6'-cyano modified locked nucleosides, nucleotides and nucleic acid polymers

Abstract

The application provides a 6'-cyano modified locked nucleoside, nucleotide and nucleic acid polymer, 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 unmodified or other modified nucleic acid polymers, and reduces 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 have significant application value in the field of nucleic acid drugs.The preparation method of the above-mentioned locked nucleoside, nucleotide and nucleic acid polymer provided by the application is efficient, stable and practical, and can be compatible with various substituted or unsubstituted bases.

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. 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 synthesis. 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...

[0005] The main toxic 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. Simultaneously, toxicity is positively correlated with the 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 2'-methoxyethyl (2'-MOE) has a 30-fold lower affinity for intracellular proteins than 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.

[0006] 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.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The primary objective of this invention is to provide 6'-cyano-modified nucleosides, nucleotides, and nucleic acid polymers with R or S configurations.

[0009] 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. This method is simple, has a high yield, and can solve at least one of the aforementioned problems.

[0010] 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.

[0011] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0012] In a first aspect, the present invention provides a 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:

[0013]

[0014] Wherein: Bx is selected from substituted or unsubstituted: adenine, guanine, thymine, cytosine, uracil or their respective salts;

[0015] Z is a cyano group;

[0016] W1 and W2 are independently selected from H or hydroxyl protecting groups; the hydroxyl protecting groups include, but are not limited to: acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, di(2-acetoxyethoxy)methyl, 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylmethylsilyl, [(triisopropylsilyl)oxy]methyl, benzoylcarboxylate, chloroacetyl, trichloroacetyl, trifluoroacetyl, tertvalyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, trimethoxytriphenylmethyl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl, 9-phenylxanthine-9-yl, 9-(p-methoxyphenyl)xanthine-9-yl or 2-naphthylmethyl;

[0017] The isomers include: 6'-cyano-modified nucleosyl groups with the Z group in the R configuration, having the following structural formula: Alternatively, a 6'-cyano-modified nucleoside with the Z group in an S configuration, having the structural formula as follows:

[0018] In a second aspect, the present invention provides a nucleotide comprising the 3'-active phosphorus group derivative of the above-mentioned 6'-cyano-modified nucleoside;

[0019] 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.

[0020] As a further technical solution, the nucleotide is selected from compounds having the structure shown in Formula 2, their salts, or isomers thereof:

[0021]

[0022] 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:

[0023] Thirdly, the present invention provides the application of the above-mentioned nucleotides in reducing the interaction between nucleic acid polymers and intracellular proteins.

[0024] Fourthly, the present invention provides a nucleic acid polymer having monomers with the structure shown in Formula 3:

[0025]

[0026] Wherein, Bx is selected from substituted or unsubstituted: adenine, guanine, thymine, cytosine, uracil or their respective salts;

[0027] Z is a cyano group;

[0028] W3 and W4 are each independently an H or hydroxyl protecting group or an internucleotide linking group that links the monomer to other parts of the nucleic acid polymer; and at least one of W3 and W4 is an internucleotide linking group that links the monomer to other parts of the nucleic acid polymer.

[0029] The hydroxyl protecting groups include: acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, di(2-acetoxyethoxy)methyl, 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and triphenylmethylsilyl. [(triisopropylsilyl)oxy]methyl, benzoylcarbamate, chloroacetyl, trichloroacetyl, trifluoroacetyl, p-valeryl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, trimethoxytriphenylmethyl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl, 9-phenylxanthine-9-yl, 9-(p-methoxyphenyl)xanthine-9-yl or 2-naphthylmethyl.

[0030] As a further technical solution, the monomer includes: a monomer with the Z group in the R configuration, having the structural formula as follows: Or a monomer with the Z group in an S configuration, with the structural formula as follows:

[0031] As a further technical solution, the nucleic acid polymer is ribonucleic acid, deoxyribonucleic acid, or a copolymer of ribonucleotides and deoxyribonucleotides.

[0032] Fifthly, the present invention provides the application of the above-mentioned nucleic acid polymer in the preparation of nucleic acid diagnostic agents or nucleic acid therapeutic agents.

[0033] 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.

[0034] 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.

[0035] Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or their respective salts.

[0036] As a further technical solution, the aforementioned The synthesis method includes: using the S configuration The reaction was carried out sequentially by methanesulfonation, despinning, and acetylation to obtain Then, glycosylation and nucleophilic substitution reactions were performed to synthesize the R configuration.

[0037] The The synthesis method includes: using the R configuration The reaction was carried out sequentially by methanesulfonation, despinning, and acetylation to obtain Then, glycosylation and nucleophilic substitution reactions were performed to synthesize the S configuration.

[0038] 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.

[0039] As a further technical solution, the S-configuration The synthetic method includes: 5-O-(tert-butyldiphenylsilyl)-4-C-hydroxymethyl-1,2-O-isopropylidene-3-O-(2-naphthylmethyl)-α-D-ribofuranoside The primary alcohol is oxidized to an aldehyde group, and then the aldehyde group is allylated to obtain the S configuration.

[0040] Preferably, 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.

[0041] 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.

[0042] 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.

[0043] 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;

[0044] The reaction medium for the terminal olefin isomerization includes methanol, ethanol, n-butanol, or toluene, preferably ethanol;

[0045] The reaction temperature for the terminal olefin isomerization is 60-100℃, preferably 60-80℃;

[0046] The reaction time for the terminal olefin isomerization is 12-72 hours.

[0047] Seventhly, the present invention provides a method for synthesizing an R-configuration or S-configuration C6'-position cyanolocked nucleic acid T-phosphamide monomer, wherein the method for preparing the R-configuration C6'-position cyanolocked nucleic acid T-phosphamide monomer includes: taking the R-configuration... The 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0048] The preparation method of the S-configuration C6'-position cyanolocked nucleic acid T phosphoramide monomer includes: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] The 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0049] Eighthly, the present invention provides a method for synthesizing a phosphoramidite monomer of R-configuration or S-configuration C6'-cyanolocked nucleic acid A, wherein the R-configuration C6'-cyanolocked nucleic acid A phosphoramidite monomer comprises: [the following steps are described in the original text, but the translation is incomplete and requires further context.] To obtain by protecting the bases Then, the 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0050] S-configuration C6'-cyanolocked nucleic acid A phosphoramide monomer includes: S-configuration To obtain by protecting the bases Then, the 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0051] Ninthly, the present invention provides a method for synthesizing an R-configuration or S-configuration C6'-position cyanolocked nucleic acid G phosphoramide monomer, wherein the method for preparing the R-configuration C6'-position cyanolocked nucleic acid G phosphoramide monomer includes: taking the R-configuration... The methoxy group was demethylated to obtain Then, base protection is performed to obtain... Then, the 3'-hydroxyl and 5'-hydroxyl groups were deprotected to obtain The 5'-hydroxyl group was then protected with DMTr, and the 3'-hydroxyl group underwent a phosphoramidation reaction to obtain the R configuration 8.

[0052] The preparation method of the S-configuration C6'-position cyanolocked nucleic acid G phosphoramide monomer includes: ... The methoxy group was demethylated to obtain Then, base protection is performed to obtain... Then, the 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0053] In a tenth aspect, the present invention provides a method for synthesizing a C6'-cyano-modified dC phosphoramide monomer of R-configuration or S-configuration, wherein the method for preparing the R-configuration C6'-cyano-modified dC phosphoramide monomer includes: taking the R-configuration... The 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0054] The preparation method of the S-configuration C6'-cyanolocked nucleic acid C phosphoramide monomer includes: [The text abruptly ends here, so the translation also ends here.] The 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0055] In the eleventh aspect, the present invention provides a method for synthesizing nucleic acid polymers, wherein monomers are subjected to polymerization reactions to prepare nucleic acid polymers;

[0056] The monomer includes the 6'-cyano-modified nucleoside or the nucleotide.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] 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.

[0059] This invention provides a universal preparation method for the above-mentioned R-configuration or S-configuration 6'-cyano-modified nucleosides, nucleotides and nucleic acid polymers. This preparation method is stable, efficient and has a high yield.

[0060] Furthermore, the CN-LNA-modified nucleic acid sequence obtained by the synthesis method of this invention can significantly reduce interference with intracellular protein distribution and reduce the effect of inducing apoptosis compared with LNA modification, thus having a significant advantage in the development of highly efficient and safe nucleic acid drugs. Attached Figure Description

[0061] 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.

[0062] Figure 1 This is the mass spectrum of the nucleic acid ON1;

[0063] Figure 2 This is the mass spectrum of the nucleic acid ON2;

[0064] Figure 3 This is the mass spectrum of the nucleic acid ON3;

[0065] Figure 4 Figure 60× shows the aggregation of parafoil protein P54nrb induced by ON1, ON2, and ON3.

[0066] Figure 5 Results for Caspase 3 / 7 activity

[0067] Figure 6 This is the mass spectrum of nucleic acid ON4;

[0068] Figure 7 The enzymatic stability of 5′-d(TTTTTTTTT)-3′ against snake venom phosphodiesterase (SVPDE) was determined. Detailed Implementation

[0069] 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.

[0070] 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 in the polymer to form the nucleic acid is 2, 3, or more. It can be an oligonucleotide with fewer than 20 nucleotides, or a polymer with more than 20 nucleotides.

[0071] In a first aspect, the present invention provides a 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:

[0072]

[0073] Wherein: Bx is selected from substituted or unsubstituted: adenine, guanine, thymine, cytosine, uracil or their respective salts;

[0074] Z is a cyano group;

[0075] W1 and W2 are independently selected from H or hydroxyl protecting groups; the hydroxyl protecting group is selected from, but not limited to, the following: acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, di(2-acetoxyethoxy)methyl, 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilane. methyl, tert-butyldiphenylsilyl, triphenylmethylsilyl, [(triisopropylsilyl)oxy]methyl, benzoylcarboxylate, chloroacetyl, trichloroacetyl, trifluoroacetyl, tert-valeryl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, trimethoxytriphenylmethyl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl, 9-phenylxanthine-9-yl, 9-(p-methoxyphenyl)xanthine-9-yl or 2-naphthylmethyl;

[0076] The isomers include: 6'-cyano-modified nucleosyl groups with the Z group in the R configuration, having the following structural formula: Alternatively, a 6'-cyano-modified nucleoside with the Z group in an S configuration, having the structural formula as follows:

[0077] 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.

[0078] In a second aspect, the present invention provides a nucleotide comprising the 3'-active phosphorus group derivative of the above-mentioned 6'-cyano-modified nucleoside;

[0079] 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.

[0080] The nucleotides provided by this invention can improve the nuclease tolerance of nucleic acid polymers prepared using them as monomers and reduce the interaction between nucleic acid polymers and intracellular proteins.

[0081] In some embodiments, the nucleotide is selected from compounds having the structure shown in Formula 2, their salts, or isomers thereof:

[0082]

[0083] 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:

[0084] Thirdly, the present invention provides the application of the above-mentioned nucleotides in reducing the interaction between nucleic acid polymers and intracellular proteins.

[0085] 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.

[0086] Fourthly, the present invention provides a nucleic acid polymer having monomers with the structure shown in Formula 3:

[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] W3 and W4 are each independently an H or hydroxyl protecting group or an internucleotide linking group that links the monomer to other parts of the nucleic acid polymer; and at least one of W3 and W4 is an internucleotide linking group that links the monomer to other parts of the nucleic acid polymer.

[0091] The hydroxyl protecting group is selected from, but is not limited to: acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, di(2-acetoxyethoxy)methyl, 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylmethylsilyl Alkyl, [(triisopropylsilyl)oxy]methyl, benzoylcarbamate, chloroacetyl, trichloroacetyl, trifluoroacetyl, p-valeryl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, methanesulfonate, p-toluenesulfonate, triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, trimethoxytriphenylmethyl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl, 9-phenylxanthine-9-yl, 9-(p-methoxyphenyl)xanthine-9-yl or 2-naphthylmethyl.

[0092] The nucleic acid polymer provided by this invention has better nuclease tolerance than unmodified or other modified nucleic acid polymers. This nucleic acid polymer can reduce interaction with intracellular proteins, reduce the impact on the subcellular distribution of intracellular proteins, and reduce apoptosis.

[0093] In some embodiments, the monomer comprises: a monomer with a Z group in the R configuration, having the structural formula as follows: Or a monomer with the Z group in an S configuration, with the structural formula as follows:

[0094] In some embodiments, the nucleic acid polymer is ribonucleic acid, deoxyribonucleic acid, or a copolymer of ribonucleotides and deoxyribonucleotides.

