WO2021000380A1 - Modified nucleoside, nucleotide and modified nucleic acid polymer, and preparation method therefor and application thereof - Google Patents

Abstract

The present disclosure relates to the technical field of nucleic acid medicines, in particular to a modified nucleoside, a nucleotide and a modified nucleic acid polymer, as well as a preparation method therefor and an application thereof. The modified nucleoside is selected from a compound having the following structure or a salt thereof: formula (1). According to the modified nucleoside of the present disclosure, an azido group, an amino group or an amine group and the like are introduced at a 4' position by means of modifying the nucleoside, and a novel modified nucleoside structure is obtained. The modified nucleoside may be further modified to obtain the nucleotide and the modified nucleic acid polymer, and has good ribozyme tolerance.

Description

Modified nucleosides, nucleotides and modified nucleic acid polymers, and preparation methods and applications thereof

Cross references to related applications

This application requires the priority of the Chinese patent application filed with the State Intellectual Property Office of China with the application number 201910586273.1, titled "Modified Nucleosides, Nucleotides and Modified Nucleic Acid Polymers and Their Preparation Methods and Applications" on July 1, 2019 Right, the entire contents of which are incorporated in this application by reference.

Technical field

The present disclosure relates to the technical field of nucleic acid medicines, in particular to a modified nucleoside, nucleotide and modified nucleic acid polymer, and a preparation method and application thereof.

Background technique

Nucleic acid chemical modification is a key technology and bottleneck for the development of nucleic acid drugs. Compared with traditional small molecule and protein drugs, nucleic acid drugs have the advantages of fast design, universal target, high specificity, can play a role in cells, and relatively fast synthesis and preparation, which can break through the major advantages of protein targets, which are difficult to make drugs. Disease treatment, especially in response to special cases and emerging infectious diseases, the rapid design and preparation of nucleic acid drugs is of unique value. For example, Tekmira developed a siRNA drug TKM targeting West African Ebola virus within 8 weeks. -Ebola-Guinea.

However, for natural oligonucleotide molecules to be effective therapeutic drugs, the following problems must be overcome:

(1) The in vivo stability is poor, and the natural phosphodiester bond oligonucleotides can be rapidly degraded by various ribozymes widely existing in blood and cells in vivo.

(2) The binding affinity and specificity of the target gene are low, and the "off-target" effects are prone to occur, resulting in immune stimulation and toxic side effects, especially liver toxicity.

(3) Low bioavailability. Oligonucleotides are usually multivalent anionic macromolecules. Therefore, it is difficult to enter target organs and tissues, and it is difficult to enter cells through lipophilic cell membranes.

In response to the above problems, since the 1970s, researchers have developed many successful oligonucleotide structural modification strategies by modifying the phosphate backbone, sugar bases, and bases, such as phosphorothioate, 2'-modified 2'- OMe, 2'-OMOE, 2'-F, Morpholino, PNA, etc., greatly improve the tolerance of oligonucleotides to nucleases, the affinity and specificity of target genes, reduce immune stimulation and side effects, and promote With the development of nucleic acid drugs, most of the nucleic acid drugs that have been marketed have used corresponding nucleic acid chemical modifications.

In view of this, in order to develop a new type of nucleic acid chemical modification structure, the present disclosure is proposed.

Summary of the invention

The purpose of the present disclosure is, for example, to provide a modified nucleoside, which can be used for modification of nucleic acid drugs and the like, and helps to improve the ribozyme tolerance of nucleic acid drugs. At the same time, modified nucleosides can be modified to obtain nucleotides and modified nucleic acid polymers, which have good ribozyme tolerance.

The present disclosure provides a modified nucleoside selected from compounds having the following structures or salts thereof:

Where R _{1 is} selected from

And their respective salts;

n is selected from 1, 2, 3, 4, 5 and 6;

R _{2 is} selected from azido, amino, amine or amide;

W is selected from H or a protecting group;

X is selected from H, α-configuration OH, β-configuration OH, α-configuration F or β-configuration F.

The protecting group is 4,4'-dimethoxytrityl (DMTr).

In the modified nucleoside of the present disclosure, a novel modified nucleoside structure is obtained by modifying the nucleoside to introduce an azide group, an amino group or an amino group at the 4'position. The modified nucleoside can be further modified to obtain nucleotides and modified oligonucleotides, etc., which have good ribozyme tolerance.

In one or more embodiments, the amine group is -NR ₃ R ₄ , and R ₃ and R ₄ are each independently selected from H, an alkyl group with a carbon number of 1-6, or a fluorescent group. R ₃ and R _{4 are} not H at the same time. If R ₃ and R ₄ are H at the same time, it is amino -NH ₂ .

In one or more embodiments, the amine group can be

In one or more embodiments, the R ₃ and R ₄ are each independently selected from alkyl groups having 1 to 5 carbon atoms. In one or more embodiments, the R ₃ and R ₄ are each independently selected from alkyl groups having 1 to 4 carbon atoms.

In one or more embodiments, the amide group is -NHCOR ₅ , and R _{5 is} selected from an alkyl group with a carbon number of 1-6 or a haloalkyl group with a carbon number of 1-6. R ₅ can use -CF ₃ , -CHF ₂ , -CH ₂ F, -CH ₂ CF ₃ , -CH ₂ CHF ₂ , -CH ₂ CH ₂ F, -C ₂ H ₄ CF ₃ , -C ₂ H ₄ CHF _2. -C ₂ H ₄ CH ₂ F and so on. For example, the amide group is -NHCOCF ₃ .

In one or more embodiments, R ₂ is an amide group.

In one or more embodiments, the fluorescent group may be pyrene and its derivatives.

Wherein, when X is H, the structure of the modified nucleoside is:

When X is a hydroxyl OH in α configuration, the structure of the modified nucleoside is:

When X is the hydroxyl OH in β configuration, the structure of the modified nucleoside is:

When X is fluorine F in α configuration, the structure of the modified nucleoside is:

When X is fluorine F in β configuration, the structure of the modified nucleoside is:

In one or more embodiments, X is selected from F in α configuration or F in β configuration.

The 2'position is modified with an F atom, which can form a C-H...F-C pseudo hydrogen bond with the base adjacent to the 3'end in the modified oligonucleotide, which is easy to form a more stable double strand and can improve the affinity.

In one or more embodiments, R ₁ is

Any of them. For example, R ₁ is

The salt in the present disclosure refers to the corresponding salt of the above-mentioned modified nucleoside compound that can be conveniently or desirably prepared, purified and/or processed, for example, a pharmaceutically acceptable salt.

For example, if the modified nucleoside compound is cationic, or has a functional group that can be cationic (for example, -NH ₂ can be -NH ₃ ⁺ ), then a salt can be formed with a suitable anion. Suitable inorganic anions include, but are not limited to, anions derived from the following inorganic acids: hydrochloric acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, phosphorous acid, hydrobromic acid, hydroiodic acid, and the like. Examples of suitable organic anions include, but are not limited to, anions derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, succinic acid, p-aminobenzenesulfonic acid, tartaric acid, etc. . It is also possible to use, for example, polymer organic anions derived from sodium carboxymethyl cellulose.

Unless otherwise indicated, references to specific compounds in this disclosure also include their salt forms.

The present disclosure also provides a nucleotide, which is a 3'-phosphoramidite derivative of the above-mentioned modified nucleoside or a salt thereof.

Specifically, its structural formula can be

Or its salt form, wherein W is selected from H and a protecting group, such as 4,4'-dimethoxytrityl (DMTr).

In one or more embodiments, R _{2 is} selected from amino and amine groups.

