Recent Advances in Organic Chemistry

A Review of Recently Reported Applications Using In Situ Spectroscopy

The Next Steps in Enzymatic Catalysis

Vaso Vlachos, Mettler-Toledo AutoChem, Inc.

Enzymes, as biocatalysts, are a highly suitable, environmentally friendly alternative to heavy metal industrial catalysts. The Green Chemistry nature of enzymes as catalysts is based on a number of characteristics. Enzymes are biodegradable and typically produced through fermentation of renewable feedstocks(1) or easily mass produced through recombinant technologies(2). Enzymes also catalyze chemical reactions under mild temperature (20C-40C) and pH conditions (pH 5-8), and perform well in aqueous environments(1). Examples of enzymecatalyzed industrial-scale organic reactions include hydrolysis, oxida-

tion, reduction, addition elimination, halogenation and dehalogenation, and transesterification(1,3). ReactIR is a real-time in situ reaction analysis system that enables scientists to easily and rapidly obtain comprehensive information and understanding about reaction chemistry. ReactIR monitors reactive chemistry using well understood midIR spectroscopy. A robust ATR probe is inserted directly into the reaction vessel, providing a molecular video of the reaction. The concentration changes of all key reactive and transient species are monitored allowing for mechanism and pathway determination.

This white paper highlights three examples pharmaceutical, academic and military where ReactIR was used to monitor the enzyme-catalyzed reactions in real time, to enable researchers to understand reaction mechanisms, and determine kinetic parameters. It is not the intent to go into detailed scientific findings as these were documented in each paper and it is recommended to read the original publications for this purpose. Instead, the author highlights the context in which ReactIR was used and how this helped researchers answer key questions.

Introduction Application 1: Monitoring Product Streams and their Dispersion Application 2: Multistep Synthesis Controlled Addition of a 3rd Stream Conclusions Enabling Technology

2 3 5 7 7

Introduction The chemical, petroleum, agriculture, polymer, electronics and pharmaceutical industries rely on catalysis to optimize their chemical processes. It is estimated that at least 90% of chemicals produced rely to some extent on catalysis(4). In recent years researchers have increasingly focused their attention on green chemistry, including catalysis(5). Enzyme-mediated catalysis may be achieved through use of the isolated enzyme, or use of whole cells. Immobilized enzymes are commonly used nowadays, allowing their removal from the reaction and reuse of the enzyme, reducing costs(1,6). Adding to the environmental advantages of the use of enzymes as catalysts are a number of industrially important characteristics. Enzymes are robust and can be stable in both aqueous and organic environments. Furthermore, enzymes are highly efficient, offering faster reaction rates than chemically catalyzed reactions and at lower concentrations. Enzymes can bind to a wide range of biological and non-natural substrates and multiple enzymes can be used to effect a cascade of sequential biocatalytic reactions. Last but not least, selectivity is a critical characteristic of enzymes as industrial catalysts. Enzymes are chemo-, regio-, diastereo-, and enatioselective, targeting specific functional groups of the substrate (1,3,6).

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Chemoselective Pancreatin Powder-Catalyzed Deacetylation Reaction

Application of chemoselective pancreatin powder-catalyzed deacetylation reaction in the synthesis of key statin side chain intermediate (4R,6S)-4-(tertbutyldimethylsilyloxy)-6-(hydroxymethyl)tetrahydropyran-2-one, Vincent Troiani, Jrme Cluzeau, and Zdenko asar, Lek Pharmaceuticals, d.d., Sandoz Development Center Slovenia, API Development, Organic Synthesis Department, Kolodvorska 27, 1234 Menge, Slovenia, Organic Process Research and Development, 2011, 15:622630.

In this paper the authors from Lek Pharmaceuticals described a chemoselective biocatalytic procedure for the synthesis of the lactonized statin side chain intermediate, (4R,6S)-4-(tert-butyldimethylsilyloxy)-6-(hydroxymethyl)tetrahydro-2Hpyran-2-one, from its acetate precursor. The chemoselective enzymatic deacetylation of the acetate precursor was achieved under mild conditions in an aqueous medium, and proved to be superior to the chemical reagent processes to which it was compared in this study. ReactIR was used to study the reaction mechanism and optimize reaction conditions and catalyst load. Statins are economically the most valuable therapeutic compounds, with annual sales revenues in excess of $30 billion, and hence have engaged the interest of many researchers. Statins have a heterocyclic core attached to a chiral 3,5-dihydroxy-6-heptenoic acid side chain (Figure 1.1). It is this side chain which is predominantly responsible for the activity of statins, and so it remains an unmodified structural component from the original, naturally occurring statin form, and researchers endeavor to synthesize it efficiently and economically.

