Amonolysis reagent and application thereof in synthesis of diaminopurine oligonucleotide

Abstract

The invention discloses an ammonolysis reagent and application thereof in synthesis of diaminopurine oligonucleotide, and belongs to the technical field of oligonucleotide synthesis, and the synthesis of the oligonucleotide containing diaminopurine comprises the steps of deprotection, coupling, oxidation or vulcanization, capping and the like. The ammonolysis reagent comprises methylamine and ammonia water, and further comprises 2-mercaptobenzimidazole and a modified imidazole compound, and the modified imidazole compound has a phenylimidazole group and a pyrimidine group. The ammonolysis reagent can realize ammonolysis of oligonucleotide containing diaminopurine, shorten the ammonolysis time, improve the purity of full-length chain nucleic acid of a target product and facilitate removal of an iBu protecting group on diaminopurine N2-.

Description

Technical Field

[0001] The invention belongs to the technical field of oligonucleotide synthesis, and particularly relates to an aminolysis reagent and application thereof in the synthesis of diaminopurine oligonucleotide. Background Art

[0002] Oligonucleotides can be used as primers (Primer) and gene probes (Probe) for DNA synthesis, and have a wide range of uses in modern molecular biology research. The synthesis of oligonucleotides mostly adopts phosphoramidite solid phase synthesis technology. The phosphoramidite solid phase synthesis method uses trivalent phosphorus as a raw material, and the synthesis reaction is rapid; but due to the strong activity of trivalent phosphorus, it is very easy to cause side reactions when it comes into contact with water, acidic substances and air. After the synthesis of oligonucleotides, cleavage will occur during the removal of protective groups under alkaline conditions, resulting in the generation of short fragment impurities. At present, the commonly used aminolysis reagent in the synthesis of oligonucleotides is ammonia water, which usually needs to be aminolyzed at 55°C for more than 4 hours, but the content of degradation products in the final product is relatively high, usually more than 30%, which seriously affects the product quality.

[0003] After the synthesis of oligonucleotides containing diaminopurine is completed, ammonia water is used as a reagent for aminolysis, which usually requires a reaction at 80°C for more than 24 hours. In addition, since the amino protecting group at the N2 position of diaminopurine is iBu, this protecting group is difficult to remove under conventional aminolysis conditions, resulting in excessive by-product content in the oligonucleotide synthesis product, affecting the performance. Therefore, it is necessary to develop a new aminolysis reagent in order to improve the product quality in the process of synthesizing oligonucleotides containing diaminopurine. Summary of the invention

[0004] The purpose of the present invention is to provide an aminolysis reagent and its application in the synthesis of diaminopurine oligonucleotides. The aminolysis reagent can realize the aminolysis of diaminopurine oligonucleotides, shorten the aminolysis time, improve the purity of the full-length nucleic acid of the target product, and help remove the iBu protecting group on the N2- of diaminopurine.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A method for synthesizing an oligonucleotide containing diaminopurine, wherein the structure of the nucleoside phosphoramidite monomer containing diaminopurine is as shown in formula (1): Formula (1) The following steps are involved: (1) Deprotection: Use dichloroacetic acid or trichloroacetic acid to remove the DMT protecting group on the 5'-OH of the nucleotide attached to the solid phase support; (2) Coupling: The nucleoside phosphoramidite monomer is mixed with tetrazolium and introduced into the synthesis column, where it is coupled with the 5'-OH of the oligonucleotide connected to the solid phase support, condensed and de-tetrazole to form a phosphotriester bond; (3) Oxidation or sulfidation: Use oxidizing agents or sulfiding agents to convert phosphite into phosphotriester to obtain stable oligonucleotides; (4) Capping: Use a capping reagent to block the unreacted free 5'-OH group; (5) Repeat steps (1) to (4) to add nucleotide monomers one by one to the synthesized oligonucleotide chain; and use an aminolysis reagent to cut the oligonucleotide chain from the solid phase support; the aminolysis reagent includes methylamine and ammonia water, and also includes 2-mercaptobenzimidazole and a modified imidazole compound, wherein the modified imidazole compound has a phenylimidazole group and a pyrimidine group.

[0006] Furthermore, the oxidizing agent is a mixed solution of iodine dissolved in tetrahydrofuran, pyridine and water, wherein the concentration of iodine therein is 0.02-0.1 mol / L; based on the sum of the volumes of tetrahydrofuran, pyridine and water, the volume content of tetrahydrofuran is 60-70%, the volume percentage of pyridine is 15-30%, and the volume percentage of water is 5-20%.

[0007] The sulfiding agent is a pyridine solution of hydrogenated xanthan gum, and the mass percentage concentration of hydrogenated xanthan gum is 2-8%.

[0008] Further, the capping agent includes acetic anhydride or N-methylimidazole (NMI).

