Chiral UNA monomer and application thereof in oligonucleotide

Abstract

The invention discloses a synthesis process of chiral UNA phosphoramidite, phosphoryl chloride and phosphoramidite and application of the chiral UNA phosphoramidite, phosphoryl chloride and phosphoramidite in oligonucleotides, and particularly designs preparation and synthesis of (2S)-UNA monomer phosphoramidite, phosphoryl chloride and phosphoramidite. And the method can also be used for solid-phase synthesized oligonucleotides in the conventional 3-to-5 direction, and the influence of introduction of monomers with different chirality to design sites on the structure, the biological activity and the like is researched.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to preparation and synthesis of chiral UNA monomer phosphoramidite, phosphorochloridite and phosphoramidite and application thereof in oligonucleic acid. BACKGROUND

[0002] Oligonucleic acid drugs are specifically combined with target genes through base complementary pairing, and the drug target is expanded to the upstream mRNA of the pathogenic protein, so as to regulate or knock down the target gene and affect the expression of the target gene at the post-transcriptional level. The oligonucleic acid drug has the advantages of rich target, high specificity, high success rate of research and development, short research and development cycle, and long-lasting drug efficacy, and provides a solution for the treatment of many refractory diseases.

[0003] In order to improve the drug property of the oligonucleic acid drug, the stability of the nucleic acid molecule is improved and the immune system reaction is avoided by chemical modification at a specific position. UNA (unlocked nucleic acid) is a kind of RNA acyclic derivative, UNA lacks the chemical bond between C2' and C3' of the ribose ring of the natural nucleotide, and can still imitate the unmodified RNA when combined to the RNA double-stranded structure. The introduction of UNA in the oligonucleic acid sequence can improve the stability of the nucleic acid in the organism, and the addition of UNA at a specific position can reduce the occurrence of off-target effect.

[0004] The reported synthesis process can prepare (2R)-DMTr-UNA phosphoramidite, and the structural formula is as follows:

[0005] In the formula, Base: nucleotide base or derivative thereof, the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, N1-methyl pseudouracil.

[0006] The above (2R)-DMTr-UNA phosphoramidite is suitable for solid-phase synthesis of oligonucleic acid sequence coupling from 3' to 5' direction. For the coupling in the reverse direction of 5' to 3', due to the existence of (2R)-chiral center, the prepared oligonucleic acid sequence has a difference in the stereochemical configuration. Therefore, the phosphoramidite, phosphorochloridite and phosphoramidite of (2S)-UNA monomer are suitable for solid-phase synthesis of 5' to 3' coupling.

[0007] Phosphorodiamidate morpholino oligonucleotide (PMO) is a new generation of chemical modification of nucleic acid drugs. PMO uses morpholine ring to replace deoxyribose or ribose structure, and electrically neutral morpholine phosphorodimethylamine to replace negatively charged phosphate group, and has the characteristics of high binding affinity and strong anti-enzymatic stability. In the PMO phosphoramide monomer, the protecting group of N3' and O6'-phosphoramide are inverted with the conventional nucleotide phosphoramide, and the PMO monomer is coupled with other monomers, and in the solid phase synthesis process, the coupling mode of nucleic acid sequence from 5' to 3' direction is generally adopted. The combination of PMO and UNA two kinds of modifications is used to construct oligonucleic acid, and the latter monomer is (2S)-UNA phosphoramidite, phosphorochloridate or phosphoramide.

[0008] Therefore, the present application is proposed. SUMMARY

[0009] The synthesis process of the present application provides a preparation method of chiral inversion (2S)-UNA monomer phosphoramidite, phosphorochloridate and phosphoramide, including the following structural formula: ; ; ; Base: nucleotide base or derivative thereof, the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, N1-methyl pseudouracil; PG: protecting group, including but not limited to trityl Tr, (4-methoxyphenyl) benzhydryl MMTr, 4,4'-dimethoxytrityl DMTr, 4,4',4''-trimethoxytrityl TMTr; LG1: cyanoethyl, cyanopropyl, 3-cyanoisobutyl, (CH3)2CHCH2CN, phenethyl or allyl, etc.; LG2: fluorine, chlorine or bromine; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl, etc. acyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc. silyl; R1, R2, R3, R4: alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic group, etc.

[0010] Preferably, the phosphoramidite, phosphorochloridate and phosphoramide of the (2S)-UNA monomer include the following structural formula: ; ; ; Wherein: Base: nucleotide base or its derivative, the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, N1-methyl pseudouracil; PG: protecting group, including but not limited to triphenylmethyl Tr, (4-methoxyphenyl) diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, 4,4',4''-trimethoxytriphenylmethyl TMTr; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl and other acyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl and other silicon groups.

[0011] The synthesis route of (2S)-UNA is as follows:

[0012] Wherein: Base: nucleotide base or its derivative, the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, N1-methyl pseudouracil; PG1: silicon group, acetyl, propionyl and other acyl, Allyl, Alloc, Bz, Bn and the like; PG2: Tr, MMTr, DMTr, TMTr and the like; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl and other acyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl and other silicon groups.

[0013] Preferably, the synthesis route of (2S)-UNA is as follows:

[0014] Wherein: Base: nucleotide base or its derivative, the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, N1-methyl pseudouracil; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl and other acyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl and the like; PG: Tr, MMTr, DMTr, TMTr and the like.

[0015] Preferably, the synthesis route of (2S)-UNA is as follows:

[0016] Wherein: Base: nucleotide base or its derivative, the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, N1-methyl pseudouracil; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl acyl, alkenyl, cycloalkenyl, alkynyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl silicon; PG: Tr, MMTr, DMTr, TMTr, etc.

[0017] Preferably, the synthesis route of (2S)-UNA is as follows:

[0018] Wherein: Base: nucleotide base or its derivative, the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, N1-methyl pseudouracil; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzoyl Bz, acetyl or propionyl acyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl silicon; PG: Tr, MMTr, DMTr, TMTr, etc.

[0019] The phosphoramidite, phosphorochloridite or phosphoramidite of nucleotide monomers is embedded in the oligonucleotide chain by solid phase synthesis method, and the solid phase synthesis process includes deprotection, coupling, capping, oxidation or sulfurization, etc., to construct a modified oligonucleotide molecule; The synthesis route of the oligonucleotide molecule is as follows: 1) Deprotection: remove O3' or N3' protecting group, then clean; 2) Coupling: use activator, according to sequence design, the solution of nucleotide monomers enters the cycle for coupling, and then uses solvent to rinse; 3) Oxidation / sulfurization: oxidation uses iodine water / pyridine (v / v, 90 / 10) solution as oxidant to oxidize P(III), and then uses acetonitrile to rinse. Sulfurization uses 3% pyridine solution of hydrogenated xanthate as sulfurizing agent to sulfurize P(III), and then uses acetonitrile to rinse; 4) Capping: use capping agent to protect the unreacted active group, and then use solvent to rinse.

[0020] According to the set sequence, the monomer phosphoramidite, phosphorochloridite or phosphoramidite is repeatedly operated according to steps 1-4, if the monomer is phosphoramidite, the synthesis steps are 1, 2 and 4, to obtain the oligonucleotide product of the target sequence.

[0021] Preferably, the oligonucleic acid is selected from any one of or a combination of at least two of a small interfering nucleotide, an antisense oligonucleic acid, a microRNA, a small activating RNA, a small guide RNA, a transfer RNA, and an aptamer.

[0022] The present application provides a preparation method of (2S)-UNA monomer phosphoramidite, phosphorochloridate and phosphoramidate and its application in oligonucleic acid drugs; specifically, the O5'-protected nucleotide is oxidized to the adjacent dihydroxy of O2' and O3', the single bond between C2 and C3 of the ribose is cut off, and then the two hydroxyl groups are reduced respectively. The original O3'-hydroxyl group is bonded to Tr, MMTr, DMTr or TMTr, etc. for protection group for solid phase synthesis, the original O5'-hydroxyl group is released, and further reaction is carried out to obtain (2S)-UNA monomer phosphoramidite, phosphorochloridate and phosphoramidate which are chiral inversion of the reported (2R)-UNA monomer phosphoramidite. The (2S)-UNA monomer phosphoramidite, phosphorochloridate and phosphoramidate can be used for solid phase synthesis of oligonucleic acid from 5' to 3' direction, and can also be used for conventional solid phase synthesis of oligonucleic acid from 3' to 5' direction, and the influence of introducing monomers with different chiralities at this site on structure and biological activity is studied.

