阅读说明：本技术 2`，3`-双脱氧核苷-5`-O-(α-硫代)三磷酸的制备方法 (Preparation method of 2', 3' -dideoxynucleoside-5 ' -O- (alpha-thio) triphosphate ) 是由 邱洪健 黄成� 毛忠华 李新亮 于 2021-08-17 设计创作，主要内容包括：2',3'-双脱氧核苷-5'-O-(α-硫代)三磷酸的制备方法,属于化学合成领域。2',3'-双脱氧核苷-5'-O-(α-硫代)三磷酸的制备方法包括：使2',3'-双脱氧核苷二氯(α-硫代一磷酸)与焦磷酸反应,然后进行水解。本申请方案可以获得高纯度的产品。(A preparation method of 2', 3' -dideoxynucleoside-5 ' -O- (alpha-thio) triphosphate, belonging to the field of chemical synthesis. The preparation method of the 2', 3' -dideoxynucleoside-5 ' -O- (alpha-thio) triphosphate comprises the following steps: 2', 3' -dideoxynucleoside dichloride (. alpha. -thiomonophosphate) is reacted with pyrophosphoric acid and then hydrolyzed. The scheme of the application can obtain a high-purity product.)

1. A method for preparing 2', 3' -dideoxynucleoside-5 ' -O- (. alpha. -thio) triphosphate, comprising: 2', 3' -dideoxynucleoside dichloride (. alpha. -thiomonophosphate) is reacted with pyrophosphoric acid and then hydrolyzed.

2. The 2', 3' -dideoxynucleoside-5 ' -O- (α -thio) triphosphate according to claim 1, wherein the 2', 3' -dideoxynucleoside dichloride (α -thio-monophosphate) and the pyrophosphate are reacted in an aprotic polar solvent; optionally, the aprotic polar solvent comprises N, N-dimethylformamide.

3. The method for producing 2', 3' -dideoxynucleoside-5 ' -O- (. alpha. -thio) triphosphate according to claim 1, wherein the 2', 3' -dideoxynucleoside dichloride (. alpha. -thio-monophosphate) is produced by a phosphorylation reaction using a 2', 3' -dideoxynucleoside; optionally, the phosphorylating reagent used in the phosphorylating reaction is PSCl₃。

4. The method for producing 2', 3' -dideoxynucleoside-5 ' -O- (α -thio) triphosphate according to claim 1, wherein the 2', 3' -dideoxynucleoside dichloride (α -thio-monophosphate) is produced by: adding trichloro-sulfur phosphorus into a liquid system formed by 2', 3' -dideoxy nucleoside, trimethyl phosphate and 2,4, 6-trimethyl pyridine for reaction.

5. The method for producing 2', 3' -dideoxynucleoside-5 ' -O- (. alpha. -thio) triphosphate according to any one of claims 3 to 4, wherein said 2', 3' -dideoxynucleoside comprises 2', 3' -dideoxyadenosine, 2', 3' -dideoxycytidine, 2', 3' -dideoxyguanosine, 2', 3' -dideoxythymidine.

6. A method for preparing 2', 3' -dideoxynucleoside-5 ' -O- (. alpha. -thio) triphosphate, comprising:

mixing trimethyl phosphate, 2,4, 6-trimethylpyridine and dried 2', 3' -dideoxy nucleoside at 0-5 ℃, and then adding trichloro-sulfur at 0-10 ℃ for reaction to obtain 2', 3' -dideoxy nucleoside dichloro (alpha-sulfo-monophosphate);

in a solution of tributylamine, tributylamine pyrophosphate and N, N-dimethylformamide, 2', 3' -dideoxynucleoside dichloride (. alpha. -thiomonophosphate) is reacted at a temperature of 20 ℃ to 25 ℃, and then hydrolyzed with ice water to form 2', 3' -dideoxynucleoside-5 ' -O- (. alpha. -thio) triphosphate.

