Synthesis of 3'-RNA oligonucleotides

Abstract

The present disclosure relates to monomers and methods for synthesizing oligonucleotides containing at least one nucleoside containing a 3'-hydroxyl group.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0002] The present invention relates generally to nucleic acid chemistry and the chemical synthesis of oligonucleotides. More specifically, the present invention relates to monomers and methods for synthesizing oligonucleotides containing at least one nucleoside that contains a 3'-hydroxyl group. [Background technology]

[0003] Modified oligonucleotides are of great value in molecular biology research and therapeutic applications. Chemical synthesis of modified oligonucleotides is routine, but the ease and yield of many modified oligonucleotides is low. For example, commonly used protective groups are unstable to the conditions used to deprotect chemically synthesized oligonucleotides. This is particularly problematic when preparing oligonucleotides that contain at least one nucleoside that contains a 3'-hydroxyl group. Therefore, there remains a need in the art for monomers and methods for preparing such oligonucleotides. The present disclosure at least partially addresses this need. 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 oligonucleotide has at least one, e.g., two, three, four or more, nucleosides having a 3'-hydroxyl group. In general, the methods include 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 of defined length and sequence can be prepared by the present methods. For example, oligonucleotides containing from about 6 to about 50 nucleotides can be prepared using the methods and monomers described herein. In some embodiments, the oligonucleotides contain from about 10 to about 30 nucleotides.

[0006] In another aspect, the disclosure provides monomers, such as nucleoside phosphoramidite monomers having a triisopropylsilyl ether protected 3'-hydroxyl group. Generally, the monomers have the formula (I): [ka] It is of the following.

[0007] In formula (I), B is a modified or unmodified nucleobase; R 1 is an acid labile hydroxyl protecting group; R 2 is -Si(R 4 )3;R 3 is -P(NR 5 R 6 ) OR 7 and each R 4is independently an optionally substituted alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or cycloalkynyl; R 5 and R 6 is independently an optionally substituted alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or cycloalkynyl, or R 5 and R 6 are joined 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 is isopropyl; and R 7 is β-cyanoethyl.

[0009] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0010] [Figure 1] HPLC trace of sequence 1 with U-2'-OTBS at N17 (aUfcaaAf(U-2'-OTBS)CfAfcuuuAfuUfgaguuuc, SEQ ID NO: 1) after deprotection with ammonium hydroxide in ethanol, showing the production of FLP-2'-OTBS, FLP-OH and truncation (16mer). [Diagram 2]PLC trace of sequence 2 with U-3'-OTBS at N17 (aUfcaaAf(U-3'-OTBS)CfAfcuuuAfuUfgaguuuc, SEQ ID NO: 2) after deprotection with ammonium hydroxide in ethanol, showing the production of FLP-3'-OTBS, FLP-OH and cleavage (16mer). [Diagram 3] HPLC trace of sequence 3 bearing G-3'-OTBS at N17 (aUfcaaAf(G-3'-OTBS)CfAfcuuuAfuUfgaguuuc, SEQ ID NO: 3) after deprotection with ammonium hydroxide in ethanol, showing the production of FLP-3'-OTBS, FLP-OH and cleavage (16mer). [Figure 4] HPLC trace of sequence 4 (aUfcaaAf(U-2'-OTOM)CfAfcuuuAfuUfgaguuuc, sequence number 4) bearing U-2'-OTOM at N17 position after deprotection with ammonium hydroxide in ethanol, showing the production of FLP-2'-OTOM, FLP-OH and truncation (16mer). [Diagram 5] HPLC trace of sequence 5 (aUfcaaAf(U-3'-OTOM)CfAfcuuuAfuUfgaguuuc, sequence number 5) bearing U-3'-OTOM at N17 position after deprotection with ammonium hydroxide in ethanol, showing the production of FLP-3'-OTOM, FLP-OH and truncation (16mer). [Figure 6] HPLC trace of sequence 6 bearing U-3'-OTIPS at N17 (aUfcaaAf(U-3'-OTIPS)CfAfcuuuAfuUfgaguuuc, SEQ ID NO:6) after deprotection with ammonium hydroxide in ethanol, showing the production of FLP-3'-OTIPS, FLP-OH and truncation (16mer). [Figure 7]HPLC trace of sequence 6 (aUfcaaAf(U-3'-OTIPS)CfAfcuuuAfuUfgaguuuc, SEQ ID NO:6) with U-3'-OTIPS at N17 after deprotection with ammonium hydroxide in ethanol and HF / pyridine, showing the generation of FLP-OH. The 3'-OTPS protecting group in RNA can be efficiently cleaved using HF / pyridine treatment. [Figure 8] 1 shows the deconvoluted mass spectrum of sequence 8 (asCfsguuu(U2p)caaagcAfcUfuuauusgsa, SEQ ID NO: 8) deprotected overnight at room temperature with concentrated aqueous ammonium hydroxide. The main peak corresponds to the desired FLP (sequence 8) and the 3'-fragment (sequence 9 (caaagcAfcUfuuauusgsa, SEQ ID NO: 9)). Approximately 14% of the FLP still retains a single N-2-isobutyryl protecting group (M=7663). [Figure 9] 1 shows the deconvoluted mass spectrum of sequence 8 (asCfsguuu(U2p)caaagcAfcUfuuauusgsa, SEQ ID NO: 8) deprotected with concentrated aqueous methylamine at room temperature overnight for 2 hours. The main peak corresponds to the desired FLP (sequence 8) and the 3'-fragment (sequence 9 (caaagcAfcUfuuauusgsa, SEQ ID NO: 9)). [Figure 10] 1 shows the deconvoluted mass spectrum of sequence 8 (asCfsguuu(U2p)caaagcAfcUfuuauusgsa, SEQ ID NO: 8) deprotected overnight at room temperature with concentrated aqueous methylamine. The main peak corresponds to the desired FLP (sequence 8), the 3'-fragment (sequence 9 (caaagcAfcUfuuauusgsa, SEQ ID NO: 9)) and the 5'-fragment (sequence 10, asCfsguuu(U2p)P, SEQ ID NO: 10)). [Figure 11] 1 shows structures of some exemplary 3'-triisopropylsilyl ether (3'-TIPS) nucleoside monomers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 comprising a triisopropylsilyl ether (TIPS)-protected 3'-hydroxyl group is coupled to a free hydroxyl, such as the 5'-OH, 3'-OH or 2'-OH, preferably the 5'-OH, of a nucleoside or oligonucleotide.

