An oligonucleotide synthesis catalyst

Abstract

This invention provides an oligonucleotide synthesis catalyst and its preparation method, as well as a method for synthesizing oligonucleotides. The oligonucleotide synthesis catalyst comprises at least one of a tetrazolium, azole salt, pyridine salt, and imidazole salt compound as the main component, and a polar additive. The oligonucleotide synthesis catalyst of this invention solves the problems of current oligonucleotide synthesis processes, such as the difficulty in synthesizing complex oligonucleotide sequences (e.g., oligonucleotide chains that are too long, have high GC content, or have repetitive or continuous sequences that easily generate secondary structures), the difficulty in obtaining the target oligonucleotide fragment, or the high impurity content and poor quality of the target oligonucleotide fragment. When the oligonucleotide synthesis catalyst of this invention is applied to oligonucleotide synthesis, the success rate of synthesizing high-GC-content oligonucleotides can be increased, and the quality of oligonucleotides can be improved.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to PCT patent application No. PCT / CN2020 / 124820, filed on October 29, 2020, entitled “An Oligonucleotide Synthesis Catalyst”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of nucleotide synthesis, and more specifically, relates to an oligonucleotide synthesis catalyst and its preparation method, as well as a method for synthesizing oligonucleotides. Background Technology

[0004] Oligonucleotides, also known as oligomeric nucleotides, are often synthesized using the solid-phase phosphoramidite method. This method involves immobilizing oligonucleotides on a solid support and synthesizing the oligonucleotide chain through four cyclic steps: deprotection, coupling, capping, and oxidation. [1-2] The process involves four steps: First, deprotection: The protecting group dimethoxytriphenylmethyl (DMT) pre-mounted on a solid support is removed using trichloroacetic acid to obtain free 5′-hydroxyl groups. Second, coupling: An activated intermediate is obtained by reacting a catalyst with the phosphorusamide monomer to be coupled (referred to as the monomer). This intermediate is then coupled with the previously obtained oligonucleotide 5′-hydroxyl group via condensation. Third, capping: The remaining few unreacted 5′-hydroxyl groups are blocked using acetic anhydride and N-methylimidazole. Fourth, oxidation: Trivalent phosphorus (P) is oxidized to more stable pentavalent phosphorus under the action of an oxidant. [3-4] .

[0005] The most important process in nucleotide synthesis is coupling. Currently, methods to improve condensation efficiency mainly focus on two aspects: one is structural modification of the phosphite acyl group, as exemplified by Huang Shifu's approach. [5] A phosphorylating agent (2-nitrile ethoxy) was designed; this group promotes the growth of oligonucleotide chains. (Keith Andrew Bowman) [6] Similarly, designs were developed to link D-prolyl and its derivatives to the phosphate group of nucleosides, which can improve the selectivity of chiral oligonucleotides and increase product purity. The second aspect involves catalyst optimization, focusing on catalysts that activate oligonucleotides to produce intermediates, such as azoles, pyridinium salts, and azole salts. [7-9] Both of the above methods can improve condensation efficiency, while the structural effect of single-stranded oligonucleotides themselves... [10-11] It is highly likely to inhibit the synthesis of oligonucleotides. For example, during the synthesis of oligonucleotides, there are often situations where no product is obtained or the product purity is low. This may be due to the oligonucleotide chain being too long, the GC content being high, or the repetitive or continuous sequences being prone to generating secondary structures. However, no relevant literature has yet proposed a solution to such problems in the solid-phase synthesis of oligonucleotides. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of oligonucleotide synthesis, such as the inability to obtain products or low product purity, by optimizing the catalysts used in oligonucleotide synthesis. More specifically, it provides a catalyst more suitable for oligonucleotide synthesis in the solid-phase phosphoramidite ester method commonly used in oligonucleotide synthesis, thereby improving the condensation efficiency of intermediates and oligonucleotide 5′-hydroxyl groups.

