Tricyclo-DNA nucleoside precursors and processes for preparing same

Abstract

The present invention relates to processes for preparing tc-DNA nucleoside precursors, the resulting tc-DNA nucleosides, and oligonucleotides containing such tc-DNA nucleosides. In embodiments of the invention, the processes involve the use of carbene precursors. In one embodiment, the invention includes a process for preparing a tc-DNA nucleoside precursor of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), or Formula (VI).

Description

[Technical field]

[0001] FIELD OF THEINVENTION 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] 2. 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.In general, the principle behind antisense technology is that antisense oligomeric compounds (nucleotide or its analogue sequence) hybridize with target nucleic acid and modulate 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 variants 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 processes that allow 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 relates to a compound of formula I, formula II, formula III, formula IV, formula V or formula VI: [ka] The method includes the steps of: wherein X can be alkoxy; T 1 and T 2 are ORed 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, R 2 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 above-mentioned compound.

[0007] In one embodiment, the invention includes a method for preparing a tc-DNA nucleoside precursor of any one of formulas I-IX, the method comprising: a. preparing a carbene precursor at a carbene preparation temperature; b. Cyclopropanation of said carbene precursor at a cyclopropanation temperature to a compound of formula X, formula XI or formula XII: [ka] adding; and c. Providing said tc-DNA nucleoside precursor of one of formulas I-IX where Y can be alkoxy; T 3 and T 4 are ORed 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, R 6 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 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, 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 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 where Y can be alkoxy; T 3 and T 4 are ORed 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, R 6 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, 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 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, R 8 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, the step of 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, where the carbene additive is selected from the group consisting of substituted or unsubstituted alkyl alcohols, carboxylic acids, and phosphates.

[0012] In some embodiments, the step of 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 the carbene precursor comprises adding a Lewis acid catalyst (e.g., ZnEt) to the 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, the step of preparing a carbene precursor comprises the step of reacting 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)2P(O)OH or (aryl-O)2P(O)OH). In some embodiments, the carbene additive may be a substituted carboxylic acid of formula Q3CCO2H, 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 Q3CCO2H, where Q3C may be defined as CCl3, CHCl2, CH2Cl, or CF3. In some embodiments, the carbene additive may be a substituted alkyl alcohol of formula Q3CCH2OH, 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 Q3CCH2OH, where Q3C may be defined as CCl3, CHCl2, CH2Cl, or CF3. In some embodiments, the carbene additive may be trichloroacetic acid, 2,2,2-trifluoroethanol, trichlorophenol, or (n-BuO)2P(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; 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 2~6Alkenyl, substituted CH-C 2~6 Alkynyl and CH-(CH2) n -C(O)-R 8 (where n is 0 to 6, and R 8 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 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 description of the drawings]

[0023] [Figure 1]FIG. 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) Me2C(OMe)2 (4.4 equiv), TsOH (0.003 equiv), 50° C., 1 h; (b) Ac2O (2 equiv), pyridine, room temperature, 16 h; (c) 64% aqueous AcOH, 55° C., 4 h; (d) HC(OMe)3 (5 equiv), reflux, 1 h; (e) Ac2O (7.4 equiv), 130° C., 3 h; (f) MeOH, t-BuOK (0.4 (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, rt, 16 h, 97% yield; (i) 10% Pd / C, H2, MeOH, rt, 16 h, 97% yield; and (j) PCC (1.7 equiv), CHCl2, rt, 16 h, 85% yield. Overall yield of 10 steps from D-mannose was 20%.

[0024] [Diagram 2] FIG. 2 illustrates an alternative synthetic route to the bicyclosugar intermediate 10 from D-ribose. The reagents and conditions used in the synthetic route are as follows: (a) Br2 (1.04 equiv), NaHCO3 (2 equiv), H2O, 0-5 °C, crude; (b) DMP, acetone, H2SO4 (catalytic amount), Amberlyst, room temperature, (crystallization, 67% yield over two steps); (c) NaIO4 (1.05 equiv), NaOH (1.1 equiv), 0 °C, BaCl2 (crude, 78% yield); (d) iPrOH, PPTS (0.02 equiv), reflux (chromatography, 63% yield); (e) LiCH2(O)P(OMe)2 (1.1 equiv), THF, -78 °C to room temperature (crude, approximately 60% yield); and (f) 10% Pd / C, H2, EtOAc, room temperature (chromatography, 95% yield). Overall yield over six steps from D-ribose is 16%.

