2-Oxoalkyl caged oligonucleotides: One-electron reductive activation into emergence of ordinary hybridization property by hypoxic X-irradiation

Article abstract of DOI:10.1039/b618810a

Ionizing radiation triggers the activation of caged oligodeoxynucleotides (ODNs) with a 2-oxoalkyl leaving group to give the corresponding normal uncaged strands. We designed and synthesized ODNs caged by a 2-oxopropyl group at a given thymine N(3) positi

Full text of DOI:10.1039/b618810a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
2-Oxoalkyl caged oligonucleotides: one-electron reductive activation into
emergence of ordinary hybridization property by hypoxic X-irradiation
Kazuhito Tanabe,* Hiroshi Kanezaki, Hirotake Ishii and Sei-ichi Nishimoto*
Received 2nd January 2007, Accepted 26th February 2007
First published as an Advance Article on the web 12th March 2007
DOI: 10.1039/b618810a
Ionizing radiation triggers the activation of caged oligodeoxynucleotides (ODNs) with a 2-oxoalkyl
leaving group to give the corresponding normal uncaged strands. We designed and synthesized ODNs
caged by a 2-oxopropyl group at a given thymine N(3) position (doxoT) to evaluate their one-electron
reduction characteristics. Upon hypoxic X-radiolysis in aqueous solution, the caged ODNs released the
2-oxopropyl group to produce the corresponding uncaged ODNs. Digestion by a restriction enzyme
Swa I revealed that caged ODN pre-irradiated in hypoxia could form an ordinary duplex with its
complementary strand.
be one-electron reduced upon hypoxic X-irradiation, releasing
Introduction
the 2-oxopropyl group and producing the corresponding uncaged
Artificial oligonucleotides (ODNs) that can be externally stimu-
ODNs, which could hybridize into an ordinary duplex with their
lated to regulate their hybridization properties have been explored
complementary strand.
for possible application to DNA biosensors, bionanodevices and
gene therapy.1–7 With this view, various attempts were made to
either enhance or suppress the bioactivity of ODNs by regulating
Results and discussion
their recognition ability, e.g. binding of a metal ion to mismatched2
or modified nucleobases,3 addition of borax and boronic acid to
modified riboses,4 and photochemical methods with nitrobenzene5
or azobenzene6 functionality linked to an ODN.
High-energy ionizing radiation seems to be among the potential
external triggers for artificial ODNs with bioactive functionality,
generating reactive transient species such as ion radicals, free
radicals and excited molecules that induce formation or cleavage
of chemical bonding.8 The advantage of ionizing irradiation is
its capability to control spatially and temporally the chemical
reactions without any additives. Thus, ionizing radiation is an
attractive trigger for the control of DNA functions, however, it
has not yet been applied to regulation of DNA hybridization
properties.
The synthetic routes to doxoT and artificial ODNs are shown in
Scheme 1. Diacetyl thymidine 1 was coupled with a-bromoacetone
and subsequently hydrolyzed under basic conditions to produce
doxoT. The resulting doxoT was incorporated into DNA via phos-
phoramidite 4, using a DNA synthesizer. The structures of syn-
thesized ODNs as confirmed by MALDI-TOF mass spectrometry
are summarized in Table 1.
We initially conducted one-electron reduction of 5-mer ODN
1 by the X-radiolysis of an argon-purged aqueous solution con-
taining excess 2-methyl-2-propanol as the scavenger of oxidizing
hydroxyl radicals.9b Under these radiolysis conditions, reducing
hydrated electrons (eaq−) are generated as the major active species.
