2apos; application of threose nucleic acid with oxygen and phosphoramidite connected at intervals in oligonucleotides

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses an application of threose nucleic acid with 2-oxygen and phosphoramidite connected through a spacer group in oligonucleotides, and a chemical modification strategy of the application comprises the following steps: preparing a threose nucleic acid phosphoramidite monomer with O2'and P (III) connected at intervals; performing solid-phase synthesis on the modified threose nucleic acid phosphoramidite to construct a target nucleic acid molecule; forming an O2 '-spacer group-P (V) fragment between O2' of threose nucleic acid and phosphonate P (V) in a phosphoric acid skeleton of the nucleic acid sequence; the prepared nucleic acid sequence contains a threose nucleic acid unit of an O2 '-spacer group-P (V), the stability of modified nucleic acid molecules can be effectively improved, and meanwhile the biological performance of nucleic acid is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a threonine with a 2´-oxygen and a phosphonamide intercalating spacer group in the modification of oligonucleotides. Background Technology

[0002] Oligonucleotide drugs bind specifically to target genes through Watson-Crick base complementarity, expanding the drug target to upstream mRNA of pathogenic proteins. They exhibit high specificity, regulating or knocking down target genes and influencing 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] To improve the stability of oligonucleotide molecules, groups of different sizes and polarities are often introduced at the 2'-position. Common examples include 2'-methoxy (or long-chain alkoxy), 2'-methoxyethoxy, and 2'-fluoro. By protecting the 2'-hydroxyl group from hydrolysis, the stability of the nucleic acid molecule is maintained.

[0004] However, existing oligonucleotide drugs obtained through modification methods face challenges in balancing resistance to exonuclease degradation and biological activity. For example, the presence of a 2'-methoxy group at a specific position on the guide chain may reduce activity, while 2'-fluorination may introduce potential toxicity. Therefore, there is a desire to develop novel chemically modified nucleotide monomers and introduce them into nucleic acid drugs to more effectively improve drug stability and enhance their pharmacological, pharmacokinetic, and other biological activities.

[0005] As a highly biostable RNA analog, threonine (TNA) has a simple chemical structure, possesses base-pairing properties, and can fold to form a functional tertiary structure, making it a potential RNA precursor. TNA is characterized by a backbone containing the unconventional four-carbon sugar α-L-threose, with phosphodiester bonds linked near the 2' and 3' positions of the sugar ring. This protects it from nuclease cleavage, enhancing the stability of the oligonucleotide structure and improving its biological activity.

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

[0007] The purpose of this invention is to provide a novel chemical modification strategy for the application of threonines linked to O2'-oxygen and phosphonamide via a spacer group in oligonucleotides. Specifically, this involves the O2' of the threonine being linked to the phosphonamide via a spacer group. This modified phosphonamide is then used in the solid-phase synthesis of nucleic acid drugs. To address the aforementioned problems in the prior art, this invention is achieved through the following technical solution: The application of a threonine linked to a 2´-oxygen and a phosphonamide spacer in oligonucleotides, using chemical modification strategies including: Preparation of modified threonine phosphine amide monomers linked to O2' and P(III) spacers; The modified threonine phosphonamide monomer was incorporated into the oligonucleotide chain via solid-phase synthesis to construct oligonucleotide molecules; An O2'-YP(V) segment is formed by inserting a Y group between the O2' and phosphonate P(V) of the oligonucleotide phosphate backbone: ; Wherein, Base is a nucleotide base such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine and its derivatives; X is oxygen (O) or sulfur (S); Y is one or more methylene (CH2), alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl with a branched structure; R, R', R1, R2, and R3 include, but are not limited to, hydrogen (H), hydroxyl (OH), halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkoxyalkoxy, carbonylalkyl, carbonylalkyloxy, alkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylmercapto, selenyl, and alkylselenoyl.

