Origin of Spectral Features and AcidâBase Properties of 3,7

Article pubs.acs.org/JPCA

Origin of Spectral Features and Acid−Base Properties of 3,7-Dihydroxyflavone and Its Monofunctional Derivatives in the Ground and Excited States Illia E. Serdiuk,*,†,‡ Alexander D. Roshal,‡ and Jerzy Błazė jowski† †

Department of Chemistry, University of Gdańsk, 80-308 Gdańsk, Poland Institute of Chemistry, V. N. Karazin Kharkiv National University, 61022 Kharkiv, Ukraine

‡

S Supporting Information *

ABSTRACT: Comprehensive spectral investigations of 3,7-dihydroxyflavone and its two derivatives, which each contain a methylblocked hydroxyl group, reveal complex radiation absorption in the 300−450 nm range and emission in the 370−650 nm range. The absorption and fluorescence characteristics of these compounds depend on the pH/H0 of the water/methanol media, which is caused by the existence of the compounds in various protolytic (cationic, neutral, anionic) and tautomeric forms. Combined analysis of steady-state, time-dependent and fluorescence decay spectral data enabled the identification of the emitting species, determination of their lifetimes with respect to radiative and nonradiative deactivation processes, fluorescence quantum yields, protolytic and tautomeric abilities under various conditions, and acidic dissociation constants of the cationic, neutral, and anionic forms of the compounds. Results of calculations carried out at the DFT and TD DFT levels of theory generally confirmed the experimental findings concerning tautomeric/protolytic transformations and equilibria. Computational methods also provided insight into possible tautomerization pathways. Electronically excited molecules are generally much more susceptible to tautomerization and acidic dissociation than ground-state ones. 3,7-Dihydroxyflavone exhibits distinguishable features among the compounds investigated and can be considered as potential spectral indicator of properties (polarity, hydrophobicity, hydrogen-bonding ability) and acidity/basicity of liquid environments.

■

Chart 1. Canonical Structures of 3,7-Dihydroxy-2-phenyl4H-chromen-4-one (1), 7-Hydroxy-3-methoxy-2-phenyl-4Hchromen-4-one (2), and 3-Hydroxy-7-methoxy-2-phenyl-4Hchromen-4-one (3) with the Numbering of Atoms Indicated.a

INTRODUCTION Organic fluorophores in which the excited-state proton transfer (ESPT) occurs have been shown to be useful spectral indicators in various chemical,1 environmental,2−4 and biological investigations.5−11 The use of the compounds in this role emerges from their ability to occur in the electronically excited state in various structural forms (tautomeric, rotameric, protolytic) each of which have different fluorescent features (emission wavelengths, lifetimes) which depend on the properties of the medium (such as polarity, acidity/basicity, hydrogen-bonding ability) or biological environment. One group of compounds that are widely used as ESPT fluorophores are hydroxyflavones (hydroxy-2-phenyl-4H-chromen-4-ones),12−19 which represent the target compounds in the present investigation. Hydroxyflavones, besides possessing mentioned above distinctive spectral properties, exhibit antioxidative features20−23 and low cytotoxicity,24 which are essential for their use in in vivo investigations. Here we report the results of studies on the synthesis, structure, and physicochemical (mainly spectral) features of 3,7-dihydroxy2-phenyl-4H-chromen-4-one (3,7-dihydroxyflavone, 1) and its two derivatives, which contain methyl-blocked hydroxyl groups at positions 3 (compound 2) and 7 (compound 3) (Chart 1). We have particularly focused our attention on 3,7-dihydroxyflavone © 2016 American Chemical Society

a 1

R , R2 = OH, OH (1); OCH3, OH (2); OH, OCH3 (3).

because this compound contains two hydroxyl groups that have the potential to take part in ESPT, and one of these groups, at position 3, can undergo excited-state intramolecular proton transfer (ESIPT), a very well-known phenomenon occurring in 3-hydroxyflavones.25−27 Moreover, the presence of two hydroxyl groups in compound 1 enhances its ability to participate in interactions with surrounding molecules and to contribute to acid/base Received: March 31, 2016 Revised: May 31, 2016 Published: June 2, 2016 4325

DOI: 10.1021/acs.jpca.6b03290 J. Phys. Chem. A 2016, 120, 4325−4337

Article

The Journal of Physical Chemistry A

hydroxyl group at position 7 in spectral features of 1 and in its interactions with the environment is still not well recognized, despite the fact that it is more acidic than the group at position 3.23,29 Thus, the investigations presented here create an opportunity to extend our knowledge regarding these cognitively and potentially applicatively interesting hydroxyflavone compounds.

equilibria when settled in media of various pH/H0. The latter two features of 1 extend the potential for its use as a spectral indicator. Compounds 2 and 3 should reflect the behavior of the complementary monohydroxy-substituted flavones related to 1, and for these reasons they were selected for investigation. The features of 2 and 3 are expected to reflect those of 1, to some extent. First, we aimed to reveal how the spectral behavior of 1 is related to that of molecules containing only one hydroxyl group, i.e., compounds 2 and 3. We further aimed to disclose the protolytic/tautomeric equilibria occurring in water/methanol media with various pH/H0 and the nature of the absorbing/ emitting entities existing under these conditions. Lastly, we attempted to outline how various features of the protolytic (cationic, neutral, and anionic) and tautomeric forms of these compounds (polarity, hydrophilicity, ability to participate in interactions; Chart 2) can affect the potential for the use of 1 as a spectral indicator.

■

EXPERIMENTAL SECTION Reagents. Reagents of relevant grade for synthesis and spectral grade for spectroscopic investigations were purchased from Sigma-Aldrich. Syntheses. The compounds investigated (1, 2, and 3) were synthesized starting from 1-(2,4-dihydroxyphenyl)ethanone (4) according to Scheme 1. First, the hydroxyl group at position 4′ of 4 was protected with a benzyl or methyl group by reaction of the compound with an equimolar amount of benzyl chloride or dimethyl sulfate, respectively, in the presence of K2CO3, in boiling acetone.36 The resulting compounds (1-[4-(benzyloxy)2-hydroxyphenyl]ethanone (5a) and 1-(2-hydroxy-4-methoxyphenyl)ethanone (5b)) were purified by flash chromatography (eluent, 1% i-PrOH in CHCl3) and subjected to condensation with benzaldehyde, accompanied by the elimination of water, in alkaline (KOH) 1-methylpyrrolidin-2-one (5a) or methanol (5b) at room temperature (RT),37 leading to the production of the respective chalcones: (2E)-1-[4-(benzyloxy)-2-hydroxyphenyl]-3-phenylprop-2-en-1-one (6a) or (2E)-1-[4-(methoxy)-2-hydroxyphenyl]-3-phenylprop-2-en-1-one (6b). Chalcones isolated from acidified reaction mixtures by filtration were next purified by flash chromatography (eluent, 1% i-PrOH in CHCl3) and subjected to oxidative heterocyclization, accompanied by the elimination of water, in the presence of hydrogen peroxide and K2CO3 in methanol (RT), leading to the production of the flavonols 7-(benzyloxy)-3-hydroxy-2-phenyl-4Hchromen-4-one (7) or 3-hydroxy-7-methoxy-2-phenyl-4H-chromen-4-one (3). Compound 3 was further investigated whereas 7, which was obtained with a reasonably high yield (57%),37 was used as a precursor for the synthesis of 1 and 2. Compounds 1 (3,7-dihydroxy-2-phenyl-4H-chromen-4-one) and 2 (7-hydroxy3-methoxy-2-phenyl-4H-chromen-4-one) were obtained by hydrolysis of 7 and 8 (7-benzyloxy-3-methoxy-2-phenyl-4H-chromen-4one), respectively, with HBr dissolved in acetic acid (RT) (typically: 0.2 g of 7 or 8 was dissolved in 4 mL of a 33% solution of HBr in acetic acid, the mixture was stirred at room temperature for 4 h, then water was added and the precipitate filtered off and subjected to purification). Compound 8 was synthesized by alkylation of 7 with dimethyl sulfate in an acetonitrile/K2CO3 mixture (RT) (typically: 1 mmol (0.344 g) of 7 was suspended in 10 mL of acetonitrile; next 2.2 mmol (0.276 g) of K2CO3 and 2.1 mmol (0.252 g) of dimethyl sulfate were added, and the suspension was stirred for 10 h at RT; then water was added and the precipitate filtered off and recrystallized from methanol). The target compounds were purified by repeated crystallization from suitable aqueous methanol mixtures (1, pale-yellow crystals, mp 254−256 °C (capillary method); 2, colorless crystals, mp 227− 229 °C; 3, pale-yellow crystals, mp 175−176 °C). The purity of the target compounds and some precursors was controlled by TLC (Merck 60 F254 plates), and their identities were confirmed by NMR (1H 200 or 500 MHz spectrometers, DMSO-d6 as solvent, chemical shifts (δ) determined with TMS as internal standard) and MS (MALDI-TOF)). 3,7-Dihydroxy-2-phenyl-4H-chromen-4-one (1). 1H NMR 200 MHz (δ, ppm; J, Hz; s, singlet; d, doublet; m, multiplet; bd,

