Dynamic covalent chemistry enables formation of antimicrobial peptide quaternary assemblies in a completely abiotic manner


Naturally occurring peptides and proteins often use dynamic disulfide bonds to impart defined tertiary/quaternary structures for the formation of binding pockets with uniform size and function. Although peptide synthesis and modification are well established, controlling quaternary structure formation remains a significant challenge. Here, we report the facile incorporation of aryl aldehyde and acyl hydrazide functionalities into peptide oligomers via solid-phase copper-catalysed azide–alkyne cycloaddition (SP-CuAAC) click reactions. When mixed, these complementary functional groups rapidly react in aqueous media at neutral pH to form peptide–peptide intermolecular macrocycles with highly tunable ring sizes. Moreover, sequence-specific figure-of-eight, dumbbell-shaped, zipper-like and multi-loop quaternary structures were formed selectively. Controlling the proportions of reacting peptides with mismatched numbers of complementary reactive groups results in the formation of higher-molecular-weight sequence-defined ladder polymers. This also amplified antimicrobial effectiveness in select cases. This strategy represents a general approach to the creation of complex abiotic peptide quaternary structures.

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Figure 1: Synthesis of DC–peptides.
Figure 2: Formation of DC–peptide quaternary structures.
Figure 3: Tricine SDS–PAGE of DC–peptide assemblies.
Figure 4: Vernier templated DC–peptide ladder polymerization.
Figure 5: Hydrodynamic radii of DC–peptides and quaternary assemblies.
Figure 6: Antimicrobial activity of DC–peptides and assemblies against the Gram-positive bacterium S. aureus.


  1. 1

    Hubscher, U., Maga, G. & Spadari, S. Eukaryotic DNA polymerases. Annu. Rev. Biochem. 71, 133–163 (2002).

    CAS  Article  Google Scholar 

  2. 2

    Bader, G. D., Betel, D. & Hogue, C. W. V. BIND: the biomolecular interaction network database. Nucleic Acids Res. 31, 248–250 (2003).

    CAS  Article  Google Scholar 

  3. 3

    Hobert, O. Gene regulation by transcription factors and microRNAs. Science 319, 1785–1786 (2008).

    CAS  Article  Google Scholar 

  4. 4

    Fischbach, M. A. & Walsh, C. T. Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms. Chem. Rev. 106, 3468–3496 (2006).

    CAS  Article  Google Scholar 

  5. 5

    Dixon, R. A. Natural products and plant disease resistance. Nature 411, 843–847 (2001).

    CAS  Article  Google Scholar 

  6. 6

    Esko, J. D. & Selleck, S. B. Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu. Rev. Biochem. 71, 435–471 (2002).

    CAS  Article  Google Scholar 

  7. 7

    Wells, L., Vosseller, K. & Hart, G. W. Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science 291, 2376–2378 (2001).

    CAS  Article  Google Scholar 

  8. 8

    Lasica, A. M. & Jagusztyn-Krynicka, E. K. The role of Dsb proteins of Gram-negative bacteria in the process of pathogenesis. FEMS Microbiol. Rev. 31, 626–636 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Kivirikko, K. I. & Myllyharju, J. Prolyl 4-hydroxylases and their protein disulfide isomerase subunit. Matrix Biol. 16, 357–368 (1998).

    CAS  Article  Google Scholar 

  10. 10

    Carlini, A. S., Adamiak, L. & Gianneschi, N. C. Biosynthetic polymers as functional materials. Macromolecules 49, 4379–4394 (2016).

    CAS  Article  Google Scholar 

  11. 11

    Niu, J., Hili, R. & Liu, D. R. Enzyme-free translation of DNA into sequence-defined synthetic polymers structurally unrelated to nucleic acids. Nat. Chem. 5, 282–292 (2013).

    CAS  Article  Google Scholar 

  12. 12

    Rosenbaum, D. M. & Liu, D. R. Efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid aldehydes. J. Am. Chem. Soc. 125, 13924–13925 (2003).

