The triplet state of a N-phenylphthalimidine with high intersystem crossing efficiency: Characterization by transient absorption spectroscopy and DNA sensitization properties

Article abstract of DOI:10.1021/jp0498926

The detailed triplet state characteristics of 2-(4-acetylphenyl)isoindolin- 1-one (kINP), a N-phenylphthalimidine (PPI) derivative, have been studied in fluid solution at room temperature. The attachment of an acetyl group to the N-phenyl moiety of PPI has permitted to enhance the intersystem crossing quantum yield, generally low for such compounds. Upon 308-nm laser flash photolysis of kINP in acetonitrile, a triplet-triplet transition has been evidenced ( λ_max = 440 nm). Further characterization of this transient at 440 nm gave a lifetime ?? = 11 μs, a molar absorption coefficient ?μ = 22 000 M^-1 ?? cm^-1, and an intersystem crossing quantum yield of 0.89. Moreover, a pi; pi; z.ast; nature has been found for this triplet state that lies at ca. 290 kJ ?? mol^-1 above the ground state. In addition to providing fundamental information on the triplet state properties of PPI derivatives, its importance during a photobiological process has been evidenced. kINP is the key compound involved in thymine dimers formation during the photosensitization of DNA by indoprofen, a nonsteroidal antiinflammatory drug.

Full text of DOI:10.1021/jp0498926

14148
J. Phys. Chem. B 2004, 108, 14148-14153
The Triplet State of a N-Phenylphthalimidine with High Intersystem Crossing Efficiency:
Characterization by Transient Absorption Spectroscopy and DNA Sensitization Properties
Virginie Lhiaubet-Vallet,†,‡ Je´roˆme Trzcionka,‡ Susana Encinas,† Miguel A. Miranda,*,† and
Nadia Chouini-Lalanne*,‡
Instituto de Tecnolog´ıa Qu´ımica UPV-CSIC, UniVersidad Polite´cnica de Valencia,
AVenida de los Naranjos s/n, 46022 Valencia, Spain and Laboratoire des Interactions Mole´culaires et
Re´actiVite´ Chimique et Photochimique, UMR 5623 au CNRS, UniVersite´ Paul Sabatier,
118 Route de Narbonne, 31062 Toulouse Cedex 4, France
ReceiVed: January 9, 2004; In Final Form: May 4, 2004
The detailed triplet state characteristics of 2-(4-acetylphenyl)isoindolin-1-one (kINP), a N-phenylphthalimidine
(PPI) derivative, have been studied in fluid solution at room temperature. The attachment of an acetyl group
to the N-phenyl moiety of PPI has permitted to enhance the intersystem crossing quantum yield, generally
low for such compounds. Upon 308-nm laser flash photolysis of kINP in acetonitrile, a triplet-triplet transition
has been evidenced (λmax ) 440 nm). Further characterization of this transient at 440 nm gave a lifetime τ )
11 µs, a molar absorption coefficient ꢀ ) 22 000 M-1 × cm-1, and an intersystem crossing quantum yield
of 0.89. Moreover, a ππ* nature has been found for this triplet state that lies at ca. 290 kJ × mol-1 above the
ground state. In addition to providing fundamental information on the triplet state properties of PPI derivatives,
its importance during a photobiological process has been evidenced. kINP is the key compound involved in
thymine dimers formation during the photosensitization of DNA by indoprofen, a nonsteroidal antiinflammatory
drug.
Introduction
CHART 1: Structures of Benzanilide (BA),
N-Phenylphthalimidine (PPI), Indoprofen (INP), and Its
Photoproduct kINP
The intriguing emission behavior of benzanilide (BA) (Chart
1) and its derivatives has received considerable attention.1-5 In
solution, at room temperature, BA is very weakly fluorescent
in nonpolar media and essentially nonfluorescent in polar media.