[0095] Fifthly, the present invention provides the application of the above-mentioned nucleic acid polymer in the preparation of nucleic acid diagnostic agents or nucleic acid therapeutic agents.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] Bx is selected from substituted or unsubstituted adenine, guanine, thymine, cytosine, uracil, or their respective salts.

[0100] This invention does not impose specific limitations on the methods used for terminal olefin isomerization, dihydroxylation, oxidative cleavage, and the conversion of aldehydes to cyanides; any reaction method well-known to those skilled in the art can be used. The R-configuration or S-configuration C6'-cyano-modified nucleosides synthesized in 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.

[0101] 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.

[0102] In some embodiments, the The synthesis method includes: using the S configuration The reaction was carried out sequentially by methanesulfonation, despinning, and acetylation to obtain Then, glycosylation and nucleophilic substitution reactions were performed to synthesize the R configuration.

[0103] The The synthesis method includes: using the R configuration The reaction was carried out sequentially by methanesulfonation, despinning, and acetylation to obtain Then, glycosylation and nucleophilic substitution reactions were performed to synthesize the S configuration.

[0104] 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.

[0105] The synthesis method provided by this invention is stable and practical. 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.

[0106] In some embodiments, the S-configuration The synthetic method includes: 5-O-(tert-butyldiphenylsilyl)-4-C-hydroxymethyl-12-O-isopropylidene-3-O-(2-naphthylmethyl)-α-D-ribofuranoside The primary alcohol is oxidized to an aldehyde group, and then the aldehyde group is allylated to obtain the S configuration.

[0107] Preferably, 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.

[0108] 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.

[0109] 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.

[0110] 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;

[0111] The reaction medium for the terminal olefin isomerization includes methanol, ethanol, n-butanol, or toluene, preferably ethanol;

[0112] The reaction temperature for the terminal olefin isomerization is 60-100℃, preferably 60-80℃;

[0113] The reaction time for the terminal olefin isomerization is 12-72 hours.

[0114] 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 without the need for anhydrous and oxygen-free operation.

[0115] Seventhly, the present invention provides a method for synthesizing an R-configuration or S-configuration C6'-position cyanolocked nucleic acid T-phosphamide monomer, wherein the method for preparing the R-configuration C6'-position cyanolocked nucleic acid T-phosphamide monomer includes: taking the R-configuration... The 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0116] The preparation method of the S-configuration C6'-position cyanolocked nucleic acid T phosphoramide monomer includes: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] The 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0117] This synthetic method is stable and efficient, and can be used universally for the synthesis of T phosphoramide monomers with cyano groups modified at the C6' position in either the R or S configuration.

[0118] Eighthly, the present invention provides a method for synthesizing a phosphoramidite monomer of R-configuration or S-configuration C6'-cyanolocked nucleic acid A, wherein the R-configuration C6'-cyanolocked nucleic acid A phosphoramidite monomer comprises: [the following steps are described in the original text, but the translation is incomplete and requires further context.] To obtain by protecting the bases Then, the 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0119] S-configuration C6'-cyanolocked nucleic acid A phosphoramide monomer includes: S-configuration To obtain by protecting the bases Then, the 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0120] This synthetic method is stable and efficient, and can be used universally for the synthesis of phosphoramide monomers of R-configuration or S-configuration C6'-cyanolocked nucleic acids A.

[0121] Ninthly, the present invention provides a method for synthesizing an R-configuration or S-configuration C6'-position cyanolocked nucleic acid G phosphoramide monomer, wherein the method for preparing the R-configuration C6'-position cyanolocked nucleic acid G phosphoramide monomer includes: taking the R-configuration... The methoxy group was demethylated to obtain Then, base protection is performed to obtain... Then, the 3'-hydroxyl and 5'-hydroxyl groups were deprotected to obtain The 5'-hydroxyl group was then protected with DMTr, and the 3'-hydroxyl group underwent a phosphoramidation reaction to obtain the R configuration 8.

[0122] The preparation method of the S-configuration C6'-position cyanolocked nucleic acid G phosphoramide monomer includes: ... The methoxy group was demethylated to obtain Then, base protection is performed to obtain... Then, the 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0123] This synthetic method is stable and efficient, and can be universally used for the synthesis of R-configuration or S-configuration C6'-position cyanolocked nucleic acid G phosphoramide monomer.

[0124] In a tenth aspect, the present invention provides a method for synthesizing a C6'-cyano-modified dC phosphoramide monomer of R-configuration or S-configuration, wherein the method for preparing the R-configuration C6'-cyano-modified dC phosphoramide monomer includes: taking the R-configuration... The 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0125] The preparation method of the S-configuration C6'-cyanolocked nucleic acid C phosphoramide monomer includes: [The text abruptly ends here, so the translation also ends here.] The 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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.

[0126] This synthetic method is stable and efficient, and can be used universally for the synthesis of C-phosphamide monomers of R-configuration or S-configuration C6'-cyanolocked nucleic acids.

[0127] In the eleventh aspect, the present invention provides a method for synthesizing nucleic acid polymers, wherein monomers are subjected to polymerization reactions to prepare nucleic acid polymers;

[0128] The monomer includes the 6'-cyano-modified nucleoside or the nucleotide.

[0129] 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.

[0130] 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.

[0131] Example 1: Stereoselective Synthesis of Cyano-locked Nucleic Acids

[0132] Part 1: Synthesis of R-6'-CN-LNA

[0133] Synthesis of R-6'-allyl-LNA

[0134]

[0135] The reaction conditions were as follows: (i) (1) 2-iodobenzoic acid, acetonitrile, reflux, 5 h; (2) allyltrimethylsilane, boron trifluoride diethyl ether solution, dichloromethane, -40℃, 4 h; (b) methanesulfonyl chloride, DMAP, pyridine, room temperature, 16 h; (c) (1) FeCl3·6H2O, dichloromethane, 0℃-room temperature, room temperature, 9 h; (2) Ac2O, DMAP, pyridine, dichloromethane, room temperature, 2 h. 2

[0137] Compound 1 (100.0 g, 0.167 mol) was dissolved in 500 mL of acetonitrile, and 2-iodobenzoic acid (56 g, 0.20 mol) was added. The mixture was heated to reflux for 5 h, and the reaction was monitored by TLC until complete (petroleum ether / ethyl acetate = 5 / 1). The reaction was stopped, and after cooling to room temperature, the mixture was filtered through diatomaceous earth. The filter cake was washed twice with acetonitrile, the solvent was evaporated, and the solution was dried under vacuum to obtain 101 g of a pale yellow liquid. No purification was required, and the mixture was directly used for the next reaction.

[0138] The above aldehyde compound was dissolved in 500 mL of dichloromethane, cooled to -40 °C, and boron trifluoride diethyl ether solution (22 mL, 0.217 mol) was added. The mixture was stirred for 5 min. Then, allyltrimethylsilane (42.7 mL, 0.217 mol) was added dropwise, and the mixture was stirred while maintaining the temperature for 4 h. The reaction was monitored by TLC until complete (petroleum ether / ethyl acetate = 5 / 1). The reaction solution was quickly quenched in 1 L of saturated sodium bicarbonate aqueous solution. The solution was extracted with dichloromethane, washed with saturated brine, dried over anhydrous NaSO4, filtered, and concentrated under reduced pressure to give 107 g of a pale yellow viscous substance 2, with a yield of 100%. No purification was required, and the reaction could proceed directly to the next step. 1 H NMR(CDCl3,400MHz)δ7.86-7.78(m,4H),7.60-7.57(m,2H),7.53-7.46(m,5H),7.41-7.27(m,6H),5.87-5.80(m,2H),5.05-4.97(m, 3H),4.76(t,J=5.2Hz,1H),4.70-4.66(m,2H),4.47(dd,J=10.7,1.7Hz,1H),3.96(d,J=11.2Hz,1H),3.80(d,J=11.2Hz,1H),3.44(br s,1H),2.54-2.49(m,1H),1.93-1.85(m,1H),1.63(s,3H),1.39(s,3H),0.90(s,9H); 13 C NMR(CDCl3,100MHz)δ136.41,135.58,135.50,134.29,133.30,133.22,133. 13,132.99,129.76,129.62,128.76,127.99,127.78,127.75,127.70,127.31 ,126.38,126.32,125.71,116.26,113.78,104.70,88.22,79.18,78.11,73.0 9,72.56,62.46,34.65,27.09,26.73,26.54,19.15; ESI-MS(m / z)637.37[MH] - . 3

[0140] At room temperature, compound 2 (100.0 g, 0.157 mol) and DMAP (1.9 g, 15.7 mmol) were dissolved in 500 mL of pyridine. Methanesulfonyl chloride (18 mL, 0.235 mol) was added dropwise, and the reaction was allowed to proceed for 16 h at room temperature. The reaction was monitored by TLC until complete (petroleum ether / ethyl acetate = 5 / 1). 100 mL of methanol was added to the reaction solution, and the reaction was quenched by stirring for 10 min. The reaction solution was then concentrated under reduced pressure to remove excess pyridine, diluted with ethyl acetate, and washed successively with water, 1 N HCl solution, and saturated brine. The solution was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by flash column chromatography (gradient elution: petroleum ether / ethyl acetate = 0-25%) to give 108.0 g of colorless viscous substance 3, with a yield of 96%.

[0141] 1 H NMR(DMSO-d6,400MHz)δ7.95-7.85(m,4H),7.56-7.36(m,13H),5.80-5.69(m,2 H),5.40(dd,J=7.6,3.2Hz,1H),5.03-4.93(m,4H),4.69(d,J=12.4Hz,1H),4.4 0(d,J=5.3Hz,1H),3.75(d,J=11.0Hz,1H),3.60(d,J=11.0Hz,1H),2.97(s,3H) ,2.85-2.81(m,1H),2.58-2.52(m,1H),1.53(s,1H),1.33(s,1H),0.78(s,9H); 13 C NMR(CDCl3,100MHz)δ135.59,135.52,135.29,134.59,133.25,133.19,132.8 8,132.84,130.39,130.36,128.59,128.34,128.29,128.24,128.09,127.33,1 26.70,126.55,126.34,118.60,113.27,105.00,87.47,81.43,78.15,76.97, 71.93,64.30,36.19,26.83,26.57,26.33,19.04; ESI-MS(m / z)734.39[M+NH4] + 739.34 [M+Na] + . 4

[0143] A 600 mL solution of compound 3 (108 g, 0.15 mol) in dichloromethane was cooled to 0 °C, and ferric chloride hexahydrate (16.3 g, 0.06 mol) was added. The reaction was carried out at room temperature for 9 h, and TLC was used to monitor the reaction until complete (petroleum ether / ethyl acetate = 4 / 1). The reaction solution was poured into 1.5 L of water and extracted with dichloromethane. The organic layer was washed successively with water, saturated sodium bicarbonate solution, and saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and dried under vacuum. The resulting viscous substance was dissolved in 500 mL of dichloromethane, and pyridine (72 mL, 0.9 mol), DMAP (2.75 g, 22.5 mol), and acetic anhydride (85 mL, 0.9 mol) were added dropwise. The reaction was carried out at room temperature for 2 h, and TLC was used to monitor the reaction until complete (petroleum ether / ethyl acetate = 4 / 1). The reaction mixture was poured into 2 L of water and extracted with dichloromethane. The organic layer was washed successively with water, 1 N HCl solution, saturated sodium bicarbonate solution, and saturated brine. The mixture was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: petroleum ether / ethyl acetate = 0-60%) to give 68 g of a mixture of diacetyl compound 4 epimers, a colorless viscous substance, in 60% yield. ESI-MS (m / z) 778.39 [M+NH4) + 783.44 [M+Na] + .

