Tricyclo-DNA nucleoside precursors and processes for preparing the same

Abstract

To provide tricyclo-DNA nucleoside precursors and processes for preparing the same.SOLUTION: The present invention relates to a process for preparing tc-DNA nucleoside precursors, the resulting tc-DNA nucleosides, and oligonucleotides comprising such tc-DNA nucleosides. In an embodiment of the invention, the process includes use of a carbene precursor. In an embodiment, the invention includes processes for preparing a tc-DNA nucleoside precursor of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V) or Formula (VI).SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to tricyclo-deoxyribonucleic acid (tc-DNA) nucleoside precursor compounds and processes for preparing such compounds as building blocks for oligomers. [Background technology]

[0002] Background of the Invention Antisense technology is an effective means for reducing the expression of specific gene products, and therefore can be useful in therapeutic, diagnostic and research applications.Generally, the principle behind antisense technology is that antisense oligomeric compound (nucleotide or its analogue sequence) hybridizes with target nucleic acid and modulates the activity or function of gene expression, such as transcription and / or translation.

[0003] Antisense oligomeric compounds may be prepared from chemically modified antisense oligonucleotides, which may contain a variety of different structural variations depending on the therapeutic strategy. For example, tricyclo-deoxyribonucleic acid (tc-DNA) is a conformationally restricted DNA analog.

[0004] There is a need in the art for a process that allows for the bulk preparation of tc-DNA nucleoside precursors that can be used as building blocks for tc-DNA-containing antisense oligonucleotide-based therapies. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention In one embodiment, the present invention provides a compound of formula I, formula II, formula III, formula IV, formula V or formula VI: [ka] a process for preparing a tc-DNA nucleoside precursor of wherein X can be alkoxy; T 1 and T 2 are OR 1 where R 1 is H or a hydroxyl protecting group, q 1 , q 2 , q 3 , q 4 and q 5 are each independently hydrogen, halogen, or C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 2~6 Alkenyl, substituted C 2~6 Alkynyl, and -(CH2) n -C(O)-R 2 (where n is 0 to 6, and R 2 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 alkyl); z 1 and z 2 are each independently hydrogen, C 1~6 Alkyl, C 1~6 Alkoxyl, OC 2~6 Alkenyl, OC 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 1~6 Alkoxy, substituted OC 2~6 Alkenyl and substituted OC 2~6 Alkynyl halogen may be selected from the group consisting of:

[0006] In one embodiment, the present invention provides a tc-DNA nucleoside precursor of Formula VII, Formula VIII, or Formula IX: [ka] The present invention includes a process for preparing the

[0007] In one embodiment, the invention includes a method for preparing a tc-DNA nucleoside precursor of any one of Formulas I-IX, said method comprising: a. preparing a carbene precursor at a carbene preparation temperature; b. cyclopropanation of said carbene precursor with a compound of Formula X, Formula XI or Formula XII at cyclopropanation temperatures: [ka] adding; and c. providing the tc-DNA nucleoside precursor of one of formulas I-IX, wherein Y can be alkoxy; T 3 and T 4 are OR 5 where R 5 can be H or a hydroxyl protecting group, q 6 , q 7 and q 8 are each independently hydrogen, halogen, or C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 2~6 Alkenyl, substituted C 2~6 Alkynyl, and -(CH2) n -C(O)-R 6 (where n is 0 to 6, and R 6 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 alkyl); z 3 and z 4 are each independently hydrogen, C 1~6 Alkyl, C 1~6 Alkoxyl, OC 2~6 Alkenyl, OC 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 1~6 Alkoxy, substituted OC 2~6 Alkenyl and substituted OC2~6 Alkynyl Halogens The compound may be selected from the group consisting of:

[0008] In one embodiment, the invention includes a method for preparing a tc-DNA nucleoside precursor of any one of Formulas I-IX, said method comprising: a. preparing a carbene precursor at a carbene preparation temperature; b. A compound of Formula X, Formula XI or Formula XII: [ka] preparing a solution of c. adding the solution of the compound of Formula X, Formula XI, or Formula XII to the carbene precursor at a cyclopropanation temperature; and d. providing the tc-DNA nucleoside precursor of one of formulas I-IX, wherein Y can be alkoxy; T 3 and T 4 are OR 5 where R 5 can be H or a hydroxyl protecting group, q 6 , q 7 and q 8 are each independently hydrogen, halogen, or C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 2~6 Alkenyl, substituted C 2~6 Alkynyl, and -(CH2) n -C(O)-R 6 (where n is 0 to 6, and R 6 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 alkyl); z 3 and z 4 are each independently hydrogen, C 1~6 Alkyl, C 1~6Alkoxyl, OC 2~6 Alkenyl, OC 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 1~6 Alkoxy, substituted OC 2~6 Alkenyl and substituted OC 2~6 Alkynyl halogen may be selected from the group consisting of:

[0009] In one embodiment, the step of preparing the carbene precursor comprises the step of reacting a Lewis acid catalyst (e.g., ZnEt) and R 7 In some embodiments, R 7 CH2, CH-C 1~6 Alkyl, CH-C 2~6 Alkenyl, CH-C 2~6 Alkynyl, substituted CH-C 1~6 Alkyl, substituted CH-C 2~6 Alkenyl, substituted CH-C 2~6 Alkynyl and CH—(CH2) n -C(O)-R 8 (where n is 0 to 6, and R 8 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 In some embodiments, R 7 is alkyl. In some embodiments, R 7 is CH2.

[0010] In some embodiments, preparing a carbene precursor comprises reacting a Lewis acid catalyst (e.g., ZnEt) and R 7 adding a carbene additive to the mixture of I2, wherein the carbene additive is selected from the group consisting of substituted or unsubstituted alkyl alcohols, carboxylic acids, and phosphates.

[0011] In some embodiments, preparing a carbene precursor comprises reacting a carbene additive and R 7The method includes adding a Lewis acid catalyst (e.g., ZnEt2) to a mixture of I2, wherein the carbene additive is selected from the group consisting of substituted or unsubstituted alkyl alcohols, carboxylic acids, and phosphates.

[0012] In some embodiments, preparing the carbene precursor comprises reacting a mixture of a Lewis acid catalyst (e.g., ZnEt) and a carbene additive with R 7 adding I2, wherein the carbene additive is selected from the group consisting of substituted or unsubstituted alkyl alcohols, carboxylic acids, and phosphates.

[0013] In some embodiments, preparing a carbene precursor comprises adding a Lewis acid catalyst (e.g., ZnEt) to a carbene additive and R 7 adding I2, wherein the carbene additive is selected from the group consisting of substituted or unsubstituted alkyl alcohols, carboxylic acids, and phosphates.

[0014] In some embodiments, preparing a carbene precursor comprises using a Lewis acid catalyst (e.g., ZnEt), R 7 The method includes combining I2 and a carbene additive, wherein the carbene additive is selected from the group consisting of substituted or unsubstituted alkyl alcohols, carboxylic acids, and phosphates.

[0015] In some embodiments, the carbene additive may be an aliphatic alcohol (e.g., a substituted or unsubstituted alkyl alcohol), an aromatic alcohol (e.g., a substituted or unsubstituted phenol), a substituted or unsubstituted carboxylic acid (e.g., trichloroacetic acid), or a substituted or unsubstituted phosphate (e.g., (alkyl-O)P(O)OH or (aryl-O)P(O)OH). In some embodiments, the carbene additive may be a substituted carboxylic acid of formula QCCOH, where each Q may be independently selected from the group consisting of H, Cl, Br, and F. In some embodiments, the carbene additive may be a substituted carboxylic acid of formula QCCOH, where QC may be defined as CCl, CHCl, CHCl, or CF. In some embodiments, the carbene additive may be a substituted alkyl alcohol of formula QCCHOH, where each Q may be independently selected from the group consisting of H, Cl, Br, and F. In some embodiments, the carbene additive may be a substituted carboxylic acid of formula QCCHOH, where QC may be defined as CCl, CHCl, CHCl, or CF. In some embodiments, the carbene additive may be trichloroacetic acid, 2,2,2-trifluoroethanol, trichlorophenol, or (n-BuO)P(O)OH.

[0016] In some embodiments, the carbene precursor is Q3CCO2ZnR 7 I, Q3CCH2OZnR 7 I, (n-BuO)2P(O)OZnR 7 I, (alkyl-O)2P(O)OZnR 7 I, (aryl-O)2P(O)OZnR 7 I or 2,4,6-Cl3C6H2OZnR 7 I, where each Q is independently selected from the group consisting of H, Cl, Br, and F; 7 CH2, CH-C 1~6 Alkyl, CH-C 2~6 Alkenyl, CH-C 2~6 Alkynyl, substituted CH-C 1~6 Alkyl, substituted CH-C 2~6Alkenyl, substituted CH-C 2~6 Alkynyl and CH—(CH2) n -C(O)-R 8 (where n is 0 to 6, and R 8 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 alkyl).

[0017] In some embodiments, the carbene precursor is CCl3CO2ZnR 7 I, CF3CH2OZnR 7 I, (n-BuO)2P(O)OZnR 7 I or 2,4,6-Cl3C6H2OZnR 7 I.

[0018] In some embodiments, the carbene precursor is CCl3CO2ZnCH2I, CF3CH2OZnCH2I, (n-BuO)2P(O)OZnCH2I, or 2,4,6-Cl3C6H2OZnCH2I.

[0019] In one embodiment, the invention includes tc-DNA nucleoside precursors prepared by the methods described herein.

[0020] In one embodiment, the present invention includes tc-DNA nucleosides prepared from the tc-DNA nucleoside precursors described herein.

[0021] In one embodiment, the invention includes tc-DNA-containing oligonucleotides that include the tc-DNA nucleosides described herein.

[0022] The foregoing summary, as well as the following detailed description of the present invention, will be better understood when read in conjunction with the appended drawings. [Brief explanation of the drawings]

[0023] [Figure 1]Figure 1 illustrates a synthetic route from D-mannose to bicyclosugar intermediate 10. The reagents and conditions used in the synthetic route are as follows: (a) MeC(OMe) (4.4 equiv), TsOH (0.003 equiv), 50 °C, 1 h; (b) AcO (2 equiv), pyridine, room temperature, 16 h; (c) 64% aqueous AcOH, 55 °C, 4 h; (d) HC(OMe) (5 equiv), reflux, 1 h; (e) AcO (7.4 equiv), 130 °C, 3 h; (f) MeOH, t-BuOK (0.4 (equivalent), room temperature, 46% yield from D-mannose; (g) NaH (1.7 equiv), DMSO (3 equiv), BrCH2P(Ph)3 (2 equiv), THF, 65 °C, 4 h, 60% yield; (h) Grubbs I (0.005 equiv), CHCl2, room temperature, 16 h, 97% yield; (i) 10% Pd / C, H2, MeOH, room temperature, 16 h, 97% yield; and (j) PCC (1.7 equiv), CHCl2, room temperature, 16 h, 85% yield. Overall yield from D-mannose was 20% over 10 steps.

[0024] [Figure 2] FIG. 2 illustrates an alternative synthetic route to bicyclosugar intermediate 10 from D-ribose. The synthetic route used the following reagents and conditions: (a) Br (1.04 equiv.), NaHCO (2 equiv.), HO, 0–5°C, crude; (b) DMP, acetone, HSO (catalytic amount), Amberlyst, room temperature (crystallization, 67% overall yield for the two steps); (c) NaIO (1.05 equiv.), NaOH (1.1 equiv.), 0°C, BaCl (crude, 78% yield); (d) iPrOH, PPTS (0.02 equiv.), reflux (chromatography, 63% yield); (e) LiCH(O)P(OMe) (1.1 equiv.), THF, −78°C to room temperature (crude, approximately 60% yield); and (f) 10% Pd / C, H, EtOAc, room temperature (chromatography, 95% yield). The overall yield for the six steps from D-ribose was 16%.

