Cyclization reagent, its use and method for chemical synthesis of circular oligonucleotides

Abstract

The application discloses a cyclization reagent and application and a chemical synthesis method of a cyclic oligonucleotide, and belongs to the technical field of chemical synthesis. The cyclization reagent comprises the following components: a divalent metal salt, an imidazole salt and cyanogen halide, or a divalent metal salt and a cyanoimidazole, or a divalent metal salt, an imidazole salt and a cyanoimidazole. The chemical synthesis method comprises the following steps: adding each component of the aforementioned cyclization reagent into a linear oligonucleotide solution to perform a cyclization reaction, and obtaining a cyclic oligonucleotide. The cyclization reagent can realize the synthesis of linear to cyclic oligonucleotides, and the synthesis method is simple and does not introduce non-natural groups. Compared with the prior art, the synthesis method is simple, low in cost, high in cyclic efficiency, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis technology, and relates to a cyclizing reagent and its application, and a chemical synthesis method for cyclic oligonucleotides. Background Technology

[0002] Oligonucleotides, as a novel gene therapy drug, have broad application prospects in clinical practice. Oligonucleotides are usually synthesized using the phosphoramide solid-phase synthesis method, but the oligonucleotides produced are all linear. In contrast to linear oligonucleotides, cyclic oligonucleotides have important biological functions and application value due to their unique topological, enzymatic properties and biological stability (Zhu Wenxuan et al., Research progress on the synthesis and application of cyclic oligonucleotides [J], Pharmaceutical Progress, 2024, 48(8), 592-604).

[0003] Cyclic oligonucleotide synthesis methods mainly include enzymatic synthesis and chemical synthesis. Enzymatic synthesis is the preferred method due to the good biocompatibility of enzymes and high cyclization yield, but it generally suffers from low yield. Chemical synthesis involves the jointing of the two ends of oligonucleotides through mild, simple, and efficient chemical reactions.

[0004] Currently, there are few studies and reports on the chemical synthesis of cyclic oligonucleotides. The main synthetic methods include cyclization via azide-alkyne click chemistry and cyclization via carboxyl and amino amidation reactions. Although existing chemical synthesis methods can compensate for the insufficient yield of enzymatic synthesis, they also have problems such as introducing non-natural groups and complex operation.

[0005] Therefore, developing a simple and easy-to-operate chemical synthesis method for cyclic oligonucleotides that does not introduce non-natural groups is of great significance for the research of cyclic oligonucleotides. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a cyclizing reagent, its application, and a method for the chemical synthesis of cyclic oligonucleotides. The cyclizing reagent of this invention is simple to operate, achieves high cyclization yields, and does not introduce non-natural groups during the chemical synthesis of cyclic oligonucleotides.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] In a first aspect, the present invention provides a cyclizing reagent comprising the following components: a divalent metal salt, an imidazole salt, and a cyanide halide; or a divalent metal salt and a cyanoimidazole; or a divalent metal salt, an imidazole salt, and a cyanoimidazole.

[0009] In some embodiments, the divalent metal salt is a water-soluble salt, including any one of chloride, nitrate, phosphate, and sulfate.

[0010] Preferably, the divalent metal includes at least one of zinc, magnesium, manganese, nickel, and cadmium.

[0011] In some embodiments, the imidazole salt is one of imidazole hydrochloride or imidazole nitrate.

[0012] In some embodiments, the cyanide halide is at least one of cyanide fluoride, cyanide chloride, cyanide bromide, and cyanide iodide.

[0013] Each component of the cyclizing reagent of the present invention is prepared independently and then mixed separately before use.

[0014] In some embodiments, the cyclizing agent comprises the following components: 50-400 μmol / mL divalent metal salt, 10-200 μmol / mL imidazole salt, and 10-200 μmol / mL cyanide halide.

[0015] In some embodiments, the cyclizing agent comprises the following components: 50-400 μmol / mL divalent metal salt and 10-200 μmol / mL cyanoimidazole.

[0016] In some embodiments, the cyclizing agent comprises the following components: 50-400 μmol / mL divalent metal salt, 10-200 μmol / mL imidazole salt, and 10-200 μmol / mL cyanoimidazole.

[0017] Secondly, the present invention provides the application of the above-mentioned cyclizing reagent in the synthesis of cyclic oligonucleotides.

[0018] Thirdly, the present invention provides a method for the chemical synthesis of cyclic oligonucleotides, comprising the following steps:

[0019] Cyclic oligonucleotides are obtained by adding cyclizing reagents to the components of a linear oligonucleotide solution and performing a cyclization reaction.

