Cap reagent, preparation method, cap reaction method and application

Abstract

The invention provides a cap reagent, a preparation method, a cap reaction method and application, and relates to the technical field of nucleic acid chemical synthesis. The composition comprises a first composition and a second composition, the first composition comprises acetic anhydride and acetonitrile; the second composition is prepared from 4-dimethylaminopyridine and an organic solvent; the organic solvent comprises a solvent A and a solvent B; the solvent A is 2, 6-dimethyl pyridine; the solvent B is at least one of acetonitrile and tetrahydrofuran.

Description

[0001] The present invention relates to the field of nucleic acid chemical synthesis technology, and in particular, to a capping reagent, a preparation method, a capping reaction method and applications thereof. Background Art

[0002] Nucleic acid chemical synthesis technology is an important branch in the fields of molecular biology and biotechnology, which allows scientists to synthesize DNA or RNA molecules with specific sequences in the laboratory. This technology is crucial for multiple fields such as gene research, drug development and diagnostic tests. The chemical synthesis of oligonucleotides began in the 1950s. With the development of the phosphoramidite triester chemical synthesis method, oligonucleotide synthesis technology has been significantly developed and further applied to high-throughput synthesis technology in the 1990s.

[0003] The solid-phase phosphoramidite triester method is currently the mainstream method for oligonucleotide chemical synthesis. This method realizes the step-by-step nucleotide extension of the oligonucleotide chain by cyclically performing four steps: deprotection, coupling, capping and oxidation. Although certain progress has been made in synthesis efficiency with this method, it is still difficult to achieve 100% efficiency in the chemical synthesis of oligonucleotides. Especially when synthesizing oligonucleotides with complex modified groups or long-chain RNA / DNA, the reaction efficiency will be further reduced.

[0004] The defects of the prior art are mainly reflected in the reaction efficiency and the control of side reactions. In the solid-phase phosphoramidite triester method, if the 5'-OH site that does not participate in the coupling reaction is not blocked, it will continue to participate in the reaction in the next coupling reaction, resulting in unexpected extensions in the synthesis product. In addition, the acylation reaction effects of capping agent A (Cap A) and capping agent B (Cap B) used in the capping step directly affect the product quality. Incomplete acylation reaction will result in more n-1 impurities in the product, while too long acylation reaction time may increase the acetylation reaction at unexpected positions and the risk of forming acid due to acetic anhydride and water.

[0005] Poor capping reactions will lead to more impurities in the oligonucleotide product, which not only increases the difficulty of purification and analysis, but also seriously affects the quality and yield of the final product. Therefore, the prior art has obvious limitations in the efficiency of oligonucleotide synthesis, the control of side reactions and the product quality, and these defects limit the potential of chemically synthesized oligonucleotides in broader applications.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The object of the present invention is to provide a capping reagent, a preparation method, a capping reaction method and an application. When the capping reagent is used in the solid-phase phosphoramidite method for synthesizing oligonucleotides, it can improve the capping efficiency, reduce n-1 impurities, avoid side reactions of long-term acetylation reaction, and compared with Cap B of the traditional formula, reduce the reagent dosage and shorten the synthesis time.

[0008] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: In a first aspect, the present invention provides a capping reagent, comprising: a first composition and a second composition; The first composition includes acetic anhydride and acetonitrile; The second composition includes 4-dimethylaminopyridine and an organic solvent; the organic solvent includes a solvent A and a solvent B; the solvent A is 2,6-dimethylpyridine; the solvent B is acetonitrile and / or tetrahydrofuran.

[0009] In an optional embodiment, in the first composition, the volume ratio of acetic anhydride to acetonitrile is 1:9.

[0010] In an optional embodiment, the concentration of 4-dimethylaminopyridine in the second composition is 0.04 mol / L to 0.12 mol / L.

[0011] In an optional embodiment, the volume ratio of the solvent A to the solvent B is (0.8 to 1.5):(8.5 to 9.5).

