Method for large-scale preparation of acetylated cytidine triphosphate
By reacting compound I with phosphorus oxychloride and trin-n-butylammonium pyrophosphate in aqueous solvent, the problems of low conversion rate and many by-products in the synthesis of acetylated cytidine triphosphate were solved, and efficient preparation of acetylated cytidine triphosphate is achieved, which is suitable for large-scale industrial production.
Patent Information
- Application Number
- CN202311863784.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-29
- Publication Date
- 2025-07-01
AI Technical Summary
The prior art is difficult to efficiently synthesize acetylated cytidine triphosphate, which has problems with low conversion rates and many by-products, especially in the process of large-scale preparation.
Compound I and phosphorus trichloride are used to react in an aqueous solvent to produce compound II, and then react with trin-n-butylammonium pyrophosphate to produce acetylcytidine triphosphate. The conversion rate is increased by controlling the reaction temperature and solvent composition, including the use of specific ratios of trimethyl phosphate, water and trin-n-butylamine, and subsequent reactions in an inert solvent.
It achieves a high conversion rate (about 80%) and a stable preparation process, which is suitable for large-scale industrial production.
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Figure CN120230157A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biochemistry, and particularly relates to a method for synthesizing acetylated cytidine triphosphate. Background Art
[0002] AcyNTPs, as PCR chain terminators, have the furanose moiety substituted with 2-hydroxyethoxymethyl. This enables wider applications of acyNTPs. Experimental results show that the error rate of acyNTPs substrates occurs much lower than that of the corresponding dNTP or ddNTP substrates. Therefore, it is very useful in applications that usually use dideoxynucleotides, such as DNA sequencing and SNP detection. AcyNTPs are applied as PCR chain terminators in a time-of-flight mass spectrometry platform.
[0003] However, synthesizing gram-scale acetylthymidine triphosphate is a great challenge because an acetylthymidine triphosphate has multiple chemical groups, such as primary hydroxyl, secondary hydroxyl, and amino groups, etc. Their different activities and selectivities in synthesis will result in a large number of by-products, which is also a big problem for purification.
[0004] Therefore, those skilled in the art are committed to developing a preparation process for acetylated cytidine triphosphate (acyCTP) with high reaction conversion rate, low by-product content, and stable process. Summary of the Invention
[0005] The purpose of the present invention is to provide a method for large-scale synthesis of acetylated cytidine triphosphate.
[0006] In the first aspect of the present invention, a method for preparing acetylated cytidine triphosphate (acyCTP) is provided, and the method includes the steps:
[0007] (S1) In an aqueous solvent, compound I (N-acetylcytidine (acyC)) reacts with phosphorus oxychloride to form compound II, and the reaction formula is as follows:
[0008]
[0009] In another preferred example, the method further includes the step:
[0010] (S2) Compound II reacts with tributylammonium pyrophosphate to form compound III, and the reaction formula is as follows:
[0011]
[0012] In another preferred example, the aqueous solvent in step (S1) is an aqueous inert solvent.
[0013] In another preferred example, the aqueous solvent in step (S1) includes: trimethyl phosphate, water, and tri-n-butylamine.
[0014] In another preferred example, the aqueous solvent consists of trimethyl phosphate, water and tri-n-butylamine.
[0015] In another preferred example, the aqueous solvent includes:
[0016] Trimethyl phosphate 500 - 1000 parts by volume;
[0017] Water 1 - 5 parts by volume;
[0018] Tri-n-butylamine 200 - 400 parts by volume.
[0019] In another preferred example, the aqueous solvent includes:
[0020] Trimethyl phosphate 900 parts by volume;
[0021] Water 3 parts by volume;
[0022] Tri-n-butylamine 250 parts by volume.
[0023] In another preferred example, the reaction in step (S1) is carried out under the protection of nitrogen or inert gas.
[0024] In another preferred example, the reaction temperature of step (S1) is -10°C to 5°C; preferably -10°C to 0°C; more preferably about -5°C to 0°C.
[0025] In another preferred example, the reaction time of step (S1) is 0.5 - 3 h; preferably about 1 - 2 h.
[0026] In another preferred example, in step (S1), compound I is first dissolved in trimethyl phosphate, and then water and tri-n-butylamine are added; preferably, the mass-to-volume ratio of compound I to trimethyl phosphate is 3 - 10%; preferably about 5%.
[0027] In another preferred example, the mass-to-volume ratio of compound I to phosphorus oxychloride in step (S1) is 50:50 - 80; preferably about 50:60.
[0028] In another preferred example, the reaction in step (S2) is carried out in an inert solvent; preferably, the inert solvent includes anhydrous acetonitrile and tri-n-butylamine; preferably, the volume ratio of anhydrous acetonitrile to tri-n-butylamine is 900:200 - 300 (such as 900:250).
[0029] In another preferred example, the reaction temperature of step (S2) is -10°C to 20°C; preferably about -5°C to 20°C; more preferably about -5°C to 15°C.
[0030] In another preferred example, in the step (S2), tri-n-butylammonium pyrophosphate is first dissolved in an inert solvent; preferably, the mass-volume ratio of tri-n-butylammonium to the inert solvent is 20%-40%; preferably, after dissolution, it is pre-cooled at -20°C.
[0031] In another preferred example, after the reaction in the step (S2) is completed, water is added to quench the reaction.
[0032] In another preferred example, the method further includes a step of purifying the compound I II.
[0033] In another preferred example, the method includes the steps:
[0034] (1) Weigh 50 g of N-acetylcytidine (acyC), add it to a reactor, and insert a thermometer for temperature monitoring;
[0035] (2) Add 900 ml of trimethyl phosphate to dissolve acyC, then add 3 ml of water and 15 ml of tri-n-butylamine. After purging with N2, pre-cool it at -5°C for 10 min;
[0036] (3) Slowly add 60 ml of phosphorus oxychloride, control the reaction temperature not to exceed 0°C, and react for 1.5 h;
[0037] (4) Weigh 290 g of tri-n-butylammonium pyrophosphate, add 900 ml of anhydrous acetonitrile and 250 ml of tri-n-butylamine. After tri-n-butylammonium pyrophosphate is dissolved clearly, pre-cool it at -20°C for 1.5 h. After the reaction in (3) is completed, quickly add the clarified mixed solution to the reactor and react for 10 min;
[0038] After the reaction in (4) is completed, take 6000 ml of aqueous solution to quench the reaction. After quenching, extract with DCM, separate the layers to obtain an aqueous solution, then add 600 ml of 2 M TEAB solution to the aqueous solution, and obtain the target compound after rotary evaporation.
[0039] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings
[0040] Figure 1 Shows the HPLC detection results of the method of Example 1;
[0041] Figure 2 Shows the HPLC detection results of the method of Example 2;
[0042] Figure 3 Shows the HPLC detection results of the method of Example 3. Detailed implementation mode
[0043] The present invention provides a method for synthesizing acetylcytidine triphosphate (acyCTP). The method of the present invention for preparing acetylcytidine triphosphate (acyCTP) is more efficient, the process is stable, and the synthesis conversion rate reaches about 80%.
[0044] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.
[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when referring to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
[0046] Although any methods and materials similar or equivalent to those described in the present invention may be used in the practice or testing of the present invention, preferred methods and materials are exemplified herein.
[0047] In a preferred embodiment of the present invention, according to the method for synthesizing acetylcytidine triphosphate (acyCTP) of the present invention, the method comprises the steps:
[0048] (1) Weigh 50 g of N-acetylcytidine (acyC), add it to a reactor, and insert a thermometer for temperature monitoring;
[0049] (2) Add 900 ml of trimethyl phosphate to dissolve acyC, then add 3 ml of water and 15 ml of tri-n-butylamine. After N2 replacement, pre-cool it at -5°C for 10 min;
[0050] (3) Slowly add 60 ml of phosphorus oxychloride, control the reaction temperature not to exceed 0°C, and react for 1.5 h;
[0051] (4) Weigh 290 g of tri-n-butylammonium pyrophosphate, add 900 ml of anhydrous acetonitrile and 250 ml of tri-n-butylamine. After the tri-n-butylammonium pyrophosphate is dissolved and clarified, pre-cool it at -20°C for 1.5 h. After the reaction in (3) is completed, quickly add the clarified mixed solution to the reactor and react for 10 min;
[0052] After the reaction of (4) is completed, take 6000 ml of aqueous solution to quench the reaction. After quenching, extract with DCM, separate the layers to obtain an aqueous solution. Then add 600 ml of 2 M TEAB solution to the aqueous solution, and obtain the target compound after rotary evaporation.
