| D,L-S(6,8)-methyllipoic acid methyl ester triflate salt (D,L- S-methyllipoic acid methyl ester) was designed and synthesized as a model for S-protonated lipoic acid, which is the hypothetical active form of lipoic acid in the reductive acylation catalyzed by the E1 and E2 enzymes of the 2-oxoacid dehydrogenase multienzyme complexes. While in an earlier study lipoic acid could only trap the enamine/C2alpha-carbanion intermediate in an intramolecular model, and with the assistance of a mercury compound to shift the equilibrium to the products (Chiu et al., 1996), D,L-S-methyllipoic acid methyl ester could trap the enamine derived from C2alpha-methoxybenzyl-3,4,5-trimethylthiazolium salt in an intermolecular reaction in the absence of a mercury compound, and with a rate constant of 6.6 x 104 M-1s -1. A tetrahedral adduct at the C2alpha-position formed between the enamine and D,L-S-methyllipoic acid methyl ester was. isolated and characterized. The reaction likely takes place by two-electron nucleophilic attack, since no evidence was found for C2alpha-linked homodimers, expected from a free-radical mechanism. The results suggest that, in the reductive acyl transfer, there is nucleophilic attack by the enamine probably at S8 of the sulfur atoms of the lipoic acid according to Frey and his coworkers (Frey et al., 1989), while there is concomitant electrophilic catalysis by a proton juxtaposed at S6 via a general acid catalyst located on the E1 enzyme.;The enamine derived from C2alpha-hydroxyethyl-3,4,5-trimethylthiazolium salt, which is a direct analogue of the enamine from the substrate pyruvic acid in pyruvate oxidase, was oxidized by ethyl isoalloxazinebutyrate with a second-order rate constant of 472 M-1s-1 . The two-electron transfer mechanism of the enamine oxidation proposed in the study of the C2alpha-hydroxybenzylenamine model was supported by the kinetic results from the C2alpha-hydroxyethylenamine model. Two biscoenzyme models were designed and synthesized to mimic the intramolecular oxidation of the enamine by a flavin analogue. 2-[2-(1'-Hydroxybenzyl)-3,4,5-trimethyl-thiazolo-5-)ethyl 10-methyl-isoalloxazine-7-butyrate utilized a butyrate linker to bind the thiazolium and isoalloxazine moieties, but showed no significant effect in assisting the redox reaction to go through an intramolecular pathway. 2-(10-Methylisoalloxazino)ethyl 2-[(1'-hydoxyethyl)-3,4,5-trimethyl-thiazolo-5-]ethyl 2,7-di-tert-butyl-9,9-dimethyl-4,5-xanthenedicarboxylate triflate salt has a more constrained xanthenedicarboxylate scaffold to push the thiazolium and isoalloxazine moieties close to each other. The kinetic data of the redox reaction between the second biscoenzyme model and base was analyzed by computer simulation. The xanthene-based biscoenzyme model gave a first-order rate constant of 0.338 s-1 for the intramolecular redox reaction, which is about 1200 times slower than redox step, and about 200 times slower than the turnover number of pyruvate oxidase. |
