The Stereochemical Fate of Chiral-Methyl Valines in Cephalosporin C Biosynthesis

Samples of D,L-valine bearing chiral-methyl groups in the 3-pro(R) and 3-pro(S) positions have been prepared to investigate the stereochemical course of the alpha-ketoglutarate-dependent dioxygenase-catalyzed ring expansion of penicillin N to deacetoxycephalosporin C and the 3'-hydroxylation of the latter to deacetylcephalosporin C, respectively. The orientation of tritium at the diastereotopic C-2 methylene positions of cephalosporin C has been determined in a kinetic assay involving its conversion to N-t-butoxycarbonyldeacetylcephalosporin C-1beta-oxide and monitoring the loss of tritium at constant pH, ionic strength, and temperature. Control experiments are described that demonstrate the validity of treating tritium losses from the methylene as parallel pseudo-first-order processes. An equal distribution to tritium between the two C-2 loci was observed accompanied by a large intrinsic isotope effect. It was concluded that the ring expansion takes place with complete epimerization. Parallel stereochemical examination of the cephem 3'-hydroxylation was carried out by oxidative degradation of cephalosporin C to obtain samples of acetylglycolate. After saponification to glycolate, these specimens were assayed with glycolate oxidase to reveal the overall stereochemical course of hydroxylation as retention.