Synthesis of 5-mercaptonucleotide-derived phosphamide and application of 5-mercaptonucleotide-derived phosphamide in oligonucleotides
By introducing a 5'-thiol nucleotide-derived phosphoramide modification structure into oligonucleotides, the problem of poor in vivo stability of traditional oligonucleotide drugs has been solved, achieving higher biological stability and activity.
Patent Information
- Application Number
- CN202511450308.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-11
- Publication Date
- 2026-02-10
AI Technical Summary
Traditional oligonucleotide drugs have poor stability in vivo, which affects their biological activity.
An oligonucleotide synthesis process using 5´-thiol nucleotide-derived phosphoramide-modified phosphoramide was employed. By preparing nucleotide phosphoramides, specific phosphoramide modification structures were introduced to improve the stability and bioactivity of oligonucleotides.
It improves the biostability and anti-enzymatic degradation activity of oligonucleotides, thereby enhancing their biological activity.
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Figure CN121494900A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of oligonucleic acid, in particular to a synthesis of 5'-mercapto nucleotide derived phosphoramidate and its application in oligonucleic acid drugs. BACKGROUND
[0002] Oligonucleic acid drugs are specifically combined with target genes through base complementary pairing, and the expression of target genes is affected from the post-transcriptional level by regulating or knocking down the upstream mRNA of pathogenic proteins. Oligonucleic acid drugs have the advantages of rich target points, short research and development cycle, high success rate of research and development, and long-lasting drug efficacy, which provide solutions for the treatment of many refractory diseases.
[0003] In addition, oligonucleic acids are widely used in molecular biology research, disease diagnosis and treatment, such as PCR primers, gene editing tools or targeted drugs.
[0004] The oligonucleic acid sequence composed of traditional nucleotide monomers has the problem of poor stability, which is easily degraded in vivo and affects the exertion of its biological activity.
[0005] Therefore, the present application is proposed. SUMMARY
[0006] The purpose of the present application is to provide a 5'-mercapto nucleotide derived phosphoramidate modified oligonucleic acid and its synthesis process. Through the preparation of nucleotide phosphoramidate, it has a specific phosphoramidate modified nucleotide structure, which can improve the stability of the modified oligonucleic acid and improve its biological activity.
[0007] In order to achieve the above purpose of the present application, the following technical solutions are adopted: The present application provides a 5'-mercapto nucleotide derived phosphoramidate modified oligonucleic acid in the first aspect, which includes a nucleotide structural unit shown in formula I or formula II: …… Formula I; In formula I: Base is a nucleotide base or its derivative, and the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methyl pseudouracil and the like nucleotide base and its derivative; R1 and R2 are respectively alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic group; R3 is hydrogen, alkyl (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl); ...Formula II; In Formula 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. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, ester, acyloxy, amide, aminoacyl, etc., respectively.
[0008] The synthesis of the 5'-thiol nucleotide-derived phosphoramide-modified oligonucleotides utilizes nucleotide phosphoramide monomers represented by Formula III or Formula IV: ...Formula III; In Formula III: 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, silyl (including trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl). R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl). ...Form IV; In Equation IV: 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, silyl (including trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl). R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, ester, acyloxy, amide, aminoacyl, etc., respectively.
[0009] Preferably, the protecting group is selected from one of triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl.
[0010] Preferably, the nucleotide phosphoramide monomer comprises: , ; Where: 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. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl). R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, ester, acyloxy, amide, aminoacyl, etc., respectively.
[0011] Preferably, the nucleotide phosphoramide monomer comprises: , , , , , , , , , , , , , , Wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively; R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl).
[0012] Preferably, the nucleotide phosphoramide monomer comprises: , , , , , , , , , , , , , , Wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic groups, respectively.
[0013] Preferably, the nucleotide phosphoramide monomer comprises: , , , , , , , , , , , , , , Wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic groups, respectively.
[0014] Preferably, the O3'-protecting group 4,4'-dimethoxytriphenylmethyl (DMTr) in the nucleotide phosphoramide monomer described above is replaced with triphenylmethyl (Tr), 4-methoxytriphenylmethyl (MMTr) or 4,4',4''-trimethoxytriphenylmethyl (TMTr).
[0015] The nucleotide monomers described above are prepared by reacting nucleotides with a 5´-mercapto-O3'-protecting group and phosphoramide dichloride in the liquid phase.