[0023] Of course, the implementation of any product of the present application does not necessarily require the simultaneous achievement of all the advantages described above. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed for the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0025] Figure 1 The solid phase synthesis route map of 6'-Up-Auna-3' nucleic acid of the present application; Figure 2 The solid phase synthesis route map of 5'-Tuna-Ame-P NMe2 -Up-3' nucleic acid of the present application; Figure 3 The solid phase synthesis route map of 5'-Tme-Ps-Auna-P NMe2 -Up-3' nucleic acid of the present application. DETAILED DESCRIPTION

[0026] With reference to the drawings of the embodiments of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0027] Example 1: (2S)-N6-Bz-Auna phosphoramidite, phosphoramidite and phosphorous chloridite

[0028] 4.86 grams of O5'-TBDMS-N6-Bz-A (10 mmol, 1.0 eq.) was dissolved in 150 mL of 1,4-dioxane and 25 mL of water, and stirred at room temperature. A solution of 2.35 grams of NaIO4(11.0 mmol, 1.10 eq.) dissolved in 25 mL of water was added dropwise. After 1 hour, 100 mL of 1,4-dioxane was added, and stirring was continued for 15 minutes or more. The resulting filtrate was filtered, and the solid was washed with 50 mL of 1,4-dioxane. After mixing the liquid phase, 0.43 grams of NaBH4(11.3 mmol, 1.13 eq.) was slowly added under nitrogen protection, and stirring was continued at room temperature for 30 minutes or more. A solution of 10 mL of pyridine and acetic acid (1:1, v / v) was added, and after stirring and mixing for 30 minutes, most of the solvent was removed under reduced pressure. 100 mL of dichloromethane was added, and the mixture was washed with 50 mL of saturated aqueous sodium bicarbonate solution three times. After the organic layer was washed with saturated brine and dried over solid sodium sulfate, it was concentrated, and column chromatography on silica gel yielded 3.98 grams of white solid A-1 (yield 82%).

[0029] 3.80 grams of A-1 (7.8 mmol, 1.0 eq.) was dissolved in 100 mL of dry pyridine, and 20 mg of DMAP was added. After stirring at room temperature, 2.88 grams of DMTCl (8.5 mmol, 1.1 eq.) was added, and stirring was continued overnight. TLC detection showed that the starting material was completely consumed. The solvent was removed under reduced pressure, and 100 mL of saturated aqueous sodium bicarbonate solution was added to the residue, which was extracted with 100 mL of dichloromethane three times. After the combined organic layers were washed with saturated brine and dried over solid sodium sulfate, they were concentrated, and column chromatography on silica gel yielded 3.76 grams of solid A-2 (yield 61%).

[0030] 3.66 g of A-2 (4.6 mmol, 1.0 eq.) was dissolved in 30 mL of dry pyridine, and 10 mg of DMAP was added. The temperature was controlled between 0-5 °C while 0.64 mL of benzoyl chloride (0.77 g, 5.5 mmol, 1.2 eq.) was added. The temperature was slowly increased to room temperature and the stirring was continued overnight. TLC test showed that the starting material was completely consumed. The solvent was removed under reduced pressure, and 50 mL of saturated aqueous sodium bicarbonate solution was added to the residue, which was extracted with 50 mL of dichloromethane three times. The combined organic layers were washed with saturated brine, dried over solid sodium sulfate, and concentrated. Column chromatography on silica gel gave 3.83 g of solid A-3 (yield 93%).

[0031] 3.60 g of A-3 (4.0 mmol, 1.0 eq.) was dissolved in 40 mL of tetrahydrofuran, and the temperature was controlled between 0-5 °C while stirring. 10 mL of tetrabutylammonium fluoride tetrahydrofuran solution (TBAF, 1.0 mol / L) was slowly added to the mixed solution under nitrogen protection. The temperature was slowly increased to room temperature and the stirring was continued overnight. TLC test showed that the starting material was completely consumed. The solvent was removed under reduced pressure, and 50 mL of saturated aqueous sodium bicarbonate solution was added to the residue, which was extracted with 50 mL of dichloromethane three times. The combined organic layers were washed with saturated brine, dried over solid sodium sulfate, and concentrated. Column chromatography on silica gel gave 3.01 g of solid A-4 (yield 97%).

[0032] Further;

[0033] 312 mg of N6-Bz-A nucleotide A-4 (0.4 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL of dichloromethane and 7 mL of acetonitrile) under nitrogen protection, and the temperature was controlled at 0 °C. 70 mg of lithium bromide (0.8 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.12 mL of DBU (0.8 mmol, 2.0 eq.) and 0.08 mL of N,N-dimethyl phosphoraminodichloride (0.6 mmol, 1.5 eq.) dissolved in 0.5 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0 °C for 40 minutes under nitrogen protection. The reaction solvent was removed under reduced pressure, and 30 mL of ethyl acetate was added to the residue, which was washed with 10 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. Column chromatography on silica gel was used for separation, and the mobile phase was selected as a gradient solvent of hexane / ethyl acetate, to obtain 206 mg of the target product A-5 (purity >99%, yield 56%).

[0034] Further;

[0035] Under nitrogen protection, 312 mg of N6-Bz-A nucleotide A-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA in acetonitrile, temperature controlled at 0°C. Under nitrogen protection, 0.13 mL of 2-cyanoethyl N,N-diisopropyl phosphoramidite (0.6 mmol, 1.5 eq.) was injected into a solution of 2 mL of dry acetonitrile. After the mixed solution was warmed to room temperature under nitrogen protection, it was continuously stirred for 40 minutes. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, which was washed with 10 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product A-6 was separated by silica gel chromatography using a gradient solvent of hexane / ethyl acetate as the mobile phase, 263 mg of which was obtained (purity >99%, yield 72%).

[0036] Further;

[0037] Under nitrogen protection, 312 mg of N6-Bz-A nucleotide A-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA in acetonitrile, temperature controlled at 0°C. Under nitrogen protection, 0.13 mL of 2-cyanoethyl N,N-diisopropyl phosphoramidite (0.6 mmol, 1.5 eq.) was injected into a solution of 2 mL of dry acetonitrile. After the mixed solution was warmed to room temperature under nitrogen protection, it was continuously stirred for 40 minutes. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, which was washed with 10 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product A-6 was separated by silica gel chromatography using a gradient solvent of hexane / ethyl acetate as the mobile phase, 263 mg of which was obtained (purity >99%, yield 72%).

[0038] Example 2: (2S)-iBu-Guna phosphoramidite, phosphoramidite and phosphorous chloride

[0039] 4.68 g of O5'-TBDMS-G (10 mmol, 1.0 eq.) was dissolved in 150 mL of 1,4-dioxane and 25 mL of water and stirred at room temperature. A solution of 2.35 g of NaIO4(11.0 mmol, 1.10 eq.) in 25 mL of water was added dropwise. After 1 h, 100 mL of 1,4-dioxane was added and stirring was continued for 15 min. The resulting filtrate was filtered and the solid was washed with 50 mL of 1,4-dioxane. After mixing the solution, 0.43 g of NaBH4(11.3 mmol, 1.13 eq.) was added slowly under nitrogen protection and stirred at room temperature for 30 min. A solution of 10 mL of pyridine and acetic acid (1:1, v / v) was added and the mixture was stirred for 30 min. Most of the solvent was removed under reduced pressure. 100 mL of dichloromethane was added and the mixture was washed with 50 mL of saturated aqueous NaHC03three times. The organic layer was washed with saturated brine, dried over solid Na2S04and concentrated. Silica gel column chromatography gave 3.63 g of G-1 as a white solid (77% yield).

[0040] 3.29 g of G-1 (7.0 mmol, 1.0 eq.) was dissolved in 100 mL of dry pyridine and 20 mg of DMAP was added. The mixture was stirred at room temperature and 2.61 g of DMTCl (7.7 mmol, 1.1 eq.) was added. The mixture was stirred overnight. TLC detection showed that the starting material was completely consumed. The solvent was removed under reduced pressure and 100 mL of saturated aqueous NaHC03was added to the residue. The mixture was extracted with 100 mL of dichloromethane three times. The combined organic layer was washed with saturated brine, dried over solid Na2S04and concentrated. Silica gel column chromatography gave 3.58 g of G-2 as a solid (66% yield).