[0144]

[0145] The reaction conditions were as follows: (a) thymine, N,O-bis(trimethoxyacetamide) (BSA), TMSOTf, acetonitrile, 80℃, 3h; (b) K2CO3, methanol, room temperature, 16h; (c) N6-benzoyladenine, BSA, TMSOTf, toluene, 100℃, 3h; (d) 6-chloroguanine, BSA, TMSOTf, toluene, 100℃, 3h. 5

[0147] BSA (29 mL, 120 mmol) was added to 50 mL of acetonitrile suspension of thymine (7.6 g, 60 mmol), and stirred at room temperature for 20 min until the thymine dissolved. After the system became clear, 200 mL of acetonitrile solution of diacetyl sugar 4 (23 g, 30 mmol) was added, followed by rapid dropwise addition of TMSOTf (8.3 mL, 45 mmol). After the addition was complete, the temperature was raised to 80 °C, and the reaction was stirred for 3 h. The reaction was monitored by TLC to ensure it was complete (petroleum ether / ethyl acetate = 5 / 1). Heating was stopped, and the reaction solution was allowed to cool to room temperature. The solution was then poured into ethyl acetate, and a semi-saturated NaHCO3 solution (400 mL) was added. The mixture was stirred for 20 min, and the insoluble solids were filtered off with diatomaceous earth. The organic layer was separated, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and vacuum dried. The residue was dissolved in 250 mL of methanol, and K2CO3 (12.4 g, 90 mmol) was added. The mixture was reacted at room temperature for 16 h, and TLC was used to confirm complete reaction (dichloromethane / ethyl acetate = 10 / 3). The reaction solution was concentrated, and the residue was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-40%) to obtain 8 g of locked nucleic acid derivative 5, a white foamy solid, with a yield of 39%. 1 H NMR(CDCl3,400MHz)δ8.72(br s,1H),7.82-7.74(m,3H),7.72(s,1H),7.68-7.63(m,4H),7.59(s,1H),7.75-7.28(m ,9H),5.80-5.69(m,1H),5.66(s,1H),5.11-5.00(m,2H),4.87(d,J=11.4Hz,1H),4.7 4-4.71(d,J=11.4Hz,1H),4.57(s,1H),4.24-4.20(m,2H),4.03(d,J=12.0Hz,1H),3. 91(d,J=12.0Hz,1H),2.49-2.42(m,1H),2.26-2.22(m,1H),1.56(s,3H),1.09(s,9H); 13C NMR(CDCl3,100MHz)δ163.60,149.72,135.44,135.27,134.23,134.16,133.6 4,133.11,132.70,132.25,130.10,130.06,128.42,127.98,127.85,127.73, 126.88,126.39,126.25,125.74,117.88,110.42,89.20,86.82,79.97,77.05 ,76,47,72.53,58.80,33.76,26.97,19.44,12.04; ESI-MS(m / z)689.41[M+H] + 711.38 [M+Na] + . 6

[0149] Add BSA (29 mL, 120 mmol) to a 50 mL toluene suspension of N6-benzoyladenine (14 g, 60 mmol), heat to 60 °C and react until N6-benzoyladenine dissolves and the system becomes clear. Cool the reaction solution to room temperature, add a 170 mL toluene solution of diacetyl sugar 4 (23 g, 30 mmol), and then rapidly add TMSOTf (8.3 mL, 45 mmol). After the addition is complete, heat to 100 °C and stir for 3 h. Monitor the reaction by TLC to ensure it is complete (petroleum ether / ethyl acetate = 5 / 1). Heating was stopped, and the reaction solution was allowed to cool to room temperature. The solution was then poured into ethyl acetate, and a semi-saturated NaHCO3 solution (400 mL) was added. The mixture was stirred for 20 min, and the insoluble solids were filtered off with diatomaceous earth. The organic layer was separated, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and dried under vacuum. The residue was dissolved in 250 mL of methanol, and K2CO3 (20.7 g, 150 mmol) was added. The mixture was reacted at room temperature for 16 h, and TLC was used to confirm complete reaction (dichloromethane / ethyl acetate = 10 / 3). The reaction solution was concentrated, and the residue was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-100%) to obtain 7.4 g of locked nucleic acid derivative 6, a white powder solid, with a yield of 35%. 1H NMR(CDCl3,400MHz)δ8.28(s,1H),8.15(s,1H),7.80-7.64(m,8H),7.47-7.28(m,9H),6.06(s,1H),5.93-5.91(m,2H),5.82-5.75(m,1H),5.13(d,J=17.1Hz,1H),5.04(d,J=10.2Hz,1H),4.81-4.72(m,3H),4.52(s,1H),4.32-4.29(m,1H),4.02(d,J=12.0Hz,1H),3.92(d,J=12.0Hz,1H),2.55-2.49(m,1H),2.34-2.27(m,1H),1.07(s,9H); 13 CNMR(CDCl3,100MHz)δ155.46,153.16,148.82,135.59,135.49,130.02,129.99,128.36,127.96,127.92,127.83,127.70,126.78,126.29,120.09,117.83,88.57,86.16,80.45,78.74,76.64,72.75,58.82,33.90,26.79,19.22;ESI-MS(m / z)698.36[M+H] + ,720.35[M+Na] + . 7

[0151] Add BSA (29 mL, 120 mmol) to 50 mL of a toluene suspension of 6-chloroguanine (10.2 g, 60 mmol), heat to 60 °C and react until 6-chloroguanine dissolves and the system becomes clear. Cool the reaction solution to room temperature and add a 170 mL toluene solution of diacetyl sugar 4 (23 g, 30 mmol). Then, rapidly add TMSOTf (8.3 mL, 45 mmol). After the addition is complete, heat to 100 °C and stir for 3 h. Monitor the reaction by TLC until it is complete (petroleum ether / ethyl acetate = 5 / 1). Heating was stopped, and the reaction solution was allowed to cool to room temperature. The solution was then poured into ethyl acetate, and a semi-saturated NaHCO3 solution (400 mL) was added. The mixture was stirred for 20 min, and the insoluble solids were filtered off with diatomaceous earth. The organic layer was separated, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and vacuum dried. The residue was dissolved in 250 mL of methanol, and K2CO3 (20.7 g, 150 mmol) was added. The mixture was reacted at room temperature for 16 h, and TLC was used to confirm complete reaction (dichloromethane / ethyl acetate = 10 / 3). The reaction solution was concentrated, and the residue was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-30%) to obtain 12.2 g of locked nucleic acid derivative 7, a white foamy solid, with a yield of 56%. 1 H NMR(CDCl3,400MHz)δ7.98(s,1H),7.82-7.67(m,8H),7.49-7.36(m,7H),7. 31-7.27(m,2H),5.92(s,1H),5.84-5.74(m,1H),5.14-5.01(m,2H),4.95(br s,2H),4.79(dd,J=17.7,11.5Hz,2H),4.58(s,1H),4.53(s,1H),4.31(dd,J=8.6,5.0Hz,1H),4.08(s,3 H),4.00(d,J=12.0Hz,1H),3.90(d,J=12.0Hz,1H),2.56-2.48(m,1H),2.34-2.27(m,1H),1.07(s,9H); 13C NMR(CDCl3,100MHz)δ161.49,159.36,142.45,136.44,135.62,134.47,133 .94,133.14,133.08,132.53,132.47,129.97,128.34,127.94,127.90,127. 69,126.67,126.25,126.12,125.65,117.75,116.09,88.31,85.93,80.46, 78.93,72.80,58.86,53.91,33.93,26.78,19.17; ESI-MS(m / z)728.39[M+H] + .

[0152] Synthesis of R-6'-CN-LNA

[0153]

[0154] The reaction conditions were as follows: (a) Tris(triphenylphosphine)carbonyl ruthenium(II) hydrochloride (Wilkinson's catalyst), EtOH, 80℃, 48h; (b) Potassium osmium dihydrate, 50% NMO, t-BuOH, H2O, 60℃, 8h; (c) NaIO4, EtOH-THF (2 / 1 v / v), H2O, room temperature, 12h; (d) NH4OH, I2, room temperature, 24h. 8

[0156] Tris(triphenylphosphine)carbonylruthenium(II) hydrochloride (345 mg, 0.36 mmol, 5% mol) was added to 50 mL of anhydrous ethanol solution of allyl nucleoside derivative 5 (5.0 g, 7.26 mmol), and the mixture was heated to 80 °C and reacted for 48 h. The reaction solution was directly concentrated, and the residue was dissolved in 40 mL of tetrahydrofuran. Then, 5 mL of tert-butanol and 5 mL of water, potassium osmium tetroxide dihydrate (26.7 mg, 72.6 μmol, 1% mol) and 50% N-methylmorpholine-N-oxide (2.3 mL, 10.9 mmol) were added sequentially, and the mixture was heated to 60 °C and reacted for 8 h. The reaction was confirmed to be complete by TLC (dichloromethane / methanol = 10 / 1). After quenching the reaction with saturated sodium sulfite solution, the product was extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate containing 5% methanol / dichloromethane = 0-80%) to obtain 5g of the dihydroxylated isomer mixture.

[0157] The above-mentioned dihydroxylated product was dissolved in 60 mL of ethanol-tetrahydrofuran solution, and 10 mL of NaIO4 (2.33 g, 10.9 mmol) aqueous solution was added dropwise. The mixture was stirred at room temperature for 12 h. After filtering off the insoluble solid, 25% ammonia (5.5 mL, 72.6 mmol) was added to the filtrate, followed by the addition of I2 (1.84 g, 7.26 mmol) in portions. The reaction was allowed to proceed at room temperature for 24 h. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate containing 5% methanol / dichloromethane = 0-60%) to give 3.3 g of a slightly gray, foamy solid product 8, with a yield of 48%. 1 H NMR(CDCl3,400MHz)δ8.99,7.85-7.76(m,3H),7.68-7.64(m,5H),7.52-7.42(m,5H),7.38-7.33(m,5H),5.84(s,1H),4.87(s,1H),4.83( s,1H),4.82(d,J=6.6Hz,1H),4.71(d,J=6.6Hz,1H),4.24(s,1H),4.17(d,J=6.9Hz,1H),4.01(d,J=6.9Hz,1H),1.63(s,3H),1.10(s,9H); 13 C NMR(CDCl3,100MHz)δ163.50,149.61,135.53,135.32,133.63,133.25,133.2 1,133.05,132.10,131.80,130.36,130.32,128.68,128.15,128.10,127.86, 127.80,127.15,126.63,126.56,125.58,115.47,111.18,89.24,87.30,78.0 6,76.26,72.81,70.55,58.15,26.86,19.43,12.24; ESI-MS(m / z)674.28[M+H] + 796.25 [M+Na] + . 9

[0159] Add 475 mg, 0.5 mmol, 5% mol) of tris(triphenylphosphine)carbonylruthenium(II) hydrochloride to 70 mL of anhydrous ethanol solution of allyl nucleoside derivative 6 (7.0 g, 10 mmol), and react at 80 °C for 48 h. Concentrate the reaction solution directly, dissolve the residue in 50 mL of tetrahydrofuran, and add 10 mL of tert-butanol, 10 mL of water, potassium osmium tetroxide dihydrate (37 mg, 0.1 mmol, 1% mol), and 50% N-methylmorpholine-N-oxide (3.13 mL, 15 mmol) sequentially. React at 60 °C for 8 h, and TLC analysis confirms complete reaction (dichloromethane / methanol = 10 / 1). After quenching the reaction with saturated sodium sulfite solution, the product was extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-10%) to obtain 7.5 g of the dihydroxylated isomer mixture.

[0160] The above-mentioned dihydroxylated product was dissolved in 60 mL of ethanol-tetrahydrofuran solution, and 15 mL of NaIO4 (3.2 g, 10.9 mmol) aqueous solution was added dropwise. The mixture was stirred at room temperature for 12 h. After filtering off the insoluble solid, 25% ammonia (7.6 mL, 100 mmol) was added to the filtrate, followed by the addition of I2 (2.54 g, 10 mmol) in portions. The reaction was allowed to proceed at room temperature for 24 h. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by flash column chromatography with ethyl acetate / dichloromethane as the eluent (0-40%) to give 1.2 g of a slightly yellow, foamy solid product 9, with a yield of 26%. 1 H NMR(DMSO-d6,400MHz)δ10.71(s,1H),7.92-7.81(m,5H),7.66-7.61(m,4H),7.54-7.36(m,9H),6.66(br s,2H),6.04(s,1H),5.30(s,1H),5.13(s,1H),4.92(d,J=12.0Hz,1H),4.84(d,J=12 .0Hz,1H),4.79(s,1H),4.21(d,J=12.2Hz,1H),4.00(d,J=12.2Hz,1H),0.94(s,9H); 13C NMR(DMSO-d6,100MHz)δ157.14,154.49,151.10,135.62,135.28,134.48,132.15,130.53,128.44,128.15,128.07,128.76,126 .64,126.57,126.17,117.74,116.98,88.23,84.86,78.84,78.44,71.98,71.22,59.76,26.77,19.19; ESI-MS(m / z)683.30[M+H] + 705.27 [M+Na] + . 10

[0162] To a 120 mL anhydrous ethanol solution of allyl nucleoside derivative 7 (11.0 g, 15.1 mmol), tris(triphenylphosphine)carbonylruthenium(II) hydrochloride (718 mg, 0.76 mmol, 5% mol) was added, and the mixture was heated to 80 °C and reacted for 48 h. The reaction solution was directly concentrated, and the residue was dissolved in 80 mL tetrahydrofuran. Then, 10 mL tert-butanol, 10 mL water, potassium osmium tetroxide dihydrate (56 mg, 0.15 mmol, 1% mol), and 50% N-methylmorpholine-N-oxide (4.7 mL, 22.7 mmol) were added sequentially, and the mixture was heated to 60 °C and reacted for 8 h. The reaction was confirmed to be complete by TLC (dichloromethane / methanol = 10 / 1). After quenching the reaction with saturated sodium sulfite solution, the product was extracted with ethyl acetate, washed with water, washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-60%) to obtain 8 g of dihydroxylated isomer mixture.

