Synthesis method of deoxyadenosine triphosphate
By reacting 2'-deoxyadenosine with phosphorus oxychloride and trin-butylammonium pyrophosphate in an aqueous solvent, the synthetic conversion and purity of deoxyadenosine triphosphate (dATP) was successfully improved, and the problems of low reaction conversion and high by-product content in the prior art were solved, achieving the stability of the process and the effect of suitable industrial production.
Patent Information
- Application Number
- CN202311748751.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-18
- Publication Date
- 2025-06-20
AI Technical Summary
In the prior art, the chemical synthesis reaction conversion rate of deoxyadenosine triphosphate (dATP) is low, the by-product content is high, and the process is unstable, making it difficult to achieve industrial production.
In an aqueous solvent, compound I (2'-deoxyadenosine) is reacted with phosphorus oxychloride to form compound II and reacted with trin-butylammonium pyrophosphate to form deoxyadenosine triphosphate (dATP). The process includes specific reaction steps and conditions, such as using a solvent composed of trimethyl phosphate, water and trin-butylamine, to control the reaction temperature and time to improve the reaction conversion and purity.
It achieves a high conversion rate of deoxyadenosine triphosphate (dATP) (about 80%), has low by-product content, stable process, and is suitable for industrial production.
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Figure CN120173039A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biochemistry, and particularly relates to a method for synthesizing deoxyadenosine triphosphate. Background Art
[0002] Deoxyadenosine triphosphate (dATP) is a basic component for synthesizing DNA molecules and has important therapeutic and diagnostic applications. It has been applied to various molecular biology applications such as PCR, real-time PCR, cDNA synthesis, primer extension, DNA sequencing, DNA labeling, etc. The quality and purity of dNTPs are crucial for their successful application in molecular biology.
[0003] Chemically synthesizing gram-scale deoxyadenosine triphosphate is a great challenge because a deoxyadenosine triphosphate has multiple chemical groups such as primary hydroxyl, secondary hydroxyl, and amino groups, etc. Their different activities and selectivities in the synthesis will result in a large number of by-products, which is also a big problem for purification.
[0004] The "one-pot three-step method" proposed by Ludwig is a currently commonly used method. However, when using the "one-pot three-step method" proposed by Ludwig, the reaction conversion rate is low, the content of by-products is high, and the conversion rate is unstable during the experimental process, making it impossible to carry out stable industrial production.
[0005] Therefore, those skilled in the art are committed to developing a preparation process for deoxyadenosine triphosphate (dATP) with high reaction conversion rate, low by-product content, and stable process. Summary of the Invention
[0006] The purpose of the present invention is to provide a preparation process for deoxyadenosine triphosphate (dATP) with high reaction conversion rate, low by-product content, and stable process.
[0007] In the first aspect of the present invention, there is provided a method for preparing deoxyadenosine triphosphate (dATP), the method comprising the steps of:
[0008] (S1) In an aqueous solvent, compound I (2'-deoxyadenosine (dA)) reacts with phosphorus oxychloride to form compound II, and the reaction formula is as follows:
[0009]
[0010] In another preferred example, the method further comprises the step of:
[0011] (S2) Compound II reacts with tributylammonium pyrophosphate to form compound III, and the reaction formula is as follows:
[0012]
[0013] In another preferred example, the aqueous solvent in the step (S1) is an inert aqueous solvent.
[0014] In another preferred example, the aqueous solvent in the step (S1) includes: trimethyl phosphate, water and tri-n-butylamine.
[0015] In another preferred example, the aqueous solvent consists of trimethyl phosphate, water and tri-n-butylamine.
[0016] In another preferred example, the aqueous solvent includes:
[0017] 50 - 100 parts by volume of trimethyl phosphate;
[0018] 0.1 - 0.5 parts by volume of water;
[0019] 10 - 20 parts by volume of tri-n-butylamine.
[0020] In another preferred example, the aqueous solvent includes:
[0021] 100 parts by volume of trimethyl phosphate;
[0022] 0.3 parts by volume of water;
[0023] 15 parts by volume of tri-n-butylamine.
[0024] In another preferred example, the reaction in the step (S1) is carried out under the protection of nitrogen or inert gas.
[0025] In another preferred example, the reaction temperature of the step (S1) is -50°C to 5°C; preferably -20°C to 0°C; more preferably about -10°C to 0°C.
[0026] In another preferred example, the reaction time of the step (S1) is 0.3 - 3 h; preferably about 0.5 - 1 h.
[0027] In another preferred example, in the 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%.
