Cyclic reagent and application thereof, and chemical synthesis method of cyclic oligonucleotide

Abstract

The invention discloses a cyclization reagent and application thereof and a chemical synthesis method of cyclic oligonucleotide, and belongs to the technical field of chemical synthesis, the cyclization reagent comprises the following components: divalent metal salt, imidazolium salt and cyanogen halide, or divalent metal salt and cyano imidazole, or divalent metal salt, imidazolium salt and cyano imidazole; the chemical synthesis method comprises the following steps: adding the components of the cyclization reagent into a linear oligonucleotide solution, and carrying out cyclization reaction to obtain the cyclic oligonucleotide. The cyclization reagent can realize synthesis of linear-to-cyclic oligonucleotide, the synthesis method is simple, and non-natural groups are not introduced. Compared with the prior art, the synthesis method disclosed by the invention is simple, low in cost and high in cyclic efficiency, and has a very good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical synthesis, and relates to a cyclization reagent and its application, as well as a chemical synthesis method of cyclic oligonucleotides. Background Art

[0002] As a new type of gene therapy drug, oligonucleotides have broad application prospects in clinical practice. Oligonucleotides are usually synthesized by phosphoramidite solid-phase synthesis, but the produced oligonucleotides are all linear; compared with linear oligonucleotides, cyclic oligonucleotides have important biological functions and application values 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], Progress in Pharmaceutical Sciences, 2024, 48(8), 592 - 604).

[0003] The synthesis methods of cyclic oligonucleotides mainly include enzymatic synthesis and chemical synthesis. Enzymatic synthesis has become the preferred method due to the good biocompatibility of enzymes and high cyclization yields, but the problem of generally low yields exists in enzymatic synthesis. Chemical synthesis is to connect the two ends of oligonucleotides head-to-tail through mild, simple and efficient chemical reactions.

[0004] At present, there are few studies and reports on the chemical synthesis of cyclic oligonucleotides. The synthesis methods mainly include azide-alkyne click chemical reaction cyclization, carboxyl and amino amidation reaction cyclization, etc. Although the existing chemical synthesis methods can make up for the defect of insufficient yield in enzymatic synthesis, they also have problems such as introducing non-natural groups and complex operations.

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

[0006] In order to solve the above technical problems, the present invention provides a cyclization reagent and its application, as well as a chemical synthesis method of cyclic oligonucleotides. The cyclization reagent of the present invention is simple to operate, has a high cyclization yield, and does not introduce non-natural groups during the chemical synthesis process of cyclic oligonucleotides.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions.

[0008] In the first aspect, the present invention provides a cyclization reagent, which comprises the following components: divalent metal salt, imidazole salt and cyanogen halide, or divalent metal salt and cyanoimidazole, or divalent metal salt, imidazole salt and cyanoimidazole.

[0009] In some embodiments, the divalent metal salt is a water-soluble salt, including any one of chlorides, nitrates, phosphates and sulfates.

[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 cyanogen halide is at least one of cyanogen fluoride, cyanogen chloride, cyanogen bromide, and cyanogen iodide.

[0013] Each component of the cyclization reagent of the present invention is independently formulated and then mixed separately when in use.

[0014] In some embodiments, the cyclization reagent includes the following components: 50 - 400 μmol / mL divalent metal salt, 10 - 200 μmol / mL imidazole salt, and 10 - 200 μmol / mL cyanogen halide.

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

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

[0017] In a second aspect, the present invention provides the application of the above cyclization reagent in the synthesis of cyclic oligonucleotides.

[0018] In a third aspect, the present invention provides a chemical synthesis method of cyclic oligonucleotides, including the following steps:

[0019] Adding each component of the cyclization reagent to the linear oligonucleotide solution for cyclization reaction to obtain cyclic oligonucleotides.

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

[0021] In some embodiments, the preparation method of the linear oligonucleotide solution is: dissolving the linear oligonucleotide in water, heating to 90 ± 2 °C, and then cooling to room temperature.

[0022] Preferably, the heating is: heating the solution to 90 ± 2 °C at a rate of 1 - 2 °C / min.

