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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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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  1. 1.

    Westheimer, F. H. The magnitude of the primary kinetic isotope effect for compounds of hydrogen and deuterium. Chem. Rev. 61, 265–273 (1961).

  2. 2.

    Cook, P. F. & Cleland, W. W. Mechanistic deductions from isotope effects in multireactant enzyme mechanisms. Biochemistry 20, 1790–1796 (1981).

  3. 3.

    LeMaster, D. M. Uniform and selective deuteration in two-dimensional NMR of proteins. Annu. Rev. Biophys. Biophys. Chem. 19, 243–266 (1990).

  4. 4.

    Groff, D., Thielges, M. C., Cellitti, S., Schultz, P. G. & Romesberg, F. E. Efforts toward the direct experimental characterization of enzyme microenvironments: tyrosine100 in dihydrofolate reductase. Angew. Chem. Int. Ed. Engl. 48, 3478–3481 (2009).

  5. 5.

    Adhikary, R., Zimmermann, J., Dawson, P. E. & Romesberg, F. E. IR probes of protein microenvironments: utility and potential for perturbation. Chemphyschem 15, 849–853 (2014).

  6. 6.

    Liu, D., Xu, R. & Cowburn, D. Segmental isotopic labeling of proteins for nuclear magnetic resonance. Methods Enzymol. 462, 151–175 (2009).

  7. 7.

    Cremeens, M. E., Zimmermann, J., Yu, W., Dawson, P. E. & Romesberg, F. E. Direct observation of structural heterogeneity in a beta-sheet. J. Am. Chem. Soc. 131, 5726–5727 (2009).

  8. 8.

    Markley, J. L., Putter, I. & Jardetzky, O. High-resolution nuclear magnetic resonance spectra of selectively deuterated staphylococcal nuclease. Science 161, 1249–1251 (1968).

  9. 9.

    Feeney, J. et al. 1H nuclear magnetic resonance studies of the tyrosine residues of selectively deuterated Lactobacillus casei dihydrofolate reductase. Proc. R. Soc. Lond. B. Biol. Sci. 196, 267–290 (1977).

  10. 10.

    Feeney, J., Birdsall, B., Ostler, G., Carr, M. D. & Kairi, M. A novel method of preparing totally alpha-deuterated amino acids for selective incorporation into proteins. Application to assignment of 1H resonances of valine residues in dihydrofolate reductase. FEBS Lett. 272, 197–199 (1990).

  11. 11.

    Chin, J. K., Jimenez, R. & Romesberg, F. E. Direct observation of protein vibrations by selective incorporation of spectroscopically observable carbon-deuterium bonds in cytochrome c. J. Am. Chem. Soc. 123, 2426–2427 (2001).

  12. 12.

    Xia, B., Jenk, D., LeMaster, D. M., Westler, W. M. & Markley, J. L. Electron-nuclear interactions in two prototypical [2Fe-2S] proteins: selective (chiral) deuteration and analysis of 1H and 2H NMR signals from the alpha and beta hydrogens of cysteinyl residues that ligate the iron in the active sites of human ferredoxin and Anabaena 7120 vegetative ferredoxin. Arch. Biochem. Biophys. 373, 328–334 (2000).

  13. 13.

    Markley, J. L. Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. I. Reinvestigation of the histidine peak assignments. Biochemistry 14, 3546–3554 (1975).

  14. 14.

    Ortega, M. A. & van der Donk, W. A. New insights into the biosynthetic logic of ribosomally synthesized and post-translationally modified peptide natural products. Cell Chem. Biol. 23, 31–44 (2016).

  15. 15.

    Marchand, D. H., Remmel, R. P. & Abdel-Monem, M. M. Biliary excretion of a glutathione conjugate of busulfan and 1,4-diiodobutane in the rat. Drug Metab. Dispos. 16, 85–92 (1988).

  16. 16.

    Wang, J., Schiller, S. M. & Schultz, P. G. A biosynthetic route to dehydroalanine-containing proteins. Angew. Chem. Int. Ed. Engl. 46, 6849–6851 (2007).

  17. 17.

    Chalker, J. M. et al. Methods for converting cysteine to dehydroalanine on peptides and proteins. Chem. Sci. 2, 1666–1676 (2011).

  18. 18.

    Morrison, P. M., Foley, P. J., Warriner, S. L. & Webb, M. E. Chemical generation and modification of peptides containing multiple dehydroalanines. Chem. Commun. 51, 13470–13473 (2015).

