Metal-free, Zn, Cu, and Rh analogues of vitamin B12 were synthesized to further characterize structural requirements for the binding to human intrinsic factor, transcobalamin I and transcobalamin II. Binding affinities of the various analogues were studied by competition against cyano[57Co]cobalamin. When albumin-coated charcoal was used for the separation of free and bound corrinoids, the relative 50% inhibition indexes were determined. The influence of metal substitution was similar among the 3 binding proteins. For analogues with a strong coordinative linkage between the heterocyclic base and the central metal ion, similar to that with Co (e.g., zincobalamin and cyanorhodibalamin), the indexes range from 0.65-2.35 for all 3 binding proteins. Analogues in which corrdination is impossible (hydrogenobalamin and dicyanorhodibalamin) exhibit markedly reduced binding with indexes between 10-160. Cupribalamin shows 50% inhibition indexes ranging from 2.3-5.0, thus suggesting a weak coordinative bond between the Cu and the 5.6-dimethylbenzimidazole moiety. The importance of the coordinative linkage between the central metal ion and the nucleotide moiety for optimal recognition by vitamin B12 binding proteins is emphasized.
