2'-oh deprotection process of ribonucleic acid

Abstract

2'-OH Deprotection process for preparing an RNA using HF in the presence of a base, i.e., imidazole or pyridine. This process offers more efficient deprotection of RNA 2'-OH silyl ether protecting group (e.g., TBDMS) than the existing process which uses TBAF. Moreover, the process described herein simplifies the RNA ammonolysis and desilylation processes by enabling a one-pot deprotection strategy, which eliminates the need to isolate the crude intermediate from the ammonolysis step prior to the desilylation.

Description

123429-126202'-0H DEPROTECTION PROCESS OF RIBONUCLEIC ACIDRELATED APPLICATION

[0001] This application claims the benefit of the filing date, under 35 U.S.C. § 119(e), of U.S. Provisional Application No. 63 / 764,985, filed on February 28, 2025, the entire contents of which are incorporated herein by reference.FIELD OF THE INVENTION

[0002] The present invention relates to a 2'-OH deprotection process for preparing a ribonucleic acid ("RNA").BACKGROUND

[0003] RNA-based medicines have emerged as a prominent area of new drug discovery, with several RNA-based drugs approved and hundreds more under development and clinical trials.

[0004] The RNA solid phase synthetic method was modeled on the method of DNA synthesis, but the RNA synthesis is more challenging due to the 2’-hydroxyl group of the ribose. Currently, the TBDMS group is the most widely used 2’ -OH protecting group in RNA synthesis. The use of TBDMS protecting group however has the shortcomings of 1) 2’- OH TBDMS desilylation needs a large amount of tetrabutylammonium fluoride solution (TBAF); and 2) as TBAF is a strong base, the cleavage of the 3’ -5’ phosphodiester bond was detected during RNA 2’-O-TBDMS group deprotection. Accordingly, this synthesis strategy leads to low reaction yield and poor quality of the final product.

[0005] Hence, there is a need for novel reagents and robust methods for synthesizing RNAs that have high efficiency and purity.SUMMARY OF THE INVENTION

[0006] The present disclosure describes a 2'-OH deprotection process for preparing an RNA using HF in the presence of a base, / .< ., imidazole. This process offers more efficient deprotection of RNA 2’-OH silyl ether protecting group (e.g., TBDMS) than the existing process which uses TBAF. Moreover, the process described herein simplifies the RNA ammonolysis and desilylation processes by enabling a one-pot deprotection strategy, which eliminates the need to isolate the crude intermediate from the ammonolysis step prior to the desilylation.

[0007] The present disclosure is directed to a process for preparing a compound of formula (I):1MEI 52290585v.1123429-12620or a salt thereof by a deprotection process, wherein the deprotection process comprises deprotecting a compound of formula (II) with HF in the presence of a base:or a salt thereof, to form the compound of formula (I), or a salt thereof;wherein R1, R2, R2, R4, R15, q, and X are defined below.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 shows comparative HPLC traces for converting compound 1 to compound la under TBAF conditions (left) and HF / imidazole conditions (right). UV 245nm signal is used to estimate the conversion of the starting material SM (compound 1) to product P (compound la).

[0009] FIG. 2 shows comparative HPLC traces for converting compound 2 to compound 2a under TBAF conditions (top) and HF / imidazole conditions (bottom). UV 245nm signal is used to estimate the conversion of the starting material SM (compound 2) to product P (compound 2a).

[0010] FIG. 3 shows comparative HPLC traces for converting compound 3 to compound 3 a under TBAF conditions (top) and HF / imidazole conditions (bottom). UV 245nm signal is2MEI 52290585v.1123429-12620used to estimate the conversion of the starting material SM (compound 3) to product P (compound 3a).[OH] FIG. 4A is a LV-UV chromatograph showing the UV trace of a blank sample.

[0012] FIG. 4B is a LV-UV chromatograph showing the UV trace of a starting material SM (6-mer (Us)sdT, compound 4) that was cleaved and deprotected by incubating the dried solid support in a (1 : 1) solution of ammonium hydroxide 30% in H2O / 30% MeNFL in H2O (AMA) at 65 °C for 30 min.

[0013] FIG. 4C is a LV-UV chromatograph showing the UV trace of a desilylation product P (compound 4a) under a HF / imidazole condition (10 eq. HF / imidazole, rt, 15min).

[0014] FIG. 4D is a LV-UV chromatograph showing the UV trace of a desilylation product P (compound 4a) under a TBAF condition (30 eq. TBAF, rt, 24 hr).

[0015] FIG. 4E is a LV-UV chromatograph showing the UV trace of a desilylation product P (compound 4a) under a TBAF condition (30 eq. TBAF, 65 °C, 1 hr).

[0016] FIG. 5A is a LV-UV chromatograph showing the UV trace of a desilylation product P (compound 4a) under a HF / imidazole condition (15 eq. HF / imidazole, rt, 1 hr).

[0017] FIG. 5B is a LV-UV chromatograph showing UV trace of a desilylation reaction under a TBAF condition (30 eq. TBAF, rt, 24 hr).

[0018] FIG. 5C is a LV-UV chromatograph showing UV trace of a desilylation product P (compound 4a) under a TBAF condition (30 eq. TBAF, 65 °C, 1 hr).

[0019] FIG. 6A is a LV-UV chromatograph showing the UV peak of a starting material SM (compound 5).

[0020] FIG. 6B is a LV-UV chromatograph showing UV peaks of a desilylation reaction under a HF / imidazole condition (30 eq. HF / imidazole, 25 °C, 20 hr).

[0021] FIG. 6C is a LV-UV chromatograph showing UV peaks of a desilylation reaction under a TBAF condition (30 eq. TBAF, 25 °C, 20 hr).

[0022] FIG. 7A is a mass spectrum of compound 5.

[0023] FIG. 7B is a mass spectrum of compound 5a.

[0024] FIG. 8 is a synthetic scheme for preparing compound 6a.

[0025] FIG. 9A is a LV-UV chromatography showing the UV trace of a starting material SM (compound 6).

[0026] FIG. 9B is a LV-UV chromatograph showing UV trace of a desilylation reaction under a TBAF condition (10 eq. TBAF, 25 °C, 10 hr).

[0027] FIG. 9C is a LV-UV chromatograph showing UV trace of a desilylation reaction under a HF / imidazole condition (10 eq. HF / imidazole, 25 °C, 20 hr).3MEI 52290585v.1123429-12620

[0028] FIG. 10A is a mass spectrum of compound 6.

[0029] FIG. 1 OB is a mass spectrum of compound 6a.

[0030] FIG. 11 A is a LV-UV chromatograph showing UV trace of a desilylation reaction of a fully protected dimer.

[0031] FIG. 1 IB shows the structures of by-products of desilylation reaction of the fully protected dimer

[0032] FIG. 12 is a LV-UV chromatograph showing UV trace of a desilylation reaction of the Dimer-SM-CE Off.DETAILED DESCRIPTIONDefinitions

[0033] The term “nucleobase” means the heterocyclic base portion of a nucleoside.Nucleobases may be naturally occurring or may be modified. In certain embodiments, a nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a nucleobase of another nucleic acid. In particular, the nucleobase is a heterocyclic base, typically purines and pyrimidines. In addition to “unmodified” or “natural” nucleobases such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), many modified nucleobases or nucleobase mimetics known to those skilled in the art are amenable to incorporation into the compounds synthesized by the method described herein. In certain embodiments, a modified nucleobase is a nucleobase that is fairly similar in structure to the parent nucleobase, such as for example a 7-deaza purine, a 5-methyl cytosine, or a G-clamp. In certain embodiments, nucleobase mimetic include more complicated structures, such as for example a tricyclic phenoxazine nucleobase mimetic. Methods for preparation of the above noted modified nucleobases are well known to those skilled in the art. The term “nucleobase” in the present disclosure also includes the nucleobase that is protected by a suitable protecting group. In some embodiments, the NH2 group of a nucleobase (e.g., cytosine, guanine, adenine, 7-methylguanine, 5-methylcytosine, or 5-hydroxymethylcytosine) is protected by an amine protecting group. Examples of a protected nucleobase include, but are not limited to, N-benzoyl- or N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, or N-acetyl-or N-isobutyrylguanine.

[0034] The term “nucleoside” means a compound comprising a heterocyclic base moiety and a sugar moiety, which can be modified at the 2’-end.4MEI 52290585v.1123429-12620

[0035] The term “nucleotide” means a nucleoside comprising a phosphate or thiophosphate or dithiophosphate linking group.

[0036] The term "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA).

[0037] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon moiety. In some embodiments, the alkyl comprises 1 to 30 carbon atoms, 1 to 20 carbon atoms, 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In some embodiments, an alkyl comprises from 6 to 20 carbon atoms.Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3 -dimethylpentyl, n-heptyl, n-octyl, n-nonyl, or n-decyl.

[0038] As used herein, the term "alkenyl" refers to a straight or branched chain hydrocarbon containing from 2 to 30 carbons, unless otherwise specified, and containing at least one carbon-carbon double bond. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2 -m ethyl- 1 -heptenyl, 3 -decenyl, and 3, 7-dimethylocta-2, 6-dienyl.

[0039] As used herein, the term "alkoxy" or "alkoxyl" refers to an O-alkyl group wherein alkyl is as defined above.

[0040] As used herein, "carbocyclyl" refers to a saturated or unsaturated monocyclic, bicyclic or tricyclic (e.g., fused, bridged or spiro ring systems) ring system which has from 4- to 12-ring members, all of which are carbon. The term “carbocyclyl” encompasses cycloalkyl groups, cycloalkenyl group and aromatic groups (i.e., aryl). “Cycloalkyl” refers to completely saturated monocyclic hydrocarbon groups of 3-7 carbon atoms, including cyclopropyl, cyclobutyl, cyclpentyl, cyclohexyl and cyclopentyl; and “cycloalkyenyl” refers to unsaturated non-aromatic monocyclic hydrocarbon groups of 3-7 carbon atoms, including cyclpenteneyl, cyclohexenyl and cyclopentenyl.