7. The method of claim 6, wherein the method is performed by a one-pot method.

8. The method of preparing a 2', 3' -dideoxynucleoside-5 ' -O- (α -thio) triphosphate according to claim 7, wherein the method comprises: the hydrolysis is followed by a purification treatment.

9. The method for producing 2', 3' -dideoxynucleoside-5 ' -O- (α -thio) triphosphate according to claim 8, wherein the purification treatment comprises: the product of the hydrolysis was extracted with dichloromethane and the upper aqueous phase was subjected to column chromatography.

10. The method for producing 2', 3' -dideoxynucleoside-5 ' -O- (α -thio) triphosphate according to claim 8, wherein the purification treatment comprises:

the product resulting from the hydrolysis was extracted with dichloromethane and the upper aqueous phase was subjected to column chromatography by: sequentially passing through an LX-650 resin column and an HZ201 resin column, and eluting with a lithium chloride aqueous solution to obtain a lower column collecting solution;

concentrating the collected liquid from the lower column to 100-200 OD/ml under reduced pressure in a water bath at 28 ℃, purifying by using a C18 column, wherein the mobile phase is an acetonitrile water solution, and concentrating the collected liquid from the lower column under reduced pressure in the water bath at 28 ℃ to form a concentrated solution;

and sequentially carrying out ultrafiltration and volume fixing on the concentrated solution to obtain a liquid containing lithium salt type 2', 3' -dideoxynucleoside-5 ' -O- (alpha-thio) triphosphate.

Technical Field

The application relates to the field of chemical synthesis, in particular to a preparation method of 2', 3' -dideoxynucleoside-5 ' -O- (alpha-thio) triphosphate.

Background

Antisense drugs are mainly drugs prepared using Antisense DNA and Antisense RNA, such as Antisense nucleic acids/Antisense Oligonucleotides (ODNs). The artificially synthesized DNA or RNA single-stranded fragment mainly comprises antisense DNA (AS-ODN), antisense RNA (ASON), Polypeptide Nucleic Acid (PNA), ribozyme (ribozyme) and the like.

Traditional drugs, such as small molecule chemical drugs, large molecule biological drug antisense technology, etc., generally act directly on pathogenic proteins themselves, while antisense drugs act on genes that produce proteins. Antisense drugs are capable of hybridizing to specific genes at the gene level to interfere with the production of pathogenic proteins. In other words, the antisense drug utilizes the synthetic, naturally occurring complementary oligonucleotide fragment to bind with the specific sequence of the target gene (single-stranded, double-stranded DNA) or messenger ribonucleic acid (mRNA) according to the base complementary pairing principle and the nucleic acid hybridization principle, and regulates the expression of the target gene from the level of gene replication, transcription, splicing, transport translation, etc., and interferes with the transmission of genetic information from nucleic acid to protein, thereby achieving the purpose of inhibiting, blocking or destroying the target gene. Therefore, compared with the traditional medicine, the antisense medicine has more selectivity, thus having higher efficiency and lower toxicity.

Disclosure of Invention

The application provides a simple and easy-to-implement preparation method of 2', 3' -dideoxynucleoside-5 ' -O- (alpha-thio) triphosphate, which is different from the prior modes of microbial fermentation, enzymatic reaction, enzymatic decomposition and the like.

The application is realized as follows:

in a first aspect, the present application provides a method for preparing a 2', 3' -dideoxynucleoside-5 ' -O- (. alpha. -thio) triphosphate, comprising: 2', 3' -dideoxynucleoside dichloride (. alpha. -thiomonophosphate) is reacted with pyrophosphoric acid and then hydrolyzed.

According to some examples of the present application, 2', 3' -dideoxynucleoside dichloro (α -thio-monophosphate) is reacted with pyrophosphoric acid in an aprotic polar solvent; alternatively, the aprotic polar solvent comprises N, N-dimethylformamide.

