Reactions of Escherichia coli TEM beta-lactamase with cephalothin and with C10-dipeptidyl cephalosporin esters.

Abstract

Two novel C10-(dipeptidyl)cephalosporin esters (3-(beta-chloro-L-alanyl-beta-chloro-L-alanyloxymethyl)-7 beta-(2-thienylacetamido)-3-cephem-4-carboxylic acid (7) and sodium 3-(L-alanyl-L-alanyloxymethyl)-7 beta-(2-thienylacetamido)-3-cephem-4-carboxylate, toluene-sulfonic acid salt (18] were synthesized, and their reactions with Escherichia coli TEM beta-lactamase were examined. Kinetic parameters determined for the enzymatic reactions of 7 (Km = 0.32 mM; Vmax = 338 mumol min-1 (mg protein)-1) and of 18 (Km = 0.33 mM, Vmax = 338 mumol min-1 (mg protein)-1) demonstrate that both of the peptidyl esters are good substrates for the lactamase. In fact, the Vmax rates for 7 and 18 are each more than 4-fold greater than that obtained for cephalothin, 1 (Vmax = 78 mumol min-1 (mg protein)-1), a well characterized substrate for the lactamases. Analysis of the enzymatic reactions by high field (500 MHz) 1H NMR revealed similar patterns for fragmentation of the cephem nucleus of 1, 7, and 18. However, while hydrolysis of 1 produces acetate, cleavage of 7 and 18 releases beta Cl-LAla-beta Cl-LAla and LAla-LAla, respectively, from the dipeptidyl cephalosporin esters. Based on these findings, a strategy for co-opting the beta-lactamases of Gram-negative bacteria for "delivery" of bactericidal agents is described, and an explanation for the previously reported (Mobashery, S., Lerner, S.A., and Johnston, M. (1986) J. Am. Chem. Soc. 108, 1685) antibacterial activity of 7 is offered.