Using standard synthesis methods, the above-mentioned nucleotide compounds can be inserted into the oligonucleotide sequence to obtain modified oligonucleotides. For example, a standard phosphoramidite method can be used to insert the above-mentioned nucleotide compound into the oligonucleotide sequence through an automatic synthesizer to obtain a modified oligonucleotide.

The present disclosure also provides a modified nucleic acid polymer, which includes at least one modified nucleotide having the following structure:

Wherein Y is selected from O or S, and R _{3 is} selected from aryl, methyl, substituted alkyl or alkenyl. As the aryl group, phenyl, benzyl, halogenated phenyl and the like can be used. As the substituted alkyl group, ethyl, propyl, isopropyl, haloalkyl, 2-cyanoethyl and the like can be used.

In one or more embodiments, the modified nucleic acid polymer includes ribonucleic acid, deoxyribonucleic acid, or a copolymer of ribonucleotide and deoxyribonucleotide. For example, the modified nucleic acid polymer is an oligonucleotide.

The nucleic acid polymer may refer to any nucleic acid molecule, including but not limited to DNA, RNA and hybrids thereof, including but not limited to single-stranded and double-stranded, etc. The number of nucleotides polymerized to form a nucleic acid is 2, 3 or more, and it can be an oligonucleotide with 20 or less nucleotides, or a polymer with 20 or more nucleotides.

Wherein, the part between the wavy lines in the modified nucleic acid polymer is a structure embedded in the sequence of the nucleic acid polymer, and the part outside the wavy line represents other sequences in the nucleic acid polymer.

In one or more embodiments, the oligonucleotide is selected from one or more of the following sequences:

SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.

SEQ ID NO: 1 is GCGTTTTTTGCT, SEQ ID NO: 2 is GCGTTGTTTGCT, and SEQ ID NO: 3 is GCGTTATTTGCT.

In one or more embodiments, the modified oligonucleotide is selected from one or more of the following sequences:

Modification at the 6th position of the 5'end of SEQ ID NO: 1 is GCGTT T TTTGCT, and modification at the 6th and 8th positions of the 5'end of SEQ ID NO: 1 is GCGTT T T T TGCT, in SEQ ID NO ：The 4th, 6th and 8th positions of the 5'end of 1 are modified, namely GCG T T T T T T TGCT, which is carried out at the 5th, 7th and 9th positions of the 5'end of SEQ ID NO:1 Modification is GCGT T T T T T T GCT, and modification is GCGT T GTTTGCT at position 5 of the 5'end of SEQ ID NO: 2, and modification is GCGTTG T TTGCT at position 7 of 5'end of SEQ ID NO: 2, Modification at position 5 of the 5'end of SEQ ID NO: 3 is GCGT T ATTTGCT, and modification at position 7 of the 5'end of SEQ ID NO: 3 is GCGTTA T TTGCT; the underlined bases are modified by replacement The site, T , is modified with nucleotide substitutions of the present disclosure.

The present disclosure also provides a method for preparing modified nucleosides, including the following steps:

When W is H and R ₂ is an azido group, the preparation method includes: compound I undergoes iodination reaction under the action of a catalyst to obtain compound II; compound II undergoes elimination reaction under alkaline conditions to obtain compound III; compound III, 2 -Azidoalkyl alcohol and iodine are added to obtain compound IV; 3'-OH of compound IV is protected with benzoyl to obtain compound V; 5'-I of compound V is oxidized by m-chloroperoxybenzoic acid, Aminolysis to obtain compound VI;

When W is a protecting group and R ₂ is an azido group, the preparation method includes: protecting the 5'-OH of compound VI obtained by the above method with a protecting group to obtain compound VII;

When W is a protecting group and R ₂ is an amino group, the preparation method includes: reducing the azide group in compound VII to obtain compound VIII;

When W is a protecting group, R ₂ is an amino group, R _{3 is} selected from H, and R _{4 is} selected from an alkyl group with a carbon number of 1-6, the preparation method includes: compound VIII is nucleophilic with R ₄ Z ₂ Substitution reaction; when W is a protecting group and R ₂ is an amino group, R ₃ and R ₄ are each independently selected from alkyl groups having 1-6 carbon atoms, compound VIII reacts with R ₃ Z ₁ and then with R ₄ Z ₂ reacts to obtain compound IX, wherein Z ₁ and Z ₂ are each independently selected from an electronegative leaving group; the electronegative leaving group includes a p-toluenesulfonate group

Mesylate group

Triflate group

Any one of I, Br, Cl;

When W is a protecting group and R ₂ is an amide group, the preparation method includes: performing an ammonolysis reaction between compound VIII and R ₅ COOR ₆ to obtain compound XI; R _{6 is} selected from an alkyl group with a carbon number of 1-6;

Among them, the structural formula of each compound is as follows:

When W is H and R ₂ is azido, the synthetic route is as follows:

In some specific embodiments of the present disclosure, in step a, compound I reacts with elemental iodine under the action of imidazole and triphenylphosphine. In one or more embodiments, the molar ratio of Compound I to the elemental iodine is 1:(3-8). In one or more embodiments, the molar ratio of compound I to iodine is 1: (5-7). The solution of elemental iodine is added dropwise under low temperature conditions such as ice bath, and then reacted at room temperature. The reaction time can be adjusted according to the actual TLC monitoring, for example, 3-5h.

In step b, compound II is eliminated in a sodium methoxide-methanol solution. In one or more embodiments, the molar ratio of sodium methoxide to compound II is 1:1. In one or more embodiments, step b is reacted under reflux conditions.

In step c, a solution of elemental iodine is added dropwise to the mixture of compound III, lead carbonate, and 2-azidoalkyl alcohol. In one or more embodiments, the molar ratio of compound III and 2-azidoalkyl alcohol is 1:(3-7). For example, the molar ratio of compound III and 2-azidoalkyl alcohol is 1:(4-6), such as 1:5. In one or more embodiments, the molar ratio of compound III to elemental iodine is 1:(1-2). For example, the molar ratio of compound III to iodine is 1:1.5. In one or more embodiments, the reaction of step c is carried out under ice bath conditions.

In step d, in the presence of an acid binding agent, the compound IV is reacted with benzoyl chloride at room temperature. In one or more embodiments, the molar ratio of compound IV to benzoyl chloride is 1: (1.1-2). The acid binding agent can be pyridine or the like.

In step e, in the dichloromethane-water system, compound V and m-chloroperoxybenzoic acid are reacted for 4-8 hours at room temperature, then the excess m-chloroperoxybenzoic acid is removed, and the ammonia-methanol solution is used for the ammonolysis , To obtain compound VI. In one or more embodiments, the molar ratio of compound V to m-chloroperoxybenzoic acid is 1:(2-4).

When W is a protecting group and R is an azido group, the synthetic route is as follows:

In step f, under the action of an acid binding agent, compound VI is reacted with 4,4'-dimethoxytrityl chloride at room temperature for 10-14 hours. In one or more embodiments, the molar ratio of compound VI to 4,4'-dimethoxytrityl chloride is 1:(1.1-2).

When W is a protecting group and R is an amino group, the synthetic route is as follows:

In step g, in tetrahydrofuran-aqueous solution, compound VII is reacted under reflux under the action of triphenylphosphorus to obtain compound VIII. Among them, the molar ratio of compound VII to triphenylphosphorus is 1: (1.1-2).