[0020] In some embodiments, the linear oligonucleotide contains a phosphate group at its 3' or 5' end.

[0021] In some embodiments, the linear oligonucleotide solution is prepared by dissolving the linear oligonucleotide in water, heating it to 90±2℃, and then cooling it to room temperature.

[0022] Preferably, the heating is performed by heating the solution to 90±2℃ at a rate of 1-2℃ / min.

[0023] Preferably, the cooling is performed by cooling the solution to room temperature at a rate of 1-2 °C / min.

[0024] Preferably, the concentration of the linear oligonucleotide solution is 0.05-5 μmol / mL.

[0025] In some embodiments, the molar ratio of the linear oligonucleotide to the divalent metal salt, imidazole salt, or cyanide halide is 1:50-100:100-200:100-200.

[0026] In some embodiments, the molar ratio of the linear oligonucleotide to the divalent metal salt, imidazole salt, or cyanoimidazole is 1:50-100:100-200:100-200.

[0027] In some embodiments, the molar ratio of the linear oligonucleotide to the divalent metal salt and cyanoimidazole is 1:50-100:100-200.

[0028] In some embodiments, the cyclization reaction is carried out at room temperature.

[0029] The beneficial effects of this invention are:

[0030] (1) The cyclization reagent of the present invention can realize the synthesis of linear oligonucleotides into cyclic oligonucleotides, and the synthesis method is simple and does not introduce non-natural groups.

[0031] (2) The present invention is specially designed for linear oligonucleotide chains, so that multiple nucleotides at the 3' and 5' ends can undergo base complementary pairing, which further enhances the efficiency of cyclization.

[0032] (3) Compared with the prior art, the synthesis method of the present invention is simple, low in cost, and has high cyclic efficiency, and has a good application prospect. Attached Figure Description

[0033] Figure 1 The mass spectra of the 76nt linear oligonucleotide before and after cyclization in Example 2-1 are shown. Detailed Implementation

[0034] The following description of the embodiments is merely to aid in understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the scope of the claims. The following description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not limited to the embodiments shown herein, but can be applied to a wider scope consistent with the principles and novel features disclosed herein.

[0035] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] In this invention, "room temperature" refers to ambient temperature, ranging from 10°C to 40°C. In some embodiments, "room temperature" refers to a temperature ranging from 20°C to 30°C; in other embodiments, "room temperature" refers to a temperature ranging from 25°C to 30°C; and in still other embodiments, "room temperature" refers to 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, etc. Unless otherwise specified, all temperatures mentioned in this invention refer to room temperature.

[0037] In this invention, "cyclic oligonucleotide" and "cyclic oligonucleotide" have the same meaning. All reagents used in this invention are commercially available or can be prepared using the methods described herein.

[0038] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to provide a more detailed description of the present invention.

[0039] The linear oligonucleotide in this invention is a linear oligonucleotide chain prepared by solid-phase synthesis. The 3' or 5' end of the oligonucleotide chain must contain a phosphate group. The imidazole salt is imidazole hydrochloride, purchased from Aladdin.

[0040] Example 1: Preparation of linear oligonucleotide solution

[0041] The linear oligonucleotide (76nt) was dissolved in water to prepare a 0.2 μmol / mL linear oligonucleotide solution. The linear oligonucleotide solution was heated from room temperature to 90±2℃ at a heating rate of 1-2℃ / min, and then cooled back to room temperature at a cooling rate of 1-2℃ / min.

[0042] Example 2-1

[0043] Cycloning reagents composed of divalent metal salts, imidazole salts, and cyanide halides

[0044] 200 μmol / mL nickel chloride solution, for later use;

[0045] Prepare a 100 μmol / mL imidazole salt solution and adjust the pH to 6 with hydrochloric acid for later use.

[0046] Prepare a 100 μmol / mL cyanogen bromide solution for later use.

[0047] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (5 mL) of 0.2 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, then add 50 μmol (250 μL) of 200 μmol / mL nickel chloride solution, and vortex until homogeneous; then add 100 μmol (1 mL) of 100 μmol / mL imidazole salt solution, and vortex until homogeneous; finally add 100 μmol (1 mL) of 100 μmol / mL cyanogen bromide solution, and vortex until homogeneous. Place the reaction flask in a shaker and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to test the purity of the cyclization product until the cyclization yield reaches the expected target.

[0048] Figure 1 The images show the mass spectra before and after cyclization. As can be seen from the molecular weights in the images, the molecular weight decreased by approximately 18 Da after cyclization, and no non-natural groups were introduced.

