Process for the synthesis of acetylated deoxyadenosine triphosphate
The one-pot, three-step synthesis of acetyl-adenosine triphosphate (ATP) solves the problems of low conversion rate and high by-product content, achieving efficient and stable synthesis of ATP, which is suitable for industrial applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DAAN GENE CO LTD
- Filing Date
- 2024-12-09
- Publication Date
- 2026-06-09
Smart Images
Figure CN122167490A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biochemistry, specifically relating to a method for synthesizing acetylated deoxyadenosine triphosphate. Background Technology
[0002] AcyNTPs, used as PCR chain terminators, have their furanose moiety replaced by a 2-hydroxyethoxymethyl group. This allows for wider applications of acyNTPs. Experimental results show that the error rate of acyNTP substrates is significantly lower than that of corresponding dNTP or ddNTP substrates. Therefore, they are very useful in applications that commonly use dideoxynucleotides, such as DNA sequencing and SNP detection. AcyNTPs are also used as PCR chain terminators in time-of-flight mass spectrometry platforms.
[0003] Synthesizing acetyl-adenosine triphosphate is a significant challenge because an acetyl-adenosine triphosphate has multiple chemical groups, such as primary hydroxyl, secondary hydroxyl, and amino groups, which have different activities and selectivities during synthesis, resulting in a large number of byproducts and posing a significant problem for purification.
[0004] The "one-pot three-step process" proposed by Ludwig is a commonly used method. However, the "one-pot three-step process" proposed by Ludwig has a low reaction conversion rate, a high content of by-products, and an unstable conversion rate during the experiment, making it impossible to carry out stable industrial production.
[0005] Therefore, those skilled in the art are dedicated to developing a process for preparing acetyl adenosine triphosphate (acyATP) with high reaction conversion rate, low by-product content, and stable process. Summary of the Invention
[0006] The purpose of this invention is to provide a preparation process for acetyl adenosine triphosphate (acyATP) with high reaction conversion rate, low by-product content, and stable process.
[0007] In a first aspect of the present invention, a method for preparing acetyl adenosine triphosphate (acyATP) is provided, the method comprising the steps of:
[0008] (S1) In an anhydrous solvent, compound I (2'-acetyladenosine (acyA)) reacts with 2-chloro-1,3,2-benzodioxaphosphazene-4-one to form compound II, as shown in the following reaction formula:
[0009]
[0010] In another preferred embodiment, the method further includes the step of:
[0011] (S2) Compound II reacts with tri-n-butylammonium pyrophosphate to form compound III, as shown in the following reaction formula:
[0012]
[0013] In another preferred embodiment, the method further includes the step of:
[0014] (S3) Compounds I and II react in the presence of iodine to form compound IV, as shown in the following reaction:
[0015]
[0016] In another preferred embodiment, the anhydrous solvent in step (S1) is an anhydrous inert solvent.
[0017] In another preferred embodiment, the anhydrous solvent in step (S1) comprises anhydrous pyridine and dioxane.
[0018] In another preferred embodiment, the anhydrous solvent is composed of anhydrous pyridine and dioxane.
[0019] In another preferred embodiment, the anhydrous solvent comprises:
[0020] 100-300 parts by volume of anhydrous pyridine;
[0021] Dioxane 300-800 parts by volume.
[0022] In another preferred embodiment, the anhydrous solvent comprises:
[0023] 200 parts by volume of anhydrous pyridine;
[0024] 450-550 parts by volume of dioxane.
[0025] In another preferred embodiment, step (S1) is carried out under the protection of nitrogen or an inert gas.
[0026] In another preferred embodiment, 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.
[0027] In another preferred embodiment, the reaction time for step (S1) is 0.5-3 hours; preferably about 1-2 hours.
[0028] In another preferred embodiment, in step (S1), compound I is first dissolved with pyridine; preferably, the mass-to-volume ratio of compound I to pyridine is 1-3%; more preferably, about 2.5%.
[0029] In another preferred embodiment, the mass-to-volume ratio of compound I and 2-chloro-1,3,2-benzodioxophosphazene-4-one in step (S1) is 5:5-6; preferably about 5:5.16.
[0030] In another preferred embodiment, step (S2) is carried out in an inert solvent; preferably, the inert solvent comprises anhydrous N,N-dimethylformamide and N,N-diisopropylethylamine; preferably, the volume ratio of anhydrous N,N-dimethylformamide and N,N-diisopropylethylamine is 75:20-35 (e.g., 75:25).