When W is a protecting group and R is an amino group, the synthetic route is as follows:

In step h, in an acetonitrile solution, compound VIII is reacted with R ₃ Z ₁ and/or R ₄ Z ₂ in the presence of a base to obtain compound IX. Wherein, when R _{3 is} selected from H and R _{4 is} selected from alkyl with carbon number of 1-6, compound VIII reacts with R ₄ Z ₂ in the presence of a base to obtain

When R ₃ and R ₄ are independently selected from alkyl groups having 1-6 carbon atoms and R ₃ and R _{4 are} not the same, compound VIII reacts with R ₃ Z ₁ and then reacts with R ₄ Z ₂ to obtain

When R ₃ and R _{4 are} selected from alkyl groups having 1-6 carbon atoms and R ₃ and R ₄ are the same, compound VIII reacts with R ₃ Z ₁ in the presence of a base to obtain

Wherein, when R ₃ and R _{4 are} not the same, the molar ratio of R ₄ Z ₂ or R ₃ Z ₁ to compound VIII in each step is (1.1-2):1. When R ₃ and R ₄ are the same, the molar ratio of R ₃ Z ₁ to compound VIII in each step is (1.1-2):1. The above reaction can be carried out at room temperature.

When W is a protecting group and R ₂ is an amide group, the synthetic route is as follows:

In step i, compound VIII and R ₅ COOR ₆ undergo an ammonolysis reaction at room temperature for 4-6 hours. Wherein, the molar ratio of compound VIII to R ₅ COOR ₆ is 1:(2-5). In one or more embodiments, R ₅ COOR ₆ is ethyl trifluoroacetate.

Conventional purification methods such as column chromatography can be used for the purification of the above steps.

The present disclosure also provides a method for preparing nucleotides, including the following steps:

Under the action of tetrazolium, the modified nucleoside reacts with phosphorus reagents such as 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite to obtain phosphoramidite monomer.

The synthetic route is as follows:

In one or more embodiments, the molar ratio of the modified nucleoside to the phosphorous reagent is 1: (1.1-2). For example, the molar ratio of modified nucleoside to phosphorous reagent is 1:1.5. The reaction temperature can be room temperature.

The modified oligonucleotide of the present disclosure uses the 3'-phosphoramidite derivative of the modified nucleoside or its salt as the raw material, and uses the standard phosphoramidite method on the automatic DNA synthesizer to embed the raw material into the oligonucleotide sequence . In actual operation, according to the actual needs of the reaction, the reaction time can be adjusted, or an activator can be added to increase the reaction rate.

Compared with the prior art, the beneficial effects of the present disclosure are:

In the modified nucleoside of the present disclosure, a novel modified nucleoside structure is obtained by modifying the nucleoside to introduce an azide group, an amino group or an amino group at the 4'position. The modified nucleoside can be further modified to obtain nucleotides and oligonucleotides, and nucleic acid polymers such as oligonucleotides with good ribozyme resistance can be obtained, which is useful for the development of nucleic acid drugs, nucleic acid primers, and nucleic acid diagnostics. Probes, etc. provide a more stable modified structure.

Description of the drawings

In order to more clearly describe the specific embodiments of the present disclosure or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the specific embodiments or the description of the prior art. Obviously, the appendix in the following description The drawings are some embodiments of the present disclosure. For those of ordinary skill in the art, other drawings may be obtained based on these drawings without creative work.

Figure 1 shows the secondary conformation of oligonucleotide ON1-3 when hybridized with complementary ssRNA provided in an embodiment of the disclosure;

Figure 2 shows the secondary conformation of the oligonucleotide ON5-8 provided by the embodiments of the disclosure when hybridized with complementary ssRNA;

FIG 3 disclosed in the present embodiment the oligonucleotide 5'-d (TTTTTTTT T T) -3 ' i.e. ON4 of snake venom phosphodiesterase (SVPDE) provided in the hydrolysis resistance;

FIG 4 is disclosed embodiment the oligonucleotide 5'-d (TTTTTTTT T T) -3 ' ON13 i.e. venom phosphodiesterase (SVPDE) provided in the hydrolysis resistance.

Detailed ways

The technical solutions of the present disclosure will be clearly and completely described below in conjunction with the drawings and specific implementations. However, those skilled in the art will understand that the embodiments described below are part of the embodiments of the present disclosure, rather than all of them. It is only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. Based on the embodiments in the present disclosure, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present disclosure. If specific conditions are not indicated in the examples, it shall be carried out in accordance with conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.

The "nucleotides" referred to in the present disclosure include "bases" (or "nucleobases" or "nitrogen-containing bases") and "sugars" (specifically 5-carbon sugars, such as ribose or 2-deoxyribose) And one or more phosphate groups (for example, monophosphate, diphosphate, triphosphate, tetraphosphate, etc., each composed of 1, 2, 3, 4, or more linked phosphates). In the absence of a phosphate moiety, nucleobases and sugars constitute "nucleosides." Nucleotides contain purines (such as in the nucleotides adenine and guanine) or pyrimidine bases (such as in the nucleotides cytosine, thymine, and uracil). Some nucleotides contain unnatural bases. Ribonucleotides are nucleotides in which the sugar is ribose. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.

The "nucleic acid polymer" referred to in the present disclosure may refer to any nucleic acid molecule, including but not limited to DNA, RNA, and hybrids thereof, including but not limited to single-stranded and double-stranded, etc. The nucleic acid bases forming the nucleic acid molecule may be the bases A, C, G, T and U and their derivatives.

The structure of the modified nucleoside of the present disclosure may include any of the following structural formulas:

and many more. Among them, 5'-OH can be replaced with the protecting group -ODMTr. 2'-F can be replaced with α-configuration fluorine atom F, α-configuration hydroxyl OH, β-configuration hydroxyl OH.

The structure of the nucleotides of the present disclosure may include phosphating treatment of 3'-OH on the basis of the above-mentioned modified nucleosides to obtain 3'-phosphoramidite monomers.

The structure of the modified oligonucleotide of the present disclosure includes embedding and modifying the above-mentioned at least one nucleotide in the oligonucleotide sequence.

Example 1

The synthetic route of the modified nucleoside of this embodiment is as follows:

The specific preparation method includes the following steps:

(a) Synthesis of compound II ₁

Add compound I ₁ (6.5g, 26.4mmol), imidazole (3.6g, 52.8mmol), triphenylphosphonium (10.4g, 39.6mmol), and tetrahydrofuran (100mL) into a three-necked flask successively, and use 100mL for dripping under ice bath The elemental iodine (10.08g, 39.6mmol) dissolved in tetrahydrofuran was reacted at room temperature for 4h after dripping. Add anhydrous sodium sulfite solution to the reaction solution until the color becomes lighter, add part of the water, extract with ethyl acetate, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, filter, concentrate, and flash column chromatography (mobile phase: dichloro chloride / methanol = 10/1 gradient elution) to give a compound of gray-black powder compound II ₁ 8g, yield ^{78%; 1 H NMR (400MHz} , methanol _{-d 4) δ7.72 (dd, J} = 8.1,1.7 Hz, 1H, H-4), 6.23 (dd, J = 19.9, 3.4 Hz, 1H, H-5), 5.71 (d, J = 8.2 Hz, 1H, H-1'), 5.07-5.06 (m, 1H,H-4'),4.31-4.27(m,1H,H-2'),3.91-3.90(m,1H,H-3'),3.76-3.74(m,2H,H-5'); ESI-MS (m/z) 357.1 [M+H] ⁺ , 379.1 [M+Na] ⁺ .

(b) Synthesis of compound III ₁

Compound II ₁ (7.3 g, 20.5 mmol), a mixture of sodium methoxide (4.43 g, 20.5 mmol) and 100 mL methanol were added to the three-necked flask, and the oil bath was heated to 65° C. to reflux for 3 h. Neutralized with glacial acetic acid to pH=7, concentrated, and flash column chromatography to obtain yellow powdery compound III ₁ 3.9g, yield: 85%; ¹ H NMR (400MHz, methanol-d ₄ ) 7.48 (dd, J=8.4 ,2.0Hz,1H,H-6),6.52(dd,J=19.6,3.2Hz,1H,H-10),5.72(d,J=8.0Hz,1H,H-5),5.10-4.96(m ,1H,H-2'), 4.67(d,J＝10.0,2.8Hz,2H,H-5'), 4.46(d,J＝2.4Hz,1H,3'-OH); ESI-MS(m /z) 229.2[M ⁺ H] ⁺ .

