Reagents and processes for preparing oligonucleotides

Abstract

The present disclosure describes novel reagents and processes for preparing oligonucleotides, which have two or more nucleotides. In one embodiment, the reagent is represented by Formula I. wherein: W is O or NH; R1 is -H or a solid support; R2 is a silyl hydroxyl protecting group or an alloc protecting group, R3 is a nucleoside or a group represented by (II) wherein (III) represents the point of attachment for R3.

Description

123429-12220 REAGENTS AND PROCESSES FOR PREPARING OLIGONUCLEOTIDES RELATED 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 / 644,768, filed on May 9, 2024, the entire contents of which is incorporated herein by reference. FIELD OF THE INVENTION

[0002] The present invention relates to reagents and processes for preparing oligonucleotides.BACKGROUND

[0003] Oligonucleotides are short DNA or RNA oligomers that can be chemicallysynthesized for a wide range of applications. Recent developments in utilizing synthetic oligonucleotides as therapeutic agents have increased demand for synthetic methods that can produce oligonucleotides in large quantities with high efficiency and purity.

[0004] Traditionally, oligonucleotides were synthesized by a solid phase automatedsynthesizer utilizing phosphoramidite chemistry, limited to a scale of less than 2 moles. Thus, the solid phase synthesis is insufficient for the production of materials needed for clinical development and commercialization of oligonucleotide drugs in large indications. In addition, the solid phase synthesis often requires the use of excess reagents and consequently increases the cost associated with the production of the target oligonucleotides. Additional challenges and issues associated with the solid phase synthesis ("SPS"), for example, include: 1) the oligonucleotide from the current SPS process suffered from the elevated level of n-1st impurities, generated due to the lower coupling efficiency in the first cycle, which is also difficult to remove in purification; 2) the SPS overall yield (65-75%) needs improvement, which is hindered by the fact that the formation mechanism of the early eluting impurities (EEIs, accounting 20% of the synthesis output) is unknown due to the unselective one pot ammonolysis deprotection condition; 3) synthesis of clinically useful di-, tri-oligonucleotides through the current SPS process is challenging since it usually only provides low overall purity with the requirement of huge excess of amidites.

[0005] Recently, liquid phase synthesis (LPS) of oligonucleotides were developed to helpsolve these probelms. For example, WO2020 / 227618 discloses a convergent liquid phase synthesis of oligonucleotides. WO2022 / 103842 discloses reagents and methods for the LPS of oligonucleotides. While the LPS provides a flexible and beneficial tool in the large scale synthesis, it has its own limitation which mainly related to efficiency. For example, an 1 ME133307051v.1123429-12220 olioguneucleotide fragment (4-5mer) synthesis process development is time-consuming, requiring 2-3 weeks even without any interruption. In addition, one ASO sequence needs to prepare 20-30 impurity markers for analytical method development and validation. Preparing those impurity markers from LPS takes lots of time since every impurity marker needs de novo synthesis.

[0006] Hence, there is a need for novel reagents and robust methods for synthesizingoligonucleotides that are suitable for large scale manufacturing process with high efficiency and purity. SUMMARY OF THE INVENTION

[0007] The present disclosure provides novel reagents / resins with the following features: 1)pre-installed first nucleoside on the resin to avoid the low coupling efficiency issue in the first cycle; 2) orthogonal resin cleavage condition other than widely used ammonolysis condition. This new cleavage condition would keep the real EEIs structure intact, which can be then be used to understand the EEI formation mechanism, laying the foundation for reducing EEIs to increase the overall SPS yield. Moreover, coupling two or three single strands of oligonucleotides would be easier to achieve since the oligonucleotides remain protected, providing more reaction conditions to explore other than the aqueous conditions needed when oligonucleotides are deprotected after ammonolysis.

[0008] One aspect of the present disclosure is directed to a compound of Formula I:, I or a salt thereof; wherein ring W, R1, R2, and R3are defined below.

[0009] One aspect of the present disclosure is directed to a process for preparing anoligonucleotide of formula (II), 2 ME138157184v.1123429-12220or a salt thereof, comprising the steps of: 1) deprotecting a compound of formula (IIA):or a salt thereof, to form a compound of formula (IIB):2) reacting the compound of formula (IIB), or a salt thereof, with a compound of formula (IIC):or a salt thereof, to form a compound of formula (IID), 3 ME138157184v.1123429-12220salt thereof; 3) sulfurizing or oxidizing the compound of formula (IID), or a salt thereof, with a sulfurization or oxidation agent to form a compound of formula (IIE):salt thereof; 4) deprotecting the compound of formula (IIE), or a salt thereof to form a compound of formula (IIF):(IIF), or a salt thereof; 4 ME138157184v.1123429-12220 5) when q is equal or greater than 2, starting with the compound of formula (IIF), repeating steps 2), 3) and 4) for q-2 times, followed by steps 2) and 3) to yield the oligonucleotide of formula (II), or a salt thereof, wherein R8, R9, R10, R11, R12, R13a, R13b, q, X, and Z are defined below. BRIEF DESCRIPTION OF THE FIGURES

[0010] FIG. 1 shows a retro-synthesis scheme for preparing an 18-mer oligonucleotide.

[0011] FIG. 2 shows a HPLC chart and a MS / UV spectrum of the CCCA oligonucleotidefragment prepared from Silyl resin or NittoPhase HL Unylinker resin. The HPLC chart shows the purity profiles of the oligonucleotide fragments while the MS spectrum shows the MS profile of the oligonucleotide fragment.

[0012] FIG. 3 shows a HPLC chart and a MS / UV spectrum of the CCATT oligonucleotidefragment prepared from Silyl resin or NittoPhase HL Unylinker resin. The HPLC chart shows the purity profiles of the oligonucleotide fragment while the MS / UV spectrum shows the MS profile of the oligonucleotide fragment.

[0013] FIG. 4 shows a HPLC chart and a MS / UV spectrum of the CTTU oligonucleotidefragment prepared from Silyl resin or NittoPhase HL Unylinker resin. The HPLC chart shows the purity profiles of the oligonucleotide fragment while the MS / UV spectrum shows the MS profile of the oligonucleotide fragment.

[0014] FIG. 5 shows a HPLC chart and a MS / UV spectrum of the UGCC oligonucleotidefragment prepared from Silyl resin or NittoPhase HL Unylinker resin. The HPLC chart shows the purity profiles of the oligonucleotide fragment while the MS / UV spectrum shows the MS profile of the oligonucleotide fragment.

[0015] FIG. 6 shows two HPLC charts that compare the purity profiles of the CCCAoligonucleotide fragment prepared from Silyl resin or liquid phase synthesis.

[0016] FIG. 7 shows two HPLC charts that compare the purity profiles of the CCATToligonucleotide fragment prepared from Silyl resin or liquid phase synthesis.

[0017] FIG. 8 shows two HPLC charts that compare the purity profiles of the CTTUoligonucleotide fragment prepared from Silyl resin or liquid phase synthesis.

[0018] FIG. 9 shows two HPLC charts that compare the purity profiles of the UGCColigonucleotide fragment prepared from Silyl resin or liquid phase synthesis.

[0019] FIG. 10 shows two MS spectrums that compare the MS profiles of the UGCColigonucleotide fragment prepared from Silyl resin or liquid phase synthesis. 5 ME138157184v.1123429-12220

[0020] FIG. 11 shows a HPLC chart and a MS / UV spectrum of the 18 mer oligonucleotideprepared from Silyl resin. The HPLC chart shows the purity profiles of the 18 mer oligonucleotide fragment while the MS / UV spectrum shows the MS profile of the 18 mer oligonucleotide.

[0021] FIG. 12 shows a HPLC chart and a MS / UV spectrum of the 18 mer oligonucleotideprepared from Liquid-phase synthesis. The HPLC chart shows the purity profiles of the 18 mer oligonucleotide fragment while the MS / UV spectrum shows the MS profile of the 18 mer oligonucleotide. DETAILED DESCRIPTION Definitions

[0022] The term "about" is used herein to mean approximately, roughly, around, or in theregions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower).

[0023] The term “solid support” refers to any support that is compatible with oligonucleotidesynthesis 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 hydroxyl functional group. In some embodiments, the solid support of the present disclosure is selected from cross-linked polystyrene beads with hydroxyl functional group or controlled pore glass beads with hydroxyl functional group.

[0024] The term “nucleobase” means the heterocyclic base portion of a nucleoside.Nucleobases may be naturally occurring or may be modified. In certain embodiments, a 6 ME138157184v.1123429-12220 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.

[0025] The term “nucleoside” means a compound comprising a heterocyclic base moiety anda sugar moiety, which can be modified at the 2’-end.

[0026] The term “nucleotide” means a nucleoside comprising a phosphate or thiophosphateor dithiophosphate linking group.

[0027] The term "oligonucleotide" refers to a compound comprising a plurality of linkednucleosides. 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).

[0028] As used herein, “target oligonucleotide” refers to the oligonucleotide product that canbe prepared based on the reagents and the processes of the present disclosure. In certain embodiments, the target oligonucleotide comprises at least 10 or at least 15 nucleotides. In certain embodiments, the target oligonucleotide has 10 to 500, 15 to 500, 15 to 200, 15 to 100, 15 to 50, 15 to 40, 15 to 30 or 16 to 30 nucleotides.

[0029] As used herein, “oligonucleotide fragments” refers to short oligonucleotides that areassembled to make the target oligonucleotide. In certain embodiments, the oligonucleotide fragment has 3 to 10, 3 to 8, 3 to 6 or 4 to 6 nucleotides. In certain embodiments, the oligonucleotide fragment has 4 or 5 nucleotides.

[0030] As used herein, the term "alkyl" refers to a fully saturated branched or unbranchedhydrocarbon 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, iso- 7 ME138157184v.1123429-12220 propyl, 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.

[0031] As used herein, the term "alloc protecting group" refers to a protecting groupconprising a moiety of allyloxycarbonyl, which is capable of protecting amines, hydroxyl, or amino acids. In one embodiment, the alloc protecting group is.

[0032] The term "aryl" refers to monocyclic, bicyclic or tricyclic aromatic hydrocarbongroups 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.

[0033] The term "aryl" also refers to a bicyclic or tricyclic group in which at least one ring isaromatic and is fused to one or two non-aromatic hydrocarbon ring(s). Nonlimiting examples include tetrahydronaphthalene, dihydronaphthalenyl and indanyl.

[0034] 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 7-ring members, or 3- to 6- 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 quaternized, 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 heterocyclyls 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, 8 ME138157184v.1123429-12220 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, oxadiazolyl, 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.

[0035] "Halogen" or "halo" may be fluoro, chloro, bromo or iodo.

[0036] As used herein, a “hydroxyl protecting group” refers to a group that is suitable forprotecting 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).

[0037] In certain embodiments, the hydroxyl protecting groups can be selected from, forexample, acetyl (Ac); benzoyl (Bz); benzyl (Bn); β-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, trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS), tert-butoxydiphenylsilyl (TBoDPS), triphenylsilyl (TPS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers); methyl ethers, and ethoxyethyl ethers (EE).

[0038] In certain embodiments, the hydroxyl protecting group protects the 2’ –hydroxyl of anucleoside (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-butoxydiphenylsilyl. In certain embodiments, the 2’- hydroxyl protecting group is TBDPS. 9 ME138157184v.1123429-12220

[0039] The suffic “yl” added to the end of a chemical name indicates that the named moietyis 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.

[0040] In certain embodiments, the hydroxyl protecting group protects the 5’ –hydroxyl of anucleoside (referred to as 5’-hydroxyl protecting group). Exemplary 5’-hydoxyl groups include, but are not limited to those as described herein (e.g., R11in any of the aspects or 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.

[0041] 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, triethylamine, diisopropylethylamine, 2-methylpropan-2-amine), 8-diazabicyclo[5.4.0]undec-7-ene (DBU), imidazole, N-methylimidazole, pyridine and 3-picoline.

[0042] 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.

