Excited-State Intermolecular Proton Transfer of Firefly Luciferin V

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Excited-State Intermolecular Proton Transfer of Firefly Luciferin V. Direct Proton Transfer to Fluoride and Other Mild Bases Itay Presiado, Rinat Gepshtein, Yuval Erez, and Dan Huppert* Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel

bS Supporting Information ABSTRACT: We studied the direct proton transfer (PT) from electronically excited Dluciferin 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.

’ INTRODUCTION D-Luciferin (shown in Scheme 1) is the natural substrate of the firefly luciferase. It was first isolated and purified by McElroy and co-workers,1,2 who continued to advance its research during the 1960s and 1970s of the 20th century.38 In the firefly, oxidation of D-luciferin to oxyluciferin is catalyzed by luciferase in the presence of ATP and magnesium ions, and accompanied by production of carbon dioxide. The following deactivation of oxyluciferin from its singlet electronically excited-state is accompanied by light emission. The bioluminescence produced by the firefly is in the greenred part of the visible spectrum.914 Although current studies15 claim that the firefly bioluminescence reaction’s quantum yield is less than half the value previously reported16 it remains higher than that of other bioluminescent reactions (41.0 ( 7.4%). The luciferinluciferase system was studied spectroscopically by techniques such as (time integrated) steadysteady and timeresolved fluorescence and also the femtosecond pumpprobe technique.1721 Chemiluminescent study of D-luciferin, despite being clearly motivated by the importance of its role in the firefly, reveals that it possesses many interesting properties as a photoacidic molecule. Photoacids are molecules that are mild acids in their ground state and strong acids in their excited state.2225 The protonated form of a photoacid generally transfers a proton not only to water or alcohol but also to mild bases such as acetate, H2PO4, HCO3, and many others. This PT reaction produces the conjugated acid of these bases. In recent works2629 the direct PT from a photoacid to a base in solution was reported. Both Nibbering and Pines and coworkers and Bakker and co-workers used short UV (400 nm) femtosecond pulse to excite photoacids and near IR pulses to probe the proton transfer reaction. 8-Hydroxy-1,3,6-pyrenetrisulfonate r 2011 American Chemical Society

(HPTS) was used in most studies as the photoacid of choice. The base used mostly was acetate but also the weaker bases, chloroacetate and trichloroacetate. Pines and Nibbering found that at very high concentrations of acetate a substantial part of the ROH population reacted within an even shorter time than the time resolution of their experimental setup, i.e., less than 150 fs. The explanation they provided for this observation is that an ultrafast PT reaction rate occurs between the ROH and Ac in contact ion pairs, ROH* 3 3 3 Ac, which preexisted the short pulse excitation, τpulse = 150 fs. They also found an intermediate decay time of 6 ps that they attributed to a HPTS acetate complex, in which one water molecule bridges between the two reactants. Bakker’s data on this system are comparable with those of Pines and Nibbering, but their interpretations are different. Both interpretations are briefly explained in the Discussion section. Bhattacharya and co-workers30 studied ESPT from HPTS to acetate in methanol. The rate constant of direct PT from HPTS to acetate was calculated to be ∼1 109 M1 s1. This is slower by about 2 orders of magnitude than that in bulk water at 4 M acetate 8 1010 M1 s1. This is due to poor stabilization of the ejected proton and the deprotonated form. At base concentrations lower than 0.5 M the direct PT reaction from HPTS to a base is rather rare, since the fraction of photoacidbase pairs in close contact is small. Most of the ESPT process occurs by PT to the solvent or to a base molecule that diffused and reached the encounter sphere whose radius is 7 Å, even when the size of the photoacid molecule is smaller than that. Bakker and coworkers suggested that in solution the PT to a base can occur at much longer distances than the actual contact ion pair distance Received: April 14, 2011 Revised: May 29, 2011 Published: June 01, 2011 7591

dx.doi.org/10.1021/jp203487j | J. Phys. Chem. A 2011, 115, 7591–7601

The Journal of Physical Chemistry A

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Scheme 1

Figure 1. Normalized steady-state emission spectra of D-luciferin in aqueous 00.85 M KF solutions.

