Synthetic method of oligonucleotide

Abstract

The invention discloses a synthesis method of oligonucleotide, and belongs to the technical field of solid-phase synthesis oligonucleotide coupling. The synthesis method comprises the following steps: carrying out chemical modification on a phosphoramidite nucleotide monomer by using MMT-hexylamino connection phosphoramidite, synthesizing an oligonucleotide fragment by using a phosphoramidite method under the action of a coupling activator, carrying out a decyanation ethyl reaction by using a triethylamine solution, adding a deprotection group reagent, and carrying out a reaction to obtain the phosphoramidite oligonucleotide. An oligonucleotide end product is obtained; the coupling activator comprises deionized water and at least one of 5-ethylthiotetrazole and an amido derivative. The invention provides a synthetic method of oligonucleotide with high purity and high yield.

Description

Technical Field

[0001] The invention relates to the technical field of solid phase synthesis oligonucleotide coupling, and in particular to a method for synthesizing oligonucleotide. Background Art

[0002] Deoxyribonucleic acid (DNA) synthesis is the underlying technology of synthetic biology, and DNA synthesis relies on oligonucleotide fragments as building blocks. With the rapid development of synthetic biology, the demand for long-fragment, high-fidelity, and low-cost oligonucleotide synthesis is increasing. Today, the mainstream method for oligonucleotide synthesis is still the solid-phase phosphoramidite four-step method. Due to the inherent limitations of chemical reactions, the length of commercially synthesized oligonucleotides is generally controlled below 100nt. There are two main factors that affect oligonucleotide synthesis: first, each cycle in oligonucleotide synthesis cannot react completely, and the coupling efficiency is less than 100%. As the oligonucleotide sequence increases, the total yield continues to decrease; second, side reactions such as depurination and cyanoethyl addition will further reduce the yield of oligonucleotides and lead to synthesis errors. Therefore, new chemical synthesis methods are needed to minimize side reactions and increase yields to obtain high-quality long-fragment oligonucleotides. Summary of the invention

[0003] The object of the present invention is to provide a method for synthesizing an oligonucleotide, which can effectively reduce the generation of by-products, thereby improving the purity and yield of the oligonucleotide final product.

[0004] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A method for synthesizing an oligonucleotide, comprising: S1, connecting the phosphoramidite nucleotide monomer and MMT-hexylamino group to a phosphoramidite reaction to obtain a phosphoramidite nucleotide monomer; S2, using the phosphoramidite method, reacting the phosphoramidite nucleotide monomer with an oligonucleotide synthesis reagent to obtain a reaction solution containing the oligonucleotide fragment; S3, first adding a triethylamine solution to the reaction solution containing the oligonucleotide fragment to carry out a decyanoethylation reaction, and then adding a deprotection reagent to carry out a reaction to obtain an oligonucleotide final product; The oligonucleotide synthesis reagents include a detrityl reagent, a coupling activator, an oxidizing reagent, a sulfurizing reagent and a capping reagent; the coupling activator includes deionized water and at least one of 5-ethylthiotetrazolyl and an amide derivative, wherein the amide derivative has an amide group and a benzene ring.

[0005] The present invention uses MMT-hexylamino to connect phosphoramidites to chemically modify phosphoramidite nucleotide monomers, synthesizes oligonucleotide fragments by phosphoramidite method under the action of coupling activator, first uses triethylamine solution to carry out decyanoethylation reaction, then adds deprotection reagent to react, and obtains oligonucleotide final product. The synthetic method of oligonucleotide of the present invention reduces the amount of by-products and the generation of by-products by adjusting the order of deprotection groups, thereby improving the purity and yield of oligonucleotides.

[0006] Preferably, the amido derivative is prepared by reacting 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and 3-chloropropionic acid [3-(3-chloropropionylamino)phenyl] ester.

[0007] Preferably, the usage ratio of the phosphoramidite nucleotide monomer and the MMT-hexylamino-linked phosphoramidite is 1 mg: 1.5-3 mL.

[0008] Preferably, the detritylation agent comprises dichloroacetic acid and dichloromethane, and the usage ratio of dichloroacetic acid to dichloromethane is 1 g: 10-50 mL.

[0009] Preferably, the oxidizing agent comprises iodine, pyridine and water, the mass ratio of iodine to pyridine is 1:1-2, and the dosage ratio of iodine to water is 1g:5-20mL.

[0010] Preferably, the sulfurizing agent comprises hydrogenated xanthan gum and pyridine, and the usage ratio of hydrogenated xanthan gum to pyridine is 1 g: 10-50 mL.

[0011] Preferably, the capping reagent comprises anhydride and acetonitrile, and the usage ratio of the anhydride to the acetonitrile is 1 g: 2-10 mL.

