Visible-light photochemistry of 6-formyl-7,8-dihydropterin in aqueous solution

Article abstract of DOI:10.1016/j.jphotochem.2009.11.003

Pterins are a family of heterocyclic compounds present in a wide range of living systems and are involved in different photobiological processes. 6-Formyl-7,8-dihydropterin (H₂Fop), is a product of oxidation of several natural occurring pterin derivatives, and its absorption spectrum presents as special feature an intense band in the visible region. Studies of the photochemistry of H₂Fop in aqueous solutions under visible radiation and room temperature were performed. The photochemical reactions were followed by UV-vis spectrophotometry, electrochemical measurement of dissolved O₂, enzymatic methods for H₂O₂ determination and HPLC. When H₂Fop in air-equilibrated solution was exposed to light, O₂ was consumed, the reactant was oxidized to 6-formylpterin (Fop), and H₂O₂ released. When the photolysis took place in the absence of O₂, a red compound was generated. This product was rapidly oxidized to Fop on admission of O₂. The quantum yields of H₂Fop disappearance and of photoproducts formation are reported and the effect of pH is analyzed. The potential biological implications of the results obtained are also discussed.

Full text of DOI:10.1016/j.jphotochem.2009.11.003

Journal of Photochemistry and Photobiology A: Chemistry 209 (2010) 104–110
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Visible-light photochemistry of 6-formyl-7,8-dihydropterin in aqueous solution
a
a
b,∗
a,∗
M. Laura Dántola , Andrés H. Thomas , Esther Oliveros , Carolina Lorente
a
Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Departamento de Química, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, CCT La Plata-CONICET, Casilla de Correo 16, Sucursal 4, 1900 La Plata, Argentina
Laboratoire des IMRCP, UMR CNRS 5623, Université de Toulouse (Paul Sabatier), 118, route de Narbonne, F-31062 Toulouse cédex 9, France
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 August 2009
Received in revised form 26 October 2009
Accepted 2 November 2009
Available online 10 November 2009
Pterins are a family of heterocyclic compounds present in a wide range of living systems and are involved
in different photobiological processes. 6-Formyl-7,8-dihydropterin (H2Fop), is a product of oxidation of
several natural occurring pterin derivatives, and its absorption spectrum presents as special feature an
intense band in the visible region. Studies of the photochemistry of H2Fop in aqueous solutions under
visible radiation and room temperature were performed. The photochemical reactions were followed
by UV–vis spectrophotometry, electrochemical measurement of dissolved O2, enzymatic methods for
H2O2 determination and HPLC. When H2Fop in air-equilibrated solution was exposed to light, O2 was
consumed, the reactant was oxidized to 6-formylpterin (Fop), and H2O2 released. When the photolysis
took place in the absence of O2, a red compound was generated. This product was rapidly oxidized to
Fop on admission of O2. The quantum yields of H2Fop disappearance and of photoproducts formation are
reported and the effect of pH is analyzed. The potential biological implications of the results obtained are
also discussed.
Keywords:
6
-Formyl-7,8-dihydropterin
Visible light
ROS
©
2009 Elsevier B.V. All rights reserved.
1
. Introduction
[5,6]. However, natural occurring dihydropterins, such as H Bip,
2
H Nep and dihydrofolic acid (H PteGlu), undergoes slow oxidation
2
2
Pterins are a family of heterocyclic compounds that occur in
by O2 to yield dihydroxanthopterin (H Xap) and 6-formyl-7,8-
2
a wide range of living systems and participate in relevant bio-
logical functions [1]. The most common pterin derivatives are
dihydropterin (H Fop) [7]. These compounds are also formed in the
2
reaction between H PteGlu and hydrogen peroxide (H O ) [8]. Both
2
2
2
6
-substituted compounds. The molecular weight and functional
H Fop and H Xap are stable in air-equilibrated aqueous solution
2
2
groups of these substituents vary considerably, e.g. pterins may
have substituents with one carbon atom, with a short hydrocar-
bon chain or larger substituents containing a p-aminobenzoic acid
moiety. Pterins can exist in different oxidation states and be divided
into two classes according to this property: (a) oxidized or aromatic
pterins and (b) reduced pterins. Within the latter group, 7,8-
dihydropterins and 5,6,7,8-tetrahydropterins (denoted throughout
as dihydropterins and tetrahydropterins, respectively) are the most
important derivatives due to their biological activity, e.g. dihy-
[7].
