Synthesis of Enantiomerically‐Pure [¹³C]Aristeromycylcobalamin and Its Reactivity in Dioldehydratase, Glyceroldehydratase, Ethanolamine Ammonia‐Lyase and Methylmalonyl‐CoA Mutase Reactions

Abstract

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'-(13)C]aristeromycin. Both the unlabelled and (13)C-labelled product was coupled to vitamin B(12) to form aristeromycylcobalamin. This carbocyclic analogue of coenzyme B(12) 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 ammonia-lyase. 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'-(13)C]aristeromycylcobalamin. We conclude that the lifetime of this radical is still too short to be observed.

Homoadenosylcobalamins as probes for exploring the active sites of coenzyme B12‐dependent diol dehydratase and ethanolamine ammonia‐lyase

“…Ribonucleotide reductase shows more strict specificity for the ribose moiety than diol dehydratase. For Aristeromycylcobalamin (AdoCbl analogue in which the ribosyl oxygen atom is replaced by ‐CH 2 ‐) is 36–44% and 38% as active as AdoCbl in diol dehydratase [27,43,44] and glycerol dehydratase [44], respectively, but it serves as a strong competitive inhibitor for ethanolamine ammonia‐lyase [44] and ribonucleotide reductase [29] and a weak competitive inhibitor for methylmalonyl‐CoA mutase [44]. 2′‐DeoxyAdoCbl is 31%, 17%, and 5–13% as active as AdoCbl for diol dehydratase [38,45], glycerol dehydratase [41], and ribonucleotide reductase [29,35], respectively, but shows only 1–2% activity for methylmalonyl‐CoA mutase [46] or no activity for glutamate mutase [45].…”

Chemistry and Enzymology of Vitamin B12

“…Despite the fact that aristeromycylcobalamin (AriCbl, Figure ) is an active coenzyme for DD and GD (see above), it is completely inactive with EAL …”

The enzymatic activation of coenzyme B12

“…In stark contrast, the class I glutamate mutase is nearly absolutely specific for AdoCbl itself with only one of 23 structural analogs (many of which are partially active coenzymes with class II enzymes) investigated showing any activity at all, 31 and MMCoA mutase can utilize a single coenzyme analog with an altered axial nucleotide structure, 32 but cannot function with analogs altered in the adenosyl moiety. 33 These observations suggest that the mechanism of activation may not be the same for all AdoCbldependent enzymes, a surprising possibility that was not even dreamed of just a few years ago.…”