CN118546193A - Oxidizing agent for oxidizing phosphite triester and application thereof - Google Patents

Abstract

The present invention generally relates to the technical field of chemical synthesis of nucleic acids, and specifically, to an oxidant for oxidizing triester phosphite and its application. The oxidant for oxidizing triester phosphite is an iodine solution, and the solvent contains tetrahydrofuran (THF), N,N-dimethylformamide (DMF) and water in a volume ratio of (7-9): (0.8-1.2): (0.8-1.2); and the oxidant also contains (1-3) mL/L of an organic base with a pKa of 9-14.

Description

Oxidant for oxidation of triester phosphite and its application

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application No. 202310171405.0 filed on February 27, 2023, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention generally relates to the technical field of chemical synthesis of nucleic acids, and in particular to an oxidant for oxidizing phosphite triester and application thereof.

Background Art

Primers are short, single-stranded DNA or RNA molecules composed of oligonucleotides, which serve as the starting point of DNA replication in organisms. Since the general structure of nucleic acids was proposed in the 1950s and the chemical synthesis of DNA was promoted, large-scale and inexpensive oligonucleotides synthesized by chemical methods have been widely used. Including polymerase chain reaction (PCR), probes, cloning and DNA sequencing adapters, etc., it has greatly promoted the research and development of life sciences such as synthetic biology, protein engineering, genome engineering, and metabolic engineering. However, unlike the unique primer polymerase in organisms that can synthesize from the 5'→3' end of nucleotides under mild conditions, the complex environment of chemical synthesis is often accompanied by many unwanted by-products. Therefore, developing a stable and efficient strategy for synthesizing oligonucleotide chains has always been a hot topic in the current research field.

The solid phase triester phosphite method has become the preferred route in the field of commercial oligonucleotide synthesis since the 1980s due to its fast and efficient coupling and stable starting reactants. It usually includes a four-step cyclic synthesis pathway. First, the dimethoxytrityl (DMT) of the nucleotide connected to the solid phase support column (CPG) is deblocked by acid; then, the target phosphoramidite-protected monomer is mixed with tetrazole to obtain a 3' activated nucleoside-phosphite activated intermediate, which undergoes a condensation reaction with the free 5' terminal hydroxyl (5'-OH); thereafter, the 5'-OH that does not participate in the reaction is capped by acetylation reaction of maleic anhydride and N-methylimidazole with the hydroxyl group to prevent it from participating in subsequent reactions; finally, the unstable trivalent triester phosphite is oxidized to a stable pentavalent triester phosphite with an iodine solution. After these four steps, a target base is grafted on the solid phase support, and then the above cycle is repeated in sequence until the target base is synthesized in sequence. Among them, the condensation efficiency of the base can be judged by the removal color of the DMT cation during the deblocking process. At present, due to the inherent limitation that the chemical synthesis efficiency cannot reach 100%, the length of oligonucleotides synthesized based on phosphoramidite chemistry is generally limited to 200 nucleotides, and theoretically cannot exceed 300 nucleotides. For the synthesis of long DNA, DNA assembly is required to assemble short oligonucleotides into long DNA. It can be seen that the efficient reaction of each step in the four-step cycle of the solid phase triester phosphite method is the decisive factor restricting the quality of commercial oligonucleotide chains.

Currently, there are many approaches to improve the efficiency of solid phase triester phosphite synthesis, mainly focusing on replacing the tetrazole activator in the condensation step to improve the condensation efficiency; however, few people have mentioned the defects of the iodine/tetrahydrofuran/pyridine/water ( _I2 /THF/pyridine/ _H2O ) reagent formula used in the common oxidation step in the actual production process.

Summary of the invention

In one aspect of the present invention, it relates to an oxidant for oxidizing triester phosphite, which is an iodine solution, wherein the solvent comprises tetrahydrofuran (THF), N,N-dimethylformamide (DMF) and water in a volume ratio of (7-9):(0.8-1.2):(0.8-1.2);

The oxidant also contains (1-3) mL/L of an organic base with a pKa of 9-14.

