Protocol | Published:

Solid-phase synthesis of short α-helices stabilized by the hydrogen bond surrogate approach

Nature Protocols volume 5, pages 18571865 (2010) | Download Citation

Abstract

Stabilized α-helices and nonpeptidic helix mimetics have emerged as powerful molecular scaffolds for the discovery of protein–protein interaction inhibitors. Protein-protein interactions often involve large contact areas, which are often difficult for small molecules to target with high specificity. The hypothesis behind the design of stabilized helices and helix mimetics is that these medium-sized molecules may pursue their targets with higher specificity because of a larger number of contacts. This protocol describes an optimized synthetic strategy for the preparation of stabilized α-helices that feature a carbon-carbon linkage in place of the characteristic N-terminal main-chain hydrogen bond of canonical helices. Formation of the carbon-carbon bond is enabled by a microwave-assisted ring-closing metathesis reaction between two terminal olefins on the peptide chain. The outlined strategy allows the synthesis and purification of a hydrogen bond surrogate (HBS) α-helix in 1 week.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain. Proc. Natl Acad. Sci. USA 37, 205–211 (1951).

  2. 2.

    & Protein-protein interactions—a review of protein dimer structures. Prog. Biophys. Mol. Biol. 63, 31–65 (1995).

  3. 3.

    & Assessment of helical interfaces in protein-protein interactions. Mol. Biosyst. 5, 924–926 (2009).

  4. 4.

    & Design and synthesis of alpha-helical peptides and mimetics. Org. Biomol. Chem. 5, 3577–3585 (2007).

  5. 5.

    , & Contemporary strategies for the stabilization of peptides in the alpha-helical conformation. Curr. Opin. Chem. Biol. 12, 692–697 (2008).

  6. 6.

    & Theory of the phase transition between helix and random coil in polypeptide chains. J. Chem. Phys. 31, 526–535 (1959).

  7. 7.

    & On the theory of helix-coil transitions in polypeptides. J. Chem. Phys. 34, 1963–1974 (1961).

  8. 8.

    & Helix-coil theories: a comparative study for finite length preferences. J. Phys. Chem. 96, 3987–3994 (1992).

  9. 9.

    , & An all-hydrocarbon cross-linking system for enhancing the helicity and metabolic stability of peptides. J. Am. Chem. Soc. 122, 5891–5892 (2000).

  10. 10.

    , & Enhanced metabolic stability and protein-binding properties of artificial alpha-helices derived from a hydrogen-bond surrogate: application to Bcl-xL. Angew. Chem. Int. Ed. Engl. 44, 6525–6529 (2005).

  11. 11.

    et al. A template for stabilization of a peptide α-helix: synthesis and evaluation of conformational effects by circular dichroism and nmr. J. Am. Chem. Soc. 119, 6461–6472 (1997).

  12. 12.

    , & Helix capping propensities in peptides parallel those in proteins. Proc. Natl Acad. Sci. USA 90, 11332–11336 (1993).

  13. 13.

    , , , & Studies of N-terminal templates for a-helix formation. Synthesis and conformational analysis of (2S,5S,8S,11S)-1-acetyl-1,4-diaza-3-keto-5-carboxy-10-thiatricyclo[2.8.1.04,8]-tridecane (Ac-Hel1-OH). J. Org. Chem. 56, 6672–6682 (1991).

  14. 14.

    & Stereochemical control of peptide folding. Bioorg. Med. Chem. 7, 105–117 (1999).

  15. 15.

    , , & α-Helix stabilization by natural and unnatural amino acids with alkyl side chains. Proc. Natl Acad. Sci. USA 88, 5317–5320 (1991).

  16. 16.

    & Highly efficient synthesis of covalently cross-linked peptide helices by ring-closing metathesis. Angew. Chem. Int. Ed. Engl. 37, 3281–3284 (1998).

  17. 17.

    & Secondary structure nucleation in peptides—transition-metal ion stabilized alpha-helices. J. Am. Chem. Soc. 112, 1630–1632 (1990).

  18. 18.

    , , , & General-approach to the synthesis of short alpha-helical peptides. J. Am. Chem. Soc. 113, 9391–9392 (1991).

  19. 19.

