Novel 6-formylpterin derivatives: Chemical synthesis and O2 to ROS conversion activities

Article abstract of DOI:10.1039/b602778d

6-Formylpterin (6FP) has been demonstrated to have strong neuroprotective effects against transient ischemia-reperfusion injury in gerbils. Also it has been shown that in rats, 6FP protected retinal neurons even when it was administered after the ischemic insult. Since there is a significant need for such a compound that effectively suppresses the events caused by the lack of oxygen supply, 6FP has attracted further investigation. Unfortunately, however, 6FP is hardly soluble in water at neutral pH and in organic solvents because of its self-assembling ability. Although a several mM solution of 6FP is available in alkaline water, it is unstable. In the present study, a novel chemical derivatization of 6FP has been developed which maintains the formyl group on the 6-position of 6FP, which is essential for the physiological activities of 6FP, and increases solubility in water and organic solvents. In the method, the 2- and 3-positions of 6FP were modified by a three component coupling reaction: 6FP was subjected to the reaction with acid chloride and N,N-dimethylformamide. The derivatives synthesized here, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3- pivaloylpteridine-4-one 1, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3- isobutyrylpteridine-4-one 2, and 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3- o-toluoylpteridine-4-one 3, showed high solubility in water (1.0-5.6 mM) and organic solvents. The O₂ conversion property has also been determined for the derivative 1. Using an oxygen electrode, it has been found that O ₂ is consumed in the presence of 1 and NADH at around pH 7.4 and that the rate of O₂ consumption is enhanced by UV-A irradiation. Electron paramagnetic resonance (EPR) analysis coupled with DMPO spin trapping has also revealed that in the presence of NADH, 1 converts O₂ to O ₂^-, which is further reduced to OH. By UV-A illumination in the analogous systems, ¹O₂ formation was observed. These results are similar to those reported previously for 6FP. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b602778d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Novel 6-formylpterin derivatives: chemical synthesis and O to ROS
2
conversion activities
Mitsuru Nonogawa, Toshiyuki Arai, Nobuyuki Endo, Seung Pil Pack, Tsutomu Kodakia,e and
Keisuke Makino*
a
b
c
d,e
d,e
Received 23rd February 2006, Accepted 13th March 2006
First published as an Advance Article on the web 3rd April 2006
DOI: 10.1039/b602778d
6
-Formylpterin (6FP) has been demonstrated to have strong neuroprotective effects against transient
ischemia-reperfusion injury in gerbils. Also it has been shown that in rats, 6FP protected retinal
neurons even when it was administered after the ischemic insult. Since there is a significant need for
such a compound that effectively suppresses the events caused by the lack of oxygen supply, 6FP has
attracted further investigation. Unfortunately, however, 6FP is hardly soluble in water at neutral pH
and in organic solvents because of its self-assembling ability. Although a several mM solution of 6FP is
available in alkaline water, it is unstable. In the present study, a novel chemical derivatization of 6FP has
been developed which maintains the formyl group on the 6-position of 6FP, which is essential for the
physiological activities of 6FP, and increases solubility in water and organic solvents. In the method, the
2
- and 3-positions of 6FP were modified by a three component coupling reaction: 6FP was subjected to
the reaction with acid chloride and N,N-dimethylformamide. The derivatives synthesized here, 2-(N,N-
dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridine-4-one 1, 2-(N,N-dimethylamino-
methyleneamino)-6-formyl-3-isobutyrylpteridine-4-one 2, and 2-(N,N-dimethylamino-
methyleneamino)-6-formyl-3-o-toluoylpteridine-4-one 3, showed high solubility in water (1.0–5.6 mM)
and organic solvents. The O
an oxygen electrode, it has been found that O
pH 7.4 and that the rate of O consumption is enhanced by UV-A irradiation. Electron paramagnetic
2
conversion property has also been determined for the derivative 1. Using
2
is consumed in the presence of 1 and NADH at around
2
resonance (EPR) analysis coupled with DMPO spin trapping has also revealed that in the presence of
•
−
•
NADH, 1 converts O
2
to O
2
, which is further reduced to OH. By UV-A illumination in the analogous
1
systems, O
2
formation was observed. These results are similar to those reported previously for 6FP.
