VOLUMEN DE TRÁNSITO

The ability of an enzyme to choose one or other enantiomer (resolution) is termed enantioselectivity and is defined by the enantiomeric ratio, E (Equation 1 Sih and Chen,

1984).

E = R/S = (kcai/KM)R/(kcat/KM)s

Resolution o f racemic alcohols, acids and esters via hydrolase-catalysed estérifications and transestérifications in microaqueous organic solvents is a well known procedure, especially for compounds unstable or poorly soluble in water (Langrand et aL, 1985; Chen and Sih, 1989; De Amici et aL, 1989; Santaniello et aL, 1992). However it is only recently that the effects of the reaction conditions (nature o f the organic solvent, enzyme form, temperature and water concentration) on enzyme enantioselectivity have started to be investigated.

Temperature

It is commonly perceived that enzymatic reactions exhibit optimal stereoselectivity at low temperatures. This is because at temperatures below their optimal activity the structures o f enzymes are known to be more rigid than at higher temperatures and steric hindrance results in large groups being excluded from the active site. However both an increase (Lam et aL, 1986; Willaert et aL, 1988; Keinan et aL, 1986) and a decrease (Boutelje et aL, 1988) o f the selectivity with a decrease in temperature have been observed. It has also been reported that the temperature had no effect on the enantioselectivity (Barton et aL, 1990). However after studying Barton’s results it was reported that there was a slight increase in enantioselectivity at lower temperatures (Holmberg and Huit, 1991). The theory of temperature effects on stereochemistry was reviewed by Phillips, (1992).

The increase in stereoselectivity with temperature is highlighted in the following examples. Secondary alcohol dehydrogenase (SADH) from the thermophilic bacterium,

Thermoanaerohacter ethanolicus exhibited activity with a wide range o f ketones,

normally forming (S)-alcohols with enantiomeric purity. With 2-butanone, the preferred product was (S)-2-butanol below 26°C, and (R)-2-butanol at temperatures above 26°C (Pham and Phillips, 1990). In contrast, in the reduction o f 2-pentanone, the formation of (S)-2-pentanol decreased in enantiomeric purity as the reaction temperature was increased (Pham and Phillips; 1990, Zheng and Phillips, 1992).

Thus decreasing the temperature is one way to modulate the enantiomeric excess from such enzyme-catalysed reactions. However conducting enantioselective resolutions at very low temperatures may be uneconomic for industrial purposes.

W ater content of the reaction

The effect of water concentration on enantioselectivity is somewhat contradictory since both increases (Holmberg and Huit, 1990; Kitaguchi et aL, 1990; van der Lugt et aL,

1992) and decreases (Wickli et aL, 1992) in enzyme enantioselectivity as a function of water content in the reaction medium have been reported. In the above studies water concentrations rather than water activities were quoted, whereas it has been

demonstrated that it is water activity (âw) rather than concentrations that determines

enzyme properties (Goderis et aL, 1987; Klibanov, 1989; Aldercreutz, 1991; Valivety et aL, 1992; Hailing, 1993). Bovara and co-workers (1993) investigated water activity in enantioselectivity and found it to have no influence in the estérification of racemic sulcatol with vinyl acetate with either Lipase PS from Pseudomonas cepacia or lipoprotein lipase from Pseudomonas sp.

Solvent effects

Fitzpatrick and Klibanov (1991) found that enzymatic transestérification reactions catalysed by subtilisin in organic media can have a dramatically altered stereoselectivity in solvents of different polarity. The addition of 25-50% DMSO improved the stereoselectivity o f esterase-catalysed hydrolysis o f methyl alkyl dimethylmalonates (Bjorkling et al.y 1986). In contrast, Jones and Mehes, (1979) reported that chymotrypsin exhibited diminished enantiospecificity for the hydrolysis of phenylalanine esters in the presence of organic solvents.

