Squaryl Group as Mimic of Phosphate Group
A R T I C L E S
(pH 6.0) at a flow rate of 1.0 mL/min. ESI mass spectra were measured
on Mariner (PerSeptive Biosystems Inc.). MALDI-TOF mass spectra
were measured on Voyager RP (Applied Biosystems Inc.). UV spectra
were measured by a U-2000 spectrophotometer (Hitachi Co., Ltd.).
Thymidine was purchased from Yamasa Co., Ltd. Snake venom
phosphodiesterase was purchased from Boehringer Mannheim Bio-
chemica Co., Ltd., and alkaline phosphatase was purchased from Takara
Shuzou Co., Ltd. Dry THF was purchased from Wako Pure Chemical
Industries, Ltd. Triethylamine was distilled from CaH2 and stored over
Molecular Sieves 4A.
115.82, 116.38, 127.18, 142.27, 156.82, 156.86, 169.87, 173.41, 174.94,
189.00. ESIMS Calcd: C24H29N6O10 [MH]+ 561.5287. Found: 561.5207.
Thymidine Dimer Building Unit (9). To a stirred solution of 7
(250 mg, 0.29 mmol) and triethylamine (120 µL, 0.87 mmol) in 2.9
mL of dry THF was added chloro(2-cyanoethoxy)(N,N-diisopropy-
lamino)phosphine (78 µL, 0.35 mmol) at r.t.. After being stirred for
3.5 h, the mixture was diluted with CHCl3 and extracted with 5%
NaHCO3 aq. The organic layers were collected and dried over Na2-
SO4. After being filtered, the mixture was evaporated under reduced
pressure. The residue was purified by silica gel column chromatography
(C-200, 5 g, methanol/chloroform 0-2%). The fractions containing the
product were combined and evaporated under reduced pressure, and
the residue was dissolved in 1.0 mL of CHCl3 and precipitated into 20
5′-O-(4,4′-Dimethoxytrityl)-3′-(2-ethoxy-3,4-dioxocyclobuten-1-
yl)amino-3′-deoxythymidine (5). A solution of 3′-amino-5′-O-(4,4′-
dimethoxytrityl)-3′-deoxythymidine (4)26-28 (0.1 g, 0.18 mmol) and
diisopropylethylamine (16 µL, 92 µmol) in 2 mL of ethanol was stirred
at room temperature (r.t.), and 3,4-diethoxy-3-cyclobuten-1, 2-dione
(47 mg, 0.28 mmol) was added and stirred for 1 h. The reaction solution
was evaporated, and the residue was purified by silica gel column
chromatography (C-200, 6 g, ethyl acetate/hexane 0-80%) to give 5
as a foam (0.12 g, 98%). 1H NMR (CDCl3, δ) 1.21 (3H, t, CH2CH3, J
) 6.76 Hz), 1.29 (3H, s, thymine CH3), 2.48-2.62 (2H, m, 2′H), 3.42-
3.59 (2H, m, 5′H), 3.79 (6H, s, OCH3), 4.15 (1H, m, 4′H), 4.51, 4.54
1
mL of hexane to give 9 as a white solid (222 mg, 72%). H NMR
(CDCl3, δ) 1.16, 1.18 (12H, d, CH3 of i-Pr, J ) 6.26 Hz), 1.49, 1.86
(each 3H, s, CH3 of thymines), 2.31-2.73 (6H, m, 2′H, CE), 3.37-
4.17 (18H, m, 3′H, 4′H, 5′H, OCH3, i-Pr, CE), 4.49, 5.02 (each 1H, s,
NH of squaryl amide), 6.01, 6.30 (each 1H, m, 1′H), 6.78-7.49 (15H,
m, DMTr, 6H); 13C NMR (CDCl3, δ) 11.80, 12.26, 20.00, 20.37, 22.85,
22.94, 24.46, 24.52, 37.57, 39.00, 43.14, 43.32, 45.20, 45.29, 55.04,
55.15, 57.73, 58.13, 63.33, 84.00, 86.35, 86.88, 111.34, 112.69, 113.07,
117.86, 118.24, 126.83, 127.76, 127.95, 129.91, 135.01, 135.23, 144.11,
150.42, 150.72, 158.05, 158.35, 163.88, 166.93, 167.70, 182.24, 183.07;
31P NMR (CDCl3, δ) 149.34, 149.90. ESIMS Calcd: C54H62N8O13PNa
[M + Na]+ 1085.0960. Found: 1085.0545.