[0043] As used herein, the term “controlled pore glass” refers to a high silica glass thatincludes pores with a particular size distribution. Porous glasses can be fabricated into a wide variety of geometric forms (such as beads, frit, plates, rods and hollow spheres), and pore sizes can be tuned precisely from the range of angstroms to millimeters. 1. Reagent

[0044] In a first aspect, the present disclosure provides reagents for facilitating the synthesisof oligonucleotides.

[0045] In a first embodiment of the first aspect, the present disclosure provides a compoundof Formula I: 10 ME138157184v.1123429-12220 ,or a salt thereof, wherein: W is O or NH; R1is -H or a solid support; R2is a silyl hydroxyl protecting group or an alloc protecting group; R3is a nucleoside or a group represented bywherein represents the point of attachment for R3.

[0046] In a specific embodiment, the present disclosure provides the compound according tothe first embodiment, or a salt thereof, wherein W is O. The definitions of the remaining variables are provided in the first embodiment.

[0047] In another specific embodiment, the present disclosure provides the compoundaccording to the first embodiment, or a salt thereof, wherein W is NH. The definitions of the remaining variables are provided in the first embodiment.

[0048] In a second embodiment, the present disclosure provides the compound according tothe first embodiment, or a salt thereof, wherein R1is H. The definitions of the remaining variables are provided in the first embodiment or any specific embodiments described therein.

[0049] In a third embodiment, the present disclosure provides a compound according to thefirst embodiment, or a salt thereof, wherein R1is a solid support. The definitions of the remaining variables are provided in the first embodiment or any specific embodiments described therein.

[0050] In a fourth embodiment, the present disclosure provides a compound according to thethird embodiment, or a salt thereof, wherein the solid support is a cross-linked polystyrene bead with a hydroxyl functional group or a controlled pore glass bead with a hydroxyl 11 ME138157184v.1123429-12220 functional group. The definitions of the remaining variables are provided in the third embodiment.

[0051] In a specific embodiment, the present disclosure provides a compound according tothe third embodiment, or a salt thereof, wherein the solid support is a cross-linked polystyrene bead with an amino functional group or a controlled pore glass bead with an amino functional group. The definitions of the remaining variables are provided in the third embodiment.

[0052] In a fifth embodiment, the present disclosure provides a compound according to thefourth embodiment, or a salt thereof, wherein the beads have a loading in the range of about 100 to about 500 µmol / gram. The definitions of the remaining variables are provided in the fourth embodiment. In some embodiments, the loading range is about 100 µmol / gram to about 400 µmol / gram, about 150 µmol / gram to about 400 µmol / gram, or about 200 µmol / gram to about 300 µmol / gram.

[0053] In a sixth embodiment, the present disclosure provides a compound according to thefifth embodiment, or a salt thereof, wherein the beads have a loading of about 250 µmol / gram. The definitions of the remaining variables are provided in the fifth embodiment.

[0054] In a seventh embodiment, the present disclosure provides a compound according toany one of the first through the sixth embodiments, or a salt thereof, wherein R2is a silyl hydroxyl protecting group selected from the following:12 ME138157184v.1123429-12220 ,-O-TBDAS-2; wherein represents the point of attachment for R2; and R5, R6and R7are each independently H, C1-30alkyl, or C1-30alkoxy. The definitions of the remaining variables are provided in any one of the first through the sixth embodiments or any specific embodiments described therein.

[0055] In an eighth embodiment, the present disclosure provides a compound according toany one of the first through the seventh embodiments, or a salt thereof, wherein R2is selected from the group consisting of –O-TBDMS, -O-TIPS, -O-TBDPS, -O-TBoDPS, and –O- TBDAS:,, , , and .13 ME138157184v.1123429-12220 The definitions of the remaining variables are provided in any one of the first through the seventh embodiments or any specific embodiments described therein.

[0056] In a ninth embodiment, the present disclosure provides a compound according to anyone of the first through the eighth embodiments, or a salt thereof, wherein R2is. The definitions of the remaining variables are provided in any one of the first through the eighth embodiments or any specific embodiments described therein.

[0057] In a tenth embodiment, the present disclosure provides a compound according to anyone of the first through the sixth embodiments, or a salt thereof, wherein R2is an alloc protecting group. The definitions of the remaining variables are provided in any one of the first through the sixth embodiments or any specific embodiments described therein.

[0058] In an eleventh embodiment, the present disclosure provides a compound according toany one of the first through the sixth and the tenth embodiments, or a salt thereof, wherein the alloc protecting group is, whereinrepresents the point of attachment for R2. The definitions of the remaining variables are provided in any one of the first through the sixth and the tenth embodiments or any specific embodiments described therein.

[0059] In a twelfth embodiment, the present disclosure provides a compound according toany one of the first through the eleventh embodiments, or a salt thereof, wherein R3is a nucleoside. The definitions of the remaining variables are provided in the first through the eleventh embodiments or any specific embodiments described therein.

[0060] In a thirteenth embodiment, the present disclosure provides a compound according tothe twelfth embodiment, or a salt thereof, wherein the nucleoside is represented by Formula (A): 14 ME138157184v.1123429-12220wherein R8is selected from the group consisting of H, halo, OH, and C1-6alkoxy optionally substituted with C1-6alkoxy or –C(O)NHC1-3alkyl; wherein the OH group is optionally protected by a hydroxyl protecting group; R9is a nucleobase, wherein the NH2of the nucleobase, if present, is protected by an amine protecting group; R10is H or forms a ring with the alkoxy group of R8; R11is a hydroxy protecting group. The definitions of the remaining variables are provided in the twelfth embodiment.

[0061] In certain embodiments, the hydroxyl protecting group of R8 is a silyl protectinggroup. In certain embodiments, the silyl protecting group is selected from the group consisting of 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-butoxydiphenylsilyl.

[0062] In a fourteenth embodiment, the present disclosure provides a compound according tothe thirteenth embodiment, or a salt thereof, wherein R8is selected from the group consisting of H, F, and C1-4alkoxy optionally substitutedwith C1-4alkoxy or –C(O)NHCH3;R10is H or forms a ring with the alkoxy group of R8; wherein the ring is a 5 or 6- membered ring optionally substituted with 1 to 3 C1-4alkyl groups; and R11is a trityl hydroxy protecting group (DMTr). The definitions of the remaining variables are provided in the thirteenth embodiment.

[0063] In a fifteenth embodiment, the present disclosure provides a compound according tothe thirteenth or the fourteenth embodiment, or a salt thereof, wherein R8is selected from H and C1-4alkoxy optionally substituted with C1-4alkoxy; R10is H; and R11is 4,4'-dimethoxytrityl. 15 ME138157184v.1123429-12220 The definitions of the remaining variables are provided in the thirteenth or the fourteenth embodiment.

[0064] In a sixteenth embodiment, the present disclosure provides a compound according toany one of the thirteenth through the fifteenth embodiments, or a salt thereof, wherein R8is selected fromThe definitions of the remaining variables are provided in any one of the thirteenth through the fifteenth embodiments.

[0065] In a seventeenth embodiment, the present disclosure provides a compound accordingto any one of the thirteenth through the sixteenth embodiments, or a salt thereof, wherein the nucleobase is 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 NH2group of the nucleobase, if present, is protected by PhCO-, CH3CO-, iPrCO-, Me2N-CH=, or Me2N-CMe=. The definitions of the remaining variables are provided in any one of the thirteenth through the sixteenth embodiments.

[0066] In an eighteenth embodiment, the present disclosure provides a compound accordingto any one of the thirteenth through the seventeenth embodiments, or a salt thereof, wherein the nucleobase is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, and 5-methylcytosine, wherein the NH2group of the nucleobase, if present, is protected by PhCO-, CH3CO-, iPrCO-, Me2N-CH=, or Me2N-CMe=. The definitions of the remaining variables are provided in any one of the thirteenth through the seventeenth embodiments.

[0067] In a nineteenth embodiment, the present disclosure provides a compound according toany one of the first through the twelfth embodiments, or a salt thereof, wherein R3is represented by the following formula: 16 ME138157184v.1123429-12220wherein Bz is PhCO- and DMTr is dimethoxytrityl. The definitions of the remaining variables are provided in any one of the first through the twelfth embodiments or any specific embodiments described therein.

[068] In a twentieth embodiment, the present disclosure provides the compounds depicted inTable 1 and prepared in the Exemplification, both the neutral form and salts thereof. Table 117 ME138157184v.1123429-1222018 ME138157184v.1123429-12220* is solid support. 2. Process for Preparing Oligonucleotide Fragment

[069] In a second aspect, the present disclosure describes a process of preparing anoligonucleotide on a solid support using the reagent described in this disclosure.

[070] In a twenty-first embodiment, the present disclosure provides a process for preparingan oligonucleotide of formula (II), 19 ME138157184v.1123429-12220or a salt thereof, comprising the steps of: 1) deprotecting a compound of formula (IIA):or a salt thereof, to form a compound of formula (IIB):2) reacting the compound of formula (IIB), or a salt thereof, with a compound of formula (IIC):or a salt thereof, to form a compound of formula (IID), 20 ME138157184v.1123429-12220salt thereof; 3) sulfurizing or oxidizing the compound of formula (IID), or a salt thereof, with a sulfurization or oxidation agent to form a compound of formula (IIE):salt thereof; 4) deprotecting the compound of formula (IIE), or a salt thereof to form a compound of formula (IIF):(IIF), or a salt thereof; 21 ME138157184v.1123429-12220 5) when q is equal or greater than 2, starting with the compound of formula (IIF), repeating steps 2), 3) and 4) for q-2 times, followed by steps 2) and 3) to yield the oligonucleotide of formula (II), or a salt thereof, wherein: R9, for each occurrence, is independently a nucleobase, wherein when the nucleobase comprises an NH2 group, the NH2 of the nucleobase is protected by an amine protecting group; R8, for each occurrence, is independently selected from the group consisting of H, halo, OH, and C1-6alkoxy optionally substituted with C1-6alkoxy or –C(O)NHC1-3alkyl; wherein the OH group is optionally protected by a hydroxyl protecting group; R10, for each occurrence, is independently H or forms a ring with the alkoxy group of R8; R11is a hydroxyl protecting group; R12, for each occurrence, is independently C1-6alkyl group, C2-6alkenyl group, phenyl or benzyl group, each of which is optionally substituted with –CN, -NO2 or halogen; orR13aand R13bare independently C1-6alkyl; q is an integer from 1 to 20; X, for each occurrence, is independently O or S; Z is a group represented by Formula I*or Formula I**,22 ME138157184v.1123429-12220; wherein represents the point of attachment for Z; W is O or NH; R1is a solid support; and R2is a silyl hydroxyl protecting group or an alloc protecting group.

[0071] In a specific embodiment, the present disclosure provides a process according to thetwenty-first embodyment, wherein W is O. The definitions of the remaining variables are provided in the twenty-first embodyment.

[0072] In a specific embodiment, the present disclosure provides a process according to thetwenty-first embodyment, wherein W is NH. The definitions of the remaining variables are provided in the twenty-first embodyment.

[0073] In certain embodiments, the hydroxyl protecting group of R8 is a silyl protectinggroup. In certain embodiments, the silyl protecting group is selected from the group consisting of 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-butoxydiphenylsilyl.

[0074] In a twenty-second embodiment, the present disclosure provides a process accordingto the twenty-first embodyment, wherein Z is a group represented by formula I*. The definitions of the remaining variables are provided in the twenty-first embodyment or any specific embodiments desribed therein.

[0075] In a twenty-third embodiment, the present disclosure provides a process according tothe twenty-first or twenty-second embodyment, wherein R2is a silyl hydroxyl protecting group selected from the following: 23 ME138157184v.1123429-12220 ,and-O-TBDAS-2; wherein represents the point of attachment for R2; and R5, R6 and R7 are each independently H, C1-30alkyl, or C1-30alkoxy. The definitions of the remaining variables are provided in the twenty-first or twenty-second embodyment or any specific embodiments described therein.

