26
silica gel to give 14 (289 mg, 74.1%, as a colorless solid): [α]D
(6ЈaS,7ЈS,8ЈR,9ЈaR)-2-(4-tert-Butylphenoxy)-N-{8Ј-[2-cyano-
Ϫ172.3 (c 0.50 in CHCl3); δH (DMSO-d6) 1.25 (9H, s, 3 × CH3),
1.85–1.95 (1H, m, 2Јa-H), 2.08–2.18 (1H, m, 4Ј-H), 2.31–2.37
(1H, m, 2Јb-H), 3.57–3.80 (3H, m, 3Ј-H, 5Јa-H and 5Јb-H), 4.75
(1H, t, J 5.2, OH), 4.87 (2H, s, ArOCH2), 5.14–5.18 (2H, m,
1Ј-H and OH), 5.95 (1H, t, J 6.9, 6Ј-H), 6.85–6.90 (2H, m,
tBPA-Ha), 7.28–7.33 (2H, m, tBPA-Hb), 8.47 (1H, s, 2-H),
10.50 (1H, br, NH); m/z (EI) 453.2011 (Mϩ. C23H27N5O5
requires 453.2010).
ethoxy(diisopropylamino)phosphinoxy]-7Ј-(4,4Ј-dimethoxy-
trityloxymethyl)-7Ј,8Ј,9Ј,9Јa-tetrahydro-6ЈaH-cyclopenta[4,5]-
oxazolo[3,2-e]purin-4Ј-yl}acetamide (18)
Yield 89.7%; δP (CDCl3) 145.54, 145.79; m/z (SIMS) 956.4478
(Mϩ ϩ 1. C53H63N7O8P requires 956.4472).
Synthesis of oligonucleotides
LCAA-CPG carrying a cU or cA nucleoside unit was syn-
thesized according to the procedure reported by Damha et al.36
Oligoribo- and oligodeoxyribonucleotides were synthesized on
an Applied Biosystems Model 392 DNA/RNA synthesizer with
the 1.0 µmol scale. Deprotection of oligonucleotides were
carried out according to the manufacturer’s instructions apart
from deprotection and cleavage from the solid support of
modified oligonucleotides, which were treated with 28% aque-
ous ammonia at room temperature for 2 h. The crude oligo-
nucleotides were purified with reversed phase HPLC. Finally,
the purified oligonucleotides were desalted on a Sep-Pak Plus
cartridge (Waters Corporation, Milford, MA, USA)
(5aR,7R,8S,8aS )-8-(4,4Ј-Dimethoxytrityloxymethyl)-5a,7,8,8a-
tetrahydro-7-hydroxy-2H,6H-cyclopenta[4,5]oxazolo[3,2-c]-
pyrimidine-2,4(3H)-dione (15)
To a solution of 1 (528 mg, 2.2 mmol) in anhydrous pyridine
(10 ml) was added 4,4Ј-dimethoxytrityl chloride (894 mg,
2.2 mmol), and the mixture was stirred at room temperature
for 2 h. After addition of EtOH, the solvent was evaporated
under reduced pressure. The residue was extracted with CHCl3
(70 ml) and the organic layer was washed with saturated
aqueous NaHCO3 (50 ml × 2). After the organic layer was dried
with Na2SO4 and concentrated, the residue was purified by
column chromatography on silica gel. After evaporation of the
solvent, the residue was dissolved with small amounts of
CHCl3, and the solution was added dropwise to n-hexane :
diethyl ether = 3 : 1 (v/v, 200 ml) to give 15 (1.21 g, 94.1%,
as a colorless powder): δH (CDCl3) 1.88–1.99 (1H, m, 2Јa-H),
2.21–2.31 (1H, m, 4Ј-H), 2.55–2.63 (1H, m, 2Јb-H and OH),
3.45–3.60 (2H, m, 5Јa-H and 5Јb-H), 3.80 (6H, s, 2 × OCH3),
4.00–4.09 (1H, m, 3Ј-H), 4.86 (1H, t, J 7.1, 1Ј-H), 4.94
(1H, d, J 1.7, 5-H), 5.32 (1H, dd, J 5.5 and 7.1, 6Ј-H), 6.82–
6.87 (4H, m, DMT-Ha), 7.22–7.43 (9H, m, DMT-Hb), 8.07
(1H, br, NH); m/z (EI) 542.2045 (Mϩ. C31H30N2O7 requires
542.2051).
MALDI-TOF MS Analysis of modified oligonucleotides
1 OD unit of purified oligonucleotides was dissolved in 15 µl of
distilled water. Aliquots (1 µl) of each solution were mixed with
1 µl of saturated aqueous solution of 3-hydroxy-2-picolinic acid
as a matrix. TOF MS spectra were obtained by a Perkin-Elmer
Applied Biosystems Voyager-Linear DE spectrometer in
negative ion mode.
cU1: m/z: 3585.40; C120H154N24O82P11 (M Ϫ 1)Ϫ requires
3585.36. cU2: m/z: 3583.34; C120H152N24O82P11 (M Ϫ 1)Ϫ requires
3583.34. cU12: m/z: 3563.64; C120H132N24O82P11 (M Ϫ 1)Ϫ
requires 3563.19. cA1: m/z: 3707.26; C121H144N60O58P11 (M Ϫ 1)Ϫ
requires 3707.55. cA2: m/z: 3719.59; C122H144N60O58P11 (M Ϫ 1)Ϫ
requires 3719.56. cA12: m/z: 3839.67; C132H144N60O58P11 (M Ϫ 1)Ϫ
requires 3839.67.
