N 6 Azide-modified nucleotide analogs, their synthesis methods and applications

Abstract

The application discloses a kind of N 6 Azide modified nucleotide analogue N 6 -(6-azidohexyl)-adenosine-5'-triphosphate or N 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate and its synthesis method and application, which is added in 6-chloropurine nucleoside or 6-chloropurine 2'-deoxynucleoside and 6-azidohexane-1-amine base and organic solvent, reflux reaction, after reaction, reaction product is purified by silica gel column, elution, concentration, drying, triphosphorylation reaction is carried out to the obtained product again.The yield of intermediate N 6 -(6-azidohexyl)-adenosine / N 6 -(6-azidohexyl)-2'-deoxyadenosine is improved, N 6 -(6-azidohexyl)-adenosine-5'-triphosphate and N 6 -(6-azidohexyl)-2'-deoxyadenosine-5 '-triphosphate High-efficiency synthesis, simple flow, mild condition, has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, to the field of nucleic acid synthesis technology, and more specifically, to an N... 6 Azide-modified nucleotide analogs (N 6 -(6-azidohexyl)-adenosine-5'-triphosphate or N 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate) and its synthesis methods and applications. Background Technology

[0002] Nucleic acids (RNA / DNA), as the core genetic material of life activities, are crucial for understanding gene expression regulation mechanisms, viral infection pathways, cell proliferation and differentiation patterns, and the screening of disease diagnostic biomarkers. Studies have shown that there is a close synergistic effect between the epigenetic regulation of DNA and RNA; DNA determines the range of gene activation, while RNA dynamically adjusts expression levels. Simultaneous monitoring of both is essential for accurately understanding life processes and disease mechanisms. With the rapid development of space omics, single-cell multi-omics, and in vivo imaging technologies, there is a comprehensive demand for tool molecules that can simultaneously label RNA and DNA, requiring "dual targeting, high specificity, excellent compatibility, and high sensitivity." Developing novel labeled probes that can cover all nucleic acid types and standardized synthesis techniques has become a core research direction in molecular biology and clinical testing.

[0003] Traditional nucleic acid labeling methods are mainly divided into two categories: radioactive labeling and non-radioactive labeling. While radioactive labeling technology boasts high sensitivity, it suffers from inherent drawbacks such as radiation hazards, short half-life, complex procedures, and inability to be used for real-time monitoring of live cells, significantly limiting its application in in vivo research and clinical settings. Non-radioactive labeling technologies, with their advantages in safety and stability, are gradually becoming mainstream. Among these, metabolic labeling methods based on nucleotide analogs are widely used in research such as nucleic acid synthesis activity tracking, cell cycle analysis, and transcriptome dynamic monitoring because they can directly embed into nascent nucleic acid chains and reflect the dynamic process of nucleic acid synthesis.

[0004] In non-radioactive metabolic labeling techniques, bioorthogonal reaction-mediated labeling strategies exhibit unique advantages, especially the azide-alkyne cycloaddition reaction (click chemistry). Due to its mild reaction conditions, high specificity, low cytotoxicity, and lack of interference with in vivo physiological processes, it has become one of the core technologies in nucleic acid labeling. The core of this strategy is to incorporate nucleotide analogs containing azide (-N3) or alkyne groups into the nucleic acid chain through the cell's own metabolic mechanisms, and then use click chemistry to couple them with reporter molecules such as fluorescent dyes and biotin, achieving visualized tracking and quantitative analysis of nucleic acids. Currently, various azide-modified nucleotide analogs have been developed, but most are designed for single nucleic acid types. For example, azide-modified UTP analogs (AMUTP) are only suitable for imaging newly formed RNA, and γ-azidohexyl-modified ATP is mostly used for phosphorylation reaction monitoring. There is a lack of probe combinations that can simultaneously label RNA and DNA. Furthermore, existing compounds still have room for optimization in terms of modification site rationality, biocompatibility, and targeting specificity, making it difficult to meet the needs of nucleic acid co-detection and simultaneous multi-omics analysis.

