6’-Cyano-modified locked 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 the priority of a Chinese patent application filed with the China National Intellectual Property Administration on May 18, 2023, with the application number CN202310565102.7 and the title of the invention "6'-Cyano-Modified Lock Nucleosides, Nucleotides, and Nucleic Acid Polymers", and all of its contents are incorporated herein by reference.

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

Background Art

[0003] Nucleic acid drugs are at the forefront of the development of biopharmaceuticals and are the third type of drug after small molecule drugs and protein drugs. Nucleic acid drugs mainly include antisense nucleic acids (ASO), small interfering RNAs (siRNA), etc. Compared with conventional small molecule drugs and protein drugs, they have the advantages of rapid design, universal targets, high specificity, the ability to act intracellularly, relatively rapid synthesis, etc., and have important value in the treatment of many chronic, refractory severe diseases, rare diseases, etc. that are difficult to develop drugs for protein targets. By 2022, 15 nucleic acid drugs (10 ASO and 5 siRNA) have been approved worldwide, and more than 400 nucleic acid drugs are in clinical trials.

[0004] However, on the one hand, the drug discovery potential of unmodified oligonucleotide drugs is usually not ideal. They have low drug discovery properties such as low stability, being easily degraded by nucleases, high polarity, difficulty in entering cells, low distribution characteristics, and low binding affinity for target mRNA. In order to achieve clinical efficacy, oligonucleotides must be chemically modified, and all commercially available nucleic acid drugs use corresponding nucleic acid chemical modifications. On the other hand, chemically modified antisense nucleic acid drugs cause a certain degree of toxicity in highly exposed organs (liver and kidney). In particular, chemically modified nucleic acid drugs such as high-affinity locked nucleic acids (LNA) and constrained ethyl locked nucleic acids (cEt-LNA) on the phosphorothioate (PS) backbone may 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 next-generation nucleic acid chemical modification technologies remain important technologies and bottlenecks in the development of nucleic acid drugs.

[0005] In recent years, antisense nucleic acid drugs have made important progress in terms of toxicity mechanisms (Non-Patent Document 1). According to research, the main toxicity mechanism of antisense nucleic acid drugs is that chemically modified thio-antisense nucleic acid drugs (PS-ASOs) bind to and interfere with the intracellular distribution of intracellular proteins (such as P54nrb), inducing apoptosis and causing toxicity. At the same time, the toxicity is positively correlated with the binding ability of ASO-protein. The stronger the binding ability, the greater the potential toxicity. Such binding ability is closely related to the water solubility (LogS) of the chemical modification structure. The stronger the hydrophobic effect, the higher the binding affinity. From experimental results, it has been 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 determining factor in the therapeutic effect of antisense nucleic acid drugs. Since structure determines properties, in order to develop an efficient and safe new nucleic acid chemical modification structure, the inventors previously disclosed a novel cyano-locked 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, from further chemical property calculations (Chemdraw calculations), CN-LNA has good hydrophilicity, and its LogS value is -0.032, which is approximately 2 times, 4 times, and 20 times that of general 2'-methoxyethyl (MOE, LogS value is -0.073), locked nucleic acid (LNA, LogS value is -0.122), and 2'-fluorine (LogS value is -0.661) respectively. It has been shown that it may reduce the binding affinity between PS-ASO and intracellular proteins, further reduce the influence on the distribution of intracellular proteins, reduce toxicity, and improve the therapeutic effect.

[0006] However, the previously disclosed CN-LNA synthesis method cannot stereoselectively synthesize both the R-configuration and S-configuration C6'-epimers, and cannot further explain the differences in the drug discovery properties of the C6'-epimers for the modified nucleic acids, thus failing to meet the need to establish a new generation of efficient and low-toxic nucleic acid chemical modification technology.

[0007] In view of this, the present invention is provided.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0010] The first object of the present invention is to provide 6'-cyano-modified locked nucleosides, nucleotides and nucleic acid polymers in R-configuration or S-configuration. The second object of the present invention is to provide a method for preparing 6'-cyano-modified locked nucleosides, nucleotides and nucleic acid polymers in R-configuration or S-configuration, which is simple, has a high yield, and can solve at least one of the above problems. A third object of the present invention is to provide use in the preparation of a nucleic acid diagnostic agent or a nucleic acid therapeutic agent of the above nucleic acid polymer.

Means for Solving the Problems

[0011] In order to achieve the above object of the present invention, the following technical solutions are particularly used.

[0012] In a first aspect, the present invention provides a 6'-cyano-modified locked nucleoside, and the 6'-cyano-modified locked nucleoside is selected from a compound having a structure represented by Formula 1, a salt thereof, or an isomer thereof, In Formula 1 of JPEG2025523325000002.jpg3551, Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or a salt thereof, Z is a cyano group, W1 and W2 are independently selected from H or a hydroxy protecting group, and the hydroxy protecting group includes 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, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl, 9-phenylxanthin-9-yl, 9-(p-methoxyphenyl)xanthin-9-yl, or 2-naphthylmethyl, The isomer includes a 6'-cyano-modified locked nucleoside with the Z group in the R configuration, and the structural formula is JPEG2025523325000003.jpg3343, or includes a 6'-cyano-modified locked nucleoside with the Z group in the S configuration, and the structural formula is JPEG2025523325000004.jpg3747.

[0013] In a second aspect, the present invention provides a nucleotide, which includes a 3'-active phosphorus group derivative of the above 6'-cyano-modified locked nucleoside, The active phosphorus group is selected from phosphoramidite, derivatives of phosphoramidite, H-phosphonate, derivatives of H-phosphonate, phosphate triesters, and derivatives of phosphate triesters.

[0014] As a further technical solution, the nucleotide is selected from a compound having the structure represented by Formula 2, a salt thereof, or an isomer thereof, In JPEG2025523325000005.jpg4863 Formula 2, the types of Bx, W2, and Z are consistent with Formula 1, The isomer includes a nucleotide with the Z group in the R configuration, and the structural formula is JPEG2025523325000006.jpg4744, or includes a nucleotide with the Z group in the S configuration, and the structural formula is JPEG2025523325000007.jpg4343.

[0015] In a third aspect, the present invention provides the use of the above nucleotide in reducing the interaction between a nucleic acid polymer and an intracellular protein, or the use in the preparation of a medicament for reducing the interaction between a nucleic acid polymer and an intracellular protein.

[0016] In a fourth aspect, the present invention provides a nucleic acid polymer, and the nucleic acid polymer has a monomer with the structure represented by Formula 3, In JPEG2025523325000008.jpg3453, Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or salts thereof, 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 another part of the nucleic acid polymer, and at least one of W3 and W4 is an internucleoside linking group that links the monomer to another part of the nucleic acid polymer, The hydroxy protecting group includes 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, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-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 with the Z group in the R configuration, and the structural formula is JPEG2025523325000009.jpg3345, or includes a monomer with the Z group in the S configuration, and the structural formula is JPEG2025523325000010.jpg3342.

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

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

[0020] In a sixth aspect, the present invention provides a method for synthesizing a cyano-modified nucleoside at the C6' position in the R configuration or the S configuration, and the cyano-modified nucleoside at the C6' position in the R configuration is JPEG2025523325000011.jpg3344, and the synthesis method is Isomerize the terminal olefin of JPEG2025523325000012.jpg3353 to Obtain JPEG2025523325000013.jpg3146, and then perform a dihydroxylation reaction to Obtain JPEG2025523325000014.jpg3556, and then, by an oxidative cleavage reaction, the aldehyde group compound Obtain JPEG2025523325000015.jpg3254, and finally, Convert the aldehyde group of JPEG2025523325000016.jpg3554 to a cyano group to synthesize the cyano-modified nucleoside at the C6' position in the R configuration JPEG2025523325000017.jpg3151, including The cyano-modified nucleoside at the C6' position in the S configuration is JPEG2025523325000018.jpg4063, and the synthesis method is Isomerize the terminal olefin of JPEG2025523325000019.jpg3355 to Obtain JPEG2025523325000020.jpg3456, and then perform a dihydroxylation reaction to Obtain JPEG2025523325000021.jpg3960, and then, by an oxidative cleavage reaction, the aldehyde group compound Obtain JPEG2025523325000022.jpg3757, and finally, Convert the aldehyde group of JPEG2025523325000023.jpg3658 to a cyano group to synthesize a cyano-modified nucleoside at the C6' site with the S configuration including synthesizing JPEG2025523325000024.jpg3957, wherein Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil or salts thereof.

[0021] As a further technical solution, the synthesis method of JPEG2025523325000025.jpg3451 is to sequentially perform a mesylation reaction, a de-subunit and an acetylation reaction on JPEG2025523325000026.jpg3857 to obtain JPEG2025523325000027.jpg3653, and then perform a glycosylation reaction and a nucleophilic substitution reaction to synthesize JPEG2025523325000028.jpg3349 with the R configuration, including The synthesis method of the JPEG2025523325000029.jpg3249 is to sequentially perform a mesylation reaction, a de-subunit and an acetylation reaction on JPEG2025523325000030.jpg3554 to obtain JPEG2025523325000031.jpg3354, and then perform a glycosylation reaction and a nucleophilic substitution reaction to synthesize JPEG2025523325000032.jpg3758 with the S configuration, including The glycosylation reaction is to react JPEG2025523325000033.jpg43152 with thymine, N6-benzoyladenine or 6-chloroguanine at 50-100 °C under activator conditions, where the activator includes BSA and TMSOTf, and the reaction medium includes acetonitrile, 1,2-dichloroethane or toluene. including synthesizing JPEG2025523325000032.jpg3758, The glycosylation reaction is carried out under activator conditions, reacting JPEG2025523325000033.jpg43152 with thymine, N6-benzoyladenine or 6-chloroguanine at 50-100 °C, where the activator includes BSA and TMSOTf, and the reaction medium includes acetonitrile, 1,2-dichloroethane or toluene.

[0022] As a further technical solution, the above S configuration The synthesis method of JPEG2025523325000034.jpg3555 is 5-O-(tert-butyldiphenylsilyl)-4-C-hydroxymethyl-1,2-O-isopropylidene-3-O-(2-naphthylmethyl)-α-D-ribofuranose Oxidize the primary alcohol of JPEG2025523325000035.jpg3551 to an aldehyde group, and then perform an allylation reaction on the aldehyde group to obtain the S configuration including obtaining JPEG2025523325000036.jpg3966, Preferably, the above R configuration The synthesis method of JPEG2025523325000037.jpg3149 is Oxidize the secondary alcohol of JPEG2025523325000038.jpg3966 to a ketone, and then reduce the ketone to a secondary alcohol to obtain the R configuration including obtaining JPEG2025523325000039.jpg3553, Here, in the process of reducing the ketone to a secondary alcohol, the reducing agent used includes at least one of lithium aluminum hydride, lithium borohydride, lithium chloride or sodium borohydride, the reaction medium includes at least one of dichloromethane, tetrahydrofuran, methanol or ethanol, and the reaction temperature is -78 to 0 °C, Preferably, the reaction of reducing the ketone to a secondary alcohol is to react 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 the above terminal olefin isomerization is a ruthenium catalyst, a palladium catalyst, a rhodium catalyst or an iridium catalyst, preferably including carbonylchlorohydridotris(triphenylphosphine)ruthenium(II), The reaction medium for the isomerization of the terminal olefin contains methanol, ethanol, n-butanol, or toluene, preferably ethanol, The reaction temperature for the isomerization of the terminal olefin is 60 to 100 °C, preferably 60 to 80 °C, The reaction time for the isomerization of the terminal olefin is 12 to 72 hours.

[0024] In a seventh aspect, the present invention provides a method for synthesizing a cyano-locked nucleic acid T phosphoramidite monomer at the C6' site in the R configuration or S configuration. The method for synthesizing a cyano-locked nucleic acid T phosphoramidite monomer at the C6' site in the R configuration involves deprotecting the 3'-hydroxy group and 5'-hydroxy group of JPEG2025523325000040.jpg4976 to obtain JPEG2025523325000041.jpg4775, protecting the 5'-hydroxy group with DMTr, and performing a phosphoramidation reaction on the 3'-hydroxy group to obtain R configuration JPEG2025523325000042.jpg6365, The method for synthesizing a cyano-locked nucleic acid T phosphoramidite monomer at the C6' site in the S configuration involves deprotecting the 3'-hydroxy group and 5'-hydroxy group of JPEG2025523325000043.jpg3553 to obtain JPEG2025523325000044.jpg3655, protecting the 5'-hydroxy group with DMTr, and performing a phosphoramidation reaction on the 3'-hydroxy group to obtain S configuration JPEG2025523325000045.jpg5658.

[0025] In an eighth aspect, the present invention provides a method for synthesizing a cyano-locked nucleic acid A phosphoramidite monomer at the C6' site in the R configuration or S configuration. The method for synthesizing a cyano-locked nucleic acid A phosphoramidite monomer at the C6' site in the R configuration involves protecting the base of JPEG2025523325000046.jpg3575 to Obtain JPEG2025523325000047.jpg3582, and then deprotect the 3'-hydroxy group and 5'-hydroxy group to Obtain JPEG2025523325000048.jpg3477, then protect the 5'-hydroxy group with DMTr, and perform a phosphoramidation reaction on the 3'-hydroxy group to obtain the R configuration Including obtaining JPEG2025523325000049.jpg4265, The method for synthesizing a cyano-locked nucleic acid A phosphoramidite monomer at the C6' site with the S configuration is the S configuration Protect the base of JPEG2025523325000050.jpg3172 to Obtain JPEG2025523325000051.jpg3588, and then deprotect the 3'-hydroxy group and 5'-hydroxy group to Obtain JPEG2025523325000052.jpg3080, then protect the 5'-hydroxy group with DMTr, and perform a phosphoramidation reaction on the 3'-hydroxy group to obtain the S configuration Including obtaining JPEG2025523325000053.jpg4265.

