A R T I C L E S
Tanaka et al.
(6-(2-(6-(4′-Methyl-2,2′-bipyridin-4-yl)hexanamido)ethylamino)-
9H-purin-9-yl)-5′-O-dimethoxytrityl-2′-deoxyriboside (2). To a solu-
tion of (6-chloro-9H-purin-9-yl)-5′-O-dimethoxytrityl-2′-deoxyriboside
(11.0 g, 19.2 mmol) in DMF (50 mL) were added diisopropylethylamine
(23 mL) and 1 (18.1 g, 55.4 mmol) in DMF (50 mL) at room
temperature, and the mixture was stirred at 75 °C for 12 h. After diluted
with ethyl acetate (1 L), the resulting mixture was washed with brine,
dried over Na2SO4, filtered, and evaporated under reduced pressure.
The crude product was purified by silica gel column chromatography
(chloroform/methanol ) 50:1 containing 1% NH4OH) to yield 2 (15.8
g, 18.3 mmol, 95%) as a white solid: 1H NMR (400 MHz, CD3OD) δ
8.41 (dd, 2H, J ) 4.8, 10.1 Hz), 8.17 (s, 1H), 8.12 (s, 1H), 8.04 (s,
2H), 7.33 (d, 2H, J ) 7.3 Hz), 7.20 (d, 4H, J ) 8.8 Hz), 7.15-7.08
(m, 5H), 6.71 (dd, 4H, J ) 4.0, 8.4 Hz), 6.38 (t, 1H, J ) 6.2 Hz), 4.60
(quartet, 1H, J ) 4.2 Hz), 4.10 (quartet, 1H, J ) 4.4 Hz), 3.66 (s, 6H),
3.43 (t, 2H, J ) 5.9 Hz), 3.32-3.27 (m, 2H), 2.83 (dt, 1H, J ) 6.2,
13.5 Hz), 2.56 (t, 2H, J ) 7.5 Hz), 2.44 (ddd, 1H, J ) 4.6, 6.2, 13.4
Hz), 2.36 (s, 3H), 2.11 (t, 2H, J ) 7.1 Hz), 1.61-1.50 (m, 4H), 1.29-
1.23 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 176.3 160.0, 157.0,
156.3, 154.7, 153.8, 150.3, 150.0, 149.9, 146.2, 140.4, 137.10, 137.06,
131.23, 131.20, 129.3, 128.7, 127.8, 126.0, 125.4, 123.6, 122.9, 114.0,
87.9, 87.6, 85.8, 72.6, 65.0, 55.7, 40.8, 40.3, 36.9, 36.0, 30.9, 29.6,
26.5, 21.2; FABMS (NBA/CH3OH) m/z 863 ([(M + H)+]), HRMS
calcd for C50H55O6N8 ([(M + H)+]) 863.4245, found 863.4235.
5′-O-Dimethoxytrityl-6-N-[9-(4′-methyl-2,2′-bipyridin-4-yl)-3-oxa-
2-azanonyl]-2′-deoxyadenine 3′-(2-cyanoethyl N,N-diisopropylphos-
phoramidite) (3). To a solution of 2 (432 mg, 0.5 mmol) and 1H-
tetrazole (35 mg, 0.5 mmol) in anhydrous dichloromethane (2.5 mL)
was added 2-cyanoethyl tetraisopropylphosphorodiamidite (151 mg, 0.6
mmol) under nitrogen. The mixture was stirred at room temperature.
After stirring for 1 h, the reaction mixture was diluted with dichlo-
romethane (50 mL) and then washed with saturated sodium bicarbonate
solution (50 mL × 2). An aqueous layer was back extracted by
dichloromethane (50 mL). Combined organic layer was dried with
sodium sulfate and then evaporated. The crude mixture was dissolved
in ethyl acetate and reprecipitated into hexane. Precipitate was filtrated
and redissolved in dry acetonitrile then evaporated in vacuo to yield 3
(two diastereomeric isomers) as a pale yellow foam (420 mg, 80%).
