A chemoselective strategy for late-stage functionalization of complex small molecules with polypeptides and proteins

Abstract

Conjugates between proteins and small molecules enable access to a vast chemical space that is not achievable with either type of molecule alone; however, the paucity of specific reactions capable of functionalizing proteins and natural products presents a formidable challenge for preparing conjugates. Here we report a strategy for conjugating electron-rich (hetero)arenes to polypeptides and proteins. Our bioconjugation technique exploits the electrophilic reactivity of an oxidized selenocysteine residue in polypeptides and proteins, and the electron-rich character of certain small molecules to provide bioconjugates in excellent yields under mild conditions. This conjugation chemistry enabled the synthesis of peptide–vancomycin conjugates without the prefunctionalization of vancomycin. These conjugates have an enhanced in vitro potency for resistant Gram-positive and Gram-negative pathogens. Additionally, we show that a 6 kDa affibody protein and a 150 kDa immunoglobulin-G antibody could be modified without diminishing bioactivity.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: The conjugation of oxidized selenocysteine to electron-rich small molecules is a one-pot chemoselective reaction for the covalent attachment of natural products and pharmaceuticals to polypeptides and proteins.
Fig. 2: Selenocysteine-based conjugation of vancomycin to antibacterial peptides enables the discovery of potent Gram-positive and Gram-negative antibacterial agents.
Fig. 3: The conjugation of vancomycin to affibody generates conjugates with retained structures and binding affinities.
Fig. 4: Conjugation of genistain to the antibody leads to conjugates with a retained target binding affinity.
Fig. 5: Selenocysteine-based conjugation of peptides to (hetero)arenes, natural products and pharmaceuticals proceeded in moderate-to-high yields.

Data availability

All the data generated or analysed during this study are included in this published article (and in the Supplementary Information). Further details are available from the corresponding authors upon request.

References

  1. 1.

    Drake, P. M. & Rabuka, D. An emerging playbook for antibody–drug conjugates: lessons from the laboratory and clinic suggest a strategy for improving efficacy and safety. Curr. Opin. Chem. Biol. 28, 174–180 (2015).

    CAS  Article  Google Scholar 

  2. 2.

    Ueda, T. Next-generation optimized biotherapeutics—a review and preclinical study. Biochim. Biophys. Acta 1844, 2053–2057 (2014).

    CAS  Article  Google Scholar 

  3. 3.

    Hamann, P. R. et al. Gemtuzumab ozogamicin, a potent and selective anti-CD33 antibody–calicheamicin conjugate for treatment of acute myeloid leukemia. Bioconjug. Chem. 13, 47–58 (2002).

    CAS  Article  Google Scholar 

  4. 4.

    Wang, L., Amphlett, G., Blättler, W. A., Lambert, J. M. & Zhang, W. Structural characterization of the maytansinoid–monoclonal antibody immunoconjugate, huN901–DM1, by mass spectrometry. Protein Sci. 14, 2436–2446 (2005).

    CAS  Article  Google Scholar 

  5. 5.

    Lewis Phillips, G. D. et al. Targeting HER2-positive breast cancer with trastuzumab–DM1, an antibody–cytotoxic drug conjugate. Cancer Res. 68, 9280–9290 (2008).

    CAS  Article  Google Scholar 

  6. 6.

    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).

    CAS  Article  Google Scholar 

  7. 7.

    Boswell, C. A. et al. Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody–drug conjugates in rats. Bioconjug. Chem. 22, 1994–2004 (2011).

    CAS  Article  Google Scholar 

  8. 8.

    Pillow, T. H. et al. Site-specific trastuzumab maytansinoid antibody–drug conjugates with improved therapeutic activity through linker and antibody engineering. J. Med. Chem. 57, 7890–7899 (2014).

    CAS  Article  Google Scholar 

  9. 9.

    Akkapeddi, P. et al. Construction of homogeneous antibody–drug conjugates using site-selective protein chemistry. Chem. Sci. 7, 2954–2963 (2016).

    CAS  Article  Google Scholar 

  10. 10.

    Zou, L.-H., Reball, J., Mottweiler, J. & Bolm, C. Transition metal-free direct C–H bond thiolation of 1,3,4-oxadiazoles and related heteroarenes. Chem. Commun. 48, 11307–11309 (2012).

    CAS  Article  Google Scholar 

  11. 11.