Introduction
Ischemia-reperfusion injury (IRI) occurs in cardiac infarction,
brain infarction, organ transplantation, and so on, resulting in
1
apoptotic cell death. Protective agents against IRI have been
important research targets in pharmaceutical studies over the
past two decades. We have demonstrated that 6-formylpterin
Scheme 1 Structures of pteridine derivatives.
2
(
6FP), which is known as a xanthine oxidase inhibitor, has potent
6
FP generates reactive oxygen species (ROS) from molecular
3a
neuroprotective effects against transient IRI in gerbils. Also we
have shown the similar effects of 6FP in rat retinal IRI.
Many pteridines are endogenous: they are important electron
transfer compounds in biological systems. They act as coenzymes
and participate in important biological functions. 6FP is one of
the pteridine derivatives (Scheme 1).
6
oxygen (O
2
) in the presence of reducing agents such as NAD(P)H.
3b
1
Recently 6FP was shown to produce singlet oxygen ( O
irradiation of long-wavelength ultraviolet light (UV-A) in aqueous
solutions.
Pteridines are generally insoluble compounds. This nature is due
to strong intermolecular hydrogen bonds which lead to the self-
assembling structure of pteridines. To overcome this drawback,
2
) under
7
4,5
8
9,10
a number of trials have been reported.
The intermolecular
a
Institute of Advanced Energy, Kyoto University, Gokasyo, Uji 611-0011,
hydrogen bonds are formed by amino and amide functions on
the pteridine ring and it is known that protection of the N- or
O-atoms of the amino or the amide function increases solubility
in water and organic solvents. Similarly, acylation of the amino
group affects the solubility. 6FP is also poorly soluble in water at
neutral pH and in organic solvents for the same reason. Although a
several mM solution of 6FP is available in alkaline water, it decom-
poses into pterin-6-carboxylic acid and 6-hydroxymethylpterin
Japan
b
Department of Anesthesia, Kyoto University Hospital, Sakyo-ku, Kyoto
6
06-8507, Japan
c
9
Wakasa Wan Energy Research Center, Tsuruga 914-0129, Japan
d
International Innovation Center, Kyoto University, Gokasho, Uji 611-0011,
Japan. E-mail: kmak@iae.kyoto-u.ac.jp; Fax: +81-774-38-3524; Tel: +81-
10
7
74-38-3517
e
CREST, JST (Japan Science and Technology Agency), Kyoto University,
Gokasho, Uji 611-0011, Japan
This journal is © The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 1811–1816 | 1811

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by the Cannizzaro reaction or photooxidation. For the clinical
application of 6FP, 6FP must be modified, possibly by the
introduction of bulky groups onto the ring, so that it becomes
soluble in water. Also, it is worthwhile to explore the detailed
chemical properties of 6FP to reveal the relationship between its
physiological activities and its chemical natures.
Scheme 2 The coupling reaction of 6FP with pivaloyl chloride and DMF
in the presence of N,N-diisopropylethylamine.
We assume that maintaining the formyl group at the 6-position
is essential for the notable physiological activities of 6FP, and
in the present study, therefore, we developed novel chemical
derivatization methods which not only improve 6FP solubility
but maintain its physiological activity.
Next, we used isobutyryl chloride, propionyl chloride and o-
toluoyl chloride instead of pivaloyl chloride in the analogous
reaction. The results are summarized in Table 1.
The reaction of isobutyryl chloride with 6FP and DMF
resulted in 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-
isobutyrylpteridine-4-one 2 in a yield of 88% (Entry 2).
The reaction of o-toluoyl chloride with 6FP and DMF
gave rise to 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-
o-toluoylpteridine-4-one 3 in a yield of 52% (Entry 3). When N,N-
diisopropylethylamine or acid chloride was absent, or when DMF
was absent and 2-butanone was used as a solvent, 6FP also did
not react. When the reaction was stopped in 20 minutes, 2-(N,N-
Results and discussion
First, a coupling reaction of three components was performed.
When 6FP (1.0 mmol) was subjected to the reaction with
pivaloyl chloride (3.5 mmol) and N,N-dimethylformamide (DMF)
(
12 ml) in the presence of N,N-diisopropylethylamine (4.5 mmol)
at room temperature, 2-(N,N-dimethylaminomethyleneamino)-6-
formyl-3-pivaloylpteridine-4-one 1 was obtained in a yield of
11
dimethylaminomethyleneamino)-6-formylpteridine-4-one 4 was
isolated by column chromatography. Additionally, although the
reaction of 4 with pivaloyl chloride produced 1 in the presence of
N,N-diisopropylethylamine in DMF, the reaction did not proceed
9
5% as a 2- and 3-position modified 6FP derivative as shown
1
13
in Scheme 2. 1 was identified by MS, IR, H-NMR, C-NMR,
and elemental analysis.
