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Chiral synthesis of LSD1 inhibitor GSK2879552 enabled by directed evolution of an imine reductase

Abstract

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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Fig. 1: Manufacturing route to GSK2879552.
Fig. 2: Key stages in the evolution of IR-46 to optimal variant M3.
Fig. 3: Homology model of IR-46 highlighting mutations incorporated throughout evolution rounds.
Fig. 4: Progress towards the desired operating conditions and improved green metrics of new route.
Fig. 5

Data availability

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.

Code availability

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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Acknowledgements

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.

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Contributions

M.S., C.M., A.A.O., S.C., D.K., J.H., J.L., M.J.B.B., M.J.S. and G.-D.R. performed the experimental work and analysed the results. D.K., M.J.S. and J.H. carried out enzyme process development work and performed the scale-up reactions. M.S., C.M., A.A.O., S.C., G.-D.R., D.F. and L.A.F.I. participated in the planning, designing and analysis of the directed evolution experiments. All authors co-wrote the manuscript.

Corresponding author

Correspondence to Gheorghe-Doru Roiban.

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Competing interests

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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Supplementary Information

Supplementary Information

Supplementary Methods, Tables 1–17, Figs. 1–14 and references.

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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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