New results on the photochemistry of biopterin and neopterin in aqueous solution

Article abstract of DOI:10.1111/j.1751-1097.2008.00450.x

New photochemical studies of the reactivity of biopterin (BPT) and neopterin (NPT) in acidic (pH = 5.5) and alkaline (pH = 10.5) aqueous solutions at 350 nm and room temperature were performed. The photochemical properties of BPT are of particular interes

Full text of DOI:10.1111/j.1751-1097.2008.00450.x

Photochemistry and Photobiology, 2009, 85: 365–373
New Results on the Photochemistry of Biopterin and Neopterin
in Aqueous Solution
1
Mariana Vignoni , Franco M. Cabrerizo , Carolina Lorente and Andres H. Thomas*
2
1
1
´
1
´
´
Instituto de Investigaciones Fisicoquımicas Teoricas y Aplicadas (INIFTA), CCT-La Plata-CONICET,
Departamento de Quımica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata,
´
La Plata, Argentina
2
´
´
Centro de Investigaciones en Hidratos de Carbono (CIHIDECAR), Departamento de Quımica Organica,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, CONICET, Ciudad Universitaria,
Buenos Aires, Argentina
Received 2 May 2008, accepted 17 August 2008, DOI: 10.1111/j.1751-1097.2008.00450.x
The participation of pterins in different photobiological
ABSTRACT
processes has been suggested or demonstrated in past decades,
and interest in the photochemistry and photophysics of this
group of compounds has subsequently increased. Under UVA
excitation (320–400 nm), these biomolecules can fluoresce,
undergo photo-oxidation to produce different photoproducts
and generate reactive oxygen species (ROS) such as singlet
oxygen (1O2) (5). Moreover, pterins are able to photoinduce
oxidation of DNA and its components (6–8).
Biopterin and other oxidized and reduced pterin derivatives
accumulate in the white skin patches of patients affected by
vitiligo (9), where hydrogen peroxide (H2O2) is present in high
concentrations. In this disease, 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 the lack of melanin. Therefore, the photo-
chemistry of pterins is of particular interest to this disease.
Moreover, 6-carboxypterin (CPT), a product of photolysis of
BPT (vide infra) that is not synthesized in the skin cells, has
been isolated from the skin of patients suffering vitiligo (10),
thus proving that photo-oxidation of pterins occurs in vivo
under pathological conditions.
It is known since many years ago that photolytic degrada-
tion of BPT under aerobic conditions leads to the formation of
CPT (11,12). In the late seventies, Pfleiderer et al. studied the
photolysis of BPT and NPT in alkaline aqueous solution
(13,14) (phosphate buffer, pH = 10) and demonstrated that
under anaerobic conditions a red compound is formed, which
was proposed to be 6-formyl-5,8-dihydropterin. This com-
pound reacts very fast with oxygen to yield 6-formylpterin
(FPT). In addition, it was demonstrated that photolysis of
BPT and NPT in air-equilibrated solutions leads to FPT as the
first product, which is transformed into CPT on further
photo-oxidation. In recent works, it was proposed that
6-formyl-5,8-dihydropterin is also generated during photolysis
of 6-hydroxymethylpterin (HPT) in the absence of O2 (15,16).
In these studies, H2O2 was detected in irradiated HPT
solutions. This ROS could be generated in the reaction of
6-formyl-5,8-dihydropterin with O2. Finally, photolysis of
BPT has been recently proposed as an additional source for
H2O2 generation in vitiligo (10). The reactions proposed in the
studies performed up to now are summarized in Scheme 1.
New photochemical studies of the reactivity of biopterin (BPT)
and neopterin (NPT) in acidic (pH = 5.5) and alkaline
(pH = 10.5) aqueous solutions at 350 nm and room temperature
were performed. The photochemical properties of BPT are of
particular interest because the photolysis of this compound takes
place in the white skin patches of patients affected by vitiligo.
The photochemical reactions were followed by UV ⁄ VIS spec-
trophotometry, HPLC, electrochemical measurement of dis-
solved O2 and enzymatic methods for hydrogen peroxide (H2O2)
and superoxide anion (O2•)) determinations. When BPT or NPT
are exposed to UVA radiation, a red intermediate, very likely
6-formyl-5,8-dihydropterin, is generated in an O2-independent
process. That product is rapidly oxidized on admission of O2 to
yield 6-formylpterin and H2O2. When the photolysis takes place
in aerobic conditions, no additional pathways exist. On the other
hand, in the absence of O2, the intermediate generated is not
stable and leads to the formation of many products. O2•) is also
generated during photo-oxidation of BPT and NPT. The
quantum yields of reactant consumption depends on the O2
concentration: the higher the O2 concentration, the lower the
quantum yields. This behavior is discussed in connection with the
excited state of the pterins.
INTRODUCTION
From a biological point of view, biopterin (BPT) and
neopterin (NPT) are probably the most important unconju-
gated pterins. 5,6,7,8-Tetrahydrobiopterin acts as a coenzyme
in hydroxylation reactions of the metabolism of some amino
acids (1) and is also relevant in nitric oxide metabolism (2).
Neopterin, a metabolite in the biosynthesis of tetrahydrobio-
pterin, is synthesized mainly in activated macrophages (3). The
determination of the concentrations of this derivative in body
fluids has proven its clinical value; for example, high levels of
NPT are related to activity in cell-mediated immune responses
and are particularly apparent during infections caused by
viruses and intracellular bacteria and parasites (4).
