Synthesis of 5-nucleotide dithiophosphamide and application of 5-nucleotide dithiophosphamide in oligonucleotide

Abstract

The invention discloses synthesis of 5-nucleotide phosphorodithioamide and application of the 5-nucleotide phosphorodithioamide in oligonucleotide. The oligonucleotide modified by the phosphorodithioamide comprises nucleotide structural units shown in a formula I or a formula II,..., the formula I,..., the formula II. According to the invention, through specific limitation of the structure of a nucleotide dithiophosphoramide monomer, the prepared oligonucleotide comprises a 5-nucleotide dithiophosphoramide structural unit. Compared with a traditional oligonucleotide molecule, the oligonucleotide molecule modified by nucleotide dithiophosphoramide shows better enzymatic degradation resistance and pharmaceutical stability. In addition, the binding performance between the modified oligonucleotide molecules and environmental ions can be improved through nucleotide dithiophosphamide modification, and a novel chemical modification strategy is provided for research, development and application of oligonucleotide drugs.

Description

Technical Field

[0001] This invention relates to the field of oligonucleotide technology, specifically to the synthesis of 5'-nucleotide dithiophosphoramide and its application in oligonucleotides. Background Technology

[0002] Oligonucleotide drugs bind specifically to target genes through base pairing, regulating or knocking down upstream mRNAs of pathogenic proteins, thus affecting target gene expression at the post-transcriptional level. Oligonucleotide drugs offer advantages such as abundant targets, long-lasting efficacy, short development cycles, and high success rates, providing solutions for the treatment of many intractable diseases.

[0003] In addition, oligonucleotides are widely used in molecular biology research, disease diagnosis and treatment, such as as PCR primers, gene editing tools or targeted drugs.

[0004] Traditional oligonucleotide sequences composed of nucleotide monomers have poor stability and are easily degraded in organisms, affecting their biological activity.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a 5'-dithionucleotide-derived phosphoramide-modified oligonucleotide and its synthesis process. By preparing nucleotide thiophosphoramide, it can be made to have a specific dithiophosphoramide-modified nucleotide structure, which can improve the stability and biological activity of the modified oligonucleotide.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: The first aspect of this invention provides a 5'-dithionucleotide-derived phosphoramide-modified oligonucleotide, wherein the dithiophosphoramide-modified oligonucleotide comprises a nucleotide structural unit represented by Formula I or Formula II: ...Formula I; In Formula I: Base is a nucleotide base or its derivative, wherein the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, etc., or its derivatives. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R3 is hydrogen, alkyl (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl); ...Formula II; In Formula II: Base is a nucleotide base or its derivative, wherein the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, etc., or its derivatives. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, etc., respectively.

[0008] The synthesis of the 5´-nucleotide dithiophosphoramide-modified oligonucleotides uses nucleotide dithiophosphoramide monomers including those shown in Formula III or Formula IV: ...Formula III; In Formula III: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, and its derivatives. PG is triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl). R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl). ...Form IV; In Equation IV: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, and its derivatives. PG is triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl). R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, etc., respectively.

[0009] Preferably, the protecting group is selected from one of triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl.

[0010] Preferably, the nucleotide dithiophosphoramide monomer comprises: , ; Where: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, and its derivatives. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl). R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, etc., respectively.

[0011] Preferably, the nucleotide dithiophosphoramide monomer comprises: , , , , , , , , , , , , , , Wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively; R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl).

[0012] Preferably, the nucleotide dithiophosphoramide monomer comprises: , , , , , , , , , , , , , , Wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic groups, respectively.

[0013] Preferably, the nucleotide dithiophosphoramide monomer comprises: , , , , , , , , , , , , , , Wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic groups, respectively.

[0014] Preferably, the O3'-protecting group 4,4'-dimethoxytriphenylmethyl (DMTr) in the nucleotide phosphoramide monomer described above is replaced with triphenylmethyl (Tr), 4-methoxytriphenylmethyl (MMTr) or 4,4',4''-trimethoxytriphenylmethyl (TMTr).

[0015] The nucleotide monomers described above are prepared by reacting nucleotides with 5'-mercapto-O3'-protecting groups and thiophosphoramide dichloride in the liquid phase.