[0031] In another preferred embodiment, the reaction temperature of step (S2) is -20°C to 20°C; preferably about -10°C to 20°C; more preferably about -5°C to 15°C.
[0032] In another preferred embodiment, in step (S2), tri-n-butylammonium pyrophosphate is first dissolved in an inert solvent; preferably, the mass-volume ratio of tri-n-butylammonium to inert solvent is 20%-40%; preferably, the solution is pre-cooled at -20°C after dissolution.
[0033] In another preferred embodiment, step (S3) is carried out in an aqueous solvent; preferably, the aqueous solvent comprises water and pyridine; preferably, the volume ratio of water to pyridine is 10:400-600 (e.g., 10:490).
[0034] In another preferred embodiment, the reaction temperature of step (S3) is -10°C to 20°C; preferably about -5°C to 20°C; more preferably about -5°C to 15°C.
[0035] In another preferred embodiment, in step (S3), iodine is first dissolved in an aqueous solvent; preferably, the mass-volume ratio of iodine to aqueous solvent is 0.5%-4%; preferably, the solution is pre-cooled at 10°C after dissolution.
[0036] In another preferred embodiment, after the reaction in step (S3) is completed, ammonia is added to quench the reaction.
[0037] In another preferred embodiment, the method further includes a step of purifying compound IV.
[0038] In another preferred embodiment, the method includes the steps of:
[0039] (1) Weigh 5g of 2'-acetyl adenosine (acyA), add it to the reactor, and insert a thermometer to monitor the temperature;
[0040] (2) Add 200 ml of anhydrous pyridine to the reactor to dissolve 2'-acetyl adenosine, then add 500 ml of dioxane, replace with N2, and pre-cool at -5℃ for 10 min.
[0041] (3) Weigh 5.16g of 2-chloro-1,3,2-benzodioxophosphazene-4-one, dissolve it in 20mL of dioxane, slowly add it to the reactor, control the reaction temperature to not exceed 0℃, and react for 1.5h;
[0042] (4) Weigh 27.5g of tri-n-butylammonium pyrophosphate, add 75ml of anhydrous N,N-dimethylformamide and 25ml of N,N-diisopropylethylamine, and after the tri-n-butylammonium pyrophosphate is dissolved, pre-cool at -20℃ for 1.5h. After the reaction in (3) is completed, quickly add the dissolved mixed solution into the reactor and react for 10min.
[0043] (5) After the reaction in (4) is completed, weigh 5.5g of iodine, dissolve it in 490mL of pyridine and 10mL of water, and add it to the reactor. React for 30min.
[0044] (6) Take 5000ml of ammonia solution to quench the reaction. After quenching, extract with 1000ml of DCM, separate the liquid and obtain an aqueous solution. After rotary evaporation, obtain the target compound.
[0045] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description
[0046] Figure 1 The HPLC detection results of Example 1 are shown;
[0047] Figure 2 The HPLC detection results of Example 2 are shown;
[0048] Figure 3 The HPLC detection results of Example 3 are shown. Detailed Implementation
[0049] This invention relates to the field of pharmaceutical and chemical technology, specifically to a method for synthesizing acetyl-adenosine triphosphate (AAP), a raw material for the development of biological reagent kits. The method of this invention makes the synthesis of AAP more efficient and stable, achieving a conversion rate of approximately 85%, and maintaining a purity of over 80% in the prepared AAP.
[0050] Before describing this invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be limiting; the scope of the invention will be limited only by the appended claims.
[0051] 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 pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. 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.).
[0052] While any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention, preferred methods and materials are exemplified herein.
[0053] The compound structure of acetyl adenosine triphosphate involved in this invention is as follows:
[0054]
[0055] In a preferred embodiment of the present invention, the method for synthesizing acetyl-adenosine triphosphate according to the present invention includes the following steps:
[0056] (1) Weigh 5g of 2'-acetyl adenosine (acyA) and add it to a 2000ml three-necked flask. Insert a thermometer to monitor the temperature.
