Synthesis method of 3′-ONH2-ribonucleotide

Abstract

The present invention provides a method for synthesizing 3′-ONH2-ribonucleotide. The above-mentioned synthesis method of the present application adopts an electrophilic NH2 donor reagent (diphenylphosphorylhydroxylamine) to introduce an ‑ONH2 protecting group at the 3′-position through a nucleophilic substitution reaction, and then sequentially performs a first deprotection reaction and a second deprotection reaction and the reaction of step S6 (the G group is connected to 3′-ONH2). Finally, a triphosphate part is constructed at the 5′-position through a phosphorylation reaction (step S7), and finally the protection of the G group is removed to obtain the target product 3′-ONH2 ribonucleotide, i.e., a compound of the structure shown in formula (IX). The target product obtained by the above-mentioned synthesis method of the present application has a high yield and a short synthesis route.

Description

Technical Field

[0001] The invention relates to the technical field of organic synthesis, and in particular to a method for synthesizing 3'-ONH2-ribonucleotide. Background Art

[0002] 3′-position protected triphosphate monomers can be used for the synthesis of oligonucleotides catalyzed by polymerases (e.g., existing documents US7544794B1, CN113195720A). A suitable easily leaving 3′-position protecting group is a necessary condition to avoid self-coupling or multiple coupling at the 3′-end. Among them, the 3′-ONH2 group is the most commonly used because of its small size, enzyme recognition, and easy removal. The synthesis of 3′-ONH2-deoxyribonucleotides has been reported in many literature reports and patents, such as Xiaoyun Lu et al. Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase. ACS Catal. 2022, 12, 2988-2997. and US8034923B1, etc. Existing documents US7544794B1 and US8212020B2 disclose methods for synthesizing 3′-O-amino-2′-deoxyribonucleotides using 2′-deoxyribonucleotides as raw materials. The method includes several steps, the most notable of which is: after protecting the 5′-hydroxyl group, the 3′-hydroxyl group is flipped using Mitsunobu conditions, and then the -ONH2 group with a protecting group is inserted at the 3′-position in the form of O-phthalimide, and after deprotection, NH2 is protected in the form of oxime, and then phosphorylated at the 5′-position, and finally the amino protecting group is removed. For ribonucleotides (RNA monomers), the presence of a hydroxyl group at the 2′-position makes it more difficult to obtain the corresponding 3′-ONH2 ribonucleotide. U.S. Patent US20220041643A1 discloses a method for an amino group at the 3′-position of an RNA monomer, but the yield of the method is very low and the route is relatively long, and the corresponding triphosphate monomer is not applied. Therefore, the strategy used for deoxyribonucleotides (DNA monomers) is not applicable to ribonucleotides. In addition, there are currently no reports on the synthesis of 3′-ONH2-ribonucleotides.

[0003] On this basis, it is of great significance to study and develop a method for synthesizing 3′-ONH2-ribonucleotide with high yield and short synthetic route. Summary of the invention

[0004] The main purpose of the present invention is to provide a method for synthesizing 3′-ONH2-ribonucleotides to solve the problems that the synthesis strategy used for 3′-ONH2-deoxyribonucleotides in the prior art is not applicable to ribonucleotides, and the synthesis route of 3′-ONH2-ribonucleotides is long and the yield is low.

[0005] In order to achieve the above object, the present invention provides a method for synthesizing 3′-ONH2-ribonucleotide, which comprises:

[0006] Step S1, in a first solvent, reacting the compound represented by formula (I) with N,N-dimethylformamide dimethyl acetal to obtain a compound represented by formula (II);

[0007] ;

[0008] Step S2, in a second solvent, subjecting the compound represented by formula (II) to a second reaction with 4,4'-dimethoxytrityl chloride to obtain a compound represented by formula (III);

[0009] ;

[0010] Step S3, in a third solvent, in the presence of a basic compound, subjecting the compound represented by formula (III) to a substitution reaction with diphenylphosphorylhydroxylamine to obtain a compound represented by formula (IV);

[0011] ;

[0012] Step S4, in a fourth solvent, subjecting methylamine to a first deprotection reaction with the compound represented by formula (IV) to obtain a compound represented by formula (V);

[0013] ;