[0008] A threonine phosphonamide monomer with O2' and P(III) spacers was prepared, with the spacer group represented by Y. After the O2'-hydroxyl group of the threonine underwent a substitution reaction with sulfonyloxyphosphonate, the phosphonate was further converted to a phosphonate ester. After activation, it reacted with amines or imines to obtain a threonine monomer with a Y-intercalated group between O2' and P(III), the structural formula of which is: ; Wherein: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and its derivatives; PG is a protecting group, including but not limited to triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, and tert-butyldimethylsilyl. The following are silyl groups: alkyl or tert-butyldiphenylsilyl; Y is one or more methylene CH2, alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl; LG1 is halogen, amino, cyano, azide, alkyloxy, etc.; LG2 is cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R, R', R1, R2, R3 include but are not limited to hydrogen (H), hydroxyl (OH), halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkoxyalkoxy, carbonylalkyl, carbonylalkyloxy, alkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylselenoyl, etc.

[0009] Preferably, a threononucleotide phosphonamide monomer modified by embedding methylene, ethylene, isopropyl, cyclopropyl, etc. between O2' and P(III) is prepared, wherein Y in the synthetic route is methylene, ethylene, isopropyl, or cyclopropyl; after the O2'-hydroxyl group of threononucleotide undergoes a substitution reaction with sulfonyloxyphosphonate, the phosphonate is condensed with 3-hydroxypropionitrile to obtain a phosphonate ester, which is then activated and reacted with an amine or imine to obtain a threononucleotide phosphonamide monomer modified by embedding methylene, ethylene, isopropyl, cyclopropyl, etc. between O2' and P(III).

[0010] Preferably, the structure of the phosphonamide monomer modifying the nucleotide contains a methylene group, an ethylene group, an isopropyl group, and a cyclopropyl group inserted between O2' and P(III), and its structural formula is as follows: , , , , , ; Wherein: Base: nucleotide bases and their derivatives containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine; PG: silyl groups such as triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, 4,4',4''-trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl; R1, R2: alkyl, alkenyl, alkynyl, cycloalkyl, or aromatic groups.

[0011] Phosphine amide monomers that modify nucleotides are embedded in oligonucleotide chains via solid-phase synthesis. The solid-phase synthesis process includes deprotection, coupling, oxidation or sulfidation, capping, etc., to construct modified oligonucleotide molecules.

[0012] Synthetic routes for constructing oligonucleotide molecules: 1) Deprotection: 3% dichloroacetic acid toluene solution is used to remove the protecting groups such as triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, and 4,4',4''-trimethoxytriphenylmethyl TMTr, and then acetonitrile is used for washing; 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.

[0013] Following the set sequence, phosphorylamine is introduced and the above steps are repeated to obtain the oligonucleotide product of the target sequence.

[0014] Preferably, an oligonucleotide molecule with a modified nucleotide structure is constructed by coupling a nucleotide phosphoryl (phosphonyl)amine or other type of monomer phosphoryl (phosphonyl)amine, phosphoryl (phosphonyl)chloride or phosphoryl (phosphonyl)amine, phosphoryl (phosphonyl)chloride with an embedded spacer group through solid-phase synthesis.

[0015] An oligonucleotide comprising modified threonine, wherein one or more methylene CH2, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, alkynyl, or alkoxyalkyl groups are inserted between O2' and P(III) in the threonine fragment, and the chimeric modified threonine phosphonamide is suitable for preparing such modified oligonucleotide molecules.

[0016] Preferably, the nucleotide structure contains an O2' spacer linked to a phosphonate ester, and the O3' protecting group is deprotected to release the hydroxyl group at that position, and is coupled with other types of monomers such as phosphorylamine, phosphoryl chloride, or phosphorylamine and phosphoryl chloride as required by the sequence design.

[0017] Preferably, the O3' protecting group of the modified threonine nucleic acid is of the same type as the protecting group at the coupling site of the other monomers phosphoryl (phosphonyl)amine, phosphoryl (phosphonyl)chloride or phosphoryl (phosphonyl)amine, phosphoryl (phosphonyl)chloride. By synthesizing nucleic acid molecules in a solid phase, it is convenient to use the same or similar deprotection conditions and monitor the coupling efficiency of this round online.

[0018] The prepared oligonucleotide sequence includes a threonine structure with O2' and phosphonate ester spacers. This modified structure can improve the biostability and anti-enzymatic degradation activity of oligonucleotides and enhance the bioactivity of oligonucleotide drugs.

[0019] 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, primer RNAs, transfer RNAs, and aptamers.