[0025] [Figure 3]Figure 3 illustrates the synthetic route from intermediate 10 to alcohol intermediate 15. The reagents and conditions used in the synthetic route are as follows: (a) (EtO)P(O)CHCOEt (1 equivalent), THF, 0°C to room temperature, 88% yield; (b) MCPBA (2.2 equivalents), CHCl, 0 to 40°C, 18 hours; (c) LiAlH (1.5 equivalents), THF, 0°C to room temperature, 1 hour (chromatography, 86% yield); (d) IBX, THF / DMSO, room temperature, 2.5 hours, 91% yield; and (e) MeOH, Amberlite IR-120 (H), room temperature for 16 hours, then 60°C for 2 hours (chromatography, 77% yield).

[0026] [Figure 4] 4 illustrates a synthetic route to tert-butyldimethylsilyl enol ether intermediate 17. The reagents and conditions used in the synthetic route are as follows: (a) TCCA (0.35 equivalents), TEMPO (0.007 equivalents), AcONa (3 equivalents), CHCl, acetone, −15 to 0° C. for 1 hour; and (b) TBDMSCl (1.4 equivalents), DBU (1.6 equivalents), KI (0.1 equivalents), THF, room temperature for 2 hours. Alternatively, the reagents and conditions used in the synthetic route can be (a) DMP, CHCl, room temperature; and (b) (1) LDA, −65° C., followed by (2) TBDMSCl, THF, −65° C. to 0° C. DETAILED DESCRIPTION OF THE INVENTION

[0027] Detailed Description of the Invention 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.The headings used herein are merely for convenience and should not be interpreted as limiting the disclosure of any aspect and embodiment of the present invention.All patents and publications referenced herein are incorporated by reference in their entirety. definition

[0028] The term "oligomeric compound," as used herein, refers to a compound comprising preferably eight or more monomeric subunits linked by internucleoside linking groups, wherein at least two of said eight or more monomeric subunits are tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides.

[0029] The term "monomer subunit," as used herein, refers to α-D-ribonucleosides, β-D-ribonucleosides, α-D-2'-deoxyribonucleosides, β-D-2'-deoxyribonucleosides, naturally occurring nucleosides, modified nucleosides, herein, in particular, tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides and 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy2'-fluoro-arabinonucleosides, 2'-deoxy- ... The term "nucleotides" is meant to include all manner of monomer units suitable for oligomer synthesis, including, typically and preferably, monomer subunits such as nucleotides, hexitol nucleic acid (HNA) nucleosides; and phosphorodiamidate morpholino (PMO) nucleosides, nucleoside mimetics, naturally occurring nucleotides, modified nucleotides, and in this context, particularly, tricyclo-deoxyribonucleic acid (tc-DNA) nucleotides and 2'-modified ribonucleic acid (2'-modified-RNA) nucleotides, and nucleotide mimetics. Typically and preferably, the term "monomer subunit", as used herein, refers to naturally occurring nucleosides and modified nucleosides, and herein particularly refers to ribonucleosides, deoxyribonucleosides, tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonucleosides, hexitol nucleic acid (HNA) nucleosides and phosphorodiamidates. It refers to morpholino (PMO) nucleosides, as well as naturally occurring and modified nucleotides, and herein refers in particular to ribonucleotides, deoxyribonucleotides, tricyclo-deoxyribonucleic acid (tc-DNA) nucleotides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleotides, locked nucleic acid (LNA) nucleotides, peptide nucleic acid (PNA) nucleotides, 2'-deoxy 2'-fluoro-arabinonucleotides, hexitol nucleic acid (HNA) nucleotides, and phosphorodiamidate morpholino (PMO) nucleotides.More preferably, the term "monomer subunit" as used herein refers to modified nucleotides, and here in particular to tricyclo-deoxyribonucleic acid (tc-DNA) nucleotides and 2'-modified ribonucleic acid (2'-modified-RNA) nucleotides.

[0030] The term "alkyl phosphate moiety" as used herein means a C 3~32 alkyl-OP(O)(OH)-O- group, 3~32 Alkyl is independently a C as defined herein. 3~32 alkyl.

[0031] The term "alkylphosphonate moiety," as used herein, refers to a C 1~32 alkyl-OP(O)-O- group, 1~32 Alkyl is independently a C as defined herein. 1~32 alkyl.

[0032] The term "alkyl," as used herein, refers to a straight- or branched-chain hydrocarbon radical containing no unsaturation, consisting solely of carbon and hydrogen atoms, having from 1 to 32 carbon atoms (e.g., (C 1~32 ) alkyl or C 1~32alkyl), which may be, or typically is, attached to the remainder of the molecule by a single bond. Whenever it appears herein, a numerical range such as "1 to 32" refers to each integer within the given range. For example, "1 to 32 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to a maximum of 32 carbon atoms, although this definition is also intended to cover occurrences of the term "alkyl" where no numerical range is specifically specified. Typical alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (used interchangeably with isopropyl; abbreviated interchangeably herein as iPr or Pri), n-butyl, isobutyl, sec-butyl, isobutyl, tert-butyl (used interchangeably with 1,1-dimethylethyl or tert-butyl), n-pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl, and decyl. Unless otherwise specified specifically in the specification, alkyl groups are optionally substituted with one or more substituents which are independently alkenyl, alkoxy, carboxy (-COOH), heteroalkyl, heteroalkenyl, hydroxyl, phosphate (-OP(O)(OH)O-), phosphonate (-OP(O)O-), halogen, preferably iodine, or a phenyl group (-CH) optionally substituted with a carboxy group. Preferably, the term "alkyl," as used herein, refers to an unsubstituted alkyl as defined herein.

[0033] The term "alkylene," as used herein, refers to a straight or branched chain hydrocarbon biradical derived from an alkyl, as defined herein, where one hydrogen of said alkyl is cleaved to produce a second radical of said alkylene. Examples of alkylene are, by way of illustration, -CH-, -CH-CH-, -CH(CH)-, -CH-CH-CH-, -CH(CH)-CH-, or -CH(CHCH)-.

[0034] The term "alkenyl," as used herein, refers to a straight- or branched-chain hydrocarbon radical group, consisting solely of carbon and hydrogen atoms, containing at least one double bond and having from 2 to 32 carbon atoms (i.e., (C 2~32 ) alkenyl or C 2~32 Alkenyl), which may be, or typically is, attached to the remainder of the molecule by a single bond. Whenever it appears herein, a numerical range such as "2 to 32" refers to each integer within the given range; for example, "2 to 32 carbon atoms" means that the alkenyl group can consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, etc., up to a maximum of 32 carbon atoms. Typical alkenyl groups include, but are not limited to, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, and penta-1,4-dienyl. Where applicable, e.g., whenever substituted, each double bond can be in either the (E)- or (Z)-configuration. Thus, alkenyl, where applicable, each of said double bonds can include either its (E)-configuration, its (Z)-configuration, and mixtures thereof in any ratio. Unless otherwise specified herein, Unless otherwise specified, alkenyl groups are optionally substituted by one or more substituents which are independently alkenyl, alkoxy, carboxy (-COOH), heteroalkyl, heteroalkenyl, hydroxyl, phosphate (-OP(O)(OH)O-), phosphonate (-OP(O)O-), halogen, preferably iodine, or a phenyl group (-CH) optionally substituted with a carboxy group. Preferably, the term "alkenyl," as used herein, refers to an unsubstituted alkenyl as defined herein.

[0035] The term "alkenylene," as used herein, refers to a straight or branched chain hydrocarbon biradical derived from an alkenyl, as defined herein, where one hydrogen of said alkenyl has been cleaved to generate a second radical of said alkenylene.

[0036] The term "alkynyl" refers to a straight- or branched-chain hydrocarbon radical group, consisting solely of carbon and hydrogen atoms, containing at least one triple bond and having from 2 to 10 carbon atoms (i.e., (C 2~32 ) alkynyl or C 2~32 Alkynyl). Whenever it appears herein, a numerical range such as "2 to 32" refers to each integer within the given range; for example, "2 to 32 carbon atoms" means that the alkynyl group can consist of 2 carbon atoms, 3 carbon atoms, etc., up to a maximum of 32 carbon atoms. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl. Unless otherwise specified specifically in the specification, an alkynyl group is optionally substituted with one or more substituents, independently, an alkenyl, a carboxy group (-COOH), a heteroalkyl, a heteroalkenyl, a phosphate group (-OP(O)(OH)O-), a phosphonate group (-OP(O)O-), a halogen, preferably iodine, or a phenyl group (-CH) optionally substituted with a carboxy group. Preferably, the term "alkynyl," as used herein, refers to an unsubstituted alkynyl as defined herein.

[0037] The term "alkynylene," as used herein, refers to a straight or branched chain hydrocarbon biradical derived from an alkynyl, as defined herein, where one hydrogen of said alkynyl has been cleaved to generate a second radical of said alkynylene.

[0038] The term "alkoxy" refers to groups of -O-alkyl, including straight, branched, and combinations thereof, of 1 to 32 carbon atoms attached to the parent structure through an oxygen. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, and cyclohexyloxy. "Lower alkoxy" refers to an alkoxy group containing 1 to 6 carbons, and also includes (C1~6 )Alkoxy or OC 1~6 Also called alkyl.

[0039] The term "substituted alkoxy" refers to an alkoxy in which the alkyl component is substituted (i.e., -O-(substituted alkyl)). Unless otherwise stated specifically in the specification, the alkyl portion of an alkoxy group is optionally substituted with one or more substituents which are independently alkenyl, carboxyl (-COOH), heteroalkyl, heteroalkenyl, phosphate (-OP(O)(OH)O-), phosphonate (-OP(O)O-), halogen, preferably iodine, or a phenyl group (-CH) optionally substituted with a carboxyl group.

[0040] The term "acyl" refers to the groups (alkyl)-C(O)-, (aryl)-C(O)-, (heteroaryl)-C(O)-, and (heteroalkyl)-C(O)-, which are attached to the parent structure through a carbonyl functionality. Unless otherwise stated specifically in the specification, the alkyl, aryl, or heteroaryl portions of the acyl group independently represent: Optionally substituted with one or more substituents which are alkenyl, carboxy group (-COOH), heteroalkyl, heteroalkenyl, phosphate group (-OP(O)(OH)O-), phosphonate group (-OP(O)O-), halogen, preferably iodine, or a phenyl group (-CH) optionally substituted with a carboxy group.

[0041] Unless otherwise specified herein, the term “amino” or “amine” refers to —N(R a )2 radical groups, each R a are independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl. a ) Two groups have two R aWhen substituted, these may be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, —N(R a )2 is meant to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. Unless otherwise specified specifically in the specification, amino or amine groups are optionally substituted with one or more substituents which are independently alkenyl, carboxy (-COOH), heteroalkyl, heteroalkenyl, phosphate (-OP(O)(OH)O-), phosphonate (-OP(O)O-), halogen, preferably iodine, or a phenyl group (-CH) optionally substituted with a carboxy group.

[0042] The term "aromatic" or "aryl" or "Ar" refers to a carbocyclic (e.g., phenyl, fluorenyl, and naphthyl) aromatic radical having 6 to 10 ring atoms (e.g., C6-C8) with at least one ring having a conjugated pi-electron system. 10 Aromatic or C6-C 10 (aryl). Divalent radicals formed from substituted benzene derivatives and having a free valence on a ring atom are named substituted phenylene radicals. Divalent radicals derived from monovalent polycyclic hydrocarbon radicals, named "-yl" by removing one hydrogen atom from the free valence carbon atom, are named by adding "-idene" to the name of the corresponding monovalent radical; for example, a naphthyl group with two points of attachment is called naphthylidene. Whenever appearing herein, numerical ranges such as "6 to 10" refer to individual integers within the given range; for example, "6 to 10 ring atoms" means that the aryl group may consist of 6 ring atoms, 7 ring atoms, etc., up to a maximum of 10 ring atoms. The term includes monocyclic or fused-ring polycyclic (i.e., rings sharing adjacent pairs of ring atoms) groups.

