6'-Cyano-modified loc nucleosides, nucleotides, and nucleic acid polymers

Abstract

The present invention provides 6'-cyano-modified locked nucleosides, nucleotides and nucleic acid polymers, and relates to the field of biotechnology. The 6'-cyano-modified locked nucleosides provided by the present invention are in the R configuration or the S configuration, and are further modified to obtain nucleotides and nucleic acid polymers. According to the research of the inventors, the 6'-cyano-modified nucleic acid polymers have better nuclease resistance than unmodified or other modified nucleic acid polymers, and reduce or regulate the interaction between nucleic acid polymers and intracellular proteins. Therefore, it has been discovered that the 6'-cyano-modified locked nucleosides, nucleotides and nucleic acid polymers provided by the present invention have significant use value in nucleic acid pharmaceuticals. The preparation methods of the above-mentioned locked nucleosides, nucleotides and nucleic acid polymers provided by the present invention are efficient, stable and practical, and can be compatible with various substituted or unsubstituted bases.

Description

[Technical Field]

[0001] This application claims priority to the Chinese patent application filed with the Chinese National Intellectual Property Office on 18 May 2023, application number CN202310565102.7, with the title of the invention "6'-cyanomodified loc nucleosides, nucleotides and nucleic acid polymers," all of which are incorporated herein by reference.

[0002] The present invention relates to the field of biotechnology, and more particularly to 6'-cyano-modified rock nucleosides, nucleotides, and nucleic acid polymers. [Background technology]

[0003] Nucleic acid drugs are at the forefront of biopharmaceutical development and represent the third type of drug, following small molecule drugs and protein drugs. Nucleic acid drugs mainly include antisense nucleic acids (ASOs) and small interfering RNAs (siRNAs). Compared to conventional small molecule drugs and protein drugs, they have advantages such as rapid design, universal targeting, high specificity, intracellular action, and relatively rapid synthesis. They hold significant value in treating many chronic, intractable, severe, and rare diseases that are difficult to treat with protein-targeted drugs. By 2022, 15 types of nucleic acid drugs (10 types of ASOs and 5 types of siRNAs) had been approved worldwide, and more than 400 types of nucleic acid drugs were in clinical trials.

[0004] However, on the one hand, the drug potential of unmodified oligonucleotide drugs is generally not ideal. They have poor drug-related characteristics such as low stability, susceptibility to degradation by nucleases, high polarity, poor cell entry, poor distribution characteristics, and low binding affinity to target mRNA. To achieve clinical efficacy, oligonucleotides must be chemically modified, and all commercially available nucleic acid drugs utilize corresponding nucleic acid chemical modifications. On the other hand, chemically modified antisense nucleic acid drugs can cause some degree of toxicity to highly exposed organs (liver and kidney), and chemically modified nucleic acid drugs such as high-affinity locked nucleic acids (LNA) and restricted ethyl-locked nucleic acids (cEt-LNA) on phosphorothioate (PS) backbone can cause hepatotoxicity or nephrotoxicity, including significant increases in liver ALT and AST, or damage such as necrosis, degeneration / regeneration of renal tubules. Therefore, efficient and safe new-generation nucleic acid chemical modification technologies remain a crucial technology and bottleneck in the development of nucleic acid drugs.

[0005] In recent years, antisense nucleic acid drugs have made significant progress in terms of their toxicity mechanisms (Non-Patent Literature 1). Studies have shown that the main toxicity mechanism of antisense nucleic acid drugs is that chemically modified thioantisense nucleic acid drugs (PS-ASOs) bind to and interfere with the intracellular distribution of intracellular proteins (e.g., P54nrb), inducing apoptosis and causing toxicity. Simultaneously, toxicity is positively correlated with the binding ability of the ASO-protein, with stronger binding affinity indicating greater potential toxicity. This binding ability is closely related to the water solubility (LogS) of the chemically modified structure, with stronger hydrophobicity leading to higher binding affinity. Experimental results have shown that the affinity of 2'-methoxyethyl (2'-MOE) for intracellular proteins can be reduced by 30 times compared to 2'-fluorine (2'-F). Therefore, the interaction between antisense nucleic acids and proteins is a determinant of the therapeutic effect of antisense nucleic acid drugs, and the structure determines the properties. In order to develop a novel nucleic acid chemical modification structure that is efficient and safe, the present inventors previously disclosed a novel cyanoloc nucleic acid (CN-LNA) modification structure (Patent Document 1), which showed high nuclease resistance and good target gene affinity and selectivity. At the same time, further chemical property calculations (Chemdraw calculations) showed that CN-LNA has good hydrophilicity, with a LogS value of -0.032, which is about 2 times, 4 times, and 20 times higher than the common 2'-methoxyethyl (MOE, LogS value of -0.073), loc nucleic acid (LNA, LogS value of -0.122), and 2'-fluorine (LogS value of -0.661), respectively. This indicates that it may reduce the binding affinity between PS-ASO and intracellular proteins, further reduce the impact on intracellular protein distribution, reduce toxicity, and improve therapeutic effect.

[0006] However, previously disclosed CN-LNA synthesis methods cannot stereoselectively synthesize both R and S configuration C6'-epimers, nor can they further explain the differences in drug-related properties of the C6'-epimers compared to their modified nucleic acids, thus failing to meet the need to establish a new generation of efficient and low-toxicity nucleic acid chemical modification technologies.

[0007] In view of this, the present invention is provided. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Specification of Chinese Registered Patent No. 20190914759.3 [Non-patent literature]

[0009] [Non-Patent Document 1] Nucleic Acids Res.2016,44,3892; [Non-Patent Document 2] Nature Biotech., 2017, 35, 230; [Non-Patent Document 3] Nucleic Acids Res.2018,46,2204; [Non-Patent Document 4] Nature Biotech., 2019, 37, 640; [Non-Patent Document 5] Nucleic Acids Res.2019,47,10865; [Non-Patent Document 6] J.Am.Chem.Soc.,2020,142,14754; [Non-Patent Document 7] J.Am.Chem.Soc.2020,142,7456 [Overview of the project] [Problems that the invention aims to solve]

[0010] The first object of the present invention is to provide 6'-cyano-modified loc nucleosides, nucleotides, and nucleic acid polymers having an R or S configuration. A second object of the present invention is to provide a simple, high-yielding method for preparing R-configured or S-configured 6'-cyano-modified rock nucleosides, nucleotides, and nucleic acid polymers that can solve at least one of the above problems. A third object of the present invention is to provide the use of the above-mentioned nucleic acid polymer in the preparation of nucleic acid diagnostic agents or nucleic acid therapeutic agents. [Means for solving the problem]

[0011] In order to achieve the above objectives of the present invention, the following technical solutions are used in particular.

[0012] In a first aspect, the present invention provides a 6'-cyanomodified rock nucleoside, which is selected from a compound having the structure represented by formula 1, a salt thereof, or an isomer thereof. In formula 1, JPEG0007898764000001.jpg3551, Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or salts thereof. Z is a cyano group, W1 and W2 are independently selected from H or a hydroxy protecting group, and the hydroxy protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl, 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzyl Contains silyl, [(triisopropylsilyl)oxy]methyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidine-4-yl, 9-phenylxanthin-9-yl, 9-(p-methoxyphenyl)xanthin-9-yl, or 2-naphthylmethyl. The isomers include a 6'-cyano-modified loc nucleoside in which the Z group is in the R configuration, and the structural formula is JPEG0007898764000002.jpg3343, or the Z group contains a 6'-cyano-modified loc nucleoside in an S configuration, and the structural formula is The filename is JPEG0007898764000003.jpg3747.

[0013] In a second embodiment, the present invention provides a nucleotide comprising a 3'-active phosphorus group derivative of the 6'-cyano-modified rock nucleoside described above. The active phosphorus group is selected from phosphoramidites, derivatives of phosphoramidites, H-phosphonates, derivatives of H-phosphonates, triesters of phosphate, and derivatives of triesters of phosphate.

[0014] As a further technical solution, the nucleotide is selected from a compound having the structure represented by formula 2, its salt, or its isomer. In JPEG0007898764000004.jpg4863, the types of Bx, W2, and Z are the same as those in Equation 1. The isomer contains a nucleotide in which the Z group is in the R configuration, and its structural formula is JPEG0007898764000005.jpg4744, or a nucleotide with a Z group in an S configuration, and the structural formula is The filename is JPEG0007898764000006.jpg4343.

[0015] In a third aspect, the present invention provides the use of the above-mentioned nucleotides in reducing the interaction between nucleic acid polymers and intracellular proteins, or in the preparation of pharmaceuticals for reducing the interaction between nucleic acid polymers and intracellular proteins.

[0016] In a fourth embodiment, the present invention provides a nucleic acid polymer having a monomer having a structure represented by formula 3, In formula 3 of JPEG0007898764000007.jpg3453, Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil or their salts. Z is a cyano group, W3 and W4 are each independently H, a hydroxy protecting group, or an internucleoside linking group that links the monomer to other parts of the nucleic acid polymer, and at least one of W3 and W4 is an internucleoside linking group that links the monomer to other parts of the nucleic acid polymer. The aforementioned hydroxy protecting groups include acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl, 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzylsilyl, [(triisopropylsilyl Contains hydroxymethyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidine-4-yl, 9-phenylxanthin-9-yl, 9-(p-methoxyphenyl)xanthin-9-yl, or 2-naphthylmethyl.

[0017] As a further technical solution, the monomer includes a monomer in which the Z group is in an R configuration, and the structural formula is JPEG0007898764000008.jpg3345, or the Z group contains a monomer in an S configuration, and the structural formula is The filename is JPEG0007898764000009.jpg3342.

[0018] As a further technical solution, the nucleic acid polymer is ribonucleic acid, deoxyribonucleic acid, or a copolymer of ribonucleotide and deoxyribonucleotide.

[0019] In a fifth embodiment, the present invention provides the use of the nucleic acid polymer in the preparation of nucleic acid diagnostic agents or nucleic acid therapeutic agents.

[0020] In a sixth embodiment, the present invention provides a method for synthesizing cyanomodified nucleosides of the C6' moiety in R configuration or S configuration, wherein the cyanomodified nucleosides of the C6' moiety in R configuration are The file is JPEG0007898764000010.jpg3344, and its composition method is: The terminal olefin of JPEG0007898764000011.jpg3353 isomerized. JPEG0007898764000012.jpg3146 was obtained, and then the dihydroxylation reaction was carried out. JPEG0007898764000013.jpg3556 is obtained, and then the aldehyde group compound is obtained by an oxidative cleavage reaction. JPEG0007898764000014.jpg3254 is obtained, and finally, Convert the aldehyde group of JPEG0007898764000015.jpg3554 to a cyano group, and modify the cyano-modified nucleoside at the R-configured C6' site. This includes compositing JPEG0007898764000016.jpg3151, The cyano-modified nucleoside at the C6' site in the S configuration is The file is JPEG0007898764000017.jpg4063, and its composition method is: The terminal olefin of JPEG0007898764000018.jpg3355 is isomerized. JPEG0007898764000019.jpg3456 was obtained, and then the dihydroxylation reaction was carried out. JPEG0007898764000020.jpg3960 is obtained, and then an aldehyde group compound is obtained by an oxidative cleavage reaction. JPEG0007898764000021.jpg3757 is obtained, and finally, Convert the aldehyde group of JPEG0007898764000022.jpg3658 to a cyano group, and modify the cyano-modified nucleoside at the S-configured C6' site. This includes compositing JPEG0007898764000023.jpg3957, Here, Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or salts thereof.

[0021] As a further technical solution, The composition method for JPEG0007898764000024.jpg3451 is S-arrangement. Mesylation, desubtraction, and acetylation reactions are performed sequentially on JPEG0007898764000025.jpg3857. JPEG0007898764000026.jpg3653 was obtained, and then glycosidication and nucleophilic substitution reactions were carried out to obtain the R configuration. This includes compositing JPEG0007898764000027.jpg3349, The aforementioned The composition method for JPEG0007898764000028.jpg3249 is R-arrangement. Mesylation, desubtraction, and acetylation reactions are performed sequentially on JPEG0007898764000029.jpg3554. JPEG0007898764000030.jpg3354 was obtained, and then glycosidication and nucleophilic substitution reactions were carried out to obtain the S configuration. This includes compositing JPEG0007898764000031.jpg3758, The glycosidication reaction described above proceeds under activator conditions. The reaction involves reacting JPEG0007898764000032.jpg43152 with thymine, N6-benzoyladenine, or 6-chloroguanine at 50-100°C, where the activator comprises BSA and TMSOTf, and the reaction medium comprises acetonitrile, 1,2-dichloroethane, or toluene.

[0022] As a further technical solution, the S arrangement The synthesis method for JPEG0007898764000033.jpg3555 is 5-O-(tert-butyldiphenylsilyl)-4-C-hydroxymethyl-1,2-O-isopropylidene-3-O-(2-naphthylmethyl)-α-D-ribofuranose The primary alcohol in JPEG0007898764000034.jpg3551 is oxidized to an aldehyde group, and then an allylation reaction is performed on the aldehyde group to obtain the S configuration. This includes obtaining JPEG0007898764000035.jpg3966, Preferably, the R configuration The method for creating JPEG0007898764000036.jpg3149 is: The secondary alcohol in JPEG0007898764000037.jpg3966 is oxidized to a ketone, and then the ketone is reduced back to a secondary alcohol to obtain the R configuration. This includes obtaining JPEG0007898764000038.jpg3553, Here, in the process of reducing ketones to secondary alcohols, the reducing agent used comprises at least one of lithium aluminum hydride, lithium borohydride, lithium chloride, or sodium borohydride, the reaction medium comprises at least one of dichloromethane, tetrahydrofuran, methanol, or ethanol, and the reaction temperature is -78 to 0°C. Preferably, the reaction to reduce the ketone to a secondary alcohol is carried out at -40 to 0°C using sodium borohydride and lithium chloride as reducing agents and tetrahydrofuran and methanol as solvents.

[0023] As a further technical solution, the catalyst for terminal olefin isomerization comprises a ruthenium catalyst, a palladium catalyst, a rhodium catalyst, or an iridium catalyst, preferably a carbonylchlorohydride tris(triphenylphosphine)ruthenium(II), The reaction medium for isomerization of the terminal olefin comprises methanol, ethanol, n-butanol, or toluene, preferably ethanol. The reaction temperature for the terminal olefin isomerization is 60 to 100°C, preferably 60 to 80°C. The reaction time for the terminal olefin isomerization is 12 to 72 hours.

[0024] In a seventh aspect, the present invention provides a method for synthesizing a cyanoloc nucleic acid T phosphoramidite monomer with an R-configuration or S-configuration C6' site, wherein the method for synthesizing a cyanoloc nucleic acid T phosphoramidite monomer with an R-configuration C6' site is R-configuration Deprotect the 3'-hydroxyl and 5'-hydroxyl groups of JPEG0007898764000039.jpg4976. JPEG0007898764000040.jpg4775 was obtained, the 5'-hydroxyl group was protected with DMTr, and the 3'-hydroxyl group was subjected to a phosphoramidite reaction to form the R configuration. This includes obtaining JPEG0007898764000041.jpg6365, The method for synthesizing cyanolock nucleic acid T phosphoramidite monomers with an S configuration at the C6' site is as follows: Deprotect the 3'-hydroxyl group and 5'-hydroxyl group of JPEG0007898764000042.jpg3553 JPEG0007898764000043.jpg3655 was obtained, the 5'-hydroxyl group was protected with DMTr, and the 3'-hydroxyl group was subjected to a phosphoramidite reaction to form the S configuration. This includes obtaining JPEG0007898764000044.jpg5658.

[0025] In the eighth aspect, the present invention provides a method for synthesizing a cyanolocic nucleic acid A phosphoramidite monomer of the R-configuration or S-configuration C6' site, wherein the method for synthesizing a cyanolocic nucleic acid A phosphoramidite monomer of the R-configuration C6' site is R-configuration Protect the base of JPEG0007898764000045.jpg3575 JPEG0007898764000046.jpg3582 is obtained, and then the 3'-hydroxyl group and 5'-hydroxyl group are deprotected. JPEG0007898764000047.jpg3477 is obtained, and then the 5'-hydroxyl group is protected with DMTr, and the 3'-hydroxyl group is subjected to a phosphoramidite reaction to form the R configuration. This includes obtaining JPEG0007898764000048.jpg4265, The method for synthesizing the S-configured C6' site of the cyanolock nucleic acid A phosphoramidite monomer is as follows: Protect the base of JPEG0007898764000049.jpg3172 JPEG0007898764000050.jpg3588 is obtained, and then the 3'-hydroxyl group and 5'-hydroxyl group are deprotected. JPEG0007898764000051.jpg3080 is obtained, then the 5'-hydroxyl group is protected with DMTr, and the 3'-hydroxyl group is subjected to a phosphoramidite reaction to form the S configuration. This includes obtaining JPEG0007898764000052.jpg4265.

[0026] In a ninth embodiment, the present invention provides a method for synthesizing a cyanoloc nucleic acid G phosphoramidite monomer with an R-configuration or S-configuration C6' site, wherein the method for synthesizing a cyanoloc nucleic acid G phosphoramidite monomer with an R-configuration C6' site is R-configuration Demethylate the methoxy group of JPEG0007898764000053.jpg3465 JPEG0007898764000054.jpg3352 is obtained, and then base protection is performed. JPEG0007898764000055.jpg3664 is obtained, and then the 3'-hydroxyl group and 5'-hydroxyl group are deprotected. JPEG0007898764000056.jpg3354 was obtained, the 5'-hydroxyl group was protected with DMTr, and phosphoramidite was performed on the 3'-hydroxyl group to form the R configuration. This includes obtaining JPEG0007898764000057.jpg4152, The method for synthesizing cyanolocic nucleic acid G phosphoramidite monomers with the S configuration at the C6' site is as follows: Demethylate the methoxy group in JPEG0007898764000058.jpg3361 JPEG0007898764000059.jpg3570 is obtained, and then base protection is performed. JPEG0007898764000060.jpg3269 is obtained, and then the 3'-hydroxyl group and 5'-hydroxyl group are deprotected. JPEG0007898764000061.jpg3473 was obtained, the 5'-hydroxyl group was protected with DMTr, and the 3'-hydroxyl group was subjected to a phosphoramidite reaction to form the S configuration. This includes obtaining JPEG0007898764000062.jpg4458.

