Biocatalytic Steroidal 9α-Hydroxylation and Fragmentation Enable the Concise Chemoenzymatic Synthesis of 9,10-Secosteroids.

9,10-Secosteroids are an important group of marine steroids with diverse biological activities. Herein, we report a chemoenzymatic strategy for the concise, modular, and scalable synthesis of ten naturally occurring 9,10-secosteroids from readily available steroids in three to eight steps. The key feature lies in utilizing a Rieske oxygenase-like 3-ketosteroid 9α-hydroxylase (KSH) as the biocatalyst to achieve efficient C9-C10 bond cleavage and A-ring aromatization of tetracyclic steroids through 9α-hydroxylation and fragmentation. With synthesized 9,10-secosteroides, structure-activity relationship was evaluated based on bioassays in terms of previously unexplored anti-infective activity. This study provides experimental evidence to support the hypothesis that the biosynthetic pathway through which 9,10-secosteroids are formed in nature shares a similar 9α-hydroxylation and fragmentation cascade. In addition to the development of a biomimetic approach for 9,10-secosteroid synthesis, this study highlights the great potential of chemoenzymatic strategies in chemical synthesis.

Engineering of an ene-reductase for producing the key intermediate of antiepileptic drug Brivaracetam.

(R)-4-Propyldihydrofuran-2(3H)-one (R-PDFO) is the key chiral intermediate for the antiepileptic drug Brivaracetam. Lacking a simple and economical method to approaching R-PDFO, the production of R-PDFO also remains environmentally unfriendly. Here, we developed a straightforward bioreduction way from easily synthesized 4-propylfuran-2(5H)-one (PFO) using ene-reductases. After screened with 27 ene-reductases, E116 stood out with 25.7% yield and 97% ee (R) as the starting enzyme. To improve the catalytic efficiency of E116, several rounds of directed evolution were first carried out. Through rational design, alanine scanning and random mutagenesis, engineered ene-reductase E116-M3 was obtained, with a 2.63-fold improvement in yields over WT, a 12.6-fold improvement in kcat/Km over WT, and stereoselectivity increased to 99% (R). To further improve the yield of R-PDFO, the reaction conditions were then optimized. The catalytic activity of the optimized reaction system was increased again by 2.3 times and the turnover number (TON) of E116-M3 reached 705. Subsequently, whole cells harboring E116-M3 were also shown to have similar capabilities of synthesizing R-PDFO. Finally, E116-M3 was employed in the 50-mL-scale synthesis of R-PDFO under 20 mM of PFO loading to achieve 81% isolated yield and 99% ee. In conclusion, this new approach of engineered ene-reductase catalyzing the asymmetric reduction of PFO could be a green alternative for the efficient synthesis of R-PDFO. KEY POINTS: • An ene-reductase library was first used to screen the bioreduction of PFO. • Rational design contributed to the enhanced R-stereoselectivity of PFO reduction. • E116-M3 was obtained with high activity and stereoselectivity for R-PDFO.