In some embodiments, the volume ratio of THF, DMF and water in the solvent is (7.5-8.5):(0.9-1.1):(0.9-1.1); preferably, the volume ratio is 8:1:1. In some embodiments, the concentration of iodine in the iodine solution is 0.015M-0.03M.

In some embodiments, the organic base is selected from one or more of triethylamine, tert-butylamine, n-propylamine, N,N-diisopropylethylamine, N-methylimidazole and 1,8-diazabicyclo[5.4.0]undec-7-ene; preferably, the organic base is triethylamine.

In some embodiments, the oxidizing agent does not contain pyridine.

Another aspect of the present invention relates to a kit for synthesizing oligonucleotides using the solid phase phosphite triester method, which contains the oxidizing agent as described above.

Another aspect of the present invention relates to a method for preparing the oxidant as described above, comprising:

a) adding iodine to a mixed solution of THF, DMF and water and mixing;

b) mixing an organic base with the solution obtained in a).

In some embodiments, the preparation of the oxidant is carried out under light-protecting conditions.

Another aspect of the present invention relates to a method for synthesizing an oligonucleotide, comprising the following steps: oxidizing an intermediate having a phosphite triester bond into a pentavalent phosphotriester intermediate in the presence of an oxidizing agent;

The oxidant is an iodine solution, the solvent comprises THF, DMF and water in a volume ratio of (7-9):(0.8-1.2):(0.8-1.2), and the oxidant also contains (1-3) mL/L of an organic base with a pKa of 9-14.

Another aspect of the present invention relates to the use of the above-mentioned oxidizing agent or the above-mentioned kit in the synthesis of oligonucleotides by solid phase triester phosphite method.

In some embodiments, the oligonucleotide is selected from a primer, a probe, a siRNA, a miRNA, an mRNA, a sgRNA, a nucleic acid aptamer or an antisense nucleic acid.

The oxidant provided by the present invention effectively avoids the defects of the traditional I ₂ /THF/pyridine/H ₂ O oxidant formula, such as iodine consumption and crystal formation, and is used to achieve efficient oxidation of phosphite triester intermediates into pentavalent phosphate triester intermediates.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is an LC-MS mass spectrum of the oligonucleotide obtained under the synthesis conditions of Example 1;

FIG2 is an LC-MS mass spectrum of the oligonucleotide obtained under the synthesis conditions of Example 2;

FIG3 is an LC-MS mass spectrum of the oligonucleotide obtained under the synthesis conditions of Example 3;

FIG. 4 is an LC-MS mass spectrum of oligonucleotides obtained under comparative example synthesis conditions.

DETAILED DESCRIPTION

References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2%, it can allow fluctuations within ±0.1%. For values that are large or do not require too fine control, it is also allowed to include greater fluctuations. For example, 100mM, it can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, it is allowed to include fluctuations of ±10%.

In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

In the present invention, "oligonucleotide" refers to a compound obtained by connecting two or more nucleotides to a nucleoside. It should be noted that "oligonucleotide" also includes: a phosphorothioate-type oligonucleotide in which the oxygen atom of the phosphate group is replaced by a sulfur atom, an oligonucleotide in which -O- of the phosphate group is replaced by -NH-, and an oligonucleotide in which the hydroxyl group (-OH) in the phosphate group is replaced by -OY (where Y represents an organic group). The number of nucleosides in the oligonucleotide in the present invention is not particularly limited, and the longer the oligonucleotide synthesized by solid phase, the greater the probability of problems. Therefore, the preferred oligonucleotide length does not exceed 150 bases, such as 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 bases, preferably no more than 80 bases, preferably 3 to 50, and more preferably 5 to 30. .

