A locked nucleoside cap analogue and its application

Abstract

The present invention discloses a locked nucleoside cap analogue, which contains a bridged ring or a substituted bridged ring structure on the first sugar ring and / or the second sugar ring. While the bridged ring itself has at least one O, the substituted bridged ring is a bridged ring containing at least one O and has a substituent such as OH, NH2, alkyl, O-alkyl, alkyl-O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or halogen. The locked nucleoside cap analogue can improve the translation efficiency of the capped mRNA while maintaining or even further improving the capping efficiency of the capped analogue and reducing hepatotoxicity.

Description

Technical Field

[0001] The present invention belongs to the technical field of mRNA capping, and particularly relates to a locked nucleoside cap analogue and its application. Background Art

[0002] In eukaryotic cells, the 5' end of most messenger RNA (mRNA) is closed, or "capped". The cap contains a 5'-5' triphosphate bond between the two nucleoside moieties and a 7-methyl group on the distal guanine ring. The capping of mRNA promotes its normal function in the cell. Synthesizing mRNA by in vitro transcription has become an important tool for introducing exogenous genes and expressing proteins, and is widely used in the treatment and prevention of diseases. In vitro transcription of mRNA allows workers to prepare RNA molecules that perform appropriately in various biological applications. Capping analogs is a key process in the mRNA preparation process. At the same time, the stability and liver toxicity of mRNA are also closely related to the cap structure.

[0003] In eukaryotic cells, in addition to identifying the start of protein synthesis, the 5'-end cap structure also acts as a protective group against 5'-to-3' exonuclease cleavage, meaning it resists degradation by 5'-exonucleases. During protein synthesis, the cap structure also serves as a unique identifier for recruiting protein factors for pre-mRNA splicing, polyadenylation, and nuclear export. It also serves as an anchor for recruiting initiation factors, facilitating ribosome recognition and binding to mRNA, enabling the correct initiation of translation.

[0004] Natural structure cap analogs can be recognized and hydrolyzed by the decapping enzyme (DCP2), which reduces the stability of mRNA in the body and ultimately reduces the translation efficiency of the target mRNA.

[0005] To improve mRNA translation efficiency, studies have reported that cap analogs with unsubstituted bridged ring structures can improve mRNA translation efficiency, but these structures have some hepatotoxicity. Therefore, it is of great significance to develop novel cap analogs that can increase mRNA stability and translation efficiency while reducing hepatotoxicity. Summary of the Invention

[0006] To address the existing problems of low in vitro transcription yield, insufficient capping efficiency, cap structure stability, and certain hepatotoxicity, the present invention provides a locked nucleoside cap analog and its application. The locked nucleoside cap analog of the present invention improves capping efficiency and in vitro transcription efficiency while also exhibiting very low hepatotoxicity.

[0007] The locked nucleoside cap analog of the present invention contains a substituted bridge ring structure on the first sugar ring and / or the second sugar ring, and its general structure is as follows:

[0008]

[0009] In the above structural formula, R a is the first sugar ring containing a substituted bridge ring; R b is a second sugar ring containing a substituted bridge ring;

[0010] The R a and R b The structures of the substituted bridge rings are each independent, and the substituted bridge rings are located at the oxygen-adjacent position of the sugar ring and at the position separated by one C from the oxygen-adjacent position. The bridge ring of the substituted bridge ring contains at least one O, at least one C or N, and O is connected to the C on the sugar ring; the substituent of the substituted bridge ring is OH, NH2, alkyl, O-alkyl, alkyl-O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or halogen; the number of C in the alkyl, O-alkyl, alkyl-O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, or cycloalkenyl group is not greater than 7.

[0011] The first and second sugar rings have a substituted bridge ring, and the bridge ring itself has at least one O. At the same time, the bridge ring has OH, NH2, alkyl, O-alkyl, alkyl-O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, halogen and other substituents, which can ensure or even further improve the capping efficiency of the capped analogue while improving the translation efficiency of the capped mRNA and reducing liver toxicity.

[0012] Preferably, the locked nucleoside cap analog has the general structural formula:

[0013] R b for or R a for or

[0014] And when R b for When R a for When R a for Yes, R. b for

[0015] Wherein, R1 is H, OH, NH2, alkyl, O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or halogen; the number of carbon atoms in the alkyl, O-alkyl, alkyl-O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, or cycloalkenyl group is not greater than 6.

[0016] X1 is CH2 or does not exist;

[0017] X2 is C or N; when X1 does not exist, X2 is directly connected to O

[0018] R2 and R3 are independently H, OH, NH2, alkyl, O-alkyl, alkyl-O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or halogen;

[0019] When X2 is N, only one of R2 and R3 exists; when X1 does not exist and X2 is C, R2 and R3 are not H at the same time.

[0020] R7 and R8 are independently H, OH, NH2, alkyl, O-alkyl, alkyl-O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or halogen;

[0021] R4 is H, OH, NH2, alkyl, O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or halogen;

[0022] X3 is CH2 or does not exist,

[0023] X4 is C or N. When X3 does not exist, X4 is directly connected to O;

[0024] R5 and R6 are H, OH, NH2, alkyl, O-alkyl, alkyl-O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or halogen;

[0025] When X4 is N, R5 and R6 can only be any one of them; when X3 does not exist and X4 is C, R5 and R6 are not H at the same time.

[0026] B1, B2, and B3 are independently natural, modified, or non-natural nucleobases.

[0027] In an alternative embodiment, when X1 is absent and X2 is C, R2 and R3 are not H at the same time.

[0028] In an alternative embodiment, when X3 is absent and X4 is C, R5 and R6 are not H at the same time.

