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A two-component 'double-click' approach to peptide stapling

Nature Protocols volume 10, pages 585–594 (2015)Cite this article

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Abstract

Peptide cyclization is a useful strategy for the stabilization of short flexible peptides into well-defined bioactive conformations, thereby enhancing their ability to interact with proteins and other important biomolecules. We present an optimized procedure for the stabilization of linear diazido peptides in an α-helical conformation upon reaction with dialkynyl linkers under Cu(I) catalysis. As this procedure generates side chain–cyclized peptides bearing a bis-triazole linkage, it is referred to as 'double-click' stapling. Double-click stapling can enhance the binding affinity, proteolytic stability and cellular activity of a peptide inhibitor. A distinguishing feature of double-click stapling is the efficiency with which peptides bearing different staple linkages can be synthesized, thus allowing for modular control over peptide bioactivity. This protocol describes the double-click reaction between a 1,3-dialkynylbenzene linker and peptides that contain azidoornithine. Subsequent peptide purification and confirmation steps are also described. The entire double-click stapling protocol can be completed in ∼48 h, including two overnight lyophilization steps.

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Figure 1: Double-click stapling is a two-component reaction, in which the peptide and linker are separate moieties.
Figure 2: Dialkynyl staple linkers bearing different functionalities.
Figure 3: The experimental setup for double-click stapling (shown at Step 11).
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8: HPLC chromatographs of unstapled starting peptides and corresponding crude reaction mixtures for stapled peptides 1 and 2.
Figure 9: IR spectra of stapled peptide 2 (black), unstapled peptide 2 (red) and wild-type p5317-29 (blue).
Figure 10: Mass chromatograph of stapled peptide 1 showing the isotope peaks for [M+2H]2+ separated by ∼0.5 AMU, corresponding to peptides containing different numbers of the 13C isotope.

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Acknowledgements

This work was supported by the European Union, the Engineering and Physical Sciences Research Council, the Biotechnology and Biological Sciences Research Council, the Medical Research Council and the Wellcome Trust. Y.H.L. acknowledges a scholarship from the Cambridge Trusts. P.d.A. thanks the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. [279337/DOS].

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  1. University Chemical Laboratory, University of Cambridge, Cambridge, UK

    Yu Heng Lau, Yuteng Wu, Peterson de Andrade, Warren R J D Galloway & David R Spring

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Contributions

Y.H.L., Y.W., P.d.A. and D.R.S. designed the experiments. Y.H.L., Y.W. and P.d.A. performed the experiments. All the authors contributed to writing the manuscript.

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Correspondence to David R Spring.

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Lau, Y., Wu, Y., de Andrade, P. et al. A two-component 'double-click' approach to peptide stapling. Nat Protoc 10, 585–594 (2015). https://doi.org/10.1038/nprot.2015.033

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