A method for synthesizing acetylated deoxyguanosine triphosphate
The one-pot, three-step synthesis of acetylated deoxyguanosine triphosphate solves the problems of low conversion rate and numerous by-products in existing technologies, enabling the industrial production of high-purity and stable acetylated deoxyguanosine triphosphate.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DAAN GENE CO LTD
- Filing Date
- 2024-12-03
- Publication Date
- 2026-06-05
AI Technical Summary
In the existing technology, the synthesis of acetylated deoxyguanosine triphosphate has problems such as low reaction conversion rate, high by-product content and unstable process, making it difficult to achieve large-scale industrial production.
A one-pot, three-step method was used to synthesize acetylated deoxyguanosine triphosphate, which involved reacting compound I with 2-chloro-1,3,2-benzodioxophosphazene-4-one in an anhydrous solvent to generate compound II, followed by reaction with tri-n-butylammonium pyrophosphate to generate compound III, and then generating compound IV in the presence of iodine. The purity was improved by purification steps.
The synthesis of acetylated deoxyguanosine triphosphate with high yield and high purity was achieved, with product purity exceeding 80%. The reaction showed good stability in multiple batches and is suitable for industrial production.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biochemistry, specifically relating to a method for synthesizing acetylated deoxyguanosine triphosphate. Background Technology
[0002] AcyNTPs, used as PCR chain terminators, have their furanose moiety replaced by a 2-hydroxyethoxymethyl group. This allows for a wider range of applications. The error rate of acyNTP substrates is significantly lower than that of corresponding dNTP or ddNTP substrates. Therefore, they are of great value in applications where dideoxynucleotides are commonly used, such as in DNA sequencing and SNP detection. AcyNTPs can also be used as PCR chain terminators in time-of-flight mass spectrometry platforms.
[0003] Currently, the large-scale synthesis of acetylated deoxyguanosine triphosphate remains a significant challenge because an acetylated deoxyguanosine 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 acetylated deoxyguanosine 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 acetylated deoxyguanosine triphosphate (acyGTP) with high yield, high product purity, and stable process.
[0007] In a first aspect of the present invention, a method for preparing acetylated deoxyguanosine triphosphate (acyGTP) is provided, the method comprising the steps of:
[0008] (S1) In an anhydrous solvent, compound I (2'-acetylguanosine (acyG)) 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) Compound III reacts with iodine in the presence of 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] 500 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 1-5%; 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 75:15-35 (e.g., 75:25).
[0031] In another preferred embodiment, the reaction temperature of step (S2) is -20°C to 20°C; preferably about -5°C to 15°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 27.5%); 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 solvent comprises water and pyridine; preferably, the volume ratio of water to pyridine is 10:450-550 (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'-acetylguanosine (acyG), add it to the reactor, and insert a thermometer to monitor the temperature;
[0040] (2) Add 200ml of anhydrous pyridine to dissolve 2'-acetylguanosine, then add 500ml of dioxane, replace with N2, and pre-cool at -5℃ for 10min;
[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 into a single-necked flask, 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 step (3) is completed, add the tri-n-butylammonium pyrophosphate solution into the reactor and react for 10min.
[0043] (5) Weigh 5.5g of iodine and dissolve it in 490mL of pyridine and 10mL of water. After the reaction in step (4) is completed, add the dissolved iodine solution into the reactor and react for 30min.
[0044] (6) Take 5000 ml of ammonia solution and add it to the reactor to quench the reaction. After quenching, extract with 1000 ml of DCM to 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 acetylated deoxyguanosine triphosphate. The method of this invention produces a high-purity acetylated guanosine triphosphate, with an HPLC purity exceeding 80%. Furthermore, even with multiple batches of feed, the conversion rate of acetylated guanosine triphosphate remains stable at around 85%.
[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 acetylguanosine triphosphate involved in this invention is as follows:
[0054]
[0055] In a preferred embodiment of the present invention, the method for synthesizing acetylguanosine triphosphate according to the present invention includes the following steps:
[0056] (1) Weigh 5g of 2'-acetylguanosine (acyG) 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 acyG, 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] After the reaction (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] Take 5000ml of ammonia solution to quench the reaction. After quenching, extract with 1000ml of DCM, separate the liquid, and obtain an aqueous solution, which is then rotary evaporated.
