Preparation method of 2 'OMe modified pseudouridine and N1 methyl pseudouridine

Abstract

The invention provides a preparation method of 2 'OMe modified pseudouridine and N1 methyl pseudouridine, the 2' OMe modified pseudouridine and N1 methyl pseudouridine are respectively as shown in a formula I and a formula II, and the method comprises the following steps: protecting hydroxyl on pseudouridine ribose; protecting a ring nitrogen atom which is not substituted by a non-hydrogen substituent on a pseudouridine base; a step of deprotecting the protected hydroxyl group on the ribose; # imgabs0 #, a step for blocking hydroxyl groups at the 3'and 4 'positions, a step for introducing a methyl group at the 2' position, a step for removing a protecting group on a nitrogen atom of a ring of a pseudouridine base, and a step for removing a protecting group at the 3 'and 4' positions.

Description

Technical Field

[0001] The invention relates to a preparation method of 2'OMe modified pseudouridine and N1 methyl pseudouridine. Background Art

[0002] As a highly potential drug form, mRNA has attracted strong attention from major pharmaceutical companies and research institutions around the world. However, some defects of mRNA itself also limit its wider clinical application. As a long-chain RNA molecule, mRNA has poor stability, short half-life in vivo, and high transient expression. Therefore, at this stage, mRNA is mainly used in the development of infectious disease vaccines. For application scenarios such as antibody replacement therapy and CAR-T drugs that require long-term stable expression, the use of mRNA is subject to certain restrictions.

[0003] In the early stages of RNAi drug development, researchers also tried to use various modified nucleotides to improve the stability of nucleic acid molecules. Methylating the pentose 2'-OH of nucleotides to form 2'-OMe is a very common modification method. 2'-OMe, as a naturally occurring modification method, can inhibit the base-catalyzed degradation effect of the RNA chain itself, greatly improving the stability of the RNA chain without affecting the function of the RNA itself. Studies have also shown that 2'-OMe can inhibit the activation of the TLR7 system in cells, reduce the immunogenicity of RNA molecules, inhibit the cellular immune system, and enable RNA molecules to function in cells for a long time (15). The use of 2'-OMe modification in siRNA can improve the loading efficiency of the RISC complex, increase the silencing activity of siRNA, and further improve the anti-tumor activity of siRNA molecules. The large-scale use of 2'-OMe modification has also directly contributed to the marketing of several RNAi drugs such as Patisiran, Givosiran, and Inclisiran. Summary of the invention

[0004] The present invention provides a method for preparing 2'OMe-modified pseudouridine or N1-methyl pseudouridine, the method comprising the following steps:

[0005] (1) a step of protecting the hydroxyl group on the ribose;

[0006] (2) a step of protecting the ring nitrogen atoms on the pseudouridine base that are not substituted by non-hydrogen substituents;

[0007] (3) a step of deprotecting the protected hydroxyl group on the ribose;

[0008] (4) blocking the hydroxyl groups at the 3' and 4' positions;

[0009] (5) a step of introducing a methyl group at the 2' position;

[0010] (6) a step of removing the protecting group on the pseudouridine base; and

[0011] (7) A step of removing the protecting groups at the 3' and 4' positions.

[0012] The detailed description of each step of the present invention is shown below. DETAILED DESCRIPTION

[0013] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0014] The first aspect of the present invention provides a method for preparing 2'OMe-modified pseudouridine or N1-methyl pseudouridine, the method comprising the following steps:

[0015] (1) a step of protecting the hydroxyl group on the ribose;

[0016] (2) a step of protecting the ring nitrogen atoms on the pseudouridine base that are not substituted by non-hydrogen substituents;

[0017] (3) a step of deprotecting the protected hydroxyl group on the ribose;

[0018] (4) blocking the hydroxyl groups at the 3' and 4' positions;

[0019] (5) a step of introducing a methyl group at the 2' position;

[0020] (6) a step of removing the protecting group on the pseudouridine base; and

[0021] (7) A step of removing the protecting groups at the 3' and 4' positions.

[0022] Herein, the nucleoside is pseudouridine or N1-methyl pseudouridine, wherein pseudouridine has the following structure:

[0023]

[0024] N1-methylpseudouridine has the following structure:

[0025]

[0026] Herein, an acyl group may be introduced into the ribose portion of a nucleoside to protect the hydroxyl group. The acyl group may be C 1-6 Acyl groups, such as formyl, acetyl, propionyl, etc. In some embodiments, the nucleoside can be reacted with compound R in the presence of an organic solvent. 2 O reacts to introduce acyl groups on the three hydroxyl groups of ribose for protection, where R is C 1-6Acyl. In some embodiments, R is acetyl (Ac). The solvent may include anhydrous DMF and 4-dimethylaminopyridine, etc. The molar ratio of 4-dimethylaminopyridine to nucleoside may be 1:1 to 3. The amount of anhydrous DMF may be used as needed. For example, the amount of anhydrous DMF (in mL) may be 20 to 40 times the weight of the nucleoside (in g). R 2 The molar ratio of O to nucleoside can be 3 to 10:1.

[0027] Typically, the nucleoside can be dissolved in anhydrous DMF, cooled to below 10°C, for example, to 0-10°C or 0-4°C, and then 4-dimethylaminopyridine is added, and then R 2 O. After the addition is complete, the mixture is heated to room temperature and stirred. After the reaction is fully reacted, an alcohol such as methanol can be added to terminate the reaction. After purification, compound 1 in which the three hydroxyl groups on the ribose are protected by acyl groups can be obtained.

[0028] Herein, benzyloxy C 1-4 Alkyl groups are used to protect the nitrogen atoms on the nucleoside base ring that are not replaced by non-hydrogen substituents. 2 CO 3 and an organic solvent such as anhydrous MeCN, wherein the three hydroxyl groups on the aforementioned ribose are protected by acyl groups and the halogenated C 1-4 The alkoxymethylbenzene is reacted to prepare a compound in which both the hydroxyl group and the unsubstituted nitrogen atom are protected. Herein, halogen includes F, Cl, Br and I. In some embodiments, the halogenated C 1-4 Alkoxymethylbenzene is a chlorinated C 1-4 Alkoxymethylbenzene, more preferably chloromethoxymethylbenzene:

[0029]

[0030] The amount of organic solvent used can be as required. For example, the amount of organic solvent used (in mL) can be 15 to 30 times the weight of the nucleoside (in g). 1-4 The molar ratio of alkoxymethylbenzene can be 1:1 to 3, and K 2 CO 3 The molar ratio can be 1:2 to 5. The reaction can be carried out at room temperature. After the reaction is completed, the organic phase can be extracted and purified by conventional methods to obtain compound 2 in which the hydroxyl group on the ribose and the unsubstituted nitrogen atom in the base ring are protected.

