A method for synthesizing 2-tbs uridine monomer
By employing low-temperature selective deprotection and simple post-processing methods in the synthesis of 2-TBS uridine monomers, the problems of difficult impurity removal and low yield in existing technologies have been solved, achieving efficient synthesis process optimization.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- KANG YU LIFE SCI TECH (SUZHOU) CO LTD
- Filing Date
- 2023-10-19
- Publication Date
- 2026-05-08
AI Technical Summary
Existing methods for synthesizing 2-TBS uridine monomers suffer from problems such as the formation of double DMTr protective impurities when protecting 5'-OH with DMTrCl, which are difficult to remove, and the formation of 3'-OTBS isomers when protecting 2'-OH with TBSCl, which are also difficult to remove. These problems result in cumbersome processes and low yields.
The synthesis route was optimized to improve selectivity and yield by selectively removing the 3'-OH and 5'-OH disilicon protection under low temperature conditions, combined with simple post-processing methods such as extraction and crystallization.
It effectively avoids the generation of 3'-OTBS impurities, simplifies the operation process, improves the yield of each step, and reduces the synthesis cost.
Smart Images
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Abstract
Description
Technical Field
[0001] This application relates to the field of small nucleic acid monomer raw materials for pharmaceutical and chemical industries, and in particular to a method for synthesizing 2-TBS uridine monomer. Background Technology
[0002] 2-TBS uridine monomer, also known as 5'-O-(4,4-dimethoxytriphenylmethyl)-2'-O-[(tert-butyl)dimethylsilyl]uridine-3'-(2-cyanoethyl-N,N-diisopropyl)2-TBS uridine monomer, has great potential as a small nucleic acid raw material monomer in the field of medicinal chemistry research and plays a pivotal role in the application of mRNA research.
[0003] The common synthetic method currently uses uridine as the starting material, first protecting the 5'-OH with DMTrCl, then protecting the 2'-OH with TBSCl, and finally performing a phospholipidation reaction to obtain the target product.
[0004] However, the above synthesis methods currently have several problems, such as: (1) the DMTrCl protection of 5'-OH stage will produce double DMTr protection impurities that are difficult to remove and require column chromatography; (2) the TBSCl protection of 2'-OH is not selective, and while obtaining the product, a large number of 3'-OTBS isomers will also be obtained, which are difficult to remove; therefore, the existing process is not only cumbersome and produces impurities that are difficult to remove, but also has a low yield. Summary of the Invention
[0005] To address the problems of existing processes, such as the difficulty in removing impurities, cumbersome processes, and low yields, this application provides a method for synthesizing 2-TBS uridine monomers.
[0006] The method for synthesizing 2-TBS uridine monomer provided in this application adopts the following technical solution:
[0007] A method for synthesizing 2-TBS uridine monomers, the synthetic route is as follows:
[0008]
[0009] In one specific feasible implementation, in Step 1, DMF, uridine, and imidazole are mixed, and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane is added while maintaining the temperature at 20-25°C and stirring continuously. After 1-5 hours, the mixture is tested until the raw materials are completely consumed. Then, the mixture is extracted with ethyl acetate, and the organic phase is washed with water 1-3 times. After solvent concentration, S1 is obtained.
[0010] In one specific implementation, the molar ratio of imidazole to uridine is 3 to 5:1.
[0011] In one specific feasible implementation, the molar ratio of 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane to uridine is 1.1 to 2:1.
[0012] In one specific feasible implementation, in Step 2, DMF, S1 and imidazole are stirred, the temperature is controlled at 20-25℃, tert-butyldimethylchlorosilane is added and stirred continuously, and the test is performed after 1-5 hours until the raw materials are completely consumed; then, the mixture is extracted with methyl tert-butyl ether, the organic phase is washed with water 1-3 times, the solvent is concentrated and removed, and S2 is obtained.
[0013] In one specific implementation, the molar ratio of imidazole to S1 is 3 to 5:1.
[0014] In one specific implementation scheme, the molar ratio of tert-butyldimethylchlorosilane to S1 is 1 to 2:1.
[0015] In one specific feasible implementation, in Step 3, tetrahydrofuran and S2 are mixed, cooled to 0-10°C and stirred while adding TBAF and stirring continuously; after 1-5 hours, the mixture is tested until the raw materials are completely consumed; then ethyl acetate is added, water extraction is performed to separate the layers, the organic phase is washed with water 1-3 times, dried, concentrated at 20-25°C to remove the solvent, and then slurryed with methyl tert-ether to obtain S3.
[0016] In one specific implementation scheme, the molar ratio of TBAF to S2 is 1 to 1.5:1.
