Preparation method of dideoxy open-ring ribonucleotide triphosphate and intermediate

Abstract

The present invention relates to a method for preparing a triphosphate dideoxy ring-opening ribonucleoside and an intermediate acetoxy dideoxy ring-opening ribonucleoside, comprising the following steps: (2-acetoxyethoxy)-methyl bromide and borate undergo coupling reaction, and then react with pyrimidine and derivatives or purine and derivatives to obtain acetoxy dideoxy ring-opening ribonucleoside; and then react with amino alcohol, phosphorus oxychloride, and tributylammonium pyrophosphate to obtain triphosphate dideoxy ring-opening ribonucleoside. The method of the present invention has a simple process route, high efficiency, and easy operation, and can quickly produce triphosphate dideoxy ring-opening ribonucleoside in large quantities, and is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the field of biological detection, and in particular to a preparation process of dideoxy open-ring ribonucleotide triphosphate and an intermediate. Background Art

[0002] Nucleic acid mass spectrometry technology, as an innovative platform in the field of molecular diagnosis, is developed on the basis of matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). It combines the high efficiency of multiplex polymerase chain reaction (PCR) and the high sensitivity of mass spectrometry analysis, providing strong technical support for precision medicine. Nucleic acid mass spectrometry technology effectively fills the market gap of medium-throughput detection needs with its stable and reliable detection results and low cost. This technology can not only accurately detect single nucleotide polymorphisms (SNPs) and gene mutations, but also conveniently perform DNA methylation and copy number variation (CNV) analysis, further enhancing its competitiveness in the field of genetic testing, especially showing great development potential in the field of clinical diagnosis.

[0003] The main core technology in nucleic acid mass spectrometry is single-base extension technology, which relies on ddNTP (dideoxynucleotide triphosphate) as the extension terminator, and analyzes the extension products formed by the single-base extension reaction, thereby achieving the purpose of analyzing the nucleotide sequence. The molecular weight of commercialized ddNTP is: ddATP is 297.21 Da, ddGTP is 313.2 Da, ddTTP is 288.1 Da, and ddCTP is 273.18 Da. The minimum molecular weight difference is 9.21 Da, and the resolution of several nucleic acid mass spectrometers that have been launched on the market is about 10Da. When the detected SNP is an A>T mutation, it will be found that the two peaks have a very low resolution, or even completely overlap, resulting in unsuccessful detection. This problem has become one of the technical barriers to the clinical application of nucleic acid mass spectrometry.

[0004] ACYddNTP is a modified ddNTP suitable for use in nucleic acid mass spectrometry single base extension technology. Its molecular weight difference is higher than the resolution of the mass spectrometer (10Da), which has become the main direction for solving the clinical application of nucleic acid mass spectrometry. Summary of the invention

[0005] The invention aims to provide a method for synthesizing dideoxy ring-opening ribonucleotide triphosphates, which has a simple process route, high efficiency, and is easy to operate, and can rapidly produce dideoxy ring-opening ribonucleotide triphosphates in large quantities, and is suitable for factory production.

[0006] The first aspect of the present invention provides a method for preparing acetoxy dideoxy ring-opening ribonucleoside, comprising the following steps:

[0007] (2-acetoxyethoxy)-methyl bromide is coupled with a boric acid ester and then reacted with a pyrimidine and its derivatives of formula i or a purine and its derivatives of formula ii to obtain an acetoxy dideoxy ring-opening ribonucleoside; the boric acid ester is a bis-pinacol boric acid ester or a bis-boronic acid neopentyl glycol ester;

[0008] .

[0009] In the present invention, (2-acetoxyethoxy)-methyl bromide is coupled with bis-pinacol borate to obtain (2-acetoxyethoxy)-methyl borate, and then the product is coupled with pyrimidine or purine to obtain acetoxy dideoxy open-ring ribonucleoside. In this reaction, the selectivity of the reaction can be improved by borate esterification, and the open-ring ribose is directed to react at the 1st position of pyrimidine or the 9th position of purine, thereby improving the yield and purity of the product. The reaction formula is as follows:

[0010] .