Table 1 Coupling reactions of 6FP with acid chlorides and DMF and the water solubility of 6FP and the 6FP derivatives obtained
Entry
1
Acid chloride
Product
Yield (%)
95
Water solubility/mM
3.8
2
3
88
52
5.6
1.0
4
5
2.3
0.02
1
812 | Org. Biomol. Chem., 2006, 4, 1811–1816
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in CHCl . According to these results, the first step of this reaction
3
would be the activation of DMF and probably the Vilsmeyer
type activated complex is involved. The reaction of the activated
complex with the amino group on the 2-position of 6FP would
give 4. Then the reaction of the complex with the 3-position of 4
proceeds and gives 1 (Scheme 3).
These 6FP derivatives showed high solubility in methanol,
ethanol, and other organic solvents. The water solubilities of these
derivatives are summarized in Table 1, indicating that the 2- and
3
-position modified 6FP derivatives have high water solubility
and are more soluble than 4 in which only the 2-position is
modified. These results suggest that the strong intermolecular
hydrogen bonds between 6FP molecules are eliminated by these
modifications. 3 showed lower solubility than 1, 2 and 4 probably
because of the hydrophobic nature of the toluoyl substituent
Fig. 1
O
2
consumption in the solution containing 6FP (2 mM) or 1
(
2 mM) and NADH (6 mM). The time course of the changes in oxygen
(
Entry 3).
concentration measured by oxygen electrodes is shown. a: Mixture of 1 and
NADH in PBS (pH 7.4); b: mixture of 6FP and NADH in PBS (pH 8.3).
Arrow 1 indicates the addition of NADH to the solutions a and b. Arrow 2
indicates irradiation with fluorescence light. Arrow 3 indicates irradiation
with UV-A light.
We further examined the reaction of 6FP derivative 1 with O .
2
In a previous paper, we have shown that the oxygen concentration
of the solution containing NADH decreased linearly and dose
6b
dependently when 6FP was added to the solution. Using a similar
method, we measured the O consumption activity of 1 using
oxygen electrodes. In Fig. 1, a comparison of the O consumption
2
2
We investigated the mechanism for the above reaction by the
EPR spin trapping technique using 5,5-dimethyl-1-pyrroline-N-
oxide (DMPO) as a spin trap. EPR spectra obtained from the
reaction of 1 in the presence of NADH are shown in Fig. 2.
After adding 1 to the solution containing NADH, prominent EPR
spectra were obtained (Fig. 2(a)), consisting of a quartet with
a 1 : 2 : 2 : 1 intensity ratio with hyperfine splitting constants
(hfsc’s) of a(N) = 1.49 mT and a(bH) = 1.49 mT which is
by 1 with that by 6FP is depicted. The solution a contained 2 mM
of 1 (pH 7.4) and the solution b contained 2 mM of 6FP (pH 8.3).
Arrow 1 indicates the time of NADH addition to the solution.
Arrows 2 and 3 indicate the time of irradiation with fluorescence
light and UV-A light, respectively. The rate of O
by 1 was lower than that by 6FP. However, both O
2
consumption
consumption
2
rates were accelerated by irradiation with fluorescence light and
UV-A light. In these experiments, the rate of O consumption by
at physiological pH (pH 7.4) showed the same tendency as that
of 6FP.
12
2
assignable to DMPO-OH, indicative that the signals were the
results of trapping hydroxyl radicals ( OH). These signals were
totally quenched by superoxide dismutase (SOD) which converts
•
1
Scheme 3 A plausible reaction mechanism for the conversion of 6FP to 1 in the presence of pivaloyl chloride and N,N-diisopropylethylamine in DMF.
Org. Biomol. Chem., 2006, 4, 1811–1816 | 1813
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water solubility of the derivatives was hundreds of times larger
than that of 6FP. Water solubility is an important factor for the
practical use of 6FP as a pharmacologically active substance in
living systems. We ascertained that the derivatives reduce O to
2
generate reactive oxygen species in the same way as 6FP. We
believe that these functions are deeply related to the physiological
activity of 6FP. Other modifications of 6FP and their effects on
the physiological and pharmacological activities of the derivatives
are now under investigation.
