Monomers and Methods for the Synthesis of Modified Oligonucleotides

Abstract

The present disclosure relates to monomers and methods for synthesizing modified oligonucleotides. TIFF2025522811000198.tif73170

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 357,379, filed on June 30, 2022, under 35 U.S.C. § 119(e). The content of this U.S. provisional patent application is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted in XML format by the Patent Center, which is hereby incorporated by reference in its entirety. The XML copy created on June 29, 2023, is named "ALN - 459 - WO.xml" and has a size of 440,737 bytes.

[0003] Technical Field The present disclosure generally relates to monomers and methods for synthesizing modified oligonucleotides, such as monomers for synthesizing single - stranded oligonucleotides and dsRNA containing such monomers and methods.

Background Art

[0004] Background RNA interference, namely "RNAi", is a term first coined by Fire and his co-workers to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Fire et al. (1998) Nature 391, 806-811 (Non-Patent Document 1), Elbashir et al. (2001) Genes Dev. 15, 188-200 (Non-Patent Document 2)). Short dsRNA directs gene-specific post-transcriptional silencing in many organisms including vertebrates and has become a new tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multi-component nuclease that destroys messenger RNA homologous to the silencing trigger. Although it is known that RISC contains short RNAs (approximately 22 nucleotides) derived from double-stranded RNA triggers, the protein components of this activity have remained unknown.

[0005] In the art, there is still a need for effective nucleotide or chemical motifs for dsRNA molecules that are advantageous for inhibiting target gene expression. The present invention is directed to that endeavor.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0007] Summary In one aspect, the present specification provides a compound of formula (I): TIFF2025522811000002.tif32128 is provided.

[0008] In the compound of formula (I), R 2 , R 3 , R 4 and R 5 at least one of which is R MA . Optionally, only one of R 2 , R 3 , R 4 and R 5 is R MA . Thus, in some embodiments of various aspects described herein, only one and only one of R 2 , R 3 , R 4 and R 5 is R MA .

[0009] In various aspects described herein, R MA is -O(CH2) m1 -X M' -R M' or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ), where Y M is O or S, m1 is an integer from 1 to 10, and n1 is an integer from 1 to 10. In some embodiments, R MA is -O(CH2) m1 -X M' -R M' . In some other embodiments, R MA is, or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ).

[0010] In various aspects described herein, X M' is N(R MX ), O or S, where R MX is hydrogen or R M' . Thus, in some embodiments of any one of the aspects described herein, X M' is O. In some other embodiments of any one of the aspects described herein, XM' is S. In some further alternative embodiments, X M' is N(R MX ).

[0011] In various aspects described herein, R M' is optionally substituted C 6~30 alkyl, optionally substituted C 6~30 alkenyl, optionally substituted C 6~30 alkynyl, optionally substituted 3- to 8-membered heterocyclyl C 3~30 alkyl, optionally substituted C 3~10 cycloalkyl C 3~30 alkyl; optionally substituted aryl C 3~30 alkyl, optionally substituted heteroaryl C 3~30 alkyl, optionally substituted C 1~30 alkoxy C 1~30 alkyl, -(CH2CH2O) mq -R MQ , a lipid, a ligand (e.g., a targeting ligand (e.g., GalNac) or a pharmacokinetic modulating substance), a linker, or a linker to one or more ligands, where mq is an integer selected from 1 to 10, and R MQ is hydrogen or C 1~6 alkyl. In some embodiments, mq is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0012] In some embodiments of any one of the aspects described herein, R M' is a lipid, a ligand, a linker, or a linker to one or more ligands. For example, R M' is a ligand, or a linker to one or more ligands. In some embodiments, R M' is a ligand, or a linker to one or more ligands.

[0013] In some embodiments of any one of the aspects described herein, R M'is optionally substituted C 6~30 alkyl, optionally substituted C 6~30 alkenyl, optionally substituted C 6~30 alkynyl, or optionally substituted C 3~30 cycloalkyl. For example, R M' is optionally substituted C 6~30 alkyl or optionally substituted C 6~30 alkenyl. In some embodiments, R M' is optionally substituted C 6~30 alkyl. For example, R M' is optionally substituted C 6~30 alkyl, where the alkyl is substituted with at least one substituent.

[0014] In some embodiments of any one of the aspects described herein, R M' is substituted at the terminus with an anionic group or a cationic group. For example, R M' is C 6~30 alkyl, C 6~30 alkenyl, or C 6~30 alkynyl, where C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl are each optionally further substituted at the terminal position with an anionic group or a cationic group, and C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl can each optionally be further substituted. Exemplary anionic groups include, but are not limited to, carboxylate, carbonate, thiocarbonate, dithiocarbonate, phosphate, phosphonate, sulfate, sulfonate, nitrate, and borate. Exemplary cationic groups include, but are not limited to, amine, ammonium group, guanidinium group, histidines, polyamine, pyridinium group, and sulfonium group.