According to some examples of this application, 2', 3' -dideoxynucleoside dichloro (. alpha. -thiomonophosphate) is by using 2'The 3' -dideoxynucleoside is prepared by phosphorylation reaction; alternatively, the phosphorylating reagent used in the phosphorylating reaction is PSCl₃。

According to some examples of the present application, the 2', 3' -dideoxynucleoside dichloride (α -thio-monophosphate) is prepared by: adding trichloro-sulfur phosphorus into a liquid system formed by 2', 3' -dideoxy nucleoside, trimethyl phosphate and 2,4, 6-trimethyl pyridine for reaction.

According to some examples of the application, the 2', 3' -dideoxynucleosides include 2', 3' -dideoxyadenosine, 2', 3' -dideoxycytidine, 2', 3' -dideoxyguanosine, 2', 3' -dideoxythymidine.

In a second aspect, the present application illustratively provides a method for preparing 2', 3' -dideoxynucleoside-5 ' -O- (. alpha. -thio) triphosphate, comprising:

mixing trimethyl phosphate, 2,4, 6-trimethylpyridine and dried 2', 3' -dideoxy nucleoside at 0-5 ℃, and then adding trichloro-sulfur at 0-10 ℃ for reaction to obtain 2', 3' -dideoxy nucleoside dichloro (alpha-sulfo-monophosphate);

in a solution of tributylamine, tributylamine pyrophosphate and N, N-dimethylformamide, 2', 3' -dideoxynucleoside dichloride (. alpha. -thiomonophosphate) is reacted at a temperature of 20 ℃ to 25 ℃, and then hydrolyzed with ice water to form 2', 3' -dideoxynucleoside-5 ' -O- (. alpha. -thio) triphosphate.

According to some examples of the present application, the preparation method is carried out by a one-pot method.

According to some examples of the present application, the method of making comprises: the hydrolysis is followed by a purification treatment.

According to some examples of the present application, the purification process comprises: the product of the hydrolysis was extracted with dichloromethane and the upper aqueous phase was subjected to column chromatography.

According to some examples of the present application, the purification process comprises:

the product produced by hydrolysis was extracted with dichloromethane and the upper aqueous phase was subjected to column chromatography by the following means: the product resulting from the hydrolysis was extracted with dichloromethane and the upper aqueous phase was subjected to column chromatography by: sequentially passing through an LX-650 anion resin column and an HZ201 resin column, and eluting with a lithium chloride aqueous solution to obtain a lower column collecting solution;

concentrating the collected liquid under reduced pressure to 100-200 OD/ml in a water bath at 28 ℃, purifying by using a C18 column, wherein the mobile phase is an acetonitrile water solution, and concentrating the collected liquid under reduced pressure in the water bath at 28 ℃ to form a concentrated solution;

and sequentially carrying out ultrafiltration and volume fixing on the concentrated solution to obtain a liquid containing lithium salt type 2', 3' -dideoxynucleoside-5 ' -O- (alpha-thio) triphosphate.

In the implementation process, the preparation method of the 2', 3' -dideoxynucleoside-5 ' -O- (. alpha. -thio) triphosphate provided by the embodiment of the application can obtain a high-purity product, and the method is a simple and easy-to-implement scheme. Furthermore, it can also act as an inhibitor of viral reverse transcriptase and viral replication.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments or the prior art of the present application, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 HLPC map of ddNTP- α -S in example 1 of the present application;

FIG. 2 MS map of ddNTP- α -S in example 1 of the present application;

FIG. 3 of ddNTP- α -S in example 1 of the present application³¹P-NMR；

FIG. 4 HLPC map of ddNTP- α -S in example 2 of the present application;

FIG. 5 MS map of ddNTP- α -S in example 2 of the present application;

FIG. 6 method for producing ddNTP- α -S in example 2 of the present application³¹P-NMR；

FIG. 7 HLPC map of ddNTP- α -S in example 3 of the present application;

FIG. 8 MS map of ddNTP- α -S in example 3 of the present application;