[0025] [Diagram 3]3 illustrates a synthetic route from intermediate 10 to alcohol intermediate 15. The reagents and conditions used in the synthetic route are as follows: (a) (EtO)2P(O)CH2CO2Et (1 eq), THF, 0° C. to room temperature, 88% yield; (b) MCPBA (2.2 eq), CHCl2, 0 to 40° C., 18 h; (c) LiAlH4 (1.5 eq), THF, 0° C. to room temperature, 1 h (chromatography, 86% yield); (d) IBX, THF / DMSO, room temperature, 2.5 h, 91% yield; and (e) MeOH, Amberlite IR-120 (H+), room temperature for 16 h, then 60° C. for 2 h (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 eq), TEMPO (0.007 eq), AcONa (3 eq), CHCl, acetone, −15 to 0° C., 1 h; and (b) TBDMSCl (1.4 eq), DBU (1.6 eq), KI (0.1 eq), THF, room temperature, 2 h. Alternatively, the reagents and conditions used in the synthetic route can be: (a) DMP, CHCl, room temperature; and (b) (1) LDA, −65° C., then (2) TBDMSCl, THF, −65° C. to 0° C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 of the aspects and embodiments of this 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, at least two of said eight or more monomeric subunits being 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'-deoxy 2'-fluoro-arabinonu ... It is meant to include all manner of monomer units suitable for oligomer synthesis, including, typically and preferably referring to, monomer subunits such as nucleotides, hexitol nucleic acid (HNA) nucleosides; and phosphorodiamidate morpholino (PMO) nucleosides, nucleoside mimetics, naturally occurring nucleotides, modified nucleotides, here in particular 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 and modified nucleosides, and herein, in particular, ribonucleosides, deoxyribonucleic acid, 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 specifically refers 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 herein 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 3~32 The alkyl group is represented by the formula: 3~32 Alkyl is independently a C as defined herein. 3~32 is selected from alkyl.

[0031] The term "alkylphosphonate moiety" as used herein means 1~32 The alkyl group is represented by the formula: 1~32 Alkyl is independently a C as defined herein. 1~32 is selected from alkyl.

[0032] The term "alkyl," as used herein, refers to a straight or branched chain hydrocarbon radical containing no unsaturation, having from 1 to 32 carbon atoms, and consisting solely of carbon and hydrogen 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 "1-32" refers to each integer within the given range. For example, "1-32 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 32 carbon atoms, although this definition is also intended to cover the occurrence of the term "alkyl" where no numerical range is specifically specified. Exemplary 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, tertiary butyl (used interchangeably with 1,1-dimethylethyl or tert-butyl), n-pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl, and decyl. Unless otherwise specified in the specification, an alkyl group is optionally substituted with one or more substituents which are independently alkenyl, alkoxy, carboxy group (-COOH), heteroalkyl, heteroalkenyl, hydroxyl, 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. 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, in which one hydrogen of said alkyl is broken off to generate a second radical of said alkylene. Examples of alkylene are, illustratively, -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 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 may consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, etc., up to 32 carbon atoms. Exemplary 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. Whenever applicable, e.g., substituted, each double bond may be in either the (E)- or (Z)-configuration. Thus, alkenyl, when applicable, each of said double bonds may include either its (E)-configuration, its (Z)-configuration, and mixtures thereof in any ratio. Unless otherwise specified in the specification, an alkenyl group is optionally substituted by one or more substituents which are independently alkenyl, alkoxy, a carboxy group (-COOH), a heteroalkyl, a heteroalkenyl, a hydroxyl, 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 "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 32 carbon atoms. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl. Unless otherwise specified herein, an alkynyl group is optionally substituted with one or more substituents that are 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 (-CH4) 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 the alkynyl has been cleaved to generate a second radical of the alkynylene.

[0038] The term "alkoxy" refers to groups of -O-alkyl, including linear, branched configurations 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 includes the alkoxy groups (C 1~6)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 specifically stated in the specification, the alkyl portion of the alkoxy group is optionally substituted with one or more substituents which are 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.

[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 in the specification, the alkyl, aryl, or heteroaryl portion of the acyl group is optionally substituted with one or more substituents, which are 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.

[0041] Unless otherwise specified in this specification, the term “amino” or “amine” refers to —N(R a )2 radical groups, each R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl. a ) 2 groups have 2 R in addition to hydrogen aWhen substituted, they can 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 specifically stated in the specification, amino or amine groups are optionally substituted with one or more substituents which are 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 (-CH4) optionally substituted with a carboxy group.

[0042] The term “aromatic” or “aryl” or “Ar” refers to an aromatic radical having 6 to 10 ring atoms (e.g., C6-C8), with at least one ring having a conjugated pi-electron system, which is carbocyclic (e.g., phenyl, fluorenyl, and naphthyl). 10 Aromatic or C6-C 10 aryl). Divalent radicals formed from substituted benzene derivatives and having a free valence at a ring atom are named substituted phenylene radicals. Divalent radicals derived from monovalent polycyclic hydrocarbon radicals, named with the suffix "-yl" by the removal of one hydrogen atom from the free valence carbon atom, are named by adding "-idene" to the name of the corresponding monovalent radical, e.g., a naphthyl group having two points of attachment is called naphthylidene. Whenever it appears herein, numerical ranges such as "6-10" refer to respective integers within the given range, e.g., "6-10 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, where 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. Cycloalkyl groups are groups 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, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, etc.

[0046] The term "fluoroalkyl" refers to an alkyl radical, as defined above, substituted by one or more fluoro radicals, as defined above, such as 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 the 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, 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 may consist of 5 ring atoms, 6 ring atoms, etc., up to 18 ring atoms. Divalent radicals derived from monovalent heteroaryl radicals, named with the suffix "-yl" by the removal of one hydrogen atom from the free valence atom, are named by adding "-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 compound is not a mirror image of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and chemical and biological reactivity. Mixtures of diastereomers can be separated under high resolution analytical procedures such as electrophoresis and chromatography.

[0052] "Enantiomers" refers 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 McRaw-Hiff Dictionary of Chemical Terms (1984), edited by S.P. Parker, 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 a 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 multifunctional compound so that a chemical reaction can be selectively carried out at an otherwise unprotected reactive site, and which can then be easily removed or deprotected after the selective reaction is complete. A variety of protecting groups are disclosed, for example, in Protecting Groups in Organic Synthesis, TW Greene and PGM 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", as used interchangeably herein, are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd Edition, John Wiley & Sons, New York (1999), Greene's Protective Groups in Organic Synthesis, PGM Wuts, 5th Edition, John Wiley & Sons (2014) and Current Protocols in Nucleic Acid Chemistry, eds. 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-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-methylethyl carbamate (Met ... -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 a hydroxyl group" or "hydroxyl protecting group", as used interchangeably herein, are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd Edition, John Wiley & Sons, New York (1999); Greene's Protective Groups in Organic Synthesis, PGM Wuts, 5th Edition, John Wiley & Sons (2014) and Current Protocols in Nucleic Acid Chemistry, eds. S.L. Beaucage et al., June 2012, herein in particular Chapter 2. In certain embodiments, the "hydroxyl protecting groups" of the present invention include, typically and preferably independently selected from each occurrence, 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-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers, and other silyl ethers; methyl ethers, ethoxyethyl ethers (EE).

[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), 4,4'-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- and each occurrence independently selected from among 9-(p-methoxyphenyl)xanthin-9-yl (MOX), 9-phenylxanthen-9-yl (pixyl), or 9-(p-methoxyphenyl)xanthin-9-yl (MOX).

[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, 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, and 6-azo 8-substituted purine bases such as 8-halo-, 8-amino-, 8-thiol-, 8-thioalkyl-, 8-hydroxyl-adenine or guanine, 5-substituted pyrimidine bases such as 5-halo-, especially 5-bromo-, 5-trifluoromethyl-uracil or -cytosine; 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, hydrophobic bases, promiscuous bases, size-extended bases, or fluorinated bases.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, pp. 273-302). The term "nucleobase derivative" also includes those in which the purine or pyrimidine base is replaced with a moiety corresponding to a spacer of the invention, in particular a moiety for linking said one or more lipid moieties within said oligomeric compound, preferably said oligonucleotide. The specific linkage of said moiety corresponding to a spacer is known to those skilled in the art.Preferred nucleobase derivatives include methylated adenine, guanine, uracil and cytosine, and nucleobase derivatives, preferably (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 linkage, preferably further linkage of said tricyclo-deoxyribonucleic acid (tc-DNA) nucleoside, either to an additional tc-DNA nucleoside, a nucleoside other than a tc-DNA nucleoside, or a non-nucleoside, including a peptide or a protein. Representative patents which teach such possible linking groups include, but are not limited to, U.S. Pat. No. 5,034,506; U.S. Pat. No. 5,166,315; U.S. Pat. No. 5,185,444; U.S. Pat. No. 5,214,134; U.S. Pat. No. 5,216,141; U.S. Pat. No. 5,235,033; U.S. Pat. No. 5,264,562; U.S. Pat. No. 5,264,564; U.S. Pat. No. 5,405,938; U.S. Pat. No. 5,434,257; U.S. Pat. No. 5,466,677; U.S. Pat. No. 5,470,967; U.S. Pat. No. 5,471,967; U.S. Pat. No. 5,472,967; U.S. Pat. No. 5,473,967; U.S. Pat. No. 5,474,967; U.S. Pat. No. 5,475,967; U.S. Pat. No. 5,476,967; U.S. Pat. No. 5,477,967; U.S. Pat. No. 5,478,967; U.S. Pat. No. 5,479,967; U.S. Pat. No. 5,477,967; U.S. Pat. No. 5,479 ... Nos. 5,677, 5,541,307, 5,561,225, 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. Non-phosphorus linking groups do 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 of the other, optionally substituted with cyano, nitro, halogen.Typically and preferably, the internucleoside linking group is a phosphorus linking group, and the phosphorus linking group is P. III or P V It refers to a moiety containing a phosphorus atom in the valence state of 1. 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, 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, the phosphonate being preferably an H-phosphonate linking group or a methylphosphonate linking group.