[0029] As a preferred structure with lower hepatotoxicity, in the structure of the locked nucleoside cap analogue: when X2 is C, R2 and R3 can be connected to form a ring through a chemical bond; or when X4 is C, R5 and R6 can be connected to form a ring through a chemical bond.

[0030] Furthermore, the structure of the locked nucleoside cap analog is a combination of any proportion of the following three structures: this structure has an anti-reverse transcription effect, and the prepared target mRNA has higher stability and lower hepatotoxicity.

[0031]

[0032]

[0033] Furthermore, in order to improve the capping efficiency of the locked nucleoside cap analog while further reducing its hepatotoxicity, in the structure of the locked nucleoside cap analog, the substituent of the substituted bridge ring is an alkyl group, and the number of carbon atoms in the alkyl group is not greater than 3.

[0034] Preferably, the structure of the locked nucleoside cap analog is any combination of the following three structures: this structure has higher translation efficiency and anti-reverse transcription effect, and the prepared target mRNA has higher stability and lower hepatotoxicity.

[0035]

[0036]

[0037] Furthermore, while improving the capping efficiency of the locked nucleoside cap analog, while improving the stability of mRNA translation, its hepatotoxicity is also reduced. In the structure of the locked nucleoside cap analog, the substituent of the substituted bridge ring is an alkoxy group, and the number of carbon atoms in the alkyl group is not greater than 3.

[0038] Preferably, the locked nucleoside cap analog is any combination of the following three structures: this structure has higher translation efficiency and anti-reverse transcription effect, and the prepared target mRNA has higher stability and lower hepatotoxicity.

[0039]

[0040] The structure of the locked nucleoside cap analog is that when X3 does not exist and X4 is C, R5 and R6 are both H; and / or X1 does not exist and X2 is C, R2 and R3 are both H, and at the same time, X3 does not exist and X4 is C, R5 and R6 are both H.

[0041] The locked nucleoside cap analog is used in a method for capping RNA in an in vitro transcription reaction, the method comprising the following steps:

[0042] Applications of the above locked nucleoside cap analogs: for in vitro transcription preparation of linear mRNA;

[0043] Step (1): preparing a DNA template;

[0044] Step (2): performing an in vitro transcription reaction, wherein the reaction system contains RNA polymerase, nucleoside triphosphates and the capping analog as claimed in claim 1.

[0045] The locked nucleoside cap analogs have the following advantages:

[0046] 1. Anti-reverse transcription effect: The locked nucleoside cap analog replaces the original five-membered sugar ring structure with a bridge ring structure. After the replacement, the bridge ring structure cannot serve as the start site of transcription, and has a good anti-reverse transcription effect during in vitro transcription of mRNA.

[0047] 2. Improve the stability of mRNA. Compared with non-natural cap analogs, cap analogs cannot be recognized by the decapping enzyme (DCP2), thereby improving the stability of mRNA.

[0048] 3. Improve the translation effect of the target mRNA. The replaced bridged nucleoside structure further stabilizes the conformation of the five-membered sugar ring, making it easier for the cap-binding protein (EIF4E) to bind, thereby improving the translation efficiency of the target mRNA.

[0049] 4. Compared with unsubstituted bridged ring cap analogs, substituted bridged ring structures can reduce hepatotoxicity. This is because substituted bridged ring structure nucleosides are more easily metabolized by the liver, reducing hepatotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 : Comparative histogram of cell activity of Examples 1-11 and Comparative Examples 1-3. DETAILED DESCRIPTION

[0051] Example 1: Synthesis of an initial capped oligonucleotide primer containing a 2',4'-locked loop structure using intermediates A and B as raw materials

[0052] Intermediate A (2.0 mol) was suspended in a DMF solution containing ZnCl2 (20.0 mol), and then intermediate B (1.8 mol) was added to the reaction solution. After stirring at room temperature for 24 hours, the reaction was terminated with 10 L of 0.25 M EDTA-Na2 solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M sodium chloride aqueous solution, nanofiltered to remove salt, and concentrated to obtain the desired product. The reaction scheme is as follows:

[0053]

[0054] Wherein, compound A is obtained by the following steps:

[0055] (1) 5.0 g of 2',4'-lock-2'-spirocyclopropene-guanosine was weighed and dissolved in 50.0 mL of trimethyl phosphate. The reaction solution was cooled to 0°C and phosphorus oxychloride (1.8 eq.) was slowly added dropwise under a nitrogen atmosphere. After stirring at 0°C for 4 hours, the reaction was quenched by adding water. The reaction mixture was washed twice with dichloromethane to remove most of the trimethyl phosphate. The residue was concentrated under reduced pressure to remove the organic solvent and then purified by reverse phase preparative liquid chromatography to obtain intermediate A1.

[0056] (2) Intermediate A1, triphenylphosphine (2.0 eq.), 2,2'-disulfide dipyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under nitrogen for 15 hours. After the reaction, the reaction solution was slowly added to a 4 M sodium perchlorate acetone solution to precipitate a solid. The filter cake was then filtered and thoroughly washed with acetone to obtain Intermediate A2.

[0057] (3) Triethylamine phosphate (3.0 eq.) and zinc chloride (8.0 eq.) were suspended in anhydrous DMF and stirred at room temperature for 5 minutes. Intermediate A2 was added to the reaction mixture in batches and stirred at room temperature for 5 hours. After the reaction was completed, the reaction was terminated with 10 volumes of 0.25 M EDTA-Na2 solution. The reaction mixture was purified by ion chromatography to obtain intermediate A3.

[0058] (4) Intermediate A3 was dissolved in 20 volumes of purified water. The reaction solution was cooled to 4°C, and dimethyl sulfate (6.0 eq.) was slowly added dropwise. During the process, the pH was adjusted to no more than 5 with 2 M sodium hydroxide. The reaction was monitored by HPLC. After the reaction was completed, the solution was washed with dichloromethane, and the aqueous phase was purified by ion chromatography to obtain intermediate A4.

