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

Article abstract of DOI:10.1016/j.jphotochem.2014.08.009

The absorption spectra for β-naphthoflavone (1) reveal a solvatochromic red shift in polar solvents which is consistent with the π,π? 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 (¹O₂) 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.

Full text of DOI:10.1016/j.jphotochem.2014.08.009

Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 121–129
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Laser flash photolysis study of the reactivity of ␤-naphthoflavone
triplet: Hydrogen abstraction and singlet oxygen generation
Nanci C. de Lucasa,∗, Guilherme L.C. Santosa, Caio S. Gaspara, Simon J. Gardena,
José Carlos Netto-Ferreirab
a Laboratório de Fotoquímica, Instituto de Química – Universidade Federal do Rio de Janeiro, Cidade Universitária, RJ, Brazil
b 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
a b s t r a c t
Article history:
Received 22 July 2014
Received in revised form 21 August 2014
Accepted 22 August 2014
Available online 1 September 2014
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 tech-
nique 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 exper-
iments 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 (1O2) 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 spec-
trum consistent with the triplet transient generated by LFP. Excellent correlation of the calculated and
experimental spectra was achieved using the conventional PBE0 hybrid functional.
Keywords:
Naphthoflavone
Triplet excited state
Hydrogen abstraction
Singlet oxygen
TDDFT calculations
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Photophysical and photochemical studies on flavonoids have
revealed important characteristics which may be related to their
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 biosyn-
thetic pathway [2]. The efficient absorbance of UV irradiation has
implied their possible involvement in the protection against UV-
induced damage in plants [3,4].
photobiological behavior. Photodimerization is the main photore-
action 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 absorp-
tion 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 deriva-
tive 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].
∗
Corresponding author. Tel.: +55 21 3938 72 78.
E-mail addresses: nancicl@iq.ufrj.br, nanci.garden@gmail.com (N.C. de Lucas).
http://dx.doi.org/10.1016/j.jphotochem.2014.08.009
1010-6030/© 2014 Elsevier B.V. All rights reserved.

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Recently Mitsumori et al. shows that 1 induced the sustained pro-
duction 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, apop-
totic or necrotic signaling pathways to cell death can be induced by
the combined use of a photo-activated drug, called the photosen-
sitizer, 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 pro-
cess toward biomolecules [22,23]. More general photosensitizing
mechanisms can involve either photo-oxidation of nucleic acid
components by the sensitizer, yielding the corresponding radi-
cal 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
(1ꢀg) 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 reac-
tion of Trp and Tyr with photosensitizers has received considerable
attention in the field of proteins photo-oxidation.
2.3. Laser flash photolysis
The laser flash photolysis (LFP) experiments were carried out
on a LuzChem Instrument model mLFP122. Samples were con-
tained in a 10 mm × 10 mm cell made from Suprasil tubing and
were deaerated by bubbling with argon for about 20 min. The sam-
ples were irradiated with the third harmonic of a Nd/YAG Surelite
laser (ꢁ = 355 nm). All laser flash photolysis experiments were per-
formed 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 appro-
priate 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]
(1)
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
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 reac-
tion mechanisms of deactivation could aid in the understanding of
the pharmacological action of this class of molecule.
The quantum efficiency of singlet oxygen (1O2) formation was
determined using a spectrofluorometer (FS920 Edinburgh Instru-
ments) with a TMS300 monochromator. The detection system was
equipped with a NIR Hamamatsu model H1033-45 photomulti-
plier. 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 phos-
phorescence 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 peri-
naphthenone emission intensity, used as standard, and ˚PER is the
quantum yield of singlet oxygen formation from perinaphthenone.
2. Materials and methods
Isample
˚
=
× ˚PER
(2)
sample
2.1. General
IPER
Acetonitrile, 2-propanol, cyclohexane and 1-methylna-
phthalene (all spectroscopic grade), were provided by
Sigma–Aldrich. Water was milli-Q grade. Phenol, 4-
methoxyphenol, 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 l-
tryptophan methyl ester, from Aldrich, were used as received
(purity >99%).
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 sol-
vated 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 ꢀS2ꢁ 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).
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 Shi-
matzu model UV-2450 UV–vis spectrophotometer.

