Cofactor Regeneration Engineering Enables the Enzymatic Synthesis of Chiral Intermediates for Erythrina Alkaloids

To address the challenges of regioselective oxidation at the C2 position and precise control of stereochemistry at the C5 position in the total synthesis of Erythrina alkaloids, a relay oxidation system comprising P450 BM3 and SjAKR was constructed. However, the distinct cofactor requirements of P450 BM3 (for NADPH) and SjAKR (for NADP⁺) posed a bottleneck for the enzymatic synthesis of chiral intermediates. To achieve cofactor regeneration in this relay oxidation system, this study systematically evaluated three cofactor regeneration strategies: self-recycling, competitive, and independent formats. Results indicated that constructing independent cofactor regeneration systems for each enzymatic step was the most efficient approach. Among the enzymes tested, BstFDH_G164M/A287G was identified as the optimal NADPH-regenerating enzyme for the P450 BM3 catalytic cycle, while SmNOX was the best NADP⁺-regenerating enzyme to support the kinetic resolution catalyzed by SjAKR. After optimizing the reaction conditions, a "one-pot" enzymatic reaction was performed. The process involved an initial hydroxylation catalyzed by the P450 BM3/BstFDH_G164M/A287G system, followed by kinetic resolution via the SjAKR/SmNOX system. This strategy afforded the chiral intermediates (2R, 5S)-3 and (5R)-4 with isolated yields of 32% and 28%, and optical purities of 99% ee and 97% ee, respectively. Through systematic optimization of cofactor regeneration, this study achieved the efficient and highly selective synthesis of chiral intermediates, providing crucial technical support for the chemoenzymatic total synthesis of Erythrina alkaloids.