In the present invention, the term "nucleic acid" or "nucleotide" refers to a linear compound (oligonucleotide) in which nucleotides are linked via phosphodiester bonds, and is considered to include DNA, RNA, etc. The nucleic acid may be single-stranded or double-stranded. Because it allows efficient synthesis using a nucleic acid synthesizer, the nucleic acid is preferably single-stranded. In this specification, "nucleic acid" includes not only oligonucleotides containing purine bases such as adenine (A), guanine (G), etc. and pyrimidine bases such as thymine (T), cytosine (C), uracil (U), etc., but also modified oligonucleotides containing other modified heterocyclic bases.

In the present invention, the term "oxidation of phosphite triester" refers to a process of oxidizing unstable trivalent phosphite triester into stable pentavalent phosphate triester in a solid phase phosphite triester process.

The invention relates to an oxidant for oxidizing triester phosphite, which is an iodine solution, wherein the solvent comprises THF, DMF and water in a volume ratio of (7-9):(0.8-1.2):(0.8-1.2);

The oxidant also contains (1-3) mL/L of an organic base with a pKa of 9-14.

This product has long-term stability and selective oxidation properties, and the synthesized oligonucleotides are of higher purity.

In some embodiments, the volume ratio of THF, DMF and water in the solvent is (7.5-8.5):(0.9-1.1):(0.9-1.1).

In some embodiments, the volume ratio of THF, DMF and water in the solvent is (7.7-8.3):(0.95-1.05):(0.95-1.05).

In some embodiments, the volume ratio of THF, DMF and water in the solvent is 8:1:1.

In some embodiments, the concentration of iodine in the iodine solution is 0.015M to 0.03M, for example, 0.02M, 0.025M.

The organic base is usually in liquid form, and its content is (1-3) mL/L, such as 1.3 mL/L, 1.5 mL/L, 1.8 mL/L, 2 mL/L, 2.3 mL/L, 2.5 mL/L, 2.8 mL/L. The pKa of the organic base is 9-14, such as 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5.

In general, organic bases contain nitrogen atoms, such as amine compounds and nitrogen-containing heterocyclic compounds. In some embodiments, the organic base is selected from one or more of triethylamine, tert-butylamine, n-propylamine, N,N-diisopropylethylamine, N-methylimidazole and 1,8-diazabicyclo[5.4.0]undec-7-ene. In a preferred embodiment, the organic base is triethylamine.

In some embodiments, the oxidizing agent does not contain pyridine.

The oxidant provided by the present invention can avoid the introduction of harmful reagent pyridine, has no obvious crystallization and pinhole blockage in the actual synthesis process, and has no obvious difference in quality between the obtained product and the traditional oxidant.

The present invention also relates to a kit for solid phase synthesis of oligonucleotides by the phosphite triester method, which contains the oxidizing agent as described above.

The kit may also contain other components commonly used in the solid phase triester phosphite method, such as cleaning solution, buffer, blocking reagent for blocking free hydroxyl groups, sulfiding agent, acid and other components, as well as optional containers for holding the components, experimental instructions and the like.

According to another aspect of the present invention, it also relates to a method for preparing the oxidant as described above, comprising:

a) adding iodine to a mixed solution of THF, DMF and water and mixing;

b) mixing an organic base with the solution obtained in a).

Firstly, a stable iodine mixed solution of I ₂ /THF/DMF/H ₂ O is prepared, and then an organic base TEA is added in a corresponding proportion according to the volume of the solution to prepare an oxidant having a higher I ₂ concentration than a traditional iodine solution formula.

In some embodiments, the preparation method is carried out under light-proof conditions.

The present invention also relates to a method for synthesizing an oligonucleotide, comprising the following steps: oxidizing an intermediate having a phosphite triester bond into a pentavalent phosphotriester intermediate in the presence of an oxidant;

The oxidant is an iodine solution, the solvent comprises THF, DMF and water in a volume ratio of (7-9):(0.8-1.2):(0.8-1.2), and the oxidant also contains (1-3) mL/L of an organic base with a pKa of 9-14.

The present invention also relates to the use of the above-mentioned oxidizing agent or the above-mentioned kit in the solid phase synthesis of oligonucleotides by the phosphite triester method.