[0011] In a specific embodiment, the pyrimidine and its derivatives include cytosine, uracil, thymine and 2-thiouracil; the purine and its derivatives include guanine or acetylguanine, adenine or acetyladenine.

[0012] In a specific embodiment, the reaction is carried out in a reaction solvent, and the reaction solvent includes dioxane and / or ethylene glycol dimethyl ether.

[0013] In a specific embodiment, the reaction temperature is 60°C to reflux temperature, such as 60°C, 70°C, 80°C, 90°C, 100°C, reflux temperature. Preferably, the reaction temperature is 90°C to reflux temperature.

[0014] Research has found that the higher the reaction temperature, the more favorable it is for improving the selectivity of the product and reducing the generation of isomers. It can also shorten the reaction time, especially at reflux temperature, where the shortest reaction time can achieve a higher yield.

[0015] In a specific embodiment, the reaction is carried out under the action of a catalyst, and the catalyst is a palladium catalyst; preferably, the palladium catalyst includes at least one of palladium acetate, bis(triphenylphosphine)palladium acetate, triphenylphosphine palladium acetate, diphenylphosphinocene palladium dichloride, tetrakistriphenylphosphine palladium, dichlorobistriphenylphosphine palladium, and palladium on carbon;

[0016] In a specific embodiment, the molar ratio of the palladium acetate catalyst to the molar ratio of the pyrimidine and derivatives of formula I or the purine and derivatives of formula II is 0.05-0.15:1. For example, 0.05:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.12:1, 0.14:1, preferably 0.09-0.12:1.

[0017] The (2-acetoxyethoxy)-methyl bromide of the present invention can be obtained by common methods in the art, including but not limited to, by reacting 1,3-dioxolane with acetyl bromide.

[0018] The second aspect of the present invention provides a method for preparing dideoxy ring-opening ribonucleotide triphosphate, comprising the following steps:

[0019] (1) (2-acetoxyethoxy)-methyl bromide reacts with bis-pinacol borate through coupling reaction, and then reacts with pyrimidine and derivatives of formula I or purine and derivatives of formula II to obtain acetoxy dideoxy open-ring ribonucleoside;

[0020] (2) The acetoxy dideoxy ring-opening ribonucleoside is hydrolyzed with ammonia methanol to obtain the dideoxy ring-opening ribonucleoside;

[0021] (3) Dideoxy ring-opened nucleoside reacts with phosphorus oxychloride and tributylammonium pyrophosphate to prepare dideoxy ring-opened ribonucleotide triphosphate.

[0022] The first step of the method is the method for preparing acetoxy dideoxy ring-opening ribonucleoside according to the first aspect of the present invention.

[0023] In a specific embodiment, in order to improve the reaction efficiency and yield, the molar ratio of the acetoxy dideoxy ring-opening ribonucleoside to ammonia methanol in step (2) is 1:3-7; for example, 1:3, 1:4, 1:5, 1:6, 1:7; preferably, the molar ratio of the intermediate 1 to ammonia methanol in step (2) is 1:5.

[0024] In a specific embodiment, step (3) is carried out in trimethyl phosphate solvent.

[0025] In a specific embodiment, the reaction temperature in step (3) is 0-5°C, and the addition temperature of phosphorus oxychloride and tributylammonium pyrophosphate is -10-0°C.

[0026] The acetoxy dideoxy ring-opening ribonucleoside prepared by the above method of the present invention is applied to the preparation of dideoxy ring-opening ribonucleoside triphosphate.

[0027] The third aspect of the present invention provides the use of the above dideoxy ring-opening ribonucleotide triphosphate in the preparation of nucleic acid mass spectrometry reagents.

[0028] The present invention has achieved the following positive effects: the synthesis method of acetoxy dideoxy ring-opening ribonucleoside and triphosphate dideoxy ring-opening ribonucleotide provided by the present invention has a simple process route, high efficiency, and easy operation, and can quickly produce triphosphate dideoxy ring-opening ribonucleotide in large quantities, and is suitable for factory production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 .LC-MS chart of acetoxy dideoxy-opened acetyl cytidine prepared in Example 1.

