Amonolysis reagent and application thereof in synthesis of base-free oligonucleotide

Abstract

The invention discloses an ammonolysis reagent and application thereof in synthesis of base-free oligonucleotide, and belongs to the technical field of oligonucleotide synthesis, and the base-free oligonucleotide synthesis comprises the steps of deprotection, coupling, oxidation or vulcanization, capping and the like. The ammonolysis reagent comprises methylamine and ammonia water, and further comprises indole-3-propanol and an indole compound I, and the indole compound I has an aminophenyl group. The ammonolysis reagent can realize ammonolysis of oligonucleotides at normal temperature, shorten ammonolysis time, improve the content of full-length chain nucleic acid of a target product, and help to reduce the content of degradation products.

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 base-free oligonucleotide synthesis. Background Art

[0002] Oligonucleotide drugs are a class of drugs composed of single-stranded or double-stranded oligonucleotides synthesized by artificial chemical synthesis. The production of oligonucleotide raw materials uses phosphoramidite solid-phase synthesis technology, which has a relatively high yield. However, the yield of oligonucleotides will gradually decrease with the increase of oligonucleotide length, and there are high barriers in process development, process scale-up and quality control.

[0003] The phosphoramidite solid phase synthesis method uses trivalent phosphorus as a raw material, and the synthesis reaction is rapid; however, 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. During the synthesis process, the reaction conditions must be strictly controlled. After the oligonucleotide is synthesized, cleavage will occur during the removal of the protecting group under alkaline conditions, resulting in the generation of short fragment impurities. At present, the commonly used aminolysis reagent in the oligonucleotide synthesis process 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.

[0004] Therefore, it is necessary to develop a new aminolysis reagent in order to improve the quality of oligonucleotide products. Summary of the invention

[0005] The object of the present invention is to provide an aminolysis reagent and its application in the synthesis of base-free oligonucleotides, wherein the aminolysis reagent can realize the aminolysis of oligonucleotides at room temperature, shorten the aminolysis time, increase the content of the full-length nucleic acid of the target product, and help reduce the content of degradation products.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A method for synthesizing an abasic oligonucleotide, wherein the abasic nucleoside phosphoramidite monomer structure includes 1'-OH and 2'-OMe or F modification; comprising the following steps: (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 indole-3-propanol and indole compound I, indole compound I has an aminophenyl group.

[0007] Preferably, in the ammonolysis reagent, the volume ratio of methylamine to aqueous ammonia is 1:0.5-3; the volume ratio of methylamine to indole-3-propanol is 1:0.5-2; and the volume mass ratio of methylamine to indole compound I is 1 mL:0.5-2 mg.

[0008] In the process of synthesizing oligonucleotides, methylamine and ammonia water are used as ammonolysis reagents, which can realize the ammonolysis reaction at room temperature and shorten the ammonolysis time to less than 4 hours. It can not only increase the content of the target product full-length nucleic acid (FL), but also help to reduce the content of degradation products. In particular, the addition of indole-3-propanol and indole compound I with an aminophenyl group to the ammonolysis reagent can further increase the FL content and reduce the content of degradation products. This may be because, compared with ammonia water, the alkalinity of the ammonolysis reagents added with methylamine, indole-3-propanol and indole compound I changes, thereby reducing the impurity products in the ammonolysis process; on the other hand, the presence of the amino group in methylamine, the hydroxyl group in indole-3-propanol, the aminophenyl group in indole compound I and the indole group protect the bases on the oligonucleotide chain, especially the bases with a hydroxyl group at the 1' position of the abasic monomer, which can reduce the probability of degradation of the structure of the bases with a hydroxyl group at the 1' position of the abasic monomer under room temperature conditions. Moreover, when the aminolysis reagent containing methylamine, indole-3-propanol and indole compound I is used for aminolysis at room temperature, the content of impurities in the product can be effectively reduced, which not only ensures the quality of the final product, but also helps to save energy and reduce costs. This may be because in the process of the aminolysis reagent cutting the oligonucleotide from the carrier, the presence of the amino group in methylamine and the aminophenyl group and indole group in indole compound I also protects the PO bond on the oligonucleotide chain, reducing the probability of oligonucleotide chain breakage.

