| Abstract [eng] |
About three hundred different amino acids and their derivatives are found in nature. Mercapto amino acids, which are related to significant physiological activities in the human organism, are very important in biological objects and their systems of various complexity. There are three mercapto biomolecules of the similar structure – cysteine, homocysteine and glutathione – that play crucial roles. It has been established that their alternations in concentration levels in cells have been linked to a number of diseases. Therefore, detection of these mercapto biomolecules in biological samples is of crucial importance. For that purpose, a fluorescent Michael addition method employing a fragment of the maleimide as biothiols receptor is widely used. The main goal of this project was to synthesize dyes (probes) with a maleimide fragment, which could react selectively with mercapto amino acids and identify them. Cyanine dyes (probes) with different number of double bonds in the conjugated chain connecting the electron donor and electron acceptor parts of dyes have been synthesized in this project. They were used as precursors in the synthesis of fluorescent probes for detection of all three biothiols – cysteine, homocysteine and glutathione. In the fluorescence spectra of the novel probes, the shifts in emission maximum have revealed that the probe with two double bonds in chromophore Z2 identifies selectively mercapto amino acids in 1,4-dioxane and tetrahydrofuran. Fluorescent cysteine probe Z2-Cys prepared from this marker showed the highest fluorescence intensity, which was 9 times higher than that of the initial cyanine die and quantum yield was 7.5 % in dimethyl sulfoxide. It has been established that the extension of the conjugated chain of double bonds shifts the emission maximum batochromically. The fluorescence maximum for the probe with three double bonds in chromophore Z3 was shifted to a region of electromagnetic radiation 815 nm, which is a particularly attractive for identification of mercapto amino acids. This is an exellent result for the identification of thiols, since no tada is provided for probes, which emission maximum would be further than 800 nm in the region of electromagnetic radiation, in the scientific literature. The probe Z3 identifies selectively mercapto amino acids in dimethyl sulfoxide. Fluorescent cysteine probe Z3-Cys prepared from this marker showed even better fluorescence intensity results, which were even 12 times higher than that of the initial cyanine die and quantum yield was 9.7 %. As a conclusion, the synthesized probes Z2 and Z3 can be used succesfully for the identification of mercapto amino acids in biological samples. |