[0014] Step S5, in a fifth solvent, subjecting hydrochloric acid to a second deprotection reaction with the compound of formula (V) in the presence of dodecanethiol to obtain a compound of formula (VI);

[0015] ;

[0016] Step S6, reacting the compound represented by formula (VI) with di-tert-butyl dicarbonate in a sixth solvent to obtain a compound represented by formula (VII);

[0017] ;

[0018] Step S7, in the presence of trimethyl phosphate, 1,8-bisdimethylaminonaphthalene is subjected to a first phosphorylation reaction with the compound represented by formula (VII) and phosphorus oxychloride to obtain an intermediate product system; the intermediate product system is subjected to a second phosphorylation reaction with tributylammonium pyrophosphate or tri-n-butylamine to obtain an intermediate product represented by formula (VIII), and the pH is adjusted to 8 to 9 to obtain a 3′-ONH2-ribonucleotide, wherein the 3′-ONH2-ribonucleotide has a structure represented by formula (IX);

[0019] ;

[0020] Wherein, R is selected from hydrogen atom, C1-C6 alkyl, silicon group or ;

[0021] QH is selected from any one of the following structures:

[0022] , , , , ;

[0023] QG is selected from any one of the following structures:

[0024] , , , , ;

[0025] G is selected from any one of the following structures:

[0026] , , , .

[0027] Further, in step S3, the molar ratio of the alkaline compound to the compound represented by formula (III) is (2 to 2.5):1; the molar ratio of diphenylphosphorylhydroxylamine to the compound represented by formula (III) is (1.5 to 2.5):1; preferably, the alkaline compound is selected from one or more of the group consisting of sodium hydride, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium phosphate and potassium phosphate; preferably, the third solvent is selected from one or more of the group consisting of toluene, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone and tetrahydrofuran.

[0028] Furthermore, in step S3, the temperature of the substitution reaction is 0 to 40° C., and the time is 12 to 24 hours.

[0029] Furthermore, in step S4, the molar ratio of methylamine to the compound represented by formula (IV) is (70-80):1; preferably, the fourth solvent is selected from water; preferably, the temperature of the first deprotection reaction is 20-30° C., and the time is 2-3 h.

[0030] Further, in step S5, the molar ratio of hydrochloric acid to the compound of formula (V) is (8-11):1, and the molar ratio of dodecanethiol to the compound of formula (V) is (1.5-3):1; preferably, the fifth solvent is selected from one or more of the group consisting of dichloromethane, 1,4-dioxane and ethyl acetate; preferably, the temperature of the second deprotection reaction is 0-3°C, and the time is 0.5-2h.

[0031] Furthermore, in step S6, the molar ratio of di-tert-butyl dicarbonate to the compound represented by formula (VI) is (1.5-2):1; preferably, the reaction in step S6 is carried out under alkaline conditions of pH 9-10; more preferably, triethylamine is used to adjust the pH in step S6; preferably, the sixth solvent is selected from DMF.

[0032] Furthermore, in step S7, the weight ratio of trimethyl phosphate to the compound represented by formula (VII) is (10-12):1; the molar ratio of 1,8-bisdimethylaminonaphthalene to the compound represented by formula (VII) is (1.5-2):1; the molar ratio of phosphorus oxychloride to the compound represented by formula (VII) is (2-2.5):1, and the molar ratio of tributylammonium pyrophosphate or tri-n-butylamine to the compound represented by formula (VII) is (3-4):1.

[0033] Furthermore, in step S1, the molar ratio of N,N-dimethylformamide dimethyl acetal to the compound represented by formula (I) is (2.5-3):1; preferably, the temperature of the first reaction is 50-60°C, and the time is 15-20h; preferably, the first solvent is selected from DMF.

[0034] Furthermore, in step S2, the molar ratio of 4,4'-dimethoxytrityl chloride to the compound represented by formula (II) is (0.95-1):1; preferably, the second solvent is selected from pyridine.

[0035] Furthermore, the temperature of the second reaction is 20-30° C., and the time is 1-3 hours.