The solvent effects on the estérification of phenoxypropionic acids with n-butanol using

Candida cylindracea lipase have been studied (Ueji et aL, 1992). The enantioselectivity

was shown to be inverted in non-polar solvents such as carbon tetrachloride from that in polar solvents such as acetone. For example, in carbon tetrachloride there was a 48% e.e o f the R enantiomer o f the butyl ester (a useful herbicide) whereas in acetone there was a 39% e.e of the S enantiomer. When the mole fraction of acetone in carbon tetrachloride was investigated the ratios of the R enantiomers in their ester products decreased with an increase in the mole fraction of acetone. The inversion o f the enantioselectivity from “R” to “S” occurred around 0.5 mole fraction of acetone, and also the conversion decreased and the estérification became significantly slower. The change in enantioselectivity was attributed to a conformation of the lipase arising from specific interactions between the solvent and the lipase (Ueji et aL, 1992).

Bosetti et aL, (1993) also studied the effects of cosolvents on the enantioselectivity of lipase catalysed resolution o f isopropylidene glycerol (an important building block for the preparation of enantiomerically pure biologically active compounds, such as p-blockers).

They found that cosolvents had a marked influence on the hydrolysis of the aromatic

esters, and was correlated to the percentage of the added solvents and to the nature of the organic media expressed through their physical and chemical properties (log P, e, |i).

The lipase displayed the highest enzymatic activity in solvents miscible (logP<0) or immiscible (logP>1.5) in water. The enzyme stereoselectivity was inversely related to the logP o f the solvent. In a similar study involving the estérification of 2-substituted propionic acids it was shown that the logP values showed a linear correlation with the enantioselectivity as well as the initial reaction rate (Gubicza and Szakacs-Schmidt,

1993). The enantioselectivity slightly decreased while the reaction rates increased with increasing log P values.

It has been shown that shifts in substrate specificity as well as in enantioselectivity take place when enzymes are suspended in microaqueous solvents (Zaks and Klibanov, 1984b; Martinek and Semenov, 1981a, b and c). Elimination of bulk water resulted in the increased rigidity of the enzyme structure with the inability to accommodate large substrates. This was also shown in the transestérification reaction involving porcine pancreatic lipase in microaqueous and anhydrous tributyrin (Zaks and Klibanov, 1984b). Only the “wet” lipase catalysed the transestérification between tributyrin and tertiary alcohols whereas the smaller secondary and primary alcohols were substrates for either enzyme preparation.

H ow ever in the systematic investigation into the influence of the nature of organic solvents on the resolutions of several primary and secondary alcohols catalysed by: PPL, lipase PS, Lipo protein lipase, chromobacteriwn viscosum lipase and mucor miehei

lipase no correlation between enantioselectivity and physico-chemical properties of the solvents was found. A rationale based on the formation o f solvent-enzyme complexes was proposed to explain these results. This rationale was also supported by the finding

that enantiomeric solvents showed different effects on enzyme activity and regioselectivity (Carrea gf a/., 1993; Ottolina er a/., 1993).

Chemical modification

The chemical modification of enzymes has been a valuable tool for investigating the nature of the active site residues and to identify those amino acids that participate in catalysis and substrate binding. This technique has been used to alter enzyme specificity (Kaiser et aL, 1985) and to improve the enantioselectivity o f Candida cylindracea lipase (B-form) (Gu and Sih, 1992). The lipase was treated with the classical chemoselective reagent tetranitromethane (TNM) which nitrated specific tyrosyl residues. The chemically modified TNM-lipase showed remarkably improved enantioselectivity in the hydrolysis of a series o f aryloxypropionic and arylpropionic esters, the enantioselectivity

E was raised from 1.5 (native enzyme) to 33. The marked improvement in enantioselectivity could be attributed to the change in the values for the S-enantiomer which decreased over ten-fold as the TNM concentration was increased. In contrast, the

kcai values for the R-enantiomer decreased only two fold under the same conditions. It has also been shown that the enantioselecdvities o f H. lanuginosa and R. miehi lipases can be manipulated by site directed mutagenesis and chemical modifications o f their respective ampipathic helices (Huit et a l , 1993).

The relatively few published papers on this subject suggest that chemical modification of enzymes as a way of altering their specificity and enantiospecificity is not generally applicable.