Kinetics and mechanism of beta-lactamase inhibition by phosphonamidates: the quest for a proton.

Four phosphonamidates were synthesized as potential beta-lactamase inhibitors. Three were methanephosphonamidates [CH3PO2-NHR/Ar, where R/Ar = 4-methoxybenzyl (3a), phenyl (3b), and m-nitrophenyl (3c)], while the fourth, PhCH2OCONHCH2PO2-NHPh (2a), also contained a beta-lactamase active site-directed amido side chain. The pH-rate profiles for the hydrolyses of these compounds in the absence of enzyme demonstrated the necessity of nitrogen protonation in the transition state; the reactive neutral form was the zwitterion, IH. The four phosphonamidates irreversibly inhibited the class C beta-lactamase of Enterobacter cloacae P99 by phosphonylation of the active-site serine hydroxyl group, but they displayed strikingly different inhibition pH-rate profiles. The pH profile and inhibition rates of the N-alkyl derivative 3a could be understood in terms of a direct reaction between IH and EH, the form of the enzyme reactive with substrates and phosphonate monoester inhibitors. The pH profile for 2a also indicated that EH was the reactive enzyme form, but its direct reaction with IH is unlikely because of the low concentration of the latter, stemming from its low nitrogen pKa. In this case, proton uptake from solution subsequent to phosphonamidate anion binding probably accounts for the observed rates. The anilides 3b and 3c were weak inhibitors with respect to 2a and 3a. Their major inhibitory activity, observed at above neutral pH in contrast to that of 2a and 3a, probably involves modes of binding not typical of substrate analogs but which allow access to protons. Inhibition by 3c was interpreted to involve rate-determining protonation at high pH. At and above neutral pH, phosphonamidates will generally be less effective inhibitors than phosphonate p-nitrophenyl monoesters. Below pH 7, enzyme-specific phosphonamidates, especially N-alkyl derivatives, will become more effective than the esters. The results are consistent with the view that, because of the specific geometry of the phosphonyl-transfer transition state, the effectiveness of phosphonic acid derivatives as beta-lactamase inhibitors is limited by the absence of a suitably positioned general acid catalyst at the active site.