Excited-State Intermolecular Proton Transfer of the Firefly's

Jul 15, 2010 - Steady-state absorption and emission as well as time-resolved emission spectroscopies were employed to study the photophysics and ...
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J. Phys. Chem. A 2010, 114, 8075–8082

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Excited-State Intermolecular Proton Transfer of the Firefly’s Chromophore D-Luciferin Yuval Erez and Dan Huppert* Raymond and BeVerly Sackler Faculty of Exact Sciences, School of Chemistry, Tel AViV UniVersity, Tel AViV 69978, Israel ReceiVed: April 12, 2010; ReVised Manuscript ReceiVed: May 23, 2010

Steady-state absorption and emission as well as time-resolved emission spectroscopies were employed to study the photophysics and photochemistry of D-luciferin, the firefly active bioluminescent compound. In aqueous solution the electronically excited-state protonated D-luciferin compound undergoes an efficient process of proton transfer to the solvent, with a rate constant kPT ) 3.0 × 1010 s-1. We found a kinetic isotope effect of about 2.5 for this process. The deprotonated form of D-luciferin in the excited state recombines irreversibly with the geminate proton. Hence, the fluorescence decay of the deprotonated form is nonexponential, and the fluorescence quantum yield is low. SCHEME 1

Introduction D-Luciferin is the chromophore found in Lampyridae, a family of winged insects. D-Luciferin is responsible for the bioluminescence that gives these Lampyridae their more familiar name, fireflies. Fireflies are known to make crepuscular use of bioluminescence, believed to play a role in sexual attraction between the adults. The light is produced by organs located on the underside of the abdomen.1 In fireflies, males, females, and larvae emit a heatless light, with no infrared or ultraviolet frequencies, in the green-yellow to red-orange visible wavelengths of the spectrum from 510 to 670 nm. Synchronized flashing is characteristic of some tropical species, particularly in Southeast Asia. In some fields, this phenomenon is explained as phase synchronization2,3 and spontaneous order. Bioluminescence in the firefly is a complex phenomenon that consists of a set of chemical reactions which D-luciferin (shown in Scheme 1) undergoes. Firefly luciferase catalyzes the oxidation of D-luciferin in the presence of ATP and magnesium ions. The product of the reaction is oxyluciferin in a singlet electronically excited state, and its deactivation is accompanied by a yellow-green bioluminescence. Luciferases from different species of fireflies differ in their amino acid sequences. Much of the early work on the chemistry of the firefly luminescence was made by McElroy and co-workers.4-11 D-Luciferin was first isolated and purified in 1949.6 Steadysteady and time-resolved techniques such as pump-probe and fluorescence were used to study the bioluminescent luciferinluciferase system.12-16 For many years17-29 intermolecular excited-state proton transfer (ESPT) to a solvent or to a base in a liquid solution, and more recently in ice, has been widely researched. In the past couple decades we extensively studied the reversible and irreversible photoprotolytic cycle of a photoacid. We used a proton transfer model that accounts for the diffusion-assisted geminate recombination of the transferred proton with the deprotonated form of the photoacid.23,30,31 In the current study we measure the steady-state (timeintegrated) emission, excitation, and absorption spectra, as well

* To whom correspondence should be addressed. E-mail: huppert@ tulip.tau.ac.il. Phone: 972-3-6407012. Fax: 972-3-6407491.

