Synthesis and Structural Properties of New Oligodeoxynucleotide Analogues
FULL PAPER
1 H, 5 H), 6.10Ϫ6.12 (m, 1 H, 1ЈH), 6.73Ϫ6.79 (m, 4 H, DMTr), evaporated under reduced pressure, and the residue was dissolved
7.13Ϫ7.39 (m, 9 H, DMTr), 7.79, 7.82 (d, J ϭ 8.24 Hz, 1 H, 6 H) in CHCl3 (1.0 mL). The CHCl3 solution was poured into hexane
ppm. 13C NMR (CDCl3): δ ϭ 15.97, 30.98, 34.20, 55.28, 63.67,
(20 mL) to give 10 as a white solid (0.26 g, 75%). 1H NMR
64.54, 70.19, 78.11, 87.05, 88.23, 103.06, 113.23, 123.68, 127.07, (CDCl3): δ ϭ 1.15Ϫ1.30 (m, 12 H, CH3 of iPr), 1.87 (s, 3 H, CH3
127.66, 127.92, 127.96, 128.32, 128.50, 129.02, 129.97, 131.87,
132.02, 133.19, 134.97, 135.18, 135.97, 139.60, 144.13, 149.53,
150.89, 158.55, 162.87, 171.82, 178.04 ppm. ESIMS calcd.
C36H35N3O9Na [M ϩ Na]ϩ 676.2271, found 676.2219.
of thymine), 2.25Ϫ2.73 (m, 8 H, U5Ј-3ЈH, T3Ј-2ЈH, CE), 3.31Ϫ4.14
(m, 16 H, U5Ј-2ЈH, 4ЈH, 5ЈH, T3Ј-3ЈH, 4ЈH, 5ЈH, OCH3, iPr), 4.43,
4.69 (s, each 1 H, NH of squaryl amide), 5.31Ϫ5.39 (m, 1 H, U5Ј-
5 H), 5.95 (2m, U5Ј-1ЈH, T3Ј-1ЈH), 6.80Ϫ6.83 (m, 4 H, DMTr),
7.12Ϫ7.40 (m, 10 H, DMTr, T3Ј-6 H), 7.86, 7.89 (d, J ϭ 8.57 Hz,
1 H, U5Ј-6 H) ppm. 13C NMR (CDCl3): δ ϭ 12.36, 20.53, 21.57,
24.63, 24.73, 26.20, 37.81, 43.26, 43.43, 45.08, 45.92, 55.27, 57.97,
63.90, 84.22, 86.87, 100.03, 103.25, 113.21, 118.16, 126.96, 127.90,
128.08, 130.04, 134.99, 135.19, 144.09, 149.98, 151.32, 164.45,
168.51, 176.55, 176.56, 182.24, 182.25 ppm. 31P NMR (CDCl3):
δ ϭ 149.37, 149.86 ppm. ESIMS calcd. C53H61N8O14PNa [M ϩ
Na]ϩ 1087.3943, found 1087.3945.
