Use of oligonucleotides with a spacer group intercalated between the 5'-terminal oxygen and the phosphate

Abstract

This invention relates to the field of biomedical technology, specifically disclosing the application of an oligonucleotide with a spacer group embedded between the oxygen and phosphate at the 5'-terminus; the application of a nucleotide with a spacer group embedded between the O5'-oxygen and phosphate and phosphorous acid and O3'-hydroxyl group bound to phosphorous acid in oligonucleotides, the chemical modification strategy including: preparing a phosphorous acid monomer of a modified nucleotide with a spacer group embedded between the O5'-oxygen and phosphate P(V); constructing the target oligonucleotide molecule by solid-phase synthesis of the phosphorous acid monomer of the modified nucleotide; embedding a spacer group between the oxygen and phosphate P(V) at the 5'-terminus of the oligonucleotide to form a 5'-terminal modified phosphate structure; since this type of terminal modified phosphate is a non-phosphatase substrate, the modification can resist exonuclease degradation and enhance the in vivo silencing effect of siRNA.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of an oligonucleic acid in which a spacer group is embedded between the oxygen and phosphate at the 5'-terminus. 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, high development success rates, short development cycles, high specificity, and long-lasting efficacy, providing solutions for the treatment of many intractable diseases.

[0003] Chemical modification is an effective strategy 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. 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, pre-phosphorylation strategies via chemical synthesis can be used to create metabolically stable phosphate analogs. Summary of the Invention

[0004] In this invention, a spacer group is added between the nucleotide O5'-oxygen and phosphate P(V), and the O3'-hydroxyl group is converted into phosphorous acid to obtain a modified nucleotide monomer. This modified nucleotide monomer is introduced into the 5'-terminus of an oligonucleotide via solid-phase synthesis for terminal modification of siRNA. This terminally modified phosphate not only increases the affinity of siRNA for Ago2, but also, because it is a non-phosphatase substrate, resists exonuclease degradation and enhances the in vivo silencing effect of siRNA.

[0005] The objective of this invention can be achieved through the following technical solutions: Applications of a nucleotide with an intercalating spacer group between O5´-oxygen and phosphate P(V) in oligonucleotides, including chemical modification strategies, such as: Phosphoramide monomers for preparing modified nucleotides with spacer groups inserted between O5'-oxygen and phosphate P(V); Oligonucleotide molecules were constructed by introducing phosphoramide monomers that modify nucleotides into oligonucleotide chains using a solid-phase synthesis method. An intercalating spacer group Y is inserted between the 5'-terminal oxygen of the oligonucleotide molecule and the phosphate ester P(V), forming O5'-Y-PO3. 2- One of the following structures: , ; Wherein: 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 groups, cycloalkyl groups, alkoxyalkyl groups, carbonylalkyl groups, etc. Benzyl (Bn), benzoyl (Bz), acetyl or propionyl, etc., acyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R1, R2: halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.

[0006] Preferably, a methylene group Y is inserted between the 5'-terminal oxygen and the phosphate ester P(V) to form O5'-CH2-PO3. 2- One of the following structures: , ; Wherein: Base: nucleotide bases such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine and their derivatives; R: acyl groups such as hydrogen, alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; R1, R2: halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylselenoyl, etc.

[0007] The synthesis of phosphoramidite monomers for preparing modified nucleotides with spacer group Y inserted between O5' and phosphate ester P(V) involves the substitution reaction of the O5'-hydroxyl group of the nucleotide monomer with sulfonyloxyphosphonate, followed by the conversion of phosphate ester P(V) into other esters or other leaving groups as required by the design, the removal of the O3' protecting group, and phosphoramidization at that position to obtain the phosphoramidite monomer of the modified nucleotide.