  19. 19.

    Wever, W. J., Bogart, J. W. & Bowers, A. A. Identification of pyridine synthase recognition sequences allows a modular solid-phase route to thiopeptide variants. J. Am. Chem. Soc. 138, 13461–13464 (2016).

  20. 20.

    Metanis, N., Keinan, E. & Dawson, P. E. Traceless ligation of cysteine peptides using selective deselenization. Angew. Chem. Int. Ed. Engl. 49, 7049–7053 (2010).

  21. 21.

    Campbell, S., Rodgers, M. T., Marzluff, E. M. & Beauchamp, J. L. Deuterium exchange reactions as a probe of biomolecule structure. Fundamental studies of gas phase H/D exchange reactions of protonated glycine oligomers with D2O, CD3OD, CD3CO2D, and ND3. J. Am. Chem. Soc. 117, 12840–12854 (1995).

  22. 22.

    Bernardes, G. J., Chalker, J. M., Errey, J. C. & Davis, B. G. Facile conversion of cysteine and alkyl cysteines to dehydroalanine on protein surfaces: versatile and switchable access to functionalized proteins. J. Am. Chem. Soc. 130, 5052–5053 (2008).

  23. 23.

    Chalker, J. M., Lercher, L., Rose, N. R., Schofield, C. J. & Davis, B. G. Conversion of cysteine into dehydroalanine enables access to synthetic histones bearing diverse post-translational modifications. Angew. Chem. Int. Ed. Engl. 51, 1835–1839 (2012).

  24. 24.

    Wang, Z. U. et al. A facile method to synthesize histones with posttranslational modification mimics. Biochemistry 51, 5232–5234 (2012).

  25. 25.

    Timms, N. et al. Structural insights into the recovery of aldolase activity in N-acetylneuraminic acid lyase by replacement of the catalytically active lysine with γ-thialysine by using a chemical mutagenesis strategy. Chembiochem 14, 474–481 (2013).

  26. 26.

    Nathani, R. I. et al. A novel approach to the site-selective dual labelling of a protein via chemoselective cysteine modification. Chem. Sci. 4, 3455–3458 (2013).

  27. 27.

    Chooi, K. P. et al. Synthetic phosphorylation of p38α recapitulates protein kinase activity. J. Am. Chem. Soc. 136, 1698–1701 (2014).

  28. 28.

    Haj-Yahya, N. et al. Dehydroalanine-based diubiquitin activity probes. Org. Lett. 16, 540–543 (2014).

  29. 29.

    Rowan, F., Richards, M., Widya, M., Bayliss, R. & Blagg, J. Diverse functionalization of Aurora-A kinase at specified surface and buried sites by native chemical modification. PLoS. ONE. 9, e103935 (2014).

  30. 30.

    Meledin, R., Mali, S. M., Singh, S. K. & Brik, A. Protein ubiquitination via dehydroalanine: development and insights into the diastereoselective 1,4-addition step. Org. Biomol. Chem. 14, 4817–4823 (2016).

  31. 31.

    Palioura, S., Sherrer, R. L., Steitz, T. A., Söll, D. & Simonovic, M. The human SepSecS-tRNASec complex reveals the mechanism of selenocysteine formation. Science 325, 321–325 (2009).

  32. 32.

    Wright, T. H. et al. Posttranslational mutagenesis: a chemical strategy for exploring protein side-chain diversity. Science 354, 597 (2016).

  33. 33.

    Holmes, T. J. Jr & Lawton, R. G. Cysteine modification and cleavage of proteins with 2-methyl-N1-benzenesulfony-N4-bromoacetylquinonediimide. J. Am. Chem. Soc. 99, 1984–1986 (1977).

  34. 34.

    Nathani, R., Moody, P., Smith, M. E. B., Fitzmaurice, R. J. & Caddick, S. Bioconjugation of green fluorescent protein via an unexpectedly stable cyclic sulfonium intermediate. Chembiochem 13, 1283–1285 (2012).

  35. 35.

    Franzen, V. & Mertz, C. Zum Mechanismus Der Hofmann-Eliminierung Bei Sulfoniumsalzen. Chem. Ber. 93, 2819–2824 (1960).

  36. 36.

    Cristol, S. J. & Stermitz, F. R. Mechanisms of elimination reactions. XXIL. Some cis- and trans-2-phenylcyclohexyl derivatives. The Hoffmann elimination. J. Am. Chem. Soc. 82, 4692–4699 (1960).

