Thymidine radical formation via one-electron transfer oxidation photoinduced by pterin: Mechanism and products characterization

Article abstract of DOI:10.1016/j.freeradbiomed.2016.04.196

UV-A radiation (320-400 nm), recognized as a class I carcinogen, induces damage to the DNA molecule and its components through different mechanisms. Pterin derivatives are involved in various biological functions, including enzymatic processes, and it has been demonstrated that oxidized pterins may act as photosensitizers. In particular, they accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder. We have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to photosensitize the degradation of the pyrimidine nucleotide thymidine 5′-monophosphate (dTMP) in aqueous solutions under UV-A irradiation. Although thymine is less reactive than purine nucleobases, our results showed that Ptr is able to photoinduce the degradation of dTMP and that the process is initiated by an electron transfer from the nucleotide to the triplet excited state of Ptr. In the presence of molecular oxygen, the photochemical process leads to the oxidation of dTMP, whereas Ptr is not consumed. In the absence of oxygen, both compounds are consumed to yield a product in which the pterin moiety is covalently linked to the thymine. This compound retains some of the spectroscopic properties of Ptr, such as absorbance in the UV-A region and fluorescence properties.

Full text of DOI:10.1016/j.freeradbiomed.2016.04.196

Free Radical Biology and Medicine 96 (2016) 418–431
Contents lists available at ScienceDirect
Free Radical Biology and Medicine
journal homepage: www.elsevier.com/locate/freeradbiomed
Thymidine radical formation via one-electron transfer oxidation
photoinduced by pterin: Mechanism and products characterization
Mariana P. Serrano a, Mariana Vignoni a, Carolina Lorente a, Patricia Vicendo b,
Esther Oliveros b, Andrés H. Thomas a,
n
a Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de
La Plata (UNLP), CCT La Plata-CONICET, Diagonal 113 y 64, 1900 La Plata, Argentina
b Laboratoire des Interactions Moléculaires et Réactivité Chimique et Photochimique (IMRCP), UMR 5623-CNRS/UPS, Université Toulouse III (Paul Sabatier),
118, route de Narbonne, F-31062 Toulouse cédex 9, France
a r t i c l e i n f o
a b s t r a c t
Article history:
UV-A radiation (320–400 nm), recognized as a class I carcinogen, induces damage to the DNA molecule
and its components through different mechanisms. Pterin derivatives are involved in various biological
functions, including enzymatic processes, and it has been demonstrated that oxidized pterins may act as
photosensitizers. In particular, they accumulate in the skin of patients suffering from vitiligo, a chronic
depigmentation disorder. We have investigated the ability of pterin (Ptr), the parent compound of oxi-
dized pterins, to photosensitize the degradation of the pyrimidine nucleotide thymidine 5′-monopho-
sphate (dTMP) in aqueous solutions under UV-A irradiation. Although thymine is less reactive than
purine nucleobases, our results showed that Ptr is able to photoinduce the degradation of dTMP and that
the process is initiated by an electron transfer from the nucleotide to the triplet excited state of Ptr. In the
presence of molecular oxygen, the photochemical process leads to the oxidation of dTMP, whereas Ptr is
not consumed. In the absence of oxygen, both compounds are consumed to yield a product in which the
pterin moiety is covalently linked to the thymine. This compound retains some of the spectroscopic
properties of Ptr, such as absorbance in the UV-A region and fluorescence properties.
Received 10 December 2015
Received in revised form
26 April 2016
Accepted 29 April 2016
Available online 3 May 2016
Keywords:
Photosensitization
Thymine
Pterins
Type I photooxidation
& 2016 Elsevier Inc. All rights reserved.
1. Introduction
therapy (PDT), a medical procedure for the treatment of aged-re-
lated macular degeneration, cancer and other pathologies where-
A photosensitized reaction is defined as a photochemical al-
teration occurring in one molecular entity as a result of the initial
absorption of radiation by another molecular entity called photo-
sensitizer [1]. The biological and medical importance of photo-
sensitized reactions is mostly related to their participation in
processes involved in the generation of skin cancer [2]. Most of the
solar UV energy incident on the Earth surface corresponds to UV-A
radiation (320–400 nm), which is not significantly absorbed by
DNA, but penetrates deeper in human skin than the more en-
ergetic UV-B radiation (280–320 nm). UV-A radiation acts in-
directly through photosensitized reactions and is now recognized
as a class I carcinogen [3]. Moreover, epidemiological evidence has
shown that exposure of humans to artificial UV-A radiation (i.e.
sun lamps and tanning beds) is a major risk factor for melanoma
induction [4–6]. Photosensitization is also important in relation to
several applications in disinfection [7,8] and photodynamic
by undesired tissue (e.g. tumor) can be destroyed in situ [9,10].
The chemical changes in biological components resulting from
photosensitized reactions can take place through different me-
chanisms. Photosensitized oxidations involve the generation of
radicals (type I), e.g., via electron transfer or hydrogen abstraction,
and/or the production of singlet molecular oxygen (O2(1
g), de-
Δ
noted throughout as 1O2) (type II) [11]. In particular, 1O2 is one of
the main reactive oxygen species (ROS) responsible for the da-
maging effects of light on biological systems (photodynamic ef-
fects) and plays a key role in the mechanism of cell death in PDT
[12–14]. This ROS reacts selectively with guanine components at
the exclusion of other nucleobases and the 2-deoxyribose moiety
[15,16]. On the other hand, all the nucleobases may undergo one-
electron oxidation, with corresponding ionization potentials in the
following order: guanineoadenineocytosineꢀthymine [17–19].
It is accepted that whereas ionizing radiation is able to ionize all of
the four main DNA bases with similar efficiency, most type I
photosensitizers produce damage only in guanine base [16]. In
addition, hole transfer has been shown to occur in double-stran-
ded DNA [20–22], principally from pyrimidine and adenine radical
n
Corresponding author.
E-mail address: athomas@inifta.unlp.edu.ar (A.H. Thomas).
http://dx.doi.org/10.1016/j.freeradbiomed.2016.04.196
0891-5849/& 2016 Elsevier Inc. All rights reserved.

M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
419
cations to guanine, which acts as a sink. As a consequence, the
12
10
8
O
4
thymidine 5'-monophosphate
amount of modified pyrimidine bases in a DNA molecule is lower
than the total amount of pyrimidine bases that underwent elec-
tron transfer.
(dTMP)
H3C
5
3NH
It is well documented that direct excitation of pyrimidine bases
by absorption of UV photons gives rise to cyclobutane pyrimidine
dimers with a predominance of thymine dimers [23,24]. But there
is a lack of information about reactions photoinduced by en-
dogenous and exogenous photosensitizers that lead to the ioni-
zation of pyrimidine bases. However, the photooxidation of the
thymine moiety in different substrates via a type I mechanism has
been proven using menadione [25] and benzophenone [26]. In
these studies, two pathways leading to different products are
2
6
1
O
O
OH
N
5'
4'
3'
1'
O
P
O
2'
OH
OH
O
6
5
N
3
4
6
7
HN
4
proposed: the formation of
a 5-(uracilyl) methyl radical
2
(−CH2•) derived from the methyl group of the thymine moiety and
the hydration of the thymine radical cation.
H2N
N
N
1
8
2
Pterins are present in the human epidermis because 5,6,7,8-
tetrahydrobiopterin (H4Bip) is an essential cofactor in the hydro-
xylation of the aromatic amino acids [27] and participates in the
regulation of melanin biosynthesis [28]. Several dihydro and tet-
rahydropterins are involved in the metabolism of H4Bip and,
hence, are also present in the human skin [29]. Vitiligo is a skin
disorder [30] in which the protection against UV radiation fails due
to the lack of melanin, the main pigment of skin. In addition, the
H4Bip metabolism is altered [31] and unconjugated oxidized or
aromatic pterins accumulate in the affected tissues. These com-
pounds are photochemically reactive in aqueous solution and,
upon UV-A excitation, can fluoresce, undergo photooxidation to
produce different photoproducts, generate ROS, such as 1O2, and
photosensitize the oxidation of biomolecules [32]. Therefore, the
photochemistry of pterins and, especially their photosensitizing
properties, are of particular interest for the study of this disease.
In the late 1990s it was reported that oxidized pterins are ef-
ficient 1O2 photosensitizers [33,34], and that UV-A excitation of
pterins induces DNA damage [35]. Later studies provided addi-
tional evidence on the photosensitizing capability of pterins to
degrade DNA, but contradictory mechanisms were proposed
[36,37]. In the context of our investigations on the photosensitiz-
ing properties of pterins in aqueous solutions, we have previously
demonstrated that pterin (Ptr), the parent unsubstituted com-
pound of oxidized pterins (Fig. 1), and the vitiligo-related pterin
derivatives (biopterin, formylpterin and carboxypterin) are effi-
cient photosensitizers inducing the degradation of purine nu-
cleotides (2′-deoxyguanosine 5′-monophosphate (dGMP) [38–40]
and 2′-deoxyadenosine 5′-monophosphate (dAMP) [41,42]). In the
case of the oxidation of dAMP and dGMP photoinduced by the
neutral form of pterins (neutral and acidic media), the pre-
dominant mechanism is type I and involves an initial electron
transfer from the nucleotide to the triplet excited state of pterins.
On the other hand, in alkaline media, where monoanionic forms of
pterins are present, the main mechanism for the photosensitized
oxidation of dGMP involves 1O2 as the reactive intermediate.
In the present work we report our investigations on the Ptr-
photosensitized degradation of thymine in aqueous solutions un-
der UV-A radiation, using the pyrimidine nucleotide thymidine 5′-
monophosphate (dTMP, Fig. 1). This compound is highly soluble in
water and may be quantified by chromatographic methods. The
main objectives of this work were to evaluate the capability of
pterins to photoinduce chemical changes in thymine and elucidate
the mechanisms involved. We have evaluated the role of molecular
oxygen (O2), and analyzed the products formed under different
experimental conditions. The potential biological implications of
the results and the proposed mechanisms are discussed.
pterin (Ptr)
0
200 250 300 350 400 450 500 550
(nm)
Fig. 1. Molecular structures of Ptr and dTMP, and corresponding absorption spectra
in air-equilibrated aqueous solutions at pH 5.5; solid line: dTMP; dashed line: Ptr.
2. Materials and methods
2.1. General
Pterin (Ptr) was purchased from Schircks Laboratories (Jona,
Switzerland) and used without further purification. Thymidine 5′-
monophosphate (dTMP), formic acid, KI (purity 499%), super-
oxide dismutase (SOD) from bovine erythrocytes (lyophilized
powder, Z95% biuret, Z3000 units per mg of protein) and other
chemicals were provided by Sigma-Aldrich and used without
further purification.
All the experiments were carried out in aqueous solutions and
the pH measurements were performed with a pH-meter sen-
sIONþpH31 GLP combined with a pH electrode 5010T (Hach). The
pH of the aqueous solutions was adjusted by adding very small
aliquots (a few
tions using a micropipette.
μ
L) of concentrated (0.1–2 M) HCl or NaOH solu-
2.2. Steady-state irradiation
Aqueous solutions containing Ptr and dTMP were irradiated
in 1 ꢁ 0.4 cm fluorescence quartz cells at room temperature, using
3 Rayonet 3500RPR lamps emitting at 350 nm (bandwidthE
20 nm) (Southern N.E. Ultraviolet Co.). In most of the experiments
the Ptr concentration was in the micromolar range because in
diseased skin cells such concentrations of pterins have been
determined [43]. The concentration of dTMP was about 1 mM,
which is much higher than that found in biological systems,
but is necessary to obtain products in suitable amounts for their
analysis.
The experiments were performed in the presence and absence
of dissolved O2 in the solutions. Experiments with air-equilibrated
solutions were performed in open quartz cells without bubbling
during irradiation to prevent light scattering. However, to avoid
that the consumption of O2 might lead to hypoxic conditions, the
irradiation was interrupted every 30 min and the sample was
bubbled with air for 10 min. Argon and oxygen-saturated solutions
were obtained by bubbling for 20 min with these gases (Linde,

