Oligonucleotide with thiophosphoramide modified nucleotide structure and synthesis process of oligonucleotide

Abstract

The invention discloses oligonucleotide with a sulfo-modified nucleotide structure and a synthesis process of the oligonucleotide. The oligonucleotide with the sulfo-modified nucleotide structure comprises a nucleotide structural unit as shown in a formula I or a formula II, through specific limitation of a thio-modified nucleotide monomer structure, the synthesized and prepared oligonucleotide contains a thiophosphoramide structural unit; compared with a traditional oligonucleotide molecule, the oligonucleotide sequence molecule with the sulfo-modified nucleotide structure has more excellent effects on pharmaceutical parameters such as drug enzymatic degradation resistance, stability and combinability, and a novel chemical modification strategy is provided for research, development and application of oligonucleotide drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of oligonucleic acid, in particular to a kind of oligonucleic acid of thio-modified nucleotide structure and its synthesis process. BACKGROUND

[0002] Oligonucleotide is a biological molecule formed by short chain nucleic acid (usually 2-20) connected by phosphodiester bond, which can be divided into DNA oligonucleotide and RNA oligonucleotide. They are widely used in molecular biology research, disease diagnosis and treatment, such as PCR primer, gene editing tool or targeted drug.

[0003] At present, the oligonucleotide sequence synthesized by traditional nucleotide monomer has the problem of poor stability, which is easy to be degraded in human serum and human liver microsomes.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] The present application provides a kind of oligonucleic acid of thio-modified nucleotide structure and its synthesis process, the oligonucleic acid of thio-modified nucleotide structure in the present application is synthesized by using thio-phosphoryl amide nucleotide monomer, so that it has specific thio-modified nucleotide structure, which can improve the stability of synthesized oligonucleic acid and improve its biological activity.

[0006] In order to achieve the above-mentioned purpose of the present application, the following technical solutions are adopted:

[0007] The present application provides a kind of oligonucleic acid of thio-modified nucleotide structure, the oligonucleic acid of thio-modified nucleotide structure includes nucleotide structure unit shown in formula I or formula II;

[0008]

[0009] In formula I:

[0010] Base is nucleotide base or its derivative, and the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine or xanthine;

[0011] R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl or aromatic group;

[0012] R3 is hydrogen, alkyl (including methyl, ethyl, long chain alkyl), cycloalkyl, alkenyl, alkynyl, silicon group (including trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl and t-butyldiphenylsilyl), silicon oxymethylene, alkoxyalkyl, benzyl or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl);

[0013]

[0014] In Formula II:

[0015] Base is a nucleotide base or derivative thereof, which is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine or xanthine;

[0016] R is hydrogen, halogen, alkyl, thiol, alkylthiol, amine, azido or cyano;

[0017] R1and R2are each alkyl, alkenyl, alkynyl, cycloalkyl or aryl.

[0018] Preferably, the nucleotide monomers used in the synthesis of the oligonucleic acid of the thio-modified nucleotide structure include Formula III or Formula IV:

[0019]

[0020] In Formula III:

[0021] Base is a nucleotide base or derivative thereof, which is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine or xanthine;

[0022] PG is trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, t-butyldimethylsilyl or t-butyldiphenylsilyl);

[0023] R is hydrogen, halogen, alkyl, thiol, alkylthiol, amine, azido or cyano;

[0024] R1and R2are each alkyl, alkenyl, alkynyl, cycloalkyl or aryl.

[0025]

[0026] In Formula IV:

[0027] Base is a nucleotide base or derivative thereof, which is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine or xanthine;

[0028] PG is trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, t-butyldimethylsilyl or t-butyldiphenylsilyl);

[0029] R1and R2are each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl;

[0030] R3is alkyl (including methyl, ethyl, long chain alkyl), cycloalkyl, alkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl), siloxy methylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl).

[0031] Preferably, the protecting group is selected from one of trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl.

[0032] Preferably, the nucleotide monomer comprises:

[0033] wherein:

[0034] R1and R2are each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl;

[0035] R3is alkyl (including methyl, ethyl, long chain alkyl), cycloalkyl, alkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl), siloxy methylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl).

[0036] Preferably, the nucleotide monomer comprises:

[0037] wherein:

[0038] R1and R2are each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl.