N.C. de Lucas et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 121–129
123
Fig. 1. Absorbance spectra for 1 (5.8 × 10−4 mol L−1) in different solvents.
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 pres-
ence 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 spec-
trum 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 solu-
tions 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 concentra-
tion; from the slope of a plot of first-order rate constants for the
decay versus the concentration of 1 a self-quenching rate con-
stant 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 absorp-
tion 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.
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.
The triplet of
1
)
was quenched by 1-methylnaphthalene
in acetonitrile with rate constant of
corresponding to its triplet state (ꢁmax = 500 nm; ꢂ = 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 pro-
cesses 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
(ET = 60.6 kcal mol−1
a
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 (ꢂ = 20 ␮s), besides the absorption

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N.C. de Lucas et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 121–129
Fig. 4. Transient absorption spectra obtained upon 355 nm excitation of
0.37 mol L−1 of 1,4-cyclohexadiene. Inset: kinetic traces at 410 and 500 nm.
1 in
Fig. 6. Transient absorption spectra obtained upon 355 nm excitation of 1 in ace-
tonitrile. (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.
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.
reaction of the triplet 1 with 2-propanol, suggesting that this reac-
tion 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).
.
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].
(3)
(4)
The triplet of 1 was quenched by 1,4-cyclohexadiene in acetoni-
trile 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
125
presence of 2.1 × 10−3 mol L−1 NATME, in acetonitrile. This tran-
sient 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 second-
order rate constants for the quenching process of triplet 1 by this
quencher.
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
)
5.46 ( 0.09) × 107
a2.3 × 108
Phenol
2.07 ( 0.03) × 107
a9.9 × 107
4-Cyanophenol
4-Methoxyphenol
2.78 ( 0.03) × 108
a3.7 × 109
a
kq values for the triplet of 2 [26].
4. Singlet oxygen formation
Singlet oxygen can be formed through an energy transfer pro-
cess from a suitable donor. A tiny fraction of 1O2 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 1O2 detection (Eq. (6)).
3S ∗ +O2 → S0
+
1O2
(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 (1O2) forma-
tion 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
2.1 × 10−3 mol L−1 of NATME in acetonitrile.
1 in
The vertical transition UV–vis spectra of 1 were simulated in
acetonitrile by using the TD-DFT-IEFPCM method and the follow-
ing density functionals: B3LYP, CAM-B3LYP, and PBE0. The initial
geometry of 1 was first optimized with solvent simulation using
370–505 nm region, that can be easily detected by LFP experiments
[26,44,46,47].
(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 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 (6-
31Gd v’s 6-31 + Gd) to calculate the initial ground state geometry
is also included in Table 2 for the calculation involving the CAM-
B3LYP 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 max-
ima 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 spec-
trum (maxima and minima) are blue shifted by about 20–30 nm
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

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N.C. de Lucas et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 121–129
Both the spectra calculated using the CAM-B3LYP and PBE0 den-
sity 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 nat-
ural 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/6-
311 + +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 con-
templated 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 UCAM-
B3LYP 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
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 (᭹).
relative to the latter method. However, the PBE0 functional better
reproduces the experimental spectrum in that the maxima are cal-
culated 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 delocal-
ized excited states (for example aromatic ketones that absorb in the
visible region of the spectrum) may be better described by CAM-
B3LYP (n–␲* transitions) whilst chromophores involving a ␲–␲*
transition are better described by the PBE0 functional [52–55].
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)
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
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-B3LYP
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
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
PBE0c
a
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.
b
c

N.C. de Lucas et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 121–129
127
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 [//DFT-
IEFPCM 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 insen-
sitive 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 exper-
imental 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.

128
N.C. de Lucas et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 121–129
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, sol-
vent = acetonitrile. UV–vis peak half-width at half height = 0.25 eV.
6. Conclusion
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The triplet state of 1 was detected by laser flash photoly-
sis 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 for-
mation was measured for
1 (ꢃꢀ = 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 calcu-
lation 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. Addi-
tionally, 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
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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 Tec-
nológico), and FAPERJ (Fundac¸ ão de Amparo a Pesquisa do Estado
do Rio de Janeiro).
tive stress responses and the induction of preneoplastic lesions in
a
diethylnitrosamine-initiated hepatocarcinogenesis model in partially hepatec-
tomized rats, Toxicology 244 (2008) 179–189.
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