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 all-hydrocarbon stapled α-helical peptides by ring-closing olefin metathesis

Nature Protocols volume 6, pages 761–771 (2011)Cite this article

Subjects

Abstract

This protocol provides a detailed procedure for the preparation of stapled α-helical peptides, which have proven their potential as useful molecular probes and as next-generation therapeutics. Two crucial features of this protocol are (i) the construction of peptide substrates containing hindered α-methyl, α-alkenyl amino acids and (ii) the ring-closing olefin metathesis (RCM) of the resulting resin-bound peptide substrates. The stapling systems described in this protocol, namely bridging one or two turns of an α-helix, are highly adaptable to most peptide sequences, resulting in favorable RCM kinetics, helix stabilization and promotion of cellular uptake.

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: Schematic representation of three stapled peptides.
Figure 2: Design of stapled peptides.
Figure 3: Overall synthetic scheme for the preparation of stapled peptides using Fmoc-based solid-phase peptide synthesis.
Figure 4
Figure 5: HPLC profiles of RCM reactions of a resin-bound model peptide (Fmoc-EWAS5TAAS5KFLAAHA8,18) monitored at 280 nm after (a) 0 min, (b) 10 min, (c) 30 min, (d) 60 min, (e) 120 min and (f) 240 min.
Figure 6

Similar content being viewed by others

References

  1. Verdine, G.L. & Walensky, L.D. The challenge of drugging undruggable targets in cancer: lessons learned from targeting BCL-2 family members. Clin. Cancer Res. 13, 7264–7270 (2007).

    Article  CAS  Google Scholar 

  2. Mimna, R., Tuchscherer, G. & Mutter, M. Toward the design of highly efficient, readily accessible peptide N-caps for the induction of helical conformations. Int. J. Pept. Res. Ther. 13, 237–244 (2007).

    Article  CAS  Google Scholar 

  3. Henchey, L.K., Jochim, A.L. & Arora, P.S. Contemporary strategies for the stabilization of peptides in the alpha-helical conformation. Curr. Opin. Chem. Biol 12, 692–697 (2008).

    Article  CAS  Google Scholar 

  4. Schafmeister, C.E., Po, J. & Verdine, G.L. An all-hydrocarbon cross-linking system for enhancing the helicity and metabolic stability of peptides. J. Am. Chem. Soc. 122, 5891–5892 (2000).

    Article  CAS  Google Scholar 

  5. Blackwell, H.E. & Grubbs, R.H. Highly efficient synthesis of covalently crosslinked peptide helices by ring-closing metathesis. Angew. Chem. Int. Ed. 37, 3281–3284 (1998).

    Article  CAS  Google Scholar 

  6. Walensky, L.D. et al. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix. Science 305, 1466–1470 (2004).

    Article  CAS  Google Scholar 

  7. Bhattacharya, S., Zhang, H.T., Debnath, A.K. & Cowburn, D. Solution structure of a hydrocarbon stapled peptide inhibitor in complex with monomeric C-terminal domain of HIV-1 capsid. J. Biol. Chem. 283, 16274–16278 (2008).

    Article  CAS  Google Scholar 

  8. Zhang, H. et al. A cell-penetrating helical peptide as a potential HIV-1 inhibitor. J. Mol. Biol. 378, 565–580 (2008).

    Article  CAS  Google Scholar 

  9. Bernal, F., Tyler, A.F., Korsmeyer, S.J., Walensky, L.D. & Verdine, G.L. Reactivation of the p53 tumor suppressor pathway by a stapled p53 peptide. J. Am. Chem. Soc. 129, 2456–2457 (2007).

    Article  CAS  Google Scholar 

  10. Danial, N.N. et al. Dual role of proapoptotic BAD in insulin secretion and beta cell survival. Nat. Med. 14, 144–153 (2008).

    Article  CAS  Google Scholar 

  11. Gavathiotis, E. et al. BAX activation is initiated at a novel interaction site. Nature 455, 1076–1081 (2008).

    Article  CAS  Google Scholar 

  12. Moellering, R.E. et al. Direct inhibition of the NOTCH transcription factor complex. Nature 462, 182–188 (2009).

    Article  CAS  Google Scholar 

  13. Stewart, M.L., Fire, E., Keating, A.E. & Walensky, L.D. The MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis sensitizer. Nat. Chem. Biol. 6, 595–601 (2010).

