Vitamin B12 monocarboxylic acids: unambiguous isomer assignments by modern two-dimensional NMR spectroscopy.
The three cyanocobalaminmonocarboxylic acid isomers known to be produced by the mild acid hydrolysis of the b-, d-, and e-propionamide side chains of vitamin B12 have been unambiguously assigned by modern 2D NMR methods. Previously, structural assignments had been made by less definitive NMR methods, and both X-ray and neutron diffraction studies had failed to locate unambiguously the position of the carboxyl group. The b and e isomers were structurally assigned in this study, on the basis of the assignment of the 13C NMR signal of the carboxyl group from HMBC (1H-detected heteronuclear multiple-bond correlation) spectra. The carboxyl group resonances exhibited the greatest changes in chemical shift between the protonated (pH 2) and deprotonated (pH greater than 7) forms of the acids. The d isomer was assigned by difference. Since the HMBC experiments required the assignments of side-chain CH2 signals, homonuclear Hartmann-Hahn, 2D homonuclear correlation, 2D nuclear Overhauser effect, 1H-detected heteronuclear multiple quantum coherence, and HMBC spectroscopies were used to assign completely the 1H and 13C NMR spectra of the b and e isomers at pH approximately 7. By comparison with the 13C NMR spectra of the b and e isomers, nearly one-fourth of the resonances of the 13C NMR spectrum of vitamin B12 have been reassigned. The sites of incorporation of 13C-labeled precursors in B12 biosynthesis found in previous studies have been verified by a comparison of 13C assignments. The results of studies using cobalamins modified at the b-, d-, and e-propionamide side chains in which the incorrect structural assignments were used (before 1980), particularly studies of B12-dependent enzymes, require reinterpretation using the correct structural assignments.
Pagano
T G
TG
Department of Chemistry, Emory University, Atlanta, Georgia 30322.
Marzilli
L G
LG
D002264:Carboxylic Acids / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D014805:Vitamin B 12 / Q000031:analogs & derivatives* / Q000032:analysis*
D002264:Carboxylic Acids; C061904:cyanocobalamin-e-monocarboxylic acid; C061902:cyanocobalamin-b-monocarboxylic acid; D014805:Vitamin B 12