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.半胱氨酸（Cys）残基的侧链钉合（side chain stapling）技术，可为制备兼具增强生物活性与生物利用度的受限肽提供便捷途径。尽管该技术已广泛应用于α螺旋、PPII及环型构象的约束，但通过链内侧链Cys钉合稳定β折叠结构的策略仍未得到充分探索。本研究表明，采用E-丁烯基、丁炔基与间二甲苯基连接臂的i→i+2位钉合，可显著提升两种不同β发夹模型肽的折叠态占比。高分辨率核磁共振（NMR）结构分析显示，此类钉合结构可维持典型β折叠的主链二面角，并稳定链间相互作用。借助完整主链氢键边缘的保留特性，我们将i→i+2侧链大环化策略应用于源自tau蛋白的受限β拱形肽的设计中。研究证实，对一段不易聚集的肽段进行链内钉合，可促进其自组装为类β折叠纤维。所得纤维还可在基于细胞的实验中，以大环化合物和序列依赖的方式，诱导内源性tau蛋白的聚集。本研究确立双Cys i→i+2钉合技术作为一种通用且合成便捷的方法，可用于稳定β折叠结构，并调控具备成种能力的淀粉样肽的自组装。