[0076] In a twenty-fourth embodiment, the present disclosure provides a process according toany one of the twenty-first through the twenty-third embodiments, wherein R2is selected from the group consisting of –O-TBDMS, -O-TIPS, -O-TBDPS, -O-TBoDPS, and –O- TBDAS: 24 ME138157184v.1123429-12220The definitions of the remaining variables are provided in any one of the twenty-first through the twenty-third embodiments or any specific embodiments described therein.

[0077] In a twenty-fifth embodiment, the present disclosure provides a process according toany one of the twenty-first through the twenty-fourth embodiments, wherein R2is. The definitions of the remaining variables are provided in any one of the twenty-first through the twenty-fourth embodiments or any specific embodiments described therein.

[0078] In a twenty-sixth embodiment, the present disclosure provides a process according tothe twenty-first or twenty-second embodyment, wherein R2is an alloc protecting group. The definitions of the remaining variables are provided in the twenty-first or twenty-second embodyment or any specific embodiments described therein.

[0079] In a twenty-seventh embodiment, the present disclosure provides a process accordingto any one of the twenty-first, the twenty-second, and the twenty sixth embodiments, wherein the alloc protecting group is, whereinrepresents the point of attachment for R2. The definitions of the remaining variables are provided in any one of the twenty-first, the twenty-second, and the twenty sixth embodiments or any specific embodiments described therein.

[0080] In a twenty-eighth embodiment, the present disclosure provides a process according toany one of the twenty-first through twenty seventh embodiments, wherein further comprising a cleavage step of the oligonucleotide of formula (II): 25 ME138157184v.1123429-12220to form an oligonucleotide of formula (IIG) when Z is represented by Formula I*form an oligonucleotide of formula (IIG’) when Z is represented by Formula I** 26 ME138157184v.1123429-12220The definitions of the remaining variables are provided in any one of the twenty-first through the twenty-seventh embodiments or any specific embodiments described therein.

[0081] In a twenty-ninth embodiment, the present disclosure provides a process according tothe twenty-eighth embodiment, wherein Z is represented by Formula I* and the process further comprises a cleavage step of the oligonucleotide of Formula (II) to form an oligonucleotide of Formula (IIG). The definitions of the remaining variables are provided in the twenty-eighth embodiment.

[0082] In a thirtieth embodiment, the present disclosure provides a process according to thetwenty-eighth embodiment, wherein Z is presented by Formula I** and the process further comprises a cleavage step of the oligonucleotide of Formula (II) to form an oligonucleotide of Formula (IIG’). The definitions of the remaining variables are provided in the twenty- eighth embodiment.

[0083] In a thirty-first embodiment, the present disclosure provides a process according tothe thirtieth embodiment, wherein the process further comprises reacting the oligonucleotide of Formula (IIG’) with NH4OH to form the deprotected oligonucleotide of Formula (IIGdep): 27 ME138157184v.1123429-12220, wherein: R9’, for each occurrence, is independently a nucleobase; R8, for each occurrence, is independently selected from the group consisting of H, halo, OH, and C1-6alkoxy optionally substituted with C1-6alkoxy or –C(O)NHC1-3alkyl; wherein the OH group is optionally protected by a hydroxyl protecting group; R10, for each occurrence, is independently H or forms a ring with the alkoxy group of R8; R11is a hydroxyl protecting group; q is an integer from 1 to 20; and X, for each occurrence, is independently O or S. The definitions of the remaining variables are provided in the thirtieth embodiment.

[0084] In a thirty-second embodiment, the present disclosure provides a process according tothe twenty-eighth embodiment or the twenty-ninth embodiment, wherein when R2is defined in any one of the twenty-third through the twenty-fifth embodyments, the cleavage step is carried out by reacting the oligonucleotide of formula (II) with HF in the presence of a base. The definitions of the remaining variables are provided in the twenty-eighth embodiment or the twenty-ninth embodiment.

[0085] In a thirty-third embodiment, the present disclosure provides a process according tothe thirty-second embodiment, wherein the base is imidazole, pyridine or a combination thereof, wherein the imidazole or pyridine are optionally substituted. The definitions of the remaining variables are provided in the thirty-second embodiment. In some embodiments, the base is selected from the group consisting of 28 ME138157184v.1123429-12220.

[0086] In a thirty-fourth embodiment, the present disclosure provides a process according tothe thirty-third embodiment, wherein the cleavage step is carried out by reacting the oligonucleotide of formula (II) with HF in the presence of pyridine and imidazole. The definitions of the remaining variables are provided in the thirty-third embodiment.

[0087] In a thirty-fifth embodiment, the present disclosure provides a process according tothe thirty-fourth embodiment, wherein the molar ratio of imidazole to HF is in the range of 0.5:1 to 10:1. The definitions of the remaining variables are provided in the thirty-fourth embodiment.

[0088] In a thirty-sixth embodiment, the present disclosure provides a process according tothe thirty-fifth 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 thirty-fifth embodiment.

[0089] In a thirty-seventh embodiment, the present disclosure provides a process according tothe thirty-sixth embodiment, wherein the molar ratio of imidazole to HF is 2:1. The definitions of the remaining variables are provided in the thirty-sixth embodiment.

[0090] In a thirty-eighth embodiment, the present disclosure provides a process according toany one of the thirty-fourth through the thirty-seventh embodiments, wherein the molar ratio of pyridine to HF is in the range of 100:1 to 1:100, 10:1 to 1:10, 5:1 to 1:5 or 2:1 to 1:2. The definitions of the remaining variables are provided in any one of the thirty-fourth through the thirty-seventh embodiments. In one embodiment, the molar ratio of pyridine to HF is in the range of 100:1 to 1:1.

[0091] In a thirty-ninth embodiment, the present disclosure provides a process according toany one of the thirty-fourth through the thirty-eighth embodiments, wherein the molar ratio of 29 ME138157184v.1123429-12220 pyridine to HF is 1:1. The definitions of the remaining variables are provided in any one of the thirty-fourth through the thirty-eighth embodiments.

[0092] In some embodiments, the present disclosure provides a process according to thethirty-third embodiment, wherein the cleavage step is carried out by reacting the oligonucleotide of formula (II) with HF in the presence of pyridine and imidazole, wherein the molar ratio of pyridine:HF is 1:1 and the molar ratio of imidazole to HF is 2:1.

[0093] In a fortieth embodiment, the present disclosure provides a process according to thetwenty-eighth embodiment, wherein when R2is an alloc protecting group as defined according to the twenty-sixth embodiment or the twenty-seventh embodiment, the cleavage step is carried out by 1) reacting the oligonucleotide of formula (II) with a palladium catalyst in the presence of an acid; and 2) adding imidazole to the reaction. The definitions of the remaining variables are provided in the twenty-eighth embodiment. In some embodiments, the acid is a barbituric acid.

[0094] In a forty-first embodiment, the present disclosure provides a process according to thefortieth embodiment, wherein the molar ratio of imidazole to the oligonucleotide of formula (II) is in the range of 1:1 to 10:1, 1:1 to 5:1 or 1:1 to 3:1. The definitions of the remaining variables are provided in the fortieth embodiment. In some embodiments, the molar ratio of imidazole to the oligonucleotide of formula (II) is about 2:1.

[0095] In a forty-second embodiment, the present disclosure provides a process according tothe fortieth embodiment or the forty-first embodiment, wherein the molar ratio of palladium catalyst to the oligonucleotide of formula (II) is about 1:100 to about 1:10. The definitions of the remaining variables are provided in the fortieth embodiment or the forty-first embodiment.

[0096] In a forty-third embodiment, the present disclosure provides a process according toany one of the fortieth through the forty-second embodiments, wherein the molar ratio of palladium catalyst to the oligonucleotide of formula (II) is about 1:50. The definitions of the remaining variables are provided in any one of the fortieth through the forty-second embodiments.

[0097] In a forty-fourth embodiment, the present disclosure provides a process according toany one of the fortieth through the forty-third embodiments, wherein the palladium catalyst is 30 ME138157184v.1123429-12220 tetrakis(triphenylphosphine) palladium(0). The definitions of the remaining variables are provided in any one of the fortieth through the forty-third embodiments.

[0098] In a forty-fifth embodiment, the present disclosure provides a process according toany one of the fortieth through the forty-fourth embodiments, wherein the barbituric acid is dimethyl barbituric acid. The definitions of the remaining variables are provided in any one of the fortieth through the forty-fourth embodiments.

[0099] In a forty-sixth embodiment, the present disclosure provides a process according toany one of the twenty-first through the forty-fifth embodiments, wherein q is 2 to 5. The definitions of the remaining variables are provided in any one of the the twenty-first through the forty-fifth embodiments.

[0100] In a forty-seventh embodiment, the present disclosure provides a process according tothe forty-sixth embodiment, wherein q is 4. The definitions of the remaining variables are provided in the forty-sixth embodiment.

[0101] In a forty-eighth embodiment, the present disclosure provides a process according toany one of the twenty-first through the forty-seventh embodiments, wherein all of the P=X groups in the nucleotide or oligonucleotide are P=S. The definitions of the remaining variables are provided in any one of the twenty-first through the forty-seventh embodiments.

[0102] In a forty-ninth embodiment, the present disclosure provides a process according toany one of the twenty-first through the forty-seventh embodiments, wherein all of the P=X groups in the nucleotide or oligonucleotide are P=O. The definitions of the remaining variables are provided in any one of the twenty-first through the forty-seventh embodiments.

[0103] In a fiftieth embodiment, the present disclosure provides a process according to anyone of the twenty-first through the forty-seventh embodiments, wherein greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the P=X groups in the oligonucleotide are P=S. The definitions of the remaining variables are provided in any one of the twenty-first through the forty-seventh embodiments.

[0104] In a fifty-first embodiment, the present disclosure provides a process according to anyone of the twenty-first through the forty-seventh 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 twenty-first through the forty-seventh embodiments.

[0105] In a fifty-second embodiment, the present disclosure provides a process according toany one of the twenty-first through the fifty-first embodiments, wherein the nucleobase is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, 31 ME138157184v.1123429-12220 hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5- hydroxymethylcytosine, wherein the NH2 group of the nucleobase, if present, is protected by PhCO-, CH3CO-, iPrCO-, Me2N-CH=, or Me2N-CMe=. The definitions of the remaining variables are provided in any one of the twenty-first through the fifty-first embodiments.

[0106] In a fifty-third embodiment, the present disclosure provides a process according toany one of the twenty-first through the fifty-second embodiments, wherein the nucleobase is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, and 5- methylcytosine, wherein the NH2 group of the nucleobase, if present, is protected by PhCO-, CH3CO-, iPrCO-, Me2N-CH=, or Me2N-CMe=. The definitions of the remaining variables are provided in any one of the twenty-first through the fifty-second embodiments.

[0107] In a fifty-fourth embodiment, the present disclosure provides a process according toany one of the twenty-first through the fifty-third embodiments, wherein: each R8is independently selected from the group consisting of H, F, and C1-4alkoxy optionally substituted with C1-4alkoxy or –C(O)NHCH3; each R10is independently H or forms a ring with the alkoxy group of R8, wherein the ring is a 5 or 6-membered ring optionally substituted with 1 to 3 C1-4alkyl groups; each R11is a 4,4’-dimethoxytirtyl group; R12is –CH2CH2CN; and R13aand R13bare independently C1-4alkyl. The definitions of the remaining variables are provided in any one of the twenty-first through the fifty-third embodiments.

[0108] In a fifty-fifth embodiment, the present disclosure provides a process according to anyone of the twenty-first through the fifty-fourth embodiments, wherein each R8is independently selected from the group consisting of H, F, -OCH3, –OCH2CH2OCH3, -OCH2C(O)NHCH3 and -OTBDMS; and each R10is independently H or forms a ring with the alkoxy group of R8, wherein the ring is a 5-membered ring. The definitions of the remaining variables are provided in any one of the twenty-first through the fifty-fourth embodiments.

