chromen-4-ones (Flavonols) - American Chemical Society

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Tautomerism and Behavior of 3-Hydroxy-2-Phenyl-4H-Chromen-4Ones (Flavonols) and 3,7-Dihydroxy-2,8-Diphenyl-4H,6Hpyrano[3,2-g] Chromene-4,6-Diones (Diflavonols) in Basic Media: Spectroscopic and Computational Investigations Valery V. Moroz, Andrey G. Chalyi, Illia E. Serdiuk, Alexander D Roshal, Beata Zadykowicz, Vasyl G. Pivovarenko, Agnieszka Wróblewska, and Jerzy Blazejowski J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/jp403487w • Publication Date (Web): 29 Aug 2013 Downloaded from http://pubs.acs.org on September 1, 2013

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The Journal of Physical Chemistry

Tautomerism and Behavior of 3-Hydroxy-2-Phenyl-4H-Chromen-4-Ones (Flavonols) and 3,7-Dihydroxy-2,8-Diphenyl-4H,6H-pyrano[3,2-g] Chromene-4,6-Diones (Diflavonols) in Basic Media: Spectroscopic and Computational Investigations Valery V. Moroz,† Andrey G. Chalyi,† Illia E. Serdiuk,† Alexander D. Roshal,† Beata Zadykowicz,‡ Vasyl G. Pivovarenko,§ Agnieszka Wróblewska,‡ and Jerzy BłaŜejowski*,‡

†

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

Faculty of Chemistry, Kyiv Taras Shevchenko National University, Volodymyrska 64, 01601 Kyiv, Ukraine

RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) *

Corresponding author.

Phone: +48 58 523 51 11. Fax: +48 58 523 50 12. e-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: Absorption and emission spectroscopic investigations and computational predictions have shown that neutral molecules of flavonols and diflavonols can exist in the ground and excited states in one or two tautomeric forms stabilized by intramolecular (in aprotic media) or intermolecular (with solvent molecule(s), in protic media) hydrogen bonds. Electronic excitation creates conditions for the transformation of tautomeric forms, accompanied by proton transfer, reflected in fluorescence spectra. Proton transfer is also probable in monoanions of diflavonols in protic media. The OH groups involved in hydrogen bonds exhibit a protondonating ability characterized by the respective acidity constants. The electronically excited diflavonols are relatively strong acids if they lose one proton. With increasing basicity of the medium, anionic forms occur, which exhibit spectral characteristics and emission abilities different from those of neutral molecules. These features open up possibilities for the analytical use of these compounds as spectral probes sensitive to the properties of liquid phases – from neutral to strongly basic. The less-intensively studied diflavonols seem to be more promising than flavonols for these purposes, since they are more lipophilic, polarizable, polar and sensitive to basic features of the environment.

Keywords: Flavonols; Tautomeric phenomena; Influence of basic environment; Spectroscopy and theory; Prospects of analytical applications


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INTRODUCTION Flavonols (Chart 1) are a topic of interest owing to their unique sensitivity to properties of the environment,1–7 as well as their biological significance and activity.8–15 Owing to the close proximity of the OH and carbonyl groups, an asymmetrical intramolecular hydrogen bond occurs in these compounds16–18 within which proton transfer can take place.19–22 The occurrence of such a process has been proven in the excited state and the term Excited-State Intramolecular Proton Transfer (ESIPT), first introduced by Sengupta and Kasha,19 is now used to describe this phenomenon.7,21–23 As a result of such proton transfer, two tautomeric forms (N and T; Chart 1S, Supporting Information) are possible in flavonols.7,21,22 Electronic excitation of the N tautomer of these compounds, which are stable in the ground state, leads to two excited forms – N* and T*.7,21,22 Intramolecular proton transfer in the much less intensively studied diflavonols (Chart 1), which have two proton-donor-acceptor sites, makes three tautomeric forms (NN, NT and TT; Chart 1S, Supporting Information) possible here.24–26 Hitherto, it has been believed that diflavonols exist in the NN form in the ground state and in two forms, NN* and NT*, in the excited state.25–27 However, this picture is not true if protic molecules (water, alcohols) are present in the environment of flavonols or diflavonols.28,29 In such a case the formation of intramolecular hydrogen bonds is interrupted by intermolecular ones with protic molecules (selected canonical structures of complexes of flavonols and diflavonols with water/methanol reflecting such interactions are shown in Chart 2S, Supporting Information). With increasing pH, flavonols convert to the anionic form,30 whereas diflavonols convert first to the monoanionic (which can exist as two tautomers) and then to the dianionic form (Charts 1S and 2S, Supporting Information).26 The variety of forms in which the latter compounds potentially occur may make them more sensitive to the features of the environment and convenient spectral probes of a medium’s properties. These features of flavonols have been utilized in investigations of acid-


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base properties,1,31,32 complexation phenomena,3,33–37 the constitution of mixed liquid phases,38,39 the nature of micelles,40–42 as well as the properties of liposomes,43–45 proteins46,47 and DNA.48,49 Labels containing flavonol fluorophores have a number of potential bioanalytical applications.50– 53

