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The Journal of Physical Chemistry
Excited-State Intramolecular Proton Transfer of the Natural Product Quercetin Ron Simkovitch and Dan Huppert* Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel *Corresponding author: Dan Huppert E-mail: [email protected] Phone: 972-3-6407012 Fax: 972-3-6407491
Abstract Intramolecular proton-transfer dynamics in the lowest excited state (ESIHT) were studied in the natural product quercetin. We found that in all seven solvents used in this study, the ESIHT rate is ultrafast. We estimate that the ESIHT rate is about 70 fs or less. We found that in deuterated protic solvents, like methanol-d or ethanol-d, the ESIHT rate is slower and the proton-transfer time constant is about 110 fs. The tautomeric form fluorescence quantum yield of quercetin is very low, of the order of the normal form.
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Introduction Quercetin, shown in Scheme 1, is a flavonol and is widely distributed in vegetables, leaves and fruit in substantial amounts. It has recently been used as a food supplement, probably because of its reported anticancer1,2, antiviral3 and antiinflammatory4 properties.
B A
C
Scheme 1: Molecular structure and numbering pattern of quercetin
In the field of photochemistry one often encounters excited-state proton-transfer processes5. These photoprotolytic processes can be divided into two categories. The first is excited-state intermolecular proton transfer (ESPT)6,7,8,9,10,11,12. In such a reaction a proton is transferred to a solvent molecule, most commonly water. Molecules in which this process occurs are called photoacids. In their ground state, these molecules are weak acids with values of pKa that range between 5-10. When photoacids are raised to their first excited state, there is a large change in acidity and the value of their pKa* is lower than that of their pKa by about 7-11 orders of magnitude. The values of pKa* of photoacids range from 3 to ~-8. The ESPT rate constant for compounds with pKa*~-8 is kPT~1013s-1 (τPT~100fs). Because of the radiative lifetime of singlet states, the smallest ESPT rate that can be measured by time-resolved fluorescence is about 107s-1 and corresponds to compounds with pKa* of 3.3. The second category of compounds undergoes an intramolecular hydrogen transfer (ESIHT)13,14,15,16,17,18,19,20,21,22,23,24. The structure of these compounds consists of a proton acceptor and a proton donor in close proximity. Usually the proton donor is a hydroxyl group of a hydroxyaryl moiety and the proton acceptor is a heterocyclic nitrogen atom, or a carbonyl oxygen atom in the keto form. Several compounds are considered prototype compounds for ESIHT. Two ESIHT compounds possess heterocyclic nitrogen as a proton acceptor, 10-
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hydroxybenzoquinoline (10-HBQ) and hydroxyphenyl benzothiazol (HBT). Recent studies on HBT and 10-HBQ in protic solvents, using ultrashort laser pulses of τpulse≈30fs have shown that ESIHT occurs in both compounds within the time resolution of the laser apparatus, i.e ~30fs. Riedle and coworkers15 reported a time constant of ~30fs for an ESIHT in HBT. Similar conclusions were drawn for the ESIHT of 10-HBQ by Tahara and coworkers14. Another class of excited-state proton transfer occurs between a hydroxyaryl group next to an aromatic carbonyl group. An example of molecules of this type is the flavonols. 3-hydroxy-flavone (3-HF) 25,26,27,28,29,30 and, to a lesser extent, 5-hydroxyflavone (5-HF) 31,32,33,34 were extensively studied. Both steady-state fluorescence and time-resolved techniques were employed to study the ESIHT process of these two molecules. In the current work we studied the photophysics and photochemistry of quercetin in solution. For that purpose we used steady-state and time-resolved fluorescence techniques We found an ESIHT process that is ultrafast in polar nonprotic and protic solvents. The proton (hydrogen) transfer occurs with a time constant of about 70fs. In deuterated protic solvents like methanol-d and ethanol-d the ESIHT is slower and the kinetic isotope effect is about 1.5. The quantum efficiency of the tautomeric-form fluorescence of quercetin is rather small.
Materials and Methods Quercetin (of purity >95.5%) was purchased from Sigma-Aldrich, no further purification was done to the samples. All measurements were carried out with fresh solutions of Quercetin. Dichloromethane, DMSO, acetonitrile, methanol, ethanol and other solvents used in this study were of HPLC or analytical grade and were purchased from Sigma-Aldrich as well. A fluorescence up-conversion technique was used to measure the timeresolved emission of quercetin at room temperature. A cavity-dumped Ti:Sapphire femtosecond laser (Mira, Coherent) is used as an excitation source for the fluorescence up-conversion measurements. This laser output consists of 120fs, pulses at about 800nm. The cavity dumper operated at a repetition rate of 800 kHz. A 3 Environment ACS Paragon Plus
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fluorescence up-conversion system (FOG-100, CDP, Russia) was used to measure the time-resolved fluorescence of quercitin The response time of the up-conversion system is measured by monitoring the Raman-Stokes line of water. We found that the full width at half maximum of the signal is 240 fs. To avoid photochemistry, we placed the samples in a rotating optical cell. The sample degradation during the measurement was rather small. For the collection of the steady-state fluorescence and the absorption spectra we used a Horiba Jobin Yvon FluroMax-3 spectrofluorometer and a Cary 5000 spectrometer. Results Steady-State Measurements The absorption spectra of quercitin in protic solvents depend on the solvent pH. This is explained by Momic et al.35 and others as arising from the possibility of several protonation deprotonation states of the 5 hydroxyl groups of quercetin (see scheme 1). Figure S1 the supporting-information (SI) section shows the absorption spectrum of quercitin in methanol and in methanolic solutions containing also NaOH (in water) of mM concentrations (pH>7). As seen in the figure, the spectra are sensitive to the solution pH. The absorption band peak assigned to S0S1 transition for the protonated form is at about 380nm. When the solution pH increases, the deprotonation of the first two protons leads to a red shift of the S0S1 transition. The deprotonation of the third proton at about pH~11 shifts the S0S1 transition to the blue and the band's peak position is at 340 nm. A detailed analysis of the absorption spectra as a function of pH is given by Momic et al35. Figure 1a shows the normalized steady-state fluorescence spectrum of quercetin in dichloromethane (DCM) excited at three wavelengths in the spectral region of 350390nm. The fluorescence quantum yield of quercetin is low as was also found for similar ESIHT compounds 3-HF and 5-HF.
