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


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.

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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


  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

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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).

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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.

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Correspondence to Gregory L Verdine.

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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.

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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).

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