Tetrabutylammonium nucleoside triphosphate salt and preparation method thereof

Abstract

The invention relates to a nucleoside triphosphate tetrabutylammonium salt and a preparation method thereof. The nucleoside triphosphate tetrabutylammonium salt is prepared by reaction with nucleoside triphosphate and tetrabutylammonium hydroxide as raw materials. The invention prepares the nucleoside triphosphate tetrabutylammonium salt by using simple and readily available raw materials, has low requirements on process equipment, and can prepare milligram-level products.

Description

Technical Field

[0001] The present invention relates to nucleosides or modified nucleotide salts, in particular to tetrabutylammonium salts of nucleoside triphosphates or deoxynucleoside triphosphates and modified nucleosides thereof. Background Art

[0002] Modified nucleotides or special salts can be used in downstream enzyme reaction systems, and nucleoside or deoxynucleoside tetrabutylammonium triphosphate salts can be used in IVD fields for PCR, mNGS sequencing, highly amplified high-throughput sequencing, LAMP, multiple amplification, etc. However, the price of nucleoside or deoxynucleoside tetrabutylammonium triphosphate salts is relatively high, especially dTTP tetrabutylammonium salt is a relatively expensive compound, so its application is subject to certain restrictions. In addition, due to the complex preparation process, the reaction conditions and purification process need to be strictly controlled to ensure its purity and quality. Therefore, dTTP tetrabutylammonium salt requires specific processes for preparation and desalting during the research stage, and strict control of product quality.

[0003] Modified nucleotides or special salts require a small amount of product in the pilot stage, usually in the milligram range, which makes it difficult to operate modified nucleotide products in terms of salt transfer and desalting. Due to the good water solubility and high polarity of nucleotide monomer products, it is difficult to completely remove the original salt form by beating or crystallization to obtain ionic compounds with new salt forms. The molecular weight of modified nucleotide monomers is relatively small, usually around 500 Da, which makes it difficult to achieve liquid exchange and desalting using membrane packages or hollow fibers commonly used for nucleic acids or proteins. Summary of the invention

[0004] The invention aims to solve the technical problems of high production cost, difficult operation of salt conversion and desalting of nucleoside or deoxynucleoside and modified nucleoside tetrabutylammonium triphosphate salts. The invention aims to provide a rapid and easy-to-operate nucleoside tetrabutylammonium triphosphate salt and a preparation method thereof, which is suitable for the preparation of expensive modified nucleotides or deoxynucleotides, can control the treated sample to milligram level, and has a higher yield.

[0005] The first aspect of the present invention provides a tetrabutylammonium nucleoside triphosphate, which is prepared by reacting nucleoside triphosphate and tetrabutylammonium hydroxide as raw materials; the nucleoside triphosphate includes ribonucleoside triphosphate, deoxyribonucleoside triphosphate, modified ribonucleoside triphosphate, and modified deoxyribonucleoside triphosphate.

[0006] In a specific embodiment, the raw material nucleoside triphosphate is sodium nucleoside triphosphate, and or;

[0007] Nucleoside triphosphates include one or more sodium salts of deoxythymidine triphosphate (dTTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP).

[0008] The second aspect of the present invention provides a method for preparing the above-mentioned tetrabutylammonium nucleoside triphosphate salt, wherein an anion exchange column is used as a preparation column, nucleoside triphosphate is loaded as a sample, tetrabutylammonium hydroxide aqueous solution is used as mobile phase A, tetrabutylammonium chloride aqueous solution is used as mobile phase B, and elution is performed to obtain an eluent containing tetrabutylammonium nucleoside triphosphate; the nucleoside triphosphate includes ribonucleoside triphosphate, deoxyribonucleoside triphosphate, modified ribonucleoside triphosphate, and modified deoxyribonucleoside triphosphate.

[0009] In a specific embodiment, the mobile phase A is a tetrabutylammonium hydroxide aqueous solution with a concentration of 5-15 mM, such as 5 mM, 7 mM, 10 mM, 12 mM, 15 mM; preferably 10 mM.

