Published: March 09, 2011

r 2011 American Chemical Society 2456 dx.doi.org/10.1021/jp1102687 | J. Phys. Chem. A 2011, 115, 2456–2464

ARTICLE

pubs.acs.org/JPCA

Fluorescence Solvatochromism in Lumichrome and Excited-StateTautomerization: A Combined Experimental and DFT StudyN. Shaemningwar Moyon and Sivaprasad Mitra*

Department of Chemistry, North-Eastern Hill University, Shillong 793 022, India

bS Supporting Information

1. INTRODUCTION

Lumichrome (7,8-dimethylalloxazine, LC, structure I inScheme 1) represents a group of heterocyclic compounds relatedto lumazines and biologically important flavins. Although iso-alloxazines (structure II in Scheme 1), especially flavins, are veryclosely related compounds to alloxazines, the spectral andphotophysical properties of these two groups of compoundsare distinctly different. Particularly, the fluorescence intensityand the excited-state lifetime for flavins are substantially higherthan the corresponding alloxazines because of the presence ofyellow chromophore characteristics of flavoproteins, enzymesoccurring widely in animals and plants. The ground- and excited-state properties of flavins and its representatives like riboflavin,flavin mononucleotide (FMN), and flavin adenine dinucleotide(FAD) are well characterized;1-5 however, alloxazines havereceived relatively little attention. The interest in LC and othersubstituted alloxazines has intensified recently because of theirimportant role in different biological systems.6-10 For example,LC is known as a triplet photosensitizer to generate singletoxygen (1O2), which initiates the oxidation of many biologicalsubstrates like enzymes, proteins, nucleic acids, hormones, and soforth.11,12 Alloxazine group of ligands are also found to be veryeffective in binding to several proteins and nucleic acids.13-16

Furthermore, LC is known to inhibit the flavin reductase in livingEscherichia coli cells, the riboflavin uptake by human-derived livercells Hep G2, colonic epithelial NCM460 cells, and also Caco-2human intestinal epithelial cells.7,8 The application of LC has

been reported in photodegradation of polyamidehydroxyur-ethane polymers in aqueous solution,12 polymerization of 2-hy-droxyehtyl methacrylate,17,18 as an optical transistor device,19

and so forth.Alloxazine nucleosides, for which the hydrogen-bonding char-

acteristics resemble thymidine, can further be used as a fluores-cence probe.20 It has already been reported that hydrogenbonding with acetic acid or pyridine derivatives promotes thetautomerization in alloxazine scaffold to produce isoalloxazinetype of structure.21,22 In an earlier report, it was also suggestedthat solvent hydrogen bonding plays an important role on thephotophysics of LC.23More particularly, both the absorption andthe emission maxima show red shift, emission quantum yieldincreases, and also the fluorescence state becomes more stable inpolar protic environments when compared with the aproticmedium. However, to the best of our knowledge, so far, nosystematic study of LC solvatochromism with solvent hydrogenbonding ability is available in the literature.

The nature of the interaction of a solute molecule withamphiprotic solvents like water, alcohol, and so forth can havefar-reaching consequences in terms of solubility as well as inchemical properties of the solute. In some cases, the solvents canfunction as mere supporting matrixes with their physical

Received: October 27, 2010Revised: February 7, 2011

ABSTRACT: Fluorescence solvatochromism of lumichrome(LC) was studied by steady-state and time-resolved fluores-cence spectroscopy. The excited-state properties of LC do notshow any correlation with solvent polarity, however, reasonablygood correlation with solvent ET(30) parameter was observed.A quantitative estimation of contribution from different solva-tochromic parameters, like solvent polarizability (π*), hydro-gen bond donor (R), and hydrogen bond acceptor (β) ability ofthe solvent, was made using linear free energy relationship onthe basis of Kamlet-Taft equation. The analysis reveals thathydrogen bond donating ability (acidity) of the solvent is themost important parameter that characterizes the excited-statebehavior of lumichrome. Quantum mechanical calculationsusing density functional theory (DFT) were done to studythemost stable structure and excited-state tautomerization process of LC toward the formation of isoalloxazines. Charge localizationin the excited state and formation of hydrogen-bonded cluster through solvent hydrogen bond donation on the N10 atom ofalloxazine moiety were predicted to be the key step toward this water-catalyzed tautomerization process.

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parameters like dielectric constant, viscosity, refractive index,polarizability, and so forth. However, in many cases, the interac-tions of heterocyclic organic compounds with amphiproticsolvents involve creation of new molecular complexes throughhydrogen-bond formation. The chemical nature of the solute ismostly controlled by the properties of the hydrogen-bondedcomplex. Hydrogen bonding can occur in different modesdepending on the structure of the solute and solvent. Thesituation becomes more complicated when a solute molecule,like LC, possesses multiple hydrogen bonding sites, and thesolvent molecule, like water or acetic acid, can act both as aproton donor as well as a proton acceptor. Under this condition,the competition among different molecular species resultingfrom hydrogen-bonding interaction between the solute and thesolvent molecules becomes inevitable. In a recent publication, wehave discussed water-assisted hydrogen-bonding phenomenonin luminol with steady-state fluorescence spectroscopy as well ashigh-level density functional theory (DFT) calculation.24 LC alsoprovides an equally important example to study the hydrogen-bonding effect because the molecule itself can exist in more thanone prototropic species having multiple hydrogen-bonding sites(chart 1). Themost stable structure and associated spectroscopicproperties will depend strongly on the relative abundance ofseveral species as well as on their hydrogen-bonding mode withthe solvent. Furthermore, the efficacy of hydrogen-bond forma-tion in the excited state and consequently the tautomerizationprocessmay change because of charge redistribution after excitation.

