Enzymatic Resolution of Racemates Contaminated by Racemic Product

Enzyme-catalyzed kinetic resolution is sometimes performed starting with substrate already containing small amounts of the racemic product. Then the determination of the enantiomeric ratio may be seriously disturbed when this parameter is calculated from the degree of conversion and the enantiomeric excess of either the substrate or the product (Chen et al., 1982, 1987) or when it is calculated directly from the enantiomeric excess of substrate and product (Rakels et al., 1993).

This paper presents modifications of these methods in order to correctly determine the enantiomeric ratio as well as the amount of racemic product in the substrate. The theoretical predictions were verified for the hydrolysis of racemic ethyl 2-chloropropionate, catalyzed by carboxylesterase NP. Despite the presence of racemic product in the substrate, accurate and reliable values for the enantiomeric ratio were obtained by using the modified methods.     

Notes on Technic: Simple, Reliable Detection of Latex Microspheres in High Quality Tissue Sections

Latex microspheres used in biological research have been visualized by light microscopy in mounts of cell suspensions, disrupted cells, or cleared tissues (Mishima et al 1987, Koonce et al 1986, LeFevre et al 1978); in unembedded coverslip monolayers (Koerten et al 1980); in fixed (Cornwall and Phillipson 1988) or unfixed (Wells et al 1988) frozen sections; in paraffin sections cleared and deparaffinized with n-butyl alcohol (Callebaut and Meeussen 1989); and in tissues embedded in resins suitable for transmission electron microscopy, such as Spurr's (Hampton et al 1987), Epon (Herzog and Miller 1979), or Ladd Low Viscosity Epon (LeFevre et al 1985). Paraffin embedding, and some plastic embedments, are impractical for demonstration of latex beads because the beads are dissolved by such organic solvents as xylene, dioxane, or chloroform (Van Furth and Diesselhoff-Den Dulk 1980), propylene oxide (Lentzen et al 1984), amyl acetate (Okada et al 1981), or toluene, the solvent in commonly used mounting media such as Fisher Permount (personal observation). The space remaining after dissolution of a bead is not maintained with paraffin embedding as it is with resin embedding. Even after plastic embedding, the resolving power of light microscopes may be inadequate to distinguish such spaces from other spaces found in and between cells. Latex beads are stable in methanol (Van Furth and Diesselhoff-Den Dulk 1980), ethanol. and n-butyl alcohol (Callebaut and Meeussen 1989).     

Reactions of Nitric Oxide with Nitronyl Nitroxides and Oxygen: Prediction of Nitrite and Nitrate Formation by Kinetic Simulation

Nitric oxide reacts with nitronyl nitroxides (NNO) to form imino nitroxides (INO) and this transformation can be monitored using electron spin resonance spectroscopy. Recently, Akaike et al., reported that NNO such as 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) and its derivatives (e.g., carboxy-PTIO) react with nitric oxide (·NO) in a 1:1 stoichiometry forming 2-phenyl-4,4,5,5-tetra-methylimidazoline-1-oxyl (PTI) or the respective product (e.g., carboxy-PTI) together with nitrite and nitrate (Akaike et al., Biochemistry 32, 827-832, 1993). In this paper, we reevaluate their results and show that the stoichiometry of the reaction between PTIO and ·NO is 0.63 ± 0.06:1.0. The reason for this discrepancy is due to an erroneous assumption by Akaike et al., that the stoichiometry for the reaction between ·NO and O₂ is 2:1 in aqueous solution. If the data reported by Akaike et al., were recalculated using a 4:1 stoichiometry established for the aqueous oxidation of ·NO, the reaction between ·NO and PTIO would give a stoichiometry of 0.5:1.0 in closer agreement with our data. We propose a mechanism for the reaction between PTIO and ·NO in aqueous solution. This mechanism predicts that the stoichiometry between carboxy-PTIO and ·NO is dependent on the rate of generation of ·NO and is 1:1 only at low rates of ·NO generation (i.e., 10^-13 M/s). However the stoichiometry approaches 0.5:1.0 at higher rates of ·NO production or when it is added as a bolus. The ratio between nitrite and nitrate also varies as a function of the rate of generation of ·NO. The model agrees with previous experimental observations that the aqueous oxidation of ·NO in air saturated solutions will exclusively form nitrite and predicts that ·NO will only generate substantial amounts of nitrate if it is released at a rate less than 10^-17 M/s. This may have important consequences in cellular systems where the concentration of ·NO is typically measured from nitrite production.     