[0043] The term "aralkyl" or "arylalkyl" refers to an (aryl)alkyl-radical, wherein the aryl and alkyl are optionally substituted with one or more substituents disclosed herein and described as suitable substituents for aryl and alkyl, respectively.

[0044] The terms "carboxyl" or "carboxylic" are used interchangeably herein and refer to the -(C=O)OH radical.

[0045] The term "cycloalkyl" refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen and can be saturated or partially unsaturated. A cycloalkyl group is a group having 3 to 10 ring atoms (i.e., (C 3~10 ) cycloalkyl or C 3~10 Whenever it appears herein, a numerical range such as "3 to 10" refers to each integer within the given range, for example, "3 to 10 carbon atoms" means that the cycloalkyl group consists of 3 carbon atoms, etc., and can contain up to 10 carbon atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohex ... cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, and the like.

[0046] The term "fluoroalkyl" refers to an alkyl radical, as defined above, substituted by one or more fluoro radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, etc. The alkyl portion of the fluoroalkyl radical can be optionally substituted as defined above for an alkyl group.

[0047] The term "halogen" as used herein refers to fluorine, chlorine, bromine, or iodine, preferably iodine. In a preferred embodiment, the halogen substituent is iodine.

[0048] The terms "heteroalkyl" and "heteroalkenyl," as used herein, refer to optionally substituted alkyl and alkenyl radicals having one or more skeletal chain atoms selected from atoms other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, or combinations thereof. Numerical ranges may be given, referring to total chain length; e.g., C1-C4 heteroalkyl, in this example, is 4 atoms long.

[0049] The term "heteroaryl" or "heteroaromatic" or "HetAr" refers to a 5- to 18-membered aromatic radical (e.g., C5-C6) containing one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. 13 Heteroaryl refers to a heteroaryl group (heteroaryl), which may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system. Whenever it appears herein, a numerical range such as "5 to 18" refers to each integer within the given range; for example, "5 to 18 ring atoms" means that the heteroaryl group can contain 5 ring atoms, 6 ring atoms, etc., up to a maximum of 18 ring atoms. Divalent radicals derived from monovalent heteroaryl radicals, designated by the suffix "-yl" by the removal of one hydrogen atom from the free valence atom, are designated by the addition of "-idene" to the name of the corresponding monovalent radical; for example, a pyridyl group with two points of attachment is a pyridylidene.

[0050] 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.

[0051] "Diastereomer" refers to a stereoisomer having two or more centers of chirality, where the compounds are not mirror images 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 separate under high-resolution analytical procedures such as electrophoresis and chromatography.

[0052] "Enantiomers" refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0053] Stereochemical definitions and conventions used herein generally follow those in S.P. Parker, ed., McRaw-Hiff Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994.

[0054] (in the chemical formula * The symbols (), (#), and (§) designate i) points of attachment, ii) radicals, and / or iii) unshared electrons.

[0055] The term "antisense oligonucleotide (AON)" as used herein refers to an oligonucleotide or oligomeric compound capable of altering gene expression by interacting with and / or hybridizing to pre-mRNA or mRNA having a complementary nucleotide sequence.

[0056] The term "protecting group," as used herein, is intended to mean a group that selectively blocks one or more reactive sites of a polyfunctional compound so that a chemical reaction can be selectively carried out at another, unprotected reactive site, and which can then be easily removed or deprotected after the selective reaction is complete. Various protecting groups are disclosed, for example, in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3rd Edition, John Wiley & Sons, New York, 1999.

[0057] The terms "protecting group for amino", "protecting group for amino group" or "amino protecting group" used interchangeably herein are well known in the art and are described in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3rd Edition, John Wiley & Sons, New York (1999), Greene's Protective Groups in Organic Synthesis, P.G.M. Wuts, 5th Edition, John Wiley & Sons (2000), and others. 14) as well as those described in detail in Current Protocols in Nucleic Acid Chemistry, edited by S.L. Beaucage et al., June 2012, herein in particular 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 (Teoc), 2-methyl- ... -phenylethyl carbamate (hZ), 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz) and 2,4,6-trimethylbenzyl carbamate, (4-methoxyphenyl)diphenylmethyl (MMTr); as well as formamide, acetamide, benzamide, typically and preferably independently selected from these at each occurrence.

[0058] The terms "protecting group for hydroxyl", "protecting group for hydroxyl group" or "hydroxyl protecting group", used interchangeably herein, are well known in the art and are described in Protecting Groups in Organic Synthesis, T.W. Greene and P. G. M. Wuts, 3rd ed., John Wiley & Sons, New York (1999); Greene's Protective Groups in Organic Synthesis, PGM Wuts, 5th ed., John Wiley & Sons (2014) and Current Protocols in Nucleic Acid Chemistry, edited by S.L. Beaucage et al., June 2012, including in particular those detailed in Chapter 2. In certain embodiments, "hydroxyl protecting groups" of the present invention include 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), t-butyldiphenylsilyl ether (TBDPS), trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM), and triisopropylsilyloxymethyl (TOM). silyl ethers such as propylsilyl (TIPS) ether; methyl ether, ethoxyethyl ether (EE), and are typically and preferably independently selected from these at each occurrence.

[0059] Preferred examples of the "hydroxyl protecting group" of the present invention include 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-fluorenylmethylcarbonate, mesylate, tosylate, triflate, 4-monomethoxytrityl (MMTr), 4-methyl ... ,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, 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl phenyl, 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) xanthin-9-yl (MOX), and independently selected from these at each occurrence.

[0060] The term "nucleobase," as used herein, is abbreviated as Bx, and refers to unmodified or naturally occurring nucleobases as well as modified or non-naturally occurring nucleobases and their synthetic mimetics. A nucleobase is any heterocyclic base that contains one or more atoms or groups of atoms that are capable of hydrogen bonding to the heterocyclic base of a nucleic acid.

[0061] Typical and preferred examples of nucleobases are purine bases or pyrimidine bases, and preferably, the purine bases are purines or substituted purines, and the pyrimidine bases are pyrimidines or substituted pyrimidines. More preferably, the nucleobases are (i) adenine (A), (ii) cytosine (C), (iii) 5-methylcytosine (MeC), (iv) guanine (G), (v) uracil (U), or (vi) 5-methyluracil (MeU), or derivatives of (i), (ii), (iii), (iv), (v) or (vi). The terms "derivatives of (i), (ii), (iii), (iv), (v) or (vi)" and "nucleobase derivatives" are used interchangeably herein. Derivatives of (i), (ii), (iii), (iv), (v) or (vi) and nucleobase derivatives are known to those skilled in the art, and are described, for example, in Sharma VK et al., Med. Chem. Commun., 2014, vol. 5, pp. 1454-1471, and examples thereof include, but are not limited to, 5-hydroxymethylcytosine, xanthine, hypoxanthine, alkyl adenines such as 2-aminoadenine, 6-methyladenine, and 2-propyladenine, alkyl guanines such as 6-methylguanine and 2-propylguanine, alkynyl pyrimidine bases such as 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C=C-CH3) uracil, and 5-propynyl (-C=C-CH3) cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, pseudo Uracil (pseudo-uracil), 4-thiouracil; 8-halo-, 8-amino-, 8-thio 8-substituted purine bases such as 8-hydroxyl-, 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-extended bases, or fluoride salts. In certain embodiments, the nucleobase includes, but is 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, ST, ed., 2008); The Concise Encyclopedia of Polymer Science And Engineering, Kroschwitz, JI, ed., John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, Vol. 30(6), pp. 613-623; Sanghvi, YS, Antisense Research and Applications, Crooke, ST and Lebleu, B., eds., CRC Press, 1993). (J. Chem. Soc., 2004, pp. 273-302). The term "nucleobase derivative" also includes those in which a purine or pyrimidine base is replaced with a moiety corresponding to a spacer of the present invention, particularly a moiety for linking one or more lipid moieties within the oligomeric compound, preferably the oligonucleotide. Specific linkages of the moieties corresponding to a spacer are known to those skilled in the art. 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. The preparation of modified nucleobases is known in the art and is described in U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; Nos. 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,750,692; 5,830,653; 5,763,588; 6,005,096; and 5,681,941.

[0062] The term "internucleoside linking group," as used herein, refers to any linking group known in the art that allows for further linkage, preferably, the tricyclo-deoxyribonucleic acid (tc-DNA) nucleoside to be further linked, either to an additional tc-DNA nucleoside, a nucleoside other than a tc-DNA nucleoside, or a non-nucleoside, including a peptide or protein. Representative patents that teach such possible linking groups include, but are not limited to, U.S. Pat. No. 5,034,506; U.S. Pat. No. 5,166,333; No. 15; U.S. Patent No. 5,185,444; U.S. Patent No. 5,214,134; U.S. Patent No. 5,216,141; U.S. Patent No. 5,235,033; U.S. Patent No. 5,264,562; U.S. Patent No. 5,264,564; U.S. Patent No. 5,405,938; U.S. Patent No. 5,434,257; U.S. Patent No. 5,466,677; U.S. Patent No. 5,470,967; U.S. Patent No. 5,489,677; U.S. Patent No. 5,541,307; U.S. Patent No. 5,5 Nos. 5,61,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,677,439; 5,646,269 and 5,792,608. Thus, the term "internucleoside linking group" includes phosphorus and non-phosphorus linking groups. The non-phosphorus linking group does not contain a phosphorus atom, and examples of non-phosphorus linking groups include, typically and preferably selected from, alkyl, aryl, preferably phenyl, benzyl or benzoyl, cycloalkyl, alkylenearyl, alkylenediaryl, alkoxy, alkoxyalkylene, alkylsulfonyl, alkyne, ether, carboxyl, amide, amine, amino, imine, thiol, sulfide, sulfoxide, sulfone, sulfamate, sulfonate, sulfonamide, siloxane, or mixtures thereof, each independently optionally substituted with cyano, nitro, or halogen. Typically and preferably, the internucleoside linking group is a phosphorus linking group, and the phosphorus linking group is P III or P V" refers to a moiety containing a phosphorus atom in a valence state of . More preferably, the internucleoside linking group is a phosphorus linking group. Again more preferably, the internucleoside 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, preferably an H-phosphonate linking group or a methylphosphonate linking group; a phosphonothioate linking group, preferably an H-phosphonothioate linking group or a methylphosphonothioate linking group; a phosphinate linking group, a phosphorthioamidate linking group, a phosphoramidate linking group, or a phosphite linking group. In another highly preferred embodiment, the internucleoside 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 preferably an H-phosphonate linking group or a methylphosphonate linking group.

[0063] As used herein, the term "nucleoside" refers to a compound comprising a nucleobase and a sugar covalently linked to the nucleobase. Furthermore, the term "nucleoside" is intended to include all types of naturally occurring or modified nucleosides, or nucleoside mimetics, that can be incorporated into oligomers using natural or chemical oligomer synthesis. Typically and preferably, the term "nucleoside" as used herein refers to naturally occurring nucleosides, modified nucleosides, or nucleoside mimetics. The term "modified nucleoside" is intended to include modifications made to the sugar and / or nucleobase of the nucleoside known to those skilled in the art and described herein. The term "nucleoside mimetics" is intended to include those structures that are used to replace sugars and nucleobases. Examples of nucleoside mimetics include nucleosides in which the nucleobase is replaced with a phenoxazine moiety (e.g., a 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one group) and the sugar moiety is replaced with a cyclohexenyl or bicyclo[3.1.0]hexyl moiety. The term "nucleoside" also includes combinations of modifications, such as modifications of two or more nucleobases, modifications of two or more sugars, or modifications of at least one nucleobase and at least one sugar.