Fig. 1 shows a representative reaction profile of the radiolytic
−
reduction of ODN 1 by eaq under hypoxic conditions. The
We herein report X-radiolytic activation of ODNs caged by a
removable 2-oxoalkyl group, which was previously demonstrated
to be an effective functionality for selective hypoxic-radiation
activated 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-
FdUrd) prodrugs.9 An activation mechanism has been proposed
by which the 2-oxoalkyl group undergoes one-electron reduction
appearance of single new peak in Fig. 1 was attributable to
formation of the corresponding uncaged ODN (5ꢀ-AATAA-3ꢀ)
according to the molecular weight [(M − H)−] of the fractionated
sample measured by MALDI-TOF mass spectroscopy11 and
overlap injection of authentic samples in the HPLC analysis. The
G values (the number of molecules produced or changed per
1 J of radiation energy absorbed by the reaction system) were
137 nmol J−1 for the decomposition of ODN 1 and 113 nmol J−1
for the formation of corresponding uncaged ODN.
−
10
by hydrated electrons (eaq
)
generated via radiolysis of water
to form the corresponding p* anion radical, followed by thermal
activation into the r* anion radical that has a weakened N–C bond
between the 5-fluorouracil unit and the 2-oxoalkyl group and is
readily hydrolyzed to release the 2-oxoalkyl group.9b,9e In this study,
we designed and synthesized ODNs caged by a 2-oxopropyl group
at a given thymine N(3) position (doxoT), which has a characteristic
structure similar to the previous radiolytic reduction activated 5-
FdUrd prodrug. These doxoT-bearing ODNs were confirmed to
In contrast to the hypoxic X-radiolysis, the removal of 2-
oxopropyl group from doxoT of ODN 1 to give uncaged ODN
became considerable less efficient in the X-radiolysis under aerobic
conditions: the G values were evaluated as 17.6 nmol J−1 for the
decomposition of ODN 1 and 2.4 nmol J−1 for the formation
of uncaged ODN. In view of the well documented evidence that
molecular oxygen efficiently captures reducing species of eaq− into
Department of Energy and Hydrocarbon Chemistry, Faculty of Engineer-
ing, Kyoto University, Katsura campus, Kyoto, 615-8510, Japan. E-mail:
tanabeka@scl.kyoto-u.ac.jp, nishimot@scl.kyoto-u.ac.jp; Fax: +81-75-383-
2501; Tel: +81-75-383-2500
superoxide anion radical (O2 •) and thereby inhibits radiolytic
−
reduction,9d,12 the radiolytic removal of 2-oxopropyl group from
−
doxoT is likely to occur via one-electron reduction by eaq almost
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Scheme 1 Reagents and conditions: (a) NaH, bromoacetone, DMF, 0 ◦C, 94%; (b) LiOH·H2O, methanol–water (2 : 1), 0 ◦C, 94%; (c) 4,4ꢀ-dimethoxytrityl
chloride, pyridine, 0 ◦C, 83%; (d) 2-cyanoethyl tetraisopropylphosphorodiamidite, 1H-tetrazole, acetonitrile, room temperature, 40 min, quant.
(e) automated DNA synthesis.
Table 1 The oligodeoxynucleotides (ODNs) used in this study
Sequencesa
ODN 1
ODN 2
ODN 3
ODN 4
ODN 5
ODN 6
ODN 7
ODN 8
5ꢀ-d(AAoxoTAA)-3ꢀ
5ꢀ-d(AAAAoxoTAAAA)-3ꢀ
5ꢀ-d(TTTTATToxoTAAATTTTTT)-3ꢀ
5ꢀ-d(AAAAAATTTAAATAAAAA)-3ꢀ
5ꢀ-d(oxoTA)-3ꢀ
5ꢀ-d(oxoTG)-3ꢀ
5ꢀ-d(oxoTT)-3ꢀ
5ꢀ-d(oxoTC)-3ꢀ
Fig. 1 HPLC profiles for the one-electron reduction of ODN 1 (110 lM)
in the hypoxic X-radiolyses (0, 300 and 900 Gy) of aqueous solution
containing 10% 2-methyl-2-propanol.
a Recognition sites of restriction enzyme Swa I are shown in italic.