[0063] As used herein, the term "nucleoside" refers to a compound that comprises a nucleobase and a sugar that is covalently linked to said nucleobase.Furthermore, the term "nucleoside" is meant to include all manner of naturally occurring or modified nucleosides, or nucleoside mimetics that can be incorporated into oligomers using natural or chemical oligomer synthesis.Exemplarily 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 described herein, as known to those skilled in the art.The term "nucleoside mimetics" is intended to include those structures that are used to replace sugar and nucleobase. 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 includes, but is not limited to, a monocyclic, bicyclic or tricyclic ring system, preferably a tricyclic or bicyclic system, or a monocyclic ribose or de(s)oxyribose. Modifications of the sugar further include, but are not limited to, a modified stereochemical configuration, substitution of at least one group, or deletion of at least one group. The modified sugar is typically and preferably a modified version of a ribosyl moiety (i.e., a furanosyl moiety) naturally occurring in RNA and DNA, such as a bicyclic sugar, a tetrahydropyran, a 2'-modified sugar, a 3'-modified sugar, a 4'-modified sugar, a 5'-modified sugar or a 4'-substituted sugar. 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-alkyl, 2'-O-(2-aminoethyl)ethyl, 2'-O-(2-chloroethoxy)ethyl, 2'-O-(2-aminoethyl ... ,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 the 2'-O-methyl modification 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 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 a modified morpholino group, including 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, e.g., the sugar moiety has a 2'-O-CH(alkyl)-4' or 2'-O-CH2-4' group, locked nucleic acid (LNA), xylo-LNA, alpha-L-LNA, beta-D-LNA, cEt (2'-O,4'-C constrained ethyl) LNA, cMOEt (2'-O,4'-C constrained methoxyethyl) LNA, 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, the oligomeric compound is an oligonucleotide.The term "oligonucleotide" as used herein refers to a compound that comprises at least two nucleosides that are linked to each other by internucleoside linking group.Therefore, the term "oligonucleotide" as used herein includes, typically and preferably refers to, an oligomeric compound that comprises at least two nucleosides that are linked 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 compounds are double-stranded (i.e., duplex). In a preferred embodiment, the oligomeric compounds are single-stranded.

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

[0069] The terms "natural" or "naturally occurring," as used interchangeably herein, refer to a compound that is 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 by traditional Watson-Crick base pairing or other non-traditional types of pairing (e.g., Hoogsteen or reversed Hoogsteen hydrogen bonds) 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 and 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 does not require 100% complementarity with a target nucleic acid sequence to be specifically hybridizable. That is, two or more nucleic acid molecules may not be perfectly complementary. Complementarity may 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% complementarity, respectively. "Perfectly" or "completely" 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, whether both nucleic acid molecules have the same number of nucleotides (i.e., 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 access of spliceosomes to one or more splice donor or splice acceptor sites, or any other sites in exons or introns involved in the definition of splicing, the oligonucleotides can prevent the splicing reaction and cause the deletion of exons from fully processed mRNA.Exon skipping is achieved in the nucleus during the maturation process of pre-mRNA.Exon skipping involves the masking of important sequences involved in the splicing of targeted exons by using antisense oligonucleotides that are complementary to splice donor sequences in pre-mRNA. For example, the compositions of the invention comprising the oligomeric compounds provided herein can be suitably used for exon skipping by masking splice sites at intron / exon junctions in 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 events that take 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. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or on the human subject and disease state to be treated (e.g., the subject's weight, age, and sex), the severity of the disease state, the mode of administration, and the like, which can be readily determined by one of ordinary skill in the art. The term also applies to a dose that induces a particular response in the target cells (e.g., reduced platelet adhesion and / or cell migration). The particular 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 to which the compound is delivered.

[0076] A "therapeutic effect" as the term is used herein 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 salts" refers to salts derived from various organic and inorganic counterions known in the art. Pharmaceutically acceptable acid addition salts can be formed with 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 with 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. The 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 said oligomeric compound of the invention, preferably in said oligonucleotide of the invention, typically and preferably bound to P(III) or P(V) and 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 typically and preferably as part of said internucleoside linking group selected from phosphorothioate or phosphorodiester, each of said hydroxyl group (OH) or thiol group (SH) can be present, independently of one another, as said OH group or in its ionic state, such as O-anion and a pharma- ceutically acceptable cation, or as said SH group or in its ionic state, such as S-anion and a pharma- ceutically acceptable cation. Additionally, any combination and any equilibrium state between the aforementioned situations in the compositions of the present invention are 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 are 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 the hydrogen is located at the oxygen, thus #-NH-C 2~12 All alkylene-OP(OH)(S)-§ and pharma- ceutically acceptable salts thereof are included herein.