[0017] In one specific feasible implementation, in Step 4, S3 and pyridine are mixed, then DMTrCl is added and stirred at a temperature of 20-25°C for 1-5 hours; then methyl tert-butyl ether is added, followed by water extraction and separation, and the organic phase is washed with water; pyridine is washed away with citric acid aqueous solution, dried and concentrated, and then S4 is obtained by column chromatography.
[0018] In one specific implementation scheme, the molar ratio of DMTrCl to S3 is 1 to 1.3:1.
[0019] In one specific feasible implementation, the volume ratio of pyridine to S3 is 5 to 20:1.
[0020] In one specific feasible implementation, in Step 5, under controlled anhydrous and oxygen-free conditions, DCM, S4, tetrazolium, and CTPPA are mixed and stirred. A dichloromethane solution containing S4 is slowly added while maintaining the temperature at 20–25°C and continuously stirring. After 1–5 hours, the mixture is tested until the raw materials are completely consumed. The dichloromethane is concentrated and removed at a controlled temperature of 10–20°C. Methyl ether, DMF, and water are added, and the mixture is extracted and separated. DMF and water are added, and the organic phase is washed 1–3 times. The mixture is dried and concentrated, and then added dropwise to n-heptane to crystallize and obtain the final product S5, namely 2-TBS uridine monomer.
[0021] In one specific feasible implementation, the molar ratio of tetrazolium to S4 is 1 to 2:1.
[0022] In one specific implementation, the molar ratio of CTPPA to S4 is 1 to 2:1.
[0023] In summary, this application includes at least one of the following beneficial technical effects:
[0024] (1) Existing technologies cannot avoid the generation of 3'-OTBS impurities, while the core of this invention is: step 3 selectively removes the dual silicon protection of 3'-OH and 5'-OH at low temperature, which effectively avoids the generation of 3'-OTBS impurities;
[0025] (2) Most of the post-processing uses simple methods such as extraction, crystallization, and pulping, which are easy to operate and can be scaled up for production.
[0026] (3) Compared with existing technologies, the present invention improves the yield of each step and reduces the synthesis cost through a completely new process design. Attached Figure Description
[0027] Figure 1 This is the HPLC chromatogram of this application. Detailed Implementation
[0028] The following is in conjunction with the appendix Figure 1 This application will be described in further detail.
[0029] This application discloses a method for synthesizing 2-TBS uridine monomer.
[0030] Example 1
[0031] A method for synthesizing 2-TBS uridine monomer, the specific steps of which are as follows:
[0032] Step 1: Add 500 mL of DMF, 100 g of uridine, and 55.76 g of imidazole to a 2000 mL reactor. Stir mechanically (100-150 r / min) and keep stirring. Slowly add 142.08 g of 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane at 20 °C and continue stirring for 5 h. HPLC is used to detect the reaction until the raw materials are completely consumed.
[0033] Post-processing: Extracted with ethyl acetate, the organic phase was washed once with water, concentrated with solvent, and S1 was obtained with a yield of 88%, a purity of 97%, and a mass of 175.39 g.
[0034] Step 2: Add 500 mL of DMF (N,N-dimethylformamide), 100 g of S1 and 55.94 g of imidazole to a 2000 mL reactor. Use mechanical stirring (100-150 r / min) to maintain stirring. Slowly add 46.45 g of tert-butyldimethylchlorosilane at 20 °C. Maintain the temperature at 20 °C and continue stirring for 5 h. HPLC analysis is performed until the raw materials are completely consumed.
[0035] Post-processing: Extracted with methyl ether, the organic phase was washed once with water, concentrated with solvent, and S2 was obtained with a yield of 85%, a purity of 95%, and a mass of 104.96 g.
[0036] Step 3: Add 1000 mL of tetrahydrofuran and 100 g of S2 to a 2000 mL jacketed reactor, cool to 0 °C while stirring, and slowly add 43.5 g of TBAF (tetrabutylammonium fluoride) using a peristaltic pump at 0 °C; keep stirring at 0 °C for 5 h, and check the reaction is complete by HPLC.
[0037] After the reaction was complete, 1000 mL of ethyl acetate was added, followed by 1000 mL of water for extraction and separation. The organic phase was washed once with water, dried over MgSO4, concentrated at 20 °C to remove the solvent, and then slurried with methyl tert-methyl ether at 0 °C to obtain S3 with a yield of 75%, a purity of 98%, and a mass of 44.74 g.
