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

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.

Structural basis for mammalian vitamin B 12 transport by transcobalamin

“…4). The upper axial ligand position of the Co ion was successfully used as well (14) despite the absence of the His coordination.…”

“…Cbl contains several functional groups that are suitable for the synthesis of bioconjugates (13,14). Because rapidly proliferating tissues take up proportionally greater amounts of Cbl, the tissue-specific delivery of both imaging agents and cytotoxic drugs via Cbl-based bioconjugates has been suggested and in part performed (15,16).…”

New Derivatives of Vitamin B12 Show Preferential Targeting of Tumors

“…In the literature, cobalamin-biotin conjugates that vary in the position of cobalamin coupling have been described. Biotin derivatives at the b-side chain showed little interaction with TCII and were thought to be used for applications where decreased binding with transport proteins is desired (17). Thus, we synthesized and tested a set of cobalamin derivatized at the b-side chain with increasing chain length.…”

“…Because the radiation dose to the kidney is often the dose-limiting factor for application of radiopharmaceuticals, we set out to investigate the possibility of preventing high organ uptake by disrupting the binding of cobalamin to its transport protein TCII and, therefore, inhibit uptake mediated by the receptors TCII-R and megalin. By abolishing binding of cobalamin to TCII (17), a decreased nontargeted organ uptake can be expected. Furthermore by interfering with the binding of cobalamin with circulating TCII, radioactive cobalamin as a small molecule should clear much faster from the blood than the proteinbound cobalamin, resulting in lower systemic toxicity.…”

Induction of Apoptosis in neoplastic cells by depletion of vitamin B12

“…We are developing`growth blockers' aimed at antagonising the uptake of vitamin B 12 via the proliferation-associated B 12 / TCII receptor. We are using two approaches: one called receptor modulation (Pathare et al, 1996) aimed at interfering with the expression and intracellular routing of B 12 receptors (and thereby delivery of B 12 to intracellular methionine synthase). The second approach uses monoclonal antibodies to TCII McLean et al, 1996), the B 12 serum transport protein, to inhibit binding to the cell receptor and thereby producing a depletion of B 12 .…”