Thioflavin T derivatives for the characterization of insulin and lysozyme amyloid fibrils in vitro: Fluorescence and quantum-chemical studies

Two charged Thioflavin T (ThT) derivatives, referred to here as ICT2 and ICT3, showed higher fluorescence response, association constants and the blue-shifted emission maxima in the presence of lysozyme fibrils compared to insulin aggregates. In turn, the other two ThT derivatives, ICT4 and ICT5, possessed much weaker sensitivity to amyloid fibrils. Furthermore, a direct correlation was found between the “light-up” ability of the fibril-bound fluorophores and those observed in concentrated dichlormethane or glycerol solutions. To explain this behavior, the ground and lowest non-relaxed excited state properties of the dyes were evaluated with the 6-31G(d,p) basis set, using DFT and the CIS method. The excited state energy dependences along the torsion angle between the benzothiazole and phenyl moieties of the ICT4, ICT5 turned out to have three directly observed minima, corresponding to the locally excited (LE) and twisted intramolecular charge transfer (TICT) states. Thus, stronger stabilization of the ICT4, ICT5 LE states resulted in significantly greater quantum yield of these dyes in buffer solution and the absence of the “light-up” feature in the presence of insulin amyloid fibrils, compared to ICT2 and ICT3.