[0012] Preferably, the mass concentration of the triethylamine solution is 2-20%, and the volume ratio of the reaction solution containing the oligonucleotide fragments to the triethylamine solution is 1:1-2.

[0013] Preferably, the deprotecting reagent comprises trifluoroacetic acid and dichloromethane, and the usage ratio of trifluoroacetic acid to dichloromethane is 1 g: 50-200 mL.

[0014] Preferably, the volume ratio of the triethylamine solution to the deprotecting group reagent is 1:1-5.

[0015] Preferably, the preparation method of the amide derivative comprises: Weigh 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and 3-chloropropionic acid [3-(3-chloropropionylamino)phenyl] ester, add chloroform and stir to dissolve, react at 70°C for 24 hours, add acetone and recrystallize three times to obtain an amide derivative.

[0016] More preferably, the molar ratio of 2-cyano-N-[2-(dimethylamino)ethyl]acetamide to 3-chloropropionic acid [3-(3-chloropropionylamino)phenyl] ester is 1:0.2-1.

[0017] More preferably, the usage ratio of 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and chloroform is 1 mol: 100-300 mL.

[0018] More preferably, the volume ratio of chloroform to acetone is 1:1-5.

[0019] Preferably, the coupling activator comprises 5-ethylthiotetrazolyl and deionized water.

[0020] More preferably, the usage ratio of 5-ethylthiotetrazolyl and deionized water is 1 g:10-50 mL.

[0021] Preferably, the coupling activator includes 5-ethylthiotetrazolyl, amide derivatives and deionized water. The amide derivatives have a large number of active groups, which expose the active groups of the phosphoramidite nucleotide monomers and react with 5-ethylthiotetrazolyl to generate active intermediates, effectively improving the monomer coupling efficiency during solid phase synthesis, thereby improving the purity and yield of oligonucleotides.

[0022] More preferably, the mass ratio of 5-ethylthiotetrazolyl to the amide derivative is 1:0.15-0.3.

[0023] More preferably, the usage ratio of 5-ethylthiotetrazolyl and deionized water is 1 g:10-50 mL.

[0024] Preferably, the coupling activator includes 5-ethylthiotetrazolyl, amide derivatives, benzotriazole derivatives and deionized water. The benzotriazole derivatives can further enhance the activity of phosphoramidite nucleotide monomers and enhance the coupling activity of phosphoramidite nucleotide monomers, thereby enhancing the purity and yield of oligonucleotides.

[0025] More preferably, the mass ratio of 5-ethylthiotetrazolyl to the amide derivative is 1:0.15-0.3.

[0026] More preferably, the mass ratio of 5-ethylthiotetrazolyl to the benzotriazole derivative is 1:0.05-0.1.

[0027] More preferably, the usage ratio of 5-ethylthiotetrazolyl and deionized water is 1 g:10-50 mL.

[0028] More preferably, the preparation method of the benzotriazole derivative is specifically, Weigh cinnamaldehyde, add carbon tetrachloride, stir evenly, slowly drop epichlorohydrin and carbon tetrachloride at 40-80°C, react for 8-12h, add ice water, add ether to extract 2-5 times, extract the organic phase, add 5-azabenzimidazole, sodium hydride, anhydrous ethanol, stir and reflux for 1-5h, reflux reaction for 12-36h, add pure water and toluene to extract 2-5 times, extract the organic phase, filter, and dry to obtain a benzotriazole derivative.

[0029] More preferably, the usage ratio of cinnamaldehyde to carbon tetrachloride is 1 mmol:1-5 mL.

[0030] More preferably, the molar ratio of cinnamaldehyde to epichlorohydrin is 1:0.5-2.

[0031] More preferably, the usage ratio of cinnamaldehyde to carbon tetrachloride is 1 mmol:0.1-0.5 mL.

[0032] More preferably, the temperature of the ice water is 0-10° C., and the usage ratio of cinnamaldehyde to ice water is 1 mmol:0.5-2 mL.

[0033] More preferably, the usage ratio of cinnamaldehyde to ether is 1 mmol:0.5-2 mL.

[0034] More preferably, the molar ratio of cinnamaldehyde to 5-azabenzimidazole is 1:0.1-0.5.

[0035] More preferably, the molar ratio of cinnamaldehyde to sodium hydride is 1:1-5.

[0036] More preferably, the usage ratio of cinnamaldehyde to anhydrous ethanol is 1 mmol:1-5 mL.

[0037] More preferably, the usage ratio of cinnamaldehyde to pure water is 1 mmol:0.5-2 mL.

[0038] More preferably, the usage ratio of cinnamaldehyde to toluene is 1 mmol:0.5-2 mL.