    Article  CAS  Google Scholar 

  14. Bird, G.H. et al. Hydrocarbon double-stapling remedies the proteolytic instability of a lengthy peptide therapeutic. Proc. Natl. Acad. Sci. USA 107, 14093–14098 (2010).

    Article  CAS  Google Scholar 

  15. Walensky, L.D. et al. A stapled BID BH3 helix directly binds and activates BAX. Mol. Cell 24, 199–210 (2006).

    Article  CAS  Google Scholar 

  16. Bernal, F. et al. A stapled p53 helix overcomes HDMX-mediated suppression of p53. Cancer Cell. 18, 411–422 (2010).

    Article  CAS  Google Scholar 

  17. Kim, Y.W., Kutchukian, P.S. & Verdine, G.L. Introduction of all-hydrocarbon i,i+3 staples into alpha-helices via ring-closing olefin metathesis. Org. Lett. 12, 3046–3049 (2010).

    Article  CAS  Google Scholar 

  18. Kim, Y.W. & Verdine, G.L. Stereochemical effects of all-hydrocarbon tethers in i,i+4 stapled peptides. Bioorg. Med. Chem. Lett. 19, 2533–2536 (2009).

    Article  CAS  Google Scholar 

  19. Williams, R.M. & Im, M.N. Asymmetric-synthesis of monosubstituted and alpha,alpha-disubstituted alpha-amino-acids via diastereoselective glycine enolate alkylations. J. Am. Chem. Soc. 113, 9276–9286 (1991).

    Article  CAS  Google Scholar 

  20. Belokon, Y.N., Tararov, V.I., Maleev, V.I., Savel'eva, T.F. & Ryzhov, M.G. Improved procedures for the synthesis of (S)-2-[N-(N′-benzylprolyl)amino]benzophenone (BPB) and Ni(II) complexes of Schiff's bases derived from BPB and amino acids. Tetrahedron: Asymmetry 9, 4249–4252 (1998).

    Article  CAS  Google Scholar 

  21. Qiu, W., Soloshonok, V.A., Cai, C.Z., Tang, X.J. & Hruby, V. Convenient, large-scale asymmetric synthesis of enantiomerically pure trans-cinnamylglycine and -alpha-alanine. Tetrahedron 56, 2577–2582 (2000).

    Article  CAS  Google Scholar 

  22. Bird, G.H., Bernal, F., Pitter, K. & Walensky, L.D. Synthesis and biophysical characterization of stabilized alpha-helices of BCL-2 domains. in Programmed Cell Death, The Biology and Therapeutic Implications of Cell Death, Part B. Methods Enzymol. 446, 369–386 (2008).

    Article  CAS  Google Scholar 

  23. Berman, H.M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).

    Article  CAS  Google Scholar 

  24. Marder, O., Shvo, Y. & Albericio, F. HCTU and TCTU. New coupling reagents: development and industrial aspects. Chim. Oggi-Chem. Today 20, 37–41 (2002).

    CAS  Google Scholar 

  25. Sabatino, G. et al. Assessment of new 6-Cl-HOBt based coupling reagents for peptide synthesis. Part 1: coupling efficiency study. Lett. Pept. Sci. 9, 119–123 (2002).

    CAS  Google Scholar 

  26. Hood, C.A. et al. Fast conventional Fmoc solid-phase peptide synthesis with HCTU. J. Pept. Sci. 14, 97–101 (2008).

    Article  CAS  Google Scholar 

  27. Subiros-Funosas, R. et al. PyClocK, the phosphonium salt derived from 6-Cl- HOBt. Chim. Oggi-Chem. Today 26, 10–12 (2008).

    CAS  Google Scholar 

  28. Carpino, L.A. 1-hydroxy-7-azabenzotriazole—an efficient peptide coupling additive. J. Am. Chem. Soc. 115, 4397–4398 (1993).

    Article  CAS  Google Scholar 

  29. Albericio, F., Bofill, J.M., El-Faham, A. & Kates, S.A. Use of onium salt-based coupling reagents in peptide synthesis. J. Org. Chem. 63, 9678–9683 (1998).

