Laser flash photolysis study of the reactivity of β-naphthoflavone triplet: Hydrogen abstraction and singlet oxygen generation

Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 121–129

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Laser flash photolysis study of the reactivity of ␤-naphthoflavone triplet: Hydrogen abstraction and singlet oxygen generation Nanci C. de Lucas a,∗ , Guilherme L.C. Santos a , Caio S. Gaspar a , Simon J. Garden a , José Carlos Netto-Ferreira b a b

Laboratório de Fotoquímica, Instituto de Química – Universidade Federal do Rio de Janeiro, Cidade Universitária, RJ, Brazil Departamento de Química – Universidade Federal Rural do Rio de Janeiro, Antiga Rio São Paulo, RJ, Brazil

a r t i c l e

i n f o

Article history: Received 22 July 2014 Received in revised form 21 August 2014 Accepted 22 August 2014 Available online 1 September 2014 Keywords: Naphthoflavone Triplet excited state Hydrogen abstraction Singlet oxygen TDDFT calculations

a b s t r a c t The absorption spectra for ␤-naphthoflavone (1) reveal a solvatochromic red shift in polar solvents which is consistent with the ␲,␲* character of the S0 → S1 electronic transition. The laser flash photolysis technique has been used to characterize and study the reactivity of the triplet excited state of 1. Excitation (355 nm) of degassed solutions of 1, in acetonitrile, resulted in the formation of its corresponding triplet excited state. Addition of hydrogen donors, such as 2-propanol and 1,4-cyclohexadiene, led to triplet quenching and formation of a new transient, which was assigned to the corresponding ketyl radical obtained from a hydrogen abstraction reaction by triplet 1. This ketyl radical was characterized by experiments with methylviologen. The triplet excited state of 1 was efficiently quenched by phenols and N-acetyl l-tryptophan methyl ester. In all cases new transients were formed in the quenching process, which were assigned to the corresponding radical pair resulting from an initial electron transfer from the quencher to the excited naphthoflavone, followed by a fast proton transfer. Singlet oxygen (1 O2 ) is formed from the triplet of 1, and a quantum yield of 0.51 was measured. TDDFT calculations with implicit solvation (IEF-PCM) were used to calculate the ground state UV–vis absorption spectrum, from which the nature of the lowest energy transitions were characterized, and the triplet–triplet absorption spectrum consistent with the triplet transient generated by LFP. Excellent correlation of the calculated and experimental spectra was achieved using the conventional PBE0 hybrid functional. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Flavones are a large class of natural pigments which contain a 2-phenyl-4-benzopyrone unit and belong to the general class of flavonoids. A large number of derivatives result from various combinations of multiple hydroxyl and/or methoxyl substituents bonded to the flavonoid skeleton, which gives them potential antioxidant activity toward free radical species. These compounds are usually found in the tissues of higher plants, mainly in tropical regions [1]. Photocontrol of their production is well documented and seems to operate at many different points along the biosynthetic pathway [2]. The efficient absorbance of UV irradiation has implied their possible involvement in the protection against UVinduced damage in plants [3,4].

∗ Corresponding author. Tel.: +55 21 3938 72 78. E-mail addresses: [email protected], [email protected] (N.C. de Lucas). http://dx.doi.org/10.1016/j.jphotochem.2014.08.009 1010-6030/© 2014 Elsevier B.V. All rights reserved.

Photophysical and photochemical studies on flavonoids have revealed important characteristics which may be related to their photobiological behavior. Photodimerization is the main photoreaction of the parent flavone in 2-propanol [5]. Time-resolved spectroscopy experiments suggest that flavone photochemistry is mainly mediated by the triplet excited state [6,7], as confirmed by its high ISC quantum yield (∼0.9) [8]. The transient absorption spectrum of triplet flavone shows bands at 365–370 nm and 640–650 nm, and this transient has been shown to be reactive toward hydrogen donors [2,6–9]. The phosphorescence spectra and lifetime measured for flavone at 77 K indicates a mixed n,␲*–␲,␲* character for the lowest triplet excited state irrespective of the solvent polarity [10] and a triplet energy of 62 kcal mol−1 [8,10]. ␤-Naphthoflavone (1) or 5,6-benzoflavone is a synthetic derivative of a naturally occurring flavonoid and has the potential to strongly induce cytochrome P450 1A enzymes via activation of aryl hydrocarbon receptor [11,12]. It has been shown that 1 has the property to increase the liver tumor-promoting activity [13].