[0039] Preferably, the nucleotide monomer comprises:

[0040] wherein:

[0041] R1and R2are each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl.

[0042] Preferably, the 4,4'-dimethoxytrityl (DMTr) in the nucleotide monomer is replaced with trityl (Tr), 4-methoxytrityl (MMTr), or 4,4',4"-trimethoxytrityl (TMTr).

[0043] Preferably, the nucleotide monomer is prepared by reacting O5' nucleotide molecule containing a protecting group and thiophosphoramidite dichloride in liquid phase.

[0044] Preferably, the thio-modified nucleotide structure in the thio-modified oligonucleic acid can improve the biological stability and anti-enzyme degradation activity of the oligonucleic acid.

[0045] The second aspect of the present application provides a synthesis process of the above-mentioned thio-modified oligonucleic acid, and the synthesis process comprises the following steps:

[0046] (a) loading the nucleotide monomer on the solid carrier, removing the O5' protecting group of the nucleotide monomer and washing;

[0047] (b) pumping the nucleotide monomer into the system after washing for coupling, and then performing capping, removing the O5' protecting group of the nucleotide monomer and washing, wherein the nucleotide monomer is a thiophosphoramidite nucleotide monomer or a phosphoramidite nucleotide monomer; if the nucleotide monomer is a phosphoramidite nucleotide monomer, after the coupling reaction, the trivalent phosphoric acid diester is converted into a pentavalent phosphoric acid diester by oxidation or sulfurization, and then capping, removing the O5' protecting group of the nucleotide monomer and washing are performed;

[0048] (c) repeating the step (b) to obtain the thio-modified oligonucleic acid with the thio-modified nucleotide structure.

[0049] Compared with the prior art, the present application has at least the following beneficial effects:

[0050] By specifically limiting the structure of the thio-modified nucleotide monomer, the present application realizes the synthesis of the oligonucleic acid containing the thiophosphoramidite structural unit. Compared with the traditional oligonucleic acid molecule, the oligonucleic acid sequence molecule with the thio-modified nucleotide structure of the present application has more excellent effects on the pharmaceutical parameters such as anti-enzyme degradation, stability and binding, and provides a new chemical modification strategy for the research and application of oligonucleic acid drugs. DETAILED DESCRIPTION

[0051] The embodiments of the technical solutions of the present application will be described in detail below with reference to the examples. The following examples are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, but cannot limit the protection scope of the present application.

[0052] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in the present application should be understood as the usual meanings understood by the skilled person in the field of the present application.

[0053] In the present application, the corresponding substances of the letter abbreviations are as follows:

[0054] CPG: controlled pore glass; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene; PS: polystyrene; Alkyl: alkyl; Allyl: allyl; Alloc: allyloxycarbonyl; Bn: benzyl; Bz: benzoyl; DMTr: dimethoxytrityl; Me: methyl; MMTr: methoxytrityl; Tr: trityl; TMTr: trimethoxytrityl; iBu: 2-isobutyryl; NPE: 4-nitrophenethyloxy; Ph: aromatic; TMS: trimethylsilyl; TBDMS: tert-butyldimethylsilyl; TBDPS: tert-butyldiphenylsilyl; DCM: dichloromethane; DMF: N,N-dimethylformamide; DMAP: 4-dimethylaminopyridine; TBAF: tetra-n-butylammonium fluoride; THF: tetrahydrofuran; TFA: trifluoroacetic acid; iPrOH: isopropanol; TLC: thin layer chromatography; A: Adenine; G: Guanine; C: Cytosine; T: Thymine; U: Uracil; I: hypoxanthine; Xan: xanthine; m5C: 5-methylcytosine.

[0055] Example 1

[0056] This example is a method for synthesizing a nucleotide monomer [adenine thiophosphoramidate, A-1: O-((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-methoxytetrahydrofuran-3-yl)dimethylphosphoramidochloridothioate] for synthesizing an oligonucleic acid having a thio-modified nucleotide structure, and the synthetic reaction formula is

[0057]

[0058] The method for synthesis includes:

[0059] Under nitrogen protection, 688 mg of O2'-Me-O5'-DMTr-N6-Bz-A (A-0, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), temperature was controlled at 0°C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoramide dichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added to the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product A-1 (purity of more than 99%, yield of 71%) was obtained by silica gel chromatography separation, and the mobile phase was selected as a mixture of dichloromethane / acetone / methanol.