[0059] (5) Intermediate A4, triphenylphosphine (2.0 eq.), 2,2'-disulfide dipyridine (2.0 eq.), imidazole (8.0 eq.) and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under nitrogen atmosphere for 10 hours. After the reaction, the reaction solution was slowly added to a 4 M sodium perchlorate acetone solution to precipitate a solid, which was then filtered and the filter cake was thoroughly washed with acetone to obtain Intermediate A. The reaction route is as follows:

[0060] The reaction process of compound A is as follows:

[0061]

[0062] Wherein, compound B is obtained by the following steps:

[0063] (1) Weigh 200.0 g of 2'OMe-rA phosphoramidite monomer and N 21-Isobutyryl-2',3'-acetylguanosine (1.0 eq.) was dissolved in 2.0 L of dichloromethane in a single-necked flask. Tetrazole (2.1 eq.) was added under nitrogen purge and allowed to react at 25°C for 3 hours. After monitoring the reaction, a 70% aqueous solution of tert-butyl hydroperoxide was added dropwise to the reaction solution and allowed to react at 25°C for 1 hour. After monitoring the reaction, a solution of trichloroacetic acid (4.0 eq.) in dichloromethane was added dropwise to the reaction solution and allowed to react at room temperature for 1 hour. After monitoring the reaction, the reaction solution was washed with 10% aqueous sodium sulfite, 10% aqueous sodium bicarbonate, and saturated brine, respectively. The organic phase was concentrated and purified by column chromatography to obtain Intermediate B1.

[0064] (2) Dissolve the intermediate B1 in acetonitrile (10V), add 1.8eq. of bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite and 1.8eq. of tetrazole, and stir at room temperature for 2 hours under nitrogen atmosphere. After the reaction, add 70% tert-butyl hydroperoxide aqueous solution (1.2eq.) dropwise to the reaction solution and react at room temperature for 1 hour. After monitoring the reaction, spin dry, add methanol and concentrated ammonia water (10V, 1:1) to the spin flask, react at room temperature for 14 hours, monitor the reaction, and spin dry after the reaction. The crude product is purified by ion chromatography and concentrated to obtain intermediate B. The reaction route flow is as follows:

[0065]

[0066] Example 2: Synthesis of an initial capped oligonucleotide primer containing a 2',4'-locked loop structure using intermediates C and D as raw materials

[0067] Using intermediates C and D as raw materials, the starting capped oligonucleotide primer of Example 2 was obtained by referring to the synthesis method of the target product in Example 1. The reaction scheme is as follows:

[0068]

[0069] Wherein, compound C is obtained by the following steps:

[0070] (1) 5.0 g of guanosine was weighed and dissolved in 50.0 mL of trimethyl phosphate. The reaction solution was cooled to 0°C and phosphorus oxychloride (1.8 eq.) was slowly added dropwise under a nitrogen atmosphere. After stirring at 0°C for 4 hours, the reaction was quenched by adding water. The reaction mixture was washed twice with dichloromethane to remove most of the trimethyl phosphate. The residue was concentrated under reduced pressure to remove the organic solvent and then purified by reverse phase preparative liquid chromatography to obtain intermediate C1.

[0071] (2) Intermediate C1, triphenylphosphine (2.0 eq.), 2,2'-dithiodipyridine (2.0 eq.), imidazole (8.0

[0072] eq.) and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under nitrogen for 15 hours. After the reaction, the reaction solution was slowly added to a 4 M sodium perchlorate acetone solution to precipitate a solid. The filter cake was then filtered and thoroughly washed with acetone to obtain intermediate C2.

[0073] (3) Triethylamine phosphate (3.0 eq.) and zinc chloride (8.0 eq.) were suspended in anhydrous DMF and stirred at room temperature for 5 minutes. Intermediate C2 was added to the reaction mixture in batches and stirred at room temperature for 5 hours. After the reaction was completed, the reaction was terminated with 10 volumes of 0.25 M EDTA-Na2 solution. The reaction mixture was purified by ion chromatography to obtain intermediate C3.

[0074] (4) Intermediate C3 was dissolved in 20 volumes of purified water. The reaction solution was cooled to 4°C, and dimethyl sulfate (6.0 eq.) was slowly added dropwise. During the process, the pH was adjusted to no more than 5 with 2 M sodium hydroxide. The reaction was monitored by HPLC. After the reaction was completed, the solution was washed with dichloromethane, and the aqueous phase was purified by ion chromatography to obtain intermediate C4.

[0075] (5) Intermediate C4, triphenylphosphine (2.0 eq.), 2,2'-dithiodipyridine (2.0 eq.), imidazole (8.0

[0076] eq.) and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under nitrogen atmosphere for 10 hours. After the reaction, the reaction solution was slowly added to a 4M sodium perchlorate acetone solution to precipitate a solid, which was then filtered and the filter cake was thoroughly washed with acetone to obtain intermediate C. The reaction scheme is as follows:

[0077]

[0078] Wherein, compound D is obtained by the following steps:

[0079] (1) Weigh 200.0 g of 2',4'-lock-2'-spirocyclopropene-rA phosphoramidite monomer and N 2 -isobutyryl-2',3'

[0080] -Acetylguanosine (1.0 eq.) was dissolved in 2.0 L of dichloromethane in a single-necked flask. Tetrazole (2.1 eq.) was added under nitrogen purge and allowed to react at 25°C for 3 hours. After monitoring the reaction, a 70% aqueous solution of tert-butyl hydroperoxide was added dropwise to the reaction solution and allowed to react at 25°C for 1 hour. After monitoring the reaction, a dichloromethane solution of trichloroacetic acid (4.0 eq.) was added dropwise to the reaction solution and allowed to react at room temperature for 1 hour. After monitoring the reaction, the reaction solution was washed with a 10% aqueous sodium sulfite solution, a 10% aqueous sodium bicarbonate solution, and saturated brine, respectively. The organic phase was concentrated and purified by column chromatography to obtain intermediate D1.