[0163] The above-mentioned dihydroxylated product was dissolved in 70 mL of ethanol-tetrahydrofuran solution, and 15 mL of NaIO4 (3.4 g, 10.9 mmol) aqueous solution was added dropwise. The mixture was stirred at room temperature for 12 h. After filtering off the insoluble solid, 25% ammonia (7.9 mL, 105 mmol) was added to the filtrate, followed by the addition of I2 (2.67 g, 10.5 mmol) in portions. The reaction was allowed to proceed at room temperature for 24 h. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-40%) to give 4 g of slightly yellow, foamy solid product 10, with a yield of 37%. 1H NMR(CDCl3,400MHz)δ7.82-7.76(m,3H),7.71-7.16(m,6H),7.51-7.30(m,9H),6.10(s,1H),4.95(s,1H),4.92(s,1H),4.77(br s,2H),4.73(d,J=2.7Hz,2H),4.50(s,1H),4.14(d,J=12.0Hz,1H),4.07(s,3H),4.02(d,J=12.0Hz,1H),1.10(s,9H); 13 C NMR(CDCl3,100MHz)δ161.60,159.29,152.25,136.27,135.67,135.54,133.6 0,133.16,133.05,132.09,132.07,130.16,128.60,128.03,127.97,127.86, 127.73,126.88,126.49,126.42,125.43,116.04,115.82,88.54,86.26,78.1 9,77.97,72.96,71.05,58.73,54.02,26.69,19.20; ESI-MS(m / z)713.40[M+H] +

[0164] Synthesis of R-6'-CN-LNA phosphoramide monomer

[0165] Synthesis of R-6'-CN-LNA-T phosphoramide monomer

[0166]

[0167] The reaction conditions were as follows: (a)(1) DDQ, dichloromethane-H2O (20 / 1), room temperature, 24h; (2) Et3N, Et3N·3HF, THF, room temperature, 9h; (b) DMTrCl, pyridine, room temperature, 12h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphine diamine, 1H-tetrazole, room temperature, 5h. 11

[0169] At room temperature, DDQ (420 mg, 1.86 mmol) was added to 10.5 mL of a dichloromethane-water (20:1) mixture containing nucleoside derivative 8 (500 mg, 0.74 mmol). The reaction was allowed to proceed for 24 h at room temperature, and TLC analysis showed that the reaction was complete (dichloromethane / methanol = 10 / 1). The reaction solution was concentrated under reduced pressure, extracted with ethyl acetate, washed successively with saturated sodium sulfite solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was dissolved in 10 mL of tetrahydrofuran, and triethylamine (260 μL, 1.86 mmol) and triethylamine trihydrofluoric acid (363 μL, 2.23 mmol) were added sequentially. The reaction was allowed to proceed for 9 h at room temperature, and TLC analysis showed that the reaction was complete (dichloromethane / methanol = 10 / 1). 600 mg of sodium bicarbonate solid was added to the reaction solution and stirred until no bubbles were generated. The reaction solution was concentrated under reduced pressure and purified directly by flash column chromatography (gradient elution: methanol / dichloromethane = 0-20%) to obtain 310 mg of colorless amorphous solid 11, with a yield of 46%. 1 H NMR (CD3OD, 400MHz) δ7.70(s,1H),5.69(s,1H),4.93(s,1H),4.46(s,1H),4.27(s,1H),3.96(s,2H),1.89(s,3H); 13 C NMR(CD3OD,100MHz)δ164.99,150.36,134.88,116.13,109.72,89.51,86.69,80.73,69.97,69.38,55.23,11.26; ESI-MS(m / z)296.13[M+H] + . 12

[0171] To a 50 mL pyridine solution of cyano-locked nucleoside derivative 11 (8.0 g, 27.1 mmol), 4,4'-dimethoxytriphenylmethyl chloride (13.8 g, 40.6 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 13.53 g of 5'-O-DMTr protected nucleoside 12 as a pale yellow powder, yield 84%. 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] + . 13

[0173] To a 40 mL solution of nucleoside 12 (4.0 g, 6.7 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonamide (3.0 g, 10.0 mmol) in dichloromethane, 1H-tetrazole (376 mg, 5.36 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 4.4 g of phosphoramide 13 as a white, foamy solid, in 82% yield. 31 P NMR(152MHz,DMSO-d6)δ148.80,148.75; ESI-MS(m / z)798.32[M+H] + .

[0174] Synthesis of R-6'-CN-LNA-A phosphoramide monomer

[0175]

[0176] The reaction conditions were as follows: (a) BzCl, pyridine, room temperature, 6 h; (b)(1) DDQ, dichloromethane-H2O (20 / 1), room temperature, 24 h; (2) Et3N, Et3N·3HF, THF, room temperature, 12 h; (b) DMTrCl, pyridine, room temperature, 12 h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphine diamine, 1H-tetrazole, room temperature, 8 h. 14

[0178] Benzoyl chloride (1.52 mL, 13.2 mmol) was added dropwise to 20 mL of pyridine solution containing nucleoside 9 (3.0 g, 4.4 mmol) at room temperature. The reaction was allowed to proceed for 6 h at room temperature, and TLC was used to confirm the completeness of the reaction (dichloromethane / ethyl acetate = 10 / 1). 15% NaOH solution was added dropwise to adjust the pH to 8-9, and the reaction was allowed to proceed for 4 h at room temperature. The reaction solution was diluted with ethyl acetate, washed successively with water, 1N HCl solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-60%) to give 1.45 g of benzoyl-protected nucleoside 14, a pale yellow solid, in 42% yield. 1 H NMR (CDCl3, 400MHz) δ8.62 (s, 1H), 8.20 (s, 1H), 8.07 (d, J = 7.0Hz, 2H), 7.82-7.75 (m, 2H), 7.69-7.32 (m, 18H), 6.25 (s, 1H), 5.11(s,1H),4.98(s,1H),4.77(s,2H),4.51(d,J=5.8Hz,1H),4.16(d,J=12.5Hz,1H),4.04(d,J=12.5Hz,1H),1.07(s,9H); 13 C NMR(CDCl3,100MHz)δ164.60,152.64,150.42,149.70,140.19,135.55,133.53,133 .42,133.09,132.99,132.12,132.09,130.15,128.94,128.52,128.00,127.92,127. 91,127.85,127.69,126.80,126.38,126.28,125.42,123.29,114.42,90.04,86.43 ,78.19,77.54,72.97,70.17,59.42,29.70,26.70,19.26; ESI-MS(m / z)787.32[M+H] + . 15

[0180] At room temperature, DDQ (1.03 g, 3.81 mmol) was added to 21 mL of a dichloromethane-water (20:1) mixture containing nucleoside derivative 14 (1.2 g, 1.52 mmol). The reaction was allowed to proceed for 24 h at room temperature, and TLC analysis showed that the reaction was complete (dichloromethane / methanol = 10 / 1). The reaction solution was concentrated under reduced pressure, extracted with ethyl acetate, washed successively with saturated sodium sulfite solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was dissolved in 15 mL of tetrahydrofuran, and triethylamine (530 μL, 3.81 mmol) and triethylamine trihydrofluoric acid (743 μL, 4.56 mmol) were added sequentially. The reaction was allowed to proceed for 12 h at room temperature, and TLC analysis showed that the reaction was complete (dichloromethane / methanol = 10 / 1). Add 1.15g of sodium bicarbonate solid to the reaction solution and stir until no bubbles are generated. Concentrate the reaction solution under reduced pressure and purify directly by flash column chromatography (gradient elution: dichloromethane / methanol = 0-15%) to obtain 400mg of cyano-locked nucleoside 15, yield 64%. 1 H NMR (CD3OD, 400MHz) δ8.72(s,1H),8.55(s,1H),8.07(d,J=4.2Hz,2H),7.66(t,J=4.2Hz,1H ),7.57(t,J=4.2Hz,2H),6.30(s,1H),5.05(s,1H),4.88(s,1H),4.64(s,1H),4.02(s,2H); 13 C NMR(CD3OD,100MHz)δ166.76,152.06,151.09,149.81,141.56,132.46,132.57,128.38,12 8.06,123.91,116.25,89.47,86.11,80.84,70.77,70.43,55.86; ESI-MS(m / z)409.15[M+H] + . 16

[0182] At room temperature, 4,4'-dimethoxytriphenylmethylchloro (481 mg, 1.42 mmol) was added to 5 mL of a pyridine solution containing cyano-O-DMTr. The mixture was stirred at room temperature for 12 h, and the reaction was monitored by TLC to ensure complete reaction (dichloromethane / methanol = 10 / 1). The reaction was quenched with 1 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: ethyl acetate containing 5% methanol / dichloromethane = 0-45%) to give 500 mg of 5'-O-DMTr-protected cyano-O-DMTr-protected cyano-O-DMTr-protected cyano-O-DMTr as a pale yellow solid, with a yield of 94%. 1H NMR(DMSO-d6,400MHz)δ12.26(s,1H),8.79(s,1H),8.59(s,1H),8.06(d,J=8.4Hz,2H ),7.67(t,J=7.4Hz,1H),7.58(t,J=7.8Hz,2H),7.46(d,J=7.4Hz,1H),7.33-7.20(m,7 H),6.89(d,J=8.4Hz,4H),6.33(s,1H),6.30(d,J=4.3Hz,1H),5.12(s,1H),4.95(s,1H ),4.83(d,J=4.2Hz,1H),3.73(s,6H),3.68(d,J=11.2Hz,1H),3.40(d,J=11.2Hz,1H); 13 C NMR(DMSO-d6,100MHz)δ166.09,158.65,152.31,152.02,151.00,144.95,142.63,135.66,135.42,133.77,132.98,130.34,130.28,128.98, 128.97,128.33,128.96,127.26,126.02,118.26,113.71,88.34,86.8 3,85.69,80.94,72.59,71.24,60.23,55.51; ESI-MS(m / z)711.36[M+H] + . 17

[0184] At room temperature, 1H-tetrazole (35 mg, 0.51 mmol) was added to 10 mL of a dichloromethane solution containing nucleoside 12 (450 mg, 0.63 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonamide (382 mg, 1.27 mmol). The reaction was allowed to proceed for 8 h at room temperature, and TLC was used to confirm the completeness of the reaction (dichloromethane / ethyl acetate = 5 / 2). The reaction solution was then purified directly by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-40%) to give 480 mg of phosphoramide 17 as a slightly yellow, foamy solid, in 83% yield. 31 P NMR(152MHz,DMSO-d6)δ149.19,149.04; ESI-MS(m / z)909.38[MH] - .

