Synthesis of 3'-rna oligonucleotides

Abstract

To provide monomers and methods for preparing oligonucleotides with improved yield and reduced impurities.SOLUTION: The present disclosure relates to monomers and methods for synthesizing oligonucleotides comprising at least one nucleoside including a 3'-hydroxyl group.SELECTED DRAWING: None

Description

Cross - reference to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 941,153, filed on November 27, 2019, under 35 U.S.C.§119(e), the content of which is incorporated herein by reference in its entirety.

[0002] The present invention generally relates to nucleic acid chemistry and chemical synthesis of oligonucleotides. More particularly, the present invention relates to monomers and methods for synthesizing oligonucleotides comprising at least one nucleoside containing a 3'-hydroxyl group. BACKGROUND OF THE INVENTION

[0003] Modified oligonucleotides are of great value in molecular biology research and therapeutic applications. The chemical synthesis of modified oligonucleotides is routine, but the ease and yield of many modified oligonucleotides are low. For example, commonly used protecting groups are unstable to the conditions used to deprotect chemically synthesized oligonucleotides. This is particularly problematic when preparing oligonucleotides comprising at least one nucleoside containing a 3'-hydroxyl group. Thus, in the art, monomers and methods for preparing such oligonucleotides are still needed. The present disclosure addresses this need at least in part. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0004] The present disclosure provides monomers and methods for preparing oligonucleotides with improved yields and fewer impurities, where the oligonucleotides have at least one, e.g., two, three, four, or more nucleosides having a 3'-hydroxyl group. Generally, the method involves coupling a free hydroxyl group of a nucleoside or oligonucleotide with a nucleoside phosphoramidite monomer having a triisopropylsilyl ether (TIPS) protected 3'-hydroxyl group. The coupling forms a phosphite triester intermediate, which can be oxidized or sulfurized to form a phosphate triester or phosphorothioate intermediate.

[0005] Oligonucleotides having a predetermined length and sequence can be prepared by this method. For example, oligonucleotides containing about 6 to about 50 nucleotides can be prepared using the methods and monomers described herein. In some embodiments, the oligonucleotide contains about 10 to about 30 nucleotides.

[0006] In another aspect, the present disclosure provides a monomer, e.g., a nucleoside phosphoramidite monomer having a triisopropylsilyl ether protected 3'-hydroxyl group. Generally, the monomer has the formula (I):

Chemical formula

[0007] In formula (I), B is a modified or unmodified nucleic acid base; R 1 is a hydroxyl protecting group labile to acids; R 2 is -Si(R 4 )3; R 3 is -P(NR 5 R 6 )OR 7 ; each R 4is, independently, optionally substituted alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl; R 5 and R 6 are, independently, optionally substituted alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl, or R 5 and R 6 are linked to form a heterocyclyl; and R 7 is optionally substituted alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl.

[0008] In some monomers of formula (I), B is adenine, guanine, cytosine or uracil; R 1 is dimethoxytrityl; R 4 , R 5 and R 6 are isopropyl; and R 7 is β-cyanoethyl.

[0009] This patent or application file contains at least one drawing created in color. Copies of this patent or patent application publication that include color drawings will be provided by the authority upon request and payment of the required fees.

Brief Description of the Drawings

[0010]

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DETAILED DESCRIPTION OF THE INVENTION

[0011] In one aspect, the present disclosure provides an improved method for preparing an oligonucleotide comprising at least one nucleoside having a 3'-hydroxyl group. A nucleoside phosphoramidite monomer having a triisopropylsilyl ether (TIPS) protected 3'-hydroxyl group is coupled to a free hydroxyl of a nucleoside or oligonucleotide, such as a 5'-OH, 3'-OH or 2'-OH, preferably a 5'-OH.

[0012] Methods and reagents for coupling nucleoside phosphoramidite monomers to hydroxyl groups are well known in the art. Thus, oligonucleotides can be prepared using procedures and apparatus known to those of ordinary skill in the art. For example, a glass reactor such as a flask can be suitably used. Preferably, solid-phase synthesis procedures and solid supports such as controlled pore glass are used. Even more preferably, the methods of the present invention can be carried out using an automated DNA synthesizer. Suitable solid-phase techniques, including automated synthesis techniques, are described in F. Eckstein (ed.), Oligonucleotides and Analogues, a Practical Approach, Oxford University Press, New York (1991).

[0013] In addition, oligonucleotides can be prepared on a small or large scale. For example, oligonucleotides can be prepared on a μmol scale or an mg scale.

[0014] The coupling step and the oxidation / sulfurization step can be carried out in a common solvent. For example, coupling and oxidation / sulfurization can be carried out in acetonitrile.

