Macrocyclization has emerged as a versatile approach in drug discovery to enhance the potential of natural α-peptides, overcome some of their limitations – such as metabolic stability, cell permeability – and convert them into valuable pharmacologic tools and drugs (> 40 available drugs are cyclic peptides). This strategy allows the stabilization of the peptide bioactive conformation to mimic structured regions of proteins – e.g. helices – involved in protein-protein interactions or recognition of nucleic acids. However, current side chain crosslinking strategies to constrain helical peptides – also called ‘staples’ – often rely on hydrophobic linkers and require additional cationic modifications (e.g., cell-penetrating peptides (CPPs)) to achieve intracellular delivery, at the cost of increased molecular weight and reduced drug-likeness.

Inspired by the frequent involvement of arginine in protein–protein and protein–DNA interactions, the Guichard team aimed to expand the “toolbox” of peptide-stapling strategies and developed a new guanidinium-based macrocyclization approach, which combines structural preorganization, charge optimization and molecular recognition in a single staple.

 Figure 1. General scheme of guanidinium stapling strategies to stabilize helical peptide conformation, yielding a positively charged crosslink by intramolecular macrocyclization or a double positively charged crosslink by using a two-component reaction.

In the two recently published studies, selective and high-yielding solid-phase synthesis (SPS) routes to (i,i+4) and (i,i+7) stapled peptides containing one or two guanidinium moieties are reported. One advantage of this approach is the synthetic accessibility of the guanidinium crosslink from lysine or any other (un)natural α-amino acid residues with alkyl amine side chains (ornithine, diaminobutyric acid…) allowing an easy modulation of the ring size and position of the staple in the ring.

This strategy was applied to the design of new stapled peptides targeting two biologically-relevant protein-protein interactions. Results showed that such peptides displayed tight – and in some cases significantly improved – binding to their respective protein target, confirming the potential of this approach. The Guichard team and their collaborators solved four x-ray co-crystal structures of these peptide-protein complexes. In all cases, the stapled peptides adopted a helical structure upon binding to the protein target, proving that the guanidinium staple successfully locks the peptide into the intended bioactive conformation. When located near the protein interface, the staple was shown to engage directly in binding, through hydrogen bonds or van der Waals contacts, highlighting its dual role as both a conformational constraint and an effective mimic of arginine that actively reinforces peptide–protein interactions.

Figure 2. X-ray co-crystal structures of guanidinium stapled peptides in complex with proteins of interest. Peptide residues located in protein hydrophobic pockets are colored in dark grey and the guanidinium staple in blue spheres in all structures.

By identifying, for the first time, the guanidinium moiety as an effective helical peptide stapling moiety, this research significantly expands the repertoire of α-helix stapling techniques for the design of useful protein mimics. While these findings are promising, further studies are needed to globally assess the impact of guanidinium stapling on peptide resistance to degradation and cell permeability. Additionally, the versatility of this synthetic route suggests that it could be extended to prepare more intricate and diverse constrained peptides, such as sequences containing multiple guanidinium staples in a row.

More information :

Guanidinium-Stapled Helical Peptides for Targeting Protein-Protein Interactions.

C. Perdriau, A. Luton, K. Zimmeter, M. Neuville, C. Saragaglia, C. Peluso-Iltis, J. Osz, B. Kauffmann, G. W. Collie, N. Rochel, G. Guichard & M. Pasco

Angew. Chem. Int. Ed. 2025, 64, e202416348

https://doi.org/10.1002/anie.202416348

A General Synthesis Approach to Double-Guanidinium Stapled Peptides and Foldamers.

M. Neuville, M.M. Bornez, M. Bourgeais, H. Samueli, L. Mauran, S.R. Goudreau, A.-M. Khatib, G. Guichard & M. Pasco

Chem. Eur. J., 2025, 31, e02273

https://doi.org/10.1002/chem.202502273