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Peptide ligation by chemoselective aminonitrile coupling in water

An Author Correction to this article was published on 07 February 2020

This article has been updated


Amide bond formation is one of the most important reactions in both chemistry and biology1,2,3,4, but there is currently no chemical method of achieving α-peptide ligation in water that tolerates all of the 20 proteinogenic amino acids at the peptide ligation site. The universal genetic code establishes that the biological role of peptides predates life’s last universal common ancestor and that peptides played an essential part in the origins of life5,6,7,8,9. The essential role of sulfur in the citric acid cycle, non-ribosomal peptide synthesis and polyketide biosynthesis point towards thioester-dependent peptide ligations preceding RNA-dependent protein synthesis during the evolution of life5,9,10,11,12,13. However, a robust mechanism for aminoacyl thioester formation has not been demonstrated13. Here we report a chemoselective, high-yielding α-aminonitrile ligation that exploits only prebiotically plausible molecules—hydrogen sulfide, thioacetate12,14 and ferricyanide12,14,15,16,17 or cyanoacetylene8,14—to yield α-peptides in water. The ligation is extremely selective for α-aminonitrile coupling and tolerates all of the 20 proteinogenic amino acid residues. Two essential features enable peptide ligation in water: the reactivity and pKaH of α-aminonitriles makes them compatible with ligation at neutral pH and N-acylation stabilizes the peptide product and activates the peptide precursor to (biomimetic) N-to-C peptide ligation. Our model unites prebiotic aminonitrile synthesis and biological α-peptides, suggesting that short N-acyl peptide nitriles were plausible substrates during early evolution.

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Fig. 1: Sulfide-mediated α-aminonitrile ligation

Data availability

All data supporting the findings of this study are available within the main text, Extended Data Tables 15, Extended Data Fig. 1 and the Supplementary Information (which contains Supplementary Discussion, Supplementary Figs. 1296, Supplementary Tables 116, experimental details and compound characterization data).

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  • 07 February 2020

    An Amendment to this paper has been published and can be accessed via a link at the top of the paper.


  1. Constable, D. J. C. et al. Key green chemistry research areas—a perspective from pharmaceutical manufacturers. Green Chem. 9, 411–420 (2007).

    Article  CAS  Google Scholar 

  2. Isidro-Llobet, A., Álvarez, M. & Albericio, F. Amino acid-protecting groups. Chem. Rev. 109, 2455–2504 (2009).

    Article  CAS  Google Scholar 

  3. Pattabiraman, V. R. & Bode, J. W. Rethinking amide bond synthesis. Nature 480, 471–479 (2011).

    Article  ADS  CAS  Google Scholar 

  4. Kulkarni, S. S., Sayers, J., Premdjee, B. & Payne, R. J. Rapid and efficient protein synthesis through expansion of the native chemical ligation concept. Nat. Rev. Chem. 2, 0122 (2018).

    Article  CAS  Google Scholar 

  5. Lee, D. H., Granja, J. R., Martinez, J. A., Severin, K. & Ghadiri, M. R. A self-replicating peptide. Nature 382, 525–528 (1996).

    Article  ADS  CAS  Google Scholar 

  6. Weber, A. L. & Pizzarello, S. The peptide-catalyzed stereospecific synthesis of tetroses: a possible model for prebiotic molecular evolution. Proc. Natl Acad. Sci. USA 103, 12713–12717 (2006).

    Article  ADS  CAS  Google Scholar 

  7. Adamala, K. & Szostak, J. W. Competition between model protocells driven by an encapsulated catalyst. Nat. Chem. 5, 495–501 (2013); corrigendum 5, 634 (2013).

    Article  CAS  Google Scholar 

  8. Patel, B. H., Percivalle, C., Ritson, D. J., Duffy, C. D. & Sutherland, J. D. Common origins of RNA, protein and lipid precursors in a cyanosulfidic protometabolism. Nat. Chem. 7, 301–307 (2015).

    Article  CAS  Google Scholar 

  9. Semenov, S. N. et al. Autocatalytic, bistable, oscillatory networks of biologically relevant organic reactions. Nature 537, 656–660 (2016).