In this paper, we use steady-state and picosecond time-resolved fluorescence spectral properties in a series of puresolvents with varying polarity as well as hydrogen-bond donorand acceptor abilities to find quantitative information about theirrelative contribution on LC solvatochromism. Furthermore,solution-phase spectral properties of LC were theoreticallymodeled by using density functional approach to gather theinformation on the molecular origin of specific solvent effect andon the driving force toward the excited-state tautomerizationprocess.

2. MATERIALS AND METHODS

2.1. Chemicals. Lumichrome (LC) was received from Sigma-Aldrich Chemical Pvt. Ltd. (product no. 103217) and was usedwithout any further purification. The organic solvents used wereof spectroscopic grade (>99.5%) as received from Alfa Aesar and,in some cases, from Aldrich Chemical Co. The analytical gradetype II water, also used as solvent, was obtained from Elix 10water purification system (Millipore India Pvt. Ltd.). Thechromophore concentration (6-8 μM) was very low to avoidany aggregation and was kept constant during spectral measure-ments in different solvents.2.2. Experimental Procedure. Steady-state absorption spec-

tra were recorded on a Perkin-Elmer model Lambda25

absorption spectrophotometer. Fluorescence spectra were takenin aHitachi model FL4500 spectrofluorimeter, and all the spectrawere corrected for the instrument response function. Quartzcuvettes of 10 mm optical path length received from PerkinEl-mer, United States, (part no. B0831009) and Hellma, Germany,(type 111-QS) were used for measuring absorption and fluores-cence spectra, respectively. Fluorescence quantum yields (φf)were calculated by comparing the total fluorescence intensityunder the whole fluorescence spectral range with that of astandard (quinine bisulfate in 0.5 M H2SO4 solution, φf

s =0.54625) with the following equation using adequate correctionfor solvent refractive index (n).26

φif ¼ φs

f 3Fi

Fs 31- 10-As

1- 10-Ai 3ni

ns

!2

ð1Þ

where Ai and As are the optical density of the sample andstandard, respectively, and ni is the refractive index of solventat 293 K. The relative experimental error of the measuredquantum yield was estimated within (10%.Fluorescence decay analysis was performed using time-corre-

lated single-photon-counting (TCSPC) technique as implemen-ted in time-resolved spectrofluorimeter FL-920 (EdinburgInstruments, United Kingdom), details of which have beendescribed elsewhere.27

2.3. Theoretical Calculations. Density functional theory(DFT) has successfully been applied to study the hydrogenbonding in various systems, and the results are found to be ingood agreement even with the results of several other computa-tionally costlier methods likeMøller-Plesset (MP2) or coupled-cluster (CC) calculations.28-31 Myshakina et al. used B3LYPfunctional along with 6-311þþG(d,p) basis set to estimate theimpact of hydrogen bonding on amide frequency for modelpeptides,32 whereas in a recent paper, the effectiveness of DFTmethod using a similar level of calculations to predict the dativebond energy of Lewis adducts was checked to be within∼1 kcal/mol of the benchmark results.33 Several recent reports are alsoavailable in literature dealing with ab initio or DFT calculation onisolated alloxazines and related compounds23,34,35 along withsome earlier quantum-mechanical (QM)/molecular-mechanics(MM) results.36-39 The success of the DFT method in elucidat-ing the complex phenomenon like hydrogen bonding prompts usto use it further in studying the energetic parameters of isolatedLC and its complex with water molecules. Several conformers ofLC are possible, and the structures are given in Chart 1. Toelucidate the lowest energy conformer, full geometry optimiza-tion was performed for all the structures of LC both in isolated

Scheme 1. Tautomerization of Lumichrome (I) and Isoal-loxazine (II)

Chart 1. Possible Isomeric Structures of Lumichrome (I)

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and in their hydrated complexes using the B3LYP method andthe 6-311þþG(d,p) basis set as implemented in Gaussian03program package.40 Frequency calculations were done in eachstationary point to characterize the minimum energy equilibriumstructure. Vertical transition energies up to the first 10 singletexcited states and corresponding oscillator strengths for all thestructures were estimated using time-dependent DFT method(TD-DFT) at the same level of calculation. The effect of solventon the energy parameters was incorporated by self-consistentreaction field calculation using polarizable continuum model(SCRF-PCM) as implemented in Gaussian03 software. Similarmethodology was successfully applied recently for characterizinghydrogen-bonded structure and the spectroscopic properties oflarge organic heterocyclic systems.24,41-43