(c) Synthesis of compound IV ₁

Under ice bath, to compound III ₁ (5.0g, 22mmol), 2-azidoethanol (8.4mL, 110mmol), lead carbonate (8.8g, 33mmol) in 50mL of tetrahydrofuran, dropwise add iodine element (8.35g, 33mmol) 60mL tetrahydrofuran. After 1h, TLC detected (dichloromethane/methanol=15/1) that a new product was formed and the reaction was complete. Anhydrous sodium sulfite solution was added to it. A white solid precipitated out. Filtered with Celite. Ethyl acetate was extracted until the aqueous layer had no product, washed with saturated sodium sulfite, saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and solid precipitated, filtered directly, washed with methanol, and dried to obtain 3.05g of white solid product IV ₁ , The yield is 31%.

¹ H-NMR(400MHz,DMSO-d ₆ )δ11.51(s,1H,NH), 7.68(d,J=8.4Hz,1H,H-6), 6.03(dd,J=21.2, 1.4Hz, 1H,H-1'), 5.69(d,J=8.0Hz,1H,H-5), 5.45(d,J=8.4Hz,1H,OH), 5.31(ddd,J=54.4,6.4,1.6Hz ,1H,H-2'),4.56(ddd,J=21.2,8.0,6.0Hz,1H,H-3'),3.80-3.40(m,6H,CH ₂ -5',N ₃ CH ₂ CH ₂ O); ¹³ C-NMR (100MHz, CDCl ₃ ) δ 162.71,149.69,142.18,103.50,101.81,92.24,90.35,89.51,89.14,71.95,71.78,60.43,49.99,5.03;ESI-MS(m/z)463.98 [M+Na] ⁺ .

(d) Synthesis of compound V ₁

Compound IV ₁ (3.0 g, 6.8 mmol) was added to 20 mL of dry dichloromethane, and dry pyridine (1.64 mL, 20.4 mmol) was added to it. After cooling in an ice bath, benzoyl chloride (1.18 mL, 10.2 mmol), react at room temperature for 12h after the dropwise addition is complete, TLC detects (dichloromethane/methanol=15/1) the reaction is complete, add a small amount of methanol to the reaction solution to quench the reaction, extract with dichloromethane, wash with water, and wash with saturated brine , Dried with anhydrous sodium sulfate, filtered, concentrated, solids precipitated out, filtered and sucked dry to obtain 2.8 g of white solid product V ₁ with a yield of 76%.

¹ H-NMR(400MHz, CDCl ₃ )δ9.07(br s,1H,NH), 8.17-7.46(m,5H,BzH), 7.35(d,J=8.4Hz,1H,H-6), 5.90 (dd,J=17.2,1.6Hz,1H,H-1'), 5.85(dd,J=8.0,2.4Hz,1H,H-5), 5.76(dd,J=18.0,6.4Hz,1H,H -3'), 5.67(ddd,J=53.6,6.4,1.6Hz,1H,H-2'),3.95-3.78(m,2H,CH ₂ -5'),3.64-3.49(m,4H,N ₃ CH ₂ CH ₂ O); ¹³ C-NMR (100MHz, CDCl ₃ ) δ164.53,163.08,150.01,143.37,133.78,129.57,128.63,128.43,104.01,102.01,91.60,91.21,90.85,88.96,73.40,73.25, 60.89, 50.24, 4.45; ESI-MS (m/z) 568.00 [M+Na] ⁺ .

(e) Synthesis of compound VI ₁

Compound V ₁ (3.0 g, 5.5 mmol) was dissolved in 100 mL of dichloromethane, 10 mL of water was added, m-CPBA (3.35 g, 16.5 mmol) was added in batches under ice-cooling, and the reaction was at room temperature for 6 hours. TLC detection (dichloro Methane/methanol=10/1), add sodium sulfite solution to the reaction solution, extract with dichloromethane, wash with saturated sodium bicarbonate water, saturated sodium chloride solution, dry with anhydrous sodium sulfate, filter, and concentrate at room temperature. Add 30 mL of ammonia methanol to it, react at room temperature for 8 hours, TLC detection (dichloromethane/methanol=10/1), the reaction is complete, flash column purification (dichloromethane/methanol=10/1), 1.52g white solid product VI ₁ , The yield is 83%.

¹ H-NMR (400MHz, CD ₃ OD) δ 7.84 (d, J = 8.0 Hz, 1H, H-6), 6.14 (dd, J = 18.8 Hz, 1H, H-1'), 5.69 (d, J=8.0Hz,1H,H-5), 5.14(dd,J=53.6,5.6Hz,1H,H-2'), 4.60(dd,J=22.8,5.6Hz,1H,H-3'), 3.94-3.37(m,6H,CH ₂ -5',N ₃ CH ₂ CH ₂ O); ¹³ C-NMR(100MHz, CDCl ₃ )δ166.10,151.84,143.19,180.10,102.86,94.61,92.73,91.55,91.18 , 71.13, 70.96, 62.52, 60.91, 52.22; ESI-MS (m/z) 354.07 [M+Na] ⁺ .

(f) Synthesis of compound VII ₁

To compound VI ₁ (1.2g, 3.6mmol) in 15mL pyridine mixture at room temperature, 4,4'-bismethoxytrityl chloride (1.47g, 4.4mmol) was added, the reaction was carried out at room temperature for 12h, TLC detection (two Chloromethyl/methanol=15/1) The reaction is complete, the reaction is quenched with methanol, concentrated under reduced pressure, extracted with ethyl acetate, washed with water, washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (2 Methyl chloride/methanol=10/1), 1.8 g of white solid product 7 was obtained, with a yield of 79%.

¹ H-NMR(400MHz, CDCl ₃ )δ9.10(br s,1H,NH), 7.65(d,J=8.0Hz,1H,H-6'),7.40-6.83(m,13H,DMTrH), 6.13(d,J=19.6Hz,H-5), 5.41(dd,J=8.4,2.4Hz,1H,H-1'), 5.06(dd,J=53.2,6.4Hz,H-2'), 4.80-4.70(m,H-3'),3.82-3.24(m,12H,OCH ₃ ×2,CH ₂ -5',N ₃ CH ₂ CH ₂ O), 2.86(m,1H,3'-OH ), ¹³ C-NMR (100MHz, CDCl ₃ ) δ162.90,158.74,158.71,149.63,143.97,140.48,134.76,134.65,130.14,130.03,128.02,127.21,113.32,105.89,102.88,93.24,91.33,89.97,89.61, 87.43, 71.08, 70.91, 61.69, 60.94, 55.22, 50.89; ESI-MS (m/z) 656.20 [M+Na] ⁺ .

(g) Synthesis of compound VIII ₁

At room temperature, add 5mL water to 15mL tetrahydrofuran solution of compound VII ₁ (1.6g, 2.53mmol), then add triphenylphosphonium (1.0g, 3.79mmol), reflux reaction for 24h, TLC detection (dichloromethane/methanol= 10/1) The reaction is complete, stop the reaction, concentrate under reduced pressure and purify by flash column (dichloromethane/methanol=6/1) to obtain 1.4 g of white solid product 8 with a yield of 91%.

¹ H-NMR(400MHz,DMSO-d ₆ )δ7.78(d,J=8.4Hz,1H,H-6),7.40-7.21(m,13H,DMTrH),6.00(d,J=20.8Hz, 1H,H-1'), 5.37(d,J=8.0Hz,1H,H-5), 5.26(dd,J=54.4,6.0Hz,1H,H-2'), 4.96(br s,2H, NH ₂ ), 4.70(dd,J=25.2,6.0Hz,H-3'),3.74(s,6H,OCH ₃ ×2),3.48-2.54(m,6H,CH ₂ -5',NCH ₂ CH ₂ O); ¹³ C-NMR (100MHz, DMSO-d ₆ ) δ163.34,158.17,150.15,144.51,141.85,135.08,135.05,129.80,127.94,127.75,126.84,113.26,105.36,101.70,93.14,91.28,89.66, 89.30, 86.05, 70.58, 70.41, 63.96, 61.47, 55.05, 54.95, 41.61; ESI-MS (m/z) 630.23 [M+Na] ⁺ .

(i) Synthesis of compound XI ₁

To the 15mL tetrahydrofuran solution of compound VIII ₁ (1.0g, 1.65mmol) was added ethyl trifluoroacetate (0.9mL, 7.6mmol), reacted at room temperature for 5h, TLC detection (dichloromethane/methanol=10/1) the reaction was complete, The reaction was stopped, concentrated under reduced pressure and purified by flash column (dichloromethane/methanol=10/1) to obtain 1.0 g of white solid product XI ₁ with a yield of 86%.

¹ H-NMR(400MHz,CDCl ₃ )δ8.95(br s,1H,NH), 7.68(d,J=8.4Hz,1H,H-6), 7.38-6.83(m,14H,DMTrH,NH) ,6.02(d,J=18.0Hz,1H,H-1'),8.34(dd,J=8.0,2.0Hz,1H,H-5),5.07(dd,J=53.6,5.6Hz,1H,H -2'), 4.79 (m, 1H, H-3'), 3.79 (s, 6H, OCH ₃ × 2), 3.73-3.40 (m, 6H, CH ₂ -5', NCH ₂ CH ₂ O), 3.10 (d, J=11.5Hz, 1H, 3'-OH); ¹³ C-NMR (100MHz, CDCl ₃ ) δ163.05,158.77,158.73,149.68,143.85,140.25,134.62,134.51,130.11,130.02,129.09,128.08 ,127.98,127.28,113.35,113.11,105.84,102.88,93.78,91.89,89.50,89.14,87.56,70.88,70.72,60.74,60.46,55.22,39.38; ESI-MS(m/z)726.21[M+Na] ⁺ .

Example 2

The synthetic route of the nucleotide in this example is as follows:

The specific preparation method includes the following steps:

Under the protection of nitrogen, to compound XI ₁ (1.0g, 1.42mmol), 1H-tetrazole (100mg, 1.42mmol) in 10mL dichloromethane solution, add 2-cyanoethyl-N,N,N',N '-Tetraisopropyl phosphorodiamidite (640mg, 2.13mmol) in 10mL dichloromethane solution, react at room temperature for 5h, TLC detection (dichloromethane/methanol=10/1) the reaction is complete, add saturated to the reaction solution Sodium bicarbonate aqueous solution, dichloromethane extraction, saturated brine washing, anhydrous sodium sulfate drying, filtration, concentration, flash column purification (dichloromethane/methanol/triethylamine = 10/1/0.1), 1.1 g White solid product, yield 86%.

³¹ P-NMR (162MHz, CDCl ₃ ) δ 152.84, 152.78, 152, 12, 152.07; ¹⁹ F-NMR (376 MHz, CDCl ₃ ) δ-75.80, -75.83, -193.01 (m, CF ₃ ), -193.44 (m, CF ₃ ); ESI-HRMS(m/z)904.3294[M+H] ^- ,926.3109[M+Na] ⁺ .

Example 3

The synthetic route of the modified nucleoside of this embodiment is as follows:

The specific preparation method refers to the preparation method of Example 1. The main difference is that the raw material compound I ₁ in Example 1 is replaced with compound I ₂ .

The yield of compound IV ₂ prepared in step (c) of this example is 32%, and the structural characterization data are as follows:

¹ H-NMR(400MHz, CDCl ₃ )δ9.17(br s,1H,NH), 7.47(dd,J=8.0,2.4Hz,1H,H-6),6.47(dd,J=8.0,4.0Hz ,1H,H-1'), 5.80(d,J=8.0Hz,1H,H-5), 5.18(ddd,J=52.0,3.6,2.4Hz,1H,H-2'), 4.62(ddd, J=20.0,6.4,2.0Hz,1H,H-3'),3.92-3.84(m,2H,5'-CH ₂ ),3.55-5.50(m,4H,N ₃ CH ₂ CH ₂ O), 3.00 (br s,1H,3'-OH); ¹³ C-NMR (100MHz, CDCl ₃ )δ163.00,150.03,140.70,140.66,102.49,102.39,102.34,95.89,93.98,82.88,82.71,78.94,78.66,61.65, 50.38, 2.65; ESI-MS (m/z) 442.04 [M+H] ⁺ .

The yield of compound V ₂ prepared in step (d) of this example is 81%, and the structural characterization data are as follows:

¹ H-NMR(400MHz,CDCl ₃ )δ8.79(s,1H,NH), 8.10(d,J=7.2Hz,2H,BzH), 7.70(dd,J=8.0,2.0Hz,1H,H- 6'),7.66-7.47(m,3H,BzH),6.57(dd,J=15.6,4.4Hz,1H,H-1'),5.97(dd,J=24.8,2.8Hz,1H,H-3 '), 5.86(dd,J=8.4,2.0Hz,1H,H-5),5.56(ddd,J=53.2,4.4,3.2Hz,1H,H-2'),3.89-3.38(m,6H, CH ₂ -5', N ₃ CH ₂ CH ₂ O); ¹³ C-NMR (100MHz, CDCl ₃ ) δ165.69,163.04,150.33,141.11,141.08,134.19,130.38,128.87,128.57,102.94,102.14,102.07,94.68 , 92.73, 82.07, 81.89, 79.47, 79.18, 61.70, 50.69, 3.62; ESI-MS (m/z) 546.01 [M+H] ⁺ , 568.00 [M+Na] ⁺ .

The yield of compound VI ₂ prepared in step (e) of this example is 64%, and the structural characterization data are as follows:

¹ H-NMR(400MHz, methanol-d ₃ )δ7.85(dd,J=8.0,1.2Hz,1H,H-6'), 6.45(t,J=6.4Hz,1H,H-1'), 5.72(d,J=8.0Hz,1H,H-5),5.34(dt,J=55.2,6.4Hz,1H,H-2'),4.56(dd,J=24.4,6.0Hz,1H,H- 3'), 3.93-3.34 (m, 6H, CH ₂ -5', N ₃ CH ₂ CH ₂ O); ¹³ C-NMR (100MHz, methanol-d ₃ ) δ165.89,151.99,142.51,105.88,105.77,102.46 , 97.58, 95.65, 82.36, 82.20, 75.38, 75.14, 62.75, 60.11, 51.91; ESI-MS (m/z) 354.07 [M+Na] ⁺ .

The yield of compound VII ₂ prepared in step (f) of this example is 82%, and the structural characterization data are as follows:

¹ H-NMR(400MHz, CDCl ₃ )δ9.17(br s,1H,NH), 7.55(dd,J=8.4,1.6Hz,1H,H-6),7.41-6.85(m,13H,DMTrH) ,6.47(dd,J＝12.0,5.2Hz,1H,H-1'),5.52(dd,J＝8.0,1.2Hz,1H,H-5), 5.25(dt,J＝53.6,4.8Hz,1H ,H-2'), 4.79(ddd,J=21.6,8.0,3.6Hz,1H,H-3'), 3.80(s,6H,OCH ₃ ×2),3.78-2.85(m,6H,CH ₂ -5',N ₃ CH ₂ CH ₂ O); ¹³ C-NMR (100MHz, CDCl ₃ ) δ163.10,159.02,150.33,144.11,140.74,134.94,130.32,130.26,128.36,128.22,127.54,113.61,103.74,103.66 , 102.64, 96.19, 94.25, 87.52, 81.93, 81.76, 76.65, 62.26, 61.05, 55.51, 50.87; ESI-MS (m/z) 656.20 [M+Na] ⁺ .

The yield of compound VIII ₂ prepared in step (g) of this example is 87%, and the structural characterization data are as follows:

¹ H-NMR(400MHz,DMSO-d ₆ )δ7.77(d,J=8.4Hz,1H,H-6),7.38-6.91(m,13H,DMTrH),6.34(t,J=6.0Hz, 1H,H-1'), 5.47(dt,J=55.2,6.4Hz,1H,H-2'), 4.62(dd,J=24.8,6.8Hz,1H,H-3'), 3.74(s, 6H,OCH ₃ ×2),3.45-2.59(m,6H,CH ₂ -5',N ₃ CH ₂ CH ₂ O); ¹³ C-NMR(100MHz,DMSO-d ₆ )δ162.84,158.27,150.21,144.31 ,134.90,134.72,129.84,128.06,127.73,127.02,113.38,103.26,103.13,101.44,95.80,93.89,86.45,80.12,79.96,74.68,74.44,63.90,61.04,55.10,41.13; ESI-MS(m/z )630.24[M+Na] ⁺ .

The yield of compound XI ₂ prepared in step (i) of this example is 95%, and the structural characterization data are as follows:

¹ H-NMR(400MHz, CDCl ₃ )δ8.21(t,J=4.8Hz,1H,NH), 7.62(dd,J=8.0,1.2Hz,1H,H-6),7.39-6.82(m, 13H,DMTrH), 6.37(dd,J=11.6,5.2Hz,1H,H-1'), 5.48(d,J=8.4Hz,1H,H-5),5.30(dt,J=53.2,4.8Hz ,1H,H-2'),4.66(dd,J=22.0,3.2Hz,1H,H-3'),3.79(s,6H,OCH ₃ ×2),3.69-3.36(m,6H,CH ₂ -5', NCH ₂ CH ₂ O); ¹³ C-NMR (100MHz, CDCl ₃ ) δ 163.25, 158.71, 150.25, 143.91, 140.70, 134.69, 130.03, 130.00, 129.08, 128.05, 127.94, 127.21, 113.31, 113.09, 103.56 ,103.49,102.26,95.90,93.97,87.17,81.54,81.38,76.34,76.10,62.26,61.17,60.65,55.20,45.72,39.60; ESI-MS(m/z)726.24[M+Na] ⁺ .

Example 4

The synthetic route of the nucleotide in this example is as follows:

The specific preparation method includes the following steps:

Under the protection of nitrogen, to compound XI ₂ (1.0g, 1.42mmol), 1H-tetrazole (100mg, 1.42mmol) in 10mL dichloromethane solution, add 2-cyanoethyl-N,N,N',N '-Tetraisopropyl phosphorodiamidite (640mg, 2.13mmol) in 10mL dichloromethane solution, react at room temperature for 5h, TLC detection (dichloromethane/methanol=10/1) the reaction is complete, add saturated to the reaction solution Sodium bicarbonate aqueous solution, dichloromethane extraction, saturated brine washing, anhydrous sodium sulfate drying, filtration, concentration, flash column purification (dichloromethane/methanol/triethylamine = 10/1/0.1), 1.06 g white solid product, yield 83%.

³¹ P-NMR (162MHz, CDCl ₃ ) δ 153.46, 153.43, 152.22, 152.19; ¹⁹ F-NMR (376 MHz, CDCl ₃ ) δ-75.60, -75.65, -197.82--198.05 (m, CF ₃ ), -198.41- -198.61 (m, CF ₃ ); ESI-HRMS (m/z) 904.3298 [M+H] ^- , 926.3120 [M+Na] ⁺ .

Example 5

The synthetic route of the modified nucleoside of this embodiment is as follows:

The specific preparation method refers to the preparation method of Example 1, the difference lies in step (h):

(h) Synthesis of compound IX ₁

At room temperature, to the 5mL acetonitrile solution of compound VIII ₁ (100mg, 0.165mmol), add triethylamine (46μL, 0.33mmol) and methyl p-toluenesulfonate (37mg, 1.2mmol) in sequence, react for 6h, TLC detection ( Dichloromethane/methanol=10/3) the reaction is complete, stop the reaction, slowly pour the reaction solution into 50mL of water, a white solid precipitates out, let stand, filter, dry and purify with a flash column (dichloromethane/methanol=10 /4) The product was collected, and after concentration, 30 mg of white solid product IX _{1 was} obtained with a yield of 29%. ¹ H-NMR(400MHz,DMSO-d ₆ ) δ7.76(d,J=8.0Hz,1H,H-6),7.38-6.88(m,13H,DMTrH),6.05(d,J=21.0Hz, 1H,H-1'), 5.38(d,J=8.4Hz,1H,H-5), 5.25(dd,J=54.4,6.0,1H,H-2'), 4.67(dd,J=24.8, 6.0Hz, 1H, H-3'), 3.74 (s, 6H, OCH ₃ × 2), 3.56-3.35 (m, 2H, NC-CH ₂ O), 3.28-3.11 (m, 2H, 5'-CH ₂ ), 2.39-2.28 (m, 2H, N-CH ₂ CO), 2.09 (s, 6H, N(CH ₃ ) ₂ ); ¹³ C-NMR (100MHz, DMSO-d ₆ ) δ 163.19, 158.19, 150.06, 144.51,142.03,135.08.135.05,129.84,129.80,127.93,127.75,126.85,113.25,105.60,101.72,92.93.91.08,89.89.89.53,86.05,70.67,70.51,61.85,59.92,58.65,55.06,45.37; ESI- MS (m/z) 658.24 [M+Na] ⁺ , 684.23 [M+K] ⁺ .

Example 6

1. This example refers to the preparation method of Example 1, except that the raw material compound I _{1 is} replaced with

2. This example refers to the preparation method of Example 3, the difference is: the raw material compound I _{1 is} replaced with

Example 7

1. This example refers to the preparation method of Example 1, except that the raw material compound I _{1 is} replaced with

2. This example refers to the preparation method of Example 3, the difference is: the raw material compound I _{1 is} replaced with

Example 8

1. This example refers to the preparation method of Example 1, except that the raw material compound I _{1 is} replaced with

2. This example refers to the preparation method of Example 3, the difference is: the raw material compound I _{1 is} replaced with

Example 9

1. This example refers to the preparation method of Example 1, except that the raw material compound I _{1 is} replaced with

2. This example refers to the preparation method of Example 3, the difference is: the raw material compound I _{1 is} replaced with

Example 10

This example refers to the preparation method of Example 1, except that the raw material compound I _{1 is} replaced with

Example 11

This example refers to the preparation method of Example 1, except that the raw material compound I _{1 is} replaced with

Example 12

This example refers to the preparation method of Example 1, except that the raw material compound I _{1 is} replaced with

Example 13

Using the phosphoramidite monomers of Examples 2 and 4 as raw materials, the standard phosphoramidite method was used in the automatic DNA synthesis to insert the phosphoramidite monomers obtained in the above examples into the corresponding oligonucleotide sequences to obtain The following oligonucleotides ON1-14 are shown in Table 1.

