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 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 Mg 2+ -and Mn 2+ -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 Mg 2+ , but not with Mn 2+ . 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 ¼ 0.7 min).Keywords: phosphoenolpyruvate analogues; chemical synthesis; inhibition; irreversible inhibitor; PEP-utilizing enzymes.Phosphoenolpyruvate (PEP) is a small and highly functionalized molecule that plays a central role in metabolism. It is not only important because of its high phosphate group-transfer potential (DG ¼ )61.9 kJAEmol )1 ), but also because it is a versatile C 3-synthon in C-C, C-P and C-O bond-formation reactions [1]. Representative examples of the first function are the synthesis of ATP catalysed by pyruvate kinase, and the transport with concomitant phosphorylation of carbohydrates across the bacterial membrane, mediated by the PEP:sugar phosphotransferase system (PTS) [2]. Examples of the second function are the fixation of CO 2 in plants (mediated by PEP carboxylase) [3], the generation of natural phosphonates (PEP mutase) [4], the first step in peptidoglycan cell-wall biosynthesis (catalysed by UDP-GlcNAc enolpyruvyl transferase) and the biosynthesis of aromatic amino acids (3-deoxy-D-arabinoheptulosonate-7-phosphate synthase and 5-enolpyruvylshikimate-3-phosphate synthase) [1].Because of its pivotal role in metabolism, PEP has been the subject of extensive chemical modification. Most of the pseudosubstrates or competitive inhibitors discovered so far differed from PEP by the presence of...