[0109] In a fifty-sixth embodiment, the present disclosure provides a process according toany one of the twenty-first through the fifty-fifth embodiments, wherein each R10is independently H or together with the alkoxy group of R8form –CH2-O-. The definitions of 32 ME138157184v.1123429-12220 the remaining variables are provided in any one of the twenty-first through the fifty-fifth embodiments.

[0110] In a fifty-seventh embodiment, the present disclosure provides a process of any one ofthe twenty-first through the fifty-sixth embodiments, wherein each R8is independently selected from H, –OCH2CH2OMe and –OCH2C(O)NHMe; each R10is H; each R11is a 4,4’-dimethoxytirtyl group; R12is –CH2CH2CN; and R13aand R13bare both -CH(CH3)2. The definitions of the remaining variables are provided in any one of the twenty-first through the fifty-sixth embodiments.

[0111] In certain embodiments, for the process described therein, R12 is one of the following:See 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.

[0112] In certain embodiments, for the process described therein, the 5’-OH deprotection (ordetritylation) reaction is carried out in the presence of a drying agent. Any suitable drying agents can be used in the deprotection reaction. In some embodiments, the drying agent is selected from calcium chloride, potassium chloride, sodium sulfate, calcium sulfate, magnesium sulfate and molecular sieves.

[0113] In certain embodiments, for the process described herein, the drying agent ismolecular sieves. 33 ME138157184v.1123429-12220

[0114] In certain embodiments, for the process described in herein, the size of molecularsieves is 3Å or 4Å. In one embodiment, the size of molecular sieves is 3Å.

[0115] In certain embodiments, for the process described herein, the anhydrous orsubstantially anhydrous solution for the deprotection reaction is obtained by removing water using azeotropic distillation prior to the deprotection reaction.

[0116] Alternatively, solvents, acids or acid solutions, and other reagents or solutionscomprising the reagents to be used in the detritylation reaction, substrates or substrate solutions to be subjected to detritylation reaction, and the reaction vessels can be dried individually or combined prior to the detritylation reaction.

[0117] In certain embodiments, for the process described herein, the deprotection reaction iscarried out in the presence of a scavenger selected from a cation scavenger comprising a –SH group, a silane scanveger (such as HSiPh3, HSiBu3, triisopropylsilane etc.), siloxane, polystyrene, furan, pyrrole and indole.

[0118] In certain embodiments, the deprotection reaction is carried out in the presence of ascavenger selected from 1-dodecanethiol, cyclohexanethiol, 1-octanethiol, triisopropylsilane, indole, 2,3-dimethylfuran, diphenylsilane, 2-mercaptoimidazole, diphenylmethylsilane, phenylsilane, 5-methoxyindole, methylphenylsilane, chlorodimethylsilane, 1,1,3,3- tetramethyldisiloxane, 1-thioglycerol, triphenylsilane, tert-butyldimethylsilane, butylsilane, methyldiethoxysilane, 1,1,3,3,5,5-hexamethyltrisiloxane, hexylsilane, (mercaptomethyl)polystyrene, or dimethylphenylsilane.

[0119] In certain embodiments, the cation scavenger is a compound of formula RSH, whereinR is an alkyl, a cycloalkyl, a heterocycloalkyl, an aryl or a heteroaryl group, each of which is optionally substituted.

[0120] In certain embodiments, the cation scavenger is CH3(CH2)5SH, CH3(CH2)11SH,cyclohexanethiol (CySH), or CH3CH2OC(=O)CH2CH2SH.

[0121] In certain embodiments, for the process described herein, R11 is a 4,4’-dimethoxytrityl(DMT) group.

[0122] In certain embodiments, for the process described herein, the deprotection reaction iscarried out by reacting the compound of formula (IIA) with a detritylation reagent. Any suitable detritylation reagent can be used.

[0123] In certain embodiments, the detritylation reagent is a strong organic acid.

[0124] In certain embodiments, the detritylation reagent is selected from CF3COOH,CCl3COOH, CHCl2COOH, CH2ClCOOH, H3PO4, methanesulfonic acid (MSA), benzenesulfonic acid (BSA), CClF2COOH, CHF2COOH, PhSO2H (phenylsulfinic acid) etc. 34 ME138157184v.1123429-12220 In a preferred embodiment, the detritylation reagent is CH2ClCOOH. In another specific embodiment, the detritylation reagent is CF3COOH. In yet another specific embodiment, the detritylation reagent is CHCl2COOH.

[0125] In certain embodiments, the detritylation reagent is citric acid. In certainembodiments, the detritylation reagent is saturated citric acid solution.

[0126] In certain embodiments, for the process described herein, the coupling reaction of step2) can be carried out in the presence of an activator described herein (e.g. activators described in the thirty-ninth embodiment). In certain embodiments, the activator is 4,5- dicyanoimidazole (DCI) or 5-ethylthio-1H-tetrazole (ETT).

[0127] In certain embodiments, for the process described herein, the sulfurization reaction ofstep 3) is carried out using a sulfurizing agent, such as 3-amino-1,2,4-dithiazole-5-thione (xanthane hydride or ADTT), 3-(N,N-dimethylamino-methylidene)amino)-3H-1,2,4- dithiazole (DDTT), phenylacetyl disulfide (PADS), 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage Reagent), or phenyl-3H-1,2,4-dithiazol-3-one (POS). In a specific embodimet, the sulfurizing agent is DDTT. In a specific embodiment, the sulfurizing agent is xanthane hydride. In certain embodiments, the sulfurization reaction is carried out in the presence of a base as described herein. In certain embodiments, the base is pyridine or imidazole. In certain embodiments, the sulfurization reaction of step 3) is carried out in the presence of DDTT and 4,5-dicyanoimidazole (DCI).

[0128] In certain embodiments, for the process described herein, the oxidation reaction ofstep 3) is carried out by using standard oxidizing agents known in the literature. Exemplary oxidizing agents include, but are not limited to, tert-butylhydroperoxide ( t-BuOOH), (1S)- (+)-(10-camphorsulfonyl)oxaziridine (CSO), (1R)-(−)-(10-camphorsulfonyl)oxaziridine (enantiomer of CSO), I2, and iodine-pyridine-water oxidizer solution. In a specific embodimet, the oxidizing agent is t-BuOOH.

[0129] In certain embodiments, for the process described herein, thecoupling / oxidation / detritylation steps are carried out in a one pot reaction. In certain embodiments, the oxidation reagents in the one pot reaction is BPO or tBuOOH:. 35 ME138157184v.1123429-12220

[0130] In certain embodiments, for a process described herein, when X is S, thephosphorothiolate group can have S-configuration, R-configuration or a mixture thereof (e.g., a racemic mixture). EXEMPLIFICATION Abbreviation ACN = acetonitrile DBU = 8-diazabicyclo[5.4.0]undec-7-ene DCM = dichloromethane DDTT = 3-(N,N-dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole DCI = 4,5-dicyanoimidazole DMT or DMTr = 4,4'-dimethoxytrityl or bis-(4-methoxyphenyl)phenylmethyl DMAP = 4-Dimethylaminopyridine DMSO = dimethyl sulfoxide EtOAc or EA = ethyl acetate ETT = 5-ethylthio-1H-tetrazole h or hr = hour iPrOH = isopropyl alcohol MeOH = methanol MOE = methoxyethyl MS = molecular sieve MTBE or TBME = methyl tert-butyl ether NMI = N-methylimidazole Rf = retention factor RT = retention time TEA = triethylamine TEAB = tetraethylammonium bromide TFA = trifluoroacetic acid THF = tetrahydrofuran TLC = thin layer chromatography Example 1. Synthesis of Silyl-resin

[0131] a. The general procedure of preparing Silyl-resin and prepared Silyl-resins36 ME138157184v.1123429-12220resin and B is a nucleobase.

[0132] b. Procedures for Synthesis of Compound 7

[0133] Synthetic Route for Compound 7

[0135] To a solution of compound 3 (300 g, 1.83 mol, 1.00 eq) in DMF (1500 mL) wasadded DBU (556 g, 3.66 mol) and DPPA (754 g, 2.74 mol) at 0-5oC. The mixture was stirred 37 ME138157184v.1123429-12220 at 20-25 °C for 3 h. TLC (Petroleum ether: Ethyl acetate = 0:1, product Rf = 0.7) indicated compound 3 was consumed completely. The reaction mixture was quenched by addition H2O (5000 ml) and diluted with ethyl acetate (5000 ml). The organic layer was washed with brine (5000 ml), dried over Na2SO4, and concentrated under vacuum to afford a light yellow liquid. The crude was used for the next step reaction without a further purification.1H NMR (400 MHz, DMSO-d6) δ = 7.88 – 7.86 (m, 2H), 7.67 (s, 1H), 7.58 - 7.56 (m, 1H), 5.43 (s, 2H), 4.66 (s, 2H).

[0136] Step 2: Synthesis of Compound 5

[0137] To a solution of compound 4 (449 g, 2.37 mol, 1.00 eq) in THF (2250 mL) was addedNaOH (284 g, 7.12 mol) in H2O (550 mL) at 20-25oC. The mixture was stirred at 20-25 °C for 2 h. TLC (Petroleum ether: Ethyl acetate = 1:1, product Rf = 0.2) indicated compound 4 was consumed completely. The reaction mixture was quenched by addition 1 mol / L HCl to PH 5-6. The mixture was extracted with ethyl acetate (2500 ml). The organic layer was washed with brine (2500 ml), dried over Na2SO4, and concentrated under vacuum to afford a light-yellow oil. The crude was used for the next step reaction without a further purification.1H NMR (400 MHz, DMSO-d6) δ = 7.88 – 7.86 (m, 2H), 7.71 (s, 1H), 7.32 - 7.30 (m, 1H), 5.27 (s, 1H), 4.85 (s, 2H), 4.54 (s, 2H).

[0138] Step 3: Synthesis of Compound 6

[0139] To a solution of compound 5 (296 g, 1.43 mol, 1.00 eq) in DCM (1800 mL) wasadded imidazole (292 g, 4.29 mol) add TBDPSCl (982 g, 3.57 mol) at 20-25oC. The mixture was stirred at 20-25 °C for 3 h. TLC (Petroleum ether: Ethyl acetate = 3:1, product Rf = 0.7) 38 ME138157184v.1123429-12220 indicated compound 5 was consumed completely. The reaction mixture was diluted with 5000 ml DCM. The organic layer was washed with H2O (5000 ml x 2) and brine (5000 ml), dried over Na2SO4, and concentrated under vacuum to afford a colorless oil. The crude was used for the next step reaction without a further purification.

[0140] Step 4: Synthesisof Compound 7

[0141] To a solution of compound 6 (815 g, 1.19 mol, 1.00 eq) in THF (1440 mL) andMeOH (160 mL) was added K2CO3(741 g, 5.36 mol) in H2O (800 ml) at 20-25oC. The mixture was stirred at 20-25 °C for 1 h. TLC (Petroleum ether: Ethyl acetate = 3:1, product Rf = 0.5) indicated compound 6 was consumed completely. The reaction mixture was quenched by addition KHSO4to PH 4-6 and diluted with ethyl acetate (5000 ml). The organic layer was washed with brine (5000 ml), dried over Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=20 / 1 to 1 / 1) to yield 500-gram product 7 (94% yield). Compound 7: 500 gram, 94% yield.1H NMR (400 MHz, DMSO-d6) δ = 7.95 (s, 1H), 7.90 - 7.67 (m, 1H), 7.66 - 7.64 (m, 4H), 7.40-7.46 (m, 7H), 5.13 (s, 2H), 4.60 (s, 2H), 1.06 (s, 9H).

