Generation of Reactive Oxygen Species during the Photolysis of 6-(Hydroxymethyl)pterin in Alkaline Aqueous Solutions

Article abstract of DOI:10.1002/hlca.200490032

Photochemical studies of the reactivity of 6-(hydroxymethyl)pterin (=2-amino-6-(hydroxymethyl)pteridin-4(1H)-one; HPT) in alkaline aqueous solutions (pH 10.2-10.8) at 350 nm and room temperature were performed. The photochemical reactions were followed by UV/VIS spectrophotometry, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and an enzymatic method for H₂O₂ determination. In the presence of O₂, 6-formylpterin (=2-amino-3,4-dihydro-4-oxopteridine-6-carboxaldehyde; FPT) was the only photoproduct detected. In the absence of O₂, we observed a compound with an absorbance maximum at 480 nm, which was oxidized very rapidly by O ₂ in a dark reaction to yield FPT. The quantum yields of substrates disappearance and of photoproducts formation were determined. The formation of H₂O₂ during photooxidation was monitored, and the number of mol of H₂O₂ released per mol of HPT consumed corresponded to a 1: 1 stoichiometry. HPT was also investigated for efficiency of singlet-oxygen (¹O₂) production and quenching in aqueous solution. The quantum yield of ¹O₂ production (Φ_Δ = 0.21 ± 0.01) was determined by measurements of the ¹O₂ luminescence in the near-IR (1270 nm) upon continuous excitation of the sensitizer. The rate constant of ¹O ₂ total quenching by HPT was determined (k_t = 3.1 · 10⁶ M^-1 s^-1), indicating that this compound was able to quench ¹O₂. However, ¹O₂ did not participate in the photooxidation of HPT to FPT.

Full text of DOI:10.1002/hlca.200490032

Helvetica Chimica Acta Vol. 87 (2004)
349
Generation of Reactive Oxygen Species during the Photolysis of
6-(Hydroxymethyl)pterin in Alkaline Aqueous Solutions
by Franco M. Cabrerizoa), Andre¬s H. Thomas*a), Carolina Lorenteb), Marıa L. Da¬ntolaa),
¬
Gabriela Petrosellia), Rosa Erra-Balsellsb), and Alberto L. Capparelli*a)
a
¬
) Instituto de Investigaciones FisicoquÌmicas Teoricas y Aplicadas (INIFTA), Departamento de QuÌmica,
Facultad de Ciencias Exactas, Universidad Nacional de La Plata, CONICET, CIC, Casilla de Correo 16,
Sucursal 4, (1900) La Plata, Argentina
(Tel/fax: 0054-221-424-2333; e-mail: alcappa@dalton.quimica.unlp.edu.ar / athomas@inifta.unlp.edu.ar)
b) CHIDECAR CONICET, Departamento de QuÌmica Organica, Facultad de Ciencias Exactas y Naturales,
¬
¬
Universidad de Buenos Aires, Pabellon 2, 3p, Ciudad Universitaria, 1428 Buenos Aires, Argentina
Photochemical studies of the reactivity of 6-(hydroxymethyl)pterin (ˆ2-amino-6-(hydroxymethyl)pter-
idin-4(1H)-one; HPT) in alkaline aqueous solutions (pH 10.2 10.8) at 350 nm and room temperature were
performed. The photochemical reactions were followed by UV/VIS spectrophotometry, thin-layer chromatog-
raphy (TLC), high-performance liquid chromatography (HPLC), and an enzymatic method for H2O2
determination. In the presence of O2, 6-formylpterin (ˆ2-amino-3,4-dihydro-4-oxopteridine-6-carboxaldehyde;
FPT) was the only photoproduct detected. In the absence of O2, we observed a compound with an absorbance
maximum at 480 nm, which was oxidized very rapidly by O2 in a dark reaction to yield FPT. The quantum yields
of substrates disappearance and of photoproducts formation were determined. The formation of H2O2 during
photooxidation was monitored, and the number of mol of H2O2 released per mol of HPT consumed
corresponded to a 1 :1 stoichiometry. HPTwas also investigated for efficiency of singlet-oxygen (1O2) production
1
and quenching in aqueous solution. The quantum yield of O2 production (FD ˆ 0.21 Æ 0.01) was determined by
1
measurements of the O2 luminescence in the near-IR (1270 nm) upon continuous excitation of the sensitizer.
The rate constant of 1O2 total quenching by HPT was determined (kt ˆ 3.1 ¥ 106 mÀ1 sÀ1), indicating that this
compound was able to quench 1O2. However, 1O2 did not participate in the photooxidation of HPT to FPT.
Introduction. Pterins (ˆ2-aminopteridin-4(1H)-ones) are a family of heterocyclic
compounds [1][2] present in a wide variety of biological systems. Although they are
present in small amounts in living systems, it is well-known that pterins participate in
important biological functions [1 3]. The light sensitivity of pterins has been known
since several decades [4][5], and interest in photochemistry and photophysics of this
group of compounds has been shown since participation of pterins in different
photobiological processes has been established or suggested. A derivative of folic acid,
a conjugated pterin, is the light-harvesting chromophore of DNA photolyases [6][7],
enzymes involved in DNA repair processes that take place after UV irradiation. It has
been suggested that pterins may play some role in photosynthesis [8], and they have
been found in photosensitive organs such as the eyes of different animals [9]. Pterins
are also interesting as possible photosensitizers in photochemical processes that take
place in organisms exposed to UV-A radiation; e.g., pterins act as sensitizers in
photochemical reactions that induce DNA damage [10][11] and are able to generate
singlet oxygen (1O2) [12][13]. Some biomedical studies suggest that pterins are
involved in pathogenesis of vitiligo, a depigmentation disorder [14][15]. Moreover, the

