Synthesis of 5-terminal phosphonic acid nucleotide and application of 5-terminal phosphonic acid nucleotide in oligonucleotide

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses synthesis of 5-terminal phosphonic acid nucleotide and application of the 5-terminal phosphonic acid nucleotide in oligonucleotide. The chemical modification strategy comprises the following steps: preparing a 5 '-phosphonite modified nucleotide phosphoramidite monomer and a 5'-phosphonite modified nucleotide phosphoramidite monomer; the modified nucleotide phosphoramidite is used for preparing a target oligonucleotide molecule, carbon-phosphonic acid C-PO32-chemical bond connection is constructed at the 5-terminal of the target oligonucleotide molecule, a 5 '-terminal phosphonic acid structure in a non-natural form is formed and does not belong to a natural substrate of phosphatase, the modified oligonucleotide can resist nuclease degradation, the affinity of the oligonucleotide with specific protein can be improved, and the oligonucleotide can be used for preparing the target oligonucleotide. And the in-vivo biological activity of the siRNA is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the preparation of a 5'-terminal phosphonate-modified nucleotide monomer and its application in oligonucleotides. Background Technology

[0002] Oligonucleotide drugs bind specifically to target genes through base complementarity, expanding the drug target to upstream mRNA of pathogenic proteins. They regulate or knock down target genes, affecting their 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] Chemical modification is one of the effective strategies for enhancing oligonucleotide drug delivery and is widely used to improve drug properties to enhance delivery efficiency. Exonucleases act on the phosphate groups at the ends of nucleic acid chains, leading to the degradation of small nucleic acid drugs. Therefore, 5'-terminal modification of nucleic acid sequences can increase drug resistance to nucleases.

[0004] In siRNA double strands, the 5'-terminus of the antisense strand must contain a phosphate group to specifically bind to the side chain residues of the MID domain in the RNA-induced silencing complex Argonaute2 (Ago2) effector protein, which is crucial for RNAi activity. Currently, phosphate analogs that are pre-phosphorylated at the 5' end through chemical synthesis can be used as metabolically stable phosphate analogs.

[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 synthesis of 5'-terminal phosphononucleotides and their application in oligonucleotides. Specifically, a novel modification strategy is used to phosphonate the 5'-terminus of the nucleotide, and the modified phosphoramidite monomer is used to prepare nucleic acid drugs.

[0007] To address the aforementioned problems in the prior art, the present invention is achieved through the following technical solution: Synthesis of a 5'-terminal phosphonate-modified nucleotide and its application in oligonucleotides, the chemical modification strategies used include: Preparation of nucleotide phosphoramid monomers with C4' linked to phosphonates via spacer group Y; Preparation of nucleotide phosphoramid monomers with C4' linked to phosphonic acid via spacer group Y; The two modified nucleotide phosphoramide monomers mentioned above were used in the preparation of oligonucleotides to construct 5'-terminal phosphonic acid modified oligonucleotide molecules; At the 5'-terminus of the oligonucleotide molecule, the C4' end is linked to a phosphonic acid via a spacer group Y, forming a C4'-YP(V) fragment, as shown in Formula 1 or Formula 2: Formula 1; Formula 2; in: Base: Nucleotide bases such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine and their derivatives; Y: One or more methylene CH2 groups, alkyl groups with branched structures, alkylene groups, mesylate groups, cycloalkyl groups, alkenyl groups, cycloalkenyl groups, alkynyl groups, alkoxyalkyl groups; R: Hydrogen, alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, thiocarbonylalkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl, etc., thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R´, R´´: Hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.

[0008] Prepare nucleotide monomers in which C4' is linked to phosphonite P(III) via a spacer group, denoted by Y, including structures shown in Formula 3 or Formula 4: Formula 3; Equation 4; in: Base: Nucleotide bases containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; Y: One or more methylene CH2 groups, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups; LG1, LG2, LG3: Cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenyl. Silicon-based groups include: LG4: halogen, amino, cyano, azide, alkyloxy, etc.; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl, thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R´, R´´: hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.

[0009] Preferably, after the 5'-end of the nucleotide monomer undergoes a substitution reaction with hypophosphite, the 3'-hydroxyl group of the phosphonate-modified nucleotide binds to phosphorimide to prepare a 5'-phosphonate-modified nucleotide phosphorimide monomer, including the structure shown in Formula 5 or Formula 6: Formula 5; Formula 6; in: Base: Nucleotide bases containing protecting groups such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; Y: One or more methylene CH2 groups, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups; LG1, LG2: Cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl, etc. R: Alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl, thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R´, R´´: Hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.; R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic groups, respectively.

[0010] Prepare nucleotide monomers with C4' linked to phosphonic acid P(V) via a spacer group, denoted by Y, including structures shown in Formula 7 or Formula 8: Formula 7; Formula 8; in: Base: Nucleotide bases containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; Y: One or more methylene CH2 groups, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups; LG1, LG2, LG3: Cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenyl. Silicon-based groups include: LG4: halogen, amino, cyano, azide, alkyloxy, etc.; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl, thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R´, R´´: hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.