[0036] Applying the technical solution of the present invention, the above-mentioned synthesis method of the present application adopts an electrophilic NH2 donor reagent (diphenylphosphohydroxylamine, ) introduces the -ONH2 protecting group at the 3′-position by a nucleophilic substitution reaction, and then sequentially performs the first deprotection reaction and the second deprotection reaction and the reaction of step S6 (the G group is connected to 3′-ONH2). Finally, the triphosphate part is constructed at the 5′-position by a phosphorylation reaction (step S7), and finally the protection of the G group is removed to obtain the target product 3′-ONH2 ribonucleotide, i.e., the compound of the structure shown in formula (IX). The target product obtained by the above-mentioned synthesis method of the present application has a high yield and a short synthesis route. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0038] Figure 1 The compound 3 prepared in Example 1 of the present application is shown 1 H NMR spectrum;

[0039] Figure 2 The compound 5 prepared in Example 1 of the present application is shown 1 H NMR spectrum;

[0040] Figure 3 The compound 7 prepared in Example 1 of the present application is shown 1 H NMR spectrum. DETAILED DESCRIPTION

[0041] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0042] As described in the background technology, the existing synthesis strategy for 3′-ONH2-deoxyribonucleotides is not applicable to ribonucleotides, and the synthesis route of 3′-ONH2-ribonucleotides is long and the yield is low. In order to solve the above technical problems, the present application provides a method for synthesizing 3′-ONH2-ribonucleotides, which comprises:

[0043] Step S1, in a first solvent, reacting the compound represented by formula (I) with N,N-dimethylformamide dimethyl acetal (DMF-DMA) to obtain a compound represented by formula (II);

[0044] ;

[0045] Step S2, in a second solvent, reacting the compound represented by formula (II) with 4,4'-dimethoxytrityl chloride (DMTrCl) to obtain a compound represented by formula (III);

[0046] ;

[0047] Step S3, in a third solvent, in the presence of a basic compound, subjecting the compound represented by formula (III) to a substitution reaction with diphenylphosphorylhydroxylamine to obtain a compound represented by formula (IV);

[0048] ;

[0049] Step S4, in a fourth solvent, subjecting methylamine to a first deprotection reaction with the compound represented by formula (IV) to obtain a compound represented by formula (V);

[0050] ;

[0051] Step S5, in a fifth solvent, subjecting hydrochloric acid to a second deprotection reaction with the compound of formula (V) in the presence of dodecanethiol to obtain a compound of formula (VI);

[0052] ;

[0053] Step S6, reacting the compound represented by formula (VI) with di-tert-butyl dicarbonate in a sixth solvent to obtain a compound represented by formula (VII);

[0054] ;

[0055] Step S7, in the presence of trimethyl phosphate, 1,8-bisdimethylaminonaphthalene and the compound represented by formula (VII) are subjected to a first phosphorylation reaction with phosphorus oxychloride to obtain an intermediate product system; the intermediate product system is subjected to a second phosphorylation reaction with tributylammonium pyrophosphate or tri-n-butylamine to obtain an intermediate product represented by formula (VIII), and the pH is adjusted to 8 to 9 to obtain a 3′-ONH2-ribonucleotide, wherein the 3′-ONH2-ribonucleotide has a structure represented by formula (IX);

[0056] ;

[0057] Wherein, R is selected from hydrogen atom, C1-C6 alkyl, silicon group or ;

[0058] QH is selected from any one of the following structures:

[0059] , , , , ;

[0060] QG is selected from any one of the following structures:

[0061] , , , , ;

[0062] G is selected from any one of the following structures:

[0063] , , , .

[0064] The synthesis strategy used for 3′-ONH2-deoxyribonucleotides in the prior art is not applicable to ribonucleotides. However, the present application achieves modification of the -ONH2 protecting group at the 3′-position of ribonucleoside through the above-mentioned synthesis method to obtain the compound shown in formula (VI), thereby providing a new route for the synthesis of 3′-ONH2 ribonucleotides.

[0065] The above-mentioned synthesis method of the present application adopts an electrophilic NH2 donor reagent (diphenylphosphohydroxylamine, ) introduces the -ONH2 protecting group at the 3′-position by a nucleophilic substitution reaction, and then sequentially performs the first deprotection reaction and the second deprotection reaction and the reaction of step S6 (the G group is connected to 3′-ONH2). Finally, the triphosphate part is constructed at the 5′-position by a phosphorylation reaction (step S7), and finally the protection of the G group is removed to obtain the target product 3′-ONH2 ribonucleotide, i.e., the compound of the structure shown in formula (IX). The target product obtained by the above-mentioned synthesis method of the present application has a high yield and a short synthesis route.