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Recently Mitsumori et al. shows that 1 induced the sustained production of oxidative stress in the livers of rats [14]. The authors suggest that secondary effects of 1, which include enhancement of reactive oxygen species (ROS), oxidative DNA damage and lipid peroxidation, may contribute to its liver tumor promotion activity [14–16]. Oxidative damage to biological systems, either accidental or intentional, is a major cause of cell death [17]. In particular, apoptotic or necrotic signaling pathways to cell death can be induced by the combined use of a photo-activated drug, called the photosensitizer, and per se harmless visible light. This process involves the generation of reactive oxygen species (ROS) capable of inflicting damage to susceptible cell components such as proteins [18,19], membrane lipids [20], and nucleic acids [21]. Several mechanisms account for the photosensitization process toward biomolecules [22,23]. More general photosensitizing mechanisms can involve either photo-oxidation of nucleic acid components by the sensitizer, yielding the corresponding radical pair and ultimately leading to sensitizer-protein photobinding (type I photosensitizers), or a triplet–triplet energy transfer to molecular oxygen, resulting in formation of singlet oxygen O2 (1 g ) and other reactive oxygen species, such as superoxide anion, hydrogen peroxide and hydroxyl radical (type II photosensitizers). Aromatic amino acid residues in proteins, especially tryptophan (Trp) and tyrosine (Tyr), are easily oxidized [24,25]. Thus, the reaction of Trp and Tyr with photosensitizers has received considerable attention in the field of proteins photo-oxidation. Recently, characterization and reactivity of triplet ␣naphthoflavone (2), an isomer of 1, was studied by us via laser flash photolysis and theoretical calculations with density functional theory (DFT) and atoms in molecules (AIM) [26]. In this sense, a study of the photochemistry of ␤-naphthoflavone (1), characterizing its triplet excited state and explaining its reaction mechanisms of deactivation could aid in the understanding of the pharmacological action of this class of molecule.

2.3. Laser flash photolysis The laser flash photolysis (LFP) experiments were carried out on a LuzChem Instrument model mLFP122. Samples were contained in a 10 mm × 10 mm cell made from Suprasil tubing and were deaerated by bubbling with argon for about 20 min. The samples were irradiated with the third harmonic of a Nd/YAG Surelite laser ( = 355 nm). All laser flash photolysis experiments were performed in acetonitrile solution, unless otherwise indicated in the text. The concentration of 1 was adjusted to yield an absorbance of ∼0.3 at the excitation wavelength (355 nm). Stock solutions of quenchers were prepared so that it was only necessary to add microliter volumes to the sample cell in order to obtain appropriate concentrations of the quencher. The rate constants for the reaction of triplet 1 with the different quenchers employed in this work were obtained from Stern–Volmer plots, following Eq. (1). kobs = ko + kq[Q ]

where ko is the triplet decay rate constant in the absence of quencher; kq is the triplet decay rate constant in the presence of the quencher and [Q] is the quencher concentration in mol L−1 . The decay trace at 500 nm was used to determine the quenching rate constants. 2.4. Singlet oxygen measurements The quantum efficiency of singlet oxygen (1 O2 ) formation was determined using a spectrofluorometer (FS920 Edinburgh Instruments) with a TMS300 monochromator. The detection system was equipped with a NIR Hamamatsu model H1033-45 photomultiplier. The excitation source (355 nm) was provided by a CryLas Nd-YAG HP 355-50 laser (pulse width of 1.0 ns and energy <150 ␮J. Singlet oxygen formation was observed by monitoring the phosphorescence at 1270 nm upon laser excitation of optically matched solutions (A = 0.3 in air-saturated acetonitrile) of 1 relative to a standard solution of perinaphthenone, which shows singlet oxygen quantum yield of 1.0 [28]. The quantum yield for singlet oxygen formation was determined from the slope of the plots of signal intensity at zero time in 1270 nm versus laser light intensity, using a set of neutral density filters, employing Eq. (2), where Isample is the emission intensity recorded for the sample, IPER is the perinaphthenone emission intensity, used as standard, and ˚PER is the quantum yield of singlet oxygen formation from perinaphthenone.