[0041] The term "aryl" refers to monocyclic, bicyclic or tricyclic aromatic hydrocarbon groups having from 6 to 14 carbon atoms in the ring portion. In one embodiment, the term aryl refers to monocyclic and bicyclic aromatic hydrocarbon groups having from 6 to 10 carbon atoms. Representative examples of aryl groups include phenyl, naphthyl, fluorenyl, and anthracenyl.5MEI 52290585v.1123429-12620

[0042] The term "aryl" also refers to a bicyclic or tricyclic group in which at least one ring is aromatic and is fused to one or two non-aromatic hydrocarbon ring(s). Nonlimiting examples include tetrahydronaphthalene, dihydronaphthal enyl and indanyl.

[0043] The term “bridged ring system,” as used herein, is a ring system that has a carbocyclyl or heterocyclyl ring wherein two non-adjacent atoms of the ring are connected (bridged) by one or more (preferably from one to three) atoms selected from C, N, O, or S. A bridged ring system may have from 6-7 ring members.

[0044] The term “spiro ring system,” as used herein, is a ring system that has two rings each of which are independently selected from a carbocyclyl or a heterocyclyl, wherein the two ring structures having one ring atom in common. Spiro ring systems have from 5 to 7 ring members.

[0045] As used herein, the term "heterocyclyl" refers to a saturated or unsaturated, monocyclic or bicyclic (e.g., bridged or spiro ring systems) ring system which has from 3- to 12-ring members, or 3- to 9- ring members or 5- to 7- ring members, at least one of which is a heteroatom, and up to 4 e.g., 1, 2, 3, or 4) of which may be heteroatoms, wherein the heteroatoms are independently selected from O, S and N, and wherein C can be oxidized (e.g., C(O)), N can be oxidized (e.g., N(O)) or quatemized, and S can be optionally oxidized to sulfoxide and sulfone. Unsaturated heterocyclic rings include heteroaryl rings. As used herein, the term "heteroaryl" refers to an aromatic 5 or 6 membered monocyclic ring system, having 1 to 4 heteroatoms independently selected from O, S and N, and wherein N can be oxidized (e.g., N(O)) or quaternized, and S can be optionally oxidized to sulfoxide and sulfone. In one embodiment, a heterocyclyl is a 3-to 7-membered saturated monocyclic or a 3-to 6-membered saturated monocyclic or a 5-to 7-membered saturated monocyclic ring. In one embodiment, a heterocyclyl is a 3-to 7-membered monocyclic or a 3-to 6-membered monocyclic or a 5-to 7-membered monocyclic ring. In another embodiment, a heterocyclyl is a 6 or-7-membered bicyclic ring. The heterocyclyl group can be attached at a heteroatom or a carbon atom. Examples of heterocyclyl s include aziridinyl, oxiranyl, thiiranyl, oxaziridinyl, dioxiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, tetrahydrofuranyl, thiolanyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, oxathiolanyl, piperidinyl, tetrahydropyranyl, thianyl, piperazinyl, morpholinyl, thiomorpholinyl, dioxanyl, dithianyl, trioxanyl, trithianyl, azepanyl, oxepanyl, thiepanyl, dihydrofuranyl, imidazolinyl, dihydropyranyl, and heteroaryl rings including azirinyl, oxirenyl, thiirenyl, diazirinyl, azetyl, oxetyl, thietyl, pyrrolyl, furanyl, thiophenyl (or thienyl), imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, furazanyl,6MEI 52290585v.1123429-12620oxadiazolyl, thiadiazolyl, dithiazolyl, triazolyl, tetrazolyl, pyridinyl, pyranyl, thiopyranyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazinyl, thiazinyl, dioxinyl, dithiinyl, oxathianyl, triazinyl, tetrazinyl, azepinyl, oxepinyl, thiepinyl, diazepinyl, and thiazepinyl and the like. Examples of bicyclic heterocyclic ring systems include 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[3.1.1]heptanyl, 2-azaspiro[3.3]heptanyl, 2-oxa-6-azaspiro[3.3]heptanyl, and 5-azaspiro[2.3]hexanyl.

[0046] "Halogen" or "halo" may be fluoro, chloro, bromo or iodo.

[0047] As used herein, a “hydroxyl protecting group” refers to a group that is suitable for protecting a hydroxyl group, -OH, from reacting with other reagents. Examples of hydroxyl protecting groups can be found in Greene, TW et al., Protective Groups in Organic Synthesis, 4th Ed., John Wiley and Sons (2007).

[0048] In certain embodiments, the hydroxyl protecting groups can be selected from, for example, acetyl (Ac); benzoyl (Bz); benzyl (Bn); P-methoxyethoxymethyl ether (MEM); methoxymethyl ether (MOM); methoxytrityl [(4-methoxyphenyl)diphenylmethyl, MMT); 4,4'-dimethoxytrityl (DMT); methoxyethyl (MOE); p-methoxybenzyl ether (PMB); methylthiomethyl ether; pivaloyl (Piv); tetrahydropyranyl (THP); tetrahydrofuran (THF); silyl ether (including, but not limited to, trimethyl silyl (TMS), tert-butyldiphenylsilyl (TBDPS), tert-butoxy diphenyl silyl (TBoDPS), triphenyl silyl (TPS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropyl silyl (TIPS) ethers); methyl ethers, and ethoxy ethyl ethers (EE).

[0049] In certain embodiments, the hydroxyl protecting group protects the 2’ -hydroxyl of a nucleoside (referred to as 2’ -hydroxyl protecting group). In certain embodiments, the 2’-hydroxyl protecting groups include a silyl hydroxyl protecting group, such as trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethylthexylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl, di-t-butylmethylsilyl tri(trimethylsilyl)silyl, t-butylmethoxyphenylsilyl, and t-butoxy diphenyl silyl. In certain embodiments, the 2’-hydroxyl protecting group is TBDMS.

[0050] In certain embodiments, the hydroxyl protecting group protects the 5’ -hydroxyl of a nucleoside (referred to as 5 ’-hydroxyl protecting group). Exemplary 5’-hydoxyl groups include, but are not limited to those as described herein (e.g., R15in any of the embodiments). In a specific embodiment, 5’-hydoxyl protecting group is an acid-labile 4,4'-dimethoxytrityl (or bis-(4-methoxyphenyl)phenylmethyl) (DMT or DMTr) protecting group.7MEI 52290585v.1123429-12620

[0051] In certain embodiments, the hydroxyl protecting group protects the 3’ -hydroxyl of a nucleoside (referred to as 3 ’-hydroxyl protecting group). Exemplary 3’-hydoxyl groups include, but are not limited to those as described herein (e.g., Y in any of the embodiments). In a specific embodiment, 3’-hydoxyl protecting group is benzoyl (Bz) protecing group.

[0052] As used herein, the term "amine protecting group" in a compound of the present disclosure refers to a group that should protect the amino functional groups concerned against unwanted secondary reactions, such as acylations, etherifications, esterifications, oxidations, solvolysis and similar reactions. It may be removed under deprotection conditions.Depending on the protecting group employed, the skilled person would know how to remove the protecting group to obtain the free amine NH2 or NH group by reference to known procedures. These include reference to organic chemistry textbooks and literature procedures such as J. F. W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, London and New York 1973; T. W. Greene and P. G. M. Wuts, "Greene's Protective Groups in Organic Synthesis", Fourth Edition, Wiley, New York 2007; in "The Peptides"; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981; P. J. Kocienski, "Protecting Groups", Third Edition, Georg Thieme Verlag, Stuttgart and New York 2005; and in "Methoden der organischen Chemie" (Methods of Organic Chemistry), Houben Weyl, 4th edition, Volume 15 / 1, Georg Thieme Verlag, Stuttgart 1974.

[0053] In some embodiments, amine protecting groups in compounds of the disclosure generally comprise: Ci-Cealkyl (e.g., tert-butyl), preferably Ci-C4alkyl, more preferably Ci-C2alkyl, most preferably Cialkyl which is mono-, di- or tri-substituted by trialkylsilyl-Ci-C?alkoxy (e.g., trimethyl sily ethoxy), aryl, preferably phenyl, or a heterocyclic group (e.g., benzyl, cumyl, benzhydryl, pyrrolidinyl, trityl, pyrrolidinylmethyl, l-methyl-1,1-dimethylbenzyl, (phenyl)methylbenzene) wherein the aryl ring or the heterocyclic group is unsubstituted or substituted by one or more, e.g., two or three, residues, e.g., selected from the group consisting of Ci-C?alkyl, hydroxy, Ci-C?alkoxy (e.g., para-methoxy benzyl (PMB)), C2-C8- alkanoyl-oxy, halogen, nitro, cyano, and CF3, aryl-Ci-C2-alkoxycarbonyl (preferably phenyl- Ci-C2-alkoxycarbonyl (e.g., benzyloxycarbonyl (Cbz), benzyloxymethyl (BOM), pivaloyloxymethyl (POM)), Ci-Cio-alkenyloxycarbonyl, Ci-Cealkylcarbonyl (e.g., acetyl or pivaloyl), Ce-Cio-arylcarbonyl; Ci-Ce-alkoxycarbonyl (e.g., tert-butyloxycarbonyl (Boc), methylcarbonyl, trichloroethoxycarbonyl (Troc), pivaloyl (Piv), allyloxycarbonyl), Ce-C10- arylCi-Ce-alkoxycarbonyl (e.g., 9-fluorenylmethyloxycarbonyl (Fmoc)), allyl or cinnamyl, sulfonyl or sulfenyl, succinimidyl group, silyl groups (e.g., triarylsilyl, trialkylsilyl,8MEI 52290585v.1123429-12620triethylsilyl (TES), trimethylsilylethoxymethyl (SEM), trimethylsilyl (TMS), triisopropyl silyl or tertbutyldimethylsilyl).

[0054] In some embodiments, the amine protecting group in compounds of the present disclosure is selected from the group consisting of PhCO-, CH3CO-, / PrCO-, Me2N-CH=, or Me2N-CMe=.

[0055] The suffic “yl” added to the end of a chemical name indicates that the named moiety is bonded to the molecule at point. The suffix “ene” added to the end of a chemical name indicates that the named moiety is bonded to the molecule at two points.

[0056] As used herein the term “base” refers to a substance that can produce hydroxide ion (OH) in water solutions or a substance that can donate a pair of nonbonding electrons.Exemplary bases include, but are not limited to, alkaline hydroxide, alkaline earth hydroxide, alkylamines (e.g., tert-butylamine, sec-butylamine, trimethylamine, tri ethylamine, diisopropylethylamine, 2-methylpropan-2-amine), 8-diazabicyclo[5.4. 0]undec-7-ene (DBU), imidazole, N-m ethylimidazole, pyridine and 3 -picoline.

