Enhancementof Coiled-Coil Stability via InterhelicalClick Chemistry Stapling

Abstract Coiled coil peptide assemblies are useful scaffolds for biomolecular and materials design, but their function often depends on preserving the folded oligomeric state after chemical modification or exposure to demanding conditions. While many peptide-stapling strategies stabilize individual α-helices, approaches that covalently reinforce higher-order architectures remain less developed. Here, we introduce an “assemble-and-click” strategy for interhelical stapling of a designed tetramer coiled coil. Cysteine and vinyl sulfonamide handles were positioned at symmetry-related sites that are brought into proximity by coiled-coil assembly, enabling pH-triggered thiol-Michael coupling after tetramer formation. LC-MS supports rapid formation of covalently linked dimeric products, SEC-MALS indicates that the stapled peptide retains the expected tetrameric assembly state, and circular dichroism shows markedly enhanced thermal robustness, with no cooperative unfolding observed up to 90 °C under the conditions examined. This approach provides a chemically programmable route to reinforcing higher-order coiled coil assemblies while preserving their oligomeric architecture.