Fluorescent probe 4-dimethylaminochalcone: mechanism of fluorescence quenching in nonpolar media

The reasons for the high sensitivity of the fluorescent probe 4-dimethylaminochalcone (DMC) to nonpolar environment were explored. It was shown that, at room temperature, the fluorescence quantum yield in nonpolar media at 20 degrees C is lower than 0.01 (0.001 in methylcyclohexane). However, as temperature was lowered to -196 degrees C, the yield in methylcyclohexane increased more than 200 times. At the same time, the oscillator strength of absorption transition increased, and the absorption spectrum was shifted to red. These results, together with quantum chemistry calculations suggest that, for fluorescence quenching to occur, some barrier in the DMC molecule, probably the barrier of rotation about C-C bonds, should be overcome. In other words, the quenching is associated with the transition of DMC molecules from a flat conformation (energy minimum) to other, nonflat conformations through rotations about C-C bonds. The phosphorescence of DMC at low temperatures was detected. This suggests that fluorescence quenching is caused by radiationless transitions from the excited singlet level to the ground and triplet levels, and rotation about bonds facilitates these transitions.     

Kinetics of rearrangement of solvation sheath of an excited molecule of the fluorescent probe 4"-dimethylaminochalcone

Processes accompanying the quenching of the fluorescent probe 4"-dimethylaminochalcone by hydroxyl groups of the proton-donor solvent 1-butanol have been studied. The kinetics of the deactivation of the excited state of 4"-dimethylaminochalcone has been monitored from the transition absorption spectra at a time resolution of 50 fs and fluorescence decay at a time resolution of 30 ps. The data obtained allow thinking that the next picture occurs in 1-butanol. At first stage, the 4"-dimethylaminochalcone molecule in its ground state forms a hydrogen bond with an alcohol molecule. At the second stage, the absorption of light quantum and corresponding rise of the dipole moment of 4"-dimethylaminochalcone take place, the initially existing hydrogen bond is retained. The third stage consists in the rearrangement of the 4"-dimethylaminochalcone solvation shell formed by alcohol dipole molecules due to an increase of the dipole of moment 4"-dimethylaminochalcone; this rearrangement takes an energy of about 24 kJ/mol, the arrangement time constant is close to 40 ps; the initial hydrogen bond is retained. The fourth stage involves processes that lead to fluorescence quenching; their time constant is about 200 ps. Taking into account that the quenching is a much slower process than the relaxation of the solvation shell, it was supposed that the quenching is not a direct consequence of the solvation shell relaxation or the existence of the hydrogen bond formed prior to excitation. Then the fluorescence quenching of 4"-dimethylaminochalcone can be accomplished through some other processes that are observed in other fluorescent molecules: (a) rearrangement of the initial hydrogen bond from a conformation that cannot quench the fluorescence of 4"-dimethylaminochalcone to a more "effective" conformation, (b) charge transfer between the excited of molecule 4"-dimethylaminochalcone and alcohol, or (c) solvent-induced twist of the 4"-dimethylaminochalcone amino group (its withdrawal from the molecule plane) by the action of the solvent.     

Kinetics of the rearrangement of the solvation shell of an excited fluorescent probe 4″-dimethylaminochalcone

A study was made of the processes associated with the quenching of 4″-dimethylaminochalcone (DMAC) fluorescence by proton-donor solvent (1-butanol). The kinetics of deactivation of the DMAC excited state was assessed by transient absorption spectra with a time resolution about 50 fs and by fluorescence decay with ～30-ps resolution. The following sequence of events could thus be envisaged: (i) the DMAC molecule in the ground state (prior to excitation) makes a hydrogen bond with an alcohol molecule; (ii) absorption of a light quantum causes a corresponding increase of the DMAC dipole moment; the H-bond is retained; (iii) the solvation shell formed by alcohol dipoles is reorganized in response to the raise of the DMAC dipole moment, with an energy expenditure about 24 kJ/mol and a time constant about 40 ps; the initial H-bond is still retained; (iv) processes leading to fluorescence quenching occur with an effective time constant of nearly 200 ps. Since quenching is far slower than solvate rearrangement, one can suppose that it is not a direct consequence of shell relaxation or prior H-bonding. Thus, DMAC fluorescence quenching may involve different processes observed with other aromatic molecules: H-bond rearrangement from a nonquenching to a more ‘efficient’ conformation, charge transfer between the excited molecule and alcohol, or solvent-induced out-of-plane twist of the DMAC amino group.     

Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy.

The investigation in this report aimed at providing photophysical evidence that the long-lived triplet excited state plays an important role in the non-single-exponential photobleaching kinetics of fluorescein in microscopy. Experiments demonstrated that a thiol-containing reducing agent, mercaptoethylamine (MEA or cysteamine), was the most effective, among other commonly known radical quenchers or singlet oxygen scavengers, in suppressing photobleaching of fluorescein while not reducing the fluorescence quantum yield. The protective effect against photobleaching of fluorescein in the bound state was also found in microscopy. The antibleaching effect of MEA let to a series of experiments using time-delayed fluorescence spectroscopy and nanosecond laser flash photolysis. The combined results showed that MEA directly quenched the triplet excited state and the semioxidized radical form of fluorescein without affecting the singlet excited state. The triplet lifetime of fluorescein was reduced upon adding MEA. It demonstrated that photobleaching of fluorescein in microscopy is related to the accumulation of the long-lived triplet excited state of fluorescein and that by quenching the triplet excited state and the semioxidized form of fluorescein to restore the dye molecules to the singlet ground state, photobleaching can be reduced.     

Picosecond kinetics of excited-state charge separation in 4-(dimethylamino)pyridine.

4-(Dimethylamino)pyridine (DMAP) shows solvent-dependent dual fluorescence from the initially excited state B* and a highly polar TICT state A*. Room-temperature time-resolved picosecond fluorescence investigations prove the bimodal kinetics of the excited-state electron transfer reaction B*-->A* in polar aprotic media. In medium polarity solvents (such as ethyl acetate) two emitting states of DMAP are shown to reach equilibrium within 50 ps. Both emitting states originate from the same ground state. The rate of excited-state charge separation depends on polarity and proton donating ability of the surrounding medium. The effects of temperature on the quantum yields of both fluorescences of DMAP in polar aprotic media indicate the transition from the kinetic regime (at low temperatures) to the equilibrium regime (at high temperatures). The kinetic behaviour of the dual luminescence of DMAP in protic solvents is more complex than in aprotic ones. In alcohols an efficient nonradiative channel competes with excited-state charge separation.     

Effect of xenon on the excited States of phototropic receptor flavin in corn seedlings

The chemically inert, water-soluble heavy atom gas, xenon, at millimolar concentrations specifically quenches the triplet excited state of flavin in solution without quenching the flavin singlet excited state. The preferential quenching of the flavin triplet over the singlet excited state by Xe has been established by showing that the flavin triplet-sensitized photooxidation of NADH is inhibited while the fluorescence intensity and lifetime of flavin are not affected by Xe.     

A picosecond pulse train study of exciton dynamics in photosynthetic membranes.

The fluorescence decay time of spinach chloroplasts at 77 degrees K was determined at 735 nm (corresponding to the photosystem I emission) using a train of 10-ps laser pulses spaced 10 ns apart. The fluorescence lifetime is constant at congruent to 1.5 ns for up to the fourth pulse, but then decreases with increasing pulse number within the pulse train. This quenching is attributed to triplet excited states, and it is concluded that triplet excitons exhibit a time lag of about 50 ns in diffusing from light harvesting antenna pigments to photosystem I pigments. The diffusion coefficient of triplet excitons is a least 300--400 times slower than the diffusion coefficient of singlet excitons in chloroplast membranes.     

Intramolecular exciplexes based on benzoxazole: photophysics and applications as fluorescent cation sensors.

Exciplex behaviour of three benzoxazole derivatives has been detected and intensively investigated by means of steady-state and time-resolved fluorescence techniques and transient absorption spectroscopy. The fluorescence of these compounds shows the properties which are typical for the excited state charge transfer complexes (exciplexes). Besides of the short wavelength fluorescence, which is similar in spectral distribution to the fluorescence of the electron acceptor (2-p-tolyl-benzoxazole), the red shifted, broad and structureless emission band is observed in solvents of low and medium polarity. The detailed analysis of the fluorescence data shows that the ratio of the CT and LE fluorescence initially increases with increasing solvent polarity, achieves a maximum, and drops for more polar solvents (epsilon(s) = 7). Similar behaviour is observed for the exciplex fluorescence lifetimes. The overall fluorescence and the relative intersystem crossing quantum yields show the decrease of these values with increasing solvent polarity. These observations have been explained on the basis of Marcus-type theory for nonradiative charge transfer rate constants. Increasing solvent polarity strongly accelerates the back electron transfer process which recovers the whole molecule in the ground state. The probability of the compact exciplex formation (i.e. sandwich-type structures) depends on solvent viscosity and degree of freedom of the bending of the saturated linker. The compound containing crown ether as a donor subunit may be used as a fluorescent indicator of inorganic cations (barium and lithium). We found an effective complexation of the compound in the ground state with barium and lithium cations. The complex is also stable in the excited state which manifests itself in strong increase of the fluorescence intensity.     