Chart 2. Canonical Structures of Neutral (N, T3, T7), Monocationic (C, C(1)), Monoanionic (A7, A7T), and Dianionic (DA) Forms of 1 (Chart 1) Occurring in Media with Various pH/H0

It is worth mentioning that, like numerous naturally occurring and widely used flavones containing hydroxyl groups at positions 3 and 7 (e.g., fisetin, robinetin, galangin, kaempferol, myricetin, or isorhamnetin),28 the spectral and acid/base features of 1 have been scarcely investigated.29,30 Somewhat more is known about the interactions of the above-mentioned compounds with albumins,16,31 hemoglobin,32 DNA,33 cyclodextrins,34 and liposomes.35 Interpretations of the spectral changes that accompany interactions of 1 with these molecules as well as conclusions made on the character of these interactions are based on the assumption that the fluorescent features of 1 are mainly due to ESIPT occurring in the neutral form of the fluorophore and involving the hydroxyl group at position 3. The role of the 4326


Article

The Journal of Physical Chemistry A Scheme 1. Path of Synthesis of 3,7-Dihydroxyflavone and Its Derivatives

broad): 6.82−6.98 (m, 2H, ArH), 7.39−7.57 (m, 3H, ArH), 7.92 (d, 1H, J = 8.6, ArH), 8.10−8.23 (m, 2H, ArH), 9.33 (bd s, 1H, OH), 10.79 (bd s, 1H, OH). MS (m/z): 254 (M + H+), 277 (M + Na+), 293 (M + K+). 7-Hydroxy-3-methoxy-2-phenyl-4H-chromen-4-one (2). 1H NMR 200 MHz (δ, ppm; J, Hz; s, singlet; d, doublet; m, multiplet; bd, broad): 3.78 (s, 3H, OCH3), 6.87−6.97 (m, 2H, ArH), 7.51−7.58 (m, 3H, ArH), 7.92 (d, 1H, J = 9.3, ArH), 7.95−8.02 (m, 2H, ArH), 10.85 (bd s, 1H, OH). MS (m/z): 269 (M + H+), 291 (M + Na+), 307 (M + K+). 3-Hydroxy-7-methoxy-2-phenyl-4H-chromen-4-one (3): 1H NMR 200 MHz (δ, ppm; J, Hz; s, singlet; d, doublet; m, multiplet; bd, broad; dd, doublet of doublets): 3.90 (s, 3H, OCH3), 7.03 (dd, 1H, J = 8.9, J = 2.3, ArH), 7.27 (d, 1H, J = 2.3, ArH), 7.45−7.60 (m, 3H, ArH), 7.98 (d, 1H, J = 9.0, ArH), 8.20 (d, 1H, J = 8.5, ArH), 9.50 (bd s, 1H, OH). MS (m/z): 269 (M + H+), 291 (M + Na+), 307 (M + K+). 7-Benzyloxy-3-methoxy-2-phenyl-4H-chromen-4-one (8): 1 H NMR 500 MHz (δ, ppm; J, Hz; s, singlet; d, doublet; m, multiplet; t, triplet; dd, doublet of doublets): 3.82 (s, 3H, OCH3), 5.27 (s, 2H, OCH2), 7.14 (dd, 1H, J = 8.8, J = 1.9, ArH), 7.34−7.38 (m, 2H, ArH), 7.42 (t, 2H, J = 7.0, ArH), 7.49 (d, 2H, J = 7.6, ArH), 7.56−7.61 (m, 3H, ArH), 8.00 (d, 1H, J = 8.8, ArH), 8.01−8.05 (m, 2H, ArH). MS (m/z): 359 (M + H+), 381 (M + Na+), 397 (M + K+). 1 H NMR, MS, and elemental analysis data for 7 were reported previously.37 Investigations of Spectral and Acid−Base Properties. Absorption and fluorescence spectra were recorded on a Perkin Elmer Lambda UV/vis 40 spectrophotometer and Varian Cary Eclipse spectrofluorometer, respectively. Fluorescence decay curves were measured on a FluoTime 300 fluorescence lifetime spectrometer equipped with a compact emission monochromator, a TimeHarp 300E TCSPC device (minimal time resolution 4 ps), a PLS 340 LED-head for subnanosecond pulses, LDH-PC-375 and LDH-P-C-420 laser heads for picosecond pulses driven by a PDL 820 device and a MCP-PMT photomultiplier (type R3809U-50) (PicoQuant GmbH, Germany) controlled by EasyTau system software. Spectral features and behavior of the compounds were investigated in liquid phase consisting of water (deionized)/methanol (spectral grade) at a ratio of 1:4 (v/v). Concentrations of the compounds investigated were approximately 1 × 10−5 M in the absorption and fluorescence investigations and titrations. In acid−base titrations, water/methanol solutions of NaOH (0.01, 0.1, and 1.0 M) and sulfuric acid (0.005 and 0.05 M), and concentrated (96%) sulfuric acid were used as titrants. For a detailed description of the titration procedures as well as the H0

scale correction in water/methanol medium, see ref 38. The pKa (−log Ka) values of the acidic dissociation reactions in the electronically excited state were estimated by the Förster’s method39 using the experimental values of 0−0 transition energies. Changes in Gibbs free energy (Δ298G°) of the corresponding reactions in the ground (S0) and electronically excited (S1) states were obtained according to the equation Δ298G° = −RT ln K a

Determination of Fluorescence Quantum Yields. Fluorescence quantum yields at 298 K were determined relative to quinine bisulfate in aqueous 0.05 M H2SO4 (excitation wavelength 320−370 nm) or ethanolic 9-aminoacridine (excitation wavelength 400−410 nm) reference solutions according to the equation φf = φfref

n ref 2 Aref ∫ If (λf ) dλf n2 A ∫ Ifref (λf ) dλf

where subscript “f” indicates fluorescence and superscript “ref” refers to the reference solution, φf and φref f (0.52 for quinine bisulfate40 and 0.99 for 9-aminoacridine41) are fluorescence quantum yields, n and nref are refractive indices of the solvents, A (≤0.2) and Aref (≤0.1) are optical densities of the sample and reference solutions at the same excitation wavelength, and integrals denote the area (computed) of the corrected fluorescence bands. In the case of complex fluorescence spectra, φf of each form was evaluated utilizing the areas of deconvoluted bands. Conditions of determination of fluorescence quantum yields are specified in the Supporting Information. Determination of the Deactivation Rates of Electronically Excited Forms. Deconvolution of single fluorescence lifetime measurements was carried out using a procedure incorporated in the FluoFitPro software. Rate constants of radiative (kf) and nonradiative (kd) deactivation of forms C*/C(1)* (at H0 = −1.8) and DA* (at pH = 13.5) of 1, and A3* (at pH = 12.5) of 3 were determined according to the equation τ=

1 k f + kd

where τ is the fluorescence lifetime. Time-resolved fluorescence spectra and rate constants of transformations in the excited state were obtained as described elsewhere.42,43 Briefly, fluorescence decay curves were recorded every 10 nm in the UV−vis region (370−700 nm) to cover the whole fluorescence decay surface. The data were processed using a procedure incorporated in the Spectral Data Lab software44 4327


Article


(S1) electronic states, as well as H2O and H3O+ in the ground state, were carried out at the DFT/TD DFT levels of theory,46 with the B3LYP47,48 hybrid functional and the cc-pVDZ basis set, and included the solvent (water) effect (at 298.14K and standard pressure) at the level of the Polarized Continuum Model (PCM),49,50 using a GAUSSIAN 09 program package51 (optimized structures are shown in Figure S1, Supporting Information). Changes of the Gibbs free energy (Δ298G°) of the tautomeric and protolytic transformations were calculated according to the equation51

kindly provided by Prof. A. O. Doroshenko. The total fluorescence intensity at each wavelength was corrected relative to the steady-state spectra. Deconvolution of the fluorescence decay surfaces, carried out with respect to the emission bands of the relevant forms (available from deconvolution of the steadystate spectra) enabled determination of their lifetimes (τ) and “apparent” rate constants of the excited-state proton transfer (k′ESPT): τ=