[0031] In this paper, the deprotection of the hydroxyl group on the ribose can be carried out using NH 3 Specifically, the compound 2 can be dissolved in NH 3After a period of reaction in an alcohol solution, compound 3 can be obtained in which the three hydroxyl groups on the ribose are deprotected (i.e., restored to the original -OH) but the N on the base ring is still protected. 3 The equivalent concentration of the alcohol solution may be 3 to 10 N. The amount of the solution (in mL) is usually 10 to 30 times the mass (in g) of compound 3. The reaction time may be determined according to the actual reaction conditions, for example, may be 1 to 5 hours. The reaction may be carried out at room temperature.

[0032] Herein, the hydroxyl groups at the 3' and 4' positions on the ribose of compound 3 can be connected to alkyl-substituted disiloxane groups to block the hydroxyl groups at these two positions from reacting when a methyl group is introduced at the 2' position. The alkyl-substituted disiloxane group can be tetraC 1-4 Alkyl substituted disiloxane groups, such as:

[0033]

[0034] In the formula, each R is independently a C1-4 alkyl group, and the wavy lines indicate the positions of attachment to the oxygen in the hydroxyl groups at the 3' and 4' positions. 1-4 The alkyl-substituted disiloxane group is tetraisopropyl disiloxane. The alkyl-substituted disiloxane group can be connected to the hydroxyl groups at the 3' and 4' positions on the ribose of compound 3 by reacting compound 3 with the following compound 3' in an organic solvent:

[0035]

[0036] In the formula, each R is independently C 1-4 alkyl, each A is independently halogen. Preferably, the compound is TiPDSCl 2 . The organic solvent may be anhydrous dioxane and pyridine. The amount of the organic solvent (in mL) may be 30 to 50 times the mass of the compound (in g). In some embodiments, the amount of anhydrous dioxane (in mL) may be 25 to 35 times the mass of the compound (in g). In some embodiments, the amount of pyridine (in mL) may be 5 to 15 times the mass of the compound (in g). The molar ratio of compound 3 and compound 3' may be 1:2 to 5. The reaction may be carried out at room temperature. After the reaction is completed, it may be extracted with DCM, washed with water, NaHCO 3 The organic phase was obtained by washing with an aqueous solution and then purified to obtain compound 4 in which the hydroxyl groups at the 3' and 4' positions on the ribose were blocked.

[0037] Compound 4 is reacted with a methylating agent in the presence of a dehydrogenating agent to modify the 2' position of compound 4 by methylation. The dehydrogenating agent may be a dehydrogenating agent conventionally used in the art for the preparation of modified nucleosides. An exemplary dehydrogenating agent is N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine. The molar ratio of the dehydrogenating agent to compound 4 may be 5 to 15:1. The methylating agent may generally be trimethoxylium tetrafluoroborate. The molar ratio of the methylating agent to compound 4 may be 5 to 15:1. The reaction may be carried out at room temperature. The reaction time may be determined according to actual conditions such as the amount of material, and may be, for example, in the range of 2 to 10 hours. After the reaction is completed, it may be filtered, and then diluted acid may be added and stirred for several minutes, and the organic phase may be collected. The diluted acid may be diluted hydrochloric acid. The organic phase may be purified to obtain compound 5 in which the hydroxyl groups at the 3' and 4' positions are still blocked (protected), the N group on the base ring is still protected, but the hydroxyl group at the 2' position is methylated (i.e., an -OMe group is formed).

[0038] In this article, the removal of the protecting group on N can be carried out at H 2 atmosphere, in the presence of formic acid and a palladium catalyst. Specifically, for example, compound 5 can be dissolved in an aqueous alcohol solution, then mixed with formic acid and a palladium catalyst, hydrogen is introduced, and the reaction is allowed to proceed for a period of time. The alcohol solution can be an aqueous solution of isopropanol, and the volume ratio of alcohol to water can be 5 to 9:1. The amount of alcohol solution (in mL) can be 10 to 20 times the mass of compound 5 (in g). The amount of formic acid (in mL) can be 1 to 5 times the mass of compound 5 (in g). The palladium catalyst can be palladium supported on activated carbon. The reaction time can be determined according to the reaction conditions and materials, and can generally be 2 to 10 hours. After the reaction is completed, filter, and the filtrate can be dissolved in DCM, and then treated with NaHCO 3 The organic phase is washed with aqueous solution and the pH is adjusted to 7-8 to obtain an organic phase. The organic phase is purified to obtain the compound 6 of the present invention in which the N atom of the base ring is deprotected.

[0039] Herein, the removal of the protecting groups at the 3' and 4' positions can be achieved in the presence of an organic solvent, an organic acid, and TBAF. The organic solvent can be anhydrous THF. The amount of the organic solvent (in mL) can be 30 to 60 times the mass of compound 6 (in g). The organic acid is preferably a monocarboxylic acid, more preferably a monocarboxylic acid with a carbon chain length of 1 to 4 carbon atoms, such as acetic acid (acetic acid). The molar ratio of the organic acid to compound 6 can be 1 to 3:1. The molar ratio of TBAF to compound 6 can be 1 to 5:1. The reaction can be completed in a relatively short time, for example, within 30 seconds to 5 minutes. NH 3 The reaction was quenched by the alcohol solution. 3 The equivalent concentration of the alcohol solution can be 5 to 10 N. After the reaction is completed, the 2'-methylated nucleoside of the present invention is obtained by purification and separation.

[0040] In some embodiments, after the three hydroxyl groups on the ribose are protected, the base of the nucleoside compound thus obtained can be modified as desired, and then steps (2) to (7) described in the present application can be performed. For example, in an exemplary embodiment, the 1-position ring N atom of the base is methylated to prepare N1 methyl pseudouridine with three hydroxyl groups on the ribose protected. The methylation modification may include reacting compound 1 with BSA at room temperature in an inorganic solvent for a period of time, adding an alkylating agent, such as MeI, and then heating to 35-45°C for a period of time, and separating to obtain a nucleoside compound 2' with an alkylated base. The nucleoside compound 2' can be used to carry out subsequent reactions. The substituted nitrogen atom on the base ring will no longer be replaced by the benzyloxy C 1-4 The inorganic solvent can be anhydrous DCM. The amount of inorganic solvent used (in mL) can be 10 to 20 times the mass of compound 2 (in g). The molar ratio of BSA to compound 2 can be 1 to 3:1. Usually, the reaction is first reacted at room temperature for 1 to 6 hours before adding the alkylating agent. The molar ratio of the alkylating agent to the compound can be 2 to 4:1. The reaction of BSA and the compound can be carried out at 38 to 42°C and the reaction time can be 10 to 60 hours. After the reaction is completed, water, NaHCO 3 The organic phase is separated and purified to obtain the compound 2'.

