Markedly enhancing enzymatic enantioselectivity in organic solvents by forming substrate salts

A new approach is proposed to enhance enzymatic enantioselectivity in organic solvents. It is based on the presumption that the less reactive substrate enantiomer experiences greater steric hindrances in the enzyme-bound transition state than the more reactive one; enlarging the substrate by forming a salt with a bulky counterion should exacerbate these hindrances disproportionately for the less reactive enantiomer, thus increasing the enantioselectivity. This strategy was implemented and verified with several structurally diverse chiral compounds converted to salts by treatment with numerous Bronsted-Lowry acids or bases, Enzymatic transesterifications or hydrolyses of the resultant racemic salts were much more enantioselective than those of the free substrates. This effect was observed in various organic solvents (but not in water, where the salts apparently dissociate to regenerate the free substrates), using both crystalline and lyophilized enzymes (Pseudomonas cepacia lipase and the protease subtilisin Carlsberg). As demonstrated both analytically and preparatively, in some instances an enzyme would exhibit virtually no enantiopreference toward a free substrate but a profound one toward its salts. All these effects were rationalized by using molecular modeling to derive enzyme-bound transition-state structures of R and S enantiomers of both free substrates and their salts. The areas of substrate surfaces overlapping with the enzyme calculated from these models turned out to be a remarkably good predictor of enzymatic enantioselectivity (E)-the greater the difference in the area of surface overlap between the enantiomers, the higher the E value.