Reactivity of conjugated and unconjugated pterins with singlet oxygen (O2(1Δg)): Physical quenching and chemical reaction

Article abstract of DOI:10.1562/2006-09-15-RA-1041

Pterins (PTs) belong to a class of heterocyclic compounds present in a wide range of living systems. They participate in relevant biological functions and are involved in different photobiological processes. We have investigated the reactivity of conjugated PTs (folic acid [FA], 10-methylfolic acid [MFA], pteroic acid [PA]) and unconjugated PTs (PT, 6-hydroxymethylpterin [HPT], 6-methylpterin [MPT], 6,7-dimethylpterin [DPT], rhamnopterin [RPT]) with singlet oxygen (¹O₂) in aqueous solutions, and compared the efficiencies of chemical reaction and physical quenching. The chemical reactions between ¹O₂, produced by photosensitization, and PT derivatives were followed by UV-visible spectrophotometry and high-performance liquid chromatography, and corresponding rate constants (k_r) were evaluated. Whenever possible, products were identified and quantified. Rate constants of ¹O₂ total quenching by the PT derivatives investigated were obtained from steady-state ¹O₂ luminescence measurements. Results show that the behavior of conjugated PTs differs considerably from that of unconjugated derivatives, and the mechanisms of ¹O₂ physical quenching by these compounds and of their chemical reaction with ¹O₂ are discussed in relation to their structural features.

Full text of DOI:10.1562/2006-09-15-RA-1041

Photochemistry and Photobiology, 2007, 83: 526–534
Reactivity of Conjugated and Unconjugated Pterins with Singlet Oxygen
(O2(1Dg)): Physical Quenching and Chemical Reaction†
1
Franco M. Cabrerizo , M. Laura Dantola , Gabriela Petroselli , Alberto L. Capparelli ,
1
1
1
´
Andres H. Thomas , Andre M. Braun , Carolina Lorente* and Esther Oliveros*
1
2
1
2
´
´
1
´
´
´
Departamento de Quımica, Facultad de Ciencias Exactas, Instituto de Investigaciones Fisicoquımicas Teoricas y Aplicadas
(INIFTA), Universidad Nacional de La Plata, CONICET, La Plata, Argentina
¨
2
Lehrstuhl fu¨r Umweltmesstechnik, Engler-Bunte Institu¨t, Universitat Karlsruhe, Karlsruhe, Germany
Received 15 September 2006; accepted 24 October 2006; published online 31 October 2006; DOI: 10.1562 ⁄ 2006-09-15-RA-1041
sensitizer (Sens) to dissolved molecular oxygen (Reactions (1)
and (2)). Subsequently, 1O2 relaxes to its ground state 3O2
ABSTRACT
Pterins (PTs) belong to a class of heterocyclic compounds
present in a wide range of living systems. They participate in
relevant biological functions and are involved in different
photobiological processes. We have investigated the reactivity
of conjugated PTs (folic acid [FA], 10-methylfolic acid [MFA],
pteroic acid [PA]) and unconjugated PTs (PT, 6-hydroxymeth-
ylpterin [HPT], 6-methylpterin [MPT], 6,7-dimethylpterin
[DPT], rhamnopterin [RPT]) with singlet oxygen (1O2) in
aqueous solutions, and compared the efficiencies of chemical
reaction and physical quenching. The chemical reactions between
1O2, produced by photosensitization, and PT derivatives were
followed by UV-visible spectrophotometry and high-performance
liquid chromatography, and corresponding rate constants (kr)
were evaluated. Whenever possible, products were identified and
quantified. Rate constants of 1O2 total quenching by the PT
derivatives investigated were obtained from steady-state 1O2
luminescence measurements. Results show that the behavior of
conjugated PTs differs considerably from that of unconjugated
derivatives, and the mechanisms of 1O2 physical quenching by
these compounds and of their chemical reaction with 1O2 are
discussed in relation to their structural features.
through solvent induced radiationless and radiative pathways
(Reactions (3) and (4)). It may also be deactivated by a
physical quencher (Reaction (5)) and ⁄ or oxidize an acceptor
molecule (Reaction (6)). The physical mechanisms proposed
for these reactions have been recently reviewed (4).
kISC
hv
1
3
1Sens ꢀ! Sensꢁ ꢀ! Sensꢁ
ð1Þ
ð2Þ
ð3Þ
ð4Þ
ð5Þ
ð6Þ
ket
3
1
1
3Sensꢁ þ O2 ꢀ! Sens þ O2
kd
1O2 ꢀ! O2
1O2 ꢀ! O2 þ hv
3
ke
00
3
kr
1
Qþ O2 ꢀ! QO2
kq
1
3
Qþ O2 ꢀ! Qþ O2
If a biological compound is able to deactivate 1O2 efficiently
by means of physical quenching, such a compound may have a
protective role against 1O2 in vivo and, very likely, against other
reactive oxygen species. On the other hand, an efficient chemical
1
reaction with O2 may be beneficial or harmful to biological
systems, depending on the nature of the oxidized products.
1
Therefore the study of the reactivity of O2 with biomolecules
INTRODUCTION
(physical quenching and chemical reaction) is a powerful tool to
analyze their antioxidant capability. The determination of the
rate constants of O2 physical quenching and of the chemical
reaction with 1O2 (kq and kr, Reactions (5) and (6), respectively)
allows the evaluation of the efficiencies of these processes.
Product identification provides information to elucidate oxida-
tion mechanisms and to discuss potential effects in vivo.
We have recently been interested in the production and
deactivation of 1O2 by PTs, a family of heterocyclic com-
pounds derived from 2-aminopteridin-4(1H)-one (PT) and
containing the pyrazine[2,3-d]pyrimidine ring structure
(Scheme 1) (5–8). These compounds, widespread in nature,
are involved in relevant biological functions (9) and participate
in many photochemical and photobiological processes (10).
PTs present, in aqueous solution, an acid–base equilibrium
that involves an amide group (acid form) and a phenolate
group (basic form) (Scheme 1), with a pKa close to 8 (11–13).
Other functional groups of the PT moiety (e.g. 2-amino group
or ring nitrogen atoms) have pKa values lower than 2 (11).