[0016] The modification strategy proposed in this invention, which includes nucleotide structures modified with 5'-thiol-derived phosphoramide, can improve the biostability and anti-enzymatic degradation activity of oligonucleotides and enhance their bioactivity.
[0017] A second aspect of the present invention provides a synthetic process for a 5'-thiol nucleotide-derived phosphoramide-modified oligonucleotide, the synthetic process comprising 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 on the nucleotide monomer, and washing. The nucleotide monomer is a phosphoramide nucleotide monomer or a phosphorous acid nucleotide monomer. If the nucleotide monomer is a phosphorous acid nucleotide monomer, 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 on the nucleotide monomer, and washing. (c) Repeat step (b) to obtain oligonucleotides with phosphoramide-modified nucleotide structures.
[0018] Compared with the prior art, the beneficial effects of the present invention include at least the following: This invention achieves the preparation of oligonucleotides containing nucleotide 5'-thiol-bound phosphoramide structural units through specific constraints on the phosphoramide structure derived from 5'-thiol nucleotides. Compared to traditional oligonucleotide molecules, the phosphoramide-modified oligonucleotides proposed in this invention exhibit superior pharmaceutical effects in terms of resistance to enzymatic degradation, stability, and binding affinity, providing a novel chemical modification strategy for the research and application of oligonucleotide drugs. Detailed Implementation
[0019] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.
[0020] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0021] In this invention, the substances corresponding to the abbreviations are as follows: PS: Polystyrene; CPG: Controlled pore glass; DBU: 1,8-Dazabicycloundec-7-ene; P(III): Trivalent phosphorus (phosphine); P(V): Pentavalent phosphorus (phosphine); F: Fluorine; Me: Methyl; Alkyl: Alkyl; Allyl: Allyl; Alloc: Allyloxycarbonyl; Ac: Acetyl; Bn: Benzyl; Bz: Benzoyl; Tr: Triphenylmethyl; MMTr: Methoxytriphenylmethyl; DMTr: Dimethoxytriphenylmethyl; TMTr: Trimethoxytriphenylmethyl; iBu: 2-Isobutyryl; NPE: 4-Nitrophenylethoxy; Ph: Aromatic; TMS: Trimethylsilyl; TBDMS: Tert-butyldimethylsilyl; TBDPS: Tert-butyldiphenylsilyl; DCM: Dichloromethane; DMF: N,N-Dimethylformamide; DMAP: 4-Dimethylaminopyridine; TBAF: Tetrabutylammonium fluoride; THF: Tetrahydrofuran; TFA: Trifluoroacetic acid; iPrOH: Isopropanol; TLC: Thin-layer chromatography; A: Adenine Adenine; G: Guanine; C: Cytosine; T: Thymine; U: Uracil; I: Hypoxanthine; Xan: Xanthine; m5C: 5-methylcytosine; S5': 5' sulfur-substituted; HPLC: High performance liquid chromatography.
[0022] Example 1 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, adenine phosphoramide, A-1: S-(((2S,3S,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-4-methoxytetrahydrofuran-2-yl)methyl) dimethylphosphoramidochloridothioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 352 mg of O2'-Me-O3'-DMTr-5'-SH-N6-Bz-A (A-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Subsequently, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphoaminodichloride (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added. The mixture 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, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 270 mg of the target product A-1 (purity above 99%, yield 65%).
[0023] Example 2 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, uracil phosphoramide, U-1-1: S-(((2S,3S,4R,5R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)methyl) dimethylphosphoramidochloridothioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 288 mg of O2'-Me-O3'-DMTr-5'-SH-U (U-1-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Subsequently, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphoaminodichloride (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added. The mixture 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, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 220 mg of the target product U-1-1 (purity above 99%, yield 63%).
[0024] Example 3 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, thymine phosphoramide, T-1-1: S-(((2S,3S,4R,5R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-4-methoxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl)dimethylphosphoramidochloridothioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 295 mg of O2'-Me-O3'-DMTr-5'-SH-T (T-1-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Subsequently, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphoaminodichloride (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added. The mixture 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, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 202 mg of the target product T-1-1 (purity above 99%, yield 56%).
[0025] Furthermore, the following nucleotide monomers were prepared using the synthetic method described above: , , , , , , , , , , .