1 k f + kd + kESPT

k′ESPT = kESPT·

k fi·Sj

Δ298G° =

k fj·Si

where i and j indicate reaction substrates and products, respectively, and E0 + G represents the sum of electronic and Gibbs free energies (Table S1, Supporting Information). The predicted Δ298G° values of the acidic dissociation reactions in the ground and excited electronic states substantially differ from the experimental values. To facilitate comparison of the observed and calculated thermodynamic characteristics of protolytic dissociation reactions, we used Gibbs free energy values for the selected ground (S0)-state processes (experimental values of which are known, Table 1) as reference data and calculated correction constants (ΔG = Δ298G°exp − Δ298G°theor), as shown in Table S2 (Supporting Information). The correction constants were subsequently used to obtain Δ298G°corr in the ground and electronically excited (S1) states according to the equation

where kESPT is the rate constant of the excited-state proton transfer (N* → T3* for 1 and 3, A7* → A7T*, A7T* → A7*, C(1)* → T7* + H+ and T3* → A7T* + H+ for 1), i and j indicate the initial and final forms, and S is the area of the relevant fluorescence bands. The ratio Sj/Si was obtained by deconvolution of the steadystate spectra. The ratio kif/kjf for each pair of excited forms was estimated on the basis of the values of oscillator strength (f) of the S1 → S0 transition predicted at the TD DFT level of theory: k f ≈ 0.661·νf 2·f

where v is the experimental wavenumber of the S1 → S0 transition. The values of the rate constants of radiative deactivation of the initial forms C*/C(1)* (kCf *), N* (kNf *), and A7* (kAf 7*) were obtained from quantum yields, lifetimes, and kESPT values45 using the following equations: φC = τC *k fC *

(irreversible process C(1)* → T7* + H+)

φN = τN *k fN *

(irreversible process N* → T3*)

φA = 7

Δ298G°corr = Δ298G°theor + ΔG

The corrected values of Δ298G°corr, obtained using this procedure, were more comparable to the experimental values, evaluated by the Förster’s method, than the uncorrected ones (Table 1). The predicted pKa* values of the corresponding photodissociation reactions were obtained from the Δ298G°corr values. The wavelengths of the S0 → S1 and S1 → S0 transitions were predicted by the single-point TD DFT calculations using the optimized S0 and S1 state geometries.52 These predicted wavelengths were corrected by dividing by the correction coefficients rabs and rfl, respectively, as shown in Table S3 (Supporting Information).

k fA 7 *(τ A 7*)−1 (τ A 7*)−1(τA 7T *)−1 − k A 7*→ A 7T *k A 7T*→ A 7 *

(reversible process A 7* ↔ A 7T*)

where φC, φN, and φA7 are fluorescence quantum yields of the C*/C(1)*, N*, and A7* forms, respectively, τC*, τN*, τA7*, and τA7T* are lifetimes of the C*/C(1)*, N*, A7*, and A7T* forms, respectively, and kA7*→A7T* and kA7T*→A7* are rate constants of the direct and reverse transformations of A7* to A7T*, respectively. T The rate constants of radiative deactivation of forms T7* (kf 7*), T3 A7T T3* (kf *), and A7T* (kf *) were then calculated using the theoretically predicted ratios kCf */kTf 7*, kNf */kTf 3* and kAf 7*/kAf 7T*, respectively. Values of kd for each form were obtained from the relationship τ=

∑ (E0 + G)j− ∑ (E0 + G)i

■

RESULTS AND DISCUSSION Steady-State Electronic Absorption Spectra at Various pH/H0. The hydroxyflavones investigated in this study (Chart 1) can participate in various tautomeric transformations and protolytic equilibria via proton transfer and protolytic dissociation processes, respectively, which involve hydroxyl and carbonyl groups. These transformations and equilibria in the ground electronic state are reflected in the absorption spectra (Figure 1 and Table 2) and titration curves (Figure S2, Supporting Information) recorded in wide range of pH/H0. Deconvolution of these spectra enables the determination of the mole fractions of the relevant forms of the compounds at various pH/H0 and calculation of the acidic dissociation constants (Ka) of the cationic, neutral, and monoanionic (in the case of 1) forms, which are reported as pKa in Table 1. Mole fractions of the cationic, neutral, monoanionic and dianionic forms of 1, reproduced on the basis of the experimentally determined pKa values, are demonstrated in Figure 2.

1 k f + kd + kESPT

The value of kESPT for the protolytic dissociation T3* → A7T* + H+ was evaluated using an “apparent” rate constant and rate constants of radiative deactivation of the relevant forms, which were obtained as described above. Quantum-Chemical Calculations. Unconstrained geometry optimizations of the tautomeric/protolytic forms of the compounds investigated in the ground (S0) and excited singlet 4328


Article


Table 1. Experimental (exp) and Predicted (corr) Values of Gibbs Free Energy (Δ298G°, kJ/mol) and pKa of the Protolytic Dissociation Reactions of the Compounds Investigated in the S0 and S1 States S0

S1

Δ298G° expa

compd

reaction

1

C(1) + H2O → N + H3O C(1) + H2O → T7 + H3O+ C + H2O → T3 + H3O+ N + H2O → A7 + H3O+ T3 + H2O → A7T + H3O+ T7 + H2O → A7 + H3O+ A7 + H2O → DA + H3O+ A7T + H2O → DA + H3O+ C + H2O → N + H3O+ C + H2O → T7 + H3O+ N + H2O → A7 + H3O+ T7 + H2O → A7 + H3O+ C(1) + H2O → N + H3O+ C + H2O → T3 + H3O+ N + H2O → A3 + H3O+ T3 + H2O → A3 + H3O+

2

3

+

Δ298G°

pKa corrb

−11.9

expa −2.09 ± 0.05

corrc 53

69.0 33.5

31.4 12.1 5.9

8.38 ± 0.02

47.9 32.2 −33.1

7.4 5.6 −5.8

12.00 ± 0.04

68.5 41.0 −11.5

57.1 7.2

−2.01 ± 0.0453 61.3

37.1 10.7

8.30 ± 0.02

47.4 −25.4 −12.4

−11.4 −4.5

−2.18 ± 0.0353 29.7

26.3 5.2

10.84 ± 0.02

61.9

expa

24.6

9.1 4.3

pKa* corrb 23.6 23.9 −22.1 −7.8 24.9 −8.1 58.1 65.6 19.3 35.5 −24.3 −40.5 19.6 −19.8 −6.1 27.3

expa 5.5

53

1.3

10.0 6.553 −2.0 4.653 1.6

corrc 4.1 4.2 −3.9 −1.4 4.4 −1.4 10.2 11.5 3.4 6.2 −4.3 −7.1 3.4 −3.5 −1.1 4.8

Experimental pKa values obtained from spectrophotometric titrations (S0 state) or by the Forster method (S1 state); experimental Δ298G° values calculated according to the equation Δ298G° = −RT ln Ka. bPredicted values of corrected (Δ298G°corr = Δ298G°theor + ΔG) Gibbs free energy in the excited state by using correction constants (ΔG) listed in Table S2 (Supporting Information). cPredicted values of pKa* calculated according to the equation pKa* = Δ298G°corr/(2.303RT). a

