CN114425443B

CN114425443B - Oligonucleotide synthesis catalyst

Info

Publication number: CN114425443B
Application number: CN202111270458.5A
Authority: CN
Inventors: 黄东亮; 王进; 张龙学; 戴兵
Original assignee: Jiangsu Genscript Biotech Co Ltd
Current assignee: Jiangsu Genscript Biotech Co Ltd
Priority date: 2020-10-29
Filing date: 2021-10-29
Publication date: 2025-10-31
Anticipated expiration: 2041-10-29
Also published as: CN114425443A

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

Oligonucleotide synthesis catalyst

Cross Reference to Related Applications

The present application claims priority from PCT patent application No. PCT/CN2020/124820, entitled "one oligonucleotide Synthesis catalyst", filed on even 29 th 10/2020, the entire contents of which are incorporated herein by reference.

Technical Field

The invention belongs to the field of nucleotide synthesis, and in particular relates to an oligonucleotide synthesis catalyst, a preparation method thereof and a method for synthesizing an oligonucleotide.

Background

The oligonucleotide is also called as oligonucleotide, and the synthesis method adopts a solid phase phosphoramidite triester method, and the method is to fix the oligonucleotide on a solid phase carrier and complete the synthesis ^[1-2] of the oligonucleotide chain through four steps of cyclic deprotection, coupling, capping and oxidation. The first step of deprotection, namely removing a protecting group dimethoxy trityl (DMT) pre-loaded on a solid phase carrier by using trichloroacetic acid to obtain free 5' -hydroxyl, the second step of coupling, namely reacting the active intermediate with a phosphoramidite monomer (monomer for short) to be connected through a catalyst to obtain an activated intermediate, condensing the activated intermediate with the obtained oligonucleotide 5' -hydroxyl to finish coupling, the third step of capping, namely blocking the residual few unreacted 5' -hydroxyl by using acetic anhydride and N-methylimidazole, and the fourth step of oxidation, namely oxidizing trivalent phosphorus (P) into more stable pentavalent phosphorus ^[3-4] under the action of an oxidant.

The most important of the nucleotide synthesis reaction is the coupling process, so that the condensation efficiency is improved mainly from two aspects, namely, on one hand, a phosphorylating reagent (2-nitrile ethoxy) is designed for structural modification of phosphite acyl group, such as Huang Shifu ^[5], the group can promote the growth of an oligonucleotide chain, on the other hand, the base, andru, bowman ^[6] also designs that the D-prolyl and derivatives thereof are connected to phosphoric acid of the nucleotide to improve the selectivity of the chiral oligonucleotide and the purity of the product, and on the other hand, the optimization of a catalyst is realized, and the focus of the catalyst is on the generation of intermediates of the activated oligonucleotide, such as an azole, a pyridinium, an azolium and the like, as a catalyst ^[7-9]. Both of these methods can increase the efficiency of condensation, whereas the structuring effect ^[10-11] of the single stranded oligonucleotide itself is highly likely to inhibit oligonucleotide synthesis. Such as the synthesis of oligonucleotides, there are often cases where the product is not obtained or the purity of the product is not high, which may be caused by the fact that the oligonucleotide has too long a chain length, high GC content, repeated or continuous sequences, etc. are liable to generate secondary structures, but no related literature has been proposed to solve such problems in solid phase synthesis of oligonucleotides.

Disclosure of Invention

The present invention aims to solve the above-mentioned drawbacks that the product is often not obtained or the purity of the product is not high in the oligonucleotide synthesis process, and to optimize the catalyst used in the oligonucleotide synthesis process, more specifically, to provide an oligonucleotide synthesis catalyst more suitable for the solid phase phosphoramidite triester method commonly used in oligonucleotide synthesis, so as to improve the condensation efficiency of the intermediate and the 5' -hydroxyl of the oligonucleotide.

The inventors have found and confirmed that, after the solid phase synthesis of DNA (the synthesis cycle is deprotection, coupling, capping and oxidation), polar reagents such as DMSO, betaine and lithium chloride as additives can form hydrogen bonds with bases in the oligonucleotide, weaken the hydrogen bond action between the bases, reduce the steric hindrance effect, thereby improving the condensation catalysis efficiency of the oligonucleotide, and simultaneously, a DMSO nucleophilic dissociation solvent promotes the nucleophilic substitution reaction in the coupling process, thereby improving the condensation efficiency, so that the additives have high-efficiency promotion effect on the synthesis of the oligonucleotide with special structures (such as long chain length of the oligonucleotide, high GC content, repeated or continuous sequence and the like, and secondary structures are easy to generate). Based on the above findings, the present inventors have thus completed the present invention.

