The thesis dedicated to the design, synthesis of fluorescent probes based on 3-hydroxyflavone for the ATP detection and to study the structure - efficiency relationship in the determination of ATP by fluorescence spectroscopy; establishing affinity, selectivity and detection limits of ATP for synthesized compounds. A series of thirty-three previously synthesized flavonols, bis-flavonols, 3-hydroxychromones and 3-hydroxyquinolones have been investigated in the aspect of the possibility of their use as fluorescence probes to determine the concentration of ATP in aqueous solutions. It has been established that most of the investigated dyes can serve as ATP probes. Among all dyes, eight compounds exhibit the best detection range, which is well consistent with the range of ATP concentrations in cells, and the sensitivity of ATP detection is greater than that of known analogs. Only 3 of the 33 compounds were not suitable for fluorescence detection of ATP. The whole series of thirty dyes is a set for ATP detection, which allows selecting a probe with an appropriate spectrum of excitation and emission, size, hydrophobicity or charge. Since complexation occurs at neutral pH values within of the physiological concentrations limits of ATP and Mg2+, most of the investigated dyes can be used as fluorescence probes for measuring the concentration of ATP in solutions and living cells.
A series of fluorescent probes containing one and two chromophores was synthesized. The individual monochromophoric compounds have anionic, cationic, and zwitterionic nature, and also contain other substituents that change the orientation of the components in the complex, as well as affinity and ATP selectivity. The bichromophoric compounds are "molecular tweezers" that contain long (active and passive) and short linkers, which also change both the orientation of the components in the complex, and the affinity and selectivity of the tweezer to the ATP.
The results of fluorometric studies of the interaction of flavonols with nucleotide phosphates in aqueous solutions at neutral pH values indicate that complexation with nucleotides displays in the following spectral effects:
• Hypochromic effect in absorption spectra caused by stacking flavonol with a nucleic base.
• Increase in the fluorescence intensity due to the decrease in the concentration of water molecules in the environment of flavonol and elimination of fluorescence quenching by water. In cases of flavonols with electron-acceptor substituents, this effect is often masked by the known effect of fluorescence quenching on a nucleic base due to photoinduced electron transfer to flavonol. However, at the formation of 1:2 complex, such quenching is blocked due to the compensation of the electron-accepting properties of flavonol by the second nucleic base.
• The appearance of a new band in the excitation spectra of due to the powerful electrostatic effect of the nucleotide phosphate groups on flavonol chromophore.
Based on the titrations of flavonols with GTP, ADP, and AMP, it was detected compounds exhibiting selectivity to ATP and to GTP. The results indicate that promotes complex formation is not only π-π stacking interactions between aromatic rings of flavones and nucleoside phosphates, and electrostatic interactions between positively charged dipole part of 3-hydroxyflavones and phosphate anion but more hydroxyl groups in the side core 3-hydroxyflavones. Compounds containing a protected 3-hydroxy group exhibit lesser effects in fluorescence spectra and have a lower affinity for nucleotides. Consequently, the 3-OH group participates in complex formation and in the formation of the spectral response to complexation.
The synthesized molecular tweezer 30 demonstrated its effectiveness in detecting ATP. Its fluorescence properties allowed the disclosure the spatial features of the complex structure with ATP. It was established that molecular tweezers in aqueous solutions exist in the "closed" conformation as a result of stacking of planar hydrophobic parts. In the same "closed" conformation it exists in the complex with ATP - at least in the case of 20% of its population. Significant (100 nm) red shift of the excitation band of the “tweezers-ATP” complex and its small band half-width create better conditions for the selective excitation of its fluorescence in order to detect it in solutions. The presence of a separate excitation band of the second chromophore of remote from the ATP location creates convenient conditions for the ratiometric determination of the concentration of ATP. These features, together with the wider limits of ATP detection, are essential advantages of molecular tweezers compared to simple monochromophoric structures of fluorescence probes.