Synthesis and structure of super-statins: Wittig Reaction

Key lactonized statin side chain precursors:

Alcohol 2

Acetate 1

phosphonium salt of heterocycle Rosuvastatin Pitavastatin

The authors experimented with using precursors Acetate 1 and Alcohol 2 to produce the formyl substituted lactonized form 3 of the 3,5-dihydroxy-6-heptenoic acid side chain, to be used in the Wittig reaction (Figure 1.1). However, the deacetylation of Acetate 1 to Alcohol 2 remains a challenge for this complex molecule with other protected functional groups and ester moieties. The authors overcame this obstacle by comparing the tin cluster ([t-Bu2SnOH(Cl)]2) catalyzed deacetylation reaction to the highly chemoselective enzymatic hydrolysis of the acetyl group. In their investigations the authors evaluated a series of 13 known reagents proven to be efficient in the deacetylation of acetates, while preserving the acid sensitive TBS moiety as well as the base-sensitive lactone ester functionality. Reactions with only three of these reagents resulted in any yield, and specifically 9%, 20% and 51% yield (after chromatographic purification). These results encouraged the authors to
3

Figure 1.1. Retrosynthetic analysis of the synthesis of optically pure superstatins containing hept-6-enoic acid residue and structure of their key lactonized side chain precursors

explore the alternative approach of enzymatic catalysis, exploiting enzymes as highly chemoselective catalysts for the deacetylation of Acetate 1. Seven different enzymes (pancreatin powder, lipase thermocymes lanuginose, and five different lipases from Rhizopeus niveus, Candida rugosa, Aspergillus niger, wheat germ and hog pancreas) were investigated in a proof of concept study. Pancreatin powder was identified as the most suitable catalyst, producing a yield of 77% (after chromatographic purification). The focus of the research then turned to optimizing the use of pancreatin powder in an industrial process of acetate deacetylation, including optimization of the catalyst load. The authors postulated that a slow stepwise addition of Acetate 1 to the catalyst solution would increase the contact between the catalyst and substrate, keeping the catalyst load high at any time relative to the substrate. In situ ReactIR was used to monitor the reaction progress in experiments to test this theory.

0.005 Water 1640cm-1 Acetate 1 1226cm-1 Alcohol 2 1240cm-1 0.003

0.004

Peak

0.002

Acetate 1, in excess, was added stepwise over eight hours to the catalyst solution 0.000 (0.2 equiv., w/w) in phosphate buffer (pH 7.0)/dioxane mixture (4/1). -0.001 Dioxane was used to solubilize Acetate 1, which is poorly soluble in water. The 00:00 04:00 shaded section of the ReactIR spectra presented in Figure 1.2 shows that Acetate 1 was initially rapidly converted to Alcohol 2 during the stepwise addition, and that all the acetate was consumed. The alcohol concentration increased for the first 2.5 hours, and at approximately 3.75 hours began to unexpectedly decrease until eight hours into the reaction. In the meantime, ReactIR data showed that Acetate 1 began to accumulate between 3.75 and eight hours. These results suggest that the catalyst was reacting with Alcohol 2, in favor of Acetate 1, when the alcohol concentration reached a particular level relative to Acetate 1 after a prolonged exposure to the phosphate buffer/dioxane medium. The result of the undesired reaction was the degradation of Alcohol 2 by the opening of its lactone ring. Acetate 1 remained unreacted and its concentration increased during the stepwise addition. Although the alcohol reaction with the catalyst slowed down after eight hours, the catalyst did not begin to react with the acetate, and the acetate concentration remained constant (slight increase in absorbance due to solvent evaporation). This data suggests the catalyst was inhibited by the product, or a side product of the unwanted consecutive and/or parallel reaction of the lactone moiety with the catalyst. To prevent the alcohol from the consecutive reaction, it was necessary to prevent contact between the catalyst and Alcohol 2 after the alcohol was formed, enabling the favored reaction of Acetate 1. To enable this, the co-solvent dioxane was omitted to result in the immediate precipitation of the alcohol, preventing its further reaction with the catalyst. However, the progress of the reaction would be limited by the
4

0.001

08:00

12:00

16:00

20:00

Relative Time (hours)

Figure 1.2. Monitoring of Acetate 1 to Alcohol 2 conversion by inline IR. The blue curve represents water. The red curve represents the reaction profile for Acetate 1 (points on the curve represent time points when substrate was dosed to the reaction mixture in a stepwise manner). The green curve represents the reaction profile for Alcohol 2.