[0026] In the ninth aspect, the present invention provides a method for synthesizing a cyano-locked nucleic acid G phosphoramidite monomer at the C6' site with the R configuration or S configuration. The method for synthesizing a cyano-locked nucleic acid G phosphoramidite monomer at the C6' site with the R configuration is the R configuration Demethylate the methoxy group of JPEG2025523325000054.jpg3465 to Obtain JPEG2025523325000055.jpg3352, and then perform base protection to Obtain JPEG2025523325000056.jpg3664, then deprotect the 3'-hydroxy group and 5'-hydroxy group to Obtain JPEG2025523325000057.jpg3354, protect the 5'-hydroxy group with DMTr, and perform phosphoramidation on the 3'-hydroxy group to obtain the R configuration including obtaining JPEG2025523325000058.jpg4152, The synthesis method of the cyano - locked nucleic acid G phosphoramidite monomer at the C6' site in the S configuration is the S configuration demethylating the methoxy group of JPEG2025523325000059.jpg3361 to obtain JPEG2025523325000060.jpg3570, and then performing base protection to obtain JPEG2025523325000061.jpg3269, then deprotecting the 3'-hydroxy group and 5'-hydroxy group to obtain JPEG2025523325000062.jpg3473, protecting the 5'-hydroxy group with DMTr, and performing a phosphoramidation reaction on the 3'-hydroxy group to obtain the S configuration including obtaining JPEG2025523325000063.jpg4458.

[0027] In the tenth aspect, the present invention provides a method for synthesizing a cyano - locked nucleic acid C phosphoramidite monomer at the C6' site in the R configuration or S configuration. The preparation method of the cyano - locked nucleic acid C phosphoramidite monomer at the C6' site in the R configuration is the R configuration deprotecting the 3'-hydroxy group and 5'-hydroxy group of JPEG2025523325000064.jpg4668 to obtain JPEG2025523325000065.jpg3958, and then protecting the 5'-hydroxy group with DMTr to obtain JPEG2025523325000066.jpg4056, performing silanization modification on the 3'-hydroxy group, and converting the carbonyl group to an amino group to obtain JPEG2025523325000067.jpg3757, then performing base protection and deprotecting the 3'-hydroxy group to obtain JPEG2025523325000068.jpg3862, and finally performing a phosphoramidation reaction on the 3'-hydroxy group to obtain the R configuration including obtaining JPEG2025523325000069.jpg4552. The preparation method of the cyano-locked nucleic acid C phosphoramidite monomer at the C6' position in the S configuration is the S configuration Deprotect the 3'-hydroxy group and 5'-hydroxy group of JPEG2025523325000070.jpg3961 to Obtain JPEG2025523325000071.jpg3756, and then protect the 5'-hydroxy group with DMTr to Obtain JPEG2025523325000072.jpg4165. Next, perform a silylation modification on the 3'-hydroxy group and convert the carbonyl group to an amino group to Obtain JPEG2025523325000073.jpg3664. Then, perform base protection and deprotect the 3'-hydroxy group to Obtain JPEG2025523325000074.jpg4174. Finally, perform a phosphoramidation reaction on the 3'-hydroxy group to obtain the S configuration Including obtaining JPEG2025523325000075.jpg4657.

[0028] In a 11th aspect, the present invention provides a method for synthesizing a nucleic acid polymer, performing a polymerization reaction on a monomer to prepare a nucleic acid polymer, The monomer includes the 6'-cyano-modified locked nucleoside or the nucleotide.

Advantages of the Invention

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

[0030] The 6'-cyano-modified locked nucleoside provided by the present invention has an R configuration or an S configuration, and is further modified to obtain nucleotides and nucleic acid polymers. According to the research of the present inventors, the 6'-cyano-modified nucleic acid polymer has better nuclease resistance than unmodified or other modified nucleic acid polymers, reduces or regulates the interaction between the nucleic acid polymer and intracellular proteins, improves the nuclease resistance of the nucleic acid polymer, and reduces the interaction between the nucleic acid polymer and intracellular proteins. In terms of this, the 6'-cyano-modified nucleic acid polymer in the S configuration is more prominent than the R configuration. Therefore, it has been discovered that the 6'-cyano-modified locked nucleoside, nucleotides and nucleic acid polymers provided by the present invention have significant use value in nucleic acid pharmaceuticals.

[0031] The present invention provides a general preparation method for the above-mentioned 6'-cyano-modified locked nucleosides, nucleotides and nucleic acid polymers with R configuration or S configuration. The preparation method is stable, efficient and has a high yield.

[0032] In addition, the CN-LNA modified nucleic acid sequence obtained by the synthesis method of the present invention can significantly reduce the interference with the distribution of intracellular proteins compared with the LNA modification, and can reduce the apoptosis-inducing effect, and has significant advantages in the development of efficient and safe nucleic acid pharmaceuticals.

Brief Description of the Drawings

[0033] In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the drawings necessary for explaining the specific embodiments or the prior art will be briefly described below. Obviously, 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 drawings without creative effort.

[0034]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Mode for Carrying Out the Invention

[0035] Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the embodiments and examples. However, those skilled in the art should understand that the following embodiments and examples are only used to explain the present invention and should not be construed as limiting the scope of the present invention. Based on the examples of the present invention, all other examples obtained by those skilled in the art without creative efforts belong to the protection scope of the present invention. When specific conditions are not specified, operations are carried out according to normal conditions or conditions recommended by the manufacturer. When the reagents or equipment used are not specified by the manufacturer, all are ordinary products obtained by purchasing commercially available ones.

[0036] The term "nucleic acid polymer" can refer to any nucleic acid molecule including, but not limited to, DNA, RNA, and their hybrids, including single-stranded and double-stranded forms. The number of nucleotides polymerized to form a nucleic acid is 2, 3 or more, and it may be an oligonucleotide with 20 or fewer nucleotides, or a polymer with 20 or more nucleotides.

[0037] In a first aspect, the present invention provides a 6'-cyano-modified locked nucleoside, wherein the 6'-cyano-modified locked nucleoside is selected from a compound having a structure represented by Formula 1, a salt thereof, or an isomer thereof, In Formula 1, 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, [(triisopropylsilyl)oxy]methyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl, 9-phenylxanthin-9-yl, 9-(p-methoxyphenyl)xanthin-9-yl, or 2-naphthylmethyl, but is not limited thereto, The isomer includes a 6'-cyano-modified locked nucleoside in which the Z group is in the R configuration and has a structural formula of JPEG2025523325000077.jpg3743, or includes a 6'-cyano-modified locked nucleoside in which the Z group is in the S configuration and has a structural formula of JPEG2025523325000078.jpg3845.

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

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

[0040] The nucleotide provided by the present invention can improve the nuclease resistance of a nucleic acid polymer prepared using it as a monomer and reduce the interaction between the nucleic acid polymer and intracellular proteins.

[0041] In some embodiments, the nucleotide is selected from a compound having a structure represented by Formula 2, a salt thereof, or an isomer thereof, JPEG2025523325000079.jpg4775 The isomer includes a nucleotide in which the Z group is in the R configuration, and the structural formula is JPEG2025523325000080.jpg5352 or includes a nucleotide in which the Z group is in the S configuration, and the structural formula is JPEG2025523325000081.jpg5352.

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

[0043] According to the research of the present inventors, it has been discovered that a nucleic acid polymer prepared using the nucleotide provided by the present invention has a small interaction with intracellular proteins and is useful for reducing the in vivo toxicity of the nucleic acid polymer.

[0044] In a fourth aspect, the present invention provides a nucleic acid polymer, the nucleic acid polymer having monomers of the structure represented by Formula 3, In Formula 3 in JPEG2025523325000082.jpg4164, Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil or salts thereof, 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 another part of the nucleic acid polymer, and at least one of W3 and W4 is an internucleoside linking group that links the monomer to another part of the nucleic acid polymer, The hydroxy protecting group is selected from, but not limited to, 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, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl, 9-phenylxanthin-9-yl, 9-(p-methoxyphenyl)xanthin-9-yl or 2-naphthylmethyl.

[0045] The nucleic acid polymer provided by the present invention has better nuclease resistance than an unmodified or other modified nucleic acid polymer, and the nucleic acid polymer can reduce the interaction with intracellular proteins, reduce the influence on the intracellular distribution of intracellular proteins, and reduce apoptosis.

[0046] In some embodiments, the monomer includes a monomer with the Z group in the R configuration, and the structural formula is JPEG2025523325000083.jpg3946, or includes a monomer with the Z group in the S configuration, and the structural formula is JPEG2025523325000084.jpg3845.

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

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

[0049] The nucleic acid polymer provided by the present invention has better nuclease resistance than unmodified or other modified nucleic acid polymers, has low interaction with intracellular proteins, low apoptosis-inducing effect, low toxicity and side effects, and can be used in nucleic acid diagnostic agents or nucleic acid therapeutic agents.

[0050] In a sixth aspect, the present invention provides a method for synthesizing a cyano-modified nucleoside at the C6' position in the R configuration or the S configuration. The cyano-modified nucleoside at the C6' position in the R configuration is JPEG2025523325000085.jpg3753, and its synthesis method is Isomerize the terminal olefin of JPEG2025523325000086.jpg3255 to Obtain JPEG2025523325000087.jpg3561, and then perform a dihydroxylation reaction to Obtain JPEG2025523325000088.jpg3555. Next, obtain an aldehyde group compound JPEG2025523325000089.jpg3152 by an oxidative cleavage reaction, and finally, Convert the aldehyde group of JPEG2025523325000090.jpg3555 to a cyano group to synthesize a cyano-modified nucleoside at the C6' site with R configuration. Including synthesizing JPEG2025523325000091.jpg3652. The cyano-modified nucleoside at the C6' site with S configuration is JPEG2025523325000092.jpg3653, and the synthesis method thereof is Isomerize the terminal olefin of JPEG2025523325000093.jpg3357 to Obtain JPEG2025523325000094.jpg3762, and then perform a dihydroxylation reaction to Obtain JPEG2025523325000095.jpg4164, and then obtain an aldehyde group compound by an oxidative cleavage reaction Obtain JPEG2025523325000096.jpg3554, and finally Convert the aldehyde group of JPEG2025523325000097.jpg3148 to a cyano group to synthesize a cyano-modified nucleoside at the C6' site with S configuration. Including synthesizing JPEG2025523325000098.jpg3450. Here, Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil or their salts.

[0051] In the present invention, the reaction methods of isomerization of the terminal olefin, dihydroxylation, oxidative cleavage reaction and conversion of the aldehyde group to a cyano group are not specifically limited, and reaction methods well-known to those skilled in the art can be adopted. Using the cyano-modified nucleoside at the C6' site with R configuration or S configuration synthesized in the present invention as a raw material, it can be used for the synthesis of four types of cyano-modified locked nucleic acids with R configuration or S configuration.

[0052] Note that the aldehyde group compound, which is an intermediate product of the above reaction, can convert the aldehyde group into other groups containing alkanes, alcohols, carboxylic acids, alkynyl groups, alkenyl groups, amines, etc. by conventional chemical methods, and further realize various C6'-modified locked nucleic acids.

[0053] In some embodiments, the synthesis method of JPEG2025523325000099.jpg3350 includes performing a mesylation reaction, a de-subunit reaction, and an acetylation reaction sequentially on JPEG2025523325000100.jpg3351 to obtain JPEG2025523325000101.jpg3251, and then performing a glycosylation reaction and a nucleophilic substitution reaction to synthesize R-configuration JPEG2025523325000102.jpg3350. The synthesis method of JPEG2025523325000103.jpg3756 includes performing a mesylation reaction, a de-subunit reaction, and an acetylation reaction sequentially on JPEG2025523325000104.jpg3555 to obtain JPEG2025523325000105.jpg3555, and then performing a glycosylation reaction and a nucleophilic substitution reaction to synthesize S-configuration JPEG2025523325000106.jpg4264. The glycosylation reaction is to react JPEG2025523325000107.jpg37142 with thymine, N6-benzoyladenine or 6-chloroguanine at 50-100 °C under activator conditions, where the activator includes BSA and TMSOTf, and the reaction medium includes acetonitrile, 1,2-dichloroethane or toluene.

[0054] The above synthesis method provided by the present invention is stable and practical, without specifically restricting the mesylation reaction, de-Asian unit and acetylation reaction, as well as the nucleophilic substitution reaction. One may adopt the reaction methods well-known to those skilled in the art.

[0055] In some embodiments, the above S configuration The synthesis method of JPEG2025523325000108.jpg4163 is 5-O-(tert-butyldiphenylsilyl)-4-C-hydroxymethyl-1,2-O-isopropylidene-3-O-(2-naphthylmethyl)-α-D-ribofuranose Oxidize the primary alcohol of JPEG2025523325000109.jpg3551 to an aldehyde group, and then perform an allylation reaction on the aldehyde group to obtain the S configuration Including obtaining JPEG2025523325000110.jpg3350, Preferably, the above R configuration The synthesis method of JPEG2025523325000111.jpg4164 is Oxidize the secondary alcohol of JPEG2025523325000112.jpg3452 to a ketone, and then reduce the ketone to a secondary alcohol to obtain the R configuration Including obtaining JPEG2025523325000113.jpg3554, Here, in the process of reducing the ketone to a secondary alcohol, the reducing agent used includes metal hydrides such as lithium aluminum hydride, lithium borohydride, lithium chloride, or sodium borohydride. The reaction medium includes solvents such as dichloromethane, tetrahydrofuran, methanol, or ethanol. The reaction temperature is -78 to 0 °C. Preferably, the reaction of reducing the ketone to a secondary alcohol is to react 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 the terminal olefin isomerization includes a transition metal catalyst such as a ruthenium catalyst, a palladium catalyst, a rhodium catalyst or an iridium catalyst, preferably carbonylchlorohydridotris(triphenylphosphine)ruthenium(II). The reaction medium for the isomerization of the terminal olefin includes 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 research of the present inventors, it has been discovered that the isomerization of terminal olefins can be stably realized by using 2.5 to 5% mol of carbonylchlorohydridotris(triphenylphosphine)ruthenium(II) as a catalyst and ethanol as a reaction solvent without performing an anhydrous and anaerobic operation.