3. RESULTS AND DISCUSSION

3.1. Steady-State Spectral Properties in Pure Solvents.Figure 1 shows some representative absorption and emissionspectra of LC in different solvents. It is observed that LC showstwo absorption peaks; the first one is within the spectral range of280∼350 nm with a maxima at ∼330 nm, whereas the secondone is relatively structured and within the spectral range of350∼420 nm with a peak position around ∼375 nm. Fluores-cence emission spectra obtained by exciting at 330 nm showgood mirror image relationship with the absorption profilehaving a structured emission in the 350-400 nm range with aclean broad emission peak at 450 nm. Interestingly, in aqueousmedia, the structured profile is lost in both the absorption andemission spectra. Excitation at the absorption peak (∼340 nm)as well as at the shoulder (∼380 nm) gives similar structurelessand broad emission at 475 nm (Figure 1, top panel). The

excitation spectra obtained by monitoring the broad low-energyemission peak nicely resemble the absorption spectra. Multiplepeaks in the absorption spectra and structured fluorescenceindicate that the excited states of LC are vibronically coupledeven in polar solvents. However, in aqueous media, there exists astrong specific interaction of the excited states with the solventresulting in broad and structureless profile both in absorptionand emission spectra. From the relatively large absorptioncoefficient (εmax∼ 5� 104 dm3 mol-1 cm-1) of the low-energyabsorption band, it can be concluded that this transition is S1(π)rS0(π) in nature. The assignment of the absorption band as well asthe origin of the structured absorption/fluorescence band isfurther confirmed from theoretical calculations described in thefollowing sections.3.2. Solvatochromism of LC Photophysics: Importance of

Hydrogen-Bond Acidity of the Solvent. The spectroscopicbehavior of LC was studied in a series of solvents with varyingpolarity and hydrogen-bonding parameters given in Table 1.Table 2 summarizes the steady-state spectral behavior of LC inthese solvents. Although the results do not show any regularvariation of steady-state spectral properties, careful observationreveals several interesting trends. For example, fluorescencemaxima (λem) show appreciable shift in protic solvents alongwith almost 2-fold increase in fluorescence quantum yield (φf)when compared with their aprotic counterpart. Similar observa-tions were also reported in an earlier report, although the numberof solvents taken was relatively few.23 To verify the effect ofsolvent polarity, several steady-state spectral parameters (P) likeemission maxima (νem) and Stokes shift (Δνss) of LC in a varietyof solvents mentioned in Table 1 were plotted against the solventpolarity parameter Δf (ε, n) with the general form of Lippert-Mataga (LM) equation given below.44,45

Figure 1. Steady-state absorption (left panel) and fluorescence emission (right panel) spectra of∼6.0� 10-6 mol dm-3 LC solution in 1,4-dioxane (a),acetonitrile (b), and water (c). The excitation wavelengths are 330 nm (solid line) and 380 nm (open circles) for both a and b, whereas for water, thehigh-energy excitation was done at 350 nm.

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P ¼ P0 þ a� Δf ðε, nÞ ð2Þwhere the orientation polarizability,Δf(ε, n), is related to the solventdielectric constant (ε) and the refractive index (n) with the relation

Δf ðε, nÞ ¼ ε- 12εþ 1

-n2 - 12n2 þ 1

ð3Þ

P0 is the measured property in gas phase or in noninteractingsolvents like hexane, and the slope of the equation a represents a

term containing the difference in dipole moment between theground and the excited states. From the results given in Figure 2(lower panel), it is clear that the spectroscopic properties of LC donot show any regular solvatochromism behavior on the solventpolarity parameter. This observation points to the existence ofspecific solute-solvent interactions.As a first trial, the empirical solvent polarity scale, ET(30), built

with a betaine dye, was used to correlate the solvent dependenceof the steady-state spectral properties of LC. The uniparametricscale depends on both the solvent dielectric properties and thehydrogen-bonding acidity, but it does not take care of solventhydrogen-bonding acceptor basicity.46 The specificity of Lewisacid base interactions in ET(30) parameter arise from thenegative charge localized on the phenolic oxygen of the betainemolecule. As it is seen in Figure 2 (middle panel) again, arelatively better correlation (with correlation coefficient R ≈0.95) is obtained for all the spectroscopic parameters likefluorescence maxima (νem) and Stokes shift (Δνss). This clearlyindicates that apart from solvent polarity, LC solvatochromism ismostly modulated by solvent hydrogen bond donor acidity (R),whereas solvent hydrogen bond acceptor basicity (β) is relativelyless important. To confirm this prediction further, the steady-state spectral properties of LC are interpreted by means of thelinear solvation energy relationship (LSER) concept usingKamlet-Taft eq 447