Singlet molecular oxygen (O2(1Dg), denoted throughout as
1O2), the lowest electronic excited state of molecular oxygen, is
an important oxidizing intermediate in chemical processes and
one of the main reactive oxygen species responsible for the
damaging effects of light on biological systems (photodynamic
effects) (1). This activated metastable state is much more
reactive than the triplet ground state (O2(3Sg)), denoted
throughout as 3O2) and both applied and fundamental aspects
of its reactivity have been attracting considerable interest
(1–4).
1
Photosensitization is primarily responsible for the produc-
tion of 1O2 in vivo (3). In this process, 1O2 is most often
produced by energy transfer from the excited triplet state of a
†This paper is part of a symposium-in-print dedicated to Professor Eduardo A.
Lissi on the occasion of his 70th birthday.
*Corresponding author email: clorente@inifta.unlp.edu.ar (Carolina Lorente)
ꢀ 2007 American Society for Photobiology 0031-8655/07
526

Photochemistry and Photobiology, 2007, 83 527
1O2 within the general context of the mechanisms of 1O2
deactivation proposed in the literature.
MATERIALS AND METHODS
Chemicals and preparation of solutions. Pterins (Shircks Laboratories)
and rose bengal (RB; Aldrich) were used without further purifica-
tion. The pH of the aqueous solutions was adjusted by adding
drops of HCl or NaOH 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.
The pH measurements were performed using a pH-meter CG 843P
(Schott, Mainz, Germany) with a pH-combination electrode Blue-
Line 14pH (Schott).
D2O (Euriso-top; Groupe CEA, Saclay, France, minimum isotopic
purity of 99.9%), a solution of DCl (Aldrich, 99.5% D) in D2O and a
solution of NaOD (CEA) in D2O were employed for preparing
solutions in D2O. The pD values were calculated by adding 0.4 to the
apparent pH values measured with the pH meter (14).
H2O2 determination. For determination of H2O2, the cholesterol
CHOD-PAP kit from Roche Diagnostics (Germany) was used.
H2O2 was quantified by its color reaction with 4-aminophenazone
and phenol catalyzed by peroxidase. Five hundred microliters of
irradiated solution of FA was added to 1 mL of reagent. The
absorbance at 505 nm of the solution containing a mixture of
irradiated solution and the reagent was measured after 20 min of
incubation at room temperature, using the nonirradiated sample
solution as a blank. Aqueous solutions of H2O2, prepared from
commercial standards, were employed for obtaining the corres-
ponding calibration curve.
Scheme 1. Molecular structures of common pterin derivatives and the
acid–base equilibrium in aqueous solution. (a) DPT has an additional
methyl group at position 7 of the pterin moiety; (b) these compounds
contain a p-aminobenzoic acid (PABA) unit in the substituent at
position 6; (c) in these compounds, a glutamic acid unit (Glu) has been
attached to the PABA moiety.
Determination of the rate constants of 1O2 total quenching by PTs.
The rate constants of 1O2 total quenching (kt) by PTs were determined
by Stern–Volmer analysis of the 1O2 luminescence in the near-infrared
(NIR) (15). Our equipment used to monitor the 1O2 luminescence at
1270 nm upon continuous monochromatic excitation of a sensitizer
has already been described (16). For the experiments reported in this
article, a cooled ()80ꢁC) NIR photomultiplier (Hamamatsu) was used
as a 1O2 detector, instead of a Ge photodiode. Briefly, the sample
The most common PT derivatives are 6-substituted com-
pounds. The molecular weight and the functional groups of
these substituents are quite different (Scheme 1); e.g. PTs may
have substituents with one carbon atom, with a short hydro-
carbon chain or larger substituents containing a p-aminoben-
zoic acid (PABA) moiety. Derivatives with the latter type of
substituents are frequently called conjugated PTs.
We have reported previously the rate constants of 1O2 total
quenching (kt = kq + kr, Reactions (5) and (6)) by a few PT
derivatives (5–7). The rate constant kt appears to be very
dependent on the chemical nature of the 6-substituent.
However, more investigations are needed because, up to
now, kt has only been determined for one conjugated PT, folic
acid (FA) (5). Moreover, to the best of our knowledge, the rate
constants of the chemical reaction between 1O2 and PT
derivatives (kr) have been only reported for PT (8) and
6-methylpterin (MPT) (7) and the corresponding reaction
products have not been studied.
solution in
a quartz cell (1 cm · 1 cm) was irradiated with a
xenon ⁄ mercury arc through a water filter, focusing optics and a
monochromator. The 1O2 luminescence was collected with a mirror,
chopped and, after passing through a focusing lens, a cut-off filter
(1000 nm) and an interference filter (1271 nm), was measured at 90ꢁ
with respect to the incident beam.
Singlet oxygen was generated by photosensitization, using RB as a
sensitizer. Groups of experiments were carried out irradiating
solutions of PTs and RB at 547 nm, where PT derivatives do not
absorb. The RB concentration was kept constant, whereas the PT
derivative concentration was varied within a series of experiments
(Stern–Volmer analysis). Besides, the concentration of RB was such
that 1O2 total quenching by the sensitizer itself was negligible
compared with 1O2 deactivation by the solvent (17). Under conditions
where the quantum yield of 1O2 production by the sensitizer (FD) is
not affected by the presence of the quencher (a PT derivative) and
assuming a dynamic quenching of 1O2, a linear relationship between
the ratio of the signals observed in the absence (Se0) and in the
presence (Se) of quencher and the quencher concentration should be
obtained (Eq. 7).
In the present article we describe the processes of deacti-
1
vation of O2 by a series of conjugated and unconjugated PT
derivatives in aqueous solutions and analyze the effect of the
6-substituent on the quenching efficiency. To avoid a mixture
of acid and basic forms of the PT derivatives in the solution
(vide supra), we have chosen to work at alkaline pH. We have
expanded our previous studies on 1O2 total quenching (5–7) by
determining kt values for new compounds. We have deter-
mined the kr values for a series of conjugated and unconju-
gated PTs and investigated the corresponding oxidation
products. Knowing kt and kr, rate constants of 1O2 physical
quenching (kq) could be calculated. Taking into account this
ensemble of results, we compare the efficiencies of chemical
reaction and physical quenching, and discuss the effects of the
structural features of the PT derivatives on their reactivity with
S0e=Se ¼ 1 þ ktsD½Qꢃ
ð7Þ
ꢂ
sD is the 1O2 lifetime in the solvent used in the absence of Q
(sD = 1 ⁄ kd, as the radiative rate constant [ke, Reaction (4)] is negli-
gible compared to the radiationless deactivation rate constant [kd,
Reaction (5)] in most solvents) (18).