3b. hydrated form of carbamazepine

poor solubility of Acetate 1 in water. Through experimentation, the authors determined it was possible to maximize the initial substrate concentration and minimize the catalyst load by the stepwise addition of the catalyst to the substrate solution. The authors were also able to lower the catalyst load from 2 equiv. (w/w) to 0.5 equiv. (w/w), and increase the initial substrate concentration from 0.11M to 0.2M, increasing the process throughput. This method ensured the maximum concentration of substrate available to the catalyst. Since the lactone moiety is sensitive to basic conditions and the TBS protection sensitive to acidic conditions the authors experimented with pH and determined 0.28 the optimal pH was 5.0 0.5. 0.25 A LabMax synthesis workstation equipped with an in situ ReactIR and a pH probe was used to determine the optimal reaction time, avoiding unnecessary exposure of the lactone moiety to the catalyst and phosphate buffer. The reaction was performed over 21 hours at 35C in PBS, with the pH maintained between 5.6 and 5.1. The catalyst was initially added at 0.2 equiv. (w/w), and then stepwise each hour at 0.1 equiv. (w/w) until a total amount of 0.7 equivalents (w/w) was reached. ReactIR monitored the progress of the reaction and confirmed the consumption of Acetate 1. Alcohol 2 precipitated out and was hence not monitored with ReactIR.

00:41:53 04:28:53 17:43:53

Reaction Spectra [A.U.]

0.23 0.20 0.18 0.15 0.13 0.10 0.08 0.05 0.03 0.00 1900 1800 1700 1600 1500 1400 1300 1200
-1

1100

1000

900

800

700

Wavenumber (cm )
40 0.24 5.9 5.8 5.7 5.6 5.5 5.4 pH Tr FTIR

39

LabMax Thermostat_C/ln1 (pH)

0.22 0.20 0.18

1223cm-1 [A.U.]

38

0.16 0.14 0.12

Tr (C)

0.10 The IR spectral profiles for the reaction 0.08 5.3 at 42 minutes, 4.5 hours and 17.75 hours 36 0.06 5.2 shows the decrease in the Acetate concen0.04 5.1 35 tration through the reaction (Figure 0.02 5 0.00 1.3a). Trending this concentration over -0.02 4.9 34 time, the IR data in Figure 1.3b confirms 00:00 03:00 06:00 09:00 12:00 15:00 18:00 21:00 the rapid consumption of Acetate 1 upon Relative Time (hours) the pH reaching the optimal pH range. Acetate consumption reached steady state after approximately nine hours. Figure 1.3. (a.) Accumulated inline FTIR spectra at 42min, 4h 29min and 17h 44min. The process yield was only 66% Alcohol 2, confirming the prolonged exposure of the (b.) The blue curve represents the Acetate 1 product to the catalyst and phosphate buffer caused degradation of the lactone ring solubilization and consumption monitored moiety, resulting in product loss. The authors concluded that the reaction should by inline FTIR at 1223cm-1. The red curve is the reaction temperature profile. The green be stopped at approximately 9-10 hours, immediately after the acetate conversion curve is the pH of the reaction. Small pH is complete, and followed by the downstream isolation of the product to maximize leaps correspond to pH correction with 1.0M yield. In doing this, the authors reported that the process succeeded in isolating NaHCO3 solution. Formation of Alcohol 2 is Alcohol 2 at 95% yield. not observed due to the insolubility of Alcohol

37

2 in aqueous media.