[0049] Example 2-2

[0050] Cycloning reagents composed of divalent metal salts, imidazole salts, and cyanide halides

[0051] 400 μmol / mL manganese chloride solution, for later use;

[0052] Prepare a 200 μmol / mL imidazole salt solution and adjust the pH to 6 with hydrochloric acid for later use.

[0053] Prepare a 200 μmol / mL cyanogen bromide solution for later use.

[0054] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (5 mL) of 0.2 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, then add 100 μmol (250 μL) of 400 μmol / mL manganese chloride solution and vortex until homogeneous; then add 200 μmol (1 mL) of 200 μmol / mL imidazole salt solution and vortex until homogeneous; finally add 200 μmol (1 mL) of 200 μmol / mL cyanogen bromide solution and vortex until homogeneous. Place the reaction flask in a shaker and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to test the purity of the cyclized product until the cyclization yield reaches the expected target.

[0055] Example 2-3

[0056] Cycloning reagents composed of divalent metal salts, imidazole salts, and cyanide halides:

[0057] 50 μmol / mL magnesium chloride solution, for later use;

[0058] Prepare a 10 μmol / mL imidazole salt solution and adjust the pH to 6 with hydrochloric acid for later use.

[0059] Prepare a 10 μmol / mL cyanogen bromide solution for later use.

[0060] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (5 mL) of 0.2 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, then add 80 μmol (1.6 mL) of 50 μmol / mL cadmium chloride solution and vortex until homogeneous; then add 150 μmol (15 mL) of 10 μmol / mL imidazole salt solution and vortex until homogeneous; finally add 150 μmol (15 mL) of 10 μmol / mL cyanogen bromide solution and vortex until homogeneous. Place the reaction flask in a shaker and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to test the purity of the cyclized product until the cyclization yield reaches the expected target.

[0061] Comparative Example 2-1

[0062] The difference between this comparative example and Example 2-1 is that the molar ratio of linear oligonucleotides to divalent metal salts, imidazole salts, and cyanide halides is different.

[0063] Specifically:

[0064] Cycloning reagents composed of divalent metal salts, imidazole salts, and cyanide halides

[0065] 200 μmol / mL nickel chloride solution, for later use;

[0066] Prepare a 100 μmol / mL imidazole salt solution and adjust the pH to 6 with hydrochloric acid for later use.

[0067] Prepare a 100 μmol / mL cyanogen bromide solution for later use.

[0068] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (5 mL) of 0.2 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, add 200 μmol (1 mL) of 200 μmol / mL nickel chloride solution, and vortex until homogeneous; then add 50 μmol (500 μL) of 100 μmol / mL imidazole salt solution, and vortex until homogeneous; finally add 50 μmol (500 μL) of 100 μmol / mL cyanogen bromide solution, vortex until homogeneous, place the reaction flask in a shaker, and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to test the purity of the cyclization product until the cyclization yield reaches the expected target.

[0069] Comparative Example 2-2

[0070] The difference between this comparative example and Example 2-1 is that the molar ratio of linear oligonucleotides to divalent metal salts, imidazole salts, and cyanide halides is different.

[0071] Specifically:

[0072] Cycloning reagents composed of divalent metal salts, imidazole salts, and cyanide halides

[0073] 200 μmol / mL nickel chloride solution, for later use;

[0074] Prepare a 100 μmol / mL imidazole salt solution and adjust the pH to 6 with hydrochloric acid for later use.

[0075] Prepare a 100 μmol / mL cyanogen bromide solution for later use.

[0076] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (5 mL) of 0.2 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, then add 20 μmol (100 μL) of 200 μmol / mL nickel chloride solution, and vortex until homogeneous; then add 300 μmol (3 mL) of 100 μmol / mL imidazole salt solution, and vortex until homogeneous; finally add 300 μmol (3 mL) of 100 μmol / mL cyanogen bromide solution, and vortex until homogeneous. Place the reaction flask in a shaker and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to test the purity of the cyclized product until the cyclization yield reaches the expected target.

[0077] Example 3-1

[0078] Cycloning reagent composed of divalent metal salt, imidazole salt and cyanoimidazolium

[0079] 200 μmol / mL magnesium chloride solution, for later use;

[0080] Prepare a 100 μmol / mL cyanoimidazole solution for later use.

[0081] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (5 mL) of 0.2 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, add 50 μmol (250 μL) of 200 μmol / mL magnesium chloride solution, and vortex until homogeneous; then add 100 μmol (1 mL) of 100 μmol / mL cyanoimidazole solution, vortex until homogeneous, place the reaction flask in a shaker, and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to test the purity of the cyclized product until the cyclization yield reaches the expected target.

