6'-cyano modified locked nucleosides, nucleotides and nucleic acid polymers, and methods of making and use thereof

Abstract

The application provides a 6'-cyano modified locked nucleoside, nucleotide and nucleic acid polymer and a preparation method and application thereof, and relates to the technical field of biotechnology.The 6'-cyano modified locked nucleoside provided by the application is in R configuration or S configuration, and is further modified to obtain a nucleotide and a nucleic acid polymer.The inventor has found that the 6'-cyano modified nucleic acid polymer has better nuclease resistance than the unmodified or other modified nucleic acid polymer, reduces the interaction between the nucleic acid polymer and intracellular proteins, and the S configuration of the 6'-cyano modified nucleic acid polymer is more significant than the R configuration in improving the nuclease resistance of the nucleic acid polymer and reducing the interaction between the nucleic acid polymer and intracellular proteins, so that the 6'-cyano modified locked nucleoside, nucleotide and nucleic acid polymer provided by the application has significant application value in the field of nucleic acid drugs.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a 6'-cyano-modified nucleoside, nucleotide, and nucleic acid polymer, its preparation method, and its application. Background Technology

[0002] Nucleic acid drugs represent a cutting-edge field in biomedical development and are the third largest drug type after small molecule drugs and protein drugs. Nucleic acid drugs mainly include antisense nucleic acids (ASOs) and small interfering RNA (siRNAs). Compared with traditional small molecule and protein drugs, they offer advantages such as rapid design, broad target coverage, high specificity, intracellular efficacy, and relatively rapid preparation. They hold significant value in treating many chronic, refractory major diseases and rare diseases that are difficult to treat with protein targets. As of 2022, 15 nucleic acid drugs have been approved globally (10 ASOs and 5 siRNAs), and more than 400 nucleic acid drugs are in clinical trials.

[0003] However, on the one hand, unmodified oligonucleotide drugs generally have unsatisfactory drug-like properties. They possess poor drug-like characteristics, such as poor stability, susceptibility to degradation by nucleases, high polarity, difficulty in cell entry, poor distribution characteristics, and poor binding affinity to target mRNA. To achieve clinical efficacy, oligonucleotides must undergo chemical modification, and all marketed nucleic acid drugs utilize corresponding nucleic acid chemical modifications. On the other hand, chemically modified antisense nucleic acid drugs can cause toxicity to high-exposed organs (liver and kidneys) to some extent. In particular, chemically modified nucleic acid drugs with high affinity locked nucleic acids (LNA) and restricted ethyl locked nucleic acids (cEt-LNA) on the phosphate thioester (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 a key technology and bottleneck in the development of nucleic acid drugs.

[0004] In recent years, significant progress has been made in understanding the toxicity mechanisms of antisense nucleic acid drugs (Nucleic Acids Res. 2016, 44, 3892; Nature Biotech., 2017, 35, 230; Nucleic Acids Res. 2018, 46, 2204; Nature Biotech., 2019, 37, 640; Nucleic Acids Res. 2019, 47, 10865; J. Am. Chem. Soc., 2020, 142, 14754; J. Am. Chem. Soc. 2020, 142, 7456). Studies have shown that the main toxicity mechanism of antisense nucleic acid drugs lies in the fact that chemically modified thio-antisense nucleic acid drugs (PS-ASOs) bind to and interfere with the subcellular distribution of intracellular proteins (such as P54nrb), thereby inducing apoptosis and producing toxicity. Meanwhile, toxicity is positively correlated with ASO-protein binding ability; the stronger the binding force, the greater the potential toxicity. Furthermore, this binding ability is closely related to the water solubility (LogS) of the chemically modified structure; the stronger the hydrophobic interaction, the greater the binding affinity. Experimental results show that the affinity of 2'-methoxyethyl (2'-MOE) for intracellular proteins is 30 times lower than that of 2'-fluoro (2'-F). Therefore, the interaction between antisense nucleic acids and proteins has become a decisive factor in the efficacy of antisense nucleic acid drugs. Structure determines properties. To develop highly efficient and safe novel chemically modified nucleic acid structures, the inventors previously disclosed a novel cyanolocked nucleic acid (CN-LNA) modified structure (ZL 20190914759.3), which exhibits high nuclease tolerance, as well as good target gene affinity and selectivity. Meanwhile, further chemical property calculations (Chemdraw calculations) showed that CN-LNA has good hydrophilicity, with a LogS value of -0.032, which is approximately 2, 4, and 20 times that of commonly used 2'-methoxyethyl (MOE, LogS value -0.073), locked nucleic acid (LNA, LogS value -0.122), and 2'-fluoro (LogS value -0.661), respectively. It has the potential to reduce the affinity of PS-ASO for binding to intracellular proteins, thereby reducing the impact on intracellular protein distribution, reducing toxicity, and improving therapeutic efficacy.

[0005] However, the previously disclosed CN-LNA synthesis method cannot stereoselectively synthesize the C6'-epomers with both R and S configurations, and it fails to further explain the differences in the drug-like properties of the modified nucleic acids by the C6'-epomers, thus failing to meet the need to establish a new generation of highly efficient and low-toxicity nucleic acid chemical modification technology.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide 6'-cyano-modified nucleosides, nucleotides, and nucleic acid polymers with R or S configurations.

[0008] A second objective of this invention is to provide a method for preparing R-configuration or S-configuration 6'-cyano-modified nucleosides, nucleotides, and nucleic acid polymers.

[0009] A third objective of this invention is to provide the application of the above-mentioned nucleic acid polymer in the preparation of nucleic acid diagnostic agents or nucleic acid therapeutic agents.

[0010] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0011] In a first aspect, the present invention provides a 1,6'-cyano-modified nucleoside, wherein the 6'-cyano-modified nucleoside is selected from compounds having the structure shown in Formula 1, their salts, or isomers thereof:

[0012]

[0013] Wherein: Bx is selected from substituted or unsubstituted: adenine, guanine, thymine, cytosine, uracil or their respective salts;

[0014] Z is a cyano group;

[0015] W1 and W2 are independently selected from H or hydroxyl protecting groups; the hydroxyl protecting groups include: benzyl or dimethoxytriphenylmethyl;

[0016] The isomers include: 6'-cyano-modified nucleosides with the Z group in the R configuration, having the structural formula as follows: Alternatively, a 6'-cyano-modified nucleoside with the Z group in an S configuration, having the structural formula as follows:

[0017] In a second aspect, the present invention provides a nucleotide comprising a 3'-active phosphorus group derivative of the 6'-cyano-modified nucleoside;

[0018] The active phosphorus group is selected from: phosphorus amide, phosphorus amide derivatives, H-phosphate esters, H-phosphate ester derivatives, triphosphate esters, or triphosphate ester derivatives.

[0019] As a further technical solution, the nucleotide is selected from compounds having the structure shown in Formula 2, their salts, or isomers thereof:

[0020]

[0021] The isomers include: nucleotides with the Z group in the R configuration, having the structural formula as follows: Or a nucleotide with the Z group in the S configuration, with the structural formula as follows:

[0022] Thirdly, the present invention provides the application of the above-mentioned nucleotides in reducing the interaction between nucleic acid polymers and intracellular proteins.

[0023] Fourthly, the present invention provides a nucleic acid polymer having at least one monomer having the following formula:

[0024]

[0025] Or the following formula:

[0026]

[0027] Or the following formula:

[0028]

[0029] Wherein: Bx is selected from substituted or unsubstituted: adenine, guanine, thymine, cytosine, uracil or their respective salts;

[0030] Z is a cyano group;

[0031] W3 and W4 are each independently an H, a hydroxyl protecting group, a linked conjugated group, or a nucleoside linker group that links the monomer to other parts of the nucleic acid polymer; and at least one of W3 and W4 is a nucleoside linker group that links the monomer to other parts of the nucleic acid polymer.

[0032] The hydroxyl protecting group includes benzyl or dimethoxytriphenylmethyl.

[0033] As a further technical solution, at least one monomer's Z group has an R configuration represented by the following formula:

[0034]

[0035] Or the following formula:

[0036]

[0037] Or the following formula:

[0038]

[0039] Alternatively, at least one monomer's Z group has an S configuration represented by the following formula:

[0040]

[0041] Or the following formula:

[0042]

[0043] Or the following formula:

[0044]

[0045] As a further technical solution, the nucleic acid polymer is ribonucleic acid, deoxyribonucleic acid, or a copolymer of ribonucleotides and deoxyribonucleotides.

[0046] Fifthly, the present invention provides the application of the nucleic acid polymer in the preparation of nucleic acid diagnostic agents or nucleic acid therapeutic agents.

[0047] Sixthly, the present invention provides a method for synthesizing a cyano-modified nucleoside at the C6' position with either an R-configuration or an S-configuration, wherein the R-configuration cyano-modified nucleoside is... Its synthesis method includes: Isomerization of terminal olefins to obtain Then, a dihydroxylation reaction is performed to obtain... Then, aldehyde compounds are obtained through oxidative cleavage reaction. Finally The aldehyde group was converted to a cyano group to synthesize an R-configuration cyano-modified nucleoside at the C6' position.

[0048] The S-configuration C6' position cyano-modified nucleoside is Its synthesis method includes: Isomerization of terminal olefins to obtain Then, a dihydroxylation reaction is performed to obtain... Then, aldehyde compounds are obtained through oxidative cleavage reaction. Finally The aldehyde group was converted to a cyano group to synthesize an S-configuration cyano-modified nucleoside at the C6' position.

[0049] Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or their respective salts.

[0050] As a further technical solution, the aforementioned The synthesis method includes: using the S configuration The methanesulfonation, de-branching, and acetylation reactions were performed sequentially to obtain the desired product. Then, glycosylation and nucleophilic substitution reactions were performed to synthesize the R configuration.

[0051] The The synthesis method includes: using the R configuration The methanesulfonation, de-branching, and acetylation reactions were performed sequentially to obtain the desired product. Then, glycosylation and nucleophilic substitution reactions were performed to synthesize the S configuration.

[0052] The glycosylation reaction is as follows: under activating agent conditions, ... It reacts with thymine, N6-benzoyladenine or 6-chloroguanine at 50-100°C, wherein the activator includes BSA and TMSOTf, and the reaction medium includes acetonitrile, 1,2-dichloroethane or toluene.

[0053] As a further technical solution, the S-configuration The synthetic method includes: 3,5-di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranoside The primary alcohol is oxidized to an aldehyde group, and then the aldehyde group is allylated to obtain the S configuration.

[0054] The R configuration The synthesis methods include: The secondary alcohol is oxidized to a ketone, and then the ketone is reduced back to the secondary alcohol to obtain the R configuration.

[0055] The reducing agent used to reduce ketones to secondary alcohols 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.

[0056] Preferably, the reaction of reducing the ketone to a secondary alcohol is carried out by using sodium borohydride and lithium chloride as reducing agents and tetrahydrofuran and methanol as solvents, and reacting at -40 to 0°C.

[0057] As a further technical solution, the catalyst for the terminal olefin isomerization includes a ruthenium catalyst, a palladium catalyst, a rhodium catalyst or an iridium catalyst, preferably tris(triphenylphosphine)carbonyl ruthenium(II) hydrochloride;

[0058] The reaction medium for the terminal olefin isomerization includes methanol, ethanol, n-butanol, or toluene, preferably ethanol;

[0059] The reaction temperature for the terminal olefin isomerization is 60-100℃, preferably 60-80℃;

[0060] The reaction time for the terminal olefin isomerization is 12-72 hours.

[0061] In a seventh aspect, the present invention provides a method for synthesizing a G phosphorous amide monomer with an R-configuration or S-configuration C6'-cyano group modification, wherein the R-configuration C6'-cyano-modified G phosphorous amide monomer is... Its preparation method includes: R configuration The methoxy group is converted into an aldehyde group to obtain... Then, the 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain... The base and 5'-hydroxyl group are then protected, and the 3'-hydroxyl group undergoes a phosphoramidation reaction to obtain the R configuration.

[0062] The S-configuration C6'-cyano-modified G-phosphamide monomer is Its preparation method includes: taking the S configuration The methoxy group was demethylated to obtain Then, the 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain... The base and 5'-hydroxyl group are then protected, and the 3'-hydroxyl group undergoes a phosphoramidation reaction to obtain the S configuration.

[0063] Preferably, ferric chloride is used for debenzylation.