[0009] Preferably, the capping agent is a tetrahydrofuran solution containing 10-20% (v / v) acetic anhydride.

[0010] Preferably, the amount of aminolysis reagent used for 1 mmol of oligonucleotide is 40-100 mL, the aminolysis temperature is 40-60° C., and the aminolysis reaction lasts 6-24 hours.

[0011] Preferably, in the ammonolysis reagent, the volume ratio of methylamine to aqueous ammonia is 1:0.5-2, the mass ratio of 2-mercaptobenzimidazole to the modified imidazole compound is 1:0.5-3, and the mass volume ratio of the modified imidazole compound to methylamine is 3-15 mg:1 mL.

[0012] In the process of synthesizing oligonucleotides containing diaminopurine, compared with the use of ammonia water alone, the use of methylamine and ammonia water as the aminolysis reagent can complete the aminolysis reaction at a relatively low aminolysis temperature and a relatively short aminolysis time, which can not only improve the purity of the obtained full-length nucleic acid (FL), but also remove the iBu protecting group on the N2- of diaminopurine, thereby reducing the content of by-products. In particular, the addition of modified imidazole compounds and 2-mercaptobenzimidazole to the aminolysis reagent can further improve the purity of the FL obtained by aminolysis and reduce the content of by-products with a molecular weight greater than 71.0Da, that is, further improve the removal effect of the iBu protecting group on the N2- of diaminopurine. This may be because, compared with aqueous ammonia, the alkalinity of the ammonolysis reagents added with methylamine, modified imidazole compounds, and 2-mercaptobenzimidazole is weakened, thereby reducing the impurity products in the ammonolysis process; in addition, the presence of phenylimidazole groups and pyrimidine groups in modified imidazole compounds and benzimidazole groups in 2-mercaptobenzimidazole can reduce the probability of oligonucleotide chain breakage during the ammonolysis process, thereby improving the purity of FL in the final product.

[0013] According to one aspect of the present invention, there is provided an ammonolysis reagent, comprising methylamine and aqueous ammonia, wherein the volume ratio of methylamine to aqueous ammonia is 1:1.

[0014] According to one aspect of the present invention, an ammonolysis reagent is provided, comprising methylamine and aqueous ammonia, and further comprising 2-mercaptobenzimidazole and a modified imidazole compound; the modified imidazole compound has a phenylimidazole group and a pyrimidine group.

[0015] Furthermore, the volume ratio of methylamine to aqueous ammonia is 1:0.5-2, the mass ratio of 2-mercaptobenzimidazole to the modified imidazole compound is 1:0.5-3, and the mass volume ratio of the modified imidazole compound to methylamine is 3-15 mg:1 mL.

[0016] The aminolysis of oligonucleotides containing diaminopurine using an aminolysis reagent containing 2-mercaptobenzimidazole and a modified imidazole compound can complete the aminolysis reaction at a relatively low aminolysis temperature and a relatively short aminolysis time, which can not only improve the purity of the obtained FL, but also remove the iBu protecting group on the N2- of diaminopurine, thereby reducing the content of by-products.

[0017] According to one aspect of the present invention, there is provided a method for preparing a modified imidazole compound, which is prepared by a substitution reaction between 5-carbonylchloropyrimidine and 4-(2-imidazolyl)phenol, comprising the following steps: In a tetrahydrofuran solution containing triethylamine, 5-carbonylchloropyrimidine reacts with 4-(2-imidazolyl)phenol. After the reaction is completed, the filtrate is rotary evaporated and the obtained solid is dried to obtain a modified imidazole compound.

[0018] The acyl chloride group in 5-carbonylchloropyrimidine undergoes substitution reaction with the active hydrogen on the pyrrole nitrogen atom on the imidazole ring in 4-(2-imidazolyl)phenol, and the resulting modified imidazole compound has a phenylimidazole group and a carbonylpyrimidine group.

[0019] Specifically, the preparation method of the modified imidazole compound is as follows: 5-Carbonylchloropyrimidine is added to tetrahydrofuran and stirred to obtain a mixed solution A; 4-(2-imidazolyl)phenol is added to tetrahydrofuran, and triethylamine is added and stirred to obtain a mixed solution B; the mixed solution B is added to the mixed solution A and stirred to react; after the reaction is completed, the solvent in the filtrate is filtered and removed, and the obtained solid is dried to obtain a modified imidazole compound.

[0020] Furthermore, in the preparation of the modified imidazole compound, after the mixed solution B is added to the mixed solution A, the mixture is stirred at 35-55° C. and 100-500 r / min for 4-10 hours.

[0021] Furthermore, in the preparation of the modified imidazole compound, after the reaction is completed, the filtrate is filtered and evaporated, the solid is washed with deionized water and filtered again, and the filter residue is vacuum dried at 45-70° C. for 4-10 hours to obtain the modified imidazole compound.

