title: 5-enolpyruvylshikimate 3-phosphate synthase: chemical synthesis of the tetrahedral intermediate and assignment of the stereochemical course of the enzymatic reaction.
abstract: A chemical synthesis of both diastereomers of the tetrahedral intermediate involved in 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) catalysis has been accomplished. Combination of methyl dibromopyruvate with a protected shikimic acid derivative, phosphorylation, and lactonization afforded the intermediates (S)-15 and (R)-15, whose configurations were assigned by NMR. After introduction of the 3-phosphate group and deprotection, photoinitiated radical debromination of the dibromo analogues (S)-5 and (R)-5 was accomplished with tributyltin hydride in mixed aqueous solvents in the presence of surfactant to give the pyruvate ketal phosphates (R)-TI and (S)-TI, respectively. These compounds are stable at high pH, but decompose at pH 7 with a half-life of ca. 10 min. (R)-TI proved to be inert to EPSPS, while (S)-TI was converted by the enzyme to a mixture of 5-enolpyruvylshikimate 3-phosphate, shikimate 3-phosphate, and phosphoenolpyruvate. The demonstration that the enzymatic intermediate possesses the S-configuration at the ketal center confirms the mechanism as an anti addition followed by a syn elimination. Furthermore, it appears that the syn stereochemistry of the second step requires the phosphate leaving group to serve as the base in catalyzing its own elimination.
authors.affiliation: Center for New Directions in Organic Synthesis, Department of Chemistry, University of California, Berkeley, California 94720-1460, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: An
authors.forename: Uday
authors.initials: U
authors.lastname: Maitra
authors.forename: Ulf
authors.initials: U
authors.lastname: Neidlein
authors.forename: Paul A
authors.initials: PA
authors.lastname: Bartlett
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D008958:Models, Molecular; D008968:Molecular Conformation; D019906:Nuclear Magnetic Resonance, Biomolecular; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D013237:Stereoisomerism
chemical_list: C058070:5-enolpyruvoylshikimate-3-phosphate; D012765:Shikimic Acid; C042786:shikimic acid-3-phosphate; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase