Solid-Phase Synthesis of m22,2G5′ppT
under reduced pressure. The residue was chromatographed
on a column of silica gel with hexanes-ethyl acetate (80:20,
v/v) containing 0.5% pyridine to give compound 10b (6.37 g,
81%): 1H NMR (270 MHz, CDCl3) δ 2.29 (6H, s), 3.51 (2H, s),
-4.7, -4.4, -4.0, 17.9, 18.1, 18.2, 18.5, 25.7, 25.8, 26.0, 37.7,
62.6, 71.8, 74.1, 77.1, 84.4, 87.9, 88.0, 115.9, 138.2, 154.3, 153.4,
159.0, 159.3; ESI-mass m/z calcd for C30H60N5O5Si3 654.3902,
obsd [M + H] 654.3887.
3.76 (6H, s), 4.13 (2H, s), 6.81-7.50 (15H, m) 7.55 (1H, s); 13
C
2′,3′-O-Bis(tert-butyldimethylsilyl)-2-N,2-N-dimethylgua-
nosine (16). Compound 15 (6.28 g, 9.6 mmol) was dissolved
in acetic acid-THF-water (96 mL, 3:1:1, v/v/v). After being
stirred at 80 °C for 28 h, the mixture was partitioned between
CHCl3 and water. The CHCl3 layer was collected, washed with
water (50 mL) and three times with 5% NaHCO3 (50 mL),
dried over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was chromatographed on a column of
silica gel with hexanes-ethyl acetate (25:75, v/v) to give
compound 16 (3.79 g, 73%): 1H NMR (270 MHz, CDCl3) δ
-0.34 (3H, s), -0.10 (3H, s), 0.06 (3H, s), 0.07 (3H, s), 0.75
(9H, s), 0.89 (9H, s), 3.21 (6H, s), 3.63-3.88 (2H, m, 5′-H), 4.06
(1H, m), 4.25-4.27 (1H, m), 4.94-4.98 (1H, m), 5.65 (1H, d,
J1′,2′ ) 7.2 Hz), 7.60 (1H, s), 11.51 (1H, bs); 13C NMR (67.8
MHz, CDCl3) δ -5.2, -4.6, -4.5, 17.8, 18.0, 25.7, 25.8, 38.9,
62.4, 73.2, 73.2, 87.2, 89.8, 118.0, 138.6, 150.8, 153.0, 158.7;
ESI-mass m/z calcd for C24H46N5O5Si2 540.3037, obsd [M + H]
540.3060.
Triethylammonium 2′,3′-O-Bis(tert-butyldimethylsi-
lyl)-2-N,2-N-dimethylguanosine 5′-Phosphonate (17). To
a solution of diphenyl phosphonate (9.38 mL, 49 mmol) in dry
pyridine (12 mL) was added dropwise and slowly a dry pyridine
solution (12 mL) of 16 (3.77 g, 7.0 mmol) which, in advance,
was dried by coevaporation three times with dry pyridine.
After being stirred under argon at room temperature for 4 h,
the mixture was treated with triethylamine-water (28 mL,
1:1, v/v). The resulting solution was stirred for 20 min and
diluted with CHCl3-pyridine (3:1, v/v). The solution was
washed three times with 0.5 M TEAB buffer, and the organic
layer was collected, dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was chromatographed
on a column of silica gel with CHCl3-MeOH (95:5-70:30, v/v)
to give compound 17 (4.29 g, 87%): 1H NMR (270 MHz, DMSO)
δ -0.33 (3H, s), -0.08 (3H, s), 0.08 (3H, s), 0.11 (3H, s), 0.72
(9H, s), 0.89 (9H, s), 1.16 (9H, m), 3.00 (6H, m), 3.05 (6H, s),
3.81-3.94 (2H, m), 3.98 (1H, m), 4.30 (1H, m), 4.83-4.87 (1H,
m), 5.76 (1H, d, J1′,2′ ) 6.6 Hz), 6.63 (1H, d, JPH ) 596.8 Hz),
8.00 (1H, s), 10.76 (1H, bs); 13C NMR (67.8 MHz, DMSO) δ
-5.5, -4.7, -4.7, -4.6, 8.4, 17.5, 17.8, 18.8, 25.5, 25.7, 37.8,
45.2, 62.5, 72.7, 73.8, 84.2, 86.4, 116.1, 136.8, 150.6, 152.7,
157.1; 31P NMR (109 MHz, DMSO) δ 2.47; ESI-mass m/z calcd
for C30H62N6O7PSi2 705.3956, obsd [M + H] 705.3964.
NMR (67.8 MHz, CDCl3) δ 45.3, 45.4, 45.5, 45.6, 55.1, 62.9,
64.6, 86.4, 113.0, 124.3, 125.6, 126.6, 127.7, 127.9, 128.0, 129.8,
130.5, 130.9, 135.8, 136.4, 139.7, 144.6, 158.2; ESI-mass m/z
calcd for C31H33BrNO3 546.1644, obsd [M + H] 546.1604.
2-[(N,N-Dimethylamino)methyl]-5-[[(4,4′-dimethoxytri-
tyl)oxy]methyl]phenylboronic Acid (11b). To a stirred
solution of 10b (2.73 g, 5 mmol) in dry THF (10.0 mL) was
added at -78 °C a solution of n-BuLi in hexane (2.80 mL, 25.0
mmol). After the mixture was stirred at -78 °C for 30 min, a
solution of trimethyl boronate (2.80 mL, 25.0 mmol) in dry
THF (2.0 mL) was added. The resulting mixture was stirred
at -78 °C for 1.5 h and then quenched by addition of 10%
NH4Cl. The resulting solution was partitioned between ethyl
acetate (80 mL) and 10% NH4Cl. The organic layer was
collected, washed three times with 10% NH4Cl, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The
residue was chromatographed on a column of NH silica gel
with CHCl3-hexane (95:5, v/v) to give compound 11b (1.91 g,
75%): 1H NMR (270 MHz, DMSO) δ 2.37 (6H, s), 3.71 (6H, s),
3.83 (2H, s), 4.08 (2H, s), 6.81-7.43 (15H, m) 7.58 (1H, s); 13
C
NMR (67.8 MHz, DMSO) δ 44.0, 44.1, 54.7, 62.8, 65.1, 85.4,
112.7, 124.1, 125.1, 126.0, 127.1, 127.2, 129.1, 135.5, 135.8,
139.8, 144.5, 157.6; ESI-mass m/z calcd for C31H35BNO5
512.2608, obsd [M + H] 512.2589.
2′,3′-O-[2-(N,N-Dimethylamino)methyl-5-[[(4,4′-di-
methoxytrityl)oxy]methyl]phenylboronylidene]uri-
dine (14b). Uridine (48.8 mg, 0.20 mmol) was rendered
anhydrous by coevaporation three times with dry pyridine and
with dry dioxane and finally dissolved in dry dioxane (4.0 mL).
To the solution was added compound 11b (122.7 mg, 0.24
mmol). After being stirred under argon at 100 °C for 40 min,
the mixture was evaporated under reduced pressure. The
residue was dissolved in CHCl3 (0.5 mL), and the solution was
poured with vigorous stirring into isopropyl ether-ethyl
acetate (200 mL, 9:1, v/v). The resulting precipitates were
collected to give compound 14b (143.9 mg, 98%): 1H NMR (270
MHz, DMSO) δ 2.45 (6H, s), 3.545-3.69 (2H, m), 3.73 (6H, s),
3.90 (2H, s), 3.94-3.96 (1H, m), 4.05 (2H, s), 4.61-4.68 (2H,
m), 5.00 (1H, bs), 5.65 (1H, d, 5-H, J5,6 ) 7.9 Hz), 5.88 (1H, d,
J1′,2′ ) 2.3 Hz), 6.90-7.45 (15H, m), 7.76 (1H, d), 11.35
(1H, s); 13C NMR (67.8 MHz, DMSO) δ 13.5, 22.8, 44.4, 55.0,
61.5, 63.6, 66.3, 67.3, 78.6, 82.8, 85.7, 87.8, 91.7, 101.8, 113.1,
122.6, 126.1, 126.5, 127.5, 127.7, 128.5, 129.5, 135.6, 135.6,
136.9, 139.5, 141.7, 144.9, 150.2, 157.9, 162.9. Anal. Calcd for
C40H42BN3O9: C, 66.76; H, 5.88; N, 5.84. Found: C, 65.29; H,
6.21; N, 5.05.
