Y. Hari et al. / Bioorg. Med. Chem. 19 (2011) 1162–1166
1165
were used. All melting points were measured on a Yanagimoto mi-
cro melting point apparatus and are uncorrected. 1H NMR, 13C NMR
and 31P NMR spectra were recorded on a JEOL ECS400 spectrome-
ter. IR spectra were recorded on JASCO FT/IR-200 and JASCO FT/IR-
4200 spectrometers. Optical rotations were recorded on a JASCO
DIP-370 instrument. Mass spectra were measured on a JEOL JMS-
600 or JEOL JMS-700 mass spectrometer. MALDI-TOF mass spectra
were recorded on a Bruker Daltonics Autoflex II TOF/TOF mass
spectrometer. EYELA Cute Mixer CM-1000 was used as a shaker.
resin by treatment with 28% aqueous NH3 solution at room tem-
perature for 1.5 h. Additional treatment with 28% aqueous NH3
solution at 55 °C for 15 h underwent the removal of all protecting
groups on TFOs. The obtained crude TFOs were purified with Sep-
PakÒ Plus C18 cartridges (Waters) followed by reversed-phase
HPLC (Waters XTerraÒ MS C18 2.5
lm, 10 ꢂ 50 mm). The composi-
tion of the TFOs was confirmed by MALDI-TOF-MS analysis.
MALDI-TOF-MS data ([MꢁH]ꢁ) for 6: found 4395.39 (calcd
4395.91).
4.1.1. 1-Ethynyl-2-deoxy-
D
-ribose (3)
4.3. Azide synthesis
NaOMe (521 mg, 9.64 mmol) was added to a solution of 216 in
MeOH (30 mL) at room temperature and the mixture was stirred
for 3 h. The solvent was removed under reduced pressure and
the residue was purified by silica gel column chromatography
Among azide reagents used for click chemistry, 1-(3-azidophe-
nyl)urea and 1-(4-azidophenyl)urea were new compounds which
were prepared according to the following procedure.
(CHCl3/MeOH = 20:1) to give compound 317
(a:b = 3:1).
4.3.1. 1-(3-Azidophenyl)urea
4.1.2. 1-(b)-Ethynyl-5-O-(4,40-dimethoxytrityl)-2-deoxy-
D
-ribose
NaN3 (10 mg, 15.3
phenyl)urea (20 mg, 76.6
7.66 mol), sodium ascorbate (3.5 mg, 17.7
N,N0-dimethylethylenediamine (1.0
L, 9.23 mol) in DMSO–H2O
l
mol) was added to a solution of 1-(3-iodo-
mol), copper(I) iodide (1.5 mg,
mol) and
(4) and 1-(
a
)-ethynyl-5-O-(4,40-dimethoxytrityl)-2-deoxy-
D
-
l
ribose (5)
l
l
Under a nitrogen atmosphere, DMTrCl (1.21 g, 2.95 mmol) was
added to a solution of compound 3 ( :b = 3:1, 280 mg, 1.97 mmol)
l
l
a
(5:1, 1.0 mL) at room temperature and the mixture was stirred
for 10 h. The mixture was extracted with AcOEt. The organic ex-
tracts were washed with water and brine and dried over Na2SO4.
The organic layer was evaporated and the residue was purified
by flash column chromatography (toluene/acetone = 3:1) to give
compound (12.6 mg, 93%) as yellow solids. mp 137–139 °C. IR
(KBr) 3508, 3376, 3318, 3201, 3141, 3086, 2116, 1659, 1592,
in anhydrous pyridine (60 mL) at room temperature and the mix-
ture was stirred for 2 h. After addition of saturated aqueous NaH-
CO3 solution, the mixture was extracted with AcOEt. The organic
extracts were washed with water, dried over Na2SO4 and concen-
trated under reduced pressure. The residue was purified by flash
silica gel column chromatography (n-hexane/AcOEt = 4:1) to give
compound 4 (159 mg, 18%) and 5 (461 mg, 56%). Compound 418
24.4 (c 1.03, CHCl3). 1H NMR (CDCl3)
1.80 (1H, d,
:
1552, 1487, 1439, 1349, 1317, 1307 cmꢁ1 1H NMR (CD3OD) d
.
½
a 2D6
ꢀ
d
6.67 (1H, ddd, J = 2.0, 8.0 and 10 Hz), 7.04 (1H, ddd, J = 2.0, 8.5
and 10 Hz), 7.24 (1H, dd, J = 8.0 and 8.5 Hz), 7.31 (1H, dd, J = 2.0
and 2.0 Hz). 13C NMR (CD3OD) d 110.3, 113.8, 116.4, 131.1, 141.9,
142.6, 159.1. FABMS: 178 (MH+). Anal. Calcd for C7H7N5O: C,
47.46; H, 3.98; N, 39.53. Found: C, 47.61; H, 4.10; N, 39.30.