[0039] Preferably, a method for synthesizing an oligonucleotide comprises: S1, weighing a phosphoramidite nucleotide monomer, adding MMT-hexylamino to connect the phosphoramidite, and obtaining a phosphoramidite nucleotide monomer; S2, sequentially adding the phosphoramidite nucleotide monomer and the detrityl reagent, the coupling activator, the oxidizing reagent, the sulfurizing reagent and the capping reagent to the OligoPliot 100 fully automatic oligonucleotide synthesizer, and automatically synthesizing in the OligoPliot 100 fully automatic oligonucleotide synthesizer by the phosphoramidite method until the oligonucleotide fragment is obtained, thereby obtaining a reaction solution containing the oligonucleotide fragment; S3. First, add triethylamine solution to the reaction solution containing the oligonucleotide fragment at a flow rate of 50-100 cm / L, react for 5-20 minutes, then add a deprotection reagent, add an aminolysis solution, react at 30-50°C for 6-12 hours, and purify by silica gel column chromatography to obtain the oligonucleotide.

[0040] More preferably, the phosphoramidite nucleotide monomer in step S1 is a DMT-dA phosphoramidite monomer, or a DMT-dT phosphoramidite monomer, or a DMT-dC phosphoramidite monomer, or a DMT-dG phosphoramidite monomer.

[0041] More preferably, in step S1, the ratio of the phosphoramidite nucleotide monomer to the MMT-hexylamino-linked phosphoramidite is 1 mg: 1.5-3 mL.

[0042] More preferably, in step S2, the detritylation agent comprises dichloroacetic acid and dichloromethane, and the usage ratio of dichloroacetic acid to dichloromethane is 1 g: 10-50 mL.

[0043] More preferably, the coupling activator in step S2 comprises deionized water and at least one of 5-ethylthiotetrazolyl, an amide derivative and a benzotriazole derivative.

[0044] More preferably, the mass ratio of 5-ethylthiotetrazolyl to the amide derivative is 1:0.15-0.3.

[0045] More preferably, the mass ratio of 5-ethylthiotetrazolyl to the benzotriazole derivative is 1:0.05-0.1.

[0046] More preferably, the usage ratio of 5-ethylthiotetrazolyl is 1 g: 10-50 mL.

[0047] More preferably, in step S2, the oxidizing reagent comprises iodine, pyridine and water, the mass ratio of iodine to pyridine is 1:1-2, and the dosage ratio of iodine to water is 1g:5-20mL.

[0048] More preferably, in step S2, the sulfiding reagent comprises hydrogenated xanthan gum and pyridine, and the usage ratio of hydrogenated xanthan gum to pyridine is 1 g: 10-50 mL.

[0049] More preferably, in step S2, the capping reagent comprises anhydride and acetonitrile, and the usage ratio of the anhydride to the acetonitrile is 1 g: 2-10 mL.

[0050] More preferably, in step S2, the volume ratio of the phosphoramidite nucleotide monomer to the detritylation solution is 1:2-10.

[0051] More preferably, the volume ratio of the phosphoramidite nucleotide monomer to the coupling activator in step S2 is 1:1-5.

[0052] More preferably, the volume ratio of the phosphoramidite nucleotide monomer to the oxidizing agent in step S2 is 1:2-10.

[0053] More preferably, in step S2, the volume ratio of the phosphoramidite nucleotide monomer to the sulfurization reagent is 1:2-10.

[0054] More preferably, the volume ratio of the phosphoramidite nucleotide monomer to the capping agent in step S2 is 1:2-10.

[0055] More preferably, the mass concentration of the triethylamine solution in step S3 is 2-20%, and the volume ratio of the reaction solution containing the oligonucleotide fragments to the triethylamine solution is 1:1-2.

[0056] More preferably, in step S3, the deprotection reagent comprises trifluoroacetic acid and dichloromethane, and the usage ratio of trifluoroacetic acid to dichloromethane is 1 g: 50-200 mL.

[0057] More preferably, in step S3, the volume ratio of the triethylamine solution to the deprotection reagent is 1:1-5.

[0058] More preferably, the ammonolysis solution in step S3 includes aqueous ammonia and methylamine solution, the volume ratio of aqueous ammonia to methylamine solution is 1:1-5, the mass concentration of aqueous ammonia is 25-35%, and the methylamine solution includes methylamine and anhydrous ethanol, and the volume ratio of methylamine to anhydrous ethanol is 1:2-2.5.

[0059] More preferably, the volume ratio of the triethylamine solution to the ammonolysis solution in step S3 is 1:0.1-0.5.