H Bip, biopterin (Bip) and other pterin derivatives accumulate
in the skin of patients suffering from vitiligo [9], a chronic depig-
mentation disorder. In the tissues affected by this disease, H O2
2
2
is present in high concentrations and the cells undergo oxidative
stress, deactivation of enzymes of the melanin biosynthesis takes
place and the protection of the skin against UV and visible radiation
fails because of the lack of melanin. Therefore, the photochem-
istry of pterins is of particular interest to this disease. Moreover,
6-carboxypterin (Cap), a product of photolysis of Bip that is not
synthesized in the skin cells, has been isolated from the affected
skin [10], thus proving that photooxidation of pterins occurs in
vivo under pathological conditions. Although the photochemistry
of aromatic pterins in air-equilibrated aqueous solutions has been
thoroughly investigated [11,12], there is a lack of information on
the photochemical behavior of dihydropterins.
droneopterin (H Nep) is secreted during the oxidative burst of
2
stimulated macrophages [2] and dihydrobiopterin (H Bip) and
2
tetrahydrobiopterin participate in the metabolism of aminoacids
[
3]. The chemistry of dihydropterins has been studied in detail [4].
Depending on their oxidation state, pterins have totally different
reactivities towards oxidizing agents. In air-equilibrated aque-
ous solutions, tetrahydropterins are rapidly oxidized by dissolved
molecular oxygen (O ), whereas dihydropterins are more stable
In contrast to most aromatic and reduced pterin derivatives,
2
H Fop can be excited by visible light due to its intense and broad
2
absorption band in the spectral range 350–500 nm (Fig. 1). This par-
ticular spectral feature is caused by a charge transfer across the
∗ Corresponding authors. Tel.: +54 221 4257291; fax: +54 221 4254642.
E-mail addresses: oliveros@chimie.ups-tlse.fr (E. Oliveros),
clorente@inifta.unlp.edu.ar (C. Lorente).
conjugated system of the pterin moiety [4]. Although H Fop may
2
certainly be formed in vivo by oxidation of H2Bip and absorbs visible
1
010-6030/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotochem.2009.11.003

M.L. Dántola et al. / Journal of Photochemistry and Photobiology A: Chemistry 209 (2010) 104–110
105
registered on a Cary 5 (Varian) spectrophotometer using quartz
cells (l = 1 cm). A more detailed description of pKa determinations
has been described elsewhere [13,14].
2
2
.3. Steady-state irradiation
.3.1. Irradiation set-up
The continuous photolysis of H Fop solutions (50–300 ␮M)
2
was carried out in quartz cells (1 cm optical path length) at
room temperature. Solutions were irradiated at 438 nm with a
xenon/mercury arc (1 kW) through a water filter, focusing optics
and a monochromator ISA Jobin-Yvon B204 (6 nm bandwidth). Pho-
tolysis experiments were performed in the presence and in the
absence of air. Deaerated solutions were obtained by bubbling with
argon for 20 min. This irradiation set-up was used for all the exper-
iments except for those corresponding to the measurements of
dissolved O . The latter experiments were performed on an optical
2
bench equipped with a halogen lamp and a cutoff filter at 395 nm
to minimize exposure to UV radiation.
Fig. 1. Absorption spectra of air-equilibrated aqueous solution of H2Fop. Solid line:
acid form; dashed line: basic form of H2Fop. Inset: variation of the absorbance
at 465 nm (circles) as a function of pH; the solid line represents the absorbance
calculated by fitting the experimental data to Eq. (1).