[0026] Example 4 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, uracil phosphoramide, U-2-1:S-(((2S,3R,4R,5R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluorotetrahydrofuran-2-yl)methyl) dimethylphosphoramidochloridothioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 282 mg of 2'-F-O3'-DMTr-5'-SH-U (U-2-O, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Subsequently, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphoaminodichloride (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added. The mixture 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, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 220 mg of the target product U-2-1 (purity above 99%, yield 64%).
[0027] Furthermore, the following nucleotide monomers were prepared using the synthetic method described above: , , , , , , , , , , , , .
[0028] Example 5 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, thymidine phosphoramide, T-2-1: S-(((2S,3S,5R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl) dimethylphosphoramidochloridothioate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 280 mg of O3'-DMTr-5'-SH-T (T-2-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Next, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphoaminodichloride (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added sequentially. The mixture 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, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 209 mg of the target product T-2-1 (purity above 99%, yield 61%).
[0029] Furthermore, the following nucleotide monomers were prepared using the synthetic method described above: , , , , , , , , , , , , .
[0030] Furthermore, the following nucleotide monomers were prepared using the synthetic method described above: , ; in: 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. R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl). R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, ester, acyloxy, amide, aminoacyl, etc., respectively.
[0031] Furthermore, the following nucleotide monomers were prepared using the synthetic method described above: , ; in: 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 (Tr), 4-methoxytriphenylmethyl (MMTr), 4,4'-dimethoxytriphenylmethyl (DMTr), 4,4',4''-trimethoxytriphenylmethyl (TMTr), allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl). R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl). R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, ester, acyloxy, amide, aminoacyl, etc., respectively.
[0032] Example 6 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, uracil phosphoramide, U-6-1: 2S,3R,4R,5R)-2-(((bis(4-methoxyphenyl)(phenyl)methyl)thio)methyl)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluorotetrahydrofuran-3-yldimethylphosphoramidochloridate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 282 mg of 2'-F-S5'-DMTr-U (U-6-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was then added, and the mixture was stirred for 5 minutes. Subsequently, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphoaminodichloride (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added. The mixture 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, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 193 mg of the target product U-6-1 (purity above 99%, yield 56%).
[0033] Example 7 This embodiment describes a method for synthesizing a phosphoramide-modified oligonucleotide monomer, thymidine phosphoramide, T-6-1:(2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yldimethylphosphoramidochloridate. The synthetic reaction formula is as follows: ; Synthesis methods include: Under nitrogen protection, 281 mg of 2'-F-O5'-DMTr-T (T-6-0, 0.5 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL dichloromethane and 3 mL acetonitrile), and the temperature was maintained at 0 °C. Then, 87 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Next, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of N,N-dimethylphosphoaminodichloride (0.75 mmol, 1.5 eq.) dissolved in 1 mL of dry dichloromethane were added sequentially. The mixture 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, followed by washing with 30 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Separation was performed by silica gel chromatography with a mixed solvent of dichloromethane / acetone / methanol as the mobile phase, yielding 206 mg of the target product T-6-1 (purity above 99%, yield 60%).
[0034] Furthermore, the following nucleotide monomers were prepared using the synthetic method described above: , ; in: 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, silyl (including trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl). Y represents oxygen (O), sulfur (S), selenium (Se), or amine; R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R3 is an alkyl group (including methyl, ethyl, long-chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl). R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, etc., respectively.
[0035] Cross-linked polystyrene (PS) was used as the support for solid-phase synthesis. 0.050 g of the modified solid-phase synthesis resin was weighed and added to a solid-phase synthesis tube. 2.0 mL of NMP was added, and the mixture swelled at room temperature for 2 hours, then the solution was drained by filtration. The resin was washed with 3.0 mL of dichloromethane, and this washing process was repeated three times to begin solid-phase synthesis.
[0036] The monomers introduced in solid-phase synthesis are phosphoramide P(III) and phosphoramide P(V).
[0037] The process conditions for phosphorus amide monomers are as follows: Reagents and monomers: Prepare a 0.15 mol / L monomer / acetonitrile solution; the deprotecting agent for DMTr (or Tr) is a 5% dichloroacetic acid / toluene solution (v / v); the activator is a 0.25 mol / L 5-ethylthiotetrazole / acetonitrile solution; the oxidizing agent is a 0.05 mol / L iodine-water / pyridine (10 / 90, v / v) solution; the sulfiding agent is a 3% hydroflavin / pyridine solution; capping agent A is a 10% acetic anhydride / acetonitrile solution (v / v), and capping agent B is a 1-methylimidazolium / pyridine / tetrahydrofuran solution (16 / 10 / 74, v / v / v).
