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CAS No. : | 65-46-3 | MDL No. : | MFCD00006545 |
Formula : | C9H13N3O5 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | UHDGCWIWMRVCDJ-XVFCMESISA-N |
M.W : | 243.22 | Pubchem ID : | 6175 |
Synonyms : |
Cytosine β-D-riboside;Cytosine-1-β-D-ribofuranoside;NSC 20258;β-D-Cytidine
|
Signal Word: | Warning | Class: | N/A |
Precautionary Statements: | P261-P280-P301+P312-P302+P352-P305+P351+P338 | UN#: | N/A |
Hazard Statements: | H302-H315-H319-H335 | Packing Group: | N/A |
GHS Pictogram: |
* 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.
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
72% | With 1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione In N,N-dimethyl-formamide at 25℃; for 0.5 h; | Typical procedure for the bromination of unprotected nucleosides: DBH (323 mg, 1.13 mmol) was added to a stirred solution of 1d (500 mg, 2.05 mmol) in DMF (5 mL). The resulting pale-yellow solution was stirred at room temperature for 20 minutes or until TLC showed absence of starting material and formation of less polar product. Volatiles were evaporated and the residue was coevaporated with MeCN. The resulting pale solid was crystallized from hot acetone to give 2d (500 mg, 75percent) as colorless crystals with data as reported.14 |
69% | With sodium azide; bromoisocyanuric acid monosodium salt In water; acetonitrile at 20℃; for 3 h; | General procedure: 2'-O-Methyluridine (5, 0.103 g, 0.4 mmol) was dissolved in aqueous acetonitrile solution(H2O:CH3CN 1:9, 5 mL) under stirring. NaN3 (0.104 g, 1.6 mmol) was added, followed by addition of SMBI (0.101 g, 0.44 mmol) at r.t. and the mixture was stirred. Progress of the reaction was followedby TLC. On completion of the reaction after 1.5 h, the reaction mixture was filtered, evaporated todryness under reduced pressure and coevaporated with acetonitrile (2 × 2 mL). The crude reactionmixture was purified by column chromatography (4percent–6percent MeOH in DCM, v/v) to afford bromonucleoside 6 (0.117 g, 93percent) in pure form as a white solid |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
85.1% | With iodine; iodic acid; acetic acid In tetrachloromethane; water at 20 - 40℃; | Synthesis of 5-iodocytidine 191: A mixture of cytidine 190 (15.0 g, 61.7 mmol) in 225 mL of acetic acid and 225 mL of carbon tetrachioride was warmed to 40 CC, and iodine (9.6 g, 75.7 mmol) was added.To the stirred reaction mixture was added slowly a solution of iodic acid (9.6 g, 54.6 mmol) in 25 mL of water within 10 mm. The reaction mixture was stirred at 40 CC for 6 h and stirred at room temperature overnight. Upon completion of the reaction as monitored by TLC, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column using dichloromethane—methanol(15:1 to 10:1 to5:1) asgradienteluents resulting in 19.4g (85.1percent) desiredproduct 5-iodocytidine (191). |
85.1% | With iodine; iodic acid In tetrachloromethane; water; acetic acid at 40℃; for 6 h; | Synthesis of 5-iodocytidine 1 91 : A mixture of cytidine 190 (15.0 g, 61 .7 mmol) in 225 mL of acetic d and 225 mL of carbon tetrachloride was warmed to 40 °C, and iodine (9.6 g, 75.7 mmol) was added. the stirred reaction mixture was added slowly a solution of iodic acid (9.6 g, 54.6 mmol) in 25 mL of ter within 10 min. The reaction mixture was stirred at 40 °C for 6 h and stirred at room temperature ernight. Upon completion of the reaction as monitored by TLC, the reaction mixture was concentrated der reduced pressure. The residue was purified by flash chromatography on a silica gel column using hloromethane-methanol (15:1 to 1 0:1 to 5:1 ) as gradient eluents resulting in 1 9.4 g (85.1 percent) desired duct 5-iodocytidine (191 ). |
85.1% | With iodine; iodic acid; acetic acid In tetrachloromethane; water at 20 - 40℃; | Synthesis of 5-iodocytidine 191: A mixture of cytidine 190 (15.0 g, 61.7 mmol) in 225 mL of acetic acid and 225 mL of carbon tetrachloride was warmed to 40 °C, and iodine (9.6 g, 75.7 mmol) was added. To the stirred reaction mixture was added slowly a solution of iodic acid (9.6 g, 54.6 mmol) in 25 mL of water within 10 min. The reaction mixture was stirred at 40 °C for 6 h and stirred at room temperature overnight. Upon completion of the reaction as monitored by TLC, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column using dichloromethane-methanol (15:1 to 10:1 to 5:1) as gradient eluents resulting in 19.4 g (85.1 percent) desired product 5-iodocytidine (191). |
72.4% | With N-iodo-succinimide In methanol at 40 - 70℃; | The cytidine (0.66 g, 2.7 mol) was added to the three-necked flask,N-iodosuccinimide (0.60 g, 2.4 mol) and 50 ml of methanol, and the temperature was raised to 40-70 ° C. The reaction was stirred for 6-12 hours.Cooled to room temperature, filtered, added with silica gel and removed by rotary evaporation. The column chromatography gave 0.48 g of a pale yellow solid in 72.4percent yield. |
51% | With iodine; iodic acid; acetic acid In tetrachloromethane; water at 40℃; for 2 h; | General procedure: The suspension of nucleosides 2a,b (19 mmol) in water (5.7 mL) was treated with HIO3(9.7 mmol, 1.7 g), AcOH (15.2 mL) and a solution of iodine (11.22 mmol, 2.85 g) inCCl4 (3.8 mL). The resulting mixture was stirred at 40C for 2 h until the starting materialwas consumed or some by-product was formed (monitored by HPLC). After that,water (20 mL) was added. The reaction mixture was cooled to 4C and filtered. The precipitatewas washed with water (2 £ 10 mL). The combined solutions were diluted withwater (250 ml) and extracted with benzene (3 £ 150 mL). The aqueous layer was evaporatedunder reduced pressure. The product was purified by RPC in a linear gradient ofEtOH in water (0–30percent) to give the product 3a,b. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
96% | for 3 h; Reflux | In a 500 mL three-necked flask was added 50.0 g of Compound 3 powder and 250 mL of acetonitrile and 25 mL of acetic anhydride, stirred and refluxed for 3 h, cooled to room temperature and filtered with suction. The solid was washed with acetonitrile twice and the filter cake was dried to give 56.3 g of compound 4 with a yield of 96.0 percent. |
95% | for 0.0111111 h; Microwave radiation | To a solution of cytidine (1.22 g, 5.00 mmol) in DMF(40 mL) is added acetic anhydride (0.94 mL, 10.00 mmol). The solution is treated with microwave radiation (Panasonic, 1000 Watts) at full power for 40 seconds. The solution is concentrated in vacuo and the residue is co-evaporated with methanol (2.x.20 mL) to give 4-N-acetyl-cytidine (1.43 g, 95.0percent) as a white powder. MS (FAB):m/z 286 (MH+). |
87% | for 24 h; | Cytidine (100.Og, 0.41 mol) was dissolved in DMF (500 ml), acetic anhydride (42.5 ml, 45.9 g, 0.45 mol) was added and the whole was left for 24 h. Solvent was evaporated, the residue boiled with methanol (40 ml) and cooled. Crystals were filtered and dried to furnish Λ^-acetylcytidine (102 g, 87.0percent). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
95% | With toluene-4-sulfonic acid; at 20℃; for 6h;Cooling with ice; | The cytidine 250mg (1mmol) was dissolved in 10 ~ 30ml (preferably 20 ml) of acetone, p-toluenesulfonic acid 8 ~ 12mmol (preferably 10 mmol) was dissolved in 10 ~ 30ml (preferably 20 ml) of acetone. Under ice cooling, a solution of p-toluenesulfonic acid in acetone was slowly added dropwise to a solution of cytidine in acetone, stirred at room temperature 4 ~ 10 h (preferably 6H), centrifugation, the white precipitate was collected, the precipitate was washed with three to four times with acetone, dissolved in a small amount of DMF ethyl acetate was added to precipitate, membrane filtration, and dried in vacuo to give a white powdery product, yield 95%. |
25.8% | With sulfuric acid; at 50℃; for 16h;Molecular sieve; | -Step 1 : Protection of cytidine (compound 1 ) to furnish 2',3'-protected cytidine (compound 8): To a 100 mL round bottom flask fitted with a reflux condenser, cytidine (4.41 g, 18.1 mmol) suspended in dry acetone (44 mL) was added. To the stirring suspension, activated 4 A molecular sieves and H2SO4 98% (0.145 mL, 2.7 mmol, 0.15 equivalents) were added. The suspension was left stirring at 50C. After 16 hours, at r.t, NaHCOs (2 g) was added and the mixture was stirred for 0.5 hours. Then, the crude mixture was filtered in vacuo. The solid obtained was washed with MeOH/EtOH 1/1 (2 20 mL). The organic phases were combined and evaporated to furnish the 2',3'-protected cytidine (compound 8,1.33 g, 25.8 %), as a white solid. 1H-NMR (delta, ppm): 8.19 - 8.32 (m, 1 H), 6.21 (dd, J=8.07, 2.93 Hz, 1 H), 5.93 (s, 1 H), 4.95 - 5.14 (m, 1 H), 4.88 (dd, J=6.24, 2.57 Hz, 1 H), 4.38 (d, J=2.93 Hz, 1 H), 3.66 - 3.83 (m, 2 H), 1 .36 - 1 .75 (m, 6 H). |
With perchloric acid; | The synthesis of 127 can be seen in Scheme 3 (FIG. 3). First, cytidine was protected using a catalytic amount of perchloric acid in acetone to give 121. This was benzoylated at the 5? position, as well as on the exocyclic amine, using benzoyl chloride (BzCl) in pyridine to afford 122. These first two steps had high crude yields and likely are not part of the reason for the low combined yield. Fully protected 122 then underwent acidic hydrolysis using dilute hydrochloric acid (HCl) in dioxane to give 123. This gave a crude yield of approximately 50%. In the workup, the crude product was redissolved in EA and THF, though there were solubility issues. Even when DCM and MeOH are added to help this, it is possible that in the aqueous washes some product is lost. This acid hydrolysis has also been shown to be problematic with the guanosine and adenosine nucleosides, possibly causing some compound degradation. To further investigate this, 122 should be purified before the acid hydrolysis. Compound 123 was selectively silylated at the 2? position using tert-butyldimethylsilyl chloride and silver nitrate (AgNO3) in a mixture of pyridine and tetrahydrofuran. The selective protection gives preference to the formation of the desired 2? hydroxyl, however it also gives rise to the protected 3? hydroxyl and bis-protected compounds. Combined, these steps delivered 124 in a very low yield. In part, this was due to the lack of purification in between any of these reactions. Compound 122 should be purified before moving forward, and, most importantly, compound 123 should be very carefully purified so as to give a pure compound going into the selective protection. Another possibility is to deploy a scheme similar to what was adopted with guanosine where there is a transient protection of the three hydroxyl groups to start with a base protected nucleoside, removing the need to perform the isopropylidene removal. Compound 124 was then protected using dimethoxytrityl chloride in pyridine to give 125 in a crude yield of 84%, after which an attempt to selectively deprotect by using 2N sodium hydroxide (NaOH) in pyridine and methanol failed. Compound 125 should therefore be purified before moving forward to ensure there is one pure product to test the reaction with. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
> 99% | With cytidine deaminase enzyme; In aq. phosphate buffer; at 37℃; for 0.0833333h;pH 7.0;Enzymatic reaction; | Comparative Example 1 : Deamination of cytidine (compound 1 ) to uridine A 100 mM solution of the cytidine (495 muIota) in 100 mM phosphate buffer at pH 7 was mixed with 50 L of cytidine deaminase enzyme solution containing >300 AU in phosphate buffer. The reaction was performed at 37C during 5 minutes and stopped with HCI. Then, the crude reaction was filtered through a 10 KDa membrane, and a portion was diluted and analyzed by HPLC under UV-DAD (ultraviolet-diode array detection). Product identification was performed by comparison to a standard sample. Uridine was obtained in quantitative yield (>99%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With boron trifluoride diethyl etherate In acetonitrile Heating; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Streptomyces phospholipase D; Yield given; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With hydrogenchloride; Diaion WK-20 resin <Na(1+) form> 1.) H2O, CHCl3, 45 deg C, 6 h, pH 4.5, 2.) CHCl3, MeOH, H2O; Yield given. Multistep reaction; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
84% | With pyridine; at 20 - 32℃;Inert atmosphere; | Step 1: 682-1 ? 682-2'; Org. Process Dev., 4, 111 (2000); US Pat 6531584 Bl (2003); Org.Lett., 8, 55 (2006). <n="17"/>Cytidine (8.Og5 32.89mmol) was pre-dried by azeotroping with pyridine (2xl5ml), then suspended in pyridine (22ml) and the vessel purged with argon. 1,3-Dichloro-l, 1,4,4- tetraisopropyldisiloxane (12.0ml, 35.40mmol) was added dropwise at room temperature over a period of 20min. A mild exotherm to 320C was observed. A heavy white precipitate gradually settled at the bottom of the flask. This was broken up with vigorous stirring and the resulting heavy suspension stirred overnight. The mixture was poured into water (200ml) and extracted with EtOAc (3x200ml). The combined organics were washed (brine), dried (MgSO4), filtered and evaporated to a white solid. This was triturated with heptane, filtered and washed with heptane (100ml) followed by light pet ether (2x50ml). 13.46g (84%) obtained, hi the last stage of the work up, isopropyl acetate may be substituted for heptane. |
84% | With pyridine; at 20 - 32℃;Inert atmosphere; | Steo 1: 682-1 -» 682-2' ; Org. Process Dev., 4, 172 (2000); US Pat 6531584 Bl (2003); Org.Lett, 8, 55 (2006). Cytidine (8.Og, 32.89mmol) was pre-dried by azeotroping with pyridine (2x15ml), then suspended in pyridine (22ml) and the vessel purged with argon. 1,3-Dichloro- 1,1,4,4-tetraisopropyldisiloxane (12.0ml, 35.40mmol) was added dropwise at room temperature over a period of 20min. A mild exotherm to 320C was observed. A heavy white precipitate gradually settled at the bottom of the flask. This was broken up with vigorous stirring and the resulting heavy suspension stirred overnight. The mixture was poured into water (200ml) and extracted with EtOAc (3x200ml). The combined organics were washed (brine), dried (MgSO4), filtered and evaporated to a white solid. This was triturated with heptane, filtered and washed with heptane (100ml) followed by light pet ether (2x50ml). 13.46g (84%) obtained. In the last stage of the work up, isopropyl acetate may be substituted for heptane. |
With pyridine; sodium hydrogencarbonate; In dichloromethane; | Method A 3',5'-O-(1,1,3,3)-Tetraisopropyl-1,3-disiloxanediylcytidine With stirring, cytidine (40 g, 0.165 mol) and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPS-Cl, 50 g, 0.159 mol) were added to dry pyridine (250 mL). After stirring for 16 h at 25 C., the reaction was concentrated under reduced pressure to an oil. The oil was dissolved in methylene chloride (800 mL) and washed with sat'd sodium bicarbonate (2*300 mL). The organic layer was passed through a silica gel (200 g) scrub column. The product was recovered by elution with methylene chloride:methanol (97:3). The appropriate fractions were combined, evaporated under reduced pressure and dried at 25 C./0.2 mmHg for 1 h to give 59.3 g (77%) of oil (the product may be crystallized from ethyl acetate as white crystals, mp 242-244 C.); TLC purity 95% (Rf 0.59, ethyl acetate-methanol 9:1); PMR (DMSO) delta7.7 (H-6), 5.68 (H-5), 5.61 (HO-2'), 5.55 (H-1'). |
With pyridine; sodium hydrogencarbonate; In dichloromethane; | Method A 3',5'-O-(1,1,3,3)-Tetraisopropyl-1,3-disiloxanediylcytidine With stirring, cytidine (40 g, 0.165 mol) and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPS-Cl, 50 g, 0.159 mol) were added to dry pyridine (250 mL). After stirring for 16 h at 25 C., the reaction was concentrated under reduced pressure to an oil. The oil was dissolved in methylene chloride (800 mL) and washed with sat'd sodium bicarbonate (2*300 mL). The organic layer was passed through a silica gel (200 g) scrub column. The product was recovered by elution with methylene chloride:methanol (97:3). The appropriate fractions were combined, evaporated under reduced pressure and dried at 25 C./0.2 mmHg for 1 h to give 59.3 g (77%) of oil (the product may be crystallized from ethyl acetate as white crystals, mp 242-244 C.); TLC purity 95% (Rf 0.59, ethyl acetate-methanol 9:1); PMR (DMSO) delta 7.7 (H-6), 5.68 (H-5), 5.61 (HO-2'), 5.55 (H-1'). | |
With pyridine; sodium hydrogencarbonate; In dichloromethane; | Method A 3',5'-O-(1,1,3,3)-Tetraisopropyl-1,3-disiloxanediylcytidine With stirring, cytidine (40 g, 0.165 mol) and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPS-Cl, 50 g, 0.159 mol) were added to dry pyridine (250 mL). After stirring for 16 h at 25 C., the reaction was concentrated under reduced pressure to an oil. The oil was dissolved in methylene chloride (800 mL) and washed with sat'd sodium bicarbonate (2*300 mL). The organic layer was passed through a silica gel (200 g) scrub column. The product was recovered by elution with methylene chloride:methanol (97:3). The appropriate fractions were combined, evaporated under reduced pressure and dried at 25 C./0.2 mmHg for 1 h to give 59.3 g (77%) of oil (the product may be crystallized from ethyl acetate as white crystals, mp 242-244 C.); TLC purity 95% (Rf 0.59, ethyl acetate-methanol 9:1); PMR (DMSO) delta 7.7 (H-6), 5.68 (H-5), 5.61 (HO-2'), 5.55 (H-1'). | |
With pyridine; at 20℃; for 15.25h; | EXAMPLE 2Preparation of Intermediate Compound Int-2elnt-2b lnt-2cStep A - Synthesis of Compound Int-2bCytidine (Int-2a, 8.0 g, 32.89 mmol) was azeotroped with pyridine (2 xl5 mL) and then suspended in pyridine (25 mL). To the suspension was addedtetraisopropyldisiloxanedichloride (12.0 mL, 35.4 mmol) dropwise over fifteen minutes and the resulting reaction was allowed to stir for about 15 hours at room temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic extracte were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue obtained was triturated with heptane to provide 13.5 g of compound Int-2b as a white solid[M+H] = 486.5. | |
13.5 g | With pyridine; at 20℃; for 15h; | Cytidine (Int-2a, 8.0 g, 32.89 mmol) was azeotroped with pyridine (2 x15 mL) and then suspended in pyridine (25 mL). To the suspension was added tetraisopropyldisiloxanedichloride (12.0 mL, 35.4 mmol) dropwise over fifteen minutes and the resulting reaction was allowed to stir for about 15 hours at room temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic extracte were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue obtained was triturated with heptane to provide 13.5 g of compound Int-2b as a white solid [M+H] = 486.5. |
13.5g | With pyridine; at 20℃; for 15.25h; | Step A-Synthesis of Compound Int-2b [0223] Cytidine (Int-2a, 8.0 g, 32.89 mmol) was azeotroped with pyridine (2×15 mL) and then suspended in pyridine (25 mL). To the suspension was added tetraisopropyldisiloxanedichloride (12.0 mL, 35.4 mmol) dropwise over fifteen minutes and the resulting reaction was allowed to stir for about 15 hours at room temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic extracted were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue obtained was triturated with heptane to provide 13.5 g of compound Int-2b as a white solid [M+H]=486.5. |
13.5 g | With pyridine; at 20℃; for 15h; | Cytidine (Int-3a, 8.0 g, 32.89 mmol) was azeotroped with pyridine (2×15 mL) and then suspended in pyridine (25 mL). Tetraisopropyldisiloxanedichloride (12.0 mL, 35.4 mmol) was added dropwise over fifteen minutes and the reaction was allowed to stir for about 15 hours at room temperature. The reaction was diluted with water and extracted with ethyl acetate. The organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was triturated with heptane to provide 13.5 g of compound Int-3b as a white solid [M+H]=486.5. |
59.3 g (77%) | With pyridine; sodium hydrogencarbonate; In dichloromethane; | Step 1. 3',5'-O-[(1,1,3,3-Tetraisopropyl)-1,3-disiloxanediyl]cytidine With stirring, cytidine (40 g, 0.165 mol) and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPS-Cl, 50 g, 0.159 mol) were added to dry pyridine (250 mL). After stirring for 16 h at 25 C., the reaction was concentrated under reduced pressure to an oil. The oil was dissolved in methylene chloride (800 mL) and washed with sat'd sodium bicarbonate (2*300 mL). The organic layer was passed through a silica gel (200 g) scrub column. The product was recovered by elution with methylene chloride-methanol (97:3). The appropriate fractions were combined, evaporated under reduced pressure and dried at 25 C./0.2 mmHg for 1 h to give 59.3 g (77%) of oil. TLC purity 95% (Rf 0.59, ethyl acetate-methanol 9:1). |
With pyridine; at 20℃; for 16h; | Cytidine (1 0.0 g, 41 .1 mmol) was azeotroped with pyridine (2x 20 ml_) and suspended in 40.0 ml_ of pyridine. To the suspension was added tetraisopropyldisiloxanedichloride (14.3 g, 45.2 mmol) dropwise over 1 5 minutes. The resulting suspension was stirred for about 16 hours at room temperature. The reaction mixture was carefully diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2S04, filtered, and concentrated in vacuo. The residue was triturated with hexane to provide compound 20 as a white solid. ESI MS for C2i H39N306Si2 calculated 485.7, observed 486.2 [M+H]+ |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
96% | In acetonitrile; for 3.0h;Reflux; | In a 500 mL three-necked flask was added 50.0 g of Compound 3 powder and 250 mL of acetonitrile and 25 mL of acetic anhydride, stirred and refluxed for 3 h, cooled to room temperature and filtered with suction. The solid was washed with acetonitrile twice and the filter cake was dried to give 56.3 g of compound 4 with a yield of 96.0 percent. |
95% | In N,N-dimethyl-formamide; for 0.0111111h;Microwave radiation; | To a solution of cytidine (1.22 g, 5.00 mmol) in DMF(40 mL) is added acetic anhydride (0.94 mL, 10.00 mmol). The solution is treated with microwave radiation (Panasonic, 1000 Watts) at full power for 40 seconds. The solution is concentrated in vacuo and the residue is co-evaporated with methanol (2.x.20 mL) to give 4-N-acetyl-cytidine (1.43 g, 95.0percent) as a white powder. MS (FAB):m/z 286 (MH+). |
87% | In N,N-dimethyl-formamide; for 24.0h; | Cytidine (100.Og, 0.41 mol) was dissolved in DMF (500 ml), acetic anhydride (42.5 ml, 45.9 g, 0.45 mol) was added and the whole was left for 24 h. Solvent was evaporated, the residue boiled with methanol (40 ml) and cooled. Crystals were filtered and dried to furnish Lambda^-acetylcytidine (102 g, 87.0percent). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
96% | With ammonium hydroxide; chloro-trimethyl-silane; In pyridine; water; acetone; | Reference Production Example 2 Production of 5'-amino-5'-deoxycytidine (5'-NH2-Cyd) A suspension of cytidine (9.72 g, 40.0 mmol) in pyridine (200 ml) was added to trimethylchlorosilane (25.6 ml, 200 mmol). After 15 minutes of stirring, benzoyl chloride (23.2 ml, 200 mmol) was added, and the reaction was allowed to proceed at room temperature for 2 hours. The reaction mixture was then cooled on an ice bath, and water (40 ml) was added. Five minutes later, 28% aqueous ammonia (40 ml) was added, and the mixture was stirred at room temperature for 15 minutes. Then, the solvent was distilled off under reduced pressure, and acetone was added to precipitate a white solid. The precipitate was filtered off and recrystallized from water to give N4-benzoylcytidine (13.3 g, 96%). |
96% | With ammonium hydroxide; chloro-trimethyl-silane; In pyridine; water; acetone; | Reference Example 2 Production of 5'-amino-5'-deoxycytidine (5'-NH2-Cyd) A suspension of cytidine (9.72 g, 40.0 mmol) in pyridine (200 ml) was added to trimethylchlorosilane (25.6 ml, 200 mmol). After 15 minutes of stirring, benzoyl chloride (23.2 ml, 200 mmol) was added, and the reaction was allowed to proceed at room temperature for 2 hours. The reaction mixture was then cooled on an ice bath, and water (40 ml) was added. Five minutes later, 28% aqueous ammonia (40 ml) was added, and the mixture was stirred at room temperature for 15 minutes. Then, the solvent was distilled off under reduced pressure, and acetone was added to precipitate a white solid. The precipitate was filtered off and recrystallized from water to give N4-benzoylcytidine (13.3 g, 96%). |
95% | Cytidine 2b (20 mmol, 4.86 g) was co-evaporated with pyridine (3 £ 20 mL) to drynessunder reduced pressure. After that, 2b was suspended in dry pyridine (200 mL). Aftercooling of the suspension in an ice bath trimethylsilylchloride (100 mmol, 13 mL) wasadded dropwise with vigorous stirring. The ice bath was removed and the suspension wasstirred well for 1.5 h at r.t. After cooling of the suspension in an ice bath benzoyl chloride(40 mmol, 4.7 mL) was added dropwise with stirring. The ice bath was removed and thesuspension was well stirred overnight at r.t. Water (20 mL) was added to the reactionmixture cooled in the ice bath. After stirring for 30 min, the mixture was treated with28% aq. NH3 (25 mL). The ice bath was removed and the stirring was continued for 1 h.The reaction mixture was concentrated under reduced pressure. The residual solvent wasremoved by co-evaporation with toluene (3 £ 50 mL). The crude material was suspendedin acetone (300 mL). After filtration, the residual solid was treated with hot water(300 ml). After cooling, the precipitate was filtered, washed with cold water (2 £ 20 mL)and dried. The yield of 9 from 2b was 95% (19 mmol, 6.59 g). NMR data were in agreementwith those reported previously.26 1H NMR (400 MHz, DMSO-d6) d 11.24 (s, 1H,NH), 8.51 (d, J D 7.2 Hz, 1H, H6), 8.01 (app d, J D 7.5 Hz, 2H, Bz), 7.61 (app t, J D 7.3Hz, 1H, Bz), 7.50 (app t, J D 7.6, 2H, Bz), 7.38-7.29 (m, 1H, H5), 5.83 (d, J D 2.8 Hz,1H, H10), 5.54 (d, J D 4.8 Hz, 1H, 20-OH), 5.20 (t, J D 5.0 Hz, 1H, 50-OH), 5.07 (d, J D5.7 Hz, 1H, 50-OH), 4.07-3.98 (m, 2H, H30, H2 0 ), 3.96-3.91 (m, 1H, H40), 3.81-3.58 (m,2H, H50). 13C NMR (125 MHz, DMSO-d6) d 167.47, 163.09, 154.75, 145.42, 133.24,132.80, 128.52, 96.01, 90.32, 84.31, 74.65, 68.72, 59.98. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
97% | With pyridine; 1H-imidazole; at 0 - 20℃;Inert atmosphere; | To a solution of cytidine (3.60 g, 14.3 mmol) in 30 mL of anhydrous pyridine was added imidazole (1.25 g, 18.40 mmol) and tert-butyldimethylsilyl chloride (TBDMSCl, 2.40 g, 15.7 mmol) at 0 C under a N2 atmosphere. The reaction mixture was stirred at rt for 12 h and then treated with methanol (8.0 mL). After stirring for 60 min, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (hexane/EtOAc=10:1 to 1:4 v/v) to give (5.12 g, 12.5 mmol) in 97% yield. Subsequently, to a solution of protected cytidine (2.68 g, 7.50 mmol) and DMAP (5.50 g, 45.0 mmol) in 50 mL of anhydrous CH2Cl2 was added benzyl chloroformate (CbzCl, 4.76 mL, 33.74 mmol) at 0 C under N2 atmosphere. After stirring for 72 h at rt, the reaction mixture was diluted with CH2Cl2 (200 mL) and then washed with cold 1.0 M HCl aqueous solution (50 mL) then water (100 mL). The solution was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified on silica gel column chromatography (hexane/EtOAc=10:1 to 1:1 v/v) to give 3a (5.47 g, 7.20 mmol) in 93% yield. 1H NMR (400 MHz, CDCl3) delta 8.32 (d, J=8.0 Hz, 1H), 7.58 (br, 1H), 7.42-7.30 (m, 15H), 7.21 (d, J=8.0 Hz, 1H), 6.27 (d, J=3.2 Hz, 1H), 5.35 (t, J=4.0 Hz, 1H), 5.28 (t, J=5.6 Hz, 1H), 5.23 (s, 2H), 5.13 (d, J=2.4 Hz, 2H), 5.10 (d, J=7.6 Hz, 2H), 4.33 (d, J=5.6 Hz, 1H), 4.06 (d, J=10.8 Hz, 1H), 3.79 (d, J=10.8 Hz, 1H), 0.93 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H); 13C NMR (100 MHz, CDCl3) delta 162.8, 154.7, 153.9, 153.8, 152.5, 144.0, 137.2, 135.1, 134.7, 134.7, 130.7, 129.2, 128.9, 128.8, 128.7, 128.7, 128.6, 128.5, 128.4, 128.3, 117.2, 95.2, 87.8, 81.9, 77.5, 72.7, 70.4, 70.4, 67.9, 61.5, 25.9, 18.4, -5.5, -5.6; MS-ESI+ m/z 760 (M+H+). |
88% | With pyridine; at 0 - 20℃; for 4h; | To an anhydrous pyridine solution (400 mL) of cytidine (100 mmol), an anhydrous pyridine solution (100 mL) of t-butyldimethylsilyl chloride (14.3 g, 95 mmol) was slowly added at 0C using a cannula. The temperature of the obtained reaction mixture was raised to room temperature, and then the mixture was stirred for 4 hours. The reaction mixture was concentrated using an evaporator, and the obtained concentrate was dissolved or suspended in methylene chloride (200 mL). The obtained solution or suspension was added dropwise to stirred distilled water (500 mL). After the organic layer was separated, a crude product material was obtained as crystal by adding ethyl acetate thereto. Further, the crystal was subjected to recrystallization using ethyl acetate - methylene chloride to obtain the 5'-O-TBDMS derivative as the target compound (yield: 88%). |
88% | With pyridine; at 0 - 20℃; for 4h; | To an anhydrous pyridine solution (400 mL) of cytidine (100 mmol), an anhydrous pyridine solution (100 mL) of t-butyldimethylsilyl chloride (14.3 g, 95 mmol) was slowly added at 0 C. using a cannula. The temperature of the obtained reaction mixture was raised to room temperature, and then the mixture was stirred for 4 hours. The reaction mixture was concentrated using an evaporator, and the obtained concentrate was dissolved or suspended in methylene chloride (200 mL). The obtained solution or suspension was added dropwise to stirred distilled water (500 mL). After the organic layer was separated, a crude product material was obtained as crystal by adding ethyl acetate thereto. Further, the crystal was subjected to recrystallization using ethyl acetate-methylene chloride to obtain the 5'-O-TBMS derivative as the target compound (yield: 88%). |
With 1H-imidazole; In N,N-dimethyl-formamide; at 20℃; for 16h;Inert atmosphere; | Cytidine (1.00g, 4.12mmol) and imidazole (700mg, 10.3mmol) were dissolved in a minimum of DMF. Once dissolved tert-butyldimethylsilyl (TBDMS) chloride (682mg, 4.52mmol) was slowly added. The resulting mixture was stirred at rt under an atmosphere of N2 and judged to be complete by TLC after 16h. Upon completion the reaction solution was poured onto crushed ice with NaCl added, with the subsequent precipitate collected via vacuum filtration. The crude 5?-O-TBDMS-protected cytidine and DMAP (1.38g, 12.3mmol) were dissolved in CH2Cl2 and the resulting suspension was cooled to 0C. Benzyl chloroformate (1.8mL, 12.3mmol) was added dropwise and the suspension stirred at rt. After 16h the solution was diluted with CH2Cl2 and washed with H2O, followed by NaHCO3 and brine. The organic phase was collected, dried with MgSO4 and concentrated under reduced pressure. The resulting crude product was purified by column chromatography (CHCl3:EtOAc, 4:1, Rf=0.58) to produce the completely protected cytidine derivative (2.18g, 70% over two steps) as a clear solid. The clear solid (1.96g, 2.58mmol) was dissolved in THF (15mL) and glacial acetic acid (0.2mL, 3.10mmol) was added. The reaction mixture was cooled to 0C and 1.0M TBAF in THF (2.8mL, 2.84mmol) was added in a dropwise manner. The resulting reaction mixture was allowed to gradually warm to rt over 2h and left to stir at rt for an additional 16h. After such time, the volatile reactants were removed under reduced pressure with the resulting crude product was purified by column chromatography (CHCl3 : EtOAc, 4:1, Rf=0.26) to produce 23 (1.60g, 96%) as a white solid. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
> 98.3% | In DMF (N,N-dimethyl-formamide); at 20℃; for 20h; | Step 1: To a suspension of cytidine (100 g, 0.411 mol) in DMF (2.06 L) is added benzoic anhydride (102.4 g, 0.452 mol). The mixture was stirred at room temperature for 20 h. The DMF was removed in vacuo and the residue was triturated with diethyl ether. The resulting solid was collected by suction filtration and washed with diethyl ether (2 x 200 mL). Further drying in vacuo at room temperature gave the N4 benzamide (140.6 g, 98. 3%). |
98.3% | In N,N-dimethyl-formamide; at 20℃; for 20h; | To a suspension of cytidine (100 g, 0.411 mol) in DMF (2.06 L) is added benzoic anhydride (102.4 g, 0.452 mol). The mixture was stirred at room temperature for 20 hours. The DMF was removed in vacuo and the residue was triturated with diethyl ether. The resulting solid was collected by suction filtration and washed with diethyl ether (2 x 200 mL). Further drying in vacuo at room temperature gave the N4 benzamide (140.6 g, 98.3%). A portion of this material (139.3 g, 0.401 mol) was dissolved in anhydrous pyridine (1.2 L) and was treated with 1 ,3-dichloro-1 ,1 ,3,3-tetraisopropyl-disiloxane (141.4 mL, 0.441 mol) at room temperature. The solution was stirred at room temperature overnight. The mixture was concentrated to near dryness in vacuo and coevaporated with toluene (3 x 200 mL). The residue was treated with EtOAc (1.8 L) and washed with HCI (2 x 200 mL, 0.05 N), NaHC03 (5 %, 2 x 400 mL). The organic layer was washed dried (Na2S04), filtered, and evaporated to dryness. Compound 701 (256.5 g, >100%) was isolated as a white foam and used without further purification. |
98.3% | In N,N-dimethyl-formamide; at 20℃; for 20h; | a. Preparation of Compound 701 To a suspension of cytidine (100 g, 0.411 mol) in DMF (2.06 L) is added benzoic anhydride (102.4 g, 0.452 mol). The mixture was stirred at room temperature for 20 hours. The DMF was removed in vacuo and the residue was triturated with diethyl ether. The resulting solid was collected by suction filtration and washed with diethyl ether (2*200 mL). Further drying in vacuo at room temperature gave the N4 benzamide (140.6 g, 98.3%). A portion of this material (139.3 g, 0.401 mol) was dissolved in anhydrous pyridine (1.2 L) and was treated with 1,3-dichloro-1,1,3,3-tetraisopropyl-disiloxane (141.4 mL, 0.441 mol) at room temperature. The solution was stirred at room temperature overnight. The mixture was concentrated to near dryness in vacuo and coevaporated with toluene (3×200 mL). The residue was treated with EtOAc (1.8 L) and washed with HCl (2×200 mL, 0.05 N), NaHCO3 (5%, 2×400 mL). The organic layer was washed dried (Na2SO4), filtered, and evaporated to dryness. Compound 701 (256.5 g, >100%) was isolated as a white foam and used without further purification. |
98% | In methanol; for 8h;Reflux; | To the flask was added 1.3 kg of methanol, 100 g of D-cytidine and 300 g of benzoic anhydride were added under stirring, The reaction system was heated to reflux, The reaction was stopped after 8 hours of refluxing, The reaction was then cooled to 25-30 C. Filtered to give the crude product of cytidine, adding the crude product of cytidine into 1000 g of water, The mixture was stirred at room temperature for 2 hours, filtered and washed with 100 g of water. The product was dried in vacuo at 65-70C for 4 hours to give a white solid powder, 140 g of N4-benzoyl-D-cytidine product, 98% yield and 99.14% purity. |
In pyridine; methanol; water; N,N-dimethyl-formamide; | Example 62. Synthesis of N4-Benzoyl cytidine (03600014013) Synthesis of N4-Benzoylcytidine (203): To a stirred solution of cytidine (202) (2.43 g, 10 mmol) in anhydrous pyridine (80 mL) and DMF (30 mL) was added benzoic anhydride (3.39 g, 15 mmol). The reaction mixture was stirred at room temperature for 24 h, and treated with water. The volatiles were evaporated under reduced pressure, and the residue was purified by chromatographic column. The desired fractions were collected and concentrated. The product was treated with methanol giving 1.5 g final product 203 with 97% HPLC purity. 1HNMR (400 MHz, DMSO-d6) delta 11.26 (s, 1H), 8.50-8.53 (m, 1H), 7.99-8.02 (m, 1H), 7.32-7.68 (m, 4H), 5.82 (d, 1H, J = 2.8 Hz), 5.54 (d, 1 H, J = 4.8 Hz), 5.20 (t, 1 H, J = 5.2 Hz), 5.08 (d, 1 H, J = 5.6 Hz), 3.80-4.02 (m, 3H), 3.50-3.75 (m, 2H); MS (ESI) m/z 348 (M + H)+, 370 (M + Na)+, 717 (2M + Na)+. UV, lambdamax at 259.5 and 302.5 nm. | |
1.5 g | With pyridine; In N,N-dimethyl-formamide; at 20℃; for 24h; | Synthesis of N4-Benzoylcytidine (203): To a stirred solution of cytidine (202) (2.43 g, 10 mmol) in anhydrous pyridine (80 mL) and DMF (30 mL) was added benzoic anhydride (3.39 g, 15mmol). The reaction mixture was stirred at room temperature for 24 h, and treated with water. The volatiles were evaporated under reduced pressure, and the residue was purified by chromatographic column. The desired fractions were collected and concentrated. The product was treated with methanol giving 1.5g final product 203 with 97% HPLC purity. 1HNMR (400 MHz, DMSO-d5)o 11.26 (s, 1 H), 8.50-8.53 (m, 1 H), 7.99-8.02 (m, 1 H), 7.32-7.68 (m, 4H), 5.82 (d, 1 H, J= 2.8 Hz), 5.54 (d,1 H, J= 4.8 Hz), 5.20 (t, 1 H, J= 5.2 Hz), 5.08 (d, 1 H, J= 5.6 Hz), 3.80-4.02 (m, 3H), 3.50-3.75 (m,2H); MS (ESI) m/z348 (M + H), 370 (M + Na), 717 (2M + Na). UV, Amax at 259.5 and 302.5 nm. |
1.5 g | With pyridine; In N,N-dimethyl-formamide; at 20℃; for 24h; | ample 62. Synthesis of N4-Benzoyl cytidine (03600014013) o Synthesis of N4-Benzoylcytidine (203): To a stirred solution of cytidine (202) (2.43 g, 10 mol) in anhydrous pyridine (80 ml_) and DMF (30 ml_) was added benzoic anhydride (3.39 g, 15 mol). The reaction mixture was stirred at room temperature for 24 h, and treated with water. The atiles were evaporated under reduced pressure, and the residue was purified by chromatographic umn. The desired fractions were collected and concentrated. The product was treated with thanol giving 1.5 g final product 203 with 97% HPLC purity. 1HNMR (400 MHz, DMSO-d6) delta 11.26 1 H), 8.50-8.53 (m, 1 H), 7.99-8.02 (m, 1 H), 7.32-7.68 (m, 4H), 5.82 (d, 1 H, J= 2.8 Hz), 5.54 (d, J=4.8 Hz), 5.20 (t, 1 H, J = 5.2 Hz), 5.08 (d, 1H, J= 5.6 Hz), 3.80-4.02 (m, 3H), 3.50-3.75 (m, ); MS (ESI) m/z 348 (M + H)+, 370 (M + Na)+, 717 (2M + Na)+. UV, Amax at 259.5 and 302.5 nm. |
1.5 g | With pyridine; In N,N-dimethyl-formamide; at 20℃; for 24h; | Synthesis of N4-Benzoylcytidine (203): To a stirred solution of cytidine (202)(2.43 g, 10 mmol) in anhydrouspyridine (80 mL) and DMF (30 mL) was added benzoic anhydride (3.39 g, 15 mmol). The reaction mixture was stirredat room temperature for 24 h, and treated with water. The volatiles were evaporated under reduced pressure, and theresidue was purified by chromatographic column. The desired fractions were collected and concentrated. The productwas treated with methanol giving 1.5 g final product 203with 97% HPLC purity. 1HNMR (400 MHz, DMSO-d6) delta11.26(s, 1H), 8.50-8.53 (m, 1H), 7.99-8.02 (m, 1H), 7.32-7.68 (m, 4H), 5.82 (d, 1H, J =2.8 Hz), 5.54 (d, 1 H, J =4.8 Hz), 5.20(t, 1 H, J= 5.2 Hz), 5.08 (d, 1 H, J =5.6 Hz), 3.80-4.02 (m, 3H), 3.50-3.75 (m, 2H); MS (ESI) m/z348 (M + H)+, 370 (M+ Na)+, 717 (2M + Na)+. UV, lambdamax at 259.5 and 302.5 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
In water at 20℃; pH 8.5-8.8; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
100% | With pyridine; dmap; at 20℃; for 18h;Inert atmosphere; | General procedure: The nucleoside (1.0 equiv) and DMAP (0.1 equiv) were stirred with distilled pyridine (5 mL:1 g starting material), to this solution was added tert-butyldiphenylsilyl chloride (1.1 equiv) and the reaction mixture stirred under an Ar atmosphere overnight. The reaction mixture was diluted with CH2Cl2 (40 mL:1 g starting material) and extracted with a saturated NaHCO3 solution(2 40 mL:1 g starting material) and brine (40 mL:1 g starting material). The combined organics were then dried over anhydrous Na2SO4, filtered and the solvent removed in vacuo. The residue was purified as described below. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
86% | With N-chloro-succinimide; 1-ethylene glycol monomethyl ether-3-methylimidazolium methanesulfonate at 50℃; for 1h; | |
56% | With hydrogenchloride; 3-chloro-benzenecarboperoxoic acid In N,N-dimethyl acetamide for 1.5h; Ambient temperature; | |
Multi-step reaction with 2 steps 1: pyridine / anschliessendes Behandeln mit Chlor in CCl4 unter Bestrahlung mit UV-Licht 2: methanol.NH3 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
72% | With 1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione; In N,N-dimethyl-formamide; at 25.0℃; for 0.5h; | Typical procedure for the bromination of unprotected nucleosides: DBH (323 mg, 1.13 mmol) was added to a stirred solution of 1d (500 mg, 2.05 mmol) in DMF (5 mL). The resulting pale-yellow solution was stirred at room temperature for 20 minutes or until TLC showed absence of starting material and formation of less polar product. Volatiles were evaporated and the residue was coevaporated with MeCN. The resulting pale solid was crystallized from hot acetone to give 2d (500 mg, 75%) as colorless crystals with data as reported.14 |
69% | With sodium azide; bromoisocyanuric acid monosodium salt; In water; acetonitrile; at 20.0℃; for 3.0h; | General procedure: 2'-O-Methyluridine (5, 0.103 g, 0.4 mmol) was dissolved in aqueous acetonitrile solution(H2O:CH3CN 1:9, 5 mL) under stirring. NaN3 (0.104 g, 1.6 mmol) was added, followed by addition of SMBI (0.101 g, 0.44 mmol) at r.t. and the mixture was stirred. Progress of the reaction was followedby TLC. On completion of the reaction after 1.5 h, the reaction mixture was filtered, evaporated todryness under reduced pressure and coevaporated with acetonitrile (2 × 2 mL). The crude reactionmixture was purified by column chromatography (4%-6% MeOH in DCM, v/v) to afford bromonucleoside 6 (0.117 g, 93%) in pure form as a white solid |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
85.1% | With iodine; iodic acid; acetic acid; In tetrachloromethane; water; at 20 - 40℃; | Synthesis of 5-iodocytidine 191: A mixture of cytidine 190 (15.0 g, 61.7 mmol) in 225 mL of acetic acid and 225 mL of carbon tetrachioride was warmed to 40 CC, and iodine (9.6 g, 75.7 mmol) was added.To the stirred reaction mixture was added slowly a solution of iodic acid (9.6 g, 54.6 mmol) in 25 mL of water within 10 mm. The reaction mixture was stirred at 40 CC for 6 h and stirred at room temperature overnight. Upon completion of the reaction as monitored by TLC, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column using dichloromethane-methanol(15:1 to 10:1 to5:1) asgradienteluents resulting in 19.4g (85.1%) desiredproduct 5-iodocytidine (191). |
85.1% | With iodine; iodic acid; In tetrachloromethane; water; acetic acid; at 40℃; for 6h; | Synthesis of 5-iodocytidine 1 91 : A mixture of cytidine 190 (15.0 g, 61 .7 mmol) in 225 mL of acetic d and 225 mL of carbon tetrachloride was warmed to 40 C, and iodine (9.6 g, 75.7 mmol) was added. the stirred reaction mixture was added slowly a solution of iodic acid (9.6 g, 54.6 mmol) in 25 mL of ter within 10 min. The reaction mixture was stirred at 40 C for 6 h and stirred at room temperature ernight. Upon completion of the reaction as monitored by TLC, the reaction mixture was concentrated der reduced pressure. The residue was purified by flash chromatography on a silica gel column using hloromethane-methanol (15:1 to 1 0:1 to 5:1 ) as gradient eluents resulting in 1 9.4 g (85.1 %) desired duct 5-iodocytidine (191 ). |
85.1% | With iodine; iodic acid; acetic acid; In tetrachloromethane; water; at 20 - 40℃; | Synthesis of 5-iodocytidine 191: A mixture of cytidine 190 (15.0 g, 61.7 mmol) in 225 mL of acetic acid and 225 mL of carbon tetrachloride was warmed to 40 C, and iodine (9.6 g, 75.7 mmol) was added. To the stirred reaction mixture was added slowly a solution of iodic acid (9.6 g, 54.6 mmol) in 25 mL of water within 10 min. The reaction mixture was stirred at 40 C for 6 h and stirred at room temperature overnight. Upon completion of the reaction as monitored by TLC, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column using dichloromethane-methanol (15:1 to 10:1 to 5:1) as gradient eluents resulting in 19.4 g (85.1 %) desired product 5-iodocytidine (191). |
72.4% | With N-iodo-succinimide; In methanol; at 40 - 70℃; | The cytidine (0.66 g, 2.7 mol) was added to the three-necked flask,N-iodosuccinimide (0.60 g, 2.4 mol) and 50 ml of methanol, and the temperature was raised to 40-70 C. The reaction was stirred for 6-12 hours.Cooled to room temperature, filtered, added with silica gel and removed by rotary evaporation. The column chromatography gave 0.48 g of a pale yellow solid in 72.4% yield. |
51% | With iodine; iodic acid; acetic acid; In tetrachloromethane; water; at 40℃; for 2h; | General procedure: The suspension of nucleosides 2a,b (19 mmol) in water (5.7 mL) was treated with HIO3(9.7 mmol, 1.7 g), AcOH (15.2 mL) and a solution of iodine (11.22 mmol, 2.85 g) inCCl4 (3.8 mL). The resulting mixture was stirred at 40C for 2 h until the starting materialwas consumed or some by-product was formed (monitored by HPLC). After that,water (20 mL) was added. The reaction mixture was cooled to 4C and filtered. The precipitatewas washed with water (2 £ 10 mL). The combined solutions were diluted withwater (250 ml) and extracted with benzene (3 £ 150 mL). The aqueous layer was evaporatedunder reduced pressure. The product was purified by RPC in a linear gradient ofEtOH in water (0-30%) to give the product 3a,b. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With acetic anhydride; In N,N-dimethyl-formamide; | EXAMPLE 1 N4 -Acetylcytidine (2) Cytidine (1) (7.3 g, 30 mmoles) was suspended in anhydrous N,N-dimethylformamide (120 ml) and to it was added acetic anhydride (3.12 ml, 33 mmoles). The mixture was stirred at room temperature overnight. After removal of the DMF under reduced pressure, the resulting residue was triturated with excess of diethylether (~30 ml) and the crystalline product obtained collected by filtration, washed thoroughly with diethylether and air dried to get a quantitative yield of 2. A small portion was crystallized from ethanol/water mixture (1:1) to obtain crystals for analytical purposes. m.p. 210°-213° C. (dec.). UV (H2 O): lambda max 296 nm and 246 nm. IR (KBr): nu1637 (vs, CO of ring amide), 1721 (s, CO of acetamide), and 2900-3600 (NH, OH) cm-1. 1 H-NMR (DMSO-d6): delta2.10 (s, 3H, COCH3), 3.65 (m, 2H, C5' CH2), 3.91-3.97 (m, 3H,C2',3',4' H), 5.06-5.51 (m, 3H, C2',3',5' --OH), 5.78 (s, 1H, C1' H), 7.18 (d, 1H, C5 H), 8.42 (d, 1H, C6 H), and 10.90 (s, 1H, CONH). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
53% | In 1,4-dioxane; water at 80℃; for 5h; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
81% | With chloro-trimethyl-silane In toluene at 70℃; for 12.5h; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
99% | With pyridine; at 20℃;Cooling with ice; | General procedure: Compound 2a, 2b or 9 (10 mmol) was co-evaporated with pyridine (3 £ 10 mL) to drynessunder reduced pressure. The dry residue was suspended in pyridine (50 mL). Aftercooling of the suspension in an ice bath, acetic anhydride (50 mmol, 4.6 mL for 2a and9; 60 mmol, 5.7 mL for 2b) was added dropwise with vigorous stirring. The ice bath wasremoved and the reaction mixture was well stirred overnight at r.t. After completion ofthe reaction, water (20 mL) was added to quench the acetic anhydride |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
at 37℃; Enzymatic reaction; Aqueous citrate buffer; | 2.4; 2.5 The cells are then centrifuged for 15' at 4000 rpm at 4° C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at -20° C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90' at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30' at 4° C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4° C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.The measurement of the enzymatic activities is carried out as described in paragraph 4.2.5) ResultsThe transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CThe results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) “Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus” J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.The selected enzyme is therefore used in the enzymatic synthesis of 2',3'-dideoxynucleosides and 2',3'-dideoxy, 2',3'-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1β-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. | |
at 37℃; Enzymatic reaction; Aqueous citrate buffer; | 2.4; 2.5 The cells are then centrifuged for 15' at 4000 rpm at 4° C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at -20° C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90' at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30' at 4° C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4° C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.The measurement of the enzymatic activities is carried out as described in paragraph 4.2.5) ResultsThe transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CThe results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) “Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus” J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.The selected enzyme is therefore used in the enzymatic synthesis of 2',3'-dideoxynucleosides and 2',3'-dideoxy, 2',3'-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1β-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. | |
at 37℃; Enzymatic reaction; Aqueous citrate buffer; | 2.4; 2.5 The cells are then centrifuged for 15' at 4000 rpm at 4° C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at -20° C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90' at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30' at 4° C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4° C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.The measurement of the enzymatic activities is carried out as described in paragraph 4.2.5) ResultsThe transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CThe results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) “Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus” J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.The selected enzyme is therefore used in the enzymatic synthesis of 2',3'-dideoxynucleosides and 2',3'-dideoxy, 2',3'-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1β-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. |