2. Materials and methods 2.1. General Acetonitrile, 2-propanol, cyclohexane and 1-methylnaphthalene (all spectroscopic grade), were provided by Sigma–Aldrich. Water was milli-Q grade. Phenol, 4methoxyphenol, 4-cyanophenol, 1,4-cyclohexadiene and DABCO were used as received from Aldrich. ␤-naphthoflavone (Aldrich) was recrystallized from hexane:ethyl acetate 1:1. Perinaphthenone, ␤-carotene, indole, methylviologen and ltryptophan methyl ester, from Aldrich, were used as received (purity >99%). N-Acetyl l-tryptophan methyl ester (NATME) was synthesized by a standard procedure (mp = 150–152 ◦ C, Lit = 152 ◦ C; yield: 88%) [27]. 2.2. Absorption spectra Optical spectra in different solvents were measured on a Shimatzu model UV-2450 UV–vis spectrophotometer.

(1)

˚sample =

Isample IPER

× ˚PER

(2)

2.5. Theoretical calculations Geometry and energy calculations were performed at (U)B3LYP/6-311 + +G**//(U)B3LYP/6-31 + G* computational level for all structures unless otherwise stated [29–31]. The calculations were performed using Gaussian 09 [32]. The gas phase and solvated phase geometries were optimized using standard techniques [33,34], and vibrational analysis was performed to confirm that the geometries were true minima on the potential energy surface, as shown by the absence of imaginary frequencies. The DFT method was used as it reasonably estimates spin contamination. Values for S2  were typically consistent with two unpaired electrons for all triplets. Implicit solvated structures were optimized by use of IEF-PCM as implemented in Gaussian 09 [35–37]. UV–vis spectra were calculated with implicit solvation (IEF-PCM) by TD-DFT B3LYP, CAM-B3LYP [38] and PBE0 [39,40] (6-311 + +G**//6-31 + G* (nstates = 20)) using solvent optimized geometries (IEF-PCM).

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Fig. 1. Absorbance spectra for 1 (5.8 × 10−4 mol L−1 ) in different solvents.

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Fig. 2. LFP spectra observed for the transient generated upon laser excitation of 1 in nitrogen saturated acetonitrile (exc = 355 nm). Inset: decay monitored at 500 nm.

3. Results and discussion 3.1. UV–vis spectrum The absorption spectrum for 1, in cyclohexane, reveals the presence of six bands with maxima at 217, 227, 271, 281, 309 and 340 nm (Fig. 1). A very small solvatochromic red shift was observed for the absorption at longer wavelength when this flavonoid was dissolved in acetonitrile or 2-propanol, in comparison to the spectrum in cyclohexane. This is consistent with the ␲,␲* character of the S0 → S1 electronic transition. 3.2. Laser flash photolysis 3.2.1. Triplet characterization Laser flash photolysis (LFP) of deoxygenated acetonitrile solutions for 1 led to the formation of a detectable transient with a broad band with maximum at 500 nm and lifetime of 2 ␮s (Fig. 2). The decay in acetonitrile is dependent on its concentration; from the slope of a plot of first-order rate constants for the decay versus the concentration of 1 a self-quenching rate constant of 1.02(±0.03) × 109 L mol−1 s−1 was obtained. This transient is quenched by O2 and ␤-carotene. In the presence of ␤-carotene, a frequently used triplet state quencher due to its low triplet energy (19 kcal mol−1 ), the formation of a transient showing an absorption maximum at 520 nm due to the ␤-carotene triplet was readily observed [41,42]. This confirms the nature of the former transient as the triplet excited state of 1. The triplet of 1 was quenched by 1-methylnaphthalene (ET = 60.6 kcal mol−1 ) in acetonitrile with a rate constant of 2.49(±0.14) × 109 L mol−1 s−1 with the concomitant formation of the 1-methylnaphthalene triplet at 420 nm (Fig. 3) [42]. These results are consistent with a value close to 60 kcal mol−1 for the triplet energy of 1, which is in agreement with a value of 62 kcal mol−1 for flavone [8,10]. 3.3. Triplet reactivity The transient spectrum obtained after LFP experiments for 1 in the presence of 1,4-cyclohexadiene or 2-propanol showed a long lived transient at 410 nm ( = 20 ␮s), besides the absorption

