Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

π-Clamp-mediated cysteine conjugation

Nature Chemistry volume 8, pages 120–128 (2016)Cite this article

Subjects

Abstract

Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the ‘π-clamp’, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody–drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: π-Clamp-mediated cysteine conjugation as a new strategy for site-selective chemistry.
Figure 2: π-Clamp-mediated cysteine conjugation on peptides.
Figure 3: The π-clamp functions at distinct positions in polypeptides and is compatible with diverse perfluoroaryl-based probes.
Figure 4: π-Clamp-mediated site-specific conjugation on proteins with multiple cysteines.
Figure 5: π-Clamp-mediated site-specific antibody conjugation.
Figure 6: Structure and mechanism of the π-clamp.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Carrico, I. S. Chemoselective modification of proteins: hitting the target. Chem. Soc. Rev. 37, 1423–1431 (2008).

    CAS  PubMed  Google Scholar 

  2. Hackenberger, C. P. R. & Schwarzer, D. Chemoselective ligation and modification strategies for peptides and proteins. Angew. Chem. Int. Ed. 47, 10030–10074 (2008).

    CAS  Google Scholar 

  3. Rabuka, D. Chemoenzymatic methods for site-specific protein modification. Curr. Opin. Chem. Biol. 14, 790–796 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Spicer, C. D. & Davis, B. G. Selective chemical protein modification. Nature Commun. 5, 4740 (2014).

    CAS  Google Scholar 

  5. Editorial. Take aim. Nature Chem. 4, 955 (2012).

  6. Panowski, S., Bhakta, S., Raab, H., Polakis, P. & Junutula, J. R. Site-specific antibody drug conjugates for cancer therapy. MAbs 6, 34–45 (2014).

    PubMed  Google Scholar 

  7. Kochendoerfer, G. G. Site-specific polymer modification of therapeutic proteins. Curr. Opin. Chem. Biol. 9, 555–560 (2005).

    CAS  PubMed  Google Scholar 

  8. Uttamapinant, C. et al. A fluorophore ligase for site-specific protein labeling inside living cells. Proc. Natl Acad. Sci. USA 107, 10914–10919 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Krishna, O. D. & Kiick, K. L. Protein- and peptide-modified synthetic polymeric biomaterials. Peptide Sci. 94, 32–48 (2010).

    CAS  Google Scholar 

  10. Lichtor, P. A. & Miller, S. J. Combinatorial evolution of site- and enantioselective catalysts for polyene epoxidation. Nature Chem. 4, 990–995 (2012).

    CAS  Google Scholar 

  11. Wilcock, B. C. et al. Electronic tuning of site-selectivity. Nature Chem. 4, 996–1003 (2012).

    CAS  Google Scholar 

  12. Sletten, E. M. & Bertozzi, C. R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. 48, 6974–6998 (2009).

    CAS  Google Scholar 

  13. Afagh, N. A. & Yudin, A. K. Chemoselectivity and the curious reactivity preferences of functional groups. Angew. Chem. Int. Ed. 49, 262–310 (2010).

    CAS  Google Scholar 

  14. Lewis, C. A. & Miller, S. J. Site-selective derivatization and remodeling of erythromycin A by using simple peptide-based chiral catalysts. Angew. Chem. Int. Ed. 45, 5616–5619 (2006).

    CAS  Google Scholar 

  15. Chen, M. S. & White, M. C. A predictably selective aliphatic C–H oxidation reaction for complex molecule synthesis. Science 318, 783–787 (2007).

    CAS  PubMed  Google Scholar 

  16. Snyder, S. A., Gollner, A. & Chiriac, M. I. Regioselective reactions for programmable resveratrol oligomer synthesis. Nature 474, 461–466 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Pathak, T. P. & Miller, S. J. Site-selective bromination of vancomycin. J. Am. Chem. Soc. 134, 6120–6123 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Wender, P. A., Hilinski, M. K. & Mayweg, A. V. W. Late-stage intermolecular CH activation for lead diversification: a highly chemoselective oxyfunctionalization of the C-9 position of potent bryostatin analogues. Org. Lett. 7, 79–82 (2005).

    CAS  PubMed  Google Scholar 

  19. Peddibhotla, S., Dang, Y., Liu, J. O. & Romo, D. Simultaneous arming and structure/activity studies of natural products employing O–H insertions: an expedient and versatile strategy for natural products-based chemical genetics. J. Am. Chem. Soc. 129, 12222–12231 (2007).

    CAS  PubMed  Google Scholar 

  20. Saxon, E. & Bertozzi, C. R. Cell surface engineering by a modified Staudinger reaction. Science 287, 2007–2010 (2000).