[0022] Furthermore, in the preparation of the modified imidazole compound, in solution A, the mass volume ratio of 5-carbonylchloropyrimidine to tetrahydrofuran is 1 g:3-8 mL.

[0023] Furthermore, in the preparation of the modified imidazole compound, in solution B, the mass volume ratio of 4-(2-imidazolyl)phenol to tetrahydrofuran is 1 g:3-8 mL.

[0024] Furthermore, in the preparation of the modified imidazole compound, in solution B, the mass ratio of 4-(2-imidazolyl)phenol to triethylamine is 2-3:1-5.

[0025] Furthermore, in the preparation of the modified imidazole compound, the mass ratio of 5-carbonylchloropyrimidine in solution A to 4-(2-imidazolyl)phenol in solution B is 1:0.5-2.

[0026] The modified imidazole compound is combined with 2-mercaptobenzimidazole, methylamine, and ammonia water as an aminolysis reagent, and is applied to the aminolysis reaction of oligonucleotides, which can greatly improve the purity of FL in the obtained product and reduce the content of by-products. This may be because the presence of phenylimidazole groups and pyrimidine groups in the modified imidazole compounds, combined with the amino groups and other groups such as benzimidazole contained in the aminolysis reagent, helps to improve the aminolysis efficiency of the aminolysis reagent, especially the benzimidazole group in 2-mercaptobenzimidazole contains a thiol group, which further improves the aminolysis efficiency of the aminolysis reagent under the joint action of the benzimidazole group, the phenylimidazole group and the pyrimidine group. In addition, the presence of phenolic hydroxyl groups in the modified imidazole compound also helps to reduce the alkalinity of the aminolysis reagent, thereby reducing the probability of oligonucleotide breakage. On the other hand, the modified imidazole compounds and 2-mercaptobenzimidazole are used in the aminolysis reagent, and the phenylimidazole group, benzimidazole group and pyrimidine group, etc., can use their own spatial structure to form protection for the PO bond on the oligonucleotide chain, thereby reducing the probability of oligonucleotide chain breakage. In addition, the phenolic hydroxyl group, phenylimidazole group and pyrimidine group in the modified imidazole compound cooperate with the thiol group and benzimidazole group in 2-mercaptobenzimidazole, which may be conducive to removing the amino protecting group iBu from the N2 position on the diaminopurine, thereby reducing the content of by-products when the oligonucleotide is cut off from the carrier.

[0027] According to one aspect of the present invention, an ammonolysis reagent is provided, which includes methylamine, ammonia water, 2-mercaptobenzimidazole and a modified imidazole compound; the modified imidazole compound has a phenylimidazole group; the ammonolysis reagent also includes 2-amino-1H-imidazole-1-propionic acid.

[0028] Furthermore, in the ammonolysis reagent, the volume ratio of methylamine and ammonia water is 1:0.5-2, the mass ratio of 2-mercaptobenzimidazole and the modified imidazole compound is 1:0.5-3, the mass volume ratio of the modified imidazole compound to methylamine is 3-15 mg:1 mL, and the mass ratio of the modified imidazole compound to 2-amino-1H-imidazole-1-propionic acid is 1:0.5-2.

[0029] The use of 2-amino-1H-imidazole-1-propionic acid in the aminolysis reagent in combination with 2-mercaptobenzimidazole, modified imidazole compounds, etc. in the oligonucleotide aminolysis process can further improve the purity of FL in the obtained product and reduce the content of by-products. This may be because the presence of the carboxyl group in 2-amino-1H-imidazole-1-propionic acid further adjusts the alkalinity of the aminolysis reagent, which helps to avoid the breakage of the oligonucleotide; and in the process of contact between 2-amino-1H-imidazole-1-propionic acid and the modified imidazole compound and / or 2-mercaptobenzimidazole, the amino group, carboxyl group, etc. on the 2-amino-1H-imidazole-1-propionic acid can further affect the activity of the phenylimidazole group in the modified imidazole compound and the benzimidazole group and thiol group in 2-mercaptobenzimidazole, thereby increasing the probability of iBu falling off from the N2 position on diaminopurine.

[0030] Compared with the prior art, the present invention has the following beneficial effects: 1. Provided is an aminolysis reagent, comprising methylamine and ammonia water, and also comprising 2-mercaptobenzimidazole and a modified imidazole compound; the modified imidazole compound has a phenylimidazole group and a pyrimidine group. The aminolysis reagent can be used to complete the aminolysis reaction of an oligonucleotide containing diaminopurine at a relatively low aminolysis temperature and a relatively short aminolysis time, which can not only improve the purity of the obtained FL, but also remove the iBu protecting group on the N2- of diaminopurine, thereby reducing the content of by-products.