[0064] Eighthly, the present invention provides a method for synthesizing an R-configuration or S-configuration C6'-cyano-locked nucleic acid T-phosphite monomer, wherein the R-configuration C6'-cyano-locked nucleic acid T-phosphite monomer is... Its preparation method includes: R configuration The 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain The 5'-hydroxyl group is then protected with DMTr, and the 3'-hydroxyl group undergoes a phosphoramidation reaction to obtain the R configuration.

[0065] S-configuration C6' position cyanolockane T-phosphite monomer is Its preparation method includes: taking the S configuration The 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain The 5'-hydroxyl group is then protected with DMTr, and the 3'-hydroxyl group undergoes a phosphoramidation reaction to obtain the S configuration.

[0066] Preferably, ferric chloride is used for debenzylation.

[0067] Ninthly, the present invention provides a method for synthesizing an R-configuration or S-configuration C6'-cyanolocked nucleic acid C-phosphamide monomer, wherein the R-configuration C6'-cyanolocked nucleic acid C-phosphamide monomer is... Its preparation method includes: R configuration The 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain Then, the 5'-hydroxyl group was protected with DMTr to obtain The 3'-hydroxyl group is then silanized, and the carbonyl group is converted to an amino group to obtain... Then, base protection was performed, and the 3'-hydroxyl group was deprotected to obtain... Finally, the 3'-hydroxyl group was subjected to a phosphoramidation reaction to obtain the R configuration.

[0068] S-configuration C6' position cyanolockaneous nucleic acid C phosphoramide monomer is Its preparation method includes: taking the S configuration The 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain Then, the 5'-hydroxyl group was protected with DMTr to obtain The 3'-hydroxyl group is then silanized, and the carbonyl group is converted to an amino group to obtain... Then, base protection was performed, and the 3'-hydroxyl group was deprotected to obtain... Finally, the 3'-hydroxyl group was subjected to a phosphoramidation reaction to obtain the S configuration.

[0069] Preferably, ferric chloride is used for debenzylation.

[0070] In a tenth aspect, the present invention provides a method for synthesizing nucleic acid polymers, wherein nucleotide monomers are subjected to a polymerization reaction to prepare nucleic acid polymers;

[0071] The nucleotide monomer includes the 6'-cyano-modified nucleoside or the nucleotide.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] The 6'-cyano-modified nucleosides provided in this invention are in either the R or S configuration, and further modifications yield nucleotides and nucleic acid polymers. The inventors have found that the 6'-cyano-modified nucleic acid polymers exhibit better nuclease tolerance and reduced interactions between the nucleic acid polymers and intracellular proteins compared to unmodified or other modified nucleic acid polymers. Furthermore, the S-configuration of the 6'-cyano-modified nucleic acid polymer is more significant than the R-configuration in improving nuclease tolerance and reducing interactions between the nucleic acid polymers and intracellular proteins. Therefore, the 6'-cyano-modified nucleosides, nucleotides, and nucleic acid polymers provided in this invention have significant application value in nucleic acid drug development.

[0074] This invention provides a universal method for preparing the above-mentioned 6'-cyano-modified nucleosides, nucleotides, and nucleic acid polymers, which can achieve stereoselective synthesis. Attached Figure Description

[0075] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0076] Figure 1It has the crystal structure of S-6'-CN-LNA-T monomer;

[0077] Figure 2 This is the mass spectrum of the nucleic acid ON1;

[0078] Figure 3 This is the mass spectrum of the nucleic acid ON2;

[0079] Figure 4 This is the mass spectrum of the nucleic acid ON3;

[0080] Figure 5 Figure 60× shows the aggregation of parafoil protein P54nrb induced by ON1, ON2, and ON3.

[0081] Figure 6 Results for Caspase 3 / 7 activity;

[0082] Figure 7 This is the mass spectrum of nucleic acid ON4;

[0083] Figure 8 The enzymatic stability of 5′-d(TTTTTTTTT)-3′ against snake venom phosphodiesterase (SVPDE) was determined. Detailed Implementation

[0084] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0085] The term "nucleic acid polymer" can refer to any nucleic acid molecule, including but not limited to DNA, RNA, and their hybrids, including but not limited to single-stranded and double-stranded molecules. The number of nucleotides polymerized to form the nucleic acid is 2, 3, or more, and can be oligonucleotides with fewer than 20 nucleotides, or polymers with more than 20 nucleotides.

[0086] In a first aspect, the present invention provides a 1,6'-cyano-modified nucleoside, wherein the 6'-cyano-modified nucleoside is selected from compounds having the structure shown in Formula 1, their salts, or isomers thereof:

[0087]

[0088] Wherein: Bx is selected from substituted or unsubstituted: adenine, guanine, thymine, cytosine, uracil or their respective salts;

[0089] Z is a cyano group;

[0090] W1 and W2 are independently selected from H or hydroxyl protecting groups; the hydroxyl protecting group is selected from benzyl or dimethoxytriphenylmethyl.

[0091] The isomers include: 6'-cyano-modified nucleosides with the Z group in the R configuration, having the structural formula as follows: Alternatively, a 6'-cyano-modified nucleoside with the Z group in an S configuration, having the structural formula as follows:

[0092] The 6'-cyano-modified nucleoside provided by this invention can improve the nuclease tolerance of nucleic acid polymers prepared from it and reduce the interaction between nucleic acid polymers and intracellular proteins.

[0093] In a second aspect, the present invention provides a nucleotide comprising a 3'-active phosphorus group derivative of the 6'-cyano-modified nucleoside;

[0094] The active phosphorus group is selected from: phosphorus amide, phosphorus amide derivatives, H-phosphate esters, H-phosphate ester derivatives, triphosphate esters, or triphosphate ester derivatives.

[0095] The nucleotides provided by this invention can improve the nuclease tolerance of nucleic acid polymers prepared using them as monomers, reduce their interaction with intracellular proteins, reduce their impact on the subcellular distribution of intracellular proteins, and reduce apoptosis.

[0096] In some embodiments, the nucleotide is selected from compounds having the structure shown in Formula 2, their salts, or isomers thereof:

[0097]

[0098] The isomers include: nucleotides with the Z group in the R configuration, having the structural formula as follows: Or a nucleotide with the Z group in the S configuration, with the structural formula as follows:

[0099] Thirdly, the present invention provides the application of the above-mentioned nucleotides in reducing the interaction between nucleic acid polymers and intracellular proteins.

[0100] The inventors have discovered that nucleic acid polymers prepared from the nucleotides provided in this invention have minimal interaction with intracellular proteins, which helps reduce the in vivo toxicity of nucleic acid polymers.

[0101] Fourthly, the present invention provides a nucleic acid polymer having at least one monomer having the following formula:

[0102]

[0103] Or the following formula:

[0104]

[0105] Or the following formula:

[0106]

[0107] Wherein: Bxx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil or their respective salts;

[0108] Z is a cyano group;

[0109] W3 and W4 are each independently an H, a hydroxyl protecting group, a linked conjugated group, or a nucleoside linker group that links the monomer to other parts of the nucleic acid polymer; and at least one of W3 and W4 is a nucleoside linker group that links the monomer to other parts of the nucleic acid polymer.

[0110] The hydroxyl protecting group is selected from benzyl or dimethoxytriphenylmethyl.

[0111] The nucleic acid polymers provided by this invention exhibit better nuclease tolerance than unmodified or other modified nucleic acid polymers, reducing interactions with intracellular proteins, minimizing the impact on subcellular protein distribution, and decreasing apoptosis. In some embodiments, at least one monomer has a Z group in the R configuration represented by the following formula:

[0112]

[0113] Or the following formula:

[0114]

[0115] Or the following formula:

[0116]

[0117] Alternatively, at least one monomer's Z group has an S configuration represented by the following formula:

[0118]

[0119] Or the following formula:

[0120]

[0121] Or the following formula:

[0122]

[0123] In some embodiments, the nucleic acid polymer is ribonucleic acid, deoxyribonucleic acid, or a copolymer of ribonucleotides and deoxyribonucleotides.

[0124] Fifthly, the present invention provides the application of the nucleic acid polymer in the preparation of nucleic acid diagnostic agents or nucleic acid therapeutic agents.

[0125] The nucleic acid polymer provided by this invention has better nuclease tolerance than unmodified or other modified nucleic acid polymers, and has low interaction with intracellular proteins, low apoptosis-inducing effect, and few toxic side effects, and can be used as a nucleic acid diagnostic agent or nucleic acid therapeutic agent.

[0126] Sixthly, the present invention provides a method for synthesizing a cyano-modified nucleoside at the C6' position with either an R-configuration or an S-configuration, wherein the R-configuration cyano-modified nucleoside is... Its synthesis method includes: Isomerization of terminal olefins to obtain Then, a dihydroxylation reaction is performed to obtain... Then, aldehyde compounds are obtained through oxidative cleavage reaction. Finally The aldehyde group was converted to a cyano group to synthesize an R-configuration cyano-modified nucleoside at the C6' position.

[0127] The S-configuration C6' position cyano-modified nucleoside is Its synthesis method includes: Isomerization of terminal olefins to obtain Then, a dihydroxylation reaction is performed to obtain... Then, aldehyde compounds are obtained through oxidative cleavage reaction. Finally The aldehyde group was converted to a cyano group to synthesize an S-configuration cyano-modified nucleoside at the C6' position.

[0128] Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or their respective salts.

[0129] The method for preparing R-configuration or S-configuration C6'-cyano-modified nucleosides provided by this invention is stable and feasible. This invention does not impose specific limitations on the methods used for terminal olefin isomerization, dihydroxylation, oxidative cleavage, and the conversion of aldehyde groups to cyano groups; reaction methods well-known to those skilled in the art can be employed. The R-configuration or S-configuration C6'-cyano-modified nucleosides synthesized by this invention can be used as raw materials for the synthesis of four types of R-configuration or S-configuration cyano-modified locked nucleic acids.

[0130] It should be noted that the intermediate aldehyde compound of the above reaction can be converted into other groups, including alkanes, alcohols, carboxylic acids, alkynyl groups, alkenyl groups, and amines, through conventional chemical methods, thereby achieving diversified C6'-modified locked nucleic acids.

[0131] In some embodiments, the The synthesis method includes: using the S configuration The methanesulfonation, de-branching, and acetylation reactions were performed sequentially to obtain the desired product. Then, glycosylation and nucleophilic substitution reactions were performed to synthesize the R configuration.

[0132] The The synthesis method includes: using the R configuration The methanesulfonation, de-branching, and acetylation reactions were performed sequentially to obtain the desired product. Then, glycosylation and nucleophilic substitution reactions were performed to synthesize the S configuration.

[0133] The glycosylation reaction is as follows: under activating agent conditions, ... or It reacts with thymine, N6-benzoyladenine or 6-chloroguanine at 50-100°C, wherein the activator includes BSA and TMSOTf, and the reaction medium includes acetonitrile, 1,2-dichloroethane or toluene.

[0134] The synthesis method provided by this invention is stable and efficient. It does not impose specific limitations on the methanesulfonation reaction, de-branching and acetylation reaction, and nucleophilic substitution reaction. Any reaction method known to those skilled in the art can be used.

[0135] In some embodiments, the S-configuration The synthetic method includes: 3,5-di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranoside The primary alcohol is oxidized to an aldehyde group, and then the aldehyde group is allylated to obtain the S configuration.

[0136] The R configuration The synthesis methods include: The secondary alcohol is oxidized to a ketone, and then the ketone is reduced back to the secondary alcohol to obtain the R configuration.

[0137] The reducing agents used to reduce ketones to secondary alcohols include metallic hydrogen compounds such as lithium aluminum hydride, lithium borohydride, lithium chloride, or sodium borohydride. The reaction media include solvents such as dichloromethane, tetrahydrofuran, methanol, or ethanol. The reaction temperature is between -78 and 0°C.

[0138] Preferably, the reaction of reducing the ketone to a secondary alcohol is carried out by using sodium borohydride and lithium chloride as reducing agents and tetrahydrofuran and methanol as solvents, and reacting at -40 to 0°C.