    Hostier, T., Ferey, V., Ricci, G., Gomez Pardo, D. & Cossy, J. Synthesis of aryl sulfides: metal-free C–H sulfenylation of electron-rich arenes. Org. Lett. 17, 3898–3901 (2015).

    CAS  Article  Google Scholar 

  12. 12.

    Ranjit, S. et al. Copper-mediated C–H activation/C–S cross-coupling of heterocycles with thiols. J. Org. Chem. 76, 8999–9007 (2011).

    CAS  Article  Google Scholar 

  13. 13.

    Fang, X.-L., Tang, R.-Y., Zhong, P. & Li, J.-H. Iron-catalyzed sulfenylation of indoles with disulfides promoted by a catalytic amount of iodine. Synthesis 2009, 4183–4189 (2009).

    Article  Google Scholar 

  14. 14.

    Cohen, D. T., Zhang, C., Pentelute, B. L. & Buchwald, S. L. An umpolung approach for the chemoselective arylation of selenocysteine in unprotected peptides. J. Am. Chem. Soc. 137, 9784–9787 (2015).

    CAS  Article  Google Scholar 

  15. 15.

    Hall, D. G. in Boronic Acids: Preparation and Applications in Organic Synthesis and Medicine (ed. Hall, D. G.) Ch. 1 (Wiley, Hoboken, 2006).

  16. 16.

    Small, P. M. & Chambers, H. F. Vancomycin for Staphylococcus aureus endocarditis in intravenous drug users. Antimicrob. Agents Chemother. 34, 1227–1231 (1990).

    CAS  Article  Google Scholar 

  17. 17.

    Cetinkaya, Y., Falk, P. & Mayhall, C. G. Vancomycin-resistant enterococci. Clin. Microbiol. Rev. 13, 686–707 (2000).

    CAS  Article  Google Scholar 

  18. 18.

    Navon-Venezia, S., Feder, R., Gaidukov, L., Carmeli, Y. & Mor, A. Antibacterial properties of dermaseptin S4 derivatives with in vivo activity. Antimicrob. Agents Chemother. 46, 689–694 (2002).

    CAS  Article  Google Scholar 

  19. 19.

    Yount, N. Y. et al. Context mediates antimicrobial efficacy of kinocidin congener peptide RP-1. PLoS One 6, e26727 (2011).

    CAS  Article  Google Scholar 

  20. 20.

    Dijkshoorn, L., Nemec, A. & Seifert, H. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat. Rev. Microbiol. 5, 939–951 (2007).

    CAS  Article  Google Scholar 

  21. 21.

    O’Shea, M. K. Acinetobacter in modern warfare. Int. J. Antimicrob. Agents 39, 363–375 (2012).

    Article  Google Scholar 

  22. 22.

    Maragakis, L. L. & Perl, T. M. Acinetobacter baumannii: epidemiology, antimicrobial resistance, and treatment options. Clin. Infect. Dis. 46, 1254–1263 (2008).

    CAS  Article  Google Scholar 

  23. 23.

    Andreadou, I., Menge, W. M. P. B., Commandeur, J. N. M., Worthington, E. A. & Vermeulen, N. P. E. Synthesis of novel Se-substituted selenocysteine derivatives as potential kidney selective prodrugs of biologically active selenol compounds: evaluation of kinetics of β-elimination reactions in rat renal cytosol. J. Med. Chem. 39, 2040–2046 (1996).

    CAS  Article  Google Scholar 

  24. 24.

    Andreadou, I., van de Water, B., Commandeur, J. N., Nagelkerke, F. J. & Vermeulen, N. P. Comparative cytotoxicity of 14 novel selenocysteine Se-conjugates in rat renal proximal tubular cells. Toxicol. Appl. Pharmacol. 141, 278–287 (1996).

    CAS  Article  Google Scholar 

  25. 25.

    Spallholz, J. E. On the nature of selenium toxicity and carcinostatic activity. Free Radic. Biol. Med. 17, 45–64 (1994).

    CAS  Article  Google Scholar 

  26. 26.

    Löfblom, J. et al. Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett. 584, 2670–2680 (2010).

    Article  Google Scholar 

  27. 27.

    Orlova, A. et al. Tumor imaging using a picomolar affinity HER2 binding affibody molecule. Cancer Res. 66, 4339–4348 (2006).

    CAS  Article  Google Scholar 

  28. 28.