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a)
350 nm 370 nm 390 nm
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0.6 0.4 0.2 0.0 400
450
500
550
600
650
700
Wavelength [nm]
Figure 1: Normalized steady-state fluorescence spectra of quercetin in a. dichloromethane. b. DMSO c. ethanol and ethanol-d
The fluorescence spectrum consists of two emission bands. The blue band with a maximum at 440-470nm is assigned to the spectrum of the normal form, while the red band with a maximum at ~585nm is assigned to the tautomeric form. The blue-band spectrum in DCM has a vibronic substructure with about four sub bands. The red band of the enol form is broad and structureless. The band position is nearly constant when excited to the S1 state at long wavelengths (λ≥390nm). When excited at short wavelengths (λ≤370nm) to the S2 state, the emission-band position is blue-shifted by F F about 15nm. The band-intensity ratio, I red / I blue ≈ 2.5 , is nearly constant at all
excitation wavelengths, except when the excitation is at the long wavelength of λex=430nm. Figure 1b shows the normalized steady-state emission and excitation spectra of quercetin in DMSO. The fluorescence spectrum is measured at three excitation
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wavelengths. At short excitation wavelengths, (λ≤390nm), the spectrum consists of the normal-form emission band with a maximum at about 450nm and a weaker vibronic band at 472nm. The red tautomeric-form emission band is weaker by about a factor of ten and the band maximum is at 550nm. Figure 1c shows the normalized steady-state emission spectrum of quercetin in ethanol and ethanol-d. In both spectra two emission bands are observed. The blue band with a maximum around 450 nm is attributed to the "N" form of quercitin and the red band with a maximum at about 540 nm is attributed to the "T" band. The red tail of the "T" form extends to longer wavelengths than in the case of quercetin in dichloromethane shown in figure 1a. This large change in the "T" spectrum arises from the large hydrogen bond of quercetin in the carbonyl or all five hydroxyls. It may also arise from the existence of deprotonated hydroxyls like those at 4',5' and 7 positions (see scheme 1). There are two sub-bands in the "T" emission spectrum at about 610 nm and 660 nm. They can be assigned to vibrational transitions or to different deprotonated RO- form of quercetin. As seen in the figure, the amplitude ratio I "FN " / I"FT " of the two fluorescence bands of the normalized spectrum for the ethanol-d sample is about twice larger than that of the ethanol sample. This difference in the amplitude ratio can be interpreted as arising from a kinetic isotope effect (KIE) of the ESIHT process. As we will show later on based on the time-resolved measurement the KIE we estimate for this process is KIE≥1.5. Time-resolved emission Figures 2a and 2b show, on linear and semilogarithmic scales, the time-resolved fluorescence of quercetin in acetonitrile solution measured at several wavelengths over the spectral region of 450-500nm.
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0.4
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450nm 460nm 470nm 480nm 490nm 500nm
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0.2 0.01
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Time (PS)
Figure 2: Time-resolved fluorescence of quercetin in an acetonitrile solution a. Normalized linear scale, showing the IRF lamp signal b. Normalized semilogarithmic scale.
The fluorescence signals were acquired by the fluorescence up-conversion technique. The system response was determined by measuring the Raman water band. The instrument response at full-width half maximum was 240±20fs. As seen in the figure, the quercetin signal decay of the normal-form band is rather short and is close to the system response. The major ultrafast-decay component is followed by a longer decay component of much smaller amplitude. Similar results are obtained for the timeresolved emission of quercitin in DMSO, for which the results are shown in Figure S2 in the SI section. We used a multiexponential function to fit the fluorescence decay of the signals shown in Figure 2. The results are given in Table 1. λ [nm]
a1
τ1[fs]
a2
τ2[fs]
a3a
440
0.973
75
0.02
300
0.007
450
0.978
75
0.021
350
0.0011
460
0.967
78
0.032
350
0.0011
470
0.965
75
0.032
310
0.0030
480
0.959
70
0.037
320
0.0040
490
0.959
65
0.037
420
0.0040
Table 1: multiexponential-fit function parameters for the fluorescence decay of quercetin in an acetonitrile solution. b a
The amplitude of the long-time component is the sum of the amplitudes of two components in the picosecond range. b
τpulse is about 240fs, τ3=2000fs, τ4=34ps.
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The major amplitude time component is estimated to be shorter than 100fs. For the fit we use τ=70fs. The second time component is about 300fs with an amplitude of ~0.04. Longer time components with a total amplitude of less than ~0.01 also appear in the signals. The longer the monitored wavelength, the higher the amplitude of the long-time components. Figure 3 shows the time-resolved emission of quercetin in methanol solution, measured over the spectral region of 440-490nm.
440nm 450nm 460nm 470nm 480nm 490nm Lamp
0.8 0.6 0.4
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a) 1.0
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1
440nm 450nm 460nm 470nm 480nm 490nm
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Figure 3: Time-resolved fluorescence of quercetin in methanol a. Normalized linear scale, showing the IRF lamp signal b. Normalized semilogarithmic scale.
The signal decay is rather short, similar to the signals of quercetin in acetonitrile, shown in Figure 2. The difference in the signal decay is that at higher wavelengths, (λ≥480nm), the amplitude of the long-time component is much greater than in the acetonitrile sample. We conclude that the excited-state intramolecular proton transfer occurring in quercetin in both solvents is short, probably equal to or shorter than 70fs. The kinetic isotope effect Figure 4 shows the time-resolved fluorescence of quercetin in both methanol and methanol-d.