[0060] The present invention uses 5-ethylthiotetrazolyl, an amide derivative and a benzotriazole derivative to form a coupling activator to participate in the synthesis of an oligonucleotide fragment, and then uses a triethylamine solution to remove the cyanoethyl group on the phosphate group, and then uses trifluoroacetic acid and dichloromethane to remove the MMT protecting group to obtain an oligonucleotide final product, so it has the following beneficial effects: the oligonucleotide has high purity and high yield, the purity of the oligonucleotide final product is 61.5-96.0%, and the yield is 34.9-89.0%. Therefore, the present invention provides a method for synthesizing an oligonucleotide with high purity and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is the infrared spectrum of the amide derivative prepared by the present invention. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0063] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0064] Embodiment 1: A method for synthesizing an oligonucleotide comprises the following steps: S1. Weigh the phosphoramidite nucleotide monomer, add MMT-hexylamino to connect the phosphoramidite, and obtain the phosphoramidite nucleotide monomer. The concentration of MMT-hexylamino to connect the phosphoramidite is 0.1 mol / L, and the dosage ratio of the phosphoramidite nucleotide monomer and the MMT-hexylamino to connect the phosphoramidite is 1 mg: 1.5 mL. When the phosphoramidite nucleotide monomer is a DMT-dA phosphoramidite monomer, a phosphoramidite adenylate monomer is obtained; when the phosphoramidite nucleotide monomer is a DMT-dT phosphoramidite monomer, a phosphoramidite thymidine monomer is obtained; when the phosphoramidite nucleotide monomer is a DMT-dC phosphoramidite monomer, a phosphoramidite cytidine monomer is obtained; when the phosphoramidite nucleotide monomer is a DMT-dG phosphoramidite monomer, a phosphoramidite guanylate monomer is obtained.

[0065] S2. Add phosphoramidite adenylic acid monomers, phosphoramidite thymic acid monomers, phosphoramidite cytidylic acid monomers and phosphoramidite guanylic acid monomers, a detrityl reagent, a coupling activator, an oxidizing reagent, a sulfurizing reagent and a capping reagent to an OligoPliot 100 fully automatic oligonucleotide synthesizer in sequence, and automatically synthesize in the OligoPliot 100 fully automatic oligonucleotide synthesizer using the phosphoramidite method until an oligonucleotide fragment is synthesized to obtain a reaction solution containing an oligonucleotide fragment. The volume ratio of phosphoramidite adenylic acid monomer to phosphoramidite thymic acid monomer is 1:1, the volume ratio of phosphoramidite adenylic acid monomer to phosphoramidite cytidylic acid monomer is 1:1, the volume ratio of phosphoramidite adenylic acid monomer to phosphoramidite guanylic acid monomer is 1:1; the volume ratio of phosphoramidite adenylic acid monomer to detritylation solution is 1:4; the volume ratio of phosphoramidite adenylic acid monomer to coupling activator is 1:1.8; the volume ratio of phosphoramidite adenylic acid monomer to oxidizing reagent is 1:4; the volume ratio of phosphoramidite adenylic acid monomer to sulfurizing reagent is 1:4; the volume ratio of phosphoramidite adenylic acid monomer to capping reagent is 1:4. The detritylation reagent includes dichloroacetic acid and dichloromethane, and the usage ratio of dichloroacetic acid to dichloromethane is 1g:33mL; the coupling activator includes 5-ethylthiotetrazolyl and deionized water, and the usage ratio of 5-ethylthiotetrazolyl and deionized water is 1g:25mL; the oxidizing reagent includes iodine, pyridine and water, the mass ratio of iodine to pyridine is 1:1, and the usage ratio of iodine to water is 1g:8mL; the sulfiding reagent includes hydrogenated xanthine and pyridine, and the usage ratio of hydrogenated xanthine to pyridine is 1g:33mL; the capping reagent includes acid anhydride and acetonitrile, and the usage ratio of acid anhydride to acetonitrile is 1g:5mL.

[0066] S3. First, add triethylamine solution at a flow rate of 75 cm / L to the reaction solution containing the oligonucleotide fragment, react for 10 minutes, then add a deprotection reagent, add an aminolysis solution, react at 45° C. for 8 hours, and purify by silica gel column chromatography to obtain an oligonucleotide. The nucleotide sequence of the oligonucleotide fragment is shown in SEQ ID No. 1, and its nucleotide sequence is: 5'-ATGCTGGTAT-3'. The mass concentration of the triethylamine solution is 10%, and the volume ratio of the reaction solution containing the oligonucleotide fragment to the triethylamine solution is 1:1; the volume ratio of the triethylamine solution to the deprotecting group reagent is 1:2, and the deprotecting group reagent includes trifluoroacetic acid and dichloromethane, and the usage ratio of trifluoroacetic acid and dichloromethane is 1g:100mL; the volume ratio of the triethylamine solution to the aminolysis solution is 1:0.3, and the aminolysis solution includes ammonia water and methylamine solution, and the volume ratio of ammonia water and methylamine solution is 1:1, and the mass concentration of ammonia water is 25%, and the methylamine solution includes methylamine and anhydrous ethanol, and the volume ratio of methylamine and anhydrous ethanol is 1:2.5.