[0064] The sugar of the nucleoside may include, but is not limited to, a monocyclic, bicyclic, or tricyclic ring system, preferably a tricyclic or bicyclic system, or a monocyclic ribose or deoxyribose. and xylibose (de(s)oxyribose). Sugar modifications further include, but are not limited to, modified stereochemical configuration, substitution of at least one group, or deletion of at least one group. Modified sugars are typically and preferably modified versions of ribosyl moieties (i.e., furanosyl moieties) naturally occurring in RNA and DNA, such as bicyclic sugars, tetrahydropyrans, 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, but are not limited to, 2', 3', and / or 4' substituted nucleosides (e.g., 4'-S-modified nucleosides); 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, and other 2'-O-modified RNA nucleotide residues; 2'-O-(haloalkoxy)methyl, e.g., 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2-thiomethyl)ethyl, and other 2'-O-substituted nucleosides. ,2-dichloroethoxy)methyl (DCEM); 2'-O-alkoxycarbonyl, such as 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl))ethyl] (DMCE), especially 2'-O-methyl modified or 2'-O-methoxyethyl (2'-O-MOE); or other modified sugar moieties such as morpholino (PMO), cationic morpholino (PMOPlus), or modified morpholino groups such as PMO-X.The term "PMO-X" refers to a modified morpholino group, including at least one 3' or 5' end modification such as a 3'-fluorescent tag, a 3' quencher (e.g., 3'-carboxyfluorescein, 3'-Gene Tools Blue, 3'-lissamine, 3'-dabsyl), a 3'-affinity tag and a functional group for chemical linkage (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 those disclosed in WO 2011 / 150408 and U.S. Patent Application Publication No. 2012 / 0065169.

[0065] "Bicyclic sugar moiety" includes bicyclic nucleosides having two interconnected ring systems, for example, a sugar moiety having 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, ethylene-bridged nucleic acid (ENA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA) or 3'-deoxypyranosyl-DNA (p-DNA).

[0066] In a preferred embodiment, oligomeric compound is oligonucleotide.The term " oligonucleotide " as used herein refers to the compound that comprises at least two nucleosides that are respectively linked to each other by internucleoside linking group.Therefore, the term " oligonucleotide " as used herein comprises, typically and preferably refers to the oligomeric compound that comprises at least two nucleosides that are linked to each other by internucleoside linking group, and said at least two nucleosides are independently selected from naturally occurring nucleosides, modified nucleosides or nucleoside mimetics.

[0067] Oligomeric compounds can be single-stranded or double-stranded. In one embodiment, the oligomeric compound is double-stranded (i.e., duplex). In a preferred embodiment, the oligomeric compound is single-stranded.

[0068] The term "terminal" refers to the end or terminus of an oligomeric compound, and an integer (3, 5, etc.) is included in a nucleoside of the oligomeric compound. The carbon atoms of the sugar are indicated. The terms "5' terminal group" or "3' terminal group", as used herein, refer to the groups located at the 5' or 3' terminus, respectively.

[0069] The terms "natural" or "naturally occurring," used interchangeably herein, refer to compounds that are of natural origin.

[0070] The term "complementary" refers to a nucleic acid molecule that can form hydrogen bond(s) with another nucleic acid molecule, either through traditional Watson-Crick base pairing or other non-traditional types of pairing (e.g., Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleosides or nucleotides. "Complementary" (or "specifically hybridizable") is a term that indicates a sufficient degree of complementarity or precise pairing such that stable, specific binding occurs between an oligomeric compound and a pre-mRNA or mRNA target. It is understood in the art that a nucleic acid molecule need not be 100% complementary to a target nucleic acid sequence to be specifically hybridizable. That is, two or more nucleic acid molecules need not be perfectly complementary. Complementarity can be indicated by the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule. For example, if a first nucleic acid molecule has 10 nucleotides and a second nucleic acid molecule has 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules represents 50%, 60%, 70%, 80%, 90%, and 100%, respectively. "Perfectly" or "fully" complementary nucleic acid molecules mean that all consecutive residues in a first nucleic acid molecule will hydrogen bond with the same number of consecutive residues in a second nucleic acid molecule, and either both nucleic acid molecules have the same number of nucleotides (i.e., have the same length), or the two molecules have different lengths.

[0071] The term "exon skipping" refers to the modification of pre-mRNA splicing by targeting splice donor and / or splice acceptor sites in pre-mRNA with one or more complementary antisense oligonucleotides or oligomeric compounds.By blocking the spliceosome's access to one or more splice donor or splice acceptor sites, or any other site in the exon or intron involved in splicing definition, the oligonucleotide can prevent the splicing reaction and cause the exon to be deleted from the fully processed mRNA.Exon skipping is achieved in the nucleus during the maturation process of pre-mRNA.Exon skipping involves masking the important sequence involved in the splicing of the target exon by using an antisense oligonucleotide that is complementary to the splice donor sequence in pre-mRNA. For example, the compositions of the present invention comprising the oligomeric compounds provided herein can be suitably used for exon skipping by masking splice sites at intron / exon junctions within dystrophin pre-mRNA, thereby facilitating the deletion of mutant exons during processing of the pre-mRNA into mature mRNA.

[0072] The term "exon inclusion" refers to an oligonucleotide-mediated process, such as base pairing of an antisense oligonucleotide to a targeted pre-mRNA, to block exon or intron splicing enhancers, block corresponding splicing repressors, and / or disrupt unfavorable secondary structures, resulting in more efficient recognition of the exon by the spliceosome and restoration of exon expression.

[0073] The term "in vivo" refers to an event that takes place within the body of a subject.

[0074] The term "in vitro" refers to events that take place outside a subject's body. In vitro assays include cell-based assays, in which live or dead cells are used, and can also include cell-free assays, in which no intact cells are used.

[0075] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to effect the intended application, including, but not limited to, the treatment of a disease. A therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the human subject and disease state being treated (e.g., the subject's weight, age, and sex), the severity of the disease state, the mode of administration, etc., and can be readily determined by one skilled in the art. The term also applies to a dose that induces a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the bodily delivery system through which the compound is delivered.

[0076] As used herein, a "therapeutic effect" encompasses a therapeutic benefit and / or a prophylactic benefit in a human subject. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0077] The term "pharmaceutically acceptable salt" refers to salts derived from various organic and inorganic counterions known in the art. Pharmaceutically acceptable acid addition salts can be formed using inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid. Pharmaceutically acceptable base addition salts can be formed using inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins. Specific examples include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts, and preferably, the pharmaceutically acceptable salt is a sodium salt.

[0078] In the case of a hydroxyl group (OH) or a thiol group (SH) present in the oligomeric compound of the invention, preferably the oligonucleotide of the invention, typically and preferably bound to P(III) or P(V), typically and preferably present in the one or more lipid moieties as part of the B group of the one or more lipid moieties, or present in the spacer, or typically and preferably as part of the internucleoside linking group selected from phosphorothioate or phosphorodiester, each of the hydroxyl group (OH) or thiol group (SH) can exist, independently of one another, as the OH group or in its ionic state, such as an O-anion and a pharmaceutically acceptable cation, or as the SH group or in its ionic state, such as an S-anion and a pharmaceutically acceptable cation. Furthermore, any combination and any equilibrium state between the aforementioned states in the compositions of the invention is included. , particularly taking into account oxygen- or sulfur-containing groups on said P(III) or P(V), such as (=O), (=S), another OH group or SH group, which will be known to those skilled in the art. For convenience, aspects and embodiments of the present invention typically describe only one of the aforementioned situations. By way of example, a preferred spacer of the present invention is #-NH-C 2~12 As shown, but not limited to, spacers in which a hydrogen is positioned on the oxygen, thus #-NH-C 2~12 All alkylene-OP(OH)(S)-§ and pharmaceutically acceptable salts thereof are included herein.

[0079] Thus, a pharmaceutically acceptable salt in the context of a hydroxyl group (OH) and / or a thiol group (SH) present in said oligomeric compound of the invention, preferably in said oligonucleotide of the invention, typically and preferably attached to P(III) or P(V), typically and preferably present in said one or more lipid moieties as part of the B group of said one or more lipid moieties, or present in said spacer, or as part of said internucleoside linking group, typically and preferably selected from phosphorothioate or phosphorodiester, can be It refers to a composition of the invention, wherein one or more of the OH group or said SH groups are present, independently of each other, as said OH group or in its ionic state, such as an O-anion and its pharmaceutically acceptable cation, or as said SH group or in its ionic state, such as an S-anion and a pharmaceutically acceptable cation, typically and preferably, said pharmaceutically acceptable cation is selected from protonated trimethylamine, protonated diethylamine, protonated methylamine, ammonium, sodium or potassium, more preferably, said pharmaceutically acceptable cation is sodium.

[0080] "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any solvent, dispersion medium, coating agent, antibacterial and antifungal agent, isotonic and absorption delaying agent, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is contemplated for use in the therapeutic compositions of the present invention, except insofar as it is incompatible with the active pharmaceutical ingredient. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0081] Unless otherwise stated, chemical structures depicted herein are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds in which one or more hydrogen atoms are replaced by deuterium or tritium, or one or more carbon atoms are replaced by 13 C or 14 Compounds in which C is replaced by enriched carbon are within the scope of the present invention.

[0082] "Isomers" are different compounds with the same molecular formula. "Stereoisomers" are isomers that differ only in the way their atoms are arranged in space, i.e., have different stereochemical configurations. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. The term "(±)" is used to designate a racemic mixture when appropriate. "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. Absolute stereochemistry is specified by the Cahn-Ingold-Prelog RS system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon can be specified as either (R) or (S). Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextrorotatory or levorotatory) they rotate plane-polarized light at the wavelength of the sodium D line. Certain compounds of the compounds described herein may contain one or more undesired These compounds may contain asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined in terms of absolute stereochemistry as (R) or (S). The chemical entities, pharmaceutical compositions, and methods of this invention are intended to include all such possible isomers, including racemic mixtures, optically pure forms, and mixtures of intermediates. Optically active (R) and (S)-isomers may be prepared using chiral synthons or chiral reagents or resolved using conventional techniques. Where a compound described herein contains an olefinic double bond or other center of geometric asymmetry, and unless otherwise specified, the compound is intended to include both E and Z geometric isomers.

[0083] "Enantiomeric purity," as used herein, refers to the relative amount, expressed as a percentage, of a particular enantiomer relative to the other enantiomer. For example, if a compound that may have either the (R) or (S)-isomer configuration exists as a racemic mixture, the enantiomeric purity with respect to either the (R) or (S)-isomer is about 50%. If a compound has one isomeric form that predominates over the other, for example, 80% (S)-isomer and 20% (R)-isomer, the enantiomeric purity of the compound with respect to the (S)-isomer form is 80%. The enantiomeric purity of a compound can be determined by several methods known in the art, including, but not limited to, chromatography using a chiral support, polarimetric measurement of the rotation of polarized light, nuclear magnetic resonance spectroscopy using chiral shift reagents (including, but not limited to, lanthanide containing chiral complexes or Pirkle reagents), or derivatization of the compound using a chiral compound such as Mosher's acid followed by chromatography or nuclear magnetic resonance spectroscopy.

[0084] In a preferred embodiment, the enantiomerically enriched composition has a higher therapeutic utility per unit mass than the racemic mixture of that composition. The enantiomers can be isolated from the mixture by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and chiral salt formation and crystallization, or the preferred enantiomer can be prepared by asymmetric synthesis. See, for example, Jacques, et al., Enantiomers, Racemates and Resolutions, Wiley Interscience, New York (1981); E.L. Eliel, Stereochemistry of Carbon Compounds, McGraw-Hill, New York (1962); and E.L. Eliel and S.H. Wilen, Stereochemistry of Organic Compounds, Wiley-Interscience, New York (1994). thing.