−0.5 to +8.0 for 1, 2, and 3, respectively), the compounds exist mainly in neutral form. In weakly and medium basic media (pH between 7.0−10.0 for 1 and 2 and 8.0−12.0 for 3), they coexist in their neutral and monoanionic forms. In strongly basic media, compound 1 coexists in monoanionic and dianionic forms in the 10.0−13.5 pH range and exists mainly in dianionic form at pH > 13.5. Compounds 2 and 3 exist predominantly in monoanionic forms at pH above 10.0 and 12.0, respectively. Steady-State and Time-Dependent Fluorescence Spectra at Various pH/H0. The absorption of radiation by the cationic, neutral, and anionic forms of the compounds investigated existing or coexisting at various pH/H0 leads to formation of relevant electronically excited species which can undergo tautomeric or protolytic transformations and radiative and nonradiative deactivation. Various electronically excited species and some of the above-mentioned processes are reflected in the steady-state (Table 2 and Figures 3−5) and time-dependent (Figures S3−S6, Supporting Information) fluorescence data. The steady-state fluorescence spectra of 3 (Figure 3a,b,c) represent emission from the cationic forms C* and C(1)* (at 478 nm) in strongly acidic media (H0 < −3.0), neutral tautomeric forms N* (at 402 nm), and T3* (at 532 nm) in medium acidic to weakly basic environments (1.0 < pH < 8.0), and the anionic form A3* (at 517 nm) in strongly basic media (pH > 12.0). Compound 3 coexists in the electronically excited cationic and neutral forms in strongly to medium acidic environments (−3.0 < H0/pH < 1.0), and in neutral and monoanionic forms in weakly to strongly basic media (8.0 < pH < 12.0). A comparison of the fluorescence and absorption characteristics indicates that electronically excited species C*, C(1)*, N*, and A3* are generated via the absorption of radiation by the ground-state molecules present at each pH/H0. The time-dependent fluorescence spectra of 3 (Figure S3a,b, Supporting Information) demonstrate that the compound undergoes N* → T3* transformation (ESIPT) in the electronically excited state. This transformation occurs on the nanosecond scale. The situation monitored in the

It is widely known that the absorption features of hydroxyflavones are related to their structure, e.g., outlined in Charts 2 and S1 in the Supporting Information. Moreover, close maxima positions of the absorption bands can indicate structural similarities of the absorbing entities. Thus, the absorption of cationic forms of the compounds (at 380 nm (2) and 387 nm (1 and 3)), represented by single bands in the long-wavelength region in strongly acidic media (Figure 1), arise from cationic forms C (1, 2, and 3) and C(1) (1 and 3) that likely exist (2) or coexist (1 and 3) under the experimental conditions, as emerges from the results of calculations (Table S1, Supporting Information). Close maxima positions of the long-wavelength absorption bands of 1 and 2 in weakly basic media (377 and 363 nm, respectively (Figure 1 and Table 2)), suggest that both arise from the monoanionic form A7; in the case of 2 it is the only possible anionic form. The absorption of the monoanionic form A3 of 3 falls at 396 nm, and if the A3 monoanionic form of 1 occurred in the system, its absorption would appear at a similar wavelength. However, the observed absorption spectra (Figure 1 and Table 2) and the results of calculations (Table S1, Supporting Information) did not confirm the presence of 1 in such a form in the ground electronic state. In strongly basic media, absorption of 1 is centered at 401 nm, which can be ascribed to the DA form of the latter compound. Absorption of the cationic and anionic forms is always long-wavelength shifted relative to that of the neutral form N. The N form is characterized by a single band (maximum at ca. 320 nm) in the absorption spectrum of 2 and by at least two overlapping bands in the absorption spectra of 1 and 3. The batochromic shift of the long-wavelength bands of the neutral forms of 1 and 3 relative to that of 2 is associated with the presence of an OH group at position 3.53 The absorption data demonstrate that in strongly acidic media (H0 < −4.0) all three compounds occur predominantly in their protonated forms. In strongly to medium acidic media (−4.0 < H0 < −0.5) they coexist in both protonated and neutral forms. In medium acidic to weakly basic media (H0 − pH: −0.5 to +7.0, −0.5 to +7.0, and 4329


Article


Figure 1. Long-wavelength absorption spectra (upper graphs) of 1, 2, and 3 (bars indicate wavelengths of absorption maxima given in Table 2) in a water/methanol phase (concentration ca. 10−5 M) at various pH/H0 (arrows indicate pH/H0 rise) together with predicted (lower graphs) positions of absorption transitions (bars indicate corrected wavelengths of S0 → S1 transitions); N, C, C(1), A, and DA indicate the lowest energy forms of the compounds (Charts 2 and S1, and Table S1, Supporting Information).

of various forms of the compound are rather weak and their lifetimes are relatively short (Table 3). It should be noted, however, that the cationic and anionic forms are longer-living than the neutral ones. Among deactivation processes, fluorescence is always the slowest, whereas nonradiative deactivation and tautomeric transformation of the neutral forms are generally the fastest.

steady-state spectra corresponds roughly to one recorded 0.4 ns after excitation pulse. Fluorescence decay curves (Figure S5a,b,c, Supporting Information) confirm the existence or coexistence of compound 3 in various forms in the electronically excited state, identified on the basis of steady-state and time-dependent fluorescent spectra. Fluorescence 4330


Article

The Journal of Physical Chemistry A Table 2. Spectral Characteristics of the Cationic, Neutral, and Anionic Forms of the Compounds Investigated in the Ground and Excited Electronic Statesa λabs compd 1

2

3

b

λfl

c

exp

corr

C C(1) N T3 T7 A7 A7T A3 DA C N T7 A7 C C(1) N T3 A3

387

350 388 330 442 426 390 467 424 410 389 301 531 393 359 389 332 448 440

form

341

377

401 380 320 363 387 339 396

d

exp 481 406 532 545 499 545 509 480 420 568 536 478 402 532 517

corrd 435 452 413 537 576 509 577 519 500 495 410 571 561 444 464 417 545 539

λabs = position of the maximum of the long-wavelength absorption band, in nm; λfl = position of the maximum of the fluorescence band, in nm. bChart 1. cCharts 2 and S1 (Supporting Information). d TD DFT level of theory, corrected using coefficients presented in Table S3 (Supporting Information). a

Figure 2. Mole fraction of neutral (N), cationic (C/C(1)), monoanionic (A7), and dianionic (DA) forms of 1 versus pH/H0 in the S0 state reproduced basing on experimentally determined vales of pKa (Table 1). Figure 3. Experimental (upper graphs) fluorescence spectra of 3 (“au”, arbitrary units; bars, wavelength of fluorescence maxima given in Table 2) in a water/methanol phase (concentration ca. 10−5 M) at various pH/H0 (arrows, pH/H0 rise) together with predicted (lower graphs) positions of emission transitions (bars, corrected wavelength of S1 → S0 transitions). N*, T*, C*, C(1)*, and A* denote the electronically excited forms of the compound (Charts 2 and S1, Supporting Information).

In the steady-state fluorescence spectra of compound 2 (Figure 4a), a single band (at 480 nm) appearing in strongly acidic media (H0 < −4.0) arises from the electronically excited cationic form C* of the compound, which is generated via absorption of radiation by the ground-state C form, only existing under such conditions. In strongly to medium acidic environments (−4.0 < H0 < −0.5), cationic (C) and neutral (N) forms of the compound coexist in the ground state and both forms can be radiatively excited and emit radiation. Fluorescence from the N* form is, however, weak under these conditions (Figure 4b), but upon a rise of H0, a new emission appears, which can be ascribed to the neutral T7* form (at 568 nm) (Figure 4a), which is most likely produced via protolytic dissociation: C* → T7* + H+, as was reported previously for 7-hydroxyflavone.38 Emission from T7* declines together with the disappearance of the C* form. The fluorescence spectra of 2 are composed of two bands in the

pH range from 1.0 to 12.0 (Figure 4b,c). Short-wavelength emission (band centered at 420 nm) arises from the N* form, whereas the long-wavelength band represents combined emissions from the declining form T7* and occurring monoanionic form A7*, most probably via protolytic dissociation: N* → A7* + H+. The single band (at 536 nm) observed in the fluorescence spectra of compound 2 in strongly basic environments (pH > 12.0) arises from the electronically excited anionic form A7*, which is generated via absorption of radiation by the 4331


Article


Table 3. Fluorescence Quantum Yields and Kinetic Parameters of Deactivation of Electronically Excited States of the Cationic, Neutral and Anionic Forms of the Compounds Investigateda kf × 10−9 compd 1

b

pH/H0

form

H0 = −2.0 H0 = −1.1

C*/C(1)* C*/C(1)* T7* N* T3* T3* A7* A7T* DA* A7* T7* C*/C(1)* N* T3* A3*

pH = 0.9 pH = 4.5 pH = 9.5

2 3

c

pH = 13.5 pH = 4.5 H0 = −1.1 H0 = −2.0 pH = 1.5 pH = 12.5

φ

τ

exp

theord

kd × 10−9

0.078 0.051 0.004 0.004 0.008

0.63 ± 0.01

0.124

0.213/0.233

1.46

0.030 0.060 0.020

0.078 0.005 0.011 0.004

kprotolytic/kES(I)PT × 10−9 10.3 ± 1.5 (C(1)* → T7* + H+)