[0057] (2) Add 200ml of anhydrous pyridine to dissolve acyA, then add 500ml of dioxane, replace with N2, and pre-cool at -5℃ for 10min;
[0058] (3) Take a 150ml single-necked flask, weigh 5.16g of 2-chloro-1,3,2-benzodioxophosphazenecyclohexane-4-one, dissolve it in 20mL of dioxane, and slowly add it to the system. Observe the thermometer to ensure that the reaction temperature does not exceed 0℃. After the addition is complete, react for 1.5h;
[0059] (4) Take a 150ml single-necked bottle, weigh 27.5g of tri-n-butylammonium pyrophosphate into the single-necked bottle, add 75ml of anhydrous N,N-dimethylformamide and 25ml of N,N-diisopropylethylamine, and after the tri-n-butylammonium pyrophosphate is dissolved, pre-cool it at -20℃ for 1.5h. After the reaction in (3) is completed, quickly add the dissolved mixed solution into the three-necked bottle and react for 10min.
[0060] (5) After the reaction in (4) is completed, take a 1000ml single-necked flask, weigh 5.5g of iodine, dissolve it in 490mL of pyridine and 10mL of water, and quickly add the dissolved mixture into a three-necked flask. React for 30min.
[0061] (6) Take 5000ml of ammonia solution to quench the reaction. After quenching, extract with 1000ml of DCM, separate the liquid, and obtain an aqueous solution by rotary evaporation.
[0062] The main advantages of this invention are:
[0063] (1) The method of the present invention has a high conversion rate of the target product, which can reach about 85%.
[0064] (2) The target product obtained by the method of the present invention has high purity, which can reach more than 80%.
[0065] (3) The method of the present invention has good reaction stability and high repeatability in multiple batches, making it suitable for industrial production.
[0066] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Manual* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents used in the following embodiments are commercially available.
[0067] Example 1
[0068] At room temperature, weigh 5g of 2'-acetyladenosine (acyA) and add it to a 2000ml three-necked flask. Insert a thermometer to monitor the temperature, add a magnetic stir bar, then add 200ml of anhydrous pyridine to dissolve acyA, and then add 500ml of dioxane. After purging with N2, place the flask in a low-temperature cold bath with a magnetic stirrer, set the temperature to -5℃, and pre-cool for 10min. Take a 50ml single-necked flask, weigh 5.16g of 2-chloro-1,3,2-benzodioxane-4-one into the flask, add 20mL of dioxane to dissolve it, and when the thermometer shows -5℃, use a syringe to draw the dissolved 2-chloro-1,3,2-benzodioxane-4-one mixture and slowly add it to the reaction system. Observe the thermometer and control the reaction temperature to not exceed 0℃. After the addition is complete, react for 1.5h.
[0069] The reaction phenomenon is that the reaction solution changes from colorless and transparent to pale yellow, and white mist is generated above the solution in the bottle. As the reaction time increases, the white mist becomes less dense and eventually disappears.
[0070] Take a 150ml single-necked flask, weigh 27.5g of tri-n-butylammonium pyrophosphate into the flask, add 75ml of anhydrous N,N-dimethylformamide and 25ml of N,N-diisopropylethylamine. After the tri-n-butylammonium pyrophosphate is completely dissolved, pre-cool it at -20℃ for 1.5h. After the above pale yellow solution has reacted, quickly add the dissolved tri-n-butylammonium pyrophosphate mixture to a three-necked flask using a syringe and react for 10min.
[0071] The reaction phenomenon is that the temperature of the reaction system rises rapidly from -5℃ to about 15℃, the pale yellow solution turns into a yellow solution, and white fumes reappear above the solution in the bottle. As the reaction time increases, the white fumes gradually become fainter and eventually disappear.
[0072] After the reaction is complete, take a 1000ml single-necked flask, weigh 5.5g of iodine, dissolve it in 490mL of pyridine and 10mL of water, and quickly add the dissolved mixture to a three-necked flask. React for 30min.
[0073] Take 5000 ml of ammonia solution to quench the reaction. After quenching, extract with 1000 ml of DCM (dichloromethane), separate the solution using a separatory funnel, and then rotary evaporate to obtain the target product.
[0074] HPLC analysis showed a purity of 80.31% and a calculated yield of 84.8%. The HPLC results are as follows: Figure 1 As shown.