2
.3.2. Actinometry
Aberchrome 540 (Aberchromics Ltd.) was used as an
actinometer for the measurements of the incident pho-
ton flux (P0) at the excitation wavelength. Aberchrome
540 is the anhydride form of the (E)-␣-(2,5-dimethyl-3-
furylethylidene)(isopropylidene)succinic acid which, under
irradiation in the spectral range 316–366 nm leads to a cyclized
form. The reverse reaction to ring opening is induced by visible
light, to the best of our knowledge, no studies of the photochemistry
of this compound have been reported in the literature.
In the context of our investigations on the photochemistry of
pterin derivatives, we have performed a systematic study of the
−
6
−1
−1
photolysis of H Fop under visible irradiation in aqueous solutions,
light (436–546 nm). A value of 3.2 (± 0.1) × 10 Einstein L min
2
in the presence and in the absence of O . The study was carried out
was obtained for P0 at 438 nm in the irradiation set-up described
in the previous paragraph. The method for the determination of
P0 has been described in detail elsewhere [15,16]. Values of the
photon flux absorbed (Pa) were calculated from P0 according to
the Lambert–Beer law:
2
for the neutral (or acid) form, which is the relevant form from a
biological point of view, and for the monoanionic (or basic) form
(
Fig. 1). In particular, we have determined the quantum yields,
investigated the production of reactive oxygen species (ROS), and
analyzed the kinetics, under different experimental conditions. The
results obtained are evaluated in connection with their biological
implications and compared with those described for the photo-
chemistry of other 6-substituted pterins.
−A
Pa = P (1 − 10
)
(2)
0
where A is the absorbance of the reactant at the excitation wave-
length.
2
. Materials and methods
2.4. Analysis of irradiated solutions
2.1. Chemicals
2.4.1. UV–vis analysis
UV–vis absorption spectra were registered on a Varian Cary-
5 spectrophotometer or on a Hewlett-Packard 8452A diode array
spectrophotometer. Absorption spectra of the solutions were
recorded at regular intervals of irradiation time, using quartz cells
of 1 cm of optical path length. Experimental difference (ED) spec-
tra were obtained by subtracting the spectrum at time t = 0 from
the subsequent spectra recorded at different times. Each ED spec-
trum was normalized relative to the maximum absolute value
of the absorbance difference yielding the normalized experimen-
tal difference (NED) spectrum. Reference-difference (RD) spectra
and normalized-reference-difference (NRD) spectra were obtained
from aqueous solutions of commercial standards. The comparison
between NED and NRD spectra allows the characterization of the
major photolysis products. The analysis based on these difference
spectra is described elsewhere [17].
6
-Formyl-7,8-dihydropterin (H Fop), 6-formylpterin (Fop) and
2
other pterin derivatives were purchased from Schircks Laboratories
Switzerland) (purity >99.5%) and used without further purifica-
tion. Other chemicals from Sigma–Aldrich were used as received.
The pH of the aqueous solutions was adjusted, by adding drops
of HCl and NaOH solutions from a micropipette. The concentra-
tion of the acid and the base used for this purpose ranged from
(
−
3
0
.1 to 2 M. The ionic strength was approximately 10 M in all the
experiments.
2
.2. pKa determination
pKa values were determined from absorption changes. Measure-
ments were performed at room temperature. The experimental
absorption changes at a given wavelength can be fitted by the fol-
lowing equation:
2.4.2. High-performance liquid chromatography (HPLC)
Two chromatographic systems were employed for monitoring
the reactions: (I) a Series 1100 equipment from Hewlett-Packard
+
A = (
c × l) × (εa[H ] + ε Ka)
(1)
b
+
Ka + [H ]
(photodiode array detector HP 1100 DAD), with a RP 18 LiChro CART
where εa and ε are the molar absorption coefficients of the acid and
basic forms of the species involved in the acid–base equilibrium, c is
125-4 column; (II) a Prominence equipment from Shimadzu (pho-
todiode array detector SPD-M20A), with a Pinnacle-II C18 column
(250 mm × 4.6 mm, 5 ␮m; Restek). Solutions containing 0–10% of
acetonitrile and 90–100% of potassium phosphate aqueous solution
b
the total concentration of the substance (H Fop), l is the optical path
2
length and Ka the acid dissociation constant. UV–vis spectra were

1
06
M.L. Dántola et al. / Journal of Photochemistry and Photobiology A: Chemistry 209 (2010) 104–110
1
(
20 mM, pH 5.5) were used as eluents. Aqueous solutions of com-
chosen as a solvent, since the O lifetime (62 ± 3 ␮s) is much longer
2
mercial standards were employed for obtaining calibration curves
of reactants and products.
