Formation of a diimino-imidazole nucleoside from 2′-deoxyguanosine by singlet oxygen generated by methylene blue photooxidation

Article abstract of DOI:10.1016/S0968-0896(03)00124-X

Singlet oxygen (¹O₂) is capable of inducing genotoxic, carcinogenic and mutagenic effects. It has previously been reported that the reaction of ¹O₂ with 2′-deoxyguanosine, which is a major target of ¹O₂ among the DNA constituents, leads to formation of various oxidized products including 8-oxo-7,8-dihydro-2′-deoxyguanosine and spiroiminodihydantoin, amino-imidazolone and diamino-oxazolone nucleosides. In addition to these products, we report that a novel diimino-imidazole nucleoside, 2,5-diimino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-2H,5H- imidazole (dD), is formed by reaction of 2′-deoxyguanosine with ¹O₂ generated by irradiation with visible light in the presence of methylene blue under aerobic conditions. Its identification is based on identical chromatographic and spectroscopic data with an authentic compound, which we recently isolated and characterised from the reaction mixture of 2′-deoxyguanosine with reagent HOCl and a myeloperoxidase-H₂O₂-Cl^- system. The yield of dD was increased by D₂O and decreased by azide. dD was not generated from 8-oxo-7,8-dihydro-2′-deoxyguanosine. These results indicate that dD is generated by ¹O₂ directly from 2′-deoxyguanosine, but not via 8-oxo-7,8-dihydro-2′-deoxyguanosine. dD may play a role in the genotoxicity of singlet oxygen in cells.

Full text of DOI:10.1016/S0968-0896(03)00124-X

Bioorganic & Medicinal Chemistry 11 (2003) 2157–2162
Formation of a Diimino-Imidazole Nucleoside from
20-Deoxyguanosine by Singlet Oxygen Generated by
Methylene Blue Photooxidation
Toshinori Suzuki, Marlin D. Friesen and Hiroshi Ohshima*
International Agency for Research on Cancer, 150 Cours Albert Thomas, F 69372Lyon Cedex 08, France
Received 29 November 2002; accepted 18 February 2003
Abstract—Singlet oxygen (1O2) is capable of inducing genotoxic, carcinogenic and mutagenic effects. It has previously been repor-
ted that the reaction of O2 with 20-deoxyguanosine, which is a major target of O2 among the DNA constituents, leads to forma-
tion of various oxidized products including 8-oxo-7,8-dihydro-20-deoxyguanosine and spiroiminodihydantoin, amino-imidazolone
and diamino-oxazolone nucleosides. In addition to these products, we report that a novel diimino-imidazole nucleoside, 2,5-diimino-
1
1
0
1
4-[(2-deoxy-b-d-erythro-pentofuranosyl)amino]-2H,5H-imidazole (dD), is formed by reaction of 2 -deoxyguanosine with O2 gen-
erated by irradiation with visible light in the presence of methylene blue under aerobic conditions. Its identification is based on
identical chromatographic and spectroscopic data with an authentic compound, which we recently isolated and characterised from
the reaction mixture of 20-deoxyguanosine with reagent HOCl and a myeloperoxidase–H2O2–Clꢀ system. The yield of dD was
increased by D2O and decreased by azide. dD was not generated from 8-oxo-7,8-dihydro-20-deoxyguanosine. These results indicate
that dD is generated by 1O2 directly from 20-deoxyguanosine, but not via 8-oxo-7,8-dihydro-20-deoxyguanosine. dD may play a role
in the genotoxicity of singlet oxygen in cells.
# 2003 Elsevier Science Ltd. All rights reserved.
1
Introduction
It has been proposed that O2 may be involved in the
human host defense system.9 Steinbeck et al.10 reported
that about 20% of the oxygen consumed by neutrophils
was converted to singlet oxygen within the phagosomes.