[0031] 2. Compared with ammonia water, the ammonolysis reagent added with methylamine, 2-mercaptobenzimidazole and modified imidazole compounds can greatly improve the FL purity in the final product of ammonolysis; compared with ammonia water and methylamine, the ammonolysis reagent added with 2-mercaptobenzimidazole and modified imidazole compounds can also improve the FL purity in the final product of ammonolysis. This may be because the alkalinity of the ammonolysis reagent added with methylamine, 2-mercaptobenzimidazole and modified imidazole compounds changes, thereby reducing the impurity products in the ammonolysis process; on the other hand, the presence of amino groups in methylamine, phenylimidazole groups and pyrimidine groups in modified imidazole compounds, and benzimidazole groups in 2-mercaptobenzimidazole can reduce the probability of oligonucleotide chain breakage during the ammonolysis process. It may also be because the presence of phenylimidazole groups and pyrimidine groups in modified imidazole compounds, together with the amino groups and other groups such as benzimidazole contained in the ammonolysis reagent, helps to improve the ammonolysis efficiency of the ammonolysis reagent. Furthermore, the presence of phenolic hydroxyl groups in the modified imidazole compounds also helps to reduce the alkalinity of the aminolysis reagent, thereby reducing the probability of oligonucleotide breakage.

[0032] 3. The aminolysis reagents added with methylamine, 2-mercaptobenzimidazole and modified imidazole compounds can significantly reduce the content of by-products in the final product of aminolysis of oligonucleotides containing diaminopurine; this may be because the phenolic hydroxyl group, phenylimidazole group and pyrimidine group in the modified imidazole compound cooperate with the thiol group and benzimidazole group in 2-mercaptobenzimidazole, which may be beneficial to the removal of the amino protecting group iBu from the N2 position of diaminopurine, thereby reducing the content of by-products when the oligonucleotide is cut from the carrier.

[0033] 4. Further adding 2-amino-1H-imidazole-1-propionic acid to the above-mentioned aminolysis reagent can further improve the purity of FL in the obtained product and reduce the content of by-products. This may be because the presence of the carboxyl group in 2-amino-1H-imidazole-1-propionic acid further adjusts the alkalinity of the aminolysis reagent, which helps to avoid the breakage of the oligonucleotide; and in the process of contact between 2-amino-1H-imidazole-1-propionic acid and the modified imidazole compound and / or 2-mercaptobenzimidazole, the amino group, carboxyl group and other groups on it can further affect the activity of the phenylimidazole group in the modified imidazole compound and the benzimidazole group and the thiol group in 2-mercaptobenzimidazole, thereby increasing the probability of iBu falling off from the N2 position on diaminopurine. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The present invention is a flow chart of deprotection-coupling-sulfurization-capping for synthesizing oligonucleotides containing diaminopurine by the phosphoramidite solid phase synthesis method, wherein the letter B represents a base; Figure 2 The figure is a flow chart of the aminolysis reaction of synthesizing oligonucleotides containing diaminopurine by the phosphoramidite solid phase synthesis method of the present invention, wherein the letter B represents the base. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] The oligonucleotide containing diaminopurine was synthesized by the general phosphoramidite solid phase synthesis method. The specific synthesis method is shown in the attached Figure 1 , including a deprotection step (detritylation), a coupling step (coupling), an oxidation (oxidation) or thiolation (thiolation) step, and a capping step (capping).

[0037] In the deprotection step, dichloroacetic acid or trichloroacetic acid is used to remove the protecting group DMT on the 5'-OH of the nucleotide attached to the solid phase support to obtain a free 5'-OH group for the next condensation reaction.

[0038] In the coupling step, the nucleoside phosphoramidite monomer is mixed with the activator tetrazolium and introduced into the synthesis column. The nucleoside phosphoramidite monomer is protonated by the activator, undergoes affinity reaction with the free 5'-OH, condenses and removes tetrazolium to form a phosphotriester bond. At this time, the synthesized oligonucleotide chain is extended by one base.

[0039] The oxidation or sulfidation step uses an oxidizing agent or sulfidation to convert the phosphite into a phosphotriester to obtain a stable oligonucleotide; the coupling step is sensitive to moisture, and before the next coupling, the residual water must be removed by washing with acetonitrile. The oxidizing agent is a mixed solution of iodine dissolved in tetrahydrofuran, pyridine, and water, wherein the concentration of iodine therein is 0.02-0.1 mol / L; based on the sum of the volumes of tetrahydrofuran, pyridine, and water, the volume content of tetrahydrofuran is 60-70%, the volume percentage of pyridine is 15-30%, and the volume percentage of water is 5-20%. The sulfidation agent is a pyridine solution of hydrogenated flavonoids, and the mass percentage concentration of hydrogenated flavonoids is 2-8%.