[0005] Adenosine triphosphate (ATP) and deoxyadenosine triphosphate (dATP) are core raw materials for RNA and DNA synthesis, respectively. The targeted modification and synthesis of their structural analogs is key to achieving dual-target labeling of nucleic acids. 6 -(6-azidohexyl)-adenosine-5'-triphosphate (N 6 -azidohexyl-ATP) and N 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate (N 6 -Azide-hexyl-dATP) is a pair of structurally homologous and target-complementary azide-modified nucleotide analogs, both of which are generated by the N-terminus of adenosine (or deoxyadenosine). 6 The introduction of a 6-azidohexyl side chain enables synergistic function and targeted differentiation. Both retain the specific binding ability to their corresponding polymerases (RNA polymerase, DNA polymerase) and the metabolic activity of embedding into nascent nucleic acid chains, while leveraging the bioorthogonal reaction properties of the azido group for precise labeling. Simultaneously, the long-chain alkyl structure reduces the perturbation of nucleotide conformation by the modifying group, improving intracellular incorporation efficiency. Compared to traditional azido-modified nucleotides, N... 6 γ-position modification can effectively avoid the interference of γ-position modification on nucleotide chain elongation function, and the problems of decreased stability and easy degradation by nucleases caused by 8-position modification. Among them, N 6 -Azide-hexyl-ATP is suitable for tracking RNA transcriptional activity and dynamic monitoring of the transcriptome. N 6 -Azide-hexyl-dATP is adapted for DNA replication activity analysis and dynamic genome monitoring. The combination of the two can achieve synchronous RNA / DNA labeling, providing a core tool for nucleic acid synergistic regulation research and clinical DNA+RNA co-detection.

[0006] Although azide-modified nucleotides have broad application prospects, there are still challenges in targeting N... 6 Existing synthetic techniques for long-chain azido-modified ATP and dATP still face many common challenges and specific difficulties: First, the selectivity of modification sites is difficult to control, and multiple modification byproducts are easily generated, resulting in low product purity and failing to meet the requirements of high-sensitivity detection. Second, the introduction of azido groups requires harsh reaction conditions, and some reaction reagents have poor compatibility with functional groups, which affects product yield and may also destroy the active structure of nucleotides. Third, long-chain alkyl modification is prone to problems such as incomplete substitution and side chain isomerization, and subsequent purification processes are complex, making it difficult to achieve large-scale preparation. Fourth, existing technologies are mostly developed for single compounds and lack standardized synthetic procedures that can be adapted to two types of homologous compounds simultaneously, which cannot guarantee the structural consistency and performance synergy of the two, thus restricting their application in nucleic acid co-detection scenarios.

[0007] Currently, a small number of documents report N. 6 -(6-azidohexyl)-adenosine-5'-triphosphate (N 6 -azidohexyl-ATP) and N 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate (N 6 The intermediate N of -azidohexyl-dATP) 6 The synthesis of -(6-azidohexyl)-adenosine mainly focuses on two synthetic pathways: 1. 6-Chloropurinic nucleoside (500 mg) and 6-azahexane-1-amine (1.26 g) were stirred at 90°C for 3 hours in ethanol (9 ml) as solvent. After cooling, the mixture was crystallized at -18°C. The crystals were filtered, and the precipitate was washed with cold ethanol to obtain intermediate N. 6 -(6-Azide-hexyl)-adenosine (RS2). However, this method has a low conversion rate, and the product is obtained through crystallization, and the mother liquor may also contain the product; therefore, the final yield is only 75%. The reaction formula is as follows: 2. Using TBS-protected 6-chloropurine nucleoside and 6-hydroxyhexane-1-amine as raw materials, the mixture was refluxed and stirred in ethanol for 3.5 hours (yield 88%–97%), then reacted sequentially with sulfonyl chloride (yield 93–94%) and sodium azide (yield 33–94%), followed by removal of the TBS protecting group (yield 88–93%) to obtain intermediate N. 6 -(6-azidohexyl)-adenosine (i.e., RS2). Although this method greatly improves the yield, the steps are very cumbersome.

[0008] The reaction formula is as follows: Additionally, from intermediate N 6-(6-Azide-hexyl)-adenosine synthesis N 6 -(6-azidohexyl)-ATP has not yet been reported.

[0009] With the rapid development of combined spatial genomics and transcriptomics analysis, single-cell nucleic acid co-detection, and in vivo nucleic acid dynamic imaging technologies, there is a growing demand for high-purity, high-activity, dual-target synergistic N... 6 -Azide-hexyl-ATP and N 6 The demand for azidohexyl-dATP combinatorial probes is increasingly urgent. Developing an efficient, mild, and highly selective synthetic method to achieve the targeted preparation, large-scale purification, and synergistic performance regulation of these two types of compounds is of great significance. Summary of the Invention

[0010] Therefore, the purpose of this invention is to provide an efficient, mild, and highly selective method for synthesizing N. 6 -Azide-hexyl-ATP and N 6 The method of -azidohexyl-dATP.

[0011] The specific technical solutions for achieving the above-mentioned objectives are as follows.

[0012] In a first aspect, the present invention provides an N 6 The method for synthesizing -(6-azidohexyl)-adenosine-5'-triphosphate includes the following steps: (1) A base and an organic solvent were added to 6-chloropurine nucleoside and 6-azidohexane-1-amine, and the mixture was refluxed. After the reaction was completed, the reaction product was purified by silica gel column chromatography, eluted, concentrated, and dried to obtain N. 6 -(6-azidohexyl)-adenosine; the molar ratio of the 6-chloropurine nucleoside and 6-azidohexane-1-amine is 1:2~4; the molar ratio of the 6-chloropurine nucleoside to the base is 1:2~5; (2) For N obtained in step (1) 6 -(6-azidohexyl)-adenosine undergoes triphosphorylation to give N 6 -(6-azidohexyl)-adenosine-5'-triphosphate.

