J. L. L. Rakels

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 Chens 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, KEQ, were obtained for a (DL)-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.

Kinetics of the enzymatic resolution of racemic compounds in BI-BI reactions

The course of the kinetic resolution of a racemic compound by an enantioselective enzyme can often be described using Michaelis-Menten kinetics. This description (Chen et at., 1982, 1987) is formally not correct for reactions with multiple substrates or products. Van Tol et at. (1992) showed for the lipase-catalyzed resolution of glycidyl butanoate that the ping-pong kinetic mechanism has to be taken into account. This paper systematically treats the deviations from the model of Chen that may occur for bi-bi reactions obeying ping-pong or ternary complex kinetics. The course of the enantiomeric excess as a function of the degree of conversion was found to be dependent on two or three kinetic parameters (in contrast to the single E-value of Chen), on the thermodynamic equilibrium constant and on the ratio of initial concentrations of the reactants. This ratio can be used to some extent to manipulate the enantiomeric excess in a resolution process.

Enzyme Kinetics in Monophasic and Biphasic Aqueous-Organic Systems

Abstract If the rate of an enzymatic reaction is known for a dilute aqueous medium, it should be possible to calculate its rate or the rate of the reverse reaction in any other medium (monophasic non-ideal aqueous solution, monophasic organic solution, biphasic aqueous-organic solution). To this purpose a framework is presented which links thermodynamics and kinetics of enzymatic reactions in a well-defined manner. Its possible application is indicated for kinetic resolution of a chiral acid by reverse hydrolysis in which product inhibition by water occurs.

Comparison of enzymatic kinetic resolution in a batch reactor and a CSTR

Abstract The difference between a continuous stirred-tank reactor (CSTR) and a batchwise reactor has been shown for the enzymatic kinetic resolution of enantiomers. By macroscopic reactor balancing new quantitative relationships between enzyme enantioselectivity, substrate or product enantiomeric excess, and the extent of conversion have been deduced for chiral resolution in a CSTR. They were experimentally verified for the esterase-catalyzed resolution of racemic methyl 2-chloropropionate. This reaction was performed both in a batch system and in an enzyme membrane reactor functioning as a CSTR. As expected, the batchwise reaction was superior.

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 en...

Improvement of lipase-catalyzed kinetic resolution by tandem transesterification

Glycidyl acetate may be prepared enantioselectively from glycidyl butyrate by sequential enzymatic resolution. First, porcine pancreas lipase selectively forms (R)-glycidol by transesterification in dehydrated butanone. Then the lipase transesterifies (R)-glycidol selectively to glycidyl acetate. A model incorporating ping-pong bi-bi kinetics for this tandem reaction was worked out. For both reaction steps the enantiomeric ratios were 7. Other model parameters were also determined. The experimental results were adequately described by the model. The maximum enantiomeric excess of glycidyl acetate was raised from 65 to 89% by carrying out a tandem reaction instead of a single-resolution reaction. However, the model predicts that the amount of enantiopure product that may be obtained in a tandem reaction is limited as a result of equilibrium restrictions.

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.

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.

Process Strategies in Enzymatic Racemate Resolution

There is an increasing need to produce chiral building blocks, for the synthesis of pharmaceuticals, agro-chemicals and food-chemicals, rather than as racemates. Biocatalytic methods are suitable tools to accomplish the resolution of enantiomers. New strategies for enhancement of the enantiomeric purity of the product by enzymatic kinetic resolution were designed, modeled, and experimentally verified. Reaction kinetics, continuous process performance, and simultaneous multiple reactions and enzymes were considered