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.

How fast can a proton-transfer reaction be beyond the solvent-control limit?

In this article, we review the field of photoacids. The rate of excited-state proton transfer (ESPT) to solvent spans a wide range of time scales, from tens of nanoseconds for the weakest photoacids to short time scales of about 100 fs for the strongest photoacids synthesized so far. We divide the photoacid strength into four regimes. Regime I includes the weak photoacids 0 < pKa* < 3. These photoacids can transfer a proton only to water or directly to a mild-base molecule in solution. The ESPT rate to other protic solvents, like methanol or ethanol, is too small in comparison with the radiative rate. The second regime includes stronger photoacids whose pKa*'s range from -4 to 0. They are capable of transferring a proton to other protic solvents and not only to water. The third regime includes even stronger photoacids. Their pKa* is ∼ -6, and the ESPT rate constant, kPT, is limited by the orientational time of the solvent which is characterized by the average solvation correlation function ⟨S(t)⟩. The fourth regime sets a new time limit for the ESPT rate of the strongest photoacids synthesized so far. The kPT value of such photoacids is 10(13) s(-1), and τPT = 100 fs. We attribute this new time limit (beyond the solvent control) to intermolecular vibration between the two heavy atoms of the proton donor and the proton acceptor, which assist the ESPT by lowering the height and width of the potential barrier, thus enhancing the ESPT rate.

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.

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.

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.

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(+)

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.

Temperature and viscosity dependence of the nonradiative decay rates of auramine-O and thioflavin-T in glass-forming solvents.

Both auramine-O (AuO) and thioflavin-T (ThT) behave as fluorescent molecular rotors, meaning that their (non)radiative properties are markedly affected by the intramolecular rotation of the molecule. In this article, steady-state and time-resolved fluorescence of AuO and ThT were measured in three alcohols, 1-propanol, 1-butanol, and 1-pentanol, over a wide range of temperatures (86-260 K). These solvents are glass-forming liquids, and their viscosity and dielectric relaxation time increase by more than 10 orders of magnitude as the temperature is lowered from room temperature to ~100 K. Accordingly, the fluorescence nonradiative rates constants of AuO and ThT in these solvents decrease by about 3 orders of magnitude at the latter temperature range. We found very good correspondence between the temperature dependence of the nonradiative rate constant, k(nr), of both molecules and the dielectric relaxation rate of the solvents. The k(nr) values of AuO are twice those of ThT along the whole temperature range. The temperature dependence of k(nr) is consistent with the nonradiative model suggested by Glasbeek and co-workers.

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.

Ultrafast proton transfer of three novel quinone cyanine photoacids.

Steady-state and time-resolved emission techniques were used to study the photoprotolytic properties of three recently synthesized strong quinone cyanine photoacids (QCy7 and its sulfonated derivatives). The rate coefficient of the excited-state proton transfer (ESPT), k(PT), of the three dyes is roughly 1.5 × 10(12) s(-1), a high value that is comparable to the solvation dynamics rate of large polar organic molecules in H(2)O and D(2)O. It is twice as fast as the proton transfer rate between two adjacent water molecules in liquid water. We found that, as expected, two of the sulfonated photoacids geminately recombines with the proton at an elevated rate. The accelerated geminate recombination process of the sulfonated derivatives is different from a simple diffusion process of protons. The ESPT rate coefficient of these molecules is the highest recorded thus far.

Comparative study of the photoprotolytic reactions of D-luciferin and oxyluciferin.

Optical steady-state and time-resolved spectroscopic methods were used to study the photoprotolytic reaction of oxyluciferin, the active bioluminescence chromophore of the firefly's luciferase-catalyzed reaction. We found that like D-luciferin, the substrate of the firefly bioluminescence reaction, oxyluciferin is a photoacid with pK(a)* value of ∼0.5, whereas the excited-state proton transfer (ESPT) rate coefficient is 2.2 × 10(10) s(-1), which is somewhat slower than that of D-luciferin. The kinetic isotope effect (KIE) on the fluorescence decay of oxyluciferin is 2.5 ± 0.1, the same value as that of D-luciferin. Both chromophores undergo fluorescence quenching in solutions with a pH value below 3.