Therefore, knowing sD, kt can be calculated from the slope of the
Stern–Volmer plot (Eq. 7). Because of the short sD in H2O (3.8 ls),
D2O (where sD is much longer: 62
3 ls [19]) was used as a solvent in
all experiments. Singlet oxygen lifetimes were determined by time-
resolved phosphorescence detection. The laser system and the custom-
built detectors employed (a Ge photodiode, Judson, or an InGaAs
photodiode, IR Components) have already been described (20).

528 Franco M. Cabrerizo et al.
Determination of the rate constants of the chemical reaction between
1O2 and PTs (kr). The rate of disappearance of a compound Q reacting
with 1O2 is given by Eq. (8).
HPLC was equipped with a diode array detector (HP 1100 DAD) and
a software (HP ChemStation for LC) for registering and analyzing
spectra of the separated substances.
The solutions were irradiated in a 1 cm · 1 cm spectroscopic cell,
on the same optical bench as used for the determination of kt values
(vide supra). The incident photon flux (Po) at the wavelength of
excitation of the sensitizer (547 nm) were determined by actinometry,
ꢀd½Qꢃ=dt ¼ kr½1O2ꢃ½Qꢃ
ð8Þ
If 1O2 is produced by sensitization and applying the quasi-stationary
hypothesis to the concentrations of excited states (Reactions (1)–(6)),
Eq. (9) gives the steady-state concentration of 1O2,
using Aberchrome 540 as an actinometer (24) (Po = 2.8 · 10)6
–
3.6 · 10)6 E L)1 s)1 at 547 nm). Values of Pa (photon flux absorbed
by the sensitizer) were calculated from Po using the Beer–Lambert law:
½1O2ꢃ ¼ PaUD=ðkd þ kS½Sꢃ þ kQt ½QꢃÞ
ð9Þ
t
Pa ¼ Poð1 ꢀ 10ꢀA
where A is the absorbance of the sensitizer at the excitation wave-
length.
Þ
ð12Þ
where Pa (E L)1 s)1) is the photon flux absorbed by the sensitizer, FD
the quantum yield of 1O2 production by the sensitizer, [S] the con-
centration of the sensitizer, ktS the rate constant of 1O2 total quenching
by the sensitizer and ktQ the rate constant of 1O2 total quenching by Q.
Combining Eqs. (8) and (9), and assuming that there is no
interference by the oxidation product(s), Eq. (10) is obtained for the
rate of disappearance of a compound Q reacting with 1O2.
The spectral changes were registered on
a Cary 5 (Varian)
spectrophotometer. The irradiation experiments were performed using
quartz cells of 1 cm of optical path length, whereas the absorbance
measurements were carried out with quartz cells of 0.1 cm or 1 cm of
optical path length.
d½Qꢃ
dt
kr½Qꢃ
ꢀ
¼ PaUD
ð10Þ
kd þ kSt ½Sꢃ þ kQt ½Qꢃ
RESULTS AND DISCUSSION
1
Rate constants of O2 total quenching (kt) by PT derivatives
If the rate of 1O2 total quenching by the sensitizer is negligible com-
pared with deactivation by the solvent (kd >> ktS [S]), integration of
Eq. (10) leads to Eq. (11).
The values of the rate constants of 1O2 total quenching (kt)
by the series of PT derivatives investigated in this work
(Scheme 1)—three conjugated PTs (FA, 10-methylfolic acid
[MFA], pteroic acid [PA]) and five unconjugated PTs (PT, 6-
hydroxymethylpterin [HPT], MPT, 6,7-dimethylpterin [DPT],
rhamnopterin [RPT])—are listed in Table 1. These values were
determined as indicated in Materials and Methods. The Stern–
hꢂ
ꢃ
i
ꢀ
ꢁ
ꢀ
ꢁ
fð½QꢃÞ¼ln ½Qꢃ=½QꢃO
ꢀ
kQt =kd ½Qꢃ0ꢀ½Qꢃ
ð11Þ
¼ꢀPaUDðkr=kdÞt
and the plot of f([Q]) as a function of time should be linear (21). In the
cases where ktQ [Q] << kd, Eq. (11) simplifies to:
ð110 Þ
1
lnð½Qꢃ=½QꢃOÞ ¼ ꢀPaUDðkr=kdÞt
Volmer plots of the quenching of the NIR O2 luminescence
(Eq. (7)) were linear within the range of concentrations used
and are shown in Fig. 1 for MFA, PA, RPT and DPT. The
values of kt were calculated from the slopes of these plots.
Values of kt obtained in this work and in former studies (5–7)
and first-order kinetics should be observed for the disappearance of
Q.
For determining kr, groups of experiments were carried out
irradiating alkaline solutions (3 cm3, pH = 10.5) containing a PT
derivative and RB as a 1O2 sensitizer. RB was excited at 547 nm
(FD = 0.75 [22,23]). The experiments were performed in D2O. Within
each series of experiments, the initial concentrations of the PT
derivative and RB were kept constant, but the solutions were
irradiated during different periods of time.
A high-performance liquid chromatography (HPLC) equipment
(Hewlett Packard Series 1100) and an RP 18 LiChro CART 125-4
column were used for the determination of the evolution of the
concentration of the PT derivatives as a function of irradiation time
and for the analysis of the products of the reaction. Solutions
containing 0–10% of acetronitrile and 90–100% of potassium phos-
phate aqueous solution (20 mM, pH 5.5) were used as eluents. The
lie in the range 3 · 106–7 · 107 M)1 s)1
The kt value obtained for RPT (3.6
.
0.4 · 106 M)1 s)1) is
of the same order of magnitude as kt values found for PT and
HPT (approx. 3 · 106 M)1 s)1, Table 1). Among unconjugat-
ed PTs, MPT and particularly DPT have larger kt values
(8.0
than PT, HPT and RPT.