420
M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
purity 499.998%), previously saturated in water. In the case of
oxygen-saturated solutions, the same procedure as that explained
for air was carried out to ensure a high enough concentration of
the gas.
counting equipment FL3 TCSPC-SP (Horiba Jobin Yvon), described
elsewhere [47].
For a given solution, the decay curve was recorded at multiple
emission wavelengths to construct the time-resolved emission
spectra (TRES), 3D data set of counts versus time and versus wa-
velength. The Global Analysis of TRES, a fit calculation (up to
5 exponentials) performed globally on up to 100 separate decay
curves, was carried out using the DAS6 Fluorescence Decay Ana-
lysis software.
2.3. Analysis of irradiated solutions
2.3.1. UV–vis spectrophotometry
Electronic absorption spectra were recorded on a Shimadzu
UV-1800 spectrophotometer, using quartz cells of 0.4 cm optical
path length. The absorption spectra of the solutions were recorded
at regular intervals of irradiation time.
2.6. Singlet oxygen (1O2) detection
The experiments were carried out at room temperature using
D2O as a solvent since the lifetime of 1O2
(
τΔ) is much longer in
2.3.2. High-performance liquid chromatography
D2O than in H2O [48,49]. The sample solution (0.8 mL) in a quartz
cell (1 cm ꢁ 0.4 cm) was irradiated with a pulsed LED source
(SpectraLED, maximum emission at 560 nm, light pulse duration
A high-performance liquid chromatography equipment Pro-
minence from Shimadzu (solvent delivery module LC-20AT, on-
line degasser DGU-20A5, communications bus module CBM-20,
auto sampler SIL-20A HT, column oven CTO-10AS VP, photodiode
array (PDA) detector SPD-M20A and fluorescence (FL) detector RF-
20A) was employed for monitoring the reaction. A Synergi Polar-
RP column (ether-linked phenyl phase with polar endcapping,
200
s). The 1O2 emission at 1270 nm was registered and analyzed
μ
using the equipment described elsewhere [40].
3. Results and discussion
150 ꢁ 4.6 mm, 4
μ
m, Phenomenex) was used for product separa-
tion. A solution of 25 mM formic acid (pH¼3.2) was used as mo-
3.1. Photosensitization of dTMP by pterin in air-equilibrated
solutions
bile phase.
2.3.3. Mass spectrometry analysis
The first aim of this work was to find out if upon UV-A irra-
diation pterin (Ptr) was able to photoinduce damage, not only in
the purine nucleotides as previously published [38–42], but also in
the pyrimidine nucleotides which are less susceptible to oxidation,
partly due to their higher redox potential. Therefore air-equili-
brated aqueous solutions containing Ptr and thymidine 5′-mono-
phosphate (dTMP) were exposed to UV-A radiation (350 nm) for
different periods of time. The pH range (5.5–6.0) was chosen so
that Ptr was present at more than 99% in its acid form (pKa 7.9).
The corresponding absorption spectra of Ptr and dTMP (Fig. 1)
show that only Ptr was excited under these experimental condi-
tions. The samples were analyzed by UV–vis spectrophotometry
and HPLC using the absorbance detector (HPLC-PDA, Section 2).
The concentration of dTMP decreased significantly as a function of
the irradiation time, whereas the decrease of the Ptr concentration
was very slow (Fig. 2, dTMP (●) and Ptr (▼)). Irradiation of Ptr
solutions in the absence of dTMP showed that the consumption of
Ptr was identical, within experimental error, to that registered in
The liquid chromatography equipment coupled to mass spec-
trometry (LC/MS) system consisted of an UPLC chromatograph
(ACQUITY UPLC from Waters) coupled to a quadrupole time-of-
flight mass spectrometer (Xevo G2-QTof-MS from Waters) (UPLC-
QTof-MS). UPLC analyses were performed using an Acquity UPLC
BEH C18 (1.7
μm; 2.1 ꢁ 50 mm) column (Waters), and isocratic
elution with 25 mM formic acid (pH¼3.2) at a flow rate of
0.2 mL minꢂ1. The mass spectrometer was operated in the nega-
tive ion mode. Therefore the samples were injected into the
chromatograph, the components were separated and then the
mass spectra were registered for each peak of the corresponding
chromatograms. In addition, mass chromatograms, i.e. re-
presentations of mass spectrometry data as chromatograms (the
x-axis representing time and the y-axis signal intensity), were
registered using different scan ranges.
2.4. Electron paramagnetic resonance-spin trapping experiments
the presence of the nucleotide (Fig. 2, Ptr ( )). Consequently, the
Δ
Electron paramagnetic resonance (EPR) experiments were
performed in order to detect the dTMP radical cation. EPR spectra
were collected on a Bruker ESP 500E spectrometer. Samples were
irradiated with Rayonet RPR3500 lamps. The following instru-
mental settings were employed for the measurements: microwave
power, 20 mW; field modulation amplitude, 0.1 mT; field mod-
ulation frequency,100 kHz; microwave frequency, 9.77 GHz.
Nitrones are common reagents for the detection and identifi-
cation of transient radicals due to their ability to form persistent
radical adducts that are detectable and fingerprintable by EPR
spectroscopy [44,45]. In our experiments, 5,5-dimethyl-1-pyrro-
line-N-oxide (DMPO) from Sigma was used as the spin trap [46].
consumption of the photosensitizer was due to its own photolysis.
Additional control experiments showed that no consumption of
the nucleotide was detected in dTMP solutions irradiated in the
absence of Ptr (Fig. 2, dTMP (○)), thus excluding the possibility of
product formation by spurious direct excitation of dTMP. More-
over, dTMP degradation was not observed in solutions containing
Ptr and dTMP that were kept in the dark. The products formed in
solutions containing Ptr and dTMP under irradiation were ana-
lyzed by HPLC and mass spectrometry (vide infra).
3.2. Photosensitization of dTMP by pterin in O2-free solutions
Samples (1 mL) contained 180 M Ptr, 1.3 mM dTMP, buffer Tris/
μ
In the experiments carried out in the absence of O2, under
otherwise the same experimental conditions, the dTMP concentration
also decreased as a function of irradiation time (Fig. 3). However, the
rate of consumption of dTMP was slower than that observed under
aerobic conditions (Fig. 2). In addition, a significant consumption of
Ptr was also observed (Fig. 3) and such consumption cannot be at-
tributed to the photochemistry of Ptr itself since, as previously re-
ported, this compound is photostable in O2-free solutions [32].
Moreover, a control experiment carried out in the absence of dTMP
HCl (pH 7.0), and DMPO (50 mM). The O2-free solutions were ir-
radiated at room temperature in sealable quartz cells of 0.4 cm
optical path length. EPR spectra were recorded every minute since
the beginning of the irradiation up to 15 min.
2.5. Fluorescence measurements
Steady-state and time-resolved fluorescence measurements
were performed at room temperature using a single-photon-