[0041] In some embodiments, the present invention provides a method for preparing 2'OMe-modified pseudouridine as shown in the following formula I:

[0042]

[0043] The method comprises the following steps:

[0044] (1) the step of protecting the hydroxyl group on the pseudouridine ribose comprises: reacting the pseudouridine with Ac in the presence of anhydrous DMF and 4-dimethylaminopyridine; 2 O reacts to introduce acyl groups on the three hydroxyl groups of the pseudouridine ribose moiety for protection, thereby preparing compound 1 shown in the following formula:

[0045]

[0046] The molar ratio of 4-dimethylaminopyridine to pseudouridine is 1:1-3, the amount of anhydrous DMF in mL is 20-40 times the weight of pseudouridine in g, and R 2 The molar ratio of O to pseudouridine is 3 to 10:1;

[0047] Preferably, pseudouridine is dissolved in anhydrous DMF, cooled to below 10°C, 4-dimethylaminopyridine is added, and then Ac2 O, then the obtained mixed solution is heated to room temperature, stirred, and after sufficient reaction, an alcohol such as methanol is added to terminate the reaction to obtain the compound 1;

[0048] (2) protecting the ring nitrogen atoms of uracil that are not substituted by non-hydrogen substituents, comprising: 2 CO 3 and anhydrous MeCN to react compound 1 with chloromethoxymethylbenzene represented by the following formula:

[0049]

[0050] Thus, compound 2 in which both the hydroxyl group and the N nitrogen atom substituted by the non-hydrogen substituent are protected is prepared:

[0051]

[0052] The amount of anhydrous MeCN in mL is 15 to 30 times the weight of compound 1 in g; the molar ratio of compound 1 to chloromethoxymethylbenzene is 1:1 to 3, and the molar ratio of compound 1 to K 2 CO 3 The molar ratio is 1:2-5;

[0053] (3) The step of deprotecting the protected hydroxyl group on the ribose comprises: dissolving compound 2 in NH 3 After a period of reaction in a methanol solution, compound 3 was obtained in which the three hydroxyl groups on the ribose were deprotected but the N on the base ring was still protected:

[0054]

[0055] Among them, the NH 3 The equivalent concentration of the methanol solution is 3 to 10 N; the amount of the methanol solution in mL is 10 to 30 times the mass of compound 2 in g;

[0056] (4) blocking the hydroxyl groups at the 3' and 4' positions, comprising: reacting compound 3 with TiPDSCl in an organic solvent; 2 Reaction to prepare compound 4 in which the hydroxyl groups at the 3' and 4' positions of ribose are blocked:

[0057]

[0058] The organic solvent is anhydrous dioxane and pyridine; the amount of the organic solvent in mL is 30 to 50 times the mass of compound 3 in g; compound 3 and TiPDSCl 2 The molar ratio is 1:2-5;

[0059] (5) The step of introducing a methyl group at the 2' position comprises: reacting compound 4 with a methylating agent, trimethoxylium tetrafluoroborate, in the presence of a dehydrogenating agent, N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine, to obtain compound 5 in which the hydroxyl groups at the 3' and 4' positions are still blocked, the N on the base ring is still protected, but the hydroxyl group at the 2' position is methylated:

[0060]

[0061] Wherein, the molar ratio of the dehydrogenation reagent to compound 4 is 5 to 15:1, and the molar ratio of the methylation reagent to compound 4 is 5 to 15:1;

[0062] (6) removing the protecting group on the ring nitrogen atom of the pseudouridine base, comprising: dissolving compound 5 in an aqueous solution of isopropanol, then mixing with formic acid and a palladium catalyst, introducing hydrogen gas, and reacting for a period of time to obtain compound 6 in which the nitrogen atom of the base ring is deprotected:

[0063]

[0064] Wherein, the volume ratio of isopropanol to water is 5 to 9:1, the amount of isopropanol aqueous solution in mL is 10 to 20 times the mass of compound 5 in g, and the amount of formic acid in mL is 1 to 5 times the mass of compound 5 in g; and

[0065] (7) removing the protecting groups at the 3' and 4' positions, comprising: removing the protecting groups at the 3' and 4' positions of compound 6 in the presence of an organic solvent, anhydrous THF, an organic acid, acetic acid, and TBAF, thereby preparing a 2'OMe-modified pseudouridine represented by formula I; wherein the amount of the organic solvent in mL is 30 to 60 times the mass of compound 6 in g, the molar ratio of the organic acid to compound 6 is 1 to 3:1, and the molar ratio of TBAF to compound 6 is 1 to 5:1.

[0066] In some embodiments, the present invention provides a method for preparing 2'OMe-modified N1-methyl pseudouridine as shown in Formula II below:

[0067]

[0068] The method comprises the following steps:

[0069] (1-1) The step of protecting the hydroxyl group on the pseudouridine ribose comprises: reacting pseudouridine with Ac in the presence of anhydrous DMF and 4-dimethylaminopyridine. 2 O reacts to introduce acyl groups on the three hydroxyl groups of the pseudouridine ribose moiety for protection, thereby preparing compound 1 shown in the following formula:

[0070]

[0071] The molar ratio of 4-dimethylaminopyridine to pseudouridine is 1:1-3, the amount of anhydrous DMF in mL is 20-40 times the weight of pseudouridine in g, and R 2 The molar ratio of O to pseudouridine is 3 to 10:1;

[0072] Preferably, pseudouridine is dissolved in anhydrous DMF, cooled to below 10°C, 4-dimethylaminopyridine is added, and then Ac 2 O, then the obtained mixed solution is heated to room temperature, stirred, and after sufficient reaction, an alcohol such as methanol is added to terminate the reaction to obtain the compound 1;

[0073] (1-2) Compound 1 and BSA are reacted in an organic solvent, anhydrous DCM, at room temperature for a period of time, and then an alkylating agent, MeI, is added. The temperature is then raised to 35-45° C. and reacted for a period of time to obtain N1-methyl pseudouridine with three hydroxyl groups on the ribose protected, i.e., compound 2′:

[0074]

[0075] The amount of the organic solvent in mL is 10 to 20 times the mass of the compound 1 in g; the molar ratio of BSA to compound 1 is 1 to 3:1; the molar ratio of the alkylating agent to compound 1 is 2 to 4:1;