0.6 · 106 and 3.1
3 · 107 M)1 s)1, respectively)
The kt values obtained for FA, MFA and PA are about 1
order of magnitude larger than the kt values obtained for most
unconjugated PTs (Table 1). It should be noted that the
1
Table 1. Rate constants of 1O2 total (kt) and physical quenching (kq) by pterin derivatives, and rate constants of their chemical reaction with O2
(kr) in D2O (pD = 10.5).
Compound
kt (106 M)1 s)1
)
kr (106 M)1 s)1
)
kq (106 M)1 s)1
)
Unconjugated pterins
PT
HPT
RPT
MPT
DPT
2.9
3.1
3.6
8.0
31
0.3 (5)
0.4 (6)
0.4
0.6 (7)
3
0.25
1.2
2.4
4.9
10
0.03 (8)
0.1
0.2
0.7 (7)
2
2.6
1.9
1.2
3
0.3
0.5
0.6
1
21
5
Conjugated pterins
FA
MFA
PA
30
44
67
3 (5)
4
7
2.8
1.9
12
0.3
0.2
2
27
42
55
3
4
9
PT = pterin; HPT = 6-hydroxymethylpterin; RPT = rhamnopterin; MPT = 6-methylpterin; DPT = 6,7-dimethylpterin; FA = folic acid; MFA
= 10-methylfolic acid; PA = pteroic acid.

Photochemistry and Photobiology, 2007, 83 529
Figure 1. Stern–Volmer plots of the quenching of the 1O2 near-
infrared luminescence by MFA, PA, RPT and DPT (inset) in D2O
(pD = 10.5, rose bengal was used as a sensitizer, kex = 547 nm); due
to solubility problems the concentration range for DPT was limited.
Figure 2. Photosensitized oxidation of pterin derivatives in air-equil-
ibrated alkaline D2O solution: plot of f([Q]) = ln([Q] ⁄ [Q]o) ) [(ktQ ⁄ kd)
([Q]0 ) [Q])] as a function of the irradiation time (Eq. 11, Materials
and Methods). Sensitizer: rose bengal (irradiation wavelength:
547 nm), pD = 10.5, concentrations of pterin derivatives determined
by HPLC analysis. Note that the slopes of these plots ()Pa FD [kr ⁄ kd])
do not reflect directly the values of kr, as experiments were carried out
using different incident photon flux.
conjugated PTs (FA, MFA, PA) contain an additional amino
group in the PABA unit (N-10, Scheme 1) and that amines are
known to deactivate efficiently 1O2 by charge-transfer-induced
physical quenching (25–27). Therefore, the presence of the
PABA amino group in FA, MFA and PA could explain the
larger kt values observed for these compounds. The compar-
ison of kt for FA and MFA with that of PA reveals that the
amide group introduced in the former compounds by the
attachment of the glutamic acid unit (Glu) to the PABA
moiety (Scheme 1) has a negative effect on the total 1O2
quenching efficiency (Table 1). However, detailed reactivity
comparisons based on kt values only are difficult to make
because kt represents the sum of kr and kq (rate constants
corresponding to chemical reaction and physical quenching,
respectively) and these two kinetics constants may be governed
by different factors.
1
the deactivation of O2 by PT is mainly a physical quenching
process (kq = 2.6
0.3 · 106 M)1 s)1 is an order of magni-
tude larger than kr). The UV-visible spectrophotometric
analysis of the irradiated solutions and the HPLC analysis of
1
the products of the reaction between O2 and PT showed that
the PT moiety was oxidized and cleaved yielding a group of
nonpterinic products; i.e. the characteristic absorption band of
the PT in the UV-A region was lost (see Supplemental
Materials). The absorption also decreased at shorter wave-
length (in the UV-B and UV-C) indicating that products
formed lack aromatic character.
The same analysis performed for MPT and DPT yielded
similar results—the oxidation of these compounds by 1O2
leads to the cleavage of the PT moiety and the formation of
several nonpterinic products. As an example, the HPLC
analysis and the disappearance of the two typical absorption
bands of the PT moiety during the sensitized oxidation of
MPT, using RB as a sensitizer (irradiation at 547 nm) can be
observed in Fig. 3. Similar spectrophotometric behavior was
observed for all unconjugated PTs (see Supplemental
Materials).
1
Chemical reaction between O2 and unconjugated PTs
1
The rate constants of the chemical reaction between O2 and
the basic forms of several unconjugated PT derivatives (HPT,
DPT and RPT) (kr) in D2O were determined from HPLC
analysis of the disappearance of the PT derivative (Q) during
the photosensitized oxidation, using RB as a 1O2 sensitizer.
The values of kr listed in Table 1 were obtained from the linear
plot of f([Q]) or ln([Q] ⁄ [Q]0) as a function of the irradiation
time (Eqs. 11 or 11¢, Materials and Methods, and Fig. 2). For
comparison purposes, we have included in Table 1, the kr
values previously reported for PT (8) and MPT (7). The values
However, significant differences can be appreciated within
this series of compounds in the chemical reactivity toward 1O2.
Values of kr increase in the order kr(PT) <<
kr(MPT) < kr(DPT), which suggests that the higher electronic
density on the pyrazine ring induced by the methyl group(s)
favors the electrophilic attack of 1O2. Introduction of a methyl
substituent at position 6 (MPT) leads to an increase in kr by a
factor of 20 relative to PT (kr(MPT) = 4.9 0.7 ·
106 M)1 s)1), and introduction of a second methyl substituent
at position 7 (DPT) leads to an increase in kr by a factor of 40
1
of the rate constants of O2 physical quenching (kq) listed in
Table 1 were obtained by subtracting the kr values from the
corresponding kt values.