NЈ-[3Ј-Deoxy-5Ј-O-(4,4Ј-dimethoxytrityl)]uridin-2Ј-yl]-3,4-dioxo-N-
(thymidin-5Ј-yl)cyclobutene-1,2-diamine (8): To a solution of 6
(0.55 g, 0.84 mmol) and diisopropylethylamine (0.14 mL,
0.84 mmol) in 8 mL of CH2Cl2/ethanol (v/v, 1:1) stirred at room
temperature and 5Ј-amino-5Ј-deoxythymidine (7)[9Ϫ12] (0.2 g, 0.84
mmol) was added and stirred at 40 °C for 20 h. The solution was
evaporated under reduced pressure. The residue was purified by
dry silica gel column chromatography (C-200, 20 g ϩ N60, 3 g,
methanol/chloroform, 0 to 4%) to give 8 (0.71 g, quant.). 1H NMR
([D6]DMSO): δ ϭ 1.78 (s, 3 H, T3Ј-CH3), 2.08Ϫ2.38 (m, 4 H, U5Ј-
3ЈH, T3Ј-2ЈH), 3.20Ϫ3.38 (m, 5 H, U5Ј-5ЈH, T3Ј-5ЈH, T3Ј-4ЈH),
3.73Ϫ3.78 (m, 7 H, U5Ј-2ЈH, OCH3), 4.18 (m, 1 H, T3Ј-4ЈH), 4.42
(m, 1 H, T3Ј-3ЈH), 5.32, 5.35 (d, J ϭ 8.24 Hz, 1 H, U5Ј-5 H),
5.42Ϫ5.43 (d, J ϭ 3.63 Hz, 1 H, T3Ј-3ЈOH), 5.84Ϫ5.86 (d, J ϭ
3.96 Hz, 1 H, U5Ј-1ЈH), 6.16Ϫ6.21 (t, J ϭ 6.59, 7.26 Hz, 1 H, T3Ј-
1ЈH), 6.88Ϫ6.91 (m, 4 H, DMTr), 7.23Ϫ7.43 (m, 9 H, DMTr),
7.67, 7.70 (d, J ϭ 7.91 Hz, 1 H, U5Ј-6 H), 7.74Ϫ7.81 (m, 1 H, T5Ј-
6 H), 11.26 (br., 2 H, NH ϫ 2) ppm. 13C NMR (CDCl3): δ ϭ
15.84, 33.77, 55.20, 63.59, 64.35, 70.06, 78.15, 86.91, 88.41, 102.78,
113.15, 123.64, 126.96, 127.84, 127.90, 129.90, 131.93, 134.94,
135.14, 139.69, 144.08, 149.35, 150.78, 158.45, 163.19, 172.25,
177.81, 183.62, 189.02. ESIMS calcd. C44H44N6O12Na [M ϩ Na]ϩ
871.2915, found 871.2996.
UV Spectra: UV spectra of U2Јsq5ЈT (9) and its model compound
11 were measured in aqueous solution at room temperature on a
U-2000 spectrophotometer (Hitachi, Ltd.). The λmax. values of 9
are 270 and 292 nm; those of 11 are 276 and 293 nm.
Synthesis of Oligonucleotides: TpT and all oligodeoxynucleotides
were synthesized on an ABI DNA/RNA synthesizer 392 on a
1.0 µmol scale and released from the CPG polymer support in the
DMTr-on mode. The coupling time used for the uridine-thymidine
dimer building unit 10 was 15 min, and the coupling with the dimer
building unit proceeded in 94% yield, as estimated by DMTr cation
analysis. The protecting groups used for dC, dA, and dG were ace-
tyl, (phenoxy)acetyl, and (isopropylphenoxy)acetyl, respectively,
and they were removed by treatment with concd. NH3 (1.5 mL) at
room temperature for 5 h. The ammonia solution was filtered, and
the solvents were evaporated under reduced pressure. The residue
was purified on a Sep-PakTM reversed-phase column purchased
from Waters Co., Ltd.. After the column was immersed with
CH3CN (5.0 mL) and 0.1 NH4OAc (pH 7.0) (5.0 mL) for 1 min,
the solution was removed by filtration. The sample was loaded onto
the column. Subsequently, the failure sequences without the DMTr
group were washed out with 10Ϫ12% CH3CN/0.1 NH4OAc (pH
7.0) (10 mL). After that, the column was treated with 1.0% aqueous
TFA (5.0 mL) at room temperature for 5 min to remove the DMTr
groups of DMTr-oligodeoxynucleotides bound to the column.
Further elution was carried out with NH4OAc (0.1 , pH 7.0,
5.0 mL) and CH3CN in water (15%, 10 mL) to obtain the desired
oligodeoxynucleotide. After removal of the solvents, the residue
was purified by anion exchange HPLC with a liner gradient of
0Ϫ60% buffer B in buffer A for 45 min at a flow rate of 1 mL/min
(buffer A: 10% CH3CN/25 m phosphate buffer (pH 6.0), buffer
B: 10% CH3CN/25 m phosphate buffer (pH 6.0), 1.0 NaCl).
The isolated yields were calculated by use of ε values obtained by
Cantor’s method.[16] The ε value (ϭ 23314) of the U2Јsq5ЈT dimer
was calculated by enzyme digestion of 5Ј-d(CGCAU2Јsq5ЈTAGCC)-
3Ј with snake venom phosphodiesterase and calf intestine alkaline
phosphatase. 5Ј-d(CGCAU2Јsq5ЈTAGCC)-3Ј: MALDI-TOF Mass
calcd. for C99H121N38O56P8: 2985.57; found 2985.30.
