Enzymes enantioselectivity

Of course, the influence of organic solvents on enzyme enantioselectivity is not limited to proteases but it is a general phenomenon. Quite soon, different research groups described the results obtained with lipases [28]. For instance, the resolution of the mucolytic drug ( )-trans-sobrerol (11) was achieved by transesteriflcation with vinyl acetate catalyzed by the lipase from Pseudomonas cepacia adsorbed on celite in various solvents. As depicted in Scheme 1.3 and Table 1.5, it was found that t-amyl alcohol was the solvent of choice in this medium, the selectivity was so high ( >500) that the reaction stopped spontaneously at 50% conversion giving both +)4rans-sobrerol and (—)-trans-sobrerol monoacetate in 100% optical purity [29]. 

When discussing the role of reaction medium on enzyme enantioselectivity, the potential effects of (i) water activity [5b,13f,32], (ii) enzyme form, and (iii) pH, should... 

The second group of studies tries to explain the solvent effects on enantioselectivity by means of the contribution of substrate solvation to the energetics of the reaction [38], For instance, a theoretical model based on the thermodynamics of substrate solvation was developed [39]. However, this model, based on the determination of the desolvated portion of the substrate transition state by molecular modeling and on the calculation of the activity coefficient by UNIFAC, gave contradictory results. In fact, it was successful in predicting solvent effects on the enantio- and prochiral selectivity of y-chymotrypsin with racemic 3-hydroxy-2-phenylpropionate and 2-substituted 1,3-propanediols [39], whereas it failed in the case of subtilisin and racemic sec-phenetyl alcohol and traws-sobrerol [40]. That substrate solvation by the solvent can contribute to enzyme enantioselectivity was also claimed in the case of subtilisin-catalyzed resolution of secondary alcohols [41]. 

Several reports deal also with the effects of ionic liquids on enzyme enantioselectivity, which is the subject of this chapter. Although in several cases there was no change or even a decrease in enantioselectivity compared to organic solvents [47], in other cases improved enantioselectivity was observed [47,49-56]. In the following text, the latter cases will be examined in some detail. 

Basically, there are three ways to tune enzyme enantioselectivity by means of additives (i) the additives are placed in the reaction medium together with the organic solvent, the enzyme, and the reagents (ii) the additives are co-lyophilized with the biocatalyst before use in the organic solvent (iii) the additives are complexed with the substrates before their transformation in the organic medium. 

An intriguing influence of a cosolvent immiscible with water on the enantioselec-tivity of the enzyme-catalyzed hydrolysis was observed. It was proven that enzyme enantioselectivity is directly correlated with the cosolvent hydrophobicity. In the best example, for ethyl ether as cosolvent, the reaction proceeded with E = 55, and the target compound was obtained in 33% yield with 92.7% ee. This finding may be of great practical importance, particularly in industrial processes [24], since it will enable better optimization of enzyme-catalyzed processes. It is clear that, in future, immobilized enzymes, as heterogeneous catalysts, wiU be widely used in most industrial transformations, especially in the preparation of pharmaceuticals [25]. 

In summary, the failure of solvation-desolvation theory to explain the observed changes of enzyme enantioselectivity may well be caused by the fact that several barriers of physically different character contribute to the E-value. For additional considerations the reader is referred to [107] and [30]. 

Recently, a report appeared of what is probably a conclusive optimization of the system [102]. After isolating a (25 kDa) lipase-like enzyme from crude PPL, applying intricate immobilization methods, and carefully checking experimental conditions, the authors report E-values in the kinetic resolution of glycidyl butyrate that are still no higher than 10. However, illustrating the importance of organic solvents on enzyme enantioselectivity, when dioxane (10% v/v) is added as a cosolvent, E > 100 is found ... 

Despite the fact that solvent effects on enzyme enantioselectivity appear to resist our efforts to rationalize their outcome using commonly accepted solvent descriptors, the effects are certainly there. An impressive example is provided in a report on the successful resolution of ds/trans-( 1 R,5 R)-bicyclo[3.2.0]hept-6-ylidene-acetate ethyl esters, intermediates in the synthesis of GABA (y-aminobutyric acid) analogs, by the Pfizer Bio transformations and Global R D groups (Scheme 2.2) [136]. From a screening protocol, CaLB was identified as a reactive catalyst for the hydrolysis of the racemic mixture of / //-os lor enantiomers with approximately equal activity for the ds- and tmns-isomers and a rather modest (E = 2.7) preference for the /Z-(lR,5R)-enantiomers. Application of medium engineering resulted in a phenomenal increase in the enantioselectivity (addition of 40% acetone, E > 200), while the ds- and trans-isomers were still converted at an almost equal rate. 

In contrast to enzyme enantioselectivity, the rationalization of the regioselective outcome of reactions catalyzed by hydrolases is still a difficult task. Thus, whereas... 

Using solid/gas reactors to improve enzyme enantioselectivity by solvent engineering and changing reaction conditions... 

In some instances, a relationship between enzyme enantioselectivity and flexibility has been found while there are some positive examples in the literature... 

T. Ke and A. M. Kubanov, Markedly enhandng enzymic enantioselectivity in organic solvents by forming substrate salts,... 

To evolve useful enzymes, genetic engineering technology has been applied increasingly to improve stability of enzymes, enantioselectivity, extension of substrate specificity for kinetic resolution of racemic compounds. Novel enzymes created by this technique will be available in large quantities and varieties within a next few years. In the near future, a lot of useful enzymes will be on the market and expanding number of chemists can use enzymes more freely than present due to the improvement in the simplification of experimental procedures. 

The IL/SC-CO2 media for 1-phenylethanol resolution, catalyzed by CALB, offers a number of advantages, such as high resolution yields, simple downstream processing, guarantying an enzyme overstabilization effect and exceptional enzyme enantioselectivity with no transformation of the not desired enantiomer, albeit apparently lower conversions were registered in this medium compared to the ones, obtained in SC-CO2. 

In addition to the widely reported techniques of amide bond formation, transesterification, and hydrolysis, enzymic enantioselective oxidation is also used in the synthesis of single isomer drugs. Patel described the elficient oxidation of benzopyran (75), an intermediate in thesynthesisof potassium channel openers (123). The transformationwas ef-fected w i t h a cell suspension of MortiereUa raman-niana with glucose over a 48-h period, the isolated product (77) was obtained in a 76%yield with an optical purity of 97%and a chemical purity of 98%, as shown in Pig. 18.32. 

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