Photochemistry of dihydrobiopterin in aqueous solution

Article abstract of DOI:10.1039/b913095k

Dihydrobiopterin (H₂Bip) and its oxidized analogue, biopterin (Bip), accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder in which the protection against UV radiation fails. The photochemistry of H₂Bip was studied in neutral aqueous solutions upon UV-A irradiation (320-400 nm) at room temperature. The photochemical reactions were followed by UV/vis spectrophotometry, HPLC and enzymatic methods for hydrogen peroxide (H₂O₂) determination. Photoproducts were analyzed by means of electrospray ionization mass spectrometry. Under anaerobic conditions, excitation of H₂Bip leads to the formation of at least two isomeric dimers with molecular masses equal to exactly twice the molecular mass of the reactant. This reaction takes place from the singlet excited state of the reactant. To the best of our knowledge, this is the first time that the photodimerization of a dihydropterin is reported. In the presence of air, the dimers are again the main photoproducts at the beginning of the reaction, but a small proportion of the reactant is converted into Bip. As the reaction proceeds and enough Bip accumulates in the solution, a photosensitized process starts, where Bip photoinduces the oxidation of H₂Bip to Bip, and H ₂O₂ is formed. As a consequence, the rates of H ₂Bip consumption and Bip formation increase as a function of irradiation time, resulting in an autocatalytic photochemical process. In this process, Bip in its triplet excited state reacts with the ground state of H ₂Bip. The mechanisms involved are analyzed and the biological implications of the results are discussed. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b913095k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Photochemistry of dihydrobiopterin in aqueous solution†
a
b
a
c,d
e
Mariana Vignoni, Franco M. Cabrerizo, Carolina Lorente, Catherine Claparols, Esther Oliveros* and
a
Andr e´ s H. Thomas*
Received 2nd July 2009, Accepted 18th November 2009
First published as an Advance Article on the web 18th December 2009
DOI: 10.1039/b913095k
Dihydrobiopterin (H
suffering from vitiligo, a chronic depigmentation disorder in which the protection against UV radiation
fails. The photochemistry of H Bip was studied in neutral aqueous solutions upon UV-A irradiation
320–400 nm) at room temperature. The photochemical reactions were followed by UV/vis
spectrophotometry, HPLC and enzymatic methods for hydrogen peroxide (H ) determination.
Photoproducts were analyzed by means of electrospray ionization mass spectrometry. Under anaerobic
conditions, excitation of H Bip leads to the formation of at least two isomeric dimers with molecular
2
Bip) and its oxidized analogue, biopterin (Bip), accumulate in the skin of patients
2
(
2
O
2
2
masses equal to exactly twice the molecular mass of the reactant. This reaction takes place from the
singlet excited state of the reactant. To the best of our knowledge, this is the first time that the
photodimerization of a dihydropterin is reported. In the presence of air, the dimers are again the main
photoproducts at the beginning of the reaction, but a small proportion of the reactant is converted into
Bip. As the reaction proceeds and enough Bip accumulates in the solution, a photosensitized process
starts, where Bip photoinduces the oxidation of H
the rates of H Bip consumption and Bip formation increase as a function of irradiation time, resulting
in an autocatalytic photochemical process. In this process, Bip in its triplet excited state reacts with the
ground state of H Bip. The mechanisms involved are analyzed and the biological implications of the
2
Bip to Bip, and H
2
O
2
is formed. As a consequence,
2
2
results are discussed.
Introduction
the precursor of melanin, the pigment of human skin and the main
natural protection against the harmful effects of UV radiation.
Pterins, heterocyclic compounds derived from 2-aminopteridin-4-
Dihydrobiopterin (H Bip), biopterin (Bip) (Scheme 1) and other
2
1
(
3H)-one or pterin, are present in biological systems in multiple
pterin derivatives accumulate in the skin of patients suffering
forms, and play different roles ranging from pigments to enzymatic
cofactors for numerous redox and one-carbon transfer reactions.
5
from vitiligo, a chronic depigmentation disorder. These patients
2,3
express a characteristic fluorescence in their white skin patches
upon Wood’s light examination. This fluorescence is due to
Pterins can exist in different oxidation states and can be divided
into two classes: (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
6
the accumulation of oxidized unconjugated pterins, compounds
7,8
with high fluorescence quantum yields. In the tissues affected
by this disease, hydrogen peroxide (H
2
2
O ) 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 radiation fails because of
due to their biological activity. In particular, tetrahydrobiopterin
4
(
H
4
Bip) is involved in the metabolism of amino acids. This
compound is present in the skin of human beings acting as a
cofactor of phenylalanine hydroxylase, an enzyme that catalyzes
the oxidation of phenylalanine to tyrosine. This latter amino acid is
a
Instituto de Investigaciones Fisicoqu ´ı micas Te o´ ricas y Aplicadas (INIFTA),
Departamento de Qu ´ı mica, Facultad de Ciencias Exactas, Universidad
Nacional de La Plata, CCT La Plata-CONICET, Casilla de Correo 16, Su-
cursal 4, (1900) La Plata, Argentina. E-mail: athomas@inifta.unlp.edu.ar
b
CIHIDECAR - CONICET, Departamento de Qu ´ı mica Org a´ nica, Facultad
de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabell o´ n 2,
3
p, Ciudad Universitaria, 1428 - Buenos Aires, Argentina
c
Service Commun de Spectrom e´ trie de Masse (FR2599), Universit e´ de
Toulouse (Paul Sabatier), 118, route de Narbonne, F-31062 Toulouse c e´ dex
9
, France
d
CNRS, LCC (Laboratoire de Chimie de Coordination), 205, route de
Narbonne, F-31077, Toulouse, France
e
Laboratoire des IMRCP, UMR CNRS 5623, Universit e´ de Toulouse (Paul
Sabatier), 118, route de Narbonne, F-31062, Toulouse c e´ dex 9, France.
E-mail: oliveros@chimie.ups-tlse.fr
†
Electronic supplementary information (ESI) available: HPLC and ESI-
Scheme 1 Chemical structures of some pterin derivatives mentioned in
MS data, and Stern–Volmer plots. See DOI: 10.1039/b913095k
this work.
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00 | Org. Biomol. Chem., 2010, 8, 800–810
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the lack of melanin. Therefore, the photochemistry of pterins
is of particular interest for the study of this disease. Moreover,
H
2
Bip in human skin, especially in pathological situations of UV
protection failure, to the best of our knowledge, the photochemical
behavior of H Bip has not been studied in detail, even though
6
-carboxypterin (Cap), a product of Bip photolysis (vide infra)
2
that is not synthesized in the skin cells, has been isolated from the
affected skin, thus proving that photooxidation of pterins occurs
photochemical oxidation of H
production.
2
Bip might be a source of Bip
9
in vivo under pathological conditions.
