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-(4methoxybenzyl)-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 gramscale 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.

Engineered Monoamine Oxidase-Catalyzed Deracemization of Sterically Hindered Acylaminomethyl-tetrahydroisoquinoline: Asymmetric Synthesis of (R)-(−)-Praziquantel and Its Analogues

“…With the crystal structure of MAO-N-D3 (PDB ID: 2VVL) used as the template, the homology model of LJY-C-D1 was created at the SWISS-MODEL server by adopting similar protocols, as described previously …”

“…21 The mutant library screening was also performed according to the procedures previously shown by us. 21 More details regarding the mutant library construction and screening are available in the Supporting Information. Protein Expression, Preparation of Whole-Cell Catalysts, and Purification of N-Terminal-His 6 -Tagged Protein.…”

“…The PCR reactions for this mutagenesis were conducted with mutagenic primers shown in Table S4 by following our previously described procedures. 21 Then, Escherichia coli DH5α competent cells (100 μL) were thawed on ice for about 10 min, followed by the addition of 2 μL of the DpnIdigested PCR product. This mixture was incubated on ice for about 30 min, then heated for 90 s at 42 °C, and finally put on ice for 2 min.…”

“…Flavin-dependent monoamine oxidases (MAOs) are a class of enzymes naturally performing molecular oxygen-mediated oxidation of amines to produce imines . Over the past two decades, several MAOs identified from fungi and bacteria have been substantially engineered to result in enzyme variants displaying a remarkably extended substrate scope. − In particular, the Turner group has developed and applied a series of evolved MAO variants derived from MAO-N, a monoamine oxidase originating from Aspergillus niger, to the stereoselective synthesis of a wide range of chiral amines through a one-pot deracemization process, comprising a MAO-catalyzed enantioselective oxidation and a nonselective chemical reduction. ,,,− Notably, to the best of our knowledge, the application of monoamine oxidase to the synthesis of ( R )-PZQ or its synthetic intermediates has not yet been reported. Prompted by the success of MAO-catalyzed synthesis of chiral 1-alkyl-, 1-benzyl-, and 1-phenyl-tetrahydroisoquinolines (THIQs), ,,,,− we envisioned that stereoselective synthesis of ( R )-1-cyclohexanecarboxylaminomethyl-THIQ (( R )- 1a , Scheme B), a demonstrated key intermediate en route to ( R )-PZQ, , through MAO-catalyzed deracemization (Scheme B), could be a viable way leading to the stereoselective, chemoenzymatic synthesis of ( R )-PZQ.…”

“…Prompted by the success of MAO-catalyzed synthesis of chiral 1-alkyl-, 1-benzyl-, and 1-phenyl-tetrahydroisoquinolines (THIQs), ,,,,− we envisioned that stereoselective synthesis of ( R )-1-cyclohexanecarboxylaminomethyl-THIQ (( R )- 1a , Scheme B), a demonstrated key intermediate en route to ( R )-PZQ, , through MAO-catalyzed deracemization (Scheme B), could be a viable way leading to the stereoselective, chemoenzymatic synthesis of ( R )-PZQ. In continuation of our interest in applying enzymes, including MAOs, to the asymmetric synthesis of chiral pharmaceuticals, ,, herein we present our endeavors to develop such a stereoselective, sustainable synthesis of ( R )-PZQ enabled by an efficient, tailor-made MAO-N variant.…”