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:

One-pot chemical synthesis of small ubiquitin-like modifier protein–peptide conjugates using bis(2-sulfanylethyl)amido peptide latent thioester surrogates

Nature Protocols volume 10, pages 269–292 (2015)Cite this article

Subjects

Abstract

Small ubiquitin-like modifier (SUMO) post-translational modification (PTM) of proteins has a crucial role in the regulation of important cellular processes. This protocol describes the chemical synthesis of functional SUMO–peptide conjugates. The two crucial stages of this protocol are the solid-phase synthesis of peptide segments derivatized by thioester or bis(2-sulfanylethyl)amido (SEA) latent thioester functionalities and the one-pot assembly of the SUMO–peptide conjugate by a sequential native chemical ligation (NCL)/SEA native peptide ligation reaction sequence. This protocol also enables the isolation of a SUMO SEA latent thioester, which can be attached to a target peptide or protein in a subsequent step. It is compatible with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, and it gives access to homogeneous, reversible and functional SUMO conjugates that are not easily produced using living systems. The synthesis of SUMO–peptide conjugates on a milligram scale takes 20 working days.

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: General description of the one-pot, three-peptide segment assembly of SUMO–peptide conjugates.
Figure 2: Synthetic route for the preparation of SUMO-1–peptide conjugate 6.
Figure 3: Characterization of synthetic SUMO-1–peptide conjugate 6.
Figure 4: Reversed-phase HPLC monitoring of the one-pot three-segment assembly process leading to the formation of SUMO-1–peptide conjugate 6.

Similar content being viewed by others

References

  1. Fierz, B. et al. Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction. Nat. Chem. Biol. 7, 113–119 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Siman, P. et al. Convergent chemical synthesis of histone H2B protein for the site-specific ubiquitination at Lys34. Angew. Chem. Int. Ed. Engl. 52, 8059–8063 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Spasser, L. & Brik, A. Chemistry and biology of the ubiquitin signal. Angew. Chem. Int. Ed. Engl. 51, 6840–6862 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Kumar, K.S. et al. Total chemical synthesis of di-ubiquitin chains. Angew. Chem. Int. Ed. Engl. 49, 9126–9131 (2011).

    Article  CAS  Google Scholar 

  5. Kumar, K.S. et al. Total chemical synthesis of a 304-amino acid K48-linked tetraubiquitin protein. Angew. Chem. Int. Ed. Engl. 50, 6137–6141 (2011).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  8. Merrifield, R.B. Solid-phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149–2154 (1963).

    Article  CAS  Google Scholar 

  9. 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  PubMed  Google Scholar 

  10. El Oualid, F. et al. Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew. Chem. Int. Ed. Engl. 49, 10149–10153 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Erlich, L.A. et al. N-methylcysteine-mediated total chemical synthesis of ubiquitin thioester. Org. Biomol. Chem. 8, 2392–2396 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Mende, F. & Seitz, O. 9-Fluorenylmethoxycarbonyl-based solid-phase synthesis of peptide α-thioesters. Angew. Chem. Int. Ed. Engl. 50, 1232–1240 (2011).

    Article  CAS  PubMed  Google Scholar 

  13. Zheng, J.-S., Tang, S., Huang, Y.-C. & Liu, L. Development of new thioester equivalents for protein chemical synthesis. Acc. Chem. Res. 46, 2475–2484 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Van Treel, N.D. & Mootz, H.D. SUMOylated RanGAP1 prepared by click chemistry. J. Pept. Sci. 20, 121–127 (2013).

    Article  PubMed  CAS  Google Scholar 

  15. Sommer, S. et al. Expanded click conjugation of recombinant proteins with ubiquitin-like modifiers reveals altered substrate preference of SUMO2-modified Ubc9. Angew. Chem. Int. Ed. Engl. 50, 9888–9892 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Boll, E. et al. A novel PEG-based solid support enables the synthesis of >50 amino-acid peptide thioesters and the total synthesis of a functional SUMO-1–peptide conjugate. Chem. Sci. 5, 2017–2022 (2014).