Under these conditions, no phosphorescence is observed. The
same is true for N-phenylphthalimidine (PPI), a rigid lactam
analogue of BA where rotation about the carbonyl-nitrogen bond
is precluded.5 Basically, all the energy absorbed by both
compounds is wasted through radiationless decay, corresponding
almost exclusively to internal conversion within the singlet
manifold.1
Recent investigations on the phosphorescence of BA and its
derivatives at 77 K, both in methyltetrahydrofuran (MTHF)
glasses and in microcrystalline solids, have shown that emission
takes place from a planar ππ* triplet state.5 This is in agreement
with theoretical (ZINDO) calculations. The phosphorescence
spectra of microcrystalline BA and PPI are strongly structured,
and their maxima (460 and 430 nm, respectively) appear at
longer wavelengths than in MTHF glasses (410 and 420 nm).
biological properties of the basic N-phenylphthalimidine chro-
mophore present in the rigid derivative PPI.
This chromophore is the key substructure of the nonsteroidal
antiinflammatory drug (NSAID) indoprofen (INP, 2-[4-(1-oxo-
2-isoindolinyl)phenyl]propionic acid) that belongs to the aryl-
propionic acid family (Chart 1). Related NSAIDs are ketoprofen,
tiaprofenic acid, naproxen, and suprofen, among others. Such
NSAIDs have been repeatedly associated with photosensitivity
reactions that generally involve drug-photosensitized damages
to biomolecules.6-9 In this context, DNA photosensitization has
received special attention, as genome damage is considered to
be the primary event for many changes of biological importance
(photogenotoxicity, photogenomutagenicity, photocarcino-
genesis).
Thus, very little information is available on the triplet states
of BA and PPI. In particular, the spectroscopic characterization
and chemical behavior of these states in solution, at room
temperature, has not yet been reported. A systematic investiga-
tion of these aspects would be interesting both from the
fundamental point of view and in connection with the photo-
One of the major alterations of DNA is formation of
cyclobutane thymine dimers, which are thought to be predomi-
nant premutagenic damages.10,11 For this photolesion to be
initiated by the UVA region of sunlight, a photosensitizer must
be able to participate in a triplet-triplet energy-transfer mech-
* Corresponding authors. Miguel A. Miranda: Phone: +34-96-3877807;
fax: +34-96-3877809; e-mail: mmiranda@qim.upv.es. Nadia Chouini-
Lalanne: Phone: +33-(0)5-61556274; fax: +33-(0)5-61558155; e-mail:
lalanne@chimie.ups-tlse.fr.
† Universidad Polite´cnica de Valencia.
‡ Universite´ Paul Sabatier.
10.1021/jp0498926 CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/21/2004

The Triplet State of a N-Phenylphthalimidine
J. Phys. Chem. B, Vol. 108, No. 37, 2004 14149
anism. Only a few compounds are efficient in this process, since
they have to combine a good intersystem crossing quantum yield
with a triplet state energy higher than that of thymine (ca. 310
kJ × mol-1 in the free base or the nucleoside, but at least 10 kJ
× mol-1 lower in DNA).12,13 Thus, although DNA damage
formation has been reported for several NSAIDs derived from
arylpropionic acid,14-19 only ketoprofen or indoprofen (Chart
1) appears to photosensitize a significant amount of dimers.17-19
In INP, this was surprising since no direct triplet-triplet
absorption can be detected by laser flash photolysis (LFP).20
Thus, intersystem crossing to the triplet state does not take place
in an efficient way, as expected from the close structural
similarity between INP and PPI. An interesting hypothesis is
that one of the INP photoproducts (rather than the parent drug)
could be the key chemical entity involved in the DNA-
photosensitization reaction. In agreement with this hypothesis,
the photostable methyl ester of indoprofen, containing the same
chromophore, does not photosensitize thymine dimers.19
All the photoproducts of indoprofen contain the same PPI
substructure, but only one of them, namely, 2-(4-acetylphenyl)-
isoindolin-1-one (kINP), includes the acetophenone chromo-
phore (Chart 1). As acetophenone is able to produce thymine
photodimerization,13,21,22 kINP was considered as the potential
photosensitizer of thymine dimers during irradiation of DNA
in the presence of INP.