[0008] Preferably, a modified nucleotide monomer is prepared by inserting a spacer group Y between O5'-oxygen and phosphate P(V); the O3'-hydroxyl group of the nucleotide monomer is combined with phosphoramide to obtain the modified nucleotide, the structural formula of which is: , ; Wherein: 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, alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups with branched structures; LG: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aromatic, cyanoalkyl, cyanoalkylene, alkoxyalkyl, acyloxymethyl, acyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldimethylsilyl. Or tert-butyldiphenylsilyl or other silyl groups; R1, R2: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic groups, etc.; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl or other acyl groups, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl or other silyl groups; R3, R4: halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkyl selenyl, etc.

[0009] Preferably, the phosphate ester is 3-cyanopropyl ester; the O3'-hydroxyl group of the nucleotide monomer is combined with phosphoramide to obtain the modified nucleotide, the structural formula of which is: , ; Wherein: 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; R1, R2: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, etc.; R: alkyl group. R3, R4: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl, etc.; alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylseleno, etc.

[0010] Preferably, the nucleotide monomer with a methylene group inserted between the O5'-oxygen and the phosphate P(V) is 3-cyanopropyl ester.

[0011] Preferably, the phosphoramidite monomer that modifies the nucleotide is coupled with hydroxyl, amino, thiol or selenium groups released from the nucleic acid sequence loaded on a solid support, and subjected to operations such as oxidation or sulfidation or deprotection to construct an oligonucleotide molecule with 5´-terminal phosphorylation modification.

[0012] Preferably, the O3´-phosphorous amide of the modified nucleotide's phosphoramidite monomer is coupled to the O5´-hydroxyl, amino, thiol, or selenium groups released from the nucleic acid sequence loaded on the solid support.

[0013] Preferably, the chemical modification strategy includes preparing a phosphoramidite monomer of a modified nucleotide with a spacer group Y embedded between the O5' end and the phosphate ester P(V), preparing an oligonucleotide with a terminal modification that is sequenced with Inclisiran, introducing a spacer group such as a methylene group between the 5'-terminal oxygen and the phosphate of the modified siRNA antisense strand, comparing the dissociation constant of binding to human Ago2 protein (hAgo2) with Inclisiran, and increasing the affinity of the 5'-terminal modified siRNA for hAgo2; the oligonucleotide with a spacer group embedded between the 5'-terminus and the phosphate ester, the terminally modified phosphate not only increases the affinity of siRNA for hAgo2, but also resists exonuclease degradation and enhances the in vivo silencing effect of siRNA.

[0014] Preferably, an oligonucleotide with a terminal modification that is identical to that of Inclisiran is prepared. A spacer group, such as a methylene group, is introduced between the 5'-terminal oxygen and phosphate of the modified siRNA antisense strand. The 5'-terminal modified siRNA reduces the PCSK9 mRNA level and LDL-C content in the model animals more significantly than Inclisiran.

[0015] Preferably, the phosphoramidite monomer that modifies the nucleotide is embedded in the oligonucleotide chain by a solid-phase synthesis method. The solid-phase synthesis process includes deprotection, coupling, capping, oxidation or sulfidation, etc., to construct the modified oligonucleotide molecule.

[0016] 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.

[0017] Following the set sequence, phosphoramide was introduced and the above operation was repeated to obtain the oligonucleotide product of the target sequence.

[0018] Preferably, the oligonucleotide is selected from any one or a combination of at least two of small interfering nucleotides, antisense oligonucleotides, microRNAs, small activating RNAs, small guide RNAs, transfer RNAs, and aptamers.

[0019] The beneficial effects of this invention: This invention provides a nucleic acid modification strategy involving 5'-terminal phosphorylation, characterized by the insertion of a spacer group between the 5'-terminal oxygen of the oligonucleotide and the phosphate P(V). Since the 5'-terminus is OY-PO3... 2- Phosphate, and Y is generally a single bond structure, unlike the double bond of 5´-VP. In addition to the cis and trans configurations, the presence of a double bond restricts the PO3 at the end. 2- The interaction between the binding site and the active enzyme; the preparation method of the nucleotide phosphoramidamide monomer with O5´-oxygen and phosphate spacer group and its application in oligonucleotide drugs; the modified nucleotide monomer is embedded in the oligonucleotide chain by solid-phase synthesis method, and the constructed oligonucleotide drug has its 5´-terminal oxygen bound to phosphate through the spacer group; thus, the synthesized oligonucleotide molecule exhibits higher stability and resistance to enzyme degradation, while the terminal phosphate modification increases the affinity of RNAi for hAgo2 and enhances the in vivo silencing effect of RNAi.

