Engineering Styrene Oxide Isomerase Towards Enhanced Chemoenzymatic Synthesis of (<i>S</i>)‐Flurbiprofen

The conventional synthesis of (<i>S</i>)‐flurbiprofen, a valuable non‐steroidal anti‐inflammatory drug (NSAID), involves inefficient chiral resolution with low‐yielding multiple reaction steps, or asymmetric synthesis with low enantioselectivity and/or regioselectivity. Here, we developed high‐yielding, enantioselective, and sustainable synthesis of (<i>S</i>)‐flurbiprofen via two chemoenzymatic approaches from a simple alkene: (A) enzymatic epoxidation‐isomerization‐oxidation cascade and Pd‐catalyzed arylation; and (B) chemical epoxidation, enzymatic isomerization‐oxidation cascade, and Pd‐catalyzed arylation. Styrene oxide isomerase (SOI)‐catalyzed enantio‐retentive Meinwald rearrangement was the key step in the two pathways, but SOI suffers from low activity. SOI was engineered via two rounds of iterative saturation mutagenesis and screening with a three‐enzyme cascade assay, generating a double‐mutant SOI_DM (N99A/D95A) with 5.5‐fold improvement in catalytic efficiency (<i>k</i>_cat/<i>K</i>_M). In Pathway A, cascade biotransformation of 3‐fluoro‐4‐bromo‐α‐methylstyrene with engineered <i>E. coli</i> strain expressing SOI_DM, styrene monooxygenase (SMO) and aldehyde dehydrogenase (ALDH), and subsequent Pd‐catalyzed arylation afforded (S)‐flurbiprofen in 98% yield and 91% <i>ee</i>. In Pathway B, chemical epoxidation (Sharpless dihydroxylation and ring‐closing), cascade conversion using an engineered <i>E. coli</i> strain expressing SOI_DM, ALDH and NADH oxidase (NOX), and Pd‐catalyzed arylation produced (<i>S</i>)‐flurbiprofen in 83% yield and 98% <i>ee</i>. Compared to conventional synthesis, the developed methods reduced reaction steps, minimized waste generation and improved enantioselectivity or overall yield.