[0085] The terms "enantiomerically enriched" and "non-racemic," as used herein, refer to a composition in which the weight percent of one enantiomer exceeds the amount of that enantiomer in a control mixture of the racemic composition (e.g., greater than 1:1 by weight). For example, an enantiomerically enriched preparation of the (S)-enantiomer refers to a preparation of the compound having more than 50% by weight, e.g., at least 75% by weight, or e.g., at least 80% by weight, of the (S)-enantiomer relative to the (R)-enantiomer. In some embodiments, the enrichment can be significantly greater than 80% by weight, which can provide a "substantially enantiomerically enriched" or "substantially non-racemic" preparation, which refers to a preparation of a composition having at least 85% by weight, e.g., at least 90% by weight, or e.g., at least 95% by weight, of one enantiomer relative to the other enantiomer. The terms "enantiomerically pure" or "substantially enantiomerically pure" refer to a composition containing at least 98% of a single enantiomer and less than 2% of the opposite enantiomer.

[0086] "Moiety" refers to a specific segment or functional group of a molecule. Chemical moieties are often In this case, it is recognized as a chemical entity incorporated into or attached to a molecule.

[0087] "Tautomers" are structurally distinct isomers that interconvert via tautomerization. "Tautomerization" is a form of isomerization and includes prototropic or proton-shift tautomerization, which are considered subsets of acid-base chemistry. "Prototropic or proton-shift tautomerization" involves the migration of a proton accompanied by a change in bond order, often the exchange of a single bond with an adjacent double bond. When tautomerization is possible (e.g., in solution), a chemical equilibrium of tautomers can be reached. An example of tautomerization is keto-enol tautomerization. A specific example of keto-enol tautomerization is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridin-4(1H)-one tautomers.

[0088] Unless more specifically defined herein, "substituted" means that the referenced group can be bound to one or more additional groups, radicals, or moieties, for example, acyl, alkyl, alkylaryl, cycloalkyl, aralkyl, aryl, carbohydrate, carbonate, heteroaryl, heterocycloalkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, ester, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, oxo, perhaloalkyl, perfluoroalkyl, phosphate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, and amino, including mono- and di-substituted amino groups, and protected derivatives thereof. The substituents themselves may be substituted; for example, a cycloalkyl substituent may itself have a halide substituent on one or more of its ring carbons. The term "optionally substituted" refers to optional substitution with a specific group, radical, or moiety.

[0089] The compounds of the present invention also include crystalline and amorphous forms of the compounds, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, non-solvated polymorphs (including anhydrous forms), conformational polymorphs, and amorphous forms of the compounds, and mixtures thereof. "Crystalline form" and "polymorph" are intended to include all crystalline and amorphous forms of the compounds, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, non-solvated polymorphs (including anhydrous forms), conformational polymorphs, and amorphous forms, and mixtures thereof, unless a specific crystalline or amorphous form is referenced.

[0090] When a range is used herein to describe a physical or chemical property, such as, for example, molecular weight or chemical formula, it is intended to include all combinations and subcombinations of ranges and specific embodiments therein. The use of the term "about" when referring to a numerical value or numerical range means that the referenced numerical value or numerical range is an approximation within experimental variation (or within statistical experimental error), and thus the numerical value or numerical range may vary. The variation is typically 0% to 15%, 0% to 10%, or 0% to 5% of the stated number or numerical range. tc-DNA nucleoside precursors

[0091] In one embodiment, the present invention provides a tc-DNA nucleoside precursor of Formula I, Formula II, Formula III, Formula IV, Formula V or Formula VI: [ka] wherein X can be alkoxy; T 1 and T 2 are OR 1 where R 1 is H or a hydroxyl protecting group; q 1 , q 2 , q 3 , q 4 and q 5 are each independently hydrogen, halogen, or C 1~6 Alkyl, C2~6 Alkenyl, C 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 2~6 Alkenyl, substituted C 2~6 Alkynyl, and -(CH2) n -C(O)-R 2 (where n is 0 to 6, and R 2 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 alkyl); R 4 may be a hydroxyl protecting group; z 1 and z 2 are each independently hydrogen, C 1~6 Alkyl, C 1~6 Alkoxyl, OC 2~6 Alkenyl, OC 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 1~6 Alkoxy, substituted OC 2~6 Alkenyl and substituted OC 2~6 alkynyl halogens] The present invention includes a process for preparing the

[0092] In some embodiments, T 1 is OR 1 and R 1is a hydroxyl protecting group. In some embodiments, each hydroxyl protecting group is selected from the group consisting of 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-fluorenylmethylcarbonate, mesylate, methylsilyl ... To, 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, 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) and 9-(p-methoxyphenyl)xanthine-9-yl In some embodiments, the hydroxyl protecting group is TBDMS.

[0093] In some embodiments, q 1 , q 2 , q 3 , q4 and / or q 5 is hydrogen.

[0094] In some embodiments, z 1 and / z 2 is hydrogen.

[0095] In some embodiments, T 2 is OR 1 and R 1 is H. In some embodiments, T 2 is hydroxy.

[0096] In some embodiments, X is OR 3 and R 3 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, or hexyl. 3 is methyl. In some embodiments, X is methoxy.

[0097] In some embodiments, R 4 is TBDMS.

[0098] In one embodiment, the present invention provides a tc-DNA nucleoside precursor of Formula VII, Formula VIII, or Formula IX: [ka] The present invention includes a process for preparing the

[0099] The tc-DNA nucleoside precursors of Formulas I-IX can be used in the preparation of tc-DNA nucleosides, which contain nucleobases and are useful in the preparation of antisense oligonucleotide therapeutics. Preparation of tc-DNA nucleoside precursors

[0100] In one embodiment, the tc-DNA nucleoside precursors described herein (eg, compounds of Formulas I-IX) can be prepared by one or more of the following processes.

[0101] In one embodiment, the invention includes a method for preparing a tc-DNA nucleoside precursor of any one of Formulas I-IX, said method comprising: a. preparing a carbene precursor at a carbene preparation temperature; b. cyclopropanation of said carbene precursor with a compound of Formula X, Formula XI or Formula XII at cyclopropanation temperatures: [ka] adding; and c. Providing the tc-DNA nucleoside precursor of one of Formulas I-IX, wherein Y is alkoxy; T 3 and T 4 are OR 5 where R 5 is H or a hydroxyl protecting group, q 6 , q 7 and q 8 are each independently hydrogen, halogen, or C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 2~6 Alkenyl, substituted C 2~6 Alkynyl, and -(CH2) n -C(O)-R 6 (where n is 0 to 6, and R 6 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 alkyl); z 3 and z 4 are each independently hydrogen, C 1~6 Alkyl, C 1~6 Alkoxyl, OC 2~6 Alkenyl, OC 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 1~6 Alkoxy, substituted OC 2~6Alkenyl and substituted OC 2~6 Alkynyl halogen is selected from the group consisting of:

[0102] In one embodiment, the invention includes a method for preparing a tc-DNA nucleoside precursor of any one of Formulas I-IX, said method comprising: a. preparing a carbene precursor at a carbene preparation temperature; b. A compound of Formula X, Formula XI or Formula XII: [ka] preparing a solution of c. adding the solution of the compound of Formula X, Formula XI, or Formula XII to the carbene precursor at a cyclopropanation temperature; and d. Providing the tc-DNA nucleoside precursor of one of Formulas I-IX, wherein Y is alkoxy; T 3 and T 4 are OR 5 where R 5 is H or a hydroxyl protecting group, q 6 , q 7 and q 8 are each independently hydrogen, halogen, or C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 2~6 Alkenyl, substituted C 2~6 Alkynyl, and -(CH2) n -C(O)-R 6 (where n is 0 to 6, and R 6 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 a selected from the group consisting of: z 3 and z 4 are each independently hydrogen, C 1~6 Alkyl, C1~6 Alkoxyl, OC 2~6 Alkenyl, OC 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 1~6 Alkoxy, substituted OC 2~6 Alkenyl and substituted OC 2~6 Alkynyl halogen is selected from the group consisting of:

[0103] In one embodiment, the method may include adding a Lewis acid catalyst to a mixture of the compound of Formula X, Formula XI, or Formula XII and the carbene precursor. In some embodiments, the Lewis acid catalyst may be selected from the group consisting of EtAlCl, EtZn, ZnI, ZnCl, ZnBr, Ti(OiPr), MeAlCl, TMSOTf, TiCl, and combinations thereof.

[0104] In one embodiment, the step of preparing the carbene precursor comprises the step of reacting a Lewis acid catalyst (e.g., EtZn) and R 7 In some embodiments, R 7 CH2, CH-C 1~6 Alkyl, CH-C 2~6 Alkenyl, CH-C 2~6 Alkynyl, substituted CH-C 1~6 Alkyl, substituted CH-C 2~6 Alkenyl, substituted CH-C 2~6 Alkynyl and CH—(CH2) n -C(O)-R 8 (where n is 0 to 6, and R 8 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 In some embodiments, R 7 is alkyl. In some embodiments, R 7 is CH2.

[0105] In some embodiments, preparing a carbene precursor comprises reacting a carbene additive, a Lewis acid catalyst (e.g., ZnEt), and R 7In some embodiments, the step of preparing the carbene precursor may include combining a Lewis acid catalyst (e.g., ZnEt) and R 7 This may include adding a carbene additive to the mixture of I2.

[0106] In some embodiments, the carbene additive may be an aliphatic alcohol (e.g., a substituted or unsubstituted alkyl alcohol), an aromatic alcohol (e.g., a substituted or unsubstituted phenol), a substituted or unsubstituted carboxylic acid (e.g., trichloroacetic acid), or a substituted or unsubstituted phosphate (e.g., (alkyl-O)P(O)OH or (aryl-O)P(O)OH). In some embodiments, the carbene additive may be a substituted carboxylic acid of formula QCCOH, where each Q may be independently selected from the group consisting of H, Cl, Br, and F. In some embodiments, the carbene additive may be a substituted carboxylic acid of formula QCCOH, where QC may be defined as CCl, CHCl, CHCl, or CF. In some embodiments, the carbene additive may be a substituted alkyl alcohol of formula QCCHOH, where each Q may be independently selected from the group consisting of H, Cl, Br, and F. In some embodiments, the carbene additive may be a substituted carboxylic acid of formula QCCHOH, where QC may be defined as CCl, CHCl, CHCl, or CF. In some embodiments, the carbene additive may be trichloroacetic acid, 2,2,2-trifluoroethanol, trichlorophenol, or (n-BuO)P(O)OH.

[0107] In some embodiments, the solvent may include hexane, toluene, dichloromethane (CHCl), tetrahydrofuran (THF), acetonitrile (CHCN), dimethylformamide (DMF), diethyl ether, dimethoxyethane (DME), or a combination thereof. In some embodiments, the solvent is dichloromethane, DME, or a combination thereof. In some embodiments, the solvent may be a polar aprotic solvent. In some embodiments, the polar aprotic solvent is dichloromethane (CHCl), tetrahydrofuran (THF), acetonitrile (CHCN), dimethylformamide (DMF), diethyl ether, dimethoxyethane (DME), or a combination thereof. The solvent may include diethyl ether, dimethoxyethane (DMF), diethyl ether, dimethoxyethane (DME), or a combination thereof.