P ¼ P0 þ sπ� þ aRþ bβ ð4Þwhere P is the value of the solvent-dependent property to bemodeled and P0, s, a, and b are the coefficients determined fromthe LSER analysis. The term π* indicates the measure of solventdipolarity/polarizability,48 whereas R and β are the scale ofhydrogen bond donation acidity and acceptance basicity of thesolvent, respectively.49 The corresponding parameters for 14solvents are taken from literature50,51 and are given in Table 1.Regression analysis of fluorescence maxima (νem) and Stokes

shift (Δνss) with solvent properties results in the following

Table 1. Solvent Parameters

no. solvents Δf(ε, n)a ET(30)b

Kamlet-Taft solvent parameters

R β π*

1 benzene 0 34.3 0.0 0.10 0.59

2 toluene 0.02 33.9 0.0 0.11 0.54

3 1,4-dioxane 0.03 36 0.0 0.37 0.55

4 ethyl acetate 0.19 38.1 0.0 0.45 0.55

5 tetrahydrofuran 0.21 37.4 0.0 0.55 0.58

6 dichloromethane 0.22 40.7 0.0 0.1 0.81

7 1-pentyl alcohol 0.25 49.1

8 1-butanol 0.26 49.7 0.84 0.84 0.47

9 DMSO 0.26 45.1 0.0 0.76 0.1

10 DMF 0.27 43.2 0.0 0.69 0.88

11 1-propanol 0.27 50.7 0.84 0.90 0.52

12 isopropanol 0.27 48.4 0.76 0.95 0.48

13 acetone 0.28 42.2 0.08 0.48 0.71

14 acetonitrile 0.3 45.6 0.19 0.40 0.75

15 methanol 0.31 55.4 0.98 0.66 0.60

16 water 0.32 63.1 1.17 0.47 1.09a Polarity parameter (=[(ε- 1)/(2εþ 1)]- [(n2- 1)/(2nþ 1)]) where solvent dielectric constant and refractive indices are represented by ε and n,respectively. bReichardt solvent parameter.

Table 2. Steady-State Spectral Properties LC in Homoge-neous Solventsa

solventsb νabs/cm-1 νem/cm

-1 Δυss/cm-1

φf/10-2

1 26 316 23 419 2897 3.1

2 26 316 23 474 2842 2.3

3 26 316 23 255 3060 1.9

4 26 316 23 474 2842 5.9

5 26 247 23 474 2773 4.4

6 26 247 23 529 2717 11.3

7 25 974 22 471 3502 8.2

8 25 974 22 421 3553 9.3

9 26 042 22 779 3263 1.5

10 26 110 22 988 3121 4.8

11 25 974 22 522 3452 8.8

12 26 042 22 573 3468 5.9

13 26 316 23 310 3006 3.8

14 26 247 22 779 3468 3.5

15 26 042 22 371 3670 6.8

16 25 974 21 551 4422 9.3aAbbreviations used: ν = absorption and emission energy;Δνss = Stokesshift; φf = fluorescence quantum yield.; bThe name of the solvents arelisted in Table 1.

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correlation equations:

νemðexp t, cm-1Þ ¼ 24161:66- 1090:3π�- 1048:96R- 303:3β ð5Þ

ΔνSSðexp t, cm-1Þ ¼ 2337:47þ 880:78π� þ 874:12Rþ 42:16β ð6Þ

The correlation diagrams of the experimental values withthose calculated from the above equations are shown in theuppermost panel of Figure 2. Reasonably acceptable correlationcoefficient (R) and the value of the slope of the linear plot close tounity indicate a very good correlation of the experimentallyobserved and theoretically calculated parameters. A close lookinto the above equations reveals several interesting features forLC solvatochromism: (1) in general, the contributions from aparameter is always more significant than b beside the s para-meter indicating the importance of solvent hydrogen-bondingacidity in LC spectroscopy as indeed pointed out in the discus-sion earlier; (2) the importance of hydrogen-bonding acidity inLC spectroscopy points toward an efficient charge localization inLC upon excitation (see DFT calculation results in section3.4.2); finally, (3) the large emission spectral shift in water isdue to the negative value of a parameter and its correspondinglarger contribution. In summary, the solvatochromic analysisreveals that in polar protic solvents like water, for example,several spectroscopic species may be present because of thehydrogen-bonded donor and acceptor properties of the solventin the ground state; however, in the excited sate, the mainfluorescing species is originated because of the hydrogen-bondedcomplex formation of LC through the solvent hydrogen-bondingacidity behavior. Theoretical calculation results (see below)indeed confirm that the extensive charge localization on N10(see Scheme 1 for atom numbering) in the excited state plays a

key role toward the formation of hydrogen bond in proticmedium like water or acetic acid with LC, which subsequentlyinitiates the tautomerization leading to the formation of isoallox-azinic structure through a six-membered cyclic transition state.This prediction is supported by a two-step mechanism for acid-catalyzed proton transfer in LC proposed earlier.52

3.3. Time-Resolved Fluorescence Measurements inHomogeneous Environments. The fluorescence decay times(τf) of LC are measured in different solvents at room

Figure 2. Plot of fluorescence maxima (left panel, open squares) and Stokes shift (right panel, shaded squares) against polarity parameter, Δf(ε, n),using Lipper-Mataga plot (a), solvent ET(30) parameter (b), and theoretically calculated values using Kamlet-Taft equation (c) of LC in differentsolvents.