[0139] In some preferred embodiments, the catalyst for the terminal olefin isomerization includes transition metal catalysts such as ruthenium catalysts, palladium catalysts, rhodium catalysts or iridium catalysts, preferably tris(triphenylphosphine)carbonyl ruthenium(II) hydrochloride;

[0140] The reaction medium for the terminal olefin isomerization includes solvents such as methanol, ethanol, n-butanol, or toluene, with ethanol being preferred;

[0141] The reaction temperature for the terminal olefin isomerization is 60-100℃, preferably 60-80℃;

[0142] The reaction time for the terminal olefin isomerization is 12-72 hours.

[0143] The inventors discovered that using 2.5-5% mol of tris(triphenylphosphine)carbonyl ruthenium(II) hydrochloride as a catalyst and ethanol as a reaction solvent, the isomerization of terminal olefins can be stably achieved with high yield without the need for anhydrous or oxygen-free operation.

[0144] In a seventh aspect, the present invention provides a method for synthesizing a G phosphorous amide monomer with an R-configuration or S-configuration C6'-cyano group modification, wherein the R-configuration C6'-cyano-modified G phosphorous amide monomer is... Its preparation method includes: R configuration The methoxy group is converted into an aldehyde group to obtain... Then, the 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain... The base and 5'-hydroxyl group are then protected, and the 3'-hydroxyl group undergoes a phosphoramidation reaction to obtain the R configuration.

[0145] The S-configuration C6'-cyano-modified G-phosphamide monomer is Its preparation method includes: taking the S configuration The methoxy group was demethylated to obtain Then, the 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain... The base and 5'-hydroxyl group are then protected, and the 3'-hydroxyl group undergoes a phosphoramidation reaction to obtain the S configuration.

[0146] Preferably, ferric chloride is used for debenzylation.

[0147] Eighthly, the present invention provides a method for synthesizing an R-configuration or S-configuration C6'-cyano-locked nucleic acid T-phosphite monomer, wherein the R-configuration C6'-cyano-locked nucleic acid T-phosphite monomer is... Its preparation method includes: R configuration The 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain The 5'-hydroxyl group is then protected with DMTr, and the 3'-hydroxyl group undergoes a phosphoramidation reaction to obtain the R configuration.

[0148] S-configuration C6' position cyanolockane T-phosphite monomer is Its preparation method includes: taking the S configuration The 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain The 5'-hydroxyl group is then protected with DMTr, and the 3'-hydroxyl group undergoes a phosphoramidation reaction to obtain the S configuration.

[0149] Preferably, ferric chloride is used for debenzylation.

[0150] Ninthly, the present invention provides a method for synthesizing an R-configuration or S-configuration C6'-cyanolocked nucleic acid C-phosphamide monomer, wherein the R-configuration C6'-cyanolocked nucleic acid C-phosphamide monomer is... Its preparation method includes: R configuration The 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain Then, the 5'-hydroxyl group was protected with DMTr to obtain The 3'-hydroxyl group is then silanized, and the carbonyl group is converted to an amino group to obtain... Then, base protection was performed, and the 3'-hydroxyl group was deprotected to obtain... Finally, the 3'-hydroxyl group was subjected to a phosphoramidation reaction to obtain the R configuration.

[0151] S-configuration C6' position cyanolockaneous nucleic acid C phosphoramide monomer is Its preparation method includes: taking the S configuration The 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain Then, the 5'-hydroxyl group was protected with DMTr to obtain The 3'-hydroxyl group is then silanized, and the carbonyl group is converted to an amino group to obtain... Then, base protection was performed, and the 3'-hydroxyl group was deprotected to obtain... Finally, the 3'-hydroxyl group was subjected to a phosphoramidation reaction to obtain the S configuration.

[0152] Preferably, ferric chloride is used for debenzylation.

[0153] This invention provides a feasible method for synthesizing G phosphoramidite monomers, T phosphoramidite monomers, or C phosphoramidite monomers with a C6' cyano group modified in the R or S configuration using dibenzyl compounds as raw materials, offering a new approach for the synthesis of locked nucleic acids with a C6' cyano group modified in the R or S configuration.

[0154] In a tenth aspect, the present invention provides a method for synthesizing nucleic acid polymers, wherein nucleotide monomers are subjected to a polymerization reaction to prepare nucleic acid polymers;

[0155] The nucleotide monomer includes the 6'-cyano-modified nucleoside or the nucleotide.

[0156] In some preferred embodiments, the polymerization reaction includes: ① Chemical synthesis. Nucleic acids are synthesized entirely using organic chemical methods from nucleosides or mononucleotides, including phosphodiester method, phosphotriester method, phosphite triester method, and solid-phase synthesis. ② Enzymatic synthesis. Enzymatic reactions can link nucleic acid monomers or small chemically synthesized fragments into larger fragments.

[0157] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0158] Example 1: Synthesis of R / S-6'-CN-LNA based on bisbenzyl starting material

[0159] 1. Synthesis of R-6'-CN-LNA-G monomer

[0160]

[0161] Experimental conditions: (a)(1) 2-iodoacryloylbenzoic acid, CH3CN, reflux, 5h; (2) boron trifluoride diethyl ether solution, allyltrimethylsilane, dichloromethane, -40℃, 2h; (b) methanesulfonyl chloride, triethylamine, DCM, room temperature, 12h; (c) Ac2O, acetic acid, conc. H2SO4, room temperature, 1h; (d)(1) 6-chloroguanine, N,O-bis(trimethylsilylacetamide), TMSOTf, (1) Toluene, 100℃, 5h; (2) K2CO3, MeOH, room temperature, 24h; (e) (1) Tris(triphenylphosphine)carbonyl ruthenium(II) hydrochloride, toluene, 100℃, 48h; (2) 50% NMO, potassium osmium dihydrate, THF, t-BuOH, 60℃, 5h; (3) NaIO4, MeOH, H2O, room temperature, 12h; (4) NH4OH, I2, room temperature, 24h; (f) 2N HCl, MeOH, THF, 60℃, 12h; (g) (1) Anhydrous FeCl3, Ac2O, dichloromethane, room temperature, 4h; (2) NH3, MeOH, room temperature, 12h; (h) (1) DMFDMA, MeOH, 60℃, 4h; (2) DMTrCl, pyridine, 12h; (i) 2-Cyanoethyl N,N,N',N'-Tetraisopropylphosphine diamine, 1H-Tetraazole, dichloromethane, room temperature, 12h. 52

[0163] Sugar compound 51 (100.0 g, 0.25 mol) was dissolved in 500 mL of acetonitrile, and 2-iodobenzoic acid (104.9 g, 0.37 mol) was added. The mixture was heated to slow reflux and stirred for 5 h. The reaction was monitored by TLC until complete (PE / EtOAc = 5 / 1). The reaction was stopped, and the mixture was allowed to cool to room temperature. The mixture was filtered through diatomaceous earth, and the filter cake was washed with acetonitrile (500 mL). The filtrate was evaporated to dryness and then dried under vacuum to obtain a pale yellow viscous substance, which was then directly used for the next reaction.

[0164] The obtained aldehyde compound was dissolved in 400 mL of dichloromethane and cooled to -40 °C with stirring. Boron trifluoride diethyl ether solution (50.82 g, 0.35 mol) was added, and the mixture was stirred for 5 min. Allyltrimethylsilane (52.9 mL, 0.33 mol) was then added dropwise, and the mixture was stirred for 2 h. The reaction was monitored by TLC until complete (PE / EtOAc = 5 / 1). The reaction solution was slowly poured into 1000 mL of saturated sodium bicarbonate aqueous solution to quench the reaction. The mixture was separated, and the aqueous phase was extracted with dichloromethane (500 mL × 2). The organic phase was washed with saturated laboratory precipitate, dried over anhydrous NaSO4, and evaporated to dryness. Flash column purification (gradient elution: EtOAc / PE = 0–60%) yielded 103 g of a pale yellow viscous substance (52 g), with a yield of 86%. 1HNMR(CDCl3,400MHz)δ7.37-7.25(m,10H),5.96-5.86(m,1H),5.82(d,J=3.9Hz ,1H),5.15(d,J=17.1Hz,1H),5.08(d,J=10.1Hz,1H),4.88(d,J=11.4Hz,1H),4. 71(t,J=5.0Hz,1H),4.58-4.41(m,5H),3.84(d,J=10.7Hz,1H),3.63(d,J=10.7 Hz,1H),3.31(s,1H),2.64(m,1H),2.10-2.02(m,1H),1.61(s,3H),1.37(s,3H). 53

[0166] Compound 52 (103.0 g, 0.23 mol) was dissolved in 500 mL of DCM, and TEA (70.9 g, 0.7 mol) was added. MsCl (53.6 g, 0.47 mol) was added dropwise under ice bath. After the addition was complete, the ice bath was removed, and the mixture was allowed to warm to room temperature for 12 h. The reaction was monitored by TLC until complete (PE / EtOAc = 5 / 1). The reaction solution was quenched in 1000 mL of saturated NaHCO3 aqueous solution, and the mixture was separated. The aqueous phase was extracted with dichloromethane (500 mL × 2), and the organic phases were combined, washed with saturated brine (1 L), dried over anhydrous NaSO4, and evaporated to dryness to obtain a brown viscous substance. The substance was purified by flash column chromatography (gradient elution: EtOAc / PE = 0–60%) to give 106.0 g of mesylated sugar 53, a pale yellow viscous substance, with a yield of 90%. 1 H NMR(CDCl3,400MHz)δ7.38-7.27(m,10H),5.93(d,J=4.3Hz,1H),5.85-5.76(m,1H),5.22(dd,J=9.9, 2.0Hz,1H),5.12(d,J=9.5Hz,1H),5.05(dd,J=18.3,1.3Hz,1H),4.86-4.83(m,2H),4.58(d,J=11.8H z,1H),4.51(d,J=13.7Hz,1H),4.43(d,J=11.3Hz,1H),4.18(d,J=5.6Hz,1H),3.69(d,J=9.9Hz,1H), 3.63(d,J=9.9Hz,1H),3.17(s,3H),2.70-2.65(m,1H),2.24-2.07(m,1H),1.64(s,3H),1.42(s,3H). 54

[0168] Compound 53 (106.0 g, 0.204 mol) was dissolved in 200 mL of acetic acid, Ac₂O (125.6 g, 1.23 mol) was added, and 2.4 mL of concentrated H₂SO₄ (2.4 mL, 45 mmol) was added dropwise. The mixture was stirred at room temperature for 1 h, and the reaction was monitored by TLC until complete (PE / EtOAc = 5 / 1). Ethyl acetate (800 mL) and H₂O (800 mL) were added, and the mixture was separated. The aqueous phase was extracted once with ethyl acetate (800 mL). The organic phases were combined, washed with water (800 mL × 3), washed twice with saturated NaHCO₃ aqueous solution, dried over anhydrous NaSO₄, and evaporated to dryness to give 106.0 g of a mixture of diastereomers of acetyl compound 54, a colorless viscous substance, with a yield of 92%. 55

[0170] BSA (115.96 g, 0.57 mol) was added to a 200 mL toluene suspension of 6-chloroguanine (49.18 g, 0.29 mol). The mixture was heated to 60 °C and stirred until dissolved. After cooling to room temperature, 54 (105.48 g, 0.19 mol) dissolved in 150 mL toluene was added, followed by the dropwise addition of TMSOTf (64.46 g, 0.29 mol). The mixture was rapidly heated to 100 °C and reacted for 5 h. The reaction was monitored by TLC until complete (PE / EtOAc = 5 / 1). The reaction was stopped, and after cooling to room temperature, the mixture was extracted with ethyl acetate (1 L), washed with saturated NaHCO3 aqueous solution (500 mL), filtered off the precipitated solid with diatomaceous earth, separated from the filtrate, washed with water and saturated brine, dried over anhydrous Na2SO4, and purified by rotary evaporation flash column chromatography (gradient elution: CH3CN / CH2Cl2 = 0-50%) to obtain 89.25 g of nucleoside, with a yield of 70.82%.