between the hydroxyl group of HPTS and the base. They also suggested that a proton can travel through water-bridged complexes, in which water forms a proton wire between the donor site and the base. The Smoluchowski model describes diffusionassisted chemical reactions in solution, where one of the reactants is in large excess (it is briefly discussed in the Supporting Information). The assumption is that the reaction takes place only when the reactants are in a contact distance. In spherical symmetry terms, we can discuss a sphere surface of radius a. Usually, this radius is taken to be 57 Å in aqueous solutions. For a mildly viscous solvent, such as water, with a viscosity of ∼1 cP the diffusion coefficient of ions of the size of the acetate anion is ∼5 106 cm2/s. The diffusion-controlled rate coefficient limits the long-time rate of the reaction, when the reactants need first to diffuse toward each other to form the encounter pair prior to chemical reaction. For 1 M solution of Ac the pseudofirst order reaction rate coefficient is ∼3 109 s1 Therefore, the reaction of 1 M of Ac with HPTS should take place at times longer than 300 ps at an average rate of 3 109 s1. However, as mentioned above, it was found that a high percentage of the molecules in the ROH* form reacts with Ac* at times shorter than 10 ps. In a recent study31 we measured by time-resolved emission the reaction of acetate ions (Ac) in both H2O and D2O solutions with the excited NROH form of D-luciferin. We found that the NROH* of D-luciferin efficiently reacts with Ac ions in aqueous solutions. We found that D-luciferin and HPTS exhibit similar behavior, in that a large portion of the NROH* population of the former reacts at short times faster than predicted by the Smoluchowski model. We found three time components in the complex decay profile of the excited NROH in 1 M aqueous solution of sodium acetate or higher. We attribute the short-time component of ∼300 and 600 fs for H2O and D2O respectively to the PT reaction of a contact ion pair or to a water-bridged complex. We attribute the intermediate time component of 2 and 3 ps for H2O and D2O respectively to a complex, where the bridge is longer by one water molecule. The long-time component we attribute to the diffusion-assisted reaction, in which the proton donor (the NROH*) and the acceptor (Ac) diffuse to produce either a contact pair or a water bridged complex. According to the Smoluchowski model the time-dependent reaction rate constant, k(t), depends on both the intrinsic reaction rate and the diffusion-controlled rate.

In the current study we further explore the direct PT reaction from D-luciferin to a mild base in aqueous solution. For this purpose we used potassium fluoride (KF), potassium dihydrogen phosphate (KH2PO4), sodium acetate (NaAc) and sodium chloroacetate (NaClAc), whose anions are mild bases. In the current study we focused on the PT process from D-luciferin to F, and we found that it readily forms a contact ion pair complex with D-luciferin. Therefore, the study of direct PT to F at 0.25 M or higher was possible. We found that the direct PT rates to F, H2PO4, and Ac are comparable. We found that D-luciferin forms a contact ion pair complex with F to a far higher extent than with the other bases. The direct PT from D-luciferin to ClAc is rather slow.

’ EXPERIMENTAL SECTION Fluorescence up-conversion technique was employed in this study to measure the time-resolved emission of D-luciferin. The laser used for the fluorescence up-conversion 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 800 kHz. The second harmonic of the laser, operating over spectral ranges of 370420 nm was used to excite the D-luciferin in the liquid samples. The fluorescence up-conversion system (FOG-100, CDP, Russia) operated at 800 kHz. The samples were excited by pulses of ∼8 mW on average at the SHG frequency. The time-response of the upconversion system is evaluated by measuring the relatively strong Raman stokes line of water shifted by 3600 cm1. It was found that the full width half-maximum (fwhm) of the signal is 280 fs. Samples were placed in a rotating optical cell to avoid degradation. (4S)-2-(6-Hydroxybenzothiazole-2-yl)-4,5-dihydrothiazole4-carboxylic acid (D-Luciferin) 99.5% was purchased from Iris Biotech (Germany). Deionized water had a resistance of >10 MΩ. Potassium fluoride, monopotassium phosphate, sodium chloroacetate, and sodium acetate of analytic grade were purchased from Fluka and Aldrich. All chemicals were used without further purification. ’ RESULTS Figure 1 shows the normalized steady-state emission spectra of D-luciferin in aqueous 00.85 M KF solutions. The slightly acidic sample (pH ∼ 6) was excited at 350 nm, close to the absorption peak of the protonated form at ∼335 nm. The emission spectrum of D-luciferin in slightly acidic aqueous solution consists of weak and strong bands with peaks positioned at ∼450 and ∼530 nm. The weak intensity band is attributed to the protonated NROH* form (see Scheme 1), whereas the more intense band is assigned to the deprotonated form. Raising the KF concentration decreases the relative intensity of the NROH* band at ∼450 nm and increases the NRO* band’s intensity at 530 nm. This we explain by the reduction of the average lifetime of the excited NROH form by the fast ESPT to the fluoride ion: NROH* þ F f NRO þ HF. 7592

dx.doi.org/10.1021/jp203487j |J. Phys. Chem. A 2011, 115, 7591–7601


Figure 2. Time-resolved emission of D-luciferin measured at several wavelengths in the spectral range of 440540 nm in (a) neat aqueous solution or (b) 1 M aqueous KF solution.

We also find that the deprotonated bandwidth increases as the KF concentration increases. Figure 2a shows the time-resolved emission of D-luciferin measured at several wavelengths in the spectral range of 440540 nm in aqueous solution. The signals shown in Figure 2 were measured by the fluorescence up-conversion technique with a time resolution of ∼200 fs. The samples were excited by ∼150 fs laser pulses at 372 nm with a bandwidth of ∼7 nm and at a repetition rate of ∼800 kHz. At λ e 500 nm the signal’s rise time is controlled by the instrument response time of the upconversion system (∼300 fs) and its decay time depends on the monitored wavelength inasmuch as the longer the wavelength the slower the average decay-time. We attribute the short wavelength signals (440480 nm) to the emission of the protonated NROH* form. The peak position of the NROH* steady-state emission band is at 440 nm. The up-conversion signal at 440 and 450 nm consists of the NROH* emission as well as some contribution from the broad Raman Stokes line of water at 3600 cm1. The NROH* signals measured at 460 and 470 nm include also a significant contribution from the NRO* emission, since the two bands overlap at these wavelengths. The longer the measured wavelength the larger the amplitude of the NRO* signal becomes. The decay time of the NRO* signal is relatively