[0062] The main advantages of this invention are:
[0063] (1) The method of the present invention produces a product with high purity, which can reach about 80%.
[0064] (2) The method of the present invention has good reaction stability and high repeatability in multiple batches, and the yield of the target product is stable at over 85%, making it suitable for industrial production.
[0065] 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.
[0066] Example 1
[0067] At room temperature, weigh 5g of 2'-acetylguanosine (acyG) 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 acyG, 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.
[0068] 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.
[0069] 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.
[0070] The reaction phenomenon is that the temperature of the reaction system rises sharply from -5℃ to about 15℃, the pale yellow solution turns into a yellow solution, and white mist reappears above the solution in the bottle. As the reaction time increases, the white mist becomes less dense and eventually disappears.
[0071] 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.
[0072] 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.
[0073] HPLC analysis showed a purity of 82.98% and a yield of 86.2%. The HPLC results are as follows: Figure 1 As shown.
[0074] Example 2
[0075] At room temperature, weigh 5g of 2'-acetylguanosine (acyG) 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 acyG, 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.
[0076] 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.
[0077] Take a 150ml single-necked flask, weigh 27.5g of tri-n-butylammonium pyrophosphate into the flask, add 70ml of anhydrous N,N-dimethylformamide and 30ml 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.
[0078] 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.
[0079] After the reaction is complete, take a 1000ml single-necked flask, weigh 5.5g of iodine, dissolve it in 450mL of pyridine and 10mL of water, and quickly add the dissolved mixture to the three-necked flask. React for 30min.
[0080] 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.
[0081] HPLC analysis showed a purity of 78.04% and a yield of 88.4%. The HPLC results are as follows: Figure 2 As shown.
[0082] Example 3
[0083] At room temperature, weigh 5g of 2'-acetylguanosine (acyG) and add it to a 2000ml three-necked flask. Insert a thermometer to monitor the temperature, add a magnetic stir bar, then add 300ml of anhydrous pyridine to dissolve acyG, and then add 800ml 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 30mL 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.
[0084] 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.
[0085] 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 15ml 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.
[0086] 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.
[0087] After the reaction is complete, take a 1000ml single-necked flask, weigh 5.5g of iodine, dissolve it in 520mL of pyridine and 10mL of water, and quickly add the dissolved mixture to the three-necked flask. React for 30min.
[0088] 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.
[0089] HPLC analysis showed a purity of 80.96% and a yield of 85.9%. The HPLC results are as follows: Figure 3 As shown.
[0090] Example 4
[0091] At room temperature, weigh 5g of 2'-acetylguanosine (acyG) 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 acyG, and then add 800ml 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-benzodioxophosphazenecyclohexane-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-benzodioxophosphazenecyclohexane-4-one mixed solution 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.
[0092] 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.
[0093] 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.
[0094] The reaction phenomenon is that the temperature of the reaction system rises sharply from -5℃ to about 15℃, the pale yellow solution turns into a yellow solution, and white mist reappears above the solution in the bottle. As the reaction time increases, the white mist becomes less dense and eventually disappears.
[0095] After the reaction is complete, take a 1000ml single-necked flask, weigh 5.5g of iodine, dissolve it in 450mL of pyridine and 10mL of water, and quickly add the dissolved mixture to the three-necked flask. React for 30min.
[0096] 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.
[0097] HPLC analysis showed a purity of 80.06% and a yield of 85.0%.
[0098] 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 acetylated deoxyguanosine triphosphate, characterized in that, The method includes the following steps: (S1) In an anhydrous solvent, compound I reacts with 2-chloro-1,3,2-benzodioxophosphazene-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.
5. The method as described in claim 4, characterized in that, The anhydrous solvent in step (S1) includes pyridine and dioxane.
6. The method as described in claim 5, characterized in that, The anhydrous solvent includes: 100-300 parts by volume of pyridine; and Dioxane 300-800 parts by volume.
7. The method as described in claim 1, characterized in that, The reaction is carried out under the protection of nitrogen or an inert gas.
8. 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 includes anhydrous N,N-dimethylformamide and N,N-diisopropylethylamine.
9. The method as described in claim 3, characterized in that, The reaction is carried out in an aqueous solvent; preferably, the solvent includes water and pyridine.
10. The method as described in claim 3, characterized in that, After the reaction in step (S3) is completed, ammonia water is added to quench the reaction.