Probing structure and dynamics of DNA with 2-aminopurine: effects of local environment on fluorescence

2-Aminopurine (2AP) is an analogue of adenine that has been utilized widely as a fluorescence probe of protein-induced local conformational changes in DNA. Within a DNA strand, this fluorophore demonstrates characteristic decreases in quantum yield and emission decay lifetime that vary sensitively with base sequence, temperature, and helix conformation but that are accompanied by only small changes in emission wavelength. However, the molecular interactions that give rise to these spectroscopic changes have not been established. To develop a molecular model for interpreting the fluorescence measurements, we have investigated the effects of environmental polarity, hydrogen bonding, and the purine and pyrimidine bases of DNA on the emission energy, quantum yield, and intensity decay kinetics of 2AP in simple model systems. The effects of environmental polarity were examined in a series of solvents of varying dielectric constant, and hydrogen bonding was investigated in binary mixtures of water with 1,4-dioxane or N,N-dimethylformamide (DMF). The effects of the purine and pyrimidine bases were studied by titrating 2AP deoxyriboside (d2AP) with the nucleosides adenosine (rA), cytidine (rC), guanosine (rG), and deoxythymidine (dT), and the nucleoside triphosphates ATP and GTP in neutral aqueous solution. The nucleosides and NTPs each quench the fluorescence of d2AP by a combination of static (affecting only the quantum yield) and dynamic (affecting both the quantum yield and the lifetime, proportionately) mechanisms. The peak wavelength and shape of the emission spectrum are not altered by either of these effects. The static quenching is saturable and has half-maximal effect at approximately 20 mM nucleoside or NTP, consistent with an aromatic stacking interaction. The rate constant for dynamic quenching is near the diffusion limit for collisional interaction (k(q) approximately 2 x 10(9) M(-1) s(-1)). Neither of these effects varies significantly between the various nucleosides and NTPs studied. In contrast, hydrogen bonding with water was observed to have a negligible effect on the emission wavelength, fluorescence quantum yield, or lifetime of 2AP in either dioxane or DMF. In nonpolar solvents, the fluorescence lifetime and quantum yield decrease dramatically, accompanied by significant shifts in the emission spectrum to shorter wavelengths. However, these effects of polarity do not coincide with the observed emission wavelength-independent quenching of 2AP fluorescence in DNA. Therefore, we conclude that the fluorescence quenching of 2AP in DNA arises from base stacking and collisions with neighboring bases only but is insensitive to base-pairing or other hydrogen bonding interactions. These results implicate both structural and dynamic properties of DNA in quenching of 2AP and constitute a simple model within which the fluorescence changes induced by protein-DNA binding or other perturbations may be interpreted.     

Hemin-catalyzed generation of triplet acetone

Hemin can substitute for horseradish peroxidase as a catalyst for the aerobic oxidation of isobutanal to acetone and formate. Previous studies have shown that the chemiphosphorescent emission observed in the enzyme-catalyzed reaction is due to the production of acetone in its triplet state. Although no chemiphosphorescence is observed with the model system (hemin), generation of triplet acetone in this system is indicated by an analysis of data for energy transfer to the 9,10-dibromoanthracene-2-sulfonate ion and for interception of the excited species by the sorbate ion, a known triplet quencher. These data are compared to those obtained with triplet acetone generated by thermal cleavage of tetramethyldioxetane in aqueous solution. The results are in agreement with the hypothesis that the quenching of triplet acetone by oxygen is less efficient in the enzyme catalyzed reaction, pointing to a protective role for the apo-enzyme in that system.     