In the context of our investigations on the photochemistry of
pterins, we herein report a systematic study of the photochemistry
The photochemistry of Bip and other oxidized pterins has
10–13
been previously studied in detail.
Briefly, UV-A irradiation
of H
2
Bip under UV-A irradiation in neutral aqueous solutions,
. In particular, we have
(
320–400 nm) of Bip in aqueous media leads to oxidation of
in the presence and in the absence of O
2
the 6-substituent to yield 6-formylpterin (Fop), which in turn
is photooxidized to Cap. The formation of reactive oxygen
determined the quantum yields, identified the photoproducts,
and analyzed the kinetics under various conditions. The results
are evaluated in connection with its biological implications and
compared with those reported for the photochemistry of related
pterins.
species has been recently demonstrated. In particular, H
2
O
2
and a
∑
-
superoxide anion (O
Bip and Fop in air-equilibrated aqueous solution. Moreover,
unconjugated oxidized pterins, such as those mentioned above,
2
) are formed during the photooxidation of
11
14
1
15
are good singlet oxygen ( O
2
) photosensitizers. In addition,
Experimental
oxidized pterins are able to photoinduce oxidation of DNA and
16–18
its components.
Therefore, accumulation of oxidized pterins
General
in the skin enhances the harmful effects of UV radiation and very
likely contributes to the oxidative stress in vitiligo.
2
H Bip, Bip and other pterins were purchased from Schircks
Bip is formed in vivo from H
However, the specific chemical process that leads to this conversion
in human skin under pathological conditions is still unknown.
2
Bip by a non-enzymatic oxidation.
Laboratories (Switzerland) and used without further purification.
Other chemicals were from Sigma Chemical Co. The pH of the
aqueous solutions was adjusted by adding drops of HCl or NaOH
solutions from a micropipette. The concentrations of the acid and
base used for this purpose ranged from 0.1 to 2 M. The ionic
The formation of Bip might result from the oxidation of H
2
Bip by
19
H
2
O , but it was recently demonstrated that this reaction yields
2
20
-3
dihydroxanthopterin (H
product was obtained for the “autooxidation” of H
2
Xap) as a product. The same main
Bip (reaction
in an air-equilibrated solution). Moreover, H Xap is
recognized as the typical oxidation product of 7,8-dihydropterin
strength was approximately 10 M in all the experiments.
2
21
with O
2
2
Steady-state irradiation
22
derivatives bearing a –CHOH group linked to C6 (Scheme 1).
Irradiation set-up. The continuous photolysis of H Bip aque-
2
1
However, it has been reported that oxidation of H
2
Bip by O
2
ous solutions was carried out in quartz cells (1 cm opti-
cal path length) at room temperature. Two radiation sources
were employed: (I) Rayonet RPR lamps emitting at 350 nm
(Southern N.E. Ultraviolet Co., Branford, CT) and (II) Xe/Hg
Osram 1000 W with a monochromator ISA Jobin-Yvon B204.
Irradiation experiments were performed in the presence and
23
yields Bip.
On the other hand, non-enzymatic oxidation of H
or other oxidants leads to the formation of H Bip in vivo.
4
Bip by O
2
4
2
2
The absorption spectrum of this compound, as well as that of
Bip, has intense bands in the UV-A and UV-B (280–320 nm)
spectral regions (Fig. 1). Despite this fact and the presence of
in the absence of air. Deaerated or O -saturated solutions
2
were obtained by bubbling with Ar gas and O
respectively.
2
for 20 min,
Actinometry. Aberchrome 540 (Aberchromics Ltd.) was used
as an actinometer for the measurements of the incident photon
flux (P
determination of P
0
) at the excitation wavelength. The method for the
25,26
0
has been described in detail elsewhere.
Values of the photon flux absorbed (P
a
) were calculated from P
0
according to the Lambert–Beer law:
-
A
P
a
= P
0
(1 - 10 )
(1)
where A is the absorbance of the reactant at the excitation
wavelength.
Analysis of irradiated solutions
UV/vis analysis. Electronic absorption spectra were recorded
on diode array spectrophotometers 8452A and 8453 (Hewlett
Packard). Measurements were made using quartz cells of 1 cm
optical path length. The absorption spectra of the solutions were
recorded at regular time intervals during irradiation.
Fig. 1 Absorption spectra of H
2
Bip and Bip in aqueous solutions
(
pH 6.8–7.0); solid line: H Bip; dashed line: Bip.
2
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High-performance liquid chromatography. Two chromato-
graphic systems were employed for monitoring the reaction: (I)
a Prominence equipment from Shimadzu with a Pinnacle-II C18
column (250 ¥ 4.6 mm, 5 mm; Restek); (II) a Waters Alliance
equipment with an XTerra RP18 column (250 ¥ 4.6 mm, 5 mm,
Waters). The mobile phase consisted of 4–5% of methanol and
different conditions. Values were obtained using the following
equations:
(
(
4)
5)
9
5–96% of an aqueous solution containing 15 mM TRIS-HCl
and 1 mM NaCl (pH = 6.8) or 10 mM ammonium acetate
pH = 7). Aqueous solutions of commercial standards were em-
ployed for obtaining calibration curves of reactants and products.
In the case of H Bip, the peak of the reactant could not be well
(
where (d[R]/dt) and (d[P]/dt) are the initial rates of reactant
0
0
2
consumption and product formation, respectively, and Pa is
the photon flux absorbed by the reactant (eqn (1)). For the
determination of the initial rates, the experiments were carried out
separated from that of the corresponding oxidized product (Bip).
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:
using solutions of relatively high reactant concentration (H Bip).
2
Under these conditions, the time evolution of the concentrations of
H Bip and products followed a pseudo-zero-order rate law as long
2
as P did not change significantly. The initial rates were obtained
a
from the slope of the corresponding plots of concentration vs.
R
R
P
P
Areal1 = f l1 C + f l1
C
C
(2)
(3)
irradiation time within such time windows. The evolution of the
concentrations during irradiation was followed by HPLC (vide
supra).
R
R
P
P
Areal2 = f l2 C + f l2
where Areal1 and Areal2 are the values resulting from integration
of the chromatogram peaks at analysis wavelengths l1 and l2,
Fluorescence measurements
R
P
C and C are the concentrations of the reactant and the product,
R
P
R
P
Steady-state fluorescence measurements were performed in quartz
cells (1 cm path length) using a Perkin-Elmer LS 50B spectroflu-
orometer. Fluorescence spectra of aqueous solutions of Bip and
f
l1 , f l1 , f l2 and f l2 are the factors obtained from the calibration
curves for the reactant and the product at l1 and l2. Although
R
P
only two equations are required for calculating C and C , more
equations were used in order to check the results obtained.