Diflavonols are still awaiting on applications in various areas of natural sciences. When searching for diflavonol derivatives with potentially interesting spectral and acid-

base features, we turned our attention to 3,7-dihydroxy-2,8-di(4-methoxyphenyl)-4H,6Hpyrano[3,2-g]chromene-4,6-dione (2b) and 3,7-dihydroxy-2,8-di(4-N,N-dimethylaminophenyl)4H,6H-pyrano[3,2-g]chromene-4,6-dione (2c). These compounds were investigated together with the respective flavonol derivatives, 3-hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (1b) and 3-hydroxy-2-(4’-N,N-dimethylaminophenyl)-4H-chromen-4-one (1c) (and if necessary with the parent molecules, 3,7-dihydroxy-2,8-diphenyl-4H,6H-pyrano[3,2-g]chromene-4,6-dione (2a) and 3-hydroxy-2-phenyl-4H-chromen-4-one (1a)), to ascertain the extent to which the structural differences of diflavonols and flavonols affect their properties. The spectral features of the above diflavonols, exhibiting Cs symmetry26 and containing two potential ESIPT sites, may be expected to be more dependent on changes in the properties of the environment than those of the respective flavonols. On the other hand, the enhanced ability of 2b and 2c to convert to anionic forms in alkaline media turned out to be extended through the functionalization of diflavonol (2a) with –OCH3 and –N(CH3)2. This should reinforce the spectral response of 2b and 2c to pH and make diflavonols convenient spectral probes in investigations of acidic/basic features of liquid media. Moreover, diflavonols should be more lipophilic than flavonols, and thus more convenient probes in biological investigations. By investigating the properties of these compounds in media of different polarity, their hydrogen bonding ability and basicity, and by determining their spectral, structural and thermodynamic characteristics by combining experimental and computational methods, we intended to elucidate the differences and similarities between


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flavonols and diflavonols and point out their prospective applications, particularly of diflavonols.

MATERIALS AND METHODS Syntheses. Flavonols 1b and 1c, and diflavonols 2a – 2c were synthesized as described in refs 54 and 55. The compounds were purified by repeated crystallization from methanol (1b – yellow crystals, mp 233–234oC; 1c – red crystals, mp 191–192oC; 2b – yellow crystals, mp 308oC; 2c – red crystals, mp >330oC). The purity of the compounds was controlled by TLC. The compounds were identified by 1H NMR spectroscopy: 1b – 9.16 (1H, s), 8.13 (1H, dd, 8.0, 2.0), 7.61-7.74 (2H, m), 7.41 (1H, m), 8.19 (2H, d, 9.0), 7.13 (2H, d, 9.0), 3.81 (6H, s) in DMSO-d6; 1c – 9.13 (1H, s), 8.08 (1H, dd, 8.0, 2.0), 7.66-7.77 (2H, m), 7.43 (1H, m), 8.13 (2H, d, 9.0), 6.85 (2H, d, 9.0), 3.00 (6H, s) in DMSO-d6;44 2a – 9.77 (2H, s), 8.76 (1H, s), 8.21 (4H, d, 8.0), 8.02 (1H, s), 7.56 (4H, m) in DMSO-d6;26 2b – 9.74 (2H, s), 8.78 (1H, s), 8.21 (4H, d, 8.0), 8.09 (1H, s), 7.15 (4H, d, 8.0), 3.84 (6H, s) in DMSO-d6; 2c – 8.74 (2H, s), 8.28 (1H, s), 8.16 (4H, d, 8.0), 8.04 (1H, s), 6.85 (4H, d, 8.0), 3.04 (12H, s) in DMSO-d6. Commercially available flavonol (1a) was used in these investigations. The identification of diflavonol 2a was described earlier.26 Spectroscopic investigations. Spectral grade solvents (distilled and dried56 before use if necessary) were used throughout. Concentrations of the compounds ranged from 5×10–6 to 5×10–5 M in the absorption and fluorescence investigations, and were ca 5×10–5 M in the titrations. The optical path length in all the absorption and fluorescence experiments was 1 cm. The spectral characteristics of the particular forms of the compounds investigated were extracted from experimental absorption and fluorescence spectra using the Spectral Data Lab program.57 Fluorescence quantum yields were determined using fluorescein solution in carbonate buffer (pH = 9.93) as a standard.58 The vector differences of the dipole moments (∆µ) and angles between


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the dipole moment vectors (θs) in the ground and excited states were derived using the Bakhshiyev equation.59,60 Solid solutions in polymeric matrices were obtained by mixing dilute solutions (5×10–5 – 2×10–4 M) of the compounds in dichloromethane with solutions of polystyrene in dichloromethane. The solvent was allowed to evaporate completely for a week before measurements of the fluorescence anisotropy spectra. The fluorescence anisotropy (r0) and the angle between the dipole moment vectors in the ground and excited states (θm) were determined using the formula given in ref. 23. The concentration dissociation constants (Ka) of 2 in methanol were obtained according to the equation pKa = pKs – pKOH, where p denotes the negative base-10 logarithm, pKOH is the concentration basicity constant (representing the process: 2 (monoanion/dianion) + CH3OH → 2 (neutral/monoanion) + CH3O–), and Ks is the ionic product of the solvent (representing the process CH3OH → CH3O– + H+). The pKs for methanol (= 16.5) was taken from ref. 61 and 62. Values of pKOH were obtained by titration of 2 against potassium hydroxide. The dissociation constants of the hydroxy groups of the compounds in the excited state were estimated by Förster’s method on the basis of the positions of 0–0 transitions, as well as the long-wavelength maxima in the absorption spectra of the neutral and anionic forms.63,64 Calculations. Unconstrained geometry optimizations of isolated molecules (Chart 1S, Supporting Information) or molecular complexes (Chart 2S, Supporting Information) in the ground (S0) or excited singlet (S1) electronic states were carried out at the DFT or TDDFT levels of theory,65 respectively, using the B3LYP functional66,67 and cc-pVDZ basis sets68,69 implemented in the GAUSSIAN 09W program package.70 After completion of each optimization, the Hessian (second derivatives of the energy as a function of the nuclear coordinates) was calculated to assess whether stationary structures had been obtained.65 The


