Epimerization of cyanocobalamin. Improved synthesis of cyano-8-epicobalamin and its characterization by x-ray crystallography, and 1H, 13C, and 15N NMR

Abstract

The structure of cyano-8-epicobalamin (CN-8-epiCbl), the C8 epimer of vitamin B 12 (cyanocobalamin, CNCbl) has been determined by X-ray crystallography, complete assignment of its 1H and 13C NMR spectra, as well as observation and assignment of its amide 1H and 15N NMR spectra in DMSO- d 6. This compound was previously reported as the poorly characterized product of the amidation of its c-monocarboxylate, obtained as a minor product of the borohydride reduction of cobalamin- c-lactone. The conditions for the key epimerization step have now been changed to provide a tripling of the yield of the 8-epi- c-monocarboxylate. Along with substantial improvement in the procedures for synthesizing cobalamin- c-lactone and for amidating the monocarboxylate, this modified method for epimerization of cyanocobalamin now provides CN-8-epiCbl in sufficient quantities for thorough characterization and studies of its properties as a vitamin B 12 analog. The crystal structure of CN-8-epiCbl has been determined. The main difference between the crystal structure of CNCbl and CN-8-epiCbl is that the d side chain (at C8) is “down-wardly” axial in CNCbl, but pseudo-equatorial in CN-8-epiCbl. In addition, the B pyrrole ring in CN-8-epiCbl is twisted by about 5°, the axial benzimidazole moiety is tilled about 7° toward the A and D rings, and the corrin macrocycle fold angle along the Co … C10 axis (23.8°) is more severe than that in CNCbl (17.7°). Fourteen water molecules are well located in the crystal structure and four others occur at half-occupancy. The epimeric relationship between CN-8-epiCbl and CNCbl and the configuration at C8 are also evident in observed 1H NMR nuclear Overhauser enhancements and differences in amide 1H and 15N chemical shifts at the d amide between CNCbl and CN-8-epiCbl. Measurements of the amide proton chemical shift thermal gradients strongly suggest the presence of a hydrogen bond in CN-8-epiCbl involving the c amide syn proton and the d amide carbonyl in solution. This intramolecular hydrogen bonding is not present in the crystal structure in which the d amide carbonyl is hydrogen bonded to two water molecules and the c amide nitrogen is hydrogen bonded to the b amide carbonyl oxygen of a symmetry related molecule. Comparison of the 13C spectra of CNCbl and CN-8-epiCbl shows that significant chemical shift changes are not restricted to the B ring (the site of epimerization) but are more global in nature, reflecting the changes in corrin ring conformation.