[0075] Example 2
[0076] At room temperature, weigh 5g of 2'-acetyladenosine (acyA) and add it to a 2000ml three-necked flask. Insert a thermometer to monitor the temperature, add a magnetic stir bar, then add 250ml of anhydrous pyridine to dissolve acyA, and then add 450ml of dioxane. After purging with N2, place the flask in a low-temperature cold bath with a magnetic stirrer, set the temperature to -5℃, and pre-cool for 10min. Take a 50ml single-necked flask, weigh 5.16g of 2-chloro-1,3,2-benzodioxane-4-one into the flask, add 20mL of dioxane to dissolve it, and when the thermometer shows -5℃, use a syringe to draw the dissolved 2-chloro-1,3,2-benzodioxane-4-one mixture and slowly add it to the reaction system. Observe the thermometer and control the reaction temperature to not exceed 0℃. After the addition is complete, react for 1.5h.
[0077] The reaction phenomenon is that the reaction solution changes from colorless and transparent to pale yellow, and white mist is generated above the solution in the bottle. As the reaction time increases, the white mist becomes less dense and eventually disappears.
[0078] Take a 150ml single-necked flask, weigh 27.5g of tri-n-butylammonium pyrophosphate into the flask, add 75ml of anhydrous N,N-dimethylformamide and 35ml of N,N-diisopropylethylamine. After the tri-n-butylammonium pyrophosphate is completely dissolved, pre-cool it at -20℃ for 1.5h. After the above pale yellow solution has reacted, quickly add the dissolved tri-n-butylammonium pyrophosphate mixture to a three-necked flask using a syringe and react for 10min.
[0079] The reaction phenomenon is that the temperature of the reaction system rises rapidly from -5℃ to about 15℃, the pale yellow solution turns into a yellow solution, and white fumes reappear above the solution in the bottle. As the reaction time increases, the white fumes gradually become fainter and eventually disappear.
[0080] After the reaction is complete, take a 1000ml single-necked flask, weigh 5.5g of iodine, dissolve it in 500mL of pyridine and 10mL of water, and quickly add the dissolved mixture to the three-necked flask. React for 30min.
[0081] Take 5000 ml of ammonia solution to quench the reaction. After quenching, extract with 1000 ml of DCM (dichloromethane), separate the solution using a separatory funnel, and then rotary evaporate to obtain the target product.
[0082] HPLC analysis showed a purity of 83.35% and a calculated yield of 85.2%. The HPLC results are as follows: Figure 2 As shown.
[0083] Example 3
[0084] At room temperature, weigh 5g of 2'-acetyladenosine (acyA) and add it to a 2000ml three-necked flask. Insert a thermometer to monitor the temperature, add a magnetic stir bar, then add 250ml of anhydrous pyridine to dissolve acyA, and then add 600ml of dioxane. After purging with N2, place the flask in a low-temperature cold bath with a magnetic stirrer, set the temperature to -5℃, and pre-cool for 10min. Take a 50ml single-necked flask, weigh 5.16g of 2-chloro-1,3,2-benzodioxane-4-one into the flask, add 20mL of dioxane to dissolve it, and when the thermometer shows -5℃, use a syringe to draw the dissolved 2-chloro-1,3,2-benzodioxane-4-one mixture and slowly add it to the reaction system. Observe the thermometer and control the reaction temperature to not exceed 0℃. After the addition is complete, react for 1.5h.
[0085] The reaction phenomenon is that the reaction solution changes from colorless and transparent to pale yellow, and white mist is generated above the solution in the bottle. As the reaction time increases, the white mist becomes less dense and eventually disappears.
[0086] Take a 150ml single-necked flask, weigh 27.5g of tri-n-butylammonium pyrophosphate into the flask, add 75ml of anhydrous N,N-dimethylformamide and 20ml of N,N-diisopropylethylamine. After the tri-n-butylammonium pyrophosphate is completely dissolved, pre-cool it at -20℃ for 1.5h. After the above pale yellow solution has reacted, quickly add the dissolved tri-n-butylammonium pyrophosphate mixture to a three-necked flask using a syringe and react for 10min.
[0087] The reaction phenomenon is that the temperature of the reaction system rises rapidly from -5℃ to about 15℃, the pale yellow solution turns into a yellow solution, and white fumes reappear above the solution in the bottle. As the reaction time increases, the white fumes gradually become fainter and eventually disappear.
[0088] After the reaction is complete, take a 1000ml single-necked flask, weigh 5.5g of iodine, dissolve it in 550mL of pyridine and 10mL of water, and quickly add the dissolved mixture to the three-necked flask. React for 30min.
