Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes.
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 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 (t(1/2) = 0.7 min).
García-Alles
Luis Fernando
LF
Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland. garcia@ibc.unibe.ch
Erni
Bernhard
B
D001671:Biochemistry / Q000379:methods; D004353:Drug Evaluation, Preclinical; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D007536:Isomerism; D010728:Phosphoenolpyruvate / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000037:antagonists & inhibitors*; D010751:Phosphopyruvate Hydratase / Q000037:antagonists & inhibitors / Q000378:metabolism; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000037:antagonists & inhibitors; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; C063016:phosphoenol-3-chloropyruvate; D010728:Phosphoenolpyruvate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D011770:Pyruvate Kinase; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase; D010730:Phosphoenolpyruvate Carboxylase; D010751:Phosphopyruvate Hydratase