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+2 stapling with E-butenyl, butynyl, and m-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+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+2 stapling as a versatile and synthetically accessible
method to stabilize β-sheet structure and modulate the self-assembly
of seed-competent amyloidogenic peptides.