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λ=440nm
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λ=460nm
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Figure 4: Time-resolved fluorescence of quercetin in methanol compared to methanol-d, shown in normalized linear scale at various wavelengths.
Each panel shows the signals measured at the same wavelength for both solvents. As is clearly seen in the figure, there is a distinct difference in both the short and long times of the fluorescence decay of quercetin in methanol and methanol-d. The shorttime component of the decay time of the normal-form band of quercetin measured in the blue spectral region, 440-500nm, is 110±15fs in methanol-d and 70±10fs in methanol. The longer-time component in the methanol-d decay time is about 700fs, compared to about 300fs in methanol. The amplitude of the long-time decay component is much larger in methanol-d than in methanol or in acetonitrile. Table 2 provides the fitting parameters of the multiexponential-fit function of both methanol and methanol-d.
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λ [nm]
a1
τ1[fs]
a2
τ2[fs]
a3a
440
0.956
72
0.035
250
0.009
450
0.953
70
0.036
270
0.011
460
0.954
70
0.036
350
0.01
470
0.928
70
0.059
260
0.013
480
0.927
68
0.060
270
0.013
440
0.948
110
0.039
800
0.013
450
0.926
115
0.060
800
0.014
460
0.890
110
0.091
750
0.019
470
0.852
110
0.122
700
0.026
480
0.836
100
0.127
700
0.037
Table 2: multiexponential-fit function parameters for the fluorescence decay of quercetin in methanol and methanol-d solutions b. a
The amplitude (a3) of the long-time component is the amplitude sum of the two components in the picosecond range. b
τpulse is about 240fs, τ3=2000fs, τ4=34ps. Figure S3 in the SI section shows the time-resolved emission of quercetin in
methanol-d on linear and semilogarithmic scales. Similar results are obtained for quercetin in water/ethanol and D2O/ethanol-d samples of ratios of 1:1 by volume. Figure S4 in the SI shows on linear and semilogarithmic scales the time-resolved emission of quercetin in these solvent mixtures. Each panel shows the signals measured at the same wavelength for ethanol/H2O and ethanol-d/D2O solutions. As seen in Figure S4, the short and long decay times of quercetin in ethanol/D2O samples are longer than those of the ethanol/H2O samples. Results similar to those of quercetin in methanol and methanol-d were obtained for quercetin in ethanol and ethanol-d. Figure S5 shows the results for quercetin in ethanol and ethanol-d. The amplitude of the longer time components is much larger in the deuterated solvent mixture. The same phenomenon is also observed in Figure 4 for quercetin in methanol and methanol-d solutions. The kinetic isotope effect of the ESIHT process in quercetin dissolved in methanol, ethanol or water/ethanol mixtures of 1:1 by volume, is estimated from the
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short-time component of the normal-form decay. For all of these solvents we found decay times of 70 and 110 femtoseconds for quercitin. These decay times provide a KIE of ~1.5. The "T" state The steady-state fluorescence spectrum of quercetin in nonpolar, nonprotic polar and protic solvents shows that the fluorescence intensity of the "T" band is of the same order of magnitude of that of the "N" band. The time-resolved emission of the "N" band shows that the decay time is equal or shorter than 70 fs, the shortest time we can estimate by using an exponential fit with the fluorescence up-conversion apparatus instrument response. Figure 5a shows the fluorescence up-conversion time-resolved signals of quercetin in ethanol-d samples measured at long wavelength 480-520 nm that also covers partially the "T" band emission. 1
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Figure 5: Time-resolved fluorescence of quercetin in: a. ethanol-d, shown on a normalized semilogarithmic scale b. quercetin in DMSO, shown on a normalized linear scale. c. quercetin in DMSO, shown on a normalized semilogarithmic scale.
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At 520 nm the long time component amplitude is about 0.17 whereas the two shorter decay time components overall amplitude is 0.83. The lifetime of the "T" state is ~40 ps. We estimate that the emission cross section of the "T" state is only 0.17 or less than that of the "N" state. Figure 5b and 5c show on linear and semilogarithmic scales the time resolved emission of quercetin in DMSO measured at 520-560 nm spectral region and the time window extends up to 50 ps. As in the case of the signals of quercetin in ethanol-d, the decay time of the long time component is about 40 ps. The amplitude of the long time component at the "T" band peak at 560 nm is only ~0.1 of that of the short time components. To summarize the results obtained for the long wavelength measurements: the relative emission cross section of the "T" band emission is much smaller than that of the "N" band. When comparing the fluorescence up-conversion apperatus count rate at the "N" band peak at 450 nm and the long time component of the "T" band at ~560 nm the intensity ratio I "FN " / I"FT " is ~30. The decay time of the "T" state is about 40 ps in all solvents used in the current study. Table 3 provides the multiexponential fitting parameters of the fluorescence up-conversion signals measured at long wavelengths in methanol, ethanol-d and DMSO. λ [nm]
Solution
a1
τ1[fs]
a2
τ2[fs]
a3
τ3[ps]
500
Methanol
0.75
70
0.07
380
0.17
40
520
Ethanol-d
0.76
115
0.07
680
0.17
41
520
DMSO
0.88
70
0.10
380
0.02
42
540
DMSO
0.85
70
0.11
380
0.04
42
560
DMSO
0.65
70
0.24
380
0.10
42
Table 3: Multiexponential fit function parameters for the fluorescence decay of Quercetin in different solutions
As was also found at short wavelength measurements λ≤490 nm, the amplitude of the intermediate decay time is ~350 fs in regular solvents and ~700 fs for deuterated solvents. The relative amplitude of this component increases as the fluorescence wavelength increases. For quercetin fluorescence in DMSO at 560 nm, the
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intermediate component amplitude is 0.24 whereas at 540 nm and 520 nm it is 0.11 and 0.1 respectively. Discussion Quercetin is a flavonol with five hydroxyl groups (see Scheme 1). Two of the hydroxyl groups, at positions C3 and C5, are next to the carbonyl group at position 4. The intramolecular ESPT to form the tautomer T* can occur in excited quercetin by a proton transfer from either the 3-OH or from the 5-OH position. The ESIHT from 3 hydroxyflavone, 3-HF and 5-HF, was extensively studied by several groups25,26,27,. Barbara and coworkers28 studied the ESIHT rate of 3-HF by picosecond laser spectroscopy. With the use of a laser system of limited time resolution of ~30ps, they found, at temperatures below 210K, that the "blue emission" of the "N" (normal) form decays biexponentially. The decay consisted of about 30% of the signal's amplitude at a time constant much shorter than the time resolution of the measurement, and about 70% with a time constant of about 20ps. Later, Schwartz et al.29, using a femtosecond laser system found that the ESIHT time constant in 3HF is much faster than was estimated by Barbara et al., namely, ~240fs in nonpolar solvents and ~125fs in methanol. In a more recent study, (Matousek et al.30), it was found that the ESIHT rate of 3-HF at room temperature is faster than 100fs. Naturally occurring flavonoids have a sugar linkage at the C(3)-position. About 85% of the natural flavonoids possess an OH group at the C(5) position31. 5-hydroxyflavone, (5-HF), forms an intramolecular hydrogen bond with the carbonyl at position 4. Studies have shown that a six-member-ring intramolecular hydrogen bond occurs in 5-HF and is considered to be stronger than the five-member-ring hydrogen bond36 in 3-HF. The rings can be seen in Scheme 2.