[0009] Furthermore, the amount of aminolysis reagent used for 1 mmol of oligonucleotide is 40-100 mL.

[0010] Furthermore, the ammonolysis temperature is 20-55°C, and the ammonolysis reaction time is 2-6h.

[0011] 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%.

[0012] The sulfiding agent is a pyridine solution of hydrogenated xanthan gum, and the mass percentage concentration of hydrogenated xanthan gum is 2-8%.

[0013] Further, the capping agent includes acetic anhydride or N-methylimidazole (NMI).

[0014] Preferably, the capping agent is a tetrahydrofuran solution containing 10-20% (v / v) acetic anhydride.

[0015] An aminolysis reagent comprises methylamine and ammonia water, and also comprises indole-3-propanol and an indole compound I, wherein the indole compound I has an aminophenyl group.

[0016] Preferably, in the above-mentioned ammonolysis reagent, the volume ratio of methylamine to aqueous ammonia is 1:0.5-3; the volume ratio of methylamine to indole-3-propanol is 1:0.5-2; and the volume mass ratio of methylamine to indole compound I is 1 mL:0.5-2 mg.

[0017] Preferably, in the aminolysis reagent, the indole compound I has an aminophenyl group and a thiophene group; the thiophene group is derived from 2-(thiophen-2-yl)-1H-indole.

[0018] The presence of the thiophene group in the indole compound I helps to reduce the content of impurities and by-products during the aminolysis process. The thiophene group is introduced into the aminolysis reagent, and the activity of the aminophenyl group in the indole compound I and the NH bond on the indole group is affected by the thiophene group, thereby shortening the aminolysis time and increasing the content of the target product of the aminolysis. The thiophene group is introduced into the aminolysis reagent, and the thiophene group, the indole group and the aminophenyl group, etc., enable the indole compound I to form protection for the base-free monomer on the oligonucleotide chain due to its own spatial structure, thereby reducing the degradation of it by NH2OH in the aminolysis reagent, reducing the content of broken fragments in the final product, and increasing the FL content.

[0019] A method for preparing an indole compound I, wherein the indole compound I has an aminophenyl group, comprises the following steps: 2-Fluoronitrobenzene, NaOH and 2-(thiophen-2-yl)-1H-indole are dissolved in DMSO and stirred for reaction; then, an organic solvent is used for extraction, and after the solvent is removed from the organic phase, a primary product of indole compound I is obtained; then, the obtained primary product of indole compound I is heated to reflux with iron powder and ammonium chloride, extracted with an organic solvent, and after the solvent is removed from the organic phase, it is separated and purified again to obtain the final product of indole compound I.

[0020] Furthermore, during the extraction, ethyl acetate is used as an extractant, and anhydrous NaSO4 is added to the organic phase obtained by extraction and dried. Specifically, anhydrous NaSO4 is added to the organic phase obtained by extraction with ethyl acetate and dried, and the solvent is completely evaporated by vacuum filtration.

[0021] Furthermore, the molar ratio of 2-fluoronitrobenzene to NaOH is 1:0.5-2; the molar ratio of 2-fluoronitrobenzene to 2-(thiophen-2-yl)-1H-indole is 1:0.5-2; and 1-3 mL DMSO is used for 1 mmol of 2-fluoronitrobenzene and DMSO.

[0022] Furthermore, the molar ratio of 2-fluoronitrobenzene to iron powder and ammonium chloride is 1:2-5:0.5-2. The temperature during heating and reflux is 35-60°C, and the reflux time is 4-10h.

[0023] Preferably, the ammonolysis reagent also includes 2-hydroxyethylamine; the mass ratio of indole compound I to 2-hydroxyethylamine is 1:0.5-2.

[0024] The addition of 2-hydroxyethylamine, in which the hydroxyl group and the amino group exist, helps to further increase the content of the target product FL and reduce the content of the degradation product during the aminolysis process.