[0081] (2) Dissolve the intermediate D1 in acetonitrile (10V), add 1.8eq. of bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite and 1.8eq. of tetrazole, and stir at room temperature for 2 hours under nitrogen atmosphere. After the reaction is completed, add 70% tert-butyl hydroperoxide aqueous solution dropwise to the reaction solution and react at room temperature for 1 hour. After monitoring the reaction, spin dry, add methanol and concentrated ammonia water (10V, 1:1) to the spin flask, react at room temperature for 14 hours, monitor the reaction, and spin dry after the reaction is completed. The crude product is purified by ion chromatography and concentrated to obtain intermediate D. The reaction route flow is as follows:

[0082]

[0083] Example 3 Synthesis of an Initial Capped Oligonucleotide Primer Containing a 2',4'-Locked Loop Structure Using Intermediates A and D as Raw Materials

[0084] Using intermediates A and D as raw materials, the starting capped oligonucleotide primer of Example 3 was obtained by referring to the synthesis method of the target product in Example 1. The reaction scheme is as follows:

[0085]

[0086] Example 4 Synthesis of an Initial Capped Oligonucleotide Primer Containing a 2',4'-Locked Loop Structure Using Intermediates E and B as Raw Materials

[0087] Using intermediates E and B as raw materials, the starting capped oligonucleotide primer of Example 4 was obtained by referring to the synthesis method of the target product in Example 1. The reaction scheme is as follows:

[0088]

[0089] Wherein, compound E is obtained by the following steps:

[0090] (1) 5.0 g of 2',4'-lock-2'-ethyl-guanosine was weighed and dissolved in 50.0 mL of trimethyl phosphate. The reaction solution was cooled to 0°C and phosphorus oxychloride (1.8 eq.) was slowly added dropwise under a nitrogen atmosphere. After stirring at 0°C for 4 hours, the reaction was quenched by adding water. The reaction mixture was washed twice with dichloromethane to remove most of the trimethyl phosphate. The residue was concentrated under reduced pressure to remove the organic solvent and then purified by reverse phase preparative liquid chromatography to obtain intermediate E1.

[0091] (2) Intermediate E1, triphenylphosphine (2.0 eq.), 2,2'-dithiodipyridine (2.0 eq.), imidazole (8.0

[0092] eq.) and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under nitrogen atmosphere for 15 hours. After the reaction, the reaction solution was slowly added to a 4 M sodium perchlorate acetone solution to precipitate a solid. The filter cake was filtered and thoroughly washed with acetone to obtain intermediate E2.

[0093] (3) Triethylamine phosphate (3.0 eq.) and zinc chloride (8.0 eq.) were suspended in anhydrous DMF and stirred at room temperature for 5 minutes. Intermediate E2 was added to the reaction mixture in batches and stirred at room temperature for 5 hours. After the reaction was completed, the reaction was terminated with 10 volumes of 0.25 M EDTA-Na2 solution. The reaction mixture was purified by ion chromatography to obtain intermediate E3.

[0094] (4) Intermediate E3 was dissolved in 20 volumes of purified water. The reaction solution was cooled to 4°C, and dimethyl sulfate (6.0 eq.) was slowly added dropwise. During the process, the pH was adjusted to no more than 5 with 2 M sodium hydroxide. The reaction was monitored by HPLC. After the reaction was completed, the solution was washed with dichloromethane, and the aqueous phase was purified by ion chromatography to obtain intermediate E4.

[0095] (5) Intermediate E4, triphenylphosphine (2.0 eq.), 2,2'-dithiodipyridine (2.0 eq.), imidazole (8.0

[0096] eq.) and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under a nitrogen atmosphere for 10 hours. After the reaction, the reaction solution was slowly added to a 4M sodium perchlorate acetone solution to precipitate a solid, which was then filtered and the filter cake was thoroughly washed with acetone to obtain intermediate E. The reaction scheme is as follows:

[0097]

[0098] Example 5 Synthesis of an Initial Capped Oligonucleotide Primer Containing a 2',4'-Locked Loop Structure Using Intermediates C and F as Raw Materials

[0099] Using intermediates C and F as raw materials, the starting capped oligonucleotide primer of Example 5 was obtained by referring to the synthesis method of the target product in Example 1. The reaction scheme is as follows:

[0100]

[0101] Wherein, compound F is obtained by the following steps:

[0102] (1) Weigh 200.0 g of 2',4'-lock-2'-ethyl-rA phosphoramidite monomer and N 21-Isobutyryl-2',3'-acetylguanosine (1.0 eq.) was dissolved in 2.0 L of dichloromethane in a single-necked flask. Tetrazole (2.1 eq.) was added under nitrogen purge and allowed to react at 25°C for 3 hours. After monitoring the reaction, a 70% aqueous solution of tert-butyl hydroperoxide was added dropwise to the reaction solution and allowed to react at 25°C for 1 hour. After monitoring the reaction, a solution of trichloroacetic acid (4.0 eq.) in dichloromethane was added dropwise to the reaction solution and allowed to react at room temperature for 1 hour. After monitoring the reaction, the reaction solution was washed with 10% aqueous sodium sulfite solution, 10% aqueous sodium bicarbonate solution, and saturated brine, respectively. The organic phase was concentrated and purified by column chromatography to obtain intermediate F1.