a)
b) OH 8
HO
1
O
7
OH
2
6
4 5
OH
O
3
O H
Scheme 2: Molecular structure of quercetin showing: a. normal form b. tautomeric form
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The large fluorescence Stokes-band shift between the S0 and S1 states for 5-HF predicts ultrafast ESIHT31,32,33. The ESIHT of 5-HF was studied by Chou et al.37. They concluded that the ESIHT rate is ultrafast and could not be resolved with their femtosecond laser system with a time response of about 160fs fwhm. They found that the decay time of the tautomeric form, T*, of 5-HF is very short, about τ≈1.2ps. By contrast, in 3-HF the decay time of the tautomeric form is long and the fluorescence quantum yield in methylcyclohexane (MCH) is high, Φem=0.2427,30. The reported ESIHT studies on both 3-HF and 5-HF indicate that an ESIHT process can occur in quercetin from both the 3-OH and 5-OH hydroxyls to the carbonyl at position 4. The ESIHT from the 5-OH hydroxyl forms a six-memberedring, intramolecular hydrogen-bond complex that is considered stronger than that of 3-HF, which possesses a five-membered-ring hydrogen bond. With the limited time resolution of the fluorescence up-conversion system we used in the current study (~240fs fwhm), we found that the time constant of the ESIHT process of quercetin is equal to or shorter than 70fs in hydrogen-bonding solvents like H2O, methanol and ethanol as well as in polar, nearly nonprotic solvents like acetonitrile and DMSO. We found that in deuterated protic solvents like methanol-d or ethanol-d/D2O mixtures, the ESPT rate is smaller than in protonated solvents and the decay of the N-form is about 110fs, (see Figures 4 and S3, S4 and S5 in the SI). An intermediate time component of about 300fs is observed in quercetin in all solvents used. This intermediate time component appears with a relative amplitude of 0.05±0.02 in the decay of the normal form. This intermediate time component was also found in previous studies of 10-HBQ keto signals13,14. Tahara and coworkers assign this time component to an IVR process in the hot tautomeric form and that component was also found by Riedle and coworkers15 in a similar ESIHT prototype compound, the HBT. In the current study we find that the longer time component depends on the deuteration of quercetin. It is longer by about a factor of two in deuterated solvents. This result does not conform with assigning the longer time component to an IVR process. It may arise from a proton shuttle forward and back between the N-form and the T-form. Similar processes occur in excited-state proton transfer (ESPT) to the solvent from a photoacid. The forward process is followed by a
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diffusion-assisted proton geminate recombination process to reform the acidic form ROH*38,39. The proton can shuttle several times to the solvent and back until the excited molecule decays to the ground state or the proton diffuses away from the basic form, the RO-. The five hydroxyl groups of quercetin (see Scheme 1) in protic solvents undergo a deprotonation process that depends on the pH of the solution. In water, Momic et al.35 found the protolytic equilibria for the five hydroxyl groups as well as that of the carbonyl at position 4 (see Scheme 1). The absorption spectrum of quercetin in the spectral range 250-450nm depends on the solvent pH. The absorption bands in this spectral region could be assigned to S0S1, S0S2 and S0S3 shift to the blue, as the proton concentration in solution increases. The transition-dipole moment of these bands also depends on the solvent pH. Using a non-linear leastsquares-regression
analysis,
Momic
et
al.
found
five
pKa
values:
pK a1 = 5.5, pK a2 = 7.15, pK a3 = 8.0, pK a4 = 9.6 and pK a5 = 11.4 . For the protonation of the carbonyl group, they found a pKa of -2. They also simulated the spectra of individual forms of quercetin as well as the distribution of the ionic forms of quercetin as a function of the solution pH. At pH 7.2, three ionic forms of the hydroxyl of quercetin exist in molar ratios of 0.2 and more. They concluded that the preferential order for OH deprotonation is: 5-OH
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Excited-State Intramolecular Proton Transfer of the Natural Product Quercetin Ron Simkovitch and Dan Huppert* Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel *Corresponding author: Dan Huppert E-mail: [email protected] Phone: 972-3-6407012 Fax: 972-3-6407491
Abstract Intramolecular proton-transfer dynamics in the lowest excited state (ESIHT) were studied in the natural product quercetin. We found that in all seven solvents used in this study, the ESIHT rate is ultrafast. We estimate that the ESIHT rate is about 70 fs or less. We found that in deuterated protic solvents, like methanol-d or ethanol-d, the ESIHT rate is slower and the proton-transfer time constant is about 110 fs. The tautomeric form fluorescence quantum yield of quercetin is very low, of the order of the normal form.
