Article

pubs.acs.org/JPCA

Chemistry and Photochemistry of 2,6-Bis(2-

hydroxybenzilidene)cyclohexanone. An Example of a Compound
Following the Anthocyanins Network of Chemical Reactions
Artur J. Moro,† Ana-Maria Pana,‡ Liliana Cseh,*,‡ Otilia Costisor,‡ Jorge Parola,† L. Cunha-Silva,§
Rakesh Puttreddy,∥ Kari Rissanen,∥ and Fernando Pina*,†
†
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte de
Caparica, Portugal
‡
Institute of Chemistry Timisoara of Romanian Academy, 24 MihaiViteazul Bvd, 300223 Timisoara, Romania
§
REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal
∥
Department of Chemistry, Nanoscience Center, University of Jyväskylä, 40014 Jyväskylä, Finland
*
S Supporting Information

ABSTRACT: The kinetics and thermodynamics of the 2,6-

bis(2-hydroxybenzilidene)cyclohexanone chemical reactions
network was studied at different pH values using NMR,
UV−vis, continuous irradiation, and flash photolysis. The
chemical behavior of the system partially resembles
anthocyanins and their analogue compounds. 2,6-Bis(2-
hydroxybenzilidene)cyclohexanone exhibits a slow color
change from yellow to red styrylflavylium under extreme
acidic conditions. The rate constant for this process (5 × 10−5
s−1) is pH independent and controlled by the cis−trans
isomerization barrier. However, the interesting feature is the
appearance of the colorless compound, 7,8-dihydro-6H-chromeno[3,2-d]xanthene, isolated from solutions of acid to neutral
range, characterized by 1H NMR and single crystal X-ray diffraction. Light absorption by 2,6-bis(2-hydroxybenzilidene)-
cyclohexanone solutions immediately after preparation exclusively results in cis-isomer as photoproduct, which via hemiketal
formation yields (i) red styrylflavylium by dehydration under extremely acidic solutions (pH < 1) and (ii) colorless 7,8-dihydro-
6H-chromeno[3,2-d]xanthene by cyclization in solutions of acid to neutral range.

■ INTRODUCTION
Flavylium derivatives are an important family of compounds
that comprise anthocyanins and other natural derivatives, as
well as a variety of synthetic ones. Existence of a common
chemical reaction network is the remarkable feature in these
compounds, one such example for malvidin-3-glucoside
(oenin) is shown in Scheme 1.1−3
The flavylium cation is stable only at very acidic pH values.
Raising the pH leads to the formation of the quinoidal base via
proton transfer in competition with hydration of the flavylium
cation to originate the hemiketal. The former reaction is by far
the fastest of the network and by consequence the quinoidal
base is formed immediately after a direct pH jump, here on
defined as the addition of base to equilibrated solutions of the
flavylium cation at very low pH values. As shown by Brouillard
and Dubois,4,5 the quinoidal base does not react in acidic to
neutral medium. Consequently, the fraction of flavylium cation
that is available to form hemiketal and allow the system to go
forward (up to trans-chalcone, Ct) decreases with increasing
pH. Quinoidal base is thus a kinetic product that retards the
formation of the final equilibrium. On the other hand, the
hemiketal is in fast equilibrium with cis-chalcone, Cc (usually in
the subseconds lifetime), and the (final) equilibrium is reached
upon formation of trans-chalcone through the isomerization.
One interesting property of flavylium related compounds is the
possibility of achieving photochromism from irradiation of the
trans-chalcone form.
It was reported that besides flavylium derivatives, some
families of structurally related compounds follow an identical
network of chemical reactions as the one shown in Scheme 1. It
is the case of the so-called styrylflavylium and naphthoflavylium,
Scheme 2.6−8
In the frame of our research to identify the anthocyanins’
network of chemical reactions in other types of compounds
with the aim of discover new photochromic systems, it was
reported that 2,2′-dihydroxychalcones have the ability to form
flavylium cations through the same mechanisms. The trans-
chalcone isomerizes to the cis-chalcone, this one closes the ring
Received: June 5, 2014
Revised: July 23, 2014
Published: July 24, 2014

