Oligonucleotide conjugates containing 7'-5'-alpha-anomeric bicyclic sugar nucleosides

Abstract

The present invention provides an oligonucleotide-lipid group conjugate, the oligonucleotide comprising at least two alpha-anomeric bicycloDNA residues linked by phosphodiester bonds, the lipid group being attached to the oligonucleotide via a linker. The present invention also provides a method for modulating gene expression using the oligonucleotide-lipid group conjugate. [Selected figure] Figure 9A

Description

[Technical Field]

[0001] The present invention relates to oligonucleotide conjugates and their use for modulating gene expression. [Background technology]

[0002] Antisense oligonucleotides affect RNA processing and regulate protein expression. In certain instances, antisense compounds cause changes in the transcription or translation of the target. Such expression regulation can be achieved, for example, by inhibition based on the degradation or occupancy of the target mRNA. Oligonucleotide analogs that exhibit strong sequence-specific binding to single-stranded or double-stranded targets and are resistant to chemical degradation are potentially useful as therapeutic agents. Chemically modified oligonucleotides have been designed for therapeutic use. Summary of the Invention

[0003] The present invention provides oligonucleotides comprising abc-DNA nucleosides conjugated to a lipid group, wherein the abc-DNA nucleosides are preferably linked via phosphodiester bonds.

[0004] The present invention provides an oligonucleotide-lipid group conjugate, wherein the oligonucleotide comprises at least two alpha anomeric bicycloDNA (abc-DNA) residues linked by phosphodiester bonds, and a lipid group is covalently attached to the oligonucleotide.

[0005] In one embodiment, the lipid group is covalently attached to the oligonucleotide via a linker.

[0006] In another embodiment, the oligonucleotide comprises 12-24 residues. In another embodiment, the oligonucleotide comprises 14-20 residues. In another embodiment, the oligonucleotide comprises 14-19 residues. In another embodiment, the oligonucleotide comprises 15-19 residues. In another embodiment, the oligonucleotide comprises 15 residues. In another embodiment, the oligonucleotide comprises 16 residues. In another embodiment, the oligonucleotide comprises 17 residues. In another embodiment, the oligonucleotide comprises 18 residues. In another embodiment, the oligonucleotide comprises 19 residues.

[0007] In another embodiment, the abc-DNA residue has the formula (V): [ka] wherein, independently for each of the at least two abc-DNA residues of formula (IV), one of T3 or T4 is a nucleoside linking group, and the other of T3 and T4 is OR1, OR2, a 5'-terminal group, a 7'-terminal group, or a nucleoside linking group; R1 is H or a hydroxyl protecting group; R2 is a phosphorus moiety; Bx is a nucleobase, preferably Bx is selected from a purine base or a pyrimidine base, more preferably Bx is selected from uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.

[0008] Thus, in another embodiment, the abc-DNA residue has the formula (V): [ka] , in the formula, (I) T3 is a nucleoside linking group, T4 is a 7'-terminal group, OR1, or OR2, preferably T4 is a 7'-terminal group or OR1, or (ii) T3 is a 5'-terminal group, OR1, or OR2, preferably, T3 is a 5'-terminal group or OR2; T4 is a nucleoside linking group, or (iii) T3 and T4 are, independently of each other, a nucleoside linking group; R1 is H or a hydroxyl protecting group; R2 is a phosphorus moiety; Bx is a nucleobase, preferably Bx is selected from a purine base or a pyrimidine base, more preferably Bx is selected from uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.

[0009] In another embodiment, all of the residues are abc-DNA residues.

[0010] In another embodiment, at least two abc-DNA residues are linked to adjacent residues via phosphodiester bonds. In another embodiment, at least two abc-DNA residues are linked to adjacent residues via phosphodiester bonds, and each additional nucleoside linkage is independently selected from a phosphodiester linkage, a phosphotriester linkage, a phosphorothioate linkage, a phosphorodithioate linkage, a phosphonate linkage, a phosphonothioate linkage, a phosphinate linkage, a phosphorothioamidate linkage, or a phosphoramidate linkage.

[0011] In another embodiment, all of the residues are abc-DNA residues and are linked via phosphodiester bonds. Thus, in another embodiment, each nucleoside linkage group is a phosphodiester linkage group.

[0012] In another embodiment, the lipid group is covalently attached to a terminal residue of the oligonucleotide.

[0013] In another embodiment, the oligonucleotide comprises residues linked via a phosphorus-containing nucleoside linking group selected from the group consisting of a phosphodiester linking group, a phosphotriester linking group, a phosphorothioate linking group, a phosphorodithioate linking group, a phosphonate linking group, a phosphonothioate linking group, a phosphinate linking group, a phosphorothioamidate linking group, and a phosphoramidate linking group.

[0014] In another embodiment, the linker is a hydrocarbon linker or a polyethylene glycol (PEG) linker.

[0015] In another embodiment, the linker is selected from the group consisting of an amino-alkyl-phosphorothioate linker, an amino-PEG-phosphorothioate linker, an alpha-carboxylate-amino-alkyl phosphorothioate linker, and an alpha-carboxylate-amino-PEG-phosphorothioate linker.

[0016] In another embodiment, the linker comprises a cleavable group.

[0017] In another embodiment, the lipid group is a fatty acid-derived group.

[0018] In one embodiment, the fatty acid is saturated or unsaturated.

[0019] In another embodiment, the fatty acid has a length of 4 to 28 carbon atoms.

[0020] In another embodiment, the fatty acid-derived group comprises a carboxylic acid group.

[0021] In another embodiment, the fatty acid derived group is derived from a dicarboxylic acid.

[0022] In another embodiment, the fatty acid is selected from the fatty acids presented in Table 1 or Table 2.

[0023] In another embodiment, the fatty acid is hexadecanoic acid.

[0024] In one embodiment, the lipid group is attached to the linker via a thiophosphate group.

[0025] In one embodiment, the lipid group is attached to the oligonucleotide via a thiophosphate group.

[0026] In another embodiment, the oligonucleotide conjugate binds to a pre-mRNA corresponding to a portion of exon 51 of the Duchenne muscular dystrophy (DMD) gene.

[0027] In another embodiment, the oligonucleotide conjugate comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39, 51-55, 404, and 414-425. In another embodiment, the oligonucleotide conjugate comprises the sequence of SEQ ID NO: 417 or SEQ ID NO: 418.

[0028] In another embodiment, the oligonucleotide comprises any one of the sequences provided in Table 3.

[0029] In one embodiment, the oligonucleotide conjugate binds to a pre-mRNA corresponding to a portion of exon 53 of the DMD gene.

[0030] In one embodiment, the oligonucleotide comprises any one of the sequences provided in Table 4.

[0031] In another embodiment, the oligonucleotide conjugate binds to a pre-mRNA corresponding to a portion of exon 45 of the DMD gene.

[0032] In one embodiment, the oligonucleotide comprises any one of the sequences provided in Table 5.

[0033] The present invention also provides a pharmaceutical composition comprising an oligonucleotide-lipid group conjugate in combination with a suitable carrier, wherein the oligonucleotide comprises at least two alpha anomeric bicycloDNA (abc-DNA) residues linked by phosphodiester bonds, and a lipid group is covalently attached to the oligonucleotide.

[0034] The present invention also provides a method for altering expression of a gene by allowing hybridization of an oligonucleotide-lipid group conjugate to RNA expressed from the gene, wherein the oligonucleotide comprises at least two alpha anomeric bicycloDNA (abc-DNA) residues joined by phosphodiester bonds, a lipid group is covalently attached to the oligonucleotide, and the oligonucleotide comprises a sequence complementary to a portion of the RNA.

[0035] The present invention also provides a method for inducing exon 51 skipping of human dystrophin pre-mRNA in a subject with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), or in a cell derived from the subject, the method comprising providing an oligonucleotide-lipid group conjugate, wherein the oligonucleotide comprises at least two alpha anomeric bicycloDNA (abc-DNA) residues linked by phosphodiester bonds, and the lipid group is covalently attached to an oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39, 51-55, 404 and 414-425, preferably SEQ ID NO: 417 or SEQ ID NO: 418, wherein the oligonucleotide conjugate induces exon skipping in the subject or cell, and wherein the mRNA produced from the skipping of exon 51 of the dystrophin pre-mRNA encodes a functional dystrophin protein or a Becker-subject dystrophin protein.

[0036] The present invention also provides methods for treating Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) in a subject or in a cell derived from a subject by inducing skipping of exon 51 of human dystrophin pre-mRNA, the method comprising providing to the subject or cell a composition comprising an oligonucleotide-lipid group conjugate, wherein the oligonucleotide comprises at least two alpha anomeric bicycloDNA (abc-DNA) residues joined by phosphodiester bonds, and the lipid group is covalently attached to an oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39, 51-55, 404, and 414-425, preferably SEQ ID NO: 417 or SEQ ID NO: 418, wherein the oligonucleotide conjugate induces exon skipping in the subject or cell, and wherein the mRNA produced from the skipping of exon 51 of the dystrophin pre-mRNA encodes a functional dystrophin protein or a Becker-subject dystrophin protein.

[0037] The present invention also provides a method for inducing skipping of exon 51 of human dystrophin pre-mRNA in a subject with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), or in a cell derived from the subject, the method comprising providing an oligonucleotide-lipid group conjugate, wherein the oligonucleotide comprises at least two alpha anomeric bicycloDNA (abc-DNA) residues joined by phosphodiester bonds, and the lipid group is covalently attached to an oligonucleotide comprising any one of the sequences shown in Table 3, preferably all of the residues are abc-DNA residues, wherein the oligonucleotide conjugation induces exon skipping in the subject or cell, and wherein the mRNA produced from the skipping of exon 51 of the dystrophin pre-mRNA encodes a functional dystrophin protein or a Becker-subject dystrophin protein.

[0038] The present invention also provides methods for treating Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) in a subject or in a cell derived from a subject by inducing skipping of exon 51 of human dystrophin pre-mRNA, the method comprising providing to the subject or cell a composition comprising an oligonucleotide-lipid group conjugate, wherein the oligonucleotide comprises at least two alpha anomeric bicycloDNA (abc-DNA) residues joined by phosphodiester bonds, and the lipid group is covalently attached to an oligonucleotide comprising any one shown in Table 3, preferably all of the residues are abc-DNA residues, wherein the oligonucleotide conjugation induces exon skipping in the subject or cell, and wherein the mRNA produced from the skipping of exon 51 of the dystrophin pre-mRNA encodes a functional dystrophin protein or a Becker-subject dystrophin protein. [Brief explanation of the drawings]

[0039] [Figure 1A] Evaluation of the acidic stability of alpha-anomeric oligonucleotides by liquid chromatography-mass spectrometry (LS-MS). LS-MS chromatogram of untreated ON1. [Figure 1B] LS-MS fragmentation pattern of untreated ON1. [Figure 1C] LS-MS chromatogram of ON1 treated under acidic conditions for 24 hours. [Figure 1D] LS-MS fragmentation pattern of ON1 treated under acidic conditions for 24 hours. [Figure 2A] Evaluation of thermal stability of alpha-anomeric oligonucleotides by LS-MS. LS-MS chromatogram of untreated ON1. [Figure 2B] LS-MS fragmentation pattern of untreated ON1. [Figure 2C] LS-MS chromatogram of ON1 heated at 95°C for 60 min. [Figure 2D] LS-MS fragmentation pattern of ON1 heated at 95 °C for 60 min. [Figure 3] Assessment of biostability of alpha-anomeric oligonucleotides by 20% denaturing PAGE. PAGE of ON1 and its corresponding native oligonucleotide incubated in mouse serum. [Figure 4] Mobility shift assay of ON1 incubated with different albumin equivalents. [Figure 5] Comparison of uncomplexed ON1 incubated with different albumin equivalents. Values ​​obtained by ultrafiltration experiments. [Figure 6] Mobility shift assay of ON1 incubated with different amounts of mouse serum. [Figure 7] Intensity of nanoparticles present in ON1 solution. [Figure 8] Agarose gel showing the efficiency of mouse exon 23 skipping in C2C12 cells as detected by nesting RT-PCR. [Figure 9A] Agarose gel of human exon 51 skipping efficiency in KM155 cells detected by nesting RT-PCR. [Figure 9B] Agarose gel of human exon 51 skipping efficiency in KM155 cells detected by nesting RT-PCR. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention provides an oligonucleotide conjugate comprising at least one (one or more) alpha anomeric bicyclo-DNA (abc-DNA) nucleoside, phosphodiester groups linking the nucleosides of the oligonucleotide, and a lipid group linked to the oligonucleotide via a linker. The present invention provides an oligonucleotide comprising abc-DNA nucleosides linked via phosphodiester internucleoside linkages and conjugated to a ligand group.

[0041] The oligonucleotides of the invention modulate gene expression by interfering with transcription, translation, splicing, and / or degradation, and / or by inhibiting the function of non-coding RNA.

[0042] Definition: As used herein, "alpha anomeric bicyclic DNA (abc-DNA) nucleoside" means a nucleoside analog that contains a bicyclic sugar moiety and has the general structure shown below. [ka] The structure of 7'-5'-alpha-anomeric bicyclo-DNA is shown below. [ka]

[0043] As used herein, a "bicyclic sugar moiety" comprises two interconnected ring systems, e.g., a bicyclic nucleoside, wherein the sugar moiety has a 2'-O-CH(alkyl)-4' or 2'-O-CH2-4' group, locked nucleic acid (LNA), xylo-LNA, alpha-L-LNA, beta-D-LNA, cEt (2'-O,4'-C constrained ethyl) LNA, cMOEt (2'-O,4'-C constrained methoxyethyl) LNA, or ethylene-bridged nucleic acid.

[0044] As used herein, "nucleoside" refers to a nucleobase covalently linked to a sugar.

[0045] "Ribonucleoside" refers to a base linked to a ribose. "Deoxyribonucleoside" refers to a base linked to a 2'-deoxyribose.

[0046] As used herein, "nucleotide" means a nucleoside that further includes a phosphorus moiety covalently linked to the sugar of the nucleoside.

[0047] As used herein, the term "residue" refers to a nucleoside or nucleotide monomer that forms the unit of an oligomer (oligonucleotide polymer).

[0048] As used herein, an "oligonucleotide" refers to an oligomer that can be single-stranded or double-stranded, but binds to complementary nucleic acids in a cell or organism as a single-stranded nucleic acid molecule. An oligonucleotide comprises at least two nucleosides linked to each other by a nucleoside linking group, as defined herein. An oligonucleotide may comprise ribonucleotides, deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2' modifications, synthetic base analogs, etc.), or a combination thereof. Such modified oligonucleotides may be preferred over natural forms due to properties such as enhanced cellular uptake and increased stability in the presence of nucleases. Oligonucleotides include naturally occurring nucleotides, modified nucleotides or nucleotide mimics, as well as compounds known in the art and described herein, including oligonucleotides with modifications to sugars and / or nucleobases and / or nucleoside linking groups.

[0049] In certain embodiments, oligonucleotides of the invention have a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides, e.g., 12 to 50 nucleotides, or 12 to 40 or 12 to 24 nucleotides, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 nucleotides.

[0050] As used herein, the term "oligomer," e.g., oligonucleotide, refers to a compound comprising two or more monomer subunits linked by nucleoside linking groups. Oligomers of the invention can have a length of up to 50 monomer subunits, e.g., up to 40 monomer subunits, e.g., up to 30 monomer subunits, up to 20 monomer subunits, or up to 15 monomer subunits. Oligomers can contain 5 to 40 monomer subunits, 8 to 30 monomer subunits, 8 to 25 monomer subunits, or 8 to 20 monomer subunits.

[0051] As used herein, the term " nucleic acid " refers to single-stranded or double-stranded deoxyribonucleotide, ribonucleotide, or modified nucleotide, and their polymers.This term includes the nucleic acid that contains known nucleotide analogues or modified backbone residues or bonds, and this nucleic acid is synthetic, naturally occurring, and non-naturally occurring, has the same binding properties as reference nucleic acid, and in some cases, is metabolized in the same manner as reference nucleotide.Examples of such analogues include but are not limited to phosphorothioate, phosphoramidate, phosphorodiamidate, methylphosphonate, chiral-methylphosphonate, 2'-O-methylribonucleotide, and peptide-nucleic acid (PNA).

[0052] The present invention provides oligonucleotides conjugated to a lipid group via a covalent bond. As used herein, "lipid group" refers to any fatty acid group or fatty acid-derived group, any steroid-derived group, and any fat-soluble vitamin group. The abc-DNA oligonucleotide-lipid group conjugate can exhibit a long half-life in vivo. The lipid group can also increase the binding of the abc-DNA oligonucleotide to albumin and / or other fatty acid receptors or transporters. The structure of the oligonucleotides of the present invention conjugated to a lipid group is such that the lipid group is exposed to facilitate binding to albumin and / or other transporters. In another embodiment of the present invention, the lipid group further comprises one or two carboxylic acid groups, further increasing interaction with albumin and / or other fatty acid receptors or transporters. In one embodiment, the lipid group is a fatty acid-derived group. In another embodiment, the lipid group is a fatty acid-derived group derived from a dicarboxylic acid. The fatty acid can include saturated or unsaturated fatty acids having a hydrocarbon chain of 2 to 28 carbon atoms and can comprise one or two carboxylic acid groups. One or two fatty acid ligands can be attached to the oligonucleotide via linkers on the 5' and / or 7' ends of the abc-DNA oligonucleotides described herein. Useful lipid groups according to the present invention are provided in Tables 1 and 2.

[0053] The lipid group of the present invention may include cholesterol, vitamin E (tocopherol), or bile acids.

[0054] As used herein, "linker" refers to the moiety that connects the oligonucleotide of the present invention to the lipid group.Linkers useful according to the present invention include, but are not limited to, hydrocarbon and PEG linkers, such as amino-alkyl-phosphorothioate linkers, alpha-carboxylate-amino-alkyl-phosphorothioate linkers, amino-PEG-phosphorothioate linkers, and alpha-carboxylate-amino-PEG-phosphorothioate linkers.Linkers according to the present invention typically and preferably do not reduce or prevent the binding of oligonucleotide to its target.Linkers can include cleavable groups.

[0055] As used herein, "nucleoside linking group" refers to the linking group that connects the abc-DNA nucleosides of an oligonucleotide. The nucleoside linking groups of the present invention are primarily phosphodiester internucleoside linkages or bonds. The term "nucleoside linking group" includes phosphorus linking groups that are not phosphodiester bonds, as well as non-phosphorus linking groups.

[0056] The present invention provides an oligonucleotide conjugated with a lipid group, in which all internucleoside bonds are phosphodiester bonds.In certain embodiments, the internucleoside bond groups of the oligonucleotide conjugated with a lipid group are mainly phosphodiester bonds.As used herein, "mainly" means that 50% or more, for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% and 100% of the internucleoside bond groups are phosphodiester bonds.For example, oligonucleotide can contain one or more, and up to 50% phosphorothioate bond.The nucleoside of the oligonucleotide of the present invention is mainly abc-DNA nucleoside. Generally, when referring to abc-DNA nucleosides, it means that 50% or more of the nucleosides are abc-DNA nucleosides, for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, and 100%. For example, the oligonucleotides of the present invention can contain one or more, and up to 50%, nucleosides having sugars that are not abc-DNA nucleosides.

[0057] The present invention provides nucleosides linked via phosphorus-containing internucleoside or phosphodiester bonds. The present invention also provides nucleosides that are primarily linked via phosphodiester bonds, but include a "phosphorus-containing nucleoside linking group" selected from phosphotriester linking groups, phosphorothioate linking groups, phosphorodithioate linking groups, and phosphonate linking groups, such as H-phosphonate linking groups or methylphosphonate linking groups, phosphonothioate linking groups, such as H-phosphonothioate linking groups, methylphosphonothioate linking groups, phosphinate linking groups, phosphorothioamidate linking groups, or phosphoramidate linking groups.

[0058] As used herein, "nucleoside" or "nucleotide" includes naturally occurring or modified nucleosides or nucleoside mimics, or naturally occurring or modified nucleotides or nucleotide mimics, respectively, that can be incorporated into oligomers of the invention by chemical or non-chemical oligomer synthesis methods. As used herein, "natural" or "naturally occurring" means of natural origin.

[0059] The term "modified nucleoside" includes nucleosides having modifications in the sugar and / or nucleobase of the nucleoside, as known in the art and as described herein. The term "modified nucleotide" includes nucleotides having modifications in the sugar and / or nucleobase of the nucleotide, and / or in the nucleoside linkage group or phosphorus moiety, as known in the art and as described herein.

[0060] As used herein, "nucleoside mimics" include structures used to replace sugars and nucleobases. The term "nucleotide mimics" includes nucleotides used to replace sugars and nucleoside linkage groups. Examples of nucleotide mimics include peptide nucleic acids (PNAs) or morpholinos.

[0061] A "nucleoside" or "nucleotide" of the invention can contain a combination of modifications, e.g., two or more nucleobase modifications, two or more sugar modifications, or at least one nucleobase and at least one sugar modification.

[0062] The oligonucleotides of the present invention contain primarily nucleosides having bicyclosugars.

[0063] However, oligonucleotides can also include nucleosides with sugars that are monocyclic or tricyclic ring systems, tricyclic or bicyclic systems, or monocyclic ribose or deoxyribose. Sugar modifications further include, but are not limited to, modified stereochemical configurations, substitution of at least one group, or deletion of at least one group. Modified sugars include modified forms of the ribosyl moiety (i.e., furanosyl moiety) naturally occurring in RNA and DNA, tetrahydropyran, 2'-modified sugars, 3'-modified sugars, 4'-modified sugars, 5'-modified sugars, or 4'-substituted sugars. Examples of suitable sugar modifications are known to those of skill in the art and include 2', 3' and / or 4' substituted nucleosides (e.g., 4'-S modified nucleosides), 2'-O modified RNA nucleotide residues, such as 2'-O-alkyl or 2'-O-(substituted) alkyl, e.g., 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-(haloalkoxy)methyl, e.g., 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)ethyl, Modified sugar moieties include, but are not limited to, methyl (DCEM), 2'-O-alkoxycarbonyl, e.g., 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DMCE), e.g., 2'-O-methyl modified or 2'-O-methoxyethyl (2'-O-MOE), or other modified sugar moieties, e.g., morpholino (PMO), cationic morpholino (PMOPlus), or modified morpholino groups, e.g., PMO-X.The term "PMO-X" refers to a modified morpholino group comprising at least one 3' or 5' end modification, such as a 3'-fluorescent tag, a 3' quencher (e.g., 3'-carboxyfluorescein, 3'-GeneToolsBlue, 3'-lissamine, 3'-dabsyl), a 3'-affinity tag, and a functional group for chemical conjugation (e.g., 3'-biotin, 3'-primary amine, 3'-disulfide amide, 3'-pyridyldithio), a 5' end modification (5'-primary amine, 5'-dabsyl), a 3'-azide, a 3'-alkyne, a 5'-azide, a 5'-alkyne, or as disclosed in WO2011 / 150408 and US2012 / 0065169.

[0064] As used herein, the term "ribonucleotide" encompasses natural and synthetic unmodified and modified ribonucleotides. Modifications include changes in the sugar moiety, the base moiety, and / or the linkages between ribonucleotides in an oligonucleotide. As used herein, the term "ribonucleotide" specifically excludes deoxyribonucleotides, which are nucleotides having a single proton group at the 2' ribose ring position.

[0065] As used herein, the term "deoxyribonucleotide" encompasses natural and synthetic unmodified and modified deoxyribonucleotides. Modifications include changes in the sugar moiety, the base moiety, and / or the linkage between deoxyribonucleotides in an oligonucleotide. As used herein, the term "deoxyribonucleotide" also includes modified ribonucleotides, such as 2'-O-methylribonucleotides, phosphorothioate-modified ribonucleotide residues, and the like.

[0066] As used herein, the term "PS-NA" refers to phosphorothioate-modified nucleotide residues.Therefore, the term "PS-NA" encompasses both phosphorothioate-modified ribonucleotides ("PS-RNA") and phosphorothioate-modified deoxyribonucleotides ("PS-DNA").

[0067] As used herein, "antisense strand" refers to a single-stranded nucleic acid molecule having a sequence complementary to that of a target RNA.

[0068] As used herein, "sense strand" refers to a single-stranded nucleic acid molecule having a sequence complementary to that of the antisense strand.

[0069] The present invention also provides oligonucleotides linked to non-nucleoside compounds.

[0070] The present invention provides oligonucleotides attached to a solid support, including but not limited to, a bead, a polymer, or a resin.

[0071] In certain embodiments, oligonucleotides are modified by the covalent attachment of one or more groups to the 5' or 7' end of the oligomer, or at any position on the oligomer, in addition to the lipid group. Groups that can be conjugated to the 5' or 7' end include, but are not limited to, capping groups, diphosphates, triphosphates, labels such as fluorescent labels (e.g., fluorescein or rhodamine), dyes, oligomers, solid supports, nanoparticles, non-nucleoside groups, antibodies, or reporter groups useful for tracking the conjugated group. Generally, conjugated groups modify one or more properties of the compound to which they are attached. Such properties include, but are not limited to, nuclease stability, binding affinity, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, delivery, charge, and clearance. Conjugated groups are routinely used in chemical engineering and are attached directly or via any linking group to parent compounds, such as oligomers. The term "conjugate group" includes, but is not limited to, lipid groups, intercalators, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, lipophilic moieties, coumarins, peptides, antibodies, nanobodies, and oligosaccharides, e.g., N-acetylgalactosamine.

[0072] As used herein, "terminal" refers to the end or terminus of an oligomer, nucleic acid sequence, or any one of the compounds described herein, and the integer (e.g., 3', 5', or 7') indicates the carbon atom of the sugar contained in the nucleotide, nucleic acid sequence, or compound of the oligomer. As used herein, a "5' terminal group" or a "7' terminal group" refers to a group located at the 5' or 7' end, respectively, of the sugar contained in any one of the compounds provided herein.

[0073] In certain embodiments, an oligomer comprises at least one monomer subunit that is a compound of formula (IV), formula (V), or formula (VI) described herein. In one embodiment, an oligomer comprises at least one compound of formula (IV), (V), or (VI) and at least one ribonucleotide or deoxyribonucleotide. In another embodiment, an oligomer comprises at least one compound of formula (IV), (V), or (VI) and at least one deoxyribonucleotide.

[0074] "Complementary" or "complementarity" means that a nucleic acid can form hydrogen bonds with another nucleic acid sequence through either conventional Watson-Crick or Hoogsteen base pairing. With respect to the nucleic acid molecules of the present disclosure, the binding free energy of a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, for example, exon skipping. The determination of the binding free energy of a nucleic acid molecule is well known in the art (see, for example, Turner, et al., CSH SympQuant.Biol.LII, pp.123-133,1987; Friere et al., Proc.Nat.Acad.Sci.USA 83:9373-9377,1986; Turner et al., J.Am.Chem.Soc.109:3783-3785,1987). The percent complementarity indicates the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in a first oligonucleotide paired with a second nucleic acid sequence having 10 nucleotides indicates 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). To determine the percent complementarity is at least a particular percent, the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence is calculated and rounded to the nearest integer (e.g., 12, 13, 14, 15, 16, or 17 nucleotides out of a total of 23 nucleotides in a first oligonucleotide base pairing with a second nucleic acid sequence having 23 nucleotides represents 52%, 57%, 61%, 65%, 70%, and 74%, respectively, and has at least 50%, 50%, 60%, 60%, 70%, and 70% complementarity, respectively). As used herein, "substantially complementary" refers to complementarity between strands such that the strands are capable of hybridizing under biological conditions. Substantially complementary sequences have 60%, 70%, 80%, 90%, 95%, or even 100% complementarity.Furthermore, techniques for determining whether two strands are capable of hybridizing under biological conditions by examining their nucleotide sequences are well known in the art.

[0075] The present invention also provides wobble base pairs between two nucleotides in RNA molecules that do not follow the Watson-Crick base pairing rules. The four main wobble base pairs are guanine-uracil (GU), hypoxanthine-uracil (IU), hypoxanthine-adenine (IA), and hypoxanthine-cytosine (IC). The thermodynamic stability of wobble base pairs is comparable to that of Watson-Crick base pairs.

[0076] Single-stranded nucleic acids that form base pairs over several bases are said to "hybridize." Hybridization is typically determined under physiological or biologically relevant conditions (e.g., intracellular: pH 7.2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion). Hybridization conditions generally include monovalent cations and biologically acceptable buffers, and may or may not include divalent cations, complex anions, such as gluconic acid from potassium gluconate, uncharged species such as sucrose, and inert polymers, such as PEG, to reduce the activity of water in the sample. Such conditions include conditions under which base pairs are formed.

[0077] Hybridization is measured as the temperature at which 50% of the nucleic acid is single-stranded and 50% is double-stranded (melting temperature; Tm). m is often used as a measure of the duplex stability of an antisense compound relative to a complementary nucleic acid.

[0078] Hybridization conditions are also conditions under which base pairs are formed. Various stringency conditions can be used to determine hybridization (see, for example, Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507). Stringent temperature conditions typically include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. For hybrids expected to be less than 50 base pairs in length, the hybridization temperature should be 5-10°C lower than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following formula: For hybrids less than 18 base pairs in length, Tm (°C) = 2(# of A+T bases) + 4(# of G+C bases). For hybrids 18 to 49 base pairs in length, Tm (°C) = 81.5 + 16.6(log10[Na+]) + 0.41(%G+C) - (600 / N), where N is the number of bases in the hybrid and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1X SSC = 0.165M).

[0079] Useful variations in hybridization conditions will be readily apparent to those skilled in the art.Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180,1977); Grunstein and Hogness (Proc. Natl. Acad. Sci. USA 72:3961,1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Antisense to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0080] As used herein, "alter" refers to an increase or decrease in expression, e.g., gene expression. A decrease in expression refers to a decrease of 10% or more, e.g., 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. A decrease also refers to a decrease of 2-fold or more, e.g., 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, or 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, or more.

[0081] Increased expression refers to an increase of 10% or more, e.g., 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%. Increase also refers to an increase of 2-fold or more, e.g., 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold or more.

[0082] An increase or decrease in gene expression is relative to a control or reference level of expression, eg, the level of gene expression in the absence of an oligonucleotide lipid group conjugate of the present invention.

[0083] As used herein, "target RNA" refers to the RNA that will be modulated by the oligonucleotides of the present invention.

[0084] As used herein, "target" refers to any nucleic acid sequence whose expression or activity is modulated by an oligonucleotide of the invention.

[0085] As used herein, "reference" refers to a standard or control. As will be apparent to those skilled in the art, an appropriate reference is one in which only one element is changed to determine the effect of that element.

[0086] As used herein, a "portion of RNA" means a length having a sequence that is identical to and complementary to the sequence of the oligonucleotide to which it is bound.

[0087] The term "in vitro" has its art-recognized meaning, including, for example, purified reagents or extracts, e.g., cellular extracts. The term "in vivo" also has its art-recognized meaning, including, for example, living cells, e.g., immortalized cells, primary cells, cell lines, and / or cells in an organism.

[0088] As used herein, "increase" or "enhance" means a positive change of at least 5% compared to a reference in an assay. The change can be 5%, 10%, 25%, 30%, 50%, 75%, or even 100% compared to a reference in an assay. "Enhancing exon skipping" means increasing the amount of a particular product that results from exon skipping.

[0089] As used herein, "reduce" means to change negatively by at least 5% compared to a reference in an assay. The change can be 5%, 10%, 25%, 30%, 50%, 75%, or even 100% compared to a reference in an assay.

[0090] As used herein, "cell" is meant to include both prokaryotic (e.g., bacterial) and eukaryotic (e.g., mammalian or plant) cells. Cells can be somatic or germline derived, totipotent or pluripotent, and dividing or non-dividing. Cells can also be derived from or include gametes or embryos, stem cells, or fully differentiated cells. Thus, the term "cell" is meant to retain its normal biological meaning and can be present in any organism, such as, for example, birds, plants, and mammals, including, for example, humans, cows, sheep, apes, monkeys, pigs, dogs, and cats. In certain embodiments, the term "cell" specifically refers to mammalian cells, such as human cells.

[0091] As used herein, "animal" refers to a multicellular eukaryotic organism, including mammals, particularly humans. The method of the present invention generally comprises administering an effective amount of the oligonucleotide herein to a subject (e.g., animal, human) in need thereof, including mammals, particularly humans. Such treatment will be preferably administered to a subject, particularly humans, suffering from, suffering from, susceptible to, or at risk of a disease or its symptoms.

[0092] By "pharmaceutically acceptable carrier" is meant a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the present disclosure in the physical location most suitable for their desired activity.

[0093] Oligonucleotide agents of the invention can enhance the following attributes of such agents compared to oligonucleotide agents lacking abc-DNA nucleosides, or oligonucleotides comprising abc-DNA nucleosides but lacking the combination of phosphate internucleoside linkages and lipid groups: in vitro efficacy (e.g., potency and duration of effect), in vivo efficacy (e.g., potency, duration of effect, pharmacokinetics, pharmacodynamics, cellular uptake, reduced toxicity).

[0094] As used herein, the term "pharmacokinetics" refers to the processes by which a drug is absorbed, distributed, metabolized, and eliminated by the body.

[0095] As used herein, the term "pharmacodynamics" refers to the action or effect of a drug on an organism.

[0096] As used herein, the term "stabilization" refers to a state of enhanced persistence of an agent in a selected environment (e.g., a cell or organism). Enhanced stability can be achieved by enhanced resistance of such an agent to degradative enzymes (e.g., nucleases) or other agents.

[0097] As used herein, "modified nucleotide" refers to a nucleotide that has one or more modifications to its nucleoside, nucleic acid base, furanose ring, or phosphate group.For example, modified nucleotide excludes ribonucleotides that contain adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate, and deoxyribonucleotides that contain deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate.Modifications include naturally occurring modifications that result from modifications by enzymes that modify nucleotides, such as methyltransferases.Modified nucleotides also include synthetic or non-naturally occurring nucleotides. Synthetic or non-naturally occurring modifications in nucleotides include those with 2' modifications, such as 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, and 2'-O-(N-methylcarbamate) or base analogs.In the context of the 2'-modified nucleotides described in the present disclosure, "amino" refers to 2'-NH2 or 2'-O-NH2, which can be modified or unmodified.Such modified groups are described, for example, in Eckstein et al., U.S. Patent No. 5,672,695 and Matulic-Adamic et al., U.S. Patent No. 6,248,878.

[0098] As used herein, "base analog" refers to a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide that can be incorporated into a nucleic acid duplex (or the equivalent position in a nucleotide sugar moiety substitution that can be incorporated into a nucleic acid duplex). Base analogs are generally either purine or pyrimidine bases, with the exception of the common bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U). Base analogs can form duplexes with other bases or base analogs in dsRNA. Base analogs include those useful in the compounds and methods of the present invention, such as those disclosed in U.S. Patent Nos. 5,432,272 and 6,001,983 to Benner and U.S. Patent Publication No. 2008 / 0213891 to Manoharan, which are incorporated herein by reference. Non-limiting examples of bases include 2,6-diaminopurine, hypoxanthine (I), xanthine (X), 3-β-D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3-β-D-ribofuranosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-d-dione) (P), isocytosine (iso-C), isoguanine (iso-G), 1-β-D-ribofuranosyl-(5-nitroindole), 1-β-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds), and pyrrole-2-carbohydrate. Bardehyde (Pa), 2-amino-6-(2-thienyl)purine (S), 2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, and 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-propynylisocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,These include 6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl, tetracenyl, pentacenyl, and their structural derivatives (Schweitzer et al., J. Org. Chem. 59:7238-7242 (1994); Berger et al., Nucleic Acids Research, 28(15):2911-2914 (2000); Moran et al., J. Am. Chem. Soc. 119:2056-2057 (1997); Morales et al., J. Am. Chem. Soc. 121:2323-2324 (1999); Guckian et al., J. Am. Chem. Soc. 118:8182-8183 (1996); Morales et al., J. Am. Chem. Soc. 118:8182-8183 (1996)). al.,J.Am.Chem.Soc.122(6):1001-1007(2000);McMinn et al.,J.Am.Chem.Soc.121:11585-11586(1999);Guckian et al.,J.Org.Chem.63:9652-9656(1998);Moran et al. al.,Proc.Natl.Acad.Sci.94:10506-10511(1997);Das et al.,J.Chem.Soc.Perkin Trans.1:197-206(2002);Shibata et al.,J.Chem.Soc.Perkin Trans.1:1605-1611(2001);Wu et al. al., J. Am. Chem. Soc. 122(32):7621-7632 (2000); O'Neill et al., J. Org. Chem. 67:5869-5875 (2002); Chaudhuri et al., J. Am. Chem. Soc. 117:10434-10442 (1995); and U.S. Patent No. 6,218,108). The base analogue can also be a universal base.

[0099] As used herein, a "universal base" refers to a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide or at an equivalent position in a nucleotide sugar moiety substitution, which, when present in a nucleic acid duplex, can be positioned opposite two or more bases without altering the double helix structure (e.g., the structure of the phosphate backbone). Furthermore, a universal base does not disrupt the ability of an oligonucleotide to form a duplex with a target nucleic acid. The ability of a single-stranded nucleic acid containing a universal base to form a duplex with a target nucleic acid can be assayed by methods known in the art (e.g., UV absorbance, circular dichroism, gel shift, single-stranded nuclease sensitivity, etc.). Furthermore, the conditions under which duplex formation is observed, such as temperature, can be varied to determine duplex stability or formation, as melting temperature (Tm) correlates with the stability of a nucleic acid duplex. Compared to a reference single-stranded nucleic acid that is exactly complementary to the target nucleic acid, a single-stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid with a lower Tm than a duplex formed with the complementary nucleic acid. However, compared to a reference single-stranded nucleic acid in which the universal base has been replaced with a base resulting in a single mismatch, the single-stranded nucleic acid containing the universal base forms a duplex with a target nucleic acid that has a higher Tm than the duplex formed with the nucleic acid having the mismatched base.

[0100] Some universal bases can base pair by forming hydrogen bonds with all of the bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U) under pairing conditions. A universal base is not a base that base pairs with only one single complementary base. In a duplex, a universal base can form zero, one, or more hydrogen bonds with each of the G, C, A, T, and U bases on the opposite strand of the duplex. Preferably, a universal base does not interact with the base on the opposite strand of the duplex. In a duplex, base pairing between universal bases occurs without changing the double helix structure of the phosphate backbone. A universal base can also interact with the base of an adjacent nucleotide on the same nucleic acid strand through stacking interactions. Such stacking interactions stabilize the duplex, especially in situations where the universal base does not form hydrogen bonds with the oppositely positioned base on the opposite strand of the duplex.Non-limiting examples of universal-binding nucleotides include inosine, 1-beta-D-ribofuranosyl-5-nitroindole, and / or 1-beta-D-ribofuranosyl-3-nitropyrrole (Quay et al., U.S. Patent Application Publication No. 2007 / 0254362; Van Aerschot et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995 Nov. 11; 23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. Nucleic Acids Res. 1995 Jul. 11; 23(13):2361-6; Loakes and Brown, 5-Nitroindole as a universal base analogue. Nucleic Acids Res. Res.1994 Oct.11;22(20):4039-43).

[0101] The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0102] "Diastereomer" refers to a stereoisomer with two or more centers of chirality in which the compound is not a mirror image of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and chemical and biological reactivity. Mixtures of diastereomers can be separated under high-resolution analytical procedures such as electrophoresis and chromatography.

