Spectral, AcidâBase Properties, and Interplay of the Protolytic Forms in

Article pubs.acs.org/JPCA

7‑Hydroxyflavone Revisited: Spectral, Acid−Base Properties, and Interplay of the Protolytic Forms in the Ground and Excited States Illia E. Serdiuk,†,‡ Andrii S. Varenikov,† and Alexander D. Roshal*,† †

Institute of Chemistry, Kharkiv V.N. Karazin National University, Svoboda 4, 61077 Kharkiv, Ukraine Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-952 Gdańsk, Poland

‡

S Supporting Information *

ABSTRACT: Spectral and acid−base properties of 7-hydroxyflavone (7HF) in the ground and excited states were investigated with a purpose to enable reasonable application of this dye and its derivatives as fluorescent probes. Analysis of solvatochromic and solvatofluorochromic ability of 7HF in 20 solvents, investigations of 7HF spectral properties in the frozen solvents, spectrophotometric and spectrofluorimetric titrations in methanol− water (4:1 v/v) in the wide pH/H0 range (from pH = 11.0 to H0 = −4.5), analysis of the 3D fluorescence and time-resolved spectra, as well as quantum-chemical calculations were carried out. It has been found that 7HF can exist in three protolythic formsneutral, anion, and cationdepending on the environment acidity or basicity. In the excited state, in methanol−water solutions, there are four forms: neutral, cation and anion, which can be formed by direct excitation of the ground-state anion or by photodissociation of the neutral form depending on pH, and only one phototautomer, which appears in the H0 range from 1.3 to −4.5. It has been shown that the mechanism of the phototautomer formation depends on medium acidity. The photoautomer can be formed by cation photodissociation as well as by photoanion protonation. Finally, it was concluded which of the 7HF protolytic forms can be used for fluorescent probing.

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INTRODUCTION The overwhelming majority of biologically active flavones has a hydroxyl or a methoxyl group in the position 7 of chromone moiety, and thus, such flavones are derivatives of 7hydroxyflavone (7HF).1 Usually, the 7-hydroxy group in polyhydroxyflavones is of the highest acidity, and therefore, it is responsible for the acid−base properties of this class of flavonoid compounds.2,3 Due to the presence of the carbonyl fragment, 7-(R)-oxyflavones are also very weak bases; however, electron-donating 7-hydroxy or 7-methoxy groups increase the carbonyl basicity in comparison with other flavonoids.4,5 The most complete investigations of 7HF acid−base and spectral properties were carried out in (6). It was found that the acidity of the 7-hydroxyl group dramatically increases in the excited state what results in the 7HF photodissociation and in appearance of anionic form fluorescence in the neutral and acidic media. The increase of the carbonyl group basicity upon excitation was also concluded. Moreover, a new emission band was found in the acidity range from H0 = −3.0 to pH = 3.0. The authors suggested that this band could be due to the fluorescence of phototautomer or 7HF−solvent exciplex. Structures of some mentioned protolythic forms of 7HF are depicted in Scheme 1. It might seem that the results presented in (6) and completed by 7-methoxyflavone investigations4 give a comprehensive scheme of acid−base interaction in 7HF and its derivatives. However, the authors of (7), using transient absorption and spectroscopy of two-photon excitation, made the conclusion about formation of two phototautomers in neutral methanolic solutions. Much later emission of one of these tautomers was © 2014 American Chemical Society

Scheme 1. Protolytic Forms of 7-HF in the Ground and Excited (*) States. N − Neutral Form, A − Anion, AP* − Photoanion (the Anion Formed by Dissociation of the Excited Neutral Form), C − Cation, T* − Phototautomer of the Neutral Form

assigned to anion form.8 Moreover, two 7HF complexes with solvent molecules, 7HF·Sol and 7HF·2Sol, were found in the ground state. The excited state proton transfer in these complexes results in formation of photoanion and phototautomer, correspondingly. Received: December 17, 2013 Revised: March 30, 2014 Published: April 1, 2014 3068

dx.doi.org/10.1021/jp412334x | J. Phys. Chem. A 2014, 118, 3068−3080

The Journal of Physical Chemistry A

Article

excitation spectrum measured in fluorescence band maximum. In the case of two-band fluorescence, when both fluorescence bands were due to the same S0 → S1 transition, the Stokes shift values for these bands were calculated as it is described above. Concentrations of 7-HF in the organic solvents used for solvatochromic investigations were in the range of 0.7 × 10−6− 1.2 × 10−5 M. Acid−base investigations were carried out in methanol−water (4:1 v/v) solutions. For titration in the 1.5− 11.0 pH range, 0.05 and 0.005 M methanol−water solutions of sulfuric acid as well as 0.1 and 0.01 M NaOH methanol−water solutions were used as titrants. pH values were measured by a pH meter. Volume of the initial 7-HF solution was 100 mL, so that the volume change during the titration was negligible. The obtained dissociation constants (pKa) were corrected by a −0.06 value for the presence of methanol.17 The acidic titration of 7-HF in −4.5 to 1.5 H0 range was made by concentrated sulfuric acid in 80 vol % aqueous methanol according to the procedure described in (18). The correction of H0 scale in methanol solutions was carried out by the Hammet method.19,20 Unlike the classical Hammet’s method, where H0 scale correction is based on pKa values of aniline dyes,19,20 we used pKa values of flavone derivatives to find correlation between H0 scales in aq methanol−sulfuric acid and water−sulfuric acid solutions. The flavones were preferred in order to take into account the differences between solvation of amino- and carbonyl-containing dyes. The pKa values of flavone derivatives (Table 1S in Supporting Information) were determined by spectrophotometric titration in aqueous media.18 The ratio HB+/B in aq methanol for the Hammett eq i was determined spectrometrically by gradual addition of 96% sulfuric acid to 2 mL of the flavone solution. The acid concentration was controlled by weight. The H0 values, obtained for aq methanol medium from eq i, were plotted against the values H0 ′ obtained for aqueous solutions with the same weight concentration of H2SO4 (see Figure 1S in the Supporting Information). The found dependence ii was used for calculation of H0 in the further investigations.

It is also worthwhile to note the contradictory information about spectral properties of 7HF. Authors of (6) found two emission bands of neutral (N*) and anionic (A*) forms and presented their spectral parameters. Authors of (9) did not find fluorescence of N*; however, the phosphorescence of this form at 77 K was detected. The low temperature experiment allowed the observation of a fluorescence band at 450 nm, which correlated with anion concentration in the ground state. Temperature growth resulted in disappearance of this band and in appearance of the new long-wavelength band attributed earlier to the anion.4,6,8,9 The mentioned bands were assigned by authors to two different solvate complexes of the 7HF anion. The differences in spectral properties of these complexes were explained by uneven intensity of solvate relaxation processes at 77 K and 298 K. The presence of several fluorescence bands and their different dependence on environment properties allows using 7HF and its derivatives as fluorescent sensors. However, the lack of reliable assignment of these bands to corresponding protolytic forms of 7HF complicates adequate interpretation of the spectral data obtained in investigations of biological and other objects. For example, bands observed in 7HF fluorescence spectra in micelles10 were interpreted as emission of the N* and A* forms. In the case of 7HF complexes with human serum albumin,11 the detected bands were assigned to two different solvates of the anionic form. The same authors (12) supposed that two detected emission bands of 7HF in model membranes are due to the anion and phototautomer forms. Taking also into account growing interest to the investigations of therapeutic and antioxidant properties of 7HF,13 analysis of the acid−base properties of 7HF and interpretation of spectral behavior of its protolytic forms becomes topical. To make assignment of the detected emission bands to the corresponding protolytic forms, we have investigated spectral behavior of 7HF in the solvents of different polarity, basicity and electrophilicity at 77 K and 298 K. The analysis of spectral properties of 7HF in water−alcohol media in the wide range of pH/H0 was made using absorption spectroscopy as well as usual and 3D steady state and time-resolved fluorescence spectroscopy. To interpret the obtained experimental data, some thermodynamic and structural parameters of some 7HF protolytic forms were calculated.

