Fluorescent Probes Based on Charge and Proton Transfer for Probing Biomolecular Environment.

Fluorescent probes for sensing fundamental properties of biomolecular environment, such as polarity and hydration, help to study assembly of lipids into biomembranes, sensing interactions of biomolecules and imaging physiological state of the cells. Here, we summarize major efforts in the development of probes based on two photophysical mechanisms: (i) an excited-state intramolecular charge transfer (ICT), which is represented by fluorescent solvatochromic dyes that shift their emission band maximum as a function of environment polarity and hydration; (ii) excited-state intramolecular proton transfer (ESIPT), with particular focus on 5-membered cyclic systems, represented by 3-hydroxyflavones, because they exhibit dual emission sensitive to the environment. For both ICT and ESIPT dyes, the design of the probes and their biological applications are summarized.

Multi-parametric sensing by multi-channel molecular fluorescent probes based on excited state intramolecular proton transfer and charge transfer processes.

Considering the applications of fluorescent probes and the information they provide, their brightness of fluorescence and photostability are of paramount importance. However, in the case of steady-state fluorescence spectroscopy and fluorescence microscopy, the amount of information can be increased by the application of multi-channel probes, via a multi-band fluorophore introduced in the probe molecule. In most cases, the use of such a multi-band (or multi-channel) fluorophore can also be combined with the concomitant introduction of one or several analyte receptors.

Behavior of the Antibacterial Peptide Cyclo[RRRWFW], Explored Using a 3-Hydroxychromone-Derived Fluorescent Amino Acid.

Labeling biomolecules with fluorescent labels is an established tool for structural, biochemical, and biophysical studies; however, it remains underused for small peptides. In this work, an amino acid bearing a 3-hydroxychromone fluorophore, 2-amino-3-(2-(furan-2-yl)-3-hydroxy-4-oxo-4H-chromen-6-yl)propanoic acid (FHC), was incorporated in a known hexameric antimicrobial peptide, cyclo[RRRWFW] (cWFW), in place of aromatic residues. Circular dichroism spectropolarimetry and antibacterial activity measurements demonstrated that the FHC residue perturbs the peptide structure depending on labeling position but does not modify the activity of cWFW significantly.

Quantitative assessment of energy transfer in upconverting nanoparticles grafted with organic dyes.

Upconverting nanoparticles (UCNPs) are luminophores that have been investigated for a multitude of biological applications, notably low-background imaging, high-sensitivity assays, and cancer theranostics. In these applications, they are frequently used as a donor in resonance energy transfer (RET) pairs. However, because of the peculiarity and non-linearity of their luminescence mechanism, their behavior as a RET pair component has been difficult to predict quantitatively, preventing their optimization for subsequent applications.

A Toolbox of Chromones and Quinolones for Measuring a Wide Range of ATP Concentrations.

A series of 26 3-hydroxychromones, three bis-flavonols and four 3-hydroxyquinolones were studied to evaluate their fluorescence response to interaction with ATP in buffer. The dyes differ by the total charge, the size and number of their aromatic units, as well as the position or electron-donating ability of their substituents. All of them were suggested to form complexes with ATP of 1:1 and 1:2 stoichiometry, which can be evidenced by their bright fluorescence and their 3000-6000 cm red-shifted excitation band.

Non-coordinating anions assemble cyanine amphiphiles into ultra-small fluorescent nanoparticles.

A non-coordinating anion, fluorinated tetraphenylborate, assembles specially designed cationic cyanine amphiphiles into 7-8 nm fluorescent nanoparticles that are >40-fold brighter than a single cyanine dye. This kind of anion, combining hydrophobic and electrostatic forces in aqueous media, constitutes promising building blocks in the self-assembly of functional nanomaterials.

Fluorescent amino acid undergoing excited state intramolecular proton transfer for site-specific probing and imaging of peptide interactions.

Fluorescent amino acids bearing environment-sensitive fluorophores are highly valuable tools for site-selective probing of peptide/ligand interactions. Herein, we synthesized a fluorescent l-amino acid bearing the 4'-methoxy-3-hydroxyflavone fluorophore (M3HFaa) that shows dual emission, as a result of an excited state intramolecular proton transfer (ESIPT). The dual emission of M3HFaa was found to be substantially more sensitive to hydration as compared to previous analogues.

Monitoring penetratin interactions with lipid membranes and cell internalization using a new hydration-sensitive fluorescent probe.