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long, and thus the long-time component of the short wavelength signals shown in the figure is attributed to the NRO*. Signals at λ g 510 nm also include two contributions. The main component arises from the broad band of the deprotonated NRO* form, whose peak is at ∼520 nm. The smaller contribution is from the protonated NROH* form, which decreases further as the monitored wavelength becomes longer. The signals have a rise-time component with a large amplitude, followed by a relatively long decay time. Figure 2b shows the time-resolved emission of D-luciferin measured at several wavelengths in 1 M aqueous KF solution. The average decay times of the NROH* signals from 1 M KF solutions at 440470 nm are much shorter than in neat aqueous solution. The short time decay of the NROH* signal of up to ∼80 ps could be resolved to three time components: short, intermediate and relatively long decay times. We attribute this fact to the efficient direct ESPT process from the NROH* to the fluoride anion, the products of which are HF and NRO*. At λ g 520 nm the rise-time component of the signals from the 1 M KF solution is much faster than that from the neat solution. The rise-time of the NRO* signal corresponds to the decay of the NROH* signal measured at short wavelength. Figure 3 shows the fluorescence up-conversion signals of Dluciferin in six samples: neat water and 25 mM and 0.25, 0.5, 1, and 2 M aqueous KF solutions. Each panel shows the signals at a given wavelength. At short wavelengths the average decay time of the signals depend on the KF concentration in that the higher the concentration the larger the amplitude of the short decay-time component. We attribute this component to the simple reaction of the direct intermolecular excited-state proton transfer (ESPT), F þ NROH* f NRO* þ HF. In the Discussion section, we analyze the decay curves of the fluorescence. Our analysis of the experimental results supports the existence of direct PT from D-luciferin to F. The PT reaction takes place when F is next to the hydroxyl group of the benzothiazol ring. PT may occur when the F is at contact distance, at which case the hydrogen bond OH 3 3 3 F is formed, or at longer distances upon formation of a water bridge, OH 3 3 3 OH2 3 3 3 F. Previous studies2628 explained in detail the various possibilities of PT mechanisms between a mild base and a photoacid in solution. In a third possible mechanism a PT reaction occurs between a donoracceptor pair located at a long distance from each other. Prior to the reaction the two species diffuse toward each other, narrowing the intermolecular distance. This type of mechanism is described by the celebrated Smoluchowski model. The panels containing the long wavelength fluorescence upconversion signal show the formation of the deprotonated NRO* form. These signals yield information complementary to the one contained in the NROH* signals at short wavelengths. The decay time of the NROH* signals are similar to the rise-time of the NRO* form at long wavelengths. As seen in the figure, the higher the KF concentration the shorter the average rise-time of the NRO* signal becomes. Figure 4 shows the fluorescence up-conversion signals of D-luciferin in deuterated 1, 3, and 4 M KF solutions. In each panel the signals of the same given wavelength are displayed. The D-luciferin signals from the KF in D2O and in H2O behave similarly. As before, the higher the KF concentration the faster the average decay rates of the NROH* signals measured at 450 and 460 nm. At long wavelengths (520 and 540 nm) the same inverse dependence of the rise-time of the NRO* on the KF concentration already shown in Figure 3 also exists. 7593



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Figure 3. Fluorescence up-conversion signals of D-luciferin in six samples: neat water and 25 mM and 0.25, 0.5, 1, and 2 M aqueous KF solutions.

Figure 5a shows a comparison of the fluorescence up-conversion signals of D-luciferin in 1 M aqueous and deuterated solutions of KF . In each panel, the H2O and D2O signals at a same given wavelength are compared. As seen in the figure, the signals of the NROH* (λ e 470 nm) and NRO* (λ g 520 nm) in H2O and D2O in the presence of KF are similar, which therefore suggests a relatively weak kinetic isotope effect (KIE). Figure 5b shows a comparison of the fluorescence up-conversion signals of D-luciferin in both H2O and D2O solutions containing 2 M of KH2PO4. H2PO4 is the mild conjugate base of H3PO4, with a pKa value of ∼2.2. The fast component of the NROH* decay at 460 and 470 nm in H2O is about the same as in D2O, whereas the longer decay components are longer in D2O than in H2O. At λ g 540 nm, where the NRO* band is located, the rise-time of the D2O signal is much slower than H2O. Comparison of the fluorescence up-conversion signals of Dluciferin in H2O and D2O solutions containing H2PO4 and F (Figures 5a and 5b) reveals that in F solutions the overall kinetic isotope effect (KIE) is much smaller than in the H2PO4 solutions. The smaller KIE in the F solution is probably due to extensive formation of either one of the two following complexes: NROH* 3 3 3 F or NROH* 3 3 3 OH2 3 3 3 F. The KIE on the PT reaction in these complexes is rather small. Solvation dynamics of large molecules in protonated and deuterated solvents as well as their dielectric relaxation times are very similar. If the solvent partially controls the PT reaction, and the short-time components of the signals at 460 and 470 nm also contain to some extent solvation