FIG. 9 of ddNTP- α -S in example 3 of the present application³¹P-NMR；

FIG. 10 HLPC map of ddNTP- α -S in example 4 of the present application;

FIG. 11 MS map of ddNTP- α -S in example 4 of the present application;

FIG. 12 method for producing ddNTP- α -S in example 4 of the present application³¹P-NMR。

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following will specifically describe the preparation method of 2', 3' -dideoxynucleoside-5 ' -O- (. alpha. -thio) triphosphate according to the examples of the present application:

2', 3' -dideoxynucleoside-5 ' -O- (. alpha. -thio) triphosphate:

all are called 2', 3' -Dideoxynucleoside-5 ' -O- (. alpha. -thio) Triphosphates, abbreviated as ddNTP-. alpha. -S, and the structure thereof is shown in the following formula 1.

Wherein Base represents a Base, which may be A, C, G, T, A represents Adenine (Adenine), T represents Thymine (Thymine), C represents Cytosine (Cytosine), and G represents Guanine (Guanine).

In other examples, the ddNTP- α -S compound may be prepared in the form of a salt, for example, a lithium salt, or a potassium salt, a sodium salt, a potassium salt, an ammonium salt, or the like. Wherein the lithium salt has the following structural formula:

in the present application, the target substance is prepared by organic synthesis. In general, in the examples of the present application, a dideoxynucleoside is used as a starting material, and a 2', 3' -dideoxynucleoside dichloro (. alpha. -thio) monophosphate-based product is obtained by phosphorylation with a trichlorothiophosphorus reagent in a trimethyl phosphate system; and adding excessive tributylamine pyrophosphate and N, N-Dimethylformamide (DMF) to react to generate a compound containing 'three phosphoric acids'. The obtained reaction solution is hydrolyzed, chromatographically separated and purified to obtain the target product with the purity of more than 97 percent.

Where dideoxynucleosides may be selected differently depending on the base in ddNTP- α -S, the dideoxynucleosides thus include:

2', 3' -dideoxyadenosine,

2', 3' -dideoxycytidine,

2', 3' -dideoxy guanosine,

2', 3' -dideoxythymidine,

wherein the 2', 3' -dideoxynucleoside dichloro (. alpha. -thio) monophosphate has the following structure (formula 2).

Formula 2, wherein Base represents a Base, and the Base is selected from A, C, T and G.

The synthesis path of the present application can be briefly described as follows:

in the examples of the present application, the synthesis of the target product is carried out by a "one-pot method", i.e., various raw materials and intermediate products are not separated in the whole preparation process. After the initial substrate reacts with other raw materials, formed intermediate products, partial raw materials which are not completely reacted and the like are continuously added with raw materials, catalysts and the like in the subsequent steps to continuously react without separation until the target product (the compound shown in the formula 1) is obtained.

Because the target product is prepared by adopting a one-pot method, the target product exists in a mixture system. In order to separate the target product, the mixture after the reaction is optionally subjected to a purification treatment. In the present example, the purification treatment was mainly performed by means of column chromatography. During the purification process, the salt type target substance can also be obtained by combining the ion exchange resin and then combining the component selection of the mobile phase.

In the above-mentioned preparation reaction, since the reaction of trichlorothiophosphoryl is generally vigorous, in order to reduce the reaction severity and at the same time reduce, or even suppress, the production of by-products, it is selected to carry out the reaction at a relatively lower temperature to obtain 2', 3' -dideoxynucleoside dichloro (. alpha. -thio) monophosphoric acid. The low temperature condition may be provided by an ice bath, for example, and the temperature may be 0 ℃ to 10 ℃, further 0 ℃ to 5 ℃. In addition, in the example, trimethyl phosphate can be used in the reaction system, and can form a phosphorylation reagent with good activity with trichlorothiophosphoryl, as shown in a formula 4.

