Synthesis of 3-amino-5-nucleotide thiophosphamide and application of 3-amino-5-nucleotide thiophosphamide in oligonucleotide
By introducing thiophosphoramide-modified nucleotide structures into oligonucleotides, the problem of poor stability of traditional oligonucleotides in the human environment is solved, and their stability and bioactivity in vivo are improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-12
- Publication Date
- 2026-03-27
AI Technical Summary
Oligonucleotide sequences synthesized from traditional nucleotide monomers are unstable in human serum and human liver microsomes and are easily degraded.
By using thiophosphoramide-modified nucleotide structures, specific thiophosphoramide modification units are introduced into oligonucleotides through solid-phase synthesis processes to improve their stability and biological activity.
This study improved the resistance to enzyme degradation and stability of oligonucleotides, providing superior pharmaceutical properties for oligonucleotide drugs.
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Figure CN121736029A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of oligonucleotide technology, specifically to the synthesis of 3´-amino-5´-nucleotide thiophosphoramide and its application in oligonucleotide drugs. Background Technology
[0002] Oligonucleotide drugs bind specifically to target genes through base complementarity, extending the target to upstream mRNA of pathogenic proteins and influencing target gene expression at the post-transcriptional level. Oligonucleotide drugs offer advantages such as abundant targets, high development success rates, short development cycles, and long-lasting efficacy, providing solutions for the treatment of many intractable diseases.
[0003] In addition, oligonucleotides are widely used in molecular biology research, disease diagnosis and treatment, such as as PCR primers, gene editing tools or targeted drugs.
[0004] Currently, oligonucleotide sequences synthesized from traditional nucleotide monomers have poor stability and are easily degraded in human serum and human liver microsomes.
[0005] In view of this, the present invention is hereby proposed. Summary of the Invention
[0006] The purpose of this invention is to provide a thiophosphoramide-modified oligonucleotide and its synthesis process. By preparing thiophosphoramide nucleotides, a specific thiophosphoramide-modified nucleotide structure can be obtained, which can improve the stability and biological activity of the synthesized oligonucleotide.
[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: A first aspect of the present invention provides a thiophosphoramide-modified oligonucleotide, wherein the thiophosphoramide-modified oligonucleotide comprises at least one of the nucleotide structural units shown in Formula I, Formula II, Formula III, or Formula IV: ...Formula I; ...Formula II; In Equations I and II: Base is a nucleotide base or its derivative, wherein the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, etc., or its derivatives. R can be hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or aromatic. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, or aromatic groups, respectively. R3 is hydrogen, alkyl (including methyl, ethyl, long chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl), siloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl); … Formula III; … Formula IV; Formula III, Formula IV: Base is a nucleotide base or a derivative thereof, the nucleotide base being adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudouracil, and the like, and derivatives thereof; R is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, aryl; R1 and R2 are each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl; R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azido, amine, mercapto, alkylmercapto, seleno, alkylseleno, and the like.
[0008] The phosphorothioate-modified oligonucleic acid is synthesized using a nucleotide monomer represented by Formula V or Formula VI: … Formula V; Formula V: Base is a nucleotide base or a derivative thereof, the nucleotide base being adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudouracil, and the like, and derivatives thereof; PG is trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4''-trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, t-butyldimethylsilyl, or t-butyldiphenylsilyl); R is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, aryl; R1 and R2 are each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl; R3 is alkyl (including methyl, ethyl, long chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl), siloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl); … Formula VI; In Formula VI: Base is a nucleotide base comprising a protected adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudouracil, and the like, and derivatives thereof; PG is trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, t-butyldimethylsilyl, or t-butyldiphenylsilyl); R is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, aryl; R1and R2are each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl; R4and R5are hydrogen, halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azido, amine, thiol, alkylthiol, seleno, alkylseleno, and the like.
[0009] 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.
[0010] Preferably, the nucleotide phosphorothioamidate monomer comprises: , ; wherein: Base is a nucleotide base comprising a protected adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudouracil, and the like, and derivatives thereof; R is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, aryl; R1and R2are each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl; R3is alkyl (including methyl, ethyl, long chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl); R4and R5are hydrogen, halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azido, amine, thiol, alkylthiol, seleno, alkylseleno, and the like.