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The Journal of Physical Chemistry A Article

Scheme 1. Network of Chemical Reactions of Anthocyanins, As Exemplified for Oenin

Scheme 2. Flavylium Derived and Flavylium Analogue Compounds Following the Same Network of Chemical Reactions

Scheme 3. Formation of Two Flavylium Cations (Marked in Red and Blue) from Asymmetric 2,2′-Dihydroxychalcones, through
the Same Mechanism as the One Followed by Anthocyanins and Related Compounds7

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The Journal of Physical Chemistry A
to give hemiketal, which dehydrates, leading to the flavylium
cation. In the case of asymmetric 2,2′-dihydroxychalcones, two
different flavylium compounds are obtained, Scheme 3.7
Besides the mechanism shown in Scheme 1 (identical to
anthocyanins), kinetic evidence for the coexistence of an
alternative mechanism which involves the formation of a ketal
intermediate was achieved.7,9 This intermediate (not isolated)
was proposed to explain the interconversion of the two
styrylflavylium isomers.
Very recently some of us reported evidence for the formation
of flavylium cation from 2,6-bis(2-hydroxybenzilidene)-
cyclohexanone (hereafter referred to as DHCH), Scheme 4.10
Scheme 4. Chemical Structure of 2,6-Bis(2-
hydroxybenzilidene)cyclohexanone (DHCH)
This compound is a symmetric 2,2′-dihydroxychalcone with the
two α carbons adjacent to the ketone linked by an aliphatic
bridge that confers more rigidity to the structure. The aim of
this work is to study the kinetics and thermodynamics of the
chemical reaction network for DHCH, particularly the isolation
and characterization of the ketal intermediate, to confirm the
existence of spironaphthopyranes as intermediates in the
chemistry of 2,2′-dihydroxychalcones, thereby linking the two
families of compounds.
■ RESULTS AND DISCUSSION
The absorption spectra of DHCH as a function of pH, Figure
1A, is compatible with a diprotic acid with acidity constants
equal to pKCt/Ct− = 9.4 and pKCt−/Ct2− = 10.9. As observed in
other polyphenols, the successive deprotonation gives rise to a
red shift of the absorption bands. The solutions at basic pH
values are stable for long time periods (weeks) at room
Figure 1. (A) Titration of the trans-chalcones of DHCH in basic medium, methanol/water (1:1), 0.05 mM. (B) Spectral variations of the compound
DHCH 0.1 mM, upon dissolution in methanol/water (1:1) (natural pH = 6.5). The value of the rate constant in the range 4.2 < pH < 6.4 is the
same within the experimental error.
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temperature and by consequence the compound can be stocked
at higher pH values.
In contrast, solutions of the compound DHCH in methanol/
water (1:1) are not stable and in a slow process evolve to a
colorless species according to spectral modifications shown in
Figure 1B. The process takes place at the same rate within a pH
range of 4.2 < pH < 6.4. The spectral changes indicate that the
trans-chalcone leads to a product lacking of absorption in the
visible, suggesting a disruption of the π−π* conjugation of the
aromatic rings. This behavior has been observed in the
flavylium reaction network for the hemiketal species. Regarding
the kinetic process, its rate is relatively low and pH
independent, compatible with a kinetic step controlled by the
cis−trans isomerization.
The formation of the colorless product in the reaction
network supports the kinetics reported in Figure 1B and was
followed by 1H NMR.
The assignment of the peaks to the structure of the Ct form
of DHCH, Scheme 5, was previously reported.10 Evolution of
the NMR spectra over long periods of time (up to 2 weeks) is
shown in Figure 2. During this period, the solution evolves
from yellow to colorless, hailing crystals suitable for single
crystal X-ray analysis. Redissolution of the crystals in CD3OD
yielded spectrum (7) which, by comparison with spectrum (6),
proves that the crystals represent the thermodynamic product
of the system under these conditions. The analysis of the 13C
NMR spectrum (see Supporting Information, Figure S3) shows
only 10 signals, a clear indication that the structure is very
symmetric.
Full characterization and assignment of 1H and 13C signals
was achieved with HSQC and HMBC spectra (Table S1,
Supporting Information) and allowed us to identify a
spiropyrane-like structure linked by a propyl bridge, 7,8-
dihydro-6H-chromeno[3,2-d]xanthene (Figure 3), henceforth
named B−B.
The crystal structure of B−B solved in the orthorhombic
space group Pbcn lies on 2-fold axis supporting our 1H NMR
and 13C NMR spectra assignment. As shown in Figure 3, the
benzopyrans are connected at carbon C9 in near tetrahedral
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Figure 2. NMR spectra of DHCH in CD3OD upon dissolution and followed over time: (1) 5 min, (2) 7 h, (3) 15 h, (4) 43 h, (5) 94 h, and (6) 2