[0007] The inventors have discovered and confirmed that, after obtaining the required oligonucleotides (up to 180 bp in length) through DNA solid-phase synthesis (the synthesis cycle consists of deprotection, coupling, capping, and oxidation), polar reagents such as DMSO, betaine, and lithium chloride, acting as additives, can form hydrogen bonds with the bases in the oligonucleotides, weakening the hydrogen bonding between their own bases and reducing steric hindrance, thereby improving the catalytic efficiency of oligonucleotide condensation. Simultaneously, DMSO, as a nucleophilic dissociating solvent, promotes nucleophilic substitution reactions during coupling, further enhancing condensation efficiency. Therefore, these additives have a highly efficient promoting effect on the synthesis of oligonucleotides with special structures (e.g., excessively long oligonucleotide chains, high GC content, repetitive or continuous sequences that easily generate secondary structures). Based on the above findings, the inventors have thus completed this invention.

[0008] In one aspect, the present invention provides an oligonucleotide synthesis catalyst comprising at least one of a tetrazolium, azole salt, pyridine salt, and imidazole salt compound as a main component, and a polar additive. In some embodiments, the oligonucleotide synthesis catalyst comprises a pyridine salt and a polar additive.

[0009] In one embodiment of the present invention, the pyridine salt compound may be pyridine trifluoroacetate onium salt (PTFA). In the present invention, the amount of catalyst required for solid-phase synthesis of oligonucleotides in the art can be determined.

[0010] In one embodiment of the invention, the polar additive is selected from at least one of dimethyl sulfoxide (DMSO), betaine, and lithium chloride, preferably DMSO. In some embodiments, the polar additive is DMSO. In other embodiments, the polar additive is betaine.

[0011] In one embodiment of the invention, the polar additive is present in an amount of 0.01-15% by weight, preferably 0.1-10% by weight, based on the weight of the main component. In some embodiments, the polar additive is dimethyl sulfoxide, present in an amount of 1-10% by weight, preferably 2% by weight. In other preferred embodiments, the polar additive is dimethyl sulfoxide, present in an amount of 1-5% by weight. In some preferred embodiments, the polar additive is dimethyl sulfoxide, present in an amount of 2-5% by weight. In one specific embodiment, the polar additive is dimethyl sulfoxide, present in an amount of 2% by weight. In some embodiments, the polar additive is betaine, present in an amount of 0.01-0.5% by weight, preferably 0.1% by weight. In other embodiments, the polar additive is lithium chloride, present in an amount of 0.01-0.5% by weight, preferably 0.1% by weight.

[0012] In one embodiment of the present invention, the oligonucleotide synthesis catalyst further comprises acetonitrile as a solvent.

[0013] In one embodiment of the present invention, the oligonucleotide synthesis catalyst further comprises N-methylimidazole as a stabilizer.

[0014] In one embodiment of the present invention, the oligonucleotide synthesis catalyst is used to catalyze the coupling reaction of oligonucleotides in solid-phase synthesis.

[0015] In another aspect, the present invention also provides a method for preparing the oligonucleotide synthesis catalyst as described above, the method comprising contacting at least one of a tetrazolium, azole salt, pyridine salt, and imidazole salt compound with the polar additive. In some embodiments, the contact is carried out in acetonitrile.

[0016] In another aspect, the present invention also provides a method for synthesizing oligonucleotides, the method comprising synthesizing oligonucleotide chains by solid-phase phosphoramidite method using an oligonucleotide synthesis catalyst as described above.

[0017] In one embodiment of the present invention, the solid-phase phosphoramidite process includes cyclic deprotection, coupling, capping and oxidation steps or cyclic deprotection, coupling, oxidation and capping steps.

[0018] In one embodiment of the invention, the oligonucleotide synthesis catalyst is used in the coupling step.

[0019] Beneficial technical effects of the present invention:

[0020] The oligonucleotide synthesis catalyst of this invention solves the problems of current oligonucleotide synthesis processes, such as the difficulty in synthesizing complex oligonucleotide sequences (e.g., excessively long oligonucleotide chains, high GC content (e.g., GC content higher than 50%, 70%, or even 90%), and the tendency for repetitive or continuous sequences to generate secondary structures), the difficulty in obtaining the target oligonucleotide fragment, or the high quality of the target oligonucleotide fragment due to numerous impurities. When the oligonucleotide synthesis catalyst of this invention is applied to oligonucleotide synthesis, the success rate of synthesizing high-GC-content oligonucleotides can be increased, thus improving the quality of the oligonucleotides.