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Helvetica Chimica Acta Vol. 87 (2004)
accumulation of 6- and 7-biopterin in the epidermis of the affected patients leads to a
typical fluorescence under Wood×s light (351 nm) [14].
Pterins behave as weak acids in aqueous solution. As reported by Albert [16] for
several pterin derivatives, the dominant equilibrium at pH higher than 5 involves an
amide group (acid form) and a phenolate group (basic form). The pKa of this
equilibrium is ca. 8 for the different pterin derivatives studied [16 19]. Other
functional groups of the pterin moiety (e.g., the 2-amino group or ring N-atoms) have
pKa values lower than 2 [16].
The 6-substituted pterins, the most-common pterin derivatives found in nature,
undergo photooxidation in air-equilibrated aqueous solutions [19 25]. Thus, it has
been reported that the mechanism involved in the photochemical transformation of
these compounds is considerably affected by the nature of the substituent at position 6
of the pterin moiety, and that the photochemistry of the acidic and basic forms of the
pterin derivatives are different. Our previous studies suggested that 1O2 participates in
photooxidation of folic acid [19][20], whereas, in the mechanism involved in the
photooxidations of biopterin and neopterin studied by Pfleiderer and co-workers
[24][25], 1O2 does not participate. On the contrary, an intermediate generated in an O2-
independent process (Scheme 1) is thermally oxidized on introduction of O2. The
mechanism of the photooxidation of 6-formylpterin (ˆ2-amino-3,4-dihydro-4-oxo-
pteridine-6-carboxaldehyde; FPT) to yield 6-carboxypterin (ˆ2-amino-3,4-dihydro-4-
oxopteridine-6-carboxylic acid; CPT) [19][21] (Scheme 1) is similar to that described
for biopterin and neopterin. Finally, photochemical decomposition of CPT in the
presence of O2 [22] occurs by two main reaction pathways: decarboxylation to yield
pterin (this photochemical process is O2-independent and occurs only for the acid
form), and photooxidation of the pterin moiety to yield non-pterinic photoproducts.
Scheme 1. Photooxidation of Biopterin, Neopterin [24][25], and 6-Formylpterin [19][20]
In the present paper, we describe the photochemistry of 6-(hydroxymethyl)pterin
(ˆ2-amino-6-(hydroxymethyl)pteridin-4-(1H)-one; HPT) in alkaline aqueous solu-
tion where only the basic form of the molecule is present. Photoproducts were
identified. The quantum yield of HPT disappearance and that of the formation of

Helvetica Chimica Acta Vol. 87 (2004)
351
photoproducts were determined. In addition, the role of O2 in the mechanisms of these
photochemical reactions was analyzed, and the capability of HPT to yield activated
oxygen species is discussed. In particular, we investigated and quantified the efficiency
of 1O2 production (FD) and quenching by HPT. We also studied the production of H2O2
during the photooxidation of HPT. The results obtained in this study were compared
with those described for the photochemistry of other pterin derivatives.
Experimental. 1. Chemicals. The 6-(hydroxymethyl)pterin (HPT), 6-formylpterin (FPT), and other
pterins were purchased from Schircks Laboratories, Switzerland, and used without further purification. Propan-
2-ol and EtOH were purchased from Merck and other chemicals from Sigma-Aldrich and used as received. The
pH of the aq. solns. was adjusted by adding drops of NaOH solns. from a micropipette. The concentration of the
base used for this purpose ranged from 0.1 to 2m. The ionic strength was ca. 10À3 m in all the experiments. In
singlet oxygen (1O2) experiments, D2O (Euriso-top, Groupe CEA, Saclay, France; minimum isotopic purity of
99.9%), DCl (Aldrich, 99.5% D) soln. in D2O and NaOD (CEA) soln. in D2O were employed.
2. Determination of pKa. pKa Values were determined from absorption changes. Measurements were
performed at r.t. The experimental absorption changes at a given wavelength can be fitted by Eqn. 1, where ea
and eb are the molar absorption coefficients of the acid and basic forms of the species involved in the acid-base
equilibrium, c is the total concentration of the substance, l is the optical length, and Ka is the dissociation
constant. A more-detailed description of pKa determinations has been described elsewhere [17][18].
‡
A ˆ (c ¥ l) ¥ {ea ‡ (eb À ea) ¥ [Ka/(Ka ‡ [H ])]}
(1)
3. Continuous photolysis. 3.1. UV Irradiation. The continuous photolysis of HPT solns. were carried out in
quartz cells (1-cm path length) at r.t. Rayonet-RPR lamps emitting at 350 nm (Southern N.E. Ultraviolet Co.)
were employed for irradiating. Photolysis experiments were performed in the presence and in the absence of air.
Deaerated solns. were obtained by bubbling with Ar for 20 min.
3.2. UV/VIS Spectrophotometric Analysis. A Cary-3 spectrophotometer (Varian) and a program for
smoothing and averaging the signals of the spectra were used. The irradiations were performed in quartz cells of
1-cm optical-path length, and the absorbance measurements were made in quartz cells of 1-cm and 0.2-cm
optical-path length. Absorption spectra of the solns. were recorded at regular intervals of irradiation time.
Experimental difference (ED) spectra were obtained by subtracting the spectrum at time t ˆ 0 from the
subsequent spectra recorded at different times. Each ED spectrum was normalized relative to the maximum
absolute value of the absorbance difference yielding the normalized experimental difference (NED) spectrum.
Reference difference (RD) spectra and normalized reference difference (NRD) spectra were obtained from aq.
solns. of commercial standards. The comparison between NED and NRD spectra allows characterization of the
major photolysis products. The analysis based on these difference spectra is described elsewhere [19][22].
4. Thin-Layer Chromatography (TLC). DEAE-cellulose plates (100 mm) and 0.3% (w/v) aq. NH4Cl soln.
as eluent were used. Irradiated HPT solns. (1.5 ¥ 10À4 m) and aq. solns. of pterin-derivatives standards were
developed by exposing them to 350-nm radiation. Under these conditions, pterin derivatives show blue
fluorescent spots. The corresponding Rf values obtained for pterin and several pterin derivatives have already
been reported [19].
5. High-Performance Liquid Chromatography (HPLC). A Konic Instruments 500-B HPLCwith a C18-
Spherisorb-S5-ODS2 (250 Â 46 mm) column and a Lambda-1000 Bischoff HPLCspectrophotometer as detector
was employed for the reaction monitoring, the identification of photoproducts, and for the quantification of
HPT and photoproducts. A 5% MeCN/H2O 60 :40, 95% aq. soln. of 20 mm potassium phosphate (pH 5.5), and
2.5 mm EDTA were used as eluent. For irradiated HPT solns., HPLCruns were monitored at 340 nm.

