Anomalous H+ and D+ Excited-State Proton-Transfer Rate in H2O/D2O Mixtures.

We used the time-resolved fluorescence technique to measure the excited-state proton-transfer (ESPT) rates from 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) to solvent mixtures of H2O and D2O. We found an anomalous deviation from linear mole-fraction behavior of the ESPT rate in H2O/D2O mixtures. We provide a chemical model based on equilibrium constant of the reaction H2O + D2O ↔ 2HOD and rate constants of the ESPT process of H and D transfers from HPTS to the mixed solvent. Anomalous deviation from linear mole-fraction behavior was previously found for H+/D+ conductance in these mixtures.

New Phenol Benzoate Cyanine Picolinium Salt Photoacid Excited-State Proton Transfer.

Steady-state and time-resolved fluorescence techniques were employed to study the excited-state proton transfer (ESPT) to water and D2O of a new photoacid, phenol benzoate cyanine picolinium salt (BCyP). We found that the ground-state pKa is about 6.5, whereas the excited-state pKa* is about -4.5. The ESPT rate constant, kPT, to water is ∼0.5 × 1012s-1 (τPT ≈ 2 ps) and in D2O the rate is 0.33 × 1012 s-1. We determined that the BCyP photoacid belongs to the third regime of photoacids, the solvent-controlled regime.

Excited-State Proton Transfer and Formation of the Excited Tautomer of 3-Hydroxypyridine-Dipicolinium Cyanine Dye.

Steady-state and time-resolved fluorescence techniques and theoretical calculations were employed to study the photoprotolytic properties of a newly synthesized photoacid 3-hydroxypyridine-dipicolinium cyanine (HPPC) dye. This dye is similar to quinone cyanine 9, which we have previously studied and is the strongest photoacid currently synthesized. In this compound, we found that several proton transfer phenomena occur after excitation. We found that the excited-state proton transfer (ESPT) rate in water is ultrafast with kPT ≈ 1.5 × 10(12) s(-1). In methanol and ethanol the rate is slower by about 5 and 6 times, respectively. The fluorescence spectrum of HPPC in water consists of three bands with maxima at 520, 600, and 665 nm, whereas in monols and other protic solvents the fluorescence spectrum consists only of two emission bands at 530 and ∼700 nm. We assign the emission bands of HPPC at 520 nm to the protonated form and the 700 nm band in monols and 665 nm in water to the deprotonated form. The 600 nm band that is the most intense band in the fluorescence spectrum of HPPC in water we assign to the tautomeric form in which the proton is attached to the pyridine's nitrogen atom. On the basis of density functional calculations, we suggest that in water the proton transfer process to the pyridine's nitrogen atom occurs in a stepwise manner via a two water molecule bridge.

Excited-state proton transfer of weak photoacids adsorbed on biomaterials: proton transfer to glucosamine of chitosan.

UV-vis steady-state and time-resolved techniques were employed to study the excited-state proton-transfer process from two weak photoacids positioned next to the surface of chitosan and cellulose. Both chitosan and cellulose are linear polysaccharides; chitosan is composed mainly of d-glucosamine units. In order to overcome the problem of the high basicity of the glucosamine, we chose 2-naphthol (pKa* ≈ 2.7) and 2-naphthol-6-sulfonate (pKa* ≈ 1.7) as the proton emitters because of their ground state pKa (≈9). Next to the 1:1 cellulose:water weight ratio, the ESPT rate of these photoacids is comparable to that of bulk water. We found that the ESPT rate of 2-naphthol (2NP) and 2-naphthol-6-sulfonate (2N6S) next to chitosan in water (1:1) weight ratio samples is higher than in bulk water by a factor of about 5 and 2, respectively. We also found an efficient ESPT process that takes place from these photoacids in the methanol environment next to the chitosan scaffold, whereas ESPT is not observed in methanolic bulk solutions of these photoacids. We therefore conclude that ESPT occurs from these photoacids to the d-glucosamine units that make up chitosan.

Excited-state proton transfer of weak photoacids adsorbed on biomaterials: 8-hydroxy-1,3,6-pyrenetrisulfonate on chitin and cellulose.

Time-resolved and steady-state florescence measurements were used to study the photoprotolytic process of an adsorbed photoacid on cellulose and chitin. For that purpose we used the 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) photoacid which transfers a proton to water with a time constant of 100 ps, but is incapable of doing so in methanol or ethanol. We found that both biopolymers accept a proton from the electronically excited acidic ROH form of HPTS. The excited-state proton-transfer (ESPT) rate of HPTS adsorbed on chitin is greater than that on cellulose by a factor of 5. The ESPT on chitin also occurs in the presence of methanol or ethanol, but at a slower rate. The transferred protons can recombine efficiently with the conjugate excited base, the RO(-) form of HPTS.