[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 equipment known to those skilled in the art. For example, glass reactors such as flasks can be used appropriately. Preferably, solid-phase synthesis procedures and solid supports such as controlled pore glass are used. Even more preferably, the method 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 the μmol scale or on the mg scale.

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

[0015] The oxidation step can be carried out by contacting the phosphite triester intermediate with an oxidation reagent for a time sufficient to result in the formation of a 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, it is a mixture of an aprotic solvent and a protic or basic solvent. A preferred solvent mixture includes a mixture 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] Sulfurization (oxidation using sulfur transfer reagent) can be carried out by contacting the phosphite triester intermediate with a sulfur transfer reagent for a sufficient time to result in the formation of phosphorothioate functional groups.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-(dimethylaminomethylidene)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 synthesis, the oligonucleotides may be deprotected, for example, using methods and reagents to remove any protecting groups on the oligonucleotides to obtain the desired product. Thus, in some embodiments, the method further comprises treating the synthesized oligonucleotides with a base to remove any non-TIPS protecting groups on the oligonucleotides. 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. Treating with a base may be suitably carried out at room temperature or at an elevated temperature. "Room temperature" includes an ambient temperature of about 20°C to about 30°C. "Elevated temperature" includes temperatures above 30°C. For example, elevated temperature may be a temperature of about 32°C to about 65°C. In some embodiments, treating with a base is at about 35°C. Treatment times range from about a few minutes, such as 5, 10, 15, 20, 25, 30, 45, or 60 minutes, to several hours, such as 2, 3, 4, 5, 10, 15, 24, or more hours. In some embodiments, the base treatment is for about 15 hours. In some embodiments, the base treatment is for about 15 hours at about 35°C.