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H 0 = pK a − lg(HB+/B)

(i)

H 0 = 1.042 × H 0′ + 0.436 (r = 0.996)

(ii)

The dissociation constants of hydroxyl groups of the compounds in the excited state were estimated by the Förster’s method on the basis of 0−0 transitions positions.21,22 Polystyrene films with 7HF were prepared by mixing dichloromethane solutions of 7HF and polystyrene. The solvent was allowed to evaporate completely at room temperature. The films obtained were used in 3−4 days after their preparation. The 7HF concentration in the initial dichloromethane solution was fitted to obtain a film optical density of about 0.2 at the wavelength corresponding to the maximum of long-wavelength absorption band of 7HF (∼300 nm). Polystyrene film without 7HF was used as a reference sample. Absorption and fluorescence spectra were separated on individual bands by deconvolution procedure by means of Spectra Data Lab software.23 Further multicomponent regression analysis of the dependence of 7HF spectral characteristics on solvent parameters was carried out by the Statistica 6.0 program. Fluorescence lifetimes and time-resolved spectra were measured on a subnanosecond kinetic spectrometer, consisting of an MDR-12 monochromator (LOMO, Russia), a TimeHarp 200 TCSPC device, a PLS 340−10 ps LED driven by a PDL 800-

EXPERIMENTAL SECTION 7-Hydroxyflavone was synthesized according to (14). Structure and purity of the obtained 7HF were controlled using HPLC, 1H NMR, and a MALDI TOF mass spectrometer. The commercial solvents used for solvatochromic investigations were checked on their transparency and additionally dried according to (15). Absorption spectra were recorded using a Hitachi U3210 spectrophotometer. Fluorescence spectra (usual and 3D) were obtained with both Hitachi F4010 and PerkinElmer Lambda spectrofluorimeters. Low-temperature fluorescence and phosphorescence spectra recording as well as phosphorescence lifetime measurements were carried out using a PerkinElmer Lambda spectrofluorimeter. Stokes shifts of fluorescence (and phosphorescence) of each protolytic form were calculated as the difference between spectral position (in wavenumbers) of the long-wavelength absorption band of this form (S0 → S1 transition) and the emission band arising from this transition.16 To prove that a fluorescence band arises from a certain S0 → S1 transition, we checked similarity of absorption spectrum of a protolytic form and fluorescence 3069



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Table 1. Spectral properties of 7HF in organic media neutral form (N, NH*) medium group I hexane cyclohexane iso-octane ethyl acetate methylformate dichloromethane acetonitrile group II dioxane diisopropyl ether diethyl ether acetone tetrahydrofuran DMF DMSO group III methanol iso-propanol tert-butanol iso-butanol pentanol ethanol octanol

νabs, cm−1 (λabs, nm)

νfl, cm−1 (λfl, nm)

photoanion (Ap*) ΔνSt, cm−1


anion (A, A*)

ΔνSt, cm−1

34 365 (291) 33 555 (298) 34 245 (292) 32 785 (305) 33 360 (295) 32 470 (308) 32 680 (306)

νabs, cm−1(λabs, nm)


26 455 (378) 27 250 (367) 26 665 (375) 26 525 (377) 27 120 (368) 27 700 (361) 25 775 (388)

18 840 (531) 18 940 (528) 18 720 (534) 18 060 (554) 18 000 (556) 18 560 (539) 17 160 (583)

7615 8310 7945 8465 9120 9140 8615

17 380 (575) 17 220 (581) 18 420 (543) 17 380 (575) 17 945 (557) 18 080 (554) 17 920 (558)

9940 9975 9990 10 170 10 145 7495 7460

18 320 (546) 18 840 (531) 18 720 (534) 18 860 (530) 19 000 (527) 18 610 (537) 19 010 (526)

8780 8260 8305 8240 8180 8415 8250

34 015 (294) 33 900 (295) 35 090 (285) 33 900 (295) 34 720 (288) 32 575 (307) 33 335 (300)

25 255 (396) 25 000 (400) 25 300 (395) 24 510 (408) 25 280 (392) 24 400 (409) 24 100 (415)

8760 8900 9790 9390 9440 8175 9235

18 030 (554) 17 955 (557)

14 545 15 380

27 320 (366) 27 195 (368) 28 410 (352) 27 550 (363) 28 090 (356) 25 575 (391) 25 380 (394)

32 570 (307) 32 470 (308) 32 575 (307) 32 470 (308) 32 480 (308) 32 575 (307) 32 680 (306)

24 725 (404) 24 360 (411) 24 920 (401) 24 640 (406) 24 500 (408) 24 620 (406) 24 580 (407)

7845 8110 7655 7830 7980 7955 8100

18 080 (553) 18 400 (543) 18 640 (536) 18 640 (536) 18 540 (539) 18 380 (544) 18 860 (530)

14 490 14 070 13 935 13 830 13 940 14 195 13 820

27 100 (369) 27 100 (369) 27 025 (370) 27 100 (369) 27 180 (368) 27 025 (370) 27 260 (367)

ΔνSt, cm−1

νabs, λabs − positions of the absorption band maxima of neutral form (N) and anionic forms in the presence of DBU (A), νfl, λfl − positions of the emission band maxima of neutral hydrogen-bonded form (NH*) and photoanion (Ap*) (both are excited at 300 nm) as well as of anion or ionic pair (A*), formed by 7-HF autodissociation in S0-state or after DBU addition (excited at 350 nm). ΔνSt − the Stokes shifts of fluorescence: ΔνSt (NH*) = νabs (N*) − νfl (NH*); ΔνSt (Ap*) = νabs (N*) − νfl (Ap*); ΔνSt (A*) = νabs (A*) − νfl (A*).

Figure 1. Normalized absorption (a), fluorescence (b, dashed line), and phosphorescence (c) spectra of 7-HF in polystyrene matrix and hexane, tetrahydrofuran (THF), and methanol liquid (a, b, 298 K) and gelled (c, 77 K) solutions.

were used to obtain the molecular cavity).31,32 The calculations were carried out on the cluster of the Ukrainian-American Laboratory of Computational Chemistry (UALCC, Kharkiv, Ukraine).