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6. Quantum-Yield Determinations. The quantum yields of HPT disappearance and FPT formation were
determined in experiments performed under different conditions. Aberchrome 540 was the actinometer
employed for the measurements of photon rates [26] ((7.2 Æ 0.3) ¥ 10À4 Einstein ¥ lÀ1 ¥ minÀ1). The initial
concentration of the reactant was adjusted so that more than 99.99% of the incident radiation at 350 nm was
absorbed. The evolution of the concentrations of reactants and photoproducts during the irradiation time was
followed by HPLC. Aq. solns. of commercial standards were employed for obtaining the corresponding
calibration curves.
7. Determination of O2 Concentration. The O2 consumption during the photolysis was measured with an O2
selective electrode (Orion, model 37-08-99). The experimental set-up for these measurements was described
elsewhere [27].
8. H2O2 Determination. For determination of H2O2 the cholesterol kit from Wiener was used. H2O2 was
quantified by its color reaction with 4-aminophenazone and phenol [28][29]. The irradiated HPT soln. (400 ml)
was added to 2 ml of reagent. The absorbance at 505 nm of the resulting soln. was measured, after 30 min of
incubation at r.t., by using the reagent as a blank. Aq. solns. of H2O2, prepared from commercial standards, were
employed for obtaining the corresponding calibration curves.
9. Hydroxy-Radical Quenching. HPT Solns. (350 mm) were irradiated in the presence of propan-2-ol or
EtOH. The concentrations of the alcohols ranged from 3.5 to 350 mm. These compounds have a high reactivity
.
with hydroxy radicals (HO ) [30] diminishing its concentration during the experiment. Results of UV/VIS
.
spectrophotometric analysis were compared with those obtained in the absence of the selective HO scavenger.
1
10. Singlet Oxygen (1O2) Studies. 10.1. Determination of Quantum Yields of O2 Production. Quantum yield
1
of 1O2 production (FD) was determined by direct analysis of the weak O2 near-IR luminescence at 1270 nm
(see, e.g. [31 33]), produced during continuous irradiation of the aq. solns. of HPT. The main features of the
method and the equipment have already been described in detail [34 36]. In the present experiments, a cooled
NIR photomultiplier (Hamamatsu) as detector, instead of a Ge photodiode, was used. Briefly, the sample soln.
in a quartz cuvette was irradiated with a Xe/Hg arc through a H2O filter, focusing optics, and a monochromator.
The 1O2 luminescence was collected with a mirror, chopped, and, after passing through focusing lens, a cut-off
filter (1000 nm), and an interference filter (1271 nm), was detected at 908 with respect to the incident beam by
using the cooled NIR photomultiplier. For determining FD, the luminescence signals of a sensitizer soln. (SSe )
and a soln. of a reference sensitizer (SRe ) were measured. If the absorbances at the wavelength of irradiation are
the same for the reference sensitizer and the sample solution, the ratio of the luminescence signals, SSe/SeR is given
by Eqn. 2, where PS0/P0R is the ratio of the incident photon rates at the wavelength of excitation of the sensitizer
investigated and of the reference. If kd (the nonradiative-deactivation rate constant) and kSt (the rate constant of
total (physical and chemical) 1O2 quenching by the sensitizer itself) are known in the solvent used, FD may be
calculated from Eqn. 2 by measuring the luminescence signals of the sensitizer and the reference solns., as well
as the corresponding incident photon rates. Because of the short lifetime of 1O2 (tD ˆ 1/kd) in H2O (3.8 ms), D2O
was used as solvent in all experiments where tD is much longer (62 ms) [37][38]. Measurements with HPTwere
carried out at an excitation wavelength (lex) of 367 nm. Rose bengal (RB) in D2O (lex 547 nm, FRD ˆ 0.75
[39][40]) was used as a reference sensitizer.
SeS
PS0 FD
kd
ˆ
ꢀ2†
SRe P0R FRD kd ‡ kS‰SensŠ
t
1
1
10.2. Determination of Rate Constants of Total O2 Quenching by Pterins. The rate constants of total O2
quenching (kt) by HPTwas determined by SternÀVolmer analysis of the 1O2-luminescence quenching. The main
features of the method was described elsewhere [13][41]. 1O2 was generated by photosensitization with RB as
sensitizer. Groups of experiments were carried out in which solns. of HPT and RB were irradiated at 547 nm,
where the investigated compound does not absorb. The RB concentration was kept constant, whereas the
pterin-derivative concentration was varied within a series of experiments. Under our experimental conditions
[13], a linear relationship between the ratio of the signals observed in the absence (S0e ) and in the presence (Se)
of quencher (Q ˆ HPT) and the quencher concentration is observed (Eqn. 3), where tD is the 1O2 lifetime in the
solvent used (D2O) in the absence of Q. Therefore, knowing tD, kt can be calculated from the slope of the
SternÀVolmer plot.
S0e/Se ˆ 1 ‡ kttD[Q]
(3)