at 37℃; Enzymatic reaction; Aqueous citrate buffer; | 2.4; 2.5 The cells are then centrifuged for 15' at 4000 rpm at 4° C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at -20° C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90' at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30' at 4° C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4° C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.The measurement of the enzymatic activities is carried out as described in paragraph 4.2.5) ResultsThe transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CThe results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) “Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus” J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.The selected enzyme is therefore used in the enzymatic synthesis of 2',3'-dideoxynucleosides and 2',3'-dideoxy, 2',3'-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1β-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. |
Yield | Reaction Conditions | Operation in experiment |
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native N-deoxyribosyl transferase of Lactobacillus leichmannii LL; at 37℃;pH 6.44;Enzymatic reaction; Aqueous citrate buffer;Conversion of starting material; | The cells are then centrifuged for 15' at 4000 rpm at 4 C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at -20 C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90' at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30' at 4 C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4 C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.The measurement of the enzymatic activities is carried out as described in paragraph 4.2.5) ResultsThe transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CThe results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) ?Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus? J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.The selected enzyme is therefore used in the enzymatic synthesis of 2',3'-dideoxynucleosides and 2',3'-dideoxy, 2',3'-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1beta-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. | |
mutant N-deoxyribosyl transferase of Lactobacillus fermentum LFA15T; at 37℃;pH 6.44;Enzymatic reaction; Aqueous citrate buffer;Conversion of starting material; | The cells are then centrifuged for 15' at 4000 rpm at 4 C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at -20 C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90' at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30' at 4 C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4 C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.The measurement of the enzymatic activities is carried out as described in paragraph 4.2.5) ResultsThe transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CThe results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) ?Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus? J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.The selected enzyme is therefore used in the enzymatic synthesis of 2',3'-dideoxynucleosides and 2',3'-dideoxy, 2',3'-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1beta-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. | |
mutant N-deoxyribosyl transferase of Lactobacillus leichmannii LL G9S; at 37℃;pH 6.44;Enzymatic reaction; Aqueous citrate buffer;Conversion of starting material; | The cells are then centrifuged for 15' at 4000 rpm at 4 C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at -20 C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90' at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30' at 4 C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4 C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.The measurement of the enzymatic activities is carried out as described in paragraph 4.2.5) ResultsThe transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CThe results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) ?Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus? J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.The selected enzyme is therefore used in the enzymatic synthesis of 2',3'-dideoxynucleosides and 2',3'-dideoxy, 2',3'-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1beta-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. |
native N-deoxyribosyl transferase of Lactobacillus fermentum LF; at 37℃;pH 6.44;Enzymatic reaction; Aqueous citrate buffer;Conversion of starting material; | The cells are then centrifuged for 15' at 4000 rpm at 4 C. , washed in 50 ml of phosphate buffer then the pellet obtained after centrifugation is preserved overnight at -20 C. The bacterial pellet resuspended in 20 ml of phosphate buffer is then lysed by passage through a French press at 14000 psi. The lysate is centrifuged for 90' at 50,000 rpm. The supernatant containing the soluble proteins is then precipitated with ammonium sulphate (40% saturation). The precipitate obtained after centrifugation at 13900 rpm (20,000 g) for 30' at 4 C. is resuspended in 1 ml of 100 mM phosphate buffer, pH 7.5, 1.5 M NaCl, then deposited on a Sephacryl S200 gel filtration column (Amersham-Pharmacia). The fractions are then analyzed by SDS-PAGE gel and the enzymatic activity determined. The most active and purest fractions are dialysed overnight at 4 C. against the same buffer at pH=6.0. The protein concentration is determined by measuring the OD at 280 nm.The measurement of the enzymatic activities is carried out as described in paragraph 4.2.5) ResultsThe transforming clones of the E. coli strain PAK9, expressing the mutated ntd gene of L. fermentans were selected in glucose mineral medium with dideoxyuracil (ddR-U) and cytosine (C) added.Several transformants were obtained and are capable of carrying out the exchange: ddR-Pyr+Pur The nucleotide sequences of the different variants of ntd are identical and only differ from the wild-type gene by one mutation (indicated in bold type in Table 2 below). In both cases (L. leichmannii and L. fermentum) a neutral amino acid (glycine and alanine) is replaced by a nucleophilic amino acid (serine and threonine respectively). The conversion of N-deoxyribosyl transferase to N-dideoxyribosyl transferase or N-didehydroribosyl transferase therefore seems to require the substitution of a neutral amino acid by a nucleophilic amino acid which must contribute to the positioning of the sugar promoting its catalysis. It is interesting to note in Table 2 that all the N-deoxyribosyl transferases as well as a certain number of homologous proteins (of unknown function) possess a glycine or an alanine in this position.The enzymatic activities of the native and mutant N-deoxyribosyl transferases of L. leichmannii (LL and LL G9S) and of L. fermentum (LF and LFA15T) in the exchange reactions dT+CThe results reported in Table 3 below show that the specific activity of the mutant LFA15T is less than that of the native enzyme (LF) for the transfer of deoxyribose but that the latter is greater for the transfer of dideoxyribose or didehydroribose. For the transfer of deoxyribose, the activity is reduced by a factor of 7, whereas the latter is increased by 3 in the case of the transfer of dideoxyribose and by 35 in the case of didehydroribose.Table 4 below shows in detail the results of enzymatic activity tests for the native enzyme and the mutated enzyme of B. fermentum for each of the dT+C, ddT+C and d4T+C reactions. The first column of the table shows the affinity constant values (Km), the second the maximum reaction speed (Vmax), the third, the catalysis constant (Kcat), and the last the ratio of the affinity and catalysis constants (Km/Kcat) taking account of the effectiveness of the enzymes tested. These different values were measured according to the protocol described in the literature [P A Kaminski (2002) ?Functional cloning, heterologous expression and purification of two different N-deoxyribosyl transferases from Lactobacillus helveticus? J. Biol. Chem; vol. 277; 14400-14407]. The enzyme mutated according to the method of the invention shows a better catalytic activity on d4T and on ddT than the native enzyme. The activities are increased respectively by 60% and 54%. Moreover, the mutated enzyme LFA15T is 60 times more effective than the native enzyme LF in the ddT+X exchange and 7.5 times more effective in the d4T+X exchange.The selected enzyme is therefore used in the enzymatic synthesis of 2',3'-dideoxynucleosides and 2',3'-dideoxy, 2',3'-didehydronucleosides from natural bases ddC, ddA, ddl, d4T, d4C, d4G (Ray et al. 2002; Stuyver et al. 2002) or modified bases (Pokrovsky et al. 2001 Chong et al., 2002) such as (1beta-3'-fluoro) 2',3'-dideoxy, 2',3'-didehydro-4'-thio-Nucleosides comprising or not comprising radioelements. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
44% | With sodium hydride In N,N-dimethyl-formamide at 20℃; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1: methanol / 5 h / Heating 2: 61 percent / pyridine / 25 °C 3: AgNO3, pyridine / tetrahydrofuran / 25 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1: 73 percent / dimethyldioxirane, NH3 / CH2Cl2; acetone / 25 °C 2: 93 percent / 2N methanol. NH3 / CH2Cl2 / 25 °C | ||
Multi-step reaction with 2 steps 1: ammonia, dimethyldioxirane / CH2Cl2; acetone / 25 °C 2: ammonia / methanol / 24 h / 25 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1: methanol / Heating 2: 92 percent / pyridine / Ambient temperature | ||
Multi-step reaction with 2 steps 1.1: pyridine / 15.25 h / 20 °C 2.1: ethanol / 3 h / 65 °C 2.2: 0 °C | ||