However, the general consensus is that if singlet oxygen
is produced in neutrophils, it is only in very small
amounts.11 On the other hand, there is no doubt that
appreciable amounts of hypochlorous acid (HOCl) are
discharged into the extracellular space surrounding
neutrophils.11 HOCl generated by myeloperoxidase is of
central importance in the host defense mechanism. The
HOCl formed also has the potential to harm normal
tissue and contribute to inflammatory injury. HOCl has
been reported to react with nucleic acid bases to form
various compounds.12ꢀ17 Recently, we showed that dS
was generated from 8-oxodG and dG by HOCl as a
major product.18,19 We also found that a novel diimino-
imidazole nucleoside (dD) was generated from dG by
the reaction with reagent HOCl and a myeloperoxidase–
H2O2–Clꢀ system in addition to dIz and 8-chloro-20-
deoxyguanosine.20,21 The involvement of 1O2 in the for-
mation of these products was ruled out for the reagent
HOCl system under neutral conditions.21 However, for
the myeloperoxidase–H2O2–Clꢀ system under mildly
Singlet oxygen (1O2) is an excited state of molecular
oxygen. O2 can be produced in biological systems by
1
photoexcitation through energy transfer (type II reac-
tion) upon exposure of endogenous photosensitizers.1,2
1O2 is capable of inducing genotoxic, carcinogenic and
mutagenic effects.1,2 Among the constituents of DNA,
20-deoxyguanosine (dG) is a major target of O2.3 The
1
reaction of 1O2 with dG leads to predominant formation
of a spiroiminodihydantoin nucleoside (dS) together with
8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxodG), an
amino-imidazolone nucleoside (dIz) and its hydrolyzed
nucleoside, a diamino-oxazolone nucleoside (dZ).4,5
8-OxodG is a much better substrate than dG for the
1
reaction with O2.6 dS, dIz, dZ and other compounds
including cyanuric acid and an oxidized guanidino-
hydantoin nucleoside have been reported as products of
the reaction of 8-oxodG with 1O2.7,8 However, dS was a
minor product in this system.
*Corresponding author. Tel.: +33-4-7273-8509; fax: +33-4-7273-
8088; e-mail: ohshima@iarc.fr
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00124-X

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T. Suzuki et al. / Bioorg. Med. Chem. 11 (2003) 2157–2162
1
acidic conditions, the involvement of O2 could not be
excluded. There is a possibility that dD could be gener-
Time course of the reaction of dG with the methylene
blue photooxidation system
1
ated from dG by O2 in addition to HOCl.
We investigated the time course of the methylene blue
photooxidation of dG. Figure 3A shows the concentra-
tions of unreacted dG when 1 mM dG and 0.5 mM
methylene blue were irradiated with visible light for
various times (0–2 h) at pH 7.4 and 37 ꢁC under aerobic
conditions. The consumption of dG increased in a time-
dependent manner. Figure 3B shows the changes in
yield of the products (dD, dIz and 8-oxodG). The yields
of dD and dIz increased in a time-dependent manner.
The yield of 8-oxodG increased rapidly in the early
stage of the reaction and reached a plateau, showing a
profile characteristic of a reaction intermediate. We
could not quantify dS and dZ in the reaction mixture,
since both were eluted as broadened peaks near the
chromatographic void volume on RP-HPLC analysis
and may overlap with other peaks. For 1 h incubation,
the concentrations of the products were 1.3 mM dD,
8.6 mM dIz and 9.1 mM 8-oxodG, with 127 mM dG
consumed. Thus, the yields were 1.0% dD, 6.8% dIz
and 7.2% 8-oxodG relative to the dG consumed. The
ratio of the yield of dD to that of dIz (dD/dIz) was
0.15.
We have now studied the reaction of dG with a meth-
ylene blue photooxidation system under aerobic condi-
tions, that is, with singlet oxygen, and found that dD is
also generated in this reaction mixture.
Results
Formation of dD from dG by methylene blue
photooxidation
When the mixture obtained from the methylene blue
photooxidation of dG at pH 7.4 and 37 ꢁC for 1 h under
aerobic conditions was analyzed by RP-HPLC, several
peaks were detected on the chromatogram in addition
to unreacted dG (Fig. 1). Among the product peaks,
four were identified as dS, dZ, dIz and 8-oxodG by
coincidence of the retention time and UV spectrum of
authentic samples. In addition to these previously
reported products of the dG–methylene blue photo-
oxidation system, we observed a peak (Fig. 1; 1) with a
retention time of 7.8 min. Product 1 exhibited a UV spec-
trum with lmax 255 and 334 nm (Fig. 1, inset). The reten-
tion time and UV spectrum of product 1 were identical to
those of the authentic diimino-imidazole nucleoside (dD)
(Fig. 2), a nucleoside recently identified by our group as a
product of the reaction of dG with HOCl.21 Co-injection
of the isolated compound and authentic dD to RP-HPLC
gave a single peak with an identical UV spectrum. On the
basis of these results, we conclude that dD is generated
from dG by methylene blue photooxidation.