M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
421
(ꢂ0.670.1
μ
M minꢂ1), a result that suggests a photochemical reac-
tion with a 1:1 stoichiometry.
The results presented in this section were surprising since the
degradation of purine nucleotides photosensitized by Ptr and
other pterin derivatives does not take place under anaerobic
conditions [38–42]. The radical ions formed by electron transfer
from the purine nucleotide (dGMP or dAMP) to the triplet excited
state of Ptr (3Ptr ) (Reaction (1)) undergo an efficient back electron
n
transfer (Reaction (2)):
3
P tr + dGMP/dAMP → Ptr•− + dGMP•+ /dAMP•+
*
(1)
(2)
Ptr•− + dGMP•+ /dAMP•+ → Ptr + dGMP/dAMP
The fact that the consumption of dTMP was faster in aerobic
than in anaerobic media suggested that back electron transfer took
place in the absence of O2, but additional reactions consuming
dTMP also occurred.
Moreover, other studies demonstrated that molecular oxygen is
necessary for the degradation of free amino acids photosensitized
by Ptr [50,51]. This is therefore the first time that a photosensitized
process consuming Ptr is described. Hence, it is clear that photo-
sensitization of dTMP might involve a mechanism different from
those previously reported that deserves a more detailed analysis.
Fig. 2. Time-evolution of the dTMP (●) and Ptr (▼) concentrations under UV-A ir-
radiation of air-equilibrated aqueous solutions (pH 5.5). Control experiments: (○)
evolution of the dTMP concentration in the absence of Ptr; (Δ) evolution of the Ptr
concentration in the absence of dTMP. Concentrations determined by HPLC analysis
([Ptr]0¼140 μM), ([dTMP]0¼1170 μM).
3.3. Participation of the triplet excited state of pterin
The triplet excited states of pterins are reactive species that
initiate all the photosensitized processes reported in the literature
so far (Section 1). Therefore it is straightforward to assume that
this is also the case for the photosensitization of dTMP. A series of
experiments was conducted to confirm this hypothesis.
It has been previously demonstrated that iodide (Iꢂ) at mi-
cromolar concentrations is an efficient and selective quencher of
triplet excited states of pterins [47,52]. The results of photo-
sensitization experiments carried out in air-equilibrated and
O2-free solutions containing dTMP and Ptr in the presence of KI
(300 M) confirmed that, in both cases, the rate of dTMP con-
μ
sumption was slower in the presence of Iꢂ than in its absence
(Fig. 4). In addition, under anaerobic conditions, where a decrease
of the Ptr concentration was observed upon irradiation (Fig. 3), Iꢂ
led to an inhibition of the consumption of the photosensitizer as
well (Fig. 4b). Thus, the inhibition of the photosensitized de-
gradation of dTMP by Iꢂ strongly suggests that the process takes
3
n
place via a purely dynamic mechanism initiated by Ptr .
3.4. Mechanistic pathways: energy transfer and singlet oxygen
sensitization
Three types of mechanisms might be responsible for the ob-
served photosensitized process: triplet-triplet energy transfer
(TTET) from the photosensitizer (Ptr) to the nucleotide, and type I
and/or type II photosensitization (Section 1). The three types of
mechanisms are, in general, initiated by the triplet excited state of
the photosensitizer.
A triplet-triplet energy transfer between the donor (3Ptr ) and
n
the acceptor (dTMP) may be discarded as the energies of the tri-
plet excited states of Ptr (ETPtr) and dTMP (ETdTMP) have been es-
timated to be 243 kJ molꢂ1 and 310 kJ molꢂ1, respectively [53–55].
Since ETPtr is much lower than ETdTMP, such an endothermic energy
transfer would be highly inefficient and the TTET process can be
ruled out [56].
Fig. 3. Time-evolution of the dTMP (●) and Ptr (▼) concentrations under UV-A ir-
radiation of O2-free aqueous solutions (pH 5.5). Control experiments: (○) evolution
of the dTMP concentration in the absence of Ptr; (Δ) evolution of the Ptr con-
centration in the absence of dTMP. Concentrations were determined by HPLC
analysis ([Ptr]0¼140 μM), [dTMP]0¼1260 μM).
A type II mechanism might not be relevant since it is accepted
that thymine does not react significantly with 1O2 [15,16]. Never-
theless, to the best of our knowledge, the rate constant of the
chemical reaction (kr) between 1O2 and dTMP (Reaction (3)) and
showed that the decrease of the Ptr concentration was negligible in
the time window used (Fig. 3). Interestingly, the rate of dTMP con-
sumption (ꢂ0.770.1
μ
M minꢂ1) for the experiment shown in Fig. 3
was equal to the rate of Ptr consumption within experimental error

422
M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
analysis of the quenching of the characteristic 1O2 emission in the
NIR spectral region by dTMP (see ESI for details, Fig. S1). A kt value
of 7 (71) ꢁ 104 Mꢂ1 sꢂ1 was obtained. This value is about 250
times
lower
than
that
reported
for
dGMP
(1.7
(70.1) ꢁ 107 Mꢂ1 sꢂ1) [38].
The method for the assessment of the role of 1O2 in the pho-
tosensitized oxidation of nucleotides has been described in detail
elsewhere [38]. Briefly, for a given experiment, the initial rate of
oxidation of dTMP by 1O2 (d[dTMP]/dt)i, can be calculated using
Δ
(Eq. (5)).
⎛
⎝
⎞
⎡
⎤
⎦
d dTMP
⎣
= − kr 1O2
dTMP
⎡
⎤
⎦
⎡
⎣
⎤
⎦
⎜
⎜
⎟
⎟
⎣
i
SS
dt
⎠
(5)
i,Δ
where [1O2]ss is the steady-state concentration of 1O2 that depends
on the photon flux absorbed by the sensitizer, on its ΦΔ (0.18 in
the case of Ptr [32]) and on the value of τΔ under the experimental
conditions used.
The initial rate (d[dTMP]/dt)i, was calculated considering that
Δ
kr¼kt (maximum possible value of kr, if physical quenching is
negligible). As expected, results obtained for different initial dTMP
and Ptr concentrations showed that, in all cases, the calculated
rate (d[dTMP]/dt)i, was negligible in comparison to the experi-
Δ
mental initial rate of dTMP consumption determined by HPLC
analysis, thus indicating that a contribution of 1O2 can be dis-
carded, which is in total agreement with previous works that
demonstrated that thymine does not react with 1O2 [15,16].
3.5. Electron transfer mechanism: formation of superoxide anion and
organic radicals
A predominant mechanism via electron transfer (Type I) would
be in agreement with previous evidence on the capability of Ptr to
photosensitize the oxidation of other nucleotides and amino acids
through this type of mechanism [59]. In this case, the photo-
sensitized oxidation would be initiated by an electron transfer
from dTMP to the triplet excited state of Ptr (3Ptr ) to form the Ptr
n
) )
radical anion (Ptr•− and the dTMP radical cation (dTMP•+
(Reac-
tion (6)). The latter may deprotonate to the corresponding neutral
radical (dTMP(−H)•) (Reaction (7)).
3
P tr + dTMP → Ptr•− + dTMP•+
(6)
*
•
dTMP•+ ⇄ dTMP −H +H+
(
)
(7)
The lifetime of 3Ptr
(τ
T) is 3.9
μs and 1.4
μ
s in the absence of
n
Fig. 4. a) Time-evolution of the dTMP concentration under UV-A irradiation of Ptr:
i) in air-equilibrated solutions in the absence (●) and in the presence of KI 300 μM
(▼), and ii) in O2-saturated solutions (■); [dTMP]0¼1445 μM, [Ptr]0¼150 μM. b)
Time-evolution of the dTMP and Ptr (inset) concentrations under UV-A irradiation
of Ar-saturated solutions in the absence (●) and in the presence of KI 300 μM (▼);
[dTMP]0¼1250 μM, [Ptr]0¼155 μM. Concentrations were determined by HPLC
analysis, pH¼5.5.
O2 and in air-equilibrated solutions, respectively [40]. The rate
constant of quenching of 3Ptr by dGMP is close to the diffusion
n
controlled limit and it can be assumed that the corresponding rate
constant of quenching by dTMP should be of the same order of
magnitude. Therefore the rate of quenching of 3Ptr by dTMP
n
should be significant in our reaction system and Reaction (6)
should take place if the electron transfer process were
spontaneous.
the rate constant of 1O2 physical quenching (kq) by dTMP (Reaction
The feasibility of this electron transfer process can be evaluated
(4)) are not known.
by estimating the free energy change (
Eq. (8) [60]:
Δ
G) of the reaction using
kr
1
(3)
(4)
dTMP + O2 → dTMPO2
⎡
⎤
ΔG eV = E(dTMP+•/dTMP) − E(Ptr/Ptr
−
e2/4ΠϵR
− ΔE0,0
−•
−
(
)
(
)
)
D+A
o
⎣
⎦
(8)
k
q
dTMP + 1 O2 → dTMP + 3 O2
−•
where E(dTMP+•/dTMP) and E(Ptr/Ptr
are the standard electron
)
potentials of electron donor and acceptor, respectively.
Therefore time-resolved experiments in D2O were performed
using rose bengal (RB) as a standard 1O2 photosensitizer (quantum
yield of 1O2 production in aqueous solutions: ΦΔ¼0.75 [57,58]).
The rate constant of 1O2 total quenching by dTMP (kt¼krþkq,
Reactions (3) and (4)) was determined from the Stern-Volmer
These values have already been reported for dTMP
−•
(
E(dTMP+•/dTMP) = 1.21V vs. NHE [61]) and Ptr (E(Ptr/Ptr = −0.55V vs.
)
NHE [53]).
Δ
E0,0 is the energy of the triplet excited state of Ptr
(ET ¼2.52 eV [53]); the term eo2/4Π
εRD þA ꢂ is the solvation
Ptr
energy of an ion pair DþA‾ and can be ignored in the case of

