Deciphering the Electronic Transitions of Thiophene‐Based Donor‐Acceptor‐Donor Pentameric Ligands Utilized for Multimodal Fluorescence Microscopy of Protein Aggregates

Abstract

Anionic pentameric thiophene acetates can be used for fluorescence detection and diagnosis of protein amyloid aggregates. Replacing the central thiophene unit by benzothiadiazole (BTD) or quinoxaline (QX) leads to large emission shifts and basic spectral features have been reported [Chem. Eur. J. 2015, 21, 15133‐13137]. Here we present new detailed experimental results of solvent effects, time‐resolved fluorescence and examples employing multi‐photon microscopy and lifetime imaging. Quantum chemical response calculations elucidate how the introduction of the BTD/QX groups changes the electronic states and emissions. The dramatic red‐shift follows an increased conjugation and quinoid character of the π‐electrons of the thiophene backbone. An efficient charge transfer in the excited states S1 and S2 compared to the all‐thiophene analogue makes these more sensitive to the polarity and quenching by the solvent. Taken together, the results guide in the interpretation of images of stained Alzheimer disease brain sections employing advanced fluorescence microscopy and lifetime imaging, and can aid in optimizing future fluorescent ligand development.

Proteophenes – Amino Acid Functionalized Thiophene‐based Fluorescent Ligands for Visualization of Protein Deposits in Tissue Sections with Alzheimer's Disease Pathology

“…When bound to CAA lesions, HS‐169‐V‐V showed a blue‐shifted emission spectrum with a maximum around 615 nm, compared to the ligand in PBS, as well as a distribution of fluorescence lifetimes between 2000 ps to 4000 ps (Figure 6). Similar decays have also been observed for other D‐A‐D thiophene‐based ligands when bound to protein aggregates [48] …”

“…The two most red‐shifted bands likely arise from the π‐π* transition and the charge‐transfer transition, respectively. [ 25 , 43 , 48 ] Upon excitation corresponding to the respective absorption band, HS‐169‐V‐V displayed a similar emission with a maximum around 720 nm for both excitation wavelengths (Figure 6 A). Similar absorption‐ and emission profiles have also been observed for the pentameric D‐A‐D thiophene based ligand HS‐169.…”

“…The ligand showed three absorbance bands between 230 nm and 600 nm (Figure 6A). The two most red‐shifted bands likely arise from the π‐π* transition and the charge‐transfer transition, respectively [25,43,48] . Upon excitation corresponding to the respective absorption band, HS‐169‐V‐V displayed a similar emission with a maximum around 720 nm for both excitation wavelengths (Figure 6A).…”

Alpha-Synuclein Strain Variability in Body-First and Brain-First Synucleinopathies

“…LCOs bind to the repetitive cross–β-sheet structures of pathogenic protein aggregates and display spectral differences based on the twisting of the flexible LCO backbone. Thus, the distinct conformation or “spectral fingerprint” of the ligand upon interaction with a certain asyn aggregate reflect the 3D structure of that asyn aggregate (Klingstedt and Nilsson, 2012 ; Gustafsson et al, 2021 ). These fluorescent LCO-ligands thus identify a broader subset of pathogenic protein aggregates than conventional ligands such as ThT, as they have been established as a class of ligands for superior recognition and spectral assignment of disease-associated protein aggregates, including different polymorphic Aβ (Ellingsen et al, 2013 ; Rasmussen et al, 2017 ; Calvo-Rodriguez et al, 2019 ; Lantz et al, 2020 ; Liu et al, 2021 ), asyn (Klingstedt et al, 2019 ; Shahnawaz et al, 2020 ) aggregates, as well as toxic and non-toxic polymorphic variants of insulin, both in vitro (Psonka-Antonczyk et al, 2012 ; Mori et al, 2021 ) and in patient biopsies (Yuzu et al, 2020 ).…”

“…Owing to their electronically delocalized conjugated thiophene backbones, LCOs exhibit intrinsic conformational dependent fluorescence characteristics that can be recorded by different modes of detection such as hyperspectral- and fluorescence life-time microscopy (Lantz et al, 2020 ; Gustafsson et al, 2021 ). Since development of the first ligands, the founding lab has generated a library of chemically diverse thiophene-based ligands that can bind to distinct aggregate conformers.…”