The relative catalytic efficiency of beta-lactamase catalyzed acyl and phosphyl transfer

Phosphonamidates which bear a simple resemblance to penicillin type structu res have been synthesised as potential inhibitors of beta -lactamases: -eth yl N-(benzyloxycarbonyl) amidomethyl phosphonyl amides, PhCH2OCONHCH2P(O)(O Et)NR2, the amines HNR2 being L-proline, D-proline. L-thiazolidine, and o-a nthranilic acid. The proline derivatives completely and irreversibly inacti vated the class C beta -lactamase from Enterobacter cloacae P99. in a time- dependent manner, indicative of covalent inhibition. The inactivation was f ound to be exclusive to the class C enzyme and no significant inhibition wa s observed with any other class of beta -lactamase. The anthranilic acid de rivative exhibited no appreciable inactivation of the beta -lactamases. The phosphonyl proline and phosphonyl thioproline derivatives were separated i nto their diastereoisomers and their individual second order rate constants for inhibition were found to be 7.72 +/- 0.37 and 8.3 x 10(-2) +/- 0.004 M -1 s(-1) for the L-proline derivatives, at pH 7.0. The products of the inhi bition reaction of each individual diastereoisomer. analyzed by electrospra y mass spectroscopy, indicate that the more reactive diastereoisomers phosp honylate the enzyme by P-N bond fission with the elimination of proline. Co nversely. gas chromatographic detection of ethanol release by the less reac tive proline diastereoisomer suggests phosphonylation occurs by P-O bond fi ssion. The enzyme enhances the rate of phosphonylation with P-N fission by at least 10(6) compared with that effected by hydroxide-ion. The pH depende nce of the rate of inhibition of the p-lactamase by the more reactive diast eroisomer is consistent with the reaction of the diprotonated form of the e nzyme. EH2. with the inhibitor, I (or its kinetic equivalents EH with IH). This pH dependence and the rate enhancement indicate that the enzyme appear s to use the same catalytic apparatus for phosphonylation as that used for hydrolysis of beta -lactams. The stereochemical consequences of nucleophili c displacement at the phosphonyl centre are discussed. (C) 2001 Academic Pr ess.