Synthesis and characterization of fluorescent stilbene-based probes targeting amyloid fibrils

Abstract

Alzheimer's disease (AD) is characterized by two main protein aggregate hallmarks in the brain: extracellular deposition of the amyloid-β (Aβ) in senile plaques and intracellular neurofibrillary tangles (NFTs) consisting of hyperphosphorylated tau protein. The past decade has seen great progress in the development of imaging probes for the non-invasive detection of Aβ and tau aggregates. Here positron emission tomography (PET), single-photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI), are highly promising technologies for clinical diagnostics. However, as a research tool, optical imaging is superior because it is real-time, sensitive, inexpensive, not radioactive and that it in particular affords high-resolution studies both in vitro and in vivo. Fluorescent probes are especially useful for designing novel binding scaffolds for structure investigations of protein aggregates. This thesis describes design, synthesis and evaluation of a series of fluorescent probes for detection of amyloid fibrils, especially Aβ or tau aggregates in vitro. Firstly, trans-stilbenoid vinylbenzene-1,2-diol with benzene, naphthalene, anthracene, and pyrene are investigated with respect to their photophysical properties free in solution and when bound to amyloid fibrils, including time-resolved fluorescence measurements. It is noted that the extended conjugated systems retained the amyloid targeting properties of the probes and both the anthracene and pyrene moieties extensively enhanced the fluorescence intensity and prolonged lifetimes. Secondly, the synthesis of two molecules, Py1SA and Py2SA, based on pyrene linked to salicylic acid via a trans-stilbene C = C bond is presented. The compounds show strikingly different emission spectra when bound to preformed Aβ1-42 fibrils as well as to fibrils from four other distinct proteins. Additionally, excited state intramolecular proton transfer (ESIPT) coupled-charge transfer (ICT) is observed for the anionic form of the probes in polar solvents. This is likely the reason for the spectral differences of the probes when bound to amyloid fibrils. Moreover, the synthesis of a further development of the Congo red analogue X-34 [2,5-bis(4'-hydroxy-3'-carboxy-styryl) benzene] by rational design and synthesis is described. Full photophysical characterization was performed, including recording absorbance and fluorescence spectra, Stokes shift, quantum yield and fluorescence lifetimes. All ligands displayed high affinity towards recombinant amyloid fibrils of Aβ1-42 and tau as well as selectivity towards the corresponding disease-associated protein aggregates in human post mortem AD tissue. Lastly, the synthesis of a set of 2,1,3-benzothiadiazole (BTD)-based ligands with different conjugated spacers and variable patterns of OH substitutions of bis-styryl-BTD prototypes were developed. Aβ binding affinities (Aβ1-42 and Aβ1-40 fibrils) and the specificity towards Aβ plaques of all ligands were determined. These findings extend the structure to activity relationships of BTD...