[0058] In a seventh aspect, the present invention provides a method for synthesizing a cyano-locked nucleic acid T phosphoramidite monomer at the C6' site in the R configuration or the S configuration. The method for preparing the cyano-locked nucleic acid T phosphoramidite monomer at the C6' site in the R configuration involves deprotecting the 3'-hydroxy group and 5'-hydroxy group of JPEG2025523325000114.jpg4163 to obtain JPEG2025523325000115.jpg3148, protecting the 5'-hydroxy group with DMTr, and performing a phosphoramidation reaction on the 3'-hydroxy group to obtain JPEG2025523325000116.jpg4847. The method for preparing the cyano-locked nucleic acid T phosphoramidite monomer at the C6' site in the S configuration involves deprotecting the 3'-hydroxy group and 5'-hydroxy group of JPEG2025523325000117.jpg3250 to Obtain JPEG2025523325000118.jpg3045, protect the 5'-hydroxy group with DMTr, and perform a phosphoramidation reaction on the 3'-hydroxy group to obtain the S configuration including obtaining JPEG2025523325000119.jpg3938.

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

[0060] In the eighth aspect, the present invention provides a method for synthesizing a cyano-locked nucleic acid A phosphoramidite monomer at the C6' site with R or S configuration. The method for synthesizing a cyano-locked nucleic acid A phosphoramidite monomer at the C6' site with R configuration involves protecting the base of JPEG2025523325000120.jpg3679 to obtain JPEG2025523325000121.jpg3170, then deprotecting the 3'-hydroxy group and 5'-hydroxy group to obtain JPEG2025523325000122.jpg3373, next protecting the 5'-hydroxy group with DMTr, and performing a phosphoramidation reaction on the 3'-hydroxy group to obtain the R configuration to obtain JPEG2025523325000123.jpg3656, including The method for synthesizing a cyano-locked nucleic acid A phosphoramidite monomer at the C6' site with S configuration involves protecting the base of JPEG2025523325000124.jpg3261 to obtain JPEG2025523325000125.jpg3177, then deprotecting the 3'-hydroxy group and 5'-hydroxy group to obtain JPEG2025523325000126.jpg3376, next protecting the 5'-hydroxy group with DMTr, and performing a phosphoramidation reaction on the 3'-hydroxy group to obtain the S configuration to obtain JPEG2025523325000127.jpg4060.

[0061] This synthetic method is stable and efficient and can be widely used in the synthesis of cyano-locked nucleic acid A phosphoramidite monomers at the C6' position in the R-configuration or S-configuration.

[0062] In a ninth aspect, the present invention provides a method for synthesizing a cyano-locked nucleic acid G phosphoramidite monomer at the C6' position in the R-configuration or S-configuration. The method for preparing a cyano-locked nucleic acid G phosphoramidite monomer at the C6' position in the R-configuration involves demethylating the methoxy group of JPEG2025523325000128.jpg3355 to obtain JPEG2025523325000129.jpg3456, and then performing base protection to obtain JPEG2025523325000130.jpg3357. Next, deprotecting the 3'-hydroxy group and 5'-hydroxy group to obtain JPEG2025523325000131.jpg3160, protecting the 5'-hydroxy group with DMTr, and performing a phosphoramidation reaction on the 3'-hydroxy group to obtain the R-configuration JPEG2025523325000132.jpg4354, including The method for preparing a cyano-locked nucleic acid G phosphoramidite monomer at the C6' position in the S-configuration involves demethylating the methoxy group of JPEG2025523325000133.jpg3469 to obtain JPEG2025523325000134.jpg3465, and then performing base protection to obtain JPEG2025523325000135.jpg3366. Next, deprotecting the 3'-hydroxy group and 5'-hydroxy group to obtain JPEG2025523325000136.jpg3474, protecting the 5'-hydroxy group with DMTr, and performing a phosphoramidation reaction on the 3'-hydroxy group to obtain the S-configuration JPEG2025523325000137.jpg3852, including

[0063] This synthesis method is stable and efficient and can be widely used in the synthesis of cyano-locked nucleic acid G phosphoramidite monomers at the C6' position in the R or S configuration.

[0064] In a tenth aspect, the present invention provides a method for synthesizing a cyano-locked nucleic acid C phosphoramidite monomer at the C6' position in the R or S configuration. The method for preparing a cyano-locked nucleic acid C phosphoramidite monomer at the C6' position in the R configuration is as follows: Deprotect the 3'-hydroxy group and 5'-hydroxy group of JPEG2025523325000138.jpg3755 to Obtain JPEG2025523325000139.jpg3857, and then protect the 5'-hydroxy group with DMTr to Obtain JPEG2025523325000140.jpg4156. Next, subject the 3'-hydroxy group to a silylation modification and convert the carbonyl group to an amino group to Obtain JPEG2025523325000141.jpg3453. Then, perform base protection and deprotect the 3'-hydroxy group to Obtain JPEG2025523325000142.jpg3456. Finally, perform a phosphoramidation reaction on the 3'-hydroxy group to obtain the R configuration Including obtaining JPEG2025523325000143.jpg4347, The method for preparing a cyano-locked nucleic acid C phosphoramidite monomer at the C6' position in the S configuration is as follows: Deprotect the 3'-hydroxy group and 5'-hydroxy group of JPEG2025523325000144.jpg3658 to Obtain JPEG2025523325000145.jpg4061, and then protect the 5'-hydroxy group with DMTr to Obtain JPEG2025523325000146.jpg3555. Next, subject the 3'-hydroxy group to a silylation modification and convert the carbonyl group to an amino group to Obtain JPEG2025523325000147.jpg3254. Then, perform base protection and deprotect the 3'-hydroxy group to Obtain JPEG2025523325000148.jpg3463, and finally, perform a phosphoramidation reaction on the 3'-hydroxy group to obtain the S configuration Including obtaining JPEG2025523325000149.jpg4255.

[0065] This synthetic method is stable and efficient and can be widely used for the synthesis of cyano-locked nucleic acid C-phosphoramidite monomers with the R or S configuration at the C6' site.

[0066] In a 11th aspect, the present invention provides a method for synthesizing a nucleic acid polymer, performing a polymerization reaction on a monomer to prepare a nucleic acid polymer, The monomer includes the 6'-cyano-modified locked nucleoside or the nucleotide.

[0067] In some preferred embodiments, the polymerization reaction includes the following. (1) Chemical synthesis: Using a nucleoside or mononucleotide as a raw material, synthesizing nucleic acids by organic chemical methods including the phosphodiester method, phosphotriester method, phosphite triester method, and solid-phase synthesis method. (2) Enzyme synthesis: Nucleic acid monomers or chemically synthesized small fragments can be ligated into large fragments by enzyme reactions.

[0068] Hereinafter, the present invention will be further described by specific examples, but it should be understood that these examples are only for explaining the present invention in more detail and are not intended to limit the present invention in any form.

[0069] Example 1, Stereoselective Synthesis of Cyano-Locked Nucleic Acid

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

[0071] Synthesis of R-6'-allyl-LNA JPEG2025523325000150.jpg34165Reaction conditions: (a) (1) 2-iodoxybenzoic acid, acetonitrile, reflux, 5 h; (2) allyltrimethylsilane, boron trifluoride ethyl ether solution, dichloromethane, -40 °C, 4 h; (b) methanesulfonyl chloride, DMAP, pyridine, room temperature, 16 h; (c) (1) FeCl3·6H2O, dichloromethane, 0 °C to room temperature, 9 h; (2) Ac2O, DMAP, pyridine, dichloromethane, room temperature, 2 h.

[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, the temperature was raised to reflux and reacted for 5 h, and the completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). The reaction was stopped, and after the reaction was cooled to room temperature, it was filtered through diatomaceous earth, the filter cake was washed twice with acetonitrile, the solvent was spin-dried and vacuum-dried to obtain 101 g of a pale yellow liquid, which did not need to be purified and the next reaction was carried out directly. The above aldehyde group compound was dissolved in 500 mL of dichloromethane, the temperature was lowered to -40 °C, boron trifluoride ethyl ether solution (22 mL, 0.217 mol) was added, and the mixture was stirred and reacted for 5 min. Next, allyltrimethylsilane (42.7 mL, 0.217 mol) was added dropwise, the temperature was maintained, and the mixture was stirred and reacted for 4 h. The completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). The reaction solution was quickly poured into 1 L of saturated aqueous sodium bicarbonate solution to quench it, 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 a yield of 100%. Without purification, the next reaction was carried out directly. 11H NMR (CDCl3, 400 MHz) δ 7.86 - 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 = 5.2 Hz, 1H), 4.70 - 4.66 (m, 2H), 4.47 (dd, J = 10.7, 1.7 Hz, 1H), 3.96 (d, J = 11.2 Hz, 1H), 3.80 (d, J = 11.2 Hz, 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 13C NMR (CDCl3, 100 MHz) δ 136.41, 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, 73.09, 72.56, 62.46, 34.65, 27.09, 26.73, 26.54, 19.15; ESI-MS (m / z) 637.37 [M-H] - .

[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 carried out at room temperature for 16 h, and the completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). 100 mL of methanol was added to the reaction solution, and the reaction was quenched by stirring for 10 min. After the reaction solution was concentrated under reduced pressure to remove excess pyridine, it was diluted with ethyl acetate and washed successively 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 a yield of 96%. 11H NMR (DMSO-d6, 400 MHz) δ 7.95 - 7.85 (m, 4H), 7.56 - 7.36 (m, 13H), 5.80 - 5.69 (m, 2H), 5.40 (dd, J = 7.6, 3.2 Hz, 1H), 5.03 - 4.93 (m, 4H), 4.69 (d, J = 12.4 Hz, 1H), 4.40 (d, J = 5.3 Hz, 1H), 3.75 (d, J = 11.0 Hz, 1H), 3.60 (d, J = 11.0 Hz, 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 13C NMR (CDCl3, 100 MHz) δ 135.59, 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. 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 resulting 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 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, 1N 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] + . JPEG2025523325000151.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 a 50 mL acetonitrile suspension of thymine (7.6 g, 60 mmol) was added BSA (29 mL, 120 mmol), and the mixture was stirred at room temperature for 20 min until thymine dissolved. After the system became transparent, 200 mL of an acetonitrile solution of diacetylated sugar 4 (23 g, 30 mmol) was added, followed by the 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 h to allow the reaction to proceed. Completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). Heating was stopped, and after the reaction solution cooled to room temperature, the reaction solution was poured into ethyl acetate, semi-saturated NaHCO3 solution (400 mL) was added, and the mixture was stirred for 20 min. The insoluble solid was filtered through diatomaceous earth, the organic layer was separated, and then it was washed successively 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 reacted at room temperature for 16 h. Completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 3). After the reaction solution was concentrated, the residue was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 40%) to obtain 8 g of locked nucleic acid derivative 5 as a white foamy solid in a yield of 39%. 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),4.74-4.71(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); 1313C NMR (CDCl3, 100 MHz) δ 163.60, 149.72, 135.44, 135.27, 134.23, 134.16, 133.64, 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 a 50 mL toluene suspension of N6-benzoyladenine (14 g, 60 mmol) was added BSA (29 mL, 120 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. Then, 170 mL of a toluene solution of diacetylated sugar 4 (23 g, 30 mmol) was added, followed by the rapid dropwise addition of TMSOTf (8.3 mL, 45 mmol). After the dropwise addition was complete, 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, semi-saturated NaHCO3 solution (400 mL) was added, and the mixture was stirred for 20 min. The insoluble solid was filtered through diatomaceous earth, the organic layer was separated, and then it was washed successively with water and saturated brine, dried over anhydrous Na2SO4, filtered, concentrated, and vacuum-dried. After that, the obtained residue was dissolved in 250 mL of methanol, K2CO3 (20.7 g, 150 mmol) was added, and the mixture was reacted at room temperature for 16 h. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 3). After the reaction solution was concentrated, the residue was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous Na2SO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 100%) to obtain 7.4 g of the locked nucleic acid derivative 6 as a white powdery solid with a yield of 35%. 11H NMR (CDCl3, 400 MHz) δ 8.28 (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 = 17.1 Hz, 1H), 5.04 (d, J = 10.2 Hz, 1H), 4.81 - 4.72 (m, 3H), 4.52 (s, 1H), 4.32 - 4.29 (m, 1H), 4.02 (d, J = 12.0 Hz, 1H), 3.92 (d, J = 12.0 Hz, 1H), 2.55 - 2.49 (m, 1H), 2.34 - 2.27 (m, 1H), 1.07 (s, 9H); 13 13C NMR (CDCl3, 100 MHz) δ 155.46, 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 To a 50 mL toluene suspension of 6-chloroguanine (10.2 g, 60 mmol) was added BSA (29 mL, 120 mmol), and the mixture was heated to 60 °C and reacted until 6-chloroguanine dissolved. After the system became transparent, the reaction solution was cooled to room temperature, and 170 mL of a toluene solution of diacetyl 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 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 NaSO4, filtered, concentrated, and dried under vacuum. The resulting residue was dissolved in 250 mL of methanol, K2CO3 (20.7 g, 150 mmol) was added, and the mixture was reacted at room temperature for 16 h. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 3). After the reaction solution was concentrated, the residue was extracted with ethyl acetate, washed successively 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 locked nucleic acid derivative 7 as a white foamy solid in a yield of 56%. 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,3H),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); 1313C NMR (CDCl3, 100 MHz) δ 161.49, 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 JPEG2025523325000152.jpg97165Reaction conditions: (a) (1) Carbonylchlorohydridotris(triphenylphosphine)ruthenium(II) (Wilkinson's catalyst), EtOH, 80 °C, 48 h; (b) Potassium osmate dihydrate, 50% NMO, t-BuOH, H2O, 60 °C, 8 h; (c) NaIO4, EtOH-THF (2 / 1 v / v), H2O, room temperature, 12 h; (d) NH4OH, I2, room temperature, 24 h.