[0011] Preferably, the nucleotide thiophosphoramidate monomer comprises: , , , , , , , , , , , wherein: R is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, aryl; R1and R2are each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl; R3is alkyl (including methyl, ethyl, long chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl), siloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl).
[0012] Preferably, the nucleotide thiophosphoramidate monomer comprises: , , , , , , , , , , , wherein: R is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, aryl; R1and R2are each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl.
[0013] Preferably, the nucleotide thiophosphoramidate monomer comprises: , , , , , , , , , , , wherein: R is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, aryl; R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl or aryl.
[0014] Preferably, the 4,4'-dimethoxytrityl (DMTr) in the nucleotide monomer is replaced by trityl (Tr), 4-methoxytrityl (MMTr) or 4,4',4''-trimethoxytrityl (TMTr).
[0015] Preferably, the nucleotide monomer is prepared by reacting N3' containing a protecting group of nucleotide and thiophosphoramidite dichloride in liquid phase.
[0016] The modified oligonucleotide provided by the present application contains a nucleotide structure modified by thiophosphoramidite, which can improve the biological stability and anti-enzyme degradation activity of the oligonucleotide.
[0017] The second aspect of the present application provides a synthesis process of the above-mentioned (thio) phosphoramidite modified oligonucleotide. The coupling of the nucleotide monomer in the solid phase synthesis is in the direction of 5' to 3'. The synthesis process comprises the following steps: (a) loading the nucleotide monomer on the solid phase carrier, then removing the 3' protecting group of the nucleotide monomer and washing; (b) pumping the nucleotide monomer into the system after washing for coupling, then performing capping treatment, removing the 3' protecting group of the nucleotide monomer and washing. The nucleotide monomer is (thio) phosphoramidite nucleotide monomer or phosphoramidite nucleotide monomer. If the nucleotide monomer is phosphoramidite nucleotide monomer, after the coupling reaction, the trivalent phosphite is converted into pentavalent phosphate by oxidation or sulfurization, and then capping, removing the 3' protecting group of the nucleotide monomer and washing are performed; (c) repeating the step (b) to obtain the oligonucleotide with (thio) phosphoramidite modified nucleotide structure.
[0018] Compared with the prior art, the present application has at least the following beneficial effects: By specific limitation of the structure of the thiophosphoramidite modified nucleotide monomer, the oligonucleotide prepared contains a structural unit of thiophosphoramidite. The thiophosphoramidite modified oligonucleotide drug provided by the present application shows more excellent anti-enzyme degradation, stability, binding and other pharmaceutical properties than the traditional oligonucleotide molecule, and provides a new chemical modification strategy for the research and application of oligonucleotide drugs. DETAILED DESCRIPTION
[0019] 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.
[0020] It should be noted that the technical terms or scientific terms used in the present application should be understood as the general meanings understood by the skilled person in the field of the present application, unless otherwise specified.
[0021] In the present application, the corresponding substances of the letter abbreviations are as follows: PS: polystyrene; CPG: controlled pore glass; DBU: 1,8-diazabicycloundec-7-ene; P(III): trivalent phosphorus (phosphine); P(V): pentavalent phosphorus (phosphine); F: fluorine; O: oxygen; S: sulfur; Me: methyl; Alkyl: alkyl; Allyl: allyl; Alloc: allyloxycarbonyl; Bn: benzyl; Bz: benzoyl; Tr: trityl; MMTr: methoxytrityl; DMTr: dimethoxytrityl; TMTr: trimethoxytrityl; iBu: 2-isobutyryl; NPE: 4-nitrophenethyloxy; Ph: aromatic group; 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: isopropyl alcohol; TLC: thin layer chromatography; A: Adenine; G: Guanine; C: Cytosine; T: Thymine; U: Uracil; I: hypoxanthine; Xan: xanthine; m5C: 5-methylcytosine; HPLC: high performance liquid chromatography.