[0038] The operational sequence for the solid-phase synthesis of phosphorous amide monomers is as follows: 1) Deprotection: 5% dichloroacetic acid toluene solution was used to remove the DMTr protecting group, followed by washing with acetonitrile; 2) Coupling: Using 0.25 mol / L 5-ethylthiotetrazole as the activator, according to the sequence design, nucleotide monomers / acetonitrile solutions were fed into the cycle for coupling, followed by rinsing with acetonitrile; 3) Oxidation / Sulfidation: Oxidation was performed using a 0.05 mol / L iodine solution in water / pyridine (v / v, 90 / 10) as the oxidant, followed by rinsing with acetonitrile. Sulfidation was performed using a 3% hydroflavin pyridine solution as the sulfiding agent, followed by rinsing with acetonitrile. 4) Capping: Capping agent A and capping agent B are used as capping reagents to protect the unreacted active groups, followed by rinsing with acetonitrile.
[0039] The process conditions for applying phosphoramide monomers to the solid-phase synthesis of oligonucleotides differ from those for the phosphoramide monomers described above as follows: Coupling solvent: 2.303 g of N-ethylmorpholine was diluted to 50 mL with 1,3-dimethyl-2-imidazolidinone (DMI) to obtain an N-ethylmorpholine DMI solution (0.40 mol / L), which was used as the coupling solvent.
[0040] Lysis buffer: Weigh 0.154 g of dithiothreitol, add 5 mL of N-methyl pyrrolidone (NMP) to dissolve, add 0.739 g of triethylamine, and dilute to 10 mL with NMP to obtain an NMP solution containing 0.10 mol / L dithiothreitol and 0.73 mol / L triethylamine, which is the lysis buffer. Prepare fresh before use.
[0041] The solid-phase synthesis process of phosphoramide monomers includes: Following the base sequence of the target sequence, nucleotide monomers were coupled one by one through a cyclical process of de-DMTr, neutralization, washing, coupling, neutralization, and washing. After the last monomer molecule was coupled, the terminal DMTr was removed, and 3.0 mL of p-methoxytriphenylmethyl chloride end-capping solution (0.32 mol / L) was added to the reaction tube. After reacting at 45 °C for 90 minutes, the reaction solution was drained, and the tube was washed 8 times with 4.0 mL of NMP each time.
[0042] Repeat the above operations according to the set sequence to obtain the oligonucleotide product of the target sequence.
[0043] Deprotection involves transferring the solid-phase support loaded with the oligonucleotide product into a reactor, adding concentrated ammonia (28%), controlling the temperature at 60 °C for 12 hours of ammonolysis, cooling to room temperature, filtering, washing the solid with a mixture of purified water and ethanol, combining the filtrates, and concentrating under reduced pressure at low temperature to obtain the crude oligonucleotide product with the designed sequence.
[0044] Purification involves dissolving the deprotected crude product in purified water, purifying it using HPLC, collecting the product peak solution, and then desalting, concentrating under reduced pressure, and lyophilizing the product to obtain the final product.
[0045] The above solid-phase synthesis methods are also applicable to monomers protected by Tr, MMTr, and TMTr.
[0046] In examples of solid-phase synthesis, the nucleotide monomers used are represented by the following structures: , , , , , , , , , , , .
[0047] Example 8 This embodiment describes an oligonucleic acid with a phosphoramide-modified nucleotide structure. The nucleotide monomers loaded on the solid support are T-3-0, and the phosphoramide-modified nucleotide monomers are U-1-1 and U-4-1. The synthesis reaction formula is ; Methods for synthesizing oligonucleotides with phosphoramide-modified nucleotide structures include: Monomer T-3-0 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 O3'-DMTr was removed again 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 again 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, desalted, concentrated and freeze-dried to obtain the product, which was confirmed by detection to be the nucleic acid molecule of the target sequence.
[0049] Example 9 This embodiment describes an oligonucleic acid with a phosphoramide-modified nucleotide structure. The nucleotide monomers loaded on the solid support are T-3-0 and the phosphoramide-modified nucleotide monomers are U-2-1. The synthesis reaction formula is ; Methods for synthesizing oligonucleotides with phosphoramide-modified nucleotide structures include: Monomer T-3-0 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 O3'-DMTr was removed again using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-2-1 was pumped in for coupling, and O3'-DMTr was removed again using a 5% dichloroacetic acid-toluene solution.