Of course, the trimethyl phosphate may be selected from other alkyl type phosphoric triesters, such as triethyl phosphate, etc. Further, in order to promote the reaction, a pyridine catalyst (acid-binding) may be selected; in the examples, 2,4, 6-collidine is selected and, for example, 2-picoline, 2, 4-lutidine, 2, 6-lutidine and the like can be used.

After the above reaction process, the compound shown in formula 2 is formed, and then the compound reacts with tributyl pyrophosphate (or tributyl pyrophosphate). Tributylamine pyrophosphate is a mixture of tri-n-butylamine and pyrophosphoric acid at a molar ratio of 2:1, and may be used as a solution previously dissolved in DMF.

In order to make it easier for the skilled person to carry out the protocol of the present application, the following details are given for the preparation methods in the examples.

First step of

The dideoxynucleosides need to be dried (otherwise the subsequent purification process would be affected) to remove the water content. Wherein the drying scheme can be distillation under reduced pressure; for example, in a rotary evaporator, the organic solvent acetonitrile is suspended over dideoxynucleosides and evaporated to dryness by distillation under reduced pressure.

Second step of

Mixing the dehydrated and dried dideoxynucleoside with trimethyl phosphate and 2,4, 6-trimethylpyridine, and dropwise adding trichlorothiophosphorus for reaction under the condition of ice bath temperature control. The progress of the reaction was monitored by HPLC. When no dideoxynucleoside starting material remains, the next reaction is carried out.

The third step

To the liquid reaction system of the second step, liquid tributylamine and previously dissolved tributylamine pyrophosphate (which may be DMF/N, N-dimethylformamide) as a liquid are added. The ice bath is removed and the reaction is monitored by HPLC after the temperature is raised to, for example, room temperature (which may be 20 ℃ to 25 ℃) until no intermediate is produced and the next step is carried out.

The fourth step

After completion of the third step, ice water was added to the reaction system to conduct hydrolysis, thereby obtaining ddNTP-. alpha. -S dispersed in the reaction system. When the pH value of the reaction system is close to or neutral, the reaction can be quenched by selecting ice water hydrolysis, gas generation is avoided, the controllability of the reaction is improved, impurities are not introduced, and the purification is convenient. If the pH value of the reaction system is lower, triethyl ammonium bicarbonate can be adopted for hydrolysis quenching reaction; or after the hydrolysis quenching reaction is carried out by using ice water, the pH value is adjusted by using triethyl ammonium bicarbonate; however, this easily causes generation of carbon dioxide gas and increases the difficulty of reaction control.

The purification may be optionally performed by the following procedure in consideration of the presence of various impurities in the reaction system.

The fifth step

Since the hydrolysis was carried out with ice water and the water solubility of the nucleotide was taken into consideration, the reaction system was extracted with methylene chloride, which is a poor solvent for water, and the upper aqueous phase was collected. Therefore, ddNTP-alpha-S with higher purity can be obtained by separating and purifying the collected water phase. In the example, the method of column chromatography is selected for separation and purification.

For example, in the examples, more ion exchange resins are used for separation and purification of proteins, nucleic acids and polypeptides selected for use in biopharmaceuticals. Ion exchange resins are a class of materials having functional groups (active groups capable of exchanging ions, such as sulfonic acid groups, carboxyl groups, amine groups, etc.), networks, insoluble polymers, and are generally present in the form of spherical particles or are porous. Also, the ion exchange resin achieves its function by ion exchange in liquid and solid phases, and can be reused by regeneration since ion exchange is reversible. The ion exchange resins generally include four types of strong acid cation exchange resins, weak acid cation exchange resins, strong base anion exchange resins, and weak base anion exchange resins. Generally, acidic ion exchange resins are designated as positive; the basic ion exchange resin is designated as anionic.

The column chromatography in the examples of the present application may be carried out in a single or multiple steps, with appropriate selection depending on the purity requirements. In addition, different chromatographic columns can be selected for multi-step purification according to components in the product to be purified, so that the purity is further improved.

