Intrastrand Peptide Staples That Promote β-Sheet Folding, Self-Assembly, and Amyloid Seeding

Side chain stapling of cysteine (Cys) residues offers convenient entry into constrained peptides with enhanced bioactivity and bioavailability. Despite its widespread application in the constraint of α-helical, PPII, and loop conformations, the stabilization of β-sheet folds via intrastrand side chain Cys stapling remains largely unexplored. Here, we demonstrate that <i>i</i>→<i>i</i>+2 stapling with <i>E</i>-butenyl, butynyl, and <i>m</i>-xylyl linkers significantly enhances the folded population of two distinct β-hairpin model peptides. High-resolution NMR structures reveal that these staples support canonical β-sheet backbone torsions and stabilize cross-strand interactions. Leveraging the maintenance of intact backbone hydrogen-bonding edges, we employed <i>i</i>→<i>i</i>+2 side chain macrocyclization in the design of constrained β-arch peptides derived from the tau protein. We show that intrastrand stapling of a nonaggregation-prone segment promotes self-assembly into β-sheet-like filaments. The resulting filaments also seed the aggregation of endogenous tau in a cell-based assay in a macrocycle- and sequence-dependent manner. These findings establish di-Cys <i>i</i>→<i>i</i>+2 stapling as a versatile and synthetically accessible method to stabilize β-sheet structure and modulate the self-assembly of seed-competent amyloidogenic peptides.