[0011] Preferably, after the 5'-end of the nucleotide monomer undergoes a substitution reaction with hypophosphite, the phosphonic acid-modified nucleotide is oxidized to obtain a phosphonic acid-modified nucleotide, and the 3'-hydroxyl group is bound to phosphorimide to prepare a 5'-phosphonic acid-modified nucleotide phosphorimide monomer, including the structure shown in Formula 9 or Formula 10: Equation 9; Formula 10; in: Base: Nucleotide bases containing protecting groups such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; Y: One or more methylene CH2 groups, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups; LG1, LG2: Cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl, etc. R: Alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl, thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R´, R´´: Hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.; R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic groups, respectively.

[0012] Modified nucleotide phosphoramide monomers are embedded in oligonucleotide chains via solid-phase synthesis. The solid-phase synthesis process includes deprotection, coupling, capping, oxidation or sulfidation, etc., to construct modified oligonucleotide molecules.

[0013] Synthetic routes for constructing oligonucleotide molecules: 1) Deprotection: 3% dichloroacetic acid toluene solution was used to remove the O5' 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.

[0014] Following the set sequence, phosphorylamine is introduced and steps 1 to 5 are repeated to obtain the oligonucleotide product of the target sequence.

[0015] Oligonucleotide molecules with modified nucleotide structures can be constructed by coupling with phosphoramidites or other types of monomers such as phosphoramidites, phosphoramidites, phosphoramides, or phosphoryl chlorides that have intercalated spacer groups through solid-phase synthesis.

[0016] The 5'-terminal phosphonate-modified nucleotide structure is applied to oligonucleotides. This modified structure can improve the affinity of oligonucleotides for Ago2 protein, and at the same time improve the anti-enzymatic degradation activity and biological activity of oligonucleotide drugs.

[0017] Oligonucleotides are selected from any one or a combination of at least two of small interfering nucleotides, antisense oligonucleotides, microRNAs, small activating RNAs, guide RNAs, transfer RNAs, and aptamers.

[0018] The beneficial effects of this invention are as follows: This invention provides a method for preparing a 5'-terminal phosphonate-modified nucleotide phosphorylamide monomer and its application in oligonucleotide drugs. Specifically, the 5'-terminus is directly or indirectly linked to phosphorous acid P(III) and phosphonate P(V). This modified nucleotide monomer is applied to the preparation of oligonucleotides, and the constructed oligonucleotide drug has a phosphate backbone where the 5'-terminal phosphonate is linked to a carbon group to form a C4'-spacer group YP(V) structural unit. The prepared oligonucleotide molecule exhibits higher stability, effectively improving the stability of the modified oligonucleotide drug molecule while optimizing pharmacokinetic (PK) and pharmacodynamic (PD) performance.

[0019] Of course, any product implementing this invention does not necessarily need to achieve all the advantages described above at the same time. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Synthetic route (2´ position OR) for preparing 5'-phosphonic acid modified nucleotide phosphoramid monomers for this invention; Figure 2Synthetic route for preparing 5'-phosphonic acid modified nucleotide phosphoramid monomers for this invention (2' positions R' and R''). Figure 3 Synthetic route (2´ position OR) for preparing 5'-phosphonic acid modified nucleotide phosphoramid monomers for this invention. Figure 4 Synthetic route for preparing 5'-phosphonic acid modified nucleotide phosphoramid monomers for this invention (2' positions R' and R''). Figure 5 The oligonucleotide is 3'-T-Am-Um-Gm-Um-Um-Tm-Um-Um-A 1-5 -5' structure. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] For synthetic routes to prepare nucleotide phosphoramid monomers with 5'-terminus directly or indirectly linked to P(III), please refer to [link to relevant documentation]. Figure 1 and Figure 2 As shown, for the synthetic route to prepare nucleotide phosphoramid monomers with the 5'-end directly or indirectly linked to P(V), please refer to [reference needed]. Figure 3 and Figure 4As shown, Base: nucleotide bases containing protecting groups such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; Y: one or more methylene CH2, alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups with branched structures; PG: triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl, etc.; LG: halogen, sulfonyloxy, etc.; LG1, LG2: cyanoethyl, cyanopropyl, cyanoisobutyl, ( CH3)2CCH2CN, phenylethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzylBn, benzoylBz, acetyl or propionyl, etc., thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R´, R´´: hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylselenoyl, etc.; R1, R2: alkyl, alkenyl, alkynyl, cycloalkyl or aromatic, etc.

[0024] Example 1

[0025] C5'-phosphonocyanate-modified uracil phosphoramide

[0026] Under nitrogen atmosphere, 0.830 g of ammonium hypophosphite (10 mmol, 1.0 eq.) and 2.09 mL of hexamethyldisilazane (10 mmol, 1.0 eq.) were mixed and reacted at 110 °C for 2 h. After cooling to 0 °C, 6.705 g of U-1-0 (10 mmol, 1.0 eq.) dissolved in 30 mL of dry dichloromethane was added under nitrogen protection. The mixture was slowly heated to room temperature and reacted for 24 h. 30 mL of saturated sodium bicarbonate aqueous solution was added to separate the organic phase. The aqueous phase was extracted with 30 mL of dichloromethane, and the combined organic phases were dried over solid sodium sulfate and concentrated. Silica gel column chromatography yielded 3.410 g of solid U-1-1 (56% yield).