[0027] In a tenth embodiment, the present invention provides a method for synthesizing a cyanoloc nucleic acid C phosphoramidite monomer with an R-configuration or S-configuration C6' site, the method for preparing a cyanoloc nucleic acid C phosphoramidite monomer with an R-configuration C6' site is provided, Deprotect the 3'-hydroxyl group and 5'-hydroxyl group of JPEG0007898764000063.jpg4668 JPEG0007898764000064.jpg3958 is obtained, and then the 5'-hydroxyl group is protected with DMTr. JPEG0007898764000065.jpg4056 is obtained, the 3'-hydroxyl group is modified by silane, and the carbonyl group is converted to an amino group. JPEG0007898764000066.jpg3757 is obtained, then base protection is performed, and the 3'-hydroxyl group is deprotected. JPEG0007898764000067.jpg3862 was obtained, and finally, the 3'-hydroxyl group was subjected to a phosphoramidite reaction to obtain the R configuration. This includes obtaining JPEG0007898764000068.jpg4552, A method for preparing a cyanolock nucleic acid C phosphoramidite monomer with an S configuration at the C6' site is as follows: Deprotect the 3'-hydroxyl and 5'-hydroxyl groups of JPEG0007898764000069.jpg3961 JPEG0007898764000070.jpg3756 is obtained, and then the 5'-hydroxyl group is protected with DMTr. JPEG0007898764000071.jpg4165 is obtained, then the 3'-hydroxyl group is modified by silane, and the carbonyl group is converted to an amino group. JPEG0007898764000072.jpg3664 is obtained, then base protection is performed, and the 3'-hydroxyl group is deprotected. JPEG0007898764000073.jpg4174 is obtained, and finally, the 3'-hydroxyl group is subjected to a phosphoramidite reaction to form the S configuration. This includes obtaining JPEG0007898764000074.jpg4657.

[0028] In an eleventh embodiment, the present invention provides a method for synthesizing nucleic acid polymers, comprising carrying out a polymerization reaction with a monomer to prepare a nucleic acid polymer. The monomer comprises the 6'-cyano-modified rock nucleoside or the nucleotide. [Effects of the Invention]

[0029] Compared to the prior art, the present invention has the following beneficial effects.

[0030] The 6'-cyanomodified loc nucleosides provided by the present invention are R-configured or S-configured, and can be further modified to obtain nucleotides and nucleic acid polymers. According to our research, 6'-cyanomodified nucleic acid polymers have better nuclease resistance than unmodified or other modified nucleic acid polymers, and the S-configured 6'-cyanomodified nucleic acid polymers are more pronounced than the R-configured ones in terms of improving nuclease resistance of nucleic acid polymers and reducing the interaction between nucleic acid polymers and intracellular proteins. Therefore, it has been found that the 6'-cyanomodified loc nucleosides, nucleotides, and nucleic acid polymers provided by the present invention have significant use value in nucleic acid pharmaceuticals.

[0031] The present invention provides a universal method for preparing 6'-cyano-modified rock nucleosides, nucleotides, and nucleic acid polymers with the above-mentioned R-configuration or S-configuration, the method of preparation being stable, efficient, and yielding high yields.

[0032] Furthermore, the CN-LNA modified nucleic acid sequences obtained by the synthesis method of the present invention significantly reduce interference with intracellular protein distribution and decrease apoptosis induction effects compared to LNA modifications, offering a remarkable advantage in the efficient and safe development of nucleic acid drugs. [Brief explanation of the drawing]

[0033] To more clearly describe specific embodiments of the present invention or technical solutions in the prior art, the drawings that need to be used in describing specific embodiments or the prior art will be briefly described below. Clearly, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these without any creative effort.

[0034] [Figure 1] This is the mass spectrum of nucleic acid ON1. [Figure 2] This is the mass spectrum of the nucleic acid ON2. [Figure 3]This is the mass spectrum of the nucleic acid ON3. [Figure 4] This is a 60× figure showing the aggregation status of the paraspeckle protein P54nrb induced by ON1, ON2, and ON3. [Figure 5] This is the result of caspase 3 / 7 activity. [Figure 6] This is the mass spectrum of the nucleic acid ON4. [Figure 7] This describes the enzymatic stability of 5'-d(TTTTTTTTT)-3' against snake venom phosphodiesterase (SVPDE). [Figure 8] This is a crystal structure diagram of the S-6'-CN-LNA-T monomer (compound 36). [Modes for carrying out the invention]

[0035] The following describes in detail the embodiments and examples of the present invention, but those skilled in the art will understand that the following embodiments and examples are used solely to illustrate the present invention and should not be construed as limiting the scope of the invention. All other embodiments obtained by those skilled in the art without creative effort based on the embodiments of the present invention are within the scope of the invention. Unless otherwise specified, the conditions shall be those of the ordinary or manufacturer's recommended conditions. Unless otherwise specified, the reagents or equipment used are ordinary products obtained by purchasing commercially available products.

[0036] The term "nucleic acid polymer" can refer to any nucleic acid molecule, including but not limited to single-stranded and double-stranded DNA, RNA, and their hybrids. The number of nucleotides that polymerize to form a nucleic acid may be two, three or more, and may be oligonucleotides with 20 or fewer nucleotides, or polymers with 20 or more nucleotides.

[0037] In a first aspect, the present invention provides a 6'-cyanomodified rock nucleoside, which is selected from a compound having the structure represented by formula 1, a salt thereof, or an isomer thereof. In formula 1, JPEG0007898764000075.jpg3757, Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or salts thereof. Z is a cyano group, W1 and W2 are independently selected from H or a hydroxy protecting group, and the hydroxy protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl, 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzylsilyl, [(t Selected from, but not limited to, lysopropylsilyl)oxymethyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidine-4-yl, 9-phenylxanthin-9-yl, 9-(p-methoxyphenyl)xanthin-9-yl or 2-naphthylmethyl, The isomers include a 6'-cyano-modified loc nucleoside in which the Z group is in the R configuration, and the structural formula is JPEG0007898764000076.jpg3743, or the Z group contains a 6'-cyano-modified loc nucleoside in an S configuration, and the structural formula is The filename is JPEG0007898764000077.jpg3845.

[0038] The 6'-cyano-modified loc nucleoside provided by the present invention can improve the nuclease resistance of nucleic acid polymers prepared using it as a raw material and reduce the interaction between nucleic acid polymers and intracellular proteins.

[0039] In a second embodiment, the present invention provides a nucleotide comprising a 3'-active phosphorus group derivative of the 6'-cyano-modified rock nucleoside described above. The active phosphorus group is selected from phosphoramidite, a derivative of phosphoramidite, H-phosphonate, a derivative of H-phosphonate, a triester phosphate, or a derivative of a triester phosphate.

[0040] The nucleotides provided in this invention can improve the nuclease resistance of nucleic acid polymers prepared using them as monomers, and can reduce the interaction between nucleic acid polymers and intracellular proteins.

[0041] In some embodiments, the nucleotide is selected from compounds having the structure represented by formula 2, salts thereof, or isomers thereof. JPEG0007898764000078.jpg4775 The isomer contains a nucleotide in which the Z group is in the R configuration, and its structural formula is JPEG0007898764000079.jpg5352, or a nucleotide with a Z group in an S configuration, and the structural formula is The filename is JPEG0007898764000080.jpg5352.

[0042] In a third aspect, the present invention provides the use of the above-mentioned nucleotides in reducing the interaction between nucleic acid polymers and intracellular proteins.

[0043] According to the inventors' research, nucleic acid polymers prepared using the nucleotides provided by the present invention have minimal interaction with intracellular proteins and are found to be useful in reducing the in vivo toxicity of nucleic acid polymers.

[0044] In a fourth embodiment, the present invention provides a nucleic acid polymer having a monomer having a structure represented by formula 3, In formula 3, Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil or their salts. Z is a cyano group, W3 and W4 are each independently H, a hydroxy protecting group, or an internucleoside linking group that links the monomer to other parts of the nucleic acid polymer, and at least one of W3 and W4 is an internucleoside linking group that links the monomer to other parts of the nucleic acid polymer. The aforementioned hydroxy protecting groups include acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl, 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzylsilyl, [(triisopropylsilyl)oxy]methyl Selected from, but not limited to, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidine-4-yl, 9-phenylxanthin-9-yl, 9-(p-methoxyphenyl)xanthin-9-yl, or 2-naphthylmethyl.

[0045] The nucleic acid polymers provided by the present invention have better nuclease resistance than unmodified or otherwise modified nucleic acid polymers, and these nucleic acid polymers can reduce interactions with intracellular proteins, reduce their impact on the intracellular distribution of intracellular proteins, and reduce apoptosis.

[0046] In some embodiments, the monomer includes a monomer in which the Z group is in the R configuration, and the structural formula is JPEG0007898764000082.jpg3946, or the Z group contains a monomer in an S configuration, and the structural formula is The filename is JPEG0007898764000083.jpg3845.

[0047] In some embodiments, the nucleic acid polymer is ribonucleic acid, deoxyribonucleic acid, or a copolymer of ribonucleotide and deoxyribonucleotide.

[0048] In a fifth embodiment, the present invention provides the use of the nucleic acid polymer in the preparation of nucleic acid diagnostic agents or nucleic acid therapeutic agents.

[0049] The nucleic acid polymers provided by the present invention have superior nuclease resistance compared to unmodified or other modified nucleic acid polymers, exhibit low interaction with intracellular proteins, low apoptosis induction, and low toxicity and side effects, making them suitable for use as nucleic acid diagnostic agents or nucleic acid therapeutic agents.

[0050] In a sixth embodiment, the present invention provides a method for synthesizing cyanomodified nucleosides of the C6' moiety in R configuration or S configuration, wherein the cyanomodified nucleosides of the C6' moiety in R configuration are The file is JPEG0007898764000084.jpg3753, and its composition method is: The terminal olefin of JPEG0007898764000085.jpg3255 isomerized. JPEG0007898764000086.jpg3561 was obtained, and then the dihydroxylation reaction was carried out. JPEG0007898764000087.jpg3555 is obtained, and then an aldehyde group compound is obtained by an oxidative cleavage reaction. JPEG0007898764000088.jpg3152 is obtained, and finally, Convert the aldehyde group of JPEG0007898764000089.jpg3555 to a cyano group, and modify the cyano-modified nucleoside at the R-configured C6' site. This includes compositing JPEG0007898764000090.jpg3652, The cyano-modified nucleoside at the C6' site in the S configuration is The file is JPEG0007898764000091.jpg3653, and its composition method is: The terminal olefin of JPEG0007898764000092.jpg3357 is isomerized. JPEG0007898764000093.jpg3762 was obtained, and then the dihydroxylation reaction was carried out. JPEG0007898764000094.jpg4164 is obtained, and then an aldehyde group compound is obtained by an oxidative cleavage reaction. JPEG0007898764000095.jpg3554 is obtained, and finally, Convert the aldehyde group of JPEG0007898764000096.jpg3148 to a cyano group, and modify the cyano-modified nucleoside at the S-configured C6' site. This includes compositing JPEG0007898764000097.jpg3450, Here, Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or salts thereof.

[0051] In the present invention, the reaction methods for isomerization, dihydroxylation, oxidative cleavage, and conversion of aldehyde groups to cyano groups of terminal olefins are not specifically limited, and reaction methods well known to those skilled in the art may be employed. The cyano-modified nucleosides of the R-configuration or S-configuration C6' site synthesized in the present invention can be used as raw materials for the synthesis of four types of cyano-modified lock nucleic acids of the R-configuration or S-configuration.

[0052] Furthermore, the aldehyde group compound, which is an intermediate product of the above reaction, can be converted by conventional chemical methods to other groups including alkanes, alcohols, carboxylic acids, alkynyl groups, alkenyl groups, and amines, thereby enabling the creation of even more diverse C6'-modified located nucleic acids.

[0053] In some embodiments, the The composition method for JPEG0007898764000098.jpg3350 is S-arrangement. Mesylation, desubtraction, and acetylation reactions are performed sequentially on JPEG0007898764000099.jpg3351. JPEG0007898764000100.jpg3251 was obtained, and then glycosidication and nucleophilic substitution reactions were performed to obtain the R configuration. This includes compositing JPEG0007898764000101.jpg3350, The aforementioned The composition method for JPEG0007898764000102.jpg3756 is R-arrangement. Mesylation, desubtraction, and acetylation reactions are performed sequentially on JPEG0007898764000103.jpg3555. JPEG0007898764000104.jpg3555 was obtained, and then glycosidication and nucleophilic substitution reactions were carried out to obtain the S configuration. This includes compositing JPEG0007898764000105.jpg4264, The glycosidication reaction described above proceeds under activator conditions. The reaction involves reacting JPEG0007898764000106.jpg37142 with thymine, N6-benzoyladenine, or 6-chloroguanine at 50-100°C, where the activator comprises BSA and TMSOTf, and the reaction medium comprises acetonitrile, 1,2-dichloroethane, or toluene.

[0054] The synthesis method provided by the present invention is stable and practical, and does not specifically limit the mesylation reaction, desubtraction and acetylation reaction, or nucleophilic substitution reaction; any reaction method well known to those skilled in the art may be used.

[0055] In some embodiments, the S arrangement The synthesis method for JPEG0007898764000107.jpg4163 is 5-O-(tert-butyldiphenylsilyl)-4-C-hydroxymethyl-1,2-O-isopropylidene-3-O-(2-naphthylmethyl)-α-D-ribofuranose The primary alcohol of JPEG0007898764000108.jpg3551 is oxidized to an aldehyde group, and then an allylation reaction is performed on the aldehyde group to obtain the S configuration. This includes obtaining JPEG0007898764000109.jpg3350, Preferably, the R configuration The method for creating JPEG0007898764000110.jpg4164 is: The secondary alcohol in JPEG0007898764000111.jpg3452 is oxidized to a ketone, and then the ketone is reduced back to a secondary alcohol to obtain the R configuration. This includes obtaining JPEG0007898764000112.jpg3554, In the process of reducing ketones to secondary alcohols, the reducing agent used includes metallic hydrogen compounds such as lithium aluminum hydride, lithium borohydride, lithium chloride, or sodium borohydride; the reaction medium includes solvents such as dichloromethane, tetrahydrofuran, methanol, or ethanol; and the reaction temperature is -78 to 0°C. Preferably, the reaction to reduce the ketone to a secondary alcohol is carried out at -40 to 0°C using sodium borohydride and lithium chloride as reducing agents and tetrahydrofuran and methanol as reaction media.

[0056] In some preferred embodiments, the catalyst for terminal olefin isomerization comprises a transition metal catalyst such as a ruthenium catalyst, palladium catalyst, rhodium catalyst, or iridium catalyst, preferably a carbonylchlorohydride tris(triphenylphosphine)ruthenium(II), The reaction medium for isomerization of the terminal olefin comprises methanol, ethanol, n-butanol, or toluene, preferably ethanol. The reaction temperature for the terminal olefin isomerization is 60 to 100°C, preferably 60 to 80°C. The reaction time for the terminal olefin isomerization is 12 to 72 hours.

[0057] According to the inventors' research, it has been discovered that isomerization of terminal olefins can be stably achieved using 2.5 to 5% mol of carbonyl chlorohydride tris(triphenylphosphine)ruthenium(II) as a catalyst and ethanol as the reaction solvent, without the need for anhydrous and oxygen-free operation.

[0058] In a seventh aspect, the present invention provides a method for synthesizing a cyanoloc nucleic acid T phosphoramidite monomer of the R-configuration or S-configuration C6' site, the method for preparing a cyanoloc nucleic acid T phosphoramidite monomer of the R-configuration C6' site is provided, Deprotect the 3'-hydroxyl and 5'-hydroxyl groups of JPEG0007898764000113.jpg4163. JPEG0007898764000114.jpg3148 was obtained, the 5'-hydroxyl group was protected with DMTr, and the 3'-hydroxyl group was subjected to a phosphoramidite reaction to form the R configuration. This includes obtaining JPEG0007898764000115.jpg4847, A method for preparing a cyanolock nucleic acid T phosphoramidite monomer with an S configuration at the C6' site is as follows: Deprotect the 3'-hydroxyl and 5'-hydroxyl groups of JPEG0007898764000116.jpg3250. JPEG0007898764000117.jpg3045 was obtained, the 5'-hydroxyl group was protected with DMTr, and the 3'-hydroxyl group was subjected to a phosphoramidite reaction to form the S configuration. This includes obtaining JPEG0007898764000118.jpg3938.

[0059] This synthesis method is stable and efficient and can be used universally for the synthesis of cyano-modified T-phosphoramidite monomers with R or S configurations at the C6' site.

[0060] In an eighth aspect, the present invention provides a method for synthesizing a cyanolocic nucleic acid A phosphoramidite monomer with an R-configuration or S-configuration C6' site, wherein the method for synthesizing a cyanolocic nucleic acid A phosphoramidite monomer with an R-configuration C6' site is R-configuration Protect the base of JPEG0007898764000119.jpg3679 JPEG0007898764000120.jpg3170 is obtained, and then the 3'-hydroxyl group and 5'-hydroxyl group are deprotected. JPEG0007898764000121.jpg3373 is obtained, then the 5'-hydroxyl group is protected with DMTr, and the 3'-hydroxyl group Then the phosphoramidite reaction is carried out to form the R configuration This includes obtaining JPEG0007898764000122.jpg3656, The method for synthesizing the S-configured C6' site of the cyanolock nucleic acid A phosphoramidite monomer is as follows: Protect the base of JPEG0007898764000123.jpg3261 JPEG0007898764000124.jpg3177 is obtained, and then the 3'-hydroxyl group and 5'-hydroxyl group are deprotected. JPEG0007898764000125.jpg3376 is obtained, and then the 5'-hydroxyl group is protected with DMTr, and the 3'-hydroxyl group is subjected to a phosphoramidite reaction to form the S configuration. This includes obtaining JPEG0007898764000126.jpg4060.