[0013] A second aspect of the present invention provides an N 6 The method for synthesizing -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate includes the following steps: (1) A base and an organic solvent were added to 6-chloropurine 2'-deoxynucleoside and 6-azidohexane-1-amine, and the mixture was refluxed. After the reaction was completed, the reaction product was purified by silica gel column chromatography, eluted, concentrated, and dried to obtain N. 6-(6-azidohexyl)-2'-deoxyadenosine; the molar ratio of 6-chloropurine 2'-deoxynucleoside and 6-azidohexane-1-amine is 1:2~4; the molar ratio of 6-chloropurine 2'-deoxynucleoside to base is 1:2~5; (2) For N obtained in step (1) 6 -(6-azidohexyl)-2'-deoxyadenosine undergoes triphosphorylation to give N 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate.

[0014] A third aspect of the present invention provides N synthesized by the above method. 6 -(6-azidohexyl)-adenosine-5'-triphosphate or N 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate.

[0015] A fourth aspect of the invention provides N 6 -(6-azidohexyl)-adenosine-5'-triphosphate or N 6 Application of (6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate in RNA labeling or monitoring, and / or DNA labeling or monitoring.

[0016] The inventors of this invention have discovered that adding a certain amount of alkali to a reaction system using 6-chloropurine nucleoside / 6-chloropurine 2'-deoxynucleoside and 6-azidohexane-1-amine as reactants is more conducive to the reaction of product N. 6 -(6-azidohexyl)-adenosine / N 6 The conversion of 6-azidohexyl)-2'-deoxyadenosine results in a high conversion rate, thus requiring only a small amount of silica gel for purification. The purification process minimizes losses, ultimately significantly increasing the yield of N-methyl-2-ethylhexylene (N). 6 -(6-azidohexyl)-adenosine / N 6 Yield of -(6-azidohexyl)-2'-deoxyadenosine.

[0017] Furthermore, the present invention improves upon product N 6 -(6-azidohexyl)-adenosine and N 6 The triphosphorylation of -(6-azidohexyl)-2'-deoxyadenosine yielded the final product N for the first time. 6 -(6-azidohexyl)-adenosine-5'-triphosphate and N 6The efficient synthesis of (6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate not only fills the gap in existing nucleic acid dual-targeting labeling nucleotide modification technology and provides a superior combination of tool molecules for the precise monitoring of RNA / DNA synthesis metabolism, damage repair and synergistic regulation processes, but also promotes the further application of bioorthogonal chemistry in multi-omics research, early tumor DNA+RNA co-detection and gene therapy vector monitoring, etc., which has important academic, industrial and clinical value.

[0018] This invention synthesizes N 6 -(6-azidohexyl)-adenosine-5'-triphosphate and N 6 The method for -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate is simple, mild, easy to promote, and has broad application prospects. Attached Figure Description

[0019] Figure 1 For N 6 ESI-MS mass spectrum of (6-azidohexyl)-adenosine.

[0020] Figure 2 For N 6 -(6-azidohexyl)-adenosine-5'-triphosphate (N 6 HPLC chromatogram of (-azidohexyl-ATP).

[0021] Figure 3 For N 6 -(6-azidohexyl)-adenosine-5'-triphosphate (N 6 ESI-MS mass spectrum of (-azidohexyl-ATP).

[0022] Figure 4 For N 6 ESI-MS mass spectrum of (6-azidohexyl)-2'-deoxyadenosine.

[0023] Figure 5 For N 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate (N 6 HPLC chromatogram of (-azidohexyl-dATP).

[0024] Figure 6 For N 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate (N 6 ESI-MS mass spectrum of (-azidohexyl-dATP). Detailed Implementation

[0025] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0027] Unless otherwise specified, all examples were conducted under standard experimental conditions or as recommended in the manufacturer's instructions. All raw materials used were commercially available and readily available.

[0028] In some embodiments of the present invention, an N is disclosed. 6 The method for synthesizing -(6-azidohexyl)-adenosine-5'-triphosphate includes the following steps: (1) A base and an organic solvent were added to 6-chloropurine nucleoside and 6-azidohexane-1-amine, and the mixture was refluxed. After the reaction was completed, the reaction product was purified by silica gel column chromatography, eluted, concentrated, and dried to obtain N. 6 -(6-azidohexyl)-adenosine; the molar ratio of the 6-chloropurine nucleoside and 6-azidohexane-1-amine is 1:2~4; the molar ratio of the 6-chloropurine nucleoside to the base is 1:2~5; (2) For N obtained in step (1) 6 -(6-azidohexyl)-adenosine undergoes triphosphorylation to give N 6 -(6-azidohexyl)-adenosine-5'-triphosphate.