[0078] 8 To a 50 mL anhydrous ethanol solution of allyl nucleoside derivative 5 (5.0 g, 7.26 mmol), carbonylchlorohydridotris(triphenylphosphine)ruthenium(II) (345 mg, 0.36 mmol, 5% mol) was added. The temperature was raised to 80 °C and the reaction was carried out for 48 h. After directly concentrating the reaction solution, 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 sequentially added. The temperature was raised to 60 °C and the reaction was carried out for 8 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After quenching the reaction with saturated sodium sulfite solution, it was 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 mixture of dihydroxylated isomer products. The above dihydroxylated product was dissolved in 60 mL of ethanol-tetrahydrofuran solution, and 10 mL of an aqueous solution of NaIO4 (2.33 g, 10.9 mmol) was added dropwise, and the mixture was stirred at room temperature for 12 h. After filtering the insoluble solid, 25% aqueous ammonia (5.5 mL, 72.6 mmol) was added to the filtrate, and then I2 (1.84 g, 7.26 mmol) was added in several portions and reacted at room temperature for 24 h. 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 a yield of 48%. 11H NMR (CDCl3, 400 MHz) δ 8.99, 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 = 6.6 Hz, 1H), 4.71 (d, J = 6.6 Hz, 1H), 4.24 (s, 1H), 4.17 (d, J = 6.9 Hz, 1H), 4.01 (d, J = 6.9 Hz, 1H), 1.63 (s, 3H), 1.10 (s, 9H); 13 13C NMR (CDCl3, 100 MHz) δ 163.50, 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 a 70 mL anhydrous ethanol solution of allyl nucleoside derivative 6 (7.0 g, 10 mmol) was added carbonylchlorohydridotris(triphenylphosphine)ruthenium(II) (475 mg, 0.5 mmol, 5% mol). The temperature was raised to 80 °C and the reaction was carried out for 48 h. After directly concentrating the reaction solution, 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 sequentially added. The temperature was raised to 60 °C and the reaction was carried out for 8 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After quenching the reaction with saturated sodium sulfite solution, it was 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 - 10%) to obtain 7.5 g of a mixture of dihydroxylated isomer products. The above dihydroxylated product was dissolved in 60 mL of an ethanol-tetrahydrofuran solution, and 15 mL of an aqueous solution of NaIO4 (3.2 g, 10.9 mmol) was added dropwise. The mixture was stirred at room temperature for 12 h. After filtering the insoluble solid, 25% aqueous ammonia (7.6 mL, 100 mmol) was added to the filtrate, and then I2 (2.54 g, 10 mmol) was added in several portions. The reaction was carried out at room temperature for 24 h. 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 with ethyl acetate / dichloromethane = 0 - 40% as the eluent to obtain 1.2 g of a slightly yellowish foamy solid product in a yield of 26%. 11H NMR (DMSO-d6, 400 MHz) δ 10.71 (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 = 12.0 Hz, 1H), 4.84 (d, J = 12.0 Hz, 1H), 4.79 (s, 1H), 4.21 (d, J = 12.2 Hz, 1H), 4.00 (d, J = 12.2 Hz, 1H), 0.94 (s, 9H); 13 13C NMR (DMSO-d6, 100 MHz) δ 157.14, 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 a 120 mL anhydrous ethanol solution of allyl nucleoside derivative 7 (11.0 g, 15.1 mmol), carbonylchlorohydridotris(triphenylphosphine)ruthenium(II) (718 mg, 0.76 mmol, 5% mol) was added. The temperature was raised to 80 °C and the reaction was carried out for 48 h. After directly concentrating the reaction solution, 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 sequentially added. The temperature was raised to 60 °C and the reaction was carried out for 8 h. Completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After quenching the reaction with saturated sodium sulfite solution, it was 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 mixture of dihydroxylation isomer products. The above dihydroxylation product was dissolved in 70 mL of an ethanol-tetrahydrofuran solution, and 15 mL of an aqueous solution of NaIO4 (3.4 g, 10.9 mmol) was added dropwise, followed by stirring at room temperature for 12 h. After filtering 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 portions and reacted at room temperature for 24 h. 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 10.4 g of a slightly yellowish foamy solid product in a yield of 37%. 1 H NMR (CDCl3, 400 MHz) δ 7.82 - 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 = 2.7 Hz, 2H), 4.50 (s, 1H), 4.14 (d, J = 12.0 Hz, 1H), 4.07 (s, 3H), 4.02 (d, J = 12.0 Hz, 1H), 1.10 (s, 9H);13 13C NMR (CDCl3, 100 MHz) δ 161.60, 159.29, 152.25, 136.27, 135.67, 135.54, 133.60, 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, 78.19, 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 JPEG2025523325000153.jpg89161 Reaction conditions: (a) (1) DDQ, dichloromethane - H2O (20 / 1), room temperature, 24 h; (2) Et3N, Et3N·3HF, THF, room temperature, 9 h; (b) DMTrCl, pyridine, room temperature, 12 h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite, 1H-tetrazole, room temperature, 5 h.

[0081] 11 At room temperature, DDQ (420 mg, 1.86 mmol) was added to a 10.5 mL dichloromethane-water (20:1) mixture of nucleoside derivative 8 (500 mg, 0.74 mmol), and the reaction was carried out at room temperature for 24 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After the reaction solution was concentrated under reduced pressure, it was extracted with ethyl acetate, washed successively with saturated sodium bisulfite solution, sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained residue was dissolved in 10 mL of tetrahydrofuran, triethylamine (260 μL, 1.86 mmol) and triethylamine hydrofluoride (363 μL, 2.23 mmol) were added successively, and the reaction was carried out at room temperature for 9 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). 600 mg of solid sodium bicarbonate was added to the reaction solution, and the mixture was stirred until no bubbles were generated. The reaction solution was concentrated under reduced pressure and directly purified by flash column chromatography (gradient elution: methanol / dichloromethane = 0 - 20%) to obtain 310 mg of a colorless amorphous solid in a yield of 46%. 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 To a 50 mL pyridine solution of cyano-locked nucleoside 11 (8.0 g, 27.1 mmol) was added 4,4'-dimethoxytrityl chloride (13.8 g, 40.6 mmol), and the mixture was stirred at room temperature for 12 h. 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 successively with water and saturated brine, dried over Na2SO4, concentrated, and purified by flash column chromatography (gradient elution: methanol / dichloromethane = 0 - 10%) to obtain 13.53 g of 5'-O-DMTr-protected nucleoside 12 as a pale yellow powder in a yield of 84%. 1 H NMR (400 MHz, DMSO-d6) δ 11.48 (s, 1H), 7.47 - 6.91 (m, 14H), 6.27 (d, J = 4.0 Hz, 1H), 5.63 (s, 1H), 5.01 (s, 1H), 4.52 (s, 1H), 3.42 (d, J = 4.4 Hz, 1H), 3.74 (s, 6H), 3.65 (d, J = 11.2 Hz, 1H), 3.36 (d, J = 11.2 Hz, 1H), 1.60 (d, J = 0.8 Hz, 3H); 13 C NMR (100 MHz, DMSO-d6) δ 163.81, 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 To a 40 mL 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 added 1H-tetrazole (376 mg, 5.36 mmol), and the mixture was reacted at room temperature for 5 h. The completion of the reaction was detected by TLC (ethyl acetate / dichloromethane = 5 / 1). Saturated NaHCO3 solution was added to the reaction mixture, and the mixture was extracted with dichloromethane, washed with saturated brine, dried over MgSO4, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 20%) to obtain 4.4 g of phosphoramidite 13 as a white foamy solid in a yield of 82%. 31 P NMR (152 MHz, DMSO-d6) δ 148.80, 148.75; ESI-MS (m / z) 798.32 [M + H] + . Synthesis of R-6'-CN-LNA-A phosphoramidite monomer JPEG2025523325000154.jpg67165 Reaction conditions: (a) BzCl, pyridine, room temperature, 6 h; (b) (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'-tetraisopropyl phosphorodiamidite, 1H-tetrazole, room temperature, 8 h.

[0084] 14 At room temperature, benzoyl chloride (1.52 mL, 13.2 mmol) was added dropwise to a 20 mL pyridine solution of nucleoside 9 (3.0 g, 4.4 mmol), and the mixture was reacted at room temperature for 6 h. The 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 value to 8 - 9, and the mixture was reacted at room temperature for 4 h. The reaction mixture was diluted with ethyl acetate, washed successively with water, 1N HCl solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 60%) to obtain 1.45 g of benzoyl-protected nucleoside 14 as a pale yellow solid in a yield of 42%. 11H NMR (CDCl3, 400 MHz) δ 8.62 (s, 1H), 8.20 (s, 1H), 8.07 (d, J = 7.0 Hz, 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 = 5.8 Hz, 1H), 4.16 (d, J = 12.5 Hz, 1H), 4.04 (d, J = 12.5 Hz, 1H), 1.07 (s, 9H); 13 13C NMR (CDCl3, 100 MHz) δ 164.60, 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 carried out at room temperature for 24 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After the reaction solution was concentrated under reduced pressure, it was extracted with ethyl acetate, washed successively with saturated sodium bisulfite 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, triethylamine (530 μL, 3.81 mmol) and triethylamine hydrofluoride (743 μL, 4.56 mmol) were added successively, and the reaction was carried out at room temperature for 12 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). 1.15 g of solid sodium bicarbonate was added to the reaction solution, and the mixture was stirred until no more bubbles were generated. The reaction solution was concentrated under reduced pressure and directly purified by flash column chromatography (gradient elution: dichloromethane / methanol = 0 - 15%) to obtain 400 mg of cyanolock nucleoside 15 in a 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)δ166.76,152.06,151.09,149.81,141.56,132.46,132.57,128.38,128.06,123.91,116.25,89.47,86.11,80.84,70.77,70.43,55.86;ESI-MS(m / z)409.15[M+H] + .

[0086] 16 At room temperature, 4,4'-dimethoxytrityl chloride (481 mg, 1.42 mmol) was added to a 5 mL pyridine solution of cyanolock nucleoside 15 (300 mg, 0.75 mmol), and the mixture was stirred at room temperature for 12 h. 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 successively with water and saturated brine, dried over Na2SO4, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0 - 45%) to obtain 500 mg of 5'-O-DMTr-protected cyanolock nucleoside 16 as a pale yellow solid in a yield of 94%. 1 H NMR (DMSO-d6, 400 MHz) δ 12.26 (s, 1H), 8.79 (s, 1H), 8.59 (s, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.67 (t, J = 7.4 Hz, 1H), 7.58 (t, J = 7.8 Hz, 2H), 7.46 (d, J = 7.4 Hz, 1H), 7.33 - 7.20 (m, 7H), 6.89 (d, J = 8.4 Hz, 4H), 6.33 (s, 1H), 6.30 (d, J = 4.3 Hz, 1H), 5.12 (s, 1H), 4.95 (s, 1H), 4.83 (d, J = 4.2 Hz, 1H), 3.73 (s, 6H), 3.68 (d, J = 11.2 Hz, 1H), 3.40 (d, J = 11.2 Hz, 1H); 13 C NMR (DMSO-d6, 100 MHz) δ 166.09, 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, 86.83, 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 a 10 mL 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 carried out at room temperature for 8 h. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 5 / 2). The reaction solution was directly purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 40%) to obtain 480 mg of phosphoramidite 17 as a slightly yellowish foamy solid in a yield of 83%. 31 P NMR (152 MHz, DMSO-d6) δ 149.19, 149.04; ESI-MS (m / z) 909.38 [M - H] - . Synthesis of R-6'-CN-LNA-G phosphoramidite monomer JPEG2025523325000155.jpg74154Reaction conditions: (a) 2N HCl, THF-CH3OH (1 / 1), 60 °C, 12 h; (b) isobutyryl chloride, Et3N, DMAP, 1,4-dioxane, 100 °C, 24 h; (c) (1) DDQ, dichloromethane-H2O (20 / 1), room temperature, 24 h; (2) Et3N, Et3N·3HF, THF, room temperature, 12 h; (d) DMTrCl, pyridine, room temperature, 12 h; (e) 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite, 1H-tetrazole, room temperature, 12 h.

[0088] 18 To a 30 mL solution of cyano-locked nucleoside 10 (2.5 g, 3.5 mmol) in tetrahydrofuran-methanol (1 / 1 v / v) was added 17.5 mL of 2N hydrochloric acid solution. The temperature was raised to 60 °C and the reaction was carried out for 12 h. The precipitation of a white solid was detected by TLC (dichloromethane / ethyl acetate = 2 / 1) to indicate the completion of the reaction. The insoluble white solid was filtered off, and the filtrate was extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the filtrates were combined and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0 - 60%) to obtain 1.4 g of the 6-site demethylated nucleoside 18 as a white solid in a yield of 57%. 1 H NMR (DMSO-d6, 400 MHz) δ 10.71 (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 = 12.0 Hz, 1H), 4.84 (d, J = 12.0 Hz, 1H), 4.78 (s, 1H), 4.21 (d, J = 12.2 Hz, 1H), 4.01 (d, J = 12.2 Hz, 1H), 0.94 (s, 9H); 13 C NMR (DMSO-d6, 100 MHz) δ 157.13, 154.49, 151.10, 135.62, 135.60, 135.29, 134.48, 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, 72.02, 71.23, 59.76, 26.80, 19.20; ESI-MS (m / z) 699.39 [M + H]+, 721.48 [M + Na] + .

