Experimental
1. Materials
1.1 General Materials
All RNA oligos except for 7d8aI5 were purchased from GE Dharmacon Inc., and were purified by polyacrylamide gel electrophoresis
and desalted by SEP-PAK. RNA containing 7d8aI was synthesized by procedures described below. The concentration of all RNAs were
determined by UV absorbance at 260 nm using a NanoDrop™ instrument. HEK293-TLR8 cells were purchased from Novusbio Inc.,
together with Secreted Alkaline Phosphatase Reporter Assay Kit. DOTAP transfection reagent were purchased from Sigma. The
reagents used in synthesis were either purchased from ThermoFisher or Sigma.
1.2 Synthesis of 7-deaza-8-azainosine phosphoramidite
4-Amino-1-(β-D-ribofuranosyl)-1H-pyrazolo[3,4-d]-pyrimidine (2).
Previously prepared compound (1)25 was added to a flame dried flask (3.34 g, 5.76 mmol) and dried overnight, in vacuo. The next day,
a 33% methyl amine solution in ethanol (40 mL) was added to the flask and allowed to stir over 24 h. The reaction was then
concentrated to a slurry under reduced pressure, and 300 mL diethyl ether was added to the flask to suspend the product. The mixture
was then filtered, providing a pure white solid. (97% yield, 5.59 mmol) 1H NMR (CD3OD, 600 MHz): δ (ppm) 8.18 (s, 1H), 8.12 (s,
1H), 6.235 (d, 1H), 4.73 (t, 1H) 4.42 (t, 1H) 4.10 (m, 1H) 3.79(dd, 1H) 3.65(dd 1H). 13C NMR (CD3OD, 150 MHz): δ (ppm) 158.5,
155.47, 153.47,133.05, 100.92, 85.58, 73.94, 71.25, 62.61. ESIHRMS (m/z): calcd for C10H14N5O4 [M+H]+ 268.1046, obsd
268.1033.
1-(β-D-Ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (3).
To a 50 mL Falcon tube, (2) was added (0.5 g, 1.86 mmol) and suspended in 10 mL of NaHPO3 buffer (0.66 mM, pH 7.4). Adenosine
Deaminase (300µg, Sigma-Aldrich) was then added to the solution and the Falcon tube was allowed to rock on an orbital shaker for 6 h.
After 6 h, the solution turned clear and the reaction was analyzed using thin layer chromatography to reveal complete conversion to a
new product. The reaction was filtered through a pad of Celite with ethanol and concentrated via a rotary evaporator. Once
concentrated, the reaction product was recrystallized in ethanol to offer a pure white powder, which was filtered and dried overnight to
1
yield the target compound (99% yield, 0.5 g, 1.85 mmol. H NMR (CD3OD, 600 MHz): δ (ppm) 8.12(s, 1H), 8.06(s, 1H), 6.265 (d,
1H), 4.68 (t, 1H), 4.44(t, 2H), 4.10 (m, 1H), 4.10 (m, 1H), 3.79(dd, 1H), 3.65(dd, 1H). 13C NMR (CD3OD, 150 MHz): δ (ppm) 144.6,
136.7, 116.56, 56.15, 12.17, 127.2, 126.5, 49.4. ESIHRMS (m/z): calcd for C10H13N4O5 [M-H]- 269.0886, obsd 269.0871.
1-(β-D-Ribofuranosyl)-5’-O-(4,4’-dimethyloxytrityl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (4).
Compound (3) (100 mg, 0.373 mmol) was dried in vacuo overnight. Pyridine (2 mL) was then added to (3) and stirred in an ice bath.
Silver nitrate, crushed to a fine powder and dried in vacuo overnight, was then added to the flask (69.4 mg, 0.410 mmol). Finally,
dimethoxytrityl chloride was added to the flask (138 mg, 0.410 mmol). Stirring occurred on ice for 15 min, and then allowed to stir at
25 oC for 24 h. After 24 h, the solution was diluted in ethyl acetate and filtered through a pad of Celite. The filtrate was evaporated to
remove pyridine, resuspended in 10 mL ethyl acetate, and washed using 10% saturated sodium bicarbonate solution (2x) and brine. The
organic layer was then dried over sodium sulfate. Flash column chromatography (4% methanol/ dichloromethane) afforded the product
(4) (131 mg, 64% yield) as a white foam. 1H NMR (CD3OD, 600 MHz): δ (ppm) 8.06 (s, 1H), 8.05 (s, 1H), 7.385 (d, J = 7.0 Hz, 3H),
7.26 (m, 6H), 7.14 (d, 4H), 6.32 (d, J = 2.9 Hz, 2H), 4.79 (d, J = 5Hz, 1H), 4.6 (t, J = 5Hz, 1H), 4.18 (m, 2H), 3.72 (s, 6H), 3.27 (dd, J =
10.3, 3.2 Hz, 1H), 3.18 (dd, J = 10, 5.9 Hz, 1H) 13C NMR (CD3OD, 150 MHz): δ (ppm) 158.66, 158.54, 153.34, 147.55, 145.11,
135.96, 135.85, 135.16, 129.85, 127.95, 127.14, 126.17, 112.47, 106.38, 89.03, 85.86, 83.56, 73.83, 71.34, 64.07. ESIHRMS (m/z):
calcd for C31H29N4O7 [M-H]- 569.2036, obsd 569.2030.
1-(β-D-Ribofuranosyl)-5’-O-(4,4’-dimethyloxytrityl)-2’-O-(t-butyldimethylsilyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (5).
Compound (4) (949 mg, 1.66 mmol) was dried in vacuo overnight. Pyridine (1.2 mL 15.2 mmol) were added to the solution, followed
by anhydrous THF (12 mL). The reaction was the stirred, and finely powdered and dried silver nitrate was added to the reaction and
allowed to stir for an additional 15 min, at which time the solution became cloudy. Finally, tert-butyl dimethyl silyl chloride was added
to the flask (324 mg, 2.16 mmol) and the solution stirred for 24 h at room temperature. After 24 h, the reaction was filtered through a
pad of Celite using ethyl acetate (10 mL) and extracted with saturated sodium bicarbonate (2x) and brine. The organic layer was dried
over sodium sulfate. Flash column chromatography (50% ethyl acetate/ hexanes) afforded 364 mg (31 % yield) of the 2’ protected
compound (5). 1H NMR (CD2Cl2, 600 MHz): δ (ppm) 8.14 (s, 1H), 8.0 (s, 1H), 7.49 (d, J = 7.0 Hz, 2H), 7.36 (m, 5H), 7.29 (m, 4H),
6.79 (m, 2H) 6.28 (d, J = 5.3 Hz, 1H), 5.1 (t, J = 5Hz, 1H), 4.6 (m, 1H), 4.18 (m, 1H), 3.77 (s, 6H), 3.36 (dd, J = 10.6, 4.7 Hz, 1H), 3.15
(dd, J = 10.6, 4.7Hz, 1H) 13C NMR (CD2Cl2, 150 MHz): δ (ppm) 158.53, 158.54, 153.55, 146.68, 145.06, 136.07, 135.78, 130.10,
128.16, 127.70, 126.60, 112.98, 106.70, 88.46, 86.16, 84.20, 74.88, 71.95, 64.01, 25.33, 17.79, -5.31, -5.43. ESIHRMS (m/z): calcd for
C37H43N4O7Si [M-H]- 683.2901, obsd 683.2888.