The kr value for PT (2.5
comparison with the corresponding kt value, indicating that
0.3 · 105 M)1 s)1) is very low in
(kr(DPT) = 10
2 · 106 M)1 s)1). Therefore, taking into

530 Franco M. Cabrerizo et al.
similar (1.2–3 · 106 M)1 s)1), whereas the corresponding val-
ues of kr are quite different (2.5 · 105–5 · 106 M)1 s)1
)
(Table 1). Only for DPT, which has the largest kr value of
all unconjugated PTs investigated, kq (2.1 · 107 M)1 s)1) is
about an order of magnitude larger than kq for other PTs in
this series. Although such a large effect on kq of the 6,7-
dimethyl substitution on the PT moiety was rather surprising,
it confirms that this substitution affects considerably the
reactivity of PT derivatives with 1O2. However, if the chemical
reaction is clearly dependent on the electronic activation of the
C6–C7 double bond of the pyrazine ring, 1O2 physical
quenching does not show such a dependence and involves
most probably different sites on the PT derivative. Thus,
charge-transfer deactivation by the amino group at position 2
1
may contribute to O2 physical quenching by PT, HPT, RPT
and MPT, although primary amines may exhibit rather low kq
values (<5 · 105 M)1 s)1) (26).
1
Chemical reaction between O2 and conjugated PTs
The kr value for FA, the most important PT derivative present
in mammalians, and two related compounds, MFA and PA
(Scheme 1), have been determined (Materials and Methods
and Table 1). These values are much lower than the corres-
ponding kt values, thus indicating that the quenching of 1O2 by
conjugated PTs is mainly a physical process. Nevertheless, kr
values remain in the range similar to that obtained for C6-
substituted unconjugated PTs (Table 1).
Figure 3. Photosensitized oxidation of 6-methylpterin ([MPT]o
=
634 lM) in air-equilibrated alkaline D2O solution: Evolution of the
absorption spectra as a function of irradiation time. Sensitizer: rose
bengal (irradiation wavelength: 547 nm), pD = 10.5. Spectra were
recorded every 30 min; arrows indicate the changes observed at
different wavelengths. Optical path length: 1 cm for irradiation and
1 mm for absorbance measurements. Inset: Chromatogram obtained
in HPLC analysis at 30 min of irradiation. All the picks shown
correspond to products of the reaction. k = 230 nm.
The kr value for FA is about 1 order of magnitude larger
than that found for PT. The HPLC analysis of the products of
the reaction between 1O2 and FA revealed the formation of
6-formylpterin (FPT). Therefore, the 6-substituent on the FA
molecule is oxidized and cleaved between the methylene group
linked to the PT moiety and the amino group of the PABA
unit (Scheme 1). The mechanism proposed by Gollnick and
Lindner for the attack of 1O2 on primary and secondary
amines bearing a nitrogen atom substituted by a CH group
(31) can explain the observed reaction (Scheme 3). Accord-
ingly, we propose that the charge-transfer reaction occurring
account the known reactivity of 1O2 with C=C (28,29), the
most probable reaction of this species with the PT moiety is a
[2 + 2] cycloaddition to the C6–C7 double bond. Such a
reaction would yield a dioxetane which may subsequently
decompose, resulting in the cleavage of the pyrazine ring and
the formation of two carbonyl groups in the first step (Scheme
2). As expected in the context of such an hypothesis, kr(HPT)
(1.2
106 M)1 s)1
0.1 · 106 M)1 s)1
are higher than kr(PT), but smaller than
)
and
kr(RPT)
(2.4
0.1 ·
)
1
between the N-10 atom and O2 leads to the oxidation of the
kr(MPT), the electron donor effect of the C6-substituent of
HPT and RPT being smaller than that of a methyl group.
Moreover, the reactivity of HPT in comparison with that of
PT and MPT follows a pattern already observed in previous
studies performed on other series of compounds; e.g. the kr
value obtained for 5-hydroxymethyl-2-furaldehyde is in
between the kr values of 2-furaldehyde and 5-methyl-2-
furaldehyde (30).
amine (–CH2–NH–) to an imine (–CH = N–) with concom-
itant H2O2 elimination. Hydrolysis of the imine functional
group in aqueous solution induces cleavage of the 6-substit-
uent (–CHO + –NH2), yielding FPT and p-aminobenzoylgl-
utamic acid (PABA-Glu) as products. Formation of H2O2 was
confirmed using a colorimetric method based on the reaction
of H2O2 with 4-aminophenazone and phenol in the presence of
peroxidase (Materials and Methods).
The HPLC analysis of the reaction mixture also shows that
other products were formed, in addition to FPT, and PABA-
Glu. The spectral features of these latter products suggest that
the pterinic moiety is also oxidized to yield non pterinic
compounds. In order to evaluate the extent of the oxidation
that yields FPT as product (oxidation of the 6-substituent), the
FPT concentration was determined as a function of irradiation
time and compared with the corresponding evolution of the
FA concentration. In the experiment shown in Fig. 4a, the
initial rate of FA consumption was 1.9
whereas the initial rate of FPT production was
0.51
0.04 lM min)1. These results show that 27% of the
FA molecules consumed are transformed into FPT. Therefore
1
Physical quenching of O2 by unconjugated PTs
It is noteworthy that the rate constants of 1O2 physical
quenching (kq) obtained for PT, HPT, RPT and MPT are
0.2 lM min)1
,
Scheme 2. Mechanism proposed for the attack of 1O2 onto the
pyrazine ring of the pterin moiety.

Photochemistry and Photobiology, 2007, 83 531
Scheme 3. Mechanism proposed for the photosensitized oxidation of
FA in aqueous solution to yield FPT and PABA-Glu as main
products.
we can propose two different pathways on two different sites
of the molecule, C6–C7 double bond of the PT moiety and N-
10 of the PABA substituent (Reactions (13) and (14),
respectively).
kr1ðFAÞ
1
FAþ O2 ꢀꢀ! Non pterinic compounds
ð13Þ
ð14Þ
kr2ðFAÞ
1
FAþ O2 ꢀꢀ! FPT þ PABA ꢀ Glu
The rate constants of these reactions, kr1(FA) and kr2(FA),
were calculated from the corresponding initial rates of FA
consumption and FPT formation. Values of 2.0 · 106 and
8 · 105 M)1 s)1 were obtained, respectively (Table 2). It is
interesting to compare the value of kr1(FA) with kr values
obtained for unconjugated PTs: kr1(FA) is almost 10 times
larger than kr(PT), but lower than kr(MPT). This could be
expected, as Reaction (13) corresponds to chemical modifica-
tions similar to those undergone by other unconjugated PTs.
The methylene group of the 6-substituent should enhance the
reaction of the PT moiety with 1O2 with respect to PT, but
such an effect should be weaker than that of the methyl group
in MPT.