[0066] In a preferred embodiment, in step S3, the molar ratio of the basic compound to the compound represented by formula (III) is (2-2.5):1; the molar ratio of diphenylphosphorylhydroxylamine to the compound represented by formula (III) is (1.5-2.5):1. Compared with other ranges, limiting the amount of each raw material and reagent in step S3 to the above range is conducive to improving the efficiency of introducing the -ONH2 protecting group into the 3′-position, and is conducive to improving the yield of the compound represented by formula (IV), and is also conducive to reducing reagent waste.

[0067] In order to improve the efficiency of the nucleophilic substitution reaction, preferably, the basic compound includes but is not limited to one or more of the group consisting of sodium hydride, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium phosphate and potassium phosphate.

[0068] In order to improve the dispersibility of the raw materials and reagents in step S3 and to improve the efficiency of the nucleophilic substitution reaction, preferably, the third solvent includes but is not limited to one or more of the group consisting of toluene, acetonitrile, 1,4-dioxane, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP) and tetrahydrofuran (THF).

[0069] In a preferred embodiment, in step S3, the temperature of the substitution reaction is 0 to 40° C., and the time is 12 to 24 hours. The temperature and time of the substitution reaction include but are not limited to the above ranges, and limiting them within the above ranges is conducive to further improving the efficiency of the nucleophilic substitution reaction and inhibiting side reactions, thereby further improving the yield of the compound represented by formula (IV).

[0070] In a preferred embodiment, in step S4, the molar ratio of methylamine to the compound represented by formula (IV) is (70-80): 1. The molar ratio of methylamine to the compound represented by formula (IV) includes but is not limited to the above range, and limiting it within the above range is conducive to improving the efficiency of the first deprotection reaction, thereby facilitating improving the yield of the compound represented by formula (V).

[0071] In order to improve the dispersibility of the raw materials and reagents in step S4, preferably, the fourth solvent includes but is not limited to water.

[0072] In a preferred embodiment, the temperature of the first deprotection reaction is 20-30°C and the time is 2-3 hours. The temperature and time of the first deprotection reaction include but are not limited to the above ranges, and limiting them within the above ranges is conducive to improving the removal efficiency of the G group, thereby facilitating improving the yield of the compound represented by formula (V).

[0073] In a preferred embodiment, in step S5, the molar ratio of hydrochloric acid to the compound of formula (V) is (8-11):1, and the molar ratio of dodecanethiol to the compound of formula (V) is (1.5-3):1. The molar ratio of dodecanethiol to the compound of formula (V) includes but is not limited to the above range, and limiting it within the above range is conducive to improving the removal efficiency of the -DMTr group, thereby facilitating improving the yield of the compound of formula (VI).

[0074] In order to improve the dispersibility of the reaction raw materials and reagents in step S5, preferably, the fifth solvent includes but is not limited to one or more of the group consisting of dichloromethane, 1,4-dioxane and ethyl acetate.

[0075] In a preferred embodiment, the temperature of the second deprotection reaction is 0-3°C and the time is 0.5-2h. The temperature and time of the second deprotection reaction include but are not limited to the above ranges, and limiting them within the above ranges is conducive to further improving the removal efficiency of the -DMTr group, thereby further improving the yield of the compound of formula (VI).

[0076] In a preferred embodiment, in step S6, the molar ratio of di-tert-butyl dicarbonate to the compound represented by formula (VI) is (1.5-2): 1. The molar ratio of di-tert-butyl dicarbonate to the compound represented by formula (VI) includes but is not limited to the above range, and limiting it within the above range is conducive to improving the efficiency of the G group connecting to 3′-ONH2, thereby helping to improve the yield of the compound represented by formula (VII).

[0077] In order to further improve the yield of the compound represented by formula (VII), preferably, the reaction in step S6 is carried out under alkaline conditions of pH 9 to 10; more preferably, triethylamine is used to adjust the pH in step S6.

[0078] In order to improve the dispersibility of the raw materials and reagents in step S6, preferably, the sixth solvent includes but is not limited to DMF.