    Article  CAS  Google Scholar 

  17. Ollivier, N. et al. Bis(2-sulfanylethyl)amino native peptide ligation. Org. Lett. 12, 5238–5241 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Hou, W., Zhang, X., Li, F. & Liu, C.F. Peptidyl N,N-bis(2-mercaptoethyl)-amides as thioester precursors for native chemical ligation. Org. Lett. 13, 386–389 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. Raibaut, L., Ollivier, N. & Melnyk, O. Sequential native peptide ligation strategies for total chemical protein synthesis. Chem. Soc. Rev. 41, 7001–7015 (2012).

    Article  CAS  PubMed  Google Scholar 

  20. Ollivier, N. et al. A one-pot three-segment ligation strategy for protein chemical synthesis. Angew. Chem. Int. Ed. Engl. 51, 209–213 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Raibaut, L. et al. Highly efficient solid phase synthesis of large polypeptides by iterative ligations of bis(2-sulfanylethyl)amido (SEA) peptide segments. Chem. Sci. 4, 4061–4066 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  23. Li, S.J. & Hochstrasser, M. A new protease required for cell-cycle progression in yeast. Nature 398, 246–251 (1999).

    Article  CAS  PubMed  Google Scholar 

  24. Ollivier, N. et al. Tidbits for the synthesis of bis(2-sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters. J. Pept. Sci. 20, 92–97 (2013).

    Article  PubMed  CAS  Google Scholar 

  25. Dheur, J., Ollivier, N., Vallin, A. & Melnyk, O. Synthesis of peptide alkylthioesters using the intramolecular N,S-acyl shift properties of bis(2-sulfanylethyl)amido peptides. J. Org. Chem. 76, 3194–3202 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Raibaut, L., Seeberger, P. & Melnyk, O. Bis(2-sulfanylethyl)amido peptides enable native chemical ligation at proline and minimize deletion side-product formation. Org. Lett. 15, 5516–5519 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Ancot, F. et al. Shedding-generated Met receptor fragments can be routed to either the proteasomal or the lysosomal degradation pathway. Traffic 13, 1261–1272 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Raibaut, L. et al. Total synthesis of biotinylated N domain of human hepatocyte growth factor. Bioorg. Med. Chem. 21, 3486–3494 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Garcia-Martin, F. et al. ChemMatrix, a poly(ethylene glycol)-based support for the solid-phase synthesis of complex peptides. J. Comb. Chem. 8, 213–220 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  31. Bang, D. & Kent, S.B. A one-pot total synthesis of crambin. Angew. Chem. Int. Ed. Engl. 43, 2534–2538 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Tan, Z., Shang, S. & Danishefsky, S.J. Insights into the finer issues of native chemical ligation: an approach to cascade ligations. Angew. Chem. Int. Ed. Engl. 49, 9500–9503 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ueda, S. et al. Photolabile protection for one-pot sequential native chemical ligation. Chembiochem 6, 1983–1986 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Bang, D., Pentelute, B.L. & Kent, S.B. Kinetically controlled ligation for the convergent chemical synthesis of proteins. Angew. Chem. Int. Ed. Engl. 45, 3985–3988 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Zheng, J.S. et al. Chemical protein synthesis by kinetically controlled ligation of peptide O-esters. Chembiochem 11, 511–515 (2010).

    Article  CAS  PubMed  Google Scholar 

  36. Sato, K. et al. N-sulfanylethylanilide peptide as a crypto-thioester peptide. Chembiochem 12, 1840–1844 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Okamoto, R., Morooka, K. & Kajihara, Y. A synthetic approach to a peptide alpha-thioester from an unprotected peptide through cleavage and activation of a specific peptide bond by N-acetylguanidine. Angew. Chem. Int. Ed. Engl. 51, 191–196 (2012).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  39. Zheng, J.-S. et al. Chemical synthesis of proteins using peptide hydrazides as thioester surrogates. Nat. Protoc. 8, 2483–2495 (2013).

    Article  CAS  PubMed  Google Scholar 

  40. Yang, R. et al. Dual native chemical ligation at lysine. J. Am. Chem. Soc. 131, 13592–13593 (2009).

    Article  CAS  PubMed  Google Scholar 

  41. Yang, R. et al. Synthesis of K48-linked diubiquitin using dual native chemical ligation at lysine. Chem. Commun. 46, 7199–7201 (2010).