[0027] 1.826 g U-1-1 (3 mmol, 1.0 eq.), 1.424 g DCC (6.9 mmol, 2.3 eq.), and 0.618 g 3-hydroxypropionitrile (8.7 mmol, 2.9 eq.) were dissolved in 60 mL of anhydrous THF. The reaction was carried out under nitrogen protection and at a controlled temperature of 60–65 °C with stirring for 20 hours. After cooling to room temperature, the mixture was filtered, and the filter cake was washed with 30 mL of ethyl acetate. The organic phases were combined and concentrated. 100 mL of dichloromethane was added, followed by washing with saturated brine, drying over solid sodium sulfate, and concentration. The solution was then purified by silica gel column chromatography to obtain solid U-1-2.

[0028] 0.890 g of triphosgene (3 mmol, 1.0 eq.) was dissolved in 10 mL of dichloromethane. Under nitrogen protection and with the temperature controlled at 10 °C, a solution of 2.361 g of triphenylphosphine (9 mmol, 3.0 eq.) dissolved in 15 mL of dichloromethane was added. The mixture was slowly heated to room temperature with stirring, and then 1.09 mL of pyridine (13.5 mmol, 4.5 eq.) was added. Under nitrogen protection and with the temperature controlled at 5 °C, a solution of U-1-2 and 1.09 mL of pyridine dissolved in 30 mL of dichloromethane was added, and the mixture was stirred for 4–6 hours. The mixture was cooled to -40 °C, and a mixture of 0.640 g of 3-hydroxypropionitrile (9.0 mmol, 3.0 eq.) and 5 mL of dichloromethane was slowly added dropwise. The mixture was slowly heated to room temperature and stirred for another hour. The reaction solvent was removed under reduced pressure, and 50 mL of dichloromethane was added to the residue. The residue was washed with a saturated sodium bicarbonate aqueous solution, dried over solid sodium sulfate, concentrated, and then purified by silica gel column chromatography to obtain 1.309 g of solid U-1-3 (two-step yield 61%).

[0029] 1.286 g of U-1-3 (1.8 mmol, 1.0 eq.) was dissolved in 20 mL of dichloromethane. A mixture of 0.69 mL of trifluoroacetic acid (9.0 mmol, 5.0 eq.) and 10 mL of dichloromethane was slowly added, and the reaction was carried out at room temperature for 2 hours. 30 mL of saturated sodium bicarbonate aqueous solution was added, the organic phase was separated, and the aqueous phase was extracted with 30 mL of dichloromethane. The organic phases were combined, dried over solid sodium sulfate, concentrated, and purified by silica gel column chromatography to give 0.661 g of solid U-1-4 (yield 89%).

[0030] 0.412 g of U-1-4 (1.0 mmol, 1.0 eq.) was dissolved in 2.5 mL of dry DMF, and 0.056 g of tetrazolium (0.8 mmol, 0.8 eq.) and 0.025 g of N-methylimidazole (0.3 mmol, 0.3 eq.) were added. Under nitrogen protection and at a temperature below 20 °C, 0.5 mL of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.0 mmol, 1.0 eq.) was added. The reaction was allowed to proceed for at least 2 hours, and the reaction was then checked for completion. 25 mL of sodium bicarbonate aqueous solution (3%) was added, and the mixture was extracted twice with 30 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over sodium sulfate, and concentrated. Column chromatography was used to separate 0.313 g of solid U-1-5 (yield 51%, purity 98.9%).

[0031] Example 2

[0032] C5'-methylenephosphonic acid-modified uracil phosphoramide

[0033] Under nitrogen atmosphere, 0.830 g of ammonium hypophosphite (10 mmol, 1.0 eq.) and 2.09 mL of hexamethyldisilazane (10 mmol, 1.0 eq.) were mixed and reacted at 110 °C for 2 h. After cooling to 0 °C, 6.845 g of U-2-0 (10 mmol, 1.0 eq.) dissolved in 30 mL of dry dichloromethane was added under nitrogen protection. The mixture was slowly heated to room temperature and reacted for 24 h. 30 mL of saturated sodium bicarbonate aqueous solution was added to separate the organic phase. The aqueous phase was extracted with 30 mL of dichloromethane, and the combined organic phases were dried over solid sodium sulfate and concentrated. Silica gel column chromatography yielded 3.923 g of solid U-2-1 (63% yield).