[0023] Preferably, the cooling is: 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, and cyanogen 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, and 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 temperature of the cyclization reaction is room temperature.

[0029] Advantages of the present invention:

[0030] (1) The cyclization reagent of the present invention can achieve the synthesis of linear oligonucleotides to cyclic oligonucleotides, and the synthesis method is simple without introducing unnatural groups.

[0031] (2) The present invention makes a special design for the linear oligonucleotide chain, enabling multiple nucleotides at the 3' and 5' ends to undergo base complementary pairing, further enhancing the cyclization efficiency.

[0032] (3) Compared with the prior art, the synthesis method of the present invention is simple, low in cost, and high in cyclic efficiency, having good application prospects. Description of the Drawings

[0033] Figure 1 Mass spectrometry diagrams before and after cyclization of the 76 nt linear oligonucleotide in Example 2 - 1. Detailed Embodiments

[0034] The following description of the embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. 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 obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but can be applied to a wider range consistent with the principles and novel features disclosed herein.

[0035] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0036] In the present invention, "room temperature" refers to the ambient temperature, which is from 10°C to 40°C. In some embodiments, "room temperature" refers to the temperature from 20°C to 30°C; in other embodiments, "room temperature" refers to the temperature from 25°C to 30°C; 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, the temperatures involved in the present invention are all room temperature.

[0037] In the present invention, "cyclic oligonucleotide" and "cyclic oligonucleotide" have the same meaning. The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0038] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

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

[0040] Example 1 Linear oligonucleotide solution configuration

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

[0042] Example 2-1

[0043] Cyclization reagent composed of divalent metal salt, imidazole salt and cyanogen halide

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

[0045] 100umol / mL imidazole salt solution, and adjust the pH to 6 with hydrochloric acid for later use;

[0046] 100umol / mL cyanogen bromide solution, set aside.

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

[0048] Figure 1 Figures and

[0048] are the mass spectra before and after cyclization. It can be seen from the molecular weights in the figures that the molecular weight after cyclization is approximately 18 Da less, and no unnatural groups are introduced.

[0049] Example 2-2

[0050] Cyclization reagent composed of divalent metal salt, imidazole salt and cyanogen halide

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

[0052] 200 μmol / mL imidazole salt solution, adjust the pH to 6 with hydrochloric acid, reserved for use;

[0053] 200 μmol / mL cyanogen bromide solution, reserved for use.

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

[0055] Example 2-3

[0056] Cyclization reagent composed of divalent metal salt, imidazole salt and cyanogen halide:

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

[0058] 10 umol / mL imidazole salt solution, and adjust the pH to 6 with hydrochloric acid for standby;

[0059] 10 umol / mL cyanogen bromide solution, for standby.

[0060] Chemical synthesis method of cyclic oligonucleotide: Take 1 umol (volume is 5 mL) of 0.2 umol / mL linear oligonucleotide solution in a 5 mL glass vial, then add 80 umol (volume is 1.6 mL) of 50 umol / mL cadmium chloride solution, and shake evenly using a vortex oscillator; then add 150 umol (volume is 15 mL) of 10 umol / mL imidazole salt solution and shake evenly; finally add 150 umol (volume is 15 mL) of 10 umol / mL cyanogen bromide solution. After shaking evenly, place the reaction flask in a shaker and carry out the cyclization reaction at room temperature of 25 °C. Take samples every 2 h to detect 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 ratios of the linear oligonucleotide to the divalent metal salt, imidazole salt, and cyanogen halide are different.

[0063] Specifically:

[0064] Cyclization reagent composed of divalent metal salt, imidazole salt, and cyanogen halide

[0065] 200 umol / mL nickel chloride solution, for standby;

[0066] 100 umol / mL imidazole salt solution, and adjust the pH to 6 with hydrochloric acid for standby;

[0067] 100 umol / mL cyanogen bromide solution, for standby.