weeks after dissolution. Spectrum (7) corresponds to the 1H NMR of crystals of B−B.
Figure 3. Crystal structure of B−B with selected atom labeling.
Selected bond lengths (Å) and bond angles (deg): C1−O1 =
1.3811(15), C9−O9 = 1.4368(14), O1−C9−O1′ = 107.48(14), O1−
C9−C8′ = 107.03(6), O1−C9−C8 = 112.50(6), C8−C9−C8 =
110.35(15).
geometry [τ4 = 0.95]11 and are linked by a propylene chain at
C8 and C8′. The twist at the spiro-carbon C9 is a commonly
observed phenomena with C8−C9−C8′ angles being charac-
teristic of the alkylene chain. Hence, the C8−C9−C8′
[110.35(15)°] angles in B−B fall between the ethylene
[104.3(2)°] and butylene [118.2(3)°] and are similar to the
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propylene [110.9(1)°] bridge of previously reported spiro
aromatic pyrans.12 The benzene and the pyran ring are nearly
coplanar with a nonbonded O1−O1′ distance of ca. 2.317 Å.
Furthermore, the bond lengths and bond angles around the
spiro-carbon C9 of B−B are similar to the values reported for
spirobinapthopyran molecule with propylene bridge.12
Solutions of B−B (equilibrated at natural pH) were
submitted to pH jumps to the basic (up to pH = 12) and
acid regions (up to [H3O+] = 2 M). Although in basic to acid
medium B−B is stable, in very acidic conditions it is rapidly
transformed into a flavylium cation via formation of hemiketal
B (Scheme 5). The absorption spectra taken 2 min upon the
pH jumps from B−B at natural pH to lower pH values are
represented in Figure 4. The pH dependence of the absorbance
at 504 nm in the inset of Figure 4 is compatible with a single
acid−base equilibrium with apparent pK′a= 0.3.
The kinetic process for the transformation of B−B into the
flavylium cation was studied as a function of pH and is reported
in Figure 5.
The rate constants follow a first-order kinetic process and
have peculiar pH dependence as shown in Figure 5B. At lower
pH values there is a direct proportionality between the value of
the rate constant and the proton concentration, whereas at
higher pH values the rate of flavylium cation formation
increases with decreasing proton concentration.
To clarify this question, a solution of DHCH was brought to
pH = 0.3 and irradiated at 366 nm. An aliquot was kept in the
dark and used as reference. Though the solution in the dark
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Scheme 5. Proposed Kinetic Scheme for the DHCH Network of Chemical Reactions
Figure 4. Spectral variations observed upon pH jumps from
equilibrated solutions of 0.1 mM B−B in water/methanol (1:1) at
natural pH to lower pH values. The spectra were collected 2 min after
the mixing. No significant changes on the absorption spectra were
observed after 1 day.
Figure 5. (A) Trace for the flavylium formation upon a pH jump of B−B at natural pH to pH = 0.6. (B) Representation of the observed rate
constants for AH+ appearance from B−B as a function of pH as determined upon pH jumps (●) and from flash photolysis of Ct (○). Fitting was
achieved with eq A6 for Ka < 10−4 M, Kh = 3.3 × 10−3 M, k−b < 0.001 s−1, kb/k−h < 3 × 10−4, kH−b = 0.065 M−1 s−1, kHb = 0.05 M−1 s−1.
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slowly evolves to flavylium cation with a rate constant of 7 ×
10−5 s−1 at pH = 0.5 (in good agreement with the thermal rate
constant in Figure 1B for the cis−trans isomerization barrier),
the flavylium cation is formed after a few seconds of irradiation,
Figure 6A. Moreover, by irradiating a fresh solution of DHCH
at natural pH, the photoproduct exhibits the same absorption
spectrum of the cyclic ketal B−B, Figure 6B. This result is
compatible with a photochemical overcoming of the cis−trans
isomerization barrier and supports that the rate-determining
step of the flavylium formation from DHCH is the cis−trans
isomerization.
Additional information was obtained by means of flash
photolysis experiments, Figure 7. In these experiments, freshly
prepared solutions of DHCH are submitted to a flash and the
absorbance is monitored at two wavelengths where flavylium
cation (500 nm) and trans-chalcone (360 nm) absorb.
Based on the experimental data regarding flavylium
compounds and according to other results reported in
literature13,14 concerning the photoinduced cis−trans isomer-
ization, it is reasonable to consider that Cc is formed
immediately after the flash. According to Figure 7 two
successive kinetic processes take place after the flash. The
bleaching of the absorption at 360 nm can in principle be
attributed to the disappearance of Cc. This process is
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Figure 6. (A) Spectral variations upon irradiation of DHCH at 365 nm, 0.05 mM pH = 0, reaction quantum yield, Φ = 0.31. (B) The same at natural
pH, Φ = 0.43. I0 = 2.6 × 10−7 einstein min−1.