With this background, the purpose of the present work has
been to perform a detailed photophysical and photochemical
study on kINP. From the fundamental point of view, attachment
of the acetyl substituent to the N-phenyl ring of PPI should
greatly enhance intersystem crossing, providing an efficient route
to the triplet state; this could in principle allow characterization
of this excited state in solution, at room temperature, by LFP.
As regards the photobiological properties, a combination of time-
resolved studies on kINP in the presence of triplet quenchers
(including DNA and its components) with gel sequencing
experiments using repair enzymes was expected to provide a
valuable tool to investigate the mechanism of DNA damage
upon photosensitization with INP.
solution of kINP (2 × 10-5 M) and biphenyl (10-3 M) in
acetonitrile was studied; under these conditions, more than 95%
of kINP triplet state was quenched by biphenyl. Then eq 1 was
used:
ꢀ(3kINP) ) ꢀ(3Biph(360)) ×
∆A(3kINP(440))/∆A(3Biph(360)) (1)
where ∆A(3kINP(440)) and ∆A(3Biph(360)) refer to the transient
absorption of kINP triplet state (λ ) 440 nm) at the beginning
of the reaction and to the absorbance of biphenyl triplet state
(λ ) 360 nm) at the end of the process, respectively; ꢀ(3Biph-
(360)) corresponds to the molar absorption coefficient of the
biphenyl triplet at 360 nm, which is 42 800 M-1 × cm-1 25
.
The kINP triplet quantum yield φisc(kINP) was obtained by
comparing the amount of 3kINP populated after laser excitation
with that for naphthalene (Naph) at the same absorbance. Thus,
φisc was calculated by using eq 2:
φisc (kINP) ) φisc(Naph) × ∆A(3kINP(440)) ×
ꢀ(3Naph(415))/[∆A(3Naph(415)) × ꢀ(3kINP(440))] (2)
where φisc(Naph) corresponds to the intersystem crossing
quantum yield of naphthalene; ꢀ(3Naph(415)) refers to the molar
absorption coefficient of naphthalene triplet at 415 nm;
∆A(3kINP(440)) and ∆A(3Naph(415)) refer to the transient
3
absorption of kINP at 440 nm and naphthalene triplet at 415
nm, respectively. The values of ꢀ(3Naph(415)) and φisc(Naph)
were 24 500 M-1 × cm-1 and 0.8, respectively.24,25
Phosphorescence Spectroscopy. Emission spectra were
obtained on a Perkin-Elmer LS-50B spectrofluorimeter equipped
with a xenon source (flash duration 8 µs) and a Hamamatsu
R928 photomultiplier tube with the low-temperature accessory
L2250136. The apparatus was operated in time-resolved mode,
with a delay time of 0.15 ms. Excitation and emission
monochromator band-passes of 4 nm were used. The sample
was put in a capillary tube (2-mm diameter) and cooled to 77
K in a liquid nitrogen bath. Dry nitrogen circulation in the cell
compartment avoided water condensation on the cell walls. The
emission was obtained by exciting the sample at 308 nm in
ethanol, using an absorbance of about 0.05 in a 10 mm cell.