[0068] Chemical synthesis method of cyclic oligonucleotide: Take 1 umol (volume is 5 mL) of 0.2 umol / mL linear oligonucleotide solution in a 5 mL glass vial, then add 200 umol (volume is 1 mL) of 200 umol / mL nickel chloride solution, and shake evenly using a vortex oscillator; then add 50 umol (volume is 500 uL) of 100 umol / mL imidazole salt solution and shake evenly; finally add 50 umol (volume is 500 uL) of 100 umol / mL cyanogen bromide solution. After shaking evenly, place the reaction flask in a shaker and carry out the cyclization reaction at room temperature of 25 °C. Take samples every 2 h to detect the purity of the cyclized 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 lies in that the molar ratios of the linear oligonucleotide to the divalent metal salt, imidazole salt, and cyanogen halide are different.

[0071] Specifically:

[0072] The cyclization reagent composed of divalent metal salt, imidazole salt, and cyanogen halide

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

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

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

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

[0077] Example 3-1

[0078] The cyclization reagent composed of divalent metal salt, imidazole salt, and cyanoimidazole

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

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

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

[0082] Example 3-2

[0083] Cyclization reagent composed of divalent metal salt, imidazole salt and cyanoimidazole

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

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

[0086] Chemical synthesis method of cyclic oligonucleotide: Take 1 μmol (volume is 5 mL) of 0.2 μmol / mL linear oligonucleotide solution in a 5 mL glass vial, then add 50 μmol (volume is 250 μL) of 400 μmol / mL zinc chloride solution, and shake evenly using a vortex oscillator; then add 100 μmol (volume is 1 mL) of 200 μmol / mL cyanoimidazole solution, after shaking evenly, place the reaction flask in a shaker, and carry out the cyclization reaction at room temperature of 25 °C. Take samples every 2 h for purity detection of the cyclized product until the cyclization yield reaches the expected target.

[0087] Example 3-3

[0088] Cyclization reagent composed of divalent metal salt, imidazole salt and cyanoimidazole

[0089] 50 μmol / mL magnesium chloride solution, for later use;

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

[0091] Chemical synthesis method of cyclic oligonucleotide: Take 1 μmol (volume is 5 mL) of 0.2 μmol / mL linear oligonucleotide solution in a 5 mL glass vial, then add 80 μmol (volume is 1.6 mL) of 50 μmol / mL magnesium chloride solution, and shake evenly using a vortex oscillator; then add 150 μmol (volume is 15 mL) of 10 μmol / mL cyanoimidazole solution, after shaking evenly, place the reaction flask in a shaker, and carry out the cyclization reaction at room temperature of 25 °C. Take samples every 2 h for purity detection 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 linear oligonucleotide to divalent metal salt and cyanoimidazole is different.

[0094] Specifically:

[0095] Cyclization reagent composed of divalent metal salt, imidazole salt and cyanoimidazole

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

[0097] 100 umol / mL cyanimidazole solution, for standby.

[0098] Chemical synthesis method of cyclic oligonucleotide: Take 1 umol (volume 5 mL) of 0.2 umol / mL linear oligonucleotide solution in a 5 mL glass vial, then add 200 umol (volume 1 mL) of 200 umol / mL magnesium chloride solution, and shake evenly using a vortex oscillator; then add 50 umol (volume 500 uL) of 100 umol / mL cyanimidazole solution, after shaking evenly, place the reaction flask in a shaker, and carry out cyclization reaction at room temperature of 25 °C, sample once every 2 h to detect 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 ratios of linear oligonucleotide to divalent metal salt and cyanimidazole are different.

[0101] Specifically:

[0102] Cyclization reagent composed of divalent metal salt, imidazole salt and cyanimidazole

[0103] 200 umol / mL magnesium chloride solution, for standby;

[0104] 100 umol / mL cyanimidazole solution, for standby.

[0105] Chemical synthesis method of cyclic oligonucleotide: Take 1 umol (volume 5 mL) of 0.2 umol / mL linear oligonucleotide solution in a 5 mL glass vial, then add 40 umol (volume 200 uL) of 200 umol / mL magnesium chloride solution, and shake evenly using a vortex oscillator; then add 250 umol (volume 2.5 mL) of 100 umol / mL cyanimidazole solution, after shaking evenly, place the reaction flask in a shaker, and carry out cyclization reaction at room temperature of 25 °C, sample once every 2 h to detect the purity of the cyclized product until the cyclization yield reaches the expected target.