[0089] Take 5000 ml of ammonia solution to quench the reaction. After quenching, extract with 1000 ml of DCM (dichloromethane), separate the solution using a separatory funnel, and then rotary evaporate to obtain the target product.
[0090] HPLC analysis showed a purity of 84.09% and a calculated yield of 84.1%. The HPLC results are as follows: Figure 3 As shown.
[0091] Example 4
[0092] At room temperature, weigh 5g of 2'-acetyladenosine (acyA) and add it to a 2000ml three-necked flask. Insert a thermometer to monitor the temperature, add a magnetic stir bar, then add 100ml of anhydrous pyridine to dissolve acyA, and then add 300ml of dioxane. After purging with N2, place the flask in a low-temperature cold bath with a magnetic stirrer, set the temperature to -5℃, and pre-cool for 10min. Take a 50ml single-necked flask, weigh 5.16g of 2-chloro-1,3,2-benzodioxane-4-one into the flask, add 20mL of dioxane to dissolve it, and when the thermometer shows -5℃, use a syringe to draw the dissolved 2-chloro-1,3,2-benzodioxane-4-one mixture and slowly add it to the reaction system. Observe the thermometer and control the reaction temperature to not exceed 0℃. After the addition is complete, react for 1.5h.
[0093] The reaction phenomenon is that the reaction solution changes from colorless and transparent to pale yellow, and white mist is generated above the solution in the bottle. As the reaction time increases, the white mist becomes less dense and eventually disappears.
[0094] Take a 150ml single-necked flask, weigh 27.5g of tri-n-butylammonium pyrophosphate into the flask, add 75ml of anhydrous N,N-dimethylformamide and 20ml of N,N-diisopropylethylamine. After the tri-n-butylammonium pyrophosphate is completely dissolved, pre-cool it at -20℃ for 1.5h. After the above pale yellow solution has reacted, quickly add the dissolved tri-n-butylammonium pyrophosphate mixture to a three-necked flask using a syringe and react for 10min.
[0095] The reaction phenomenon is that the temperature of the reaction system rises rapidly from -5℃ to about 15℃, the pale yellow solution turns into a yellow solution, and white fumes reappear above the solution in the bottle. As the reaction time increases, the white fumes gradually become fainter and eventually disappear.
[0096] After the reaction is complete, take a 1000ml single-necked flask, weigh 5.5g of iodine, dissolve it in 550mL of pyridine and 10mL of water, and quickly add the dissolved mixture to the three-necked flask. React for 30min.
[0097] Take 5000 ml of ammonia solution to quench the reaction. After quenching, extract with 1000 ml of DCM (dichloromethane), separate the solution using a separatory funnel, and then rotary evaporate to obtain the target product.
[0098] HPLC analysis showed a purity of 75.89% and a calculated yield of 79.3%.
[0099] Example 5
[0100] At room temperature, weigh 5g of 2'-acetyladenosine (acyA) and add it to a 2000ml three-necked flask. Insert a thermometer to monitor the temperature, add a magnetic stir bar, then add 250ml of anhydrous pyridine to dissolve acyA, and then add 600ml of dioxane. After purging with N2, place the flask in a low-temperature cold bath with a magnetic stirrer, set the temperature to -5℃, and pre-cool for 10min. Take a 50ml single-necked flask, weigh 5.16g of 2-chloro-1,3,2-benzodioxane-4-one into the flask, add 20mL of dioxane to dissolve it, and when the thermometer shows -5℃, use a syringe to draw the dissolved 2-chloro-1,3,2-benzodioxane-4-one mixture and slowly add it to the reaction system. Observe the thermometer and control the reaction temperature to not exceed 0℃. After the addition is complete, react for 1.5h.
[0101] The reaction phenomenon is that the reaction solution changes from colorless and transparent to pale yellow, and white mist is generated above the solution in the bottle. As the reaction time increases, the white mist becomes less dense and eventually disappears.
[0102] Take a 150ml single-necked flask, weigh 27.5g of tri-n-butylammonium pyrophosphate into the flask, add 75ml of anhydrous N,N-dimethylformamide and 20ml of N,N-diisopropylethylamine. After the tri-n-butylammonium pyrophosphate is completely dissolved, pre-cool it at -20℃ for 1.5h. After the above pale yellow solution has reacted, quickly add the dissolved tri-n-butylammonium pyrophosphate mixture to a three-necked flask using a syringe and react for 10min.
