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

Nature Protocols volume 5, pages 1857–1865 (2010)Cite this article

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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.

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Figure 1
Figure 2: Synthesis of HBS α-helices on solid phase (Knorr or Rink amide resins).
Figure 4: Reaction setup for the triphosgene-mediated coupling step.
Figure 5: Representative analytical HPLC profile of the triphosgene-mediated coupling reaction (Step 4A(xix–xxiv)).
Figure 6: Synthesis of HBS helix 5 with method B.
Figure 3: Synthesis of HBS helix 5 with method A.
Figure 7: Reaction setup for the metathesis step.
Figure 8: A representative HPLC chromatogram of the olefin metathesis reaction (Step 6).

References

  1. Pauling, L., Corey, R.B. & Branson, H.R. The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain. Proc. Natl Acad. Sci. USA 37, 205–211 (1951).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Jochim, A.L. & Arora, P.S. Assessment of helical interfaces in protein-protein interactions. Mol. Biosyst. 5, 924–926 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. 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 

  10. Wang, D., Liao, W. & Arora, P.S. 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).

    Article  CAS  Google Scholar 

  11. Austin, R.E. 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).

    Article  CAS  Google Scholar 

  12. Chakrabartty, A., Doig, A.J. & Baldwin, R.L. Helix capping propensities in peptides parallel those in proteins. Proc. Natl Acad. Sci. USA 90, 11332–11336 (1993).

    Article  CAS  Google Scholar 

  13. Kemp, D.S., Curran, T.P., Davis, W.M., Boyd, J.G. & Muendel, C. 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).

    Article  CAS  Google Scholar 

  14. Kaul, R. & Balaram, P. Stereochemical control of peptide folding. Bioorg. Med. Chem. 7, 105–117 (1999).

    Article  CAS  Google Scholar 

  15. Lyu, P.C., Sherman, J.C., Chen, A. & Kallenbach, N.R. α-Helix stabilization by natural and unnatural amino acids with alkyl side chains. Proc. Natl Acad. Sci. USA 88, 5317–5320 (1991).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Jackson, D.Y., King, D.S., Chmielewski, J., Singh, S. & Schultz, P.G. General-approach to the synthesis of short alpha-helical peptides. J. Am. Chem. Soc. 113, 9391–9392 (1991).

    Article  CAS  Google Scholar 

  19. Osapay, G. & Taylor, J.W. 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).

    Article  CAS  Google Scholar 

  20. Phelan, J.C., Skelton, N.J., Braisted, A.C. & McDowell, R.S. A general method for constraining short peptides to an alpha-helical conformation. J. Am. Chem. Soc. 119, 455–460 (1997).

    Article  CAS  Google Scholar 

  21. Harrison, R.S. 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).

    Article  CAS  Google Scholar 

  22. Shepherd, N.E., Hoang, H.N., Abbenante, G. & Fairlie, D.P. Left- and right-handed alpha-helical turns in homo- and hetero-chiral helical scaffolds. J. Am. Chem. Soc. 131, 15877–15886 (2009).

    Article  CAS  Google Scholar 

  23. Ma, M.T., Hoang, H.N., Scully, C.C., Appleton, T.G. & Fairlie, D.P. Metal clips that induce unstructured pentapeptides to be alpha-helical in water. J. Am. Chem. Soc. 131, 4505–4512 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Cabezas, E. & Satterthwait, A.C. 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).

    Article  CAS  Google Scholar 

  26. 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 

  27. Wang, D., Chen, K., Kulp, J.L. III & Arora, P.S. Evaluation of biologically relevant short alpha-helices stabilized by a main-chain hydrogen-bond surrogate. J. Am. Chem. Soc. 128, 9248–9256 (2006).

    Article  CAS  Google Scholar 

  28. Liu, J., Wang, D., Zheng, Q., Lu, M. & Arora, P.S. Atomic structure of a short alpha-helix stabilized by a main chain hydrogen-bond surrogate. J. Am. Chem. Soc. 130, 4334–4337 (2008).

    Article  CAS  Google Scholar 

  29. Henchey, L.K. 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).

    Article  CAS  Google Scholar 

  30. 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 

  31. Dimartino, G., Wang, D., Chapman, R.N. & Arora, P.S. Solid-phase synthesis of hydrogen-bond surrogate-derived alpha-helices. Org. Lett. 7, 2389–2392 (2005).

    Article  CAS  Google Scholar 

  32. Patgiri, A., Witten, M.R. & Arora, P.S. 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).

    Article  CAS  Google Scholar 

  33. Trost, B.M. & Van Vranken, D.L. Asymmetric transition metal-catalyzed allylic alkylations. Chem. Rev. 96, 395–422 (1996).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. 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 

  36. Chan, W.C. & White, P.D. Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Oxford University Press, 2000).

  37. Kaiser, E., Colescot, R.L., Bossinger, C.D. & Cook, P.I. Color test for detection of free terminal amino groups in solid-phase synthesis of peptides. Anal. Biochem. 34, 595–598 (1970).

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

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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.

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

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

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  1. Anupam Patgiri
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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.

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Correspondence to Paramjit S Arora.

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Patgiri, A., Menzenski, M., Mahon, A. et al. Solid-phase synthesis of short α-helices stabilized by the hydrogen bond surrogate approach. Nat Protoc 5, 1857–1865 (2010). https://doi.org/10.1038/nprot.2010.146

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