[0108] In some embodiments, the carbene precursor is Q3CCO2ZnR 7 I, Q3CCH2OZnR 7 I, (n-BuO)2P(O)OZnR 7 I, (alkyl-O)2P(O)OZnR 7 I, (aryl-O)2P(O)OZnR 7 I or 2,4,6-Cl3C6H2OZnR 7 I, where each Q may be independently selected from the group consisting of H, Cl, Br, and F. In some embodiments, the carbene precursor is QCCOZnR 7 I, Q3CCH2OZnR 7 I, (n-BuO)2P(O)OZnR 7 I, (alkyl-O)2P(O)OZnR 7 I, (aryl-O)2P(O)OZnR 7 I or 2,4,6-Cl3C6H2OZnR 7 I, where QC may be defined as CCl, CHCl, CHCl, or CF. In some embodiments, the carbene precursor is CClCOZnR 7 I, CF3CH2OZnR 7 I, (n-BuO)2P(O)OZnR 7 I or 2,4,6-Cl3C6H2OZnR 7In some embodiments, the carbene precursor is CClCOZnCHI, CFCHOZnCHI, (n-BuO)P(O)OZnCHI, or 2,4,6-ClCHOZnCHI.

[0109] In some embodiments, the step of preparing a solution of a compound of Formula X, Formula XI, or Formula XII can include adding ZnEt2 to a compound of Formula X, Formula XI, or Formula XII.

[0110] In some embodiments, the carbene preparation temperature may be a temperature selected from the range of about −80° C. to about 0° C. In some embodiments, the carbene preparation temperature may be about −80° C. to about 0° C. In some embodiments, the carbene preparation temperature may be −80° C., −79° C., −78° C., −77° C., −76° C., −75° C., −74° C., −73° C., −72° C., −71° C., −70° C., −69° C., −68° C., −67° C., −66° C., −65° C., −64° C., −63° C., −62° C., −61° C., −60° C., −59° C., −58° C., −57° C., −56° C., −55° C., −54° C., −53° C., −52° C., −51° C., −50° C., −49° C., −48° C., −47° C., −46° C., −45° C., −44° C., −43° C., − The temperature may be higher than 42°C, -41°C, -40°C, -39°C, -38°C, -37°C, -36°C, -35°C, -34°C, -33°C, -32°C, -31°C, -30°C, -29°C, -28°C, -27°C, -26°C, -25°C, -24°C, -23°C, -22°C, -21°C, -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11°C, -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, -2°C, -1°C, or -0°C. In some embodiments, the carbene preparation temperature is -80°C, -79°C, -78°C, -77°C, -76°C, -75°C, -74°C, -73°C, -72°C, -71°C, -70°C, -69°C, -68°C, -67°C, -66°C, -65°C, -64°C, -63°C, -62°C, -61°C, -60°C, -59°C, -58°C, -57°C, -56°C, -55°C, -54°C, -53°C, -52°C, -51°C, -50°C, -49°C, -48°C, -47°C, -46°C, -45°C, -44°C, -43°C, The temperature may be lower than 42°C, -41°C, -40°C, -39°C, -38°C, -37°C, -36°C, -35°C, -34°C, -33°C, -32°C, -31°C, -30°C, -29°C, -28°C, -27°C, -26°C, -25°C, -24°C, -23°C, -22°C, -21°C, -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11°C, -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, -2°C, -1°C, or -0°C.In some embodiments, the carbene preparation temperature is about −80° C., about −79° C., about −78° C., about −77° C., about −76° C., about −75° C., about −74° C., about −73° C., about −72° C., about −71° C., about −70° C., about −69° C., about −68° C., about −67° C., about −66° C., about −65° C., about −64° C., about −63° C., about −62° C., about −61° C., about −60° C., about −59° C., about −58° C., about −57° C., about −56° C., about −55° C., about −54° C., about −53° C., about −52° C., about −51° C., about −50° C., about −49° C., about −48° C., about −47° C., about −4. 6°C, approximately -45°C, approximately -44°C, approximately -43°C, approximately -42°C, approximately -41°C, approximately -40°C, approximately -39°C, approximately -38°C, approximately -37°C, approximately -36°C, approximately -35°C, approximately -34°C, approximately -33°C, approximately -32°C, approximately -31°C, approximately -30°C, approximately -29°C, approximately -28°C, approximately -27°C, approximately -26°C, approximately -25°C, approximately -24°C, approximately -2 The temperature may be about -3°C, about -22°C, about -21°C, about -20°C, about -19°C, about -18°C, about -17°C, about -16°C, about -15°C, about -14°C, about -13°C, about -12°C, about -11°C, about -10°C, about -9°C, about -8°C, about -7°C, about -6°C, about -5°C, about -4°C, about -3°C, about -2°C, about -1°C, or about -0°C.

[0111] In some embodiments, the carbene preparation temperature may include a first carbene preparation temperature and a second carbene preparation temperature, and the temperature of the reaction begins at the first carbene preparation temperature and is increased to a second carbene preparation temperature, where the first carbene preparation temperature is lower than the second carbene preparation temperature. However, in some embodiments, the temperature of the reaction begins at the first carbene preparation temperature and is decreased to a second carbene preparation temperature, where the first carbene preparation temperature is higher than the second carbene preparation temperature.

[0112] In some embodiments, the cyclopropanation temperature may be a temperature selected from the range of about -30°C to about room temperature (i.e., 25°C). In some embodiments, the cyclopropanation temperature may be about -30°C to about room temperature (i.e., 25°C). In some embodiments, the cyclopropanation temperature may be -30°C, -29°C, -28°C, -27°C, -26°C, -25°C, -24°C, -23°C, -22°C, -21°C, -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11°C, -10°C, -9°C, -8°C, -7°C, -6°C, It may be greater than -5°C, -4°C, -3°C, -2°C, -1°C, -0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C. In some embodiments, the cyclopropanation temperature is -30°C, -29°C, -28°C, -27°C, -26°C, -25°C, -24°C, -23°C, -22°C, -21°C, -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11°C, -10°C, -9°C, -8°C, -7°C, -6°C, It may be lower than -5°C, -4°C, -3°C, -2°C, -1°C, -0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.In some embodiments, the cyclopropanation temperature is about −30° C., about −29° C., about −28° C., about −27° C., about −26° C., about −25° C., about −24° C., about −23° C., about −22° C., about −21° C., about −20° C., about −19° C., about −18° C., about −17° C., about −16° C., about −15° C., about −14° C., about −13° C., about −12° C., about −11° C., about −10° C., about −9° C., about −8° C., about −7° C., about −6° C., about −5° C., The temperature may be about -4°C, about -3°C, about -2°C, about -1°C, about -0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C or about 30°C.

[0113] In some embodiments, the cyclopropanation temperature may include a first cyclopropanation temperature and a second cyclopropanation temperature, where the temperature of the reaction begins at a first cyclopropanation temperature and is increased to a second cyclopropanation temperature, where the temperature of the first cyclopropanation is lower than the temperature of the second cyclopropanation. However, in some embodiments, the temperature of the reaction begins at a first cyclopropanation temperature and is decreased to a second cyclopropanation temperature, where the temperature of the first cyclopropanation is higher than the temperature of the second cyclopropanation.

[0114] In some embodiments of the methods described herein, the carbene precursor is added to the compound of Formula X, Formula XI, or Formula XII in an amount of about 1.0 to about 10 molar equivalents of carbene precursor relative to the compound of Formula X, Formula XI, or Formula XII.

[0115] In some embodiments of the methods described herein, the carbene precursor is added to the compound of Formula X, Formula XI, or Formula XII in an amount of less than 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 6, 7, 8, 9, or 10 molar equivalents of the carbene precursor relative to the compound of Formula X, Formula XI, or Formula XII.

[0116] In some embodiments of the methods described herein, the carbene precursor is added to the compound of Formula X, Formula XI, or Formula XII in an amount greater than 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 6, 7, 8, 9, or 10 molar equivalents of carbene precursor relative to the compound of Formula X, Formula XI, or Formula XII.

[0117] In some embodiments of the methods described herein, the carbene precursor is reacted with a compound of Formula X, Formula XI, or Formula XII at a concentration of about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, or about 2.7 relative to the compound of Formula X, Formula XI, or Formula XII. , about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 6, about 7, about 8, about 9 or about 10 molar equivalents of carbene precursor. tc-DNA nucleosides prepared from tc-DNA nucleoside precursors

[0118] In one embodiment, tc-DNA nucleosides can be prepared from the tc-DNA nucleoside precursors described herein.

[0119] In one embodiment, the present invention provides a method for the preparation of tc-DNA nucleoside precursors of one or more of Formulas I-IX into compounds of Formula XIII, Formula XIV or Formula XV: [ka] (wherein Bx is a nucleobase; T 5 and T 6 is an internucleoside linking group, and T 5 and T 6 The other is OR 9 , OR 10 , a 5'-terminal group, a 3'-terminal group, or an internucleoside linking group, where R 9 is H or a hydroxyl protecting group, and R 10 is the phosphorus moiety); q 9 , q 10 , q 11 , q 12 and q 13 are independently hydrogen (H), halogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 2~6 Alkenyl, substituted C 2~6 Alkynyl, and -(CH2) n -C(O)-R 11 (where n is 0 to 6, and R 11 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 alkyl); z 5 and z 6 are each independently H, halogen, or C 1~6 Alkyl, C 1~6 Alkoxyl, OC 2~6Alkenyl, OC 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 1~6 Alkoxy, substituted OC 2~6 Alkenyl and substituted OC 2~6 alkynyl) The present invention also includes a method for preparing the compound.

[0120] In one embodiment, a tc-DNA nucleoside described herein comprises a compound of Formula XIII, Formula XIV, or Formula XV, where Bx is selected from the group consisting of thymine, adenine, guanine, and cytosine. In one embodiment, a tc-DNA nucleoside described herein comprises a compound of Formula XIII, Formula XIV, or Formula XV, where Bx is a modified base. In one embodiment, a tc-DNA nucleoside described herein comprises a compound of Formula XIII, Formula XIV, or Formula XV, where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-bromouracil, inosine, and 2,6-diaminopurine.

[0121] In one embodiment, the present invention comprises tc-DNA nucleoside-containing oligomeric compounds comprising one or more of the tc-DNA nucleosides of Formula XIII, Formula XIV, Formula XV, or a pharmaceutically acceptable salt thereof.