[0016] The modification strategy proposed in this invention, in which oligonucleotides include nucleotide structures modified with 5´-dithiophosphoramide, can improve the anti-enzymatic degradation activity and biological stability of oligonucleotides, and enhance their biological activity.

[0017] A second aspect of the present invention provides a synthetic process for 5'-dithiophosphoramide-modified oligonucleotides, the synthetic process comprising the following steps: (a) Load nucleotide monomers onto a solid support, then remove the 3' protecting group from the nucleotide monomers and wash them; (b) A nucleotide monomer is pumped into the washed system for coupling, followed by capping, removal of the 3' protecting group on the nucleotide monomer, and washing. The nucleotide monomer is a phosphoramide nucleotide monomer or a phosphorous acid nucleotide monomer. If the nucleotide monomer is a phosphorous acid nucleotide monomer, after the coupling reaction, the trivalent phosphite is converted to a pentavalent phosphate ester by oxidation or sulfidation, followed by capping, removal of the 3' protecting group on the nucleotide monomer, and washing. (c) Repeat step (b) to obtain oligonucleotides with phosphoramide-modified nucleotide structures.

[0018] Compared with the prior art, the beneficial effects of the present invention include at least the following: This invention achieves the preparation of oligonucleotides containing a 5'-thiol-bound thiophosphoramide structural unit by specifically defining the structure of a 5'-dithionucleotide-derived phosphoramide. Compared with traditional oligonucleotide molecules, the dithiophosphoramide-modified oligonucleotides proposed in this invention exhibit superior pharmaceutical effects in terms of resistance to enzymatic degradation, stability, and binding, providing a novel chemical modification strategy for the research and application of oligonucleotide drugs. Detailed Implementation

[0019] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0020] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] In this invention, the substances corresponding to the abbreviations are as follows: PS: Polystyrene; CPG: Controlled pore glass; DBU: 1,8-Dazabicycloundec-7-ene; P(III): Trivalent phosphorus (phosphine); P(V): Pentavalent phosphorus (phosphine); F: Fluorine; Me: Methyl; Alkyl: Alkyl; Allyl: Allyl; Alloc: Allyloxycarbonyl; Ac: Acetyl; Bn: Benzyl; Bz: Benzoyl; Tr: Triphenylmethyl; MMTr: Methoxytriphenylmethyl; DMTr: Dimethoxytriphenylmethyl; TMTr: Trimethoxytriphenylmethyl; iBu: 2-Isobutyryl; NPE: 4-Nitrophenylethoxy; Ph: Aromatic; TMS: Trimethylsilyl; TBDMS: Tert-butyldimethylsilyl; TBDPS: Tert-butyldiphenylsilyl; DCM: Dichloromethane; DMF: N,N-Dimethylformamide; DMAP: 4-Dimethylaminopyridine; TBAF: Tetrabutylammonium fluoride; THF: Tetrahydrofuran; TFA: Trifluoroacetic acid; iPrOH: Isopropanol; TLC: Thin-layer chromatography; A: Adenine Adenine; G: Guanine; C: Cytosine; T: Thymine; U: Uracil; I: Hypoxanthine; Xan: Xanthine; m5C: 5-methylcytosine; S5´: 5´-thiosubstituted; HPLC: High performance liquid chromatography.

[0022] Example 1 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, adenine phosphoramide, A-1:((2S,3S,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-4-methoxytetrahydrofuran-2-yl)methyl dimethylphosphoramidochloridodithioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 352 mg of O2'-Me-O3'-DMTr-5'-SH-N6-Bz-A (A-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Subsequently, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphothiocarbamate (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added. The mixture was stirred at 0 °C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure. 50 mL of ethyl acetate was added to the residue, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 259 mg of the target product A-1 (purity above 99%, yield 61%).

[0023] Example 2 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, uracil phosphoramide, U-1-1:((2S,3S,4R,5R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)methyl dimethylphosphoramidochloridodithioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 288 mg of O2'-Me-O3'-DMTr-5'-SH-U (U-1-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Subsequently, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphothiocarbamate (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added. The mixture was stirred at 0 °C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure. 50 mL of ethyl acetate was added to the residue, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 233 mg of the target product U-1-1 (purity above 99%, yield 65%).

[0024] Example 3 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, thymine phosphoramide, T-1-1:((2S,3S,4R,5R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-4-methoxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl dimethylphosphoramidochloridodithioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 295 mg of O2'-Me-O3'-DMTr-5'-SH-T (T-1-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Subsequently, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphothiocarbamate (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added. The mixture was stirred at 0 °C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure. 50 mL of ethyl acetate was added to the residue, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 223 mg of the target product T-1-1 (purity above 99%, yield 61%).

[0025] Furthermore, the following nucleotide dithiophosphoramide monomers were prepared using the synthetic method described above: , , , , , , , , , , .

[0026] Example 4 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, uracil phosphoramide, U-2-1:((2S,3R,4R,5R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluorotetrahydrofuran-2-yl)methyl dimethylphosphoramidochloridodithioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 282 mg of 2'-F-O3'-DMTr-5'-SH-U (U-2-O, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Subsequently, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphothiocarbamate (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added. The mixture was stirred at 0 °C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure. 50 mL of ethyl acetate was added to the residue, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 212 mg of the target product U-2-1 (purity above 99%, yield 60%).

[0027] Furthermore, the following nucleotide dithiophosphoramide monomers were prepared using the synthetic method described above: , , , , , , , , , , , , .

[0028] Example 5 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, thymidine phosphoramide, T-2-1:((2S,3S,5R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl dimethylphosphoramidochloridodithioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 280 mg of O3'-DMTr-5'-SH-T (T-2-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Next, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphothiocarbamate (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added sequentially. The mixture was stirred at 0 °C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure. 50 mL of ethyl acetate was added to the residue, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 229 mg of the target product T-2-1 (purity above 99%, yield 65%).

[0029] Furthermore, the following nucleotide dithiophosphoramide monomers were prepared using the synthetic method described above: , , , , , , , , , , , , .

[0030] Furthermore, the following nucleotide dithiophosphoramide monomers were prepared using the synthetic method described above: , ; in: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, and its derivatives. R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl). R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, etc., respectively.

[0031] Furthermore, the following nucleotide dithiophosphoramide monomers were prepared using the synthetic method described above: , ; in: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, and its derivatives. PG is triphenylmethyl (Tr), 4-methoxytriphenylmethyl (MMTr), 4,4'-dimethoxytriphenylmethyl (DMTr), 4,4',4''-trimethoxytriphenylmethyl (TMTr), allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl). R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl). R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, ester, acyloxy, amide, aminoacyl, etc., respectively.

[0032] Example 6 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, uracil phosphoramide, U-4-1:O-(((2R,3R,4R,5R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)methyl) dimethylphosphoramidochloridothioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 280 mg of O2'-Me-O3'-DMTr-U (U-4-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Next, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphothiocarbamate (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added sequentially. The mixture was stirred at 0 °C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure. 50 mL of ethyl acetate was added to the residue, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 232 mg of the target product U-4-1 (purity above 99%, yield 66%).

[0033] Furthermore, the following nucleotide monomers were prepared using the synthetic method described above: , ; in: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, and its derivatives. PG is triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl). R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl). R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, etc., respectively.

[0034] Example 7 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, uracil phosphoramide, U-6-1: O-((2S,3R,4R,5R)-2-(((bis(4-methoxyphenyl)(phenyl)methyl)thio)methyl)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluorotetrahydrofuran-3-yl) dimethylphosphoramidochloridothioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 282 mg of 2'-F-S5'-DMTr-U (U-6-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Next, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphothiocarbamate (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added sequentially. The mixture was stirred at 0 °C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure. 50 mL of ethyl acetate was added to the residue, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 211 mg of the target product U-6-1 (purity above 99%, yield 60%).

[0035] Example 8 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, thymidine phosphoramide, T-6-1: O-((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-methoxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl) dimethylphosphoramidochloridothioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 287 mg of O2'-Me-O5'-DMTr-T (T-6-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Next, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphothiocarbamate (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added sequentially. The mixture was stirred at 0 °C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure. 50 mL of ethyl acetate was added to the residue, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 202 mg of the target product T-6-1 (purity above 99%, yield 56%).