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11. Continuous Photolysis in D2O. Solns. of HPT were prepared in H2O and D2O. Couples of both types of
solns. containing HPT at the same concentration were irradiated under identical experimental conditions. The
effect of D2O was evaluated by comparing results of UV/VIS spectrophotometric analysis.
12. Calculation of Molecular Properties. The ground-state geometry, heat of formation, and HOMO and
LUMO energies of HPT and 6-formyl-5,8-dihydropterin were calculated by using the semiempirical para-
metrized PM3 method as implemented in the 2002 version of the HyperChem program [42], which has been
shown to be effective in studies of molecules containing heteroatoms, compared with other methods such as
MINDO/3 or MNDO.
Results and Discussion. Determination of pKa of 6-(Hydroxymethyl)pterin. To the
best of our knowledge, the pKa value of the amide group in 6-(hydroxymethyl)pterin
(HPT) has not been reported in the literature. We determined this pKa, and a value of
8.1 Æ 0.1 was found. This value is quite similar to those previously determined for other
related pterin derivatives (pKa(pterin) ˆ 7.9 [17], pKa(6-formylpterin) ˆ 7.3 [19], and
pKa(6-carboxypterin) ˆ 7.9 [17]). Taking into account the pKa of HPT, we chose to use
HPT solutions with pH > 10, so that only the basic form of HPTwas present in the stock
solutions.
Photolysis in the Presence of Oxygen. The spectrum of air-equilibrated solutions of
HPT changes significantly with irradiation time, as shown in Fig. 1,a. An isosbestic
point at 268 nm is observed during at least 12 min of irradiation. No further changes are
detected in irradiated solutions that were stored in the dark. ED and NED spectra were
obtained as described in the experimental section. The NED spectrum is similar to the
NRD spectrum, obtained from standard solutions of HPT and 6-formylpterin
(Fig. 1,b). This result suggests that under these experimental conditions, a new
absorbing species with UV-absorption spectrum similar to that of FPT is formed. At
irradiation times higher than 15 min, the NED spectrum changes and the isosbestic
point at 268 nm disappears.
TLCAnalysis shows the presence of three fluorescent substances in irradiated HPT
solutions (t ˆ 0, 10, 30, and 55 min). The obtained Rf values correspond to HPT, FPT,
and 6-carboxypterin (CPT). Experiments performed at different irradiation times
reveal that CPT appears at a later stage than FPT. This is in good agreement with
previous results reported on photochemistry of FPT in alkaline aqueous solutions [21],
i.e., in the presence of O2, FPT undergoes photooxidation to yield CPT.
In a similar way, the presence of FPT and CPT in irradiated solutions of HPT is
confirmed by HPLCexperiments. The HPT, FPT, and CPT concentrations were
measured as a function of irradiation time by means of two sets of HPLCexperiments.
In the first group of experiments, concentrated solutions of HPT (350 mm) were
irradiated. In the second group, irradiations were performed with solutions containing a
mixture of HPT and FPT.
The first group of experiments shows that during the first 5 min of irradiation, the
evolutions of the concentrations of HPT and FPT follow a zero-order rate law (Fig. 2).
The rate of degradation of HPT is 13.7 Æ 0.8 mm/min, whereas the rate of formation of
FPT is 12.9 Æ 0.9 mm/min. In this first stage, no significant increase in CPT concentration
is observed. Thus, HPT seems to absorb the whole incident radiation in this time range.
The quantum yield associated with the disappearance of HPT is 1.8 ¥ 10À2. As a value of
1.6 ¥ 10À2 is obtained for the quantum yield of FPT formation, the consumption of HPT
to yield FPT would be the only process that takes place in the analyzed time range.