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harmonic vibrational frequencies were then derived from the numerical values of these second derivatives and used to obtain the enthalpy and Gibbs’ free energy contributions at 298.15 K and standard pressure (o) with the aid of a built-in computational program of statistical thermodynamics routines.71 The solvent effect was included in the DFT (TDDFT) calculations at the level of the Polarized Continuum Model (PCM) (UAHF radii were used to obtain the molecular cavity).72,73 The wavelengths of S0→S1 and S1→S0 transitions corresponding to optimized ground (S0) and excited (S1) singlet state geometries were predicted by the single point time-depend DFT or TDDFT calculations, respectively.74 Enthalpies of formation and differences of enthalpy or Gibbs’ free energy between relevant tautomeric forms were calculated by following the basic rules of thermodynamics.26,75 Calculations were carried out on the computers of the Tri-City Academic Network Computer Centre (TASK) in Gdańsk (Poland) and the Wroclaw Centre for Networking and Supercomputing (WCSS).

RESULTS AND DISCUSSION Electronic Absorption and Emission Spectroscopy. The long-wavelength electronic absorption of flavonols and diflavonols appears between 330 and 440 nm (Table 1). The positions of the absorption maxima depend only slightly and non-systematically on the properties of the organic solvents. Absorption maxima in diflavonols are red shifted on average 32 nm relative to the maxima in flavonols (the differences in the positions of the maxima are the greatest in the case of 1c and 2c). In the majority of cases, the fluorescence spectra of flavonols and diflavonols are a superposition of the emissions from two electronically excited states (Figure 1 and Table 1). Deconvolution of the fluorescence spectra provides information on the position of the emission bands and their corresponding fluorescence quantum yields (which were assumed to be


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proportional to the area under the deconvoluted curves).

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Long-wavelength fluorescence is

usually stronger than short-wavelength fluorescence; the latter was not detected in the case of 2a dissolved in chloroform. Fluorescence is the strongest if the compounds are dissolved in chloroform; it is the weakest when they are dissolved in methanol. Diflavonols generally fluoresce more efficiently than flavonols. Stokes shifts relevant to short-wavelength emission are on average 820 cm–1 lower for flavonols than for diflavonols. In the case of long-wavelength emission, Stokes shifts are on average 550 cm–1 higher for flavonols than for diflavonols. The Stokes shifts corresponding to two fluorescence bands are separated by roughly 4300 cm–1 in flavonols and by 2900 cm–1 in diflavonols. By plotting the Stokes shifts corresponding to the short-wavelength band against media polarity we obtain straight lines (Figure 1S, Supporting Information); according to the Bakhshiyev approach,59,60 these yield the vector difference of dipole moments in the ground and excited states. Values of this quantity are shown in Table 2. This table also lists the scalar differences of the dipole moments and the angles between dipole moment vectors in the ground and excited states. Analysis of the dipole moments in the ground and excited states demonstrates that electronic excitation enhances non-uniform charge distribution in neutral molecules of flavonols and diflavonols. This effect is the greatest for 1c and 2c, and the least for 1a and 2a, when comparing flavonols and diflavonols, and generally higher for the latter compounds. The angles between the dipole moment vectors in the ground and excited states are usually larger in diflavonols (for 1c and 2c they are comparable). The fluorescence of flavonols and diflavonols dispersed in polystyrene matrices is, as in solutions, the result of the superposition of the emissions from two excited states (Tables 3).26 This was demonstrated by the deconvolution of experimental fluorescence spectra, as well as by the recording of fluorescence anisotropy spectra. The pattern of these spectra indicates that each


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fluorescence band is attributable to only one fluorophore. The fluorescence anisotropies are usually higher in the case of flavonols, whereas the angles between the dipole moment vectors of the fluorophore and chromophore are higher for diflavonols. The Stokes shifts corresponding to the two fluorescence bands are separated by roughly 4400 cm–1 in flavonols and by 5500 cm–1 in diflavonols. This is a substantial difference in the values of this characteristic in comparison with those for diflavonols in solutions. Spectrophotometric titration of diflavonols with potassium hydroxide in methanolic solution leads to the appearance of new bands in the long-wavelength region of the absorption spectra with increasing alkali concentration (Figures 2 and 3, Figure 3 in ref. 26). These bands can be attributed to the monoanionic and dianionic forms of the compounds. Deconvolution of these complex spectra enables the positions of the absorption maxima to be determined (Tables 1 and 4). Spectrofluorometric titration of diflavonols with potassium hydroxide in methanolic solution initially decreases the intensity of emission but subsequently increases it with a shift of the maximum toward the short-wavelength region (Figures 2–4). Comparison of the spectrophotometric and spectrofluorometric curves shows that the only emitting forms are the neutral and dianionic ones, while the fluorescence of the monoanionic form is negligible; this was demonstrated in detail earlier in the case of 2a.26 Deconvolution of the spectrophotometric and spectrofluorometric titration curves enabled the positions of the absorption and fluorescence maxima of the neutral, monoanionic and dianionic forms to be calculated (Tables 1 and 4). The Stokes shifts calculated for dianionic forms are less than those of neutral diflavonols. Proton-Donating Ability. The equilibrium constants corresponding to the detachment of a proton from neutral diflavonols (Ka(1)) and their monoanionic forms (Ka(2)) in the ground and excited (*) electronic states are shown in Table 5. The values of Ka(1)* are much higher than