Effect of D2O and NaN3 on the reaction of dG with the
methylene blue photooxidation system
To identify the reactive species generating dD from dG
with the methylene blue photooxidation system, the
reaction was performed in the presence of D2O, an
enhancer of singlet oxygen (1O2) reactions and NaN3, a
1
quencher of O2. As shown in Table 1, the consump-
tion of dG and the yields of dD and dIz increased when
Figure 1. RP-HPLC chromatogram of a reaction mixture of dG with methylene blue photooxidation. A solution (1 mL) of 1 mM dG containing 0.5
mM methylene blue and 100 mM sodium phosphate buffer (pH 7.4) in an NMR tube (5 mm i.d.) was irradiated with visible light (550 nm cut-off
filter) at 37 ꢁC for 1 h under aerobic conditions. RP-HPLC analysis was conducted immediately after the reaction. Inset is an on-line UV spectrum of
product 1. The peak indicated by an asterisk is an impurity present in the starting dG.

T. Suzuki et al. / Bioorg. Med. Chem. 11 (2003) 2157–2162
2159
Formation of dD by reaction of dG with other
photooxidation systems
We examined the formation of dD from dG with other
photooxidation systems. As shown in Table 3, rose
bengal with visible light, another type II (1O2, energy
transfer) dominant photooxidation system, generated
dD with a dD/dIz ratio (0.10) similar to that in the
methylene blue system. For type I (hydrogen atom
abstraction or electron transfer) dominant photooxida-
tion systems, we used riboflavin and benzophenone with
UV light. Although dD was generated by the riboflavin
system, the dD/dIz ratio (0.019) was one order of mag-
nitude smaller than for the type II photooxidation sys-
tems. dD was not detected in the benzophenone system.
Figure 2. The structure of product 1, a diimino-imidazole nucleoside,
2,5-diimino-4-[(2-deoxy-b-d-erythro-pentofuranosyl)amino]-2H,5H-
imidazole (dD).
the reactions were carried out in the presence D2O and
decreased in the presence of NaN3. The dD/dIz ratio
was increased by D2O and decreased by NaN3 (Table
1
1). These results suggest that O2 is involved in the for-
mation of dD from dG with the methylene blue photo-
oxidation system.
Reaction of dD with the photooxidation systems
We have reported that dD is not stable in a neutral
solution, hydrolysing gradually with a half-life of 4.9 h
at pH 7.4 and 37 ꢁC, to give rise to dIz and its base
moiety (Iz).21 To examine whether dD reacts with pho-
tooxidation systems and disappears readily from the
reaction mixture, authentic dD was incubated with
photooxidation systems and the reaction was monitored
by RP-HPLC. Figure 4 shows the change in dD con-
centration when 18.5 mM dD was incubated in 100 mM
sodium phosphate buffer at pH 7.4 and 37 ꢁC for 0–2 h
with or without photooxidation systems. The methylene
blue photooxidation system did not affect the rate of dD
Reaction of 8-oxodG with the methylene blue
photooxidation system
In order to determine whether dD is generated via 8-
oxodG, we analyzed by RP-HPLC the products of
methylene blue photooxiation of 8-oxodG at pH 7.4
and 37 ꢁC for 1 h under aerobic conditions. As shown in
Table 2, dD was not detected (detection limit, 0.03 mM),
while a large amount of dIz was formed. The reaction of
8-oxodG was enhanced by D2O and suppressed by
NaN3; in neither case was dD detected.
Figure 3. Time course of methylene blue photooxidation of dG. (A) concentration of unreacted dG (open circles). (B) Formation of dD (closed
circles), dIz (open squares), and 8-oxodG (open triangles). A solution (1 mL) of 1 mM dG containing 0.5 mM methylene blue and 100 mM sodium
phosphate buffer (pH 7.4) in an NMR tube (5 mm i.d.) was irradiated with visible light (550nm cut-off filter) at 37 ꢁC for 0–2 h under aerobic
conditions. The concentrations were determined by RP-HPLC analysis, conducted immediately after the reaction. MeansꢂSD (n=3) are presented.