Fast photoinduced reactions in the condensed phase are nonexponential.

Time-resolved measurements of photoinduced reactions reveal that many ultrafast reactions in the femto- to picosecond time scale are nonexponential. In this article we provide several examples of reactions that exhibit a nonexponential rate. We explain the wide range of the nonexponential reaction by the lack of time separation between τ(s), the characteristic fast equilibration time of the population in the reactant potential well, and the longer time τ(e), the characteristic time to cross the energy barrier between the reactant and the product.

Insight into the structure and the mechanism of the slow proton transfer in the GFP double mutant T203V/S205A.

Mutations near the fluorescing chromophore of the green fluorescent protein (GFP) have direct effects on the absorption and emission spectra. Some mutants have significant band shifts and most of the mutants exhibit a loss of fluorescence intensity. In this study we continue our investigation of the factors controlling the excited state proton transfer (PT) process of GFP, in particular to study the effects of modifications to the key side chain Ser205 in wt-GFP, proposed to participate in the proton wire. To this aim we combined mutagenesis, X-ray crystallography, steady-state spectroscopy, time-resolved emission spectroscopy and all-atom explicit molecular dynamics (MD) simulations to study the double mutant T203V/S205A. Our results show that while in the previously described GFP double mutant T203V/S205V the PT process does not occur, in the T203V/S205A mutant the PT process does occur, but with a 350 times slower rate than in wild-type GFP (wt-GFP). Furthermore, the kinetic isotope effect in the GFP double mutant T203V/S205A is twice smaller than in the wt-GFP and in the GFP single mutant S205V, which forms a novel PT pathway. On the other hand, the crystal structure of GFP T203V/S205A does not reveal a viable proton transfer pathway. To explain PT in GFP T203V/S205A, we argue on the basis of the MD simulations for an alternative, novel proton-wire pathway which involves the phenol group of the chromophore and water molecules infrequently entering from the bulk. This alternative pathway may explain the dramatically slow PT in the GFP double mutant T203V/S205A compared to wt-GFP.

Optical spectroscopy of molecular-rotor molecules adsorbed on cellulose.

Steady-state and time-resolved emission techniques were used to study the fluorescence properties of two molecular rotors, thioflavin-T and auramine-O adsorbed on cellulose powder. Molecular rotors are known for their weak fluorescence intensity and short fluorescence lifetime when dissolved in liquids of low viscosity. We found that these molecular-rotor molecules when adsorbed on cellulose exhibit a rather strong steady-state fluorescence spectrum as well as long emission lifetime. We explain these results by the inhibition of segmental intramolecular rotation when these molecules are adsorbed on cellulose.

Excited-state proton transfer from quinone-cyanine 9 to protic polar-solvent mixtures.

Steady-state and time-resolved emission techniques were used to study the excited-state proton-transfer (ESPT) process of quinone cyanine 9 (QCy9) in solvent mixtures. We found that the ESPT rate from QCy9 in water/methanol mixtures is independent of the mixture composition and the rate constant is k(PT) ∼ 10(13) s(-1). In ethanol/trifluoroethanol (TFE) mixtures the ESPT rate strongly depends on the solvent-mixture composition. We observe two ESPT rates rather than one over a wide range of solvent-mixture compositions. The average ESPT rate decreases as the mole fraction of TFE increases.

Acid effect on photobase properties of curcumin.

Femtosecond UV-vis pump-probe spectroscopy was employed to study the acid effect on curcumin in the excited state. Curcumin in solutions of weak acids was found to be a photobase forming a protonated curcumin within a few tens of picoseconds from the time of excitation. The excited-state protonation reaction is also observed in the steady-state emission spectrum as a new red emission band with a maximum at 620 nm in the presence of weak acids. The transient pump-probe spectrum consists of four spectral bands, two emission bands, and two absorption bands. We assign a transient absorption band at ∼600 nm and an emission band at ∼540 nm to the neutral ROH form of curcumin. An absorption band at ∼500 nm and an emission band at 620 nm are assigned to the protonated ROH2(+) form of curcumin.

Effect of acid on the ultraviolet-visible absorption and emission properties of curcumin.