The solid phase synthesis method of triester phosphite is well known. The improvement of the present invention lies in the process of oxidizing unstable trivalent triester phosphite into stable pentavalent triester phosphite. Therefore, it is easy to understand that related variations other than this process or which have no substantial effect on the oxidation of triester trivalent phosphite are not excluded from the content of the above-mentioned "solid phase synthesis method of triester phosphite".

As an example, a typical process of "phosphite triester solid phase synthesis" may include:

1) providing a nucleoside monomer, nucleotide or oligonucleotide (a), wherein the 5'-hydroxyl group is protected by a substituted or unsubstituted dimethoxytrityl group (DMT), and at least one nucleoside monomer, nucleotide or oligonucleotide is bound to a solid support;

removing the substituted or unsubstituted trityl group under the action of acid to obtain free hydroxyl groups, and washing the solid phase support;

2) condensing the nucleoside monomer, nucleotide or oligonucleotide (b) with the nucleoside monomer, nucleotide or oligonucleotide (a) after the reaction in step 1) to obtain a phosphite triester;

wherein the 3' position of the nucleoside monomer, nucleotide or oligonucleotide (b) has a phosphoramidite group and is pre-activated by an acidic activating agent;

3) using an oxidant, or an oxidant + a sulfiding agent to convert the phosphite triester into a phosphate triester or a thiophosphate triester.

Steps 1) to 3) may be repeated multiple times to extend the oligomeric compound successively, and after the oligomeric compound reaches the desired length, the oligomeric compound may further include step 4): excising the obtained oligomeric compound from the solid phase, removing all its protecting groups, and optionally purifying it. The methods for excising from the solid phase and purifying may be performed using methods known in the art, and the preferred purification methods are desalting, BioRP/OPC purification, HPLC or PAGE purification.

The solid phase used in the solid phase triester phosphite method is not particularly limited in the present invention, and it is preferably porous particles, and further preferably porous particles with functional groups that contribute to nucleic acid synthesis. "Functional groups that contribute to nucleic acid synthesis" refers to functional groups that can become the starting point of nucleic acid synthesis and can be added with joints. In particular, amino, hydroxyl, etc. can be mentioned. The shape of the above-mentioned porous solid phase carrier is not particularly limited, and can be a flat plate, particle, fiber, etc. in any shape. Because the filling efficiency of the synthesis reaction vessel can be enhanced, the reaction vessel is not easy to destroy, and thus preferably has a porous synthetic polymer in particle shape. The term "particle" in the specification does not mean precise spherical, but means having any constant shape (for example, roughly spherical, such as elliptical spherical, polygonal, cylindrical, amorphous form, etc.). Although the size (volume) of a particle of porous synthetic polymer particles is not particularly limited, when the average particle size obtained when measuring porous particles by laser diffraction (scattering type) is less than 1 micron, it will be inconvenient when it is packaged in a column, and the back pressure becomes too high or the solution delivery speed decreases when used. On the other hand, when the average particle size is greater than 1000 μm, the spaces between the carrier particles become larger, and it becomes difficult to effectively pack the carrier particles in a column having a predetermined volume. Therefore, it is preferably 1 to 1000 μm, more preferably 5 to 500 μm, and further preferably 10 to 200 μm.

Examples of solid phase carriers include rigid functional supports such as polystyrene supports, polydimethylacrylamide encapsulated with diatomaceous earth, silica or microporous glass; the solid phase resin matrix can be composed of amphiphilic polystyrene-PEG resin or PEG-polyamide or PEG-polyester resin; as solid phase carriers, Wang-PEG resin or Rink-amide PEG resin are also included. The most commonly used solid phase carrier is controlled pore glass beads (CPG). The pore size of CPG depends on the length of the synthesized oligonucleotide. Generally, when the synthetic chain length is less than 60mer, a pore size of 500 angstroms CPG is selected; when the chain length is greater than 60mer, 1000 angstroms CPG is used. The coupling efficiency of CPG is as high as 98%-99.9%, which can meet the conditions for synthesizing oligonucleotides up to 175mer. CPG is covalently bonded to the hydroxyl group of the initial nucleotide through a linker, and the 5'-OH of the nucleotide is protected with DMT. In some embodiments, the CPG is an unmodified naked CPG having hydroxyl groups on its surface, which are covalently linked to an oligonucleotide/one or several nucleotides.