[0185] Synthesis of R-6'-CN-LNA-G phosphoramide monomer

[0186]

[0187] The reaction conditions were as follows: (a) 2N HCl, THF-CH3OHH (1 / 1), 60℃, 12h; (b) isobutyryl chloride, Et3N, DMAP, 1,4-dioxane, 100℃, 24h; (c)(1) DDQ, dichloromethane-H2O (20 / 1), room temperature, 24h; (2) Et3N, Et3N·3HF, THF, room temperature, 12h; (d) DMTrCl, pyridine, room temperature, 12h; (e) 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite, 1H-tetrazole, room temperature, 12h. 18

[0189] 17.5 mL of 2N hydrochloric acid solution was added to 30 mL of tetrahydrofuran-methanol solution (1 / 1 v / v) containing cyano-demethylated nucleoside 10 (2.5 g, 3.5 mmol). The mixture was heated to 60 °C and reacted for 12 h. A white solid precipitated, and the reaction was confirmed to be complete by TLC (dichloromethane / ethyl acetate = 2 / 1). The insoluble white solid was filtered off, and the filtrate was extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the filtrates were combined. The filtrates were purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane containing 5% methanol = 0-60%) to give 1.4 g of 6-demethylated nucleoside 18 as a white solid, yield 57%. 1 H NMR(DMSO-d6,400MHz)δ10.71(s,1H),7.92-7.81(m,5H),7.66(m,4H),7.54-7.49(m,2H),7.47-7.36(m,7H),6.66(br s,2H),6.04(s,1H),5.29(s,1H),5.13(s,1H),4.92(d,J=12.0Hz,1H),4.84(d,J=12 .0Hz,1H),4.78(s,1H),4.21(d,J=12.2Hz,1H),4.01(d,J=12.2Hz,1H),0.94(s,9H); 13CNMR(DMSO-d6,100MHz)δ157.13,154.49,151.10,135.62,135.60,135.2 9,134.48,133.17,133.01,132.46,132.46,130.52,128.44,128.15,128 .07,126.75,126.65,126.56,126.17,117.70,117.02,88.24,84.89,78. 86,78.48,72.02,71.23,59.76,26.80,19.20; ESI-MS(m / z)699.39[M+H] + 721.48 [M+Na] + . 19

[0191] DMAP (105 mg, 0.86 mmol) and triethylamine (718 μL, 5.16 mmol) were added to 12 mL of a 1,4-dioxane solution of guanosine 18 (1.2 g, 1.72 mmol), followed by isobutyryl chloride (550 μL, 5.16 mmol). The mixture was heated to 100 °C and reacted for 24 h. The reaction was confirmed to be complete by TLC (dichloromethane / methanol = 10 / 1). Heating was stopped, and the reaction solution was allowed to cool to room temperature. The reaction was quenched by adding 1 mL of methanol. The solution was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-80%) to obtain 1.1 g of isobutyryl-protected guanosine 19, a slightly yellow, foamy solid, in 83% yield. 1 H NMR(CDCl3,400MHz)δ12.00(s,1H),9.02(s,1H),7.78-7.74(m,3H),7.69-7.63 (m,5H),7.52(s,1H),7.47-7.28(m,9H),5.86(s,1H),4.87(s,1H),4.76(d,J=11 .6Hz,1H),4.66(d,J=11.6Hz,1H),4.47(s,1H),4.33(s,1H),4.13(d,J=12.0Hz, 1H), 4.02 (d, J = 12.0Hz, 1H), 2.70-2.66 (m, 1H), 1.29-1.24 (m, 6H), 1.08 (s, 9H); 13C NMR (CDCl3, 100MHz) δ178.83,155.24,147.84,146.82,135.64,135.49,135.11,133. 37,133.11,132.95,131.99,131.93,130.25,130.22,128.67,128.07,128.01,127.74 ,127.59,127.25,126.61,126.53,125.70,131.62,115.87,86.71,86.12,78.40,73. 33,71.03,58.74,36.40,26.71,19.78,19.21,18.99; ESI-MS(m / z)870.60[M+Et3N+H] - . 20

[0193] At room temperature, DDQ (702 mg, 3.64 mmol) was added to 10.5 mL of a dichloromethane-water (20:1) mixture containing nucleoside derivative 19 (800 mg, 1.04 mmol). The reaction was allowed to proceed for 24 h at room temperature, and TLC analysis showed that the reaction was complete (dichloromethane / methanol = 10 / 1). The reaction solution was concentrated under reduced pressure, extracted with ethyl acetate, washed successively with saturated sodium sulfite solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was dissolved in 10 mL of tetrahydrofuran, and triethylamine (363 μL, 2.60 mmol) and triethylamine trihydrofluoric acid (509 μL, 3.12 mmol) were added sequentially. The reaction was allowed to proceed for 12 h at room temperature, and TLC analysis showed that the reaction was complete (dichloromethane / methanol = 10 / 1). 786 mg of sodium bicarbonate solid was added to the reaction solution and stirred until no more bubbles were generated. The reaction solution was concentrated under reduced pressure and purified directly by flash column chromatography (gradient elution: dichloromethane / methanol = 0-25%) to obtain 230 mg of cyano-locked nucleoside 20, with a yield of 57%. 1 H NMR (CD3OD, 400MHz) δ8.09(s,1H),6.08(s,1H),5.01(s,1H),4.73(s,1H),4.54(s,1H),3.99(s,2H),2.72-2.68(m,1H),1.23-1.21(m,6H); 13 C NMR(CD3OD,100MHz)δ180.35,148.67,148.20,136.57,120.03,116.22,89.37,85.62,8 0.91,70.56,70.35,55.80,46.48,35.58,17.92,17.90,7.84; ESI-MS(m / z)391.08[M+H] +413.05 [M+Na] + . twenty one

[0195] At room temperature, 4,4'-dimethoxytriphenylmethyl chloride (260 mg, 0.77 mmol) was added to 5 mL of pyridine solution of cyano-O-DMTr. The mixture was stirred at room temperature for 12 h, and the reaction was monitored by TLC to ensure complete reaction (dichloromethane / methanol = 5 / 1). The reaction was quenched with 2 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: ethyl acetate / dichloromethane containing 5% methanol = 0-40%) to obtain 270 mg of 5'-O-DMTr-protected cyano-O-DMTr-protected cyano-O-DMTr-protected cyano-O-DMTr as a pale yellow powder, with a yield of 100%. 1 H NMR(DMSO-d6,400MHz)δ12.14(s,1H),11.85(s,1H),8.11(s,1H),7.46(d,J=7.4 Hz,2H),7.33-7.21(m,7H),6.90(dd,J=8.8,1.4Hz,4H),6.26(d,J=4.2Hz,1H),6. 08(s,1H),5.08(s,1H),4.77(s,1H),4.57(d,J=4.2Hz,1H),3.74(s,6H),3.68(d, J=11.2Hz,1H),3.39(d,J=11.2Hz,1H),2.82-2.75(m,1H),1.14(d,J=6.8Hz,6H); 13 C NMR(DMSO-d6,100MHz)δ180.73,158.66,155.22,148.89,148.58,144.97,136.87,135.59,135.40,130.34,130.31,128.33,128.21,12 7.27,120.65,118.31,113.71,88.30,86.87,84.93,80.95,72.28,71.18,60.22,55.51,35.27,19.33,19.28; ESI-MS(m / z)691.36[MH] - . twenty two

[0197] At room temperature, 1H-tetrazole (15 mg, 0.21 mmol) was added to 10 mL of a dichloromethane solution containing nucleoside 21 (180 mg, 0.26 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonamide (157 mg, 0.52 mmol). The reaction was allowed to proceed for 12 h at room temperature, and TLC was used to confirm the completeness of the reaction (dichloromethane / ethyl acetate = 2 / 1). The reaction solution was then purified directly by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-40%) to give 180 mg of phosphoramide 22, a slightly yellow, waxy solid, in 78% yield. 31 P NMR(152MHz,DMSO-d6)δ149.26,149.23; ESI-MS(m / z)893.40[M+H] + .

[0198] R-6'-CN-LNA- m Synthesis of C phosphoramide monomer

[0199]

[0200] 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

[0202] 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); 13 C NMR(DMSO-d6,100MHz)δ166.09,158.81,158.79,154.95,144.78,136.54,135.38,135.17,130.30,130.14,128.43,128.07,127.46,11 7.58,113.81,113.77,101.92,88.29,87.59,86.82,80.77,71.60,71.09,58.50,55.55,14.01,6.78,4.42; ESI-MS(m / z)733.31[M+Na] + . twenty four

[0204] 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, the pH was adjusted to 10 with 15% NaOH, 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

[0206] 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. 31P NMR(152MHz,DMSO-d6)δ149.20,148.47; ESI-MS(m / z)901.46[M+H] + .

[0207] Part Two: Synthesis of S-6'-CN-LNA

[0208] Synthesis of S-6'-Allyl-LNA

[0209]

[0210] The reaction conditions were as follows: (a) 2-iodobenzoic acid, acetonitrile, reflux, 11 h; (b) NaBH4, LiCl, MeOH, -40℃, 8 h; (c) methanesulfonyl chloride, DMAP, pyridine, room temperature, 32 h; (d) (1) FeCl3·6H2O, dichloromethane, 0℃-room temperature, 3 h; (2) Ac2O, DMAP, pyridine, dichloromethane, room temperature, 4 h. 26

[0212] 2-Iodobenzoic acid (69 g, 0.25 mol) was added to 500 mL of acetonitrile solution of compound 2 (105 g, 0.164 mol), and the mixture was refluxed for 6 h. Then, 13.8 g, 0.05 mol of 2-iodobenzoic acid was added again, and the mixture was refluxed for another 5 h. The reaction was confirmed by TLC (petroleum ether / ethyl acetate = 5 / 1). The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated and purified by flash column chromatography (gradient elution: ethyl acetate / petroleum ether = 0-20%) to give 93 g of colorless viscous substance 26, yield 89%. 1 H NMR(CDCl3,400MHz)δ7.85-7.70(m,4H),7.60-7.53(m,4H),7.48-7.45(m,2H),7.40-7.27( m,7H),6.08(d,J=4.0Hz,1H),5.98-5.83(m,1H),5.13-5.01(m,2H),4.90(d,J=12.0Hz,1H) ,4.86(t,J=8.0,4.0Hz,1H),4.63(d,J=12.0Hz,1H),4.23(d,J=8.0Hz,1H),3.92(d,J=12.0 Hz,1H),3.72(d,J=12.0Hz,1H),3.66-3.55(m,2H),1.61(s,3H),1.43(s,3H),0.98(s,9H); 13CNMR(CDCl3,100MHz)δ207.44,135.53,134.92,134.83,133.23,133.06,13 2.49,132.46,130.73,129.99,129.91,128.12,127.97,127.87,127.75,127 .71,126.57,126.09,125.93,125.82,118.44,114.93,106.94,96.44,81.14 ,80.15,73.55,44.95,27.61,26.81,26.68,19.13; ESI-MS(m / z)635.40[MH] - . 27

[0214] At room temperature, 200 mL of a methanol solution containing 18 g of lithium chloride (0.424 mol) was added to 400 mL of a tetrahydrofuran solution of compound 26 (108 g, 0.17 mol). The reaction solution was cooled to -40 °C, and sodium borohydride (4.3 g, 0.113 mol) was added. After reacting for 4 h, sodium borohydride (4.3 g, 0.113 mol) was added again, and the reaction was continued for another 4 h. The reaction was confirmed to be complete by TLC (petroleum ether / ethyl acetate = 5 / 1). The reaction solution was poured into a beaker, and the reaction was quenched with 100 mL of saturated ammonium chloride solution. The mixture was stirred until no bubbles were generated, extracted with 1 L of ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / petroleum ether = 0-20%) to give 90 g of a colorless viscous substance 27, with a yield of 83%. 1 H NMR(CDCl3,400MHz)δ7.86-7.79(m,4H),7.59-7.57(m,2H),7.51-7.46(m,5H),7.41-7.28( m,6H),5.91-5.83(m,2H),5.01-4.97(m,3H),4.78(dd,J=5.3,3.9Hz,1H),4.65(d,J=12.0Hz ,1H),4.51(d,J=5.4Hz,1H),4.19(dd,J=10.0,2.1Hz,1H),3.76(d,J=10.7Hz,1H),3.54(d,J =10.7Hz,1H),2.36-2.31(m,1H),2.09-2.02(m,1H),1.67(s,3H),1.39(s,3H),0.91(s,9H); 13C NMR(CDCl3,100MHz)δ136.40,135.53,135.51,134.80,133.22,132.16,1 32.88,132.74,129.85,129.78,128.41,127.94,127.78,126.85,126.29 ,126.14,125.67,161.13,114.38,104.73,90.15,79.74,78.12,72.80,7 1.04,64.51,35.91,26.98,26.81,26.75,19.11; ESI-MS(m / z)637.41[MH] - . 28