Gel Sequencing Experiments. Oligonucleotides 1 and 2 and
their complementary strands were synthesized and purified by
polyacrylamide gel electrophoresis by Genosys (Cambridgeshire,
U.K.). Phage T4 endonuclease V was purchased from TEBU
(Le Perray-En-Yvelines, France):
Materials and Methods
Synthesis of kINP. Although kINP was available in small
amounts by photolysis of indoprofen, it was prepared at higher
scale by reaction of 4-aminoacetophenone with 2-carboxybenz-
aldehyde in methanol, followed by reduction with NaBH4.23 Its
characterization was in agreement with the previously published
data.20
Laser Flash Photolysis Measurements. A pulsed Excimer
Laser Systems with Xe/HCl/Ne mixture was used for excitation
at 308 nm. The single pulses were ∼17 ns duration and the
energy was e100 mJ/pulse. A pulsed Lo255 Oriel xenon lamp
was employed as detecting light source. The laser flash
photolysis apparatus consisted of the pulsed laser, the Xe lamp,
a 77200 Oriel monochromator, an Oriel photomultiplier tube
(PMT) system made up of a 77348 side-on PMT tube, 70680
PMT housing, and a 70705 PMT power supply. The oscilloscope
was a TDS-640A Tektronix. The output signal from the
oscilloscope was transferred to a personal computer. Indoprofen
was dissolved in acetonitrile or in phosphate buffer (PBS) (10
mM in NaCl)/20% ethanol mixtures (to increase kINP solubility)
and had an absorbance of 0.4 (∼2 × 10-5 M) at 308 nm. All
the solutions were deaerated by nitrogen bubbling.
25-mer 1: 5′TGAGCGTTAGTTTAAGTCGGCTATC-3′
20-mer 2: 5′-TGATCGGTGCGTCTGAGACT-3′
Preparation of 32P End-Labeled Oligonucleotides. 25-mer
1 and 20-mer 2 were radiolabeled at the 5′-end using standard
procedures. Typically, 15 µCi of [γ32P] ATP and 5 pmol of
oligonucleotide were added to the Ready-to-Go T4 Polynucle-
otide Kinase. After 30-min incubation at 37 °C, the reaction
was stopped by addition of 5 µL of 250 mM ethylenediamine
tetraacetic acid. Then, to remove free [γ32P] ATP, the reaction
mixture was purified using G-25 Microspin columns. Double-
stranded DNA was prepared by hybridization of the 5′-end-
labeled oligonucleotide with the same amount of its unlabeled
complementary strand, heating 10 min at 70 °C and cooling
slowly to room temperature.
Photosensitization Experiments. Frank and Alkali-Labile
Breaks. All the solutions were prepared in 5 mM phosphate
buffer with 10 mM NaCl, pH 7.4. Samples containing 5 µL of
radiolabeled double-stranded oligonucleotides (5000 cpm/µL),
The molar absorption coefficient of triplet-triplet transition
of kINP (ꢀ (3kINP)) was determined by the energy-transfer
method using biphenyl (Biph) as triplet energy acceptor.24
A

14150 J. Phys. Chem. B, Vol. 108, No. 37, 2004
Lhiaubet-Vallet et al.
TABLE 1: Bimolecular Rate Constants for the Interaction
of KINP Triplet with Several Quenchers of Different Triplet
Energy, in Acetonitrile
triplet state energy
quencher
oxygen
(kJ × mol-1
)
kq (M-1 × s-1
)
95
255
260
275
285
290
300
6.0 × 109
4.0 × 109
7.8 × 109
3.3 × 109
3.4 × 109
<5 × 107
1.6 × 106
naphthalene
styrene
biphenyl
fluorene
benzophenonea
norbornene
Figure 1. Transient absorption spectrum obtained 0.45 µs after laser
excitation of 2 × 10-5 M kINP in acetonitrile (absorbance ∼0.4 at
308 nm) under nitrogen. Insert: Decay of the signal at 440 nm.
a Because of the significant absorption of benzophenone at 308 nm,
accurate kINP triplet lifetime measurements were not possible at
quencher concentrations higher than 10-3 M.
5 µL of drug, 5 µL of unlabeled double-stranded oligonucleo-
tides, and 5 µL of buffered solution were irradiated at 25 °C
using a xenon lamp (Muller 450 W) equipped with a long pass
filter λ > 320 nm (Oriel, WG-320). After irradiation, samples
used for the detection of frank breaks were lyophilized. Alkali-
labile sites were revealed using 20 µL of freshly prepared 1 M
aqueous piperidine solution at 90 °C during 30 min. Piperidine
was then removed by lyophilization.