[0079] In a preferred embodiment, in step S7, the weight ratio of trimethyl phosphate to the compound represented by formula (VII) is (10-12):1; the molar ratio of 1,8-bisdimethylaminonaphthalene to the compound represented by formula (VII) is (1.5-2):1; the molar ratio of phosphorus oxychloride to the compound represented by formula (VII) is (2-2.5):1, and the molar ratio of tributylammonium pyrophosphate or tri-n-butylamine to the compound represented by formula (VII) is (3-4):1. The amount ratio of each raw material in step S7 includes but is not limited to the above range. Limiting it within the above range is beneficial to improving the efficiency of the phosphorylation reaction, and at the same time, it is beneficial to improving the utilization rate of each raw material, thereby facilitating improving the yield of the phosphorylation reaction product.

[0080] In a preferred embodiment, in step S1, the molar ratio of N,N-dimethylformamide dimethyl acetal (DMF-DMA) to the compound represented by formula (I) is (2.5-3): 1. The molar ratio of DMF-DMA to the compound represented by formula (I) includes but is not limited to the above range. Limiting it within the above range is beneficial to the reaction efficiency of the first reaction, and is also beneficial to improving the utilization rate of the reaction raw materials, thereby improving the yield of the compound represented by formula (II), and providing more favorable conditions for the subsequent synthesis of the target product 3′-ONH2 ribonucleotide.

[0081] In a preferred embodiment, the temperature of the first reaction is 50-60°C and the time is 15-20 hours. The temperature and time of the first reaction include but are not limited to the above ranges, and limiting them within the above ranges is conducive to suppressing side reactions, thereby facilitating improving the yield of the compound represented by formula (II).

[0082] In order to improve the dispersibility of the raw materials in step S1 and facilitate the first reaction, preferably, the first solvent includes but is not limited to DMF.

[0083] In a preferred embodiment, in step S2, the molar ratio of 4,4'-dimethoxytrityl chloride (DMTrCl) to the compound represented by formula (II) is (0.95-1):1. The molar ratio of 4,4'-dimethoxytrityl chloride to the compound represented by formula (II) includes but is not limited to the above range. Limiting it within the above range is beneficial to improving the efficiency of the second reaction, thereby helping to improve the yield of the compound represented by formula (III).

[0084] In order to improve the dispersibility of the raw materials in step S2, preferably, the second solvent includes but is not limited to pyridine.

[0085] In a preferred embodiment, the temperature of the second reaction is 20-30°C and the time is 1-3 hours. The temperature and time of the second reaction include but are not limited to the above ranges, and limiting them within the above ranges is conducive to suppressing the occurrence of side reactions, thereby facilitating the improvement of the yield of the compound represented by formula (III).

[0086] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0087] Example 1

[0088] A method for synthesizing 3′-ONH2-ribonucleotide, comprising:

[0089] (1) At 25°C, 11 g of compound 1 was mixed with 176 mL of dry DMF, stirred and dissolved, 13.70 g of N,N-dimethylformamide dimethyl acetal (DMF-DMA) was added, the temperature was raised to 50°C and stirred for 18 h. After the reaction was completed by HPLC, the system was concentrated to obtain a concentrated system containing compound 2, which was directly used for the next step. The synthetic route of compound 2 is as follows:

[0090] ;

[0091] (2) At 25°C, 154 mL of pyridine was added to the above concentrated system to obtain a reaction system containing pyridine; 12.99 g of 4,4'-dimethoxytrityl chloride (DMTrCl) was dissolved in 44 L of dichloromethane, and the mixture was slowly dripped into the reaction system containing pyridine within 30 min and stirred for 2 h. When HPLC showed that the raw material was completely reacted, 110 mL of 10% sodium bicarbonate solution was added and stirred for 15 min. The aqueous phase was extracted with dichloromethane, and the organic phase was dried with anhydrous sodium sulfate. The dried organic phase was added with dichloromethane and stirred to dissolve to obtain a dichloromethane solution. The dichloromethane solution was dripped into isopropyl ether to precipitate a white solid, which was filtered and dried to obtain 20.8 g of compound 3. The synthetic route of compound 3 is as follows:

[0092] ;