[0034] 3.736 g U-2-1 (6 mmol, 1.0 eq.), 2.847 g DCC (13.8 mmol, 2.3 eq.), and 1.237 g 3-hydroxypropionitrile (17.4 mmol, 2.9 eq.) were dissolved in 100 mL of anhydrous THF. The reaction was carried out under nitrogen protection and at a controlled temperature of 60–65 °C with stirring for 20 h. After cooling to room temperature, the mixture was filtered, and the filter cake was washed with 50 mL of ethyl acetate. The combined organic phases were concentrated. 100 mL of dichloromethane was added, and the mixture was washed with saturated brine to separate the organic phase. The aqueous phase was extracted with 100 mL of dichloromethane. The combined organic phase was dried over sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain solid U-2-2.

[0035] 1.781 g of triphosgene (6 mmol, 1.0 eq.) was dissolved in 20 mL of dichloromethane. Under nitrogen protection and with the temperature controlled at 10 °C, a solution of 4.721 g of triphenylphosphine (18 mmol, 3.0 eq.) dissolved in 30 mL of dichloromethane was added. The mixture was slowly heated to room temperature with stirring, and then 2.18 mL of pyridine (27 mmol, 4.5 eq.) was added. Under nitrogen protection and with the temperature controlled at 5 °C, a solution of U-2-2 and 2.18 mL of pyridine dissolved in 50 mL of dichloromethane was added, and the mixture was stirred for 4–6 hours. The mixture was cooled to -40 °C, and a mixture of 1.279 g of 3-hydroxypropionitrile (18.0 mmol, 3.0 eq.) and 10 mL of dichloromethane was slowly added dropwise. The mixture was slowly heated to room temperature and stirred for another hour. The reaction solvent was removed under reduced pressure, and 50 mL of dichloromethane was added to the residue. The residue was washed with a saturated sodium bicarbonate aqueous solution, dried over solid sodium sulfate, concentrated, and then purified by silica gel column chromatography to obtain 2.931 g of solid U-2-3 (67% yield in two steps).

[0036] 1.312 g of U-2-3 (1.8 mmol, 1.0 eq.) was dissolved in 20 mL of dichloromethane. A mixture of 0.69 mL of trifluoroacetic acid (9.0 mmol, 5.0 eq.) and 10 mL of dichloromethane was slowly added, and the reaction was carried out at room temperature for 2 hours. 30 mL of saturated sodium bicarbonate aqueous solution was added, the organic phase was separated, and the aqueous phase was extracted with 30 mL of dichloromethane. The organic phases were combined, dried over sodium sulfate, concentrated, and purified by silica gel column chromatography to give 0.699 g of solid U-2-4 (91% yield).

[0037] 0.426 g of U-2-4 (1.0 mmol, 1.0 eq.) was dissolved in 2.5 mL of dry DMF, and 0.056 g of tetrazolium (0.8 mmol, 0.8 eq.) and 0.025 g of N-methylimidazole (0.3 mmol, 0.3 eq.) were added. Under nitrogen protection and at a temperature below 20 °C, 0.5 mL of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.0 mmol, 1.0 eq.) was added. The reaction was allowed to proceed for at least 2 hours, and the reaction was then checked for completion. 25 mL of sodium bicarbonate aqueous solution (3%) was added, and the mixture was extracted twice with 30 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over sodium sulfate, and concentrated. Column chromatography was used to separate 0.333 g of solid U-2-5 (yield 53%, purity 99.1%).

[0038] Furthermore, the following nucleotide monomers were prepared using the synthetic method described above, wherein n = 1~4: , , , , , , , , , , , , , .

[0039] Furthermore, the following nucleotide monomers were prepared using the synthetic method described above, wherein n = 1~4: , , , , , , , , , , , , , .

[0040] Furthermore, the following nucleotide monomers were prepared using the synthetic method described above, wherein n = 1~4: , , , , , , , , , , , , , .

[0041] Furthermore, similar synthetic procedures are applicable to the following molecular structures in which one or more CH2 groups are inserted between C4' and phosphonate P(III) in the structure of the nucleotide phosphoramidide monomer, including one of the following structures: , ; Where: n = 1~10; Base: Nucleotide bases containing protecting groups, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; LG1, LG2: Cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl, etc.; R: Alkyl, cycloalkyl, alkyl alkyl oxy, carbonyl alkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl acyl groups, thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl silyl groups, etc.; R´, R´´: hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.

[0042] Furthermore, similar synthetic procedures are applicable to the following molecular structures in which the C4' of the nucleotide phosphoramidide monomer is linked to the phosphonite P(III) via a spacer group Y, including one of the following structures: , ; in: Base: Nucleotide bases containing protecting groups such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; Y: One or more methylene CH2 groups, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups; LG1, LG2: Cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl, etc. R: Alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl, thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R´, R´´: Hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.; R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic groups, respectively.

[0043] Example 3

[0044] C5'-methylenephosphonic acid-modified uracil phosphoramide

[0045] 1.457 g of U-2-3 (2.0 mmol, 1.0 eq.) was dissolved in 30 mL of dichloromethane, and 0.38 mL of tert-butylhydrogen peroxide was added. The reaction was carried out at room temperature for 0.5 h, and the reaction was quenched by adding 30 mL of sodium sulfite aqueous solution (5% concentration). The organic phase was separated, and the aqueous phase was extracted twice with 30 mL of dichloromethane. The combined organic phases were washed with saturated brine, dried over solid sodium sulfate, concentrated, and purified by silica gel column chromatography to give 1.430 g of solid U-2-6 (96% yield).