[0076] Preferably, after adding BSA, the alkylating agent is added after stirring at room temperature for 1 to 6 hours, and then the reaction is carried out at 38 to 42°C for 10 to 60 hours; after the reaction is completed, water and NaHCO are added. 3 The solution was then extracted with DCM, and an organic phase was obtained by separation, and compound 2' was obtained from the organic phase;

[0077] (2) protecting the ring nitrogen atoms of uracil that are not substituted by non-hydrogen substituents, comprising: 2 CO 3 and anhydrous MeCN to react compound 2' with chloromethoxymethylbenzene represented by the following formula:

[0078]

[0079] Thus, compound 2 in which both the hydroxyl group and the N nitrogen atom substituted by the non-hydrogen substituent are protected is prepared:

[0080]

[0081] The amount of anhydrous MeCN in mL is 15 to 30 times the weight of compound 2' in g; the molar ratio of compound 2' to chloromethoxymethylbenzene is 1:1 to 3, and the molar ratio of compound 2' to K2 CO 3 The molar ratio is 1:2-5;

[0082] (3) The step of deprotecting the protected hydroxyl group on the ribose comprises: dissolving compound 2 in NH 3 After a period of reaction in a methanol solution, compound 3 was obtained in which the three hydroxyl groups on the ribose were deprotected but the N on the base ring was still protected:

[0083]

[0084] Among them, the NH 3 The equivalent concentration of the methanol solution is 3 to 10 N; the amount of the methanol solution in mL is 10 to 30 times the mass of compound 2 in g;

[0085] (4) blocking the hydroxyl groups at the 3' and 4' positions, comprising: reacting compound 3 with TiPDSCl in an organic solvent; 2 Reaction to prepare compound 4 in which the hydroxyl groups at the 3' and 4' positions of ribose are blocked:

[0086]

[0087] The organic solvent is anhydrous dioxane and pyridine; the amount of the organic solvent in mL is 30 to 50 times the mass of compound 3 in g; compound 3 and TiPDSCl 2 The molar ratio is 1:2-5;

[0088] (5) The step of introducing a methyl group at the 2' position comprises: reacting compound 4 with a methylating agent, trimethoxylium tetrafluoroborate, in the presence of a dehydrogenating agent, N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine, to obtain compound 5 in which the hydroxyl groups at the 3' and 4' positions are still blocked, the N on the base ring is still protected, but the hydroxyl group at the 2' position is methylated:

[0089]

[0090] Wherein, the molar ratio of the dehydrogenation reagent to compound 4 is 5 to 15:1, and the molar ratio of the methylation reagent to compound 4 is 5 to 15:1;

[0091] (6) removing the protecting group on the ring nitrogen atom of the pseudouridine base, comprising: dissolving compound 5 in an aqueous solution of isopropanol, then mixing with formic acid and a palladium catalyst, introducing hydrogen gas, and reacting for a period of time to obtain compound 6 in which the nitrogen atom of the base ring is deprotected:

[0092]

[0093] Wherein, the volume ratio of isopropanol to water is 5 to 9:1, the amount of isopropanol aqueous solution in mL is 10 to 20 times the mass of compound 5 in g, and the amount of formic acid in mL is 1 to 5 times the mass of compound 5 in g; and

[0094] (7) removing the protecting groups at the 3' and 4' positions, comprising: removing the protecting groups at the 3' and 4' positions of compound 6 in the presence of an organic solvent, anhydrous THF, an organic acid, acetic acid, and TBAF, thereby preparing a 2'OMe-modified pseudouridine represented by formula I; wherein the amount of the organic solvent in mL is 30 to 60 times the mass of compound 6 in g, the molar ratio of the organic acid to compound 6 is 1 to 3:1, and the molar ratio of TBAF to compound 6 is 1 to 5:1.

[0095] The typical reaction routes for preparing 2'OMe-modified pseudouridine and N1-methyl pseudouridine are shown in the following reaction formulas:

[0096]

[0097]

[0098] In the pentose structure of pseudouridine and its similar nucleoside derivatives, the methylation activity of the three active OH groups is 5'-OH>3'-OH=2'-OH. However, only 3'-OMe is the target product. Therefore, when the methylation reagent is directly used for pentose methylation, a large amount of by-products will be generated. For example, in the method disclosed by BSROSS et al. (Synthesis And Incorporation of 2'-O-Methyl-Pseudouridine into Oligonucleotides, Natural Products, Vol. 29, No. 9, March 3, 1998), the yields of 3'-OMe-modified pseudouridine and pseudouridine modified with 2'-OMe were 18% and 7%, respectively; in the method disclosed by MJROBINS et al. (Nucleic Acid Related Compounds. 12. The Facile and High-Yield Stannous Chloride Catalyzed Monomethylation of the Cis-Glycol System of Nucleosides by Diazomethane, Natural Products, Vol. 5, No. 44, November 5, 1974), the yields of 3'-OMe-modified pseudouridine and pseudouridine modified with 2'-OMe were 27% and 10%, respectively. The method of the present application can inhibit unnecessary methylation products such as 3'-OMe and 5'-OMe, and can increase the synthesis yields of 2'-OMe-modified pseudouridine and 2'-OMe-modified N1-methylpseudouridine to 68% and 26%, respectively, which are much higher than the yields obtained by existing methods.

[0099] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0100] Except for those specifically prepared in the examples, all other reagents are commercially available.

[0101] Example 1

[0102]

[0103] Pseudouridine (20 g, 81.9 mmol) was dissolved in anhydrous DMF (600 mL), cooled to 0°C, and DMAP (4-dimethylaminopyridine) (10 g, 81.9 mmol) was added. 2O (46.2mL, 491.4mmol) was slowly added dropwise to the reaction solution. After the addition, the reaction was slowly warmed to room temperature and stirred overnight. MeOH (50mL) was added and stirred for 10 minutes. The reaction solution was spin-dried. The crude product was dissolved in DCM (dichloromethane) and washed with a small amount of water. The organic phase was dried and spin-dried. The crude product was purified by column chromatography (DCM / EA (dichloromethane / ethyl acetate) = 1 / 2) to obtain (2R, 3R, 4S, 5S)-2-(acetoxymethyl)-5-(2,4-dicarbonyl-1,2,3,4-tetrahydropyrimidin-5-yl)tetrahydrofuran-3,4-diyl diacetate (30.0g, yield 99%) as a white solid.

[0104] ESI-MS m / z calcd for C 15 H 17 N 2 O 9 - [MH] - 369.1;found.369.0.

