unless otherwise stated. Microwave reactions were conducted
using the CEM Discover as a focused microwave unit. Calf
intestine alkaline phosphatase (AP) (1,500 units) was purchased
from Promega and Roch. Nuclease P1 (500 units) was purchased
from Yamasa. Reactions were monitored on TLC plates precoated
with Merck silica gel 60 F254. Kanto Chemical Silica Gel 60 N
was used for silica gel column chromatography. ODNs were
synthesized on an Applied Biosystems 3400 DNA Synthesizer.
Reverse phase HPLC was performed on a Cosmosil 5C18AR-II
(Nacalai Tesque) column (4.6 ¥ 150 mm) or a CHEMCOBOND
5-ODS-H (Chemco) column (4.6 ¥ 150 mm) with a JASCO PU-
980, HG-980–31, DG-980–50 system equipped with a JASCO
UV 970 spectrometer at 260 nm. Irradiation was performed by
a transilluminator (Funakoshi TR-366 nm). Mass spectra were
recorded on a Voyager-DE PRO-SF, Applied Biosystems.
acetonitrile and coevaporated three times in vacuo. After substitu-
tion with nitrogen, 2-cyanoethyl N, N, N¢, N¢-tetraisopropyl phos-
phorodiamidite (120 ml, 0.38 mmol) in dry acetonitrile (1.5 ml),
and 0.5 M tetrazole (0.95 ml) were added, and the reaction mixture
was stirred at ambient temperature for 2 h. The reaction mixture
was then extracted with AcOEt (10 ml x 3) and water (15 ml).
The organic layer was collected, dried over anhydrous Mg2SO4,
filtered, and evaporated to dryness under reduced pressure. Then,
the crude product cyanoethylphosphoramidite of 4b (316 mg,
0.38 mmol, quant.) in a sealed bottle with septum was dissolved
in dry acetonitrile and coevaporated three times, and was used for
automated DNA synthesis without further purification.
Synthesis and characterization of HU-containing ODN
VUderivatives-containing ODN were synthesized by the auto-
mated solid-phase phosphoramidite method as reported. [S1] After
automated synthesis, the oligomer was deprotected by incubation
with 28% ammonia for 4 h at 65 ◦C and was purified on a Chem-
cobond 5-ODS-H column (4.6 ¥ 150 mm) by reverse phase HPLC;
elution was with 0.05M ammonium formate containing 3–20%
acetonitrile, linear gradient (30 min) at a flow rate of 1.0 mL/min,
30 ◦C. Preparation of oligonucleotides was confirmed by MALDI-
TOF-MS analysis.
Preparation of 5-heptenyl-2¢-deoxyuridine (2b). 5-Iodo-2¢-
deoxyuridine (500 mg, 1.41 mmol) was dissolved in DMF in a
microwave tube with a stirring bar. To this was added palla-
dium (II) acetate (33 mg, 0.15 mmol), tri-n-butylamine (340 ml,
1.41 mmol) and 1-heptene (5 ml, 3.53 mmol). The reaction tube
was sealed and reacted in a microwave reactor for 20 min at
100 ◦C with continuous stirring. The reaction mixture was filtered
to remove the resulting precipitate, and extracted with EtOAc
(20 ml x 3) and water (30 ml). The organic layer was collected,
dried over anhydrous Na2SO4, filtered, and evaporated to dryness
under reduced pressure. The crude product was purified by silica
gel column chromatography (CHCl3/MeOH 9:1) to afford 2b
MALDI-TOF MS: calcd. 7114.95 for ODN (VU) [(M + H)+],
found 7114.20).
MALDI-TOF MS: calcd. 7185.08 for ODN (HU) [(M + H)+],
found 7184.95).
1
(326 mg, 1.36 mmol, 97%). H NMR (300 MHz, CDCl3): d 7.58
MALDI-TOF MS: calcd. 7197.09 for ODN(HVU) [(M + H)+],
found 7197.76).