[0015] The oxidation step can be carried out by contacting the phosphite intermediate with an oxidizing reagent for a time sufficient to effect the formation of the phosphotriester functional group. Solvent systems suitable for use in the oxidation of the phosphite intermediates of the present invention include mixtures of two or more solvents. Preferably, they are mixtures of an aprotic solvent and a protic or basic solvent. Preferred solvent mixtures include mixtures of acetonitrile and a weak base. For example, the oxidation step can be carried out in the presence of a weak base. Exemplary bases include, but are not limited to, pyridine, lutidine, picoline or collidine. In some embodiments, the oxidation step can be carried out in the presence of I2 / H2O.

[0016] Sulfidation (oxidation using a sulfur transfer reagent) can be carried out by contacting the phosphite intermediate with a sulfur transfer reagent for a time sufficient to effect the formation of the phosphorothioate functional group. Exemplary sulfur transfer reagents for use in oligonucleotide synthesis include, but are not limited to, phenylacetyl disulfide, arylacetyl disulfide and aryl-substituted phenylacetyl disulfide. For example, the sulfur transfer reagent can be 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-3-thione (DDTT) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent).

[0017] After completion of the synthesis, the oligonucleotide can be deprotected, for example, using methods and reagents for removing any protecting groups on the oligonucleotide to obtain the desired product. Thus, in some embodiments, the method further comprises treating the synthesized oligonucleotide with a base to remove any non-TIPS protecting groups on the oligonucleotide. Exemplary bases for use in removing non-TIPS protecting groups used in the synthesis of oligonucleotides include, but are not limited to, ammonium hydroxide, methylamine, and mixtures thereof. Treatment with a base can be appropriately carried out at room temperature or elevated temperature. "Room temperature" includes ambient temperatures of about 20°C to about 30°C. "Elevated temperature" includes temperatures higher than 30°C. For example, the elevated temperature can be a temperature of about 32°C to about 65°C. In some embodiments, treatment with a base is at about 35°C. The treatment time can range from approximately a few minutes, such as 5, 10, 15, 20, 25, 30, 45, or 60 minutes, etc., to several hours, such as 2, 3, 4, 5, 10, 15, 24 hours, or more. In some embodiments, treatment with a base is for about 15 hours. In some embodiments, treatment with a base is for about 15 hours at about 35°C.

[0018] After the non-TIPS protecting group is removed, the TIPS protecting group can be removed by treating the partially deprotected oligonucleotide with a deprotection reagent effective to convert the TIPS-protected hydroxyl group to a free hydroxyl group. Methods and reagents for removing silyl-containing hydroxyl protecting groups are well known in the art. Generally, the deprotection reagent contains a fluoride anion. One exemplary deprotection reagent for removing the TIPS protecting group is HF·pyridine. The deprotection step for removing the TIPS group can be appropriately carried out at room temperature or elevated temperature. For example, the deprotection step can be carried out at a temperature of 35 °C to about 65 °C. In some embodiments, the deprotection step is carried out at about 50 °C. The deprotection time is from approximately several minutes, such as 5, 10, 15, 20, 25, 30, 45 or 60 minutes, etc., to several hours, such as 2 hours, 3 hours, 4 hours or 5 hours, etc. In some embodiments, the oligonucleotide is treated with the deprotection reagent for about 1 hour.

[0019] After deprotection, the desired product can be isolated and purified using methods known in the art for isolation and purification of oligonucleotides. Such methods include, but are not limited to, filtration and / or HPLC purification.

[0020] In another aspect, the present disclosure provides a nucleoside monomer having a triisopropylsilyl ether (TIPS)-protected 3'-hydroxyl group, such as formula (I):

Chemical formula

[0021] In the monomer of formula (I), B is a modified or unmodified nucleobase. Optionally, the nucleobase can contain one or more protecting groups. Exemplary nucleobases include adenine, guanine, cytosine, uracil, thymine, inosine, xanthine, hypoxanthine, nebularine, isoguanosine, tubercidin, as well as substituted or modified analogs of adenine, cytosine, and uracil, such as 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl, and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkylcytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N 4-Acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine or O-alkylated bases are included, but not limited thereto. Further purines and pyrimidines include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990 and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

[0022] In some embodiments, the nucleobase is adenine, guanine, cytosine, uracil, thymine, inosine, xanthine, hypoxanthine, nebularine, isoguanosine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N 6 -(isopentyl)adenine, N 6 -(methyl)adenine, N 6 、N 6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4 -(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazol-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(Methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxo)-naphthalene, inosine, xanthine, hypoxanthine, tubercidin, toyocamycin, isoguanosine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyryl, 5-(methyl)isocarbostyryl, 3-(methyl)-7-(propynyl)isocarbostyryl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidazopyridinyl, 9-(methyl)-imidazopyridinyl, pyrrolopyridinyl, isocarbostyryl, 7-(propynyl)isocarbostyryl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthrenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N, 2 -substituted purine, N6 - Replacement purine, O 6 - It may be selected from the group consisting of replacement purine, replacement 1, 2, 4 - triazole and any O - alkylated or N - alkylated derivatives thereof. In some embodiments, the nucleobase is selected from the group consisting of adenine, guanine, cytosine and uracil.