[0079] Thus, a pharma- ceutically 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 bound to P(III) or P(V) and 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 present in said oligonucleotide of the invention 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 O-anion and its pharma- ceutically acceptable cation, or as said SH group or in its ionic state, such as S-anion and its pharma- ceutically acceptable cation, typically and preferably, said pharma- ceutically acceptable cation is selected from protonated trimethylamine, protonated diethylamine, protonated methylamine, ammonium, sodium or potassium, more preferably said pharma- ceutically 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 retarding 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 composition of the present invention, except where 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 which 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 the atoms are arranged in space, i.e., are isomers with 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 racemic mixtures 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 described herein contain one or more asymmetric centers and may therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined with respect to absolute stereochemistry as (R) or (S). The chemical entities, pharmaceutical compositions, and methods of the present 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 can be prepared using chiral synthons or chiral reagents or resolved using conventional techniques. In cases where the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise specified, the compounds are intended to include both E and Z geometric isomers.

[0083] "Enantiomeric purity" as used herein refers to the relative amount of a particular enantiomer present relative to the other enantiomer, expressed as a percentage. For example, when a compound that may have the configuration of (R) or (S)-isomer exists as a racemic mixture, the enantiomeric purity for either (R) or (S)-isomer is about 50%. When 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 for the (S)-isomer form is 80%. The enantiomeric purity of a compound can be determined in a number of ways 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 reagent), or derivatization of the compound with a chiral compound such as Mosher's acid followed by chromatography or nuclear magnetic resonance spectroscopy.

[0084] In a preferred embodiment, the enantiomer-enriched composition has a higher therapeutic utility per unit mass than the composition has as a racemic mixture. The enantiomer can be isolated from the mixture by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts, 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); EL Eliel, Stereochemistry of Carbon Compounds, McGraw-Hill, New York (1962); and EL Eliel and SH Wilen, Stereochemistry of Organic Compounds, Wiley-Interscience, New York (1994).

[0085] The terms "enantiomerically enriched" and "non-racemic" as used herein refer to a composition in which the weight percentage of one enantiomer exceeds the amount of the other enantiomer in a control mixture of the racemic composition (e.g., greater than 1:1 by weight). For example, an enantiomerically enriched preparation of an (S)-enantiomer refers to a preparation of a 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 that contains 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. A chemical moiety is often recognized as a chemical entity that is incorporated into or appended to a molecule.

[0087] "Tautomers" are structurally distinct isomers that interconvert by tautomerization. "Tautomerization" is a form of isomerization and includes prototropic or proton-transfer tautomerization, which is considered a subset of acid-base chemistry. "Prototropic or proton-transfer tautomerization" involves the transfer 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 referred group may be bound to one or more additional groups, radicals or moieties, each of which is independently selected from, 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" means optional substitution with a specified 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), conformational polymorphs and amorphous forms of the compounds, and mixtures thereof. "Crystalline forms" and "polymorphs" are intended to include all crystalline and amorphous forms of the compounds, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, non-solvated polymorphs (including anhydrous), conformational polymorphs and amorphous forms, and mixtures thereof, unless a specific crystalline or amorphous form is mentioned.

[0090] When a range is used herein to describe a physical or chemical property, such as, for example, a molecular weight or a chemical formula, it is intended to include all combinations and subcombinations of the range and specific embodiments therein. The use of the term "about" when referring to a numerical value or numerical range means that the numerical value or numerical range referred to 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%, 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 ORed 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, R 2 OH, NH2, OC 1~32 Alkyl and NH-C 1~32 alkyl); R 4 can 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 above-mentioned compound.

[0092] In some embodiments, T 1 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, tosylate, triflate, 4-monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr), and the like. ) 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 nyl, 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)xanthin-9-yl (MOX). In some embodiments, the hydroxyl protecting group is TBDMS.

[0093] In some embodiments, q 1 , q 2 , q 3 , q 4 and / or q 5is hydrogen.

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

[0095] In some embodiments, T 2 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 above-mentioned compound.

[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, the method comprising: a. preparing a carbene precursor at a carbene preparation temperature; b. Cyclopropanation of said carbene precursor at a cyclopropanation temperature to a compound of formula X, formula XI or formula XII: [ka] and c. Providing said tc-DNA nucleoside precursor of one of formulas I-IX wherein Y is alkoxy; T 3 and T 4 are ORed 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, R 6 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 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, 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 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 ORed 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, R 6 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 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 Et2AlCl, Et2Zn, ZnI2, ZnCl2, ZnBr2, Ti(OiPr)4, Me2AlCl, TMSOTf, TiCl4, 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, R 8 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)2P(O)OH or (aryl-O)2P(O)OH). In some embodiments, the carbene additive may be a substituted carboxylic acid of formula Q3CCO2H, 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 Q3CCO2H, where Q3C may be defined as CCl3, CHCl2, CH2Cl, or CF3. In some embodiments, the carbene additive may be a substituted alkyl alcohol of formula Q3CCH2OH, 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 Q3CCH2OH, where Q3C may be defined as CCl3, CHCl2, CH2Cl, or CF3. In some embodiments, the carbene additive may be trichloroacetic acid, 2,2,2-trifluoroethanol, trichlorophenol, or (n-BuO)2P(O)OH.