[0082] Example 3-2

[0083] Cycloning reagent composed of divalent metal salt, imidazole salt and cyanoimidazolium

[0084] 400 μmol / mL zinc chloride solution, for later use;

[0085] Prepare a 200 μmol / mL cyanoimidazole solution for later use.

[0086] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (5 mL) of 0.2 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, then add 50 μmol (250 μL) of 400 μmol / mL zinc chloride solution, and vortex until homogeneous; then add 100 μmol (1 mL) of 200 μmol / mL cyanoimidazole solution, vortex until homogeneous, place the reaction flask in a shaker, and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to test the purity of the cyclized product until the cyclization yield reaches the expected target.

[0087] Example 3-3

[0088] Cycloning reagent composed of divalent metal salt imidazole salt and cyanoimidazolium

[0089] A 50 μmol / mL magnesium chloride solution is prepared for later use.

[0090] Prepare a 10 μmol / mL cyanoimidazole solution for later use.

[0091] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (5 mL) of 0.2 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, then add 80 μmol (1.6 mL) of 50 μmol / mL magnesium chloride solution, and vortex until homogeneous; then add 150 μmol (15 mL) of 10 μmol / mL cyanoimidazole solution, vortex until homogeneous, place the reaction flask in a shaker, and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to test the purity of the cyclized product until the cyclization yield reaches the expected target.

[0092] Comparative Example 3-1

[0093] The difference between this comparative example and Example 3-1 is that the molar ratio of the linear oligonucleotide to the divalent metal salt and cyanoimidazole is different.

[0094] Specifically:

[0095] Cycloning reagent composed of divalent metal salt, imidazole salt and cyanoimidazolium

[0096] 200 μmol / mL magnesium chloride solution, for later use;

[0097] Prepare a 100 μmol / mL cyanoimidazole solution for later use.

[0098] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (5 mL) of 0.2 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, then add 200 μmol (1 mL) of 200 μmol / mL magnesium chloride solution, and vortex until homogeneous; then add 50 μmol (500 μL) of 100 μmol / mL cyanoimidazole solution, vortex until homogeneous, place the reaction flask in a shaker, and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to test the purity of the cyclized product until the cyclization yield reaches the expected target.

[0099] Comparative Example 3-2

[0100] The difference between this comparative example and Example 3-1 is that the molar ratio of the linear oligonucleotide to the divalent metal salt and cyanoimidazole is different.

[0101] Specifically:

[0102] Cycloning reagent composed of divalent metal salt, imidazole salt and cyanoimidazolium

[0103] 200 μmol / mL magnesium chloride solution, for later use;

[0104] Prepare a 100 μmol / mL cyanoimidazole solution for later use.

[0105] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (5 mL) of 0.2 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, then add 40 μmol (200 μL) of 200 μmol / mL magnesium chloride solution, and vortex until homogeneous; then add 250 μmol (2.5 mL) of 100 μmol / mL cyanoimidazole solution, vortex until homogeneous, place the reaction flask in a shaker, and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to test the purity of the cyclized product until the cyclization yield reaches the expected target.

[0106] Example 4-1

[0107] Cycloning reagent composed of divalent metal salt, imidazole salt and cyanoimidazolium

[0108] 200 μmol / mL cadmium chloride solution, for later use;

[0109] Prepare a 100 μmol / mL imidazole salt solution and adjust the pH to 6 with hydrochloric acid for later use.

[0110] Prepare a 100 μmol / mL cyanoimidazole solution for later use.

[0111] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (volume 2 mL) of 0.5 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, then add 50 μmol (volume 250 μL) of 200 μmol / mL cadmium chloride solution, and vortex until homogeneous; then add 100 μmol (volume 1 mL) of 100 μmol / mL imidazole salt solution, and vortex until homogeneous; finally add 100 μmol (volume 1 mL) of 100 μmol / mL cyanoimidazole solution, and vortex until homogeneous. Place the reaction flask in a shaker and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to detect the cyclization efficiency until the cyclization yield reaches the expected target.

[0112] Example 4-2

[0113] Cycloning reagent composed of divalent metal salt, imidazole salt and cyanoimidazolium

[0114] 400 μmol / mL cadmium chloride solution, for later use;

[0115] Prepare a 200 μmol / mL imidazole salt solution and adjust the pH to 6 with hydrochloric acid for later use.

[0116] Prepare a 200 μmol / mL cyanoimidazole solution for later use.