    Article  ADS  CAS  Google Scholar 

  10. Lipmann, F. Attempts to map a process evolution of peptide biosynthesis. Science 173, 875–884 (1971).

    Article  ADS  CAS  Google Scholar 

  11. De Duve, C. Blueprint for a Cell: The Nature and Origin of Life (Neil Patterson Publishers, 1991).

  12. Liu, R. & Orgel, L. E. Oxidative acylation using thioacids. Nature 389, 52–54 (1997).

    Article  ADS  CAS  Google Scholar 

  13. Weber, A. L. Prebiotic amino acid thioester synthesis: thiol-dependent amino acid synthesis from formose substrates (formaldehyde and glycolaldehyde) and ammonia. Orig. Life Evol. Biosph. 28, 259–270 (1998).

    Article  ADS  CAS  Google Scholar 

  14. Bowler, F. R. et al. Prebiotically plausible oligoribonucleotide ligation facilitated by chemoselective acetylation. Nat. Chem. 5, 383–389 (2013).

    Article  CAS  Google Scholar 

  15. Keefe, A. D. & Miller, S. L. Was ferrocyanide a prebiotic reagent? Orig. Life Evol. Biosph. 26, 111–129 (1996).

    Article  ADS  CAS  Google Scholar 

  16. Maurel, M.-C. & Orgel, L. E. Oligomerization of α-thioglutamic acid. Orig. Life Evol. Biosph. 30, 423–430 (2000).

    Article  ADS  CAS  Google Scholar 

  17. Leman, L., Orgel, L. & Ghadiri, M. R. Carbonyl sulfide-mediated prebiotic formation of peptides. Science 306, 283–286 (2004).

    Article  ADS  CAS  Google Scholar 

  18. Islam, S., Bučar, D.-K. & Powner, M. W. Prebiotic selection and assembly of proteinogenic amino acids and natural nucleotides from complex mixtures. Nat. Chem. 9, 584–589 (2017).

    Article  CAS  Google Scholar 

  19. Stairs, S. et al. Divergent prebiotic synthesis of pyrimidine and 8-oxo-purine ribonucleotides. Nat. Commun. 8, 15270 (2017).

    Article  ADS  CAS  Google Scholar 

  20. Islam, S. & Powner, M. W. Prebiotic systems chemistry: complexity overcoming clutter. Chem 2, 470–501 (2017).

    Article  CAS  Google Scholar 

  21. Roberts, S. J. et al. Selective prebiotic conversion of pyrimidine and purine anhydronucleosides into Watson–Crick base-pairing arabino-furanosyl nucleosides in water. Nat. Commun. 9, 4073 (2018).

    Article  ADS  Google Scholar 

  22. Chadha, M. S., Replogle, L., Flores, J. & Ponnamperuma, C. Possible role of aminoacetonitrile in chemical evolution. Bioorg. Chem. 1, 269–274 (1971).

    Article  Google Scholar 

  23. Paventi, M. & Edward, J. T. Preparation of α-aminothioamides from aldehydes. Can. J. Chem. 65, 282–289 (1987).

    Article  CAS  Google Scholar 

  24. Sheehan, J. C. & Johnson, D. A. The synthesis and reactions of N-acyl thiol amino acids. J. Am. Chem. Soc. 74, 4726–4727 (1952).

    Article  CAS  Google Scholar 

  25. Leman, L. J. & Ghadiri, M. R. Potentially prebiotic synthesis of α-amino thioacids in water. Synlett 28, 68–72 (2017).

    Article  CAS  Google Scholar 

  26. Okamoto, R. et al. Regioselective α-peptide bond formation through oxidation of amino thioacids. Biochemistry 58, 1672–1678 (2019).

    Article  CAS  Google Scholar 

  27. Steinberg, S. M. & Bada, J. L. Peptide decomposition in the neutral pH region via the formation of diketopiperazines. J. Org. Chem. 48, 2295–2298 (1983).

    Article  CAS  Google Scholar 

  28. Radzicka, A. & Wolfenden, R. Rates of uncatalyzed peptide bond hydrolysis in neutral solution and the transition state affinities of proteases. J. Am. Chem. Soc. 118, 6105–6109 (1996).