than in H O (3.8 ␮s), leading to stronger luminescence signals
2
[23]. Measurements were carried out under continuous irradia-
tion of the solutions using the irradiation set-up described before
(xenon/mercury lamp). The main features of the method and equip-
ment have already been described in detail [24,25]. Briefly, the
O luminescence was collected with a mirror, chopped, and after
passing through a focusing lens, a cutoff filter (1000 nm), and an
interference filter (1271 nm) was detected at 90 with respect to
the incident beam using a cooled NIR photomultiplier (Hamamatsu
R5509-42).
It should be noted that in the case of H Fop the peak of the
2
reactant could not be well separated from that of the correspond-
ing oxidized product (Fop). Therefore, integrations of the peaks at
different wavelengths were performed. Assuming that the peak
considered is only due to the reactant and one known product, the
concentration of both compounds can be calculated by resolving
sets of equations as follows:
1
2
◦
Areaꢀ1 = f CR + f CP
Areaꢀ2 = f CR + f CP
R
P
(3)
(4)
ꢀ1
ꢀ1
R
ꢀ2
P
ꢀ2
3. Results and discussion
where Areaꢀ1 and Areaꢀ2 are the values resulting from integration
R
of the chromatogram peaks at analysis wavelengths ꢀ1 and ꢀ2, C
3.1. Determination of the pKa
P
R
and C are the concentrations of the reactant and the product, f 1,
ꢀ
P1, f and f P are the factors obtained from the calibration curves
R
ꢀ2
Pterins exhibit several dissociation equilibria (Fig. 1), but above
pH 4 the lactam (amide) group is the only relevant ionizable group
f
ꢀ
ꢀ2
for the reactant and the product at ꢀ1 and ꢀ2. Although only two
R
P
(Fig. 1) [26]. The pK values of the other functional groups of the
equations are required for calculating C and C , more equations
a
pterin moiety, e.g. 2-amino group, are lower than 2. For several
were used in order to check the results obtained.
dihydropterin derivatives, the pKa value of the lactam group was
reported to be higher than 10 [4]. However, to the best of our knowl-
edge, the corresponding pKa value for 6-formyl-7,8-dihydropterin
2
.4.3. H O2 determination
2
For determination of H O , a Cholesterol Kit (Wiener Lab-
2
2
(
H Fop) has not been reported in the literature. Therefore we deter-
oratories) was used. H O2 was quantified after reaction with
2
2
mined this pKa and a value of 9.68 ± 0.04 was found (Fig. 1), which
is a little lower than those reported for other dihydropterins [4,27].
This fact is very likely due to the presence of the formyl group.
Moreover, a similar behavior was observed for aromatic pterins,
i.e. 6-formylpterin (Fop) is more acidic (pKa = 7.3) [17] than other
aromatic pterin derivatives (pKa ∼ 8) [14].
4
-aminophenazone and phenol [18,19]. Briefly, 400 ␮L of irradiated
solution of H Fop was added to 1.8 mL of reagent. The absorbance
2
at 505 nm of the resulting mixture was measured after 30 min at
room temperature, using the reagent as a blank. Aqueous solutions
of H O , prepared from commercial standards, were employed for
2
2
obtaining the corresponding calibration curves.
Taking into account these results and to avoid interferences
between acid and basic forms, we performed our experiments at
2.4.4. Determination of the concentration of O2
pH < 7.5, where H Fop is present at more than 99% in the acid (neu-
The O2 consumption during irradiation was measured with an
2
tral) form, and in the range 10.8–11.8, where H Fop is present
O -selective electrode (Orion 37-08-99). The solutions and the elec-
trode were placed in a closed glass-cell of 130 mL.