0.104 ± 0.009 0.030 ± 0.004 0.168 ± 0.004 1.31 ± 0.02 1.45 ± 0.01 0.08 ± 0.01 0.58 ± 0.01 ∼0.11 0.47 ± 0.08 0.024 ± 0.004 0.202 ± 0.008 1.95 ± 0.07

0.057 0.131 0.060

0.098 0.291 0.134

9.56 10.8 5.89

0.019 0.027 0.250

0.090 0.127 0.143

0.023 0.549 12.3

0.166 0.224 0.103 0.002

0.194/0.215 0.288 0.136 0.105

1.96 17.9 4.90 0.511

22.4 ± 2.5 (N* → T3*) 0.34 ± 0.05 (T3* → A7T*+ H+) 0.72 ± 0.02 (A7* → A7T*) 0.11 ± 0.02 (A7T * → A7*)

23.5 ± 1.5 (N* → T3*)

φ = fluorescence quantum yield; τ = lifetime of the electronically excited state (ns) calculated according to the formula τ = (kf + kd + kprotolytic/ kES(I)PT)−1; kf = rate constant of radiative deactivation (s−1); kd = rate constant of nonradiative deactivation with the exception of protolytic and ES(I)PT processes (s−1); kprotolytic/kES(I)PT = rate constant of protolytic and ES(I)PT processes (s−1). bChart 1. cCharts 2 and S1, Supporting Information. dCalculated according to the formula kf ≈ 0.661·(λf)−2·f, where f is the S1 → S0 transition oscillator strength predicted at the TD DFT(B3LYP)/cc-pVDZ (PCM/water) level of theory. a

neutral environments (Figure 2), leads to the N* species that fluoresce together with T3*, which is produced via ESIPT (Figures 5c,d, S4c,d and S6c, Supporting Information), in a manner similar to that previously described for compound 3. At pH > 1.0, the fluorescence of both neutral forms (N* and T3*) is, however, accompanied by the emission from two additional species, most probably A7* (at 499 nm) and A7T*(at 545 nm), which are both identified in basic environments (Figures 5c,d,e, S4e,f,g,h and S6d,e, Supporting Information). In the 1.0−8.0 pH range, the monoanionic entities can be formed via two protolytic processes, T3* → A7T* + H+ and N* → A7* + H+, followed by ESIPT A7T* ↔ A7*. Because the presence of two monoanionic emitting forms is confirmed by the fluorescence decay data, it is not likely that other protolytic processes that would lead to the formation of only one emitting form A3*, namely, N*/T3* → A3* + H+, would take place. In medium basic environments, compound 1 coexists in neutral (N) and monoanionic (A7) forms in the ground state (Figure 2); thus, the fluorescence spectra in this region result from the combination of decreasing emissions from N* and T3* and increasing emissions from the A7* and A7T* forms as pH increase (Figure 5d,e). The time-dependent fluorescence spectra (Figure S4i,j,k,l, Supporting Information) and fluorescence decay curves (Figure S6f, Supporting Information) indicate that the A7T* species are most probably formed via tautomeric A7* ↔ A7T* (ESIPT) transformation. Molecules of 1, coexisting in the A7 and dianionic (DA) forms in medium to strongly basic environments and existing exclusively in the DA form in strongly basic media in the ground state (Figure 2), emit radiation arising from A7*, A7T*, and DA* (at 509 nm) species (Figure 5e). The existence and coexistence of electronically excited anionic forms is confirmed by the fluorescence decay data (Figure S6g, Supporting Information). Processes that involve the participation of the electronically excited tautomeric/protolytic forms of compound 1 generally occur on the nanosecond time scale (Table 3). Emission of various forms of the compound is quite weak and their lifetimes are relatively short. Among the deactivation processes, fluorescence is relatively slow, whereas nonradiative processes, including

ground-state form A7, only existing under such conditions. Fluorescence data do not provide any evidence for the proton transfer in the excited state. The fluorescence data do, however, demonstrate that the anionic form A7* appears in medium acidic environments, with pH > 1.5. Low stability of the cationic, neutral, and anionic forms of compound 2 in the electronically excited state makes it difficult to gain information regarding their behavior and decay. Rough measurements revealed, however, that the excited anionic form is longer living than the neutral form and that the lifetimes of both of these species are comparable to those of the other two compounds investigated (Table 3). The single band (at 481 nm) in the steady-state fluorescence spectra of 1 (Figure 5a,b) appearing in strongly acidic media (H0 < −4.0) arises from the electronically excited cationic forms C* or C(1)* (fluorescence decay of which is shown in Figure S6a, Supporting Information) which are generated via the absorption of radiation by the ground-state forms C or C(1), which are predominant under such conditions (Figure 2). In strongly to medium acidic environments (−4.0 < H0 < −0.5), the cationic (C/C(1)) and neutral (N) forms of the compound 1 coexist in the ground state and they both absorb and emit radiation. Upon an increase of H0 in this region, the emissions from the cationic C* and C(1)* forms decrease and the emissions from the neutral N* (at 406 nm) and T3* (at 532 nm) forms increase (Figure 5a); the latter species are formed via N* → T3* (ESIPT) tautomeric transformation. Similar to compound 2, compound 1’s long-wavelength emission (at 545 nm) accompanying the fluorescence of C*/C(1)* can be observed in strongly to medium acidic media (Figures 5b and S4a,b, Supporting Information), which arise from the form T7* produced via protolytic dissociation of C(1)*:C(1)* → T7* + H+. Formation of T7* via tautomeric transformation N* → T7* is not likely to occur because it would require the proton transfer between remote sites of the molecule. Emission from T7* (fluorescence decay, which is demonstrated in Figure S6b, Supporting Information) thus disappears, if the cationic forms are not present in the system. Electronic excitation of the neutral molecules of compound 1 (N), which exist predominantly in medium acidic to 4332


Article


been reported for other flavonolic systems so far.54 The fluorescence quantum yield (φ) of the A7* + A7T* forms of 1 is 7.5 times higher than that of the N* + T3* forms, whereas for compound 3 the tendency is reversed, i.e., φ of A3* is 3.75 times lower than that of N* + T3* (Table 3). The relatively high intensity of dual fluorescence of the monoanionic forms of 1 seems to be a unique feature of the compound and was not observed in similar ESIPT fluorophores dissolved in aqueous media (e.g., compound 3). The above-described feature of 1 is most likely the consequence of a difference in the rates of nonradiative deactivation of the monoanionic and neutral forms of the compound. The predicted coexistence of the electronically excited monoanionic forms (A7* and A7T*) of 1 (Table 4) is confirmed by the time-dependent fluorescence spectra, which demonstrate that equilibrium between both forms is reached after roughly 4 ns (Figure S4i,k, Supporting Information). This implies that ESIPT processes in monoanionic forms are much slower than the corresponding processes in neutral forms (Table 3). Such a slow ESIPT rate is a rather rare phenomenon and is characteristic for tautomeric transformations in electron-rich species, like flavonols containing electron-releasing dialkylamino groups.55−57 To summarize of the discussion concerning fluorescent features of 1, Figure 6 illustrates the presence of different electronically excited tautomeric/protolytic forms of the compound in a wide range of environments, from strongly acidic to strongly basic. It is interesting to note that the cationic forms are present at H0 < −0.5 and the dianionic form is present at pH > 12.5, whereas the neutral forms occur in the range −2.0 < H0/pH < 9.5, and the monoanionic forms occur at pH between 1.0 and 13.5. This means that the fluorescence of compound 1 in the whole pH/H0 range investigated arise from one to four emitting tautomeric/protolytic forms. This information is essential for determining the potential applications of the compound (e.g., as fluorescent pH/H0 probe). Protolytic/Tautomeric Transformations at Various pH/ H0. Possible protolytic/tautomeric transformations of compound 1 in the ground and excited electronic states are outlined in Schemes 2 and 3, respectively. These schemes were constructed on the basis of the information compiled in Tables 2 and 4 and presented in the text. Coexistence of the cationic forms C and C(1) in the ground electronic state of 1 (Scheme 2) and 3, predicted by taking into account results of calculations (Table 4, molar ratio of C(1):C should be 0.41 and 0.14 for 1 and 3, respectively), is difficult to notice in the absorption spectra (Figure 1). This may be a result of the overlapping absorption of both tautomeric forms due to the energetic proximity of relevant electronic transitions. However, such an explanation is not confirmed by the results of calculations (Table 2). Upon an increase in pH/H0, two cationic forms of 1 and 3, and the only possible cationic C form of 2 are transformed into the neutral form N because other tautomeric forms (T3 (1 and 3) or T7 (1 and 2)) are thermodynamically less stable (Table 4). Protolytic transformation of neutral (N) to monoanionic (A7 (1 and 2) or A3 (3)) forms, and further of monoanionic to dianionic forms of 1, complete the picture of acid−base equilibria in the ground electronic state, which occur in the compounds investigated. Absorption of radiation by coexisting in the ground state the C and C(1) tautomeric forms of 1 and 3 or the C form of 2 leads to the electronically excited species, which can emit radiation or undergo tautomeric transformation (1 and 3) to the thermodynamically more stable C(1)* form (Scheme 3, Table 4; molar