[0025] 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 indole-3-propanol and indole compound I, wherein the indole compound I has an aminophenyl group. In the process of synthesizing oligonucleotides, the aminolysis reagent can be used to achieve an aminolysis reaction at room temperature and shorten the aminolysis time to less than 4 hours, which can not only increase the content of the target product full-length nucleic acid (FL), but also help reduce the content of degradation products.

[0026] 2. Compared with ammonia water, the ammonolysis reagent with methylamine, indole-3-propanol and indole compound I added can significantly increase the content of the target product FL and reduce the content of degradation products; compared with ammonia water and methylamine, the ammonolysis reagent with indole-3-propanol and indole compound I added can also increase the content of the target product FL and reduce the content of degradation products. This may be because the alkalinity of the ammonolysis reagent with methylamine, indole-3-propanol and indole compound I added changes, thereby reducing the impurity products in the ammonolysis process; on the other hand, the presence of the amino group in methylamine, the hydroxyl group in indole-3-propanol, and the aminophenyl group and indole group in indole compound I protect the bases on the oligonucleotide chain, especially the bases with a hydroxyl group at the 1' position of the abasic monomer, which can reduce the probability of degradation of the structure of the bases with a hydroxyl group at the 1' position of the abasic monomer under room temperature conditions.

[0027] 3. Indole compound I has an aminophenyl group and a thiophene group; the thiophene group comes from 2-(thiophene-2-yl)-1H-indole. When the thiophene group is introduced into the ammonolysis reagent, the activity of the aminophenyl group in the indole compound I and the NH bond on the indole group are affected by the thiophene group, thereby shortening the ammonolysis time and increasing the content of the target product of ammonolysis. When the thiophene group is introduced into the ammonolysis reagent, the thiophene group, the indole group and the aminophenyl group, etc., enable the indole compound I to form protection for the abasic monomer on the oligonucleotide chain due to its own spatial structure, thereby reducing the degradation of it by NH2OH in the ammonolysis reagent, reducing the content of broken fragments in the final product, and increasing the FL content. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The flowchart of the deprotection-coupling-sulfurization-capping of the full-length oligonucleotide synthesized by the phosphoramidite solid phase synthesis method of the present invention, wherein the letter B represents the base; Figure 2 The figure is a flow chart of the aminolysis reaction of synthesizing full-length sequence oligonucleotides by the phosphoramidite solid phase synthesis method of the present invention, wherein the letter B represents the base. DETAILED DESCRIPTION

[0029] 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.

[0030] Full-length oligonucleotides containing abasic monomers were synthesized using a universal phosphoramidite solid-phase synthesis method.

[0031] See attached Figure 1The synthesis method specifically includes a deprotection step (detritylation), a coupling step (coupling), an oxidation (oxidation) or a thiolation (thiolation) step, and a capping step (capping).

[0032] 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.

[0033] 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.

[0034] 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%.

[0035] 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.

[0036] 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.

[0037] Example 1 In this example, an OligoPilot™ oligonucleotide synthesizer (manufacturer: Cytiva) was used to synthesize abasic oligonucleotides, and the synthesis column was FineLINE 35 (manufacturer: Cytiva); wherein the abasic nucleoside phosphoramidite monomer structure used included 1'-OH and 2'-OMe or F modification, as shown in formula (1) and formula (2):

[0038] Formula (1) Formula (2) The resulting sequence of the abasic oligonucleotide is: AmCmGm UfUmCf GmGmCm AmAmUm CfGfGfAmAmAm Uf, wherein m=2'-OMe; f=2'-F.

[0039] The synthesis method of abasic oligonucleotides is as follows: (1) Deprotection: Use trichloroacetic acid to remove the DMT protecting group on the 5'-OH of the nucleotide attached to the solid phase support.

[0040] (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.

[0041] (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.

[0042] (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.

[0043] (5) Using a washing liquid to remove the residual reagents in steps (1) to (4), the washing liquid is tetrahydrofuran.

[0044] (6) Repeat steps (1) to (5).

[0045] (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.