B device (PicoQuant GmbH, Germany), and a Hamamatsu H5783P PMT (Hamamatsu, Japan). Unconstrained geometry optimizations of isolated 7HF molecules and their complexes with solvent molecules in the ground (S0) or excited singlet (S1) electronic states were carried out at the DFT or TDDFT levels of theory,24 respectively, using the B3LYP functional25−27 and cc-pVDZ basis sets28,29 implemented in the GAUSSIAN 09 program package.30 Enthalpy and the Gibbs’ free energy contributions at 298.15 K and standard pressure were made by built-in computational program of statistical thermodynamics routines. The solvent effect was included in the DFT (TDDFT) calculations at the level of the Polarized Continuum Model (PCM) (UAHF radii

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EXPERIMENTAL AND THEORETICAL RESULTS Solvatochromic and Solvatofluorochromic Properties of 7HF. Theoretical modeling of spectral behavior of the 7HF neutral form (N) in nonaqueous solvents was discussed in (33). A linear dependence of quantum-chemistry calculated energies of long-wavelength transition (λabs) on the environment polarity was demonstrated. However, analysis of experimental data evidenced the dependence of λabs on the Kamlet−Taft 3070



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the authors of (6) to the emission of the form N*. The regression analysis shows that the position of this band depends on solvent polarity (Y), as well as on solvent basicity when the Kamlet−Taft parameters are being used:

parameters α and β,34 which characterize specific interaction between 7HF and solvent molecules. Our investigations of 7HF solvatochromism show that the dependence λabs = f (α, β) disappears when the number of used solvents increases. The values of absorption band maxima obtained by deconvolution of the 7HF absorption spectra in long-wavelength range are listed in Table 1. The spectra in some media are depicted in Figure 1a. Analysis of the data showed that all the solvents used can be divided into three groups depending on spectral behavior of N. The first group (I) contains solvents with mainly nonspecific character of interaction with N and having a weak acid or basic properties: these are hexane, cyclohexane, iso-octane, dichloromethane, ethyl acetate, methyl formate, and acetonitrile. Basic aprotic solvents able to form strong hydrogen bonds with flavone 7-hydroxy group were placed in the second group (II): dioxane, diethyl, and diisopropyl ethers, tetrahydrofuran, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF). The third group (III) consists of solvents, which have both strong protondonating and proton-accepting properties, such as methanol, ethanol, propanol, iso-butanol, tert-butanol, octanol, and water. In solvents of the group I, the long-wavelength absorption band of N undergoes the bathochromic shift from 290 to 310 nm (Δνabs ≈ 1900 cm−1) with an increase of the medium polarity. The regression analysis shows a linear dependence of the maximum position of N long-wavelength band on the Kamlet− Taft parameter π*, characterizing the summary influence of nonspecific interactions of the solvent and solute. The linear dependence is also found on the Kirkwood constant Y (KoppelPalm parameters35), characterizing a solvent polarity: νabs = −1791.2 × π * + 34 045 (r = 0.906)

(1a)

νabs = −5208.0 × Y + 35 050 (r = 0.880)

(1b)

(2a)

νabs = −6088.1 × Y + 3.83 × B + 35 145 (r = 0.747)

(2b)

(3a)

νfl = −3788.6 × Y + 26 330 (r = 0.760)

(3b)

The eqs (3) demonstrate that the increase of solvent polarity and basicity stabilizes N* in the relaxed excited state. Fluorescence of the 7HF neutral form has high values of the Stokes shifts (ΔνSt), from 7650 to 9790 cm−1, which denote the significant structural relaxation of N* in the excited state. Specific interaction with a solvent hinders the structural relaxation that follows from the equation: ΔνSt = − 2950 × β + 10 500 (r = 0.750)

(4)

The presence of basicity parameter β in correlation equations obtained for the solvents of groups II and III demonstrate the relationship between spectral properties of N and N* and the strength of a hydrogen bond between hydrogen atom of 7hydroxy group and nucleophile atoms of the solvent molecules (>X···H−O−). It is significant that fluorescence of N* is absent in acetonitrile, which forms a less strong hydrogen bond with 7hydroxy group than the solvents of II and III groups. This allows us to conclude that the observed emission is due to the hydrogenbonded neutral form − NH*. In the case of group III solvents, the lack of relationship between positions of absorption and emission bands and α or E parameters (responsible for electrophile interaction of the solvent with 7HF) was detected. It shows that the hydrogen bond between hydroxyl group of a solvent molecule and carbonyl group of 7HF (>CO···H−O−, the bond c on Figures 3bS, 8b), as well as hydrogen bond between hydroxyl group of a solvent molecule and 7-hydroxy group (H > O···H−O−, the bond b in Figure 3bS, 8b), do not influence significantly the spectral properties of NH*. In the polar solvents of the group II, DMSO and DMF, the second emission band of low intensity in the range of 550−560 nm was detected. This band corresponds to fluorescence of the anionic form A*. The three-dimensional (3D) fluorescence spectrum of 7HF solution in DMF (Figure 2) shows that the excited anion arises from two different protolytic forms in the ground state. The wavelength of emission peak a in Figure 2

Such dependence is not observed in the solvents of group II. The long-wavelength absorption band shifts with changing not only polarity but also basicity of a solvent. The shift of N band in solvents of the group II is higher than that in solvents of the group I and reaches Δνabs = 2225 cm−1. The regression analysis of νabs(N) in the solvents of joined I and II groups evidences multiple correlation of νabs with parameters of polarity and basicity. The dependencies obtained using the Kamlet−Taft and Koppel−Palm parameters are the following: νabs = −3030.0 × π * + 2456.6 × β + 34 259 (r = 0.806)

νfl = −1023.4 × π * − 1159.5 × β + 26 085 (r = 0.810)

These equations show a different influence of the solvent polarity and basicity on the spectral behavior of N. The increase of medium polarity results in the batochromic shift of the longwavelength band, whereas the increase of the medium basicity leads to the hypsochromic shift. No correlation between νabs and solvent parameters was found in the case of alcohols (group III), because their Kamlet−Taft or Koppel−Palm parameters are similar. The long-wavelength band of N is always in the range of 306−308 nm in alcohols and at 319 nm in aqueous methanol. In solvents of the group I, 7HF has no fluorescence, but it phosphoresces at low temperatures.9 The spectral behavior of 7HF at 77 K is described in the next section. In solvents of the groups II and III, 7HF has a very weak fluorescence in the short-wavelength range, which is between 390 and 410 nm (Δνfl = 4595 cm−1). This band was attributed by

Figure 2. Three-dimensional fluorescence spectrum of 7-HF in N,Ndimethylformamide (projection on λexc:λem plane). 3071



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To estimate the relationship between the spectral properties of A* and solvent nature, we added DBU in 7HF solutions with a ratio of DBU/7HF as approximately 1.5:1 mol/mol. In the nonpolar solvents of the group I, where anion and cation are weakly solvated, DBU and 7HF could form an intimate ionic pair DBU-H+·7F−. In this case, we detected an absorption band of A with maximum in the range of 360−380 nm (26 455−27 700 cm−1) and emission band of A*with a maximum in the range of 530−555 nm (18 950−18 000 cm−1). The Stokes shift of fluorescence was between 7615 and 9140 cm−1 increasing with growth of medium polarity:

corresponds to the maximum of absorption/excitation band of the neutral form (∼310 nm), which provides evidence for its dissociation in the excited state. Peak b corresponds to the maximum of absorption/excitation band of the anionic form (∼390 nm), which indicates partial dissociation of 7HF in DMF and the presence of free anions in the ground state. Thus, the detected emission of the form A* is due to both the photodissociation of the neutral form resulting in photoanion (Ap*) formation NH → NH* → Ap* and the direct excitation of the anion existing in the ground state NH ⇆ A → A*. Addition of a small amount of DBU to the 7HF solutions in DMF and DMSO leads to an increase of A* emission intensity (when exciting the anionic form in the ground state at 350 nm) and to a decrease of NH* and Ap* intensity (when exciting the neutral form in the ground state at 300 nm). This phenomenon is due to shift of the ground state equilibrium NH ⇆ A toward the anion formation. The same spectral behavior of 7HF was detected also in all solvents of the group III. When exciting the neutral form NH, we have obtained a fluorescence spectrum with two emission bands of NH* and Ap*. The excitation of the anion A gives a fluorescence spectrum with only the A* emission band. Position of the photoanion emission band in solvents of the group III and in DMF and DMSO linearly depends on the solvent polarity: νfl(A p*) = − 1231.3 × π * + 19 090 (r = 0.883) νfl(A p*) = − 16 159 × Y + 25 845 (r = 0.947)