[0046] 1.312 g of U-2-6 (1.8 mmol, 1.0 eq.) was dissolved in 20 mL of dichloromethane. A mixture of 0.69 mL of trifluoroacetic acid (9.0 mmol, 5.0 eq.) and 10 mL of dichloromethane was slowly added, and the reaction was carried out at room temperature for 2 hours. 30 mL of saturated sodium bicarbonate aqueous solution was added, and the organic phase was separated. The aqueous phase was extracted with 30 mL of dichloromethane. The organic phases were combined, dried over sodium sulfate, concentrated, and purified by silica gel column chromatography to give 0.686 g of solid U-2-7 (86% yield).

[0047] 0.442 g of U-2-7 (1.0 mmol, 1.0 eq.) was dissolved in 2.5 mL of dry DMF, and 0.056 g of tetrazolium (0.8 mmol, 0.8 eq.) and 0.025 g of N-methylimidazole (0.3 mmol, 0.3 eq.) were added. Under nitrogen protection and at a temperature below 20 °C, 0.5 mL of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.0 mmol, 1.0 eq.) was added. The reaction was allowed to proceed for at least 2 hours, and the reaction was then checked for completion. 25 mL of sodium bicarbonate aqueous solution (3%) was added, and the mixture was extracted twice with 30 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over sodium sulfate, and concentrated. Column chromatography was used to separate 0.360 g of solid U-2-8 (yield 56%, purity 98.7%).

[0048] Furthermore, similar synthetic procedures are applicable to the following molecular structures in which one or more CH2 groups are inserted between C4' and phosphonic acid P(V) in the structure of the nucleotide phosphoramidide monomer, including one of the following structures: , ; Where: n = 1~10; Base: Nucleotide bases containing protecting groups, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; LG1, LG2: Cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl, etc.; R: Alkyl, cycloalkyl, alkyl alkyl oxy, carbonyl alkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl acyl groups, thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl silyl groups, etc.; R´, R´´: hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.

[0049] Furthermore, similar synthetic procedures are applicable to the following molecular structures in which the C4' of the nucleotide phosphoramidide monomer is linked to the phosphonic acid P(V) via a spacer group Y, including one of the following structures: , ; in: Base: Nucleotide bases containing protecting groups such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; Y: One or more methylene CH2 groups, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups; LG1, LG2: Cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl, etc. R: Alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl, thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R´, R´´: Hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.; R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic groups, respectively.

[0050] 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.

[0051] Solid-phase synthesis of phosphorous amide monomers incorporating C4´-spacer groups Y, under the following process conditions: Reagents and monomers: Prepare a 0.15 mol / L monomer / acetonitrile solution; the deprotecting agent for DMTr (or Tr) is a 3% 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).

[0052] The sequence of operations for solid-phase synthesis is as follows: 1) Deprotection: 3% 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.

[0053] Repeat the above operations according to the set sequence to obtain the oligonucleotide product of the target sequence.

[0054] 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.

[0055] Remove TBDMS or other silyl ether protecting groups: Add TBAF / THF solution to the oligonucleotide product containing silyl ether protecting groups and treat at room temperature for 2-6 hours.

[0056] 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.

[0057] The above solid-phase synthesis methods are also applicable to monomers protected by Tr, MMTr, and TMTr.

[0058] In examples of solid-phase synthesis, the nucleotide monomers used are represented by the following structures: , , , , , , , , , , , , , , .

[0059] Example 4:

[0060] The monomer O5'-DMTr-O2'-Bz-T (Tbz) was loaded onto crosslinked polystyrene PS, followed by monomers A in sequence. m U m G m U m U m T m U m U m Following the cyclical operation of deDMTr, coupling, oxidation, and capping, after the cycle is completed, the oligonucleotide molecule 3'-T-Am-Um-Gm-Um-Um-Tm-Um-Um-A is obtained after ammonolysis and deprotection treatment. The filtrate is concentrated and purified by HPLC to obtain the designed sequence. 1-5 -5', please refer to the appendix for the structure. Figure 5 .

[0061] The above solid-phase synthesis is also applicable to the C4´-group Y-phosphonic acid modified nucleotide phosphoramid monomers of the present invention, wherein Y is one or more methylene CH2, alkyl, alkylene, mesylate, cycloalkyl, alkoxyalkyl, etc. with branched chain structure.

[0062] Example 5: The monomer O5'-DMTr-O2'-Bz-T (Tbz) was loaded onto crosslinked polystyrene PS, followed by monomers A in sequence. m U m G m U m U m T m U m U m Following the cyclical operation of deDMTr, coupling, oxidation, and capping (A-1-8), after the cycle was completed, ammonolysis and deprotection were performed. The filtrate was concentrated and purified by HPLC to obtain the oligonucleotide molecule with the designed sequence. Analysis confirmed that it had the same structure as in Example 4. Please refer to the appendix for the structure. Figure 5 .