as the time-resolved emission properties of excited D-luciferin. We find that D-luciferin has dual emission bands when excited from its neutral form (the protonated form, ROH*). The bands correspond to emission from the ROH* (band maximum at 450 nm) and from the deprotonated (530 nm) species RO-* of D-luciferin. The decay of the time-resolved emission of ROH* is nonexponential and fast, i.e., about 25 ps at room temperature. The time-resolved emission signal of RO-* is complex because the proton geminate recombination process is irreversible and thus enhances the quenching of the RO- fluorescence. We interpret these observations as arising from an ESPT to the solvent process. Experimental Section Time-correlated single-photon-counting (TCSPC) and fluorescence up-conversion techniques were employed in this study to measure the time-resolved emission of D-luciferin. For sample excitations we used a Ti:sapphire femtosecond laser, Mira, Coherent, which provides short, 150 fs, pulses. The laser’s second harmonic, operating over spectral ranges of 380-420 nm, was used to excite the D-luciferin in the liquid samples. The up-conversion system (FOG-100, CDP, Russia) operated at 76 MHz. The samples were excited by a train of pulses of ∼20 mW average power at the SHG frequency. The upconversion system’s time response is evaluated by measuring the relatively strong water’s Raman Stokes line shifted by 3600 cm-1. It was found that the full width at half-maximum (fwhm) of the signal is 280 fs. Samples were placed in a rotating optical cell to avoid degradation. The laser used with TCSPC was a cavity-dumped Ti:sapphire femtosecond laser, Mira, Coherent, which provides short, 150 fs, pulses at around 800 nm. The cavity dumper operated with a relatively low repetition rate of 500 kHz. The TCSPC detection system was based on a Hamamatsu 3809U photomultiplier and Edinburgh Instruments TCC 900 computer module for TCSPC.

10.1021/jp103264y  2010 American Chemical Society Published on Web 07/15/2010

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Figure 1. Absorption, excitation, and emission spectra of D-luciferin: (a) absorption and emission spectra in neutral and basic aqueous solutions, (b) excitation and emission spectra in H2O and D2O, (c) emission spectra in ethanol and acetonitrile and their water mixtures.

The overall instrumental response was about 35 ps (fwhm). The excitation pulse energy was reduced to about 10 pJ by neutral density filters. (4S)-2-(6-Hydroxybenzothiazol-2-yl)-4,5-dihydrothiazole-4carboxylic acid (D-luciferin), 99.5%, was purchased from Iris Biotech (Germany). Deionized water had a resistance of >10 MΩ. Methanol, ethanol, and acetonitrile of analytical grade were purchased from Fluka. All chemicals were used without further purification. Results Steady State. Figure 1a shows the absorption and emission spectra of D-luciferin in neutral and basic aqueous solution. When D-luciferin in aqueous solution at pH < pKa is excited at 330 nm, the absorption peak of ROH, the emission spectrum consists of two bands, the band of the protonated form ROH, with a maximum at about 440 nm, and the deprotonated form RO- at about 530 nm. As seen in the figure the intensity of the ROH band is much smaller than that of the RO- band. In general, if the solution pH is lower by about one pK unit than the ground-state pKa of the photoacid and the excited-state lifetimes of both ROH and RO- are about the same, then the steady-state fluorescence intensity ratio can be related to the proton transfer rate constant:

IRO-/IROH ) kPTτ

(1)

where IROH and IRO- are the peak intensities of the steady-state ROH and RO- bands, kPT is the proton transfer rate constant, and τ is the excited-state lifetime. For a lifetime of 5 ns (the lifetime of the deprotonated state) the estimated proton transfer rate constant is 1.2 × 1010 s-1; i.e., τPT ≈ 80 ps. As we will show, the time-resolved emission data provide a larger rate constant by about a factor of 3. Figure 1b shows the excitation and steady-state emission spectra of D-luciferin in H2O and D2O samples. The emission spectra of both samples are normalized to the RO- peak. The ROH band intensity of the deuterated sample is larger by a factor of ∼2.5 than that of the H2O signal. The kinetic isotope effect (KIE) is therefore estimated to be 2.5. For many reversible photoacids it was found that the kinetic isotope effect is around 3. The large ESPT rate constant of D-luciferin evinces that it is a strong photoacid with a KIE somewhat smaller than that of strong reversible photoacids. Figure 1c shows the normalized steady-state emission of D-luciferin in two neat solvents and their water mixtures. Several photoacids, such as hydroxyflavone (HF)32 and 2-(2′-hydroxyphenyl)benzothiazole (HBT), exhibit an efficient intramolecular ESPT process.33-35 In enol-keto proton transfer it was found that the intramolecular proton transfer is ultrafast, kPT > 1013 s-1. As seen in Figure 1c, in neat acetonitrile the steady-state emission spectrum of D-luciferin consists of only the ROH band, with a maximum at 430 nm. The band has a highly asymmetric broad band with a large red tail. This spectrum indicates that

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Figure 2. Time-resolved emission of the D-luciferin ROH and ROforms in neutral pH H2O and D2O solution measured by the TCSPC technique.