[0061] This synthesis method is stable and efficient and can be used universally for the synthesis of cyanolocic nucleic acid A phosphoramidite monomers with R or S configurations at the C6' site.

[0062] In a ninth embodiment, the present invention provides a method for synthesizing a cyanoloc nucleic acid G phosphoramidite monomer of the R-configuration or S-configuration C6' site, the method for preparing a cyanoloc nucleic acid G phosphoramidite monomer of the R-configuration C6' site, the R-configuration Demethylate the methoxy group of JPEG0007898764000127.jpg3355 JPEG0007898764000128.jpg3456 is obtained, and then base protection is performed. JPEG0007898764000129.jpg3357 is obtained, and then the 3'-hydroxyl group and 5'-hydroxyl group are deprotected. JPEG0007898764000130.jpg3160 was obtained, the 5'-hydroxyl group was protected with DMTr, and the 3'-hydroxyl group was subjected to a phosphoramidite reaction to form the R configuration. This includes obtaining JPEG0007898764000131.jpg4354, A method for preparing a cyanolock nucleic acid G phosphoramidite monomer with the S configuration at the C6' site is as follows: Demethylate the methoxy group of JPEG0007898764000132.jpg3469 JPEG0007898764000133.jpg3465 is obtained, and then base protection is performed. JPEG0007898764000134.jpg3366 is obtained, and then the 3'-hydroxyl group and 5'-hydroxyl group are deprotected. JPEG0007898764000135.jpg3474 was obtained, the 5'-hydroxyl group was protected with DMTr, and the 3'-hydroxyl group was subjected to a phosphoramidite reaction to form the S configuration. This includes obtaining JPEG0007898764000136.jpg3852.

[0063] This synthesis method is stable and efficient and can be used universally for the synthesis of cyanolocic nucleic acid G phosphoramidite monomers with R or S configurations at the C6' site.

[0064] In a tenth embodiment, the present invention provides a method for synthesizing a cyanoloc nucleic acid C phosphoramidite monomer with an R-configuration or S-configuration C6' site, the method for preparing a cyanoloc nucleic acid C phosphoramidite monomer with an R-configuration C6' site is provided, Deprotect the 3'-hydroxyl and 5'-hydroxyl groups of JPEG0007898764000137.jpg3755. JPEG0007898764000138.jpg3857 is obtained, and then the 5'-hydroxyl group is protected with DMTr. JPEG0007898764000139.jpg4156 is obtained, then the 3'-hydroxyl group is modified by silane, and the carbonyl group is converted to an amino group. JPEG0007898764000140.jpg3453 is obtained, then base protection is performed, and the 3'-hydroxyl group is deprotected. JPEG0007898764000141.jpg3456 is obtained, and finally, the 3'-hydroxyl group is subjected to a phosphoramidite reaction to form the R configuration. This includes obtaining JPEG0007898764000142.jpg4347, A method for preparing a cyanolock nucleic acid C phosphoramidite monomer with an S configuration at the C6' site is as follows: Deprotect the 3'-hydroxyl and 5'-hydroxyl groups of JPEG0007898764000143.jpg3658. JPEG0007898764000144.jpg4061 is obtained, and then the 5'-hydroxyl group is protected with DMTr. JPEG0007898764000145.jpg3555 is obtained, then the 3'-hydroxyl group is modified by silaneization and the carbonyl group is converted to an amino group. JPEG0007898764000146.jpg3254 is obtained, then base protection is performed, and the 3'-hydroxyl group is deprotected. JPEG0007898764000147.jpg3463 is obtained, and finally, the 3'-hydroxyl group is subjected to a phosphoramidite reaction to form the S configuration. This includes obtaining JPEG0007898764000148.jpg4255.

[0065] This synthesis method is stable and efficient and can be used universally for the synthesis of cyanolocic nucleic acid C phosphoramidite monomers with R or S configurations at the C6' site.

[0066] In an eleventh embodiment, the present invention provides a method for synthesizing nucleic acid polymers, comprising carrying out a polymerization reaction with a monomer to prepare a nucleic acid polymer. The monomer comprises the 6'-cyano-modified rock nucleoside or the nucleotide.

[0067] In some preferred embodiments, the polymerization reaction includes: (1) Chemical synthesis: Nucleic acids are synthesized using nucleosides or mononucleotides as raw materials by organic chemical methods including phosphate diestering, phosphate triestering, phosphite triestering, and solid-phase synthesis. (2) Enzymatic synthesis: Nucleic acid monomers or small chemically synthesized fragments can be linked to larger fragments by enzymatic reactions.

[0068] The present invention will be further described below with reference to specific examples, but it should be understood that these examples are merely for the purpose of illustrating the present invention in more detail and should not be interpreted as not limiting the present invention in any way.

[0069] Example 1: Stereoselective synthesis of cyanoloc nucleic acids

[0070] Part 1: Synthesis of R-6'-CN-LNA

[0071] Synthesis of R-6'-allyl-LNA JPEG0007898764000149.jpg34165 Reaction conditions: (a) (1) 2-iodoxybenzoic acid, acetonitrile, reflux, 5h; (2) allyltrimethylsilane, boron trifluoride ethyl ether solution, dichloromethane, -40℃, 4h; (b) methanesulfonyl chloride, DMAP, pyridine, room temperature, 16h; (c) (1) FeCl3·6H2O, dichloromethane, 0℃~room temperature, 9h; (2) Ac2O, DMAP, pyridine, dichloromethane, room temperature, 2h.

[0072] 2 Compound 1 (100.0 g, 0.167 mol) was dissolved in 500 mL of acetonitrile, 2-iodoxybenzoic acid (56 g, 0.20 mol) was added, and the mixture was heated to reflux and reacted for 5 hours. Completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). The reaction was stopped, and after the reaction cooled to room temperature, the mixture was filtered through diatomaceous earth, the filter cake was washed twice with acetonitrile, the solvent was spin-dried, and the mixture was vacuum-dried to obtain 101 g of a pale yellow liquid. Purification was not necessary, and the next reaction was carried out directly. The above aldehyde compound was dissolved in 500 mL of dichloromethane, cooled to -40°C, and a solution of boron trifluoride ethyl ether (22 mL, 0.217 mol) was added. The mixture was stirred for 5 minutes to allow the reaction to proceed. Next, allyltrimethylsilane (42.7 mL, 0.217 mol) was added dropwise, the temperature was maintained, and the mixture was stirred for 4 hours to allow the reaction to proceed. The completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). The reaction mixture was rapidly poured into 1 L of saturated sodium bicarbonate aqueous solution, quenched, extracted with dichloromethane, washed with saturated brine, dried over anhydrous NaSO4, filtered, and concentrated under reduced pressure to obtain 107 g of a pale yellow viscous substance in 100% yield. The following reaction was carried out directly without purification. 1H NMR(CDCl3,400MHz)δ7.86-7.78(m,4H),7.60-7.57(m,2H),7.53-7.46(m,5H),7.41-7.27(m,6H),5.87-5.80(m,2H),5.05-4.97(m, 3H),4.76(t,J=5.2Hz,1H),4.70-4.66(m,2H),4.47(dd,J=10.7,1.7Hz,1H),3.96(d,J=11.2Hz,1H),3.80(d,J=11.2Hz,1H),3.44(br s,1H),2.54-2.49(m,1H),1.93-1.85(m,1H),1.63(s,3H),1.39(s,3H),0.90(s,9H); 13 C NMR(CDCl3,100MHz)δ136.41,135.58,135.50,134.29,133.30,133.22,133. 13,132.99,129.76,129.62,128.76,127.99,127.78,127.75,127.70,127.31 ,126.38,126.32,125.71,116.26,113.78,104.70,88.22,79.18,78.11,73.0 9,72.56,62.46,34.65,27.09,26.73,26.54,19.15;ESI-MS(m / z)637.37[MH] - .

[0073] 3 At room temperature, compound 2 (100.0 g, 0.157 mol) and DMAP (1.9 g, 15.7 mmol) were dissolved in 500 mL of pyridine, and methanesulfonyl chloride (18 mL, 0.235 mol) was added dropwise. The reaction was allowed to proceed at room temperature for 16 hours, and completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). 100 mL of methanol was added to the reaction mixture, and the reaction was quenched by stirring for 10 minutes. The reaction mixture was then concentrated under reduced pressure to remove excess pyridine, diluted with ethyl acetate, sequentially washed with water, 1N HCl solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by flash column (gradient elution: petroleum ether / ethyl acetate = 0-25%) to obtain 108.0 g of colorless viscous substance 3 in 96% yield. 1H NMR(DMSO-d6,400MHz)δ7.95-7.85(m,4H),7.56-7.36(m,13H),5.80-5.69(m,2H),5.40(dd,J=7.6,3.2Hz,1H),5.03-4.93(m,4H),4.69(d,J=12.4Hz,1H),4.40(d,J=5.3Hz,1H),3.75(d,J=11.0Hz,1H),3.60(d,J=11.0Hz,1H),2.97(s,3H),2.85-2.81(m,1H),2.58-2.52(m,1H),1.53(s,1H),1.33(s,1H),0.78(s,9H); 13 C NMR(CDCl3,100MHz)δ135.59,135.52,135.29,134.59,133.25,133.19,132.88,132.84,130.39,130.36,128.59,128.34,128.29,128.24,128.09,127.33,126.70,126.55,126.34,118.60,113.27,105.00,87.47,81.43,78.15,76.97,71.93,64.30,36.19,26.83,26.57,26.33,19.04;ESI-MS(m / z)734.39[M+NH4] + ,739.34[M+Na] + .

[0074] 4 A 600 mL dichloromethane solution of compound 3 (108 g, 0.15 mol) was cooled to 0 °C, iron(III) chloride hexahydrate (16.3 g, 0.06 mol) was added, and the mixture was reacted at room temperature for 9 h. The completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 4 / 1). The reaction solution was poured into 1.5 L of water and extracted with dichloromethane. The organic layer was washed successively with water, saturated sodium bicarbonate solution, and saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and dried in vacuo. The obtained viscous substance was dissolved in 500 mL of dichloromethane, pyridine (72 mL, 0.9 mol) and DMAP (2.75 g, 22.5 mol) were added successively, and then acetic anhydride (85 mL, 0.9 mol) was added dropwise. The mixture was reacted at room temperature for 2 h, and the completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 4 / 1). The reaction solution was poured into 2 L of water and extracted with dichloromethane. The organic layer was washed successively with water, 1 N HCl solution, saturated sodium bicarbonate solution, and saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: petroleum ether / ethyl acetate = 0 - 60%) to obtain 68 g of an epimer mixture of diacetyl compound 4 as a colorless viscous substance in a yield of 60%. ESI-MS (m / z) 778.39 [M+NH4] + ,783.44 [M+Na] + . JPEG0007898764000150.jpg103170Reaction conditions: (a) thymine, N,O-bis(trimethylsilyl)acetamide (BSA), TMSOTf, acetonitrile, 80 °C, 3 h; (b) K2CO3, methanol, room temperature, 16 h; (c) N6-benzoyladenine, BSA, TMSOTf, toluene, 100 °C, 3 h; (d) 6-chloroguanine, BSA, TMSOTf, toluene, 100 °C, 3 h.

[0075] 5 To 50 mL of acetonitrile suspension of thymine (7.6 g, 60 mmol), BSA (29 mL, 120 mmol) was added and the mixture was stirred at room temperature for 20 minutes until the thymine dissolved. After the system became clear, 200 mL of acetonitrile solution of diacetylated sugar 4 (23 g, 30 mmol) was added, followed by rapid dropwise addition of TMSOTf (8.3 mL, 45 mmol). After the addition was complete, the temperature was raised to 80°C and the mixture was stirred for 3 hours to allow the reaction to proceed. Completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). After stopping the heating and allowing the reaction mixture to cool to room temperature, the reaction mixture was poured into ethyl acetate, a semi-saturated NaHCO3 solution (400 mL) was added, and the mixture was stirred for 20 minutes. The insoluble solid was filtered through diatomaceous earth to separate the organic layer, which was then washed sequentially with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and vacuum-dried. The resulting residue was dissolved in 250 mL of methanol, K2CO3 (12.4 g, 90 mmol) was added, and the mixture was allowed to react at room temperature for 16 hours. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 3). After concentrating the reaction mixture, the residue was extracted with ethyl acetate, washed sequentially with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-40%) to obtain 58 g of the loc nucleic acid derivative as a white foamy solid in 39% yield. 1 H NMR(CDCl3,400MHz)δ8.72(br s,1H),7.82-7.74(m,3H),7.72(s,1H),7.68-7.63(m,4H),7.59(s,1H),7.75-7.28(m ,9H),5.80-5.69(m,1H),5.66(s,1H),5.11-5.00(m,2H),4.87(d,J=11.4Hz,1H),4.7 4-4.71(d,J=11.4Hz,1H),4.57(s,1H),4.24-4.20(m,2H),4.03(d,J=12.0Hz,1H),3. 91(d,J=12.0Hz,1H),2.49-2.42(m,1H),2.26-2.22(m,1H),1.56(s,3H),1.09(s,9H); 13C NMR(CDCl3,100MHz)δ163.60,149.72,135.44,135.27,134.23,134.16,133.6 4,133.11,132.70,132.25,130.10,130.06,128.42,127.98,127.85,127.73, 126.88,126.39,126.25,125.74,117.88,110.42,89.20,86.82,79.97,77.05 ,76.47,72.53,58.80,33.76,26.97,19.44,12.04;ESI-MS(m / z)689.41[M+H] + ,711.38[M+Na] + .

[0076] 6 To 50 mL of a toluene suspension of N6-benzoyladenine (14 g, 60 mmol), BSA (29 mL, 120 mmol) was added and heated to 60°C until the N6-benzoyladenine dissolved. After the system became clear, the reaction mixture was cooled to room temperature, and 170 mL of a toluene solution of diacetylated sugar 4 (23 g, 30 mmol) was added. Subsequently, TMSOTf (8.3 mL, 45 mmol) was rapidly added dropwise. After the addition was complete, the temperature was raised to 100°C and the mixture was stirred for 3 hours. The completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). After stopping the heating and allowing the reaction mixture to cool to room temperature, the reaction mixture was poured into ethyl acetate, a semi-saturated NaHCO3 solution (400 mL) was added, and the mixture was stirred for 20 minutes. The insoluble solid was filtered through diatomaceous earth to separate the organic layer, which was then washed sequentially with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and vacuum-dried. The resulting residue was dissolved in 250 mL of methanol, K2CO3 (20.7 g, 150 mmol) was added, and the mixture was allowed to react at room temperature for 16 hours. Completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 3). After concentrating the reaction mixture, the residue was extracted with ethyl acetate, washed sequentially with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-100%) to obtain 7.4 g of LOCK nucleic acid derivative 6 as a white powder in 35% yield. 1H NMR(CDCl3,400MHz)δ8.28(s,1H),8.15(s,1H),7.80-7.64(m,8H),7.47-7.28(m,9H),6.06(s,1H),5.93-5.91(m,2H),5.82-5.75(m,1H),5.13(d,J=17.1Hz,1H),5.04(d,J=10.2Hz,1H),4.81-4.72(m,3H),4.52(s,1H),4.32-4.29(m,1H),4.02(d,J=12.0Hz,1H),3.92(d,J=12.0Hz,1H),2.55-2.49(m,1H),2.34-2.27(m,1H),1.07(s,9H); 13 C NMR(CDCl3,100MHz)δ155.46,153.16,148.82,135.59,135.49,130.02,129.99,128.36,127.96,127.92,127.83,127.70,126.78,126.29,120.09,117.83,88.57,86.16,80.45,78.74,76.64,72.75,58.82,33.90,26.79,19.22;ESI-MS(m / z)698.36[M+H] + ,720.35[M+Na] + .