[0012] In an optional embodiment, in the second composition, the volume ratio of 4-dimethylaminopyridine, the solvent A and the solvent B is (4.5 to 8.5):(0.8 to 2.0):(8.0 to 9.5).

[0013] In a second aspect, the present invention provides a preparation method of the capping reagent according to any one of the foregoing embodiments, comprising: Preparing the first composition by mixing acetic anhydride and acetonitrile; and mixing the solvent A and the solvent B to obtain an organic solvent; adding 4-dimethylaminopyridine to the organic solvent until it is dissolved to obtain the second composition; Preferably, the mixing temperature condition of the pyridine mixture is 25±5°C; Preferably, the mixing humidity condition of the pyridine mixture is not more than 30%.

[0014] In a third aspect, the present invention provides a capping reaction method, comprising: Determining the addition amounts of the first composition and the second composition in the capping reagent according to any one of the foregoing embodiments according to the synthesis scale; Based on the determined addition amounts, the first composition and the second composition are simultaneously delivered into a reaction vessel for a capping reaction.

[0015] In an alternative embodiment, the reaction conditions for the capping reaction include: Reaction temperature: 25 ± 5°C; Reaction humidity: not more than 30%.

[0016] In an alternative embodiment, the reaction time for the capping reaction is 15 seconds to 50 seconds.

[0017] In a fourth aspect, the present invention provides an application of the capping reagent as described in any one of the foregoing embodiments in the synthesis of oligonucleotides by the solid-phase phosphoramidite method, where the oligonucleotides include at least one of a fluorescent probe, siRNA, sgRNA, aptamer, and antisense nucleic acid.

[0018] The present invention provides a capping reagent, comprising: a first composition and a second composition; the first composition includes acetic anhydride and acetonitrile; the second composition includes 4-dimethylaminopyridine and an organic solvent; the organic solvent includes a solvent A and a solvent B; the solvent A is 2,6-dimethylpyridine; the solvent B is at least one of acetonitrile and tetrahydrofuran. The capping reagent provided by the present invention can achieve a more efficient reaction in the capping step during the synthesis of oligonucleotides by the solid-phase phosphoramidite method. On the one hand, it can reduce the proportion of n-1 impurities in the product and avoid side reactions that may be caused by a long-term acetylation reaction; on the other hand, compared with the traditional Cap B with an n-methylimidazole formulation, it can effectively reduce the reagent usage amount and the synthesis reaction time. Description of the Drawings

[0019] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1 LC-MS spectrum of the oligonucleotide product obtained in Example 1 of the present application; Figure 2 LC-MS spectrum of the oligonucleotide product obtained in Example 2 of the present application; Figure 3 LC-MS spectrum of the oligonucleotide product obtained in Example 3 of the present application; Figure 4 LC-MS spectrum of the oligonucleotide product obtained in the comparative example of the present application. Detailed Embodiments

[0021] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, the operations are carried out under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not specified by the manufacturer can be obtained through commercial purchase and are conventional products.

[0022] An end-capping reagent is provided in an embodiment of the present application, including: a first composition and a second composition; wherein, the first composition includes acetic anhydride and acetonitrile; the second composition includes 4-dimethylaminopyridine and an organic solvent. The organic solvent includes solvent A and solvent B.

[0023] Solvent A is 2,6-dimethylpyridine; solvent B is acetonitrile and / or tetrahydrofuran.

[0024] As described above, an end-capping reagent is provided in this embodiment, which is used for the synthesis of oligonucleotides based on the solid-phase phosphoramidite method. This reagent consists of two parts, namely the first composition and the second composition.

[0025] In the first composition (Cap A), acetic anhydride and acetonitrile are included. Acetic anhydride serves as the main component of the capping agent A (Cap A) here, and is responsible for closing the 5'-OH sites that did not participate in the coupling reaction during the oligonucleotide synthesis process to prevent these sites from participating in subsequent reactions. Acetonitrile acts as a solvent, facilitating the mixing and reaction of acetic anhydride.

[0026] In the second composition (Cap B), 4-dimethylaminopyridine and an organic solvent are included. The organic solvent is a mixed solvent, which includes solvent A, 2,6-dimethylpyridine, and solvent B. Solvent B can be at least one of acetonitrile and tetrahydrofuran. For example, the second composition can be 2,6-dimethylpyridine and acetonitrile.

[0027] 4-dimethylaminopyridine serves as the main component of the capping agent B (Cap B) here and is an effective catalyst for promoting the acylation reaction of acetic anhydride and accelerating the end-capping process. 2,6-dimethylpyridine also acts as a solvent and, together with acetonitrile, constitutes the solvent system of Cap B, facilitating the dissolution and reaction of 4-dimethylaminopyridine.

[0028] The design of the capping reagent provided in this embodiment aims to improve the efficiency and product quality of oligonucleotide synthesis by the solid-phase phosphoramidite method. By precisely controlling the concentrations of acetic anhydride and 4-dimethylaminopyridine and their ratios to the corresponding solvents, the conditions of the capping reaction can be optimized, the occurrence of side reactions can be reduced, and the generation of n-1 impurities can be lowered, thereby improving the purity and yield of oligonucleotide synthesis. In addition, this reagent combination also helps to reduce the amount of reagent used and shorten the synthesis time, improving the economy and efficiency of the synthesis process.

[0029] In some embodiments, in the first composition, the volume ratio of acetic anhydride to acetonitrile is 1:9.

[0030] In some embodiments, the concentration of 4-dimethylaminopyridine in the second composition is 0.04 mol / L to 0.12 mol / L. For example, the concentration can be 0.04 mol / L, 0.05 mol / L, 0.08 mol / L, 0.10 mol / L, 0.11 mol / L, 0.12 mol / L, and so on.

[0031] In some embodiments, the volume ratio of the A solvent to the B solvent is (0.8 to 2.0):(8.0 to 9.5). In this volume ratio, the proportion of the A solvent (such as 2,6-dimethylpyridine) can be, for example, 0.8, 0.9, 1.0, 1.2, 1.4, 1.5, 1.7, 2.0, and so on. The proportion of the B solvent (such as acetonitrile) can be 8.0, 8.5, 9.0, 9.5, and so on.

[0032] In some embodiments, in the second composition, the volume ratio of 4-dimethylaminopyridine, the A solvent, and the B solvent is (4.5 to 8.5):(0.8 to 2.0):(8.0 to 9.5). In this volume ratio, the proportion of 4-dimethylaminopyridine can be, for example, 4.5, 5.0, 6.0, 7.0, 8.0, 8.5, and so on; the proportion of the A solvent (such as 2,6-dimethylpyridine) can be, for example, 0.8, 0.9, 1.0, 1.2, 1.4, 1.5, 1.7, 2.0, and so on. The proportion of the B solvent (such as acetonitrile) can be 8.0, 8.5, 9.0, 9.5, and so on.

[0033] This application provides a method for preparing the capping reagent according to any one of the foregoing embodiments, including: Preparing a first composition by mixing acetic anhydride and acetonitrile; and mixing the A solvent and the B solvent to obtain an organic solvent; adding 4-dimethylaminopyridine to the organic solvent until it dissolves to obtain the second composition; In some embodiments, the mixing temperature condition of the pyridine mixture is 25 ± 5 °C; In some embodiments, the mixing humidity condition of the pyridine mixture is not greater than 30%.

[0034] The above method is a preparation method of a capping reagent. First, a first composition (Cap A) is prepared by mixing acetic anhydride and acetonitrile. Acetic anhydride serves as the active ingredient, and acetonitrile serves as the solvent. The two are mixed to form the first composition for the capping reaction.

[0035] Then, solvent A and solvent B are mixed to obtain an organic solvent. Then, 4-dimethylaminopyridine is added to this mixture until it is completely dissolved to form a second composition (Cap B). 4-dimethylaminopyridine serves as a catalyst, and 2,6-dimethylpyridine and acetonitrile serve as solvents, jointly constituting the second composition.

[0036] The above preparation method is to optimize the composition and reaction conditions of the capping reagent to improve the efficiency of oligonucleotide synthesis and the product quality. By precisely controlling the proportions of each component and the reaction conditions, the efficiency and specificity of the capping reaction can be ensured, and the generation of side reactions and impurities can be reduced.

[0037] Based on the capping reagent, the principle of the capping reaction is to introduce an acetyl group at the 5'-OH site of the oligonucleotide chain to block these sites and prevent them from participating in the reaction in subsequent coupling steps. Acetic anhydride provides the acetyl group in Cap A, while 4-dimethylaminopyridine serves as a catalyst in Cap B to accelerate the acetylation reaction. 2,6-dimethylpyridine and acetonitrile serve as solvents, which help to improve the solubility of the reactants and the reaction rate.

[0038] The preparation method provided in this example effectively improves the efficiency of oligonucleotide synthesis and the product quality by precisely controlling the component proportions and reaction conditions of the capping reagent. This method can accelerate the capping reaction rate, thereby improving the overall synthesis efficiency; at the same time, by optimizing the reaction conditions and component proportions, the generation of impurities such as n-1 is reduced, and the product purity is improved. Compared with the traditional n-methylimidazole formulation, the new formulation reduces the amount of reagent used, which helps to reduce costs. In addition, the optimized reaction conditions also shorten the capping reaction time and speed up the entire synthesis process. In terms of environmental control, maintaining a temperature of 25 ± 5°C and a humidity condition of not greater than 30% for mixing helps to maintain the stability and repeatability of the reaction and reduce the adverse effects of hydration on the reaction. Generally speaking, this preparation method aims to improve the performance of the capping reagent through fine regulation, and then improve the efficiency of oligonucleotide synthesis and the product quality.

[0039] An embodiment of the present application provides a capping reaction method, including: Step S1, determining the addition amounts of the first composition and the second composition in the capping reagent as described in any one of the foregoing embodiments according to the synthesis scale.

[0040] Step S2, based on the determined addition amounts, simultaneously transport the first composition and the second composition into a reaction vessel for a capping reaction.

[0041] The above capping reaction method involves determining the addition amounts of the capping reagents and simultaneously transporting the first composition (Cap A) and the second composition (Cap B) into a reaction vessel for a capping reaction. Cap A mainly consists of acetic anhydride and acetonitrile, while Cap B consists of 4-dimethylaminopyridine, 2,6-dimethylpyridine, and acetonitrile. The volume ratios and concentrations of these components are precisely designed to optimize the reaction conditions.

[0042] The capping reaction method is to block the unreacted 5'-OH sites during the solid-phase phosphoramidite method for synthesizing oligonucleotides, preventing these sites from participating in subsequent reactions, thereby reducing the generation of impurities such as n-1. The capping step is a very crucial step in oligonucleotide synthesis, which directly affects the purity and quality of oligonucleotides. The principle lies in chemically blocking the unreacted 5'-OH sites using the active components in Cap A and Cap B. The acetic anhydride in Cap A provides an acetyl group, while 4-dimethylaminopyridine in Cap B acts as a catalyst to accelerate the acetylation reaction and form an ester bond with the 5'-OH site to achieve the blocking effect.

[0043] In some embodiments, the reaction conditions for the capping reaction include: a reaction temperature of 25 ± 5°C and a reaction humidity of not more than 30%.

[0044] For the above capping reaction method, by precisely controlling the component ratios and reaction conditions, this method can accelerate the capping reaction rate, thereby improving the overall efficiency of oligonucleotide synthesis; the optimized reaction conditions and component ratios help reduce the generation of impurities such as n-1, thereby improving the product purity; compared with the traditional n-methylimidazole formulation, the new formulation reduces the amount of reagents used, effectively reducing costs; in addition, the optimized reaction conditions can also shorten the capping reaction time and accelerate the entire synthesis process. In terms of environmental control, maintaining a temperature of 25 ± 5°C and a humidity of not more than 30% for mixing helps control the stability and repeatability of the reaction and reduces the adverse effects of hydration on the reaction. Generally speaking, this capping reaction method aims to improve the efficiency and product quality of oligonucleotide synthesis, reduce the generation of impurities, and optimize the economic cost through fine regulation.

