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.

Chemical synthesis of proteins using peptide hydrazides as thioester surrogates

Nature Protocols volume 8, pages 2483–2495 (2013)Cite this article

Subjects

Abstract

This protocol provides a detailed procedure for the chemical synthesis of proteins through native chemical ligation of peptide hydrazides. The two crucial stages of this protocol are (i) the solid-phase synthesis of peptide hydrazides via Fmoc chemistry and (ii) the native chemical ligation of peptide hydrazides through in situ NaNO2 activation and thiolysis. This protocol may be of help in the synthesis of proteins that are not easily produced by recombinant technology and that include acid-sensitive modifications; it also does not involve the use of hazardous HF. The utility of the protocol is shown for the total synthesis of 140-aa-long α-synuclein, a protein that has an important role in the development of Parkinson's disease. The whole synthesis of the target protein α-synuclein in milligram scale takes ∼30 working days.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: General description for the native chemical ligation of peptide hydrazides.
Figure 2: Synthetic route for the preparation of α-synuclein 8.
Figure 3: Characterization of synthetic α-synuclein 8.
Figure 4: RP-HPLC traces for the native chemical ligation of α-synuclein(1–29)-NHNH2 1 with α-synuclein(30–68, A30C)-NHNH2 2.

References

  1. Kent, S.B.H. Total chemical synthesis of proteins. Chem. Soc. Rev. 38, 338–351 (2009).

    Article  CAS  Google Scholar 

  2. Nilsson, B.L., Soellner, M.B. & Raines, R.T. Chemical synthesis of proteins. Annu. Rev. Biophys. Biomol. Struct. 34, 91–118 (2005).

    Article  CAS  Google Scholar 

  3. Yeates, T.O. & Kent, S.B.H. Racemic protein crystallography. Annu. Rev. Biophys. 41, 41–61 (2012).

    Article  CAS  Google Scholar 

  4. Dawson, P.E., Muir, T.W., Clark-Lewis, I. & Kent, S.B.H. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994).

    Article  CAS  Google Scholar 

  5. Johnson, E.C.B. & Kent, S.B.H. Insights into the mechanism and catalysis of the native chemical ligation reaction. J. Am. Chem. Soc. 128, 6640–6646 (2006).

    Article  CAS  Google Scholar 

  6. Schnolzer, M., Alewood, P., Jones, A., Alewood, D. & Kent, S.B.H. In situ neutralization in Boc-chemistry solid phase peptide synthesis. Int. J. Pept. Res. Ther. 13, 31–44 (2007).

    Article  CAS  Google Scholar 

  7. Shin, Y. et al. Fmoc-based synthesis of peptide-thioesters: application to the total chemical synthesis of a glycoprotein by native chemical ligation. J. Am. Chem. Soc. 121, 11684–11689 (1999).

    Article  CAS  Google Scholar 

  8. Huse, M., Holford, M.N., Kuriyan, J. & Muir, T.W. Semisynthesis of hyperphosphorylated type I TGF-β receptor: addressing the mechanism of kinase activation. J. Am. Chem. Soc. 122, 8337–8338 (2000).

    Article  CAS  Google Scholar 

  9. Tolbert, T.J. & Wong, C.-H. Intein-mediated synthesis of proteins containing carbohydrates and other molecular probes. J. Am. Chem. Soc. 122, 5421–5428 (2000).

    Article  CAS  Google Scholar 

  10. Ingenito, R., Bianchi, E., Fattori, D. & Pessi, A. Solid phase synthesis of peptide C-terminal thioesters by Fmoc/t-Bu chemistry. J. Am. Chem. Soc. 121, 11369–11374 (1999).

    Article  CAS  Google Scholar 

  11. Kawakami, T. & Aimoto, S. Peptide ligation using a building block having a cysteinyl prolyl ester (CPE) autoactivating unit at the carboxy terminus. Chem. Lett. 36, 76–77 (2007).

    Article  CAS  Google Scholar 

  12. Blanco-Canosa, J.B. & Dawson, P.E. An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation. Angew. Chem. Int. Ed. 47, 6851–6855 (2008).