Experimental
Materials
6FP was obtained from Sankyo Kasei Kogyo (Hiratsuka,
Japan). N,N-Diisopropylethylamine, o-toluoyl chloride and 4-
oxo-2,2,6,6-tetramethyl-4-piperidine (4-oxo-TEMP) were pur-
chased from SIGMA Chemical Co. (St. Louis, MO, USA), N,N-
dimethylformamide (DMF) and isobutyryl chloride from Wako
(
Osaka, Japan), pivaloyl chloride from Tokyo Kasei (Tokyo,
Fig. 2 (a), (b) and (c): EPR spectra obtained from a PBS solution
containing 1 (2 mM), NADH (6 mM) and DMPO (1.47 M) under
fluorescence light. (a) Mixture of 1, NADH and DMPO; (b) mixture
Japan), NADH from Oriental Yeast (Tokyo, Japan), and 5,5-
dimethyl-1-pyrroline-N-oxide (DMPO) from Labotec Co. (Tokyo,
Japan).
−
1
of 1, NADH and DMPO in the presence of SOD (2000 units ml ); (c)
−
1
4
mixture of 1, NADH, DMPO, SOD (2000 units ml ), FeSO (0.1 mM) and
Synthesis
DTPA (1 mM) as a chelating agent. (d), (e) and (f): EPR spectra obtained
under UV-A irradiation from a PBS solution containing 1 (50 lM) and
2
-(N,N -Dimethylaminomethyleneamino)-6-formyl-3-pivaloyl-
4
-oxo-TEMP (1 mM). (d) Without 1; (e) mixture of 1 and 4-oxo-TEMP;
pteridine-4-one (1). 6FP (191.1 mg, 1.0 mmol), N,N-
diisopropylethylamine (780 ll, 4.5 mmol) and pivaloyl chloride
430 ll, 3.5 mmol) were mixed in DMF (12 ml) with stirring
1
(
f) mixture of 1, 4-oxo-TEMP, and NaN
3
(100 lM) as a scavenger of O
2
.
2+
Signals appearing at both high and low field correspond to Mn installed
in the EPR cavity as a reference.
(
under nitrogen for 2 h at room temperature. During this time,
the suspended solid 6FP dissolved. The mixture was evaporated
in vacuo and the resultant brown oil was purified by column
chromatography eluting with chloroform–methanol (99 : 1) to
•
−
the superoxide anion radical ( O
2
) to H
2
O in the presence of a
2
proton source (Fig. 2(b)), indicating that the signals were generated
•
•
−
by the DMPO-trapping of OH converted from O
was added to the reaction mixture of 1, NADH and SOD, a
: 2 : 2 : 1 quartet of hydroxyl radical signals was observed
Fig. 2(c)). It should be noted that in the presence of ferrous ion
Fe ), OH is generated from H
reaction. Consequently these results show that O
2
. When FeSO
4
give the title compound 1 as a yellow solid (312.4 mg, 95%),
◦
mp 178–179 C. d
(
H
(600 MHz; CDCl
3
) 10.21 (1H, s, CHO), 9.32
), 3.18 (3H, s,
) 191.1, 183.8,
1
1H, s, 7-H), 9.02 (1H, s, NCHN), 3.29 (3H, s, CH
3
(
(
t
2
+
•
CH
3
) and 1.43 (9H, s, Bu). d
C
(150 MHz; CDCl
3
2
2
O via the iron-catalyzed Fenton
13
•
−
160.5, 159.7, 157.9, 157.8, 148.9, 143.1, 130.3, 44.1, 42.2, 36.2
2
generated
+
and 27.9. (Found: C, 54.77; H, 5.46; N, 25.15; O, 14.33%; M ,
by the reaction of 1 and NADH under light illumination was
3
30.1448. C15
H
18
N
6
O requires C, 54.54; H, 5.49; N, 25.44; O,
3
converted to H
NADH, O was converted to O
of 1 with O
2
O
2
by SOD. In summary, in the presence of 1 and
−
1
•
−
•
14.53%; M, 330.3421). mmax/cm 1766, 1693 and 1639 (CO). The
water solubility (3.8 mM) was determined by UV-spectroscopy.