[0057] As used herein, the term “salt” refers to an organic or inorganic salt of a compound, nucleotide or oligonucleotide described herein. In certain embodiments, the salt is a pharmaceutically acceptable salt thereof. The phrase “pharmaceutically acceptable” indicates that the substance or composition must be compatible chemically and / or toxicologically, with the other ingredients comprising a formulation, and / or the mammal being treated therewith. In certain embodiments, the salt of the compound, nucleotide or oligonucleotide described herein is a sodium salt, a potassium salt or an ammonium salt. In certain embodiments, the salt is a sodium salt or ammonium salt.

[0058] As used herein, the term “solid support” refers to any support that is compatible with oligonucleotide, i.e., RNA, synthesis including, for example, glass, controlled pore glass, polymeric materials, polystyrene, beads, coated glass and the like. Such materials are known in the art and include, for example, beads, pellets, disks, fibers, gels, or particles such as cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene and optionally grafted with polyethylene glycol, poly-acrylamide beads, latex beads, dimethylacrylamide beads optionally cross-linked with N,N'-bis-acryloyl ethylene diamine, glass particles coated with hydrophobic polymer, and material having a rigid or semi-rigid surface. The solid supports optionally have functional groups such as amino, hydroxy, carboxy, or halo groups. In some embodiments, the solid support of the present disclosure is selected from cross-linked polystyrene beads or controlled pore glass beads. In some embodiments, the solid support of the present disclosure comprises a hydroxyl9MEI 52290585v.1123429-12620functional group. In some embodiments, the solid support of the present disclosure is selected from cross-linked polystyrene beads with hydroxyl functional groups or controlled pore glass beads with hydroxyl functional groups.

[0059] In some embodiments, the solid support of the present disclosure comprises a linker. In some embodiments, the linker is a chemical moiety that connects oligonucleotide, / .< ., RNA, with the solid support described above. In some embodiments, a linker comprises a linear or branched carbon chain comprising from 2 to 40 carbon atoms or a group that comprises one or more rings, which chain optionally includes one or more double or triple bonds, and which chain is optionally substituted with one or more hydroxy or oxo groups, and wherein one or more of the carbon atoms in the chain is optionally replaced with a nonperoxide -O-, -S- or - NH-. Non-limiting examples of such include polyethylene glycol (PEG). In some embodiments, the linker comprises a group represented by:

[0060] In some embodiment, the solid support having a linker is represented by the following:

[0061] In some embodiment, the solid support having a linker is represented bywherein 1 indicates the point of attachment to the oligonucleotide described herein.

[0062] In a first embodiment, the present disclosure provides a process for preparing a compound of formula (I):10MEI 52290585v.1123429-12620or a salt thereof by a deprotection process, wherein the deprotection process comprises deprotecting a compound of formula (II) with HF in the presence of a base:or a salt thereof, to form the compound of formula (I), or a salt thereof, wherein:R1, for each occurrence, is independently a nucleobase;R2, for each occurrence, is independently selected from the group consisting of H and OH, and at least one R2is OH;R2, for each occurrence, is independently selected from the group consisting of H and OR2a, and at least one R2is OR2a; wherein R2ais a silyl hydroxyl protecting group;R4, for each occurrence, is independently H or forms a ring with the H group of R2or R2;R15is H or a hydroxyl protecting group;q is an integer from 0 to 200;11MEI 52290585v.1123429-12620X, for each occurrence, is independently O or S; andY is H or a hydroxyl protecting group.

[0063] In a second embodiment, the present disclosure provides a process according to the first embodiment, wherein the deprotection reaction is carried out in water. The definitions of the remaining variables are provided in the first embodiment or any specific embodiments described therein.

[0064] In a third embodiment, the present disclosure provides a process according to the first or the second embodiment, wherein the base is an optionally substituted imidazole or an optionally substituted pyridine. The definitions of the remaining variables are provided in the first or the second embodiment or any specific embodiments described therein.

[0065] In a fourth embodiment, the present disclosure provides a process according to any one of the first through the third embodiments, wherein the deprotection reaction is carried out by reacting the compound of formula (II) with HF in the presence of pyridine and imidazole. The definitions of the remaining variables are provided in the first through the third embodiments or any specific embodiments described therein.

[0066] In a fifth embodiment, the present disclosure provides a process according to any one of the first through the fourth embodiments, wherein excess amount of base relative to HF is used. The definitions of the remaining variables are provided in the first through the fourth embodiments or any specific embodiments described therein.

[0067] In a sixth embodiment, the present disclosure provides a process according to any one of the first through the fifth embodiments, wherein the base is imidazole. The definitions of the remaining variables are provided in the first through the fifth embodiments or any specific embodiments described therein.

[0068] In a seventh embodiment, the present disclosure provides a process according to the sixth embodiment, wherein the molar ratio of imidazole to HF is in the range of 1.1 : 1 to 5 : 1. The definitions of the remaining variables are provided in the sixth embodiment or any specific embodiments described therein.

[0069] In an eighth embodiment, the present disclosure provides a process of according to the seventh embodiment, wherein the molar ratio of imidazole to HF is 2:1. The definitions of the remaining variables are provided in the seventh embodiment or any specific embodiments described therein.

[0070] In a ninth embodiment, the present disclosure provides a process according to any one of the first through the fifth embodiments, wherein the base is pyridine. The definitions of the12MEI 52290585v.1123429-12620remaining variables are provided in the first through the fifth embodiments or any specific embodiments described therein.

[0071] In a tenth embodiment, the present disclosure provides a process according to the ninth embodiment, wherein the molar ratio of pyridine to HF is in the range of 1.1 : 1 to 20: 1. The definitions of the remaining variables are provided in the ninth embodiment or any specific embodiments described therein.

[0072] In an eleventh embodiment, the present disclosure provides a process according to any one of the first through the tenth embodiments, wherein the molar ratio of HF to every R2aof the compound of formula (II) is in the range of 1 : 1 to 30: 1. The definitions of the remaining variables are provided in any one of the first through the tenth embodiments or any specific embodiments described therein. In some embodiments, the molar ratio of HF to every R2aof the compound of formula (II) is in the range of 1 : 1 to 25: 1, 1:1 to 20: 1, 1:1 to 15:1, 1:1 to 10:1, 1:1 to 5:1, or 2:1 to 4:1. In some embodiments, the molar ratio of HF to every R2aof the compound of formula (II) is 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[0073] In a twelfth embodiment, the present disclosure provides a process according to any one of the first through the eleventh embodiments, wherein the molar ratio of HF to every R2aof the compound of formula (II) is in the range of 1 : 1 to 10: 1. The definitions of the remaining variables are provided in any one of the first through the eleventh embodiments or any specific embodiments described therein. In some embodiments, the molar ratio of HF to every R2aof the compound of formula (II) is 10:1.

[0074] In a thirteenth embodiment, the present disclosure provides a process according to any one of the first through the twelfth embodiments, wherein the molar ratio of HF to every R2aof the compound of formula (II) is in the range of 1 : 1 to 5 : 1. The definitions of the remaining variables are provided in any one of the first through the twelfth embodiments or any specific embodiments described therein. In some embodiments, the molar ratio of HF to every R2aof the compound of formula (II) is 5:1.

[0075] In a fourteenth embodiment, the present disclosure provides a process according to any one of the first through the thirteenth embodiments, wherein the molar ratio of HF to every R2aof the compound of formula (II) is in the range of 2: 1 to 4: 1. The definitions of the remaining variables are provided in any one of the first through the thirteenth embodiments or any specific embodiments described therein. In some embodiments, the molar ratio of HF to every R2aof the compound of formula (II) is 3 : 1.13MEI 52290585v.1123429-12620

[0076] In a fifteenth embodiment, the present disclosure provides a process according to any one of the first through the fourteenth embodiments, wherein R2ais a silyl protecting group selected from TBDPS, TBoDPS and TBDMS:TBDPS TBoDPS TBDMSThe definitions of the remaining variables are provided in any one of the first through the fourteenth embodiments or any specific embodiments described therein.

[0077] In a sixteenth embodiment, the present disclosure provides a process according to the fifteenth embodiment, wherein R2ais TBDMS. The definitions of the remaining variables are provided in the fifteenth embodiment or any specific embodiments described therein.

[0078] In a seventeenth embodiment, the present disclosure provides a process according to the fifteenth embodiment, wherein R2ais TBDPS. The definitions of the remaining variables are provided in the fifteenth embodiment or any specific embodiments described therein.

[0079] In an eighteenth embodiment, the present disclosure provides a process according to any one of the first through the seventeenth embodiments, wherein R15is H or 4,4’-dimethoxytrityl group. The definitions of the remaining variables are provided in any one of the first through the seventeenth embodiments or any specific embodiments described therein.

[0080] In a nineteenth embodiment, the present disclosure provides a process according to any one of the first through the seventeenth embodiments, wherein R15is 4, 4’-dimethoxytrityl group. The definitions of the remaining variables are provided in any one of the first through the seventeenth embodiments or any specific embodiments described therein.

[0081] In a twentieth embodiment, the present disclosure provides a process according to any one of the first through the nineteenth embodiments, wherein R4is H. The definitions of the remaining variables are provided in any one of the first through the nineteenth embodiments or any specific embodiments described therein.

[0082] In a twenty-first embodiment, the present disclosure provides a proces according to any one of the first through the nineteenth embodiments, wherein14MEI 52290585v.1123429-12620each R4is independently H or forms a ring with the H group of R2, wherein the ring is a 5 or 6-membered ring optionally substituted with 1 to 3 Ci-4 alkyl groups.The definitions of the remaining variables are provided in any one of the first through the nineteenth embodiments or any specific embodiments described therein. In some embodiments, the 5 or 6-membered ring formed by R2and R4is a 5 or 6-membered carbocyclyl ring optionally substituted with 1 to 3 C1-2 alkyl groups. In some embodiments, the 5 or 6-membered ring formed by R2and R4is a 5 or 6-membered heterocyclyl ring optionally substituted with 1 to 3 C1-2 alkyl groups.