[0021] Furthermore, the current mainstream method for gene production involves designing appropriate overlaps using short-chain oligonucleotides, and then splicing these oligonucleotides into the target DNA fragment via PCR. In other words, for DNA fragments of the same length, because the oligonucleotides synthesized by this invention are longer, the number of oligonucleotide fragments that need to be designed will be reduced accordingly, thus saving synthesis costs. Attached Figure Description

[0022] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0023] Figure 1 A schematic diagram of the oligonucleotide synthesis cycle is shown;

[0024] Figure 2 PAGE electrophoresis patterns of oligonucleotides 1, 2, and 3 synthesized by PTFA solutions with different concentrations of DMSO, betaine, and lithium chloride are shown (a. DMSO content in PTFA is 0%; b. DMSO content in PTFA is 1%; c. DMSO content in PTFA is 2%; d. DMSO content in PTFA is 10%; e. Betaine content in PTFA is 0.1%; f. Betaine content in PTFA is 1%; g. Lithium chloride content in PTFA is 0.1%; h. Lithium chloride content in PTFA is 1%).

[0025] Figure 3 MS spectra of oligonucleotide 1 are shown (a. PTFA with 2% DMSO catalyst; b. PTFA catalyst).

[0026] Figure 4 MS spectra of oligonucleotide 2 are shown (a. PTFA with 2% DMSO catalyst; b. PTFA catalyst).

[0027] Figure 5 MS spectra of oligonucleotide 3 are shown (a. PTFA with 2% DMSO catalyst; b. PTFA catalyst).

[0028] Figure 6 MS spectra of oligonucleotide 1 are shown (a. PTFA with 0.1% betaine content as catalyst; b. PTFA with 1% betaine content as catalyst);

[0029] Figure 7 MS spectra of oligonucleotide 2 are shown (a. PTFA with 0.1% betaine content as catalyst; b. PTFA with 1% betaine content as catalyst);

[0030] Figure 8 MS spectra of oligonucleotide 3 are shown (a. PTFA with 0.1% betaine content as catalyst; b. PTFA with 1% betaine content as catalyst);

[0031] Figure 9 MS spectra of oligonucleotide 1 are shown (a. PTFA with 0.1% lithium chloride as catalyst; b. PTFA with 1% lithium chloride as catalyst);

[0032] Figure 10 MS spectra of oligonucleotide 2 are shown (a. PTFA with 0.1% lithium chloride catalyst; b. PTFA with 1% lithium chloride catalyst); and

[0033] Figure 11 MS spectra of oligonucleotide 3 are shown (a. PTFA with 0.1% lithium chloride as catalyst; b. PTFA with 1% lithium chloride as catalyst). Detailed Implementation

[0034] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0035] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0036] Before describing the invention in detail, it should be understood that the terminology used herein is for describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the appended claims. For a more complete understanding of the invention described herein, the following terms are used, and their definitions are as follows. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0037] In one aspect, the present invention provides an oligonucleotide synthesis catalyst comprising at least one of tetrazolium, azole salts, pyridine salts, and imidazole salts as a major component (e.g., in a content greater than 50%, 70%, or even not less than 90%), and a polar additive. The inventors have discovered that in conventional solid-phase oligonucleotide synthesis methods, the catalysts used typically fall into the categories of tetrazolium, azole salts, pyridine salts, and imidazole salts.

[12] However, these catalysts may result in impurities and poor quality of the target oligonucleotide fragments when applied to oligonucleotide synthesis. This is particularly true when synthesizing complex oligonucleotides (e.g., those with excessively long chains, high GC content, or repetitive or continuous sequences prone to secondary structures), where obtaining the target oligonucleotide fragment may be difficult. The addition of polar additives can form hydrogen bonds with the bases in the oligonucleotide, weakening the hydrogen bonding between its own bases and reducing steric hindrance, thereby improving the catalytic efficiency of oligonucleotide condensation. In this invention, the oligonucleotide synthesis refers to a method of synthesizing oligonucleotides using a solid-phase support, such as the solid-phase phosphoramidite method, which involves completing the synthesis of the oligonucleotide chain on a solid-phase support through four steps: cyclic deprotection, coupling, capping, and oxidation.