*Corresponding author email: athomas@inifta.unlp.edu.ar (Andres H. Thomas)
´
ꢀ 2008 The Authors. JournalCompilation. The AmericanSocietyofPhotobiology 0031-8655/09
365

366 Mariana Vignoni et al.
Figure 1. Acid–base equilibrium and absorption spectra in
air-equilibrated aqueous solutions; solid line: acid form of BPT
(pH = 5.5); dashed line: basic form of BPT (pH = 10.5). Spectra of
NPT solutions are almost identical to those shown for BPT.
tigations on the photochemistry of pterins, we herein report a
systematic study of the photochemistry of both acid–base
forms of BPT and NPT under UVA irradiation in aqueous
solutions, in the presence and in the absence of O2. In
particular, we have determined the quantum yields, investi-
gated the production of ROS (H2O2 and superoxide anion
[O2•)]), studied the stability of 6-formyl-5,8-dihydropterin
(Scheme 1), and analyzed the kinetics, under different exper-
imental conditions. The results obtained are evaluated in
connection with its biological implications and compared with
those described for the photochemistry of other 6-substituted
pterins.
MATERIALS AND METHODS
Chemicals. Biopterin, neopterin and other pterins were purchased from
Schircks Laboratories (Jona, Switzerland) and used without further
purification. Cytochrome c (Cyt) from horse heart, and SOD from
bovine erythrocytes and other chemicals were obtained from Sigma-
Aldrich (St. Louis, MO) and they were used as received. 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 strength was
approximately 10)3 M in all the experiments.
UV irradiation. The continuous photolysis of BPT and NPT
aqueous solutions was carried out in quartz cells (1 cm path length)
at room temperature. Rayonet RPR lamps emitting at 350 nm
(Southern N.E. Ultraviolet Co., Branford, CT) were employed for
irradiating. Photolysis experiments were performed in the presence
and in the absence of air. Deaerated and O2–equilibrated solutions
were obtained by bubbling with Ar gas and O2 for 20 min,
respectively.
UV ⁄ VIS analysis. Electronic spectra were recorded on a Varian
Cary-3 spectrophotometer or in a Hewlett Packard 8452A Diode
Array Spectrophotometer. Measurements were made using quartz
cells of 0.2 and 1 cm optical path length. The absorption spectra of
the solutions were recorded at regular intervals of irradiation time.
Experimental-difference (ED) spectra were obtained by subtracting
the spectrum at time t = 0 from the subsequent spectra recorded at
different times t. Each ED spectrum was normalized, yielding the
Scheme 1. Photochemistry of BPT, NPT and HPT in aqueous solution.
Pterins behave as weak acids in aqueous solution. In
general, the dominant equilibrium at pH > 5 involves the
lactam group (pyrimidine ring) (17) (Fig. 1). The pKa of this
equilibrium is 8.1 and 8.0 for BPT and NPT, respectively (18).
Other functional groups of the pterin moiety (e.g. the 2-amino
group or ring N-atoms) have pKa values <2 (17). In H2O,
absorption spectra of BPT and NPT strongly depend on the
pH; i.e. spectra of the acid forms are different from those
corresponding to the basic forms (Fig. 1). However, both acid–
base forms present intense absorption bands in the UVA
range. On the other hand, absorption spectra of the acid and
the basic forms of BPT are almost identical to those of the acid
and the basic forms of NPT, respectively.
Although several studies on photochemistry of BPT and
NPT have been performed (vide supra), many interesting
points remain still unknown. In the context of our inves-

Photochemistry and Photobiology, 2009, 85 367
normalized experimental-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.
Superoxide anion assay. Photogenerated superoxide anion radicals
(O2•)) were detected with the aid of the SOD-inhibitable Cyt reduction
(22). Solutions of BPT or NPT were irradiated in the presence of 14 lM
Cyt. The extent of Cyt reduction was determined by monitoring the
increase in the A at 550 nm. The absorption spectra of oxidized and
reduced Cyt are very characteristic, and this spectral change is a
sensitive assay for electron-transfer reactions. This assay has been used
High-performance liquid chromatography.
A high-performance
liquid chromatograph Prominence 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 and photodiode array detector SPD-M20A) was
•)
to detect not only O2 in aerated solutions, but also electron transfer
from excited sensitizers under anaerobic conditions (23).
employed for monitoring the reaction.
A
Pinnacle-II C18
column (250 · 4.6 mm, 5 lm; Restek) was used for product
separation. Solutions containing 4–5% of methanol and 95–96%
of 15 mM TRIS–HCl—1 mM NaCl (pH = 6.8) were used as mobile
phase.
Quantum yield determinations. The quantum yields of reactant
disappearance (FR) and photoproduct formation (FP) were deter-
mined in experiments performed under different conditions. Values
were obtained using the following equations:
RESULTS AND DISCUSSION
Photolysis in the presence of O2
Acidic (pH = 5.5) and alkaline (pH = 10.5) air-equilibrated
aqueous solutions of BPT were irradiated at 350 nm during
different periods of time. Fast changes in the absorption
spectra of the solutions were observed during irradiation
(Fig. 2). No further changes were detected for irradiated
solutions stored in the dark for several hours. NRD spectrum
(see Materials and Methods) was obtained by subtracting the
spectrum of a standard solution of BPT from that of a
standard solution containing FPT. At short irradiation times,
at both pH conditions, the NED spectra were almost equal to
the NRD spectrum (Fig. 2). A similar set of experiments and
spectral analysis performed using NPT solutions, showed
exactly the same results (data not shown). This suggests that
FPT is the main photoproduct of photolysis of both acid–base
forms of BPT and NPT.