M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
423
strong polar solvents. The calculated
Δ
G
value was
10
5
ꢂ100.4 J molꢂ1, thus indicating that electron transfer from
a) Ptr+DMPO
dTMP to 3Ptr can spontaneously occur.
n
tirr= 0 min
•
To investigate the formation of dTMP(–H) (pKao4) [62] in the
irradiated solutions, a series of EPR experiments was performed in
the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide
(DMPO), which reacts with a variety of organic radicals and re-
active oxygen species to form stable radical adducts [63], with
characteristic EPR spectra.
0
-5
The experiments were performed under Ar (bubbled for 20 min
before irradiation) to avoid the interference of the superoxide
Ptr+DMPO
b)
tirr= 5 min
•−
5
0
anion (O2
)
in the investigation of the dTMP radical. Indeed, it has
been reported that an aqueous solution of Ptr exposed to UV-A
•−
produces O2 [64]. The process involves an electron transfer be-
tween Ptr in its ground state and 3Ptr leading to the corre-
n
sponding pair of radical ions (Ptr•+ and Ptr•−) (Reaction (9)), fol-
-5
Ptr•−
lowed by electron transfer from
to O2 and superoxide for-
•−
mation (Reaction (10)). DMPO reacts very efficiently with O2 to
form the adduct DMPO• − OOH (Reaction (11)), which decays to
the more stable adduct DMPO• − OH with a characteristic EPR
spectrum [65].
-10
3460
3480
3500
3520
Field (G)
Ptr + Ptr → Ptr•+ + Ptr•–
10
5
*
(9)
(10)
(11)
c) Ptr+dTMP+DMPO
tirr= 0 min
•−
Ptr•– + O2 → Ptr+O2
DMPO+O2•− +H+ → DMPO• − OOH
0
Before irradiation, no EPR signal was detected in Ar-bubbled
solutions (pH 7.2) containing Ptr (180 M) and DMPO (50 mM) in
-5
μ
the absence of nucleotide (Fig. 5a). However, irradiation led to the
immediate formation of an EPR signal (Fig. 5b), characterized by
hyperfine coupling constants AN¼AH¼ 14.9 G and corresponding
to the adduct DMPO• − OH [65]. This result shows that, despite Ar
d) Ptr+dTMP+DMPO
t3=46105 min
3480
3500
3520
irr
10
5
*
*
•−
*
X Data
*
bubbling, some O2 was formed due to the remaining O2.
*
*
A more complex EPR signal consisting of two superimposed EPR
spectra was registered after irradiation of solutions bubbled with Ar
0
and containing Ptr (180 M), dTMP (1.3 mM) and DMPO (50 mM)
μ
(Fig. 5c and d). One of the EPR spectra corresponds to the adduct
DMPO• − OH, which was expected considering the results of the
control carried out in the absence of the nucleotide (Fig. 5b). The
other spectrum was composed of six lines (Fig. 5d) having hyperfine
coupling constants of aN¼16.3 G, aH¼23.7 G (g¼2.0060), which is
typical of a DMPO adduct obtained from a carbon-centered radical.
Moreover, the recorded spectrum is identical, within experimental
error, to that reported for an adduct DMPO• − thymidine [66].
Therefore, we have demonstrated that an electron transfer
-5
-10
-15
DMPO -OH
DMPO -dTMP
*
3460
3480
3500
3520
Field (G)
from the nucleotide to 3Ptr is thermodynamically feasible and
n
Fig. 5. EPR experiments performed in Ar-bubbled solutions (buffer Tris/HCl,
pH¼7.2), using DMPO (50 mM) as a spin trap. Signals registered in solutions of Ptr
(180 μM) before (a) and after 5 min of UV-A irradiation (b). Signals registered in
solutions of Ptr (180 μM) and dTMP (1.3 mM) before (c) and after 15 min of UV-A
irradiation (d).
that, in fact, dTMP radicals are formed when a solution containing
both Ptr and the nucleotide is irradiated. Hence, it can be con-
cluded that the process observed takes place via a type I me-
chanism and is initiated by Reaction (6).
Comparative photolysis experiments carried out in air-equili-
brated and O2-saturated solutions clearly showed that the rate of
dTMP disappearance was much greater in the former than in the
latter solutions (Fig. 4a), which apparently contradicts the fact that
the photosensitized reaction is faster in air-equilibrated than in Ar-
saturated solutions (vide supra). It has been demonstrated that, in
3
n
O2 concentration quenches Ptr decreasing the efficiency of
electron transfer.
•−
As mentioned above, O2 is formed in our reaction system in
the presence of O2 (Reaction (10)). In the case of dGMP, it has been
reported that O2 reacts with oxidizing guanine radical very fast
according to two main competitive mechanisms: chemical repair
with the restoration of the guanine through electron transfer
(Reaction (12)) [67,68] and addition, leading to the predominant
formation of 2,5-diamino-4H-imidazolone (Iz) (Iz 5′-monopho-
sphate (IzMP) for guanine in nucleotides) (Reaction (13)) [69,70].
•−
contrast to the singlet excited state of Ptr (1Ptr ), its triplet excited
n
3
n
state ( Ptr ) is efficiently quenched by O2 [32,40]. Therefore the
decrease in the rate of nucleotide consumption in O2-saturated
solutions may be interpreted as the result of the competition be-
tween the reaction of 3Ptr with dTMP and its quenching by O2.
n
The overall behavior can be interpreted assuming that there is an
optimal concentration of O2 for the rate of the dTMP consumption:
lack of O2 favors back electron transfer to recover dTMP and high
•
dGMP −H +O •− /HO2 → dGMP +O2
•
(
)
2
(12)

424
M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
dGMP −H •+O•− /HO2 → IzMP
(
)
(13)
(14)
2
dGMP•+ + HO− → dGMP(ox)
Due to the former process (Reaction (12)) and secondary oxi-
dation pathways (e.g. Reaction (14)), the oxidation of guanine
photosensitized by Ptr is faster in the presence of superoxide
dismutase (SOD), an enzyme that catalyzes the conversion of
•−
O2 into H2O2 and O2 [71], than in its absence. To investigate the
•−
participation of O2 in the mechanism of photosensitization of
dTMP, we performed experiments in the presence of SOD. The data
showed that the rate of dTMP consumption was not affected when
SOD was present in the solution (ESI, Fig. S2). These results in-
•−
dicate that
O2
)
does not react significantly with
•
dTMP•+/dTMP −H and, hence, is not involved in the photo-
(
sensitized process.
3.6. Analysis of the photoproducts formed in the presence of O2
Solutions containing Ptr and dTMP, before and after irradiation,
were analyzed by HPLC-PDA and HPLC coupled to the fluorescence
detector (HPLC-FL) (Section 2.3.2). Several photoproducts were de-
tected by the former technique with retention times (tr) lower and
higher than that corresponding to the intact nucleotide. None of the
products absorbed above 320 nm. Analysis with the fluorescence de-
tector showed that the only fluorescent compound in the treated so-
lutions, when excited at 340 nm, was the photosensitizer, indicating
that no fluorescent products were formed. This point is relevant in the
context of the results that will be presented in the next section.
To characterize the photoproducts a qualitative analysis was
carried out by means of UPLC coupled to mass spectrometry
(UPLC-QTof-MS, see Section 2.3.3). The solutions containing dTMP
and Ptr at pH 5.5 were analyzed in both positive and negative ion
modes (ESIþ and ESIꢂ, respectively). As expected, the signals
corresponding to the intact molecular ion of Ptr as [MþH]þ and
[MꢂH]ꢂ species at m/z 164.1 Da and 162.1 Da, respectively, were
observed. However, in the case of dTMP, the resolution was much
better in ESIꢂ than in ESIþ mode. Therefore all the results pre-
sented in this section correspond to mass spectrometry analysis
carried out in the former mode. In this way, the signal corre-
sponding to the intact molecular ion of dTMP as [MꢂH]ꢂ species
at m/z 321.05 Da was registered for the analysis of the dTMP peak.
The MS/MS spectrum in the ESIꢂ mode of dTMP was recorded
(Fig. 6) and the observed fragmentation was in agreement with
previous results obtained using soft ionization MS methods [72].
Briefly, the loss of the thymine (Thy) base is a prominent reaction
and occurs via a 1,2-elimination, yielding the base as a deproto-
nated anion ([ThyꢂH]ꢂ, m/z¼125.03 Da) and a fragment corre-
sponding to the 2′-deoxyribose 5′-phosphate ([MꢂThyꢂ2H]ꢂ, m/
z¼195.01 Da) (Fig. 6). Following the loss of base, the 3′-C–OH and
4′-C–H bonds are cleaved to yield the [MꢂThy–2HꢂH2O]ꢂ ion at
m/z 177.00 Da. Finally, the typical species [PO3]ꢂ and [PH2O4]ꢂ at
m/z 78.96 and 96.97 Da were also detected.
Fig. 6. MS/MS spectrum of dTMP recorded in ESIꢂ mode and fragmentation of
dTMP via a 1,2-elimination reaction obtained using soft ionization MS methods.
Arrows indicate the peaks corresponding to each fragment.
weight of 338 Da. To get more information on the structure of
these products, their MS/MS spectra were registered and com-
pared to that corresponding to dTMP.
The MS/MS spectra of the two products P338 were similar, al-
though not identical and a signal corresponding to Thy containing
an additional oxygen atom in its structure was observed at m/z
141.03 Da ([ThyþOꢂH]ꢂ) (Fig. 7). A peak at m/z 294.04 Da, re-
sulting from the loss of a fragment CO-NH from the base, was
registered. The typical fragments corresponding to the 2′-deoxyr-
ibose 5′-phosphate moiety, [MꢂThyꢂ2H]ꢂ at m/z 195.01 Da and
[MꢂThy–2HꢂH2O]ꢂ at m/z 177.0 Da, and to [PO3]ꢂ at m/z 79.0
and [PH2O4]ꢂ at m/z 97.0 Da, were also detected. Therefore, the
oxidation took place on the Thy moiety and resulted in the in-
corporation of one oxygen atom. To the best of our knowledge, this
is the first time that this type of products is described for the
photosensitization of dTMP.
However, the oxidation of thymidine (Thd) photosensitized by
benzophenone has been studied and 5-(hydroxymethyl)-2′-ur-
idine (5-HmdUrd) has been reported as an oxidation product
[26,56,73]. Similarly, in our case, one of the products with MW
338 Da might be the equivalent of 5-HmdUrd; i.e. 5-(hydro-
xymethyl)-2′-deoxyuridine 5′-monophosphate (5-HmdUMP), re-
sulting from the oxidation of the Thy methyl group to hydro-
xymethyl. It has been reported that loss of H2O from the hydro-
xymethyl group is a typical fragmentation of 5-HmdUrd [74]. The
MS/MS spectrum of the product with tr 2.8 min showed two peaks
that were not registered for the product with tr 2.2 min (Fig. 7).
One of them is a small peak at m/z 319 Da ([MꢂHꢂH2O]ꢂ) and
another one at m/z 276 Da ([MꢂH–CONH–H2O]ꢂ), that is, result-
ing from the loss of H2O from the fragment at 294 Da. These re-
sults strongly suggest that the product P338 with tr 2.8 min is
5-HmdUMP.
Irradiated solutions were analyzed in the same way and the
mass spectra corresponding to the chromatographic peaks of the
photoproducts were registered. Although many products could be
detected, three main compounds were registered, all of them
having molecular weights higher than that corresponding to the
intact nucleotide and could be attributed to the following species:
[MþOꢂH]ꢂ at m/z¼337.04 Da, [MþOꢂ3H]ꢂ at m/z¼335.03 Da
and [Mþ2OþH]ꢂ at m/z¼355.06 Da, with M¼dTMP. Therefore
these products will be named as P338, P336 and P356, respec-
tively. It is worth mentioning that two well defined chromato-
graphic peaks presented m/z¼337.0450 Da (tr¼2.2 and 2.8 min),
revealing that two isomeric products are formed with a molecular