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

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

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

[0021] In the monomer of formula (I), B is a modified or unmodified nucleobase.Optionally, the nucleobase can include one or more protecting groups.Exemplary nucleobases include adenine, guanine, cytosine, uracil, thymine, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, and substituted or modified analogs of adenine guanine, 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-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyl uracil, 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, uracil, substituted 1,2,4-triazole, 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 4Examples of purines and pyrimidines include, but are not limited to, -acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine or O-alkylated bases. Additional purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JI, 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, nubularine, isoguanisine, 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, cytosine, 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-(guanidinium alkyl)uracil, 5-(1,3-diazole-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-(methoxycarbonylmethyl)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 Douracil, 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 Uracil, 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-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinium alkyl) 7-(guanidinium alkyl hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinium alkyl hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinium alkyl hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deazainosinyl indolyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl , 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, 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 purines, N6 -substituted purines, O 6 -substituted purines, substituted 1,2,4-triazoles, and any O- 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. The protecting group conventionally used for protection of the nucleoside 5'-hydroxyl is 4,4'-dimethoxytrityl ("DMT"). However, any hydroxyl protecting group known and used in the art for oligonucleotide synthesis can be used. Such protecting groups include, but are not limited to, monomethoxytrityl ("MMT"), 9-fluorenylmethylcarbonate ("Fmoc"), o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, and 5'-(α-methyl-2-nitropiperonyl)oxycarbonyl ("MeNPOC"). Preferably, R is a hydroxyl protecting group. 1 is an acid labile hydroxyl protecting group, such as DMT or MMT. In some embodiments, R 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, each of which can be optionally substituted, for example, with one, two, three, four or more independently selected substituents. For example, each R 4 R can be, independently, an optionally substituted C1-C6 alkyl. 4 Exemplary alkyl for include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, 2-methylpropyl, t-butyl, and pentyl. 4 is isopropyl.

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

[0026] R 7 is alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or cycloalkynyl, each of which can be optionally substituted, e.g., with 1, 2, 3, 4 or more independently selected substituents. For example, each R 7R can be, independently, an optionally substituted C1-C6 alkyl. 7 Exemplary alkyl for includes, but is not limited to, optionally substituted methyl, ethyl, propyl, isopropyl, butyl, 2-methylpropyl, t-butyl, and pentyl. In some embodiments, R 7 is β-cyanoethyl.

[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 an optionally substituted C1-C6 alkyl; R 3 is H, and R 7 is an 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 an optionally substituted C1-C6 alkyl; R 5 and R 6 is independently an optionally substituted C1-C6 alkyl, or R 5 and R 5 are joined to form a 4- to 8-membered heterocyclyl; and R 7 is an 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 described 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 a 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 synthesis steps are carried out on 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 1-3, wherein the oxidizing is in the presence of a weak base.

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

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

[0036] Embodiment 7: The method of any one of embodiments 1-6, wherein the 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-(dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione (DDTT) or 3H-1,2-benzodithiol-3-one 1,1-dioxide.

[0038] Embodiment 9: The method of 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 of embodiment 9 or 10, wherein said treating with base is at room temperature or at an elevated temperature.

[0041] Embodiment 12: The method of any one of embodiments 9-11, wherein the treating with base is at a temperature of 30° C. or greater.

[0042] Embodiment 13: The method of any one of embodiments 9-12, wherein the treating with base is for at least 30 minutes.

[0043] Embodiment 14: The method of any one of embodiments 9-13, wherein the treating with base is for at least 4 hours.

[0044] Embodiment 15: The method of any one of embodiments 9-14, 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 comprises a fluoride anion.

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

[0047] Embodiment 18: The method of any one of embodiments 15-17, wherein the treating with a deprotecting reagent is at a temperature of 30° C. or greater.

[0048] Embodiment 19: The method of any one of embodiments 1 to 18, wherein the oligonucleotide comprises from about 6 to about 50 nucleotides.

[0049] Embodiment 20: The method of any one of embodiments 1 to 19, wherein the oligonucleotide comprises from about 10 to about 30 nucleotides.

[0050] Embodiment 21: Formula (I): [ka] where 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 and each R 4 is independently an optionally substituted alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or cycloalkynyl; R 5 and R 6 is independently an optionally substituted alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or cycloalkynyl, or R 5 and R 6 are joined to form a heterocyclyl; and R 7 is optionally substituted alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, or cycloalkynyl. A nucleoside monomer having the structure:

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

[0052] Embodiment 23: Each R 4 is independently an optionally substituted C1-C6 alkyl.

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

[0054] Embodiment 25: R 5 and R 6 The nucleoside monomer of any one of embodiments 21-24, wherein is independently an optionally substituted C1-C6 alkyl.

[0055] Embodiment 26:R 5 and R 6 The nucleoside monomer of any one of embodiments 21-25, wherein is isopropyl.

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

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

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

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

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

[0061] Unless otherwise defined, 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 can be used in the practice or testing of the present invention, methods, devices, and materials related thereto are described herein.

