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:

Maleimide–thiol adducts stabilized through stretching

Nature Chemistry volume 11, pages 310–319 (2019)Cite this article

Subjects

Abstract

Maleimide–thiol reactions are widely used to produce protein–polymer conjugates for therapeutics. However, maleimide–thiol adducts are unstable in vivo or in the presence of thiol-containing compounds because of the elimination of the thiosuccinimide linkage through a retro-Michael reaction or thiol exchange. Here, using single-molecule force spectroscopy, we show that applying an appropriate stretching force to the thiosuccinimide linkage can considerably stabilize the maleimide–thiol adducts, in effect using conventional mechanochemistry of force-accelerated bond dissociation to unconventionally stabilize an adjacent bond. Single-molecule kinetic analysis and bulk structural characterizations suggest that hydrolysis of the succinimide ring is dominant over the retro-Michael reaction through a force-dependent kinetic control mechanism, and this leads to a product that is resistant to elimination. This unconventional mechanochemical approach enabled us to produce stable polymer–protein conjugates by simply applying a mechanical force to the maleimide–thiol adducts through mild ultrasonication. Our results demonstrate the great potential of mechanical force for stimulating important productive chemical transformations.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Single-molecule force spectroscopy of maleimide–thiol adducts.
Fig. 2: Pre-stretching experiment of maleimide–thiol conjugates.
Fig. 3: Chemical characterization of the force-induced hydrolysis of the maleimide–thiol conjugates.
Fig. 4: Ultrasonication increases the stability of maleimide–thiol-based antibody–PEG conjugates.

Similar content being viewed by others

Code availability

Igor procedures for single-molecule experiment analyses of this study are available from the corresponding author upon request.

Data availability

All data generated and analysed during this study are included in this article and its Supplementary Information, and are also available from the authors upon reasonable request.

References

  1. Hoyle, C. E. & Bowman, C. N. Thiol-ene click chemistry. Angew. Chem. Int. Ed. 49, 1540–1573 (2010).

    Article  CAS  Google Scholar 

  2. Mather, B. D., Viswanathan, K., Miller, K. M. & Long, T. E. Michael addition reactions in macromolecular design for emerging technologies. Prog. Polym. Sci. 31, 487–531 (2006).

    Article  CAS  Google Scholar 

  3. Jung, H. S., Chen, X., Kim, J. S. & Yoon, J. Recent progress in luminescent and colorimetric chemosensors for detection of thiols. Chem. Soc. Rev. 42, 6019–6031 (2013).

    Article  CAS  Google Scholar 

  4. George, N., Pick, H., Vogel, H., Johnsson, N. & Johnsson, K. Specific labeling of cell surface proteins with chemically diverse compounds. J. Am. Chem. Soc. 126, 8896–8897 (2004).

    Article  CAS  Google Scholar 

  5. Qin, B. et al. Supramolecular interfacial polymerization: a controllable method of fabricating supramolecular polymeric materials. Angew. Chem. Int. Ed. 56, 7639–7643 (2017).

    Article  CAS  Google Scholar 

  6. Houseman, B. T., Gawalt, E. S. & Mrksich, M. Maleimide-functionalized self-assembled monolayers for the preparation of peptide and carbohydrate biochips. Langmuir 19, 1522–1531 (2003).

    Article  CAS  Google Scholar 

  7. Phelps, E. A. et al. Maleimide cross‐linked bioactive peg hydrogel exhibits improved reaction kinetics and cross‐linking for cell encapsulation and in situ delivery. Adv. Mater. 24, 64–70 (2012).

    Article  CAS  Google Scholar 

  8. Baldwin, A. D. & Kiick, K. L. Reversible maleimide–thiol adducts yield glutathione-sensitive poly(ethylene glycol)–heparin hydrogels. Polym. Chem. 4, 133–143 (2013).

    Article  CAS  Google Scholar 

  9. Tsurkan, M. V. et al. Defined polymer–peptide conjugates to form cell‐instructive starPEG–heparin matrices in situ. Adv. Mater. 25, 2606–2610 (2013).

    Article  CAS  Google Scholar 

  10. Chudasama, V., Maruani, A. & Caddick, S. Recent advances in the construction of antibody–drug conjugates. Nat. Chem. 8, 114–119 (2016).