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Introduction Quercetin, shown in Scheme 1, is a flavonol and is widely distributed in vegetables, leaves and fruit in substantial amounts. It has recently been used as a food supplement, probably because of its reported anticancer1,2, antiviral3 and antiinflammatory4 properties.
B A
C
Scheme 1: Molecular structure and numbering pattern of quercetin
In the field of photochemistry one often encounters excited-state proton-transfer processes5. These photoprotolytic processes can be divided into two categories. The first is excited-state intermolecular proton transfer (ESPT)6,7,8,9,10,11,12. In such a reaction a proton is transferred to a solvent molecule, most commonly water. Molecules in which this process occurs are called photoacids. In their ground state, these molecules are weak acids with values of pKa that range between 5-10. When photoacids are raised to their first excited state, there is a large change in acidity and the value of their pKa* is lower than that of their pKa by about 7-11 orders of magnitude. The values of pKa* of photoacids range from 3 to ~-8. The ESPT rate constant for compounds with pKa*~-8 is kPT~1013s-1 (τPT~100fs). Because of the radiative lifetime of singlet states, the smallest ESPT rate that can be measured by time-resolved fluorescence is about 107s-1 and corresponds to compounds with pKa* of 3.3. The second category of compounds undergoes an intramolecular hydrogen transfer (ESIHT)13,14,15,16,17,18,19,20,21,22,23,24. The structure of these compounds consists of a proton acceptor and a proton donor in close proximity. Usually the proton donor is a hydroxyl group of a hydroxyaryl moiety and the proton acceptor is a heterocyclic nitrogen atom, or a carbonyl oxygen atom in the keto form. Several compounds are considered prototype compounds for ESIHT. Two ESIHT compounds possess heterocyclic nitrogen as a proton acceptor, 10-
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hydroxybenzoquinoline (10-HBQ) and hydroxyphenyl benzothiazol (HBT). Recent studies on HBT and 10-HBQ in protic solvents, using ultrashort laser pulses of τpulse≈30fs have shown that ESIHT occurs in both compounds within the time resolution of the laser apparatus, i.e ~30fs. Riedle and coworkers15 reported a time constant of ~30fs for an ESIHT in HBT. Similar conclusions were drawn for the ESIHT of 10-HBQ by Tahara and coworkers14. Another class of excited-state proton transfer occurs between a hydroxyaryl group next to an aromatic carbonyl group. An example of molecules of this type is the flavonols. 3-hydroxy-flavone (3-HF) 25,26,27,28,29,30 and, to a lesser extent, 5-hydroxyflavone (5-HF) 31,32,33,34 were extensively studied. Both steady-state fluorescence and time-resolved techniques were employed to study the ESIHT process of these two molecules. In the current work we studied the photophysics and photochemistry of quercetin in solution. For that purpose we used steady-state and time-resolved fluorescence techniques We found an ESIHT process that is ultrafast in polar nonprotic and protic solvents. The proton (hydrogen) transfer occurs with a time constant of about 70fs. In deuterated protic solvents like methanol-d and ethanol-d the ESIHT is slower and the kinetic isotope effect is about 1.5. The quantum efficiency of the tautomeric-form fluorescence of quercetin is rather small.
Materials and Methods Quercetin (of purity >95.5%) was purchased from Sigma-Aldrich, no further purification was done to the samples. All measurements were carried out with fresh solutions of Quercetin. Dichloromethane, DMSO, acetonitrile, methanol, ethanol and other solvents used in this study were of HPLC or analytical grade and were purchased from Sigma-Aldrich as well. A fluorescence up-conversion technique was used to measure the timeresolved emission of quercetin at room temperature. A cavity-dumped Ti:Sapphire femtosecond laser (Mira, Coherent) is used as an excitation source for the fluorescence up-conversion measurements. This laser output consists of 120fs, pulses at about 800nm. The cavity dumper operated at a repetition rate of 800 kHz. A 3 Environment ACS Paragon Plus
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fluorescence up-conversion system (FOG-100, CDP, Russia) was used to measure the time-resolved fluorescence of quercitin The response time of the up-conversion system is measured by monitoring the Raman-Stokes line of water. We found that the full width at half maximum of the signal is 240 fs. To avoid photochemistry, we placed the samples in a rotating optical cell. The sample degradation during the measurement was rather small. For the collection of the steady-state fluorescence and the absorption spectra we used a Horiba Jobin Yvon FluroMax-3 spectrofluorometer and a Cary 5000 spectrometer. Results Steady-State Measurements The absorption spectra of quercitin in protic solvents depend on the solvent pH. This is explained by Momic et al.35 and others as arising from the possibility of several protonation deprotonation states of the 5 hydroxyl groups of quercetin (see scheme 1). Figure S1 the supporting-information (SI) section shows the absorption spectrum of quercitin in methanol and in methanolic solutions containing also NaOH (in water) of mM concentrations (pH>7). As seen in the figure, the spectra are sensitive to the solution pH. The absorption band peak assigned to S0S1 transition for the protonated form is at about 380nm. When the solution pH increases, the deprotonation of the first two protons leads to a red shift of the S0S1 transition. The deprotonation of the third proton at about pH~11 shifts the S0S1 transition to the blue and the band's peak position is at 340 nm. A detailed analysis of the absorption spectra as a function of pH is given by Momic et al35. Figure 1a shows the normalized steady-state fluorescence spectrum of quercetin in dichloromethane (DCM) excited at three wavelengths in the spectral region of 350390nm. The fluorescence quantum yield of quercetin is low as was also found for similar ESIHT compounds 3-HF and 5-HF.