[0053] The main advantages of the present invention are as follows:
[0054] (1) The conversion rate of preparing acetylcytidine triphosphate (acyCTP) by the method of the present invention is high, and the conversion rate can reach about 80%.
[0055] (2) The reaction stability of preparing acetylcytidine triphosphate (acyCTP) by the method of the present invention in multiple batches is good, and the repeatability is high, which is suitable for large-scale industrial production.
[0056] The present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specified detailed conditions in the following embodiments are usually carried out under conventional conditions such as those described in "Molecular Cloning: A Laboratory Manual" by Sambrook.J et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or under the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.
[0057] Example 1
[0058] At room temperature, weigh 50 g of N-acetylcytidine (acyC), add it to a 5000 ml three-necked flask, insert a thermometer for temperature monitoring, add a magnetic stir bar, then add 900 ml of trimethyl phosphate to dissolve acyC, add 3 ml of water and 250 ml of tri-n-butylamine. After purging with N2, place it in a low-temperature cooling bath magnetic stirrer, set the temperature to -5 °C, and pre-cool for 10 min. When the temperature in the thermometer shows -5 °C, draw 60 ml of phosphorus oxychloride with a syringe and slowly add it to the reaction system. Observe the thermometer and control the reaction temperature not to exceed 0 °C. After adding, react for 1.5 h.
[0059] The reaction phenomenon is that the reaction solution changes from colorless and transparent to light yellow, and white mist appears above the solution in the bottle. As the reaction time prolongs, the white mist turns from thick to light until it disappears.
[0060] Take a 2000 ml single-necked flask, weigh 290 g of tri-n-butylammonium pyrophosphate in the single-necked flask, add 900 ml of anhydrous acetonitrile and 250 ml of tri-n-butylamine. After the tri-n-butylammonium pyrophosphate is dissolved clearly, place it at -20 °C and pre-cool for 1.5 h. After the above light yellow solution reaction is completed, draw the dissolved tri-n-butylammonium pyrophosphate mixed solution with a syringe and quickly add it to the three-necked flask, and react for 10 min.
[0061] The reaction phenomenon was that the reaction system heated up violently, rising from -5°C to about 15°C. The light yellow solution turned into a yellow solution, and white fog appeared again above the solution in the bottle. As the reaction time extended, the white fog became thicker and then thinner until it disappeared.
[0062] After the reaction ended, 6000 ml of aqueous solution was taken to quench the reaction. After quenching, it was extracted with 1000 ml of DCM (dichloromethane) using a separatory funnel to obtain an aqueous solution. Then, 600 ml of 2M TEAB (triethylamine-carbonate buffer solution) was added to the aqueous solution, and the target product was obtained after rotary evaporation.
[0063] Detected by HPLC, the purity was 68%. The calculated yield was 83.4%. The HPLC detection results are as Figure 1 shown.
[0064] Example 2
[0065] At room temperature, 50 g of N-acetylcytidine (acyC) was weighed and added to a 5000-ml three-necked flask. A thermometer was inserted for temperature monitoring, a magnetic stir bar was added, and then 900 ml of trimethyl phosphate was added to dissolve acyC. Then, 4 ml of water and 300 ml of tri-n-butylamine were added. After purging with N2, it was placed in a low-temperature cooling bath magnetic stirrer, and the temperature was set to -5°C and pre-cooled for 10 min. When the temperature in the thermometer showed -5°C, 60 ml of phosphorus oxychloride was drawn with a syringe and slowly added to the reaction system. Observe the thermometer and control the reaction temperature not to exceed 0°C. After adding, react for 1.5 h.
[0066] The reaction phenomenon was that the reaction solution changed from colorless and transparent to a light yellow solution, and white fog appeared above the solution in the bottle. As the reaction time extended, the white fog became thicker and then thinner until it disappeared.
[0067] Take a 2000-ml single-necked flask, weigh 300 g of tri-n-butylammonium pyrophosphate in the single-necked flask, add 900 ml of anhydrous acetonitrile and 250 ml of tri-n-butylamine. After the tri-n-butylammonium pyrophosphate was dissolved clearly, it was pre-cooled at -20°C for 1.5 h. After the above light yellow solution reaction ended, the dissolved tri-n-butylammonium pyrophosphate mixed solution was drawn with a syringe and quickly added to the three-necked flask, and reacted for 10 min.
