(Z)-3-(fluoromethyl)phosphoenolpyruvate: synthesis and enzymatic studies

(Z)-3-(Fluoromethyl)phosphoenolpyruvate has been synthesized in nine chemical steps from glyoxylic acid. The compound is stable at pH 3, but at pH 8 it decomposes within seconds to give 2-oxo-3-butenoate. When 3-(fluoromethyl)phosphoenolpyruvate is added to a solution of phosphoenolpyruvate carboxylase or pyruvate kinase, the enzyme is inactivated over the course of an hour. Identical kinetics of inactivation are observed whether the reaction is initiated by addition of 3-(fluoromethyl)-phosphoenolpyruvate, preformed 2-oxo-3-butenoate, or 4-fluoro-2-oxobutanoate (which rapidly undergoes elimination of fluoride ion to form 2-oxo-3-butenoate). The inactivating species in all cases is believed to be 2-oxo-3-butenoate. The inactivation is completely prevented by the presence of dithiothreitol, which reacts rapidly with 2-oxo-3-butenoate. Studies with competitive inhibitors of both enzymes indicate that inactivation does not occur at the active site.