[0063] The above solid-phase synthesis is also applicable to the C4´-group γ-phosphonic acid modified nucleotide phosphoramide monomers of the present invention, wherein γ is one or more methylene CH2, alkyl, alkylene, mesylate, cycloalkyl, alkoxyalkyl, etc. with branched chain structure.

[0064] Example 6:

[0065] The sense and antisense strands of inclisiran were prepared using a solid-phase synthesis method, and their structures are shown below (labeled as IN-RNA):

[0066] Example 7:

[0067] In solid-phase synthesis, the nucleotide monomer A-1-5 of this invention is used to replace the 5'-terminal Am of the Inclisiran antisense strand to prepare a 5'-terminal PO3-carrying structure. 2- The antisense strand of the Inclisiran homologue is paired equimolarly with the sense strand of the Inclisiran to obtain the siRNA structure shown below (labeled IN-RNA-PO3):

[0068] The structure of the last two nucleotides at the 5' end of the IN-RNA-PO3 antisense strand is shown below:

[0069] Wherein Af: 2'-fluoroadenine nucleoside; Cf: 2'-fluorocytosine nucleoside; Gf: 2'-fluoroguanine nucleoside; Am: O2'-methyladenine nucleoside; Cm: O2'-methylcytosine nucleoside; Gm: O2'-methylguanine nucleoside; Um: O2'-methyluracil nucleoside; dT: deoxythymidine nucleoside; L96: triantennary GalNAc (N-acetyl-galactosamine); s: thiolated phosphate skeleton.

[0070] Example 8:

[0071] In solid-phase synthesis, the nucleotide monomer A-2-5 of this invention is used to replace the 5'-terminal Am of the Inclisiran antisense strand to prepare a 5'-terminal PO3-carrying structure. 2- The antisense strand of the Inclisiran homologue is paired equimolarly with the sense strand of Inclisiran to obtain the siRNA structure shown below (labeled as IN-RNA-CH2-PO3):

[0072] The structure of the last two nucleotides at the 5' end of the antisense strand of IN-RNA-CH2-PO3 is shown below:

[0073] Wherein Af: 2'-fluoroadenine nucleoside; Cf: 2'-fluorocytosine nucleoside; Gf: 2'-fluoroguanine nucleoside; Am: O2'-methyladenine nucleoside; Cm: O2'-methylcytosine nucleoside; Gm: O2'-methylguanine nucleoside; Um: O2'-methyluracil nucleoside; dT: deoxythymidine nucleoside; L96: triantennary GalNAc (N-acetyl-galactosamine); s: thiolated phosphate skeleton.

[0074] Example 9:

[0075] In solid-phase synthesis, the nucleotide monomer A-2-8 of this invention is used to replace the 5'-terminal Am of the Inclisiran antisense strand to prepare a 5'-terminal PO3-carrying structure. 2- The antisense strand of the inclisiran homologue was analyzed and confirmed to have the same structure as the antisense strand of Example 8. Equimolarly paired with the inclisiran sense strand, the resulting siRNA structure is shown below (labeled IN-RNA-CH2-PO3):

[0076] The structure of the last two nucleotides at the 5' end of the antisense strand of IN-RNA-CH2-PO3 is shown below:

[0077] Wherein Af: 2'-fluoroadenine nucleoside; Cf: 2'-fluorocytosine nucleoside; Gf: 2'-fluoroguanine nucleoside; Am: O2'-methyladenine nucleoside; Cm: O2'-methylcytosine nucleoside; Gm: O2'-methylguanine nucleoside; Um: O2'-methyluracil nucleoside; dT: deoxythymidine nucleoside; L96: triantennary GalNAc (N-acetyl-galactosamine); s: thiolated phosphate skeleton.

[0078] Example 10: Affinity Test The dissociation constants of IN-RNA, IN-RNA-PO3, and IN-RNA-CH2-PO3 binding to hAgo2 protein were tested separately. Affinity assays showed that the affinities of IN-RNA-PO3 and IN-RNA-CH2-PO3 were 23.2±1.5 and 20.6±2.0 times that of IN-RNA, respectively. This invention utilizes a terminal phosphorylation modification strategy, with 5'-terminal CH2-PO3... 2- The modified siRNA increased its affinity for hAgo2.

[0079] Example 11: 5'-terminal CH2-PO3 2- Activity assay of modified siRNA In mouse models, siRNA drugs were administered single doses of 5 mg / kg, including IN-RNA, IN-RNA-CH2-PO3, and IN-RNA-CH2-PO3. The effects of siRNA on PCSK9 mRNA levels and LDL-C content were analyzed. The study found that in mice, IN-RNA-PO3 and IN-RNA-CH2-PO3 were more effective than IN-RNA in reducing PCSK9 mRNA levels. Compared with IN-RNA, IN-RNA-PO3 improved LDL-C levels in mice at 10, 20, and 40 days of age by 32%, 29%, and 28%, respectively; while IN-RNA-CH2-PO3 improved LDL-C levels by 35%, 26%, and 27%, respectively.

