Construction of Chiral Tricyclic Structures Using Whole‐Cell Biocatalysts: A Strategy via the Cyclopropanation of Unsaturated π‐Systems

Comprehensive Summary Chiral tricyclic compounds represent a significant class of biologically active substances with wide‐ranging applications in materials science, medicinal chemistry, as well as the food and fragrance industries. Here, we combine CAST (combinatorial active site saturation test) and bacterial surface display technology to develop a laboratory‐evolved whole‐cell catalyst, designated SD‐VHb Tric (surface display of vitreoscilla hemoglobin). This novel carbene‐transferase is based on the engineered VHb and employs a strategy for the cyclopropanation of unsaturated π‐systems to synthesize various chiral tricyclic structures. SD‐VHb Tric exhibits unparalleled stereocontrol (up to 99.9% de and 99% ee ) and good reactivity, enabling the synthesis of chiral tricyclic structures with diverse aromatic or heterocyclic ring scaffolds from simple starting materials. Additionally, computational studies were conducted to elucidate the crucial role of intermolecular hydrophobic interactions in regulating the reaction, while also demonstrating the significant impact of adaptive changes in the active pocket size on the orientation of the ligand.