dynamics components, then the short-time components in H2O and D2O should have similar decay times. Their degree of complexation is higher in the F solution than in the H2PO4 and Ac solutions. Consequently, the overall difference between fluorescence up-conversion signals from H2O and D2O KF solutions is small. Comparison of ESPT Process from D-Luciferin to Several Mild Bases. In this subsection we present our study of the ESPT from the hydroxyl group of the benzothiazole ring of D-luciferin not only to the fluoride but also to other mild bases. Figure 6a shows the fluorescence up-conversion signal of the protonated form of D-luciferin measured at 460 nm in aqueous solutions containing 2 M of several mild bases, all of which are conjugate bases of weak acids and univalent anions. In this figure we compared the fluorescence up-conversion signals from 2 M solutions of F, H2PO4, acetate and chloroacetate. In addition, the signals from a 20 mM aqueous solution of NaAc were added for comparison. At this low acetate concentration the direct PT to the acetate is very rare. The proton is first transferred from the excited D-luciferin to the water, and is then scavenged by the acetate to form the acetic acid. This secondary reaction reduces the geminate recombination of the NRO* with the proton. In previous studies we found that this geminate recombination reaction leads to fluorescence quenching, and thus introduction of 20 mM of NaAc decreases the effective decay rate of both the protonated and deprotonated forms. As seen in the figure, the fluorescence decay rate of the NROH* form in the presence of 2 M of chloroacetate is distinctively slower than the decay rates of the signals in the 7594



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Figure 4. Fluorescence up-conversion signals of D-luciferin in deuterated 1, 2, 3, and 4 M KF solutions.

F, Ac, or H2PO4 solutions. This phenomenon was also observed in the time-resolved pumpprobe IR measurements of Pines, Nibbering and co-workers. The ESPT rate from HPTS to chloroacetate is much slower than to acetate. As seen in the figure, this is also true for D-luciferin. They explained their result by that the chloroacetate is a much weaker base than the acetate anion. Consequently, the PT reaction rate not only depends on the strength of the photoacid but also on that of the base. The average decay time is the fastest for the KF solution. The fastest decay time of the NROH* signal is about the same for F, Ac and H2PO4. The average decay time is determined not only by the decay-time of each individual component but also by their relative amplitudes. The complexation constant of fluoride with D-luciferin is larger than that of other bases. This translates to a faster average decay component in the fluorescence up-conversion signal. Figure 6b shows the fluorescence up-conversion signals of the deprotonated NRO* form of D-luciferin measured at 520 nm in the same solutions, whose NROH* signals are given in Figure 6a. At t < 40 ps the signals consist of a fast rise component followed by a relatively slow decay component. All signals decay with nearly the same rate at longer times, i.e., t > 40 ps. The amplitude of the fastest rising component is the smallest for chloroacetate and the largest for the fluorideanion.

Main Findings

1 Direct intermolecular ESPT from D-luciferin to fluoride occurs efficiently in aqueous KF solutions with concentration of 0.25 M or higher. 2 Three major decay components can be resolved in the fluorescence up-conversion signal of the NROH* in aqueous solution: a short time component of e2 ps, an intermediate component of ∼10 ps and a relatively long-time component of 3050 ps with a time coefficient that depends on the fluoride concentration. 3 The KIE on the direct ESPT to the fluoride is rather small and independent of the F concentration. 4 A comparison of direct ESPT rates from D-luciferin to several mild bases reveals that the fastest average rate occurs in a KF solution. The ESPT average rates to the dihyrogen phosphate and acetate ions are similar but slightly longer. The ESPT rate to chloroacetate anion is the slowest of the four. Data Analysis of the Fluorescence Up-Conversion Experimental Results. We used a multiexponential fitting procedure to fit the fluorescence up-conversion signal of Dluciferin in solution in the presence of a base, measured at short and long wavelengths. For the best fit of the short wavelength signal we used four exponential terms. We used 7595



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Figure 5. Fluorescence up-conversion signals of D-luciferin in H2O and D2O solutions containing (a) 1 M KF or (b) 2 M KH2PO4. 7596



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Figure 7. Fluorescence up-conversion signals of D-luciferin in several KF solutions measured at 460 nm (dots) and their multiexponential fits (solid lines).

Figure 6. Fluorescence up-conversion signal of D-luciferin in aqueous solutions containing 2 M of several mild bases measured at (a) 460 or (b) 540 nm.