ESPT does not occur in acetonitrile and therefore rules out a possible intramolecular proton transfer process, such as was found to exist in HF and HBT. However, this does not rule out a solvent-assisted intramolecular proton transfer, via one or more solvent molecules. The deprotonated band appears in the spectrum with the addition of water to the sample. With the increase in water concentration, the deprotonated band intensity increases and the protonated band decreases. The figure also shows that the emission spectrum of D-luciferin in neat ethanol consists of both the ROH and RO- bands, where the ROH band intensity is twice as large as that of the RO- band. The emission band maximum of the ROH band is shifted by 20 nm to the red, with respect to its position in neat acetonitrile. This fact demonstrates the importance of the hydrogen bonds in the energetics and stabilization of D-luciferin in the excited state. When water is added to the sample, the RO- band intensity respectively increases, whereas the ROH band intensity decreases. It is wellknown that the proton transfer rate of a photoacid increases by several orders of magnitude in water in comparison to alcohols such as methanol, ethanol, or propanol.19,23 Equation 1 explains that the bands ratio is directly related to the proton transfer rate, and therefore, in water mixtures kPT is much larger. Time-Resolved Measurements. We used two techniques to time resolve the emission of both the ROH and RO- bands. For the short times we used the fluorescence up-conversion technique with a time resolution of about 100 fs. For longer times and a large dynamic range we used the TCSPC technique. Figure 2 shows on a semilogarithmic scale the time-resolved emission of the ROH and RO- forms of D-luciferin in both H2O and D2O samples, measured by the TCSPC technique. The figure shows the TCSPC signals of both ROH and RO-, measured at 450 and 540 nm, respectively. The instrument response function of the TCSPC technique has a full width at half-maximum of 35 ps. As seen in the figure, the decay of the ROH signal is fast, whereas the RO- signal exhibits a fast but distinctive rise time followed by a decay which is in the subnanosecond range. The fast decay and fast rise of the ROH and RO- signals, respectively, are attributed to an excited-state intermolecular proton transfer process of D-luciferin. The TSCPC technique’s relatively slow time response somewhat limits the accurate fine details of the signals at times shorter than ∼50 ps. The signal measured at 540 nm shows at short times a fast nonexponential