[0045] In some embodiments, the reaction time for the capping reaction is 15 seconds to 50 seconds. For example, the reaction time can be 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, and so on.

[0046] In an embodiment of the present application, there is provided an application of the capping reagent as described in any one of the foregoing embodiments in the solid-phase phosphoramidite method for synthesizing oligonucleotides, and the oligonucleotides include at least one of a fluorescent probe, siRNA, sgRNA, an aptamer, and an antisense nucleic acid.

[0047] Example 1: In this example, the capping reagent was prepared.

[0048] Experimental method: (1) First, accurately measure 900 mL of anhydrous acetonitrile, and then measure 100 mL of acetic anhydride and mix it evenly with anhydrous acetonitrile. This reagent is used as Cap A (solvent A).

[0049] (2) First, accurately measure 100 mL of 2,6-dimethylpyridine and 900 mL of anhydrous acetonitrile, mix them evenly, and then weigh 6.5 g of 4-dimethylaminopyridine and add it to the prepared solution. After fully dissolving and mixing, it is used as Cap B (solvent B).

[0050] Example 2 In this example, the capping reagent was prepared.

[0051] Experimental method: The method used in this example is basically the same as that in Example 1, except that: in step (2), 12.2 g of 4-dimethylaminopyridine was added.

[0052] Example 3 In this example, the capping reagent was prepared.

[0053] Experimental method: The method used in this example is basically the same as that in Example 1, except that: The anhydrous acetonitrile in step (2) was replaced with tetrahydrofuran.

[0054] Comparative Example 1 In this comparative example, a conventional capping reagent was prepared.

[0055] Experimental method: Cap A is acetic anhydride:tetrahydrofuran:pyridine with a volume ratio of 1:8:1 Cap B is N-methylimidazole:pyridine:tetrahydrofuran with a volume ratio of 1:1:8.

[0056] Comparative test experiment: The capping reagents provided in Examples 1 to 3 and Comparative Example 1 were used for experiments using a Platinum 192 high-throughput oligonucleotide synthesizer.

[0057] Experimental method: (1)Deprotection: The deprotection reagent is a toluene solution of 3 w / v% trichloroacetic acid. The deprotection reaction lasts for 60 s, and then acetonitrile is used for cleaning.

[0058] (2)Coupling: For coupling, a solution containing 5-ethylthiotetrazole and phosphoramidite monomer is simultaneously delivered into the reaction column and waits for 60 s for the reaction.

[0059] (3)Oxidation: 0.02 M iodine solution is used as the oxidant. After the reaction for 50 s, acetonitrile is used for cleaning.

[0060] (4)Capping: It is simultaneously delivered into the reaction column and reacts for 30 s. Finally, acetonitrile is used for cleaning.

[0061] After the synthesis is completed, the solid support is transferred to a gas-phase ammonolysis reactor. The ammonolysis conditions are set at 90 °C and 120 min for cleavage and deprotection. After the ammonolysis cleavage is completed, the solid support is washed twice with anhydrous acetonitrile, and then 200 μL of TE buffer is added to elute the synthesized product from the solid support. The eluted product obtained in the previous step is taken for LC-MS detection. The synthesis quality of the oligonucleotide is analyzed according to the LC-MS spectrum.

[0062] Experimental results: Table 1. Comparative mass spectrometry analysis of the synthesized products in the examples and comparative examples

[0063] Analysis: (1)In the analysis of the proportion of the target product, Example 1 showed excellent performance, with the proportion of the target product as high as 99%. This result highlights the excellent effect of the capping reagent formulation in this example in improving the product purity. In contrast, the proportion of the target product in Example 2 was 96%, slightly lower than that in Example 1, but the purity still remained at a relatively high level.

[0064] The proportion of the target product in the comparative example was only 87%, the lowest among all examples. This finding indicates that the new formulation can achieve higher efficiency in synthesizing oligonucleotides compared with the capping reagent with the traditional formulation.