Table 1. Fitting Parameters of Up-Conversion Fluorescence of D-Luciferin in H2O with KF at Several Wavelengthsa,b,c KF concn [M] 1

0.5

0.25

λ [nm]

a1

τ1d

a2

440

0.50

1000

0.20

450

0.44

1300

0.20

460 470

0.36 0.31

2000 2700

440

0.44

450

0.34

460

τ2d

a3

τ3d

a4

10

0.26

22

0.038

12

0.31

22

0.039

0.35 0.27

9 12

0.26 0.33

40 50

0.028 0.083

1200

0.20

12

0.31

22

0.039

1700

0.20

12

0.41

30

0.038

0.25

1800

0.29

9

0.41

40

0.041

470

0.22

2700

0.24

13

0.49

50

0.055

440

0.23

2200

0.29

10

0.47

30

0.008

450

0.23

2000

0.25

12.5

0.51

31

0.007

460 470

0.16 0.09

2000 2700

0.28 0.30

13.5 13.5

0.55 0.59

33 41

0.008 0.020

y = ∑ai exp[(t/τi)Ri]. b R1 = 0.9; R2 = 0.8; R3 = 0.75; R4 = 1. c τ4 = 430 ps. d Values are in picoseconds. a

stretched exponents for better results in the process of fitting the fast and intermediate time components. The best fitting parameters to the NROH* decay in F solution are given in Table 1. Figure 7 shows the experimental up-conversion

signals of D-luciferin in several KF solutions measured at 460 nm along with multiexponential fits. Table 1 shows the fitting parameters of the fluorescence upconversion signals measured at 440, 450, 460, and 470 nm from 0.25, 0.5, and 1 M KF solutions. We were able to obtain good fits for the experimental data by using a four-exponential fit (see Figure 7). The long-time decay component is assigned to the deprotonated NRO* form that overlaps to some extent with the protonated NROH* form’s band. The amplitude of this component is rather small, i.e., a4 e 0.1 (see Table 1), and its time coefficient is g400 ps. The amplitude of the NRO* becomes smaller as the monitored wavelength gets shorter. At 440 nm the amplitude is smaller than 0.04. The three other components, on the other hand, are rather short, τ e 70 ps. We used stretched exponents for these decay components to get the best fits. The stretch factors are in the range of 0.750.9. The longest component of the three ranges from 20 to 50 ps. Two other factors that affect the decay rate are the wavelength and base concentration: average decay time increases with longer detection wavelengths and decreases the higher the KF concentration becomes. The amplitude of this relatively long component is ∼1/3 of the total signal at 1 M, but increases when the F concentration decreases. The amplitude slightly increases with the wavelength. We attribute this component to the PT to the solvent and PT to the fluoride by a transport mechanism, in which a fluoride anion located at long distance from the photoacid, diffuses toward it. When it reaches a distance close enough, it forms a water-bridged complex with the D-luciferin molecule. D-Luciferin then transfers the proton via these water molecules, which results in the formation of the NRO* form and HF. This diffusion-assisted process could be formulated by the Smoluchowski model (briefly detailed in the Supporting Information). Since most of the decay does not follow the diffusion-assisted reaction scheme, we used a simple stretched exponent fitting procedure instead. The intermediate decay time component has an amplitude of roughly 0.3 of the signal and a time coefficient of 12 ( 1.5 ps. We assign this component to the PT from F via a water-bridged complex, NROH* 3 3 3 OH2 3 3 3 F, though we have no evidence to substantiate the existence of such complexes. Nibbering, Pines, and co-workers advocate their existence.2628 Bakker, 7597



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Table 2. Fitting Parameters of Up-Conversion Fluorescence of D-Luciferin in H2O with NaAc at Several Wavelengthsa,b,c NaAc concn [M] λ [nm] 1

τ1d

a1

τ2d

a2

a3

τ3d

a4

440

0.30 1200 0.42 13

0.23

28

0.055

450 460

0.23 1800 0.34 13 0.39 0.18 1800 0.36 13.5 0.39

30 33

0.037 0.065

470

0.16 1800 0.26 13.5 0.51

37

0.074

y = ∑ai exp[(t/τi)Ri]. b R1 = 0.9; R2 = 0.8; R3 = 0.75; R4 = 1. c τ4 = 430 ps. d Values are in picoseconds. a

Table 3. Fitting Parameters of Up-Conversion Fluorescence of D-Luciferin in H2O with KH2PO4 at Several Wavelengthsa,b,c KH2PO4 concn [M] λ [nm] 2

a1

τ1d

a2

τ2d

a3

τ3d

a4

440

0.54

750

0.24

14

0.18

55

0.04

450

0.39

850

0.31

14

0.26

65

0.05

460

0.40 1100 0.29

15

0.27

70

0.03

470

0.26 1300 0.34

15

0.36

80

0.03

a y = ∑ai exp[(t/τi)Ri]. b R1 = 0.75; R2 = 0.85; R3 = 0.9; R4 = 0.9. c τ4 = 430 ps. d Values are in picoseconds.