[0038] Furthermore, among the tetrazolium, azole, pyridine, and imidazole salts that are the main components of catalysts, pyridine trifluoroacetate onium salt (PTFA), as a pyridine salt, has the advantages of low cost and excellent catalytic performance, and is therefore more suitable for large-scale industrial synthesis of oligonucleotides. Therefore, in a preferred embodiment of the present invention, the pyridine salt compound may be pyridine trifluoroacetate onium salt (PTFA), but the types of oligonucleotide synthesis catalysts of the present invention are not limited to this, and may include combinations of multiple types of tetrazolium, azole, pyridine, and imidazole salt compounds.

[0039] According to the present invention, there are no particular restrictions on the type and amount of polar additives contained in the oligonucleotide synthesis catalyst, as long as they can effectively form hydrogen bonds with the bases in the oligonucleotide, thereby weakening the hydrogen bonding between their own bases.

[0040] In a preferred embodiment of the present invention, the polar additive may be selected from at least one of DMSO, betaine, and lithium chloride. In a more preferred embodiment, the polar additive may be DMSO. DMSO is an aprotic polar solvent that is soluble in both water and organic solvents, and is known as a "universal solvent." DMSO plays a dual role as a reaction solvent and a reaction reagent in some chemical reactions. Some reactions that are difficult to achieve can proceed smoothly in DMSO, which has an accelerating and catalytic effect, and can improve the reaction rate and yield. As an aprotic solvent, DMSO can also act as a nucleophilic dissociation solvent in nucleophilic substitution reactions to accelerate the reaction, making the nucleophilic substitution reaction rate faster than that of conventional aprotic solvents.

[0041] In another preferred embodiment of the present invention, the content of the polar additive may be 0.01-10% by weight, based on the weight of the main component, for example, 0.05% by weight, 0.1% by weight, 0.5% by weight, 1% by weight, 2% by weight, 3% by weight, 4% by weight, or 5% by weight. Furthermore, the amount of polar additive contained in the oligonucleotide synthesis catalyst of the present invention may also depend on the type of polar additive; therefore, the effective content of different polar additives may only be a portion of the aforementioned 0.01-10% by weight, for example, 0.01-0.1% by weight, 0.1-1% by weight, 0.1-2% by weight, 1-5% by weight, 2-10% by weight, or 1-10% by weight.

[0042] According to the present invention, the oligonucleotide synthesis catalyst of the present invention may also contain other adjuvants commonly used in the art, such as solvents and stabilizers, as needed, as long as the adjuvant does not significantly alter or even enhance the catalytic performance of the oligonucleotide synthesis catalyst. In a preferred embodiment of the present invention, the oligonucleotide synthesis catalyst may further contain, for example, acetonitrile as a solvent; in another preferred embodiment of the present invention, the oligonucleotide synthesis catalyst may further contain, for example, N-methylimidazole as a stabilizer.

[0043] In another aspect, the present invention also provides a method for preparing the oligonucleotide synthesis catalyst as described above, the method comprising contacting at least one of a tetrazolium, azole salt, pyridine salt, and imidazole salt compound with the polar additive.

[0044] In a more specific embodiment of the preparation method of the present invention, at least one of the tetrazolium, azole salts, pyridine salts, and imidazole salts, the polar additive, acetonitrile, and N-methylimidazole may be contacted. The contacting of the above components can be carried out in any order, such as simultaneously, separately, or sequentially. Therefore, in a preferred embodiment of the present invention, the oligonucleotide synthesis catalyst in solution form can be prepared using PTFA as a catalyst, acetonitrile as a solvent, N-methylimidazole as a stabilizer, and DMSO as an additive.