[0060] Example 2

[0061] This example is a synthesis method of a nucleotide monomer [guanine adenine thiophosphoramidate, G-1-1: O-((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-methoxytetrahydrofuran-3-yl)dimethylphosphoramidochloridothioate] for the synthesis of oligonucleic acid with a thio-modified nucleotide structure, and the synthesis reaction formula is

[0062] The synthesis method comprises:

[0063] Under nitrogen protection, 670 mg of O2'-Me-O5'-DMTr-N2-iBu-G (G-1-0, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), temperature was controlled at 0°C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoraminodichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added into the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product G-1-1 (purity of more than 99%, yield of 61%) was separated by silica gel chromatography using dichloromethane / acetone / methanol mixed solvent as the mobile phase.

[0064] Example 3

[0065] This example is a synthesis method of a nucleotide monomer [guanine adenine thiophosphoramidate, G-2-1: O-((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-isobutyramido-6-(4-nitrophenethoxy)-9H-purin-9-yl)-4-methoxytetrahydrofuran-3-yl)dimethylphosphoramidochloridothioate] for the synthesis of oligonucleic acid with a thio-modified nucleotide structure. The synthesis reaction formula is as follows:

[0066] The synthesis method comprises:

[0067] Under nitrogen protection, 819 mg of O2'-Me-O5'-DMTr-N2-iBu-O6-NPE-G (G-2-0, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), temperature was controlled at 0°C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoramide dichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added to the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product G-2-1 (purity of more than 99%, yield of 67%) was separated by silica gel chromatography using a dichloromethane / acetone / methanol mixed solvent as the mobile phase.

[0068] Example 4

[0069] This embodiment is a synthesis method of a nucleotide monomer [guanine adenine thiophosphoramidate, G-3-1: O-((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(((E)-(dimethylamino)methylene)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-methoxytetrahydrofuran-3-yl)dimethylphosphoramidochloridethioate] for the synthesis of oligonucleic acid with a thio-modified nucleotide structure. The synthesis reaction formula is as follows:

[0070]

[0071] The synthesis method comprises:

[0072] Under nitrogen protection, 655 mg of O2'-Me-O5'-DMTr-N2-DMF-G (G-3-0, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), temperature was controlled at 0°C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoramide dichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added into the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product G-3-1 (purity of more than 99%, yield of 56%) was separated by silica gel chromatography, and the mobile phase was selected as a mixture of dichloromethane / acetone / methanol.

[0073] Example 5

[0074] This embodiment is a synthesis method of a nucleotide monomer [guanine adenine thiophosphoramidate, G-4-1: O-((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(((E)-(dimethylamino)methylene)amino)-6-(4-nitrophenethoxy)-9H-purin-9-yl)-4-methoxytetrahydrofuran-3-yl)dimethylphosphoramidochloridothioate] for the synthesis of oligonucleic acid with a thio-modified nucleotide structure. The synthesis reaction formula is as follows:

[0075]

[0076] The synthesis method comprises:

[0077] Under nitrogen protection, 804 mg of O2'-Me-O5'-DMTr-N2-DMF-O6-NPE-G (G-4-0, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), temperature was controlled at 0°C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoramide dichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added to the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product G-4-1 (purity of more than 99%, yield of 66%) was obtained by silica gel chromatography separation, and the mobile phase was selected as a mixture of dichloromethane / acetone / methanol.

[0078] Example 6

[0079] This embodiment is a synthesis method of a nucleotide monomer [cytosine thiophosphoramidate, C-1: O-((2R,3R,4R,5R)-5-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-methoxytetrahydrofuran-3-yl)dimethylphosphoramidochloridothioate] for the synthesis of oligonucleic acid with a thio-modified nucleotide structure. The synthesis reaction formula is as follows:

[0080]

[0081] The synthesis method comprises:

[0082] Under nitrogen protection, 664 mg of O2'-Me-O5'-DMTr-N4-Bz-C(C-O, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), temperature was controlled at 0°C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoraminodichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added to the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product C-1 (purity of more than 99%, yield of 70%) was obtained by silica gel chromatography separation, and the flow phase was selected as a mixture of dichloromethane / acetone / methanol.