For example, in the examples of the present application, column chromatography (e.g., LX-650 resin) is first performed using a strong anion exchange resin to remove a portion of impurities, and then elution is performed using a strongly basic ion exchange resin HZ201 with, for example, an aqueous lithium chloride solution as a mobile phase.

In the elution process, the collected elution product liquid (the product purity can reach 85%) can be further purified by column chromatography so as to improve the purity of the elution product liquid. Optionally concentrating before column chromatography; for example, a water bath is selected for concentration under reduced pressure. Then, the aforementioned eluate was subjected to column chromatography on a C18 silica gel column. Namely, bonded silica gel in which an alkane chain having 18 carbons is bonded to silica gel is used as a stationary phase, and a liquid phase composed of acetonitrile and water is used as a mobile phase for elution. The product is purified and has a purity of, for example, 98% as described above.

Namely, ddNTP-alpha-S in the form of lithium salt is obtained, and the structure is as shown in the following formula 5:

and (5) formula. Wherein Base represents a Base.

The lithium chloride may be replaced by sodium chloride, which in turn gives ddNTP- α -S in the form of its sodium salt. The required salt form is prepared by the method, so that impurities can be prevented from being introduced, and the working procedures can be reduced. The direct addition of a salt solution (e.g., perchlorate) results in the presence of a non-target salt type product, resulting in reduced purity and yield, as compared to the solutions exemplified herein. For example, direct mixing of the lower C18 column with the lithium perchlorate solution results in a portion of ddNTP- α -S being present as the ammonium salt (non-target salt form) and, correspondingly, a reduction in the product in the form of its lithium salt (target salt form).

The present application is described in further detail with reference to examples below.

Example 1

One-step chemical synthesis of 2', 3' -dideoxyadenosine-5 ' -O- (. alpha. -thiophosphoric acid).

1. 44g (187 mmol) of dideoxyadenosine/2 ', 3' -dideoxyadenosine were dried 2 times with acetonitrile by distillation under reduced pressure on a rotary evaporator, and the amount of acetonitrile used was 440 ml/time. Then, 880ml of trimethyl phosphate was added, followed by 38.5g of 2,4, 6-trimethylpyridine. The temperature is controlled to 0 ℃ in an ice bath, and 63.4g of PSCl is added dropwise₃. After the dropwise addition, the temperature is controlled to be 0 ℃ for reaction in the whole reaction process. After reacting for 2h, monitoring HPLC every hour, ending the reaction without raw material residue, and entering the next step without treatment of the reaction solution.

2. 45g of tributylamine and 410g of tributylamine pyrophosphate (DMF was dissolved in advance) were added to the reaction mixture, and the temperature was raised to 20 ℃. And timing the reaction, reacting for 0.5h, monitoring by HPLC, and ending the reaction until no intermediate exists. The reaction solution was hydrolyzed with 4L of ice water and extracted once with 5L of dichloromethane, and the upper aqueous phase was collected as a solution to be chromatographed.

3. Column chromatography:

and carrying out column chromatography on the liquid to be chromatographed according to the following mode.

Filling 0.88L of LX-650 strong anion resin column, loading chromatographic solution to the resin column with a conductance value of 3500-4000 us/c, and passing through the resin column, then washing with water and collecting lower column solution; and (3) performing column chromatography on the collected liquid by using HZ201 chlorine type resin column, and performing gradient elution by using 0.1-0.5M lithium chloride aqueous solution to obtain a product liquid with the purity of more than 85%, namely a lower column collected liquid.

C18 preparative column purification:

and (3) concentrating the collected liquid of the lower column to 100OD/ml under reduced pressure at 28 ℃ in a water bath, preparing and purifying by using a C18 preparation column, separating a product liquid with the purity of more than 98% by using a mobile phase of acetonitrile aqueous solution (0-5% acetonitrile/water solution), merging, and concentrating under reduced pressure at 28 ℃ in the water bath.

5. The concentrated solution was collected and ultrafiltered to obtain a final volume of 33ml of 100mM product solution (3.3 mmol).

