Synthesis and Characterization of 3‘,4‘-Anhydroadenosylcobalamin: A Coenzyme B12 Analogue with Unusual Properties

Abstract

The question of how coenzyme B12-dependent enzymes facilitate the cleavage of the Co−C bond of the cofactor is of interest. We have synthesized an analogue of 5‘-deoxyadenosylcobalamin (AdoCbl1) designed to stabilize the 5‘-deoxyadenosyl radical (5‘-deoxyadenosine-5‘-yl) that is produced upon homolysis 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 nucleoside analogue is stabilized by allylic delocalization. The compound, 5‘-deoxy- 3‘,4‘-anhydroadenosylcobalamin (3‘,4‘-anAdoCbl) was synthesized by chemical and enzymatic methods. The final step was coupling of cob(I)alamin and 3‘,4‘-anhydroATP catalyzed by CobA, an ATP:corrinoid adenosyltransferase. 3‘,4‘-anAdoCbl displays interesting properties. The compound has not been purified to homogeneity due to its thermal and oxygen sensitivity. It was characterized by UV−vis spectroscopy, ESI-MS, and NMR spectroscopy. The bond dissociation energy of the Co−C bond of the analogue was measured by radical trapping techniques. A significantly weaker bond (24 ± 2 kcal/mol) as compared to AdoCbl (30 kcal/mol) was observed, as was homolytic cleavage at ambient temperature. Photolysis experiments conducted under anaereobic conditions reveal no formation of cob(II)alamin, whereas the compound breaks down rapidly under aerobic conditions as measured by cob(III)alamin formation. We postulate that the weak Co−C bond is cleaved reversibly by photolysis, where recombination of the allylic radical and cob(II)alamin occurs efficiently in the absence of a radical scavanger. Activation of the coenzyme B12-dependent enzymes diol dehydrase and ethanolamine ammonia-lyase was observed with the cofactor analogue. The measured activity was low and no formation 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 with diol dehydrase and ethanolamine ammonia-lyase suggest that cleavage of the Co−C bond is facilitated by enzyme-coenzyme binding contacts that are remote from the Co−C bond.