[0083] Example 7

[0084] This example is a synthesis method of a nucleotide monomer [5-methyl adenine thiophosphoramidate, m5C-1: O-((2R,3R,4R,5R)-5-(4-benzamido-5-methyl-2-oxo pyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-methoxytetrahydrofuran-3-yl)dimethylphosphoramidochloridothioate] for the synthesis of oligonucleic acid with a thio-modified nucleotide structure, and the synthesis reaction formula is

[0085] The synthesis method comprises:

[0086] Under nitrogen protection, 678 mg of O2'-Me-O5'-DMTr-N4-Bz-m5C (m5C-O, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), temperature was controlled at 0 °C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 min. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoramide dichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0 °C for 40 min under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added to the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product m5C-1 (purity >99%, yield 72%) was separated by silica gel chromatography using dichloromethane / acetone / methanol mixed solvent as the mobile phase.

[0087] Example 8

[0088] This example is a synthesis method of a nucleotide monomer [uracil thiophosphoramidate, U-1-1: O-((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl)dimethylphosphoramidochloridothioate] for the synthesis of oligonucleic acid with a thio-modified nucleotide structure, and the synthesis reaction formula is

[0089]

[0090] The synthesis method comprises:

[0091] Under nitrogen protection, 561 mg of O2'-Me-O5'-DMTr-U (U-1-0, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), temperature was controlled at 0°C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoramide dichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added to the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product U-1-1 (purity of more than 99%, yield of 60%) was obtained by silica gel chromatography separation, and the mobile phase was selected as a mixture of dichloromethane / acetone / methanol.

[0092] Example 9

[0093] This example is a synthesis method of a nucleotide monomer [thymine thiophosphoramidate, T-1-1: O-((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-methoxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)dimethylphosphoramidochloridothioate] for the synthesis of oligonucleic acid with a thio-modified nucleotide structure, and the synthesis reaction formula is

[0094]

[0095] The synthesis method comprises:

[0096] Under nitrogen protection, 575 mg of O2'-Me-O5'-DMTr-T (T-1-0, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), temperature was controlled at 0°C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoramide dichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added into the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product T-1-1 (purity of more than 99%, yield of 58%) was obtained by silica gel chromatography separation using dichloromethane / acetone / methanol mixed solvent as the mobile phase.

[0097] Example 10

[0098] This example is a synthesis method of a nucleotide monomer [thiophosphoramidite of inosine, I-1-1: O-((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-methoxy-5-(6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)dimethylphosphoramidochloridothioate] for the synthesis of oligonucleic acid with a thio-modified nucleotide structure, and the synthesis reaction formula is

[0099]

[0100] The synthesis method comprises:

[0101] Under nitrogen protection, 585 mg of O2'-Me-O5'-DMTr-I (I-1-0, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), temperature was controlled at 0°C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoramide dichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added into the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product I-1-1 (purity of more than 99%, yield of 53%) was obtained by silica gel chromatography separation, and the mobile phase was selected as a mixed solvent of dichloromethane / acetone / methanol.

[0102] Example 11

[0103] This example is a synthesis method of a nucleotide monomer [thio-modified nucleotide structure, I-2-1: O-((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-methoxy-5-(6-(4-nitrophenethoxy)-9H-purin-9-yl)tetrahydrofuran-3-yl)dimethylphosphoramidochloridothioate] for the synthesis of oligonucleic acid, and the synthesis reaction formula is as follows:

[0104]

[0105] The synthesis method comprises:

[0106] Under nitrogen protection, 734 mg of O2'-Me-O5'-DMTr-O6-NPE-I (I-2-0, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), temperature was controlled at 0°C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoramide dichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added into the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product I-2-1 (purity of more than 99%, yield of 62%) was separated by silica gel chromatography using dichloromethane / acetone / methanol mixed solvent as the mobile phase.

[0107] Example 12

[0108] This example is a synthesis method of a nucleotide monomer [uracil thiophosphoramidate, U-2-1: O-((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluorotetrahydrofuran-3-yl)dimethylphosphoramidochloridothioate] for the synthesis of oligonucleic acid with a thio-modified nucleotide structure, and the synthesis reaction formula is as follows:

[0109]

[0110] The synthesis method comprises:

[0111] Under nitrogen protection, 549 mg of 2'-F-O5'-DMTr-U (U-2-0, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), and the temperature was controlled at 0°C. 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoraminodichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added to the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. Silica gel chromatography was used for separation, and the mobile phase was selected as a mixture of dichloromethane / acetone / methanol to obtain 386 mg of the target product U-2-1 (purity >99%, yield 55%).