[0083] In a twenty-second embodiment, the present disclosure provides a process according to any one of the first through the twenty-first embodiments, wherein q is an integer from 1 to 50. The definitions of the remaining variables are provided in any one of the first through the twenty-first embodiments or any specific embodiments described therein.

[0084] In a twenty-third embodiment, the present disclosure provides a process according to any one of the first through the twenty-second embodiments, wherein q is an integer from 1 to 20. The definitions of the remaining variables are provided in any one of the first through the twenty-second embodiments or any specific embodiments described therein.

[0085] In a twenty-fourth embodiment, the present disclosure provides a process according to any one of the first through the twenty -third embodiments, wherein q is an integrer from 10 to 20. The definitions of the remaining variables are provided in any one of the first through the twenty-third embodiments or any specific embodiments described therein.

[0086] In a twenty-fifth embodiment, the present disclosure provides a process according to any one of the first through the twenty-fourth embodiments, wherein all of the P=X groups in the compound or oligonucleotide are P=S. The definitions of the remaining variables are provided in any one of the first through the twenty -fourth embodiments or any specific embodiments described therein.

[0087] In a twenty-sixth embodiment, the present disclosure provides a process according to any one of the first through the twenty-fourth embodiments, wherein all of the P=X groups in the compound or oligonucleotide are P=O. The definitions of the remaining variables are provided in any one of the first through the twenty -fourth embodiments or any specific embodiments described therein.

[0088] In a twenty-seventh embodiment, the present disclosure provides a process according to any one of the first through the twenty -fourth embodiments, wherein greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the P=X groups in the compound or 15MEI 52290585v.1123429-12620oligonucleotide are P=S. The definitions of the remaining variables are provided in any one of the first through the twenty -fourth embodiments or any specific embodiments described therein.

[0089] In a twenty-eighth embodiment, the present disclosure provides a process according to any one of the first through the twenty-fourth embodiments, wherein 10-90%, 20-80%, 30-70% or 40-60% of the P=X groups in the compound or oligonucleotide are P=S. The definitions of the remaining variables are provided in any one of the first through the twentyfourth embodiments or any specific embodiments described therein.

[0090] In a twenty-ninth embodiment, the present disclosure provides a process according to any one of the first through the twenty-eighth embodiments, wherein the nucleobase represented by R1is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine, wherein the NH2 group of said cytosine, guanine, adenine, 7-methylguanine, 5-methylcytosine, or 5-hydroxymethylcytosine represented by R1is not protected. The definitions of the remaining variables are provided in any one of the first through the twenty-eighth embodiments or any specific embodiments described therein.

[0091] In a thirtieth embodiment, the present disclosure provides a process according to any one of the first through the twenty-eighth embodiments, wherein the nucleobase represented by R1is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, 7-methylguanine, and 5-methylcytosine, wherein the NH2 group of said cytosine, guanine, adenine, 7-methylguanine, or 5-methylcytosine represented by R1is not protected. The definitions of the remaining variables are provided in any one of the first through the twentyeighth embodiments or any specific embodiments described therein.

[0092] In a thirty-first embodiment, the present disclosure provides a process according to any one of the first through the twenty-eighth embodiments, wherein the nucleobase represented by R1is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, and 5-methylcytosine, wherein the NH2 group of said cytosine, guanine, adenine, or 5-methylcytosine represented by R1is not protected. The definitions of the remaining variables are provided in any one of the first through the twenty-eighth embodiments or any specific embodiments described therein.

[0093] In some embodiments, the present disclosure provides a process according to any one of the first through the twenty-eighth embodiments, wherein the nucleobase represented by R1is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil,16MEI 52290585v.1123429-12620hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine, wherein the NH2 group of said cytosine, guanine, adenine, 7-methylguanine, 5-methylcytosine, or 5-hydroxymethylcytosine represented by R1is protected by an amine protecting group. In some embodiments, the amine protecting group is PhCO-, CH3CO-, zPrCO-, Me2N-CH=, or Me2N-CMe=. The definitions of the remaining variables are provided in any one of the first through the twenty-eighth embodiments or any specific embodiments described therein.

[0094] In some embodiments, the present disclosure provides a process according to any one of the embodiments described herein, wherein Y is H. In some embodiments, Y is PhCO-. The definitions of the remaining variables are provided in any one of specific embodiments described therein.

[0095] In a thirty-second embodiment, the present disclosure provides a process according to any one of the first through the thirty-first embodiments, whereinR2, for each occurrence, is OH;R2, for each occurrence, is OR2a; wherein R2ais TBDMS; andR15is 4,4’-dimethoxytrityl group.The definitions of the remaining variables are provided in any one of the first through the thirty-first embodiments or any specific embodiments described therein.

[0096] In a thirty-third embodiment, the present disclosure provides a process according to any one of the first through the thirty-second embodiments, wherein the reaction time is less than 10 hours. The definitions of the remaining variables are provided in any one of the first through the thirty-second embodiments or any specific embodiments described therein.

[0097] In a thirty-fourth embodiment, the present disclosure provides a process according to any one of the first through the thirty-third embodiments, wherein the reaction time is about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 0.5 hour, or about 0.2 hour. The definitions of the remaining variables are provided in any one of the first through the thirty-third embodiments or any specific embodiments described therein.

[0098] In a thirty-fifth embodiment, the present disclosure provides a process according to any one of the first through the thirty-fourth embodiments, wherein the reaction time is about 6 hours. The definitions of the remaining variables are provided in any one of the first through the thirty- fourth embodiments or any specific embodiments described therein.

[0099] In a thirty-sixth embodiment, the present disclosure provides a process according to any one of the first through the thirty-fifth embodiments, wherein the compound of formula 17MEI 52290585v.1123429-12620(II) is prepared by an ammonolysis process; wherein the ammonolysis process comprises reacting a compound of formula (III) or a salt thereof with a base:(III),whereinR1A, for each occurrence, is independently a nucleobase, wherein the NH2 of the nucleobase, if present, is protected by an amine protecting group;R2, for each occurrence, is independently selected from the group consisting of H and OR2a, and at least one R2is OR2a; wherein R2ais a silyl hydroxyl protecting group;R4, for each occurrence, is independently H or forms a ring with the H group of R2; R15is H or a hydroxyl protecting group;R16A, for each occurrence, is independently Ci-ealkyl group, C2-ealkenyl group, phenyl or benzyl group, each of which is optionally substituted with -CN, -NO2 or halogen; orZ is H, a hydroxyl protecting group, or a solid support.The definitions of the remaining variables are provided in any one of the first through the thirty- fifth embodiments or any specific embodiments described therein.

[0100] In some embodiments, the present disclosure provides a process according to the thirty-sixth embodiment, wherein the base is NH4OH or EtsN. In some embodiments, the18MEI 52290585v.1123429-12620base is NH4OH. The definitions of the remaining variables are provided in the thirty-sixth embodiment or any specific embodiments described therein.

[0101] In a thirty-seventh embodiment, the present disclosure provides a process according to the thirty-sixth embodiment, wherein the reaction between the compound of Formula (III) or a salt thereof and NH4OH is carried out in the presence of methylamine. The definitions of the remaining variables are provided in the thirty-sixth embodiment or any specific embodiments described therein.

[0102] In a thirty-eighth embodiment, the present disclosure provides a process according to the thirty-sixth embodiment, wherein the ammonolysis process is carried out at a temperature in the range between 40°C and 75°C. The definitions of the remaining variables are provided in the thirty-sixth embodiment or any specific embodiments described therein.

[0103] In a thirty-ninth embodiment, the present disclosure provides a process according to the thirty-sixth or the thirty-eighth embodiment, wherein the compound of Formula (II) prepared by the ammonolysis process is used without purification before the deprotection process to prepare the compound of Formula (I). The definitions of the remaining variables are provided in the thirty-sixth or the thirty-eighth embodiment or any specific embodiments described therein.

[0104] In a fortieth embodiment, the present disclosure provides a process according to the thirty-ninth embodiment, wherein the deprotection process and the ammonolysis process are carried out in one pot. The definitions of the remaining variables are provided in the thirtyninth embodiment or any specific embodiments described therein.

[0105] In a forty-first embodiment, the present disclosure provides a process according to the thirty-sixth embodiment, wherein the deprotection process and the ammonolysis process are carried out in stepwise. The definitions of the remaining variables are provided in the thirtysixth embodiment or any specific embodiments described therein.

[0106] In a forty-second embodiment, the present disclosure provides a process according to any one of the thirty-sixth through the forty-first embodiments, wherein Z is a solid support. The definitions of the remaining variables are provided in any one of the thirty-sixth through the forty-first embodiments or any specific embodiments described therein.

[0107] In some embodiments, the present disclosure provides a process according to any one of the thirty-sixth embodiment through forty-first embodiments, wherein Z is PhCO-. The definitions of the remaining variables are provided in any one of the thirty-sixth embodiment through forty-first embodiments or any specific embodiments described therein.19MEI 52290585v.1123429-12620

[0108] In a forty -third embodiment, the present disclosure provides a process according to any one of the thirty-sixth through the forty-second embodiments, wherein the nucleobase represented by R1Ais selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine, wherein the NH2 group of said cytosine, guanine, adenine, 7-methylguanine, 5-methylcytosine, or 5-hydroxymethylcytosine represented by R1Ais protected by PhCO-, CH3CO-, zPrCO-, Me2N-CH=, or Me2N-CMe=. The definitions of the remaining variables are provided in any one of the thirty-sixth through the forty-second embodiments or any specific embodiments described therein.

[0109] In a forty-fourth embodiment, the present disclosure provides a process according to any one of the thirty-sixth through the forty -third embodiments, wherein the nucleobase represented by R1Ais selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, 7-methylguanine, and 5-methylcytosine, wherein the NH2 group of said cytosine, guanine, adenine, 7-methylguanine, or 5-methylcytosine represented by R1Ais protected by PhCO-, CH3CO-, / PrCO-, Me2N-CH=, or Me2N-CMe=. The definitions of the remaining variables are provided in any one of the thirty-sixth through the forty -third embodiments or any specific embodiments described therein.

