Phosphonamidate analogues of dipeptides with carboxypeptidase A and β-lactamase-inhibitory activity: elucidation of the mechanism of β-lactamase inhibition by electrospray mass spectrometry

A series of phosphonamidate compounds with different P1´ amino acid residues have been shown to be irreversible inactivators of the serine β-lactamase from Enterobacter cloacae P99. The efficiency of inhibition (based on k2/K values) of P99 by these derivatives, ordered in decreasing potency, is: β-phenyl-β-Ala> L-Phe> β-Ala> Gly>D-Phe>D-Pro> D-thiazolidine. The D- and L-Phe compounds also inhibit carboxypeptidase A. The proline and thiazolidine derivatives were phosphonamidate methyl esters, whereas the others were salts of diacids. Electrospray mass spectrometry showed that equimolar mixtures of the P99 enzyme with each of the following derivatives, Gly, D-Phe, L-Phe, β-Ala and β-phenyl-β-Ala, effected efficient adduct formation (70–95% of enzyme modified), illustrating the particularly active nature of some of these compounds. All the primary amino acid derivatives gave a similar mass increment, which suggests the displacement of the variable P1´ part of the molecule. This observation provides evidence that the compounds phosphonylate the active-site serine, with the phosphonamidate bond as the scissile bond and the amino acid as the leaving group. The thiazolidine derivative (phosphonamidate methyl ester) also appeared to work by the same mechanism. The comparable proline derivatives caused lower than expected mass shifts of 227–229, and therefore it is proposed that with these compounds both the amino acid and the phosphonamidate ester methoxy group were displaced at the phosphorus atom during the inhibition process. Therefore, electrospray mass spectrometry has provided both a measure of potency and a rationale for the mechanism of inhibition of P99 by these compounds.