Table 1 Different modified oligonucleotide sequences and corresponding mass spectra

ON	Phosphoramidite monomer	Sequence (5’-3’)	MS calculated value	MS measured value
ON1	Compound XI 1	GCGTT T TTTGCT	3696.4	3,698.9
ON2	Compound XI 1	GCGTT T T T TGCT	3759.4	3762.9
ON3	Compound XI 1	GCG T T T T T TGCT	3,822.5	3824.2
ON4	Compound XI 1	TTTTTTTT T T	3043.0	3043.3
ON5	Compound XI 2	GCGTT T TTTGCT	3696.4	3,695.5
ON6	Compound XI 2	GCGTT T T T TGCT	3759.4	3758.6
ON7	Compound XI 2	GCG T T T T T TGCT	3,822.5	3,821.7
ON8	Compound XI 2	GCGT T T T T T GCT	3,822.5	3,821.7
ON9	Compound XI 2	GCGT T GTTTGCT	3721.4	3,720.6
ON10	Compound XI 2	GCGTTG T TTGCT	3721.4	3,720.8
ON11	Compound XI 2	GCGT T ATTTGCT	3705.4	3704.7
ON12	Compound XI 2	GCGTTA T TTGCT	3705.4	3704.5
ON13	Compound XI 2	TTTTTTTT T T	3043.0	3,042.5
ON14	Compound I 1	GCGTTT T TTGCT	3637.3	3637.4
ON15	Compound I 2	GCGTTT T TTGCT	3637.3	3637.2

Experimental example 1

In order to compare and illustrate the properties of modified nucleosides and nucleotides obtained in different embodiments of the present disclosure, the properties of different modified oligonucleotides obtained are tested.

The experimental method includes: annealing buffer: 10 mM Na ₃ PO ₄ , 100 mM NaCl, pH 7.2. Annealing method: dilute the two oligonucleotide single strands with annealing buffer to the final concentration of 2μM, heat in a water bath at 95°C for 5min, slowly cool to room temperature, and place overnight in a refrigerator at 4°C. T _m measurement method: add 800μL of the sample to be tested in the cuvette and cover it with a thermally insulated lid. Choose 15 ℃ as the starting temperature, 90 ℃ as the end temperature, the temperature rise rate is 0.5 ℃/min, the A260 reading rate is 1 time/℃, and finally the T _m value is given by the instrument. The measurement was repeated 3 times for each sample, and the average value was taken as the final result.

Thermal denaturation experiment

Table 2 The melting temperature T _m value (℃) of oligonucleotide ON1-18 and corresponding complementary DNA/RNA to form a double strand

Note: 1. The complementary sequence of ON1-3, ON5-8, ON14-16 is 5'-d(AGCAAAAAACGC)-3' or 5'-r(AGCAAAAAACGC)-3'; the complementary sequence of ON9-10 and ON17 is 5'-d(AGCAAACAACGC)-3' or 5'-r(AGCAAACAACGC)-3'; the complementary sequence of ON11-12 and ON18 is 5'-d(AGCAAATAACGC)-3' or 5'-r(AGCAAAUAACGC)- 3'; 2. ΔT _m = T _m (modified)-T _m (unmodified); 3. All T _m values are the average of three measurements.

From Table 2 above, it can be seen that all ON1-3 modified by compound XI ₁ can maintain binding affinity to complementary RNA strands, which is better than that of compound I ₁ modified sequence ON14. At the same time, compared with the natural DNA sequence ON16, a single modified pair ΔT _m /mod<1.05℃, and can improve the binding selectivity to ssRNA, the selectivity of the three modified ON3 to complementary RNA strands reaches 4.6℃. The T _m values of ON5-8 and ON15 are equivalent, and the ΔT _m values are both less than 0.7°C. Among them, the T _m values of ON9 and ON11 with T G and T A steps and complementary RNA strands are higher than those of the corresponding ON10 and ON12 with G T and A T steps. The F with 2'β configuration can interact with purines. False hydrogen bonds are formed between C8-H, which improves the affinity of modified oligonucleotides with complementary RNA. Compound XI ₂ was modified, may enhance the binding selectivity ssRNA sequences, and sequence-dependent, and three modified ON7 ON8 selectivity to complementary RNA strand is 1.8 deg.] C, T G containing the step sequence of the RNA binding ON9 The selectivity reaches 4.1°C.

Mismatch experiment

Table 3 The melting temperature T _m and mismatch value △T _m (℃) of the hybridization of oligonucleotide ON1 and ON5 with single-stranded DNA/RNA

Note: 1. All T _m values are the average of three measurements; 2. ΔT _m = T _{m (mismatch)} -T _{m (match)} .

From Table 3 above, it can be seen that ON1 and ON5 have good binding specificity with complementary RNA, and the ability of ON5 to recognize mismatched bases is equivalent to that of the natural sequence ON16.

Experimental example 2

Circular dichroism experiment

Experimental method: Take the same annealing buffer containing the hybrid double-strand to be tested as in the thermal denaturation experiment, measure it with a circular dichrograph, scan range 200～400nm, scan speed 50nm/min, scan interval 0.5nm, cuvette optical path 1mm, measuring temperature 20℃. After each sample is scanned three times in a row, the average value is automatically taken, and the graph is made after smoothing through the software of the instrument. The test results are shown in Figure 1-2.

From the CD figure, it can be seen that the DNA-RNA double strands bound by the oligonucleotide ON1-3 complementary RNA have typical A-form conformation characteristics, with the largest negative absorption peak (trough) near ~210nm, between 260nm-280nm There is the largest positive absorption peak (wave crest), and the peak signal is strong. The modification of nucleotide XI _{1 of} the present disclosure also does not affect the double-strand formation ability of antisense oligonucleotide and its complementary RNA strand (ssRNA). Oligonucleotides ON5-8, especially the three-base modified ON7-8, DNA-RNA double strands combined with complementary RNA have typical A-form conformation characteristics, with the largest negative absorption peak (trough) near ~210nm , There is a maximum positive absorption peak (peak) between 260nm-280nm. The XI ₂ modified oligonucleotide of the present disclosure has good hybridization ability with its complementary RNA strand (ssRNA).

Experimental example 3

Nuclease stability

experimental method:

Buffer system: 50mM Tris-HCl, 10mM MgCl ₂ , pH 8.0. HPLC analysis conditions: Waters type HPLC, flow rate: 1mL/min; injection volume: 10μL; mobile phase A: water, mobile phase B: methanol; the gradient is set from A:B=98:2 ( v/v) to A:B=92:8 (v/v); UV detector wavelength: 260nm.

Enzyme stability determination: Take 20μL of 1μg/μL sample (about 7nmol based on molecular weight ~3000) and dissolve it in 375μL buffer, and add 0.02μg/μL SVPDE 5μL or 5μL high purity water as a blank control (total volume 400μL), All were incubated at 37°C. At 0 min, 2 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 μL of incubation solution, 150 μL of methanol to precipitate the protein were taken out, and 150 μL of supernatant was taken after centrifugation at 10000 rpm for 10 min. After draining, 150 μL of water was added, and the content of the sample was detected by HPLC. According to the measured percentage of undegraded samples, a content-time curve of the sample is made. The test result is shown in Figure 3-4.

Under the experimental conditions, the modification of compound XI ₁ can significantly enhance the tolerance of oligonucleotide ON4 to nuclease. At 40 min, ON4 only degrades less than 20%, while its corresponding natural oligonucleotide ON19 (natural DNA- dT, TTTTTTTTTT) have all been degraded. Where ON20 stands for 3'-phosphorothioate-T(Ts).

