Imine reductases catalyse the reductive amination of aldehydes or ketones with amines to produce chiral amines—a key transformation in the preparation of fine chemicals and active pharmaceutical ingredients. Although significant progress has been recently made in the field, their industrial application has not been demonstrated. Herein, we describe a wild-type imine reductase that was engineered to perform reductive amination with concomitant substrate amine resolution to give a commercially relevant manufacturing process to lysine-specific demethylase-1 inhibitor GSK2879552. Three rounds of evolution resulted in an enzyme variant showing a >38,000-fold improvement over wild type. The engineering of a more stable and active enzyme variant enabled process optimization to an economic, high quality and sustainable operating space. Using the evolved enzyme, kilogram quantities of a key intermediate to GSK2879552 were produced in 84% yield, at 99.9% purity and >99.7% enantiomeric excess, with improved process mass intensity.
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Methods, synthesis of starting materials, enzyme identification, backbone selection and fermentation, directed evolution, selected scale-up examples, redox-neutral enzymatic cascade conversion of 2 to (1R,2S)-4, sequence listing, UPLC analysis and selected chromatograms, NMR spectra, Supplementary Tables and Supplementary Figures are available in the Supplementary Information. All other data are available from the corresponding author upon reasonable request.
Scikit-learn was used to spatially cluster the beneficial mutations from evolution round one. DBSCAN clustering script is given in the Supplementary Information. CodeEvolver protein engineering technology platform software (for example, Harvester, MOSAIC) can be in-licensed from Codexis, Inc.
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The authors thank A. Ochen for the preparation of aldehyde 3 and T. Kubowicz for help with achiral and chiral high-performance liquid chromatography HPLC method development. The authors also thank A. Dann for enzyme fermentation trials, C. Boudet and R. Dean for downstream processing, K. Honicker for enzyme outsource and A. Roberts for help with spectra characterization. We thank P.W. Sutton, R. Snajdrova, P. Lyn Tey, M. Woods and K. Ruffell for valuable discussions and initial scoping experiments. Thanks to S. Fenner and K. Ruffell for calculation of the green metrics.
Johnson, N. W. and Kasparec, J. have filed a patent application (US patent application no. 8853408B2), which is the current assignee GlaxoSmithKline Intellectual Property (No. 2) Ltd, published on 4 October, 2014. Its application status is active, and it discloses the structure of the active pharmaceutical ingredient GSK2879552. Also, one or more authors hold shares at GlaxoSmithKline plc.
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Schober, M., MacDermaid, C., Ollis, A.A. et al. Chiral synthesis of LSD1 inhibitor GSK2879552 enabled by directed evolution of an imine reductase. Nat Catal 2, 909–915 (2019). https://doi.org/10.1038/s41929-019-0341-4
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