[0020] The beneficial effects of this invention are as follows: This invention provides a method for preparing a threonium phosphonamide monomer with O2' and phosphonate P(III) spacer linkage and its application in oligonucleotide drugs; specifically, the oxygen at the O2' position is linked to phosphonamide P(III) through a spacer group, and the modified threonium monomer is embedded in the oligonucleotide chain through solid-phase synthesis; the constructed oligonucleotide drug has O2' of the threonium fragment in its phosphate backbone linked to phosphonate P(V) through a spacer group to form an O2'-spacer group-P(V) fragment; since O2' and phosphonate diester P(V) do not directly form an ester bond, it belongs to a non-natural form of phosphate (phosphonate) ester, thus the synthesized oligonucleotide molecule exhibits higher stability, which can effectively improve the stability of the modified oligonucleotide drug molecule while optimizing pharmacokinetic (PK) and pharmacodynamic (PD) performance.

[0021] 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

[0022] 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.

[0023] Figure 1 This invention provides a synthetic route for preparing a threonine phosphine amide monomer with O2' and P(III) linked by a spacer group; Figure 2 The structure of the oligonucleotide ON-11-1 synthesized from the nucleotide monomers of this invention in the solid phase (control sequence); Figure 3 The solid-phase synthesis of the oligonucleotide ON-11-2 structure (complementary sequence) of the nucleotide monomer of this invention is shown. Figure 4 The structure of the oligonucleotide ON-11-C0 synthesized from the nucleotide monomer of this invention is shown. Figure 5 The structure of the oligonucleotide ON-11-C1 synthesized from the nucleotide monomer of this invention is shown. Figure 6 The structure of the oligonucleotide ON-11-C2 is obtained by solid-phase synthesis of the nucleotide monomer of this invention. Detailed Implementation

[0024] 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.

[0025] This invention relates to the application of threonine nucleic acid with 2´-oxygen and phosphonamide intercalation spacer groups in the modification of oligonucleotides; specifically, through a novel chemical modification strategy for nucleic acid drugs, in the phosphate backbone of the nucleic acid sequence, the O2' of the threonine nucleic acid fragment is linked to the phosphonate P(V) through a spacer group.

[0026] In the phosphate backbone of the nucleic acid sequence, a spacer group Y is inserted between the O2' of the threononucleotide fragment and the phosphonate P(V), forming the O2'-YP(V) fragment (the structure is shown below). This can effectively improve the stability of the modified oligonucleotide drug molecule and is also used to optimize the pharmacokinetic (PK) and pharmacodynamic (PD) performance.

[0027] ; Wherein, Base is a nucleotide base such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine and its derivatives; X is oxygen (O) or sulfur (S); Y is one or more methylene (CH2), alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl with a branched structure; R, R', R1, R2, and R3 include, but are not limited to, hydrogen (H), hydroxyl (OH), halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkoxyalkoxy, carbonylalkyl, carbonylalkyloxy, alkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylmercapto, selenyl, and alkylselenoyl.

[0028] For the synthetic route to prepare the threonine phosphine amide monomer with O2' and P(III) linked by the spacer group Y, please refer to [link to synthetic route]. Figure 1 As shown, Base: nucleotide bases containing protecting groups such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudouracil; PG: protecting groups such as triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, and 4,4',4''-trimethoxytriphenylmethyl TMTr; Y: - One or more methylene groups (CH2), branched alkyl, alkylene, trialkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl; R1, R2, R3: alkyl, alkenyl, alkynyl, cycloalkyl or aromatic groups, etc.; LG: cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.

[0029] The process for preparing O2'-hydroxymethylenethreonine nucleic acid phosphamide by liquid-phase synthesis is as follows: Example 1: O2'-hydroxymethylene adenine threonine nucleosides phosphonamide

[0030] 3.219 g A-0 (5 mmol, 1.0 eq.) and 2.041 g sodium phosphonate P-Cl-Ts (7.5 mmol, 1.5 eq.) were dissolved in 50 mL of dry DMF. The mixture was cooled to 0 °C and stirred. Under nitrogen protection, 0.440 g NaH (60% dispersed in mineral oil, 11 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 2.383 g of a white solid A-1-1 (66% yield).

