BF₂-Functionalized Benzothiazole Amyloid Markers: Effect of Donor Substituents on One- and Two-Photon Properties

Investigation of amyloids with the aid of fluorescence microscopy provides crucial insights into the development of numerous diseases associated with the formation of aggregates. Here, we present a series of BF₂-functionalized benzothiazoles with electron-donating methoxy group(s), which are tested as amyloid fluorescent markers. We evaluate how the position of donor functional group(s) influences optical properties (fluorescence lifetime (τ) and fluorescence quantum yield (FQY)) in a solution and upon binding to amyloids. We elucidate the importance of surrounding environmental factors (hydrogen-bonding network, polarity, and viscosity) on the observed changes in FQY and evaluate how the localization of a donor influences radiative and nonradiative decay pathways. We conclude that a donor attached to the benzothiazole ring contributes to the increment of radiative decay pathways upon binding to amyloids (k_r), while the donor attached to the flexible part of a molecule (with rotational freedom) contributes to a decrease in nonradiative decay pathways (k_nr). We find that the donor-acceptor-donor architecture allows us to obtain 58 times higher FQY of the dye upon binding to bovine insulin amyloids. Finally, we measure two-photon absorption (2PA) cross sections (σ₂) of the dyes and their change upon binding by the two-photon excited fluorescence (2PEF) technique. Measurements reveal that dyes that exhibit the increase/decrease of σ₂ values when transferred from highly polar solvents to CHCl₃ present a similar behavior upon amyloid binding. Our 2PA experimental values are supported by quantum mechanics/molecular mechanics (QM/MM) simulations. Despite this trend, the values of σ₂ are not the same, which points out the importance of two-photon absorption measurements of amyloid-dye complexes in order to understand the performance of 2P probes upon binding.