[0216] Compound 27 (90 g, 0.14 mol) and DMAP (1.71 g, 14 mmol) were dissolved in 400 mL of pyridine. Methanesulfonyl chloride (16.4 mL, 0.21 mmol) was added dropwise, and the mixture was reacted at room temperature for 20 h. Then, methanesulfonyl chloride (5.5 mL, 0.07 mol) was added, and the reaction was continued for another 12 h. The reaction was confirmed to be complete by TLC (petroleum ether / ethyl acetate = 5 / 1). The reaction was quenched with 50 mL of methanol, concentrated under reduced pressure to remove excess pyridine, and the residue was diluted with ethyl acetate. The residue was washed successively with water, 1 N HCl solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 100 g of methanesulfonyl compound 28, a slightly yellow viscous substance, with a yield of 100%. 1 HNMR(DMSO-d6,400MHz)δ7.95-7.87(m,4H),7.59-7.38(m,13H),5.85(d,J=4.0Hz,1H),5. 68-5.58(m,1H),5.13(dd,J=9.8,1.4Hz,1H),5.07(t,J=5.0Hz,1H),4.98-4.91(m,3H),4.6 9(d,J=11.8Hz,1H),4.47(d,J=5.4Hz,1H),3.76(d,J=10.8Hz,1H),3.71(d,J=10.8Hz,1H), 3.08(s,3H),2.83-2.78(m,1H),2.16-2.08(m,1H),1.58(s,3H),1.37(s,3H),0.88(s,9H); 13C NMR(CDCl3,100MHz)δ135.54,135.59,135.30,134.36,133.24,133.15,132.7 2,132.68,130.50,130.47,128.58,128.43,128.35,128.21,127.32,126.79, 126.61,126.48,118.25,124.07,105.02,88.46,85.65,79.89,77.62,72.51, 63.50,36.01,27.06,26.95,26.82,26.58,19.07; ESI-MS(m / z)734.39[M+NH4] + 739.34 [M+Na] + . 29

[0218] A 1000 mL solution of compound 28 (100 g, 0.14 mol) in dichloromethane was cooled to 0 °C. Ferric chloride hexahydrate (15.1 g, 56 mmol) was added, and the mixture was allowed to react at room temperature for 3 h. The reaction was monitored by TLC until complete (petroleum ether / ethyl acetate = 4 / 1). The reaction mixture was poured into 2 L of water and extracted with dichloromethane. The organic layer was washed successively with water, saturated sodium bicarbonate solution, and saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and dried under vacuum. The resulting viscous substance was dissolved in 1 L of dichloromethane, and pyridine (113 mL, 1.4 mol), DMAP (2.56 g, 21 mol), and acetic anhydride (79 mL, 0.84 mol) were added dropwise. The mixture was allowed to react at room temperature for 4 h, and the reaction was monitored by TLC until complete (petroleum ether / ethyl acetate = 4 / 1). The reaction mixture was poured into 2 L of water and extracted with dichloromethane. The organic layer was washed successively with water, 1 N HCl solution, saturated sodium bicarbonate solution, and saturated brine. The mixture was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: petroleum ether / ethyl acetate = 0-60%) to give 95 g of a mixture of diacetyl compound 29 epimers, a colorless viscous liquid, in 89% yield. ESI-MS (m / z) 778.39 [M+NH4] + 783.44 [M+Na] + .

[0219]

[0220] The reaction conditions were as follows: (a) thymine, N,O-bis(trimethoxyacetamide) (BSA), TMSOTf, acetonitrile, 80℃, 3h; (b) K2CO3, MeOH, room temperature, 16h; (c) N6-benzoyladenine, BSA, TMSOTf, toluene, 100℃, 3h; (d) 6-chloroguanine, BSA, TMSOTf, toluene, 100℃, 3h. 30

[0222] BSA (29 mL, 120 mmol) was added to 50 mL of acetonitrile suspension of thymine (7.6 g, 60 mmol), and stirred at room temperature for 20 min until the thymine dissolved. After the system became clear, 200 mL of acetonitrile solution of diacetyl sugar 29 (23 g, 30 mmol) was added, followed by rapid dropwise addition of TMSOTf (8.3 mL, 45 mmol). After the addition was complete, the temperature was raised to 80 °C, and the reaction was stirred for 3 h. The reaction was monitored by TLC to ensure it was complete (petroleum ether / ethyl acetate = 5 / 1). Heating was stopped, and the reaction solution was allowed to cool to room temperature. The solution was then poured into ethyl acetate, and a semi-saturated NaHCO3 solution (400 mL) was added. The mixture was stirred for 20 min, and the insoluble solids were filtered off with diatomaceous earth. The organic layer was separated, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and vacuum dried. The residue was dissolved in 250 mL of methanol, and K2CO3 (12.4 g, 90 mmol) was added. The mixture was reacted at room temperature for 16 h, and TLC was used to confirm complete reaction (dichloromethane / ethyl acetate = 10 / 3). The reaction solution was concentrated, and the residue was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-25%) to obtain 15.4 g of locked nucleic acid derivative 30, a white foamy solid, with a yield of 75%. 1H NMR(CDCl3,400MHz)δ8.92(br s,1H),7.84-7.64(m,8H),7.51-7.29(m,10H),5.77-5.59(m,1H),5.65(s,1H),5.01-4.96(m,2H),4.84(d,J=11.4Hz,1H),4 13C NMR(CDCl3,100MHz)δ163.79,149.75,135.59,135.35,134.36,134.13,134.01,1 33.14,133.10,132.80,132.34,130.11,130.06,128.45,127.99,127.95,127.87, 127.75,126.91,126.45,126.30,125.75,117.38,110.34,89.58,87.12,84.02,7 6.90,72.53,59.27,35.33,29.72.26.94,19.44,12.15; ESI-MS(m / z)689.43[M+H] + 711.34 [M+Na] + . 31

[0224] BSA (17 mL, 68 mmol) was added to 25 mL of a toluene suspension of N6-benzoyladenine (6.1 g, 25.5 mmol), and the mixture was heated to 60 °C until N6-benzoyladenine dissolved and the system became clear. The reaction solution was then cooled to room temperature, and 70 mL of a toluene solution of diacetyl sugar 29 (13 g, 17 mmol) was added. TMSOTf (4.63 mL, 25.5 mmol) was then rapidly added dropwise. After the addition was complete, the temperature was raised to 100 °C, and the reaction was stirred for 3 h. The reaction was monitored by TLC until it was complete (petroleum ether / ethyl acetate = 5 / 1). Heating was stopped, and the reaction solution was allowed to cool to room temperature. The solution was then poured into ethyl acetate, and a semi-saturated NaHCO3 solution (400 mL) was added. The mixture was stirred for 20 min, and the insoluble solids were filtered off using diatomaceous earth. The organic layer was separated, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and vacuum dried. Flash column purification (gradient elution: ethyl acetate / dichloromethane = 0-40%) yielded 9.1 g of the nucleoside product, with a yield of 74%. ESI-MS (m / z) 940.04 [M+H] + .

[0225] The above product (8.5 g, 9 mmol) was dissolved in 90 mL of methanol, and K2CO3 (6.25 g, 45 mmol) was added. The mixture was reacted at room temperature for 16 h, and the reaction was confirmed to be complete by TLC (dichloromethane / ethyl acetate = 10 / 3). The reaction solution was concentrated, and the residue was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-100%) to give 4.9 g of locked nucleic acid derivative 31, a white powder solid, with a yield of 78%. 1 HNMR(CDCl3,400MHz)δ8.27(s,1H),7.98(s,1H),7.81-7.67(m,7H),7.62(s,1H),7.49-7 .31(m,9H),6.02(s,1H),5.86-5.76(m,3H),5.06-5.02(m,2H),4.95(s,1H),4.74(d,J=1 1.6Hz,1H),4.66(d,J=11.6Hz,1H),4.27(s,1H),4.18(dd,J=9.3,4.6Hz,1H),4.09(d,J= 11.9Hz,1H),4.05(d,J=11.9Hz,1H),2.77-2.69(m,1H),2.36-2.30(m,1H),1.07(s,9H); 13 C NMR(CDCl3,100MHz)δ155.16,152.74,148.84,138.22,135.69,135.63,134.35, 134.40,133.10,133.06,132.79,132.62,129.99,129.97,128.36,127.92,127. 85,127.82,127.71,126.92,126.32,126.19,125.64,120.14,117.16,89.04,86 .48,84.57,78.06,72.72,59.50,35.31,26.78,19.27; ESI-MS(m / z)698.36[M+H] + . 32

[0227] BSA (13 mL, 52.6 mmol) was added to a 25 mL toluene suspension of 6-chloroguanine (3.34 g, 19.7 mmol), and the mixture was heated to 60 °C until 6-chloroguanine dissolved and the system became clear. The reaction solution was then cooled to room temperature, and a 75 mL toluene solution of diacetyl sugar 29 (10 g, 13.1 mmol) was added. TMSOTf (3.6 mL, 19.7 mmol) was then rapidly added dropwise. After the addition was complete, the temperature was raised to 100 °C, and the reaction was stirred for 3 h. The reaction was monitored by TLC until it was complete (petroleum ether / ethyl acetate = 5 / 1). Heating was stopped, and the reaction solution was allowed to cool to room temperature. The solution was then poured into ethyl acetate, and a semi-saturated NaHCO3 solution (400 mL) was added. The mixture was stirred for 20 min, and the insoluble solids were filtered off with diatomaceous earth. The organic layer was separated, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and vacuum dried. The residue was dissolved in 100 mL of methanol, and K2CO3 (7.25 g, 52.6 mmol) was added. The mixture was reacted at room temperature for 16 h, and TLC was used to confirm complete reaction (dichloromethane / ethyl acetate = 10 / 3). The reaction solution was concentrated, and the residue was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous NaSO4, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0–30%) to obtain 6.07 g of locked nucleic acid derivative 32, a white foamy solid, with a yield of 63%. 1 H NMR(CDCl3,400MHz)δ7.82-7.65(m,9H),7.49-7.30(m,9H),5.91(s,1H),5.85-5.76(m,1H),5.06-5.02(m,2H),4.86(br s,2H),4.81(s,1H),4.73(d,J=11.5Hz,1H),4.64(d,J=11.5Hz,1H),4.27(s, 1H),,4.09-4.01(m,5H),2.76-2.69(m,1H),2.35-2.28(m,1H),1.07(s,9H); 13C NMR(CDCl3,100MHz)δ161.48,159.29,152.47,135.70,135.55,134.47,124.36 ,133.12,133.04,132.74,132.58,129.98,129.95,128.34,127.91,127.88,127 .84,127.70,126.60,125.59,117.15,116.13,88.77,86.19,84.49,78.23,77. 15,72.78,55.50,53.90,35.31,29.75,26.76,19.24; ESI-MS(m / z)728.39[M+H] + .

[0228] Synthesis of S-6'-CN-LNA

[0229]

[0230] The reaction conditions were as follows: (a)(1) tris(triphenylphosphine)carbonyl ruthenium(II) hydrochloride, EtOH, 80℃, 12-48h; (b) potassium osmium tetroxide dihydrate, 50% NMO, THF, t-BuOH, H2O, 60℃, 8-12h; (c) NaIO4, EtOH-THF (2 / 1 v / v), H2O, room temperature, 12h; (d) NH4OH, I2, room temperature, THF, EtOH, 24h. 33

[0232] To a 120 mL anhydrous ethanol solution of allyl nucleoside derivative 30 (10.0 g, 14.5 mmol), tris(triphenylphosphine)carbonylruthenium(II) hydrochloride (345 mg, 0.36 mmol, 2.5% mol) was added, and the mixture was heated to 80 °C and reacted for 12 h. The reaction solution was directly concentrated, and the residue was dissolved in 80 mL tetrahydrofuran. Then, 10 mL tert-butanol, 10 mL water, potassium osmium tetroxide dihydrate (26.7 mg, 72.6 μmol, 0.5% mol), and 50% N-methylmorpholine-N-oxide (4.53 mL, 10.9 mmol) were added sequentially, and the mixture was heated to 60 °C and reacted for 8 h. The reaction was confirmed to be complete by TLC (dichloromethane / methanol = 10 / 1). After quenching the reaction with saturated sodium sulfite solution, the product was extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-60%) to obtain 9.3 g of the dihydroxylated isomer mixture.