[0031] 2.165 g of A-1-1 (3 mmol, 1.0 eq.), 1.424 g of DCC (6.9 mmol, 2.3 eq.), and 0.618 g of 3-hydroxypropionitrile (8.7 mmol, 2.9 eq.) were dissolved in 60 mL of dry 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 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 with solid sodium sulfate, and then concentration followed by silica gel column chromatography to give a white solid, A-1-2.

[0032] 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 1.1 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 A-1-2 and 1.10 mL of pyridine dissolved in 30 mL of dichloromethane was added, and the mixture was stirred for 4–6 hours. The temperature was lowered to -40 °C, and a mixture of 2.4 mL of diisopropylamine (17.1 mmol, 5.7 eq.) and 3 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. 30 mL of ethyl acetate was added to the residue, and after washing with 10 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 hexane / ethyl acetate mixed solvent (containing 0.5% triethylamine) as the mobile phase, yielding 1.262 g of the target product A-1-3 (purity 99.1%, two-step yield 49%).

[0033] Example 2: O2'-hydroxymethyleneuracil threonine phosphinoamide

[0034] 2.583 g U-0 (5 mmol, 1.0 eq.) and 2.041 g sodium phosphonate P-Cl-Ts (7.5 mmol, 1.5 eq.) were dissolved in 50 mL of dry DMF. The mixture was cooled to 0 °C and stirred. Under nitrogen protection, 0.440 g NaH (60% dispersed in mineral oil, 11 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 1.872 g of white solid U-1-1 (63% yield).

[0035] 1.784 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 dry 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 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 with solid sodium sulfate, and then concentration followed by silica gel column chromatography to give a white solid U-1-2.

[0036] 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 1.1 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 1.10 mL of pyridine dissolved in 30 mL of dichloromethane was added, and the mixture was stirred for 4–6 hours. The temperature was lowered to -40 °C, and a mixture of 2.4 mL of diisopropylamine (17.1 mmol, 5.7 eq.) and 3 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. 30 mL of ethyl acetate was added to the residue, and after washing with 10 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 hexane / ethyl acetate mixed solvent (containing 0.5% triethylamine) as the mobile phase, yielding 1.163 g of the target product U-1-3 (purity 99.0%, two-step yield 53%).

[0037] Furthermore, the same synthetic procedure applies to the following molecular structures, i.e., the group between O2' and P(III) in the modified threonine phosphonamide monomer is methylene CH2, and its structural formula includes: , , , , , , , , , , , .

[0038] The process for preparing O2'-hydroxyethylidene threonine nucleic acid phosphonamide by liquid-phase synthesis is as follows: Example 3: O2'-hydroxyethylidene adenine threonine nucleoside phosphonamide

[0039] 3.219 g of A-0 (5 mmol, 1.0 eq.) and 2.147 g of sodium phosphonate P-C2-Ts (7.5 mmol, 1.5 eq.) were dissolved in 50 mL of dry DMF. The mixture was cooled to 0 °C and stirred. Under nitrogen protection, 0.440 g of NaH (60% dispersed in mineral oil, 11 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 2.501 g of a white solid A-2-1 (68% yield).

[0040] 2.207 g A-2-1 (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 dry 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 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 with solid sodium sulfate, and then concentration followed by silica gel column chromatography to give a white solid, A-2-2.

[0041] 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 1.1 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 A-2-2 and 1.10 mL of pyridine dissolved in 30 mL of dichloromethane was added, and the mixture was stirred for 4–6 hours. The temperature was lowered to -40 °C, and a mixture of 2.4 mL of diisopropylamine (17.1 mmol, 5.7 eq.) and 3 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. 30 mL of ethyl acetate was added to the residue, and after washing with 10 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 hexane / ethyl acetate mixed solvent (containing 0.5% triethylamine) as the mobile phase, yielding 1.466 g of the target product A-2-3 (purity 99.0%, two-step yield 56%).

[0042] Example 4: O2'-hydroxyethylideneuracil threonine nuclease phosphonamide

[0043] 2.583 g U-0 (5 mmol, 1.0 eq.) and 2.147 g sodium phosphonate P-C2-Ts (7.5 mmol, 1.5 eq.) were dissolved in 50 mL of dry DMF. The mixture was cooled to 0 °C and stirred. Under nitrogen protection, 0.440 g NaH (60% dispersed in mineral oil, 11 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 1.979 g of white solid U-2-1 (65% yield).