selectively under hypoxic conditions. Similar reductive removal
of the 2-oxopropyl group in a hypoxia selective manner was
also confirmed for 9 mer ODN 2, although reaction efficiency
was lower than that of 5-mer ODN 1 (Table 2). The lowered
efficiency is probably due to the increased competitive reaction of
nucleobases around doxoT structure with eaq−. Furthermore, as seen
in Table 2, a trend seems to be apparent that the radiolytic reductive
conversion of caged ODN consisting of dT, dA and doxoT into the
corresponding uncaged analog becomes substantially less efficient
upon increasing the ODN chain length. The radiolytic one-
electron reduction of ODN 1–3 by eaq− may produce primary anion
radicals of dT and dA that could transfer the electron to the caged
base doxoT. However, the results shown in Table 2 indicate that
this may not occur significantly, thus suggesting that irreversible
processes are occurring such as irreversible protonation of the
anion radicals. This is in accord with the fact that the thymine
anion radical protonates irreversibly at C-6 and the adenine anion
radical protonates irreversibly at C-2 and C-8.13
Table 2 G-values for the decomposition of caged ODNs bearing doxoT and for the formation of the corresponding uncaged ODNs in the X-radiolysis
under hypoxic and aerobic conditions
Hypoxic conditions
Aerobic conditions
Decomposition/nmol J−1
Formation/nmol J−1
Decomposition/nmol J−1
Formation/nmol J−1
ODN 1
ODN 2
ODN 3
ODN 5
ODN 6
ODN 7
ODN 8
137
106
(<62)a
167
167
181
69
113
45
17.6
12
2.4
0.8
NDb
30
15
11
(<50)a
149
134
130
49
(<10)a
35
24
35
19
11
a Since the signals assigned to ODN 3 and the corresponding uncaged ODN considerably overlapped each other on the HPLC chart, the G values could
not be quantified with sufficient accuracy. b The formation of uncaged ODN was not detected.
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To confirm the removal of the 2-oxoalkyl group from caged
ODN with longer chains upon hypoxic irradiation and to char-
acterize its hybridization property, we performed the enzymatic
digestion of 18-mer caged ODN 3 pre-irradiated in hypoxia, which
had doxoT at the center of the Swa I recognition site.14 An equimolar
amount of 5ꢀ-32P-end labeled ODN 4, which is complementary to
the uncaged normal analog of ODN 3, was added to a solution
of caged ODN 3 after hypoxic X-irradiation and the resulting
duplex was digested by Swa I at 25 ◦C for 15 min. Fig. 2
shows a representative gel electrophoresis for elucidating strand
cleavage behavior. Without X-irradiation, no cleavage of ODN 4
was observed (lane 2), indicating that the enzymatic cleavage of
ODN 3–ODN 4 duplex by Swa I was inhibited by the presence
of obstructive 2-oxopropyl group on the caged ODN 3 strand.
In contrast, the cleavage efficiency of ODN 4 in the presence of
caged ODN 3 pre-irradiated in hypoxia was remarkably enhanced
(lane 3). This result indicates that hypoxic X-irradiation removed
2-oxopropyl group on caged ODN 3, thereby resulting in the
formation of an ordinary duplex with ODN 4 that can be a
substrate for Swa I. Consistent with the inefficient removal of
2-oxopropyl group upon aerobic X-irradiation, the enzymatic
cleavage of the duplex derived from caged ODN 3 pre-irradiated
under aerobic conditions was suppressed to a background level
(lane 5).
processes involved in cytosine anion radical may be competitive
with electron transfer from one-electron reduced cytosine to the
neighboring doxoT in ODN 8.
Conclusion
In summary, we have demonstrated activation of caged ODNs
possessing a hindered doxoT unit by X-radiolytic one-electron
reduction. The removal of the 2-oxopropyl group on caged ODN
occurred efficiently upon hypoxic X-irradiation, while the reaction
efficiency was dramatically decreased upon aerobic irradiation.
Artificial DNA derivatives that can be activated to regulate DNA
and RNA recognition properties with the aid of external triggers
are useful for a variety of applications. As an example, we are trying
to make a next generation of DNAzyme and antisense molecules,
whose function can be regulated by hypoxic irradiation.