[0105]

[0106] (2R,3R,4S,5S)-2-(Acetoxymethyl)-5-(2,4-dicarbonyl-1,2,3,4-tetrahydropyrimidin-5-yl)tetrahydrofuran-3,4-diyl diacetate (17.8 g, 48.1 mmol) was dissolved in anhydrous MeCN (400 mL), and ((chloromethoxy)methyl)benzene (9.8 g, 62.6 mmol) and K 2 CO 3 (20.0 g, 144.3 mmol). The reaction was stirred at room temperature overnight. After the reaction solution was concentrated, DCM and water were added thereto and the liquids were separated and extracted. The organic phase was dried and then spin-dried. The crude product was purified by column chromatography (DCM / EA=3 / 1) to give (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(1,3-((benzyloxy)methyl)-2,4-dicarbonyl-1,2,3,4-tetrahydropyrimidin-5-yl)tetrahydrofuran-3,4-diyl diacetate (28.7 g, yield 98%).

[0107] ESI-MS m / z calcd for C 31 H 35 N 2 O 11 + [M+H] + 611.2;found.611.0.

[0108]

[0109] (2R,3R,4S,5S)-2-(Acetoxymethyl)-5-(1,3-((benzyloxy)methyl)-2,4-dicarbonyl-1,2,3,4-tetrahydropyrimidin-5-yl)tetrahydrofuran-3,4-diyl diacetate (16.7 g, 27.3 mmol) was dissolved in 7N NH 3 / MeOH (250 mL). The reaction was stirred at room temperature for 3 hours. The reaction solution was spin-dried. The crude product was purified by column chromatography (DCM / MeOH=9 / 1) to give 1,3-((benzyloxy)methyl)-5-((2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2,4(1H,3H)-dione (12.7 g, yield 96%).

[0110] ESI-MS m / z calcd for C 25 H 29 N 2 O 8 + [M+H] + 485.1;found.485.5.

[0111]

[0112] 1,3-((Benzyloxy)methyl)-5-((2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2,4(1H,3H)-dione (12.8 g, 26.4 mmol) was dissolved in anhydrous dioxane (400 mL) and pyridine (100 mL) was added. TiPDSCl 2 (25.0 g, 79.3 mmol) was added. The reaction was stirred at room temperature overnight. The reaction solution was vortexed to dryness. The crude product was dissolved in DCM, and water and NaHCO were added. 3 The organic phase was dried and then spin-dried. The crude product was purified by column chromatography (DCM / EA=1 / 1) to give 1,3-((benzyloxy)methyl)-5-((6aR,8S,9S,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)-1-methylpyrimidine-2,4(1H,3H)-dione (17.3 g, yield 90%).

[0113]

[0114] 1,3-((Benzyloxy)methyl)-5-((6aR,8S,9S,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)-1-methylpyrimidine-2,4(1H,3H)-dione (7.6 g, 10.5 mmol) was dissolved in anhydrous DCM (130 ml), and N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (22.4 g, 104.7 mmol) and trimethoxylium tetrafluoroborate (13.9 g, 94.2 mmol) were added respectively. The reaction was stirred at room temperature for 6 hours. After the reaction solution was filtered, dilute hydrochloric acid was added thereto and stirred for 2 minutes. The organic phase was collected, dried and spin-dried. The crude product was purified by column chromatography (DCM / EA=9 / 1) to give 1,3-((benzyloxy)methyl)-1-methyl-5-((6aR,8S,9S,9aR)-2,2,4,4-tetraisopropyl-9-methoxytetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)pyrimidine-2,4(1H,3H)-dione (7.13 g, yield 92%).

[0115]

[0116] 1,3-((Benzyloxy)methyl)-5-((6aR,8S,9S,9aR)-2,2,4,4-tetraisopropyl-9-methoxytetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (6.2 g, 8.4 mmol) was dissolved in a mixed solution of iPrOH / H2O (v: 9 / 1, 100 mL), and formic acid (10 mL) and wet Pd / C (600 mg) were added respectively. The reaction was carried out under H 2 The reaction mixture was stirred for 6 hours under the same conditions. The reaction mixture was filtered through celite, and the filtrate was concentrated and then DCM was added to dissolve. NaHCO 3 Aqueous solution was added thereto and the pH was adjusted to 7-8. The organic phase was dried and then spin-dried. The crude product was purified by column chromatography (DCM / EA=3 / 1) to give 1-methyl-5-((6aR,8S,9S,9aR)-2,2,4,4-tetraisopropyl-9-methoxytetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (3.8 g, yield 93%).

[0117] ESI-MS m / z calcd for C 24 H 43 N 2 O 7 Si 2+ [M+H] + 501.7;found.501.6.

[0118]

[0119] 5-((6aR,8S,9S,9aR)-2,2,4,4-tetraisopropyl-9-methoxytetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (2.7 g, 5.3 mmol) was dissolved in anhydrous THF (100 mL), and acetic acid (315 mg, 5.3 mmol) was added. TBAF·3H 2 O (3.3 g, 10.5 mmol) was added and stirred at room temperature for 1 minute. 7N NH 3 / MeOH (10mL) was added to quench the reaction. The mixed solution was quickly spin-dried and mixed with DCM / MeOH (9 / 1) system. The crude product was purified by column chromatography (DCM / MeOH=9 / 1), and the product was concentrated and then washed with a large amount of DCM, and finally dried to obtain 5-((2S,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)-1-methylpyrimidine-2,4(1H,3H)-dione (1.3 g, yield 96%), which was a white solid powder.

[0120] ESI-MS m / z calcd for C 11 H 17 N 2 O 6 [M+H] + 273.1;found.273.0.

[0121] Example 2

[0122]

[0123] (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(2,4-dicarbonyl-1,2,3,4-tetrahydropyrimidin-5-yl)tetrahydrofuran-3,4-diyl diacetate was prepared according to the method of Example 1. (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(2,4-dicarbonyl-1,2,3,4-tetrahydropyrimidin-5-yl)tetrahydrofuran-3,4-diyl diacetate (25.5 g, 68.9 mmol) was dissolved in anhydrous DCM (400 mL), and BSA (bis(trimethylsilyl)acetamide) (28.0 g, 137.7 mmol) was added. The reaction was stirred at room temperature for 3 hours, and MeI (iodomethane) (9.4 mL, 151.5 mmol) was added. The reaction was heated to 40°C and stirred for 2 days. Water was added, and NaHCO 3 Aqueous solution, adjust pH to 7-8. DCM is added thereto and extracted. The organic phase is dried and then spin-dried. The crude product is purified by column chromatography (DCM / EA=3 / 2) to obtain (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(1-methyl-2,4-dicarbonyl-1,2,3,4-tetrahydropyrimidin-5-yl)tetrahydrofuran-3,4-diyl diacetate (23.8 g, yield: 90%)

[0124] 1 H-NMR (400 MHz, DMSO-d 6 )δ11.41(br.s,1H),7.75(s,1H),5.37(t,J=5.4Hz,1H),5.24(t,J=5.8Hz,1H),4.66(d,J= 5.0Hz,1H),4.35–4.25(m,1H),4.17–4.03(m,2H),3.25(s,3H),2.08–2.00(m,9H).ESI-MS m / z calcd for C 16 H 21 N 2 O 9 + [M+H] + 385.1;found.385.0.