[0044] 1.826 g U-2-1 (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 dry 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 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 with solid sodium sulfate, and then concentration followed by silica gel column chromatography to give a white solid U-2-2.

[0045] 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 1.1 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-2-2 and 1.10 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 2.4 mL of diisopropylamine (17.1 mmol, 5.7 eq.) and 3 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. 30 mL of ethyl acetate was added to the residue, and after washing with 10 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 hexane / ethyl acetate mixed solvent (containing 0.5% triethylamine) as the mobile phase, yielding 1.141 g of the target product U-2-3 (purity 99.2%, two-step yield 51%).

[0046] Furthermore, the same synthetic procedure applies to the following molecular structures, i.e., the group between O2' and P(III) in the modified threonine phosphonamide monomer is ethylene CH2CH2, and its structural formula includes: , , , , , , , , , , , .

[0047] Furthermore, similar synthetic procedures are applicable to the following molecular structure, namely the modified threonine phosphonamide, with the following structural formula: , , , , , ; Wherein: Base: Nucleotide bases and their derivatives containing adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine.

[0048] Furthermore, similar synthetic procedures are applicable to the following molecular structure: a threonine phosphonamide with O2´ linked to P(III) via a spacer group, with the following structural formula: ; 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; R1, R2: alkyl, alkenyl, alkynyl, cycloalkyl, or aromatic groups, etc.

[0049] Furthermore, similar synthetic procedures are applicable to the following molecular structures, namely, threonine monomers in which O2´ is linked to P(III) via a spacer group, with the following structural formula: ; Wherein: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and its derivatives; PG is a protecting group, including but not limited to triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl; Y is one or more methylene CH2, branched alkyl, alkylene, mesylate, cycloalkyl, or alkenyl groups. 1. Cycloalkenyl, alkynyl, alkoxyalkyl; LG1 is halogen, amino, cyano, azide, alkyloxy, etc.; LG2 is cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R, R', R1, R2, R3 include but are not limited to hydrogen (H), hydroxyl (OH), halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkoxyalkoxy, carbonylalkyl, carbonylalkyloxy, alkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylselenoyl, etc.

[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 phosphorylated (phosphonyl)amine monomers was performed 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] 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.

[0056] The above solid-phase synthesis methods are also applicable to monomers protected by Tr, MMTr, and TMTr.

[0057] The nucleotide monomers used in the solid-phase synthesis examples are represented by the following structures: , , , , , , , , , , , , , .

[0058] Example 5:

[0059] ; Monomer U-3-0 was loaded onto cross-linked polystyrene (PS). O5'-DMTr was removed using a 3% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-0-3 was pumped in for coupling. A 3% hydroxanthin solution in pyridine converted the phosphite to a thiophosphate ester, and the system was capped. O3'-DMTr was removed using a 3% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-3-3 was pumped in for coupling. A 0.05 mol / L iodine solution in water / pyridine oxidized the phosphite to a phosphate ester, and O5'-DMTr was removed using a 3% dichloroacetic acid-toluene solution.

[0060] 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.

[0061] Example 6:

[0062] ; Monomer U-3-0 was loaded onto cross-linked polystyrene (PS). O5'-DMTr was removed using a 3% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-1-3 was pumped in for coupling. A 3% hydroxanthin pyridine solution converted the phosphonite to a thiophosphonate, and the system was capped. O3'-DMTr was removed using a 3% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-3-3 was pumped in for coupling. A 0.05 mol / L iodine-water / pyridine solution oxidized the phosphonite to a phosphate ester, and O5'-DMTr was removed using a 3% dichloroacetic acid-toluene solution.

[0063] 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.

[0064] Example 7:

[0065] ; Monomer U-3-0 was loaded onto cross-linked polystyrene (PS). O5'-DMTr was removed using a 3% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-1-3 was pumped in for coupling. A 0.05 mol / L iodine-water / pyridine solution oxidized the phosphonite to phosphonate, and the system was capped. O3'-DMTr was removed using a 3% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-2-3 was pumped in for coupling. A 0.05 mol / L iodine-water / pyridine solution oxidized the phosphonite to phosphonate, and O5'-DMTr was removed using a 3% dichloroacetic acid-toluene solution.

[0066] 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.