[0045] In another aspect, the present invention also provides a method for synthesizing oligonucleotides, the method comprising synthesizing oligonucleotide chains via a solid-phase phosphoramidite process using an oligonucleotide synthesis catalyst as described above. In a preferred embodiment of the invention, the contact can be carried out in acetonitrile.

[0046] As previously described, the oligonucleotide synthesis catalyst of the present invention can react with the phosphorusamide monomer to be coupled in the second coupling step to obtain an activated intermediate, which is then coupled with the oligonucleotide 5′-hydroxyl group via condensation. Therefore, in a preferred embodiment of the present invention, the solid-phase phosphorusamide trimerization process may include cyclic steps of deprotection, coupling, capping, and oxidation, or cyclic steps of deprotection, coupling, oxidation, and capping. In another preferred embodiment of the present invention, the oligonucleotide synthesis catalyst may be used in the coupling step.

[0047] The inventors have discovered that the oligonucleotide synthesis catalyst of this invention solves the problems of current oligonucleotide synthesis processes, such as the difficulty in synthesizing complex oligonucleotide sequences (e.g., oligonucleotide chains that are too long, have high GC content, or have repetitive or continuous sequences that easily generate secondary structures), the difficulty in obtaining the target oligonucleotide fragment, or the high quality of the target oligonucleotide fragment due to numerous impurities. When the oligonucleotide synthesis catalyst of this invention is applied to oligonucleotide synthesis, it can increase the success rate of synthesizing high-GC-content oligonucleotides and improve the quality of oligonucleotides.

[0048] The effects of the specific oligonucleotide synthesis catalyst of the present invention will be described in detail below through examples.

[0049] Example

[0050] Example 1: Preparation of Instruments and Materials

[0051] Pyridine, trifluoroacetic acid, N-methylimidazole, DMSO, lithium chloride, betaine, acetonitrile, iodine, tetrahydrofuran (analytical grade, Sinopharm Group); TCA solution (Suzhou Kelama Biotechnology Co., Ltd.); CAP-A solution, CAP-B solution (Anhui Shilian Special Solvents Co., Ltd.); 50 nmol universal synthetic carrier (Beijing Dinaxingke Biotechnology Co., Ltd.); DMT-dA(bz)-phosphite, DMT-dC(ac)-phosphite, DMT-dG(dmf)-phosphite and DMT-dT-phosphite were all analytical grade and purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., 4150 g.

[0052] Example 2: Preparation of the oligonucleotide synthesis catalyst and oxidizing agent of the present invention

[0053] (1) Preparation of PTFA solutions with different DMSO contents: 0 mL, 40 mL, 80 mL and 400 mL of DMSO were mixed with 0.88 mol 69.5 g pyridine, 0.88 mol 100 g trifluoroacetic acid and 0.44 mol 36 g N-methylimidazole and dissolved in 5338 mL, 5282 mL, 5226 mL and 4779 mL of acetonitrile respectively to prepare PTFA solutions with DMSO contents of 0%, 1%, 2% and 10%;

[0054] (2) Preparation of PTFA solutions with different betaine contents: 0.0285 mol 3.35 g and 0.289 mol 33.82 g of betaine were mixed with 0.88 mol 69.5 g of pyridine, 0.88 mol 100 g of trifluoroacetic acid and 0.44 mol 36 g of N-methylimidazole and dissolved in 4000 mL of acetonitrile to prepare PTFA solutions with betaine contents of 0.1% and 1% respectively;

[0055] (3) Preparation of PTFA solutions with different lithium chloride contents: 0.079 mol (3.35 g) and 0.798 mol (33.82 g) of lithium chloride were respectively mixed with 0.88 mol (69.5 g) of pyridine, 0.88 mol (100 g) of trifluoroacetic acid, and 0.44 mol (36 g) of N-methylimidazole and dissolved in 4000 mL of acetonitrile to prepare PTFA solutions with lithium chloride contents of 0.1% and 1%; and

[0056] (4) Preparation of oxidizing agent: Mix 0.06 mol 15.24 g iodine, 3120 mL tetrahydrofuran, 80 mL water and 800 mL pyridine and stir magnetically for 30 min to prepare oxidizing agent.