    Article  CAS  Google Scholar 

  42. Greenfield, N. & Fasman, G.D. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry 8, 4108–4116 (1969).

    Article  CAS  PubMed  Google Scholar 

  43. Bayer, P. et al. Structure determination of the small ubiquitin-related modifier SUMO-1. J. Mol. Biol. 280, 275–286 (1998).

    Article  CAS  PubMed  Google Scholar 

  44. Muller, S., Hoege, C., Pyrowolakis, G. & Jentsch, S. SUMO, ubiquitin's mysterious cousin. Nat. Rev. Mol. Cell Biol. 2, 202–210 (2001).

    Article  CAS  PubMed  Google Scholar 

  45. Melnyk, O. & Agouridas, V. Perhydro-1,2,5-dithiazepine. e-EROS http://dx.doi.org/10.1002/047084289X.rn01723 (20 October 2014).

  46. Vojkovsky, T. Detection of secondary amines on solid phase. Pept. Res. 8, 236–237 (1995).

    CAS  PubMed  Google Scholar 

  47. 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  PubMed  Google Scholar 

  48. Hancock, W.S. & Battersby, J.E. A new micro-test for the detection of incomplete coupling reactions in solid-phase peptide synthesis using 2,4,6-trinitrobenzenesulphonic acid. Anal. Biochem. 71, 260–264 (1976).

    Article  CAS  PubMed  Google Scholar 

  49. Yung, A., Papworth-Smith, J. & Wilkinson, S.M. Occupational contact urticaria from the solid-phase peptide synthesis coupling agents HATU and HBTU. Contact Dermatitis 49, 108–109 (2003).

    Article  CAS  PubMed  Google Scholar 

  50. Dheur, J., Ollivier, N. & Melnyk, O. Synthesis of thiazolidine thioester peptides and acceleration of native chemical ligation. Org. Lett. 13, 1560–1563 (2011).

    Article  CAS  PubMed  Google Scholar 

  51. Mutter, M. et al. Pseudo-prolines (psi Pro) for accessing 'inaccessible' peptides. Pept. Res. 8, 145–153 (1995).

    CAS  PubMed  Google Scholar 

  52. 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 

  53. Wôhr, T. & Mutter, M. Pseudo-prolines in peptide synthesis: direct insertion of serine and threonine derived oxazolidines in dipeptides. Tetrahedron Lett. 36, 3847–3848 (1995).

    Article  Google Scholar 

  54. 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).

  55. Ball, H.L. et al. Engineering the prion protein using chemical synthesis. J. Pept. Res. 58, 357–374 (2001).

    Article  CAS  PubMed  Google Scholar 

  56. Cardona, V. et al. Application of Dmb-dipeptides in the Fmoc SPPS of difficult and aspartimide-prone sequences. Int. J. Pept. Res. Ther. 14, 285–292 (2008).

    Article  CAS  Google Scholar 

  57. Bourel, L., Carion, O., Gras-Masse, H. & Melnyk, O. The deprotection of Lys(Mtt) revisited. J. Pept. Sci. 6, 264–270 (2000).

    Article  CAS  PubMed  Google Scholar 

  58. Alnabari, M. & Bittner, S. Quinonic enaminones; Synthesis of new dialkylaminovinyl and bis(dialkylaminovinyl) derivatives of quinones. Synthesis 8, 1087–1090 (2000).

    Article  Google Scholar 

  59. Alnabari, M. & Bittner, S. New quinone-amino acid conjugates linked via a vinylic spacer. Amino Acids 20, 381–387 (2001).

    Article  CAS  PubMed  Google Scholar 

  60. Ellman, G.L. A colorimetric method for determining low concentrations of mercaptans. Arch. Biochem. Biophys. 74, 443–450 (1958).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported by the Centre National de la Recherche Scientifique (CNRS), the University of Lille, Institut Pasteur de Lille and the Site de Recherche Intégrée sur le Cancer (SIRIC) OncoLille.