    CAS  Article  Google Scholar 

  13. 13

    Xi, W. et al. Clickable nucleic acids: sequence-controlled periodic copolymer/oligomer synthesis by orthogonal thiol-X reactions. Angew. Chem. Int. Ed. 54, 14462–14467 (2015).

    CAS  Article  Google Scholar 

  14. 14

    Porel, M., Thornlow, D. N., Phan, N. N. & Alabi, C. A. Sequence-defined bioactive macrocycles via an acid-catalysed cascade reaction. Nat. Chem. 8, 590–596 (2016).

    CAS  Article  Google Scholar 

  15. 15

    Porel, M. & Alabi, C. A. Sequence-defined polymers via orthogonal allyl acrylamide building blocks. J. Am. Chem. Soc. 136, 13162–13165 (2014).

    CAS  Article  Google Scholar 

  16. 16

    Barnes, J. C. et al. Iterative exponential growth of stereo- and sequence-controlled polymers. Nat. Chem. 7, 810–815 (2015).

    CAS  Article  Google Scholar 

  17. 17

    Gutekunst, W. R. & Hawker, C. J. A general approach to sequence-controlled polymers using macrocyclic ring opening metathesis polymerization. J. Am. Chem. Soc. 137, 8038–8041 (2015).

    CAS  Article  Google Scholar 

  18. 18

    Ura, Y., Beierle, J. M., Leman, L. J., Orgel, L. E. & Ghadiri, M. R. Self-assembling sequence-adaptive peptide nucleic acids. Science 325, 73–77 (2009).

    CAS  Article  Google Scholar 

  19. 19

    Wei, T., Jung, J. H. & Scott, T. F. Dynamic covalent assembly of peptoid-based ladder oligomers by vernier templating. J. Am. Chem. Soc. 137, 16196–16202 (2015).

    CAS  Article  Google Scholar 

  20. 20

    Wilson, A., Gasparini, G. & Matile, S. Functional systems with orthogonal dynamic covalent bonds. Chem. Soc. Rev. 43, 1948–1962 (2014).

    CAS  Article  Google Scholar 

  21. 21

    Rowan, S. J., Cantrill, S. J., Cousins, G. R. L., Sanders, J. K. M. & Stoddart, J. F. Dynamic covalent chemistry. Angew. Chem. Int. Ed. 41, 899–952 (2002).

    CAS  Article  Google Scholar 

  22. 22

    Sadownik, J. W. & Ulijn, R. V. Dynamic covalent chemistry in aid of peptide self-assembly. Curr. Opin. Biotechnol. 21, 401–411 (2010).

    CAS  Article  Google Scholar 

  23. 23

    Dirksen, A., Dirksen, S., Hackeng, T. M. & Dawson, P. E. Nucleophilic catalysis of hydrazone formation and transimination: implications for dynamic covalent chemistry. J. Am. Chem. Soc. 128, 15602–15603 (2006).

    CAS  Article  Google Scholar 

  24. 24

    Ruff, Y., Garavini, V. & Giuseppone, N. Reversible native chemical ligation: a facile access to dynamic covalent peptides. J. Am. Chem. Soc. 136, 6333–6339 (2014).

    CAS  Article  Google Scholar 

  25. 25

    Sadownik, J. W., Mattia, E., Nowak, P. & Otto, S. Diversification of self-replicating molecules. Nat. Chem. 8, 264–269 (2016).

    CAS  Article  Google Scholar 

  26. 26

    Krishnan-Ghosh, Y. & Balasubramanian, S. Dynamic covalent chemistry on self-templating peptides: formation of a disulfide-linked β-hairpin mimic. Angew. Chem. Int. Ed. 42, 2171–2173 (2003).

    CAS  Article  Google Scholar 

  27. 27

    Lam, R. T. S. et al. Amplification of acetylcholine-binding catenanes from dynamic combinatorial libraries. Science 308, 667–669 (2005).

