PHOSPHONAMIDATE ANALOGS OF DIPEPTIDES WITH CARBOXYPEPTIDASE-A AND BETA-LACTAMASE-INHIBITORY ACTIVITY - ELUCIDATION OF THE MECHANISM OF BETA-LACTAMASE INHIBITION BY ELECTROSPRAY MASS-SPECTROMETRY

A series of phosphonamidate compounds with different P-1' amino acid r esidues have been shown to be irreversible inactivators of the serine beta-lactamase from Enterobacter cloacae P99. The efficiency of inhibi tion (based on k(2)/K values) of P99 by these derivatives, ordered in decreasing potency, is: beta-phenyl-beta-Ala > L-Phe > beta-Ala > Gly > D-Phe > D-Pro > D-thiazolidine. The D- and L-Phe compounds also inhi bit carboxypeptidase A. The proline and thiazolidine derivatives were phosphonamidate methyl esters, whereas the others were salts of diacid s. Electrospray mass spectrometry showed that equimolar mixtures of th e P99 enzyme with each of the following derivatives, Gly, D-Phe, L-Phe , beta-Ala and beta-phenyl-beta-Ala effected efficient adduct formatio n (70-95% of enzyme modified), illustrating the particularly active na ture of some of these compounds. All the primary amino acid derivative s gave a similar mass increment, which suggests the displacement of th e variable P-1' part of the molecule. This observation provides eviden ce that the compounds phosphonylate the active-site serine, with the p hosphonamidate bond as the scissile bond and the amino acid as the lea ving group. The thiazolidine derivative (phosphonamidate methyl ester) also appeared to work by the same mechanism. The comparable proline d erivatives caused lower than expected mass shifts of 227-229, and ther efore it is proposed that with these compounds both the amino acid and the phosphonamidate ester methoxy group were displaced at the phospho rus atom during the inhibition process. Therefore, electrospray mass s pectrometry has provided both a measure of potency and a rationale for the mechanism of inhibition of P99 by these compounds.