2′,3′,5′-O-Tris(tert-butyldimethylsilyl)-2-N,2-N-dimeth-
ylguanosine (15). 2′,3′,5′-O-Tris(tert-butyldimethylsilyl)gua-
nosine (12.52 g, 20 mmol) was dissolved in acetic acid (200
mL). To the solution were added paraformaldehyde (1.80 g,
60 mmol) and NaBH3CN (3.77 g, 60 mmol). The mixture
was vigorously stirred under argon at 45 °C. Six times at
8 h intervals, paraformaldehyde (1.80 g, 60 mmol) and
NaBH3CN (3.77 g, 60 mmol) were added to the suspension and
the mixture was vigorously stirred at the same temperature.
The reaction was quenched by addition of water (100 mL), and
CHCl3 (100 mL) was added. The CHCl3 layer was collected,
washed three times with water (50 mL) and five times with
5% NaHCO3 (50 mL), dried over Na2SO4, filtered, and evapo-
rated under reduced pressure. The residue was chromato-
graphed on a column of silica gel with hexanes-ethyl acetate
(70:30, v/v) to give compound 15 (6.54 g, 50%): 1H NMR (270
MHz, CDCl3) δ -0.18 (3H, s), -0.05 (3H, s), 0.08 (3H, s), 0.09
(6H, s), 0.10 (3H, s), 0.80 (9H, s), 0.91 (9H, s), 0.92 (9H, s),
3.21 (6H, s), 3.74-3.93 (2H, m), 4.02-4.06 (1H, m), 4.25-4.28
(1H, m), 4.45-4.49 (1H, m), 5.86 (1H, d, J1′,2′ ) 5.3 Hz), 7.80
(1H, s), 10.96 (1H, bs); 13C NMR (67.8 MHz, CDCl3) δ -5.4,
Triethylammonium 2-N,2-N-Dimethylguanosine 5′-
Phosphonate (18). Compound 17 (4.30 g, 6.1 mmol) was
dissolved in formic acid-water (4:1, v/v, 60 mL). After being
stirred at room temperature for 46 h, the mixture was diluted
with CHCl3. The CHCl3 solution was extracted 10 times with
0.1 M TEAB buffer. The aqueous extracts were collected and
evaporated under reduced pressure. The residue was chro-
-
matographed on a column of DEAE Sephadex A-25 (HCO3
form) with 0-1 M NH4HCO3 to give 18 (2.4 g, 84%): 31P NMR
(109 MHz, DMSO) δ 2.60, 2.74; ESI-mass m/z calcd for
C18H34N6O7P 477.2227, obsd [M + H] 477.2228.
Triethylammonium 2′,3′-O-[2-(N,N-Dimethylamino)-
methyl]-5-[[(4,4′-dimethoxytrityl)oxy]methyl]phen-
ylboronylidene]-2-N,2-N-guanosine 5′-Phosphonate (19b).
Compound 18 (187.6 mg, 0.5 mmol) was rendered by coevapo-
ration three times with dry pyridine and finally dissolved in
dry dioxane (10 mL). To the solution was added 2-[(N,N-
dimethylamino)methyl]-5-[(4,4′-dimethoxytrityl)oxy]methyl]-
phenylboronic acid (341.1 mg, 0.6 mmol). After being stirred
under argon at 100 °C for 1 h, the solution was evaporated
under reduced pressure. The residue was dissolved in CHCl3
(500 µL), and the solution was added to a solution of ether-
ethyl acetate (300 mL, 9:1, v/v) to give 19b as a white
precipitate (468.4 mg, 98%): 1H NMR (270 MHz, DMSO) δ
1.11 (9H, m), 2.46 (6H, s), 2.91 (6H, s), 2.95-3.01 (6H, m), 3.73
(6H, s), 3.84 (2H, m), 3.90 (2H, s), 4.04 (2H, s), 4.13-4.14 (1H,
J. Org. Chem, Vol. 70, No. 21, 2005 8407