In one aspect, the present invention provides an oligonucleotide synthesis catalyst comprising at least one of tetrazole, azolium salt, pyridinium salt, and imidazolium salt compounds as a main component, and a polar additive. In some embodiments, the oligonucleotide synthesis catalyst comprises a pyridinium salt and a polar additive.

In one embodiment of the present invention, the pyridinium compound may be Pyridinium Trifluoroacetate (PTFA). In the present invention, it may be determined according to the amount of catalyst required for solid phase synthesis of oligonucleotides in the art.

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.

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

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

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

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

In another aspect, the invention also provides a method of preparing an oligonucleotide synthesis catalyst as described above, the method comprising contacting at least one of tetrazole, azolium, pyridinium, and imidazolium compounds with the polar additive. In some embodiments, the contacting is performed in acetonitrile.

In another aspect, the present invention also provides a method for synthesizing an oligonucleotide, the method comprising performing a solid phase phosphoramidite triester method for synthesizing an oligonucleotide chain using the oligonucleotide synthesis catalyst described above.

In one embodiment of the invention, the solid phase phosphoramidite triester process comprises or is cycled through the steps of deprotecting, coupling, capping and oxidizing.

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

The beneficial technical effects of the invention are as follows:

The oligonucleotide synthesis catalyst solves the problems that the existing oligonucleotide synthesis technology has great difficulty in synthesizing the complex oligonucleotide sequences (for example, the oligonucleotide chain length is overlong, the GC content is high (for example, the GC content is higher than 50%, 70% or even 90%), secondary structures are easy to generate in repeated or continuous sequences and the like), and the target oligonucleotide fragments or the target oligonucleotide fragments have poor impurity quality. When the oligonucleotide synthesis catalyst is applied to oligonucleotide synthesis, the success rate of synthesizing the oligonucleotide with high GC content can be increased, and the quality of the oligonucleotide can be improved.

In addition, the mainstream method of gene production today is to design appropriate overlap by short-chain oligonucleotides, and splice the oligonucleotides into a DNA fragment of interest by PCR reaction. In other words, for the same length of DNA fragments, the length of the oligonucleotides which can be synthesized by the invention is longer, and the number of designed oligonucleotide fragments is correspondingly reduced, so that the synthesis cost is saved.

Drawings

The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:

FIG. 1 shows a schematic diagram of a synthesis cycle of an oligonucleotide;

FIG. 2 shows PAGE electropherograms of synthesized oligonucleotides 1, 2 and 3 catalyzed by PTFA solutions of different concentrations of DMSO, betaine, and lithium chloride (a.0% in PTFA, b.1% in PTFA, c.2% in PTFA, d.10% in PTFA, e.0.1% in PTFA, f.1% in PTFA, 0.1% in g.0.1% in PTFA, and 1% in h.1% in PTFA);

FIG. 3 shows the MS spectra of oligonucleotide 1 (a. Catalyst is PTFA with DMSO content of 2%; b. Catalyst is PTFA);

FIG. 4 shows the MS spectra of oligonucleotide 2 (a. Catalyst is PTFA with DMSO content of 2%; b. Catalyst is PTFA);

FIG. 5 shows the MS spectra of oligonucleotide 3 (a. Catalyst is PTFA with DMSO content of 2%; b. Catalyst is PTFA);

FIG. 6 shows the MS spectra of oligonucleotide 1 (a. Catalyst is PTFA with betaine content of 0.1%; b. Catalyst is PTFA with betaine content of 1%);

FIG. 7 shows the MS spectra of oligonucleotide 2 (a. Catalyst is PTFA with betaine content of 0.1%; b. Catalyst is PTFA with betaine content of 1%);

FIG. 8 shows the MS spectra of oligonucleotide 3 (a. Catalyst is PTFA with betaine content of 0.1%; b. Catalyst is PTFA with betaine content of 1%);

FIG. 9 shows the MS spectra of oligonucleotide 1 (a. Catalyst is PTFA with lithium chloride content of 0.1%; b. Catalyst is PTFA with lithium chloride content of 1%);

FIG. 10 shows the MS spectra of oligonucleotide 2 (a. Catalyst is PTFA with lithium chloride content of 0.1%; b. Catalyst is PTFA with lithium chloride content of 1%; and

FIG. 11 shows the MS spectra of oligonucleotide 3 (a. Catalyst is PTFA with lithium chloride content of 0.1%; b. Catalyst is PTFA with lithium chloride content of 1%).