Detection of Pyrimidine Dimers. The samples were prepared
by the procedure described above but in a Tris/HCl buffer (5
mM in EDTA, 5 mM in MgCl2, pH 7.5). After irradiation, they
were incubated for 1 h at 37 °C with an excess of T4
endonuclease V and lyophilized.
Electrophoresis Analysis. Before electrophoresis, the samples
were resuspended in 10 µL of formamide sequencing buffer.
Frank breaks and alkali- and endonuclease-sensitive lesions were
separated by 20% denaturating (7 M urea) polyacrylamide gel
electrophoresis and visualized after exposure of autoradiograms
overnight at -70 °C (Hyperfilm ECL). G26 and T . G27
markers were prepared as described in the literature.
triplet energy-transfer mechanism at the origin of this efficient
population of the quencher triplet state evidenced the triplet
nature of the 440-nm transient.
3
To evaluate the triplet state energy of kINP, kINP lifetime
was measured in the presence of different amounts of several
quenchers, and the corresponding quenching rate constants (kq)
were obtained by means of the Stern-Volmer plots. A clear
effect of triplet state energy on the bimolecular rate constant
3
was observed (Table 1) and would permit to situate kINP by
respect with the energy of triplet quenchers.
Representation of the logarithm of kq against the triplet state
energy of the quenchers (Figure 3) showed a “plateau” for
energies up to 275-285 kJ × mol-1; over this value the rate of
energy transfer sharply decreased. As triplet-triplet energy
transfer is nearly diffusion-controlled when the triplet energy
of the donor is higher than that of the acceptor by at least 10 kJ
3
× mol-1 28
,
the energy of kINP was situated in the range of
285-295 kJ × mol-1. Furthermore, kINP phosphorescence
emission in ethanol at 77 K (see Supporting Information)
permitted to estimate a similar value (ca. 285 kJ × mol-1). Thus,
the triplet energy of kINP must be very close to that of
benzophenone, but still somewhat lower. Actually, when a 10:1
mixture of benzophenone and kINP was submitted to LFP at
355 nm (where most of the energy is absorbed by the former
compound) only the T-T absorption of kINP at λmax ) 440
nm was observed 1 µs after the laser pulse.
Results
The Triplet of kINP in Acetonitrile. Laser flash photolysis
of kINP in acetonitrile was performed at 308 nm with a pulsed
excimer laser. A well-defined transient absorption spectrum with
a maximum at 440 nm was observed (Figure 1). The decay
monitored at 440 nm (Figure 1, insert) showed a first-order
kinetic corresponding to a lifetime of ca. 11 µs. It was tentatively
assigned to the kINP triplet state by comparison with the
spectrum obtained upon photosentization of the triplet state of
indoprofen by acetone in aqueous medium (λmax ) 450 nm, τ
) 25 µs).20
This assignment was confirmed by quenching experiments.
As expected for a triplet, it was efficiently quenched by oxygen.
Moreover, experiments in the presence of naphthalene or
biphenyl showed clearly formation of the corresponding triplet
states (at λ ) 410 and 360 nm, respectively) concomitantly with
the decay of the 3kINP signal (Figure 2a and 2b). The triplet-
3
For further characterization of kINP, its molar absorption
coefficient was determined by using the energy-transfer method
described in the Materials and Methods section (see eq 1) and
is 22 000 M-1 × cm-1 (at λ ) 440 nm). On the basis of this
value, a high intersystem crossing quantum yield (φisc ) 0.89)
was obtained by means of the comparative method also
described in the Experimental Section (see eq 2). Hence,
although INP does not produce significant amounts of triplet
upon direct photolysis, attachment of the acetyl substituent to
the phenyl ring results in a dramatic enhancement of intersystem
crossing efficiency.