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Fig. 1. a) Evolution of the absorption spectra of irradiated solutions of 6-(hydroxymethyl)pterin (150 mm at
pH 10.5) as a function of time (experiment performed in the presence of O2, spectra recorded every 2 min;
arrows indicate the changes observed at different wavelengths). b) NED Spectrum (––) obtained by
subtracting the initial 6-(hydroxymethyl)pterin spectrum from the corresponding spectrum after 14 min of
photolysis; NRD spectrum
(
) obtained by subtracting the spectrum of a standard solution of 6-
(hydroxymethyl)pterin from the spectrum of a standard solution of 6-formylpterin, at the same concentration
and pH.
After 5 min of irradiation, important increases in the rate of disappearance of HPT
and in the rate of formation of FPT are observed (see Fig. 3). These changes coincide
temporally with an increase of CPT concentration. As mentioned above, CPT is a
photoproduct generated from FPT [21]. Therefore, after ca. 5 min, some significant
proportion of radiation is absorbed by FPT. Thus, the chemical transformation from
HPT to FPT seems to be enhanced through the participation of excited FPT. The
formation of an FPT HPT exciplex could be the cause of the enhancement observed.
As can be seen in Fig. 3, during the first 20 min of irradiation, the total concentration of
pterins (sum of HPT, FPT, and CPT concentrations) is almost constant, indicating that
FPT and CPT are the main photoproducts.

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Fig. 2. Evolution of 6-(hydroxymethyl)pterin and 6-formylpterin concentrations in an irradiated solution of 6-
(hydroxymethyl)pterin (350 mm at pH 10.5) as a function of irradiation time. HPLCAnalysis during the first
4 min of photolysis.
In the second group of experiments, HPT absorbs a low proportion of the initial
incident radiation, whereas a higher proportion is absorbed by FPT. As shown in
Fig. 4,a solutions prepared as a mixture of 47 mm HPT and 315 mm FPT solutions were
irradiated under air or He-atmosphere. Taking into account the quantum yield
previously calculated (1.8 ¥ 10À2) and that the rate of photons absorbed by HPT is 8% of
the incident photon rate, a theoretical rate of consumption of HPT in the irradiated
mixture can be estimated (À1.2 mm/min). However, an experimental rate of À 5.3 Æ
0.3 mm/min is obtained for the first 4 min of photolysis of the mixture in the presence of
O2. These results suggest that the absorption of radiation by FPT induces the
conversion of HPT. As shown in Fig. 4,b similar results are obtained with solutions
prepared as a mixture of 200 mm HPT and 310 mm FPT solutions.
The evolution of the O2 concentration during irradiation was monitored with an O2
electrode in a closed cell. As shown in Fig. 5, O2 concentration decreases with the
irradiation time. Moreover, after a few minutes of irradiation, an increase on the rate of
consumption of O2 is observed. The degradation of HPT induced by FPT and the
photooxidation of FPT itself may explain this observation.
H2O2 was detected in irradiated solutions of HPT, indicating that photooxidation of
HPT generates this reactive oxygen species. Concentration of H2O2 was determined at
different times during the irradiation of a HPT solution (360 mm, pH 10.2) (Fig. 6). The
estimated rate of production of H2O2 is 14 Æ 2 mm/min in the first 4 min of irradiation.
The quantum yield of H2O2 formation is calculated from that rate value and taking into

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Fig. 3. Evolution of 6-(hydroxymethyl)pterin, 6-formylpterin, and 6-carboxypterin concentrations in an
irradiated solution of 6-(hydroxymethyl)pterin (350 mm at pH 10.5) as a function of time. HPLCAnalysis
*
during 20 min of photolysis; ( ) sum of the 6-(hydroxymethyl)pterin, 6-formylpterin, and 6-carboxypterin
concentrations in mm, at each time.
account the incident photon rate (see details in the Exper. Part). Thus a value of 1.7 ¥
10À2 is obtained. The comparison of the quantum yield of H2O2 formation and the
quantum yields of HPT consumption and FPT formation suggest that for each molecule
of HPT consumed, one molecule of H2O2 is generated.
Generation of H2O2 during the photooxidation of other pterin derivatives
(biopterin, sepiapterin, and FPT) has previously been reported [15]. Generation of
H2O2 in photolysis of pterins is important from a biomedical point of view and is
particularly relevant for skin diseases. Thus, H2O2 participates in the pathogenesis of
vitiligo [43], and the accumulation of pterins in the skin of patients that suffer that
disease has been established [15][44].
.
To investigate whether the hydroxyl radical (HO ) is involved in the mechanism of
photooxidation of HPT, photolysis in the presence of propan-2-ol or EtOH, selective
.
HO scavengers [30], were carried out. HPT Solutions (350 mm, pH 10.5) were
irradiated in the presence of different concentrations of alcohol (3.5 350 mm).
Absorption spectra of the solutions were recorded at regular intervals of irradiation
time during 15 min, and ED and NED spectra were obtained. These results were
.
compared with those obtained in the absence of the selective HO scavenger, and no
.
significant differences are detected, thus suggesting that OH does not participate in the
photochemical processes studied.