NЈ-(3Ј-Deoxyuridin-2Ј-yl)-3,4-dioxo-N-(thymidin-5Ј-yl)cyclobutene-
1,2-diamine (9): Compound 8 (0.16 g, 0.19 mmol) was dissolved in
acetic acid (80%,5.0 mL), and the mixture was stirred at room tem-
perature for 3 h. The solution was extracted with CHCl3/H2O, and
the aqueous layer was extracted with CHCl3 (2ϫ). The aqueous
layer was evaporated under reduced pressure and coevaporated
three times with water to remove the last traces of acetic acid to
1
give 9 as a white solid (83 mg, 81%). H NMR ([D6]DMSO): δ ϭ
1.78 (s, 3 H, T3Ј-CH3), 2.09Ϫ2.29 (m, 4 H, U5Ј-3ЈH, T3Ј-2ЈH),
3.47Ϫ3.89 (m, 5 H, U5Ј-2ЈH, 5ЈH, T3Ј-5ЈH), 4.18, 4.27 (m, 2 H,
U5Ј-4ЈH, T3Ј-4ЈH), 4.62 (m, 1 H, T3Ј-3ЈH), 5.16 (br., 1 H, 5ЈOH),
5.41 (br., 1 H, 3ЈOH), 6.62, 6.65 (d, J ϭ 7.91 Hz, 1 H, U5Ј-5 H),
5.82Ϫ5.84 (d, J ϭ 4.29 Hz, 1 H, U5Ј-1ЈH), 6.15Ϫ6.20 (t, J1Ј,2Ј
ϭ
6.27, 6.60 Hz, 1 H, T3Ј-1ЈH), 7.43 (s, 1 H, T3Ј-6 H), 7.70 (br., 1 H,
NH of squarylamide), 7.88, 7.91 (d, J ϭ 7.91 Hz, 1 H, U5Ј-6 H),
8.04 (br., 1 H, NH of squarylamide), 11.23 (br., 2 H, NH of bases)
ppm. 13C NMR ([D6]DMSO): δ ϭ 12.10, 32.75, 45.42, 57.78, 62.55,
70.53, 78.99, 83.49, 84.94, 88.78, 101.71, 109.83, 135.81, 140.31,
150.25, 150.37, 162.89, 163.45, 166.86, 167.72, 181.56, 182.68 ppm.
ESIMS calcd. C23H27N6O10 [M ϩ H]ϩ 547.1789, found 547.1791.
3Ј-Deoxyuridine-Thymidine Dimer Building Unit 10: Chloro(2-
cyanoethoxy)(diisopropylamino)phosphane (0.12 mL, 0.53 mmol)
was added at room temperature to a stirred solution of 8 (0.3 g,
0.35 mmol) and triethylamine (0.29 mL, 1.77 mmol) in dry THF Enzyme Analysis of Modified Oligodeoxynucleotides: The enzy-
(3.5 mL). After the mixture had been stirred for 3.5 h, methanol matic digestion was performed with a modified oligodeoxynucleo-
(1 mL) was added, and the mixture was diluted with CHCl3 and tide (0.5 OD), snake venom phosphodiesterase (4 µL), and calf
extracted with 5% aq. NaHCO3. The organic layers were collected intestine alkaline phosphatase (2 µL) in 50 µL of alkaline phospha-
and dried with Na2SO4. After filtration, the solvents were evapo- tase buffer (pH 9.0) at 37 °C for 2 h. After the enzymes had been
rated under reduced pressure. The residue was purified by silica
gel column chromatography (N60, 6 g, methanol/chloroform, 0 to
1.5%). The fractions containing the product were combined and
deactivated by heating at 100 °C for 1 min, the solution was diluted
and filtered (0.45 µm filter, Millex-HV, MILLIPORE). This solu-
tion was analyzed by reversed-phase HPLC with a linear gradient
Eur. J. Org. Chem. 2004, 2142Ϫ2150
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2149