Effect of Confinement on Excited-State Proton Transfer of Firefly's

Article pubs.acs.org/JPCB

Effect of Confinement on Excited-State Proton Transfer of Firefly’s Chromophore D‑Luciferin in AOT Reverse Micelles Jagannath Kuchlyan, Debasis Banik, Niloy Kundu, Surajit Ghosh, Chiranjib Banerjee, and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India ABSTRACT: Excited-state intermolecular proton transfer of D-luciferin in reverse micelles has been investigated using steady-state and time-resolved fluorescence spectroscopy measurement. The different polar cores have been chosen for the study of proton transfer dynamics in aerosol−OT (AOT) reverse micelles. It is shown that aqueous reverse micelle is the suitable environment for the photoprotolytic reaction of D-luciferin. The neutral form of the chromophore is present both in ground and excited state at W0 = 0. The proton transfer in nanometer size water pool of water/AOT/n-heptane begins at W0 = 8 and increases with increasing W0 values. However, the intermolecular excited-state proton transfer (ESPT) of D-luciferin is inhibited in nonaquous reverse micelles with DMF and DMSO as a polar core. Thus, the requirement of ESPT of D-luciferin to take place in reverse micelles consists of polar protic solvent like water as a polar core.

resolved fluorescence spectroscopy measurement.12,13 The electronically excited and protonated D-luciferin transfer a proton to aqueous solution with a rate constant of kPT = 3.0 × 1010 S−1. However, in methanol the proton transfer rate constant is 3 times slower than in water. The neutral form of Dluciferin (the protonated form, NROH) shows dual emission bands upon excitation. In bulk water D-luciferin gives an intense emission peak at 530 nm corresponds to the deprotonated form NRO− and a weak emission peak at 440 nm corresponds to the protonated NROH form. Huppert et al.12 found that D-luciferin undergoes an intermolecular ESPT to the solvent not an intramolecular to its strong photobase of the nearest cyclic nitrogen heteroatom. Upon excitation of the neutral form of Dluciferin, the bezothiazole hydroxyl proton is transferred to the solvent. From computational studies it was suggested that an efficient bioluminescence reaction occurs after the deprotonation of the hydroxyl-group of D-luciferin.21,22 The proton transfer time scale of D-luciferin in water is about 27 ps which is measured by femtosecond fluorescence upconversion techniques.12 D-Luciferin can transfer a proton to methanol due to its strong photoacid nature. However, HPTS (8-hydroxy-1,3,6pyrene trisulfonate) does not transfer a proton to methanol or other alcohols within the excited state lifetime.13 Recently it is shown that the nature of the spectra and emission maxima of Dluciferin is altered in different acidic solution.12−14 These interesting features of this phenomenon would be helpful for the development of pharmaceutical, biomedical, and bioanalytical analysis.23−25 ESPT is governed by solvation and dynamics of the ion pair. One of the main factors that markedly affect the intermolecular ESPT rate of D-luciferin is the hydrogen-

1. INTRODUCTION Excited-state proton transfer reaction in confined medium is a current research topics because of its wide range of applications in the field of chemistry and biology.1−3 Proton transfer plays a vital role in fuel cell operation and in many enzymatic reactions.4,5 Detailed studies of intermolecular excited-state proton transfer (ESPT) in confined media have increased dramatically for understanding the basic proton transfer dynamics.6−9 Studies of different excited-state photoacids provide several pathways of excited state proton transfer reaction and roles of environment on proton transfer dynamics.6−10 Douhal et al. demonstrated on the utilization of intermolecular ESPT chromophores to study the protein conformations and binding site polarity.11 Few studies are there about intermolecular ESPT of D-luciferin in different solvents of varying polarity, pH and hydrogen-bonding ability.12−14 However, there is no report on intermolecular ESPT dynamics of D-luciferin in organized media. D-Luciferin, a substrate of bioluminescence reactions, is found in Lampyridae which is a family of winged insects.15,16 DLuciferin plays an important role in firefly bioluminescence processes. Its oxidation in the presence of ATP and magnesium ions results the formation of the singlet-excited oxyluciferin. Oxyluciferin is an extremely unstable compound due to rapid condensation reaction of its neutral form. It exists as six chemical forms and the exact chemical form depends on various conditions during the chemiluminescence and bioluminescence reactions.17The deactivation of oxyluciferin is accompanied by the emission of visible light of wavelength from 510 to 670 nm.18−20 As a photoacid D-luciferin has a much lower pKa in its first electronic excited state (∼0) than in its ground state (∼8).12 ESPT of D-luciferin has been investigated in aqueous solution and aqueous methanolic solution using steady-state and time© 2014 American Chemical Society

