KINETICS AND MECHANISM OF BETA-LACTAMASE INHIBITION BY PHOSPHONAMIDATES - THE QUEST FOR A PROTON

Four phosphonamidates were synthesized as potential beta-lactamase inh ibitors. Three were methanephosphonamidates [CH3PO2-NHR/Ar, where R/Ar = 4-methoxybenzyl (3a), phenyl (3b), and m-nitrophenyl (3c)], while t he fourth, PhCH2OCONHCH2PO2-NHPh (2a), also contained a beta-lactamase active site-directed amido side chain. The pH-rate profiles for the h ydrolyses of these compounds in the absence of enzyme demonstrated the necessity of nitrogen protonation in the transition state; the reacti ve neutral form was the zwitterion, IH. The four phosphonamidates irre versibly inhibited the class C beta-lactamase of Enterobacter cloacae P99 by phosphonylation of the active-site serine hydroxyl group, but t hey displayed strikingly different inhibition pH-rate profiles. The pH profile and inhibition rates of the N-alkyl derivative 3a could be un derstood in terms of a direct reaction between IH and EH, the form of the enzyme reactive with substrates and phosphonate monoester inhibito rs. The pH profile for 2a also indicated that EH was the reactive enzy me form, but its direct reaction with IH is unlikely because of the lo w concentration of the latter, stemming from its low nitrogen pK(a). I n 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 substr ate analogs but which allow access to protons. Inhibition by 3c was in terpreted to involve rate-determining protonation at high pH. At and a bove neutral pH, phosphonamidates will generally be less effective inh ibitors 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 v iew 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 posi tioned general acid catalyst at the active site.