The evolution of O2 concentration during irradiation was
monitored with an O2 electrode in a closed cell. In photolysis
of both pterin derivatives and at both pH conditions,
significant decrease in O2 concentration with irradiation time
was observed (Fig. 3). This result confirms that O2 present in
solution was consumed during photolysis.
HPLC analysis revealed the presence of both FPT and CPT
in irradiated solutions of BPT under both pH conditions.
Concentration profiles obtained by HPLC (Fig. 4) showed
that, as expected, CPT is not formed at short irradiation times.
In contrast, CPT appears in irradiated solutions and its
concentration increases after FPT is accumulated. This is in
agreement with the pathways depicted in Scheme 1. Similar
results were obtained in experiments performed irradiating
NPT solutions. In all cases, the sum of the concentrations of
the reactant (BPT or NPT), FPT and CPT remain constant
during the experiments. This indicates that FPT and CPT are
the main photoproducts, with no other substances being
formed in significant amounts.
In accordance with the previous reports (10), H2O2 was
detected in irradiated solutions of BPT and NPT, indicating that
photo-oxidation generates this ROS. In all cases, the H2O2
concentration increased with irradiation time (Fig. 4) and, for
short times (t < 120 s), was almost equal to the FPT concen-
tration. On the other hand, at longer irradiation times, the H2O2
formed is higher than the FPT generated and is even higher than
the consumption ofthe reactant. Thisfact verylikely isdue to the
photo-oxidation of FPT to yield CPT and H2O2 (24).
A similar set of experiments was performed using solutions
saturated with O2. In this case, the behavior was comparable
to that observed in experiments performed under aerobic
conditions: FPT is the main photoproduct, CPT was formed
after FPT was accumulated, and the H2O2 concentration
increased with irradiation time and, for short times, was
ꢀðd½Rꢁ=dtÞ0
UR
¼
ð1Þ
Pa
(d½Pꢁ=dtÞ0
UP
¼
ð2Þ
Pa
where (d[R] ⁄ dt)0 and (d[P] ⁄ dt)0 are the initial rates of reactant
consumption and photoproduct formation, respectively and Pa is the
photon flux absorbed by the reactant. For determining the initial
rates, the experiments were carried out using solutions of relatively
high concentration of a given reactant. Under these conditions the
time evolution of the concentrations of reactants and products
followed a zero-order rate law during the period of time within
which the change of Pa is negligible. In our experiments, this con-
dition was fulfilled at irradiation times lower than 60 and 40 s for
acidic and alkaline media, respectively. The initial rates were
obtained from the slope of the corresponding plots of concentration
vs. irradiation time within such time windows. For calculating the
quantum yields of photoproduct formation the secondary photolysis
was taken into account, and in all cases, in the period of time
where the initial rate of photoproduct formation was determined,
the concentration of secondary photoproducts was negligible
(vide infra).
Aberchrome 540 (Aberchromics Ltd.) was used as an actinometer
for the measurements of the incident photon flux (P0) at the excitation
wavelength. Aberchrome 540 is the anhydride form of the (E)a-(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. The
method for the determination of P0 has been described in detail
elsewhere (19). Values of Pa were calculated from P0 according to the
Lambert–Beer law:
Pa ¼ P0ð1 ꢀ 10ꢀA
Þ
ð3Þ
where A is the absorbance of the reactant at the excitation
wavelength.
The evolution of the concentrations of reactants and photoproducts
during irradiation was followed by HPLC (vide supra). Aqueous
solutions of commercial standards were employed for obtaining the
corresponding calibration curves.
Determination of the concentration of O2. The O2 consumption
during irradiation was measured with an O2-selective electrode (Orion
37-08-99). The solutions and the electrode were placed in a closed
glass-cell of 130 mL.
Detection and quantification of H2O2. For the determination of
H2O2, a Cholesterol Kit (Wiener Laboratorios S.A.I.C.) was used.
H2O2 was quantified after reaction with 4-aminophenazone and
phenol (20,21). Briefly, 400 lL of irradiated solution were added to
1.8 mL of reagent. The A at 505 nm of the resulting mixture was
measured after 30 min at room temperature, using the reagent as a
blank. Aqueous H2O2 solutions prepared from commercial stan-
dards were employed for obtaining the corresponding calibration
curves.

368 Mariana Vignoni et al.
(a)
Figure 3. Time evolution of the O2 concentration in acidic and
alkaline BPT solutions (150 lM). Control experiment: irradiation of
H2O.
(b)
sensitizers upon UVA excitation (25), the participation of this
reactive species must be assessed. Although oxidations of BPT
1
and NPT by O2 have not been studied, reactions with other
oxidized pterin derivatives have been studied in detail (26).
These reactions are very slow and lead to the formation of
nonpterinic compounds. Therefore 1O2 can be discarded as
intermediate in the fast oxidation of the 6-substituents of BPT
and NPT to yield FPT.