Received: January 8, 2014 Revised: February 28, 2014 Published: March 1, 2014 3401

dx.doi.org/10.1021/jp500210n | J. Phys. Chem. B 2014, 118, 3401−3408

The Journal of Physical Chemistry B

Article

Scheme 1. Chemical Structures of NaAOT and D-Luciferin (NROH)

luciferin have a vital role in firefly bioluminescence reaction. Previously, we have investigated ESPT of different proton transfer probes inside polar core of AOT RMs and various micellar assemblies.45,46 The dynamics of proton transfer depends on the reorganization and solvation of the ion pair formed in the reaction. Thus, we want to investigate the effect of the intermolecular ESPT process of D-luciferin inside RMs. Lastly, we have revealed that the intermolecular ESPT process of D-luciferin is significantly affected by the confined environment.

bonding ability of the solvent.12−14It is expected that ESPT is dramatically affected in nanoconfined environment due to constrained mobility, slower solvation and closeness of the ion pair.26−30 Reverse micelles (RMs) consist of surfactants (such as aerosol−OT) dispersed in a nonpolar solvent and an inner polar core surrounded by surfactant monolayers. The unique confinement effect of RMs has been used as a model system of various bioaggregates.6,31−35RMs have been extensively used in enzymatic reactions, chemical catalysis and drug delivery.31−38The general properties of reverse micelle and different miceller size have been extensively studied by the several groups.6,38 The size of the micelle is characterized by the molar ratio of polar solvent to surfactant. W0 =

[polar solvent] [AOT]

2. EXPERIMENTAL SECTION 2.1. Materials and Method. Sodium bis(2-ethylhexyl) sulfosuccinate (AOT) was purchased from Sigma-Aldrich. The spectroscopic grade n-heptane, DMF and DMSO were obtained from Spectrochem and used as received. The NaAOT was used after drying above 30 h with a vacuum pump. We have used n-heptane as nonpolar solvent for the preparation of reverse micelle. AOT reverse micelles solutions are prepared by dissolving a quantitative amount of solid NaAOT in required amount of n-heptane solvent. Then Milli-Q water or DMF or DMSO is added to the solutions to get the desired W0 values. For all experiments the AOT concentration was kept at 0.9 mol dm−3 and the W0 values varies from 0 to 45. The D-luciferin, (4S)-2-(6-hydroxybenzothiazole-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (99%), was purchased from Sigma-Aldrich. The chemical structures of the AOT and Dluciferin are given in Scheme 1. 2.2. Spectroscopic Techniques. The steady-state absorption and fluorescence spectra were obtained using a Shimadzu (model UV 2450) absorption spectrophotometer and a Hitachi (model F 7000) spectrofluorimeter, respectively. All timeresolved emission spectra of D-luciferin were recorded using a picoseconds pulsed diode laser based time-correlated single photon counting (TCSPC) spectrometer. The instrumental details have been described in our earlier publication.47In our experiment the samples were excited at 336 nm and the emission was collected in magic angle (54.7°) polarization using Hamamatsu MCP PMT (3809U). The full width at halfmaximum of our system response function is about 800 ps. The analysis of fluorescence decays were performed using IBH DAS-6 decay analysis software. The anisotropy decay function, r(t), was calculated from I∥ and I⊥ decays using the following equation:

(1)