[0029] In one embodiment, the molar ratio of 6-chloropurine nucleoside and 6-azidohexane-1-amine in step (1) is 1:2~3.

[0030] In one embodiment, the molar ratio of 6-chloropurine nucleoside to base in step (1) is 1:2.0~2.2.

[0031] In one embodiment, the base is N,N-diisopropylethylamine or triethylamine.

[0032] In one embodiment, the organic solvent is anhydrous ethanol, anhydrous acetonitrile, or N,N-dimethylformamide.

[0033] In one embodiment, the reflux reaction temperature is 80°C to 90°C.

[0034] In one embodiment, the reflux reaction temperature is 85°C to 90°C.

[0035] In one embodiment, the reflux reaction time is 3 to 6 hours.

[0036] In one embodiment, the reflux reaction time is 4 to 6 hours.

[0037] In one embodiment, the weight ratio of the silica gel to the reaction product is 4 to 10:1.

[0038] In one embodiment, the weight ratio of the silica gel to the reaction product is 4 to 6:1.

[0039] In one embodiment, the eluent used for elution is methanol and dichloromethane in a volume ratio of 1:10~15.

[0040] In one embodiment, step (1) is as follows: N,N-diisopropylethylamine and anhydrous ethanol are added to 6-chloropurine nucleoside and 6-azidohexane-1-amine, and the mixture is refluxed at 85°C to 90°C for 3 to 6 hours. After the reaction is completed, the reaction product is purified by silica gel column chromatography, eluted, concentrated, and dried to obtain N. 6 -(6-azidohexyl)-adenosine; the molar ratio of the 6-chloropurine nucleoside and 6-azidohexane-1-amine is 1:2~3; the molar ratio of the 6-chloropurine nucleoside to N,N-diisopropylethylamine is 1:2.0~2.2; the weight ratio of the silica gel to the reaction product is 4~6:1.

[0041] In one embodiment, the triphosphorylation reaction in step (2) includes the following steps: (a) Dissolving N using trimethyl phosphate 6 -(6-azidohexyl)-adenosine, phosphorus oxychloride was added under ice bath conditions of -10℃ to 5℃, and the reaction was carried out for 1 h to 2 h. (b) Add alkali and organic solvent to tributylammonium pyrophosphate and incubate in an ice bath at -10°C to 5°C for 4 to 6 minutes; (c) Add the reaction solution from step (b) to the reaction solution from step (a), react for 1 h to 2 h under ice bath conditions of -10℃ to 5℃, and then add TEAB buffer solution to react for 1 h to 2 h. (d) Dilution and purification of the reaction product in step (c): Elute the reaction product with TEAB buffer solution, collect the reaction product with a purity ≥95%, concentrate and freeze dry.

[0042] In one embodiment, N is dissolved in trimethyl phosphate in step (a). 6 -(6-azidohexyl)-adenosine and 1,8-bis(dimethylaminonaphthalene).

[0043] In one embodiment, the base in step (b) is N,N-diisopropylethylamine or triethylamine.

[0044] In one embodiment, the organic solvent in step (b) is anhydrous ethanol, anhydrous acetonitrile, or N,N-dimethylformamide.

[0045] In one embodiment, the concentration of the TEAB buffer solution in step (c) is 0.8 M to 1.2 M.

[0046] In one embodiment, the concentration of the TEAB buffer solution in step (d) is 0M~1M.

[0047] In other embodiments of the present invention, N synthesized by the above method is disclosed. 6 -(6-azidohexyl)-adenosine-5'-triphosphate.

[0048] In other embodiments of the present invention, an N is disclosed. 6 The method for synthesizing -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate includes the following steps: (1) A base and an organic solvent were added to 6-chloropurine 2'-deoxynucleoside and 6-azidohexane-1-amine, and the mixture was refluxed. After the reaction was completed, the reaction product was purified by silica gel column chromatography, eluted, concentrated, and dried to obtain N. 6 -(6-azidohexyl)-2'-deoxyadenosine; the molar ratio of 6-chloropurine 2'-deoxynucleoside and 6-azidohexane-1-amine is 1:2~4; the molar ratio of 6-chloropurine 2'-deoxynucleoside to base is 1:2~5; (2) For N obtained in step (1) 6 -(6-azidohexyl)-2'-deoxyadenosine undergoes triphosphorylation to give N 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate.

[0049] In one embodiment, the molar ratio of 6-chloropurine 2'-deoxynucleoside and 6-azidohexane-1-amine in step (1) is 1:2~3.

[0050] In one embodiment, the molar ratio of 6-chloropurine 2'-deoxynucleotide to base in step (1) is 1:2.0~2.2.

[0051] In one embodiment, the base is N,N-diisopropylethylamine or triethylamine.