[0030] Figure 2 . LC-MS chart of intermediate C2 obtained in Example 1-P step 2.

[0031] Figure 3 . LC-MS chart of ACYddCTP prepared in Example 1-P step 3.

[0032] Figure 4 .LC-MS graph of acetoxydideoxy-open-ring uridine prepared in Example 2.

[0033] Figure 5 .The LC-MS graph of acetoxy dideoxy-opened acetyl adenine nucleoside obtained in Example 6. DETAILED DESCRIPTION

[0034] The present invention is described in detail below in conjunction with specific implementations, but it should be noted that the protection scope of the present invention is not limited by these specific implementations and principle explanations, but is determined by the claims.

[0035] In the present invention, except for the contents explicitly described, any matters or items not mentioned are directly applicable to those known in the art without any changes. Moreover, any embodiment described herein can be freely combined with one or more other embodiments described herein, and the technical solutions or technical ideas formed thereby are regarded as part of the original disclosure or original record of the present invention, and should not be regarded as new contents not disclosed or anticipated herein, unless a person skilled in the art considers that the combination is obviously unreasonable.

[0036] All features disclosed in the present invention may be arbitrarily combined, and these combinations should be understood as contents disclosed or recorded in the present invention, unless a person skilled in the art considers that the combination is obviously unreasonable.

[0037] The numerical points disclosed in this specification include not only the numerical points specifically disclosed in the embodiments, but also the endpoints of each numerical range in the specification. Any combination of these numerical points should be regarded as the scope disclosed or recorded in the present invention.

[0038] The technical and scientific terms in the present invention shall be based on their definitions if they are defined, and shall be understood according to the general meaning in the art if no definitions are given.

[0039] At present, acetoxy dideoxy ring-opening ribonucleoside is mainly made by heating hexamethyldisilazane (HMDS), p-toluenesulfonic acid and acyclovir side chain at high temperature to make ring-opening ribose and base linking, and then through hydrolysis, triphosphate connection and other reaction steps. First, when HMDS or p-toluenesulfonic acid and acyclovir side chain are heated at high temperature to make ring-opening ribose and base linking, the efficiency is low, the reaction yield is not high, and the selectivity is poor, and by-products connected to different base positions will be produced. The properties of this by-product are similar to those of the product and are extremely difficult to remove.

[0040] In order to solve the single base extension technology and ddNTP resolution problems in the process of nucleic acid mass spectrometry detection, the present application has specially developed a preparation process for modified triphosphate dideoxy open-ring ribonucleoside and its intermediate acetoxy dideoxy open-ring ribonucleoside. In the first step of the reaction, this process creatively uses boric acid esters, such as double pinacol borate for boric acid esterification, which improves the selectivity of the reaction, and through systematic process development, improves the yield and purity of the product.

[0041] The present application provides a method for preparing an acetoxy dideoxy ring-opening ribonucleoside, comprising the following steps:

[0042] 1,3-dioxolane reacts with acetyl bromide to obtain (2-acetoxyethoxy)-methyl bromide, which is then coupled with a boric acid ester and then reacted with a pyrimidine and derivative of formula i or a purine and derivative of formula ii to obtain an acetoxy dideoxy open-ring ribonucleoside; the boric acid ester is a bis-pinacol boric acid ester or a bis-boronic acid neopentyl glycol ester;

[0043] .

[0044] The pyrimidine and its derivatives include cytosine, uracil, thymine and 2-thiouracil; the purine and its derivatives include guanine or acetylguanine, adenine or acetyladenine.

[0045] Example 1 Preparation of acetoxy dideoxy open-ring acetyl cytidine (corresponding to i-2)

[0046] Take 10g (0.135mol) of 1,3-dioxolane, add 16.6g (0.135mol) of acetyl bromide, react for 1h, add 100ml of dioxane, 0.3g (0.014mol) of palladium acetate catalyst and 34.3g (0.135mol) of bis-pinacol borate, reflux for 2h, add 15.0g (0.135mol) of cytosine and 13.8g (0.135mol) of acetic anhydride for reflux reaction, quench with water after the reaction is completed, extract with DCM (dichloromethane), and dry the organic phase to obtain 31.3g of intermediate C1, i.e., acetoxy dideoxy-opened acetyl cytidine, with a yield of 86%.