[0089] 19 To a 12 mL 1,4-dioxane solution of guanosine 18 (1.2 g, 1.72 mmol) were added DMAP (105 mg, 0.86 mmol) and triethylamine (718 μL, 5.16 mmol). Next, isobutyryl chloride (550 μL, 5.16 mmol) was added, and the temperature was raised to 100 °C and reacted for 24 h. Completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After stopping heating and cooling the reaction solution to room temperature, 1 mL of methanol was added to quench the reaction, and it was extracted with ethyl acetate, washed successively 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 a yield of 83%. 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 a 10.5 mL mixture of dichloromethane - water (20:1) of nucleoside derivative 19 (800 mg, 1.04 mmol), and the reaction was carried out at room temperature for 24 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After the reaction solution was concentrated under reduced pressure, it was extracted with ethyl acetate, washed successively with saturated sodium bisulfite solution, sodium hydrogen carbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained residue was dissolved in 10 mL of tetrahydrofuran, triethylamine (363 μL, 2.60 mmol) and triethylamine hydrofluoride (509 μL, 3.12 mmol) were added successively, and the reaction was carried out at room temperature for 12 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). 786 mg of solid sodium hydrogen carbonate was added to the reaction solution, stirred until no bubbles were generated, the reaction solution was concentrated under reduced pressure, and directly purified by flash column chromatography (gradient elution: dichloromethane / methanol = 0 - 25%) to obtain 230 mg of cyanolock nucleoside 20 in a yield of 57%. 1 1H NMR (CD3OD, 400 MHz) δ 8.09 (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 13C NMR (CD3OD, 100 MHz) δ 180.35, 148.67, 148.20, 136.57, 120.03, 116.22, 89.37, 85.62, 80.91, 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] 21 At room temperature, 4,4'-dimethoxytrityl chloride (260 mg, 0.77 mmol) was added to a 5 mL pyridine solution of cyanolock nucleoside 20 (150 mg, 0.384 mmol), and the mixture was stirred at room temperature for 12 h. 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 successively with water and saturated brine, dried over Na2SO4, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0 - 40%) to obtain 270 mg of 5'-O-DMTr-protected cyanolock nucleoside 21 as a pale yellow powder in a yield of 100%. 1 H NMR (DMSO-d6, 400 MHz) δ 12.14 (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, 1.4 Hz, 4H), 6.26 (d, J = 4.2 Hz, 1H), 6.08 (s, 1H), 5.08 (s, 1H), 4.77 (s, 1H), 4.57 (d, J = 4.2 Hz, 1H), 3.74 (s, 6H), 3.68 (d, J = 11.2 Hz, 1H), 3.39 (d, J = 11.2 Hz, 1H), 2.82 - 2.75 (m, 1H), 1.14 (d, J = 6.8 Hz, 6H); 13 C NMR (DMSO-d6, 100 MHz) δ 180.73, 158.66, 155.22, 148.89, 148.58, 144.97, 136.87, 135.59, 135.40, 130.34, 130.31, 128.33, 128.21, 127.27, 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 [M - H] - .

[0092] 22 At room temperature, 1H-tetrazole (15 mg, 0.21 mmol) was added to a 10 mL dichloromethane solution of nucleoside 21 (180 mg, 0.26 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (157 mg, 0.52 mmol), and the reaction was carried out at room temperature for 12 h. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 2 / 1). The reaction solution was directly purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 40%) to obtain 180 mg of phosphoramidite 22 as a slightly yellowish wax-like solid in a yield of 78%. 31 P NMR (152 MHz, DMSO-d6) δ 149.26, 149.23; ESI-MS (m / z) 893.40 [M + H] + . R-6'-CN-LNA- m Synthesis of C-phosphoramidite monomer JPEG2025523325000156.jpg91170Reaction conditions: (a) (1) TESCl, Et3N, acetonitrile, 0 °C to room temperature, 3 h; (2) 1,2,4-triazole, POCl3, acetonitrile, 0 °C to room temperature, 2 h; (3) NH4OH, 1,4-dioxane, room temperature, 2 h; (b) (1) Bz2O, acetonitrile, room temperature, 20 h; (2) TBAF, THF, room temperature, 12 h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite, 1H-tetrazole, dichloromethane, room temperature, 5 h.

[0093] 23 At 0 °C, triethylsilyl chloride (7.0 mL, 41.8 mmol) was added to a solution of nucleotide 12 (5.0 g, 8.37 mmol) and triethylamine (23.3 mL, 167 mmol) in acetonitrile (100 mL), and the mixture was reacted at room temperature for 3 h. 1,2,4-Triazole (8.67 g, 125.55 mmol) was added to the reaction solution, and after stirring for 10 min, the mixture was cooled to 0 °C. POCl3 (2.34 mL, 25.11 mmol) was added dropwise to the reaction solution, and the reaction was continued at room temperature for 2 h. Completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). The reaction solution was poured into 300 mL of ice water and extracted with ethyl acetate. The organic layer was washed successively with water, saturated sodium bicarbonate solution, and saturated brine, 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 h. After concentration under reduced pressure, the reaction solution 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 chromatography (gradient elution: methanol / dichloromethane = 0 - 15%) to obtain 5.5 g of aminonucleoside 23 as a pale yellow solid in a yield of 92%. 1 H NMR (DMSO-d6, 400 MHz) δ 7.52 - 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 = 11.2 Hz, 1H), 3.41 (d, J = 11.2 Hz, 1H), 1.72 (s, 3H), 0.78 (t, J = 7.9 Hz, 9H), 0.53 - 0.41 (m, 6H); 13 C NMR (DMSO-d6, 100 MHz) δ 166.09, 158.81, 158.79, 154.95, 144.78, 136.54, 135.38, 135.17, 130.30, 130.14, 128.43, 128.07, 127.46, 117.58, 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] 24 To a solution of aminonucleoside 23 (5.5 g, 7.73 mmol) in acetonitrile (60 mL) was added benzoic anhydride (3.85 g, 12 mmol). After stirring at room temperature for 20 h, the reaction was quenched with 5 mL of water. TBAF (4.38 g, 19.3 mmol) was added to the reaction mixture. After stirring at room temperature for 12 h, 15% NaOH was added to adjust the pH to 10, and stirring was continued for 3 h. The reaction solution was diluted with ethyl acetate, washed with water, and 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 a yield of 57%. 1 H NMR (DMSO-d6, 400 MHz) δ 13.06 (br s, 1H), 8.19 (s, 2H), 7.69 - 7.25 (m, 13H), 6.94 (d, J = 8.3 Hz, 4H), 6.32 (s, 1H), 5.70 (s, 1H), 5.06 (s, 1H), 4.53 (s, 1H), 4.38 (d, J = 4.1 Hz, 1H), 3.73 (d, J = 11.0 Hz, 1H), 3.40 (d, J = 11.0 Hz, 1H), 1.84 (s, 3H); 13 C NMR (DMSO-d6, 100 MHz) δ 178.64, 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] 25 To a 20 mL anhydrous dichloromethane solution of nucleoside 24 (1.7 g, 2.42 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (1.1 g, 3.63 mmol) was added 1H-tetrazole (135 mg, 1.94 mmol). After reacting at room temperature for 5 h, saturated NaHCO3 solution was added to the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was washed with saturated brine, dried over MgSO4, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 20%) to obtain 1.83 g of phosphoramidite 25 as a white foamy solid in a yield of 84%. 31 P NMR (152 MHz, 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 JPEG2025523325000157.jpg84166 Reaction conditions: (a) 2-iodoxybenzoic acid, acetonitrile, reflux, 11 h; (b) NaBH4, LiCl, MeOH, -40 °C, 8 h; (c) methanesulfonyl chloride, DMAP, pyridine, room temperature, 32 h; (d) (1) FeCl3·6H2O, dichloromethane, 0 °C to room temperature, 3 h; (2) Ac2O, DMAP, pyridine, dichloromethane, room temperature, 4 h.

[0098] 26 To a 500 mL acetonitrile solution of compound 2 (105 g, 0.164 mol) was added 2-iodoxybenzoic acid (69 g, 0.25 mol). The temperature was raised and the mixture was refluxed for 6 h. Then, 2-iodoxybenzoic acid (13.8 g, 0.05 mol) was added and the reflux reaction was continued for 5 h. Completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). The reaction solution was filtered through diatomaceous earth, and after concentrating the filtrate, it was purified by flash column chromatography (gradient elution: ethyl acetate / petroleum ether = 0 - 20%) to obtain 93 g of colorless viscous substance 26 in a yield of 89%. 11H NMR (CDCl3, 400 MHz) δ 7.85 - 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 = 4.0 Hz, 1H), 5.98 - 5.83 (m, 1H), 5.13 - 5.01 (m, 2H), 4.90 (d, J = 12.0 Hz, 1H), 4.86 (t, J = 8.0, 4.0 Hz, 1H), 4.63 (d, J = 12.0 Hz, 1H), 4.23 (d, J = 8.0 Hz, 1H), 3.92 (d, J = 12.0 Hz, 1H), 3.72 (d, J = 12.0 Hz, 1H), 3.66 - 3.55 (m, 2H), 1.61 (s, 3H), 1.43 (s, 3H), 0.98 (s, 9H); 13 13C NMR (CDCl3, 100 MHz) δ 207.44, 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, 200 mL of a methanol solution of lithium chloride (18 g, 0.424 mol) was added to 400 mL of a tetrahydrofuran solution of compound 26 (108 g, 0.17 mol). After cooling the reaction solution to -40 °C, sodium borohydride (4.3 g, 0.113 mol) was added, and the reaction was carried out for 4 h. Then, sodium borohydride (4.3 g, 0.113 mol) was added again, and the reaction was continued for 4 h. The completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). The reaction solution was poured into a beaker, quenched with saturated ammonium chloride solution (100 mL), stirred until no 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 with a yield of 83%. 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)δ136.40,135.53,135.51,134.80,133.22,132.16,132.88,132.74,129.85,129.78,128.41,127.94,127.78,126.85,126.29,126.14,125.67,161.13,114.38,104.73,90.15,79.74,78.12,72.80,71.04,64.51,35.91,26.98,26.81,26.75,19.11;ESI - MS(m / z)637.41[M - H] - .

[0100] 28 Compound 27 (90 g, 0.14 mol) and DMAP (1.71 g, 14 mmol) were dissolved in 400 mL of pyridine, methanesulfonyl chloride (16.4 mL, 0.21 mmol) was added dropwise, and the mixture was reacted at room temperature for 20 h. Then, methanesulfonyl chloride (5.5 mL, 0.07 mol) was added and the reaction was continued for 12 h. The completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). 50 mL of methanol was added to the reaction solution to quench the reaction, and the mixture was concentrated under reduced pressure to remove the excess pyridine. The residue was diluted with ethyl acetate and washed successively with water, 1N HCl solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and 100 g of methanesulfonyl compound 28 was obtained as a slightly yellow viscous substance in a yield of 100%. 1 H NMR (DMSO-d6, 400 MHz) δ 7.95 - 7.87 (m, 4H), 7.59 - 7.38 (m, 13H), 5.85 (d, J = 4.0 Hz, 1H), 5.68 - 5.58 (m, 1H), 5.13 (dd, J = 9.8, 1.4 Hz, 1H), 5.07 (t, J = 5.0 Hz, 1H), 4.98 - 4.91 (m, 3H), 4.69 (d, J = 11.8 Hz, 1H), 4.47 (d, J = 5.4 Hz, 1H), 3.76 (d, J = 10.8 Hz, 1H), 3.71 (d, J = 10.8 Hz, 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, 100 MHz) δ 135.54, 135.59, 135.30, 134.36, 133.24, 133.15, 132.72, 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 A 1000 mL dichloromethane solution of Compound 28 (100 g, 0.14 mol) was cooled to 0 °C. After adding iron(III) chloride hexahydrate (15.1 g, 56 mmol), the mixture was reacted at room temperature for 3 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 successively washed 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 1 L of dichloromethane, pyridine (113 mL, 1.4 mol) and DMAP (2.56 g, 21 mol) were successively added, and then acetic anhydride (79 mL, 0.84 mol) was added dropwise. The mixture was reacted at room temperature for 4 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 successively washed with water, 1N 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 95 g of a diacetyl compound 29 as an epimer mixture as a colorless viscous substance in a yield of 89%. ESI-MS (m / z) 778.39 [M+NH4] + ,783.44 [M+Na] + . JPEG2025523325000158.jpg105166Reaction conditions: (a) thymine, N,O-bis(trimethylsilyl)acetamide (BSA), TMSOTf, acetonitrile, 80 °C, 3 h; (b) K2CO3, MeOH, room temperature, 16 h; (c) N6-benzoyladenine, BSA, TMSOTf, toluene, 100 °C, 3 h; (d) 6-chloroguanine, BSA, TMSOTf, toluene, 100 °C, 3 h;

