Engineered Cyclohexylamine Oxidase with Improved Activity and Stereoselectivity for Asymmetric Synthesis of a Bulky Dextromethorphan Precursor and Its Analogues

Abstract Cyclohexylamine oxidase CHAO CCH12‐C2 was previously discovered and utilized in the chemo‐enzymatic synthesis of ( S )‐1‐(4‐methoxybenzyl)‐1,2,3,4,5,6,7,8‐octahydroisoquinoline (( S )‐ 1 a ), the key precursor for the industrial production of antitussive dextromethorphan. Herein, structure‐guided semi‐rational engineering and random mutagenesis were applied to CHAO CCH12‐C2 , resulting in an evolved variant WXF‐FM which possessed five point mutations: H68Q/E198G/L200V/I201L/V209S and displayed >15‐fold higher k cat and improved stereoselectivity towards ( R )‐ 1 a relative to the WT enzyme. WXF‐FM‐catalyzed deracemization of 200 mM of rac ‐ 1 a was achieved at gram‐scale under Turner's deracemization conditions, affording ( S )‐ 1 a in 76 % isolated yield with 97 % ee, demonstrating the effectiveness and great potential of this enzyme in the practical, green synthesis of dextromethorphan. Two bulky analogues of ( S )‐ 1 a were also afforded with much higher optical purities in WXF‐FM‐catalyzed reactions than those obtained in WT enzyme‐catalyzed reactions. Through conducting complete deconvoluting experiments, presence of strong cooperative effect was revealed, and the mutational effect of H68Q on ( R )‐ 1 a was suggested to be possibly applicable to related cyclohexylamine oxidases. Molecular dynamics (MD) simulations indicated the enlargement of binding and entrance cavities, and the formation of a new hydrogen‐bonding between FAD and ( R )‐ 1 a both likely contributed to the enhanced catalytic activity.