In this master’s thesis, we focused on the optimization of the synthetic route to the bisbenzimidazole DNA label Hoechst 33258 and on the preparation of its derivatives for further functionalization with fluorophores. Hoechst 33258 binds specifically to the minor groove of DNA at AT-rich sequences, but its application in live-cell imaging is limited by excitation in the ultraviolet region. Nevertheless, Hoechst dyes remain indispensable in fluorescence microscopy because they readily cross the plasma membrane and efficiently stain DNA in living cells. Their fluorescence intensity increases markedly upon binding to DNA, which is a consequence of rigidization of the bisbenzimidazole scaffold and changes in the polarity of the surrounding environment; in practice, this results in lower background signal and an improved signal-to-noise ratio. Like most compounds that interact with DNA, Hoechst dyes are to some extent toxic, but at the concentrations used their toxicity is acceptable and significantly lower than that of probes that intercalate between base pairs. Their main drawback remains excitation with phototoxic UV light; therefore, analogues that could be excited with visible light and exhibit a larger Stokes shift would be highly valuable for research applications. The main aim of this thesis was to optimize the synthetic route to the Hoechst label, prepare its structural analogues suitable for further functionalization, and develop new probes with improved photophysical properties. In the experimental part, we repeated the multistep synthesis of the bisbenzimidazole scaffold of Hoechst 33258 and optimized individual reaction steps in order to improve yields. The most demanding step was the formation of the second benzimidazole unit, where condensation between a diamine and an aldehyde proved to be the most suitable approach after several attempts. Using this reaction, we successfully introduced various functional groups and prepared a series of new probes.
The optical properties of the compounds were evaluated by measuring excitation and emission spectra in the presence and absence of deoxyribonucleic acid. Upon binding to the target, the fluorescence signal increased, and in some derivatives a spectral shift toward the visible region was also observed. Biological evaluation in a metastatic pancreatic adenocarcinoma cell line showed that some of the newly prepared probes efficiently crossed the cell membrane and selectively stained the cell nucleus.
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