[0110] In a forty-fifth embodiment, the present disclosure provides a process according to any one of the thirty-sixth through the forty -fourth embodiments, wherein the nucleobase represented by R1Ais selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, and 5-methylcytosine, wherein the NH2 group of said cytosine, guanine, adenine, or 5-methylcytosine represented by R1Ais protected by PhCO-, CH3CO-, / PrCO-, Me2N-CH=, or Me2N-CMe=. The definitions of the remaining variables are provided in any one of the thirty-sixth through the forty-fourth embodiments or any specific embodiments described therein.[OlH] In certain embodiments, for the process described therein, R16Ais one of the following:MEI 52290585v.1123429-12620ee Nat Biotechnol. 2017 Sep;35(9):845-851; J. Org. Chem. 1999, 64, 7515-7522; Biopolymers (Peptide Science), 2001, 60, 3, each of which is incorporated herein by reference. In some embodiments, R16Ais -CH2CH2CN.EXEMPLIFICATIONAbbreviationDBU = 8-diazabicyclo[5.4. 0]undec-7-eneDDTT = 3-(N,N-dimethylamino-methylidene)amino)-3H-l,2,4-dithiazole DCI = 4,5-dicyanoimidazoleDMT or DMTr = 4,4'-dimethoxytrityl or bis-(4-methoxyphenyl)phenylmethyl DMSO = dimethyl sulfoxideEDCI = l-ethyl-3-(3-dimethylaminopropyl)carbodiimideEtOAc or EA = ethyl acetateh or hr = hourMOE = methoxyethylTHF = tetrahydrofuranMEI 52290585v.1123429-12620Example 1. Comparative Study of Two 2'-OH Deprotection Processes of RNA

[0112] Preparation of compound 1. A mixture of DMT-2'-O-TBDMS-U-CE-Phosphoramidite (2.13 g, 2.47 mmol, 1.2 equiv) and 5-hydroxypentyl benzoate (0.43 g, 2.1 mmol, 1.0 equiv) in CH3CN-CH2Q2 (25 mL, 2:1, v / v) was stirred at 20-25 °C for 10 min. DCI (0.37 g, 3.1 mol, 1.5 equiv) was added and the reaction mixture was stirred at 20-25 °C for 0.5 h. DDTT (0.45 g, 2.26 mmol, 1.1 equiv) was added and the mixture was stirred at 20-25 °C for 10 min. The reaction mixture was diluted with EtOAc (50 mL) and was washed with 4% NaHCO3 aqueous solution (30 mL) and saturated brine (30 ml), dried over MgSCU (2 g), filtered, and concentrated. The crude product was dissolved in EtsN / CEECN (30 mL, 1:1, v / v) and stirred at 20-25 °C for 7 h. The mixture was concentrated under vacuum, and the crude product was purified by a short silica gel column chromatography (CEECh-EtOAc, 10:1 to 1:1, v / v) to afford compound 1 (1.26 g, 65% yield). 'H NMR (400 MHz, CDCI3) 5 ppm -0.1 - 0.1 (m, 6 H), 0.69 - 0.72 (m, 9 H), 1.15 - 1.55 (m, 6 H), 3.15 - 3.90 (m, 10 H), 3.99 - 4.62 (m, 4 H), 4.75 - 4.90 (m, 1 H), 5.80 - 5.90 (m, 1 H), 6.64 - 6.68 (m, 4 H), 6.99 -7.38 (m, 12 H), 7.62 - 7.71 (m, 1 H), 7.73 - 7.74 (m, 2 H), 11.9 (s, 1 H). 13C NMR (125 MHz, DMSO-d6) 8 ppm - 2.7, - 2.0, 20.5, 24.8, 28.2, 30.6, 32.5, 57.7, 65.9, 66.1, 66.9, 67.3, 67.4, 76.1, 76.2, 77.3, 85.3, 85.7, 88.9, 89.4, 89.8, 104.5, 115.4, 115.9, 129.5, 130.4, 130.6, 131.4, 131.8, 132.6, 135.9, 137.6, 137.9, 142.2, 147.3, 153.3, 160.5, 160.9, 165.5, 168.4. HRMS calcd for C48H60N2O12PSSi+ [M+H]+ 947.3369, found: 947.3387.

[0113] Preparation of compound 2. A mixture of DMT-2'-O-TBDMS-A(Bz)-CE-Phosphoramidite (5.7 g, 5.8 mmol, 1.2 equiv) and 5-hydroxypentyl benzoate (1.0 g, 4.8 mmol, 1.0 equiv) in CH3CN-CH2Q2 (60 mL, 2:1, v / v) was stirred at 20-25 °C for 10 min.22MEI 52290585v.1123429-12620DCI (0.88 g, 7.2 mol, 1.5 equiv) was added and the reaction mixture was stirred at 20-25 °C for 0.5 h. tert-Butyl hydroperoxide (1.66 mL, 12.0 mmol, 2.5 equiv) was added and the mixture was stirred at 20-25 °C for 30 min. The reaction mixture was diluted with EtOAc (50 mL) and was washed with 4% NaHCCh aqueous solution (30 mL) and saturated brine (30 ml), dried over MgSCU (2 g), filtered, and concentrated. The crude product was dissolved in EtsN / CHsCN (50 mL, 1:1, v / v) and stirred at 20-25 °C for 7 h. The mixture was concentrated under vacuum, and the crude product was purified by a short silica gel column chromatography (CEECh-EtOAc, 10:1 to 1:1, v / v) to afford compound 2 (2.7 g, 53% yield). 'HNMR (400 MHz, DMSO-d6) 8 ppm -0.03 - 0.02 (m, 6 H), 0.87 - 0.89 (m, 9 H), 1.36 -1.45 (m, 2 H), 1.53 - 1.61 (m, 2 H), 1.62 - 1.72 (m, 2 H), 3.29 - 3.40 (m, 2 H), 3.59 - 3.74 (m, 2 H), 4.23 - 4.27 (m, 3 H), 4.66 - 4.72 (m, 1 H), 5.01 - 5.03 (m, 1 H), 6.10 (d, J = 7.0 Hz, 1 H), 6.82 - 6.94 (m, 4 H), 7.20 - 7.35 (m, 7 H), 7.40 - 7.49 (m, 2 H), 7.51 - 7.71 (m, 6 H), 7.95 - 8.04 (m, 2 H), 8.09 - 8.11 (m, 2 H), 8.67 - 8.73 (m, 2 H), 11.28 (s, 1 H).13C NMR (125 MHz, DMSO- d6) 6 ppm - 2.9, - 2.7, 20.3, 24.7, 27.9, 28.0, 28.5, 30.6, 30.7, 32.7, 32.8, 57.7, 66.4, 66.6, 66.9, 67.0, 67.3, 75.2, 76.4, 76.6, 85.7, 85.8, 86.2, 88.3, 88.4, 89.9, 90.4, 115.8, 129.3, 130.4, 130.5, 131.2, 131.4, 131.8, 132.4, 132.5, 135.9, 136.0, 138.2, 147.6, 153.2, 154.3, 154.9, 160.7, 168.3, 168.4. HRMS calcd for CsefLsNsOnPSL [M+H]+1058.4131, found: 1058.4189.

[0114] Preparation of compound 3. A mixture of DMT-2'-O-TBDMS-A(Bz)-CE-Phosphoramidite (3.77 g, 3.8 mmol, 1.2 equiv) and 5'-HO-Deoxy-T-3'-OBz (1.1 g, 3.2 mmol, 1.0 equiv) in CH3CN-CH2C12 (40 mL, 1:1, v / v) was stirred at 20-25 °C for 10 min. DCI (0.75 g, 6.4 mol, 2.0 equiv) was added and the reaction mixture was stirred at 20-25 °C for 0.5 h. DDTT (0.70 g, 3.5 mmol, 1.1 equiv) was added and the mixture was stirred at 20-25 °C for 10 min. The reaction mixture was diluted with EtOAc (100 mL) and was washed with 4% NaHCO3 aqueous solution (50 mL) and saturated brine (50 ml), dried over MgSO423MEI 52290585v.1123429-12620(5 g), filtered, and concentrated. The crude product was dissolved in Et3N / CH3CN (100 mL, 1:1, v / v) and stirred at 20-25 °C for 7 h. The mixture was concentrated under vacuum, and the crude product was purified by a short silica gel column chromatography (CH2C12-EtOAc, 10: 1 to 1 : 1, v / v) to afford compound 3 (2.4 g, 63% yield).JH NMR (400 MHz, DMSO-d6) 6 ppm -0.28 - -0.17 (m, 3 H), -0.05 - 0.0 (m, 3 H), 0.59 - 0.67 (m, 9 H), 1.83 - 1.86 (m, 3 H), 2.26 (br s, 1 H), 2.21 - 2.26 (m, 1 H), 2.35 - 2.38 (m, 1 H), 3.37 - 3.43 (m, 2 H), 3.63 - 3.73 (m, 6 H), 3.90 - 4.15 (m, 2 H), 4.24 - 4.30 (m, 1 H), 4.62 - 4.70 (m, 1 H), 4.92 - 5.03 (m, 1 H), 5.17 - 5.19 (m, 1 H), 5.52 - 5.62 (m, 1 H), 6.10 - 6.15 (m, 1 H), 6.31 - 6.35 (m, 1 H), 6.76 - 6.83 (m, 4 H), 7.14 - 7.30 (m, 7 H), 7.39 - 7.45 (m, 2 H), 7.50 - 7.57 (m, 4 H), 7.61 - 7.69 (m, 2 H), 7.96 - 8.06 (m, 5 H), 8.53 - 8.55 (m, 2 H), 11.19 (s, 1 H), 11.29 - 11.31 (m, 1 H).13C NMR (125 MHz, DMSO-d6) 8 ppm -5.14, -5.12, 12.6, 18.1, 36.8, 36.9, 55.4, 64.4, 64.5, 65.0, 65.7, 74.1, 74.5, 74.8, 77.0, 77.4, 83.6, 83.7, 84.0, 84.2, 84.4, 86.1, 87.7, 87.8, 110.8, 110.9, 113.2, 113.4, 126.3, 128.2, 128.3, 128.9, 129.0, 129.3, 129.8, 130.3, 132.9, 134.0, 135.9, 136.8, 143.6, 145.4, 150.9, 151.1, 152.0, 152.7, 158.5, 164.3, 165.6, 166.1. HRMS calcd for C6iH67N70i4PSSi+ [M+H]+ 1212.3968, found: 1212.4040.