[0022] Example 1 This example is a method for synthesizing a nucleotide monomer for the synthesis of a thiophosphoramidate-modified oligonucleic acid, adenine thiophosphoramidate, A-1: O-(((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-((bis(4-methoxyphenyl)(phenyl)methyl)amino)-4-methoxytetrahydrofuran-2-yl)methyl) dimethylphosphoramidochloridothioate. The synthesis reaction formula is as follows: ; The synthesis method comprises: Under nitrogen protection, 687 mg of O2'-Me-N3'-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), 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-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. Silica gel chromatography was used for separation, and the mobile phase was selected as a mixture of dichloromethane / acetone / methanol to obtain 423 mg of the target product A-1 (purity of more than 99%, yield of 51%).
[0023] Example 2 This example is a method for synthesizing a thiophosphoramidate-modified oligonucleotide nucleotide monomer, uracil thiophosphoramidate, U-1-1: O-(((2S,3R,4R,5R)-3-((bis(4-methoxyphenyl)(phenyl)methyl)amino)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)methyl) dimethylphosphoramidochloridothioate. The synthesis reaction formula is as follows: ; The synthesis method comprises: Under nitrogen protection, 560 mg of O2'-Me-N3'-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), 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 410 mg of the target product U-1-1 (purity of more than 99%, yield of 58%).
[0024] Further, the following nucleotide monomers were prepared using the above-described synthesis method: 、 、 、 、 、 、 、 、 .
[0025] Example 3 This example is a synthesis method of a thiophosphoramidate-modified oligonucleotide nucleotide monomer, uracil thiophosphoramidate, U-2-1: O-(((2S,3R,4R,5R)-3-((bis(4-methoxyphenyl)(phenyl)methyl)amino)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluorotetrahydrofuran-2-yl)methyl) dimethylphosphoramidochloridothioate. The synthesis reaction formula is as follows: ; The synthesis method comprises: Under nitrogen protection, 548 mg of 2'-F-N3'-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), 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 U-2-1 (purity >99%, yield 55%) was obtained by silica gel chromatography separation using dichloromethane / acetone / methanol mixed solvent as the mobile phase.
[0026] Further, the following nucleotide monomers were prepared using the above-described synthesis method: 、 、 、 、 、 、 、 、 、 .
[0027] Further, the following nucleotide monomers were prepared using the above-described synthesis method: 、 、 、 、 、 、 、 、 、 、 .
[0028] Example 4 The present embodiment is a method for synthesizing a nucleotide monomer for the synthesis of a phosphorothioamidite-modified oligonucleic acid, uracil phosphorothioamidite, U-3-1: O-(((2S,3R,4R,5R)-3-((bis(4-methoxyphenyl)(phenyl)methyl)(methyl)amino)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)methyl) dimethylphosphoramidochloridothioate. The synthesis reaction formula is as follows: ; The synthesis method comprises the following steps: Under nitrogen protection, 574 mg of O2'-Me-N3'-Me-N3'-DMTr-U (U-3-0, 1.0 mmol, 1.0 eq.) is added into a dry mixed solvent (5 mL of dichloromethane and 5 mL of acetonitrile), and the temperature is controlled at 0°C. Then 174 mg of lithium bromide (2.0 mmol, 2.0 eq.) is added, and the mixture is stirred for 5 minutes. Then 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 are added in sequence. The mixed solution is stirred at 0°C for 40 minutes under nitrogen protection. The solvent is removed under reduced pressure, 50 mL of ethyl acetate is added to the residue, and after washing with 30 mL of cold water, the organic phase is dried over sodium sulfate and concentrated under reduced pressure. Silica gel chromatography is used for separation, and a dichloromethane / acetone / methanol mixed solvent is selected as the mobile phase to obtain 402 mg of the target product U-3-1 (purity of more than 99%, yield of 56%).
[0029] Further, the following nucleotide monomers are prepared by using the above-mentioned synthesis method: 、 ; Among them: Base is a nucleotide base containing a protected group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil, and derivatives thereof; R is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, aryl; R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R3 is alkyl (including methyl, ethyl, long chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl), siloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl); R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azido, amine group (amino), mercapto, alkylmercapto, seleno, alkylseleno, etc.
[0030] Further, the above-mentioned synthetic method is used to prepare the following nucleotide monomers: , ; wherein: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudouracil, and derivatives thereof; PG is trityl (Tr), 4-methoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr), 4,4',4''-trimethoxytrityl (TMTr), allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, t-butyldimethylsilyl, or t-butyldiphenylsilyl); R is alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, aryl; R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl, or aryl; R3 is alkyl (including methyl, ethyl, long chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl), siloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl); R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azido, amine group (amino), mercapto, alkylmercapto, seleno, alkylseleno, etc.
