* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
General procedure: 15mM solutions of selected activated nucleotides, ImpN (N = A, U, C, G, dA)2 were dissolved in 1M sodium fluoride (Sigma). The reaction mixtures were allowed to stand at 24 °C for 8-10 days with periodic monitoring by HPLC using a reverse phase column. After completion of the reaction, each product mixture comprised the corresponding phosphorofluoridate together with a small amount of the NMP hydrolysis product. The materials were separated using a semi-preparative reverse phase Alltima C-18, 5 (10 mm x 300 mm)column (Alltech, Grace Davison) under isocratic conditions (88percent of 0.2percent aqueous formic acid (Sigma) and 12percent of 30percent acetonitrile in water with 0.2percent formic acid) at flow rate of mL/minute. The individual samples collected were analyzed by mass spectrometry.
Reference:
[1] Journal of Biological Chemistry, 1934, vol. 106, p. 113,115, 118
[2] Zeitschrift fuer Naturforschung, 1962, vol. 17b, p. 291,293
[3] Journal of the Chemical Society, 1949, p. 2476,2484
[4] Journal of the American Chemical Society, 1954, vol. 76, p. 5056,5058
[5] Journal of the American Chemical Society, 1955, vol. 77, p. 1871,1872, 1873
[6] Journal of the Chemical Society, 1958, p. 1957,1961
[7] Organic Letters, 2005, vol. 7, # 10, p. 1999 - 2002
[8] Tetrahedron Letters, 1987, vol. 28, # 20, p. 2259 - 2262
[9] Patent: DE1119278, 1958, ,
3
[ 27908-36-7 ]
[ 58-97-9 ]
[ 26184-65-6 ]
Yield
Reaction Conditions
Operation in experiment
7.2 mg
at 20℃; for 48 h;
The mixture containing compound 7 (16.3 mg, 44.0 μmol) was co-evaporated with pyridine (3 × 1.0 mL) and dissolved in pyridine (1.0 mL). The solution was added to UMP-morpholidate (43.5 mg, 63.4 μmol), which evaporated with pyiridine (3 × 1.0 mL), in the reaction flask. Moreover, 1H-tetrazole (9.9 mg, 142 μmol) was co-evaporated with pyridine (3 × 1.0 mL) and dissolved in pyridine (300 μL). The solution was transferred to the reaction flask by a syringe and the mixture was then stirred at room temperature for 48 h. The mixture was evaporated and the residue was purified by silica gel chromatography with 7:1 (v/v/v) acetonitrile:water. The absorbance of each fraction was measured at 262 nm and the combined fractions were evaporated. The residue was purified by silica gel chromatography with 7:3:1 (v/v/v) ethyl acetate:methanol:water to give 9 (7.2 mg, 31percent) as a syrup: Rf 0.38 (acetonitrile:water = 4:1) ; 1H NMR δ (D2O, 400 MHz) 8.02 (d, 1H, J5'', 6'' = 2.8 Hz, H-6''), 5.99-5.96 (m, 2 H, H-1', H-5''), 5.76 (br-d, 1 H, J1, 2eq = 4.4 Hz, H-1), 5.31 (m, 1 H, J2eq, 3 = 5.6 Hz, J3, 4 = 9.6 Hz, H-3), 4.97 (t, 1 H, H-4), 4.45 (dd, 1 H, J5, 6a = 2.8 Hz, J6a, 6b = 12.4 Hz, H-6a), 4.36-4.18 (m, 6 H, H-2', 3', 4', 5, 5'), 4.11 (dd, 1 H, J5, 6b = 2.0 Hz, H-6b), 2.38 (br-dd, 1 H, J2ax, 2eq = 13.0 Hz, H-2eq), 2.10-1.99 (m, 9 H, Acetyl × 3), 1.99-1.90 (m, 1 H, H-2ax); 13C NMR δ (D2O, 100 MHz) 173.74 (4C, C=O), 173.32, 173.14, 166.15 (C-4''), 151.71 (C-2''), 141.72 (C-6''), 102.52 (C-5''), 94.18 (C-1), 88.62 (C-1'), 83.00 (C-4'), 73.96 (C-2'), 69.38-68.60 (C-3, 3', 4, 5), 65.93 (C-5'), 61.94 (C-6), 34.64 (C-2), 20.32 (3C, CH3), 20.14, 20.08; 31P NMR δ (D2O, 162 MHz) -10.36 (m, 1P, P-2), -12.70 (m, 1P, P-1); ESI-HRMS m/z calcd for C21H29N2O19P2- [M-H]-: 675.0845, found: 675.0858.