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Fig. 4. Evolution of 6-(hydroxymethyl)pterin concentration in irradiated solutions containing a mixture of 6-
(hydroxymethyl)pterin and 6-formylpterin as a function of time: a) 47 mm 6-(hydroxymethyl)pterin and 315 mm 6-
formylpterin (pH 10.5); b) 200 mm 6-(hydroxymethyl)pterin and 310 mm 6-formylpterin (pH 10.5). HPLC
Analysis; the experiments were performed in the presence and in the absence of O2;
ˆ theoretical rate of
consumption of 6-(hydroxymethyl)pterin, estimated taking into account the intensity of the incident photon
rate absorbed by 6-(hydroxymethyl)pterin and the quantum yield of consumption calculated in the absence of
6-formylpterin.
Fig. 5. Evolution of the O2 concentration in an irradiated solution of 6-(hydroxymethyl)pterin (150 mm at
pH 10.2) as a function of irradiation time

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Fig. 6. Evolution of the H2O2 concentration in an irradiated solution of 6-(hydroxymethyl)pterin (360 mm at
pH 10.2) as a function of irradiation time
Moreover, the concentration of H2O2 was determined at different times during the
irradiation of a HPT solution (350 mm, pH 10.2) in the presence of propan-2-ol
(350 mm). The rate of H2O2 production is 11 Æ 2 mm/min in the first 4 min of irradiation.
This rate value is similar, within the experimental error, to that obtained under the
.
same experimental conditions but in the absence of the HO scavenger (14 Æ 2 mm/
.
min). These results indicate that HO is not involved in the oxidative process that yields
H2O2.
Photolysis in the Absence of Oxygen. To elucidate the role of O2 in the
photochemistry of HPT, solutions previously bubbled with Ar were irradiated. Under
these conditions, visible changes in the color of the solutions were observed. As shown
in Fig. 7, anaerobic photolysis of HPT leads to the formation of a compound with a
longwave absorbing band of low molar absorption coefficient at 480 nm. This
compound will be named throughout as −red intermediate× (see its possible structure
in Scheme 2, below). Commercially available pterin-derivative standards do not
present such spectrophotometrical characteristics and, therefore, the NED spectrum of
this photolysis does not agree with the NRD spectra obtained from commercial
standard solutions.
The −red intermediate× obtained in O2-free solutions suffers a thermal reaction in
the dark as soon as air or O2 is admitted into the cell. The band around 480 nm

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359
Fig. 7. Evolution of the absorption spectra of irradiated solutions of 6-(hydroxymethyl)pterin (150 mm at
pH 10.1) as a function of time. Experiment performed in the absence of O2; spectra were recorded every 10 min,
arrows indicate the changes observed at different wavelengths.
Scheme 2. Photooxidation of 6-(Hydroxymethyl)pterin in Alkaline Aqueous Solutions (pH 10.0 10.5) upon
Irradiation with UV-A Radiation (l 350 nm)
disappears. The NED spectrum, obtained by subtracting the spectrum of a non-
irradiated solution of HPT from that of an irradiated and then immediately aerated
solution, matches with the NRD spectrum obtained from standard solutions of HPT
and FPT. This fact suggests that an FPT-like chromophore is the final oxidized stable
structure of the −red intermediate× (Scheme 2).

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Helvetica Chimica Acta Vol. 87 (2004)
TLCAnalysis of the irradiated HPT solutions under anaerobic conditions and then
aerated show a fluorescent spot having an Rf identical to that of FPT. The intensity of
the spot of HPT decreases with the irradiation time. No other product could be
detected by this procedure.
The results obtained by HPLCanalysis are in good agreement with those obtained
by TLC, i.e., FPT is the only photoproduct found in HPT solutions irradiated in the
absence of O2 and then immediately aerated. In the first 4 min of irradiation the rate of
consumption of HPT is 13.6 Æ 1.5 mm/min (quantum yield of 1.8 ¥ 10À2), whereas the rate
of formation of FPT is 12.5 Æ 1.2 mm/min (quantum yield of 1.8 ¥ 10À2). The quantum
yield obtained under these experimental conditions is equal to that measured in the
presence of O2 (1.8 ¥ 10À2). Fig. 8 shows that no increase in the rate of photo-
disappearance of HPTand of the rate of formation of FPT is observed during 20 min of
photolysis, confirming that the acceleration of the photolysis observed in the presence
of O2 is not due to the accumulation of the −red intermediate×. On the contrary,
accumulation of some reactive intermediate formed during the photolysis of FPT could
be the cause of the observed acceleration.
Fig. 8. Evolution of 6-(hydroxymethyl)pterin and 6-formylpterin concentrations in an irradiated solution of 6-
(hydroxymethyl)pterin (350 mm at pH 10.5) as a function of time. Experiment performed in the absence of O2;
&
HPLCanalysis during 20 min of photolysis;
ˆ sum of 6-(hydroxymethyl)pterin and 6-formylpterin
concentrations in mm, at each irradiation time.
Measurements of H2O2 in solutions of HPT (360 mm, pH 10.2) immediately aerated
after different times of irradiation in anaerobic conditions reveal that H2O2 is generated

