A systematic theoretical derivation of bioresolution-inversion process was made. An equation was derived between the maximum ee value of final product (eef(max)) and enantiomeric ratio (E) of a reaction. The corresponding equations of conv.(max), eep(max), ees(max) versus E-value were also derivative and the interrelationships among eef(max), conv.(max), eep(max) and ees(max) were deduced. Furthermore, a simple equation was de-veloped to predict the enantiomeric excess of substrate (ees) at any other time of the whole reaction course based on the ees value which was determined at a certain reaction time. This equation of ees versus time was verified by three different experiments. Based on the equation of ees versus time, a new equation for predicting the time (t(max)) needed to reach the maximum enantiomeric excess of the final product (eef (max)) after the resolution-inversion was developed.
Of all the types of enzyme-catalyzed reactions, hydrolytic transformations involving amide and ester bonds are the easiest to perform using proteases, esterases, or lipases. The key features that have made hydrolases the favorite class of enzymes for organic chemists during the past two decades are their lack of sensitive cofactors (which otherwise would need to be recycled) and the large number of readily available enzymes possessing relaxed substrate specificities to choose from. About half of the total research in the field of biotransformations has been performed using hydrolytic enzymes of this type [1, 2]. The reversal of the reaction, giving rise to ester or amide synthesis, has been particularly well investigated using enzymes in organic solvent systems. The special methodologies involved in this latter type of reaction are described in Sect. 3.1.
Of all the types of enzyme-catalyzed reactions, hydrolytic transformations involving amide and ester bonds are the easiest
to perform using proteases, esterases, or lipases. The key features that have made hydrolases the favorite class of enzymes
for organic chemists during the past two decades are their lack of sensitive cofactors (which otherwise would need to be recycled)
and the large number of readily available enzymes possessing relaxed substrate specificities to choose from. About two-thirds
of the total research in the field of biotransformations has been performed using hydrolytic enzymes of this type [1, 2].
We describe herein a modified quick E to access the enantioselectivity (E) of epoxide hydrolases using chiral fluorogenic probes. Accessing the true E values of the same biocatalytic reaction with chiral HPLC validated this methodology.
A recently published curve fitting method of determining E values was put to the test using a computer and statistical software. Using idealized data and curves, the methodology worked reasonably well. Confidence limits generated by the ideal data were still large. However, when real data were employed, this methodology was not satisfactory in generating reliable E values due to inherent errors in the progress curves generated. This approach does not appear to be suitable for determining E values from one dimensional progress curves even when powerful statistical software and a computer are used to evaluate the data.
Kinetic resolution is an important method in organic chemistry; catalytic kinetic resolution is especially attractive because a smaller amount of the optically active material is required. In principle, a naturally occurring catalyst (enzyme) or a synthetic catalyst can be employed. Possible disadvantages of an enzymatic catalyst include limited scope of reactions and substrates; therefore, there is an increasing effort targeted at the design of chemical catalysts for kinetic resolution. The most useful parameter in comparing different catalysts is the selectivity factor S, which is the ratio of the rate constants for the reaction of the catalyst with the two enantiomers of the substrate (S = (k_1/k_2), see below). Mathematical treatment of kinetic resolution should provide a way to calculate S from experimental observables, and for enantiomerically pure catalysts the equations are well-known from the work of Kagan and others.
A systematic study of the enzymatic activity of immobilized lipase from Rhizomucor miehei (Lipozyme IM) in the enantioselective esterification of 2-arylpropionic acids has been carried out. The main variables controlling the process (enzyme amount, water amount, temperature, stirring speed, and type of organic solvent) were studied using factorial analysis. The negative effect of water amount is explained by means of water activity (aw) considerations. A new and easy to calculation parameter (Enantiomeric Factor, EF) is defined for evaluating the enantioselectivity of the reaction. Influence of the alcohol and acid moieties is also considered. Lipozyme IM shows S-(+) enantiorecognition in all cases, except for (R,S)-Ketoprofen, where the R-(−) stereobias is confirmed using pure enantiomers (VR/VS = 8). An explanation for this different enantiopreference is suggested by means of MD calculus.
Biocatalytic resolution of 3-(2′-nitrophenoxy)propylene oxide (1a), 3-(3′-nitrophenoxy)propylene oxide (1b) and 3-(4′-nitrophenoxy)propylene oxide (1c) were exploited by using lyophilized cells of yeast Trichosporon loubierii ECU1040 with epoxide hydrolase (EH) activity, which preferentially hydrolyzes (S)-enantiomers of the epoxides (1a–c), yielding (S)-diols and (R)-epoxides. The activity increased as the nitro group in the phenyl ring was shifted from 4′-position (1c) to 2′-position (1a). When the substrate concentration of 1a was increased from 10 to 80 mM, the E-value increased at first, until reaching a peak at 40 mM, and then decreased at higher concentrations (>40 mM). The optically active epoxide (R)-1a was prepared at gram-scale (97% ee, 41% yield). Furthermore, a simple method was developed to predict the enantiomeric excess of substrate (ees) at any time of the whole reaction course based on the ees value determined at a certain reaction time at a relatively lower substrate concentration. This will be helpful for terminating the reaction at a proper time to get both higher optical purity and higher yield of the remaining epoxides.