In such systems, the polar solvent finds its way into the polar core of the reverse micelle forming a nanoscopic polar solvent pool. With increasing W0 values, the solvent pool diameter usually increases with concomitant changes in the local environment within the pools. For water/AOT reverse micelles, the radius (rmax) is 2.3W0 Å.39 Confined water of RMs have different properties than the bulk water.28,39−42 The water core of AOT RMs are similar to water pockets present in the bioaggregates. To understand the dynamics of proton transfer processes in biological water, many group studied ESPT in AOT reverse micelle.40,41,43,44 Douhal et al. investigated the slower proton-transfer in RMs due to lower fluidity of confined water which is related to H-bond network dynamics within RMS.44 To understand the biological and chemical processes it is essential to explore the dynamics of proton transfer in confined water molecules. Photophysical properties of Dluciferin in RMs will be a model system of this chromophore inside the biological membrane. In this work, we have taken up the study of ground and electronically excited state of D-luciferin in the nanoscopic polar domains within AOT/n-heptane RMs by using steady-state and time-resolved fluorescence spectroscopy measurement. Three different polar solvents namely Polar protic water and polar aprotic N,N-dimethylformamide (DMF) and dimethyl sulphoxide (DMSO) are used to prepare the RMs. The photoprotolytic properties of D-luciferin in these organized assemblies is essential due to the structural alternation of RMs by changing the different solvents and the amount of solvents inside the polar core.40The study of this molecule is important as D3402


The Journal of Physical Chemistry B r (t ) =

Article

I (t ) − GI⊥(t ) I (t ) + 2GI⊥(t )

(2)

where G is the “correction factor for detector sensitivity to the polarization direction of the emission” and I∥ and I⊥ are the fluorescence decays polarized parallel and perpendicular, respectively, to the polarization of the excitation light. In our set up, the G value is 0.6. For the calculation of average fluorescence lifetimes for the decay curves we have used the following equation τav = τ = a1τ1 + a 2τ2

(3)

where τ1 and τ2 are the first and second components of the decay times and a1 and a2 are the corresponding relative amplitudes of these components.

Figure 2. Emission spectra of D-luciferin (λex = 336 nm) at different W0 values for water/AOT reverse micelles.

3. RESULTS AND DISCUSSION 3.1. Steady-State Absorption and Fluorescence Studies. It is already reported that the absorption spectra of D-luciferin appear at ∼330 nm in neutral and ∼390 nm in basic aqueous solution.12The maxima of the spectra at ∼330 and ∼390 nm correspond to protonated and deprotonated form of D-luciferin, respectively. Figure 1 depicts the absorption spectra

at low water content (W0 = 2). Similar observation was observed at W0 = 4 with emission of neutral form at 435 nm. The changes in fluorescence emission intensity and position of λmaxem are due to the confined environment of RMs. The red shift and enhancement of emission intensity after addition of water to “dry” AOT (W0 = 0) reflects that D-luciferin undergoes a more polar region compared to W0 = 0.0 which may be due to the chromophore molecules move from a nonpolar to a polar domain of RMs.43 This indicates that the hydrogen bonds plays an important role for stabilization of D-luciferin in the excited state. With increasing water content in RMs the extent of Hbonding interaction of D-luciferin increases. This leads to the change in energetic with stabilization of excited states of Dluciferin. Interestingly with further addition of water (W0 = 8) to RMs the emission of neutral form appeared at 438 nm with enhancement of intensity which is almost similar to that reported in neutral water.12 Furthermore, at W0 = 8 there is a shoulder band in the higher wavelength region (around 535 nm) which corresponds to the emission maximum of the anionic form of D-luciferin. With consequent increase in W0 values (from W0 = 12) the emission intensity of anionic species at 535 nm increases with decreasing the emission intensity from the neutral form of the compound at 440 nm. Similar result also observed when water is added to ethanol solution of Dluciferin.12 It is reported that the rate of proton transfer of a photoacid is always higher in water than in alcohols.49 Valeur et al. also observed similar results for HPTS (8-hydroxy-1,3,6pyrenetrisulfonate) molecule in AOT RMs.50 Such behavior is usually expected when intermolecular ESPT is involved in water pool of RMs. Water is the vital solvent for proton transfer reaction due to its proton accepting and conducting properties. Water has also been recognized as an active participant in the deprotonation step, stabilization of ion pair and in the transport mechanism of the proton.51 The proton transfer rate of a strong photoacid increases with increasing the size of the AOT RMs.50,52It is well reported that the number of the free water molecules inside the pool of the RMs increases with increasing the W0 values.32,35,37 The structural variation and physical parameters of water inside RMs of various W0 values are well reported by Biswas et al.34 ESPT of D-luciferin occurs when sufficient water molecules (from at W0 = 8) stabilize the polar transition state (TS) and the products (i.e., proton and anion). The stabilization of the hydrated proton (H30+) increases with its hydration number in liquid water.50Under the condition where the number of available water molecules are reduced and/or where the structure of water is partially broken the