[0102] 30 To a 50 mL acetonitrile suspension of thymine (7.6 g, 60 mmol) was added BSA (29 mL, 120 mmol), and the mixture was stirred at room temperature for 20 min until thymine dissolved. After the system became transparent, 200 mL of an acetonitrile solution of diacetyl sugar 29 (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 80 °C and the mixture was stirred for 3 h to allow the reaction to proceed. Completion of the reaction was detected by TLC (petroleum ether / ethyl acetate = 5 / 1). Heating was stopped, and after the reaction solution had 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 NaSO4, filtered, concentrated, and dried under vacuum. The resulting residue was dissolved in 250 mL of methanol, K2CO3 (12.4 g, 90 mmol) was added, and the mixture was reacted at room temperature for 16 h. Completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 3). After the reaction solution was concentrated, the residue was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 25%) to obtain 15.4 g of the locked nucleic acid derivative 30 as a white foamy solid in a yield of 75%. 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 was 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, semi-saturated NaHCO3 solution (400 mL) was added, and the mixture was stirred for 20 min. 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, vacuum dried, and then purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 40%) to obtain 9.1 g of the nucleoside product 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 carried out at room temperature for 16 h. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 3). After concentrating the reaction solution, the residue was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 100%) to obtain 4.9 g of the locked nucleic acid derivative 31 as a white powdery solid in a yield of 78%. 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 = 11.6Hz,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 a 25 mL toluene suspension of 6-chloroguanine (3.34 g, 19.7 mmol) was added BSA (13 mL, 52.6 mmol), and the mixture was heated to 60 °C and reacted until 6-chloroguanine dissolved. After the system became transparent, the reaction solution was cooled to room temperature. Then, 75 mL of a toluene solution of diacetyl sugar 29 (10 g, 13.1 mmol) was added, followed by the rapid dropwise addition of TMSOTf (3.6 mL, 19.7 mmol). 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 dried under vacuum. The obtained residue was dissolved in 100 mL of methanol, K2CO3 (7.25 g, 52.6 mmol) was added, and the mixture was reacted at room temperature for 16 h. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 3). After the reaction solution was concentrated, the residue was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous Na2SO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 30%) to obtain 6.07 g of locked nucleic acid derivative 32 as a white foamy solid with a yield of 63%. 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 JPEG2025523325000159.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 a 120 mL anhydrous ethanol solution of allyl nucleoside derivative 30 (10.0 g, 14.5 mmol) was added carbonylchlorohydridotris(triphenylphosphine)ruthenium(II) (345 mg, 0.36 mmol, 2.5% mol). The temperature was raised to 80 °C and the reaction was carried out for 12 h. After directly concentrating the reaction solution, the residue was dissolved in 80 mL of tetrahydrofuran. Then 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 sequentially added. The temperature was raised to 60 °C and the reaction was carried out for 8 h. Completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After quenching the reaction with saturated sodium sulfite solution, it was 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 9.3 g of a mixture of dihydroxylation isomer products. The above dihydroxylation product was dissolved in 60 mL of an ethanol-tetrahydrofuran solution. A 10 mL aqueous solution of NaIO4 (3.32 g, 15.5 mmol) was added dropwise, and the mixture was stirred at room temperature for 12 h. After filtering 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 carried out at room temperature for 24 h. 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 in a yield of 37%. 11H NMR (CDCl3, 400 MHz) δ 8.87 (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 = 1.2 Hz, 1H), 5.62 (s, 1H), 4.93 (d, J = 11.8 Hz, 1H), 4.85 (s, 1H), 4.76 (d, J = 11.8 Hz, 1H), 4.69 (s, 1H), 4.30 (d, J = 12.2 Hz, 1H), 4.25 (d, J = 12.2 Hz, 1H), 4.07 (s, 1H), 1.58 (d, J = 1.2 Hz, 3H), 1.08 (s, 9H); 13 13C NMR (CDCl3, 100 MHz) δ 163.31, 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 a 50 mL ethanol solution of allyl nucleoside derivative 31 (4.9 g, 7.0 mmol), carbonylchlorohydridotris(triphenylphosphine)ruthenium(II) (167 mg, 0.175 mmol, 2.5% mol) was added, and the temperature was raised to 80 °C and reacted for 36 h. After directly concentrating the reaction solution, the residue was dissolved in 40 mL of tetrahydrofuran, 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 sequentially added, and the temperature was raised to 60 °C and reacted for 8 h. After quenching the reaction with saturated sodium sulfite solution, it was 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 containing 5% methanol = 0 - 100%) to obtain 3.6 g of a mixture of dihydroxylated isomer products. The above dihydroxylated product was dissolved in 40 mL of an ethanol-tetrahydrofuran solution, 8 mL of an aqueous solution of NaIO4 (1.58 g, 7.4 mmol) was added dropwise, and stirred at room temperature for 12 h. After filtering the insoluble solid, 25% aqueous ammonia (3.05 mL, 49.2 mmol) was added to the filtrate, and then I2 (1.25 g, 4.92 mmol) was added in several portions and reacted at room temperature for 24 h. 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 a white foamy solid product in a yield of 48%. 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); 1313C NMR (CDCl3, 100 MHz) δ 155.43, 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 a 120 mL anhydrous ethanol solution of allyl nucleoside derivative 32 (7.2 g, 10 mmol), carbonylchlorohydride tris(triphenylphosphine)ruthenium(II) (238 mg, 0.25 mmol, 2.5% mol) was added, and the temperature was raised to 80 °C and reacted for 48 h. After directly concentrating the reaction solution, the residue was dissolved in 50 mL of tetrahydrofuran, 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 sequentially added, and the temperature was raised to 60 °C and reacted for 8 h. Completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After quenching the reaction with saturated sodium bisulfite solution, it was 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 5.6 g of a mixture of dihydroxylation isomers. The above dihydroxylated product was dissolved in 60 mL of an ethanol-tetrahydrofuran solution, and 10 mL of an aqueous solution of NaIO4 (3.54 g, 7.35 mmol) was added dropwise. The mixture was stirred at room temperature for 12 h. After filtering off 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. The reaction was allowed to proceed at room temperature for 24 h. 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 chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 40%) to obtain 2.5 g of a white foamy solid product in a yield of 48%. 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),4.81-4.76(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)δ161.63,159.37,152.27,136.01,135.60,135.58,133.75,133.11,132.19,132.16,130.13,128.49,127.99,127.96,127.69,126.67,126.31,126.23,125.38,115.99,114.61,89.67,86.10,78.31,77.64,72.91,70.05,59.41,53.97,26.70,19.25;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 JPEG2025523325000160.jpg92165Reaction 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'-tetraisopropylphosphorodiamidite, 1H-tetrazole, room temperature, 5 h.

[0108] 36 At room temperature, DDQ (420 mg, 1.86 mmol) was added to a 42 mL mixture of dichloromethane-water (20:1) of nucleoside derivative 33 (3.6 g, 5.34 mmol), and the reaction was carried out at room temperature for 24 h. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 10 / 1). After the reaction solution was concentrated under reduced pressure, it was extracted with ethyl acetate, washed successively with saturated sodium bisulfite solution, sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained residue was dissolved in 50 mL of tetrahydrofuran, triethylamine (1.78 mL, 13.35 mmol) and triethylamine trihydrofluoride (2.61 mL, 16 mmol) were added successively, and the reaction was carried out at room temperature for 12 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). 10 g of solid sodium bicarbonate was added to the reaction solution, and the mixture was stirred until no bubbles were generated. The reaction solution was concentrated under reduced pressure and directly purified by flash column chromatography (gradient elution: methanol / dichloromethane = 0-20%) to obtain 1.1 g of a slightly yellowish white solid 36 in a yield of 70%. 1 1H-NMR (400 MHz, MeOH-d4) δ 7.62 (s, 1H), 5.86 (s, 1H), 4.78 (s, 1H), 4.53 (s, 1H), 4.22 (s, 1H), 4.17 (d, J = 12.0 Hz, 1H), 4.13 (d, J = 12.0 Hz, 1H), 1.89 (s, 3H); 13 13C-NMR (100 MHz, MeOH-d4) δ 164.97, 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 a 15 mL pyridine solution of cyano-locked nucleoside derivative 36 (2.0 g, 6.77 mmol) was added 4,4'-dimethoxytrityl chloride (3.4 g, 10 mmol), and the mixture was stirred at room temperature for 12 h. 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 successively with water and saturated brine, dried over Na2SO4, concentrated, and purified by flash column chromatography (gradient elution: methanol / dichloromethane = 0 - 10%) to obtain 2.6 g of 5'-O-DMTr protected nucleoside 37 as a white solid in a yield of 64%. 1 1H-NMR (400 MHz, DMSO-d6) δ 11.44 (s, 1H), 7.46 - 7.24 (m, 10H), 6.92 (dd, J = 6.0, 1.4 Hz, 4H), 6.30 (s, 1H), 5.53 (s, 1H), 5.11 (s, 1H), 3.79 (d, J = 7.9 Hz, 1H), 3.74 (s, 6H), 3.49 (d, J = 7.4 Hz, 1H), 1.60 (s, 3H); 13 13C-NMR (100 MHz, DMSO-d6) δ 162.66, 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-MS 620.20 (m / z) [M+Na] + .

[0110] 38 To a 20 mL 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 added 1H-tetrazole (230 mg, 3.35 mmol), and the reaction was carried out at room temperature for 5 h. The completion of the reaction was detected by TLC (ethyl acetate / dichloromethane = 5 / 1). Saturated NaHCO3 solution was added to the reaction solution, extracted with dichloromethane, washed with saturated brine, 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 a yield of 79%. 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 JPEG2025523325000161.jpg69164Reaction conditions: (a) BzCl, pyridine, room temperature, 6 h; (b) (1) DDQ, dichloromethane-H2O (20 / 1), room temperature, 24 h; (2) Et3N, Et3N·3HF, THF, room temperature, 12 h; (b) DMTrCl, pyridine, room temperature, 8 h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite, 1H-tetrazole, room temperature, 8 h.

[0111] 39 At room temperature, benzoyl chloride (422 μL, 3.66 mmol) was added dropwise to a 5 mL pyridine solution of nucleoside 34 (1.0 g, 1.46 mmol), and the reaction was carried out at room temperature for 4 h. Then, benzoyl chloride (100 μL, 0.87 mmol) was added to the reaction solution and the reaction was continued for 2 h. 15% NaOH was added dropwise to the reaction solution to adjust the pH value to 8, and the mixture was stirred at room temperature for 2 h. Diluted with ethyl acetate, washed successively with water, 1N HCl solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (gradient elution: ethyl acetate / dichloromethane = 0 - 45%) to obtain 1.05 g of a slightly yellowish foamy solid in a yield of 91%. 11H NMR (CDCl3, 400 MHz) δ 9.02 (br s, 1H), 8.65 (s, 1H), 8.03 (d, J = 7.2 Hz, 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 = 12.2 Hz, 1H), 4.82 (d, J = 12.2 Hz, 1H), 4.34 - 4.31 (m, 2H), 4.19 (d, J = 12.2 Hz, 1H), 1.06 (s, 9H); 13 13C NMR (CDCl3, 100 MHz) δ 164.60, 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 a 10 mL mixture of dichloromethane - water (20:1) of nucleoside derivative 39 (900 mg, 1.14 mmol), and the reaction was carried out at room temperature for 24 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After the reaction solution was concentrated under reduced pressure, it was extracted with ethyl acetate, washed successively with saturated sodium bisulfite solution, sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained residue was dissolved in 15 mL of tetrahydrofuran, triethylamine (400 μL, 2.85 mmol) and triethylamine hydrofluoride (550 μL, 3.42 mmol) were added successively, and the reaction was carried out at room temperature for 12 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). 1.0 g of solid sodium bicarbonate was added to the reaction solution, and it was stirred until no bubbles were generated. The reaction solution was concentrated under reduced pressure and directly purified by flash column chromatography (gradient elution: dichloromethane / methanol = 0 - 20%) to obtain 340 mg of cyanolock nucleoside 40 in a 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, 4,4'-dimethoxytrityl chloride (373 mg, 1.1 mmol) was added to a 5 mL pyridine solution of cyanolock nucleoside 40 (300 mg, 0.735 mmol), and the mixture was stirred at room temperature for 8 h. 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 successively with water and saturated brine, dried over Na2SO4, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0 - 80%) to obtain 330 mg of 5'-O-DMTr protected cyanolock nucleoside 41 as a pale yellow solid in a yield of 64%. 11H NMR (DMSO-d6, 400 MHz) δ 11.26 (br s, 1H), 8.79 (s, 1H), 8.54 (s, 1H), 8.06 (d, J = 7.3 Hz, 2H), 7.67 (t, J = 7.3 Hz, 1H), 7.57 (t, J = 7.8 Hz, 2H), 7.46 (d, J = 7.4 Hz, 2H), 7.34 - 7.24 (m, 6H), 7.25 (t, J = 7.2 Hz, 1H), 6.90 (d, J = 8.9 Hz, 4H), 6.33 (d, J = 3.5 Hz, 1H), 6.23 (s, 1H), 5.36 (s, 1H), 4.92 (s, 1H), 4.70 (d, J = 3.5 Hz, 1H), 3.85 (d, J = 11.1 Hz, 1H), 3.74 (s, 6H), 3.47 (d, J = 11.1 Hz, 1H); 13 13C NMR (DMSO-d6, 100 MHz) δ 166.10, 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 a 10 mL dichloromethane solution of nucleoside 41 (240 mg, 0.34 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (203 mg, 0.75 mmol), and the mixture was reacted at room temperature for 8 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 15 / 1). The reaction solution was directly purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 40%) to obtain 280 mg of phosphoramidite 42 as a slightly pale yellow foamy solid in a yield of 90%. 31 31P NMR (152 MHz, DMSO-d6) δ 149.56, 149.25; ESI-MS (m / z) 909.38 [M - H] - . Synthesis of S-6'-CN-LNA-G Phosphoramidite Monomer JPEG2025523325000162.jpg79166Reaction conditions: (a) 2N HCl, THF-CH3OH (1 / 1), 60 °C, 24 h; (b) isobutyryl chloride, Et3N, DMAP, toluene, 100 °C, 12 h; (c) (1) DDQ, dichloromethane-H2O (20 / 1), room temperature, 24 h; (2) Et3N, Et3N·3HF, THF, room temperature, 12 h; (d) DMTrCl, pyridine, room temperature, 12 h; (e) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite, 1H-tetrazole, room temperature, 6 h.

[0115] 43 15.4 mL of 2N hydrochloric acid solution was added to 24 mL of a tetrahydrofuran-methanol solution (1 / 1 v / v) of cyanolocked nucleoside 35 (2.2 g, 3.08 mmol), and the temperature was raised to 60 °C and reacted for 24 h. Completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 2 / 1). Extracted with ethyl acetate, washed with water and saturated brine, 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 a yield of 69%. 1 H NMR (CDCl3, 400 MHz) δ 11.94 (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 = 12.2 Hz, 1H), 1.03 (s, 9H); 1313C NMR (CDCl3, 100 MHz) δ 158.96, 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, 127.66, 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 a 12 mL toluene solution of guanosine 43 (1.2 g, 1.72 mmol) were added DMAP (105 mg, 0.86 mmol) and triethylamine (718 μL, 5.16 mmol), and then isobutyryl chloride (550 μL, 5.16 mmol) was added. The temperature was raised to 100 °C and the reaction was carried out for 12 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After stopping the heating and cooling the reaction solution to room temperature, 1 mL of methanol was added to quench the reaction. The mixture was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 80%) to obtain 1.1 g of isobutyryl-protected guanosine 44 as a slightly yellowish foamy solid in a yield of 83%. 1 1H NMR (CDCl3, 400 MHz) δ 11.97 (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 = 12.0 Hz, 1H), 4.69 (s, 1H), 4.65 (d, J = 12.0 Hz, 1H), 4.62 (s, 1H), 4.27 (d, J = 12.3 Hz, 1H), 4.18 - 4.14 (m, 2H), 2.68 - 2.61 (m, 1H), 1.28 - 1.24 (m, 6H), 1.06 (s, 9H); 1313C NMR (CDCl3, 100 MHz) δ 178.64, 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 [M-H] - .

