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.

  • Protocol
  • Published:

Synthesis of N-methylated cyclic peptides

Nature Protocols volume 7, pages 432–444 (2012)Cite this article

Subjects

Abstract

This protocol presents a detailed description of the synthesis of N-methylated cyclic peptides. N-methylation is a powerful technique to modulate the physicochemical properties of peptides by introducing one or more methyl groups into the peptidic amide bonds. Together with peptide cyclization, this procedure confers unprecedented pharmacokinetic properties to the peptides, including metabolic stability, membrane permeability and even oral bioavailability. Here we describe two simplified methods of N-methylation of linear peptides on solid supports, which can be performed in less than 2 h and are applicable to any amino acid. Finally, we also describe two methods of peptide cyclization, which can be used to obtain the N-methylated cyclic peptide and which are not limited to specific peptide sequences. With this protocol, multiply N-methylated cyclic peptides can be synthesized in as little as 4–5 d.

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

Figure 1
Figure 2: Conformational modulation of penta-alanine cyclic peptide backbone by successive multiple N-methylation (mono-, di- and tri-N-methylation) of the peptide bonds.
Figure 3: Stereoimage of a metabolically stable, orally bioavailable tri-N-methylated somatostatin analog.
Figure 4: Photographs showing the setup used for the synthesis of the linear peptide on solid support.
Figure 5: Schematic representation of the synthesis of a cyclic peptide cyclo(-PFMewMeKTMeF-) (17) via two different linear sequences.
Figure 6
Figure 7: Analytical HPLC profile of crude cyclo(-PFMewMeKTMeF-).

Similar content being viewed by others

References

  1. Gilon, C., Dechantsreiter, M.A., Burkhart, F., Friedler, A. & Kessler, H. in Houben-Weyl Methods of Organic Chemistry, Vol. E22c (eds. Goodman, M., Felix, A., Moroder, L. & Tonolio, C.) 215–291 (Georg Thieme Verlag, 2002).

  2. Hamman, J.H., Enslin, G.M. & Kotze, A.F. Oral delivery of peptide drugs: barriers and developments. Biodrugs 19, 165–177 (2005).

    Article  CAS  Google Scholar 

  3. Kessler, H. Peptide Conformations. 19. Conformation and biological activity of cyclic-peptides. Angew. Chem. Int. Ed. 21, 512–523 (1982).

    Article  Google Scholar 

  4. Hess, S. et al. Backbone cyclic peptidomimetic melanocortin-4 receptor agonist as a novel orally administrated drug lead for treating obesity. J. Med. Chem. 51, 1026–1034 (2008).

    Article  CAS  Google Scholar 

  5. Shemyakin, M.M., Ovchinnikov, Y.A. & Ivanov, V.T. Topochemical investigation of peptide systems. Angew. Chem. Int. Ed. Engl. 8, 492–499 (1969).

    Article  CAS  Google Scholar 

  6. Goodman, M. & Chorev, M. Concept of linear modified retro-peptide structures. Acc. Chem. Res. 12, 1–7 (1979).

    Article  CAS  Google Scholar 

  7. Giannis, A. Peptidomimetics for receptor ligands-discovery, development, and medical perspectives. Angew. Chem. Int. Ed. 32, 1244–1267 (1993).

    Article  Google Scholar 

  8. Lee, H.K. et al. Anticonvulsant Met-enkephalin analogues containing backbone spacers reveal alternative non-opioid signaling in the brain. ACS Chem. Biol. 4, 659–671 (2009).

    Article  CAS  Google Scholar 

  9. Simon, R.J. et al. Peptoids—a modular approach to drug discovery. Proc. Natl. Acad. Sci. USA 89, 9367–9371 (1992).

    Article  CAS  Google Scholar 

  10. Kessler, H. Peptoids—a new approach to the development of pharmaceuticals. Angew. Chem. Int. Ed. Engl. 32, 543–544 (1993).

    Article  Google Scholar 

  11. White, C.J. & Yudin, A.K. Contemporary strategies for peptide macrocyclization. Nat. Chem. 3, 509–524 (2011).

    Article  CAS  Google Scholar 

  12. Rothbard, J.B. et al. Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation. Nat. Med. 6, 1253–1257 (2000).

    Article  CAS  Google Scholar 

  13. Wender, P.A. et al. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc. Natl. Acad. Sci. USA 97, 13003–13008 (2000).

