Synthesis method of acetylated guanosine triphosphate

Abstract

The invention provides the synthesis method of acetylated guanosine triphosphate, the acetylated guanosine triphosphate can be efficiently prepared by using the method provided by the invention, the stability of batch-to-batch production is good, the conversion rate of the acetylated guanosine triphosphate is high, the purity of a target product is high, and the method is suitable for large-scale industrial preparation.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry, and particularly relates to a method for synthesizing acetylated guanosine triphosphate. Background Art

[0002] AcyNTPs are used as PCR chain terminators, and the furanose moiety is replaced by 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 acetyl guanosine triphosphate is a great challenge because an acetyl guanosine triphosphate has multiple chemical groups, such as primary hydroxyl, secondary hydroxyl, and amino groups, etc. Their activities and selectivities are different during synthesis, which will result in a large number of by-products and pose a great problem for purification.

[0004] Therefore, those skilled in the art are committed to developing a preparation process for acetyl guanosine triphosphate (acyGTP) 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 synthesizing acetylated guanosine triphosphate.

[0006] In the first aspect of the present invention, a method for preparing acetyl guanosine triphosphate (acyGTP) is provided. The method includes the steps:

[0007] (S1) In an aqueous solvent, compound I (N-acetyl guanosine (acyG)) reacts with phosphorus oxychloride to form compound II. 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. The reaction formula is as follows:

[0011]

[0012] In another preferred example, the aqueous solvent in step (S1) is an inert aqueous 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 100 - 200 parts by volume.

[0019] In another preferred example, the aqueous solvent includes:

[0020] Trimethyl phosphate 800 parts by volume;

[0021] Water 3 parts by volume;

[0022] Tri-n-butylamine 125 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 6%.

[0027] In another preferred example, the mass-to-volume ratio (grams: milliliters) of compound I to phosphorus oxychloride in step (S1) is 50:50 - 100; preferably about 50:75.

[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 750:500 - 1000 (such as 750:600).

[0029] In another preferred example, the reaction temperature of step (S2) is -20°C to 20°C; preferably about -15°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 10%-30%; 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 of:

[0034] (1) Weigh 50 g of N-acetylguanosine (acyG), add it to a reactor, and insert a thermometer for temperature monitoring;

[0035] (2) Add 800 mL of trimethyl phosphate to dissolve N-acetylguanosine, then add 3 mL of water and 125 mL of tri-n-butylamine. After replacing with N2, pre-cool it at -5°C for 10 min;

[0036] (3) Slowly add 75 mL of phosphorus oxychloride, control the reaction temperature not to exceed 0°C, and react for 1.5 h;

[0037] (4) Weigh 250 g of tri-n-butylammonium pyrophosphate in a single-necked flask, add 750 mL of anhydrous acetonitrile and 600 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 1000 mL of DCM, separate the liquid to obtain an aqueous solution, and then add 600 mL of 2M 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 repeated 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 3Shows the HPLC detection results of the method of Example 3. Detailed implementation

[0043] The present invention provides a method for synthesizing acetylated guanosine triphosphate. The method of the present invention has a higher efficiency in synthesizing acetylated guanosine triphosphate, a stable process, and is suitable for large-scale preparation of acetylated guanosine triphosphate.

[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 can 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 acetylated guanosine triphosphate of the present invention, the method comprises the steps of:

[0048] (1) Weigh 50 g of N-acetylguanosine (acyG), add it to a reactor, and insert a thermometer for temperature monitoring;

[0049] (2) Add 800 mL of trimethyl phosphate to dissolve N-acetylguanosine, then add 3 mL of water and 125 mL of tri-n-butylamine. After N2 replacement, pre-cool it at -5 °C for 10 min;

[0050] (3) Slowly add 75 mL of phosphorus oxychloride, control the reaction temperature not to exceed 0 °C, and react for 1.5 h;

[0051] (4) Weigh 250 g of tri-n-butylammonium pyrophosphate into a single-neck flask, add 750 mL of anhydrous acetonitrile and 600 mL of tri-n-butylamine. After the 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;

[0052] After the reaction of (4) is completed, 6000 mL of aqueous solution is taken to quench the reaction. After quenching, it is extracted with 1000 mL of DCM, separated, and the aqueous solution is obtained. Then, 600 mL of 2 M TEAB solution is added to the aqueous solution, and the target compound is obtained after rotary evaporation.

[0053] The main advantages of the present invention are as follows:

[0054] (1) The conversion rate of preparing acetylguanosine triphosphate (acyGTP) by the method of the present invention is high, and the conversion rate can reach about 80%.

[0055] (2) The method of the present invention has good stability between batches in the multi-batch preparation of acetylguanosine triphosphate (acyGTP), and the product has high purity, 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 specific conditions noted in the following embodiments are usually carried out according to the conditions described in the conventional conditions such as "Molecular Cloning: A Laboratory Manual" (translated by Huang Peitang et al., Beijing: Science Press, 2002) written 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 embodiments can be obtained from commercial channels without special instructions.