[0041] 3.32 g of G-2 (4.3 mmol, 1.0 eq.) was dissolved in 30 mL of dry pyridine and 10 mg of DMAP was added. The mixture was stirred at a temperature of 0-5 °C and 0.60 mL of benzoyl chloride (0.72 g, 5.2 mmol, 1.2 eq.) was added. The temperature was slowly increased to room temperature and the mixture was stirred overnight. TLC detection showed that the starting material was completely consumed. The solvent was removed under reduced pressure and 50 mL of saturated aqueous NaHC03was added to the residue. The mixture was extracted with 50 mL of dichloromethane three times. The combined organic layer was washed with saturated brine, dried over solid Na2S04and concentrated. Silica gel column chromatography gave 3.35 g of G-3 as a solid (89% yield).

[0042] 3.15 g of G-3 (3.6 mmol, 1.0 eq.) was dissolved in 40 mL of THF and stirred at a controlled temperature between 0-5 °C. 10 mL of TBAF (1.0 mol / L) in THF was added slowly into the mixture solution under nitrogen protection. The temperature was slowly increased to room temperature and the stirring was continued overnight. TLC test showed that the starting material was completely consumed. The solvent was removed under reduced pressure and 50 mL of saturated NaHC03 aqueous solution was added into the residue. The mixture was extracted with 50 mL of dichloromethane for three times. The combined organic layer was washed with saturated brine, dried over solid Na2S04 and concentrated. The product was obtained by silica gel column chromatography (2.61 g, 93% yield).

[0043] Further;

[0044] 305 mg of iBu-G nucleotide G-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA in acetonitrile under nitrogen protection at a controlled temperature of 0 °C. 0.13 mL of 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.6 mmol, 1.5 eq.) in 2 mL of dry acetonitrile was injected into the mixture under nitrogen protection. The mixture was increased to room temperature and stirred for 40 minutes. The reaction solvent was removed under reduced pressure and 30 mL of ethyl acetate was added into the residue. The mixture was washed with 10 mL of cold water and the organic phase was dried over solid Na2S04 and concentrated under reduced pressure. The product was obtained by silica gel column chromatography (244 mg, 99% purity, 68% yield) using a gradient of hexane / ethyl acetate as the mobile phase.

[0045] Further;

[0046] 305 mg of iBu-G nucleotide G-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA in acetonitrile under nitrogen protection at a controlled temperature of 0 °C. 0.13 mL of 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.6 mmol, 1.5 eq.) in 2 mL of dry acetonitrile was injected into the mixture under nitrogen protection. The mixture was increased to room temperature and stirred for 40 minutes. The reaction solvent was removed under reduced pressure and 30 mL of ethyl acetate was added into the residue. The mixture was washed with 10 mL of cold water and the organic phase was dried over solid Na2S04 and concentrated under reduced pressure. The product was obtained by silica gel column chromatography (244 mg, 99% purity, 68% yield) using a gradient of hexane / ethyl acetate as the mobile phase.

[0047] Further;

[0048] Under nitrogen protection, 305 mg of iBu-G nucleotide G-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA in acetonitrile, and the temperature was controlled at 0 °C. Under nitrogen protection, 0.19 mL of bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.6 mmol, 1.5 eq.) was injected into a solution of 2 mL of dry acetonitrile. After the mixed solution was allowed to warm to room temperature under nitrogen protection, it was continuously stirred for 40 min. The reaction solvent was removed under reduced pressure, and 30 mL of ethyl acetate was added to the residue. After washing with 10 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product G-7 was obtained by silica gel column chromatography using a gradient solvent of hexane / ethyl acetate (purity >99%, yield 62%).

[0049] Example 3: (2S)-Luna phosphoramidites, phosphoramidites and phosphorous oxychloride

[0050] 3.83 g of O5'-TBDMS-G (10 mmol, 1.0 eq.) was dissolved in 150 mL of 1,4-dioxane and 25 mL of water, and stirred at room temperature. A solution of 2.35 g of NaIO4 (11.0 mmol, 1.10 eq.) in 25 mL of water was added dropwise. After 1 hour, 100 mL of 1,4-dioxane was added, and stirring was continued for 15 min or more. The resulting filtrate was filtered, and the solid was washed with 50 mL of 1,4-dioxane. After mixing the liquid phases, 0.43 g of NaBH4 (11.3 mmol, 1.13 eq.) was slowly added under nitrogen protection, and stirring was continued at room temperature for 30 min or more. A solution of 10 mL of pyridine and acetic acid (1:1, v / v) was added, and after stirring for 30 min, most of the solvent was removed under reduced pressure. 100 mL of dichloromethane was added, and the mixture was washed with 50 mL of saturated aqueous sodium bicarbonate solution three times. After washing the organic layer with saturated brine, it was dried over solid sodium sulfate and concentrated. Silica gel column chromatography yielded 3.32 g of white solid I-1 (yield 86%).

[0051] 3.10 g of I-1 (8.0 mmol, 1.0 eq.) was dissolved in 100 mL of dry pyridine, and 20 mg of DMAP was added. The mixture was stirred at room temperature, and 2.98 g of DMTCl (8.8 mmol, 1.1 eq.) was added. The mixture was stirred overnight. TLC analysis showed that the starting material was consumed completely. The solvent was removed under reduced pressure, and 100 mL of saturated aqueous NaHC03was added to the residue. The mixture was extracted with 100 mL of dichloromethane three times. The combined organic layers were washed with saturated aqueous NaCl, dried over solid Na2S04, and concentrated. The residue was purified by column chromatography on silica gel to give 3.46 g of solid I-2 (63% yield).

[0052] 3.17 g of I-2 (4.6 mmol, 1.0 eq.) was dissolved in 30 mL of dry pyridine, and 10 mg of DMAP was added. The mixture was stirred at a controlled temperature between 0-5 °C, and 0.64 mL of benzoyl chloride (0.77 g, 5.5 mmol, 1.2 eq.) was added. The mixture was slowly warmed to room temperature and stirred overnight. TLC analysis showed that the starting material was consumed completely. The solvent was removed under reduced pressure, and 50 mL of saturated aqueous NaHC03was added to the residue. The mixture was extracted with 50 mL of dichloromethane three times. The combined organic layers were washed with saturated aqueous NaCl, dried over solid Na2S04, and concentrated. The residue was purified by column chromatography on silica gel to give 3.38 g of solid I-3 (93% yield).

[0053] 3.16 g of I-3 (4.0 mmol, 1.0 eq.) was dissolved in 40 mL of tetrahydrofuran, and the mixture was stirred at a controlled temperature between 0-5 °C. 10 mL of a solution of tetrabutylammonium fluoride in tetrahydrofuran (TBAF, 1.0 mol / L) was added dropwise to the mixture under nitrogen. The mixture was slowly warmed to room temperature and stirred overnight. TLC analysis showed that the starting material was consumed completely. The solvent was removed under reduced pressure, and 50 mL of saturated aqueous NaHC03was added to the residue. The mixture was extracted with 50 mL of dichloromethane three times. The combined organic layers were washed with saturated aqueous NaCl, dried over solid Na2S04, and concentrated. The residue was purified by column chromatography on silica gel to give 2.46 g of solid I-4 (91% yield).

[0054] Further;

[0055] Under nitrogen protection, 271 mg of nucleotide I-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA acetonitrile solution, and the temperature was controlled at 0°C. Under nitrogen protection, 0.13 mL of 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.6 mmol, 1.5 eq.) was injected into a solution of 2 mL of dry acetonitrile. After the mixed solution was raised to room temperature under nitrogen protection, it was continuously stirred for 40 min. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, and after washing with 10 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. Silica gel chromatography was used for separation, and the gradient solvent of hexane / ethyl acetate was selected as the mobile phase to obtain 230 mg of the target product I-6 (purity >99%, yield 68%).

[0056] Further;

[0057] Under nitrogen protection, 271 mg of nucleotide I-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA acetonitrile solution, and the temperature was controlled at 0°C. Under nitrogen protection, 0.13 mL of 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.6 mmol, 1.5 eq.) was injected into a solution of 2 mL of dry acetonitrile. After the mixed solution was raised to room temperature under nitrogen protection, it was continuously stirred for 40 min. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, and after washing with 10 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. Silica gel chromatography was used for separation, and the gradient solvent of hexane / ethyl acetate was selected as the mobile phase to obtain 230 mg of the target product I-6 (purity >99%, yield 68%).