[0052] In one embodiment, the organic solvent is anhydrous ethanol, anhydrous acetonitrile, or N,N-dimethylformamide.

[0053] In one embodiment, the reflux reaction temperature is 80°C to 90°C.

[0054] In one embodiment, the reflux reaction temperature is 85°C to 90°C.

[0055] In one embodiment, the reflux reaction time is 3 to 6 hours.

[0056] In one embodiment, the reflux reaction time is 4 to 6 hours.

[0057] In one embodiment, the weight ratio of the silica gel to the reaction product is 4 to 10:1.

[0058] In one embodiment, the weight ratio of the silica gel to the reaction product is 4 to 6:1.

[0059] In one embodiment, the eluent used for elution is methanol and dichloromethane in a volume ratio of 1:10~15.

[0060] In one embodiment, step (1) is as follows: N,N-diisopropylethylamine and anhydrous ethanol are added to 6-chloropurine 2'-deoxynucleoside and 6-azidohexane-1-amine, and the mixture is refluxed at 85°C to 90°C for 3 to 6 hours. After the reaction is completed, the reaction product is purified by silica gel column chromatography, eluted, concentrated, and dried to obtain N 6 -(6-azidohexyl)-2'-deoxyadenosine; the molar ratio of 6-chloropurine 2'-deoxynucleoside and 6-azidohexane-1-amine is 1:2~3; the molar ratio of 6-chloropurine 2'-deoxynucleoside to N,N-diisopropylethylamine is 1:2.0~2.2; the weight ratio of silica gel to the reaction product is 4~6:1.

[0061] In one embodiment, the triphosphorylation reaction in step (2) includes the following steps: (a) Dissolving N using trimethyl phosphate 6 -(6-azidohexyl)-2'-deoxyadenosine, phosphorus oxychloride was added under ice bath conditions of -10℃ to 5℃, and the reaction was carried out for 1 h to 2 h; (b) Add alkali and organic solvent to tributylammonium pyrophosphate and incubate in an ice bath at -10°C to 5°C for 4 to 6 minutes; (c) Add the reaction solution from step (b) to the reaction solution from step (a), react for 1 h to 2 h under ice bath conditions of -10℃ to 5℃, and then add TEAB buffer solution to react for 1 h to 2 h. (d) Dilution and purification of the reaction product in step (c): Elute the reaction product with TEAB buffer solution, collect the reaction product with a purity ≥95%, concentrate and freeze dry.

[0062] In one embodiment, N is dissolved in trimethyl phosphate in step (a). 6 -(6-azidohexyl)-2'-deoxyadenosine and 1,8-bis(dimethylaminonaphthalene).

[0063] In one embodiment, the base in step (b) is N,N-diisopropylethylamine or triethylamine.

[0064] In one embodiment, the organic solvent in step (b) is anhydrous ethanol, anhydrous acetonitrile, or N,N-dimethylformamide.

[0065] In one embodiment, the concentration of the TEAB buffer solution in step (c) is 0.8 M to 1.2 M.

[0066] In one embodiment, the concentration of the TEAB buffer solution in step (d) is 0M~1M.

[0067] In other embodiments of the present invention, N synthesized by the above method is disclosed. 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate.

[0068] In other embodiments of the present invention, the above-mentioned N is disclosed. 6 -(6-azidohexyl)-adenosine-5'-triphosphate or N 6 Application of (6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate in RNA labeling or monitoring, and / or DNA labeling or monitoring.

[0069] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0070] Example 1 N 6 -(6-azidohexyl)-adenosine-5'-triphosphate (N 6 Synthesis of -azidohexyl-ATP This embodiment provides N 6 -(6-azidohexyl)-adenosine-5'-triphosphate (N 6 The method (-azidohexyl-ATP) has the following reaction formula: Specifically, the following steps are included: 1. N 6 Synthesis of -(6-azidohexyl)-adenosine (RS2) Take a 10 ml reaction flask, add a stir bar, and weigh out 286 mg (1 mmol) of 6-chloropurine nucleoside (Annegi, trade code: E080172) and 357 mg (2.5 mmol) of 6-azidohexane-1-amine (Annegi, B01005348-1g). Measure 2 ml of ethanol and add it to the flask, then add 348 μL (2 mmol) of N,N-diisopropylethylamine. Install a reflux apparatus and heat to 90°C for 3 hours until the reaction is complete. Concentrate the product and purify using a 2 g silica gel column (silica gel to product weight ratio approximately 5:1). Elute the product with methanol:dichloromethane = 1:10, collect the eluent, concentrate, and dry to obtain 372 mg of solid, yield 95%.

[0071] Its ESI-MS mass spectra are as follows Figure 1 As shown, the results indicate m / z = 392.99 [MH]- (theoretical exact mass 392.5), confirming that the product is N. 6 -(6-azidohexyl)-adenosine (RS2).