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those of Ka(1), which means that excited diflavonols in methanol are much stronger acids than neutral ones. This feature may explain the relatively low fluorescence quantum yields in methanol (Table 1) as a result of the partial dissociation of diflavonols to non-emitting radiation monoanions (Table 4). The values of Ka(2)* are somewhat lower than those of Ka(2), which indicates that monoanionic forms of diflavonols are stronger acids in the ground than in the excited state. However, the relatively low values of both Ka(2) and Ka(2)* imply that the ability of diflavonols to lose a second proton is low in both the ground and excited electronic states. If we compare the ability of diflavonols to lose a proton in the ground state, it is about one order of magnitude higher for neutral than for monoanionic forms. In the case of the excited state, detachment of the first proton from diflavonols is quite easy, whereas their ability to lose a second proton is about 10 orders of magnitude less (the lowest is for 2b, the highest for 2c). The implication of this analysis is that diflavonols exhibit only a slight ability to lose one or two protons in the ground state, whereas they are relatively susceptible to losing the first proton in the excited state. This feature can have an influence on intra- or intermolecular proton transfer in the excited state.24–26 The trends of the acidic features of flavonols and diflavonols, relevant to the detachment of one (the first one in the case of diflavonols) proton are similar (Table 5): compounds belonging to both groups are stronger acids in the excited than the ground state. In the ground state, flavonols exhibit stronger acidic properties than diflavonols; in the electronically excited state, there are no unequivocal relations between the acidities of compounds of both types. Only in the case of electronically excited diflavonols are the computationally predicted and experimental values of equilibrium constants relevant to the detachment of the first proton roughly comparable (Table 5). In the remaining cases predicted pKa are much higher than the experimental values. Note, however, that the trends in pKa changes are similar for both


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experimental and computed characteristics. The ability of flavonols and diflavonols to lose protons, particularly in the excited state, makes these compounds potential spectral (fluorescent) indicators of media basicity. In such cases the relations between the above-mentioned ability, reflected by pKa values, and the features of these molecules may be of some interest. The relations (in some cases linear) between pKa (experimental and predicted) and logPC (isooctane-water partition coefficient (PC) computed using the ACDLabs program76), volume, polarizability and dipole moments (extracted from data files after DFT and TDDFT geometry optimizations), shown in Figures 2S–5S (Supporting Information), imply that diflavonols are more sensitive to pH changes, voluminous, polarizable, polar and lipophilic than flavonols, and can therefore be regarded as more convenient spectral indicators of global or local environmental basicity than flavonols. Structure and Physicochemical Features. To gain insight into the absorption and emission phenomena and the interpretation of the relevant spectra of flavonols and diflavonols and their anionic forms, we considered isolated molecules of these compounds (Chart 1, Supporting Information)24 and the molecular complexes they can form with water or methanol (Chart 2S, Supporting Information). The structural data for isolated entities supplied in the Supporting Information (Table 1S and Figure 6S) indicate that the carbonyl O atom and OH group in flavonols or two carbonyl O atoms and two OH groups in diflavonols (one O atom and one OH group in the case of monoanions) may be involved in hydrogen bonding interactions. The hydrogen bonds are asymmetrical and the H atom(s) is (are) always moved to one of the O atoms involved in the interactions. Values of the OHO angle are not far from that of a straight angle, which may imply that the interactions are rather weak. The geometry of the hydrogen bonds changes substantially following conversion from one tautomeric form to another and as a result of electronic


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excitation. The T, NT, and TT forms are characterized by higher enthalpies and Gibbs free energies than are the N and NN forms in the gaseous phase; the latter are thus predominant in the ground electronic state (S0) (Table 6 and Figure 7S (Supporting Information)). Molecules of diflavonols in the NN form exhibit Cs symmetry, which is reflected in the symmetrical pattern of the experimental 1H NMR signals (Materials and Methods section). In the first excited state (S1), the enthalpies and Gibbs free energies of the T* and NT* forms are always lower than those characterizing the N*, NN*, and TT* forms. This means that flavonols in the T* form and diflavonols in the NT* form are easily attainable following the intramolecular transfer of one proton within the originally excited N* or NN* form. None of the excited state forms of neutral diflavonols are symmetrical, since the electronic transitions bring about changes in electron density distribution in the molecules. It has been noted by other researchers that protic molecules (H2O, CH3OH) can interrupt intramolecular hydrogen bonds in flavonols and diflavonols and influence their spectral features.28,29 We investigated such an effect by considering the formation of 1:1 molecular complexes of flavonols and 1:1 or 1:2 complexes of diflavonols with the above-mentioned protic molecules (Chart 2, Supporting Information). The formation of molecular complexes, whose structures are presented in Figure 8S (Supporting Information), is predicted to be energetically and thermodynamically favorable (negative values of ∆r,298Ho and ∆r,298Go in majority of cases) in the gaseous phase (Table 2S, Supporting Information). Such complexes should thus predominate in protic media (H2O, CH3OH), affecting tautomeric transformations in the ground and excited states and consequently the spectral characteristics of flavonols and diflavonols. Comparison of the thermodynamic data shown in Tables 6 and 2S (Supporting Information) shows that the enthalpy and Gibbs’ free energy differences between relevant tautomeric forms of