[0058] Further;

[0059] Under nitrogen protection, 271 mg of nucleotide I-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA in acetonitrile at 0 °C. Under nitrogen protection, 0.19 mL of bis(diisopropylamino)(2-cyanoethoxy) phosphine (0.6 mmol, 1.5 eq.) was injected into a solution of 2 mL of dry acetonitrile. After the mixed solution was warmed to room temperature under nitrogen protection, it was stirred for 40 min. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, which was washed with 10 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product I-7 was obtained by silica gel column chromatography using a gradient of hexane / ethyl acetate as the mobile phase (226 mg, purity >99%, yield 62%).

[0060] Example 4: (2S)-N-Bz-Cuna phosphoramidite, phosphoramidite and phosphorous oxychloride

[0061] 4.62 g of N-Bz-O5'-TBDMS-C (10 mmol, 1.0 eq.) was dissolved in 150 mL of 1,4-dioxane and 25 mL of water, and stirred at room temperature. 2.35 g of NaIO4(11.0 mmol, 1.10 eq.) was added dropwise to a solution of 25 mL of water. After 1 hour, 100 mL of 1,4-dioxane was added, and stirring was continued for 15 min. The resulting filtrate was filtered, and the solid was washed with 50 mL of 1,4-dioxane. After mixing the liquid phases, 0.43 g of NaBH4(11.3 mmol, 1.13 eq.) was slowly added under nitrogen protection, and stirring was continued at room temperature for 30 min. 10 mL of a mixture of pyridine and acetic acid (1:1, v / v) was added, and after stirring for 30 min, most of the solvent was removed under reduced pressure. 100 mL of dichloromethane was added, and the mixture was washed with 50 mL of saturated aqueous sodium bicarbonate solution three times. The organic layer was washed with saturated brine, dried over solid sodium sulfate, and concentrated. Silica gel column chromatography gave 4.13 g of white solid C-1 (yield 89%).

[0062] 3.71 g of C-1 (8 mmol, 1.0 eq.) was dissolved in 100 mL of dry pyridine, and 20 mg of DMAP was added. After stirring at room temperature, 2.88 g of DMTCl (8.8 mmol, 1.1 eq.) was added, and stirring was continued overnight. TLC detection showed that the starting material was completely consumed. The solvent was removed under reduced pressure, 100 mL of saturated aqueous sodium bicarbonate solution was added to the residue, and the mixture was extracted with 100 mL of dichloromethane three times. The combined organic layers were washed with saturated brine, dried over solid sodium sulfate, and concentrated. Silica gel column chromatography gave 3.62 g of solid C-2 (yield 59%).

[0063] 3.29 g of C-2 (4.3 mmol, 1.0 eq.) was dissolved in 30 mL of dry pyridine, 10 mg of DMAP was added. The temperature was controlled between 0-5 °C while 0.60 mL of benzoyl chloride (0.72 g, 5.2 mmol, 1.2 eq.) was added. The temperature was slowly increased to room temperature and the stirring was continued overnight. TLC test showed that the starting material was completely consumed. The solvent was removed under reduced pressure and 50 mL of saturated aqueous sodium bicarbonate solution was added to the residue, which was extracted with 50 mL of dichloromethane three times. The combined organic layers were washed with saturated brine, dried over solid sodium sulfate and concentrated. Column chromatography on silica gel gave 3.33 g of solid C-3 (yield 89%).

[0064] 3.13 g of C-3 (3.6 mmol, 1.0 eq.) was dissolved in 40 mL of tetrahydrofuran, the temperature was controlled between 0-5 °C while 10 mL of tetrabutylammonium fluoride tetrahydrofuran solution (TBAF, 1.0 mol / L) was slowly added to the mixture under nitrogen protection. The temperature was slowly increased to room temperature and the stirring was continued overnight. TLC test showed that the starting material was completely consumed. The solvent was removed under reduced pressure and 50 mL of saturated aqueous sodium bicarbonate solution was added to the residue, which was extracted with 50 mL of dichloromethane three times. The combined organic layers were washed with saturated brine, dried over solid sodium sulfate and concentrated. Column chromatography on silica gel gave 2.58 g of solid C-4 (yield 95%).

[0065] Further;

[0066] 303 mg of nucleotide C-4 (0.4 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL of dichloromethane and 7 mL of acetonitrile) under nitrogen protection, the temperature was controlled at 0 °C, 70 mg of lithium bromide (0.8 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.12 mL of DBU (0.8 mmol, 2.0 eq.) and 0.08 mL of N,N-dimethyl phosphoraminodichloride (0.6 mmol, 1.5 eq.) dissolved in 0.5 mL of dry dichloromethane were added in turn. The mixture was stirred at 0 °C for 40 minutes under nitrogen protection. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, washed with 10 mL of cold water, and the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The product was separated by silica gel chromatography, and the mobile phase was selected as a gradient solvent of hexane / ethyl acetate, to obtain 216 mg of the target product C-5 (purity >99%, yield 61%).

[0067] Further;

[0068] Under nitrogen protection, 303 mg of nucleotide C-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 ml of dry 20% DIPEA acetonitrile solution, temperature was controlled at 0°C. Under nitrogen protection, 0.13 ml of 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.6 mmol, 1.5 eq.) was injected into a solution of 2 ml of dry acetonitrile. After the mixed solution was raised to room temperature under nitrogen protection, it was continuously stirred for 40 minutes. The reaction solvent was removed under reduced pressure, 30 ml of ethyl acetate was added to the residue, and after washing with 10 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product C-6 (purity >99%, yield 73%) was separated by silica gel chromatography with a gradient solvent of hexane / ethyl acetate.

[0069] Further;

[0070] Under nitrogen protection, 303 mg of nucleotide C-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 ml of dry 20% DIPEA acetonitrile solution, temperature was controlled at 0°C. Under nitrogen protection, 0.13 ml of 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.6 mmol, 1.5 eq.) was injected into a solution of 2 ml of dry acetonitrile. After the mixed solution was raised to room temperature under nitrogen protection, it was continuously stirred for 40 minutes. The reaction solvent was removed under reduced pressure, 30 ml of ethyl acetate was added to the residue, and after washing with 10 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product C-6 (purity >99%, yield 73%) was separated by silica gel chromatography with a gradient solvent of hexane / ethyl acetate.

[0071] Example 5: (2S)-N-Bz-m5Cuna phosphoramidite, phosphoramidite and phosphorous chloride

[0072] 4.76 g of N-Bz-O5'-TBDMS-m5C (10 mmol, 1.0 eq.) was dissolved in 150 mL of 1,4-dioxane and 25 mL of water and stirred at room temperature. A solution of 2.35 g of NaIO4(11.0 mmol, 1.10 eq.) in 25 mL of water was added dropwise. After 1 h, 100 mL of 1,4-dioxane was added and stirring was continued for 15 min. The resulting filtrate was filtered and the solid was washed with 50 mL of 1,4-dioxane. After mixing the solution, 0.43 g of NaBH4(11.3 mmol, 1.13 eq.) was added slowly under nitrogen and stirred at room temperature for 30 min. A solution of 10 mL of pyridine and acetic acid (1:1, v / v) was added and the mixture was stirred for 30 min. Most of the solvent was removed under reduced pressure. The residue was dissolved in 100 mL of dichloromethane and washed with 50 mL of saturated aqueous NaHC03three times. The organic layer was washed with saturated brine, dried over solid Na2S04and concentrated. Purification by column chromatography on silica gel gave 4.05 g of m5C-1 as a white solid (85% yield).

[0073] 3.82 g of m5C-1 (8.0 mmol, 1.0 eq.) was dissolved in 100 mL of dry pyridine and 20 mg of DMAP was added. The mixture was stirred at room temperature and 2.98 g of DMTCl (8.8 mmol, 1.1 eq.) was added. The mixture was stirred overnight. TLC analysis showed that the starting material was completely consumed. The solvent was removed under reduced pressure and the residue was dissolved in 100 mL of saturated aqueous NaHC03. The mixture was extracted with 100 mL of dichloromethane three times. The combined organic layer was washed with saturated brine, dried over solid Na2S04and concentrated. Purification by column chromatography on silica gel gave 3.75 g of m5C-2 as a solid (60% yield).