Figure 2. Time-dependent transient absorption spectra of nitrogen-purged solutions of 2 × 10-5 M kINP in acetonitrile in the presence of 10-4
M
naphthalene (a) or 10-3 M biphenyl (b). Insert Figure 2b: Decay of 3kINP at λ ) 440 nm (;) and growth of biphenyl triplet at λ ) 360 nm (‚‚‚).

The Triplet State of a N-Phenylphthalimidine
J. Phys. Chem. B, Vol. 108, No. 37, 2004 14151
The two oligomers (8 µM in base pair) were UVA-irradiated
in the presence of kINP (8 µM) and submitted to different
treatments; the oligonucleotide 1 was treated with a specific
DNA repair enzyme (T4 endonuclease V) to reveal thymine
dimers, while alkali-labile products of guanine oxidation were
studied by piperidine treatment of the oligonucleotide 2.
Without piperidine or enzymatic treatment no direct cleavage
of DNA occurred (Figure 4, lanes D1 and K1). However, after
incubation with T4 endonuclease V, DNA irradiation in the
presence of kINP gave rise to fragments at the TTT site (Figure
4, lane D2) corresponding to an efficient formation of thymine
dimers. Alkali-labile treatment revealed that kINP is also
responsible for a specific guanine oxidation (Figure 4, lane K2)
in the 5′ site of the GG sequence. The efficiency of these
reactions was similar to that observed using benzophenone and
its derivatives as photosensitizers.17
Figure 3. Plot of the logarithm of the bimolecular quenching rate
constants of 3kINP by different quenchers vs their triplet state energy.
Finally, the reactivity toward hydrogen abstraction was
Discussion
investigated as an indicator of the electronic nature (nπ* vs ππ*)
3
of kINP. Thus, laser flash photolysis of kINP in acetonitrile
Overall, the obtained results have provided the first evidence
for the triplet state of an isoindolinone derivative in fluid solution
and at room temperature. Characterization by laser flash
photolysis of this transient at 440 nm gave a τ of ca. 11 µs
solution was performed in the presence of increasing amounts
of 2-propanol as hydrogen donor (from equimolar to 200-fold
molar ratio). Even when the highest 2-propanol excess was used,
no significant 3kINP quenching was observed; the bimolecular
with a molar absorption coefficient ꢀ ) 22 000 M-1 × cm-1
,
rate constant was <8 × 105 M-1 × s-1
.
an intersystem crossing quantum yield of 0.89, an energy of
290 kJ × mol-1, and a ππ* nature in acetonitrile.
It is interesting that the triplet state is directly available from
excitation of kINP; this is in sharp contrast with the case of
indoprofen where acetone photosensitization was needed.20
Hence, kINP may be at the origin of the thymine dimers
formation previously described during photosensitization of
DNA by INP.19
The Triplet of kINP in PBS. The generation and reactivity
3
of kINP was also studied in PBS to determine its potential to
induce DNA photosensitization in aqueous media. The same
transient as in acetonitrile was observed at λmax ) 440 nm, but
with a longer lifetime (τ ) 35 µs). This transient was efficiently
quenched by oxygen and n×aphthalene, with bimolecular rate
constants of 4 × 109 M-1 s-1 and 1.5 × 109 M-1 × s-1
,
respectively.
3
The spectroscopic properties of kINP have been studied at
3
Further, the interaction of kINP with key substructures of
DNA was investigated; thymidine (dThd) and 2′-deoxygua-
nosine (dGuo) were chosen as model nucleosides because of
their particular properties in DNA. Thus, thymine is the
nucleobase with the lowest triplet energy,12 being the most
favorable acceptor site for an energy-transfer mechanism. By
contrast, guanine is the preferred site for oxidation in DNA,
owing to its low oxidation potential.29,30 Laser flash photolysis
of kINP was performed in PBS, in the presence of increasing
amounts of dThd and dGuo. The lifetime of the resulting
transient 3kINP was determined under different conditions, and
then the Stern-Volmer plots were recorded as usually. Operat-
room temperature and compared with results reported for similar
compounds.1,5,20 First, the similarity of the transient spectrum
(λmax ) 440 nm) to that of the photosensitized triplet state of
INP together with the similar values of the triplet state energy
of kINP and PPI (i.e., 280 kJ × mol-1)5 reveal the minor
influence of substitution at the N-phenyl group on these
properties. As triplet states of composite molecules (unlike
singlets) prefer to be localized, it seems that the delocalized
ππ* singlet state undergoes intersystem crossing to form the
acetophenone nπ* triplet, which decays to the lower energy (N-
phenyl)-isoindolinone ππ* triplet.