[0077] 7 50 mL of a toluene suspension of 6-chloroguanine (10.2 g, 60 mmol) was mixed with BSA (29 mL, 120 mmol) and heated to 60°C until the 6-chloroguanine dissolved. After the system became clear, the reaction mixture was cooled to room temperature, and 170 mL of a toluene solution of diacetylated sugar 4 (23 g, 30 mmol) was added. Subsequently, TMSOTf (8.3 mL, 45 mmol) was rapidly added dropwise. After the addition was complete, the temperature was raised to 100°C and the mixture was stirred for 3 hours. The completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). After stopping the heating and allowing the reaction mixture to cool to room temperature, the reaction mixture was poured into ethyl acetate, a semi-saturated NaHCO3 solution (400 mL) was added, and the mixture was stirred for 20 minutes. The insoluble solid was filtered through diatomaceous earth to separate the organic layer, which was then washed sequentially with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and vacuum-dried. The resulting residue was dissolved in 250 mL of methanol, K2CO3 (20.7 g, 150 mmol) was added, and the mixture was allowed to react at room temperature for 16 hours. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 3). After concentrating the reaction mixture, the residue was extracted with ethyl acetate, washed sequentially with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-30%) to obtain 12.2 g of LOCK nucleic acid derivative 7 as a white foamy solid in 56% yield. 1 H NMR(CDCl3,400MHz)δ7.98(s,1H),7.82-7.67(m,8H),7.49-7.36(m,7H),7. 31-7.27(m,2H),5.92(s,1H),5.84-5.74(m,1H),5.14-5.01(m,2H),4.95(br s,2H),4.79(dd,J=17.7,11.5Hz,2H),4.58(s,1H),4.53(s,1H),4.31(dd,J=8.6,5.0Hz,1H),4.08(s,3 H),4.00(d,J=12.0Hz,1H),3.90(d,J=12.0Hz,1H),2.56-2.48(m,1H),2.34-2.27(m,1H),1.07(s,9H); 13C NMR(CDCl3,100MHz)δ161.49,159.36,142.45,136.44,135.62,134.47,133 .94,133.14,133.08,132.53,132.47,129.97,128.34,127.94,127.90,127. 69,126.67,126.25,126.12,125.65,117.75,116.09,88.31,85.93,80.46, 78.93,72.80,58.86,53.91,33.93,26.78,19.17;ESI-MS(m / z)728.39[M+H] + . Synthesis of R-6'-CN-LNA JPEG0007898764000151.jpg97165 Reaction conditions: (a) (1) Carbonyl chlorohydride tris(triphenylphosphine)ruthenium(II) (Wilkinson's catalyst), EtOH, 80℃, 48h; (b) Potassium osmate dihydrate, 50% NMO, t-BuOH, H2O, 60℃, 8h; (c) NaIO4, EtOH-THF(2 / 1 v / v), H2O, room temperature, 12h; (d) NH4OH, I2, room temperature, 24h.

[0078] 8 To 50 mL of an anhydrous ethanol solution of allyl nucleoside derivative 5 (5.0 g, 7.26 mmol), carbonyl chlorohydride tris(triphenylphosphine)ruthenium(II) (345 mg, 0.36 mmol, 5% mol) was added, and the mixture was heated to 80°C and reacted for 48 hours. After directly concentrating the reaction mixture, the residue was dissolved in 40 mL of tetrahydrofuran, and 5 mL of tert-butanol, 5 mL of water, potassium osmate dihydrate (26.7 mg, 72.6 μmol, 1% mol), and 50% N-methylmorpholine-N-oxide (2.3 mL, 10.9 mmol) were added sequentially. The mixture was heated to 60°C and reacted for 8 hours, and completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0-80%) to obtain 5 g of a mixed product of dihydroxylated isomers. The above dihydroxylation product was dissolved in 60 mL of ethanol-tetrahydrofuran solution, and 10 mL of aqueous solution of NaIO4 (2.33 g, 10.9 mmol) was added dropwise, and the mixture was stirred at room temperature for 12 hours. After filtering out the insoluble solid, 25% aqueous ammonia (5.5 mL, 72.6 mmol) was added to the filtrate, followed by the addition of I2 (1.84 g, 7.26 mmol) in several portions, and the reaction was allowed to proceed at room temperature for 24 hours. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0-60%) to obtain 3.3 g of a slightly grayish foamy solid product in 48% yield. 1H NMR(CDCl3,400MHz)δ8.99,7.85-7.76(m,3H),7.68-7.64(m,5H),7.52-7.42(m,5H),7.38-7.33(m,5H),5.84(s,1H),4.87(s,1H),4.83(s,1H),4.82(d,J=6.6Hz,1H),4.71(d,J=6.6Hz,1H),4.24(s,1H),4.17(d,J=6.9Hz,1H),4.01(d,J=6.9Hz,1H),1.63(s,3H),1.10(s,9H); 13 C NMR(CDCl3,100MHz)δ163.50,149.61,135.53,135.32,133.63,133.25,133.21,133.05,132.10,131.80,130.36,130.32,128.68,128.15,128.10,127.86,127.80,127.15,126.63,126.56,125.58,115.47,111.18,89.24,87.30,78.06,76.26,72.81,70.55,58.15,26.86,19.43,12.24;ESI-MS(m / z)674.28[M+H] + ,796.25[M+Na] + .

[0079] 9 To 70 mL of an anhydrous ethanol solution of allyl nucleoside derivative 6 (7.0 g, 10 mmol), carbonyl chlorohydride tris(triphenylphosphine)ruthenium(II) (475 mg, 0.5 mmol, 5% mol) was added, and the mixture was heated to 80°C and reacted for 48 hours. After directly concentrating the reaction mixture, the residue was dissolved in 50 mL of tetrahydrofuran, and 10 mL of tert-butanol, 10 mL of water, potassium osmate dihydrate (37 mg, 0.1 mmol, 1% mol), and 50% N-methylmorpholine-N-oxide (3.13 mL, 15 mmol) were added sequentially. The mixture was heated to 60°C and reacted for 8 hours, and completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column elution (gradient elution: ethyl acetate / dichloromethane = 0-10%) to obtain 7.5 g of a mixed product of dihydroxylated isomers. The above dihydroxylation product was dissolved in 60 mL of ethanol-tetrahydrofuran solution, and 15 mL of aqueous solution of NaIO4 (3.2 g, 10.9 mmol) was added dropwise, and the mixture was stirred at room temperature for 12 hours. After filtering out the insoluble solid, 25% aqueous ammonia (7.6 mL, 100 mmol) was added to the filtrate, followed by the addition of I2 (2.54 g, 10 mmol) in several portions, and the reaction was allowed to proceed at room temperature for 24 hours. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by flash column to obtain 1.2 g of a slightly yellowish foamy solid product with ethyl acetate / dichloromethane = 0-40% as the eluent, in 26% yield. 1H NMR(DMSO-d6,400MHz)δ10.71(s,1H),7.92-7.81(m,5H),7.66-7.61(m,4H),7.54-7.36(m,9H),6.66(br s,2H),6.04(s,1H),5.30(s,1H),5.13(s,1H),4.92(d,J=12.0Hz,1H),4.84(d,J=12.0Hz,1H),4.79(s,1H),4.21(d,J=12.2Hz,1H),4.00(d,J=12.2Hz,1H),0.94(s,9H); 13 C NMR(DMSO-d6,100MHz)δ157.14,154.49,151.10,135.62,135.28,134.48,132.15,130.53,128.44,128.15,128.07,128.76,126.64,126.57,126.17,117.74,116.98,88.23,84.86,78.84,78.44,71.98,71.22,59.76,26.77,19.19;ESI-MS(m / z)683.30[M+H] + ,705.27[M+Na] + .

[0080] 10 To 120 mL of an anhydrous ethanol solution of allyl nucleoside derivative 7 (11.0 g, 15.1 mmol), carbonyl chlorohydride tris(triphenylphosphine)ruthenium(II) (718 mg, 0.76 mmol, 5% mol) was added, and the mixture was heated to 80°C and reacted for 48 hours. After directly concentrating the reaction mixture, the residue was dissolved in 80 mL of tetrahydrofuran, and 10 mL of tert-butanol, 10 mL of water, potassium osmate dihydrate (56 mg, 0.15 mmol, 1% mol), and 50% N-methylmorpholine-N-oxide (4.7 mL, 22.7 mmol) were added sequentially. The mixture was heated to 60°C and reacted for 8 hours, and completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-60%) to obtain 8 g of a mixed product of dihydroxylated isomers. The above dihydroxylation product was dissolved in 70 mL of ethanol-tetrahydrofuran solution, and 15 mL of aqueous solution of NaIO4 (3.4 g, 10.9 mmol) was added dropwise. The mixture was stirred at room temperature for 12 hours. After filtering out the insoluble solid, 25% aqueous ammonia (7.9 mL, 105 mmol) was added to the filtrate, and then I2 (2.67 g, 10.5 mmol) was added in several batches. The reaction was allowed to proceed at room temperature for 24 hours. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-40%) to obtain 10⁴ g of a slightly yellowish foamy solid product in 37% yield. 1 H NMR(CDCl3,400MHz)δ7.82-7.76(m,3H),7.71-7.16(m,6H),7.51-7.30(m,9H),6.10(s,1H),4.95(s,1H),4.92(s,1H),4.77(br s,2H),4.73(d,J=2.7Hz,2H),4.50(s,1H),4.14(d,J=12.0Hz,1H),4.07(s,3H),4.02(d,J=12.0Hz,1H),1.10(s,9H);13 C NMR(CDCl3,100MHz)δ161.60,159.29,152.25,136.27,135.67,135.54,133.6 0,133.16,133.05,132.09,132.07,130.16,128.60,128.03,127.97,127.86, 127.73,126.88,126.49,126.42,125.43,116.04,115.82,88.54,86.26,78.1 9,77.97,72.96,71.05,58.73,54.02,26.69,19.20;ESI-MS(m / z)713.40[M+H] + Synthesis of R-6'-CN-LNA phosphoramidite monomer Synthesis of R-6'-CN-LNA-T phosphoramidite monomer JPEG0007898764000152.jpg89161 Reaction conditions: (a) (1) DDQ, dichloromethane-H2O(20 / 1), room temperature, 24h; (2) Et3N, Et3N·3HF, THF, room temperature, 9h; (b) DMTrCl, pyridine, room temperature, 12h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite, 1H-tetrazole, room temperature, 5h.

[0081] 11 At room temperature, DDQ (420 mg, 1.86 mmol) was added to 10.5 mL of a dichloromethane-water (20:1) mixture of nucleoside derivative 8 (500 mg, 0.74 mmol), and the reaction was allowed to proceed at room temperature for 24 hours. Completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After concentrating the reaction mixture under reduced pressure, it was extracted with ethyl acetate, washed sequentially with saturated sodium sulfite solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was dissolved in 10 mL of tetrahydrofuran, and triethylamine (260 μL, 1.86 mmol) and triethylamine hydrofluoric acid (363 μL, 2.23 mmol) were added sequentially. The reaction was allowed to proceed at room temperature for 9 hours, and completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). 600 mg of sodium bicarbonate solid was added to the reaction mixture and stirred until no more bubbles were generated. The reaction mixture was then concentrated under reduced pressure and directly purified using a flash column (gradient elution: methanol / dichloromethane = 0-20%) to obtain 11,310 mg of colorless amorphous solid in 46% yield. 1 H NMR(CD3OD,400MHz)δ7.70(s,1H),5.69(s,1H),4.93(s,1H),4.46(s,1H),4.27(s,1H),3.96(s,2H),1.89(s,3H); 13 C NMR(CD3OD,100MHz)δ164.99,150.36,134.88,116.13,109.72,89.51,86.69,80.73,69.97,69.38,55.23,11.26;ESI-MS(m / z)296.13[M+H] + .

[0082] 12 50 mL of a pyridine solution of cyanoloc nucleoside derivative 11 (8.0 g, 27.1 mmol) was mixed with 4,4'-dimethoxytrityl chloride (13.8 g, 40.6 mmol) and stirred at room temperature for 12 hours. The reaction was detected by TLC to confirm completion (dichloromethane / methanol = 10 / 1). The reaction was quenched with 50 mL of methanol, concentrated under reduced pressure, the residue was diluted with ethyl acetate, washed sequentially with water and saturated saline, dried over Na2SO4, concentrated, and purified by flash column (gradient elution: methanol / dichloromethane = 0-10%) to obtain 13.53 g of 5'-O-DMTr protected nucleoside 12 as a pale yellow powder in 84% yield. 1 H NMR(400MHz,DMSO-d6)δ11.48(s,1H),7.47-6.91(m,14H),6.27(d,J=4.0Hz,1H),5.63(s,1H),5.01(s,1H),4.52( s,1H),3.42(d,J=4.4Hz,1H),3.74(s,6H),3.65(d,J=11.2Hz,1H),3.36(d,J=11.2Hz,1H),1.60(d,J=0.8Hz,3H); 13 C NMR(100MHz,DMSO-d6)δ163.81,158.27,149.91,144.50,135.07,134.84,134.00,129.91,129.84,128.00,127.71,126. 94,117.41,113.34,109.01,87.99,86.38,86.35,80.37,70.52,70.33,58.47,55.09,12.35;ESI-MS(m / z)620.20[M+Na] + .

[0083] 13 40 mL of a dichloromethane solution of nucleoside 12 (4.0 g, 6.7 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (3.0 g, 10.0 mmol) was mixed with 1H-tetrazole (376 mg, 5.36 mmol) and reacted at room temperature for 5 hours. The completion of the reaction was detected by TLC (ethyl acetate / dichloromethane = 5 / 1). A saturated NaHCO3 solution was added to the reaction mixture, extracted with dichloromethane, washed with saturated saline solution, dried over MgSO4, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-20%) to obtain 4.4 g of phosphoramidite 13 as a white foamy solid in 82% yield. 31 P NMR(152MHz,DMSO-d6)δ148.80,148.75;ESI-MS(m / z)798.32[M+H] + . Synthesis of R-6'-CN-LNA-A phosphoramidite monomer JPEG0007898764000153.jpg67165 Reaction conditions: (a) BzCl, pyridine, room temperature, 6h; (b) (1) DDQ, dichloromethane-H2O(20 / 1), room temperature, 24h; (2) Et3N, Et3N·3HF, THF, room temperature, 12h; (b) DMTrCl, pyridine, room temperature, 12h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite, 1H-tetrazole, room temperature, 8h.

[0084] 14 At room temperature, 20 mL of pyridine solution of nucleoside 9 (3.0 g, 4.4 mmol) was mixed with benzoyl chloride (1.52 mL, 13.2 mmol) dropwise, and the reaction was allowed to proceed at room temperature for 6 hours. Completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 1). 15% NaOH solution was added dropwise to the reaction mixture to adjust the pH to 8-9, and the reaction was allowed to proceed at room temperature for 4 hours. The reaction mixture was diluted with ethyl acetate, washed sequentially with water, 1N HCl solution, and saturated saline solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-60%) to obtain 1.45 g of benzoyl-protected nucleoside 14 as a pale yellow solid in 42% yield. 1H NMR(CDCl3,400MHz)δ8.62(s,1H),8.20(s,1H),8.07(d,J=7.0Hz,2H),7.82-7.75(m,2H),7.69-7.32(m,18H),6.25(s,1H),5.11(s,1H),4.98(s,1H),4.77(s,2H),4.51(d,J=5.8Hz,1H),4.16(d,J=12.5Hz,1H),4.04(d,J=12.5Hz,1H),1.07(s,9H); 13 C NMR(CDCl3,100MHz)δ164.60,152.64,150.42,149.70,140.19,135.55,133.53,133.42,133.09,132.99,132.12,132.09,130.15,128.94,128.52,128.00,127.92,127.91,127.85,127.69,126.80,126.38,126.28,125.42,123.29,114.42,90.04,86.43,78.19,77.54,72.97,70.17,59.42,29.70,26.70,19.26;ESI-MS(m / z)787.32[M+H] + .

[0085] 15 At room temperature, DDQ (1.03 g, 3.81 mmol) was added to 21 mL of a dichloromethane-water (20:1) mixture of nucleoside derivative 14 (1.2 g, 1.52 mmol), and the reaction was allowed to proceed at room temperature for 24 hours. Completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After concentrating the reaction mixture under reduced pressure, it was extracted with ethyl acetate, washed sequentially with saturated sodium sulfite solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was dissolved in 15 mL of tetrahydrofuran, and triethylamine (530 μL, 3.81 mmol) and triethylamine hydrofluoric acid (743 μL, 4.56 mmol) were added sequentially. The reaction was allowed to proceed at room temperature for 12 hours, and completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). 1.15 g of sodium bicarbonate solid was added to the reaction mixture, and the mixture was stirred until no more bubbles were generated. The reaction mixture was then concentrated under reduced pressure and purified directly using a flash column (gradient elution: dichloromethane / methanol = 0-15%) to obtain 400 mg of cyanoloc nucleoside 15 in yield of 64%. 1 H NMR(CD3OD,400MHz)δ8.72(s,1H),8.55(s,1H),8.07(d,J=4.2Hz,2H),7.66(t,J=4.2Hz,1H ),7.57(t,J=4.2Hz,2H),6.30(s,1H),5.05(s,1H),4.88(s,1H),4.64(s,1H),4.02(s,2H); 13 C NMR(CD3OD,100MHz)δ166.76,152.06,151.09,149.81,141.56,132.46,132.57,128.38,12 8.06,123.91,116.25,89.47,86.11,80.84,70.77,70.43,55.86;ESI-MS(m / z)409.15[M+H] + .

[0086] 16 At room temperature, 5 mL of a pyridine solution of cyanoloc nucleoside 15 (300 mg, 0.75 mmol) was mixed with 4,4'-dimethoxytrityl chloride (481 mg, 1.42 mmol), stirred at room temperature for 12 hours, and the completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). The reaction was quenched with 1 mL of methanol, concentrated under reduced pressure, the residue was diluted with ethyl acetate, washed sequentially with water and saturated saline, dried over Na2SO4, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0-45%) to obtain 500 mg of 5'-O-DMTr-protected cyanoloc nucleoside 16 as a pale yellow solid in 94% yield. 1 H NMR(DMSO-d6,400MHz)δ12.26(s,1H),8.79(s,1H),8.59(s,1H),8.06(d,J=8.4Hz,2H ),7.67(t,J=7.4Hz,1H),7.58(t,J=7.8Hz,2H),7.46(d,J=7.4Hz,1H),7.33-7.20(m,7 H),6.89(d,J=8.4Hz,4H),6.33(s,1H),6.30(d,J=4.3Hz,1H),5.12(s,1H),4.95(s,1H) ),4.83(d,J=4.2Hz,1H),3.73(s,6H),3.68(d,J=11.2Hz,1H),3.40(d,J=11.2Hz,1H); 13 C NMR(DMSO-d6,100MHz)δ166.09,158.65,152.31,152.02,151.00,144.95,142.63,135.66,135.42,133.77,132.98,130.34,130.28,128.98, 128.97,128.33,128.96,127.26,126.02,118.26,113.71,88.34,86.8 3,85.69,80.94,72.59,71.24,60.23,55.51;ESI-MS(m / z)711.36[M+H] + .