[0171] The above product was dissolved in 540 mL of methanol, and K2CO3 (89.88 g, 0.65 mol) was added. The reaction was carried out at room temperature for 24 h, and the reaction was monitored by TLC until complete (CH2Cl2 / EtOAC = 5 / 1). The solvent was removed by concentration under reduced pressure, and the product was extracted with ethyl acetate (500 mL). The product was washed successively with water and saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated (gradient elution: CH3CN / CH2Cl2 = 0-50%) to give 51 g of allyl nucleoside 55, with a yield of 75%. 1H NMR(CDCl3,400MHz)δ7.86(s,1H),7.40-7.24(m,10H),5.91-5.85(m,2H),5. 24(d,J=17.1Hz,1H),5.16(d,J=10.2Hz,1H),5.01(s,1H),4.72-4.53(m,4H), 4.50(s,1H),4.42(s,1H),4.34(dd,J=8.3,5.3Hz,1H),4.09(s,3H),3.82(d, J=11.2Hz,1H),3.75(d,J=11.2Hz,1H),2.61-2.54(m,1H),2.45-2.38(m,1H); 13 C NMR(CDCl3,100MHz)δ161.52,159.41,152.54,137.58,137.09,136.53,133.98,128.59,128.46,128.04, 127.91,127.73,127.58,117.83,115.98,87.14,85.69,80.53,78.83,73.74,72.50,64.55,53.91,33.84. 56

[0173] Add 4.71 g, 4.95 mmol, 5% mol) of tris(triphenylphosphine)carbonylruthenium(II) hydrochloride to a 300 mL toluene solution of allyl nucleoside 55 (51 g, 98.92 mmol), purged with nitrogen three times, and heated to 100 °C for 48 h. Stop the reaction and evaporate the solvent. Dissolve the residue in tetrahydrofuran (300 mL) and tert-butanol (75 mL), add 48 mL, 0.23 mol, of 50% NMO, and slowly add potassium osmium tetroxide dihydrate (0.94 g, 0.99 mmol, 1% mol). Heat to 60 °C and react for 5 h. Monitor the reaction by TLC until complete (CH₂Cl₂ / MeOH = 10 / 1). The reaction was quenched with sodium sulfite solution, extracted with ethyl acetate, washed with water, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: EtOAc / CH2Cl2 = 0-40%) to give 44 g of a mixture of dihydroxylated isomers, yield 79%. ESI-MS (m / z) 585.16 [M+Na] + .

[0174] The dihydroxylated mixture was dissolved in MeOH (300 mL), H₂O (40 mL) was added, followed by NaIO₄ (20.0 g, 95.3 mmol). The reaction was carried out at room temperature for 12 h. The insoluble solids were filtered off, and ammonia (117 mL) was added to the filtrate. Then, I₂ (24.3 g, 0.1 mol) was added in portions, and the reaction was carried out at room temperature for 24 h. The reaction was monitored by TLC to indicate completion (CH₂Cl₂ / MeOH = 10 / 1). The reaction solution was poured into an aqueous sodium sulfite solution (400 mL), and water (600 mL) and ethyl acetate (600 mL) were added. The mixture was stirred for 10 min and separated. The aqueous phase was extracted with ethyl acetate (300 mL), and the organic phases were combined. The organic phases were washed successively with saturated brine (600 mL), dried over anhydrous sodium sulfate, evaporated to dryness, and purified by flash column chromatography (EtOAc / CH₂Cl₂ = 0–80%) to obtain 13.2 g of cyano-locked nucleoside 56, with a yield of 27%. 1 H NMR(CDCl3,400MHz)δ7.75(s,1H),7.38-7.21(m,10H),6.11(s,1H),4.93-4.88(m,4H),4. 68-4.59(m,4H),4.49(s,1H),4.09(s,3H),3.97(d,J=11.4Hz,1H),3.88(d,J=11.4Hz,1H); 13 C NMR (CDCl3, 100MHz) δ161.64,159.49,152.38,137.00,136.42,136.34,128.66,128.65,128. 44,128.13,127.77,115.96,115.82,87.57,86.07,78.28,77.95,74.08,72.71,63.87,53.40. 57

[0176] Cyano-mononucleotide 56 (15 g, 29.2 mmol) was dissolved in THF / MeOH (100 mL / 100 mL), and 2N HCl (146 mL) was added. The mixture was heated to 60 °C and reacted for 12 h. The reaction was monitored by TLC until complete (CH2Cl2 / MeOH = 10 / 1). Water (400 mL) and ethyl acetate (500 mL) were added, and the mixture was stirred for 10 min. The mixture was separated into liquid and liquid phases. The aqueous phase was extracted again with ethyl acetate (300 mL × 2). The organic phases were combined and washed successively with saturated sodium bicarbonate (500 mL) and saturated brine (500 mL), and dried over anhydrous sodium sulfate. The product was then evaporated to dryness to give 14.0 g of pale yellow solid product 57, with a yield of 96%. 1H NMR(DMSO-d6,400MHz)δ10.73(s,1H),7.81(s,1H),7.37-7.27(m,10H),6.67(br s,2H),5.98(s,1H),5.23(s,1H),5.05(s,1H),4.73(s,3H),4.61(s,3H),4.03(d,J=12.0Hz,1H),3.91(d,J=12.0Hz,1H); 13 C NMR(DMSO-d6,100MHz)δ157.11,154.49,151.11,138.22,137.78,128.77,128.73,128.27,128 .09,128.02,127.83,117.66,116.93,87.54,84.70,78.88,78.69,73.30,71.88,71.34,65.25. 58

[0178] Anhydrous FeCl3 (29.2 g, 180.0 mmol) was dissolved in dichloromethane (110 mL), and acetic anhydride (17 mL, 180 mmol) was added dropwise. After the addition was complete, nucleoside 56 (10.0 g, 20.0 mmol) was added in portions at room temperature, and the reaction was allowed to proceed for 4 h at room temperature. The reaction was monitored by TLC until it was complete (CH2Cl2 / MeOH = 10 / 1). After the reaction was complete, the mixture was cooled in an ice bath, and methanol (50 mL) was added dropwise, which was exothermic. After the addition was complete, the reaction solution was poured into ice water (600 mL), stirred for 10 min, and passed through diatomaceous earth to remove flocculent matter. The aqueous phase was extracted with dichloromethane (200 mL × 2), the organic phases were combined, washed with water (500 mL), dried over anhydrous sodium sulfate, and evaporated to dryness. NH3-MeOH (70 mL) was added to the concentrate, and the mixture was stirred at room temperature for 12 h. A white solid precipitated out. The solid was filtered, dried, and 5 g of guanosine 58 was obtained as a white solid, with a yield of 78%. 1 H NMR (DMSO-d6, 400MHz) δ7.83 (s, 1H), 6.67 (br s, 2H), 5.02 (s, 1H), 4.62 (s, 1H), 4.47 (s, 1H), 3.89 (d, J = 13.0Hz, 1H), 3.80 (d, J = 13.0Hz, 1H); 13 C NMR(DMSO-d6,100MHz)δ157.22,154.53,150.92,134.41,118.04,117.06,89.54,84.68,81.14,71.21,80.80,56.48,56.48. 59

[0180] DMFDMA (3.5 mL, 26.23 mmol) was added to 70 mL of methanol containing guanosine 58 (1.4 g, 4.37 mmol), and the mixture was heated to 60 °C and reacted for 4 h. The reaction was stopped, the reaction solution was concentrated under reduced pressure, dried under vacuum, and the residue was dissolved in 20 mL of pyridine. 4,4'-dimethoxytriphenylchloromethane (2.22 g, 6.56 mmol) was added, and the mixture was reacted at room temperature for 6 h. 15 mL of methanol was added to the reaction solution, and the mixture was stirred for 5 min. The reaction solution was concentrated under reduced pressure, and 20 mL of ethyl acetate and 20 mL of water were added to the residue. The mixture was stirred, and a white solid precipitated. The solid was filtered, washed with water, and the filter cake was dried under vacuum. Flash column purification (gradient elution: MeOH / DCM = 0-10%) was performed to give 1.83 g of white solid product 59, yield 62%. 1 H NMR(DMSO-d6,400MHz)δ11.46(s,1H),8.61(s,1H),7.91(s,1H),7.63-6.88(m,13H),6.10(s,1H),6.07(br s,1H),4.80(s,1H),4.75(s,1H),4.20(s,1H),3.75(s,6H),3.62(d,J=11.0Hz,1H),3.36(d,J=11.0Hz),3.16(s,3H),3.05(s,3H); 13 CNMR(DMSO-d6,100MHz)δ171.23,148.75,158.59,158.07,158.00,149.26,145.31,135.86,135.82,135.23,130.20,130.13,128.35,128 .12,127.24,120.57,113.71,113.68,88.43,85.86,85.24,80.29,80.45,71.76,57.84,55.51,41.17,35.14; ESI-MS(m / z)696.25[M+H3O] + . 60

[0182] To a 40 mL dichloromethane solution of guanosine 59 (1.8 g, 2.66 mmol) and tetrazolium diisopropylammonium salt (455 mg, 2.66 mmol), 1.3 mL of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonic diamine (1.3 mL, 4.0 mmol) was added, and the reaction was carried out at room temperature for 12 h. The reaction was quenched by adding 15 mL of saturated sodium bicarbonate solution, extracted with dichloromethane, washed with water and saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: CH3CN / DCM = 10%-75%) to give 1.84 g of white solid product 60, yield 79%. 31P-NMR(152MHz,DMSO-d6)δ148.25,147.66; ESI-MS(m / z)896.47[M+H3O] + .

[0183] 2. Synthesis of S-6'-CN-LNA-T monomer

[0184]

[0185] Reaction conditions: (a)(1) 2-iodobenzoic acid, CH3CN, reflux, 1.5h; (2) LiAlH4, THF, -78℃, 8h; (b) methanesulfonyl chloride, TEA, dichloromethane, 6h; (c) conc. H2SO4, Ac2O, HOAc, room temperature, 1h; (d) thymine, N,O-bis(trimethylsilylacetamide), TMSOTf, CH3CN, 80℃, 2h; (e) K2CO3, MeOH, room temperature, 12h; (f)(1) tris(triphenylphosphine)carbonylruthenium(II) hydrochloride, EtOH, 80℃, 24h; (2) 50% NMO, potassium osmium tetroxide dihydrate, THF, t-BuOH, H2O, 60℃, 12h; (3) NaIO4, MeOH, H2O, room temperature, 5h; (4) NH4OH, I2, room temperature, 48h; (g) (1) Anhydrous FeCl3, Ac2O, dichloromethane, room temperature, 3h; (2) NH3, MeOH, room temperature, 12h.

[0186] Compound 52 (52.8 g, 0.12 mol) was dissolved in 500 mL of acetonitrile, and 2-iodobenzoic acid (50.34 g, 0.18 mol) was added. The mixture was heated to 60 °C and stirred for 1.5 h. The reaction was monitored by TLC until complete (PE / EtOAc = 5 / 1). The reaction was stopped, and the mixture was allowed to cool to room temperature. The mixture was filtered through diatomaceous earth, and the filter cake was washed twice with acetonitrile (100 mL × 2). The solvent was evaporated, and the mixture was dried under vacuum. The residue was dissolved in 300 mL of anhydrous THF and cooled to -78 °C with stirring. Lithium aluminum hydride (8.45 g, 0.223 mol) was added in portions, and the mixture was stirred for 8 h. The reaction was monitored by TLC until it was complete (PE / EtOAc = 5 / 1). Water (8.45 mL) and 15% NaOH solution (8.45 mL) were added dropwise to the system, and then water (25 mL) was added to completely quench the reaction. The reaction solution was filtered through diatomaceous earth, the filtrate was evaporated to dryness, and purified by flash column chromatography (EtOAc / PE = 0-20%) to obtain 41.45 g of colorless viscous substance 61, with a yield of 75%. 1H NMR (CDCl3, 400MHz) δ7.37-7.25 (m, 10H), 5.99-5.88 (m, 1H), 5.86 (d, J = 4.0Hz, 1H), 5. 09-5.03(m,2H),4.84(d,J=11.6Hz,1H),4.74(dd,J=5.3,4.1Hz,1H),4.55-4.42(m,3H) ,4.32(d,J=5.4Hz,1H),4.27(d,J=10.2Hz,1H),3.60(d,J=10.2Hz,1H),3.48(d,J=10. 2Hz,1H),2.95(s,1H),2.48-2.43(m,1H),2.15-2.09(m,1H),1.64(s,3H),1.38(s,3H). 62