    Article  CAS  Google Scholar 

  13. Tofteng, A.P., Sorensen, K.K., Conde-Frieboes, K.W., Hoeg-Jensen, T. & Jensen, K.J. Fmoc solid-phase synthesis of C-terminal peptide thioesters by formation of a backbone pyroglutamyl imide moiety. Angew. Chem. Int. Ed. 48, 7411–7414 (2009).

    Article  CAS  Google Scholar 

  14. Kang, J., Richardson, J.P. & Macmillan, D. 3-Mercaptopropionic acid-mediated synthesis of peptide and protein thioesters. Chem. Commun. 2009, 407–409 (2009).

    Article  Google Scholar 

  15. Tsuda, S., Shigenaga, A., Bando, K. & Otaka, A. N-to-S acyl-transfer-mediated synthesis of peptide thioesters using anilide derivatives. Org. Lett. 11, 823–826 (2009).

    Article  CAS  Google Scholar 

  16. Zheng, J.S., Cui, H.K., Fang, G.M., Xi, W.X. & Liu, L. Chemical protein synthesis by kinetically controlled ligation of peptide O-esters. ChemBioChem 11, 511–515 (2010).

    Article  CAS  Google Scholar 

  17. Ollivier, N., Dheur, J., Mhidia, R., Blanpain, A. & Melnyk, O. Bis(2-sulfanylethyl)amino native peptide ligation. Org. Lett. 12, 5238–5241 (2010).

    Article  CAS  Google Scholar 

  18. Zheng, J.S., Chang, H.N., Wang, F.L. & Liu, L. Fmoc synthesis of peptide thioesters without post-chain-assembly manipulation. J. Am. Chem. Soc. 133, 11080–11083 (2011).

    Article  CAS  Google Scholar 

  19. Fang, G.M. et al. Protein chemical synthesis by ligation of peptide hydrazides. Angew. Chem. Int. Ed. 50, 7645–7649 (2011).

    Article  CAS  Google Scholar 

  20. Fang, G.M., Wang, J.X. & Liu, L. Convergent chemical synthesis of proteins by ligation of peptide hydrazides. Angew. Chem. Int. Ed. 51, 10347–10350 (2012).

    Article  CAS  Google Scholar 

  21. Zheng, J.S., Tang, S., Guo, Y., Chang, H.N. & Liu, L. Synthesis of cyclic peptides and cyclic proteins via ligation of peptide hydrazides. ChemBioChem 13, 542–546 (2012).

    Article  CAS  Google Scholar 

  22. Tang, S., Zheng, J.S., Yang, K. & Liu, L. Synthesis of cyclic tetrapeptides via ligation of peptide hydrazides. Acta Chim. Sinica 70, 1471–1476 (2012).

    Article  CAS  Google Scholar 

  23. Li, Y.M. et al. Ligation of expressed protein α-hydrazides via genetic incorporation of an α-hydroxy acid. ACS Chem. Biol. 7, 1015–1022 (2012).

    Article  CAS  Google Scholar 

  24. Siman, P., Karthikeyan, S.V., Nikolov, M., Fischle, W. & Brik, A. Convergent chemical synthesis of histone H2B protein for the site-specific ubiquitination at Lys34. Angew. Chem. Int. Ed. 52, 8059–8063 (2013).

    Article  CAS  Google Scholar 

  25. Cremades, N. et al. Direct observation of the interconversion of normal and toxic forms of α-synuclein. Cell 149, 1048–1059 (2012).

    Article  CAS  Google Scholar 

  26. Luk, K.C. et al. Pathological α-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science 338, 949–953 (2012).

    Article  CAS  Google Scholar 

  27. Lashuel, H.A., Overk, C.R., Oueslati, A. & Masliah, E. The many faces of α-synuclein: from structure and toxicity to therapeutic target. Nat. Rev. Neurosci. 14, 38–48 (2013).

    Article  CAS  Google Scholar 

  28. Paleologou, K.E. et al. Phosphorylation at S87 is enhanced in synucleinopathies, inhibits α-synuclein oligomerization, and influences synuclein-membrane interactions. J. Neurosci. 30, 3184–3198 (2010).

    Article  CAS  Google Scholar 

  29. Braithwaite, S.P., Stock, J.B. & Mouradian, M.M. α-Synuclein phosphorylation as a therapeutic target in Parkinson's disease. Rev. Neurosci. 23, 191–198 (2012).