[0020] 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

[0021] 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.

[0022] Figure 1 Synthetic route for phosphorous amide with O5´-oxygen and phosphate spacer group (2´-oxygen). Figure 2 Synthetic route for phosphorous amides with O5´-oxygen and phosphate spacer group (2´-other groups); Figure 3 This is the route for solid-phase synthesis; Figure 4It has the structure of oligonucleotide 3'-T-Am-Um-Gm-Um-Um-Tm-Um-Um-A6-5'. Detailed Implementation

[0023] 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.

[0024] Please see Figure 1 As shown, the present invention relates to the application of an oligonucleotide in which a spacer group is embedded between the oxygen and phosphate at the 5'-terminus; it can be used as a metabolically stable phosphate analog by pre-phosphorylation strategy through chemical synthesis.

[0025] Example 1: Preparation of O5'-methylene phosphate nucleotide monomer The synthetic route for phosphorous amide of O5´-methylene phosphate guanine is shown in the figure below:

[0026] 4.996 g of O2'-Me-O3'-TBDMS-N6-Bz-A (A-0, 10 mmol, 1.0 eq.) and 4.414 g of Ts-O-PO(OMe)2 (15 mmol, 1.5 eq.) were dissolved in 50 mL of dry DMF. The mixture was cooled to 0 °C and stirred. Under nitrogen protection, 0.880 g of NaH (60% dispersed in mineral oil, 22 mmol, 2.2 eq.) was added, and the mixture was slowly heated to room temperature and reacted overnight. Under nitrogen protection, 10 mL of 3% ammonium chloride aqueous solution was slowly added dropwise. The solvent was distilled under reduced pressure, and then 200 mL of dichloromethane was added. The organic phase was washed with 100 mL of 3% ammonium chloride aqueous solution, dried over solid sodium sulfate, concentrated, and purified by silica gel column chromatography to give 4.786 g of solid A-1 (77% yield).

[0027] 4.352 g of A-1 (7 mmol, 1.0 eq.) was dissolved in 20 mL of dry dichloromethane. Under nitrogen protection at room temperature, 2.3 mL of pyridine (28 mmol, 4.0 eq.) and 2.5 mL of trimethyliodosilane (TMSI) (17.5 mmol, 2.5 eq.) were added. After 1 hour of reaction, the reaction was confirmed to be complete. The solvent was removed by vacuum distillation, and the mixture was washed with 100 mL of ethyl acetate and 50 mL of dilute hydrochloric acid solution (0.1 mol / L). The organic phase was separated, dried over sodium sulfate, and concentrated to give 3.622 g of solid A-2 (87% yield).

[0028] 3.265 g of A-2 (5.5 mmol, 1.0 eq.) was dissolved in 14 mL of dry dichloromethane. 6.777 g of 2,6-di-tert-butyl-4-methylpyridine (33 mmol, 6.0 eq.), 2.100 g of oxaloyl chloride (16.5 mmol, 3.0 eq.), and the catalyst amount DMF were added. The mixture was stirred overnight at room temperature under nitrogen protection for 3 h. After removing the solvent under reduced pressure, 30 mL of dry dichloromethane was added. 2.36 g of 3-hydroxypropionitrile (33 mmol, 6.0 eq.) was added, and the mixture was stirred overnight at room temperature under nitrogen protection. 50 mL of sodium bicarbonate aqueous solution (3%) was slowly added, and the mixture was mixed and the organic phase was separated. The organic phase was washed with 30 mL of saturated brine, dried over sodium sulfate, and concentrated. Column chromatography yielded 1.273 g of solid A-4 (33% yield).