M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
425
Fig. 8. MS/MS spectra recorded in ESIꢂ mode of the product P336 formed by Ptr
photosensitization of dTMP in the presence of O2. The chemical structure of the
photoproduct proposed, 5-formyl-2′-deoxyuridine 5′-monophosphate (5-For-
dUMP), is depicted (M¼dTMP).
Fig. 7. MS/MS spectra recorded in ESIꢂ mode of the products P338 formed by Ptr
photosensitization of dTMP in the presence of O2. a) product with tr¼2.8 min; b)
product with tr¼2.2 min. The chemical structure proposed for the former,
5-HmdUMP (5-(hydroxymethyl)-2′-deoxyuridine 5′-monophosphate), is depicted
(M¼dTMP).
[MꢂThyꢂ2H]ꢂ at m/z 195.01 Da and [MꢂThy–2HꢂH2O]ꢂ at m/z
177.0 Da, and to phosphate, [PO3]ꢂ at m/z 79.0 and and [PH2O4]ꢂ at
m/z 97.0 Da, were detected. These results show that the oxidation
takes place again in the Thy moiety. Taking into account that the
increase in the m/z value of the fragment corresponding to the base
was 14 Da and the previous characterization of the products of the
photosensitized oxidation of Thd [26,56], we propose that P336 is
5-formyl-2′-deoxyuridine 5′-monophosphate (5-FordUMP) (Fig. 8).
Two products were found with molecular ions at m/z
355.06 Da (products P356), which suggests that two oxygen atoms
were incorporated in the structure of dTMP. Products of photo-
sensitized reactions containing two oxygen atoms in the Thy
moiety have been reported [26,56]. The MS/MS spectra were
identical for the two products P356 (Fig. 9). The comparison of
these spectra with that corresponding to oxidation products of the
Thy moiety reported for the treatment of oligodeoxynucleotides
with osmium tetraoxide [76] suggests that these products are
diastereoisomers of 5,6-dihydroxy-5,6-dihydrothymidine 5′-
monophophate (thymidine glycol 5′-monophophate, dTMPGly).
Beside the phosphate fragments ([PO3]ꢂ and [PH2O4]ꢂ), four other
peaks corresponding to the reported fragmentation of thymidine
glycol (ThdGly) were observed at m/z values of 142.01 Da
Position 6 of the base moiety is also available for oxidation, and
6-hydroxythymidine 5′-monophophate (6-OHdTMP), or its keto
tautomer (6-oxo-dTMP) containing the 6-oxo-thymine moiety (5-
methylbarbituric acid), could be formed in the process. However,
we can discard this product P338 with tr 2.2 min is 6-oxo-dTMP
because it has been reported that its fragmentation is quite dif-
ferent from that registered in our experiments [74]. The generation
of 5-(hydroperoxymethyl)-2′-deoxyuridine 5′-monophosphate (5-
HPmdUMP) might alternatively be considered since 5-(hydro-
peroxymethyl)-2′-deoxyuridine was reported to be formed in the
photosensitization of Thd by 2-methyl-1,4-naphthoquinone, a type
I photosensitizer, and partly decompose into 5-HmdUrd [75]. In
our case, weak signals corresponding to a species [Mþ2OꢂH]ꢂ
(m/z ¼353.04 Da) were registered in the MS spectra at tr values
coinciding with the second product P338. These results suggest
that the hydroperoxide 5-HPmdUMP is very likely formed and the
species assigned to a product P338 with tr 2.2 min could have been
produced by decomposition of 5-HPmdUMP. Since the amount of
hydroperoxide present in the solution was quite low, no further
significant changes could be detected in the composition of the
irradiated solution kept in the dark.
([Thyþ2OH-NH3-H]),
194.02 Da
([M-ThyþNH2-H-H2O]),
212.03 Da ([M-ThyþNH2-H]) and 238.01 Da ([M-ThyþNCO-H])
(Fig. 9).
Finally, two other products were detected in the MS spectra,
with signals at m/z¼371.05 Da and 325.04 Da, both of them having
weak signals so that no suitable MS/MS spectra could be recorded.
The MS/MS spectrum of P336 did not show the typical fragment
corresponding to the base at m/z¼125.03 Da, and a signal corre-
sponding to Thy containing an oxygen atom in its structure was
observed at m/z 139.03 Da ([ThyþOꢂ3H]ꢂ) (Fig. 8). The fragments
corresponding to the 2′-deoxyribose 5′-phosphate moiety,
The former can be attributed to
a
species [Mþ3Oꢂ3H]ꢂ
(M¼dTMP), which may correspond to 5-hydroperoxy-6-hydroxy-
5,6-dihydrothymidine 5′-monophosphate (5-HP-6-OHdTMP).
The equivalent hydroperoxide derived from Thd has been also

426
M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
3.7. Analysis of the photoproducts formed in the absence of O2
HPLC-PDA analysis of irradiated O2-free solutions showed that
a main product was formed, with a retention time (tR) value higher
than those corresponding to both dTMP and Ptr. Its absorption
spectrum presented a band similar to the typical low-energy band
of pterins (Fig. 10a). HPLC-FL analysis revealed that the main
product found using HPLC-PDA was fluorescent and emitted at
450 nm when excited at 340 nm (Fig. 10b), which is compatible
with the fluorescence properties of pterins. Considering the de-
crease in the Ptr concentration, these results suggested that the
main product detected contains the Ptr moiety. The other chro-
matographic peaks observed could correspond to minor products
formed under anaerobic conditions or products of the aerobic
photosensitization at very low concentrations due to traces of
remaining O2. It is worth mentioning that non-fluorescent pro-
ducts absorbing below 300 nm, which is expected for dTMP de-
gradation products, were not detected.
The absorption and emission properties of the irradiated so-
lutions were analyzed and compared to those of solutions kept in
the dark. The absorption spectrum of a solution containing dTMP
(1255
after 2 h of irradiation, although the Ptr concentration, determined
by HPLC, decreased significantly (to 105.4 M). This fact is in
μ
M) and Ptr (155
μM) at pH¼5.5 almost did not change
μ
agreement with the HPLC-PDA analysis and confirms that the re-
action leads to a product with spectral properties similar to those
of Ptr itself.
In another set of experiments, excitation-emission matrices of
irradiated and non-irradiated solutions were recorded after a 1:10
dilution (ESI, Fig. S3) and it was observed that irradiation led to a
decrease in the overall emission of the solution. Spectra obtained
by excitation into the low-energy pterin band showed that the
emission intensity decreased, but the maximum remained un-
changed (Fig. 11). This suggests that the photoproduct containing
the pterin moiety presents an emission spectrum similar to Ptr,
but with a lower fluorescence quantum yield.
Fig. 9. MS/MS spectra recorded in ESIꢂ mode of the product P356 formed by Ptr
photosensitization of dTMP in the presence of O2. The chemical structure of the
photoproduct proposed is depicted (M¼dTMP).
identified as a photoproduct of photosensitization of the nucleo-
side by 2-methyl-1,4-naphthoquinone [75], which partially de-
composes to 5-hydroxy-5-methylhydantoin and ThdGly. The
Time-resolved experiments were carried out on irradiated and
non-irradiated O2-free solutions ([Ptr]0¼ 15.5
μM, [dTMP]0¼
equivalent
1-(2-deoxy-beta-D-erythro-pentofuranosyl-5-phos-
125.5 M, pH 5.5). The study was performed by excitation at
μ
phate)-5-hydroxy-5-methylhydantoin (5-OH-5MHMP) has a mo-
lecular weight of 326.04 Da and therefore the photoproduct with a
signal at m/z 325.04 Da may be assigned to this compound.
The formation of the photoproducts found can be explained
taking into account reactions of dTMP•+ previously reported
[75,77,78]. The two main competitive reactions of dTMP•+ are
deprotonation (Reaction (7)) and hydration (Reaction (15))
(Scheme 1).
341 nm and the corresponding fluorescence decays were recorded
at 450 nm. First-order kinetics were observed for the decays be-
fore irradiation, with fluorescence lifetime ( F) of 8.470.4 ns,
τ
which is in agreement with the reported τF of Ptr in aqueous so-
lution at pH 5.5 [32]. In contrast, the emission decays of solutions
irradiated for 2 h were clearly bi-exponential with a short-lived
component, with a τF about 2 ns, and a long-lived component,
with a τF value equal to that of Ptr, within experimental error
(Fig. 12). To separate the emission of the two components, the
remaining Ptr and the fluorescent photoproduct(s), time resolved
emission spectra (TRES) were registered, by exciting at 341 nm and
recording the corresponding decays in the wavelength range 360–
560 nm. The global analysis of TRES also revealed the presence of
two fluorescent components. The corresponding spectra were
obtained from the corrected plots of the pre-exponential factors as
a function of the wavelength (Fig. 12). The spectrum and the τF
value of the long-lived component matched very well those pre-
viously published for Ptr [32], and, therefore, it was assigned to the
remaining Ptr. A τF value of 2.0 ns was determined for the short-
lived component and its spectrum was similar to that of Ptr. This
component was assigned to the fluorescent product.
dTMP•+ + HO− → dTMP(OH)•
(15)
The former pathway leads mainly to the formation of the (5-
uracilyl)-methyl radical, which traps oxygen on the methylene
group, the resulting peroxyl radical yielding 5-HmdUMP and
5-FordUMP (Scheme 1), through tetroxide formation and decom-
position [79,80]. Alternatively, reduction of the peroxyl radical
leads to the corresponding hydroperoxide, 5-HPmdUMP, which
can decompose into HmdUMP and 5-FordUMP (Scheme 1).
Hydration, in turn, leads predominantly to the 6-hydroxy-5,6-
dihydrothym-5-yl radical that traps oxygen on position 5 [75,81]
to yield the corresponding peroxyl radical, which by reduction
gives rise to a group of diastereomers of the hydroperoxide 5-HP-
6-HOdTMP (Scheme 1). These hydroperoxides decomposes to
5-HO-5MHMP and dTMPGly (Scheme 1) [75]. It has also been re-
ported that the peroxyl radical yields 5-OH-5MHMP and dTMPGly,
without 5-HP-6-OHdTMP as an intermediate (Scheme 1) [25,82].
Analysis by means of UPLC-QTof-MS was carried out and a
signal at m/z 482,08 Da was registered for the chromatographic
peak of the main photoproduct. The m/z value found corresponds
to the molecular ion of a compound bearing both the photo-
sensitizer and the substrate moieties ([PtrþdTMP–3H]–). In the
MS/MS spectrum of the product (Fig. 13), the typical fragments