[0038] Step 4: In a 1000mL reactor, dissolve 60g of S3 in 300mL of pyridine, add 56.57g of DMTrCl to the system and stir. The stirring temperature is 20℃ and the stirring time is 5h.
[0039] Post-processing: Add 600 mL of methyl tert-butyl ether, extract and separate the liquid with 600 mL of water, wash the organic phase once with water; add 5% citric acid aqueous solution to wash away pyridine, dry and concentrate the organic phase, and then obtain S4 by column chromatography (n-heptane / ethyl acetate = 1:1), with a yield of 73%, purity of 99%, and a mass of 80.73 g.
[0040] Step 5: Under anhydrous and oxygen-free conditions, add 400 mL of DCM (dichloromethane), 80 g of S4, 8.47 g of tetrazolium, and 36.49 g of CTPPA (bis(diisopropylamino)(2-cyanoethoxy)phosphine) to a 500 mL reactor and keep stirring. Dissolve 80 g of S4 in 800 mL of dichloromethane solution and slowly add the solution to the system. Keep the temperature at 20 °C and stir for 5 hours. HPLC analysis is performed until the raw materials are completely consumed.
[0041] Post-processing: Dichloromethane was concentrated and removed at 5℃. 800 mL of methyl ether, 400 mL of DMF, and 400 mL of water were added. The mixture was extracted and separated. The organic phase was washed once with 400 mL of DMF and 400 mL of water. The solution was dried and concentrated to 400 mL. The solution was then added dropwise to 2400 mL of n-heptane to crystallize and obtain the final product S5, namely 2-TBS uridine monomer, with a yield of 75%, a purity of 98%, and a mass of 78.19 g.
[0042] Example 2
[0043] A method for synthesizing 2-TBS uridine monomer, the specific steps of which are as follows:
[0044] Step 1: Add 500 mL of DMF, 100 g of uridine, and 111.52 g of imidazole to a 2000 mL reactor. Stir mechanically (100-150 r / min) and keep stirring. Slowly add 258.33 g of 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane at 25 °C and continue stirring for 5 h. Analyze the mixture using HPLC until all the raw materials are consumed.
[0045] Post-processing: Extracted with ethyl acetate, the organic phase was washed three times with water, concentrated with solvent, and S1 was obtained with a yield of 88%, a purity of 97%, and a mass of 179.37 g.
[0046] Step 2: Add 500 mL of DMF (N,N-dimethylformamide), 100 g of S1 and 83.90 g of imidazole to a 2000 mL reactor. Use mechanical stirring (100-150 r / min) to maintain stirring. Slowly add 77.42 g of tert-butyldimethylchlorosilane at 25 °C. Maintain the temperature at 25 °C and continue stirring for 5 h. HPLC analysis is performed until the raw materials are completely consumed.
[0047] Post-processing: Extracted with methyl ether, the organic phase was washed three times with water, concentrated with solvent, and S2 was obtained with a yield of 89%, a purity of 95%, and a mass of 109.9 g.
[0048] Step 3: Add 1000 mL of tetrahydrofuran and 100 g of S2 to a 2000 mL jacketed reactor, cool to 10 °C while stirring, and slowly add 62.25 g of TBAF (tetrabutylammonium fluoride) using a peristaltic pump while maintaining the temperature at 10 °C; keep stirring at 10 °C for 5 h, and check the reaction is complete by HPLC.
[0049] After the reaction was completed, 1000 mL of ethyl acetate was added, followed by 1000 mL of water for extraction and separation. The organic phase was washed three times with water, dried over MgSO4, concentrated at 25 °C to remove the solvent, and then slurried with methyl tert-methyl ether at 10 °C to obtain S3 with a yield of 64%, a purity of 98%, and a mass of 38.18 g.
[0050] Step 4: In a 1000mL reactor, dissolve 60g of S3 in 1200mL of pyridine, add 73.54g of DMTrCl to the system and stir. The stirring temperature is 25℃ and the stirring time is 5h.
[0051] Post-processing: Add 600 mL of methyl tert-butyl ether, extract and separate the liquid with 600 mL of water, wash the organic phase once with water; add 5% citric acid aqueous solution to wash away pyridine, dry and concentrate the organic phase, and then obtain S4 by column chromatography (n-heptane / ethyl acetate = 1:1), with a yield of 75%, purity of 99%, and a mass of 82.95 g.
[0052] Step 5: Under anhydrous and oxygen-free conditions, add 1600 mL of DCM (dichloromethane), 80 g of S4, 16.95 g of tetrazolium, and 72.98 g of CTPPA (1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane) to a 500 mL reactor while stirring. Dissolve 80 g of S4 in 800 mL of dichloromethane solution and slowly add the solution to the system. Maintain the temperature at 25 °C and stir for 5 hours. HPLC analysis is performed until the raw materials are completely consumed.