[0062] Furthermore, the practice of the present invention may employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (FMA Usubel 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] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is included within the scope of the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the scope of the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0064] Certain ranges are presented herein with numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that the term precedes and a number that is close to or approximately the number that the term precedes. In determining whether a number is close to or approximately a specifically recited number, the near or approximate unrecited number may be a number that, in the context in which it is presented, provides a substantial equivalent to the specifically recited number.

[0065] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods and their respective components that are essential to the present invention, but also embrace the inclusion of unspecified elements, whether essential or not.

[0066] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude optional elements. Thus, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely," "only," and the like, or for use of a "negative" limitation in connection with the recitation of claim elements.

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

[0068] The term "optionally substituted" means that the specified group or moiety is unsubstituted or substituted with one or more (usually 1, 2, 3, 4, 5 or 6 substituents) independently selected from the group of substituents described below or otherwise defined in the definition of "substituent". The term "substituent" refers to a "substituted" group on the substituted group at any atom of the substituted group. Suitable substituents include, but are not limited to, halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, 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 between 70% and 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 between 90% and 100%. In some embodiments, the term "essentially" can include 100%.

[0070] As will be apparent to one of skill in the art upon reading this disclosure, each of the individual aspects depicted and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the invention. Any method described can be carried out in the order of events depicted or in any other order which is logically possible.

[0071] This invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents cited throughout this application are hereby expressly incorporated 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. may 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 do not limit the present invention in any way.

[0073] Example 1: Synthesis of phosphoramidites bearing TIPS protecting groups [ka] Compound 2: To a stirred solution of 5'-ODMTr uridine 1 (50 g, 91.48 mmol) in anhydrous pyridine (450 mL) were added sequentially imidazole (24.91 g, 365.92 mmol) and chloro(triisopropyl)silane (47.0 mL, 220 mmol). After stirring at 50 °C for 24 h, the volatiles were removed under reduced pressure. The residue was combined with saturated aqueous NaHCO3 (400 mL) and EtOAc (500 mL) and stirred for 5 min. The mixture was transferred to a separatory funnel, the layers were separated, and the organic layer was washed with saturated aqueous NaHCO3 and brine. The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by ISCO automated column. Dissolved in minimal DCM and loaded onto a 120 g silica gel column using 0-30% EtOAc in hexane as eluent to give compound 2 (26.1 g, 41%). 1 H NMR(500MHz,acetonitrile-d3)δ 7.70(d,J=8.2Hz,1H),7.45-7.37(m,2H),7.35-7.19(m,8H),6.93-6.84(m,4H),5.82(d,J=3.9Hz,1H),5.37(d,J=8.1Hz,1H),4.4 2(t,J=5.4Hz,1H),4.17(td,J=5.3,3.9Hz,1H),3.77(s,6H),3.49(dd,J=10.9,2.7Hz,1H),3.31-3.23(m,2H),1.06-0.90(m,22H). LRMS(ESI) Calculated for C39H50N2O8Si [M+H] + m / z=703.34, actual value 703.4.

[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 added sequentially 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 sequentially 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 ISCO automated column using 0-40% EtOAc in hexanes as eluent to give compound 3 (26.5 g, 79%). 1 H NMR(500MHz,CD3CN)δ 8.73(s,1H),7.59(d,J=8.1Hz,1H),7.44-7.41(m,2H),7.36-7.28(m,7H),6.89-6.85 (m,4H),6.06(d,J=5.4Hz,1H),5.51(d,J=8.1Hz,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=10.9,3.7Hz,1H),3.28(dd ,J=11.0,4.2Hz,1H),2.57(t,J=6.2Hz,2H),1.16-1.11(m,11H),1.04-0.95(m,23H). 31 P NMR(202MHz,CD3CN)δ 150.83,150.80,149.64,149.61. LRMS(ESI) Calculated for C48H67N4O9PSi [M+Na] + m / z=902.44, actual value 925.2.

