Catalysis with engineered enzymes has provided more efficient routes for the production of active pharmaceutical agents. However, the potential of biocatalysis to assist in early-stage drug discovery campaigns remains largely untapped. In this study, we have developed a biocatalytic strategy for the construction of sp³-rich polycyclic compounds via the intramolecular cyclopropanation of benzothiophenes and related heterocycles. Two carbene transferases with complementary regioisomer selectivity were evolved to catalyse the stereoselective cyclization of benzothiophene substrates bearing diazo ester groups at the C2 or C3 position of the heterocycle. The detailed mechanisms of these reactions were elucidated by a combination of crystallographic and computational analyses. Leveraging these insights, the substrate scope of one of the biocatalysts could be expanded to include previously unreactive substrates, highlighting the value of integrating evolutionary and rational strategies to develop enzymes for new-to-nature transformations. The molecular scaffolds accessed here feature a combination of three-dimensional and stereochemical complexity with ‘rule-of-three’ properties, which should make them highly valuable for fragment-based drug discovery campaigns.

工程酶催化为活性药物的生产提供了更有效的途径。然而，生物催化协助早期药物发现活动的潜力在很大程度上尚未开发。在这项研究中，我们开发了一种生物催化策略，通过苯并噻吩和相关杂环的分子内环丙烷化来构建富含sp ³的多环化合物。两种具有互补区域异构体选择性的卡宾转移酶被进化来催化在杂环的C2或C3位置带有重氮酯基团的苯并噻吩底物的立体选择性环化。通过晶体学和计算分析的结合阐明了这些反应的详细机制。利用这些见解，一种生物催化剂的底物范围可以扩大到包括以前不反应的底物，突出了整合进化和理性策略来开发用于新的自然转化的酶的价值。这里获得的分子支架具有三维和立体化学复杂性与“三规则”特性的结合，这使得它们对于基于片段的药物发现活动非常有价值。