[0036] Furthermore, the following nucleotide monomers were prepared using the synthetic method described above: , ; in: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, and its derivatives. PG is triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl). Y represents oxygen (O), sulfur (S), selenium (Se), or amine; R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl). R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, etc., respectively.

[0037] Cross-linked polystyrene (PS) was used as the support for solid-phase synthesis. 0.050 g of the modified solid-phase synthesis resin was weighed and added to a solid-phase synthesis tube. 2.0 mL of NMP was added, and the mixture swelled at room temperature for 2 hours, then the solution was drained by filtration. The resin was washed with 3.0 mL of dichloromethane, and this washing process was repeated three times to begin solid-phase synthesis.

[0038] The monomers introduced in solid-phase synthesis are phosphoramide P(III) and phosphoramide P(V).

[0039] The process conditions for phosphorus amide monomers are as follows: Reagents and monomers: Prepare a 0.15 mol / L monomer / acetonitrile solution; the deprotecting agent for DMTr (or Tr) is a 5% dichloroacetic acid / toluene solution (v / v); the activator is a 0.25 mol / L 5-ethylthiotetrazole / acetonitrile solution; the oxidizing agent is a 0.05 mol / L iodine-water / pyridine (10 / 90, v / v) solution; the sulfiding agent is a 3% hydroflavin / pyridine solution; capping agent A is a 10% acetic anhydride / acetonitrile solution (v / v), and capping agent B is a 1-methylimidazolium / pyridine / tetrahydrofuran solution (16 / 10 / 74, v / v / v).

[0040] The operational sequence for the solid-phase synthesis of phosphorous amide monomers is as follows: 1) Deprotection: 5% dichloroacetic acid toluene solution was used to remove the DMTr protecting group, followed by washing with acetonitrile; 2) Coupling: Using 0.25 mol / L 5-ethylthiotetrazole as the activator, according to the sequence design, nucleotide monomers / acetonitrile solutions were fed into the cycle for coupling, followed by rinsing with acetonitrile; 3) Oxidation / Sulfidation: Oxidation was performed using a 0.05 mol / L iodine solution in water / pyridine (v / v, 90 / 10) as the oxidant, followed by rinsing with acetonitrile. Sulfidation was performed using a 3% hydroflavin pyridine solution as the sulfiding agent, followed by rinsing with acetonitrile. 4) Capping: Capping agent A and capping agent B are used as capping reagents to protect the unreacted active groups, followed by rinsing with acetonitrile.

[0041] The process conditions for applying phosphoramide monomers to the solid-phase synthesis of oligonucleotides differ from those for the phosphoramide monomers described above as follows: Coupling solvent: 2.303 g of N-ethylmorpholine was diluted to 50 mL with 1,3-dimethyl-2-imidazolidinone (DMI) to obtain an N-ethylmorpholine DMI solution (0.40 mol / L), which was used as the coupling solvent.

[0042] Lysis buffer: Weigh 0.154 g of dithiothreitol, add 5 mL of N-methyl pyrrolidone (NMP) to dissolve, add 0.739 g of triethylamine, and dilute to 10 mL with NMP to obtain an NMP solution containing 0.10 mol / L dithiothreitol and 0.73 mol / L triethylamine, which is the lysis buffer. Prepare fresh before use.

[0043] The solid-phase synthesis process of phosphoramide monomers includes: Following the base sequence of the target sequence, nucleotide monomers were coupled one by one through a cyclical process of de-DMTr, neutralization, washing, coupling, neutralization, and washing. After the last monomer molecule was coupled, the terminal DMTr was removed, and 3.0 mL of p-methoxytriphenylmethyl chloride end-capping solution (0.32 mol / L) was added to the reaction tube. After reacting at 45 °C for 90 minutes, the reaction solution was drained, and the tube was washed 8 times with 4.0 mL of NMP each time.

[0044] Repeat the above operations according to the set sequence to obtain the oligonucleotide product of the target sequence.