    & Multicyclic polypeptide model compounds. 2. synthesis and conformational properties of a highly alpha-helical uncosapeptide constrained by 3 side-chain to side-chain lactam bridges. J. Am. Chem. Soc. 114, 6966–6973 (1992).

  20. 20.

    , , & A general method for constraining short peptides to an alpha-helical conformation. J. Am. Chem. Soc. 119, 455–460 (1997).

  21. 21.

    et al. Downsizing human, bacterial, and viral proteins to short water-stable alpha helices that maintain biological potency. Proc. Natl Acad. Sci. USA 107, 11686–11691 (2010).

  22. 22.

    , , & Left- and right-handed alpha-helical turns in homo- and hetero-chiral helical scaffolds. J. Am. Chem. Soc. 131, 15877–15886 (2009).

  23. 23.

    , , , & Metal clips that induce unstructured pentapeptides to be alpha-helical in water. J. Am. Chem. Soc. 131, 4505–4512 (2009).

  24. 24.

    , & A hydrogen bond surrogate approach for stabilization of short peptide sequences in alpha-helical conformation. Acc. Chem. Res. 41, 1289–1300 (2008).

  25. 25.

    & The hydrogen bond mimic approach: solid-phase synthesis of a peptide stabilized as an alpha-helix with a hydrazone link. J. Am. Chem. Soc. 121, 3862–3875 (1999).

  26. 26.

    , & A highly stable short alpha-helix constrained by a main-chain hydrogen-bond surrogate. J. Am. Chem. Soc. 126, 12252–12253 (2004).

  27. 27.

    , , & Evaluation of biologically relevant short alpha-helices stabilized by a main-chain hydrogen-bond surrogate. J. Am. Chem. Soc. 128, 9248–9256 (2006).

  28. 28.

    , , , & Atomic structure of a short alpha-helix stabilized by a main chain hydrogen-bond surrogate. J. Am. Chem. Soc. 130, 4334–4337 (2008).

  29. 29.

    et al. Inhibition of hypoxia inducible factor 1–transcription coactivator interaction by a hydrogen bond surrogate alpha-helix. J. Am. Chem. Soc. 132, 941–943 (2010).

  30. 30.

    & Optimized synthesis of hydrogen-bond surrogate helices: surprising effects of microwave heating on the activity of grubbs catalysts. Org. Lett. 8, 5825–5828 (2006).

  31. 31.

    , , & Solid-phase synthesis of hydrogen-bond surrogate-derived alpha-helices. Org. Lett. 7, 2389–2392 (2005).

  32. 32.

    , & Solid phase synthesis of hydrogen bond surrogate derived alpha-helices: resolving the case of a difficult amide coupling. Org. Biomol. Chem. 8, 1773–1776 (2010).

  33. 33.

    & Asymmetric transition metal-catalyzed allylic alkylations. Chem. Rev. 96, 395–422 (1996).

  34. 34.

    & oNBS-SPPS: a new method for solid-phase peptide synthesis. J. Am. Chem. Soc. 120, 2690–2691 (1998).

  35. 35.

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

  36. 36.

    & Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Oxford University Press, 2000).

  37. 37.

    , , & Color test for detection of free terminal amino groups in solid-phase synthesis of peptides. Anal. Biochem. 34, 595–598 (1970).

  38. 38.

    Detection of secondary amines on solid-phase. Peptide Res. 8, 236–237 (1995).

Download references

Acknowledgements

We are grateful for financial support from the NIH (GM073943). We also thank the National Science Foundation for equipment Grant CHE-0958457 and the NIH National Center for Research Resources (NIRR) for Research Facilities Improvement Grant C06 RR-16572.

Author information

Affiliations

  1. Department of Chemistry, New York University, New York, USA.

    • Anupam Patgiri
    • , Monica Z Menzenski
    • , Andrew B Mahon
    •  & Paramjit S Arora

Authors

  1. Search for Anupam Patgiri in:

  2. Search for Monica Z Menzenski in:

  3. Search for Andrew B Mahon in:

  4. Search for Paramjit S Arora in:

Contributions

A.P. carried out the experiments as reported in the main paper; M.Z.M. tested the protocol; and A.P., M.Z.M., A.B.M. and P.S.A. wrote the article.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Paramjit S Arora.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2010.146

Further reading

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.