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.

中文翻译：

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工程化苯乙烯氧化物异构酶以增强 (S)-氟比洛芬的化学酶法合成


(<i>S</i>)-氟比洛芬（一种有价值的非甾体抗炎药 (NSAID)）的常规合成涉及低效的手性拆分和低产率的多个反应步骤，或者具有低对映选择性和/或区域选择性的不对称合成。在这里，我们通过两种化学酶法从简单的烯烃中开发了高产、对映选择性和可持续的 (<i>S</i>)-氟比洛芬合成：(A) 酶促环氧化-异构化-氧化级联和Pd催化的芳基化； (B) 化学环氧化、酶促异构化-氧化级联和 Pd 催化芳基化。苯乙烯氧化物异构酶 (SOI) 催化的对映保留 Meinwald 重排是这两条途径的关键步骤，但 SOI 的活性较低。 SOI 通过两轮迭代饱和诱变和三酶级联分析筛选进行工程设计，生成双突变体 SOI_DM (N99A/D95A)，催化效率提高 5.5 倍（<b1005 ></b1005>k</i>_猫/<i>K</i>_M）。在途径 A 中，3-氟-4-溴-α-甲基苯乙烯与工程 <i>E 进行级联生物转化。表达 SOI_DM、苯乙烯单加氧酶 (SMO) 和乙醛脱氢酶 (ALDH) 的大肠杆菌</i>菌株，随后 Pd 催化的芳基化得到 (S)-氟比洛芬，产率 98%，产率 91 ％<i>ee</i>。在途径 B 中，化学环氧化（Sharpless 二羟基化和闭环），使用工程 <i>E 进行级联转化。表达 SOI_DM、ALDH 和 NADH 氧化酶 (NOX) 的大肠杆菌</i>菌株，并产生 Pd 催化的芳基化 (<i>S</i> )-氟比洛芬，产率为 83%，<i>ee</i> 为 98%。与传统合成相比，所开发的方法减少了反应步骤，最大限度地减少了废物的产生，并提高了对映选择性或总产率。