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

    CAS  Article  Google Scholar 

  29. 29.

    Russo, M. et al. Understanding genistein in cancer: the ‘good’ and the ‘bad’ effects: a review. Food Chem. 196, 589–600 (2016).

    CAS  Article  Google Scholar 

  30. 30.

    Thyer, R., Robotham, S. A., Brodbelt, J. S. & Ellington, A. D. Evolving tRNASec for efficient canonical incorporation of selenocysteine. J. Am. Chem. Soc. 137, 46–49 (2015).

    CAS  Article  Google Scholar 

  31. 31.

    Hofer, T., Thomas, J. D., Burke, T. R. & Rader, C. An engineered selenocysteine defines a unique class of antibody derivatives. Proc. Natl Acad. Sci. USA 105, 12451–12456 (2008).

    CAS  Article  Google Scholar 

  32. 32.

    Beasley, R., Pearce, N., Crane, J. & Burgess, C. Withdrawal of fenoterol and the end of the New Zealand asthma mortality epidemic. Int. Arch. Allergy Immunol. 107, 325–327 (1995).

    CAS  Article  Google Scholar 

  33. 33.

    Miyamoto, Y., Sakamoto, Y., Yoshida, N. & Baba, H. Efficacy of S-1 in colorectal cancer. Expert. Opin. Pharmacother. 15, 1761–1770 (2014).

    CAS  Article  Google Scholar 

  34. 34.

    Wiśniewski, K. & Car, H. (S)-3,5-DHPG: a review. CNS Drug. Rev. 8, 101–116 (2002).

    Article  Google Scholar 

  35. 35.

    Chan, G. M., Sharma, R., Price, D., Hoffman, R. S. & Nelson, L. S. Phentolamine therapy for cocaine-associated acute coronary syndrome (CAACS). J. Med. Toxicol. 2, 108–111 (2006).

    Article  Google Scholar 

  36. 36.

    Wong, G. W. K., . & Boyda, H. N. & Wright, J. M. Blood pressure lowering efficacy of partial agonist beta blocker monotherapy for primary hypertension. Cochrane Database Syst. Rev. CD007450 (2014).

  37. 37.

    Lesch, K.-P. et al. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 274, 1527–1531 (1996).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

Financial support was provided by NIH Awards GM46059 (S.L.B.), F32GM108294 (D.T.C.), F32GM122204 (L.H.), F30HD093358 (C.M.F.) and GM110535 (B.L.P.) and also by MIT startup funds, a Damon Runyon Cancer Research Foundation Award and a Sontag Distinguished Scientist Award for B.L.P. C.Z. is a recipient of a George Büchi Summer Research Fellowship, a Koch Graduate Fellowship in Cancer Research and a Bristol-Myers Squibb Fellowship in Synthetic Organic Chemistry. A.J.M. 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) and CD spectrometer (NSF-0070319). We thank Y.-M. Wang and M. Pirnot (MIT) for help in preparing this article. We also acknowledge S. Bano (Merck) for Cu ICP-MS analysis of the purified peptides 13c, 13d, 13e and 13f.

Author information

Affiliations

Authors

Contributions

D.T.C. and B.L.P. conceived the work and designed the experiments. D.T.C., C.Z. and C.M.F. performed the peptide and protein labelling experiments. D.T.C., C.M.F. and A.J.M. developed the seleno–affibody synthesis. L.H. and S.J.M. performed the NMR characterization to assign regioselectivity for the selenocysteine conjugation. K.D.J., Z.S. and O.P. carried out the cytotoxicity and haemolysis assays on 23v analogues. D.T.C., C.Z., C.M.F. and B.L.P. wrote the manuscript, with input from all other authors.

Corresponding authors

Correspondence to Daniel T. Cohen or Bradley L. Pentelute.

Ethics declarations

Competing interests

K.D.J., Z.S. and O.P. are employees of Visterra Inc. D.T.C., C.Z., S.L.B. and B.L.P. are inventors on a patent filed by MIT-TLO to cover this work (US patent application no. 15,187,169 and international application (PCT/US16/38372).

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 Materials, Methods, and References

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Cohen, D.T., Zhang, C., Fadzen, C.M. et al. A chemoselective strategy for late-stage functionalization of complex small molecules with polypeptides and proteins. Nature Chem 11, 78–85 (2019). https://doi.org/10.1038/s41557-018-0154-0

Download citation

Further reading

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