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

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
orientations of naphthyl groups that are different from those of the
aromatic side chains of the phenylalanine analogs.