[0106] Example 4-1

[0107] Cyclization reagent composed of divalent metal salt, imidazole salt and cyanimidazole

[0108] 200 umol / mL cadmium chloride solution, for standby;

[0109] 100 umol / mL imidazole salt solution, and adjust the pH to 6 with hydrochloric acid, for standby;

[0110] 100 umol / mL cyanimidazole solution, for standby.

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

[0112] Example 4-2

[0113] Cyclization reagent composed of divalent metal salt, imidazole salt and cyanoimidazole

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

[0115] 200 μmol / mL imidazole salt solution, and adjust the pH to 6 with hydrochloric acid, for standby;

[0116] 200 μmol / mL cyanoimidazole solution, for standby.

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

[0118] Example 4-3

[0119] Cyclization reagent composed of divalent metal salt, imidazole salt and cyanoimidazole

[0120] 50 μmol / mL cadmium chloride solution, for standby;

[0121] 10 μmol / mL imidazole salt solution, and adjust the pH to 6 with hydrochloric acid, for standby;

[0122] 10 μmol / mL cyanoimidazole solution, for standby.

[0123] Chemical synthesis method of cyclic oligonucleotide: Take 1 μmol (volume: 2 mL) of a 0.5 μmol / mL linear oligonucleotide solution in a 5 mL glass vial, then add 60 μmol (volume: 1.2 mL) of a 50 μmol / mL cadmium chloride solution, and shake well using a vortex oscillator; then add 120 μmol (volume: 12 mL) of a 10 μmol / mL imidazole salt solution and shake well; finally, add 120 μmol (volume: 12 mL) of a 10 μmol / mL cyanoimidazole solution. After shaking well, place the reaction flask in a shaker and carry out a cyclization reaction at room temperature (25 °C). Take samples every 2 h for cyclization efficiency detection 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] Yield % 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 h of cyclization.

[0128] The method of the present invention has been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate modifications and combinations to the methods and applications described herein within the content, spirit, and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A cyclization reagent, characterized in that The invention comprises the following components: a divalent metal salt, an imidazole salt and a cyanogen halide, or a divalent metal salt and a cyanoimidazole, or a divalent metal salt, an imidazole salt and a cyanoimidazole.

2. The cyclization reagent according to claim 1, characterized in that The divalent metal includes at least one of zinc, magnesium, manganese, nickel and cadmium.

3. The cyclization reagent according to claim 1, characterized in that The cyanogen halide is at least one of cyanogen fluoride, cyanogen chloride, cyanogen bromide and cyanogen iodide.

4. The cyclization reagent according to any one of claims 1 to 3, characterized in that The cyclization reagent comprises the following components: 50-400umol / mL divalent metal salt, 10-200umol / mL imidazole salt and 10-200umol / mL cyanogen halide, or 50-400umol / mL divalent metal salt and 10-200umol / mL cyanoimidazole, or 50-400umol / mL divalent metal salt, 10-200umol / mL imidazole salt and 10-200umol / mL cyanoimidazole.

5. Use of the cyclization reagent according to any one of claims 1 to 4 in the synthesis of circular oligonucleotides.

6. A method for chemically synthesizing a circular oligonucleotide, characterized in that: The method comprises the following steps: adding the components of the cyclization reagent according to any one of claims 1 to 4 to a linear oligonucleotide solution to carry out a cyclization reaction to obtain a cyclic oligonucleotide.

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

8. The chemical synthesis method according to claim 7, characterized in that The preparation method of the linear oligonucleotide solution is: dissolving the linear oligonucleotide in water, heating to 90±2° C., and then cooling to room temperature.

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

10. The chemical synthesis method according to any one of claims 6 to 9, characterized in that: The molar ratio of the linear oligonucleotide to the divalent metal salt, the imidazole salt, and the cyanogen halide is 1:50-100:100-200:100-200; or the molar ratio of the linear oligonucleotide to the divalent metal salt and the cyanoimidazole is 1:50-100:100-200; Or the molar ratio of the linear oligonucleotide to the divalent metal salt, the imidazole salt and the cyanoimidazole is 1:50-100:100-200:100-200.

Images