[0107] In some embodiments, the solvent may include hexane, toluene, dichloromethane (CH2Cl2), tetrahydrofuran (THF), acetonitrile (CH3CN), dimethylformamide (DMF), diethyl ether, dimethoxyethane (DME), or combinations thereof. In some embodiments, the solvent is dichloromethane, DME, or combinations thereof. In some embodiments, the solvent may be a polar aprotic solvent. In some embodiments, the polar aprotic solvent may include dichloromethane (CH2Cl2), tetrahydrofuran (THF), acetonitrile (CH3CN), dimethylformamide (DMF), diethyl ether, dimethoxyethane (DME), or combinations 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 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 Q3C may be defined as CCl3, CHCl2, CH2Cl, or CF3. 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 7I. In 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 the compound of formula X, formula XI, or formula XII can include adding ZnEt2 to the 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, It 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 -46°C, about -45°C, about -44°C, about -43°C, about -4 2°C, about -41°C, about -40°C, about -39°C, about -38°C, about -37°C, about -36°C, about -35°C, about -34°C, about -33°C, about -32°C, about -31°C, 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 -2 It may be 1°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 temperature of the cyclopropanation 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 the 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 the 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 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, 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 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8. , 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 formulas I-IX from one or more of the 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, 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, 9 is H or a hydroxyl protecting group, and R 10 is a phosphorus moiety); q 9 , q 10 , q 11 , q 12 and q 13 are each independently hydrogen (H), 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 11 (where n is 0 to 6, R 11 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 includes a method for preparing the above compound.

[0120] In one embodiment, the tc-DNA nucleosides described herein include 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, the tc-DNA nucleosides described herein include a compound of formula XIII, formula XIV, or formula XV, where Bx is a modified base. In one embodiment, the tc-DNA nucleosides described herein include 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 invention includes tc-DNA nucleoside-containing oligomeric compounds comprising one or more of the tc-DNA nucleosides of formula XIII, formula XIV, formula XV, or a pharma- ceutically 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. EXAMPLES

[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 the 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 routes shown in FIGS. Example 4 - Cyclopropanation of compound 17 using carbenoids 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 purified α-anomer (3.736 mmol) of 17 was dissolved in 37 ml of dry CHCl and cooled to 0° C. (ice). Then, 22.3 ml (22.3 mmol, 6 eq.) of 1.0 M EtZn (Aldrich) in hexane was added dropwise and stirred at 0° C. for 30 min under Ar. Then, 3.02 ml (37.2 mmol, 10 eq.) of CHI was added dropwise over 15 min at the same temperature and stirred at 0° C. for another 2 h. Then, the cooling bath was removed and the mixture was stirred at ambient temperature for another 21 h. TLC showed a significant amount of unreacted α-17. It was diluted by addition of 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; particle size approximately 0.5 mm, immediately etched with 0.1 M HCl, washed with H2O 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 washed with AcOH (40 ml) and dry Et2O (3×40 ml). The now dark grey 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) was added CHI2 (8.41 g, 31.4 mmol) 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, 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 grey 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 x 50 ml) and the aqueous phase was extracted with Et2O (50 ml). The combined organic phases were dried (MgSO4), 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 stable carbenoids prepared from CH2I2 and Et2Zn and (n-BuO)2P(O)OH Preparation of (n-BuO)2P(O)OZnCH2I solution

[0130] To 8 ml of toluene at -10°C (dry ice / acetone), 2.4 ml (2.4 mmol) of 1.0 M diethylzinc in toluene (Acros) was added dropwise. Then, 0.5 ml (0.53 g, 2.44 mmol) of 97% (n-BuO)2P(O)OH (Aldrich) diluted in 5 ml of toluene was added dropwise slowly. The mixture was stirred for 20 min at -10°C under Ar. Then, 0.20 ml (0.65 g, 2.44 mmol) of CH2I2 (Alfa Aesar) was added dropwise and stirred for 30 min at -10°C under Ar. 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)2P(O)OZnCH2I prepared by the procedure described above. The mixture was stirred for the times 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 completion of the reaction.

[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)OZnCH2I 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)2P(O)OZnCH2I prepared by the procedure described above. The mixture was stirred at 0-10 °C for the times given in Table 2, followed by an extractive aqueous workup. The ratio of 17 / 18 was estimated from the crude 1H NMR.