Figure 4. Evolution of pterin derivative and product concentrations as
a function of irradiation time during sensitized photooxidation.
Chemical reaction between 1O2 and: (a) FA, (b) PA. Conditions:
sensitizer, RB; irradiation wavelength, 547 nm; solvent, D2O; pD,
10.5; concentrations of FA, PA and FPT determined by HPLC
analysis.
hinder the quenching of 1O2 by the PABA moiety (N-10 atom).
Otherwise, PA is expected to undergo similar reactions with
1O2 (Reactions (15) and (16)) as already proposed for FA
(Reactions (13) and (14)).
kr1ðPAÞ
1
PAþ O2 ꢀꢀ! Non pterinic compounds
ð15Þ
ð16Þ
The kr and kt values found for PA are significantly larger
than corresponding values obtained for FA (Table 1). The
only structural difference between PA and FA is the lack of the
glutamic (Glu) unit attached to the PABA substituent of PA.
Therefore steric effects due to the Glu unit present in FA might
kr2ðPAÞ
1
PAþ O2 ꢀꢀ! FPT þ PABA
Indeed, FPT is a product of the reaction between 1O2 and PA.
Therefore, similar to FA (Scheme 3), the amino group of the

532 Franco M. Cabrerizo et al.
Table 2. Rate constants of the chemical reaction of conjugated pterins
with 1O2 in D2O (pD = 10.5). kr1 and kr2: reaction of 1O2 with the
pterin moiety and the PABA substituent, respectively.
quenching) is quantitatively more important than the latter
ones (chemical reactions) (4). In agreement, the singlet exciplex
formed by interaction between the N-10 atom of FA and PA
with 1O2 leads mainly to 1O2 physical quenching
(kq(FA) = 2.7 · 107 and kq(PA) = 5.5 · 107 M)1 s)1), the
corresponding chemical reaction (Scheme 3) contributing to a
much lesser extent to the deactivation (kr2(FA) = 8 · 105 and
kr2(PA) = 9.5 · 106 M)1 s)1). kq(PA) is larger than kq(FA)
Compound
kr1 (106 M)1 s)1
)
kr2 (106 M)1 s)1
)
FA
MFA
PA
2.0
1.9
ꢄ2.4
0.4
0.2
0.8
9.5
0.1
1
–
1
by a factor of 2, showing that O2 physical quenching is less
sensitive than the chemical reaction to steric effects of the Glu
unit present in FA. The case of MFA emphasizes the
important contribution of the amine group of the PABA unit
to the physical deactivation of 1O2: although the chemical
reaction can only take place on the PT moiety as for
unconjugated PTs, kq(MFA) (4.2 · 107 M)1 s)1) is much
higher than kq of the latter compounds, and is comparable
to kq values for PA and FA. As expected for a charge-transfer-
PABA = p-aminobenzoic acid; FA = folic acid; MFA = 10-
methylfolic acid; PA = pteroic acid.
1
PABA unit of PA reacts with O2, leading to the cleavage of
the PABA substituent and the formation of FPT and PABA
(Reaction (16) and Scheme 3). The evolution of FPT and PA
concentrations as a function of irradiation time is shown in
Fig. 4b. The initial rate of PA consumption was
0.5 lM min)1, whereas that of FPT production was
1
11.6
9.2
induced O2 quenching, kq for MFA (N-10 tertiary amine) is
larger than kq for FA (N-10 secondary amine).
0.4 lM min)1. These results show that, in this case,
approximately 80% of the PA molecules consumed were
transformed into FPT. The value of kr2(PA) was calculated
from the latter rate to be 9.5 · 106 M)1 s)1 (Table 2). This
value is 1 order of magnitude higher than that of kr2(FA),
indicating that the chemical reaction of 1O2 with the N-10
atom of the PABA substituent is much more efficient for PA
than for FA. As mentioned above, steric effects of the Glu unit
in FA may explain this result. Indeed, in a study on a series of
22 amines, Monroe has shown that 1O2 quenching is highly
sensitive to steric effects, and is inhibited in sterically hindered
amines (26). As expected, the rate constants for the chemical
reaction of 1O2 with the PT moiety (kr1(PA) and kr1(FA),
Table 2) are close (2.4 · 106 and 2.0 · 106 M)1 s)1, respect-
ively) and similar to those observed for C-6 substituted
unconjugated PTs (Table 1).
6-Formylpterin was not detected as a product of the reaction
between 1O2 and MFA. In contrast to the case of FA and PA,
only non pterinic compounds were found as products by HPLC
analysis. These results strongly support the mechanism pro-
posed in Scheme 3 for FA. Indeed such a sequence of reactions
as proposed in this Scheme is not possible if the N-10 atom
bears a methyl group, as it is the case for MFA. Moreover, the
kr value obtained for MFA (Table 1) is similar to kr1(FA) and
kr1(PA) (reaction with the PT moiety).
CONCLUSIONS
The reactivity of a series of conjugated (FA, MFA, PA) and
unconjugated PTs (PT, HPT, DPT, RPT) (Scheme 1) with
singlet oxygen (1O2) in alkaline aqueous solutions has been
investigated.
The rate constant of the chemical reaction between 1O2 and
the basic forms of unconjugated PT derivatives (kr) is strongly
affected by the chemical nature of the 6-substituent; and the
values lie in a wide range (2.5 · 105–107 M)1 s)1, Table 1).
These values increase with the electronic activation of the C6–C7
double bond of the pyrazinic ring of the PT moiety, suggesting
that the electrophilic attack of 1O2 takes place preferentially on
this bond. The PT moiety was oxidized and broken yielding a
group of non pterinic products; e.g. the characteristic absorption
band of PTs in the UV-A region was lost. In contrast to kr, the
rate constants of 1O2 physical quenching (kq in the range
1.2–3 · 106 M)1 s)1 for PT, HPT, RPT and MPT) do not
depend on the electronic activation of the C6–C7 double bond of
the pyrazine ring. The exception in the series of unconjugated
PTs investigated is the 6,7-dimethyl derivative (DPT) with a kq
value an order of magnitude larger (2.1 · 107 M)1 s)1).