Conclusion In situ ReactIR provided the authors with the information to understand their biocatalytic reaction mechanism to prevent the the undesired competing reaction. They were also able to monitor reaction progress and optimize the reaction conditions to achieve an optimized procedure for the deprotection of acetyl moiety in((2S,4R)-4-(tert-butyldimethylsilyloxy)-6-oxotetrahydro-2H-pyran-2-yl) methyl acetate to provide a key lactonized statin side chain precursor (4R,6S)4-(tert-butyldimethylsilyloxy)-6-(hydroxymethyl)-tetrahydro-2 H-pyran-2-one. The enzymatic catalysis employed under mild conditions proved to be high yielding and economically favorable.

a.
Reaction Spectra [A.U.]

0.28 0.25 0.23 0.20 0.18 0.15 0.13 0.10 0.08 0.05 0.03 0.00 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 00:41:53 04:28:53 17:43:53

b.
40

Wavenumber (cm-1)
0.24 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5 4.9 00:00 03:00 06:00 09:00 12:00 15:00 18:00 21:00 pH Tr FTIR

39

LabMax Thermostat_C/ln1 (pH)

0.22 0.20 0.18

1223cm-1 [A.U.]

38

0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 -0.02

Tr (C)

37

36 35

34

Relative Time (hours)

Figure 1.3.

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Monitoring of Baeyer-Villiger Biotransformations of Ketones to Lactones

Pseudomonad cyclopentadecanone monooxygenase displaying an uncommon spectrum of Baeyer-Villiger oxidations of cyclic ketones Hiroaki Iwaki,1 Shaozhao Wang,2 Stephan Grosse,2 Hlne Bergeron,2 Ayako Nagahashi,1 Jittiwud Lertvorachon,2 Jianzhong Yang,2 Yasuo Konishi,2 Yoshie Hasegawa,1 and Peter C. K. Lau2 (1. Department of Biotechnology, Faculty of Engineering and High Technology Research Center, Kansai University, Suita, Osaka 564-8680, Japan; 2. Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, Canada), Applied and Environmental Microbiology, 2006, 72: 27072720. Monitoring of Baeyer-Villiger biotransformation kinetics and fingerprinting using ReactIR 4000 spectroscopy Jianzhong Yang, Marie-Jose Lorrain, Denis Rho, and Peter C.K. Lau, Biotechnology Research Institute, National Research Council Canada BRINRCC, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada, Industrial Biotechnology, 2006, 2:138-142.
Cyclododecanone

O O NADPH CPDMO NADP+ O

O2

H2O
Lauryl lactone

Cyclopentadecanone monooxygenase (CPDMO), a Baeyer-Villiger monooxygenase (BVMO), is an enzyme proven to be effective in the biotransformation of large and small cyclic ketones to lactones and offers high chemo-, regio-, and enantioselectivity. Naturally produced by Pseudomonas spp., CPDMO is now expressed in Escherichia coli(2,8,9). The work described here shows how ReactIR was used to monitor reaction progress in real time and determine the reaction kinetics of the enzyme catalyzed Baeyer-Villiger oxidation of cyclododecanone (CDD) to lauryl lactone (LL) (Figure 2.1). Iwaki et al.(8) cloned, sequenced and overexpressed CPDMO in E. coli in order to characterize the enzyme, and study its catalytic ability. In proof-of-concept work, reaction progress of purified CPDMO (200mU) and CDD substrate to produce product LL was monitored at 0, 5, 10, 20 and 60 minutes using ReactIR. ReactIR spectral data clearly shows the depletion of CDD (1714cm-1) while LL (1741cm-1) is formed as the reaction progressed (Figure 2.2). In subsequent studies by Yang et al.(2, 9), ReactIR was used to monitor the reaction progress of the whole cell Bayer-Villiger biotransformation of CDD to LL, catalyzed by recombinant CPDMO expressed by E. coli BL21. E. coli was grown at 30C, pH7 1, in a 1L fed-batch culture medium. At an OD600 of 1, 5.0g of substrate (CDD) in 20mL solvent (hexadecane) was added. Similarly to the work of Iwaki and co-workers, ReactIR spectral data (Figure 2.3a) confirms the conversion of CDD to LL by the decrease in absorbance of the substrate CDD (1713.8cm-1), while the absorbance of the product LL (1740.9cm-1) increased as the reaction progressed. Through quantitative modeling (based on authentic CDD and LL standards), the principle components of the reaction are shown
7

Figure 2.1. Biotransformation of cyclododecanone (CDD) to lauryl lactone (LL) catalyzed by a recombinant NADPH-dependent cyclopentadecanone monooxygenase (CPDMO)

0.017

Lauryl Lactone
0 min 5 min 10 min 20 min 60 min

0.016

Cyclododecanone
0.015

Absorbance

0.014

0.013

0.012

0.011 1700

1720

1740

1760

Relative Time (hours)

Figure 2.2. Overlaid infrared spectra of a time course monitoring of cyclododecanone conversion to lauryl lactone catalyzed by CPDMO using ReactIR

a.
0.05 0.04

b.