[0067] Embodiment 2: The difference between this embodiment and embodiment 1 is only the component of the coupling activator in step 2.

[0068] The coupling activator of this embodiment includes 5-ethylthiotetrazolyl, an amide derivative and deionized water, the mass ratio of 5-ethylthiotetrazolyl and the amide derivative is 1:0.3, and the amount ratio of 5-ethylthiotetrazolyl and deionized water is 1g:25mL. The preparation method of the amide derivative includes the following steps: Weigh 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and 3-chloropropionic acid [3-(3-chloropropionylamino)phenyl] ester, add chloroform, stir and dissolve, react at 70°C for 24 hours, add acetone and recrystallize three times to obtain an amide derivative. The molar ratio of 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and 3-chloropropionic acid [3-(3-chloropropionylamino)phenyl] ester is 1:0.5; the dosage ratio of 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and chloroform is 1 mol:200 mL; the volume ratio of chloroform and acetone is 1:3.

[0069] Embodiment 3: The difference between this embodiment and embodiment 1 is only the component of the coupling activator in step 2.

[0070] The coupling activator of this embodiment includes 5-ethylthiotetrazolyl, an amide derivative and deionized water, the mass ratio of 5-ethylthiotetrazolyl and the amide derivative is 1:0.15, and the amount ratio of 5-ethylthiotetrazolyl and deionized water is 1g:25mL. The preparation method of the amide derivative includes the following steps: Weigh 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and 3-chloropropionic acid [3-(3-chloropropionylamino)phenyl] ester, add chloroform, stir and dissolve, react at 70°C for 24 hours, add acetone and recrystallize three times to obtain an amide derivative. The molar ratio of 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and 3-chloropropionic acid [3-(3-chloropropionylamino)phenyl] ester is 1:0.5; the dosage ratio of 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and chloroform is 1 mol:200 mL; the volume ratio of chloroform and acetone is 1:3.

[0071] Embodiment 4: The difference between this embodiment and embodiment 1 is only the component of the coupling activator in step 2.

[0072] The coupling activator in this embodiment includes 5-ethylthiotetrazolyl, amide derivatives, benzotriazole derivatives and deionized water, the mass ratio of 5-ethylthiotetrazolyl and amide derivatives is 1:0.3, the mass ratio of 5-ethylthiotetrazolyl and benzotriazole derivatives is 1:0.1; the dosage ratio of 5-ethylthiotetrazolyl and deionized water is 1g:25mL. The preparation methods of amide derivatives and benzotriazole derivatives are as follows: The preparation method of the amide derivative comprises the following steps: Weigh 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and 3-chloropropionic acid [3-(3-chloropropionylamino)phenyl] ester, add chloroform, stir and dissolve, react at 70°C for 24 hours, add acetone and recrystallize three times to obtain an amide derivative. The molar ratio of 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and 3-chloropropionic acid [3-(3-chloropropionylamino)phenyl] ester is 1:0.5; the dosage ratio of 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and chloroform is 1 mol:200 mL; the volume ratio of chloroform and acetone is 1:3.

[0073] The preparation of benzotriazole derivatives comprises the following steps: Weigh cinnamaldehyde, add carbon tetrachloride, stir evenly, slowly add epichlorohydrin and carbon tetrachloride dropwise at 60°C, react for 8 hours, add ice water, extract with ether three times, extract the organic phase, add 5-azabenzimidazole, sodium hydride, and anhydrous ethanol, stir and reflux for 2 hours, reflux reaction for 24 hours, add pure water and toluene to extract three times, extract the organic phase, filter, and dry to obtain a benzotriazole derivative. The molar ratio of cinnamaldehyde to carbon tetrachloride is 1mmol:1mL; the molar ratio of cinnamaldehyde to epichlorohydrin is 1:1.1; the molar ratio of cinnamaldehyde to carbon tetrachloride is 1mmol:0.25mL; the temperature of ice water is 4°C, and the molar ratio of cinnamaldehyde to ice water is 1mmol:0.75mL; the molar ratio of cinnamaldehyde to ether is 1mmol:0.75mL; the molar ratio of cinnamaldehyde to 5-azabenzimidazole is 1:0.25; the molar ratio of cinnamaldehyde to sodium hydride is 1:1; the molar ratio of cinnamaldehyde to anhydrous ethanol is 1mmol:1mL; the molar ratio of cinnamaldehyde to pure water is 1mmol:0.75mL; the molar ratio of cinnamaldehyde to toluene is 1mmol:0.75mL.