(1H, 2H, m, q, 3′H, CH2CH3, J ) 6.76 Hz), 6.78 (1H, t, 1′H, J1′,2′
)
7.91 Hz), 6.84 (4H, m, DMTr), 7.26-7.40 (11H, m, DMTr), 7.67 (1H,
s, 6H), 9.03, 9.07 (1H, br, 3′NH), 10.19, 10.41 (1H, br, thymine NH);
13C NMR (CDCl3, δ) 11.62, 15.94, 55.29, 56.62, 63.63, 70.02, 84.51,
86.17, 87.21, 113.01, 113.15, 127.27, 127.90, 128.14, 134.55, 134.73,
134.85, 143.73, 151.75, 158.68, 162.95, 170.40, 178.12, 181.10, 190.56.
ESIMS Calcd: C37H36N3O9Na [M + Na]+ 689.6975. Found: 689.6871.
UV Spectra. UV spectra of TsqT (8) and its model compound 10
were measured in aqueous solution at r.t. by a U-2000 spectrophotom-
eter (Hitachi, Co., Ltd.). The λmax values of 8 are 270 and 292 nm.
Those of 10 are 276 and 293 nm.
N-(Thymidin-5′-yl)-N′-[5′-O-(4,4′-dimethoxytrityl)]thymidin-3′-
yl]-3,4-dioxocyclobuten-1,2-diamine (7). To a solution of 5 (50 mg,
75 µmol) in 2 mL of ethanol was added 5′-amino-5′-deoxythymidine
(6)28,43-45 (18 mg, 75 µmol). The mixture was stirred at 40 °C for 24
h. The solution was evaporated under reduced pressure. The residue
was purified by silica gel column chromatography (C-200, 4 g,
methanol/chloroform 0-8%) to give 7 (64 mg, 99%). 1H NMR (DMSO-
d6, δ) 1.44 (3H, s, T5′-CH3), 1.70 (3H, s, T3′-CH3), 2.02-2.29 (4H,
m, T5′-2′H, T3′-2′H), 3.12-3.73 (11H, m, OCH3, T5′-5′H, T3′-5′H,
T3′-4′H), 3.89 (1H, m, T5′-4′H), 4.11 (1H, m, T3′-3′H), 4.74 (1H, br,
T5′-3′H), 5.36 (1H, br, OH), 6.14 (2H, t, T5′-1′H, T3′-1′H), 6.77, 6.80
(4H, m, DMTr), 7.15-7.37 (10H, m, DMTr, T3′-6H), 7.47 (1H, s,
T5′-6H), 7.73 (1H, br, NH), 8.24 (1H, s, NH), 11.26 (2H, br, T5′-NH,
T3′-NH); 13C NMR (DMSO-d6, δ) 11.87, 12.09, 45.58, 53.50, 54.98,
62.92, 70.46, 79.10, 83.25, 83.58, 84.94, 85.90, 109.54, 109.82, 113.05,
126.59, 127.50, 127.70, 129.56, 134.99, 135.16, 135.60, 135.85, 144.40,
150.12, 150.20, 157.90, 163.40, 181.81, 182.54. ESIMS Calcd: C45H45N6-
O12Na [M + Na]+ 884.8753. Found: 884.8174.
N-(Thymidin-5′-yl)-N′-(thymidin-3′-yl)-3,4-dioxocyclobuten-1,2-
diamine (8). Compound 7 (86 mg, 0.1 mmol) was dissolved in 2.0
mL of 80% acetic acid, and the mixture was stirred at r.t. for 1.5 h.