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non-complexed and complexed flavonols and diflavonols are comparable. Therefore, complexation should not substantially influence the thermodynamics of electronic transitions in these compounds. This conclusion is corroborated by the computational data in Figure 5, which show that the Gibbs’ free energy differences for absorption and fluorescence transitions are comparable for non-complexed and complexed 2a in a methanolic environment, as well as by the experimental spectral data (Figure 9S, Supporting Information) demonstrating that the addition of methanol to aprotic dichloromethane changes the absorption and fluorescence characteristics only slightly. There is a substantial difference, however, between the relative enthalpies and Gibbs’ free energies of the NT* and TT* forms of diflavonols: in non-complexed molecules NT* forms are more stable than TT* ones, whereas the situation is reversed in complexed diflavonols. This could imply that in the excited state of complexed diflavonols single or double proton transfer could occur via a bridge of two hydrogen bonds formed between the latter molecules and the protic ones (H2O, CH3OH). This effect could lower the intensity of fluorescence in the visible region arising from NT* tautomers; emission from TT* forms is expected to occur in the infrared region (Figure 5).26 Detachment of one proton from flavonols and one or two protons from diflavonols lowers the enthalpies of formation and increases the energetic stability of the relevant anionic entities in both the ground and excited states (Tables 6 and 2S (Supporting Information)). Of the two possible monoanionic forms of isolated 2, NA/NA* or TA/TA*, the former is always the energetically more stable one, which means that molecules in the NA/NA* form should predominate in the ground and excited states. If 2 are complexed with protic molecules (H2O, CH3OH), the NA form predominates in the ground state, while TA* one in the excited state. The dianionic forms of diflavonols are energetically less stable than the monoanionic ones and, like the corresponding neutral forms, are symmetrical in the ground state (Cs symmetry). Electronic


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excitation of dianionic forms of diflavonols changes the electron density distribution in them. The dipole moments of the most stable N form of flavonols and the NN form of diflavonols increase during electronic excitation (Table 6), which is consistent with experimental data (Table 2) and which indicates that the process enhances non-uniform electron density distribution. Proton transfer in the excited state usually leads to the less polar T* or NT* forms. The computationally predicted angles between the vectors of the dipole moments in the ground and excited states (Table 6) are not correlated with those found on the basis of experimental data (Tables 2 and 3). Nevertheless, the values of this quantity indicate that the dipole moment changes location and sometimes direction following electronic excitation. Origin of Light Absorption and Emission. According to the results of computations (Tables 6 and 2S (Supporting Information)) and the discussion in the previous chapter, the non-complexed or complexed with protic solvents N form of flavonols and NN form of diflavonols are the ones that absorb radiation. This is confirmed by the similarities in the positions of the longwavelength absorption maxima in various solvents (Figures 2 and 3). Absorption converts N (flavonols) and NN (diflavonols) to the non-relaxed NFC* and NNFC* forms, which, on losing vibrational energy, attain the relaxed N* or NN* states from which fluorescence takes place (N*→NNR, NN*→NNNR) – if proton transfer does not occur (Figures 5 and 7S (Supporting Information)). However, as the data in Tables 1 and 3 indicate, intra- or intermolecular proton transfer (N*→T* or NN*→NT* transfer) does take place, since fluorescence occurs from two excited states, i.e. the relaxed parent N* or NN* states and the T* or NT* states generated by this process. The long-wavelength fluorescence, characterized by an atypically large Stokes shift (Tables 1 and 3),24,26 is relatively strong in aprotic solvents, but weak in protic methanol. Emission from the T* and NT* states predominates in the majority of cases. If we compare the data for the compounds dissolved in chloroform and acetonitrile on the one hand, and in


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methanol on the other, we find that the fluorescence quantum yield decreases strongly; this can be explained by the formation of intermolecular hydrogen bonding complexes with solvent molecules.26−29 In such complexes, single or double intermolecular proton transfer can occur, as a result of which either NT* or TT* states are generated: the former emits in the visible, the latter in the infrared region. In a methanolic environment, therefore, the possible quenching of fluorescence by the solvent may be enhanced by the transformation of excited molecules to nonemitting ones in the visible region. Non-complexed monoanionic diflavonols should exist in only one (NA/NA*) tautomeric form in the ground and excited states, since the relevant TA/TA* form is thermodynamically much less stable (Tables 6); the NA form thus absorbs and the NA* form emits radiation (Figures 3 and 4). Complexed with protic molecules (H2O, CH3OH) diflavonols should exist in the NA tautomeric form in the ground state and NA* and/or TA* forms in the excited state (Table 2S, Supporting Information); the NA form thus absorbs and two NA* and/or TA* forms can emit radiation (Figures 3 and 4). Dianionic diflavonols exist in one form (AA/AA*) in the ground and excited states, which participates in the absorption and emission of radiation. Experimental absorption and emission data for 2a,26 2b (Figure 3) and 2c (Figure 4) are qualitatively in accordance with the computationally predicted positions of transitions. The latter characteristics are useful in the interpretation and deconvolution of experimental data and form a convenient framework for understanding the features of electronic transitions. The contours of the HOMO and LUMO orbitals of neutral and anionic forms of noncomplexed flavonols and diflavonols in the S0 and S1 states (Figure 10S, Supporting Information) indicate that electronic absorption and emission always accompany the electron density shift, in the case of excitation from the phenyl fragment to the chromene-one fragment of flavonols or from phenyl fragment(s) to the chromene-dione fragment of diflavonols. The extent


15


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Page 16 of 42

of the electron density shift is an individual feature of the compounds investigated. This effect is qualitatively reflected in the experimentally found and computationally predicted dipole moment changes (Tables 2 and 6).