A simple method to determine the enantiomeric ratio in enantioselective biocatalysis

The enantiomeric ratio (E) is commonly used to characterize the enantioselectivity in enzyme-catalyzed kinetic resolution. In this paper this parameter is directly derived from the enantiomeric excess of substrate and product. This is formally more correct than using Chen's equation after calculating the degree of conversion from both ee values using the relation of Sih and Wu. New expressions and useful graphs have been generated for reversible and irreversible uni-uni reactions. The theoretical predictions have been verified experimentally for various reactions. Values for E and the thermodynamic equilibrium constant,K_EQ, were obtained for a ( -dehalogenase-catalyzed dehalogenation, a hydrolysis reaction by porcine pancreatic lipase, and for C. Cylindracea lipase-catalyzed esterification and transesterification. In view of the current developments in the field of chiral analysis, this method is an easily available tool in the quantitative treatment of enzyme-catalyzed resolution of enantiomers.     

Overexpression of Serratia marcescens lipase in Escherichia coli for efficient bioresolution of racemic ketoprofen

Lipase from Serratia marcescens ECU1010 was cloned and overexpressed in E. coli. After optimization, the maximum lipase activities reached 5000–6000 U/l and this recombinant lipase could enantioselectively hydrolyze (S)-ketoprofen esters into (S)-ketoprofen. Among six alkyl esters of racemic ketoprofen investigated, this lipase showed the best enantioselectivity for the kinetic resolution of ketoprofen ethyl ester, with an ee_p (enantiomeric excess of product) of 91.6% and E-value of 63 obtained at 48.2% conversion. Twelve nonionic surfactants were tested for enhancing the enantioselectivity of this lipase in the bioresolution of ketoprofen ethyl ester. A very high E-value of 1084 was achieved, with an optical purity of >99% ee_p and a yield of 42.6% in the presence of 3% Brij 92V. Further studies showed that the selectivity of the lipase was improved with the increase of Brij 92V concentration. The substrate (ketoprofen ethyl ester) does not inhibit the lipase activity, while the product (S)-ketoprofen inhibits the lipase activity to some extent. These results indicate that the S. marcescens lipase is very useful for biocatalytic production of chiral profens such as (S)-ketoprofen.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine catalyzed by Lipase B from Candida antarctica

A biotransformation process has been developed for the production of (S)-N-(2-ethyl-6-methylphenyl) alanine by enantioselective hydrolysis of racemic methyl ester in the presence of Candida antarctica lipase B (CAL-B). However, the enantioselectivity of CAL-B towards the resolution is not high enough to obtain enantiomerically pure product. In order to improve the enantioselectivity of the enzyme, the effects of surfactants on CAL-B-catalyzed hydrolysis were tested. The results suggest that surfactants can influence the microenvironment of the enzyme, and the addition of Tween-80, in particular, to the reaction medium markedly enhanced the selectivity of CAL-B towards the substrate used, with the enantiomeric ratio (E-value) increasing from 11.3 to 60.1.     

Mass balancing in kinetic resolution: calculating yield and enantiomeric excess using chiral balance

When kinetic resolution is applied for the production of enantiomerically pure compounds, process options may be used which involve more than one chiral substrate and one chiral product, such as sequential or parallel enzymatic kinetic resolutions or hydrolysis of diastereomers. Although the relation between the yields (y) of the chiral compounds is straightforward in these cases, the relation between their enantiomeric excess (ee) values is not. Combining mass balances into a so-called chiral balance (Sigma y . ee(R) = 0) provides the relation between enantiomeric excess values in a useful manner. This chiral balance easily shows which nonmeasured enantiomeric excess values and yields can be calculated from measured values. The chiral balance is only valid when configurations at chiral centers are conserved. (c) 1995 John Wiley & Sons, Inc.     

Mathematical Modelling of Lipase Catalysed Resolution of O-Acetyl and O-Formyl Derivatives of Secondary Alcohols

Lipase catalysed transesterification of O-acetyl and O-formyl derivatives of several sterically hindered secondary alcohols with n-butanol is studied. The reaction is mathematically modelled and the rate constants in the model are evaluated by transient parameter estimation procedure. Several models are considered for predicting the enantiomeric excess (ee) of the product and compared with experimental observations. It is found that the maximum ee depends only on the rate constants of the two competing reactions and not on the order of reaction. A second order model is found to predict the observed behaviour well. When the data is fitted to an equilibrium type of model developed by Chen et al. (1987), it is found that the rate of forward reaction is several orders of magnitude larger than the rate of reverse reaction. The effect of acetyl and formyl groups on the reactivity and selectivity are also discussed.     