Figure 1. UV−vis absorption spectra of D-luciferin at different W0 values for water/AOT reverse micelles.

of D-luciferin in water/AOT reverse micelles with different W0 values. The absorption spectra were obtained with maximum of 334 nm at W0 = 0.0 and slight red shift in other W0 values. The slight red shift of D-luciferin is ascribed to grater stabilization of the π -electron of D-luciferin in the ground state by specific Hbonding interaction.48 Thus, only deprotonated form of the compound exists in the ground state. It is already reported that deprotonation of the hydroxyl-benzothiazole group of Dluciferin is not feasible even in polar environment as water.12The red-shift along with deprotonation of D-luciferin in ground state occurs only at basic pH (pH ∼10). D-Luciferin is less soluble in nonpolar heptane solvent but in the presence of AOT solubility is enhanced significantly. So it is expected that D-luciferin occupies at the interfacial domain of the AOT RMs toward the water pool. The steady-state fluorescence spectrum of D-luciferin in AOT RMs at W0 = 0.0 exhibits a single emission peak at around 411 nm corresponding to the emission from the excited protonated form (NROH*) of the D-luciferin. Figure 2 depicts the emission spectra of D-luciferin in water/AOT reverse micelles at different W0 values. The increase in fluorescence intensity of the neutral form with a huge red shift (∼19 nm) was observed 3403



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Figure 3. variation of (a) I411−440 and (b) I535/440 with different W0 values of water/AOT reverse micelles. I411−440 and I535/440 were measured at the wavelength where the fluorescence intensity is maxima.

probability of transfer is reduced.53 Ladanyi et al.37 suggested the various structural parameters of water inside RMs based on theoretical simulation studies. They also found that water pool of RMs behave as bulk-like nature above W0 = 7.5. Thus, the nature of water confined within the RMs at higher W0 is similar as in bulk water.49 From this fact it is indicated that the Dluciferin molecule occupies in the water pool of the RMs. Similar results are also observed in previous reports.50 Spry et al.6 found that the degree of excited-state deprotonation of HPTS molecule is almost similar as in pure water when water pool of the RMs is greater than W0 = 10. The same group also found that up to a certain W0 values the HPTS molecule resides in the interfacial region of AOT RMs. At small W0 value the water pool cannot able to solvate the charge pair formed in the proton transfer process and reduces the extent of ESPT. Considering the fluorescence behavior the emission around 411−440 nm region is attributed to the neutral form of Dluciferin in AOT RMs. Figure 3 (a) shows that how emission intensity of the neutral form varies with the size of the RMs (W0 values). It clearly indicates that up to W0 = 8 the intensity of neutral form increases and after that it gradually decreases. The variation of the ratio of the fluorescence intensities of anionic form at 535 nm and neutral form at 411−440 nm is shown in Figure 3 (b). From this Figure we can see that the ratio of intensities of the two species varies linearly with W0. The steady-state emission spectra for D-luciferin in DMF/ AOT/n-heptane and DMSO/AOT/n-heptane solutions at different W0 values are depicted in Figure 4 and Figure 5, respectively. In both the polar core D-luciferin does not show any ESPT process. It is previously reported that D-luciferin

Figure 5. Emission spectra of D-luciferin (λex = 336 nm) at different W0 values for DMSO/AOT reverse micelles.

shows only the NROH emission band with a maxima at 430 nm in neat acetonitrile solvent.12 D-Luciferin also exhibits only one emission band in neat DMF at 440 nm and in DMSO at 445 nm. Thus, in polar aprotic solvent like DMF and DMSO Dluciferin does not show any ESPT process and so it rules out intramolecular ESPT. Aprotic organic solvents like DMF and DMSO do not facilitate the proton transfer process of Dluciferin due to the inability to accept and conduct protons.54 This emphasizes the importance of the hydrogen bonded network in the proton conduction. Although many of the photoacids like hydroxyflavone (HF) and 2-(2′-hydroxyphenyl) benzothiazole (HBT) shows ESPT in aprotic polar solvents.55−57 With gradual addition of DMF to the RMs there occurs an enhancement of fluorescence intensity of neutral form with a red shift. In this case there are 19 nm red shift from W0 = 0.0 to W0 = 4.5 in DMF/AOT reverse micelles. Similarly in case of DMSO/AOT reverse micelles enhancement of intensity of NROH with 19 nm red shift is observed from W0 = 0.0 to W0 = 1.5. 3.2. Time-Resolved Fluorescence Lifetime Emission Measurements. The time-resolved fluorescence decays of Dluciferin in “dry” AOT and after addition of different polar solvents (such as water, DMF and DMSO) were recorded at corresponding emission maxima of protonated and deprotonated form with excitation at 336 nm. The time-resolved fluorescence data further support the results obtained from steady-state experiment. The time-resolved fluorescence decays of neutral form in water/AOT RMs at their corresponding emission maxima are depicted in Figure 6. The corresponding lifetime values and their amplitudes are given in Table 1. We