Photolysis in the absence of O2
In acidic and alkaline solutions, anaerobic photolysis of BPT
and NPT led to the formation of a compound with a long-
wavelength band and a low molar-absorption coefficient at
480 nm (see Supporting Information). As soon as air was let
into the cell, a fast thermal reaction in the dark was
observed, and the mentioned UV ⁄ VIS absorption band
disappeared (see Supporting Information). This behavior
was similar to those described by Pfleiderer et al. for
alkaline media (13) and, according to these authors, the
Figure 2. Time evolution of the absorption spectra of air-equilibrated
solutions of BPT. (a) [BPT]0 = 150 lM, pH = 5.5, spectra were
recorded at 0, 30, 60, 90, 120, 180 and 300 s. Optical path
length = 10 mm. (b) [BPT]0 = 200 lM, pH = 10.5, spectra were
recorded at 0, 30, 60, 90, 150 s. Optical path length = 2 mm. Arrows
indicate the changes observed at different wavelengths. Insets: NED
spectrum obtained by subtracting the initial BPT spectrum from the
corresponding spectrum of an irradiated solution (t = 120 and 150 s
for pH 5.5 and 10.5, respectively), and NRD spectrum obtained by
subtracting the spectrum of a standard solution of BPT from the
spectrum of a standard solution of FPT, at the same concentration and
pH.
mentioned
intermediate
is
6-formyl-5,8-dihydropterin
(Scheme 1). The NED spectra, obtained by subtracting the
spectra of nonirradiated solutions of the reactants (BPT and
NPT) from those of irradiated and then immediately aerated
solutions, are comparable to the NRD spectrum obtained
from standard solutions of a given reactant and FPT (see
Supporting Information). Therefore, FPT seems to be the
main final product after letting O2 in irradiated free
O2-solutions of BPT and NPT. However, the differences
between NED and NRD spectra, although small, are
significant and suggest the formation of other products at
low concentrations.
HPLC analysis showed that, for both BPT and NPT, FPT is
the main photoproduct of photolysis in the absence of O2,
followed by immediate aeration. CPT is also formed under
these experimental conditions, but in amounts lower than in
photolysis performed in aerated solutions. This is logical
almost equal to the FPT concentration. However, for a given
reactant and a given pH, the rate of the photochemical
oxidation was slower in O2 than in air. This fact will be
analyzed in more detail in the section dealing with quantum
yields (vide infra).
Taking into account that the processes described in this
section are photo-oxidations and pterins are good 1O2

Photochemistry and Photobiology, 2009, 85 369
Figure 4. Time evolution of reactant and photoproducts concentrations in air-equilibrated aqueous solutions under UVA irradiation (350 nm).
(a) [BPT]0 = 150 lM, pH = 5.5; (b) [BPT]0 = 155 lM, pH = 10.5; (c) [NPT]0 = 145 lM, pH = 5.5; (d) [NPT]0 = 150 lM, pH = 10.5. (¤) sum
of the reactant (BPT or NPT), FPT and CPT concentrations in lM, at each time.
considering that CPT is formed mainly from aerobic photo-
lysis of FPT. The corresponding concentration profiles are
shown in Fig. 5. In agreement with spectral analysis, chroma-
tograms showed the presence of small peaks corresponding to
additional products. It is clear that under these experimental
conditions 1O2 cannot participate as oxidizing agent. Very
likely, the mechanism under both aerobic and anaerobic
conditions is the same and the only difference is that in the
former case, the red intermediate is oxidized by O2 immedi-
ately after it is formed.
The determination of H2O2 in solutions of BPT and NPT at
pH 5.5 and 10.5, immediately aerated after different times of
irradiation under anaerobic conditions, revealed that this ROS
is also generated (Fig. 5). H2O2 cannot be produced during
anaerobic photolysis, therefore, these results demonstrate that
H2O2 is formed in the thermal reaction between O2 and the red
intermediate, as suggested in previous studies (10). In all cases,
except for NPT in alkaline media the amount of H2O2
generated was similar to that of reactant consumption, within
experimental error. In experiments performed using NPT at
pH 10.5, the rate of H2O2 formation was lower than the rates
of NPT consumption and FPT formation. The explanation for
this surprising result, confirmed in several experiments,
requires further investigation.
It has been reported that 5,8-dihydropterins are not stable
in anaerobic conditions and tautomerize to 7,8-dihydropterin
derivatives (27). In addition, the photoproduct generated
during the anaerobic irradiation of FPT, which was proposed
to be 6-carboxy-5,8-dihydropterin (28,29), undergoes, in the
absence of O2, a thermal reaction, in which H2O2 is not
generated (24). The corresponding products were not identi-
fied. A set of experiments was carried out in order to check if
the intermediates detected in our reactions followed the same
behavior. For a given reaction, we observed that the typical
band at 480 nm disappeared when the irradiated solutions
were kept in the dark and in anaerobic conditions at room
temperature, indicating that the red intermediate is not stable
in such conditions. That means that the red intermediate not
only reacts with O2 to yield the corresponding oxidized
derivative (FPT) and H2O2, but also undergoes a thermal
O2-independent process. Figure 6 shows the time evolution of
the absorption band centered at 480 nm after interrupting the
irradiation. The kinetic analysis of the decays of the A at
480 nm shows that the reaction follows a first-order law for
both studied compounds and under both pH conditions.