Figure 3. Time-resolved fluorescence decay profile (open circles) andsimulated data (solid line) of ∼5.0 � 10-6 mol dm-3 LC solution indifferent solvents. IRF indicates the instrument response function. Themagnitude of τf in different solvents is tabulated in the inset. Solventnumbers are the same as mentioned in Table 1.

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temperature using time-correlated single-photon-countingmethod. Some of the representative decay traces are shown inFigure 3, and the results are provided in the inset table. All decayscould be reproduced with single exponential decay function.Fluorescence decay times measured in this study are in very goodagreement with some of the data available in literature.23 Ingeneral, the fluorescence decay time decreases with decrease insolvent hydrogen-bonding acidity (e.g., 0.24 ns in DMSOcompared to 2.69 ns in aqueous buffer medium). This indicatesthat specific interaction through solvent hydrogen bond dona-tion stabilizes the fluorescing states of LC. The increase in τfvalues, that is, the stabilization of the fluorescing state in thepresence of hydrogen bond donor, is quite obvious from thediscussion made above as well as from the theoretical calculationdiscussed below. However, the reasons of almost 3-fold decreaseof τf in DMSO (0.24 ns) when compared with noninteractingsolvent, for example, dichloroethane (0.61 ns23), are not clear.3.4. Theoretical Calculation Using Density Functional

Theory. 3.4.1. Energetics of Different Conformers in the GroundState. Full geometry optimization of different conformers of LC(structures are given in Chart 1) in isolated condition as well as indifferent solvents with SCRF/PCM model was done usingB3LYP/6-311þþG(d,p) methodology. The fully optimizedstructures along with all the geometrical parameters are shownin Figure 1S of the Supporting Information. The correspondingenergies are listed in Table 3.It is clear that structure I is the most stable conformer in the

isolated condition as well as in different solvent systems.

Therefore, the ground-state population of LC is mainly con-tributed by structure I, and we will confine our discussion ofspectral properties on the basis of this structure only. To under-stand the absorption spectral behavior, we have carried out TD-DFT calculation of conformer I in different solvents for the first10 excited singlet states. The contributions of different orbitals inthe corresponding excited states within the experimentally ob-served spectral range (280-420 nm) are given in Table 4. Thecalculated excitation wavelengths are in very good agreementwith the absorption spectra shown in Figure 1. From the table, itis clear that the excited state of LC is mainly contributed from theHOMO (H) f LUMO (L) transition with a little contributionfrom H- 1 and Lþ 1 orbitals also. Careful analysis of electron-density distribution in all these orbitals (see below) confirms theπ* r π nature of transition involved in optical absorptionspectroscopy of LC.3.4.2. Analysis of Frontier Orbitals: Charge Localization in the

Excited State. All the four MOs involved in electronic transitionof LC are depicted in Figure 4. From the discussion made in theprevious section and also from the results given in Table 4, it isclear that the major contribution for the electronic absorptionarises from the transition to the lowest unoccupied molecularorbital (LUMO). Careful analysis of this orbital and comparingwith the occupied molecular orbitals reveals that the chargeredistribution of LC occurs in the excited state. It is indeedobserved that the charge localization to a greater extent occurs onthe N10 atom, whereas N1 hydrogen becomes strongly acidic onexcitation. Therefore, it is highly probable that excited-state

Table 3. Ground-State Energy Parameters (in Hartree) of Different Possible Conformers of LC Obtained by Full GeometryOptimization at the B3LYP/6-311þþG(d,p) Level of Calculation in the Gas Phase as well as in Some Representative Solvents

conformera gas phase 1,4-dioxane acetonitrile water

I -833.0499109 -833.076708227 -833.082797005 -833.084065647

Ia -833.0132127 -833.040351540 -833.046950983 -833.048484606

Ib -833.0275746 -833.055739194 -833.062265591 -833.06809155

Ic -833.0249842 -833.054456108 -833.061430244 -833.063097388a Structures are given in Chart 1.

Table 4. Energy of Absorption (nm), Its Oscillator Strength (f), and Contribution of Different Orbitals in the Transition to theCorresponding Excited States (ES) of LC (Structure I) in Gas Phase as well as in Some Representative Solvents Obtained by TD-DFT Calculation at B3LYP/6-311þþG(d,p) Level of Calculationa

ES gas phase 1,4-dioxane acetonitrile water

1 365.8 nm (f = 0.0626) 381 nm (f = 0.0626) 383.2 nm (f = 0.0626) 383.5 nm (f = 0.0626)

62 f 64 0.1279 62 f 64 0.1231 62 f 64 0.1303 62 f 64 0.1319

62 f 65 0.1315 62 f 65 0.1095 62 f 65 0.1084 62 f 65 0.1083

63 f 64 0.6475 63 f 64 0.6538 63 f 64 0.6521 63 f 64 0.6517

2 362.3 nm (f = 0.0014) 353.6 nm (f = 0.0018) 351.6 nm (f = 0.0016) 351 nm (f = 0.0019)

60 f 64 0.1169 60 f 64 0.1034 60 f 64 0.1026 60 f 64 0.018

61 f 64 0.6748 61 f 64 0.6799 61 f 64 0.6807 61 f 64 0.6809

3 321.4 nm (f = 0.2173) 333.7 nm (f = 0.3472) 334.8 nm (f = 0.3407) 335.0 nm (f = 0.3398)

