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Biocatalytic reductive amination from discovery to commercial manufacturing applied to abrocitinib JAK1 inhibitor

Nature Catalysis volume 4, pages 775–782 (2021)Cite this article

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Abstract

Enzymatic reductive amination, being a direct, selective and green methodology, has attracted significant interest in a short period of time and is emerging as a powerful tool for the synthesis of chiral alkylated amines. The discovery of an increasing number of imine reductases with reductive aminase (RedAm) activity has enabled mechanistic and substrate profiling studies. However, their potential for commercial applications has not been realized. Here, we report the discovery of RedAm activity in an imine reductase enzyme for the direct reductive amination of a cyclic ketone with methylamine. We also investigate engineering the enzyme to access a cis-cyclobutyl-N-methylamine for the manufacturing of a late-stage drug candidate, Janus kinase 1 (JAK1) inhibitor abrocitinib. The engineered enzyme, SpRedAm-R3-V6, showed >200-fold improvement in performance over the wild-type enzyme and was successfully used to develop a commercial manufacturing process with 73% isolated yield at 99.5% purity and high selectivity (>99:1 cis:trans). This process has been successfully used to manufacture multi-metric tons of the amine, demonstrating the potential of RedAm technology for commercial manufacturing.

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Fig. 1: Various synthetic routes for the synthesis of amine 1.
Fig. 2: Screening data from enzyme engineering round 1 SSM libraries.
Fig. 3: Evolution of enzyme and screening conditions.
Fig. 4: Active-site view of the cofactor NADPH-bound SpRedAm-R3-V6.
Fig. 5: Performance of best variants from different rounds of enzyme engineering.
Fig. 6: Scale-up performance of engineered enzyme SpRedAm-R3-V6.

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Data availability

Additional data supporting the findings reported in this paper are available as Supplementary Information. All other data are available from the authors upon reasonable request.

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Acknowledgements

We would like to thank Pfizer leadership L. Handanyan, S. Caron, N. Thomson, J. Nelson and C. McWilliams for their support for the work and publication. We would also like to thank R. Lewis and S. France for proof-reading the manuscript.

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Authors and Affiliations

  1. Chemical Research & Development, Pfizer Worldwide Research & Development, Groton, CT, USA

    Rajesh Kumar, Michael J. Karmilowicz, Michael P. Burns, Christina G. Connor, Eric Cordi, Nga M. Do, Chad A. Lewis, Carlos A. Martinez, Emma L. McInturff, Robert Pearson, Jeremy Steflik, Anil Rane & John Weaver

  2. Pfizer Global Supply, Ringaskiddy, Ireland

    Dylan Burke, Leslie A. Clark & David Mangan

  3. Analytical Research & Development, Pfizer Worldwide Research & Development, Groton, CT, USA

    Kevin M. Doyle & Steve Hoagland

  4. Chemical Research & Development, Pfizer Worldwide Research and Development, Sandwich, UK

    Kevin Meldrum

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Contributions

R.K., M.J.K., J.S., M.P.B. and C.A.M. contributed to enzyme engineering and enzyme process development work. R.K., E.L.M., N.M.D., C.G.C. and E.C. performed process engineering and process development. D.B., L.A.C., K.M., D.M. and S.H. ran the manufacturing scale reactions. C.A.L., K.M.D., R.P. and J.W. provided analysis, sourcing support and participated in discussion. R.K., M.J.K. and J.S. wrote the paper and the other co-authors provided information and feedback.

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Correspondence to Rajesh Kumar.

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Peer review information Nature Catalysis thanks John Woodley and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Methods, Notes 1 and 2, Figs. 1–4 and Tables 1–4.

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Kumar, R., Karmilowicz, M.J., Burke, D. et al. Biocatalytic reductive amination from discovery to commercial manufacturing applied to abrocitinib JAK1 inhibitor. Nat Catal 4, 775–782 (2021). https://doi.org/10.1038/s41929-021-00671-5

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