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a)
350 nm 370 nm 390 nm
1.0
Norm. Signal
norm. Signal
b)
1.0
350 nm 370 nm 390 nm
0.8
0.8 0.6 0.4
0.6 0.4 0.2
0.2 0.0 400
450
500
550
600
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0.0 400
450
c)
500
550
600
Wavelength (nm)
Wavelength (nm)
Ethanol Ethanol-d
1.0 0.8
Norm.Signal
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0.6 0.4 0.2 0.0 400
450
500
550
600
650
700
Wavelength [nm]
Figure 1: Normalized steady-state fluorescence spectra of quercetin in a. dichloromethane. b. DMSO c. ethanol and ethanol-d
The fluorescence spectrum consists of two emission bands. The blue band with a maximum at 440-470nm is assigned to the spectrum of the normal form, while the red band with a maximum at ~585nm is assigned to the tautomeric form. The blue-band spectrum in DCM has a vibronic substructure with about four sub bands. The red band of the enol form is broad and structureless. The band position is nearly constant when excited to the S1 state at long wavelengths (λ≥390nm). When excited at short wavelengths (λ≤370nm) to the S2 state, the emission-band position is blue-shifted by F F about 15nm. The band-intensity ratio, I red / I blue ≈ 2.5 , is nearly constant at all
excitation wavelengths, except when the excitation is at the long wavelength of λex=430nm. Figure 1b shows the normalized steady-state emission and excitation spectra of quercetin in DMSO. The fluorescence spectrum is measured at three excitation
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wavelengths. At short excitation wavelengths, (λ≤390nm), the spectrum consists of the normal-form emission band with a maximum at about 450nm and a weaker vibronic band at 472nm. The red tautomeric-form emission band is weaker by about a factor of ten and the band maximum is at 550nm. Figure 1c shows the normalized steady-state emission spectrum of quercetin in ethanol and ethanol-d. In both spectra two emission bands are observed. The blue band with a maximum around 450 nm is attributed to the "N" form of quercitin and the red band with a maximum at about 540 nm is attributed to the "T" band. The red tail of the "T" form extends to longer wavelengths than in the case of quercetin in dichloromethane shown in figure 1a. This large change in the "T" spectrum arises from the large hydrogen bond of quercetin in the carbonyl or all five hydroxyls. It may also arise from the existence of deprotonated hydroxyls like those at 4',5' and 7 positions (see scheme 1). There are two sub-bands in the "T" emission spectrum at about 610 nm and 660 nm. They can be assigned to vibrational transitions or to different deprotonated RO- form of quercetin. As seen in the figure, the amplitude ratio I "FN " / I"FT " of the two fluorescence bands of the normalized spectrum for the ethanol-d sample is about twice larger than that of the ethanol sample. This difference in the amplitude ratio can be interpreted as arising from a kinetic isotope effect (KIE) of the ESIHT process. As we will show later on based on the time-resolved measurement the KIE we estimate for this process is KIE≥1.5. Time-resolved emission Figures 2a and 2b show, on linear and semilogarithmic scales, the time-resolved fluorescence of quercetin in acetonitrile solution measured at several wavelengths over the spectral region of 450-500nm.
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a) 1.0
b)
0.6
Norm. Signal
450nm 460nm 470nm 480nm 490nm 500nm Lamp
0.8
Norm. Signal
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0.4
1
450nm 460nm 470nm 480nm 490nm 500nm
0.1
0.2 0.01
0.0 0.0
0.5
1.0
0.0
1.5
0.5
1.0
Time (PS)
Time (PS)
Figure 2: Time-resolved fluorescence of quercetin in an acetonitrile solution a. Normalized linear scale, showing the IRF lamp signal b. Normalized semilogarithmic scale.
The fluorescence signals were acquired by the fluorescence up-conversion technique. The system response was determined by measuring the Raman water band. The instrument response at full-width half maximum was 240±20fs. As seen in the figure, the quercetin signal decay of the normal-form band is rather short and is close to the system response. The major ultrafast-decay component is followed by a longer decay component of much smaller amplitude. Similar results are obtained for the timeresolved emission of quercitin in DMSO, for which the results are shown in Figure S2 in the SI section. We used a multiexponential function to fit the fluorescence decay of the signals shown in Figure 2. The results are given in Table 1. λ [nm]
a1
τ1[fs]
a2
τ2[fs]
a3a
440
0.973
75
0.02
300
0.007
450
0.978
75
0.021
350
0.0011
460
0.967
78
0.032
350
0.0011
470
0.965
75
0.032
310
0.0030
480
0.959
70
0.037
320
0.0040
490
0.959
65
0.037
420
0.0040
Table 1: multiexponential-fit function parameters for the fluorescence decay of quercetin in an acetonitrile solution. b a
The amplitude of the long-time component is the sum of the amplitudes of two components in the picosecond range. b
τpulse is about 240fs, τ3=2000fs, τ4=34ps.
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The major amplitude time component is estimated to be shorter than 100fs. For the fit we use τ=70fs. The second time component is about 300fs with an amplitude of ~0.04. Longer time components with a total amplitude of less than ~0.01 also appear in the signals. The longer the monitored wavelength, the higher the amplitude of the long-time components. Figure 3 shows the time-resolved emission of quercetin in methanol solution, measured over the spectral region of 440-490nm.
440nm 450nm 460nm 470nm 480nm 490nm Lamp
0.8 0.6 0.4
b)
Norm. Signal
a) 1.0
Norm. Signal
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1
440nm 450nm 460nm 470nm 480nm 490nm
0.1
0.01
0.2 0.0 0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
Time (ps)
1.0
1.5
2.0
2.5
Time (ps)
Figure 3: Time-resolved fluorescence of quercetin in methanol a. Normalized linear scale, showing the IRF lamp signal b. Normalized semilogarithmic scale.
The signal decay is rather short, similar to the signals of quercetin in acetonitrile, shown in Figure 2. The difference in the signal decay is that at higher wavelengths, (λ≥480nm), the amplitude of the long-time component is much greater than in the acetonitrile sample. We conclude that the excited-state intramolecular proton transfer occurring in quercetin in both solvents is short, probably equal to or shorter than 70fs. The kinetic isotope effect Figure 4 shows the time-resolved fluorescence of quercetin in both methanol and methanol-d.