62 f 64 0.1169 62 f 64 0.6316 62 f 64 0.6290 62 f 64 0.6287

63 f 65 -0.2230 63 f 65 -0.1852 63 f 65 -0.1878 63 f 65 -0.1878

4 310.2 nm (f = 0.0002) 299.4 nm (f = 0.0001) 292.2 nm (f = 0.0001) 296.7 nm (f = 0.0001)

57 f 64 -0.1364 57 f 64 -0.1383 57 f 64 -0.1451 57 f 64 -0.1469

59 f 64 0.6218 60 f 64 0.6643 60 f 64 0.6640 60 f 64 0.6638

63 f 65 0.2259aThe highest occupied and lowest unoccupied MOs are represented by numbers 63 and 64, respectively.

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photophysics of LC will be strongly modulated by hydrogenbond donation of the solvent with N10 atom; on the other hand,hydrogen bond accepting solvents like DMSO mainly actsthrough proton abstraction from the N1 atom. Analysis ofsolvent dependence of LC fluorescence discussed earlier hasshown that the fluorescence properties show good correlationeven with solvent ET(30) parameter that mainly takes care ofsolvent hydrogen bond donation ability (acidity). Consequently,it is reasonable to believe that in an amphoteric medium likewater or acetic acid, the key step involves the formation ofhydrogen bond through solvent hydrogen bond acidity at theN10 atom. Once this hydrogen bond formation occurs, geo-metric rearrangement of the solvent occurs to form a cyclic six-membered structure through solvent hydrogen bond basicitywith N1 proton of LC. A similar mechanism was proposed earlierby Sikorska et al. for proton transfer in LC-acetic acidcomplex.52 The authors termed the second step as the formationof an appropriate structure to account for the slow protontransfer following the very fast initial hydrogen bond donatedcomplex at the N10 position of LC with acetic acid. However,contrary to the hypothesis proposed by the authors, we believethat the first hydrogen-bonded complex is not available in theground state. Charge localization in the excited state is the originof this specific hydrogen bond to occur at the N10 of LC whichserves as the catalytic pathway for the excited-state protontransfer toward the formation of isoalloxazine (II).3.4.3. Water-Assisted Proton Transfer in LC: Analysis of

Transition State. The ground-state energy parameters of thealloxazine (I) and isoalloxazine (II) structures in gas phase aswell as in different solvents calculated using SCRF/PCM modelat B3LYP/6-311þþG(d,p) level are listed in Table 1S of theSupporting Information. It is found that although structure II isless stable by 54.06 kJ mol-1 energy relative to that of structure Iin the gas phase, the energy difference decreases substantially inthe presence of solvents (for example, ∼26.27 kJ mol-1 in thepresence of water and ∼26.30 kJ mol-1 in the presence ofacetonitrile). In contrast, full geometry optimization with specific

hydrogen-bonded structures of I and II with water moleculespredicts an energy difference of ∼34.0 kJ mol-1 between them.These results indicate the importance of specific hydrogen bondformation in the proton-transfer process and confirm that onlysolvent physical parameters like polarity, as imposed by SCRF/PCM model, are not sufficient to catalyze the process. Ratherthan acting merely as a matrix with dielectric continuum, thesolvent directly takes part in the chemical transformationthrough hydrogen-bonding interaction. The optimized struc-tures along with the geometrical and energy parameters for thesetwo conformers, specifically hydrogen bonded with water mole-cule, are given in Figure 2S of the Supporting Information. Onlyprimary hydrogen-bonded complex of I and II with water wasconsidered to give different complexes. It is obvious that addi-tional solvent molecules will combine to give secondary watercluster and that the actual hydrated structure is far more complexthan what is considered for these calculations. However, we

Figure 4. Frontier orbital diagram of differentMOs for LC (I). The block arrows in LUMO indicate charge accumulation (filled arrow) removal (emptyarrow) on N10 and N1, respectively, due to electronic excitation.

Figure 5. Ground- and excited-state potential energy surface for water-catalyzed conversion of I to II.

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restrict ourselves only in the first hydration layer as the effect ofinternal hydrogen bonding (IHB) is expected to diminish veryfast from the center of origin. Consequently, it is reasonable tobelieve that any further addition of water structure will haveinsignificant contribution toward the relative energy parameters.The calculated energy difference between I and II in the

ground state is ∼34.0 kJ mol-1. The potential energy surface(PES) (Figure 5), constructed by intrinsic reaction coordinate(IRC) calculation from the transition state (TS), indicates thatthe water-assisted conversion of I to II is associated with a largeactivation barrier of∼89.88 kJ mol-1. Thus, in ground state, thepossibility of proton transfer is very less, both from kinetic andthermodynamic points of view. However, TD-DFT calculationresults show that the relative energy difference between I and II isvery small in the first excited state (∼1.8 kJ mol-1) and that thebarrier height for the proton transfer also reduces substantiallyto ∼68.5 kJ mol-1. As a result, the water-catalyzed proton-transfer process becomes highly feasible in the excited state. TheTS structure (Figure 6) is confirmed by the presence of animaginary frequency (∼1590.35 cm-1) which is active toward theproton translocationwithinN1 andN10 atoms of alloxazinemoiety.