decay followed by an exponential decay with a lifetime of about 5 ns. We attribute the nonexponential decay to the irreversible proton geminate recombination process which leads to formation of the ground-state ROH form; see Scheme 2. The fluorescence quenching process is denoted by the diagonal arrow and the rate constant kq. In an excited reversible geminate recombination process the RO- signal exhibits a convex shape at short times, while at intermediate and long times the signal decays exponentially. As mentioned above this is not the case with D-luciferin, where the RO- decay is concave. 1-Naphthol and 1-naphtholsulfonate derivatives are good examples of photoacids for which the geminate recombination is irreversible and the proton reaction quenches the fluorescence. On the other hand, 2-naphtholsulfonate derivatives are reversible photoacids in which the proton geminate recombination regenerates the excited-state ROH form. Strictly speaking, both the reversible and irreversible processes coexist in most of the photoacids. This becomes clear when noting that at least two possible sites for proton recombination exist in such large molecules. The hydroxyl site is designated to the reversible process, and all other existing sites belong to the irreversible process. Usually heterocyclic nitrogens are proton binding sites that quench the fluorescence. The D-luciferin RO- signal decay is biphasic, a relatively fast nonexponential decay component being followed by a long nearly exponential decay component. This kind of decay pattern is attributed to a fluorescence quenching process by the geminate recombination of the emitted proton. Upon excitation of ROH, the hydroxy proton is first transferred to the solvent. The proton then diffuses in the liquid and has a finite probability to recombine with several molecular sites of the D-luciferin, which is negatively charged and thus attracts the positively charged proton by Coulomb potential. In addition to the hydroxy group, there are two nitrogens that, in the excited state, can also be considered as strong basic groups, capable of efficiently reacting with protons. We propose that proton recombination with a nitrogen causes a nonradiative process, leading to a groundstate ROH form. Figure 3 shows the TCSPC signals of the deprotonated form of D-luciferin in basic H2O and D2O solutions. In a basic solution where the sample is excited in its deprotonated form RO-, the fluorescence decay is almost exponential and the fast component seen in Figure 2 is almost completely missing. This is due to the fact that when the sample is excited as RO-, the ESPT and recombination processes are altogether avoided. Figure 4a shows on a semilogarithmic scale the fluorescence up-conversion signal of a basic aqueous solution of D-luciferin, in which the ground-state population of D-luciferin is in its deprotonated form RO-. The sample was excited by ∼150 fs, 387 nm pulses of the second harmonic of the mode-locked Ti: sapphire laser operating at 76 MHz. The overall instrument response function full width at half-maximum was 280 fs. The signal consists of two time components, of short and long decay times. The short time component is attributed to the red shift (Stokes shift) of the emission band, which is attributed to solvation dynamics. The water solvation correlation

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Figure 3. Time-resolved emission of the deprotonated form of D-luciferin measured at 530 nm in basic samples of H2O and D2O.

function is known to be fast and nonexponential.36,37 The slowest component is about 800 fs. After a short photon pulse excitation, an emission band is usually red-shifted with time. Thus, timeresolved measurements at wavelengths shorter than the steadystate emission maximum will show a fast decaying component with a relative amplitude that progressively decreases with the increase in detection wavelength. Such a behavior is clearly seen in Figure 4a. At wavelengths longer than the emission band maximum, the solvation contribution to the signal shows a distinct rise time. This is also seen in the RO- form of D-luciferin up-conversion fluorescence signals at λ g 520 nm. The fluorescence long time component of RO- in basic solution is nearly exponential with a long lifetime of about 5 ns. In a short time window of 250 ps of the up-conversion signal, the 5 ns decay time component only weakly affects the signal. Figure 4b shows, on a semilogarithmic scale, the fluorescence up-conversion signals of a neutral pH aqueous D-luciferin sample measured at several wavelengths in the spectral range of 440-550 nm. The signals at short wavelengths, 440-500 nm, exhibit a complex decay pattern. At these wavelengths, the signal consists of short, intermediate, and long time decay components. The short time decay is attributed to solvation dynamics. When ROH is excited, the solvation dynamics process red shifts the emission band as in the RO- case (basic solution). In water (as mentioned above) the solvation dynamics is fast and the slowest component is on the order of 800 fs. This component is clearly seen in the figure. At intermediate times, 2 ps < t < 250 ps, and short wavelengths, 440-480 nm, the signal decays nonexponentially with an average time constant of 25 ps. At long wavelengths, λ g 520 nm, the RO- emission band shows a component with a relative amplitude of 0.4 of a rise with a time constant of about 20 ps. The 25 ps decay component, measured at the ROH band maximum (440-460 nm measurements), is attributed to the ESPT process, which takes place when ROH is excited (see Scheme 2). Figure 4c shows the fluorescence up-conversion signal of D-luciferin in a neutral pH D2O sample at several wavelengths in the spectral region 440-540 nm. As in the case of the H2O neutral pH sample, the solvation dynamics is fast. The solvation dynamics of the D2O sample is almost the same as that in H2O, whereas the ESPT rate in D2O is much slower than that in H2O due to a relatively mild kinetic isotope effect, KIE ≈ 2.5. Parts a and b of Figure 5 show a comparison of the fluorescence up-conversion signals on linear and semilogarith-