trying to explain the PT from HPTS to acetate, advocates waterbridged complexes of up to 4 water molecules. In a recent publication with Agmon they used an extended Smoluchowski model,39 in which PT takes place over a wide range of distances and not just at the contact radius (∼5 Å). They used a distance distribution function for the various water-bridged complexes and a distance-dependent rate constant. In Table 1 the amplitude of the intermediate component and its time-coefficient are almost independent of the F concentration or the measured wavelength of the signal. The amplitude of the shortest decay component of the Dluciferin signal from aqueous KF solution is relatively large and it inversely depends on the length of the measured wavelength. In a 1 M KF solution at 440 nm the amplitude is ∼0.5, whereas at 470 nm it is only 0.33. Moreover, decreasing the fluoride concentration decreases the amplitude of this component. We assign this component to PT to F that is positioned prior to photoexcitation next to the hydroxyl group, and is already hydrogen-bonded within the complex. The short-time component of the NROH* signal also includes a time-dependent Stokes shift. This shift depends on both the solvent and the presence of the fluoride anion next to the D-luciferin molecule. In a 1 M KF solution the decay times of the short-time component are 1, 1.3, 2, and 2.7 ps for the signals measured at 440, 450, 460, and 470 nm, respectively. The large differences in both the amplitudes and lifetimes of this component between signals measured at different wavelengths are usually attributed to a time-dependent Stokes shift. Since the ESPT rate is probably somewhat slower than solvation dynamics, we are left to conclude that it is limited by the solvent rearrangement prior to actual PT. We further discuss this issue in the following section. Table 2 shows the fitting parameters for D-luciferin signals from 1 M NaAc aqueous solution. The analysis procedure is the same as that for the fluoride solutions. Interestingly, even though the amplitude of the short-time component is much smaller than in the 1 M KF solution, the decay times are slightly shorter, ∼1.8 ps at 460 and 470 nm. This is explained by that the fluoride

Figure 8. Fluorescence up-conversion signals of D-luciferin in basic aqueous NaOH solution, measured at several wavelengths.

ion is more readily inclined to form hydrogen bond complexes with D-luciferin prior to excitation than the acetate salt. The actual ESPT rate is slightly faster for the acetate ion, since it is a stronger base. The fitting parameters for the 2 M KH2PO4 solution are given in Table 3. The findings are quite similar to those of the NaAc solution.

’ DISCUSSION As above-mentioned, the fluorescence up-conversion decay curves of D-luciferin are strongly dependent on wavelength in aqueous KF, NaAc, and KH2PO4 solutions. At the short wavelength region and upon a short laser pulse excitation, this is attributed to the time of solvent reorganization around the newly formed excited NROH* complexated with F. Usually, the solvent reorganization process around a large molecule, excited by a shorter pulse is observed by time-resolved emission studies. Molecules like coumarin dyes were routinely used to study solvation dynamics of electronically excited molecules. Maroncelli and co-workers32,33 used the fluorescence up-conversion technique to measure the solvation correlation function, S(t), of many solvents such as water, alcohols, nitriles etc. S(t) of protic associative liquids decays nonexponentially with a time component ranging from tens of femtoseconds to tens of picoseconds in small linear alcohols. The analysis of the decay curves of D-luciferin in aqueous KF solutions and other bases measured at 440, 450, 460, and 470 nm clearly shows that the average decay time (derived from the decay curve at the first 20 ps) strongly depends on the wavelength in that the shorter the measured wavelength the shorter the average lifetime. A hallmark of a solvation process occurring around molecules with larger dipole moments at their electronically excited-state is a time-dependent red-shift of the emission spectra. The aforementioned wavelength dependence of the up-conversion decay curves is manifested in their solvation component, which is fast, nonexponential. This is exactly how the fluorescence up-conversion signals from KF aqueous solutions behave in the spectral region of the NROH* band (440480 nm). In basic solution, only the ground-state NRO species exists, thus, the steady-state emission spectrum consists only of the NRO* band whose peak is positioned at ∼530 nm. We measured the fluorescence up-conversion signals of the NRO* 7598


The Journal of Physical Chemistry A band in basic NaOH solution at several wavelengths in the range of 440560 nm. Figure 8 shows the normalized signals of the NRO* band in a basic solution with a pH value of ∼9.2. At λ e 500 nm, the signal consists of a short-time decay component followed by a long-time component. The relative amplitude of the short-time component decreases as the wavelength increases. The decay time of the short-time component also depends on the wavelength. The decay-times of the signal measured at 440, 450, 460, and 480 nm are rather short: 250, 350, 600, and 1000 fs, respectively. We attributed the short-time component to the solvation dynamics of the excited NRO. Interestingly, there is a relatively large dynamic Stokes shift in the NRO* emission as opposed to the time-resolved emission of the NROH* form at 430470 nm in neat water, which does not have appreciable ultrafast components of less than 1 ps that can be assigned to dynamic Stokes shift (see Figure 2a). The signals of the NROH* band from a slightly acidic solution (pH ∼ 6) in the presence of KF at 440480 nm consist of a short-time decay component with a wavelength-dependent lifetime followed by a long-time decay component (see Figure 2b). This short time-component, however, we attribute to the PT process: NROH* þ F f NRO* þ HF. This process is controlled by solvent dynamics. First, the solvent rearranges in the best configuration for a direct PT to KF, and only then the PT reaction takes place. This model explains the wavelength dependence of the fast decaying component of the fluorescence signal. Ernsting and co-workers34,35 studied by femtosecond timeresolved technique the ESPT process from N-methyl-6-hydroxyquinolinium (NM6HQþ) superphotoacid to water. The timeresolved spectra of NM6HQþ measured at several times showed a relatively large dynamic Stokes shift in the protonated form’s emission. The time-dependent shift of the emission band in acetonitrile and protic solvents was about the same as that previously found for coumarin 153 and 343, the dyes that were frequently used in the determination of the solvation correlation function, S(t), in many polar liquids. They found that the ESPT dynamics is roughly twice as slow as the solvation correlation function. They concluded that the ESPT from NM6HQþ to the solvent is controlled by the solvation dynamics and thus, the energy barrier of the PT is rather small, on the order of 2 kJ/mol. They also concluded that in water, a negatively charged oxygen and two water molecules must form hydrogen bonds before PT can occur. Our main finding in the current study is that the direct ESPT process from D-luciferin to the fluoride ion occurs on an ultrafast time scale, and that it also exhibits the same characteristic pattern of the time-dependent red-shift of the NROH* band, which we assign to specific solvation dynamics. As mentioned above, the NROH* signals at 440470 nm in neat water do not have a solvation dynamics component shorter than 1 ps, while these components are easily time-resolved in the NROH* signals from solutions containing g0.25 M KF and other bases. The reaction rate is therefore partially limited by the time it takes the hydrogen bond network that includes the F to reorganize prior to the actual PT process. We suggest that the formation and reorganization of the hydrogen bond connecting the F and the hydroxyl group on the benzothiazole ring play an important part in the ESPT process in general, and in the determination of the ESPT rate in particular. This interpretation of the experimental data relies on the work of Ernsting, Kovalenko and their co-workers.34,35 Previous Ultrafast Studies of PhotoacidBase Reactions. Pines and Nibbering research groups extensively studied the