[0028] In another preferred example, the mass-to-volume ratio of compound I to phosphorus oxychloride in the step (S1) is 1:1 - 1.2; preferably about 1:1.
[0029] In another preferred example, the reaction in the 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 50:20 - 40 (such as 50:30).
[0030] In another preferred example, the reaction temperature in step (S2) is -30°C to 20°C; preferably about -20°C to 10°C.
[0031] In another preferred example, in 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.
[0032] In another preferred example, after the reaction in step (S2) is completed, water is added to quench the reaction.
[0033] In another preferred example, the method further includes a step of purifying compound III.
[0034] In another preferred example, the method includes the steps of:
[0035] (1) Weigh 5 g of 2'-deoxyadenosine (dA), add it to a 250 ml three-necked flask, and insert a thermometer for temperature monitoring;
[0036] (2) Add 100 ml of trimethyl phosphate to dissolve dA, then add 0.3 ml of water and 15 ml of tri-n-butylamine. After purging with N2, pre-cool it at -5°C for 10 min;
[0037] (3) Slowly add 5 ml of phosphorus oxychloride, observe the thermometer to ensure that the reaction temperature does not exceed 0°C. After addition, react for 1.5 h;
[0038] (4) Take a 150 ml single-necked flask, weigh 20 g of tri-n-butylammonium pyrophosphate in the single-necked flask, add 50 ml of anhydrous acetonitrile and 30 ml of tri-n-butylamine. After 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 three-necked flask and react for 10 min;
[0039] After the reaction in (4) is completed, take 600 ml of aqueous solution to quench the reaction. After quenching, extract with 100 ml of DCM, separate the layers to obtain an aqueous solution, and then add 60 ml of 2 M TEAB solution to the aqueous solution, and obtain the target compound after rotary evaporation.
[0040] It should be understood that within the scope of the present invention, the above 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 repeated one by one here. Description of the Drawings
[0041] Figure 1 Shows the HPLC detection results of the method in Example 1;
[0042] Figure 2 Shows the HPLC detection results of the method of Example 2;
[0043] Figure 3 Shows the HPLC detection results of the method of Example 3. Detailed implementation mode
[0044] The present invention provides a method for synthesizing deoxyadenosine triphosphate. Through extensive and in-depth research, the inventors unexpectedly found that using a water-added solvent in the synthesis step makes the synthesis of deoxyadenosine triphosphate more efficient and stable. The synthesis conversion rate of deoxyadenosine triphosphate reaches about 80%, and with multiple batches of feeding, the synthesis conversion rate of deoxyadenosine triphosphate is stable.
[0045] 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 only intended to describe specific embodiments and are not intended to be restrictive. The scope of the present invention will be limited only by the appended claims.
[0046] 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.).
[0047] Although any methods and materials similar or equivalent to those described in the present invention can be used in the practice or testing of the present invention, preferred methods and materials are exemplified herein.
[0048] The chemical structure of the deoxyadenosine triphosphate involved in the present invention is as follows:
[0049]
[0050] In a preferred embodiment of the present invention, according to the method for synthesizing deoxyadenosine triphosphate of the present invention, it includes the steps:
[0051] (1) Weigh 5 g of 2'-deoxyadenosine (dA) and add it to a 250 ml three-necked flask, insert a thermometer for temperature monitoring;
[0052] (2) Add 100 ml of trimethyl phosphate to dissolve dA, then add 0.3 ml of water and 15 ml of tri-n-butylamine. After N2 replacement, pre-cool it at -5°C for 10 min;
[0053] (3) Slowly add 5 ml of phosphorus oxychloride, observe the thermometer, and keep the reaction temperature not exceeding 0 °C. After the addition is complete, react for 1.5 h;
[0054] (4) Take a 150-ml single-necked flask, weigh 20 g of tributylammonium pyrophosphate into the single-necked flask, add 50 ml of anhydrous acetonitrile and 30 ml of tributylamine. After the tributylammonium pyrophosphate is dissolved and clarified, place it in a -20 °C bath for pre-cooling for 1.5 h. After the reaction in (3) is completed, quickly add the clarified mixed solution to the three-necked flask and react for 10 min;
[0055] After the reaction in (4) is completed, take 600 ml of aqueous solution to quench the reaction. After quenching, extract with 100 ml of DCM, separate the layers to obtain the aqueous solution, and then add 60 ml of 2 M TEAB solution to the aqueous solution. After rotary evaporation, the target compound is obtained.