[0103] The reaction phenomenon is that the temperature of the reaction system rises rapidly from -5℃ to about 15℃, the pale yellow solution turns into a yellow solution, and white fumes reappear above the solution in the bottle. As the reaction time increases, the white fumes gradually become fainter and eventually disappear.
[0104] After the reaction is complete, take a 1000ml single-necked flask, weigh 5.5g of iodine, dissolve it in 550mL of pyridine and 20mL of water, and quickly add the dissolved mixture to the three-necked flask. React for 30min.
[0105] Take 5000 ml of ammonia solution to quench the reaction. After quenching, extract with 1000 ml of DCM (dichloromethane), separate the solution using a separatory funnel, and then rotary evaporate to obtain the target product.
[0106] HPLC analysis showed a purity of 73.28% and a calculated yield of 70.6%.
[0107] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.
Claims
1. A method for preparing acetyl adenosine triphosphate (acyATP), characterized in that, The method includes the following steps: (S1) In an anhydrous solvent, compound I (2'-acetyladenosine (acyA)) reacts with 2-chloro-1,3,2-benzodioxaphosphazene-4-one to form compound II, as shown in the following reaction formula:
2. The method as described in claim 1, characterized in that, The method further includes the following steps: (S2) Compound II reacts with tri-n-butylammonium pyrophosphate to form compound III, as shown in the following reaction formula:
3. The method as described in claim 2, characterized in that, The method further includes the following steps: (S3) Compound III reacts with iodine in the presence of compound IV, as shown in the following reaction:
4. The method as described in claim 1, characterized in that, The anhydrous solvent in step (S1) is an anhydrous inert solvent; preferably, the anhydrous solvent in step (S1) includes anhydrous pyridine and dioxane.
5. The method as described in claim 1, characterized in that, The reaction is carried out under the protection of nitrogen or an inert gas.
6. The method as described in claim 1, characterized in that, In step (S1), the mass-to-volume ratio of compound I and 2-chloro-1,3,2-benzodioxophosphazenecyclohexane-4-one is 5:5-6; preferably about 5:5.
16.
7. The method as described in claim 2, characterized in that, The reaction in step (S2) is carried out in an inert solvent; preferably, the inert solvent comprises anhydrous N,N-dimethylformamide and N,N-diisopropylethylamine; preferably, the volume ratio of anhydrous N,N-dimethylformamide and N,N-diisopropylethylamine is 75:20-35 (e.g., 75:25).
8. The method as described in claim 3, characterized in that, The reaction in step (S3) is carried out in an aqueous solvent; preferably, the aqueous solvent includes water and pyridine; preferably, the volume ratio of water to pyridine is 10:400-600 (e.g., 10:490).
9. The method as described in claim 8, characterized in that, After the reaction in step (S3) is completed, ammonia water is added to quench the reaction.
10. The method as described in claim 1, characterized in that, The method includes the following steps: (1) Weigh 5g of 2'-acetyl adenosine (acyA), add it to the reactor, and insert a thermometer to monitor the temperature; (2) Add 200 ml of anhydrous pyridine to the reactor to dissolve 2'-acetyl adenosine, then add 500 ml of dioxane, replace with N2, and pre-cool at -5℃ for 10 min. (3) Weigh 5.16g of 2-chloro-1,3,2-benzodioxophosphazene-4-one, dissolve it in 20mL of dioxane, slowly add it to the reactor, control the reaction temperature to not exceed 0℃, and react for 1.5h; (4) Weigh 27.5g of tri-n-butylammonium pyrophosphate, add 75ml of anhydrous N,N-dimethylformamide and 25ml of N,N-diisopropylethylamine, and after the tri-n-butylammonium pyrophosphate is dissolved, pre-cool at -20℃ for 1.5h. After the reaction in (3) is completed, quickly add the dissolved mixed solution into the reactor and react for 10min. (5) After the reaction in (4) is completed, weigh 5.5g of iodine, dissolve it in 490mL of pyridine and 10mL of water, and add it to the reactor. React for 30min. (6) Take 5000ml of ammonia solution to quench the reaction. After quenching, extract with 1000ml of DCM, separate the liquid and obtain an aqueous solution. After rotary evaporation, obtain the target compound.