[0103] "Enantiomers" refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0104] The stereochemical definitions and rules used herein generally follow S.P. Parker, Ed., McRaw-Hiff Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Elie, I.E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994.

[0105] Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art to which this invention belongs.The following references provide those skilled in the art with the general definitions of many terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed.1988); The Glossary of Genetics, 5th ed. R. Rieger et al. (eds.) Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991).As used herein, the following terms have the meanings that are ascribed to them below, unless otherwise specified.

[0106] The present invention may be understood more readily by reference to the following detailed description of the invention and the examples included therein.

[0107] All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the publication dates provided herein may be different from the actual publication dates, which may require independent confirmation.

[0108] Alpha-anomeric bicyclo-DNA (abc-DNA) nucleosides Alpha bicyclic ("abc") DNA is a nucleoside analog containing a bicyclic sugar moiety that is useful in antisense oligonucleotides (AONs), for example, to treat disease by inducing exon skipping. abc-DNA nucleosides have the following general structure: [ka]

[0109] The structure of 7'-5'-alpha-anomeric bicyclo-DNA is shown below. [ka]

[0110] In addition to high selectivity for RNA, the 7'5'-abc-DNA modification improves mismatch discrimination compared to DNA, is compatible with phosphorothioate modifications, confers complete biostability, induces low complement activation, and exhibits high in vitro exon skipping.

[0111] The present invention provides oligonucleotides comprising any one of the abc-DNA nucleosides and having any of the substituents disclosed herein.

[0112] The present invention provides an oligonucleotide comprising at least one compound of formula (I): [ka] wherein one of T1 and T2 is OR1 or OR2; the other of T1 and T2 is OR1 or OR2, R1 is H or a hydroxyl protecting group; R2 is a phosphorus moiety; Bx is a nucleobase.

[0113] In one embodiment, the compound of formula (I) of the present invention is a compound of formula (II): [ka] During the ceremony, (i) T1 is OR1 and T2 is OR1 or OR2, or (II) T1 is OR1 or OR2, and T2 is OR1; T1 is OR1 or OR2, and T2 is OR1.

[0114] The compounds of formula (II) are alpha anomers or alpha anomeric monomers in which the spatial arrangement of Bx at the chiral center of the first carbon at the 1' end is different from that of the beta anomer.

[0115] In another embodiment, the compound of formula (I) is a compound of formula (III): [ka] During the ceremony, (i) T1 is OR1 and T2 is OR1 or OR2, or (II) T1 is OR1 or OR2, and T2 is OR1; T1 is OR1, and T2 is OR1 or OR2.

[0116] The compound of formula (III) is a beta anomer or beta anomeric monomer in that the spatial arrangement of Bx at the chiral center of the first carbon at the 1' end is different from that of the alpha anomer.

[0117] In another embodiment, in a compound of Formula (I) or (II), Bx is selected from a purine base or a pyrimidine base, and Bx is selected from (i) adenine (A), (ii) cytosine (C), (iii) 5-methylcytosine (MeC), (iv) guanine (G), (v) uracil (U), (vi) thymine, or (vii) 2,6-diaminopurine, or a derivative of (i), (ii), (iii), (iv), (v), (vi), or (vii). In another embodiment, in a compound of Formula (I), (II), or (III), Bx is selected from thymine, 5-methylcytosine, uracil, adenine, or guanine. In another embodiment, in a compound of Formula (I), (II), or (III), Bx is selected from thymine, 5-methylcytosine, adenine, or guanine.

[0118] As used herein, B x The term "nucleobase," abbreviated as "nucleobase," refers to unmodified or naturally occurring nucleobases, as well as modified or non-naturally occurring nucleobases and their synthetic mimics. A nucleobase is any heterocyclic base that contains one or more atoms or groups of atoms that can hydrogen bond to the heterocyclic base of a nucleic acid.

[0119] In one embodiment, the nucleobase is a purine base or a pyrimidine base, preferably the purine base is a purine or a substituted purine, and the pyrimidine base is a pyrimidine or a substituted pyrimidine.More preferably, the nucleobase is (i) adenine (A), (ii) cytosine (C), (iii) 5-methylcytosine (MeC), (iv) guanine (G), (v) uracil (U), or (vi) 5-methyluracil (MeU), or a derivative of (i), (ii), (iii), (iv), (v), or (vi).The terms "derivative of (i), (ii), (iii), (iv), (v), or (vi)" and "nucleobase derivative" are used interchangeably herein.Derivatives and nucleobase derivatives of (i), (ii), (iii), (iv), (v), or (vi), respectively, are known to those skilled in the art and are described, for example, in Sharma VK et al., Med. Chem. Commun. 2014, 5, 1454-1471, and include, but are not limited to, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, alkyl adenines such as 6-methyladenine, 2-propyladenine, alkyl guanines such as 6-methylguanine, 2-propylguanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil, 5-halocytosine, alkynylpyrimidine bases such as 5-propynyl(-C=C-CH3)uracil, 5-propynyl(-C=C-CH3)cytosine, 6-azouracil, 6-azocytosine, 6-aminoadenine, 5-hydroxymethylcytosine, 5-aminoaden ... -azothymine, pseudo-uracil, 4-thiouracil, 8-substituted purine bases such as 8-halo-, 8-amino-, 8-thiol-, 8-thioalkyl-, 8-hydroxyl-adenine or guanine, 5-substituted pyrimidine bases such as 5-halo-, especially 5-bromo-, 5-trifluoromethyl-uracil or -cytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, hydrophobic bases, promiscuous bases, size-expanded bases, or fluorinated bases. In certain embodiments, nucleobases include, but are not limited to, tricyclic pyrimidines, such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one, or 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp). The term "nucleobase derivative" also includes those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, or 2-pyridone.Additional nucleobases of the present invention include, but are not limited to, those known to those skilled in the art (e.g., U.S. Pat. No. 3,687,808; Swayze et al., The Medicinal Chemistry of Oligonucleotides in Antisense a Drug Technology Chapter, 6 pp. 143-182 (Crooke S.T. ed. 2008); The Concise Encyclopedia of Polymer Science and Engineering Kroschwitz J.I. Ed. John Wiley & Sons, 1990 pp. 858-859; Englisch et al., Angewandte Chemie International Edition, 1991 Vol. 30(6) pp. 613-623; Sanghvi Y.S. Antisense Research and Applications Crooke S.T. and Lebleu B. Eds. CRC Press, 1993 pp. 273-302).

[0120] Preferred nucleobase derivatives include methylated adenine, guanine, uracil, and cytosine, and nucleobase derivatives, preferably nucleobase derivatives of (i), (ii), (iii), or (iv), wherein each amino group, preferably the exocyclic amino group, is protected by an acyl protecting group or a dialkylformamidino, preferably dimethylformamidino (DMF), and further include nucleobase derivatives such as 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, and pyrimidine analogs such as pseudoisocytosine and pseudouracil.

[0121] In a further preferred embodiment, the nucleobase derivative is selected from methylated adenine, methylated guanine, methylated uracil, and methylated cytosine, and nucleobase derivatives of (i), (ii), (iii), or (iv), each of which amino group, preferably exocyclic amino group, is protected by a protecting group.

[0122] In a further preferred embodiment, the nucleobase derivative is selected from methylated adenine, methylated guanine, methylated uracil and methylated cytosine, and from nucleobase derivatives of (i), (ii), (iii), or (iv), wherein each amino group, preferably the exocyclic amino group, is protected by an acyl protecting group or a dialkylformamidino, preferably dimethylformamidino (DMF).

[0123] In a further preferred embodiment, the nucleobase derivative is selected from the nucleobase derivatives (i), (ii), (iii), or (iv), and each amino group, preferably an exocyclic amino group, is protected by a protecting group.

[0124] In a further preferred embodiment, the nucleobase derivative is a nucleobase derivative of (i), (ii), (iii), or (iv), wherein the exocyclic amino group is protected by an acyl protecting group or a dialkylformamidino, preferably dimethylformamidino (DMF).

[0125] In a further highly preferred embodiment, the acyl protecting group of the exocyclic amino group of the nucleobase derivative of (i), (ii), (iii), or (iv) above is —C(O)—R 11 and, independently of each other, R 11 is C1-C 10 Alkyl, C6-C 10 Aryl, C6-C 10 Aryl C1-C 10 Alkylene, or C6-C 10 Aryloxy C1-C 10 alkylene, and the dialkylformamidino protecting group is selected from ═C(H)—NR 12 R 13 and R 12 and R 13 are independently selected from C1-C4 alkyl.

[0126] In a further highly preferred embodiment, the acyl protecting group of the exocyclic amino group of the nucleobase derivative of (i), (ii), (iii), or (iv) is —C(O)—R 14 and, independently of each other, R 14 is selected from C1-C4 alkyl, phenyl, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, phenyl substituted with C1-C4 alkoxy, benzyl, benzyl substituted with halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C4 alkoxy, or phenyloxyC1-C2 alkylene optionally substituted with halogen, C1-C6 alkyl, C1-C4 alkoxy, and the dialkylformamidino protecting group is ═C(H)—NR 12 R 13 and R 12 and R 13 are independently selected from C1-C4 alkyl.

[0127] In a further highly preferred embodiment, the acyl protecting group of the exocyclic amino group of the nucleobase derivative of (i), (ii), (iii), or (iv) is —C(O)—R 15 and, independently of each other, R 15 is selected from C1-C4 alkyl, phenyl, halogen, C1-C4 alkyl, C5-C6 cycloalkyl, phenyl substituted with C1-C4 alkoxy, benzyl, benzyl substituted with halogen, C1-C4 alkyl, C1-C4 alkoxy, or phenyloxymethylene (CH2-OC6H5), wherein phenyl is optionally substituted with halogen, C1-C4 alkyl, C5-C6 cycloalkyl, C1-C4 alkoxy, and the dialkylformamidino protecting group is ═C(H)—NR 12 R 13 and R 12 and R 13 are independently selected from C1-C4 alkyl.

[0128] In a further highly preferred embodiment, the acyl protecting groups of the exocyclic amino groups of the nucleobase derivatives of (i), (ii), (iii), or (iv) are, independently of each other, R16 is selected from C1-C3 alkyl, phenyl, C1-C3 alkyl, phenyl substituted with methoxy, benzyl, C1-C3 alkyl, benzyl substituted with methoxy, or phenyloxymethylene (CH2-OC6H5), wherein C6H5 is optionally substituted with C1-C3 alkyl, methoxy, and the dialkylformamidino protecting group is ═C(H)—NR 12 R 13 and R 12 and R 13 are independently selected from C1-C4 alkyl.

[0129] In a further highly preferred embodiment, the acyl protecting group of the exocyclic amino group of the nucleobase derivative of (i), (ii), (iii), or (iv) is —C(O)—R 17 and, independently of each other, R 17 is selected from C1-C3 alkyl, phenyl, C1-C3 alkyl, phenyl substituted with methoxy, benzyl, C1-C3 alkyl, benzyl substituted with methoxy, or phenyloxymethylene (CH2-OC6H5), wherein C6H5 is optionally substituted with C1-C3 alkyl, methoxy, and the dialkylformamidino protecting group is dimethylformamidino (DMF).

[0130] In a further highly preferred embodiment, the acyl protecting group of the exocyclic amino group of the nucleobase derivative of (i), (ii), (iii), or (iv) is —C(O)—R 18 and, independently of each other, R 18 is selected from methyl, isopropyl, phenyl, benzyl, or phenyloxymethylene (CH2-OC6H5), where C6H5 is optionally substituted with C1-C3 alkyl, methoxy, and the dialkylformamidino protecting group is dimethylformamidino (DMF).

[0131] In a further highly preferred embodiment, the acyl protecting group of the exocyclic amino group of the nucleobase derivative of (i), (ii), (iii), or (iv) is —C(O)—R 19and, independently of each other, R 19 is selected from methyl, isopropyl, phenyl, benzyl, or phenyloxymethylene (CH2-OC6H5), where C6H5 is optionally substituted with methyl, isopropyl, and the dialkylformamidino protecting group is dimethylformamidino (DMF).

[0132] As used herein, the term "dialkylformamidino" refers to ==C(H)-NR 12 R 13 refers to R 12 and R 13 are each independently selected from C1-C4 alkyl. In a preferred embodiment, the dialkylformamidino is a protecting group for the exocyclic amino group of the nucleobase derivative of (i), (ii), (iii), or (iv). The resulting compound may be in either the (E) or (Z) configuration, and mixtures thereof in any ratio are within the scope of the present invention. In a preferred embodiment, the compound of the present invention comprises a dialkylformamidino, preferably dimethylformamidino (DMF), in the (Z) configuration.

[0133] According to one embodiment, Bx is selected from uracil, thymine, cytosine, 5-methylcytosine, adenine, and guanine. Preferably, Bx is selected from thymine, 5-methylcytosine, adenine, and guanine. According to one embodiment, Bx is an aromatic heterocyclic moiety capable of forming base pairs when incorporated into a DNA or RNA oligomer in place of the bases uracil, thymine, cytosine, 5-methylcytosine, adenine, and guanine.

[0134] As used herein, the term "phosphorus moiety" is independently selected in each instance from a moiety derived from a phosphonate, a phosphite triester, a monophosphate, a diphosphate, a triphosphate, a phosphate triester, a phosphate diester, a thiophosphate ester, a dithiophosphate ester, or a phosphoramidite.

[0135] In another embodiment, in the compound of formula (I), the phosphorus moiety R2 is selected from a phosphate moiety, a phosphoramidate moiety, and a phosphoramidite moiety. In another embodiment, in the compound of formula (II), the phosphorus moiety R2 is selected from a phosphate moiety, a phosphoramidate moiety, and a phosphoramidite moiety. In another embodiment, in the compound of formula (III), the phosphorus moiety R2 is selected from a phosphate moiety, a phosphoramidate moiety, and a phosphoramidite moiety.

[0136] As used herein, the term "phosphorus moiety" refers to P III or P V refers to a moiety comprising a phosphorus atom in a valence state and represented by formula (VII): [ka] , in the formula, W represents O, S, or Se, or W represents an electron pair, or W represents BH; R3 and R4 are, independently of one another, H, halogen, OH, OR5, NR6R7, SH, SR8, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C6 aminoalkyl, and R5 are, each independently of one another, cyano, nitro, halogen, —NHC(O)C1-C3 alkyl, —NHC(O)C1-C3 haloalkyl, C1-C9 alkyl, C1-C6 alkoxy optionally substituted with C1-C3 alkylsulfonyl; each independently of one another, cyano, nitro, halogen, C aryl, C1-C6 alkylenearyl, C1-C6 alkylenediaryl optionally substituted with 1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, NHC(O)C1-C3 alkyl, NHC(O)C1-C3 haloalkyl, C1-C3 alkylsulfonyl; acetyl; hydroxyl protecting groups, wherein R6 and R7 are each independently hydrogen, cyano, nitro, halogen, C2-C6 alkenyl, C3-C6 cycloalkyl, C1-C9 alkyl optionally substituted with C1-C3 alkoxy; Cyano, nitro, halogen, C 1 -C 3 Alkyl, C 1 -C 3 aryl optionally substituted with alkoxy; are amino protecting groups or form a heterocycle together with the nitrogen atom to which they are attached, the heterocycle being selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, and homopiperazine, the heterocycle being optionally substituted with C1-C3 alkyl, R8 is a thiol protecting group, and the wavy line indicates the bond to the oxygen of the OR2 group. When W represents O, S, or Se, the P atom in the phosphorus moiety is P V If W represents an electron pair, the P atom in the phosphorus moiety is P III The moiety of formula (VII) includes any possible stereoisomers. Further included in the moiety represented by formula (VII) are salts of formula (VII), which can be formed upon treatment with an inorganic base or amine, and can be salts derived from reaction with OH or SH groups which are (independently of each other) R3 and R4. Inorganic bases or amines which result in salt formation with OH or SH groups are well known in the art and include trimethylamine, diethylamine, methylamine, or ammonium hydroxide. These phosphorus moieties included in the present invention are, where appropriate, referred to as "O - HB + ", also abbreviated as HB + refers to the counter cation formed.

[0137] In one embodiment, in the "phosphorus moiety," R and R are, independently of one another, H, OH, OR, NR, R, C-C alkyl, C-C alkyl, C-C haloalkyl, C-C alkoxy, C-C haloalkoxy, C-C aminoalkyl, where R is C-C alkyl optionally substituted with cyano, nitro, halogen; aryl, C-C alkylenearyl, each independently of the other, optionally substituted with cyano, nitro, halogen; acetyl; a hydroxyl protecting group, where R and R are, independently of one another, hydrogen, C-C alkyl optionally substituted with cyano, nitro, halogen; aryl optionally substituted with cyano, nitro, halogen, C-C alkyl, C-C alkoxy; an amino protecting group, and R is a thiol protecting group, where the wavy line indicates the attachment of the OR group to the oxygen.

[0138] As used herein, the term "phosphorus moiety" includes a moiety derived from a phosphonate, phosphite triester, monophosphate, diphosphate, triphosphate, phosphate triester, phosphate diester, thiophosphate ester, dithiophosphate ester, or phosphoramidite.

[0139] Thus, in one embodiment, OR2, at each occurrence, is selected from a phosphonate, a phosphite triester, a monophosphate, a diphosphate, a triphosphate, a phosphate triester, a phosphate diester, a thiophosphate ester, a dithiophosphate ester, or a phosphoramidite, and OR2 is a phosphoramidite or a phosphate triester.

[0140] In one embodiment, the phosphorus moiety is derived from a phosphonate represented by formula (VII), wherein W is O; R is selected from C-C alkyl, C-C haloalkyl, C-C alkoxy, C-C haloalkoxy, C-C aminoalkyl; and R is OH or O. - HB +wherein the wavy line indicates the bond to the oxygen of the OR group. In another embodiment, the phosphorus moiety of formula (VII) is an H-phosphonate, where W is O, R is hydrogen, and R is OH or O. - HB + and O - HB + is HNEt3 + In a further embodiment, the phosphorus moiety of formula (VII) is an alkylphosphonate, where W is O, R3 is alkyl, and R4 is OH or O - HB + and O - HB + is HNEt3 + In one embodiment, the phosphorus moiety of formula (VII) is a methylphosphonate, where W is O, R3 is hydrogen, and R4 is OH or O - HB + and O - HB + is HNEt3 + In another embodiment, the phosphorus moiety of formula (VII) is a phosphonocarboxylate, where R3 or R4 is, independently of each other, a carboxylic acid. The phosphonocarboxylate can be phosphonoacetic acid or phosphonoformic acid. In a further embodiment, the phosphorus moiety of formula (VII) is 2-aminoethyl-phosphonate.

[0141] In another embodiment, R and R of the phosphorus moiety of formula (VII) are, independently of each other, H, OH, halogen, OR, NR, R, SH, SR, C-C alkyl, e.g., C-C alkyl, C-C haloalkyl, C-C haloalkyl, C-C alkoxy, C-C alkoxy, C-C haloalkoxy, C-C haloalkoxy, C-C aminoalkyl, C-C aminoalkyl, and R are each independently of each other. C-C alkyl, e.g., C-C alkyl, optionally substituted independently with cyano, nitro, halogen, NHC(O)C-C alkyl, NHC(O)C-C haloalkyl, C-C alkylsulfonyl; each independently of the others, cyano, nitro, halogen, C-C alkoxy, C-C haloalkyl, C-C haloalkoxy, NHC(O)C-C alkyl, NHC(O)C-C haloalkyl, C-C aryl, C1-C3 alkylenearyl, C1-C3 alkylenediaryl optionally substituted with C1-C3 alkylsulfonyl; acetyl; a hydroxyl protecting group, wherein R6 and R7 are, independently of one another, hydrogen, C1-C6 alkyl, e.g., C1-C4 alkyl, each independently of the other, optionally substituted with cyano, nitro, halogen, C2-C4 alkenyl, C3-C6 cycloalkyl, C1-C3 alkoxy; aryl optionally substituted with cyano, nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy; an amino protecting group, or together with the nitrogen atom to which they are attached, form a heterocycle, the heterocycle being selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, and homopiperazine, the heterocycle being optionally substituted with C1-C3 alkyl; R8 is a thiol protecting group, and the wavy line indicates the bond of the OR2 group to the oxygen.

[0142] In another embodiment, R or R of the phosphorus moiety of formula (VII) are independently in each occurrence and independently of each other a halogen, e.g., chlorine, or OR, where R is a hydroxyl protecting group. Additional phosphorus moieties for use in the present invention are disclosed in Tetrahedron Report Number 309 (Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-2311).

[0143] As used herein, the term "phosphorus moiety" includes P III or P V an R group comprising a phosphorus atom in a valence state and represented in each instance independently by either formula (VIII), formula (IX), or formula (X); [ka] wherein Y is O, S, or Se, and R and R 5’are independently in each occurrence and independently of one another, hydrogen, each independently of one another, cyano, nitro, halogen, —NHC(O)C-C alkyl, —NHC(O)C-C haloalkyl, C-C alkyl optionally substituted with C-C alkylsulfonyl, C-C alkoxy; aryl, C-C alkylenearyl, C-C alkylenediaryl, each independently of one another, optionally substituted with cyano, nitro, halogen, C-C alkoxy, C-C haloalkyl, C-C haloalkoxy, —NHC(O)C-C alkyl, NHC(O)C-C haloalkyl, C-C alkylsulfonyl; a hydroxyl protecting group; R6 and R7 are, independently of each other, hydrogen, cyano, nitro, halogen, C2-C6 alkenyl, C3-C6 cycloalkyl, C1-C9 alkyl optionally substituted with C1-C3 alkoxy; aryl, e.g., phenyl, optionally substituted with cyano, nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy; or an amino protecting group, or together with the nitrogen atom to which they are attached, form a heterocycle, the heterocycle being selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, and homopiperazine, the heterocycle being optionally substituted with C1-C3 alkyl; R8 is a thiol protecting group, and the wavy line indicates the bond of the OR2 group to the oxygen.

[0144] In one embodiment, the phosphorus moiety R2 is represented by formula (VIII): [ka] wherein Y is O, S, or Se, Y is O or S, or Y is O, and R and R 5’are independently in each occurrence and independently of one another, hydrogen, C-C alkyl, C-C alkoxy, each independently of one another optionally substituted with cyano, nitro, halogen, —NHC(O)C-C alkyl, —NHC(O)C-C haloalkyl, C-C alkylsulfonyl; aryl, C-C alkylenearyl, C-C alkylenediaryl, each independently of one another optionally substituted with cyano, nitro, halogen, C-C alkoxy, C-C haloalkyl, C-C haloalkoxy, —NHC(O)C-C alkyl, NHC(O)C-C haloalkyl, C-C alkylsulfonyl; a hydroxyl protecting group; P(O)(OR)(OR 9’ ), P(O)OP(O)(OR9)(OR 9’ ) and R9 and R 9’ are independently at each occurrence and independently of one another, hydrogen, cyano, nitro, halogen, —NHC(O)C-C alkyl, —NHC(O)C-C haloalkyl, C-C alkylsulfonyl optionally substituted; aryl, C-C alkylenearyl, C-C alkylenediaryl, each independently of one another, optionally substituted with cyano, nitro, halogen, C-C alkoxy, C-C haloalkyl, C-C haloalkoxy, —NHC(O)C-C alkyl, NHC(O)C-C haloalkyl, C-C alkylsulfonyl; a hydroxyl protecting group, wherein the wavy line indicates the attachment of the OR group to the oxygen.

[0145] In another embodiment, R and R of formula (VIII) 5’are independently in each occurrence and independently of one another, hydrogen, C-C alkyl, C-C alkyl, C-C alkoxy, C-C alkoxy, each independently of one another optionally substituted with cyano, nitro, halogen, —NHC(O)C-C alkyl, —NHC(O)C-C haloalkyl, C-C alkylsulfonyl; aryl, for example, phenyl, C-C alkylenearyl, C-C alkylenediaryl, each independently of one another optionally substituted with cyano, nitro, halogen, C-C alkoxy, C-C haloalkyl, C-C haloalkoxy, —NHC(O)C-C alkyl, NHC(O)C-C haloalkyl, C-C alkylsulfonyl; a hydroxyl protecting group.

[0146] In another embodiment, R and R of formula (VIII) 5’ are each independently C1-C4 alkyl or aryl, for example, phenyl. In another embodiment, R5 and R6 of formula (VIII) 5’ are each independently methyl or ethyl. In another embodiment, R and R of formula (VIII) 5’ are, independently of each other, phenyl or benzyl. In another embodiment, R and R 5’ are independently in each occurrence and independently of each other, hydrogen or a hydroxyl protecting group. In another embodiment, in formula (VIII), R and R 5’ are independently in each occurrence and independently of each other hydrogen, C1-C9 alkyl, C1-C6 alkoxy, each independently of each other optionally substituted with cyano, nitro, halogen; aryl, C1-C6 alkylenearyl, each independently of each other optionally substituted with cyano, nitro, halogen; or a hydroxyl protecting group. In one embodiment, the phosphorus moiety R2 represented by formula (VIII) is referred to herein as a "phosphate moiety."

[0147] In one embodiment, the phosphorus moiety R2 is represented by formula (IX): [ka] During the ceremony, Y is O, S, or Se; Y is O or S; R5 is independently at each occurrence hydrogen, C1-C9 alkyl, C1-C6 alkoxy, each independently of one another optionally substituted with cyano, nitro, halogen, -NHC(O)C1-C3 alkyl, -NHC(O)C1-C3 haloalkyl, C1-C3 alkylsulfonyl; aryl, C1-C6 alkylenearyl, C1-C6 alkylenediaryl, each independently of one another optionally substituted with cyano, nitro, halogen, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, -NHC(O)C1-C3 alkyl, NHC(O)C1-C3 haloalkyl, C1-C3 alkylsulfonyl; a hydroxyl protecting group; R and R are independently hydrogen, cyano, nitro, halogen, C2-C6 alkenyl, C3-C6 cycloalkyl, C1-C9 alkyl optionally substituted with C1-C3 alkoxy; aryl, e.g., phenyl, optionally substituted with cyano, nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy; an amino protecting group; or together with the nitrogen atom to which they are attached, form a heterocycle, wherein the heterocycle is selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, and homopiperazine, and the heterocycle is optionally substituted with C1-C3 alkyl. The wavy line indicates the bond to the oxygen of the OR2 group. In one embodiment, the phosphorus moiety R2 represented by formula (IX) is referred to herein as a "phosphoramidate moiety" or, interchangeably, a "phosphoramidate moiety."

[0148] In another embodiment, the phosphorus moiety R2 is represented by formula (X): [ka] , in the formula, R5 is hydrogen, C1-C9 alkyl, C1-C6 alkoxy, each independently of the others optionally substituted with cyano, nitro, halogen, -NHC(O)C1-C3 alkyl, -NHC(O)C1-C3 haloalkyl, C1-C3 alkylsulfonyl; aryl, C1-C6 alkylenearyl, C1-C6 alkylenediaryl, each independently of the others optionally substituted with cyano, nitro, halogen, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, -NHC(O)C1-C3 alkyl, NHC(O)C1-C3 haloalkyl, C1-C3 alkylsulfonyl, hydroxyl protecting group; R6 and R7 are each independently hydrogen, cyano, nitro, halogen, C2-C6 alkenyl, C3-C6 cycloalkyl, C1-C3 alkoxy optionally substituted, C1-C9 alkyl, cyano, nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy optionally substituted, aryl, e.g., phenyl, or together with the nitrogen atom to which they are attached form a heterocycle, wherein the heterocycle is selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, and homopiperazine, and the heterocycle is optionally substituted with C1-C3 alkyl. The wavy line indicates the bond to the oxygen of the OR2 group. Typically, and as described above, the phosphorus moiety R2 represented by formula (X) is referred to herein as a "phosphoramidite moiety" or, interchangeably, as a "phosphoramidite moiety."

[0149] In another embodiment, in formula (IX), Y is O; R5 is independently, at each occurrence, hydrogen; C1-C9 alkyl; C1-C6 alkoxy, each independently of one another, optionally substituted with cyano, nitro, halogen; aryl; C1-C6 alkylenearyl, each independently of one another, optionally substituted with cyano, nitro, halogen; a hydroxyl protecting group; and R6 and R7 are independently hydrogen; C1-C9 alkyl optionally substituted with cyano, nitro, halogen, C2-C6 alkenyl; aryl optionally substituted with cyano, nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy; an amino protecting group, where the wavy line indicates the bond of the OR2 group to the oxygen.

[0150] In one embodiment, in Formula (X), R5 is independently at each occurrence hydrogen, C1-C9 alkyl, C1-C6 alkoxy, each independently of one another optionally substituted with cyano, nitro, halogen; aryl, C1-C6 alkylenearyl, each independently of one another optionally substituted with cyano, nitro, halogen; a hydroxyl protecting group; R6 and R7 are independently hydrogen, C1-C9 alkyl optionally substituted with cyano, nitro, halogen, C2-C6 alkenyl; aryl optionally substituted with cyano, nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy; an amino protecting group, and the wavy line indicates the bond of the OR2 group to the oxygen.

[0151] In another embodiment, the phosphorus moiety R2 is independently at each occurrence selected from a phosphate moiety, a phosphoramidate moiety, and a phosphoramidite moiety.

[0152] In another embodiment, R5 is independently at each occurrence selected from the group consisting of hydrogen, C-C alkyl, C-C alkyl, C-C alkoxy, each independently of one another optionally substituted with cyano, nitro, halogen, —NHC(O)C-C alkyl, —NHC(O)C-C haloalkyl, C-C alkylsulfonyl; aryl, C-C alkylenearyl, C-C alkylene, each independently of one another optionally substituted with cyano, nitro, halogen, C-C alkoxy, C-C haloalkyl, C-C haloalkoxy, —NHC(O)C-C alkyl, NHC(O)C-C haloalkyl, C-C alkylsulfonyl. diaryl; a hydroxyl protecting group, wherein R6 and R7 are, independently of each other, hydrogen, cyano, nitro, halogen, C2-C4 alkenyl, C3-C6 cycloalkyl, C1-C6 alkyl optionally substituted with C1-C3 alkoxy; aryl optionally substituted with cyano, nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy; an amino protecting group, or together with the nitrogen atom to which they are attached, form a heterocycle, wherein the heterocycle is selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, and homopiperazine, wherein the heterocycle is optionally substituted with C1-C3 alkyl, and the wavy line indicates the bond to the oxygen of the OR2 group.

[0153] In another embodiment, R5 is C1-C3 alkyl optionally substituted with cyano, chlorine, fluorine, or bromine; aryl, C1-C3 alkylenearyl, C1-C3 alkylenediaryl, each independently of the others, optionally substituted with cyano, nitro, chlorine, fluorine, bromine, C1-C2 alkoxy, or C1 haloalkyl. In another embodiment, R5 is C1-C3 alkyl optionally substituted with cyano, chlorine, fluorine, or bromine. In another embodiment, R5 is cyano-substituted C2 alkyl, e.g., -CH2CH2-CN.

[0154] In another embodiment, R5 is C1-C4 alkyl, such as methyl or ethyl; aryl, such as phenyl or benzyl; chloride, or a hydroxyl protecting group. In another embodiment, R5 is methyl or a hydroxyl protecting group.

[0155] In another embodiment, R5 is C1-C6 alkoxy optionally substituted with cyano, chlorine, fluorine, or bromine.

[0156] In another embodiment, R and R are, independently of each other, H or C-C alkyl, or together with the nitrogen atom to which they are attached, form a heterocycle, wherein the heterocycle is selected from pyrrolidinyl, piperidinyl, morpholinyl, and piperazinyl, and the heterocycle is optionally substituted with methyl. In one embodiment, R and R are, independently of each other, C-C alkyl, alkoxy, or aryl, wherein aryl is phenyl or benzyl optionally substituted with cyano, nitro, chlorine, fluorine, or bromine. In another embodiment, R is hydrogen, and R is (i) C-C alkyl or (ii) aryl, wherein (i) or (ii) is optionally substituted with cyano, nitro, halogen, or aryl, and R is C-C alkyl, phenyl, or benzyl.

[0157] In another embodiment, R6 and R7 are independently selected from methyl, ethyl, isopropyl, or isobutyl. In another embodiment, R6 and R7 are independently isopropyl.

[0158] In another embodiment, the phosphorus moiety R2 is represented by formula (X), wherein R5 is (i) C1-C9 alkyl, (ii) aryl, e.g., phenyl, or (iii) (i) or (ii) optionally substituted with cyano, nitro, halogen, aryl, and R6 and R7 are, independently of each other, C1-C9 alkyl, e.g., isopropyl.

[0159] In another embodiment, the phosphorus moiety R2 is represented by formula (X), wherein R5 is C1-C9 alkyl optionally substituted with cyano, nitro, halogen, —NHC(O)C1-C3 alkyl, —NHC(O)C1-C3 haloalkyl, C1-C3 alkylsulfonyl; aryl, C1-C6 alkylenearyl, C1-C6 alkylenedia, each independently of the others, optionally substituted with cyano, nitro, halogen, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, —NHC(O)C1-C3 alkyl, —NHC(O)C1-C3 haloalkyl, C1-C3 alkylsulfonyl. R and R are, independently of one another, phenyl optionally substituted with cyano, nitro, halogen, C-C alkenyl, C-C cycloalkyl, C-C alkoxy, C-C alkyl, cyano, nitro, halogen, C-C alkyl, C-C alkoxy, or together with the nitrogen atom to which they are attached, form a heterocycle, the heterocycle being selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, and homopiperazine, the heterocycle being optionally substituted with C-C alkyl, and the wavy line indicates the bond to the oxygen of the OR group.

[0160] In another embodiment, the phosphorus moiety R2 is represented by formula (X), wherein R5 is C1-C9 alkyl optionally substituted with cyano, nitro, chlorine, fluorine, bromine, —NHC(O)C1-C3 alkyl, —NHC(O)C1-C3 haloalkyl; aryl, C1-C6 alkylenearyl, C1-C6 alkylenediaryl, each independently of the others optionally substituted with cyano, nitro, chlorine, fluorine, bromine, C1-C4 alkoxy, C1-C4 haloalkyl.

[0161] In another embodiment, the phosphorus moiety R2 is represented by formula (X), wherein R5 is C1-C3 alkyl optionally substituted with cyano, chlorine, fluorine, and bromine; aryl, C1-C3 alkylenearyl, C1-C3 alkylenediaryl, each independently of the others, optionally substituted with cyano, nitro, chlorine, fluorine, bromine, C1-C2 alkoxy, C1 haloalkyl.

[0162] In another embodiment, the phosphorus moiety R2 is represented by formula (X), wherein R5 is C1-C3 alkyl, 2-cyanoethyl, 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, —(CH2) n NHC(O)CF3 (n=3-6); each independently of the others is cyano, nitro, chlorine, fluorine, bromine, C1-C2 alkoxy, phenyl, C1-C3 alkylenephenyl, benzhydryl optionally substituted with -CF3.

[0163] In another embodiment, the phosphorus moiety R2 is represented by formula (X), wherein R5 is methyl, ethyl, 2-cyanoethyl, such as 2-cyanoethyl (CH2)2CN).

[0164] In another embodiment, the phosphorus moiety R2 is represented by formula (X), wherein R6 and R7, independently of each other, are C1-C3 alkyl or together with the nitrogen atom to which they are attached, form a heterocycle, wherein the heterocycle is selected from pyrrolidine, piperidine, morpholine, wherein the heterocycle is optionally substituted with C1-C3 alkyl, and wherein the heterocycle is optionally substituted with methyl.

[0165] In another embodiment, the phosphorus moiety R2 is represented by formula (X), wherein R6 is equal to R7, and R6 and R7 are isopropyl or methyl.

[0166] In another embodiment, the phosphorus moiety R2 is represented by formula (X), wherein R5 is methyl, ethyl, 2-cyanoethyl, R6 is equal to R7, and R6 and R7 are isopropyl or methyl.

[0167] Each alkyl moiety, alone or as part of a larger group such as alkoxy or alkylene, can be straight or branched, and can be =C1-C6 alkyl, e.g., C1-C3 alkyl. Examples include methyl, ethyl, n-propyl, prop-2-yl (isopropyl; herein abbreviated interchangeably as iPr or Pri in particularly depicted chemical formulas), n-butyl, but-2-yl, 2-methyl-prop-1-yl, or 2-methyl-prop-2-yl. Examples of alkoxy include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neo-pentoxy, and n-hexoxy. As described herein, alkoxy can include additional substituents, such as halogen atoms, resulting in haloalkoxy moieties.

[0168] Each alkylene moiety is straight or branched, for example, -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(CH3)-CH2-, or -CH(CH2CH3)-.

[0169] Each alkenyl moiety, alone or as part of a larger group such as alkenyloxy or alkenylene, is straight-chain or branched, and is C2-C6 alkenyl, e.g., C2-C4 alkenyl. Each moiety can be in either the (E) or (Z) configuration. Examples include vinyl and allyl. Thus, compounds of the invention containing alkenyl moieties include, where applicable, compounds having alkenyl moieties in the (E) configuration, compounds having alkenyl moieties in the (Z) configuration, and mixtures thereof in any ratio.

[0170] Each alkynyl moiety, alone or as part of a larger group such as alkynyloxy, is straight or branched, for example, C2-C6 alkynyl, or C2-C4 alkynyl. Examples are ethynyl and propargyl.

[0171] Halogen is fluorine, chlorine, bromine, or iodine.

[0172] Each haloalkyl moiety, alone or as part of a larger group such as haloalkoxy, is an alkyl group substituted with one or more of the same or different halogen atoms. Examples include difluoromethyl, trifluoromethyl, chlorodifluoromethyl, and 2,2,2-trifluoroethyl.

[0173] In another embodiment, the compound of formula (I) or (II) is bound to a non-nucleoside compound, for example, a solid phase.

[0174] In another embodiment, the compound of formula (I) is selected from: [ka] [ka] .

[0175] The present invention provides an oligonucleotide comprising at least one compound of formula (IV): [ka] wherein, independently for each of the at least one compound of formula (IV): one of T3 or T4 is a nucleoside linking group; T3 and T4 are OR1, OR2, the 5'-terminal group, the 7'-terminal group, or the nucleoside linking group; R1 is H or a hydroxyl protecting group; R2 is a phosphorus moiety; and Bx is a nucleobase.

[0176] In another embodiment, the oligonucleotide of the invention comprises at least one compound of formula (IV), wherein the compound of formula (IV) is a compound of formula (V): [ka] During the ceremony, (I) T3 is a nucleoside linking group, T4 is a 7'-terminal group or OR1, or OR2, preferably T4 is a 7'-terminal group or OR1, or (ii) T3 is a 5' terminal group, OR1, or OR2, preferably T3 is a 5' terminal group or OR2, and T4 is a nucleoside linking group; or (iii) T3 and T4 are, independently of each other, a nucleoside linking group.

[0177] In another embodiment, the oligonucleotide of the present invention comprises at least one compound of formula (IV), wherein said compound of formula (IV) is a compound of formula (VI): [ka] During the ceremony, (I) T3 is a nucleoside linking group, T4 is the 7'-terminal group, OR1, or OR2, preferably T4 is the 7'-terminal group or OR2, or (ii) T3 is a 5'-terminal group, OR1, or OR2, preferably T3 is a 5'-terminal group or OR1; T4 is a nucleoside linking group, or (iii) T3 and T4 are, independently of each other, a nucleoside linking group.

