Synthesis of phosphoenolpyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes

The synthesis of 10 new phosphoenol pyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z )- and (E )-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the (3) J (H-C) coupling constants between the carboxylate carbons and the vinyl protons. All of these and other known PEP-analogues were tested as reversible and irreversible inhibitors of Mg2+ - and Mn2+ - activated PEP-utilizing enzymes: enzyme I of the phosphoenol pyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant<linkr rid="q1"> </linkr> to such modifications. Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+ , but not with Mn2+ . For the irreversible inhibition of these enzymes, (Z )-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I (t (1/2) = 0.7 min).