Synthesis of enantiomerically-pure [13C]aristeromycylcobalamin and its reactivity in dioldehydratase, glyceroldehydratase, ethanolamine ammonia-lyase and methylmalonyl-CoA mutase reactions

We describe a novel enantioselective synthesis of aristeromycin, the carbocyclic analogue of adenosine. The seven-step synthesis is also suitable for the preparation of specifically -labelled [6'-C-13]aristeromycin. Both the unlabelled and 13 C-labelled product was coupled to vitamin B-12 to form aristeromycylcobalamin. This carbocyclic analogue of coenzyme B, was examined for its coenzymic activity with several adenosylcobalamin-dependent enzymes. For glyceroldehydratase and dioldehydratase, the reaction rate (k(cat)) was 38 and 44 % of that measured with adenosylcobalamin as coenzyme. In contrast, aristeromycylcobalamin showed no detectable activity with methylmalonyl-CoA mutase and ethanolamine ammonialyase. Instead, it was a weak inhibitor of the former and a strong inhibitor of the latter enzyme. The slower turnover rate with glyceroldehydratase raised the hope of detecting the 6'-deoxyaristeromycyl radical intermediate. Comparison of the EPR spectra of the intermediates in the glyceroldehydratase reaction, which used adenosyl- and aristeromycylcobalamines, respectively, as coenzyme, revealed a significant shift and this suggests a different geometric position of these cofactors at the binding site during the cleavage of the carbon-cobalt bond. However, we found no evidence for the existence of a 6-deoxyaristeromycyl radical during the reaction with [6'-C-13]aristeromycylcobalamin. We conclude that the lifetime of this radical is still too short to be observed.