Erez and Huppert mic scales, respectively, of D-luciferin in H2O and D2O measured at 460 nm (ROH band). At short times (t e 2 ps) the signals are almost identical, whereas at intermediate and long times (t g 2 ps) the decay of the D2O signal is slower than that of the H2O signal. As seen in the figure, the solvation component t < 2 ps is almost identical in its amplitude and decay time in both H2O and D2O. The intermediate and long time components of the fluorescence up-conversion signal are attributed to the ESPT photoprotolytic processes, where at the first step the proton transfers from the hydroxyl group to the solvent. This step depends on the isotopic constitution of the solvent, and therefore, the D2O decay rate is much slower. The subsequent steps of the photoprotolytic cycle include proton diffusion in the liquid surrounding the D-luciferin and proton reversible and irreversible recombination processes with the RO- form. If the recombination rate constants are larger than the diffusion-controlled rate, then the overall reaction is partially controlled by the proton diffusion constant. In general, the Debye-Smoluchowski equation can be used to quantify the time-resolved emission signal, incorporating the kinetic rate constants, the diffusion constant, and the Coulomb potential as well as the excited-state lifetimes. Figure 6 shows a comparison between samples in neutral pH H2O and D2O of the RO- fluorescence up-conversion signal of D-luciferin measured at 540 nm. The H2O signal shows a relatively large amplitude of a rise component, whereas the D2O sample does not. According to the irreversible diffusioninfluenced proton geminate recombination model, after the step of proton transfer to the solvent, a diffusion-assisted irreversible proton geminate recombination process occurs, in which the transferred proton presumably reacts with the heterocyclic nitrogens of the D-luciferin. The ESPT rate to the solvent is slower by a factor of 2.5 in D2O than it is in H2O, whereas the overall irreversible recombination rate is only slightly slower in D2O samples since DH+ ≈ 1.4DD+. Therefore, the opposite contributions cancel each other, and the signal of RO- in the case of D2O lacks the rise time seen in H2O samples. Figure 7 shows a fit of the fluorescence up-conversion signal of D-luciferin in neutral pH aqueous solution measured at 460 nm. The solid line in the plot is a multistretched exponent fit of the experimental data shown by dots. A total of 85% of the signal could be fitted by a single stretched exponent, exp[(-t/τ)R], where τ ) 30 ps and R ) 0.67. Table 1 contains the multiexponential fitting parameters of several time-resolved emission signals. As we will show in a more detailed analysis, this major nonexponential component is attributed to the photoprotolytic process and can also be fitted by the ESPT photoprotolytic model based on the Debye-Smulochowski equation. Discussion This study deals with the photophysics and photochemistry of D-luciferin, the firefly active bioluminescent chromophore. The results show that this molecule is a photoacid. The photophysical and photochemical properties of D-luciferin are examined in light of what we know about the properties of a typical photoacid. (1) In the ground state photoacids are weak acids with pKa values in the range of 5-10. That is why both acidic and basic forms of these molecules should be manifested in their absorption spectra, where the basic form’s band is red-shifted with respect to the acidic form. Fo¨rster38 related the difference between the ground- and excited-state band shifts with the change in acidity in the ground and excited states of a photoacid.

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Figure 4. Fluorescence up-conversion signals of D-luciferin in (a) basic aqueous solution, (b) neutral pH aqueous solution, and (c) neutral pH D2O solution.

Figure 5. Fluorescence up-conversion signals of D-luciferin in neutral pH H2O and D2O solutions: (a) linear scale, (b) semilogarithmic scale.

Figure 1 shows that the absorption spectra indeed indicate that D-luciferin is a weak acid in the ground state (pKa ≈ 8). (2) The steady-state emission of the acidic form of a photoacid should have two emission bands: one that is attributed to the acidic form and another attributed to the basic form, as is shown in Figure 2 for D-luciferin. If both forms have a large fluorescence quantum yield, i.e., the nonradiative rates are small with respect to the ESPT rate, then the ratio between the bands’

intensities and the excited-state lifetimes provides an estimate for the proton transfer rate constant.17 (3) When an excited-state intermolecular proton transfer takes place, the time-resolved emission of the protonated form decays rapidly in water. The deprotonated (basic) form’s emission signal should at early times have a rise time, indicating the formation of the deprotonated form by a photochemical reaction (Scheme 2). In D-luciferin we found that an efficient irreversible proton

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Figure 6. Fluorescence up-conversion signals of the D-luciferin ROform in neutral pH H2O and D2O solutions.