[0050] After ammonolysis and deprotection treatment, the crude product of the loaded oligonucleotide molecule was concentrated under reduced pressure and purified by HPLC. The main fraction was collected, desalted, concentrated and freeze-dried to obtain the product, which was confirmed by detection to be the nucleic acid molecule of the target sequence.
[0051] Example 10 This embodiment uses an oligonucleic acid with the same structure as in Example 9. The nucleotide monomers loaded on the solid support are U-6-0, and the phosphoramide-modified nucleotide monomers are U-6-1 and T-6-1. The synthesis reaction formula is ; Methods for synthesizing oligonucleotides with phosphoramide-modified nucleotide structures include: Monomer U-6-0 was loaded onto cross-linked polystyrene (PS). S5'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-6-1 was pumped in for coupling. The system was then capped, and S5'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-6-1 was pumped in for coupling, and O5'-DMTr was removed using a 5% dichloroacetic acid-toluene solution.
[0052] After ammonolysis and deprotection treatment, the crude product of the loaded oligonucleotide molecule was concentrated under reduced pressure and purified by HPLC. The main fraction was collected, desalted, concentrated and freeze-dried to obtain the product, which was confirmed by detection to be the nucleic acid molecule of the target sequence.
[0053] Example 11 This embodiment describes an oligonucleic acid with a phosphoramide-modified nucleotide structure. The nucleotide monomers loaded on the solid support are T-3-0, the phosphoramide-modified nucleotide monomers are U-1-1, and the nucleotide phosphorylate is Af-1. The synthesis reaction formula is ; Methods for synthesizing oligonucleotides with phosphoramide-modified nucleotide structures include: Monomer T-3-0 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 O3'-DMTr was removed again using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer Af-1 was pumped in for coupling, followed by iodine / pyridine oxidation, and O3'-DMTr was removed again using a 5% dichloroacetic acid-toluene solution.
[0054] After ammonolysis and deprotection treatment, the crude product of the loaded oligonucleotide molecule was concentrated under reduced pressure and purified by HPLC. The main fraction was collected, desalted, concentrated and freeze-dried to obtain the product, which was confirmed by detection to be the nucleic acid molecule of the target sequence.
[0055] Example 12 This embodiment is an oligonucleotide with a phosphoramide-modified nucleotide structure. During the preparation process, the nucleotide monomer loaded on the solid support is U-3-0, and the phosphoramide-modified nucleotide monomers are U-1-1 and T-1-1.
[0056] The structural formula of oligonucleotides is ; Methods for synthesizing oligonucleotides with phosphoramide-modified nucleotide structures include: Monomer U-3-0 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. After capping, O3'-DMTr was removed again using a 5% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer U-1-1 was pumped in, and this cycle was repeated five times. After washing with acetonitrile, monomer T-1-1 was pumped in for coupling, and O3'-DMTr was removed using a 5% dichloroacetic acid-toluene solution. Following solid-load shearing, deprotection, HPLC purification, and desalting, a phosphoramide-modified oligonucleotide molecule with the designed sequence structure was obtained, denoted as ON7-PS6-2.
[0057] Example 13 This comparative example is an oligonucleotide with the same base sequence as ON7-PS6-2. During preparation, the nucleotide monomer loaded on the solid-phase support was T-2-0, and the nucleotide monomer used was nucleotide phosphorylate U-5-1. The structural formula of the oligonucleotide is as follows: This oligonucleotide, denoted as ON7, was synthesized using a conventional solid-phase synthesis method.
[0058] Example 14 The isoelectric points of the oligonucleotides ON7-PS6-2 prepared in Example 12 and ON7 prepared in Example 13 were tested. The results showed that the isoelectric point of the oligonucleotide ON7 was 5.03 (±0.12), and the isoelectric point of the phosphoramide-modified oligonucleotide ON7-PS6-2 was 7.59 (±0.15). It can be seen that phosphoramide modification can improve the binding ability of the modified oligonucleotide to anions.
[0059] Example 15 The stability of ON7-PS6-2 prepared in Example 12 and ON7 prepared in Example 13 in human serum and human liver microsomes were tested respectively.