The solution was evaporated under reduced pressure and coevaporated
twice with water to remove the last traces of acetic acid. The residue
was diluted with ethyl acetate and extracted with water. The aqueous
layer was evaporated under reduced pressure to give 8 as a white solid
(49 mg, 87%). 1H NMR (DMF-d7, δ) 1.78, 1.80 (each 3H, s, T5′-CH3,
T3′-CH3), 2.14-2.52 (4H, m, T5′-2′H, T3′-2′H), 3.78-3.99 (6H, m,
T5′-4′H, 5′H, T3′-4′H, 5′H), 4.37 (1H, m, T5′-3′H), 4.73 (1H, m, T3′-
3′H), 5.31 (1H, br, 5′OH), 5.52 (1H, br, 3′OH), 6.27 (2H, t, t, 1′H,
J1′,2′ ) 6.27, 6.60 Hz), 7.54 (1H, s, 6H), 7.76 (1H, br, NH of squaryl
amide), 7.87 (1H, s, 6H), 7.97 (1H, br, NH of squaryldiamide), 11.19
(2H, br, NH, NH of thymines); 13C NMR (DMF-d7, δ) 17.89, 18.10,
44.76, 44.88, 52.02, 60.00, 67.27, 77.32, 89.90, 90.30, 91.70, 92.19,
NMR Spectra of 8 in the Presence of Mg2+. NMR spectra were
obtained on a JEOL GX-270. The 1H NMR spectra of 8 were obtained
at various concentrations of MgCl2.
Synthesis of Oligonucleotides. TpT and all oligodeoxynucleotides
were synthesized by 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 prescribed for the thymidine dimer building
unit 9 was 15 min. The coupling using the dimer building unit proceeded
in >99% yield, which was estimated by the DMTr cation analysis.
The protecting groups of dC, dA, and dG used were acetyl, phenoxy-
acetyl, and isopropylphenoxyacetyl, respectively, and were deprotected
by treatment with concentrated NH3 (1.5 mL) at r.t. for 2 h. The
ammonia solution was filtered and evaporated under reduced pressure.
The residue was purified by a Sep-Pak reverse-phase column purchased
from Waters Co., Ltd. After the column was immersed with CH3CN
(5.0 mL) and 0.1 M NH4OAc (pH 7.0) (5.0 mL) for 1 min, the solution
was removed by filtration. The sample was charged on the column.
Subsequently, the failure sequences without the DMTr group were
washed out by 10-12% CH3CN/0.1 M NH4OAc (pH 7.0) (10 mL).
After that, the column was treated with 1.0% aqueous TFA (5.0 mL)
at r.t. for 5 min to remove the DMTr group of DMTr-oligodeoxy-
nucleotides bound to the column. Further elution was carried out by
0.1 M NH4OAc (pH 7.0) (5.0 mL) and 15% CH3CN in water (10 mL)
to obtain the desired oligodeoxynucleotide. After removal of the
solvents, the residue was purified by reverse-phase HPLC using a linear
gradient of 0-15% CH3CN in 0.1 M NH4OAc (pH 7.0) for 20 min at
the flow rate of 1 mL/min. If necessary, anion-exchange HPLC using
a linear gradient of 0-60% buffer B in buffer A for 45 min at the
flow rate 1 mL/min was used (buffer A, 10% CH3CN/25 mM phosphate
buffer (pH 6.0); buffer B, 10% CH3CN/25 mM phosphate buffer (pH
6.0), 1.0 M NaCl).
The isolated yields were calculated by using the ꢀ values obtained
by the method of Cantor.46 The ꢀ value ()22386) of the TsqT dimer
was calculated by the enzyme digestion of 5′-d(GACGCATsqTAGC-
(43) Hata, T.; Yamamoto, I.; Sekine, M. Chem. Lett. 1976, 601-604.
(44) Yamomoto, I.; Sekine, M.; Hata, T. J. Chem. Soc., Perkin Trans. 1 1980,
306-310.
(46) Fasman, G. D., Ed. Handbook of Biochemistry and Molecular Biology,
(45) Lin, T.-S.; Prusoff, W. H. J. Med. Chem. 1978, 21, 109-112.
3rd ed.; CRC Press: Cleveland, 1977; Vol. 1.
9
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