CATALYSIS OF CARBAMATE HYDROLYSIS BY VANCOMYCIN AND SEMISYNTHETIC DERIVATIVES

The semisynthesis of vancomycin (1a) derivatives bearing propyl (1b), histaminyl (1c), and 3-aminopropyl (1d) groups attached to the C-terminus is reported. This work was undertaken as part of a program to create new biomimetic catalysts and to ultimately test the relative potencies of antibiotics that act stoichiometrically versus catalytically against a common target. 1b-d were prepared by dicyclohexylcarbodiimide-mediated condensations between vancomycin's carboxyl group and amine coupling partners and were found to bind to N(alpha),N(omega)-diacetyl-L-Lys-D-Ala-D-Ala (2a, a tripeptide analogue of the C-terminus of the Staphylococcus aureus cell wall precursor) with affinities similar to that observed with 1a. 1a-d exhibit novel carbamate hydrolase activity, catalyzing the hydrolysis of N(alpha)-(p-nitrophenyl-oxycarbonyl-Gly)-N(omega)-acetyl-L-Lys-D-Ala-D-Ala (2b) with demonstrated substrate turnover, specificity, and inhibition by 2a. Catalytic rate constants (k(cat)) for hydrolysis of 2b were 0.55, 0.97, 1.14, and 1.48 min-1 for 1a-d, respectively. These rate constants correspond to accelerations of 4-11-fold above the buffer-catalyzed reaction (k(cat)/k'buf) and 2200-5900-fold above the uncatalyzed reaction (k(cat)/k'uncat) with effective molarities (k(cat)/k(buf)) of 0.085-0.23 M. Differences in k(cat) among the series la-d were small and did not correlate with the presence or absence of C-terminal catalytic functional groups. The variation in rate constants did correlate with increases in positive charge at the C-terminus of 1a-d. The microenvironment in the region where hydrolysis occurs was probed using the ligand N(alpha)-(fluoresceinylthiocarbamoyl)-N(omega)-acetyl-L-Lys-D-Ala-D-Ala (2c), which has a pH-dependent absorption spectrum. The spectral changes observed upon complexation of 2c to 1a-d were consistent with an increase in microscopic pH and/or a decrease in pK(a) in this region relative to the bulk solution. Trends in DELTApH/DELTApK(a) correlate with the trend in k(cat) for 1a-d although the apparent increase in microscopic hydroxide ion concentration could not quantitatively account for the observed rate accelerations above the buffer-catalyzed reaction.