A new fluorescent label N-[4′-(dimethylamino)-3-hydroxyflavone-7-yl]-N-methyl-β-alanine (7AF) was synthesized. Due to two electron donor groups at the opposite ends of the chromophore, an excited state intramolecular proton transfer (ESIPT) resulting in a dual emission was observed even in highly polar media and its fluorescence quantum yield was found to be remarkably high in a broad range of solvents including water. As a consequence, this label exhibits a remarkable sensitivity to the hydration of its environment, which is observed as a color switch between the emission of the ESIPT product (T* form) and that of the normal N* form.

Dual-fluorescence L-amino acid reports insertion and orientation of melittin peptide in cell membranes.

Monitoring insertion and orientation of peptides in situ on cell membranes remains a challenge. To this end, we synthesized an l-amino acid (AFaa) containing a dual-fluorescence dye of the 3-hydroxyflavone family, as a side chain. In contrast to other labeling approaches using a flexible linker, the AFaa fluorophore, introduced by solid phase synthesis into desired position of a peptide, is attached closely to its backbone with well-defined orientation, and, therefore, could reflect its localization in the membrane.

Tautomerism and behavior of 3-hydroxy-2-phenyl-4H-chromen-4-ones (flavonols) and 3,7-dihydroxy-2,8-diphenyl-4H,6H-pyrano[3,2-g]chromene-4,6-diones (diflavonols) in basic media: spectroscopic and computational investigations.

Absorption and emission spectroscopic investigations and computational predictions have shown that neutral molecules of flavonols and diflavonols can exist in the ground and excited states in one or two tautomeric forms stabilized by intramolecular (in aprotic media) or intermolecular (with solvent molecule(s), in protic media) hydrogen bonds. Electronic excitation creates conditions for the transformation of tautomeric forms, accompanied by proton transfer, reflected in fluorescence spectra. Proton transfer is also probable in monoanions of diflavonols in protic media.

Two-color fluorescent l-amino acid mimic of tryptophan for probing peptide-nucleic acid complexes.

Non-natural amino acids are important tools for site-selective probing of peptide properties and interactions. Here, for the first time a fluorescent l-amino acid, exhibiting excited-state intramolecular proton transfer (ESIPT) and hydration-sensitive dual emission, was synthesized. It is an analogue of l-tryptophan bearing a slightly larger 2-(2-furyl)-3-hydroxychromone aromatic moiety instead of indole.

Quantification of local hydration at the surface of biomolecules using dual-fluorescence labels.

By using four labels of the 3-hydroxyflavone family displaying selective sensitivity to hydrogen bond (HB) donors and poor response to other polar molecules, we developed an approach for measuring local water concentration [H(2)O](L) (or partial volume of water: W(A) = [H(2)O](L)/55.6) in the label surrounding both in solvent mixtures and in biomolecules by the intensity ratio of two emissive forms of the label, N*/T*. Using a series of binary water/solvent mixtures with limited preferential solvation effects, a linear dependence of log(N*/T*) on the local concentration of HB donor was obtained and then used as a calibration curve for estimating the W(A) values in the surroundings of the probes conjugated to biomolecules.

2-(Furan-2-yl)-3-hy-droxy-4H-chromen-4-one.

In the crystal structure of the title compound, C(13)H(8)O(4), the inversely oriented mol-ecules form inversion dimers through pairs of O-H⋯O hydrogen-bonding inter-actions. An intramolecular O-H⋯O hydrogen bond occurs. In the packing of the mol-ecules, the nearly planar 2-(furan-2-yl)-4H-chromene units [dihedral angle between the chromene and furan rings = 3.

3-Hy-droxy-2-(4-hy-droxy-phen-yl)-4H-chromen-4-one.

In the title compound, C(15)H(10)O(4), the benzene ring is twisted at an angle of 20.7 (1)° relative to the 4H-chromene skeleton. In the crystal, adjacent mol-ecules are linked via a network of O-H⋯O and C-H⋯O hydrogen bonds.

3-Hy-droxy-1-methyl-2-[4-(piperidin-1-yl)phen-yl]quinolin-4(1H)-one.

There are two structurally similar but crystallographically independent mol-ecules (A and B) in the asymmetric unit of the title compound, C(21)H(22)N(2)O(2), which are linked via two O-H⋯O hydrogen bonds. An intramolecular O-H⋯O hydrogen bond also occurs in each molecule. In the crystal, the A and B mol-ecules are further linked through C-H⋯O inter-actions.