Figure 4. Experimental (upper graphs) fluorescence spectra of 2 (“au”, arbitrary units; bars, wavelength of fluorescence maxima given in Table 2) in a water/methanol phase (concentration ca. 10−5 M) at various pH/H0 (arrows, pH/H0 rise) together with predicted (lower graphs) positions of emission transitions (bars, corrected wavelength of S1 → S0 transitions). N*, T*, C*, and A* denote the electronically excited forms of the compound (Chart 2).

tautomeric/protolytic transformations, are faster and processes involving cationic, neutral and dianionic forms of the compound are the fastest. All deactivation processes involving electronically excited monoanionic forms are slow and thus their lifetimes are relatively long. The most distinguishable feature of 1 is its relatively intense dual fluorescence in basic solutions, a characteristic that has not 4333


Article


Figure 5. Experimental (upper graphs) fluorescence spectra of 1 (“au”, arbitrary units; bars, wavelength of fluorescence maxima given in Table 2) in a water/methanol phase (concentration ca. 10−5 M) at various pH/H0 (arrows, pH/H0 rise) together with the predicted (lower graphs) positions of emission transitions (bars, corrected wavelength of S1 → S0 transitions). N*, T*, C*, C(1)*, A*, AT*, and DA* denote the electronically excited forms of the compound (Chart 2).

undergo tautomeric transformation to the T3* form; the latter species could also be formed via direct protolytic dissociation of C* generated upon absorption of radiation by dominating in the ground-state C form. The contribution of the latter two processes is rather minor, as in the case of compound 2, whose cationic form undergo dissociation mainly to the T7* tautomeric form when the media is changed from strongly to medium acidic (Figure 4). The absorption of radiation by the neutral forms N of the compounds investigated lead to the electronically excited N*

ratio of C(1)*:C* is predicted to be 9.1 and 10.9 for 1 and 3, respectively). The presence of two fluorescing forms is not confirmed by the fluorescence data of 1 and 3 (Figures 3, 5 and S3, S4 (Supporting Information)). This may be due to the energetic proximity of the relevant fluorescence transitions (Table 2) and the overlapping emissions of both tautomeric forms. Protolytic dissociation of the C(1)* form of 1 leads to the least thermodynamically stable neutral form T7* emitting radiation under strongly to medium acidic conditions (Figure 6). C(1)* could also dissociate to the N* form and subsequently 4334


Article

The Journal of Physical Chemistry A Table 4. Theoretically Predicted Values of Δ298G° (kJ/mol) of the Tautomeric Transformations of Compounds 1, 2, and 3 in the S0 and S1 States compd

reaction

S0

S1

1

C → C(1) N → T3 N → T7 A7 → A7T A7 → A3 N → T7 C → C(1) N → T3

2.2 43.2 80.9 27.5 28.8 72.8 4.8 37.3

−5.5 −40.2 0.4 −7.5 (−4.7)a 8.1 16.2 −5.9 −33.5

2 3

Scheme 3. Protolytic/Tautomeric Transformations of Compound 1 in the Electronically Excited Statea

a

For tautomeric transformations, the predicted and experimental (in parentheses) values of Δ298G° (kJ/mol) are indicated (Table 4). For protolytic reactions, the predicted values of Δ298G° (corrected) are shown above the arrows and the pKa* values are present below the arrows. The corresponding experimental values are shown in parentheses (Table 1). The symbols in circles represent canonical structures of the compounds as described in Chart 2.

Obtained on the basis of experimental rates of direct (A7* → A7T*) and reverse (A7T* → A7*) reactions (Table 3) according to equation Δ298G° = −RT ln(kA7*→A7T*/kA7T*→A7*). a

For tautomeric transformation, the predicted values of Δ298G° (kJ/mol) are indicated (Table 4). For protolytic reactions, the predicted values of Δ298G° (corrected) are placed above the arrows and pKa values are below the arrows. The corresponding experimental values (from Table 1) are shown in parentheses. Symbols in the circles represent canonical structures of the compounds, as described in Chart 2.

T3* leads to the formation of the monoanionic form A7T*. The coexistence of the monoanionic form A7T* with the A7* one in the electronically excited state (Figure 6) is confirmed by the time-dependent fluorescence spectra (Figure S4i,k, Supporting Information). According to the results of calculations (Table 3), protolytic dissociation of N* to A7* is also a thermodynamically spontaneous process; however, the intramolecular tautomeric transformation N* → T3* is thermodynamically (Table 4) and kinetically favorable (according to the timedependent fluorescence spectra presented in Figure S4e,g, Supporting Information). Therefore, transformations of compound 1 in medium acidic to neutral media can be described by the scheme N* → T3* → A7T* ↔ A7* (Scheme 3), which explains the presence of monoanionic forms of the compound under these conditions. In the case of compound 2, dissociation of N* (which is formed as a result of the absorption of radiation by neutral molecules) and T7* (which is generated via protolytic dissociation of electronically excited cations) to A7* occurs in medium acidic media (Figure 4, Table 3). Under highly basic conditions, the dianionic form of compound 1 is present and absorbs and emits radiation. Monoanionic forms of compounds 2 (A7*) and 3 (A3*) occur upon electronic excitation of the compounds in alkaline media. The predicted values of the thermodynamic characteristics for protolytic dissociation processes obtained by assuming the involvement of water molecule are, in some cases, different from the experimental values (Table 3). Nevertheless, due to the lack of experimental data, predicted values form a useful framework on which to consider protolytic transformations in excited molecules and to explain experimental findings. The computationally predicted energies and wavelengths of the S1−S0 electronic transitions of cationic, neutral, and anionic forms of 1 are much lower than that of the S2−S0 transitions (Table S4, Supporting Information). These findings confirm the suggestion that S1 is the emittive state in all the tautomeric/ protolytic forms. The frontier orbitals involved in S0−S1 and S1−S0 transitions are generally localized on the whole molecule (Figure S7, Supporting Information).

form which in the case of 1 and 3 undergo ESIPT to produce a thermodynamically more stable T3* form (Table 4, coexistence of pairs of relevant tautomers is reflected in the relevant fluorescence spectra, Figures 3 and 5). Protolytic dissociation of

CONCLUSIONS On the basis of the current investigations and quantum-chemical calculations on 3,7-dihydroxyflavone and its related monohydroxy derivatives, we demonstrate that in the ground state, this

Figure 6. Fluorescence intensities (in arbitrary units, au), corresponding to maxima (indicated, in nm, in parentheses (Table 2)) in deconvoluted spectra, of tautomeric/protolytic forms of 1 (Chart 2) at various pH/H0.