Strategies in the Preparation of Homochiral Compounds Using Combined Enantioselective Enzymes

In order to obtain a homochiral product from a racemic substrate, different strategies can be followed using a moderately enantioselective enzymatic catalyst. Two new strategies are presented, involving the simultaneous use of two enzymes, parallel or consecutive. In the parallel system, the substrate enantiomer yielding the unwanted product enantiomer is enantioselectively converted by the second enzyme. In the consecutive system, the substrate enantiomer yielding the desired product enantiomer is itself the preferred product of another enantioselective enzymatic reaction.

For irreversible pseudo-first order enzyme kinetics, a relationship was found which describes the dependency of the yield and enantiomeric excess for these systems on the E-values of the separate enzymes and on the ratio of their concentrations. For Michaelis-Menten kinetics, these relationships usually give good approximations.

According to these calculations, the yield and enantiomeric excess obtainable with the concepts of combined enzymes exceed significantly those obtainable with the separate enzymes, and also those obtainable with the strategy of product recirculation.     

酮基布洛芬拆分用酯酶产生菌的筛选及其催化特性

从土壤中筛选获得一株可以高对映选择性水解酮基布洛芬乙酯的酵母KET4,经鉴定为芸苔丝孢酵母（Trichosporon brassicae）。研究了该菌的生长和产酶过程,考察了其静息细胞对酮基布洛芬乙酯水解的催化特性。用该菌催化酯水解时,转化率为41%时,产物的对映体过量值为91%,对映选择率达到45。     

Sequential Kinetic Resolution by two Enantioselective Enzymes

Enhancement of the enantioselectivity by simultaneous use of two enzymes in a sequential kinetic resolution process is presented. The model system consisted of carboxylesterase NP catalyzed hydrolysis of racemic methyl 2-chloropropionate, followed by dehalogenation of the enantiomerically enriched 2-chloropropionate by DL-dehalogenase into lactate. Optimal results are shown to be attained when the conversion rates of both faster reacting enantiomers are the same. An optimization parameter D for sequential resolutions is introduced. The kinetics of both reaction steps were investigated separately by progress curve analysis, and the enantioselectivity of the enzymes was determined. From a quantitative kinetic model we could formulate the sequential resolution, which yielded the predicted improvements of product enantiomeric excess.     

Enantioselective lipase-catalyzed kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine

The enzyme (BSL2), a highly active lipase expressed from newly constructed strain of Bacillus subtilis BSL2, is used in the kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine from the corresponding racemic methyl ester. Reaction conditions are optimized to enhance the enantioselectivity. The effects of various racemic alkyl esters, substrate concentration, operating temperature, pH of the aqueous medium and organic solvents on activity and enantioselectivity of BSL2 for kinetic resolution are also studied. A high enantiomeric ratio (E = 60.7) is reached in diisopropyl ether/water (10%, v/v) and the enantioselectivity is about 22-fold higher than that in pure buffered aqueous solution. The results show that the reaction medium greatly influences BSL2 reaction and its enantioselectivity in the hydrolysis of racemic methyl ester.     

Microbial deracemisation of aromatic β-hydroxy acid esters

Aromatic β-hydroxy acid esters were found to undergo deracemisation using whole cells of Candida parapsilosis. The conditions for the deracemisation reaction were optimised where 75% isolated yield and >95% enantiomeric excess of the product was achieved. The effect of electron donating as well as electron withdrawing groups present in the standard substrate, ethyl 3-hydroxy 3-phenyl propionate was studied to establish the generality of the reaction. The enantiomeric excess of the product remains high (>95%) irrespective of the different substituents in the para position but substitution at the ortho position obstructs the process. Similarly, ethyl and methyl esters of the standard substrate undergo deracemisation reaction giving high ee of the product, but the benzyl ester of the standard substrate did not undergo deracemisation.     

Improved Kinetic Resolution by Enzyme-Catalyzed Parallel Reactions

Competitive parallel reactions with opposite enantioselectivity are presented as a strategy to enhance the enantiomeric product purity in enzymatic kinetic resolution. Lipase-catalyzed simultaneous hydrolysis and amidation of racemic methy 12-chloropropionate led to significantly improved amide yield and enantiomeric excess. Process results can be controlled by changing the hydrolysis/amidation reaction rates through variation of the solvent and the initial amine concentration. This is described by a kinetic model.     

