Synthesis and characterization of 3 ',4 '-anhydroadenosylcobalamin: A coenzyme B-12 analogue with unusual properties

The question of how coenzyme B-12-dependent enzymes facilitate the cleavage of the Co-C bond of the cofactor is of interest. We have synthesized an an alogue of 5'-deoxyadenosylcobalamin (AdoCbl(1)) designed to stabilize the 5 '-deoxyadenosyl radical (5'-deoxyadenosine-5'-yl) that is produced upon hom olysis of the Co-C bond. By replacement of the upper axial ligand of AdoCbl by a 3',4'-anhydro-5'-deoxyadenosyl moiety, the radical formed on the nucl eoside analogue is stabilized by allylic delocalization. The compound, 5'-d eoxy- 3',4'-anhydroadenosylcobalamin (3',4'-anAdoCbl) was synthesized by ch emical and enzymatic methods. The final step was coupling of cob(I)alamin a nd 3',4'-anhydroATP catalyzed by CobA, an ATP: corrinoid adenosyltransferas e. 3',4'-anAdoCbl displays interesting properties. The compound has not bee n purified to homogeneity due to its thermal and oxygen sensitivity. It was characterized by UV-vis spectroscopy, ESI-MS, and NMR spectroscopy. The bo nd dissociation energy of the Co-C bond of the analogue was measured by rad ical trapping techniques. A significantly weaker bond (24 +/- 2 kcal/mol) a s compared to AdoCbl (30 kcal/mol) was observed, as was homolytic cleavage at ambient temperature. Photolysis experiments conducted under anaereobic c onditions reveal no formation of cob(II)alamin, whereas the compound breaks down rapidly under aerobic conditions as measured by cob(In)alamin formati on. We postulate that the weak Co-C bond is cleaved reversibly by photolysi s, where recombination of the allylic radical and cob(II)alamin occurs effi ciently in the absence of a radical scavanger. Activation of the coenzyme B it-dependent enzymes diol dehydrase and ethanolamine ammonia-lyase was obse rved with the cofactor analogue. The measured activity was low and no forma tion of cob(II)alamin could be detected in the steady-state of the reaction for either enzyme. Comparative interactions of AdoCbl and 3',4'-anAdoCbl w ith diol dehydrase and ethanolamine ammonia-lyase suggest that cleavage of the Co-C bond is facilitated by enzyme-coenzyme binding contacts that are r emote from the Co-C bond.