Asymmetric Synthesis of a Key Dextromethorphan Intermediate and Its Analogues Enabled by a New Cyclohexylamine Oxidase: Enzyme Discovery, Reaction Development, and Mechanistic Insight.

()-1-(4-Methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline [()-1-(4-methoxybenzyl)-OHIQ, ()-] is a key synthetic intermediate in the industrial production of dextromethorphan, one of the most widely used over-the-counter antitussives. We report here that a new cyclohexylamine oxidase discovered by genome mining, named CHAO, was able to completely deracemize 100 mM under Turner's deracemization conditions to afford ()- in 80% isolated yield and 99% ee at a semipreparative scale (0.4 mmol). When this biocatalytic reaction was scaled up to a gram scale (5.8 mmol), without reaction optimization ()- was still isolated in 67% yield and 96% ee. The relatively higher determined for CHAO was rationalized as one major factor rendering this enzyme capable of oxidizing effectively at elevated substrate concentrations. Protein sequence alignment, analysis of our co-crystal structure of CHAO complexed with the product 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline [1-(4-methoxybenzyl)-HHIQ, ], and the structure-guided mutagenesis study together indicated L295 is one of the critical residues for this efficient enzymatic oxidation process and supported the presence of two cavities as well as a catalytically important "aromatic cage" formed by F342, Y433, and FAD. The synthetic applicability of CHAO was further underscored by the stereoselective synthesis of various enantioenriched 1-benzyl-OHIQ derivatives of potential pharmaceutical importance at a semipreparative scale.