(5a) (5b)

(6a)

(7a)

Δνfl(A*) = −13 660 × Y − 3.80 × B + 25 850 (r = 0.937)

(7b)

The Stokes shift of anion A* in alcohol also linearly depend on π* and Y:

ΔνSt(A*) = 5795 × Y + 5680 (r = 0.718)

(8b)

ΔνSt(A*) = 378 × π * + 9860 (r = 0.700)

(10a)

ΔνSt(A*) = 1050 × Y + 9670 (r = 0.914)

(10b)

(11)

The intercept values in eqs 9a, 10a, and 11 (7955, 9860, and 7315 cm−1) indicate structural relaxation energies of the free or ion-pair bonded anion. Comparison of these intercepts shows that the minimal structural relaxation is typical for free solvated anions (i.e., in polar media) where the 7HF dissociation is possible. The maximal structural relaxation was found for anions in the loose ion pairs, that is probably due to participation in the relaxation processes of the one solvent molecule bound with DBU ion. The inverse relationship was observed for sensitivity of the anions to medium polarity, which is characterized by the parameter π*. The minimal value of this parameter is typical for the loose ion-pairs, the maximal value is in the case of free anions. Spectral Properties of 7HF at Low Temperatures. The low temperature experiments were carried out in polystyrene films and in the frozen 7HF solutions in hexane, dichloromethane, tetrahydrofuran, and methanol at the temperature of boiling nitrogen (77 K). The investigations of 7HF spectral properties in the polystyrene films showed absence of any fluorescence at both temperatures 298 K and 77 K. The same spectral behavior was observed in the frozen solvents of the group I in dichloromethane and hexane. The excitation at λex ≈ 300 nm (absorption maximum of NH) allowed us to detect a weak NH* emission in tetrahydrofuran (the group II) and two emission bands, NH* and AP* in methanol (the group III). The emission of A*, when

Surprisingly, in alcohols the positions of photoanion Ap* emission bands differ from positions of bands A* obtained by direct ground-state anion excitation. In the last case, the emission band is shifted to the red spectral region by 80−440 cm−1 (2−13 nm). The position of emission band A* in the neutral alcohol solutions and in the presence of DBU also depends on solvent polarity:

(8a)

(9b)

ΔνSt(A*) = 2237 × π * + 7315 (r = 0.890)

(6b)

ΔνSt(A*) = 1473.4 × π * + 7635 (r = 0.917)

ΔνSt(A*) = 4683 × Y + 7025 (r = 0.900)

In the solvents of group III , as well as in polar acetonitrile, DMF, and DMSO, in which cation and anion are solvated and complete dissociation of the loose ion pair DBU-H+∥7F− is probable, the free solvated anions absorb and emit at λmax/νmax, 367−394 nm (25 380−27 260 cm−1) and 525−583 nm (17 160−19 000 cm−1), correspondingly. The Stokes shifts are in the range of 7460−8780 cm−1 and linearly depend on π* parameter:

ΔνSt(A p*) = 13 035 × Y + 6.58 × B + 6565 (r = 0.949)

νfl(A*) = − 1696.7 × π * + 19 560 (r = 0.954)

(9a)

In the basic solvents of the group II, DBU cation could be solvated, DBU-H+·Solv, that in our opinion, has to result in the formation of the loose ion pair, DBU-H+∥7F−. In nonpolar basic solvents, the absorption and emission maxima are at 350−370 nm (27 195−28 410 cm−1) and 545−580 nm (17 220−18 420 cm−1), correspondingly. The Stokes shifts of fluorescence are substantially higher than in solvents of the group I − 9940− 10170 cm−1. In addition, ΔνSt(A*) are also sensitive to medium polarity:

Anomalously large Stokes shifts for the Ap* emission band (13800−14550 cm−1) indicate that this protolytic form is a result of the photodissociation process. The value of ΔνSt(Ap*) linearly depends on solvent polarity and basicity (when the Koppel− Palm solvent parameters were used): ΔνSt(A p*) = 2107.7 × π * + 13 047 (r = 0.943)

ΔνSt(A*) = 1461 × π * + 7955 (r = 0.901)

Comparison of eqs 4 and 6a, 6b shows that in spite of some difference in νfl of A* and AP*, the dependences of νfl(Ap*) and νfl(A*) are similar. The authors tend to explain the differences between the spectral characteristics of Ap* and A* in alcohols by the small differences in solvate sphere depending on the way of the anion formation. 3072



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Table 2. Spectral Properties of 7-HF at Low Temperature fluorescence neutral forms N*, NH* medium hexane dichloromethane tetrahydrofuran methanol polystyrene

νfl77,

−1

cm nm)

(λfl77,

26 315 (380) 26 040 (384)

νfl77−νfl298, −1 cm

1035 1315

photoanion, AP* ΔνSt77, −1 cm

−1

νfl , cm (λfl , nm)

8405 6530

77

77

νfl77−νfl298, −1 cm

21 100 (474)

3020

phosphorescence ΔνSt77, −1 cm

11 470

νph, cm−1 (λph, nm)

ΔνSt,ph, cm−1

22 570 (443) 21 880 (457) 23 310 (429) 20 660 (484) 20 450 (489)

11 795 10 590 11 410 11 910 12 020

νfl77, λfl77, νph, λph − positions of the fluorescence and phosphorescence emission bands at 77 K, νfl77−νfl298 − shifts of the fluorescence emission bands under sample heating from 77 K up to 298 K, ΔνSt77 − the Stokes shift of fluorescence, ΔνSt,ph − the Stokes shift of phosphorescence calculated relative to a position of absorption band.

Table 3. Spectral Parameters of 7-HF Protolytic Forms in the Ground and Excited States in Methanol−Water (4:1 v/v) Medium pH/H0 range form

νabs, cm−1 (λabs, nm)


νex, cm−1 (λex, nm)

ΔνSt, cm−1

τ, ns

S0

C, C* NH, NH* A, A* AP* T*

27 250 (367) 32 360 (309) 27 680 (361)

21 550 (464) 24 600 (407) 18 360 (545)

27 330 (366) 32 450 (308) 27 700 (361) 32 450 (308) 32 260 (310)b 27 030 (370)c

5700 7760 9320 14 000 14 710 9480

1.11 ± 0.03 2.0 ± 0.3 0.35 ± 0.03

7

17550 ± 310a (570 ± 10)a

0.14 ± 0.05

S1 −1.15 −4.5 to 1.3

τ − form’s lifetime in the excited state; νabs, λabs − positions of the long-wavelength absorption band maxima; νfl, λfl − positions of the emission band maxima; νex, λex − positions of the fluorescence excitation band maxima detected at the form’s emission maximum; ΔνSt − the Stokes shifts of fluorescence. Last column shows acidity diapason, where every protolytic form is registered. athe emission maximum of T* form shifts batochromically with acidity growth. bat H0 = 0 (excitation of the neutral form NH) cat H0 = −2.6 (excitation of the cation C)

Figure 3. Changes in 7-HF absorption spectra in methanol−water solutions: (a) in pH range from 6.5 to 11.0, (b) in H0 range from −2.15 to 1.04. Arrows show changing absorption bands when increasing pH (a) and decreasing H0 (b). Titration curves in pH (c) and H0 (d) ranges at 375 nm.

exciting at λex ≈ 360 nm (absorption maximum of A), was not detected.