[0072] 2. N 6 Synthesis of -(6-azidohexyl)-adenosine-5'-triphosphate (RS3) (1) Take 25 ml reaction flask 1, add a stir bar, and weigh 372 mg (0.95 mmol) N. 6 -(6-azidohexyl)-adenosine (RS2), 406 mg (1.9 mmol) 1,8-bis(dimethylaminonaphthalene) (proton sponge, Anaiji, B010047-25g), 5 ml trimethyl phosphate, after nitrogen purging, placed in an ice bath, and after 5 minutes, slowly added 121 uL (1.42 mmol) phosphorus oxychloride (Anaiji, W611039-100 g), and reacted for 1 hour.

[0073] (2) Take another 25 ml reaction flask 2, add 1045 mg (2 mmol) of tributylammonium pyrophosphate (Annegi, A01078619-25g), 0.82 ml (5 mmol) of N,N-diisopropylethylamine, and 5 mL of acetonitrile, and incubate on ice for 5 minutes.

[0074] (3) Quickly transfer the liquid from reaction flask 1 to reaction flask 2, react for 1 hour, add 5 ml of 1 M TEAB buffer solution and continue reacting for half an hour, then take a sample for HPLC to check if the reaction is complete. The HPLC conditions are as follows: mobile phase: ACN-0.1M TEAB, elution gradient: 0 min-3 min 2%, 3 min-15 min 2%-60%, 15 min-16 min 60%-90%, 16 min-17 min 90%-2%, 17 min-20 min 2%-2%.

[0075] (4) Dilute the reaction product from step (3) with pure water to 100 ml, purify with DEAE ion exchange resin, and perform gradient elution with 1 MTEAB buffer and pure water, elute impurities with 0.2 M TEAB buffer, and elute the product with 0.4 M TEAB buffer. Collect the product in test tubes, and send a sample from each tube for HPLC analysis. Collect samples with a purity ≥95% together. Concentrate to 5 mL using a rotary evaporator, purify using a preparative liquid chromatography column, and collect the eluent containing the product peak. HPLC analysis shows a purity of 99.60% (…). Figure 2 (HPLC conditions as above). Concentrate to 5 mL, transfer to a 15 mL centrifuge tube, pre-freeze, and then freeze-dry for 48 h to obtain 590 mg of white solid with a purity greater than 99.20% and a yield of 60%.

[0076] Its ESI-MS mass spectra are as follows Figure 3 As shown, the results indicate m / z = 630.91 [MH]- (theoretical exact mass 632.04), confirming that the product is N. 6 -(6-azidohexyl)-adenosine-5'-triphosphate (RS3).

[0077] Example 2 N 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate (N 6 Synthetic methods of -azidohexyl-dATP This embodiment provides an N 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate (N 6 The method for (-azidohexyl-dATP) has the following reaction formula: Specifically, the following steps are included: 1. N 6 Synthesis of 6-azidohexyl)-2'-deoxyadenosine (DS2) Take a 10 ml reaction flask, add a stir bar, and weigh out 270 mg (1 mmol) of 6-chloropurine 2'-deoxynucleoside (Bide BD02507996-1 g) and 357 mg (2.5 mmol) of 6-azidohexane-1-amine (Annegi, B01005348-1 g). Measure 2 ml of ethanol and add it to the flask, then add 348 μL (2 mmol) of N,N-diisopropylethylamine. Install a reflux apparatus and heat to 90°C for 4 hours until the reaction is complete. Concentrate the product and purify using a 2 g silica gel column (silica gel to product weight ratio approximately 5:1). Elute the product with methanol:dichloromethane = 1:15, collect the eluent, concentrate, and dry to obtain 353 mg of solid, yield 94%.

[0078] Its ESI-MS mass spectra are as follows Figure 4 As shown, the results indicate m / z = 377.05 [MH]- (theoretical exact mass 376.04), confirming that the product is N. 6 -(6-azidohexyl)-2'-deoxyadenosine (DS2).

[0079] 2. N 6 Synthesis of -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate (DS3) (1) Take 25 ml reaction flask 1, add stir bar, and weigh 353 mg (0.94 mmol) N. 6 -(6-azidohexyl)-2'-deoxyadenosine (DS2), 402 mg (1.88 mmol) of 1,8-bis(dimethylaminonaphthalene) (proton sponge, Anaiji, B010047-25g), and 5 ml of trimethyl phosphate were purged with nitrogen and placed in an ice bath. After 5 minutes, 120 uL (1.41 mmol) of phosphorus oxychloride (Anaiji, W611039-100 g) was slowly added, and the reaction was carried out for 1 hour.

[0080] (2) Take another 25 ml reaction flask 2, add 1040 mg (1.88 mmol) tributylammonium pyrophosphate (Annegi, A01078619-25g), 0.82 ml (4.7 mmol) N,N-diisopropylethylamine, 5 mL acetonitrile, and incubate on ice for 5 minutes.