[0023] R 1 is a hydroxyl protecting group. A protecting group conventionally used for protecting the nucleoside 5'-hydroxyl is 4,4'-dimethoxytrityl ("DMT"). However, for oligonucleotide synthesis, any hydroxyl protecting group known and used in the art can be used. Such protecting groups include, but are not limited to, monomethoxytrityl ("MMT"), 9 - fluorenylmethyl carbonate ("Fmoc"), o - nitrophenylcarbonyl, p - phenylazophenylcarbonyl, phenylcarbonyl, p - chlorophenylcarbonyl and 5'-(α - methyl - 2 - nitropiperonyl) oxycarbonyl ("MeNPOC"). Preferably, R 1 is an acid - labile hydroxyl protecting group, such as DMT or MMT. In some embodiments, R1 is DMT.

[0024] Each R 4 can be independently selected from the group consisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl, and these can be optionally substituted by, for example, one, two, three, four or more substituents independently selected. For example, each R 4 can independently be optionally substituted C1 - C6 alkyl. Exemplary alkyls for R 4 include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, 2 - methylpropyl, t - butyl and pentyl. In some embodiments, each R 4 is isopropyl.

[0025] R 3 is H or -P(NR 5 R 6 )OR 7 and can be. In some embodiments, R 3 is H. In some other embodiments, R 3 is -P(NR 5 R 6 )OR 7 . When R 3 is -P(NR 5 R 6 )OR 7 , R 5 and R 6 can be independently selected from the group consisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl and cycloalkynyl, which can each be optionally substituted by, for example, one, two, three, four or more substituents independently selected, or R 5 and R 6 can be linked to form a heterocyclyl, which can be optionally substituted by, for example, one, two, three, four or more substituents independently selected. For example, R 5 and R 6 can be independently optionally substituted C1-C6 alkyl. Exemplary alkyls for R 5 and R 6 include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, 2-methylpropyl, t-butyl and pentyl. In some embodiments, R 5 and R 6 are isopropyl.

[0026] R 7 is alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl, which can each be optionally substituted by, for example, one, two, three, four or more substituents independently selected. For example, each R 7can be independently optionally substituted C1-C6 alkyl. R 7 Exemplary alkyls for 7 include, but are not limited to, optionally substituted methyl, ethyl, propyl, isopropyl, butyl, 2-methylpropyl, t-butyl and pentyl. In some embodiments, R

[0027] In some embodiments of the monomer of formula (I), B is adenine, guanine, cytosine, thymine or uracil; R 1 is monomethoxytrityl or dimethoxytrityl; R 4 is independently optionally substituted C1-C6 alkyl; R 3 is H, and R 7 is optionally substituted C1-C6 alkyl. For example, B is adenine, guanine, cytosine, thymine or uracil; R 1 is dimethoxytrityl; R 4 is independently isopropyl; and R 3 is H.

[0028] In some embodiments of the monomer of formula (I), B is adenine, guanine, cytosine, thymine or uracil; R 1 is monomethoxytrityl or dimethoxytrityl; R 4 is independently optionally substituted C1-C6 alkyl; R 5 and R 6 are independently optionally substituted C1-C6 alkyl or R 5 and R 6 are linked to form a 4- to 8-membered heterocyclyl; and R 7 is optionally substituted C1-C6 alkyl. For example, B is adenine, guanine, cytosine, uracil or thymine; R 1 is dimethoxytrityl; R 4 , R 5 and R 6is isopropyl; and R 7 is β-cyanoethyl.

[0029] Exemplary embodiments may be illustrated by the following numbered embodiments.

[0030] Embodiment 1: A method for synthesizing an oligonucleotide having at least one nucleoside having a 3'-OH group, comprising: (i) coupling a free hydroxyl group on the nucleoside or oligonucleotide with a nucleoside phosphoramidite monomer having a triisopropylsilyl ether (TIPS)-protected 3'-hydroxyl group to form a phosphite triester intermediate; and (ii) oxidizing or sulfurizing the phosphite triester intermediate to form a protected intermediate.

[0031] Embodiment 2: The method of Embodiment 1, wherein all synthetic steps are carried out in an automated oligonucleotide synthesizer.

[0032] Embodiment 3: The method of Embodiment 1 or 2, wherein the oligonucleotide is synthesized on a large scale.

[0033] Embodiment 4: The method of any one of Embodiments 1 to 3, wherein said oxidizing is in the presence of a weak base.

[0034] Embodiment 5: The method of Embodiment 4, wherein said weak base is pyridine, lutidine, picoline or collidine.

[0035] Embodiment 6: The method of any one of Embodiments 1 to 5, wherein said oxidizing is in the presence of I2 / H2O.