[0080] This invention provides a method for preparing a class of 5'-terminal phosphonate-modified nucleotide phosphoramidite monomers and their application in solid-phase synthesis of nucleic acid drugs. Introducing the modified nucleotide unit provided by this invention can improve the affinity between nucleic acid molecules and the hAgo2 protein, and also helps to improve the biological activity of nucleic acid drugs in vivo.

[0081] 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.

[0082] The substances corresponding to the abbreviations in this invention are as follows: DCC: N,N'-Dicyclohexylcarbodiimide; P-Ts: sodium((tosyloxy)methyl)phosphinate; CPG: controlled pore glass; HPLC: high performance liquid chromatography; P(III): trivalent phosphorus (phosphine); P(V): pentavalent phosphorus (phosphine); PS: polystyrene; CH2: methylene; F: fluorine; DIPEA: N,N-diisopropylethylamine; Alkyl: alkyl; Allyl: allyl; Alloc: allyloxycarbonyl; Bn: benzyl; Bz: benzoyl; DMTr: dimethoxytriphenylmethyl; MMTr: methoxytriphenylmethyl Benzyl; Tr: Triphenylmethyl; TMTr: Trimethoxytriphenylmethyl; iBu: 2-Isobutyryl; NPE: 4-Nitrophenylethoxy; Ph: Aromatic; TBDMS: Tert-Butyldimethylsilyl; TBDPS: Tert-Butyldiphenylsilyl; Ts: p-Toluenesulfonyl; DCM: Dichloromethane; DMF: N,N-Dimethylformamide; TBAF: Tetrabutylammonium fluoride; THF: Tetrahydrofuran; TMSI: Trimethyliodosilane; A: Adenine Adenine; G: Guanine; C: Cytosine; T: Thymine; U: Uracil; I: Hypoxanthine; Xan: Xanthine; m5C: 5-methylcytosine; Ψ: Pseudouracil; m1Ψ: N1-methylpseudouracil; Af: 2'-fluoroadenine nucleoside; Cf: 2'-fluorocytosine nucleoside; Gf: 2'-fluoroguanine nucleoside; Am: O2'-methyladenine nucleoside; Cm: O2'-methylcytosine nucleoside; Gm: O2'-methylguanine nucleoside; Um: O2'-methyluracil nucleoside; dT: Deoxythymidine nucleoside; L96: triantennary GalNAc (N-acetyl-galactosamine); mRNA: messenger ribonucleic acid; LDL-C: low-density lipoprotein cholesterol; PCSK9: proprotein convertase subtilisin 9.

[0083] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to mutually. Each embodiment focuses on describing the differences from other embodiments. In particular, the embodiments of apparatus, devices, and non-volatile computer storage media are basically similar to the method embodiments, so the descriptions are relatively simple; relevant parts can be referred to the descriptions of the method embodiments.

[0084] The foregoing has described specific embodiments of this specification. Other embodiments are within the scope of the appended documents. In some cases, the actions or steps described in this application may be performed in a different order than that shown in the embodiments and still achieve the desired results. Furthermore, the processes depicted in the drawings do not necessarily require the specific or sequential order shown to achieve the desired results. In some embodiments, multitasking and parallel processing are also possible or may be advantageous.

[0085] The above description is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the concept of the invention or exceed the scope defined in this application, they should all fall within the protection scope of the present invention.

Claims

1. A synthesis of a 5'-terminal phosphononucleotide and its application in oligonucleotides, characterized in that, The chemical modification strategy includes 5'-terminus via carbon-phosphonic acid C-PO3. 2- Chemical bonds with phosphonic acid PO3 2- Linkage, including the nucleotide structure shown in Formula 1 or Formula 2: Formula 1; Formula 2; in: Base: Nucleotide bases such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine and their derivatives; Y: One or more methylene CH2 groups, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl groups; R: Hydrogen, alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, thiocarbonylalkyl, benzyl Bn , Benzoyl Bz, Acyl or propionyl and other acyl groups, Thioacyl, Alkenyl, Cycloalkenyl, Alkynyl, Allyl, Allyloxycarbonyl, Trimethylsilyl, Triethylsilyl, Triisopropylsilyl, Tert-butyldimethylsilyl or Tert-butyldiphenylsilyl and other silyl groups; R´, R´´: Hydrogen, Halogen, Alkyl, Cycloalkyl, Alkoxy, Alkoxyalkyl, Alkenyl, Cycloalkenyl, Alkynyl, Cyano, Azide, Amino (Amino), Mercapto, Alkyl Mercapto, Selenyl, Alkyl Selenyl, etc.