[0087] 17 At room temperature, 1H-tetrazole (35 mg, 0.51 mmol) was added to 10 mL of a dichloromethane solution of nucleoside 16 (450 mg, 0.63 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (382 mg, 1.27 mmol), and the reaction was allowed to proceed at room temperature for 8 hours. Completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 5 / 2). The reaction mixture was directly purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-40%) to obtain 480 mg of phosphoroamidite 17 as a slightly yellowish foamy solid in 83% yield. 31 P NMR(152MHz,DMSO-d6)δ149.19,149.04;ESI-MS(m / z)909.38[MH] - . Synthesis of R-6'-CN-LNA-G phosphoramidite monomer JPEG0007898764000154.jpg74154 Reaction conditions: (a) 2N HCl, THF-CH3OH (1 / 1), 60℃, 12h; (b) Isobutyryl chloride, Et3N, DMAP, 1,4-dioxane, 100℃, 24h; (c) (1) DDQ, dichloromethane-H2O (20 / 1), room temperature, 24h; (2) Et3N, Et3N·3HF, THF, room temperature, 12h; (d) DMTrCl, pyridine, room temperature, 12h; (e) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite, 1H-tetrazole, room temperature, 12h.

[0088] 18 To 30 mL of a tetrahydrofuran-methanol solution of cyanoloc nucleoside 10 (2.5 g, 3.5 mmol), 17.5 mL of 2N hydrochloric acid solution was added (1 / 1 v / v), and the mixture was heated to 60°C and reacted for 12 hours. A white solid precipitated, and the completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 2 / 1). The insoluble white solid was filtered, the filtrate was extracted with ethyl acetate, washed with water and saturated saline solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the filtrates were combined and purified by flash column (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0-60%) to obtain 1.4 g of 6-position demethylated nucleoside 18 as a white solid in 57% yield. 1 H NMR(DMSO-d6,400MHz)δ10.71(s,1H),7.92-7.81(m,5H),7.66(m,4H),7.54-7.49(m,2H),7.47-7.36(m,7H),6.66(br s,2H),6.04(s,1H),5.29(s,1H),5.13(s,1H),4.92(d,J=12.0Hz,1H),4.84(d,J=12 .0Hz,1H),4.78(s,1H),4.21(d,J=12.2Hz,1H),4.01(d,J=12.2Hz,1H),0.94(s,9H); 13 C NMR(DMSO-d6,100MHz)δ157.13,154.49,151.10,135.62,135.60,135.29,13 4.48,133.17,133.01,132.46,132.46,130.52,128.44,128.15,128.07,126 .75,126.65,126.56,126.17,117.70,117.02,88.24,84.89,78.86,78.48,7 2.02,71.23,59.76,26.80,19.20;ESI-MS(m / z)699.39[M+H]+,721.48[M+Na] + .

[0089] 19 To 12 mL of a 1,4-dioxane solution of guanosine 18 (1.2 g, 1.72 mmol), DMAP (105 mg, 0.86 mmol) and triethylamine (718 μL, 5.16 mmol) were added, followed by isobutyryl chloride (550 μL, 5.16 mmol). The mixture was heated to 100°C and allowed to react for 24 hours. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After stopping the heating and allowing the reaction mixture to cool to room temperature, 1 mL of methanol was added to quench the reaction. The mixture was extracted with ethyl acetate, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-80%) to obtain 1.1 g of isobutyryl-protected guanosine 19 as a slightly yellowish foamy solid in 83% yield. 1 H NMR(CDCl3,400MHz)δ12.00(s,1H),9.02(s,1H),7.78-7.74(m,3H),7.69-7.63 (m,5H),7.52(s,1H),7.47-7.28(m,9H),5.86(s,1H),4.87(s,1H),4.76(d,J=11 .6Hz,1H),4.66(d,J=11.6Hz,1H),4.47(s,1H),4.33(s,1H),4.13(d,J=12.0Hz, 1H),4.02(d,J=12.0Hz,1H),2.70-2.66(m,1H),1.29-1.24(m,6H),1.08(s,9H); 13 C NMR(CDCl3,100MHz)δ178.83,155.24,147.84,146.82,135.64,135.49,135.11,133. 37,133.11,132.95,131.99,131.93,130.25,130.22,128.67,128.07,128.01,127.74 ,127.59,127.25,126.61,126.53,125.70,131.62,115.87,86.71,86.12,78.40,73. 33,71.03,58.74,36.40,26.71,19.78,19.21,18.99;ESI-MS(m / z)870.60[M+Et3N+H] - .

[0090] 20 At room temperature, DDQ (702 mg, 3.64 mmol) was added to 10.5 mL of a dichloromethane-water (20:1) mixture of nucleoside derivative 19 (800 mg, 1.04 mmol), and the reaction was allowed to proceed at room temperature for 24 hours. Completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After concentrating the reaction mixture under reduced pressure, it was extracted with ethyl acetate, washed sequentially with saturated sodium sulfite solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was dissolved in 10 mL of tetrahydrofuran, and triethylamine (363 μL, 2.60 mmol) and triethylamine hydrofluoric acid (509 μL, 3.12 mmol) were added sequentially. The reaction was allowed to proceed at room temperature for 12 hours, and completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). 786 mg of sodium bicarbonate solid was added to the reaction mixture, and the mixture was stirred until no more bubbles were generated. The reaction mixture was then concentrated under reduced pressure and directly purified using a flash column (gradient elution: dichloromethane / methanol = 0-25%) to obtain 230 mg of cyanoloc nucleoside 20 in yield of 57%. 1 H NMR(CD3OD,400MHz)δ8.09(s,1H),6.08(s,1H),5.01(s,1H),4.73(s,1H),4.54(s,1H),3.99(s,2H),2.72-2.68(m,1H),1.23-1.21(m,6H); 13 C NMR(CD3OD,100MHz)δ180.35,148.67,148.20,136.57,120.03,116.22,89.37,85.62,8 0.91,70.56,70.35,55.80,46.48,35.58,17.92,17.90,7.84;ESI-MS(m / z)391.08[M+H] + ,413.05[M+Na] + .

[0091] twenty one At room temperature, 5 mL of a pyridine solution of cyanoloc nucleoside 20 (150 mg, 0.384 mmol) was mixed with 4,4'-dimethoxytrityl chloride (260 mg, 0.77 mmol), stirred at room temperature for 12 hours, and the completion of the reaction was detected by TLC (dichloromethane / methanol = 5 / 1). The reaction was quenched with 2 mL of methanol, concentrated under reduced pressure, the residue was diluted with ethyl acetate, washed sequentially with water and saturated saline, dried over Na2SO4, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0-40%) to obtain 270 mg of 5'-O-DMTr-protected cyanoloc nucleoside 21 as a pale yellow powder in 100% yield. 1 H NMR(DMSO-d6,400MHz)δ12.14(s,1H),11.85(s,1H),8.11(s,1H),7.46(d,J=7.4 Hz,2H),7.33-7.21(m,7H),6.90(dd,J=8.8,1.4Hz,4H),6.26(d,J=4.2Hz,1H),6. 08(s,1H),5.08(s,1H),4.77(s,1H),4.57(d,J=4.2Hz,1H),3.74(s,6H),3.68(d, J=11.2Hz,1H),3.39(d,J=11.2Hz,1H),2.82-2.75(m,1H),1.14(d,J=6.8Hz,6H); 13 C NMR(DMSO-d6,100MHz)δ180.73,158.66,155.22,148.89,148.58,144.97,136.87,135.59,135.40,130.34,130.31,128.33,128.21,12 7.27,120.65,118.31,113.71,88.30,86.87,84.93,80.95,72.28,71.18,60.22,55.51,35.27,19.33,19.28;ESI-MS(m / z)691.36[MH] - .

[0092] twenty two At room temperature, 10 mL of a dichloromethane solution of nucleoside 21 (180 mg, 0.26 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (157 mg, 0.52 mmol) was mixed with 1H-tetrazole (15 mg, 0.21 mmol), and the reaction was allowed to proceed at room temperature for 12 hours. Completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 2 / 1). The reaction mixture was directly purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-40%) to obtain 180 mg of phosphoramidite 22 as a slightly yellowish waxy solid in 78% yield. 31 P NMR(152MHz,DMSO-d6)δ149.26,149.23;ESI-MS(m / z)893.40[M+H] + . R-6'-CN-LNA- m Synthesis of C phosphoramidite monomers JPEG0007898764000155.jpg91170 Reaction conditions: (a) (1) TESCl, Et3N, acetonitrile, 0°C to room temperature, 3h; (2) 1,2,4-triazole, POCl3, acetonitrile, 0°C to room temperature, 2h; (3) NH4OH, 1,4-dioxane, room temperature, 2h; (b) (1) Bz2O, acetonitrile, room temperature, 20h; (2) TBAF, THF, room temperature, 12h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite, 1H-tetrazole, dichloromethane, room temperature, 5h.

[0093] twenty three At 0°C, triethylsilyl chloride (7.0 mL, 41.8 mmol) was added to a 100 mL solution of nucleotide-12 (5.0 g, 8.37 mmol) and triethylamine (23.3 mL, 167 mmol) in acetonitrile, and the reaction was allowed to proceed at room temperature for 3 hours. 1,2,4-triazole (8.67 g, 125.55 mmol) was added to the reaction mixture, and after stirring for 10 minutes, the mixture was cooled to 0°C. POCl3 (2.34 mL, 25.11 mmol) was added dropwise to the reaction mixture, and the reaction was allowed to proceed at room temperature for 2 hours. Completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). The reaction mixture was poured into 300 mL of ice water, extracted with ethyl acetate, and washed sequentially with water, saturated sodium bicarbonate solution, and saturated saline solution. The mixture was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was dissolved in 60 mL of 1,4-dioxane, 25% aqueous ammonia (6.25 mL, 92 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After concentrating the reaction mixture under reduced pressure, it was diluted with ethyl acetate, the organic layer was washed with water and saturated brine, dried over anhydrous Na2SO4, filtered, concentrated, and purified by flash column (gradient elution: methanol / dichloromethane = 0-15%) to obtain 5.5 g of aminonucleoside 23 as a pale yellow solid in 92% yield. 1 H NMR(DMSO-d6,400MHz)δ7.52-7.25(m,12H),6.96-6.91(m,6H),5.66(s,1H),4.93(s,1H),4.45(s,1H),4.40(s,1H), 3.75(s,6H),3.54(d,J=11.2Hz,1H),3.41(d,J=11.2Hz,1H),1.72(s,3H),0.78(t,J=7.9Hz,9H),0.53-0.41(m,6H); 13 C NMR(DMSO-d6,100MHz)δ166.09,158.81,158.79,154.95,144.78,136.54,135.38,135.17,130.30,130.14,128.43,128.07,127.46,11 7.58,113.81,113.77,101.92,88.29,87.59,86.82,80.77,71.60,71.09,58.50,55.55,14.01,6.78,4.42;ESI-MS(m / z)733.31[M+Na] + .

[0094] twenty four To a solution of aminonucleoside 23 (5.5 g, 7.73 mmol) in acetonitrile (60 mL), benzoic anhydride (3.85 g, 12 mmol) was added and the mixture was stirred at room temperature for 20 hours, after which the reaction was quenched with 5 mL of water. TBAF (4.38 g, 19.3 mmol) was added to the reaction mixture and the mixture was stirred at room temperature for 12 hours, after which 15% NaOH was added to adjust the pH to 10, and stirring was continued for 3 hours. The reaction solution was diluted with ethyl acetate and washed with water. The organic layer was washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-40%) to obtain 3.1 g of 5'-O-DMTr protected nucleoside 24 as a white foamy solid in 57% yield. 1 H NMR(DMSO-d6,400MHz)δ13.06(br s,1H),8.19(s,2H),7.69-7.25(m,13H),6.94(d,J=8.3Hz,4H),6.32(s,1H),5.70(s,1H),5.06(s,1 H),4.53(s,1H),4.38(d,J=4.1Hz,1H),3.73(d,J=11.0Hz,1H),3.40(d,J=11.0Hz,1H),1.84(s,3H); 13 C NMR(DMSO-d6,100MHz)δ178.64,159.43,158.75,147.49,144.93,137.11,135.53,135.32,133.07,130.37,130.32,129.85,128.83, 128.46,128.21,127.41,117.77,113.82,110.12,88.75,87.28,80.55,71.06,70.70,58.88,55.56,13.66;ESI-MS(m / z)701.32[M+H] + .

[0095] twenty five To 20 mL of an anhydrous dichloromethane solution of nucleoside 24 (1.7 g, 2.42 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (1.1 g, 3.63 mmol), 1H-tetrazole (135 mg, 1.94 mmol) was added and the mixture was reacted at room temperature for 5 hours. Then, saturated NaHCO3 solution was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with saturated brine, dried over MgSO4, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-20%) to obtain 1.83 g of phosphoramidite 25 as a white foamy solid in 84% yield. 31 P NMR(152MHz,DMSO-d6)δ149.20,148.47;ESI-MS(m / z)901.46[M+H] + .

[0096] Part 2: Synthesis of S-6'-CN-LNA

[0097] Synthesis of S-6'-allyl-LNA JPEG0007898764000156.jpg84166 Reaction conditions: (a) 2-iodoxybenzoic acid, acetonitrile, reflux, 11h; (b) NaBH4, LiCl, MeOH, -40℃, 8h; (c) methanesulfonyl chloride, DMAP, pyridine, room temperature, 32h; (d) (1) FeCl3·6H2O, dichloromethane, 0℃~room temperature, 3h; (2) Ac2O, DMAP, pyridine, dichloromethane, room temperature, 4h.

[0098] 26 500 mL of acetonitrile solution of compound 2 (105 g, 0.164 mol) was mixed with 2-iodoxybenzoic acid (69 g, 0.25 mol), heated, and refluxed for 6 hours. Then, 2-iodoxybenzoic acid (13.8 g, 0.05 mol) was added, and refluxing was continued for 5 hours. Completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated and purified by flash column (gradient elution: ethyl acetate / petroleum ether = 0-20%) to obtain 93 g of colorless viscous substance 26 in yield 89%. 1H NMR(CDCl3,400MHz)δ7.85-7.70(m,4H),7.60-7.53(m,4H),7.48-7.45(m,2H),7.40-7.27(m,7H),6.08(d,J=4.0Hz,1H),5.98-5.83(m,1H),5.13-5.01(m,2H),4.90(d,J=12.0Hz,1H),4.86(t,J=8.0,4.0Hz,1H),4.63(d,J=12.0Hz,1H),4.23(d,J=8.0Hz,1H),3.92(d,J=12.0Hz,1H),3.72(d,J=12.0Hz,1H),3.66-3.55(m,2H),1.61(s,3H),1.43(s,3H),0.98(s,9H); 13 C NMR(CDCl3,100MHz)δ207.44,135.53,134.92,134.83,133.23,133.06,132.49,132.46,130.73,129.99,129.91,128.12,127.97,127.87,127.75,127.71,126.57,126.09,125.93,125.82,118.44,114.93,106.94,96.44,81.14,80.15,73.55,44.95,27.61,26.81,26.68,19.13;ESI-MS(m / z)635.40[M-H] - .

[0099] 27 At room temperature, 400 mL of tetrahydrofuran solution of compound 26 (108 g, 0.17 mol) was added to 200 mL of methanol solution of lithium chloride (18 g, 0.424 mol). After cooling the reaction mixture to -40°C, sodium borohydride (4.3 g, 0.113 mol) was added and the reaction was allowed to proceed for 4 hours. Then, sodium borohydride (4.3 g, 0.113 mol) was added and the reaction was continued for another 4 hours, and completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). The reaction mixture was poured into a beaker, the reaction was quenched with saturated ammonium chloride solution (100 mL), stirred until no more bubbles were generated, extracted with ethyl acetate (1 L), washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / petroleum ether = 0-20%) to obtain 90 g of colorless viscous substance 27 in 83% yield. 1 H NMR(CDCl3,400MHz)δ7.86-7.79(m,4H),7.59-7.57(m,2H),7.51-7.46(m,5H),7.41-7.28( m,6H),5.91-5.83(m,2H),5.01-4.97(m,3H),4.78(dd,J=5.3,3.9Hz,1H),4.65(d,J=12.0Hz ,1H),4.51(d,J=5.4Hz,1H),4.19(dd,J=10.0,2.1Hz,1H),3.76(d,J=10.7Hz,1H),3.54(d,J =10.7Hz,1H),2.36-2.31(m,1H),2.09-2.02(m,1H),1.67(s,3H),1.39(s,3H),0.91(s,9H); 13 C NMR(CDCl3,100MHz)δ136.40,135.53,135.51,134.80,133.22,132.16,1 32.88,132.74,129.85,129.78,128.41,127.94,127.78,126.85,126.29 ,126.14,125.67,161.13,114.38,104.73,90.15,79.74,78.12,72.80,7 1.04,64.51,35.91,26.98,26.81,26.75,19.11;ESI-MS(m / z)637.41[MH] - .