H
2
Bip were obtained by excitation at different wavelengths (lexc
=
330–390 nm) and were recorded between (lexc + 10) nm and
Detection and quantification of H
of H , a Cholesterol Kit (Wiener Laboratorios S.A.I.C.) was
used. H was quantified after reaction with 4-aminophenazone
and phenol.
2
O
2
. For the determination
6
50 nm. The spectra were corrected for differences in instrumental
2
O
2
response and light scattering. For determining the quenching of
fluorescence of Bip by iodide (I ), emission spectra of Bip and
O
2
-
2
27,28
Briefly, 400 mL of irradiated solution was added
H
2
Bip solutions (25 mM) were recorded in the absence and in the
to 1.8 mL of reagent. The absorbance at 505 nm of the resulting
mixture was measured after 30 min at room temperature, using
-
presence of I (0–10 mM). The total fluorescence intensities (I
F
)
were calculated by integration of the corresponding fluorescence
bands.
the reagent as a blank. Aqueous H
2
2
O solutions prepared from
commercial standards were employed for calibration.
Mass spectrometry analysis. An LC/MS system, equipped
with an HPLC chromatograph (Agilent 1100) and a triple
quadrupole mass spectrometer (Q TRAP Applied Biosystems),
was employed. HPLC analyses were performed by using an
XTerra RP18 column (vide supra), and isocratic elution with
Results
Photolysis of H Bip in aqueous solution in the absence of O
2
2
Aqueous solutions containing H
2
Bip (50–200 mM, pH = 7.0),
previously saturated with Ar, were irradiated at 335 and 350 nm
using both radiation sources described in the Experimental
section. The irradiation-induced spectral changes (Fig. 2a) re-
vealed that the characteristic low energy band of H Bip, cen-
tered at 330 nm, decreased in intensity, whereas a product (or
products) absorbing at wavelengths lower than 300 nm was
9
6% ammonium acetate (10 mM) and 4% methanol at a flow
-
1
rate of 0.6 mL min . The mass spectrometer was equipped with
an electrospray ion (ESI) source (Turbo Ion Spray (TIS)) and
was operated in both positive and negative ion modes. Nitrogen
served as auxiliary, collision gas and nebulizer gas. The nitrogen
2
◦
temperature of the TIS source was 450 C and the declustering
(
were) formed. In addition, the same isosbestic points could be
potential (DP) 60 V. The detection was scan mode with a step
size of 0.1 atomic mass units (amu) and a scan range of 150–500
amu. Mass chromatograms, representations of mass spectrometry
data as chromatograms (the x-axis represents time and the y-axis
represents signal intensity) were registered using different scan
ranges.
observed for more than 10 min of irradiation, suggesting that the
same photochemical transformation occurred during that time
window.
HPLC analysis of the irradiated H
the H Bip concentration decreased as a function of irradiation
time (Fig. S1, ESI†). A value of (5.3 ± 0.3) ¥ 10 was obtained for
the quantum yield of H Bip consumption (U
lower than the quantum yields associated with the disappearance
2
Bip solutions showed that
2
-
2
2
H
2Bip). This value is
Quantum yield determinations
11
The quantum yields of reactant disappearance (U
R
) and product
of Bip (UBip = 0.10 ± 0.01) and the related derivative neopterin,
formation (U ) were determined in experiments performed under
P
under similar conditions.
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02 | Org. Biomol. Chem., 2010, 8, 800–810
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Fig. 3 Chromatogram (lan = 245 nm) and absorption spectra obtained
2
by HPLC analysis of Ar-saturated solutions of H Bip after 10 min
of irradiation. [H
using irradiation source I (350 nm) and chromatographic system I
Experimental).
2
Bip]
0
= 100 mM, pH = 7.0. Experiments performed
(
Photolysis of H
2
Bip in aqueous solution in the presence of O
2
Air-equilibrated aqueous solutions of H
2
Bip (50–200 mM, pH =
7
.0) were irradiated at 350 nm (radiation source I, Experimental).
The spectral changes registered were very different from those
observed in the absence of O (Fig. 2b). In this case, whereas
the higher energy band of H Bip, centered at 230 nm, rapidly
2
2
decreased in intensity, an absorbance increase at l > 340 nm,
where oxidized pterins show a typical band, was observed (Fig. 2).
In addition, no isosbestic points could be detected, thus indicating
the presence of several products.
HPLC analysis of the irradiated H Bip solutions revealed the
2
presence of 6 photoproducts. Comparison of retention times and
spectra showed that 3 of them are the same photoproducts as
those detected in anaerobic experiments (P1, P2 and P3, Fig. 3).
The retention times and the spectra of the other 3 additional
products are identical to those obtained for standard solutions of
Bip, Fop and Cap. The formation of these compounds explains
the increase in the absorbance above 340 nm and reveals the
existence of an oxidative pathway. Taking into account that Cap is
Fig. 2 Evolution of the absorption spectra of irradiated solutions of
Bip ([H Bip]
Spectra were recorded at 0, 1, 2, 3, 4, 6, 8 and 10 min. Optical path
length: 10 mm, irradiation: source I (350 nm). Arrows indicate the changes
observed at different wavelengths. (a) Ar-saturated aqueous solutions;
H
2
2
0
= 100 mM, pH = 7.0) as a function of irradiation time.
(
b) air-equilibrated aqueous solutions.
29
a photooxidation product of Fop, and Fop is, in turn, formed by
10,11
As H
2
Bip was consumed, at least 3 photoproducts (P1, P2,
photooxidation of Bip,
this latter compound can be proposed
P3, Fig. 3), with retention times lower than that of the reactant,
were formed. Analysis of the chromatograms obtained at different
wavelengths showed that the areas of the P2 and P3 peaks increased
simultaneously, and were much larger than that corresponding to
P1, suggesting that this latter compound was probably a minor
photoproduct. The changes in the peak area of P1 were difficult
to quantify due to relatively large errors in integrating very small
peaks. The spectra of the photoproducts P2 and P3 were also
quite similar, with no absorption above 320 nm and a band
centered at 246 nm (Fig. 3). These results are consistent with
the absorption spectra shown in Fig. 2. As expected, neither Bip
as the primary photoproduct in the oxidative pathway.
The concentration profiles of the reactant and the photoprod-
ucts, showed that the rate of H Bip consumption, as well as the
2
rate of Bip production, increased with irradiation time (Fig. 4a).
In the experiment shown in Fig. 4a, Bip concentration reached
a maximum and then decreased due to its own photooxidation
yielding Fop. Accordingly, Fop formation occurred later. Similarly
to Bip, Fop concentration reached a maximum and then decreased
due to its photooxidation yielding Cap. Cap was the last oxidized
pterin appearing in the irradiated solution. On the other hand,
the area profiles of chromatographic peaks of P2 and P3 showed
a steady increase until reaching a plateau. Therefore, (i) the
production of P2 and P3 does not undergo any acceleration,
nor H
2
2
O were detected by HPLC and the enzymatic method,
respectively.