[0075] [ka] Compound 5: To a stirred solution of compound 4 (2.0 g, 3.0 mmol, 1 eq.) in anhydrous pyridine (15.0 mL) was added imidazole (1.62 g, 23.7 mmol, 8 eq.) and chloro(triisopropyl)silane (1.52 mL, 7.12 mol, 2.4 eq.) sequentially. After stirring at 50 °C for 24 h, saturated aqueous NaHCO3 (50 mL) and Et2O were added, the resulting mixture was transferred to a separatory funnel, the layers were separated, and the aqueous layer was extracted with Et2O (50 mL x 2). The combined organic layers were dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by an ISCO automated column using 0-40% EtOAc in hexanes as eluent to give compound 5 (0.78 mg, 31%). 1 H NMR(500MHz,DMSO-d6)δ 11.22(s,1H),8.64(d,J=8.0Hz,2H),8.08-8.02(m,2H),7.67-7.61(m,1H),7.57-7.52(m ,2H),7.39-7.32(m,2H),7.28-7.16(m,8H),6.88-6.80(m,4H),6.06(d,J=5.5Hz,1H),5. 50(d,J=6.2Hz,1H),4.96(q,J=5.6Hz,1H),4.65-4.59(m,1H),4.15(q,J=4.6Hz,1H),3.7 2(s,6H),3.41(dd,J=10.5,4.6Hz,1H),3.20(dd,J=10.5,5.1Hz,1H),1.14-0.93(m,24H). 13 C NMR(101MHz,DMSO)δ 166.15,158.59,152.51,151.85,151.01,145.26,144.51,135.91,135.88,133.87,132.92,130.16,128.98,128.94,128.22,1 28.09,127.15,126.62,113.60,113.58,88.78,86.22,84.61,72.88,72.65,63.83,55.51,40.03,18.34,18.11,18.01,12.27. LRMS(ESI) Calculated for C47H56N5O7Si [M+H] + m / z=830.39, actual value 830.4.

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

[0077] [ka] Compound 8: To a stirred solution of compound 7 (20.0 g, 30.5 mmol) in anhydrous pyridine (150.0 mL) were added sequentially imidazole (16.61 g, 0.24 mol) and chloro(triisopropyl)silane (26.1 mL, 0.12 mol). After stirring at 50 °C for 24 h, the volatiles were removed under reduced pressure. The residue was combined with saturated aqueous NaHCO3 (100 mL) and EtOAc (500 mL) and stirred for 10 min. The mixture was transferred to a separatory funnel, the layers were separated, and the organic layer was washed with saturated aqueous NaHCO3 (50 mL) and brine (50 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by an ISCO automated column using 0-70% EtOAc in hexane as eluent to give compound 8 (9.85 g, 40%). The column was equilibrated with hexane containing 1% NEt3. 1H NMR(400MHz,CDCl3)δ 11.96(s,1H),7.88-7.81(m,1H),7.54-7.48(m,1H),7.42-7.36(m,1H),7.30-7.18(m,1H),6.85- 6.76(m,1H),5.70(d,J=6.3Hz,1H),4.95-4.88(m,1H),4.62-4.57(m,1H),4.54-4.50(m,1H),4.1 7-4.13(m,1H),3.77(d,J=3.5Hz,9H),3.59-3.52(m,1H),3.27-3.16(m,1H),3.14-3.05(m,1H),1 .72-1.62(m,1H),1.33-1.20(m,1H),1.01-0.88(m,1H),0.72(d,J=6.9Hz,4H),0.60-0.45(m,3H). LRMS(ESI) Calculated for C44H57N5O8Si [M+H] + m / z=811.40, measured value 812.2.

[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 x 1000 mL) and brine (2 x 1000 mL), dried over Na2SO4, and then 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 P NMR(202MHz,CDCl3)δ 150.72,149.33. LRMS(ESI) Calculated for C53H75N7O9PSi [M+H] + m / z=1012.5, actual value 1012.4.

