Probing the Specificity of the Serine Proteases Subtilisin Carlsberg and α-Chymotrypsin with Enantiomeric 1-Acetamido Boronic Acids. An Unexpected Reversal of the Normal “<scp>l</scp>”-Stereoselectivity Preference

Abstract

Enantiomeric 1-acetamido boronic acids, which are N -acetyl transition state analog inhibitor analogs of l - and d -forms of the amino acids alanine, phenylalanine, p -fluorophenylalanine, p -chlorophenylalanine, and 1-naphthylalanine, have been evaluated as inhibitors of the serine proteases subtilisin Carlsberg (SC) and α-chymotrypsin (CT). All of the boronic acids are powerful competitive inhibitors of both enzymes, with, as expected, the l -enantiomers being generally more potent than the d -enantiomers. However, a dramatic reversal of the normal stereoselectivity preference was observed in the inhibition of CT by [1-acetamido-2-(1-naphthyl)ethyl]boronic acid, with the d -enantiomer becoming a 25-fold more potent inhibitor than the l -enantiomer. Furthermore, the K I of 127 nM for CT inhibition by this d -enantiomer is the lowest of any of the boronic acids evaluated. Molecular modeling analyses of the possible binding modes of the inhibitors suggest that the stereoselectivity reversal is due to S 1 -pocket orientations of naphthyl groups that are different from those of the aromatic side chains of the phenylalanine analogs.