[0188] Compound 61 (4.0 g, 9.1 mmol) was dissolved in 20 mL of dichloromethane, and triethylamine (3.8 mL, 27.3 mmol) was added. Methanesulfonyl chloride (1.4 mL, 18.2 mmol) was added dropwise under ice bath conditions. After the addition was complete, the ice bath was removed, and the mixture was allowed to warm to room temperature for 6 h. The reaction was monitored by TLC until complete (hexane / acetone = 10 / 1). The reaction was quenched by adding 50 mL of saturated NaHCO3 aqueous solution. The mixture was separated, and the aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed twice with saturated brine, dried over anhydrous NaSO4, and evaporated to dryness. The solution was then purified by flash column chromatography (EtOAc / PE = 0-40%) to give 3.75 g of colorless viscous compound 62, with a yield of 79%. 1 H NMR(CDCl3,400MHz)δ7.41-7.27(m,10H),5.92(d,J=4.3Hz,1H),5.85-5.77(m,1H),5.22(dd,J=9.8 4,1.80Hz,1H),5.12(d,J=10.2Hz,1H),5.05(d,J=17.0Hz,1H),4.86-4.81(m,2H),4.58(d,J=11.8Hz ,1H),4.52(d,J=11.8Hz,1H),4.43(d,J=11.3Hz,1H),4.17(d,J=5.5Hz,1H),3.68(d,J=9.9Hz,1H), 3.59(d,J=9.9Hz,1H),3.17(s,3H),2.69-2.64(m,1H),2.23-2.15(m,1H),1.63(s,3H),1.41(s,3H); 13C NMR(CDCl3,100MHz)δ137.45,137.12,133.39,128.56,128.47,128.06,128.02,127.98,1 27.66,118.34,114.78,105.76,88.39,85.49,81.36,73.85,73.11,70.59,27.39,26.73. 63

[0190] Compound 62 (3.5 g, 6.75 mmol) was dissolved in 20 mL of HOAc, and Ac2O (2.83 mL, 40.5 mmol) was added. 80 μL of concentrated H2SO4 was added dropwise, and the mixture was stirred at room temperature for 1.0 h. The reaction was monitored by TLC until complete (PE / EtOAc = 5 / 1). The solution was diluted with 100 mL of ethyl acetate, washed with water to weakly acidic conditions, washed with a saturated NaHCO3 aqueous solution, washed twice with saturated brine, dried over anhydrous NaSO4, evaporated to dryness, and dried under vacuum to give 3.7 g of a mixture of acetylated isomers, 63, as a colorless viscous liquid, with a yield of 97%. ESI-MS (m / z) 585.16 [M+Na] + . 64

[0192] N,O-bis(trimethylsilylacetamide) (10 mL, 40.5 mmol) was added to 50 mL of acetonitrile suspension of thymine (2.56 g, 20.3 mmol), and the mixture was stirred for 1 h until the solution was dissolved. Then, 63 (3.8 g, 6.75 mmol) and TMSOTf (1.84 mL, 10.1 mmol) were added sequentially, and the mixture was heated to 80 °C for 2 h. The reaction solution was slowly poured into 400 mL of water, and the pH was adjusted to neutral with saturated NaHCO3 solution. The solution was extracted with ethyl acetate, washed with water, washed with saturated sodium chloride, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: EtOAc / CH2Cl2 = 0-40%) to obtain 3.72 g of nucleoside 64 as a foamy solid, with a yield of 88%. 1H NMR (CDCl3, 400MHz) δ8.51 (s, 1H), 7.42-7.28 (m, 11H), 6.35 (d, J = 6.8Hz, 1H), 5. 70-5.64(m,2H),5.15-5.08(m,2H),5.07(d,J=17.4Hz,1H),4.76(d,J=11.0Hz,1 H),4.61(s,2H),4.51-4.45(m,2H),3.86(d,J=10.0Hz,1H),3.71(d,J=10.0Hz,1 H),3.00(s,3H),2.49-2.44(m,1H),2.29-2.21(m,1H),2.11(s,3H),1.55(s,3H); 13 C NMR(CDCl3,100MHz)δ170.25,163.35,150.36,136.80,136.53,133.04,128.82,128.69,128.43,128.39,12 8.08,127.75,118.73,87.36,86.01,83.39,78.93,75.03,74.53,73.99,69.85,39.09,35.69,20.74,12.09. 65

[0194] K₂CO₃ (3.07 g, 22.3 mmol) was added to 40 mL of a methanol solution of nucleoside 64 (3.5 g, 5.57 mmol), and the reaction was carried out at room temperature for 12 h. The reaction was monitored by TLC until complete. The reaction was stopped, filtered through diatomaceous earth, the solvent was removed by rotary evaporation under reduced pressure, extracted with ethyl acetate, washed with water, washed with saturated NaCl aqueous solution, dried over anhydrous Na₂SO₄, filtered, concentrated, and purified by flash column chromatography (gradient elution: EtOAc / CH₂Cl₂ = 0-60%) to give 2.2 g of allyl nucleoside 65, a white solid, in 80% yield. 1 H NMR(CDCl3,400MHz)δ9.16(br s,1H),7.47(s,1H),7.41-7.28(m,10H),5.86-5.79(m,1H),5.63(s,1H),5.15(d,J=4.6Hz,1H),5.11(s,1H),4.69-4. 49(m,5H),4.08(dd,J=9.0,5.0Hz,1H),3.94(s,1H),3.88(s,2H),2.74-2.66(m,1H),2.32-2.26(m,1H),1.64(s,3H); 13C NMR(CDCl3,100MHz)δ163.97,149.77,137.48,136.78,134.96,128.61,128.52,128.11,127.89, 127.71,117.41,110.13,87.95,86.93,84.15,76.82,76.44,73.93,72.33,64.85,35.21,12.35. 66

[0196] To a 15 mL ethanol solution of allyl 65 (1.0 g, 2.04 mmol), ruthenium(II) tris(triphenylphosphine)carbonyl hydrochloride (48 mg, 2.5% mol) was added, and the mixture was heated to 80 °C and reacted for 24 h. The reaction solution was directly concentrated, and the residue was dissolved in 20 mL of tetrahydrofuran. Then, 2 mL of tert-butanol, potassium osmium tetroxide dihydrate (7.5 mg, 1% mol), and 50% N-methylmorpholine-N-oxide (1.43 mL, 6.12 mmol) were added sequentially, and the mixture was heated to 60 °C and reacted for 12 h. After quenching the reaction with saturated sodium sulfite solution, the mixture was extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by flash column chromatography (gradient elution: MeOH / CH₂Cl₂ = 0-10%) to give 560 mg of the dihydroxylated isomer mixture, yield 52%.

[0197] The above-mentioned dihydroxylated product (500 mg, 0.95 mmol) was dissolved in 20 mL of methanol, and 4 mL of water and NaIO4 (306 mg, 1.43 mmol) were added. The mixture was stirred at room temperature for 5 h. After filtering off the insoluble solid, 25% ammonia (1.3 mL, 19 mmol) was added to the filtrate, followed by the addition of I2 (723 mg, 2.85 mmol) in portions. The reaction was carried out at room temperature for 48 h. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by flash column chromatography (gradient elution: EtOAc / DCM = 0-60%) to give 150 mg of cyano-locked nucleoside 66 as a white foamy solid, with a yield of 32%. 1 H NMR(CDCl3,400MHz)δ9.18(br s,1H),7.37-7.28(m,11H),5.63(s,1H),4.78-4.74(m,2H),4.67-4.63(m,5H),4.41(d,J=11.2Hz,1H),4.08-4.05(m,2H),1.66(s,3H); 13C NMR(CDCl3,100MHz)δ163.62,149.63,136.83,136.03,134.05,128.70,128.61,128.38,127.92,127.8 9,114.16,111.00,88.51,86.99,78.02,75.80,74.26,72.60,69.23,63.99; ESI-MS(m / z)498.17[M+Na] + . 36

[0199] At room temperature, acetic anhydride (9 mL, 95 mmol) was slowly added to 50 mL of a dichloromethane suspension of anhydrous FeCl3 (9.2 g, 57 mmol). After stirring until the FeCl3 dissolved, 66 (4.5 g, 9.5 mmol) was added in portions, and the mixture was stirred at room temperature for 3 h. The reaction solution was then poured into 100 mL of a methanol-ice-water (v / v, 1:1) mixture. The precipitate at the bottom of the flask was also diluted with 100 mL of the methanol-ice-water mixture and extracted with dichloromethane. The organic layers were combined, washed with water and brine, dried over MgSO4, and concentrated. The obtained acetyl intermediate was dissolved in methanol and deprotected with 45 mL of 2N NH3-methanol solution. After stirring at room temperature for 12 h, the insoluble matter was removed by filtration. The resulting filtrate was concentrated and purified by rapid column chromatography (gradient elution: MeOH / DCM = 0-15%) to give 1.8 g of deprotected thymine-modified cyano-locked nucleoside monomer as a white solid, in 64% yield. 1 H-NMR(400MHz,MeOH-d4)δ7.62(s,1H),5.86(s,1H),4.78(s,1H),4.53(s,1H ),4.22(s,1H),4.17(d,J=12.0Hz,1H),4.13(d,J=12.0Hz,1H),1.89(s,3H); 13 C-NMR(100MHz,MeOH-d4)δ164.97,150.36,134.89,114.74,109.74,90.36,86.75,80.78,69.56,68.45,55.87.11.19; ESI-MS(m / z)296.13[M+H] + .

[0200] The crystal structure of the S-6'-CN-LNA-T monomer is as follows: Figure 1 As shown.

[0201] Synthesis of S-6'-CN-LNA-T phosphoramide monomer

[0202]

[0203] The reaction conditions were: (a) DMTrCl, pyridine, room temperature, 12 h; (b) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphine diamine, 1H-tetrazolium tetrazolium, room temperature, 5 h. 37

[0205] To a 15 mL pyridine solution of cyano-locked nucleoside derivative 36 (2.0 g, 6.77 mmol), 4,4'-dimethoxytriphenylmethyl chloride (3.4 g, 10 mmol) was added, and the mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC until complete (dichloromethane / methanol = 10 / 1). The reaction was quenched with 50 mL of methanol, concentrated under reduced pressure, and 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 give 2.6 g of 5'-O-DMTr protected nucleoside 37 as a white solid, yield 64%. 1 H-NMR(400MHz,DMSO-d6)δ11.44(s,1H),7.46-7.24(m,10H),6.92(dd,J=6.0,1.4Hz,4H),6.30(s,1H),5.5 3(s,1H),5.11(s,1H),3.79(d,J=7.9Hz,1H),3.74(s,6H),3.49(d,J=7.4Hz,1H),1.60(s,3H); 13C-NMR(10 0MHz,DMSO-d6)δ162.66,157.16,148.78,143.44,134.16,133.74,132.93,128.73,128.65,126.87,126.5 4,126.80,114.35,112.20,107.85,87.62,85.06,84.89,79.26,68.84,67.42,57.44,53.97,11.15; ESI-MS 620.20(m / z)[M+Na] + . 38

[0207] To a 20 mL solution of nucleoside 37 (2.0 g, 3.35 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonamide (1.51 g, 5 mmol) in dichloromethane, 1H-tetrazole (230 mg, 3.35 mmol) was added, and the mixture was reacted at room temperature for 5 h. The reaction was confirmed to be complete by TLC (ethyl acetate / dichloromethane = 5 / 1). A saturated NaHCO3 solution was added to the reaction mixture, followed by extraction with dichloromethane, washing with saturated brine, drying to MgSO4, concentration, and flash column purification (gradient elution: ethyl acetate / dichloromethane = 0-20%) to give 2.1 g of phosphoramide 38 as a white, foamy solid, in 79% yield.31 P-NMR(152MHz,DMSO-d6)δ148.95,148.35; ESI-MS(m / z)798.33[M+H] + .