[0112] The following nucleotide monomers were synthesized by using the above-mentioned similar synthesis method:

[0113]

[0114] Example 13

[0115] This example is a synthesis method of a nucleotide monomer [thymine thiophosphoramidate, T-2-1: O-((2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)dimethylphosphoramidochloridothioate] for the synthesis of oligonucleic acid with a thio-modified nucleotide structure, and the synthesis reaction formula is

[0116]

[0117] The synthesis method comprises:

[0118] Under nitrogen protection, 545 mg of O5'-DMTr-T(T-2-0, 1.0 mmol, 1.0 eq.) was added into dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), temperature was controlled at 0°C, 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.30 mL of DBU (2.0 mmol, 2.0 eq.) and 0.20 mL of N,N-dimethylthiophosphoramide dichloride (1.5 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The solvent was removed under reduced pressure, 50 mL of ethyl acetate was added into the residue, and after washing with 30 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product T-2-1 (purity of more than 99%, yield of 63%) was obtained by silica gel chromatography separation using dichloromethane / acetone / methanol mixed solvent as the mobile phase.

[0119] The following nucleotide monomers were synthesized by using the similar synthesis method as described above:

[0120]

[0121]

[0122] Example 14

[0123] The present embodiment is a kind of oligonucleic acid synthesis of thio modified nucleotide structure nucleotide monomer, the synthesis method of the nucleotide monomer uses the similar method in the above-mentioned embodiments 1-3, and the structural formula of the nucleotide monomer includes:

[0124]

[0125]

[0126] The preparation and synthesis of the above-mentioned O5' position protection group 4,4'-dimethoxytrityl (DMTr) of thiophosphoramide nucleotide monomer is also applicable to the replacement of the O5' position protection group of the nucleotide monomer with trityl (Tr), 4-methoxytrityl (MMTr) and 4,4',4"-trimethoxytrityl (TMTr).

[0127] Example 15

[0128] The present embodiment is a kind of oligonucleic acid The nucleotide monomer loaded on the solid phase carrier is The thio modified nucleotide monomer is

[0129]

[0130] The synthesis reaction formula is

[0131]

[0132] The synthesis method of the oligonucleic acid with thio-modified nucleotide structure comprises:

[0133] The monomer O5'-DMTr-O2'-Me-T is loaded on the cross-linked polystyrene PS, and O5'-DMTr is removed by using 3% dichloroacetic acid toluene solution. After acetonitrile washing, the monomer U-1-1 is pumped in for coupling. The system is capped, O5'-DMTr is removed by using 3% dichloroacetic acid toluene solution, and the conversion rate is detected online as 98.2%. After acetonitrile washing, the monomer U-1-1 is pumped in for coupling. O5'-DMTr is removed by using 3% dichloroacetic acid toluene solution, and the conversion rate is detected online as 98.0%.

[0134] After the loaded oligonucleic acid molecule is subjected to ammonolysis and deprotection treatment, the crude product concentrated under reduced pressure is separated and purified by HPLC, the main fraction is collected, and the product obtained after concentration and freeze-drying is detected and confirmed as the nucleic acid molecule of the target sequence.

[0135] Example 16

[0136] The example is an oligonucleic acid with thio-modified nucleotide structure The nucleotide monomer loaded on the solid-phase carrier is The thio-modified nucleotide monomer is

[0137]

[0138] The synthesis reaction formula is

[0139]

[0140] The synthesis method of the oligonucleic acid with thio-modified nucleotide structure comprises:

[0141] The monomer O5'-DMTr-O2'-Me-T is loaded on the cross-linked polystyrene PS, and O5'-DMTr is removed by using 3% dichloroacetic acid toluene solution. After acetonitrile washing, the monomer U-1-1 is pumped in for coupling. The system is capped, O5'-DMTr is removed by using 3% dichloroacetic acid toluene solution, and the conversion rate is detected online as 98.2%. After acetonitrile washing, the monomer U-1-1 is pumped in for coupling. O5'-DMTr is removed by using 3% dichloroacetic acid toluene solution, and the conversion rate is detected online as 98.0%.

[0142] After the loaded oligonucleic acid molecule is subjected to ammonolysis and deprotection treatment, the crude product concentrated under reduced pressure is separated and purified by HPLC, the main fraction is collected, and the product obtained after concentration and freeze-drying is detected and confirmed as the nucleic acid molecule of the target sequence.