[0065] (2)In terms of the analysis of the proportion of pre-impurities, Example 1 further confirmed the effectiveness of its capping reagent formulation with a pre-impurity proportion of only 1%, significantly lower than that of other examples and comparative examples. The pre-impurity proportion of Example 2 was 4%, although it increased, it was still at a relatively low level.

[0066] The comparative example had the highest pre-impurity proportion, reaching 13%, which was consistent with the analysis result of the proportion of the target product, indicating that the capping reagent with the traditional formulation was less effective in controlling side reactions than the new formulation. These data together indicate that the new capping reagent formulation has obvious advantages in reducing by-products and improving synthesis purity.

[0067] In summary, the capping reagent formulations of Examples 1-3 performed relatively well in oligonucleotide synthesis, achieving the highest target product ratio and the lowest pre-impurity ratio, and significantly improving the purity and efficiency of synthesis. This result may be attributed to the precise ratio of acetic anhydride and 4-dimethylaminopyridine in the formulation and the preferred solvent, which together improve the efficiency and specificity of the capping reaction. The traditional capping reagent formulation in the comparative example has a ratio of acetic anhydride and N-methylimidazole and a solvent selection that are not conducive to improving the reaction efficiency and specificity, resulting in lower synthesis efficiency and more by-products. Therefore, it can be concluded that the capping reagent formulation in the examples can achieve a higher target product ratio and a lower pre-impurity ratio when synthesizing oligonucleotides, thereby significantly improving the quality and efficiency of oligonucleotide synthesis.

[0068] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A capping reagent, characterized in that, Comprising: A first composition and a second composition; The first composition comprises acetic anhydride and acetonitrile; The second composition comprises 4-dimethylaminopyridine and an organic solvent; the organic solvent comprises a solvent A and a solvent B; the solvent A is 2,6-dimethylpyridine; the solvent B is acetonitrile and / or tetrahydrofuran.

2. The capping reagent according to claim 1, wherein In the first composition, the volume ratio of acetic anhydride to acetonitrile is 1:

9.

3. The capping reagent according to claim 1, wherein The concentration of 4-dimethylaminopyridine in the second composition is 0.04 mol / L to 0.12 mol / L.

4. The capping reagent according to claim 1, wherein The volume ratio of the solvent A to the solvent B is (0.8 - 2.0):(8.0 - 9.5).

5. The capping reagent according to claim 1, wherein In the second composition, the volume ratio of 4-dimethylaminopyridine, the solvent A and the solvent B is (4.5 - 8.5):(0.8 - 2.0):(8.0 - 9.5).

6. A method for preparing the capping reagent according to any one of claims 1-5, characterized in that, Comprising: By mixing acetic anhydride and acetonitrile, a first composition is prepared; and, by mixing the solvent A and the solvent B, an organic solvent is obtained; 4-Dimethylaminopyridine is added to the organic solvent until dissolved, thus obtaining the second composition; Preferably, the mixing temperature condition of the pyridine mixture is 25 ± 5 °C; Preferably, the mixing humidity condition of the pyridine mixture is not more than 30%.

7. A capping reaction method, characterized in that, Comprising: Determine the addition amounts of the first composition and the second composition in the capping reagent according to any one of claims 1 - 5 based on the synthesis scale; Based on the determined addition amounts, the first composition and the second composition are simultaneously delivered into a reaction vessel for a capping reaction.

8. The capping reaction method according to claim 7, wherein, The reaction conditions of the capping reaction include: Reaction temperature 25 ± 5 °C; Reaction humidity not more than 30%.

9. The capping reaction method according to claim 7, characterized in that, The reaction time of the capping reaction is 15 seconds to 50 seconds.

10. Use of a capping reagent according to any one of claims 1-5 in the solid-phase phosphoramidite method for synthesizing oligonucleotides, characterized in that, The oligonucleotide comprises at least one of a primer, a probe, siRNA, sgRNA, an aptamer and an antisense nucleic acid.

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