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hand, the estimation of the quantum yield of Bip formation led to
-
3
a value at least one order of magnitude lower (UBip < 5 ¥ 10 ). This
fact is logical considering that, as previously pointed out (Fig. 4b),
the initial rate of Bip production is negligible in comparison with
the H
2
Bip consumption to yield P2 and P3.
was detected in irradiated air-equilibrated H
tions, its concentration increasing with irradiation time. As shown
H
2
O
2
2
Bip solu-
previously, H
2
O
2
is also produced during the photooxidation of
11
14
Bip and Fop in a 1 : 1 stoichiometry (one molecule of H
2
O
2
generated for each molecule of substrate consumed). However,
during the photolysis of H Bip, H was detected during the first
minutes of irradiation, when photolysis of Bip was still negligible,
thus suggesting that H was generated together with Bip.
In addition, the mass balance was calculated in larger time
windows (Fig. 5). If H present in the irradiated H Bip solutions
were generated only during the photooxidations of Bip and Fop, its
2
2
O
2
2
O
2
2
O
2
2
Fig. 4 Time evolution of reactant and photoproduct concentrations
in air-equilibrated aqueous solutions under UV-A irradiation. (a) Total
irradiation time = 15 min, [H
2
Bip]
0
= 100 mM. (b) Total irradiation
time = 2 min, [H
2
Bip]
0
= 128 mM. Insets: time evolution of the areas
of chromatographic peaks corresponding to photoproducts P2 and P3.
Experiments performed at pH 7.0 using irradiation source I (350 nm) and
chromatographic system I (Experimental).
(
ii) after the reactant is completely consumed, their formation
stops and (iii) these compounds do not participate in subsequent
reactions. The same behavior was observed independently of the
initial H Bip concentration.
2
We performed a more detailed HPLC analysis within the first
min of irradiation, where the secondary photolysis of Bip was still
2
negligible. The results (Fig. 4b) clearly showed that, whereas the
reaction forming Bip underwent an autocatalytic effect, the rate
of reactions yielding P2 and P3 remained constant, as observed
under anaerobic conditions. The initial quantum yield of H Bip
2
consumption was evaluated within the time window where the rate
of reactant disappearance is constant (ca. 1 min). In spite of the
relatively large error in the calculation due to the small conversion,
the value obtained, (5 ± 1) ¥ 10 , was equal within the experimental
error to that determined under anaerobic conditions. On the other
Fig. 5 Time evolution of H
UV-A irradiation and comparison with oxidized product formation. (a)
Air-equilibrated solutions. [H Bip] -saturated solutions,
= 100 mM. (b) O
2 2 2
O concentration in H Bip solutions under
2
0
2
-
2
[H Bip] = 150 mM. Experiments performed at pH 7.0 using irradiation
2
0
source I (350 nm) and chromatographic system I (Experimental).
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04 | Org. Biomol. Chem., 2010, 8, 800–810
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concentration at a given time should be equal to [Fop] + 2[Cap].
On the other hand, if H were also produced with the same
stoichiometry in the photochemical conversion of H Bip into Bip,
its concentration, at a given time, should be equal to [Bip] + 2[Fop]
3[Cap]. The concentration profiles shown in Fig. 5 show that the
latter hypothesis is the correct one, and confirm that H was
also generated in the same reaction in which Bip was formed from
Bip.
The results presented so far reveal that upon irradiation in air-
equilibrated solutions, two main pathways of H Bip consumption
exist. The first one is O -independent and is the same as that
occurring under anaerobic conditions. The second one only takes
place in the presence of O , undergoes acceleration after some time,
and is very interesting from a biological point of view, because it
proves that Bip can be generated photochemically from H Bip. In
addition, H , in part responsible for the inhibition of enzymes of
2
O
2
2
+
2
O
2
H
2
2
2
2
2
2
O
2
30,31
the melanin biosynthesis in vitiligo
in this process.
(Introduction), is released
Photolysis of H
and O
2
Bip in aqueous solution in the presence of Bip
2
The acceleration of the O -dependent process deserves a deeper
2
analysis. It seems that when Bip accumulates in the solution, in
some way this compound causes an increase in the rate of its own
production. Since a solution containing H Bip and Bip is stable
2
in the dark, this effect must be photochemical. Therefore, excited
states of Bip seem to be needed for the acceleration. In order to
investigate this issue in more detail, solutions containing H Bip
2
and Bip at different concentrations were irradiated and analyzed
by HPLC.
In the first group of experiments, solutions were irradiated at
3
50 nm. Fig. 6a shows an example of results obtained using
equimolar initial concentrations of H Bip and Bip. An initial
increase in Bip concentration was observed as a consequence of
a fast oxidation of H Bip. After almost all of the H Bip was
consumed, the photooxidation of Bip itself was dominant and
its concentration started to decrease. The initial rate of H Bip
= (11 ± 1) mM min ) was higher
than that calculated from the initial quantum yield of H Bip
2
2
2
Fig. 6 Concentration profiles of reactants and photoproducts during
2
irradiation of air-equilibrated aqueous solutions containing initially H
and Bip. pH = 7.0. (a) [H Bip] = 65 mM, [Bip] = 65 mM, experiments
performed using irradiation source I (350 nm) and chromatographic
system I. (b) [H Bip]
2
Bip
-
1
consumption ((-d[H
2
Bip]/dt)
0
2
0
0
2
-
1
consumption in the absence of Bip ((4.6 ± 0.9) mM min ).
Moreover, the initial rate of H Bip oxidation, measured as
d([Bip]+[Fop]+[Cap])/dt) ((7.2 ± 0.6) mM min ), instead of being
negligible as in the absence of Bip (Fig. 4), was of the same order of
magnitude as (-d[H Bip]/dt) . In addition, the initial proportion
of H Bip converted into products P2 and P3, assessed through
2
0
= 145 mM, [Bip] = 70 mM, experiments performed
0
2
using irradiation source II (380 nm) and chromatographic system II
(Experimental).
-
1
(
0
2
0
indicate that when Bip is excited, the oxidation of H
Bip takes place even in the absence of radiation absorbed by
the reactant (H Bip), which means that this is a photosensitized
2
Bip into
2
the areas of the corresponding peaks, was much lower than that
registered in the experiments performed in the absence of an initial
concentration of Bip.
Another set of experiments was performed using the irradiation
source II (Experimental section), under conditions where the
2
process. A control experiment was performed under the same
conditions, but in the absence of air. After irradiation, the solutions
were aerated and then analyzed. As expected, the consumption
of H
presence of air. These results demonstrated that O
for the photosensitized formation of Bip, i.e. no reaction occurs
between excited Bip and H Bip under anaerobic conditions.
