Diverse N ‐Oxidation of Primary Aromatic Amines Controlled by Engineered P450 Peroxizyme Variants Facilitated by Dual‐Functional Small Molecule

Abstract Amine oxidation is an important organic reaction for the production of high‐value N ‐containing compounds. However, it is still challenging to control the reactivity of active N ‐centered radicals to selectively access N ‐oxidation products. Herein, this study reports the engineering of cytochrome P450BM3 into multifunctional N ‐oxidizing enzymes with the assistance of dual‐functional small molecules (DFSM) to selectively produce N ‐oxygenation (i.e., p ‐nitrosobenzene, p ‐nitrobenzene, and azoxybenzene) and one‐electron oxidation products (i.e., oligomeric quinones and azobenzene) from aromatic amines. The best mutant, F87A/T268V/V78T/A82T, exclusively gives p ‐nitrosobenzene (up to 98% selectivity), whereas the selectivity for p ‐nitrobenzene is >99% using the mutant F87A/T268V/A82T/I263L. Crystal structure analysis reveals that key mutations and DFSM exert synergistic effects on catalytic promiscuity by controlling the substrate orientation in active center. This study highlights the potential of DFSM‐facilitated P450 peroxygenase and peroxidase for the synthesis of N ‐containing compounds via the controllable oxidation of aromatic amines, substantially expanding the chemical space of P450 enzymes.