The effect of a mild base on curcumin in methanol and ethanol.

Steady-state and time-resolved emission techniques were employed to study the effect of acetate, a mild base, on the luminescence of curcumin in methanol and ethanol. We found that the steady-state emission intensity as well as the average fluorescence decay time are reduced by a factor of 5 when the acetate concentration is raised to about 1.8 M. We attribute this large effect to an excited-state proton transfer (ESPT) from the acidic groups of curcumin to the acetate anion. We analyze the experimental data in terms of an ESPT reaction occurring between a photoacid and a base.

Ultrafast excited-state intermolecular proton transfer of cyanine fluorochrome dyes.

Steady-state and time-resolved emission spectroscopy techniques were employed to study the excited-state proton transfer (ESPT) to water and D(2)O from QCy7, a recently synthesized near-infrared (NIR)-emissive dye with a fluorescence band maximum at 700 nm. We found that the ESPT rate constant, k(PT), of QCy7 excited from its protonated form, ROH, is ~1.5 × 10(12) s(-1). This is the highest ever reported value in the literature thus far, and it is comparable to the reciprocal of the longest solvation dynamics time component in water, τ(S) = 0.8 ps. We found a kinetic isotope effect (KIE) on the ESPT rate of ~1.7. This value is lower than that of weaker photoacids, which usually have KIE value of ~3, but comparable to the KIE on proton diffusion in water of ~1.45, for which the average time of proton transfer between adjacent water molecules is similar to that of QCy7.

Structure and excited-state proton transfer in the GFP S205A mutant.

To further explore excited state proton transfer (ESPT) pathways within green fluorescent protein (GFP), mutagenesis, X-ray crystallography, and time-resolved and steady-state optical spectroscopy were employed to create and study the GFP mutant S205A. In wild type GFP (wt-GFP), the proton transfer pathway includes the hydroxyl group of the chromophore, a water molecule, Ser205, and Glu222. We found that the ESPT rate constant of S205A is smaller by a factor of 20 than that of wt-GFP and larger by a factor of 2 in comparison to the ESPT rate of S205V mutant which we previously characterized. (1) High resolution crystal structures reveal that in both S205A and S205V mutants, an alternative proton transfer pathway is formed that involves the chromophore hydroxyl, a bridging water molecule, Thr203 and Glu222. The slow PT rate is explained by the long (∼3.2 Šand presumably weak) hydrogen bond between Thr203 and the water molecule, compared to the 2.7 Šnormal hydrogen bond between the water molecule and Ser205 in wt-GFP. For data analysis of the experimental data from both GFP mutants, we used a two-rotamer kinetic model, assuming only one rotamer is capable of ESPT. Data analysis supports an agreement with the underlying assumption of this model.

Temperature dependence of the fluorescence properties of curcumin.

Steady-state and time-resolved techniques were employed to study the nonradiative process of curcumin dissolved in ethanol and 1-propanol in a wide range of temperatures. We found that the nonradiative rate constants at temperatures between 175-250 K qualitatively follow the same trend as the dielectric relaxation times of both neat solvents. We attribute the nonradiative process to solvent-controlled proton transfer. We also found a kinetic isotope effect on the nonradiative process rate constant of ~2. We propose a model in which the excited-state proton transfer breaks the planar hexagonal structure of the keto-enol center of the molecule. This, in turn, enhances the nonradiative process driven by the twist angle between the two phenol moieties.

Excited-state intermolecular proton transfer of firefly luciferin V. Direct proton transfer to fluoride and other mild bases.

We studied the direct proton transfer (PT) from electronically excited D-luciferin to several mild bases. The fluorescence up-conversion technique is used to measure the rise and decay of the fluorescence signals of the protonated and deprotonated species of D-luciferin. From a base concentration of 0.25 M or higher the proton transfer rates to the fluoride, dihdyrogen phosphate or acetate bases are fast and comparable. The fluorescence signals are nonexponential and complex. We suggest that the fastest decay component arises from a direct proton transfer process from the hydroxyl group of D-luciferin to the mild base. The proton donor and acceptor molecules form an ion pair prior to photoexcitation. Upon photoexcitation solvent rearrangement occurs on a 1 ps time-scale. The PT reaction time constant is ∼2 ps for all three bases. A second decay component of about 10 ps is attributed to the proton transfer in a contact pair bridged by one water molecule. The longest decay component is due to both the excited-state proton transfer (ESPT) to the solvent and the diffusion-assisted PT process between a photoacid and a base pair positioned remotely from each other prior to photoexcitation.