Steady-state and time-resolved emission techniques were employed to study the acid-base effects on the UV-vis spectrum of curcumin in several organic solvents. The fluorescence-decay rate of curcumin increases with increasing acid concentration in all of the solvents studied. In methanol and ethanol solutions containing about 1 M HCl, the short-wavelength fluorescence (λ < 560 nm) decreases by more than an order of magnitude. (The peak fluorescence intensity of curcumin in these solvents is at 540 nm.) At longer wavelengths (λ ≥ 560 nm) the fluorescence quenching is smaller by a factor of ∼3. A new fluorescence band with a peak at about 620 nm appears at an acid concentration of about 0.2 M in both methanol and ethanol. The 620 nm/530 nm band intensity ratio increases with an increase in the acid concentration. In trifluoroethanol and also in acetic acid in the presence of formic acid, the steady-state emission of curcumin shows an emission band at 620 nm. We attribute this new emission band in hydrogen-bond-donating solvents to a protonated curcumin ROH2(+) form. At high acid concentrations in acetic acid and in trifluoroethanol, the ground state of curcumin is also transformed to ROH2(+) which absorbs at longer wavelengths with a band peak at ∼530 nm compared to 420 nm in neutral-pH samples or 480 nm in basic solutions. In hydrogen-bond-accepting solvents such as dimethyl sulfoxide and also in methanol and ethanol, curcumin does not accept a proton to form the ground-state ROH2(+)

Comprehensive study of ultrafast excited-state proton transfer in water and D2O providing the missing RO(-)···H(+) ion-pair fingerprint.

Steady-state and time-resolved optical techniques were employed to study the photoprotolytic mechanism of a general photoacid. Previously, a general scheme was suggested that includes an intermediate product that, up until now, had not been clearly observed experimentally. For our study, we used quinone cyanine 7 (QCy7) and QCy9, the strongest photoacids synthesized so far, to look for the missing intermediate product of an excited-state proton transfer to the solvent. Low-temperature steady-state emission spectra of both QCy7 and QCy9 clearly show an emission band at T < 165 K in H2O ice that could be assigned to ion-pair RO(-)*···H3O(+), the missing intermediate. Room-temperature femtosecond pump-probe spectroscopy transient spectra at short times (t < 4 ps) also shows the existence of transient absorption and emission bands that we assigned to the RO(-)*···H3O(+) ion pair. The intermediate dissociates on a time scale of 1 ps and about 1.5 ps in H2O and D2O samples, respectively.

Theoretical photodynamic study of the photoprotolytic cycle of firefly oxyluciferin.

Firefly oxyluciferin presents a pH-sensitive fluorescence in aqueous solutions. Its fluorescence spectra are composed of two green peaks at different pH values, despite the enolate anion being the only emitter. A computational approach was used to further elucidate the photoprotolytic cycle of oxyluciferin and investigate its pH sensitivity. It was found that oxyluciferin forms π-π stacking complexes both in the ground and excited states, at basic and acidic/neutral pH. However, at different pH values, these complexes adopt a different conformation, which explains the lower energy of the emission at acidic/neutral pH, in comparison with the emission at basic pH.

Oxyluciferin photoacidity: the missing element for solving the keto-enol mystery?

The oxyluciferin family of fluorophores has been receiving much attention from the research community and several systematic studies have been performed in order to gain more insight regarding their photophysical properties and photoprotolytic cycles. In this minireview, we summarize the knowledge obtained so far and define several possible lines for future research. More importantly, we analyze the impact of the discoveries on the firefly bioluminescence phenomenon made so far and explain how they re-open again the discussion regarding the identity (keto or enol species) of the bioluminophore.

Temperature dependence of the excited-state proton-transfer reaction of quinone-cyanine-7.

Steady-state and time-resolved fluorescence techniques were used to study the temperature dependence of the photoprotolytic process of quinone-cyanine-7 (QCy7), a very strong photoacid, in H2O and D2O ice, over a wide temperature range, 85-270 K. We found that the excited-state proton-transfer (ESPT) rate to the solvent decreases as the temperature is lowered with a very low activation energy of 10.5 ± 1 kJ/mol. The low activation energy is in accord with free-energy-correlation theories that predict correlation between ΔG of reaction and the activation energy. At very low temperatures (T < 150 K), we find that the emission band of the RO(-)*, the deprotonated form of QCy7, is blue-shifted by ~1000 cm(-1). We attributed this band to the RO(-)*···H3O(+) ion pair that was suggested to be an intermediate in the photoprotolytic process but has not yet been identified spectroscopically.

Ultrafast excited-state proton transfer to the solvent occurs on a hundred-femtosecond time-scale.

