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Peptide ligation by chemoselective aminonitrile coupling in water

Nature volume 571, pages 546–549 (2019)Cite this article

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

This article has been updated

Abstract

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

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Data availability

All data supporting the findings of this study are available within the main text, Extended Data Tables 1–5, Extended Data Fig. 1 and the Supplementary Information (which contains Supplementary Discussion, Supplementary Figs. 1–296, Supplementary Tables 1–16, 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.

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Acknowledgements

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

  1. Department of Chemistry, University College London, London, UK

    Pierre Canavelli, Saidul Islam & Matthew W. Powner

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  1. Pierre Canavelli
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Contributions

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.

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Correspondence to Matthew W. Powner.

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The authors declare no competing interests.

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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. 112–114). 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. 145–151). 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
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Extended Data Table 2 α-Aminonitrile ligation in the presence of nucleophilic competitors
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Extended Data Table 3 α-Aminonitrile ligation at various concentrations and temperatures
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Extended Data Table 4 Chemoselective synthesis of N-acetyl dipeptidyl amides
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Extended Data Table 5 Chemoselective synthesis of N-acetyl dipeptidyl nitriles
Full size table

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). https://doi.org/10.1038/s41586-019-1371-4

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