’CONCLUSIONS

The fluorescence behavior and the water-catalyzed excited-state tautomerization process of lumichrome have been studiedby steady-state and time-resolved fluorescence spectroscopy aswell as by DFT calculations. The observed solvatochromism oflumichrome fluorescence is rationalized by multiparametricapproach using Kamlet-Taft equation. It has been found thatthe photoluminescence behavior of lumichrome is stronglymodulated by specific solute-solvent interaction through sol-vent hydrogen bond donation. These observations have alsobeen supported by DFT calculations on lumichrome in isolatedcondition as well as in the presence of some representativesolvents. The results predict that extensive charge localization onthe N10 atom and subsequent hydrogen bond formation with

the solvent are the key processes in the excited state, whichessentially explains the dominance of solvent hydrogen bondacidity toward the lumichrome fluorescence behavior.

’ASSOCIATED CONTENT

bS Supporting Information. Energy parameters of I and IIin gas phase as well as in some representative solvents (Table 1S),full optimized ground-state geometrical parameters in gas phaseof I, Ia, Ib, and Ic (Figure 1S), and specific hydrogen-bondedwater complex of I and II (Figure 2S). This material is availablefree of charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*Phone: (91)-364-2722634. Fax: (91)-364-2550076. E-mail:smitra@nehu.ac.in, sivaprasadm@yahoo.com.

’ACKNOWLEDGMENT

Financial support through research project 2009/37/26/BRNS from Board of Research in Nuclear Sciences (BRNS),Government of India, is gratefully acknowledged. The authorsthank Dr. A. Bhasikuttan of Bhaba Atomic Research Center(BARC) for helpful discussion. Thanks are also due to AIRF,JNU for their help in TCSPC measurement.

’REFERENCES

(1) Dutta Choudhury, S.; Mohanty, J.; Bhasikuttan, A. C.; Pal, H. J.Phys. Chem. B 2010, 114, 10717–10727.

(2) Chosrowjan, H.; Taniguchi, S.; Mataga, N.; Nakanishi, T.;Haruyama, Y.; Sato., S.; Kitamura, M.; Tanaka, F. J. Phys. Chem. B2010, 114, 6175–6182.

(3) Murakami, M.; Ohkubo, K.; Fukuzumi, S. Chem.—Eur. J. 2010,16, 7820–7832.

Figure 6. Ground-state structure of the transition state (TS) for water-catalyzed I f II conversion obtained by IRC calculation using B3LYP/6-311þþG(d,p) method. The bond lengths are given in angstroms, and the angles are in degrees.

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(4) Radoszkowicz, L.; Huppert, D.; Nachliel, E.; Gutman, M. J. Phys.Chem. A 2010, 114, 1017–1022.(5) Kao, Y. T.; Saxena, C.; He, T. F.; Guo, L.; Wang, L.; Sancar, A.;

Zhong, D. J. Am. Chem. Soc. 2008, 130, 13132–13139.(6) Chastain, J.; McCormick, D. B. In Chemistry and Biochemistry of

Flavoenzymes, 1; Muller, F., Ed.; CRC Press: Boston, MA, 1991; pp196-200.(7) Said, H. M.; Ortiz, A.; Moyer, M. P.; Yanagawa, N. Am. J. Physiol.

Cell Physiol. 2000, 278, C270–C276.(8) Cunningham, O.; Gore, M. G.; Mantle, T. J. Biochem. J. 2000,

345, 393–399.(9) Tyagi, A.; Penzkofer, A.; Mathes, T.; Hegemann, P. J. Photochem.

Photobiol., B: Biol. 2010, 101, 76–88.(10) van den Berg, P. A.; van Hoek, A.; Walentas, C. D.; Perham,

R. N.; Visser, A. J. Biophys. J. 1998, 74, 2046–2058.(11) Sikorska, E.; Sikorski, M.; Steer, R. P.; Wilkinson, F.; Worall,

D. R. J. Chem. Soc., Faraday Trans 1998, 94, 2347–2353.(12) Onu, A.; Palamaru, M.; Tutovan, E.; Ciobanu, C. Polym.

Degrad. Stab. 1998, 60, 465–470.(13) Li, H.; Jiang, Z. Q.; Zhang, R. H. Chin. Sci. Bull. 2010,

55, 2829–2834.(14) Rajendar, B.; Nishizawa, S.; Teramae, N. Org. Biomol. Chem.

2008, 21, 670–673.(15) Grininger, M.; Zeth, K.; Oesterhelt, D. J. Mol. Biol. 2006,

357, 842–857.(16) Rhee, H. W.; Choi, S. J.; Yoo, S. H.; Jang, Y. O.; Park, H. H.;

Pinto, R. M.; Cameselle, J. C.; Sandoval, F. J.; Roje, S.; Han, K.; Chung,D. S.; Suh, J.; Hong, J. I. J. Am. Chem. Soc. 2009, 131, 10107–10112.(17) Bertolotti, S. G.; Previtali, C. M.; Rufs, A. M.; Encinus, M. V.