[0047] LC-MS: m / z = [M + H]: 270.0 LC-MS results are shown in Figure 1 .

[0048] The system temperature was adjusted to 60°C, 80°C, and 100°C, respectively, and the corresponding reaction time was extended until the reaction was complete, and the yields of the products were 55%, 61%, and 86%, respectively.

[0049] The different reaction conditions and results of acetoxy dideoxy ring-opening acetylcytidine are shown in Table 1.

[0050]

[0051] Example 1-P Preparation of dideoxy open-ring cytosine triphosphate nucleotide

[0052] This example takes the synthesis route of dideoxy open-ring cytosine triphosphate (ACYddCTP) as an example, and the route map is as follows:

[0053] .

[0054] Step 1: Take 10g (0.135mol) of 1,3-dioxolane, add 16.6g (0.135mol) of acetyl bromide, react for 1h, then add 100ml of dioxane, 0.3g (0.014mol) of palladium acetate catalyst and 34.3g (0.135mol) of bis-pinacol borate, reflux for 2h, add 15.0g (0.135mol) of cytosine and 13.8g (0.135mol) of acetic anhydride, reflux for reaction, add water to quench after the reaction is completed, extract with DCM (dichloromethane), and dry the organic phase to obtain 31.3g of intermediate C1 with a yield of 86%.

[0055] LC-MS: m / z=[M+H]: 269.9.

[0056] Step 2: Add 31.3 g of step 1 product into 131 ml of ammonia methanol solution, stir at room temperature for 12-20 h, monitor the reaction, and after completion, shrink the system to dryness, add ethyl acetate to slurry, filter, and dry the filter cake to obtain 17.1 g of intermediate C2 with a yield of 80%.

[0057] LC-MS: m / z = [M + H]: 186.1, LC-MS: Results see Figure 2 .

[0058] Step 3: Add 17.1g of the product from step 2 to a 1000ml three-necked flask that was dried and cooled to room temperature, then add 200ml of trimethyl phosphate and N,N-diisopropylethylamine (23.1g, 178.44mmol) to mix and dissolve. Replace nitrogen three times through a double-row tube. Start stirring the system under nitrogen protection. After 5 minutes, the system is dissolved and clarified, and then replace nitrogen three times again. Then, start cooling to -10~0℃ under nitrogen protection, and then use a constant pressure dropping funnel to slowly add phosphorus oxychloride (POCl3) (21.88g, 142.76mmol) to the system. The phosphorus oxychloride is added dropwise in about 30 minutes, and then the reaction system is slowly heated to 0℃ and reacted for 3 hours (first reaction). Wait for HPLC to detect the reaction volume. After the reaction of the raw materials is completed, the reaction system is cooled to -10~0°C, and a 50% acetonitrile solution of tributylammonium pyrophosphate (TBAP) (103.44 g, 142.76 mmol) is slowly added dropwise, and then the reaction system is maintained at 0°C for 8 hours (second reaction), and samples are taken and sent to HPLC to monitor the reaction progress until the reaction is completed; post-treatment: after the reaction is completed, the solvent is dried with a vacuum oil pump, 400 ml of water is added to the system to dissolve, and 400 ml of dichloromethane is used to backwash five times. After the aqueous phase is dried at 40°C, it is slurried again with methyl tert-butyl ether to obtain 26.97 g of an off-white solid, namely ACYddCTP, with a yield of 64.64%.

[0059] LC-MS: m / z = [M + H]: 425.9 LC-MS: Results see Figure 3 .