Scavenging of photogenerated oxidative species by antimuscarinic drugs: atropine and derivatives

The quenching ability of photogenerated oxidative species by some antimuscarinic drugs generically named atropines (e.g. atropine [I] eucatropine [II], homatropine [III] and scopolamine [IV]) have been investigated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Using Rose Bengal as a dye sensitiser for singlet molecular oxygen, O(2)((1)Delta(g)), generation, compounds I-IV behave as moderate chemical plus physical quenchers of the oxidative species. Correlation between kinetic and electrochemical data indicates that the process is possibly driven by a charge-transfer interaction. The situation is somewhat more complicated employing the natural pigment riboflavin (Rf) as a sensitiser. Compounds I and II complex Rf ground state, diminishing the quenching ability towards singlet and triplet excited state of the pigment. On the other hand, compounds III and IV effectively quench Rf excited states, protecting the pigment against photodegradation. Under anaerobic conditions, semireduced Rf (Rf(.-)) is formed through quenching of excited triplet Rf. Nevertheless, although Rf(.-) is a well-known generator of the reactive species superoxide radical anion by reductive quenching in the presence of oxygen, the process of O(2)((1)Delta(g)) production prevails over superoxide radical generation, due to the relatively low rate constants for the quenching of triplet Rf by the atropines (in the order of 10(7) M(-1)s(-1) for compounds III and IV) in comparison to the rate constant for the quenching by ground state oxygen, approximately two orders of magnitude higher, yielding O(2)((1)Delta(g)). Compound I is the most promising O(2)((1)Delta(g)) physical scavenger, provided that it exhibits the higher value for the overall quenching rate constant and only 11% of the quenching process leads to its own chemical damage.     

Photoprocesses of chlorin e6 glucose derivatives

The ground and excited state processes of chlorin e6, the monomethyl ester C1, the glucose derivative C2 and the 3-heptylchlorin-glucose C3, were studied in solvents of lower and higher polarity. The excited singlet and lowest triplet states of C1-C3 were characterized by spectroscopic methods for several conditions. The quantum yields of formation of singlet molecular oxygen and the other triplet properties of the three chlorins and chlorin e6 are similar, whereas the fluorescence quantum yield decreases on going from C1 to C3. Time-resolved optoacoustic experiments revealed a ca. 30 kJ mol(-1) higher triplet level for C3 with respect to C1/2.     

Solvent polarity effect on excited-state lifetime of carotenoids and some dyes

The theory demonstrating the role of medium at the fluorescence quenching of polar compounds in solutions is briefly presented. It has been shown, that the rate of S(1) --> X(n) nonradiative conversion between the intramolecular charge transfer states depends on the permanent dipole moments in the ground (S(0)) and excited (S(1), X(n)) states as well as on solvent polarity. A relation for the rate of nonradiative excited-state energy conversion has been obtained and employed to test the known literature data for solvent effect on the S(1)-state lifetime of some biologically significant carotenoids and dyes (phthalimides). For phthalimides, the solvent isotope effect on the S(1)-state energy conversion, when hydrogen is replaced by deuterium in the OH groups of alcohols and water, has been analyzed. Based on the data for fluorescence quenching in solvents of different polarity, the dipole moments in the intermolecular charge transfer S(1) state have been obtained for carotenoids (peridinin, fucoxanthin, uriolide acetate) and for hydrogen-bonding complexes, which are formed by 4-amino-, 4-methylamino-, and 4-dimethylamino-N-methylphthalimides in alcohols and water.     

On mechanisms of fluorescence quenching by water

Mechanisms of fluorescence quenching of aromatic chromophores by water are reviewed. The mechanisms include polarity of chromophore environment, proton or electron transfer between the excited chromophore and water. A hypothesis is proposed that the quenching can be a result of chromophore-solvent hydrogen bond breaking in the excited state.     

Intramolecular charge transfer processes in donor-acceptor substituted vinyltetrahydropyrenes.

Two novel donor-acceptor-substituted vinyltetrahydropyrene derivatives, 2-N,N-dimethylamino-7-(1-carbethoxyvinyl)-4,5,9,10-tetrahydropyrene, , and 2-N,N-dimethylamino-7-(1,1-dicyanovinyl)-4,5,9,10-tetrahydropyrene, , were synthesized and their photophysical properties investigated in solvents of different polarities. Our studies revealed the existence of intramolecular charge transfer excited states in these molecules. For both compounds the fluorescence maxima exhibited solvent polarity-dependent red shifts. These were quantitatively analysed by the Lippert-Mataga and Liptay equations to obtain the excited state dipole moments. Our results indicated that in the case of , emission takes place from a planar (1)CT state in all non-protic solvents. In the case of , the nature of the excited state depends on the solvent. A fast relaxation to a triplet state is proposed in cyclohexane. The emitting state in medium polar solvents is a planar (1)CT state. In highly polar solvents a twisted (1)CT state is invoked to explain the low fluorescence quantum yield. For both compounds CT nature of the emitting states were further confirmed by studies in acidic medium. The ground and excited state pK(a) values for and were determined using absorption and emission spectral changes observed in the presence of protic acids.     