Study of thioflavin-T immobilized in porous silicon and the effect of different organic vapors on the fluorescence lifetime.

Steady-state and time-resolved emission techniques have been employed to study the fluorescence properties of thioflavin-T (ThT) adsorbed on oxidized porous silicon (PSi) surfaces, with an average pore size of ∼10 nm. We found that the average fluorescence decay time of ThT, when it is adsorbed on the PSi surface, is rather long, τ(av) = 1.3 ns. We attribute this relatively long emission lifetime to the effect of the immobilization of ThT on the PSi surface, which inhibit the rotation of the aniline with respect to the benzothiazole moieties of ThT. We also measured the fluorescence properties of ThT in PSi samples in equilibrium with vapors of several liquids, such as methanol, acetonitrile, and water. We found that the fluorescence intensity drops by a factor of 10, and the average decay time, measured by a time-correlated single-photon counting technique, decreases by a factor of 3. We explain these results in terms of liquid condensation of the vapors in the PSi pores, which leads to partial dissolution of the ThT molecules in the liquid pools.

Pressure effect on the nonradiative process of thioflavin-T.

Time-resolved emission techniques were employed to study the nonradiative process of thioflavin-T (ThT) in 1-propanol, 1-butanol, and 1-pentanol as a function of the hydrostatic pressure. Elevated hydrostatic pressure increases the alcohol viscosity, which in turn strongly influences the nonradiative rate of ThT. A diamond-anvil cell was used to increase the pressure up to 2.4 GPa. We found that the nonradiative rate constant, k(nr), decreases with pressure. We further found a remarkable linear correlation between a decrease in k(nr) (increase in the nonradiative lifetime, τ(nr)) and an increase in the solvent viscosity. The viscosity was varied by a factor of 1000 and k(nr) was measured at high pressures, at which the nonradiative rate constant of the molecules decreased from (7 ps)(-1) to (13 ns)(-1), (13 ps)(-1) to (17 ns)(-1) and (17 ps)(-1) to (15 ns)(-1) for 1-propanol, 1-butanol, and 1-pentanol, respectively. The viscosity-dependence of k(nr) is explained by the excited-state rotation rate of the two-ring systems, with respect to each other.

Temperature dependence of the fluorescence properties of thioflavin-T in propanol, a glass-forming liquid.

Steady-state and time-resolved emission techniques were employed to study the nonradiative process of Thioflavin-T (ThT) in 1-propanol as a function of temperature. We found that the nonradiative rate, k(nr), decreased by about 3 orders of magnitude when the temperature was lowered to 88 K. We found remarkably good correspondence between the temperature dependence of k(nr) of ThT and the dielectric relaxation times of the 1-propanol solvent.

Excited-state intermolecular proton transfer of firefly luciferin IV. Temperature and pH dependence.

Time-resolved emission as well as steady-state UV-vis techniques were employed to study the photoprotolytic processes that d-luciferin, the natural substrate of the firefly luciferase, undergoes in both acidic aqueous solutions and ice. The emission spectrum of D-luciferin in a 20 mM HCl aqueous solution or higher has an additional emission band at 590 nm red-shifted with respect to the strongest emission band positioned at 530 nm of the deprotonated NRO(-*) form in a pH-neutral aqueous solution. We attribute this emission band to the zwitterion form designated as (+)HNRO(-). The time-resolved emission signals show that the NRO(-*) emission band at 530 nm and the zwitterion emission band at 590 are strongly quenched by a recombination process with a proton in an acidic solution and in ice. In ice, the quenching rate is 10 times faster than in the liquid state. We attribute the fast quenching rate to the high value of the proton diffusion constant in ice.