[0056] The main advantages of the present invention are as follows:
[0057] (1) In the method of the present invention, the conversion rate of the product is high, and the conversion rate can reach about 80%.
[0058] (2) The method of the present invention has good reaction stability in multiple batches, high repeatability, and is suitable for industrial production.
[0059] The present invention will be further described in detail below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are usually carried out under conventional conditions such as those described in "Molecular Cloning: A Laboratory Manual" (translated by Huang Peitang et al., Beijing: Science Press, 2002) by Sambrook.J et al. in the United States, or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels without special instructions.
[0060] Example 1
[0061] At room temperature, weigh 5 g of 2'-deoxyadenosine (dA), add it to a 250-ml three-necked flask, insert a thermometer for temperature monitoring, add a magnetic stirrer, then add 100 ml of trimethyl phosphate to dissolve dA, and then add 0.3 ml of water and 15 ml of tributylamine. After purging with N2, place it in a low-temperature cooling bath magnetic stirrer, set the temperature to -20 °C, and pre-cool for 10 min. When the temperature in the thermometer shows -20 °C, use a syringe to draw 5 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 the addition is complete, react for 0.5 h.
[0062] The reaction phenomenon is that the reaction solution changes from colorless and transparent to light yellow, and white fog appears above the solution in the bottle. As the reaction time extends, the white fog turns from thick to light until it disappears.
[0063] Take a 150 ml single-necked flask, weigh 20 g of tributylammonium pyrophosphate into the single-necked flask, add 50 ml of anhydrous acetonitrile and 30 ml of tributylamine. After the tributylammonium pyrophosphate dissolves clearly, pre-cool it at -20 °C for 1.5 h. After the above-mentioned light yellow solution reaction ends, draw the clear tributylammonium pyrophosphate mixed solution through a syringe and quickly add it to the three-necked flask, and react for 10 min.
[0064] The reaction phenomenon is that the reaction system heats up violently, rising from -20 °C to about 10 °C, the light yellow solution turns into a yellow solution, and white fog appears above the solution in the bottle again. As the reaction time extends, the white fog turns from thick to light until it disappears.
[0065] After the reaction ends, take 600 ml of aqueous solution to quench the reaction. After quenching, extract with 100 ml of DCM (dichloromethane), separate with a separating funnel to obtain the aqueous solution, and then add 60 ml of 2 M TEAB (triethylamine-carbonate buffer solution) to the aqueous solution, and obtain the target product after rotary evaporation.
[0066] Detected by HPLC, the purity is 75.8%. The conversion rate of the product reaches 84.7%. The HPLC detection results are as Figure 1 shown.
[0067] Example 2
[0068] At room temperature, weigh 5 g of 2'-deoxyadenosine (dA), add it to a 250 ml three-necked flask, insert a thermometer for temperature monitoring, add a magnetic stir bar, then add 100 ml of trimethyl phosphate to dissolve dA, then add 0.2 ml of water and 10 ml of tributylamine. After replacing with N2, place it in a low-temperature cooling bath magnetic stirrer, set the temperature to -20 °C, and pre-cool for 10 min. When the temperature in the thermometer shows -20 °C, draw 5 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 0.5 h.
[0069] The reaction phenomenon is that the reaction solution changes from colorless and transparent to light yellow, and white fog appears above the solution in the bottle. As the reaction time extends, the white fog turns from thick to light until it disappears.
[0070] Take a 150-ml single-necked flask, weigh 25 g of tributylammonium pyrophosphate into the single-necked flask, add 60 ml of anhydrous acetonitrile and 30 ml of tributylamine. After the tributylammonium pyrophosphate is dissolved and clarified, pre-cool it at -20 °C for 1.5 h. After the above light yellow solution reacts, draw the clarified tributylammonium pyrophosphate mixed solution with a syringe and quickly add it to a three-necked flask, and react for 10 min.
[0071] The reaction phenomenon is that the reaction system heats up violently, rising from -20 °C to about 10 °C. The light yellow solution turns into a yellow solution, and white mist appears again above the solution in the flask. As the reaction time extends, the white mist turns from thick to light until it disappears.
[0072] After the reaction is completed, take 600 ml of aqueous solution to quench the reaction. After quenching, extract with 100 ml of DCM (dichloromethane) using a separating funnel to obtain an aqueous solution. Then add 60 ml of 2 M TEAB (triethylamine-carbonate buffer solution) to the aqueous solution, and obtain the target product after rotary evaporation.
[0073] Detected by HPLC, the purity is 78.3%. The conversion rate of the product reaches 80.2%. The HPLC detection results are as Figure 2 shown.