Experimental
General
1H NMR spectra and 13C NMR spectra were measured with JEOL
JNM-AL 300 (300 MHz) or JEOL JMN-EX-400 (400 MHz)
spectrometers. Coupling constants (J values) are reported in
Hertz. The chemical shifts are expressed in ppm downfield from
tetramethylsilane, using residual chloroform (d = 7.24 in 1H
NMR, d = 77.0 in 13C NMR) and residual dimethyl sulfoxide
1
(d = 2.49 in H NMR, d = 39.5 in 13C NMR) as an internal
standard. FAB Mass spectra were recorded on a JEOL JMS-
SX102A spectrometer. A Rigaku RADIOFLEX-350 was used for
X-radiolysis. High-performance liquid chromatography (HPLC)
was performed using a HITACHI L-7100 equipped with a Intersil
ODS-3 column (GL science Inc.), and the elution peaks were
detected using a UV-Vis detector L-7455 at 260 nm wavelength. A
Wakogel C-200 was used for silica gel chromatography. Precoated
TLC plates Merck silica gel 60 F254 were used for monitoring the
reactions and also for preparative TLC. Restriction enzyme, Swa
I, was purchased from New England BioLabs. The reagents for
the DNA synthesizer were purchased from Glen Research. N,N-
Dimethylformamide (DMF) was distilled under reduced pressure.
All other reagents and solvents were used as received.
Fig. 2 Autoradiograms of the denaturing sequencing gel for enzymatic
digestion of ODN 4 in the presence of ODN 3 pre-irradiated under hypoxic
or aerobic conditions. The pre-irradiated ODN 3 was added to the solution
of 5ꢀ-32P labeled ODN 4, and then digested by Swa I (10 U) at 25 ◦C for
15 min. Lane 1, control duplex (5ꢀ-TTTTTATTTAAATTTTTT-3ꢀ)–ODN
4; lanes 2, 3 ODN 3 pre-irradiated under hypoxic conditions (0 Gy and
100 Gy)–ODN 4; lanes 4, 5 ODN 3 (0 and 100 Gy) pre-irradiated under
aerobic conditions–ODN 4.
2ꢀ-Deoxy-3ꢀ,5ꢀ-di-O-acetyl-3-(2ꢀ-oxopropyl)thymidine (2)
2ꢀ-Deoxy-3ꢀ,5ꢀ-di-O-acetylthymidine 1 (2.78 g, 8.53 mmol) was
added to a suspension of sodium hydride (614 mg, 25.6 mmol)
in anhydrous DMF (20 ml) at 0 ◦C and the mixture was stirred at
0 ◦C for 15 min. To the resulting mixture was added bromoacetone
(3.51 g, 25.6 mmol) and the mixture was stirred at 0 ◦C for
2.5 h. The reaction mixture was diluted with water and extracted
with ethyl acetate. The extract was washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo. The crude product
was purified by column chromatography (SiO2, 33% hexane–ethyl
acetate) to give 2 (2.61 g, 80%) as a yellow oil: 1H NMR (CDCl3,
400 MHz) d 7.25 (s, 1H), 6.22 (dd, 1H, J = 7.6, 6.1 Hz), 5.14 (m,
1H), 4.69–4.68 (2H), 4.30–4.28 (2H), 4.19 (m, 1H), 2.42 (ddd, 1H,
J = 14.6, 6.1, 1.5 Hz), 2.19 (s, 3H), 2.15 (dd, 1H, J = 14.6, 7.6
Hz), 2.06 (s, 3H), 2.03 (s, 3H), 1.89 (s, 3H); 13C NMR (CDCl3,
100 MHz) d 200.2, 170.2, 170.1, 162.5, 150.3, 133.0, 110.3, 85.6,
82.1, 74.0, 63.7, 49.8, 37.5, 27.1, 20.7, 20.7, 13.2; FABMS (NBA)
To identify the effect of base sequence on the radiolytic reduc-
tion of intramolecular doxoT, we carried out a comparative one-
electron reduction of dinucleotides ODN 5–8 consisting of doxo
T
and four types of natural nucleobase upon hypoxic X-irradiation.