[0074] 3.60 g of m5C-2 (4.6 mmol, 1.0 eq.) was dissolved in 30 mL of dry pyridine and 10 mg of DMAP was added. The mixture was stirred at a controlled temperature between 0-5 °C and 0.64 mL of benzoyl chloride (0.77 g, 5.5 mmol, 1.2 eq.) was added. The temperature was slowly increased to room temperature and the mixture was stirred overnight. TLC analysis showed that the starting material was completely consumed. The solvent was removed under reduced pressure and the residue was dissolved in 50 mL of saturated aqueous NaHC03. The mixture was extracted with 50 mL of dichloromethane three times. The combined organic layer was washed with saturated brine, dried over solid Na2S04and concentrated. Purification by column chromatography on silica gel gave 3.70 g of m5C-3 as a solid (91% yield).

[0075] 3.55 g of m5C-3 (4.0 mmol, 1.0 eq.) was dissolved in 40 mL of tetrahydrofuran, and the temperature was controlled between 0-5 °C with stirring. 10 mL of tetrabutylammonium fluoride tetrahydrofuran solution (TBAF, 1.0 mol / L) was slowly added to the mixed solution under nitrogen protection. The temperature was slowly increased to room temperature, and the stirring was continued overnight. TLC detection showed that the starting material completely disappeared. The solvent was removed under reduced pressure, 50 mL of saturated aqueous sodium bicarbonate solution was added to the residue, and the mixture was extracted with 50 mL of dichloromethane three times. After the organic layer was washed with saturated brine, it was dried over solid sodium sulfate and concentrated. Silica gel column chromatography gave 2.86 g of solid m5C-4 (yield 93%).

[0076] Further;

[0077] Under nitrogen protection, 308 mg of nucleotide m5C-4 (0.4 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL of dichloromethane and 7 mL of acetonitrile), and the temperature was controlled at 0 °C. 70 mg of lithium bromide (0.8 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 min. 0.12 mL of DBU (0.8 mmol, 2.0 eq.) and 0.08 mL of N,N-dimethylphosphor amidodichloride (0.6 mmol, 1.5 eq.) dissolved in 0.5 mL of dry dichloromethane were added in turn. The mixed solution was stirred at 0 °C for 40 min under nitrogen protection. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, and the organic phase was dried over sodium sulfate and concentrated under reduced pressure. Silica gel chromatography was used for separation, and the gradient solvent of hexane / ethyl acetate was selected as the mobile phase to obtain 230 mg of the target product m5C-5 (purity >99%, yield 64%).

[0078] Further;

[0079] Under nitrogen protection, 308 mg of nucleotide m5C-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA acetonitrile solution, and the temperature was controlled at 0 °C. 0.13 mL of 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.6 mmol, 1.5 eq.) dissolved in 2 mL of dry acetonitrile was injected under nitrogen protection. After the mixed solution was increased to room temperature, it was continued to stir for 40 min under nitrogen protection. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, and the organic phase was dried over sodium sulfate and concentrated under reduced pressure. Silica gel chromatography was used for separation, and the gradient solvent of hexane / ethyl acetate was selected as the mobile phase to obtain 256 mg of the target product m5C-6 (purity >99%, yield 71%).

[0080] Further;

[0081] Under nitrogen protection, 308 mg of nucleotide m5C-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA in acetonitrile, and the temperature was controlled at 0 °C. Under nitrogen protection, 0.19 mL of bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.6 mmol, 1.5 eq.) was injected into a solution of 2 mL of dry acetonitrile. After the mixed solution was allowed to warm to room temperature under nitrogen protection, it was continuously stirred for 40 min. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, and after washing with 10 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product m5C-7 was obtained by silica gel column chromatography using a gradient solvent of hexane / ethyl acetate (purity >99%, yield 61%).

[0082] Example 6: (2S)-Uuna phosphoramidites, phosphoramidites and phosphorous oxychloride

[0083] 3.58 g of O5'-TBDMS-U (10 mmol, 1.0 eq.) was dissolved in 150 mL of 1,4-dioxane and 25 mL of water, and stirred at room temperature. A solution of 2.35 g of NaIO4(11.0 mmol, 1.10 eq.) in 25 mL of water was added dropwise. After 1 hour, 100 mL of 1,4-dioxane was added, and stirring was continued for 15 min or more. The resulting filtrate was filtered, and the solid was washed with 50 mL of 1,4-dioxane. After mixing the liquid phases, 0.43 g of NaBH4(11.3 mmol, 1.13 eq.) was slowly added under nitrogen protection, and stirring was continued at room temperature for 30 min or more. A solution of 10 mL of pyridine and acetic acid (1:1, v / v) was added, and after stirring for 30 min, most of the solvent was removed under reduced pressure. 100 mL of dichloromethane was added, and washing was performed three times with 50 mL of saturated aqueous sodium bicarbonate solution. After washing the organic layer with saturated brine, it was dried over solid sodium sulfate and concentrated. Silica gel column chromatography yielded 3.15 g of white solid U-1 (yield 87%).

[0084] 2.88 g of U-1 (8.0 mmol, 1.0 eq.) was dissolved in 100 mL of dry pyridine, and 20 mg of DMAP was added. The mixture was stirred at room temperature, and 2.98 g of DMTCl (8.8 mmol, 1.1 eq.) was added. The mixture was stirred overnight. TLC test showed that the starting material was consumed completely. The solvent was removed under reduced pressure, and 100 mL of saturated aqueous NaHC03was added to the residue. The mixture was extracted with 100 mL of dichloromethane three times. The combined organic layers were washed with saturated brine, dried over solid Na2S04, and concentrated. The residue was purified by column chromatography on silica gel to give 3.13 g of U-2 as a solid (yield 59%).

[0085] 3.00 g of U-2 (4.5 mmol, 1.0 eq.) was dissolved in 30 mL of dry pyridine, and 10 mg of DMAP was added. The mixture was stirred at a temperature between 0-5 °C, and 0.63 mL of benzoyl chloride (0.76 g, 5.4 mmol, 1.2 eq.) was added. The mixture was allowed to warm up to room temperature, and stirred overnight. TLC test showed that the starting material was consumed completely. The solvent was removed under reduced pressure, and 50 mL of saturated aqueous NaHC03was added to the residue. The mixture was extracted with 50 mL of dichloromethane three times. The combined organic layers were washed with saturated brine, dried over solid Na2S04, and concentrated. The residue was purified by column chromatography on silica gel to give 3.26 g of U-3 as a solid (yield 93%).

[0086] 3.07 g of U-3 (4.0 mmol, 1.0 eq.) was dissolved in 40 mL of tetrahydrofuran, and the mixture was stirred at a temperature between 0-5 °C. 10 mL of tetrabutylammonium fluoride in tetrahydrofuran solution (TBAF, 1.0 mol / L) was added dropwise to the mixture under nitrogen protection. The mixture was allowed to warm up to room temperature, and stirred overnight. TLC test showed that the starting material was consumed completely. The solvent was removed under reduced pressure, and 50 mL of saturated aqueous NaHC03was added to the residue. The mixture was extracted with 50 mL of dichloromethane three times. The combined organic layers were washed with saturated brine, dried over solid Na2S04, and concentrated. The residue was purified by column chromatography on silica gel to give 2.48 g of U-4 as a solid (yield 95%).

[0087] Further;

[0088] Under nitrogen protection, 262 mg of nucleotide U-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA in acetonitrile, and the temperature was controlled at 0°C. Under nitrogen protection, 0.13 mL of 2-cyanoethyl N,N-diisopropyl phosphoramidite (0.6 mmol, 1.5 eq.) dissolved in 2 mL of dry acetonitrile was injected. After the mixed solution was raised to room temperature under nitrogen protection, it was continuously stirred for 40 min. The reaction solvent was removed under reduced pressure, and 30 mL of ethyl acetate was added to the residue. After washing with 10 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 gradient solvent of hexane / ethyl acetate was selected as the mobile phase to obtain 227 mg of the target product U-6 (purity >99%, yield 72%).