[0125]

[0126] (2R,3R,4S,5S)-2-(Acetoxymethyl)-5-(1-methyl-2,4-dicarbonyl-1,2,3,4-tetrahydropyrimidin-5-yl)tetrahydrofuran-3,4-diyl diacetate (18.5 g, 48.1 mmol) was dissolved in anhydrous MeCN (acetonitrile) (400 mL), and ((chloromethoxy)methyl)benzene (9.8 g, 62.6 mmol) and K were added respectively.2 CO 3 (13.3 g, 96.3 mmol). The reaction was stirred at room temperature overnight. After the reaction solution was concentrated, DCM and water were added thereto and the liquids were separated and extracted. The organic phase was dried and then spin-dried. The crude product was purified by column chromatography (DCM / EA=3 / 1) to give (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(3-((benzyloxy)methyl)-1-methyl-2,4-dicarbonyl-1,2,3,4-tetrahydropyrimidin-5-yl)tetrahydrofuran-3,4-diyl diacetate (23.7 g, yield 98%).

[0127] ESI-MS m / z calcd for C 16 H 21 N 2 O 9 + [M+H] + 385.1;found.385.0.

[0128]

[0129] (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(3-(benzyloxy)methyl)-1-methyl-2,4-dione-1,2,3,4-tetrahydropyrimidin-5-yl)tetrahydrofuran-3,4-diacetate (14 g, 27.3 mmol) was dissolved in 7N NH3 / MeOH (250 mL). The reaction was stirred at room temperature for 3 hours. The reaction solution was spin-dried. The crude product was purified by column chromatography (DCM / MeOH=9 / 1) to give 3-((benzyloxy)methyl)-5-((2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1-methylpyrimidine-2,4(1H,3H)-dione (10 g, yield 92%).

[0130] 1 H-NMR (400 MHz, DMSO-d 6 )δ7.81(d,J=1.0Hz,1H),7.40–7.22(m,5H),5.33(s,2H),4.96(d,J=4.9Hz,1H),4.83–4.72(m,2H),4.59(s,2H),4.54(d ,J=4.0,0.9Hz,1H),3.96–3.84(m,2H),3.77–3.70(m,1H),3.70–3.62(m,1H),3.55–3.45(m,1H),3.29(s,3H).ESI-MSm / z calcd for C 18 H 23 N 2 O7 + [M+H] + 379.1;found.379.1.

[0131]

[0132] 3-((Benzyloxy)methyl)-5-((2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1-methylpyrimidine-2,4(1H,3H)-dione (10 g, 26.4 mmol) was dissolved in anhydrous dioxane (400 mL) and pyridine (100 mL) was added. TiPDSCl2 (1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane) (25.0 g, 79.3 mmol) was added. The reaction was stirred at room temperature overnight. The reaction solution was dried by vortexing. The crude product was dissolved in DCM and water and NaHCO were added. 3 The organic phase was dried and then spin-dried. The crude product was purified by column chromatography (DCM / EA=1 / 1) to give 3-((benzyloxy)methyl)-5-((6aR,8S,9S,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)-1-methylpyrimidine-2,4(1H,3H)-dione (14.6 g, yield 90%).

[0133] 1 H NMR (400MHz, DMSO-d6) δ7.49(d,J=1.2Hz,1H),7.38–7.25(m,5H),5.35(s,2H),5.13(d,J=4.3Hz,1H),4.60(s,2H),4.57(s ,1H),4.18–4.11(m,1H),4.11–4.03(m,1H),4.01–3.95(m,1H),3.94–3.84(m,2H),3.29(s,3H),1.11–0.94(m,28H).ESI-MS m / zcalcd for C 30 H 49 N 2 O 8 Si 2 + [M+H] + 621.3;found.621.2.

[0134]

[0135] 3-((Benzyloxy)methyl)-5-((6aR,8S,9S,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)-1-methylpyrimidine-2,4(1H,3H)-dione (6.5 g, 10.5 mmol) was dissolved in anhydrous DCM (130 ml), and N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine (22.4 g, 104.7 mmol) and trimethoxylium tetrafluoroborate (13.9 g, 94.2 mmol) were added respectively. The reaction was stirred at room temperature for 6 hours. After the reaction solution was filtered, dilute hydrochloric acid was added thereto and stirred for 2 minutes. The organic phase was collected, dried and spin-dried. The crude product was purified by column chromatography (DCM / EA=9 / 1) to give 3-((benzyloxy)methyl)-1-methyl-5-((6aR,8S,9S,9aR)-2,2,4,4-tetraisopropyl-9-methoxytetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)pyrimidine-2,4(1H,3H)-dione (5.9 g, yield 89%).

[0136] 1 H NMR(400MHz, DMSO-d6)δ7.47(d,J=1.2Hz,1H),7.41–7.26(m,5H),5.36(s,2H),4.66–4.63(m,1H),4.62(s,2H),4.27–4.20(m,1H),4 .13–4.07(m,1H),3.94–3.86(m,1H),3.86–3.81(m,1H),3.66(d,J=4.6Hz,1H),3.52(s,3H),3.29(s,3H),1.10–0.99(m,28H).ESI-MS m / z calcd for C 31 H 51 N 2 O 8 Si 2 + [M+H] + 635.3;found.635.7.

[0137]

[0138] 3-((Benzyloxy)methyl)-1-methyl-5-((6aR,8S,9S,9aR)-2,2,4,4-tetraisopropyl-9-methoxytetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (5.3 g, 8.4 mmol) was dissolved in a mixed solution of iPrOH / H2O (v:9 / 1, 100 mL), and formic acid (10 mL) and wet Pd / C (600 mg) were added respectively. The reaction was stirred under H2 for 6 hours. The reaction solution was filtered through celite, and the filtrate was concentrated and then dissolved in DCM. NaHCO 3 Aqueous solution was added thereto and the pH was adjusted to 7-8. The organic phase was dried and then spin-dried. The crude product was purified by column chromatography (DCM / EA=3 / 1) to give 1-methyl-5-((6aR,8S,9S,9aR)-2,2,4,4-tetraisopropyl-9-methoxytetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (4.0 g, yield 93%).