[0060] Example 2 Preparation of acetoxy dideoxy open-ring uridine

[0061] Take 10g (0.135mol) of 1,3-dioxolane, add 16.6g (0.135mol) of acetyl bromide, react for 1h, then add 100ml of dioxane, 0.3g (0.014mol) of palladium acetate catalyst and 34.3g (0.135mol) of bis-pinacol borate, reflux for 1h, then add 15.0g (0.135mol) of uracil for reflux reaction, detect that the reaction is complete, quench with water, extract with DCM (dichloromethane), dry the organic phase to obtain 26.4g, with a yield of 86%.

[0062] LC-MS: m / z = [M + H]: 229.0 LC-MS: Results see Figure 4 .

[0063] The system temperature was adjusted to 60°C, 80°C, and 100°C, respectively, and the corresponding reaction time was extended until the reaction was complete, and the yields of the products were 62%, 80%, and 84%, respectively.

[0064] Example 3 Preparation of acetoxy dideoxythymidine

[0065] Take 10g (0.135mol) of 1,3-dioxolane, add 16.6g (0.135mol) of acetyl bromide, react for 1h, add 100ml of dioxane, 0.3g (0.014mol) of palladium acetate catalyst and 34.3g (0.135mol) of bis-pinacol borate, reflux for 1h, add 17.0g (0.135mol) of thymine and reflux for reaction. After the reaction is completed, add water to quench, extract with DCM (dichloromethane), and dry the organic phase to obtain 26.4g, with a yield of 86%.

[0066] The system temperature was changed to 60°C, 80°C, and 100°C, respectively, and the corresponding time was extended until the reaction was complete, and the yields of the products were 48%, 72%, and 81%, respectively.

[0067] The different reaction conditions and results of acetoxy dideoxy thymidine are shown in Table 2.

[0068]

[0069] Example 4 Preparation of acetoxy dideoxy ring-opening thiouridine

[0070] Take 10g (0.135mol) of 1,3-dioxolane, add 16.6g (0.135mol) of acetyl bromide, react for 1h, add 100ml of dioxane, 0.3g (0.014mol) of palladium acetate catalyst and 34.3g (0.135mol) of bis-pinacol borate, reflux for 1h, add 17.3g (0.135mol) of 2-thiouracil and reflux for reaction. After the reaction is completed, add water to quench, extract with DCM (dichloromethane), and dry the organic phase to obtain 32.65g, with a yield of 85%.

[0071] The system temperature was changed to 60°C, 80°C, and 100°C, respectively, and the corresponding reaction time was extended until the reaction was complete, and the yields of the products were 49%, 67%, and 79%, respectively.

[0072] The different reaction conditions and results of acetoxy dideoxy ring-opening thiouridine are shown in Table 3.

[0073]

[0074] Example 5 Preparation of acetoxy dideoxy ring-opened acetylguanine nucleoside

[0075] Take 10g (0.135mol) of 1,3-dioxolane, add 16.6g (0.135mol) of acetyl bromide, react for 1h, then add 100ml of dioxane, 0.3g (0.014mol) of palladium acetate catalyst and 34.3g (0.135mol) of bis-pinacol borate, reflux for 1h, then add 26.1g (0.135mol) of acetylguanine and reflux for reaction. After the reaction is completed, add water to quench, extract with DCM (dichloromethane), and dry the organic phase to obtain 31.8g, with a yield of 76%.

[0076] When the system temperature was adjusted to 60°C, 80°C, and 100°C, the corresponding time was extended to complete the reaction, and the yields of the products were 43%, 55%, and 68%, respectively.

[0077] The different reaction conditions and results of acetoxy dideoxy ring-opening acetylguanosine nucleoside are shown in Table 4.

[0078]

[0079] Example 6 Preparation of acetoxy dideoxy ring-opened acetyl adenine nucleoside

[0080] Take 10g (0.135mol) of 1,3-dioxolane, add 16.6g (0.135mol) of acetyl bromide, react for 1h, then add 100ml of dioxane, 0.3g (0.014mol) of palladium acetate catalyst and 34.3g (0.135mol) of bis-pinacol borate, reflux for 1h, then add 23.9g (0.135mol) of acetyladenine and reflux for reaction. After the reaction is completed, add water to quench, extract with DCM (dichloromethane), and dry the organic phase to obtain 26.5g, with a yield of 67%.