[0142] c. Procedures for Synthesis of Silyl Linker

[0143] c1. Synthetic Route for Silyl linker –dT39 ME138157184v.1123429-12220

[0144] Step 1: Synthesis of Compound 9

[0145] To a solution of compound 7 (35 g, 78.5 mmol, 1.00 eq) in DCM (900 mL) was addedDMAP (14.4 g, 118 mmol), EDCI (30.1 g, 157 mmol), and compound 8 (55.6 g, 102 mmol) at 0-5oC. The mixture was warmed to 20-25 °C and stirred at 20-25 °C for 12 hours. HPLC indicated compound 7 was consumed completely. The reaction mixture was diluted with DCM (1000 ml). The organic layer was washed with brine (1000 ml X 3), dried over Na2SO4, and concentrated under vacuum. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=10 / 1 to 0 / 1) to yield 70-gram product 9 (91.6 % yield). Compound 9: 70 gram, 91.6% yield.

[0146] Step 2: Synthesis of Compound 10

[0147] To a solution of compound 9 (70 g, 72 mmol, 1.00 eq) in ethyl acetate (280 mL) wasadded Ph3P (37.7 g, 144 mmol) at 0-5oC. The mixture was warmed to 20-25 °C and stirred at 20-25 °C for 9 hours. H2O (140 ml) was added at 20-25 °C and continue stirred 4 hours. HPLC indicated compound 9 was consumed completely. The reaction mixture was diluted with ethyl acetate (250 ml). The organic layer was washed with brine (200 ml X 2), dried over Na2SO4, and concentrated under vacuum. The residue was purified by prep HPLC (Column: Xtimate C18 10u 250mm*80mm; mobile phase: [water (10 mM NH4HCO3)- ACN]; B%: 70%-100%, 17 min) to yield 55-gram product 10 (80.7 % yield). Compound 10: 55 gram, 80.7% yield.1H NMR (400 MHz, DMSO-d6) δ = 7.88 – 7.86 (m, 2H), 7.63 - 7.61 (m, 4H), 7.52 (s, 1H), 7.42 - 7.33(s, 9H), 7.24 – 7.20 (m, 7H), 6.84 - 6.82 (m, 4H), 5.42(s, 1H), 5.09 (s, 2H), 4.09 – 4.08 (m, 1H), 3.82 (s, 1H), 3.69 (s, 6H), 3.21 – 3.18 (m, 2H), 2.33 – 2.37 (m, 1H), 1.45 (s, 3H), 1.04 – 1.01 (s, 9H).

[0148] Step 3: Synthesis of Silyl linker –dT

[0149] To a solution of compound 10 (53 g, 56 mmol, 1.00 eq) in DCM (300 mL) was addedTEA (28.3 g, 280 mmol) and compound 11 (11.2 g, 112 mmol) at 20-25oC. The mixture was 40 ME138157184v.1123429-12220 stirred at 20-25 °C for 4 hours. HPLC indicated compound 10 was consumed completely. The reaction mixture was diluted with DCM (300 ml). The organic layer was washed with brine (200 ml X 3), dried over Na2SO4, and concentrated under vacuum. The residue was used without further purification. Silyl linker-dT: 66 gram.1H NMR (400 MHz, DMSO-d6) δ = 11.38 (s, 1H) 8.62 – 8.61 (m, 1H) 7.90 – 7.88 (m, 1H), 7.80 (s, 1H) 7.62 - 7.60 (m, 4H), 7.52 (s, 1H), 7.41 - 7.39 (m, 9H), 7.33 – 7.19 (m, 7H) 6.84 - 6.82 (m, 4H), 5.41 – 5.40 (m, 1H), 5.07 (s, 2H), 4.38 – 4.37 (d, J = 8 Hz, 1H), 3.82 (s, 1H), 3.69 (s, 8H), 3.31 – 3.27 (m, 2H), 2.47 – 2.45 (m, 4H), 1.45 (s, 3H), 1.04 (s, 9H).

[0150] c2. Synthetic Route for Silyl Linker -MOE U

[0151] Silyl linker MOE U was prepared based on the similar procedures for preparing Silyllinker –dT.

[0152] Compound 13 ((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3- yl 5-(aminomethyl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzoate) -1H NMR (400 MHz, DMSO-d6) δ = 7.94 (s, 1H), 7.91 - 7.87 (m, 1H), 7.62 - 7.60 (m, 4H), 7.51 (s, 1H), 7.40-7.32 (m, 10H), 7.21-7.18 (m, 8H), 6.82 - 6.80 (m, 4H), 5.84 - 5.82 (d, J = 8 Hz, 1H), 5.37 - 5.35 41 ME138157184v.1123429-12220 (m, 1H), 5.17 – 5.03 (m, 2H), 4.43 – 4.40 (m, 1H), 4.14 - 4.13 (m, 8H), 3.85 (s, 2H), 3.69 (s, 6H), 3.54 – 3.50 (m, 1H), 3.22 – 3.19 (m, 4H), 2.98 (s, 3H), 3.05 (s, 5H), 1.05 (s, 9H).

[0153] Silyl linker MOE U (4-((4-((((2R,3S,4S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)oxy)carbonyl)-3-(((tert- butyldiphenylsilyl)oxy)methyl)benzyl)amino)-4-oxobutanoic acid) -1H NMR (400 MHz, DMSO-d6) δ = 11.45 (drs, 1H), 8.81 (s, 1H), 7.90 – 7.88 (m, 2H), 7.62 – 7.60 (m, 5H), 7.51 (m, 1 H), 7.39 – 7.31 (m, 10H), 7.20 – 7.18 (m, 8H), 6.82 – 6.80 (m, 4H), 5.81 – 5.82 (d, J = 4 Hz, 1H), 5.36 – 5.33 (m, 1H), 5.15 – 5.05 (m, 2H), 4.42 – 4.39 (m, 3H), 4.42 – 4.40 (m, 1H), 3.68 (s, 6H), 3.50 – 3.49 (m, 1H), 3.24 – 3.22 (m, 1H), 3.19 – 3.17 (m, 4H), 2.97 (s, 3H), 2.45 – 2.23 (m, 7H), 1.47 (s, 3H), 1.00 (s, 9H).

[0154] c3. Synthetic Route for Silyl Linker -MOE C

[0155] Silyl linker MOE C was prepared based on the similar procedures for preparing Silyllinker –dT.

[0156] Compound 16 ((2R,3R,4R,5R)-5-(4-benzamido-5-methyl-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)tetrahydrofuran-3- 42 ME138157184v.1123429-12220 yl 5-(aminomethyl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzoate) -1H NMR (400 MHz, DMSO-d6) δ = = 8.61 – 8.55 (m, 2H), 8.04 – 8.04 (m, 2H), 7.95 – 7.95 (m, 2=1H), 7.88 (s, 1H), 7.65 – 7.63 (m, 5 H), 7.39 – 7.37 (m, 11H), 7.19 – 7.15 (m, 7H), 6.77 – 6.75 (m, 4H), 6.15 – 6.14 (d, J = 4 Hz, 1H), 5.62 – 5.60 (m, 1H), 5.16 – 5.04 (m, 2H), 4.30 – 4.25 (m, 1H), 3.67 (s, 6H), 3.40 – 3.37 (m, 4H), 3.29 – 3.27 (m, 6H), 3.11 – 3.10 (m, 2H), 2.84 (s, 3H), 2.06 (s, 2H), 1.05 (s, 9H).

[0157] Silyl linker MOE C (4-((4-((((2R,3S,4S,5R)-5-(4-benzamido-5-methyl-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2- methoxyethoxy)tetrahydrofuran-3-yl)oxy)carbonyl)-3-(((tert- butyldiphenylsilyl)oxy)methyl)benzyl)amino)-4-oxobutanoic acid) -1H NMR (400 MHz, DMSO-d6) δ = 8.58 – 8.56 (m, 1H), 8.15 – 8.13 (m, 2H), 7.89 – 7.85 (m, 2H), 7.61(s, 1H), 7.61 – 7.59 (m, 5 H), 7.36 – 7.32 (m, 11H), 7.22 – 7.19 (m, 7H), 6.83 – 6.79 (m, 4H), 6.86 – 6.85 (d, J = 4 Hz, 1H), 5.73 (s, 1H), 5.35 – 5.34 (m, 1H), 5.16 – 5.04 (m, 2H),4.46 – 4.40 (m, 3H), 4.20 – 4.12 (m, 1H), 3.69 – 3.57 (m, 7H), 3.42 – 3.26 (m, 3H), 3.25 – 3.20 (m, 3H), 3.19 – 3.18 (m, 5H), 2.95 (s, 3H), 2.54 – 2.52 (m, 2H), 2.07 (s, 3H) 1.69 (s, 3H), 1.05 (s, 9H).

[0158] c4. Synthetic Route for Silyl Linker -dC43 ME138157184v.1123429-12220

[0159] Silyl linker-dC was prepared based on the similar procedures for preparing Silyllinker –dT.

[0160] Compound 19 ((2R,3S,5R)-5-(4-benzamido-5-methyl-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl 5-(aminomethyl)-2- (((tert-butyldiphenylsilyl)oxy)methyl)benzoate) -1H NMR (400 MHz, DMSO-d6) δ = 8.58 – 8.56 (m, 1H), 8.15 – 8.13 (m, 2H), 7.89 – 7.85 (m, 2H), 7.61(s, 1H), 7.61 – 7.59 (m, 5 H), 7.36 – 7.32 (m, 11H), 7.22 – 7.19 (m, 7H), 6.83 – 6.79 (m, 4H), 6.86 – 6.85 (d, J = 4 Hz, 1H), 5.73 (s, 1H), 5.35 – 5.34 (m, 1H), 5.16 – 5.04 (m, 2H),4.46 – 4.40 (m, 3H), 4.20 – 4.12 (m, 1H), 3.69 – 3.57 (m, 7H), 3.42 – 3.26 (m, 3H), 3.25 – 3.20 (m, 3H), 3.19 – 3.18 (m, 5H), 2.95 (s, 3H), 2.54 – 2.52 (m, 2H), 2.07 (s, 3H) 1.69 (s, 3H), 1.05 (s, 9H).

[0161] Silyl linker dC (4-((4-((((2R,3R,5R)-5-(4-benzamido-5-methyl-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3- yl)oxy)carbonyl)-3-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)amino)-4-oxobutanoic acid) -1H NMR (400 MHz, DMSO-d6) δ = 8.57 – 8.56 (m, 1H), 8.16 – 8.14 (m, 2H), 7.84 – 7.82 (m, 1H), 7.81 (s, 2H), 7.62 – 7.61 (m, 5 H), 7.41 – 7.40 (m, 11H), 7.33 – 7.20 (m, 7H), 6.85 – 6.82 (m, 4H), 6.22 – 6.19 (m, 1H), 5.44 – 5.42 (m, 1H), 5.07 (s, 2H), 4.40 – 4.38 (m, 3H), 4.15 – 4.14 (m, 1H), 3.69 (m, 6H), 3.68 – 3.24 (m, 6H), 2.53 – 2.51 (m, 2H), 2.06 (s, 2H) 1.69 (s, 3H), 1.04 (s, 9H).

[0162] c5. Synthetic Route for Silyl Linker – MOE A44 ME138157184v.1123429-12220

[0163] Silyl linker-MOE A was prepared based on the similar procedures for preparing Silyllinker –dT.

[0164] Compound 22 ((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)tetrahydrofuran-3-yl 5- (aminomethyl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzoate) -1H NMR (400 MHz, DMSO-d6) δ = 8.64 – 8.61 (m, 2H), 8.06 – 8.01 (m, 4H), 7.66 – 7.63 (m, 5H), 7.55 – 7.37 (m, 11H), 7.20 – 7.15 (m, 7 H), 6.78 – 7.72 (m, 4H), 6.17 – 6.16 (m, 2H), 5.65 – 5.63 (m, 1H), 5.21 – 5.10 (m, 2H), 4.65 (s, 2H), 4.28 – 4.27 (m ,1H), 3.67 (s, 6H), 3.49 – 3.31 (m, 4H), 3.11 – 3.10 (m, 2H), 2.83 (s, 3H), 1.07 (s, 9H).