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λ=440nm
1.0
Norm. Signal
λ=460nm
1.0
MeOH Methanol-d
0.8
MeOH Methanol-d
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0.4
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λ=490nm
λ=480nm Norm. Signal
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0.6
0.6
MeOH Methanol-d
0.4
0.4
0.2
0.2
0.0
MeOH Methanol-d
0.0 0.0
0.5
1.0
1.5
0.0
Time (ps)
0.5
1.0
1.5
Time (ps)
Figure 4: Time-resolved fluorescence of quercetin in methanol compared to methanol-d, shown in normalized linear scale at various wavelengths.
Each panel shows the signals measured at the same wavelength for both solvents. As is clearly seen in the figure, there is a distinct difference in both the short and long times of the fluorescence decay of quercetin in methanol and methanol-d. The shorttime component of the decay time of the normal-form band of quercetin measured in the blue spectral region, 440-500nm, is 110±15fs in methanol-d and 70±10fs in methanol. The longer-time component in the methanol-d decay time is about 700fs, compared to about 300fs in methanol. The amplitude of the long-time decay component is much larger in methanol-d than in methanol or in acetonitrile. Table 2 provides the fitting parameters of the multiexponential-fit function of both methanol and methanol-d.
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MeOD
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λ [nm]
a1
τ1[fs]
a2
τ2[fs]
a3a
440
0.956
72
0.035
250
0.009
450
0.953
70
0.036
270
0.011
460
0.954
70
0.036
350
0.01
470
0.928
70
0.059
260
0.013
480
0.927
68
0.060
270
0.013
440
0.948
110
0.039
800
0.013
450
0.926
115
0.060
800
0.014
460
0.890
110
0.091
750
0.019
470
0.852
110
0.122
700
0.026
480
0.836
100
0.127
700
0.037
Table 2: multiexponential-fit function parameters for the fluorescence decay of quercetin in methanol and methanol-d solutions b. a
The amplitude (a3) of the long-time component is the amplitude sum of the two components in the picosecond range. b
τpulse is about 240fs, τ3=2000fs, τ4=34ps. Figure S3 in the SI section shows the time-resolved emission of quercetin in
methanol-d on linear and semilogarithmic scales. Similar results are obtained for quercetin in water/ethanol and D2O/ethanol-d samples of ratios of 1:1 by volume. Figure S4 in the SI shows on linear and semilogarithmic scales the time-resolved emission of quercetin in these solvent mixtures. Each panel shows the signals measured at the same wavelength for ethanol/H2O and ethanol-d/D2O solutions. As seen in Figure S4, the short and long decay times of quercetin in ethanol/D2O samples are longer than those of the ethanol/H2O samples. Results similar to those of quercetin in methanol and methanol-d were obtained for quercetin in ethanol and ethanol-d. Figure S5 shows the results for quercetin in ethanol and ethanol-d. The amplitude of the longer time components is much larger in the deuterated solvent mixture. The same phenomenon is also observed in Figure 4 for quercetin in methanol and methanol-d solutions. The kinetic isotope effect of the ESIHT process in quercetin dissolved in methanol, ethanol or water/ethanol mixtures of 1:1 by volume, is estimated from the
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short-time component of the normal-form decay. For all of these solvents we found decay times of 70 and 110 femtoseconds for quercitin. These decay times provide a KIE of ~1.5. The "T" state The steady-state fluorescence spectrum of quercetin in nonpolar, nonprotic polar and protic solvents shows that the fluorescence intensity of the "T" band is of the same order of magnitude of that of the "N" band. The time-resolved emission of the "N" band shows that the decay time is equal or shorter than 70 fs, the shortest time we can estimate by using an exponential fit with the fluorescence up-conversion apparatus instrument response. Figure 5a shows the fluorescence up-conversion time-resolved signals of quercetin in ethanol-d samples measured at long wavelength 480-520 nm that also covers partially the "T" band emission. 1
b)
480nm 500nm 520nm
Norm.Signal
a)
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0.1
0.01 0
10
20
30
40
50
Time [ps]
Figure 5: Time-resolved fluorescence of quercetin in: a. ethanol-d, shown on a normalized semilogarithmic scale b. quercetin in DMSO, shown on a normalized linear scale. c. quercetin in DMSO, shown on a normalized semilogarithmic scale.