[0057] Example 3: Synthesis of oligonucleotides 1-3

[0058] This invention utilizes an Oligo 768 DNA synthesizer (Biolytic Lab Performance Inc. SN: BLP-30285) to synthesize designed oligonucleotides 1, 2, and 3 via deprotection, coupling, capping, and oxidation cycles (oligonucleotide sequences are shown in Table 1 below; exemplary synthesis methods are as follows). Figure 1 As shown in the figure, 160 μL of trichloroacetic acid (TCA) deprotectant was automatically added to the solid support to remove the protecting group dimethoxytriphenylmethyl (DMT). After deprotection, 22 μL of various PTFA solutions and 18 μL of 0.035 mol / L acetonitrile solution of phosphite monomer were added to couple with the deprotected 5′-hydroxyl group. After coupling, 20 μL of a self-made oxidizing agent was added to react with the phosphite triester to form a phosphate triester for oxidation. Finally, 20 μL of acetic anhydride (CAP-A) and 20 μL of N-methylimidazole (CAP-B) were added to acetylate the free 5′-hydroxyl group to complete the capping. The cycle was repeated to synthesize the desired oligonucleotide. The product was then treated with ammonia at 80 °C for 6 h to obtain the crude product, which was analyzed by PAGE electrophoresis and MS.

[0059] Table 1 Sequences of oligonucleotides 1-3

[0060]

[0061] Example 4: PAGE electrophoresis analysis

[0062] The PAGE image of the product obtained in the example is shown below. Figure 2 As shown, Figure 2 a is the PAGE analysis diagram of the product obtained by PTFA catalysis without additives, which shows that the target product cannot be obtained without additives. Figure 2 b, 2c, and 2d are the products obtained at DMSO concentrations of 1%, 2%, and 10%, respectively. Figure 2 Oligonucleotide 1 in c is more Figure 2 In b and 2d, product 1 has a clear band, but still contains faint impurities. Figure 2 The bands of oligonucleotides 2 and 3 in b, 2c, and 2d are all... Figure 2 c has a clear main band and low impurity band content. Therefore, among the three methods, 2% DMSO is most favorable for oligonucleotide synthesis. (Note: The designed sequence has a high GC content in the first band, and its own structure causes a slower electrophoresis speed than normal oligonucleotides during PAGE).

[0063] Figure 2 e and 2f are PAGE spectra of oligonucleotides obtained with 0.1% and 1% betaine, respectively. Figure 2 As shown in e, 0.1% betaine can yield the target oligonucleotide fragment, while Figure 2 The desired oligonucleotide cannot be obtained by using 1% betaine in f; Figure 2 g and 2h are PAGE patterns of oligonucleotides obtained with 0.1% and 1% lithium chloride, respectively. The target oligonucleotide could not be obtained with 1% lithium chloride, while the target oligonucleotide fragment could be obtained with 0.1% lithium chloride.

[0064] Compare Figure 2 The PAGE spectra of the oligonucleotides obtained from c, 2e, and 2g show that... Figure 2 e and Figure 2 g contains a relatively high amount of impurities, while Figure 2 The main band in c is clear, therefore, a DMSO concentration of 2% is most favorable for oligonucleotide synthesis.

[0065] Example 5: MS Result Analysis

[0066] The oligonucleotide coupling reaction using 2% DMSO and PTFA solution as catalyst yielded the highest quality products. Therefore, products 1, 2, and 3 were analyzed by MS. To enhance data comparability, products obtained from the reaction using PTFA solution without additives were analyzed by MS in the same manner. The analytical spectra are shown below. Figures 3 to 5 As shown. Figure 3 Oligonucleotide 1, obtained by the PTFA solution catalytic reaction without additives, has a maximum MS value of 21145.2, which does not match the theoretical molecular weight of 24869.87. Figure 3 The oligonucleotides obtained by the PTFA solution catalyzed by the addition of 2% DMSO had an MS value of 24873, which is approximately consistent with the theoretical value; similarly, Figure 4 b represents oligonucleotide 2 obtained from the PTFA solution catalytic reaction without additives. The maximum MS value was 13717.3, which differs from the theoretical value of 22349.3. Figure 4 The MS value obtained by the PTFA solution with 2% DMSO added was 22348.9, which is consistent with the theoretical value. Figure 5 b represents oligonucleotide 3 obtained from the PTFA solution catalytic reaction without additives. Its maximum MS value is 7073.1, which is less than the theoretical value of 27525.8. Figure 5 The oligonucleotide obtained by the reaction catalyzed by PTFA solution with 2% DMSO added had an MS value of 27523.8, which is consistent with the theoretical value.