Figure 4. Emission spectra of D-luciferin (λex = 336 nm) at different W0 values for DMF/AOT reverse micelles. 3404



Figure 6. Fluorescence decay profiles of corresponding NROH emission maxima.

Article

D-luciferin

(λex = 336 nm) in varying W0 values for water/AOT reverse micelles monitoring at

Table 1. Fluorescence Emission Maxima and Lifetime Data of D-Luciferin (λex = 336 nm) in Water/AOT Reverse Micelles with an Increase of W0 from 0 to 45

a

W0

λem max

a1

τ1 (ns)

a2

τ2 (ns)

⟨τav⟩a (ns)

0 2 4 8 12 20 32 45

411 430 435 438 440 440 440 440

0.93 0.61 0.55 0.36 0.34 0.33 0.30 0.26

0.20 0.41 0.49 0.62 0.59 0.55 0.52 0.42

0.07 0.39 0.45 0.64 0.66 0.67 0.70 0.73

2.04 2.25 2.49 2.57 2.44 2.19 1.98 1.86

0.33 1.13 1.39 1.87 1.81 1.65 1.54 1.47

Error in experimental data ±5%

Figure 7. Fluorescence decay profiles of D-luciferin (λex = 336 nm) in varying W0 values for water/AOT reverse micelles monitoring at NRO− emission maximum.

observed that upon addition of water (up to W0 = 8.0) the lifetime of the neutral species increases and after that decreases. The result indicates the occurrence of photoprotolytic processes leading to the formation of the anionic species in the excited state. The decay profile collected at emission maxima of neutral species in aqueous solution of D-luciferin is very fast i.e. around 45 ps.13 At W0 = 0.0 the decay is biexponential in nature with short component 0.20 ns (93%) and long component 2.04 ns (7%). With addition of water to AOT/n-heptane (up to W0 = 8.0) the short component increases from 0.20 to 0.62 ns and further addition of water it decreases from 0.62 to 0.42 ns with a concomitant decrease in amplitude. The results indicate that the neutral species of Dluciferin may form complexes with AOT head groups by specific H-bonding interaction. After addition of water (up to W0 = 8.0) the neutral species gradually move to water pool of RMs for the stabilization of their excited state in more polar environments. With subsequent addition of water to AOT RMs the excited neutral species start photoprotolytic reaction in water pool of RMs and it increases with increasing the water content. The similar fact also obtained from the steady-state measurement. At higher W0 values the free water molecules inside the RMs nanopool increases. We can expect that the anion formation will be more favorable at higher W0 values.50 However the long component increases up to W0 = 8.0 then decreases with increasing their corresponding amplitudes. The long component reflects some neutral species at the center of the AOT RMs which is stabilized by water pool of the RMs up to W0 = 8.0. After W0 = 8.0 the long components decreases due to bulk-like nature of the water nanopool at higher W0 values. We have also measured the decay profiles monitored at 535 nm for the anionic species of D-luciferin in AOT RMs which is

shown in Figure 7. The decay profile at W0 = 12.0 is biexponential in nature with long component 4.27 ns (62%) and short component 1.02 ns (38%). The long component corresponds to the species in water pool of RMs and the short component in less polar environment of AOT reverse micelle. Huppert et al. also observed that the lifetime of the anionic species in neat methanol and neutral aqueous solution is ∼5 ns.12,13 At higher water contain (W0 = 45) long component increases with increasing its amplitude from 4.27 ns (62%) to 4.57 ns (79%) indicating that anionic species increase after addition of water to AOT RMs. The increase of lifetime may be due to stabilization of ion pair (anion and proton) by more water molecules at high W0 values. Furthermore, we have shown that the lifetime of the anionic species of D-luciferin is more than that of neutral species indicating more stabilization of the anionic species by water molecules than the normal form through the solvation. However the short component remains almost unchanged with addition of water. The decay components and their amplitude at W0 = 12 and W0 = 45 are shown in Table 2. Table 2. Lifetime Data of D-Luciferin (λex = 336 nm) Monitored at Emission Maxima of 535 nm in Water/AOT Reverse Micelles with Different W0 Values

a

3405

W0

a1

τ1 (ns)

a2

τ2 (ns)