[0067] Example 8:

[0068] ; Monomer U-3-0 was loaded onto cross-linked polystyrene (PS). O5'-DMTr was removed using a 3% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-1-3 was pumped in for coupling. A 0.05 mol / L iodine-water / pyridine solution oxidized the phosphonite to phosphonate, and the system was capped. O3'-DMTr was removed using a 3% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-2-3 was pumped in for coupling. A 3% hydroxanthin-pyridine solution converted the phosphonite to thiophosphonate, and O2'-DMTr was removed using a 3% dichloroacetic acid-toluene solution.

[0069] 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.

[0070] Example 9:

[0071] Monomer U-3-0 was loaded onto cross-linked polystyrene (PS). Monomers U-3-3, A-3-3, A-3-3, C-3-3, U-3-3, U-3-3, C-3-3, A-3-3, U-3-3, and U-3-3 were subjected to a cyclical process of de-DMTr, coupling, oxidation, and capping. After the cycle was completed, ammonolysis and deprotection were performed. The concentrated filtrate was then purified by HPLC, and the main fraction was collected, desalted, concentrated, and lyophilized to obtain the oligonucleotide molecule 3'-UUAACUUCAUU-5' with the designed sequence, labeled ON-11-1. The structure is shown in the appendix. Figure 2 .

[0072] Example 10: Monomer A-3-0 was loaded onto cross-linked polystyrene (PS). Monomers A-3-3, U-3-3, G-3-3, A-3-3, A-3-3, G-3-3, U-3-3, U-3-3, A-3-3, and A-3-3 were subjected to a cyclical process of de-DMTr, coupling, oxidation, and capping. After the cycle was completed, ammonolysis and deprotection were performed. The concentrated filtrate was then purified by HPLC, and the main fraction was collected, desalted, concentrated, and lyophilized to obtain the oligonucleotide molecule 3'-AAUGAAGUUAA-5' with the designed sequence, labeled ON-11-2. The structure is shown in the appendix. Figure 3 .

[0073] Example 11: Monomer U-3-0 was loaded onto cross-linked polystyrene (PS). Monomers U-0-3, A-0-3, A-3-3, C-3-3, U-3-3, U-3-3, C-3-3, A-0-3, U-0-3, and U-3-3 were subjected to a cyclical process of de-DMTr, coupling, oxidation, and capping. After the cycle was completed, ammonolysis and deprotection were performed. The concentrated filtrate was then purified by HPLC. The main fraction was collected, desalted, concentrated, and lyophilized to obtain an oligonucleotide molecule with the same nucleotide base sequence as in Example 9, labeled ON-11-CO. The structure is shown in the appendix. Figure 4 .

[0074] Example 12: Monomer U-3-0 was loaded onto cross-linked polystyrene (PS). Monomers U-1-3, A-1-3, A-3-3, C-3-3, U-3-3, U-3-3, C-3-3, A-1-3, U-1-3, and U-3-3 were subjected to a cyclical process of de-DMTr, coupling, oxidation, and capping. After the cycle was completed, ammonolysis and deprotection were performed. The concentrated filtrate was then purified by HPLC. The main fraction was collected, desalted, concentrated, and lyophilized to obtain an oligonucleotide molecule with the same nucleotide base sequence as in Example 9, labeled ON-11-C1. The structure is shown in the appendix. Figure 5 .

[0075] Example 13: Monomer U-3-0 was loaded onto cross-linked polystyrene (PS). Monomers U-2-3, A-2-3, A-3-3, C-3-3, U-3-3, U-3-3, C-3-3, A-2-3, U-2-3, and U-3-3 were subjected to a cyclical process of de-DMTr, coupling, oxidation, and capping. After the cycle was completed, ammonolysis and deprotection were performed. The concentrated filtrate was then purified by HPLC. The main fraction was collected, desalted, concentrated, and lyophilized to obtain an oligonucleotide molecule with the same nucleotide base sequence as in Example 9, labeled ON-11-C2. The structure is shown in the appendix. Figure 6 .