In some embodiments, the oligonucleotide is selected from a primer, a probe, a siRNA, a miRNA, an mRNA, a sgRNA, a nucleic acid aptamer or an antisense nucleic acid.

The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

Example 1

Preparation of a new formula of I ₂ /THF/DMF/H ₂ O iodine mixture and an oxidant containing organic base TEA

(1) First, 100 mL of DMF and 100 mL of H ₂ O were added to a brown glass bottle containing 800 mL of THF and mixed thoroughly;

(2) adding the weighed _I2 element to the solution prepared in step (1) and mixing evenly to obtain a 0.03 M iodine mixed solution;

(3) Finally, 1 mL of TEA solution was pipetted into the iodine solution prepared in step (2) and mixed thoroughly to obtain a new oxidant.

Example 2

Preparation of a new formula of I ₂ /THF/DMF/H ₂ O iodine mixture and an oxidant containing organic base TEA

(1) First, 100 mL of DMF and 100 mL of H ₂ O were added to a brown glass bottle containing 800 mL of THF and mixed thoroughly;

(2) adding the weighed _I2 element to the solution prepared in step (1) and mixing evenly to obtain a 0.03 M iodine mixed solution;

(3) Finally, 2 mL of TEA solution was pipetted into the iodine solution prepared in step (2), and the mixture was thoroughly mixed to obtain a novel oxidant.

Example 3

Preparation of a new formula of I ₂ /THF/DMF/H ₂ O iodine mixture and an oxidant containing organic base TEA

(1) First, 100 mL of DMF and 100 mL of H ₂ O were added to a brown glass bottle containing 800 mL of THF and mixed thoroughly;

(2) adding the weighed _I2 element to the solution prepared in step (1) and mixing evenly to obtain a 0.03 M iodine mixed solution;

(3) Finally, 3 mL of TEA solution was pipetted into the iodine solution prepared in step (2) and mixed thoroughly to obtain a new oxidant.

Comparative Example

Preparation of I ₂ /THF/pyridine/H ₂ O iodine mixed solution oxidant with common market formula

(1) First, weigh 200 mL of pyridine and 100 mL of H ₂ O respectively and add them into a brown glass bottle containing 700 mL of THF and mix them thoroughly;

(2) Add the weighed _I2 element to the solution prepared in step (1) and mix well to obtain a 0.03 M iodine mixed solution, which is then fully mixed to obtain an oxidant.

Experimental example

In the specific embodiment of the present invention, the recognition performance evaluation is carried out according to the following method: oligonucleotide synthesis is performed using a Dr.Oligo 192 high-throughput oligonucleotide synthesizer. A 50nmol solid phase CPG synthesis carrier column is inserted into a 96-well synthesis plate, and the target oligonucleotide sequence is introduced. The detailed synthesis steps are shown in Table 1. After the synthesis, the oligonucleotide carrier column is placed in a gas phase pot and cut and deprotected by ammonia gas phase aminolysis (90°C, 60min). After the nucleotide cleavage is completed, acetonitrile is used for washing, and the oligonucleotide is eluted from the synthesis carrier with 80μL TE buffer; the eluate from the previous step is taken according to Table 2 for LC-MS detection, OD ₂₆₀ value is determined using an enzyme marker, and high performance liquid chromatography (HPLC) purification analysis detection, and the influence of different ratios on the quality of oligonucleotide synthesis is analyzed according to purity and MS spectrum.

