An Enzymatic Platform for Aniline Synthesis Through Oxidative Amination

Aniline motifs are commonly found in natural products and synthetic molecules. While chemists have developed numerous methods for constructing C(sp²)─N bonds, their biocatalytic counterparts in nature are primarily limited to P450-based protein machineries. To address this limitation, we developed a biocatalytic platform for aniline synthesis based on oxidative amination of cyclohexanones. Through directed evolution of a flavin-dependent enzyme PtOYE, we identified several protein catalysts (e.g., OYE_G3 and OYE_M3) that exhibited activity across a broad array of substrates, enabling the preparation of 40 different secondary and tertiary anilines with various substitution patterns in up to 91% GC conversion. Mechanistic investigations revealed the improved kinetic performance of the evolved variants on the desaturation of imines. Additionally, mutations introduced through protein engineering further reduced the propensity for phenol formation. This enzymatic platform represents a highly promising application of flavin-dependent enzymes, showcasing their great potential in organic synthesis and drug development.