[0081] (3) Quickly transfer the liquid from reaction flask 1 to reaction flask 2, react for 1 hour, add 5 ml of 1 M TEAB buffer solution and continue reacting for half an hour, then take a sample for HPLC to check if the reaction is complete. The HPLC conditions are as follows: mobile phase: ACN-0.1M TEAB, elution gradient: 0 min-3 min 2%, 3 min-15 min 2%-60%, 15 min-16 min 60%-90%, 16 min-17 min 90%-2%, 17 min-20 min 2%-2%.

[0082] (4) Dilute the reaction product from (3) with pure water to 100 ml, purify with DEAE ion exchange resin, and perform gradient elution with 1M TEAB buffer and pure water, elute impurities with 0.2M TEAB buffer, and elute the product with 0.4M TEAB buffer. Collect in test tubes, send a sample from each tube for HPLC analysis, and collect samples with a purity ≥95% together. Concentrate to 5 mL using a rotary evaporator, purify using a preparative liquid chromatography column, collect the eluent containing the product peak, and determine the purity to be 99.28% by HPLC. Figure 5 (HPLC conditions as above). Concentrate to 5 mL, transfer to a 15 mL centrifuge tube, pre-freeze, and then freeze-dry for 48 h to obtain 594 mg of white solid with a purity greater than 99.20% and a yield of 62%.

[0083] Its ESI-MS mass spectra are as follows Figure 6 As shown, the results indicate m / z = 614.99 [MH]- (theoretical exact mass 616.04), confirming that the product is N. 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate (DS3).

[0084] Example 3: Effects of different reaction conditions on N 6 Effect of -(6-azidohexyl)-adenosine (RS2) yield This embodiment compares the results in N. 6 The effect of different reaction conditions on the yield of -(6-azidohexyl)-adenosine (RS2) in the synthesis method.

[0085] Except for the changes in reaction conditions (Table 1), all other operations are the same as step 1 of Example 1.

[0086] Table 1 As can be seen from the results in Table 1: In the synthesis of N 6 When -(6-azidohexyl)-adenosine (RS2) is added, compared to not adding a base, the addition of a base such as triethylamine or N,N-diisopropylethylamine is more conducive to the conversion to the product. 6 The yield of -(6-azidohexyl)-adenosine (RS2) was greatly improved. When triethylamine was added, the reaction time was extended, and N... 6 -(6-azidohexyl)-adenosine (RS2) yielded higher yields when N,N-diisopropylethylamine was added, using anhydrous ethanol as a solvent. 6 The yield of -(6-azidohexyl)-adenosine (RS2) was significantly higher.

[0087] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0088] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A kind of N 6 The method for synthesizing 5'-(6-azidohexyl)-adenosine-5'-triphosphate is characterized by, Includes the following steps: (1) A base and an organic solvent were added to 6-chloropurine nucleoside and 6-azidohexane-1-amine, and the mixture was refluxed. After the reaction was completed, the reaction product was purified by silica gel column chromatography, eluted, concentrated, and dried to obtain N. 6 -(6-azidohexyl)-adenosine; the molar ratio of the 6-chloropurine nucleoside and 6-azidohexane-1-amine is 1:2~4; the molar ratio of the 6-chloropurine nucleoside to the base is 1:2~5; (2) For N obtained in step (1) 6 -(6-azidohexyl)-adenosine undergoes triphosphorylation to give N 6 -(6-azidohexyl)-adenosine-5'-triphosphate.

2. The synthesis method according to claim 1, characterized in that, The base mentioned in step (1) is N,N-diisopropylethylamine or triethylamine; And / or, the organic solvent in step (1) is anhydrous ethanol, anhydrous acetonitrile or N,N-dimethylformamide.

3. The synthesis method according to claim 1, characterized in that, The molar ratio of 6-chloropurine nucleoside to 6-azidohexane-1-amine in step (1) is 1:2~3; And / or, the molar ratio of 6-chloropurine nucleoside to base in step (1) is 1:2.0~2.

2.

4. The synthesis method according to claim 1, characterized in that, The reflux reaction temperature in step (1) is 80℃~90℃, and the reflux reaction time is 3 hours~6 hours; And / or, the weight ratio of the silica gel to the reaction product in step (1) is 4~10:1; And / or, the eluent used in step (1) is methanol and dichloromethane in a volume ratio of 1:10~15.