    Article  CAS  Google Scholar 

  11. Doronina, S. O. et al. Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat. Biotechnol. 21, 778–784 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Chapman, A. P. et al. Therapeutic antibody fragments with prolonged in vivo half-lives. Nat. Biotechnol. 17, 780–783 (1999).

    Article  CAS  Google Scholar 

  14. Greenwald, R. B., Choe, Y. H., McGuire, J. & Conover, C. D. Effective drug delivery by PEGylated drug conjugates. Adv. Drug Deliv. Rev. 55, 217–250 (2003).

    Article  CAS  Google Scholar 

  15. Scott, A. M., Wolchok, J. D. & Old, L. J. Antibody therapy of cancer. Nat. Rev. Cancer 12, 278–287 (2012).

    Article  CAS  Google Scholar 

  16. Alley, S. C., Okeley, N. M. & Senter, P. D. Antibody–drug conjugates: targeted drug delivery for cancer. Curr. Opin. Chem. Biol. 14, 529–537 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Verma, S. et al. Trastuzumab emtansine for HER2-positive advanced breast cancer. New Engl. J. Med. 367, 1783–1791 (2012).

    Article  CAS  Google Scholar 

  19. Younes, A. et al. Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. New Engl. J. Med. 363, 1812–1821 (2010).

    Article  CAS  Google Scholar 

  20. Younes, A. et al. Results of a pivotal phase II study of brentuximab vedotin for patients with relapsed or refractory Hodgkin’s lymphoma. J. Clin. Oncol. 30, 2183–2189 (2012).

    Article  CAS  Google Scholar 

  21. Senter, P. D. & Sievers, E. L. The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat. Biotechnol. 30, 631–637 (2012).

    Article  CAS  Google Scholar 

  22. Pasut, G. & Veronese, F. M. Polymer–drug conjugation, recent achievements and general strategies. Prog. Polym. Sci. 32, 933–961 (2007).

    Article  CAS  Google Scholar 

  23. Baldwin, A. D. & Kiick, K. L. Tunable degradation of maleimide–thiol adducts in reducing environments. Bioconjugate Chem. 22, 1946–1953 (2011).

  24. Alley, S. C. et al. Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjugate Chem. 19, 759–765 (2008).

  25. Ryan, C. P. et al. Tunable reagents for multi-functional bioconjugation: reversible or permanent chemical modification of proteins and peptides by control of maleimide hydrolysis. Chem. Commun. 47, 5452–5454 (2011).

    Article  CAS  Google Scholar 

  26. Lyon, R. P. et al. Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody–drug conjugates. Nat. Biotechnol. 32, 1059–1062 (2014).

    Article  CAS  Google Scholar 

  27. Shen, B.-Q. et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody–drug conjugates. Nat. Biotechnol. 30, 184–189 (2012).

    Article  CAS  Google Scholar 

  28. Kalia, J. & Raines, R. T. Catalysis of imido-group hydrolysis in a maleimide conjugate. Bioorg. Med. Chem. Lett. 17, 6286–6289 (2007).

    Article  CAS  Google Scholar 

  29. Tumey, L. N. et al. Mild method for succinimide hydrolysis on ADCs: impact on ADC potency, stability, exposure and efficacy. Bioconjugate Chem. 25, 1871–1880 (2014).

  30. Fontaine, S. D., Reid, R., Robinson, L., Ashley, G. W. & Santi, D. V. Long-term stabilization of maleimide–thiol conjugates. Bioconjugate Chem. 26, 145–152 (2015).

  31. Sohma, J. Mechanochemistry of polymers. Prog. Polym. Sci. 14, 451–596 (1989).

    Article  CAS  Google Scholar 

  32. Li, J., Nagamani, C. & Moore, J. S. Polymer mechanochemistry: from destructive to productive. Acc. Chem. Res. 48, 2181–2190 (2015).

    Article  CAS  Google Scholar 

  33. Piermattei, A., Karthikeyan, S. & Sijbesma, R. P. Activating catalysts with mechanical force. Nat. Chem. 1, 133–137 (2009).

    Article  CAS  Google Scholar 

  34. May, P. A. & Moore, J. S. Polymer mechanochemistry: techniques to generate molecular force via elongational flows. Chem. Soc. Rev. 42, 7497–7506 (2013).

