Fluorescent sondes have become an indispensable tool in researching cell dynamics, identifying biomolecules, and monitoring intercellular interactions in real time. Despite their widespread use, only a limited set of sondes with high photostability, selectivity, and biocompatibility are commercially available.
These markers utilise the phenomenom of fluorescence. When a phorophore is illuminated with light of a certain wavelenght, it absorbs a photon and transitions to an excited state, then emits light at a longer wavelenght. The difference between the absorbed and emitted wavelengths is known as the Stokes shift. Fluorophores with a large Stokes shift, high photostability, and specificity are particularly suitable for confocal microscopy.
This master's thesis aimed to develop new fluorescent markers based on coumarin ring containing a trifluoromethyl group at position 4 and an electrophilic group at position 3, enabling reactions with compounds containing a thiol group. We studied various synthetic routes to introduce different electrophilic groups.. We measured the excitation and emission spectra of three prepared markers (11, 18, 19) and confirmed the suitability of their spectral properties. We then tested their labelling ability, potential cytotoxicity and photostability using confocal microscopy on a mouse lung epithelial cell line (LA-4). Based on the physicochemical properties of the markers, we expected selective labeling of lipid droplets.
We did not observe any cytotoxic effects during measurements. Sonde 11 demonstrated the lowest photostability, while sonde 19 formed aggregates during measurements. None of the sondes showed exclusive selectivity for lipid droplets, as they also labeled other cellular organelles. Nevertheless, the synthesized compounds represent useful intermediates for the development of various fluorescent probes.
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