Vasyl G. Pivovarenko

Novel Two-Band Ratiometric Fluorescence Probes with Different Location and Orientation in Phospholipid Membranes

3-hydroxyflavone (3-HF) derivatives are very attractive fluorescence sensors due to their ability to respond to small changes in their microenvironment via a dramatic alteration of the relative intensities of their two well-separated emission bands. We developed fluorescence probes with locations at different depths and orientations of 3-HF moiety in the phospholipid bilayer, which determine their fluorescence behavior. While the spectral shifts of the probes correlate with their binding site polarity, the intensity ratio is a complex parameter that is also sensitive to the local hydration. We demonstrate that even the deeply located probes sense this hydration effect, which can be modulated by the charge of the lipid heads and is anisotropic with respect to the bilayer plane. Thus the two-band ratiometric fluorescence probes can provide multiparametric information on the properties of lipid membranes at different depths.

Modulation of the solvent-dependent dual emission in 3-hydroxychromones by substituents

3-Hydroxychromones (3HCs) are fluorescent dyes, which respond to solvent perturbations by shifts and changes in the relative intensity of the two well-separated bands in the emission spectra. These bands originate from an excited state intramolecular proton transfer (ESIPT) reaction, which can be modulated by different factors, including modifications in the 3HC chromophore. In view of the great importance of 3HCs as prospective basic elements of molecular sensors, we have performed the first systematic study on the correlation between 3HC structure and spectroscopic properties. Two series of known and newly synthesized 2-phenyl-3-hydroxychromones and 2-(2-benzo[b]furanyl)-3-hydroxychromones with varied electron-donor substituents, introduced on opposite sides of the chromophore, were compared in solvents of different polarities. The substitution of 2-phenyl for 2-(2-benzo[b]furanyl) and introduction of electron donors on the 2-aryl group not only shift the absorption and fluorescence spectra to the red, but also strongly modulate the ESIPT behavior, resulting in a dramatic increase of the intensity ratio of the two emission bands, IN*/IT*. In contrast, introduction of a 7-methoxy group results in exactly the opposite spectroscopic effects. All the studied 3HC dyes demonstrate a linear increase in ln(IN*/IT*) with the solvent polarity parameter ET(30). Substitution of 2-phenyl for 2-(2-benzo[b]furanyl) or introduction of electron donors on the 2-aryl group in 3HC increases the sensitivity of their IN*/IT* ratio to solvent polarity and shifts the optimal range of ratiometric polarity sensing to less polar solvents. The opposite effects are observed for 7-methoxy derivatives. These results allow a new generation of two-band fluorescent sensors based on 3HC that operate by the ESIPT mechanism to be proposed. By proper substituents their photophysical and sensing properties can be tuned over broad ranges.

A 3-hydroxychromone with dramatically improved fluorescence properties

A new 3-hydroxychromone derivative, 2-(6-diethylaminobenzo(b)furan-2-yl)-3-hydroxychromone, has been synthesized by a concise route. Possessing dual emission common for 3-hydroxyflavones, it exhibits strong red shifts of both absorption and fluorescence spectra, which makes it the longest wavelength fluorescent dye among all known chromones. It also demonstrates a significant increase in fluorescence quantum yield in aprotic solvents and shift in solvent-polarity-dependent switch between normal and tautomer emissive forms. This derivative offers new possibilities in designing novel molecular sensors.

Flavonols as metal-ion chelators: complex formation with Mg2+ and Ba2+ cations in the excited state

The excited state complexation of flavonol derivatives with alkali and alkali-earth metal ions were investigated. It was found that in the excited state chelating magnesium complexes are more stable than in the ground state, and they are produced both from normal and from phototautomer forms of flavonols. The ‘external’ barium complexes with flavonols containing electron donor groups in 4 0 -position behave similarly in the excited state. The ‘external’ barium complexes of unsubstituted and 4 0 -methoxy substituted flavonols dissociate in the excited state. Their disruption occurs as a result of the excited state intramolecular proton transfer (ESIPT) in flavonol molecules. Ejection of metal ion from the crown-cycle was found for the magnesium and barium complexes of azacrown-flavonol ‐ M2L. In the case of Ba2L complex the process of cation ejection results in the formation of a complex which does not exist in the ground state. The authors assumed that the short-wavelength band in the emission spectrum of unsubstituted flavonol in acetonitrile might be due to the fluorescence of flavonol molecules forming a hydrogen bond with the solvent. ©1999 Elsevier Science S.A. All rights reserved.