M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
427
.
+
hh
O
O
H3C
OH
P
= R
NH
O
O
OH
HO
O
N
R
a
.
+
dTMP
H+
HO
hydration
deprotonation
O
O
.
H3C
H2C
.
NH
NH
O
O
N
HO
N
R
.
dTMP(-H)
R
O2
O2
.
O
O
O
O
H3C
H2C
NH
NH
.
O
O
O
O
HO
N
R
N
R
reduction
reduction
O
O
O
O
O
O
O
CH2
H2C
HOOH2C
O
NH
HN
NH
H3C
NH
O
O
N
R
O
N
R
N
R
HOO
HO
O
N
R
O2
5-HPmdUMP
5-HP-6-OHdTMP
OH
H2C
O
HC
O
O
O
O
H3C
NH
NH
O
NH
NH
HO
HO
H3C
HO
O
O
O
N
R
N
R
N
R
N
R
5-FordUMP
5-OH-5-MHMP
dTMPGly
5-HmdUMP
Scheme 1. Pathways proposed for the formation of the photoproducts found upon irradiation of aqueous solutions of dTMP in the presence of Ptr and O2.
corresponding to the 2′-deoxyribose 5′phosphate moiety at m/z
195.01 Da and 177.0 Da were observed, thus indicating that the
chemical changes did not take place in this part of the molecule.
The intensity of the typical fragment of the nucleobase at m/
z¼125.03 Da was almost negligible and no ion that could be as-
signed to the Ptr moiety was observed. However, an intense signal
at m/z 286.07 was present, which can be attributed to a fragment
bearing both the Ptr and the Thy moieties ([PtrþThyꢂ3H]ꢂ).
These results strongly suggest that after the electron transfer
step (Reaction (6)), the radicals ions (or neutral radicals formed by
protonation/deprotonation) combine to yield an adduct where the
pterinic moiety is attached to the nucleobase (Reaction (16)).
the product and that the low fluorescence quantum yield is due to
the attachment to the nucleotide that acts as internal quencher.
Therefore positions 6 or 7 of the Ptr moiety (Fig. 1) are the most
suitable for the link to dTMP. Taking into account that the analyzed
product has
a mass corresponding to Ptr-dTMP(-2H), that
dTMP(−H)• may be stabilized on the methyl group of the base
(methylene radical) and that no conjugation between the two ring
systems was observed, we propose the chemical structure de-
picted in Fig. 13 for this product. A mechanism for its formation is
presented in Scheme 2.
After the electron transfer step (Reaction (6)) in the absence of
O2, the coupling of the deprotonated form of dTMP•+ (dTMP(−H)•)
neutral methylene radical derived from the methyl group of the
thymine moiety, with the protonated radical anion of Ptr (PtrH•)
may be proposed as the main reaction. Possible tautomeric forms
for PtrH• are C-centered radicals on the C-6 or C-7 position of the
pterin moiety, as a result of the protonation of one of the N atoms
in the pyrazine ring. Coupling of these radicals would yield a
•
Ptr•− /PtrH• + dTMP•+/dTMP −H
→
→
Ptr−dTMP −2H
(
)
(
)(
)
(16)
Moreover, as indicated above, the analyzed product has ab-
sorption as well as emission spectra similar to those of Ptr, but
with a lower fluorescence quantum yield. These findings are an
additional indication that the chemical structure of Ptr is intact in

428
M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
Fig. 12. Emission decay recorded at 450 nm (λexc¼341 nm). Upper inset: residual
analysis. Lower inset: Spectra obtained by global analysis of TRES for the short-lived
and long-lived components [dTMP]0¼125.5 μM and
(λexc¼341 nm).
[Ptr]0¼15.5 μM, Ar-saturated aqueous solutions at pH¼5.5 irradiated for 2 h.
Fig. 10. a) Chromatograms obtained by HPLC-PDA analysis at 280 nm, before and
after 2 h of irradiation. Inset: absorption spectrum of the main photoproduct. b)
Chromatograms obtained by HPLC-FL analysis (λexc¼340 nm, λem¼450 nm) nm,
after 2 h of irradiation. Inset: evolution of the peak area of the main photoproduct.
[Ptr]0¼151 μM, [dTMP]0¼1600 μM, pH¼5.5.
Fig. 11. Corrected fluorescence spectra (λexc¼340 nm) of an aqueous solution
(pH¼5.5) of dTMP (125.5 μM) and Ptr (15.5 μM) before and after 2 h of irradiation.
Fig. 13. MS/MS spectra recorded in ESIꢂ mode of the main product formed by Ptr
photosensitization of dTMP in the absence of O2. The proposed chemical structure
of the product and its fragmentation are shown. Arrows indicate the peaks corre-
sponding to each fragment.
primary
adduct
containing
a
dihydropterin
moiety
(H2Ptr-dTMP(-H)) where the dTMP unit is linked through the CH2
group to position 6 or 7 of the 5,6-(or 7,8-)dihydropyrazine ring
(Scheme 2). This adduct has the same mass as the sum of the
masses of Ptr and dTMP. Re-aromatization when the solution is
exposed to air would yield the observed fluorescent product Ptr-
dTMP(-2H) where the Ptr conjugated structure is regenerated. It
should be noted that very low amounts of the coupling product
Ptr-dTMP(-2H) have been detected in the presence of O2.

M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
429
H+
O
O
N
O
O
N
H
N
H
N
CH2
N
.
N
N
HC
NH
N
H
.
-
H2N
N
N
O
H2N
N
N
R
H2N
N
.
.
-
Ptr
PtrH
H2Ptr-dTMP(-H)
O
O2
.
H2C
O
OH
= R
NH
O
O
P
O
O
N
CH2
O
N
R
OH
N
HO
NH
.
N
N
H2N
O
N
R
dTMP(-H)
(Ptr-dTMP)(-2H)
Scheme 2. Mechanism proposed for the formation of the coupling product Ptr-dTMP(-2H); note that the radical in PtrHꢃ could also be on the C-7 position.
4. Conclusions
h
The degradation of the thymidine 5′-monophosphate (dTMP)
photosensitized by pterin (Ptr), the parent compound of oxidized
pterins, in aqueous solution under UV-A irradiation was in-
vestigated. The mechanism of the photodegradation, summarized
in Scheme 3, depends on the presence of dissolved oxygen in the
solution. After excitation of Ptr and formation of its triplet excited
COUPLING
state (3Ptr ), three reaction pathways compete for its deactivation:
n
i) intersystem crossing to the singlet ground state, ii) energy
transfer to O2 leading to regeneration of Ptr and formation of 1O2,
Primary Adduct
and iii) electron transfer reaction from dTMP to 3Ptr , which leads
n
to the formation of the Ptr radical anion (Ptr•−
)
and the dTMP
radical cation (dTMP•+
)
. The latter is in equilibrium with its de-
protonated form (dTMP(−H)•) whereas the radical anion
may
•−
Ptr
•. Although Ptr produces 1O under UV-
Final Adduct
(fluorescent)
be protonated to yield
PtrH
2
A irradiation, we have shown that only the electron transfer
pathway (iii) is responsible for the dTMP degradation, both under
aerobic and anaerobic conditions. Further reactions of the dTMP
and Ptr radical ions (or radicals) compete with back electron
Scheme 3. Main pathways in the mechanism of the degradation of dTMP photo-
sensitized by Ptr.
to dTMP•+ (or eventually proton coupled
Ptr•−
transfer from
electron transfer from
Ptr.
to (dTMP(−H)•) to recover dTMP and
PtrH•
336 Da), 5-(hydroxymethyl)-2′-deoxyuridine 5′-monophosphate
(5-HmdUMP, MW 338 Da) and 5-(hydroperoxymethyl)-2′-deox-
yuridine 5′-monophosphate (5-HPmdUMP, MW 354 Da).
Under aerobic conditions, the electron transfer from Ptr•−/PtrH•
to O2 regenerates Ptr and yields O2•−/HO2•, which in turn dis-
proportionate to H2O2. This reaction of O2 with Ptr•−/PtrH• pre-
vents back electron transfer to dTMP•+/dTMP(−H)• to regenerate
dTMP, leading to the oxidation of the nucleotide, whereas Ptr is
not consumed. Based on HPLC and mass spectrometry analyses
and literature results on the photosensitized oxidation of thymi-
dine (Thd), structures could be proposed for the dTMP oxidation
products, taking into account the two main competitive reactions
of dTMP•+: deprotonation (Reaction (7)) and hydration (Reaction
(15)). Following hydration and trapping by O2 of the resulting ra-
dical on the Thy moiety (Scheme 1), the following stable products
may be formed: i) diastereoisomers of 5,6-dihydroxy-5,6-dihy-
drothymidine 5′-monophophate (thymidine glycol 5′-mono-
phophate, dTMPGly), compounds with identical MS/MS spectra
and incorporation of two O-atoms into the Thy moiety (MW
In the absence of O2, experimental evidence of the formation of
an adduct where the pterinic moiety is attached to the nucleobase
was obtained. In this case, coupling of the radical PtrH• (C-centered
radical on the C-6 or C-7 position of the pterin moiety) and
(dTMP(−H)•) (neutral methylene radical) is the only pathway
competing with back electron transfer to regenerate Ptr and dTMP
(Scheme 3). Consequently, the consumption of dTMP is slower
than in the presence of O2, but Ptr is also consumed. When ex-
posed to air, the adduct formed by the coupling reaction yields the
observed product Ptr-dTMP(-2H), where the Ptr conjugated
structure is regenerated. Consequently, Ptr-dTMP(-2H) has an ab-
sorption spectrum similar to that of Ptr, although its fluorescence
efficiency is lower.
The results obtained in this work may be important from a
biological point of view because they demonstrate that thymine,
which is frequently assumed as a non-reactive nucleobase, can be
damaged under UV-A radiation (a component of solar and artificial
radiation), when compounds (e.g., aromatic pterins) are present in
the skin due to specific pathological conditions. Moreover, the
photosensitized damages can take place at low O2 concentration
and even under anaerobic conditions. This fact should be
356 Da), ii) 1-(2-deoxy-beta-
D-erythro-pentofuranosyl-5-phos-
phate)-5-hydroxy-5-methylhydantoin
(5-OH-5MHMP, MW
326 Da) and iii) 5-hydroperoxy-6-hydroxy-5,6-dihydrothymidine
5′-monophosphate (5-HP-6-OHdTMP, MW 372 Da). Besides, trap-
ping by O2 of the (5-uracilyl)-methyl radical resulting from the
deprotonation of dTMP•+ may explain the subsequent formation of
5-formyl-2′-deoxyuridine 5′-monophosphate (5-FordUMP, MW