    CAS  Article  Google Scholar 

  28. 28

    Haney, C. M., Loch, M. T. & Horne, W. S. Promoting peptide α-helix formation with dynamic covalent oxime side-chain cross-links. Chem. Commun. 47, 10915–10917 (2011).

    CAS  Article  Google Scholar 

  29. 29

    Haney, C. M. & Horne, W. S. Dynamic covalent side-chain cross-links via intermolecular oxime or hydrazone formation from bifunctional peptides and simple organic linkers. J. Pept. Sci. 20, 108–114 (2014).

    CAS  Article  Google Scholar 

  30. 30

    Rocard, L., Berezin, A., De Leo, F. & Bonifazi, D. Templated chromophore assembly by dynamic covalent bonds. Angew. Chem. Int. Ed. 54, 15739–15743 (2015).

    CAS  Article  Google Scholar 

  31. 31

    Fernandez-Lopez, S. et al. Antibacterial agents based on the cyclic D,L-α-peptide architecture. Nature 412, 452–456 (2001).

    CAS  Article  Google Scholar 

  32. 32

    Kennedy, A. D. et al. Epidemic community-associated methicillin-resistant Staphylococcus aureus: recent clonal expansion and diversification. Proc. Natl Acad. Sci. USA 105, 1327–1332 (2008).

    CAS  Article  Google Scholar 

  33. 33

    Jin, Y., Yu, C., Denman, R. J. & Zhang, W. Recent advances in dynamic covalent chemistry. Chem. Soc. Rev. 42, 6634–6654 (2013).

    CAS  Article  Google Scholar 

  34. 34

    Ladame, S. Dynamic combinatorial chemistry: on the road to fulfilling the promise. Org. Biomol. Chem. 6, 219–226 (2008).

    CAS  Article  Google Scholar 

  35. 35

    Lehn, J.-M. & Eliseev, A. V. Dynamic combinatorial chemistry. Science 291, 2331–2332 (2001).

    CAS  Article  Google Scholar 

  36. 36

    Schagger, H. Tricine-SDS–PAGE. Nat. Protoc. 1, 16–22 (2006).

    Article  Google Scholar 

  37. 37

    Heinis, C. Drug discovery: tools and rules for macrocycles. Nat. Chem. Biol. 10, 696–698 (2014).

    CAS  Article  Google Scholar 

  38. 38

    Driggers, E. M., Hale, S. P., Lee, J. & Terrett, N. K. The exploration of macrocycles for drug discovery—an underexploited structural class. Nat. Rev. Drug. Discov. 7, 608–624 (2008).

    CAS  Article  Google Scholar 

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Financial support for the presented work was provided by the DARPA Fold-Fx programme (N66001-14-2-4051) and the Welch Regents Chair (F-0046). The authors thank M. Persons of the Proteomics facility at University of Texas (UT) at Austin for aid with MALDI–TOF MS acquisition, S. Sorey of the NMR facility at UT Austin for aid with 2D-DOSY-NMR acquisition and I. Riddington of the Mass Spectrometry facility at UT Austin for aid with HRMS acquisition.

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J.F.R. and E.V.A. devised the conducted experiments. J.F.R. wrote the manuscript, collected the MALDI–TOF MS data, collected NMR data and conducted SDS–PAGE experiments. J.L.D. and M.W. designed and conducted the high-throughput luminescence assay for determination of antibiotic efficiencies. J.F.R., I.V.K., D.V.U. and R.G. contributed to the synthesis and purification of all peptides and small-molecule precursors reported. E.T.H. aided in the training for peptide synthesis and HPLC purification. All authors edited the manuscript.

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Correspondence to Marvin Whiteley or Eric V. Anslyn.

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The authors declare no competing financial interests.

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Reuther, J., Dees, J., Kolesnichenko, I. et al. Dynamic covalent chemistry enables formation of antimicrobial peptide quaternary assemblies in a completely abiotic manner. Nature Chem 10, 45–50 (2018). https://doi.org/10.1038/nchem.2847

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