[0093] Compound 3 1 H NMR spectrum Figure 1 As shown;

[0094] Its content was confirmed by NMR to be 92%, and the separation efficiency of step (1) and step (2) was 78.77%;

[0095] (3) Under nitrogen protection, 1 g of compound 3 was mixed with 30 mL of dry tetrahydrofuran (THF), stirred to dissolve, cooled to 0°C, added with 156.55 mg of 60 wt% sodium hydride, stirred for 2 h, and slowly heated to room temperature, added with 730 mg of diphenylphosphorylhydroxylamine, and the substitution reaction was carried out for 16 h. The reaction was completed by HPLC to obtain a product system containing compound 4. The synthetic route of compound 4 is as follows:

[0096] ;

[0097] (4) 10 mL of water was added to the product system containing compound 4, and 10 mL of methylamine ethanol solution was added. The mixture was stirred at 25 °C for 2 h and then monitored by HPLC until the first deprotection reaction was confirmed to be complete. 10 mL of DCM was added to extract the liquid, and the organic phase was dried with anhydrous sodium sulfate and purified by column to obtain 0.75 g of a white solid (compound 5). The synthetic route of compound 5 is as follows:

[0098] ;

[0099] Compound 5 1 H NMR spectrum Figure 2 As shown;

[0100] Its content was confirmed by NMR to be 87% and its separation efficiency was 91.28%;

[0101] (5) 5.6 g of compound 5 was mixed with 16 mL of dichloromethane (DCM), 2.84 g of dodecanethiol was added, the mixture was cooled to 0°C in an ice-water bath, 28 mL of a 4 mol / L hydrochloric acid solution of 1,4-dioxane was added, and the mixture was stirred at 0°C for 1 h. The reaction was completed by HPLC monitoring, 224 mL of dichloromethane and 224 mL of water were added, and the aqueous phase was separated and collected. The aqueous phase was spin-dried to obtain a red solid (compound 6), which was blown dry with nitrogen. The synthetic route of compound 6 is as follows:

[0102] ;

[0103] (6) The red solid obtained in step (5) was mixed with 34 mL of DMF. The system was dissolved and 2.84 g of triethylamine was added. The pH was adjusted to 10. Di-tert-butyl dicarbonate (Boc)2O was added and stirred overnight at 25°C. The reaction was completed after HPLC monitoring. Water was added and extracted with ethyl acetate three times. The organic phase was dried and purged with nitrogen overnight to obtain 2.69 g of compound 7. The synthetic route of compound 7 is as follows:

[0104] ;

[0105] Compound 7 1 H NMR spectrum Figure 3 As shown;

[0106] Its internal standard content was confirmed to be 68.65% by NMR, and the separation yield was 65.61%;

[0107] (7) 200 mg of the product obtained in step (6) was mixed with 162 mg of 1,8-bisdimethylaminonaphthalene and 2 mL of trimethyl phosphate, and the mixture was cooled to 0°C in an ice-water bath. 154.7 mg of phosphorus oxychloride was added to generate a white solid (intermediate product). After stirring in an ice-water bath for 30 min, 3 mL of an acetonitrile solution containing 374.0 mg of tri-n-butylamine and 550.0 mg of tributylammonium pyrophosphate was added, and the mixture was stirred in an ice-water bath for 1 h. After the reaction was complete as detected by LCMS, 8 mL of concentrated hydrochloric acid was added, and the mixture was stirred for 30 min. Concentrated aqueous ammonia was added to adjust the pH to 8. After purification, the target product 3′-ONH2-ribonucleotide was obtained. The column used for purification was AQC18, and the mobile phases were: A was 10 mM sodium dihydrogen phosphate, and B was acetonitrile.

[0108] The synthetic route of compound 9 is as follows:

[0109] .

[0110] Example 2

[0111] The difference from Example 1 is that the temperature of the substitution reaction in step (3) is 40° C. and the time is 12 h.

[0112] Example 3

[0113] The difference from Example 1 is that the time of the substitution reaction in step (3) is 24 hours.

[0114] Example 4

[0115] The difference from Example 1 is that the time of the substitution reaction in step (3) is 3 h.

[0116] Example 5

[0117] The difference from Example 1 is that in step (3), the molar ratio of sodium hydride to compound 3 is 2:1, and the molar ratio of diphenylphosphorylhydroxylamine to compound 3 is 1.5:1.