  37. 37.

    Franzen, V. & Schmidt, H. J. Zum Mechanismus Der Hofmann-Eliminierung Bei Sulfoniumsalzen. 2. Chem. Ber. 94, 2937–2942 (1961).

  38. 38.

    Weygand, F. & Daniel, H. Fragmentierung Von S-Methyl-Thiolanium-Jodid Mit Phenyllithium Zu Athylen Und Methyl-Vinyl-Sulfid. Chem. Ber. 94, 3145–3146 (1961).

  39. 39.

    Banait, N. S. & Jencks, W. P. Elimination reactions: experimental confirmation of the predicted elimination of (β-cyanoethyl)sulfonium ions through a concerted, E2 mechanism. J. Am. Chem. Soc. 112, 6950–6958 (1990).

  40. 40.

    Grob, C. A. & Schiess, P. W. Heterolytic fragmentation. A class of organic reactions. Angew. Chem. Intl. Ed. 6, 1–15 (1967).

  41. 41.

    Bordwell, F. G. Equilibrium acidities in dimethyl-sulfoxide solution. Acc. Chem. Res. 21, 456–463 (1988).

  42. 42.

    Omura, K. & Swern, D. Oxidation of alcohols by activated dimethyl-sulfoxide - preparative, steric and mechanistic study. Tetrahedron 34, 1651–1660 (1978).

  43. 43.

    Chalker, J. M., Bernardes, G. J. L., Lin, Y. A. & Davis, B. G. Chemical modification of proteins at cysteine: opportunities in chemistry and biology. Chem. Asian J. 4, 630–640 (2009).

  44. 44.

    Dawson, P. E. Native chemical ligation combined with desulfurization and deselenization: A general strategy for chemical protein synthesis. Isr. J. Chem. 51, 862–867 (2011).

  45. 45.

    Kinnaman, C. S., Cremeens, M. E., Romesberg, F. E. & Corcelli, S. A. Infrared line shape of an α-carbon deuterium-labeled amino acid. J. Am. Chem. Soc. 128, 13334–13335 (2006).

  46. 46.

    Adhikary, R., Zimmermann, J. & Romesberg, F. E. Transparent window vibrational probes for the characterization of proteins with high structural and temporal resolution. Chem. Rev. 117, 1927–1969 (2017).

  47. 47.

    Kay, L. E., Ikura, M., Tschudin, R. & Bax, A. Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. J. Magn. Reson. 89, 496–514 (1990).

  48. 48.

    Darland, G. K. et al. Oxidative and defluorinative metabolism of fludalanine, 2-2H-3-fluoro-d-alanine. Drug Metab. Dispos. 14, 668–673 (1986).

  49. 49.

    Timmins, G. S. Deuterated drugs: where are we now? Expert Opin. Ther. Pat. 24, 1067–1075 (2014).

  50. 50.

    Hermanson, G. T. Bioconjugate Techniques (Academic Press, 1995).

  51. 51.

    Yasuda, S. K. & Lambert, J. L. Preparation and properties of anhydrous trisodium and tripotassium monothiophosphates. J. Am. Chem. Soc. 76, 5356–5356 (1954).

  52. 52.

    Zhao, Y. & Truhlar, D. G. The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals. Theor. Chem. Acc. 120, 215–241 (2008).

  53. 53.

    Barone, V. & Cossi, M. Quantum calculation of molecular energies and energy gradients in solution by a conductor solvent model. J. Phys. Chem. A 102, 1995–2001 (1998).

  54. 54.

    Cossi, M., Rega, N., Scalmani, G. & Barone, V. Energies, structures, and electronic properties of molecules in solution with the C-PCM solvation model. J. Comput. Chem. 24, 669–681 (2003).

  55. 55.

    Hodgson, D. M., Charlton, A., Paton, R. S. & Thompson, A. L. C-alkylation of chiral tropane- and homotropane-derived enamines. J. Org. Chem. 78, 1508–1518 (2013).

  56. 56.

    Simón, L. & Paton, R. S. Origins of asymmetric phosphazene organocatalysis: computations reveal a common mechanism for nitro- and phospho-aldol additions. J. Org. Chem. 80, 2756–2766 (2015).

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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.

Author information


  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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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.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Vijay Chudasama or Benjamin G. Davis.

Supplementary information

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    Synthetic procedures and small molecule reactions

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