However, these Pd-mediated stapling reactions were performed only on an analytical scale and the secondary structures of the cyclic peptides were not studied. They can further be applied for peptide macrocyclisation of the two above mentioned side chain residues of the natural amino acids. The approaches by Buchwald and Pentelute are suitable for selective, bioorthogonal labelling of cysteine- and lysine-containing peptides and proteins using stochiometric amounts of pre-formed Pd(II)-aryl complexes. The groups of Buchwald, Pentelute, and Ackermann pioneered the development of Pd-mediated arylation chemistry of biomolecules. Moreover, it has been shown that Pd-mediated cross-couplings can be successfully employed in the generation of cyclic and conformationally stabilised peptides. The content of helicity can moreover be changed by the introduction of a photo-switchable azobenzene staple.

Since then, many other reactions have been investigated for macrocyclisation with the objective of peptide stapling including lactam-, disulfide-, thioether-, triazole-, oxime- and hydrazone formation as well as multicomponent reactions such as the Ugi- or Petasis reaction. By this robust and reliable approach, a library of stapled peptides was generated influencing diverse α-helical dominated protein–protein interactions (PPI) spanning pathways involved in cancer, infectious diseases, metabolic diseases and neurological disorders, which had been considered undruggable for a long time due to their large contact area and shallow surface. The optimised protocol for these so-called hydrocarbon-stapled peptides uses α-methyl-, α-alkenylglycines in a distance of i, i + 3/ i + 4 for one helix turn or i, i + 7 for two helix turns, respectively, followed by Ru-catalysed cross-linking. This technique is called peptide stapling and the most prominent methodology was developed by the groups of Grubbs and Verdine using ring-closing metathesis (RCM). Cross-linking of side chain residues results in constrained conformations and can be used to stabilise α-helical secondary structures. Hence, peptide-based drugs became of high interest because of their high selectivity combined with low toxicity. Moreover, cyclic peptides are more stable against proteolytic digestion and can provide an improved membrane permeability. Peptide cyclisation emerged as a popular approach to limit conformational mobility in order to enhance the binding affinity towards a biological target.

Stapled peptide