[0118] Example 6

[0119] The difference from Example 1 is that in step (3), the molar ratio of sodium hydride to compound 3 is 2.5:1, and the molar ratio of diphenylphosphorylhydroxylamine to compound 3 is 2.5:1.

[0120] Example 7

[0121] The difference from Example 1 is that in step (3), the molar ratio of sodium hydride to compound 3 is 1:1, and the molar ratio of diphenylphosphorylhydroxylamine to compound 3 is 1.5:1.

[0122] Example 8

[0123] The difference from Example 1 is that in step (4), the molar ratio of methylamine to compound 4 is 70:1.

[0124] Example 9

[0125] The difference from Example 1 is that in step (4), the molar ratio of methylamine to compound 4 is 80:1.

[0126] Example 10

[0127] The difference from Example 1 is that in step (4), the molar ratio of methylamine to compound 4 is 20:1.

[0128] Embodiment 11

[0129] The difference from Example 1 is that in step (4), the temperature of the first deprotection reaction is 20° C. and the time is 3 h.

[0130] Example 12

[0131] The difference from Example 1 is that in step (4), the temperature of the first deprotection reaction is 30° C. and the time is 2 h.

[0132] Example 13

[0133] The difference from Example 1 is that in step (4), the temperature of the first deprotection reaction is 0°C.

[0134] Embodiment 14

[0135] The difference from Example 1 is that in step (5), the molar ratio of hydrochloric acid to compound 5 is 8:1.

[0136] Embodiment 15

[0137] The difference from Example 1 is that in step (5), the molar ratio of hydrochloric acid to compound 5 is 11:1.

[0138] Example 16

[0139] The difference from Example 1 is that in step (5), the molar ratio of hydrochloric acid to compound 5 is 4:1.

[0140] Comparative Example 1

[0141] 3′-ONH2-deoxyribonucleotide is prepared by existing technology, and its synthesis route is as follows:

[0142] .

[0143] The yield of the product in each step is shown in Table 1.

[0144] Table 1

[0145]

[0146] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the above synthesis method of the present application uses an electrophilic NH2 donor reagent (diphenylphosphorylhydroxylamine) to introduce an -ONH2 protective group at the 3′-position through a nucleophilic substitution reaction, and then sequentially performs the first deprotection reaction and the second deprotection reaction and the reaction of step S6 (the G group is connected to 3′-ONH2). Finally, a triphosphate part is constructed at the 5′-position through a phosphorylation reaction (step S7), and finally the protection of the G group is removed to obtain the target product 3′-ONH2 ribonucleotide, i.e., a compound of the structure shown in formula (IX). The target product obtained by the above synthesis method of the present application has a high yield and a short synthesis route.

[0147] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the terms used in this way can be interchangeable where appropriate, so that the embodiments of the present application described herein can be implemented in an order other than those described herein, for example.

[0148] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for synthesizing 3′-ONH2-ribonucleotide, characterized in that: The synthesis method comprises: Step S1, in a first solvent, reacting the compound represented by formula (I) with N,N-dimethylformamide dimethyl acetal to obtain a compound represented by formula (II); ； Step S2, in a second solvent, subjecting the compound represented by formula (II) to a second reaction with 4,4'-dimethoxytrityl chloride to obtain a compound represented by formula (III); ； Step S3, in a third solvent, in the presence of a basic compound, subjecting the compound represented by formula (III) to a substitution reaction with diphenylphosphorylhydroxylamine to obtain a compound represented by formula (IV); ； Step S4, in a fourth solvent, subjecting methylamine to a first deprotection reaction with the compound represented by formula (IV) to obtain a compound represented by formula (V); ； Step S5, in a fifth solvent, subjecting the compound of formula (V) to a second deprotection reaction with hydrochloric acid in the presence of dodecanethiol to obtain a compound of formula (VI); ； Step S6, reacting the compound represented by formula (VI) with di-tert-butyl dicarbonate in a sixth solvent to obtain a compound represented by formula (VII); ； Step S7, in the presence of trimethyl phosphate, 1,8-bisdimethylaminonaphthalene is subjected to a first phosphorylation reaction with the compound represented by the formula (VII) and phosphorus oxychloride to obtain an intermediate product system; the intermediate product system is subjected to a second phosphorylation reaction with tributylammonium pyrophosphate or tri-n-butylamine to obtain an intermediate product represented by the formula (VIII), and the pH is adjusted to 8 to 9 to obtain the 3′-ONH2-ribonucleotide, wherein the 3′-ONH2-ribonucleotide has a structure represented by the formula (IX); ； Wherein, R is selected from C1-C6 alkyl, silicon or ; QH is selected from any one of the following structures: ； QG is selected from any one of the following structures: ； G is selected from the following structures: .