Fig. 3. LFP spectra observed upon excitation of 1 in the presence of 8.9 × 10−4 mol L−1 of 1-methylnaphthalene (MN), in ACN (exc = 355 nm). Inset: Kinetic trace at 500 nm in the presence and in the absence of MN.

corresponding to its triplet state (max = 500 nm;  = 2 ␮s). Fig. 4 shows the spectra and kinetic traces for the LFP of 1 in 0.37 mol L−1 of 1,4-cyclohexadiene in acetonitrile as an example. The inset of Fig. 4 shows the formation of the transient at 410 nm and the triplet decay at 500 nm, with the rate constants for these two processes having the same order of magnitude under these conditions. This new transient was assigned to the ketyl radical derived from 1 formed by a hydrogen abstraction (HA) reaction by the triplet state of 1 (Eq. (3)), which was confirmed by experiments with methylviologen. Ketyl radicals are known to be excellent electron donors, and in the presence of methylviologen they can generate the methylviologen cation radical, which shows absorption bands at 398 nm and 603 nm (Eq. (4) and Fig. 5) [43–45]. Similar behavior

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Fig. 4. Transient absorption spectra obtained upon 355 nm excitation of 1 in 0.37 mol L−1 of 1,4-cyclohexadiene. Inset: kinetic traces at 410 and 500 nm.

Fig. 5. Transient absorption spectra obtained upon 355 nm excitation of 1 in the presence of 1,4-cyclohexadiene (0.7 mol L−1 ) in acetonitrile, and an excess of methylviologen.

was observed when the reaction was performed in neat 2-propanol. It is noteworthy that the triplet and the ketyl radical derived from ␣naphthoflavone (2) show absorption maximum at 430 and 470 nm, respectively [26].

Fig. 6. Transient absorption spectra obtained upon 355 nm excitation of 1 in acetonitrile. (A) In 1.62 × 10−3 mol L−1 of 4-methoxyphenol at a short time scale. Inset: growth at 400 nm. (B) In 1.01 × 10−3 mol L−1 of 4-methoxyphenol at a long time scale. Inset: decay at 400 nm.

reaction of the triplet 1 with 2-propanol, suggesting that this reaction occurs with values of kq <105 L mol−1 s−1 . In order to verify if 1 can act as a type I photosensitizer we performed quenching experiments of its triplet with phenols and N-Acetyl l-tryptophan methyl ester (NATME).

(3)

(4) The triplet of 1 was quenched by 1,4-cyclohexadiene in acetonitrile with a rate constant of 1.30(±0.05) × 106 L mol−1 s−1 . It was not possible to determine the second order rate constant for the

The hydrogen abstraction reaction of triplet 1 from phenols leads to the formation of the corresponding phenoxyl radicals (Eq. (5)). These radicals have strong absorption bands in the

N.C. de Lucas et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 121–129 Table 1 Second-order rate constants for the quenching of the triplet of 1 by phenols, in acetonitrile. Quencher

kq (L mol−1 s−1 )

Phenol

a

4-Cyanophenol 4-Methoxyphenol a

5.46 (±0.09) × 107 2.3 × 108 2.07 (±0.03) × 107 a 9.9 × 107 2.78 (±0.03) × 108 a 3.7 × 109

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presence of 2.1 × 10−3 mol L−1 NATME, in acetonitrile. This transient was attributed to an indolyl-like radical as indicated in the literature [49,50]. In fact, an experiment using indole as quencher shows the formation of a similar transient, i.e. the indolyl radical, which reveals H-N hydrogen abstraction from the indolyl group. As the indolyl-like radical from NATME shows absorption in the same region as the triplet 1 it was not possible to obtain secondorder rate constants for the quenching process of triplet 1 by this quencher.

kq values for the triplet of 2 [26].