[0074] Embodiment 5: The difference between this embodiment and embodiment 1 is only the component of the coupling activator in step 2.

[0075] The coupling activator in this embodiment includes 5-ethylthiotetrazolyl, amide derivatives, benzotriazole derivatives and deionized water, the mass ratio of 5-ethylthiotetrazolyl and amide derivatives is 1:0.3, the mass ratio of 5-ethylthiotetrazolyl and benzotriazole derivatives is 1:0.05; the dosage ratio of 5-ethylthiotetrazolyl and deionized water is 1g:25mL. The preparation methods of amide derivatives and benzotriazole derivatives are as follows: The preparation method of the amide derivative comprises the following steps: Weigh 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and 3-chloropropionic acid [3-(3-chloropropionylamino)phenyl] ester, add chloroform, stir and dissolve, react at 70°C for 24 hours, add acetone and recrystallize three times to obtain an amide derivative. The molar ratio of 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and 3-chloropropionic acid [3-(3-chloropropionylamino)phenyl] ester is 1:0.5; the dosage ratio of 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and chloroform is 1 mol:200 mL; the volume ratio of chloroform and acetone is 1:3.

[0076] The preparation of benzotriazole derivatives comprises the following steps: Weigh cinnamaldehyde, add carbon tetrachloride, stir evenly, slowly add epichlorohydrin and carbon tetrachloride dropwise at 60°C, react for 8 hours, add ice water, extract with ether three times, extract the organic phase, add 5-azabenzimidazole, sodium hydride, and anhydrous ethanol, stir and reflux for 2 hours, reflux reaction for 24 hours, add pure water and toluene to extract three times, extract the organic phase, filter, and dry to obtain a benzotriazole derivative. The molar ratio of cinnamaldehyde to carbon tetrachloride is 1mmol:1mL; the molar ratio of cinnamaldehyde to epichlorohydrin is 1:1.1; the molar ratio of cinnamaldehyde to carbon tetrachloride is 1mmol:0.25mL; the temperature of ice water is 4°C, and the molar ratio of cinnamaldehyde to ice water is 1mmol:0.75mL; the molar ratio of cinnamaldehyde to ether is 1mmol:0.75mL; the molar ratio of cinnamaldehyde to 5-azabenzimidazole is 1:0.25; the molar ratio of cinnamaldehyde to sodium hydride is 1:1; the molar ratio of cinnamaldehyde to anhydrous ethanol is 1mmol:1mL; the molar ratio of cinnamaldehyde to pure water is 1mmol:0.75mL; the molar ratio of cinnamaldehyde to toluene is 1mmol:0.75mL.

[0077] Comparative Example 1: Compared with Example 1, the difference between this comparative example and Example 1 is only in step 3: Firstly, a deprotecting agent is added to the oligonucleotide fragment, and then a triethylamine solution is added at a flow rate of 75 cm / L, the reaction is carried out for 10 minutes, an ammonolysis solution is added, the reaction is carried out at 45°C for 8 hours, and the oligonucleotide is purified by silica gel column chromatography. The mass concentration of the triethylamine solution is 10%, and the volume ratio of the reaction solution containing the oligonucleotide fragment to the triethylamine solution is 1:1; the volume ratio of the triethylamine solution to the deprotecting agent is 1:2, the deprotecting agent includes trifluoroacetic acid and dichloromethane, and the dosage ratio of trifluoroacetic acid and dichloromethane is 1g:100mL; the volume ratio of the triethylamine solution to the ammonolysis solution is 1:0.3, the ammonolysis solution includes ammonia water and methylamine solution, the volume ratio of ammonia water and methylamine solution is 1:1, the mass concentration of ammonia water is 25%, the methylamine solution includes methylamine and anhydrous ethanol, and the volume ratio of methylamine and anhydrous ethanol is 1:2.5.

[0078] Comparative Example 2: The difference between this comparative example and Example 1 is only the component of the coupling activator in step 2.