IPTG CDD Sugar

6 30 5 4

Absorbance

0.03 0.02 0.01 0.00

20 OD600 lauryl lactone concentration CDD concentration 3 2 1 0.0 -5 0 5 10 15 20 0

Time (h)

10 5 0 1780 1720 1700

-1

10

1760

Wavenumber (cm )

1740

1680

Time (h)

Figure 2.3. (a) A fingerprint of CDD bioconversion to LL, obtained by ReactIR 4000 under conditions shown in right panel. (b) Cyclododecanone (CDD) biotransformation profile as a function of cell growth in a fed-batch culture: E. coli BL21 (pCD201) cells were grown at 30C, pH 7.0 0.1, in 1L of culture medium, in a 3L bioreactor (Biobundles; Applikon Biotechnology, Foster City, California). At an OD 600 of 1, the bacterial cells were induced with 0.1mM isopropyl--D-thiogalactopyranoside (IPTG). Hexadecane (20ml) was used as the solvent carrier for both the substrate (5.0g CDD) and the extractant for the LL product. A glucose solution (500g/L) fortified with MgSO4 (10g/L) was fed at a rate of 10g/h when the OD600 of cell culture reached 10.

in as a function of time (Figure 2.3b). At approximately nine hours, the LL concentration reached steady state, indicating reaction completion. The authors estimated the limits of detection at 0.2mM for product LL. Reaction kinetics was studied with ReactIR in a microscale bioreactor system. Product formation rates at various substrate concentrations and four cell densities were determined. The Lineweaver-Burk plot, Figure 2.4, shows the reaction followed the Michaelis-Menton model. Using the Lineweaver-Burk plot the maximum (apparent) production rate Vmax-app and K m at different cell concentrations were determined (Table 2.1). This research group extended their study to improve the yield of water insoluble LL product through Baeyer-Villiger biotransformation of water-insoluble CDD by E. coli whole cell biocatalysts, harboring BVMO, in organic-aqueous two-phase bioreactor systems (TPPB) in semi-continuous mode. ReactIR was instrumental in determining the optimal solvent for the biotransformation of CDD and monitoring the formation of product LL in the organic phase.(9) LL yield increased from 2.4g in batch mode to 10~16g in semi-continuous mode. Conclusion ReactIR proved to be a useful tool for the rapid monitoring and quantification of enzyme catalyzed Bayer-Villiger biotransformations of CDD to LL, in situ. The simplest calibration mode could be applied without interference from the complex cell culture medium.

2.0

1.25 OD600 2.50 OD600 5.00 OD600 10.00 OD600

1.5

1/V (U-1)

1.0

0.5

0.0 0.000 0.005 0.010

-1

0.015

1/Cr (M )
Figure 2.4. Lineweaver-Burk plot to obtain the maximum (apparent) production rate Vmax-app and Km at different cell concentrations. V= reaction rate, Cr=initial reaction concentration.

Cell concentration (g DCW/L) Vmax-app (U) Specific Vmax-app (U/g DCW) Km (M)

0.54 4.35 8.1 587

1.08 9.43 8.8 548

2.15 18.86 8.8 550

Table 2.1. Kinetic parameters of cyclododecanone biotransformation using resting cells

CDD and LL (g/L)

4.3 44.84 10.4 591

OD600

Enzymatic Hydrolysis of Toxic Organophosphates and Organophosphonates

Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy. Jrgen Gba,b, Marco Melzera,c, Kai Kehed, Andr Richardte, Marc-Michael Bluma, a. BlumScientific Services, 80331 Munich, Germany; b. Institute of Pharmaceutical Chemistry, Philipps University Marburg, 35032 Marburg, Germany; c. Institute of Pathology, Johannes Gutenberg University Mainz, 55101 Mainz, Germany; d. Bundeswehr Institute for Pharmacology and Toxicology, 80937 Munich,Germany; e. Armed Forces Scientific Institute for Protection TechnologiesNBC Protection, 29633, Munster, Germany, Analytical Biochemistry 2009, 385, 187-193

