Fluorescence is one of the key phenomena in modern cell biology, as it enables sensitive and spatially resolved visualization of biomolecules, organelles, and other cellular structures. It relies on the use of fluorophores or fluorescent probes, whose optical properties allow the monitoring of biological processes in cells. Different classes of fluorophores, including merocyanines, differ in their absorption and emission wavelengths, Stokes shifts, photostability, and environmental sensitivity, which enables the development of probes for labeling specific cellular organelles, such as mitochondria and lipid droplets.
In this master’s thesis, we synthesized a series of merocyanine fluorophores and successfully reproduced the synthesis of compounds previously described in the literature. The synthetic route was then optimized in terms of reagent availability and cost-effectiveness by replacing the more expensive cesium carbonate with cheaper and more readily available potassium carbonate. Various functional groups, including carboxyl, azide, alkyne, and trifluoromethyl groups, were introduced into the structures of the merocyanine fluorophores. The introduction of the trifluoromethyl group increased the Stokes shift, which is advantageous for fluorescent probes because it reduces overlap between the excitation and emission signals. The alkyne group enables further derivatization by copper-catalyzed azide–alkyne cycloaddition, which was used to prepare fluorescent probe 18 for mitochondrial labeling.The prepared compounds were characterized by determining their basic photophysical properties, including excitation and emission spectra and Stokes shifts. These data enabled the selection of promising compounds for biological evaluation and the optimization of confocal fluorescence microscopy settings. Probe 18, which contains a 3,5-diphenylpyridinium cation, was evaluated at the Max Planck Institute for Multidisciplinary Sciences in Göttingen, Germany. Despite the expected mitochondrial localization, no visible accumulation of the compound in mitochondria was observed under the experimental conditions used. Since the compound concentration and incubation time were limited, the results do not allow definitive conclusions regarding its mitochondrial localization; therefore, further studies should optimize the labeling conditions.
In addition to the mitochondrial probe, probes 3 and 5 were prepared for lipid droplet labeling. Confocal fluorescence microscopy showed that compound 3 effectively and selectively labels lipid droplets, which was further confirmed by Pearson’s correlation coefficient and Manders’ overlap coefficients M1 and M2. In contrast, compound 5 did not effectively label lipid droplets under the same conditions. The results indicate that merocyanine fluorophores are a useful platform for the development of fluorescent probes, with compound 3 emerging as the most promising probe for lipid droplet labeling.
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