[0079] The coupling activator of this embodiment includes 5-ethylthiotetrazolyl, a benzotriazole derivative and deionized water, the mass ratio of 5-ethylthiotetrazolyl and the benzotriazole derivative is 1:0.1; the amount ratio of 5-ethylthiotetrazolyl and deionized water is 1g:25mL. The preparation method of the benzotriazole derivative includes the following steps: Weigh cinnamaldehyde, add carbon tetrachloride, stir evenly, slowly add epichlorohydrin and carbon tetrachloride dropwise at 60°C, react for 8 hours, add ice water, extract with ether three times, extract the organic phase, add 5-azabenzimidazole, sodium hydride, and anhydrous ethanol, stir and reflux for 2 hours, reflux reaction for 24 hours, add pure water and toluene to extract three times, extract the organic phase, filter, and dry to obtain a benzotriazole derivative. The molar ratio of cinnamaldehyde to carbon tetrachloride is 1mmol:1mL; the molar ratio of cinnamaldehyde to epichlorohydrin is 1:1.1; the molar ratio of cinnamaldehyde to carbon tetrachloride is 1mmol:0.25mL; the temperature of ice water is 4°C, and the molar ratio of cinnamaldehyde to ice water is 1mmol:0.75mL; the molar ratio of cinnamaldehyde to ether is 1mmol:0.75mL; the molar ratio of cinnamaldehyde to 5-azabenzimidazole is 1:0.25; the molar ratio of cinnamaldehyde to sodium hydride is 1:1; the molar ratio of cinnamaldehyde to anhydrous ethanol is 1mmol:1mL; the molar ratio of cinnamaldehyde to pure water is 1mmol:0.75mL; the molar ratio of cinnamaldehyde to toluene is 1mmol:0.75mL.

[0080] Experimental example: 1. Material Characterization The infrared spectrum of the amide derivative prepared by the present invention was measured by Fourier transform infrared spectrometer using KBr tablet method at a wave number of 400-4000cm -1Measurement within the range, resolution 0.06cm -1 , scanned 32 times.

[0081] Figure 1 This is the infrared spectrum of the amide derivative prepared by the present invention, 3400cm -1 The absorption peak of NH appears near 2920cm -1 、2850cm -1 Saturated alkanes -CH 3 and -CH 2 The absorption peak is 2240cm -1 The absorption peak of cyano group appears near 1750cm -1 There is a C=O absorption peak near 1620cm -1 The absorption peak of C=C appears near 1170cm -1 The absorption peak of COC appears nearby.

[0082] 2. Determination of purity The purity of the oligonucleotides prepared in Examples 1-5 and Comparative Examples 1-2 was determined by HPLC. Purity (%) = (target protein peak area / target protein peak height) / (total peak area / total peak height) × 100%. The results are shown in Table 1.

[0083] Table 1 Purity of oligonucleotides (%)

[0084] As can be seen from Table 1, the purity of Example 1 of the present invention is higher than that of Comparative Example 1. This is because in the synthesis of the oligonucleotide, Example 1 first uses a triethylamine solution to remove the cyanoethyl group on the phosphate group, and then uses a deprotecting group reagent to remove the MMT protecting group on the group; while Comparative Example 1 first uses a deprotecting group reagent to remove the MMT protecting group on the group, and then uses a triethylamine solution to remove the cyanoethyl group on the phosphate group; this shows that the use of the deprotecting group sequence of Example 1 of the present invention to synthesize oligonucleotides can improve the purity of the oligonucleotides.

[0085] The purity of Example 2-3 of the present invention is higher than that of Example 1 because, in the synthesis of the oligonucleotide, the coupling activator used in Example 1 includes 5-ethylthiotetrazole and deionized water, and the coupling activator used in Example 2-3 includes 5-ethylthiotetrazole, an amide derivative and deionized water; the purity of Example 2 is higher than that of Example 3 because the amount of the amide derivative used in the coupling activator is different; this shows that the purity of the oligonucleotide can be improved by additionally adding an amide derivative to the coupling activator in the present invention.

[0086] The purity of Examples 4-5 of the present invention is higher than that of Example 2 because, in the coupling activator, Examples 4-5 further add a benzotriazole derivative; the purity of Example 4 is higher than that of Example 5 because the amount of the benzotriazole derivative used in the coupling activator is different; the purity of Example 4 is higher than that of Comparative Example 2 because, in the coupling activator, Comparative Example 2 only adds a benzotriazole derivative, but does not add an amide derivative; this shows that the synergistic use of amide derivatives and benzotriazole derivatives in the coupling activator can further improve the purity of the oligonucleotide.

[0087] 3. Determination of yield The yields of the oligonucleotides prepared in Examples 1-5 and Comparative Examples 1-2 were determined by HPLC, where yield = final yield of oligonucleotide / theoretical yield of oligonucleotide × 100%. The results are shown in Table 2.