[0079] [ka] Compound 11: To a stirred solution of compound 10 (0.5 g, 0.85 mmol, 1 eq.) in anhydrous CHCl (2.8 mL) was added anhydrous diisopropylamine (0.72 mL, 5.1 mmol, 6 eq.) and chloro(triisopropyl)silane (0.55 mL, 2.5 mmol, 3 eq.) sequentially. After stirring at room temperature for 4 days, methanol (3 mL) was added and the resulting solution was stirred for 15 min. The mixture was diluted with DCM (10 mL) and the layers were separated. The organic layer was washed with water (10 mL x 2), dried over NaSO, filtered and evaporated to dryness. The residue was purified by an ISCO automated column (column equilibrated with hexane containing 1% NEt) using 0-60% EtOAc in hexane as eluent to give compound 11 (287 mg, 45%). 1 H NMR(500MHz,DMSO-d6)δ 10.89(s,1H),8.36(d,J=7.5Hz,1H),7.40-7.18(m,10H),7.04(d,J=7.5Hz,1H),6.8 9(dq,J=8.3,3.2Hz,4H),5.84(d,J=2.5Hz,1H),5.47(d,J=5.7Hz,1H),4.28(dd,J=7. 1,4.8Hz,1H),4.12-4.08(m,1H),4.07-4.04(m,1H),3.75(d,J=0.8Hz,6H),3.54(dd ,J=11.0,2.9Hz,1H),3.24(dd,J=11.0,3.8Hz,1H),2.10(s,3H),1.05-0.82(m,24H). 13 C NMR(101MHz,DMSO)δ 170.97,170.30,162.35,158.24,158.23,154.47,144.69,144.19,134.98,134.93,129.81,129.78,127.82,126.91,113 .17,113.13,95.34,91.03,86.20,82.34,74.15,70.29,61.98,59.73,55.01,39.52,24.34,20.74,17.74,14.07,11.63. LRMS(ESI) Calculated for C41H53N3O8SiNa [M+Na] + m / z=766.35, actual value 766.3.

[0080] Compound 12: To a stirred solution of compound 11 (1.0 eq.) in anhydrous DCM (8 V) was added pyridine (6.5 eq), 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (1.3 eq) and DCI (1.2 eq). 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 eluent to give compound 12 (yield: 76.6%). 31 P NMR(202MHz,CDCl3)δ 151.96,148.56. LRMS(ESI) Calculated for C50H71N5O9PSi [M+H] + m / z=944.4, actual value 944.1.

[0081] Example 2: Synthesis of uridine with 3'-TOM and POM protecting groups [ka] Compound 13: A solution containing compound 2 (7 g, 13.1 mmol) and N-ethyl-N-isopropyl-propan-2-amine (8.01 mL, 46.01 mmol) in THF (50 mL) was treated with dibutyl(dichloro)stannane (4.58 g, 14.46 mmol, 3.36 mL) and stirred at room temperature for 1 h. The reaction mixture was heated to 66° C., then chloromethoxy(triisopropyl)silane (4.13 g, 15.77 mmol, 4.31 mL) was added and stirred at 66° C. for 40 min. The reaction mixture was cooled to room temperature and the volatiles were removed under reduced pressure. The crude residue was partitioned between DCM and saturated NaHCO3 solution, the layers were separated and the organic layer was washed with aqueous NaHCO3, brine and dried over Na2SO4. The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by ISCO automated column using 0-40% EtOAc in hexanes as eluent to give compound 13 (3.48 g, 37%). 1H NMR(400MHz,CDCl3)δ 7.77(d,J=8.2Hz,1H),7.39-7.22(m,1H),6.87-6.80(m,1H),5.96(d,J=4.4Hz,1H),5.39(d,J=8.1Hz,1H),5.06(d,J=4.9Hz,1H),4.90 (d,J=4.9Hz,1H),4.35-4.22(m,1H),3.80(s,6H),3.59-3.51(m,1H),3.43-3.36(m,1H),2.05(s,2H),1.60(s,2H),1.13-1.01(m,2H). LRMS(ESI) Calculated for C40H52N2O9Si [M+Na] + m / z=732.34, actual value 755.4.

[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 added sequentially 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 sequentially 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 ISCO automated column using 0-40% EtOAc in hexanes as eluent to give compound 14 (3.26 g, 71%). 1H NMR(400MHz,CD3CN)δ 7.69(dd,J=9.7,8.2Hz,1H),7.46(dd,J=7.2,1.1Hz,2H),7.36-7.21(m,7H ),6.90(dd,J=7.6,1.3Hz,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),3.9 1-3.58(m,11H),3.47-3.33(m,2H),2.68-2.61(m,2H),1.25-0.94(m,36H). 31 P NMR(162MHz,CD3CN)δ 150.61,150.55. LRMS(ESI) Calculated for C49H69N4O10PSi [M+H] + m / z=932.45, measured value 955.5 (M+Na).