Coming back to the general analysis of the H Bip photolysis,
we have shown that an O -dependent process yields Bip as the
2
Bip was negligible in comparison to that observed in the
proportion of radiation absorbed by H
2
Bip was very low (Fig. 1),
2
is needed
i.e. only Bip was excited (wavelengths of irradiation >370 nm).
The results obtained in one of these experiments are illustrated in
2
Fig. 6b. The initial rate of H
than those obtained in experiments performed in the absence of
Bip. Moreover, the proportion of H Bip converted into Bip was
2
Bip consumption was much higher
2
2
2
main product. This oxidation involves, as a matter of fact, two
different pathways. One of them consists of the reaction of excited
very high (>80%). Accordingly, the amounts of products P1, P2
and P3 formed were undetectable or very low. These results clearly
states of H
2
Bip with dissolved O
2
, does not need the presence of
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excited Bip and is responsible for the very slow initial formation of
Bip during the irradiation of air-equilibrated H Bip solutions. The
other pathway is a photosensitized process, in which excited states
of Bip induce the oxidation of H Bip to yield the same product.
This second pathway explains the acceleration in the oxidation of
the reactant observed in solutions initially containing only H Bip,
as well as the high rates of H Bip consumption and Bip formation
2
2
2
2
in experiments performed with mixture solutions containing both
pterin derivatives.
In order to find out if the photosensitized oxidation of H
also takes place using other pterin derivatives as photosensitizers,
air-equilibrated solutions containing H Bip and an oxidized pterin
different from Bip were irradiated at 380 nm. In order to avoid
2
Bip
2
32
interferences and to simplify the analysis, pterin (Ptr) and
33
6
-methylpterin (Mep) were chosen for their low quantum yields
of consumption under UV-A irradiation. Fig. 7 shows the results in
the case of Mep (similar results were obtained with Ptr). Whereas
Mep was not consumed significantly, a strong consumption of
H Bip was observed with a significant production of Bip, and
2
negligible amounts of P1, P2 and P3. In control experiments
performed in the absence of Mep, no significant amount of
Bip was detected. These results clearly demonstrate that the
photosensitized conversion of H Bip into Bip takes place not only
2
with Bip as sensitizer, but also with Mep, Ptr, and probably other
oxidized pterins, acting as sensitizers.
Fig. 8 Time evolution of reactant and photoproduct concentrations
in O -saturated aqueous solutions under UV-A irradiation. (a) Total
2
irradiation time = 10 min. (b) Total irradiation time = 2 min. Insets:
time evolution of the areas of chromatographic peaks corresponding to
photoproducts P2 and P3. [H
pH 7.0 using irradiation source I (350 nm) and chromatographic system I
Experimental).
2
Bip]
0
= 146 mM, experiments performed at
(
Fig. 7 Concentration profiles of reactants and photoproducts during
showed that H
of H Bip. From the analysis of the first 2 min of irradiation
Fig. 8b), a value of (5 ± 1) ¥ 10 was obtained for the quantum
yield of H Bip consumption, i.e. equal within experimental error
to that calculated under anaerobic and air-equilibrated conditions.
However, kinetic analysis of concentration profiles showed
different results from those expected. The initial rate of Bip
formation was not higher than that measured in air-equilibrated
2
O
2
was also a photoproduct of the photooxidation
irradiation of air-equilibrated aqueous solutions containing initially H
and Mep. Inset: time evolution of the areas of chromatographic peaks
corresponding to photoproducts P2 and P3. [H Bip]
= 100 mM, [Mep]
0 mM, pH = 7.0, experiments performed using irradiation source II
380 nm) and chromatographic system II (Experimental).
2
Bip
2
-
2
(
2
0
0
=
4
2
(
Experiments were carried out to assess the effect of the O
concentration, by bubbling O before irradiation (Fig. 8). HPLC
2
2
solutions. Moreover, the consumption of H Bip did not undergo
2
analysis showed that the results were similar to those obtained
under air-equilibrated solutions. The concentrations of products
a strong acceleration and, accordingly, the rate of oxidized pterins
formation hardly increased. Therefore the oxidative pathway of
P1, P2 and P3 increased with irradiation time (O
photolysis), and, as expected, the oxidative pathway also occurred
formation of Bip). The corresponding mass balance (Fig. 5b)
2
-independent
H
2
Bip photolysis was not enhanced in O
the contrary, the photosensitized oxidation of H
inhibited by O at high concentrations (vide infra).
2
-saturated solutions. On
2
Bip seemed to be
(
2
8
06 | Org. Biomol. Chem., 2010, 8, 800–810
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Mass spectrometry analysis of photoproducts
Solutions of H Bip were analyzed by HPLC coupled to elec-
2
trospray ionization (ESI) mass spectrometry, before and after
irradiation. The mass analysis was carried out in both positive
+
-
and negative ion modes (ESI and ESI , respectively). An H Bip
2
standard solution was analyzed and, as expected, the signal
-
corresponding to the intact molecular ion of H Bip as [M - H]
2
-
species at m/z 238.2 Da was observed in ESI mode (data not
+
shown). In addition, in ESI mode the intact molecular H
2
Bip
ion as [M + H] and the adduct [M + Na] were detected at m/z
40.2 Da and 262.2 Da, respectively (Fig. S2, ESI†). The mass
chromatograms of H Bip solutions irradiated under anaerobic
+
+
2
2
conditions were registered for large mass windows and specific ion
masses, corresponding to the intact molecular ion of each detected
compound (H Bip, P1, P2 and P3, data not shown). Whereas
2
the signals of photoproducts P2 and P3 were intense, the signal
of P1 was very weak, thus suggesting again that P1 is a minor
photoproduct.
+
The mass spectra of P2 and P3 obtained in ESI mode are
shown in Fig. S2 (ESI†). Notice that the more intense peaks of
the spectra of H
and correspond to the mass of the intact ion of H
2
Bip, P2 and P3 are identical (m/z = 240.2 Da),
+
Scheme 2 Hypothetical structures for the dimers formed by photolysis
of H Bip.
2
2
Bip ([M + H] ).
However, both P2 and P3 have an additional peak at m/z = 479
+
Da, which corresponds to [M
2
+ H] = 2 ¥ 239 + 1 Da. Therefore,
reactant, P1 and the dimers, products with retention times and
spectra matching those of Bip, Fop and Cap were also detected.