[0046] In this embodiment, the aminolysis reagent is NH2OH, the amount of the aminolysis reagent corresponding to 1 mmol of oligonucleotide is 50 mL, the aminolysis temperature is 25°C, and the reaction is carried out for 2 hours.

[0047] Example 2 The difference between this embodiment and embodiment 1 is that the ammonolysis reagent used in step (7) includes methylamine and ammonia water, and the volume ratio of methylamine to ammonia water is 1:2. The other conditions and steps are the same.

[0048] Example 3 Compared with Example 1, the difference between this example and Example 1 is that the aminolysis reagent used in step (7) includes methylamine, ammonia water, indole-3-propanol and indole compound I, and indole compound I has an aminophenyl group. The volume mass ratio of methylamine, ammonia water and indole compound I is 1 mL:2 mL:0.5 mg. The volume ratio of methylamine to ammonia water is 1:2; the volume ratio of methylamine to indole-3-propanol is 1:0.5; the volume mass ratio of methylamine to indole compound I is 1 mL:0.5 mg. The remaining conditions and steps are the same.

[0049] Wherein, the preparation method of indole compound I is as follows: 2-Fluoronitrobenzene, NaOH and 2-(thiophene-2-yl)-1H-indole are dissolved in DMSO and stirred at 25°C for 4 hours; then an organic solvent is used for extraction, and after the organic phase removes the solvent, a primary product of indole compound I is obtained. Specifically, 3 volumes of ethyl acetate are used as an extractant during extraction, and after 3 extractions, the organic phases are combined and dried by adding anhydrous NaSO4, filtered, and the solvent is completely evaporated to obtain a primary product of indole compound I.

[0050] Then the obtained indole compound I primary product was heated to reflux with iron powder and ammonium chloride, extracted with an organic solvent, and after the organic phase was freed of the solvent, the final product of indole compound I was separated and purified again. Specifically, distilled water was added according to the ratio of 1mmol 2-fluoronitrobenzene to 5mL water, and then iron powder and ammonium chloride were added, and the mixture was stirred and refluxed at 55°C for 6h. After the reaction solution was cooled to 25°C, it was extracted with 3 volumes of ethyl acetate. After 3 extractions, the organic phases were combined and dried by adding anhydrous NaSO4, filtered and evaporated to remove the solvent, and the obtained product was separated and purified by silica gel column chromatography, with ethyl acetate / petroleum ether (1:1, v / v) as the stationary phase and dichloromethane / petroleum ether (1:1, v / v) as the mobile phase to obtain the final product.

[0051] The molar ratio of 2-fluoronitrobenzene to NaOH is 1:1; the molar ratio of 2-fluoronitrobenzene to 2-(thiophen-2-yl)-1H-indole is 1:1; and 2 mL of DMSO is used for 1 mmol of 2-fluoronitrobenzene and DMSO.

[0052] The molar ratio of 2-fluoronitrobenzene to iron powder and ammonium chloride is 1:3:1.

[0053] Example 4 Compared with Example 1, the present embodiment is different in that the aminolysis reagent used in step (7) includes methylamine, aqueous ammonia, indole-3-propanol and indole compound I, the volume ratio of methylamine to aqueous ammonia is 1:2; the volume ratio of methylamine to indole-3-propanol is 1:0.5; and the volume mass ratio of methylamine to indole compound I is 1 mL:1 mg. The remaining conditions and steps are the same.

[0054] Wherein, the preparation method of indole compound I is the same as that in Example 3.

[0055] Example 5 Compared with Example 1, the difference between this example and Example 1 is that the aminolysis reagent used in step (7) includes methylamine, ammonia water, indole-3-propanol and indole compound I, the volume ratio of methylamine to ammonia water is 1:2; the volume ratio of methylamine to indole-3-propanol is 1:0.5; and the volume mass ratio of methylamine to indole compound I is 1 mL:1.5 mg. The other conditions and steps are the same.

[0056] Wherein, the preparation method of indole compound I is the same as that in Example 3.