[0031] The carrier for solid phase synthesis is selected as cross-linked polystyrene PS; 0.050 grams of modified solid phase synthesis resin is weighed and added into a solid phase synthesis tube; after 2.0 milliliters of NMP is added, it is swelled at room temperature for 2 hours, and then filtered and emptied of the solution. 3.0 milliliters of dichloromethane is used to wash the resin, and this washing process is repeated 3 times, and the solid phase synthesis is started.
[0032] The monomers are introduced in solid phase synthesis as phosphoramidite P(III) and (thio)phosphoramidite P(V).
[0033] The process conditions for the phosphoramidite monomers are as follows: Reagents and monomers: 0.15 mol / L monomer / acetonitrile solution was prepared; the deprotection reagent was 5% dichloroacetic acid / toluene solution (v / v) for removing the DMTr (or Tr) protecting group; the activator was 0.25 mol / L 5-ethylthiotetrazole / acetonitrile solution; the oxidizing reagent was 0.05 mol / L iodine / water / pyridine (10 / 90, v / v) solution; the sulfurizing reagent was 3% hydrogenated xanthate / pyridine solution; cap A was 10% acetic anhydride / acetonitrile solution (v / v), and cap B was 1-methylimidazole / pyridine / tetrahydrofuran solution (16 / 10 / 74, v / v / v).
[0034] The operation sequence for the solid phase synthesis of the phosphoramidite monomers is as follows: 1) Deprotection: 5% dichloroacetic acid / toluene solution was used to remove the DMTr protecting group, and then acetonitrile was used for washing.
[0035] 2) Coupling: 0.25 mol / L 5-ethylthiotetrazole was used as the activator, and the nucleotide monomer / acetonitrile solution was introduced into the cycle according to the sequence design for coupling, and then acetonitrile was used for washing.
[0036] 3) Oxidation / sulfurization: 0.05 mol / L iodine / water / pyridine (v / v, 90 / 10) solution was used as the oxidizing reagent to oxidize P(III), and then acetonitrile was used for washing. 3% hydrogenated xanthate / pyridine solution was used as the sulfurizing reagent to sulfurize P(III), and then acetonitrile was used for washing.
[0037] 4) Capping: cap A and cap B were used as the capping reagents to protect the unreacted active groups, and then acetonitrile was used for washing.
[0038] The process conditions for the application of (thio)phosphoramidite monomers to solid phase synthesis of oligonucleic acids are as follows: Coupling solvent: 2.303 grams of N-ethylmorpholine was dissolved in 1,3-dimethyl-2-imidazolidinone (DMI) to make up to 50 milliliters, obtaining an N-ethylmorpholine DMI solution (0.40 mol / L), which was used as the coupling solvent.
[0039] Lysis solution: 0.154 g dithiothreitol was weighed and dissolved in 5 mL N-methyl pyrrolidone (NMP), 0.739 g triethylamine was added, and the volume was made up to 10 mL with NMP to obtain a solution containing 0.10 mol / L dithiothreitol and 0.73 mol / L triethylamine in NMP, which was used immediately.
[0040] The process operation of solid-phase synthesis of (thio)phosphoramidite monomers includes: According to the base sequence of the target sequence, the nucleotide monomers were coupled one by one through the cycle operation process of DMTr removal, neutralization, washing, coupling, neutralization, and washing. After the coupling of the last monomer molecule was completed, the terminal DMTr was removed, and 3.0 mL of p-methoxytrityl chloride capping solution (0.32 mol / L) was continuously added in the reaction tube. After reaction at 45°C for 90 minutes, the reaction solution was emptied, and NMP was used for washing 8 times, each time 4.0 mL.
[0041] The above operation was repeated according to the set sequence to obtain the target sequence oligonucleotide product.
[0042] Deprotection is to transfer the solid-phase carrier loaded with the oligonucleotide product to the reactor, add concentrated ammonia water (28%), control the temperature at 60°C for ammoniaolysis for 12 hours, then reduce to room temperature, filter, and rinse the solid with a mixture of purified water and ethanol. The combined filtrate was concentrated under reduced pressure at low temperature to obtain the crude oligonucleotide of the designed sequence.