[0103] (2) Dissolve the intermediate F1 in acetonitrile (10V), add 1.8eq. of bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite and 1.8eq. of tetrazole, and stir at room temperature for 2 hours under nitrogen atmosphere. After the reaction, add 70% tert-butyl hydroperoxide aqueous solution dropwise to the reaction solution and react at room temperature for 1 hour. After monitoring the reaction, spin dry, add methanol and concentrated ammonia water (10V, 1:1) to the spin flask, react at room temperature for 14 hours, monitor the reaction, and spin dry after the reaction. The crude product is purified by ion chromatography and concentrated to obtain intermediate F. The reaction route flow is as follows:

[0104]

[0105] Example 6 Synthesis of an Initial Capped Oligonucleotide Primer Containing a 2',4'-Locked Loop Structure Using Intermediates E and F as Raw Materials

[0106] Using intermediates E and F as raw materials, the starting capped oligonucleotide primer of Example 6 was obtained by referring to the synthesis method of the target product in Example 1. The reaction scheme is as follows:

[0107]

[0108] Example 7 Synthesis of an Initial Capped Oligonucleotide Primer Containing a 2',4'-Locked Loop Structure Using Intermediates G and B as Raw Materials

[0109] Using intermediates G and B as raw materials, the starting capped oligonucleotide primer of Example 7 was obtained by referring to the synthesis method of the target product in Example 1. The reaction scheme is as follows:

[0110]

[0111] Wherein, compound G is obtained by the following steps:

[0112] (1) 5.0 g of 2',4'-lock-2'-methoxyethyl-guanosine was weighed and dissolved in 50.0 mL of trimethyl phosphate. The reaction solution was cooled to 0°C and phosphorus oxychloride (1.8 eq.) was slowly added dropwise under a nitrogen atmosphere. After stirring at 0°C for 4 hours, the reaction was quenched by adding water. The reaction mixture was washed twice with dichloromethane to remove most of the trimethyl phosphate. The residue was concentrated under reduced pressure to remove the organic solvent and then purified by reverse phase preparative liquid chromatography to obtain intermediate G1.

[0113] (2) Intermediate G1, triphenylphosphine (2.0 eq.), 2,2'-dithiodipyridine (2.0 eq.), imidazole (8.0

[0114] eq.) and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under nitrogen for 15 hours. After the reaction, the reaction solution was slowly added to a 4 M sodium perchlorate acetone solution to precipitate a solid. The filter cake was then filtered and thoroughly washed with acetone to obtain intermediate G2.

[0115] (3) Triethylamine phosphate (3.0 eq.) and zinc chloride (8.0 eq.) were suspended in anhydrous DMF and stirred at room temperature for 5 minutes. Intermediate G2 was added to the reaction mixture in batches and stirred at room temperature for 5 hours. After the reaction was completed, the reaction was terminated with 10 volumes of 0.25 M EDTA-Na2 solution. The reaction mixture was purified by ion chromatography to obtain intermediate G3.

[0116] (4) Intermediate G3 was dissolved in 20 volumes of purified water. The reaction solution was cooled to 4°C, and dimethyl sulfate (6.0 eq.) was slowly added dropwise. During the process, the pH was adjusted to no more than 5 with 2 M sodium hydroxide. The reaction was monitored by HPLC. After the reaction was completed, the solution was washed with dichloromethane, and the aqueous phase was purified by ion chromatography to obtain intermediate G4.

[0117] (5) Intermediate G4, triphenylphosphine (2.0 eq.), 2,2'-dithiodipyridine (2.0 eq.), imidazole (8.0

[0118] eq.) and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under a nitrogen atmosphere for 10 hours. After the reaction, the reaction solution was slowly added to a 4M sodium perchlorate acetone solution to precipitate a solid, which was then filtered and the filter cake was thoroughly washed with acetone to obtain intermediate G. The reaction scheme is as follows:

[0119]

[0120] Example 8 Synthesis of an Initial Capped Oligonucleotide Primer Containing a 2',4'-Locked Loop Structure Using Intermediates C and H as Raw Materials

[0121] Using intermediates C and H as raw materials, the starting capped oligonucleotide primer of Example 8 was obtained by referring to the synthesis method of the target product in Example 1. The reaction scheme is as follows:

[0122]

[0123] Wherein, compound H is obtained by the following steps:

[0124] (1) Weigh 200.0 g of 2',4'-lock-2'-methoxyethyl-rA phosphoramidite monomer and N 2 1-Isobutyryl-2',3'-acetylguanosine (1.0 eq.) was dissolved in 2.0 L of dichloromethane in a single-necked flask. Tetrazole (2.1 eq.) was added under nitrogen purge and allowed to react at 25°C for 3 hours. After monitoring the reaction, a 70% aqueous solution of tert-butyl hydroperoxide was added dropwise to the reaction solution and allowed to react at 25°C for 1 hour. After monitoring the reaction, a solution of trichloroacetic acid (4.0 eq.) in dichloromethane was added dropwise to the reaction solution and allowed to react at room temperature for 1 hour. After monitoring the reaction, the reaction solution was washed with 10% aqueous sodium sulfite, 10% aqueous sodium bicarbonate, and saturated brine, respectively. The organic phase was concentrated and purified by column chromatography to obtain intermediate H1.