As shown in Table 2, the G values for the decomposition of caged
ODN 5–7, in which doxoT is linked to A, G, or T, respectively,
and the formation of corresponding uncaged dinucleotides were
similar levels. In contrast, dinucleotides ODN 8 with a linkage
between doxoT and C showed a considerably lower G value, relative
to the other three dinucleotides, suggesting that the counterpart
base unit of C could effectively capture eaq− and thereby inhibit the
one-electron reduction of doxoT to generate dT. It has been reported
−
that cytosine derivatives are reduced by eaq faster than the other
bases.15 Furthermore, as suggested by the recent studies on excess
electron transfer through DNA,16 rapid irreversible protonation
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m/e 383 [(M + H)+]; HRMS calcd. for C17H23N2O8 [(M + H)+]
383.1449, found 383.1449.
2ꢀ,3ꢀ,4ꢀ-trihydroxyacetophenone as matrix, using T8 ([M − H]−
2370.61) and T17 ([M − H]− 5108.37) as an internal standard; ODN
1, 5ꢀ-d(AAoxoTAA)-3ꢀ, m/z 1494.09 (calcd for [M − H]− 1494.05);
ODN 2, 5ꢀ-d(AAAAoxoTAAAA)-3ꢀ, m/z 2803.37 (calcd for [M −
H]− 2802.94); ODN 3, 5ꢀ-d(TTTTTATToxoTAAATTTTTT)-3ꢀ,
m/z 5504.41 (calcd for [M − H]− 5504.62); ODN 5, 5ꢀ-d(oxoTA)-3ꢀ,
m/z 610.65 (calcd for [M − H]− 610.49); ODN 6, 5ꢀ-d(oxoTG)-3ꢀ,
m/z 625.72 (calcd for [M − H]− 626.49); ODN 7, 5ꢀ-d(oxoTT)-3ꢀ,
m/z 602.24 (calcd for [M − H]− 601.48); ODN 8, 5ꢀ-d(oxoTC)-3ꢀ,
m/z 586.13 (calcd for [M − H]− 586.47).
2ꢀ-Deoxy-3-(2ꢀ-oxopropyl)thymidine (doxoT)
To a solution of 2 (2.61 g, 6.83 mmol) in methanol (13.4 ml)
and water (6.7 ml) was added lithium hydroxide monohydrate
(631 mg, 15.04 mmol) and the mixture was stirred at 0 ◦C for
30 min. The reaction mixture was concentrated in vacuo and the
crude product was purified by column chromatography (SiO2, 9%
MeOH–CHCl3) to give doxoT (1.91 g, 94%) as a yellow solid: mp
56–57 ◦C; 1H NMR (CDCl3, 400 MHz) d 7.30 (s, 1H), 6.07 (t, 1H,
J = 6.9 Hz), 4.69 (s, 2H), 3.90 (m, 1H), 3.87 (dd, 1H, J = 11.6, 2.8
Hz), 3.75 (dd, 1H, J = 11.6, 2.8 Hz), 2.44–2.36 (1H), 2.24 (ddd,
1H, J = 13.4, 6.9, 3.9 Hz), 2.18 (s, 3H), 1.87 (s, 3H); 13C NMR
(CDCl3, 100 MHz) d 201.0, 162.7, 150.5, 135.2, 110.1, 87.0, 86.9,
71.1, 62.2, 50.0, 40.2, 27.4, 13.2; FABMS (NBA) m/e 299 [(M +
H)+]; HRMS calcd. for C13H19N2O6 [(M + H)+] 299.1243, found
299.1242.