Mass spectra confirmed that the pathway yielding dimers also
takes place under aerobic conditions. In the mass spectra obtained
from the chromatographic peaks of Bip, Fop and Cap, the signals
corresponding to the intact molecular ions were detected in both
the molecular mass of both P2 and P3 should be equal to exactly
twice the molecular mass of H Bip. These results strongly suggest
that P2 and P3 are dimers of H Bip and that the most efficient
2
2
pathway for fragmentation during the electrospray ionization
process is the cleavage of the dimers to yield the corresponding
monomers. P2 and P3 have the same molecular weight and similar
absorption spectra, but their structures are different enough to
produce a variation in the retention time in the chromatographic
runs, which allows their separation and independent analysis.
Most probably, photodimerization occurs through the enone-
type C=C bond (C9=C10) at the junction of the pyrazine and
+
-
ESI and ESI modes. Therefore, these results also confirm the
formation of the oxidized pterins (Bip, Fop and Cap) in the aerobic
photolysis of H Bip.
2
Mechanistic analysis of the oxidative pathway
20,23,36,37
pyrimidine rings. This type of dimerization was reported in the
case of thymine and uracil (pyrimidine derivatives).
of the photodimerization of H Bip, four dimers may be formed by
syn or anti combinations: for each syn or anti dimer, the head-to-
head (h,h) and head-to-tail (h,t) isomers are possible (Scheme 2).
The products denoted above as P2 and P3 might correspond to
the anti and syn isomers.
As dihydropterins are reactive towards oxidizing species
34,35
In the case
and Bip, and other oxidized pterins produce reactive oxygen
1
1,15
species,
chemical production of such species by Bip and the subsequent
reaction of these species with H Bip to yield more Bip. One
hypothesis involves H ; however, this species can be discarded
because it was already demonstrated that the product of the
one of the mechanisms to be considered is the photo-
2
2
2
O
2
20
The MS/MS spectra of H
2
Bip and of the dimers (m/z =
corresponding reaction is not Bip (Introduction).
The pterins Bip, Fop and Cap are good O sensitizers upon
+
1
2
40.2 Da) were registered in the ESI mode (Fig. S3, ESI†).
2
15
1
In the fragmentation of H
2
Bip, the main peak (m/z = 165 Da)
UV-A excitation, and the oxidation of H
2
Bip by O
as products; therefore, the participation
of this species was assessed more carefully. In the case of the
2
is fast,
23
corresponds to the loss of the 6-substituent ([M - (CHOH)
2
CH
3
yielding Bip and H
2
O
2
+
+
H] ) and the peak at m/z = 222 Da corresponds to the loss
+
2 2
1
of H
O ([M - H
O + H] ). It is difficult to speculate about
oxidation of H
Bip (D[Bip]/D[H
relationship between the H
(D[H ]/D[H Bip]) was equal to 0.17 ± 0.03. Analysis of
mass balances, such as those shown in Fig. 5, indicate that
D[H ]/D[H Bip] is very close to 1 for experiments performed
in both air-equilibrated and O -saturated solutions. Similarly,
calculation of D[Bip]/D[H Bip] gave values significantly higher
than 0.42. This is particularly evident in experiments where the
Bip consumption via the oxidative pathway is much higher than
2
Bip by O
Bip]) was reported to be 0.42 ± 0.03, whereas the
released and the H Bip consumed
2
, the fraction of H
2
Bip converted to
the rest of the fragments and, to the best of our knowledge,
this is the first time H Bip has been analyzed by ESI mass
spectrometry. The fragmentations registered, under the same
conditions, for P2 and P3 (Fig. S3, ESI†) are exactly the same
2
2
O
2
2
2
23
2
O
2
2
as those for H
fragments formed during ionization of products P2 and P3 are
Bip. These results confirmed the dimeric nature of P2 and
P3.
Irradiated air-equilibrated H
2
Bip. Therefore, it can be concluded that the main
2
O
2
2
2
H
2
2
Bip solutions were also analyzed
H
2
2
by HPLC-ESI mass spectrometry. As expected, besides the
via the pathway yielding the dimers (Fig. 6). Hence, oxidation via
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1
O
2
cannot be a significant pathway for H
2
Bip consumption and
is not responsible for the acceleration phenomenon.
Quenching of the triplet state of Bip by O
studied by time-resolved and steady-state methods. On the
2
has already been
38
15
other hand, although it was demonstrated that O does not
2
7,39
deactivate singlet excited states of several oxidized pterins, no
previous studies have been reported for Bip. Therefore, emission
spectra of Ar-, air- and O
centration were registered. No differences in the total fluorescence
intensities were detected, thus indicating that O does not quench
the singlet excited state of Bip. For this reason, the inhibition of
the photosensitized process observed at high O concentrations is
due to the quenching of the Bip triplet state by O
2
-saturated Bip solutions at the same con-
2
2
2
.
In order to investigate this issue further, experiments in the
-
presence of iodide (I ) were performed. This anion is able to
interact with both singlet and triplet excited states of organic
compounds. The resulting effects on the photophysical behaviour
of a given compound depend on the relative rates of the different
deactivation pathways (non-radiative decays to ground state,
Fig. 9 Time evolution of concentrations of reactant and photoproduct
40
intersystem crossing). Therefore, in some cases, the presence
during the photolysis of H
KI (600 mM). Inset: time evolution of the areas of chromatographic
peaks corresponding to photoproducts P2 and P3. [H Bip]
2
Bip, in the absence and in the presence of
-
of I causes an increase of the quantum yields of triplet state
formation, whereas in others, a decrease is observed. The efficiency
2
0
= 115 mM,
-
of I in quenching flavin triplet states is much higher than the
experiments performed in air-equilibrated aqueous solution at pH 7.0
using irradiation source I (350 nm) and chromatographic system I
(Experimental).
efficiency of quenching the corresponding excited singlet states.
This property has been used to investigate the role of the excited
41,42
states of flavin molecules in photochemical mechanisms.
The
same approach was used to evaluate the participation of triplet
generated and would react with O
latter one, an electron transfer from the ground state of H
to the triplet state of Bip would take place. The capability of
pterins to act as photosensitizers via electron transfer processes
has already been proven. In the case of the oxidation of purine
2
to yield Bip and H
2
O
2
. In the
Bip
43
excited states of pterins in their photoreduction. Furthermore,
in studies of the phosphorescence of pterins adsorbed on paper
at room temperature, it was observed that the non-radiative decay
2
-
44
from the lowest triplet state of pterins was enhanced by I .
17,18
We applied this methodology to our system, and evaluated the
nucleotides photoinduced by pterin,
the photosensitizer in its
-
capability of I to deactivate the S
1
states of Bip and H Bip by
2
triplet excited state reacts in a first step with the substrate to yield
∑
-
fluorescence quenching experiments (Fig. S4, ESI†). For both
compounds, only moderate quenching was registered within the
the pterin radical anion (Ptr ) and the nucleotide radical cation.
∑
-
∑-
In a second step, Ptr reacts with O
2
and O
2
is formed.