Table 1. Effects of D2O and NaN3 on the reaction of dG with the methylene blue photooxidation systema
Additives
dD (mM)
dIz (mM)
8-oxodG (mM)
dG (mM)
dD/dIz
None
+D2O (75%)
+NaN3 (10 mM)
1.3ꢂ0.1
5.4 ꢂ0.0
0.06ꢂ0.01
8.6ꢂ0.2
19.3 ꢂ0.1
0.6ꢂ0.1
9.1ꢂ0.2
7.2 ꢂ0.1
1.1ꢂ0.1
873ꢂ7
647ꢂ8
996ꢂ6
0.15
0.28
0.10
aA solution containing 1 mM dG, 0.5 mM methylene blue, and 100 mM sodium phosphate buffer (pH 7.4) was irradiated with visible light (550 nm
cut-off filter) in the presence of 75% D2O or 10mM NaN at 37 ꢁC for 1 h under aerobic conditions. The reaction mixture was analyzed by RP-
3
HPLC immediately after the reaction. MeansꢂSD (n=3) are presented.

2160
T. Suzuki et al. / Bioorg. Med. Chem. 11 (2003) 2157–2162
Table 2. Effects of D2O and NaN3 on the reaction of 8-oxodG with
the methylene blue photooxidation systema
Additives
dD (mM)
dIz (mM)
8-oxoG (mM)
None
+D2O (75%)
+NaN3 (10mM)
<0.03
<0.03
<0.03
39.1ꢂ4.4
129.3ꢂ5.5
2.5ꢂ0.2
679ꢂ14
283ꢂ18
931ꢂ10
aA solution containing 1 mM 8-oxodG, 0.5 mM methylene blue, and
100 mM sodium phosphate buffer (pH 7.4) was irradiated with visible
light (550nm cut-off filter) in the presence of 75% D 2O or 10 mM
NaN3 at 37 ꢁC for 1 h under aerobic conditions. The reaction mixture
was analyzed by RP-HPLC immediately after the reaction.
MeansꢂSD (n=3) are presented.
hydrolysis. The riboflavin photooxidation system accel-
erated the hydrolysis of dD, but not greatly.
Figure 4. Effect of photooxidation on the hydrolysis of dD. Con-
centrations of dD without sensitizer, light and air bubbling (closed
circles), with methylene blue, visible light and air bubbling (open
squares), with riboflavin, UV light and air bubbling (open triangles),
are plotted against the incubation time. A solution (1 mL) of 18.5 mM
dD and 100 mM sodium phosphate buffer (pH 7.4) with or without
0.5 mM photosensitizers was incubated in the NMR tube at 37 ꢁC for
0–2 h. The solution was irradiated with visible light (>550nm) for
methylene blue photooxidation and UV light (366 nm) for riboflavin
photooxidation under aerobic conditions. The concentrations of dD
were determined by RP-HPLC analysis, conducted immediately after
the reaction. MeansꢂSD (n=3) are presented.
Discussion
In the present study, we identified one of the products
formed from dG by methylene blue photooxidation as a
dD, which was recently reported as a novel product in
the reaction of dG with HOCl.21 The yield of dD from
dG with the methylene blue photooxidation system
increased in the presence of D2O and was decreased by
addition of azide. No dD was formed from 8-oxodG by
the methylene blue photooxidation system. The ribo-
flavin photooxidation system generated only a small
amount of dD relative to that of dIz. dD does not react
with the methylene blue photooxidation system. dD
reacted with the riboflavin photooxidation system, but
only slowly. On the basis of these combined observa-
tions, we conclude that dD is formed by singlet oxygen
directly from dG, not via 8-oxodG. A possible reaction
path is shown in Scheme 1
in aqueous medium and should be generated directly
from dG.4,5 In the present study, we showed another
product, dD, is formed from dG by the reaction with
1O2, but not via 8-oxodG, while dS can be generated not
only from dG but also from 8-oxodG.7 The direct for-
mation of dD from dG may provide new information
concerning the mechanism of the reaction of dG with
1O2.