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At 520 nm the long time component amplitude is about 0.17 whereas the two shorter decay time components overall amplitude is 0.83. The lifetime of the "T" state is ~40 ps. We estimate that the emission cross section of the "T" state is only 0.17 or less than that of the "N" state. Figure 5b and 5c show on linear and semilogarithmic scales the time resolved emission of quercetin in DMSO measured at 520-560 nm spectral region and the time window extends up to 50 ps. As in the case of the signals of quercetin in ethanol-d, the decay time of the long time component is about 40 ps. The amplitude of the long time component at the "T" band peak at 560 nm is only ~0.1 of that of the short time components. To summarize the results obtained for the long wavelength measurements: the relative emission cross section of the "T" band emission is much smaller than that of the "N" band. When comparing the fluorescence up-conversion apperatus count rate at the "N" band peak at 450 nm and the long time component of the "T" band at ~560 nm the intensity ratio I "FN " / I"FT " is ~30. The decay time of the "T" state is about 40 ps in all solvents used in the current study. Table 3 provides the multiexponential fitting parameters of the fluorescence up-conversion signals measured at long wavelengths in methanol, ethanol-d and DMSO. λ [nm]
Solution
a1
τ1[fs]
a2
τ2[fs]
a3
τ3[ps]
500
Methanol
0.75
70
0.07
380
0.17
40
520
Ethanol-d
0.76
115
0.07
680
0.17
41
520
DMSO
0.88
70
0.10
380
0.02
42
540
DMSO
0.85
70
0.11
380
0.04
42
560
DMSO
0.65
70
0.24
380
0.10
42
Table 3: Multiexponential fit function parameters for the fluorescence decay of Quercetin in different solutions
As was also found at short wavelength measurements λ≤490 nm, the amplitude of the intermediate decay time is ~350 fs in regular solvents and ~700 fs for deuterated solvents. The relative amplitude of this component increases as the fluorescence wavelength increases. For quercetin fluorescence in DMSO at 560 nm, the
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intermediate component amplitude is 0.24 whereas at 540 nm and 520 nm it is 0.11 and 0.1 respectively. Discussion Quercetin is a flavonol with five hydroxyl groups (see Scheme 1). Two of the hydroxyl groups, at positions C3 and C5, are next to the carbonyl group at position 4. The intramolecular ESPT to form the tautomer T* can occur in excited quercetin by a proton transfer from either the 3-OH or from the 5-OH position. The ESIHT from 3 hydroxyflavone, 3-HF and 5-HF, was extensively studied by several groups25,26,27,. Barbara and coworkers28 studied the ESIHT rate of 3-HF by picosecond laser spectroscopy. With the use of a laser system of limited time resolution of ~30ps, they found, at temperatures below 210K, that the "blue emission" of the "N" (normal) form decays biexponentially. The decay consisted of about 30% of the signal's amplitude at a time constant much shorter than the time resolution of the measurement, and about 70% with a time constant of about 20ps. Later, Schwartz et al.29, using a femtosecond laser system found that the ESIHT time constant in 3HF is much faster than was estimated by Barbara et al., namely, ~240fs in nonpolar solvents and ~125fs in methanol. In a more recent study, (Matousek et al.30), it was found that the ESIHT rate of 3-HF at room temperature is faster than 100fs. Naturally occurring flavonoids have a sugar linkage at the C(3)-position. About 85% of the natural flavonoids possess an OH group at the C(5) position31. 5-hydroxyflavone, (5-HF), forms an intramolecular hydrogen bond with the carbonyl at position 4. Studies have shown that a six-member-ring intramolecular hydrogen bond occurs in 5-HF and is considered to be stronger than the five-member-ring hydrogen bond36 in 3-HF. The rings can be seen in Scheme 2.
a)
b) OH 8
HO
1
O
7
OH
2
6
4 5
OH
O
3
O H
Scheme 2: Molecular structure of quercetin showing: a. normal form b. tautomeric form
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The Journal of Physical Chemistry
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The large fluorescence Stokes-band shift between the S0 and S1 states for 5-HF predicts ultrafast ESIHT31,32,33. The ESIHT of 5-HF was studied by Chou et al.37. They concluded that the ESIHT rate is ultrafast and could not be resolved with their femtosecond laser system with a time response of about 160fs fwhm. They found that the decay time of the tautomeric form, T*, of 5-HF is very short, about τ≈1.2ps. By contrast, in 3-HF the decay time of the tautomeric form is long and the fluorescence quantum yield in methylcyclohexane (MCH) is high, Φem=0.2427,30. The reported ESIHT studies on both 3-HF and 5-HF indicate that an ESIHT process can occur in quercetin from both the 3-OH and 5-OH hydroxyls to the carbonyl at position 4. The ESIHT from the 5-OH hydroxyl forms a six-memberedring, intramolecular hydrogen-bond complex that is considered stronger than that of 3-HF, which possesses a five-membered-ring hydrogen bond. With the limited time resolution of the fluorescence up-conversion system we used in the current study (~240fs fwhm), we found that the time constant of the ESIHT process of quercetin is equal to or shorter than 70fs in hydrogen-bonding solvents like H2O, methanol and ethanol as well as in polar, nearly nonprotic solvents like acetonitrile and DMSO. We found that in deuterated protic solvents like methanol-d or ethanol-d/D2O mixtures, the ESPT rate is smaller than in protonated solvents and the decay of the N-form is about 110fs, (see Figures 4 and S3, S4 and S5 in the SI). An intermediate time component of about 300fs is observed in quercetin in all solvents used. This intermediate time component appears with a relative amplitude of 0.05±0.02 in the decay of the normal form. This intermediate time component was also found in previous studies of 10-HBQ keto signals13,14. Tahara and coworkers assign this time component to an IVR process in the hot tautomeric form and that component was also found by Riedle and coworkers15 in a similar ESIHT prototype compound, the HBT. In the current study we find that the longer time component depends on the deuteration of quercetin. It is longer by about a factor of two in deuterated solvents. This result does not conform with assigning the longer time component to an IVR process. It may arise from a proton shuttle forward and back between the N-form and the T-form. Similar processes occur in excited-state proton transfer (ESPT) to the solvent from a photoacid. The forward process is followed by a
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The Journal of Physical Chemistry
diffusion-assisted proton geminate recombination process to reform the acidic form ROH*38,39. The proton can shuttle several times to the solvent and back until the excited molecule decays to the ground state or the proton diffuses away from the basic form, the RO-. The five hydroxyl groups of quercetin (see Scheme 1) in protic solvents undergo a deprotonation process that depends on the pH of the solution. In water, Momic et al.35 found the protolytic equilibria for the five hydroxyl groups as well as that of the carbonyl at position 4 (see Scheme 1). The absorption spectrum of quercetin in the spectral range 250-450nm depends on the solvent pH. The absorption bands in this spectral region could be assigned to S0S1, S0S2 and S0S3 shift to the blue, as the proton concentration in solution increases. The transition-dipole moment of these bands also depends on the solvent pH. Using a non-linear leastsquares-regression
analysis,
Momic
et
al.
found
five
pKa
values:
pK a1 = 5.5, pK a2 = 7.15, pK a3 = 8.0, pK a4 = 9.6 and pK a5 = 11.4 . For the protonation of the carbonyl group, they found a pKa of -2. They also simulated the spectra of individual forms of quercetin as well as the distribution of the ionic forms of quercetin as a function of the solution pH. At pH 7.2, three ionic forms of the hydroxyl of quercetin exist in molar ratios of 0.2 and more. They concluded that the preferential order for OH deprotonation is: 5-OH