1
In the chemical reaction of O2 with conjugated PTs, two
different processes have been observed: (1) the attack of 1O2 on
the PT moiety is responsible for the formation of nonpterinic
products as in the case of unconjugated derivatives (corres-
1
Physical quenching of O2 by conjugated PTs
1
The results presented above on the reactivity of O2 with FA,
1
ponding rate constants are comparable); (2) the attack of O2
MFA and PA confirm the important contribution of the amine
group of the PABA unit to the deactivation of 1O2 through
charge-transfer-induced quenching. Several studies have
shown that the singlet exciplex (1(QÆÆÆO2)) formed by interac-
tion of 1O2 with the N-lone electron pair of aliphatic and
aromatic amines is stabilized by the transfer of charge from the
amine to O2 (4,25,27,32), and may follow three different
pathways: (1) decay by intersystem-crossing to a triplet
ground-state complex (3(QÆÆÆO2)), which finally dissociates to
the quencher and 3O2, without charge separation; (2) chemical
reaction due to the formation of a chemical bond between a
nitrogen atom of the quencher and the O2 molecule (oxygen-
ation); (3) dissociation with charge separation, i.e. formation
of free radical ions. In most cases, the first pathway (physical
on the secondary amine group (N-10 atom) of the PABA unit
leads to the cleavage of the 6-substituent and formation of 6-
formyl pterin (FTP, Scheme 3). The kr value for this latter
reaction (kr2) is 1 order of magnitude smaller for FA
(8 · 105 M)1 s)1) than for PA (9.5 · 106 M)1 s)1), due to
steric effects of the Glu unit attached to the PABA substituent
of FA. In the case of MFA, the N-10 substitution by a methyl
group prevents this reaction, confirming the mechanism
proposed in Scheme 3.
In contrast to unconjugated PTs, rate constants of 1O2
physical quenching by conjugated derivatives are larger than
corresponding rate constants for the chemical reaction. Values
of kq for conjugated PTs are also at least 1 order of magnitude
larger than those for unconjugated PTs. These results show

Photochemistry and Photobiology, 2007, 83 533
methylpterin in alkaline aqueous solutions. Helv. Chim. Acta 87,
349–365.
7. Cabrerizo, F. M., C. Lorente, M. Vignoni, R. Cabrerizo, A. H.
Thomas and A. L. Capparelli (2005) Photochemical behaviour of
6-methylpterin in aqueous solutions: Generation of reactive oxy-
gen species. Photochem. Photobiol. 81, 793–801.
that the amine group of the PABA unit of the former
compounds plays a dominant role in the physical deactivation
of 1O2 through charge-transfer-induced quenching, in agree-
ment with published data for different types of amines (25,26).
Finally the biological relevance of the results obtained can
also be evaluated. At physiological pH (ꢄ7.4) both acid and
basic forms of PTs are present. Several unconjugated PTs
accumulate in white skin patches of patients affected by vitiligo,
zones of the skin where the protection against UV radiation
fails due to the lack of melanin (33). In addition, unconjugated
PTs are good singlet oxygen photosensitizers. Therefore the
reactions studied in this work could also take place in vivo.
8. Cabrerizo, F. M., M. L. Dantola, A. H. Thomas, C. Lorente,
´
A. M. Braun, E. Oliveros and A. L. Capparelli (2004) Photooxi-
dation of pterin in aqueous solutions: Biological and biomedical
implications. Chem. Biodiv. 1, 1800–1811.
9. Nichol, C. A., G. K. Smith and D. S. Duch (1985) Biosynthesis
and metabolism of tetrahydrobiopterin and molybdopterin. Annu.
Rev. Biochem. 54, 729–764.
10. Lorente, C. and A. H. Thomas (2006) Photophysics and photo-
chemistry of pterins in aqueous solution. Acc. Chem. Res. 39, 395–
402.
Acknowledgements—The present work was partially supported by
11. Albert, A. (1953) Quantitative studies of the acidity of naturally
occurring substances for trace metals. Biochem. J. 54, 646–654.
12. Thomas, A. H., C. Lorente, A. L. Capparelli, M. R. Pokhrel, A.
M. Braun and E. Oliveros (2002) Fluorescence of pterin, 6-form-
ylpterin, 6-carboxypterin and folic acid in aqueous solutions: pH
effects. Photochem. Photobiol. Sci. 1, 421–426.
13. Cabrerizo, F. M., G. Petroselli, C. Lorente, A. L. Capparelli, A.
H. Thomas, A. M. Braun and E. Oliveros (2005) Substituent
effects on the photophysical properties of pterin derivatives in
acidic and alkaline aqueous solutions. Photochem. Photobiol. 81,
1234–1240.
14. Salomaa, P., L. L. Schaleger and F. A. Long (1964) Solvent
deuterium isotope effects on acid–base equilibria. J. Am. Chem.
Soc. 86, 1–7.
15. Tournaire, C., S. Croux, M.-T. Maurette, I. Beck, M. Hocquaux,
A. M. Braun and E. Oliveros (1993) Antioxidant activity of flav-
onoids: Efficiency of singlet oxygen (1Dg) quenching. J. Photo-
chem. Photobiol. B, Biol. 19, 205–215.
Consejo Nacional de Investigaciones Cientificas y Te
CET-PIP 02470 ⁄ 00 and 6301 ⁄ 05), Agencia de Promocio
Tecnologica (ANPCyT Grant PICT 06-12610) and Universidad
Nacional de La Plata (UNLP). A.L.C., A.M.B. and E.O. thank the
Secretarıa de Ciencia, Tecnologıa e Innovacion Productiva (SECyT,
´
cnicas (CONI-
´
n Cientıfica y
´
´
´
´
´
Argentina) and Bundesministerium fur Forschung und Bildung
(BMFB, Germany) for financial support of their project EVI ⁄ 013.
F.M.C. and C.L. thank the Fundacion Antorchas (Grant 4248-70).
´
F.M.C., C.L., G.P. and M.L.D. thank CONICET for graduate and
postdoctoral research fellowships. A.H.T., C.L., F.M.C. and M.L.D.
thank the Deutscher Akademischer Austauschdienst (DAAD) for
research fellowships. A.H.T., C.L. and A.L.C. are research members of
CONICET.
16. Oliveros, E., P. Murasecco-Suardi, A. M. Braun and H.-J. Hansen
(1992) Efficiency of singlet oxygen quenching by carotenoids
measured by near-infrared steady-state luminescence. In Methods
in Enzymology, Carotenoids, Vol. 213 (Edited by A. L. Packer), pp.
