(5′R)‐2H1)Adenosine [(5′R):(5′S) = 85:15] was prepared by a procedure which involved inter alia the reduction of 6‐N‐benzoyl‐2′,3′‐O‐isopropylidene 5′‐oxoadenosine with a reagent obtained from LiAl2H4 and (‐)‐ isoborneol. (5′S)‐(5′‐2H1)AdoCbl= 5′S):(5′R) = 74:26] (AdoCbl = 5′ deoxyadenosylcoalamin) was synthesised by reacting cobal(I)amin with (5′R)‐2′‐3′‐O‐isopropylidene‐5′‐tosyl‐(5′‐2H1) adenosine followed by acid hydrolysis to remove the isopropylidene protective group. (5′R)‐2H1)AdpCbl [(5′S):(5′R) = 77:23] was prepared by reacting cobal (I) amin with (5′S)‐5′‐chloro 5′‐(5′‐2H1) deoxyadenosine [(5′S):(5′R= 80:20] obtained in turn from (5′R)‐(5′‐2H1)adenosine. The reaction sequence involved two consecutive inversions at the C‐5′ atom of adenosine. Comparison of the 500‐MHz 1H‐NMR spectra of unlaelled, (5′S)‐ and (5′R)‐(5′2H1)AdCbl allowed assignment of the triplet at 0.58 ppm and the doublet at 1.525 ppm to the diastereotopic 5′‐HRe and 5′‐HSt atoms, respectively. On acidification, these two protons gave rise to two triplets at 0.11 ppm and 1.78 ppm indicating that torsion had occurred around the C‐4′‐C‐5′ bond. Samples of (5′R)‐ and (5′S)‐(5′2H1)AdoCbl were incubated with metylmalonyl0‐CoA mutase from Propioniacterium shermanii Examination by 1H‐NMR spectroscopy at 500 MHz revealed partial los and stereochemical scrambling of the deuterium at the 5′ position. This indicates transient conversion of the C‐5′ atom into a torsiosymmetric group and hence cleavage of the cobalt‐carbon bond during interaction with the enzyme. The mechanism by which deuterium is lost remains to be elucidated.

1. When (methyL2H 3)methylmalonyl-CoA was reacted with partially purified methylmalonyl-CoA mutase, 'H-NMR revealed that about 24% of the migrating deuterium was lost after 88% conversion.2. When [methyf-3H]methylmalonyl-CoA was incubated with highly purified methylmalonyl-CoA mutase, tritium exchange with the medium depended on added methylmalonyl-CoA epimerase. 3. With highly purified preparations of methylmalonyl-CoA mutase, effective tritium exchange from [5'-3H]adenosylcobalamin to water required the addition of methylmalonyl-CoA epimerase and of substrate (e.g. succinyl-CoA).4. By addition of ['4C]succinyl-CoA to a partially purified preparation of methylmalonyl-CoA mutase, it was shown that the mutase binds one substrate molecule very tightly.5. Coupling the mutase reaction with the transcarboxylase reaction and using variously labelled succinyl-CoA as substrate, revealed that only (2R)-and not (2S)-methylmalonyl-CoA will be formed by the mutase with a kinetic isotope effect of 3.5 using ('H4)succinyl-CoA.6 . When (I -I3C) propionyl-CoA was reacted with a mixture of highly purified methylmalonyl-CoA carboxylase, epimerase and mutase, 13C-NMR signals were obtained for the thioester carbonyl of succinyl-CoA (relative intensity 100%) and of methylmalonyl-CoA (5%) as well as for the carboxyl of free succinic acid (27%) and of succinyl-CoA (< 4.5%). Thus very little, if any, migration of the CoA from one carboxyl to the other appears to take place.(1 ,4-13C2)Succinic acid and (1 ,4-'3C2)succinyl-CoA were synthesised and their I3C-NMR chemical shifts were exactly determined. 7. Evidence is provided for a strict stereospecificity of the mutase toward the (2R)-epimer of methylmalonylCoA and for an incomplete stereospecificity toward the two diastereotopic 3-H atoms of succinyl-CoA. The latter, combined with a high intramolecular isotope discrimination, causes rapid washing-out of the migrating 2H and 3 H to water and slow washing-in from the medium. Whenever migration of protium from the sterically less preferred 3-pro(S)-position of succinyl-CoA occurs and simultaneously a heavy isotope is maneuvered from the migratable 3-pro(R)-position into the labile a-position of methylmalonyl-CoA, the substitution by the COSCoA group takes place with inversion of configuration. When the sterically preferred 3-pro(R)-hydrogen atom migrates, the previously reported stereochemical retention occurs.A mechanistic and stereochemical scheme is discussed that fully accounts for all observations.