With methanol; tributyl-amine In 1,4-dioxane; N,N-dimethyl-formamide
Stage #1: 5'-Uridylic Acid With tributyl-amine In water at 20℃; for 0.333333h;
Stage #2: 1,1'-carbonyldiimidazole In N,N-dimethyl acetamide at 20℃; for 1h;
6
Tributylamine (0.25 ml, 1.1 mmol) was added to a 2.05 M aqueous UMP free solution (0.49 ml, 1.0 mmol), andthe mixed solution was stirred at room temperature for 20 minutes. Then, dimethylacetamide (1.5 ml) was further added,and the mixed solution was azeotropically dehydrated three times. The resulting UMP-tributylamine salt solution wasdissolved in dimethylacetamide (1.5 ml), and carbonyldiimidazole (486 mg, 3.0 mmol) was added thereto, then themixture was stirred at room temperature for 1 hour. Water (0.2 ml) was added to the mixture under ice cooling, andfurther, a 0.5 M aqueous hydrochloric acid solution (4.0 ml) was added dropwise (pH 7.2). The mixture was concentratedunder reduced pressure to prepare a UMP-imidazolide solution.
With tributyl-amine In water; sodium hydrogencarbonate; N,N-dimethyl-formamide
2 Method for the Production of Diuridine Tetraphosphate Tetraammonium Salt Using Uridine 5'-Monophosphate
EXAMPLE 2 Method for the Production of Diuridine Tetraphosphate Tetraammonium Salt Using Uridine 5'-Monophosphate Uridine 5'-monophosphate (Sigma, Milwaukee, 3.0 g, 9.26 mmol) was dissolved in dry DMF (10 mL) and tributylamine (Aldrich, 2 mL). The solution was evaporated in vacuo at 40° C. to an oil. The residue was dissolved in dry DMF (Aldrich, 8 mL) to form a solution. Carbonyldilmidazole (Aldrich, 1.65 g, 10.18 mmol) was added to this solution. The reaction was heated at 50° C. for one hour. Uridine 5'-triphosphate (Yamasa, 5.60 g, 10.18 mmol) prepared as the anhydrous tributylammonium salt in DMF (5 mL) and tributylamine (2 mL), as described in Example 3 below, was added to the reaction solution. The mixture was allowed to stir at 50° C. for three days when the solution was evaporated in vacuo to an oil, redissolved in water (5 mL) and purified by column (300*50 mm) chromatography (Sephadex DEAE-A25, 40-120μ, Aldrich, pre-swollen in 1.0 M NaHCO3 and washed with 2 column volumes of deionized H2O (H2O→0.03 M NH4HCO3 gradient). The pure fractions were concentrated in vacuo at 35° C., and H2O added and reevaporated 5 times to obtain diuridine tetraphosphate tetraammonium salt as a white solid (2.37 g, 30% yield): 92.11% pure by HPLC with the same retention time as the standard. In addition, the tetraammonium salt was analyzed by FABMS to give a mass of [C18H25N4O23P4 (M-H+)-: calculated 788.9860] 788.9857, confirming a parent formula of C18H26N4O23P4 for the free acid].