Detailed Description

The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

Before describing the present invention in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. For a more complete understanding of the invention described herein, the following terms are used and their definitions are shown below. Unless defined otherwise, 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 belongs.

In one aspect, the invention provides an oligonucleotide synthesis catalyst comprising as a major component (e.g., greater than 50%, 70% or even no less than 90%) at least one of tetrazole, azolium salt, pyridinium salt, and imidazolium salt compounds, and a polar additive. The present inventors have found that in conventional methods for solid phase synthesis of oligonucleotides, the catalysts used can be generally classified into tetrazolium, azolium, pyridinium, and imidazolium compounds ^[12], but these catalysts may result in a large number of impurities and poor quality of the target oligonucleotide fragment when applied to oligonucleotide synthesis, and particularly, it may be difficult to obtain the target oligonucleotide fragment when applied to complex (e.g., oligonucleotide chain length is too long, GC content is high, repeated or continuous sequences are liable to generate secondary structures), while the addition of polar additives can form hydrogen bonds with bases in the oligonucleotide, weaken hydrogen bonding between self bases, reduce steric hindrance effects, and thus improve the oligonucleotide condensation catalytic efficiency. In the present invention, the oligonucleotide synthesis means a method for synthesizing an oligonucleotide by using a solid phase carrier, such as a solid phase phosphoramidite triester method, comprising the steps of circularly deprotecting, coupling, capping and oxidizing the solid phase carrier to complete the synthesis of an oligonucleotide chain.

Among tetrazolium, azolium, pyridinium, and imidazolium compounds as the main components of the catalyst, pyridinium Trifluoroacetate (PTFA) as a pyridinium compound has the advantages of low cost and excellent catalytic performance, and is thus more suitable for industrial mass synthesis of oligonucleotides. Thus, in a preferred embodiment of the present invention, the pyridinium compound may be Pyridinium Trifluoroacetate (PTFA), but the kind of the oligonucleotide synthesis catalyst of the present invention is not limited thereto, and may include a combination of plural kinds of tetrazolium, azolium, pyridinium, and imidazolium compounds.

According to the present invention, the kind and amount of the polar additive contained in the oligonucleotide synthesis catalyst are not particularly limited as long as it can effectively form hydrogen bonds with bases in the oligonucleotide, thereby weakening hydrogen bonding between the bases themselves.

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 has the dual functions of a reaction solvent and a reaction reagent in some chemical reactions, and some reactions which are difficult to realize can be smoothly carried out in DMSO, and the DMSO has the functions of acceleration and catalysis, so that the reaction speed and the yield can be improved. DMSO is used as an aprotic solvent, but can be used as a nucleophilic dissociating solvent in nucleophilic substitution reaction to accelerate the reaction, so that the nucleophilic substitution reaction speed is faster than that of a conventional aprotic solvent.

In another preferred embodiment of the present invention, the polar additive may be contained in an amount of 0.01 to 10 wt%, for example, 0.05 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, etc., based on the weight of the main component. In addition, the amount of the polar additive contained in the oligonucleotide synthesis catalyst of the present invention may also depend on the kind of the polar additive, and thus the effective content of the different polar additives may be taken only from a part of the above-mentioned 0.01 to 10% by weight, for example, 0.01 to 0.1% by weight, 0.1 to 1% by weight, 0.1 to 2% by weight, 1 to 5% by weight, 2 to 10% by weight, 1 to 10% by weight, or the like.

According to the present invention, for the oligonucleotide synthesis catalyst of the present invention, it may further contain other adjuvants commonly known in the art, such as solvents and stabilizers, etc., according to actual needs, as long as the adjuvants do not significantly alter, or even promote, the catalytic performance of the oligonucleotide synthesis catalyst. In a preferred embodiment of the present invention, the oligonucleotide synthesis catalyst may further comprise acetonitrile, for example, as a solvent, and in another preferred embodiment of the present invention, the oligonucleotide synthesis catalyst may further comprise N-methylimidazole, for example, as a stabilizer.

In another aspect, the invention also provides a method of preparing an oligonucleotide synthesis catalyst as described above, the method comprising contacting at least one of tetrazole, azolium, pyridinium, and imidazolium compounds with the polar additive.