[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 carried out at room temperature for 24 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). After the reaction solution was concentrated under reduced pressure, it was extracted with ethyl acetate, washed successively with saturated sodium bisulfite solution, sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained residue was dissolved in 10 mL of tetrahydrofuran, and triethylamine (453 μL, 3.25 mmol) and triethylamine hydrofluoride (636 μL, 3.9 mmol) were added successively, and the reaction was carried out at room temperature for 12 h. The completion of the reaction was detected by TLC (dichloromethane / methanol = 10 / 1). 700 mg of solid sodium bicarbonate was added to the reaction solution, and the mixture was stirred until no bubbles were generated. The reaction solution was concentrated under reduced pressure and directly purified by flash column chromatography (gradient elution: dichloromethane / methanol = 0-25%) to obtain 380 mg of cyanolock nucleoside 45 in a yield of 75%. 1 1H NMR (MeOH-d4, 400 MHz) δ 8.03 (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 = 6.9 Hz, 6H); 1313C NMR (MeOH-d4, 100 MHz) δ 181.72, 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 [M-H] - .

[0118] 46 At room temperature, 4,4'-dimethoxytrityl chloride (390 mg, 1.15 mmol) was added to a 5 mL pyridine solution of cyanolock nucleoside 45 (300 mg, 0.77 mmol), and the mixture was stirred at room temperature for 12 h. 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 successively with water and saturated brine, dried over Na2SO4, concentrated, and purified by flash column chromatography (gradient elution: methanol / dichloromethane = 0 - 10%) to obtain 520 mg of 5'-O-DMTr-protected cyanolock nucleoside 46 as a slightly yellowish foamy solid in a yield of 97%. 1 1H NMR (DMSO-d6, 400 MHz) δ 12.13 (s, 1H), 11.86 (s, 1H), 8.06 (s, 1H), 7.45 - 7.22 (m, 9H), 8.91 (d, J = 8.8 Hz, 4H), 6.29 (d, J = 3.5 Hz, 1H), 5.95 (s, 1H), 5.29 (s, 1H), 4.75 (s, 1H), 4.48 (d, J = 3.5 Hz, 1H), 3.86 (d, J = 11.2 Hz, 1H), 3.74 (s, 6H), 3.46 (d, J = 11.2 Hz, 1H), 2.82 - 2.75 (m, 1H), 1.13 (d, J = 6.8 Hz, 6H); 13 13C NMR (DMSO-d6, 100 MHz) δ 180.74, 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 [M-H] -.

[0119] 47 At room temperature, 1H-tetrazole (31 mg, 0.44 mmol) was added to a 10 mL 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 reaction was carried out at room temperature for 6 h. The completion of the reaction was detected by TLC (dichloromethane / ethyl acetate = 2 / 1). The reaction solution was directly purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 50%) to obtain 380 mg of phosphoramidite 47 as a slightly yellowish wax-like solid in a yield of 77%. 31 P NMR (152 MHz, DMSO-d6) δ 149.69, 149.09; ESI-MS (m / z) 891.42 [M - H] - . S-6'-CN-LNA- m Synthesis of C-phosphoramidite monomer JPEG2025523325000163.jpg85165Reaction conditions: (a) (1) TESCl, Et3N, acetonitrile, 0 °C to room temperature, 3 h; (2) 1,2,4-triazole, POCl3, acetonitrile, 0 °C to room temperature, 2 h; (3) NH4OH, 1,4-dioxane, room temperature, 2 h; (b) (1) Bz2O, acetonitrile, room temperature, 20 h; (2) TBAF, THF, room temperature, 12 h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite, 1H-tetrazole, dichloromethane, room temperature, 5 h.

[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 h. 1,2,4-Triazole (5.9 g, 85.35 mmol) was added to the reaction solution and stirring was continued for 10 min. Next, POCl3 (1.6 mL, 17.07 mmol) was added dropwise at 0 °C, and the reaction was carried out at room temperature for 2 h. The reaction solution was poured into 300 mL of ice water, extracted with ethyl acetate, washed successively with water, saturated NaHCO3, and saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was dissolved in 30 mL of 1,4-dioxane, aqueous ammonia (4.25 mL) was added, and the mixture was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated. Purification by flash column chromatography (gradient elution: methanol / dichloromethane = 0 - 15%) gave 3.9 g of amino nucleoside 48 as a pale yellow solid in a yield of 96%. 1 H NMR (DMSO-d6, 400 MHz) δ 7.50 - 7.44 (m, 4H), 7.37 - 7.25 (m, 8H), 6.93 (d, J = 8.6 Hz, 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 = 11.2 Hz, 1H), 3.53 (d, J = 11.2 Hz, 1H), 1.70 (s, 3H), 0.81 - 0.74 (m, 9H), 0.55 - 0.43 (m, 6H); 13 C NMR (DMSO-d6, 100 MHz) δ 166.07, 158.80, 158.77, 154.96, 144.83, 136.61, 135.63, 135.18, 130.24, 130.08, 128.43, 128.03, 127.44, 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) was added benzoic anhydride (2.73 g, 12 mmol). After stirring at room temperature for 20 h, 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 reacted at room temperature for 12 h. Then, 15% NaOH was added to adjust the pH to 10, and stirring was continued for 3 h. The mixture was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous Na2SO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 40%) to obtain 3.2 g of nucleoside 49 as a white foamy solid in a yield of 83%. 1 H NMR (DMSO-d6, 400 MHz) δ 13.08 (s, 1H), 8.21 (d, J = 7.6 Hz, 1H), 7.62 - 7.27 (m, 12H), 6.95 (d, J = 8.6 Hz, 4H), 6.40 (d, J = 3.2 Hz, 1H), 5.61 (s, 1H), 5.18 (s, 1H), 4.57 (s, 1H), 4.31 (d, J = 3.2 Hz, 1H), 3.86 (d, J = 11.2 Hz, 1H), 3.76 (s, 6H), 3.53 (d, J = 11.2 Hz, 1H), 1.86 (s, 3H); 13 C NMR (DMSO-d6, 100 MHz) δ 178.62, 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 To a 25 mL anhydrous dichloromethane solution of nucleoside 49 (2.3 g, 3.28 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (1.2 g, 3.93 mmol), 1H-tetrazole (230 mg, 3.28 mmol) was added, and the mixture was reacted at room temperature for 5 h. Saturated NaHCO3 solution was added to the reaction solution, and the mixture was extracted with dichloromethane, washed with saturated brine, dried over anhydrous MgSO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0 - 20%) to obtain 2.13 g of phosphoramidite 50 as a white foamy solid in a yield of 72%. 31 P-NMR (152 MHz, DMSO-d6) δ149.23, 147.95; ESI-MS (m / z) 901.46 [M+H] + .

[0123] Test Example 1 Cyanolocked nucleic acid-modified 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 single-stranded oligonucleotides 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 in a 4°C refrigerator overnight. Tm measurement method: 100 μL of the sample to be measured was added to a cuvette and covered tightly with a heat-insulating cover. The starting temperature for measurement was selected as 15°C, the ending temperature was 90°C, the temperature increase rate was 0.5°C / min, and the A260 reading rate was once per °C. Finally, the instrument gave the T m value. Each sample was measured 3 times repeatedly, and the average value was taken as the final result.

[0127] Immunofluorescence experiment: (1) Cell preparation: The day before, HeLa cells were seeded at 2×10 5Inoculated at the cell density of (2) Arrangement of PS-ASOs: Powders of ON1, ON2 and ON3 were dissolved in water free of RNase and DNase enzymes to a solution of 100 μM. (3) Transfection: 10 μL of Lipofectamine 2000 (Solarbio) was aspirated and added to Opti-MEM® Medium to prepare 250 μL of transfection reagent. Next, 250 μL of the Lipofectamine 2000 dilution was uniformly mixed at a transfection amount of 4 μg / dish, and after 5 min, 500 μL of the mixed solution was added to each dish. The mixed solution of the transfection reagent was aspirated and removed, and the cells were washed 3 times with PBS. Next, 500 μL of DMEM (SIGMA) as the cell maintenance medium was added to each well, and the 20 mm confocal culture dish was placed in a CO2 incubator and transfected for 5 min, 2 h, and 4 h, respectively. (4) Fixation: The waste liquid was discarded, and the cells were washed 3 times with PBS, then fixed with 4% paraformaldehyde at room temperature for 30 min, and then permeated with 0.1% Triton X-100 (prepared with PBS) for 5 min. (5) Blocking: The fixed cells were blocked with blocking buffer (prepared with 1 mg / ml BSA and PBS) at room temperature for 30 min. (6) Binding of primary antibody: The primary antibody (NONO, Cell signaling) was diluted with blocking buffer, incubated at room temperature for 1 h, then placed in a refrigerator at 4 °C overnight, and washed 3 times with washing buffer (prepared with 0.1% Tween in PBS and washed once every 5 min). (7) Binding of secondary antibody: The secondary antibody (Anti-rabbit IgG, Cell signaling) was diluted with blocking buffer, incubated at room temperature for 1 h, and finally the cells were washed 3 times with washing buffer. (8) Enclosure and detection: Prolong anti-fading agent (Cell signaling) containing 4',6-diamidino-2-phenylindole (DAPI) was dropped onto the culture dish, and finally, it was observed under an inverted fluorescence microscope and photographed.

[0128] Caspase 3 / 7 activity experimental method (1) Cell preparation: The day before, HeLa cells (Beina Biology) were inoculated into a 96-well cell culture plate at a cell density of 5×10 4 . Next, the 96-well cell culture plate was placed in a CO2 incubator and cultured. (2) Preparation of PS ASOs: ON1, ON2, and ON3 powders were dissolved in RNase- and DNase-free water to a 10 μM solution. 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® Medium to prepare 100 μL of transfection reagent. Next, 100 μL of PS ASOs solutions at different concentrations were aspirated and uniformly mixed with 100 μL of the Lipofectamine 2000 dilution. After 5 min, 50 μL of the mixed solution was added to each well. After 4 h of transfection, the mixed solution of the transfection reagent was aspirated and removed, and the cells were washed 3 times with PBS. Next, 100 μL of DMEM (Thermofisher) as the cell maintenance medium was added to each well, and the 96-well cell culture plate was placed in a CO2 incubator and cultured for another 8 h. (4) Detection: The 96-well cell plate was taken out of the CO2 incubator, 100 μL of Caspase-Glo 3 / 7 Reagent (Promega) was added to each well, and after incubating for 30 min, the luminescence signal of each well was read with a multifunctional fluorescence microplate reader (Promega).

[0129] Experimental content

[0130] Intracellular ASO-protein interactions are closely related to the drug discovery properties of PS ASOs. 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 cyano-locked nucleotide-modified ASO on the interaction with intracellular proteins, the inventors selected and studied the toxic 3-10-3 gapmer PS ASO sequence 449093 (5'- TTC AGTCATGACT TCC -3' (SEQ ID NO: 1)) as the template sequence (Nature Biotech., 2019, 37, 640). T and m C corresponding to the synthesized R-CN-LNA and S-CN-LNA were introduced at both ends of the 449093 sequence by the solid-phase phosphoramidite method to synthesize ON1 and ON2 respectively, and 449093 (ON3) was modified with LNA as a control. The mass spectrometry identification data are shown in Table 1, and the corresponding mass spectra are shown in Figures 1 to 3.

[0131] First, when measuring the hybridization characteristics of ON1-3 with the target RNA, their Tm values were 67.04 °C, 63.96 °C, and 66.59 °C respectively. Compared with the PS ASO ON3 modified with LNA, the PS-ASOs ON1-2 modified with CN-LNA were shown to still be able to maintain good affinity with the target RNA.

[0132] Next, using the paraspeckle protein P54nrb in cells as a model protein, the effect of cyano-locked nucleic acid-modified ASO on the interaction with intracellular proteins was investigated. The paraspeckle protein P54nrb binds to toxic ASOs (binding affinity is at the low nmol level), changes its distribution, and can enter the nucleolus and aggregate. Its effect is obvious and is positively correlated with ASO toxicity. The test results are shown by the aggregation status of the 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 uniformly 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, for the R-CN-LNA modified sequence ON1, no significant aggregation of the P54nrb protein in the nucleolus was observed at 2 h, and the red fluorescence was uniformly 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 uniformly distributed in the cytoplasm.

[0134] [Table 1] Oligonucleotide sequences ON1-4 and their corresponding mass spectrometry data JPEG2025523325000164.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 at 10 min, 30 min, 1 h, 2 h, and 4 h in each group of LNA, R-CN-LNA, and S-CN-LNA with respect to 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% even 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 effect 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 JPEG2025523325000165.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 transfection for 4 h, the cells were continuously incubated for 8 h, and caspase 3 / 7 activity was detected using a fluorescence microplate reader. As shown in Figure 5, the experimental results are as follows: (1) At low concentrations (0 - 62.5 nM), no significant change in caspase activity was observed in the three groups. (2) The LNA modification group started at a concentration of 125 nM, and with the increase in the administered concentration, the caspase activity level increased rapidly. The two groups with CN-LNA modification increased slowly only from 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 could reduce the increase in caspase activity by more than twofold compared to the LNA modification, and the S configuration was superior to the R configuration. The above results indicated that R / S-CN-LNA modification could significantly reduce the PS ASO-induced apoptotic toxicity compared to the LNA modification, which was consistent with the results of the immunofluorescence experiment.

[0139] In short, the introduction of a cyano group into LNA can reduce the lipophilicity of LNA and improve the water solubility of the modified structure. Cyano locked nucleic acid modification can significantly reduce the impact of PS ASO on intracellular proteins, induce apoptotic toxicity, improve the therapeutic effect of PS ASO, have significant application value for nucleic acid drugs, and provide support for nucleic acid drugs with next-generation chemical modification technologies.