Figure 7. Multiexponential fit of the up-conversion signal of the D-luciferin ROH form measured at 460 nm.

geminate recombination process takes place, strongly reducing the RO- emission band intensity and modifying the early time of the time-resolved emission signal of the RO- form. (4) The intermolecular ESPT kinetics of D-luciferin shows a mild KIE value of ∼2.5. Figure 5 shows the time-resolved emission of the protonated form of D-luciferin in both H2O and D2O. As seen in the figure, the ROH decay in D2O is indeed much slower than that in H2O. The KIE is somewhat smaller than 3, the KIE value that was found for many reversible photoacids. (5) Careful consideration of the steady-state spectra of both the ROH* and RO-* emission bands on one hand and the timeresolved emission decay of ROH* when the D-luciferin molecules were excited as ROH (slightly acidic samples) on the other reveals a fast nonradiative decay rate of ROH* that competes with the ESPT and hence decreases both of these bands’ fluorescence quantum yields. Reversible and Irreversible Photoprotolytic Cycle of Photoacids. Excitation of a solution at pH values lower than the ground-state pKa of photoacids usually generates within 100 fs a vibrationally relaxed, electronically excited ROH molecule (denoted by ROH*). Intermolecular relaxation (solvation dynamics in general occurs at a slower rate than intramolecular relaxation processes) further decreases the solute solvent energy and optimizes the hydrogen-bonding network prior to the photoprotolytic cycle (see Scheme 2).

Erez and Huppert Proton dissociation, with an intrinsic rate constant kPT, leads to the formation of the contact ion pair R*O- · · · H+, whereas reversible (adiabatic) recombination with a rate constant ka reforms the excited acid ROH*. In general, back-protonation may also proceed by an irreversible (nonadiabatic) pathway, involving fluorescence quenching of RO-* by a proton with a rate constant kq, forming the ground-state ROH. 2-Naphtol-6,8-disulfonate (2N68DS) and 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) are considered reversible photoacids, for which the proton quenching rate constant, kq, is much smaller than the reversible proton geminate recombination rate constant, ka. For this class of photoacids, geminate proton-RO-* recombination repopulates the ROH* form. As a consequence of this process, the fluorescence of ROH* does not decay exponentially, but rather consists of short and long time components. In the neutral pH samples the long time fluorescence tail decays nonexponentially. 1-Naphthol and its derivatives are known to exhibit large fluorescence quenching of the deprotonated form RO- in both neutral and acidic aqueous solutions. When excess protons are introduced to a solution of an irreversible photoacid, the ROlifetime decreases and the nonexponential decay component at short times remains almost unaffected. Separation of an ion pair from the contact radius, a, to infinity is described by the transient numerical solution of the Debye-Smoluchowski equation (DSE).23 In addition, the fluorescence lifetimes of all excited species are considered, with 1/k0 ) τ0 for the acid and 1/k0′ ) τ0′ for the conjugate base. Generally, k0′ and k0 are much smaller than both the proton reaction and the proton diffusion-controlled rates. The RO-* fluorescence signal in the irreversible process is strongly affected by the presence of excess protons in the sample. Figure 8 shows the time-resolved emission of D-luciferin in neutral aqueous solution, measured by the TCSPC system at the ROH and RO- bands and the fitting using the ESPT extended model that includes the diffusion-assisted proton geminate recombination process as described above. For the numeric fit, we used a user-friendly graphic program, SSDP (version 2.63), of Krissinel and Agmon.39 As seen in the figure, the fits are rather good at all times. The fitting parameters are given in Table 2. The important kinetic parameters of D-luciferin are the proton transfer rate constant, kPT ) 3.0 × 1010 s-1, and the irreversible proton recombination rate, kq ) 6 × 1011 M-1 s-1, which is larger than the diffusion-controlled recombination rate constant, kD ) 5 × 1010 M-1 s-1, and indicates a very efficient quenching of the RO- band by a proton. Referring to Scheme 2, the excited protonated molecule transfers a proton to the solvent. The proton then diffuses in the solvent and exhibits a finite probability to geminately recombine back to re-form the protonated ground-state molecule denoted by ROH(g). Figure 9 shows the TCSPC signal of the RO- form of D-luciferin, excited at ∼385 nm, in several aqueous solutions with different pH values. In neutral solution, where the sample is excited in its ROH form, we found that the signal decay of RO- consists of two components: a fast nonexponential component with a large amplitude and an average decay time of about 200 ps and a long exponentially decaying component of 5 ns. In a basic solution of pH ≈ 9, D-luciferin is excited as RO- and the fast component almost disappears. We attribute the fast nonexponentially decaying component to the irreversible proton geminate recombination process:

RO-* + H+ f ROH(g) for which the ground-state ROH is the reaction product.

(2)

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TABLE 1: Fitting Parameters of Time-Resolved Emission of D-Luciferin at Several Wavelengths (nm)a,b,c wavelength b

460 470b 520a,c,d 540a,c,d 560a,c,d d

R1

a1

τ1 (fs)

R2

a2

τ2 (ps)

R3

a3

τ3 (ps)

0.8 0.8 1 1 1

0.13 0.1 0.31 0.39 0.39

1200 1200 0.5 0.5 0.5

0.67 0.67 1 1 1

0.77 0.84 0.68 0.6 0.6

35 35 18 17 17

0.9 0.9

0.1 0.06

300 300

a11

a22

0.39 0.39 0.54

0.61 0.61 0.46

a y ) a11y1 + a22y2. b y1 ) a1 exp[(-t/τ1)a1] + a2 exp[(-t/τ2)a2] + a3 exp[(-t/τ3)a3]. c y2 ) a1[1 - exp[(-t/τ1)a1]] + a2[1 - exp[(-t/τ2)a2]]. All values of R, a, and τ given are the y2 rise parameters. The y1 value used is that of 460 nm.

Figure 8. Fit (solid line) of the TCSPC signals (dots) of the ROH and RO- forms of D-luciferin in neutral pH aqueous solution, using the ESPT geminate recombination model (see the text): (a) ROH signal, (b) RO- signal.

TABLE 2: SSDP Fitting Parameters 1

solvent

kPT (1010 s-1)

kr (Å/ps)

kq (ns-1)

/τ (ns-1)

D (10-5 cm2/s)

H 2O D2O

35 17

10 10

66 44

0.2 0.17

9.1 6.6

To verify this explanation, we measured the TCSPC signal of both the ROH and RO- forms of D-luciferin in several acidic aqueous solutions. As seen in Figure 9, the long time component of the RO- TCSPC signal strongly depends on the acid concentration. Thus, homogeneous excess protons introduced to the aqueous solution react with the excited RO- form, and the final product is ROH(g). As seen in the decay plots shown in Figure 9, the fast decaying component attributed to the ejected proton irreversible geminate recombination process is only

Figure 9. TCSPC signals of the D-luciferin RO- form measured at 530 nm in several acidic and basic aqueous solutions.