General procedure: 15mM solutions of selected activated nucleotides, ImpN (N = A, U, C, G, dA)2 were dissolved in 1M sodium fluoride (Sigma). The reaction mixtures were allowed to stand at 24 °C for 8-10 days with periodic monitoring by HPLC using a reverse phase column. After completion of the reaction, each product mixture comprised the corresponding phosphorofluoridate together with a small amount of the NMP hydrolysis product. The materials were separated using a semi-preparative reverse phase Alltima C-18, 5 (10 mm x 300 mm)column (Alltech, Grace Davison) under isocratic conditions (88% of 0.2% aqueous formic acid (Sigma) and 12% of 30% acetonitrile in water with 0.2% formic acid) at flow rate of mL/minute. The individual samples collected were analyzed by mass spectrometry.
double stranded RNA (polyadenylic acid-polyuridylic acid)[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
42%
With lithium chloride In water at 85℃; for 8h; Inert atmosphere;
7 EXAMPLE 7: Increasing polymer yield synthesis- based on manuscript
As previously, two monomers were chosen adenosine 5’-monophosphate (AMP) and uridine 5’-monophosphate (UMP) in their acid forms rather than as sodium salts (Sigma-Aldrich). When dissolved in water at 10 mM concentration the pH of the solution is 2.5. Commercial polyadenylic acid (polyA) and polyuridylic acid (polyU) were used as polynucleotide control standards (Sigma-Aldrich). These were mixed in 1:1 mole ratios with respect to the bases to produce double stranded RNA (polyA-polyU). The effects on oligomerization of a variety of monovalent salts, including LiC1, NaC1, KC1, and NH4C1 weretested. During evaporation, the salts formed crystalline films when their solubility was exceeded. The growing crystals excluded other solutes such as the mononucleotides, producing highly concentrated eutectic phases within the salt matrix. A laboratory simulation of HD cyclesSimulations were carried out using glass slides with two wells on each slide that hold0.1 mL of the reaction mixture. Four slides were arranged on a laboratory hot plate set at thedesired temperature range, and a plastic flow box with 8 small holes (1 mm diameter) was set on the slides. Each hole was placed directly over a well so that carbon dioxide gas flowed onto the mixture at approximately 1 cc/sec into each well. The gas was used to exclude oxygen, but also to carry away water vapor from condensation reaction as ester bondsformed, thereby preventing hydrolytic back reactions. Reaction mixturesMononucleotides, AMP (10 mM) and UMP (10 mM), were initially mixed in a 1:1 volume ratio. The mononucleotides solution and 0.1 M monovalent salts were mixed in a 2:1 volume ratio so that the initial concentrations were 3.3 mM AMP and UMP, together with0.033 M salt. Because water evaporated during dehydration, these dilute solutions become highly concentrated and finally dry, so it is the ratios that are significant rather than the initial concentrations. In a typical experiment, the reactants were exposed to 1-16 cycles of wetting and drying. The temperature (85° C) and flow of carbon dioxide caused drying within 1 - 2 minutes. After each dehydration phase of 30 minutes, the samples were dispersed in 0.1 mLof 1.0 mM HC1 to maintain acidity, followed by the next dehydration cycle. Variable experimental parameters included initial pH, temperature, the time given to each cycle and the numbers of cycles. At the end of the cycle series, the samples were dissolved in 0.1 mL of water. Isolation of productsThe polymer products were isolated by standard precipitation in ethanol (2.5 X volume ethanol 100%, 1/10 volume sodium acetate 3 M pH 5.2, 1.6 jiL linear acrylamide 5 mg/mL (Fischer scientific) for 700 jiL of reaction mixtures, followed by incubation at - 20°C overnight). The pellets were consistent with polymers that behaved like RNA. Quantitative analysis was performed by UV absorbance with a NanoVue instrumentcalibrated for RNA to estimate yields of products. Depending on the conditions, typical yields ranged from 1% to 40% expressed as the fraction of the total weight of