    Article  CAS  Google Scholar 

  35. Caruso, M. M. et al. Mechanically-induced chemical changes in polymeric materials. Chem. Rev. 109, 5755–5798 (2009).

    Article  CAS  Google Scholar 

  36. Beyer, M. K. & Clausen-Schaumann, H. Mechanochemistry: the mechanical activation of covalent bonds. Chem. Rev. 105, 2921–2948 (2005).

    Article  CAS  Google Scholar 

  37. Takacs, L. The historical development of mechanochemistry. Chem. Soc. Rev. 42, 7649–7659 (2013).

    Article  CAS  Google Scholar 

  38. Zhang, H. et al. Multi-modal mechanophores based on cinnamate dimers. Nat. Commun. 8, 1147 (2017).

    Article  Google Scholar 

  39. Hickenboth, C. R. et al. Biasing reaction pathways with mechanical force. Nature 446, 423–427 (2007).

    Article  CAS  Google Scholar 

  40. Klukovich, H. M. et al. Tension trapping of carbonyl ylides facilitated by a change in polymer backbone. J. Am. Chem. Soc. 134, 9577–9580 (2012).

    Article  CAS  Google Scholar 

  41. Kryger, M. J. et al. Masked cyanoacrylates unveiled by mechanical force. J. Am. Chem. Soc. 132, 4558–4559 (2010).

    Article  CAS  Google Scholar 

  42. Klukovich, H. M., Kean, Z. S., Iacono, S. T. & Craig, S. L. Mechanically induced scission and subsequent thermal remending of perfluorocyclobutane polymers. J. Am. Chem. Soc. 133, 17882–17888 (2011).

    Article  CAS  Google Scholar 

  43. Kean, Z. S., Black Ramirez, A. L., Yan, Y. & Craig, S. L. Bicyclo[3.2.0]heptane mechanophores for the non-scissile and photochemically reversible generation of reactive bis-enones. J. Am. Chem. Soc. 134, 12939–12942 (2012).

    Article  CAS  Google Scholar 

  44. Chen, Y. et al. Mechanically induced chemiluminescence from polymers incorporating a 1,2-dioxetane unit in the main chain. Nat. Chem. 4, 559–562 (2012).

    Article  CAS  Google Scholar 

  45. Karthikeyan, S., Potisek, S. L., Piermattei, A. & Sijbesma, R. P. Highly efficient mechanochemical scission of silver-carbene coordination polymers. J. Am. Chem. Soc. 130, 14968–14969 (2008).

    Article  CAS  Google Scholar 

  46. Davis, D. A. et al. Force-induced activation of covalent bonds in mechanoresponsive polymeric materials. Nature 459, 68–72 (2009).

    Article  CAS  Google Scholar 

  47. Larsen, M. B. & Boydston, A. J. ‘Flex-activated’ mechanophores: using polymer mechanochemistry to direct bond bending activation. J. Am. Chem. Soc. 135, 8189–8192 (2013).

    Article  CAS  Google Scholar 

  48. Lenhardt, J. M. et al. Trapping a diradical transition state by mechanochemical polymer extension. Science 329, 1057–1060 (2010).

    Article  CAS  Google Scholar 

  49. Huang, W. et al. Single molecule study of force-induced rotation of carbon–carbon double bonds in polymers. ACS Nano 11, 194–203 (2017).

    Article  CAS  Google Scholar 

  50. Wang, J. et al. Inducing and quantifying forbidden reactivity with single-molecule polymer mechanochemistry. Nat. Chem. 7, 323–327 (2015).

    Article  CAS  Google Scholar 

  51. Akbulatov, S. et al. Experimentally realized mechanochemistry distinct from force-accelerated scission of loaded bonds. Science 357, 299–303 (2017).

    Article  CAS  Google Scholar 

  52. Zhang, W. & Zhang, X. Single molecule mechanochemistry of macromolecules. Prog. Polym. Sci. 28, 1271–1295 (2003).

    Article  CAS  Google Scholar 

  53. Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H. & Gaub, H. E. How strong is a covalent bond? Science 283, 1727–1730 (1999).

  54. Merkel, R., Nassoy, P., Leung, A., Ritchie, K. & Evans, E. Energy landscapes of receptor–ligand bonds explored with dynamic force spectroscopy. Nature 397, 50–53 (1999).

    Article  CAS  Google Scholar 

  55. Alegre-Cebollada, J., Kosuri, P., Rivas-Pardo, J. A. & Fernández, J. M. Direct observation of disulfide isomerization in a single protein. Nat. Chem. 3, 882–887 (2011).