4. Singlet oxygen formation Singlet oxygen can be formed through an energy transfer process from a suitable donor. A tiny fraction of 1 O2 molecules undergoes radiative decay, thereby emitting a photon in the near infrared (NIR). This extremely weak phosphorescence, centered at 1270 nm, provides the means for the most direct and unambiguous method for 1 O2 detection (Eq. (6)). 3

S ∗ +O2 → S0 + 1 O2

(6)

A representative decay for the singlet oxygen phosphorescence generated by energy transfer from 1 is shown in Fig. 8. Linear plots for the singlet oxygen phosphorescence intensity versus energy dependence were obtained for perinaphthenone (standard) and ␤naphthoflavone (inset Fig. 8). From the slopes of these plots one could calculate the quantum yield of singlet oxygen (1 O2 ) formation and a value of 0.51 was found for 1. This indicates that 1 can act efficiently as a type II photosensitizer. 5. Theoretical calculations Fig. 7. Transient absorption spectra obtained upon 355 nm excitation of 1 in 2.1 × 10−3 mol L−1 of NATME in acetonitrile.

370–505 nm region, that can be easily detected by LFP experiments [26,44,46,47].

The vertical transition UV–vis spectra of 1 were simulated in acetonitrile by using the TD-DFT-IEFPCM method and the following density functionals: B3LYP, CAM-B3LYP, and PBE0. The initial geometry of 1 was first optimized with solvent simulation using

(5) Fig. 6 shows the spectrum of 1 in acetonitrile in the presence of 4-methoxyphenol. In this spectrum the ketyl radical is probably hidden by the phenoxyl radical absorption. On a short time scale one can see the formation of the transient (phenoxyl radical) at 400 nm (Fig. 6A) and on a long time scale its corresponding decay (Fig. 6B). The reactivity of triplet 1 with phenols (Table 1) is, as expected, greater than the reactivity with 2-propanol and 1,4-cyclohexadiene and can be attributed to the low bond dissociation energy and the ease of oxidation of the phenols [26,44,46–48]. The reactivity of phenols toward the triplet 1 is observed to be dependent upon the nature of the phenolic substituent, i.e. phenols with an electron donating substituent (4-MeO) react faster than phenols with an electron withdrawing substituent (4-CN), as shown in Table 1. A similar reactivity pattern has been observed in previous studies of ␣-naphthoflavone [26]. Interestingly, the reactivity of 1 is lower than 2, which may be justified due to a steric effect on the approach of the hydrogen donor to the carbonyl group in the excited state. When N-acetyl l-tryptophan methyl ester (NATME) was employed as a quencher, a new long lived transient with absorption at 450–500 nm was observed. Fig. 7 shows the transient absorption spectrum recorded upon laser excitation of a solution of 1 in the

the DFT-IEFPCM method, the density functional to be used for the TD-DFT-IEFPCM calculation, and a 6-31Gd or a 6-31 + Gd basis set. For the calculation of the vertical transitions a minimal basis set polarized with d orbitals and augmented with a diffuse function (6-31 + Gd) has been shown in previous studies to be effective for the reliable calculation of the electronic transitions [45,51,52]. In the present study we have used the larger basis set 6-311 + +Gdp. The results are presented in Table 2. The effect of the basis set (631Gd v’s 6-31 + Gd) to calculate the initial ground state geometry is also included in Table 2 for the calculation involving the CAMB3LYP density functional. The inclusion of the diffuse function in the basis set for ground state geometry optimization results in about a 0.5 nm red shift for the calculated UV–vis spectrum. The notable features of the UV–vis spectrum of 1 are the maxima at approximately 220, 275, 310 nm and the valleys close to 245 and 295 nm in acetonitrile as well as the ondulated structure of the first band (Fig. 1). Calculations using the B3LYP functional did not reliably reproduce the essential features of the UV–vis spectrum of 1 (Fig. 9a) whereas the use of the functionals CAM-B3LYP and PBE0 gave spectra (Fig. 9b and c) with a more credible resemblance to the experimental spectrum. In the former case the features of the spectrum (maxima and minima) are blue shifted by about 20–30 nm