Multiparametric Color-Changing Fluorescence Probes

1053-0509/03/0700-0291/0

Perturbation of planarity as the possible mechanism of solvent-dependent variations of fluorescence quantum yield in 2-aryl-3-hydroxychromones

In order to understand the unexpectedly low quantum yields of 3-hydroxyflavones (3-HFs) in certain solvents, such as acetonitrile or ethyl acetate, the comparative study of solvent-dependent properties of parent 3-HF, 2-furyl-3-hydroxychromone and 2-benzofuryl-3-hydroxychromone derivatives have been performed. The results suggest that the formation of intermolecular hydrogen bond of 3-hydroxy group with the solvent favors non-planar conformations of phenyl group with respect to chromone system. This steric hindrance is not observed in the case of furan- and benzofuran-substituted 3-hydroxychromones (3-HCs). These results suggesting a new strategy for dramatic improvement of fluorescence properties of 3-HCs as two-wavelength ratiometric fluorescence probes.

Fluorescence quenching of the ketocyanine dyes in polar solvents: anti-TICT behavior

Spectral properties and fluorescence decay in a series of differently N-substituted ketocyanine dyes belonging to the dibenzalcyclopentanone type were studied in solvents of various polarity and hydrogen bonding ability. The significant solvent-induced fluorescence quenching was explained by the increase of internal conversion rate owing to the lowering of the energy gap between S1 and S0 states in polar solvents. The alternative model of non-luminescent TICT states formation was shown to be less probable in the discussed series.

Bis-Azacrown Derivative of Di-Benzilidene-Cyclopentanone as Alkali Earth Ion Chelating Probe: Spectroscopic Properties, Proton Accepting Ability and Complex Formation with Mg2+ and Ba2+ Ions

Abstract1,3- bis-[4-(N-aza-15-crown-5)-benzylidene]cyclopentanone-2 ( I), a ketocyanine dye, was synthesized. The electronic absorption and emission spectra, protolytic interactions in aqueous alcohol media and complex formation with alkali earth metals in acetonitrile were studied for the dye ( I) and for several model compounds. It was shown that protonation of both dialkylamino groups of all compounds studied takes place at closely similar pH values. The complexation of alkali earth metals with azacrown derivative ( I) takes place at azacrown moieties and the carbonyl group. The sequence of binding to these sites is different for Mg2+ and Ba2+ ions. The effective ejection of Ba2+ ion out of the azacrown cycle was observed in the excited state, though, as in the case of Mg2+ ion, such process occurred only partially. The results obtained suggest that this azacrown derivative of dibenzylidene cyclopentanone is sensitive to alkali earth ions and has prospects for different biologically oriented applications.

Bands separation in fluorescence spectra of ketocyanine dyes: evidence for their complex formation with monohydric alcohols

Abstract In the studies of binary solvent systems containing non-polar (toluene) and polar proton-donating components (monohydric alcohols) using ketocyanine dyes of 2,5-di-benzylidene-cyclopentanone-1 type as solvent polarity probes, we found that in addition to common solvent polarity-dependent shifts of fluorescence spectra, at low alcohol concentrations there appear two new well-resolved spectral bands. They are attributed to the emission of hydrogen bonded complexes of 1:1 and 1:2 type. Effective constants for hydrogen bond complex formation were estimated for them from the fluorescence titration data.

Modulation of dual fluorescence in a 3-hydroxyquinolone dye by perturbation of its intramolecular proton transfer with solvent polarity and basicity

A representative of a new class of dyes with dual fluorescence due to an excited state intramolecular proton transfer (ESIPT) reaction, namely 1-methyl-2-(4-methoxy)phenyl-3-hydroxy-4(1H)-quinolone (QMOM), has been studied in a series of solvents covering a large range of polarity and basicity. A linear dependence of the logarithm of its two bands intensity ratio, log(I(N*)/I(T*)), upon the solvent polarity expressed as a function of the dielectric constant, (epsilon- 1)/(2epsilon + 1), is observed for a series of protic solvents. A linear dependence for log(I(N*)/I(T*)) is also found in aprotic solvents after taking into account the solvent basicity. In contrast, the positions of the absorption and the two emission bands of QMOM do not noticeably depend on the solvent polarity and basicity, indicating relatively small changes in the transition moment of QMOM upon excitation and emission. Time-resolved experiments in acetonitrile, ethyl acetate and dimethylformamide suggest an irreversible ESIPT reaction for this dye. According to the time-resolved data, an increase of solvent basicity results in a dramatic decrease of the ESIPT rate constant, probably due to the disruption of the intramolecular H-bond of the dye by the basic solvent. Due to this new sensor property, 3-hydroxyquinolones are promising candidates for the development of a new generation of environment-sensitive fluorescence dyes for probing interactions of biomolecules.