    CAS  PubMed  Google Scholar 

  21. Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective ‘ligation’ of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596–2599 (2002).

    CAS  Google Scholar 

  22. Agard, N. J., Prescher, J. A. & Bertozzi, C. R. A strain-promoted [3+2] azide−alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126, 15046–15047 (2004).

    CAS  PubMed  Google Scholar 

  23. Song, W., Wang, Y., Qu, J. & Lin, Q. Selective functionalization of a genetically encoded alkene-containing protein via ‘photoclick chemistry’ in bacterial cells. J. Am. Chem. Soc. 130, 9654–9655 (2008).

    CAS  PubMed  Google Scholar 

  24. Blackman, M. L., Royzen, M. & Fox, J. M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels–Alder reactivity. J. Am. Chem. Soc. 130, 13518–13519 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Saxon, E., Armstrong, J. I. & Bertozzi, C. R. A ‘traceless’ Staudinger ligation for the chemoselective synthesis of amide bonds. Org. Lett. 2, 2141–2143 (2000).

    CAS  PubMed  Google Scholar 

  26. Lin, C.-W. & Ting, A. Y. Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells. J. Am. Chem. Soc. 128, 4542–4543 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Wollack, J. W., Silverman, J. M., Petzold, C. J., Mougous, J. D. & Distefano, M. D. A minimalist substrate for enzymatic peptide and protein conjugation. ChemBioChem 10, 2934–2943 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Yin, J. et al. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc. Natl Acad. Sci. USA 102, 15815–15820 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Cull, M. G. & Schatz, P. J. Biotinylation of proteins in vivo and in vitro using small peptide tags. Methods Enzymol. 326, 430–440 (2000).

    CAS  PubMed  Google Scholar 

  30. Fernandez-Suarez, M. et al. Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nature Biotechnol. 25, 1483–1487 (2007).

    CAS  Google Scholar 

  31. Popp, M. W., Antos, J. M., Grotenbreg, G. M., Spooner, E. & Ploegh, H. L. Sortagging: a versatile method for protein labeling. Nature Chem. Biol. 3, 707–708 (2007).

    CAS  Google Scholar 

  32. Whitford, D. Proteins: Structure and Function (Wiley, 2005).

    Google Scholar 

  33. Walsh, C. Enabling the chemistry of life. Nature 409, 226–231 (2001).

    CAS  PubMed  Google Scholar 

  34. Giles, N. M., Giles, G. I. & Jacob, C. Multiple roles of cysteine in biocatalysis. Biochem. Biophys. Res. Commun. 300, 1–4 (2003).

    CAS  PubMed  Google Scholar 

  35. Weerapana, E. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 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).

    CAS  PubMed  Google Scholar 

  37. 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).

    CAS  PubMed  Google Scholar 

  38. Hermanson, G. T. in Bioconjugate Techniques 3rd edn (ed Hermanson, G. T.) 1–125 (Academic, 2013).

    Google Scholar 

  39. Sun, M. M. C. et al. Reduction–alkylation strategies for the modification of specific monoclonal antibody disulfides. Bioconjug. Chem. 16, 1282–1290 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Griffin, B. A., Adams, S. R. & Tsien, R. Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    CAS  PubMed  Google Scholar 

  41. Adams, S. R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).

    CAS  PubMed  Google Scholar 

  42. Wilson, P. et al. Organic arsenicals as efficient and highly specific linkers for protein/peptide–polymer conjugation. J. Am. Chem. Soc. 137, 4215–4222 (2015).

    CAS  PubMed  Google Scholar 

  43. Stroffekova, K., Proenza, C. & Beam, K. G. The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins. Pflügers Arch. 442, 859–866 (2001).

    CAS  PubMed  Google Scholar 

  44. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).

    CAS  PubMed  Google Scholar 

  45. Rush, J. S. & Bertozzi, C. R. New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo. J. Am. Chem. Soc. 130, 12240–12241 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Spokoyny, A. M. et al. A perfluoroaryl-cysteine SNAr chemistry approach to unprotected peptide stapling. J. Am. Chem. Soc. 135, 5946–5949 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Zhang, C., Spokoyny, A. M., Zou, Y., Simon, M. D. & Pentelute, B. L. Enzymatic ‘click’ ligation: selective cysteine modification in polypeptides enabled by promiscuous glutathione S-transferase. Angew. Chem. Int. Ed. 52, 14001–14005 (2013).