[0057] Example 1

[0058] At room temperature, 50 g of N-acetylguanosine (acyG) is weighed and added to a 5000 mL three-necked flask. A thermometer is inserted for temperature monitoring, a magnetic stirrer is added, and then 800 mL of trimethyl phosphate is added to dissolve acyG. Then, 3 mL of water and 125 mL of tributylamine are added. After purging with N2, it is placed in a low-temperature cooling bath magnetic stirrer, the temperature is set to -5 °C, and it is pre-cooled for 10 min. When the temperature in the thermometer shows -5 °C, 75 mL of phosphorus oxychloride is 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.

[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 flask. 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 250 g of tributylammonium pyrophosphate into the single-necked flask, add 750 mL of anhydrous acetonitrile and 600 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 reaction is completed, draw the clarified tributylammonium pyrophosphate mixed solution through a syringe and quickly add it to the three-necked flask, and react for 10 min.

[0061] 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 again above the solution in the flask. As the reaction time prolongs, the white mist turns from thick to light until it disappears.

[0062] After the reaction is completed, take 6000 mL of aqueous solution to quench the reaction. After quenching, extract with 1000 mL of DCM (dichloromethane), separate with a separating funnel 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.

[0063] Detected by HPLC, the purity is 65.38%. Calculated based on N-acetylguanosine, the yield is 84.8%. The HPLC detection results are as Figure 1 shown.

[0064] Example 2

[0065] At room temperature, weigh 50 g of N-acetylguanosine (acyG), add it to a 5000 mL three-necked flask, insert a thermometer for temperature monitoring, add a magnetic stirrer, then add 900 mL of trimethyl phosphate to dissolve acyG, and then add 3.5 mL of water and 130 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, draw 75 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.

[0066] 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 prolongs, the white mist turns from thick to light until it disappears.

[0067] Take a 2000 mL single-necked flask, weigh 250 g of tributylammonium pyrophosphate into the single-necked flask, add 750 mL of anhydrous acetonitrile and 600 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 reaction is completed, draw the clarified tributylammonium pyrophosphate mixed solution through a syringe and quickly add it to the three-necked flask, and react 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 mist appeared above the solution in the bottle again. As the reaction time extended, the white mist became thicker and then thinner until it disappeared.

[0069] 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 separating funnel to obtain the 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.

[0070] Detected by HPLC, the purity was 65.46%. Calculated based on N-acetylguanosine, the yield was 83.2%. The HPLC detection results were as Figure 2 shown.

[0071] Example 3

[0072] At room temperature, 50 g of N-acetylguanosine (acyG) 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, 700 mL of trimethyl phosphate was added to dissolve acyG, followed by 2.5 mL of water and 110 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 and pre-cooled for 10 min. When the temperature in the thermometer showed -5°C, 75 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.

[0073] 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.

[0074] Take a 2000 mL single-necked flask, weigh 250 g of tri-n-butylammonium pyrophosphate in the single-necked flask, add 800 mL of anhydrous acetonitrile and 550 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.

[0075] 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 mist appeared above the solution in the bottle again. As the reaction time extended, the white mist became thicker and then thinner until it disappeared

[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 separatory 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 66.57%. Calculated based on N-acetylguanosine, the yield is 85.1%. The HPLC detection results are as Figure 3 shown.

[0078] Example 4

[0079] At room temperature, weigh 50 g of N-acetylguanosine (acyG) and add it to a 5000 mL three-necked flask. Insert a thermometer for temperature monitoring, add a magnetic stirrer, then add 800 mL of trimethyl phosphate to dissolve acyG, and then add 3 mL of water and 130 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 75 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 changes from thick to light until it disappears.

[0081] Take a 2000 mL single-necked flask, weigh 250 g of tri-n-butylammonium pyrophosphate in the single-necked flask, add 750 mL of anhydrous acetonitrile and 550 mL of tri-n-butylamine. After the tri-n-butylammonium pyrophosphate is dissolved and clarified, place it in a -20 °C bath and pre-cool for 1.5 h. After the above light yellow solution reaction is completed, draw the clarified tri-n-butylammonium 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 changes from thick to light until it disappears

[0083] 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 separatory 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.

[0084] Detected by HPLC, the purity is 65.36%. Calculated based on N-acetylguanosine, the yield is 81.7%.

[0085] All documents mentioned in this invention are cited herein for reference as if each individual document was cited for reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for preparing acetyl guanosine triphosphate (acyGTP), characterized in that, The method includes the steps of: (S1) In an aqueous solvent, compound I (N-acetylguanosine (acyG)) 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 of: (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 41, wherein The aqueous solvent includes: 500-1000 parts by volume of trimethyl phosphate; 1-5 parts by volume of water; 100-200 parts by volume of tributylamine.

7. The method according to claim 2, wherein The reaction in the step (S2) is carried out in an inert solvent; preferably, the inert solvent includes anhydrous acetonitrile and tributylamine.

8. The method according to claim 7, wherein In the step (S2), tributylammonium pyrophosphate is first dissolved in an inert solvent; preferably, the mass-volume ratio of tributylammonium to the inert solvent is 10%-30%.

9. The method according to claim 2, wherein After the reaction in the step (S2) is completed, water is added to quench the reaction.

10. The method according to claim 2, wherein The method further includes the step of purifying compound III.

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