mononucleotides present, and over 55% if additional monomers were added during cycling.Characterization of productsAs described above, double-stranded polynucleotide structure was shown by ethidiumbromide, alkaline hydrolysis, RNase hydrolysation, hypochromicity, nanopore analysis andmicroscopy. Effect of monovalent cations on polymerizationWhen the HD cycles were run with monovalent salts in the reaction mixture, yields ofpolymer were dramatically increased compared to absence of salts. Furthermore, the productswere stained by ethidium bromide, an intercalating dye, suggesting that base stacking was present. Sodium, potassium and ammonium chloride all promoted synthesis of polymers containing AMP and UMP as monomers. Products ranging from 10 to 300 nucleotides with a peak around 100 mers were detected. NH4C1 had the greatest effect, but products from LiC1produced only a weak band in the gel even though the yield measured by ethanol precipitation was in the same range as NH4C1 (Table 2). The A260/A280 ratio provides an estimate of how much of the absorbance is due to polymers and how much to monomers. A ratio of 2 corresponds to RNA while a ratio of 3.4 is observed for monomers. The high ratio with LiC1 indicates that the product has relatively short strands of oligomer lacking base stacking compared with the other salts.Mixtures of AMP 10 mM + UMP 10 mM + monovalent salt 0.1 M (LiC1, KC1, NaC1 and NH4C1) in 1:1:1 volume ratio were submitted to 16 HD cycles of 30 minutes. Table 2 below shows yields of polymers synthesized and ratio A260/A280 measured by UV absorbance with a NanoVue instrument. Yields are values from duplicate samples, and were calculated as the percent by weight of the original AMP and UMP present in the mixtureTable 2. Effect of monovalent salts on polymerization.SaltYield (%)A260/A280LiC138; 423.4NaC116; 182.1KC125; 292.0NH4C134; 372.0Yields were highest with LiC1, NH4C1, KC1 and NaC1, in that order, but the LiC1product was less stained by ethidium, probably because the oligomers were shorter withdecreased base stacking.Cycling increases yield of polymersThe optimum conditions for the polymerization process were determined by performing a set of experiments using a variety of controls and conditions including thenumber of cycles, duration of the cycles, pH and temperature. The synthesis of polymers is the most efficient at high temperature (around 85 °C), at acidic pH (3) and in the presence of CO2 stream. Without wishing to be bound by any theory, the above suggests that synthesis of the ester bond is an acid catalyzed mechanism and that CO2 plays an essential role in the polymerization process. Most of the product appeared to be polymers from 10 to 300nucleotides long. Finally, the dehydration phase appeared to be essential for the polymerization process since a minimum of 30 minutes of drying at each cycle is necessary to synthesize the 300 nt species (data not shown). Role of NH4 cations in promoting polymerizationBecause NH4C1 seemed to have the greatest effect on yields of polymers, a series of further experiments were conducted. Figure 3 shows results with different ammonium salts, including ammonium phosphate, ammonium molybdate, and ammonium formate. Only the ammonium formate yielded polymers ranging from 10 to 300 nucleotides in length but inlesser amounts compared to ammonium chloride. The importance of the chemical effect of the ammonium cation in this polymerization process was also tested by substituting tetramethylammonium chloride for ammonium chloride. Fig. 14 shows polymer synthesis after 8 hours of 30 minutes HD cycles. Yields are normalized for comparison, taking the products in the presence of NH4C1 as 1.0. Salts, lane A: NH4C1; lane B: C19H42BrN; lane C:HCO2NH4 lane D: (NH4)6Mo7O244H2O; lane E: NH4H2PO4. Fig.14 shows that tetramethylammonium chloride also produced polymers ranging from 10 to 300 nucleotides in length but with lower efficiency than ammonium chloride. This suggests that NH4 might have chemical effects induced by its protons coupled to the ordering effects on the mononucleotides.Kinetics