gamma-Glutamyl transpeptidase (GGT, EC 2.3.2.2) catalyzes the hydrolysis and transpeptidation of extracellular glutathione and plays a central role in glutathione homeostasis. We report here the synthesis and evaluation of a series of hydrolytically stable gamma-(monophenyl)phosphono glutamate analogues with varying electron-withdrawing para substituents on the leaving group phenols as mechanism-based and transition-state analogue inhibitors of Escherichia coli and human GGTs. The monophenyl phosphonates caused time-dependent and irreversible inhibition of both the E. coli and human enzymes probably by phosphonylating the catalytic Thr residue of the enzyme. The inactivation rate of E. coli GGT was highly dependent on the leaving group ability of phenols with electron-withdrawing groups substantially accelerating the rate (Bronsted beta(Ig), = - 1.4), whereas the inactivation of human GGT was rather slow and almost independent on the nature of the leaving group. The inhibition potency and profiles of the phosphonate analogues were compared to those of acivicin, a classical inhibitor of GGT, suggesting that the phosphonate-based glutamate analogues served as a promising candidate for potent and selective GGT inhibitors. (c) 2006 Elsevier Ltd. All rights reserved.