    Article  CAS  Google Scholar 

  30. Boal, A.K. et al. Facile and E-selective intramolecular ring-closing metathesis reactions in 3(10)-helical peptides: a 3D structural study. J. Am. Chem. Soc. 129, 6986–6987 (2007).

    Article  CAS  Google Scholar 

  31. Chapman, R.N., Dimartino, G. & Arora, P.S. A highly stable short alpha-helix constrained by a main-chain hydrogen-bond surrogate. J. Am. Chem. Soc. 126, 12252–12253 (2004).

    Article  CAS  Google Scholar 

  32. Chapman, R.N. & Arora, P.S. Optimized synthesis of hydrogen-bond surrogate helices: surprising effects of microwave heating on the activity of Grubbs catalysts. Org. Lett. 8, 5825–5828 (2006).

    Article  CAS  Google Scholar 

  33. Toniolo, C., Crisma, M., Formaggio, F. & Peggion, C. Control of peptide conformation by the Thorpe-Ingold effect (C-alpha-tetrasubstitution). Biopolymers 60, 396–419 (2001).

    Article  CAS  Google Scholar 

  34. Urnes, P. & Doty, P. Optical rotation and the conformation of polypeptides and proteins. Adv. Protein Chem. 16, 401–544 (1961).

    Article  CAS  Google Scholar 

  35. Sohma, Y., Sasaki, M., Hayashi, Y., Kimura, T. & Kiso, Y. Novel and efficient synthesis of difficult sequence-containing peptides through O-N intramolecular acyl migration reaction of O-acyl E isopeptides. Chem. Commun. 124–125 (2004).

  36. Johnson, T., Quibell, M., Owen, D. & Sheppard, R.C. A reversible protecting group for the amide bond in peptides—use in the synthesis of difficult sequences. J. Chem. Soc. Chem. Commun. 369–372 (1993).

  37. Haack, T. & Mutter, M. Serine derived oxazolidines as secondary structure disrupting, solubilizing building-blocks in peptide-synthesis. Tetrahedron Lett. 33, 1589–1592 (1992).

    Article  CAS  Google Scholar 

  38. Lauer, J.L., Fields, C.G. & Fields, G.B. Sequence dependence of aspartimide formation during 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. Lett. Pept. Sci. 1, 197–205 (1994).

    Article  Google Scholar 

  39. Crabb, J.W., West, K.A., Dodson, W.S. & Hulmes, J.D. Amino acid analysis. Curr. Protoc. Protein Sci. 11.9.42 (2001).

  40. Gasteiger, E. et al. Protein identification and analysis tools on the ExPASy server. in The Proteomics Protocols Handbook (ed. Walker, J.M.) 571–607 (Humana Press, 2005).

Download references

Acknowledgements

This research was supported by the Harvard and Dana-Farber Program in Cancer Chemical Biology. T.N.G. is grateful for a fellowship from Deutsche Akademie der Naturforscher Leopoldina (LPDS 2009-2).

Author information

Author notes

  1. Young-Woo Kim

    Present address: Present address: College of Pharmacy, Dongguk University, Goyang-si, Seoul, Korea (Y.-W.K.).,

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Young-Woo Kim, Tom N Grossmann & Gregory L Verdine

  2. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA

    Young-Woo Kim, Tom N Grossmann & Gregory L Verdine

  3. Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Young-Woo Kim & Gregory L Verdine

  4. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA

    Gregory L Verdine

Authors
  1. Young-Woo Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tom N Grossmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gregory L Verdine
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.L.V. directed the research and, with Y.-W.K., conceptualized the experiments. Y.-W.K. performed the experiments. All authors discussed the results and wrote the manuscript.

Corresponding author

Correspondence to Gregory L Verdine.

Ethics declarations

Competing interests

G.L.V. is a shareholder in and a paid consultant of Aileron Therapeutics, which has been granted a license from Harvard University and the Dana-Farber Cancer Institute to develop the stapled peptide technology.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, YW., Grossmann, T. & Verdine, G. Synthesis of all-hydrocarbon stapled α-helical peptides by ring-closing olefin metathesis. Nat Protoc 6, 761–771 (2011). https://doi.org/10.1038/nprot.2011.324

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.324

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