[0117] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (volume 2 mL) of 0.5 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, then add 50 μmol (volume 250 μL) of 400 μmol / mL cadmium chloride solution, and vortex until homogeneous; then add 100 μmol (volume 1 mL) of 200 μmol / mL imidazole salt solution, and vortex until homogeneous; finally add 100 μmol (volume 1 mL) of 200 μmol / mL cyanoimidazole solution, and vortex until homogeneous. Place the reaction flask in a shaker and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to detect the cyclization efficiency until the cyclization yield reaches the expected target.

[0118] Example 4-3

[0119] Cycloning reagent composed of divalent metal salt, imidazole salt and cyanoimidazolium

[0120] Prepare a 50 μmol / mL cadmium chloride solution for later use;

[0121] Prepare a 10 μmol / mL imidazole salt solution and adjust the pH to 6 with hydrochloric acid for later use.

[0122] Prepare a 10 μmol / mL cyanoimidazole solution for later use.

[0123] Chemical synthesis method of cyclic oligonucleotides: Take 1 μmol (volume 2 mL) of 0.5 μmol / mL linear oligonucleotide solution into a 5 mL glass vial, then add 60 μmol (volume 1.2 mL) of 50 μmol / mL cadmium chloride solution, and vortex until homogeneous; then add 120 μmol (volume 12 mL) of 10 μmol / mL imidazole salt solution, and vortex until homogeneous; finally add 120 μmol (volume 12 mL) of 10 μmol / mL cyanoimidazole solution, and vortex until homogeneous. Place the reaction flask in a shaker and carry out the cyclization reaction at room temperature (25 °C). Take samples every 2 hours to detect the cyclization efficiency until the cyclization yield reaches the expected target.

[0124] The cyclization results of the cyclic oligonucleotides prepared in each example and comparative example are shown in Table 1.

[0125] Table 1

[0126] Example 2-1 10±2％ Example 2-2 8±2％ Example 2-3 15±2％ Comparative Example 2-1 5±2％ Comparative Example 2-2 5±2％ Example 3-1 15±3％ Example 3-2 20±3％ Example 3-3 25±3％ Comparative Example 3-1 8±2％ Comparative Example 3-2 10±2％ Example 4-1 40±5％ Example 4-2 50±5％ Example 4-3 55±5％

[0127] Note: The cyclization yield in the table is the data after 24 hours of cyclization.

[0128] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A cyclizing reagent, characterized in that, It includes the following components: 50-400 μmol / mL divalent metal salt, 10-200 μmol / mL imidazole salt and 10-200 μmol / mL cyanoimidazole; The divalent metal salt is cadmium chloride; When the cyclizing agent is used to synthesize cyclic oligonucleotides, the molar ratio of the linear oligonucleotide to the divalent metal salt, imidazole salt, and cyanoimidazole is 1:50-100:100-200:100-200.

2. The cyclizing reagent according to claim 1, characterized in that, It includes the following components: 50 μmol / mL divalent metal salt, 10 μmol / mL imidazole salt and 10 μmol / mL cyanoimidazole.

3. The cyclizing reagent according to claim 1, characterized in that, When the cyclizing agent is used to synthesize cyclic oligonucleotides, the molar ratio of the linear oligonucleotide to the divalent metal salt, imidazole salt, and cyanoimidazole is 1:60:120:

120.

4. The use of the cyclizing agent according to any one of claims 1-3 in the synthesis of cyclic oligonucleotides.

5. A method for the chemical synthesis of a cyclic oligonucleotide, characterized in that, The method includes the following steps: adding each component of the cyclizing reagent according to any one of claims 1-3 to a linear oligonucleotide solution to carry out a cyclization reaction to obtain a cyclic oligonucleotide.

6. The chemical synthesis method according to claim 5, characterized in that, The linear oligonucleotide contains a phosphate group at its 3' or 5' end.

7. The chemical synthesis method according to claim 6, characterized in that, The linear oligonucleotide solution is prepared by dissolving the linear oligonucleotide in water, heating it to 90±2℃, and then cooling it to room temperature.

8. The chemical synthesis method according to claim 7, characterized in that, The heating is performed by heating the solution to 90±2℃ at a rate of 1-2℃ / min; the cooling is performed by cooling the solution to room temperature at a rate of 1-2℃ / min.

9. The chemical synthesis method according to any one of claims 5-8, characterized in that, The molar ratio of the linear oligonucleotide to the divalent metal salt, imidazole salt, and cyanoimidazole is 1:50-100:100-200:100-200.

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