[0081] LC-MS: m / z=[M+H]: 294.0LC-MS results are shown in Figure 5 .

[0082] The system temperature was adjusted to 60°C, 80°C, and 100°C, respectively, and the corresponding reaction time was extended until the reaction was complete, and the yields of the products were 34%, 45%, and 51%, respectively.

[0083] The different reaction conditions and results of acetoxy dideoxy ring-opening acetyl adenine nucleoside are shown in Table 5.

[0084]

[0085] Comparative Example 1

[0086] The difference from Example 1 is that the palladium acetate catalyst is replaced by an equal molar amount of cuprous iodide catalyst and no catalyst is added, and the remaining steps and process parameters are referred to Example 1, and the yields of the products are 30% and 0%, respectively.

[0087] Comparative Example 2

[0088] The difference from Example 1 is that the dioxane is dissolved in toluene, acetonitrile, tetrahydrofuran, etc. in the same volume as the solvent, and the remaining steps and process parameters are the same as step (1) of Example 1. The yields of the products are 5%, 30% and 13% respectively.

[0089] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing acetoxy dideoxy ring-opening ribonucleoside, comprising the following steps: (2-acetoxyethoxy)-methyl bromide is coupled with a boric acid ester and then reacted with a pyrimidine and its derivatives of formula i or a purine and its derivatives of formula ii to obtain an acetoxy dideoxy ring-opening ribonucleoside; the boric acid ester is a bis-pinacol boric acid ester or a bis-boronic acid neopentyl glycol ester; The reaction is carried out in a reaction solvent, and the reaction solvent includes dioxane and / or ethylene glycol dimethyl ether; The reaction is carried out under the action of a catalyst, and the catalyst is a palladium catalyst; 。 2. The preparation method according to claim 1, characterized in that: The reaction temperature of the reaction is 60°C to reflux temperature.

3. The preparation method according to claim 1, characterized in that The palladium catalyst comprises at least one of palladium acetate, bis(triphenylphosphine)palladium acetate, triphenylphosphine palladium acetate, diphenylphosphinocene palladium dichloride, tetrakistriphenylphosphine palladium, dichlorobistriphenylphosphine palladium, and palladium on carbon; and / or, the ratio of the molar amount of the palladium acetate catalyst to the molar amount of the pyrimidine and derivatives of formula I or the purine and derivatives of formula II is (0.05~0.15):

1.

4. The preparation method according to claim 1, characterized in that: The (2-acetoxyethoxy)-methyl bromide is prepared by reacting 1,3-dioxolane with acetyl bromide.

5. A method for preparing dideoxy ring-opening ribonucleotide triphosphate, comprising the following steps: (1) Acetoxy dideoxy ring-opening ribonucleoside is prepared according to the method according to any one of claims 1 to 4; (2) reacting the acetoxy dideoxy ring-opened ribonucleoside obtained in step (1) with ammonia methanol to obtain the dideoxy ring-opened ribonucleoside; (3) Dideoxy ring-opened ribonucleoside is reacted with phosphorus oxychloride and tributylammonium pyrophosphate to prepare dideoxy ring-opened ribonucleotide triphosphate.

6. The preparation method according to claim 5, characterized in that: The molar ratio of the acetoxy dideoxy ring-opening ribonucleoside to ammonia methanol in step (2) is 1:3-7.

7. The preparation method according to claim 5, characterized in that: The reaction temperature in step (3) is 0-5°C, and the addition temperature of phosphorus oxychloride and tributylammonium pyrophosphate is -10-0°C.

8. The acetoxy dideoxy ring-opening ribonucleoside prepared according to any one of claims 1 to 4 is used in the preparation of dideoxy ring-opening ribonucleoside triphosphate.

9. Use of the dideoxy-open-ring ribonucleotide triphosphate obtained according to any one of claims 5 to 7 in the preparation of nucleic acid mass spectrometry reagents.

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