[0125] (2) Dissolve the intermediate H1 in acetonitrile (10V), add 1.8eq. of bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite and 1.8eq. of tetrazole, and stir at room temperature for 2 hours under nitrogen atmosphere. After the reaction is completed, add 70% tert-butyl hydroperoxide aqueous solution dropwise to the reaction solution and react at room temperature for 1 hour. After monitoring the reaction, spin dry, add methanol and concentrated ammonia water (10V, 1:1) to the spin flask, react at room temperature for 14 hours, monitor the reaction, and spin dry after the reaction is completed. The crude product is purified by ion chromatography and concentrated to obtain intermediate H. The reaction route flow is as follows:

[0126]

[0127] Example 9 Synthesis of an Initial Capped Oligonucleotide Primer Containing a 2',4'-Locked Loop Structure Using Intermediates G and H as Raw Materials

[0128] Using intermediates G and H as raw materials, the starting capped oligonucleotide primer of Example 9 was obtained by referring to the synthesis method of the target product in Example 1. The reaction scheme is as follows:

[0129]

[0130] Example 10: Synthesis of an Initial Capped Oligonucleotide Primer Containing a 2',4'-Locked Loop Structure Using Intermediates C and I as Raw Materials

[0131] Using intermediates C and I as raw materials, the starting capped oligonucleotide primer of Example 10 was obtained by referring to the synthesis method of the target product in Example 1. The reaction scheme is as follows:

[0132]

[0133] Wherein, compound I is obtained by the following steps:

[0134] (1) Weigh 200.0 g of 2',4'-locked ring-rA phosphoramidite monomer and N 2 1-Isobutyryl-2',3'-acetylguanosine (1.0 eq.) was dissolved in 2.0 L of dichloromethane in a single-necked flask. Tetrazole (2.1 eq.) was added under nitrogen purge and allowed to react at 25°C for 3 hours. After monitoring the reaction, a 70% aqueous solution of tert-butyl hydroperoxide was added dropwise to the reaction solution and allowed to react at 25°C for 1 hour. After monitoring the reaction, a solution of trichloroacetic acid (4.0 eq.) in dichloromethane was added dropwise to the reaction solution and allowed to react at room temperature for 1 hour. After monitoring the reaction, the reaction solution was washed with 10% aqueous sodium sulfite solution, 10% aqueous sodium bicarbonate solution, and saturated brine, respectively. The organic phase was concentrated and purified by column chromatography to obtain intermediate I1.

[0135] (2) The intermediate I1 was dissolved in acetonitrile (10V), 1.8eq. of bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite and 1.8eq. of tetrazole were added, and the mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. After the reaction, 70% aqueous tert-butyl hydrogen peroxide solution was added dropwise to the reaction solution, and the mixture was reacted at room temperature for 1 hour. After monitoring the reaction, the mixture was dried by spin-drying, and methanol and concentrated ammonia water (10V, 1:1) were added to the spin-drying bottle, and the mixture was reacted at room temperature for 14 hours. The reaction was monitored and the mixture was dried by spin-drying after the reaction was completed. The crude product was purified by ion chromatography and concentrated to obtain the intermediate I. The reaction route flow is as follows:

[0136]

[0137] Example 11 Synthesis of an Initial Capped Oligonucleotide Primer Containing a 2',4'-Locked Loop Structure Using Intermediates J and I as Raw Materials

[0138] Using intermediates J and I as raw materials, the starting capped oligonucleotide primer of Example 11 was obtained by referring to the synthesis method of the target product in Example 1. The reaction scheme is as follows:

[0139]

[0140] Wherein, compound J is obtained by the following steps:

[0141] (1) 5.0 g of 2',4'-locked guanosine was weighed and dissolved in 50.0 mL of trimethyl phosphate. The reaction solution was cooled to 0°C and phosphorus oxychloride (1.8 eq.) was slowly added dropwise under a nitrogen atmosphere. After stirring at 0°C for 4 hours, the reaction was quenched by adding water. The reaction mixture was washed twice with dichloromethane to remove most of the trimethyl phosphate. The residue was concentrated under reduced pressure to remove the organic solvent and then purified by reverse phase preparative liquid chromatography to obtain intermediate J1.

[0142] (2) Intermediate J1, triphenylphosphine (2.0 eq.), 2,2'-disulfide dipyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under nitrogen atmosphere for 15 hours. After the reaction, the reaction solution was slowly added to a 4 M sodium perchlorate acetone solution to precipitate a solid. The filter cake was then filtered and thoroughly washed with acetone to obtain Intermediate J2.

[0143] (3) Triethylamine phosphate (3.0 eq.) and zinc chloride (8.0 eq.) were suspended in anhydrous DMF and stirred at room temperature for 5 minutes. Intermediate J2 was added to the reaction mixture in batches and stirred at room temperature for 5 hours. After the reaction was completed, the reaction was terminated with 10 volumes of 0.25 M EDTA-Na2 solution. The reaction mixture was purified by ion chromatography to obtain intermediate J3.

[0144] (4) Intermediate J3 was dissolved in 20 volumes of purified water. The reaction solution was cooled to 4°C, and dimethyl sulfate (6.0 eq.) was slowly added dropwise. During the process, the pH was adjusted to no more than 5 with 2 M sodium hydroxide. The reaction was monitored by HPLC. After the reaction was completed, the solution was washed with dichloromethane, and the aqueous phase was purified by ion chromatography to obtain intermediate J4.