2
2
at more than 90% in the basic form (monoanion). No experiment
was carried out at pH values above this range because, in strongly
alkaline solutions, H Fop undergoes a slow thermal reaction, thus
indicating that the reactant is not stable under such pH conditions.
2.5. Quantum yield determinations
2
The quantum yields of reactant disappearance (˚−R) and prod-
uct formation (˚P) were determined in experiments performed
under different conditions. Values were obtained using the follow-
ing equations:
3
.2. Photolysis of H Fop in the presence of O
2
2
Neutral or slightly acidic air-equilibrated aqueous solutions of
H Fop (pH = 5–7) were irradiated at 438 nm during different peri-
(
d[R]/dt)0
Pa
2
˚
−R = −
(5)
(6)
ods of time. Fast changes in the absorption spectra of the solutions
were observed during irradiation (Fig. 2a), with isosbestic points at
278 and 379 nm. The resulting spectrum at long irradiation times,
e.g. more than 90 min in the experiment shown in Fig. 2a, was
similar to that corresponding to Fop. Moreover, the normalized
experimental difference (NED) spectra obtained at different irradia-
tion times (Section 2.4, UV–vis analysis) were almost identical to the
NRD spectrum (obtained by subtracting the spectrum of a standard
solution of H2Fop from that of a standard solution containing Fop)
(Fig. 2a). Therefore spectral analysis strongly suggests that Fop is
the main photoproduct of photolysis of H2Fop. No further changes
were detected when irradiated solutions were stored in the dark
for several hours, thus indicating that no thermal reactions took
place after interrupting the irradiation.
(
d[P]/dt)0
Pa
˚P =
where (d[R]/dt) and (d[P]/dt) are the initial rates of reactant con-
0
0
sumption and product formation, respectively, and Pa is the photon
flux absorbed by the reactant (Eq. (2)). For determining the initial
rates, the experiments were carried out using solutions of relatively
high concentration of reactant (H Fop). Under these conditions
the time evolution of the concentrations of H Fop and products
followed a pseudo-zero order rate law during the period of time
within which the change of Pa was negligible. The initial rates were
obtained from the slope of the corresponding plots of concentra-
tion vs. irradiation time within such time windows. The evolution
of the concentrations during irradiation was followed by HPLC (vide
supra).
2
2
HPLC analysis revealed that Fop was present in H2Fop solutions
exposed to light and that its concentration steadily increased as
a function of irradiation time. In several experiments carried out
with different initial concentrations of the reactant, the rate of
1
2
.6. Singlet oxygen ( O ) measurements
2
H Fop consumption was equal, within experimental error, to the
2
The photosensitized production of 1O2 by H Fop in D O
corresponding rate of Fop formation and, accordingly, the sum of
the concentrations of the reactant and the product remained con-
stant during the experiments (Fig. 3a). In agreement with these
2
2
1
solutions was determined by direct analysis of the weak O near-
2
infrared (NIR) phosphorescence at 1270 nm [20–22]. D O was
2

M.L. Dántola et al. / Journal of Photochemistry and Photobiology A: Chemistry 209 (2010) 104–110
107
Fig. 2. Time evolution of the absorption spectra of solutions of the acid form of
H2Fop (47.3 ␮M) exposed to visible light (438 nm). Optical path length = 10 mm;
spectra recorded every 10 min; arrows indicate the changes observed at differ-
ent wavelengths. (a) Air-equilibrated solution, and (b) argon-equilibrated solution.
Inset: NED spectrum obtained by subtracting the initial H2Fop spectrum from the
corresponding spectrum of an irradiated solution, and NRD spectrum obtained by
subtracting the spectrum of a standard solution of H2Fop from the spectrum of a
standard solution of Fop, at the same concentration and pH.