420–429. Academic Press, San Diego, CA.
17. Tanielian, C., L. Golder and C. Wolff (1984) Production and
quenching of singlet oxygen by the sensitizer in dye-sensitized
photo-oxygenations. J. Photochem. 25(2-4), 117–125.
18. Scurlock, R. D., S. Nonell, S. E. Braslavsky and P. R. Ogilby
(1995) Effect of solvent on the radiative decay of singlet molecular
oxygen (a1Dg). J. Phys. Chem. 99, 3521–3526.
19. Wilkinson, F., H. P. Helman and A. B. Ross (1995) Rate con-
stants for the decay and reactions of the lowest electronically
singlet state of molecular oxygen in solution. An expanded and
revised compilation. J. Chem. Phys. Ref. Data 24, 663–677.
20. Martinez, L. A., C. G. Martinez, B. B. Klopotek, J. Lang, A.
Neuner, A. M. Braun and E. Oliveros (2000) Non radiative and
radiative deactivation of singlet molecular oxygen in micellar
media and microemulsions. J. Photochem. Photobiol. B, Biol. 58,
94–107.
21. Croux, S., M.-T. Maurette, M. Hocquaux, A. Ananides, A. M.
Braun and E. Oliveros (1990) Kinetic parameters of the reactivity
of dihydroxy naphthalenes with singlet oxygen. New J. Chem. 14,
161–167.
SUPPLEMENTAL MATERIALS
Additional figures are available for this article:
Evolution of the absorption spectra as a function of
irradiation time.
Sensitizer: rose bengal (irradiation wavelength: 547 nm),
pD = 10.5.
This material is available as part of the online article from:
http://www.blackwell-synergy.com/doi/full/10.1111/j.10.1562/
2006-09-15-RA-1041
Please note: Blackwell Publishing are not responsible for the
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REFERENCES
1. DeRosa, M. C. and R. J. Crutchley (2002) Photosensitized singlet
oxygen and its applications. Coor. Chem. Rev. 233–234, 351–371.
2. Braun, A. M., M. T. Maurette and E. Oliveros (1991) Photo-
chemical Technology, Chapter 11 (Translated by D. Ollis and
N. Serpone), pp. 445–499. Wiley, Chichester.
3. Tarr, M. and D. P. Valenzeno (2003) Singlet oxygen: The rele-
vance of extracellular production mechanisms to oxidative stress
in vivo. Photochem. Photobiol. Sci. 2, 355–361.
22. Murasecco-Suardi, P., E. Gassmann, A. M. Braun and E. Oliveros
(1987) Solvent deuterium isotope effects on acid–base equilibria.
Helv. Chim. Acta 70, 1760–1773.
23. Neckers, D. C. (1989) Rose bengal. J. Photochem. Photobiol. A,
Chem. 47, 1–29.
24. Braun, A. M., M. T. Maurette and E. Oliveros (1991) Photo-
chemical Technology, Chapter 2 (Translated by D. Ollis and N.
Serpone), pp. 85–88. Wiley, Chichester.
4. Schweitzer, C. and R. Schmidt (2003) Physical mechanisms of
generation and deactivation of singlet oxygen. Chem. Rev. 103,
1685–1757.
25. Darmanyan, A. P., W. S. Jenks and P. Jardon (1998) Charge-
transfer quenching of singlet oxygen (O2(1Dg)) by amines and
aromatic hydrocarbons. J. Phys. Chem. A 102, 7420–7426 [Erra-
tum: p. 9308].
5. Thomas, A. H., C. Lorente, A. L. Capparelli, C. G. Martınez, A.
´
M. Braun and E. Oliveros (2003) Singlet oxygen (1Dg) production
by pterin derivatives in aqueous solutions. Photochem. Photobiol
Sci. 2, 245–250.
26. Monroe, B. M. (1977) Quenching of singlet oxygen by aliphatic
amines. J. Phys. Chem. 81, 1861–1864.
6. Cabrerizo, F. M., A. H. Thomas, C. Lorente, M. L. Dantola, G.
´
Petroselli, R. Erra-Balsells and A. L. Capparelli (2004) Generation
of reactive oxygen species during the photolysis of 6-hydroxy-

534 Franco M. Cabrerizo et al.
27. Ouannes, C. and T. Wilson (1968) Quenching of singlet oxygen by
tertiary aliphatic amines. Effect of DABCO (1,4-diazabicy-
clo[2.2.2]octane). J. Am. Chem. Soc. 90, 6527–6528.
28. Foote, C. S. and E. L. Clennan (1995) Properties and reactions of
singlet dioxygen. In Active Oxygen in Chemistry, Vol. 2 (Edited by
C. S. Foote, J. S. Valentine, A. Greenberg and J. F. Liebman), pp.
105–140. Chapman & Hall, New York.
31. Gollnick, K. and J. H. E. Lindner (1973) Photosensibilisierte
Oxygenierung von Aminen. Mechanismus der Reaktion von Sin-
gulett-Sauerstoff mit Aminen. Tetrahedron Lett. 21, 1903–1906.
32. Aubry, J.-M., B. Mandard-Cazin, M. Rougee and R. V. Bensas-
son (1995) Kinetic studies of singlet oxygen [4 + 2]-cycloadditions
with cyclic 1,3-dienes in 28 solvents. J. Am. Chem. Soc. 117, 9159–
9164.
29. Clennan, E. L. and A. Pace (2005) Advances in singlet oxygen
chemistry. Tetrahedron 61, 6665–6691.
30. Cottier, L., G. Descotes, H. Nigay, J.-C. Parron and B. Gregoire
33. Schallreuter, K. U., J. M. Wood, M. R. Pittelkow, M. Gutlich, K.
R. Lemke, W. Rodl, N. N. Swanson, K. Hitzemann and I. Ziegler
(1994) Phosphatidylinositol 4-kinase: Gene structure and
requirement for yeast cell viability. Science 263, 1444–1448.
(1986) Photo-oxygenation des derives de l’hydroxymethyl-5 furf-
´ ´ ´ ´
ural-2. Bull. Soc. Chim. Fr. 5, 844–850.