When the reaction was stopped after 20 min, 2-(N,N-
dimethylaminomethyleneamino)-6-formylpteridine-4-one 4 was
2
2
and then to OH. The reaction
2
under UV-A irradiation was also investigated by
analogous EPR analysis. When the solution containing 1 and 4-
oxo-2,2,6,6-tetramethylpiperidine (4-oxo-TEMP) was irradiated
with UV-A light, a 1 : 1 : 1 triplet (a(N) = 1.61 mT) of
isolated by column chromatography as
a
yellow solid.
SO) 12.26 (1H, bs, NH), 10.02 (1H, s, CHO),
.13 (1H, s, 7-H), 8.91 (1H, s, NCHN), 3.28 (3H, s, CH ) and
requires M,
d
H
(600 MHz; (CD
)
3 2
4
-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO) was
9
3
observed (Fig. 2(e)). It should be noted that 4-oxo-TEMP has
+
1
14
3.15 (3H, s, CH
3
). (Found: M , 246.0874. C10
H
10
N
6
O
2
been used as a
O
2
detector. These signals were completely
1
246.0865). The water solubility (2.3 mM) was determined by UV-
spectroscopy.
quenched by sodium azide, a scavenger of O
result demonstrates that the signals were derived from the reaction
of 4-oxo-TEMP with produced O
2
(Fig. 2(f)). This
1
2
, and that 1 has the ability to
2
-(N,N-Dimethylaminomethyleneamino)-6-formyl-3-isobutyryl-
1
generate O
2
under UV-A light irradiation.
pteridine-4-one (2). 6FP (192.0 mg, 1.0 mmol), N,N-diiso-
propylethylamine (780 ll, 4.5 mmol) and isobutyryl chloride
(
370 ll, 3.5 mmol) were mixed in DMF (6 ml) with stirring
Conclusions
under nitrogen for 2 h at room temperature. During this time,
the suspended solid 6FP dissolved. The mixture was evaporated
in vacuo and the resultant brown oil was purified by column
chromatography eluting with chloroform–methanol (99 : 1) to
We synthesized novel 6FP derivatives and succeeded in increasing
the water and organic solvent solubility of 6FP. The NH and
NH
2
of 6FP were modified in one single action in one pot. The
1
814 | Org. Biomol. Chem., 2006, 4, 1811–1816
This journal is © The Royal Society of Chemistry 2006

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1
give the title compound 2 as a yellow solid (277.7 mg, 88%), mp
Measurements of O
2
production under UV-A irradiation in an
◦
1
92–193 C. d
H
(600 MHz; CDCl
3
) 10.22 (1H, s, CHO), 9.32
), 3.21 (1H, m,
) and 1.37 (6H, d, J 7.2, iPr). d (150 MHz;
) 191.1, 180.1, 160.4, 159.4, 157.9, 157.7, 148.8, 143.1,
30.5, 42.0, 40.3, 35.9 and 18.3. (Found: C, 52.99; H, 5.14; N,
aqueous solution of 1
(
1H, s, 7-H), 9.00 (1H, s, NCHN), 3.29 (3H, s, CH
CH), 3.16 (3H, s, CH
CDCl
3
1
To examine whether 1 produces O
spectroscopy was used and 4-oxo-2,2,6,6-tetramethylpiperidine
4-oxo-TEMP), which reacts with
nitroxide radical, 4-oxo-2,2,6,6-tetramethyl-4-piperidinyloxy (4-
2
under UV-A irradiation, EPR
3
C
3
1
(
O
2
and produces a stable
1
2
+
6.58; O, 15.00%; M , 316.1290. C14
H
16
N
6
O requires C, 53.16;
3
−
1
1
oxo-TEMPO), was employed as a O detector. The solution of 1
2
H, 5.10; N, 26.57%; M, 316.3155). mmax/cm 1778, 1697 and
643 (CO). The water solubility (5.6 mM) was determined by
UV-spectroscopy.
