Abstract

Isotopic replacement has long-proven applications in small molecules. However, applications in proteins are largely limited to biosynthetic strategies or exchangeable (for example, N–H/D) labile sites only. The development of postbiosynthetic, C–1H → C–2H/D replacement in proteins could enable probing of mechanisms, among other uses. Here we describe a chemical method for selective protein α-carbon deuteration (proceeding from Cys to dehydroalanine (Dha) to deutero-Cys) allowing overall 1H→2H/D exchange at a nonexchangeable backbone site. It is used here to probe mechanisms of reactions used in protein bioconjugation. This analysis suggests, together with quantum mechanical calculations, stepwise deprotonations via on-protein carbanions and unexpected sulfonium ylides in the conversion of Cys to Dha, consistent with a ‘carba-Swern’ mechanism. The ready application on existing, intact protein constructs (without specialized culture or genetic methods) suggests this C–D labeling strategy as a possible tool in protein mechanism, structure, biotechnology and medicine.

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Data availability

All key MS data supporting figures are given in the Supplementary Information, and all related raw data are available on request. All primary numerical data for graphical plots in figures will be deposited as spreadsheets in the Oxford open access depository ‘ORA-data’; https://doi.org/10.5287/bodleian:NG0gbEEzP.

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Acknowledgements

We thank BBSRC/AstraZeneca (S.G.), EU Horizon 2020 program under the Marie Sklodowska-Curie (700124, J.D.), the EPSRC Centres for Doctoral Training in Theory and Modelling in Chemical Sciences (EP/L015722/1) and in Synthesis for Biology and Medicine (EP/ L015838/1), Leverhulme Trust (RPG-2017-288, A/N: 176274, V.C.), A*STAR Singapore (X.Z.) and the Croucher Foundation (W.-L.N.) for funding. We thank G. Karunanithy, S. Nadal, R. Raj, R. Nathani, J. Willwacher, G. Pairaudeau, J. Read, A. Breeze and A. Baldwin for useful discussions.

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Affiliations

  1. Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UK

    • Sébastien R. G. Galan
    • , James R. Wickens
    • , Jitka Dadova
    • , Wai-Lung Ng
    • , Xinglong Zhang
    • , Robert A. Simion
    • , Robert Quinlan
    • , Elisabete Pires
    • , Robert S. Paton
    •  & Benjamin G. Davis
  2. Department of Chemistry, Colorado State University, Fort Collins, CO, USA

    • Robert S. Paton
  3. Department of Chemistry, University College London, London, UK

    • Stephen Caddick
    •  & Vijay Chudasama

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Contributions

S.R.G.G., J.D., W.-L.N., V.C. and R.Q. conducted chemical experiments; X.Z., R.A.S. and R.S.P. conducted computational experiments; S.R.G.G., J.R.W. and E.P. conducted mass-spectrometric experiments; S.R.G.G., J.R.W., R.S.P., S.C., V.C. and B.G.D. designed the experiments and analyzed the data; S.R.G.G., V.C. and B.G.D. wrote the paper; all authors read and commented on the paper.

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The authors declare no competing interests.

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Correspondence to Vijay Chudasama or Benjamin G. Davis.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Tables 1–3, Supplementary Figures 1–31

  2. Reporting Summary

  3. Supplementary Note 1

    Synthetic procedures and small molecule reactions

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https://doi.org/10.1038/s41589-018-0128-y