[0089] Further;

[0090] Under nitrogen protection, 262 mg of nucleotide U-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA in acetonitrile, and the temperature was controlled at 0°C. Under nitrogen protection, 0.13 mL of 2-cyanoethyl N,N-diisopropyl phosphoramidite (0.6 mmol, 1.5 eq.) dissolved in 2 mL of dry acetonitrile was injected. After the mixed solution was raised to room temperature under nitrogen protection, it was continuously stirred for 40 min. The reaction solvent was removed under reduced pressure, and 30 mL of ethyl acetate was added to the residue. After washing with 10 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 gradient solvent of hexane / ethyl acetate was selected as the mobile phase to obtain 227 mg of the target product U-6 (purity >99%, yield 72%).

[0091] Further;

[0092] Under nitrogen, 262 mg of nucleotide U-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 mL of dry 20% DIPEA in acetonitrile at 0 °C. Under nitrogen, 0.19 mL of bis(diisopropylamino)(2-cyanoethoxy) phosphine (0.6 mmol, 1.5 eq.) was injected as a solution in 2 mL of dry acetonitrile. After the mixture was allowed to warm to room temperature under nitrogen, stirring was continued for 40 min. The reaction solvent was removed under reduced pressure, and the residue was taken up in 30 mL of ethyl acetate and washed with 10 mL of cold water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using a gradient of hexanes / ethyl acetate as the mobile phase to give 218 mg of the desired product U-7 (purity >99%, yield 64%).

[0093] Example 7: (2S)-Tuna phosphoramidites, phosphoramidites and phosphorous chlorides

[0094] 3.73 g of O5'-TBDMS-T (10 mmol, 1.0 eq.) was dissolved in 150 mL of 1,4-dioxane and 25 mL of water and stirred at room temperature. A solution of 2.35 g of NaIO4(11.0 mmol, 1.10 eq.) in 25 mL of water was added dropwise. After 1 h, 100 mL of 1,4-dioxane was added and stirring was continued for 15 min. The resulting filtrate was filtered and the solid was washed with 50 mL of 1,4-dioxane. The liquid phases were combined and 0.43 g of NaBH4(11.3 mmol, 1.13 eq.) was added slowly under nitrogen. The mixture was stirred at room temperature for 30 min. A solution of 10 mL of pyridine and acetic acid (1:1, v / v) was added and the mixture was stirred for 30 min. Most of the solvent was removed under reduced pressure. The residue was taken up in 100 mL of dichloromethane and washed with 50 mL of saturated aqueous NaHC03three times. The organic layer was washed with saturated aqueous NaCl and dried over solid Na2S04. The solvent was removed under reduced pressure and the residue was purified by silica gel chromatography to give 3.22 g of T-1 as a white solid (yield 86%).

[0095] 3.00 g of T-1 (8.0 mmol, 1.0 eq.) was dissolved in 100 mL of dry pyridine and 20 mg of DMAP was added. The mixture was stirred at room temperature and 2.98 g of DMTCl (8.8 mmol, 1.1 eq.) was added. The mixture was stirred overnight. TLC indicated that the starting material was completely consumed. The solvent was removed under reduced pressure and the residue was taken up in 100 mL of saturated aqueous NaHC03. The mixture was extracted with 100 mL of dichloromethane three times. The combined organic layers were washed with saturated aqueous NaCl and dried over solid Na2S04. The solvent was removed under reduced pressure and the residue was purified by silica gel chromatography to give 3.36 g of T-2 as a solid (yield 62%).

[0096] 3.11 g of T-2 (4.6 mmol, 1.0 eq.) was dissolved in 30 mL of dry pyridine, 10 mg of DMAP was added. The temperature was controlled between 0-5 °C with stirring, 0.64 mL of benzoyl chloride (0.77 g, 5.5 mmol, 1.2 eq.) was added. The temperature was slowly increased to room temperature, and the stirring was continued overnight. TLC detection showed that the starting material was completely consumed. The solvent was removed under reduced pressure, 50 mL of saturated aqueous sodium bicarbonate solution was added to the residue, and the mixture was extracted with 50 mL of dichloromethane three times. The combined organic layers were washed with saturated brine, dried over solid sodium sulfate, and concentrated. Silica gel column chromatography gave 3.23 g of solid T-3 (yield 90%).

[0097] 3.12 g of T-3 (4.0 mmol, 1.0 eq.) was dissolved in 40 mL of tetrahydrofuran, and the temperature was controlled between 0-5 °C with stirring. 10 mL of tetrabutylammonium fluoride tetrahydrofuran solution (TBAF, 1.0 mol / L) was slowly added to the mixture under nitrogen protection. The temperature was slowly increased to room temperature, and the stirring was continued overnight. TLC detection showed that the starting material was completely consumed. The solvent was removed under reduced pressure, 50 mL of saturated aqueous sodium bicarbonate solution was added to the residue, and the mixture was extracted with 50 mL of dichloromethane three times. The combined organic layers were washed with saturated brine, dried over solid sodium sulfate, and concentrated. Silica gel column chromatography gave 2.61 g of solid T-4 (yield 98%).

[0098] Further;

[0099] 267 mg of nucleotide T-4 (0.4 mmol, 1.0 eq.) was added to a dry mixed solvent (3 mL of dichloromethane and 7 mL of acetonitrile) under nitrogen protection, and the temperature was controlled at 0 °C. 70 mg of lithium bromide (0.8 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.12 mL of DBU (0.8 mmol, 2.0 eq.) and 0.08 mL of N,N-dimethyl phosphoraminodichloride (0.6 mmol, 1.5 eq.) dissolved in 0.5 mL of dry dichloromethane were added in sequence. The mixture was stirred at 0 °C for 40 minutes under nitrogen protection. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, and 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 gradient solvent of hexane / ethyl acetate, to obtain 191 mg of the target product T-5 (purity >99%, yield 60%).

[0100] Further;

[0101] Under nitrogen protection, 267 mg of nucleotide T-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 ml of dry 20% DIPEA acetonitrile solution, and the temperature was controlled at 0°C. Under nitrogen protection, 0.13 ml of 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.6 mmol, 1.5 eq.) was injected into a solution of 2 ml of dry acetonitrile. After the mixed solution was raised to room temperature under nitrogen protection, it was continuously stirred for 40 minutes. The reaction solvent was removed under reduced pressure, 30 ml of ethyl acetate was added to the residue, and after washing with 10 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product T-6 (purity >99%, yield 75%) was obtained by silica gel chromatography separation using a gradient solvent of hexane / ethyl acetate.

[0102] Further;

[0103] Under nitrogen protection, 267 mg of nucleotide T-4 (0.4 mmol, 1.0 eq.) was dissolved in 10 ml of dry 20% DIPEA acetonitrile solution, and the temperature was controlled at 0°C. Under nitrogen protection, 0.13 ml of 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.6 mmol, 1.5 eq.) was injected into a solution of 2 ml of dry acetonitrile. After the mixed solution was raised to room temperature under nitrogen protection, it was continuously stirred for 40 minutes. The reaction solvent was removed under reduced pressure, 30 ml of ethyl acetate was added to the residue, and after washing with 10 ml of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product T-6 (purity >99%, yield 75%) was obtained by silica gel chromatography separation using a gradient solvent of hexane / ethyl acetate.

[0104] The following monophosphoramidites were prepared using the above synthesis method: ; ; ; ; ; ; ; ; ; ; ; ; ; ; Further, the monophosphoramidites prepared by the above similar synthesis method have the following structure: ; wherein: Base: a nucleotide base or derivative thereof, the nucleotide base being adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, N1-methylpseudouracil; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl and the like acyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl and the like silyl; PG: a protecting group, including but not limited to trityl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytrityl DMTr, 4,4',4''-trimethoxytrityl TMTr.

[0105] The following monomer phosphorous chlorides are prepared using the above synthesis method: ; ; ; ; ; ; ; ; ; ; ; ; ; ; The following monomer phosphorous chlorides are prepared using the above synthesis method: ; wherein: Base: a nucleotide base or derivative thereof, the nucleotide base being adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, N1-methylpseudouracil; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl and the like acyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl and the like silyl; PG: a protecting group, including but not limited to trityl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytrityl DMTr, 4,4',4''-trimethoxytrityl TMTr.

[0106] The following monomer phosphorous amides are prepared using the above synthesis method: ; ; ; ; ; ; ; ; ; ; ; ; ; ; Further, the monomer phosphoramidite prepared by the similar synthesis method has the following structure: ; wherein: Base: nucleotide base or its derivative, the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, N1-methylpseudouracil; R: 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; PG: protecting group, including but not limited to trityl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytrityl DMTr, 4,4',4''-trimethoxytrityl TMTr.