[0178] In another embodiment, the oligonucleotide comprises a compound of formula (V). In another embodiment, the oligonucleotide comprises a compound of formula (VI). In another embodiment, the oligonucleotide comprising at least one compound of formula (IV), (V), or (VI) is DNA or RNA.

[0179] The wavy line in formulas (I) and (IV) depicting the bond between Bx and the bicyclic core of the compounds of the invention indicates that any spatial orientation of the nucleobase Bx is encompassed by formula (I) or (IV), meaning that formulas (I) and (IV) encompass either the alpha or beta conformation of the compounds of the invention, or any mixture of the alpha and beta anomers.

[0180] The term "aryl" as used herein refers to an aromatic ring system having 6 to 14 carbon atoms (C6-C8) derived by removing one hydrogen atom from a single carbon atom of a parent aromatic ring system. 14 ) and aryl, as defined above, optionally substituted independently with one or more substituents, typically and preferably one or two substituents as described below. Aryl includes bicyclic groups fused to a saturated, partially unsaturated ring, aromatic ring, or aromatic carbocyclic or heterocyclic ring. Aryl groups are optionally substituted independently with one or more substituents, typically one or two substituents, which are independently, in each occurrence, C1-C4 alkyl, halogen, CF3, OH, C1-C3 alkoxy, NR 20 R 21 , C6H5, C6H5 substituted with halogen, C1-C3 alkyl, C1-C3 alkoxy, NR 20 R 21 Selected from R 20 , R 21 is independently at each occurrence H, C1-C3 alkyl. Typical aryl groups include, but are not limited to, groups derived from benzene (phenyl), substituted phenyl, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and the like. As used herein, the term "aryl" preferably refers to a group having 1 to 3 R 22 R refers to phenyl optionally substituted with 22 is independently at each occurrence halogen, —OH, C1-C3 alkyl optionally substituted with one or two OH, C1-C2 fluoroalkyl, C1-C2 alkoxy, C1-C2 alkoxyC1-C3 alkyl, C3-C6 cycloalkyl, —NH2, NHCH3, or N(CH3)2.

[0181] The terms "amino-protecting group", "amino group protecting group", or "amino protecting group", which are used interchangeably herein, are well known in the art and are described in, for example, Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Butts, 3 rd edition,John Wiley & Sons,1999,Greene's Protective Groups in Organic Synthesis,PGMWuts,5 th edition, John Wiley & Sons, 2014, and current protocols in Nucleic Acid Chemistry, edited by SLBeaucage et al. 06 / 2012, and particularly those described in detail in Chapter 2. Suitable "amino protecting groups" for the present invention include methyl carbamate, ethyl carbamate, 9-fluorenylmethylcarbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacylcarbamate (Phenoc), 2,2,2-trichloroethylcarbamate (Troc), 2-trimethylsilylethylcarbamate (Troc), 2-trimethylsilylethylcarbamate (Tm ... Typically and preferably, these include, and are independently in each instance selected from, methylcarbamate (Teoc), 2-phenylethylcarbamate (hZ), 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethylcarbamate (TCBOC), benzylcarbamate (Cbz), p-methoxybenzylcarbamate (Moz), and 2,4,6-trimethylbenzylcarbamate, as well as formamide, acetamide, benzamide.

[0182] The terms "hydroxyl protecting group," "hydroxyl group protecting group," or "hydroxyl protecting group," used interchangeably herein, are well known in the art and are described in, for example, Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Butts, 3 rd edition,John Wiley & Sons,1999;Greene's Protective Groups in Organic Synthesis,PGMWuts,5 th edition John Wiley & Sons, 2014, and Nucleic Acid Chemistry, edited by SLBeaucage et al. 06 / 2012, including current protocols, and especially those detailed in Chapter 2. In certain embodiments, a "hydroxyl protecting group" of the invention is independently, at each occurrence, selected from acetyl, benzoyl, benzyl, β-methoxyethoxymethyl ether (MEM), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMTr), methoxymethyl ether (MOM), methoxytrityl[(4-methoxyphenyl)diphenylmethyl] (MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ethers such as t-butyldiphenylsilyl (TBDPS), trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers, methyl ethers, ethoxyethyl ethers (EE).

[0183] In one embodiment, a "hydroxyl protecting group" of the present invention is, independently at each occurrence, acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl(trityl), 4,4'-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl (TBDMS), t-Butyldiphenylsilyl (TBDPS), triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, 4-monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr) and 4,4',4''-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl and selected from 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4',4''-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (Pixyl), or 9-(p-methoxyphenyl)xanthine-9-yl (MOX).

[0184] In some embodiments, the hydroxyl protecting group is independently at each occurrence selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trityl, 4-monomethoxytrityl, 4,4'-dimethoxytrityl (DMTr), 4,4',4-trimethoxytrityl (TMTr), 9-phenylxanthin-9-yl (pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX).

[0185] In some embodiments, the hydroxyl protecting group is independently at each occurrence selected from triphenylmethyl (trityl), 4-monomethoxytrityl, 4,4′-dimethoxytrityl (DMTr), 4,4′,4″-trimethoxytrityl (TMTr), 9-phenylxanthin-9-yl (pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX).

[0186] In further embodiments, the hydroxyl protecting groups are independently at each occurrence selected from trityl, 4-monomethoxytrityl, and 4,4'-dimethoxytrityl groups.

[0187] In another embodiment, the hydroxyl protecting groups are independently at each occurrence selected from triphenylmethyl (trityl), 4-monomethoxytrityl, 4,4'-dimethoxytrityl (DMTr), 4,4',4''-trimethoxytrityl (TMTr), 9-phenylxanthin-9-yl (pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX).

[0188] In another embodiment, the hydroxyl protecting group of the present invention is acetyl, dimethoxytrityl (DMTr), tert-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM), or t-butyldiphenylsilyl ether (TBDPS). In another embodiment, the hydroxyl protecting group is independently selected at each occurrence from 4,4'-dimethoxytrityl (DMTr) or 4-monomethoxytrityl. In another embodiment, the hydroxyl protecting group is 4,4'-dimethoxytrityl (DMTr).

[0189] When a group is said to be optionally substituted, there can be 1 to 5 substituents, 1 to 3 substituents, or 1 or 2 substituents. When a group is said to be optionally substituted, and there are more than one substituent, the two or more substituents can be the same or different.

[0190] Internucleoside phosphorus-containing linking groups The oligonucleotides of the invention comprise predominantly phosphodiester internucleoside linkages, e.g., 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, and 100% of the internucleoside linkage groups are phosphodiester linkage groups.

[0191] The oligonucleotides of the invention, in addition to predominantly phosphodiester internucleoside linkages, can also contain nucleoside linkage groups selected from phosphotriester, phosphorothioate, phosphorodithioate, phosphonate, phosphonothioate, phosphinate, phosphorothioamidate, or phosphoramidate linkages.

[0192] The term "nucleoside linking group" includes phosphorus linking groups and non-phosphorus linking groups.

[0193] In one embodiment, the nucleoside linking group is a phosphorus linking group, and the phosphorus linking group is selected from a phosphodiester linking group, a phosphotriester linking group, a phosphorothioate linking group, a phosphorodithioate linking group, a phosphonate linking group, e.g., an H-phosphonate linking group or a methylphosphonate linking group, a phosphonothioate linking group, e.g., an H-phosphonothioate linking group, a methylphosphonothioate linking group, a phosphinate linking group, a phosphorthioamidate linking group, a phosphoramidate linking group, or a phosphorodiamidate linking group. In another embodiment, the nucleoside linking group is a phosphorus linking group, and the phosphorus linking group is selected from a phosphodiester linking group, a phosphotriester linking group, a phosphorothioate linking group, or a phosphonate linking group, and the phosphonate is an H-phosphonate linking group or a methylphosphonate linking group.

[0194] In another embodiment, the nucleoside linking group is a phosphorus linking group and the phosphorus linking group is a phosphodiester linking group. In another embodiment, the nucleoside linking group is a phosphorus linking group and the phosphorus linking group is a phosphorothioate linking group.

[0195] The phosphorus linking group can be selected from an alkyl phosphodiester linking group, an alkylene phosphodiester linking group, a thionoalkyl phosphodiester linking group or an aminoalkyl phosphodiester linking group, an alkyl phosphotriester linking group, an alkylene phosphotriester linking group, a thionoalkyl phosphotriester linking group or an aminoalkyl phosphotriester linking group, an alkyl phosphonate linking group, an alkylene phosphonate linking group, an aminoalkyl phosphonate linking group, a thionoalkyl phosphonate linking group or a chiral phosphonate linking group. The nucleoside linking groups of the present invention comprise a phosphorus linking group, which may be a phosphodiester linking group -OP(=O)(OH)O- or a counterion [HB + ] -OP(=O)(O - )O-, with phosphorothioate -OP(=S)(OH)O- or [HB + ] -OP(=S)(O -)O—, methylphosphonate, —OP(═O)(CH3)O—. Included are various salts, mixed salts, and free acid forms of the phosphorus-linked groups.

[0196] A nucleoside linking group can link a nucleoside, nucleotide, or oligonucleotide to another nucleoside, nucleotide, or oligonucleotide.

[0197] Non-phosphorus linking groups do not contain a phosphorus atom, and examples of non-phosphorus linking groups include alkyl, aryl, preferably phenyl, benzyl, or benzoyl, cycloalkyl, alkylenearyl, alkylenediaryl, alkoxy, alkoxyalkylene, alkylsulfonyl, alkyne, ether, each optionally substituted, independently of the other, with cyano, nitro, halogen, carboxyl, amido, amine, amino, imine, thiol, sulfide, sulfoxide, sulfone, sulfamate, sulfonate, sulfonamide, siloxane, or mixtures thereof. Non-phosphorus linking groups include aminopropyl, long chain alkylamine groups, vinyl, acetylamido, aminomethyl, formacetal, thioformacetal, thioformacetyl, riboacetyl, methyleneimino, methylenehydrazino, or neutral non-ionic nucleoside linking groups, such as amido-3 (3'-CH2-C(=O)-N(H)-5') or amido-4 (3'-CH2-N(H)-C(=O)-5'). Non-phosphorus linking groups include compounds selected from alkyl, aryl, preferably phenyl, benzyl, or benzoyl, cycloalkyl, alkylene, alkylenediaryl, alkoxy, alkoxydialkylene, alkylsulfonyl, alkyne, or ether, wherein the compound comprises C1-C9, C1-C6, or C1-C4.

[0198] lipid group The present invention provides an oligonucleotide comprising abc-DNA nucleoside and a lipid group linked via a linker.The structure of the oligonucleotide conjugated with the lipid group is such that the hydrocarbon chain of the lipid group, for example, fatty acid, is exposed, thereby allowing the hydrocarbon chain to interact with albumin and / or fatty acid receptor or transporter, thereby providing the oligonucleotide with a long half-life in vivo.The lipid group is conjugated to the hydroxyl group at the 5'-end or 7'-end of the oligonucleotide via a linker.

[0199] In certain embodiments, the lipid group is a fatty acid-derived group. In certain embodiments, the fatty acid-derived group comprises a carboxy group. Fatty acids include any saturated or unsaturated fatty acid having a hydrocarbon chain of 4 to 28 carbon atoms, and the fatty acid may comprise one or two carboxylic acid groups. A fatty acid comprising two carboxylic acid groups is a dicarboxylic acid. One or two fatty acid ligands can be attached to the oligonucleotide via linkers on the 5' and / or 7' ends of the abc-DNA oligonucleotide, as described herein.

[0200] In certain embodiments, the lipid group is a fatty acid-derived group, and the fatty acid is any one of the fatty acids shown in Tables 1 and 2. [Table 1] [Table 2]

[0201] Additional lipid groups useful in accordance with the present invention include cholesterol, vitamin E (tocopherol), and bile acids.

[0202] In one embodiment, the lipid group is a saturated fatty acid-derived group having a hydrocarbon chain of 8 to 24 carbon atoms. In a specific embodiment, the lipid group is a saturated fatty acid-derived group, and the fatty acid is selected from the group consisting of octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid, and tetracosanoic acid. In one embodiment, the lipid group is a saturated fatty acid-derived group, and the fatty acid is hexadecanoic acid. In one embodiment, the fatty acid-derived group is attached to the oligonucleotide via a linker at the 5'-end of the abc-DNA oligonucleotide. In one embodiment, the fatty acid-derived group is attached to the oligonucleotide via a linker at the 7'-end of the abc-DNA oligonucleotide.

[0203] In one embodiment, the lipid group is derived from an unsaturated fatty acid having a hydrocarbon chain of 8 to 24 carbon atoms. In a particular embodiment, the lipid group is derived from an unsaturated fatty acid selected from the group consisting of myristoleic acid, palmitic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolenic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

[0204] Linker The oligonucleotide of the present invention is linked to the lipid group via a linker. In some embodiments, the linker is linked to the lipid group via an amide bond. In the case of a hydrocarbon linker, the linker contains 2 to 20 carbons, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbons. In the case of a polyethylene glycol (PEG) linker, the linker contains 1 to 20 ethylene glycol subunits, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ethylene glycol repeats. The linker can be a hydrocarbon linker or a polyethylene glycol (PEG) linker. A linker according to the present invention, in which abcDNA is attached to the phosphorus moiety of the linker and a lipid group, e.g., a fatty acid-derived group, is attached to Y, can have, for example, the general structure shown below: [ka] During the ceremony, When Y=NH, the fatty acid-derived group is attached via an amide bond; When n=1, R1 can be, for example, CO2H and R2 can be, for example, H; T' can be -CH-CH-O, where m is the number of ethylene glycol repeats; T can be a biocleavable moiety such as a disulfide group, k is equal to 1, and in certain embodiments, X can be oxygen or NH; Z can be O or S; WR5 can be OH or SH.

[0205] Linkers useful according to the present invention include, but are not limited to: Amino-alkyl-phosphorothioate linkers: [ka] In the formula, n is preferably an integer of 2 to 12, preferably an integer of 4 to 10. In one embodiment, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In one embodiment, n is 6. Alpha-carboxylate-amino-alkyl-phosphorothioate linker: [ka] In the formula, n is preferably an integer of 2 to 12, preferably an integer of 4 to 10. In one embodiment, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In one embodiment, n is 6. Amino-PEG-phosphorothioate linker: [ka] In the formula, n is preferably an integer of 1 to 8. In one embodiment, n is an integer of 1, 2, 3, 4, 5, 6, 7, or 8. and Alpha-carboxylate-amino-PEG-phosphorothioate linker: [ka] In the formula, n is preferably an integer of 1 to 8. In one embodiment, n is an integer of 1, 2, 3, 4, 5, 6, 7, or 8.

[0206] Thus, in one embodiment, the linker is selected from the group consisting of: (i) amino-alkyl-phosphorothioate linkers; (ii) alpha-carboxylate-amino-alkyl-phosphorothioate linker; (iii) an amino-PEG-phosphorothioate linker, and (iv) alpha-carboxylate-amino-PEG-phosphorothioate linker All as defined above in the formula provided.

[0207] In one embodiment, the linker is an amino-alkyl-phosphorothioate linker of the formula: [ka] In the formula, n is an integer of 2 to 12, preferably an integer of 4 to 10. In one embodiment, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In one embodiment, n is 6.

[0208] Thus, the present invention provides an aminoalkyl phosphorothioate linker having the structure shown below: [ka] (wherein n is an integer of 2 to 12, preferably an integer of 4 to 10.) In one embodiment, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In one embodiment, n is 6.

[0209] An example of an oligonucleotide of the invention comprising SEQ ID NO: 10 attached to a lipid group via an aminoalkyl phosphorothioate linker has the following structure: [ka] (wherein n is an integer of 2 to 12, preferably an integer of 4 to 10, more preferably n is 6.) Preferably, all of the residues of SEQ ID NO: 10 are abc-DNA residues corresponding to SEQ ID NO: 418.

[0210] Another example of an oligonucleotide of the invention (SEQ ID NO: 412) attached to a lipid group via an aminoalkyl phosphorothioate linker has the following structure: [ka]

[0211] The present invention also provides amino-PEG-phosphorothioate linkers having the structures provided below: [ka] (wherein n is preferably an integer of 1 to 8.) In one embodiment, n is an integer of 1, 2, 3, 4, 5, 6, 7, or 8.

[0212] An example of an oligonucleotide of the invention comprising SEQ ID NO: 10 attached to a lipid group via an amino-PEG-phosphorothioate linker has the following structure: [ka] (wherein n is preferably an integer from 1 to 8). In one embodiment, n is an integer of 2, 3, 4, 5, 6, 7, or 8. Preferably, all of the residues of SEQ ID NO: 10 are abc-DNA residues corresponding to SEQ ID NO: 418.

[0213] The present invention also provides an alpha-carboxylate-amino-alkyl-phosphorothioate linker having the structure provided below: [ka] (wherein n is preferably an integer of 2 to 12, preferably an integer of 4 to 10.) In one embodiment, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In one embodiment, n is 6.

[0214] An example of an oligonucleotide of the invention comprising SEQ ID NO: 10 attached to a lipid group via an alpha-carboxylate-amino-alkyl-phosphorothioate linker has the following structure: [ka] (wherein n is an integer of 2 to 12, preferably an integer of 4 to 10, more preferably n is 6.) Preferably, all of the residues of SEQ ID NO: 10 are abc-DNA residues corresponding to SEQ ID NO: 418.

[0215] The present invention also provides an alpha-carboxylate-amino-PEG-phosphorothioate linker having the structure provided below: [ka] (wherein n is preferably an integer of 1 to 8.) In one embodiment, n is an integer of 2, 3, 4, 5, 6, 7, or 8.

[0216] An example of an oligonucleotide of the invention comprising SEQ ID NO: 10 attached to a lipid group via an alpha-carboxylate-amino-PEG-phosphorothioate linker has the following structure: [ka] (wherein n is preferably an integer from 1 to 8). In one embodiment, n is an integer of 2, 3, 4, 5, 6, 7, or 8. Preferably, all of the residues of SEQ ID NO: 10 are abc-DNA residues corresponding to SEQ ID NO: 418. or having the following structure: [ka] (wherein n is preferably an integer from 1 to 8). In one embodiment, n is an integer of 2, 3, 4, 5, 6, 7, or 8. Preferably, all of the residues of SEQ ID NO: 10 are abc-DNA residues corresponding to SEQ ID NO: 418.

[0217] In one embodiment, the present invention provides conformationally restricted linkers, for example, based on hydroxyproline. [ka] (wherein n is preferably an integer of 1 to 8.) In one embodiment, n is an integer of 2, 3, 4, 5, 6, 7, or 8.

[0218] Linker can be bound to the 5' and / or 7' terminal OH group of oligonucleotide, for example, via thiophosphate group.In one embodiment, linker is bound to the 5' terminal OH group of oligonucleotide, for example, via thiophosphate group.In one embodiment, linker is bound to the 7' terminal OH group of oligonucleotide, for example, via thiophosphate group.Another group that can be used to bind linker to oligonucleotide includes phosphate group.

[0219] In some embodiments, fatty acid conjugated phosphoramidites can be used to attach fatty acids to abc-DNA at either the 5' end, the 7' end, or both the 5' and 7' ends. An example of a phosphoramidite that can be used to attach fatty acids to abc-DNA has the following structure: [ka] where R-CO is a fatty acid moiety.

[0220] In other embodiments, the linker is an alpha-carboxylate-amino linker, for example, having the following structure: [ka] (wherein n is preferably an integer of 1 to 8.) In one embodiment, n is an integer of 2, 3, 4, 5, 6, 7, or 8.

[0221] In its simplest form, the linker is a 2-amino-6-hydroxy-4-oxahexanoic acid linker, where n=1. Alternatively, a linker having the above structure, in which the stereochemistry at C2 matches that of serine, is the O-(2-hydroxyethyl)-L-serine linker. In the context of abcDNA fatty acid conjugates, the hydroxyl function of the linker is attached to the abcDNA via a phosphorothioate bond, and the amino group is attached to the carboxyl group of the fatty acid moiety via an amide bond.

[0222] In another embodiment, the fatty acid conjugated α-carboxylate-amino-PEG phosphoramidite reagent has the following structure, where R is a suitable protecting group, such as 2-chlorotrityl, used in the final step of the solid phase synthesis of the abcDNA-linker-fatty acid conjugate: [ka] (wherein n is preferably an integer of 1 to 8.) In one embodiment, n is an integer of 2, 3, 4, 5, 6, 7, or 8.

[0223] In another embodiment, a phosphoramidite that can be used to attach a fatty acid to abc-DNA has the following structure (AM Chemicals, LLC, Oceanside, CA): [ka] where R is a fatty acid moiety.

[0224] In some embodiments, a fatty acid conjugate solid support can be used to attach a fatty acid to abc-DNA at the 5' end. An example of a solid support that can be used to attach a fatty acid to abc-DNA has the following structure: [ka] where R-CO is the fatty acid moiety and the shaded circle is the solid support.

[0225] In another embodiment, a solid support that can be used for binding fatty acids to abc-DNA has the following structure (AM Chemicals, LLC, Oceanside, CA): [ka] where R is the fatty acid moiety and the shaded circle is the solid support.

[0226] In certain embodiments, the linker comprises a cleavable bond, for example, a disulfide bond, an acid-cleavable hydrazone bond, or a protease-cleavable moiety.

[0227] Synthesis method Use well-known synthetic methods in the art to synthesize abc-DNA nucleoside and the oligonucleotide that comprises abc-DNA nucleoside.In some embodiments, for the oligonucleotide that is conjugated to lipid group at 5'-end, the linker of the present invention is added to solid support before the synthesis and addition of oligonucleotide.In certain embodiments, for the oligonucleotide that is conjugated to lipid group at 7'-end of oligonucleotide, conjugation occurs during solid-phase synthesis.In other embodiments, for the oligonucleotide that is conjugated to lipid group at 7'-end of oligonucleotide, conjugation occurs after synthesis is completed.

[0228] General Procedure All reactions are carried out in dry glassware and under an inert atmosphere of argon. Anhydrous solvents for reactions are obtained by filtration through activated alumina or by storage over molecular sieves (4 Å). Column chromatography (CC) is performed on silica gel (Silia Flash P60, 40-63 μm, 60 Å). Methanol used for CC is HPLC grade; all other solvents used for CC are technical grade and distilled before use. Thin-layer chromatography is performed on silica gel plates (Macherey-Nagel, precoated TLC plates silG-25UV254). Compounds are visualized under UV light or by immersion in p-anisaldehyde staining solution [p-anisaldehyde (3.7 mL), glacial acetic acid (3.7 mL), concentrated sulfuric acid (5 mL), ethanol (135 mL)] followed by heating with a heat gun. NMR spectra are obtained at 300 or 400 MHz ( 1 H), 75 or 101MHz ( 13 C), and 122MHz ( 31 Chemical shifts (δ) are recorded at 1000 kJ / cm² / cm² (P). Chemical shifts (δ) are recorded at 1000 kJ / cm² ... 1 H), 77.16 ppm ( 13 C);CD3OD:3.31 ppm( 1 H), 49.00 ppm ( 13 C). Signal assignments are reported for APT and DEPT and 1 H, 1 H, and 1 H, 13 High-resolution mass data are based on C correlation experiments (COSY, HSQC, HMBC). Electrospray ionization in positive mode (ion trap, ESI) + ) is obtained by

[0229] Melting temperature UV melting experiments were recorded on a Varian Cary Bio 100 UV / vis spectrophotometer. Experiments were performed at two concentrations of 2 μM: 10 mM NaH2PO4, 0 M to 150 mM NaCl (alpha anomer) or 0.05 M to 1.00 M NaCl (beta anomer), and pH adjusted to 7.0. A methylpolysiloxane cover layer protected the sample from evaporation. Absorbance was monitored at 260 nm. All experiments involved three cooling-heating cycles with a temperature ramp of 0.5 °C / min. The maximum of the first derivative of the curve was calculated using Varian WinUV software, and Tm values ​​were reported as the average of six ramps.

[0230] Circular dichroism spectroscopy CD spectra are recorded on a Jasco J-715 spectropolarimeter equipped with a Jasco PFO-350S temperature controller. Sample conditions are the same as for the UV melting experiments. Spectra are recorded from 210 to 320 nm at a rate of 50 nm / min, and the temperature is measured directly from the sample. For each experiment, a blank containing the same salt concentration as the sample is recorded. The reported spectrum is obtained by taking the smoothed average of three scans and subtracting the corresponding blank spectrum.

[0231] Synthesis of abcDNA nucleosides Bicyclic scaffolds 7 and 10, intended for subsequent nucleoside synthesis, are constructed from the previously described intermediate 1 (Tarkoy, M.; Bolli, M.; Schweizer, B.; Leumann, C. Helv. Chim. Acta 1993, 76, 481) (Scheme 1). The epoxide ring in 1 is efficiently opened by LiHMDS-mediated intramolecular elimination at −78 °C to generate unsaturated ester 2 in good yield. Subsequent nickel-catalyzed NaBH reduction of 2 proceeds stereospecifically from the convex side of the bicyclic core structure, affording ester 3 as the only identifiable diastereoisomer. Protection of the hydroxyl function in 3 with TBDPS affords 4 in quantitative yield. The resulting intermediate 4 is reduced to aldehyde 5 with DIBAL at −78 °C. The acetonide protecting group of 5 is then hydrolyzed in a mixture of MeCN and HO under mild conditions using In(OTf) as a catalyst (Golden, KC; Gregg, BT; Quinn, JF Tetrahedron Lett. 2010, 51, 4010), and the resulting bicyclic hemiacetal is converted to the methyl glycoside 6 by simply changing the solvent to MeOH. Compound 6 is then acetylated to generate the protected precursor 7, which is used in the synthesis of the corresponding purine nucleoside via Vorbruggen chemistry. [ka] Scheme 1: (a) LiHMDS, THF, -78 °C, 2 hours, 74%, (b) NaBH4, NiCl2, EtOH, 0 °C → room temperature, 2 hours, 90%, (c) TBDPSCl, I2, N-methylimidazole, THF, room temperature, 3 hours, quantitative, (d) DiBAL-H, CH2Cl2, -78 °C, 90 minutes, 89%, (e) i) In(OTf)3, MeCN / H2O, room temperature, 48 hours, ii) MeOH, 6 hours, 81%, (f) Ac2O, DMAP, DCM, room temperature, 2 hours, 96%, (g) i) TMSOTf, 2,6-lutidine, DCM, room temperature, 60 minutes, ii) TBAF, THF, 0°C, 20 minutes, 92%, (h) DMTr-Cl, AgOTf, DCM / lutidine, room temperature, 4 hours, 93%, (i) TBAF, THF, room temperature, 20 hours, quantitative.

[0232] The synthesis of the pyrimidine nucleosides of the present invention consists of the well-established application of the β-stereoselective NIS-induced addition of nucleobases to the corresponding bicyclic glycans (Medvecky, M.; Istrate, A.; Leumann, CJJ Org. Chem. 2015, 80, 3556; Dugovic, B.; Leumann, CJJ Journal of Organic Chemistry 2014, 79, 1271; Lietard, J.; Leumann, CJJ Org. Chem. 2012, 77, 4566). First, to introduce the thymine nucleobase, N-iodosuccinimide (NIS)-induced nucleosidation is carried out on the direct precursor of glycan 8 (where R = TMS), which is easily obtained from 6 by treatment with TMSOTf alone. This approach resulted in the stereoselective formation of the corresponding β-nucleoside, albeit with significant contamination of the 7% α-anomer, which remained unresolved by standard chromatographic techniques. It is conceivable that β-selectivity could be enhanced by increasing steric bulk at R1 and decreasing steric bulk at R2, as in glycal 10. This would favor the initial α-attack of the electrophilic iodine at C(4). To this end, compound 6 was converted to glycal 8 with TMSOTf, followed by a brief treatment with TBAF to selectively remove the newly introduced TMS group. Intermediate 8 was then elaborated to dimethoxytrityl compound 9, which was finally subjected to removal of the TBDPS protecting group with TBAF to yield the desired sugar component 10.

[0233] In situ NIS nucleosidation on TMS-protected glycal 10 followed by radical reduction of the iodide intermediate with Bu3SnH afforded only trace amounts of ( 1The DMTr-protected thymidine derivative 11, containing less than 2% of the α-anomer by H-NMR, was obtained in good yield (Scheme 2). Final phosphitylation with 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite afforded the thymidine phosphoramidite building block 12. The synthesis of 5-methylcytosine nucleosides was achieved by conversion of basic thymines. To this end, nucleoside 11 was protected with TMS and converted to the corresponding triazolidine by treatment with 1,2,4-triazole and POCl3. Subsequent treatment of this triazolidine in a mixture of ammonia and 1,4-dioxane afforded the corresponding 5-methylcytosine nucleoside, which was directly protected with BzO to give 13 in 88% yield over three steps. The phosphoramidite 14 was obtained by the above-described phosphitylation. [ka] Scheme 2: (a) i) thymine, BSA, NIS, DCM, rt, 7 h, ii) Bu3SnH, AIBN, toluene, 70 °C, 30 min, 73%, (b) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite, ETT, DCM, rt, 30 min, 70% for 12, 75% for 14, (c) i) BSA, triazole, POCl3, Et3N, CH3CN, rt, 5 h, ii) 1,4-dioxane / NH4OH, rt, 2 h, iii) Bz2O, Et3N, DMF, rt, 20 h, 88%.

[0234] To introduce the purine nucleobase, classical Vorbruggen nucleosidation was applied, which generally leads to the widespread presence of α-nucleosides. 6-Conversion with either benzoyladenine or 2-amino-6-chloropurine yields inseparable anomeric mixtures 15 and 20 in α / β ratios of 4:1 and 7:3, respectively (Scheme 3). Separation of the anomers is possible after deacetylation, yielding the pure β-anomers 16 and 21. From this, standard dimethoxytritylation (→17), followed by TBAF-mediated cleavage of the silyl protecting group (→19) and phosphitylation, affords the adenine building block 19. Synthesis of the guanine building block requires conversion of the 2-amino-6-chloropurine nucleobase. This is achieved by treating 21 with 3-hydroxypropionitrile and TBD, followed by protection of the 2-amino group with DMF, yielding the protected guanosine derivative 22. Following the same chemical pathway as above, synthesis of the guanine building block 25 is achieved by dimethoxytritylation (→23), followed by removal of the silyl protecting group (→24) and phosphitylation. [ka] Scheme 3: (a) N 6 -benzoyladenine, BSA, TMSOTf, MeCN, 70°C, 20 min, 64%, (b) NaOH, THF / MeOH / H2O, 0°C, 20 min, 69%, (c) DMTr-Cl, pyridine, room temperature, 24 h, 87%, (d) TBAF, THF, room temperature, 48 h, 87%, (e) CEP-Cl, DIPEA, THF, room temperature, 2 h, 71%, (f) 2-amino-6-chloropurine, BSA, TMSOTf, MeCN, 55°C, 50 min, 77%, (g) NaOH, T HF / MeOH / HO, 0°C, 20 min, 85%, (i) i) TBD, 3-hydroxypropionitrile, DCM, 48 h, ii) N,N-dimethylformamide dimethyl acetal, DMF, 55°C, 2 h, 73%, (j) DMTr-Cl, pyridine, rt, 18 h, 70%, (k) TBAF, THF, rt, 7 h, 87%, (l) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite, ETT, DCM, rt, 50 min, 69%.

[0235] Starting from protected sugar 7, the synthesis of four preferred phosphoramidite building blocks of the present invention was developed. Treatment of a mixture of sugar 7 and in situ silylated thymine with TMSOTf afforded a preferred anomeric ratio α / β ( 1 Nucleoside 35 with a 5'-dimer structure (determined by H-NMR) is rapidly formed (Scheme 4). The chemical route leading to a thymidine phosphoramidite bearing a DMTr group at the 5' position does not allow for the separation of the anomers by standard chromatography. Therefore, to introduce a polarity-reversal modification into the DNA chain, a DMTr group is introduced at the 7' position. To this end, the silyl group of 35 is removed by a brief treatment with TBAF (→ 36), followed by standard dimethoxytritylation (→ 37). After standard deacetylation, the two anomers can be separated, yielding the pure α-anomer 38. Phosphitylation with 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite in the presence of 5-(ethylthio)-1H-tetrazole finally affords the thymidine building block 39. Intermediate 38 also provides a short route to 5-methylcytosine nucleosides by converting the in situ TMS-protected nucleoside 38 to the corresponding triazolide using POCl3 and 1,2,4-triazole, followed by treatment with a mixture of ammonia and 1,4-dioxane. Direct protection with BzO in DMF efficiently forms nucleoside 40, cleaving the labile silyl protecting group in the process. Final phosphorylation under the conditions described above affords 5-methylcytidine phosphoramidite 41. [ka] Scheme 4: (a) Thymine, BSA, TMSOTf, MeCN, rt, 18 h, 82%, (b) TBAF, THF, 2 h, 75%, (c) DMTr-Cl, pyridine, rt, 24 h, 96%, (d) K2CO3, MeOH, 3 h, 86%, (e) 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite, ETT, DCM, rt, 1 h, 81% for 39, 30 min, 80% for 41, f) i) BSA, 1,2,4-triazole, POCl3, Et3N, MeCN, rt, 7 h, ii) 1,4-dioxane / NH4OH, rt, 3 h, iii) Bz2O, Et3N, DMF, rt, 18 h, 83%.

[0236] In the case of purine nucleobases, the introduction of purines is 6- A short nucleosidation reaction using either benzoyladenine or 2-amino-6-chloropurine at slightly elevated temperatures affords nucleosides 15 and 20 in α / β ratios of 4:1 and 7:3, respectively (Scheme 5). To separate the anomers, the acetyl group is removed under mild conditions to give the pure α-anomers 42 and 48. Formation of the adenosine building block continues with reintroduction of the acetyl protecting group (→ 43), removal of the TBDPS protecting group with TBAF (→ 44), followed by standard dimethoxytritylation (→ 45). Selective deprotection of the acetyl group (→ 46), followed by phosphorylation under the conditions described above, affords the adenine building block 47.

[0237] For the guanine building block, after separation of the two anomers, 6-chloropurine was converted to the guanine nucleobase by treatment with TBD and 3-hydroxypropionitrile to give guanosine nucleoside 49. Acetylation for 48 h allowed simultaneous protection of the 5'-hydroxy and 2-amino groups, yielding the protected nucleoside 50. As above, removal of the silyl protecting group with TBAF (→ 51) followed by dimethoxytritylation (→ 52) introduced the DMTr group. The two acetyl groups were removed by treatment with K2CO3, and the resulting polar product was directly protected with DMF to give guanosine nucleoside 53. Final phosphorylation afforded building block 54. [ka] Scheme 5: a) N 6- Benzoyladenine, BSA, TMSOTf, MeCN, 70°C, 20 min, 64%, (b) NaOH, THF / MeOH / H2O, 0°C, 20 min, 51% α-anomer, 18% β-anomer, (c) Ac2O, DMAP, DCM, rt, 18 h, 90%, (d) TBAF, THF, rt, 3.5 h, 90%, (e) DMTr-Cl, pyridine, rt, 24 h, 89%, (f) NaOH, THF / MeOH / H2O, 0°C, 30 min, 94%, (g) 2-cyanoethyl N,N,N',N'-tetraisopropyl-phosphordiamidite, ETT, DCM, rt, 1 h, 77% for 47, 50 min , 67% for 54, (h) 2-amino-6-chloropurine, BSA, TMSOTf, MeCN, 55 °C, 50 min, 77%, (i) NaOH, THF / MeOH / H2O, 0 °C, 20 min, 85%, (j) TBD, 3-hydroxypropionitrile, DCM, 48 h, 87% (k) Ac2O, DMAP, DCM, rt, 48 h, 76%, (l) TBAF, THF, rt, 4 h, 87%, (m) DMTr-Cl, pyridine, rt, 48 h, 99%, (n) i) K2CO3, MeOH, rt, 7 h, ii) N,N-dimethylformamide dimethyl acetal, DMF, 55 °C, 2 h, 77%.

[0238] Ethyl (E and Z, 1'R,5'S,7'R)-(7'-hydroxy-3',3'-dimethyl-2',4'-dioxabicyclo[3.3.0]oct-6'-ylidene)acetate (2a / b) [ka] A solution of epoxide 1 (4.46 g, 18.4 mmol) in dry THF (100 mL) is cooled to −78 °C. LiHMDS (1 M in THF, 22.1 mL, 22.1 mmol) is then slowly added. The solution is stirred at −78 °C for 2 h, then warmed to room temperature, and neutralized by the addition of 1 M aqueous HCl (22.1 mL). The mixture is then diluted with EtOAc (100 mL), and the THF is removed under reduced pressure. The mixture is then washed with 0.5 M NaH2PO4 (50 mL), and the aqueous phase is extracted with EtOAc (2 × 50 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (EtOAc / hexane 3:1) to give two isomers 2a / b (3.30 g, 74%) as a pale yellow solid.

[0239] Data for 2a:R f =0.37(EtoAc / Hexane 1:1): 1 H NMR (300 MHz, CDCl3)δ 6.07-5.98(m, 1H, HC(2)), 5.59(d, J=6.0 Hz, 1H, HC(5')), 4.94-4.81(m, 1H, HC(1')), 4.65(t, J=5.6 Hz, 1H, HC(7')), 4.18(q, J=7.1 Hz, 2H, CH3CH2), 2.67(br, 1H, OH), 2.37(dd, J=13.5, 7.5 Hz, 1H, HC(8')), 1.55-1.42(m, 1H, HC(8')), 1.40, 1.33(2s, 6H, (CH3)2C), 1.26(t, J=7.1 Hz, 3H, CH2CH3). 13C NMR (75 MHz, CDCl3)δ 165.75(C(1)), 161.61(C(6')), 116.53(C(2)), 110.69(C(3')), 76.55(C(5')), 75.52(C(1') )), 71.63(C(7')), 60.51(CH2CH3), 37.46(C(8')), 26.44, 24.11((CH3)2C), 14.27(CH2CH3). C 12 H 19 O5([M+H] + ) ESI+-HRMS m / z calculated 243.1227, found 243.1231. Data for 2b:R f =0.52 (EtoAc / Hexane 1:1): 1 H NMR (300 MHz, CDCl3)δ 6.15-6.05(m, 1H, HC(2)), 5.37-5.02(m, 2H, HC(5'), OH), 4.87(d, J=3.4 Hz, 1H, HC(1')), 4.67(t, J=4.9 Hz, 1H, HC(7')), 4.20(qd, J=7.1, 0.9 Hz, 2H, CH3CH2), 2.55(dd, J=14.6, 8.1 Hz, 1H, HC(8')), 1.94-1.77(m, 1H, HC(8')), 1.39-1.25(m, 9H, (CH3)2C, CH2CH3). 13 C NMR (75 MHz, CDCl3)δ 167.91(C(1)), 167.43(C(6')), 120.13(C(2)), 111.75(C(3')), 81.62(C(5')), 78.08(C(1') )), 70.85(C(7')), 61.25(CH2CH3), 36.53(C(8')), 27.38, 25.45((CH3)2C), 14.19(CH2CH3). C 12 H 19 O5([M+H] + ) ESI+-HRMS m / z calculated 243.1227, found 243.1227.