Macromolecules 1999, 32, 2920–2924.(18) Bairi, P.; Roy, B.; Nandi, A. K. J. Phys. Chem. B 2010,

114, 11454–11461.(19) Zen, Y. H.; Wang, C. M. J. Chem. Soc., Chem. Commun.

1994, 2625–2626.(20) Wang, Z. W.; Rizzo, C. J. Org. Lett. 2000, 2, 227–230.(21) Song, P.-S.; Sun, M.; Koziolowa, A.; Koziol, J. J. Am. Chem. Soc.

1974, 96, 4319–4323.(22) Koziol, J. Photochem. Photobiol. 1966, 5, 41–54.(23) Sikorska, E.; Khmelinskii, I. V.; Prukala, W.; Wiliams, S. L.;

Patel, M.;Worall, D. R.; Bourdelande, J. L.; Koput, J.; Sikorski, M. J. Phys.Chem. A 2004, 108, 1501–1508.(24) Moyon, N. S.; Chandra, A. K.; Mitra, S. J. Phys. Chem. A 2010,

114, 60–67.(25) Meech, S. R.; Phillips, D. J. Photochem. 1983, 23, 193–217.(26) Mataga, N.; Kubota, T. In Molecular Interactions and Electronic

Spectra; Marcel Dekker Inc.: New York, 1970.(27) Moyon, N. S.; Mitra, S. Chem. Phys. Lett. 2010, 498, 178–183.(28) Ireta, J.; Neugebauer, J.; Scheffler, M. J. Phys. Chem. A 2004,

108, 5692–5698.(29) Ni, W.; Mosquera, R. A.; P�erez-Juste, J.; Liz-Marz�an, L. M. J.

Phys. Chem. Lett. 2010, 1, 1181–1185.(30) Hao, M.-H. J. Chem. Theory Comput. 2006, 2, 863–872.(31) Chen, X.; Brauman, J. I. J. Am. Chem. Soc. 2008,

130, 15038–15046.(32) Myshakina, N. S.; Ahmed, Z.; Asher, S. A. J. Phys. Chem. B 2008,

112, 11873–11877.(33) Potter, R. G.; Camaioni, D. M.; Vasiliu, M.; Dixon, D. A. Inorg.

Chem. 2010, 49, 10512–10521.(34) Klaum€unzer, B.; Kr€oner, D.; Saalfrank, P. J. Phys. Chem. B 2010,

114, 10826–10834.(35) Salzmann, S.; Marian, C. M. Photochem. Photobiol. Sci. 2009,

8, 1655–1666.(36) Salzmann, S.; Silva-Junior, M. R.; Thiel, W.; Marian, C. M. J.

Phys. Chem. B 2009, 113, 15610–15618.(37) Szymusiak, H.; Konarshi, J.; Kozioz, J. J. Chem. Soc., Perkin

Trans. 1990, 2, 229–236.(38) Komasa, J.; Rychlewski, J.; Kozioz, J. J. Mol. Struc.

(THEOCHEM) 1988, 170, 205–212.

(39) Sun, M.; Moore, T. A.; Song, P. S. J. Am. Chem. Soc. 1972,94, 1730–1740.

(40) Frisch, M. J. et al. Gaussian 03, revision D.01; Gaussian, Inc.:Wallingford, CT, 2004.

(41) Mitra, S.; Singh, T. S.; Mandal, A.; Mukherjee, S. Chem. Phys.2007, 342, 309–317.

(42) Singh, T. S.; Mitra, S.; Chandra, A. K.; Tamai, N.; Kar, S. J.Photochem. Photobiol., A: Chem. 2008, 197, 295–305.

(43) Moyon, N. S.; Singh, T. S.; Mitra, S. Biophys. Chem. 2008,138, 55–62.

(44) Mataga, N.; Kaifu, Y.; Koizumi, M. Bull. Chem. Soc. Jpn. 1956,29, 465–470.

(45) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd Edition;Springer: New York, 2006; pp 208-210.

(46) Reichardt, C. Chem. Rev. 1994, 94, 2319–2358.(47) Kamlet, M. J.; Abboud, J. M.; Taft, R. W. Prog. Phys. Org. Chem.

1981, 13, 485–630.(48) Abboud, J. M.; Kamlet,M. J.; Taft, T.W. J. Am. Chem. Soc. 1977,

99, 8325–8327.(49) Miller, J. V.; Bartick, E. G.Appl. Spectrosc. 2001, 55, 1729–1732.(50) Marcus, Y. Chem. Soc. Rev. 1993, 22, 409–416.(51) Kamlet, M. J.; Abboud, J. M.; Abraham, M. H.; Taft, R. W. J.

Org. Chem. 1983, 48, 2877–2887.(52) Sikorska, E.; Koziozowa, A.; Sikorski, M.; Siemiarczuk, A. J.

Photochem. Photobiol., A: Chem. 2003, 157, 5–14.