Development of bifunctional, Raman active diyne-girder stapled alpha-helical peptides

Abstract

Stapled peptides are a unique class of cyclic alpha-helical peptides that are conformationally constrained via their amino acid side-chains. They have been transformative to the field of chemical biology and peptide drug discovery through addressing many of the physicochemical limitations of linear peptides. However, there are several issues with current chemical strategies to produce stapled peptides. For example, two distinct unnatural amino acids (R8 and S5) are required for synthesis of i, i + 7 alkene stapled peptides, leading to high cost of production. Furthermore, low purified yields are obtained due to cis/trans isomers being produced during the key ring-closing metathesis macrocyclisation step. Here we report the development of a new i, i + 7 diyne-girder stapling strategy that addresses these issues. The asymmetric synthesis of nine unnatural Fmoc-protected alkyne-amino acids facilitated a systematic study to determine the optimal (S,S)-stereochemistry and 14-carbon diyne-girder bridge length. Diyne-girder stapled T-STAR peptide 29 was demonstrated to have excellent helicity, was cell permeable and stable to protease degradation. Finally, we demonstrate that the diyne-girder constraint is a bifunctional Raman chromophore with potential use in Raman cell microscopy. Development of this highly effective, bifunctional diyne-girder stapling strategy leads us to believe that it can be used to produce other stapled peptide probes and therapeutics.