Synthesis and properties of a highly soluble dihydoxo(tetra-tert-butylphthalocyaninato)antimony(V) complex as a precursor toward water-soluble phthalocyanines

The title complex cation, [Sb(tbpc)(OH)(2)](+) (where tbpc denotes tetra(tert-butyl)phthalocyaninate, C(48)H(48)N(8)(2-)), has been prepared by oxidizing [Sb(tbpc)]I(3) with tert-butyl perbenzoate in CH(2)Cl(2), CHCl(3), o-dichlorobenzene and also without solvent in the range of 20-80 degrees C. This species has been isolated as I(3)(-) salt and characterized by elemental analysis, ESI-MS, FT-IR, optical absorption and emission, and magnetic circular dichroism spectroscopy. This compound is quite well soluble in common polar organic solvents (e.g., CH(2)Cl(2), acetonitrile, acetone) without detectable aggregation at least up to ca. 10(-4)M while much less soluble (e.g., benzene, chloronaphthalene) or insoluble (hexane) in non-polar solvents. Although this compound is insoluble in water, it makes hydrophilic colloids in acetone-water mixtures. The most intense absorption band (Q-band) in a specific solvent red-shifts with an increase in the refractive index of the solvent. However, considerable deviation of the Q-band positions in donor-solvents from linear correlation between the positions and Onsager's solvent polarity function suggests that there are significant specific chemical interactions between the axial hydroxyl groups and the surrounding donor molecules. The low fluorescence quantum yield (ca. 0.01) for [Sb(tbpc)(OH)(2)](+) suggests that the singlet excited state of this species is considerably quenched by the presence of antimony ion in the chromophore.     

Enols of aldehydes in the peroxidase/oxidase-promoted generation of excited triplet species

General (acid and base) or specific (fluoride ion) catalysis generates the enol of isobutanal and propanal from the corresponding trimethylsilyl enol ethers. The enols are directly rapidly oxidized by peroxidase (acting as an oxidase) to triplet acetone or triplet acetaldehyde, respectively, and formic acid. Due to the faster rate of reaction and the absence of quenching by excess aldehyde, the excited carbonyl emits more strongly than when the aldehyde itself is the substrate. With both enols the emission is pure phosphorescence. Both triplet acetone and triplet acetaldehyde are generated within the enzyme, as shown by the different quenching by D- and L-tryptophan, and are somewhat protected from oxygen quenching, as attested by the very fact that phosphorescence is observed. The use of enol precursors as substrates opens wide possibilities for photochemical investigations in the absence of light over a much broader range of experimental conditions.     

Proton transfer kinetics in the lowest excited state of 1,N6-ethenoadenosine as revealed by fluorescence lifetime measurements

Fluorescence lifetimes have been examined of 1, N6-ethenoadenosine (epsilon Ado), 1-methyl-ethenoadenosine (m1 epsilon Ado+), 9-methyl-ethenoadenosine (m9 epsilon Ado+), N1-deazaethenoadenosine (N1-deaza-epsilon Ado, and 9-methyl-N1-deazaethenoadenosine (m9N1-deaza epsilon Ado) at various pH's in the 7.5 approximately 1.5 region and at 20 degrees C. From an analysis we have reached the following conclusions. (1) The observed fluorescence of epsilon Ado is caused by the unprotonated, neutral, excited species epsilon Ado not not only at pH 7 but also at pH 2. (2) pKa of the excited epsilon Ado is lower than that of the ground state epsilon Ado. (3) The rate constant of protonation in the excited state is about 10(10) sec-1 M-1.     

Unusual fluorescent properties of N-9-anthroyl derivatives of aromatic amines

9-Anthroyl derivatives of some aromatic amines exhibit unusual fluorescence characteristics. In solvents of low and medium polarity (hexane, chloroform, DMF, and tert-butanol), their emission maxima are shifted to longer wavelengths as compared to the spectra recorded in polar solvents (ethanol and methanol); the red shift is accompanied by an increase in the fluorescence quantum yield. Possible reasons of such an anomalous spectral shift are discussed.