HETERONUCLEAR NMR-STUDIES OF COBALAMINS .12. FURTHER-STUDIES OF DICYANOCOBAMIDES AND THE COMPLETE PROTON, CARBON, AND AMIDE NITROGEN NMR ASSIGNMENTS OF DICYANOCOBALAMIN

From a combination of P-31-H-1 chemical shift correlated, homonuclear J-correlated, absorption-mode hypercomplex NOE, hypercomplex homonuclear Hartmann-Hahn, H-1-detected heteronuclear, and H-1-detected multiple-bond heteronuclear multiple-quantum coherence NMR spectroscopies, the complete H-1 and C-13 NMR assignments of dicyanocobalamin in D2O have been made. From these proton assignments in conjunction with the NOESY map, the H-1 spectrum of dicyanocobalamin in DMSO-d6 could be nearly completely assigned from a NOESY experiment in this solvent. In DMSO-d6, the amide proton resonances are visible, and these could be unambiguously assigned from observation of numerous NOE's to side-chain methylene and corrin ring protons. Along with our previous determination of the amide N-15 chemical shifts and amide proton-nitrogen connectivities from H-1-detected H-1, N-15 multiple-quantum coherence spectroscopy these assignments permitted, for the first time, the complete assignment of the amide N-15 resonances. Comparisons of the C-13 resonances of the nucleotides of dicyanocobalamin, the dicyano derivatives of the cobalamin b, d, and e monocarboxylate analogues, and the dicyano derivative of the C13 epimer of cobalamin among each other and with the free base of the detached nucleotide are consistent with the persistance of the previously postulated tuck-in species of base-off dicyanocobalamin, in which the benzimidazole nitrogen B3 is hydrogen bonded to a side-chain amide, in each of the above cobalamin analogues. These comparisons eliminate the b,d, and e amides as possible hydrogen-bond donors in the tuck-in species. Methylation of the benzimidazole B3 nitrogen was shown to prevent formation of the tuck-in species in the dicyano derivative of the trimethylbenzimidazolyl analogue by comparison of its C-13 spectrum to that of the detached, N-methylated nucleotide methyl ester. Taken together with previous N-15 NMR results, numerous NOE's observed between the benzimidazole B2, B4, and B7 protons and protons on the corrin side chains, ring and ring methyl groups strongly suggest that the g side-chain amide is the hydrogen-bond donor in the tuck-in species.