Fig. 3. Time evolution of H2Fop and Fop concentrations in irradiated solutions of
H2Fop (300 ␮M, pH 7, ꢀirr = 438 nm). (a) Air-equilibrated solutions and (b) argon-
equilibrated solutions; (ꢀ) sum of the H2Fop and Fop concentrations in ␮M, at each
time (concentrations were determined by HPLC analysis).
results, no additional product was detected by HPLC analysis. Under
−
3
these experimental conditions, values of 9.9 (± 0.8) × 10 and 9.6
−
3
(
± 0.8) × 10 were obtained for the quantum yields of H Fop con-
2
sumption (˚−H Fop) and Fop formation (˚Fop), respectively.
The evolution of the O2 concentration during irradiation was
monitored with an O2 electrode in a closed cell. In these experi-
2
corresponding aromatic analogue and H O (Reaction (7)):
2
2
hꢂ,O2
H Fop −→ Fop + H O
(7)
2
2
2
ments, the concentration of H Fop was measured by HPLC before
2
and after irradiation. A significant decrease in O2 concentra-
tion with irradiation time was observed (Fig. 4), thus indicating
Since H Fop can be present in the skin (Section 1), it is important
2
to evaluate the biological implications of this reaction. The pho-
that O2 was consumed during the photooxidation of H Fop. In
tochemical generation of H O2 may be deleterious because this
2
2
addition, the relationship between O2 and H Fop consumptions
reactive oxygen species (ROS) is in part responsible for the inhi-
bition of enzymes of the melanin biosynthesis in vitiligo [28,29].
2
(
ꢁ[O ]/ꢁ[H Fop]) was calculated for different irradiation times
2 2
and values close to 1 were obtained.
In addition, the photooxidation of H Fop produced H O . Its
concentration was determined at different times during the irradi-
In addition, in contrast to H Fop [30], Fop is a good singlet oxygen
2
1
( O ) photosensitizer upon irradiation in the UV-A spectral region
2
2
2
2
1
(320–400 nm) [31].The lowest electronic excited state of O ( ꢁg) is
2
ation of a H Fop solution (120 ␮M, pH 7.0). The rate of production
an important oxidizing intermediate in chemical processes and one
of the main chemical species responsible for the damaging effects
2
of H O (1.3 (± 0.1) ␮M/min) was very close to the rate of H Fop
2
2
2
consumption (1.4 (± 0.1) ␮M/min), showing that for each molecule
of light on biological systems (photodynamic effects) (see e.g. [32]).
1
of H Fop consumed, one molecule of H O was generated.
The main source of O production in vivo is photosensitization (see
2
2
2
2
Results presented so far demonstrate that the photolysis of the
acid form of H Fop in air-equilibrated solutions consists in a pro-
e.g. [33]).
Therefore, if H Fop were irradiated in the UV-A, its photooxida-
2
2
cess in which the reactant is oxidized by dissolved O to yield the
tion product (Fop) would also absorb part of the incident radiation,
2

1
08
M.L. Dántola et al. / Journal of Photochemistry and Photobiology A: Chemistry 209 (2010) 104–110
Table 1
Quantum yields of H2Fop consumption (˚−H2Fop) and Fop formation (˚Fop) deter-
mined under different experimental conditions.
Acid form
Basic form
Air
Argon
Air
Argon
˚
˚
−H2Fop (10−3)
9.9(± 0.8) 11.3 (± 0.9)
1.1 (± 0.1)
1.2 (± 0.1)
−
3
Fop (10
)
9.6(± 0.8) 9.8 (± 0.6)
0.57(± 0.05) 0.59 (± 0.05)
comparison between the rates of disappearance and formation sug-
gests that the photochemical process according to Reaction (7) can
explain only part of the H Fop consumption. For comparative pur-
2
poses, the values of the corresponding quantum yields (˚−H Fop
2
and ˚Fop) are presented in Table 1, together with those correspond-
ing to the acid form of H Fop. It should be noted that ˚−H Fop is
2
2
about one order of magnitude lower for the basic form, revealing
that this species is much less photosensitive than the correspond-
ing acid form. In addition, values obtained for ˚−H Fop and ˚Fop
2
Fig. 4. Time evolution of O2 concentration during irradiation of a H2Fop aqueous
solution (162 ␮M, pH 7, halogen lamp and a cutoff filter at 395 nm).
indicate that the formation of Fop corresponds to approximately
50% of the reactant consumption for the basic form.
resulting in its excitation and the subsequent production of 1O .