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Fig. 8. Decay for the singlet oxygen phosphorescence emission generated upon excitation (exc = 355 nm) of a sample of 1 in acetonitrile monitored at 1270 nm. Inset: plots for singlet oxygen phosphorescence emission versus light intensity for solutions of perinaphthenone () (standard for comparison) and 1 (䊉).

relative to the latter method. However, the PBE0 functional better reproduces the experimental spectrum in that the maxima are calculated to be 228, 281 and 324 nm with the valleys close to 250 and 300 nm. The difference in performance of the two functionals can be attributed to the nature of the chromophore where highly delocalized excited states (for example aromatic ketones that absorb in the visible region of the spectrum) may be better described by CAMB3LYP (n–␲* transitions) whilst chromophores involving a ␲–␲* transition are better described by the PBE0 functional [52–55].

Both the spectra calculated using the CAM-B3LYP and PBE0 density functionals reveal a weakly allowed first excited state that is close in energy to the strongly allowed second excited state. As for the higher excited states, the first excited state is composed of a mixture of significant pairs of canonical orbitals. Therefore a natural transition orbital (NTO) analysis was performed [56]. Fig. 10 reveals the nature of the first two excited states in the form of the natural transition orbitals (particle and hole orbitals – P and H, respectively) for both the CAM-B3LYP and PBE0 calculations. The results are equivalent and show that the first, weakly allowed, excited state corresponds to a n → ␲* transition (the respective CI coefficients are  = 0.986 and 0.997) whilst the second excited state corresponds to a strongly allowed ␲ → ␲* transition ( = 0.867 and 0.952, respectively). The optimized T1 state of 1 was calculated by DFT (UB3LYP/6311 + +G**//6-31G*) and indicated that the T1 state is about 59 kcal mol−1 (H298 ) more energetic than the ground S0 state. This value is in good agreement with theoretical results for the isomer ␣-naphthoflavone 2 [26] and for the experimental results obtained using triplet quenchers in the current study, as well as from the literature value for flavone (62 kcal mol−1 ) [8,10]. As the DFT method can be used to reliably calculate the T1 state we contemplated the possibility of calculating the T1 → Tn spectrum of 1, the results of which can be compared with the spectrum from the LFP experiments (see Fig. 2). Calculations were performed in an analogous manner to the previous calculations for the S0 state. The T1 state was fully optimized using UCAM-B3LYP and UPBE0 density functionals with a 6-31 + Gd basis set and vertical transitions to the second triplet state (T2 ) from this geometry were calculated using the same density functional and basis set. The calculated spectra are shown in Fig. 11. Of the two methods used to calculate the T1 → Tn spectra the method using the UPBE0 density functional bears an incredible resemblance to the triplet–triplet spectrum generated by LFP as shown in Fig. 2 whereas the spectrum calculated using the UCAMB3LYP functional is blue shifted. The experimental spectrum shows valleys at 400 and 650 nm, to either side of a maximum at about 500 nm, and non-zero absorption at greater than 650 nm. Literature

Table 2 The principal calculated vertical transitions for 1 using different DFT functionals.a DFT functional (func.)b

Excited state

Energy (eV)

 (nm)

Oscillator strength (f)