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

[0141] Stability against nucleases is one of the important parameters of nucleotide chemical modification. In this experiment, snake venom phosphodiesterase (SVPDE) was used to study the oligonucleotide with S-CN-LNA-T modification (5'-TTTTTTTT T T-3' (SEQ ID NO: 2), T=The resistance of S-6'-CN-LNA)ON4 to nuclease was examined and compared with the sequences of R-CN-LNA(ON5), LNA(ON6), thio-modified(ON7) and natural(ON8). The corresponding nucleic acid sequences (7 nM) were digested with SVPDE (1.0 μg / mL) in a buffer system of 50 mM Tris-HCl, 10 mM MgCl2, pH 8.0 under physiological conditions at 37 °C. The incubation solution was taken out at different time points (0, 2, 5, 10, 20, 30, 40 min) and quantified by HPLC to obtain the corresponding content-time curve. From the results of the nuclease resistance experiment (Figure 7, where T = S-CN-LNA (rectangle) modified ON4, R-CN-LNA (diamond) modified ON5, LNA (triangle) modified ON6, 3'-thio-T (Ts, cross) modified ON7 and natural T (Natural-T, star) modified ON8), under the condition of 1.0 μg / mL of SVPDE, the R-6'-CN-LNA modified sequence ON7 was slowly degraded, and more than 40% was still not degraded at 40 min, more than 50% of the S-6'-CN-LNA modified sequence ON8 was not degraded, and less than 10% of the LNA modified sequence was not degraded, indicating that 6'-CN-LNA can significantly improve the resistance of oligonucleotides to nuclease, is clearly superior to LNA, and the S configuration is superior to the R configuration.

[0142] Obviously, the cyano group is relatively small, but it can enhance the stability of LNA against nuclease, which is consistent with the results of other C6'-modified LNAs. Notably, the reported R- and S-configured cEt-LNAs in the literature have similar nuclease resistance, but for CN-LNA, the S configuration is better than the R configuration in terms of nuclease resistance, which seems to indicate that CN has a more complex effect on phosphate considering that the cyano group in the S configuration faces the phosphate. If so, the interaction between CN-LNA modified ASO and protein is affected not only by electrostatic interaction but also more significantly.

[0143] Finally, the following should be noted. Each of the above embodiments is merely for explaining the technical solution of the present invention and does not limit it. 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 each of the above embodiments, or perform equivalent substitutions for some or all of the technical features therein. These modifications or substitutions do not deviate from the essence of the corresponding technical solutions from the scope of the technical solutions of each embodiment of the present invention.

Claims

1. A 6'-cyano-modified locked nucleoside, wherein the 6'-cyano-modified locked nucleoside is selected from a compound having a structure represented by Formula 1, a salt thereof, or an isomer thereof, In Formula 1, Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or a salt thereof, Z is a cyano group, W １ and W ２ is independently selected from H or a hydroxy protecting group, and the hydroxy protecting group includes 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, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl, 9-phenylxanthin-9-yl, 9-(p-methoxyphenyl)xanthin-9-yl or 2-naphthylmethyl, The isomer includes a 6'-cyano-modified locked nucleoside in which the Z group has an R configuration, and the structural formula is or includes a 6'-cyano-modified locked nucleoside in which the Z group has an S configuration, and the structural formula is characterized in that 6'-cyano-modified locked nucleoside.

2. The 6'-cyano-modified locked nucleoside in which the Z group has an R configuration is and the 6'-cyano-modified locked nucleoside in which the Z group has an S configuration is characterized in that the 6'-cyano-modified locked nucleoside according to Claim 1.

3. A nucleotide, wherein the nucleotide includes a 3'-active phosphorus group derivative of the 6'-cyano-modified locked nucleoside according to Claim 1 or 2, The active phosphorus group is selected from phosphoramidite, a derivative of phosphoramidite, H-phosphonate, a derivative of H-phosphonate, phosphoric acid triester, and a derivative of phosphoric acid triester, characterized in that nucleotide.

4. The nucleotide is selected from a compound having a structure represented by Formula 2, a salt thereof, or an isomer thereof, In Formula 2, the types of Bx, W ２ and Z are consistent with Formula 1, The isomer includes a nucleotide in which the Z group has an R configuration, and the structural formula is or includes a nucleotide in which the Z group has an S configuration, and the structural formula is characterized in that the nucleotide according to Claim 3.

5. The nucleotide in which the Z group has an R configuration is and the nucleotide in which the Z group has an S configuration is characterized in that the nucleotide according to Claim 4.

6. Use of the nucleotide according to Claim 3, 4 or 5 in reducing the interaction between a nucleic acid polymer and an intracellular protein, or use in the preparation of a medicament for reducing the interaction between a nucleic acid polymer and an intracellular protein.

7. Use of the nucleotide according to Claim 3, 4 or 5 in regulating the interaction between a nucleic acid polymer and an intracellular protein, or use in the preparation of a medicament for regulating the interaction between a nucleic acid polymer and an intracellular protein.

8. A nucleic acid polymer, wherein the nucleic acid polymer has 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 salts thereof, Z is a cyano group, W ３ and W ４ is, independently of each other, H, a hydroxy protecting group, or an internucleoside linking group that links the monomer to another part of the nucleic acid polymer, and W ３ and W ４ at least one of which is an internucleoside linking group that links the monomer to another part of the nucleic acid polymer, The hydroxy protecting group includes 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, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl, 9-phenylxanthin-9-yl, 9-(p-methoxyphenyl)xanthin-9-yl, or 2-naphthylmethyl. Nucleic acid polymer.

9. The monomer includes a monomer in which the Z group has an R configuration, and the structural formula is or includes a monomer in which the Z group has an S configuration, and the structural formula is It is characterized in that The nucleic acid polymer according to claim 8.

10. The nucleic acid polymer is characterized in that it is a ribonucleic acid, deoxyribonucleic acid, or a copolymer of ribonucleotides and deoxyribonucleotides. The nucleic acid polymer according to claim 9.

11. Use in the preparation of a nucleic acid diagnostic agent or nucleic acid therapeutic agent of the nucleic acid polymer according to any one of claims 8 to 10.

12. A method for synthesizing a cyano-modified nucleoside at the C6' position with an R configuration or an S configuration. The cyano-modified nucleoside at the C6' position with an R configuration is and its synthesis method is Isomerize the terminal olefin of to obtain Then, carry out a dihydroxylation reaction to obtain Next, obtain an aldehyde group compound by an oxidative cleavage reaction, and finally Convert the aldehyde group of to a cyano group to synthesize a cyano-modified nucleoside at the C6' position with an R configuration , including The cyano-modified nucleoside at the C6' position in the S configuration is and the synthesis method thereof is isomerize the terminal olefin of to obtain and then perform a dihydroxylation reaction to obtain Next, an aldehyde group compound is obtained by an oxidative cleavage reaction, and finally the aldehyde group of is converted to a cyano group to synthesize the cyano-modified nucleoside at the C6' position in the S configuration including wherein Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil or salts thereof, characterized in that A method for synthesizing a cyano-modified nucleoside at the C6' position in the R configuration or S configuration.

13. The synthesis method of the above sequentially performs a mesylation reaction, a deazetization unit and an acetylation reaction on the S configuration to obtain and then performs a glycosylation reaction and a nucleophilic substitution reaction to synthesize the R configuration including The synthesis method of the above sequentially performs a mesylation reaction, a deazetization unit and an acetylation reaction on the R configuration to obtain and then performs a glycosylation reaction and a nucleophilic substitution reaction to synthesize the S configuration including The glycosylation reaction is to react with thymine, N6-benzoyladenine or 6-chloroguanine at 50-100 °C under activator conditions, where the activator includes BSA and TMSOTf, and the reaction medium includes acetonitrile, 1,2-dichloroethane or toluene, characterized in that The synthesis method according to claim 12.

14. The synthesis method of the above S configuration oxidizes the primary alcohol of to an aldehyde group, and then performs an allylation reaction on the aldehyde group to obtain the S configuration including The synthesis method of the above R configuration oxidizes the secondary alcohol of to a ketone, and then reduces the ketone to a secondary alcohol to obtain the R configuration including wherein in the process of reducing the ketone to a secondary alcohol, the reducing agent used includes at least one of lithium aluminum hydride, lithium borohydride, lithium chloride or sodium borohydride, the reaction medium includes at least one of dichloromethane, tetrahydrofuran, methanol or ethanol, and the reaction temperature is -78 to 0 °C, characterized in that The synthesis method according to claim 13.

15. The reaction method for reducing the ketone to a secondary alcohol is characterized in that sodium borohydride and lithium chloride are used as reducing agents, tetrahydrofuran and methanol are used as reaction media, and the reaction is carried out at -40 to 0 °C. The synthesis method according to claim 14.

16. The catalyst for the terminal olefin isomerization includes a ruthenium catalyst, a palladium catalyst, a rhodium catalyst or an iridium catalyst. The reaction medium for the terminal olefin isomerization includes methanol, ethanol, n-butanol or toluene. The reaction temperature for the terminal olefin isomerization is 60 °C to 100 °C. The reaction time for the terminal olefin isomerization is characterized by being 12 to 72 hours. The synthesis method according to claim 12.

17. The catalyst for the isomerization of the terminal olefin is carbonylchlorohydridotris(triphenylphosphine)ruthenium(II), the reaction medium for the terminal olefin isomerization is ethanol, and the reaction temperature for the terminal olefin isomerization is 60 to 80 °C. The synthesis method according to claim 12.

18. A method for synthesizing a cyano-locked nucleic acid T phosphoramidite monomer at the C6' site in the R configuration or S configuration. The method for synthesizing the cyano-locked nucleic acid T phosphoramidite monomer at the C6' site in the R configuration is to deprotect the 3'-hydroxy group and 5'-hydroxy group of the R configuration synthesized in claim 12 to obtain, and then protect the 5'-hydroxy group with DMTr and perform a phosphoramidation reaction on the 3'-hydroxy group to obtain the R configuration including obtaining. The method for preparing the cyano-locked nucleic acid T phosphoramidite monomer at the C6' site in the S configuration is to deprotect the 3'-hydroxy group and 5'-hydroxy group of the S configuration synthesized in claim 12 to obtain, and then protect the 5'-hydroxy group with DMTr and perform a phosphoramidation reaction on the 3'-hydroxy group to obtain the S configuration including obtaining. The method for synthesizing a cyano-locked nucleic acid T phosphoramidite monomer at the C6' site in the R configuration or S configuration is characterized by including obtaining. The method for synthesizing a cyano-locked nucleic acid T phosphoramidite monomer at the C6' site in the R configuration or S configuration.

19. A method for synthesizing a cyano-locked nucleic acid A phosphoramidite monomer at the C6' site in the R configuration or S configuration. The method for synthesizing the cyano-locked nucleic acid A phosphoramidite monomer at the C6' site in the R configuration is to protect the base of the R configuration synthesized in claim 12 to obtain. ​ is obtained, and then the 3'-hydroxy group and 5'-hydroxy group are deprotected to is obtained, next, the 5'-hydroxy group is protected with DMTr, and a phosphoramidation reaction is carried out on the 3'-hydroxy group to obtain the R configuration including obtaining The method for synthesizing a cyano-locked nucleic acid A phosphoramidite monomer at the C6' site in the S configuration is the S configuration synthesized in claim 12 protecting the base of is obtained, and then the 3'-hydroxy group and 5'-hydroxy group are deprotected to is obtained, next, 5'-hydroxy is protected with DMTr, and a phosphoramidation reaction is carried out on 3'-hydroxy to obtain the S configuration characterized by including obtaining A method for synthesizing a cyano-locked nucleic acid A phosphoramidite monomer at the C6' site in the R configuration or S configuration.

20. A method for synthesizing a cyano-locked nucleic acid G phosphoramidite monomer at the C6' site in the R configuration or S configuration. The method for synthesizing a cyano-locked nucleic acid G phosphoramidite monomer at the C6' site in the R configuration is to demethylate the methoxy group of the R configuration synthesized in claim 12 to obtain and then perform base protection to obtain, next, deprotect the 3'-hydroxy group and 5'-hydroxy group to obtain, protect the 5'-hydroxy group with DMTr, and perform a phosphoramidation reaction on the 3'-hydroxy group to obtain the R configuration including obtaining The method for synthesizing a cyano-locked nucleic acid G phosphoramidite monomer at the C6' site in the S configuration is to demethylate the methoxy group of the S configuration synthesized in claim 12 to obtain and then perform base protection to obtain, next, deprotect the 3'-hydroxy group and 5'-hydroxy group to obtain, protect the 5'-hydroxy group with DMTr, and perform a phosphoramidation reaction on the 3'-hydroxy group to obtain the S configuration characterized by including obtaining A method for synthesizing a cyano-locked nucleic acid G phosphoramidite monomer at the C6' site in the R configuration or S configuration.

21. A method for synthesizing a cyano-locked nucleic acid C phosphoramidite monomer at the C6' site in the R configuration or S configuration. The method for synthesizing a cyano-locked nucleic acid C phosphoramidite monomer at the C6' site in the R configuration is to deprotect the 3'-hydroxy group and 5'-hydroxy group of the R configuration synthesized in claim 12 to obtain and then protect the 5'-hydroxy group with DMTr to obtain, perform silylation modification on the 3'-hydroxy group, and convert the carbonyl group to an amino group to obtain, then perform base protection and deprotect the 3'-hydroxy group is obtained, and finally, a phosphoramidation reaction is carried out on the 3'-hydroxy group to obtain the R configuration including obtaining A method for synthesizing a cyano-locked nucleic acid C-phosphoramidite monomer at the C6' site in the S configuration is the S configuration synthesized in claim 12 deprotect the 3'-hydroxy group and 5'-hydroxy group of to obtain Next, protect the 5'-hydroxy group with DMTr to obtain The 3'-hydroxy group is subjected to silanization modification, and the carbonyl group is converted to an amino group to obtain After that, base protection is carried out, and the 3'-hydroxy group is deprotected to obtain Finally, a phosphoramidation reaction is carried out on the 3'-hydroxy group to obtain the S configuration A method for synthesizing a cyano-locked nucleic acid C-phosphoramidite monomer at the C6' site in the R configuration or S configuration, characterized by including

22. A method for synthesizing a nucleic acid polymer, which comprises performing a polymerization reaction on a monomer to prepare a nucleic acid polymer The monomer includes the 6'-cyano-modified locked nucleoside according to claim 1 or 2 or the nucleotide according to claim 3, 4 or 5, characterized in that A method for synthesizing a nucleic acid polymer

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