[0043] Purification is to dissolve the crude product after deprotection with purified water, purify it by HPLC, collect the product peak solution, and obtain the product by reducing pressure concentration and freeze-drying.
[0044] The above solid-phase synthesis is also applicable to Tr, MMTr, and TMTr-protected monomers.
[0045] In the example of solid-phase synthesis, the nucleotide monomers used are represented by the following structures: 、 、 、 、 、 、 、 、 、 .
[0046] Example 5 This example is a kind of oligonucleotide (DNA) with thio-phosphoramidite modified nucleotide structure The nucleotide monomers loaded on the solid support are T-1-0, and the nucleotide monomers modified with thiophosphoramide are U-1-1 and U-4-1.
[0047] The synthesis reaction formula is ; Methods for synthesizing oligonucleotides with thiophosphoramide-modified nucleotide structures include: Monomer T-1-0 was loaded onto cross-linked polystyrene (PS). O3'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-1-1 was pumped in for coupling. The system was then capped, and N3'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-4-1 was pumped in for coupling, and O3'-DMTr was removed using a 5% dichloroacetic acid-toluene solution.
[0048] After ammonolysis and deprotection treatment, the crude product of the loaded oligonucleotide molecule was concentrated under reduced pressure and purified by HPLC. The main fraction was collected, concentrated, and freeze-dried to obtain the product, which was then confirmed to be the nucleic acid molecule of the target sequence.
[0049] Example 6 This embodiment is an oligonucleotide with a thiophosphoramide-modified nucleotide structure. The nucleotide monomers loaded on the solid support are T-1-0 and thiophosphoramide-modified nucleotide monomers are U-2-1.
[0050] The synthesis reaction formula is ; Methods for synthesizing oligonucleotides with thiophosphoramide-modified nucleotide structures include: Monomer T-1-0 was loaded onto cross-linked polystyrene (PS). O3'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-2-1 was pumped in for coupling. The system was then capped, and N3'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-2-1 was pumped in for coupling, and N3'-DMTr was removed using a 5% dichloroacetic acid-toluene solution.
[0051] After ammonolysis and deprotection treatment, the crude product of the loaded oligonucleotide molecule was concentrated under reduced pressure and purified by HPLC. The main fraction was collected, concentrated, and freeze-dried to obtain the product, which was then confirmed to be the nucleic acid molecule of the target sequence.
[0052] Example 7 This embodiment is an oligonucleotide with a thiophosphoramide-modified nucleotide structure. The nucleotide monomers loaded on the solid support are T-1-0, the thiophosphoramide-modified nucleotide monomers are U-1-1, and the nucleotide phosphorylate is Af-1.
[0053] The synthetic reaction formula is ; The synthesis method of the oligonucleic acid with the thio-phosphoramide modified nucleotide structure comprises: The monomer T-1-0 is loaded on the cross-linked polystyrene PS, and the O3'-DMTr is removed by using 5% dichloroacetic acid toluene solution. After acetonitrile washing, the monomer U-1-1 is pumped in for coupling. The system is capped, and the N3'-DMTr is removed by using 5% dichloroacetic acid toluene solution. After acetonitrile washing, the monomer Af-1 is pumped in for coupling, iodine / pyridine oxidation, and removal of the O3'-DMTr by using 5% dichloroacetic acid toluene solution.
[0054] 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 using HPLC, the main fraction is collected, and the product obtained after concentration and freeze-drying is confirmed as the nucleic acid molecule of the target sequence through detection.
[0055] Example 8 The present example is an oligonucleic acid with a thio-phosphoramide modified nucleotide structure, wherein the nucleotide monomer loaded on the solid phase carrier is T-1-0, and the thio-phosphoramide modified nucleotide monomer is U-3-1 and U-4-1. The synthetic reaction formula is
[0056] ; The synthesis method of the oligonucleic acid with the thio-phosphoramide modified nucleotide structure comprises: The monomer T-1-0 is loaded on the cross-linked polystyrene PS, and the O3'-DMTr is removed by using 5% dichloroacetic acid toluene solution. After acetonitrile washing, the monomer U-3-1 is pumped in for coupling. The system is capped, and the N3'-DMTr is removed by using 5% dichloroacetic acid toluene solution. After acetonitrile washing, the monomer U-4-1 is pumped in for coupling. The system is capped, and the O3'-DMTr is removed by using 5% dichloroacetic acid toluene solution.