[0115] Preparation of 1.0 M HF / Imidazole = 1:1 (equiv. : equiv.) CfbCN solution: in a high-density polyethylene vial, HF-py (2.86 g, 0.10 mol HF, -70% HF in pyridine) was slowly added the solution of imidazole (6.81 g, 0.10 mol,) in CH3CN (100 mL) at -5-5 °C, and the mixture was stirred at -5-5 °C for another 30 min to yield a homogeneous solution.

[0116] Preparation of compound la. In 10 mL vial, HF-py (39 pL, 1.5 mmol HF, -70% HF in pyridine) was slowly added the solution of imidazole (102 mg, 1.5 mmol) in CH3CN (2.5 mL) at 20-25 °C, and the mixture was stirred at 20-25 °C for another 10 min to yield a homogeneous solution. Compound 1 (473 mg, 0.50 mmol) was added to the HF / Imidazole CH3CN solution in one portion, and the resulting mixture was stirred at 20-25 °C for 30 minutes (HPLC indicated the reaction conversion > 99.0%).

[0117] The reaction mixture was diluted with EtOAc (20 mL) and water (20 mL) to yield two layers. The organic layer (upper layer) was washed with 4% NaHCCh (20 mL), water (20 mL), and brine (20 mL), dried over MgSCU (1 g), filtered, and concentrated. The crude 24MEI 52290585v.1123429-12620product was purified by a short silica gel column chromatography (ClbCh-EtOAc, 10:1 to 1 :2, v / v) to afford compound la (303 mg, 73% yield).1H NMR (500 MHz, DMSO-tL) 6 ppm 1.32 - 1.44 (m, 2 H), 1.50 - 1.59 (m, 2 H), 1.61 - 1.72 (m, 2 H), 3.18 (t,J= 11.0 Hz, 1 H), 3.25 - 3.33 (m, 1 H), 3.62 - 3.64 (m, 1 H), 3.72 (s, 6 H), 3.75 - 3.85 (m, 1 H), 4.09 - 4.03 (m, 1 H), 4.19 - 4.24 (m, 2 H), 4.27 - 4.30 (m, 1 H), 4.65 - 4.71 (m, 1 H), 4.84 - 4.90 (m, 1 H), 5.33 - 5.41 (m, 1 H), 5.80 (dd, J= 17.6, 5.2 Hz, 1 H), 6.10 - 6.15 (m, 1 H), 6.85 - 6.89 (m, 4 H), 7.20 - 7.33 (m, 7 H), 7.37 - 7.42 (m, 2 H), 7.48 - 7.52 (m, 2 H), 7.61 -7.63 (m, 1 H), 7.67 - 7.75 (m, 1 H), 7.95 (d, J= 7.6 Hz, 2 H), 11.43 (s, 1 H).13C NMR (125 MHz, DMSO-t / 6)) 6 ppm 22.4, 28.3, 30.0, 55.5, 63.0, 63.6, 65.1, 65.4, 73.2, 73.4, 73.6, 73.9, 82.1, 82.2, 82.5, 86.4, 86.5, 88.3, 88.9, 102.2, 113.7, 127.2, 128.1, 128.3, 129.2, 129.5, 130.2, 133.6, 135.5, 135.8, 140.8, 145.1, 151.1, 158.6, 163.5, 166.2. HRMS calcd for C42H46N2Oi2PS+[M+H]+833.2504, found: 833.2522.

[0118] Desilylation of compound 1 through TBAF. In a 10 mL vial, Compound 1 (473 mg, 0.50 mmol) was added to a mixture of CH3CN (1 ml) and 1.5 ml TBAF / THF (1.0 M / L THF) solution, and the resulting mixture was stirred at 20-25 °C for 30 -180 minutes. HPLC indicated the reaction conversion was 74.6% at 60 min and 98.8% at 180 min.Table 1

[0119] Preparation of compound 2a. In 10 mL vial, HF-py (39 pL, 1.5 mmol HF, -70% HF in pyridine) was slowly added the solution of imidazole (102 mg, 1.5 mmol) in CH3CN (2.5 mL) at 20-25 °C, and the mixture was stirred at 20-25 °C for another 10 min to yield a homogeneous solution. Compound 2 (528 mg, 0.50 mmol) was added to the HF / Imidazole25MEI 52290585v.l123429-12620CH3CN solution in one portion, and the resulting mixture was stirred at 20-25 °C for 30 minutes (HPLC indicated the reaction conversion > 99.0%).

[0120] The reaction mixture was diluted with EtOAc (20 mL) and water (20 mL) to yield two layers. The organic layer (upper layer) was washed with 4% NaHCCh (20 mL), water (20 mL), and brine (20 mL), dried over MgSCU (1 g), filtered, and concentrated. The crude product was purified by a short silica gel column chromatography (C LCh-EtOAc, 10:1 to 1 :2, v / v) to afford compound 2a (330 mg, 70% yield).JH NMR (500 MHz, DMSO-tL) 6 ppm 1.34 - 1.39 (m, 2 H), 1.49 - 1.54 (m, 2 H), 1.62 -1.67 (m, 2 H), 3.25 - 3.31 (m, 2 H), 3.63 -3.65 (m, 1 H), 3.71 - 3.75 (m, 7 H), 4.17 - 4.26 (m, 3 H), 4.62 - 4.65 (m, 1 H), 4.96 - 4.99 (m, 1 H), 6.08 (d, J= 6.7 Hz, 1 H), 6.83 - 6.88 (m, 4 H), 7.18 - 7.22 (m, 1 H), 7.23 - 7.28 (m, 6 H), 7.40 (d, J= 7.6 Hz, 2 H), 7.45 - 7.58 (m, 4 H), 7.60 - 7.68 (m, 2 H), 7.96 (d, J= 7.3 Hz, 2 H), 8.07 (d, J= 7.6 Hz, 2 H), 8.64 (d, J= 12.0 Hz, 2 H), 8.67 (s, 1 H), 11.28 (s, 1 H).13C NMR (125 MHz, DMSO- de) 6 ppm 22.4, 28.4, 30.4, 55.5, 64.2, 64.7, 64.8, 65.1, 73.1, 74.2, 83.5, 86.2, 88.2, 113.6, 126.3, 128.1, 128.3, 128.9, 129.0, 129.2, 129.6, 130.2, 130.3, 133.7, 133.8, 136.0, 145.2, 150.9, 152.2, 152.8, 158.6, 166.3. HRMS calcd for C5oH5iN5Oi2P+[M+H]+944.3267, found: 944.3299.

[0121] Desilylation of compound 2 through TBAF. In a 10 mL vial, Compound 1 (528 mg, 0.50 mmol) was added to a mixture of CH3CN (1 ml) and 1.5 ml TBAF / THF (1.0 M / L THF) solution, and the resulting mixture was stirred at 20-25 °C for 30 -120 minutes. HPLC indicated the reaction conversion was 92.5% at 60 min and 98.8% at 120 min.Table 2MEI 52290585v.1123429-12620

[0122] Preparation of compound 3a. In 10 mL vial, HF-py (65 pL, 2.5 mmol HF, -70% HF in pyridine) was slowly added the solution of imidazole (170 mg, 1.5 mmol) in CH3CN (2.5 mL) at 20-25 °C, and the mixture was stirred at 20-25 °C for another 10 min to yield a homogeneous solution. Compound 3 (605 mg, 0.50 mmol) was added to the HF / Imidazole CH3CN solution in one portion, and the resulting mixture was stirred at 20-25 °C for 30 minutes (HPLC indicated the reaction conversion > 99.0%).

[0123] The reaction mixture was diluted with EtOAc (20 mL) and water (20 mL) to yield two layers. The organic layer (upper layer) was washed with 4% NaHCCh (20 mL), water (20 mL), and brine (20 mL), dried over MgSCU (1 g), filtered, and concentrated. The crude product was purified by a short silica gel column chromatography (CH2C12-EtOAc, 10:1 to 1 :2, v / v) to afford compound 3a (412mg, 75% yield).1H NMR (500 MHz, DMSO-tL) 6 ppm 1.85 - 1.90 (m, 3 H), 2.37 - 2.56 (m, 2 H), 3.29 - 3.42 (m, 2 H), 3.74 (s, 6 H), 4.03 - 4.48 (m, 4 H), 4.94 - 5.13 (m, 2 H), 5.62 (dd, J= 19.6, 5.4 Hz, 1 H), 6.13 - 6.19 (m, 1 H), 6.40 - 6.45 (m, 1 H), 6.83 - 6.88 (m, 4 H), 7.15 - 7.33 (m, 6 H), 7.44 (t, J= 8.4 Hz, 2 H), 7.57 - 7.62 (m, 4 H), 7.67 - 7.75 (m, 2 H), 8.00 - 8.12 (m, 5 H), 8.62 - 8.69 (m, 2 H), 11.27 - 11.44 (m, 2 H).13C NMR (125 MHz, DMSO- de) 6 ppm 12.5, 12.9, 36.9, 55.4, 63.9, 64.3, 65.8, 65.9, 72.7, 72.8, 74.6, 76.9, 77.2, 83.3, 83.5, 83.6, 83.7, 83.8, 84.2, 86.1, 87.8, 88.3, 110.8, 110.9, 113.6, 121.8, 126.3, 128.2, 128.9, 129.0, 129.3, 129.8, 130.2, 132.9, 133.8, 135.9, 136.4, 143.6, 145.2, 150.9, 151.2, 152.0, 152.7, 158.5, 164.3, 165.7, 166.2. HRMS calcd for C55H53N7O14SP+[M+H]+1098.3104, found: 1098.3156.

