Cyclopeptide-Based Fluorescent Conjugates for Monitoring Prefibrillar Aβ Nanostructures

Amyloid-β (Aβ1–42) prefibrillar aggregates are considered the most neurotoxic amyloid species, yet their transient and heterogeneous nature makes selective detection challenging. Many fluorescent probes also fail to discriminate Aβ from homologous peptides such as IAPP, leading to poor specificity. We report a peptide-guided late-stage diversification strategy to generate BODIPY-based probes highly selective for prefibrillar Aβ1–42. A rationally engineered cyclic peptide derived from the C-terminal region of Aβ1–42 provides conformational rigidity and precise molecular recognition. Conjugation to BODIPY fluorophores afforded peptide–dye hybrids systematically evaluated for selectivity and photophysical response. A controlled aggregation protocol enabling reproducible generation of prefibrillar Aβ species was established to validate probe performance. A Sonogashira-derived conjugate (probe 8) showed strong fluorescence turn-on and selective affinity for prefibrillar Aβ1–42, with no response to IAPP aggregates. In neuronal cells, probe 8 outperformed conventional antibodies, supporting its potential for mechanistic studies and early Alzheimer’s disease diagnostics.β淀粉样蛋白（Amyloid-β, Aβ1–42）原纤维前聚集体被视为神经毒性最强的淀粉样蛋白物种，但其瞬时性与异质性使得选择性检测极具挑战。诸多荧光探针亦无法区分Aβ与同源肽类（如胰岛淀粉样多肽（IAPP）），导致特异性欠佳。本研究报道了一种肽导向的后期多样化策略，用于构建对原纤维前Aβ1–42具有高选择性的基于硼二吡咯亚甲基（BODIPY）的荧光探针。源自Aβ1–42 C端区域的理性设计环肽，可赋予构象刚性与精准的分子识别能力。将其与BODIPY荧光团共轭得到肽-染料杂合物，并对其选择性与光物理响应进行了系统评估。本研究建立了可重复制备原纤维前Aβ物种的可控聚集方案，用于验证探针性能。经薗头耦合反应（Sonogashira）衍生得到的共轭物（探针8）展现出显著的荧光开启效应与对原纤维前Aβ1–42的选择性亲和性，且对IAPP聚集体无任何响应。在神经元细胞实验中，探针8的性能优于常规抗体，证实其在阿尔茨海默病的机制研究与早期诊断中具备应用潜力。