Design, synthesis and characterization of fluorescent cobalamin analogs with a cyclohexane rigid linker

Three fluorescent analogs of cyanocobalamin were synthesized by using a cyclohexane ring to thrust the pendant fluorophore away from the corrin ring of cobalamin. Fluorescein, Rhodamine 6G, and Cy5 were attached to the 5'-OH-alpha-ribofuranose of cyanocobalamin. The rigid molecular scaffold minimizes spatial intramolecular interactions between the two moieties, thereby minimizing the dipolar quenching of the fluorophore and increasing the overall fluorescence quantum yield. All three fluorescent cobalamin analogs exhibit a 10--100 fold increase in the respective fluorescence quantum yield when compared to the analog constructed with a flexible alkyl linker, thereby suggesting that intramolecular collisional quenching has been minimized.;The binding kinetics of cobalamin-fluorescein (CBC205) and cobalamin-Rhodamine 6G (CBC318) analogs with two essential cobalamin transport proteins, intrinsic factor and transcobalamin, were measured using a stopped-flow spectrofluorimetric assay. An increase in fluorescence intensity upon binding to the transport proteins indicates that the fluorescent cobalamin analogs binding to their corresponding transport proteins and derivatization of the cobalamin 5'-OH moiety of ribose does not significantly affect its recognition and transport by the binding proteins and the cell surface receptors. Preliminary in vitro studies using murine fibroblast NIH3T3 cells indicate a successful uptake by transcobalamin-dependent receptors on the surface of the fibroblast cells, followed by successful cellular internalization of CBC318. The increased transport and sequestration of cobalamin by the rapidly proliferating cells suggests the fluorescent cobalamin analogs may be suitable as cancer diagnostic and imaging agents.;Quantum mechanical calculations using density functional theory confirmed the rigidity of the cyclohexane linker. Computer modeling suggests that the cyclohexane linker is locked in a rigid chair conformation, and the pendant corrin ring is folded underneath the cobalamin molecule. The fluorescence lifetime of all three cobalamin analogs with a rigid linker remains the same as in the unconjugated fluorophore, thus providing additional evidence that intramolecular collisional quenching is no longer the dominant interaction that quenches the excited electronic state of the fluorophore.