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HPTSAc system.36,37 They used 400 nm femtosecond laser pulses to pump the HPTS to S1, and mid-IR femtosecond pulses to probe it. The IR probing is advantageous in that it allows probing of the photoexcitation events, and the monitoring of not only HPTS but also acetate and H3Oþ. They found that in addition to the long-time reaction, ROH þ B, controlled by the diffusion process of both species, there are two kinds of ROH*Ac complexes reacting within the short-time regime. One kind of complex is a contact pair, where one of the oxygen atoms of the acetate is in close proximity to the hydrogen atom of the hydroxyl group of HPTS, and it reacts within 150 fs, which is the time resolution of the experimental system. The other kind of complex is a water-bridged complex, ROH 3 3 (H2O)n 3 3 Ac, for which the reaction rate is much slower, and depends on the number of water molecules in the “wire”. When one water molecule was in the complex they found that the PT timecoefficient was 6 ps. Also, they noticed that there is a time lag between the detachment of the proton from the hydroxyl group of the ROH* and the formation of the HAc. Bakker and co-worker38,39 also used UV pump mid-IR probe femtosecond technique to study the HPTS acetate system. Their results show only intermediate and long time components. Their interpretation of the experimental data differs from that of Pines and Nibbeirng. At high base concentrations the PT process to the Ac occurs via proton wires of various lengths comprised of hydrogen bonded water molecules. Typical length of these wires varies from one to four water molecules. The proton moves along the wires by a concerted Grotthus like mechanism. F as a Base. HF is known as a relatively weak acid with pKa value of ∼3.2. In contrast, other halides form much stronger acids with pKa e 6. The hydrogen bonds in gaseous HF are stronger than those in H2O, i.e., 6.8 kcal/mol in HF vs 4.4 kcal/mol in water.40 The strong hydrogen bond may lead to that a fairly large fraction from contact pairs such as NROH* 3 3 3 For NROH* 3 3 H2O 3 3 F. The PT from the NROH* to F, may then be of particular interest. HF on the other hand, is known to be a relatively stronger acid in ice than other weak acids.41 In a previous study,42 we found that strong acids are still strong in ice, whereas weak organic acids like acetic acids become weaker in ice by ∼0.6 pKa units. HF maintains its strength in ice, and its pKa value is estimated to be the same as in water at room temperature. When a low concentration of KF salt is introduced into ice, it probably creates mobile orientational L-defects.41 We found that photoacids in KF-doped ice transfer protons faster than in undoped ice. The following reaction explains this phenomenon, ROH* þ L f RO* þLHþ. The L orientation defect mobility in ice is five times as slow as that of the proton. We found that proton mobility in ice at T > 240 K is 10 times higher than in liquid water.43 L-defects are therefore capable of reaching a photoacid molecule from an average distance of a few tens of angstroms within 5 ns and the reaction within the ROH 3 3 3 L contact pair takes place, whereby the photoacid transfers a proton to the L-defect. Thus, in ice the L-defect, generated by F, is a strong and mobile enough base to which a proton is transferred from the hydroxyl group of the photoacid. The most widely studied ESPT reaction to a base in solution is that of HPTS to acetate. Acetic acid is a much weaker acid than HF (pKa = 4.75). The strength of a base is inversely proportional to the strength of its conjugate acid. Therefore, we expect that the basicity of F to be much weaker than that of the acetate ion, and comparable or slightly stronger than that of the chloroacetate anion (pKa ≈ 2.9). According to the Marcus model, in the 7599