[0045] Deprotection involves transferring the solid-phase support loaded with the oligonucleotide product into a reactor, adding concentrated ammonia (28%), controlling the temperature at 60 °C for 12 hours of ammonolysis, cooling to room temperature, filtering, washing the solid with a mixture of purified water and ethanol, combining the filtrates, and concentrating under reduced pressure at low temperature to obtain the crude oligonucleotide product with the designed sequence.

[0046] Purification involves dissolving the deprotected crude product in purified water, purifying it using HPLC, collecting the product peak solution, and then desalting, concentrating under reduced pressure, and lyophilizing the product to obtain the final product.

[0047] The above solid-phase synthesis methods are also applicable to monomers protected by Tr, MMTr, and TMTr.

[0048] In examples of solid-phase synthesis, the nucleotide monomers used are represented by the following structures: , , , , , , , , , , , .

[0049] Example 9 This embodiment describes an oligonucleic acid with a phosphoramide-modified nucleotide structure. The nucleotide monomers loaded on the solid support are T-3-0, and the phosphoramide-modified nucleotide monomers are U-1-1 and U-4-1. The synthesis reaction formula is ; Methods for synthesizing oligonucleotides with phosphoramide-modified nucleotide structures include: Monomer T-3-0 was loaded onto cross-linked polystyrene (PS). O3'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-1-1 was pumped in for coupling. The system was then capped, and O3'-DMTr was removed again using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-4-1 was pumped in for coupling, and O3'-DMTr was removed again using a 5% dichloroacetic acid-toluene solution.

[0050] After ammonolysis and deprotection treatment, the crude product of the loaded oligonucleotide molecule was concentrated under reduced pressure and purified by HPLC. The main fraction was collected, desalted, concentrated and freeze-dried to obtain the product, which was confirmed by detection to be the nucleic acid molecule of the target sequence.

[0051] Example 10 This embodiment describes an oligonucleic acid with a phosphoramide-modified nucleotide structure. The nucleotide monomers loaded on the solid support are T-3-0 and the phosphoramide-modified nucleotide monomers are U-2-1. The synthesis reaction formula is ; Methods for synthesizing oligonucleotides with phosphoramide-modified nucleotide structures include: Monomer T-3-0 was loaded onto cross-linked polystyrene (PS). O3'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-2-1 was pumped in for coupling. The system was then capped, and O3'-DMTr was removed again using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-2-1 was pumped in for coupling, and O3'-DMTr was removed again using a 5% dichloroacetic acid-toluene solution.

[0052] After ammonolysis and deprotection treatment, the crude product of the loaded oligonucleotide molecule was concentrated under reduced pressure and purified by HPLC. The main fraction was collected, desalted, concentrated and freeze-dried to obtain the product, which was confirmed by detection to be the nucleic acid molecule of the target sequence.

[0053] Example 11 This embodiment uses an oligonucleic acid with the same structure as that in Example 10. The nucleotide monomers loaded on the solid support are U-6-0, and the phosphoramide-modified nucleotide monomers are U-6-1 and T-6-1. The synthesis reaction formula is ; Methods for synthesizing oligonucleotides with phosphoramide-modified nucleotide structures include: Monomer U-6-0 was loaded onto cross-linked polystyrene (PS). S5'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-6-1 was pumped in for coupling. The system was then capped, and S5'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-6-1 was pumped in for coupling, and O5'-DMTr was removed using a 5% dichloroacetic acid-toluene solution.

[0054] After ammonolysis and deprotection treatment, the crude product of the loaded oligonucleotide molecule was concentrated under reduced pressure and purified by HPLC. The main fraction was collected, desalted, concentrated and freeze-dried to obtain the product, which was confirmed by detection to be the nucleic acid molecule of the target sequence.

