Introduction

One of the most challenging aspects in the synthesis of chiral molecules is the achievement of products with high enantiopurity. This can be sought through asymmetric synthesis or resolution of a racemic form into its components (1). The resolution of racemic mixtures via enzyme catalysis is a highly studied and practiced methodology, and alcohols, acids and amines are routinely resolved with hydrolases in aqueous, aqueous/cosolvent, or organic media. The hydrolysis of esters in aqueous media is a procedure that has been utilized for a long time and changing the pH, temperature, or ionic strength of the medium can modify enzyme enantioselectivity. Another way to modify enzyme enantioselectivity is to add substantial proportions of organic cosolvents such as dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), i-BuOH, acetone, and acetonitrile (2) to the aqueous buffer.

Since the early eighties, it has been demonstrated that several enzymes are active in pure organic media and that changing the nature of solvents can modify the enantioselectivity of the enzymes (3-5). The opportunity to modify enzyme enantioselectivity by changing the nature of the medium has been mainly exploited for hydrolytic enzymes such as lipases and proteases and, in the literature, there is a wealth of examples referring to these enzymes (for review articles, see refs. 4 and 5). Sometimes, the influence of the solvent nature is so remarkable that not only can it induce dramatic increases of enantioselectivity (6) but even reverse enzyme enantiopreference (7).

Many attempts have been made trying to correlate enzyme enantioselectivity and solvent physico-chemical properties (polarity, hydrophobicity, dielectric

From: Methods in Biotechnology, Vol. 15: Enzymes in Nonaqueous Solvents: Methods and Protocols Edited by: E. N. Vulfson, P. J. Halling, and H. L. Holland © Humana Press Inc., Totowa, NJ

constant, and so forth) (4), but it seems that in most cases, these macroscopic characteristics cannot address the scope (5,8). More recently, computational approaches have been proposed to rationalize the phenomenon, but, in addition to being demanding and time-consuming, they do not provide results of general applicability (9,10).

Therefore, in our opinion the "trial- and-error" approach is still that of choice because, in principle, any solvent could be successfully used. Experience suggests the avoidance of solvents such as DMSO, DMF, ethanol, and methanol because they tend to denature enzymes. One remarkable exception is subtilisin, which is active in DMF. Other water-miscible solvents such as dioxane, acetonitrile, pyridine, tetrahydrofuran (THF), triethylamine, and acetone are widely used, but, with them, the control of water activity can be difficult. Instead, with water-immiscible solvents, it is relatively simple to set the water activity at the desired value, and the solvents most commonly used are benzene, CCl4, methyl i-butyl ether, CH2Cl2, CHCl3, nitrobenzene, 3-pentanone, i-amyl alcohol, hexane, and dodecane. The reaction medium can also be made of vinyl acetate or other vinyl esters that act both as solvent and acylating agent.

In addition to solvent properties, the water content in the reaction medium is another important factor to be considered. The parameter that better describes the effects of water on such enzyme properties as KM, Vmax, stability, and conformation is the thermodynamic water activity (11,12). The effects of water activity on enzyme enantioselectivity are, however, controversial because an increase, decrease, or no variation in selectivity as a function of the water present in the reaction medium have been reported (13-15). Therefore, when facing a resolution process, it is advisable to carry it out at different water activity values (see Note 1).

The resolution of a secondary alcohol via a transesterification reaction is a kinetic resolution where both enantiomers are transformed, but at different rates (see Fig. 1). For sufficiently long reaction times, all of the substrate will be converted into the product.

This means that the enantiomeric excess of the substrate increases as the conversion increases, whereas the enantiomeric excess of the product decreases as the conversion increases. The fact that the enantiomeric excess is related to the degree of conversion makes this parameter unsuitable for measurement of enzyme enantioselectivity in different solvents. A parameter that better describes enzyme enantioselectivity is the enantiomeric ratio (E), which is the ratio between the specificity constants of the two competing enantiomers. The enantiomeric ratio is commonly calculated from the conversion (c) and the enantiomeric excess of either the substrate (eeS) or the product (eep) (16):

Fig. 1. Schematic representation of a generic secondary alcohol resolution through a transesterification reaction.

Fig. 1. Schematic representation of a generic secondary alcohol resolution through a transesterification reaction.

where 0 < c < 1, 0 < eeS <1, and 0 < eeP < 1.

To exemplify the influence of organic solvents on enzyme enantioselectivity, the resolution of (±) trans-sobrerol [(±) trans-5-(1-hydroxyl-1-methylethyl)-2-methyl-2-cyclohexen-1-ol] by transesterification with vinyl acetate catalyzed by lipase from Pseudomonas cepacia (Lipase PS) (see Fig. 2) will be described (6).

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