[0233] The above-mentioned dihydroxylated product was dissolved in 60 mL of ethanol-tetrahydrofuran solution, and 10 mL of NaIO4 (3.32 g, 15.5 mmol) aqueous solution was added dropwise. The mixture was stirred at room temperature for 12 h. After filtering off the insoluble solid, 25% ammonia (5.5 mL, 72.6 mmol) was added to the filtrate, followed by the addition of I2 (1.84 g, 7.26 mmol) in portions. The reaction was allowed to proceed at room temperature for 24 h. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-30%) to give 3.6 g of a slightly yellow, foamy solid product 33, with a yield of 37%. 1 H NMR(CDCl3,400MHz)δ8.87(br s,1H),7.82-7.74(m,4H),7.68-7.63(m,4H),7.50-7.29(m,9H),7.18(d,J=1.2Hz,1H),5.62(s,1H),4.93(d,J=11.8Hz,1H),4.85(s,1H ),4.76(d,J=11.8Hz,1H),4.69(s,1H),4.30(d,J=12.2Hz,1H),4.25(d,J=12.2Hz,1H),4.07(s,1H),1.58(d,J=1.2Hz,3H),1.08(s,9H); 13 C NMR(CDCl3,100MHz)δ163.31,149.51,135.49,135.36,133.37,133.33,133 .14,133.11,132.21,131.87,130.27,130.23,128.55,128.01,127.72,126 .90,126.43,126.35,125,44,114.18,111.17,90.08,87.22,78.12,76.00, 72.69,69.48,60.40,58.82,26.83,19.40,12.18; ESI-MS(m / z)674.28[M+H] + . 34

[0235] To a 50 mL ethanol solution of allyl nucleoside derivative 31 (4.9 g, 7.0 mmol), tris(triphenylphosphine)carbonylruthenium(II) hydrochloride (167 mg, 0.175 mmol, 2.5% mol) was added, and the mixture was heated to 80 °C and reacted for 36 h. The reaction solution was directly concentrated, and the residue was dissolved in 40 mL tetrahydrofuran. Then, 5 mL tert-butanol, 5 mL water, potassium osmium tetroxide dihydrate (26 mg, 70 μmol, 1% mol), and 50% N-methylmorpholine-N-oxide (2.2 mL, 10.5 mmol) were added sequentially, and the mixture was heated to 60 °C and reacted for 8 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: ethyl acetate containing 5% methanol / dichloromethane = 0-100%) to give 3.6 g of the dihydroxylated isomer mixture. The above-mentioned dihydroxylated product was dissolved in 40 mL of ethanol-tetrahydrofuran solution, and 8 mL of NaIO4 (1.58 g, 7.4 mmol) aqueous solution was added dropwise. The mixture was stirred at room temperature for 12 h. After filtering off the insoluble solid, 25% ammonia (3.05 mL, 49.2 mmol) was added to the filtrate, followed by the addition of I2 (1.25 g, 4.92 mmol) in portions. The reaction was allowed to proceed at room temperature for 24 h. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate containing 5% methanol / dichloromethane = 0-60%) to give 1.6 g of white foamy solid product 34, yield 48%. 1 H NMR(CDCl3,400MHz)δ8.22(s,1H),7.80-7.75(m,3H),7.70-7.66(m,6H),7.48-7.31(m,9H),5.96(s,1H),5.85(br s,2H),5.19(s,1H),4.88-4.77(m,3H),4.36(s,1H),4.32(d,J=12.2Hz,1H),4.19(d,J=12.2Hz,1H),1.06(s,9H); 13 C NMR (CDCl3, 100MHz) δ155.43,153.10,148.62,135.58,130.12,128.48,127.98,127.91,127.89,127.70,126.83,126. 33,125.47,119.91,114.52,89.84,86.36,78.22,75.52,72.86,70.11,59.39,29.70,19.27; ESI-MS(m / z)683.35[M+H] + . 35

[0237] To a 120 mL anhydrous ethanol solution of allyl nucleoside derivative 32 (7.2 g, 10 mmol), ruthenium(II) tris(triphenylphosphine)carbonyl hydrochloride (238 mg, 0.25 mmol, 2.5% mol) was added, and the mixture was heated to 80 °C and reacted for 48 h. The reaction solution was directly concentrated, and the residue was dissolved in 50 mL tetrahydrofuran. Then, 10 mL tert-butanol and 10 mL water, potassium osmium tetroxide dihydrate (36 mg, 10 μmol, 1% mol) and 50% N-methylmorpholine-N-oxide (4.0 mL, 20 mmol) were added sequentially, and the mixture was heated to 60 °C and reacted for 8 h. The reaction was confirmed to be complete by TLC (dichloromethane / methanol = 10 / 1). After quenching the reaction with saturated sodium sulfite solution, the product was extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-40%) to give 5.6 g of the dihydroxylated isomer mixture.

[0238] The above-mentioned dihydroxylated product was dissolved in 60 mL of ethanol-tetrahydrofuran solution, and 10 mL of NaIO4 (3.54 g, 7.35 mmol) aqueous solution was added dropwise. The mixture was stirred at room temperature for 12 h. After filtering off the insoluble solid, 25% ammonia (5.5 mL, 73.5 mmol) was added to the filtrate, followed by the addition of I2 (1.87 g, 7.35 mmol) in portions. The reaction was allowed to proceed at room temperature for 24 h. The reaction was quenched with saturated sodium sulfite solution, extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-40%) to give 2.5 g of white foamy solid product 35, yield 48%. 1 HNMR(CDCl3,400MHz)δ7.80-7.66(m,8H),7.58(s,1H),7.47-7.31(m,9H),5.86(s,1H),5.03(s,1H),4. 81-4.76(m,5H),4.34(s,1H),4.31(d,J=12.2Hz,1H),4.19(d,J=12.2Hz,1H),4.06(s,3H),1.06(s,9H); 13C NMR(CDCl3,100MHz)δ161.63,159.37,152.27,136.01,135.60,135.58,1 33.75,133.11,132.19,132.16,130.13,128.49,127.99,127.96,127.69, 126.67,126.31,126.23,125.38,115.99,114.61,89.67,86.10,78.31,77 .64,72.91,70.05,59.41,53.97,26.70,19.25; ESI-MS(m / z)713.40[M+H] + .

[0239] Synthesis of S-6'-CN-LNA phosphoramide monomer

[0240] Synthesis of S-6'-CN-LNA-T phosphoramide monomer

[0241]

[0242] The reaction conditions were as follows: (a)(1) DDQ, dichloromethane-H2O (20 / 1), room temperature, 24h; (2) Et3N, Et3N·3HF, THF, room temperature, 12h; (b) DMTrCl, pyridine, room temperature, 12h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphine diamine, 1H-tetrazolium tetrazolium, room temperature, 5h. 36

[0244] At room temperature, DDQ (420 mg, 1.86 mmol) was added to 42 mL of a dichloromethane-water (20:1) mixture containing nucleoside derivative 33 (3.6 g, 5.34 mmol). The reaction was allowed to proceed for 24 h at room temperature, and TLC analysis showed that the reaction was complete (dichloromethane / ethyl acetate = 10 / 1). The reaction solution was concentrated under reduced pressure, extracted with ethyl acetate, washed successively with saturated sodium sulfite solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was dissolved in 50 mL of tetrahydrofuran, and triethylamine (1.78 mL, 13.35 mmol) and triethylamine trihydrofluoric acid (2.61 mL, 16 mmol) were added sequentially. The reaction was allowed to proceed for 12 h at room temperature, and TLC analysis showed that the reaction was complete (dichloromethane / methanol = 10 / 1). 10g of sodium bicarbonate solid was added to the reaction solution and stirred until no bubbles were generated. The reaction solution was concentrated under reduced pressure and purified directly by flash column chromatography (gradient elution: methanol / dichloromethane = 0-20%) to obtain 1.1g of slightly yellow white solid 36, with a yield of 70%. 1H-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] + . 37

[0246] 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

[0248] 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] + .

[0249] Synthesis of S-6'-CN-LNA-A phosphoramide monomer

[0250]

[0251] The reaction conditions were: (a) BzCl, pyridine, room temperature, 6 h; (b)(1) DDQ, dichloromethane-H2O (20 / 1), room temperature, 24 h; (2) Et3N, Et3N·3HF, THF, room temperature, 12 h; (b) DMTrCl, pyridine, room temperature, 8 h; (c) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphine diamine, 1H-tetrazole, room temperature, 8 h. 39

[0253] Benzoyl chloride (422 μL, 3.66 mmol) was added dropwise to 5 mL of pyridine solution containing nucleoside 34 (1.0 g, 1.46 mmol) at room temperature. After reacting for 4 h at room temperature, benzoyl chloride (100 μL, 0.87 mmol) was added to the reaction solution, and the reaction was continued for 2 h. 15% NaOH was added dropwise to adjust the pH to 8, and the mixture was stirred at room temperature for 2 h. The solution was diluted with ethyl acetate, washed successively with water, 1N HCl solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-45%) to give 1.05 g of a slightly yellow, foamy solid, in 91% yield. 1H NMR (CDCl3, 400MHz) δ9.02 (brs, 1H), 8.65 (s, 1H), 8.03 (d, J = 7.2Hz, 2H), 7.99 (s, 1H), 7.79-7.34 (m, 20H), 6.03 (s, 1H), 5.19 (s,1H),4.89(s,1H),4.87(d,J=12.2Hz,1H),4.82(d,J=12.2Hz,1H),4.34-4.31(m,2H),4.19(d,J=12.2Hz,1H),1.06(s,9H); 13 C NMR(CDCl3,100MHz)δ164.60,152.64,150.42,149.70,140.19,135.55,133.53,133 .42,133.09,132.99,132.12,132.09,130.15,128.94,128.52,128.00,127.92,127. 91,127.85,127.69,126.80,126.38,126.28,125.42,123.29,114.42,90.04,86.43 ,78.19,77.54,72.97,70.17,59.42,29.70,26.70,19.26; ESI-MS(m / z)787.32[M+H] + . 40

[0255] At room temperature, DDQ (780 mg, 3.44 mmol) was added to 10 mL of a dichloromethane-water (20:1) mixture containing nucleoside derivative 39 (900 mg, 1.14 mmol). The reaction was allowed to proceed for 24 h at room temperature, and TLC analysis showed that the reaction was complete (dichloromethane / methanol = 10 / 1). The reaction solution was concentrated under reduced pressure, extracted with ethyl acetate, washed successively with saturated sodium sulfite solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was dissolved in 15 mL of tetrahydrofuran, and triethylamine (400 μL, 2.85 mmol) and triethylamine trihydrofluoric acid (550 μL, 3.42 mmol) were added sequentially. The reaction was allowed to proceed for 12 h at room temperature, and TLC analysis showed that the reaction was complete (dichloromethane / methanol = 10 / 1). Add 1.0 g of sodium bicarbonate solid to the reaction solution and stir until no bubbles are generated. Concentrate the reaction solution under reduced pressure and purify directly by flash column chromatography (gradient elution: dichloromethane / methanol = 0-20%) to obtain 340 mg of cyano-locked nucleoside 40, yield 73%. 1 H NMR(CD3OD,400MHz)δ; ESI-MS(m / z)409.19[M+H] + 431.16 [M+Na]+ . 41

[0257] At room temperature, 4,4'-dimethoxytriphenylmethylchloro (373 mg, 1.1 mmol) was added to 5 mL of a pyridine solution containing cyano-O-DMTr. The mixture was stirred at room temperature for 8 h, and the reaction was monitored by TLC until complete (dichloromethane / methanol = 10 / 1). The reaction was quenched with 1 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: ethyl acetate containing 5% methanol / dichloromethane = 0-80%) to give 330 mg of 5'-O-DMTr-protected cyano-O-DMTr-protected cyano-O-DMTr-protected cyano-O-DMTr as a pale yellow solid, yield 64%. 1 H NMR(DMSO-d6,400MHz)δ11.26(brs,1H),8.79(s,1H),8.54(s,1H),8.06(d,J=7.3Hz,2H),7. 67(t,J=7.3Hz,1H),7.57(t,J=7.8Hz,2H),7.46(d,J=7.4Hz,2H),7.34-7.24(m,6H),7.25(t ,J=7.2Hz,1H),6.90(d,J=8.9Hz,4H),6.33(d,J=3.5Hz,1H),6.23(s,1H),5.36(s,1H),4.92 (s,1H),4.70(d,J=3.5Hz,1H),3.85(d,J=11.1Hz,1H),3.74(s,6H),3.47(d,J=11.1Hz,1H); 13 C NMR(DMSO-d6,100MHz)δ166.10,158.64,152.33,152.02,150.97,145.07,142.33,135.88,135.38,130.25,130.19,128.98,128.96, 128.36,128.12,126.03,116.03,113.73,113.69,89.29,86.26,85.39,81.12,71.78,69.40,60.09,55.52; ESI-MS(m / z)711.37[M+H] + . 42

[0259] At room temperature, 1H-tetrazole (21 mg, 0.3 mmol) was added to 10 mL of a dichloromethane solution containing nucleoside 41 (240 mg, 0.34 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonamide (203 mg, 0.75 mmol). The reaction was allowed to proceed for 8 h at room temperature, and TLC was used to confirm the completeness of the reaction (dichloromethane / methanol = 15 / 1). The reaction solution was then purified directly by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-40%) to give 280 mg of phosphoramide 42, a slightly yellow, foamy solid, in 90% yield. 31 P NMR(152MHz,DMSO-d6)δ149.56,149.25; ESI-MS(m / z)909.38[MH] - .