[0055] Example 12 This embodiment describes an oligonucleic acid with a phosphoramide-modified nucleotide structure. The nucleotide monomers loaded on the solid support are T-3-0, the phosphoramide-modified nucleotide monomers are U-1-1, and the nucleotide phosphorylate is Af-1. The synthesis reaction formula is ; Methods for synthesizing oligonucleotides with phosphoramide-modified nucleotide structures include: Monomer T-3-0 was loaded onto cross-linked polystyrene (PS). O3'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-1-1 was pumped in for coupling. The system was then capped, and O3'-DMTr was removed again using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer Af-1 was pumped in for coupling, followed by iodine / pyridine oxidation, and O3'-DMTr was removed again using a 5% dichloroacetic acid-toluene solution.

[0056] After ammonolysis and deprotection treatment, the crude product of the loaded oligonucleotide molecule was concentrated under reduced pressure and purified by HPLC. The main fraction was collected, desalted, concentrated and freeze-dried to obtain the product, which was confirmed by detection to be the nucleic acid molecule of the target sequence.

[0057] Example 13 This embodiment is an oligonucleotide with a phosphoramide-modified nucleotide structure. During the preparation process, the nucleotide monomer loaded on the solid support is U-3-0, and the phosphoramide-modified nucleotide monomers are U-1-1 and T-1-1.

[0058] The structural formula of oligonucleotides is ; Methods for synthesizing oligonucleotides with phosphoramide-modified nucleotide structures include: Monomer U-3-0 was loaded onto cross-linked polystyrene (PS). O3'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-1-1 was pumped in for coupling. After capping, O3'-DMTr was removed again using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-1-1 was pumped in, and this cycle was repeated five times. After washing with acetonitrile, monomer T-1-1 was pumped in for coupling, and O3'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. Following solid-load shearing, deprotection, HPLC purification, and desalting, a phosphoramide-modified oligonucleotide molecule with the designed sequence structure was obtained, denoted as ON7-P2S6-2.

[0059] Example 14 This comparative example is an oligonucleotide with the same base sequence as ON7-P2S6-2. During preparation, the nucleotide monomer loaded on the solid-phase support was T-2-0, and the nucleotide monomer used was nucleotide phosphorylate U-5-1. The structural formula of the oligonucleotide is as follows: This oligonucleotide, denoted as ON7, was synthesized using a conventional solid-phase synthesis method.

[0060] Example 15 The isoelectric points of the oligonucleotides ON7-P2S6-2 prepared in Example 13 and ON7 prepared in Example 14 were tested. The results showed that the isoelectric point of the oligonucleotide ON7 was 5.03 (±0.12), and the isoelectric point of the phosphoramide-modified oligonucleotide ON7-P2S6-2 was 7.78 (±0.23). This indicates that phosphoramide modification can improve the binding affinity of the modified oligonucleotide to anions.

[0061] Example 16 The stability of ON7-P2S6-2 prepared in Example 13 and ON7 prepared in Example 14 in human serum and human liver microsomes were tested respectively.

[0062] Test results show that: The oligonucleotide molecule ON7 without phosphoramide modification showed significant degradation, with the proportion of undegraded nucleic acid sequences falling below 5% after 4 hours. In contrast, the proportion of undegraded oligonucleotide chain ON7-P2S6-2 containing phosphoramide-modified nucleotides was greater than 80% after 24 hours of incubation in human serum or human liver microsomes, indicating that the modified oligonucleotide molecule has a stronger resistance to biological enzyme degradation.

[0063] 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 therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. The synthesis of 5'-nucleotide phosphorodithioamides and their use in oligonucleotides, characterized in that, The phosphorodithioate-modified oligonucleic acid comprises a nucleotide structural unit shown in Formula I or Formula II: … Formula I; In Formula I: Base is a nucleotide base or its derivative, which is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil, and the like, and its derivative; R is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, aryl; R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aryl; R3 is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl, siloxymethylene, alkoxyalkyl, benzyl or acyl; … Formula II; In Formula II: Base is a nucleotide base or its derivative, which is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil, and the like, and its derivative; R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aryl; R4 and R5 are respectively hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amine, thiol, alkylthio, seleno, alkylseleno, ester, acyloxy, amido, amidoacyl, and the like.