The high intersystem crossing quantum yield indicates that
the acetophenone moiety still plays an important role. Actually,
the studies on INP and PPI described in the literature have
shown that population of their triplets is quite ineffective; no
transient was detected during the “direct” flash photolysis of
INP,20 while PPI has a low triplet yield (φisc < 0.05).1 It is
clear that the well-known behavior of ketones plays a key role
in kINP, where substitution on the isoindolinone ring by
acetophenone (φisc close to 1)25 facilitates intersystem crossing
for the whole system.
The electronic nature of 3kINP was studied by investigating
its ability to abstract hydrogen atoms. In this context, efficient
quenching of the triplet state by H-donors is associated with
nπ* triplet character (i.e., benzophenone kq ) 2.9 × 108 M-1
× s-1);31 by contrast, ππ* states exhibit low bimolecular
quenching rates (i.e., 4-methoxybenzophenone kq ) 5 × 105
M-1 × s-1).32 On the other hand, the isoindolinone ring has
been reported to be a H-donating (rather than H-accepting) group
in photochemical reactions.33 Thus, the low bimolecular rate
constant found for quenching of 3kINP by 2-propanol (ca. 8 ×
105 M-1 × s-1) points to the ππ* nature of kINP triplet state.
ing in this way, bimolecular rate constants of 7.8 × 106 M-1
×
s-1 and 1.6 × 109 M-1 × s-1 were obtained for quenching of
3kINP by dThd and dGuo, respectively. The same procedure
was employed to study quenching of 3kINP by calf thymus DNA
in aqueous medium. This process occurred with a rate constant
of ca. 2 × 107 M-1 × s-1, a value intermediate between those
obtained for dThd and dGuo.
DNA Photosensitization. The photosensitizing properties of
kINP toward DNA were investigated to conclude about its
possible role during DNA damage upon irradiation in the
presence of indoprofen. The experiments were carried out on
two different 5′-32P end-labeled synthetic double-stranded
oligonucleotides, each of them containing specific sites in
relation with the potentially involved mechanism:
(i) oligonucleotide 1 included adjacent thymines to investigate
the possibility of energy transfer at the origin of the formation
of thymine dimers,
(ii) oligonucleotide 2 contained several guanines included in
different sequences to study the participation of electron transfer
from a guanine base to the excited drug.

14152 J. Phys. Chem. B, Vol. 108, No. 37, 2004
Lhiaubet-Vallet et al.
Figure 4. A 20% polyacrylamide gel showing DNA photosensitization by kINP. All experiments were carried out in phosphate buffer, 10 mM
NaCl (pH 7.4), and the samples were UVA-irradiated (λ > 320 nm) for 50 min. (a) Duplex oligonucleotide 1 was submitted or not to enzymatic
treatment to reveal thymine dimers formation. (b) Duplex oligonucleotide 2 was treated or not with hot piperidine to reveal guanine oxidations.
Lanes A, H: nonirradiated; Lanes B, I: irradiated alone; Lanes C, J: nonirradiated in the presence of 8 µM kINP; Lanes D, K: irradiated in the
presence of 8 µM kINP. G and T are guanine and thymine markers, respectively. The subscripts 1 and 2 refer to nontreated and treated (endonuclease
or piperidine) DNA samples.
