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

Nature Catalysis volume 2, pages 909–915 (2019)Cite this article

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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

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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.

References

  1. Breuer, M. et al. Industrial methods for the production of optically active intermediates. Angew. Chem. Int. Ed. 43, 788–824 (2004).

    Article  CAS  Google Scholar 

  2. Nugent, T. C. Chiral Amine Synthesis: Methods, Developments and Applications (Wiley, 2010).

  3. Mutti, F. G., Knaus, T., Scrutton, N. S., Breuer, M. & Turner, N. J. Conversion of alcohols to enantiopure amines through dual-enzyme hydrogen-borrowing cascades. Science 349, 1525 (2015).

    Article  CAS  Google Scholar 

  4. Chen, H. C. et al. Engineered imine reductases and methods for the reductive amination of ketone and amine compounds. US patent US20130302859 A1 (2013).

  5. Abrahamson, M. J., Vázquez-Figueroa, E., Woodall, N. B., Moore, J. C. & Bommarius, A. S. Development of an amine dehydrogenase for synthesis of chiral amines. Angew. Chem. Int. Ed. 51, 3969–3972 (2012).

    Article  CAS  Google Scholar 

  6. Ghislieri, D. & Turner, N. J. Biocatalytic approaches to the synthesis of enantiomerically pure chiral amines. Top. Catal. 57, 284–300 (2014).

    Article  CAS  Google Scholar 

  7. Grogan, G. Synthesis of chiral amines using redox biocatalysis. Curr. Opin. Chem. Biol. 43, 15–22 (2018).

    Article  CAS  Google Scholar 

  8. Höhne, M. & Bornscheuer, U. T. Biocatalytic routes to optically active amines. Chem. Cat. Chem. 1, 42–51 (2009).

    Google Scholar 

  9. Savile, C. K. et al. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329, 305–309 (2010).

    Article  CAS  Google Scholar 

  10. Mayol, O. et al. A family of native amine dehydrogenases for the asymmetric reductive amination of ketones. Nat. Catal. 2, 324–333 (2019).

    Article  CAS  Google Scholar 

  11. Matzel, P., Krautschick, L. & Höhne, M. Photometric characterization of the reductive amination scope of the imine reductases from streptomyces tsukubaensis and streptomyces ipomoeae. Chem. Bio. Chem. 18, 2022–2027 (2017).

    Article  CAS  Google Scholar 

  12. Lenz, M., Borlinghaus, N., Weinmann, L. & Nestl, B. M. Recent advances in imine reductase-catalyzed reactions. World J. Microb. Biot. 33, 199 (2017).

    Article  Google Scholar 

  13. Schrittwieser, J. H., Velikogne, S. & Kroutil, W. Biocatalytic imine reduction and reductive amination of ketones. Adv. Synth. Catal. 357, 1655–1685 (2015).

    Article  CAS  Google Scholar 

  14. Aleku, G. A. et al. A reductive aminase from Aspergillus oryzae. Nat. Chem. 9, 961 (2017).

    Article  CAS  Google Scholar 

  15. Roiban, G.-D. et al. Efficient biocatalytic reductive aminations by extending the imine reductase toolbox. Chem. Cat. Chem. 9, 4475–4479 (2017).

    CAS  Google Scholar 

  16. France, S. P. et al. Identification of novel bacterial members of the imine reductase enzyme family that perform reductive amination. Chem. Cat. Chem. 10, 510–514 (2018).

    CAS  Google Scholar 

  17. Wetzl, D. et al. Expanding the imine reductase toolbox by exploring the bacterial protein-sequence space. Chem. Bio. Chem. 16, 1749–1756 (2015).

    Article  CAS  Google Scholar 

  18. Gröger, H., Zumbrägel, N., Wetzl, D. & Iding, H. Asymmetric biocatalytic reduction of cyclic imines: design and application of a tailor-made whole-cell catalyst. Heterocycles 95, 1261–1271 (2017).

    Article  Google Scholar 

  19. Zumbragel, N., Merten, C., Huber, S. M. & Groger, H. Enantioselective reduction of sulfur-containing cyclic imines through biocatalysis. Nat. Commun. 9, 1949 (2018).

    Article  Google Scholar 

  20. Velikogne, S., Resch, V., Dertnig, C., Schrittwieser, J. H. & Kroutil, W. Sequence-based in-silico discovery, characterisation, and biocatalytic application of a set of imine reductases. Chem. Cat. Chem. 10, 3236–3246 (2018).

    CAS  PubMed  Google Scholar 

  21. Johnson, N. W., Kasparec, J. Cycloproplylamines as LSD1 inhibitors. US patent 8853408 B2 (2014).

  22. Alder, C. M. et al. Updating and further expanding GSK’s solvent sustainability guide. Green. Chem. 18, 3879–3890 (2016).

    Article  CAS  Google Scholar 

  23. Matzel, P., Gand, M. & Höhne, M. One-step asymmetric synthesis of (R)- and (S)-rasagiline by reductive amination applying imine reductases. Green. Chem. 19, 385–389 (2017).

    Article  CAS  Google Scholar 

  24. Scheller, P. N., Lenz, M., Hammer, S. C., Hauer, B. & Nestl, B. M. Imine reductase-catalyzed intermolecular reductive amination of aldehydes and ketones. Chem. Cat. Chem. 7, 3239–3242 (2015).

    CAS  Google Scholar 

  25. Li, H., Luan, Z.-J., Zheng, G.-W. & Xu, J.-H. Efficient synthesis of chiral indolines using an imine reductase from Paenibacillus lactis. Adv. Synth. Catal. 357, 1692–1696 (2015).

    Article  CAS  Google Scholar 

  26. Aleku, G. A. et al. Stereoselectivity and structural characterization of an imine reductase (IRED) from Amycolatopsis orientalis. ACS Catal. 6, 3880–3889 (2016).

    Article  CAS  Google Scholar 

  27. Cope, G. A. Methods, systems, and software for identifying bio-molecules with interacting components. US patent 9665694 B2 (2017).

  28. Jimenez-Gonzalez, C., Ponder, C. S., Broxterman, Q. B. & Manley, J. B. Using the right green yardstick: why process mass intensity is used in the pharmaceutical industry to drive more sustainable processes. Org. Proc. Res. Dev. 15, 912–917 (2011).

    Article  CAS  Google Scholar 

  29. Montgomery, S. L. et al. Direct alkylation of amines with primary and secondary alcohols through biocatalytic hydrogen borrowing. Angew. Chem. Int. Ed. 56, 10491–10494 (2017).

    Article  CAS  Google Scholar 

Download references

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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Author notes

  1. These authors contributed equally: Markus Schober, Chris MacDermaid.

Authors and Affiliations

  1. Synthetic Biochemistry, Medicinal Science & Technology, GlaxoSmithKline Medicines Research Centre, Stevenage, UK

    Markus Schober, Joseph Hosford, Jonathan Latham, Murray J. B. Brown & Gheorghe-Doru Roiban

  2. Molecular Design, Computational and Modelling Sciences, GlaxoSmithKline, Collegeville, PA, USA

    Chris MacDermaid

  3. Synthetic Biochemistry, Medicinal Science & Technology, GlaxoSmithKline, Collegeville, PA, USA

    Anne A. Ollis, Sandy Chang, Leigh Anne F. Ihnken & Douglas Fuerst

  4. Chemical Development, Medicinal Science & Technology, GlaxoSmithKline, Medicines Research Centre, Stevenage, UK

    Diluar Khan & Mahesh J. Sanganee

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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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