[0260] Synthesis of S-6'-CN-LNA-G phosphoramide monomer

[0261]

[0262] The reaction conditions were as follows: (a) 2N HCl, THF-CH3OH (1 / 1), 60℃, 24h; (b) Isobutyryl chloride, Et3N, DMAP, toluene, 100℃, 12h; (c)(1) DDQ, dichloromethane-H2O (20 / 1), room temperature, 24h; (2) Et3N, Et3N·3HF, THF, room temperature, 12h; (d) DMTrCl, pyridine, room temperature, 12h; (e) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphine diamine, 1H-tetrazole, room temperature, 6h. 43

[0264] 15.4 mL of 2N hydrochloric acid solution was added to 24 mL of tetrahydrofuran-methanol solution (1 / 1 v / v) containing cyano-mononucleotide 35 (2.2 g, 3.08 mmol), and the mixture was heated to 60 °C and reacted for 24 h. The reaction was confirmed to be complete by TLC (dichloromethane / ethyl acetate = 2 / 1). The solution was extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: methanol / dichloromethane = 0-10%) to give 1.48 g of guanosine 43 as a white solid, yield 69%. 1 H NMR(CDCl3,400MHz)δ11.94(br s,1H),7.72-7.64(m,9H),7.48-7.16(m,10H),6.53(br s,2H),5.75(s,1H),4.81-4.70(m,3H),4.28-4.25(m,2H),4.17(d,J=12.2Hz,1H),1.03(s,9H); 13C NMR(CDCl3,100MHz)δ158.96,153.85,150.40,135.56,133.07,133.03,132.17,132.13,130.15,129.06,128.41,128.25,128.02,127.91,127.6 6,126.67,126.34,126.26,125.41,125.33,117.21,114.58,89.76,85. 91,78.28,72.87,69.98,59.39,26.72,19.28; ESI-MS(m / z)699.29[M+H] + . 44

[0266] DMAP (105 mg, 0.86 mmol) and triethylamine (718 μL, 5.16 mmol) were added to 12 mL of a toluene solution of guanosine 43 (1.2 g, 1.72 mmol), followed by isobutyryl chloride (550 μL, 5.16 mmol). The mixture was heated to 100 °C and reacted for 12 h. The reaction was confirmed to be complete by TLC (dichloromethane / methanol = 10 / 1). Heating was stopped, and the reaction solution was allowed to cool to room temperature. The reaction was quenched by adding 1 mL of methanol. The solution was extracted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-80%) to obtain 1.1 g of isobutyryl-protected guanosine 44, a slightly yellow, foamy solid, in 83% yield. 1 H NMR(CDCl3,400MHz)δ11.97(s,1H),8.78(s,1H),7.75-7.60(m,9H),7.44-7.32(m,9H),5.60(s,1H),4.79(d,J=12.0Hz,1H),4.69(s,1 H),4.65(d,J=12.0Hz,1H),4.62(s,1H),4.27(d,J=12.3Hz,1H),4.18-4.14(m,2H),2.68-2.61(m,1H),1.28-1.24(m,6H),1.06(s,9H); 13C NMR(CDCl3,100MHz)δ178.64,155.20,147.76,146.66,135.53,132.02,128.45,128.04,127.97,127.78,127.57,126.81,126.49 ,126.38,125.47,114.47,89.81,86.06,78.45,73.25,69.89,59.26,36.40,26.70,19.25,19.08,18.85; ESI-MS(m / z)767.39[MH] - . 45

[0268] At room temperature, DDQ (885 mg, 3.9 mmol) was added to 16 mL of a dichloromethane-water (20:1) mixture containing nucleoside derivative 19 (1.0 g, 1.3 mmol). The reaction was allowed to proceed for 24 h at room temperature, and TLC analysis showed that the reaction was complete (dichloromethane / methanol = 10 / 1). The reaction solution was concentrated under reduced pressure, extracted with ethyl acetate, washed successively with saturated sodium sulfite solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was dissolved in 10 mL of tetrahydrofuran, and triethylamine (453 μL, 3.25 mmol) and triethylamine trihydrofluoric acid (636 μL, 3.9 mmol) were added sequentially. The reaction was allowed to proceed for 12 h at room temperature, and TLC analysis showed that the reaction was complete (dichloromethane / methanol = 10 / 1). 700 mg of sodium bicarbonate solid was added to the reaction solution and stirred until no bubbles were generated. The reaction solution was concentrated under reduced pressure and purified directly by flash column chromatography (gradient elution: dichloromethane / methanol = 0-25%) to obtain 380 mg of cyano-locked nucleoside 45, with a yield of 75%. 1 H NMR(MeOH-d4,400MHz)δ8.03(s,1H),5.91(s,1H),4.88(s,1H),4.78(s,1H),4.45(s,1H),4.17(s,2H),2.74-2.67(m,1H),1.23(d,J=6.9Hz,6H); 13 C NMR(MeOH-d4,100MHz)δ181.72,150.01,149.42,137.97,121.52,116.12,91. 68,87.21,82.33,71.84,70.25,57.76,36.36,19.28; ESI-MS(m / z)389.09[MH] - . 46

[0270] At room temperature, 4,4'-dimethoxytriphenylmethylchloro (390 mg, 1.15 mmol) was added to 5 mL of pyridine solution containing cyano-O-DMTr. The mixture was stirred at room temperature for 12 h, and the reaction was monitored by TLC to ensure completeness (dichloromethane / MeOH = 5 / 1). The reaction was quenched with 2 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 obtain 520 mg of 5'-O-DMTr-protected cyano-O-DMTr-protected cyano-O-DMTr-protected cyano-O-DMTr as a slightly yellow, foamy solid, with a yield of 97%. 1 H NMR(DMSO-d6,400MHz)δ12.13(s,1H),11.86(s,1H),8.06(s,1H),7.45-7.22(m,9H),8.91(d,J=8.8Hz,4H),6.29(d,J=3.5Hz,1H),5.95(s,1H),5.2 9(s,1H),4.75(s,1H),4.48(d,J=3.5Hz,1H),3.86(d,J=11.2Hz,1H),3.74 (s,6H),3.46(d,J=11.2Hz,1H),2.82-2.75(m,1H),1.13(d,J=6.8Hz,6H); 13 C NMR(DMSO-d6,100MHz)δ180.74,158.67,155.20,148.86,145.08,136.74,135.82,135.37,130.26,130.21,128.35,128.11,127 .29,120.82,115.96,113.72,113.68,81.22,86.29,80.07,71.35,69.42,60.01,55.52,35.24,19.31; ESI-MS(m / z)691.36[MH] - . 47

[0272] At room temperature, 1H-tetrazole (31 mg, 0.44 mmol) was added to 10 mL of a dichloromethane solution containing guanosine 46 (380 mg, 0.55 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonamide (330 mg, 1.1 mmol). The reaction was allowed to proceed for 6 h at room temperature, and TLC was used to confirm the completeness of the reaction (dichloromethane / ethyl acetate = 2 / 1). The reaction solution was then purified directly by flash column chromatography (gradient elution: ethyl acetate / dichloromethane = 0-50%) to give 380 mg of phosphoramide 47, a slightly yellow waxy solid, in 77% yield. 31P NMR(152MHz,DMSO-d6)δ149.69,149.09; ESI-MS(m / z)891.42[MH] - .

[0273] S-6'-CN-LNA- m Synthesis of C phosphoramide monomer

[0274]

[0275] 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. 48

[0277] 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: methanol / dichloromethane = 0-15%) yielded 3.9 g of aminonucleoside 48, a pale yellow solid, in 96% yield. 1H 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

[0279] 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: ethyl acetate / dichloromethane = 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); 13C 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

[0281] 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: ethyl acetate / dichloromethane = 0-20%) to give 2.13 g of phosphoramide 50, a white, foamy solid, in 72% yield. 31 P-NMR(152MHz,DMSO-d6)δ149.23,147.95; ESI-MS(m / z)901.46[M+H] + .

[0282] Experimental Example 1: Cyanolocked Nucleic Acid Modification and ASO-Protein Interaction

[0283] Experimental methods

[0284] Thermal denaturation test (T) m value)

[0285] 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.

[0286] Immunofluorescence assay:

[0287] 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.

[0288] 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.

[0289] 3) Transfection: Add 10 μL of Lipofectamine 2000 (Soleb) to... 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.

[0290] 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.

[0291] 5) Blocking: The fixed cells were blocked at room temperature for 30 min with blocking buffer (prepared with 1 mg / ml BSA and PBS).

[0292] 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).

[0293] 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.

[0294] 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.

[0295] Caspase 3 / 7 activity assay

[0296] 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.

[0297] 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... 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.

[0298] 3) Transfection: Add 2 μL of Lipofectamine 2000 (Thermofisher) to... 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.

[0299] 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).

[0300] Experimental content

[0301] 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 1-3 .

[0302] 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.

[0303] 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.

[0304] 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 4(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.

[0305] Table 1. Oligonucleotide sequences ON1-4 and their corresponding mass spectrometry data

[0306]

[0307] 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.

[0308] Table 2. Percentage of cells in which LNA-ASO, R-CN-LNA-ASO, and S-CN-LNA-ASO significantly aggregated P54nrb to the nucleolus.

[0309]

[0310]

[0311] 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.

[0312] 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 5 As 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.

[0313] 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 can significantly reduce the impact of PS ASO on intracellular proteins and induce apoptosis toxicity, thereby improving the therapeutic effect of PS ASO. It has significant application value for nucleic acid drugs and can provide next-generation chemical modification technology support for nucleic acid drugs.

[0314] Experiment 2: Nuclease Tolerance Test of S-CN-LNA-T

[0315] 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), TThe 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 7 The 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.

[0316] 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.

[0317] 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 reaction was carried out sequentially by methanesulfonation, despinning, and acetylation to obtain Then, glycosylation and nucleophilic substitution reactions were carried out to synthesize the R configuration. ; The The synthesis method includes: using the R configuration The reaction was carried out sequentially by methanesulfonation, despinning, and acetylation to obtain Then, glycosylation and nucleophilic substitution reactions were carried out to synthesize the S configuration. ; The S configuration The synthesis methods include: 5- O -(tert-butyldiphenylsilyl)-4- C -Hydroxymethyl-1,2- O -Isopropylidene-3- O -(2-Naphthylmethyl)-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 60-80℃.

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 an R-configuration or S-configuration C6'-cyano-locked nucleic acid, characterized in that, The preparation method of the R-configuration C6'-cyanolocked nucleic acid T-phosphamide monomer includes: synthesizing the R-configuration using the synthetic method of claim 1. The resulting R configuration will then be synthesized. The 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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. ; The preparation method of the S-configuration C6'-position cyanolocked nucleic acid T-phosphamide monomer 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 deprotected 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. .

5. A method for synthesizing a phosphoramide monomer of a C6'-cyano-locked nucleic acid with R or S configuration, characterized in that, The method for preparing the R-configuration C6'-cyanolocked nucleic acid A phosphoramide monomer includes: synthesizing the R-configuration using the synthetic method of claim 1. The resulting R configuration will then be synthesized. To obtain by protecting the bases Then, the 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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. ; The preparation method of the S-configuration C6'-position cyanolocked nucleic acid A phosphoramide monomer includes: synthesizing the S-configuration using the synthesis method of claim 1. The synthesized S configuration will then be... To obtain by protecting the bases Then, the 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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. .

6. A method for synthesizing a phosphoramidite monomer of a C6'-cyano-locked nucleic acid with R or S configuration, characterized in that, The preparation method of the R-configuration C6'-cyanolocked nucleic acid G phosphoramide monomer includes: synthesizing the R-configuration using the synthetic method of claim 1. The resulting R configuration will then be synthesized. The methoxy group was demethylated to obtain Then, base protection is performed to obtain Then, the 3'-hydroxyl and 5'-hydroxyl groups were deprotected to obtain Then, the 5'-hydroxyl group is protected with DMTr, and the 3'-hydroxyl group is subjected to phosphoramidation to obtain the R configuration 8. ; The preparation method of the S-configuration C6'-position cyanolocked nucleic acid G phosphoramide monomer 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, base protection is performed to obtain Then, the 3'-hydroxyl and 5'-hydroxyl groups were deprotected 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. A method for synthesizing a C6'-cyanophosphamide monomer of an R-configuration or S-configuration cyanolocked nucleic acid, characterized in that, The method for preparing the R-configuration C6'-cyano-modified dC phosphoramide monomer 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 deprotected 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. ; The preparation method of the S-configuration C6'-cyanolocked nucleic acid C-phosphamide monomer 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 deprotected 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. .

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