Spectral parameters of the emission bands are listed in Table 2. The data show that the absence of solvent relaxation in the solid 3073



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solvent results in hypsochromic shifts of the both formsNH* and AP* . It is worthwhile to note that phosphorescence of the neutral forms N* and NH* (Figure 1c) takes place at 77 K in all the solutions, independent of medium properties. The phosphorescence bands have pronounced vibronic structure; the focal points of these bands are in the range of 430−490 nm. The growth of solvent polarity leads to the bathochromic shift of phosphorescence band, except for basic tetrahydrofuran, where a blue shift of the band was detected. Acid−Base Properties of 7HF in Alcohol−Water Solutions. As was noted above, the physicochemical parameters of equilibria between 7HF protolytic forms as well as their spectral parameters were obtained by acid−base titration. Spectral parameters of all forms are listed in Table 3. Changes of the spectral curves in pH range from 6.5 to 11.0 are shown in Figure 3a. When increasing the pH value, disappearance of the NH band and growth of the anion band intensity are detected. In the pH range from 6.5 to 1.5, only the absorption band of NH form is detected. In the H0 region (Figure 3b), the intensity of the NH band decreases, and a new absorption band of the cation C appears. The NH and C bands do not cross at isosbestic point due to changing the concentrations of sulfuric acid in the solvent mixtures of different H0. In this case, calculations of equilibrium constants were made according to (36). Corresponding titration curves at 375 nm are depicted in Figure 3c,d. Analysis of the absorption spectra shows that, in the ground state, 7HF exists in three protolytic forms. The equilibrium constants for these forms are presented in Table 4. The obtained

correspond to the absorption spectra of the neutral 7HF. According to this, the detected bands can be assigned to emission of NH* and of photoanion AP*, which is formed according to scheme NH → NH* → Ap*. The further acidity growth results in decrease of the NH* and Ap* bands’ intensity and in the appearance of two new bands: a long-wavelength band with maximum at 590 nm (at H0 < 1.3) and a short-wavelength band with maximum at 480 nm (at H0 < 0.4). When H0 < −4.5, fluorescence spectra contain only one short-wavelength emission band, and the corresponding fluorescence excitation spectra is similar to the cation absorption spectrum. Therefore, the fluorescence with λmax ≈ 480 nm detected in the solutions of high acidity can be assigned to the cation form emission − C* (Figure 5b). The dependence of fluorescence intensity for each emission band on pH/H0 values are depicted in Figure 4c. The figure shows that, in contrast to the ground state, the excited 7HF has not three but four protolytic forms. The Förster’s estimation of acid−base properties of 7HF protolytic forms in the excited state indicated the inversion of values of carbonyl and 7-hydroxy groups (Table 4). In this case, the proton transfer and phototautomer formation in the excited state are possible. According to the estimated pKa for the excited state, the maximal acidity range, where phototautomer could be formed, is from H0 ≈ −3 to pH ≈ 8 (i.e., between acidity ranges of the 7HF cation and photoanion). The emission band at 590 nm is observed in the range H0 from −4.5 to 1.3. This range overlaps with those of the cation and anion forms. That is why we assigned this band to that of the phototautomer, T*. This supposition corresponds to data of (6), where authors also observed the appearance of a new emission band in the solutions of high acidity. Two mechanisms of the phototautomer formation can be proposed: by the photoanion protonation AP* → T* and by dissociation of the cationic form C* → T*. In the first case, the fluorescence excitation spectra at λfl = 590 nm have to be similar to the excitation spectrum of the photoanion and correspond to the absorption spectrum of the neutral form. In the second case, the fluorescence excitation spectrum must correspond to the absorption spectrum of the cationic form. There is a possibility of the phototautomer formation by both these ways depending on pH/H0 values. To determine the mechanism of T* formation, 3D steady-state and time-resolved fluorescence spectra were used. A projection of 3D fluorescence spectra of 7HF at H0 = −1.95 is depicted in Figure 6. The H0 value used is in the acidity range, where only C* and T* forms are present. The figure shows that the cation and phototautomer have similar excitation spectra that evidence the tautomer formation according to C* → T* mechanism. Figure 7a shows the 7HF time-resolved fluorescence spectra at H0 = −2.6 with different delay after the excitation pulse. A spectrum obtained with 40 ps delay corresponds to that of the form C*, however, when increasing the delay the formation of T* is detected. At H0 = −0.7, the fluorescence spectrum contains both the bands of T* and AP*; however, it is impossible to record the 3D spectrum of high quality because of the low intensity of these bands. The time-resolved spectra at H0 = −0.7 are depicted in Figure 7b. The first fluorescence spectrum recorded with 40 ps delay contains only photoanion emission; however, the next spectra obtained with higher delays demonstrate also the emission band of the phototautomer.

Table 4. 7-HF Dissociation Constants in the Ground and Excited States and Phototautomerization Rate Constantsa C ⇆ NH NH ⇆ A

pKa

pKa*

−1.01 ± 0.04 (−0.79)18 8.91 ± 0.02 (8.122, 7.396)

6.6 (6.3)6 −1.5 (−2.3)6

kT × 10−9, s−1 C* → T* AP* → T*

12.3 ± 0.2 (H0 = −2.2) 3.1 ± 0.3 (H0 = −1.2)

pKa and pKa* − AH ⇆ A− + H+ equilibrium constants in the ground and excited state, correspondingly, kT − the rate constant of tautomer formation in the excited state, measured at H0 indicated in parentheses.

a

values correlate with the ones obtained in other alcohol−water media.2,6,18 The dependence of molar fractions of protolytic forms on pH/H0 values is depicted in Figure 4a. The 7HF fluorescence spectra in basic solutions have the one emission band with the maximum at 545 nm (Figure 5a). The fluorescence excitation spectrum recorded at this wavelength corresponds to the absorption spectrum of the anionic form. Therefore, the observed emission can be assigned to the anion A*, which is formed by excitation of the ground-state anion: A → A*. When pH is lowering to ∼6.0, the fluorescence intensity decreases by almost two times. The intensity of A* emission correlates with concentration of the form A in the ground state. In solutions with pH from ∼6.0 to 2.0, the intensity of the anion emission remains approximately the same. Moreover, at pH < 8, a very weak emission band of the form NH* (∼420 nm) appears. Its intensity is comparable with the one of the Raman scattering band at ∼380 nm (Figure 5a). The excitation spectra at both fluorescence maxima 420 and 545 nm in pH range from 6.0 to 2.0 3074



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Figure 4. pH/H0 ranges of 7-HF protolytic forms in the ground (a) and excited (b, c) states. (a) Molar fractions of C, NH, and A forms in the ground state calculated from experimental pKa values. (b) Molar fractions of C* and A* forms in the excited state calculated from pKa* values estimated by the Forster method. (c) Intensities of emission band maxima of C*, T*, NH*, and A* (AP*) forms.

Figure 5. Changes in 7-HF fluorescence spectra in methanol−water solutions: (a) in pH range from 11.4 to 2.2, (b) in H0 range from 1.3 to −4.8. Arrows show changing emission bands when decreasing pH (a) and H0 (b). The emission bands of T* form and AP* form obtained by deconvolution of the fluorescence spectrum at H0 = 1.3 are depicted by dashed lines.