Steady-state and ultrafast time-resolved techniques were used to study a newly synthesized photoacid, phenol-carboxyether dipicolinium cyanine dye, QCy9. We found that the excited-state proton transfer (ESPT) to water occurs at the remarkably short time of about 100 fs, k(PT) ≈ 1 × 10(13) s(-1), the fastest rate reported up to now. On the basis of the Förster-cycle, the pK(a)* value is estimated to be -8.5 ± 0.4. In previous studies, we reported the photoacidity of another superphotoacid, the QCy7 for which we found an ESPT rate constant of ~1.25 × 10(12) s(-1), one-eighth that of the QCy9 compound. We found a kinetic isotope effect of the ESPT of about two.

Excited-state proton transfer of firefly dehydroluciferin.

Steady-state and time-resolved emission techniques were used to study the protolytic processes in the excited state of dehydroluciferin, a nonbioluminescent product of the firefly enzyme luciferase. We found that the ESPT rate coefficient is only 1.1 × 10(10) s(-1), whereas those of d-luciferin and oxyluciferin are 3.7 × 10(10) and 2.1 × 10(10) s(-1), respectively. We measured the ESPT rate in water-methanol mixtures, and we found that the rate decreases nonlinearly as the methanol content in the mixture increases. The deprotonated form of dehydroluciferin has a bimodal decay with short- and long-time decay components, as was previously found for both D-luciferin and oxyluciferin. In weakly acidic aqueous solutions, the deprotonated form's emission is efficiently quenched. We attribute this observation to the ground-state protonation of the thiazole nitrogen, whose pK(a) value is ~3.

Excited-State Proton Transfer of Phenol Cyanine Picolinium Photoacid.

Steady-state and time-resolved fluorescence techniques as well as quantum-mechanical calculations were used to study the photophysics and photochemistry of a newly synthesized photoacid-the phenol cyanine picolinium salt. We found that the nonradiative rate constant k nr of the excited protonated form of the photoacid is larger than that of the excited-state proton transfer (ESPT) to the solvent, k ESPT. We estimate that the quantum efficiency of the ESPT process is about 0.16. The nonradiative process is explained by a partial trans-cis isomerization reaction, which leads to the formation of a "dark" excited state that can cross to the ground state by nonadiabatic coupling. Moreover, the ESPT process is coupled to the photo-isomerization reaction, as this latter reaction enhances the photoacidity of the studied compound, as a result of photoinduced charge transfer. To prevent trans-cis isomerization of the cyanine bridge, we conducted experiments of PCyP adsorbed on cellulose in the presence of water. We found that the steady-state fluorescence intensity increased by about a factor of 50 and the lifetime of the ROH band increased by the same factor. The fluorescence intensity of the RO- band with respect to that of the ROH band was the same as in aqueous solution. This explains why inhibiting the photo-isomerization reaction by adsorbing the PCyP on cellulose does not lead to a higher ESPT rate.

Photoprotolytic Processes of Lumazine.

Steady-state and time-resolved UV-vis spectroscopies were used to study the photoprotolytic properties of lumazine, which belongs to a class of biologically important compounds-the petridines. We found that in water an excited-state proton transfer occurs with a time constant of ∼70 ps and competes with a nonradiative rate of about the same value. The nonradiative rate of the protonated form of lumazine in polar and nonpolar solvents is large knr ≥ 1.5 × 1010s-1. The fluorescence properties indicate that in water, the ground-state neutral form of lumazine is already stable in two tautomeric forms. The fluorescence of the deprotonated form is quenched by protons in acidic solutions with a diffusion-controlled reaction rate. We conclude that the neutral form of lumazine is an irreversible mild photoacid.

Irradiation by blue light in the presence of a photoacid confers changes to colony morphology of the plant pathogen Colletotrichum gloeosporioides.

We used the photoacid 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) that converts blue photons to acidic protons in water, with an efficiency of close to 100%, and determined that this treatment conferred changes to colony morphology of the plant pathogen Colletotrichum gloeosporioides. The time elapsed until hyphal collapse is noticed depends on both the laser intensity in mW/cm2, and the concentration of HPTS in the Agar hydrogel. The time elapsed until hyphal collapse is noticed varies by only ±8% at HPTS concentrations of 500µM and at lower concentrations of HPTS the variance increases as the inverse of the concentration. We found that the effect on C. gloeosporioides was photoacid concentration and irradiation dose dependent. In the presence of 500µM of HPTS within the agar hydrogel-based medium, hyphae collapsed after 37±3.5min of irradiation at 405nm at an intensity of 25mW/cm2. We propose two mechanisms for such photo-alteration of C. gloeosporioides. One is based on the pH drop in the extracellular environment by the photo-protolytic process that the photoacid molecule undergoes. The second mechanism is based on an intracellular mechanism in which there is an uptake of HPTS into the interior of the fungus. We suggest that both mechanisms for photo-alteration which we found in this study may occur in plants during fungal infection.