High-yield on-resin synthesis of doubly crosslinked α-helical peptides with significantly improved protease resistance using an isophthalic acid-based crosslinker.

We describe doubly crosslinked α-helical peptides possessing an isophthalic acid-based crosslinker. The peptides can be obtained in good overall yields by conventional solid-phase peptide synthesis using natural and semi-natural amino acids, followed by double-crosslinking with a readily available isophthalic acid derivative. The doubly crosslinked peptide, including the binding domain sequence from A-kinase anchoring proteins (AKAPs), showed a significantly high α-helicity and a strong binding affinity for protein kinase A, regulatory subunit Iα (PKA-RIα). In addition, the peptide displayed higher proteolytic stability in human serum than the corresponding singly crosslinked ones. The doubly crosslinked peptide with a cell-penetrating peptide was internalized into cells and effectively inhibited the protein-protein interaction between AKAP and PKA-RIα in living cells.