Thus, we can conclude that two mechanisms of the phototautomer formation are possible, and the resultant mechanism depends on medium acidity. Rates of the corresponding tautomerization reactions are listed in Table 4. Theoretical Predictions of 7HF Acid−Base Properties. Estimations of proton donating and accepting ability, geo-

metrical and thermodynamic parameters of 7HF in the ground and excited state in nonaqueous media were carried out by quantum-chemical calculations. The energies of 7-HF and its complexes were calculated in vacuum, diethyl ether, and in methanol, which corresponds to the solvents of groups I, II, and III. The optimized structures of 7HF-solvent complexes in the 3075



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negative charge on the carbonyl oxygen atom by 0.059 e̅ and hence to an increase in basicity in the excited state. The analysis of the Mulliken charges mentioned above allows us to conclude that the hydrogen bond formation additionally polarizes the hydroxyl and carbonyl groups, which explains the substantial growth of their acidity and basicity in the excited state. A bond between the 7-hydroxyl group and the protonaccepting oxygen atom of diethyl ether in the 7HF-ether complex (bond a in Table 5) shortens insignificantly upon excitation, and changes in the Gibbs energy of formation are also small (less than 0.8 kJ/mol). The 7HF−methanol complex is stronger: the hydrogen bond a is shorter than in the ether complex, and its ΔGf,298 is higher by ∼10 kJ/mol. In the excited state, the length of the hydrogen bond a decreases, whereas the length of the O−H bond in the hydroxy group increases, corresponding to geometry of a complex for the first step of the excited-state proton transfer. The hydrogen bond c between methanol molecule and 7HF carbonyl group is longer and less stable than the bond a. The length of the former one slightly decreases upon excitation. The hydrogen bond b is the weakest and the longest among the other mentioned hydrogen bonds. Moreover, its length increases in the excited state. Probably, this explains the lack of solvatochromism and solvatofluorochromism when changing the medium electrophilicity. It would seem that a decrease in the hydrogen bond length upon excitation would be accompanied by an increase of negative values of ΔGf,298. However, the data obtained by the DFT method demonstrate a weak opposite tendency. The greatest decrease of ΔGf,298 is in the case of the hydrogen bond a. Probably, the correspondence between geometrical and thermodynamic values could be more adequate if one took into account changing length and ΔGf,298 not only of hydrogen bonds but also of O−H and CO bonds in the hydroxyl and carbonyl groups. The decrease of ΔGf,298 could be also due to errors of the method of ΔGf,298 calculation, described in detail in Supporting materials.

Figure 6. Three-dimensional fluorescence spectrum of 7-HF at H0 = −1.95 (projection on λexc:λem plane).

ground state are shown in Figure 8, and the excited state geometry of the complexes is presented in Supporting Information (Figure 2S). Structures of the model complexes used in ΔGf,298 calculation are depicted in Figure 3S (Supporting Information). Changes of acidity and basicity of 7-HF upon excitation can be estimated by analysis of the Mulliken charges on oxygen atoms of 7-hydroxyl and carbonyl groups.37 The data in Table 5 show the increase of negative charge on the hydroxyl oxygen atom under solvation and increase of the medium polarity. This implies that the acidity of the 7-hydroxy group must decrease with going from vacuum to methanol solution. Negative charge on the oxygen atom decreases upon excitation indicating the growth of hydroxyl proton mobility and, thus, the increase of 7HF acidity in the excited state. The negative charge decrease is minimal for 7HF in vacuum (0.005 e), ̅ but it is higher in the case of 7HF−diethyl ether complex (0.013 e)̅ and maximal for 7HF−methanol complex (0.082 e). ̅ Hence, the maximal acidity growth is theoretically predicted for methanol medium, in which photodissociation of the 7-hydroxyl group is observed. The data listed in Table 5 show that negative charge on the carbonyl oxygen also increases from vacuum to methanol solutions, indicating the growth of 7HF basicity. It can be seen that the basic properties of the carbonyl group decrease upon excitation, except for the methanol complex, in which the carbonyl fragment is bound to the solvent molecule. In this case, the formation of a hydrogen bond results in an increase of

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DISCUSSION The 7HF molecule contains acidic and basic fragments. In the ground state, 7HF demonstrates weak proton-donating and proton-accepting properties. As mentioned above, the excitation of 7HF results in a dramatic growth of basicity and acidity. In the result, the behavior of excited 7HF protolytic forms differs substantially from the behavior of these forms in the ground

Figure 7. Fluorescence time-resolved spectra of 7-HF at (a) H0 = −2.6 (C* → T* transformation) and (b) H0 = −0.7 (AP* → T* transformation). 3076



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Figure 8. Structures of NH forms used for quantum-chemical calculations. (a) H-bonded complex 7-HF with diethyl ether, (b) H-bonded complex 7-HF with methanol (R = CH3).

Table 5. Theoretically Predicted Atomic Charges, Geometrical and Thermodynamic Parameters of 7HFa charges on oxygen atoms, e̅ structures calculated N in vacuum complex N and diethyl ether (Figure 8a) in diethyl ether medium complex N and methanol (Figure 8b) in methanol medium a

lengths of hydrogen bonds, Å

state

7-hydroxy group

carbonyl group

length of 7-hydroxy group, Å

a

S0 S1 S0 S1 S0 S1

−0.168 −0.163 −0.216 −0.203 −0.245 −0.163

−0.235 −0.132 −0.282 −0.170 −0.333 −0.392

0.969 0.969 0.993 0.994 1.000 1.033

1.689 1.685 1.642 1.507

b

1.950 2.168

Gibbs energy of hydrogen bond formation (ΔGf,298), kJ/mol

c

a

b

c

1.769 1.711

−39.04 −38.28 −49.41 −42.76

−25.69 −23.77

−38.03 −36.36

Hydrogen bonds a, b, and c are depicted in Figures 8 and 3S.

formation of hydrogen bond between the 7-hydroxyl group and solvent molecules is not possible. Because the dipole moment of N increases upon excitation, the growth of solvent polarity leads to a bathochromic shift of the long-wavelength absorption band. As mentioned above, the deactivation mechanism of the excited neutral form N* is radiationless. In the aprotic basic media and in alcohols (the solvents of II and III groups), neutral form is present as a hydrogen-bonded complex with the solvent molecules − NH. The formation of such complexes is confirmed by the dependence of the NH absorption band in the solvents of group II on parameters of solvent basicity − B and β. Evidence for the high stability of the complexes was obtained from the results of quantum-chemical calculations. Analysis of theoretical ΔGf showed that hydrogen bonds between 7HF and the solvent molecules are more stable in the ground state than in the excited one. This explains a hypsochromic shift of the long-wavelength absorption band of NH when increasing solvent basicity. The form NH has a very weak fluorescence depending on polarity and basicity of solvents. At low temperatures N and NH demonstrate also phosphorescence bands of low intensity with pronounced vibrational structure. The absence of fluorescence and the presence of phosphorescence of N* indicates that the lowest excited state of this form is of nπ* type. Therefore, spectral properties of neutral 7HF are similar to those of flavone and isoflavone.40−42 The rise of fluorescence in the case of NH* complexes shows that formation of the hydrogen bonds with the solvent molecules leads to appearance of a channel of fluorescent deactivation that is possible when ππ* excited state becomes energetically close to the nπ* type. A weak fluorescence of NH* and the presence of phosphorescence in the solvents of groups II and III evidence that the energy gap between the lowest excited states ππ* and