Ethyl 1,4-benzodioxan-2-carboxylate is used as an intermediate compound for the production of drug doxazosin mesylate. The title compound was kinetically resolved to get S-enantiomer of ethyl 1,4-benzodioxan 2-carboxylate in a simple lipase catalyzed transesterification reaction. Ethyl acetate was used as reaction medium as well as acyl donor. The influence of the enzyme source and time of reaction on the enantio selectivity of product were studied. Lipase from Candida antartica-B (Novozyme A/S) catalyzed transesterification reaction with good enantio selectivity towards S-enantiomer. The high enantiomeric ratio, E = 160, provided S-2 an acceptable chemical yield (50%) and enantiomeric excess (>95%).
A complete and exact kinetic analysis of the phenomenon of dynamic kinetic resolution is presented. This analysis is applicable to reactions of stable stereoisomeric substrates whose ratios can be controlled and is valid for any set of kinetic conditions within the constraint of first-order and pseudo-first-order processes. Two new linear relationships are found for the dependence of the initial product ratio on the initial substrate ratio and for the dependence of the final product excess on the initial substrate excess. These relationships yield the minimum number of rate constant ratios needed to characterize the energetics of a chemical system exhibiting dynamic kinetic resolution completely. A distinct experimental advantage of this method is that it is based entirely on product studies. A simple graphical representation of the second linear relationship depicts visually the limiting Curtin-Hammett and anti-Curtin-Hammett conditions. From these conditions, a new parameter is defined that characterizes the efficiency of dynamic kinetic resolution and Curtin-Hammett efficiency. Simulations based on enantiomeric substrates illustrate how reactions may be optimized using this graphical treatment. An extension of this analysis to related kinetic schemes of varying degrees of complexity shows that the above linear relationships are universal. Results from these treatments are compared with Noyori's quantitative work on the stereoselective hydrogenation of -ketoesters. Implications of this new analysis are also discussed in light of previous work done on the applicability of the Winstein-Holness and Curtin-Hammett approximations to reactions of substrates that are interconverting conformers. For these cases, an alternate definition of Curtin-Hammett efficiency is proposed that is based on the experimental determination of the initial and final product ratios and the equilibrium constant for substrate interconversion. This unified analysis can be readily applied to a wide variety of synthetic and mechanistic problems in organic chemistry where dynamic kinetic resolution is applicable.
The enantiomeric ratio ) () offers a concise representation of the enantioselective properties of an enzyme in reactions that involve chiral compounds. Both as a measure of the intrinsic selectivity of the catalyst, and as a parameter to model the performance of enzymatic processes for the production of enantiopure fine-chemicals, its merits have been well-recognized.Several methods for the determination of E exist. The scope and limitations of these methods are evaluated in terms of accuracy and feasibility. There appears to be no single method that is both reliable and readily applicable in all cases. Complementary methods, however, are available.The outstanding characteristics of the enantiomeric ratio as a quantitative measure of the effects of physical and chemical conditions on the intrinsic enantioselectivity of enzymes are presented in terms of the difference in Gibbs energies of the diastereomeric enzyme-substrate transition states. The prospects of molecular modeling strategies for the prediction of E are discussed.
Development of effective chemical catalysts is a key concern in organic chemistry. Therefore, convenient screening systems for chemical catalysts are required, and although some fluorescence-based HTS systems have been developed, little attempt has been made to apply them to asymmetric catalysts. Therefore, we tried to develop a chiral fluorescence probe which can evaluate the reactivity and enantioselectivity of asymmetric catalysts. We focused on kinetic resolution catalysts as a target of our novel fluorescence probe, employing β-elimination following acylation of nitroaldol. Once the hydroxyl group of nitroaldol is acylated, β-elimination occurs immediately, affording nitro olefin. Therefore, we designed and synthesized a fluorescence probe with an asymmetric nitroaldol moiety. Its fluorescence intensity decreases dramatically upon β-elimination, so the fluorescence decrease is an indicator of the reaction yield. Thus, the enantioselectivity of kinetic resolution catalysts can be assessed simply by measuring the fluorescence intensities of the reaction mixtures of the two enantiomers; it is not necessary to purify the product. This fluorescence probe revealed that benzotetramisole is a superior catalyst for kinetic resolution of nitroaldol. Furthermore, we established an HTS system for asymmetric catalysts, using a fluorescence probe and benzotetramisole. To our knowledge, this is the first fluorescence-based HTS system for asymmetric catalysts.
Many drugs are chiral—that is, they exist in right- and left-handed molecular forms called enantiomers. Although structurally almost identical, enantiomers can differ considerably in their pharmacological effects. Today, growing awareness of some of the problems and complexities arising from the use of chiral drugs is raising concerns about their development and marketing by pharmaceutical firms and their application in clinical medicine. "This is an important issue faced by the pharmaceutical industry now" says Bernard Testa, director of the School of Pharmacy of Université de Lausanne, Switzerland. "Many drugs exist as enantiomers, but in most cases it's not a single enantiomer that's being marketed, but a mixture of two enantiomers, the racemate." In the past few years, evidence has grown that the use of racemic drugs can cause problems when each enantiomer acts differently in the body. For example, one enantiomer may be a pharmacologically active agent, whereas the other may be inactive or even act ...
Chiral norbornane-type alcohols of high optical purity were prepared via enzymatic resolution of their racemic esters using lipases from Candida cylindracea and Pseudomonas sp. This method presents a short and efficient access to a number of chiral building blocks on a molar scale for the synthesis of optically active cyclopentane systems.
Equations and useful graphs for the quantitative treatment of biochemical kinetic resolution data have been developed. These expressions have been verified experimentally, and they possess predictive values in relating the parameters of the extent of conversion of racemic substrate (c),the optical purity expressed as enantiomeric excess (ee), and the enantiomeric ratio (E).