[0040] The capping step uses acetic anhydride or N-methylimidazole (NMI) to block the unreacted free 5'-OH group to prevent a small amount of unreacted 5'-OH attached to the solid phase carrier from entering the next cycle, which can greatly improve the purity of the final product. After that, this cycle is repeated once for each base to finally produce the desired oligonucleotide.

[0041] After the above four steps, the oligonucleotides are added one by one to the synthesized oligonucleotide chain. Finally, the oligonucleotides are cut off from the solid phase support with an aminolysis reagent to remove the protecting groups on the bases and phosphate groups, followed by purification and quantification.

[0042] Example 1 In this example, an oligonucleotide containing diaminopurine was synthesized using an OligoPilot™ oligonucleotide synthesizer (manufacturer: Cytiva), and the synthesis column was FineLINE 35 (manufacturer: Cytiva); wherein the structure of the nucleoside phosphoramidite monomer containing diaminopurine used is shown in formula (1):

[0043] Formula (1) The sequence of the obtained diaminopurine-containing oligonucleotide is: AmoeCmoeGmoe UmoeUmoeCmoeGmoeGmoeCmoe AmoeAmoeUmoe AmoeAmoeAmoe UmoeUmoeUmoe, wherein moe=2'-O-MOE Synthesis of diaminopurine-containing oligonucleotides (1) Deprotection: Use trichloroacetic acid to remove the DMT protecting group on the 5'-OH of the nucleotide attached to the solid phase support.

[0044] (2) Coupling: The nucleoside phosphoramidite monomer is mixed with tetrazolium and introduced into the synthesis column, where it is coupled with the 5'-OH of the oligonucleotide connected to the solid phase carrier, condensed and de-tetrazole to form a phosphotriester bond. At this time, the synthesized oligonucleotide chain is extended by one base.

[0045] (3) Sulfidation: Use a sulfiding agent to convert the phosphite into a phosphotriester to obtain a stable oligonucleotide; the sulfiding agent is a pyridine solution of hydrogenated xanthine, the mass percentage concentration of hydrogenated xanthine is 3%, and the amount of sulfiding agent used is 1 reaction column volume.

[0046] (4) Capping: The unreacted free 5'-OH group is blocked with a capping reagent, wherein the capping reagent is a tetrahydrofuran solution containing 15% (v / v) acetic anhydride.

[0047] (5) Using a washing liquid to remove the residual reagents in steps (1) to (4), the washing liquid is tetrahydrofuran.

[0048] (6) Repeat steps (1) to (5).

[0049] (7) Following the above steps, oligonucleotides are added one by one to the synthesized oligonucleotide chain, and finally an aminolysis reagent is used to cut the oligonucleotide from the solid phase support, remove the protecting group, and obtain the target product.

[0050] In this embodiment, methylamine and ammonia water are included, and the volume ratio of methylamine to ammonia water is 1:1; the amount of aminolysis reagent corresponding to 1 mmol of oligonucleotide is 80 mL, the aminolysis temperature is 45° C., and the reaction is carried out for 8 hours.

[0051] Example 2 Compared with Example 1, the difference between this example and Example 1 is that in the synthesis process of the oligonucleotide containing diaminopurine, the aminolysis reagent used in step (7) includes methylamine, ammonia water, 2-mercaptobenzimidazole and a modified imidazole compound, and the modified imidazole compound has a phenylimidazole group and a pyrimidine group. Among them, the volume ratio of methylamine to ammonia water is 1:1, the mass ratio of 2-mercaptobenzimidazole to the modified imidazole compound is 1:2, and the mass volume ratio of the modified imidazole compound to methylamine is 5 mg:1 mL. The remaining conditions and steps are the same.

[0052] The preparation method of the modified imidazole compound is as follows: 5-Carbonylchloropyrimidine was added to tetrahydrofuran, stirred at 200r / min for 30min to obtain a mixed solution A; 4-(2-imidazolyl)phenol was added to tetrahydrofuran, stirred at 200r / min for 30min, and then triethylamine was added, stirred at 200r / min for 10min, and mixed evenly to obtain a mixed solution B. Mixed solution B was added dropwise to mixed solution A, sealed, stirred at 40°C, 300r / min for 6h. After the reaction was completed, the mixture was filtered, the filtrate was rotary evaporated, the obtained solid was washed with deionized water and filtered again, and the residue was vacuum dried at 55°C for 8h to obtain a modified imidazole compound.

[0053] In solution A, the mass volume ratio of 5-carbonylchloropyrimidine to tetrahydrofuran is 1 g:5 mL; in solution B, the mass volume ratio of 4-(2-imidazolyl)phenol to tetrahydrofuran is 1 g:5 mL; in solution B, the mass ratio of 4-(2-imidazolyl)phenol to triethylamine is 3:2; the mass ratio of 5-carbonylchloropyrimidine in solution A to 4-(2-imidazolyl)phenol in solution B is 1:1.