3.3. Photolysis of H2Fop in the absence of O2
2
In order to check this assumption, the 1O2 emission in the near-
infrared (NIR) was followed during the irradiation of H Fop (Section
The spectral changes observed in H2Fop solutions after irradia-
tionunderanaerobic conditions were different fromthose observed
in air-equilibrated solutions (Fig. 2b). In the absence of oxidant, the
formation of Fop as a photoproduct could in any case be discarded.
In the anaerobic experiments, the solutions initially yellow took up
an orange hue. Time evolution of the spectra indicates that this fact
is due to the formation of a compound absorbing above 450 nm,
with a broad band partially superimposed to the low energy band
of H2Fop. In this case, the NED spectra obtained at different irra-
diation times were quite different to the NRD spectrum, obtained
from standard solutions of H2Fop and Fop (Fig. 2b).
As soon as air was introduced into the cell, a fast reaction
occurred in the dark, which was observed by the disappearance of
the absorption above 450 nm (Fig. 6). The NED spectra, obtained by
subtracting the spectra of non-irradiated solutions of the reactant
from those of irradiated and then immediately aerated solutions,
are comparable to the NRD spectrum obtained from standard solu-
tions of H2Fop and Fop (Fig. 6). Therefore, the latter compound
2
2
.6). Experiments were performed irradiating H Fop solutions in
2
D O at the same concentration and pH (90 ␮M, pD 7.0) at two dif-
2
ferent wavelengths: (i) 438 nm, where Fop does not absorb, and (ii)
3
72 nm, where both reactant and product absorb. Upon irradiation
at the former wavelength, the signal was very low indicating a neg-
ligible production of 1O , as expected because H Fop is not a O2
1
2
2
sensitizer and Fop hardly absorbed at 438 nm. On the other hand,
upon irradiation at 372 nm a fast increase in the NIR emission was
observed (Fig. 5), thus revealing that, under these conditions, 1O2
was produced and its steady-state concentration increased with
irradiation time.
Although the basic form of H Fop is not relevant from a bio-
2
logical point of view, some experiments at pH values higher than
1
0.8 (Section 3.1) were performed in order to characterize its pho-
tochemical behavior. The results in air-equilibrated solutions were
comparable to those found for the acid form: H Fop and O2 were
2
consumed, whereas Fop and H O2 were produced. However, the
2
appears to be the main final product after introducing O in H Fop
2
2
solutions irradiated under argon.
These results indicate that irradiation under anaerobic condi-
tions leads to the formation of a “red product” (RP) with a broad
absorption band in the visible region that reacts very rapidly
with O2 to yield Fop. This behavior is similar to that previously
described for the photolysis of a group of aromatic pterins bearing
a CHOH group (Bip, Nep and 6-hydroxymethylpterin (Hmp)) or a CO
group on the first C-atom of the substituent [17,34,35]. Taking into
account these studies, the photoproduct formed in the absence of
O (RP) could be 6-formyl-5,8-dihydropterin. Further investigation
2
is needed to unambiguously identify this unstable compound.
HPLC analysis confirmed that Fop is the main product obtained
by photolysis of H Fop in the absence of O and followed by imme-
2
2
diate aeration. An additional product was also detected, with a
spectrum compatible with an aromatic pterin derivative. However,
its corresponding retention time did not match with any of the
available standards. In particular, the formation of Hmp and 6-
carboxypterin (Cap) was discarded. In agreement with the presence
of an additional product, the rates of Fop production were a little
lower than the corresponding rates of H Fop consumption (Fig. 3b).