[0139] 1 H NMR (400MHz, DMSO-d6) δ11.41(br.s,1H),7.46(d,J=1.2Hz,1H),4.60(s,1H),4.30–4.24(m,1H),4.12–4.04(m,1H) ,3.95–3.87(m,1H),3.85–3.79(m,1H),3.70(d,J=4.7Hz,1H),3.50(s,3H),3.24(s,3H),1.14–0.97(m,28H).ESI-MS m / z calcd forC 24 H 43 N 2 O 7 Si 2 + [M+H] + 515.7;found.515.6.

[0140]

[0141] 1-Methyl-5-((6aR,8S,9S,9aR)-2,2,4,4-tetraisopropyl-9-methoxytetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (2.7 g, 5.3 mmol) was dissolved in anhydrous THF (tetrahydrofuran) (100 mL), and acetic acid (315 mg, 5.3 mmol) was added. TBAF·3H 2O (tetrabutylammonium fluoride) (3.3 g, 10.5 mmol) was added and stirred at room temperature for 1 minute. 7N NH 3 / MeOH (10mL) was added to quench the reaction. The mixed solution was quickly spin-dried and mixed with DCM / MeOH (9 / 1) system. The crude product was purified by column chromatography (DCM / MeOH=9 / 1), and the product was concentrated and then washed with a large amount of DCM, and finally dried to obtain 5-((2S,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)-1-methylpyrimidine-2,4(1H,3H)-dione (1.3 g, yield 90%), which was a white solid powder.

[0142] 1 H NMR(400MHz, Deuterium Oxide)δ7.71(d,J=0.8Hz,1H),4.72–4.70(m,1H),4.22(t,J=5.5Hz,1H),3.98–3. 91(m,2H),3.85–3.76(m,1H),3.74–3.65(m,1H),3.43(s,3H),3.33(s,3H).ESI-MS m / z calcd for C 11 H 17 N 2 O 6 [M+H] + 273.1;found.273.0.

[0143] Application Examples

[0144]

[0145] The method is based on the classic Yoshikawa Procedure. Different nucleoside compounds A, namely, 2'OMe-modified pseudouridine prepared in Example 1 or 2'OMe-modified N1-methyl pseudouridine prepared in Example 2, are dissolved in methyl triphosphate and cooled to 10°C. After slowly dropping 1.5eq of phosphorus oxychloride, the reaction system is heated to 15°C and stirred until the reaction raw materials completely disappear and are converted into the corresponding intermediate product B. Subsequently, 5eq of tri-n-butylamine pyrophosphate and 6eq of tri-n-butylamine pre-cooled to -10°C are added, and the reaction system is heated to 15°C and stirred until the raw material B is completely converted into the target product D. The reaction is quenched with ultrapure water pre-cooled to 4°C, and the pH is adjusted to 6 and extracted three times with dichloromethane. The aqueous phase system after the extraction is concentrated by vacuum distillation to obtain a crude product of product C as a light yellow-green oil. The final product is purified by AKTA or preparative HPLC to obtain pure 2'OMe-Pseudo UTP and 2'OMe-1-Me-Pseudo UTP.

[0146] ESI-MS m / z calcd for C 10 H 16 N 2 O 15 P 3 - [MH] - 497.0;found.496.9.

[0147] ESI-MS m / z calcd for C 11 H 18 N 2 O 15 P 3 - [MH] - 511.0;found.510.9.

[0148] This example shows that the 2'OMe-modified pseudouridine and 2'OMe-modified N1-methyl pseudouridine prepared by the present invention can be used for the synthesis of NTPs through the classical Yoshikawa pyrophosphorylation reaction, and thus can be used for the in vitro transcription reaction of mRNA.

Claims

1. A method for preparing 2'OMe-modified pseudouridine of formula I or 2'OMe-modified N1-methyl pseudouridine of formula II from pseudouridine: It is characterized in that The method comprises the following steps: (1) a step of protecting the hydroxyl group on the pseudouridine ribose; (2) a step of protecting the ring nitrogen atoms on the pseudouridine base that are not substituted by non-hydrogen substituents; (3) a step of deprotecting the protected hydroxyl group on the ribose; (4) blocking the hydroxyl groups at the 3' and 4' positions; (5) a step of introducing a methyl group at the 2' position; (6) a step of removing the protecting group on the ring nitrogen atom of the pseudouridine base; and (7) A step of removing the protecting groups at the 3' and 4' positions.

2. The method according to claim 1, characterized in that In step (1), an acyl group is introduced into the ribose portion of pseudouridine to protect the hydroxyl group; preferably, the acyl group is C 1-6 acyl group; Preferably, the step (1) comprises: reacting pseudouridine with a compound R2O in the presence of an organic solvent, thereby introducing acyl groups on the three hydroxyl groups of the pseudouridine ribose moiety for protection, wherein R is C 1-6 Acyl, preferably R is acetyl; preferably, the solvent comprises anhydrous DMF and 4-dimethylaminopyridine; preferably, the molar ratio of 4-dimethylaminopyridine to nucleoside is 1:1-3, the amount of anhydrous DMF in mL is 20-40 times the weight of pseudouridine in g, and the molar ratio of R2O to pseudouridine is 3-10:1; The compound prepared by step (1) in which the three hydroxyl groups on the ribose are protected by acyl groups R is preferably a compound of the following formula:

3. The method according to claim 2, characterized in that Pseudouridine is dissolved in anhydrous DMF, cooled to below 10°C, 4-dimethylaminopyridine is added, and then R2O is added. The resulting mixture is then heated to room temperature and stirred. After sufficient reaction, an alcohol such as methanol is added to terminate the reaction, thereby obtaining a compound in which the three hydroxyl groups on the ribose are protected by acyl groups R.