slightly affected by the excess protons introduced to the solution. This type of a TCSPC signal behavior is expected since the proton geminate recombination process in low acid concentration efficiently competes with the excess protons. Thus, both the acid effect on the long time decay of the ROsignal and the nearly exponential signal in basic solution, seen in Figure 9, strongly indicate that the fast decay component of RO-, when the sample is excited as ROH, arises from the irreversible proton geminate recombination process. Direct Proton Transfer via a Solvent Bridge. The diffusionassisted proton geminate recombination model, described in detail above, may not be the only proton recombination pathway that takes place in excited D-luciferin. We now provide an alternative explanation to the exceptionally large and efficient RO- quenching by the proton geminate recombination process experimentally observed. The hydroxyl group is distant from the thiazole’s nitrogen, and thus, classical enol-keto intramolecular proton transfer is impossible. In HBT, the hydroxyl group of the phenol is in close proximity to the thiazole’s nitrogen. It was found that in HBT an efficient intramolecular proton transfer with a rate constant of 1.6 × 1013 s-1 takes place in nonprotic solvents.33-35 We propose that a solvent bridge consisting of about three protic solvent molecules may form an efficient proton wire, connecting the hydroxyl group (the proton donor) and the proton-accepting thiazole’s nitrogen. Such a solvent bridge should have a unique hydrogen-bonding structure that assists the proton shuttle from the donor to the acceptor. A proton wire as described can easily transfer a proton on a picosecond time scale. In many recent studies such a solvent bridge was suggested to explain relatively fast proton transfers from a photoacid to a proton acceptor (a mild base introduced in solution). In many previous studies it was found that the presence of acetate anion in aqueous solutions enhances the proton transfer rate of a photoacid. Pines, Nibbering, and co-workers20,26,40 and

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more recently Bakker and co-workers25 extensively studied the proton transfer rate from HPTS to acetate and chloroacetates20 in aqueous solutions containing high and moderate concentrations of these substances at room temperature by using femtosecond UV-pump IR-probe spectroscopy. They concluded that a wide range of water bridge complexes among the proton donor, the hydroxyl group, and the acetate enable the efficient ESPT process. The longer the water bridge, the slower the proton transfer rate. Hydroxyquinolines are bifunctional photoacids, where the hydroxyl is the acidic group and the imine nitrogen is a strong photobase. Bardez and co-workers41,42 suggested that in 7-hydroxyquinoline a solvent bridge assists the direct proton shuttle from the hydroxyl to the imine nitrogen. In a previous work, we studied the ESPT process of 6- and 7-hydoxyquinolines (HQs). We examined a possible zwitterion formation mechanism, where the released hydroxyl proton reaches the nitrogen via a water bridge. However, in light of the experimental results, we concluded that such a water-bridged proton transfer is not the main process in the case of HQs, when the neutral form is excited. Summary The photophysics and photochemistry of D-luciferin were extensively studied in the 1970s and 1980s and much less later on. Only a few studies have emphasized the acid-base properties of D-luciferin in both the ground and excited states. In the current study we employed time-resolved emission techniques such as fluorescence up-conversion with a time resolution of 100 fs and time-correlated single-photon counting with much lower time-resolution capabilities but a much higher detection sensitivity and a much larger dynamic range. We conducted systematic research to study the photoprotoytic cycle of the protonated form of D-luciferin. The D-luciferin molecule is constructed of two subunits. The main subunit consists of a 6-hydroxy-benzothiazole, a two-ring system that undergoes an efficient deprotonation process of the hydroxyl group in the excited state. In the current study we found that ESPT to water is fast, kPT ≈ 3.0 × 1010 s-1. We also found that the kinetic isotope effect is about 2.5, similar to that of other strong photoacids. A unique property of the photoprotolytic cycle of D-luciferin is a very large fluorescence quenching of the deprotonated form by the irreversible proton geminate recombination process with the excited deprotonated form, schematically shown in Scheme 2. Such an irreversible process also occurs in many other photoacids such as 1-naphthol and its derivatives. The efficiency of the process is very large, considering that the deprotonated form is only singly charged and thus the Coulomb attraction between the ion pair RO-,H+ is relatively small. For D-luciferin in water (εS ) 80), the Debye radius in which the Coulomb attraction equals the thermal energy kBT is only RD ≈ 7 Å, compared to 14 Å for 1-naphthol-4-sulfonate with two charges. We found that the intrinsic nonradiative proton geminate recombination rate for D-luciferin is much larger than for 1-naphtol-4-sulfonate. We attribute the large quenching efficiency to the proton recombination with nitrogens of the two thiazole rings.

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