    CAS  Google Scholar 

  48. Zhang, C., Dai, P., Spokoyny, A. M. & Pentelute, B. L. Enzyme-catalyzed macrocyclization of long unprotected peptides. Org. Lett. 16, 3652–3655 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Zou, Y. et al. Convergent diversity-oriented side-chain macrocyclization scan for unprotected polypeptides. Org. Biomol. Chem. 12, 566–573 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Sievers, E. L. & Senter, P. D. Antibody–drug conjugates in cancer therapy. Annu. Rev. Med. 64, 15–29 (2013).

    CAS  PubMed  Google Scholar 

  51. Junutula, J. R. et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nature Biotechnol. 26, 925–932 (2008).

    CAS  Google Scholar 

  52. Junutula, J. R. et al. Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs. J. Immunol. Methods 332, 41–52 (2008).

    CAS  PubMed  Google Scholar 

  53. Piccart-Gebhart, M. J. et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N. Engl. J. Med. 353, 1659–1672 (2005).

    CAS  PubMed  Google Scholar 

  54. Spangler, J. B., Manzari, M. T., Rosalia, E. K., Chen, T. F. & Wittrup, K. D. Triepitopic antibody fusions inhibit cetuximab-resistant BRAF and KRAS mutant tumors via EGFR signal repression. J. Mol. Biol. 422, 532–544 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Perrotte, P. et al. Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice. Clin. Cancer Res. 5, 257–265 (1999).

    CAS  PubMed  Google Scholar 

  56. Artamkina, G. A., Egorov, M. P. & Beletskaya, I. P. Some aspects of anionic σ complexes. Chem. Rev. 82, 427–459 (1982).

    CAS  Google Scholar 

  57. Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40, 2004–2021 (2001).

    CAS  Google Scholar 

  58. Jewett, J. C. & Bertozzi, C. R. Cu-free click cycloaddition reactions in chemical biology. Chem. Soc. Rev. 39, 1272–1279 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Lang, K. & Chin, J. W. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. 114, 4764–4806 (2014).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank S. Buchwald (Massachusetts Institute of Technology, MIT) and K. Dane Wittrup (MIT) for comments and suggestions regarding the manuscript. This work was supported by an MIT start-up fund, the National Institutes of Health (NIH; R01GM110535) and the Sontag Foundation Distinguished Scientist Award (to B.L.P.). C.Z. is a recipient of the George Büchi Research Fellowship, the Koch Graduate Fellowship in Cancer Research of MIT, and the Bristol-Myers Squibb Graduate Fellowship in Synthetic Organic Chemistry. M.W. is a National Science Foundation Graduate Research Fellow. The authors acknowledge the Biological Instrument Facility of MIT for providing the Octet BioLayer Interferometry System (NIH S10 OD016326), the MIT Center for Environmental Health Sciences (CEHS) Instrument Facility for providing the flow cytometer (P30-ES002109) and R.J. Collier (Harvard) for contributing laboratory equipment used in this study.

Author information

Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Chi Zhang, Matthew Welborn, Tianyu Zhu, Troy Van Voorhis & Bradley L. Pentelute

  2. Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Nicole J. Yang & Michael S. Santos

Authors
  1. Chi Zhang
    View author publications

    Search author on:PubMed Google Scholar

  2. Matthew Welborn
    View author publications

    Search author on:PubMed Google Scholar

  3. Tianyu Zhu
    View author publications

    Search author on:PubMed Google Scholar

  4. Nicole J. Yang
    View author publications

    Search author on:PubMed Google Scholar

  5. Michael S. Santos
    View author publications

    Search author on:PubMed Google Scholar

  6. Troy Van Voorhis
    View author publications

    Search author on:PubMed Google Scholar

  7. Bradley L. Pentelute
    View author publications

    Search author on:PubMed Google Scholar

Contributions

C.Z. and B.L.P. conceived the work and designed the experiments. C.Z. performed the peptide and protein labelling experiments. M.W., T.Z. and T.V.H. performed the computational studies. N.J.Y. helped with antibody expression and purification. M.S.S. and C.Z. performed the flow cytometer and cell viability assays. C.Z. and B.L.P. wrote the manuscript, with input from all other authors.

Corresponding author

Correspondence to Bradley L. Pentelute.

Ethics declarations

Competing interests

A patent application covering this work has been filed by MIT-TLO (US patent application no. 14/278060, International patent application no. PCT/US15/031082).

Supplementary information

Supplementary information

Supplementary information (PDF 4297 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, C., Welborn, M., Zhu, T. et al. π-Clamp-mediated cysteine conjugation. Nature Chem 8, 120–128 (2016). https://doi.org/10.1038/nchem.2413

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/nchem.2413

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing