Guanidinium‐Stapled Helical Peptides for Targeting Protein‐Protein Interactions

Peptide stapling has emerged as a versatile approach in drug discovery to reinforce secondary structure elements especially α‐helices and improve properties of linear bioactive peptides. Inspired by the prevalence of arginine in protein‐protein and protein‐DNA interfaces, we investigated guanidinium‐stapling as a means to constrain helical peptides. Guanidinium stapling was readily achieved on solid support, utilizing two orthogonally protected lysine or unatural α‐amino acid residues with an amino function. This method allows for easy modulation of the nature and size of the staple as well as helix propensity. Evaluating a set of guanidinium‐stapled peptides for their interaction with different protein targets identified several binders with increased target affinity. X‐ray structure determination of four complexes revealed that all stapled peptides adopt a helical conformation upon protein binding. Notably, the disubstituted guanidinium generally exhibits a distinct cis/trans conformation and, in one instance, retains a conserved hydrogen bond with the protein surface. By identifying, for the first time, the guanidinium moiety as an effective helical peptide stapling group, this research significantly expands the repertoire of α‐helix stapling techniques for the creation of useful protein mimics. Guanidinium‐stapling is a versatile and straightforward macrocyclization strategy that is performed selectively on solid support by crosslinking two amino side chains. It does not only stabilize the helical structure of a peptide, but also adds a positive charge within the ring and can serve as a constrained mimic of arginine, thereby contributing to specific protein surface interactions, as demonstrated by several co‐crystal structures with proteins of interest.