-
range of I concentration used; i.e., a decrease of less than 50% of
Taking into account these reactions and the results presented so
far, the following mechanism can be proposed for the oxidation of
H Bip:
2
-
the fluorescence was measured at a concentration of 10 mM of I .
-
At I concentrations lower than 1 mM, the fluorescence quenching
was negligible (Fig. S4 insets, ESI†).
(
6)
Photolysis experiments performed in the presence and in the
-
absence of different I concentrations were compared. The results
1
H
2
Bip* + H
2
Bip → (H
2
Bip)
2
(7)
(8)
-
showed that, whereas I did not affect the rate of dimer formation,
the acceleration of the H
2
Bip consumption was not observed any
3
H
2
Bip* + O
2
→ Bip + H
2
O
2
more and, consequently, formation of Bip was very slow (Fig. 9).
-
Therefore, I inhibited the photosensitized oxidation of H
2
Bip. In
(
9)
addition, during the first 4 min of irradiation, the rate of formation
of dimers was identical to the control, thus indicating that I did
not inhibit this pathway.
These results are in agreement with experiments performed in
-
3
1
Bip* + O
2
→ Bip + O
2
(10)
(11)
(12)
(13)
(14)
(15)
(16)
3
Bip* + O
2
→ Fop + H
2
O
2
O -saturated solutions (Fig. 8) and strongly suggest that dimers are
2
3
Bip + Bip∑-
∑+
formed from the singlet excited states of H
although it is difficult to assess the effect of I on the initial non-
2
Bip. On the other hand,
H
2
Bip + Bip* → H
2
-
∑
-
∑-
photosensitized oxidation of H
of the triplet state of Bip by I inhibits the photosensitized process,
2
Bip, it is clear that the quenching
Bip + O
2
→ Bip + O
2
-
∑
+
∑-
+
thus preventing the acceleration of H
resulting fast Bip formation.
2
Bip consumption and the
H
2
Bip + O
2
→ Bip + O
2
+ 2H
∑
-
+
∑
The oxidation of H
transfer or electron transfer from the triplet excited state of Bip.
In the former mechanism, the triplet state of H Bip would be
2
Bip could be photoinduced via energy
O
2
+ H
3
O ꢀ HO
2
+ H
2
O
∑
∑-
-
2
HO
2
+ O
2
→ HO
2
+ O
2
8
08 | Org. Biomol. Chem., 2010, 8, 800–810
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View Article Online
Conclusions
2 W. Pfleiderer, in Chemistry and Biology of Pteridines and Folates,
Plenum Press, New York, 1993, pp. 1–16.
3
4
T. J. Kappock and J. P. Caradonna, Chem. Rev., 1996, 96, 2659–2756.
In this work, we have studied the photochemistry of dihydro-
C. A. Nichol, G. K. Smith and D. S. Duch, Annu. Rev. Biochem., 1985,
biopterin (H
Under anaerobic conditions, excitation of H
2
Bip) in aqueous solution upon UV-A irradiation.
Bip leads to the
5
4, 729.
2
5
K. U. Schallreuter, J. Moore, J. M. Wood, W. D. Beazley, E. M. Peters,
L. K. Marles, S. C. Behrens-Williams, R. Dummer, N. Blau and B.
Th o¨ ny, J. Invest. Dermatol., 2001, 116, 167–174.
formation of isomeric dimers with molecular masses equal to
exactly twice the molecular mass of the reactant. This reaction
takes place from the singlet excited state of the reactant and the
6
K. U. Schallreuter, J. M. Wood, M. R. Pittelkow, M. G u¨ tlich, K. R.
Lemke, W. R o¨ dl, N. N. Swanson, K. Hitzemann and I. Ziegler, Science,
1994, 263, 1444–1446.
corresponding quantum yield of H
2
Bip consumption U
equal to (5.3 ± 0.3) ¥ 10 . The formation of dimers by photolysis
of H Bip has so far never been reported. In the presence of air,
Bip is initially consumed with the same quantum yield and
H
2Bip was
-
2
7 A. H. Thomas, C. Lorente, A. L. Capparelli, M. R. Pokhrel, A. M.
Braun and E. Oliveros, Photochem. Photobiol. Sci., 2002, 1, 421–426.
C. Lorente, A. L. Capparelli, A. H. Thomas, A. M. Braun and E.
Oliveros, Photochem. Photobiol. Sci., 2004, 3, 167–173.
2
8
H
2
the dimers are again the main photoproducts. However, a small
proportion of the reactant is converted into its oxidized analogue,
biopterin (Bip). As the reaction proceeds and a certain amount of
Bip accumulates in the solution, a new pathway appears. This is a
photosensitized process in which Bip photoinduces the oxidation
9 H. Rokos, W. D. Beazley and K. U. Schallreuter, Biochem. Biophys.
Res. Commun., 2002, 292, 805–811.
1
0 R. Mengel, W. Pfleiderer and W. R. Knappe, Tetrahedron Lett., 1977,
1
8, 2817–2820.
11 M. Vignoni, F. M. Cabrerizo, C. Lorente and A. H. Thomas,
Photochem. Photobiol., 2009, 85, 365–373.
1
2 G. Su a´ rez, F. M. Cabrerizo, C. Lorente, A. H. Thomas and A. L.
Capparelli, J. Photochem. Photobiol., A, 2000, 132, 53–57.
of H
as a sensitizer of its own production. As a consequence, the rates
of H Bip consumption and Bip formation increase as a function
of irradiation time, resulting in an autocatalytic photochemical
reaction. In this process, in which no excitation of H Bip is needed,
Bip in its triplet excited state reacts with the ground state of H Bip.
The first step could involve an electron transfer from H Bip to the
triplet excited state of Bip. Other oxidized pterins can also act
2
Bip to Bip, and H
2
O
2
is formed, i.e. the photoproduct acts
1
3 C. Lorente and A. H. Thomas, Acc. Chem. Res., 2006, 39, 395–402.
2
14 G. Petroselli, J. M. Bartsch and A. H. Thomas, Pteridines, 2006, 17,
8
2–89.
1
5 A. H. Thomas, C. Lorente, A. L. Capparelli, C. G. Mart ´ı nez, A. M.
2
Braun and E. Oliveros, Photochem. Photobiol. Sci., 2003, 2, 245–
2
2
50.
16 K. Ito and S. Kawanishi, Biochemistry, 1997, 36, 1774–1781.
2
1
7 G. Petroselli, R. Erra-Balsells, F. M. Cabrerizo, C. Lorente, A. L.
Capparelli, A. M. Braun, E. Oliveros and A. H. Thomas, Org. Biomol.
Chem., 2007, 5, 2792–2799.
as photosensitizers of H
mechanism.