In a more specific embodiment of the preparation method of the present invention, at least one of tetrazole, azolium salt, pyridinium salt and imidazolium salt compounds, the polar additive, acetonitrile and N-methylimidazole may be further contacted. The contacting of the above components may be performed in any order, for example simultaneously, separately or sequentially. Thus, in a preferred embodiment of the present invention, the oligonucleotide synthesis catalyst may be formulated in solution with PTFA as catalyst, acetonitrile as solvent, N-methylimidazole as stabilizer, DMSO as additive.

In another aspect, the present invention also provides a method for synthesizing an oligonucleotide, the method comprising performing a solid phase phosphoramidite triester method for synthesizing an oligonucleotide chain using the oligonucleotide synthesis catalyst described above. In a preferred embodiment of the invention, the contacting may be performed in acetonitrile.

As previously mentioned, the oligonucleotide synthesis catalysts of the present invention are capable of reacting with the phosphoramidite monomer to be ligated in a second coupling step to provide an activated intermediate, such that the intermediate is condensed with the 5' -hydroxyl of the oligonucleotide to complete the coupling. Thus, in a preferred embodiment of the present invention, the solid phase phosphoramidite triester process may comprise the steps of cyclic deprotection, coupling, capping and oxidation or the steps of cyclic deprotection, coupling, oxidation and capping. In another preferred embodiment of the present invention, the oligonucleotide synthesis catalyst may be used in the step of coupling.

The research of the inventor shows that the oligonucleotide synthesis catalyst solves the problems that the existing oligonucleotide synthesis technology has great difficulty in synthesizing complex (for example, the oligonucleotide chain length is overlong, the GC content is high, the repeated or continuous sequence and the like are easy to generate secondary structures) oligonucleotide sequences, and the target oligonucleotide fragments or the target oligonucleotide fragments have poor impurity quality. When the oligonucleotide synthesis catalyst is applied to oligonucleotide synthesis, the success rate of synthesizing the oligonucleotide with high GC content can be increased, and the quality of the oligonucleotide can be improved.

Hereinafter, the effect of the specific oligonucleotide synthesis catalyst of the present invention will be described in detail by way of examples.

Examples

Example 1 preparation of instruments and materials

Pyridine, trifluoroacetic acid, N-methylimidazole, DMSO, lithium chloride, betaine, acetonitrile, iodine, tetrahydrofuran (analytical grade, national drug Co., ltd.), TCA solution (St. No. Ke Lema Biotechnology Co., ltd.), CAP-A solution, CAP-B solution (Anhui time series specialty solvents Co., ltd.), 50nmol of a general synthetic support (Beijing Di Nemack Biotechnology Co., ltd.), DMT-dA (bz) -phosphoramidite, DMT-dC (ac) -phosphoramidite, DMT-dG (dmf) -phosphoramidite and DMT-dT-phosphoramidite are all analytical grade, available from Sigma Aldrich (Shanghai) trade Co., ltd.4150 g.

EXAMPLE 2 preparation of oligonucleotide Synthesis catalyst and Oxidation reagent of the invention

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

(2) Preparation of PTFA solutions with different betaine contents A PTFA solution with betaine contents of 0.1% and 1% was prepared by mixing 0.0285mol of 3.35g and 0.289mol of 33.82g of betaine with 0.88mol of 69.5g of pyridine, 0.88mol of 100g of trifluoroacetic acid and 0.44mol of 36g of N-methylimidazole, respectively, and dissolving in 4000mL of acetonitrile, respectively;

(3) PTFA solutions with different lithium chloride contents were prepared by mixing 0.079mol of 3.35g and 0.798mol of 33.82g of lithium chloride with 0.88mol of 69.5g of pyridine, 0.88mol of 100g of trifluoroacetic acid and 0.44mol of 36g of N-methylimidazole, respectively, in 4000mL of acetonitrile to prepare PTFA solutions with lithium chloride contents of 0.1% and 1%, respectively, and

(4) Preparation of oxidizing reagent 0.06mol of 15.24g iodine, 3120mL tetrahydrofuran, 80mL water and 800mL pyridine were mixed and magnetically stirred for 30min to prepare an oxidizing agent.

