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Stereoselective C–C bond formation catalysed by engineered carboxymethylproline synthases

Nature Chemistry volume 3, pages 365–371 (2011)Cite this article

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Abstract

The reaction of enol(ate)s with electrophiles is used extensively in organic synthesis for stereoselective C–C bond formation. Protein-based catalysts have had comparatively limited application for the stereoselective formation of C–C bonds of choice via enolate chemistry. We describe protein engineering studies on 5-carboxymethylproline synthases, members of the crotonase superfamily, aimed at enabling stereoselective C–C bond formation leading to N-heterocycles via control of trisubstituted enolate intermediates. Active site substitutions, including at the oxyanion binding site, enable the production of substituted N-heterocycles in high diastereomeric excesses via stereocontrolled enolate formation and reaction. The results reveal the potential of the ubiquitous crotonase superfamily as adaptable catalysts for the control of enolate chemistry.

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Figure 1: CarB and ThnE are crotonase superfamily enzymes catalysing enolate alkylation.
Figure 2: 1H NMR spectra for purified products of CMPS catalysis, produced under standard conditions, showing results for variants displaying a high degree of diastereoselectivity.
Figure 3: Analyses of stereoselective CMPS-catalysed reactions.
Figure 4: Crotonyl–CoA carboxylase reductase (Ccr)–CMPS coupled incubations.
Figure 5: Proposed mechanism for the CMPS-catalysed formation and reaction of enolates.

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Acknowledgements

The authors acknowledge the Biotechnology and Biological Sciences Research Council, the Ministry of Higher Education (Egypt, R.B.H.), CONACyT and FIDERH (Mexico, R.G.C.), Cancer Research UK (A.T.) and the Deutsche Akademie der Naturforscher Leopoldina (Germany, C.D.) for financial support. The ccr/pET3d construct was a kind gift from B.E. Alber at the Ohio State University.

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Author notes

  1. Christian Ducho

    Present address: Present address: Department of Chemistry, Institute of Organic and Biomolecular Chemistry, Georg-August-University Göttingen, Göttingen, Germany,

  2. J. Ruben Gomez-Castellanos and Armin Thalhammer: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK

    Refaat B. Hamed, J. Ruben Gomez-Castellanos, Armin Thalhammer, Daniel Harding, Christian Ducho, Timothy D. W. Claridge & Christopher J. Schofield

  2. Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, 71256, Egypt

    Refaat B. Hamed

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  1. Refaat B. Hamed
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Contributions

R.B.H. and D.H. identified and prepared CMPSs. R.G.C. prepared Ccr. R.B.H., D.H. and R.G.C. carried out enzyme assays and analysed the results. R.B.H. purified and ran NMR analysis of the products of enzyme catalysis. R.B.H. and T.D.W.C. analysed the NMR data. A.T. synthesized the (2S,5S,6R)-6-ethylcarbapenam methyl ester. C.D. synthesized protected L-AASA (2). R.B.H. and C.J.S. designed the study and wrote the manuscript. All authors commented on the manuscript.

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Correspondence to Christopher J. Schofield.

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The authors declare no competing financial interests.

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Hamed, R., Gomez-Castellanos, J., Thalhammer, A. et al. Stereoselective C–C bond formation catalysed by engineered carboxymethylproline synthases. Nature Chem 3, 365–371 (2011). https://doi.org/10.1038/nchem.1011

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