[0057] 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 using HPLC, the main fraction is collected, and the product obtained after concentration and freeze-drying is confirmed as the nucleic acid molecule of the target sequence through detection.
[0058] Example 9 The present example is an oligonucleic acid with a thio-phosphoramide modified nucleotide structure, wherein the nucleotide monomer loaded on the solid phase carrier is U-5-0, the thio-phosphoramide modified nucleotide monomer is U-1-1, and the 3'-terminal nucleotide thio-phosphoramide monomer is T-5-1.
[0059] The structural formula of the oligonucleic acid is ; The method for synthesizing the oligonucleic acid with the structure of the phosphorothioamidate modified nucleotide comprises the following steps: The monomer U-5-0 is loaded on the cross-linked polystyrene PS, and the O3'-DMTr is removed by using 5% dichloroacetic acid toluene solution. After acetonitrile washing, the monomer U-1-1 is pumped in for coupling. After the system is capped, the N3'-DMTr is removed by using 5% dichloroacetic acid toluene solution. After acetonitrile washing, the monomer U-1-1 is pumped in, and the above cycle operation is repeated five times. After the system is capped, the N3'-DMTr is removed by using 5% dichloroacetic acid toluene solution. After acetonitrile washing, the monomer T-5-1 is pumped in for coupling. After shearing solid loading, deprotection, HPLC purification, desalination and other treatments, the oligonucleic acid molecule with the sequence structure of the phosphorothioamidate modification is obtained, which is denoted as ON7-PN6-2.
[0060] Example 10 The present comparative example is an oligonucleic acid with the same base sequence as ON7-PN6-2. In the preparation process, the nucleotide monomer loaded on the solid support is T-2-0, and the nucleotide monomer used is nucleotide phosphoramidite U-6-1. The structural formula of the oligonucleic acid is , which is denoted as ON7. The oligonucleic acid is synthesized by using the conventional solid-phase synthesis method, and the coupling of the nucleotide is synthesized in the direction of 3' to 5'.
[0061] Example 11 The isoelectric point test of the nucleic acid is performed on the ON7-PN6-2 prepared in Example 9 and the ON7 prepared in Example 10, respectively. The test results show that the isoelectric point of the oligonucleic acid ON7 is 5.03 (±0.12), and the isoelectric point of the oligonucleic acid ON7-PN6-2 with the phosphorothioamidate modification is 7.78 (±0.21). It can be seen that the phosphorothioamidate modification can improve the binding capacity of the oligonucleic acid with anions.
[0062] Example 12 The stability of the oligonucleic acid ON7-PN6-2 prepared in Example 9 and the oligonucleic acid ON7 prepared in Example 10 in human serum and human liver microsomes is tested, respectively.
[0063] The test results show that: The oligonucleic acid molecule ON7 without the phosphorothioamidate modification is obviously degraded, and the proportion of the undegraded nucleic acid sequence is less than 5% after 4 hours. In contrast, the proportion of the undegraded oligonucleic acid chain ON7-PN6-2 with the phosphorothioamidate modified nucleotide is greater than 80% after 18 hours of incubation in the human serum or the human liver microsomes. The data results show that the phosphorothioamidate modification improves the biological enzyme resistance of the oligonucleic acid molecule.
[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that the technical solutions recorded in the foregoing embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements 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 description of the present application.
Claims
1. The synthesis of a 3´-amino-5´-nucleotide thiophosphoramide and its application in oligonucleotides, characterized in that, The thiophosphoramide-modified oligonucleotide includes at least one of the nucleotide structural units shown in Formula I, Formula II, Formula III, or Formula IV: ...Formula I; ...Formula II; In Equations I and II: Base is a nucleotide base or its derivative, wherein the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, etc., or its derivatives. R can be hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or aromatic. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, or aromatic groups, respectively. R3 is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl, silyloxymethylene, alkoxyalkyl, benzyl, or acyl. ...Formula III; ...Form IV; In Equations III and IV: Base is a nucleotide base or its derivative, wherein the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, etc., or its derivatives. R can be hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or aromatic. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, or aromatic groups, respectively. R4 and R5 can be hydrogen, halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkyl selenyl, etc.