Improved hydration-sensitive dual-fluorescence labels for monitoring peptide-nucleic acid interactions.

Environmentally sensitive labels constitute a new, attractive tool for monitoring biomolecular interactions. 3-Hydroxychromone derivatives are of particular interest because they undergo excited-state intramolecular proton transfer (ESIPT) showing dual emission highly sensitive to environmental hydration. To overcome the drawbacks of the previously developed label for sensing protein-DNA interactions based on 2-furanyl-3-hydroxychromone (FC), a series of hydration-sensitive labels based on 3-hydroxy-4'-methoxyflavone have been synthesized.

Dual-fluorescence probe of environment basicity (hydrogen bond accepting ability) displaying no sensitivity to polarity.

3-Hydroxyquinolones (3HQs) are a new class of water soluble dual fluorescence probes that can monitor both polarity and basicity (H-bond accepting ability) parameters. Both parameters play an important role in proteins and lipid membranes. Nevertheless, no method exists actually to measure the basicity parameter separately from the polarity.

Modulation of excited-state intramolecular proton transfer by viscosity in protic media.

3-Hydroxyquinolones undergo excited-state intramolecular proton transfer (ESIPT), resulting in a dual emission highly sensitive to H-bonding perturbations. Here, we report on the strong effect of viscosity on the dual emission of 2-(2-thienyl)-3-hydroxyquinolone in protic solvents. An increase in viscosity significantly decreases the formation of the ESIPT product, thus changing dramatically the ratio of the two emission bands.

Steric control of the excited-state intramolecular proton transfer in 3-hydroxyquinolones: steady-state and time-resolved fluorescence study.

3-Hydroxyquinolones (3HQs), similarly to their 3-hydroxychromone analogs, undergo excited state intramolecular proton transfer (ESIPT) resulting in dual emission. In the ground state, 2-phenyl-3HQ derivatives are not flat due to a steric hindrance between the 2-phenyl group and the 3-OH group that participates in the ESIPT reaction. To study the effect of this steric hindrance on the ESIPT reaction, a number of 3HQ derivatives have been synthesized and characterized in different organic solvents by steady-state and time-resolved fluorescence techniques.

Sensing of adenosine-5'-triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye.

The current work demonstrates the formation of complexes between the tetraanion adenosine-5'-triphosphate (ATP) and the flavone derivative 3-hydroxy-4'-(dimethylamino)flavone (FME). Two kinds of complexes are evidenced. The higher affinity ATP-FME complex corresponds to a stacking of the two aromatic molecules and leads to a strong hypochromicity of the absorption spectrum of the dye.

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.

Influence of freezing and low molecular weight cryoprotectants on microsomal membrane structure: a study by multiparametric fluorescent probe.

The influence of low molecular weight cryoprotectants (CPs) such as glycerol (GL), 1,2-propanediol (PD) and dimethylsulfoxide (DMSO) on the structure of rat liver microsomal membranes on the stages of equilibration and upon freezing up to -196 degrees C was studied using a multiparametric fluorescent probe of flavonol nature. It was estimated that the studied CPs have individual concentration ranges defining low amplitude of their action on biomembranes. An exceeding of these ranges strongly increases the violation of membrane native structure already at the stage of incubation with CPs, strengthening it during the freezing procedure.

Fluorometric detection of adenosine triphosphate with 3-hydroxy-4'-(dimethylamino)flavone in aqueous solutions.

An effect of appearance of new band in the excitation spectra of 3-hydroxy-4'-(dimethylamino)flavone (FME probe) in presence of adenosine triphosphate (ATP) is described. Considerable shift of new band up to the red and increase of fluorescence intensity points to the formation of FME-ATP associate, in which FME molecule undergoes to a strong electrostatic stabilization by tetra-charged ATP anion. It is shown the FME anion formation is possible under influence of ATP in the studied conditions.

Solvent influence on excited-state intramolecular proton transfer in 3-hydroxychromone derivatives studied by cryogenic high-resolution fluorescence spectroscopy.

High-resolution Shpol'skii spectra (recorded at 10 K in n-octane) of 3-hydroxychromone (3HC) substituted at the 2-position with a furan (3HC-F), a benzofuran (3HC-BF) or a naphthofuran group (3HC-NF) are presented. Being close analogues of 3-hydroxyflavone (3HF), these compounds can undergo excited-state intramolecular proton transfer (ESIPT). Luminescence can occur from the normal N* state (blue) or from the tautomeric T* state (green).

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).