Example 3 Synthesis of oligonucleotides 1-3

The invention synthesizes designed oligonucleotide 1, oligonucleotide 2 and oligonucleotide 3 by a Oligo-768 DNA synthesizer (company: biolytic Lab Performance Inc. SN: BLP-30285) through deprotection, coupling, capping and oxidation circulation modes (the oligonucleotide sequences are shown in the following table 1, an exemplary synthesis method is shown in figure 1), 160 mu L of trichloroacetic acid (TCA) deprotection agent is automatically added into a solid-phase carrier through the synthesizer to complete the removal process of a protecting group dimethoxy trityl (DMT), 22 mu L of various PTFA solutions and 18 mu L of acetonitrile solution of 0.035mol/L of phosphoramidite monomer are added after the deprotection is coupled with 5 '-hydroxyl obtained after the deprotection, 20 mu L of self-made oxidation agent is added to react with phosphite triester to form phosphotriester to complete oxidation after the coupling is finished, and finally 20 mu L of acetic anhydride (CAP-A) and 20 mu L of N-methylimidazole (CAP-B) are added to acetylate the free 5' -hydroxyl CAP to complete. The cycle was repeated to synthesize the desired oligonucleotide, the product was further treated with aqueous ammonia at 80℃for 6 hours to give a crude product, and the crude product was subjected to PAGE electrophoresis and MS analysis.

TABLE 1 sequence of oligonucleotides 1-3

EXAMPLE 4 PAGE electrophoretic analysis

The PAGE patterns of the products obtained in the examples are shown in FIG. 2, FIG. 2a is a photograph of the PAGE analysis of the product obtained in the presence of PTFA catalyst without additives, and it can be seen that the desired product was not obtained without additives, and FIGS. 2b, 2c and 2d are the products obtained at DMSO concentrations of 1%, 2% and 10%. Oligonucleotide 1 in FIG. 2c is more clear than the product 1 in FIGS. 2b and 2d, but still contains weak bands, and the bands of oligonucleotides 2 and 3 in FIGS. 2b, 2c and 2d are all clear bands with less main band in FIG. 2 c. Thus 2% DMSO among the three modes is most favourable for oligonucleotide synthesis. (remark: the first GC content of the designed sequence is high, and its own structure results in slower electrophoresis speed than that of normal oligonucleotides during PAGE electrophoresis).

FIG. 2e and 2f are, respectively, a PAGE profile of oligonucleotides obtained with 0.1% and 1% betaine, and it is seen from FIG. 2e that 0.1% betaine gives the target oligonucleotide fragment, whereas FIG. 2f shows that 1% betaine gives no target oligonucleotide, FIG. 2g and 2h are, respectively, a PAGE profile of oligonucleotides obtained with 0.1% and 1% lithium chloride, whereas 1% lithium chloride gives no target oligonucleotide, whereas 0.1% lithium chloride gives the target oligonucleotide fragment.

Comparing the PAGE patterns of the oligonucleotides obtained in FIGS. 2c, 2e and 2g, it can be seen from the figures that FIG. 2e and FIG. 2g have a higher impurity content, whereas the main band in FIG. 2c is clear, so that the synthesis of the oligonucleotide is most favored when the DMSO concentration is 2%.

EXAMPLE 5 MS results analysis

The products obtained from the oligonucleotide coupling reaction of the PTFA solution with 2% DMSO as a catalyst were analyzed by MS in the same manner as those obtained from the catalyst reaction of the PTFA solution without additives for enhancing data comparison, and the analysis patterns are shown in FIGS. 3 to 5. FIG. 3b shows oligonucleotide 1 obtained by solution-catalyzed reaction of PTFA with no additive added thereto, wherein the MS value is at most 21145.2 which does not correspond to theoretical molecular weight 24869.87, while the MS value of oligonucleotide obtained by solution-catalyzed reaction of PTFA with 2% DMSO added in FIG. 3a is 24873 which corresponds approximately to theoretical value, while FIG. 4b shows oligonucleotide 2 obtained by solution-catalyzed reaction of PTFA with no additive added thereto, the MS value is at most 13717.3 which does not correspond to theoretical value 22349.3, while the MS value obtained by solution-catalyzed reaction of PTFA with 2% DMSO added in FIG. 4a is 22348.9 which corresponds to theoretical value. FIG. 5b shows that the maximum MS value of oligonucleotide 3 obtained by PTFA solution catalyzed reaction without additive is 7073.1 less than theoretical 27525.8, while the MS value of oligonucleotide obtained by PTFA solution catalyzed reaction with 2% DMSO in FIG. 5a is 27523.8 consistent with theoretical.