Concluding Remarks In the ground electronic state flavonols and diflavonols, non-complexed and complexed with protic molecules, exist in only one tautomeric form (N and NN, respectively), which absorbs radiation, whereas in the excited state they exist in two or three forms, two of which, namely N* and T*, and NN* and NT*, respectively, emit visible radiation. T* and NT* occur as a result of excited-state intramolecular or intermolecular (in the latter case in molecular complexes of flavonols and diflavonols with protic molecules) proton transfer. The extent of proton transfer, as well as the fluorescence quantum yield, depends on the properties of the medium. Non-complexed monoanionic flavonols and diflavonols exist in only one tautomeric form in the ground and excited states. Monoanionic flavonols and diflavonols complexed with protic molecules (H2O, CH3OH) exist in one tautomeric form (NA) in the ground state and one or two tautomeric forms (NA* and/or TA*) in the excited state. Monoanionic diflavonols absorb, but do not emit radiation. Dianionic diflavonols existing in only one form absorb radiation more strongly than the corresponding monoanionic species. Electronically excited dianionic diflavonols fluoresce less strongly than neutral entities. Neutral diflavonols exhibit acidic properties that arise from their ability to detach protons from the OH groups present in the molecules. This acidity is relatively low in the ground state, but increases considerably in the excited state. The acidity in this latter state follows the order 2c < 2a < 2b.


16

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The distinctive influence of the properties of the medium (polarity, hydrogen bonding ability, basicity) on the spectral behavior of flavonols and diflavonols makes these compounds interesting spectral probes for analytical applications. Comparison of flavonols and diflavonols from this point of view reveals a greater differentiation and enhancement of spectral features in the latter compounds. Furthermore, diflavonols are more susceptible to changes in the properties of the medium. Generally, they should be more selective in the case of basicity changes.

Acknowledgment. The financial support of this work by State Funds for Scientific Research

(grants

DS/530-8220-D184-3

and

BMN/538-8220-B004-13)

is

gratefully

acknowledged.

Supporting Information Available: Computationally predicted structures of tautomeric forms of neutral, monoanionic and dianionic flavonols and diflavonols in the ground and excited states, non-complexed and complexed with protic molecules. Selected thermodynamic and spectral characteristics of flavonols, diflavonols and their anionic forms, non-complexed and complexed with protic molecules. Plots of Stokes shifts versus orientational polarizability. Plots of base-10 logarithms of acidity constants versus molecular characteristics. Computationally predicted geometries of intramolecular hydrogen bonds in flavonols, diflavonols and monoanionic diflavonols. HOMO and LUMO orbitals in non-complexed entities. This material is available free of charge via the Internet at http://pubs.acs.org.

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TABLE 1. Spectral Data for Flavonols and Diflavonols in Liquid Phasesa compound (Chart 1)

medium

λabs

λfl (I)

∆νSt (I)

φ (I)

λfl (II)

∆νSt (II)

φ (II)

1a

chloroform

344

413

4860

< 0.1

530

10200

22.0

acetonitrile

339

404

4750

< 0.1

526

10500

5.4

methanol

342

413

5030

0.4

538

10600

2.3

chloroform

356

419

4200

0.1

532

9290

9.7

acetonitrile

355

422

4470

0.1

537

9550

4.2

methanol

355

433

5070

1.8

539

9620

1.4

1b


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1c

2a

2b

2c

Page 28 of 42

chloroform

405

466

3230

5.5

560

6830

18.4

acetonitrile

397

526

6180

6.5

578

7890

5.4

methanol

404

541

6270

0.3

609

8330

3.2

chloroform

378

598

9730

27.1

acetonitrile

379b

511b

6780b

0.2

610b

9980b

25.0b

methanol

380b

520b

7100b

< 0.1

573b

8860b

< 0.1b

chloroform

380

464

4760

598

9590

42.8

acetonitrile

371

510

7350

1.1

608

10500

4.2

methanol

384

500

6040

0.3

568

8440

1.5

chloroform

438

543

4410

46.0

634

7060

30.6

acetonitrile

431

538

4610

0.6

635

7450

2.7

methanol

435

535

4300

0.1

583

5830

0.3

a

λabs = position of the maximum of the long-wavelength absorption band, in nm; λfl (I) and λfl (II) = positions of the maxima, in nm, of fluorescence bands I and II (I corresponds to the N* or NN* form, and II – to the T* or NT* form); ∆νSt (I) and ∆νSt (II) = Stokes shifts (1/λabs – 1/λfl) for fluorescence bands I and II, in cm–1; φ (I) and φ (II) = fluorescence quantum yields of forms N* or NN* and T* or NT*, respectively, in %. b Ref. 26.


28

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TABLE 2. Electronic Transition Characteristics Obtained from Absorption and Emission Spectraa compound (chart 1) 1a 1b 1c 2a 2b 2c

∆µ

θs

∆µ*

1.1 1.1 2.9 2.4 4.1 4.9

17 26 26 52 37 25

2.0 2.8 6.1 6.4 10.5 10.3

a

∆µ = vector difference of dipole moments in the ground and excited states obtained using the Bakhshiyev equations,59,60 in D; θs = angle between dipole moment vectors in the ground and excited states, in deg; ∆µ* = scalar difference of dipole moments in the ground and excited states obtained using the Lippert equation,23 in D.