If this thermal reaction takes place in the absence of O2,
H2O2 should not be a product. Therefore, a new series of
experiments was performed to check this issue. Deaerated BPT

370 Mariana Vignoni et al.
Figure 5. Time evolution of reactant and photoproducts concentrations in Ar-equilibrated aqueous solutions under UVA irradiation (350 nm).
(a) [BPT]0 = 150 lM, pH = 5.5; (b) [BPT]0 = 150 lM, pH = 10.5; (c) [NPT]0 = 148 lM, pH = 5.5; (d) [NPT]0 = 148 lM, pH = 10.5. (¤) sum
of the reactant (BPT or NPT), FPT and CPT concentrations in lM, at each time.
Figure 7. Time evolution of the H2O2 concentration in irradiated
solutions of BPT after interrupting the irradiation. BPT solutions
Figure 6. Time evolution of the absorption band corresponding to the
red intermediate after interrupting the irradiation in the absence of O2.
After irradiation, the solutions were kept in the dark and in anaerobic
conditions (steady flow of Ar). Inset: decays of the A at 480 nm.
[BPT] = 150 lM, pH = 10.5.
(150 lM) were irradiated in anaerobic conditions at pH 5.0 and 10.5 for
105 s. After irradiation the solutions were kept in the dark and in the
absence of air (steady flow of Ar). At different times the solutions were
bubbled with air for 2 min and the H2O2 concentration was deter-
mined.

Photochemistry and Photobiology, 2009, 85 371
and NPT (140–200 lM) solutions at pH 5.5 and 10.5 were
irradiated for ca. 100 s. After stopping the irradiation, the
solutions were kept in the dark for different times. During
these periods of time, the solutions were bubbled with Ar to
guarantee anaerobic conditions. Finally, the solutions were
bubbled with air for 2 min and the concentration H2O2 was
determined. In both acidic and alkaline media, the amount of
H2O2 formed decreased as a function of time (Fig. 7). These
results indicate that the thermal O2-independent reaction, as
expected, does not generate H2O2 as a product.
HPLC analysis of O2-free solutions irradiated and kept in
the dark and in anaerobic conditions at room temperature
until the red intermediate is completely consumed revealed
the presence of several products: FPT, CPT, 6-formyl-7,
8-dihydropterin (FDHPT) and other compounds that could
not be identified. FDHPT was quantified and, surprisingly, the
proportion of reactant consumption converted into this com-
pound was very low (<10%) in all cases. This is in disagree-
ment with the hypothesis that 5,8-dihydropterins tautomerize
to the corresponding 7,8-dihydropterin derivatives.
extent of Cyt reduction was higher than in the presence of
air, i.e. the plateau was reached at a higher DA550 value.
Thus, the reduction of Cyt, photosensitized by BPT, can
occur in the absence of O2 by means of a direct electron
transfer. The photoreduction of Cyt in the absence of O2
has been observed before for other heterocyclic compounds
in aqueous solution (23).
Control experiments were performed to investigate thermal
or photochemical processes that might interfere with the assay.
No reduction of Cyt was observed in solutions containing
75 lM of reactant (BPT or NPT) and 14 lM of Cyt at pH 5.5 or
10.5, when kept in the dark for more than 10 min. When
solutions containing only Cyt were exposed to UVA radiation,
no changes were observed in the spectra.
Taking into account the studies reported in the literature on
the photo-oxidation of different pterin derivatives (see Intro-
duction and Scheme 1), in combination with the results
presented in the previous sections, it can be concluded that
the intermediate(s) generated after irradiation of BPT and
NPT is (are) able to transfer one electron either to O2 to yield
O2•), or directly to Cyt. In air-equilibrated solutions, where
there is a large excess of O2 over Cyt, the first process takes
place. However, in the absence of O2, the latter occurs.
Superoxide anion investigation
•)
When air-equilibrated solutions containing 75 lM of BPT and
14 lM of cytochrome c (Cyt) at pH 5.5 or 10.5 were irradiated,
an increase in the A at 550 nm was observed. The difference in
A (DA550) steadily increased with irradiation time, reaching a
plateau after ca. 120 s (Fig. 8). The corresponding ED spectra
in the range 500–600 nm were in good agreement with those
reported in the literature for the reduction of Cyt (30).
Photoreduction of Cyt was inhibited in similar experiments
performed in the presence of SOD (200 U ⁄ mL) (Fig. 8). These
results show that, under the present conditions, O2•), gener-
ated photochemically by BPT, is the actual species that
transfers an electron to Cyt.
Thereby, the observed H2O2 is produced from O2 by the
following reactions:
ꢂ
H
þ þ O2ꢂꢀ ) HO2 pKa ¼ 4:6931
*
ð4Þ
ð5Þ
2HOꢂ2 ꢀ! H2O2 þ O2
Quantum yields
The quantum yields of reactants consumption (FR) and
photoproducts formation (FP) were determined in acidic and
alkaline media (see Materials and Methods). The experiments
were carried out using Ar-equilibrated, air-equilibrated and
O2-equilibrated solutions. In the first case, the irradiated
solutions were bubbled with air immediately after interrupting
the irradiation.
