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

(S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline ((S)-1-(4-methoxybenzyl)-OHIQ, (S)-1a) is a key synthetic intermediate in the industrial production of dextromethorphan, one of the most widely used over-the-counter antitussives. We report here a new cyclohexylamine oxidase discovered by genome mining, named CHAOCCH12-C2, was able to completely deracemize 100 mM of 1a under Turner's deracemization conditions to afford (S)-1a in 80% isolated yield and 99% ee at semi-preparative scale (0.4 mmol). When this biocatalytic reaction was up-scaled to gram scale (5.8 mmol), without reaction optimization (S)-1a was still isolated in 67% yield and 96% ee. The relatively higher kcat determined of CHAOCCH12-C2 was rationalized as one major factor rendering this enzyme capable of oxidizing 1a effectively at elevated substrate concentrations. Protein sequence alignment, analysis of our co-crystal structure of CHAOCCH12-C2 complexed with the product 1-(4-methoxybenzyl)- 3,4,5,6,7,8-hexahydroisoquinoline (1-(4-methoxybenzyl)-HHIQ, 2a), and the structure-guided mutagenesis study together indicated L295 as 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 residues F342, Y433, and FAD. The synthetic applicability of CHAOCCH12-C2 was further underscored by the stereoselective synthesis of various enantioenriched 1-benzyl-OHIQ derivatives of potential pharmaceutical importance at semi-preparative scale.