Proline‐to‐cysteine cyclization for generating conformationally constrained cyclic peptides

Macrocyclic peptides have received increasing attention throughout the pharmaceutical industry as attractive scaffolds for the development of new therapeutics. Here, we describe the development of a new proline‐to‐cysteine (PTC) peptide cyclization reaction. Peptide sequences flanked by an N‐terminal proline and a C‐terminal cysteine were reacted with α,α′‐dibromo‐m‐xylene to furnish cyclic peptides bearing a tertiary amine embedded within the macrocycle backbone. Macrocyclization proceeded efficiently in solution and on‐resin with peptides of different sequence lengths (5‐10 amino acids) and amino acid compositions. This approach was also applied for peptide bicyclization. Liquid chromatography mass spectrometry (LC‐MS)/MS analysis of a fingerprint ion related to the PTC linkage that was present throughout the substrate scope expedited confirmation of the product cyclic topologies. Conformational studies by variable‐temperature NMR revealed PTC macrocycles can adopt a rigid structure and display an intramolecular hydrogen‐bonding pattern that differs significantly from their cysteine‐to‐cysteine linked counterparts, further highlighting the value of this alternative cyclization approach. Due to its compatibility with library‐based peptide display and selection technologies, the described approach could offer significant utility in drug discovery campaigns.