[0208] 3. Synthesis of R-6'-CN-LNA-T monomer

[0209]

[0210] Reaction conditions: (a) Thymine, N,O-bis(trimethylsilylacetamide) (BSA), TMSOTf, CH3CN, 80℃, 3h; (b) K2CO3, MeOH, room temperature, 12h; (c) (1) Tris(triphenylphosphine)carbonylruthenium(II) hydrochloride, EtOH, 100℃, 28h; (2) 50% NMO, potassium osmium tetroxide dihydrate, THF, H2O, 80℃, 12h; (3) NaIO4 ,EtOH,H2O,room temperature,5h;(4)NH4OH,I2,EtOH,room temperature,48h;(d)(1)Anhydrous FeCl3,Ac2O,dichloromethane,room temperature,3h;(2)NH3,MeOH,room temperature,12h;(e)DMTrCl,pyridine,room temperature,12h;(f)2-CyanoethylN,N,N',N'-Tetraisopropylphosphinediamine,1H-Tetrazolium tetrazolium,room temperature,5h; 67

[0212] Thymine T (30.3 g, 0.24 mol) was dissolved in acetonitrile (300 mL), BSA (122.1 g, 0.60 mol) was added, and the mixture was heated to 50 °C until the system was completely dissolved. The heating was removed and the mixture was cooled to room temperature. An acetonitrile solution (100 mL) of compound 54 (112.8 g, 0.20 mol) was added with stirring, followed by the dropwise addition of TMSOTf (57.8 g, 0.26 mol). The mixture was heated to 80 °C and stirred for 3 h. The reaction was monitored by TLC until it was complete (DCM / MeOH = 1 mL / 2 drops). After the reaction was complete, the system was cooled to room temperature and quenched by pouring in saturated sodium bicarbonate solution (1000 mL), which produced a large number of bubbles. Ethyl acetate (1000 mL) was added, and the mixture was stirred for 15 min. After standing, the liquid was separated, and the aqueous phase was extracted with EA (400 mL × 1). The organic phases were combined and washed successively with saturated brine (1000 mL), dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 135 g of yellow viscous substance 67. No purification was required before proceeding to the next step of the reaction. 1H NMR(CDCl3,400MHz)δ8.37(s,1H),7.42-7.33(m,11H),6.30(d,J=6.8Hz,1H),6.87-5.77(m,1H),5.52(t,J=6.2Hz,1H),5.32-5.12(m,3H),4.68-4 .57(m,5H),3.86(d,J=10.2Hz,1H),3.69(d,J=10.2Hz,1H),2.99(s,3H), 2.85-2.79(m,1H),2.60-2.52(m,1H),2.11(s,3H),1.53(d,J=1.2Hz,3H). 68

[0214] The product 67 obtained in the previous step was dissolved in anhydrous methanol (800 mL), and anhydrous K₂CO₃ (83.1 g, 0.6 mol) was added. The reaction was carried out at room temperature for 12 h, and the reaction was monitored by TLC until completion (DCM / MeOH = 1 mL / 2 drops). After the reaction was complete, the mixture was filtered, and the filter cake was washed with ethyl acetate (100 mL). The filtrate was evaporated to dryness, and the product was separated by flash column chromatography (MeOH / DCM = 0-15%) to give 50.0 g of product 68, with a yield of 48.54%. 1 HNMR (CDCl3, 400MHz) δ8.54 (s, 1H), 7.57 (d, J = 1.2Hz, 1H), 7.40-7.28 (m, 10H), 5.89-5 .79(m,1H),5.64(s,1H),5.32(dd,J=17.1,1.4Hz,1H),5.15(d,J=10.2Hz,1H),4.70-4. 55(m,4H),4.41(s,1H),4.30(dd,J=7.8,5.9Hz,1H),4.10(s,1H),3.86(d,J=11.0Hz,1H ),3.76(d,J=11.0Hz,1H),2.57-2.49(m,1H),2.38-2.31(m,1H),1.60(d,J=1.2Hz,1H); 13 C NMR(CDCl3,100MHz)δ163.88,149.81,137.48,136.88,134.93,133.66,128.61,128.50,128.16,1 28.11,127.86,127.68,118.06,110.17,87.63,86.54,79.85,77.20,76.36,73.86,72.34,64.58. 69

[0216] To a 300 mL ethanol solution of allyl 68 (38.0 g, 77 mmol), ruthenium(II) tris(triphenylphosphine)carbonyl hydrochloride (1.8 g, 2.5 mol) was added, and the mixture was heated to 80 °C and reacted for 28 h. The reaction solution was directly concentrated, and the residue was dissolved in 250 mL of tetrahydrofuran. Then, 125 mL of tert-butanol, potassium osmium tetroxide dihydrate (280 mg, 1 mol), and 50% N-methylmorpholine-N-oxide (48 mL, 228 mmol) were added sequentially, and the mixture was heated to 60 °C and reacted for 12 h. After quenching the reaction with saturated sodium sulfite solution, the mixture was extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by flash column chromatography (gradient elution: MeOH / DCM = 0-10%) to give 26 g of a dihydroxylated isomer mixture, yield 64%.

[0217] The above-mentioned dihydroxylated product (26 g, 50 mmol) was dissolved in 300 mL of ethanol, and 100 mL of an aqueous solution of NaIO4 (15.9 g, 74 mmol) was added dropwise. The mixture was stirred at room temperature for 5 h. After filtering off the insoluble solids, the reaction solution was concentrated to a minimum volume, extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue did not require further purification and could be directly used for the next step. ESI-MS (m / z) 477.12 [MH] - .

[0218] The above aldehyde derivative was dissolved in 200 mL of ethanol, and 37.4 mL (500 mmol) of 25% ammonia solution was added. Then, I2 (12.6 g, 2.85 mmol) was added in portions, and the reaction was carried out at room temperature for 48 h. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by flash column chromatography (gradient elution: EtOAc / DCM = 0-60%) to give 6 g of cyano-locked nucleoside 69, a white foamy solid, with a yield of 25%. 1 H-NMR(400MHz,MeOH-d4)δ8.99(s,1H),7.58(d,J=1.2Hz,1H),7.38-7.28(m,10 H),5.89-5.79(m,1H),5.64(s,1H),5.32-5.15(m,2H),4.70-4.55(m,4H),4.43 (s,1H),4.30(dd,J=7.8,5.9Hz,1H),4.10(s,1H),3.86(d,J=11.0Hz,1H),3.76 (d,J=11.0Hz,1H),2.55-2.49(m,1H),2.38-2.32(m,1H),1.60(d,J=1.2Hz,1H). 70

[0220] 50 mL of dichloromethane was added to a suspension of anhydrous FeCl3 (20.5 g, 126 mmol), followed by the slow addition of acetic anhydride (12 mL, 126 mmol) at room temperature. The mixture was stirred until the FeCl3 dissolved, and then 69 (10 g, 21 mmol) was added in portions. After stirring at room temperature for 3 h, the precipitate at the bottom of the flask was separated from the reaction solution. The reaction solution was then poured into a 300 mL methanol-ice-water (v / v, 1:1) mixture, and the bottom precipitate was diluted with another 300 mL methanol-ice-water mixture and extracted with dichloromethane. The organic layers were mixed and washed with water and brine, dried over MgSO4, and concentrated. The resulting acetic acid intermediate was then deprotected in methanol solution with 100 mL of 2N NH3. After stirring at room temperature for 12 h, the undissolved solid was filtered off and concentrated. Purification was achieved by rapid dissolution chromatography (gradient elution: MeOH / DCM, 0–15%) to give 5 g of deprotected nucleoside 70 as a white solid, in 80% yield. 1 H-NMR (400MHz, MeOH-d4) δ7.69(d,J=1.1Hz,1H),5.69(s,1H),4.92(s,1H),4.45(s,1H),4.27(s,1H),3.96(s,1H),1.89(d,J=1.1Hz,3H); 13 C-NMR(100MHz,MeOH-d4)δ165.86,151.26,135.77,117.02,110.65,90.40,87.61,81.65,70.88,70.33,56.16,12.12; ESI-MS(m / z)296.13[M+H] + . 71

[0222] 50 mL of pyridine and 13.8 g of 4,4'-dimethoxytriphenylmethyl chloride (40.6 mmol) were added to a solution of 70 (8.0 g, 27.1 mmol). After stirring at room temperature for 12 h, the reaction mixture was quenched with 50 mL of methanol and concentrated under reduced pressure. The resulting residue was then diluted with ethyl acetate. The organic layer was washed with water and brine, dried over Na2SO4, and concentrated. Purification by rapid dissolution chromatography (gradient elution: MeOH / CH2Cl2, 0–10%) yielded 13.53 g of 5'-O-DMTr protected nucleoside 71 as a pale yellow powder, in 84% yield. 1H-NMR (400MHz, DMSO-d6) δ11.48(s,1H),7.47-6.91(m,14H),6.27(d,J=4.0Hz,1H),5.63(s,1H),5.01(s,1H),4.52 (s,1H),3.42(d,J=4.4Hz,1H),3.74(s,6H),3.65(d,J=11.2Hz,1H),3.36(d,J=11.2Hz,1H),1.60(d,J=0.8Hz,3H); 13 C-NMR(100MHz,DMSO-d6)δ163.81,158.27,149.91,144.50,135.07,134.84,134.00,129.91,129.84,128.00,127.71,126 .94,117.41,113.34,109.01,87.99,86.38,86.35,80.37,70.52,70.33,58.47,55.09,12.35; ESI-MS(m / z)620.20[M+Na] + . 72

[0224] To a solution of 71 (4.0 g, 6.7 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonamide (3.0 g, 10.0 mmol), 1H-tetraazole (376 mg, 5.36 mmol) was added. After stirring at room temperature for 5 h, a saturated NaHCO3 solution was added, and the reaction mixture was extracted with dichloromethane. The organic layer was washed with brine, dried over MgSO4, and concentrated. Rapid chromatographic purification (gradient eluent: EtOAc / CH2Cl2, 0–20%) yielded 4.4 g of phosphoramide 10a, a white foam, in 82% yield. 31 P-NMR(152MHz,DMSO-d6)δ148.80,148.75; ESI-MS(m / z)798.32[M+H] + .

[0225] 4.R-6'-CN-LNA- m Synthesis of C phosphoramide monomer

[0226]

[0227] The reaction conditions were as follows: (a)(1) TESCl, Et3N, acetonitrile, 0℃-room temperature, 3h; (2) 1,2,4-triazole, POCl3, acetonitrile, 0℃-room temperature, 2h; (3) NH4OH, 1,4-dioxane, room temperature, 2h; (b)(1) Bz2O, acetonitrile, room temperature, 20h; (2) TBAF, THF, room temperature, 12h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphine diamine, 1H-tetrazole, dichloromethane, room temperature, 5h. twenty three

[0229] 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 after the addition was complete. 1,2,4-triazole (8.67 g, 125.55 mmol) was added to the reaction solution, and the mixture was stirred for 10 min. The mixture was then cooled to 0°C, and POCl3 (2.34 mL, 25.11 mmol) was added dropwise. The reaction was continued at room temperature for 2 h, and TLC analysis confirmed completeness (dichloromethane / methanol = 10 / 1). The reaction solution was poured into 300 mL of ice water, extracted with ethyl acetate, 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, and 6.25 mL (92 mmol) of 25% ammonia solution was added. The mixture was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure, diluted with ethyl acetate, and the organic layer was washed with water and saturated brine. The solution was dried over anhydrous Na₂SO₄, filtered, concentrated, and purified by flash column chromatography (gradient elution: methanol / dichloromethane = 0-15%) to give 5.5 g of pale yellow solid aminonucleoside 23, with a yield of 92%. 1 H NMR(DMSO-d6,400MHz)δ7.52-7.25(m,12H),6.96-6.91(m,6H),5.66(s,1H),4.93(s,1H),4.45(s,1H),4.40(s,1H), 3.75(s,6H),3.54(d,J=11.2Hz,1H),3.41(d,J=11.2Hz,1H),1.72(s,3H),0.78(t,J=7.9Hz,9H),0.53-0.41(m,6H); 13C NMR(DMSO-d6,100MHz)δ166.09,158.81,158.79,154.95,144.78,136.54,135.38,135.17,130.30,130.14,128.43,128.07,127.46,11 7.58,113.81,113.77,101.92,88.29,87.59,86.82,80.77,71.60,71.09,58.50,55.55,14.01,6.78,4.42; ESI-MS(m / z)733.31[M+Na] + . twenty four

[0231] Benzoic anhydride (3.85 g, 12 mmol) was added to a solution of aminonucleoside 23 (5.5 g, 7.73 mmol) in acetonitrile (60 mL). 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, and after stirring at room temperature for 12 h, 15% NaOH was added to adjust the pH to 10, and stirring was continued for another 3 h. The reaction solution was diluted with ethyl acetate, washed with water, and the organic layer was washed again with water and saturated brine. The solution was dried over anhydrous Na₂SO₄, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-40%) to give 3.1 g of 5'-O-DMTr protected nucleoside 24 as a white, foamy solid, in 57% yield. 1 HNMR(DMSO-d6,400MHz)δ13.06(br s,1H),8.19(s,2H),7.69-7.25(m,13H),6.94(d,J=8.3Hz,4H),6.32(s,1H),5.70(s,1H),5.06(s,1 H),4.53(s,1H),4.38(d,J=4.1Hz,1H),3.73(d,J=11.0Hz,1H),3.40(d,J=11.0Hz,1H),1.84(s,3H); 13 C NMR(DMSO-d6,100MHz)δ178.64,159.43,158.75,147.49,144.93,137.11,135.53,135.32,133.07,130.37,130.32,129.85,128.83, 128.46,128.21,127.41,117.77,113.82,110.12,88.75,87.28,80.55,71.06,70.70,58.88,55.56,13.66; ESI-MS(m / z)701.32[M+H] + . 25

[0233] To a 20 mL solution of nucleoside 24 (1.7 g, 2.42 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonamide (1.1 g, 3.63 mmol) in anhydrous dichloromethane, 1H-tetrazole (135 mg, 1.94 mmol) was added. After reacting at room temperature for 5 h, a saturated NaHCO3 solution was added to the reaction mixture, followed by extraction 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 give 1.83 g of phosphoramide 25, a white foamy solid, in 84% yield. 31 P NMR(152MHz,DMSO-d6)δ149.20,148.47; ESI-MS(m / z)901.46[M+H] + .