Figure 7. (A) Flash photolysis of DHCH at pH = 1.49. In both cases the process is biexponential. Fitting was achieved for the rate constants 0.46
and 0.06 s−1. (B) pH dependence of the faster rate constant. Fitting was achieved for 0.06 + 20[H+] s−1. The slowest process is fitted according to
the right branch of Figure 5B.

biexponential. At the same time, the trace at 500 nm reveals the
appearance of flavylium cation followed by its disappearance.
Both traces (at 500 and 360 nm) can be fitted with the same
two rate constants. These results can be interpreted by
considering that in the fast process flavylium is formed from
Cc via B. The question is which of these two steps,
tautomerization or hydration, is the rate-determining step.
Representation of the pH dependence of the faster process of
the flash photolysis is shown in Figure 7B. In the case of a rate-
determining step by tautomerization, the pH dependence
would be due to the acidic catalysis, whereas in the case of
hydration, the rate would be controlled by the term k−h[H+].
The data shown above suggest that the process is controlled by
the hydration reaction. Fitting can be achieved for kh = 0.06 s−1
and k−h = 20 M−1 s−1, leading to the respective equilibrium
constant Kh= 3.3 × 10−3 M. At this point the species AH+, B,
and Cc are in pseudoequilibrium. The subsequent disappear-
ance of the flavylium cation can be explained if a new (and
slower) channel appears. A possible explanation is the
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formation of B−B from B. One significant aspect is the
coincidence of the slowest process of the flash photolysis with
the right branch of the kinetic step represented in Figure 5B. In
other words, the disappearance of the flavylium cation during
the slowest kinetic step of the flash photolysis to form B−B is
the same as the appearance of flavylium cation from B−B upon
a pH jump.
These results can be interpreted if the following sequence of
reactions is considered AH+ ⇌ B (Cc) ⇌ B−B. A kinetic
expression can be deduced considering the steady state
hypothesis for the species B (Cc), eq 1 (see Supporting
Inofrmation for detailed description).
[H+]
K
[H+] + K a h
+ (k −b + k −Hb[H+])[H+]
kobs =
k b + k bH[H+]
[H+] + k −h (1)
The data reported in Figure 5B can be fitted with the
equilibrium and rate constants shown in Table 1.
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Table 1. Rate and Equilibrium Constants of DHCH Network