⟨τav⟩a (ns)

12 45

0.38 0.21

1.02 1.05

0.62 0.79

4.27 4.57

3.04 3.83

Error in experimental data ±5% dx.doi.org/10.1021/jp500210n | J. Phys. Chem. B 2014, 118, 3401−3408


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The picosecond decay curves for D-luciferin in AOT/nheptane solutions containing DMF and DMSO as polar solvents are displayed in Figure 8 and Figure 9, respectively.

Table 3. Fluorescence Emission Maxima and Lifetime Data of D-Luciferin (λex = 336 nm) DMF/AOT Reverse Micelles with an Increase of W0 from 0 to 4.5

a

W0

λem max

a1

τ1 (ns)

a2

τ2 (ns)

⟨τav⟩a (ns)

0.0 0.5 1.5 2.5 3.5 4.5

411 415 422 425 428 430

0.93 0.84 0.59 0.32 0.22 0.17

0.20 0.22 0.28 0.45 0.62 0.74

0.07 0.16 0.41 0.68 0.78 0.83

2.04 2.07 2.25 2.36 2.44 2.52

0.33 0.52 1.09 1.75 2.04 2.22


Table 4. Fluorescence Emission Maxima and Lifetime Data of D-Luciferin (λex = 336 nm) in DMSO/AOT Reverse Micelles with an Increase of W0 from 0 to 1.5

Figure 8. Fluorescence decay profiles of D-luciferin (λex = 336 nm) in varying W0 values for DMF/AOT reverse micelles monitoring at corresponding NROH emission maxima. a

W0

λem max

a1

τ1 (ns)

a2

τ2 (ns)

⟨τav⟩a (ns)

0.0 0.5 1.0 1.5

411 417 426 430

0.93 0.67 0.32 0.23

0.20 0.26 0.70 0.97

0.07 0.33 0.68 0.77

2.04 2.19 2.54 2.74

0.33 0.89 1.95 2.33


about the location of the probe molecules. However, for better understanding time-resolved fluorescence anisotropy measurements of D-luciferin in AOT RMs were performed using picoseconds TCSPC set up. It provides important information about the rotational motion of the chromophores in this confined medium. Guchhait et al.58,59 studied anisotropy decays of proton transfer probes in different biological microenvironments and found important structural environments of supramolecular assemblies and the special activity of probe molecules in these systems. In this work we measured the rotational motion of neutral excited-state D-luciferin in water/ AOT reverse micelles at W0 = 2 and W0 = 12 which is shown in Figure 10. Still now there is no study on the rotational motion

Figure 9. Fluorescence decay profiles of D-luciferin (λex = 336 nm) in varying W0 values for DMSO/AOT reverse micelles monitoring at corresponding NROH emission maxima.

From the steady-state experiment it is shown that there was no ESPT of D-luciferin in polar core of DMF/AOT and DMSO/ AOT reverse micelles. The decays profile monitored at their corresponding emission maxima at different W0 values of DMF/AOT and DMSO/AOT reverse micelles are biexponential with short and long components which is almost similar to emission of neutral species in water RMs. With increasing DMF and DMSO the short component increases with decreasing their amplitudes which is almost similar for water reverse micelle up to W0 = 8.0. The results also indicate that probe molecule moves from the interface to polar core of RMs for the stabilization of excited neutral species in more polar (DMF and DMSO) environment. After addition of DMF and DMSO the long component of neutral species located at the center of the pool of RMs increases with increasing their amplitude. With addition of the DMF to AOT RMs the average lifetime increases from 0.33 to 2.22 ns and in DMSO RMs increases from 0.33 to 2.33 ns. The results indicate that excited-state of NROH is stabilized in the pool of the polar core of RMs. All decay parameters in DMF/AOT and DMSO/AOT RMs are shown in Table 3 and Table 4, respectively. 3.3. Time-Resolved Fluorescence Anisotropy Measurements. From the steady-state and time-resolved fluorescence decay measurements we have obtained an information