2. The synthesis of a 5'-terminal phosphononucleotide according to claim 1 and its application in oligonucleotides, characterized in that, The chemical modification strategy also includes preparing modified nucleotide phosphoramide monomers linked to the 5'-end of the phosphonate via a carbon-phosphonic acid chemical bond, including the nucleotide structures shown in Formula 3 or Formula 4: Formula 3; Equation 4; in: Base: Nucleotide bases containing protecting groups such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; Y: One or more methylene CH2 groups, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups; LG1, LG2, LG3: Cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl groups; LG4: Halogen, amino, cyano, azide, alkyloxy, etc.; R: Alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzylBn, benzoylBz, acetyl Acyl groups such as alkyl or propionyl, thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R´, R´´: hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.

3. The synthesis of a 5'-terminal phosphononucleotide according to claim 2 and its application in oligonucleotides, wherein the 5'-terminal phosphonate-modified nucleotide monomer comprises the structure shown in Formula 5 or Formula 6: Formula 5; Formula 6; in: Base: Nucleotide bases containing protecting groups such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; Y: One or more methylene CH2 groups, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups; LG1, LG2: Cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl, etc.; R: Alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl, etc., thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, etc. Silicon groups include allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl; R´, R´´: hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylmercapto, selenyl, alkylselenoyl, etc.; R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively.

4. The synthesis of a 5'-terminal phosphononucleotide according to claim 1 and its application in oligonucleotides, characterized in that, The chemical modification strategy also includes preparing modified phosphoramid monomers of nucleotides linked to the 5'-end of a phosphonate ester via a carbon-phosphonic acid bond, including nucleotide structures shown in Formula 7 or Formula 8: Formula 7; Formula 8; in: Base: Nucleotide bases containing protecting groups such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; Y: One or more methylene CH2 groups, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups; LG1, LG2, LG3: Cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl groups; LG4: Halogen, amino, cyano, azide, alkyloxy, etc.; R: Alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzylBn, benzoylBz, acetyl Acyl groups such as alkyl or propionyl, thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R´, R´´: hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.

5. The synthesis of a 5'-terminal phosphonic nucleotide according to claim 4 and its application in oligonucleotides, wherein the 5'-terminal phosphonic acid-modified nucleotide monomer comprises the structure shown in Formula 9 or Formula 10: Equation 9; Formula 10; in: Base: Nucleotide bases containing protecting groups such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and their derivatives; Y: One or more methylene CH2 groups, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups; LG1, LG2: Cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl, etc.; R: Alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl, etc., thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, etc. Silicon groups include allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl; R´, R´´: hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylmercapto, selenyl, alkylselenoyl, etc.; R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively.

6. The synthesis of a 5'-terminal phosphononucleotide as described in any one of claims 2-3 and its application in oligonucleotides, characterized in that, The synthesis of the 5'-phosphonite-modified nucleotide phosphoramidite monomer involves a substitution reaction between the 5'-terminus and hypophosphite, followed by the condensation of phosphonite with a hydroxyl substrate to obtain a phosphonite ester. The release of the 3'-hydroxyl group then binds to the phosphoramidite to yield the 5'-phosphonite-modified nucleotide phosphoramidite monomer.

7. The synthesis of a 5'-terminal phosphononucleotide as described in any one of claims 4-5 and its application in oligonucleotides, characterized in that, The synthesis of the 5'-phosphonic acid modified nucleotide phosphoramidite monomer involves a substitution reaction between the 5'-terminus and hypophosphite, followed by the condensation of phosphonic acid with a hydroxyl substrate to obtain a phosphonate ester. This phosphonate ester is then oxidized to a phosphonate ester, releasing a 3'-hydroxyl group that binds to phosphoramidite to obtain the 5'-phosphonic acid modified nucleotide phosphoramidite monomer.

8. The synthesis of a 5'-terminal phosphononucleotide according to claim 1 and its application in oligonucleotides, characterized in that, The chemical modification strategy described herein includes synthesizing nucleotide phosphoramide monomers with 5'-terminal C-phosphonates or C-phosphonates chemically linked, to prepare oligonucleotides with 5'-terminal phosphonophosphate modification that are identical in sequence to Inclisiran. Compared with the dissociation constant of Ago2 protein bound by Inclisiran, the terminal phosphonophosphate modification proposed in this invention, whether directly linked to the phosphonate at C5' or linked to the phosphonate via a spacer methylene group, improves the affinity between the modified siRNA and Ago2.

9. The synthesis of a 5'-terminal phosphononucleotide according to claim 1 and its application in oligonucleotides, characterized in that, Oligonucleotides with 5'-terminal phosphonate modification and identical sequences to Inclisiran were prepared. Whether C5' was directly linked to phosphonate or linked to a methylene group between C5' and phosphonate, the 5'-terminal phosphonate-modified siRNA significantly reduced PCSK9 mRNA levels and LDL-C content in model animals compared to Inclisiran.

10. A modified oligonucleotide, characterized in that, The oligonucleotide structural unit includes a nucleotide with the modified structure of any one of claims 1 to 7, wherein the oligonucleotide is selected from any one or a combination of at least two of small interfering nucleotides, antisense oligonucleotides, microRNAs, small activating RNAs, guide RNAs, transfer RNAs and aptamers.

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