3. TYPES OF CLICK CHEMISTRY REACTION

Cycloaddition reaction usually joins unsaturated reactants to form a

(PPh3)3Br, sodium ascorbate reduced CuSO4, Cu and Cu reduced CuSO4 [30,

2) Nucleophilic ring-opening reaction

The nucleophilic ring-opening reaction is realized mainly by the

Figure 1. Mechanism of Cu (I) catalysis for terminal azide-alkyne coupling.

Epoxy compounds contain a three-membered ring with great tension,

3) Carbonyl condensation reaction

Figure 2. The ring-opening reaction of epoxides by amine attack.

Figure 3. Regioselectivity of oxirane opening.

The reactions of aldehydes or ketones with 1, 3-diols to produce 1, 3-

4) Addition reaction of carbon-carbon multibonds

The click reaction of thiol–ene/yne is a typical addition reaction of carbon-

Figure 4. Acetals of D-isoascorbic acid containing 1, 3-dioxolane via the carbonyl

Figure 5. Mechanism of thiol-ene click chemistry.

4. APPLICATION OF CLICK CHEMISTRY REACTION

Click chemistry has been rapidly developed and applied in a variety of

free radical polymerization methods have their advantages and disadvantages.

for complex molecule synthesis. Journal of the American Chemical

[14] Tornoe, CW; Christensen, C; Meldal, M. Peptidotriazoles on solid

[25] Martinek, M; Filipova, L; Galeta, J; Ludvikova, L; Klan, P.

[34] Markiewicz, KH; Wilczewska, AZ; Chernyaeva, O; Winkler, K. Ring-

[75] Fugier, E; Dumont, A; Malleron, A; Poquet, E; Pons, JM; Baron, A;

Yasuhito Koyama1, and Toshikazu Takata2,†

the reported self-reactions including (i) dimerization to furoxan, (ii)

application of nitrile N-oxide-based click agents, such as homoditopic nitrile

Nitrile N-oxide can be generated from various precursors. Scheme 1

Scheme 1. Preparation of nitrile N-oxide.

The other preparation methods of nitrile N-oxide include the dehydration

Scheme 2. Retro-1,3-dipolar cycloaddition of strained furoxan to give vic-nitrile N-

2. REACTION OF NITRILE N-OXIDE

shows the representative 1,3-dipolar cycloaddition reactions of nitrile N-oxide

Scheme 3. 1,3-Dipolar cycloaddition reactions of nitrile N-oxide to unsaturated bonds.

research has demonstrated that the isomerization rate is strongly dependent on

Scheme 4. Possible self-decompositions (I-III) of nitrile N-oxide and the structures of

Scheme 5. Intramolecular cycloaddition of nitrile N-oxide to non-strained, monocyclic

In the structural optimization of the aromatic nitrile N-oxide agents, we

Table 1. Intramolecular cycloaddition of 2-phenoxynitrile N-oxides

entry 1 R1 R2 R3 2 yield/% time/h 3 yield/%

We next investigated the electronic effect of the benzene ring on the

3. STRUCTURE OF NITRILE N-OXIDE

Scheme 6. Synthesis of 13C-labeled nitrile N-oxide 7 and 8.

Thus, we investigated the synthesis of 13C-labeled nitrile N-oxide and the

The VT-UV-vis spectra of 7 and 8 provided clear information concerning

Figure 6. Possible temperature-dependent structural change of nitrile N-oxide in

These phenomena observed in the VT-13C NMR and VT-UV-vis spectra

4. SYNTHESIS AND APPLICATIONS OF

Scheme 7. Synthesis of homoditopic nitrile N-oxide 9 and the structure of 10.

Both 9 and 10 served as a catalyst-free chemical ligation tool between