No information is available with regard to the forma-
tion and occurrence of dD in cells. The half-life of dD
was 4.9 h at pH 7.4 and 37 ꢁC, giving rise to dIz and its
base moiety, Iz (Scheme 1).21 If a similar conversion
occurs in DNA, it will result in a dIz moiety and an
abasic site in the DNA strand. An abasic site is poten-
tially mutagenic.26 It has been reported that dIz is
hydrolyzed in neutral solution with a half-life of 2.0h
(pH 7.4 and 37 ꢁC) to form dZ (Scheme 1), which is
stable under physiological conditions.21,27 dZ in an oli-
godeoxynucleotide induces mainly dAMP insertion by
DNA polymerases.28 Thus, the formation of dZ in
DNA should lead to a G to T transversion if the lesion
is not repaired. On the other hand, no information is
available on genotoxic effects of dD.
1
The reaction mechanisms of dG and 8-oxodG with O2
were studied by Foote’s group exclusively by means of
direct measurements of NMR signals for intermediates
in organic solvents at low temperature.22ꢀ25 In the reac-
tion of 8-oxodG with O2, diastereomers of an endo-
peroxide intermediate were formed, which upon
warming were converted to a variety of products.23 The
1
1
mechanism of the reaction between dG and O2 is not
fully known. No endoperoxide intermediate was detec-
ted, while two carbamic acids were observed as inter-
mediates.25 However, the carbamic acids were not
converted to dS in the organic solvents upon warming.
dS is a remarkable product with respect to the reaction
1
mechanism of dG with O2, since it is a major product
Table 3. Yields of dD, dIz and 8-oxodG in the reaction of dG with various photooxidation systemsa
Photooxidation systems
dD (mM)
dlz (mM)
8-oxodG (mM)
dG (mM)
dD/dlz
0.15
Methylene blue (550nm)
Rose bengal (550nm)
Riboflavin (366 nm)
1.3 ꢂ0.1
0.15 ꢂ0.03
1.7ꢂ0.1
<0.03
8.6ꢂ0.2
1.5ꢂ0.1
88.4ꢂ3.0
2.0ꢂ0.5
9.1ꢂ0.2
<0.03
<0.03
<0.03
873ꢂ7
.10
951ꢂ200
287ꢂ9
0.019
<0.015
Benzophenone (366 nm)
944ꢂ11
aA solution containing 1 mM dG, 0.5 mM photosensitizers (except for benzophenone, used as a saturated solution), and 100 mM sodium phosphate
buffer (pH 7.4) was irradiated with visible light (550nm cut-off filter) for methylene blue and rose bengal or with UV light (366 nm) for riboflavin
and benzophenone at 37 ꢁC for 1 h under aerobic conditions. The reaction mixture was analyzed by RP-HPLC immediately after the reaction.
MeansꢂSD (n=3) are presented.

T. Suzuki et al. / Bioorg. Med. Chem. 11 (2003) 2157–2162
2161
USA). For reversed-phase HPLC (RP-HPLC), an
Ultrasphere octadecylsilane (ODS) column (4.6ꢃ250
mm with a particle size of 5 mm, Beckman, CA, USA)
was used. The eluent used was 20mM sodium phos-
phate buffer (pH 7.0) containing acetonitrile. The ace-
tonitrile concentration was increased from 0to 12.5%
over 15 min in a linear gradient mode. The column
temperature was 30 ꢁC and the flow rate 1 mL/min.
Reaction conditions
A solution (1 mL) of 1 mM dG or 8-oxodG contain-
ing 0.5 mM photosensitizers (except for benzophe-
none, used as a saturated solution) in 100 mM sodium
phosphate buffer (pH 7.4) in an NMR tube (5 mm
i.d.) was irradiated with visible light generated by a
10W tungsten lamp through a 550nm cut-off filter
or with UV light of wavelength 366 nm at 37 ꢁC for 1
h. During the irradiation, the solution was kept satu-
rated with oxygen by continuous air bubbling (7 mL/
min). RP-HPLC analysis was conducted immediately
after the reaction. All experiments were carried out in
triplicate.