[0088] Table 2 Oligonucleotide yield (%)

[0089] As can be seen from Table 2, the yield of Example 1 of the present invention is higher than that of Comparative Example 1. This is because in the synthesis of the oligonucleotide, Example 1 first uses a triethylamine solution to remove the cyanoethyl group on the phosphate group, and then uses a deprotecting group reagent to remove the MMT protecting group on the group; while Comparative Example 1 first uses a deprotecting group reagent to remove the MMT protecting group on the group, and then uses a triethylamine solution to remove the cyanoethyl group on the phosphate group; this shows that the use of the deprotecting group sequence of Example 1 of the present invention to synthesize oligonucleotides can improve the yield of oligonucleotides.

[0090] The yield of Example 2-3 of the present invention is higher than that of Example 1 because, in the synthesis of the oligonucleotide, the coupling activator used in Example 1 includes 5-ethylthiotetrazole and deionized water, and the coupling activator used in Example 2-3 includes 5-ethylthiotetrazole, an amide derivative and deionized water; the yield of Example 2 is higher than that of Example 3 because the amount of the amide derivative used in the coupling activator is different; this shows that the additional addition of an amide derivative to the coupling activator in the present invention can increase the yield of the oligonucleotide.

[0091] The yield of Examples 4-5 of the present invention is higher than that of Example 2 because in the coupling activator, Examples 4-5 further add a benzotriazole derivative; the yield of Example 4 is higher than that of Example 5 because the amount of the benzotriazole derivative used in the coupling activator is different; the yield of Example 4 is higher than that of Comparative Example 2 because in the coupling activator, only a benzotriazole derivative is added to the Comparative Example 2, but no amide derivative is added; this shows that the synergistic use of amide derivatives and benzotriazole derivatives in the coupling activator can further improve the yield of oligonucleotides.

[0092] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0093] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for synthesizing an oligonucleotide, comprising: S1, connecting the phosphoramidite nucleotide monomer and MMT-hexylamino group to a phosphoramidite reaction to obtain a phosphoramidite nucleotide monomer; S2, using the phosphoramidite method, reacting the phosphoramidite nucleotide monomer with an oligonucleotide synthesis reagent to obtain a reaction solution containing the oligonucleotide fragment; S3, first adding a triethylamine solution to the reaction solution containing the oligonucleotide fragment to carry out a decyanoethylation reaction, and then adding a deprotection reagent to carry out a reaction to obtain an oligonucleotide final product; The oligonucleotide synthesis reagents include a detrityl reagent, a coupling activator, an oxidizing reagent, a sulfurizing reagent and a capping reagent; the coupling activator includes deionized water and at least one of 5-ethylthiotetrazolyl and an amide derivative, wherein the amide derivative has an amide group and a benzene ring.

2. A method for synthesizing an oligonucleotide according to claim 1, characterized in that: The amide derivative is prepared by reacting 2-cyano-N-[2-(dimethylamino)ethyl]acetamide and 3-chloropropionic acid [3-(3-chloropropionylamino)phenyl] ester.

3. A method for synthesizing an oligonucleotide according to claim 1, characterized in that: The usage ratio of the phosphoramidite nucleotide monomer and the MMT-hexylamino linked phosphoramidite is 1 mg: 1.5-3 mL.

4. A method for synthesizing an oligonucleotide according to claim 1, characterized in that: The detritylation reagent comprises dichloroacetic acid and dichloromethane, and the usage ratio of dichloroacetic acid to dichloromethane is 1g:10-50mL.

5. A method for synthesizing an oligonucleotide according to claim 1, characterized in that: The oxidizing agent comprises iodine, pyridine and water, the mass ratio of iodine to pyridine is 1:1-2, and the dosage ratio of iodine to water is 1g:5-10mL.

6. A method for synthesizing an oligonucleotide according to claim 1, characterized in that: The sulfurizing agent includes hydrogenated xanthan gum and pyridine, and the usage ratio of hydrogenated xanthan gum to pyridine is 1g:10-50mL.

7. A method for synthesizing an oligonucleotide according to claim 1, characterized in that: The capping reagent includes acid anhydride and acetonitrile, and the usage ratio of acid anhydride to acetonitrile is 1g:2-10mL.

8. A method for synthesizing an oligonucleotide according to claim 1, characterized in that: The mass concentration of the triethylamine solution is 2-20%, and the volume ratio of the reaction solution containing the oligonucleotide fragments to the triethylamine solution is 1:1-2.

9. A method for synthesizing an oligonucleotide according to claim 1, characterized in that: The deprotection reagent comprises trifluoroacetic acid and dichloromethane, and the usage ratio of trifluoroacetic acid to dichloromethane is 1g:50-200mL.

10. The method for synthesizing an oligonucleotide according to claim 1, characterized in that: The volume ratio of the triethylamine solution to the deprotection reagent is 1:1-5.

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