29


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Page 30 of 42

TABLE 3. Emission Data for Flavonols and Diflavonols in Polystyrene Matricesa compound (chart 1)

λexc

1a

348

1b

359

1c

410

2a

393

2b

384

2c

446

fluorescence band I II I II I II I II I II I II

λfl

∆νSt

r0

θm

437 536 437 542 457 571

5860 10300 4940 9400 2500 6860

0.278 0.232 0.332 0.304 0.239 0.232

27.0 32.0 19.6 23.6 31.2 31.9

598b 436 602 504 635

8720b 3120 9420 2540 6640

0.061 0.278 0.060 0.041 0.066

59.0b 26.8 48.8 50.7 48.3

a

λexc = position of the maximum in the fluorescence excitation spectrum (excitation wavelength), in nm; λfl = positions of the maxima, in nm, of the fluorescence bands I and II (I corresponds to the N* or NN* form, II to the T* or NT* form); ∆νSt = Stokes shifts (1/λexc – 1/λfl) for fluorescence bands I and II, in cm–1; r0 = fluorescence anisotropy; θ m = angle between dipole moment vectors in the ground and excited states, in deg. 59,60 b Ref. 26.


30

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TABLE 4. Spectral Characteristics of Anionic Forms of Diflavonols in Methanolic Solutionsa compound (Chart 1) 2ab 2b 2c

form monoanionic dianionic monoanionic dianionic monoanionic dianionic

λabs 459 454 464 433 461 458

λfl

∆νSt

ϕ

529

3120

3.20

524

4010

0.17

555

3820

0.07

a λabs = position of the long-wavelength absorption band maximum, in nm; λfl = position of the fluorescence band maximum, in nm; ∆νSt = Stokes shift (1/λabs – 1/λfl), in cm–1; ϕ = fluorescence quantum yield, in %. b Ref. 26.


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Page 32 of 42

TABLE 5. Dissociation Constants of Diflavonols in Methanol in the Ground and Excited (*) States compound (Chart 1) 1aa 1ba 1ca 2ab 2b 2c

method expc theor exp

d

c d

S0 pKa (1)

pKa (2)

S1 ∆pKa

pKa (1)*

9.1

–1.2

27.6

11.5

9.6

4.5

28.1

14.8

expc

10.2

6.7

theord

29.3

19.2

theor

exp

c

theor

d

expc theor exp

d

c

theor

d

pKa (2)*

∆pKa*

∆pKa (1)e ∆pKa (2)e –16.1 –13.3 –10.1

13.5±0.1 14.3±0.1 23.8 27.3

0.8

4.9

15.0

10.1

–8.6

0.7

3.5

4.2

33.4

29.2

–19.6

6.1

13.6±0.1 14.5±0.1 27.2 28.0

0.9

3.4

17.8

14.4

–10.2

3.3

0.8

4.6

31.6

27.0

–22.6

3.6

13.9±0.1 14.9±0.1 29.5 31.7

1.0

8.3

16.0

7.7

–5.6

1.1

2.2

5.1

35.7

30.6

–24.4

4.0

a

Experimental values, ref. 28. b Experimental values, ref. 26. c pKa = –log Ka; Ka(1) corresponds to the equilibrium 1 (2) ↔ monoanionic 1 (2) + H+, Ka(2) corresponds to the equilibrium monoanionic 2 ↔ dianionic 2 + H+. d pKa = ∆r,298Go/(2.303×298.14×R), where R is the gas constant and ∆r,298Go corresponds to reactions: 1 (2) ↔ monoanionic 1 (2) + H+(CH3OH)2 (1) and monoanionic 2 ↔ dianionic 2 + H+(CH3OH)2 (2); Gibbs free energies of reactants (298Go) were obtained by geometry optimization at the DFT(PCM−methanol) (S0 state) or TDDFT(PCM−methanol) (S1 state) level of theory. e ∆pKa = pKa (2) – pKa (1), ∆pKa* = pKa (2)* – pKa (1)*, ∆pKa (1) = pKa (1)* – pKa (1), ∆pKa (2) = pKa (2)* – pKa (2).


32

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TABLE 6. Computationally Predicted Thermochemical and Physicochemical Data for Flavonols and Diflavonols, and their Anionic Forms in the Gaseous Phasea compound

entity

(Chart 1) 1a

(Chart 1S)b N T A N T T(1) A N T T(1) A NN NT TT NA TA AA NN NT NT(1) NT(2) TT TT(1) TT(2) TT(3) NA TA TA(1) AA NN NT NT(1) NT(2) TT TT(1) TT(2) TT(3) NA TA

1b

1c

2a

2b

2c

S0 state (DFT method) ∆f.298Ho −4.4 6.5 −25.9 −35.4 −24.5 50.1 −54.2 1.8 12.2 55.9 −15.2 −40.1 −28.2 −15.8 −69.0 −60.6 −45.8 −102.0 −90.4 7.3 −18.9 −78.4 9.4 −8.1 68.5 −127.4 −118.8 −33.2 −102.1 −28.2 −17.2 51.1 24.6 −5.8 52.9 35.1 81.2 −49.8 −41.1

∆t.298Ho

S1 state (TDDFT method)