Helvetica Chimica Acta Vol. 87 (2004)
361
under these experimental conditions. The value obtained for the rate of H2O2
production is 15 Æ 1 mm/min for the first 3 min of irradiation. This result suggests that
H2O2 is a product of the thermal reaction between the −red intermediate× and O2.
The −red intermediate×, detected in the absence of O2, seems to participate in an
active way in the oxidation of HPT to FPT. The similar quantum yields measured in the
presence and the absence of O2 support this hypothesis. Under air, the −red
intermediate× oxidizes immediately to FPT, explaining why the −red intermediate×
can be detected only under anaerobic conditions. If this is the reaction pathway, 1O2 is
not necessary in the later step of the formation of FPT from HPT, because the yield of
FPT from the −red intermediate× takes place in the absence of UV radiation. The role of
1O2 will be analyzed in detail in the next section.
The mechanism involved in the photolysis of HPT seems to be similar to that found
in photooxidation of biopterin, neopterin [24][25] and FPT [19][21] (see Introduction).
It has been proposed that the intermediates detected in these photooxidations, which
have spectral features and reactivities similar to those of the so-called −red
intermediate×, are 6-acyl-5,8-dihydropterins. Therefore, the −red intermediate× could
be a 6-formyl-5,8-dihydropterin, one of the isomeric forms of HPT (Scheme 2), which is
easily formed in the absence of O2 from the electronically excited HPT. Further
investigations to obtain more evidence on this are in progress in our laboratory.
To compare the stability of HPT and its isomer, 6-formyl-5,8-dihydropterin, their
ground-state geometry, and heat of formation (DHf) were calculated by using the
semiempirical parameterized PM3 method. The loss of the aromatic character of the
pterin moiety on going from the former to the latter structure (see Scheme 2) agrees
with the different calculated DHf and the experimental finding that the 6-formyl-5,8-
dihydropterin is stable only under inert atmosphere conditions, and it is formed from
the electronically excited state of HPT.
The molecular-orbital energies were computed to compare the relative reactivity of
HPT and of its isomer, the 6-formyl-5,8-dihydropterin, with O2. As shown in the Table,
on going from HPT to its isomer, the difference calculated between the HOMO and
LUMO levels is in between 7.70 and 6.42 eV, and clearly the energy of the highest
occupied molecular orbital (HOMO) increases from À4.50 to À3.70 eV. Since the
energy of the HOMO is regarded as a criterion of nucleophilicity, this finding indicates
that the 6-formyl-5,8-dihydropterin has the highest nucleophilicity among the two isomers.
Table. Calculated DHf and HOMO and LUMO Energy
DHf/kcal molÀ1
HOMO/eV
LUMO/eV
D (LUMO À HOMO)/eV
6-(Hydroxymethyl)pterina)
6-Formyl-5,8-dihydropterina)
À 81.98
À 75.11
À 4.51
À 3.70
3.19
2.73
7.70
6.42
a) Anionic form, net charge À1 (see Scheme 2)
The Role of Singlet Oxygen (1O2). The value of the quantum yields of 1O2
production (FD) by HPT was determined in O2-saturated and air-equilibrated D2O
solutions, by monitoring the near-IR 1O2 luminescence. Most pterins have a relatively
high fluorescence quantum yield (FF) [45]. Therefore, control experiments in Ar-
saturated solutions were carried out to check possible tailing of the fluorescence

362
Helvetica Chimica Acta Vol. 87 (2004)
emission of HPT in the near-IR. No luminescence at 1270 nm can be detected under
those experimental conditions.
Experiments for determining FD were performed at pD 10.5. Significant 1O2
emission is detected, and a value of 0.21 Æ 0.01 is obtained in air-equilibrated solutions.
No significant differences in FD values are observed in experiments carried out in O2-
saturated solutions. The value of FD obtained for HPT is a little lower than those
previously found for FPT, CPT, and pterin [13] under similar experimental conditions.
1O2 relaxes to ground-state 3O2 by both radiationless and radiative pathways
(Eqns. 4 and 5, resp.).
If a substance in solution is able to quench or trap 1O2, chemical reaction (Eqn. 6)
and physical quenching (Eqn. 7) must be considered.
kd
3
1O2 À! O2
(4)
(5)
(6)
(7)
ke
3
1O2 À! O2 ‡ hn0
kr
Q ‡ 1O2 À! QO2
kq
Q ‡ 1O2 À! Q ‡ 3O2
1
The values of the rate constants of total O2 quenching (kt ˆ kr ‡ kq) by HPT were
determined as indicated in the Exper. Part. Fig. 9 reveals that the Stern-Volmer plots of
the quenching of the near-IR 1O2 luminescence (Eqn. 3) are linear within the range of
concentrations used. The value of kt is calculated from the slope of this plot, taking a
1
value of 62 ms for the O2 lifetime (tD) in D2O [37][38]. Under our experimental
conditions, a value of 3.1 ¥ 106 mÀ1 sÀ1 is calculated. It is important to take into account
that this value is similar to those previously reported for pterin and FPT [13] as well as
1
that O2 is not involved in the mechanisms of their photooxidations [19][21]. On the
other hand, this value is significantly lower than that reported for folic acid [13], where
1O2 seems to be involved in the photooxidation pathways [19][20].
1
Eqn. 8 expresses the rate of the chemical reaction between O2 and HPT. The
1
steady-state concentration of O2 during the irradiation of a HPT aqueous solution is
given by Eqn. 9, where Ia and FD are the rate of absorption of photons by HPT and the
1
quantum yield of O2 production by HPT, respectively, and kd is the nonradiative-
deactivation rate constant in H2O (tD ˆ 1/kd ˆ 3.8 ms). ke is not included in Eqn. 9
because kd is much higher than ke [31][37]. The maximum possible value of kr is kt (if
kr >> kq). Therefore, assuming that kt in H2O is equal to that obtained in D2O (no
1
isotope effect) and taking into account the steady-state concentration of O2 and the
initial HPT concentration, the maximum possible value for the rate of the reaction
1
between O2 and HPT in H2O can be calculated ((d[HPT]/dt)1 ˆ Àkt [1O2] [HPT]).
Under the same experimental conditions used for the determination of the quantum
yields associated with the disappearance of HPT and the formation of FPT, a value of
0.66 mm/min is calculated for (d[HPT]/dt)1. This value is much lower than that obtained
for the experimental rate of degradation of HPT (13.7 Æ 0.8 mm/min, see above).
Therefore, these results suggest that although HPT is a good 1O2 sensitizer and is able to