Multi-step reaction with 2 steps 1: pyridine / 15 h / 20 °C 2: ethanol / 3 h / 65 °C |
Multi-step reaction with 2 steps 1: pyridine / 15.25 h / 20 °C 2: ethanol / 3 h / 65 °C | ||
Multi-step reaction with 2 steps 1: pyridine / 15 h / 20 °C 2: ethanol / 3 h / 65 °C | ||
Multi-step reaction with 2 steps 1: pyridine / 16 h / 20 °C 2: ethanol / 3 h / Reflux |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Stage #1: (S-trityl-6-mercaptohexyl)-(2-cyanoethyl)-(N,N-diisopropyl)phosphoramidite; G; CYTIDINE; thymidine; adenosine With 1H-tetrazole; 5-amino-3H-1,2,4-dithiazole-3-thione In pyridine; triethylammonium acetate; acetonitrile Stage #2: With ammonia In water at 58℃; for 16h; Stage #3: With acetic acid In water | 18 Synthesis of 5'-C6-disulfide-TCGTCGA (4): Synthesis of 5'-C6-disulfide-TCGTCGA (4): The 5'-C6-disulfide-TCGTCGA is synthesised using a Perseptive Biosystems Expedite 8909 automated DNA synthesizer using the manufacturer's protocol for 1 umol phosphorothioate DNA. The nucleoside monomers and the thiol-modifier C6 S-S (Glen Research, Sterling, Va.) are dissolved in anhydrous acetonitrile to a final concentration of 0.1 M. The thio-modifier is placed in an auxiliary monomer site on the instrument.The instrument is programmed to add the nucleotide monomers and the thiol modifier in the desired order, with synthesis of the nucleic acid moieties occurring in the 3' to 5' direction. 1.Use a 3'-support bound "A" solid support 2.Synthesis of 5'-TCGTCG-3' moiety 3.Addition of the thiol modifier precursor (S-trityl-6-mercaptohexyl)-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) The synthesis cycle consists of a detritylation step, a coupling step (phosphoramidite monomer plus 1H-tetrazole), a capping step, a sulfurization step using 0.02 M 3-amino-1,2,4-dithiazole-5-thione (ADTT) in 9:1 acetonitrile:pyridine, and a final capping step.At the completion of assembly, the 'trityl-on' compound is cleaved from the controlled-pore glass and the bases are deprotected with concentrated aqueous ammonia at 58° C. for 16 hours.The compound is purified by HPLC on a Hamilton PRP-1 column using an increasing gradient of acetonitrile in 0.1 M triethylammonium acetate.The purified compound is concentrated to dryness, the 4,4'-dimethoxytrityl group is removed with 80% aqueous acetic acid, and then the compound is precipitated two times from 1 M aqueous sodium chloride with 2.5 volumes of 95% ethanol.The compound is dissolved in Milli Q water and the yield is determined from the absorbance at 260 nm.Finally, the compound is lyophilized to a powder. The compound is characterized by capillary gel electrophoresis, electrospray mass spectrometry, and RP-HPLC to confirm composition and purity. An endotoxin content assay (LAL assay, Bio Whittaker) is also conducted, showing endotoxin levels were <5 EU/mg compound. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Stage #1: 4,4'-O-dimethoxytritylpropyl-O-(N,N-diisopropyl)-2-cyanoethylphosphoramidite; G; CYTIDINE; thymidine With 1H-tetrazole; 5-amino-3H-1,2,4-dithiazole-3-thione In acetone; acetonitrile Stage #2: With ammonia In water at 58℃; for 16h; Stage #3: With acetic acid In water | 12 C-13: 5'-TCGTCG-3'-(C3)15-5'-T-3' The C-13 molecule was synthesized on a Perseptive Biosystems Expedite 8909 automated DNA synthesizer using the manufacturers protocol for 1 umol phosphorothioate DNA. The nucleoside monomers and the spacer moiety precursor, 4,4'-O-dimethoxytrityl-propyl-O-(N,N-diisopropyl) 2-cyanoethylphosphoramidite (obtained from Glen Research, Sterling, Va.) were dissolved in anhydrous acetonitrile to a final concentration of 0.1 M. The C3 spacer was placed in an auxiliary monomer site on the instrument. The instrument was programmed to add the nucleotide monomers and C3 spacers in the desired order, with synthesis of the nucleic acid moieties occurring in the 3' to 5' direction. [0563] 1. Use a 3'-support bound “T” solid support [0564] 2. Addition of 15 C3 spacers [0565] 3. Synthesis of 5'-TCGTCG-3' moiety [0566] The synthesis cycle consisted of a detritylation step, a coupling step (phosphoramidite monomer plus 1H-tetrazole), a capping step, a sulfurization step using 0.02 M 3-amino-1,2,4-dithiazole-5-thione (ADTT) in 9:1 acetonitrile:pyridine, and a final capping step. At the completion of assembly, the ‘trityl-on’ compound was cleaved from the controlled-pore glass and the bases were deprotected with concentrated aqueous ammonia at 58° C. for 16 hours. The compound was purified by HPLC on a Hamilton PRP-1 column using an increasing gradient of acetonitrile in 0.1 M triethylammonium acetate. The purified compound was concentrated to dryness, the 4,4'-dimethoxytrityl group was removed with 80% aqueous acetic acid, and then the compound was precipitated two times from 1 M aqueous sodium chloride with 2.5 volumes of 95% ethanol. The compound was dissolved in Milli Q water and the yield was determined from the absorbance at 260 nm. Finally, the compound was lyophilized to a powder. [0567] The compound was characterized by capillary gel electrophoresis, electrospray mass spectrometry, and RP-HPLC to confirm composition and purity. An endotoxin content assay (LAL assay, Bio Whittaker) was also conducted, showing endotoxin levels were <5 EU/mg compound. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 3 steps 1.1: ethanol / 60 °C 2.1: pyridine / 1 h / 0 °C / Inert atmosphere 3.1: trifluoromethylsulfonic anhydride / tetrahydrofuran / 2 h / 20 °C / Inert atmosphere 3.2: 10 h / -20 °C / Inert atmosphere |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
In N-methyl-acetamide; methanol; ethanol; dichloromethane; | Step 1. 2'-O-methylcytidine Cytidine (100 g, 0.41 mol) was dissolved in warm dimethylformamide (65 C., 1125 mL). The solution was cooled with stirring to 0 C. A slow, steady stream of nitrogen gas was delivered throughout the reaction. Sodium hydride (60% in oil, washed thrice with hexanes, 18 g, 0.45 mol) was added and the mixture was stirred at 0 C. for 45 min. A solution of methyl iodide (92.25 g, 40.5 mL, 0.65 mol) in dimethylformamide (400 mL) was added in portions over 4 h at 0 C. The mixture was stirred for 7 h at 25 C. and then filtered. The filtrate was concentrated to dryness under reduced pressure followed by co-evaporation with methanol (2*200 mL). The residue was dissolved in methanol (350 mL). The solution was adsorbed on silica gel (175 g) and evaporated to dryness. The mixture was slurried in dichloromethane (500 mL) and applied on top of a silica gel column (1 kg). The column was eluted with a gradient of dichloromethane-methanol (10:1?2:1). The less polar 2',3'-dimethyl side product was removed and the co-eluding 2' and 3'-O-methyl product containing fractions were combined and evaporated under reduced pressure to a syrup. The syrup was dissolved in a minimum of hot ethanol (ca. 150 mL) and allowed to cool to 25 C. The resulting precipitate (2' less soluble) was collected, washed with ethanol (2*25 ml) and dried to give 15.2 g of pure 2'-O-methylcytidine; mp 252-254 C. mp 252-254 C.); TLC homogenous (Rf 0.50, dichloromethane-methanol 3:1, (Rf of 3' isomer 0.50 and the dimethyl product 0.80). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
In <i>N</i>-methyl-acetamide; propan-1-ol; di-isopropyl ether | 4.a 4(a) 4(a) N4 -palmitoylcytidine A mixture of 24.3 g (100 mmole) of cytidine and 54.34 g (110 mmole) of palmitic anhydride was placed in a round-bottomed flask, and 60 ml of dimethylformamide were added to the mixture. The resulting mixture was stirred in an oil bath kept at 100° C. for 4 hours and then cooled to room temperature, after which 500 ml of diisopropyl ether were added, with stirring. The crystals thus obtained were collected by filtration and recrystallized from 500 ml of propanol. The resulting crystals were again collected by filtration and dried in a desiccator, to give 44.85 g of the title compound as a colorless powder. Nuclear Magnetic Resonance Spectrum (hexadeuterated dimethyl sulfoxide, 270 MHz) δ ppm: 10.81 (1H, singlet); 8.40 (1H, doublet, J=7.32 Hz); 7.20 (1H, doublet, J=7.33 Hz); 5.76 (1H, doublet, J=2.44 Hz); 5.45 (1H, singlet); 5.08 (2H, doublet, J=29.78 Hz); 3.90 (1H, singlet); 3.95 (2H, triplet, J=4.88 Hz); 3.66 (2H, doublet of doublets, J=12.21 & 40.00 Hz). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
46.6% | EXAMPLE 18 The reaction and the treatment were carried out in the same manner as in Example 16 but using cytidine (10 mmol) in place of cytosine to give 5-fluorouridine (1.22 g). Yield, 46.6%. M.P. 183 to 185 C. UV absorption spectrum: lambdamaxpH 2 268 nm; lambdamaxpH 10.0 268 nm. Thin layer chromatography: Rf 0.40. Paper chromatography: Rf 0.37. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 5 steps 1.1: 1H-imidazole / pyridine / Molecular sieve; Inert atmosphere 2.1: sodium periodate; ammonium biborate tetrahydrate / methanol / 1.5 h / 20 °C / Molecular sieve; Inert atmosphere 2.2: 2 h / Molecular sieve; Inert atmosphere 3.1: triethylamine / N,N-dimethyl-formamide / 1 h / 0 °C / Molecular sieve; Inert atmosphere 4.1: triethylamine / tetrahydrofuran / 1.5 h / 0 - 20 °C / Molecular sieve; Inert atmosphere 5.1: tetrabutyl ammonium fluoride / tetrahydrofuran / 1.5 h / 20 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
84% | With hydroxyammonium sulfate In water at 70℃; for 5h; | |
60% | With hydroxylamine acetate at 37℃; for 15h; | |
50% | With hydroxylamine acetate In water at 40℃; for 48h; |