∆t.298Go

µ

10.9

11.3

2.86 3.97

10.9 85.5

11.2 84.4

4.50 4.94 19.83

10.6 54.7

5.29 5.00 20.80

11.9 24.3

12.0 24.7

4.69 4.87 4.18

8.4

8.6

11.6 109.3 83.1 23.6 111.4 93.9 170.5

11.3 107.8 81.9 23.8 110.2 92.8 167.9

8.6 94.2

8.7 92.7

10.4 54.1

11.0 79.3 52.8 22.4 81.1 63.3 109.4

10.7 80.2 53.5 22.2 82.2 64.1 110.0

8.7

8.9

8.50 8.56 37.78 23.90 8.49 35.25 25.19 29.82

10.03 10.20 39.27 24.70 10.44 34.78 25.53 33.21

∆f.298Ho 69.7 60.9 22.1 36.1 29.4

∆t.298Go

µ

θ

−8.8

−8.7

3.95 1.91

69.9 4.0

−6.7

−6.6

8.23 3.27

52.4 23.7

−3.0

−3.3

13.76 5.03

29.8 25.2

−4.6 0.6

−4.3 1.5

16.03 7.88 2.79

69.3 90.0 89.0

9.5

10.4

−3.1

−3.1

20.31 11.10

150.8 74.2

−41.3

1.1

1.5

7.04

179.2

−111.8 −103.3

8.5

9.3

57.1 52.3

52.7

−7.1 68.0 65.0 31.9 21.4 16.8 22.0 −53.6 −44.1 −2.8 −42.4 −45.5

∆t.298Ho

−59.3 27.7 26.9

−0.8

−0.9

24.57 13.57

31.5

3.8

4.7

15.49

−33.8 −20.3

13.5

14.2


33


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13.4 −24.4

TA(1) AA

63.2

Page 34 of 42

62.8 17.4

a ∆f.298Ho denotes standard (o) enthalpy of formation, in kcal/mol; ∆t.298Ho and ∆t.298Go denote standard (o) enthalpy and Gibbs’ free energy of tautomerization (t) (i.e. of one of the processes: N→T, NN→NT or TT, NA→TA), both in kcal/mol; µ is the dipole moment, in D; θ is the angle between the dipole moment vectors in the ground (S0) and excited (S1) states, in deg. b Supporting Information.

Figure Captions CHART 1. Canonical Structures of 3-Hydroxy-2-phenyl-4H-chromen-4-ones (flavonols) (1) and 3,7-Dihydroxy-2,8-diphenyl-4H,6H-pyrano[3,2-g]chromene-4,6-diones (diflavonols) (2) with the Numbering of Atoms Indicated. AR = H (a), OCH3 (A=O) (b), N(CH3)2 (A=N) (c). FIGURE 1.

Experimental (upper graph) fluorescence spectra (solid lines) of 2a and 2c (the

bars represent the values given in Table 1) in acetonitrile (deconvoluted (dashed lines) in the case of 2c) with predicted (lower graph) positions of fluorescence transitions for the NN* and NT* forms (Chart 1S, Supporting Information) at the TDDFT(PCM) level of theory. FIGURE 2. Changes in absorption (A) and fluorescence (C) of 2b (the bars represent the values given in Tables 1 and 4) in methanol (concentration ~10–5 M) with increasing basicity of the solution (KOH concentration from 1.0 × 10–7 to 0.3 M), together with predicted (TDDFT(PCM) level of theory) long wavelength absorption (B) and fluorescence (D) transitions for the relevant tautomeric forms (Charts 1S and 2S, Supporting Information) of the compound. FIGURE 3. Changes in absorption (A) and fluorescence (C) of 2c (the bars represent the values given in Tables 1 and 4) in methanol (concentration ~10–5 M) with increasing basicity of the solution (KOH concentration from 1.0 × 10–7 to 0.3 M), together with predicted (TDDFT(PCM) level of theory) long wavelength absorption (B) and fluorescence (D) transitions for the relevant tautomeric forms (Charts 1S and 2S, Supporting Information) of the compound. FIGURE 4. Normalized fluorescence intensity of NN* ( ), NT* ( ) and AA* ( ) emitting entities (upper graph) and mole fraction of neutral ( ), monoanionic ( ) and dianionic ( ) forms (lower graph) of 2c (concentration ~10–5 M) versus negative base-10 logarithm of KOH concentration in methanolic media. FIGURE 5. Gibbs free energy changes predicted at the DFT(PCM) and TDDFT(PCM) levels of theory following excitation, proton transfer in the excited state, fluorescence, and relaxation of non-complexed (Chart 1, Supporting Information; upper graph) and complexed (Chart 2,


34

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Supporting Information; lower graph) neutral tautomeric forms of 2a in a methanolic environment (the subscripts FC and NR denote non-relaxed excited Franck-Condon and ground states, respectively).

Table of Contents (TOC) Image:


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Page 36 of 42

CHART 1.

AR 8

O

2

4

3

7 6 5

O

AR

RA

O

O

H

H

8

O

7

6

1

10

2

4

3

5

O

O

2


O

O H

Page 37 of 42

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FIGURE 1



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

FIGURE 2


Page 38 of 42

Page 39 of 42

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FIGURE 3


Page 40 of 42

1.00

0.75

0.50

0.25

0.00 7.0

5.0

3.0

1.0

5.0

3.0

1.0

1.00

0.75

Mole fraction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Normalized fluorescence intensity


0.50

0.25

0.00 7.0

– log (KOH concentration)

FIGURE 4


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FIGURE 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents (TOC) Image:


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chromen-4-ones (Flavonols) - American Chemical Society

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