[0010] In a specific embodiment, the mobile phase B is tetrabutylammonium chloride with a concentration of 0.5-2M, such as 0.5M, 0.8M, 1.0M, 1.2M, 1.5M, 1.8M, 2M; preferably 0.8-1.8M. The concentration of mobile phase B is very critical for the preparation of the product. When the concentration is too low, such as less than 0.5M, the product cannot be eluted and the target peak cannot be detected in the spectrum; when the concentration of the mobile phase is too high, such as greater than 2M, the salt concentration is high, the operating pressure is high, the material requirements for the equipment prepared are high, and the production cost will increase accordingly.

[0011] In a specific embodiment, the sample weight of the nucleoside triphosphate is 1:(10-100) of the weight of the anion exchange column filler, for example, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, preferably 1:(30-100).

[0012] In a specific embodiment, the nucleoside triphosphate includes, but is not limited to, one or more sodium salts of deoxythymidine triphosphate (dTTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP).

[0013] In a specific embodiment, the purity of the sodium nucleoside triphosphate sample is HPLC purity ≥ 99%, the conductivity is not higher than 10ms / cm, and the ratio of the injection amount (content) to the filler is 1 / 100-1 / 20.

[0014] The anion exchange column of the present invention can be any anion exchange column that can adsorb nucleoside triphosphates as understood in the art. In a specific embodiment, the anion exchange column contains -CH2N + A strong anion exchange column with (CH3)3 ion exchange functional group.

[0015] In a specific embodiment, the elution conditions are:

[0016] .

[0017] Preferably, the elution conditions are:

[0018] .

[0019] In the present invention, the method for preparing the tetrabutylammonium nucleoside triphosphate salt further comprises the step of purifying the eluent:

[0020] A hydrophilic chromatography (Hilic) column is used as a purification column, the eluent obtained in the preparation step is used as a sample for loading, purified water is used as mobile phase A, and acetonitrile is used as mobile phase B. The ratio of the mobile phase A to the mobile phase B mixture is 30-70:70-30, preferably 50:50, and the main fraction obtained by elution is tetrabutylammonium nucleoside triphosphate.

[0021] In a specific embodiment, during the purification process, multiple injections are performed, such as 2-8 times, and each injection is followed by balancing with a balancing solution; the ratio of the mobile phase A and mobile phase B mixture in the balancing solution is 1-3:99-97 (e.g., 1:99, 2:98, 3:97); and then the mobile phase A and mobile phase B mixture is used for elution to obtain a pure product of tetrabutylammonium triphosphate nucleoside.

[0022] Research has found that when using a hydrophilic chromatography column for purification, a large amount of injection can be performed. Taking a 120g filler column as an example, 20ml can be injected each time, and 2-8 injections can be performed continuously. With a large injection volume, desalting can save a lot of time and operating costs. Taking 3 injections as an example, the equilibrium injection conditions are:

[0023] .

[0024] In a specific embodiment, the purification step further includes the step of collecting pure products, including washing with a mixture of mobile phase A and mobile phase B, receiving a main peak fraction, and obtaining a pure product of tetrabutylammonium nucleoside triphosphate.

[0025] The nucleoside tetrabutylammonium salt can also be prepared into a solid form with better stability, especially a powder form, according to needs. The specific method includes rapidly freezing the pure nucleoside tetrabutylammonium triphosphate salt eluted from the HILIC chromatography column, discarding the acetonitrile layer when the product is layered, retaining the water layer and continuing to freeze, and when it is frozen into a solid, starting a freeze-drying procedure to obtain a freeze-dried powder.

[0026] The nucleoside triphosphate tetrabutylammonium salt obtained by the preparation method of the present invention can be used in PCR, mNGS sequencing, highly amplified high-throughput sequencing, LAMP, and multiple amplification.

[0027] The present invention has achieved the following positive effects: the present invention prepares the tetrabutylammonium salt of nucleoside triphosphate through simple and readily available raw materials, and the preparation method adopts ion exchange column chromatography for salt conversion, the filler price is low, the equipment requirements are low, the sample loading capacity is high, the single needle operation time is short, and the tetrabutylammonium salt of nucleoside can be quickly obtained. The Hilic chromatography continuous injection method can be used to increase the injection volume by large-scale loading, which can solve the problem that the modified nucleotide has a large polarity and cannot be hung on the C18 column, and can overcome the disadvantage that the Hilic column cannot load a large volume of aqueous solution. Through continuous injection of the Hilic column, the free tetrabutylammonium salt can be removed to obtain the tetrabutylammonium salt of nucleoside triphosphate. The method can effectively process a small amount of rare salt forms of modified nucleotides, with low cost and fast time, and can process milligram-level products with a high yield; the method of the present invention can prepare 1 mg to 100 g of tetrabutylammonium salt of nucleoside triphosphate according to demand. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 .Ion chromatography chromatogram obtained in Example 1.1.

