INVESTIGATIONS OF B-12 DERIVATIVES WITH INORGANIC LIGANDS USING 2D NMR-SPECTROSCOPY - LIGAND-RESPONSIVE SHIFTS SUGGEST THAT THE DEOXYADENOSYL MOIETY IN COENZYME B-12 HAS A STERIC TRANS INFLUENCE

The H-1 and C-13 NMR spectra of aquocobalamin (B-12a, H2OCbl) and azid ocobalamin (N3Cbl) have been assigned unambiguously by modern homonucl ear and heteronuclear 2D NMR techniques. In addition, H-1-detected mul tiple-bond heteronuclear multiple quantum coherence spectroscopy (HMBC ) has been used to assign the H-1 spectra and to evaluate past C-13 sp ectral assignments of hydroxocobalamin (B-12b, OHCbl), as well as to e valuate the H-1 and C-13 assignments of vitamin B-12 (cyanocobalamin, CNCbl). Ligand-responsive trends in H-1, C-13, and P-31 NMR spectra fo r a series of cobalamins (Cbls) with R or X axial ligands were compare d with analogous trends available for B,2 model compounds, such as the recently reported lariat-type (C1py) organocobalt derivatives. Moreov er, the ligand-responsive spectroscopic results for Cbls were compared to electronic and steric parameters for the axial ligand R (or X). In general, the pattern of C-13 shifts of most of the 5,6-dimethylbenzim idazole (DMBz) and ribose carbons and some of the corrin ring carbons can be understood if the C-13 NMR shifts are primarily influenced by i nductive electronic effects. P-31 shifts have been reported to reflect changes in phosphodiester conformation as the axial ligand R (or X) c hanges. Even though the P-31, C-13, and H-1 NMR shift changes probably are influenced by different factors (e.g., inductive vs conformationa l effects), good correlations are observed between the P-31 shifts and many of the Cbl C-13 shifts, suggesting that these signals respond to the trans influence of the axial ligand. The chemical shifts of equat orial C's, DMBz C's, and P-31 of coenzyme B-12 (5'-deoxyadenosylcobala min, AdoCbl) are downfield, similar, and upfield, respectively, to shi fts of methylcobalamin (CH3Cbl, the other biologically important B-12 coenzyme). The most reasonable explanation of this shift pattern rests on a trans steric influence of the Ado moiety (which lengthens the Co -N(DMBz) bond) and on a smaller electron-donating ability of Ado compa red to CH3. The weaker electron-donating Ado moiety explains the relat ive downfield shifts of the corrin C's. Likewise, the longer bond betw een DMBz and the Co decreases electron donation from DMBz to Co to the level found in CH3Cbl, explaining the similar shifts of DMBz C's for both coenzymes. The longer Co-DMBz bond in AdoCbl compared to that in CH3Cbl explains the upfield shift of P-31 in AdoCbl. Thus, the shift p atterns provide strong evidence for a steric trans influence of Ado in coenzyme B-12. The net lower electron density at Co in AdoCbl vs CH3C bl indicated by electrochemical studies is consistent with the steric trans influence. Steric lengthening of both the Co-C and Co-N axial bo nds will together facilitate Co-C bond homolysis, a key step in holoen zyme function.