430
M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
emphasized since the intracellular O2 concentration in tissues is
much lower than that corresponding to air-equilibrated aqueous
solutions. Additionally, the spectroscopic properties of the adduct,
in particular its fluorescence, might have some applications as
fluorescent DNA probe. More studies are currently undertaken to
find out if the Ptr moiety of the Ptr-dTMP(-2H) adduct is photo-
chemically active and able to produce radicals and reactive oxygen
species (ROS) upon irradiation.
Chem. Soc. Rev. 39 (2010) 3181–3209.
[15] J.-L. Ravanat, C. Saint-Pierre, P. Di Mascio, G.R. Martinez, M.H.G. Medeiros,
J. Cadet, Damage to isolated DNA mediated by singlet oxygen, Helv. Chim. Acta
84 (2001) 3702–3709.
[16] J. Cadet, J.R. Wagner, DNA base damage by reactive oxygen species, oxidizing
agents, and UV radiation, Cold Spring Harb. Perspect. Biol. 5 (2013) a12559.
[17] S. Steenken, S.V. Jovanovic, How easily oxidizable is DNA? One-electron re-
duction potentials of adenosine and guanosine radicals in aqueous solution, J.
Am. Chem. Soc. 119 (1997) 617–618.
[18] H. Yanagawa, Y. Ogawa, M. Ueno, Redox ribonucleosides. Isolation and char-
acterization of 5-hydroxyuridine, 8-hydroxyguanosine, and 8-hydro-
xyadenosine from Torula yeast RNA, J. Biol. Chem. 267 (1992) 13320–13326.
[19] C.J. Burrows, J.G. Muller, Oxidative nucleobase modifications leading to strand
scission, Chem. Rev. 98 (1998) 1109–1152.
Acknowledgements
[20] D.B. Hall, R.E. Holmlin, J.K. Barton, Oxidative DNA damage through long-range
electron transfer, Nature 382 (1996) 731–735.
The present work was partially supported by Consejo Nacional
de Investigaciones Científicas y Técnicas (CONICET-Grant PIP 112-
200901-00425), Agencia de Promoción Científica y Tecnológica
(ANPCyT-Grant PICT-2012-0508), Universidad Nacional de La Plata
(UNLP-Grant X712). The authors thank the Centre National de la
Recherche Scientifique (CNRS) and CONICET for supporting their
collaboration through a Programme de Coopération Scientifique
(CONICET-CNRS/PICS No. 05920). M.P.S. thanks CONICET for doc-
toral research fellowship. M.V., C.L. and A.H.T. are research mem-
bers of CONICET. P.V and E.O are research members of CNRS. The
authors also thank Lionel Rechignat of the Service des Mesures
Magnétiques du Laboratoire de Chimie de Coordination (LCC,
CNRS, Toulouse, France) and Nathalie Martins-Froment of Service
Commun de Spectrométrie de Masse (FR2599), Université de
Toulouse III (Paul Sabatier) for their valuable help with the EPR
and mass spectrometry measurements, respectively.
[21] T. Melvin, Guanine is the target for direct ionisation damage in DNA, as de-
tected using excision enzymes, Nucleic Acids Res. 26 (1998) 4935–4942.
[22] B. Giese, Long-distance charge transport in DNA: the hopping mechanism, Acc.
Chem. Res. 33 (2000) 631–636.
[23] A. Banyasz, I. Vayá, P. Changenet-Barret, T. Gustavsson, T. Douki, D. Markovitsi,
Base pairing enhances fluorescence and favors cyclobutane dimer formation
induced upon absorption of UVA radiation by DNA, J. Am. Chem. Soc. 133
(2011) 5163–5165.
[24] J. Cadet, A. Grand, T. Douki, Solar UV radiation-induced DNA Bipyrimidine
photoproducts: formation and mechanistic insights, Top Curr. Chem. 356
(2015) 249–275.
[25] C. Decarroz, J.R. Wagner, J.E. van Lier, C.M. Krishna, P. Riesz, J. Cadet, Sensitized
photo-oxidation of thymidine by 2-methylꢂ1,4-naphthoquinone. Character-
ization of the stable photoproducts., Int. J. Radiat. Biol. Relat. Stud. Phys. Chem.
Med. 50 (1986) 491–505.
[26] T. Delatour, T. Douki, C. D’Ham, J. Cadet, Photosensitization of thymine nu-
cleobase by benzophenone through energy transfer, hydrogen abstraction and
one-electron oxidation, J. Photochem. Photobiol. B Biol. 44 (1998) 191–198.
[27] I. Ziegler, Production of pteridines during hematopoiesis and T-lymphocyte
proliferation: potential participation in the control of cytokine signal trans-
mission, Med. Res. Rev. 10 (1990) 95–114.
[28] K.U. Schallreuter, J.M. Wood, M.R. Pittelkow, M. Gütlich, K.R. Lemke, W. Rödl,
et al., Regulation of melanin biosynthesis in the human epidermis by tetra-
hydrobiopterin, Science 263 (1994) 1444–1446.
[29] C.A. Nichol, G.K. Smith, D.S. Duch, Biosynthesis and metabolism of tetra-
hydrobiopterin and molybdopterin, Annu. Rev. Biochem. 54 (1985) 729–764.
[30] S.J. Glassman, Vitiligo, reactive oxygen species and T-cells, Clin. Sci. 120 (2011)
99–120.
[31] K.U. Schallreuter, J. Moore, J.M. Wood, W.D. Beazley, E.M.J. Peters, L.K. Marles,
et al., Epidermal H2O2 accumulation alters tetrahydrobiopterin (6BH4) re-
cycling in vitiligo: identification of a general mechanism in regulation of all
6BH4-dependent processes? J. Invest. Dermatol. 116 (2001) 167–174.
[32] C. Lorente, A.H. Thomas, Photophysics and photochemistry of pterins in aqu-
eous solution, Acc. Chem. Res. 39 (2006) 395–402.
[33] K. Neverov, E.A. Mironov, T.A. Lyudnikova, Phosphorescence analysis of the
triplet state of pterins in connection with their photoreceptor function in
biochemical systems, Biochemistry 61 (1996) 1149–1155.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.freeradbiomed.
2016.04.196.
References
[1] S.E. Braslavsky, Glossary of terms used in photochemistry, Pure Appl. Chem. 3
(79) (2007) 293–465.
[2] J. Cadet, T. Douki, J.-L. Ravanat, P. Di Mascio, Sensitized formation of oxida-
tively generated damage to cellular DNA by UVA radiation, Photochem. Pho-
tobiol. Sci. 8 (2009) 903–911.
[34] S.Y. Egorov, A.A. Krasnovsky, M.Y. Bashtanov, E.A. Mironov, T.A. Ludnikova, M.
S. Kritsky, Photosensitization of singlet oxygen formation by pterins and fla-
vins. Time-resolved studies of oxygen phosphorescence under laser excitation,
Biochemistry 64 (1999) 1117–1121.
[3] F. El Ghissassi, R. Baan, K. Straif, Y. Grosse, B. Secretan, V. Bouvard, et al., A
review of human carcinogens – Part D: radiation, Lancet Oncol. 10 (2009)
751–752.
[35] K. Ito, S. Kawanishi, Photoinduced hydroxylation of deoxyguanosine in DNA by
pterins: sequence specificity and mechanism, Biochemistry. 36 (1997)
1774–1781.
[36] K. Hirakawa, H. Suzuki, S. Oikawa, S. Kawanishi, Sequence-specific DNA da-
mage induced by ultraviolet A-irradiated folic acid via its photolysis product,
Arch. Biochem. Biophys. 410 (2003) 261–268.
[37] T. Offer, B.N. Ames, S.W. Bailey, E.A. Sabens, M. Nozawa, J.E. Ayling, 5-Me-
thyltetrahydrofolate inhibits photosensitization reactions and strand breaks in
DNA, FASEB J. 21 (2007) 2101–2107.
[38] G. Petroselli, M.L. Dántola, F.M. Cabrerizo, A.L. Capparelli, C. Lorente,
E. Oliveros, et al., Oxidation of 2′-deoxyguanosine 5′-monophosphate photo-
induced by pterin: type I versus type II mechanism, J. Am. Chem. Soc. 130
(2008) 3001–3011.
[4] T.B.H. Buckel, A.M. Goldstein, M.C. Fraser, B. Rogers, M.A. Tucker, Recent tan-
ning bed use, Arch. Dermatol. 142 (2006) 485–488.
[5] W. Ting, K. Schultz, N.N. Cac, M. Peterson, H.W. Walling, Tanning bed exposure
increases the risk of malignant melanoma, Int. J. Dermatol. 46 (2007)
1253–1257.
[6] J. Moan, A.C. Porojnicu, A. Dahlback, Ultraviolet radiation and malignant
melanoma, Adv. Exp. Med. Biol. 624 (2008) 104–116.
[7] F. Manjón, L. Villén, D. García-Fresnadillo, G. Orellana, On the factors influ-
encing the performance of solar reactors for water disinfection with photo-
sensitized singlet oxygen, Environ. Sci. Technol. 42 (2008) 301–307.
[8] F. Manjón, D. García-Fresnadillo, G. Orellana, Water disinfection with Ru(II)
photosensitisers supported on ionic porous silicones, Photochem. Photobiol.
Sci. 8 (2009) 926–932.
[39] M.P. Serrano, C. Lorente, F.E.M. Vieyra, C.D. Borsarelli, A.H. Thomas, Photo-
sensitizing properties of biopterin and its photoproducts using 2′-deox-
yguanosine 5′-monophosphate as an oxidizable target, Phys. Chem. Chem.
Phys. 14 (2012) 11657–11665.
[9] A.P. Castano, P. Mroz, M.R. Hamblin, Photodynamic therapy and anti-tumour
immunity, Nat. Rev. Cancer. 6 (2006) 535–545.
[40] M.P. Serrano, C. Lorente, C.D. Borsarelli, A.H. Thomas, Unraveling the de-
gradation mechanism of purine nucleotides photosensitized by pterins: the
role of charge-transfer steps, ChemPhysChem 16 (2015) 2244–2252.
[41] G. Petroselli, R. Erra-Balsells, F.M. Cabrerizo, C. Lorente, A.L. Capparelli, A.
M. Braun, et al., Photosensitization of 2′-deoxyadenosine-5′-monophosphate
by pterin, Org. Biomol. Chem. 5 (2007) 2792–2799.
[10] P. Babilas, S. Schreml, M. Landthaler, R.-M. Szeimies, Photodynamic therapy in
dermatology: state-of-the-art, Photodermatol. Photoimmunol. Photomed. 26
(2010) 118–132.
[11] C.S. Foote, Definition of type I and type II photosensitized oxidation, Photo-
chem. Photobiol. 54 (1991) 659–659.
[12] M.C. DeRosa, R.J. Crutchley, Photosensitized singlet oxygen and its applica-
tions, Coord. Chem. Rev. 233–234 (2002) 351–371.
[13] J. Cadet, J.-L. Ravanat, G.R. Martinez, M.H.G. Medeiros, P. Di Mascio, Singlet
oxygen oxidation of isolated and cellular DNA: product formation and me-
chanistic insights, Photochem. Photobiol. 82 (2006) 1219–1225.
[14] P.R. Ogilby, Singlet oxygen: there is indeed something new under the sun,
[42] M.P. Serrano, C.D. Borsarelli, A.H. Thomas, Type I photosensitization of 2′-
deoxyadenosine 5′-monophosphate (5′-dAMP) by biopterin and its photo-
product formylpterin, Photochem. Photobiol. (2013) 1456–1462.
[43] K.U. Schallreuter, J. Moore, J.M. Wood, W.D. Beazley, E.M.J. Peters, L.K. Marles,
S.C. Behrens-Williams, R. Dummer, N. Blau, B.J. Thöny, Epidermal H2O2 accu-
mulation alters tetrahydrobiopterin (6BH4) recycling in vitiligo: identification