    Article  CAS  Google Scholar 

  14. Richard, J.P. et al. Cell-penetrating peptides—a re-evaluation of the mechanism of cellular uptake. J. Biol. Chem. 278, 585–590 (2003).

    Article  CAS  Google Scholar 

  15. Jones, S.W. et al. Characterisation of cell-penetrating peptide-mediated peptide delivery. Br. J. Pharmacol. 145, 1093–1102 (2005).

    Article  CAS  Google Scholar 

  16. Borel, J.F., Feurer, C., Gubler, H.U. & Stahelin, H. Biological effects of Cyclosporin-a—new antilymphocytic agent. Agents Actions 6, 468–475 (1976).

    Article  CAS  Google Scholar 

  17. Pettit, G.R. et al. Isolation of Dolastatins 10–15 from the marine mollusk Dolabella auricularia. Tetrahedron 49, 9151–9170 (1993).

    Article  CAS  Google Scholar 

  18. Cruz, L.J. et al. IB-01212, a new cytotoxic cyclodepsipeptide isolated from the marine fungus Clonostachys sp. ESNA-A009. J. Org. Chem. 71, 3335–3338 (2006).

    Article  CAS  Google Scholar 

  19. Plaza, A. et al. Mutremdamide A and koshikamides C-H, peptide inhibitors of HIV-1 entry from different Theonella species. J. Org. Chem. 75, 4344–4355 (2010).

    Article  CAS  Google Scholar 

  20. Lipinski, C.A., Lombardo, F., Dominy, B.W. & Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46, 3–26 (2001).

    Article  CAS  Google Scholar 

  21. Sugano, H., Higaki, K. & Miyoshi, M. Synthesis and biological-activity of peptides related to eledoisin. 1. Hexapeptide amides containing alpha-hydroxy acids. Bull. Chem. Soc. Japan 46, 226–230 (1973).

    Article  CAS  Google Scholar 

  22. Dechantsreiter, M.A. et al. N-methylated cyclic RGD peptides as highly active and selectiveα(v)β(3) integrin antagonists. J. Med. Chem. 42, 3033–3040 (1999).

    Article  CAS  Google Scholar 

  23. Mas-Moruno, C., Rechenmacher, F. & Kessler, H. Cilengitide: the first anti-angiogenic small molecule drug candidate design, synthesis and clinical evaluation. Anticancer Agents Med. Chem. 10, 753–768 (2011).

    Article  Google Scholar 

  24. Haubner, R., Finsinger, D. & Kessler, H. Stereoisomeric peptide libraries and peptidomimetics for designing selective inhibitors of the αvβ3 integrin for a new cancer therapy. Angew. Chem. Int. Ed. Engl. 36, 1374–1389 (1997).

    Article  CAS  Google Scholar 

  25. Gordon, D.J., Tappe, R. & Meredith, S.C. Design and characterization of a membrane permeable N-methyl amino acid-containing peptide that inhibits A β(1–40) fibrillogenesis. J. Pept. Res. 60, 37–55 (2002).

    Article  CAS  Google Scholar 

  26. Kessler, H. Detection of intramolecular mobility by NMR spectroscopy. 13. Detection of hindered rotation and inversion by NMR spectroscopy. Angew. Chem. Int. Ed. 9, 219–235 (1970).

    Article  CAS  Google Scholar 

  27. Chatterjee, J., Gilon, C., Hoffman, A. & Kessler, H. N-methylation of peptides: a new perspective in medicinal chemistry. Acc. Chem. Res. 41, 1331–1342 (2008).

    Article  CAS  Google Scholar 

  28. Ovadia, O. et al. Improvement of drug-like properties of peptides: the somatostatin paradigm. Expert Opin. Drug Discov. 5, 655–671 (2010).

    Article  CAS  Google Scholar 

  29. Chatterjee, J., Mierke, D.F. & Kessler, H. Conformational preference and potential templates of N-methylated cyclic pentaalanine peptides. Chem. Eur. J. 14, 1508–1517 (2008).