[0145] (5) Intermediate J4, triphenylphosphine (2.0 eq.), 2,2'-dithiodipyridine (2.0 eq.), imidazole (8.0

[0146] eq.) and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under a nitrogen atmosphere for 10 hours. After the reaction, the reaction solution was slowly added to a 4M sodium perchlorate acetone solution to precipitate a solid, which was then filtered and the filter cake was thoroughly washed with acetone to obtain Intermediate J. The reaction scheme is as follows:

[0147]

[0148] Comparative Example 1 Synthesis of an Initial Capped Oligonucleotide Primer Containing a 2',4'-Locked Loop Structure Using Intermediates J and B as Raw Materials

[0149] Using intermediates J and B as raw materials, the starting capped oligonucleotide primer of Comparative Example 1 was obtained by referring to the synthesis method of the target product in Example 1. The reaction scheme is as follows:

[0150]

[0151] Comparative Example 2 Synthesis of Initial Capping Oligonucleotide Primers Using Intermediates C and B as Raw Materials

[0152] Using intermediates C and B as raw materials, the starting capped oligonucleotide primer of Comparative Example 2 was obtained by referring to the synthesis method of the target product in Example 1. The reaction scheme is as follows:

[0153]

[0154] Comparative Example 3

[0155] The starting capping oligonucleotide primer of Comparative Example 3 was obtained by referring to the synthesis method of the target product in Example 1.

[0156]

[0157] Application and biological activity testing

[0158] Application Example 1: Determination of mRNA in vitro transcription yield and capping efficiency

[0159] In vitro synthesis of mRNA using capping analogs: first linearize the plasmid with plasmid linearization enzyme and digest it at 4°C overnight; extract the DNA template; and synthesize mRNA by in vitro transcription, using the capping analogs of Examples 1-11 and Comparative Examples 1-3 as cap structures, respectively.

[0160] The reaction system is shown in Table 1:

[0161] Table 1 In vitro transcription reaction system

[0162]

[0163]

[0164] Note: During the experiment, first calculate the required volume of materials for the system and then add the sample. First, add sterile enzyme-free water to the system, followed by 10X buffer, NTPs, and cap analogs. Mix well and centrifuge gently. Then add nuclease inhibitors, inorganic pyrophosphatase, T7 RNA polymerase, and linearized DNA template. Mix thoroughly and centrifuge gently. Incubate at 37°C. After 2 hours, add 1U of DNase I and continue incubation at 37°C for 30 minutes to remove the DNA template. RNA is then purified, usually using magnetic beads. The purified mRNA is dissolved in sterile enzyme-free water and then quantified using Nanodrop One.

[0165] Liquid chromatography-mass spectrometry (LC-MS) is used to measure the IVT capping rate of mRNA with different initiation cap analogs. First, a labeled DNA probe is designed that matches the start base of the transcript mRNA, typically with a biotin tag. After washing, the streptavidin-labeled magnetic beads are incubated with the synthesized DNA probe, mRNA, and 10× RNase H reaction buffer at room temperature for 30 minutes, gently mixing. Subsequently, 20 μl of RNase H (5 U / μl) is added and incubated at 37°C for 3 hours, mixing every half hour. After incubation, the beads are washed and 100 μl of 75% methanol heated to 80°C is added. The mixture is heated to 80°C on a hot plate for 3 minutes, then placed on a magnetic rack, the supernatant is aspirated, and dried in an evaporative centrifuge at room temperature for 45 minutes to a volume of 10 μl. The sample is then resuspended in 50 μl of 100 μM EDTA / 1% MeOH for LC-MS analysis to determine the capping status of the transcription reaction. Since there is a significant difference in molecular weight between capped and non-capped bases, the difference in molecular mass can be used to determine the capping rate of mRNA transcription initiated by different cap analogs.

[0166] Table 2 In vitro transcription yield and capping rate of mRNA

[0167]

[0168]

[0169] Conclusion As shown in Table 2, the locked nucleoside cap analogs of Examples 1-11 can transcribe the corresponding target mRNA. The yield and capping rate of the locked nucleoside cap analogs of Examples 1-11 are better than those of Comparative Examples 1 to 3.

[0170] Among them, Examples 1-3 having a cycloalkyl structure on the sugar ring, Examples 4-6 having a methyl structure on the sugar ring, and Examples 7-9 having a -CH2-O-CH3 structure on the sugar ring have a trend of gradually increasing yields and capping rates.

[0171] Test Example 2: Stability of the cap structure by decapping enzyme

[0172] 30 pmol of RNA, purified by polyacrylamide gel electrophoresis (PAGE), was reacted with 50 U of mRNA decapping enzyme (New England Biolabs) and 1× MDE buffer at 37°C for 45 min. The enzymatic reaction was subjected to PAGE electrophoresis, stained with SYBR Green II (Lonza), and the gel image was visualized on a Typhoon FLA 7000 (GE Healthcare). The decapping efficiency of the decapping enzyme was calculated by analyzing the ratio of electrophoretic band intensities of capped and decapped RNA using Image Quant software (GE Healthcare). Statistical analysis was performed using the Dunnett test using KaleidaGraph (Synergy) software.

[0173] Table 3 Decapping rate detection after decapping enzyme treatment

[0174]

[0175]

[0176] From the data in Table 3, it can be seen that the bridged ring structure cap analogs Examples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 have lower decapping rates after decapping enzyme treatment than that of Comparative Example 2; among them, Examples 1-3 having a cycloalkyl structure on the sugar ring, Examples 4-6 having a methyl structure on the sugar ring, and Examples 7-9 having a -CH2-O-CH3 structure on the sugar ring, have a trend of decreasing DCP2 enzyme decapping rates.

[0177] Test Example 3: Cell protein expression test

[0178] The eGFP coding sequence was used as a DNA template and the cap analogs of Examples 1-11 and Comparative Examples 1-2 were used as the starting point for in vitro transcription. The different mRNA products obtained were then used to transfect 293T cells. 293T cells were cultured at a density of (0.5-1)×10 5 Cells were plated (24-well plates). It is recommended to use cells within 50 generations for transfection experiments. The cells were required to be passaged again 24 hours before transfection. Adding antibiotics to the culture medium has no effect on the transfection effect. During transfection, the cell density is generally 60-80% as the best. 2μg mRNA was transfected per well. The transfection reagent used was Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) and its usage instructions were referred to. The transfected cells were placed in a 37°C, CO2 incubator. After 4-6 hours of transfection, fresh complete culture medium was replaced. After incubation in a 37°C CO2 incubator for 24 hours, the fluorescence intensity of GFP was observed under a fluorescence microscope.