[0068] The reaction phenomenon was that the reaction system heated up violently, rising from -5°C to about 15°C. The light yellow solution turned into a yellow solution, and white fog appeared again above the solution in the bottle. As the reaction time extended, the white fog became thicker and then thinner until it disappeared
[0069] After the reaction is completed, take 6000 ml of aqueous solution to quench the reaction. After quenching, extract with 1000 ml of DCM (dichloromethane) using a separating funnel to obtain an aqueous solution. Then add 600 ml of 2 M TEAB (triethylamine-carbonate buffer solution) to the aqueous solution and obtain the target product after rotary evaporation.
[0070] Detected by HPLC, the purity is 65%. The calculated yield is 81.6%. The HPLC detection results are as Figure 2 shown.
[0071] Example 3
[0072] At room temperature, weigh 50 g of N-acetylcytidine (acyC) and add it to a 5000-ml three-necked flask. Insert a thermometer for temperature monitoring, add a magnetic stir bar, then add 900 ml of trimethyl phosphate to dissolve acyC, then add 2.5 ml of water and 200 ml of tri-n-butylamine. After purging with N2, place it in a low-temperature cooling bath magnetic stirrer and set the temperature to -5°C. Pre-cool for 10 min. When the temperature in the thermometer shows -5°C, use a syringe to draw 60 ml of phosphorus oxychloride and slowly add it to the reaction system. Observe the thermometer and control the reaction temperature not to exceed 0°C. After adding, react for 1.5 h.
[0073] The reaction phenomenon is that the reaction solution changes from colorless and transparent to light yellow, and white mist appears above the solution in the flask. As the reaction time extends, the white mist changes from thick to light until it disappears.
[0074] Take a 2000-ml single-necked flask, weigh 250 g of tri-n-butylammonium pyrophosphate in the single-necked flask, add 900 ml of anhydrous acetonitrile and 250 ml of tri-n-butylamine. After the tri-n-butylammonium pyrophosphate is dissolved and clarified, place it at -20°C for pre-cooling for 1.5 h. After the above light yellow solution reaction is completed, draw the clarified tri-n-butylammonium pyrophosphate mixed solution through a syringe and quickly add it to the three-necked flask, and react for 10 min.
[0075] The reaction phenomenon is that the reaction system heats up violently, rising from -5°C to about 15°C. The light yellow solution turns into a yellow solution, and white mist appears above the solution in the flask again. As the reaction time extends, the white mist changes from thick to light until it disappears
[0076] After the reaction is completed, take 6000 ml of aqueous solution to quench the reaction. After quenching, extract with 1000 ml of DCM (dichloromethane) using a separating funnel to obtain an aqueous solution. Then add 600 ml of 2 M TEAB (triethylamine-carbonate buffer solution) to the aqueous solution and obtain the target product after rotary evaporation.
[0077] Detected by HPLC, the purity is 71%. The calculated yield is 83.8%. The HPLC detection results are as Figure 3 shown.
[0078] Example 4
[0079] At room temperature, weigh 50 g of N-acetylcytidine (acyC) and add it to a 5000-ml three-necked flask. Insert a thermometer for temperature monitoring, add a magnetic stir bar, then add 900 ml of trimethyl phosphate to dissolve acyC. Then add 3.5 ml of water and 260 ml of tributylamine. After purging with N2, place it in a low-temperature cooling bath magnetic stirrer, set the temperature to -5 °C, and pre-cool for 10 min. When the temperature in the thermometer shows -5 °C, use a syringe to draw 60 ml of phosphorus oxychloride and slowly add it to the reaction system. Observe the thermometer and control the reaction temperature not to exceed 0 °C. After adding, react for 1.5 h.
[0080] The reaction phenomenon is that the reaction solution changes from colorless and transparent to light yellow, and white mist appears above the solution in the flask. As the reaction time extends, the white mist turns from thick to light until it disappears.
[0081] Take a 2000-ml single-necked flask, weigh 250 g of tributylammonium pyrophosphate in the single-necked flask, add 900 ml of anhydrous acetonitrile and 250 ml of tributylamine. After the tributylammonium pyrophosphate is dissolved clearly, place it in a -20 °C bath and pre-cool for 1.5 h. After the above light yellow solution reaction ends, draw the dissolved tributylammonium pyrophosphate mixed solution with a syringe and quickly add it to the three-necked flask, and react for 10 min.
[0082] The reaction phenomenon is that the reaction system heats up violently, rising from -5 °C to about 15 °C, the light yellow solution turns into a yellow solution, and white mist appears above the solution in the flask again. As the reaction time extends, the white mist turns from thick to light until it disappears.