[0240] Ethyl (1'R,5'S,6'S,7'R)-(7'-hydroxy-3',3'-dimethyl-2',4'-dioxabicyclo[3.3.0]oct-6'-yl)acetate (3) [ka] To a solution of alcohol 2a / b (12.65 g, 52.2 mmol) and nickel chloride hexahydrate (2.48 g, 10.4 mmol) in EtOH (300 mL) is added sodium borohydride (9.88 g, 261 mmol) portionwise at 0 °C. The resulting dark solution is stirred at 0 °C for 30 min and at room temperature for 90 min. EtOH is then carefully removed under reduced pressure, and the resulting solid is diluted with EtOAc (200 mL), and excess NaBH4 is quenched by the addition of water (100 mL) at 0 °C, followed by stirring at room temperature for 30 min. The two phases are then separated. The organic phase is washed with water (100 mL). The aqueous phases are then combined, filtered, and extracted with EtOAc (2 × 100 mL). The combined organic phases are dried over MgSO4, filtered, and concentrated. The crude product is purified by CC (EtOAc / hexane 2:1) to give 3 (11.4 g, 90%) as a white solid.

[0241] Data for 3:R f =0.40 (EtOAc / hexane 1:1): 1 H NMR (300 MHz, CDCl3)δ 4.65-4.52(m, 2H, HC(1'), HC(5')), 4.15(qd, J=7.1, 1.4 Hz, 2H, CH3CH2), 4.05(ddd, J=10.0, 9.99, 6.2 Hz. Hz, 1H.HC(6')), 1.56(ddd, J=13.9, 10.2, 5.5 Hz, 1H, HC(8')), 1.38(s, 3H, (CH3)2C), 1.30~1.21(m, 6H, (CH3)2C, CH2CH3). 13C NMR (75 MHz, CDCl3)δ 174.38(C(1)), 109.06(C(3')), 79.65(C(5')), 77.19(C(1'), 74.32(C(7'), 60.80(CH2CH 3), 46.66(C(6')), 40.38(C(8')), 32.43(C(2)), 26.00, 23.69((CH3)2C), 14.17(CH2CH3). C 12 H 21 O5([M+H] + ) ESI+-HRMS m / z calculated 245.1384, found 245.1388.

[0242] Ethyl (1'R,5'S,6'S,7'R)-(7'-(tert-butyldiphenylsilyl)oxy)-3',3'-dimethyl-2',4'-dioxabicyclo[3.3.0]oct-6'-yl)acetate (4) [ka] To a solution of alcohol 3 (2.50 g, 10.2 mmol), N-methylimidazole (12.6 g, 153 mmol), and iodine (7.80 g, 30.6 mmol) in dry THF (60 mL) is added dropwise tert-butyl(chloro)diphenylsilane (3.0 mL, 11.2 mmol) at room temperature (rt). The solution is stirred at room temperature for 3 h, then the THF is evaporated, and the mixture is diluted with EtOAc (50 mL) and washed with 10% aqueous Na2O3S2 solution (2 × 40 mL). The aqueous phases are then combined and extracted with EtOAc (50 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (EtOAc / hexane 1:10) to give 4 (5.01 g, quantitative yield) as a white solid.

[0243] Data for 4:R f =0.87(DCM / MeOH 10:1): 1H NMR (300 MHz, CDCl3)δ 7.77-7.59(m, 4H, H-arom), 7.51-7.32(m, 6H, H-arom), 4.61(t, J=5.7 Hz, 1H, HC(5')), 4.49(t, J=5.7 Hz, 1H, HC(1')), 4.15(q, J=6.9 Hz, 2H, CH3CH2), 3.96(dd, J=15.5, 9.5 Hz, 1H, HC(7')), 2.64-2.32(m, 2H, HC(2)), 2.15(tt, J=9.0, 4.3 Hz, 1H, HC(6')), 1.83(dd, J=12.7, 5.2 Hz, 1H, HC(8')), 1.61-1.45(m, 1H, HC(8')), 1.27(td, J=7.1, 1.9 Hz, 3H, CH2CH3), 1.18(s, 6H, (CH3)2C), 1.09, 1.08(2s, 9H, (CH3)3-C-Si) 13 C NMR (75 MHz, CDCl3)δ 173.07(C(1)), 135.87, 135.85(CH-arom), 134.08, 133.73(C-arom), 129.80, 129.7 5, 127.67, 127.58(CH-arom), 108.82(C(3')), 77.92(C(5')), 76.96(C(1')), 74.93 (C(7')), 60.24(CH2CH3), 47.27(C(6')), 40.27(C(8')), 31.10(C(2)), 27.04(CH3) 3-C-Si), 25.86((CH3)2C), 23.83((CH3)2C), 19.23(CH3)3-C-Si), 14.24(CH2-CH3). C 28 H 39 O5Si([M+H] + ) ESI + -HRMS m / z calculated 483.2561, found 483.2562.

[0244] (1'R,5'S,6'S,7'R)-(7'-(tert-butyldiphenylsilyl)oxy)-3',3'-dimethyl-2',4'-dioxabicyclo[3.3.0]oct-6'-yl)acetaldehyde (5) [ka] A solution of ester 4 (8.56 g, 16.3 mmol) in dry DCM (120 mL) is cooled to -78 °C, and then DiBAL-H (1 M in cyclohexane, 18 mL, 18 mmol) is slowly added. The solution is stirred for an additional 90 min at -78 °C before warming to room temperature. The reaction is quenched by adding 0.5 M aqueous NaH2PO4 (100 mL). The organic phase is separated, and the aqueous phase is further extracted with DCM (2 x 100 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (2:10 to 2:1 EtOAc / hexane) to give aldehyde 5 (6.36 g, 89%) and alcohol 34 (0.637 g, 9%).

[0245] Data for 5:R f =0.65 (EtOAc / hexane 2:1): 1 H NMR (300 MHz, CDCl3)δ 9.72(s, 1H, HC(1)), 7.65(td, J=8.0, 1.6 Hz, 4H, H-arom), 7.47-7.33(m, 6H, H-arom), 4.57(t, J=5.7 Hz, 1H, HC(5')), 4.51(t, J=5.7 Hz, 1H, HC(1')), 3.99(td, J=10.0, 5.9 Hz, 1H, HC(7')), 2.58-2.43(m, 2H, HC(2)), 2.20-2.08(m, 1H, HC(6')), 1.87(dd, J=13.5, 5.9 Hz, 1H, HC(8')), 1.53(ddd, J=13.5, 10.1, 5.5 Hz, 1H, HC(8')), 1.16(d, J=3.5 Hz, 6H, ((CH3)2C), 1.05(s, 9H, (CH3)3-C-Si). 13C NMR (75 MHz, CDCl3) δ 201.87(C(1)), 135.93, 135.90(CH-arom), 133.96, 133.73(C-arom), 129. 96, 129.89, 127.79, 127.68(CH-arom), 108.89(C(3')), 77.76(C(5')), 77. 17(C(1')), 74.96(C(7'), 45.44(C(6')), 41.31(C(2)), 40.16(C(8')), 27. 08(CH3)3-C-Si), 25.87((CH3)2C), 23.79((CH3)2C), 19.25(CH3)3-C-Si). C 26 H 35 O4Si([M+H] + ) ESI + -HRMS m / z calculated 439.2299, found 439.2297.

[0246] (3aR,4R,6R,6aS)-4-((tert-butyldiphenylsilyl)oxy)-2-methoxyhexahydro-2H-cyclopenta[b]furan-6-ol (6) [ka] To a solution of aldehyde 5 (13.73 g, 31.31 mmol) in MeCN (170 mL) and HO (19 mL) is added indium(III) trifluoromethanesulfonate (703 mg, 1.25 mmol). The solution is stirred for a further 48 h, then the solvent is removed under reduced pressure and coevaporated with toluene. The residue is dissolved in dry MeOH and stirred for 6 h. After evaporation of the solvent, the crude product is purified by CC (EtOAc / hexane 3:10) to obtain the anomeric ratio α / β. [ka] A 4:1 mixture of 6 (10.50 g, 81%) is obtained as a colorless oil.

[0247] Data for 6:R f =0.53 (EtOAc / hexane 1:1): 1H NMR(300 MHz、CDCl3)δ 7.63(dd、J=7.1、0.6 Hz、4H、H-arom)、7.46-7.34(m6H、H-arom)、4.98(d、J=4.8 Hz、0.8H、HC(2))、4.91(dd、J=5.9、1.3 Hz、0.2 H, HC(2)), 4.63-4.54(m, 1H, HC(6a)), 4.53-4.37(m, 1H, HC(6)), 4.09(m, 0.2 H, HC(4)), 3.92(br, 0.8). H、HC(4)), 3.29, 3.27(2s, 3 H, MeO), 2.79(dd, J=17.0, 8.2 Hz, 0.8H, HC(3a)), 2.64-2.51(m, 0.2 H, HC(3a)), 2.29(d, J=8.1 Hz, 1H, OH), 2.10-1.80(m, 2.4 H, HC(3), HC(5)), 1.65(ddd, J=13.2, 9.1, 4.4 Hz, 0.8 H, HC(5)), 1.44-1.34(m, 0.2 H, HC(3)), 1.22(ddd, J=13.2, 8.1, 4.9 Hz, 0.8 H, HC(3)), 1.05(s, 9H, (CH3)3-C-Si). 13 C NMR(75 MHz、CDCl3)δ 135.78, 135.74(CH-arom), 133.96, 133.84(C-arom), 129.78, 127.72(CH-ar om) 107.21 106.50(C(2)) 85.37 81.76(C(6a)) 78.11 77.19(C(4)) 03, 72.44(C(6)), 55.30, 54.46(MeO), 50.91, 49.67(C(3a)), 41.13, 40.29(C (3)) 38.16 37.98(C(5)) 26.96 26.92(CH3)3-C-Si) 19.07(CH3)3-C-Si). C 26 H 35 O4Si([M+H] + )についてのESI + -HRMS m / z designation 435.1962.

[0248] (3aR,4R,6R,6aS)-4-((tert-butyldiphenylsilyl)oxy)-2-methoxyhexahydro-2H-cyclopenta[b]furan-6-yl acetate (7) [ka] To a solution of sugar 6 (3.35 g, 8.12 mmol) and 4-dimethylaminopyridine (1.29 g, 10.6 mmol) in dry DCM (100 mL) is added acetic anhydride (3.8 mL, 41 mmol) at room temperature. After stirring for 2 h, the reaction is quenched by the slow addition of saturated NaHCO3 (10 mL). The mixture is then diluted with saturated NaHCO3 (50 mL) and extracted with DCM (3 x 50 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (1:2 EtOAc / hexane) to obtain the anomeric ratio α / β. [ka] A 4:1 mixture of 7 (3.53 g, 96%) is obtained as a colorless oil.

[0249] Data for 7:R f =0.42 (EtOAc / hexane 1:2): 1H NMR(400 MHz、CDCl3)δ 7.70-7.59(m、4H、H-arom)、7.48-7.34(m、6H、H-arom)、5.41(dt、J=11.0、5.6). Hz、0.8H、HC(6))、5.28(ddd、J=11.7、6.6、5.2 Hz、0.2H、HC(6))、4.99(d、J=4.8 Hz、0.8H、HC(2)),4.89-4.81(m、0.4H、HC(2)、HC(6a)),4.76-4.69(m、0.8H、HC(6a))、4.11(d、J=5.1). Hz、0.2H、HC(4))、3.90(d、J=4.0 Hz、0.8H、HC(4)),3.27、3.24(2s、3H、MeO)、2.81(dd、J=16.67.6). Hz、0.8H、HC(3a))、2.60(dd、J=10.1、7.0 Hz、0.2H、HC(3a))、2.30-2.18(m、0.2 H、HC(5))、2.12、2.10(J=7.7)). Hz、3H、MeCO2)、2.07-1.82(m、2.8H、HC(5)、HC(3))、1.24(ddd、J=12.9、7.6、3.7 Hz、1H、HC(3))、1.07(s、9H、(CH3)3-C-Si). 13 C NMR(75 MHz、CDCl3)δ 170.75、170.66(MeCO2)、135.77、135.73、135.72(CH-arom)、133.75、133.65(C-arom)、129.82、1 29.74、127.76、127.75、127.71(CH-arom)、106.19、106.15(C(2))、83.17、79.80(C(6a))、77.49、 76.46(C(4))、75.64、74.41(C(6))、54.34、54.25(MeO)、51.48、50.17(C(3a))、38.05、37.98(C(3a)) ))、36.96、36.21(C(5))、26.95、26.90(CH3)3-C-Si)、21.09、21.04(MeCO2)、19.04(CH3)3-C-Si). C 26 H 34 O5NaSi([M+Na] + )についてのESI +-HRMS m / z calculated 477.2068, found 477.2063.

[0250] (3aR,4R,6R,6aS)-4-((tert-butyldiphenylsilyl)oxy)-3a,5,6,6a-tetrahydro-4H-cyclopenta[b]furan-6-ol (8) [ka] To a solution of sugar 6 (2.08 g, 5.04 mmol) in dry DCM (35 mL) is added 2,6-lutidine (2.95 mL, 25.2 mmol) at 0 °C. After stirring at 0 °C for 20 min, TMSOTf (2.73 mL, 15.1 mmol) is added dropwise, and the solution is then warmed to room temperature and stirred for an additional 60 min. The reaction is then quenched by adding saturated NaHCO (40 mL). The organic phase is separated, and the aqueous phase is further extracted with DCM (3 × 30 mL). The combined organic phases are dried over MgSO, filtered, and evaporated.

[0251] The resulting product is dissolved in dry THF (35 mL), cooled to 0 °C, and TBAF (1 M in THF, 5.6 mL, 5.6 mmol) is added. The solution is stirred for 10 min, then diluted with saturated NaHCO (30 mL) and extracted with DCM (4 × 40 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (EtOAc / hexane 1:4) to give glycal 8 (1.76 g, 92%).

[0252] Data for 8:R f =0.49 (EtOAc / hexane 1:2): 1H NMR (300 MHz, CDCl3) δ 7.66 (m, 4H, H-arom), 7.42 (m, 6H, H-arom), 6.22 (t, J=2.1 Hz, 1H, HC(2)), 4.91 (dd, J=8.2, 5.3 Hz, 1H, HC(3)), 4.70(dt, J=11.1, 5.6 Hz, 1H, HC(6)), 4.56(t, J=2.8 Hz, 1H, HC(6a)), 3.97(d, J=4.0 Hz, 1H, HC(4)), 3.24(d, J=8.2 Hz, 1H, HC(3a)), 2.30(br, 1H, OH), 2.03(dd, J=12.6, 5.4 Hz, 1H, HC(5)), 1.51(ddd, J=12.7, 11.2, 4.2 Hz, 1H, HC(5)), 1.08(s, 9H, (CH3)3-C-Si). 13 C NMR (75 MHz, CDCl3)δ 146.24(C(2)), 135.72, 135.69(CH-arom), 134.03, 133.74(C-arom), 129.80, 129.78, 127.73(CH-arom), 101.84(C(3) ), 84.59(C(6a)), 76.79(C(4)), 74.10(C(6)), 55.56(C(3a)), 39.38(C(5)), 26.93(CH3)3-C-Si), 19.08(CH3)3-C-Si). C 23 H 29 O3Si([M+H] + ) ESI + -HRMS m / z calculated 381.1880, found 381.1893.

[0253] (((3aR,4R,6R,6aS)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-3a,5,6,6a-tetrahydro-4H-cyclopenta[b]furan-4-yl)oxy)(tert-butyl)diphenylsilane (9) [ka] To a solution of glycal 8 (1.34 g, 3.52 mmol) and DMTr-Cl (1.43 g, 4.23 mmol) in a mixture of dry DCM (15 mL) and dry 2,6-lutidine (15 mL) was added silver triflate (1.13 g, 4.40 mmol) in portions, resulting in a dark red suspension. After stirring at room temperature for 2 h, an additional portion of DMTr-Cl (239 mg, 0.705 mmol) was added. The suspension was stirred for another 2 h and then filtered. The organic phase was washed with saturated NaHCO3 (100 mL), and the aqueous phase was extracted with DCM (3 × 30 mL). The combined organic phases were dried over MgSO4, filtered, and evaporated. The crude product was purified by CC (1:7 EtOAc / hexane + 0.5% Et3N) to give the protected glycal 9 (2.24, 93%) as a white foam.

[0254] 9 Data:R f =0.59 (EtOAc / hexane 1:2): 1 H NMR (400 MHz, CDCl3)δ 7.76(d, J=7.4 Hz, 2H, H-arom), 7.69-7.60(m, J=9.3, 5.9, 4.6 Hz, 8H, H-arom), 7.56-7.39(m, 8H, H-arom), 7.33(t, J=7.3 Hz, 1H, H-arom), 7.00-6.93(m, 4H, H-arom), 6.47-6.37(m, 1H, HC(2)), 4.67- 4.58(m, 1H, HC(6)), 4.58-4.50(m, 2H, HC(3), HC(6a)), 3.86, 3.85(2s, 6H, MeO ), 3.82(d, J=4.0 Hz. 13C NMR (75 MHz, CDCl3)δ 158.67(MeO-C-arom), 147.61(C(2)), 146.26, 137.36, 137.21(C-arom), 135.81, 135.78(CH-arom) m), 134.17, 134.04(C-arom), 130.48, 129.83, 129.81, 128.37, 127.98, 127.76, 127.73, 126.79, 1 13.32, 113.28(CH-arom), 100.29(C(3)), 86.96(C(Ph)3), 84.95(C(6a)), 76.17(C(6)), 76.07(C (4)), 55.26(MeO-DMTr), 55.11(C(3a)), 37.32(C(5)), 27.04(CH3)3-C-Si), 19.21(CH3)3-C-Si). C 44 H 46 05NaSi([M+Na] + ) ESI + -HRMS m / z calculated 705.3007, found 705.3021.

[0255] (3aS,4R,6R,6aS)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-3a,5,6,6a-tetrahydro-4H-cyclopenta[b]furan-4-ol (10) [ka] To a solution of glycal 9 (2.23 g, 3.27 mmol) in dry THF (20 mL) is added TBAF (1 M in THF, 20 mL, 20 mmol) at room temperature. The solution is stirred for 20 h, then diluted with saturated NaHCO3 (100 mL) and extracted with DCM (3 × 80 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (0.5% MeOH in DCM + 0.5% Et3N) to give 10 (1.45 g, quantitative) as a white foam.

[0256] 10 Data:R f =0.44 (EtOAc / hexane 1:1): 1H NMR (300 MHz, CDCl3)δ 7.53-7.46(m, 2H, H-arom), 7.43-7.35(m, 4H, H-arom), 7.21(dd, J=10.7, 5.3 Hz, 2 H, H-arom), 7.16-7.08(m, 1H, H-arom), 6.80-6.71(m, 4H, H-arom), 6.30(t, J=2.1 Hz, 1H, HC(2)), 4.68(t, J=2.8 Hz, 1H, HC(3)), 4.29-4.14(m, 2H, HC(6), HC(6a)), 3.71(s, 6H, MeO), 3.65(d, J=3.5 Hz, 1H, HC(4)), 2.87(d, J=7.9 Hz, 1H, HC(3a)), 1.59(ddd, J=13.2, 11.6, 4.3 Hz, 1H, HC(5)), 1.05-0.95(m, 2H, HC(5), OH). 13 C NMR (75 MHz, CDCl3)δ 158.54(MeO-C-arom), 147.64(C(2)), 145.82, 137.12, 137.08(C-arom), 130.26, 128.29, 127.81, 126.71, 113.13(CH-arom), 100.17(C(3)), 86.75(C(Ph)3), 84.42 C(6a)), 75.54(C(6)), 74.59(C(4)), 55.22(MeO-DMTr), 54.25(C(3a)), 37.56(C(5)). C 30 H 27 O5([M+H] + ) ESI+-HRMS m / z calculated 467.1853, found 467.1844.

[0257] (3'S,5'R,7'R)-1-{2',3'-dideoxy-3',5'-ethano-7'-hydroxy-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}thymine (11) [ka] To a solution of glycal 10 (1.45 g, 3.27 mmol) in dry DCM (45 mL) is added dropwise BSA (2.0 mL, 8.18 mmol) at 0 °C, and the solution is then warmed to room temperature. After stirring for 45 min, thymine (595 mg, 4.91 mmol) is added, and the reaction is stirred for an additional 60 min at room temperature. The mixture is then cooled to 0 °C, and N-iodosuccinimide (875 mg, 3.92 mmol) is added. After stirring for 3 h at 0 °C and 4 h at room temperature, the reaction mixture is diluted with EtOAc (100 mL) and subsequently washed with 10% aqueous NaSO (100 mL) and saturated NaHCO (100 mL). The aqueous phases are combined and extracted with DCM (3 × 50 mL). The combined organic phases are dried over MgSO, filtered, and evaporated.

[0258] The crude product is dissolved in dry toluene (45 mL), and then Bu3SnH (1.32 mL, 4.91 mmol) and azoisobutyronitrile (AIBN, 53 mg, 0.33 mmol) are added at room temperature. After heating at 70 °C for 30 min, the mixture is cooled to room temperature, and TBAF is added (1 M in THF, 6.5 mL, 6.5 mmol). The solution is stirred for an additional 25 min, diluted with saturated NaHCO3 (100 mL), and extracted with DCM (4 × 70 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (3% MeOH in DCM + 0.5% Et3N) to give 11 (1.45 g, 73% over two steps) as a white foam.

[0259] 11 Data:R f =0.29(6%MeOH in DCM): 1H NMR(400 MHz、CDCl3)δ 9.37(br、1H、H-N(3))、7.83(d、J=1.1 Hz、1H、H-C(6))、7.58-7.52(m、2H、H-arom)、7.48-7.41(m、4H、H-arom)、7.28(t、J=7.7 Hz、2H、H-arom)、7.21(t、J=7.2 Hz、1H、H-arom)、6.84(dd、J=8.9、1.2 Hz、4H、H-arom)、5.91(dd、J=8.0、5.5 Hz、1H、H-C(1’))、4.25(dt、J=10.8、6.0 Hz、1H、H-C(5’))、4.13-4.08(m、1H、H-C(4’))、3.86(d、J=3.4 Hz、1H、H-C(7’)、3.79(s、6H、MeO)、2.70(ddd、J=12.8、10.2、5.5 Hz、1H、H-C(2’))、2.61(dd、J=16.9、8.2 Hz、1H、H-C(3’))、1.84(d、J=0.8 Hz、3H、Me-C(5))、1.80(br、1H、OH)、1.60(ddd、J=14.2、10.5、4.2 Hz、1H、H-C(6’))、1.33(dt、J=12.9、8.0 Hz、1H、H-C(2’))、1.14(dd、J=13.7、6.1 Hz、1H、H-C(6’)). 13 C NMR(101 MHz、CDCl3)δ 164.17(C(4))、158.64(MeO-C-arom)、150.47(C(2))、145.65、136.85、136.71(C-arom)、135.52(C(6))、130.20、128.12、127.91、126.90、113.22、113.21(CH-arom)、110.69(C(5))、87.21(C(Ph)3)、86.57(C(1’))、82.02(C(4’))、74.19(C(5’))、74.13(C(7’))、55.25(MeO-DMTr)、49.40(C(3’))、38.51(C(6’))、37.64(C(2’))、12.58(Me-C(5)). C 33 H 34 O7N2Na([M+Na] + )についてのESI +-HRMS m / z calculated 593.2258, found 593.2250.

[0260] (3'R,5'R,7'R)-1-{7'-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}thymine (12) [ka] To a solution of nucleoside 11 (232 mg, 0.406 mmol) and 5-(ethylthio)-1H-tetrazole (90 mg, 0.69 mmol) in dry DCM (10 mL) was added 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (0.26 mL, 0.81 mmol) dropwise at room temperature. After stirring for 30 min, the reaction mixture was diluted with DCM (50 mL) and washed with saturated NaHCO3 (2 x 30 mL) and saturated NaCl (30 mL). The aqueous phases were combined and extracted with DCM (50 mL). The combined organic phases were dried over MgSO4, filtered, and evaporated. The crude product was purified by CC (1.8% MeOH in DCM + 0.5% Et3N) to give 12 (219 mg, mixture of two isomers, 70%) as a white foam.

[0261] 11 Data:R f =0.68(6%MeOH in DCM): 1H NMR(300 MHz、CDCl3)δ 8.93(br、1H、H-N(3))、7.85(d、J=1.2 Hz、1H、H-C(6))、7.65-7.52(m、2H、H-arom)、7.52-7.40(m、4H、H-arom)、7.40-7.21(m、3H、H-arom)、6.96-6.81(m、4H、H-arom)、6.00、5.94(2dd、J=8.3、5.2 Hz、1H、H-C(1’))、4.29-4.17(m、1H、H-C(5’))、4.12-3.89(m、2H、H-C(4’)、H-C(7’))、3.85、3.84(2s、6H、MeO)、3.81-3.63(m、2H、OCH2CH2CN)、3.56-3.41(m、2H、(Me2CH)2N)、2.88-2.69(m、2H、H-C(3’)、H-C(2’))、2.61、2.56(dt、J=12、9 6.3 Hz、2H、OCH2CH2CN)、1.92、1.82(2d、J=0.8 Hz、3H、Me-C(5))、1.75-1.56(m、1H、H-C(6’))、1.52-1.36(m、2H、H-C(6’)、H-C(2’))、1.22-1.01(m、12H、(Me2CH)2N). 13 C NMR(101 MHz、CDCl3)δ 163.86(C(4))、158.66、158.64(MeO-C-arom)、150.29、150.27(C(2))、145.58、145.52、136.76、136.71、136.69、136.60(C-arom)、135.49、135.35(C(6))、130.21、130.16、128.17、128.13、127.88、126.91、126.89(CH-arom)、117.49(OCH2CH2CN)、113.18(CH-arom)、110.74(C(5))、87.27、87.25(C(Ph)3)、86.58、86.45(C(1’))、81.79、81.68(C(4’))、76.02、75.50(J C、P =16.5、15.7 Hz、C(7’))、74.22(C(5’))、58.26、58.06、57.87(OCH2CH2CN)、55.26、55.22(MeO-DMTr)、48.85、48.62(J C、P=2.6, 5.0 Hz, C(3')), 43.10, 43.04(J C、P =12.3, 12.4 Hz(Me2CH)2N), 37.78(J C、P =5.3 Hz C(6')), 37.62, 37.48(C(2')), 37.41(J C、P =3.6 Hz C(6')), 24.57, 24.53, 24.50, 24.46, 24.44, 24.39, 24.37(Me2CH)2N), 20.35, 20.25(J C、P =7.1, 7.0 Hz, OCH2CH2CN), 12.58, 12.41(7s, Me-C(5)). 31 P NMR(122 MHz, CDCl3)δ 147.32, 146.98. C 42 H 52 O8N4P([M+H] + ) ESI + -HRMS m / z calculated 771.3517, found 771.3512.

[0262] (3'S,5'R,7'R)-N 4 -Benzoyl-1-{2',3'-dideoxy-3',5'-ethano-7'-hydroxy-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}-5-methylcytosine (13) [ka] To a solution of nucleoside 11 (302 mg, 0.530 mmol) in dry MeCN (5 mL) was added BSA (0.31 mL, 1.27 mmol) dropwise at 0 °C, and the solution was stirred overnight at room temperature. In a separate flask, a suspension of 1,2,4-triazole (1.28 g, 18.55 mmol) in dry MeCN (50 mL) was cooled to 0 °C, and POCl (0.40 mL, 4.2 mmol) and EtN (2.96 mL, 21.2 mmol) were added. The suspension was stirred at 0 °C for 30 min, and then the previously prepared solution of silylated compound 11 was added to the suspension, and the mixture was further stirred at room temperature for 5 h. The reaction was quenched by adding saturated NaHCO (10 mL), the MeCN was removed under reduced pressure, and the resulting mixture was diluted with saturated NaHCO (35 mL) and extracted with DCM (3 × 40 mL). The combined organic phases are dried over MgSO4, filtered and evaporated.

[0263] The crude product is then dissolved in a mixture of 1,4-dioxane (10 mL) and concentrated NH4OH (10 mL). After stirring at room temperature for 2 h, the mixture is reduced to half its volume in vacuo, diluted with saturated NaHCO3 (30 mL), and extracted with DCM (4 x 30 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated.

[0264] The crude product is then dissolved in dry DMF (13 mL), EtN (90 μL, 0.64 mmol) is added followed by BzO (300 mg, 1.33 mmol) at room temperature, and the solution is stirred overnight. The resulting brown solution is quenched by careful addition of saturated NaHCO (50 mL) and extracted with DCM (4 × 50 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (hexane / EtOAc 1:2, +0.5% EtN) to give 13 (315 mg, 88%) as a white foam.

[0265] 13 Data:R f =0.57(4%MeOH in DCM): 1H NMR(300 MHz、CDCl3)δ 13.39(br、1H、NH)、8.46-8.26(m、2H、H-arom)、8.13(d、J=0.5 Hz、1H、C(6))、7.61(d、J=7.3 Hz、2H、H-arom)、7.58-7.43(m、7H、H-arom)、7.34(t、J=7.4 Hz、2H、H-arom)、7.30-7.23(m、1H、H-arom)、6.89(d、J=8.8 Hz、4H、H-arom)、5.96(dd、J=7.5、5.8 Hz、1H、H-C(1’))、4.38-4.25(m、1H、H-C(5’))、4.22-4.12(m、1H、H-C(4’))、3.90(d、J=3.6 Hz、1H、H-C(7’))、3.83(s、6H、MeO)、2.82(ddd、J=13.3、10.2、5.7 Hz、1H、H-C(2’))、2.66(dd、J=17.0、8.1 Hz、1H、H-C(3’))、2.08(s、3H、Me-C(5))、1.77(br、1H、OH)、1.71-1.57(m、1H、H-C(6’))、1.49-1.36(m、1H、H-C(2’))、1.21(dd、J=13.7、6.2 Hz、1H、H-C(6’)). 13 C NMR(75 MHz、CDCl3)δ 179.56(CONH)、160.01(C(4))、158.70(MeO-C-arom)、147.96(C(2))、145.65(C-arom)、137.26(C(6))、136.99、136.83、136.71(C-arom)、132.41、130.22、129.89、128.16、128.14、127.95、126.94、113.25(CH-arom)、111.57(C(5))、87.34(C(Ph)3)、87.32(C(1’))、82.57(C(4’))、74.30(C(5’))、74.16(C(7’))、55.27(MeO-DMTr)、49.56(C(3’))、38.52(C(6’))、38.00(C(2’))、13.63(Me-C(5)). C 40 H 40 O7N3([M+H] +) ESI+-HRMS m / z calculated 674.2861, found 674.2862.

[0266] (3'R,5'R,7'R)-N 4 -Benzoyl-1-{7'-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}-5-methylcytosine (14) [ka] To a solution of nucleoside 13 (276 mg, 0.409 mmol) and 5-(ethylthio)-1H-tetrazole (69 mg, 0.53 mmol) in dry DCM (10 mL) is added 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (0.20 mL, 0.61 mmol) dropwise at room temperature. After stirring for 60 min, the reaction mixture is diluted with DCM (50 mL) and washed with saturated NaHCO3 (2 x 30 mL) and saturated NaCl (30 mL). The aqueous phases are combined and extracted with DCM (50 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (2:3 EtOAc / hexanes + 0.5% Et3N) to give 14 (268 mg, mixture of two isomers, 75%) as a white foam.

[0267] 14 Data:R f =0.77(5%MeOH in DCM): 1H NMR(400 MHz、CDCl3)δ 13.32(s、1H、NH)、8.41-8.28(m、2H、H-arom)、8.13-8.04(m、1H、C(6))、7.61-7.51(m、3H、H-arom)、7.51-7.40(m、6H、H-arom)、7.37-7.29(m、2H、H-arom)、7.29-7.20(m、1H、H-arom)、6.92-6.82(m、4H、H-arom)、6.07-5.87(m、1H、H-C(1’))、4.24(dq、J=11.7、5.8 Hz、1H、H-C(5’))、4.13-4.00(m、1H、H-C(4’))、3.94(ddd、J=14.5、10.5、2.8 Hz、1H、H-C(7’))、3.83、3.82(2s、6H、MeO)、3.69(m、2H、OCH2CH2CN)、3.53-3.40(m、2H、(Me2CH)2N)、2.91-2.70(m、2H、H-C(2’)、H-C(3’))、2.57、2.53(2t、J=6.3 Hz、2H、OCH2CH2CN)、2.08、1.99(2d、J=0.6 Hz、3H、Me-C(5))、1.72-1.56(m、1H、H-C(6’))、1.54-1.36(m、2H、H-C(2’)、H-C(6’))、1.10(m、12H、(Me2CH)2N). 13 C NMR(101 MHz、CDCl3)δ 179.54(CONH)、159.98(C(4))、158.69(MeO-C-arom)、147.90(C(2))、145.58、145.54(C-arom)、137.30、136.93(C(6))、136.81、136.80、136.73、136.70、136.67、136.60(C-arom)、132.37、132.35、130.22、130.17、129.89、128.17、128.15、128.11、127.93、126.94(CH-arom)、117.49(OCH2CH2CN)、113.23(CH-arom)、111.60(C(5))、87.36、87.35(C(Ph)3)、87.33、87.25(C(1’))、82.33、82.25(C(4’))、76.05、75.52(J C、P=16.4, 15.6 Hz, C(7')), 74.32(C(5')), 58.18, 57.98(J C、P =19.5 Hz OCH2CH2CN), 55.28, 55.24(MeO-DMTr), 48.93, 48.72(J C、P =2.7, 4.9 Hz, C(3')), 43.11, 43.05(J C、P =12.4 Hz(Me2CH)2N), 38.02, 37.88(C(2')), 37.74, 37.40(J C、P =5.3, 3.4 Hz, C(6')), 24.58, 24.54, 24.50, 24.47, 24.40, 24.38(6s, Me2CH)2N), 20.36, 20.26(J C、P =7.1 Hz, OCH2CH2CN), ), 13.66, 13.49(Me-C(5)). 31 P NMR(122 MHz, CDCl3)δ 147.37, 147.07. C 49 H 57 O8N5P([M+H] + ) ESI + -HRMS m / z calculated 874.3939, found 874.3937.

[0268] (3'R,5'R,7'R)-N 6 -Benzoyl-9-{5'-O-acetyl-7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α,β-D-ribofuranosyl}adenine (15) [ka] Sugar 7 (1.86 g, 4.10 mmol) and N in dry MeCN (40 mL) 6To a suspension of -benzoyladenine (1.96 g, 8.20 mmol) is added BSA (4.00 mL, 16.4 mmol) at room temperature. After stirring for 25 min, the suspension becomes a clear solution and is then heated to 70 °C. TMSOTf (1.48 mL, 8.20 mmol) is added dropwise and the solution is stirred for another 20 min at 70 °C. The solution is then cooled to room temperature, quenched by the addition of saturated NaHCO3 (100 mL) and extracted with EtOAc (4 x 50 mL). The combined organic phases are dried over MgSO4, filtered and evaporated. The crude product is purified by CC (2% MeOH in DCM) to obtain the anomeric ratio α / β. [ka] A 4:1 mixture of 15 (1.74 g, 64%) is obtained as a white foam.

[0269] 15 Data:R f =0.33 (EtOAc / hexane 4:1): 1H NMR(400 MHz、CDCl3)δ 9.33(br、1H、NH)、8.68(d、J=5.4 Hz、0.8H、H-C(2))、8.64(d、J=5.6 Hz、0.2H、H-C(2))、8.10(d、J=1.5 Hz、0.2H.H-C(8))、7.99(dJ=7.3 Hz、2H、H-arom)、7.95(s、0.8H、H-C(8))、7.63(t、J=8.7 Hz、4H、H-arom)、7.55(dd、J=13.0、6.4 Hz、1H、H-arom)、7.50-7.34(m、8H、H-arom)、6.20(dd、J=6.3、2.5 Hz、0.8H、H-C(1’))、6.05(t、J=6.5 Hz、0.2H、H-C(1’))、5.43-5.32(m、1H、H-C(5’))、5.03-4.97(m、0.8H、H-C(4’))、4.83(t、J=6.0 Hz、0.2H、H-C(4’))、4.14(br、0.2H、H-C(7’))、4.08(d、J=3.7 Hz、0.8H、H-C(7’))、3.02(dd、J=16.1、6.6 Hz、0.8H、H-C(3’))、2.83(dd、J=16.9、7.7 Hz、0.2H、H-C(3’))、2.59-2.39(m、1H、H-C(2’))、2.18-2.11(m、1H、H-C(6’))、2.07(d、J=1.6 Hz、2.4H、MeCO2)、2.02(d、J=1.9 Hz、0.6H、MeCO2)、2.01-1.92(m、1H、H-C(6’))、1.91-1.80(m、1H、H-C(3’))、1.07(s、9H、(CH3)3-C-Si). 13C NMR (101 MHz, CDCl3)δ 170.57, 170.49(MeCO2), 164.82(CONH), 152.50(C(2)), 151.27(C(4)), 149.56(C(6)), 141.37, 141.06(C(8)), 135.72, 13 5.68, 135.66 (CH-arom), 133.67, 133.57, 133.24, 133.22 (C-arom), 132.73, 130.03, 129.98, 128.80, 128.78, 127.92, 127. 86, 127.85(CH-arom), 123.61(C(5)), 87.19, 86.17(C(1')), 83.22, 80.96(C(4'), 76.50, 76.04(C(7')), 74.38(C(5')), 51 .07(C(3')), 37.29, 37.15, 36.80, 36.60(C(2'), C(6')), 26.89(CH3)3-C-Si), 20.97, 20.90(MeCO2), 19.01(CH3)3-C-Si). C 37 H 40 O5N5Si([M+H] + ) ESI + -HRMS m / z calculated 662.2793, found 662.2787.

[0270] (3'R,5'R,7'R)-N 6 -benzoyl-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-β-D-ribofuranosyl}adenine (16): [ka] Nucleoside 15 (1.74 g, 2.64 mmol) was dissolved in 0.15 M NaOH in THF / MeOH / HO (5:4:1, 80 mL) at 0 °C. The reaction was stirred for 20 min and quenched by adding NH Cl (1.06 g). The solvent was then removed under reduced pressure, and the product was purified by CC (5% isopropanol in DCM) to give 16 (287 mg, 18%) and its corresponding α anomer (836 mg, 51%) as white foams.

[0271] 16th century:R f =0.44(DCMin 6%MeOH): 1 H NMR(300 MHz、CDCl3)δ 8.70(s、1H、HC(2)), 8.09-7.98(m、2H、H-arom), 7.97(s、1H、HC(8)), 7.63(ddd, J=7.4, 5.7, 1.5 Hz、4H、H-arom)、7.59-7.55(m、1H、H-arom)、7.51(m、2H、H-arom)、7.44-7.33(m、6H、H-arom)、6.02(dd、J=9.4、5.5 Hz Hz, 1H, HC(5')) 4.26(br, 1H, HC(7')) 2.78 (q, J=8.9 Hz、1H、HC(3')) 2.32-1.80(m、5H、HC(2')、HC(6')、OH) 1.06(s、9H、(CH3)3-C-Si). 13 C NMR(101 MHz、CDCl3)δ 164.85(CONH) 152.56(C(2)) 151.17(C(4)) 149.86(C(6)) 141.25(C(8)) (CH-arom) 133.87 133.39(C-arom) 132.78 129.92 128.78 128.01 127.78(CH-ar om) 123.51(C(5)) 87.65(C(1')) 82.91(C(4')) 76.66(C(7')) 72.54(C(5')) 44(C(3′)), 41.42(C(6′)), 36.17(C(2′)), 26.89(CH3)3-C-Si), 19.03(CH3)3-C-Si). C 35 H 38 O4N5Si([M+H] + )についてのESI + -HRMS m / z format 620.2688.