Gb and co-workers studied the enzymatic hydrolysis of organophosphates and organophosphonates, including toxic nerve agents, with in situ ReactIR. Using ReactIR the authors were able to quantitatively monitor reaction progress and obtain reaction kinetics data. The inline approach also enabled the use of small chemical quantities, avoided cross-contamination and significantly reduced the researchers risk of toxic exposure.

a.
O O P F DFP O P F Cyclosarin (GF) O N O O P F Sarin (GB) O P CN Tabun (GA) O O O P F Soman (GD) O

The phosphotriesterase, diisopropyl fluorophosb. phatase (DFPase), isolated from the squid Loligo O O vulgaris, effectively hydrolyzes diisopropyl fluoroDFPase + + + H 2O phosphates (DFP) and the G-type nerve agents R1 P R2 R1 P R 2 + X + 2H (+HCN + 1H for Tabun) (Figure 3.1a), tabun (GA), sarin (GB), soman X O(GD) and cyclosarin (GF), which still pose a threat to both military and civilian populations. R 1 = Methyl or O-Alkyl (DFP) or N,N-Dimethylamino (Tabun) DFPase is a highly stable enzyme which can be R 2 = O-Alkyl X = F (DFP, Sarin, Soman, Cyclosarin) or CN (Tabun) easily expressed in bulk quantities, making it suitable for the decontamination of these nerve agents. Detoxification of the organophosphorus Figure 3.1. (a.) Chemical structures of DFP and the G-type nerve agents sarin (GB), compounds occurs by the hydrolysis of the P-N bond (or P-CN bond in tabun), soman (GD), cyclosarin (GF), and tabun producing a phosphate or phosphonate, protons and anions of the leaving group (GA). (b.) General outline of the hydrolysis (Figure 3.1b). reaction catalyzed by the enzyme DFPase. Previously reported methods of monitoring this reaction included titrimetric pH-stat method and the application of ion-sensitive electrodes, but these methodologies had limitations in this application. In this particular hydrolysis, the release of protons requires the use of buffers to prevent inactivation of the enzyme, which is not possible with the time consuming and labor intensive pH-stat method. Fluoride sensitive probes require a specific ionic strength of solution, and cannot be used with tabun, limiting the application of this method. In situ ReactIR FTIR spectroscopy, was investigated as an alternative technology as it would not have these limitations. ReactIR is a probe technology, monitoring reaction progression in real time and avoiding the need for sampling.
9

ReactIR with an AgX fiber conduit and DiComp ATR probe was used to quantitatively monitor the progression of the enzymatic hydrolysis of DFP and 4 toxic nerve agents by DFPase. Substrate concentrations were in the range of 1-8mM, and working volumes were less than 1mL. DFPase was expressed in Escherichia coli.
Absorbance

0.008 DFP Diisopropylphosphate 0.006

P-O-R

0.004

Depletion of DFP and the formation of diisopropylphosphate were monitored by ReactIR, as depicted in the spectra of Figure 3.2 and the decreasing and increasing arrows, respectively. The strong band at 1030cm-1 shifted to 1065cm-1 in the formation of diisopropylphosphate, and represents the P-O-R stretch. Quantitative data was obtained by first calibrating the system by the total hydrolysis of DFP at 7 different concentrations, with all other components kept constant. Exact DFP concentrations were determined by pH-stat titration, and ReactIR absorbances were measured for concentration ranges 1-8mM. Plotting a concentration versus absorbance curve the data shows a straight line, a linear dependency (Figure 3.3). Enzyme activity measurements with ReactIR were made and validated by comparison to pH-stat results. Measurements of activity were made after starting the reaction by adding a defined amount of DFPase to DFP samples in the range of 1.1mM to 8.3mM. The average slope of three individual measurements was converted to enzyme activity or reaction rate (to determine kinetic constants), using the calibration curve. Intraday validation (averaging three consecutive reactions) and interday validation (averaging three experiments obtained over three single days) showed ReactIR and pH-stat values to be comparable, and no significant difference in interday and intraday values (Figure 3.4). ReactIR data values are slightly lower than those of pH-stat; this is likely due to the slightly different reaction conditions [pH-stat experiments were conducted in the absence of buffers, changing the ionic strength of the mixture]. The kinetic constants of the reaction were determined by using ReactIR to measure reaction rates at varying concentrations of DFP. The Lineweaver-Burk plot (Figure 3.5) shows good agreement between the slope and individual data points. The values obtained for DFP were K M = 3.7mM and kcat = 2.11s-1.

