Abstract
To further expand the substrate range of the cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans, a library of diverse mutants was created and assayed toward a group of structurally diverse substrates. Among them, mutants T198A and M226A exhibited enhanced activity relative to wt CHAO for most (S)-enantiomers of primary amines and some secondary amines. While mutants T198I, L199I, L199F, M226I and M226T were more active than wt CHAO toward the primary amines, mutants T198F, L199T, Y321A, Y321T, Y321I and Y321F enhanced the enzyme activity toward the secondary amines. In particular, mutant Y321I displayed an enhanced catalytic efficiency toward 1-(4-methoxybenzyl)-1, 2, 3, 4, 5, 6, 7, 8-octahydroisoquinoline (13). Whereas a double mutant, Y321I/M226T, acted on (S)-N-(prop-2-yn-1-yl)-2, 3-dihydro-1H-inden-1-amine [(S)-8]. Since (R)-8 is an irreversible inhibitor of monoamine oxidase and (S)-13 is an intermediate of dextromethorphan, a cough suppressant drug, deracemizations of 8 and 13 were carried out with crude enzyme extracts of the respective mutants. This resulted in 51% and 78% isolated yields of (R)-8 and (S)-13, respectively, each with high enantiomeric excess (93% and 99% ee). The results demonstrated the application potential of the evolved CHAO mutants in drug synthesis requiring chiral secondary amines.
Highlights
Using either kinetic resolution or deracemization protocol[14]
The solved two-domain structures of cyclohexylamine oxidase (CHAO) consisting of a cofactor-binding domain and a substrate-binding domain provided a framework for the choice of amino acid residues for mutagenesis[24]
This cage-like structure in CHAO is similar to that of the human monoamine oxidase B (MAO B) formed by the side chains of Y398 and Y435 which serve to orient the amine substrates and promote catalysis[25]
Summary
Using either kinetic resolution or deracemization protocol[14]. The wild-type CHAO (wt CHAO) exhibited low or no activity toward secondary and tertiary amines. The substrate 2-methyl-1, 2, 3, 4-tetrahydroquinoline (10) was deracemized by a CHAO triple mutant (T198F/ L199S/M226F) leading to the production of (R)-2-methyl-1, 2, 3, 4-tetrahydroquinoline (THQ) with 76% yield and 98% ee. Vergne-Vaxelaire et al reported a high throughput orthogonal screening of putative nitrilases against 25 structurally diverse substrates that resulted in over one hundred new nitrilases from a genomic library of prokaryotic origin[23]. This prompted us to adopt a similar strategy to further explore the substrate specificity of CHAO. Y321I/M226T and Y321I were applied to deracemization of N-(prop-2-yn1-yl)-2, 3-dihydro-1H-inden-1-amine (8) and 1-(4-methoxybenzyl)-1, 2, 3, 4, 5, 6, 7, 8-octahydroisoquinoline (13), respectively, as a proof of concept for the synthetic potential of the biocatalysts
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