[0107] Phosphorodiamidate morpholino oligonucleotide (PMO) uses morpholine ring instead of deoxyribose or ribose structure, in the PMO phosphoramide monomer, the protecting group of N3' and O6'-phosphoramide are inverted with the traditional nucleotide phosphoramidite, the PMO monomer and other monomers in the oligonucleic acid are coupled, and in the solid phase synthesis process, the coupling mode of the nucleic acid sequence from 5' to 3' direction is generally used. The combination of PMO and UNA two kinds of modifications is used to construct oligonucleic acid, and the latter monomer is (2S)-UNA phosphoramidite, phosphorochloridate or phosphoramide.

[0108] The carrier for solid phase synthesis is selected from 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 swollen at room temperature for 2 hours, and then filtered and emptied. 3.0 milliliters of dichloromethane is used to wash the resin, and the washing process is repeated 3 times, and the solid phase synthesis is started.

[0109] The monomers introduced in the solid phase synthesis are phosphoramidite P(III) and phosphoramide P(V).

[0110] The process conditions of the phosphoramidite monomer are as follows: Reagents and monomers: 0.15 mol / L monomer / acetonitrile solution was prepared; the deprotection reagent for DMTr (or Tr) protecting group was 3% dichloroacetic acid / toluene solution (v / v); the activating reagent 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% hydorxanthone / pyridine solution; the capping reagent A was 10% acetic anhydride / acetonitrile solution (v / v), and the capping reagent B was 1-methylimidazole / pyridine / tetrahydrofuran solution (16 / 10 / 74, v / v / v).

[0111] The operation sequence of the solid-phase synthesis of the phosphoramidite monomer is as follows: 1) Deprotection: 3% dichloroacetic acid / toluene solution was used as the deprotection reagent for DMTr protecting group, and then acetonitrile was used for washing.

[0112] 2) Coupling: 0.25 mol / L 5-ethylthiotetrazole was used as the activating reagent, 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.

[0113] 3) Oxidation / sulfurization: 0.05 mol / L iodine / water / pyridine (v / v, 90 / 10) solution was used as the oxidizing reagent for oxidizing P(III), and then acetonitrile was used for washing. 3% hydorxanthone / pyridine solution was used as the sulfurizing reagent for sulfurizing P(III), and then acetonitrile was used for washing.

[0114] 4) Capping: the capping reagent A and the capping reagent B were used as the capping reagents for protecting the unreacted active groups, and then acetonitrile was used for washing.

[0115] The process conditions of the phosphoramidite monomer for the solid-phase synthesis of oligonucleic acids are as follows: The deprotection solution: 5.355 g of 4-cyanopyridine was added into 100.0 mL of dichloromethane, and then 4.900 g of trifluoroacetic acid was added under stirring at room temperature. After stirring for 1 hour, 4.5 mL of ethanol, 100 mL of trifluoroethanol, and dichloromethane were added to make up to 500 mL. A 4-cyanopyridinium trifluoroacetate (2%) and ethanol (0.9%) trifluoroethanol / dichloromethane solution was prepared as the deprotection solution.

[0116] The neutralization solution: 25.0 mL of diisopropylethylamine and 125.0 mL of isopropanol were added, and dichloromethane was added to make up to 500 mL. A diisopropylethylamine (5%) isopropanol / dichloromethane solution was prepared as the neutralization solution.

[0117] Coupling solvent: 2.303 g of N-ethylmorpholine was dissolved in 1,3-dimethyl-2-imidazolidinone (DMI) to make 50 mL of N-ethylmorpholine DMI solution (0.40 mol / L) as the coupling solvent.

[0118] Lysis solution: 0.154 g of dithiothreitol was dissolved in 5 mL of N-methyl pyrrolidone (NMP), 0.739 g of triethylamine was added, and the solution was diluted with NMP to 10 mL to obtain a NMP solution containing 0.10 mol / L of dithiothreitol and 0.73 mol / L of triethylamine, which was prepared immediately before use.

[0119] The process operation of the phosphoramidite monomer solid-phase synthesis includes: According to the base sequence of the target sequence, the nucleotide monomer molecules 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 added to 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.

[0120] The above operation was repeated according to the set sequence to obtain the oligonucleotide product of the target sequence.

[0121] Deprotection is to transfer the solid-phase carrier loaded with the oligonucleotide product to a reactor, add concentrated ammonia water (28%), control the temperature at 60°C for ammoniaolysis for 12 hours, then reduce the temperature 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.

[0122] TBDMS or other silyl ether protecting group removal: To the oligonucleotide product containing the silyl ether protecting group, TBAF / THF solution was added and treated at room temperature for 2-6 hours.

[0123] Purification: The crude product after deprotection was dissolved in purified water, and HPLC purification was performed. The product peak solution was collected, and the product peak solution was concentrated under reduced pressure and freeze-dried to obtain the product.

[0124] The above solid-phase synthesis is also applicable to Tr, MMTr, and TMTr-protected monomers.

[0125] Example 8:

[0126] Monomer N3'-DMTr-Up was loaded on cross-linked polystyrene PS, and N3'-DMTr was removed by trichloroacetic acid solution. After washing, monomer A-5 was pumped in for coupling. After the loaded nucleic acid molecule was treated by ammonolysis and deprotection, the crude product concentrated under reduced pressure was separated and purified by HPLC. The main fraction was collected, concentrated, and lyophilized to obtain the product. The product was detected and confirmed to be 6'-Up-P NMe2 -Auna-3' nucleic acid molecule.

[0127] Example 9: The operation steps of solid-phase synthesis are shown in the attached Figure 1 Monomer N3'-DMTr-Up was loaded on cross-linked polystyrene PS, and N3'-DMTr was removed by trichloroacetic acid solution. After washing, monomer A-6 was pumped in for coupling, capping, and oxidation. After the loaded nucleic acid molecule was treated by ammonolysis and deprotection, the crude product concentrated under reduced pressure was separated and purified by HPLC. The main fraction was collected, concentrated, and lyophilized to obtain the product. The product was detected and confirmed to be 6'-Up-Auna-3' nucleic acid molecule of the target sequence.

[0128] Example 10:

[0129] Monomer T4 was loaded on cross-linked polystyrene PS, and the protecting group DMTr was removed by dichloroacetic acid solution. After washing, monomer Ap was pumped in for coupling. After the loaded oligonucleic acid molecule was treated by ammonolysis and deprotection, the crude product concentrated under reduced pressure was separated and purified by HPLC. The main fraction was collected, concentrated, and lyophilized to obtain the product. The product was detected and confirmed to be 5'-Tuna-P NMe2 -Ap-3' nucleic acid molecule.

[0130] Example 11: The operation steps of solid-phase synthesis are shown in the attached Figure 2 Monomer T4 was loaded on cross-linked polystyrene PS, and the protecting group DMTr was removed by dichloroacetic acid solution. After washing, monomer O2´-Me-O3´-DMTr-O5´-OCE-A was pumped in for coupling, capping, and oxidation. DMTr was removed by dichloroacetic acid solution, and monomer Up phosphoramidite was pumped in for coupling. After the loaded oligonucleic acid molecule was treated by ammonolysis and deprotection, the crude product concentrated under reduced pressure was separated and purified by HPLC. The main fraction was collected, concentrated, and lyophilized to obtain the product. The product was detected and confirmed to be 5'-Tuna-Ame-P NMe2 -Up-3' nucleic acid molecule.

[0131] Example 12: The operation steps of solid-phase synthesis are shown in the attached Figure 3. Monomer O2'-Me-O3'-DMTr-T is loaded on cross-linked polystyrene PS, O3'-DMTr is removed by dichloroacetic acid solution, after washing, monomer A-7 is pumped in for coupling, capping and oxidation. The loaded oligonucleic acid molecule is treated by ammonolysis and deprotection, 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 molecular detection as the 3'-Tme-Auna-Uuna-Uuna-Guna-Uuna-Auna-Uuna-Uuna-Guna-5' nucleic acid molecule of the target sequence. NMe2 -Up-3' nucleic acid molecule.