[0036] Embodiment 7: The method of any one of Embodiments 1 to 6, wherein said sulfurizing is in the presence of a sulfur transfer reagent.

[0037] Embodiment 8: The method of Embodiment 7, wherein the sulfur transfer reagent is 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-3-thione (DDTT) or 3H-1,2-benzodithiol-3-one 1,1-dioxide.

[0038] Embodiment 9: The method according to any one of Embodiments 1 to 8, further comprising the step of deprotecting the protected intermediate with a base.

[0039] Embodiment 10: The method of Embodiment 9, wherein the base is ammonium hydroxide, methylamine, or a mixture of ammonium hydroxide and methylamine.

[0040] Embodiment 11: The method according to Embodiment 9 or 10, wherein the treatment with the base is carried out at room temperature or high temperature.

[0041] Embodiment 12: The method according to any one of Embodiments 9 to 11, wherein the treatment with the base is carried out at a temperature of 30°C or higher.

[0042] Embodiment 13: The method according to any one of Embodiments 9 to 12, wherein the treatment with the base lasts for at least 30 minutes.

[0043] Embodiment 14: The method according to any one of Embodiments 9 to 13, wherein the treatment with the base lasts for at least 4 hours.

[0044] Embodiment 15: The method according to any one of Embodiments 9 to 14, further comprising treating the base-treated intermediate with a deprotecting reagent effective to convert the TIPS-protected hydroxyl group to a free hydroxyl group.

[0045] Embodiment 16: The method of Embodiment 15, wherein the deprotecting reagent contains a fluoride anion.

[0046] Embodiment 17: The method according to Embodiment 15 or 16, wherein the deprotecting reagent is HF.pyridine.

[0047] Embodiment 18: The treatment with the deprotection reagent is carried out at a temperature of 30 °C or higher, and is the method according to any one of Embodiments 15 to 17.

[0048] Embodiment 19: The oligonucleotide contains about 6 to about 50 nucleotides, and is the method according to any one of Embodiments 1 to 18.

[0049] Embodiment 20: The oligonucleotide contains about 10 to about 30 nucleotides, and is the method according to any one of Embodiments 1 to 19.

[0050] Embodiment 21: Formula (I):

Chemical formula

[0051] Embodiment 22: The hydroxyl protecting group is selected from the group consisting of 4,4'-dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-fluorenylmethyl carbonate (Fmoc), o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl and 5'-(α-methyl-2-nitropiperonyl)oxycarbonyl (MeNPOC), the nucleoside monomer of Embodiment 21.

[0052] Embodiment 23: Each R 4 is independently optionally substituted C1-C6 alkyl, the nucleoside monomer of Embodiment 21 or 22.

[0053] Embodiment 24: Each R 4 is isopropyl, the nucleoside monomer of any one of Embodiments 21-23.

[0054] Embodiment 25: R 5 and R 6 are independently optionally substituted C1-C6 alkyl, the nucleoside monomer of any one of Embodiments 21-24.

[0055] Embodiment 26: R 5 and R 6 are isopropyl, the nucleoside monomer of any one of Embodiments 21-25.

[0056] Embodiment 27: R 7 is optionally substituted C1-C6 alkyl, the nucleoside monomer of any one of Embodiments 21-26.

[0057] Embodiment 28: R 7 is methyl or β-cyanoethyl, the nucleoside monomer of any one of Embodiments 21-27.

[0058] Embodiment 29: B is adenine, guanine, cytosine, thymine or uracil; R 1is monomethoxytrityl or dimethoxytrityl; R 4 is independently optionally substituted C1-C6 alkyl; R 5 and R 6 are independently optionally substituted C1-C6 alkyl or R 5 and R 6 are linked to form a 4- to 8-membered heterocyclyl; and R 7 is optionally substituted C1-C6 alkyl, a nucleoside monomer of any one of embodiments 21 to 28.

[0059] Embodiment 30: B is adenine, guanine, cytosine or uracil; R 1 is dimethoxytrityl; R 4 , R 5 and R 6 are isopropyl; and R 7 is β-cyanoethyl, a nucleoside monomer of any one of embodiments 1 to 29.

[0060] Some selected definitions For convenience, the specific terms used herein, in the examples and in the claims are summarized here. Unless otherwise specified or implicit from the context, the following terms and phrases include the meanings provided below. Unless otherwise explicitly stated or not apparent from the context, the following terms and phrases do not exclude the meaning obtained by the term or phrase in its relevant technical field. The definitions are provided to assist in the description of specific embodiments and are not intended to limit the claimed invention as the scope of the invention is limited only by the claims. Further, unless otherwise required by the context, singular terms shall include the plural and plural terms shall include the singular.

[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the present invention, the methods, devices, and materials relevant thereto are described herein.

[0062] Furthermore, the practice of the present invention can use conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art unless otherwise described. Such techniques are fully described in the literature such as “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M.J. Gait, ed., 1984); “Animal Cell Culture” (R.I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F.M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001).