Figure 10. Fluorescence anisotropy decays of D-luciferin (λex = 336 nm) in water/AOT reverse micelles at W0 = 2 (at 430 nm) and W0 = 12 (at 440 nm).

of D-luciferin inside AOT RMs. As in bulk water the fluorescence intensity of NROH at 440 nm is very low, the anisotropy decay of D-luciferin was measured at 535 nm (NRO− emission). In bulk water D-luciferin shows a rotational time constant about 569 ps. The anisotropy decays of Dluciferin at W0 = 2 (1.37 ns) monitored at 430 nm and at W0 = 3406



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(2) Chattoraj, M.; King, B. A.; Bublitz, G. U.; Boxer, S. G. Ultra-fast Excited State Dynamics in Green Fluorescent Protein: Multiple States and Proton Transfer. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8362− 8367. (3) Miskovsky, P. Hypericin - A New Antiviral and Antitumor Photosensitizer: Mechanism of Action and Interaction with Biological Macromolecules. Curr. Drug Targets 2002, 3, 55−84. (4) Kreuer, K. D. Proton Conductivity: Materials and Applications. Chem. Mater. 1996, 8, 610−641. (5) Ruitenberg, M.; Kannt, A.; Bamberg, E.; Fendler, K.; Michel, H. Reduction of Cytochrome c oxidase by a Second Electron Leads to Proton Translocation. Nature 2002, 417, 99−102. (6) Spry, D. B.; Goun, A.; Glusac, K.; Moilanen, D. E.; Fayer, M. D. Proton Transport and the Water Environment in Nafion Fuel Cell Membranes and AOT Reverse Micelles. J. Am. Chem. Soc. 2007, 129, 8122−8130. (7) Tielrooij, K. J.; Cox, M. J.; Bakker, H. J. Effect of Confinement on Proton-Transfer Reactions in Water Nanopools. Chemphyschem 2009, 10, 245−251. (8) Mondal, T.; Das, A. K.; Sasmal, D. K.; Bhattacharyya, K. Excited State Proton Transfer in Ionic Liquid Mixed Micelles. J. Phys.Chem. B 2010, 114, 13136−13142. (9) Mojumdar, S. S.; Mondal, T.; Das, A. K.; Dey, S.; Bhattacharyya, K. Ultrafast and Ultraslow Proton Transfer of Pyranine in an Ionic Liquid Microemulsion. J. Chem. Phys. 2010, 132, 194505−194512. (10) Tran-Thi, T. H.; Gustavsson, T.; Prayer, C.; Pommeret, S.; Hynes, J. T. Primary Ultrafast Events Preceding the Photoinduced Proton Transfer from Pyranine to Water. Chem. Phys. Lett. 2000, 329, 421−430. (11) Carmona, N. A.; Cohen, B.; Organero, J. A.; Douhal, A. Excited State Intermolecular Proton and Energy Transfer of 1-hydroxypyrene Interacting With the Human Serum Albumin Protein. J. Photochem. Photobiol. A: Chem. 2012, 234, 3−11. (12) Erez, Y.; Huppert, D. Excited-State Intermolecular Proton Transfer of the Firefly’s Chromophore D-Luciferin. J. Phys. Chem. A 2010, 114, 8075−8082. (13) Presiado, I.; Erez, Y.; Huppert, D. Excited-State Intermolecular Proton Transfer of the Firefly’s Chromophore D-Luciferin. 2. WaterMethanol Mixtures. J. Phys. Chem. A 2010, 114, 9471−9479. (14) Erez, Y.; Presiado, I.; Gepshtein, R.; Huppert, D. Excited-State Intermolecular Proton Transfer of Firefly Luciferin IV. Temperature and pH Dependence. J. Phys. Chem. A 2011, 115, 1617−1626. (15) The Columbia Encyclopedia, 6th ed.; Columbia University Press: New York, 2008. (16) Strehler, B. L.; McElroy, W. D. J. Purification of Firefly Luciferin. Cell. Physiol. 1949, 34, 457−466. (17) Naumov, P.; Ozawa, Y.; Ohkubo, K.; Fukuzumi, S. Structure and Spectroscopy of Oxyluciferin, the Light Emitter of the Firefly Bioluminescence. J. Am. Chem. Soc. 2009, 131, 11590−11605. (18) McElroy, W. D.; Ballentine, R. The Mechanism of Bioluminescence. Proc. Natl. Acad. Sci. U.S.A. 1944, 12, 377−382. (19) Shimomura, O. In Bioluminescence: Chemical Principles and Methods; World Scientific Publishing: Singapore, 2006; pp 1−29. (20) Pinto da Silva, L.; Esteves da Silva, J. C. G. Firefly Luciferin as a Multifunctional Chemiluminescence Molecule. Photochem. Photobiol. Sci. 2013, 12, 1615−1621. (21) Pinto da Silva, L.; Santos, A. J. M.; Esteves da Silva, J.C. G. Efficient Firefly Chemi/Bioluminescence: Evidence for Chemiexcitation Resulting from the Decomposition of a Neutral Firefly Dioxetanone Molecule. J. Phys. Chem. A 2013, 117, 94−100. (22) Anselmi, M.; Marocchi, S.; Aschi, M.; Amadei, A. Theoretical Modeling of the Spectroscopic Absorption Properties of Luciferin and Oxyluciferin: A Critical Comparison with Recent Experimental Studies. Chem. Phys. 2012, 392, 205−214. (23) Roda, A.; Guardigli, M. Analytical Chemiluminescence and Bioluminescence: Latest Achievements and New Horizons. Anal. Bioanal. Chem. 2012, 402, 69−76. (24) Marques, S. M.; Peralta, F.; Esteves da Silva, J. C. G. Optimized Chromatographic and Bioluminescent Methods for Inorganic