2. The synthesis of a 3'-amino-5'-nucleotide thiophosphoramide according to claim 1 and its application in oligonucleotides, characterized in that, The synthesis of the thiophosphoramide-modified oligonucleotides uses nucleotide thiophosphoramide monomers represented by formula V or formula VI: ...Form V; In formula V: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, and its derivatives. PG is triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl. R can be hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or aromatic. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, or aromatic groups, respectively. R3 is a alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl, acyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc. ……Formula VI; In formula VI: Base is a nucleotide base containing a protecting group, such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil, or N1-methylpseudorabies, and its derivatives. PG is triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl. R can be hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or aromatic. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, or aromatic groups, respectively. R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl mercapto, selenyl, alkyl selenyl, etc.
3. The synthesis of a 3'-amino-5'-nucleotide thiophosphoramide according to claim 2 and its application in oligonucleotides, characterized in that, The nucleotide monomers include: 、 、 、 、 、 、 、 、 、 、 ， in: R can be hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or aromatic. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, or aromatic groups, respectively. R3 is an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl, silyloxymethylene, alkoxyalkyl, benzyl, or acyl group.
4. The synthesis of a 3'-amino-5'-nucleotide thiophosphoramide according to claim 2 and its application in oligonucleotides, characterized in that, The nucleotide monomers include: 、 、 、 、 、 、 、 、 、 、 ， in: R can be hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or aromatic. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, or aromatic groups, respectively.
5. The synthesis of a 3'-amino-5'-nucleotide thiophosphoramide according to claim 2 and its application in oligonucleotides, characterized in that, The nucleotide monomers include: 、 、 、 、 、 、 、 、 、 、 ， in: R can be hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or aromatic. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, or aromatic groups, respectively.
6. The thiophosphoramide-modified oligonucleic acid according to any one of claims 3 to 5, characterized in that, In the nucleotide monomer, 4,4'-dimethoxytriphenylmethyl (DMTr) is replaced with triphenylmethyl (Tr), 4-methoxytriphenylmethyl (MMTr), or 4,4',4''-trimethoxytriphenylmethyl (TMTr).
7. The synthesis of a 3'-amino-5'-nucleotide thiophosphoramide according to claim 2 and its application in oligonucleotides, characterized in that, The nucleotide monomer is prepared by reacting a nucleotide molecule containing a protecting group (N3') with dichlorothiophosphoramide.
8. The synthesis of a 3'-amino-5'-nucleotide thiophosphoramide according to claim 1 and its application in oligonucleotides, characterized in that, The modified oligonucleotides include nucleotide structures modified with 3´-amino and 5´-thiophosphoramide, which not only improves the biostability and anti-enzymatic degradation activity of the oligonucleotides, but also improves the binding performance between the modified oligonucleotide molecules and anions through thiophosphoramide modification.
9. The synthesis process of the thiophosphoramide-modified oligonucleic acid according to any one of claims 1 to 8, characterized in that, In solid-phase synthesis, the coupling of nucleotide monomers occurs along a 5' to 3' direction, and the synthetic process includes the following steps: (a) Load nucleotide monomers onto a solid support, then remove the 3' protecting group from the nucleotide monomers and wash them; (b) A nucleotide monomer is pumped into the washed system for coupling, followed by capping, removal of the 3' protecting group from the nucleotide monomer, and washing. The nucleotide monomer is a nucleotide monomer of (thio)phosphoramide or a nucleotide monomer of phosphorous acid. If the nucleotide monomer is a nucleotide monomer of phosphorous acid, after the coupling reaction, the trivalent phosphite is converted to a pentavalent phosphate ester by oxidation or sulfidation, followed by capping, removal of the 3' protecting group from the nucleotide monomer, and washing. (c) Repeat step (b) to obtain oligonucleotides with (thio)phosphoramide-modified nucleotide structures.
10. The oligonucleic acid according to claim 1, characterized in that, The oligonucleotide is selected from any one or a combination of at least two of small interfering nucleotides, antisense oligonucleotides, microRNAs, small activating RNAs, small guide RNAs, transfer RNAs, and aptamers.