J = 3.3 Hz), 2.15 (1H, ddd, J = 3.0, 6.6 and 17.2 Hz), 2.24 (1H, ddd,
J = 6.0, 8.7 and 17.2 Hz), 2.46 (1H, d, J = 2.1 Hz), 3.15 (1H, dd,
J = 6.0 and 12.8 Hz), 3.26 (1H, dd, J = 4.5 and 12.8 Hz), 3.78 (6H,
s), 3.86–3.91 (1H, m), 4.35–4.38 (1H, m), 4.76 (1H, m), 6.78–6.83
(4H, m), 7.16–7.69 (9H, m). Compound 5: ½a D25
ꢀ
0.926 (c 0.98,
CHCl3). IR mmax (KBr) 3280, 2934, 2044, 1611, 1509, 1251, 1221,
4.3.2. 1-(4-Azidophenyl)urea
1177, 1085, 1034 cmꢁ1
.
1H NMR (CDCl3) d 2.07 (1H, dt, J = 3.6
NaN3 (20 mg, 30.5
phenyl)urea (40 mg, 153
15.3 mol), sodium ascorbate (6.5 mg, 32.8
N,N0-dimethylethylenediamine (1.7
L, 15.6 mol) in DMSO-H2O
l
mol) was added to a solution of 1-(4-iodo-
mol), copper(I) iodide (2.9 mg,
mol) and
and 13.4 Hz), 2.11 (1H, d, J = 6.7 Hz), 2.45 (1H, ddd, 6.7, 7.9 and
13.4 Hz), 2.51 (1H, d, J = 1.8 Hz), 3.10 (1H, dd, J = 5.5 and 9.8 Hz),
3.16 (1H, dd, J = 4.3 and 9.8 Hz), 3.72 (6H, s), 4.09–4.12 (1H, m),
4.21–4.25 (1H, m), 4.80–4.85 (1H, m), 6.78–6.83 (4H, m),
7.12–7.40 (9H, m). 13C NMR (CDCl3) d 41.8, 55.2, 64.0, 67.3, 73.9,
74.6, 84.0, 86.2, 113.1, 126.8, 127.8, 128.0, 130.0, 135.8, 144.7,
158.4. HRMS (FAB) m/z calcd for C28H28NaO5 [M+Na]+: 467.1834;
found, 467.1840.
l
l
l
l
l
(5:1, 3.0 mL) at room temperature and the mixture was stirred
for 12 h. The mixture was extracted with AcOEt. The organic ex-
tracts were washed with water and brine and dried over Na2SO4.
The organic layer was evaporated and the residue was purified
by flash column chromatography (n-hexane/acetone = 4:1) to give
compound (23.8 mg, 88%) as yellow solids. mp 174–177 °C. IR
(KBr) 3418, 3351, 3318, 2575, 2491, 2449, 2141, 1613, 1581,
4.1.3. 1-(b)-Ethynyl-3-O-[2-
cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,40-
1534, 1512, 1485 cmꢁ1 1H NMR (CD3OD)
. d 6.96 (2H, d,
dimethoxytrityl)-2-deoxy-
D
-ribose (1)
J = 8.0 Hz), 7.38 (2H, d, J = 8.0 Hz). 13C NMR (CD3OD) d 120.3,
121.9, 135.5, 138.1, 159.3. FABMS: 178 (MH+). Anal. Calcd for
C7H7N5O: C, 47.46; H, 3.98; N, 39.53. Found: C, 47.44; H, 4.09; N,
39.13.
Under a nitrogen atmosphere, 2-cyanoethyl N,N,N0,N0-tetra-
isopropylphosphordiamidite (0.10 mL, 0.33 mmol) was added to
a solution of compound 4 (77 mg, 0.17 mmol) and diisopropylam-
monium tetrazolide (35 mg, 0.17 mmol) in anhydrous MeCN–THF
(3:1, 2.4 mL) at room temperature and the mixture was stirred
for 4 h. The solvent was removed under reduced pressure and
the residue was purified by flash silica gel column chromatography
(n-hexane/AcOEt = 5:1) to give compound 119 (80 mg, 69%) as a
colorless oil. 31P NMR (CDCl3) d 148.5, 149.1.
4.4. Click chemistry: Typical procedure
A solution of 1-(4-azidephenyl)urea (10 mM in DMSO, 3
was added to a mixture of CuSO4 (2 mM in H2O, 3 L), TBTA
(2 mM in DMSO, 6 L), sodium ascorbate (10 mM in H2O, 3 L), 6
(0.9 mM in H2O, 3.3 L) and H2O (8.7 L) in a 1.5-mL Eppendorf
lL)
l
l
l
l
l
4.2. Oligonucleotide synthesis
tube. The mixture was shaken at room temperature for 20 h using
a shaker (1000 rpm). The whole was purified by reversed-phase
The synthesis of 6 was performed on a 0.2-
l
mol scale on an
HPLC [column: Waters XTerraÒ MS C18 2.5
l
m, 4.6 ꢂ 50 mm; gra-
automated DNA synthesizer (Applied Biosystems Expedite™
8909) using the common phosphoramidite protocol except for a
prolonged coupling time of 5 min for unnatural phosphoramidites.
TFOs synthesized on DMTr-ON mode were cleaved from the CPG
dient: 8–20% acetonitrile in 0.1 M triethylammonium acetate buf-
fer (pH 7.0) for 30 min; flow rate: 1.0 mL/min] to give the desired
TFO 7r, the composition of which was confirmed by MALDI-TOF-
MS analysis.