[0029] 1.120 g of A-4 (1.60 mmol, 1.0 eq.) was dissolved in 5 mL of THF, and 3.7 mL of TBAF / THF solution (1.0 mol / L, 2.3 eq.) was added. The reaction was carried out at room temperature for at least 6 hours, and the solvent was removed by evaporation under reduced pressure. 30 mL of sodium bicarbonate aqueous solution (3%) was added, and the mixture was extracted twice with 30 mL of dichloromethane. The combined organic phases were washed with saturated brine, dried over sodium sulfate, and concentrated. Column chromatography was used to separate 0.661 g of solid A-5 (71% yield).

[0030] 0.586 g of A-5 (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 the solid A-6 to obtain 0.443 g.

[0031] The same synthetic procedure applies to the following molecular structure, namely, the phosphoramidite monomer modified with O2´-Me-O5´-CH2-P(V), whose structural formula is: , , , , , , , , , .

[0032] The same synthetic procedure applies to the following molecular structure, namely, the phosphoramidite monomer modified with 2´-F-O5´-CH2-P(V), whose structural formula is: , , , , , , , , , , .

[0033] The same synthetic procedure applies to the following molecular structure, namely, the O5´-CH2-P(V) modified phosphoramide monomer, whose structural formula is: , , , , , , , , , , .

[0034] Furthermore, the same synthetic procedures apply to the following molecular structures: , ; Wherein: 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, alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl groups with branched structures; LG: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aromatic, cyanoalkyl, cyanoalkylene, alkoxyalkyl, acyloxymethyl, acyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldimethylsilyl. Or tert-butyldiphenylsilyl or other silyl groups; R1, R2: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic groups, etc.; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl or other acyl groups, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl or other silyl groups; R3, R4: halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkyl selenyl, etc.

[0035] Example 2: Solid-phase synthesis Reagents and monomers: Prepare a 0.15 mol / L monomer / acetonitrile solution; the reagent for removing the DMTr (or Tr) protecting group 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 solution of water / pyridine (10 / 90, v / v); the sulfiding agent is a 3% hydroxanthin / 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).

[0036] Solid support: cross-linked polystyrene (PS), and the operation sequence 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.

[0037] Repeat the above operations according to the set sequence to obtain the oligonucleotide product of the target sequence.

[0038] Deprotection involves transferring the solid-phase support loaded with the oligonucleotide product into a reactor, adding concentrated ammonia (25-28%), controlling the temperature at 60℃ 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.

[0039] Purification involves dissolving the deprotected crude product in purified water, purifying it using HPLC, collecting the product peak solution, and then concentrating and lyophilizing the product peak solution under reduced pressure to obtain the final product.

[0040] The above solid-phase synthesis methods are also applicable to monomers protected by Tr, MMTr, and TMTr.

[0041] The 5'-terminus is coupled with the O5'-CH2-phosphate modified nucleotide monomer of the present invention. The synthesis process is described in the appendix. Figure 3 .

[0042] In examples of solid-phase synthesis, the nucleotide monomers used are represented by the following structures: , , , , , , , , , .

[0043] Example 1: The monomer O5'-DMTr-O3'-OCE-T(Tsi) is supported on crosslinked polystyrene PS, followed by monomers A in sequence. m U m U m G m U m U m T m U m Following a cyclical process of de-DMTr, coupling, oxidation, and capping, after the cycle was completed, the oligonucleotide molecule 3'-T-Am-Um-Gm-Um-Um-Tm-Um-Um-A6-5' was obtained after ammonolysis and deprotection treatment. The filtrate was concentrated and purified by HPLC to obtain the designed sequence. (See attached diagram for structure.) Figure 4 .

[0044] The above solid-phase synthesis is also applicable to the nucleotide monomers modified with the O5´-spacer group Y-phosphate of the present invention, wherein Y: one or more methylene CH2, alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, etc. with branched structure.