[0100] 28 Compound 27 (90 g, 0.14 mol) and DMAP (1.71 g, 14 mmol) were dissolved in 400 mL of pyridine, and methanesulfonyl chloride (16.4 mL, 0.21 mmol) was added dropwise. The reaction was allowed to proceed at room temperature for 20 hours, then methanesulfonyl chloride (5.5 mL, 0.07 mol) was added, and the reaction was continued for 12 hours. Completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). 50 mL of methanol was added to the reaction mixture to quench the reaction, and the mixture was concentrated under reduced pressure. Excess pyridine was removed, the residue was diluted with ethyl acetate, and the mixture was sequentially washed with water, 1N HCl solution, and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 100 g of methanesulfonyl compound 28 as a slightly yellowish viscous substance in 100% yield. 1 H NMR(DMSO-d6,400MHz)δ7.95-7.87(m,4H),7.59-7.38(m,13H),5.85(d,J=4.0Hz,1H),5.6 8-5.58(m,1H),5.13(dd,J=9.8,1.4Hz,1H),5.07(t,J=5.0Hz,1H),4.98-4.91(m,3H),4.69 (d,J=11.8Hz,1H),4.47(d,J=5.4Hz,1H),3.76(d,J=10.8Hz,1H),3.71(d,J=10.8Hz,1H), 3.08(s,3H),2.83-2.78(m,1H),2.16-2.08(m,1H),1.58(s,3H),1.37(s,3H),0.88(s,9H); 13 C NMR(CDCl3,100MHz)δ135.54,135.59,135.30,134.36,133.24,133.15,132.7 2,132.68,130.50,130.47,128.58,128.43,128.35,128.21,127.32,126.79, 126.61,126.48,118.25,124.07,105.02,88.46,85.65,79.89,77.62,72.51, 63.50,36.01,27.06,26.95,26.82,26.58,19.07;ESI-MS(m / z)734.39[M+NH4] + ,739.34[M+Na] + .

[0101] 29 1000 mL of a dichloromethane solution of compound 28 (100 g, 0.14 mol) was cooled to 0°C, and iron(III) chloride hexahydrate (15.1 g, 56 mmol) was added. The reaction was allowed to proceed at room temperature for 3 hours, and completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 4 / 1). The reaction mixture was poured into 2 L of water and extracted with dichloromethane. The organic layer was sequentially washed with water, saturated sodium bicarbonate solution, and saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and vacuum dried. The resulting viscous substance was dissolved in 1 L of dichloromethane, pyridine (113 mL, 1.4 mol) and DMAP (2.56 g, 21 mol) were added sequentially, and then acetic anhydride (79 mL, 0.84 mol) was added dropwise. The reaction was allowed to proceed at room temperature for 4 hours, and completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 4 / 1). The reaction mixture was poured into 2 L of water and extracted with dichloromethane. The organic layer was sequentially washed with water, 1N HCl solution, saturated sodium bicarbonate solution, and saturated brine. The mixture was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by flash column (gradient elution: petroleum ether / ethyl acetate = 0-60%) to obtain a 299 g epimer mixture of diacetyl compound 29 as a colorless viscous substance in 89% yield. ESI-MS (m / z) 778.39 [M+NH4] + ,783.44[M+Na] + . JPEG0007898764000157.jpg105166 Reaction conditions: (a) Thymine, N,O-bistrimethylsilylacetamide (BSA), TMSOTf, acetonitrile, 80°C, 3h; (b) K2CO3, MeOH, room temperature, 16h; (c) N6-benzoyladenine, BSA, TMSOTf, toluene, 100°C, 3h; (d) 6-chloroguanine, BSA, TMSOTf, toluene, 100°C, 3h;

[0102] 30 To 50 mL of acetonitrile suspension of thymine (7.6 g, 60 mmol), BSA (29 mL, 120 mmol) was added and the mixture was stirred at room temperature for 20 minutes until the thymine dissolved. After the system became clear, 200 mL of acetonitrile solution of diacetylated sugar 29 (23 g, 30 mmol) was added, followed by rapid dropwise addition of TMSOTf (8.3 mL, 45 mmol). After the addition was complete, the temperature was raised to 80°C and the mixture was stirred for 3 hours to allow the reaction to proceed. Completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). After stopping the heating and allowing the reaction mixture to cool to room temperature, the reaction mixture was poured into ethyl acetate, a semi-saturated NaHCO3 solution (400 mL) was added, and the mixture was stirred for 20 minutes. The insoluble solid was filtered through diatomaceous earth to separate the organic layer, which was then washed sequentially with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and vacuum-dried. The resulting residue was dissolved in 250 mL of methanol, K2CO3 (12.4 g, 90 mmol) was added, and the mixture was allowed to react at room temperature for 16 hours. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 3). After concentrating the reaction mixture, the residue was extracted with ethyl acetate, washed sequentially with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-25%) to obtain 15.4 g of LOCK nucleic acid derivative 30 as a white foamy solid in 75% yield. 1 H NMR(CDCl3,400MHz)δ8.92(br s,1H),7.84-7.64(m,8H),7.51-7.29(m,10H),5.77-5.59(m,1H),5.65(s,1H),5.01-4.96(m,2H),4.84(d,J=11.4Hz,1H) ,4.73(s,1H),4.67(d,J=11.4Hz,1H),4.07-3.99(m,4H),2.68-2.60(m,1H),2.26-2.19(m,1H),1.58(s,3H),1.09(s,9H); 1313C NMR (CDCl3, 100 MHz) δ 163.79, 149.75, 135.59, 135.35, 134.36, 134.13, 134.01, 133.14, 133.10, 132.80, 132.34, 130.11, 130.06, 128.45, 127.99, 127.95, 127.87, 127.75, 126.91, 126.45, 126.30, 125.75, 117.38, 110.34, 89.58, 87.12, 84.02, 76.90, 72.53, 59.27, 35.33, 29.72, 26.94, 19.44, 12.15; ESI-MS (m / z) 689.43 [M+H] + , 711.34 [M+Na] + .

[0103] 31 To a 25 mL toluene suspension of N6-benzoyladenine (6.1 g, 25.5 mmol) was added BSA (17 mL, 68 mmol), and the mixture was heated to 60 °C and reacted until N6-benzoyladenine dissolved. After the system became transparent, the reaction solution was cooled to room temperature, and 70 mL of a toluene solution of diacetylated sugar 29 (13 g, 17 mmol) was added. Subsequently, TMSOTf (4.63 mL, 25.5 mmol) was rapidly added dropwise. After the addition, the temperature was raised to 100 °C and the mixture was stirred and reacted for 3 h. The completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). Heating was stopped, and after the reaction solution was cooled to room temperature, the reaction solution was poured into ethyl acetate, and semi-saturated NaHCO3 solution (400 mL) was added. The mixture was stirred for 20 min, and the insoluble solid was filtered through diatomaceous earth. The organic layer was separated, then washed successively with water and saturated brine, dried over anhydrous Na2SO4, filtered, concentrated, and vacuum dried. After purification by flash column (gradient elution: ethyl acetate / dichloromethane = 0 - 40%), 9.1 g of the nucleoside product was obtained in a yield of 74%. ESI-MS (m / z) 940.04 [M+H] + . The above product (8.5 g, 9 mmol) was dissolved in 90 mL of methanol, K2CO3 (6.25 g, 45 mmol) was added, and the reaction was allowed to proceed at room temperature for 16 hours. Completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 3). After concentrating the reaction mixture, the residue was extracted with ethyl acetate, washed sequentially with water and saturated saline, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-100%) to obtain 4.9 g of LOCK nucleic acid derivative 31 as a white powder in 78% yield. 1 H NMR(CDCl3,400MHz)δ8.27(s,1H),7.98(s,1H),7.81-7.67(m,7H),7.62(s,1H),7.49-7 .31(m,9H),6.02(s,1H),5.86-5.76(m,3H),5.06-5.02(m,2H),4.95(s,1H),4.74(d,J=1 1.6Hz,1H),4.66(d,J=11.6Hz,1H),4.27(s,1H),4.18(dd,J=9.3,4.6Hz,1H),4.09(d,J= 11.9Hz,1H),4.05(d,J=11.9Hz,1H),2.77-2.69(m,1H),2.36-2.30(m,1H),1.07(s,9H); 13 C NMR(CDCl3,100MHz)δ155.16,152.74,148.84,138.22,135.69,135.63,134.35, 134.40,133.10,133.06,132.79,132.62,129.99,129.97,128.36,127.92,127. 85,127.82,127.71,126.92,126.32,126.19,125.64,120.14,117.16,89.04,86 .48,84.57,78.06,72.72,59.50,35.31,26.78,19.27;ESI-MS(m / z)698.36[M+H] + .

[0104] 32 To 25 mL of a toluene suspension of 6-chloroguanine (3.34 g, 19.7 mmol), BSA (13 mL, 52.6 mmol) was added and heated to 60°C until the 6-chloroguanine dissolved. After the system became clear, the reaction mixture was cooled to room temperature, and 75 mL of a toluene solution of diacetylated sugar 29 (10 g, 13.1 mmol) was added. Subsequently, TMSOTf (3.6 mL, 19.7 mmol) was rapidly added dropwise, and after addition, the temperature was raised to 100°C and the mixture was stirred for 3 hours. The completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). After stopping the heating and allowing the reaction mixture to cool to room temperature, the reaction mixture was poured into ethyl acetate, a semi-saturated NaHCO3 solution (400 mL) was added, and the mixture was stirred for 20 minutes. The insoluble solid was filtered through diatomaceous earth to separate the organic layer, which was then washed sequentially with water and saturated brine. The mixture was dried over anhydrous NaSO4, filtered, concentrated, and vacuum-dried. The resulting residue was dissolved in 100 mL of methanol, K2CO3 (7.25 g, 52.6 mmol) was added, and the mixture was allowed to react at room temperature for 16 hours. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 3). After concentrating the reaction mixture, the residue was extracted with ethyl acetate, washed sequentially with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-30%) to obtain 6.07 g of LOCK nucleic acid derivative 32 as a white foamy solid in 63% yield. 1 H NMR(CDCl3,400MHz)δ7.82-7.65(m,9H),7.49-7.30(m,9H),5.91(s,1H),5.85-5.76(m,1H),5.06-5.02(m,2H),4.86(br s,2H),4.81(s,1H),4.73(d,J=11.5Hz,1H),4.64(d,J=11.5Hz,1H),4.27(s, 1H),,4.09-4.01(m,5H),2.76-2.69(m,1H),2.35-2.28(m,1H),1.07(s,9H); 1313C NMR (CDCl3, 100 MHz) δ 161.48, 159.29, 152.47, 135.70, 135.55, 134.47, 124.36, 133.12, 133.04, 132.74, 132.58, 129.98, 129.95, 128.34, 127.91, 127.88, 127.84, 127.70, 126.60, 125.59, 117.15, 116.13, 88.77, 86.19, 84.49, 78.23, 77.15, 72.78, 55.50, 53.90, 35.31, 29.75, 26.76, 19.24; ESI-MS (m / z) 728.39 [M+H] + . Synthesis of S-6'-CN-LNA JPEG0007898764000158.jpg99165Reaction conditions: (a) (1) Carbonylchlorohydridotris(triphenylphosphine)ruthenium(II), EtOH, 80 °C, 12 - 48 h; (b) Potassium osmate dihydrate, 50% NMO, THF, t-BuOH, H2O, 60 °C, 8 - 12 h; (c) NaIO4, EtOH-THF (2 / 1 v / v), H2O, room temperature, 12 h; (d) NH4OH, I2, room temperature, THF, EtOH, 24 h.

[0105] 33 To 120 mL of an anhydrous ethanol solution of allyl nucleoside derivative 30 (10.0 g, 14.5 mmol), carbonyl chlorohydride tris(triphenylphosphine)ruthenium(II) (345 mg, 0.36 mmol, 2.5% mol) was added, and the mixture was heated to 80°C and reacted for 12 hours. After directly concentrating the reaction mixture, the residue was dissolved in 80 mL of tetrahydrofuran, and 10 mL of tert-butanol, 10 mL of water, potassium osmate dihydrate (26.7 mg, 72.6 μmol, 0.5% mol), and 50% N-methylmorpholine-N-oxide (4.53 mL, 10.9 mmol) were added sequentially. The mixture was heated to 60°C and reacted for 8 hours, and completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column elution (gradient elution: ethyl acetate / dichloromethane = 0-60%) to obtain 9.3 g of a mixed product of dihydroxylated isomers. The above dihydroxylation product was dissolved in 60 mL of ethanol-tetrahydrofuran solution, and 10 mL of aqueous solution of NaIO4 (3.32 g, 15.5 mmol) was added dropwise, and the mixture was stirred at room temperature for 12 hours. After filtering out the insoluble solid, 25% aqueous ammonia (5.5 mL, 72.6 mmol) was added to the filtrate, followed by the addition of I2 (1.84 g, 7.26 mmol) in several portions, and the reaction was allowed to proceed at room temperature for 24 hours. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-30%) to obtain 3.6 g of a slightly yellowish foamy solid product 33 in 37% yield. 1H NMR(CDCl3,400MHz)δ8.87(br s,1H),7.82-7.74(m,4H),7.68-7.63(m,4H),7.50-7.29(m,9H),7.18(d,J=1.2Hz,1H),5.62(s,1H),4.93(d,J=11.8Hz,1H),4.85(s,1H),4.76(d,J=11.8Hz,1H),4.69(s,1H),4.30(d,J=12.2Hz,1H),4.25(d,J=12.2Hz,1H),4.07(s,1H),1.58(d,J=1.2Hz,3H),1.08(s,9H); 13 C NMR(CDCl3,100MHz)δ163.31,149.51,135.49,135.36,133.37,133.33,133.14,133.11,132.21,131.87,130.27,130.23,128.55,128.01,127.72,126.90,126.43,126.35,125,44,114.18,111.17,90.08,87.22,78.12,76.00,72.69,69.48,60.40,58.82,26.83,19.40,12.18;ESI-MS(m / z)674.28[M+H] + .

[0106] 34 To 50 mL of an ethanol solution of allyl nucleoside derivative 31 (4.9 g, 7.0 mmol), carbonyl chlorohydride tris(triphenylphosphine)ruthenium(II) (167 mg, 0.175 mmol, 2.5% mol) was added, and the mixture was heated to 80°C and reacted for 36 hours. After directly concentrating the reaction mixture, the residue was dissolved in 40 mL of tetrahydrofuran, and 5 mL of tert-butanol, 5 mL of water, potassium osmate dihydrate (26 mg, 70 μmol, 1% mol), and 50% N-methylmorpholine-N-oxide (2.2 mL, 10.5 mmol) were added sequentially, and the mixture was heated to 60°C and reacted for 8 hours. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue purified by flash column (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0-100%) to obtain 3.6 g of a mixed product of dihydroxylated isomers. The above dihydroxylated product was dissolved in 40 mL of ethanol-tetrahydrofuran solution, and 8 mL of aqueous solution of NaIO4 (1.58 g, 7.4 mmol) was added dropwise, and the mixture was stirred at room temperature for 12 hours. After filtering out the insoluble solid, 25% aqueous ammonia (3.05 mL, 49.2 mmol) was added to the filtrate, followed by the addition of I2 (1.25 g, 4.92 mmol) in several portions, and the mixture was reacted at room temperature for 24 hours. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0-60%) to obtain 1.6 g of the white foamy solid product 34 in 48% yield. 1 H NMR(CDCl3,400MHz)δ8.22(s,1H),7.80-7.75(m,3H),7.70-7.66(m,6H),7.48-7.31(m,9H),5.96(s,1H),5.85(br s,2H),5.19(s,1H),4.88-4.77(m,3H),4.36(s,1H),4.32(d,J=12.2Hz,1H),4.19(d,J=12.2Hz,1H),1.06(s,9H); 13C NMR(CDCl3,100MHz)δ155.43,153.10,148.62,135.58,130.12,128.48,127.98,127.91,127.89,127.70,126.83,126. 33,125.47,119.91,114.52,89.84,86.36,78.22,75.52,72.86,70.11,59.39,29.70,19.27;ESI-MS(m / z)683.35[M+H] + .

[0107] 35 To 120 mL of an anhydrous ethanol solution of allyl nucleoside derivative 32 (7.2 g, 10 mmol), carbonyl chlorohydride tris(triphenylphosphine)ruthenium(II) (238 mg, 0.25 mmol, 2.5% mol) was added, and the mixture was heated to 80°C and reacted for 48 hours. After directly concentrating the reaction mixture, the residue was dissolved in 50 mL of tetrahydrofuran, and 10 mL of tert-butanol, 10 mL of water, potassium osmate dihydrate (36 mg, 10 μmol, 1% mol), and 50% N-methylmorpholine-N-oxide (4.0 mL, 20 mmol) were added sequentially. The mixture was heated to 60°C and reacted for 8 hours, and completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column elution (gradient elution: ethyl acetate / dichloromethane = 0-40%) to obtain 5.6 g of a mixed product of dihydroxylated isomers. The above dihydroxylation product was dissolved in 60 mL of ethanol-tetrahydrofuran solution, and 10 mL of aqueous solution of NaIO4 (3.54 g, 7.35 mmol) was added dropwise, and the mixture was stirred at room temperature for 12 hours. After filtering out the insoluble solid, 25% aqueous ammonia (5.5 mL, 73.5 mmol) was added to the filtrate, followed by the addition of I2 (1.87 g, 7.35 mmol) in several portions, and the reaction was allowed to proceed at room temperature for 24 hours. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-40%) to obtain 2.5 g of the white foamy solid product 35 in 48% yield. 1 H NMR(CDCl3,400MHz)δ7.80-7.66(m,8H),7.58(s,1H),7.47-7.31(m,9H),5.86(s,1H),5.03(s,1H),4.8 1-4.76(m,5H),4.34(s,1H),4.31(d,J=12.2Hz,1H),4.19(d,J=12.2Hz,1H),4.06(s,3H),1.06(s,9H); 13 C NMR(CDCl3,100MHz)δ161.63,159.37,152.27,136.01,135.60,135.58,1 33.75,133.11,132.19,132.16,130.13,128.49,127.99,127.96,127.69, 126.67,126.31,126.23,125.38,115.99,114.61,89.67,86.10,78.31,77 .64,72.91,70.05,59.41,53.97,26.70,19.25;ESI-MS(m / z)713.40[M+H] + . Synthesis of S-6'-CN-LNA phosphoramidite monomer Synthesis of S-6'-CN-LNA-T phosphoramidite monomer JPEG0007898764000159.jpg92165 Reaction conditions: (a) (1) DDQ, dichloromethane-H2O(20 / 1), room temperature, 24 h; (2) Et3N, Et3N·3HF, THF, room temperature, 12 h; (b) DMTrCl, pyridine, room temperature, 12 h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite, 1H-tetrazole, room temperature, 5 h.