state. The ground-state and excited forms exist at different pH ranges, and the quantity of these forms is also different. In this section, we systematize the information about protolytic forms described earlier. Investigations of the spectral behavior of 7HF in nonaqueous media at 298 K and 77 K, as well as the detailed analysis of changing spectral properties of 7HF solutions in the wide pH/H0 range, showed the presence of three protolytic forms in the ground state: neutral form − N (or NH), cation − C, and anion − A. In the excited state, the emission of four forms was detected. NH* and C* can be obtained by excitation of corresponding NH and C ground-state forms. The phototautomer T* forms in the excited state from other protolytic forms. The anionic form can appear by two ways: by direct excitation of ground-state anion − A* or by photodissociation of neutral form − AP*. Probable structures of these forms are depicted in Scheme 1. The appropriate models for investigations of the spectral behavior of 7HF are 6-hydroxyquinoline (6HQ)38 and βnaphtol.39 The distance between the acidic and basic centers of 6HQ is similar to that of 7HF. Moreover, 6HQ has the same protolytic forms in the ground and excited states and can be useful for analysis of cation−phototautomer transformations. However, the basicity of 7HF is much lower than that of 6HQ, and in the result, 7HF demonstrates amphoteric properties in much shorter range of medium acidity. The best model for the simulation of 7HF acid−base properties in the pH range from ∼2.0 to ∼11.0 is considered to be β-naphtol, which allows the analysis of peculiarities of the 7-hydroxyl group dissociation and photodissociation. Neutral Form. The investigations of absorption and fluorescence spectra of 7HF in different media showed that, depending on the environment properties, two different neutral forms can exist in the ground and excited states. The first form N exists in the aprotic solvents of low basicity, in which the 3077



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relaxation of AP* in methanol is substantial and reaches 3020 cm−1. The Stokes shift of A* which appears from the ground-state anion in alkaline water−methanol solution at 298 K is also very high at 9040 cm−1. When freezing this solution, the anion emission band undergoes a hypsochromic shift of 3960 cm−1, and the Stokes shift values reduces to 5280 cm−1. This shows that the contribution of solvent relaxation in spectral parameters of the anion A* obtained by excitation of the ground-state anion is substantially higher than in the case of the photoanion AP* formation. According to (39), photodissociation is a two-step process, and dependent on its dynamics, the fluorescence decay can demonstrate mono- or polyexponential character. In the methanol−water medium, we found monoexponential decay of the photoanion fluorescence. The obtained lifetime of AP* (and of A* in alkaline solutions) is 350 ps. The lifetime of an intermediate second form, if it exists, is probably less than 100 ps (below the detection limits of the equipment used). Taking into account the known mechanisms of photoacid dissociation the emission of 7HF in methanol solution attributed by the authors of (7) to two phototautomers or to a mixture of the photoanion and phototautomer8, seems to us to be rather the photoanion emission. Phototautomer. Formation of the phototautomer T* is possible due to the excited-state inversion of pKa* values for the cation and anion. The fluorescent titration allowed us to find the phototautomer emission in more acidic range of H0 from 1.3 to −4.5 (Figure 4c). The mismatch between the results of the fluorescence titration and the data estimated by the Förster method (see the section entitled Acid−Base Properties of 7HF in Alcohol−Water Solutions) takes place because the latter method predicts pKa* values supposing the presence of an equilibrium between protolytic forms in the excited state, which is hardly possible in a real system. The data mentioned above corroborate the conclusion of (refs 4 and 6) that the phototautomer appears in acidic media and do not correspond to results of the authors (refs 7 and 8), which detect phototautomer in neutral methanolic solutions. The maximum of the phototautomer emission band is at ∼570 nm. The data listed in Table 3 show that T* is very sensitive to solvent properties: the decrease of H0 results in bathochromic shift of the emission band. The form T* is short-living, and its lifetime is estimated at approximately 140 ps. We have proposed above two mechanisms of the phototautomer formation: dissociation of 7-hydroxy group of the cation C* → T* and photoanion protonation AP* → T*. The second mechanism, in fact, involves the double proton transfer NH* → AP* → T*. The experimental data presented in the previous part show that both mechanisms of phototautomer formation are realized. The pathway of phototautomer formation is switched by pH/H0 value of the solution. It should be noted, however, that the rate of phototautomerization by the “cationic” pathway is four times higher than that of the “anionic” type. On the basis of the obtained data, we can propose a general scheme of interplay of 7HF protolytic forms depicted in Scheme 2.

nπ* is very small, and they demonstrate a strong coupling even at 77 K. Table 1 shows abnormally high Stokes shifts of NH* fluorescence. Suppression of solvate relaxation by freezing of 7HF solutions and their cooling up to 77 K results in hypsochromic shift of the emission band. However, even in the case of the absence of solvate relaxation effects, the Stokes shift values are between 6500 and 8500 cm−1 that indicates a significant contribution of the structural relaxation processes. Cationic Form. 7HF is a weak base with pKa = −1.01. Negative charge on the oxygen atom of the carbonyl group substantially increases upon excitation, which results in the growth of carbonyl basicity in the excited state. The estimation of 7HF basicity in the S1 state made by the Förster method predicts increasing pKa* up to 6.0. However, the cation emission dramatically decays with decreasing the concentration of sulfuric acid, and at H0 = −4.5, the long-wavelength emission band of phototautomer appears. Thus, the differences between the Förster basicity and the results of fluorimetric titration are due to transformation of the cationic form to the phototautomer. Anionic Form. The pKa value for methanol−water (4:1 v/v) solution is 8.91, and this value correlates well with the constants obtained for other water−alcohol mixtures.2 As discussed above, the acid−base behavior of 7HF in neutral and alkaline solutions is similar to that for β-naphtol. It is known that the acidity of βnaphtol dramatically increases upon excitation, and the pKa* value of hydroxyl group reaches 2.8.39 The presence of an electron-withdrawing substituent in the naphthalene moiety results in additional decrease of pKa* up to values between 0.2 and −4.5, which is typical for “super” photoacids. 7HF also has the electron-withdrawing carbonyl fragment, and the pKa* of the 7HF hydroxyl group reaches a lower value than that of β-naphtol, −1.5 (Figure 4b, Table 4). Because of that, 7HF can also be assigned to the “super” photoacids. The fluorescent titration of 7HF showed the presence of anionic form in the wide pH/H0 range, up to H0 = −1.15, which proves high acidity of 7-hydroxyl group in the excited state. The obtained titration curve (Figure 4c) has two inflection points. The first one corresponds to the pKa of the 7-hydroxyl group in the ground state. At values pH ≥ pKa more than a half of A* arise from the excitation of the ground-state anion A. The second inflection point indicates the dissociation constant in the excited state − Ka*. In the range between pKa and pKa*, most of the anionic form (AP*) is generated by the photoacid (i.e., by the excited neutral form). In water−alcohol solutions (methanol, ethanol, DMSO, and DMF) A* and AP* have similar spectral parameters; therefore, in this case, the designations A* and AP* indicate only different ways of formation of the same anion. Because the pKa value of the 7-hydroxyl group is relatively low, some quantity of anion exists in the neutral methanol−water solutions, polar alcohols, DMSO, and DMF in the ground state. Therefore, the anion fluorescence appears upon the excitation of both the neutral form (AP*) and the ground-state anion (A*). Obviously, the ground-state dissociation of 7HF is absent at 77 K, because in frozen solution, only AP* fluorescence can be detected. The anionic form has the highest Stokes shift of fluorescence in comparison with other protolytic forms of 7HF. The Stokes shift for photoanion AP* (relative to the absorption band maximum of N) is 13 800−14 500 cm−1. In the frozen methanol, ΔνSt decreases to 11 470 cm−1, thus the contribution of solvate