[0045] Example 2: 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):

[0046] In solid-phase synthesis, the nucleotide monomer A-6 of this invention is used to replace the 5'-terminal Am of the antisense strand of Inclisiran to prepare a 5'-terminal CH2-PO3-carrying material. 2- The antisense strand of Inclisiran is paired equimolarly with the sense strand of Inclisiran to obtain the siRNA structure shown below (labeled as IN-RNA-CH2-PO3):

[0047] The structure of the last two nucleotides at the 5' end of the antisense strand is shown below:

[0048] 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.

[0049] Example 3: 5'-terminal CH2-PO3 2- Activity assay of modified siRNA The dissociation constants of IN-RNA and IN-RNA-CH2-PO3 binding to hAgo2 (human Ago2) protein were tested separately. Affinity assays showed that the latter had an affinity 11.8 times greater than the former. The terminal modification strategy of this invention, 5'-terminal CH2-PO3... 2- The induced modification of siRNA increased its affinity for hAgo2.

[0050] Model mice were administered IN-RNA-CH2-PO3 and IN-RNA at a dose of 5 mg / kg, respectively, as a single dose. The effects of these two treatments on PCSK9 mRNA levels and LDL-C content were analyzed. The study found that in mice, IN-RNA-CH2-PO3 was more effective than IN-RNA in reducing PCSK9 mRNA levels. At 10, 20, and 40 days of age, IN-RNA-CH2-PO3 improved LDL-C levels by 32%, 29%, and 28%, respectively, compared to IN-RNA.

[0051] 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.

[0052] The substances corresponding to the abbreviations in this invention are as follows: CPG: Controlled pore glass; PS: Polystyrene; F: Fluorine; DIPEA: N,N-Diisopropylethylamine; Alkyl: Alkyl; Allyl: Allyl; Alloc: Allyloxycarbonyl; Bn: Benzyl; Bz: Benzoyl; DMTr: Dimethoxytriphenylmethyl; MMTr: Methoxytriphenylmethyl; Tr: Triphenylmethyl; TMTr: Trimethoxytriphenylmethyl; iBu: 2-Isobutyryl; NPE: 4-Nitrophenylethoxy; Ph: Aromatic; TBDMS: Tert-Butyldimethylsilyl; TBDPS: Tert-Butyldiphenylsilyl; DCM: Dichloromethane; DMF: N,N-Dimethylformamide; TBAF: Tetrabutylammonium fluoride; THF: Tetrahydrofuran; TMSI: Trimethyliodosilane; TBAF: Tetrabutylammonium fluoride Ammonium; A: 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; HPLC: high-performance liquid chromatography.

[0053] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to interchangeably. Each embodiment focuses on describing the differences from other embodiments. In particular, the embodiments for 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.

[0054] 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.

[0055] The above content 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. Use of an oligonucleotide having a 5'-end with an intercalated spacer group between the oxygen and the phosphate, characterized in that, The chemical modification strategy includes a modification of O5' and a phospho PO3 2- group Y between the phospho PO3 group Y between the phospho PO3 group Y between the phospho PO3 group Y between the phospho PO3 group Y between the phospho PO3 group Y between the phospho PO3 group Y between the phospho PO3 group Y between the phospho PO3 group Y between the phospho PO3 Formula I Formula II wherein: Base: adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil and the like nucleotide bases and their derivatives; Y: one or more methylene CH2, branched alkyl, alkylene, alkynylene, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl; R: hydrogen, alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl and the like acyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl and the like silyl groups; R1, R2: halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azido, amine group (amino), mercapto, alkylmercapto, seleno, alkylseleno and the like.