    Article  CAS  Google Scholar 

  56. Oesterhelt, F., Rief, M. & Gaub, H. E. Single molecule force spectroscopy by AFM indicates helical structure of poly(ethylene-glycol) in water. New J. Phys. 1, 6.1–6.11 (1999).

    Article  Google Scholar 

  57. Cao, Y. & Li, H. Polyprotein of GB1 is an ideal artificial elastomeric protein. Nat. Mater. 6, 109–114 (2007).

    Article  CAS  Google Scholar 

  58. Evans, E. & Ritchie, K. Dynamic strength of molecular adhesion bonds. Biophys. J. 72, 1541–1555 (1997).

    Article  CAS  Google Scholar 

  59. Bell, G. I. Models for the specific adhesion of cells to cells. Science 200, 618–627 (1978).

    Article  CAS  Google Scholar 

  60. Wang, J. et al. A remote stereochemical lever arm effect in polymer mechanochemistry. J. Am. Chem. Soc. 136, 15162–15165 (2014).

    Article  CAS  Google Scholar 

  61. Lenhardt, J. M., Black, A. L. & Craig, S. L. gem-Dichlorocyclopropanes as abundant and efficient mechanophores in polybutadiene copolymers under mechanical stress. J. Am. Chem. Soc. 131, 10818–10819 (2009).

    Article  CAS  Google Scholar 

  62. Li, Y., Qin, M., Li, Y., Cao, Y. & Wang, W. Single molecule evidence for the adaptive binding of DOPA to different wet surfaces. Langmuir 30, 4358–4366 (2014).

    Article  CAS  Google Scholar 

  63. Oberbarnscheidt, L., Janissen, R. & Oesterhelt, F. Direct and model free calculation of force-dependent dissociation rates from force spectroscopic data. Biophys. J. 97, L19–L21 (2009).

    Article  CAS  Google Scholar 

  64. Serpe, M. J. et al. A simple and practical spreadsheet-based method to extract single-molecule dissociation kinetics from variable loading-rate force spectroscopy data. J. Phys. Chem. C 112, 19163–19167 (2008).

    Article  CAS  Google Scholar 

  65. Wiggins, K. M., Brantley, J. N. & Bielawski, C. W. Methods for activating and characterizing mechanically responsive polymers. Chem. Soc. Rev. 42, 7130–7147 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research is supported mainly by the National Natural Science Foundation of China (grants nos. 21522402, 11674153, 11374148, 21774057 and 11334004) and the Fundamental Research Funds for the Central Universities (grant no. 020414380080). The authors thank Y. Li for discussions.

Author information

Authors and Affiliations

  1. Collaborative Innovation Center of Advanced Microstructures, National Laboratory of Solid State Microstructure, Department of Physics, Nanjing University, Nanjing, China

    Wenmao Huang, Xin Wu, Xiang Gao, Yifei Yu, Hai Lei, Zhenshu Zhu, Meng Qin, Wei Wang & Yi Cao

  2. State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China

    Yi Shi

  3. Tianjin Key Laboratory of Molecular Optoelectronic Science, Department of Chemistry, Tianjin University, Tianjin, China

    Yulan Chen

  4. Institute for Brain Sciences, Nanjing University, Nanjing, China

    Wei Wang & Yi Cao

Authors
  1. Wenmao Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xin Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiang Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yifei Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hai Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhenshu Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yi Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yulan Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Meng Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Wei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yi Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Yi.C., W.H. and X.G. conceived the project and designed the experiments. W.H., X.G. and H.L. performed the single-molecule experiments and analysed the data. W.H., Y.Y., Z.Z., Y.L.C. and Y.S. performed the ultrasound experiments and analysed the data. W.H., X.W. and Y.Y. performed the antibody stability experiments. Yi.C., W.W. and M.Q. supervised the project. W.H. and Yi.C. wrote the paper with contributions from all authors.

Corresponding authors

Correspondence to Wei Wang or Yi Cao.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Notes, Supplementary Figures 1–27, Supplementary Tables 1 and 2.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Huang, W., Wu, X., Gao, X. et al. Maleimide–thiol adducts stabilized through stretching. Nat. Chem. 11, 310–319 (2019). https://doi.org/10.1038/s41557-018-0209-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-018-0209-2

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