M.P. Serrano et al. / Free Radical Biology and Medicine 96 (2016) 418–431
431
of a general mechanism in regulation of all 6BH4-dependent processes? J
Invest. Dermatol. 116 (2001) 167–174.
[64] M.L. Dántola, M. Vignoni, C. González, C. Lorente, P. Vicendo, E. Oliveros, et al.,
Electron-transfer processes induced by the triplet state of pterins in aqueous
solutions, Free Radic. Biol. Med. 49 (2010) 1014–1022.
[65] E. Finkelstein, G.M. Rosen, E.J. Rauckman, Spin trapping of superoxide and
hydroxyl radical: practical aspects, Arch. Biochem. Biophys. 200 (1980) 1–16.
[66] V. Maurel, J.-L. Ravanat, S. Gambarelli, Detection of reactive free radicals de-
rived from nucleosides by liquid chromatography coupled to tandem mass
spectrometry of DMPO spin trapping adducts, Rapid Commun. Mass Spec-
trom. 20 (2006) 2235–2242.
[67] R. Misiaszek, C. Crean, A. Joffe, N.E. Geacintov, V. Shafirovich, Oxidative DNA
damage associated with combination of guanine and superoxide radicals and
repair mechanisms via radical trapping, J. Biol. Chem. 279 (2004)
32106–32115.
[68] M. Al-Sheikhly, The reactivity of adenyl and guanyl radicals towards oxygen,
Radiat. Phys. Chem. 44 (1994) 297–301.
[69] J. Cadet, T. Douki, J.L. Ravanat, Oxidatively generated damage to the guanine
moiety of DNA: mechanistic aspects and formation in cells, Acc. Chem. Res. 41
(2008) 1075–1083.
[70] V. Shafirovich, J. Cadet, D. Gasparutto, A. Dourandin, N.E. Geacintov, Nitrogen
dioxide as an oxidizing agent of 8-oxo-7,8-dihydroꢂ2′-deoxyguanosine but
not of 2′-deoxyguanosine, Chem. Res. Toxicol. 14 (2001) 233–241.
[71] I. Fridovich, Superoxide radicals, superoxide dismutases and the aerobic life-
style, Photochem. Photobiol. 28 (1978) 733–741.
[44] E.G. Janzen, Spin trapping, Acc. Chem. Res. 4 (1971) 31–40.
[45] F.A. Villamena, J.L. Zweier, Detection of reactive oxygen and nitrogen species
by EPR spin trapping, Antioxid. Redox Signal. 6 (2004) 619–629.
[46] F.A. Villamena, Superoxide radical anion adduct of 5,5-dimethyl-1-pyrroline
N-oxide. 5. Thermodynamics and kinetics of unimolecular decomposition, J.
Phys. Chem. A 113 (2009) 6398–6403.
[47] M.P. Denofrio, P.R. Ogilby, A.H. Thomas, C. Lorente, Selective quenching of
triplet excited states of pteridines, Photochem. Photobiol. Sci. 13 (2014) 1058.
[48] P.R. Ogilby, C.S. Foote, Chemistry of singlet oxygen. 42. Effect of solvent, sol-
vent isotopic substitution, and temperature on the lifetime of singlet mole-
cular oxygen (1
Δg), J. Am. Chem. Soc. 105 (1983) 3423–3430.
[49] F. Wilkinson, W.P. Helman, A.B. Ross, Rate constants for the decay and reac-
tions of the lowest electronically excited singlet state of molecular oxygen in
solution. An expanded and revised compilation, J. Phys. Chem. Ref. Data 24
(1995) 663.
[50] A.H. Thomas, M.P. Serrano, V. Rahal, P. Vicendo, C. Claparols, E. Oliveros, et al.,
Tryptophan oxidation photosensitized by pterin, Free Radic. Biol. Med. 63
(2013) 467–475.
[51] C. Castaño, M.L. Dántola, E. Oliveros, A.H. Thomas, C. Lorente, Oxidation of
tyrosine photoinduced by pterin in aqueous solution, Photochem. Photobiol.
89 (2013) 1448–1455.
[52] M.S. Kritsky, T.A. Lyudnikova, E.A. Mironov, I.V. Moskaleva, The UV radiation-
driven reduction of pterins in aqueous solution, J. Photochem. Photobiol. B Biol
39 (1997) 43–48.
[53] Q.-H. Song, K.C. Hwang, Direct observation for photophysical and photo-
chemical processes of folic acid in DMSO solution, J. Photochem. Photobiol. A
Chem 185 (2007) 51–56.
[54] P.D. Wood, R.W. Redmond, Triplet state interactions between nucleic acid
bases in solution at room temperature: intermolecular energy and electron
transfer, J. Am. Chem. Soc. 118 (1996) 4256–4263.
[55] F. Bosca, V. Lhiaubet-Vallet, M.C. Cuquerella, J.V. Castell, M.a. Miranda, The
triplet energy of thymine in DNA, J. Am. Chem. Soc. 128 (2006) 6318–6319.
[56] M.C. Cuquerella, V. Lhiaubet-Vallet, J. Cadet, M.A. Miranda, Benzophenone
photosensitized DNA damage, Acc. Chem. Res. 45 (2012) 1558–1570.
[57] P. Murasecco-Suardi, E. Gassmann, A.M. Braun, E. Oliveros, Determination of
the quantum yield of intersystem crossing of Rose Bengal, Helv. Chim. Acta. 70
(1987) 1760–1773.
[72] S.A. Mcluckey, G.J. Berkel, G.L. Glish, Tandem mass spectrometry of small,
multiply charged oligonucleotides, J. Am. Soc. Mass Spectrom. 3 (1992) 60–70.
[73] N. Belmadoui, S. Encinas, M.J. Climent, S. Gil, M.A. Miranda, Intramolecular
interactions in the triplet excited states of benzophenone–thymine dyads,
Chem. – Eur. J. 12 (2006) 553–561.
[74] P. Wang, D. Fisher, A. Rao, R.W. Giese, Nontargeted nucleotide analysis based
on benzoylhistamine labeling-MALDI-TOF/TOF-MS: discovery of putative
6-oxo-thymine in DNA, Anal. Chem. 84 (2012) 3811–3819.
[75] J.R. Wagner, J.E. van Lier, M. Berger, J. Cadet, Thymidine hydroperoxides:
structural assignment, conformational features, and thermal decomposition in
water, J. Am. Chem. Chem. Soc 116 (1994) 2235–2242.
[76] Y. Wang, J.-S. Taylor, M.L. Gross, Isolation and mass spectrometric character-
ization of dimeric adenine photoproducts in oligodeoxynucleotides, Chem.
Res. Toxicol. 14 (2001) 738–745.
[77] J. Cadet, J.R. Wagner, V. Shafirovich, N.E. Geacintov, One-electron oxidation
reactions of purine and pyrimidine bases in cellular DNA, Int. J. Radiat. Biol. 90
(2014) 423–432.
[78] J. Cadet, J.R. Wagner, Oxidatively generated base damage to cellular DNA by
hydroxyl radical and one-electron oxidants: similarities and differences, Arch.
Biochem. Biophys. 557 (2014) 47–54.
[58] D.C. Neckers, Rose Bengal, J. Photochem. Photobiol. A Chem 47 (1989) 1–29.
[59] C. Lorente, G. Petroselli, M.L. Dántola, E. Oliveros, A.H. Thomas, Electron
transfer initiated reactions photoinduced by pterins, Pteridines 22 (2011)
111–119.
[60] D. Rehm, A. Weller, Kinetics of fluorescence quenching by electron and
H-atom transfer, Isr. J. Chem. 8 (1970) 259–271.
[61] S. Fukuzumi, H. Miyao, K. Ohkubo, T. Suenobu, Electron-transfer oxidation
properties of DNA bases and DNA oligomers, J. Phys. Chem. A 109 (2005)
3285–3294.
[62] Y. Shi, C. Huang, W. Wang, J. Kang, S. Yao, N. Lin, et al., Electron transfer from
purine deoxynucleotides to deoxynucleotides deprotonated radical cations,
Radiat. Phys. Chem. 58 (2000) 253–260.
[63] L.P. Candeias, P. Wardman, R.P. Mason, The reaction of oxygen with radicals
from oxidation of tryptophan and indole-3-acetic acid, Biophys. Chem. 67
(1997) 229–237.
[79] C. von Sonntag, H.P. Schuchmann, Elucidation of peroxyl radical reactions in
aqueous solution with radiation chemistry technology, Angew. Chem. Int. Ed.
Engl. 30 (1991) 1229–1253.
[80] C. von Sonntag, H.P. Schuchmann, Peroxyl radicals in aqueous solutions, in: Z.
B. Alfassi (Ed.), Peroxyl Radicals, John Wiley and Sons, New York, 1997,
pp. 173–234.
[81] C. Murali Krishna, C. Decarroz, J.R. Wagner, J. Cadet, P. Riesz, Menadione
sensitized photooxidation of nucleic acid and protein constituents. An ESR and
spin-trapping study, Photochem. Photobiol. 46 (1987) 175–182.
[82] J.-L. Ravanat, T. Douki, J. Cadet, Direct and indirect effects of UV radiation on
DNA and its components, J. Photochem. Photobiol. B Biol. 63 (2001) 88–102.