and 4-oxo-TEMP was placed in a flat quartz EPR aqueous cell,
which was fixed in the cavity of the EPR spectrometer. The EPR
spectrum recording started after 10 s of UV-A irradiation in a
microwave cavity with a light focused from a RUVF-203F xenon
arc-lamp (Radical Research Co., Tokyo, Japan) with a 360 nm
band path filter operating at 200 mW. The EPR spectra were
recorded with the following EPR parameters: microwave power:
5 mW; field: 329 ± 5 mT (9.2331 GHz); modulation: 0.079 mT;
time constant: 0.03 s; amplitude: 320; and sweep time: 2 min. The
intensity of 4-oxo-TEMPO and the hfsc’s were calculated in a
similar way to that shown above. In some experiments, sodium
1
2
-(N,N-Dimethylaminomethyleneamino)-6-formyl-3-o-toluoyl-
pteridine-4-one (3). 6FP (190.0 mg, 1.0 mmol), N,N-
diisopropylethylamine (780 ll, 4.5 mmol) and o-toluoyl chloride
(
460 ll, 3.5 mmol) were mixed in DMF (6 ml) with stirring
under nitrogen for 2 h at room temperature. During this time,
the suspended solid 6FP dissolved. The mixture was evaporated
in vacuo and the resultant brown oil was purified by column
chromatography eluting with chloroform–methanol (99 : 1) to
1
2
give the title compound 3 as a yellow solid (187.8 mg, 52%),
azide was used as a scavenger of O .
◦
mp 140–144 C. d
H
(600 MHz; CDCl ) 10.23 (1H, s, CHO),
3
9
.34 (1H, s, 7-H), 8.94 (1H, s, NCHN), 7.59 (1H, d, J 7.8,
Ph), 7.48 (1H, t, J 7.2, Ph), 7.37 (1H, d, J 7.2, Ph), 7.23 (1H,
t, J 7.5, Ph), 3.20 (3H, s, CH ), 2.88 (3H, s, CH ) and 2.77
(150 MHz; CDCl ) 191.2, 168.7, 160.7, 159.2,
58.1, 158.0, 148.9, 143.1, 142.5, 133.7, 132.2, 131.2, 131.1, 130.6,
Acknowledgements
3
3
This work is partially supported by the Grants-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science
and Technology, Japan to K.M. (no. 15350099), and by the Center
of Excellence (COE) program for the "Establishment of COE
on Sustainable-Energy System", Grant-in-aid for Science and
Technology Promotion from the Ministry of Education, Science,
Sports and Culture, Japan. This work was also supported by
CREST of the Japan Science and Technology Corporation.
(
3H, s, PhCH
3
). d
C
3
1
1
+
26.4, 41.9, 35.6 and 21.9. (Found: M , 364.1286. C18
H
16
N
6
O
3
−
1
requires M, 364.3582). mmax/cm 1743, 1695 and 1633 (CO). The
water solubility (1.0 mM) was determined by UV-spectroscopy.
Oxygen consumption measurements with an oxygen electrode
The O consumption during the reaction of 1 in the presence
2
of NADH was measured polarographically using a Clarke oxy-
gen electrode (model 5300; Yellow Springs Instruments; Yellow
Springs, OH, USA) at 37 C in a 4 ml reaction mixture. The
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EPR spectra obtained from 1 in the presence of NADH
To determine whether 1 produces ROS in the presence of NADH
and what species are generated, EPR spectroscopy combined
with spin trapping with DMPO was employed. For the sample
preparation, a solution containing DMPO (1.47 M) was employed.
Diethylenetriaminepentaacetic acid (DTPA) (1 mM) was added
to the solution to avoid baseline contamination. The EPR spectra
were recorded on a Model JES-TE300 spectrometer (JEOL Ltd.;
Tokyo, Japan). The EPR settings were as follows: microwave
power: 5 mW; field: 334.5 ± 5 mT (9.4160 GHz); modulation:
4
(
1
c) C. A. Nichol, G. K. Smith and D. S. Duch, Annu. Rev. Biochem.,
985, 54, 729.
5
6
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Ishii, K. Hirota, K. Makino and K. Fukuda, Free Radical Biol. Med.,
0
2
.079 mT; time constant: 0.03 s; amplitude: 500; and sweep time:
min. Sample solutions were placed in a flat quartz EPR aqueous
2
001, 30, 248.
cell fixed in the cavity of the EPR spectrometer. The hyperfine split-
7
A. H. Thomas, C. Lorente, A. L. Capparelli, C. G. Martinez,
A. M. Braun and E. Oliveros, Photochem. Photobiol. Sci., 2003, 2,
245.
2
+
ting constants (hfsc’s) were calculated based on the Mn marker,
which was inserted into the cavity of the EPR spectrometer.
This journal is © The Royal Society of Chemistry 2006
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8
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1
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