Environment-sensitive quinolone demonstrating long-lived fluorescence and unusually slow excited-state intramolecular proton transfer kinetics.

A new small fluorescent dye based on 3-hydroxybenzo[g]quinolone, a benzo-analogue of Pseudomonas quinolone signal species, has been synthesized. The dye demonstrates interesting optical properties, with absorption in the visible region, two band emission due to an excited-state intramolecular proton transfer (ESIPT) reaction and high fluorescence quantum yield in both protic and aprotic media. Time-resolved fluorescence spectroscopy shows that the ESIPT reaction time is unusually long (up to 8 ns), indicating that both forward and backward ESIPT reactions are very slow in comparison to other 3-hydroxyquinolones. In spite of these slow rate constants, the ESIPT reaction was found to show a reversible character as a result of the very long lifetimes of both N* and T* forms (up to 16 ns). The ESIPT reaction rate is mainly controlled by the hydrogen bond donor ability in protic solvents and the polarity in aprotic solvents. Using large unilamellar vesicles and giant unilamellar vesicles of different lipid compositions, the probe was shown to preferentially label liquid disordered phases.

Monitoring membrane binding and insertion of peptides by two-color fluorescent label.

Herein, we developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. For this purpose, we labeled the N-terminus of three synthetic peptides, melittin, magainin 2 and poly-l-lysine capable to interact with lipid membranes. Binding of these peptides to lipid vesicles induced a strong fluorescence increase, which enabled to quantify the peptide-membrane interaction. Moreover, the dual emission of the label in these peptides correlated well with the depth of its insertion measured by the parallax quenching method. Thus, in melittin and magainin 2, which show deep insertion of their N-terminus, the label presented a dual emission corresponding to a low polar environment, while the environment of the poly-l-lysine N-terminus was rather polar, consistent with its location close to the bilayer surface. Using spectral deconvolution to distinguish the non-hydrated label species from the hydrated ones and two photon fluorescence microscopy to determine the probe orientation in giant vesicles, we found that the non-hydrated species were vertically oriented in the bilayer and constituted the best indicators for evaluating the depth of the peptide N-terminus in membranes. Thus, this label constitutes an interesting new tool for monitoring membrane binding and insertion of peptides.

[Fluorescent probes for examination of spermatozoa in cryoprotective mediums].

By fluorescent spectroscopy and microscopy methods the possibility of fluorescent probes DSM, E-176, 3-DAB and FME application for study of cryoprotective agents' influence on the dog spermatozoa are investigated. It is established that FME and 3-DAD dyes are suitable for the posed problem solving, and the DSM and E-176 probes have restrictions owing to enough strong fluorescence from cryoprotectant solutions. It is shown that the fluorescent probes investigated influence the cells motility to different degree. The perspectives of investigated dyes application for study of cryoprotective agent' finfluence on spermatozoa are considered.

Dipole moments of 4'-aminoflavonol fluorescent probes in different solvents.

Electrooptical absorption measurements (EOAM) were used to measure the dipole moments of the normal form of 4'-(dimethylamino)-3-hydroxyflavone (FME), and 4' N-(15-azacrown-5)-3-hydroxyflavone (FCR) in 1,4-dioxane, toluene, and cyclohexane. With these probes excited-state intramolecular proton transfer (ESIPT) takes place. For comparison, the dipole moments of 4'-(dimethylamino)-3-metoxyflavone (FME3ME), for which ESIPT is lacking, were measured, too. For all three probes the ground (micro(g)) and excited Franck-Condon state (micro(e)FC) electrical dipole moments are parallel to each other and also parallel to the transition dipole moment. The electrical dipole moments of FCR, FME, and FME3ME in their ground state have values within the range (12.0-17.7) x 10(-30) C m. Upon optical excitation, the dipole moments increase by (41.9-52.9) x 10(-30) C m. The value of the change of the dipole moment vector delta(a)micro with excitation to the Franck-Condon state and the value of the vector micro(e)FC for FCR and FME are practically independent on the solvent polarity. From this point of view and due to large values of the dipole moments FCR and FME are very promising probes for the investigation of the distribution of the local polarity in biological systems using site-selective excitation of the different sites. Our steady-state fluorescence studies on FME and FCR have demonstrated a high spectral sensitivity of the normal form to such solvent characteristics as polarity.

Spectral and acid-base features of 3,7-dihydroxy-2,8-diphenyl-4H,6H-pyrano[3,2-g]chromene-4,6-dione (diflavonol)--a potential probe for monitoring the properties of liquid phases.

Diflavonol is a molecule that can exist in neutral or anionic form and in several tautomeric forms in ground and excited states. Absorption and emission spectroscopy combined with theoretical calculations have shown that only one tautomer of neutral diflavonol exists in the ground state, but two exist in the excited state. In the latter case, one is the tautomer originating from the ground state tautomer, which exists in strongly protic solvents, the other is the phototautomer occurring in weakly protic or aprotic solvents as a result of the intramolecular transfer of one proton. The OH groups present in diflavonol and involved in weak intramolecular hydrogen bonds exhibit a proton-donating ability reflected by the experimental values of acidity constants or theoretical enthalpies and free energies of proton detachment. The electronically excited molecule is a relatively strong acid when it loses one proton. With increasing basicity of the medium, monoanionic and dianionic forms occur which exhibit spectral characteristics and an emission ability different from those of neutral diflavonol. These interesting features of diflavonol open up possibilities for the analytical use of the compound and its application as a spectral probe sensitive to the properties of liquid phases.

Flavonols--new fluorescent membrane probes for studying the interdigitation of lipid bilayers.

Two flavonols, 3-hydroxy-4'-dimethylaminoflavone (FME) and 3-hydroxy-4'-(15-azacrown-5) flavone (FRC) have been investigated as new fluorescence probes for studying the formation of the interdigitated gel phase in lipid bilayers. The formation of the interdigitated gel phase in the saturated symmetrical phosphatidylcholines (PCs) and phosphatidylethanol (Peth) in the presence of ethanol has been well studied. The present study examines the behavior of these new probes in PC-ethanol and Peth-ethanol systems, as well as in PC-cholesterol and Peth-cholesterol vesicles. The present results demonstrate that both flavonols give distinctively different spectra in interdigitated lipid compared to non-interdigitated lipids, when examined in lipids in which the interdigitation behavior is known. This makes them useful for determinations of the structural state of unknown lipids, and for following the transitions between interdigitated and non-interdigitated phases. However, in the presence of cholesterol, only FCR gave appropriate indications of interdigitation. The results with FME in the presence of cholesterol were not consistent with the known behavior of the lipids examined; instead, FME appears to be located preferentially in the cholesterol-rich non-interdigitated regions of the bilayer.