Helvetica Chimica Acta Vol. 87 (2004)
363
Fig. 9. SternÀVolmer plot of the quenching of the 1O2 near-IR luminescence by 6-(hydroxymethyl)pterin in D2O.
Rose bengal was used as a sensitizer, lex 547 nm.
quench 1O2, the chemical reaction between both species is not quantitatively relevant
as a HPT photooxidation pathway.
(d[HPT]/dt)1 ˆ Àkr[1O2] [HPT]
(8)
(9)
IaFD
‰1O2Š ˆ
kd ‡ kt‰HPTŠ
Photolysis experiments were performed in D2O to evaluate the effect of D2O on the
photooxidation pathways of HPT. Air-equilibrated solutions of HPT (260 mm) at
pH 10.5 in H2O and D2O were irradiated. Evolutions of spectra as a function of the
irradiation time were recorded for both photolysis experiments. Within experimental
error, the NED spectra obtained in both photolysis experiments and the rates of
absorbance changes at different wavelengths are quite similar (results not shown).
These results indicate that 1O2 does not participate in the main mechanism of
photooxidation of HPT, being in good agreement with results presented above in this
section.
Conclusions.
Photooxidation of the basic form of 6-(hydroxymethyl)pterin
(HPT) shows similar patterns to those observed in the photooxidation of 6-
formylpterin (FPT) [19][21] in alkaline medium. Photooxidation of HPT to FPT

364
Helvetica Chimica Acta Vol. 87 (2004)
occurs through, at least, two steps. In the first one, after absorption of a photon and
without participation of O2, a −red intermediate× is generated. In the second step, the
intermediate reacts with O2 yielding FPT. Due to its spectral features and reactivity, the
−red intermediate× could be 6-formyl-5,8-dihydropterin (Scheme 2).
During photooxidation of HPT, H2O2 is generated. H2O2 is probably a product of
the thermal reaction between the −red intermediate× and O2. During the HPT! FPT
.
process, the hydroxy radical (HO ) does not seem to participate actively. Generation of
H2O2 by HPT is important from a biomedical point of view, since this activated oxygen
species is involved in pathogenesis of vitiligo [43], and the accumulation of different
pterin derivatives in the epidermis of the affected patients has been established.
Evidence is presented showing that HPT is a good singlet-oxygen (1O2) sensitizer
under UV-A irradiation in alkaline aqueous solutions (FD ˆ 0.21 Æ 0.01). This result, in
good agreement with previous studies reported on other pterin derivatives [13],
suggests that the participation of pterins in biological oxidative photodynamic
processes should be taken into account.
The rate constant kt of total 1O2 quenching by HPT is 3.1 ¥ 106 mÀ1 sÀ1, indicating that
1
this compound is able to trap O2. However, kinetic analysis and experiments in the
1
presence of D2O indicate that O2 does not participate in the main pathway of the
photooxidation of HPT to FPT.
¬
The present work was partially supported by the Consejo Nacional de Investigaciones Cientificas y Tecnicas
¬
¬
¬
(CONICET-PIP 4354), the Agencia de Promocion Cientifica y Tecnologica (ANPCyT, grant N8 PICT 06-
¬
03531), and the Universidad Nacional de La Plata. R. E.-B. and A. L. C. thank the Fundacion Antorchas (grant
# 14116-207/03). We deeply thank Prof. Dr. A. M. Braun and E. Oliveros for the facilities given to A. H. T. and
C. L. during their stay at the Engler-Bunte Institute, University of Karlsruhe, Germany. A. H. T. wishes to thank
CONICET for a research post-doctoral fellowship and the Deutsche Akademische Austauschdienst (DAAD) for
¬
a research fellowship. C. L. and F . M. C.thank the Fundacion Antorchas and CONICET, respectively for their
¬
graduate fellowships. M. L. D. thanks the Fundacion Antorchas for an undergraduate grant. The authors thank
the authorities of CINDECA for the HPLCfacilities as well as Lic. R. Martino for his technical assistance.
A. H. T., R. E.-B., and A. L. C. are research members of CONICET.
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Received August 7, 2003