2. The method for synthesizing 3′-ONH2-ribonucleotide according to claim 1, characterized in that: In the step S3, the molar ratio of the alkaline compound to the compound represented by the formula (III) is (2-2.5):1; the molar ratio of the diphenylphosphohydroxylamine to the compound represented by the formula (III) is (1.5-2.5):1; and / or, The alkaline compound is selected from one or more of the group consisting of sodium hydride, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium phosphate and potassium phosphate; and / or, The third solvent is selected from one or more of the group consisting of toluene, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone and tetrahydrofuran.

3. The method for synthesizing 3′-ONH2-ribonucleotide according to claim 1, characterized in that: In the step S3, the temperature of the substitution reaction is 0 to 40° C., and the time is 12 to 24 hours.

4. The method for synthesizing 3′-ONH2-ribonucleotide according to any one of claims 1 to 3, characterized in that: In the step S4, the molar ratio of the methylamine to the compound represented by the formula (IV) is (70-80):1; and / or, The fourth solvent is selected from water; and / or, The temperature of the first deprotection reaction is 20-30° C. and the time is 2-3 hours.

5. The method for synthesizing 3′-ONH2-ribonucleotide according to claim 4, characterized in that: In the step S5, the molar ratio of the hydrochloric acid to the compound of formula (V) is (8-11):1, and the molar ratio of the dodecanethiol to the compound of formula (V) is (1.5-3):1; and / or, The fifth solvent is selected from one or more of the group consisting of dichloromethane, 1,4-dioxane and ethyl acetate; and / or, The temperature of the second deprotection reaction is 0-3°C and the time is 0.5-2h.

6. The method for synthesizing 3′-ONH2-ribonucleotide according to claim 5, characterized in that: In the step S6, the molar ratio of di-tert-butyl dicarbonate to the compound represented by formula (VI) is (1.5-2):1; and / or, The reaction in step S6 is carried out under alkaline conditions of pH 9 to 10; and / or, The sixth solvent is selected from DMF.

7. The method for synthesizing 3′-ONH2-ribonucleotide according to claim 6, characterized in that: The pH in step S6 is adjusted using triethylamine.

8. The method for synthesizing 3′-ONH2-ribonucleotide according to any one of claims 1 to 3, characterized in that: In the step S7, the weight ratio of the trimethyl phosphate to the compound represented by the formula (VII) is (10-12):1; the molar ratio of the 1,8-bisdimethylaminonaphthalene to the compound represented by the formula (VII) is (1.5-2):1; the molar ratio of the phosphorus oxychloride to the compound represented by the formula (VII) is (2-2.5):1, and the molar ratio of the tributylammonium pyrophosphate or the tri-n-butylamine to the compound represented by the formula (VII) is (3-4):

1.

9. The method for synthesizing 3′-ONH2-ribonucleotide according to any one of claims 1 to 3, characterized in that: In the step S1, the molar ratio of the N,N-dimethylformamide dimethyl acetal to the compound represented by the formula (I) is (2.5-3):1; and / or, The temperature of the first reaction is 50-60° C. and the time is 15-20 hours; and / or, The first solvent is selected from DMF.

10. The method for synthesizing 3′-ONH2-ribonucleotide according to any one of claims 1 to 3, characterized in that: In the step S2, the molar ratio of the 4,4'-dimethoxytrityl chloride to the compound represented by the formula (II) is (0.95-1):1; and / or, The second solvent is selected from pyridine.

11. The method for synthesizing 3′-ONH2-ribonucleotide according to claim 10, characterized in that: The temperature of the second reaction is 20-30° C. and the time is 1-3 hours.

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