4. The method according to any one of claims 1 to 3, characterized in that In the method for preparing 2'OMe-modified N1-methyl pseudouridine shown in formula II, after obtaining a compound in which the hydroxyl groups on the ribose are protected, the 1-ring N atom of the base of the compound is methylated to prepare N1-methyl pseudouridine in which the three hydroxyl groups on the ribose are protected; Preferably, the methylation modification comprises reacting a compound in which the hydroxyl groups on the ribose are protected by acyl groups R with BSA in an organic solvent at room temperature for a period of time, adding an alkylating agent, and then heating to 35-45° C. to react for a period of time, and separating and obtaining N1 methyl pseudouridine in which the three hydroxyl groups on the ribose are protected; Preferably, the organic solvent is anhydrous DCM; Preferably, the alkylating agent is MeI; Preferably, the amount of the organic solvent in mL is 10 to 20 times the mass of the compound in which the hydroxyl group on the ribose is protected by the acyl group R in g; the molar ratio of BSA to the compound in which the hydroxyl group on the ribose is protected by the acyl group R is 1 to 3:1; the molar ratio of the alkylating agent to the compound in which the hydroxyl group on the ribose is protected by the acyl group R can be 2 to 4:1 Preferably, after adding BSA, the mixture is stirred at room temperature for 1 to 6 hours, then an alkylating agent is added, and then the reaction is carried out at 38 to 42° C. for 10 to 60 hours. After the reaction is completed, water and NaHCO3 solution are added, and then DCM is added for extraction, and an organic phase is separated, and then N1 methyl pseudouridine with three hydroxyl groups on the ribose protected is separated from the organic phase.

5. The method according to claim 1, characterized in that In the step (2), benzyloxy C 1-4 The alkyl group protects the nitrogen atom on the nucleoside base ring that is not substituted by a non-hydrogen substituent; Preferably, the compound in which the hydroxyl group on the ribose obtained in step (1) is protected by an acyl group is reacted with a halogenated C 1-4 Alkoxymethylbenzene reacts to prepare a compound in which both the hydroxyl group and the N nitrogen atom substituted by the non-hydrogen substituent are protected; Preferably, the halogenated C 1-4 Alkoxymethylbenzene is a chlorinated C 1-4 Alkoxymethylbenzene, more preferably chloromethoxymethylbenzene: Preferably, the amount of organic solvent used in mL is 15 to 30 times the weight of the nucleoside in g; the compound with acyl protected hydroxyl group on ribose obtained in step (1) and halogenated C 1-4 The molar ratio of alkoxymethylbenzene is 1:1-3, and the molar ratio of alkoxymethylbenzene to K2CO3 is 1:2-5.

6. The method according to claim 1, characterized in that In the step (3), an alcohol solution of NH3 (e.g., methanol) is used to remove the hydroxyl group on the ribose; Preferably, the compound obtained in step (2) can be dissolved in an alcohol solution of NH3, and after a period of reaction, a compound in which the three hydroxyl groups on the ribose are all deprotected but the N on the base ring is still protected is obtained; Preferably, the equivalent concentration of the alcoholic solution of NH3 used is 3-10N; the amount of the alcoholic solution used in mL is 10-30 times the mass of the compound in which the three hydroxyl groups on the ribose are deprotected but the N on the base ring is still protected in g.

7. The method according to claim 1, characterized in that In the step (4), the hydroxyl groups at the 3' and 4' positions of the ribose are connected to an alkyl-substituted disiloxane group so as to block the hydroxyl groups at these two positions from reacting when a methyl group is introduced at the 2' position; Preferably, the alkyl-substituted disiloxane group is a tetra-C 1-4 Alkyl substituted disiloxane groups: In the formula, each R is independently a C1-4 alkyl group, and the wavy lines indicate the positions of attachment to the oxygen in the hydroxyl groups at the 3' and 4' positions; Preferably, the compound obtained in step (3) is reacted with the compound of the following formula 3' in an organic solvent, and the hydroxyl groups at the 3' and 4' positions on the ribose of the compound obtained in step (3) are connected to alkyl-substituted disiloxane groups: In the formula, each R is independently C 1-4 Alkyl, each A is independently halogen; preferably, the compound is TiPDSCl2; Preferably, the organic solvent is anhydrous dioxane and pyridine; preferably, the amount of the organic solvent in mL is 30 to 50 times the mass of the compound obtained in step (3) in g; the molar ratio of the compound obtained in step (3) to compound 3' is 1:2 to 5; Preferably, the reaction is carried out at room temperature; after the reaction is completed, the mixture is extracted with DCM, washed with water and NaHCO3 aqueous solution, and the organic phase is obtained and purified to obtain a compound in which the hydroxyl groups at the 3' and 4' positions on the ribose are blocked.

8. The method according to claim 1, characterized in that In the step (5), the compound obtained in the step (4) is reacted with a methylating agent in the presence of a dehydrogenating agent to perform methylation modification on the 2' position of the compound, thereby obtaining a compound in which the hydroxyl groups at the 3' and 4' positions are still blocked, the N on the base ring is still protected, but the hydroxyl group at the 2' position is methylated; Preferably, the dehydrogenation agent is N1,N1,N8,N8-tetramethylnaphthalene-1,8-diamine; Preferably, the methylating agent is trimethoxylium tetrafluoroborate; Preferably, the molar ratio of the dehydrogenation reagent to the compound obtained in step (4) is 5 to 15:1; Preferably, the molar ratio of the methylating agent to the compound obtained in step (4) is 5 to 15:

1.

9. The method according to claim 1, characterized in that In the step (6), the removal of the protecting group on N is carried out in a H2 atmosphere in the presence of formic acid and a palladium catalyst; Preferably, the compound obtained in step (5) is dissolved in an aqueous alcohol solution, then mixed with formic acid and a palladium catalyst, hydrogen is introduced, and the reaction is carried out for a period of time to obtain a compound in which the N atom of the base ring is deprotected; Preferably, the alcohol is isopropanol; Preferably, the volume ratio of alcohol to water is 5 to 9:1; Preferably, the amount of the alcohol solution in mL is 10 to 20 times the mass of the compound obtained in step (5) in g; Preferably, the amount of formic acid used in mL is 1 to 5 times the mass of the compound obtained in step (5) in g.

10. The method according to claim 1, characterized in that In the step (7), the protecting groups at the 3' and 4' positions of the compound obtained in the step (6) are removed in the presence of an organic solvent, an organic acid and TBAF, thereby preparing a 2'OMe-modified pseudouridine as shown in formula I or a 2'OMe-modified N1-methyl pseudouridine as shown in formula II; Preferably, the organic solvent is anhydrous THF; Preferably, the amount of organic solvent used in mL is 30 to 60 times the mass of the compound obtained in step (6) in g; Preferably, the organic acid is a monocarboxylic acid, more preferably a monocarboxylic acid with a carbon chain length of 1 to 4 carbon atoms, such as acetic acid; Preferably, the molar ratio of the organic acid to the compound obtained in step (6) is 1 to 3:1; Preferably, the molar ratio of TBAF to the compound obtained in step (6) is 1 to 5:1.

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