Structurally diverse cyclisation linkers impose different backbone conformations in bicyclic peptides

Combinatorial repertoires of structurally diverse peptide macrocycles offer a rich source for the development of high-affinity ligands to targets of interest. In this work, we developed linkers for the generation of genetically encoded bicyclic peptides and tested whether the peptides cyclised by them have significant variations in their backbone conformations. Two new cyclisation reagents, both having three thiol-reactive groups, efficiently and selectively cyclised linear peptides containing three cysteines. When the mesitylene linker of the bicyclic peptide PK15, a potent inhibitor of plasma kallikrein (Ki = 2 nM), was replaced by the new linkers, its inhibitory activity dropped by more than a factor 1000, suggesting that the linkers impose different conformations onto the peptide. Indeed, structural analysis by solution-state NMR revealed different NOE constraints in the three bicyclic peptides, indicating that the relatively small linkers at the centre of bicyclic peptides significantly influence the conformations of the peptides. These results demonstrate the prominent structural role of linkers in peptide macrocycles and suggest that applying different cyclisation linkers in a combinatorial fashion could be an attractive means for generating topologically diverse macrocycle libraries.