Scheme 2. Protolytic/Tautomeric Transformations of Compound 1 in the Ground Statea

a

■

4335



■

compound exists in four protolytic forms, cationic, neutral, anionic, and dianionic, whereas in the electronically excited state, three former species undergo various types of proton transfer in a wide range of pH/H0. For these reasons, 3,7-dihydroxyflavone exhibits multiband fluorescence under a wide pH/H0 range (including physiological pH) arising from various protolytic/ tautomeric forms (cationic, three neutral tautomeric, two monoanionic tautomeric and dianionic) coexisting in the excited state via proton transfer reactions. From the cognitive point of view, the most interesting observation is the fluorescent behavior of the compound in neutral and low-acidic solutions, as in such media two neutral and two monoanionic tautomeric, electronically excited, forms coexist via protolytic dissociation (T3* → A7T* + H+) and intramolecular (N* → T3*, A7T* ↔ A7*) proton transfer. In the 8.4 < pH < 12.0 range, two monoanionic forms coexist via one of the slowest ESIPT process reported so far. Under these conditions, 3,7-dihydroxyflavone exhibits one of the most intensive dual fluorescence recorded in water/methanol media, which makes the compound particularly interesting from the cognitive and possibly applicative point of view. Prospectively, the compound can be used as a multiparametric fluorescence probe to measure pH and concentration of molecules with low deprotonating abilities. The spectral features of the monohydroxyflavones investigated (compounds 2 and 3) are very similar to those previously reported for 7-hydroxyflavone38 and 3-hydroxyflavone.54,58

■

REFERENCES

(1) Demchenko, A. P. Introduction to Fluorescence Sensing; Springer: Netherlands, 2009. (2) Miao, Q.; Wu, Z.; Hai, Z.; Tao, C.; Yuan; Gong, Y.; Guan, Y.; Jiang, J.; Liang, G. Bipyridine Hydrogel for Selective and Visible Detection and Absorption of Cd2+. Nanoscale 2015, 7, 2797−2804. (3) Xiao, K.; Nie, H.; Gong, C.; Ou, X.; Tang, Q.; Chow, C. A. Colorimetric and Fluorescent Dual-Channel Cyanide Ion Probe Using Crosslinked Polymer Microspheres Functionalized with Protonated Brooker’s Merocyanine. Dyes Pigm. 2015, 116, 82−88. (4) Chen, G.; Liua, J.; Liua, M.; Lia, G.; Sun, Z.; Zhang, S.; Song, C.; Wang, H.; Suo, Y.; Youa, J. Sensitive, Accurate and Rapid Detection of Trace Aliphatic Amines in Environmental Samples with UltrasonicAssisted Derivatization Microextraction Using a New Fluorescent Reagent for High Performance Liquid Chromatography. J. Chromatography A 2014, 1352, 8−19. (5) Joshi, H.; Sengupta, A.; Gavvala, K.; Hazra, P. Unraveling the Mode of Binding of the Anticancer Drug Topotecan with dsDNA. RSC Adv. 2014, 4, 1015−1024. (6) Paredes, J. M.; Giron, M. D.; Ruedas-Rama, M. J.; Orte, A.; Crovetto, L.; Talavera, E. M.; Salto, R.; Alvarez-Pez, J. M. Real-Time Phosphate Sensing in Living Cells Using Fluorescence Lifetime Imaging Microscopy (FLIM). J. Phys. Chem. B 2013, 117, 8143−8149. (7) De, D.; Santra, K.; Datta, A. Prototropism of [2,2′-Bipyridyl]-3,3′diol in Albumin−SDS Aggregates. J. Phys. Chem. B 2012, 116, 11466− 11472. (8) Cohen, B.; Alvarez, C. M.; Carmona, N. A.; Organero, J. A.; Douhal, A. Proton-Transfer Reaction Dynamics within the Human Serum Albumin Protein. J. Phys. Chem. B 2011, 115, 7637−7647. (9) Sarangi, M. K.; Mitra, A.; Basu, S. Prototropic Interactions of Pyrimidine Nucleic Acid Bases with Acridine: A Spectroscopic Investigation. J. Phys. Chem. B 2012, 116, 10275−10282. (10) Sardar, P. S.; Samanta, S.; Maity, S. S.; Dasgupta, S.; Ghosh, S. Energy Transfer Photophysics from Serum Albumins to Sequestered 3Hydroxy-2-Naphthoic Acid, an Excited State Intramolecular ProtonTransfer Probe. J. Phys. Chem. B 2008, 112, 3451−3461. (11) Dumas, A.; Luedtke, N. W. Site-Specific Control of N7−Metal Coordination in DNA by a Fluorescent Purine Derivative. Chem. - Eur. J. 2012, 18, 245−254. (12) Strizhak, A. V.; Postupalenko, V. Y.; Shvadchak, V. V.; Morellet, N.; Guittet, E.; Pivovarenko, V. G.; Klymchenko, A. S.; Mély, Y. TwoColor Fluorescent L-Amino Acid Mimic of Tryptophan for Probing Peptide-Nucleic Acid Complexes. Bioconjugate Chem. 2012, 23, 2434− 2443. (13) Postupalenko, V. Y.; Zamotaiev, O. M.; Shvadchak, V. V.; Strizhak, A. V.; Pivovarenko, V. G.; Klymchenko, A. S.; Mely, Y. DualFluorescence L-Amino Acid Reports Insertion and Orientation of Melittin Peptide in Cell Membranes. Bioconjugate Chem. 2013, 24, 1998−2007. (14) Klymchenko, A. S.; Shvadchak, V. V.; Yushchenko, D. A.; Namrata, J.; Mély, Y. Excited-State Intramolecular Proton Transfer Distinguishes Microenvironments in Single- and Double-Stranded DNA. J. Phys. Chem. B 2008, 112, 12050−12055. (15) Shynkar, V. V.; Klymchenko, A. S.; Kunzelmann, C.; Duportail, G.; Muller, C. D.; Demchenko, A. P.; Freyssinet, J.-M.; Mely, Y. Fluorescent Biomembrane Probe for Ratiometric Detection of Apoptosis. J. Am. Chem. Soc. 2007, 129, 2187−2193. (16) Ma, J.; Liu, Y.; Chen, L.; Xie, Y.; Wang, L.; Xie, M. Spectroscopic Investigation on the Interaction of 3,7-Dihydroxyflavone with Different Isomers of Human Serum Albumin. Food Chem. 2012, 132, 663−670. (17) Xiao, J.; Cao, H.; Wang, Y.; Yamamoto, K.; Wei, X. Structure− Affinity Relationship of Flavones on Binding to Serum Albumins: Effect of Hydroxyl Groups on Ring A. Mol. Nutr. Food Res. 2010, 54, S253− S260. (18) 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. (19) Chaudhuri, S.; Basu, K.; Sengupta, B.; Banerjee, A.; Sengupta, P. K. Ground- and Excited-State Proton Transfer and Antioxidant Activity

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpca.6b03290. Selected thermodynamic and spectral characteristics of the compounds investigated: computationally predicted Gibbs free energies, information on possible protolytic dissociation reactions, information on spectral correction coefficients, canonical structures of protolytic/tautomeric forms of 1 not occurring in media of various pH/H0, computationally predicted structures, frontier orbitals and parameters of the S1−S0 and S2−S0 electronic transitions in 1, titration curves for the compounds investigated, timedependent and steady-state fluorescence spectra and fluorescence decay curves of the compounds investigated at various pH/H0, conditions of determination of fluorescence quantum yields (PDF)

■

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +48 58 523 51 13. Phone/fax: +48 58 523 50 12. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The research was financed by the Polish National Science Centre (NCN) under Grant No. 2014/13/N/ST4/04105. Quantum chemical calculations were performed on the computers of the Ukrainian-American Laboratory of Computational Chemistry (UALCC, Kharkiv, Ukraine). I.E.S. is grateful to the Polish Bureau for Academic Recognition and International Exchange (BUWiWM) for scholarship during his Ph.D. studies. 4336