[0083] After the reaction ends, take 6000 ml of aqueous solution to quench the reaction. After quenching, extract with 1000 ml of DCM (dichloromethane), use a separating funnel for liquid separation to obtain the aqueous solution, and then add 600 ml of 2 M TEAB (triethylamine-carbonate buffer solution) to the aqueous solution, and obtain the target product after rotary evaporation.
[0084] Detected by HPLC, the purity is 70%. The calculated yield is 80.2%.
[0085] Example 5
[0086] At room temperature, 50 g of N-acetylcytidine (acyC) was weighed and added to a 5000-ml three-necked flask. A thermometer was inserted for temperature monitoring, and a magnetic stir bar was added. Then, 900 ml of trimethyl phosphate was added to dissolve acyC, followed by 4.0 ml of water and 250 ml of tri-n-butylamine. After purging with N2, it was placed in a low-temperature cooling bath magnetic stirrer, and the temperature was set to -5 °C. After pre-cooling for 10 min, when the temperature in the thermometer showed -5 °C, 60 ml of phosphorus oxychloride was drawn with a syringe and slowly added to the reaction system. The thermometer was observed, and the reaction temperature was controlled not to exceed 0 °C. After the addition was completed, the reaction was carried out for 1.5 h.
[0087] The reaction phenomenon was that the reaction solution changed from colorless and transparent to a light yellow solution, and white fog was generated above the solution in the flask. As the reaction time extended, the white fog became thicker and then lighter until it disappeared.
[0088] A 2000-ml single-necked flask was taken, and 250 g of tri-n-butylammonium pyrophosphate was weighed into the single-necked flask. 900 ml of anhydrous acetonitrile and 300 ml of tri-n-butylamine were added. After the tri-n-butylammonium pyrophosphate was dissolved clearly, it was pre-cooled at -20 °C for 1.5 h. After the above light yellow solution reaction ended, the clarified tri-n-butylammonium pyrophosphate mixed solution was drawn with a syringe and quickly added to the three-necked flask, and the reaction was carried out for 10 min.
[0089] The reaction phenomenon was that the reaction system heated up violently, rising from -5 °C to about 15 °C. The light yellow solution became a yellow solution, and white fog was generated again above the solution in the flask. As the reaction time extended, the white fog became thicker and then lighter until it disappeared.
[0090] After the reaction ended, 6000 ml of aqueous solution was taken to quench the reaction. After the quenching ended, it was extracted with 1000 ml of DCM (dichloromethane), and separated with a separating funnel to obtain an aqueous solution. Then, 600 ml of 2 M TEAB (triethylamine-carbonate buffer solution) solution was added to the aqueous solution, and the target product was obtained after rotary evaporation.
[0091] Detected by HPLC, the purity was 73.5%. The yield was calculated to be 82.7%.
[0092] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.
Claims
1. A method for preparing acetylcytidine triphosphate (acyCTP), the method comprising the steps: (S1) In an aqueous solvent, compound I (N-acetylcytidine (acyC)) reacts with phosphorus oxychloride to form compound II, and the reaction formula is as follows:
2. The method according to claim 1, characterized in that The method further comprises the steps: (S2) Compound II reacts with tributylammonium pyrophosphate to form compound III, and the reaction formula is as follows:
3. The method according to claim 1, characterized in that In the step (S1), the aqueous solvent is an inert aqueous solvent.
4. The method according to claim 1, wherein In the step (S1), the aqueous solvent includes: trimethyl phosphate, water and tributylamine.
5. The method according to claim 4, wherein The aqueous solvent consists of trimethyl phosphate, water and tributylamine.
6. The method according to claim 4, wherein The aqueous solvent includes: Trimethyl phosphate 500-1000 parts by volume; Water 1-5 parts by volume; Tributylamine 200-400 parts by volume.
7. The method according to claim 1, wherein The reaction in the step (S1) is carried out under the protection of nitrogen or inert gas.
8. The method according to claim 1, characterized in that, The reaction temperature of the step (S1) is -10°C to 5°C; preferably -10°C to 0°C; more preferably about -5°C to 0°C.
9. The method according to claim 1, wherein The reaction time of the step (S1) is 0.5-3 h.
10. The method according to claim 1, wherein In the step (S1), compound I is first dissolved in trimethyl phosphate, and then water and tributylamine are added.