[0124] Desilylation of compound 3 through TBAF. In a 10 mL vial, Compound 3 (605 mg, 0.50 mmol) was added to 2.5 ml TBAF / THF (1.0 M / L THF) solution, and the resulting mixture was stirred at 20-25 °C for 30 -180 minutes. HPLC indicated the reaction conversion was 84.1% at 60 min and 98.4% at 180 min.Table 327MEI 52290585v.1123429-12620

[0125] In summary, Tables 1-3 above show that the HF / imidazole conditions deprotect the 2'-OH silyl group much faster than the conventional TBAF conditions when the same equivalent desilylation reagents are used. Furthermore, the conversion rates under the HF / imidazole conditions are higher than the ones under the TBAF conditions.Example 2. Sythetic Procedures for Preparing Compound 4a Stepwise

[0126] Desilylation of compound 4 (ammonolysis and desilylation stepwise process):

[0127] A 6-mer (Us)sdT sequence (compound 4) was synthesized on an AKTA oligopilot 100 system, 1 mmol scale with UnyLinker 350 solid support. At the end of the cycle, the sequence was cleaved and deprotected by incubating the dried solid support in a (1 : 1) solution of ammonium hydroxide 30% in H2O / 30% MeNH2 in H2O (AMA) at 65 °C for 30 min. The solid support beads were removed through filtration and the product was dried by 28MEI 52290585v.1123429-12620evaporating the solution. Treatment with AMA led to the loss of some TBDMS groups from the final sequence (Figure 4B). The dried product residue was then used to test the HF-Imidazole (1.0 M in THF) desilylation reaction and compared with TBAF (1.0 M in THF). The results show clean and efficient desilylation of 4 with HF-Imidazole (10eq) at room temperature in just 15 minutes(Figure 4C). However, TBAF (30 eq) at room temperature did not work (Figure 4D). Application of heat (65 °C) was necessary for TBAF to work (Figure 4E).Example 3. Sythetic Procedures for Preparing Compound 4a in One-Pot Preparation of compound 4a through the ammonolysis and desilylation one pot process

[0128] A 6-mer (Us)sdT sequence (compound 4S) was synthesized on an AKTA oligopilot 100 system, 1 mmol scale with UnyLinker 350 solid support. At the end of the cycle, the sequence was cleaved and deprotected by incubating the dried solid support in a (1 : 1) solution of ammonium hydroxide / methyl amine (AMA) at 65 °C for 30 min. After cooling down the reaction mixture to room temperature, HF-Imidazole (1.0 M in THF) and TBAF (1.0 M in THF) were directly added to the cleaved and deprotected product solution.Surprisingly, full desilylation of compound 4 was achieved with HF-Imidazole in one-pot (Figure 5A), but TBAF did not work under one-pot condition (Figure 5B). The application of 29MEI 52290585v.1123429-12620heat generated some of the desired product, however, the level of side products also increased significantly (Figure 5C).Example 4. Sythetic Procedures for Preparing Compound 5a in One-Pot

[0129] Compound 5 was synthesized on an AKTA oligopilot 100 system, with a 1 mmol scale and UnyLinker 350 solid support. At the end of the cycle, the sequence was cleaved and deprotected by incubating the dried solid support (500 mg) in a 1:1 solution (5 ml) of ammonium hydroxide 30% in H2O / 30% MeNFfc in H2O at 25°C for 120 min. The solid support beads were removed through filtration. 100 ul ammonolysis solution was added to 100 ul DMSO and treated with HF-Imidazole (Figure 6B) and TBAF (Figure 6C) separately. Surprisingly, full desilylation of compound 5 was achieved with HF-Imidazole in one-pot (Figure 6B), but TBAF did not work under one-pot condition (Figure 6C). Figures 7A and 7B are mass spectrums of compounds 5 and 5a, respectively.Example 5. Sythetic Procedures for Preparing Compound 6a in One-Pot

[0130] The synthetic scheme for preparing compound 6a is depicted in Figure 8.MEI 52290585v.1123429-12620

[0131] Compound 6 was synthesized on an AKTA oligopilot 100 system, with a 1 mmol scale and UnyLinker 350 solid support.

[0132] Ammonolysis: 500 mg of compound 6 was added to a mixture of 30% MeNtt in H2O (1.25 mL) and 30% NHs^fbO (1.25 mL) in one portion at 20-25 °C. The mixture was shaken at 20-25 °C for 2 hours. Then 250 ul of the reaction mixture was added to DMSO 250 ul with 10 eq HF / Imidazole (per TBDMS) or 10 eq TBAF (per TBDMS). Sitr the mixture at 25 °C and check the desilylation conversion by LCMS (Figures 9B and 9C). Surprisingly, full desilylation of compound 6 was achieved with HF-Imidazole in one-pot (Figure 9C), but the TBAF desilylation conversion was very low under one-pot condition (Figure 9B). Figures 10A and 10B are mass spectrums of compounds 6 and 6a, respectively.Example 6. Desilylation of Cyanoethyl Deprotected Dimer vs Desilylation of Fully Protected Dimer

[0133] The synthetic scheme for a 2'-OH desilylation of a fully protected dimer is depicted below:

[0134] Figue 11 A is a LV-UV chromatograph showing UV trace of a desilylation reaction of the fully protected dimer above.

[0135] As shown in Figure 11 A, a direct desilylation of fully protected RNA fragments (the base and the phosphate are protected) produced many by-products, including 3’ to 2’ phosphate migration and chain cleavage byproducts (See ATDBio, S.-P. O. S., Nucleic Acids Book, 20 pages, Published on Jul. 31, 2011; Morgan, M. A.; Kazakov, S. A.; Hecht, S. M., Phosphoryl migration during the chemical synthesis of RNA. Nucleic Acids Res. 1995, 23 (19), 3949-3953; Reese, C. B., Oligo-and poly-nucleotides: 50 years of chemical synthesis. Org. Biomol. Chem.2005, 3 (21), 3851-3868). By-products 1-5 and De-CE by-product are depicted in Figure 1 IB.

[0136] Suprisingly, the process of the present disclosure minimized these by-products. Below is a synthetic scheme for preparing a dimer without the cyanoethyl ("CE") protecting group:31MEI 52290585v.1123429-12620

[0137] The synthetic procedure is described below.

[0138] Preparation of compound Dimer-SM-CE Off. A mixture of DMT-2'-O-TBDMS-A(Bz)-CE-Phosphoramidite (3.77 g, 3.8 mmol, 1.2 equiv) and 5'-HO-Deoxy-T-3'-OBz (1.1 g, 3.2 mmol, 1.0 equiv) in CH3CN-CH2CI2 (40 mL, 1:1, v / v) was stirred at 20-25 °C for 10 min. DCI (0.75 g, 6.4 mol, 2.0 equiv) was added and the reaction mixture was stirred at 20-25 °C for 0.5 h. DDTT (0.70 g, 3.5 mmol, 1.1 equiv) was added and the mixture was stirred at 20-25 °C for 10 min. The reaction mixture was diluted with EtOAc (100 mL) and was washed with 4% NaHCOs aqueous solution (50 mL) and saturated brine (50 ml), dried over MgSCU (5 g), filtered, and concentrated. The crude product was dissolved in EtsN / CEECN (100 mL, 1:1, v / v) and stirred at 20-25 °C for 7 h. The mixture was concentrated under vacuum, and the crude product was purified by a short silica gel column chromatography (CH2Cl2-EtOAc, 10:1 to 1:1, v / v) to afford Dimer-SM-CE off (2.4 g, 63% yield). 'H NMR (400 MHz, DMSO-t / q) 6 ppm -0.28 - -0.17 (m, 3 H), -0.05 - 0.0 (m, 3 H), 0.59 - 0.67 (m, 9 H), 1.83 - 1.86 (m, 3 H), 2.26 (br s, 1 H), 2.21 - 2.26 (m, 1 H), 2.35 - 2.38 (m, 1 H), 3.37 - 3.43 (m, 2 H), 3.63 - 3.73 (m, 6 H), 3.90 - 4.15 (m, 2 H), 4.24 - 4.30 (m, 1 H), 4.62 - 4.70 (m, 1 H), 4.92 - 5.03 (m, 1 H), 5.17 - 5.19 (m, 1 H), 5.52 - 5.62 (m, 1 H), 6.10 - 6.15 (m, 1 H), 6.31 - 6.35 (m, 1 H), 6.76 - 6.83 (m, 4 H), 7.14 - 7.30 (m, 7 H), 7.39 - 7.45 (m, 2 H), 7.50 -7.57 (m, 4 H), 7.61 - 7.69 (m, 2 H), 7.96 - 8.06 (m, 5 H), 8.53 - 8.55 (m, 2 H), 11.19 (s, 1 H), 11.29 - 11.31 (m, 1 H).13C NMR (125 MHZ, DMSO-t / 6) 6 ppm -5.14, -5.12, 12.6, 18.1, 36.8, 36.9, 55.4, 64.4, 64.5, 65.0, 65.7, 74.1, 74.5, 74.8, 77.0, 77.4, 83.6, 83.7, 84.0, 84.2, 84.4, 86.1, 87.7, 87.8, 110.8, 110.9, 113.2, 113.4, 126.3, 128.2, 128.3, 128.9, 129.0, 129.3, 129.8, 130.3, 132.9, 134.0, 135.9, 136.8, 143.6, 145.4, 150.9, 151.1, 152.0, 152.7, 158.5, 164.3, 165.6, 166.1. FIRMS calcd for C61H67N7O14PSSE [M+H]+1212.3968, found: 1212.4040.

[0139] Preparation of compound Dimer-product-CE Off. In 10 mL vial, HF-py (65 pL, 2.5 mmol HF, -70% HF in pyridine) was slowly added the solution of imidazole (170 mg, 1.5 mmol) in CH3CN (2.5 mL) at 20-25 °C, and the mixture was stirred at 20-25 °C forMEI 52290585v.1123429-12620another 10 min to yield a homogeneous solution. Compound Dimer-SM-CE Off (605 mg, 0.50 mmol) was added to the HF / Imidazole CH3CN solution in one portion, and the resulting mixture was stirred at 20-25 °C for 30 minutes (HPLC indicated the reaction conversion > 99.0%).