[0053] Post-processing: Dichloromethane was concentrated and removed at 15℃. 800 mL of methyl ether, 400 mL of DMF, and 400 mL of water were added. The mixture was extracted and separated. The organic phase was washed three times with 400 mL of DMF and 400 mL of water. The solution was dried and concentrated to 400 mL. The solution was then added dropwise to 2400 mL of n-heptane to crystallize and obtain the final product S5, namely 2-TBS uridine monomer, with a yield of 75%, a purity of 98%, and a mass of 78.19 g.
[0054] Example 3
[0055] A method for synthesizing 2-TBS uridine monomer, the specific steps of which are as follows:
[0056] Step 1: Add 500 mL of DMF, 100 g of uridine, and 83.64 g of imidazole to a 2000 mL reactor. Stir mechanically (100-150 r / min) and keep stirring. Slowly add 193.75 g of 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane at 22.5 °C and continue stirring for 5 h. HPLC is used to detect the total amount of raw materials consumed.
[0057] Post-processing: Extracted with ethyl acetate, the organic phase was washed twice with water, concentrated with solvent, and S1 was obtained with a yield of 90%, a purity of 97%, and a mass of 179.37 g.
[0058] Step 2: Add 500 mL of DMF (N,N-dimethylformamide), 100 g of S1 and 69.92 g of imidazole to a 2000 mL reactor. Use mechanical stirring (100-150 r / min) to maintain stirring. Slowly add 61.94 g of tert-butyldimethylchlorosilane at a controlled temperature of 22.5 °C. Maintain the temperature at 22.5 °C and continue stirring for 5 h. HPLC analysis is performed until the raw materials are completely consumed.
[0059] Post-processing: Extracted with methyl ether, the organic phase was washed twice with water, concentrated with solvent, and S2 was obtained with a yield of 90%, a purity of 95%, and a mass of 111.14 g.
[0060] Step 3: Add 1000 mL of tetrahydrofuran and 100 g of S2 to a 2000 mL jacketed reactor, cool to 5 °C while stirring, and slowly add 54.38 g of TBAF (tetrabutylammonium fluoride) using a peristaltic pump while maintaining the temperature at 5 °C; keep stirring at 5 °C for 5 h, and check the reaction is complete by HPLC.
[0061] After the reaction was completed, 1000 mL of ethyl acetate was added, followed by 1000 mL of water for extraction and separation. The organic phase was washed twice with water, dried over MgSO4, concentrated at 22.5 °C to remove the solvent, and then slurried with methyl tert-methyl ether at 5 °C to obtain S3 with a yield of 80%, a purity of 98%, and a mass of 47.72 g.
[0062] Step 4: In a 1000mL reactor, dissolve 60g of S3 in 600mL of pyridine, add 65.05g of DMTrCl to the system and stir. The stirring temperature is 22.5℃ and the stirring time is 5h.
[0063] Post-processing: Add 600 mL of methyl tert-butyl ether, extract and separate the liquid with 600 mL of water, wash the organic phase once with water; add 5% citric acid aqueous solution to wash away pyridine, dry and concentrate the organic phase, and then obtain S4 by column chromatography (n-heptane / ethyl acetate = 1:1), with a yield of 80%, purity of 99%, and a mass of 88.47 g.
[0064] Step 5: Under anhydrous and oxygen-free conditions, add 800 mL of DCM (dichloromethane), 80 g of S4, 12.71 g of tetrazolium, and 54.73 g of CTPPA (1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane) to a 500 mL reactor. While stirring, dissolve 80 g of S4 in 800 mL of dichloromethane solution and slowly add the solution to the system. Maintain the temperature at 22.5 °C and stir for 5 hours. HPLC analysis is performed until the raw materials are completely consumed.
[0065] Post-processing: Dichloromethane was concentrated and removed at 10℃. 800 mL of methyl ether, 400 mL of DMF, and 400 mL of water were added, and the mixture was extracted and separated. The organic phase was washed twice with 400 mL of DMF and 400 mL of water. The solution was dried and concentrated to 400 mL, and then added dropwise to 2400 mL of n-heptane to crystallize and obtain the final product S5, namely 2-TBS uridine monomer, with a yield of 80%, a purity of 99.26%, and a mass of 83.40 g.
[0066] The spectrum shows two distinct absorption peaks at 11.403 min and 11.513 min, which are very close to each other and both belong to the target product. The splitting into two peaks is likely due to the target product being a chiral molecule, resulting in slightly different retention times. Therefore, the purity of the target product is 99.26%.