Article

The Journal of Physical Chemistry A of 3-Hydroxyflavone in Egg Yolk Phosphatidylcholine Liposomes: Absorption and Fluorescence Spectroscopic Studies. Luminescence 2008, 23, 397−403. (20) Neves, M. P.; Cidade, H.; Pinto, M.; Silva, A. M. S.; Gales, L.; Damas, A. M.; Lima, R. T.; Vasconcelos, M. H.; Nascimento, M. S. J. Prenylated Derivatives of Baicalein and 3,7-Dihydroxyflavone: Synthesis and Study of Their Effects on Tumor Cell Lines Growth, Cell Cycle and Apoptosis. Eur. J. Med. Chem. 2011, 46, 2562−2574. (21) Bojić, M.; Debeljak, Ž .; Tomičić, M.; Medić-Šarić, M.; Tomić, S. Evaluation of Antiaggregatory Activity of Flavonoid Aglycone Series. Nutr. J. 2011, 10, 73. (22) Ravishankar, D.; Rajora, A. K.; Greco, F.; Osborn, H. M. I. Flavonoids as Prospective Compounds for Anti-Cancer Therapy. Int. J. Biochem. Cell Biol. 2013, 45, 2821−2831. (23) Lemańska, K.; Szymusiak, H.; Tyrakowska, B.; Zieliński, R.; Soffers, A. E. M. F.; Rietjens, I. M. C. M. The Influence of pH on Antioxidant Properties and the Mechanism of Antioxidant Action of Hydroxyflavones. Free Radical Biol. Med. 2001, 31, 869−881. (24) Chieli, E.; Romiti, N.; Zampini, I. C.; Garrido, G.; Isla, M. I. Effects of Zuccagnia Punctata Extracts and Their Flavonoids on the Function and Expression of ABCB1/P-glycoprotein Multidrug Transporter. J. Ethnopharmacol. 2012, 144, 797−801. (25) Schwartz, B. J.; Peteanu, L. A.; Harris, C. B. Direct Observation of Fast Proton Transfer: Femtosecond Photophysics of 3-Hydroxyflavone. J. Phys. Chem. 1992, 96, 3591−3598. (26) Ameer-Beg, S.; Ormson, S. M.; Brown, R. G.; Matousek, P.; Towrie, M.; Nibbering, E. T. J.; Foggi, P.; Neuwahl, F. V. R. Ultrafast Measurements of Excited State Intramolecular Proton Transfer (ESIPT) in Room Temperature Solutions of 3-Hydroxyflavone and Derivatives. J. Phys. Chem. A 2001, 105, 3709−3718. (27) Strandjord, A. J. G.; Barbara, P. F. Proton-Transfer Kinetics of 3Hydroxyflavone: Solvent Effects. J. Phys. Chem. 1985, 89, 2355−2361. (28) Grotewold, E. The Science of Flavonoids; Springer-Verlag: New York, 2006. (29) 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. (30) Voicescu, M.; Ionescu, S.; Gatea, F. Effect of pH on the Fluorescence Characteristics of Some Flavones Probes. Spectrochim. Acta, Part A 2014, 123, 303−308. (31) Sengupta, B.; Banerjee, A.; Sengupta, P. K. Interactions of the Plant Flavonoid Fisetin with Macromolecular Targets: Insights from Fluorescence Spectroscopic Studies. J. Photochem. Photobiol., B 2005, 80, 79−86. (32) Chaudhuri, S.; Chakraborty, S.; Sengupta, P. K. Probing the Interactions of Hemoglobin with Antioxidant Flavonoids via Fluorescence Spectroscopy and Molecular Modeling Studies. Biophys. Chem. 2011, 154, 26−34. (33) Sengupta, B.; Pahari, B.; Blackmon, L.; Sengupta, P. K. Prospect of Bioflavonoid Fisetin as a Quadruplex DNA Ligand: A Biophysical Approach. PLoS One 2013, 8, e65383. (34) Pahari, B.; Sengupta, B.; Chakraborty, S.; Thomas, B.; McGowan, D.; Sengupta, P. K. Contrasting Binding of Fisetin and Daidzein in γCyclodextrin Nanocavity. J. Photochem. Photobiol., B 2013, 118, 33−41. (35) Chaudhuri, S.; Pahari, B.; Sengupta, B.; Sengupta, P. K. Binding of the Bioflavonoid Robinetin with Model Membranes and Hemoglobin: Inhibition of Lipid Peroxidation and Protein Glycosylation. J. Photochem. Photobiol., B 2010, 98, 12−19. (36) Arjunan, V.; Devi, L.; Subbalakshmi, R.; Rani, T.; Mohan, S. Synthesis, Vibrational, NMR, Quantum Chemical and StructureActivity Relation Studies of 2-Hydroxy-4-methoxyacetophenone. Spectrochim. Acta, Part A 2014, 130, 164−177. (37) Serdiuk, I. E.; Roshal, A. D.; Błażejowski, J. Quantum-Chemical Analysis of the Algar−Flynn−Oyamada Reaction Mechanism. Chem. Heterocycl. Compd. 2014, 50, 396−403. (38) Serdiuk, I. E.; Varenikov, A. S.; Roshal, A. D. 7-Hydroxyflavone Revisited: Spectral, Acid−Base Properties, and Interplay of the Protolytic Forms in the Ground and Excited States. J. Phys. Chem. A 2014, 118, 3068−3080.

(39) Mulder, W. H. Effect of Medium Relaxation on the Acidity Constants of Electronically Excited States Obtained by the Förster Cycle Method. J. Photochem. Photobiol., A 2003, 161, 21−25. (40) Fraser, R. D. B.; Suzuki, E. In Spectral Analysis; Blackburn, J. A., Ed.; Marcel Dekker: New York, 1970; p 171. (41) Ware, W. R.; Baldwin, B. A. Absorption Intensity and Fluorescence Lifetimes of Molecules. J. Chem. Phys. 1964, 40, 1703− 1705. (42) Doroshenko, A. O.; Posokhov, E. A. Mechanisms of Photoinduced Protolytic Interactions of 2,5-Diphenyloxazole ortho-Hydroxy Derivatives in Media of Various Acidities. High Energy Chem. 2000, 34, 93−100. (43) Doroshenko, A. O.; Kirichenko, A. V.; Mitina, V. G.; Ponomaryov, O. A. Spectral Properties and Dynamics of the Excited State Structural Relaxation of the ortho-Analogues of POPOP − Effective Abnormally Large Stokes Shift Luminophores. J. Photochem. Photobiol., A 1996, 94, 15−26. (44) Doroshenko, A. O. Spectral Data Lab software; V. N. Karazin Kharkiv National University: Kharkiv, Ukraine, 1999. (45) Maus, M.; Rettig, W. The Excited State Equilibrium Between Two Rotational Conformers of a Sterically Restricted Donor-Acceptor Biphenyl as Characterized by Global Fluorescence Decay Analysis. J. Phys. Chem. A 2002, 106, 2104−2111. (46) Labanowski, J. K. In Density Functional Methods in Chemistry; Andzelm, J. W., Ed.; Springer-Verlag: New York, 1991. (47) Becke, A. D. A New Mixing of Hartree-Fock and Local-DensityFunctional Theories. J. Chem. Phys. 1993, 98, 1372−1377. (48) Becke, A. D. Density-Functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648−5652. (49) Tomasi, J.; Persico, M. Molecular Interactions in Solution: An Overview of Methods Based on Continuous Distributions of the Solvent. Chem. Rev. 1994, 94, 2027−2094. (50) Barone, V.; Cossi, M.; Tomasi, J. A New Definition of Cavities for the Computation of Solvation Free Energies by the Polarizable Continuum Model. J. Chem. Phys. 1997, 107, 3210−3221. (51) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.; et al. Gaussian 09, Revision C.02; Gaussian, Inc.: Wallingford, CT, 2004. (52) Runge, E.; Gross, E. K. U. Density-Functional Theory for TimeDependent Systems. Phys. Rev. Lett. 1984, 52, 997−1000. (53) Varenikov, A. S.; Serdiuk, I. E.; Roshal, A. D. Spectral and Basic Properties of Flavones in the Ground and Excited States. FrenchUkrainian Journal of Chemistry 2013, 1, 153−157. (54) Moroz, V. V.; Chalyi, A. G.; Serdiuk, I. E.; Roshal, A. D.; Zadykowicz, B.; Pivovarenko, V. G.; Wróblewska, A.; Błażejowski, J. Tautomerism and Behavior of 3-Hydroxy-2-Phenyl-4H-Chromen-4ones (Flavonols) and 3,7-Dihydroxy-2,8-Diphenyl-4H,6H-pyrano[3,2g] Chromene-4,6-Diones (Diflavonols) in Basic Media: Spectroscopic and Computational Investigations. J. Phys. Chem. A 2013, 117, 9156− 9167. (55) Chou, P.-T.; Huang, C.-H.; Pu, S.-C.; Cheng, Y.-M.; Liu, Y.-H.; Wang, Y.; Chen, C.-T. Tuning Excited-State Charge/Proton Transfer Coupled Reaction via the Dipolar Functionality. J. Phys. Chem. A 2004, 108, 6452−6454. (56) Roshal, A. D.; Organero, J. A.; Douhal, A. Tuning the Mechanism of Proton-Transfer in a Hydroxyflavone Derivative. Chem. Phys. Lett. 2003, 379, 53−59. (57) Ghosh, D.; Batuta, S.; Begum, N. A.; Mandal, D. Unusually Slow Intramolecular Proton Transfer Dynamics of 4′-N,N-Dimethylamino-3hydroxyflavone in High n-Alcohols: Involvement of Solvent Relaxation. Photochem. Photobiol. Sci. 2016, 15, 266−277. (58) 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.

4337


Origin of Spectral Features and AcidâBase Properties of 3,7

Recommend Documents

Origin of Spectral Features and AcidâBase Properties of 3,7