of oligomerizationThe oligomerization process in the presence of ammonium chloride follows anexponential curve, and reaches a plateau after 30 hours of wet-dry cycles with a yield of 40%(Fig. 15). Fig. 15 shows results from Mixture of AMP 10 mM + UMP 10 mM + NH4C1 0.1M (1:1:1 volume ratio) showing the total amount and yield of products over multiple cycles.Each hour has two 30 minutes cycles, so 40 hours represents 80 cycles.Control of nucleotide concentration: nucleotide feedingTo determine whether the plateau was due to exhaustion of monomers, a feeding experiment was performed in which fresh monomers were added every 2 hours (4 cycles).An enhancement of oligomerization occurred when cycling is accompanied by regular additions of monomers. A yield of 58% is obtained after 5 feeding steps (final concentration of nucleotides equal to 60 mM) whereas for the same concentration (60 mM) present at the beginning of the experiment, the yield is 37%. This means that controlling nucleotides concentration by stepwise additions enhances the polymerization process in comparison to nucleotide pooi at an equivalent concentration.The plateau can be due to an equilibrium between synthesis and hydrolysis, although degradation of nucleotides over time may also contribute. Figure 6 shows that longerproducts accumulate in later cycles. Indeed, there is an enhancement of the production of short fragments (10 and 150 nts) after few cycles, and then their presence decreases as a function of time whereas the long polymers (700 and 1000 nts) accumulate in the later cycles. Lengthening may occur either by elongation and/or by ligation of short fragments.
Stage #1: diimidazolylpyrophosphate; 5'-Uridylic Acid With calcium chloride In N,N-dimethyl-formamide at 30℃; for 4h; Cooling with ice;
Stage #2: With hydrogenchloride; sodium chloride In water
1-3 Example 1
Prepare 2-1 imidazole pyrophosphate (, 0.2mol)DMF solution (380mL),Add a solution of uridine monophosphate (UMP, 0.4mol) in DMF (390mL) under ice bath,Calcium chloride (11.0 g, 0.1 mol) was stirred at 30 ° C for 4 h, and the reaction solution was taken for HPLC detection.Ethyl acetate (700 mL) and water (600 mL) were added to the reaction solution and stirred for 10 minutes. The aqueous phase was separated to retain the aqueous phase. The aqueous phase was added with a saturated sodium carbonate aqueous solution to adjust the pH to about 10, filtered, the filter cake was discarded, and ethanol was added to the filtrate. (1200 mL), stirred for 12 h, filtered and discarded the mother liquor.The filter cake was dissolved by adding water (400 mL), and the aqueous solution was passed through an anion exchange column (Amberlite IRA-67, chlorine type). The by-products were removed by elution with deionized water, 0.18 N hydrochloric acid, and then 0.5 N sodium chloride and 0.005 N hydrochloric acid. The target product was eluted from an aqueous solution, and the product was obtained by recrystallization from water / ethanol.Diquafosol pure product (140g, yield 80.0%), that is, purity 99%.
With 5wt.% Rh on activated alumina; hydrogen In water at 21℃; for 3h;
3.2 Hydrogenation conditions
General procedure: The hydrogenations were undertaken as described previously [10]. Briefly, the nucleoside starting material (30mg) in aqueous solution (3mL) were reacted for 3h at room temperature (21 C). Catalyst (5wt% Rh/Al2O3; 20mg) was introduced at the start of the reactions, and hydrogen gas was bubbled through at atmospheric pressure. The products were recovered by filtration to remove the catalyst, followed by lyophilization to remove the water. MALDI/MS spectra were recorded on the aqueous solutions immediately after removal of the catalyst. The lyophilized product residues were re-dissolved in deuterated water for analysis by NMR (1H NMR, 13C NMR, HSQC) without further purification. For the base-catalyzed ring opening reactions, the hydrogenated nucleosides were treated at room temperature for 18h using aqueous sodium hydroxide (0.25M). After neutralization with HCl (0.25M) the samples were analyzed by NMR and MALDI/MS.