Lactam rings are found in many biologically active natural products and pharmaceuticals, including important classes of antibiotics. Methods for the asymmetric synthesis of these molecules are therefore highly desirable, particularly through the selective functionalization of unreactive aliphatic C-H bonds. Here we show the development of a strategy for the asymmetric synthesis of β-, γ-, and δ-lactams via hemoprotein-catalysed intramolecular C-H amidation reaction with readily available dioxazolone reagents. Engineered myoglobin variants serve as excellent biocatalysts for this transformation yielding the desired lactam products in high yields, high enantioselectivity, and on preparative scale. Mechanistic and computational studies elucidate the nature of the C-H amination and enantiodetermining steps and provide insights into protein-mediated control of regioselectivity and stereoselectivity. Additionally, an alkaloid natural product and a drug molecule were synthesized chemoenzymatically in much fewer steps (7-8 vs. 11-12) than previously reported, further demonstrating the power of biosynthetic strategy for the preparation of complex bioactive molecules.

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.