Under the experimental conditions, the modification of compound XI ₂ can significantly enhance the tolerance of oligonucleotide ON13 to nuclease. At 40 min, 60% of ON13 is still degraded, while its corresponding natural oligonucleotide ON19 (natural DNA -dT, TTTTTTTTTT) has all been degraded.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, not to limit them; although the present disclosure has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand: It is still possible to modify the technical solutions described in the foregoing embodiments, or equivalently replace some or all of the technical features; these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present disclosure. range.

Industrial applicability

In the modified nucleoside of the present disclosure, a novel modified nucleoside structure is obtained by modifying the nucleoside to introduce an azide group, an amino group or an amino group at the 4'position. The modified nucleoside can be further modified to obtain nucleotides and oligonucleotides, and nucleic acid polymers such as oligonucleotides with good ribozyme resistance can be obtained, which is useful for the development of nucleic acid drugs, nucleic acid primers, and nucleic acid diagnostics. Probes, etc. provide a more stable modified structure.

Claims (15)

1. A modified nucleoside, selected from compounds with the following structures or their salts:

   

   Where R _{1 is} selected from

   

   And their respective salts;

   n is selected from 1, 2, 3, 4, 5 or 6;

   R _{2 is} selected from azido, amino, amine or amide;

   W is selected from H or a protecting group;

   X is selected from H, α-configuration OH, β-configuration OH, α-configuration F or β-configuration F.
2. The modified nucleoside according to claim 1, wherein the amine group is -NR ₃ R ₄ , R ₃ and R ₄ are each independently selected from H, an alkyl group with a carbon number of 1-6 or a fluorescent group, R ₃ and R _{4 are} not H at the same time;

   Preferably, the R ₃ and R ₄ are each independently selected from alkyl groups having 1 to 5 carbon atoms;

   More preferably, the R ₃ and R ₄ are each independently selected from alkyl groups having 1 to 4 carbon atoms.
3. The modified nucleoside of claim 2, wherein the fluorescent group is pyrene and its derivatives.
4. The modified nucleoside of claim 1, wherein the amide group is -NHCOR ₅ , and R _{5 is} selected from an alkyl group having 1 to 6 carbon atoms or a haloalkyl group having 1 to 6 carbon atoms;

   Preferably, the R _{5 is} selected from -CF ₃ , -CHF ₂ , -CH ₂ F, -CH ₂ CF ₃ , -CH ₂ CHF ₂ , -CH ₂ CH ₂ F, -C ₂ H ₄ CF ₃ ,- Any one of the group consisting of C ₂ H ₄ CHF ₂ and -C ₂ H ₄ CH ₂ F;

   More preferably, the amide group is -NHCOCF ₃ .
5. The modified nucleoside according to any one of claims 1 to 4, wherein the X is selected from any one of F in the α configuration and F in the β configuration;

   Preferably, the R _{1 is} selected from

   

   Any of

   More preferably, the R ₁ is

   
6. The modified nucleoside of any one of claims 1-5, wherein the W is 4,4'-dimethoxytrityl.
7. A nucleotide characterized in that it comprises the 3'-phosphoramidite derivative of the modified nucleoside described in any one of claims 1-6 or a salt thereof.
8. The nucleotide according to claim 7, characterized in that it is selected from compounds having the following structures or salts thereof:

   

   Preferably, the W is 4,4'-dimethoxytrityl.
9. The nucleotide according to claim 7 or 8, R2 is selected from an amino group and an amino group.
10. A modified nucleic acid polymer comprising at least one modified nucleotide having the following structure:

    

    Wherein Y is selected from O or S, and R _{3 is} selected from aryl, methyl, substituted alkyl or alkenyl;

    Preferably, the modified nucleic acid polymer includes ribonucleic acid, deoxyribonucleic acid, or a copolymer of ribonucleotide and deoxyribonucleotide;

    More preferably, the modified nucleic acid polymer is an oligonucleotide.
11. The modified nucleic acid polymer according to claim 10, wherein the nucleic acid polymer is selected from one or more of the following sequences:

    SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3;

    Preferably, the modified nucleic acid polymer is selected from one or more of the following sequences:

    The sequence obtained by modifying the 6th position at the 5'end of SEQ ID NO: 1;

    The sequence obtained by modifying the 6th and 8th positions at the 5'end of SEQ ID NO: 1;

    The sequence obtained by modifying the 4th, 6th and 8th positions at the 5'end of SEQ ID NO: 1;

    A sequence obtained by modifying positions 5, 7 and 9 at the 5'end of SEQ ID NO: 1;

    The sequence obtained by modifying position 5 at the 5'end of SEQ ID NO: 2;

    The sequence obtained by modifying position 7 at the 5'end of SEQ ID NO: 2;

    The sequence obtained by modifying position 5 at the 5'end of SEQ ID NO: 3;

    The sequence obtained by modifying position 7 at the 5'end of SEQ ID NO: 3.
12. The method for preparing modified nucleosides according to any one of claims 1-6, characterized in that it comprises the following steps:

    When W is H and R ₂ is an azido group, the preparation method includes: compound I undergoes iodination reaction under the action of a catalyst to obtain compound II; compound II undergoes elimination reaction under alkaline conditions to obtain compound III; compound III, 2 -Azidoalkyl alcohol and iodine are added to obtain compound IV; 3'-OH of compound IV is protected with benzoyl to obtain compound V; 5'-I of compound V is oxidized by m-chloroperoxybenzoic acid, Aminolysis to obtain compound VI;

    When W is a protecting group and R ₂ is an azido group, the preparation method includes: protecting the 5'-OH of compound VI obtained by the above method with a protecting group to obtain compound VII;

    When W is a protecting group and R ₂ is an amino group, the preparation method includes: reducing the azide group in compound VII to obtain compound VIII;

    When W is a protecting group, R ₂ is an amino group, R _{3 is} selected from H, and R _{4 is} selected from an alkyl group with a carbon number of 1-6, the preparation method includes: nucleophilic substitution of compound VIII with R ₄ Z ₂ Reaction to obtain compound IX; when W is a protecting group, R ₂ is an amino group, and R ₃ and R ₄ are each independently selected from alkyl groups having 1-6 carbon atoms, compound VIII is reacted with R ₃ Z ₁ and then React with R ₄ Z ₂ to obtain compound IX; wherein Z ₁ and Z ₂ are each independently selected from an electronegative leaving group;

    When W is a protecting group and R ₂ is an amide group, the preparation method includes: compound VIII and R ₅ COOR ₆ undergo an ammonolysis reaction to obtain compound XI; R _{5 is} selected from an alkyl group with a carbon number of 1-6 or a carbon number Is a haloalkyl group of 1-6, R _{6 is} selected from alkyl groups of carbon number 1-6;

    Among them, the structural formula of each compound is as follows:

    

    
13. The method according to any one of claims 7 to 9, characterized in that it comprises reacting the modified nucleoside of any one of claims 1-6 with a phosphorus reagent under the action of tetrazolium, The step of obtaining phosphoramidite monomer;

    Preferably, the phosphorous reagent is 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite.
14. The method according to claim 13, characterized in that the molar ratio of the modified nucleoside to the phosphorous reagent is 1:(1.1-2), preferably 1:1.5.
15. Application of the modified nucleic acid polymer according to claim 10 or 11 in the preparation of nucleic acid diagnostic agents and nucleic acid therapeutic agents;

    Preferably, the nucleic acid diagnostic agent includes any one or more of nucleic acid primers and nucleic acid diagnostic probes;

    Preferably, the nucleic acid therapeutic agent includes any one or more of antisense nucleic acid, small interfering RNA, and miRNA.

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