    Article  CAS  Google Scholar 

  30. Hejjaoui, M. et al. Elucidating the role of C-terminal post-translational modifications using protein semisynthesis strategies: α-synuclein phosphorylation at tyrosine 125. J. Am. Chem. Soc. 134, 5196–5210 (2012).

    Article  CAS  Google Scholar 

  31. Wan, Q. & Danishefsky, S.J. Free-radical-based, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides. Angew. Chem. Int. Ed. 46, 9248–9252 (2007).

    Article  CAS  Google Scholar 

  32. Rochet, J.C., Conway, K.A. & Lansbury, P.T. Jr. Inhibition of fibrillization and accumulation of prefibrillar oligomers in mixtures of human and mouse α-synuclein. Biochemistry 39, 10619–10626 (2000).

    Article  CAS  Google Scholar 

  33. Carpino, L.A. et al. The uronium/guanidinium peptide coupling reagents: finally the true uronium salts. Angew. Chem. Int. Ed. 41, 441–445 (2002).

    Article  CAS  Google Scholar 

  34. Palladino, P. & Stetsenko, D.A. New TFA-free cleavage and final deprotection in Fmoc solid-phase peptide synthesis: dilute HCl in fluoro alcohol. Org. Lett. 14, 6346–6349 (2012).

    Article  CAS  Google Scholar 

  35. Kaiser, E., Colescot, R.L., Bossinge, 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 

Download references

Acknowledgements

This study was supported by the National Basic Research Program of China (973 program, no. 2013CB932800), the '863' Program of the Ministry of Science and Technology (grant no. 2012AA02A700), the National Natural Science Foundation of China (grants no. 20932006), National Science Fund for Distinguished Young Scholars and the Specialized Research Fund for the Doctoral Program of Higher Education (grant no. 20120002130004). We thank T. Chu and Y. Chen for technical assistance in the biophysical characterization of synthetic α-synuclein 8.

Author information

Authors and Affiliations

  1. Department of Chemistry, Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Tsinghua University, Beijing, China

    Ji-Shen Zheng, Shan Tang, Yun-Kun Qi, Zhi-Peng Wang & Lei Liu

Authors
  1. Ji-Shen Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shan Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yun-Kun Qi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhi-Peng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.L. conceived and led the project. J.-S.Z., S.T., Y.-K.Q. and Z.-P.W. conducted the experiments and co-wrote the manuscript with L.L.

Corresponding author

Correspondence to Lei Liu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The general chemical synthetic strategy of α-synuclein 8.

Supplementary Figure 2 RP-HPLC analysis of α-synuclein(1-29)-NHNH2 running on a C4-column.

The linear gradient for analytical HPLC: 5% buffer B in buffer A to 95% B in A over 30 min at rt with flow rate 1 mL/min (Left: the crude and Right: purified).

Supplementary Figure 3 ESI-MS of α-synuclein(1-29)-NHNH2.

Supplementary Figure 4 RP-HPLC analysis of α-synuclein(30-68, A30C)-NHNH2 running on a C4-column.

The linear gradient for analytical HPLC: 5% buffer B in buffer A to 95% B in A over 30 min at rt (Left: the crude and Right: purified).

Supplementary Figure 5 ESI-MS of α-synuclein(30-68, A30C)-NHNH2.

Supplementary Figure 6 RP-HPLC analysis of α-synuclein(69-106, A69C)-NHNH2 running on a C4-column.

The linear gradient for analytical HPLC: 5% buffer B in buffer A to 95% B in A over 30 min at rt (Left: the crude and Right: purified).

Supplementary Figure 7 ESI-MS of α-synuclein(69-106, A69C)-NHNH2.

Supplementary Figure 8 RP-HPLC analysis of α-synuclein(107-140, A107C)-OH running on a C4-column.

The linear gradient for analytical HPLC: 5% buffer B in buffer A to 95% B in A over 30 min at rt (Left: the crude and Right: purified).

Supplementary Figure 9 ESI-MS of α-synuclein(107-140, A107C)-OH.

Supplementary Figure 10 SDS-PAGE analysis and circular dichroism spectrum of 8.