[0136] The examples given in Table 2 demonstrate that the solvent range for the transformation of 17, the 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)OZnCH2I solution

[0138] Diethylzinc (1.5M in toluene, 14.0 ml, 20.95 mmol) (Acros) was added dropwise to a solution of 4.3 ml (n-BuO)2P(O)OH (4.54 g, 20.95 mmol, 97%, Aldrich) in 22.8 ml CH2Cl2 at -20°C (dry ice / acetone) during 30 min. The mixture was stirred under Ar at -20°C for 30 min. Then 1.69 ml (5.61 g, 20.95 mmol) CH2I2 (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 for 15 min at the same temperature. Then, a solution of the carbenoid (n-BuO)2P(O)OZnCH2I 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, followed by an aqueous workup by extraction. The ratio of 17 / 18 was estimated from the 1H NMR of the crude.

[0140] The example shown in Table 3 demonstrates the promotion of the 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)OZnCH2I solution

[0143] To 22.8 ml of CH2Cl2 at -20°C (dry ice / acetone) was added dropwise 14.0 ml (20.95 mmol) of 1.5 M diethylzinc in toluene (Acros). Then 4.28 ml (4.54 g, 20.95 mmol) of 97% (n-BuO)2P(O)OH (Aldrich) was added dropwise slowly. The mixture was stirred under Ar at -20°C for 30 min. Then 1.69 ml (5.62 g, 20.95 mmol) of CH2I2 (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 purified α-anomer and 174 mg of β-anomer (355 mg, 1.239 mmol) of 17 were dissolved in 0.7 ml of CHCl and cooled to −10° C. (dry ice). Then, 826 μl (1.239 mmol, 1 equiv.) of 1.5 M EtZn(Acros) in toluene was added dropwise and stirred at −10° C. for 10 min under Ar. Then, 5.312 ml (2.602 mmol, 2.1 equiv.) of (n-BuO)P(O)OZnCHI solution was added dropwise and stirred at −10° C. for another 10 min. Then, 336 μl (298.5 mg, 0.619 mmol, 50 mol %) of 25 wt % diethylaluminum chloride solution in toluene (Aldrich) was slowly added dropwise. The mixture was stirred at -10°C for 2 hours and then allowed to warm slowly to room temperature in a bath. The mixture was stirred for a total of 21 hours. Experiment 6 - Cyclopropanation of compound 17 using 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, 3 equiv.) of (n-BuO)P(O)OZnCHI solution was added dropwise and stirred at −10° C. for another 10 min. Then, 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 x 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 (approximately 0.749 mmol, 102%) of a 1:1 diastereomeric mixture of compound 18 was obtained, 31 P-NMR showed the presence of "dibutyl phosphate" in the form of a yellowish oil.

[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 is formed. 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. Then, 505 μl (0.758 mmol, 1 equiv.) of 1.5 M EtZn in toluene was added dropwise and stirred at −10° C. for 10 min. Then, 3.40 ml (1.668 mmol, 2.2 equiv.) of (n-BuO)P(O)OZnCHI solution was added dropwise and stirred at −10° C. for another 10 min. Then, 412 μl (0.758 mmol, 1 equiv.) of a 25 wt. % diethylaluminum chloride solution in toluene was 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. Then, 491 μl (0.737 mmol, 1 eq.) of 1.5 M EtZn in toluene was added dropwise and stirred at −10° C. for 10 min. Then, 6.01 ml (2.211 mmol, 3 eq.) of a (n-BuO)P(O)OZnCHI solution was added dropwise and stirred at −10° C. for another 10 min. Then, 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 23 h (→ yellowish solution). 202 mg (approximately 0.672 mmol, 91%) of the α-anomer of compound 18 was obtained. 31 P-NMR showed the presence of "dibutyl phosphate" in the form of a yellowish oil.

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

[0150] Preparation of 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 h and at 0° C. for 1 h. The precipitate was filtered off using Celite, washed with cold CH3CN and the filtrate was concentrated (pyridine and toluene were coevaporated) and dried under high vacuum overnight. 146 mg of compound 18 was obtained. Example 8 – Preparation of tc-DNA nucleoside precursors