2. Use of an oligonucleotide having a 5'-end with an intercalating spacer group between the oxygen and the phosphate according to claim 1, characterized in that, The chemical modification strategy also includes preparing phosphoramidite monomers of modified nucleotides with a spacer group Y embedded between O5' and phosphate P(V), including nucleotide structures shown in Formula III or Formula IV: Formula III Formula IV wherein: Base: adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil and the like nucleotide bases and their derivatives; Y: one or more methylene CH2, branched alkyl, alkylene, alkynylene, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl; LG: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aromatic group, cyanoalkyl, cyanoalkylene, alkoxyalkyl, acyloxymethyl, acyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsiliyl or tert-butyldiphenylsilyl and the like silyl groups; Ri, R2: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkeyl or aromatic group and the like; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl benzyl Bn, benzoyl Bz, acetyl or propionyl and the acyl, alkenyl, cycloalkenyl, alkynyl, allyl group, allyloxycarbonyl, trimethylsilyl, triethylsilyl triisopropylsilyl, tert-butyldimethylsilyl or ter-butyldiphenylsilyl and the like silyl groups; R3, R4: halogen, alkyl, cycloalkyl, alkoxyalkyl, alkyenyl, cycloalkenyl, alkynyl, cyano, azido, amido (amino), mercapto, alkylmercapto, seleno, alkyseleno and the like.

3. Use of an oligonucleotide according to claim 2, wherein the 5'-end is embedded with a spacer group between the oxygen and the phosphate. The synthesis of the phosphoramidite monomers of modified nucleotides with a spacer group Y embedded between O 5'and phosphate P(V) can be converted to other esters or other leaving groups as needed by design, and the protecting group at O 3'is removed to form the phosphoramidite at this position to obtain the phosphoramidite monomers of the modified nucleotides after the substitution reaction of the O 5 '-hydroxyl group of the nucleotide monomers with the sulfonyloxy phosphate.

4. Use of an oligonucleotide according to claim 2, wherein the 5'-end is embedded with a spacer group between the oxygen and the phosphate group. The phosphoramidite monomers of the modified nucleotides are coupled, oxidized or sulfided, deprotected and the like with the hydroxyl, amine, mercapto or seleno groups and the like released from the nucleic acid sequence loaded on the solid phase carrier to construct the 5'-terminal phosphorylated modified oligonucleic acid molecules.

5. Use of an oligonucleic acid with a spacer group intercalated between the 5'-terminal oxygen and the phosphate according to claim 4, wherein the O3'-phosphoramidite of the modified nucleotide phosphoramidite monomer is coupled to the O5'-hydroxyl, amine, thiol or selenol group released from the nucleic acid sequence supported on a solid support.

6. Use of an oligonucleotide according to claim 1, wherein the 5'-end is embedded with a spacer group between the oxygen and the phosphate group. The chemical modification strategy includes preparation of a phosphoramidite monomer of a modified nucleotide with a spacer group intercalated between the O5' and the phosphate P(V), preparation of a terminally modified oligonucleic acid of the same sequence as Inclisiran, introduction of a spacer group between the 5'-terminal oxygen and the phosphate of the modified siRNA antisense strand, dissociation constant of the modified siRNA antisense strand compared to Inclisiran for binding to the Ago2 protein, 5'-terminal phosphorylation of the modified siRNA increases the affinity of the modified siRNA to Ago2.

7. Use of an oligonucleotide according to claim 1, wherein the 5'-end is embedded with a spacer group between the oxygen and the phosphate group. Preparation of a terminally modified oligonucleic acid of the same sequence as Inclisiran, introduction of a spacer group between the 5'-terminal oxygen and the phosphate of the modified siRNA antisense strand, 5'-terminal phosphorylation of the modified siRNA reduces the PCSK9 mRNA level and the LDL-C content in a model animal more significantly than Inclisiran.

8. The 5'-terminal oxygen and phosphodiester gapmer modified oligonucleic acid of claim 1, wherein, The oligonucleic acid is selected from any one or a combination of at least two of a small interfering nucleic acid, an antisense oligonucleic acid, a microRNA, a small activating RNA, a small guide RNA, a transfer RNA, and an aptamer.

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