[0108] 36 At room temperature, DDQ (420 mg, 1.86 mmol) was added to 42 mL of a dichloromethane-water (20:1) mixture of nucleoside derivative 33 (3.6 g, 5.34 mmol), and the reaction was allowed to proceed at room temperature for 24 hours. Completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 1). After concentrating the reaction mixture under reduced pressure, it was extracted with ethyl acetate, washed sequentially with saturated sodium sulfite solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was dissolved in 50 mL of tetrahydrofuran, and triethylamine (1.78 mL, 13.35 mmol) and triethylamine hydrofluoric acid (2.61 mL, 16 mmol) were added sequentially. The reaction was allowed to proceed at room temperature for 12 hours, and completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). 10 g of sodium bicarbonate solid was added to the reaction mixture and stirred until no more bubbles were generated. The reaction mixture was then concentrated under reduced pressure and purified directly using a flash column (gradient elution: methanol / dichloromethane = 0-20%) to obtain 1.1 g of a slightly yellowish-white solid 36 in 70% yield. 1 H-NMR(400MHz,MeOH-d4)δ7.62(s,1H),5.86(s,1H),4.78(s,1H),4.53(s,1H ),4.22(s,1H),4.17(d,J=12.0Hz,1H),4.13(d,J=12.0Hz,1H),1.89(s,3H); 13 C-NMR(100MHz,MeOH-d4)δ164.97,150.36,134.89,114.74,109.74,90.36,86.75,80.78,69.56,68.45,55.87.11.19;ESI-MS(m / z)296.13[M+H] +The crystal structure of the .S-6'-CN-LNA-T monomer (compound 36) is shown in Figure 8.

[0109] 37 To 15 mL of a pyridine solution of cyanoloc nucleoside derivative 36 (2.0 g, 6.77 mmol), 4,4'-dimethoxytrityl chloride (3.4 g, 10 mmol) was added and the mixture was stirred at room temperature for 12 hours. Completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). The reaction was quenched with 50 mL of methanol, concentrated under reduced pressure, the residue was diluted with ethyl acetate, washed sequentially with water and saturated saline, dried over Na2SO4, concentrated, and purified by flash column (gradient elution: methanol / dichloromethane = 0-10%) to obtain 2.6 g of 5'-O-DMTr protected nucleoside 37 as a white solid in 64% yield. 1 H-NMR(400MHz,DMSO-d6)δ11.44(s,1H),7.46-7.24(m,10H),6.92(dd,J=6.0,1.4Hz,4H),6.30(s,1H) ),5.53(s,1H),5.11(s,1H),3.79(d,J=7.9Hz,1H),3.74(s,6H),3.49(d,J=7.4Hz,1H),1.60(s,3H); 13 C-NMR(100MHz,DMSO-d6)δ162.66,157.16,148.78,143.44,134.16,133.74,132.93,128.73,128.65,126.87,126.54,126 .80,114.35,112.20,107.85,87.62,85.06,84.89,79.26,68.84,67.42,57.44,53.97,11.15;ESI-MS620.20(m / z)[M+Na] + .

[0110] 38 20 mL of a dichloromethane solution of nucleoside 37 (2.0 g, 3.35 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (1.51 g, 5 mmol) was mixed with 1H-tetrazole (230 mg, 3.35 mmol) and allowed to react at room temperature for 5 hours. The completion of the reaction was detected by TLC (ethyl acetate / dichloromethane = 5 / 1). A saturated NaHCO3 solution was added to the reaction mixture, extracted with dichloromethane, washed with saturated saline solution, dried over MgSO4, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-20%) to obtain 2.1 g of phosphoramidite 38 as a white foamy solid in 79% yield. 31 P-NMR(152MHz,DMSO-d6)δ148.95,148.35;ESI-MS(m / z)798.33[M+H] + . Synthesis of S-6'-CN-LNA-A phosphoramidite monomer JPEG0007898764000160.jpg69164 Reaction conditions: (a) BzCl, pyridine, room temperature, 6h; (b) (1) DDQ, dichloromethane-H2O(20 / 1), room temperature, 24h; (2) Et3N, Et3N·3HF, THF, room temperature, 12h; (b) DMTrCl, pyridine, room temperature, 8h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite, 1H-tetrazole, room temperature, 8h.

[0111] 39 At room temperature, 5 mL of pyridine solution of nucleoside 34 (1.0 g, 1.46 mmol) was mixed with benzoyl chloride (422 μL, 3.66 mmol) dropwise and allowed to react at room temperature for 4 hours. Then, 100 μL, 0.87 mmol of benzoyl chloride was added to the reaction mixture and the reaction was continued for 2 hours. 15% NaOH was added dropwise to the reaction mixture to adjust the pH to 8, and the mixture was stirred at room temperature for 2 hours. The mixture was diluted with ethyl acetate, washed sequentially with water, 1N HCl solution, and saturated saline solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column elution (gradient elution: ethyl acetate / dichloromethane = 0-45%) to obtain 1.05 g of a slightly yellowish foamy solid in 91% yield. 1H NMR(CDCl3,400MHz)δ9.02(br s,1H),8.65(s,1H),8.03(d,J=7.2Hz,2H),7.99(s,1H),7.79-7.34(m,20H),6.03(s,1H),5.19(s,1H),4.89(s,1H),4.87(d,J=12.2Hz,1H),4.82(d,J=12.2Hz,1H),4.34-4.31(m,2H),4.19(d,J=12.2Hz,1H),1.06(s,9H); 13 C NMR(CDCl3,100MHz)δ164.60,152.64,150.42,149.70,140.19,135.55,133.53,133.42,133.09,132.99,132.12,132.09,130.15,128.94,128.52,128.00,127.92,127.91,127.85,127.69,126.80,126.38,126.28,125.42,123.29,114.42,90.04,86.43,78.19,77.54,72.97,70.17,59.42,29.70,26.70,19.26;ESI-MS(m / z)787.32[M+H] + .

[0112] 40 At room temperature, DDQ (780 mg, 3.44 mmol) was added to 10 mL of a dichloromethane-water (20:1) mixture of nucleoside derivative 39 (900 mg, 1.14 mmol), and the reaction was allowed to proceed at room temperature for 24 hours. Completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After concentrating the reaction mixture under reduced pressure, it was extracted with ethyl acetate, washed sequentially with saturated sodium sulfite solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was dissolved in 15 mL of tetrahydrofuran, and triethylamine (400 μL, 2.85 mmol) and triethylamine hydrofluoric acid (550 μL, 3.42 mmol) were added sequentially. The reaction was allowed to proceed at room temperature for 12 hours, and completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). 1.0 g of sodium bicarbonate solid was added to the reaction mixture, and the mixture was stirred until no more bubbles were generated. The reaction mixture was then concentrated under reduced pressure and purified directly using a flash column (gradient elution: dichloromethane / methanol = 0-20%) to obtain 340 mg of cyanoloc nucleoside 40 in yield of 73%. 1 H NMR(CD3OD,400MHz)δ;ESI-MS(m / z)409.19[M+H] + ,431.16[M+Na] + .

[0113] 41 At room temperature, 5 mL of a pyridine solution of cyanoloc nucleoside 40 (300 mg, 0.735 mmol) was mixed with 4,4'-dimethoxytrityl chloride (373 mg, 1.1 mmol), stirred at room temperature for 8 hours, and the completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). The reaction was quenched with 1 mL of methanol, concentrated under reduced pressure, the residue was diluted with ethyl acetate, washed sequentially with water and saturated saline, dried over Na2SO4, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0-80%) to obtain 330 mg of 5'-O-DMTr-protected cyanoloc nucleoside 41 as a pale yellow solid in 64% yield. 1H NMR(DMSO-d6,400MHz)δ11.26(br s,1H),8.79(s,1H),8.54(s,1H),8.06(d,J=7.3Hz,2H),7.67(t,J=7.3Hz,1H),7.57 (t,J=7.8Hz,2H),7.46(d,J=7.4Hz,2H),7.34-7.24(m,6H),7.25(t,J=7.2Hz,1H),6 .90(d,J=8.9Hz,4H),6.33(d,J=3.5Hz,1H),6.23(s,1H),5.36(s,1H),4.92(s,1H), 4.70(d,J=3.5Hz,1H),3.85(d,J=11.1Hz,1H),3.74(s,6H),3.47(d,J=11.1Hz,1H); 13 C NMR(DMSO-d6,100MHz)δ166.10,158.64,152.33,152.02,150.97,145.07,142.33,135.88,135.38,130.25,130.19,128.98,128.96, 128.36,128.12,126.03,116.03,113.73,113.69,89.29,86.26,85.39,81.12,71.78,69.40,60.09,55.52;ESI-MS(m / z)711.37[M+H] + .

[0114] 42 At room temperature, 1H-tetrazole (21 mg, 0.3 mmol) was added to 10 mL of a dichloromethane solution of nucleoside 41 (240 mg, 0.34 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (203 mg, 0.75 mmol), and the reaction was allowed to proceed at room temperature for 8 hours. Completion of the reaction was detected by TLC (dichloromethane / methanol = 15 / 1). The reaction mixture was directly purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-40%) to obtain 280 mg of phosphoroamidite 42 as a slightly pale yellow foamy solid in 90% yield. 31 P NMR(152MHz,DMSO-d6)δ149.56,149.25;ESI-MS(m / z)909.38[MH] - . Synthesis of S-6'-CN-LNA-G phosphoramidite monomer JPEG0007898764000161.jpg79166 Reaction conditions: (a) 2N HCl, THF-CH3OH (1 / 1), 60℃, 24h; (b) Isobutyryl chloride, Et3N, DMAP, toluene, 100℃, 12h; (c) (1) DDQ, dichloromethane-H2O (20 / 1), room temperature, 24h; (2) Et3N, Et3N·3HF, THF, room temperature, 12h; (d) DMTrCl, pyridine, room temperature, 12h; (e) 2-Cyanoethyl N,N,N',N'tetraisopropylphosphodiamidite, 1H-tetrazole, room temperature, 6h.

[0115] 43 To 24 mL of a tetrahydrofuran-methanol solution (1 / 1 v / v) of cyanoloc nucleoside 35 (2.2 g, 3.08 mmol), 15.4 mL of 2N hydrochloric acid solution was added, and the mixture was heated to 60°C and reacted for 24 hours. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 2 / 1). The solution was extracted with ethyl acetate, washed with water and saturated saline, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (gradient elution: methanol / dichloromethane = 0-10%) to obtain 1.48 g of guanosine 43 as a white solid in 69% yield. 1 H NMR(CDCl3,400MHz)δ11.94(br s,1H),7.72-7.64(m,9H),7.48-7.16(m,10H),6.53(br s,2H),5.75(s,1H),4.81-4.70(m,3H),4.28-4.25(m,2H),4.17(d,J=12.2Hz,1H),1.03(s,9H); 13C NMR(CDCl3,100MHz)δ158.96,153.85,150.40,135.56,133.07,133.03,132.17,132.13,130.15,129.06,128.41,128.25,128.02,127.91,127.6 6,126.67,126.34,126.26,125.41,125.33,117.21,114.58,89.76,85. 91,78.28,72.87,69.98,59.39,26.72,19.28;ESI-MS(m / z)699.29[M+H] + .

[0116] 44 To 12 mL of a toluene solution of guanosine 43 (1.2 g, 1.72 mmol), DMAP (105 mg, 0.86 mmol) and triethylamine (718 μL, 5.16 mmol) were added, followed by isobutyryl chloride (550 μL, 5.16 mmol). The mixture was heated to 100°C and allowed to react for 12 hours. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After stopping the heating and allowing the reaction mixture to cool to room temperature, 1 mL of methanol was added to quench the reaction. The mixture was extracted with ethyl acetate, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-80%) to obtain 1.1 g of isobutyryl-protected guanosine 44 as a slightly yellowish foamy solid in 83% yield. 1 H NMR(CDCl3,400MHz)δ11.97(s,1H),8.78(s,1H),7.75-7.60(m,9H),7.44-7.32(m,9H),5.60(s,1H),4.79(d,J=12.0Hz,1H),4.69(s,1 H),4.65(d,J=12.0Hz,1H),4.62(s,1H),4.27(d,J=12.3Hz,1H),4.18-4.14(m,2H),2.68-2.61(m,1H),1.28-1.24(m,6H),1.06(s,9H); 13C NMR(CDCl3,100MHz)δ178.64,155.20,147.76,146.66,135.53,132.02,128.45,128.04,127.97,127.78,127.57,126.81,126.49 ,126.38,125.47,114.47,89.81,86.06,78.45,73.25,69.89,59.26,36.40,26.70,19.25,19.08,18.85;ESI-MS(m / z)767.39[MH] - .

[0117] 45 At room temperature, DDQ (885 mg, 3.9 mmol) was added to 16 mL of a dichloromethane-water (20:1) mixture of nucleoside derivative 44 (1.0 g, 1.3 mmol), and the reaction was allowed to proceed at room temperature for 24 hours. Completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After concentrating the reaction mixture under reduced pressure, it was extracted with ethyl acetate, washed sequentially with saturated sodium sulfite solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was dissolved in 10 mL of tetrahydrofuran, and triethylamine (453 μL, 3.25 mmol) and triethylamine hydrofluoric acid (636 μL, 3.9 mmol) were added sequentially. The reaction was allowed to proceed at room temperature for 12 hours, and completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). 700 mg of sodium bicarbonate solid was added to the reaction mixture, and the mixture was stirred until no more bubbles were generated. The reaction mixture was then concentrated under reduced pressure and purified directly using a flash column (gradient elution: dichloromethane / methanol = 0-25%) to obtain 380 mg of cyanoloc nucleoside 45 in 75% yield. 1 H NMR(MeOH-d4,400MHz)δ8.03(s,1H),5.91(s,1H),4.88(s,1H),4.78(s,1H),4.45(s,1H),4.17(s,2H),2.74-2.67(m,1H),1.23(d,J=6.9Hz,6H); 13C NMR(MeOH-d4,100MHz)δ181.72,150.01,149.42,137.97,121.52,116.12,91. 68,87.21,82.33,71.84,70.25,57.76,36.36,19.28;ESI-MS(m / z)389.09[MH] - .

[0118] 46 At room temperature, 5 mL of a pyridine solution of cyanoloc nucleoside 45 (300 mg, 0.77 mmol) was mixed with 4,4'-dimethoxytrityl chloride (390 mg, 1.15 mmol) and stirred at room temperature for 12 hours. The completion of the reaction was detected by TLC (dichloromethane / MeOH = 5 / 1). The reaction was quenched with 2 mL of methanol, concentrated under reduced pressure, the residue was diluted with ethyl acetate, washed sequentially with water and saturated saline, dried over Na2SO4, concentrated, and purified by flash column (gradient elution: methanol / dichloromethane = 0-10%) to obtain 520 mg of 5'-O-DMTr-protected cyanoloc nucleoside 46 as a slightly yellowish foamy solid in 97% yield. 1 H NMR(DMSO-d6,400MHz)δ12.13(s,1H),11.86(s,1H),8.06(s,1H),7.45-7.22(m,9H),8.91(d,J=8.8Hz,4H),6.29(d,J=3.5Hz,1H),5.95(s,1H),5.2 9(s,1H),4.75(s,1H),4.48(d,J=3.5Hz,1H),3.86(d,J=11.2Hz,1H),3.74 (s,6H),3.46(d,J=11.2Hz,1H),2.82-2.75(m,1H),1.13(d,J=6.8Hz,6H); 13 C NMR(DMSO-d6,100MHz)δ180.74,158.67,155.20,148.86,145.08,136.74,135.82,135.37,130.26,130.21,128.35,128.11,127 .29,120.82,115.96,113.72,113.68,81.22,86.29,80.07,71.35,69.42,60.01,55.52,35.24,19.31;ESI-MS(m / z)691.36[MH] -.

[0119] 47 At room temperature, 1H-tetrazole (31 mg, 0.44 mmol) was added to 10 mL of a dichloromethane solution of guanosine 46 (380 mg, 0.55 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (330 mg, 1.1 mmol), and the mixture was allowed to react at room temperature for 6 hours. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 2 / 1). The reaction mixture was directly purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-50%) to obtain 380 mg of phosphoroamidite 47 as a slightly yellowish waxy solid in 77% yield. 31 P NMR(152MHz,DMSO-d6)δ149.69,149.09;ESI-MS(m / z)891.42[MH] - . S-6'-CN-LNA- m Synthesis of C phosphoramidite monomers JPEG0007898764000162.jpg85165 Reaction conditions: (a) (1) TESCl, Et3N, acetonitrile, 0°C to room temperature, 3h; (2) 1,2,4-triazole, POCl3, acetonitrile, 0°C to room temperature, 2h; (3) NH4OH, 1,4-dioxane, room temperature, 2h; (b) (1) Bz2O, acetonitrile, room temperature, 20h; (2) TBAF, THF, room temperature, 12h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite, 1H-tetrazole, dichloromethane, room temperature, 5h.