The product was used for conventional solid-phase DNA synthesis
without further purification: 31P NMR (160 MHz, CDCl3) δ 149.39,
149.28; FAB HRMS (NBA) m/z calcd for C59H72N10O7P ([(M + H)+])
1063.5318, found 1063.5292.
ATP (Amersham) and T4 polynucleotide kinase using a standard
procedure. The 5′-end-labeled DNA was recovered by ethanol precipi-
tation and further purified by 15% denaturing polyacrylamide gel
electrophoresis (PAGE).
Osmium Oxidation of 32P-5′-End-Labeled DNA (Bipyridine Use).
The 5′-end-labeled DNA1(N) (10 pmol strand) to be examined was
incubated in a solution of 1 µM DNA1′(N′), 5 mM potassium osmate,
100 mM potassium hexacyanoferrate(III), 100 mM bipyridine, and 1
mM EDTA in 100 mM Tris-HCl buffer (pH 7.7) and 10% acetonitrile
at 0 °C for 5 min. The reaction mixture was ethanol precipitated with
the addition of 15 µL of 3 M sodium acetate (pH 5.0), 10 µL of salmon
sperm DNA (1 mg/mL), and 1 mL of cold ethanol. The precipitated
DNA was washed with 150 µL of 80% cold ethanol and dried in vacuo.
The precipitated DNA was redissolved in 50 µL of 10% piperidine
(v/v), heated at 90 °C for 20 min, and then evaporated to dryness by
vacuum rotary evaporation.
Osmium Oxidation of 32P-5′-End-Labeled DNA (B-Containing
DNA Use). The 5′-end-labeled DNA2(N) (10 pmol strand) to be
examined was incubated in a solution of 1 µM DNA2′, 5 mM potassium
osmate, 100 mM potassium hexacyanoferrate(III), and 1 mM EDTA
in 100 mM Tris-HCl buffer (pH 7.0) and 10% acetonitrile at 50 °C for
10 min. The reaction mixture was ethanol precipitated with the addition
of 15 µL of 3 M sodium acetate (pH 5.0), 10 µL of salmon sperm
DNA (1 mg/mL), and 1 mL of cold ethanol. The precipitated DNA
was washed with 150 µL of 80% cold ethanol and dried in vacuo. For
determination of the reaction site, the precipitated DNA was redissolved
in 50 µL of 10% piperidine (v/v), heated at 90 °C for 20 min, and then
evaporated to dryness by vacuum rotary evaporation.
Polyacrylamide Electrophoresis of Reaction Samples. The dried-
up reaction sample was resuspended in 5-20 µL of 80% formamide
loading buffer (a solution of 80% formamide (v/v), 1 mM EDTA, 0.1%
xylenecyanol, and 0.1% bromophenol blue). The samples (1 µL, 3-10
kcpm) were loaded onto 15% denaturing 19:1 acrylamide/bisacrylamide
gel containing 7 M urea, electrophoresced at 1900 V for approximately
1 h, and transferred to a cassette and stored at -80 °C with X-ray
film.
Melting Temperature (Tm) Measurements. All Tm measurements
of the DNA duplexes (2.5 µM, final duplex concentration) were made
in 50 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium
chloride. Absorbance versus temperature profiles were measured at 260
nm with a Shimadzu UV-2550 spectrophotometer equipped with a
Peltier temperature controller using a cell with a 1 cm path length.
The absorbance of the samples was monitored at 260 nm from 5 to
90 °C, with a heating rate of 1 °C/min. From these profiles, first
derivatives were calculated to determine Tm values.
