EPIMERIZATION OF CYANOCOBALAMIN - IMPROVED SYNTHESIS OF CYANO-8-EPICOBALAMIN AND ITS CHARACTERIZATION BY X-RAY CRYSTALLOGRAPHY, AND H-1, C-13, AND N-15 NMR

The structure of cyano-8-epicobalamin (CN-8-epiCbl), the C8 epimer of vitamin B-12 (cyanocobalamin, CNCbl) has been determined by X-ray crys tallography, complete assignment of its H-1 and C-13 NMR spectra, as w ell as observation and assignment of its amide H-1 and N-15 NMR spectr a in DMSO-d(6). This compound was previously reported as the poorly ch aracterized product of the amidation of its c-monocarboxylate, obtaine d as a minor product of the borohydride reduction of cobaiamin-c-lacto ne, The conditions for the key epimerization step have now been change d to provide a tripling of the yield of the 8-epi-c-monocarboxylate. A long with substantial improvement in the procedures for synthesizing c obalamin-c-lactone and for amidating the monocarboxylate, this modifie d 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 s tructure 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 d egrees, the axial benzimidazole moiety is tilted about 7 degrees towar d the A and D rings, and the corrin macrocycle fold angle along the Co ... C10 axis (23.8 degrees) is more severe than that in CNCbl (17.7 d egrees). Fourteen water molecules are well located in the crystal stru cture and four others occur at half-occupancy. The epimeric relationsh ip between CN-8-epiCbl and CNCbl and the configuration at C8 are also evident in observed H-1 NMR nuclear Overhauser enhancements and differ ences in amide H-1 and N-15 chemical shifts at the d amide between CNC bl and CN-8-epiCbl. Measurements of the amide proton chemical shift th ermal 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 C-13 spectra of CNCbl and CN-8-epiCbl shows that significant chem ical shift changes are not restricted to the B ring (the site of epime rization) but are more global in nature, reflecting the changes in cor rin ring conformation.