[0122] While preferred embodiments of the present invention have been shown and described herein, such embodiments are provided by way of example only and are not intended to otherwise limit the scope of the invention. Various alternatives to the described embodiments of the invention can be employed in practicing the invention. [Example]

[0123] The embodiments encompassed herein will now be described with reference to the following examples, which are provided for illustrative purposes only, and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather as encompassing any and all variations that become evident as a result of the teachings provided herein. Example 1 – Preparation of bicyclosugar precursors

[0124] Intermediate 10 can be prepared by the synthetic route shown in FIG. 1 starting from D-mannose. Example 2 – Preparation of bicyclosugar precursors

[0125] Intermediate 10 can be prepared by an alternative synthetic route shown in FIG. 2, starting from D-ribose. Example 3 – Preparation of tert-butyldimethylsilyl enol ether intermediate

[0126] Silyl enol ether 17 can be prepared from intermediate 10 by the synthetic route shown in FIGS. Example 4 - Cyclopropanation of Compound 17 using a carbenoid prepared from CH2I2 and Et2Zn in the absence of additives

[0127] According to the following scheme, compound 17 was converted to the tc-DNA nucleoside precursor 18 using the cyclopropanation conditions described in Examples 4-7. [ka]

[0128] 1.07 g of the purified α-anomer (3.736 mmol) of 17 was dissolved in 37 ml of dry CHCl and cooled to 0 °C (ice). 22.3 ml (22.3 mmol, 6 equiv.) of 1.0 M EtZn (Aldrich) in hexane was then added dropwise and stirred at 0 °C for 30 min under Ar. 3.02 ml (37.2 mmol, 10 equiv.) of CHCl was then added dropwise over 15 min at the same temperature and stirred at 0 °C for an additional 2 h. The cooling bath was then removed, and the mixture was stirred at ambient temperature for an additional 21 h. TLC indicated a significant amount of unreacted α-17. This was diluted with EtOAc and quenched with 50 mL of saturated aqueous NHCl. Extractive workup gave 1.79 g of crude material, which was purified by chromatography on silica gel to give 0.43 g (39%) of compound 18 and 0.49 g of a mixture of compounds 17 and 18 (approximately 20:80). Example 5 – Cyclopropanation of compound 17 using carbene prepared from CH2I2 and activated Zn

[0129] AgOAc (60 mg) was dissolved in concentrated AcOH (60 ml) at 70 °C, and Zn (10 g; approximately 0.5 mm particle size, immediately etched with 0.1 M HCl, washed with HO and EtOH, and dried at room temperature / 0.01 Torr for 15 min) was added in one portion. The mixture was stirred for 1 min, decanted, and resuspended in AcOH (40 ml) and dry EtO (3 × 40 ml). The resulting dark gray Ag-Zn couple was stored on Ag wool in anhydrous Et2O. To the Ag-Zn couple (6.3 g, 96.4 mmol) in Et2O (11 mL), CHCl (8.41 g, 31.4 mmol) was added via syringe, and the mixture was stirred at room temperature under Ar for 1 h. A solution of 17 (1.5 g, 5.23 mmol, a mixture of both anomers) in dry Et2O (11 mL) was added dropwise to the carbene solution. Within 10 min, the mixture was refluxed under Ar for 3 h. The gray suspension was then cooled to 0 °C and diluted with Et2O (30 mL), after which pyridine (6.6 mL) was added dropwise at 0 °C. The white precipitate was filtered off through Celite, the filtrate was washed with saturated NaHCO3 (2 × 50 mL), and the aqueous phase was extracted with Et2O (50 mL). The combined organic phases were dried (MgSO4) and evaporated, and the residue was purified by flash chromatography (silica gel (50 g), Et2O / hexane 1:1) to give 908 mg (58%) of compound 18 as a colorless oil in a 2:1 ratio (α / β; 1 H-NMR). Example 4 - Cyclopropanation of Compound 17 using a stable carbenoid prepared from CH2I2 and Et2Zn and (n-BuO)2P(O)OH Preparation of (n-BuO)2P(O)OZnCHI solution

[0130] To 8 ml of toluene at -10 °C (dry ice / acetone), 2.4 ml (2.4 mmol) of 1.0 M diethylzinc (Acros) in toluene was added dropwise. Then, 0.5 ml (0.53 g, 2.44 mmol) of 97% (n-BuO)2P(O)OH (Aldrich) diluted with 5 ml of toluene was slowly added dropwise. The mixture was stirred under Ar at -10 °C for 20 min. Then, 0.20 ml (0.65 g, 2.44 mmol) of CHCl2 (Alfa Aesar) was added dropwise and stirred under Ar at -10 °C for 30 min. The clear solution was stored in the freezer.

[0131] Crude, unpurified compound 17 was dissolved in the reaction solvent and mixed at room temperature with a solution of the carbenoid (n-BuO)P(O)OZnCHI prepared by the procedure described above. The mixture was stirred for the time given in Table 1, followed by an extractive aqueous workup. Where indicated, yields are after FC.

[0132] The examples given in Table 1 demonstrate that unrealistically long reaction times and excess carbenoid are required for reaction completion.

[0133] [Table 1] Example 5 - Cyclopropanation of compound 17 using stable carbenoids prepared from CH2I2 and Et2Zn and (n-BuO)2P(O)OH in various solvents Preparation of (n-BuO)2P(O)OZnCHI solution

[0134] Carbenoids were prepared by the above procedure, substituting toluene with CH2Cl2 where indicated. 1.0 M diethylzinc (Acros) in toluene was used in all experiments.

[0135] Crude unpurified compound 17 was dissolved in the reaction solvent and mixed at room temperature with a solution of the carbenoid (n-BuO)P(O)OZnCHI prepared by the procedure described above. The mixture was stirred at 0-10 °C for the time given in Table 2, followed by an extractive aqueous workup. The ratio of 17 / 18 was estimated from H NMR of the crude material.

[0136] The examples given in Table 2 demonstrate that the solvent range for the transformation of 17, cyclopropanation, was poor regardless of solvent.

[0137] [Table 2] Example 6 - Cyclopropanation of compound 17 using stable carbenoids prepared from CH2I2 and Et2Zn and (n-BuO)2P(O)OH in various solvents Preparation of (n-BuO)2P(O)OZnCHI solution

[0138] Diethylzinc (1.5 M in toluene, 14.0 ml, 20.95 mmol) (Acros) was added dropwise to a solution of 4.3 ml of (n-BuO)P(O)OH (4.54 g, 20.95 mmol, 97%, Aldrich) in 22.8 ml of CHCl at −20° C. (dry ice / acetone) over a period of 30 min. The mixture was stirred under Ar at −20° C. for 30 min. Then, 1.69 ml (5.61 g, 20.95 mmol) of CHCl (Alfa Aesar) was added dropwise and stirred under Ar at −20° C. for 30 min. The clear solution was stored in the freezer.

[0139] Crude unpurified compound 17 (0.272 g, 0.70 mmol) was dissolved in the reaction solvent, and the Lewis acid shown in Table 3 was added at 0 °C (ice bath). The mixture was stirred at the same temperature for 15 min. Then, a solution of the carbenoid (n-BuO)2P(O)OZnCHI prepared by the above procedure was added at the same temperature. The mixture was stirred at 0-10 °C for the time given in Table 3. The mixture was stirred for 1 hour, followed by an extractive aqueous workup. The 17 / 18 ratio was estimated from the crude 1H NMR.

[0140] The example shown in Table 3 demonstrates the promotion of cyclopropanation of compound 17 in the presence of a Lewis acid.

[0141] [Table 3] Example 7 Preparation of tc-DNA nucleoside precursors

[0142] [Table 4-1] [Table 4-2] Preparation of (n-BuO)2P(O)OZnCHI solution

[0143] To 22.8 ml of CHCl at −20° C. (dry ice / acetone), 14.0 ml (20.95 mmol) of 1.5 M diethylzinc (Acros) in toluene was added dropwise. Then, 4.28 ml (4.54 g, 20.95 mmol) of 97% (n-BuO)P(O)OH (Aldrich) was added dropwise slowly. The mixture was stirred under Ar at −20° C. for 30 min. Next, 1.69 ml (5.62 g, 20.95 mmol) of CHCl (Reagent Plus, 99%, Aldrich) was added dropwise and stirred under Ar at −15° C. for 30 min. The clear solution was stored in the freezer. Experiment 5 - Cyclopropanation of Compound 17 using 2.1 molar equivalents of carbene and 0.5 equivalents of Lewis acid

[0144] 181 mg of the purified α-anomer and 174 mg of the β-anomer (355 mg, 1.239 mmol) of 17 were dissolved in 0.7 mL of CHCl and cooled to −10°C (dry ice). 826 μL (1.239 mmol, 1 equiv.) of 1.5 M EtZn(Acros) in toluene was then added dropwise and stirred at −10°C for 10 min under Ar. 5.312 mL (2.602 mmol, 2.1 equiv.) of (n-BuO)P(O)OZnCHI solution was then added dropwise and stirred at −10°C for an additional 10 min. 336 μL (298.5 mg, 0.619 mmol, 50 mol%) of a 25 wt % diethylaluminum chloride solution in toluene (Aldrich) was then slowly added dropwise. The mixture was stirred at -10°C for 2 hours and then allowed to warm slowly to room temperature in the bath. The mixture was stirred for a total of 21 hours. Experiment 6 - Cyclopropanation of Compound 17 with 3 molar equivalents of carbene and 0.5 equivalents of Lewis acid

[0145] The purified α-anomer (106 mg) and β-anomer (105 mg) (211 mg, 0.737 mmol) of 17 were dissolved in 0.5 ml of CHCl and cooled to −10 °C. Then, 491 μl (0.737 mmol, 1 equiv.) of 1.5 M EtZn in toluene was added dropwise and stirred at −10 °C for 10 min. Then, 4.51 ml (2.211 mmol) of 17 was added dropwise. A solution of (n-BuO)2P(O)OZnCHCl (0.369 mmol, 3 equiv.) was added dropwise and stirred at −10° C. for another 10 min. Afterwards, 201 μl (0.369 mmol, 50 mol%) of a 25 wt. % solution of diethylaluminum chloride in toluene was slowly added dropwise. The mixture was stirred under Ar at −10° C. for 2 h and then at room temperature for 22 h. The mixture was quenched with 4 ml of saturated NH4Cl solution, taken up in MTBE, and washed with 30 ml of saturated NaCl solution. The aqueous phase was extracted with 2 × 20 ml of MTBE. The combined organic phases were dried over Na2SO4, filtered, concentrated, and briefly dried under high vacuum. The resulting yellowish oil was purified by flash chromatography on 20 g of silica gel with 3:1 hexane / EtOAc. 225 mg (approx. 0.749 mmol, 102%) of a 1:1 diastereomeric mixture of compound 18 was obtained. 31 P-NMR revealed that the product was in the form of a yellowish oil contaminated with "dibutyl phosphate."

[0146] 1 Hq-NMR (24.4 mg product, 24.9 mg dimethyl terephthalate, 2 ml CD3CH): opaque solution with white precipitate → after addition of 150 μl pyridine a clear solution with a white precipitate forms. Experiment 7 - Cyclopropanation of Compound 17 using 2.2 molar equivalents of carbene and 0.5 equivalents of Lewis acid

[0147] The purified α-anomer (117 mg) and β-anomer (100 mg) (217 mg, 0.758 mmol) of 17 were dissolved in 0.5 mL of CHCl and cooled to −10°C. 505 μL (0.758 mmol, 1 equiv.) of 1.5 M EtZn in toluene was then added dropwise and stirred at −10°C for 10 min. 3.40 mL (1.668 mmol, 2.2 equiv.) of (n-BuO)P(O)OZnCHI solution was then added dropwise and stirred at −10°C for an additional 10 min. 412 μL (0.758 mmol, 1 equiv.) of a 25 wt. % diethylaluminum chloride solution in toluene was then slowly added dropwise. The mixture was stirred under Ar at −10°C for 2 h and then at room temperature for 19 h. Experiment 9 - Cyclopropanation of Compound 17 with 3 molar equivalents of carbene and 0.5 equivalents of Lewis acid

[0148] 211 mg (0.737 mmol) of the purified α-anomer of 17 was dissolved in 0.5 ml of CHCl and cooled to −10 °C. 491 μL (0.737 mmol, 1 equiv.) of 1.5 M EtZn in toluene was then added dropwise and stirred at −10 °C for 10 min. 6.01 ml (2.211 mmol, 3 equiv.) of a (n-BuO)P(O)OZnCHCl solution was then added dropwise and stirred at −10 °C for another 10 min. 201 μL (0.369 mmol, 50 mol%) of a 25 wt % diethylaluminum chloride solution in toluene was then slowly added dropwise. The mixture was stirred under Ar at −10 °C for 2 h and then at room temperature for 23 h (→ yellowish solution). 202 mg (approximately 0.672 mmol, 91%) of the α-anomer of compound 18 was obtained. 31 P-NMR revealed that the product was in the form of a yellowish oil contaminated with "dibutyl phosphate."