    Article  CAS  Google Scholar 

  30. Laufer, B., Chatterjee, J., Frank, A.O. & Kessler, H. Can N-methylated amino acids serve as substitutes for prolines in conformational design of cyclic pentapeptides? J. Pept. Sci. 15, 141–146 (2009).

    Article  CAS  Google Scholar 

  31. Chatterjee, J. et al. Multiple N-methylation by a designed approach enhances receptor selectivity. J. Med. Chem. 50, 5878–5881 (2007).

    Article  CAS  Google Scholar 

  32. Chatterjee, J. et al. N-Methylated sst2 selective somatostatin cyclic peptide analogue as potent candidate for treating neurogenic inflammation. ACS Med. Chem. Lett. 2, 509–514 (2011).

    Article  CAS  Google Scholar 

  33. Doedens, L. et al. Multiple N-methylation of MT-II backbone amide bonds leads to melanocortin receptor subtype hMC1R selectivity: pharmacological and conformational studies. J. Am. Chem. Soc. 132, 8115–8128 (2010).

    Article  CAS  Google Scholar 

  34. Biron, E. et al. Improving oral bioavailability of peptides by multiple N-methylation: somatostatin analogues. Angew. Chem. Int. Ed. 47, 2595–2599 (2008).

    Article  CAS  Google Scholar 

  35. Ovadia, O. et al. The effect of multiple N-methylation on intestinal permeability of peptides. Mol. Pharmaceutics 8, 479–487 (2011).

    Article  CAS  Google Scholar 

  36. Biron, E., Chatterjee, J. & Kessler, H. Optimized selective N-methylation of peptides on solid support. J. Pept. Sci. 12, 213–219 (2006).

    Article  CAS  Google Scholar 

  37. Coin, I., Beyermann, M. & Bienert, M. Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nat. Protoc. 2, 3247–3256 (2007).

    Article  CAS  Google Scholar 

  38. Fukuyama, T., Jow, C.-K. & Cheung, M. 2- and 4-Nitrobenzenesulfonamides: exceptionally versatile means for preparation of secondary amines and protection of amines. Tetrahedron Lett. 36, 6373–6374 (1995).

    Article  CAS  Google Scholar 

  39. Patgiri, A., Menzenski, M.Z., Mahon, A.B. & Arora, P.S. Solid-phase synthesis of short α-helices stabilized by the hydrogen bond surrogate approach. Nat. Protoc. 5, 1857–1865 (2010).

    Article  CAS  Google Scholar 

  40. Kim, Y.W., Grossmann, T.N. & Verdine, G.L. Synthesis of all-hydrocarbon stapled α-helical peptides by ring-closing olefin metathesis. Nat. Protoc. 6, 761–771 (2011).

    Article  CAS  Google Scholar 

  41. Malesevic, M., Strijowski, U., Bächle, D. & Sewald, N. An improved method for the solution cyclization of peptides under pseudo-high dilution conditions. J. Biotechnol. 112, 73–77 (2004).

    Article  CAS  Google Scholar 

  42. Chatterjee, J., Mierke, D. & Kessler, H. N-methylated cyclic pentaalanine peptides as template structures. J. Am. Chem. Soc. 128, 15164–15172 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M.M. Baksh of the Scripps Research Institute for his help in proofreading and editing the manuscript.

Author information

Author notes

  1. Horst Kessler

    Present address: Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia. Present addresses: European Molecular Biology Laboratory, Heidelberg, Germany (J.C.); Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA (B.L.).,

Authors and Affiliations

  1. Institute for Advanced Study and Center for Integrated Protein Science at the Department Chemie, Technische Universität München, Garching, Germany

    Jayanta Chatterjee, Burkhardt Laufer & Horst Kessler

Authors
  1. Jayanta Chatterjee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Burkhardt Laufer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Horst Kessler
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.C. and B.L. conducted the experiments as reported in the original papers and tested the protocol. H.K. supervised the overall project. J.C. wrote the manuscript, B.L. and H.K. edited the manuscript.

Corresponding author

Correspondence to Horst Kessler.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chatterjee, J., Laufer, B. & Kessler, H. Synthesis of N-methylated cyclic peptides. Nat Protoc 7, 432–444 (2012). https://doi.org/10.1038/nprot.2011.450

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.450

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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