2
−
2
Under these experimental conditions, values of 1.13 (± 0.09) × 10
−
3
and 9.8 (± 0.6) × 10 were obtained for ˚−H Fop and ˚Fop, respec-
Fig. 5. Emission at 1270 nm observed from D2O solutions of H2Fop under continu-
ous irradiation at different wavelengths. [H2Fop] = 90 ␮M, pD 7.0. Inset: absorption
spectra of an air-equilibrated aqueous solution of Fop (pH 7.2).
2
tively. These values are similar to those obtained under aerobic
conditions (Table 1).

M.L. Dántola et al. / Journal of Photochemistry and Photobiology A: Chemistry 209 (2010) 104–110
109
4. Conclusion
6
-Formyl-7,8-dihydropterin (H Fop) presents in aqueous solu-
2
tion an acid–base equilibrium that involves the lactam group
(
Fig. 1), with a pKa value of 9.68 ± 0.04. We have investigated the
photochemistry of 6-formyl-7,8-dihydropterin (H Fop) in aque-
2
ous solutions exposed to visible radiation at room temperature.
Photolysis of H Fop in air-equilibrated solutions in the pH range
2
5
–7, where the neutral (acid) form is predominant, yielded 6-
formylpterin (Fop) and hydrogen peroxide (H O ) as final products.
2
2
−
3
−3
Values of 9.9 × 10 and 9.6 × 10 were obtained for the quan-
tum yields of H Fop consumption (˚−H Fop) and Fop formation
2
2
(
˚Fop), respectively. When the photolysis took place in the absence
of O , a red compound was generated. This product was rapidly
2
oxidized upon admission of O to yield Fop and H O . Under these
2
2
2
experimental conditions, the values of ˚−H Fop and ˚Fop (for the
2
combined photolysis and subsequent O oxidation) were similar to
2
those obtained for photolysis in the presence of O . In both aerobic
2
and anaerobic conditions, ˚−H Fop values were about one order of
2
magnitude lower for the basic form (pH > 10.8). In addition, the for-
mation of Fop was only about the 50% of the reactant consumption
in this latter case.
Fig. 6. (1) Spectrum of a non-irradiated solution of H2Fop; (2) spectrum of a
H2Fop solution irradiated for 25 min under anaerobic conditions; (3) spectrum of a
H2Fop solution irradiated for 25 min under anaerobic conditions, and then aerated
Acknowledgements
(
[H2Fop]0 = 220 ␮M, pH 5.5, optical path length = 10 mm). Lower inset: detail of the
The present work was partially supported by Consejo Nacional
de Investigaciones Científicas y Técnicas (CONICET-Grant PIP
6301/05), Agencia de Promoción Científica y Tecnológica (ANPCyT-
Grant PICT 33919), and Universidad Nacional de La Plata (UNLP).
M.L.D. thanks CONICET for graduate research fellowships. A.H.T.,
C.L. and M.L.D. thank the Deutscher Akademischer Austauschdi-
enst (DAAD) for research fellowships. C.L., A.H.T. and E.O. thank
Ministerio de Ciencia, Tecnología e Innovación Productiva (Min-
CyT, Argentina) and ECOS-Sud (France) for financial support of their
cooperation project A07E07. C.L. and A.H.T. are research members
of CONICET. E.O. is research member of CNRS.
spectral changes in the region 475–650 nm. Upper inset: NRD spectrum obtained
by substracting the spectrum of H2Fop from that of Fop at the same concentration
and pH, and NED spectrum obtained by subtracting the initial spectrum (1) from the
corresponding spectrum (3).
The determination of H O2 in solutions of H Fop, immedi-
2
2
ately aerated after different times of irradiation under anaerobic
conditions, revealed that this ROS is also generated. H O2 can-
not be produced during anaerobic photolysis, therefore, these
results demonstrate that this ROS is formed in the thermal reaction
between O2 and RP. The concentrations of H O2 generated and of
the reactant consumed were of the same order of magnitude.
Taking into account the results presented in this section,
Scheme 1 shows the main reaction pathway of the photolysis of
2
2
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