25% | With ammonium hydroxide; hydroxylamine; acetic acid In water at 50℃; for 40h; Sealed tube; Inert atmosphere; | 21 Example 21 EIDD-2159: A 2 N hydroxylamine (30.0 mL, 60.0 mmol) aqueous solution was made by adjusting a 50% w/w aq. H2OH solution with glacial AcOH and then diluting with water to achieve the desired concentration. A sealable pressure vessel was charged with the above solution, L-cytidine (0.486 g, 2.0 mmol), and a stir bar. The vessel was sealed and the mixture was heated at 50°C for 40 h. The mixture was cooled to rt and concentrated by rotary evaporation. The crude reside was dissolved in water, and automated reverse phase flash chromatography (100 g column, gradient of 100% water to 100% MeCN) gave 300 mg of semi pure material as a yellow flaky solid. The compound was taken up in MeOH and immobilized on Celite. Automated flash chromatography (12 g column, gradient of 10 to 25%) MeOH in DCM) gave -150 mg of a white flaky solid containing some occluded solvent. The residue was dissolved in water, frozen in a dry ice/acetone bath, and lyophilized to give the title compound (0.128 g, 0.494 mmol, 25% yield) as an off-white flocculent solid. Spectral analysis showed 90-95%> purity; the impurity was unknown and inseparable by chromatography. 1H MR (400 MHz, D20) δ 7.04 (d, J = 8.3 Hz, 1H), 5.83 (d, J = 5.7 Hz, 1H), 5.72 (d, J = 8.2 Hz, 1H), 4.27 (t, J = 5.5 Hz, 1H), 4.16 (t, J = 4.7 Hz, 1H), 4.03 (q, J = 3.9 Hz, 1H), 3.80 (dd, J = 12.9 Hz, 3.0 Hz, 1H), 3.72 (dd, J = 12.9 Hz, 4.2 Hz, 1H); 13C NMR (100 MHz, D20) δ 151.1, 146.5, 131.2, 98.6, 87.8, 83.9, 72.4, 69.7, 60.9; HRMS calcd. for C9H14N3O6 [M + H]+: 260.08771, found: 260.08734. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With NADH In aq. phosphate buffer at 37℃; for 0.383333h; Microbiological reaction; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 6 steps 1.1: N,N-dimethyl-formamide / 20 h / 20 °C 2.1: pyridine / 20 °C 3.1: dimethyl sulfoxide; trifluoroacetic anhydride / tetrahydrofuran / 2.75 h / -20 - -15 °C 3.2: 0.33 h 4.1: diethyl ether / 2 h / -78 °C / Inert atmosphere 4.2: 0.75 h / 20 °C 5.1: pyridine / 1.67 h / 0 °C / Inert atmosphere 6.1: diethylamino-sulfur trifluoride / 1 h / -20 °C / Inert atmosphere |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1.1: pyridine / 100 °C 2.1: N,N,N',N'-tetramethyl-1,8-diaminonaphthalene; trimethyl phosphite; trichlorophosphate / 2 h / 0 °C 2.2: 0.17 h / 0 °C 2.3: 1 h / 20 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
82% | With pyridine at 100℃; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Stage #1: 5'-CGGCUXUUAACCGA-3', X=2-deoxouridine With 1U P1 nuclease In aq. buffer at 37℃; for 16h; Enzymatic reaction; Stage #2: With 2U alkaline phosphatase In aq. buffer at 37℃; for 1h; Enzymatic reaction; | Enzymatic digestion Hydrolysis of desulfured S2URNA (1) was performed as follows: 20 µg of RNA suspended in 25 mM Tris pH 7.0 was digested with 1U P1 nuclease at 37 °C for 16 h. Then 2U alkaline phosphatase (Calf Intestinal, CIP) and 1x CIP buffer were added and the reaction mixture was incubated at 37 °C for 1 h. The hydrolysate was applied to RP-HPLC column (Kinetex 5µ C18 100A 250 x 4.60 mm) equilibrated with buffer A: 0.1 M CH3COONH4/H2O, and eluted at 1 mL/min with buffer A and then with buffer B: 0.1M CH3COONH4/H2O + 40% CH3CN, with gradient conditions as follows: 0-20 min, linear gradient with 100 % buffer A, 20-60 min linear gradient hold at 100 % buffer B. Each ribonucleoside was identified with the diode array UV detector. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
88% | With 1,8-diazabicyclo[5.4.0]undec-7-ene-functionalized MCM-41-coated alpha-Fe2O3 magnetic nanoparticles; In water; at 80℃; for 7.5h; | General procedure: Carbonyl compounds (2, 1 mmol), barbituric acid (4, 1 mmol,0.128 g), nucleosides (3a-d, 1 mmol) and malononitrile (5, 1 mmol,0.06 g)/barbituric acid (4, 1 mmol, 0.128 g)/dimedone (6, 1 mmol,0.14 g) were added in a 10 mL round-bottom flask contained DBU-F-MCM-41-CNSH (20 mg) and two drops of water. The resulting mixture was stirred at 80 C and the progress of the reaction was followed by TLC. After the completion of the reaction, hot ethanol (20 mL) was added and obtained mixture was boiled for 5 min. Then, the insoluble catalyst was separated from the solution by an external magnet, washed well by hot ethanol (5 mL, 2 times),dried at 120 C and kept for another use (0.019 g of the catalyst was recovered successfully). The hot solution of crude products in ethanol was kept at room temperature over the night and during this time, pure desired products were slowly precipitated. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
81% | With dmap; In acetonitrile; at 0 - 50℃; for 2h; | Cytidine (0.243 g, 1.0 mmol)and DMAP (0.183 g, 1.5 mmol) in MeCN (10 mL) were stirredslowly and cooled to 0 C, and 7 (2.0 mmol) was added slowly. Themixture was heated to 50 C and kept at this temperature for 2 h.The solvent was removed in vacuo and the residue was purified byrecrystallisation from EtOH to give 2 as a white semi-solid (0.318 g);yield 81%; m.p. 62-64 C |
at 0 - 30℃; for 2.5h; | Cytidine (10g, 41.1mmol) was added into the three-necked flask. Add solvent (50 ml). Stir. Cool to 0-5C. Add dropwise N-dichlorophosphorylmorpholine (10.0g, 49 . 3mmol). Control dropping to within 30 min. Completion of the dropping. Elevate temperature. Under the condition of keeping the temperature at room temperature (25-30 C), continue to stir reaction for 2h. After the reaction, by adding 50 ml of saturated sodium bicarbonate aqueous solution, stirring 10 minutes, for exertion of excessive two chlorine phosphinylidynes morphline. Filtered, collecting the filtrate, the solvent is distilled under reduced pressure, to obtain bombycinous, recrystallized with absolute ethanol, to obtain 5'-phosphorothio morpholino cytidine, white solid. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
77% | With sodium hydroxide; In water; at 20℃; for 4h;pH 8.0; | General procedure: Nucleoside/nucleotide (2; 100 mM) and N-acetyl imidazole (1a;10 equiv) were dissolved in water (pH 8; adjusted with 4 MNaOH). The solution was incubated at r.t. for 4 h, and NMR spectra were periodically acquired. The product was purified byreverse-phase (C18) flash coumn chromatography (eluted at pH4 with 100 mM NH4HCO2/MeCN = 98:2 to 80:20). The fractions containing 5 were lyophilised to yield a white powder. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
55% | General procedure: A mixtureof 23 (80.4 mg, 114 mol), 13 (20.4 mg, 76.3 mol) and 11c (21.7 mg, 114 mol) was co-evaporated with anhydrous pyridine (three times) and anhydrous 1,4-dioxane (three times) and dissolved in anhydrous 1,4-dioxane (760 muL). This reaction mixture was stirred under reflux conditions for 1 h and concentrated under reduced pressure. The resulting mixture was stirred with activated 4 A molecular sieves (150 mg) in anhydrous EtCN (1.50 mL) at room temperature for 30 min and then cooled to -40 C, to which p-TolSCl (30.3 L, 229 mol) and AgOTf (117.6 mg, 458 mol) were added at thesame temperature. After stirring for 1.5 h at -40 C, the reaction mixture was quenched with saturated aqueous NaHCO3, diluted with CHCl3 and filtered through Celite. The organic layer was washed withsaturated aqueous NaHCO3 and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (CHCl3/MeOH =1/0-30/1) to give beta-24 as a colorless solid (27.4 mg, 42% yield) |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
47.7% | With [1,3]-dioxolan-2-one; In N,N-dimethyl-formamide; at 82.0℃; for 2.0h; | Add 100g cytidine and 500ml N to the three-neck bottle.N-dimethylformamide,After stirring and dispersing evenly,Add 410g of ethylene carbonate,Turn on heating,Warming up to 82 C,After basic dissolution,start the timer,Reaction 2h; TLC showed that the starting material reaction was complete,Stop heating,Naturally cool and stir,When cooling to 40 C,Drop 200ml of methanol,After the addition is completed,Stir at room temperature for 1.2 h,Then the solvent was distilled off under reduced pressure; 2500 ml of methanol was added to the basic evaporation.Beating at room temperature for 3 hours,Filter under reduced pressure,Rinse twice with ethyl acetate,The amount of ethyl acetate used is 200ml each time.The filter cake was dried under vacuum at 65 C for 4 h; the gray solid powder was obtained.Weighing 51.4g,The yield is 47.7%.Based on cytidine,The purity is 79%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
40% | In 1,4-dioxane; water at 50℃; | 1 a) Method: General procedure: Anhydride of fatty-acid was dissolved in dioxane at final concentration of 50 mg.mL -1. This solution was heated to 50 °C on a water bath. An aqueous solution of cytidine (50 mg.mL -1) was then added drop by drop. The mixture was mixed under magnetic stirring at 50 °C for one night. The reaction was monitored by thin-layer chromatography (Silica gel on TLC Alu foils, Sigma - Aldrich, St Quentin-Fallavier, France). The components were eluted by dichloromethane/methanol 85/15 (v/v) and observed under UV light (UV-Light, VL-6.C, 6 W - 254 nm; Fisher Bioblock Scientific, lllkirch, France). Evaporation under vacuum was performed at 35 °C to remove reaction solvents. The residue was purified by silica gel column flash chromatography (elution with a mixture of dichloromethane/ethanol between 100/0 and 85/15 v/v). Pure fractions were gathered and evaporated under vacuum to obtain a white product, modified cytidine, as the main product. The purity of the compound was controlled by 1H NMR on an Avance DRX 500 MHz (Bruker Daltonics GmbH, Bremen, Germany) in deuterated dimethylsulfoxide. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
8% | Stage #1: Methylenediphosphonic acid; CYTIDINE With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 20℃; Stage #2: triethylamine carbonate In water; N,N-dimethyl-formamide at 20℃; for 0.5h; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
89% | With manganese(IV) oxide; C17H20N4O9P(1-)*Na(1+); oxygen In water; acetonitrile at 20℃; for 7h; Irradiation; chemoselective reaction; |
Tags: 65-46-3 synthesis path| 65-46-3 SDS| 65-46-3 COA| 65-46-3 purity| 65-46-3 application| 65-46-3 NMR| 65-46-3 COA| 65-46-3 structure
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H420 | Harms public health and the environment by destroying ozone in the upper atmosphere |
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