[0029] Figure 2 .HPLC detection chart of ion chromatography fractions obtained in Example 1.1.

[0030] Figure 3 .HILIC chromatography chromatogram obtained in Example 1.2.

[0031] Figure 4 .HPLC detection chart of HILIC chromatography fractions obtained in Example 1.2.

[0032] Figure 5 .HILIC chromatography chromatogram obtained in Example 4.2. DETAILED DESCRIPTION

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] At present, modified nucleotide tetrabutylammonium salt is mainly used in IVD field, mNGS sequencing, highly amplified high-throughput sequencing, LAMP, multiple amplification, etc. It is very expensive and has a small application volume. Since the molecular weight of nucleotides is similar to that of tetrabutylammonium salt, the roll membrane commonly used in production is difficult to use for desalting, and dextran gel is also difficult to desalt. Nucleotides have unique biological activity and high polarity, resulting in no adsorption on commonly used C18 fillers.

[0039] For nucleotide salt conversion, there are currently two main methods that are achieved through chromatography technology: Patent CN 104356224A uses reverse phase chromatography technology to prepare semaglutide in the form of acetate, and patent CN 109438569A uses reverse phase chromatography technology to prepare liraglutide in the form of acetate. Patent CN 108359006A uses gel chromatography to convert salts to obtain semaglutide in a salt-free form. However, more organic solvents are used and more organic waste liquid is generated; dextran gel salt conversion is not suitable for modifying nucleotide monomers because the molecular weight of nucleotide monomers and salts is similar and the difference is small. The sample solution after salt conversion also needs to be concentrated under reduced pressure and most of the organic solvents are removed before it can be freeze-dried. Although reverse phase chromatography and gel chromatography technologies can achieve salt conversion of semaglutide and liraglutide, they both have certain disadvantages. Another method of salt conversion is CN117024488A, which is carried out by nanofiltration concentration. Nanofiltration concentration salt conversion has the problem of incomplete salt conversion, and the sample requirement is at least 1L or more, which is not suitable for expensive modified nucleotide monomers. In addition, the polarity of the modified nucleotides is particularly high and is not retained on the reverse phase filler, resulting in the inability to increase the amount of salt transfer and desalting. Overall, for products such as modified nucleotides, reverse phase chromatography, gel chromatography and nanofiltration salt transfer and desalting have no advantages in terms of operation, recovery rate, cost, efficiency and environmental protection.

[0040] In view of the small amount of expensive modified nucleotides, the present invention converts the sodium salt of nucleoside or deoxynucleoside triphosphate into the tetrabutylammonium salt of nucleoside or deoxynucleoside triphosphate by ion exchange.

[0041] Research has found that the structure of nucleotides determines that they are very polar and difficult to retain on conventional reverse chromatographic columns. HILIC mode can be used. Hilic chromatography fillers can be used to separate highly polar and hydrophilic compounds, such as nucleosides and nucleotides, amino acids, sugars, etc. It uses a polar stationary phase and a polar mobile phase, and generally uses a solution with a lower polarity than the stationary phase, such as acetonitrile / water. However, since the sample is a high-concentration aqueous solution, a large injection volume cannot be applied. We use multiple loadings and multiple equilibrations to ensure that the sample is bound to the Hilic filler, and then equilibrate and elute to remove excess free tetrabutylammonium salts, and then rinse the nucleoside or deoxynucleoside triphosphate tetrabutylammonium salts.

[0042] Since the fractions collected by Hilic chromatography contain acetonitrile, they cannot be directly freeze-dried. In order to save time and operation, quick freezing is performed before freeze-drying. After the acetonitrile and aqueous phases are separated, the acetonitrile phase is separated and removed, and the remaining aqueous phase is freeze-dried to powder. Since the modified nucleotide tetrabutylammonium salt is mainly soluble in the aqueous phase and insoluble in acetonitrile, there is almost no loss in yield.