2
Bip oxidation, thus revealing a general
18 G. Petroselli, M. L. D a´ ntola, F. M. Cabrerizo, A. L. Capparelli, C.
Lorente, E. Oliveros and A. H. Thomas, J. Am. Chem. Soc., 2008, 130,
The quantum yields of H
2
Bip consumption are relatively high
3
001–3011.
and show that this substance can be easily photolyzed. This
is of particular importance from a biomedical point of view,
1
9 J. Moore, J. M. Wood and K. U. Schallreuter, J. Raman Spectrosc.,
2
002, 33, 610–617.
since H
2
Bip is accumulated in the skin of patients suffering
20 M. L. D a´ ntola, T. M. Schuler, M. P. Denofrio, M. Vignoni, A. L.
5
Capparelli, C. Lorente and A. H. Thomas, Tetrahedron, 2008, 64, 8692–
from vitiligo, a chronic depigmentation disorder, in which the
protection against UV radiation fails. Even more important is
8
699.
2
1 M. L. D a´ ntola, M. Vignoni, A. L. Capparelli, C. Lorente and A. H.
the photooxidation of H
2
Bip because it demonstrates that Bip,
Thomas, Helv. Chim. Acta, 2008, 91, 411–425.
a compound that generates reactive oxygen species and is toxic
22 W. Pfleiderer, in Biochemical and Clinical Aspects of Pteridines, Walter
45
de Gruyter & Co., Berlin, New York, 1987, pp. 3–21.
for melanocytes, can be formed photochemically from H
Moreover, taking into account the presence of oxidized pterins,
the fast photosensitized oxidation of H Bip should be considered
2
Bip.
2
3 M. L. D a´ ntola, A. H. Thomas, A. M. Braun, E. Oliveros and C.
Lorente, J. Phys. Chem. A, 2007, 111, 4280–4288.
24 J. C. Nixon, C. L. Lee, S. Milstien, S. Kaufman and K. Bartholom e´ ,
2
J. Neurochem., 1980, 35, 898–904.
as a very probable source of Bip in the skin.
2
5 A. M. Braun, M. T. Maurette, and E. Oliveros, Photochemical
Technology, John Wiley & Sons, Chichester, 1991, pp. 85–88.
Acknowledgements
26 H. J. Kuhn, S. E. Braslavsky and R. Schmidt, Pure Appl. Chem., 2004,
7
6, 2105–2146.
The present work was partially supported by Consejo Nacional
de Investigaciones Cient ´ı ficas y T e´ cnicas (CONICET-Grant PIP
27 C. C. Allain, L. S. Poon, C. S. G. Chan, W. Richmond and P. C. Fu,
Clin. Chem., 1974, 20, 470–475.
8 H. M. Flegg, Ann. Clin. Biochem., 1973, 10, 79–84.
9 A. H. Thomas, G. Su a´ rez, F. M. Cabrerizo, R. Martino and A. L.
Capparelli, J. Photochem. Photobiol., A, 2000, 135, 147–154.
0 J. M. Wood, B. Chavan, I. Hafeez and K. U. Schallreuter, Biochem.
Biophys. Res. Commun., 2004, 325, 1412–1417.
1 K. U. Schallreuter, S. M. A. Elwary, N. C. J. Gibbons, H. Rokos and
J. M. Wood, Biochem. Biophys. Res. Commun., 2004, 315, 502–508.
32 F. M. Cabrerizo, M. L. D a´ ntola, A. H. Thomas, C. Lorente, A. M.
2
2
6
301/05), Agencia de Promoci o´ n Cient ´ı fica y Tecnol o´ gica (AN-
PCyT Grants PICT 06-12610 and PICT 33919), and Universidad
Nacional de La Plata (UNLP). M.V. thanks CONICET for
graduate research fellowships. F.M.C., C.L., and A.H.T. are
research members of CONICET. C.L., A.H.T., M.V., and E.O.
thank Ministerio de Ciencia, Tecnolog ´ı a e Innovaci o´ n Produc-
tiva (MinCyT, Argentina) and ECOS-Sud (France) for financial
support of their cooperation project A07E07. The authors thank
Jean-Christophe Garrigues (Laboratoire des IMRCP) for his
valuable technical support and helpful discussions of HPLC-MS
experiments.
3
3
Braun, E. Oliveros and A. L. Capparelli, Chem. Biodiversity, 2004, 1,
1
800–1811.
3
3
3
3 F. M. Cabrerizo, C. Lorente, M. Vignoni, R. Cabrerizo, A. H. Thomas
and A. L. Capparelli, Photochem. Photobiol., 2005, 81, 793–801.
4 B. H. Jennings, S. C. Pastra and J. L. Wellington, Photochem. Photobiol.,
1
970, 11, 215–226.
5 B. H. Jennings, S. Pastra-Landis and J. W. Lerman, Photochem.
Photobiol., 1972, 15, 479–491.
3
3
6 R. Shen, Arch. Biochem. Biophys., 1994, 310, 60.
7 G. Weiss, D. Fuchs, A. Hausen, G. Reibnegger, E. R. Werner, G. Werner-
Felmayer, E. Semenitz, M. P. Dierich and H. Wachter, FEBS Lett.,
1993, 321, 89–92.
References
1
D. J. Brown, in The Chemistry of Heterocyclic Compounds, John Wiley
Sons, New York, 1988, pp. 1–42.
&
This journal is © The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 800–810 | 809

View Article Online
3
3
8 J. W. Ledbetter, W. Pfleiderer and J. H. Freisheim, Photochem.
Photobiol., 1995, 62, 71–81.
9 F. M. Cabrerizo, G. Petroselli, C. Lorente, A. L. Capparelli, A. H.
Thomas, A. M. Braun and E. Oliveros, Photochem. Photobiol., 2005,
42 P. A. W. Van den Berg, J. Widengren, M. A. Hink, R. Rigler and
A. J. W. G. Visser, Spectrochim. Acta, Part A, 2001, 57, 2135–
2144.
43 M. S. Kritsky, T. A. Lyudnikova, E. A. Mironov and I. V. Moskaleva,
J. Photochem. Photobiol., B, 1997, 39, 43–48.
44 R. T. Parker, Anal. Chem., 1979, 51, 1921–1926.
45 K. U. Schallreuter, G. B u¨ ttner, M. R. Pittelkow, J. M. Wood, N. N.
Swanson and C. Korner, Biochem. Biophys. Res. Commun., 1994, 204,
43–48.
8
1, 1234–1240.
¨
4
4
0 J. Widengren, U. Mets and R. Rigler, J. Phys. Chem., 1995, 99, 13368–
3379.
1 R. D. Vierstra, K. L. Poff, E. B. Walker and P.-S. Song, Plant Physiol.,
981, 67, 996–998.
1
1
8
10 | Org. Biomol. Chem., 2010, 8, 800–810
This journal is © The Royal Society of Chemistry 2010