Enhancing the asymmetric reduction activity of ene-reductases for the synthesis of a brivaracetam precursor

Brivaracetam is an effective third-generation antiepileptic drug, and its two chiral centers are a major challenge for designing an inexpensive large-scale synthesis process. Enzymatic asymmetric reduction offers an alternative biotransformation method for reducing 2-(5H)-furanone-4-propyl (3a) to (R)-4-propyldihydrofuran-2(3 H)-one (3b), which is a key intermediate in synthesizing brivaracetam. However, no enzymes have been reported to catalyze this reaction with both high activity and stereoselectivity. In this study, fourteen ene-reductases were compared, and the old yellow enzyme OYE2 was identified as having strict stereoselectivity but unsatisfactory activity. Through evaluations with structurally similar substrates and alanine-screening methods, two key residues were identified, T37 and Y82, playing critical roles in catalytic activity. The hydrogenation activity of mutants Y82W and T37C increased by 2.97 and 7.13 times, respectively. Molecular dynamics simulation indicates that mutant Y82W might shorten the distance between the electron acceptor and donor, while T37A might decrease the redox potential of FMN and then accelerate hydrogen transfer. These results indicate that tuning the redox potential properties of FMN is an important consideration for engineering FMN-dependent oxidoreductases. During the gram-scale synthesis of 3b, T37C exhibited excellent stereoselectivity (>99 % ee) and conversion (>99 %), laying a foundation for a simple one-step route in producing the chiral precursor brivaracetam.