[0057] Example 6 Compared with Example 1, this example is different in that the aminolysis reagent used in step (7) includes methylamine, ammonia water, indole-3-propanol, indole compound I and 2-hydroxyethylamine; the volume ratio of methylamine to ammonia water is 1:2, the volume ratio of methylamine to indole-3-propanol is 1:0.5, the volume mass ratio of methylamine to indole compound I is 1 mL:1.5 mg, and the mass ratio of indole compound I to 2-hydroxyethylamine is 1:1. The remaining conditions and steps are the same.

[0058] Wherein, the preparation method of indole compound I is the same as that in Example 3.

[0059] Example 7 Compared with Example 1, the present embodiment is different in that the aminolysis reagent used in step (7) includes methylamine, aqueous ammonia, indole-3-propanol, indole compound I and 2-hydroxyethylamine; the volume ratio of methylamine to aqueous ammonia is 1:2, the volume ratio of methylamine to indole-3-propanol is 1:0.5, the volume mass ratio of methylamine to indole compound I is 1 mL:1.5 mg, and the mass ratio of indole compound I to 2-hydroxyethylamine is 1:2. The remaining conditions and steps are the same.

[0060] Wherein, the preparation method of indole compound I is the same as that in Example 3.

[0061] Comparative Example 1 Compared with Example 3, this example is different in that the aminolysis reagent used in step (7) replaces the indole compound I with an equal mass of indole-3-propanol. The remaining conditions and steps are the same.

[0062] Comparative Example 2 Compared with Example 1, this example is different in that the aminolysis reagent used in step (7) is replaced by an equal mass of indole compound I in place of indole-3-propanol. The remaining conditions and steps are the same.

[0063] 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 content of the full-length nucleic acid (FL) is shown in Table 1, and the content of the degradation product is shown in Table 2.

[0064] Table 1 FL content of each embodiment and comparative example

[0065] Referring to the data in Table 1, the FL contents obtained in Examples 2-7 and Comparative Examples 1-2 are all improved relative to Example 1. Specifically, relative to Example 1, the FL contents obtained in Examples 2-7 and Comparative Examples 1-2 are increased by 12.9%, 22.6%, 27.4%, 30.6%, 32.2%, 33.9%, 16.1% and 17.7%, respectively. It can be seen that during aminolysis, compared with the use of ammonia water alone as a reagent, the use of methylamine in combination with ammonia water can greatly increase the content of the target product, i.e., the full-length nucleic acid, in the final product.

[0066] Compared with Example 2, the FL contents obtained in Examples 3-7 and Comparative Examples 1-2 were increased by 8.6%, 12.8%, 15.7%, 17.1%, 18.6%, 2.8% and 4.3%, respectively. It can be seen that the addition of indole-3-propanol and indole compound I can further increase the content of the target product, i.e., the full-length nucleic acid, in the final product compared with the use of methylamine and ammonia water as reagents during aminolysis. Combined with the data of Examples 3-5, it can be seen that the content of indole compound I in the aminolysis reagent containing indole compound I is positively correlated with the FL content in the final product.

[0067] Compared with Example 3, the FL contents obtained in Examples 6-7 were increased by 7.9% and 9.2%, respectively. It can be seen that adding 2-hydroxyethylamine to the aminolysis reagent containing indole compound I can further increase the FL content in the final product.

[0068] Compared with Example 3, the FL contents obtained in Comparative Examples 1-2 were reduced by 5.3% and 3.9%, respectively. It can be seen that in the aminolysis reagent, in addition to methylamine and ammonia water, the use of indole compound I and indole-3-propanol in combination can be more conducive to the acquisition of full-length nucleic acid in the aminolysis process than adding the two separately.

[0069] Table 2 Degradation product contents of various examples and comparative examples

[0070] Referring to the data in Table 2, the contents of degradation products obtained in Examples 2-7 and Comparative Examples 1-2 were all reduced relative to Example 1. Specifically, relative to Example 1, the contents of degradation products obtained in Examples 2-7 and Comparative Examples 1-2 were reduced by 37.9%, 51.7%, 58.6%, 65.5%, 69.0%, 72.4%, 44.8% and 48.3%, respectively. It can be seen that the use of methylamine in combination with ammonia water can significantly reduce the content of degradation products in the final product relative to the use of ammonia water alone as a reagent during ammonolysis.