0.002

1300

1200

1100
-1

1000

Wavenumber (cm )
Figure 3.2. ReactIR spectra of DFP and diisopropylphosphate. Arrows indicate increasing and decreasing bands during the course of the reaction. The peak definition used is indicated by a transparent rectangle (area under the curve) and a vertical line (peak height vs. single point baseline).

0.14 0.12 0.10

0.08 0.06 0.04 0.02 1 2 3 4 5 6 7

r2 = 0.9995

c (DFP) [mmol/L]
Figure 3.3. Representative calibration curve for DFP determined by total hydrolysis of seven DFP samples with different concentrations.

10

Specic Activity [U/mg]

Conclusion This paper reported the use of in situ ReactIR, as a novel method for the quantitation of the enzymatic degradation of DFP and organophosphorus nerve agents. ReactIR reported results that were confirmed by the titrimetric pH-stat method, and enabled the determination of the kinetic parameters, K M and kcat, of the reaction. The acknowledged benefits of in situ ReactIR included real-time monitoring of reaction progression, the technology is non-invasive and non-destructive to the chemical components of the reaction, reduction in cross-contamination and the ability to work with small quantities. Importantly, for this particular work, the use of in situ ReactIR reduced the researchers risk of toxic exposure to the nerve agent samples while monitoing the reaction progression and gaining reaction kinetics information.

250

200

FTIR pH-stat

150

100

50

DFP

GB

GD

GF

DFP

GB

GD

GF

Intraday

Interday
Figure 3.4. Intraday and interday comparison of enzyme activity as determined by the FTIR and pH-stat method against DFP and three nerve agents. GB, sarin; GD, soman; GF, cyclosarin. 1U = 1mol min-1.

60 50

1/[v(M/min)]

40 30 20 10

-0.2

0.2

0.4

0.6

0.8

1/[DFP (mM)]
Figure 3.5. LineweaverBurk plot of DFPase kinetics using DFP as a substrate. Data points are means of three individual determinations from separate experiments, and error bars indicate standard deviations.

11

General Conclusions ReactIR is an effective tool for in situ real-time monitoring of biotransformation reaction progress and kinetics, providing both qualitative and quantitative information. ReactIR enables understanding of reaction mechanism to optimize the process and significantly increase yield. The in situ mid-IR system is non-invasive to the process and non-destructive to the components of the reaction. The use of the simplest calibration mode can be applied without interference from the complex culture medium in whole cell catalysis processes. ReactIR is proven to monitor the principal components of enzymatic catalysis in aqueous, buffered, ionic and organic systems. Study Real-World Chemistry Under Actual Reaction Conditions Traditional offline methods to analyze reaction chemistry, such as HPLC, NMR, GC, etc. share a common problem when a sample is removed for analysis it may be altered or compromised resulting in significant analytical errors. ReactIR was developed for this very reason. It is ideal for studying chemistry as it actually exists in the reactor, eliminating time delays and errors resulting from grab sampling analysis. Why In Situ FTIR Analysis over Offline Methods? - A critical intermediate may be present that is lost in offline sampling - Air, accidentally introduced when a sample was removed for analysis, can change the chemistry - Reaction toxicity is significant enough to prohibit exposure - The reaction is run under pressure and/or extreme temperatures removing a sample may be unsafe, and alter the chemistry, invalidating the analysis ReactIR In Situ Probes - Temperatures from -120C to 400C - Pressures from vacuum to 350 bar - Superacids to highly basic chemistry - Slurries and reactions with larger particles/bubbles present - Highly oxidizing conditions - Submillimolar concentration reagents - Precise reaction temperature monitoring coupled with infrared monitoring - Continuous flow chemical processes - Integrated temperature sensor - reaction temperature trending with each spectrum
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References
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