One-pot chemo-enzymatic cascades using ene-reductase and alcohol dehydrogenase to produce a brivaracetam chiral precursor

Abstract

Brivaracetam is a third-generation antiepileptic drug containing two chiral centers (2S, 4 R). To achieve effective asymmetric synthesis of 4 R chiral center, a novel one-pot chemo-enzymatic cascade strategy was developed to synthesize the 4 R chiral precursor, (R)-4-propyldihydrofuran-2(3H)-one (1b). The butenolide 5-hydroxy-4-propylfuran-2(5H)-one was selected as the starting substrate due to the presence of an active electron-withdrawing group, which enabled rapid enzymatic reduction by an ene-reductase with > 99 % conversion. However,‌ this reaction generated ‌a racemization-prone intermediate‌. Subsequent reduction ‌using a newly identified alcohol dehydrogenase (YahK)‌ demonstrated high activity but ‌exhibited S-configuration stereoselectivity‌. ‌ Through semi-rational engineering, the stereoselectivity of a YahK triple mutant (G132T/T182A/M313R) was successfully reversed to favor the R-configuration. The chemo-enzymatic cascade combining ADH reduction and chemical cyclization yielded the chiral product 4R-1b with an enantiomeric excess (ee) of > 91 % (R). Notably, the one-pot mode further enhanced the ee value of the final product to > 98 %. This strategy effectively bypassed the limitations of low-reactivity alkene substrates, enabling efficient construction of the 4 R chiral center in brivaracetam.