[0043] In a preferred embodiment of the present invention, a deoxynucleoside triphosphate tetrabutylammonium salt is also provided, which is prepared using deoxynucleoside triphosphate sodium and tetrabutylammonium hydroxide as raw materials.

[0044] In a preferred embodiment of the present invention, a method for preparing tetrabutylammonium deoxynucleoside triphosphate is also provided:

[0045] S1, an anion exchange column is used as a preparative column, deoxynucleoside triphosphate sodium is loaded as a sample, 5-15mM tetrabutylammonium hydroxide aqueous solution is used as mobile phase A, 0.5-2M tetrabutylammonium chloride aqueous solution is used as mobile phase B, and an eluent containing deoxynucleoside triphosphate tetrabutylammonium salt is eluted;

[0046] The elution conditions are:

[0047] .

[0048] S2, loading the sample with an eluent containing tetrabutylammonium triphosphate of deoxynucleoside, and injecting the sample multiple times; balancing with a balance solution after each injection; eluting to obtain a pure tetrabutylammonium triphosphate of deoxynucleoside. Purified water is used as mobile phase A, acetonitrile is used as mobile phase B, the ratio of the mobile phase A to the mobile phase B in the balance solution is 1-3:99-97, and the ratio of the mobile phase A to the mobile phase B in the eluent is 30-70:70-30.

[0049] S3, quickly freezing the pure deoxynucleoside triphosphate tetrabutylammonium salt eluted from the hydrophilic chromatography column, discarding the acetonitrile layer when the product is separated into layers, and continuing to freeze the water layer. When it is frozen into a solid, start the freeze-drying process to obtain freeze-dried powder.

[0050] Terminology explanation: "M" stands for volume molar concentration (i.e., the amount of substance concentration). 1M means 1 mol / L. 1mM means 1mmol / L.

[0051] Hereinafter, the tetrabutylammonium salt of deoxythymidine triphosphate and its preparation method according to one embodiment of the present invention are further described in conjunction with examples. In each example and comparative example, the anion exchange chromatography filler used is a Nano Q strong anion exchange column; and the hydrophilic chromatography filler is a neutral amide group bonded HILIC chromatography column.

[0052] Example 1 Tetrabutylammonium salt of deoxythymidine triphosphate

[0053] 1.1. Preparation of tetrabutylammonium salt of deoxythymidine triphosphate:

[0054] a Sample pretreatment: dTTP sodium salt (Tianjin Olive Biotechnology Co., Ltd.), HPLC purity ≥ 99%. Dissolve in purified water to obtain a 50 mg / ml aqueous solution.

[0055] b Column chromatography: Equipment: medium pressure chromatography system, DAC50; Filler: anion exchange chromatography filler particle size 50um;

[0056] Mobile phase A: 10 mM tetrabutylammonium hydroxide aqueous solution; Mobile phase B: 1 M tetrabutylammonium chloride aqueous solution;

[0057] Temperature: room temperature; wavelength: 254nm; flow rate: 60ml / min;

[0058] Injection volume: product / filler (m / m) = 1 / 40

[0059] Elution conditions:

[0060] .

[0061] Anion chromatogram Figure 1 , eluent receiving: segmented receiving of the main peak eluent of the chromatogram, the purity of which can reach more than 99%, the results are shown in Figure 2 The main peak (retention time 11.378min) is tetrabutylammonium salt of deoxythymidine triphosphate.

[0062] 1.2. Desalting using HILIC mode

[0063] a. Sample: Fraction obtained by ion exchange chromatography

[0064] b. HILIC chromatography Flash column: HILIC chromatography filler, particle size 30um, 120g;

[0065] Mobile phase A: purified water; Mobile phase B: acetonitrile;

[0066] Temperature: room temperature, flow rate: 40ml / min

[0067] gradient:

[0068] HILIC column loading and elution settings:

[0069] .

[0070] As shown in the table, 20 ml was injected each time, and then the initial mobile phase was used for 4-6 minutes for equilibrium. The injection was repeated 6 times, so that the total injection volume could reach 120 ml. Then the mobile phase was balanced for 3 minutes, and then the mobile phase A: mobile phase B = 50:50 was used for flushing. The chromatogram is shown in Figure 3 . Collect the main fraction, the purity is more than 99% (see Figure 4 ). The solution conductivity is lower than 10ms / cm, and the excess free salt has been removed.