Sequences of ICON Probes and PCR Primers in Methylation
Quantification Experiments. For model RB1 study. Probes for site
59683/4, 5′-d(TGTTCBAGGTGAACCATTp)-3′ and 5′-d(CTCBAA-
CACCCAGGCGAGp)-3′. Probes for site 59695/6, 5′-d(AGGCBAG-
GTCAGAACAGGp)-3′ and 5′-d(CCTCBCCTGGGTGTTCGAp)-3′.
Primers, 5′-d(ACAGCTGTTATACCCATT)-3′ and 5′-d(GTTGTTTTGC-
TATCCGTGCA)-3′. For mouse genome study. Probes for chr11 115,-
285,676, 5′-d(GCTCBGGGACCTCGTTGGp)-3′ and 5′-d(CCCCBAGC-
CCAAAGAGGAp)-3′. Probes for chr11 115,285,805, 5′-d(ATAC-
BTCACGGGCGTGACp)-3′ and 5′-d(TGACBTATATGCAGAAGCp)-
3′. Primers, 5′-d(GGAGCCTGCTGAGGCCTGT)-3′ and 5′-d(CAGT-
GCAAAACCCAGGTTCA)-3′; “p” is a phosphate group of the 3′ end
to avoid that probes work as PCR primers.
Methylation Quantification. The model DNA duplex (100 nM or
the desired concentration) or genome DNA (20 ng) was added into a
solution (16 µL) of B-containing DNA probes (1 nM each for the sense
and antisense strands), 100 mM potassium hexacyanoferrate(III), 0.5
mM EDTA, and 1 M sodium chloride in 50 mM Tris-HCl buffer (pH
7.7). The reaction mixture was incubated at 95 °C for 5 min and then
cooled to 0 °C immediately. To the mixture was added 25 mM
potassium osmate (4 µL), and the reaction was allowed to proceed at
DNA Synthesis and Characterization. Artificial DNA was syn-
thesized by the conventional phosphoramidite method by using an
Applied Biosystems 392 DNA/RNA synthesizer. Synthesized DNA was
purified by reverse phase HPLC on a 5-ODS-H column (10 × 150
mm, elution with a solvent mixture of 0.1 M triethylammonium acetate
(TEAA), pH 7.0, linear gradient over 30 min from 5 to 20% acetonitrile
at a flow rate 3.0 mL/min). Each ODN was characterized by MALDI-
TOF MS. DNA2′, 5′-d(TGTGAGGCBCTGCCCCCACC)-3′ ([M -
H]-, calcd 6347.30, found 6347.96). Probes for RB1 59683/4 methy-
lation site, 5′-d(TGTTCBAGGTGAACCATTp)-3′ ([M - H]-, calcd
5901.97, found 5901.61), 5′-d(CTCBAACACCCAGGCGAGp)-3′ ([M
- H]-, calcd 5850.94, found 5849.51). Probes for RB1 59695/6
methylation site, 5′-d(AGGCBAGGTCAGAACAGGp)-3′ ([M - H]-,
calcd 5995.04, found 5994.40), 5′-d(CCTCBCCTGGGTGTTC-
GAp)-3′ ([M - H]-, calcd 5854.91, found 5854.81). Probes for mouse
chr11 115,285,676 methylation site, 5′-d(GCTCBGGGACCTCGT-
TGGp)-3′ ([M - H]-, calcd 5919.94, found 5919.37), 5′-d(CCCCBA-
GCCCAAAGAGGAp)-3′ ([M - H]-, calcd 5859.96, found 5859.39).
Probes for mouse chr11 115,285,805 methylation site, 5′-d(ATACBT-
CACGGGCGTGACp)-3′ ([M - H]-, calcd 5896.96, found 5895.37),
5′-d(TGACBTATATGCAGAAGCp)-3′ ([M - H]-, calcd 5920.00,
found 5919.29).
Preparation of 32P-5′-End-Labeled DNA. The DNAs (400 pmol
strand) were 5′-end-labeled by phosphorylation with 4 µL of [γ-32P]-
9
14516 J. AM. CHEM. SOC. VOL. 129, NO. 46, 2007