Radiolytic reduction
To establish hypoxia, aqueous solutions of ODN 1–8 (110 lM)
containing 10% 2-methyl-2-propanol were purged with argon for
30 min and then irradiated in a sealed glass ampoule at ambient
temperature with an X-ray source (4.05 Gy min−1). After the
irradiation, the solution was immediately subjected to HPLC
analysis.
2ꢀ-Deoxy-5ꢀ-O-dimethoxytrityl-3-(2ꢀ-oxopropyl)thymidine (3)
Melting temperature (Tm) measurement
A solution of doxoT (544 mg, 1.83 mmol) and 4,4ꢀ-dimethoxytrityl
chloride (929 mg, 2.74 mmol) was stirred in anhydrous pyridine
(3.5 ml) for 50 min at 0 ◦C. The reaction mixture was diluted with
saturated NaHCO3 and extracted with CHCl3. The extract was
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo. The crude product was purified by column chromatography
(SiO2, 9% MeOH–CHCl3) to give 3 (908 mg, 83%) as a yellow solid:
mp 93–94 ◦C; 1H NMR (CDCl3, 270 MHz) d 7.55 (s, 1H), 7.35–
7.18 (9H), 6.76 (d, 2H, J = 8.9 Hz), 6.30 (t, 1H, J = 6.4 Hz), 4.67
(d, 2H, J = 1.9 Hz), 4.49 (m, 1H), 3.96 (m, 1H), 3.71 (s, 6H), 3.40
(dd, 1H, J = 10.6, 2.9 Hz), 3.29 (dd, 1H, J = 10.6, 3.4 Hz), 2.43–
2.17 (2H), 2.16 (s, 3H), 1.40 (s, 3H); 13C NMR (CDCl3, 75 MHz) d
200.5, 162.6, 158.2, 150.1, 148.7, 144.0, 136.2, 135.1, 134.9, 134.2,
129.7, 127.7, 127.5, 126.6, 123.6, 112.8, 109.5, 86.4, 86.0, 85.2,
71.1, 63.1, 57.3, 54.7, 49.6, 40.7, 26.7, 17.8, 11.9; FABMS (NBA)
m/e 601 [(M + H)+]; HRMS calcd. for C34H37N2O8 [(M + H)+]
601.2550, found 601.2541.
After the irradiation of the oligomers possessing 2-oxoalkyl
groups, the corresponding complementary DNA was added to
the samples. Tms of the duplexes (3.0 lM, duplex concentration)
were taken in a 50 mM Tris-HCl (pH 7.9) containing 100 mM
NaCl and 10 mM MgCl2. Absorbance vs. temperature profiles
were measured at 260 nm using a JASCO V-530 UV/VIS
spectrometer connected with a ETC-505T temperature controller.
The absorbance of the samples was◦monitored at 260 nm from 5 ◦C
◦
to 70 C with a heating rate of 1 C min−1. From these profiles,
first derivatives were calculated to determine Tm values.
Preparation of 5ꢀ-32P-end labeled DNA oligomers
The DNA oligomers were 5ꢀ-end-labeled by phosphorylation with
4 lL of [c -32P]ATP and 4 lL of T4 polynucleotide kinase using
standard procedures.17 The 5ꢀ-end-labeled DNA oligomers were
recovered by ethanol precipitation and further purified by 15%
preparative nondenaturing gel electrophoresis and isolated by the
crush and soak method.18
2ꢀ-Deoxy-5ꢀ-O-dimethoxytrityl-3ꢀ-O-cyanoethyl-N,N-
diisopropylphosphoramidite-3-(2ꢀ-oxopropyl)thymidine (4)
Digestion of irradiated ODN 3 and 5ꢀ-32P-labeled ODN 4 by
restriction enzyme
A solution of 3 (218 mg, 3.63 mmol), 2-cyanoethyl tetraiso-
propylphosphorodiamidite (115 ll, 0.362 mmol), and tetrazole
(33.1 mg, 0.47 mmol) in acetonitrile (1.0 ml) was stirred at ambient
temperature for 40 min. The mixture was filtered off and used
without further purification.