Table 1 DNA oligonucleotide synthesis cycle table on solid phase synthesizer

Table 2 Product testing methods and qualified standards

Synthetic oligonucleotide sequence: GAAGGTGATGTTGAACACGA

Under the conditions of using the oxidants prepared in Experimental Examples 1/2/3 and the Comparative Example, the detection results of the synthesized oligonucleotides are shown in Table 3 below, and the obtained LC-MS mass spectra are shown in Figures 1-4 respectively;

Table 3 Oligonucleotide synthesis sequence detection results

The purity and cost comparison of oligonucleotides synthesized using different ratios of oxidants by HPLC analysis are shown in Table 4 below.

Table 4 Comparison of synthesis purity and cost of each experimental example

in conclusion:

The synthesis results of the iodine solution of the best embodiment 2 (i.e., 1L of 0.03MI ₂ /THF/DMF/H ₂ O plus 2ml TEA) as the oxidant show that except for the presence of a small amount of base pre-depurination impurity peaks, no obvious impurities are generated under the gas phase aminolysis conditions, and the purity of the synthesized oligonucleotide reaches up to 87.05%; in terms of cost, the cost of the conventional formula of the comparative example is 129.93 yuan/L, while the new formula is only about 88.17 yuan/L. The conventional technology represented by the comparative example has the problems of reduced actual iodine concentration, introduction of harmful reagent pyridine, easy crystallization of synthesizer pinholes during actual solid phase synthesis, and incomplete clogging of CPG channels by residual reagent cleaning. The experimental results show that the new oxidant provided by the present application has obvious advantages over the traditional iodine solution oxidant formula, not only has long-term stability and selective oxidation performance, but also has a higher purity of synthesized oligonucleotides. In addition, it effectively avoids the introduction of harmful reagent pyridine, greatly reducing production costs.

The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims (11)

1. An oxidant for oxidizing triester phosphite, which is an iodine solution, wherein the solvent comprises THF, DMF and water in a volume ratio of (7-9):(0.8-1.2):(0.8-1.2); The oxidant also contains (1-3) mL/L of an organic base with a pKa of 9-14. 2. The oxidant according to claim 1, wherein the volume ratio of THF, DMF and water in the solvent is (7.5-8.5):(0.9-1.1):(0.9-1.1); preferably, the volume ratio is 8:1:1. 3. The oxidant according to claim 1, wherein the concentration of iodine in the iodine solution is 0.015M to 0.03M. 4. The oxidant according to claim 1, wherein the organic base is selected from one or more of triethylamine, tert-butylamine, n-propylamine, N,N-diisopropylethylamine, N-methylimidazole and 1,8-diazabicyclo[5.4.0]undec-7-ene; preferably, the organic base is triethylamine. 5 . The oxidizing agent according to claim 1 , which does not contain pyridine. 6. A kit for solid phase synthesis of oligonucleotides by a phosphite triester method, comprising the oxidizing agent according to any one of claims 1 to 5. 7. The method for preparing the oxidant according to any one of claims 1 to 5, comprising: a) adding iodine to a mixed solution of THF, DMF and water and mixing; b) mixing an organic base with the solution obtained in a). The preparation method according to claim 7 , which is carried out under light-proof conditions. 9. A method for synthesizing an oligonucleotide, comprising the following steps: oxidizing an intermediate having a phosphite triester bond into a pentavalent phosphotriester intermediate in the presence of an oxidant; The oxidant is an iodine solution, the solvent comprises THF, DMF and water in a volume ratio of (7-9):(0.8-1.2):(0.8-1.2), and the oxidant also contains (1-3) mL/L of an organic base with a pKa of 9-14. 10. Use of the oxidizing agent according to any one of claims 1 to 5 or the kit according to claim 6 in solid phase synthesis of oligonucleotides using the phosphite triester method. 11. The use according to claim 10, wherein the oligonucleotide is selected from a primer, a probe, a siRNA, a miRNA, an mRNA, a sgRNA, a nucleic acid aptamer or an antisense nucleic acid.