[0063] When a range of values is provided, unless the context clearly indicates otherwise, each intervening value between the upper and lower limits of the range to one tenth of the unit of the lower limit and any other specified value or intervening value within the specified range is understood to be included within the scope of the invention. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges, subject to any specifically excluded limits within the specified range, and are also included within the scope of the invention. When the specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0064] In this specification, a particular range is presented with a numerical value preceded by the term "about". The term "about" is used in this specification to provide literal support for the exact number preceded by the term and for numbers close to or approximately the number preceded by the term. In determining whether a number is close to or approximately the specifically recited number, a number not described as being close or approximate may, in the context in which it is presented, be a number that provides a substantial equivalent of the specifically recited number.

[0065] As used herein, the terms "comprising" or "comprises" are used with respect to the compositions, methods, and respective components essential to the invention, and also embrace the inclusion of elements not specified, whether essential or not.

[0066] The singular terms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Further, it is noted that the claims may be drafted to exclude optional elements. Accordingly, this description is intended to serve as a basis for use of exclusive terms such as "solely", "only", etc. in connection with the description of claim elements or as a basis for use of "negative" limitations.

[0067] As used herein, the term "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA), for example, a nucleotide having a length of less than 100, 200, 300 or 400. As used herein, oligonucleotides also include dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, hexanucleotides and heptanucleotides. Further, the term "nucleotide, nucleoside, oligonucleotide or oligonucleoside" as used herein is intended to include both naturally occurring species and non-naturally occurring or modified species, as known to those skilled in the art.

[0068] The term "optionally substituted" means that a particular group or moiety is unsubstituted or substituted by one or more (usually 1, 2, 3, 4, 5 or 6 substituents) independently selected from the group of substituents described below in the definition of "substituent" or otherwise specified. The term "substituent" refers to a "substituted" group on the group being substituted at any atom of the group being substituted. Suitable substituents include, but are not limited to, halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclil, heterocyclil, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, acyloxy, cyano or ureido. Optionally, two substituents can form a ring together with the carbon to which they are attached.

[0069] As used interchangeably herein, the terms "essentially" and "substantially" mean at least about 60% or preferably at least about 70%, or at least about 80%, or at least about 90%, at least about 95%, at least about 97% or at least about 99% or more or any integer percentage from 70% to 100%. In some embodiments, the term "essentially" means at least about 90%, at least about 95%, at least about 98%, at least about 99% or more or any integer percentage from 90% to 100%. In some embodiments, the term "essentially" can include 100%.

[0070] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has distinct components and features, which can be readily separated from or combined with any of the features of some other aspects without departing from the scope or spirit of the invention. The methods described can be performed in any order of the events described or any other order that is logically possible.

[0071] The present invention is further illustrated by the following examples, which should not be construed as a further limitation. The contents of all references, pending patent applications and published patents cited throughout this application are hereby expressly incorporated herein by reference.

Examples

[0072] The following examples illustrate some embodiments and aspects of the present invention. It will be apparent to those skilled in the art that various modifications, additions, substitutions, etc. can be made without changing the spirit or scope of the present invention, and such modifications and variations are included within the scope of the present invention as defined in the following claims. The following examples are in no way intended to limit the present invention.

[0073] Example 1: Synthesis of Phosphoramidite with TIPS Protecting Group

Chem.

[0074] Compound 3: DIPEA (19.3 mL, 111 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (24.7 mL, 110.7 mmol), and N-methylimidazole (2.9 mL, 36.9 mmol) were sequentially added to a stirred solution of Compound 2 (25.93 g, 36.89 mmol) in anhydrous EtOAc (600 mL) at 0 °C. The cold bath was removed and the reaction mixture was stirred for 1 h. The reaction was quenched with a solution of triethanolamine (2.7 M, 50 mL) in MeCN / toluene and stirred for 5 min. The mixture was diluted with ethyl acetate, transferred to a separatory funnel, the layers were separated, and the organic layer was washed successively with 5% NaCl solution and brine. The organic layer was dried over Na2SO4 and evaporated to dryness. The residue was pre-adsorbed onto silica gel pretreated with triethylamine. The column was equilibrated with hexane containing 1% NEt3. The residue was purified by an ISCO automatic column using 0 - 40% EtOAc in hexane as the eluent to afford Compound 3 (26.5 g, 79%). 1 H NMR (500 MHz, CD3CN) δ 8.73 (s, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.44 - 7.41 (m, 2H), 7.36 - 7.28 (m, 7H), 6.89 - 6.85 (m, 4H), 6.06 (d, J = 5.4 Hz, 1H), 5.51 (d, J = 8.1 Hz, 1H), 4.32 - 4.23 (m, 2H), 4.11 - 4.07 (m, 1H), 3.84 - 3.67 (m, 10H), 3.67 - 3.54 (m, 3H), 3.46 (dd, J = 10.9, 3.7 Hz, 1H), 3.28 (dd, J = 11.0, 4.2 Hz, 1H), 2.57 (t, J = 6.2 Hz, 2H), 1.16 - 1.11 (m, 11H), 1.04 - 0.95 (m, 23H). 31 P NMR (202 MHz, CD3CN) δ 150.83, 150.80, 149.64, 149.61. LRMS (ESI) Calculated for C48H67N4O9PSi [M+Na] + m / z = 902.44, found 925.2.