[0149] 1 Hq-NMR (37.38 mg of product, 37.24 mg of dimethyl terephthalate, 1 ml of CDCl3)

[0150] Preparation of the Zn salt: 158 mg of contaminated compound 18, 2 ml of CH3CN, and 200 μl (2.47 mmol) of pyridine were combined. The mixture was stirred at room temperature for 1 hour and at 0° C. for 1 hour. The precipitate was filtered off using Celite, washed with cold CH3CN, and the filtrate was concentrated (pyridine and toluene were co-evaporated) and dried under high vacuum overnight. 146 mg of compound 18 was obtained. Example 8 – Preparation of tc-DNA nucleoside precursors

[0151] A 1.5 M solution of EtZn in toluene (51.49 ml, 77.24 mmol, 5 equiv.; Acros) was added dropwise to dry CHCl (19.17 ml) over 7 min at −20° C. Then, 97% dibutyl phosphate (12.63 ml, 61.79 mmol, 4 equiv.; Aldrich) was added dropwise over 12 min (exothermic reaction, temperature rises from −20° C. to −10° C.). After stirring for 30 min, diiodomethane (4.98 ml, 61.79 mmol, 4 equiv.; Aldrich, 99%) was added dropwise over 3 min, and the mixture was stirred at −20° C. for an additional 30 min. Next, a solution of crude 17 (5.0 g, 88.5% purity, 15.45 mmol, 1 equiv.) in dry CHCl (10.51 ml) was added dropwise (exothermic reaction, temperature rising from −20 to −10 °C). After stirring at −20 °C for 15 min, EtAlCl (25 wt.% solution in toluene, 4.20 ml, 7.72 mmol, 0.5 equiv.; Aldrich) was added. The reaction mixture was stirred at −10 °C for 2 h. The cooling bath was then removed, and the mixture was allowed to warm to room temperature and stirred at that temperature for 22 h. It was then diluted with hexane (400 ml) and washed with 0.2 M aqueous HCl (2 × 400 ml). The combined aqueous phases were re-extracted with hexane (300 ml). The combined organic phases were then washed with 0.2 M aqueous NaOH (2 x 400 ml), dried over Na2SO4, filtered and evaporated to give 5.09 g of TC-003 (a mixture of two anomers in a 24:76 ratio; 1 The product was obtained as a dark yellow viscous liquid with a purity of 73.5% by Hq-NMR and a yield of 81%. For example, the present invention provides the following items. (Item 1) 1. A method for preparing a tc-DNA nucleoside precursor, the method comprising: a. preparing a carbene precursor at a carbene preparation temperature; b. cyclopropanation of said carbene precursor with a compound of Formula X, Formula XI or Formula XII at cyclopropanation temperatures: [ka] adding; and c. Providing the tc-DNA nucleoside precursor of one of Formulas I-IX, wherein Y is alkoxy; T 3 and T 4 are OR 5 where each R 5 is H or a hydroxyl protecting group, q 6 , q 7 and q 8 are each independently hydrogen, halogen, or C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 2~6 Alkenyl, substituted C 2~6 Alkynyl, and -(CH2) n -C(O)-R 6 (where n is 0 to 6, and R 6 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 alkyl); z 3 and z 4 are each independently hydrogen, C 1~6 Alkyl, C 1~6 Alkoxyl, OC 2~6 Alkenyl, OC 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 1~6 Alkoxy, substituted OC 2~6 Alkenyl and substituted OC 2~6alkynyl halogens, method. (Item 2) 1. A method for preparing a tc-DNA nucleoside precursor, the method comprising: a. preparing a carbene precursor at a carbene preparation temperature; b. A compound of Formula X, Formula XI or Formula XII: [ka] preparing a solution of c. adding the carbene precursor to the solution of the compound of Formula X, Formula XI, or Formula XII at a cyclopropanation temperature; and d. Providing the tc-DNA nucleoside precursor of one of Formulas I-IX, wherein Y is alkoxy; T 3 and T 4 are OR 5 where R 5 is H or a hydroxyl protecting group, q 6 , q 7 and q 8 are each independently hydrogen, halogen, or C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 2~6 Alkenyl, substituted C 2~6 Alkynyl, and -(CH2) n -C(O)-R 6 (where n is 0 to 6, and R 6 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 alkyl); z 3 and z 4 are each independently hydrogen, C 1~6 Alkyl, C 1~6 Alkoxyl, OC 2~6 Alkenyl, OC2~6 Alkynyl, substituted C 1~6 Alkyl, substituted C 1~6 Alkoxy, substituted OC 2~6 Alkenyl and substituted OC 2~6 alkynyl halogens, method. (Item 3) The method of any of the preceding items, comprising adding a Lewis acid catalyst to the mixture of the compound of Formula X, Formula XI or Formula XII and the carbene precursor. (Item 4) 2. The method of claim 1, further comprising the step of preparing a carbene precursor comprising the step of: 7 I2 in a solvent, wherein R 7 CH2, CH-C 1~6 Alkyl, CH-C 2~6 Alkenyl, CH-C 2~6 Alkynyl, substituted CH-C 1~6 Alkyl, substituted CH-C 2~6 Alkenyl, substituted CH-C 2~6 Alkynyl and CH—(CH2) n -C(O)-R 8 (where n is 0 to 6, and R 8 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 The method of any of the preceding items, wherein the alkyl group is selected from the group consisting of alkyl. (Item 5) 5. The method of claim 4, wherein the solvent comprises hexane, toluene, dichloromethane (CH2Cl2), tetrahydrofuran (THF), acetonitrile (CH3CN), dimethylformamide (DMF), diethyl ether, dimethoxyethane (DME), or a combination thereof. (Item 6) 2. The method of claim 1, further comprising the step of preparing a carbene precursor comprising the step of: 7 adding a carbene additive to said mixture of I2, wherein said carbene additive is a substituted or is selected from the group consisting of unsubstituted alkyl alcohols, carboxylic acids and phosphates. (Item 7) The carbene precursor is Q3CCO2ZnR 7 I, Q3CCH2OZnR 7 I, (n-BuO)2P(O)OZnR 7 I, (alkyl-O)2P(O)OZnR 7 I, (aryl-O)2P(O)OZnR 7 I or 2,4,6-Cl3C6H2OZnR 7 I, where each Q is independently selected from the group consisting of H, Cl, Br, and F; 7 CH2, CH-C 1~6 Alkyl, CH-C 2~6 Alkenyl, CH-C 2~6 Alkynyl, substituted CH-C 1~6 Alkyl, substituted CH-C 2~6 Alkenyl, substituted CH-C 2~6 Alkynyl and CH—(CH2) n -C(O)-R 8 (where n is 0 to 6, and R 8 are OH, NH2, OC 1~32 Alkyl and NH-C 1~32 The method of any of the preceding items, wherein the alkyl group is selected from the group consisting of alkyl. (Item 8) 3. The method of any of the preceding items, wherein the carbene precursor is CCl3CO2ZnCH2I, CF3CH2OZnCH2I, (n-BuO)2P(O)OZnCH2I or 2,4,6-Cl3C6H2OZnCH2I. (Item 9) A tc-DNA nucleoside precursor prepared by the method according to any one of items 1 to 8. (Item 10) tc-DNA nucleosides prepared from the tc-DNA precursors described in item 9. (Item 11) 11. A tc-DNA-containing oligonucleotide comprising the tc-DNA nucleoside according to item 10.

Claims

1. A compound of any one of formulas I to III: 【Chemistry 13】 1. A method for preparing a compound comprising: a. Preparing a carbene precursor at a carbene preparation temperature by combining a first Lewis acid selected from the group consisting of Et2AlCl, ZnI2, ZnCl2, ZnBr2, Ti(OiPr)4, Me2AlCl, TMSOTf, TiCl4, and combinations thereof, with R7I2 in a solvent, where R7 is CH2, CH-C1-6 alkyl, CH-C2-6 alkenyl, CH-C2-6 alkynyl, substituted CH-C1-6 alkyl, substituted CH-C2-6 alkenyl, substituted CH-C2-6 alkynyl, and CH-(CH2)n-C(O)-R8 (where n is 0-6 and R8 is OH, NH2, O-C1-32 alkyl, or OH). alkyl and NH-C 1-32 alkyl); b. Cycling the carbene precursor at cyclopropanation temperatures to form a compound of Formula X, Formula XI, or Formula XII: 【Chemistry 14】 mixing with the c. adding a catalytic amount of a second Lewis acid to the mixture. Including, wherein X and Y are each alkoxy; T 1 , T 2 , T 3 and T 4 are each OR 5 , where each R 5 is independently H or a hydroxyl protecting group; q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 are each independently selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, substituted C 1-6 alkyl, substituted C 2-6 alkenyl, substituted C 2-6 alkynyl, and —(CH 2 ) n —C(O)—R 6 where n is 0-6 and R 6 is selected from the group consisting of OH, NH 2 , O—C 1-32 alkyl and NH—C 1-32 alkyl; q 8 is selected from the group consisting of hydrogen, halogen, C 1-3 alkyl, C 2-3 alkenyl, C 2-3 alkynyl, substituted C 1-3 alkyl, substituted C 2-3 alkenyl, substituted C 2-3 alkynyl, and —(CH 2 ) n —C(O)—R 6 where n is 0-2 and R 6 is selected from the group consisting of OH, NH 2 , O—C 1-3 alkyl, and NH—C 1-3 alkyl; Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from the group consisting of hydrogen, C 1-6 alkyl, C 1-6 alkoxyl, O—C 2-6 alkenyl, O—C 2-6 alkynyl, substituted C 1-6 alkyl, substituted C 1-6 alkoxy, substituted O—C 2-6 alkenyl and substituted O—C 2-6 alkynyl halogen; method.

2. The compound of claim 1, wherein the compound is a compound of any one of formulas IV to VI: 【Chemistry 15】 and 10. The method of claim 1, wherein R4 is a hydroxyl protecting group.

3. The compound of claim 2, wherein the compound is a compound of any one of formulas VII-IX: 【Chemistry 16】 The method of claim 1, wherein 4. The method of claim 1, wherein the solvent comprises hexane, toluene, dichloromethane (CH2Cl2), tetrahydrofuran (THF), acetonitrile (CH3CN), dimethylformamide (DMF), diethyl ether, dimethoxyethane (DME), or a combination thereof.

5. The method of claim 1, wherein the step of preparing the carbene precursor comprises adding a carbene additive to the mixture of Lewis acid catalyst and R 7 I 2 , wherein the carbene additive is selected from the group consisting of substituted or unsubstituted alkyl alcohols, carboxylic acids and phosphates.

6. The carbene precursor is Q 3 CCO 2 ZnR 7 I, Q 3 CCH 2 OZnR 7 I, (n-BuO) 2 P(O)OZnR 7 I, (alkyl-O) 2 P(O)OZnR 7 I, (aryl-O) 2 P(O)OZnR 7 I or 2,4,6-Cl 3 C 6 H 2 OZnR 7 I, wherein each Q is independently selected from the group consisting of H, Cl, Br and F, and R 7 is CH 2 , CH—C 1-6 alkyl, CH—C 2-6 alkenyl, CH—C 2-6 alkynyl, substituted CH—C 1-6 alkyl, substituted CH—C 2-6 alkenyl, substituted CH—C 2-6 alkynyl and CH—(CH 2 ) n -C(O)-R 8 , where n is 0-6 and R 8 is selected from the group consisting of OH, NH 2 , O-C 1-32 alkyl and NH-C 1-32 alkyl.

7. The method of claim 1, wherein the carbene precursor is CCl 3 CO 2 ZnCH 2 I, CF 3 CH 2 OZnCH 2 I, (n-BuO) 2 P(O)OZnCH 2 I or 2,4,6-Cl 3 C 6 H 2 OZnCH 2 I.

8. The method of claim 1, wherein the second Lewis acid is selected from the group consisting of Et2AlCl, Et2Zn, ZnI2, ZnCl2, ZnBr2, Ti(OiPr)4, Me2AlCl, TMSOTf, TiCl4, and combinations thereof.

9. The method of claim 1, wherein the second Lewis acid is Et 2 Zn.

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