[0132] Example 13: Monomer O2'-Me-O5'-DMTr-T is loaded on cross-linked polystyrene PS, O5'-DMTr is removed by dichloroacetic acid solution, after washing, monomer A-7, U-7, U-7, G-7, U-7, A-7, U-7, U-7, G-7 are pumped in for coupling, capping and oxidation. The loaded oligonucleic acid molecule is treated by ammonolysis and deprotection, 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 molecular detection as the 3'-Tme-Auna-Uuna-Uuna-Guna-Uuna-Auna-Uuna-Uuna-Guna-5' nucleic acid molecule of the target sequence.

[0133] Further, the above-mentioned solid phase synthesis is also applicable to the following UNA monomers: , , ; wherein: PG: protecting group, including but not limited to trityl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytrityl DMTr, 4,4',4''-trimethoxytrityl TMTr; R: alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl and the like acyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl and the like silyl group; R1, R2: alkyl.

[0134] Further, the above-mentioned solid phase synthesis is also applicable to the UNA, PMO and other types of nucleotide monomers protected by Tr, MMTr and TMTr.

[0135] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in the present application should be the usual meanings understood by the skilled person in the field to which the present application belongs.

[0136] In the present application, the corresponding substances of the letter abbreviations are as follows: UNA: unlocked nucleic acid; PMO: phosphorodiamidate morpholino oligonucleotide; DCC: N,N'-Dicyclohexylcarbodiimide; CPG: controlled pore glass; PS: polystyrene; F: fluoro; DIPEA: N,N-diisopropylethylamine; Alkyl: alkyl; Allyl: allyl; Alloc: allyloxycarbonyl; Bn: benzyl; Bz: benzoyl; Me: methyl; NMe2: dimethyl secondary amine; Ps: phosphorothioate; P NMe2 : phosphorodiamidate; DMTr: dimethoxytrityl; MMTr: methoxytrityl; Tr: trityl; TMTr: trimethoxytrityl; iBu: 2-isobutyryl; NPE: 4-nitrophenethoxy; Ph: aromatic; TBDMS: tert-butyldimethylsilyl; TBDPS: tert-butyldiphenylsilyl; DCM: dichloromethane; DMF: N,N-dimethylformamide; DMAP: 4-dimethylaminopyridine; TBAF: tetra-n-butylammonium fluoride; THF: tetrahydrofuran; iPrOH: isopropanol; TLC: thin layer chromatography; A: Adenine; G: Guanine; C: Cytosine; T: Thymine; U: Uracil; I: hypoxanthine; Xan: xanthine; m5C: 5-methylcytosine; Ψ: pseudouracil; m1Ψ: N1-methylpseudouracil; Auna: A UNA; Guna: G UNA; Cuna: C UNA; m5Cuna: m5C UNA; Tuna: T UNA; Uuna: U UNA; Iuna: I UNA; Ap: A PMO; Gp: G PMO; Cp: C PMO; m5Cp: m5C PMO; Tp: T PMO; Up: U PMO; Ip: I PMO; DMTCl: 4,4'-dimethoxytrityl chloride; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0137] Various embodiments described in this specification are described in the context of methods, devices, apparatus, and systems. Embodiments of the subject matter described in this specification can also be implemented in the context of computer-readable media storing computer-executable instructions for execution by a local or remote computing device. Accordingly, those skilled in the art will recognize that the various embodiments described in this specification can be implemented in a variety of ways.

[0138] The above description of specific embodiments of the application has been presented for the purpose of illustration and description. Other embodiments are within the scope of the file. In some cases, the actions or steps recited in this application can be performed in a different order and still accomplish the desired result. In addition, the processes depicted in the figures do not necessarily require the particular order shown or sequential order to achieve the desired results. In certain implementations, multitasking and parallel processing can be advantageous or necessary.

[0139] The above description is intended to be illustrative and not restrictive. Many other embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the application should, therefore, be determined not with reference to the above description, but instead with reference to the appended claims, along with their full scope of equivalents.

Claims

1. A chiral UNA monomer and its application in oligonucleotide drugs, characterized in that, The application includes chemical modification strategies, which involve preparing phosphorous amides, phosphorous chlorides, and phosphoramides of (2S)-UNA monomers, comprising nucleotide structures represented by Formula I, Formula II, or Formula III: Formula I; Formula II; Formula III; in: Base: Nucleotide base or its derivative, wherein the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, or N1-methylpseudorabies. PG: Protecting group, including but not limited to triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, and 4,4',4''-trimethoxytriphenylmethyl TMTr; LG1: Cyanoethyl, Cyanopropyl, 3-Cyanoisobutyl, (CH3)2CHCH2CN, Phenethyl or Allyl, etc.; LG2: Fluorine, chlorine, or bromine; R: Alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc. R1, R2, R3, R4: alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, etc.

2. The chiral UNA monomer according to claim 1 and its application in oligonucleotide drugs, characterized in that, The synthesis of the (2S)-UNA monomer involves oxidizing the ortho- and tho-dihydroxy groups of the O5'-protected nucleotides, cleaving the single bond between the C2 and C3 of the ribose, and then reducing them to two hydroxyl groups. The original O3'-hydroxyl group is bonded to the protecting group used in solid-phase synthesis, releasing the original O5'-hydroxyl group, and further reacting to obtain the (2S)-UNA monomer phosphorusamide, phosphorus chloride, and phosphoramide.

3. According to claim 1, a chiral UNA monomer and its application in oligonucleotide drugs, the phosphoramide, phosphoryl chloride and phosphoramide of the (2S)-UNA monomer are used in the direction of coupling extension in solid phase synthesis from 5' to 3', so that the chirality of the oligonucleotide molecule at the UNA site is consistent with that of the corresponding natural nucleotide.

4. According to claim 1, a chiral UNA monomer and its application in oligonucleotide drugs, the phosphoramide, phosphoryl chloride and phosphoramide of the (2S)-UNA monomer are used in the direction of coupling extension in solid phase synthesis from 3' to 5', constructing an oligonucleotide molecule whose chirality at the UNA site is opposite to that of the corresponding natural nucleotide, which helps to study the effect of chirality reversal on the stereoconformity, stability and bioactivity of the synthesized oligonucleotide.

5. The chiral UNA monomer according to claim 1 and its application in oligonucleotide drugs, characterized in that, The phosphoramide, phosphoryl chloride, and phosphoramide of the (2S)-UNA monomer used for solid-phase synthesis include the modified nucleotide structures shown in Formula IV, V, or VI: Formula IV; Formula V; Formula VI; in: Base: Nucleotide base or its derivative, wherein the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, or N1-methylpseudorabies. PG: Protecting group, including but not limited to triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, and 4,4',4''-trimethoxytriphenylmethyl TMTr; R: Alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl (Bn), benzoyl (Bz), acetyl or propionyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, etc.

6. The chiral UNA monomer according to claim 1 and its application in oligonucleotide drugs, characterized in that, The (2S)-UNA monomer's phosphorous amide, phosphorous chloride, and phosphoramide are directly coupled with the phosphoramide of the PMO monomer, and the oligonucleotides constructed by solid-phase synthesis include one of the following structures: ； ； ； ； Wherein: Base: nucleotide base or its derivative, wherein the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, N1-methylpseudorazine; X: Oxygen or sulfur; R: hydrogen, alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, etc.; R1, R2: Alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, etc.

7. The chiral UNA monomer according to claim 1 and its application in oligonucleotide drugs, characterized in that, The (2S)-UNA monomer's phosphorous amide, phosphorous chloride, and phosphoramide are linked to the PMO monomer's phosphoramide by one or more nucleic acid sequences, and the oligonucleotides constructed by solid-phase synthesis include one of the following structures: ； ； ； ； Wherein: Base: nucleotide base or its derivative, wherein the nucleotide base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, hypoxanthine, xanthine, pseudouracil, N1-methylpseudorazine; X: Oxygen or sulfur; Z: One or more nucleic acid sequences; R: hydrogen, alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, etc.; R1, R2: Alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, alkenyl, cycloalkenyl, alkynyl, aromatic, etc.

8. A chiral UNA monomer according to claims 6 and 7 and its application in oligonucleotide drugs, characterized in that, The (2S)-UNA monomer is coupled with the PMO monomer and other nucleotide monomers to construct an oligonucleotide molecule via phosphoramide, phosphoryl chloride or phosphoramide, wherein the protecting group is selected from one of triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, 4,4',4''-trimethoxytriphenylmethyl TMTr, allyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl.

9. The oligonucleic acid according to claims 6 and 7, 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.

Images