[0071] 1.3 Freezing treatment

[0072] a. Set the temperature of the in-situ freeze dryer partition to -25±5°C, place the Hilic chromatography-treated fraction in a 50 ml vial, and freeze quickly;

[0073] b. When the liquid in the vial is separated into layers, remove the upper layer of acetonitrile and keep the water phase to continue freezing;

[0074] c. When the vial is frozen solid, start the freeze-drying process and harvest the freeze-dried powder.

[0075] The initial sample amount was 15 mM dTTP sodium salt, and finally 14.8 mM dTTP tetrabutylammonium salt was obtained with a yield of 98.7% and a purity of 99.69%.

[0076] Example 2 Tetrabutylammonium salt of deoxythymidine triphosphate

[0077] 2.1. Preparation of tetrabutylammonium salt of deoxythymidine triphosphate:

[0078] a Sample pretreatment: dTTP sodium salt (Tianjin Olive Biotechnology Co., Ltd.), HPLC purity ≥ 99%. Dissolve in purified water to obtain a 50 mg / ml aqueous solution.

[0079] b Column chromatography: Equipment: medium pressure chromatography system, DAC50; Filler: anion exchange chromatography filler, particle size 50um;

[0080] Mobile phase A: 10 mM tetrabutylammonium hydroxide aqueous solution; Mobile phase B: 1 M tetrabutylammonium chloride aqueous solution;

[0081] Temperature: room temperature; wavelength: 254nm; flow rate: 60ml / min

[0082] Elution conditions:

[0083] .

[0084] Injection volume: product / filler (m / m) = 1 / 10. The product partially penetrates during the injection process.

[0085] 2.2 is the same as 1.2 in Example 1;

[0086] 2.3 is the same as 1.3 in Example 1, and the final qualified product yield is 61% and the purity is 99.2%.

[0087] Example 3 Tetrabutylammonium salt of deoxythymidine triphosphate

[0088] 3.1. Preparation of tetrabutylammonium salt of deoxythymidine triphosphate:

[0089] a Sample pretreatment: dTTP sodium salt (Tianjin Olive Biotechnology Co., Ltd.), HPLC purity ≥ 99%. Dissolve in purified water to obtain a 50 mg / ml aqueous solution.

[0090] b Column chromatography: Equipment: medium pressure chromatography system, DAC50; Filler: anion exchange chromatography filler particle size 50um;

[0091] Mobile phase A: 10 mM tetrabutylammonium hydroxide aqueous solution; Mobile phase B: 1 M tetrabutylammonium chloride aqueous solution;

[0092] Temperature: room temperature; wavelength: 254nm; flow rate: 60ml / min

[0093] Injection volume: product / filler (m / m) = 1 / 100

[0094] Elution conditions:

[0095] .

[0096] 3.2 is the same as 1.2 in Example 1;

[0097] 3.3 is the same as 1.3 in Example 1, and the final qualified product yield is 98.1% and the purity is 99.7%. The injection volume of the chromatography column is low, and the time consumption per unit injection volume is 5 times that of Example 1.

[0098] Example 4

[0099] 4.1. Preparation of tetrabutylammonium salt of deoxythymidine triphosphate:

[0100] Same as Example 1.1.

[0101] 4.2 Desalting using HILIC mode

[0102] a. Sample: Fraction obtained by ion exchange chromatography

[0103] b. HILIC chromatography Flash column: HILC chromatography filler, particle size 30um, 120g;

[0104] Mobile phase A: purified water; mobile phase B: acetonitrile

[0105] Temperature: room temperature; flow rate: 40ml / min

[0106] First, balance with mobile phase A: mobile phase B = 3:97 for 7 minutes, inject 20ml, then balance with initial mobile phase for 6 minutes, and then flush with mobile phase A: mobile phase B = 50:50. The results are shown below. Figure 5 .

[0107] 4.3. Same as Example 1.3

[0108] The final qualified product yield was 97.5% and the purity was 99.5%. Due to the low single needle injection volume (Hilic chromatography only injected one needle), the total time consumption was 6 times that of Example 1.

