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Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines

Nature Chemistry volume 8, pages 1076–1082 (2016)Cite this article

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Abstract

The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering.

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Figure 1: Schematic representation of the strategy pursued for the identification of novel TAs active towards bulky substrates.
Figure 2: Selected compounds of interest for the present study.
Figure 3: Quinonoid of compounds 1a and 2a accommodated in the active site of 3FCR.

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Acknowledgements

The authors thank J.F. Kabisch for preparing the M5nr database, M. Althaus and I. Duffour for developing the chiral and achiral analysis methods, J. Joerger and M. Rothe for the preparative separation of the chiral amines, C. Wyss-Gramberg for the NMR analysis of the Mosher amides, I. Menyes for support with HPLC and gas chromatography analyses and P. Meier for performing the preparative asymmetric synthesis experiments.

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Authors and Affiliations

  1. Department of Biotechnology, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Str. 4, Greifswald, D-17489, Germany

    Ioannis V. Pavlidis, Martin S. Weiß, Maika Genz & Uwe T. Bornscheuer

  2. Department of Biochemistry, University of Kassel, Heinrich-Plett-Str. 40, Kassel, D-34132, Germany

    Ioannis V. Pavlidis

  3. Process Research and Development, Biocatalysis, F. Hoffmann-LaRoche Ltd, Grenzacher Str. 124, Basel, 4070, Switzerland

    Paul Spurr, Steven P. Hanlon, Beat Wirz & Hans Iding

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  1. Ioannis V. Pavlidis
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Contributions

U.T.B., H.I. and B.W. initiated the study and directed the project. P.S. undertook the substrate and product syntheses. I.V.P. performed the bioinformatics analysis. I.V.P., M.S.W. and M.G. prepared and characterized all the variants. I.V.P, S.P.H. and H.I. performed the preparative asymmetric synthesis experiments. I.V.P., H.I. and U.T.B. prepared the manuscript, which was revised and approved by all authors.

Corresponding authors

Correspondence to Hans Iding or Uwe T. Bornscheuer.

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Competing interests

The biocatalysis group of Roche has a committed interest over the long term in establishing a set of technically applicable TAs with broad substrate acceptance to assist devising more attractive, shorter, economical and greener synthetic routes to investigational drugs and beyond.

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Pavlidis, I., Weiß, M., Genz, M. et al. Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines. Nature Chem 8, 1076–1082 (2016). https://doi.org/10.1038/nchem.2578

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