Enantioconvergent production of (R)-1-phenyl-1,2-ethanediol from styrene oxide by combining the Solanum tuberosum and an evolved Agrobacterium radiobacter AD1 epoxide hydrolases

Soluble epoxide hydrolase (EH) from the potato Solanum tuberosum and an evolved EH of the bacterium Agrobacterium radiobacter AD1, EchA-I219F, were purified for the enantioconvergent hydrolysis of racemic styrene oxide into the single product (R)-1-phenyl-1,2-ethanediol, which is an important intermediate for pharmaceuticals. EchA-I219F has enhanced enantioselectivity (enantiomeric ratio of 91 based on products) for converting (R)-styrene oxide to (R)-1-phenyl-1,2-ethanediol (2.0 +/- 0.2 micromol/min/mg), and the potato EH converts (S)-styrene oxide primarily to the same enantiomer, (R)-1-phenyl-1,2-ethanediol (22 +/- 1 micromol/min/mg), with an enantiomeric ratio of 40 +/- 17 (based on substrates). By mixing these two purified enzymes, inexpensive racemic styrene oxide (5 mM) was converted at 100% yield to 98% enantiomeric excess (R)-1-phenyl-1,2-ethanediol at 4.7 +/- 0.7 micromol/min/mg. Hence, at least 99% of substrate is converted into a single stereospecific product at a rapid rate.     

A Modified Giemsa C-Banding Technique For Hordeum Species

A Giemsa C-banding technique with a hot 1 N HCI hydrolysis step has been developed for barley chromosomes. This step makes it easy to obtain well separated C-banded chromosomes. To compare this technique with other C-banding techniques, chromosomes of H. vulgare cv. York were stained by both this technique and a modification of the technique of Kimber et al (1976). With respect to centromeric and intercalary bands, both techniques produce a similar banding pattern, but telomeric bands observed by the modified technique of Kimber et al (1976) were not detected by our procedure. This indicates that telomeric heterochromatin may be different chemically and/or structurally from the centromeric and intercalary heterochromatin and its appearance dependent upon the C-banding technique. The procedure described provides a relatively rapid technique for C-banding of barley chromosomes.     

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile via asymmetric transcyanation of acetyltrimethylsilane with acetone cyanohydrin in an aqueous/organic biphasic system catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal was successfully carried out for the first time. The optimal volume ratio of aqueous to organic phase, buffer pH value and reaction temperature were 15% (v/v), 5.0 and 30°C, respectively, under which both substrate conversion and product enantiomeric excess (ee) were 99%. Silicon atom in the substrate showed great effect on the reaction. Acetyltrimethylsilane was a much better substrate for (R)-hydroxynitrile lyase from Prunus japonica than its carbon analogue.     

Strategies in the Preparation of Homochiral Compounds Using Combined Enantioselective Enzymes

In order to obtain a homochiral product from a racemic substrate, different strategies can be followed using a moderately enantioselective enzymatic catalyst. Two new strategies are presented, involving the simultaneous use of two enzymes, parallel or consecutive. In the parallel system, the substrate enantiomer yielding the unwanted product enantiomer is enantioselectively converted by the second enzyme. In the consecutive system, the substrate enantiomer yielding the desired product enantiomer is itself the preferred product of another enantioselective enzymatic reaction.

For irreversible pseudo-first order enzyme kinetics, a relationship was found which describes the dependency of the yield and enantiomeric excess for these systems on the E-values of the separate enzymes and on the ratio of their concentrations. For Michaelis-Menten kinetics, these relationships usually give good approximations.

According to these calculations, the yield and enantiomeric excess obtainable with the concepts of combined enzymes exceed significantly those obtainable with the separate enzymes, and also those obtainable with the strategy of product recirculation.     

Preparation of both enantiomers of methyl 3-benzoyloxypentanoate by enzyme-catalysed hydrolysis of corresponding racemic nitrile and amide

Rhodococcus rhodochrous IFO 15564 enantioselectively hydrolysed racemic 3-benzoyloxypentanenitrile and 3-benzoyloxypentanamide to afford (R)-amide and (S)-car☐ylic acid with high enantiomeric excess (> 90%). In this reaction, both enantiomers of the starting nitrile were converted to the amide by nitrile hydratase, and amidase-catalysed enantioselective hydrolysis of the amide was responsible for the kinetic resolution. The lack of enantioselectivity of the nittile hydratase toward the racemic nitrile forms a marked contrast to the case of previously reported highly enantioselective conversion of prochiral 3-benzoyloxypentanedinitrile by this enzyme. since (R)-amide could be hydrolysed chemically to (R)-car☐ylic acid without any loss of its ee, the present microbial kinetic resolution serves as an effective method for preparing both enantiomers of synthetically useful 3-hydroxypentanoic acid derivatives.