[0109] Comparative Example 1 Tetrabutylammonium salt of deoxythymidine triphosphate

[0110] The difference from Example 1 is that in step 1.2,

[0111] a. Sample: Fraction obtained by ion exchange chromatography

[0112] b. AQ-C18 desalting

[0113] Flash column: AQ-C18 chromatography filler, particle size 30um, 120g;

[0114] Mobile phase A: purified water; mobile phase B: acetonitrile

[0115] Temperature: room temperature; flow rate: 40ml / min

[0116] First, balance with mobile phase A for 7 minutes, inject 20 ml, then balance with initial mobile phase for 6 minutes, and then flush with mobile phase A: mobile phase B = 50:50. The results show that the sample is flushed out within 6 minutes, there is no retention in the equilibrium stage, and it is impossible to fully desalt.

[0117] 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. Tetrabutylammonium nucleoside triphosphate, characterized in that: The invention is prepared by reacting nucleoside triphosphate and tetrabutylammonium hydroxide as raw materials. The nucleoside triphosphate includes ribonucleoside triphosphate, deoxyribonucleoside triphosphate, modified ribonucleoside triphosphate and modified deoxyribonucleoside triphosphate.

2. The nucleoside triphosphate tetrabutylammonium salt according to claim 1, characterized in that The raw material nucleoside triphosphate is sodium nucleoside triphosphate, and or; Nucleoside triphosphates include one or more sodium salts of deoxythymidine triphosphate (dTTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP).

3. A method for preparing the tetrabutylammonium nucleoside triphosphate salt according to claim 1, characterized in that: The anion exchange column is a preparative column, nucleoside triphosphate is loaded as a sample, tetrabutylammonium hydroxide aqueous solution is used as mobile phase A, tetrabutylammonium chloride aqueous solution is used as mobile phase B, and an eluent containing nucleoside triphosphate tetrabutylammonium salt is eluted to obtain the eluent; the nucleoside triphosphate includes ribonucleoside triphosphate, deoxyribonucleoside triphosphate, modified ribonucleoside triphosphate, and modified deoxyribonucleoside triphosphate.

4. The preparation method according to claim 3, characterized in that: The mobile phase A is a 5-15 mM tetrabutylammonium hydroxide aqueous solution; the mobile phase B is a 0.5-2 M tetrabutylammonium chloride aqueous solution; and / or, The loading weight of the nucleoside triphosphate sample is 1:(10-100) of the weight of the anion exchange column filler.

5. The preparation method according to claim 3, characterized in that: Nucleoside triphosphates include one or more sodium salts of deoxythymidine triphosphate (dTTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP).

6. The preparation method according to claim 3, characterized in that: The elution conditions are: 。 7. The preparation method according to claim 3, characterized in that: The step of purifying the eluate is also included: A hydrophilic chromatography column is used as a purification column, an eluent containing tetrabutylammonium triphosphate of nucleoside is loaded as a sample, purified water is used as a mobile phase A, acetonitrile is used as a mobile phase B, the ratio of the mobile phase A to the mobile phase B in the eluent is 30-70:70-30, and a pure product of tetrabutylammonium triphosphate of nucleoside is obtained by elution.

8. The preparation method according to claim 7, characterized in that: When the hydrophilic chromatography column is used for purification, the sample is injected multiple times; after each injection, the sample is balanced with a balance solution; the ratio of the mobile phase A and the mobile phase B mixed solution in the balance solution is 1-3:99-97.

9. The preparation method according to any one of claims 3 to 8, characterized in that: The method also includes preparing the pure product of tetrabutylammonium triphosphate nucleoside into a solid form, rapidly freezing the pure product of tetrabutylammonium triphosphate nucleoside obtained by washing a hydrophilic chromatography column, discarding the acetonitrile layer when the product is separated into layers, and continuing to freeze the water layer, and starting a freeze-drying procedure when the product is frozen into a solid to obtain a freeze-dried powder.

10. Use of the tetrabutylammonium nucleoside triphosphate salt obtained by the preparation method according to any one of claims 3 to 9 in PCR, mNGS sequencing, highly amplified high-throughput sequencing, LAMP, and multiplex amplification.

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