[0060] Test results show that: The oligonucleotide molecule ON7 without phosphoramide modification showed significant degradation, with the proportion of undegraded nucleic acid sequences falling below 5% after 4 hours. In contrast, the proportion of undegraded oligonucleotide chain ON7-PS6-2 containing phosphoramide-modified nucleotides was greater than 75% after 24 hours of incubation in human serum or human liver microsomes, indicating that the modified oligonucleotide molecule has a stronger resistance to biological enzyme degradation.
[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.
Claims
1. A synthesis of a 5'-mercaptonucleotide-derived phosphoramide and its application in oligonucleotides, characterized in that, The phosphoramide-modified oligonucleotide includes nucleotide structural units represented by Formula I or Formula II: ... Formula I; In Formula I: 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, cycloalkenyl, or aromatic groups, respectively. R3 is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl, silyloxymethylene, alkoxyalkyl, benzyl, or acyl. ...Formula II; In Formula 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. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, ester, acyloxy, amide, aminoacyl, etc., respectively.
2. The synthesis of a 5'-mercaptonucleotide-derived phosphoramide according to claim 1 and its application in oligonucleotides, characterized in that, The synthesis of the phosphoramide-modified oligonucleotides uses nucleotide phosphoramide monomers represented by Formula III or Formula IV: ...Formula III; In Formula III: 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. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, 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. ...Form IV; In Equation IV: 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. R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively. R4 and R5 are hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylthio, selenyl, alkylseleno, ester, acyloxy, amide, aminoacyl, etc., respectively.
3. The synthesis of a 5'-mercaptonucleotide-derived phosphoramide according to claim 2 and its application in oligonucleotides, characterized in that, The nucleotide monomers include, but are not limited to: 、 、 、 、 、 、 、 、 、 、 、 、 、 ， Wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or aromatic groups, respectively; R3 is an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl, silyloxymethylene, alkoxyalkyl, benzyl, or acyl group.
4. The synthesis of a 5'-mercaptonucleotide-derived phosphoramide according to claim 2 and its application in oligonucleotides, characterized in that, The nucleotide monomers include, but are not limited to: 、 、 、 、 、 、 、 、 、 、 、 、 、 ， Wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic groups, respectively.
5. The synthesis of a 5'-mercaptonucleotide-derived phosphoramide according to claim 2 and its application in oligonucleotides, characterized in that, The nucleotide monomers include, but are not limited to: 、 、 、 、 、 、 、 、 、 、 、 、 、 ， Wherein: R1 and R2 are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aromatic groups, respectively.
6. The synthesis of a 5'-mercaptonucleotide-derived phosphoramide according to claim 2 and its application in oligonucleotides, characterized in that, The nucleotide phosphoramide is prepared by reacting a nucleotide molecule with a 5'-mercapto-O3'-protecting group and phosphoramide dichloride; the 5'-mercaptonucleotide-derived phosphoramide-modified oligonucleotide according to any one of claims 3 to 5 is characterized in that the O3'-protecting group 4,4'-dimethoxytriphenylmethyl (DMTr) in the nucleotide monomer is replaced with triphenylmethyl (Tr), 4-methoxytriphenylmethyl (MMTr) or 4,4',4''-trimethoxytriphenylmethyl (TMTr).
7. The synthesis of a 5'-mercaptonucleotide-derived phosphoramide according to claim 1 and its application in oligonucleotides, characterized in that, The modified oligonucleotides include a 5'-thiol nucleotide-derived phosphoramide modified structure, which not only improves the biostability and anti-enzymatic degradation activity of the oligonucleotides, but also improves the binding performance between the oligonucleotide molecules and anions.
8. The synthetic process of the 5'-mercaptonucleotide-derived phosphoramide-modified oligonucleic acid according to any one of claims 1 to 7, characterized in that, The synthesis 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 on the nucleotide monomer, and washing. The nucleotide monomer is a phosphoramide nucleotide monomer or a phosphorous acid nucleotide monomer. If the nucleotide monomer is a phosphorous acid nucleotide monomer, 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 on the nucleotide monomer, and washing. (c) Repeat step (b) to obtain oligonucleotides with phosphoramide-modified nucleotide structures.
9. The synthesis of a 5'-mercaptonucleotide-derived phosphoramide according to claim 1 and its application in oligonucleotides, characterized in that, The oligonucleotide molecule prepared by coupling a nucleotide monomer with a 5´-thio-S5´-protecting group to a nucleotide monomer with an O3´-phosphoramide comprises the structural unit of Formula I or Formula II as shown in claim 1.
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, guide RNAs, and aptamers.