[0074] Example 3
[0075] At room temperature, weigh 5 g of 2'-deoxyadenosine (dA) and add it to a 250-ml three-necked flask. Insert a thermometer for temperature monitoring, add a magnetic stir bar, then add 100 ml of trimethyl phosphate to dissolve dA, and then add 0.3 ml of water and 12 ml of tributylamine. After replacing with N2, place it in a low-temperature cooling bath magnetic stirrer, set the temperature to -20 °C, and pre-cool for 10 min. When the temperature in the thermometer shows -20 °C, draw 5 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 0.5 h.
[0076] 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.
[0077] Take a 150-ml single-necked flask, weigh 20 tributylammonium pyrophosphate into the single-necked flask, add 50 ml of anhydrous acetonitrile and 28 ml of tributylamine. After the tributylammonium pyrophosphate is dissolved and clarified, pre-cool it at -20 °C for 1.5 h. After the above light yellow solution reacts, draw the clarified tributylammonium pyrophosphate mixed solution with a syringe and quickly add it to a three-necked flask, and react for 10 min.
[0078] The reaction phenomenon was that the reaction system heated up violently, rising from -20°C to about 10°C. The light yellow solution turned into a yellow solution, and white mist appeared again above the solution in the bottle. As the reaction time extended, the white mist became thicker and then thinner until it disappeared.
[0079] After the reaction ended, 600 ml of aqueous solution was taken to quench the reaction. After quenching, it was extracted with 100 ml of DCM (dichloromethane) using a separating funnel to obtain the aqueous solution. Then, 60 ml of 2M TEAB (triethylamine-carbonate buffer solution) was added to the aqueous solution, and the target product was obtained after rotary evaporation.
[0080] Detected by HPLC, the purity was 81.6%. The conversion rate of the product reached 80.7%. The HPLC detection results are as Figure 2 shown.
[0081] Example 4
[0082] At room temperature, 5 g of 2'-deoxyadenosine (dA) was weighed and added to a 250 ml three-necked flask. A thermometer was inserted for temperature monitoring, a magnetic stir bar was added, and then 100 ml of trimethyl phosphate was added to dissolve dA. Then, 0.4 ml of water and 18 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 -20°C and pre-cooled for 10 min. When the temperature in the thermometer showed -20°C, 5 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 0.5 h.
[0083] The reaction phenomenon was that the reaction solution changed from colorless and transparent to light yellow, and white mist appeared above the solution in the bottle. As the reaction time extended, the white mist became thicker and then thinner until it disappeared.
[0084] Take a 150 ml single-necked flask, weigh 20 g of tri-n-butylammonium pyrophosphate in the single-necked flask, add 40 ml of anhydrous acetonitrile and 25 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.
[0085] The reaction phenomenon was that the reaction system heated up violently, rising from -20°C to about 10°C. The light yellow solution turned into a yellow solution, and white mist appeared again above the solution in the bottle. As the reaction time extended, the white mist became thicker and then thinner until it disappeared.
[0086] After the reaction is completed, take 600 ml of aqueous solution to quench the reaction. After quenching, extract with 100 ml of DCM (dichloromethane) using a separating funnel to obtain an aqueous solution. Then add 60 ml of 2M TEAB (triethylamine-carbonate buffer solution) to the aqueous solution, and obtain the target product after rotary evaporation.
[0087] Detected by HPLC, the purity is 80.9%. The conversion rate of the product reaches 81.8%.
[0088] All documents mentioned in the present invention are cited herein as references, as if each document was individually cited as a 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 deoxyadenosine triphosphate (dATP), characterized in that, The method includes the steps: (S1) In an aqueous solvent, compound I (2'-deoxyadenosine (dA)) 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 includes 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 solvent containing water.
4. The method according to claim 3, characterized in that, In the step (S1), the aqueous solvent includes: trimethyl phosphate, water and tributylamine.
5. The method according to claim 4, characterized in that, The aqueous solvent consists of trimethyl phosphate, water and tributylamine.
6. The method according to claim 4, characterized in that, The aqueous solvent includes: 50 - 100 parts by volume of trimethyl phosphate; 0.1 - 0.5 parts by volume of water; 10 - 20 parts by volume of tributylamine.
7. The method according to claim 1, characterized in that, 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 -50°C to 5°C.
9. The method according to claim 2, characterized in that, The reaction in the step (S2) is carried out in an inert solvent.
10. The method according to claim 9, characterized in that, The reaction temperature of the step (S2) is -30°C to 20°C.