The final product obtained was subjected to HPLC analysis as shown in FIG. 1, and its peak structure is shown in Table 1 below.

TABLE 1

Mass spectra referring to figure 2 of the drawings,³¹the P-NMR chart is shown in FIG. 3.

Example 2

And chemically synthesizing 2', 3' -dideoxycytidine-5 ' -O- (alpha-thiophosphoric acid) by a one-step method.

1. 59g (279 mmol) of dideoxycytidine are weighed out and reduced with acetonitrileThe mixture is dried by pressure distillation for 2 times, and the using amount of the acetonitrile is 590 ml/time. 1180ml of trimethyl phosphate was then added, followed by 57.4g of 2,4, 6-trimethylpyridine. Then the system was cooled to 2 ℃ and 94.5g of PSCl were added dropwise₃. After the dripping is finished, controlling T_{Inner part}: and reacting at 3 ℃. After reacting for 2h, monitoring HPLC every hour, ending the reaction without raw material residue, and entering the next step without treatment of the reaction solution.

2. 67.2g of tributylamine and 610g of tributylamine pyrophosphate (DMF was dissolved in advance) were added to the reaction mixture, and the temperature was raised to 20 to 25 ℃. And timing the reaction, reacting for 0.5h, monitoring by HPLC, and ending the reaction until no intermediate exists. The reaction solution was hydrolyzed with 4L of ice water and extracted once with 5L of dichloromethane, and the upper aqueous phase was collected as a solution to be chromatographed.

3. Column chromatography:

and carrying out column chromatography on the liquid to be chromatographed according to the following mode.

Filling 1.18L of LX-650 strong anion resin column, loading a chromatographic solution onto the resin column at a conductance value of 3500-4000 us/c, and passing through the resin column, then washing with water and collecting lower column solution; and (3) performing column chromatography on the collected liquid by using HZ201 chlorine type resin column, and performing gradient elution by using 0.1-0.5M lithium chloride aqueous solution to obtain a product liquid with the purity of more than 85%, namely a lower column collected liquid.

C18 preparative column purification:

and (3) concentrating the collected liquid of the lower column to 100OD/ml under reduced pressure at 28 ℃ in a water bath, preparing and purifying by using a C18 preparation column, separating a product liquid with the purity of more than 98% by using a mobile phase of acetonitrile aqueous solution (0-5% acetonitrile/water solution), merging, and concentrating under reduced pressure at 28 ℃ in the water bath.

5. The concentrated solution was collected and ultrafiltered to a constant volume to obtain 40ml of a final 100mM product solution (4 mmol).

The final product obtained was subjected to HPLC analysis as shown in FIG. 4, and its peak structure is shown in Table 2 below.

TABLE 2

Mass spectrum referring to figure 5 of the drawings,³¹the P-NMR chart is shown in FIG. 6.

Example 3

One-step chemical synthesis of 2', 3' -dideoxyguanosine-5 ' -O- (. alpha. -thiophosphoric acid).

1. 40g (159 mmol) of dideoxyguanosine was weighed out, dried 2 times with acetonitrile by means of distillation under reduced pressure and 400 ml/time. 800ml of trimethyl phosphate are then added, followed by 32g of 2,4, 6-trimethylpyridine. The system was then cooled to 5 ℃ and 54g of PSCl3 were added dropwise. After the dripping is finished, controlling T_{Inner part}: reacting at 6 ℃. After reacting for 2h, monitoring HPLC every hour, ending the reaction without raw material residue, and entering the next step without treatment of the reaction solution.

2. 38.4g of tributylamine and 348g of tributylamine pyrophosphate (DMF pre-dissolved) were added thereto, and the temperature was raised to 20 to 25 ℃. And timing the reaction, reacting for 0.5h, monitoring by HPLC, and ending the reaction until no intermediate exists. The reaction solution was hydrolyzed with 2L of ice water and extracted once with 2L of dichloromethane, and the upper aqueous phase was collected as a solution to be chromatographed.

