Guanosine, 8-nitro-, cyclic 3',5'-(hydrogen phosphate) synthesis

Yield:913645-39-3 20.8%

Reaction Conditions:

Stage #1: 8-bromo-cyclic guanosine 3':5'-monophosphatewith hydrogenchloride;sodium nitrite in water;dimethyl sulfoxide at 70; for 120 h;
Stage #2: with sodium hydroxide in water;dimethyl sulfoxide; pH=8.5 - 9.0;

Steps:

The powdery compound (2) was dissolved in dimethylsulfoxide at a final concentration of 83 mM. 5 N hydrochloric acid was added to the solution at a final concentration of 34.5 mM. Immediately after the addition, 1 M sodium nitrite dissolved in dimethylsulfoxide was added at a final concentration of 333 mM. Reaction was performed for 5 days at 70°C, so that the nitration reaction was performed. After the completion of the reaction, pure water was added in a volume 2.3 times greater than that of the reaction solution. The solution was adjusted at pH 8.5-9.0 using IN NaOH. 1-Butanol was added to the solution to double the volume of the solution and then the solution was stirred. The thus obtained aqueous layer was condensed using a rotary evaporator. The condensed sample was filtered using a 0.45-μm filter. High-purity 8-nitroguanosine-3',5'-cyclic monophosphate was obtained through 3 instances of reverse-phase chromatography using TSK-gel ODS-80Ts (21.5 × 300 mm) performed under different mobile phase conditions. The 1^st instance of reverse-phase chromatography was performed using a mobile phase (10 mM sodium phosphate buffer (pH7.0) and 16% methanol) at a flow rate of 3.5 ml/min and then a peak fraction taken during a retention time of approximately 3 minutes (between 55 and 58 minutes) was recovered. The fraction was condensed using a rotary evaporator and then 100% ethanol cooled at -20°C was added, so that the precipitated salt was removed by centrifugation. Ethanol (100 %) was added again to the thus recovered ethanol supernatant and then the resultant was centrifuged, so that an ethanol layer was recovered. Ethanol was removed in a gas phase using a rotary evaporator via heating and condensation. An aqueous solution of 8-nitroguanosine-3',5'-cyclic monophosphate was recovered. The 2^nd instance of reverse-phase chromatography was performed using a mobile phase (10 mM sodium phosphate buffer (pH 7.0), 100 mM NaC1, and 16% methanol) at a flow rate of 3.5 ml/min and then a peak fraction taken during a retention time of approximately 8 minutes (between 59 and 67 minutes) was recovered. The fraction was condensed using a rotary evaporator and then the resultant was subjected to desalting using ethanol. The 3^rd instance of reverse phase chromatography was performed using a mobile phase (0.02% trifluoroacetic acid and 20% methanol) at a flow rate of 3.5 ml/min and then a peak fraction taken during a retention time of approximately 15 minutes (between 50 and 65 minutes) was recovered. The fraction was condensed using a rotary evaporator. The resultant was freeze-dried so that 11.1 mg (yield: 20.8%) of compound (3) in the form of powder was obtained. The thus obtained powdery compound (3) was dissolved in pure water added thereto. The solution was subjected to mass spectroscopy using LC-MS (LCMS-QP8000α) (SHIMADZU). As a result, an [M+H]⁺390 peak was detected in agreement with a peak fraction taken during the retention time between 15 and 18 minutes. Specifically, the results agreed with the theoretical values. ¹H NMR and spectrum data such as that regarding MS and UV spectra of the thus obtained compound (3), are as shown below. ¹H NMR (400 MHz, DMSO-d₆): δ: 4.06 (1H, ddd, J= 4.9, 10, 10 Hz), 4.28 (1H, ddd, J= 1.7, 10, 10 Hz), 4.43 (1H, ddd, J = 20, 10, 4.9 Hz), 4.83 (1H, d, J = 5.4 Hz), 5.02 (1H, ddd, J= 10, 5.4, 1.7 Hz), 6.00 (1H, br s) ,6.33 (1H, s), 7.05 (2H, br s), 11.3 (1H, s) MS (ESI, negative): Calculated for C₁₀H₁₁N₆O₉P ([M-H]^-): 389.02 Found: 389.10UV spectrum: λ_max=253, 275, 390 nm (solvent: CH₃OH)

References:

EP1860114,2007,A1 Location in patent:Page/Page column 5; 7