[0272] (3'R,5'R,7'R)-N 6-Benzoyl-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}adenine (17) [ka] To a solution of nucleoside 16 (307 mg, 0.495 mmol) in dry pyridine (6 mL) is added DMTr-Cl (503 mg, 1.49 mmol) at room temperature. The solution is stirred for 1 day, then diluted with saturated NaHCO (50 mL) and extracted with DCM (3 × 70 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (1.5% MeOH in DCM + 0.5% EtN) to give 17 (395 mg, 87%) as a yellow foam.

[0273] 17 Data:R f =0.65(5%MeOH in DCM): 1 H NMR (300 MHz, MeOD) δ 8.64(s, 1H, HC(2)), 8.61(s, 1H, HC(8)), 8.08(d, J=7.2 Hz, 2H, H-arom), 7.68-7.17(m, 22H, H-arom), 6.86-6.75(m, 4H, H-arom), 6.14(dd, J=7.4, 6.3 Hz, 1H, HC(1')), 4.48-4.31(m, 1H, HC(5')), 4.28-4.15(m, 1H, HC(4')), 3.88(d, J=3.8 Hz, 1H, HC(7')), 3.75, 3.74(2s, 6H, MeO), 2.67(dd, J=16.6, 6.7 Hz, 1H, HC(3')), 2.47(ddd, J=13.3, 10.2, 6.1 Hz, 1H, HC(2')), 2.15-1.94(m, 1H, HC(6')), 1.71(ddd, J=13.0, 11.3, 4.4 Hz, 1H, HC(2')), 1.11(dd, J=12.2, 4.9 Hz, 1H, HC(6')), 0.95(s, 9H, (CH3)3-C-Si). 13C NMR (75 MHz, CDCl3)δ 164.69(CONH), 158.61, 158.60(MeO-C-arom), 152.42(C(2)), 151.27(C(4)), 149.41(C(6)), 145.81(C-arom), 141.25(C(8)), 137.00, 136.85(C-arom), 135.60, 135.57(CH-arom), 133.80, 133.69, 133.43(C-arom), 132.70, 130.28, 130.25, 129.85, 129.81, 128.84, 128. 18, 127.89, 127.71, 127.65, 126.78(CH-arom), 123.52(C(5)), 113.22, 113.19(CH-arom), 87.09(C(Ph)3), 86.41(C(1')), 83.52(C(4') )), 76.05(C(7')), 74.78(C(5')), 55.20(MeO-DMTr), 50.43(C(3')), 38.10(C(2'), C(6')), 26.84(CH3)3-C-Si), 19.00(CH3)3-C-Si). C 56 H 56 O6N5Si([M+H] + ) ESI + -HRMS m / z calculated 922.3994, found 922.3953.

[0274] (3'S,5'R,7'R)-N 6- Benzoyl-9-{2',3'-dideoxy-3',5'-ethano-7'-hydroxy-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}adenine (18) [ka] To a solution of nucleoside 17 (376 mg, 0.408 mmol) in dry THF (9 mL) is added TBAF (1 M in THF, 1.22 mL, 1.22 mmol) at room temperature. The solution is stirred for 2 days, then diluted with saturated NaHCO (25 mL) and extracted with DCM (4 × 25 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (4% MeOH in DCM + 0.5% EtN) to give 18 (242 mg, 87%) as a white foam.

[0275] 18 Data:R f =0.33(5%MeOH in DCM): 1 H NMR (300 MHz, CD3CN)δ 9.35(br, 1H, NH), 8.67(s, 1H, C(2)), 8.46(s, 1H, C(8)), 8.01(d, J=7.4 Hz, 2H, H-arom), 7.54(m, 5H, H-arom), 7.35(m, 4H, H-arom), 7.30-7.17(m, 3H, H-arom), 6.84(d, J=8.9 Hz, 4H, H-arom), 6.09(dd, J=7.8, 6.2 Hz, 1H, HC(1')), 4.12(dt, J=11.2, 5.8 Hz. Hz, 1H, C(2')), 1.92-1.82(m, 1H, C(6')), 1.29-1.17(m, 1H, C(6')). 13C NMR (75 MHz, CDCl3)δ 165.03(CONH), 158.57(MeO-C-arom), 152.40(C(2)), 151.23(C(4)), 149.52(C(6)), 145.68(C-arom) m), 141.49(C(8)), 136.86, 136.84, 133.77(C-arom), 132.77, 130.22, 128.81, 128.16, 128.02, 127. 89, 126.84(CH-arom), 123.40(C(5)), 113.19(CH-arom), 87.06(C(Ph)3), 86.74(C(1')), 83.58(C(4) ')), 74.62(C(5')), 74.38(C(8')), 55.25(MeO-DMTr), 49.77(C(3')), 38.55, 38.32(C(6'), C(2')). C 40 H 38 O6N5([M+H] + ) ESI + -HRMS m / z calculated 684.2817, found 684.2830.

[0276] (3'R,5'R,7'R)-N 6 -Benzoyl-9-{7'-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}adenine (19) [ka] To a solution of nucleoside 18 (173 mg, 0.253 mmol) and N,N-diisopropylethylamine (0.18 mL, 1.0 mmol) in dry THF (8 mL) was added N,N-diisopropylchlorophosphoramidite (0.11 mL, 0.50 mmol) at room temperature. The solution was stirred for 2 h, then diluted with saturated NaHCO (40 mL) and extracted with DCM (4 × 40 mL). The combined organic phases were dried over MgSO, filtered, and evaporated. The crude product was purified by CC (EtOAc, +0.5% EtN) to give 19 (177 mg, mixture of two isomers, 71%) as a white foam.

[0277] 19 Data:R f = 0.38, 0.44 (EtOH): 1H NMR(400 MHz、CDCl3)δ 9.05(br、1H、NH)、8.70、8.70(2s、1H、H-C(2))、8.47、8.46(2s、1H、H-C(8))、7.97(d、J=7.5 Hz、2H、H-arom)、7.57-7.50(m、1H、H-arom)、7.49-7.41(m、4H、H-arom)、7.39-7.31(m、4H、H-arom)、7.24-7.17(m、5.4 Hz、2H、H-arom)、7.13(dt、J=12.5、6.2 Hz、1H、H-arom)、6.83-6.70(m、4H、H-arom)、6.14-5.97(m、1H、H-C(1’))、4.14(ddd、J=11.1、7.8、3.4 Hz、1H、H-C(5’))、3.91-3.74(m、2H、H-(4’)、H-C(7’))、3.71、3.70(2s、6H、MeO)、3.65-3.50(m、2H、OCH2CH2CN)、3.37(ddq、J=13.9、10.2、6.8 Hz、2H、(Me2CH)2N)、2.90-2.76(m、1H、H-C(2’))、2.75-2.60(m、1H、H-C(3’))、2.47、2.42(2t、J=6.3 Hz、2H、OCH2CH2CN)、2.11(dt、J=12.7、6.1 Hz、1H、H-C(2’))、1.73(ddt、J=13.6、10.4、5.1 Hz、1H、H-C(6’))、1.39(ddd、J=50.2、13.4、6.2 Hz、1H、H-C(6’))、1.10-0.89(m、12H、(Me2CH)2N). 13C NMR(101 MHz、CDCl3)δ 164.66(CONH)、158.57(MeO-C-arom)、152.46(C(2))、151.32、151.26(C(4))、149.45、149.43(C(6)16). 45.59(C-arom)、141.52、141.47(C(8))、136.88、136.83、136.81、133.78(C-arom)、132.75、1.2012.73、 .21、130.19、130.17、128.87、128.17、126.87(CH-arom)、123.59(C(2))、123.59(C(2))CH1 CH2CN)、113.17(CH-arom)、87.10、87.07(C(Ph)3)、86.72、86.68(C(1'))、83.36、83.25(C(4'J)7)、76.5. C、P =16.9、15.7 Hz、C(7'))、74.63、74.60(C(5'))、58.24、57.86(J C、P =19.1、19.2 Hz OCH2CH2CN)、55.25、55.21(MeO-DMTr)、49.29、49.08(J C、P =2.6、4.7 Hz、C(3'))、43.12、43.00(J C、P =2.4、2.3 Hz(Me2CH)2N)、38.27、38.23(C(2'))、37.41、37.22(J C、P =5.3、3.5 Hz、C(6'))24.56、24.53、24.49、24.47、24.36、24.33(2s、Me2CH5、20N.)、 C、P =7.2、7.0 Hz、OCH2CH2CN). 31 P NMR(122 MHz、CDCl3)δ 147.64、146.87. C 49 H 55 O7N7([M+H] + )についてのESI + -HRMS m / z located at

[0278] (3'R,5'R,7'R)-2-amino-6-chloro-9-{5'-O-acetyl-7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α,β-D-ribofuranosyl}purine (20) [ka] To a suspension of sugar 7 (1.75 g, 3.85 mmol) and 2-amino-6-chloropurine (1.05 g, 6.17 mmol) in dry MeCN (20 mL) is added BSA (3.80 mL, 15.4 mmol) at room temperature. The suspension is heated to 55 °C and stirred for 30 min. TMSOTf (1.05 mL, 5.78 mmol) is then added dropwise, and the solution is stirred at 55 °C for an additional 50 min. The solution is cooled to room temperature, quenched by the addition of saturated NaHCO (10 mL), diluted with EtOAc (50 mL), and filtered through a short pad of SiO. The SiO was washed with additional EtOAc. The mixture was then washed with saturated NaHCO (2 × 80 mL), and the aqueous phases were combined and extracted with EtOAc (3 × 50 mL). The combined organic phases were dried over MgSO, filtered, and evaporated. The crude product was purified by CC (2.5% MeOH in DCM) to obtain the anomeric ratio α / β [ka] A 7:3 mixture of 20 (1.77 g, 77%) is obtained as a white foam.

[0279] 20 Data:R f =0.54 (EtOAc / hexane 5:1): 1H NMR(300 MHz、CDCl3)δ 7.86(s、0.3H、H-C(8))、7.69(s、0.7H、H-C(8))、7.68-7.60(m、4H、H-arom)、7.47-7.34(m、6H、H-arom)、6.04(dd、J=6.9、3.0 Hz、0.7H、H-C(1’))、5.87(dd、J=8.0、6.2 Hz、0.3H、H-C(1’))、5.37(dt、J=14.2、4.6 Hz、1H、H-C(5’))、5.16(br、2H、NH2)、4.91(dd、J=6.5、5.1 Hz、0.7H、H-C(4’))、4.79(dd、J=6.9、5.2 Hz、0.3H、H-C(4’))、4.13(br、0.3H、H-C(7’))、4.06(d、J=4.0 Hz、0.7H、H-C(7’))、2.95(dd、J=16.3、6.6 Hz、0.7H、H-C(3’))、2.81(dd、J=17.0、7.4 Hz、0.3H、H-C(3’))、2.49-2.30(m、1H、H-C(2’))、2.14(dd、J=13.1、6.7 Hz、1H、H-C(6’))、2.08(s、2.1H、MeCO2)、2.02(s、0.9H、MeCO2)、2.02-1.91(m、1H、H-C(6’))、1.80(td、J=13.4、6.8 Hz、1H、H-C(2’))、1.07、1.06(2s、9H、(CH3)3-C-Si). 13C NMR (75 MHz, CDCl3)δ 170.55, 170.44(MeCO2), 158.98, 158.91(C(2)), 153.18, 152.95(C(4)), 151.40, 151.34(C(6)), 140.38, 140.14(C(8)), 135.73, 135.70(CH-arom), 133.78, 133.62, 133.24, 133.17(C-arom), 130.03, 130.00, 127.88, 127.86(CH-arom), 125.6 5, 125.57(C(5)), 86.59, 85.74(C(1')), 82.93, 80.99(C(4')), 76.57, 76.14(C(7')), 74.34, 74.32(C(5')), 51.15, 51.1 0(C(3')), 37.19, 36.99(C(6')), 36.70, 36.25(C(2')), 26.87(CH3)3-C-Si), 20.95, 20.86(MeCO2), 19.00(CH3)3-C-Si). C 30 H 35 O4N5ClSi([M+H] + ) ESI + -HRMS m / z calculated 592.2141, found 592.2158.

[0280] (3'R,5'R,7'R)-2-amino-6-chloro-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-β-D-ribofuranosyl}purine (22b) [ka] Nucleoside 20 (1.78 g, 3.01 mmol) is dissolved in 0.5 M NaOH in THF / MeOH / HO (5:4:1, 15 mL) at 0 °C. The reaction is stirred at 0 °C for 20 min and quenched by the addition of NH Cl (484 mg). The suspension is then diluted with saturated NaHCO (100 mL) and extracted with DCM (4 × 75 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (3% MeOH in DCM) to give 21 (428 mg, 25%) and its corresponding α anomer (992 mg, 60%) as a white foam.

[0281] 21 Data:R f =0.43(5%MeOH in DCM): 1 H NMR (300 MHz, CDCl3)δ 7.71(s, 1H, HC(8)), 7.68-7.60(m, 4H, H-arom), 7.44-7.33(m, 6H, H-arom), 5.85(dd, J=9.3, 5.8 Hz, 1H, HC(1')), 5.33(br, 2H, NH2), 4.62(dd, J=8.4, 4.9 Hz, 1H, HC(4')), 4.44(dd, J=10.7, 5.3 Hz, 1H, HC(5')), 4.40-4.15(m, 2H, HC(7'), OH), 2.79(q, J=8.7 Hz, 1H, HC(3')), 2.22(dd, J=15.2, 9.3 Hz, 1H, HC(6')), 2.11-2.02(m, 1H, HC(6')), 2.02-1.85(m, 2H, HC(2')), 1.06(s, 9H, (CH3)3-C-Si). 13C NMR (75 MHz, CDCl3) δ 158.73(C(2)), 152.78(C(4)), 151.94(C(6)), 140.70(C(8)), 135.70(CH-a rom), 133.91, 133.48(C-arom), 129.90, 127.78(CH-arom), 125.97(C(5)), 87.96(C(1')), 82.88(C(5')), 76.85(C(7')), 72.36(C(5')), 50.41(C(3') ), 41.96(C(6')), 35.73(C(2')), 26.90(CH3)3-C-Si), 19.02(CH3)3-C-Si). C 28 H 33 O3N5ClSi([M+H] + ) ESI + -HRMS m / z calculated 550.2036, found 550.2015.

[0282] (3'R,5'R,7'R)-N 2 -(N,N-Dimethylformamidino)-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-β-D-ribofuranosyl}guanine (22) [ka] To a solution of 21 (380 mg, 0.645 mmol) and 3-hydroxypropionitrile (0.22 mL, 3.23 mmol) in dry DCM (15 mL) was added 1,5,7-triazabicyclo[4.4.0]dec-5-ene (400 mg, 2.87 mmol) at 0 °C. The solution was stirred at 0 °C for 3 h and then at room temperature for 2 days. The reaction was quenched by adding silica. After evaporation of the solvent, the SiO powder was filtered, washed with MeOH, and the solvent was evaporated to give a brown foam.

[0283] The crude product is dissolved in dry DMF (5 mL) and N,N-dimethylformamide dimethyl acetal (0.43 mL, 3.2 mmol) is added. The solution is stirred at 55 °C for 2 h, and then the solvent is removed under reduced pressure. The crude product is purified by CC (6% MeOH in DCM) to give 23 (274 mg, 73%) as a yellowish foam.

[0284] 22 Data:R f =0.45(12%MeOH in DCM): 1 H NMR (300 MHz, CDCl3)δ 9.52(s, 1H, NH), 8.46(s, 1H, NCHN(CH3)2), 7.63(dd, J=7.7, 1.5 Hz, 4H, H-arom), 7.50(s, 1H, HC(8)), 7.44-7.30(m, 6H, H-arom), 5.83(dd, J=9.3, 6.0 Hz, 1H, HC(1')), 4.61(dd, J=8.7, 5.0 Hz, 1H, HC(4')), 4.43-4.32(m, 1H, HC(5')), 4.29(dd, J=7.0, 4.8 Hz, 1H, HC(7')), 3.95(d, J=5.1 Hz, 1H, OH), 2.98(s, 6H, NCHN(CH3)2), 2.79(dd, J=18.0, 7.0 Hz, 1H, HC(3')), 2.20(dt, J=12.8, 5.4 Hz, 1H, HC(6')), 2.09-1.88(m, 3H, HC(6'), HC(2')), 1.05(s, 9H, (CH3)3-C-Si)). 13C NMR (75 MHz, CDCl3)δ 158.73(C(2)), 157.79(C(6)), 156.91(NCHN(CH3)2), 149.84(C(4)), 137.00(C(8)), 135.70, 135. 67(CH-arom), 133.78, 133.60(C-arom), 129.93, 129.86, 127.78, 127.72(CH-arom), 121.61(C(5) ), 88.04(C(1')), 82.21(C(4')), 77.49(C(7')), 71.94(C(5')), 50.13(C(3')), 42.23(C(6')), 41 .20(NCHN(CH3)2), 35.50(C(2')), 34.97(NCHN(CH3)2), 26.87(CH3)3-C-Si), 19.02(CH3)3-C-Si). C 31 H 38 O4N6Si([M+H] + ) ESI + -HRMS m / z calculated 586.2718, found 586.2703.

[0285] (3'R,5'R,7'R)-N 2 -(N,N-Dimethylformamidino)-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}guanine (23) [ka] To a solution of 22 (139 mg, 0.237 mmol) in dry pyridine (2 mL) is added DMTr-Cl (240 mg, 0.708 mmol) in six portions over 3 h at room temperature. After stirring overnight, the orange solution is diluted with saturated NaHCO (20 mL) and extracted with DCM (3 × 20 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (4% MeOH in DCM + 0.5% EtN) to give 23 (148 mg, 70%) as a yellowish foam.

[0286] Data of 23: R f = 0.52 (10% MeOH in DCM): 1 H NMR (400 MHz, CDCl3) δ 9.49 (s, 1H, NH), 8.38 (s, 1H, NCHN(CH3)2), 7.80 (s, 1H, C(8)), 7.50 - 7.43 (m, 2H, H-arom), 7.42 - 7.27 (m, 10H, H-arom), 7.26 - 7.15 (m, 6H, H-arom), 7.14 - 7.08 (m, 1H, H-arom), 6.77 - 6.68 (m, 4H, H-arom), 5.78 (dd, J = 8.2, 5.9 Hz, 1H, H-C(1’)), 4.25 (dt, J = 11.0, 5.6 Hz, 1H, H-C(5’)), 4.14 - 4.03 (m, 1H, H-C(4’)), 3.70 - 3.64 (m, 7H, MeO, H-C(7’)), 3.00 (s, 3H, NCHN(CH3)2), 2.97 (s, 3H, NCHN(CH3)2), 2.43 (dd, J = 16.7, 7.5 Hz, 1H, H-C(3’)), 2.24 (ddd, J = 13.3, 10.1, 5.8 Hz, 1H, H-C(2’)), 1.62 (td, J = 13.1, 4.3 Hz, 1H, H-C(6’)), 1.43 (dt, J = 13.5, 8.0 Hz, 1H, H-C(2’)), 0.99 (dd, J = 13.3, 6.2 Hz, 1H), 0.86 (s, 9H, (CH3)3-C-Si). 13C NMR (101 MHz, CDCl3)δ 158.51, 158.49(MeO-C-arom), 158.04(C(2)), 157.91(C(6)), 156.60(NCHN(CH3)2), 149.76(C(4)), 145.83, 137.12, 136.94(C-a rom), 136.01(C(8)), 135.60, 135.59(CH-arom), 133.81, 133.47(C-arom), 130.32, 130.26, 129.77, 128.24, 127.82, 127.65, 127 .62, 126.67(CH-arom), 120.65(C(5)), 113.13, 113.09(CH-arom), 86.82(C(Ph)3), 85.01(C(1')), 82.26(C(4')), 76.14(C(7')) , 74.61(C(5')), 55.19(MeO-DMTr), 50.18(C(3')), 41.29(NCHN(CH3)2), 38.01(C(6')), 37.76(C(2')), 35.14(NCHN(CH3)2)26.81 87(CH3)3-C-Si), 19.01(CH3)3-C-Si). C 52 H 57 O6N6Si([M+H] + ) ESI + -HRMS m / z calculated 889.4103, found 889.4128.

[0287] (3'S,5'R,7'R)-N 2 -(N,N-Dimethylformamidino)-9-{2',3'-dideoxy-3',5'-ethano-7'-hydroxy-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}guanine (24) [ka] To a solution of 23 (243 mg, 0.273 mmol) in dry THF (2 mL) is added TBAF (1 M in THF, 1.65 mL, 1.63 mmol) at room temperature. The solution is stirred for 7 h, then diluted with saturated NaHCO3 (30 mL) and extracted with DCM (4 x 30 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (7% MeOH in DCM + 0.5% Et3N) to give 24 (155 mg, 87%) as a white foam still containing traces of TBAF.

[0288] 24 Data:R f =0.44(10%MeOH in DCM): 1 H NMR (400 MHz, CDCl3)δ 9.55(s, 1H, NH), 8.45(s, 1H, NCHN(CH3)2), 8.00(s, 1H, HC(8)), 7.60-7.50(m, 2H, H-arom), 7. 49-7.39(m, 4H, H-arom), 7.31-7.23(m, 2H, H-arom), 7.21-7.12(m, 1H, H-arom), 6.81(d, J=8.5 Hz, 4H, H-arom), 5.93(dd, J=7.5, 6.1 Hz, 1H, HC(1')), 4.26(dt, J=11.1, 5.8 Hz, 1H, HC(5')), 4.07-3.98(m, 1H, HC(4')), 3.91(d, J=4.3 Hz, 1H, HC(7')), 3.77(s, 6H, MeO), 3.14(s, 3H, NCHN(CH3)2), 3.04(s, 3H, NCHN(CH3)2), 2.73(ddd, J=13.3, 10.1, 6.0 Hz. 13C NMR (101 MHz, CDCl3)δ 158.52(MeO-C-arom), 158.12(C(2)), 157.88(C(6)), 156.65(NCHN(CH3)2), 149.78(C(4)), 145.69, 1 37.02, 136.99(C-arom), 136.07(C(8)), 130.26, 128.26, 127.82, 126.74(CH-arom), 120.53(C(5)), 11 3.12(CH-arom), 86.81(C(Ph)3), 85.35(C(1')), 82.64(C(4')), 74.61(C(7')), 74.48(C(5')), 55.23 (MeO-DMTr), 49.63(C(3')), 41.37(NCHN(CH3)2), 38.55(C(6')), 38.23(C(2')), 35.14(NCHN(CH3)2). C 36 H 39 O6N6([M+H] + ) ESI + -HRMS m / z calculated 651.2926, found 651.2912.

[0289] (3'R,5'R,7'R)-N 2 -(N,N-Dimethylformamidino)-9-{7'-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}guanine (25) [ka] To a solution of nucleoside 24 (143 mg, 0.220 mmol) and 5-(ethylthio)-1H-tetrazole (43 mg, 0.33 mmol) in dry DCM (10 mL) was added 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (0.12 mL, 0.38 mmol) dropwise at room temperature. After stirring for 50 min, the reaction mixture was diluted with saturated NaHCO3 (20 mL) and extracted with DCM (3 × 20 mL). The combined organic phases were dried over MgSO4, filtered, and evaporated. The crude product was purified by CC (3.5% MeOH in DCM + 0.5% Et3N) to give 25 (130 mg, mixture of two isomers, 69%) as a white foam.

[0290] 25 Data:R f =0.60(10%MeOH in DCM): 1H NMR(300 MHz、CDCl3)δ 9.54、9.47(2s、1H、NH)、8.54、8.52(2s、1H、NCHN(CH3)2)、8.02.8.00(2s、1H、H-C(8))、7.58-7.49(m、2H、H-arom)、7.46-7.36(m、4H、H-arom)、7.25(dd、J=11.0、3.5 Hz、2H、H-arom)、7.21-7.13(m、1H、H-arom)、6.80(dd、J=8.8、2.2 Hz、4H、H-arom)、6.00-5.82(m、1H、H-C(1’))、4.16(dd、J=10.7、5.4 Hz、1H、H-C(5’))、4.00-3.82(m、2H、H-C(4’)、H-C(7’))、3.77、3.77(2s、6H、MeO)、3.62(dt、J=12.2、6.1 Hz、2H、OCH2CH2CN)、3.51-3.33(m、2H、(Me2CH)2N)、3.15、3.14(2s、3H、NCHN(CH3)2)、3.07(s、3H、NCHN(CH3)2)、2.85-2.61(m、2H、C(2’)、C(3’))、2.59-2.44(m、2H、OCH2CH2CN)、2.00-1.79(m、2H、H-(C2’)、H-C(6’))、1.53-1.26(m、1H、H-C(6’))、1.10、1.01(2t、J=6.4 Hz、12H、(Me2CH)2N). 13 C NMR(101 MHz、CDCl3)δ 158.50(MeO-C-arom)、158.04、158.00(C(2))、157.93(C(6))、156.61、156.60(NCHN(CH3)2)、149.73、149.72(C(4))、145.62、145.62、136.97、136.94(C-arom)、136.14(C(8))、130.27、130.24、130.22、128.26、127.81、126.73(CH-arom)、120.81、120.76(C(5))、117.67、117.56(OCH2CH2CN)、113.10(CH-arom)、86.88、86.85(C(Ph)3)、85.58、85.37(C(1’))、82.41、82.07(C(4’))、77.08、76.01(J C、P=37.0, 15.1 Hz, C(7')), 74.52, 74.46(C(5')), 58.19, 57.74(J C、P =18.9, 19.0 Hz OCH2CH2CN), 55.25, 55.21(MeO-DMTr), 49.10, 48.83(J C、P =2.2, 4.8 Hz, C(3')), 43.12, 43.00((Me2CH)2N), 41.34, 41.33(NCHN(CH3)2), 38.48, 38.41(C(2')), 37.23, 36.92(J C、P =5.7, 3.3 Hz C(6')), 35.17((Me2CH)2N), 24.56, 24.53, 24.48, 24.47, 24.43, 25.36, 24.35(7s, Me2CH)2N), 20.39, 20.28(J C、P =7.1, 6.9 Hz, OCH2CH2CN). 31 P NMR(122 MHz, CDCl3)δ 147.69, 146.37. C 45 H 56 O7N8P([M+H] + ) ESI + -HRMS m / z calculated 851.4004, found 851.4018.

[0291] (3'S,5'R,7'R)-1-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-β-D-ribofuranosyl}uracil (26) [ka] To a solution of sugar 6 (669 mg, 1.62 mmol) in dry DCM (13 mL) is added 2,6-lutidine (0.94 mL, 8.10 mmol) at 0 °C. After stirring at 0 °C for 20 min, TMSOTf (0.89 mL, 4.86 mmol) is added dropwise, and the solution is then warmed to room temperature and stirred for an additional 3 h. The reaction is then quenched by adding saturated NaHCO (20 mL). The organic phase is separated, and the aqueous phase is further extracted with DCM (2 × 20 mL). The combined organic phases are dried over MgSO, filtered, and evaporated.

[0292] The crude product is dissolved in dry DCM (12 mL), and then uracil (545 mg, 4.86 mmol) and BSA (1.8 mL, 7.29 mmol) are added at room temperature. After stirring for 60 min at room temperature, the resulting fine suspension is cooled to 0 °C, and N-iodosuccinimide (578 mg, 2.52 mmol) is added. After stirring for 30 min at 0 °C and 4 h at room temperature, the reaction mixture is diluted with EtOAc (50 mL) and subsequently washed with 10% aqueous Na2S2O3 (30 mL) and saturated NaHCO3 (30 mL). The aqueous phases are combined and extracted with DCM (2 × 20 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated.

[0293] The crude product is dissolved in dry toluene (15 mL), and then Bu3SnH (0.65 mL, 2.43 mmol) and azoisobutyronitrile (AIBN, 13 mg, 0.081 mmol) are added at room temperature. After heating at 95 °C for 2 h, the mixture is cooled to room temperature, and MeOH (7 mL) and HCl (1 M in water, 1.6 mL, 1.6 mmol) are added. The solution is stirred for an additional 15 min, then diluted with saturated NaHCO3 (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (EtOAc / hexane 4:1) to give 26 (490 mg, 61% over three steps) as a white foam.

[0294] 26 Data:R f =0.15 (EtOAc / hexane 2:1): 1H NMR (300 MHz, CDCl3)δ 9.95(br, 1H, HN(3)), 7.69(d, J=6.4 Hz, 4H, H-arom), 7.54-7.39(m, 7H, HC(6), H-arom), 5.98(dd, J=9.3, 5.6 Hz, 1H, HC(1')), 5.71(d, J=8.1 Hz, 1H, HC(5)), 4.51(dd, J=13.7, 6.3 Hz, 2H, HC(4'), HC(5')), 4.14(br, 1H, HC(7')), 3.25(br, 1H, OH), 2.74(dd, J=17.1, 8.7 Hz, 1H, HC(3')), 2.26-1.87(m, 3H, HC(2'), HC(6')), 1.49-1.19(m, 1H, HC(2')), 1.12(s, 9H, (CH3)3-C-Si). 13 C NMR (75 MHz, CDCl3)δ 163.65(C(4)), 150.46(C(2)), 139.85(C(6)), 135.69, 135.66(CH-arom), 133 .71, 133.42(C-arom), 129.98, 129.93, 127.85, 127.81(CH-arom), 102.84(C( 5)), 86.17(C(1')), 81.83(C(4')), 76.94(C(7')), 72.45(C(5')), 50.09(C(3') ')), 40.93(C(6')), 35.83(C(2')), 26.91(CH3)3-C-Si), 19.03(CH3)3-C-Si). C 27 H 32 O5N2NaSi([M+Na] + ) ESI + -HRMS m / z calculated 515.1973, found 515.1963.

[0295] (3'S,5'R,7'R)-1-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}uracil (27) [ka] To a solution of nucleoside 26 (438 mg, 0.889 mmol) in dry pyridine (7 mL) is added DMTr-Cl (1.20 g, 3.55 mmol) at room temperature. The solution is stirred at room temperature for 1 day, then diluted with saturated NaHCO (30 mL) and extracted with DCM (3 × 40 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (1.5% MeOH in DCM + 0.5% EtN) to give 27 (601 mg, 80%) as a yellow foam.

[0296] 27 Data:R f =0.48 (EtOAc / hexane 2:1): 1 H NMR (300 MHz, CDCl3)δ 9.26(br, 1H, HN(3)), 7.84(d, J=8.1 Hz, 1H, HC(6)), 7.40-7.08(m, 19H, H-arom), 6.69(dd, J=8.8, 4.9 Hz, 4H, H-arom), 5.70(dd, J=7.8, 5.8 Hz, 1H, HC(1')), 5.49(dd, J=8.1, 1.5 Hz, 1H, HC(5)), 4.24-4.11(m, 1H, HC(5')), 4.05-3.95(m, 1H, HC(4')), 3.65(d, J=1.7 Hz, 6H, MeO), 3.62(d, J=3.0 Hz, 1H, HC(7')), 2.41(dd, J=17.2, 8.5 Hz, 1H, HC(3')), 2.24(ddd, J=13.5, 10.2, 5.7 Hz. 13C NMR (75 MHz, CDCl3)δ 163.58(C(4)), 158.66(MeO-C-arom), 150.38(C(2)), 145.61(C-arom), 139.92(C(6)), 136.71, 136.56(C-arom), 13 5.61, 135.55 (CH-arom), 133.55, 133.41 (C-arom), 130.30, 129.92, 129.84, 128.16, 127.90, 127.74, 127.67, 126.9 0, 113.19, 113.15(CH-arom), 102.12(C(5)), 87.41(C(Ph)3), 86.80(C(1')), 82.32(C4')), 75.54(C(7')), 74.41(C (5')), 55.23(MeO-DMTr), 50.05(C(3')), 38.49(C(6')), 37.53(C(2')), 26.81(CH3)3-C-Si), 18.99(CH3)3-C-Si). C 48 H 50 O7N2NaSi([M+Na] + ) ESI + -HRMS m / z calculated 817.3279, found 817.3286.

[0297] (3'S,5'R,7'R)-1-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}cytosine (28) [ka] To a suspension of 1,2,4-triazole (1.83 g, 26.5 mmol) in dry MeCN (70 mL) was added POCl (0.57 mL, 6.05 mmol) at 0 °C, followed by EtN (4.2 mL, 30.2 mmol). The suspension was stirred at 0 °C for 30 min, and then a solution of nucleoside 27 (601 mg, 0.756 mmol) in dry MeCN (4 mL) was added at 0 °C. After stirring at room temperature for 4 h, the reaction was quenched by the addition of saturated NaHCO (20 mL), the MeCN was removed under reduced pressure, and the resulting mixture was diluted with saturated NaHCO (30 mL) and extracted with DCM (3 × 60 mL). The combined organic phase was dried over MgSO, filtered, and evaporated.

[0298] The crude product is then dissolved in a mixture of 1,4-dioxane (18 mL) and concentrated NH4OH (18 mL). After stirring at room temperature for 3 h, the mixture is reduced to half its volume in vacuo, diluted with saturated NaHCO3 (30 mL), and extracted with DCM (3 x 30 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (5% MeOH in DCM + 0.5% Et3N) to give 28 (520 mg, 87%) as a white foam.

[0299] 28 Data:R f =0.41(10%MeOH in DCM): 1H NMR(300 MHz、CDCl3)δ 7.96(d、J=7.4 Hz、1H、HC(6))、7.45(d、J=7.4 Hz、2H、H-arom)、7.38-7.08(m、17H、H-arom)、6.73(dd、J=8.7、4.7 Hz、4H、H-arom)、5.73(t、J=8.6 Hz 2H HC(5) HC(1')) 4.32-4.16(m 1H HC(5')) 4.03(t J=5.6 Hz 1H HC(4')) 3.66(d J=0.9 Hz、6H、MeO)、3.61(d、J=2.9 Hz Hz, 1H, HC(6')), 0.92-0.75 (m, 10H, HC(2'), (CH3)3-C-Si). 13 C NMR(75 MHz、CDCl3)δ 165.78(C(4)), 158.59(MeO-C-arom), 155.94(C(2)), 145.88(C-arom), 140.68(C(6)), 136.93, 136.78(C-arom),13 5.59, 135.53(CH-arom), 133.60, 133.54(C-arom), 130.31, 129.86, 129.77, 128.15, 127.88, 127.71, 127.64, 126.7 9, 113.18, 113.14(CH-arom), 94.53(C(5)), 87.55(C(Ph)3), 87.22(C(1')), 82.23(C(4')), 75.76(C(7')), 74.68(C (5')) 55.21(MeO-DMTr) 50.18(C(3')) 38.25(C(6')) 38.08(C(2')) 26.83(CH3)3-C-Si) 19.00(CH3)3-C-Si). C 48 H 52 O6N3Si([M+H] + )についてのESI + -HRMS m / z registration number 794.3620.

[0300] (3'S,5'R,7'R)-N 4 -Benzoyl-1-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}cytosine (29) [ka] To a solution of nucleoside 28 (519 mg, 0.653 mmol) in dry DMF (15 mL) is added EtN (110 μL, 0.784 mmol) at room temperature, followed by BzO (370 mg, 1633 mmol), and the solution is stirred overnight. The solution is then quenched by careful addition of saturated NaHCO (60 mL) and extracted with DCM (3 × 70 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (hexane / EtOAc 2:3, +0.5% EtN) to give 29 (580 mg, 99%) as a white foam.

[0301] 29 Data:R f =0.51(EtOAc): 1 H NMR (300 MHz, CDCl3)δ 8.61(d, J=7.4 Hz, 1H, HC(6)), 7.81(d, J=7.5 Hz, 2H, H-arom), 7.49-7.13(m, 24H, H-arom, HC(5)), 6.77(dd, J=8.5, 4.4 Hz, 4H, H-arom), 5.73(t, J=6.4 Hz, 1H, HC(1')), 4.39-4.20(m, 1H, HC(5')), 4.05(t, J=6.1 Hz, 1H, HC(4')), 3.70(s, 6H, MeO), 3.63(d, J=2.3 Hz, 1H, HC(7')), 2.72-2.55(m, 1H, HC(2')), 2.48(dd, J=16.0, 8.4 Hz, 1H, HC(3')), 1.42-1.29(m, 1H, HC(6')), 1.19-1.11(m, 1H, HC(6')), 1.07-0.96(m, 1H, HC(2')), 0.85(s, 9H, (CH3)3-C-Si). 13 C NMR (75 MHz, CDCl3)δ 166.64(CONH), 162.25(C(4)), 158.70(MeO-C-arom), 154.84(C(2)), 145.71(C-arom), 144.84(C(6)), 136.74, 136.67(C-arom) , 135.59, 135.51 (CH-arom), 133.52, 133.42, 133.24 (C-arom), 133.11, 130.30, 129.92, 129.85, 129.02, 128.12, 127.97, 127.7 6, 127.68, 127.61, 126.94, 113.25, 113.22(CH-arom), 96.22(C(5)), 89.07(C(Ph)3), 87.53(C(1')), 83.46(C(4')), 75.59(C(7) ')), 74.71(C(5')), 55.24(MeO-DMTr), 50.35(C(3')), 38.61(C(6')), 38.15(C(2')), 26.82(CH3)3-C-Si), 19.00(CH3)3-C-Si). C 55 H 56 O7N3Si([M+H] + ) ESI + -HRMS m / z calculated 898.3882, found 898.3898.

[0302] (3'S,5'R,7'R)-N 4- Benzoyl-1-{-2',3'-dideoxy-3',5'-ethano-7'-hydroxy-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}cytosine (30) [ka] To a solution of 29 (580 mg, 0.648 mmol) in dry THF (14 mL) is added TBAF (1 M in THF, 3.25 mL, 3.25 mmol) at room temperature. The solution is stirred for 1 day, then diluted with saturated NaHCO3 (50 mL) and extracted with DCM (3 x 40 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (3% MeOH in DCM + 0.5% Et3N) to give 30 (366 mg, 85%) as a white foam.

[0303] 30 Data:R f =0.31(5%MeOH in DCM): 1 H NMR (300 MHz, CDCl3)δ 8.90(br, 1H, NH), 8.73(d, J=7.5 Hz, 1H, HC(6)), 7.82(d, J=7.3 Hz, 2H, H-arom), 7.55-7.31(m, 10H, H-arom, HC(5)), 7.28-7.09(m, 3H, H-arom), 6.76(dd, J=8.8, 1.7 Hz, 4H, H-arom), 5.73(t, J=6.3 Hz, 1H, HC(1')), 4.28-4.13(m, 1H, HC(5')), 3.83(t, J=6.0 Hz, 1H, HC(4')), 3.75(d, J=3.6 Hz, 1H, HC(7')), 3.70(s, 6H, MeO), 2.86(d, J=14.7 Hz, 1H, HC-(2')), 2.54(dd, J=17.4, 7.4 Hz, 1H, HC(3')), 1.68-1.55(m, 1H, HC(6')), 1.45-1.13(m, 3H, HC(2'), HC(6'), OH). 13C NMR (75 MHz, CDCl3)δ 166.63(CONH), 162.34(C(4)), 158.65(MeO-C-arom), 155.00(C(2)), 145.62(C-arom), 145 .11(C(6)), 136.72, 136.64, 133.16(C-arom), 130.25, 129.02, 128.12, 127.93, 127.61, 126 .95, 113.20(CH-arom), 96.24(C(5)), 89.20(C(Ph)3), 87.48(C(1')), 83.40(C(4')), 74.5 0, (C(5'))73.90(C(7')), 55.25(MeO-DMTr), 50.05(C(3')), 38.90(C(6')), 38.40(C(2')). C 39 H 38 O7N3([M+H] + ) ESI + -HRMS m / z calculated 660.2704, found 660.2707.

