Probing the Specificity of the Serine Proteases Subtilisin Carlsberg and α-Chymotrypsin with Enantiomeric 1-Acetamido Boronic Acids. An Unexpected Reversal of the Normal “l”-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 KI 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 S1-pocket orient...