When the same assay was carried out under anaerobic
conditions, photoreduction of Cyt was also observed, even
in the presence of SOD (200 U ⁄ mL) (Fig. 8). Moreover, the
In order to calculate correctly these quantum yields [Eqs (1)
and (2)], two experimental problems must be taken into
account. First, for determining the initial rates ((d[R] ⁄ dt)0 and
(d[P] ⁄ dt)0), the plots of concentration vs. irradiation time must
be linear. Generally, this condition is fulfilled only if the
radiation absorbed by the reactant (Pa) does not decrease
significantly (<15%). Therefore, for each experiment, a period
of time, within which this condition was satisfied, was
considered to calculate (d[R] ⁄ dt)0 and (d[P] ⁄ dt)0. Typically,
we took into account only the first 40 s for acidic media and
60 s for alkaline media. Such a difference is due to FR being
higher under the latter pH condition. The other problem is the
secondary photolysis, i.e. the photochemical oxidation of FPT
into CPT. If this reaction were significant within the analyzed
time window, the value calculated for (d[P] ⁄ dt)0) would be
incorrect because part of the FPT formed would have been
consumed. This issue was controlled and for all experiments
the concentration of CPT, and then the consumption of FPT
by the secondary process, was negligible within the period of
time considered.
Figure 8. Photoreduction of cytochrome c (14 lM) sensitized by BPT
(150 lM) in aqueous solution (pH = 5.5). The experiment was
performed in the presence (air) and in the absence of O2 (Ar), and in
the presence and in the absence of superoxide dismutase (SOD).
The values obtained for BPT and NPT under the different
experimental conditions are listed in Table 1. In all cases, as

372 Mariana Vignoni et al.
Table 1. Quantum yields of reactants (BPT or NPT) consumption (FR) and photoproduct (FPT) formation (FP), and ratios of the initial rate of
H2O2 formation to the initial rate of reactant consumption ((d[H2O2] ⁄ dt)0 ⁄ (d[R] ⁄ dt)0)
pH = 5.5
air
pH = 10.5
air
Ar
O2
Ar
O2
Photolysis of BPT
FR
0.10
0.10
0.01
0.01
0.037
0.037
0.003
0.004
0.024
0.021
0.003
0.003
0.18
0.17
0.02
0.02
0.12
0.12
0.01
0.01
0.07
0.07
0.01
0.01
FP
ðd½H2O2ꢁ=dtÞ0
ðd½Rꢁ=dtÞ0
Photolysis of NPT
0.7
0.1
1.2
0.2
1.3
0.2
1.0
0.1
1.00
0.05
1.2
0.2
FR
0.11
0.10
0.01
0.01
0.044
0.043
0.003
0.004
0.018
0.019
0.002
0.002
0.16
0.16
0.03
0.03
0.11
0.10
0.01
0.01
0.069
0.070
0.005
0.005
FP
ðd½H2O2ꢁ=dtÞ0
ðd½Rꢁ=dtÞ0
0.7
0.1
1.3
0.2
1.4
0.2
0.31
0.05
0.8
0.2
1.0
0.1
Experiments were performed in argon-saturated, air-equilibrated and oxygen-saturated aqueous solutions.
expected, the quantum yields of reactant (BPT or NPT)
consumption were equal, within the experimental error, to the
corresponding quantum yields of FPT formation.
mechanism is valid for both acid–basic forms of BPT and NPT.
The quantification of reactants and photoproducts revealed
that, when the photolysis takes place in aerobic conditions, no
additional pathways exist. On the other hand, in the absence of
O2, the intermediate generated is not stable and leads to the
formation of many products, and, surprisingly, only a little
proportion is converted into the corresponding more stable
7,8-dihydro-tautomer, 6-formyl-7,8-dihydropterin.
Another ROS, superoxide anion (O2•)), is also generated
during photo-oxidation of BPT and NPT, probably as a
consequence of the electron transfer from the red intermediate
to dissolved O2. This fact is very important from a biomedical
point of view because this radical is implicated in the etiology of
many pathological conditions. Moreover, considering that BPT
accumulates in the white skin patches of the patients affected by
vitiligo, results shown in this article suggest that photoinduced
generation of ROS by BPT could contribute to the oxidative
stress involved in the pathogenesis of that disease.
For a given reactant (BPT and NPT) and a given pH
condition, the quantum yields of reactant consumption
depends on the O2 concentration: the higher the O2 concen-
tration, the lower the quantum yields. This behavior can be
explained on the basis of a quenching of excited state of the
reactant, from which the reaction proceeds, by dissolved O2.
Taking into account the results of experiments of quenching of
pterin fluorescence by O2 and previous works, one can propose
that such excited states are triplet states.
Finally, the identity of the species that is formed upon
anaerobic photolysis of BPT and NPT is most consistent with
6-formyl-5,8-dihydropterin. However, because of the rapid
reactivity of this species towards oxygen, its structure has not
been unambiguously characterized. This characterization is of
interest and importance to a complete understanding of the
photochemical properties of pterins and is a topic being
pursued in our laboratories.
For each experiment, the (d[R] ⁄ dt)0 value was compared
with the initial rate of H2O2 production ((d[H2O2] ⁄ dt)0),
calculated within the same time window used for the determi-
nation of (d[R] ⁄ dt)0 and (d[P] ⁄ dt)0. The values of the ratio of
the initial formation of H2O2 to the initial consumption of
reactant ((d[H2O2] ⁄ dt)0 ⁄ (d[R] ⁄ dt)0) are listed in Table 1. In
agreement with concentration profiles (vide supra), except in
the case of the anaerobic photolysis of NPT at pH 10.5, these
values are around 1; i.e. for each molecule of reactant
consumed, one molecule of H2O2 was generated.