[0151] A 1.5M solution of Et2Zn in toluene (51.49 ml, 77.24 mmol, 5 equiv.; Acros) was added dropwise to dry CHCl2 (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 a period of 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 a further 30 min. A solution of crude 17 (5.0 g, 88.5% purity, 15.45 mmol, 1 equiv) in dry CHCl (10.51 ml) was then added dropwise (exothermic reaction, temperature rises 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 x 400 ml). The combined aqueous phases were re-extracted with hexane (300 ml). The combined organic phase was then washed with 0.2 M aqueous NaOH (2×400 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: (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 at a cyclopropanation temperature to a compound of formula X, formula XI or formula XII:

change

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Claims

1. Formulas I-IX: 【Chemistry 13】 A method for preparing a tc-DNA nucleoside precursor according to any one of claims 1 to 4, comprising the steps of: wherein X is alkoxy; T １ and T ２ is 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 -(CH ２ ) ｎ -C(O)-R ２ (wherein n is 0 to 6, R ２ OH, NH ２ , O.C. 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, 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; R ４ is a hydroxyl protecting group; The method comprises: a. preparing a carbene precursor at a carbene preparation temperature, wherein the preparing the carbene precursor comprises reacting a carbene additive, a first Lewis acid catalyst, and R ７ I ２ in a solvent, 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 (wherein n is 0 to 6, R ８ OH, NH ２ , O-C １～３２ Alkyl and NH-C １～３２ alkyl), wherein the first Lewis acid catalyst is selected from the group consisting of Et ２ Zn, wherein the carbene additive is selected from the group consisting of substituted or unsubstituted alkyl alcohols, carboxylic acids and phosphates; b. reacting said carbene precursor with a compound of formula X, formula XI or formula XII at cyclopropanation temperature: 【Chemistry 11】 to form a mixture; c. adding a catalytic amount of a second Lewis acid to the mixture, wherein the second Lewis acid is Et ２ AlCl, Et ２ Zn, ZnI ２ , ZnCl ２ , ZnBr ２ , Ti(OiPr) ４ , Me ２ AlCl, and combinations thereof; and d. providing said tc-DNA nucleoside precursor of one of formulas I-IX; In formulas X to XII, Y is alkoxy; T ３ and T ４ are OR'ed 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 -(CH ２ ) ｎ -C(O)-R ６ (wherein n is 0 to 6, R ６ OH, NH ２ , O-C 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, 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 halogens, method.

2. Formulas I-IX: 【Chemistry 14】 A method for preparing a tc-DNA nucleoside precursor according to any one of claims 1 to 4, comprising the steps of: wherein X is alkoxy; T １ and T ２ is 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 -(CH ２ ) ｎ -C(O)-R ２ (wherein n is 0 to 6, R ２ OH, NH ２ , O.C. 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, 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; R ４ is a hydroxyl protecting group; The method comprises: a. preparing a carbene precursor at a carbene preparation temperature, wherein the preparing the carbene precursor comprises reacting a carbene additive, a first Lewis acid catalyst, and R ７ I ２ in a solvent, 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 (wherein n is 0 to 6, R ８ OH, NH ２ , O.C. １～３２ Alkyl and NH-C １～３２ alkyl), wherein the first Lewis acid catalyst is selected from the group consisting of Et ２ Zn, wherein the carbene additive is selected from the group consisting of substituted or unsubstituted alkyl alcohols, carboxylic acids and phosphates; b. A compound of formula X, formula XI or formula XII: 【Chemistry 12】 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 to form a mixture; d. adding a catalytic amount of a second Lewis acid, wherein the second Lewis acid is Et ２ AlCl, Et ２ Zn, ZnI ２ , ZnCl ２ , ZnBr ２ , Ti(OiPr) ４ , Me ２ AlCl, and combinations thereof; and e. Providing said tc-DNA nucleoside precursor of one of formulas I-IX; In formulas X to XII, Y is alkoxy; T ３ and T ４ are OR'ed 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 -(CH ２ ) ｎ -C(O)-R ６ (wherein n is 0 to 6, R ６ OH, NH ２ , O.C. 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, 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 halogens, method.

3. The solvent is hexane, toluene, dichloromethane (CH ２ C ２ ), tetrahydrofuran (THF), acetonitrile (CH ３ CN), dimethylformamide (DMF), diethyl ether, dimethoxyethane (DME) or combinations thereof.

4. The step of preparing the carbene precursor comprises the steps of: ７ I ２ 2. The method of claim 1, comprising the step of adding a carbene additive to said mixture of, wherein said carbene additive is selected from the group consisting of substituted or unsubstituted alkyl alcohols, carboxylic acids and phosphates.

5. The carbene precursor is 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, where each Q is independently selected from the group consisting of H, Cl, Br and F; 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 (wherein n is 0 to 6 and R ８ OH, NH ２ , O.C. １～３２ Alkyl and NH-C １～３２ The method of any one of claims 1 to 4, wherein the alkyl group is selected from the group consisting of alkyl.

6. The carbene precursor is CCl ３ CO ２ ZnCH ２ I, C.F. 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 The method according to any one of claims 1 to 5, wherein I is

7. The carbene additive is 2,2,2-trifluoroethanol or (n-BuO) ２ The method according to any one of claims 1 to 6, wherein the compound is P(O)OH.

8. The second Lewis acid catalyst is Et ２ AlCl or Et ２ The method according to any one of claims 1 to 7, wherein the metal is Zn.

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