[0083] [ka] Compound 15: Compound 2 (2 g, 3.76 mmol) was added to an empty microwave tube, followed by dibutyl(oxo)tin (1.22 g, 4.88 mmol, 769.23 uL) and tetrabutylammonium bromide (1.57 g, 4.88 mmol). The tube was closed with a rubber septum and the system was flushed with Ar for 5 min. 1,2-DCE (10 mL) was added and the resulting suspension was stirred for 1 min before chloromethyl pivalate (1.41 g, 9.39 mmol, 1.35 mL) was added. The septum was quickly replaced with a microwave tube cap and the tube was heated to 75° C. in a microwave at 300 W for 2.5 h. The reaction was carried out two more times with the same amounts of reagents to give a total of 6 g of compound 2. The three combined crude reaction mixtures were combined and evaporated to dryness under reduced pressure. Samples were pre-adsorbed onto silica pretreated with triethylamine. The residue was purified by an ISCO automated column (silica pretreated with NEt3) using 0-40% EtOAc in hexanes as eluent to give compound 15 (1.68 g, 23%). 1H NMR(400MHz,CD3OD)δ 7.87(d,J=8.1Hz,1H),7.48-7.36(m,3H),7.35-7.22(m,4H),6.94-6.84(m,2H),5.89(d,J=4.7Hz,1H),5.41(d,J=6.5Hz) ,1H),5.37-5.27(m,1H),4.50-4.38(m,2H),4.23-4.17(m,1H),3.54-3.39(m,1H),3.35-3.28(m,1H),1.20-1.08(m,4H). LRMS(ESI) Calculated for C36H40N2O10 [M+H] + m / z=660.27, actual value 661.7.

[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 added sequentially 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 sequentially 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 ISCO automated column using 0-60% EtOAc in hexanes as eluent to give compound 16 (1.517 g, 74%). 1H NMR(500MHz,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=6.5Hz,1H),5.20(d,J=6.4Hz,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=6.4Hz,11H). 31 P NMR(202MHz,CD3CN)δ 150.84,150.47.

[0085] Example 3: Selective synthesis of 3'-OTIPS protected nucleosides and phosphoramidites [ka] The synthesis was initiated by the introduction of uracil at the anomeric position of sugar 17 under Vorbrüggen conditions. The resulting compound 18 was treated with potassium carbonate to cleave the acetate group to give nucleoside 19, which was protected at the 5'-O position with DMTCl to give nucleoside 2. Formation of phosphoramidite 4 was achieved using 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite under standard conditions.

[0086] [ka] Starting from nucleoside 18, the uracil nucleobase was converted to cytosine in a two-step triazolation / ammonolysis sequence to give nucleoside 20. Protection of the primary hydroxyl group with DMTCl and selective introduction of a benzoate group at the nucleobase afforded nucleoside 21. Formation of phosphoramidite 22 was achieved using 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite under standard conditions.

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

[0088] [ka] Using sugar 17 as the starting material, a two-step sequence was used to introduce the guanine moiety to give nucleoside 24. Protection of the nucleobase with isobutyric anhydride gave compound 25. Cleavage of the acetate group under basic conditions and protection of the primary hydroxyl group as the DMT ether gave nucleoside 8. 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: The synthesis of representative oligonucleotides was carried out using the parameters shown in the table below. The goal of this study was to determine optimal RNA protecting groups that would be compatible with our current cleavage and deprotection methods (including prolonged exposure to aqueous base) and minimize side reactions such as premature loss of the protecting group that can result in RNA hydrolysis / cleavage. The conditions of the synthesis 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 assess the amount of synthesis, more specifically to identify impurities resulting from premature deprotection of the RNA protecting groups. 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 ammonia / methylamine mixtures (AMA) can be used.

[0094] Step 1: 1. After synthesis, the plate containing the columns was placed on a cutting chuck over 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, after which the solution was completely removed from the columns using a vacuum. 3. Step #2 was repeated once more and the plate was sealed and shaken at RT for the specified time. 4. A sample of the crude product was diluted 100x with RODI water and analyzed using LCMS.

[0095] Step 2: 1. A small amount of dry post-synthesis support (approximately 30 mg) 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 and then the sample was aliquoted and diluted 30x with RODI water before being analysed by HPLC for initial crude analysis). 3. For the desilylation step, the crude solution was decanted and the resin was washed 3 times with 0.5 mL 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 kept 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 solids. 6. Samples were aliquoted and diluted 30x with RODI water for HPLC analysis.

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

[0097] [Table 4]

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

[0099] [Table 5]

[0100] All patents, patent applications, and publications identified are expressly incorporated herein by reference to, for example, describe and disclose methods described in such publications that may be used in conjunction with the present invention. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior invention or for any other reason. Any statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0101] These and other variations to the embodiments can be made in light of the above detailed description. In general, the terms used in the following claims should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Thus, the claims are not limited by this disclosure.