Preparation of authentic samples
The authentic dD was prepared by incubation of 1 mM
dG with 1 mM NaOCl in 100 mM sodium phosphate
buffer (pH 7.4) at 37 ꢁC for 15 min and subsequent
termination of the reaction with N-acetylcysteine.21 dD
1
Scheme 1. Proposed pathway for the reaction of dG with the methyl-
ene blue photooxidation system.
was isolated from reaction mixtures by RP-HPLC. H
NMR (DMSO-d6): d (ppm/TMS) 8.19 (br, 1H), 7.95
(br, 1H), 7.68 (br, 1H), 5.62 (dd, 1H), 4.89 (br, 2H), 4.10
(m, 1H), 3.67 (m, 1H), 3.41 (m, 2H), 2.06 (m, 1H), 1.63
(m, 1H). ESI-MS: m/z 228 (M+H). UV: lmax 255 and
334 nm (pH 7.0). The authentic dIz was prepared by
irradiating a solution of 5 mM dG containing 0.6 mM
riboflavin with UV light of wavelength 366 nm for 2 h at
room temperature under aerated conditions.27,29 The
authentic dZ was prepared by incubating a solution of
dIz in 100 mM sodium phosphate buffer (pH 7.4) at
70 ꢁC for 30min. dIz and dZ were isolated from each
reaction mixture by RP-HPLC. Authentic dS was pre-
pared by incubation of 1 mM 8-oxodG with 1 mM
NaOCl in 100 mM sodium phosphate buffer (pH 7.4) at
37 ꢁC for 15 min and was purified by normal-phase
HPLC.18 We confirmed that the on-line UV and elec-
trospray mass spectrometry spectra of dIz, dZ and dS
were consistent with reported values.
Conclusion
In the present study, we found that the reaction of dG
with singlet oxygen yielded dD in addition to dIz, dZ,
dS and 8-oxodG. dD was generated from dG directly,
not via 8-oxodG. Further studies are necessary to clarify
the mechanism of the formation of dD and to elucidate
the biological significance of dD in singlet oxygen-
mediated genotoxicity.
Experimental
Materials
dG was obtained from Fluka (Buchs, Switzerland).
8-Oxo-7,8-dihydro-20-deoxyguanosine (8-oxodG) was
purchased from Sigma (MO, USA). All other chemicals
of reagent grade were purchased from Sigma, Fluka
or Aldrich (WI, USA), and used without further
purification.
Quantitative procedures
The concentrations of products were evaluated from the
integrated peak areas of HPLC chromatograms and
molar extinction coefficients (e) at 260or 330nm. ꢀT1he
values of e at 260nm used were 1.60 ꢃ104 Mꢀ1 cm for
dD, 1.99ꢃ104 Mꢀ1 cmꢀ1 for dIz and 1.15ꢃ104 Mꢀ1
cmꢀ1 for dG.21,30 We used e values of 7.20ꢃ103 Mꢀ1
cmꢀ1 for 8-oxodG at 260nm and of 2.49 ꢃ103 Mꢀ1
cmꢀ1 for dD and 4.23ꢃ103 Mꢀ1 cmꢀ1 for dIz at 330nm,
which were calculated on the basis of the reported e
values21,31 and on-line UV spectra of 8-oxodG, dD and
dIz.
High-performance liquid chromatography (HPLC)
conditions
The HPLC system consisted of an HP1050 series
pumping system (Hewlett Packard, CA, USA). On-line
UV spectra were obtained with a Spectra Focus UV–
visible photodiode-array detector (Spectra Physics, CA,

2162
T. Suzuki et al. / Bioorg. Med. Chem. 11 (2003) 2157–2162
15. Hoyano, Y.; Bacon, V.; Summons, R. E.; Pereira, W. E.;
Acknowledgements
Halpern, B.; Duffield, A. M. Biochem. Biophys. Res. Commun.
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S. L. Free Radical Res. Commun. 1990, 9, 303.
17. Whiteman, M.; Jenner, A.; Halliwell, B. Biomarkers 1999,
4, 303.
The work reported in this paper was undertaken during
the tenure of a Special Training Award granted to T.S.
by the International Agency for Research on Cancer.
We also thank Dr. J. Cheney for editing the manuscript
and Ms. M. Wrisez and Ms. P. Collard for secretarial
assistance.
18. Suzuki, T.; Masuda, M.; Friesen, M. D.; Ohshima, H.
Chem. Res. Toxicol. 2001, 14, 1163.
19. Suzuki, T.; Ohshima, H. FEBS Lett. 2002, 516, 67.
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Cadet, J.; Pignatelli, B.; Nishino, H.; Ohshima, H. J. Biol.
Chem. 2001, 276, 40486.
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