[0143] Example 17

[0144] The oligonucleic acid of the present application is a thio-modified nucleotide structure The nucleotide monomer loaded on the solid phase carrier is The thio-modified nucleotide monomer is The nucleotide phosphoramidite 2'-F-O5'-DMTr-A-P(OCE)(N i Pr2) is

[0145]

[0146] The synthetic reaction formula is

[0147]

[0148] The synthesis method of the oligonucleic acid of the thio-modified nucleotide structure comprises:

[0149] The monomer O5'-DMTr-O2'-Me-T is loaded on the cross-linked polystyrene PS, O5'-DMTr is removed by 3% dichloroacetic acid toluene solution, acetonitrile is washed, and then the monomer U-2-1 is pumped in for coupling. The system is capped, O5'-DMTr is removed by 3% dichloroacetic acid toluene solution, and the conversion rate is detected online as 98.2%. After acetonitrile is washed, the monomer 2'-F-O5'-DMTr-A-P(OCE)(N i Pr2) is pumped in for coupling and iodine / pyridine oxidation. O5'-DMTr is removed by 3% dichloroacetic acid toluene solution, and the conversion rate is detected online as 99.3%.

[0150] After the loaded oligonucleic acid molecule is subjected to ammonolysis and deprotection treatment, the crude product concentrated under reduced pressure is separated and purified by HPLC, the main fraction is collected, concentrated and freeze-dried to obtain the product, which is confirmed by detection to be the nucleic acid molecule of the target sequence.

[0151] Example 18

[0152] The oligonucleic acid of the present application is a thio-modified nucleotide structure The nucleotide monomer loaded on the solid phase carrier is The thio-modified nucleotide monomer is

[0153]

[0154] The synthetic reaction formula is

[0155]

[0156] The synthesis method of the oligonucleic acid of the thio-modified nucleotide structure comprises:

[0157] The monomer O5'-DMTr-O2'-Me-T is loaded on the cross-linked polystyrene PS, and O5'-DMTr is removed by 3% dichloroacetic acid toluene solution. After acetonitrile washing, the monomer T-2-1 is pumped in for coupling. The system is capped, O5'-DMTr is removed by 3% dichloroacetic acid toluene solution, and the conversion rate is detected on line as 98.4%. After acetonitrile washing, the monomer T-2-1 is pumped in for coupling. The system is capped, O5'-DMTr is removed by 3% dichloroacetic acid toluene solution, and the conversion rate is detected on line as 98.5%.

[0158] After the loaded oligonucleotide molecule is subjected to ammonolysis and deprotection, the crude product concentrated under reduced pressure is separated and purified by HPLC. The main fraction is collected, concentrated, and lyophilized to obtain the product. The product is detected and confirmed as the nucleic acid molecule of the target sequence.

[0159] Example 19

[0160] The oligonucleotide of the thio-modified nucleotide structure in this example is prepared by using the nucleotide monomer loaded on the solid support as The thio-modified nucleotide monomer is (U-1-1);

[0161] The structural formula of the oligonucleotide is

[0162] The synthesis method of the oligonucleotide of the thio-modified nucleotide structure comprises:

[0163] The monomer O5'-DMTr-O2'-Me-T is loaded on the cross-linked polystyrene PS, and O5'-DMTr is removed by 3% dichloroacetic acid toluene solution. After acetonitrile washing, the monomer U-1-1 is pumped in for coupling. The system is capped, O5'-DMTr is removed by 3% dichloroacetic acid toluene solution. After acetonitrile washing, the monomer U-1-1 is pumped in, and the above cycle operation is repeated six times to obtain the oligonucleotide molecule of the sequence structure of the thio-phosphoramide modification, which is denoted as ON7-SN.

[0164] Comparative Example 1

[0165] The oligonucleotide of this comparative example has the same base sequence as ON7-SN, and is prepared by using the nucleotide monomer loaded on the solid support as The nucleotide monomer used is nucleotide phosphoramidite The structural formula of the oligonucleotide is denoted as ON7. The oligonucleotide is synthesized by using the conventional solid-phase synthesis method.

[0166] Experimental Example

[0167] The stability of ON7-SN prepared in Example 19 and ON7 prepared in Comparative Example 1 in human serum and human liver microsomes was tested.