[0067] Similarly, the oligonucleotides synthesized from betaine and lithium chloride were analyzed by MS, and the results are as follows: Figures 6 to 11 As shown; Figures 6 to 8MS spectra of oligonucleotides obtained by coupling reactions catalyzed by adding 0.1% and 1% betaine to PTFA show that the target oligonucleotide can be obtained under the catalysis of PTFA solution with a concentration of 0.1% betaine, while no target peak is observed under the catalysis of PTFA solution with a concentration of 1% betaine, indicating that the target oligonucleotide cannot be obtained. Similarly, from... Figures 9 to 11 It can be concluded that adding 0.1% lithium chloride to PTFA can catalyze the reaction to obtain the target oligonucleotide, while adding 1% lithium chloride will not produce the target peak.

[0068] MS and PAGE analyses revealed that in the coupled reaction of oligonucleotide synthesis, it was difficult to obtain the target oligonucleotide without the addition of additives to the PTFA catalyst. Adding polar additives to PTFA effectively promoted the synthesis of the target oligonucleotide fragment. However, excessively high concentrations inhibited oligonucleotide synthesis, possibly because excess polar additives formed hydrogen bonds with the active hydroxyl group at the 5′ end, hindering the attack of the hydroxyl group on the P(III) terminal to be linked. Experimental data showed that adding 2% DMSO to PTFA resulted in the optimal quality of the synthesized target oligonucleotide.

[0069] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0070] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0071] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

[0072] References

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[0074] [2]Zamecnik PC,Stephenson M L.Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide[J].Proceedings of the National Academy of Sciences,1978,75(1):280-284.

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[0076] [4] Beaucage SL, Iyer R P. Advances in the synthesis of oligonucleotides by the phosphoramidite approach [J]. Tetrahedron, 1992, 48(12): 2223-2311.

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[0081] [9] Vargeese C, Carter J, Yegge J, et al., Efficient activation of nucleosidephosphoramidites with 4,5-dicyanoimidazole during oligonucleotide synthesis[J]. Nucleic acids research, 1998, 26(4):1046-1050.]

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Claims

1. A catalyst for oligonucleotide synthesis comprising, as a main component, a pyridine trifluoroacetate onium salt, and a polar additive; in, The polar additive is dimethyl sulfoxide, with a content of 1-10% by weight; or The polar additive is betaine, with a content of 0.01-0.5% by weight; or The polar additive is lithium chloride, with a content of 0.01-0.5% by weight.

2. The catalyst for oligonucleotide synthesis according to claim 1, wherein, The polar additive is dimethyl sulfoxide, and its content is 2% by weight.

3. The catalyst for oligonucleotide synthesis according to claim 1, wherein, The polar additive is betaine, with a content of 0.1% by weight.

4. The catalyst for oligonucleotide synthesis according to claim 1, wherein, The polar additive is lithium chloride, with a content of 0.1% by weight.

5. The catalyst for oligonucleotide synthesis according to claim 1, further comprising acetonitrile as a solvent.

6. The catalyst for oligonucleotide synthesis according to claim 1, further comprising N-methylimidazole as a stabilizer.

7. A method for preparing a catalyst for oligonucleotide synthesis according to any one of claims 1-6, comprising contacting a pyridine trifluoroacetic acid onium salt with the polar additive.

8. The preparation method according to claim 7, wherein the contact is carried out in acetonitrile.

9. A method for synthesizing oligonucleotides, comprising synthesizing oligonucleotide chains by solid-phase phosphoramidite esterification using a catalyst for oligonucleotide synthesis according to any one of claims 1-6.

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