For a given reactant and a given pH condition, the quantum
yields of reactant consumption and products formation
depends on the O2 concentration (Table 1): the higher the O2
concentration, the lower the quantum yields. This behavior
suggests that the reactant excited state, from which the
reaction proceeds, is quenched by dissolved O2.
In order to investigate the quenching of singlet excited states
of BPT and NPT by O2, experiments of fluorescence were
performed. Emission spectra of Ar-equilibrated, air-equili-
brated and O2-equilibrated solutions of acidic (pH = 5.5) and
alkaline (pH = 10.5) solutions of BPT and NPT were regis-
tered (data not shown). In contrast to the behavior of the
quantum yield of the photolysis, for a given reactant and a
given pH condition, the emission (integrated fluorescence
intensity) was independent of the O2 concentration. These
results suggest that singlet excited states of the reactants do not
participate in the photochemical process. Therefore, the
participation of triplet states can be proposed. This is
reasonable taking into account that BPT and other pterin
derivatives efficiently produced triplet states (32,33) that are
1
quenched by O2 to generate O2 (25).
CONCLUSIONS
Acknowledgements—The present work was partially supported by
In this work, we have studied several aspects of the photochem-
istry of BPT and NPT in H2O that still remained unclear. When
BPT or NPT are exposed to UVA radiation, a red intermediate,
very likely 6-formyl-5,8-dihydropterin, is generated in an O2-
independent process. That product is rapidly oxidized on
admission of O2 to yield FPT and H2O2 (Scheme 1). This
Consejo Nacional de Investigaciones Cientı
(CONICET-Grant PIP 6301 ⁄ 05), Agencia de Promocio
Tecnologica (ANPCyT Grants PICT 06-12610 and PICT 33919) and
´
ficas
y
Te
´
cnicas
´
n Cientı
´
fica y
´
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.

Photochemistry and Photobiology, 2009, 85 373
(Edited by R. L. Kisliuk and G. M. Brown), pp. 13–18. Else-
vier ⁄ North-Holland, New York.
15. Cabrerizo, F. M., A. H. Thomas, C. Lorente, M. L. Dantola,
´
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Time evolution of the absorption spectra of
irradiated solutions of biopterin. Experiments performed in
the absence of O2.
Figure S2. Spectral analysis of non-irradiated solutions,
solutions irradiated under anaerobic conditions and solutions
irradiated under anaerobic conditions and aerated immedi-
ately after interrupting the irradiation.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
G. Petroselli, R. Erra-Balsells and A. L. Capparelli (2004) Gen-
eration of reactive oxygen species during the photolysis of 6-hy-
droxymethylpterin in alkaline aqueous solutions. Helv. Chim. Acta
87, 349–365.
16. Thomas, A. H., R. Cabrerizo, M. Vignoni, R. Erra-Balsells,
F. M. Cabrerizo and A. L. Capparelli (2004) Photoinduced
generation of reactive oxygen species by the acid form of
6-(hydroxymethyl)-pterin in aqueous solutions. Helv. Chim. Acta
89, 1090–1104.
17. Albert, A. (1953) Quantitative studies of the acidity of
naturally occurring substances for trace metals. Biochem. J. 54,
646–654.
18. Lorente, C., A. L. Capparelli, A. H. Thomas, A. M. Braun and
E. Oliveros (2004) Quenching of the fluorescence of
pterin derivatives by anions. Photochem. Photobiol. Sci. 3, 167–
173.
19. Braun, A. M., M. T. Maurette and E. Oliveros (1991) Photo-
chemical Technology, Chapter 2, (Edited by John Wiley & Sons),
pp. 85–88. John Wiley & Sons, Chichester.
20. Allain, C. C., L. S. Poon, C. S. G. Chan, W. Richmond and
P. C. Fu (1974) Enzymatic determination of total serum choles-
terol. Clin. Chem. 20, 470–475.
REFERENCES
1. Nichol, C. A., G. K. Smith and D. S. Duch (1985) Biosynthesis
and metabolism of tetrahydrobiopterin and molybdopterin. Annu.
Rev. Biochem. 54, 729–764.
2. Hevel, J. M. and M. A. Marletta (1992) Macrophage nitric oxide
synthase: Relationship between enzyme-bound tetrahydrobio-
pterin and synthase activity. Biochemistry 31, 7160–7165.
3. Huber, C., J. R. Batchelor, D. Fuchs, A. Hausen, A. Lang,
D. Niederwieser, G. Reibnegger, P. Swetly, J. Troppmair and
H. Wachter (1984) Immune response-associated production of
neopterin. Release from macrophages primarily under control of
interferon-gamma. J. Exp. Med. 160, 310–316.
4. Fuchs, D., A. Hausen, G. Reibnegger, E. R. Werner, M. P. Dierich
and H. Wachter (1988) Neopterin as marker for activated cell-
mediated immunity. Immunol. Today 9, 150–155.
5. Lorente, C. and A. H. Thomas (2006) Photophysics and photo-
chemistry of pterins in aqueous solution. Acc. Chem. Res. 39, 395–
402.
21. Flegg, H. M. (1973) An investigation of the determination of
serum chloresterol by an enzymatic method. Ann. Clin. Biochem.
10, 79–84.