(s, 1H, H-C(6)); 6.21–6.13 (m, 1H, H-C(1¢)); 6.04 (d, 1H, J= 15.9,
vinylic H); 5.54–5.38 (m, 1H, vinylic H); 4.58 (br. s, 1H, H-C(3¢));
4.03 (br, d, 1H, J= 3.3, H-C(4¢)); 3.95–3.80 (m, 2H, H-C(5¢));
2.45–2.32 (m, 2H, H-C(2¢)); 2.16–0.84 (m, 11H, H of heptene).
MALDI-TOF MS; calcd. for 325.70 [M + H]+; found 325.80.
MALDI-TOF MS: calcd. 7171.05 for ODN (BuVU) [(M + H)+],
found 7171.08).
Preparation of 5-heptenyl-2¢-deoxy-5¢-O-(4,4-dimethoxytrityl)-
uridine (3b). 5-Heptenyl-2¢-deoxyuridine (2b) (600 mg,
1.85 mmol) was dissolved in dry pyridine and coevaporated
three times. 4, 4¢-dimethoxytrityl chloride (752 mg, 2.22 mmol), N,
N-dimethylamino pyridine (68 mg, 0.56 mmol) and triethylamine
(310 ml, 2.22 mmol) were added to a solution of 2b in dry pyridine
(10 ml). The solution was stirred at ambient temperature under a
nitrogen atmosphere for 16 h. The reaction mixture was extracted
with EtOAc (100 ml x 3) and water (150 ml). The organic layer was
collected, dried over anhydrous Mg2SO4, filtered, and evaporated
to dryness under reduced pressure. The crude product was then
purified by silica gel column chromatography (CHCl3/EtOH
97:3) to afford 3b (863 mg, 1.38 mmol, 75%).
1H NMR (300 MHz, CDCl3): d 8.40 (br. s, 1H, H-C(6)); 7.45–
7.20 (m, 8H, arom.H); 6.83–6.80 (m, 5H, arom. H); 6.39 (t, 1H,
J= 6.9, H-C(1¢)); 6.27–6.17(m, 1H, vinylic H); 5.57–5.52 (d, 1H,
J= 15.9, vinylic H); 4.54 (br, s, 1H, H-C(3¢)); 4.05–4.01 (m, 1H,
H-C(4¢)); 3.78 (s, 6H, H of methoxyl); 3.53–3.31 (m, 2H, H-C(5¢));
2.43–2.18 (m, 2H, H-C(2¢)); 1.98–0.73 (m, 11H, H of heptene).
MALDI-TOF MS; calcd. for 649.29 [M + Na]+; found 649.29.
Photochemical detection of 5-methylcytosine on a DNA chip using
5-vinyl-2¢-deoxyuridine derivatives
To demonstrate that template-directed photoligation by using
photosensitive ODNs could be incorporated into platforms suit-
able for DNA chip technologies, we constructed the DNA chip by
attaching amino-labeled ODN containing VU derivatives onto the
aldehyde-modified glass surface. We determined the feasibility of
the template-directed photoligation through photosensitive ODNs
on a DNA chip. A glass chip spotted with 2 mM target ODN(C)
or ODN(mC) and template ODN was irradiated at 366 nm for 1 h
in 50 mM sodium cacodylate buffer (pH 7.0) and 100 mM sodium
chloride. After the chip had been washed with deionized water at
98 ◦C for 5 min, 5 mM Cy3-containing ODN(Cy3) conjugate was
added to the surface for 4 h in 50 mM sodium cacodylate buffer
(pH 7.0) and 100 mM sodium chloride, and the chip was washed
twice in PBS. Fluorescence signals were detected on a microarray
scanner.
Acknowledgements
Preparation of 5-heptenyl-2¢-deoxy-5¢-O-(4,4-dimethoxytrityl)-
uridine phosphoramidite (4b). 5-heptenyl-2¢-deoxy-5¢-O-(4,4-
dimethoxytrityl)uridine (3b) (235 mg, 0.38 mmol) in dry CH3CN
(1.5 ml) in a sealed bottle with septum was dissolved in dry
This work was supported by a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science, and
Technology, Japan.
3166 | Org. Biomol. Chem., 2009, 7, 3163–3167
This journal is
The Royal Society of Chemistry 2009
©