pKa Kh (M) kh (s−1) k−h (M−1 s−1) kb/k−h (M) kb (s−1) k−b s−1(s−1) KbH (M−1 s−1) k‑bH (M−1 s−1)
−3
>4 3.3 × 10 0.065 a
20 a
<0.0003 <0.006 <0.001 0.05 0.065
a
Determined from fast process flash photolysis. The other constants were obtained from the pH jumps of B−B and the slow process of the flash
photolysis.

Scheme 6. Thermodynamic Level of the Systema

a
The energy levels of species Ct and Cc could not be obtained but are expected to be higher than that for B.

Taking into account that Ka + Kh(1 + Kt + Kb) should be
equal to 0.3, a value of Kb + Kt = 90 is obtained. According to
the absorption spectra at the equilibrium, no absorption bands
at the wavelength region where the species Ct is expected to
absorb have been detected, indicating that Kt ≪ 1 and that Kb
is approximately equal to 90.
Using the equilibrium constant Kb, the value of k−b in Table 1
and the ratio kb/k−h, a limit value of kb < 0.09 s−1 can be
estimated. These values indicate that the kinetics between B
and B−B is slow and explains why, upon the flash, the first
process leads to flavylium cation via B and only later the
equilibrium between AH+ and B−B is established. The
following thermodynamic energy level of the species involved
The interconversion between the families of 2,2′-dihydrox-
ychalcones, spironaphthopyranes, and styrylflavylium com-
pounds can be achieved through external inputs of pH and
light.
This report opens the possibility for the development of new
photochromic systems based on these types of molecules,
where one can selectively “lock” the system in three different
species (i.e., flavylium cation/spironaphthopyran/trans-chal-
cone) that can be readily unlocked through the appropriate
stimulus.
■
*
ASSOCIATED CONTENT
S Supporting Information

can be drawn. Discussion of the thermodynamic equilibrium, synthesis of

Scheme 6 can be used to summarize all the data reported in DHCH, and absorption measurements, NMR spectra, mass
this work. Solutions of Ct are thermodynamically unstable. For spectra, and single crystal X-ray diffraction details. This material
is available free of charge via the Internet at http://pubs.acs.org.

■
example, a pH jump to pH = 2.48 is expected to form equal
amounts of AH+ and B because formation of B is faster than
AUTHOR INFORMATION
B−B. B, in pseudoequilibrium with AH+, is thus a kinetic
product that, in a second and slower process, gives B−B. Corresponding Authors
Moreover, B−B is very stable and practically the only species at *L. Cseh. E-mail: lili_cseh@yahoo.com. Tel: +40256-491818.
pH values higher than 2. Increasing the pH does not give any Fax: +40256-491824.
Ct back. However, if the equilibrium pH is very acidic, upon a *F. Pina. E-mail: fp@fct.unl.pt. Tel:+3512948355. Fax:
+3512948550.
pH jump to very basic pH values the flavylium in equilibrium
yields deprotonated B and Cc, and from these species a small Notes
The authors declare no competing financial interest.

■
fraction of deprotonated Ct can be formed.

■ CONCLUSIONS
We have proven that symmetric and asymmetric 2,2′-
dihydroxychalcones respectively may yield one or two flavylium
cations, via the formation of a spironaphthopyran intermediate.
The presence of a propylene bridge confers more rigidity to the
structure and seems to stabilize the intermediate, which was
isolated and fully characterized.
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ACKNOWLEDGMENTS
Fundaçaõ para a Ciência e Tecnologia is acknowledged through
the National Portuguese NMR Network, Grant PEst-C/EQB/
LA0006/2013 and Post-Doc grant SFRH/BPD/69210/2010.
The financial support of European Union COST Action
CM1005 is gratefully acknowledged. The Romanian authors
acknowledge the support of the Romanian Academy, Project
3.1.
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■
Article

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