[0054] Example 3 Compared with Example 1, the present embodiment is different in that, in the synthesis process of the oligonucleotide containing diaminopurine, the aminolysis reagent used in step (7) includes methylamine, ammonia water, 2-mercaptobenzimidazole and a modified imidazole compound; wherein the volume ratio of methylamine to ammonia water is 1:1, the mass ratio of 2-mercaptobenzimidazole to the modified imidazole compound is 1:2, and the mass volume ratio of the modified imidazole compound to methylamine is 7.5 mg:1 mL. The remaining conditions and steps are the same.

[0055] Wherein, the preparation method of the modified imidazole compound is the same as that in Example 3.

[0056] Example 4 Compared with Example 1, the present embodiment is different in that, in the synthesis process of the oligonucleotide containing diaminopurine, the aminolysis reagent used in step (7) includes methylamine, ammonia water, 2-mercaptobenzimidazole and a modified imidazole compound; wherein the volume ratio of methylamine to ammonia water is 1:1, the mass ratio of 2-mercaptobenzimidazole to the modified imidazole compound is 1:2, and the mass volume ratio of the modified imidazole compound to methylamine is 10 mg:1 mL. The remaining conditions and steps are the same.

[0057] The preparation method of the modified imidazole compound is the same as that in Example 2.

[0058] Example 5 Compared with Example 1, the present embodiment is different in that, in the synthesis process of the oligonucleotide containing diaminopurine, the aminolysis reagent used in step (7) includes methylamine, ammonia water, 2-mercaptobenzimidazole and a modified imidazole compound, and also includes 2-amino-1H-imidazole-1-propionic acid; wherein the volume ratio of methylamine to ammonia water is 1:1, the mass ratio of 2-mercaptobenzimidazole to the modified imidazole compound is 1:2, the mass volume ratio of the modified imidazole compound to methylamine is 5 mg:1 mL, and the mass ratio of the modified imidazole compound to 2-amino-1H-imidazole-1-propionic acid is 1:0.5. The remaining conditions and steps are the same.

[0059] The preparation method of the modified imidazole compound is the same as that in Example 2.

[0060] Example 6 Compared with Example 1, the present embodiment is different in that, in the synthesis process of the oligonucleotide containing diaminopurine, the aminolysis reagent used in step (7) includes methylamine, ammonia water, 2-mercaptobenzimidazole and a modified imidazole compound, and also includes 2-amino-1H-imidazole-1-propionic acid; wherein the volume ratio of methylamine to ammonia water is 1:1, the mass ratio of 2-mercaptobenzimidazole to the modified imidazole compound is 1:2, the mass volume ratio of the modified imidazole compound to methylamine is 5 mg:1 mL, and the mass ratio of the modified imidazole compound to 2-amino-1H-imidazole-1-propionic acid is 1:1. The remaining conditions and steps are the same.

[0061] The preparation method of the modified imidazole compound is the same as that in Example 2.

[0062] Comparative Example 1 The difference between this comparative example and Example 1 is that in the synthesis process of the oligonucleotide containing diaminopurine, the aminolysis reagent used in step (7) includes methylamine and ammonia water, and also includes a modified imidazole compound; wherein the volume ratio of methylamine to ammonia water is 1:1, and the mass volume ratio of the modified imidazole compound to methylamine is 7.5 mg:1 mL. The other conditions and steps are the same.

[0063] The preparation method of the modified imidazole compound is the same as that in Example 2.

[0064] Comparative Example 2 The difference between this comparative example and Example 1 is that in the synthesis process of the oligonucleotide containing diaminopurine, the aminolysis reagent used in step (7) includes methylamine and ammonia water, and also includes 2-mercaptobenzimidazole; wherein the volume ratio of methylamine to ammonia water is 1:1, and the mass volume ratio of 2-mercaptobenzimidazole to methylamine is 7.5 mg:1 mL. The other conditions and steps are the same.

[0065] Comparative Example 3 The difference between this embodiment and embodiment 1 is that in the synthesis process of the oligonucleotide containing diaminopurine, the aminolysis reagent used in step (7) is ammonia water. The other conditions and steps are the same.

[0066] Test example The oligonucleotides obtained in Examples 1-7 and Comparative Examples 1-2 were purified by ion exchange chromatography, elution solution A: 25 mmol sodium hydroxide, elution solution B: 25 mmol sodium hydroxide + 1.5 M sodium chloride; elution gradient: solution B 0-100%, 10-30 CV. The obtained oligonucleotides were desalted and concentrated, and then freeze-dried to obtain the final product, wherein the purity of the full-length nucleic acid (FL) is shown in Table 1, and the content of the by-product with a molecular weight greater than 71.0 Da is shown in Table 2.