[0179] Table 4 Translation and expression efficiency of intracellular mRNA

[0180]

[0181] The results are shown in Table 4, which clearly show that the mRNA expression efficiency of the present invention is significantly higher than that of the comparative example, and no significant cell death was caused. This result indicates that the capped analogs of the present invention have higher expression efficiency; that is, the capped analogs containing substituted bridged ring nucleoside structures in the present invention are significantly more efficient in mRNA synthesis than the protein translation efficiency of the cap structure in Comparative Example 2. As can be seen from the data in Table 4, the target mRNA translation efficiency of Examples 1-11 of the bridged ring capped analogs is significantly improved compared to that of Comparative Example 2.

[0182] Test Example 4: Hepatotoxicity Test

[0183] Normal human hepatocytes (L02 cells) were plated at 5000 cells (96-well plate). The optimal cell density for transfection was 60-80%. 0.5 μg of mRNA was transfected per well. Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) was used as the transfection reagent and the procedure was performed according to its instructions. The transfected cells were placed in a 37°C, CO2 incubator. After 4-6 hours of transfection, fresh complete medium was replaced. After incubation in a 37°C CO2 incubator for 48 hours, MTT was added to the cells and the cell viability was calculated by measuring the absorbance at a wavelength of 570 nm using a microplate reader. A cell viability test column was obtained for comparison, see the attached figure. Figure 1 .

[0184] From the attached Figure 1 It can be seen that the substituted bridged ring structure cap analogs of the present invention have obvious hepatotoxicity compared with comparative examples 1-3, and the hepatotoxicity of the substituted bridged ring structure cap analogs 1-9 is significantly lower than that of comparative example 1.

[0185] The specific structures of the capped analogs involved in the Examples and Comparative Examples are shown in the appendix below.

[0186] Example 1

[0187]

[0188] Example 2

[0189]

[0190] Example 3

[0191]

[0192] Example 4

[0193]

[0194] Example 5

[0195]

[0196] Example 6

[0197]

[0198] Example 7

[0199]

[0200] Example 8

[0201]

[0202] Example 9

[0203]

[0204] Example 10

[0205]

[0206] Example 11

[0207]

[0208] Comparative Example 1

[0209]

[0210] Comparative Example 2

[0211]

Claims

1. A locked nucleoside cap analogue, characterized in that: Its general structural formula is as follows: The general structural formula of the locked nucleoside cap analog is: R b for or R a for or And when R b for When R a for When R a for When R b for Wherein, R1 is H, OH, NH2, alkyl, O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, halogen; X1 does not exist; X2 is C, and X2 is directly connected to O; R2 and R3 are independently H, OH, alkyl, O-alkyl, alkyl-O-alkyl, cycloalkyl; R2 and R3 are not H at the same time; R7 and R8 are independently H, OH, alkyl, O-alkyl, alkyl-O-alkyl, or cycloalkyl; R4 is H, OH, alkyl, O-alkyl, or cycloalkyl; X3 does not exist, X4 is C, and X4 is directly connected to O; R5 and R6 are H, OH, alkyl, O-alkyl, alkyl-O-alkyl, or cycloalkyl; R5 and R6 are not H at the same time; The number of carbon atoms in the alkyl, O-alkyl, alkyl-O-alkyl, and cycloalkyl groups is not greater than 6; B1, B2, and B3 are independently natural or unnatural nucleoside bases.

2. The locked nucleoside cap analog according to claim 1, characterized in that: The R2, R3 and / or R5, R6 are chain alkyl groups with a carbon number not greater than 3.

3. A locked nucleoside cap analogue, characterized in that: The structure of the locked nucleoside cap analog is selected from:

4. The locked nucleoside cap analog according to claim 1, characterized in that: The R2, R3 and / or R5, R6 are O-alkyl groups, and the C number of the O-alkyl group is not greater than 3.

5. A locked nucleoside cap analogue, characterized in that: Its structure is selected from:

6. A locked nucleoside cap analogue, characterized in that: Its general structural formula is as follows: The general structural formula of the locked nucleoside cap analog is: R b for or R a for or And when R b for When R a for When R a for When R b for Wherein, R1 is H, OH, NH2, alkyl, O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, halogen; X1 does not exist; X2 is C, and X2 is directly connected to O; R2 and R3 are linked to form a ring via a chemical bond, and / or R5 and R6 are linked to form a ring via a chemical bond; R7 and R8 are independently H, OH, alkyl, O-alkyl, alkyl-O-alkyl, or cycloalkyl; R4 is H, OH, alkyl, O-alkyl, or cycloalkyl; X3 does not exist, X4 is C, and X4 is directly connected to O; The number of carbon atoms in the alkyl, O-alkyl, alkyl-O-alkyl, and cycloalkyl groups is not greater than 6; B1, B2, and B3 are independently natural or unnatural nucleoside bases.

7. A locked nucleoside cap analogue, characterized in that: The structure of the locked nucleoside cap analog is selected from:

8. A locked nucleoside cap analogue, characterized in that: Its structural formula is as follows:

9. The use of the locked nucleoside cap analogue according to any one of claims 1 to 8, characterized in that: Used to cap RNA in in vitro transcription reactions and for in vitro transcription preparation of linear mRNA; the steps are as follows: Step (1): preparing a DNA template; Step (2): performing an in vitro transcription reaction, wherein the reaction system contains RNA polymerase and nucleoside triphosphates.

Images