This is also in agreement with the low efficiency of INP as
photosensitizer of lipid peroxidation, a process that generally
involves abstraction of double allylic hydrogens from polyun-
saturated fatty acids, as the initiation step of a radical chain
mechanism.34 Further support in favor of the ππ* character of
3kINP was provided by the lack of quenching by norbornene.
In this case, energy transfer is not possible because of
thermodynamic reasons (ET of norbornene ) 300 kJ × mol-1);
however, triplet quenching associated with a Paterno-Bu¨chi
cycloaddition should still be observable for a carbonylic nπ*
triplet.35
state which occurs on going from mononucleotide to oligo-
nucleotide, base triplet energy levels must be lowered in
DNA.13,37 Moreover, the determination of the triplet state of
kINP permits to approach the triplet energy of thymine within
DNA, which is not yet known. This value must be lower than
that of kINP (290 kJ × mol-1) since an energy-transfer
mechanism takes place from the triplet state of kINP to thymine
residues. As a matter of fact, an analogous energy transfer takes
place from benzophenone and its derivatives,17,38,39 leading to
cyclobutadithymidine lesions in DNA. The frequency of thymine
dimers formation is also influenced by adjacent sequence and
will be favored if the 5′ neighbouring base is a thymine.40
In principle, thymine dimerization could also occur via a
singlet energy-transfer mechanism. However, this possibility
appears very unlikely in kINP, as its singlet energy (350 kJ ×
mol-1) is much lower than that of the nucleobase (417 kJ ×
mol-1).25 Accordingly, the weak fluorescence of kINP was not
quenched by addition of dThd (see Supporting Information).
In addition to this bipyrimidine lesion, the production of
piperidine alkali-labile sites essentially at 5′G of a GG step has
been evidenced. It is now well known that the presence of a
GG site in DNA will act as a “potential trap”, this specific
oxidation being relevant of a mechanism involving an electron
transfer from the guanine to the excited photosensitizer.30,41
Laser flash photolysis of kINP was also performed in PBS
to study the photosensitizing properties of 3kINP toward DNA
or some of its substructures, thymidine and 2′-deoxyguanosine.
3
The weak interaction of kINP with dThd (ca. 7.8 × 106 M-1
× s-1) can be accounted for by comparing the triplet energies
of the nucleoside (310 kJ × mol-1) and kINP (ca. 290 kJ ×
mol-1); this makes an energy-transfer mechanism endergonic
and hence quite disfavored. As regards quenching of 3kINP by
dGuo, it can be attributed to an electron transfer from dGuo to
the excited kINP. Similar guanine oxidations by phthalimide
derivatives have been reported in the literature.36
Photosensitization of DNA by kINP gave rise to the formation
of thymine dimers and guanine oxidation products. Thymine
dimerization occurs by a triplet-triplet energy-transfer mech-
anism. Although this type of lesions was detected during INP
photosensitization of DNA, the parent drug was not at the origin
of the triplet-triplet energy transfer. Accordingly, it has been
reported that no DNA damage occurs when photodecomposition
of the drug is “blocked” by esterification, while preirradiated
solutions containing the photodecomposition products give rise
to thymine dimers.19
The DNA-photosensitization experiments now reported are
in agreement with the data obtained by laser flash photolysis.
Nevertheless, the efficiency of the reactions in DNA cannot be
completely correlated with the reactivity of the isolated nucleo-
sides. It is assumed that, like the lowering of base singlet excited
Acknowledgment. We thank the Spanish MCYT (Grant
BQU2001-2725 and Ramon y Cajal project to S.E.), the
Generalitat Valenciana (Grupo 03/082), and the EU (Marie Curie
postdoctoral fellowship HFMF-CT-2001-01228 to V.L-V.) for
financial support.
Supporting Information Available: UV, phosphorescence,
and fluorescence spectra of kINP. This material is available free
of charge via the Internet at http://pubs.acs.org.
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