(5aR,7R,8S,8aS )-7-[2-Cyanoethoxy(diisopropylamino)-
phosphinoxy]-8-(4,4Ј-dimethoxytrityloxymethyl)-5a,7,8,8a-
tetrahydro-2H,6H-cyclopenta[4,5]oxazolo[3,2-c]pyrimidine-
2,4(3H)-dione (16)
Snake venom phosphodiesterase digestion
0.8 OD units of oligonucleotides were dissolved in 3 ml of
10 mM MgCl2, 50 mM Tris-HCl (pH 8.0). The solution was
transferred into a quartz cell (1 cm path length) and the tem-
perature was maintained at 37 ЊC. After addition of snake
venom phosphodiesterase (2 µl, 2 mg mlϪ1, Boehringer
Mannheim), absorbance at 260 nm was measured for 30 min.
To a solution of 15 (542 mg, 1 mmol) in anhydrous dichloro-
methane (5 ml) was added 2-cyanoethyl tetraisopropyl phos-
phorodiamidite (0.476 ml, 1.5 mmol) and diisopropyl-
ammonium tetrazolide (86 mg, 0.5 mmol), and the mixture was
stirred at room temperature for 3 h. The mixture was extracted
with CH2Cl2 (70 ml) and the organic layer was washed with
saturated aqueous NaHCO3 (50 ml × 2). After the organic layer
was dried with Na2SO4 and concentrated, the residue was puri-
fied by column chromatography on silica gel [0–2% MeOH in
benzene : triethylamine (99 : 1)]. After evaporation of the
solvent, the residue was dissolved with small amounts of
CH2Cl2, and the solution was added to n-pentane (100 ml).
After decantation, the precipitate was dissolved in CH2Cl2 and
evaporated to give 16 (716 mg, 96.3%, as a colorless foam):
δP (CDCl3) 146.32, 146.63; m/z (SIMS) 743.3209 (Mϩ ϩ 1.
C40H48N4O8P requires 743.3206).
Measurements of melting curves
The concentrations of oligonucleotide solutions were calcu-
lated by using equation and coefficients described by Borer.37
The coefficients of the modified 12mers were calculated on the
assumption that modified dimers have the same hypochromicity
as the corresponding unmodified dimers. Molar coefficients
(ε260) of cU and cA were determined experimentally as 14500
and 14200 L molϪ1 cmϪ1. Each pair of 12mers was mixed
and dissolved in a buffer containing 1 M NaCl, 10 mM
sodium phosphate (pH 7.0) at duplex concentrations of 4 µM.
After annealing, the solution was transferred to a quartz cell
(1 cm path length), and melting curves were measured at least
twice at 260 nm on a JASCO Ubest-55 spectrophotometer. The
temperature was raised at a rate of 0.5 ЊC minϪ1, and the T m
values were obtained by the first-derivative plots of the melting
curves.
(6ЈaS,7ЈS,8ЈR,9ЈaR)-2-(4-tert-Butylphenoxy)-N-[7Ј-(4,4Ј-di-
methoxytrityloxymethyl)-7Ј,8Ј,9Ј,9Јa-tetrahydro-8Ј-hydroxy-
6ЈaH-cyclopenta[4,5]oxazolo[3,2-e]purin-4Ј-yl]acetamide (17)
Yield 98.3%; [α]26 Ϫ76.3 (c 0.51 in CHCl3); δH (CDCl3) 1.30
(9H, s, 3 × CH3), 2.00–2.11 (1H, m, 2Јa-H), 2.29–2.39 (1H, m,
4Ј-H), 2.67–2.74 (1H, m, 2Јb-H), 2.92 (1H, d, J 2.2, OH), 3.56
(1H, dd, J 8.0 and 9.6, 5Јa-H), 3.73 (1H, dd, J 6.0 and 9.6,
5Јb-H), 3.79 (6H, s, 2 × OCH3), 4.15–4.24 (1H, m, 3Ј-H), 4.74
(2H, s, ArOCH2), 5.13 (1H, dd, J 6.9 and 7.1, 1Ј-H), 5.86
(1H, dd, J 6.0 and 6.9, 6Ј-H), 6.06 (1H, br, NH), 6.81–
6.98 (6H, m, DMT-Ha and tBPA-Ha), 7.20–7.41 (11H, m,
DMT-Hb and tBPA-Hb), 8.60 (1H, s, 2-H); m/z (EI) 755.3324
(Mϩ. C44H45N5O7 requires 755.3317).
Measurements of CD spectra
The same samples (duplex concentration; 4 µM in 1 M NaCl,
10 mM sodium phosphate, pH 7.0) as used for UV melting
experiments were employed for CD experiments. Measure-
ments were carried out on a JASCO J-820 spectropolarimeter at
1 ЊC.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 8 3 – 1 8 9
188