Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP‐utilizing enzymes

Abstract

The synthesis of 10 new phosphoenolpyruvate (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 3JH‐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 phosphoenolpyruvate: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 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 (t1/2 = 0.7 min).