(a) SDS-PAGE analysis and (b) circular dichroism spectra of synthetic α-synuclein 8 (proteins (∼10 μM) were dissolved in 20 mM sodium phosphate pH 7.4 and analyzed in a 1 mm quartz cell).

Supplementary Figure 11 Thioflavin T assays of purified α-synuclein 8.

The sample with 10 μM (containing 20 mM Tris, 150 mM NaCl, pH 7.5) is incubated at 37° C. Aliquots are taken and analyzed by ThT fluorescence (450 nm/482 nm) at the indicated time points. The experiments are performed in triplicate.

Supplementary information

Supplementary Figure 1

The general chemical synthetic strategy of α-synuclein 8. (PDF 282 kb)

Supplementary Figure 2

RP-HPLC analysis of α-synuclein(1-29)-NHNH2 running on a C4-column. The linear gradient for analytical HPLC: 5% buffer B in buffer A to 95% B in A over 30 min at rt with flow rate 1 mL/min (Left: the crude and Right: purified). (PDF 216 kb)

Supplementary Figure 3

ESI-MS of α-synuclein(1-29)-NHNH2. (PDF 157 kb)

Supplementary Figure 4

RP-HPLC analysis of α-synuclein(30-68, A30C)-NHNH2 running on a C4-column. The linear gradient for analytical HPLC: 5% buffer B in buffer A to 95% B in A over 30 min at rt (Left: the crude and Right: purified). (PDF 739 kb)

Supplementary Figure 5

ESI-MS of α-synuclein(30-68, A30C)-NHNH2. (PDF 79 kb)

Supplementary Figure 6

RP-HPLC analysis of α-synuclein(69-106, A69C)-NHNH2 running on a C4-column. The linear gradient for analytical HPLC: 5% buffer B in buffer A to 95% B in A over 30 min at rt (Left: the crude and Right: purified). (PDF 235 kb)

Supplementary Figure 7

ESI-MS of α-synuclein(69-106, A69C)-NHNH2. (PDF 180 kb)

Supplementary Figure 8

RP-HPLC analysis of α-synuclein(107-140, A107C)-OH running on a C4-column. The linear gradient for analytical HPLC: 5% buffer B in buffer A to 95% B in A over 30 min at rt (Left: the crude and Right: purified). (PDF 236 kb)

Supplementary Figure 9

ESI-MS of α-synuclein(107-140, A107C)-OH. (PDF 174 kb)

Supplementary Figure 10

SDS-PAGE analysis and circular dichroism spectrum of 8. (a) SDS-PAGE analysis and (b) circular dichroism spectra of synthetic α-synuclein 8 (proteins (∼10 μM) were dissolved in 20 mM sodium phosphate pH 7.4 and analyzed in a 1 mm quartz cell). (PDF 292 kb)

Supplementary Figure 11

Thioflavin T assays of purified α-synuclein 8. The sample with 10 μM (containing 20 mM Tris, 150 mM NaCl, pH 7.5) is incubated at 37° C. Aliquots are taken and analyzed by ThT fluorescence (450 nm/482 nm) at the indicated time points. The experiments are performed in triplicate. (PDF 201 kb)

Supplementary Data 1

RP-HPLC trace of native chemical ligation of 1 with 2 running on a C4- column. (PDF 550 kb)

Supplementary Data 2

RP-HPLC trace of native chemical ligation of α-synuclein(1-68, A30C)-NHNH2 with α-synuclein(68-106, A69C)-NHNH2 running on a C4-column. (PDF 496 kb)

Supplementary Data 3

RP-HPLC trace of native chemical ligation of α-synuclein(1-106, A30,69C)-NHNH2 with α-synuclein(107-140, A107C)-OH running on a C4-column. (PDF 333 kb)

Supplementary Data 4

RP-HPLC trace of desulfurization of α-synuclein(1-140, A30,69,107C)-OH running on a C4-column. (PDF 425 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zheng, JS., Tang, S., Qi, YK. et al. Chemical synthesis of proteins using peptide hydrazides as thioester surrogates. Nat Protoc 8, 2483–2495 (2013). https://doi.org/10.1038/nprot.2013.152

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.152

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