[0076] Example 14: Demothering temperature of oligonucleotide chains T m test ON-11-1 from Example 9, ON-11-C0 from Example 11, ON-11-C1 from Example 12, and ON-11-C2 from Example 13 were mixed in equimolar amounts with ON-11-2 from Example 10 and labeled as ON-11-1-D, ON-11-C0-D, ON-11-C1-D, and ON-11-C2-D, respectively. Double-stranded oligonucleotide samples were prepared in a buffer solution of 10 mmol / L sodium phosphate and 0.1 mol / L NaCl, with a double-strand concentration of 3.0 μmol / L. Using a UV-Vis spectrophotometer with a cuvette path length of 1.0 cm and a sample temperature increase of 0.5 °C per minute, the thermally induced transition sigmoid curves of the four complementary double strands were monitored at 260 nm.

[0077] Analysis of melting temperature T m It was found that the Tm of the ON-11-C0-D double strand, ON-11-C1-D double strand, and ON-11-C2-D double strand increased by 4.1 ℃, 6.4 ℃, and 7.6 ℃, respectively, compared to the ON-11-1-D double strand. Experimental data indicate that oligonucleotides containing O2´-methylene and ethylidene threonine phosphonic acid structures can effectively improve the binding affinity of modified oligonucleotide molecules to target sequences.

[0078] Example 15: The stability of the nucleic acid molecules ON-11-1 from Example 9, ON-11-C0 from Example 11, ON-11-C1 from Example 12, and ON-11-C2 from Example 13 in human serum and human liver microsomes were tested, respectively.

[0079] Compared with the control sequence ON-11-1, the results showed that after 24 hours, the proportions of undegraded nucleic acid sequences ON-11-C0, ON-11-C1, and ON-11-C2 in human serum increased by 23%, 38%, and 43%, respectively, while the proportions in human liver microsomes increased by 29%, 52%, and 49%, respectively. The experimental data indicate that oligonucleotides containing O2´-methylene and ethylidene threononucleotide phosphonic acid structures effectively improved the enzyme resistance and stability of the modified oligonucleotide molecules.

[0080] In summary, this invention provides a method for preparing a threononucleotide phosphonamide monomer with 2'-oxygen and phosphonamide spacers, and its application in the solid-phase synthesis of nucleic acid drugs. Introducing the modified nucleotide unit provided by this invention can improve the stability of nucleic acid molecules and help 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-C1-Ts: sodium ((tosyloxy)methyl)phosphinate; P-C2-Ts: sodium ((tosyloxy)ethyl)phosphinate; CPG: controlled pore glass. (glass); PS: Polystyrene; F: Fluorine; S: Sulfur; DIPEA: N,N-Diisopropylethylamine; Alkyl: Alkyl; Allyl: Allyl; Alloc: Allyloxycarbonyl; Bn: Benzyl; Bz: Benzoyl; Tr: Triphenylmethyl; MMTr: Methoxytriphenylmethyl; DMTr: Dimethoxytriphenylmethyl; 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; A: Adenine; G: Guanine; C: Cytosine; T: Thymine Thymine; U: Uracil; I: hypoxanthine; Xan: xanthine; m5C: 5-methylcytosine; Ψ: pseudouracil; m1Ψ: N1-methylpseudouracil; HPLC: high performance liquid chromatography; P(III): trivalent phosphorus (phosphine); P(V): pentavalent phosphorus (phosphine); RNA: ribonucleic acid; TNA: threonucleic acid.

[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 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. The application of a threononucleotide linked to a 2'-oxygen and a phosphonamide spacer in oligonucleotides, characterized in that, The chemical modification strategy involves linking the O2' of threonine nucleic acid to a phosphonate ester via a spacer group, including the modified threonine nucleic acid structure shown in Formula I: ... Formula I; Wherein, Base is a nucleotide base such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine and its derivatives; X is oxygen (O) or sulfur (S); Y is one or more methylene (CH2), alkyl, alkylene, mesylate, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or alkoxyalkyl with a branched structure; R, R', R1, R2, and R3 include, but are not limited to, hydrogen (H), hydroxyl (OH), halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkoxyalkoxy, carbonylalkyl, carbonylalkyloxy, alkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylmercapto, selenyl, and alkylselenoyl.

2. The application of the threononucleotide linked to 2'-oxygen and phosphonamide as described in claim 1 in oligonucleotides, characterized in that, The chemical modification strategy includes preparing a threonine monomer in which O2' and P(III) are linked by a spacer group Y, comprising the nucleotide structure shown in Formula II: ...Formula II; Wherein: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine, and its derivatives; PG is a protecting group, including but not limited to triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl; Y is one or more methylene CH2, branched alkyl, alkylene, mesylate, cycloalkyl, or alkenyl groups.