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CONCLUSION In the introduction to this article, we have mentioned the attempts to use 7HF and its derivatives as fluorescent probes and markers in biochemical and biophysical investigations.10−12 To adequately interpret changing parameters of complex biological 3078


The Journal of Physical Chemistry A Scheme 2. General Scheme of Interplay between 7-HF Protolytic Forms in the Ground and Excited States

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REFERENCES

(1) Valant-Vetschera, K. M.; Wollenweber, E. In Flavonoids: Chemistry, Biochemistry, and Applications; Andersen, Ø.M., Markham, K. R., Eds.; Taylor & Francis Group: Boca Raton, FL, 2006; pp 617−748. (2) Tyukavkina, N. A.; Pogodaeva, N. N. Ultraviolet Absorption of Flavonoids II. Ionization Constants of 7- and 4′-Hydroxy Derivatives of Flavone and Flavonol. Chem. Nat. Compd. 1971, 7, 8−11. (3) Tyukavkina, N. A.; Pogodaeva, N. N. Ultraviolet Absorption of Flavonoids VIII. Ionization Constants of Kaempferol and Quercetin. Chem. Nat. Compd. 1975, 11, 741−743. (4) Wolfbeis, O. S.; Leiner, M.; Hochmuth, P.; Geiger, H. Absorption and Fluorescence Spectra, pKa Values, and Fluorescence Lifetimes of Monohydroxyflavones and Monomethoxyflavones. Ber. Bunsenges. Phys. Chem. 1984, 88, 759−767. (5) Arnett, E. M. Quantitative Comparisons of Weak Organic Bases. In Prog. Phys. Org. Chem., Vol. 1; Cohen, S. G.; Streitwieser, A. Jr.; Taft, R. W., Eds.; John Wiley & Sons: New York, 1963; pp 223−403. (6) Schipfer, R.; Wolfbeis, O. S.; Knierzinger, A. pH-Dependent Fluorescence Spectroscopy. Part 12. Flavone, 7-Hydroxyflavone, and 7Methoxyflavone. J. Chem. Soc., Perkin Trans. 2 1981, 1443−1448. (7) Itoh, M.; Adachi, T. Transient Absorption and Two-Step Laser Excitation Fluorescence Studies of the Excited-State Proton Transfer and Relaxation in the Methanol Solution of 7-Hydroxyflavone. J. Am. Chem. Soc. 1984, 106, 4320−4324. (8) Mukaihata, H.; Nakagawa, T.; Kohtani, Sh.; Itoh, M. Picosecond and Two-Step LIF Studies of the Excited-State Proton Transfer in 3Hydroxyxanthone and 7-Hydroxyflavone Methanol Solutions: Reinvestigation of Tautomer and Anion Formations. J. Am. Chem. Soc. 1994, 116, 10612−10618. (9) Sarkar, M.; Sengupta, P. K. Luminescence Behaviour of 7Hydroxyflavone: Temperature-Dependent Effects. J. Photochem. Photobiol., A 1989, 48, 175−183. (10) Sarkar, M.; Ray, J. G.; Sengupta, P. K. Luminescence Behaviour of 7-Hydroxyflavone in Aerosol OT Reverse Micelles: Excited-State Proton Transfer and Red-Edge Excitation Effects. J. Photochem. Photobiol., A 1996, 95, 157−160. (11) Banerjee, A.; Basu, K.; Sengupta, P. K. Interaction of 7Hydroxyflavone with Human Serum Albumin: A Spectroscopic Study. J. Photochem. Photobiol., B 2008, 90, 33−40. (12) Chaudhuri, S.; Pahari, B.; Sengupta, P. K. Ground and Excited State Proton Transfer and Antioxidant Activity of 7-Hydroxyflavone in Model Membranes: Absorption and Fluorescence Spectroscopic Studies. Biophys. Chem. 2009, 139, 29−36. (13) Montaña, P.; Pappano, N.; Debattista, N.; Á vila, V.; Posadaz, A.; Bertolotti, S. G.; García, N. A. The Activity of 3- and 7-Hydroxyflavones as Scavengers of Superoxide Radical Anion Generated from PhotoExcited Riboflavin. Can. J. Chem. 2003, 81, 909−914. (14) Chi, Y. S.; Dao, T. Th.; Kim, H. P.; Kim, J.; Park, H.; Kim, S. Synthesis and Inhibitory Activity against COX-2 Catalyzed Prostaglandine Production of Chrysin Derivatives. Bioorg. Med. Chem. Lett. 2004, 14, 1165−1167. (15) Weissberger, A.; Proskauer, E. S.; Riddick, J. A.; Toops, E. E. Organic Solvents. Physical Properties and Methods of Purification. In Technique of Organic Chemistry, Vol. 7; Weissberger, A., Ed.; Interscience Publishers: New York, 1955. (16) Braslavsky, S. E. Glossary of Terms Used in Photochemistry, 3rd ed. (IUPAC Recommendations 2006); Pure Appl. Chem. 2007, 79, 293− 465. (17) Bates, R. G. Determination of pH. Theory and Practice; John Wiley & Sons: New York, 1964.

ASSOCIATED CONTENT

S Supporting Information *

H0 scale correction for the mixture methanol−water-sulfuric acid including a plot between H0 scales in methanol−water-sulfuric acid and water-sulfuric acid, as well as dissociation constants of flavones used as H0 indicators. A table containing statistical parameters of regression analysis equations for solvatochromic and solvatofluorochromic effects. Computationally predicted structures of 7HF−solvent complexes (with methanol and diethyl ether) in the ground and excited states. An information concerning calculations of Gibbs energies (ΔGf,298) of hydrogen bond formation: computationally predicted structures of model molecules in the ground and excited states, and schemes with algorithms of ΔGf,298 calculations. This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

The financial support of this work by Ukrainian State Grant 0112U007568 is gratefully acknowledged. The authors also gratefully acknowledge the Ukrainian-American Laboratory of Computational Chemistry (UALCC, Kharkiv, Ukraine).

systems using fluorescent probes, it is necessary to know what protolytic form is responsible for the detected spectral phenomenon and what physicochemical parameters of the environment influence on spectral properties of this form. Taking into account the obtained data, we can conclude that only emission of forms AP* and A* can be used in the steadystate fluorescent investigations of the biological and other complex objects. Thus, the influence of medium polarity on the ground-state equilibrium NH ⇆ A will be detected by changing emission intensity of the anion fluorescence excited at 310 nm (NH → NH* → AP*) and 360 nm (A → A*). Other protolytic forms of 7HF are out of the scope for the majority of investigations: NH* has a very weak fluorescence, and C* and T* appear in media of a very high acidity. In lifetime experiments, one could use not only AP* and A* (τ = 350 ps) but also long-living neutral form NH* with τ = 2 ns. Because photodissociation is a complex process, the use of lifetime equipment with the resolution beginning from 0.1 ps could allow the use of kinetic parameters of fluorescent deactivation of AP* in investigations of 7HF binding with biological and other substrates.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel./Fax: +38 057 707 51 30. Notes

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