According to the protocol described above, radiation (0 and
100 Gy) of ODN 4 (110 lM) was carried out. After the irradiation,
3 lL of radiated ODN 3 was added to 12 lL of 3.3 lM 32P-labeled
ODN 4. Hybridization was achieved by heating the sample at 90 ◦C
for 5 min and slowly cooling to room temperature. The resulting
duplex was incubated in 50 mM Tris-HCl (pH 7.9), 100 mM NaCl,
10 mM MgCl2, 1 mM DTT, 1 lg mL−1 BSA with Swa I (10 U)
at 25 ◦C for 15 min. After the digestion, the reaction mixture
was ethanol precipitated with 800 lL of ethanol. The precipitated
DNA was washed with 100 lL of cold ethanol and then dried
in vacuo. The radioactivity of the samples was then measured using
aloka 1000 liquid scintillation counter and the dried DNA pellets
were resuspended in 80% formamide loading buffer (a solution of
80% v/v formamide, 1 mM EDTA, 0.1% xylene cyanol and 0.1%
Caged ODN synthesis
ODNs caged by a 2-oxopropyl group were synthesized by the con-
ventional phorphoramidite method using an Applied Biosystems
392 DNA/RNA synthesizer. Synthesized ODNs were purified by
reversed phase HPLC on a Inertsil ODS-3 column (10 × 250 mm,
elution with a solvent mixture of 0.1 M triethylammonium acetate
(TEAA), pH 7.0, linear gradient over 60 min from 0% to 30%
acetonitrile at a flow rate 3.0 mL min−1). Mass spectra of ODNs
purified by HPLC were determined with MALDI-TOF mass
spectroscopy (acceleration voltage 21 kV, negative mode) with
◦
bromophenol blue). All samples were heat denature at 90 C for
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3 min and quickly chilled on ice. The samples (1 lL, 10 × 103 cpm)
were loaded onto 15% polyacrylamide and 7 M urea sequencing
gel and electrophoresed at 1900 V for approximately 30 min. The
gel was dried and exposed to X-ray film with an intensifying sheet
at −80 ◦C.
8 J. W. T. Spinks and R. J. Wood, Introduction to Radiation Chemistry,
Wiley-Interscience, New York, 3rd edn., 1990.
9 (a) Y. Shibamoto, L. Zhou, H. Hatta, M. Mori and S. Nishimoto,
Jpn. J. Cancer Res., 2000, 91, 433–438; (b) M. Mori, H. Hatta and S.
Nishimoto, J. Org. Chem., 2000, 65, 4641–4647; (c) Y. Shibamoto, L.
Zhou, H. Hatta, M. Mori and S. Nishimoto, Int. J. Radiat. Oncol.,
Biol., Phys., 2001, 49, 407–413; (d) K. Tanabe, Y. Mimasu, A. Eto, Y.
Tachi, S. Sakakibara, M. Mori, H. Hatta and S. Nishimoto, Bioorg.
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Acknowledgements
This research was partially supported by the Ministry of Edu-
cation, Science, Sports and Culture, Grant-in-Aid for Scientific
Research on Priority Areas. We thank Dr Takeo Ito and Mr
Hiroshi Hatta for their helpful discussions.
10 Radiolysis of a diluted aqueous solution at around pH 7.0 produces
primary water radicals such as oxidizing hydroxyl radicals (•OH),
reducing hydrated electrons (eaq−) and reducing hydrogen atoms (•H)
with the G values of G(•OH) = 280 nmol J−1, of G(eaq−) = 280 nmol J−1
,
and G(•H) = 60 nmol J−1, respectively.
11 The radiolytic product from caged ODN 1 was identified as the
corresponding uncaged ODN (5ꢀ-AATAA-3ꢀ, m/z 1494.08, calcd for
[M − H]−1494.05) by mass spectrometry.
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1246 | Org. Biomol. Chem., 2007, 5, 1242–1246
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