[0165] Silyl linker-MOE A (4-((4-((((2R,3S,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)tetrahydrofuran-3- yl)oxy)carbonyl)-3-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)amino)-4-oxobutanoic acid) -1H NMR (400 MHz, DMSO-d6). δ = 8.61 – 8.55 (m, 2H), 8.04 – 8.04 (m, 2H), 7.95 – 7.95 (m, 2H), 7.88 (s, 1H), 7.65 – 7.63 (m, 5 H), 7.39 – 7.37 (m, 11H), 7.19 – 7.15 (m, 7H), 6.77 – 6.75 (m, 4H), 6.15 – 6.14 (d, J = 4 Hz, 1H), 5.62 – 5.60 (m, 1H), 5.16 – 5.04 (m, 2H), 4.30 – 4.25 (m, 1H), 3.67 (s, 6H), 3.40 – 3.37 (m, 4H), 3.29 – 3.27 (m, 6H), 3.11 – 3.10 (m, 2H), 2.84 (s, 3H), 2.06 (s, 2H), 1.05 (s, 9H).

[0166] d. Procedures for Synthesis of Silyl Resin45 ME138157184v.1123429-12220

[0167] General procedure to prepare Silyl and Alloc resin (Hydroxyl functionalized solidsupport)

[0168] To a three-necked round bottom flask was successively added hydroxyl functionalizedsolid support, Silyl or Alloc Linker, HBTU, ethyldiisopropylamine and anhydrous MeCN (0.8 L). The resulting mixture was stirred for 20 h at rt. An aliquot sample was taken, filtered, washed with MeCN, dried and loading determined by UV.

[0169] FTIR data of compound B1: 759.4, 830.7, 906.3, 1030.1, 1067.8, 1112.9, 1140.2,1175.2, 1203.1, 1252.5, 1380.4, 1452.3, 1493.4, 1508.5, 1584.0, 1602.8, 1693.8, 1758.0, 2927.6, 3026.2 cm-1.

[0170] FTIR data of compound C1: 756.8, 828.2, 907.8, 1029.5, 1065.8, 1113.1, 1139.7,1172.5, 1202.2, 1251.1, 1364.9, 1452.1, 1493.4, 1507.6, 1569.5, 1601.8, 1655.6, 1759.9, 2927.7, 3060.1 cm-1.

[0171] FTIR data of compound D1: 758.3, 827.6, 908.0, 959.2, 1029.4, 1066.7, 1111.3,1138.7, 1173.0, 1204.2, 1250.5, 1364.5, 1452.1, 1493.3, 1507.6, 1568.9, 1654.6, 1712.4, 2927.8, 3026.2 cm-1.

[0172] FTIR data of compound F1: 748.7, 759.2, 829.2, 886.9, 907.9, 974.9, 1030.0, 1061.9,1074.4, 1111.7, 1140.1, 1179.2, 1251.5, 1379.6, 1427.8, 1452.4, 1493.6, 1507.9, 1603.2, 1718.6, 2928.4, 3026.3 cm-1.

[0173] General procedure to prepare Silyl and Alloc resin (amino-derivatized solid support)

[0174] To a three-necked round bottom flask was successively added amino functionalizedsolid support, Silyl or Alloc Linker, HBTU, ethyldiisopropylamine and anhydrous MeCN (0.8 L). The resulting mixture was stirred for 20 h at rt. An aliquot sample was taken, filtered, washed with MeCN, dried and loading determined by UV. 46 ME138157184v.1123429-12220 Example 2 Synthesis of Oligonucleotide through Silyl-resin

[0175] a. Synthesis of Deprotected Oligonucleotide Fragments Through Silyl-resin

[0176] A Moe and Deoxy oligonucleotide 18-mer sequence was prepared through Silyl-resins. The general synthetic scheme is shown in Fig.1. Fragment 5 can be directly obtained from Silyl linker-MOE U resin and thus didn’t require extra synthesis steps.

[0177] a1. Synthetic Scheme for Preparing Fragments:

[0178] The scheme below showed the synthesis of fragments using fragment 1 as example.The starting material Silyl Linker MOE C resin went through a 3-step solid phase synthesis (SPS) elongation process repeatedly to afford the fragment 1. The detailed procedures of 3- step solid phase synthesis are described in WO2017 / 223258, which is incorporated herein by its entirety. Other fragments were synthesized similarly using this process.

[0179] The synthesis of oligonucleotide fragments was performed on an AKTA 100 solid-phase synthesizer at 1.1 mmol scale using Si-resin (250 umol / g) described above or NittoPhase HL Unylinker (350 umol / g) as the solid support at ambient temperature through the 3-step solid phase synthesis (SPS) elongation process. After the final elongation cycle, the deprotection of phosphorus was skipped to ensure the fragments are fully protected. The resin was dried with N2 for 2h to remove residue solvent after synthesis and stored at -20C.

[0180] b. Fragment Quality Comparison

[0181] To determine the quality of the fragment synthesized by the process of the presentdisclosure, the fragment synthesized from Si-resin was compared with the one prepared from NittoPhase HL Unylinker resin. Ammonolysis was performed on both Si-resin and 47 ME138157184v.1123429-12220 NittoPhase HL Unylinker resin synthesized fragments. Oligonucleotides fragments were cleaved from resin and deprotected by subjecting to 30% NH4OH (1g resin using 10 mL NH4OH solution) at 65 °C for 6.5 h. Aliquots from the resulting mixture was taken and dried with nitrogen before dissolving in 1 mL 0.01% TEA aqueous solution. The fragments purity was checked by HPLC and the results are shown in Figs. 2-5 for each fragment in comparison of Si-resin and NittoPhase HL Unylinker resin. The MS profile for main UV peak was also compared and shown in the Figs 2-5.

[0182] From the HPLC and MS profiles of each fragment, the quality of the Si-resinsynthesized fragments is comparable to the one of the NittoPhase HL Unylinker resin synthesized fragments.

[0183] c. Preparation of Protected Oligonucleotide Fragments Through Silyl-resin

[0184] Desilylation scheme

[0185] 0.125 g resin was charged with 0.5 mL THF and stirred at room temperature. Aseparate vial was charged with 96 mg imidazole (60 eq to resin) and 0.75 mL THF.18 uL of HF-pyridine (30 eq to resin, 70% HF) was added to the vial after dissolving imidazole. The second vial was added back to the resin mixture and stirred at 0 °C for 5h before quenching with 1M aq NaHCO3solution. The resin was removed by filtration, the fragment was extracted with ethyl acetate and washed with water two times before drying with sodium sulfate. The ethyl acetate was removed under vacuum evaporation. The resulting solid was injected into HPLC for purity check in comparison with fragment samples obtained from 48 ME138157184v.1123429-12220 liquid phase synthesis ("LPS") based on the procedures described in WO2022 / 103842, which is incorporated herein by its entirety. The comparison results are summarized in Table 1 below. The HPLC traces are provided in Figs.6-9. Table 1: summary of purity comparison for Si-resin and LPS fragments

[0186] Fragments from Si-resin and liquid phase synthesis showed similar MS profiles. TheMS profiles for the UGCC oligonucleotide fragment prepared from Silyl resin or liquid phase synthesis were shown in Fig.10.

[0187] d. 18-mer Oligonucleotide

[0188] Ammonolysis condition:

[0189] The 18-mer full-length oligonucleotide was cleaved from the resin and deprotected bysubjecting to 30% NH4OH (1g resin using 10 mL NH4OH solution). Reaction temperature and time was varied to achieve full deprotection. After reaction completion, aliquots from the mixture was taken and dried with nitrogen before dissolving in 1 mL 0.01% TEA aqueous solution. The full-length product purity was checked by HPLC and the results were shown in Fig. 11. The MS profile for main UV peak was also included. Finally, liquid-phase synthesized full length product after ammonolysis was showed in Fig.12 as a comparison. Example 3. Synthesis of Alloc-linker MOE G, MOE U, and Unylinker

[0190] a. Procedures for Synthesis of Compound 53

[0191] Step 1 – Synthesis of Compound 50

[0192] To a vessel, maintained at 20-25 °C, containing 4-(azidomethyl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzoic acid (75.0 g, 168 mmol, 1.0 eq) and K2CO3(34.8 g, 49 ME138157184v.1123429-12220 252 mmol, 1.5 eq) in DMF (750 mL), PMBCl (31.6 g, 201 mmol, 27.5 ml, 1.2 eq) was added and the mixture was stirred for 2 h. LCMS indicated consumption of the starting carboxylic acid. The mixture was poured into H2O (750 mL) and EtOAc (750 mL x 3), stirred, and the phases were separated. The organic layers were washed with saturated aqueous sodium bicarbonate (750 mL) and brine (750 mL), dried over Na2SO4, then filtered and concentrated under reduced pressure at 30-40 °C. The resulting residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=50 / 1 to 20 / 1). The appropriate fractions were collected and concentrated under reduced pressure to afford Compound 50 (85.0 g, 150 mmol, 89.2% yield) as a colorless oil.

[0193] Step 2 – Synthesis of Compound 51

[0194] A vessel, maintained at at 25 °C under N2, was charged with Compound 50 (85.0 g,150 mmol, 1.0 eq) and THF (500 mL). This mixture was treated with triethylamine trihydrofluoride complex (121 g, 751 mmol, 122 ml, 5.0 eq) in THF (170 mL) in one portion and stirred at 20-25 °C for 16 h. LCMS indicated complete consumption of Compound 50. The mixture was poured into H2O (850 mL) and extracted with three portions of EtOAc (850 mL). The combined organic layers were washed with H2O (850 mL x 3), brine (850 mL), dried over Na2SO4,filtered, and concentrated under reduced pressure at 30-40 °C. The crude, colorless oil thus obtained was used for the next step without further purification (100 g, crude).

[0195] Step 3 – Synthesis of Compound 52

[0196] A vessel, maintained at 20-25 °C, containing Compound 51 (49.1 g, 150 mmol, 1.0eq) and DMAP (55.1 g, 450 mmol, 3.0 eq) in DCM (500 mL), was charged with allyl chloroformate (36.2 g, 300 mmol, 31.7 mL, 3.0 eq). After 16 hours, LCMS indicated complete consumption of Compound 51. The organic were washed with NaHCO3 (500 mL x 3), dried over Na2SO4, filtered and concentrated under reduced pressure at 30-40 °C. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 50:1to 30:1). After collecting and concentrating the appropriate fractions, Compound 52 (64.0 g,111 mmol, 74.4% yield) was obtained as a light-yellow foam.

[0197] Step 4 – Synthesis of Compound 53

[0198] A vessel, maintained at 20-25 °C, containing Compound 52 (46.0 g, 111 mmol, 1.0eq) and anisole (36.3 g, 335 mmol, 3.0 eq) in DCM (400 mL) was charged with TFA (102 g, 894 mmol, 66.2 mL, 8.0 eq). After 2 hours, LCMS analysis indicated complete consumption of Compound 52. The mixture was concentrated under reduced pressure at 30-40 °C. The resulting residue was subjected to column chromatography (SiO2, Petroleum ether / Ethyl 50 ME138157184v.1123429-12220 acetate = 20 / 1 to 10 / 1). After collecting and concentrating the appropriate fractions, Compound 53 (28.0 g, 87.5 mmol, 78.3% yield) was obtained as a light white solid.

[0199] b. Procedures for Synthesis of Alloc-linker MOE G

[0200] Step 1 – Synthesis of Compound 54

[0201] A vessel, maintained at 20-25 °C, containing Compound 53 (12.0 g, 41.2 mmol, 1.0eq), DMAP (10.1 g, 82.4 mmol, 2.0 eq) and N-(9-((2R,3R,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran- 2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (44.1 g, 61.8 mmol, 1.5 eq) dissolved in DCM (120 mL) was charged with EDCI (15.8 g, 82.4 mmol, 2.0 eq). After 4 hours, LCMS analysis indicated complete consumption of Compound 53. The mixture was washed with three portions of saturated aqueous NaHCO3(50 mL), the organics were collected dried over Na2SO4, filtered and concentrated under reduced pressure at 30-40 °C. The resulting residue was subjected to column chromatography (SiO2, Petroleum ether / Ethyl acetate = 10 / 1 to 5 / 1). After collecting and concentrating the appropriate fractions, Compound 54 (35.0 g, 35.5 mmol, 86.1% yield) was obtained as a white solid.