12 (0.94 ns) monitored at 440 nm are very slower compared to that in bulk water. Again the anisotropy decays at W0 = 12 is faster compared to that at W0 = 2. Bhattacharyya et al.9 found that the anisotropy decays of HPTS molecule in H2O/TX-100/ benzene reverse micelle are much slower compared to that in bulk water. The anisotropy decays in H2O/AOT/n-heptane reverse micelles was also studied by Douhal et al. using protontransfer probe 7-hydroxyquinoline (7HQ) at different W0 values. They found that the decays become faster at higher W0 values.60The results indicate that with addition of water to reverse micelle the chromophore moves from the interface to water pool of the RMs. In the interface of RMs the D-luciferin experiences more hindered situation than the water pool of the RMs. Thus, the water content effect insight the nanopool of RMs gives environmental structures around the probe molecule. However the initial anisotropy, r(0), values of Dluciferin in water/AOT reverse micelles are lower than the ideal 0.4. This might be due to the participation of some ultrafast process for the D-luciferin molecules.46

4. CONCLUSION Photoprotolytic reaction of D-luciferin has been studied successfully in AOT RMs using steady-state and time-resolved fluorescence measurements. The selective occurrence of ESPT in aqueous pool and its absence in the nonaqueous pool of AOT RMs has been observed. Proton transfer of D-luciferin in surfactant entrapped water pool can be considered as a model for the specific activity of this chromophore in biological water. At small W0 values (up to W0 = 4) only the deprotonated form of the chromophore is present in both the ground and the excited state. Observed results are shown that ESPT process of D-luciferin starts from W0 = 8 in water/AOT reverse micelles and it becomes more favorable at higher W0 values. Thus, the present work clearly shows that the protolysis reaction of Dluciferin in the water pool of AOT RMs strongly depends on the size of the RMs. The results reveal that foreign ions can influence significantly the confined water of RMs. In the excited state two prototropic forms of D-luciferin exist and those arise from the same neutral form of the excited chromophore solubilized in the water pool of the RMs. It is also interesting that no ESPT is observed by changing the polar protic core of reverse micelle like water with polar aprotic core like DMF and DMSO.

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AUTHOR INFORMATION

Corresponding Author

*(N.S.) E-mail: [email protected]. Fax: 91-3222255303. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.S. thanks the Council of Scientific and Industrial Research (CSIR) and Government of India, for generous research grants. J.K. and C.B. are thankful to the UGC; N.K. and D.B. are thankful to IIT Kharagpur and S.G. is thankful to CSIR for the research fellowships.

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REFERENCES

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