[0234] 5.S-6'-CN-LNA- m Synthesis of C phosphoramide monomer

[0235]

[0236] The reaction conditions were as follows: (a)(1) TESCl, Et3N, CH3CN, 0℃-room temperature, 3h; (2) 1,2,4-triazole, POCl3, CH3CN, 0℃-room temperature, 2h; (3) NH4OH, 1,4-dioxane, room temperature, 2h; (b)(1) Bz2O, CH3CN, room temperature, 20h; (2) TBAF, THF, room temperature, 12h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonic diamine, 1H-tetrazole, CH2Cl2, room temperature, 5h. 48

[0238] At 0°C, triethylsilyl chloride (4.78 mL, 28.45 mmol) was added to a solution of thymidine 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 mixture, and stirring was continued for 10 min. Then, 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 mixture 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 and 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. Flash column purification (gradient elution: MeOH / CH₂Cl₂ = 0-15%) yielded 3.9 g of aminonucleoside 48, a pale yellow solid, in 96% yield. 1 H NMR(DMSO-d6,400MHz)δ7.50-7.44(m,4H),7.37-7.25(m,8H),6.93(d,J=8.6Hz,4H),5.55(s,1H),5.09(s,1H),4.46(s,1H), 4.35(s,1H),3.75(s,6H),3.68(d,J=11.2Hz,1H),3.53(d,J=11.2Hz,1H),1.70(s,3H),0.81-0.74(m,9H),0.55-0.43(m,6H); 13 C NMR(DMSO-d6,100MHz)δ166.07,158.80,158.77,154.96,144.83,136.61,135.63,135.18,130.24,130.08,128.43,128.03,127.4 4,115.51,113.76,101.81,88.80,87.37,86.49,80.72,71.31,69.03,58.57,55.55,13.94,6.75,4.45; ESI-MS(m / z)733.31[M+Na] + . 49

[0240] Benzoic anhydride (2.73 g, 12 mmol) was added to a 20 mL acetonitrile solution of aminonucleoside 48 (3.9 g, 5.49 mmol). After stirring at room temperature for 20 h, the reaction was quenched with 5 mL of water. TBAF (3.47 g, 11 mmol) was added to the reaction mixture, and the reaction was carried out 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: EtOAc / CH2Cl2 = 0-40%) to obtain 3.2 g of nucleoside 49 as a white, foamy solid, with a yield of 83%. 1 H NMR(DMSO-d6,400MHz)δ13.08(s,1H),8.21(d,J=7.6Hz,1H),7.62-7.27(m,12H),6.95(d,J=8.6Hz,4H),6.40(d,J=3.2Hz,1H),5.61 (s,1H),5.18(s,1H),4.57(s,1H),4.31(d,J=3.2Hz,1H),3.86(d,J=11.2Hz,1H),3.76(s,6H),3.53(d,J=11.2Hz,1H),1.86(s,3H); 13 C NMR(DMSO-d6,100MHz)δ178.62,170.82,159.46,147.48,145.03,137.25,137.10,135.30,130.31,130.25,129.86,128.80,128.46, 128.12,127.39,115.87,113.78,110.13,89.52,87.06,86.50,80.60,70.34,69.06,59.99,55.55,13.62; ESI-MS(m / z)701.32[M+H] + . 50

[0242] To a solution of nucleoside 49 (2.3 g, 3.28 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonamide (1.2 g, 3.93 mmol) in 25 mL of anhydrous dichloromethane, 1H-tetrazole (230 mg, 3.28 mmol) was added, and the mixture was reacted at room temperature for 5 h. A saturated NaHCO3 solution was added to the reaction mixture, followed by extraction with dichloromethane, washing with saturated brine, drying on anhydrous MgSO4, filtration, concentration, and flash column purification (gradient elution: EtOAc / CH2Cl2 = 0-20%) to give 2.13 g of phosphoramide 50, a white, foamy solid, in 72% yield. 31P-NMR(152MHz,DMSO-d6)δ149.23,147.95; ESI-MS(m / z)901.46[M+H] + .

[0243] Since changes in the configuration of the raw materials do not affect the preparation of nucleic acid monomers, nucleotides with five bases (A, T, C, G, U) in either the R or S configuration can be obtained using the above preparation method.

[0244] Experimental Example 1: Cyanolocked Nucleic Acid Modification and ASO-Protein Interaction

[0245] Experimental methods

[0246] Thermal denaturation test (T) m value)

[0247] Annealing buffer: 10mM Na3PO4, 100mM NaCl, pH 7.2. Annealing method: Dilute the two oligonucleotide single strands with annealing buffer to a final concentration of 2μM each, heat in a 95℃ water bath for 5 min, slowly cool to room temperature, and incubate overnight at 4℃. Tm determination method: Add 100μL of the sample to a cuvette and seal tightly with a heat-insulating cap. Use 15℃ as the initial measurement temperature and 90℃ as the termination temperature. The temperature rise rate is 0.5℃ / min, the A260 reading rate is 1 time / ℃, and the instrument will give the final Tm reading. m Value. Each sample was measured three times, and the average value was taken as the final result.

[0248] Immunofluorescence assay:

[0249] 1) Cell preparation: The day before, HeLa cells were seeded into confocal culture dishes with a diameter of 20 mm at a cell density of 2 × 10⁶ cells / mL. 5 Then, the 20mm confocal culture dish was placed in a CO2 incubator for incubation.

[0250] 2) Preparation of PS-ASOs: Dissolve ON1, ON2, and ON3 powders in water free of RNase and DNase enzymes to form a 100 μM solution.

[0251] 3) Transfection: Add 10 μL of Lipofectamine 2000 (Soleb) to Opti- Medium was prepared to contain 250 μL of transfection reagent. Then, 4 μg / dish was mixed thoroughly with 250 μL of Lipofectamine 2000 dilution. After 5 min, 500 μL of the mixture was added to each dish. The transfection reagent mixture was removed, and the cells were washed three times with PBS. Then, 500 μL of DMEM (SIGMA) was added to each well as cell maintenance medium. 20 mm confocal culture dishes were placed in a CO2 incubator for transfection at 5 min, 2 h, and 4 h.

[0252] 4) Fixation: Discard the waste liquid, wash the cells 3 times with PBS, then fix them with 4% paraformaldehyde for 30 min at room temperature, and then permeate them with 0.1% Triton X-100 (prepared with PBS) for 5 min.

[0253] 5) Blocking: The fixed cells were blocked at room temperature for 30 min with blocking buffer (prepared with 1 mg / ml BSA and PBS).

[0254] 6) Primary antibody binding: Dilute the primary antibody (NONO, Cell signaling) with blocking buffer, incubate at room temperature for 1 hour, then place in a 4°C refrigerator overnight. Wash 3 times with washing buffer (0.1% Tween prepared with PBS, wash once every 5 minutes).

[0255] 7) Secondary antibody binding: Dilute the secondary antibody (Anti-rabbit IgG, Cell signaling) with blocking buffer, incubate at room temperature for 1 hour, and finally wash the cells 3 times with washing buffer.

[0256] 8) Mounting and detection: Add Prolong antiquenching agent (Cellsignaling) containing 4ˊ,6-diamidinyl-2-phenylimidazoline (DAPI) to a culture dish, and finally observe and photograph it using an inverted fluorescence microscope.

[0257] Caspase 3 / 7 activity assay

[0258] 1) Cell preparation: The day before, HeLa cells (Beina Biotechnology) were seeded into 96-well cell culture plates at a cell density of 5 × 10⁶ cells / well. 4 Then the 96-well cell culture plates were placed in a CO2 incubator for incubation.

[0259] 2) Preparation of PS ASOs: Dissolve ON1, ON2, and ON3 powders in RNase- and DNase-free water to prepare a 10 μM solution. Then use Opti- Medium (Thermofisher) diluted 10 μM solutions of ON1, ON2, and ON3 to 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, and 0 nM solutions, respectively.

[0260] 3) Transfection: Add 2 μL of Lipofectamine 2000 (Thermofisher) to Opti- Medium was prepared as a 100 μL transfection reagent. Then, 100 μL of different concentrations of PS ASOs solution was mixed with 100 μL of Lipofectamine 2000 dilution buffer. After 5 min, 50 μL of the mixture was added to each well. Four h after transfection, the transfection reagent mixture was removed, and the cells were washed three times with PBS. Then, 100 μL of DMEM (Thermofisher) was added to each well as cell maintenance medium, and the 96-well cell culture plate was placed in a CO2 incubator for another 8 h of incubation.

[0261] 4) Detection: Remove the 96-well cell plate from the CO2 incubator, add 100 μL of Caspase-Glo 3 / 7Reagent (Promega) to each well, incubate for 30 min, and then read the luminescence signal of each well using a multi-functional fluorescent microplate reader (Promega).

[0262] Experimental content

[0263] Intracellular ASO-protein interactions are closely related to the drug-like properties of PS-ASOs. Differences in chemical modification will significantly affect ASO-protein interactions, thereby affecting the subcellular distribution of binding proteins and inducing apoptosis and toxicity. To investigate the effect of cyanolocked nucleic acid modification on the interaction between ASO and intracellular proteins, the inventors selected the toxic 3-10-3 gapmer PS ASO sequence 449093 (5'- TTC AGTCATGACT TCC -3' (SEQ ID NO. 1) was used as a template sequence (Nature Biotech., 2019, 37, 640) for investigation. The corresponding T and... m C, ON1 and ON2 were synthesized by introducing phosphoramidite into 449093 via a solid-phase phosphoramide method, with LNA-modified 449093 (ON3) as a control. Mass spectrometry identification data are shown in Table 1, and the corresponding mass spectra are shown in [Table 1]. Figures 2-4 .

[0264] First, the hybridization properties of ON1-3 with the target RNA were determined, and their Tm values ​​were 67.04℃, 63.96℃, and 66.59℃, respectively. This indicates that compared with LNA-modified PS ASO ON3, CN-LNA-modified PS-ASOs ON1-2 can still maintain good affinity with the target RNA.

[0265] Secondly, using the intracellular parafocal protein P54nrb as a model protein, the effect of cyanolocked nucleic acid modification on the interaction between ASO and intracellular proteins was investigated. Parafocal protein P54nrb can bind to toxic ASO (with binding affinity at low nmol levels), altering its distribution and causing it to aggregate in the nucleolus. This effect is significant and positively correlated with ASO toxicity. Experimental results are expressed as the aggregation of parafocal protein P54nrb in the nucleolus.