B3LYP

6 7 10 13 19

3.6536 3.6726 3.9551 4.2546 4.4280

339.35 337.60 313.48 291.41 280.00

0.2614 0.1297 0.0708 0.3889 0.0772

CAM-B3LYP

1 2 4 5 10 16

4.1570 4.2088 4.7686 4.9459 5.7699 6.3049

298.25 294.58 260.00 250.68 214.88 196.65

0.0274 0.3633 0.4570 0.3123 0.4470 0.6605

CAM-B3LYPc

1 2 4 5 10 16

4.1367 4.1964 4.7600 4.9358 5.7600 6.2893

299.71 295.45 260.47 251.19 215.25 197.14

0.0249 0.3672 0.4612 0.3238 0.4405 0.7011

PBE0c

1 2 4 11 12 18

3.7602 3.8223 4.4048 5.3675 5.4331 5.7938

329.73 324.37 281.48 230.99 228.20 214.00

0.0200 0.3922 0.4495 0.2611 0.4584 0.2396

a b c

The calculated spectra are presented in Fig. 9. TD-DFT-IEFPCM func./6-311 + +G(d,p)//DFT-IEFPCM func./6-31G(d). Solvent = acetonitrile, nstates = 20. Geometry DFT-IEFPCM func./6-31 + G(d). Solvent = acetonitrile.

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Fig. 9. Comparison of the calculated UV–vis spectrum of 1. TD-DFT-IEFPCM func./6-311 + +G(d,p). func.= (a) B3LYP [//DFT-IEFPCM B3LYP/6-31G(d)]; (b) CAM-B3LYP [//DFTIEFPCM CAM-B3LYP/6-31 + G(d)]; (c) PBE0 [//DFT-IEFPCM PBE0/6-31 + G(d)]. Solvent = acetonitrile, nstates = 20, UV–vis peak half-width at half height = 0.25 eV.

precedents for the use of TDDFT (using B3LYP) for the calculation of T1 → Tn spectra indicate that the calculations are relatively insensitive to the basis set used and give good predictive results for the triplet–triplet transitions [57,58]. Further exploration of the

calculation of triplet–triplet spectra and comparison with experimental spectra of the respective transients is necessary in order to establish the generality with which such spectra may be calculated and used to investigate the structure of triplet transients.

Fig. 10. The natural transition orbitals (NTOs) for the first two excited states as calculated from the respective transition densities from the TDDFT calculations using the CAM-B3LYP and PBE0 density functionals.

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Fig. 11. TD-DFT-IEFPCM T1 → Tn vertical excitation spectra calculated using the density functionals UCAM-B3LYP and UPBE0 (6-31 + Gd//6-31 + Gd), nstates = 20, solvent = acetonitrile. UV–vis peak half-width at half height = 0.25 eV.

6. Conclusion The triplet state of 1 was detected by laser flash photolysis experiments in acetonitrile solution. In 2-propanol (or in 1,4-cyclohexadiene), the ketyl radical of 1 formed by hydrogen abstraction from the triplet state, was observed and characterized. It was shown that 1 is also able to act as a photosensitizer for the one-electron oxidation of phenols and N-acetyl-l-tryptophan methyl ester (NATME). Additionally, efficient singlet oxygen formation was measured for 1 ( = 0.51). The results clearly demonstrate that 1 is able to undergo photosensitized type I and type II mechanisms with biological substrates. A DFT-B3LYP calculation of the triplet energy of 1 gave a value consistent with the observed reactivity of 1. TDDFT and NTO calculations were used to characterize the nature of the lowest excited states. A weakly allowed n,␲* excited state was characterized in close energetic proximity to the strongly dipole-allowed ␲,␲* excited state. Additionally, TDDFT was used to calculate a triplet–triplet spectrum that was consistent with the spectrum obtained after LFP excitation of 1. Therefore further substantiating the triplet nature of the generated transient. Acknowledgements This study was supported by the Brazilian agencies: CAPES (Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), and FAPERJ (Fundac¸ão de Amparo a Pesquisa do Estado do Rio de Janeiro). References [1] M.M. Caldwell, Solar UV irradiation and the growth and development of higher plants, in: A.C. Giese (Ed.), Photophysiology, Academic Press, New York, 1971, pp. 131–177. [2] J.B. Harborne, T.W. Goodwin, Plants Pigments, Oval Road, Academic Press, London, 1987. [3] H.A. Stafford, Flavonoid evolution: an enzymic approach, Plant Physiol. 96 (1991) 680–685.

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