1. Cycloalkenyl, alkynyl, alkoxyalkyl; LG1 is halogen, amino, cyano, azide, alkyloxy, etc.; LG2 is cyanoethyl, cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, phenethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.; R, R', R1, R2, R3 include but are not limited to hydrogen (H), hydroxyl (OH), halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkoxyalkoxy, carbonylalkyl, carbonylalkyloxy, alkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkylselenoyl, etc.

3. The application of the threononucleotide linked by 2'-oxygen and phosphonamide as described in claim 2 in oligonucleotides, characterized in that, The synthesis of the threonine phosphonamide monomer, in which O2' and P(III) are linked by a spacer group Y, involves the substitution reaction of the O2'-hydroxyl group of threonine with sulfonyloxyphosphonate, followed by condensation with 3-hydroxypropionitrile to obtain a phosphonate ester, which is then activated and reacted with an amine or imine to obtain a modified nucleotide phosphonamide monomer.

4. The application of the threononucleotide linked by 2'-oxygen and phosphonamide as described in claim 2 in oligonucleotides, characterized in that, In the structure of the phosphonamide monomer of the modified nucleotide, O2' and P(III) are linked through methylene, ethylene, isopropyl, cyclopropyl, etc., and its structural formula is: 、 、 、 、 、 ； Wherein: Base: nucleotide bases and their derivatives containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorazine; PG: silyl groups such as triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, 4,4',4''-trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl; R1, R2: alkyl, alkenyl, alkynyl, cycloalkyl, or aromatic groups.

5. The application of the threononucleotide linked by 2'-oxygen and phosphonamide as described in claim 1 in oligonucleotides, characterized in that, The modified threonine phosphonamide is embedded in the oligonucleotide chain via solid-phase synthesis. After coupling, oxidation or sulfidation, and capping of the 2' end of the threonine with the solid-loaded nucleic acid chain, the 3'-oxygen protecting group is removed, releasing the 3'-hydroxyl group of the threonine, which is then coupled with other monomers such as phosphoryl (phosphonamide), phosphoryl (phosphonyl) chloride, or phosphoryl (phosphonamide), phosphoryl (phosphonyl) chloride to construct the modified nucleotide structure oligonucleotide molecule.

6. The application of the threononucleotide linked by 2'-oxygen and phosphonamide as described in claim 5 in oligonucleotides, characterized in that, The O3' protecting group of the modified threonine nucleic acid is of the same type as the protecting group at the coupling site of other monomers such as phosphorylamine, phosphoryl chloride, or phosphorylamine or phosphoryl chloride. By synthesizing nucleic acid molecules in the solid phase, it is convenient to use the same or similar deprotection conditions and monitor the coupling efficiency of this round online.

7. An oligonucleic acid, based on the application of a threononucleotide linked to a 2'-oxygen and phosphonamide spacer as described in any one of claims 1 to 6, characterized in that, The threonine contains one or more methylene CH2, branched alkyl, alkylene, mesylate, cycloalkyl, alkenyl, alkynyl, or alkoxyalkyl groups between O2' and P(III). This modified phosphonamide is suitable for preparing nucleic acid sequences including threonines with phosphonate ester modifications at the 2' end.

8. The application of the threononucleotide linked to 2'-oxygen and phosphonamide spacers according to claim 1 in oligonucleotides, characterized in that, The prepared oligonucleotide molecule contains a threonine structure with O2' and phosphonate ester spacers, which is used to improve the binding ability of the modified oligonucleotide to the target sequence.

9. The application of the threononucleotide linked by 2'-oxygen and phosphonamide spacers according to claim 1 in oligonucleotides, characterized in that, The prepared oligonucleotide molecules contain a threonine structure with O2' and phosphonate ester spacers, which is used to improve the biostability and resistance to enzyme degradation of the modified oligonucleotides.

10. The application of the threononucleotide linked to 2'-oxygen and phosphonamide spacers according to claim 1 in oligonucleotides, characterized in that, 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, primer RNAs, transfer RNAs, and aptamers.

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