[0067] Table 1 Purity of FL in each embodiment and comparative example

[0068] Referring to the data in Table 1, the FL purity obtained in Examples 1-6 and Comparative Examples 1-2 is improved relative to that in Comparative Example 3. Specifically, relative to Comparative Example 3, the FL purity obtained in Examples 1-6 and Comparative Examples 1-2 is increased by 37.9%, 45.4%, 47.3%, 49.6%, 50.1%, 51.1%, 30.0% and 30.9%, respectively. It can be seen that the use of methylamine and ammonia water in combination can greatly improve the purity of the target product, i.e., the full-length nucleic acid, in the final product relative to the use of ammonia water alone as a reagent during aminolysis.

[0069] Compared with Example 1, the FL purity obtained in Examples 2-6 and Comparative Examples 1-2 was increased by 5.5%, 6.8%, 8.4%, 8.8%, 9.6%, 5.7% and 5.1%, respectively. It can be seen that compared with the use of methylamine and ammonia water as reagents during aminolysis, the additional addition of modified imidazole compounds and 2-mercaptobenzimidazole can further improve the purity of the target product, that is, the full-length nucleic acid in the final product. Combined with the data of Examples 2-4, it can be seen that the content of the modified imidazole compound and 2-mercaptobenzimidazole in the aminolysis reagent is positively correlated with the FL purity in the final product.

[0070] Compared with Example 2, the purity of FL obtained in Examples 5-6 was increased by 3.2% and 3.9%, respectively. It can be seen that adding 2-amino-1H-imidazole-1-propionic acid to the aminolysis reagent containing the modified imidazole compound and 2-mercaptobenzimidazole can further improve the purity of FL in the final product.

[0071] Compared with Example 2, the purity of FL obtained in Comparative Examples 1-2 was reduced by 10.6% and 10.0%, respectively. It can be seen that in the aminolysis reagent, in addition to methylamine and ammonia water, the use of modified imidazole compounds and 2-mercaptobenzimidazole in combination can be more helpful in obtaining full-length nucleic acids and improving purity during the aminolysis process compared with the use of the two separately.

[0072] Table 2 Content of by-products with molecular weight greater than 71.0Da in each example and comparative example

[0073] Referring to the data in Table 2, compared with Comparative Example 3, the contents of by-products with a molecular weight greater than 71.0 Da obtained in Examples 1-6 and Comparative Examples 1-2 were reduced. Specifically, compared with Comparative Example 3, the contents of by-products with a molecular weight greater than 71.0 Da obtained in Examples 1-6 and Comparative Examples 1-2 were reduced by 40.9%, 55.4%, 63.0%, 65.9%, 68.1%, 70.6%, 43.5% and 45.6%, respectively. It can be seen that the use of methylamine in combination with ammonia water can significantly reduce the content of by-products with a molecular weight greater than 71.0 Da in the final product compared with the use of ammonia water alone as a reagent during aminolysis.

[0074] Compared with Example 1, the degradation contents obtained in Examples 2-6 and Comparative Examples 1-2 were reduced by 24.5%, 37.4%, 42.3%, 46.0%, 50.3%, 4.2% and 7.9%, respectively. It can be seen that the addition of modified imidazole compounds and 2-mercaptobenzimidazole can further reduce the content of by-products with a molecular weight greater than 71.0 Da in the final product compared with the use of methylamine and ammonia water as reagents during aminolysis. Combined with the data of Examples 2-4, it can be seen that the content of the modified imidazole compounds and 2-mercaptobenzimidazole in the aminolysis reagents is negatively correlated with the content of by-products with a molecular weight greater than 71.0 Da in the final product.

[0075] Compared with Example 2, the by-product contents of Examples 5-6 with a molecular weight greater than 71.0 Da were reduced by 28.4% and 34.1%, respectively. It can be seen that adding 2-amino-1H-imidazole-1-propionic acid to the aminolysis reagent containing the modified imidazole compound and 2-mercaptobenzimidazole can further reduce the by-product content in the final product.

[0076] Compared with Example 2, the contents of byproducts with a molecular weight greater than 71.0 Da obtained in Comparative Examples 1-2 were increased by 26.8% and 22.0%, respectively. It can be seen that in the ammonolysis reagent, in addition to methylamine and ammonia water, the use of modified imidazole compounds and 2-mercaptobenzimidazole in combination can further reduce the probability of generating byproducts with a molecular weight greater than 71.0 Da during the ammonolysis process compared with the use of the two separately.

[0077] 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.