Claims

1. 1. A method for synthesizing an oligonucleotide having at least one nucleoside having a 3′-OH group, comprising: (i) coupling a free 5'-hydroxyl group on a nucleoside or oligonucleotide with a nucleoside phosphoramidite monomer having (ia) a 2'-phosphoramidite group and (ib) a triisopropylsilyl ether (TIPS) protected 3'-hydroxyl group to form a phosphite triester intermediate; (ii) oxidizing or sulfurizing the phosphite triester intermediate to form a protected intermediate; and (iii) deprotecting the protected intermediate with a base, the treatment with the base being carried out at a temperature of 30° C. or higher. Including, The nucleoside phosphoramidite monomer has the formula (I): 【Chemistry 1】 The structure is During the ceremony, B is a modified or unmodified nucleobase; R 1 is a hydroxyl protecting group; R 2 is —Si(R 4 ) 3 ; R 3 is —P(NR 5 R 6 )OR 7 ; Each R 4 is isopropyl; R 5 and R 6 are independently optionally substituted alkyl, or R 5 and R 6 combine to form a heterocyclyl; and The process wherein R 7 is β-cyanoethyl.

2. 10. The method of claim 1, wherein all synthesis steps are carried out on an automated oligonucleotide synthesizer.

3. The method of claim 1 , wherein the oligonucleotide is synthesized on a mg scale.

4. 10. The method of claim 1, wherein the oxidizing is in the presence of a weak base.

5. 5. The method of claim 4, wherein the weak base is pyridine, lutidine, picoline or collidine.

6. The oxidizing step comprises: ２ / H ２ The method of claim 1 in the presence of O.

7. 10. The method of claim 1, wherein the sulfurizing is in the presence of a sulfur transfer reagent.

8. 8. The method of claim 7, wherein the sulfur transfer reagent is 3-(dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione (DDTT) or 3H-1,2-benzodithiol-3-one 1,1-dioxide.

9. 10. The method of claim 1, wherein the base is ammonium hydroxide, methylamine, or a mixture of ammonium hydroxide and methylamine.

10. 2. The method of claim 1, wherein the treatment with base is carried out at a temperature of from 32° C. to 65° C.

11. 11. The method of claim 10, wherein the treatment with base is carried out at a temperature of 35°C.

12. 10. The method of claim 1, wherein the treatment with base is for at least 30 minutes.

13. 13. The method of claim 12, wherein the treatment with base is for at least 4 hours.

14. 10. The method of claim 1, further comprising treating the base-treated intermediate with a deprotecting reagent effective to convert the TIPS-protected hydroxyl group to a free hydroxyl group.

15. The method of claim 14 , wherein the deprotection reagent comprises a fluoride anion.

16. 15. The method of claim 14, wherein the deprotection reagent is HF.pyridine.

17. 15. The method of claim 14, wherein the treating with the deprotecting reagent is carried out at a temperature of 30° C. or higher.

18. The method of claim 1, wherein the oligonucleotide comprises 6 to 50 nucleotides.

19. The method of claim 18, wherein the oligonucleotide comprises 10 to 30 nucleotides.

20. The method of claim 1, wherein in the nucleoside phosphoramidite monomer of formula (I), the hydroxyl protecting group is selected from the group consisting of 4,4'-dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-fluorenylmethylcarbonate (Fmoc), o-nitrophenylcarbonyl, p-phenylazophenylcarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl and 5'-(α-methyl-2-nitropiperonyl)oxycarbonyl (MeNPOC).

21. The nucleoside phosphoramidite monomer of formula (I), wherein R ５ and R ６ is independently an optionally substituted C １ ~C ６ The method of claim 1 , wherein the alkyl group is an alkyl group.

22. R ５ and R ６ The method of claim 21 , wherein is isopropyl.

23. The nucleoside phosphoramidite monomer of formula (I), wherein B is adenine, guanine, cytosine, thymine or uracil; R １ is monomethoxytrityl or dimethoxytrityl; R ５ and R ６ is independently an optionally substituted C １ ~C ６ alkyl or R ５ and R ６ The method of claim 1, wherein:

24. B is adenine, guanine, cytosine or uracil; R １ is dimethoxytrityl; R ５ and R ６ The method of claim 23, wherein is isopropyl.

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