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  30. Villain, M., Gaertner, H. & Botti, P. Native chemical ligation with aspartic and glutamic acids as C-terminal residues: scope and limitations. Eur. J. Org. Chem. 2003, 3267–3272 (2003).

    Article  Google Scholar 

  31. Danger, G. et al. 5(4H)-Oxazolones as intermediates in the carbodiimide- and cyanamide-promoted peptide activations in aqueous solution. Angew. Chem. Int. Ed. 52, 611–614 (2013).

    Article  CAS  Google Scholar 

  32. Griesser, H., Bechthold, M., Tremmel, P., Kervio, E. & Richert, C. Amino acid-specific, ribonucleotide-promoted peptide formation in the absence of enzymes. Angew. Chem. Int. Ed. 56, 1224–1228 (2017).

    Article  CAS  Google Scholar 

  33. Zhang, L. & Tam, J. P. Lactone and lactam library synthesis by silver ion-assisted orthogonal cyclization of unprotected peptides. J. Am. Chem. Soc. 121, 3311–3320 (1999).

    Article  CAS  Google Scholar 

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We thank the Engineering and Physical Sciences Research Council (EP/K004980/1, EP/P020410/1), the Simons Foundation (318881, 493895) and the Volkswagen Foundation (94743) for financial support. The authors thank K. Karu (UCL Mass Spectrometry Facility), E. Samuel (Mass Spectrometry, UCL School of Pharmacy) and A. E. Aliev (NMR spectroscopy) for assistance.

Author information

Authors and Affiliations



M.W.P. conceived the research. P.C., S.I. and M.W.P. designed and analysed the experiments. P.C. and S.I. contributed equally to the experiments. S.I. wrote the Supplementary Information. M.W.P and S.I. wrote the paper and Supplementary Discussion.

Corresponding author

Correspondence to Matthew W. Powner.

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Peer review information Nature thanks Irene Chen and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Chemoselective native peptide bond ligations of cysteine and lysine residues.

a, Ligation of Cys is notoriously challenging owing to its highly nucleophilic thiol side chain, which necessitates S-protection to prevent it outcompeting C- and/or N-terminal activation through degradation of the electrophilic activating agents. Protecting-group-free ligation of Cys (150 mM) is achieved through reaction with Ac-Gly-SH (50 mM) and K3[Fe(CN)6] (300 mM) in water (pH 9.5, room temperature), followed by thiol reduction (MeSH, 600 mM, pH 10.8, room temperature) to give Ac-Gly-Cys-OH in high yield (80%, over two steps) (Supplementary Figs. 112114). b, Lys-X coupling partners (X = CN, CONH2 or CO2H) pose greater chemoselectivity challenges because they possess two amino groups (α-NH2 and ε-NH2). However, pKa-controlled native peptide ligation of Lys-CN demonstrates the pivotal role that the unusually low α-amine pKaH of AA-CN19 can play in selective ligation. Ligation of Lys-CN (100 mM) with Ac-Gly-SH (50 mM) proceeds with unprecedented selectivity in neutral water (pH 7.5, room temperature). Little or no selectivity was observed for the corresponding α-amino amide (Lys-NH2; 150 mM) and AA (Lys; 150 mM) (Supplementary Figs. 145151). c, Selective intermolecular ligation of the C-terminal lysine residue with AA-CN coupling partner Gly-CN at near-neutral pH (pH 6.5–9.0, blue; see Supplementary Fig. 70). In the absence of Gly-CN, highly efficient intramolecular caprolactam formation is observed (red).

Extended Data Table 1 α-Amidothioacid activating agents
Extended Data Table 2 α-Aminonitrile ligation in the presence of nucleophilic competitors
Extended Data Table 3 α-Aminonitrile ligation at various concentrations and temperatures
Extended Data Table 4 Chemoselective synthesis of N-acetyl dipeptidyl amides
Extended Data Table 5 Chemoselective synthesis of N-acetyl dipeptidyl nitriles

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary Figures 1–296, Supplementary Tables 1–16, experimental details and compound characterization data.

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Canavelli, P., Islam, S. & Powner, M.W. Peptide ligation by chemoselective aminonitrile coupling in water. Nature 571, 546–549 (2019).

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