[0304] (3'S,5'R,7'R)-N 4 -Benzoyl-1-{7'-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}cytosine (31) [ka] To a solution of nucleoside 30 (67 mg, 0.101 mmol) and 5-(ethylthio)-1H-tetrazole (22 mg, 0.17 mmol) in dry DCM (3 mL) was added dropwise 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (65 μL, 0.20 mmol) at room temperature. After stirring for 40 min, the reaction mixture was diluted with DCM (20 mL) and washed with saturated NaHCO3 (2 × 15 mL) and saturated NaCl (15 mL). The combined aqueous phases were extracted with DCM (20 mL). The combined organic phases were dried over MgSO4, filtered, and evaporated. The crude product was purified by CC (EtOAc, +0.5% Et3N) to give 31 (75 mg, mixture of two isomers, 86%) as a white foam.

[0305] 31 Data:R f =0.67(4%MeOH in DCM): 1 H NMR (300 MHz, CDCl3)δ 8.88(s, 1H, NH), 8.79(d, J=7.5 Hz, 1H, HC(6)), 7.93(d, J=7.5 Hz, 2H, H-arom), 7.67-7.40(m, 10H, H-arom, HC(5)), 7.39-7.22(m, 3H, H-arom ), 6.93-6.79(m, 4H, H-arom), 5.97-5.77(m, 1H, HC(1')), 4.22(dt, J=14.5, 5.6 Hz, 1H, H-(5')), 3.98-3.84(m, 2H, HC(4'), HC(7')), 3.82(s, 6H, MeO), 3.66(ddd, J=16.8, 13.5, 6.7 Hz, 2H, OCH2CH2CN), 3.53-3.37(m, 2H, (Me2CH)2N), 3.14-2.93(m, 1H, HC(2')), 2.84-2.66(m, 1H, HC(3')), 2.53(dt, J=12.4, 6.3 Hz, 2H, OCH2CH2CN), 1.83-1.56(m, 2H, HC(6')), 1.46(td, J=14.1, 7.0 Hz, 1H, HC(2')), 1.18-0.97(m, 12H, (Me2CH)2N). 13C NMR(75 MHz、CDCl3)δ 166.70(CONH) 162.32 162.28(C(4)) 158.68(MeO-C-arom) 154.93(C(2)) 145.53(C-arom)1 44.95, 144.89(C(6)), 136.69, 136.63, 136.56, 136.52, 133.24(C-arom), 133.10, 130.24, 130.2 0、129.01、128.10、127.94、127.60、126.96(CH-arom)、117.53(OCH2CH2CN)、113.20(CH-arom)9 6.24(C(5)), 89.15, 89.10(C(Ph)3), 87.55, 87.54(C(1')), 83.11, 83.04(C(4')), 75.93, 75.37(J C、P =16.7、15.5 Hz、C(7'))、74.48(C(5'))、58.25、57.99(J C、P =17.9、18.1 Hz OCH2CH2CN)、55.27、55.24(MeO-DMTr)、49.27、49.03(J C、P =3.1、4.8 Hz、C(3'))、43.15、42.98((Me2CH)2N)、38.89、38.80(C(2'))、37.44、37.24(J C、P =5.2、3.2 Hz、C(6'))、24.58、24.54、24.48、24.45、24.35(5s、Me2CH)2N)、20.33、20.24(J C、P =5.8, 5.7 Hz, OCH2CH2CN). 31 P NMR(121 MHz, CDCl3)δ 147.19, 146.94. C 48 H 55 O8N5P([M+H] + )についてのESI + -HRMS m / z registration number 860.3783.

[0306] (3'S,5'R,7'R)-1-{2',3'-dideoxy-3',5'-ethano-7'-O-(4-nitrobenzoyl)-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}thymine (32) [ka] To a solution of nucleoside 11 (100 mg, 0.175 mmol) and 4-dimethylaminopyridine (26 mg, 0.21 mmol) in dry DCM (8 mL) was added 4-nitrobenzoyl chloride (59 mg, 0.315 mmol) at room temperature. After stirring for 6 h, the reaction was quenched by adding saturated NaHCO3 (5 mL). The mixture was then diluted with saturated NaHCO3 (15 mL) and extracted with DCM (3 × 15 mL). The combined organic phases were dried over MgSO4, filtered, and evaporated. The crude product was purified by CC (2.5% MeOH in DCM + 0.5% Et3N) to give 32 (98 mg, 78%) as a white foam containing traces of Et3N.

[0307] 32 Data:R f =0.42(5%MeOH in DCM): 1H NMR(300 MHz、CDCl3)δ 8.26(t、J=7.3 Hz、3H、H-arom.HN(3))、8.00(d、J=8.9 Hz、2H、H-arom)、7.72(d、J=1.0 Hz、1H、HC(6))、7.55(d、J=6.9 Hz、2H、H-arom)、7.44(dd、J=8.8、6.6 Hz、4H、H-arom)、7.35-7.18(m、3H、H-arom)、6.83(dd、J=9.0、2.6 Hz、4H、H-arom)、6.01(dd、J=8.2、5.2 Hz、1H、HC(1'))、4.96(d、J=3.3 Hz, 1H, HC(7')), 4.33-4.24(m, 1H, HC(4')), 4.24-4.13(m, 1H, HC(5')), 3.78 (d, J=0.9 Hz、6H、MeO) 2.92-2.72(m、2H、HC(3')、HC(2')) 1.81(d、J=0.6 Hz、3H、Me-C(5)) 1.79-1.62(m、2H、HC(6')) 1.22(d、J=5.9 Hz、1H、HC(2')). 13 C NMR(75 MHz、CDCl3)δ 164.05, 163.84(C(4), CO2R), 158.81(MeO-C-arom), 150.64, 150.52(O2N-C-arom, C(2)), 145.29,13 6.43, 136.34(C-arom), 135.18(C(6)), 130.62, 130.20, 130.17, 128.16, 128.01, 127.15, 123.58,11 3.30, 113.27(C-arom), 111.17(C(5)), 87.53(C(Ph)3), 86.29(C(1')), 81.59(C(4')), 78.65(C(7') ) 74.16(C(5')) 55.26(MeO-DMTr) 47.07(C(3')) 37.35(C(2')) 35.71(C(6')) 12.51(Me-C(5)). C40H 37 O 10 N3Na([M+Na] + )についてのESI + -HRMS m / z registration number 742.2371

[0308] (3'S,5'R,7'R)-1-{2',3'-dideoxy-3',5'-ethano-7'-O-(4-nitrobenzoyl)-β-D-ribofuranosyl}thymine (33) [ka] To a solution of 32 (60 mg, 0.083 mmol) in a mixture of dry DCM (1 mL) and MeOH (0.4 mL) is added dropwise dichloroacetic acid (0.2 mL) at room temperature. After stirring for 3 h, the mixture is diluted with saturated NaHCO (15 mL) and extracted with DCM (3 × 10 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (5% MeOH in DCM) to give 33 (29 mg, 84%) as a white foam. Crystals suitable for X-ray analysis are obtained by recrystallization in a mixture of HO / MeOH.

[0309] 33 Data:R f =0.18(5%MeOH in DCM): 1 H NMR (400 MHz, DMSO) δ 11.33(s, 1H, HN(3)), 8.34(d, J=8.8 Hz, 2H, H-arom), 8.27-8.13(m, 2H, H-arom), 7.78(s, 1H, HC(6)), 5.96(dd, J=9.3, 5.6 Hz, 1H, HC(1')), 5.18(t, J=3.8 Hz, 1H, HC(7')), 5.12(d, J=6.0 Hz, 1H, OH), 4.33(dd, J=7.3, 4.7 Hz, 1H, HC(4')), 4.27(td, J=10.5, 5.5 Hz, 1H, HC(5')), 2.90(dd, J=17.2, 8.5 Hz, 1H, HC(3')), 2.58-2.46(m, 1H, HC(2')), 2.30(ddd, J=13.8, 8.8, 5.3 Hz, 1H, HC(6')), 2.03(dd, J=9.6, 4.2 Hz, 1H, HC(6')), 1.92-1.76(m, 4H, HC(2'), Me-C(5)). 13C NMR (101 MHz, DMSO)δ 164.33, 164.23(C(4), CO2R), 150.91, 150.75(O2N-C-arom, C(2)), 136.79(C-arom), 135.69(C(6)), 131.20, 124.32(CH-arom), 1 09.89(C(5)), 85.31(C(1')), 81.48(C(4')), 80.07(C(7')), 71.72(C(5')), 47.18(C(3')), 37.77(C(6')), 35.48(C(2')), 12.66 12.58(Me-C(5)). C 19 H 20 O8N3([M+H] + ) ESI + -HRMS m / z calculated 418.1245, found 418.1242.

[0310] (3'R,5'R,7'R)-1-{5'-O-acetyl-7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α,β-D-ribofuranosyl}thymine (35) [ka] To a solution of sugar 7 (933 mg, 2.05 mmol) and thymine (372 mg, 3.08 mmol) in dry MeCN (12 mL) is added dropwise BSA (1.5 mL, 6.15 mmol) at room temperature. After stirring for 50 min at room temperature, the solution is cooled to 0 °C and TMSOTf (0.45 mL, 2.5 mmol) is added dropwise. After stirring for an additional 3 h at 0 °C and 15 h at room temperature, the reaction mixture is diluted with saturated NaHCO (100 mL) and extracted with DCM (4 × 40 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (2.5% isopropanol in DCM) to obtain the anomeric ratio α / β. [ka] An 85:15 mixture of 35 (924 mg, 82%) is obtained as a white foam.

[0311] 35 degrees:R f =0.56(DCMin 7%MeOH): 1 H NMR(300 MHz, CDCl3)δ 9.14(br, 1H, HN(3)) 7.53 (dd, J=7.7, 1.6 Hz、4H、H-arom)、7.39-7.23(m、6H、H-arom)、7.09(d、J=1.0 Hz、0.15 H、HC(6))、6.87(d、J=1.0 Hz、0.85 H, HC(6)), 5.83 (t, J=6.2 Hz, 0.85 H, HC(1')), 5.80-5.70(m, 0.15H, HC(1')), 5.36-5.04(m, 1H, HC(5')), 4.89 (dd, J=6.3, 5.2 Hz, 1H, HC(4')), 4.62(dd, J=7.1, 5.6 Hz 0.15H HC(4′)) 4.01–3.85(m 1H HC(7′)) 2.76–2.55(m 1H HC(3′)) 2.09–1.91(m 4). H, HC(6′), MeCO2), 1.90-1.58(m, 6H, HC(6′), HC(2′), Me-C(5)), 0.96(s, 9H, (CH3)3-C-Si). 13 C NMR(75 MHz、CDCl3)δ 170.70(MeCO2), 163.87(C(4)), 150.29(C(2)), 135.69, 135.67(CH-arom), 134.99(C( 6))、133.58、133.18(C-arom)、130.03、127.87(CH-arom)、111.05(C(5))、87.56(C(1') )、82.85(C(4'))、76.50(C(7'))、74.76(C(5'))、50.72(C(3'))、37.79(C(6'))、36.94( C(2')) 26.88((CH3)3-C-Si) 20.95(MeCO2) 19.01((CH3)3-C-Si) 12.63(Me-C(5)). C 30 H 37 O6N2Si([M+H] + )についてのESI + -HRMS m / z registration number 549.2415.

[0312] (3'S,5'R,7'R)-1-{5'-O-acetyl-2',3'-dideoxy-3',5'-ethano-7'-hydroxy-α,β-D-ribofuranosyl}thymine (36) [ka] To a solution of nucleoside 35 (924 mg, 1.68 mmol) in dry THF (10 mL) is added TBAF (1 M in THF, 3.4 mL, 3.4 mmol) at room temperature. After stirring at room temperature for 2 h, the reaction mixture is diluted with saturated NaHCO3 (80 mL) and extracted with EtOAc (3 × 80 mL) and DCM (2 × 80 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (5% MeOH in DCM) to give the anomeric mixture of 36 (391 mg, 75%).

[0313] 36 Data:R f =0.24(7%MeOH in DCM): 1H NMR(400 MHz、CDCl3)δ 9.66(br、0.15H、HN(3)) 9.63(br、0.85H、HN(3)) 7.27(d、J=1.0 Hz、0.15H、HC(6))、7.06(d、J=1.0 Hz、0.85H、HC(6))、6.00(t、J=6.1 Hz、0.85H、HC(1'))、5.91(dd、J=8.8、5.5 Hz, 0.15H, HC(1')), 5.26-5.10(m, 1H, HC(5')), 4.92(dd, J=6.5, 5.3 Hz, 0.85H, HC(4')), 4.65(dd, J=6.9, 5.7 Hz 0.15H HC(4')) 4.19-4.03(m 1H HC(7')) 2.91-2.72(m 2H HC(3') OH) 2.64(ddd J=13.3 9.8 5.5 Hz, 0.15H, HC(2')), 2.25-2.15(m, 1.70H, HC(2')), 2.05(s, 0.45H, MeC O2) 2.04(s 2.55H MeCO2) 2.03-1.89(m 2H HC(6')) 1.88(d J=0.7). Hz, 0.45H, Me-C(5)), 1.85(d, J=0.6 Hz, 2.55H, Me-C(5)), 1.42-1.28(m, 0.15H, HC(2')). 13 C NMR(101 MHz、CDCl3)δ 170.87(MeCO2), 164.26(C(4)), 150.66(C(2)), 135.54(C(6)), 111.22(C(5)), 87.97(C(1')), 82.97(C(4')) 75.08(C(7′)), 74.52(C(5′)), 50.07(C(3′)), 37.81(C(2′)), 37.23(C(6′)), 21.02(MeCO2), 12.67(Me-C(5)). C 14 H 19 O6N2([M+H] + )についてのESI + -HRMS m / z registration number 311.1238.

[0314] (3'S,5'R,7'R)-1-{5'-O-acetyl-2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α,β-D-ribofuranosyl}thymine (37) [ka] To a solution of nucleoside 36 (364 mg, 1.17 mmol) in dry pyridine (7 mL) is added DMTr-Cl (1.19 g, 3.51 mmol) at room temperature. The solution is stirred for 1 day, then diluted with saturated NaHCO (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (2:1 EtOAc / hexanes + 0.5% EtN) to give 37 (690 mg, 96%) as a yellow foam containing the anomeric mixture.

[0315] 37 Data:R f =0.70(8%MeOH in DCM): 1H NMR(300 MHz、CDCl3)δ 9.17(br、0.85H、HN(3))、8.56(br、0.15H、HN(3))、7.38-7.3 2(m、2H、H-arom)、7.29-7.15(m、7H、H-arom)、6.82(d、J=1.1 Hz Hz, 0.15H, HC(1')), 5.25(dd, J=10.2, 5.6 Hz, 0.15H, HC(5')), 5.21-5.11(m, 0.85H, H-(C5')), 4.78(dd, J=6.7, 4.8 Hz, 0.85H, HC(4')), 4.49(dd, J=7.1, 5.3 Hz 0.15H HC(4')) 3.84(br 1H HC(7')) 3 72 3.71(2s 6H MeO) 2.34-2.23(m、1H、HC(3'))、2.01、1.99(2s、3H、MeCO2)、1.82(d、J=0.5 Hz、Me-C(5))、1.80–1.56(m、4H、HC(2′)、HC(6′)). 13 C NMR(75 MHz、CDCl3)δ 170.69(MeCO2) 163.91(C(4)) 158.82(MeO-C-arom) 150.33(C(2)) 145.34 136.64 136 .58(C-arom)、135.00(C(6))、130.25、128.39、128.07、127.15、113.41(CH-arom)、111.04(C (5)) 87.70(C(Ph)3) 87.31(C(1')) 83.15(C(4')) 77.16(C(7')) 74.96(C(5')) 55.37( MeO-DMTr) 49.12(C(3')) 37.55(C(2')) 36.82(C(6')) 21.07(MeCO2) 12.66(Me-C(5)). C 35 H 36 O8N2([M+H] + )についてのESI + -HRMS m / z registration number 612.2466.

[0316] (3'S,5'R,7'R)-1-{2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}thymine (38) [ka] To a solution of nucleoside 37 (690 mg, 1.12 mmol) in dry MeOH (10 mL) was added KCO (467 mg, 3.36 mmol) at room temperature. The solution was stirred for 3 h, then diluted with saturated NaCl (60 mL) and extracted with DCM (3 × 60 mL). The combined organic phases were dried over MgSO, filtered, and evaporated. The crude product was purified by CC (3% isopropanol in EtO + 0.5% EtN) to give the α-anomer 38 (550 mg, 86%) as a white solid.

[0317] 38 Data:R f =0.39(5%MeOH in DCM): 1 H NMR (400 MHz, CDCl3) δ 9.37 (br, s, 1H, HN(3)), 7.39-7.31 (m, 2H, H-arom), 7.25 (d, J=8.3 Hz, 4H, H-arom), 7.20 (t, J=7.7 Hz, 2H, H-arom), 7.16-7.08(m, 1H, H-arom), 6.78(d, J=1.1 Hz, 1H, HC(6)), 6.74(d, J=8.8 Hz, 4H, H-arom), 5.91(dd, J=6.5, 4.9 Hz, 1H, HC(1')), 4.57(dd, J=7.2, 4.4 Hz, 1H, HC(4')), 4.35-4.18(m, 1H, HC(5')), 3.86(d, J=4.7 Hz, 1H, HC(7')), 3.69(s, 6H, MeO), 2.53(br, 1H, OH), 2.22(dd, J=15.3, 6.3 Hz, 1H, HC(3')), 1.85-1.69(m, 5H, Me-C(5), HC(2'), HC(6')), 1.66-1.49(m, 2H, HC(2'), HC(6')). 13C NMR (101 MHz, CDCl3)δ 163.98(C(4)), 158.67(MeO-C-arom), 150.47(C(2)), 145.48, 136.80, 136.75(C-arom) m), 134.94(C(6)), 130.19, 130.18, 128.35, 127.97, 127.01, 113.31(CH-arom), 111.0 4(C(5)), 87.82(C(Ph)3), 87.05(C(1')), 85.74(C(4')), 78.26(C(7')), 73.33(C(5') ), 55.31(MeO-DMTr), 48.81(C(3')), 40.21(C(6')), 37.68(C(2')), 12.65(Me-C(5)). C 33 H 35 O7N2([M+H] + ) ESI + -HRMS m / z calculated 571.2439, found 571.2421.

[0318] (3'S,5'R,7'R)-1-{5'-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}thymine (39) [ka] To a solution of nucleoside 38 (200 mg, 0.350 mmol) and 5-(ethylthio)-1H-tetrazole (59 mg, 0.46 mmol) in dry DCM (7 mL) was added 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (0.17 mL, 0.53 mmol) dropwise at room temperature. After stirring for 1 h, the reaction mixture was diluted with DCM (50 mL) and washed with saturated NaHCO3 (2 × 25 mL) and saturated NaCl (25 mL). The combined aqueous phases were extracted with DCM (30 mL). The combined organic phases were dried over MgSO4, filtered, and evaporated. The crude product was purified by CC (2% MeOH in DCM + 0.5% Et3N) to give 39 (220 mg, mixture of two isomers, 81%) as a white solid.

[0319] Data of 39: R f = 0.44 (4% MeOH in DCM): 1 H NMR (300 MHz, CDCl3) δ 9.03 (br, 1H, H-N(3)), 7.36 (d, J = 8.1 Hz, 2H, H-arom), 7.30 - 7.07 (m, 7H, H-arom), 6.84 (s, 1H, H-C(6)), 6.80 - 6.69 (m, 4H, H-arom), 5.95, 5.88 (2dd, J = 6.6, 4.8 Hz, 1H, H-C(1’)), 4.70, 4.61 (2dd, J = 7.3, 4.3 Hz, 1H, H-C(4’)), 4.41 - 4.20 (m, 1H, H-C(5’)), 3.94 - 3.82 (m, 1H, H-C(7’)), 3.81 - 3.62 (m, 8H, MeO, OCH2CH2CN), 3.59 - 3.40 (m, 2H, (Me2CH)2N), 2.61 - 2.46 (m, 2H, OCH2CH2CN), 2.28 (ddd, J = 14.1, 13.2, 7.3 Hz, 1H, H-C(3’)), 1.91 - 1.73 (m, 5H, Me-C(5), H-C(6’), H-C(2’)), 1.72 - 1.46 (m, 2H, H-C(6’), H-C(2’)), 1.16 - 1.00 (m, 12H, (Me2CH)2N). 13 C NMR (75 MHz, CDCl3) δ 164.01, 163.9 (C(4)), 158.70 (MeO-C-arom), 150.39, 150.17 (C(2)), 145.52, 136.84, 136.78 (C-arom), 135.44, 135.39 (C(6)), 130.21, 128.36, 128.32, 128.00, 127.03 (CH-arom), 118.02, 117.76 (OCH2CH2CN), 113.32 (CH-arom), 110.91, 110.59 (C(5)), 88.31, 88.06 (C(Ph)3), 87.11, 87.06 (C(1’)), 85.44, 85.39 (J C、P = 4.6, 3.1 Hz, C(4’)), 78.25, 78.13 (C(7’)), 74.70, 74.34 (J C、P=13.5, 18.5 Hz, C(5')), 58.73, 58.47(J C、P =18.9, 20.1 Hz, (OCH2CH2CN)), 55.35, 55.32(MeO-DMTr), 48.80, 48.64(C(3')), 43.22, 43.06(J C、P =12.4, 11.0 Hz(Me2CH)2N), 39.68, 39.63(C(6')), 38.06, 37.93(C(2')), 24.81, 24.74, 24.71, 24.68, 24.65, 24.59(6s, Me2CH)2N), 20.37, 20.35(J C、P =7.1, 6.8 Hz, OCH2CH2CN), 12.66(Me-C(5)). 31 P NMR(122 MHz, CDCl3)δ 148.18, 147.80. C 42 H 52 O8N4P([M+H] + ) ESI + -HRMS m / z calculated 771.3517, found 771.3517.

[0320] (3'S,5'R,7'R)-N 4 -Benzoyl-1-{2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}-5-methylcytosine (40) [ka] To a solution of nucleoside 38 (268 mg, 0.470 mmol) in dry MeCN (5 mL) was added BSA (0.28 mL, 1.13 mmol) dropwise at 0 °C, and the solution was stirred overnight at room temperature. In a separate flask, a suspension of 1,2,4-triazole (1.14 g, 16.5 mmol) in dry MeCN (50 mL) was cooled to 0 °C, and POCl (0.35 mL, 3.8 mmol) was added, followed by EtN (2.62 mL, 18.8 mmol). The suspension was stirred at 0 °C for 30 min, and then the previously prepared solution of silylated compound 38 was added to the suspension, and the mixture was further stirred at room temperature for 7 h. The reaction was quenched by adding saturated NaHCO (10 mL), the MeCN was removed under reduced pressure, and the resulting mixture was diluted with saturated NaHCO (30 mL) and extracted with DCM (3 × 30 mL). The combined organic phases are dried over MgSO4, filtered and evaporated.

[0321] The crude product is then dissolved in a mixture of 1,4-dioxane (10 mL) and concentrated NH4OH (10 mL). After stirring at room temperature for 3 h, the mixture is reduced to half its volume in vacuo, diluted with saturated NaHCO3 (25 mL), and extracted with DCM (4 x 30 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated.

[0322] The crude product is then dissolved in dry DMF (10 mL). EtN (80 μL, 0.56 mmol) is added followed by BzO (266 mg, 1.18 mmol) at room temperature, and the solution is stirred overnight. The resulting brownish solution is quenched by careful addition of saturated NaHCO (40 mL) and extracted with DCM (4 × 40 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (1:1 EtOAc / hexane + 0.5% EtN) to give 40 (263 mg, 83%) as a white foam.

[0323] 40 Data:R f =0.53 (EtOAc / hexane 3:1): 11H NMR (300 MHz, CDCl3) δ 13.11 (broad, 1H, NH), 8.30 - 8.10 (m, 2H, H-arom), 7.47 - 7.29 (m, 5H, H-arom), 7.28 - 7.06 (m, 7H, H-arom), 7.00 (d, J = 0.8 Hz, 1H, H-C(6)), 6.74 (d, J = 8.6 Hz, 4H, H-arom), 5.89 (dd, J = 6.3, 4.6 Hz, 1H, H-C(1’)), 4.61 (dd, J = 7.2, 4.5 Hz, 1H, H-C(4’)), 4.33 - 4.20 (m, 1H, H-C(5’)), 3.87 (broad, 1H, H-C(7’)), 3.69 (s, 6H, MeO), 2.32 - 2.13 (m, 2H, H-C(3’), OH), 1.99 (s, 3H, Me-C(5)), 1.87 - 1.73 (m, 2H, H-C(2’), H-C(6’)), 1.66 - 1.47 (m, 2H, H-C(2’), H-C(6’)). 13 13C NMR (75 MHz, CDCl3) δ 179.61 (CONH), 159.76 (C(4)), 158.74 (MeO-C-arom), 147.87 (C(2)), 145.47 (C-arom), 137.17 (C(6)), 136.77, 136.68, 136.03 (C-arom), 132.55, 130.21, 129.98, 128.34, 128.21, 128.03, 127.07, 113.35 (CH-arom), 111.81 (C(5)), 88.74 (C(Ph)3), 87.13 (C(1’)), 86.12 (C(4’)), 78.17 (C(7’)), 73.31 (C(5’)), 55.35 (MeO-DMTr), 48.63 (C(3’)), 40.35 (C(6’)), 38.06 (C(2’)), 13.78 (Me-C(5)). C 40 H 40 O7N3([M + H] + ) for ESI + -HRMS m / z calculated value 674.2861, measured value 674.2877.

[0324] (3'S,5'R,7'R)-N4-benzoyl-1-{5'-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}-5-methylcytosine (41) [ka] To a solution of nucleoside 40 (250 mg, 0.371 mmol) and 5-(ethylthio)-1H-tetrazole (73 mg, 0.56 mmol) in dry DCM (8 mL) was added 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (0.20 mL, 0.63 mmol) dropwise at room temperature. After stirring for 30 min, the reaction mixture was diluted with DCM (30 mL) and washed with saturated NaHCO3 (2 x 20 mL) and saturated NaCl (20 mL). The combined aqueous phases were extracted with DCM (20 mL). The combined organic phases were dried over MgSO4, filtered, and evaporated. The crude product was purified by CC (1:1 EtOAc / hexanes with 0.5% Et3N) to give 41 (260 mg, mixture of two isomers, 80%) as a white foam.

[0325] 41 Data:R f =0.57 (EtOAc / hexane 1:1): 1H NMR(300 MHz、CDCl3)δ 13.26(br、1H、NH)、8.32(d、J=7.2 Hz、2H、H-arom)、7.58-7.39(m、5H、H-arom)、7.38-7.14(m、8H、H-arom、H-C(6))、6.88-6.77(m、4H、H-arom)、6.01、5.96(2dd、J=6.3、4.6 Hz、1H、H-C(1’))、4.82、4.74(2dd、J=7.3、4.3 Hz、1H、H-C(4’))、4.42(td、J=10.6、6.0 Hz、1H、H-C(5’))、3.97(br、1H、H-C(7’))、3.91-3.68(m、8H、MeO、OCH2CH2CN)、3.59(dtd、J=16.7、6.7、3.4 Hz、2H、(Me2CH)2N))、2.62(dt、J=15.5、6.4 Hz、2H、OCH2CH2CN)、2.49-2.23(m、1H、H-C(3’))、2.11、2.09(2d、J=0.5 Hz、3H、Me-C(5))、2.00-1.82(m、2H、H-C(6’)、H-C(2’))、1.82-1.55(m、2H、H-C(6’)、H-C(2’))、1.17(dd、J=16.3、6.8 Hz、12H、(Me2CH)2N). 13 C NMR(101 MHz、CDCl3)δ 179.60(CONH)、159.97(C(4))、158.76(MeO-C-arom)、147.81、147.70(C(2))、145.54(C-arom)、137.34、136.83(C(6))、136.77、136.72、136.65、136.55(C-arom)、132.45、130.22、130.20、129.96、128.34、128.31、128.18、128.00、127.04(CH-arom)、117.89、117.71(OCH2CH2CN)、113.35(CH-arom)、111.60、111.36(C(5))、89.24、89.01(C(Ph)3)、87.16、87.12(C(1’))、85.78、85.62(J C、P =4.3、3.2 Hz、C(4’))、78.20、77.98(C(7’))、74.68、74.37(J C、P=13.4, 18.2 Hz, C(5')), 58.70, 58.44(J C、P =18.5, 20.0 Hz, (OCH2CH2CN)), 55.36, 55.33(MeO-DMTr), 48.65, 48.44(C(3')), 43.27, 43.14(J C、P =12.4, 12.3 Hz(Me2CH)2N), 39.87, 39.64(J C、P =3.4, 3.7 Hz(C(6')), 38.30, 38.22(C(2')), 24.80, 24.72, 24.70, 24.67, 24.63(Me2CH)2N), 20.39, 20.37(J C、P =7.2, 6.8 Hz, OCH2CH2CN), 13.72(Me-C(5)). 31 P NMR(121 MHz, CDCl3)δ 148.18, 147.96. C 49 H 57 O8N5P([M+H] + ) ESI + -HRMS m / z calculated 874.3939, found 874.3946.

[0326] (3'R,5'R,7'R)-N 6 -Benzoyl-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α-D-ribofuranosyl}adenine (42) [ka] Nucleoside 15 (1.74 g, 2.64 mmol) was dissolved in 0.15 M NaOH in THF / MeOH / HO (5:4:1, 80 mL) at 0 °C. The reaction was stirred for 20 min and quenched by adding NH Cl (1.06 g). The solvent was then removed under reduced pressure, and the product was purified by CC (5% isopropanol in DCM) to give 42 (α-anomer, 836 mg, 51%) and 16 (β-anomer, 287 mg, 18%) as white foams.

[0327] 42 Data:R f=0.35(DCMin 5%MeOH): 1 H NMR(300 MHz、CDCl3)δ 9.34(s、1H、NH)、8.71(s、1H、HC(2))、8.02(d、J=7.4 Hz、2H、H-arom))、7.92(s、1H、HC(8))、7.68-7.58(m、4H、H-arom)、7.58-7.31(m、9H、H-arom)、6.23(dd、J=6.7、2.4 Hz、1H、HC(1'))、4.74(dd、J=6.6、4.9 Hz、1H、HC(4'))、4.49(dt、J=12.5、6.3 Hz Hz 1H HC(3')) 2.52-2.35(m 1H HC(2')) 2.10-1.97(m 1H HC(6). ')) 1.94-1.77(m 2H HC(2') HC(6')) 1.06(s 9H (CH3)3-C-Si). 13 C NMR(75 MHz、CDCl3)δ 164.98(CONH), 152.65(C(2)), 151.31(C(4)), 149.69(C(6)), 140.93(C(8)), 135.74(CH-ar om) 133.82 133.68 133.39(C-arom) 132.77 130.02 129.98 128.76 128.06 127.87 127 .85(CH-arom) 123.38(C(5)) 87.16(C(1')) 85.35(C(4')) 77.40(C(7')) 72.79(C(5')) 50.63(C(3′)), 40.86(C(6′)), 37.25(C(2′)), 26.94((CH3)3-C-Si), 19.05((CH3)3-C-Si). C 35 H 38 O4N5Si([M+H] + )についてのESI + -HRMS m / z format 620.2688.

[0328] (3'R,5'R,7'R)-N 6 -Benzoyl-9-{5'-O-acetyl-7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α-D-ribofuranosyl}adenine (43) [ka] To a solution of nucleoside 42 (1.09 g, 1.75 mmol) and 4-dimethylaminopyridine (321 mg, 2.63 mmol) in dry DCM (50 mL) is added acetic anhydride (0.83 mL, 8.8 mmol) at room temperature. After stirring overnight, the reaction is quenched by adding saturated NaHCO3 (50 mL). The phases are separated and the aqueous phase is further extracted with DCM (2 x 80 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (2.5% MeOH in DCM) to give 43 (1.04 g, 90%) as a white foam.

[0329] 43 Data:R f =0.33 (EtOAc / hexane 4:1): 1H NMR(300 MHz、CDCl3)δ 8.99(br 1H HC(2)) 8.73(s 1H HC(2)) 8.09-7.99(m 2H HC(8)) 7.98(s 1H HC(8)) 7.70-7. 58(m、5H、H-arom)、7.57-7.48(m、2H、H-arom)、7.47-7.34(m、6H、H-arom)、6.22(dd、J=6.8、3.2 Hz、1H、HC(1')) 5.45-5.35(m、1H、HC(5')) 5.01(dd、J=6.7; Hz、1H、HC(7')) 3.02(dt、J=9.5、6.5 Hz、1H、HC(3'))、2.55(ddd、J=13.5、10.0、3.2 Hz, 1H, HC(2')), 2.15(dd, J=13.2, 6.2 Hz, 1H, HC(6')), 2.09(s, 3H, MeCO2), 2.01(dt, J=8.0, 3.5 Hz 1H HC(2')) 1.88(dt J=13.6 5.3 Hz 1H HC(6')) 1.08(s 9H(CH3)3-C-Si). 13 C NMR(101 MHz、CDCl3)δ 170.61(MeCO2) 164.75(CONH) 152.67(C(2)) 151.37(C(4)) 149.64(C(6)) 141.41(C(8)) .85(CH-arom) 133.71 133.38(C-arom) 132.91 130.15 130.10 128.99 128.02 127.99 127.97 (CH-arom), 123.64(C(5)), 87.37(C(1')), 83.37(C(4')), 76.63(C(7')), 74.51(C(5')), 51.19(C (3')) 37.44(C(2')) 37.32(C(6')) 27.01((CH3)3-C-Si) 21.08(MeCO2) 19.14((CH3)3-C-Si). C 37 H 40 O5N5Si([M+H] + )についてのESI + -HRMS m / z registration number 662.2793.

[0330] (3'S,5'R,7'R)-N 6 -Benzoyl-9-{5'-O-acetyl-2',3'-dideoxy-3',5'-ethano-7'-hydroxy-α-D-ribofuranosyl}adenine (44) [ka] To a solution of nucleoside 43 (990 mg, 1.50 mmol) in dry THF (50 mL) is added TBAF (1 M in THF, 3.0 mL, 3.0 mmol) at room temperature. After stirring at room temperature for 3.5 h, the solution is diluted with EtOAc (30 mL) and the THF is removed under reduced pressure. The mixture is then diluted with saturated NaHCO3 (50 mL) and extracted with DCM (4 x 50 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (6% MeOH in DCM) to give 44 (570 mg, 90%) as a white foam containing traces of TBAF.

[0331] 44 Data:R f =0.33(10%MeOH in DCM): 1 H NMR (400 MHz, CDCl3)δ 9.60(br, 1H, NH), 8.67(s, 1H, HC(2)), 8.09(s, 1H, HC(8)), 7.96(d, J=7.4 Hz, 2H, H-arom), 7.51(t, J=7.4 Hz, 1H, H-arom), 7.42(t, J=7.5 Hz, 2H, H-arom), 6.33(dd, J=6.7, 3.1 Hz, 1H, HC(1')), 5.25(ddd, J=9.7, 6.4, 5.3 Hz, 1H, HC(5')), 4.92(dd, J=6.4, 5.4 Hz, 1H, HC(1')), 4.14(br, 2H, HC(7'), OH), 3.06(dd, J=16.0, 6.6 Hz, 1H, HC(3')), 2.87(ddd, J=13.2, 9.9, 3.0 Hz, 1H, HC(2')), 2.26-2.17(m, 1H, H-C2')), 2.10-1.98(m, 5H, HC(6'), MeCO2). 13C NMR (75 MHz, CDCl3)δ 170.64(MeCO2), 165.27(CONH), 152.49(C(2)), 151.26(C(4)), 149.58(C(6)), 141.64(C(8)), 133.60(C-arom), 132.82, 128.76, 128.06(CH-ar om), 123.30(C(5)), 87.30(C(1')), 83.17(C(4')), 74.67(C(7')), 74.2 0(C(5')), 50.41(C(3')), 37.43(C(2')), 36.92(C(6')), 20.96(MeCO2). C 21 H 22 O5N5([M+H] + ) ESI + -HRMS m / z calculated 424.1615, found 424.1623.

[0332] (3'S,5'R,7'R)-N 6 -Benzoyl-9-{5'-O-acetyl-2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}adenine (45) [ka] To a solution of nucleoside 44 (570 mg, 1.35 mmol) in dry pyridine (16 mL) is added DMTr-Cl (1.37 g, 4.04 mmol) at room temperature. The solution is stirred for 1 day, then diluted with saturated NaHCO (100 mL) and extracted with DCM (3 × 80 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (2% MeOH in DCM + 0.5% EtN) to give 45 (876 mg, 89%) as a yellow foam.

[0333] 45 Data:R f =0.81(5%MeOH in DCM): 1H NMR(300 MHz、CDCl3)δ 9.42(d、J=14.6 Hz、1H、NH)、8.73(s、1H、HC(2))、8.03(d、J=7.6 Hz、2H、H-arom)、7.93(s、1H、HC(8))、7.66-7.55(m、1H、H-arom)、7. 55-7.45(m、4H、H-arom)、7.45-7.22(m、7H、H-arom)、6.87(d、J=8.7 Hz、4H、H-arom) 6.25(dd、J=6.6;2.4 Hz、1H、HC(1')) 5.47-5.33(m 1H、HC(5')) 4.89(dd、J=6.7;4.9 Hz、1H、HC(4')) 4.02(d、J=2.5 Hz、1H、HC(7')) 3.79(s、6H、MeO) 2.58(dd、J=16.0、6.9 Hz, 1H, HC(3')), 2.38(ddd, J=12.7, 10.0, 2.4 Hz, 1H, HC(2')), 2.11 (s, 3H, MeCO2), 2.09-1.87 (m, 3H, HC(2'), HC(6')). 13 C NMR(75 MHz、CDCl3)δ 170.40(MeCO2) 164.84(CONH) 158.66(MeO-C-arom) 152.45(C(2)) 151.22(C(4)) 149.51(C(6)) 23(C-arom) 141.23(C(8)) 136.51 133.65(C-arom) 132.68 130.12 128.75 128.33 127.95 127.901 27.03(CH-arom) 123.55(C(5)) 113.27(CH-arom) 87.19(C(Ph)3) 87.12(C(1')) 83.25(C(4')) 77.16 (C(7')) 74.41(C(5')) 55.23(MeO-DMTr) 49.23(C(3')) 37.61(C(2')) 36.22(C(6')) 20.98(MeCO2). C 42 H 40 O7N5([M+H] + )についてのESI + -HRMS m / z registration number 726.2922.