[0071] Compared with Example 2, the degradation contents obtained in Examples 3-7 and Comparative Examples 1-2 were reduced by 22.2%, 33.3%, 44.4%, 50.0%, 55.6%, 11.1% and 16.7%, respectively. It can be seen that the addition of indole compound I can further reduce the content of degradation products in the final product compared with the use of methylamine and ammonia water as reagents during ammonolysis. Combined with the data of Examples 3-5, it can be seen that the content of indole compound I in the ammonolysis reagent containing indole compound I is negatively correlated with the content of degradation products in the final product.

[0072] Compared with Example 3, the degradation product contents of Examples 6-7 were reduced by 35.7% and 42.8%, respectively. It can be seen that adding 2-hydroxyethylamine to the aminolysis reagent containing indole compound I can further reduce the degradation content in the final product.

[0073] Compared with Example 3, the content of degradation products obtained in Comparative Examples 1-2 increased by 14.3% and 7.1%, respectively. It can be seen that in the ammonolysis reagent, in addition to methylamine and ammonia water, the indole compound I and indole-3-propanol are used in combination, and compared with the use of the two separately, the indole compound I can further reduce the probability of generating degradation products during the ammonolysis process.

[0074] 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.

[0075] 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 abasic oligonucleotide, characterized in that: The abasic nucleoside phosphoramidite monomer structure includes 1'-OH and 2'-OMe or F modification; comprising the following steps: (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 indole-3-propanol and indole compound I, indole compound I has an aminophenyl group.

2. The method for synthesizing an abasic oligonucleotide according to claim 1, wherein: In the ammonolysis reagent, the volume ratio of methylamine to ammonia water is 1:0.5-3; the volume ratio of methylamine to indole-3-propanol is 1:0.5-2; and the volume mass ratio of methylamine to indole compound I is 1 mL:0.5-2 mg.

3. The method for synthesizing an abasic oligonucleotide according to claim 1, wherein: The amount of aminolysis reagent used for 1mmol oligonucleotide is 40-100mL; The ammonolysis temperature is 20-55°C and the ammonolysis reaction lasts 2-6h.

4. The method for synthesizing an abasic oligonucleotide according to claim 1, wherein: 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 indole-3-propanol and indole compound I. Indole compound I has an aminophenyl 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 1:0.5-3; the volume ratio of methylamine to indole-3-propanol is 1:0.5-2; and the volume mass ratio of methylamine to indole compound I is 1 mL:0.5-2 mg.

7. The ammonolysis reagent according to claim 5, characterized in that The indole compound I has an aminophenyl group and also has a thiophene group; the thiophene group is derived from 2-(thiophen-2-yl)-1H-indole.

8. A method for synthesizing an indole compound I, characterized in that: The indole compound I has an aminophenyl group, comprising the following steps: 2-Fluoronitrobenzene, NaOH and 2-(thiophen-2-yl)-1H-indole are dissolved in DMSO and stirred for reaction; then, an organic solvent is used for extraction, and after the solvent is removed from the organic phase, a primary product of indole compound I is obtained; then, the obtained primary product of indole compound I is heated to reflux with iron powder and ammonium chloride, extracted with an organic solvent, and after the solvent is removed from the organic phase, it is separated and purified again to obtain the final product of indole compound I.

9. The method for synthesizing the indole compound I according to claim 8, characterized in that: The molar ratio of 2-fluoronitrobenzene to NaOH is 1:0.5-2; the molar ratio of 2-fluoronitrobenzene to 2-(thiophen-2-yl)-1H-indole is 1:0.5-2; 1-3 mL DMSO is used for 1 mmol 2-fluoronitrobenzene and DMSO.

10. The method for synthesizing the indole compound I according to claim 8, characterized in that: The molar ratio of 2-fluoronitrobenzene to iron powder and ammonium chloride is 1:2-5:0.5-2; the temperature during heating and reflux is 35-60°C, and the reflux time is 4-10h.

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