[0120] 48 At 0°C, triethylsilyl chloride (4.78 mL, 28.45 mmol) was added to a solution of 5'-O-DMTr protected nucleoside 37 (3.4 g, 5.69 mmol) and triethylamine (15.8 mL, 113.8 mmol) in acetonitrile (50 mL) and the mixture was stirred at room temperature for 3 hours. 1,2,4-triazole (5.9 g, 85.35 mmol) was added to the reaction mixture and the mixture was stirred for 10 minutes. Next, POCl3 (1.6 mL, 17.07 mmol) was added dropwise at 0°C and the mixture was reacted at room temperature for 2 hours. The reaction mixture was poured into 300 mL of ice water, extracted with ethyl acetate, washed sequentially with water, saturated NaHCO3, and saturated saline solution, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was dissolved in 30 mL of 1,4-dioxane, water ammonia (4.25 mL) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated. Purified by flash column (gradient elution: methanol / dichloromethane = 0-15%), 3.9 g of aminonucleoside 48 was obtained as a pale yellow solid in 96% yield. 1 H NMR(DMSO-d6,400MHz)δ7.50-7.44(m,4H),7.37-7.25(m,8H),6.93(d,J=8.6Hz,4H),5.55(s,1H),5.09(s,1H),4.46(s,1H), 4.35(s,1H),3.75(s,6H),3.68(d,J=11.2Hz,1H),3.53(d,J=11.2Hz,1H),1.70(s,3H),0.81-0.74(m,9H),0.55-0.43(m,6H); 13 C NMR(DMSO-d6,100MHz)δ166.07,158.80,158.77,154.96,144.83,136.61,135.63,135.18,130.24,130.08,128.43,128.03,127.4 4,115.51,113.76,101.81,88.80,87.37,86.49,80.72,71.31,69.03,58.57,55.55,13.94,6.75,4.45;ESI-MS(m / z)733.31[M+Na] + .

[0121] 49 To a solution of aminonucleoside 48 (3.9 g, 5.49 mmol) in acetonitrile (20 mL), benzoic anhydride (2.73 g, 12 mmol) was added and the mixture was stirred at room temperature for 20 hours. Then, 5 mL of water was added to quench the reaction. TBAF (3.47 g, 11 mmol) was added to the reaction mixture and the mixture was allowed to react at room temperature for 12 hours. Next, 15% NaOH was added to adjust the pH to 10, and the mixture was stirred for 3 hours. The solution was extracted with ethyl acetate, washed sequentially with water and saturated saline, dried over anhydrous Na₂SO₄, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-40%) to obtain 3.2 g of nucleoside 49 as a white foamy solid in 83% yield. 1 H NMR(DMSO-d6,400MHz)δ13.08(s,1H),8.21(d,J=7.6Hz,1H),7.62-7.27(m,12H),6.95(d,J=8.6Hz,4H),6.40(d,J=3.2Hz,1H),5.61 (s,1H),5.18(s,1H),4.57(s,1H),4.31(d,J=3.2Hz,1H),3.86(d,J=11.2Hz,1H),3.76(s,6H),3.53(d,J=11.2Hz,1H),1.86(s,3H); 13 C NMR(DMSO-d6,100MHz)δ178.62,170.82,159.46,147.48,145.03,137.25,137.10,135.30,130.31,130.25,129.86,128.80,128.46, 128.12,127.39,115.87,113.78,110.13,89.52,87.06,86.50,80.60,70.34,69.06,59.99,55.55,13.62;ESI-MS(m / z)701.32[M+H] + .

[0122] 50 25 mL of an anhydrous dichloromethane solution of nucleoside 49 (2.3 g, 3.28 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (1.2 g, 3.93 mmol) was mixed with 1H-tetrazole (230 mg, 3.28 mmol) and reacted at room temperature for 5 hours. Saturated NaHCO3 solution was added to the reaction mixture, extracted with dichloromethane, washed with saturated brine, dried over anhydrous MgSO4, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0-20%) to obtain 2.13 g of phosphoramidite 50 as a white foamy solid in 72% yield. 31 P-NMR(152MHz,DMSO-d6)δ149.23,147.95;ESI-MS(m / z)901.46[M+H] + .

[0123] Test Example 1: Cyanolock nucleic acid modification ASO-protein interaction

[0124] Experimental method

[0125] Thermal denaturation test (T m value)

[0126] Annealing buffer: 10 mM Na3PO4, 100 mM NaCl, pH 7.2. Annealing method: The final concentration of the two oligonucleotide single strands was diluted to 2 μM with the annealing buffer, heated in a 95°C water bath for 5 min, slowly cooled to room temperature, and left overnight in a 4°C refrigerator. Tm measurement method: 100 μL of the sample to be measured was added to the cuvette and tightly covered with an insulating cover. The measurement start temperature was set to 15°C and the end temperature to 90°C, the temperature rise rate to 0.5°C / min, and the A260 reading rate to 1 reading / °C. Finally, the Tm was measured by the instrument. m A value was given. Each sample was measured three times, and the average value was used as the final result.

[0127] Immunofluorescence experiment: (1) Cell preparation: The day before, place HeLa cells in a 20mm diameter confocal culture dish in a 2x10⁶ container. 5The cells were inoculated at the specified cell density. Next, 20 mm confocal culture dishes were placed in a CO2 incubator and cultured. (2) Arrangement of PS-ASOs: ON1, ON2, and ON3 powders were dissolved in 100 μM solution of water that does not contain RNase and DNase enzymes. (3) Transfection: 10 μL of Lipofectamine 2000 (Solarbio) was aspirated and added to Opti-MEM (registered trademark) Medium to prepare 250 μL of transfection reagent. Next, 4 μg / dish was used for transfection and 250 μL of diluted Lipofectamine 2000 was uniformly mixed with the reagent. After 5 min, 500 μL of the mixed solution was added to each dish. The transfection reagent mixture was aspirated and removed, and the cells were washed three times with PBS. Next, 500 μL of DMEM (SIGMA) was added to each well as a cell maintenance solution, and the 20 mm confocal culture dishes were placed in a CO2 incubator and transfected for 5 min, 2 h, and 4 h respectively. (4) Fixation: After discarding the waste liquid, the cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 30 minutes, and then infused with 0.1% Triton X-100 (prepared with PBS) for 5 minutes. (5) Blocking: The fixed cells were blocked for 30 minutes at room temperature with blocking buffer (prepared with 1 mg / ml BSA and PBS). (6) Primary antibody binding: The primary antibody (NONO, Cell signaling) was diluted with blocking buffer, incubated at room temperature for 1 hour, then placed in a refrigerator at 4°C overnight, and washed three times with washing buffer (0.1% Tween prepared in PBS, washed once every 5 minutes). (7) Secondary antibody binding: The secondary antibody (Anti-rabbit IgG, Cell signaling) was diluted with blocking buffer and incubated at room temperature for 1 hour. Finally, the cells were washed three times with washing buffer. (8) Mounting and detection: Prolong anti-quenching agent (cell signaling) containing 4',6-diamidino-2-phenylindole (DAPI) was dropped into the culture dish, and finally, the culture was observed and photographed using an inverted fluorescence microscope.

[0128] Caspase 3 / 7 Activity Experimental Method (1) Cell preparation: The day before, place 5 × 10 HeLa cells (Kita-no Biological) into a 96-well cell culture plate. 4 The cells were inoculated at the specified cell density. Next, the 96-well cell culture plates were placed in a CO2 incubator and cultured. (2) Preparation of PS ASOs: ON1, ON2, and ON3 powders were dissolved in 10 μM solution with water that did not contain RNase and DNase enzymes. Next, the 10 μM ON1, ON2, and ON3 solutions were diluted with Opti-MEM® Medium (Thermofisher) to 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, and 0 nM solutions. (3) Transfection: 2 μL of Lipofectamine 2000 (Thermofisher) was aspirated and added to Opti-MEM (registered trademark) Medium to prepare 100 μL of transfection reagent. Next, 100 μL of PS ASOs solution of different concentrations was aspirated and uniformly mixed with 100 μL of diluted Lipofectamine 2000. After 5 min, 50 μL of the mixed solution was added to each well. After 4 hours of transfection, the transfection reagent mixture was aspirated and removed, and the cells were washed three times with PBS. Next, 100 μL of DMEM (Thermofisher) was added to each well as a cell maintenance medium, and the 96-well cell culture plate was placed in a CO2 incubator and cultured for 8 hours. (4) Detection: The 96-well cell plate was removed from the CO2 incubator, 100 μL of Caspase-Glo 3 / 7 Reagent (Promega) was added to each well, and after incubation for 30 minutes, the luminescence signal from each well was read using a multifunctional fluorescence microplate reader (Promega).

[0129] Experiment details

[0130] Intracellular ASO-protein interactions are closely related to the drug properties of PS ASOs, and differences in chemical modifications significantly affect ASO-protein interactions, and further significantly affect the intracellular distribution of binding proteins and the induction of apoptotic toxicity. To detect the effect of cyanoloc nucleotide modifications on the interaction between ASO and intracellular proteins, the inventors used the toxic 3-10-3 gapmer PS ASO sequence 449093(5'- TTC AGTCATGACT TCC -3' (Sequence ID 1) was selected as the template sequence (Nature Biotech., 2019, 37, 640) for study. The T and corresponding sequences of the synthesized R-CN-LNA and S-CN-LNA were studied. m ¹C was introduced to both ends of the 449093 sequence by solid-phase phosphoramidite spectroscopy to synthesize ON1 and ON2, respectively. 449093 (ON3) was then modified with LNA for comparison. Mass spectrometry identification data are shown in Table 1, and the corresponding mass spectra are shown in Figures 1-3.

[0131] First, when the hybridization characteristics of ON1-3 and target RNA were measured, their Tm values ​​were 67.04°C, 63.96°C, and 66.59°C, respectively. This indicated that PS-ASOs ON1-2 modified with CN-LNA still maintained good affinity with target RNA compared to PS-ASOs ON3 modified with LNA.

[0132] Next, we investigated the effect of cyanoloc nucleic acid modification on the interaction between ASOs and intracellular proteins using the intracellular paraspeckle protein P54nrb as a model protein. Paraspeckle protein P54nrb binds to toxic ASOs (low binding affinity at the nmol level), alters their distribution, and can enter the nucleolus and aggregate. This effect is clear and positively correlated with ASO toxicity. The results are shown by the aggregation status of paraspeckle protein P54nrb in the nucleolus.

[0133] Cells were transfected with ON1, ON2, and ON3, and the transfection times were set to 5 min, 2 h, and 4 h respectively. Finally, the protein localization of the P54nrb protein was observed with an inverted fluorescence microscope and photographed. As shown in Figure 4 (scale bar in the figure: 10 μm), the results are as follows. (1) At 5 min, the distribution of P54nrb in the cell nucleus was the same as that of the blank control, the nucleolus was clear, P54nrb was evenly distributed in the cell nucleus, and neither LNA nor CN-LNA modified ASO changed the distribution of P54nrb, and no aggregation in the nucleolus was observed. (2) For the LNA group, when the time was extended to 2 h and 4 h respectively, significant P54nrb aggregation appeared in the LNA group. The longer the incubation time, the more it aggregated in the nucleolus. It was shown that the binding affinity between the LNA modified ASO and intracellular proteins was strong, and it was easy to inhibit the normal distribution and function of intracellular proteins, resulting in toxicity and side effects. (3) From the R-CN-LNA group, the R-CN-LNA modified sequence ON1 showed no significant aggregation of the P54nrb protein in the nucleolus at 2 h, and the red fluorescence was evenly distributed in the cell nucleus. However, at 4 h, significant P54nrb aggregation occurred in some cells, but it was obvious that the degree of aggregation was significantly lower than that of the LNA group at the corresponding time point. (4) Regardless of whether it was 2 h or 4 h, the S-CN-LNA group showed no visible change in the distribution of P54nrb at each time point, and P54nrb was still evenly distributed in the cytoplasm.

[0134] [Table 1] Oligonucleotide sequences ON1-4 and their corresponding mass spectrum data JPEG0007898764000163.jpg90149

[0135] Based on the current immunofluorescence experiments, in order to further quantify the effects of LNA, R-CN-LNA, and S-CN-LNA modifications on the distribution of P54nrb in the cell nucleus, the inventors statistically analyzed the percentage of the number of cells in which P54nrb was "significantly aggregated" in nucleosomes in each group of LNA, R-CN-LNA, and S-CN-LNA at 10 min, 30 min, 1 h, 2 h, and 4 h respectively, out of the total number of cells. As shown in Table 2, in the LNA group, with the increase in transfection time, the number of cells in which P54nrb was significantly aggregated in nucleosomes also increased. It was found that at 4 h of LNA transfection, P54nrb was significantly aggregated in nucleosomes in approximately 92% of the transfected cells. In the R-CN-LNA group, only 5% of the cells' P54nrb was significantly aggregated in nucleosomes at 2 h of ASO transfection, and only about 8% at 4 h. For the S-CN-LNA group, there was no significant aggregation of P54nrb, and the cell ratio was 0%. The above results indicated that compared with LNA modification, CN-LNA modification could actually reduce the effect of ASO on intracellular proteins, and the S configuration had less impact and was superior to the R configuration.

[0136] [Table 2] Percentage of cells in which LNA-ASO, R-CN-LNA-ASO, and S-CN-LNA-ASO significantly aggregated P54nrb in nucleosomes JPEG0007898764000164.jpg116149

[0137] Based on the above evaluation of the effects on the intracellular distribution of intracellular proteins, the effects of CN-LNA modification on the induction of apoptotic toxicity were further investigated by detecting caspase 3 / 7 activity in HeLa cells.

[0138] Cells were transfected with ON1, ON2, and ON3 at concentrations of 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, and 0 nM, respectively. After 4 hours of transfection, the cells were incubated for 8 hours, and caspase 3 / 7 activity was detected using a fluorescence microplate reader. As shown in Figure 5, the experimental results were as follows: (1) At low concentrations (0-62.5 nM), no significant changes in caspase activity were observed in the three groups. (2) The LNA-modified group started at a concentration of 125 nM, and caspase activity levels rapidly increased with increasing dose concentration. The two CN-LNA-modified groups showed a gradual increase starting only at 250 nM, and the S configuration was lower than the R configuration. (3) At high concentrations of 250 nM and 500 nM, R-CN-LNA and S-CN-LNA modifications were able to reduce the increase in caspase activity by more than twice as much as LNA modifications, with the S configuration being superior to the R configuration. The above results show that R / S-CN-LNA modifications can significantly reduce PS ASO-induced apoptotic toxicity compared to LNA modifications, which is consistent with the results of immunofluorescence experiments.

[0139] In short, the introduction of cyano groups into LNA can reduce the lipophilicity of LNA and improve the water solubility of the modified structure. Cyanolock nucleic acid modification significantly reduces the effects of PS ASO on intracellular proteins, induces apoptosis toxicity, and improves the therapeutic effect of PS ASO. This demonstrates significant utility for nucleic acid drugs and can provide support for next-generation chemical modification technologies in nucleic acid medicine.

[0140] Test Example 2: Nuclease Resistance Experiment of S-CN-LNA-T

[0141] Nuclease stability is one of the important parameters of nucleotide chemical modification, and this experiment used snake venom phosphodiesterase (SVPDE) to analyze S-CN-LNA-T modified oligonucleotides (5'-TTTTTTTT). T T-3' (Sequence ID 2), TThe resistance of (=S-6'-CN-LNA)ON4 to nucleases was investigated and compared with the sequences of R-CN-LNA (ON5), LNA (ON6), thio-modified (ON7), and native (ON8). The corresponding nucleic acid sequences (7nM) were enzymatically digested using SVPDE (1.0 μg / mL) under physiological conditions at 37°C in a buffer system of 50 mM Tris-HCl, 10 mM MgCl2, pH 8.0. Incubation solutions were taken at different time points (0, 2, 5, 10, 20, 30, 40 min) and quantified by HPLC to obtain corresponding content-time curves. Results from nuclease resistance experiments (Figure 7, showing T=S-CN-LNA (rectangular) modified ON4, R-CN-LNA (rhomboid) modified ON5, LNA (triangular) modified ON6, 3'-thio-T (Ts, cross-shaped) modified ON7, and natural-T (star-shaped) modified ON8) indicate that under SVPDE 1.0 μg / mL conditions, the R-6'-CN-LNA modified sequence ON7 was slowly degraded, with over 40% still undegraded after 40 mins. The S-6'-CN-LNA modified sequence ON8 also showed over 50% undegraded, and less than 10% of the LNA modified sequences remained undegraded. This demonstrates that 6'-CN-LNA can significantly improve the resistance of oligonucleotides to nucleases, is clearly superior to LNA, and that the S configuration is superior to the R configuration.

[0142] Clearly, the cyano group, though relatively small, can enhance the nuclease stability of LNA, which is consistent with the results for other C6'-modified LNAs. Notably, while R and S configurations of cEt-LNA reported in the literature exhibit similar nuclease resistance, for CN-LNA, the S configuration is better for nuclease resistance than the R configuration. This suggests that CN has a more complex influence on phosphate, considering that the cyano group on the S configuration is oriented towards phosphate. If so, the interaction between CN-LNA-modified ASOs and proteins would have a greater influence than just electrostatic interactions.

[0143] Finally, the following points should be made: The above embodiments are merely for illustrating, and not limiting, the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some or all of their technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

[Claim 1] A method for synthesizing a cyano-modified nucleoside of the C6' moiety in the R configuration or S configuration, wherein the cyano-modified nucleoside of the C6' moiety in the R configuration is The method of its synthesis is The terminal olefin is isomerized After obtaining the result, the dihydroxylation reaction is carried out. Next, an aldehyde group compound is obtained by an oxidative cleavage reaction. Finally, Convert the aldehyde group to a cyano group, and modify the cyano-modified nucleoside at the C6' site of the R configuration. This includes synthesizing, The cyano-modified nucleoside at the C6' site in the S configuration is The method of its synthesis is The terminal olefin is isomerized After obtaining the result, the dihydroxylation reaction is carried out. Next, an aldehyde group compound is obtained by an oxidative cleavage reaction. Finally, Convert the aldehyde group to a cyano group, and modify the cyano-modified nucleoside at the S-configured C6' site. This includes synthesizing, Here, Bx is characterized by being selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or salts thereof. A method for synthesizing cyano-modified nucleosides with an R-configuration or S-configuration C6' site.

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