3. Column chromatography:

and carrying out column chromatography on the liquid to be chromatographed according to the following mode.

Filling 0.8L of LX-650 strong anion resin column, loading chromatographic solution to the resin column with a conductance value of 3500-4000 us/c, and passing through the resin column, then washing with water and collecting lower column solution; and (3) performing column chromatography on the collected liquid by using HZ201 chlorine type resin column, and performing gradient elution by using 0.1-0.5M lithium chloride aqueous solution to obtain a product liquid with the purity of more than 85%, namely a lower column collected liquid.

C18 preparative column purification:

and (3) concentrating the collected liquid of the lower column to 100OD/ml under reduced pressure at 28 ℃ in a water bath, preparing and purifying by using a C18 preparation column, separating a product liquid with the purity of more than 98% by using an acetonitrile aqueous solution (0-5% acetonitrile aqueous solution) as a mobile phase, combining, and concentrating under reduced pressure at 28 ℃ in the water bath.

5. The concentrated solution was collected and ultrafiltered to a constant volume to obtain 30ml of a final 100mM product solution (3 mmol).

The final product obtained was subjected to HPLC analysis as shown in FIG. 7, and its peak structure is shown in Table 3 below.

TABLE 3

Mass spectrum MS referring to figure 8 of the drawings,³¹the P-NMR chart is shown in FIG. 9.

Example 4

One-step chemical synthesis of 2', 3' -dideoxy thymidine-5 ' -O- (alpha-thiophosphoric acid).

1. 32g (141.6 mmol) of dideoxythymidine were weighed out, dried 2 times with acetonitrile by means of reduced pressure distillation and 320 ml/time. 640ml of trimethyl phosphate are then added, followed by 29g of 2,4, 6-trimethylpyridine. The system was then cooled to 4 ℃ and 48g of PSCl3 were added dropwise. After the dripping is finished, controlling T_{Inner part}: and reacting at 7 ℃. After reacting for 2h, monitoring HPLC every hour until the raw material is less than 5%, finishing the reaction, and entering the next step without processing the reaction solution.

2. 34g of tributylamine and 310g of tributylamine pyrophosphate (DMF is dissolved in advance) are added, and the temperature is raised to 20-25 ℃. And timing the reaction, reacting for 0.5h, monitoring by HPLC, and ending the reaction when no intermediate is generated in the reaction and less than 5%. The reaction solution was hydrolyzed with 3L of ice water and extracted once with 3L of dichloromethane, and the upper aqueous phase was collected as a solution to be chromatographed.

3. Column chromatography:

and carrying out column chromatography on the liquid to be chromatographed according to the following mode.

Filling 0.64L of LX-650 strong anion resin column, loading chromatographic solution to the resin column with a conductance value of 3500-4000 us/c, and then washing with water and collecting lower column solution; and (3) performing column chromatography on the collected liquid by using HZ201 chlorine type resin column, and performing gradient elution by using 0.1-0.5M lithium chloride aqueous solution to obtain a product liquid with the purity of more than 85%, namely a lower column collected liquid.

C18 preparative column purification:

and (3) concentrating the collected liquid of the lower column to 100OD/ml under reduced pressure at 28 ℃ in a water bath, preparing and purifying by using a C18 preparation column, separating a product liquid with the purity of more than 98% by using an acetonitrile aqueous solution (0-5% acetonitrile aqueous solution) as a mobile phase, combining, and concentrating under reduced pressure at 28 ℃ in the water bath.

5. The concentrated solution was collected and ultrafiltered to a constant volume to obtain a final 32ml of 100mM product solution (3.2 mmol in volume).

The final product obtained was subjected to HPLC analysis as shown in FIG. 10, and its peak structure is shown in Table 4 below.

TABLE 4

Mass spectrum MS referring to figure 11,³¹the P-NMR chart is shown in FIG. 12.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.