Facile oxygen exchanges of phosphoenolpyruvate and preparation of [18O]phosphoenolpyruvate

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Abstract
Phosphoenolpyruvate when heated in acidic solution exchanges its phosphoryl and carboxyl O rapidly and its enolic O much more slowly with O from water. The incorporation of 18O into phosphoenolpyruvate was measured by gas chromatography-mass spectrometry and 31P NMR after heating in H218O at 98.degree. C. The rates of exchange of all 6 O of phosphoenolpyruvate with water increase with increasing acidity, and the phosphoryl O exchange more rapidly than the carboxyl O. The rate of exchange of each O of the phosphoryl group is 16-fold greater than the hydrolysis rate at 1 N HCl. This provides a simple and useful method for the synthesis of [18O]phosphoenolpyruvate highly enriched in its phosphoryl-group O. An enrichment of 89% was obtained with a 50% yield. The [18O]phosphoenolpyruvate showed a binomial distribution of 18O in the phosphoryl-group O. The exchange may be explained by the reversible formation of a transient cyclic phosphate and, for exchange of the enolic O, a transient acyl phosphate. Preparation of [18O]phosphoenolpyruvate from [18O]Pi by a chemical synthesis from .beta.-chlorolactate was not satisfactory because of drastic loss of 18O during the procedures used. Some loss of 18O also occurred during an enzymic synthesis with KCNO, [18O]Pi, carbamate kinase and pyruvate kinase.