[0334] (3'S,5'R,7'R)-N 6 -Benzoyl-9-{2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}adenine (46) [ka] Nucleoside 45 (870 mg, 1.20 mmol) is dissolved in 0.1 M NaOH / MeOH / HO in THF (5:4:1, 50 mL) at 0 °C. The reaction is stirred at 0 °C for 30 min and then quenched by adding NH Cl (321 mg). The solution is diluted with saturated NaHCO (100 mL) and extracted with DCM (4 × 80 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (3% MeOH in DCM + 0.5% EtN) to give 46 (777 mg, 94%) as a white foam.

[0335] 46 Data:R f =0.26(5%MeOH in DCM): 1H NMR(300 MHz、CDCl3)δ 9.39(s、1H、NH)、8.61(s、1H、HC(2))、7.93(d、J=7.4 Hz、2H、H-arom) 7.75(s、1H、HC(8)) 7.46(t、J=7.3 Hz、1H、H-arom) 7.40-7.31(m、4H、H-arom)、7.29-7.16(m、6H、H-arom)、7.11(t、J=7.2 Hz、1H、H-arom)、6.73(d、J=8.7 Hz、4H、H-arom)、6.12(dd、J=6.5、1.9 Hz、1H、HC(1'))、4.53(dd、J=7.5、4.5 Hz、1H、HC(4')) 4.32(br、1H、HC(5')) 3.90(t、J=4.5 Hz, 1H, HC(7')), 3.66, 3.65(2s, 6H, MeO), 3.31(br, 1H, OH), 2.36, dd, J=16.5, 8.1 Hz. 13 C NMR(75 MHz、CDCl3)δ 164.92(CONH) 158.64(MeO-C-arom) 152.60(C(2)) 151.28(C(4)) 149.61(C(6)) 145.44(C-arom )、140.71(C(8))、136.77、133.65(C-arom)、132.72、130.15、130.12、128.73、128.39、128.04、127.9 6, 127.02(CH-arom), 123.27(C(5)), 113.28(CH-arom), 87.11(C(1')), 87.01(C(Ph)3), 85.60(C(4') )) 78.16(C(7')) 72.72(C(5')) 55.28(MeO-DMTr) 48.89(C(3')) 39.93(C(6')) 37.55(C(2')). C 40 H 38 O6N5([M+H] + )についてのESI + -HRMS m / z registration number 684.2817.

[0336] (3'S,5'R,7'R)-N 6 -Benzoyl-9-{5'-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}adenine (47) [ka] To a solution of nucleoside 46 (199 mg, 0.290 mmol) and 5-(ethylthio)-1H-tetrazole (57 mg, 0.44 mmol) in dry DCM (7 mL) is added 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (0.16 mL, 0.49 mmol) dropwise at room temperature. After stirring for 60 min, the reaction mixture is diluted with saturated NaHCO3 (20 mL) and extracted with DCM (3 × 20 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (EtOAc, +0.5% Et3N) to give 47 (197 mg, mixture of two isomers, 77%) as a white foam.

[0337] 47 Data:R f =0.75(5%MeOH in DCM): 1H NMR(300 MHz、CDCl3)δ 8.98(br、1H、NH)、8.68、8.67(2s、1H、C(2))、7.94(d、J=7.6 Hz、2H、H-arom)、7.90、7.84(2s、1H、C(8))、7.56-7.49(m、1H、H-arom)、7.48-7.34(m、4H、H-arom)、7.30-7.10(m、7H、H-arom)、6.80-6.69(m、4H、Harom)、6.21、6.15(2dd、J=6.8、2.2 Hz、1H、H-C(1’))、4.69、4.59(2dd、J=7.3、4.5 Hz、1H、H-C(4’))、4.44(tt、J=12.3、6.3 Hz、1H、H-C(5’))、3.90(dd、J=9.0、3.8 Hz、1H、H-C(5’))、3.82-3.63(m、8H、MeO、OCH2CH2CN)、3.59-3.43(m、2H、(Me2CH)2N)、2.61-2.49(m、2H、OCH2CH2CN)、2.47-2.07(m、2H、H-C(3’)、H-C(2’))、1.98-1.66(m、3H、H-C(2’)、H-C(6’))、1.15-1.03(m、12H、(Me2CH)2N). 13 C NMR(101 MHz、CDCl3)δ 164.67(CONH)、158.77(MeO-C-arom)、152.58(C(2))、151.34、151.29(C(4))、149.46(C(6))、145.55、145.54(C-arom)、141.58、141.50(C(8))、136.87、136.85、136.84、133.85(C-arom)、132.85、130.26、130.23、130.20、128.97、128.47、128.43、128.02、127.96、127.08(CH-arom)、123.62、123.58(C(5))、117.91、117.70(OCH2CH2CN)、113.37(CH-arom)、87.80、87.67(C(1’))、87.20、87.14(C(Ph)3)、85.29、85.22((J C、P =4.2、3.1 Hz、C(4’))、78.16、77.96(C(7’))、74.28、73.98(J C、P=14.8, 18.4 Hz, C(5')), 58.80, 58.61(J C、P =16.2, 17.3 Hz OCH2CH2CN), 55.37, 55.35(MeO-DMTr), 49.02, 48.91(C(3')), 43.29, 43.16(J C、P =8.9, 9.0 Hz, ((Me2CH)2N), 39.09(C(6')), 37.99, 37.95(C(2')), 24.82, 24.77, 24.74, 24.70, 24.64((Me2CH)2N), 20.43, 20.42(J C、P =1.4, 1.9 Hz, OCH2CH2CN). 31 P NMR(121 MHz, CDCl3)δ 148.14, 148.11. C 45 H 56 O7N8P([M+H] + ) ESI + -HRMS m / z calculated 884.3895, found 884.3904.

[0338] (3'R,5'R,7'R)-2-amino-6-chloro-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α-D-ribofuranosyl}purine (48) [ka] Nucleoside 20 (1.78 g, 3.01 mmol) is dissolved in 0.5 M NaOH in THF / MeOH / HO (5:4:1, 15 mL) at 0 °C. The reaction is stirred at 0 °C for 20 min and quenched by the addition of NH Cl (484 mg). The suspension is then diluted with saturated NaHCO (100 mL) and extracted with DCM (4 × 75 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (3% MeOH in DCM) to give 48 (α-anomer, 992 mg, 60%) and 21 (β-anomer, 428 mg, 25%) as white foams.

[0339] 48 Data:R f=0.34(5%MeOH in DCM): 1 H NMR (400 MHz, CDCl3)δ 7.71-7.60(m, 5H, H-arom, H-(C(8)), 7.49-7.34(m, 6H, H-arom), 6.08(dd, J=6.9, 2.6 Hz, 1H, HC(1')), 5.26(s, 2H, NH2), 4.70(dd, J=7.5, 4.8 Hz, 1H, HC(4')), 4.47(dt, J=10.0, 5.1 Hz, 1H, HC(5')), 4.11(t, J=3.3 Hz, 1H, HC(7')), 2.87(dd, J=16.5, 7.7 Hz, 1H, HC(3')), 2.57(br, 1H, OH), 2.27(ddd, J=14.0, 9.9, 2.6 Hz, 1H, HC(2')), 2.10-2.01(m, 1H, HC(6')), 1.92-1.76(m, 2H, HC(2'), HC(6')), 1.06(s, 9H, (CH3)3-C-Si). 13 C NMR (75 MHz, CDCl3)δ 159.09(C(2)), 153.05(C(4)), 151.46(C(6)), 139.91(C(8)), 135.71(CH-arom) , 133.96, 133.27(C-arom), 130.00, 129.96, 127.86, 127.83(CH-arom), 125.52( C(5)), 86.46(C(1')), 84.92(C(4')), 77.40(C(7')), 72.63(C(5')), 50.55(C(3') ')), 40.92(C(6')), 36.78(C(2')), 26.88((CH3)3-C-Si), 19.01((CH3)3-C-Si). C 28 H 33 O3N5ClSi([M+H] + ) ESI + -HRMS m / z calculated 550.2036, found 550.2019.

[0340] (3'R,5'R,7'R)-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α-D-ribofuranosyl}guanine (49) [ka] To a solution of nucleoside 48 (610 mg, 1.03 mmol) in dry DCM (15 mL) was added 3-hydroxypropionitrile (0.28 mL, 4.12 mmol) followed by 1,5,7-triazabicyclo[4.4.0]dec-5-ene (287 mg, 2.06 mmol) at room temperature. After stirring at room temperature for 4 h, a second portion of 3-hydroxypropionitrile (0.28 mL, 3.23 mmol) was added followed by 1,5,7-triazabicyclo[4.4.0]dec-5-ene (287 mg, 2.06 mmol). The reaction was stirred for an additional 2 days and then directly purified by CC (10% MeOH in DCM) to give 49 (500 mg, 87%) as a white foam.

[0341] 49 Data:R f =0.30(10%MeOH in DCM): 1 H NMR (400 MHz, MeOD) δ 7.73-7.61(m, 5H, H-arom, HC(8)), 7.53-7.32(m, 6H, H-arom), 6.06(dd, J=6.9, 3.7 Hz, 1H, HC(1')), 4.74(dd, J=7.0, 4.6 Hz, 1H, HC(4')), 4.46-4.36(m, 1H, HC(5')), 4.11(br, 1H, HC(7')), 2.91(dd, J=16.2, 6.6 Hz, 1H, HC(3')), 2.31(ddd, J=13.8, 10.0, 3.7 Hz, 1H, HC(2')), 1.98-1.78(m, 3H, HC(2'), HC(3')), 1.07(s, 9H, (CH3)3-C-Si). 13C NMR (101 MHz, MeOD)δ 159.30(C(2)), 155.14(C(6)), 152.38(C(4)), 137.28(C(8)), 136.93, 136.88(CH- arom), 135.13, 134.78(C-arom), 131.07, 131.06, 128.91, 128.89(CH-arom), 117. 98(C(5)), 87.72(C(1')), 86.25(C(4')), 79.21, (C(7'))73.87(C(5')), 52.13(C( 3')), 41.44(C(6')), 38.35(C(2')), 27.42((CH3)3-C-Si), 19.82((CH3)3-C-Si)). C 28 H 34 O4N5Si([M+H] + ) ESI + -HRMS m / z calculated 532.2386, found 532.2367.

[0342] (3'R,5'R,7'R)-N2-acetyl-9-{5'-O-acetyl-7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α-D-ribofuranosyl}guanine (50) [ka] To a solution of nucleoside 49 (500 mg, 0.940 mmol) and 4-dimethylaminopyridine (276 mg, 2.4 mmol) in dry DCM (15 mL) was added acetic anhydride (1.0 mL, 10.3 mmol) at room temperature. After stirring for 2 days, the reaction was quenched by adding saturated NaHCO3 (30 mL). The mixture was then extracted with DCM (3 × 30 mL). The combined organic phases were dried over MgSO4, filtered, and evaporated. The crude product was purified by CC (3.5% MeOH in DCM) to give 50 (441 mg, 76%) as a white foam.

[0343] 50 Data:R f =0.62(10%MeOH in DCM): 1H NMR (300 MHz, CDCl3)δ 12.11(br, 1H, NH-C(4)), 9.94(br, 1H, HN(1)), 7.62(d, J=6.7 Hz, 5H, H-arom, HC(8)), 7.46-7.31(m, 6H, H-arom), 6.03(dd, J=6.7, 2.7 Hz, 1H, HC(1')), 5.31(dt, J=10.3, 5.2 Hz, 1H, H-(C5')), 4.99-4.81(m, 1H, HC(4')), 4.02(d, J=3.8 Hz, 1H, HC(7')), 2.88(dd, J=16.0, 6.6 Hz, 1H, HC(3')), 2.44-2.20(m, 4H, MeCONH, HC(2')), 2.12-1.73(m, 6H, MeCO2, HC(6'), HC(2')), 1.04(s, 9H, (CH3)3-C-Si). 13 C NMR (75 MHz, CDCl3)δ 172.73(MeCONH), 170.46(MeCO2), 155.87(C(6)), 148.09(C(4)), 147.47(C(2)), 137.13(C(8)), 135.74(CH-arom), 133.62, 133.29(C-arom), 130.13, 130.09, 127.96, 127.93(CH-arom), 121.54 (C(5)), 86.47(C(1')), 82.81(C(4')), 76.60(C(7')), 74.37(C(5')), 51.23(C(3')), 37.04, 37. 01, (C(2'), C(6'))26.92((CH3)3-C-Si), 24.46(MeCONH), 21.00(MeCO2), 19.05((CH3)3-C-Si). C 32 H 38 O6N5Si([M+H] + ) ESI + -HRMS m / z calculated 616.2586, found 616.2580.

[0344] (3'S,5'R,7'R)-N 2 -Acetyl-9-{5'-O-acetyl-2',3'-dideoxy-3',5'-ethano-7'-hydroxy-α-D-ribofuranosyl}guanine (51) [ka] To a solution of nucleoside 50 (440 mg, 0.714 mmol) in dry THF (5 mL) was added TBAF (1 M in THF, 1.1 mL, 1.1 mmol) at room temperature. The solution was stirred at room temperature for 4 h and then directly purified by CC (13% MeOH in DCM) to give 51 (235 mg, 87%) as a white foam. Crystals suitable for X-ray analysis were obtained by recrystallization from a mixture of HO / MeOH.

[0345] 51 Data:R f =0.25(13%MeOH in DCM): 1 H NMR (300 MHz, MeOD)δ 8.03(s, 1H, HC(8)), 6.28(dd, J=7.0, 3.8 Hz, 1H, HC(1')), 5.21(ddd, J=9.2, 6.8, 5.1 Hz, 1H, HC(5')), 4.98(dd, J=6.7, 5.0 Hz, 1H, H-(4')), 4.13-4.05(m, 1H, HC(7')), 3.17-3.05(m, 1H, HC(3')), 2.86(ddd, J=13.8, 10.0, 3.8 Hz, 1H, HC(2')), 2.39-2.27(m, 1H, HC(2')), 2.24(s, 3H, MeCONH), 2.16-2.00(m, 5H, MeCO2, HC(6')). 13 C NMR (101 MHz, MeOD)δ 174.95(MeCONH), 172.32(MeCO2), 157.50(C(6)), 149.96(C(4)), 149.38(C(2)), 139.66(C(8)), 121.76(C(5)), 88.23(C( 1')), 84.23(C(4')), 75.83(C(5'), C(7')), 51.65(C(3')), 38.04, 37.93(C(2'), C(6')), 23.83(MeCONH), 20.71(MeCO2). C 16 H 20 O6N5([M+H] + ) ESI +-HRMS m / z calculated 378.1408, found 378.1419.

[0346] (3'S,5'R,7'R)-N 2 -Acetyl-9-{5'-O-acetyl-2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}guanine (52) [ka] To a solution of nucleoside 51 (186 mg, 0.492 mmol) in dry pyridine (10 mL) is added DMTr-Cl (501 mg, 1.48 mmol) at room temperature. The solution is stirred for 2 days, then diluted with saturated NaHCO (40 mL) and extracted with DCM (3 × 30 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (3% MeOH in DCM + 0.5% EtN) to give 52 (333 mg, 99%) as a yellow foam.

[0347] 52 Data:R f =0.56(10%MeOH in DCM): 1 H NMR (300 MHz, CDCl3)δ 12.05(br, 1H, NH-C(4)), 9.90(br, 1H, HN(1)), 7.40(s, 1H, HC(8)), 7.38-7.31(m, 2H, H-arom), 7.28-7.08(m, 7H, H-arom), 6.75(dd, J=9.0, 2.7 Hz, 4H, H-arom), 5.95-5.85(m, 1H, HC(1')), 5.30-5.10(m, 1H, HC(5')), 4.70-4.58(m, 1H, HC(4')), 3.81(br, 1H, HC(7')), 3.68, 3. 68(2s, 6H, MeO), 2.25-2.07(m, 5H, MeCONH, HC(3'), HC(2')), 1.96-1.79(m, 5H, MeCO2, HC(2'), HC(6')), 1.74-1.59(m, 1H, HC(6')). 13C NMR (75 MHz, CDCl3)δ 172.65(MeCONH), 170.42(MeCO2), 158.73, 158.70(MeO-C-arom), 155.86(C(6)), 147.96(C(4)), 147.43(C(2)) , 145.31(C-arom), 137.17(C(8)), 136.69, 136.44(C-arom), 130.32, 130.21, 128.29, 128.05, 127.09(CH-arom ), 121.53(C(5)), 113.38, 113.35(CH-arom), 87.25(C(Ph)3), 86.73(C(1')), 82.77(C(4')), 77.19(C(7')), 74 .37(C(5')), 55.38(MeO-DMTr), 49.28(C(3')), 37.25(C(2')), 36.06(C(6')), 24.40(MeCONH), 21.01(MeCO2). C 37 H 38 O8N5([M+H] + ) ESI + -HRMS m / z calculated 680.2715, found 680.2718.

[0348] (3'S,5'R,7'R)-N2-(N,N-dimethylformamidino)-9-{2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}guanine (53) [ka] To a solution of nucleoside 52 (333 mg, 0.490 mmol) in dry MeOH (10 mL) was added KCO (305 mg, 2.20 mmol) at room temperature. The suspension was stirred at room temperature for 7 h, then NHCl (78 mg, 1.46 mmol) was added and the resulting mixture was filtered through a short pad of SiO. The SiO was washed with additional MeOH, and the solvent was evaporated.

[0349] The crude product is dissolved in dry DMF (10 mL) and N,N-dimethylformamide dimethyl acetal (0.33 mL, 2.5 mmol) is added. The solution is stirred at 55 °C for 2 h, and then the solvent is removed under reduced pressure. The crude product is purified by CC (7% MeOH in DCM + 0.5% EtN) to give 53 (245 mg, 77%) as a white foam containing traces of EtN.

[0350] 53 Data:R f =0.32(12%MeOH in DCM): 1 H NMR (300 MHz, CDCl3)δ 9.75(br, 1H, HN(1)), 8.25(s, 1H, NCHN(CH3)2), 7.37(d, J=7.3 Hz, 2H, H-arom), 7.29-7.08(m, 8H, H-arom, HC(8)), 6.74(d, J=8.1 Hz, 4H, H-arom), 6.03(dd, J=6.7, 2.8 Hz, 1H, HC(1')), 4.57(dd, J=7.5, 4.6 Hz, 1H, HC(4')), 4.37-4.26(m, 1H, HC(5')), 3.89(t, J=3.9 Hz, 1H, HC(7')), 3.67, 3.67(2s, 6H, MeO), 3.24(br, 1H, OH), 2.94(s, 3H, NCHN(CH3)2), 2.87(s, 3H, NCHN(CH3)2), 2.35(dd, J=15.9, 7.6 Hz, 1H, HC(3')), 1.94-1.68(m, 4H, HC(2'), HC(6')). 13C NMR (75 MHz, CDCl3)δ 158.61(MeO-C-arom), 158.28(C(2)), 157.92(NCHN(CH3)2), 156.69(C(6)), 149.90(C(4)), 145.52, 1 36.86, 136.77(C-arom), 135.50(C(8)), 130.15, 128.32, 127.92, 126.95(CH-arom), 120.27(C(5)), 11 3.24(CH-arom), 86.92(C(Ph)3), 85.57(C(1')), 85.12(C(4')), 78.31(C(7')), 72.69(C(5')), 55.28 (MeO-DMTr), 49.28(C(3')), 41.38(NCHN(CH3)2), 39.77(C(6')), 37.58(C(2')), 35.04(NCHN(CH3)2). C 36 H 39 O6N6([M+H] + ) ESI + -HRMS m / z calculated 651.2926, found 651.2921.

[0351] (3'S,5'R,7'R)-N 2 -(N,N-Dimethylformamidino)-9-{5'-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}guanine (54) [ka] To a solution of nucleoside 53 (245 mg, 0.377 mmol) and 5-(ethylthio)-1H-tetrazole (74 mg, 0.57 mmol) in dry DCM (15 mL) was added 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (0.20 mL, 0.64 mmol) dropwise at room temperature. After stirring for 50 min, the reaction mixture was diluted with saturated NaHCO3 (25 mL) and extracted with DCM (3 × 25 mL). The combined organic phases were dried over MgSO4, filtered, and evaporated. The crude product was purified by CC (3% MeOH in DCM + 0.5% Et3N) to give 54 (212 mg, mixture of two isomers, 67%) as a white foam.

[0352] 54 Data:R f =0.42(7%MeOH in DCM): 1 H NMR (300 MHz, CDCl3)δ 9.35(br, 1H, HN(1)), 8.51, 8.49(2s, 1H, NCHN(CH3)2), 7.41-7.10(m, 10H, H-arom, HC(8)), 6 .83-6.70(m, 4H, H-arom), 6.15-6.00(m, 1H, HC(1')), 4.64-4.36(m, 2H, HC(4'), HC(5')), 3. 90-3.82(m, 1H, HC(7')), 3.80-3.62(m, 8H, MeO, OCH2CH2CN), 3.59-3.43(m, 2H, (Me2CH)2N), 3.04, 3.02(2s, 6H, NCHN(CH3)2), 2.67-2.48(m, 2H, OCH2CH2CN), 2.32(ddd, J=24.1, 15.1, 6.7 Hz, 1H, HC(3')), 2.02-1.63(m, 4H, HC(2'), HC(6')), 1.14-1.03(m, 12H, (Me2CH)2N). 13C NMR(101 MHz、CDCl3)δ 158.76(MeO-C-arom)、158.17、158.12(C(2))、158.03(NCHN(CH3)2)、156.66、156.59(C(6))、149.85 、149.79(C(4))、145.51、145.49、136.84、136.77、136.73、136.71(C-arom)、135.76、135.59(C(8)) 30.24、130.20、128.41、128.33、128.02、127.10、127.08(CH-arom)、120.74、120.70(C(5))、117.18、117.10 17.72(OCH2CH2CN)、113.34(CH-arom)、87.16、87.10(C(Ph)3)、86.00、85.72(C(1'))、84.13、84.10(J C、P =3.6、2.5 Hz、C(4'))、78.02、77.67(C(7'))、74.15、73.74(J C、P =15.3、18.7 Hz、C(5'))、58.90、58.67(J C、P =18.7、19.7 Hz OCH2CH2CN),55.38、55.36(MeO-DMTr)、49.20、49.09(C(3'))、43.20、43.15(J C、P =12.4、12.6 Hz、((Me2CH)2N),41.42、41.38(NCHN(CH3)2)、38.68、38.65(C(6'))、37.97、37.84(C(2'))、35 .25(NCHN(CH3)2)、24.83、24.75、24.68、24.60、24.53((Me2CH)2N)、20.35、20.28(OCH2CH2CN). 31 P NMR(121 MHz、CDCl3)δ 148.21、148.01. C 45 H 56 O7N8P([M+H] + )についてのESI + -HRMS m / z 851.4004 and 851.4013.

[0353] (3aR,4R,6R,6aS)-4-((Tert-butyldiphenylsilyl)oxy)-2-methoxyhexahydro-2H-cyclopenta[b]furan-6-yl(4-nitrobenzoate) (55) [ka] To a solution of sugar 6 (195 mg, 0.437 mmol) and 4-dimethylaminopyridine (70 mg, 0.568 mmol) in dry DCM (10 mL) is added 4-nitrobenzoyl chloride (158 mg, 0.850 mmol) at room temperature. After stirring overnight, the reaction is quenched by the slow addition of saturated NaHCO3 (3 mL). The mixture is then diluted with saturated NaHCO3 (15 mL) and extracted with DCM (3 x 15 mL). The combined organic phases are dried over MgSO4, filtered, and evaporated. The crude product is purified by CC (1:5 EtOAc / hexane) to obtain the anomeric ratio α / β. [ka] A 4:1 mixture of 55 (260 mg, 98%) is obtained as a white solid.

[0354] 55 Data:R f =0.62 (EtOAc / hexane 1:2): 1H NMR(300 MHz、CDCl3)δ 8.33-8.17(m、4H、H-arom) 7.72-7.61(m、4H、H-arom) 7.51-7.3 2(m、6H、H-arom)、5.65-5.47(m、1H、HC(6))、4.97(dd、J=9.2、5.6 Hz Hz, 0.8H, HC(4)), 3.21(d, J=15.1 Hz, 3H, MeO), 2.88(dd, J=16.6, 7.9). Hz, 0.8H, HC(3a)), 2.75-2.62(m, 0.2H, HC(3a)), 2.49-2.34(m, 0.2H, H C(5)) 2.24-1.83(m 2.8H H-(5) HC(3)) 1.28(ddd J=13.0 7.9 4.9 Hz、1H、HC(3))、1.09(s、9H、(CH3)3-C-Si). 13 C NMR(75 MHz、CDCl3)δ 164.46, 164.41(CO2R), 150.63(O2N-C-arom), 135.87, 135.82(CH-arom), 134.07, 133.75 133.69(CH-arom) 130.98 130.89 129.98 129.96 129.91 127.89 127.87 127.85 123 .59(CH-arom) 106.49 106.39(C(2)) 83.21 79.87(C(6a)) 76.54(C(4)) 76.09(C(6)) . 54.55, 54.47(MeO), 51.69, 50.30(C(3a), 38.07(C(3)), 37.17, 36.65(C(5)), 27.04, 26.99 90((CH3)3-C-Si)、19.14((CH3)3-C-Si). C 31 H 35 O7NaSi([M+Na] + )についてのESI + -HRMS m / z registration number 584.2075.

[0355] (3'R,5'R,7'R)-1-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-5'-O-(4-nitrobenzoate)-α,β-D-ribofuranosyl}thymine (56) [ka] To a solution of sugar 55 (260 mg, 0.463 mmol) and thymine (84 mg, 0.695 mmol) in dry MeCN (3 mL) is added dropwise BSA (0.34 mL, 1.4 mmol) at room temperature. After stirring for 30 min at room temperature, the solution is cooled to 0 °C and TMSOTf (0.10 mL, 1.3 mmol) is added dropwise. After stirring for an additional 2 h at 0 °C and 18 h at room temperature, the reaction mixture is diluted with saturated NaHCO (30 mL) and extracted with DCM (4 × 40 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (2% MeOH in DCM) to obtain the anomeric ratio α / β. [ka] A 88:12 mixture of 56 (240 mg, 79%) is obtained as a white foam.

[0356] 56 Data:R f =0.56(DCM+3%MeOH): 1H NMR(300 MHz、CDCl3)δ 9.38(br、1H、(s、1H、HN(3))、8.32-8.23(m、2H、H-arom)、8.22-8.11(m、2H、H-arom)、7.65(dd、J=7.7、1.5 Hz、4H、H-arom)、7.50-7.36(m、6H、H-arom)、6.95(d、J=0.9 Hz、1H、HC(6))、5.96(t、J=6.3 Hz 1H HC(1')) 5.55(dt J=9.9 6.0 Hz 1H HC(5')) 5.13(dd J=6.4 5.4). Hz, 1H, HC(4')), 4.20-4.05(m, 1H, HC(7')), 2.94-2.78(m, 1H, HC(3')), 2.22 (dd, J=13.3, 6.4 Hz 1H HC(6')) 2.09-1.73(m 6H HC(6') HC(2') Me-C(5)) 1.09(s 9H (CH3)3-C-Si). 13 C NMR(75 MHz、CDCl3)δ 164.32,163.79(C(4),CO2R),150.65,150.39(O2N-C-arom,C(2)),135.70,135.68(CH-arom),135 .13(C-arom) 134.83(C(6)) 133.46 133.10(C-arom) 130.91 130.73 130.11 127.93 123.60(C H-arom) 111.30(C(5)) 87.26(C(1')) 82.44(C(4')) 76.40(C(7')) 76.07(C(5')) 50.76(C(3). ')) 37.94(C(6')) 36.68(C(2')) 26.89((CH3)3-C-Si) 19.03((CH3)3-C-Si) 12.62(Me-C(5)). C 35 H 37 O8N3NaSi([M+Na] + )についてのESI + -HRMS m / z registration number 678.2242.

[0357] (3'R,5'R,7'R)-1-{2',3'-dideoxy-3',5'-ethano-7'-hydroxy-5'-O-(4-nitrobenzoyl)-α,β-D-ribofuranosyl}thymine (57) [ka] To a solution of nucleoside 56 (220 mg, 0.335 mmol) in dry THF (2 mL) is added TBAF (1 M in THF, 0.84 mL, 0.84 mmol) at room temperature. After stirring at room temperature for 4 h, the reaction mixture is diluted with saturated NaHCO (20 mL) and extracted with EtOAc (3 × 20 mL) and DCM (2 × 80 mL). The combined organic phases are dried over MgSO, filtered, and evaporated. The crude product is purified by CC (5% MeOH in DCM) to give the anomeric mixture of 57 (101 mg, 72%). Crystals suitable for X-ray analysis are obtained by recrystallization in EtOAc.

[0358] 57 Data:R f =0.50(DCM+7%MeOH): 1 H NMR (300 MHz, CDCl3)δ 8.96(br, 1H, HN(3)), 8.34-8.17(m, 4H, H-arom), 7.07(d, J=1.1 Hz, 1H, HC(6)), 6.11(t, J=6.3 Hz, 1H, HC(1')), 5.57-5.45(m, 1H, HC(5')), 5.15(dd, J=6.6, 5.4 Hz, 1H, HC(4')), 4.38-4.23(m, 1H, HC(7')), 2.96(dd, J=13.5, 6.9 Hz, 1H, HC(3')), 2.26(ddd, J=13.1, 10.3, 5.4 Hz, 4H, HC(2'), HC(6')), 1.91(d, J=0.9 Hz, 3H, Me-C(5)). C 19 H 19 O8N3Na([M+Na] + ) ESI + -HRMS m / z calculated 440.1064, found 440.1072.

[0359] Process for synthesis, deprotection, and purification of alpha anomeric oligonucleotides Oligonucleotides containing at least two alpha anomeric bicycloDNA (abc-DNA) residues linked by phosphodiester bonds can be synthesized on a synthesizer, such as a Pharmaci-Gene-Assembler-Plus DNA synthesizer, according to methods well known in the art and as described below. The steps for the synthesis of the abc-DNA oligonucleotides of the present invention are shown below: [ka]

[0360] Oligonucleotide synthesis is performed on a Pharmaci-Gene-Assembler-Plus DNA synthesizer at a 1.3 μmol scale, following the Gene Assembler manufacturer's recommended protocol. Native DNA phosphoramidites (dT, dC4bz, dG2DMF, dA6Bz) and solid support (Glen Unysupport 500) are purchased from Glen Research. Native DNA phosphoramidites are prepared as 0.1 M solutions in MeCN and coupled using a 4-min step. abc-DNA phosphoramidites are prepared as 0.1 M solutions in 1,2-dichloroethane and coupled using a 12-min extension step with 5-(ethylthio)-1H-tetrazole (0.25 M in MeCN) as the coupling agent. Detritylation of modified nucleosides is performed using a solution of 5% dichloroacetic acid in dichloroethane. Oxidation is carried out in a solution of 0.01 M iodine in MeCN / water / collidine (32:3:15) with a reaction time of 1 min. Sulfurization is carried out using a solution of 0.2 M phenylacetyl disulfide in MeCN / pyridine (1:1) with a reaction time of 3.5 min. Capping is carried out using standard conditions. Cleavage from the solid support and deprotection of the oligonucleotide are achieved by treatment with concentrated ammonia at 55 °C for 16 h. After centrifugation, the supernatant is collected, the beads are washed with additional HO (0.5 mL x 2), and the resulting solution is evaporated to dryness. The crude oligonucleotide is purified by ion-exchange HPLC (Dionex-DNA PacPA200). A buffer of 25 mM Trizma in HO, pH 8.0, was used as mobile phase "A," and 25 mM Trizma, 1.25 M NaCl in HO, pH 8.0, was used as mobile phase "B." For phosphorothioate chains, a buffer of 10 mM NaOH in HO, pH 12.0 was used as mobile phase "A," and 10 mM NaOH, 2.50 M NaCl in HO, pH 12.0 was used as mobile phase "B." The purified oligonucleotides were then desalted on Sep-pak C-18 cartridges. The concentrations were determined by measuring the absorbance at 260 nm on a Nanodrop spectrophotometer using the extinction coefficient of the corresponding native DNA oligonucleotide.Characterization of oligonucleotides was performed using ESI. - This is done by mass spectrometry or LC-MS.

[0361] Pharmaceutical Composition In certain embodiments, the present invention provides pharmaceutical compositions comprising the oligonucleotides of the present invention. The oligonucleotide sample can be appropriately formulated and introduced into the cellular environment by any means that allows a sufficient portion of the sample to enter the cell and induce an effect, such as exon skipping. In certain embodiments, the oligonucleotide is preloaded with albumin and administered as an oligonucleotide-albumin complex. Many formulations of oligonucleotides are known in the art and can be used as long as the oligonucleotide can enter and act on the target cell. For example, the oligonucleotide agents of the present invention can be formulated in buffers such as phosphate-buffered saline, liposomes, micellar structures, and capsids. Formulations of oligonucleotide agents containing cationic lipids can be used to facilitate transfection of the oligonucleotide agent into cells. For example, cationic lipids such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules such as polylysine (published PCT International Application No. WO 97 / 30731) can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals Inc., Boulder, Colorado), or FuGene 6 (Roche), all of which may be used according to the manufacturer's instructions.

[0362] Such compositions typically comprise nucleic acid molecules and pharmaceutically acceptable carriers.As used herein, the term "pharmaceutically acceptable carriers" includes physiological saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration.Auxiliary active compounds can also be incorporated into the composition.

[0363] Pharmaceutical compositions are formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal, transdermal (topical), transmucosal, intrathecal, intraventricular, intraperitoneal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: a sterile diluent, e.g., water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, e.g., benzyl alcohol or methylparaben; an antioxidant, e.g., ascorbic acid or sodium bisulfite; a chelating agent, e.g., ethylenediaminetetraacetic acid; a buffer, e.g., acetate, citrate, or phosphate; and a tonicity adjuster, e.g., sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0364] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0365] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in a suitable solvent, which contains one or combination of the above-listed components as needed, and then sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and other components that are required from the above-listed components.For the aseptic powder that is used to prepare sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which can obtain the powder of active ingredient and any additional desired components from the solution that has been previously sterile-filtered.

[0366] Oral compositions generally contain an inert diluent or edible carrier.For oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, lozenges, or capsules, such as gelatin capsules.Orally administered compositions can also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binders and / or auxiliary substances can be included as part of the composition.Tablets, pills, capsules, lozenges, etc. can contain any of the following ingredients, or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, orange flavor.

[0367] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer, including those methods described in U.S. Patent No. 6,468,798.

[0368] Systemic administration can also be via transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, surfactants, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams as generally known in the art.

[0369] The present invention also provides a dry powder delivery method.

[0370] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0371] The compounds can also be administered by transfection or infection using methods known in the art, including, but not limited to, those described in McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10) 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J. Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst. Pharm. 53(3), 325 (1996).

[0372] The compound can also be administered by any suitable method for administering nucleic acid agents such as DNA vaccines.These methods include gene guns, bioinjectors, skin patches, and needleless methods such as the microparticle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389, and needleless transdermal vaccination of mammals with powder-form vaccines disclosed in U.S. Patent No. 6,168,587.In addition, intranasal delivery is possible, particularly as described in Hamajima et al.(1998), Clin.Immunol.Immunopathol.,88(2),205-10.Liposomes (for example, as described in U.S. Patent No. 6,472,375) and microencapsulation can also be used.Biodegradable targetable microparticle delivery systems (for example, as described in U.S. Patent No. 6,471,996) can also be used.

[0373] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Such formulations can be prepared using standard techniques. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells using monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0374] The toxicity and therapeutic efficacy of such compounds can be determined, for example, by LD 50 (lethal dose for 50% of the population) and ED 50 The dose (therapeutically effective dose in 50% of the population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, and is the ratio LD 50 / ED 50 The therapeutic index can be expressed as: Compounds that exhibit a high therapeutic index are preferred. Compounds that exhibit toxic side effects can also be used, but care must be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0375] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. Such compounds can be administered at doses that produce little or no toxicity and an ED 50Preferably, the circulating concentration of the compound is within a range including the IC (IC ). The dosage may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. The IC (IC ) determined in cell culture is used. 50 A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the concentration of the test compound that achieves a half-maximal inhibition of symptoms (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0376] As defined herein, a therapeutically effective amount of a nucleic acid molecule (i.e., an effective dosage) depends on the nucleic acid selected. For example, a single dosage ranging from about 1 pg to 1000 mg may be administered, and in some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10, 30, 100, or 1000 mg may be administered. In some embodiments, 1 to 5 g of the composition may be administered. The composition may be administered once or more times daily to once or more times weekly, including every other day or once or more monthly. One of skill in the art will appreciate that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other existing diseases, may influence the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of an oligonucleotide of the invention can include a single treatment or, preferably, a series of treatments.

[0377] In certain embodiments, the dosage of the oligonucleotide according to the invention is between 5 mg / kg / week and 500 mg / kg / week, e.g., 5 mg / kg / week, 10 mg / kg / week, 15 mg / kg / week, 20 mg / kg / week, 25 mg / kg / week, 30 mg / kg / week, 35 mg / kg / week, 40 mg / kg / week, 45 mg / kg / week, 50 mg / kg / week, 55 mg / kg / week, 60 mg / kg / week, 65 In certain embodiments, the dosage of an oligonucleotide according to the invention ranges from 10 mg / kg / week to 200 mg / kg / week, 20 mg / kg / week to 150 mg / kg / week, or 25 mg / kg / week to 100 mg / kg / week. In certain embodiments, the oligonucleotide is administered once a week for a period of 2 weeks to 6 months, for example, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 26 weeks, 6 months, 8 months, 10 months, or a year or more. In certain embodiments, the oligonucleotide is administered twice a week. In other embodiments, the oligonucleotide is administered every other week. In certain embodiments, the oligonucleotide is administered intravenously.

[0378] It can be understood that the method of introducing an oligonucleotide agent into the cellular environment depends on the type of cell and the composition of its environment.For example, if cells are found in liquid, one preferred formulation is a lipid formulation such as lipofectamine, and the oligonucleotide agent can be directly added to the liquid environment of the cell.The lipid formulation can also be administered to animals by intravenous, intramuscular, or intraperitoneal injection, or orally, or by inhalation, or by other methods known in the art.If the formulation is suitable for administration to animals such as mammals, more particularly humans, the formulation is also pharmaceutically acceptable.Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used.In some cases, such as research using oocytes, it may be preferable to formulate the oligonucleotide agent in a buffer or saline solution and directly inject the formulated oligonucleotide agent into cells.Direct injection of oligonucleotides can also be performed.

[0379] It is necessary to introduce a suitable amount of oligonucleotide agent, and these amounts can be empirically determined using standard methods.Typically, the effective concentration of each oligonucleotide agent species in cellular environment will be the composition that can use a concentration of about 50 nanomoles or less, 10 nanomoles or less, or about 1 nanomoles or less.In another embodiment, the method that utilizes a concentration of about 200 picomoles or less, or even about 50 picomoles or less, about 20 picomoles or less, about 10 picomoles or less, or about 5 picomoles or less can be used in many situations.

[0380] The method can be carried out by adding the oligonucleotide agent composition to any extracellular matrix in which cells can live, provided that the oligonucleotide agent composition is formulated so that a sufficient amount of the oligonucleotide agent can enter the cells and exert its effect. For example, the method is suitable for use with cells present in a liquid, such as a liquid culture or cell growth medium, in a tissue explant, or in a whole organism, including animals, such as mammals, particularly humans.