The Journal of Physical Chemistry A “normal region” the rate of ESPT reaction from a photoacid to a base depends on the strengths of both the photoacid and the base. The model predicts that for any given photoacid the stronger the base, the faster the rate. There is a well-established relation between the reaction rate and the Gibbs’ free energy function for acidbase reactions.44 However, certain reactions may not follow this free energy relation when the solvent configuration equilibration time is rather slow, and the reaction is controlled by the solvent fluctuations response time. The dielectric relaxation may be indicative of the time scale of this solvent motion. In previous studies on the temperature dependence of ESPT from a photoacid to a solvent, in general and to water in particular, we found that the activation energy is temperature dependent. At T > 280 K, the activation energy is low, e 8 kJ/mol, and it increases monotonically to ∼20 kJ/mol in supercooled liquid water at 8°C. Previously, we used a qualitative model that accounts for the unusual temperature dependence of the ESPT.45 The protontransfer reaction depends on two coordinates; the first one depends on a generalized solvent configuration. For the alcohols used in this study, the solvent coordinate characteristic reorganization time is within the range of the dielectric relaxation time τD and the longitudinal relaxation,τL = τDε0/εs. The second coordinate is the actual proton translational motion (tunneling) along the reaction path. The model restricts the PT process to be stepwise. The proton moves to proton accepting molecule or to a water bridged complex only when the solvent configuration brings the system to the crossing point according to Kuznetsov model.46 Recently, computer simulations showed that PT process between water molecules in the liquid phase is very complex, and cannot be easily formulated by a general two-coordinate model.4749 The ESPT process from a photoexcited photoacid to a nearby mild base such as fluoride or acetate should probably be considered as an ultrafast reaction, in which the solvent reorganization controls the overall rate. Therefore, the time coefficients of these reactions in water at room temperature are on the order of 2 ps or slightly shorter than that, since the solvent reorganization time is roughly 1 ps. The reaction rates of excited D-luciferin with F , Ac , and H2PO4 are about the same since the rate limiting step is the solvent reorganization and not the actual PT. The large differences between fluorescence up-conversion signals measured at different wavelengths, and especially at short wavelengths, i.e., 440470 nm (see Figure 2b) are indicative of a solvent reorganization process prior to the actual PT.

’ SUMMARY In this work, we study direct ESPT from D-luciferin to the fluoride anion and some other mild bases. We used the fluorescence up-conversion technique to monitor the time-resolved emission of D-luciferin at several wavelengths in the spectral range of 440560 nm. We found that the average decay times of the signals at 440470 nm are considerably shorter in aqueous solutions containing 250 mM of KF or higher. Analysis of these signals reveals three time-scales in the PT to the fluoride anion. The short one lasts 2 ps. We assign this part of the signal’s decay to a direct ESPT process from the hydroxyl group to a fluoride anion that is hydrogen-bonded to it. The longer decay time of about 10 ps is assigned to an ESPT process between the hydroxyl

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group and the fluoride anion through one water molecule. Both the proton donor and acceptor are hydrogen-bonded to the same water molecule. The water molecule serves as a bridge or a proton wire for the long distance PT process. The length of the longest time component varies between 20 and 50 ps, depending on the fluoride concentration in that the lower that concentration the longer the decay time. We attribute the third component to two competing PT reactions. The first is an ESPT to the water molecules, i.e., the solvent. This reaction occurs since D-luciferin becomes a strong acid in its excited-state. The competing reaction is described by the celebrated Smoluchowski model (see Supporting Information). A distant fluoride in the solution diffuses and forms an encounter pair with the excited-photoacid. An ESPT process occurs only when the NROH*F or NROH* 3 3 OH2 3 3 3 Fcomplexes are formed. The overall time constant of the reaction is τ = τT þ τR, where τT is the transport time of encounter pair formation and τR is the reaction time. The average time depends on the fluoride concentration. The transport time depends on the mutual diffusion coefficient, D (see Supporting Information). We compared the ESPT rate from D-luciferin to F with the following mild bases: Ac, H2PO4, and chloroacetate. We found that F and H2PO4, which are weaker bases than Ac but comparable to chloroacetate, transfer a proton at about the same rate as Ac. The direct ESPT to chloroacetate is rather slow. The similarity of the values of the ESPT rate constants of the three mild bases is explained by the importance of the solvent reorganization prior to the proton translocation from the hydroxyl group to the mild base. Since solvent reorganization is a prerequisite of PT, and the PT rate is comparable to the reorganization rate, then the overall reaction rate is determined by both the PT and solvent reorganization times. Thus, this reaction is a quasi-solvent-controlled reaction. Ernsting and Kovelenko34,35 studied the ESPT process from NM6HQþ, a super photoacid, to water. The rate they found is similar to the rate we found in the current ESPT study between D-luciferin and F or Ac and H2PO4. They found that the emission band of the protonated form of NM6HQþ red-shifts during the ESPT process. We also found that a red-shift occurs 2 ps into the direct ESPT process from the NROH* form of D-luciferin to the complexated base.

’ ASSOCIATED CONTENT

bS

Supporting Information. The Smoluchowski model is presented to describe the diffusion-assisted irreversible reaction. This material is available free of charge via the Internet at http:// pubs.acs.org

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Telephone: 972-3-6407012. Fax: 972-3-6407491.

’ ACKNOWLEDGMENT This work was supported by grants from the Israel Science Foundation and from the James Franck GermanIsraeli Program in LaserMatter Interaction. Photograph of firefly in the Abstract and Table of Contents graphics courtesy of Terry Priest. 7600



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Excited-State Intermolecular Proton Transfer of Firefly Luciferin V

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