Synthesis of gem-dideuterated tetradecanoic acids and their use in investigating the enzymatic transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid

We report the preparation of the deuterated tetradecanoic acids [2,2,3,3-H-2(4)]-, [2,2,3,3,10,10-H-2(6)]-, and [2,2,3,3,13,13-H-2(6)]-tetradecanoic acids (1, 2, and 3, respectively) and their use to investigate the mechanism of the enzymatic transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid. Probes 2 and 3 were prepared from intermediate ketones 7 and 10, which were transformed into the labeled bromides 17 and 18 by reduction with NaBD4, tosylation of the resulting alcohol, replacement of the tosyloxy group by deuteride with LiAlD4, hydrolysis, and reaction with N-bromosuccinimide. The resulting bromides were converted into the alpha -acetylenic esters 21 and 22, respectively, and the additional deuterium labels were introduced by reduction of the conjugated triple bond with Mg in deuterated methanol. The same sequence of reactions starting with 11-bromoundecane afforded 27. Saponification of the labeled esters 23, 24, and 27 gave the deuterated acids 2, 3, and 1, respectively. The results of the biochemical experiments showed that C10-H removal, but not elimination of C13-H, was sensitive to deuterium substitution in the transformation of (Z)-11-tetradecenoic acid into (E,E)-10,12-tetradecadienoic acid, which is consistent with the hypothesis that this desaturase reaction involves a first slow, C10-H bond cleavage, with probable formation of an unstable allylic intermediate, followed by a second fast C13-H bond removal and concomitant rearrangement.