5. The synthesis method according to any one of claims 1 to 4, characterized in that, The triphosphorylation reaction described in step (2) includes the following steps: (a) Dissolving N using trimethyl phosphate 6 -(6-azidohexyl)-adenosine, phosphorus oxychloride was added under ice bath conditions of -10℃ to 5℃, and the reaction was carried out for 1 h to 2 h. (b) Add alkali and organic solvent to tributylammonium pyrophosphate and incubate in an ice bath at -10°C to 5°C for 4 to 6 minutes; (c) Add the reaction solution from step (b) to the reaction solution from step (a), react for 1 h to 2 h under ice bath conditions of -10℃ to 5℃, and then add TEAB buffer solution to react for 1 h to 2 h. (d) Dilution and purification of the reaction product in step (c): Elute the reaction product with TEAB buffer solution, collect the reaction product with a purity ≥95%, concentrate and freeze dry.

6. The synthesis method according to claim 5, characterized in that, In step (a), trimethyl phosphate is used to dissolve N 6 -(6-azidohexyl)-adenosine and 1,8-bis(dimethylaminonaphthalene); And / or, the base in step (b) is N,N-diisopropylethylamine or triethylamine, and the organic solvent is anhydrous ethanol, anhydrous acetonitrile or N,N-dimethylformamide; And / or, the concentration of the TEAB buffer solution in step (c) is 0.8 M to 1.2 M; And / or, the concentration of the TEAB buffer solution in step (d) is 0M~1M.

7. The N synthesized by the method according to any one of claims 1 to 6 6 -(6-azidohexyl)-adenosine-5'-triphosphate.

8. A kind of N 6 The method for synthesizing -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate, characterized in that, Includes the following steps: (1) A base and an organic solvent were added to 6-chloropurine 2'-deoxynucleoside and 6-azidohexane-1-amine, and the mixture was refluxed. After the reaction was completed, the reaction product was purified by silica gel column chromatography, eluted, concentrated, and dried to obtain N. 6 -(6-azidohexyl)-2'-deoxyadenosine; the molar ratio of 6-chloropurine 2'-deoxynucleoside and 6-azidohexane-1-amine is 1:2~4; the molar ratio of 6-chloropurine 2'-deoxynucleoside to base is 1:2~5; (2) For N obtained in step (1) 6 -(6-azidohexyl)-2'-deoxyadenosine undergoes triphosphorylation to give N 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate.

9. The synthesis method according to claim 8, characterized in that, The base mentioned in step (1) is N,N-diisopropylethylamine or triethylamine; And / or, the organic solvent in step (1) is anhydrous ethanol, anhydrous acetonitrile or N,N-dimethylformamide.

10. The synthesis method according to claim 8, characterized in that, The molar ratio of 6-chloropurine 2'-deoxynucleoside to 6-azidohexane-1-amine in step (1) is 1:2~3; And / or, the molar ratio of 6-chloropurine 2'-deoxynucleoside to base in step (1) is 1:2.0~2.

2.

11. The synthesis method according to claim 8, characterized in that, The reflux reaction temperature in step (1) is 80℃~90℃, and the reflux reaction time is 3 hours~6 hours; And / or, the weight ratio of the silica gel to the reaction product in step (1) is 4~10:1; And / or, the eluent used in step (1) is methanol and dichloromethane in a volume ratio of 1:10~15.

12. The synthesis method according to any one of claims 8 to 11, characterized in that, The triphosphorylation reaction described in step (2) includes the following steps: (a) Dissolving N using trimethyl phosphate 6 -(6-azidohexyl)-2'-deoxynucleoside-adenosine, phosphorus oxychloride was added under ice bath conditions of -10℃ to 5℃, and the reaction was carried out for 1 h to 2 h. (b) Add alkali and organic solvent to tributylammonium pyrophosphate and incubate in an ice bath at -10°C to 5°C for 4 to 6 minutes; (c) Add the reaction solution from step (b) to the reaction solution from step (a), react for 1 h to 2 h under ice bath conditions of -10℃ to 5℃, and then add TEAB buffer solution to react for 1 h to 2 h. (d) Dilution and purification of the reaction product in step (c): Elute the reaction product with TEAB buffer solution, collect the reaction product with a purity ≥95%, concentrate and freeze dry.

13. The synthesis method according to claim 12, characterized in that, In step (a), trimethyl phosphate is used to dissolve N 6 -(6-azidohexyl)-2'-deoxynucleoside-adenosine and 1,8-bis(dimethylaminonaphthalene); And / or, the base in step (b) is N,N-diisopropylethylamine or triethylamine, and the organic solvent is anhydrous ethanol, anhydrous acetonitrile or N,N-dimethylformamide; And / or, the concentration of the TEAB buffer solution in step (c) is 0.8 M to 1.2 M; And / or, the concentration of the TEAB buffer solution in step (d) is 0M~1M.

14. The N synthesized by the method according to any one of claims 8 to 13 6 -(6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate.

15. The N according to claim 7 6 -(6-azidohexyl)-adenosine-5'-triphosphate or the N as described in claim 14 6 Application of (6-azidohexyl)-2'-deoxyadenosine-5'-triphosphate in RNA labeling or monitoring, and / or DNA labeling or monitoring.

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