[0202] Step 2 – Synthesis of Compound 55

[0203] A vessel, maintained at 20-25 °C, containing Compound 54 (35.0 g, 35.4 mmol, 1.0eq) in EtOAc (350 mL) was charged with PPh3(13.9 g, 53.1 mmol, 1.5 eq). After 2 hours, LCMS analysis indicated complete consumption of Compound 54. Water (150 mL) was 51 ME138157184v.1123429-12220 added, and the mixture was allowed to stir for 16 hours at 20-25 °C. The mixture was washed with water (350 mL) and brine (350 mL). The organics were collected, dried over Na2SO4, filtered and concentrated under reduced pressure at 30-40 °C. The crude product was purified by reversed-phase HPLC (column: Welch Xtimate C18250*70mm, 10um; mobile phase: water (NH4HCO3), ACN B%: 55%-95%, 20 min). After collecting and concentrating the appropriate fractions, Compound 55 (29.0 g, 29.7 mmol, 83.9% yield) was obtained as a white solid.

[0204] Step 3 – Synthesis of Alloc-linker MOE GTo a vessel, maintained at 20-25 °C, containing Compound 55 (29.0 g, 30.1 mmol, 1.0 eq) in DCM (290 mL) was added TEA (15.3 g, 150 mmol, 21 mL, 5.0 eq) and succinic anhydride (6.04 g, 60.3 mmol, 2.0eq). After 2 hours, HPLC analysis indicated complete consumption of Compound 55. The mixture was poured into aqueous TEAB (0.5 M, 200 mL) and stirred. The organics were separated and washed with two portions of aqueous TEAB (0.5 M, 200 mL). The organics were collected, dried over Na2SO4, filtered, and concentrated under reduced pressure at 30-40 °C to give Alloc-linker MOE G (30 g, 25.1 mmol, 83.4% yield, 97.5% purity) as a light white foam.1H NMR: (CD3CN 400 MHz) δ 7.94 (d, J = 8.00 Hz, 1H), 7.86 (s, 1H), 7.79 (m, 1H), 7.49-7.16 (m, 12H), 6.80 (m, 4H), 6.03-5.85 (m, 2H), 5.66 (m, 1H), 5.55-5.41 (m, 3H), 5.35-5.16 (m, 2H), 4.94 (m, 1H), 4.59 (d, J = 4 Hz, 2H), 4.48- 4.30 (m, 4H), 3.72 (s, 6H), 3.64 (m, 2H), 3.46-3.57 (m, 1H), 3.44-3.22 (m, 3H), 3.03 (s, 3H), 2.76 (m, 7H), 2.69-2.57 (m, 1H), 2.44 (s, 4H), 1.16-0.98 (m, 16H). LCMS: m / z = 1061.41 (M+H)+

[0205] c. Procedures for Synthesis of Alloc-linker MOE U52 ME138157184v.1123429-12220

[0206] Step 1 – Synthesis of Compound 56

[0207] A vessel, maintained at 20-25 °C, containing Compound 53 (13.0 g, 44.6 mmol, 1.0eq), DMAP (10.9 g, 89.2 mmol, 2.0 eq) and 1-((2R,3R,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran- 2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (41.4 g, 66.9 mmol, 1.5 equiv) dissolved in DCM (130 mL) was charged with EDCI (17.1 g, 89.2 mmol, 2.0 eq). After 4 hours, LCMS analysis indicated complete consumption of Compound 53. The mixture was washed with three portions of saturated aqueous NaHCO3 (50 mL), the organics were collected dried over Na2SO4, filtered and concentrated under reduced pressure at 30-40 °C. The resulting residue was subjected to column chromatography (SiO2, Petroleum ether / Ethyl acetate = 10 / 1 to 5 / 1). After collecting and concentrating the appropriate fractions, Compound 56 (33.0 g, 37.0 mmol, 82.8% yield) was obtained as a white solid.

[0208] Step 2 – Synthesis of Compound 57

[0209] A vessel, maintained at 20-25 °C, containing Compound 56 (33.0 g, 37.0 mmol, 1.0eq) in EtOAc (330 mL) was charged with PPh3 (14.5 g, 55.5 mmol, 1.5 eq). After 2 hours, LCMS analysis indicated complete consumption of Compound 56. Water (150 mL) was added, and the mixture was allowed to stir for 16 hours at 20-25 °C. The mixture was washed 53 ME138157184v.1123429-12220 with water (350 mL) and brine (350 mL). The organics were collected, dried over Na2SO4, filtered and concentrated under reduced pressure at 30-40 °C. The crude product was purified by reversed-phase HPLC (column: Welch Xtimate C18 250*70mm, 10um; mobile phase: water (NH4HCO3), ACN B%: 15%-80%, 20 min). After collecting and concentrating the appropriate fractions, Compound 57 (28.0 g, 31.7 mmol, 85.8% yield) was obtained as a white solid.

[0210] Step 3 – Synthesis of Alloc-linker MOE U

[0211] To a vessel, maintained at 20-25 °C, containing Compound 57 (28.0 g, 32.3 mmol,1.0 equiv) in DCM (280 mL) was added TEA (16.3 g, 161 mmol, 22.5 mL, 5.0 equiv) and succinic anhydride (6.47 g, 64.6 mmol, 2.0 equiv). After 2 hours, HPLC analysis indicated complete consumption of Compound 57 The mixture was poured into aqueous TEAB (0.5 M, 200 mL) and stirred. The organics were separated and washed with two portions of aqueous TEAB (0.5 M, 200 mL). The organics were collected, dried over Na2SO4, filtered, and concentrated under reduced pressure at 30-40 °C to give Alloc-linker MOE U (31 g, 29.1 mmol, 89.8% yield, 97.9% purity) as a light white foam. LCMS: m / z = 964.4 (M-H+)-.1H NMR: (CDCl3400 MHz) δ 7.98-7.96 (d, J = 8.00 Hz, 1H), 7.56 (s, 1H), 7.46-7.35 (m, 4H), 7.34-7.22 (m, 8H), 6.80 (m, 4H), 5.99-5.88 (m, 2H), 5.56-5.54 (dd, J = 4.00 Hz, 1H), 5.49 (s, 2H), 5.35-5.20 (m, 2H), 4.61-4.59 (m, 1H), 4.52-4.49 (t, J = 8 Hz, 1H), 4.39-4.34 (d, J = 4.00 Hz, 2H), 4.33-4.32 (m, 1H), 3.76-3.64 (m, 9H), 3.44-3.35 (m, 5H), 3.31 (s, 3H), 2.81-2.74 (m, 5H), 2.45-2.39 (m, 4H), 1.45 (s, 3H), 1.09-1.05 (m, 7H).

[0212] d. Procedures for Synthesis of Alloc-linker Unylinker54 ME138157184v.1123429-12220

[0213] Step 1 – Synthesis of Compound 58

[0214] A vessel, maintained at 20-25 °C, containing Compound 53 (10.0 g, 34.3 mmol, 1.0eq), DMAP (8.39 g, 68.6 mmol, 2.0 eq) and (3aR,4R,5S,6S,7S,7aS)-5-(bis(4- methoxyphenyl)(phenyl)methoxy)-6-hydroxy-2-phenylhexahydro-1H-4,7-epoxyisoindole- 1,3(2H)-dione (29.7 g, 51.5 mmol, 1.5 eq) dissolved in DCM (120 mL) was charged with EDCI (15.8 g, 82.4 mmol, 2.0 eq). After 4 hours, LCMS analysis indicated completeconsumption of Compound 53. The mixture was washed with three portions of saturatedaqueous NaHCO3 (50 mL), the organics were collected dried over Na2SO4, filtered and concentrated under reduced pressure at 30-40 °C. The resulting residue was subjected to reversed-phase HPLC (column: Agela DuraShell C18250*70mm*10um; mobile phase: [H2O (10mM NH4HCO3)-ACN]; gradient: 70%-90% B over 20.0 min). After collecting and concentrating the appropriate fractions, Compound 58 (26.0 g, 30.5 mmol, 89.0% yield) was obtained as a yellow solid. 55 ME138157184v.1123429-12220

[0215] Step 2 – Synthesis of Compound 59

[0216] A vessel, maintained at 20-25 °C, containing Compound 58 (26.0 g, 30.5 mmol, 1.0eq) in EtOAc (260 mL) was charged with PPh3(12.0 g, 45.8 mmol, 1.5 eq). After 2 hours, LCMS analysis indicated complete consumption of Compound 58. Water (150 mL) was added, and the mixture was allowed to stir for 16 hours at 20-25 °C. The mixture was washed with water (350 mL) and brine (350 mL). The organics were collected, dried over Na2SO4,filtered and concentrated under reduced pressure at 30-40 °C. The crude product was purified by reversed-phase HPLC (column: Welch Xtimate C18250*70mm, 10um; mobile phase: [H2O (10mM NH4HCO3)-ACN]; gradient: 60%-80% B over 20.0 min). After collecting and concentrating the appropriate fractions, Compound 59 (24.0 g, 28.5 mmol, 92.4% yield) was obtained as a white solid.

[0217] Step 3 – Synthesis of Alloc-linker Unylinker

[0218] To a vessel, maintained at 20-25 °C, containing Compound 59 (25.0 g, 30.3 mmol,1.0 eq) in DCM (250 mL) was added TEA (15.3 g, 151 mmol, 21.1 mL, 5.0 eq) and succinic anhydride (6.07 g, 60.6 mmol, 2.0 eq). After 2 hours, HPLC analysis indicated complete consumption of Compound 59. The mixture was poured into aqueous TEAB (0.5 M, 200 mL) and stirred. The organics were separated and washed with two portions of aqueous TEAB (0.5 M, 200 mL). The organics were collected, dried over Na2SO4, filtered, and concentrated under reduced pressure at 30-40 °C to give Alloc-linker Unylinker (24 g, 25.9 mmol, 85.6% yield, 96.6% purity) as a light white foam. LCMS: m / z = 923.2 (M-H+)-, found 923.3.1H NMR: (CDCl3400 MHz) δ 8.19-8.17 (d, J = 8.00 Hz, 1H), 7.48-7.25 (s, 1H), 6.88- 6.80 (m, 4H), 5.99-5.89 (m, 1H), 5.61-5.51 (m, 1H), 5.37-5.21 (m, 3H), 4.78 (s, 1H), 4.62- 4.61 (d, J = 4.00 Hz, 2H), 4.43-4.41 (d, J = 8.00 Hz, 1H), 4.19-4.18 (d, J = 4.00 Hz, 1H), 3.75 (s, 6H), 3.18 (s, 1H), 3.09-3.07 (d, J = 8.00 Hz, 1H), 2.92-2.86 (m, 2H), 2.70-2.68 (d, J = 8.00 Hz, 1H), 2.52-2.43 (m, 4H), 1.16-1.08 (m, 3H).

[0219] The breadth and scope of the present disclosure should not be limited by any of theabove-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents. 56 ME138157184v.1

Claims

123429-12220 CLAIMS What is claimed is:

1. A compound of Formula I: ,or a salt thereof, wherein: W is O or NH; R1is -H or a solid support; R2is a silyl hydroxyl protecting group or an alloc protecting group; R3is a nucleoside or a group represented bywherein represents the point of attachment for R3.

2. The compound of claim 1, wherein W is O.

3. The compound of claim 1, wherein W is NH.

4. The compound of any one of claims 1 to 3, or a salt thereof, wherein: R1is H.

5. The compound of claim 1, or a salt thereof, wherein: R1is a solid support. 57 ME138157184v.1123429-12220 6. The compound of claim 5, or a salt thereof, wherein the solid support is a cross-linked polystyrene bead with a hydroxyl functional group or a controlled pore glass bead with a hydroxyl functional group.

7. The compound of claim 5, or a salt thereof, wherein the solid support is a cross-linked polystyrene bead with an amino functional group or a controlled pore glass bead with an amino functional group.