[0140] The reaction mixture was diluted with EtOAc (20 mL) and water (20 mL) to yield two layers. The organic layer (upper layer) was washed with 4% NaHCCh (20 mL), water (20 mL), and brine (20 mL), dried over MgSCU (1 g), filtered, and concentrated. The crude product was purified by a short silica gel column chromatography (CLLCh-EtOAc, 10:1 to 1 :2, v / v) to afford Dimer-product-CE Off (412mg, 75% yield).JH NMR (500 MHz, DMSO-tL) 6 ppm 1.85 - 1.90 (m, 3 H), 2.37 - 2.56 (m, 2 H), 3.29 - 3.42 (m, 2 H), 3.74 (s, 6 H), 4.03 - 4.48 (m, 4 H), 4.94 - 5.13 (m, 2 H), 5.62 (dd, J= 19.6, 5.4 Hz, 1 H), 6.13 - 6.19 (m, 1 H), 6.40 - 6.45 (m, 1 H), 6.83 - 6.88 (m, 4 H), 7.15 - 7.33 (m, 6 H), 7.44 (t, J= 8.4 Hz, 2 H), 7.57 - 7.62 (m, 4 H), 7.67 - 7.75 (m, 2 H), 8.00 - 8.12 (m, 5 H), 8.62 - 8.69 (m, 2 H), 11.27 - 11.44 (m, 2 H).13C NMR (125 MHz, DMSO- 1 / 6) 6 ppm 12.5, 12.9, 36.9, 55.4, 63.9, 64.3, 65.8, 65.9, 72.7, 72.8, 74.6, 76.9, 77.2, 83.3, 83.5, 83.6, 83.7, 83.8, 84.2, 86.1, 87.8, 88.3, 110.8, 110.9, 113.6, 121.8, 126.3, 128.2, 128.9, 129.0, 129.3, 129.8, 130.2, 132.9, 133.8, 135.9, 136.4, 143.6, 145.2, 150.9, 151.2, 152.0, 152.7, 158.5, 164.3, 165.7, 166.2. HRMS calcd for C55H53N7Oi4SP+[M+H]+1098.3104, found: 1098.3156.

[0141] Figure 12 is a LV-UV chromatograph showing UV trace of a desilylation reaction of the Dimer-SM-CE Off above. The chromatograph shows that the desilylation reaction of the present disclosure can produce the deprotected product with high yield and without significant amount of byproducts observed for the direct desilylation of fully protected RNA.

[0142] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.MEI 52290585v.1123429-12620

[0143] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.MEI 52290585v.1

Claims

123429-12620CLAIMSWhat is claimed is:

1. A process for preparing a compound of formula (I):or a salt thereof by a deprotection process, wherein the deprotection process comprises deprotecting a compound of formula (II) with HF in the presence of a base:or a salt thereof, to form the compound of formula (I), or a salt thereof, wherein:R1, for each occurrence, is independently a nucleobase;35MEI 52290585v.1123429-12620R2, for each occurrence, is independently selected from the group consisting of H and OH, and at least one R2is OH;R2, for each occurrence, is independently selected from the group consisting of H and OR2a, and at least one R2is OR2a; wherein R2ais a silyl hydroxyl protecting group;R4, for each occurrence, is independently H or forms a ring with the H group of R2or R2;R15is H or a hydroxyl protecting group;q is an integer from 0 to 200;X, for each occurrence, is independently O or S; andY is H or a hydroxyl protecting group.

2. The process of claim 1, wherein the deprotection reaction is carried out in water.

3. The process of claim 1 or 2, wherein the base is an optionally substituted imidazole or an optionally substituted pyridine.

4. The process of any one of claims 1-3, wherein the deprotection reaction is carried out by reacting the compound of formula (II) with HF in the presence of pyridine and imidazole.

5. The process of any one of claims 1-4, wherein excess amount of base relative to HF is used.

6. The process of any one of claims 1-5, wherein the base is imidazole.

7. The process of claim 6, wherein the molar ratio of imidazole to HF is in the range of 1.1:1 to 5:l.

8. The process of claim 7, wherein the molar ratio of imidazole to HF is 2: 1.

9. The process of any one of claims 1-5, wherein the base is pyridine.

10. The process of claim 9, wherein the molar ratio of pyridine to HF is in the range of 1.1:1 to 20:1.

11. The process of any one of claims 1-10, wherein the molar ratio of HF to every R2aof the compound of formula (II) is 30:1.36MEI 52290585v.1123429-1262012. The process of any one of claims 1-11, wherein the molar ratio of HF to every R2aof the compound of formula (II) is 10:1.

13. The process of any one of claims 1-12, wherein the molar ratio of HF to every R2aof the compound of formula (II) is 5:1.

14. The process of any one of claims 1-13, wherein the molar ratio of HF to every R2aof the compound of formula (II) is 3 : 1.

15. The process of any one of claims 1-14, wherein R2ais a silyl protecting group selected from TBDPS, TBoDPS and TBDMS:TBDPS TBoDPS TBDMS16. The process of claim 15, wherein R2ais TBDMS.

17. The process of claim 15, wherein R2ais TBDPS.

18. The process of any one of claims 1-17, wherein R15is H or 4,4’-dimethoxytrityl group.

19. The process of any one of claims 1-17, wherein R15is 4,4’-dimethoxytrityl group.

20. The process of any one of claims 1-19, wherein R4is H.

21. The process of any one of claims 1-19, whereineach R4is independently H or forms a ring with the H group of R2, wherein the ring is a 5 or 6-membered ring optionally substituted with 1 to 3 Ci-4 alkyl groups.

22. The process of any one of claims 1-21, wherein q is an integer from 1 to 50.

23. The process of any one of claims 1-22, wherein q is an integer from 1 to 20.37MEI 52290585v.1123429-1262024. The process of any one of claims 1-23, wherein q is an interger is from 10 to 20.

25. The process of any one of claims 1-24, wherein all of the P=X groups in the compound or oligonucleotide are P=S.

26. The process of any one of claims 1-24, wherein all of the P=X groups in the compound or oligonucleotide are P=O.

27. The process of any one of claims 1-24, wherein greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the P=X groups in the compound or oligonucleotide are P=S.

28. The process of any one of claims 1-24, wherein 10-90%, 20-80%, 30-70% or 40-60% of the P=X groups in the compound or oligonucleotide are P=S.

29. The process of any one of claims 1-28, wherein the nucleobase represented by R1is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine, wherein the NH2 group of said cytosine, guanine, adenine, 7-methylguanine, 5-methylcytosine, or 5-hydroxymethylcytosine represented by R1is not protected.

30. The process of any one of claims 1-28, wherein the nucleobase represented by R1is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, 7-methylguanine, and 5-methylcytosine, wherein the NH2 group of said cytosine, guanine, adenine, 7-methylguanine, or 5-methylcytosine represented by R1is not protected.

31. The process of any one of claims 1-28, wherein the nucleobase represented by R1is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, and 5-methylcytosine, wherein the NH2 group of said cytosine, guanine, adenine, or 5-methylcytosine represented by R1is not protected.

32. The process of any one of claims 1-28, wherein the nucleobase represented by R1is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine, wherein the NH2 group of said cytosine, guanine, adenine, 7-38MEI 52290585v.1123429-12620methylguanine, 5-methylcytosine, or 5-hydroxymethylcytosine represented by R1is protected by an amine protecting group.

33. The process of claim 32, wherein the amine protecting group is PhCO-, CH3CO-, zPrCO-, Me2N-CH=, or Me2N-CMe=.

34. The process of any one of claims 1-33, wherein Y is H.

35. The process of any one of claims 1-33, wherein Y is PhCO-.

36. The process of any one of claims 1-35, whereinR2, for each occurrence, is OH;R2, for each occurrence, is OR2a; wherein R2ais TBDMS; andR15is 4,4’-dimethoxytrityl group.

37. The process of any one of claims 1-36, wherein the reaction time is less than 10 hours.

38. The process of any one of claims 1-37, wherein the reaction time is about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 0.5 hour, or about 0.2 hour.

39. The process of any one of claims 1-38, wherein the reaction time is about 6 hours.

40. The process of any one of claims 1-39, wherein the compound of formula (II) is prepared by an ammonolysis process; wherein the ammonolysis process comprises reacting a compound of formula (III) or a salt thereof with a base:MEI 52290585v.1123429-12620whereinR1A, for each occurrence, is independently a nucleobase, wherein the NH2 of the nucleobase, if present, is protected by an amine protecting group;R2, for each occurrence, is independently selected from the group consisting of H and OR2a, and at least one R2is OR2a; wherein R2ais a silyl hydroxyl protecting group;R4, for each occurrence, is independently H or forms a ring with the H group of R2; R15is H or a hydroxyl protecting group;R16A, for each occurrence, is independently Ci-ealkyl group, C2-ealkenyl group, phenyl or benzyl group, each of which is optionally substituted with -CN, -NO2 or halogen; orZ is H, a hydroxyl protecting group, or a solid support.

41. The process of claim 40, wherein the base is NH4OH or EtsN.

42. The process of claim 40, wherein the base is NH4OH.

43. The process of claim 42, wherein the reaction between the compound of Formula (III) or a salt thereof and NH4OH is carried out in the presence of methylamine.

44. The process of claim 40, wherein the ammonolysis process is carried out at a temperature in the range between 40°C and 75°C.

45. The process of claim 40 or 44, wherein the compound of Formula (II) prepared by the ammonolysis process is used without purification before the deprotection process to prepare the compound of Formula (I).

46. The process of claim 45, wherein the deprotection process and the ammonolysis process are carried out in one pot.

47. The process of claim 40, wherein the deprotection process and the ammonolysis process are carried out in stepwise.40MEI 52290585v.1123429-1262048. The process of any one of claims 40-47, wherein Z is a solid support.

49. The process of any one of claims 40-47, wherein Z is PhCO-.

50. The process of any one of claims 40-49, wherein the nucleobase represented by R1Ais selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine, wherein the NH2 group ofsaid cytosine, guanine, adenine, 7-methylguanine, 5-methylcytosine, or 5-hydroxymethylcytosine represented by R1Ais protected by PhCO-, CH3CO-, / PrCO-, Me2N-CH=, or Me2N-CMe=.

51. The process of any one of claims 40-50, wherein the nucleobase represented by R1Ais selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, 7-methylguanine, and 5-methylcytosine, wherein the NH2group of said cytosine, guanine, adenine, 7-methylguanine, or 5-methylcytosine represented by R1Ais protected by PhCO-, CH3CO-, zPrCO-, Me2N-CH=, orMe2N-CMe=.

52. The process of any one of claims 40-51, wherein the nucleobase represented by R1Ais selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, and 5-methylcytosine, wherein the NH2group of said cytosine, guanine, adenine, or 5-methylcytosine represented by R1Ais protected by PhCO-, CH3CO-, zPrCO-, Me2N-CH=, or Me2N-CMe=.

53. The process of any one of claims 40-52, R16Ais -CH2CH2CN.41MEI 52290585v.1

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