[0075]

Chemical Structure

[0076] Compound 6: Pyridine (6.0 eq), 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (3.0 eq), and DCI (2.0 eq) were added to a stirred solution of Compound 5 (201.5 g, 1.0 eq) in anhydrous DCM (10 V). The mixture was stirred at 25 °C for 4 h. After work-up, the organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The crude reaction product was precipitated with DCM / hept to give Compound 6 (130 g, 52%). 31 P NMR (202 MHz, CDCl3) δ 150.82, 150.66. LRMS (ESI) Calculated for C56H73N7O8PSi [M+H] + m / z = 1031.49, found 1031.5.

[0077]

Chemical Structure

[0078] Compound 9: To a stirred solution of compound 8 (140 g, 1.0 eq.) in anhydrous DCM (1.4 L) was added 2 - cyanoethyl N,N,N’,N’ - tetraisopropylphosphorodiamidite (5.0 eq) and DCI (3.0 eq). The mixture was stirred at 25 °C for 12 h. The reaction was washed with 10% NaHCO3 (10 × 1000 mL) and brine (2 × 1000 mL), dried over Na2SO4 and concentrated at 35 °C to give the crude product (387 g) as a pale yellow oil. The crude product (386 g) was precipitated several times (8 times) in DCM / MTBE until compound 9 (81 g, 46%) was obtained as a white solid. 31 31P NMR (202 MHz, CDCl3) δ 150.72, 149.33. Calculated value for [M + H] for LRMS (ESI) C53H75N7O9PSi + m / z = 1012.5, found 1012.4.

[0079]

Chemical Structure

[0080] Compound 12: Pyridine (6.5 eq), 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (1.3 eq) and DCI (1.2 eq) were added to a stirred solution of Compound 11 (1.0 eq) in anhydrous DCM (8V). After stirring at 25 °C for 20 h, the mixture was washed with saturated NaHCO3 and brine. After work-up, the organic layer was concentrated to give crude Compound 12, which was purified by column using 0 - 50% EtOAc in n-heptane containing 1% pyridine as the eluent to give Compound 12 (yield: 76.6%). 31 P NMR (202 MHz, CDCl3) δ 151.96, 148.56. LRMS (ESI) Calculated for C50H71N5O9PSi [M+H] + m / z = 944.4, found 944.1.

[0081] Example 2: Synthesis of uridine having 3’-TOM and POM protecting groups

Chemical formula

[0082] Compound 14: DIPEA (1.7 mL, 9.8 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (2.2 mL, 9.81 mmol), and N-methylimidazole (0.39 mL, 4.9 mmol) were sequentially added to a stirred solution of compound 13 (3.5 g, 4.9 mmol) in anhydrous EtOAc (100 mL) at 0 °C. The cold bath was removed and the reaction mixture was stirred for 1 h. The reaction was quenched with a solution of triethanolamine (2.7 M, 11 mL) in MeCN / toluene and stirred for 5 min. The mixture was diluted with ethyl acetate, transferred to a separatory funnel, the layers were separated, and the organic layer was washed successively with 5% NaCl solution and brine. The organic layer was dried over Na2SO4 and evaporated to dryness. The residue was pre-adsorbed onto silica gel pretreated with triethylamine. The column was equilibrated with hexane containing 1% NEt3. The residue was purified by an ISCO automated column using hexane with 0 - 40% EtOAc as the eluent to give compound 14 (3.26 g, 71%). 11H NMR (400 MHz, CD3CN) δ 7.69 (dd, J = 9.7, 8.2 Hz, 1H), 7.46 (dd, J = 7.2, 1.1 Hz, 2H), 7.36 - 7.21 (m, 7H), 6.90 (dd, J = 7.6, 1.3 Hz, 4H), 6.00 - 5.96 (m, 1H), 5.43 - 5.35 (m, 1H), 5.12 - 4.96 (m, 2H), 4.56 - 4.48 (m, 1H), 4.42 - 4.36 (m, 1H), 4.33 - 4.25 (m, 1H), 3.91 - 3.58 (m, 11H), 3.47 - 3.33 (m, 2H), 2.68 - 2.61 (m, 2H), 1.25 - 0.94 (m, 36H). 31 31P NMR (162 MHz, CD3CN) δ 150.61, 150.55. LRMS (ESI) Calculated for C49H69N4O10PSi [M + H] + m / z = 932.45, found 955.5 (M + Na).