In the same way, the synthesized oligonucleotides of betaine and lithium chloride are subjected to MS analysis, the analysis results are shown in fig. 6 to 11, and fig. 6 to 8 are MS patterns of the oligonucleotides obtained by the catalytic coupling reaction of the PTFA with 0.1% and 1% betaine, so that the target oligonucleotides can be obtained under the catalysis of the PTFA solution with the concentration of 0.1% betaine, and the target oligonucleotides cannot be obtained without target peaks under the catalysis of the PTFA solution with the concentration of 1% betaine. Similarly, from FIGS. 9 to 11, it can be seen that PTFA can be catalyzed by the addition of 0.1% lithium chloride to obtain the target oligonucleotide, while there is no target peak when 1% lithium chloride is added.

MS and PAGE analysis show that the target oligonucleotide is difficult to obtain without adding additive in the catalyst PTFA during the coupling reaction of oligonucleotide synthesis, and the addition of polar additive in PTFA can promote the synthesis reaction effectively to obtain target oligonucleotide fragment. However, too high a concentration will inhibit oligonucleotide synthesis, probably because the excess polar additive forms hydrogen bonds with the active hydroxyl groups to be attached at the 5' end, preventing the attack of the hydroxyl groups on the P (III) to be attached. From the data obtained from the experiments, it was seen that the addition of 2% DMSO to the PTFA produced the best quality for synthesizing the target oligonucleotide.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Reference to the literature

[1]Stephenson M L,Zamecnik P C.Inhibition of Rous sarcoma viral RNA translation by aspecific oligodeoxyribonucleotide[J].Proceedings of the National Academy of Sciences,1978,75(1):285-288.[J].

[2]Zamecnik P C,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.

[3]Engels J W,Uhlmann E.Gene Synthesis[New Synthetic Methods(77)][J].Angewandte Chemie International Edition in English,1989,28(6):716-734.

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

[5] Huang Shifu, zheng Qingquan, liang Weizhou, li Nongtao a phosphitylation agent and its use in oligonucleotide synthesis.

[6] Base Andru Bowman, qian Dela Walsh, wei Charles Tang Nai Butler, et al.

[7]Gryaznov S M,Letsinger R L.Selective O-phosphitilation with nucleoside phosphoramidite reagents[J].Nucleic acids research,1992,20(8):1879-1882.

[8] Eleuteri A, CAPALDI D C, krotz A H et al ,Pyridinium trifluoroacetate/N-methylimidazole as an efficient activator for oligonucleotide synthesis via the phosphoramidite method[J].Organic Process Research&Development,2000,4(3):182-189.

[9] VARGEESE C, carter J, yegge J et al ,Efficient activation of nucleoside phosphoramidites with 4,5-dicyanoimidazole during oligonucleotide synthesis[J].Nucleic acids research,1998,26(4):1046-1050.]

[10] Zhang Xiang, zhang Zhenyu, tang Peifu et al, study of NaCl concentration and base structure in the stem-loop on the annealing effect of shRNA [ J ]. Biomedical engineering and clinic, 2010,14 (002): 157-160.

[11] Lv Zhenzhen non-labeled fluorescence detection method based on aptamer research [ D ].2014.

[12]Xia Wei.Coupling activators for the oligonucleotide synthesis via phosphoramidite approach[J].Tetrahedron,2013.

Claims

1. A catalyst for oligonucleotide synthesis comprising as a main component an onium pyridine trifluoroacetate, and a polar additive;

wherein the polar additive is dimethyl sulfoxide, and the content is 1-10wt%, or

The polar additive is betaine 0.01-0.5 wt%, or

The polar additive is lithium chloride, and the content of the polar additive is 0.01-0.5 wt%.

2. The catalyst for oligonucleotide synthesis according to claim 1, wherein the polar additive is dimethyl sulfoxide in an amount of 2 wt%.

3. The catalyst for oligonucleotide synthesis according to claim 1, wherein the polar additive is betaine in an amount of 0.1 wt%.

4. The catalyst for oligonucleotide synthesis according to claim 1, wherein the polar additive is lithium chloride in an amount of 0.1 wt%.

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 of preparing a catalyst for oligonucleotide synthesis according to any one of claims 1-6, comprising contacting an onium pyridine trifluoroacetate with the polar additive.

8. The method of claim 7, wherein the contacting is performed in acetonitrile.

9. A method for synthesizing an oligonucleotide, comprising performing a solid phase phosphoramidite triester method for synthesizing an oligonucleotide chain using the catalyst for oligonucleotide synthesis according to any one of claims 1 to 6.