22. McCord, J. M. and J. Fridovich (1969) The utility of superoxide
dismutase in studying free radical reactions. J. Biol. Chem. 244,
6049–6055.
23. Moore, D. E. and J. Wang (1998) Electron-transfer mechanisms in
photosensitization by the anti-inflammatory drug benzydamine.
J. Photochem. Photobiol. B 43, 175–180.
24. Petroselli, G., J. M. Bartsch and A. H. Thomas (2006)
Photoinduced generation of H2O2 and O2 by 6-formylpterin in
aqueous solutions. Pteridines 17, 82–89.
•)
25. Thomas, A. H., C. Lorente, A. L. Capparelli, C. G. Martınez,
´
A. M. Braun and E. Oliveros (2003) Singlet oxygen (1Dg) pro-
duction by pterin derivatives in aqueous solutions. Photochem.
Photobiol. Sci. 2, 245–250.
6. Ito, K. and S. Kawanishi (1997) Photoinduced hydroxylation of
deoxyguanosine in DNA by pterins: Sequence specificity and
mechanism. Biochemistry 36, 1774–1781.
26. Cabrerizo, F. M., M. L. Dantola, G. Petroselli, A. L. Capparelli,
´
7. Petroselli, G., R. Erra-Balsells, F. M. Cabrerizo, C. Lorente,
A. L. Capparelli, A. M. Braun, E. Oliveros and A. H. Thomas
(2007) Photosensitization of 2¢-deoxyadenosine-5¢-monophos-
phate by pterin. Org. Biomol. Chem. 5, 2792–2799.
A. H. Thomas, A. M. Braun, C. Lorente and E. Oliveros (2007)
Reactivity of conjugated and unconjugated pterins with singlet
oxygen (O2(1Dg)): Physical quenching and chemical reaction.
Photochem. Photobiol. 83, 526–534.
8. Petroselli, G., M. L. Dantola, F. M. Cabrerizo, A. L. Capparelli,
´
27. Pfleiderer, W. (1987) Chemistry and properties of
dihydropterins. In Biochemical and Clinical Aspects of
Pteridines, Vol. 5 (Edited by W. Pfleiderer, H. Wachter and
J. A. Blair), pp. 3–21. Walter de Gruyter & Co., Berlin, New
York.
C. Lorente, E. Oliveros and A. H. Thomas (2008) Oxidation of
2¢-deoxyguanosine 5¢-monophosphate photoinduced by pterin:
Type I versus type II mechanism. J. Am. Chem. Soc. 130, 3001–3011.
9. Schallreuter, K. U., J. Moore, J. M. Wood, W. D. Beazley,
E. M. Peters, L. K. Marles, S. C. Behrens-Williams, R. Dummer,
N. Blau and B. Thony (2001) Epidermal H2O2 accumulation alters
tetrahydrobiopterin (6BH4) recycling in vitiligo: Identification of a
general mechanism in regulation of all 6BH4-dependent processes?
J. Invest. Dermatol. 116, 167–174.
10. Rokos, H., W. D. Beazley and K. U. Schallreuter (2002) Oxidative
stress in vitiligo: Photo-oxidation of pterins produces H2O2 and
pterin-6-carboxylic acid. Biochem. Biophys. Res. Commun. 292,
805–811.
28. Thomas, A. H., G. Suarez, F. M. Cabrerizo, R. Martino and
´
A. L. Capparelli (2000) Study of the photolysis of folic acid and
6-formylpterin in acid aqueous solutions. J. Photochem. Photobiol.
A 135, 147–154.
29. Thomas, A. H., G. Suarez, F. M. Cabrerizo and A. L. Capparelli
´
(2001) Photochemistry of 6-formylpterin in alkaline medium.
Helv. Chim. Acta 84, 3849–3860.
30. Kuciel, R. and A. Mazurkiewicz (2004) Formation and detoxifi-
cation of reactive oxygen species. Biochem. Mol. Biol. Educ. 32,
183–186.
11. Forrest, H. S. and H. K. Mitchell (1955) Pteridines from Dro-
sophila. III. Isolation and identification of three more pteridines.
J. Am. Chem. Soc. 77, 4865–4869.
31. Pauling, L. (1979) The discovery of superoxide radical. Trends
Biochem. Sci. 4, N270–N271.
12. Viscontini, M. and H. Raschig (1958) Fluoreszierende Stoffe aus
Drosophila melanogaster. 7th Mitteilung. Helv. Chim. Acta 41,
108–113.
13. Mengel, R., W. Pfleiderer and W. R. Knappe (1977) Norrish type
II analogous photodegradation of neopterin and biopterin to
5,8-dihydropterin-6-aldehyde. Tetrahedron Lett. 32, 2817–2820.
14. Baur, R., M. Kappel, R. Mengel and W. Pfleiderer (1979) Pho-
tochemistry of pteridines. In Chemistry and Biology of Pteridines
32. Chahidi, C., M. Aubailly, A. Momzikoff, M. Bazin and R. Santus
(1981) Photophysical and photosensitizing properties of 2-amino-
4-pteridinone: A natural pigment. Photochem. Photobiol. 33, 641–
649.
33. Ledbetter, J. W., W. Pfleiderer and J. H. Freisheim (1995)
Photosensitized reduction of L-biopterin in the active ternary
complex of dihydrofolate reductase. Photochem. Photobiol. 62,
71–81.