[0168] The test results show that:

[0169] The oligonucleic acid molecule ON7 without phosphorothioamidate modification was obviously degraded, and the proportion of undegraded nucleic acid sequence was less than 5% after 4 hours. In contrast, the proportion of undegraded oligonucleic acid chain ON7-SN containing phosphorothioamidate modified nucleotides was greater than 96% after 48 hours of incubation in human serum or human liver microsomes, showing that the modified oligonucleic acid molecule has stronger resistance to biological enzyme degradation.

[0170] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent substitution for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should be covered in the scope of the claims and the specification of the present application.

Claims

1. An oligonucleic acid of a thio-modified nucleotide structure, characterized in that, The oligonucleic acid of the thio-modified nucleotide structure comprises a nucleotide structural unit shown in Formula I or Formula II; In Formula I: Base is a nucleotide base or a derivative thereof, the nucleotide base being adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine or xanthine; R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R3 is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, silyl, siloxy-methylene, alkoxyalkyl, benzyl or acyl; In Formula II: Base is a nucleotide base or a derivative thereof, the nucleotide base being adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine or xanthine; R is hydrogen, halogen, alkyl, thiol, alkylthiol, amine, azide or cyano; R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl or aryl.

2. The oligonucleotide of thio -modified nucleotide structure according to claim 1, characterized in that, The nucleotide monomer used in the synthesis of the oligonucleic acid of the thio-modified nucleotide structure comprises Formula III or Formula IV: In Formula III: Base is a nucleotide base or a derivative thereof, the nucleotide base being adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine or xanthine; PG is trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; R is hydrogen, halogen, alkyl, thiol, alkylthiol, amine, azide or cyano; R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl or aryl. In Formula IV: Base is a nucleotide base or a derivative thereof, the nucleotide base being adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine or xanthine; PG is trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl or aryl. R3 is alkyl, cycloalkyl, alkenyl, alkynyl, silyl, siloxy-methylene, alkoxyalkyl, benzyl or acyl.

3. The oligonucleotide of thio -modified nucleotide structures according to claim 2, characterized in that, The protecting group is selected from one of trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl.

4. The oligonucleotide structure of claim 2, wherein, The nucleotide monomer comprises: wherein: R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R3 is alkyl, cycloalkyl, alkenyl, alkynyl, silyl, siloxy-methylene, alkoxyalkyl, benzyl or acyl.

5. The oligonucleotide structure of claim 2, wherein, The nucleotide monomer comprises: wherein: R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl or aryl.

6. The oligonucleotide structure of claim 1, wherein, The nucleotide monomer comprises: wherein: R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl or aryl.

7. The oligonucleic acid of thio -modified nucleotide structure according to any one of claims 4 to 6, characterized in that, The 4,4'-dimethoxytrityl (DMTr) in the nucleotide monomer is replaced by trityl (Tr), 4-methoxytrityl (MMTr) or 4,4',4"-trimethoxytrityl (TMTr).

8. The oligonucleotide structure of claim 2, wherein, The nucleotide monomer is prepared by reacting O5' protected nucleotide molecule and phosphorothioamidite dichloride in liquid phase.

9. The oligonucleotide structure of claim 1, wherein, The thio-modified nucleotide structure in the oligonucleic acid can improve the biological stability and anti-enzyme degradation activity of the oligonucleic acid.

10. The process for the synthesis of a sulfur modified nucleotide structure oligonucleic acid according to any one of claims 1 to 9, characterized in that, The synthesis process comprises the following steps: (a) loading the nucleotide monomer on the solid phase carrier, removing the O5' protective group of the nucleotide monomer and washing; (b) pumping the nucleotide monomer into the system after washing for coupling, and then carrying out capping, removing the O5' protective group of the nucleotide monomer and washing, wherein the nucleotide monomer is a phosphorothioamidite nucleotide monomer or a phosphoramidite nucleotide monomer; if the nucleotide monomer is a phosphoramidite nucleotide monomer, trivalent phosphorodiseter is converted into pentavalent phosphorodiseter by oxidation or sulfurization after the coupling reaction, and then capping, removing the O5' protective group of the nucleotide monomer and washing are carried out; (c) repeating step (b) to obtain the oligonucleic acid with thio-modified nucleotide structure.

Images