[0078] The above-described embodiments provide a detailed description of the technical solution of the present invention. It should be understood that the above-described embodiments are only specific embodiments of the present invention and are not intended to limit the present invention. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A method for synthesizing an oligonucleotide containing diaminopurine, wherein the structure of the nucleoside phosphoramidite monomer containing diaminopurine is: , The following steps are involved: (1) Deprotection: Use dichloroacetic acid or trichloroacetic acid to remove the protecting group on the 5'-OH of the nucleotide attached to the solid phase support; (2) Coupling: The nucleoside phosphoramidite monomer is mixed with tetrazolium and introduced into the synthesis column, where it is coupled with the 5'-OH of the oligonucleotide connected to the solid phase support, condensed and de-tetrazole to form a phosphotriester bond; (3) Oxidation or sulfidation: Use oxidizing agents or sulfiding agents to convert phosphite into phosphotriester to obtain stable oligonucleotides; (4) Capping: Use a capping reagent to block the unreacted free 5'-OH group; (5) Repeat steps (1) to (4) to add nucleotide monomers one by one to the synthesized oligonucleotide chain; and use an aminolysis reagent to cut the oligonucleotide chain from the solid phase support; the aminolysis reagent includes methylamine and ammonia water, and also includes 2-mercaptobenzimidazole and a modified imidazole compound, wherein the modified imidazole compound has a phenylimidazole group and a pyrimidine group.

2. The method for synthesizing an oligonucleotide according to claim 1, characterized in that: In the ammonolysis reagent, the volume ratio of methylamine to ammonia water is 1:0.5-2, the mass ratio of 2-mercaptobenzimidazole to the modified imidazole compound is 1:0.5-3, and the mass volume ratio of the modified imidazole compound to methylamine is 3-15 mg:1 mL.

3. The method for synthesizing an oligonucleotide according to claim 1, characterized in that: The amount of aminolysis reagent used for 1mmol oligonucleotide is 40-100mL; The ammonolysis temperature is 40-60°C and the ammonolysis reaction lasts 6-24h.

4. The method for synthesizing an oligonucleotide according to claim 1, characterized in that: The oxidizing agent is a mixed solution of iodine dissolved in tetrahydrofuran, pyridine and water, wherein the concentration of iodine is 0.02-0.1 mol / L; based on the sum of the volumes of tetrahydrofuran, pyridine and water, the volume content of tetrahydrofuran is 60-70%, the volume percentage of pyridine is 15-30%, and the volume percentage of water is 5-20%; The sulfiding agent is a pyridine solution of hydrogenated xanthan gum, and the mass percentage concentration of hydrogenated xanthan gum is 2-8%.

5. An ammonolysis reagent, characterized in that It includes methylamine and ammonia water, and also includes 2-mercaptobenzimidazole and modified imidazole compounds, wherein the modified imidazole compound has a phenylimidazole group and a pyrimidine group.

6. The ammonolysis reagent according to claim 5, characterized in that In the ammonolysis reagent, the volume ratio of methylamine to ammonia water is 1:0.5-2, the mass ratio of 2-mercaptobenzimidazole to the modified imidazole compound is 1:0.5-3, and the mass volume ratio of the modified imidazole compound to methylamine is 3-15 mg:1 mL.

7. The ammonolysis reagent according to claim 5, characterized in that The phenylimidazole group in the modified imidazole compound is derived from 4-(2-imidazolyl)phenol, and the pyrimidine group is derived from 5-carbonylchloropyrimidine.

8. A method for synthesizing a modified imidazole compound, characterized in that: The modified imidazole compound has a phenylimidazole group and a pyrimidine group, and is prepared by a substitution reaction between 5-carbonylchloropyrimidine and 4-(2-imidazolyl)phenol.

9. The method for synthesizing a modified imidazole compound according to claim 8, characterized in that: The following steps are involved: 5-Carbonylchloropyrimidine is added to tetrahydrofuran and stirred to obtain a mixed solution A; 4-(2-imidazolyl)phenol is added to tetrahydrofuran, and triethylamine is added and stirred to obtain a mixed solution B; the mixed solution B is added to the mixed solution A and stirred to react; after the reaction is completed, the solvent in the filtrate is filtered and removed, and the obtained solid is dried to obtain a modified imidazole compound.

10. The method for synthesizing a modified imidazole compound according to claim 9, characterized in that: In solution A, the mass volume ratio of 5-carbonylchloropyrimidine to tetrahydrofuran is 1 g:3-8 mL; In solution B, the mass volume ratio of 4-(2-imidazolyl)phenol to tetrahydrofuran is 1 g:3-8 mL; In solution B, the mass ratio of 4-(2-imidazolyl)phenol to triethylamine is 2-3:1-5; The mass ratio of 5-carbonylchloropyrimidine in solution A to 4-(2-imidazolyl)phenol in solution B is 1:0.5-2.

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