[0381] Oligonucleotide agents can be formulated as pharmaceutical compositions containing a pharmacologically effective amount of an oligonucleotide agent and a pharmaceutically acceptable carrier. A pharmacologically effective amount or a therapeutically effective amount refers to the amount of an oligonucleotide agent that is effective to produce the intended pharmacological, therapeutic, or preventive result. The phrases "pharmacologically effective amount" and "therapeutically effective amount" or simply "effective amount" refer to the amount of an oligonucleotide that is effective to produce the intended pharmacological, therapeutic, or preventive result. For example, if a given clinical treatment is considered effective when a measurable parameter related to a disease or disorder is reduced by at least 20%, then the therapeutically effective amount of an agent for treating that disease or disorder is the amount required to reduce that parameter by at least 20%.

[0382] Suitably formulated pharmaceutical compositions of the present invention can be administered by any means known in the art, such as parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, respiratory (aerosol), rectal, vaginal, and topical (including buccal and sublingual) administration. In some embodiments, the pharmaceutical compositions are administered intravenously or by parenteral infusion or injection.

[0383] Generally, suitable dosage units for oligonucleotides will be in the range of 0.001 to 0.25 milligrams per kilogram of recipient body weight per day, or 0.01 to 20 micrograms per kilogram of body weight per day, or 0.01 to 10 micrograms per kilogram of body weight per day, or 0.10 to 5 micrograms per kilogram of body weight per day, or 0.1 to 2.5 micrograms per kilogram of body weight per day. In certain embodiments, dosages range from 0.1 mg / kg body weight per day to 5 mg / kg body weight per day, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 mg / kg body weight. Pharmaceutical compositions containing oligonucleotides can be administered once daily. However, therapeutic agents can also be administered in dosage units containing 2, 3, 4, 5, 6, or more subdoses administered at appropriate intervals throughout the day. In this case, the amount of oligonucleotide contained in each sub-dose must be correspondingly smaller to achieve the total daily dosage unit.The dosage unit can also be formulated for a single administration over several days, for example, by using a conventional sustained-release formulation that provides a sustained and consistent release of oligonucleotide over several days.Sustained-release formulations are well known in the art.In this embodiment, the dosage unit contains a multiple of the corresponding daily dose.Regardless of formulation, the pharmaceutical composition must contain a sufficient amount of oligonucleotide to be active, for example, to cause exon skipping or inhibit the expression of target genes in treated animals or humans.The composition can be formulated so that the sum of multiple units of oligonucleotide together contains a sufficient dose.

[0384] Data can be obtained from cell culture assays and animal studies to formulate a suitable dosage range for humans. The dosage of the compositions of the present invention can be adjusted to achieve ED with little or no toxicity. 50The therapeutically effective dose of any compound used in the methods of the present invention can be estimated initially from cell culture assays. The therapeutically effective dose is within a range of circulating concentrations that includes the IC (as determined by known methods). The dosage may vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the methods of the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. The IC (as determined in cell culture) 50 Doses can be formulated in animal models to achieve a circulating plasma concentration range of the compound that includes the target concentration (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma oligonucleotide levels can be measured by standard methods, such as high performance liquid chromatography.

[0385] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.

[0386] Treatment method The present invention provides both prophylactic and therapeutic methods of treating subjects at risk for (or susceptible to) a disease or disorder caused in whole or in part by the expression of a target RNA and / or the presence of such a target RNA.

[0387] As used herein, "treatment" or "treating" is defined as the application or administration of a therapeutic agent (e.g., an oligonucleotide agent or vector or transgene encoding same) to a patient, or to a tissue or cell line isolated from a patient with a disease or disorder, a symptom of a disease or disorder, or a predisposition to a disease or disorder, with the intent to cure, heal, alleviate, alleviate, alter, cure, ameliorate, ameliorate, or affect the disease or disorder, symptom of the disease or disorder, or predisposition to the disease or disorder.

[0388] In one aspect, the invention provides a method for preventing the above-described diseases or disorders in a subject by administering a therapeutic agent (e.g., an oligonucleotide agent or a vector or transgene encoding same) to the subject. Subjects at risk for the disease can be identified, for example, by any or a combination of the diagnostic or prognostic assays described herein. Administration of the prophylactic agent can occur, for example, prior to detection of viral particles in the subject or prior to the onset of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, its progression is delayed.

[0389] Another aspect of the invention relates to methods of therapeutically treating a subject, i.e., methods for altering the onset of symptoms of a disease or disorder. These methods can be performed in vitro (e.g., by culturing a cell with an oligonucleotide agent) or in vivo (e.g., by administering an oligonucleotide agent to a subject).

[0390] With regard to both preventive and therapeutic treatment methods, such treatments can be specifically tailored or modified based on knowledge gained from the field of pharmacogenomics. As used herein, "pharmacogenomics" refers to the application of genomics technologies, such as gene sequencing, statistical genetics, and gene expression analysis, to drugs in clinical development and on the market. More specifically, this term refers to the study of how a patient's genes determine their response to drugs (e.g., their "drug response phenotype" or "drug response genotype"). Pharmacogenomics allows clinicians or physicians to target preventive or therapeutic treatments to patients who will benefit most from treatment and avoid treating patients who experience toxic drug-related side effects.

[0391] Therapeutic agents can be tested in suitable animal models. For example, the oligonucleotide agents described herein (or expression vectors or transgenes encoding them) can be used in animal models to determine the efficacy, toxicity, or side effects of treatment with the agent. Alternatively, therapeutic agents can be used in animal models to determine the mechanism of action of such agents. For example, agents can be used in animal models to determine the efficacy, toxicity, or side effects of treatment with such agents. Alternatively, agents can be used in animal models to determine the mechanism of action of such agents.

[0392] Furthermore, the therapeutic efficacy of abc-DNA lipid-conjugated oligonucleotides will be determined by assessing muscle function, grip strength, respiratory function, cardiac function via MRI, and muscle physiology. Complement activation and blood coagulation will also be assessed to examine the negative side effects of the oligonucleotides.

[0393] disease The oligonucleotides of the present invention are useful for regulating gene expression by interfering with transcription, translation, splicing, and / or degradation, and / or inhibiting the function of non-coding RNA, for the treatment or prevention of diseases based on abnormal levels of mRNA or non-coding RNA. If one or more disease symptoms are reduced or eliminated after administration of the cells of the present invention, the subject is said to be treated for the disease.

[0394] The abc-DNA lipid group-conjugated oligonucleotides of the present invention can regulate the level or activity of a target RNA. The level or activity of a target RNA can be determined by any suitable method currently known in the art or later developed. It should be understood that the method used to measure the target RNA and / or expression of the target RNA may depend on the nature of the target RNA. For example, if the target RNA encodes a protein, the term "expression" can refer to the protein or RNA / transcript derived from the target RNA. In such cases, the expression of the target RNA can be determined by measuring the amount of RNA corresponding to the target RNA or by measuring the amount of the protein product. Proteins can be measured by protein assays such as staining or immunoblotting, or by measuring the reaction rate if the protein catalyzes a measurable reaction. All such methods are known in the art and can be used. When measuring target RNA levels, any art-recognized method for detecting RNA levels (e.g., RT-PCR, Northern blotting, etc.) can be used. Any of the above measurements can be performed on cells, cell extracts, tissues, tissue extracts, or any other suitable source material.

[0395] The abc-DNA lipid-conjugated oligonucleotides of the present invention are used to modulate the expression of microRNAs or other non-coding RNAs that regulate mRNA expression.

[0396] MicroRNAs are small, non-coding RNAs, approximately 20–25 nucleotides in length, that direct post-transcriptional regulation of gene expression. They regulate the expression of multiple target genes through sequence-specific hybridization to the 3' untranslated region of messenger RNA. These microRNAs can block translation or directly degrade target messenger RNAs.

[0397] The abc-DNA lipid group conjugate oligonucleotide of the present invention is synthesized and binds to target miRNA.These oligonucleotides are designed to bind to miRNA and prevent miRNA from binding to its target mRNA.As described in the above example, the abc-DNA lipid group conjugate oligonucleotide can be used to regulate miRNA binding in vitro and in vivo.

[0398] Long non-coding RNAs (lncRNAs) are a large and diverse class of transcribed RNA molecules greater than 200 nucleotides in length that do not encode proteins (or lack an open reading frame of more than 100 amino acids). lncRNAs are important regulators of gene expression, and lncRNAs are thought to have a wide range of functions in cellular and developmental processes. They can both inhibit and activate genes through various mechanisms. The validated functions of lncRNAs suggest that they are master regulators of gene expression, often exerting their influence through epigenetic mechanisms by modulating chromatin structure.

[0399] The abc-DNA lipid group-conjugated oligonucleotides of the present invention are synthesized to be complementary to the target lncRNA of interest. In the nucleus, they hybridize with the targeted lncRNA to form heteroduplexes.

[0400] The present invention relates to Duchenne muscular dystrophy, spinal muscular atrophy (exon 7 inclusion in the SMN2 gene), myotonic dystrophy (CAG n The present invention provides for the treatment or prevention of diseases including, but not limited to, cerebrospinal fluid (CSF)-associated leukemia (targeting CUGexp-DMPK transcripts with CAG triplet expansion), Huntington's disease (allele-selective and non-selective approaches targeting CAG triplet expansion), amyotrophic lateral sclerosis (a genetically heterogeneous disorder with several causative genes), and Pompe disease (targeted splice mutation c.-32IVS1-13T>G, found in over half of all Caucasian patients).

[0401] array The present invention provides any abc-DNA oligonucleotide having a predominantly phosphate internucleoside linkage, one or two linkers, and a lipid group. The sequence can be designed for any target. The sequences of exemplary abc-DNA oligonucleotides of the present invention are provided below.

[0402] In certain embodiments, the oligonucleotide has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides, e.g., 21-50 nucleotides, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 nucleotides. In one embodiment, the oligonucleotide has a length of 14, 15, 16, 17, 18, or 19 nucleotides. In one embodiment, the oligonucleotide has a length of 15 nucleotides. In one embodiment, the oligonucleotide has a length of 16 nucleotides. In one embodiment, the oligonucleotide has a length of 17 nucleotides. In one embodiment, the oligonucleotide has a length of 18 nucleotides. In one embodiment, the oligonucleotide has a length of 19 nucleotides.

[0403] DMD-targeting oligonucleotides Duchenne muscular dystrophy (DMD) affects 1 in 3,500 newborn boys, and Becker muscular dystrophy (BMD) affects 1 in 20,000. Both DMD and BMD are caused by mutations in the DMD gene, which is located on the X chromosome and encodes dystrophin. DMD patients suffer from progressive muscle weakness, become wheelchair-bound before the age of 13, and often die before the age of 30. BMD is generally milder, and patients are often ambulatory for over 40 years and have a longer life expectancy compared to DMD patients.

[0404] Dystrophin is an essential component of the dystrophin-glycoprotein complex (DGC). In particular, the DGC maintains the membrane stability of muscle fibers. Frameshift mutations in the DMD gene cause dystrophin deficiency in muscle cells, which, along with reduced levels of other DGC proteins, results in the severe phenotype seen in DMD patients. Mutations in the DMD gene that keep the reading frame intact generate shorter but partially functional dystrophin and are associated with less severe BMD. In Duchenne muscular dystrophy (DMD) patients, frameshift mutations in the DMD gene generate out-of-frame mRNA, producing a truncated, nonfunctional dystrophin protein. This in-frame mature mRNA encodes an in-frame dystrophin protein that is still partially functional and results in a milder Becker muscular dystrophy (BMD) phenotype.

[0405] In certain embodiments, the oligonucleotides of the invention are complementary to portions of the DMD gene, for example, exon 51, exon 53, and exon 45.

[0406] Exon 51 The sequence of exon 51 of the DMD gene (SEQ ID NO: 401) is shown below: Sequence Listing 1 TIFF0007665509000113.tif31170

[0407] The corresponding transcript sequence of the highlighted portion is as follows: 5' CC AAA CTA GAA ATG CCA TCT TCC TTG ATG T 3' (SEQ ID NO: 402).

[0408] Oligonucleotides complementary to exon 51 of the DMD gene useful according to the present invention include, but are not limited to: 5'GG TTT GAT CTT TAC GGT AGA AGG AAC TAC A 7' (SEQ ID NO: 403) and oligonucleotides provided in Table 3: [Table 3-1] [Table 3-2]

[0409] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-75. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-75, and the oligonucleotide has a length of 14 to 20 nucleotides. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-75, and the oligonucleotide has a length of 14 to 19 nucleotides. In one embodiment, the oligonucleotide has a length of 14 to 19 nucleotides. In one embodiment, the oligonucleotide has a length of 14 nucleotides. In one embodiment, the oligonucleotide has a length of 15 nucleotides. In one embodiment, the oligonucleotide has a length of 16 nucleotides. In one embodiment, the oligonucleotide has a length of 17 nucleotides. In one embodiment, the oligonucleotide has a length of 18 nucleotides. In one embodiment, the oligonucleotide has a length of 19 nucleotides. In one embodiment, the oligonucleotide has a length of 20 nucleotides.

[0410] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-75, and the oligonucleotide has a length of 19 nucleotides. In such an embodiment, the oligonucleotide is a 19-mer. In one embodiment, the oligonucleotide comprises the sequence 5'CTTTACGGTAGAAGGAACT 7' (SEQ ID NO: 404; 19-mer). In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 404.

[0411] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-75, and the oligonucleotide has a length of 18 nucleotides. In such an embodiment, the oligonucleotide is an 18-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-13. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 1-13. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 4. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 5. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 4. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 5.

[0412] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-75, and the oligonucleotide has a length of 17 nucleotides. In such an embodiment, the oligonucleotide is a 17-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 14-27. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 14-27. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 22. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 23. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 24.

[0413] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-75, and the oligonucleotide has a length of 16 nucleotides. In such an embodiment, the oligonucleotide is a 16-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 28-42. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 28-42. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 36. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 37. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 38. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 39. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 36. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 37. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 38. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO:39.

[0414] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-75, and the oligonucleotide has a length of 15 nucleotides. In such an embodiment, the oligonucleotide is a 15-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 43-58. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 43-58. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 55. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 51. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 52. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 53. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 54. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 55. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 55. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 51. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 52. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 53. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 54. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 55.

[0415] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-75, and the oligonucleotide has a length of 14 nucleotides. In such an embodiment, the oligonucleotide is a 14-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 59-75. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 59-75. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 67. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 68. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 69. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 70. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 67. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 68. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 69. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO:70.

[0416] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39, 51-55, 67-70, and SEQ ID NO: 404. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39, 51-55, 67-70, and SEQ ID NO: 404, wherein all of the residues are abc-DNA residues. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39, 51-55, 67-70, and SEQ ID NO: 404. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39, 51-55, 67-70, and SEQ ID NO: 404, wherein all of the residues are abc-DNA residues. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39, 51-55, and SEQ ID NO: 404. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39, 51-55, and SEQ ID NO: 404, wherein all of the residues are abc-DNA residues. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39, 51-55, and SEQ ID NO: 404. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39, 51-55, and SEQ ID NO: 404, wherein all of the residues are abc-DNA residues. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 417 and 418. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 417. In one embodiment, the oligonucleotide comprises the sequence of SEQ ID NO: 418. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NO: 417 and SEQ ID NO: 418. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 417. In one embodiment, the oligonucleotide consists of the sequence of SEQ ID NO: 418.

[0417] Exon 53 The sequence of exon 53 of the DMD gene (SEQ ID NO: 405) is shown below: Sequence Listing 2 TIFF0007665509000116.tif50170

[0418] The corresponding transcript sequence of the highlighted portion is as follows: Sequence Listing 3 TIFF0007665509000117.tif18170

[0419] Oligonucleotides complementary to exon 53 of the DMD gene useful according to the present invention include, but are not limited to: 5' CAT GTT CTT GTG GAA GTC TTG GCC TCC GTT GTC AAC TTA CTT TAC AAT 7' (SEQ ID NO: 407) and oligonucleotides provided in Table 4. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0420] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 76-240. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 76-240, and the oligonucleotide has a length of 14 to 20 nucleotides. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 76-240, and the oligonucleotide has a length of 14 to 19 nucleotides. In one embodiment, the oligonucleotide has a length of 14 nucleotides. In one embodiment, the oligonucleotide has a length of 15 nucleotides. In one embodiment, the oligonucleotide has a length of 16 nucleotides. In one embodiment, the oligonucleotide has a length of 17 nucleotides. In one embodiment, the oligonucleotide has a length of 18 nucleotides. In one embodiment, the oligonucleotide has a length of 19 nucleotides.

[0421] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 76-240, and the oligonucleotide has a length of 18 nucleotides. In such an embodiment, the oligonucleotide is an 18-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 76-106. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 76-106.

[0422] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 76-240, and the oligonucleotide has a length of 17 nucleotides. In such an embodiment, the oligonucleotide is a 17-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 107-138. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 107-138.

[0423] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 76-240, and the oligonucleotide has a length of 16 nucleotides. In such an embodiment, the oligonucleotide is a 16-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 139-171. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 139-171.

[0424] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 76-240, and the oligonucleotide has a length of 15 nucleotides. In such an embodiment, the oligonucleotide is a 15-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 172-205. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 172-205.

[0425] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 76-240, and the oligonucleotide has a length of 14 nucleotides. In such an embodiment, the oligonucleotide is a 14-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 206-240. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 206-240.

[0426] Exon 45 The sequence of exon 45 of the DMD gene (SEQ ID NO: 408) is shown below: Sequence Listing 4 TIFF0007665509000122.tif44170

[0427] The corresponding transcript sequence of the highlighted portion is as follows: Sequence Listing 5 TIFF0007665509000123.tif18170

[0428] Oligonucleotides complementary to exon 45 of the DMD gene useful according to the present invention include, but are not limited to: 5' CC ATAGAATGTC CTTGAGGTCC TACCGTAACC CGTCGCCGTT TGACA 7' (SEQ ID NO: 410) and any one of the sequences shown in Table 5. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0429] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 241 to 400. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 241 to 270, and the oligonucleotide has a length of 14 to 20 nucleotides. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 241 to 400, and the oligonucleotide has a length of 14 to 19 nucleotides. In one embodiment, the oligonucleotide has a length of 14 nucleotides. In one embodiment, the oligonucleotide has a length of 15 nucleotides. In one embodiment, the oligonucleotide has a length of 16 nucleotides. In one embodiment, the oligonucleotide has a length of 17 nucleotides. In one embodiment, the oligonucleotide has a length of 18 nucleotides. In one embodiment, the oligonucleotide has a length of 19 nucleotides.

[0430] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 241-400, and the oligonucleotide has a length of 18 nucleotides. In such an embodiment, the oligonucleotide is an 18-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 241-270. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 241-270.

[0431] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 241-400, and the oligonucleotide has a length of 17 nucleotides. In such an embodiment, the oligonucleotide is a 17-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 271-301. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 271-301.

[0432] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 241-400, and the oligonucleotide has a length of 16 nucleotides. In such an embodiment, the oligonucleotide is a 16-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 302-333. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 302-333.

[0433] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 241-400, and the oligonucleotide has a length of 15 nucleotides. In such an embodiment, the oligonucleotide is a 15-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 334-366. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 334-366.

[0434] In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 241-400, and the oligonucleotide has a length of 14 nucleotides. In such an embodiment, the oligonucleotide is a 14-mer. In one embodiment, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 367-240. In one embodiment, the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 367-240.

[0435] The present invention also provides oligonucleotides complementary to the intronic splicing silencer N1 (ISS-N1) in spinal muscular atrophy, for example, TCACTTTCATAATGCTGG (SEQ ID NO: 411).

[0436] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture, and transgenic biology, which are within the skill of the art. For example, Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Ausubel et al. al.,1992),Current Protocols in Molecular Biology(John Wiley&Sonsincluding periodic updates);Glover,1985,DNA Cloning(IRL PressOxford);Anand,1992;Guthrie and Fink,1991;Harlow and Lane,1988,Antibodies,(Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY);Jakoby and Pastan,1979;Nucleic Acid Hybridization(BDHames&S.J.Higgins eds.1984);Transcription And Translation(BDHames&S.J.Higgins eds.1984);Culture Of Animal Cells(RIFreshney,Alan R.Liss,Inc.,1987);Immobilized Cells And Enzymes(IRL Press,1986);B.See Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1986); Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ. of Oregon Press, Eugene, 2000).

[0437] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used to implement or test the present invention, and suitable methods and materials are described below.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will prevail.

[0438] In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting of the various embodiments of the present invention described herein. [Example]

[0439] Example 1 Affinity of alpha anomeric oligonucleotides for complementary parallel RNAs. The affinity for complementary parallel RNA was assessed for several alpha anomeric oligonucleotides by UV melting experiments (Table 1).

[0440] The melting temperatures ranged from 53.3°C to 77.0°C, indicating good affinity of the alpha anomeric oligonucleotides for their RNA complements. [Table 6]

[0441] Example 2 Stability of alpha-anomeric oligonucleotides under acidic conditions. The acidic stability of ON1 was assessed by diluting ON1 to 10 μM in acetate buffer (0.1 M, pH 4.5) and incubating the resulting solution at 37 °C for 24 h. Untreated ON1 was used as a reference. The integrity of ON1 was measured by LC-MS. No differences were observed between the chromatograms and fragmentation patterns of untreated ON1 (Figure 1A, Figure 1B) and ON1 treated under acidic conditions for 24 h (Figure 1C, Figure 1D). This experiment demonstrates the stability of alpha-anomeric oligonucleotides in acidic conditions that may be encountered, for example, in the lysosomal compartment of a cell.

[0442] Example 3 Thermal stability of alpha anomeric oligonucleotides The thermal stability of ON1 was assessed by diluting it to 10 μM in PBS solution (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO, pH 7.4) and incubating the resulting solution at 95 °C for 60 min. Untreated ON1 was used as a reference. The integrity of ON1 was measured by LC-MS. No differences were observed between the chromatograms and fragmentation patterns of untreated ON1 (Figures 2A and 2B) and ON1 heated at 95 °C (Figures 2C and 2D). This experiment demonstrates the chemical stability of alpha-anomeric oligonucleotides in aqueous solution.

[0443] Example 4 Biostability of alpha-anomeric oligonucleotides ON1 and its corresponding native oligonucleotide were diluted to 10 μM in a 1:1 mixture of HO and mouse serum (Sigma). Reactions were performed in 20 μL volumes and incubated at 37°C. Control reactions were performed by incubating 10 μM of the oligonucleotide in HO at 37°C for 24 hours. The reaction was stopped at specific times (1, 2, 4, and 24 hours) by adding 20 μL of formamide. The resulting mixtures were stored at -20°C, heat-denatured at 90°C for 5 minutes, and then analyzed by 20% denaturing PAGE (Figure 3). Visualization was performed by staining with stains-all solution according to standard protocols. The experimental results indicate that the native DNA strand was already completely digested after 4 hours, and ON1 remained completely stable even after 24 hours.

[0444] Example 5 Binding of alpha anomeric oligonucleotides conjugated to lipid groups to albumin The binding of ON1 to albumin was evaluated by a mobility shift assay (Figure 4). Test solutions were prepared by incubating ON1 at 40 μM in PBS solution (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO, pH 7.4) containing 0, 0.1, 0.2, 0.3, 0.5, 0.7, 1.0, and 1.5 albumin equivalents (mouse serum albumin, lyophilized powder, ≥96% (Sigma-Aldrich)) for 1 h at 37 °C. Ten μL of each sample was analyzed by 10% native PAGE (40 V, 170 min, 7 °C). Visualization was performed by staining all solutions according to standard protocols. The lower band indicates the presence of uncomplexed ON1, and the upper band indicates the presence of ON1 complexed to albumin. This experiment demonstrates that ON1 can efficiently bind to albumin at albumin concentrations of 0.3 equivalents or greater.

[0445] Quantification of ON1 albumin binding was assessed by ultrafiltration experiments (Figure 5). Briefly, test solutions were prepared by incubating ON1 at 55 μM in PBS solution (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO, pH = 7.4) containing 0, 0.1, 0.2, 0.3, 0.4, 0.4, 0.6, and 0.7 albumin equivalents (albumin from mouse serum, lyophilized powder, ≥96% (Sigma-Aldrich)) for 1 h at 37 °C. The solution was then filtered through a Spin Column (Amicon Ultra-0.5 Centrifugal Filter Unit (Sigma-Aldrich)). The absorbance of ON1 in the filtrate was measured using a Nanodrop spectrophotometer, and the percentage of uncomplexed ON1 was calculated by reference to a solution containing 0 equivalents of albumin. Experimental results show that at 0.3 equivalents of albumin, only 14% of the oligonucleotide remains uncomplexed in solution.

[0446] The binding of ON1 to albumin in mouse serum (Sigma-Aldrich) was evaluated by a mobility shift assay (Figure 6). Test solutions were prepared by incubating ON1 at 40 μM in PBS solutions (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO, pH 7.4) containing 25% glycerol and 0%, 1.25%, 5.0%, 12.5%, and 25.0% mouse serum for 1 h at 37°C. Control solutions were prepared by incubating ON1 at 40 μM in PBS solutions containing 25% glycerol and 80 μM mouse albumin for 1 h at 37°C. 10 μL of each sample was analyzed by 15% native PAGE (60 V, 260 min). Visualization was performed by staining all solutions according to standard protocols. The lower band indicates the presence of uncomplexed ON1, and the upper band indicates the presence of ON1 complexed with albumin. The experimental results demonstrate that ON1 can efficiently bind to albumin in serum.

[0447] Example 6 Presence and dissolution of aggregates The formation and dissolution of aggregates was analyzed using a Zetasizer Nano ZS (Figure 7). ON1 was dissolved at a concentration of 7.5 mg / mL in a PBS solution (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO, pH 7.4). The initial presence of nanoparticles was recorded (0 min). The solution was then heated to 95 °C, and the presence of nanoparticles was recorded after 10, 20, and 30 minutes of heating. The solution was then left at room temperature for 24 hours, and the presence of nanoparticles was recorded again. Initially, a strong signal was observed for particles with a size of approximately 1000 nm. Heating the solution eliminated the signal. The experimental results indicate that oligonucleotides conjugated to lipophilic moieties form aggregates in aqueous solution. However, heating the solution at 95 °C for at least 20 minutes ensures the dispersion of the aggregates. After leaving the solution at room temperature for 24 hours, the aggregates did not reappear.

[0448] Example 7 Determination of exon skipping efficiency Exon skipping involves the use of antisense oligonucleotides to remove one or more exons from mature mRNA.By using exon skipping, one or more exons can be removed from mature mRNA to obtain in-frame mature mRNA.Exon skipping can be induced by binding of antisense oligonucleotides that target one or both splice sites or internal exon sequences.Since an exon is only included in mRNA when both splice sites are recognized by the spliceosome complex, splice sites are obvious targets for antisense oligonucleotides.

[0449] To determine whether the abc-DNA lipid group conjugate oligonucleotide of the present invention causes exon skipping in the pre-mRNA of a gene of interest, cells are incubated with an oligonucleotide conjugate targeting a given exon for a certain period of time.In certain embodiments, cells are transfected with lipofectamine.Exon skipping is detected by using reverse transcription polymerase chain reaction (RT-PCR) or DNA sequencing.Total RNA is extracted from cells, and RT-PCR is performed on the entire target exon, and the size of the RT-PCR product is evaluated by gel electrophoresis.If exon skipping occurs, the product will not contain the targeted exon, and the size of this product will be shorter as expected compared to the product containing the target exon.Similarly, mature mRNA can be sequenced across the entire target exon to determine whether the sequence of the target exon is absent in mature mRNA.

[0450] To further determine the effect of the abc-DNA lipid-group conjugated oligonucleotides of the present invention, dystrophin restoration is verified by Western blot of samples taken from muscle biopsies, and the % dystrophin-positive myofibers are determined by microscopic examination.

[0451] Targeted skipping of specific exons can convert the DMD phenotype into a milder BMD phenotype. Exon skipping is detected by incubating differentiated human myoblasts, muscle cells derived from DMD patients or healthy patients with antisense abc-DNA lipid-conjugated oligonucleotides that bind to the pre-mRNA of the DMD gene. Alternatively, as described in this example, the cells are derived from MDX mice, a mouse model of DMD. In addition to comparing the level of exon skipping in normal cells with that in DMD cells, the level of exon skipping in cells derived from DMD patients or MDX mice is compared to the level of exon skipping absent in the abc-DNA lipid-conjugated oligonucleotide. In certain embodiments, the activity of the abc-DNA lipid-conjugated oligonucleotide of interest is compared to the level of exon skipping after administration of eteplirsen or drisapersen.

[0452] In this example, the concentration of antisense oligonucleotides was estimated by measuring the absorbance of diluted aliquots at 260 nm. Specific amounts of antisense oligonucleotides (AONs) were then tested for their ability to induce exon skipping in an in vitro assay, as described below.

[0453] Briefly, experiments were performed on immortalized mouse control myoblast cultures (C2C12) or human control myoblast cultures (KM155). Cells were grown and differentiated into myotubes using standard culture techniques. Cells were transfected with AONs using lipofectamine for mouse cell culture and oligofectamine for human cell culture as transfection reagents. Complementary AONs with a 2'-OMe-phosphorodithioate (2OMePS) backbone and scrambled (nonfunctional) 2OMePS AONs were used as positive and negative controls, respectively.

[0454] After 24 hours, total RNA was extracted and subjected to molecular analysis. To study the target region of dystrophin pre-mRNA or induced exon rearrangement, reverse transcriptase amplification (RT-PCR) using a two-step (nested) PCR reaction was performed.

[0455] To analyze AONs aimed at inducing exon 23 skipping, RT-PCR was performed on the region spanning exon 23. After cDNA synthesis, a first round of PCR was performed using specific primers in mouse exons 21 and 26 (region 21-26), and a second round of PCR was performed using specific primers in mouse exons 22 and 24 (region 22-24).

[0456] To analyze AONs aimed at inducing exon 51 skipping, RT-PCR was performed on the region spanning exon 51. After cDNA synthesis, a first-round PCR was performed using specific primers in human exons 48 and 53 (region 48–53), and a second-round PCR was performed using specific primers in human exons 49 and 52 (region 49–52).

[0457] The expected product sizes of unskipped and skipped products were calculated. The intensities of the reaction products were estimated using agarose gels containing size standards. This requires consideration of potential bias. This is because short or exon-skipped products tend to be amplified more efficiently than larger products, leading to an overestimation of skipping efficiency. Bands representing exon-skipped products can be measured in mouse cells (Figure 8) or human cells (Figures 9A and 9B). Experimental results demonstrate the ability of alpha-anomeric oligonucleotides to regulate gene expression in vitro. The exon-skipping ability of the conjugates of the present invention has been confirmed in vivo in the mdx mouse model of muscular dystrophy. To this end, mdx23 mice received 12 weeks of intravenous injections (50 mg / kg / week) of the abc-DNA lipid-conjugated oligonucleotide of the present invention, which has a sequence comprising SEQ ID NO: 412. After treatment, diaphragm and gastrocnemius muscle tissues were isolated and exon skipping was measured.

[0458] Example 8 Exon skipping in the hDMDdel52 / mdx mouse model The effectiveness of exon skipping is determined in the hDMDdel52 / mdx mouse model of muscular dystrophy. Mice are intravenously injected with abc-DNA lipid-conjugated oligonucleotides containing the sequence of SEQ ID NO: 418, the corresponding phosphorodiamidate morpholino oligomer (PMO), or saline. Mice are injected with the oligonucleotide 12 times a week (50 mg / kg / week). After treatment, the following tissues are isolated: heart, diaphragm, tibialis anterior, gastrocnemius, quadriceps, triceps, brain, liver, kidney, and exon skipping is determined. In certain embodiments, mice are treated with etiplasen or drisapersen instead of the abc-DNA lipid-conjugated oligonucleotide. Exon skipping is determined by RT-PCR, Western blot, immunofluorescence, and / or digital PCR.

Claims

1. An oligonucleotide-lipid group conjugate, wherein the oligonucleotide comprises residues, at least two of which are alpha anomeric bicycloDNA (abc-DNA) residues linked by phosphodiester bonds, and the lipid group is covalently attached to the oligonucleotide; the lipid group is a fatty acid-derived group having a hydrocarbon chain of 8 to 24 carbon atoms; The abc-DNA residue has the formula (V): 【Chemistry 1】 wherein, independently for each of said at least two abc-DNA residues of formula (V): T ３ Or T ４ is a nucleoside linking group, T ３ and T ４ The other is OR １ , OR ２ , a 5' end group, a 7' end group, or a nucleoside linking group; R １ is H or a hydroxyl protecting group, R ２ is represented by formula (VII): 【change】 (In the formula, W represents O, S, or Se, or W represents an electron pair; R ３ and R ４ are each independently H, a halogen, OH, OR ５ , N.R. ６ R ７ , S.H., S.R. ８ , C １ -C ６ Alkyl, C １ -C ６ Haloalkyl, C １ -C ６ Alkoxy, C １ -C ６ Haloalkoxy, C １ -C ６ is an aminoalkyl; R ５ is cyano, nitro, halogen, -NHC(O)C １ -C ３ Alkyl, -NHC(O)C １ -C ３ Haloalkyl, C １ -C ３ C optionally substituted with alkylsulfonyl １ -C ９ alkyl; each independently of the others is cyano, nitro, halogen, C １ -C ４ Alkoxy, C １ -C ４ Haloalkyl, C １ -C ４ Haloalkoxy, NHC(O)C １ -C ３ Alkyl, NHC(O)C １ -C ３ Haloalkyl, C １ -C ３ aryl, optionally substituted with alkylsulfonyl; １ -C ６ Alkylene aryl, C １ -C ６ an alkylenediaryl; an acetyl; a hydroxyl protecting group; R ６ and R ７ are each independently hydrogen; cyano, nitro, halogen, C ２ -C ６ Alkenyl, C ３ -C ６ Cycloalkyl, C １ -C ３ C optionally substituted with alkoxy １ -C ９ Alkyl; cyano, nitro, halogen, C １ -C ３ Alkyl, C １ -C ３ aryl, optionally substituted with alkoxy; an amino protecting group or, together with the nitrogen atom to which they are attached, form a heterocycle, the heterocycle being selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, and homopiperazine, the heterocycle being selected from C １ -C ３ optionally substituted with alkyl; R ８ is a thiol protecting group, The wavy line is OR ２ (showing the bond of the aryl group to the oxygen) is the phosphorus portion of Bx is a nucleobase, oligonucleotide-lipid group conjugate.

2. The oligonucleotide-lipid group conjugate of claim 1, wherein all of the residues of the oligonucleotide are abc-DNA residues.

3. 3. The oligonucleotide-lipid group conjugate of claim 1 or 2, wherein the at least two abc-DNA residues are linked to adjacent residues via phosphodiester bonds.

4. The oligonucleotide-lipid group conjugate of any one of claims 1 to 3, wherein all of the residues of the oligonucleotide are abc-DNA residues and are linked via phosphodiester bonds.

5. The oligonucleotide-lipid group conjugate of any one of claims 1 to 4, wherein the lipid group is covalently attached to a terminal residue of the oligonucleotide.

6. 4. The oligonucleotide-lipid group conjugate of any one of claims 1 to 3, wherein the oligonucleotide comprises residues linked through a phosphorus-containing nucleoside linking group selected from the group consisting of phosphodiester linking groups, phosphotriester linking groups, phosphorothioate linking groups, phosphorodithioate linking groups, phosphonate linking groups, phosphonothioate linking groups, phosphinate linking groups, phosphorothioamidate linking groups, and phosphoramidate linking groups.

7. The oligonucleotide-lipid group conjugate of any one of claims 1 to 6, wherein the lipid group is attached to the oligonucleotide via a linker.

8. the linker is a hydrocarbon linker or a polyethylene glycol (PEG) linker; or 8. The oligonucleotide-lipid group conjugate of claim 7, wherein the linker is selected from the group consisting of an amino-alkyl-phosphorothioate linker, an amino-PEG-phosphorothioate linker, an alpha-carboxylate-amino-alkyl phosphorothioate linker, and an alpha-carboxylate-amino-PEG-phosphorothioate linker.

9. 9. The oligonucleotide-lipid group conjugate of any one of claims 1 to 8, wherein the fatty acid-derived group comprises a carboxylic acid group and / or the fatty acid is hexadecanoic acid.

10. The oligonucleotide-lipid group conjugate of any one of claims 1 to 9, wherein the lipid group is attached to the oligonucleotide via a thiophosphate group.

11. The oligonucleotide-lipid group conjugate of any one of claims 1 to 10, wherein the oligonucleotide-lipid group conjugate binds to a pre-mRNA corresponding to a portion of exon 51 of the Duchenne muscular dystrophy (DMD) gene.

12. The oligonucleotide-lipid group conjugate of any one of claims 1 to 10, wherein the oligonucleotide-lipid group conjugate comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39, and 51-55.

13. The oligonucleotide-lipid group conjugates are as shown in Table 3:

11. The oligonucleotide-lipid group conjugate of any one of claims 1 to 10, comprising any one of the sequences provided in

14. The oligonucleotide-lipid group conjugate of any one of claims 1 to 10, wherein the oligonucleotide-lipid group conjugate binds to a pre-mRNA corresponding to a portion of exon 53 of the DMD gene.

15. The oligonucleotide-lipid group conjugates are as shown in Table 4:

11. The oligonucleotide-lipid group conjugate of any one of claims 1 to 10, comprising any one of the sequences provided in

16. The oligonucleotide-lipid group conjugate of any one of claims 1 to 10, wherein said oligonucleotide-lipid group conjugate binds to a pre-mRNA corresponding to a portion of exon 45 of the DMD gene.

17. The oligonucleotide-lipid group conjugates are as shown in Table 5:

11. The oligonucleotide-lipid group conjugate of any one of claims 1 to 10, comprising any one of the sequences provided in

18. The oligonucleotide-lipid group conjugate of any one of claims 1 to 17, wherein the oligonucleotide comprises from 12 to 24 residues.

19. A pharmaceutical composition comprising an oligonucleotide-lipid group conjugate according to any one of claims 1 to 18 in combination with a suitable carrier.

20. 20. Use of an oligonucleotide-lipid group conjugate according to any one of claims 1 to 18 in the manufacture of a medicament for altering expression of a gene by allowing hybridization of the oligonucleotide-lipid group conjugate to RNA expressed from said gene, wherein said oligonucleotide comprises a sequence complementary to a portion of said RNA.

21. 14. Use of the oligonucleotide-lipid group conjugate of any one of claims 1 to 13 in the manufacture of a medicament for inducing skipping of exon 51 of human dystrophin pre-mRNA in a subject with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) or in a cell derived from said subject, wherein said oligonucleotide-lipid group conjugate comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39 and 51-55, and wherein said oligonucleotide-lipid group conjugate induces skipping of said exon in said subject or said cell, and wherein the mRNA generated from the skipping of exon 51 of the human dystrophin pre-mRNA encodes a functional dystrophin protein or a Becker-subject dystrophin protein.

22. 14. Use of the oligonucleotide-lipid group conjugate of any one of claims 1 to 13 in the manufacture of a medicament for treating Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) in a subject or in a cell derived from said subject, wherein said oligonucleotide-lipid group conjugate comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 22-24, 36-39 and 51-55, wherein said oligonucleotide-lipid group conjugate induces skipping of exon 51 of human dystrophin pre-mRNA, and wherein the mRNA generated from the skipping of exon 51 of the human dystrophin pre-mRNA encodes a functional dystrophin protein or a Becker-subject dystrophin protein.

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