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'-(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.
Weigl
Ulrich
U
Institut für Organische Chemie, Lehrstuhl für Biochemie Universität Karlsruhe Kaiserstrasse 12, 76 128 Karlsruhe, Germany. biochem@ochhades.chemie.uni-karlsruhe.de
Heimberger
Martin
M
Pierik
Antonio J
AJ
Rétey
János
J
D000241:Adenosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000378:metabolism*; D002250:Carbon Radioisotopes / Q000737:chemistry; D002384:Catalysis; D003038:Cobamides / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D004981:Ethanolamine Ammonia-Lyase / Q000378:metabolism*; D006836:Hydro-Lyases / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008697:Methane / Q000031:analogs & derivatives* / Q000737:chemistry; D008765:Methylmalonyl-CoA Mutase / Q000378:metabolism*; D009595:Nitroparaffins / Q000737:chemistry; D011408:Propanediol Dehydratase / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
D002250:Carbon Radioisotopes; D003038:Cobamides; D009595:Nitroparaffins; C030034:aristeromycin; D006836:Hydro-Lyases; D011408:Propanediol Dehydratase; C020671:glycerol dehydratase; D004981:Ethanolamine Ammonia-Lyase; D008765:Methylmalonyl-CoA Mutase; C000913:cobamamide; D000241:Adenosine; D008697:Methane; C008640:nitromethane