Synthesis of Cobalamin−Biotin Conjugates That Vary in the Position of Cobalamin Coupling. Evaluation of Cobalamin Derivative Binding to Transcobalamin II

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Abstract
Six cobalamin−biotin conjugates have been prepared. The cobalamin−biotin conjugates were prepared to evaluate the effect that the location of attachment had on the binding with transcobalamin II (TCII), the cobalamin binding protein in plasma, and to evaluate their potential use for in vitro and in vivo applications. This study focused only on the effect of binding with TCII. To decrease the possibility of steric problems in binding of the cobalamin conjugates with TCII, and biotin's binding with streptavidin or avidin, moieties of 11−18 atoms in length were used as linkers. Four biotin conjugates were prepared which were attached to the corrin ring of the cobalamin molecule (on b-, c-, d-, and e-side chains). One conjugate was attached to the 5‘-OH of the ribose moiety, and another conjugate was attached at the cobalt metal (in place of the cyanide moiety of cyanocobalamin). Competitive binding studies were conducted where various amounts of the cobalamin−biotin conjugates and their precursor cobalamin derivatives competed with [57Co]cyanocobalamin for binding of recombinant human TCII (rhTCII). Evaluation of cobalamin derivatives which were conjugated at the 5‘-OH of ribose or the cobalt metal center indicated that conjugation at either of these positions had little effect on binding with rhTCII. However, conjugates where the attachment was made on the corrin ring substituents had a large variation in binding with rhTCII. Conjugates on the e-propionamide side chain had little effect (relative affinity was equal to or decreased less than a factor of 3) on binding with rhTCII, conjugates of the b-isomer had decreased binding (relative affinity decreased less than a factor of 10), conjugates of the d-propionamide had further decreased binding (relative affinity decreased between 44 and 69 times), and conjugates on the c-acetamide group had poor binding to rhTCII (relative affinity decreased between 295 and 1160 times). The significance of the side chains on the corrin ring in providing specificity and high-affinity binding with rhTCII is discussed.