[0067] In addition, the equation corresponding to Step 1 in the above embodiments is as follows:
[0068]
[0069] The equation corresponding to Step 2 is:
[0070]
[0071] The equation corresponding to Step 3 is:
[0072]
[0073] The equation corresponding to Step 4 is:
[0074]
[0075] The equation corresponding to Step 5 is:
[0076]
[0077] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.
Claims
1. A method for synthesizing 2-TBS uridine monomer, characterized in that... The synthesis route is as follows: In Step 3, tetrahydrofuran and S2 are mixed, cooled to 0-10℃ and stirred, and TBAF is added and stirred continuously. After 1-5 hours, the mixture is tested until the raw material is completely consumed. Then ethyl acetate is added, water is extracted and the layers are separated. The organic phase is washed with water 1-3 times, dried, concentrated at 20-25℃ to remove the solvent, and then slurryed with methyl tert-butyl ether to obtain S3.
2. The method for synthesizing 2-TBS uridine monomer according to claim 1, characterized in that: In Step 1, DMF, uridine, and imidazole are mixed and the temperature is controlled at 20-25°C. Then, 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane is added and stirred continuously. After 1-5 hours, the mixture is tested until the raw materials are completely consumed. Then, the mixture is extracted with ethyl acetate, and the organic phase is washed with water 1-3 times. After the solvent is concentrated, S1 is obtained.
3. The method for synthesizing 2-TBS uridine monomer according to claim 2, characterized in that: The molar ratio of imidazole to uridine is 3-5:
1.
4. The method for synthesizing 2-TBS uridine monomer according to claim 2, characterized in that: The molar ratio of 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane to uridine is 1.1 to 2:
1.
5. The method for synthesizing 2-TBS uridine monomer according to claim 1, characterized in that: In Step 2, DMF, S1 and imidazole are stirred, and the temperature is controlled at 20~25℃. Then tert-butyldimethylchlorosilane is added and stirred continuously. After 1~5 hours, the sample is tested until the raw material is completely consumed. Then, methyl tert-butyl ether is used for extraction, the organic phase is washed with water 1~3 times, and the solvent is concentrated to obtain S2.
6. The method for synthesizing 2-TBS uridine monomer according to claim 5, characterized in that: The molar ratio of imidazole to S1 is 3~5:
1.
7. The method for synthesizing 2-TBS uridine monomer according to claim 5, characterized in that: The molar ratio of tert-butyldimethylchlorosilane to S1 is 1~2:
1.
8. The method for synthesizing 2-TBS uridine monomer according to claim 1, characterized in that: The molar ratio of TBAF to S2 is 1~1.5:
1.
9. The method for synthesizing 2-TBS uridine monomer according to claim 1, characterized in that: In Step 4, S3 and pyridine are mixed, then DMTrCl is added and stirred at a temperature of 20-25°C for 1-5 hours; then methyl tert-butyl ether is added, followed by water extraction and separation, and the organic phase is washed with water. Add citric acid aqueous solution to wash away pyridine, dry and concentrate, and then obtain S4 by column chromatography.
10. The method for synthesizing 2-TBS uridine monomer according to claim 9, characterized in that: The molar ratio of DMTrCl to S3 is 1~1.3:
1.
11. The method for synthesizing 2-TBS uridine monomer according to claim 9, characterized in that: The volume ratio of pyridine to S3 is 5~20:
1.
12. The method for synthesizing 2-TBS uridine monomer according to claim 1, characterized in that: In Step 5, under anhydrous and oxygen-free conditions, DCM, S4, tetrazolium, and CTPPA are mixed and stirred. A dichloromethane solution containing S4 is slowly added while maintaining the temperature at 20-25°C and stirring continuously. After 1-5 hours, the mixture is tested until the raw materials are completely consumed. The dichloromethane is concentrated and removed at 10-20°C. Methyl tert-butyl ether, DMF, and water are added, and the mixture is extracted and separated. DMF and water are added, and the organic phase is washed 1-3 times. The mixture is dried and concentrated, and then added dropwise to n-heptane to crystallize and obtain the final product S5, namely 2-TBS uridine monomer.
13. The method for synthesizing 2-TBS uridine monomer according to claim 12, characterized in that: The molar ratio of tetrazolium to S4 is 1~2:
1.
14. The method for synthesizing 2-TBS uridine monomer according to claim 12, characterized in that: The molar ratio of CTPPA to S4 is 1~2:1.
Citation Information
Patent Citations
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US20020120129A1