[0083]

Chemical Structure

[0084] Compound 16: DIPEA (1.1 mL, 6.2 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.4 mL, 6.2 mmol) and N-methylimidazole (0.19 mL, 2.4 mmol) were sequentially added to a stirred solution of compound 15 (1.6 g, 2.5 mmol) in anhydrous EtOAc (50 mL) at 0 °C. The cold bath was removed and the reaction mixture was stirred for 1 h. The reaction was quenched with a solution of triethanolamine (2.7 M, 6 mL) in MeCN / toluene and stirred for 5 min. The mixture was diluted with ethyl acetate, transferred to a separatory funnel, the layers were separated, and the organic layer was washed successively with 5% NaCl solution and brine. The organic layer was dried over Na2SO4 and evaporated to dryness. The residue was pre-adsorbed onto silica gel pretreated with triethylamine. The column was equilibrated with hexane containing 1% NEt3. The residue was purified by an ISCO automated column using hexane with 0 - 60% EtOAc as the eluent to afford compound 16 (1.517 g, 74%). 11H NMR (500 MHz, CD3CN) δ 7.65 - 7.59 (m, 1H), 7.46 - 7.41 (m, 1H), 7.35 - 7.21 (m, 6H), 6.93 - 6.83 (m, 3H), 5.98 - 5.91 (m, 1H), 5.46 - 5.37 (m, 1H), 5.34 (d, J = 6.5 Hz, 1H), 5.20 (d, J = 6.4 Hz, 1H), 4.61 - 4.50 (m, 1H), 4.47 - 4.38 (m, 1H), 4.21 - 4.14 (m, 1H), 3.67 - 3.57 (m, 3H), 3.40 - 3.31 (m, 2H), 2.69 - 2.59 (m, 1H), 1.19 - 1.16 (m, 6H), 1.12 (t, J = 6.4 Hz, 11H). 31 31P NMR (202 MHz, CD3CN) δ 150.84, 150.47.

[0085] Example 3: Selective Synthesis of 3'-OTIPS-Protected Nucleosides and Phosphoramidites

Chemical Structure

[0086]

Chemical Structure

[0087] [Chemical] The conversion of sugar 17 to nucleoside 23 was achieved by using N-benzoyladenine under Vorbruggen conditions followed by cleavage of the acetate group under basic conditions. The primary hydroxyl of nucleoside 23 was protected as a DMT ether to give nucleoside 5, which was subsequently converted to the corresponding phosphoramidite 6 under standard conditions.

[0088] [Chemical] Using sugar 17 as a starting material, nucleoside 24 was obtained by using a two-step sequence to introduce the guanine moiety. Protection of the nucleobase with isobutyric anhydride gave compound 25. The acetate group was cleaved under basic conditions and the primary hydroxyl group was protected as a DMT ether to give nucleoside 8. The formation of phosphoramidite 9 was achieved using 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite under standard conditions.

[0089] Example 4. siRNA Synthesis with 3'-O-Protected Nucleosides Oligonucleotide synthesis: Representative oligonucleotides were synthesized using the parameters shown in the following table. The aim of this study was to determine the optimal RNA protecting groups that are compatible with the inventors' current cleavage and deprotection methods (including long-term exposure to aqueous base) and minimize side reactions such as premature loss of protecting groups that can lead to hydrolysis / cleavage of the RNA. The synthesis conditions are shown in Tables 1 and 2, and the sequences of the oligonucleotides synthesized for these studies are summarized in Table 3.

[0090] [Table 1]

[0091] [Table 2]

[0092]

Table 3

[0093] Cleavage and Deprotection: This deprotection is used to evaluate the amount of synthesis and more specifically to identify impurities resulting from premature deprotection of the RNA protecting group. Two different procedures were used depending on the scale of synthesis (Procedure 1 for small scale and Procedure 2 for large scale). For both procedures, NH4OH, NH4OH / EtOH, MeNH2 or a mixture of ammonia / methylamine (AMA) can be used.

[0094] Procedure 1: 1. After synthesis, the plate containing the columns was placed in a cleavage chuck on a 96-deep well plate. 2. Concentrated aqueous methylamine or concentrated ammonium hydroxide solution (150 μL) was added to each column and incubated at room temperature for 30 minutes. Then the solution was completely withdrawn from the column using vacuum. 3. Repeat Step #2 once more, seal the plate and shake at RT for a defined time. 4. Samples of the crude product were diluted 100× with RODI water and analyzed using LCMS.

