Probing the Abilities of Synthetically Useful Serine Proteases To Discriminate between the Configurations of Remote Stereocenters Using Chiral Aldehyde Inhibitors

The abilities of the synthetically useful serine proteases,
subtilisin Carlsberg (SC) and α-chymotrypsin
(CT), to discriminate between R- and
S-configurations of stereocenters remote from the catalytic
site have been
explored using chiral aldehyde transition state analog inhibitors as
probes. The inhibitors evaluated were (R)-
and
(S)-3-phenylbutanal and (R)- and
(S)-4-phenylpentanal, for which the stereocenters at C-3 and
C-4 respectively are
distant from the aldehyde functionality that interacts with the
catalytic serine residue. The achiral parent
compounds,
3-phenylpropanal and 4-phenylbutanal, respectively, were also assessed
for reference purposes. Each aldehyde was
found to be a competitive inhibitor for both enzymes, with CT being
significantly more potently inhibited than SC.
Within this series, the presence of an R-center methyl
group improved binding significantly over that of the
achiral
parent aldehyde for both enzymes. In contrast, the effects on
binding of S-methyl substituents in the same
positions
were modest, and generally somewhat deleterious. Furthermore, the
greater the separation of the stereocenter from
the aldehyde group, the lower the degree of configuration
discrimination. The most effective inhibition, and
the
highest degree of remote stereocenter discrimination, observed was that
by CT of (R)-3-phenylbutanal, whose
KI of
8.4 μM was 61-fold lower than that of its achiral parent
3-phenylpropanal, and 88-fold lower than the KI
of its
S-enantiomer. Molecular mechanics and molecular
dynamics calculations were performed to identify each
favored
aldehyde−enzyme complex and to reveal the binding and orientation
differences responsible for the R- and
S-enantiomer binding discriminations observed.