Author information

Authors and Affiliations

  1. Centre National de la Recherche Scientifique, Lille Institute of Biology, Institut Pasteur de Lille, University of Lille, Lille, France

    Emmanuelle Boll, Hervé Drobecq, Nathalie Ollivier, Annick Blanpain, Laurent Raibaut, Rémi Desmet, Jérôme Vicogne & Oleg Melnyk

Authors
  1. Emmanuelle Boll
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hervé Drobecq
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nathalie Ollivier
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Annick Blanpain
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Laurent Raibaut
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rémi Desmet
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jérôme Vicogne
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Oleg Melnyk
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

O.M. designed and led the project. H.D., N.O., A.B., L.R., R.D. and J.V. conducted the experiments; E.B. conducted the experiment and co-wrote the manuscript with O.M.

Corresponding author

Correspondence to Oleg Melnyk.

Ethics declarations

Competing interests

O.M. is cofounder of X'ProChem company.

Integrated supplementary information

Supplementary Figure 1 HPLC chromatogram, ESI and MALDI-TOF spectra for purified SEAoff peptide segment 1.

Figure adapted from ref.16 with permission.

Supplementary Figure 2 HPLC chromatogram, ESI and MALDI-TOF spectra for SEAoff peptide segment 3.

Figure adapted from ref.16 with permission.

Supplementary Figure 3 HPLC analysis of the exchange reaction of the bis(2-sulfanylethyl)amino group by 3-mercaptopropionic acid (MPA). Synthesis of MPA thioester peptide 2.

Figure adapted from ref.16 with permission.

Supplementary Figure 4 HPLC chromatogram, ESI and MALDI-TOF spectra for purified MPA thioester peptide 2.

Figure adapted from ref.16 with permission.

Supplementary Figure 5 HPLC chromatogram for purified SUMO-1-SEAoff protein 4.

Figure adapted from ref.16 with permission.

Supplementary Figure 6 MALDI-TOF analysis of SUMO-1-SEAoff protein 4.

Figure adapted from ref.16 with permission.

Supplementary Figure 7 MALDI-TOF analysis of the purified SUMO-1-peptide conjugate 6.

Figure adapted from ref.16 with permission.

Supplementary Figure 8 MALDI-TOF and in source fragmentation analysis of the purified SUMO-1-peptide conjugate 6 (part 1).

Figure adapted from ref.16 with permission.

Supplementary Figure 9 MALDI-TOF and in source fragmentation analysis of the purified SUMO-1-peptide conjugate 6 (part 2).

Figure adapted from ref.16 with permission.

Supplementary Figure 10 Desumoylation of SUMO-1 peptide conjugate 6 by Ulp1 protease.

Micro HPLC analysis of the desumoylation of SUMO-1 peptide conjugate 6 by Ulp1 protease.1 Trace (a) Synthetic peptide 5; Trace (b) Synthetic SUMO-1 peptide conjugate 6 in the cleavage buffer. Trace (c) Cleavage mixture few seconds after addition of the enzyme Ulp1; SUMO-1 peptide conjugate 6 (0.75 mg/ml final concentration) and Ulp1 (2 units) were reacted in 41.5 mM Tris.HCl buffer, pH 8.0, 0.17% Igepal (NP-40) and 0.83 mM DTT at room temperature (20 °C). The peaks were collected and analyzed by MALTI-TOF MS using sinapinic acid as matrix. Experimental conditions for the microLC analysis: A: deionized H2O containing 0.1% (vol/vol) TFA; B: ACN 60% containing 0.1% (vol/vol) TFA. Flow rate 50 µl/min, gradient 0-100% of B in 30 min, Waters BEH300 C18 column, 5 µm, 1 × 150 mm, UV detection at 215 nm. Figure adapted from ref.16 with permission.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Notes 1–8, Supplementary Methods 1 and 2 (PDF 2503 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Boll, E., Drobecq, H., Ollivier, N. et al. One-pot chemical synthesis of small ubiquitin-like modifier protein–peptide conjugates using bis(2-sulfanylethyl)amido peptide latent thioester surrogates. Nat Protoc 10, 269–292 (2015). https://doi.org/10.1038/nprot.2015.013

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2015.013

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