Synthesis and Evaluation of HS-336-OH and an Azide-Linked Analog as Fluorescent Probes for Protein Aggregate Detection in Alzheimer’s Disease

Alzheimer's disease is the leading cause of dementia worldwide with advancing age being the most prominent risk factor. The disease is caused by the presence of specific protein aggregates in the brain; the two most common being the amyloid-β peptide and tangles of tau proteins. The pathogenesis of the disease can be studied using fluorescent tracer ligands that selectively detect these aggregates, which could potentially serve as a molecular diagnostic tool and facilitate an early diagnosis of the disease. Previous studies have demonstrated that the thiophene-vinylene-benzothiazole-based ligand, HS-336, detects both protein aggregates and the analog, HS-336-OH, was therefore investigated. Building upon prior work indicating the potential of linker-modified ligands to improve binding performance, this study aimed to attach various linkers of differing lengths and functional groups to the HS-336-OH scaffold as well as an analogous ligand. The photophysical properties of the resulting ligands were characterized and their binding capacity was evaluated through staining of Aβ and tau aggregates in human brain tissue. Results showed that the ligand without linker, HS-336-OH, stained both Aβ- and tau aggregates, while the ligand with linker, designated E-HS-336-O-Linker-B-N3, only weakly stained Aβ aggregates and no tau. Based on these results, HS-336-OH can be regarded as a functional fluorescent ligand, while E-HS-336-O-Linker-B-N3 can be regarded as a weak Aβ-selective fluorescent ligand. The reduced binding of the latter may be attributed to the presence of the linker and the absence of a positively charged nitrogen atom, which is present in HS-336-OH. These findings contribute to the understanding of how linker chemistry influences ligand-target interactions with implications for the development of advanced diagnostic tools for Alzheimer’s disease.