[0095] Procedure 2: 1. A small amount of the dried support (about 30 mg) after synthesis was placed in a 2 mL glass screw cap vial. 2. Ammonium hydroxide solution (1 mL) was added and the vial was kept at 35 °C for 15 hours (Note: At this stage, the crude product was cooled to room temperature, then the sample was aliquoted, diluted 30× with RODI water and analyzed by HPLC for initial crude analysis). 3. For the desilylation step, the crude solution was decanted and the resin was washed three times with 0.5 mL of DMSO. The vial was vortexed and then left for 2 minutes to allow all of the resin to settle. The DMSO solution was decanted and combined with the first filtrate in a 4 mL scintillation vial, which was then cooled to 0 °C using an ice bath. 4. Pyridine*HF (Sigma Aldrich, 0.75 mL) was added to the mixture (the reaction became turbid) and the vial was held at 50 °C for 1 hour. 5. The reaction was cooled to room temperature and quenched with water (2.5 mL). The vial was vortexed to dissolve all of the solid. 6. The sample was aliquoted and diluted 30× with RODI water for HPLC analysis.

[0096] Analysis of the crude oligonucleotide mixture by HPLC: A crude analysis was performed using IPRP-LCMS with the conditions shown in Table 4.

[0097]

Table 4

[0098] Results: Seven different 23-mer oligonucleotides with different RNA protecting groups were synthesized (Table 3) and subjected to various cleavage and deprotection conditions. Where applicable, initial HPLC analysis was performed prior to HF treatment to determine the stability of the various protecting groups during base treatment. For simplicity, all HPLC and MS integrations were performed only for the four target species, the fully deprotected oligos having 3'- or 2'-hydroxyl groups protected with silyl or other groups (FLP-OX - X = TBS, TOM, TIPS or pivaloyloxymethyl), the deprotected oligo (FLP-OH), the cleaved 3'-fragment and the cleaved 5'-fragment. As shown in Table 5, the silyl protecting groups (TBS and TIPS) and the TOM protecting group are unstable to long-term base treatment, although to different extents. The 23-mer containing the TIPS protected RNA gave the best overall results, with only 3% deprotected FLP and 1% cleaved hydrolysis products. The protecting group (TIPS) can be easily removed using excess HF pyridine (Figure 7), generating FLP-OH. In addition, the generation of FLP-OH and the long-term treatment of FLP-OH under basic conditions can result in various levels of strand cleavage, as shown in Table 5 and Figures 8 - 10.

[0099]

Table 5

[0100] All patents, patent applications, and publications identified are hereby expressly incorporated by reference herein to describe and disclose, for example, the methods described in such publications that may be used in conjunction with the present invention. These publications are provided only for their disclosure prior to the filing date of the present application. In this regard, nothing herein shall be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. Statements as to the date or content of these documents are based entirely on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0101] From the perspective of the above detailed description, these and other variations to the embodiments may be made. In general, in the following claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed in this specification and the claims, but rather the claims should be construed to include all possible embodiments together with the entire scope of equivalents provided by such claims. Accordingly, the claims are not limited by the present disclosure.

Claims

Claim 1 A nucleoside monomer having the structure of formula (I): 【Chemical 1】 [wherein, B is a modified or unmodified nucleobase; R １ is a hydroxyl protecting group; R ２ is -Si(R ４ ) ３ ; R ３ is H or -P(NR ５ R ６ )OR ７ ; Each R ４ is, independently, optionally substituted alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl; R ５ and R ６ are, independently, optionally substituted alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl, or R ５ and R ６ are linked to form heterocyclyl; and R ７ is alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl, optionally substituted. Claim 2 The nucleoside monomer according to claim 1, wherein the hydroxyl protecting group is selected from the group consisting of 4,4'-dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-fluorenylmethyl carbonate (Fmoc), o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, and 5'-(α-methyl-2-nitropiperonyl)oxycarbonyl (MeNPOC). Claim 3 Each R ４ is, independently, optionally substituted C １ to C ６ alkyl, the nucleoside monomer according to claim 1. Claim 4 Each R ４ is isopropyl, the nucleoside monomer according to claim 1. Claim 5 R ５ and R ６ are, independently, optionally substituted C １ -C ６ alkyl, the nucleoside monomer according to claim 1. Claim 6 R ５ and R ６ is isopropyl, the nucleoside monomer according to claim 1. Claim 7 R ７ is optionally substituted C １ to C ６ alkyl, the nucleoside monomer according to claim 1. Claim 8 R ７ The nucleoside monomer according to claim 1, wherein R is methyl or β-cyanoethyl. Claim 9 B is adenine, guanine, cytosine, thymine or uracil; R １ is monomethoxytrityl or dimethoxytrityl; R ４ is independently optionally substituted C １ to C ６ alkyl; R ５ and R ６ are independently optionally substituted C １ to C ６ alkyl, or R ５ and R ６ are linked to form a 4- to 8-membered heterocyclyl; and R ７ is optionally substituted C １ to C ６ alkyl, the nucleoside monomer according to claim 1. Claim 10 B is adenine, guanine, cytosine or uracil; R １ is dimethoxytrityl; R ４ , R ５ and R ６ are isopropyl; and R ７ is β-cyanoethyl, the nucleoside monomer according to claim 9.

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