2. A process for the synthesis of 5'-phosphorothioate nucleotides and their use in oligonucleotide synthesis according to claim 1, characterized in that, The phosphorodithioate-modified oligonucleic acid comprises a nucleotide structural unit shown in Formula I or Formula II: … Formula III; In Formula III: Base is a nucleotide base or its derivative, which is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil, and the like, and its derivative; PG is trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4''-trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aryl; R3 is alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, and the like; … Formula IV; In Formula IV: Base is a nucleotide base or its derivative, which is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil, and the like, and its derivative; PG is trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4''-trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aryl; R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azido, amine, mercapto, alkylthio, seleno, alkylseleno, ester, acyloxy, amido, amidoacyl, and the like.

3. A synthesis of 5'-nucleotidyl dithiophosphoramidates and their use in oligonucleotides according to claim 2, characterized in that, The nucleotide monomer includes but is not limited to: 、 、 、 、 、 、 、 、 、 、 、 、 、 ， wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aryl, respectively; R3 is alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl, silyloxy methylene, alkoxyalkyl, benzyl or acyl.

4. A process for the synthesis of 5'-phosphorothioate nucleotides and their use in oligonucleotide synthesis according to claim 2, characterized in that, The nucleotide monomer includes but is not limited to: 、 、 、 、 、 、 、 、 、 、 、 、 、 ， wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aryl, respectively.

5. A process for the synthesis of 5'-phosphorothioate nucleotides and their use in oligonucleotide synthesis according to claim 2, characterized in that, The nucleotide monomer includes but is not limited to: 、 、 、 、 、 、 、 、 、 、 、 、 、 ， wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aryl, respectively.

6. A process for the synthesis of 5'-nucleotidyl dithiophosphoramidates according to claim 2 and their use in oligonucleotides, characterized in that, The nucleotide monomer includes but is not limited to:

7. A process for the synthesis of 5'-nucleotidyl dithiophosphoramidates according to claim 1 and their use in oligonucleotides, characterized in that, wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aryl, respectively.

8. The process for the synthesis of 5'-nucleotide phosphorodithioate modified oligonucleic acids according to any one of claims 1 to 7, characterized in that, The nucleotide phosphoramidite is prepared by the reaction of 5'-thio-O3'-protecting group nucleotide molecule and thiophosphoramidite dichloride; the 5'-dithio nucleotide-derived phosphoramidite according to any one of claims 3-5 is characterized in that the O3'-protecting group 4,4'-dimethoxytrityl (DMTr) in the nucleotide monomer is replaced by trityl (Tr), 4-methoxytrityl (MMTr) or 4,4',4''-trimethoxytrityl (TMTr). The modified oligonucleic acid includes a 5'-dithio nucleotide-derived phosphoramidite modified structure, which can improve the anti-enzyme degradation activity and biological stability of the oligonucleic acid, and at the same time improve the binding performance between the oligonucleic acid molecule and anions. The synthesis process includes the following steps: (a) loading the nucleotide monomer on the solid phase carrier, then removing the 3' protecting group of the nucleotide monomer and washing; 9. A synthesis of 5'-nucleotidyl dithiophosphoramidates according to claim 1 and their use in oligonucleotides, characterized in that, (b) pumping the nucleotide monomer into the system after washing for coupling, then performing capping treatment, removing the 3' protecting group of the nucleotide monomer and washing, wherein the nucleotide monomer is a phosphoramidite nucleotide monomer or a phosphoramidite nucleotide monomer; if the nucleotide monomer is a phosphoramidite nucleotide monomer, after the coupling reaction, the trivalent phosphite is converted into pentavalent phosphate by oxidation or sulfurization, then capping, removing the 3' protecting group of the nucleotide monomer and washing are performed; 10. The oligonucleic acid of claim 1, wherein (c) repeating step (b) to obtain an oligonucleic acid with a phosphoramidite modified nucleotide structure. The oligonucleic acid molecule prepared by the coupling between the 5'-thio S5'-protecting group nucleotide monomer and the O3'-thiophosphoramidite nucleotide monomer includes the structural unit of formula I or formula II shown in claim 1. The oligonucleic acid is selected from any one or a combination of at least two of small interfering nucleotides, antisense oligonucleic acids, microRNAs, small activating RNAs, small guide RNAs, transfer RNAs, guide RNAs and aptamers.

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