8. The compound of claim 6 or 7, or a salt thereof, wherein the beads have a loading in the range of about 100 to about 500 µmol / gram.

9. The compound of claim 8, or a salt thereof, wherein the beads have a loading of about 250 µmol / gram.

10. The compound of any one of claims 1 to 9, or a salt thereof, wherein R2is a silyl hydroxyl protecting group selected from the following: ,58 ME138157184v.1123429-12220-O-TBDAS-2; wherein represents the point of attachment for R2; and R5, R6 and R7 are each independently H, C1-30alkyl, or C1-30alkoxy.

11. The compound of any one of claims 1 to 10, or a salt thereof, wherein R2is selected from the group consisting of –O-TBDMS, -O-TIPS, -O-TBDPS, -O-TBoDPS, and –O- TBDAS:,, , , and .

12. The compound of any one of claims 1 to 11, or a salt thereof, wherein R2is.

13. The compound of any one of claims 1 to 9, or a salt thereof, wherein R2is an alloc protecting group. 59 ME138157184v.1123429-12220 14. The compound of any one of claims 1 to 9 and 13, or a salt thereof, wherein the alloc protecting group is, wherein represents the point of attachment for R2.

15. The compound of any one of claims 1 to 14, or a salt thereof, wherein R3is a nucleoside.

16. The compound of claim 15 or a salt thereof, wherein the nucleoside is represented by Formula (A):wherein R8is selected from the group consisting of H, halo, OH, and C1-6alkoxy optionally substituted with C1-6alkoxy or –C(O)NHC1-3alkyl; wherein the OH group is optionally protected by a hydroxyl protecting group; R9is a nucleobase, wherein the NH2 of the nucleobase, if present, is protected by an amine protecting group; R10is H or forms a ring with the alkoxy group of R8; R11is a hydroxy protecting group.

17. The compound of claim 16 or a salt thereof, wherein R8is selected from the group consisting of H, F, and C1-4alkoxy optionally substitutedwith C1-4alkoxy or –C(O)NHCH3;R10is H or forms a ring with the alkoxy group of R8; wherein the ring is a 5 or 6- membered ring optionally substituted with 1 to 3 C1-4alkyl groups; and R11is a trityl hydroxy protecting group (DMTr).

18. The compound of claim 16 or 17 or a salt thereof, wherein R8is selected from H and C1-4alkoxy optionally substituted with C1-4alkoxy; R10is H; and 60 ME138157184v.1123429-12220 R11is 4,4'-dimethoxytrityl.

19. The compound of any one of claims 16 to 18 or a salt thereof, wherein R8is selected from20. The compound of any one of claims 16 to 19 or a salt thereof, wherein the nucleobase is 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 NH2group of the nucleobase, if present, is protected by PhCO-, CH3CO-, iPrCO-, Me2N-CH=, or Me2N-CMe=.

21. The compound of any one of claims 16 to 20 or a salt thereof, wherein the nucleobase is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, and 5-methylcytosine, wherein the NH2 group of the nucleobase, if present, is protected by PhCO-, CH3CO-, iPrCO-, Me2N-CH=, or Me2N-CMe=.

22. The compound of any one of claims 1-15 or a salt thereof, wherein R3is represented23. A compound of Table 1 or a salt thereof. 61 ME138157184v.1123429-12220 24. A process for preparing an oligonucleotide of formula (II),or a salt thereof, comprising the steps of: 1) deprotecting a compound of formula (IIA):or a salt thereof, to form a compound of formula (IIB):salt thereof; 2) reacting the compound of formula (IIB), or a salt thereof, with a compound of formula (IIC):62 ME138157184v.1123429-12220 or a salt thereof, to form a compound of formula (IID),salt thereof; 3) sulfurizing or oxidizing the compound of formula (IID), or a salt thereof, with a sulfurization or oxidation agent to form a compound of formula (IIE):salt thereof; 4) deprotecting the compound of formula (IIE), or a salt thereof to form a compound of formula (IIF):(IIF), or a salt thereof; 63 ME138157184v.1123429-12220 5) when q is equal or greater than 2, starting with the compound of formula (IIF), repeating steps 2), 3) and 4) for q-2 times, followed by steps 2) and 3) to yield the oligonucleotide of formula (II), or a salt thereof, wherein: R9, for each occurrence, is independently a nucleobase, wherein when the nucleobase comprises an NH2 group, the NH2 of the nucleobase is protected by an amine protecting group; R8, for each occurrence, is independently selected from the group consisting of H, halo, OH, and C1-6alkoxy optionally substituted with C1-6alkoxy or –C(O)NHC1-3alkyl; wherein the OH group is optionally protected by a hydroxyl protecting group; R10, for each occurrence, is independently H or forms a ring with the alkoxy group of R8; R11is a hydroxyl protecting group; R12, for each occurrence, is independently C1-6alkyl group, C2-6alkenyl group, phenyl or benzyl group, each of which is optionally substituted with –CN, -NO2 or halogen; orR13aand R13bare independently C1-6alkyl; q is an integer from 1 to 20; X, for each occurrence, is independently O or S; Z is a group represented by Formula I*or Formula I**,64 ME138157184v.1123429-12220; wherein represents the point of attachment for Z; W is O or NH; R1is a solid support; and R2is a silyl hydroxyl protecting group or an alloc protecting group.

25. The process of claim 24, wherein W is O.

26. The process of claim 24, wherein W is NH.

27. The process of claim 24, wherein Z is a group represented by formula I*.

28. The process of claim 24 or 27, wherein R2is a silyl hydroxyl protecting group selected from the following: ,, 65 ME138157184v.1123429-12220-O-TBDAS-2; wherein represents the point of attachment for R2; and R5, R6 and R7 are each independently H, C1-30alkyl, or C1-30alkoxy.

29. The process of any one of claims 24 to 28, wherein R2is selected from the group consisting of –O-TBDMS, -O-TIPS, -O-TBDPS, -O-TBoDPS, and –O-TBDAS:,, , , and .

30. The process of any one of claims 24 to 29, wherein R2is 66 ME138157184v.1123429-12220.

31. The process of any one of claims 24 to 27, wherein R2is an alloc protecting group.

32. The process of any one of claims 24 to 27, and 31, wherein the alloc protecting grouprepresents the point of attachment for R2.

33. The process of any one of claims 24-32, wherein further comprising a cleavage step of the oligonucleotide of formula (II):to form an oligonucleotide of formula (IIG) when Z is represented by Formula I* 67 ME138157184v.1123429-12220form an oligonucleotide of formula (IIG’) when Z is represented by Formula I**34. The process of claim 33, wherein Z is represented by Formula I* and the process further comprises a cleavage step of the oligonucleotide of Formula (II) to form an oligonucleotide of Formula (IIG). 68 ME138157184v.1123429-12220 35. The process of claim 33, wherein Z is presented by Formula I** and the process further comprises a cleavage step of the oligonucleotide of Formula (II) to form an oligonucleotide of Formula (IIG’).

36. The process of claim 35, wherein the process further comprises reacting the oligonucleotide of Formula (IIG’) with NH4OH to form the deprotected oligonucleotide of Formula, wherein: R9’, for each occurrence, is independently a nucleobase; R8, for each occurrence, is independently selected from the group consisting of H, halo, OH, and C1-6alkoxy optionally substituted with C1-6alkoxy or –C(O)NHC1-3alkyl; wherein the OH group is optionally protected by a hydroxyl protecting group; R10, for each occurrence, is independently H or forms a ring with the alkoxy group of R8; R11is a hydroxyl protecting group; q is an integer from 1 to 20; and X, for each occurrence, is independently O or S.

37. The process of claim 33 or 34, wherein when R2is defined in any one of claims 28-30, the cleavage step is carried out by reacting the oligonucleotide of formula (II) with HF in the presence of a base.

38. The process of claim 37, wherein the base is imidazole, pyridine or a combination thereof, wherein the imidazole or pyridine are optionally substituted. 69 ME138157184v.1123429-12220 39. The process of 38, wherein the cleavage step is carried out by reacting the oligonucleotide of formula (II) with HF in the presence of pyridine and imidazole.

40. The process of claim 39, wherein the molar ratio of imidazole to HF is in the range of 0.5:1 to 10:

1.

41. The process of claim 40, wherein the molar ratio of imidazole to HF is in the range of 1.1:1 to 5:

1.

42. The process of claim 41, wherein the molar ratio of imidazole to HF is 2:

1.

43. The process of any one of claims 39 -42, wherein the molar ratio of pyridine to HF is in the range of 100:1 to 1:

1.

44. The process of any one of claims 39-43, wherein the molar ratio of pyridine to HF is 1:

1.

45. The process of claim 33, wherein when R2is defined in claim 31 or 32, the cleavage step is carried out by 1) reacting the oligonucleotide of formula (II) with a palladium catalyst in the presence of a barbituric acid; and 2) adding imidazole to the reaction.

46. The process of claim 45, wherein the molar ratio of imidazole to the oligonucleotide of formula (II) is about 2:

1.

47. The process of claim 45 or 46, wherein the molar ratio of palladium catalyst to the oligonucleotide of formula (II) is about 1:100 to about 1:

10.

48. The process of any one of claims 45 to 47, wherein the molar ratio of palladium catalyst to the oligonucleotide of formula (II) is about 1:

50. 70 ME138157184v.1123429-12220 49. The process of any one of claims 45 to 48, wherein the palladium catalyst is tetrakis(triphenylphosphine) palladium(0).

50. The process of any one of claims 45 to 49, wherein the barbituric acid is dimethyl barbituric acid.

51. The process of any one of claims 21-45, wherein q is 2 to 5.

52. The process of claim 51, wherein q is 4.

53. The process of any one of claims 24-52, wherein all of the P=X groups in the nucleotide or oligonucleotide are P=S.

54. The process of any one of claims 24-52, wherein all of the P=X groups in the nucleotide or oligonucleotide are P=O.

55. The process of any one of claims 24-52, wherein greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the P=X groups in the oligonucleotide are P=S.

56. The process of any one of claims 24-52, wherein 10-90%, 20-80%, 30-70% or 40- 60% of the P=X groups in the compound or oligonucleotide are P=S.

57. The process of any one of claims 24-56, wherein the nucleobase is 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 the nucleobase, if present, is protected by PhCO-, CH3CO-, iPrCO-, Me2N- CH=, or Me2N-CMe=.

58. The process of any one of claims 24-57, wherein the nucleobase is selected from the group consisting of cytosine, guanine, adenine, thymine, uracil, and 5-methylcytosine, wherein the NH2group of the nucleobase, if present, is protected by PhCO-, CH3CO-, iPrCO-, Me2N-CH=, or Me2N-CMe=.

59. The process of any one of claims 24-58, wherein 71 ME138157184v.1123429-12220 each R8is independently selected from the group consisting of H, F, and C1-4alkoxy optionally substituted with C1-4alkoxy or –C(O)NHCH3; each R10is independently H or forms a ring with the alkoxy group of R8, wherein the ring is a 5 or 6-membered ring optionally substituted with 1 to 3 C1-4 alkyl groups; each R11is a 4,4’-dimethoxytirtyl group; R12is –CH2CH2CN; and R13aand R13bare independently C1-4alkyl.

60. The process of any one of claims 24-59, wherein each R8is independently selected from the group consisting of H, F, -OCH3, –OCH2CH2OCH3, -OCH2C(O)NHCH3 and -OTBDMS; and each R10is independently H or forms a ring with the alkoxy group of R8, wherein the ring is a 5-membered ring.

61. The process of any one of claims 24-60, wherein each R10is independently H or together with the alkoxy group of R8form –CH2-O-.

62. The process of any one of claims 24-61, wherein each R8is independently selected from H, –OCH2CH2OMe and –OCH2C(O)NHMe; each R10is H; each R11is a 4,4’-dimethoxytirtyl group; R12is –CH2CH2CN; and R13aand R13bare both -CH(CH3)2. 72 ME138157184v.1

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