[0266] Cells were transfected with ON1, ON2, and ON3 at transfection times of 5 min, 2 h, and 4 h, respectively. Finally, the protein localization of P54nrb was observed and photographed under an inverted fluorescence microscope. Figure 5 (Scale bar in figure: 10μm) As shown, the results showed that: (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 and P54nrb was evenly distributed in the cell nucleus. Neither LNA nor CN-LNA modified ASO changed the distribution of P54nrb, and no aggregation was observed in the nucleolus; (2) For the LNA group, when the time was extended to 2 h and 4 h respectively, the LNA group showed significant P54nrb aggregation. The longer the incubation time, the more P54nrb aggregated in the nucleolus, indicating that LNA modified ASO has a strong affinity for intracellular proteins and easily interferes with the normal distribution and function of intracellular proteins, producing toxic side effects. (3) From the R-CN-LNA group, no obvious P54nrb protein aggregation was observed in the nucleolus at 2h, and the red fluorescence was evenly distributed in the cell nucleus. However, at 4h, some cells showed obvious P54nrb aggregation, but obviously, the degree of aggregation was significantly lower than that of the LNA group at the corresponding time point. (4) Regardless of 2h or 4h, no visible changes in the distribution of P54nrb were observed at any time point in the S-CN-LNA group. P54nrb was still evenly distributed in the cytoplasm.

[0267] Table 1. Oligonucleotide sequences ON1-4 and their corresponding mass spectrometry data

[0268]

[0269]

[0270] Based on current immunofluorescence experiments, to further quantify the effects of LNA, R-CN-LNA, and S-CN-LNA modifications on the distribution of P54nrb in the cell nucleus, we statistically analyzed the percentage of cells with significant P54nrb aggregation in the nucleolus at 10 min, 30 min, 1 h, 2 h, and 4 h in each of the LNA, R-CN-LNA, and S-CN-LNA groups. As shown in Table 2, in the LNA group, the number of cells with significant P54nrb aggregation in the nucleolus increased with transfection time. At 4 h after LNA transfection, approximately 92% of transfected cells showed significant P54nrb aggregation in the nucleolus. In the R-CN-LNA group, only 5% of cells after ASO transfection showed significant P54nrb aggregation in the nucleolus at 2 h, and only about 8% at 4 h. For the S-CN-LNA group, there was no significant P54nrb aggregation, with a cell proportion of 0%. The above results indicate that, compared to LNA modification, CN-LNA modification can indeed reduce the impact of ASO on intracellular proteins, and the S conformation has less impact than the R conformation.

[0271] Table 2. Percentage of cells in which LNA-ASO, R-CN-LNA-ASO, and S-CN-LNA-ASO significantly aggregated P54nrb to the nucleolus.

[0272]

[0273] Based on the above evaluation of the effects on the subcellular distribution of intracellular proteins, the effect of CN-LNA modification on the toxicity of induced apoptosis was further investigated by detecting Caspase 3 / 7 activity in HeLa cells.

[0274] Cells were transfected with ON1, ON2, and ON3 at concentrations of 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, and 0 nM, respectively. After 4 h of transfection, cells were incubated for another 8 h, and Caspase 3 / 7 activity was detected using a fluorescent microplate reader. Figure 6As shown, the experimental results indicate that: (1) at low concentrations (0-62.5 nM), no significant changes in caspase activity were observed in any of the three groups; (2) in the LNA-modified group, caspase activity increased rapidly with increasing concentration starting from 125 nM, while in the two CN-LNA-modified groups, caspase activity only increased slowly starting from 250 nM, and the S configuration was lower than the R configuration; (3) at high concentrations of 250 nM and 500 nM, compared to LNA modification, both R-CN-LNA and S-CN-LNA modifications reduced the increase in caspase activity by more than 2 times, and the S configuration was superior to the R configuration. These results indicate that, compared to LNA modification, R / S-CN-LNA modification can significantly reduce PS ASO-induced apoptosis toxicity, consistent with the results of immunofluorescence experiments.

[0275] In summary, the introduction of cyano groups into LNA can reduce the lipophilicity of LNA and improve the water solubility of the modified structure. Cyano-locked nucleic acid modification, especially the S configuration, can significantly reduce the impact of PSASO on intracellular proteins and induce apoptosis toxicity, thereby improving the therapeutic effect of PSASO. This has significant application value for nucleic acid drugs and can provide a new generation of chemical modification platform technology support for nucleic acid drugs.

[0276] Experiment 2: Nuclease Tolerance Test of S-CN-LNA-T

[0277] Stability to nucleases is one of the important parameters of nucleotide chemical modification. This experiment used snake venom phosphodiesterase (SVPDE) to investigate the stability of S-CN-LNA-T modified oligonucleotides (5'-TTTTTTTT). T T-3'(SEQ ID NO.2), T The nuclease tolerance of the (S-6'-CN-LNA)ON4 sequence was investigated and compared with that of R-CN-LNA (ON5), LNA (ON6), thio-modified (ON7), and native (ON8) sequences. Under physiological conditions of 37℃ and a buffer system of 50 mM Tris-HCl, 10 mM MgCl2, and pH 8.0, the corresponding nucleic acid sequence (7 nM) was digested using SVPDE (1.0 μg / mL). The incubation solution was collected at different time points (0, 2, 5, 10, 20, 30, 40 min), and quantification was performed by HPLC to obtain the corresponding content-time curves. Nuclease tolerance test results ( Figure 8The figure shows that under 1.0 μg / mL LSVPE, the R-6'-CN-LNA modified sequence ON7 (rectangular), R-CN-LNA modified sequence ON5 (rhomboid), LNA modified sequence ON6 (triangular), 3′-thio-T (Ts, cross) modified sequence ON7, and natural-T (star) modified sequence ON8 (star) modified sequence ON7) degraded slowly, with more than 40% remaining undegraded after 40 min. The S-6'-CN-LNA modified sequence ON8 had more than 50% remaining undegraded, while the LNA modified sequence had less than 10% remaining. This indicates that 6'-CN-LNA can significantly improve the tolerance of oligonucleotides to nucleases, which is significantly better than LNA, and the S conformation is better than the R conformation.

[0278] Clearly, despite the relatively small size of the cyano group, it can still enhance the stability of LNA to nucleases, consistent with the results of other C6'-modified LNAs. Notably, the literature reports that both R and S-configurations of cEt-LNA exhibit similar nuclease tolerance, while for CN-LNA, the S-configuration shows better nuclease tolerance than the R-configuration. Considering the cyano group on the S-configuration faces the phosphate ester, this seems to indicate that CN has a more complex effect on the phosphate ester. If so, this will have a greater impact on the protein-protein interaction of CN-LNA-modified ASO, beyond just electrostatic interactions.

[0279] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for synthesizing a cyano-modified nucleoside at the C6' position with either an R-configuration or an S-configuration, characterized in that, The R-configuration C6' position cyano-modified nucleoside is Its synthesis method includes: Isomerization of terminal olefins to obtain Then, a dihydroxylation reaction is performed to obtain Then, through an oxidative cleavage reaction, aldehyde compounds are obtained. Finally The aldehyde group was converted to a cyano group to synthesize an R-configuration cyano-modified nucleoside at the C6' position. ; The S-configuration C6' position cyano-modified nucleoside is Its synthesis method includes: Isomerization of terminal olefins to obtain Then, a dihydroxylation reaction is performed to obtain Then, through an oxidative cleavage reaction, aldehyde compounds are obtained. Finally The aldehyde group was converted to a cyano group to synthesize an S-configuration cyano-modified nucleoside at the C6' position. ; Bx is selected from adenine, guanine, thymine, cytosine, uracil, or their respective salts; The The synthesis method includes: using the S configuration The methanesulfonation, de-branching, and acetylation reactions were performed sequentially to obtain the desired product. Then, glycosylation and nucleophilic substitution reactions were carried out to synthesize the R configuration. ; The The synthesis method includes: using the R configuration The methanesulfonation, de-branching, and acetylation reactions were performed sequentially to obtain the desired product. Then, glycosylation and nucleophilic substitution reactions were carried out to synthesize the S configuration. ; The S configuration The synthesis method includes: 3,5-di- O -benzyl-4- C -Hydroxymethyl-1,2- O -Isopropylidene-α-D-ribofuranoside The primary alcohol is oxidized to an aldehyde group, and then the aldehyde group is allylated to obtain the S configuration. ; The R configuration The synthesis methods include: The secondary alcohol is oxidized to a ketone, and then the ketone is reduced back to the secondary alcohol to obtain the R configuration. ; The reducing agent used to reduce the ketone to the secondary alcohol is selected from at least one of lithium aluminum hydride, lithium borohydride, lithium chloride or sodium borohydride, the reaction medium is selected from at least one of dichloromethane, tetrahydrofuran, methanol or ethanol, and the reaction temperature is -78 to 0℃. The catalyst for the terminal olefin isomerization is tris(triphenylphosphine)carbonyl ruthenium(II) hydrochloride; the reaction medium for the terminal olefin isomerization is ethanol; and the reaction temperature for the terminal olefin isomerization is 60-100℃.

2. The synthesis method according to claim 1, characterized in that, The glycosylation reaction is as follows: under activating agent conditions, ... or It reacts with thymine, N6-benzoyladenine or 6-chloroguanine at 50-100°C, wherein the activator is selected from BSA and TMSOTf, and the reaction medium is selected from acetonitrile, 1,2-dichloroethane or toluene.

3. The synthesis method according to claim 1, characterized in that, The reaction for reducing ketones to secondary alcohols is as follows: using sodium borohydride and lithium chloride as reducing agents, and tetrahydrofuran and methanol as solvents, at -40°C... Reaction at 0℃.

4. A method for synthesizing a phosphoramidite monomer with a cyano group modified at the C6' position in either the R or S configuration, characterized in that, The R-configuration C6'-cyano-modified G-phosphamide monomer is... The preparation method includes: synthesizing the R configuration using the synthesis method of claim 1. The resulting R configuration will then be synthesized. The methoxy group is converted into an aldehyde group to obtain... Then, the 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain... Then, the base and 5'-hydroxyl group are protected, and the 3'-hydroxyl group undergoes a phosphoramidation reaction to obtain the R configuration. ; The S-configuration C6'-cyano-modified G-phosphamide monomer is The preparation method includes: synthesizing the S configuration using the synthesis method of claim 1. The synthesized S configuration will then be... The methoxy group was demethylated to obtain Then, the 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain... Then, the base and 5'-hydroxyl group are protected, and the 3'-hydroxyl group undergoes a phosphoramidation reaction to obtain the S configuration. .

5. The synthesis method according to claim 4, characterized in that, Debenzylation was performed using ferric chloride.

6. A method for synthesizing a phosphoramidite monomer with an R-configuration or S-configuration C6'-cyano-locked nucleic acid, characterized in that, R-configuration C6' position cyanolockane T phosphoramide monomer is The preparation method includes: synthesizing the R configuration using the synthesis method of claim 1. The resulting R configuration will then be synthesized. The 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain The 5'-hydroxyl group is then protected with DMTr, and the 3'-hydroxyl group undergoes a phosphoramidation reaction to obtain the R configuration. ; S-configuration C6' position cyanolockane T-phosphite monomer is The preparation method includes: synthesizing the S configuration using the synthesis method of claim 1. The synthesized S configuration will then be... The 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain The 5'-hydroxyl group is then protected with DMTr, and the 3'-hydroxyl group undergoes a phosphoramidation reaction to obtain the S configuration. .

7. The synthesis method according to claim 6, characterized in that, Debenzylation was performed using ferric chloride.

8. A method for synthesizing a C6'-cyano-locked nucleic acid C-phosphamide monomer with R or S configuration, characterized in that, R-configuration C6' position cyanolockaneous nucleic acid C phosphoramide monomer is The preparation method includes: synthesizing the R configuration using the synthesis method of claim 1. The resulting R configuration will then be synthesized. The 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain Then, the 5'-hydroxyl group was protected with DMTr to obtain Then, the 3'-hydroxyl group is silanized and the carbonyl group is converted to an amino group to obtain... Then, base protection was performed, and the 3'-hydroxyl group was deprotected to obtain Finally, the 3'-hydroxyl group was subjected to a phosphoramidation reaction to obtain the R configuration. ; S-configuration C6' position cyanolockaneous nucleic acid C phosphoramide monomer is The preparation method includes: synthesizing the S configuration using the synthesis method of claim 1. The synthesized S configuration will then be... The 3'-hydroxyl and 5'-hydroxyl groups were debenzylated to obtain Then, the 5'-hydroxyl group was protected with DMTr to obtain Then, the 3'-hydroxyl group is silanized and the carbonyl group is converted to an amino group to obtain... Then, base protection was performed, and the 3'-hydroxyl group was deprotected to obtain Finally, the 3'-hydroxyl group was subjected to a phosphoramidation reaction to obtain the S configuration. .

9. The synthesis method according to claim 8, characterized in that, Debenzylation was performed using ferric chloride.

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