[0015] In various aspects described herein, m1 is an integer from 1 to 10. For example, m1 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, m1 is 2, 3, 4, 5, 6, 7, or 8. Thus, in some embodiments of any one of the aspects described herein, m1 is 2. In some other embodiments of any one of the aspects described herein, m1 is 8.

[0016] In various aspects described herein, n1 is an integer from 1 to 10. For example, n1 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, n1 is 1 or 2. Thus, in some embodiments of any one of the aspects described herein, n1 is 1. In some other embodiments of any one of the aspects described herein, n1 is 2.

[0017] In various aspects described herein, R N' and R N'' are independently hydrogen, optionally substituted C 6~30 alkyl, optionally substituted C 6~30 alkenyl, optionally substituted C 6~30 alkynyl, or optionally substituted C 3~30 cycloalkyl, lipid, ligand (such as targeting ligand (such as GalNac) or pharmacokinetic modifier), linker, or linker to one or more ligands, provided that at least one of R N' and R N'' is not H. In some embodiments of any one of the aspects described herein, R N' and R N'' are independently hydrogen, lipid, ligand, linker, or linker to one or more ligands, provided that at least one of R N' and R N'' is not H. For example, R N' and R N'' are independently hydrogen, ligand, or linker to one or more ligands, provided thatN' and R N'' at least one of which is not H.

[0018] In some embodiments of any one of the aspects described herein, R N' and R N'' at least one of which is a lipid, a ligand, a linker, or a linker to one or more ligands. For example, R N' and R N'' at least one of at least one of which is a ligand, or a linker to one or more ligands.

[0019] In the compound of formula (I), B is an optionally modified nucleobase. For example, B can be a natural or unnatural nucleobase, each of which can optionally be modified with one or more of a functional group, a ligand, a protecting group, etc. In some embodiments, B is an unmodified nucleobase. In some other embodiments, B is a modified nucleobase.

[0020] In the compound of formula (I), R 2 is -O(CH2) m1 -X M' -R M' -O(CH2) n1 -C(Y M )N(R N' )(R N'' ), hydrogen, hydroxyl, protected hydroxyl, phosphate group, reactive phosphorus (III) group, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -O-C 4~30 alkyl-ON(CH2R 8)(CH2R 9 )、 -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 )、 a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support.

[0021] In some embodiments of any one of the aspects described herein, R 2 is -O(CH2) m1 -X M' -R M' For example, R 2 is -O(CH2) m1 -O-R M' In another non-limiting example, R 2 is -O(CH2) m1 -S-R M'

[0022] In some embodiments of any one of the aspects described herein, R 2 is -OCH2CH2-X M' -R M' For example, R 2 is -OCH2CH2-O-R M' In another non-limiting example, R 2 is -OCH2CH2-S-R M'

[0023] In some embodiments of any one of the aspects described herein, R 2 is -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) For example, R 2 is -O(CH2) n1 -C(O)N(R N' )(R N'' ) In some embodiments, R 2 is -OCH2-C(O)N(R N' )(R N'' ) In some other embodiments, R 2 ​​is -OCH2CH2-C(O)N(R N' )(R N'' ).

[0024] In some embodiments of any one of the aspects described herein, R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkoxy, a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support. For example, herein, R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support. In some embodiments of any one of the aspects described herein, R 2 is hydrogen, hydroxyl, protected hydroxyl, a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support. For example, R 2 is a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support. In some embodiments of any one of the aspects described herein, R 2 is a reactive phosphorus(III), or a linker covalently attached to a solid support. For example, R 2 is a reactive phosphorus(III) group (e.g., a phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite).

[0025] In the compound of formula (I), R 3 is -O(CH2) m1 -X M' -R M' , -O(CH2) n1 -C(Y M )N(R N' )(R N'' ), hydrogen, hydroxyl, protected hydroxyl, a phosphate group, a reactive phosphorus(III) group, halogen, optionally substituted C1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, optionally substituted C 1~30 Alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 )、-O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 )、a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support.

[0026] In some embodiments of any one of the aspects described herein, R 3 is -O(CH2) m1 -X M' -R M' . For example, R 3 is -O(CH2) m1 -O-R M' . In another non-limiting example, R 3 is -O(CH2) m1 -S-R M' .

[0027] In some embodiments of any one of the aspects described herein, R 3 is -OCH2CH2-X M' -R M' . For example, R 3 is -OCH2CH2-O-R M' . In another non-limiting example, R 3 is -OCH2CH2-S-R M' .

[0028] In some embodiments of any one of the aspects described herein, R 3 is, -O(CH2) n1 -C(YM )(R N' )(R N'' ). For example, R 3 is -O(CH2) n1 -C(O)N(R N' )(R N'' ). In some embodiments, R 3 is -OCH2-C(O)N(R N' )(R N'' ). In some other embodiments, R 3 is -OCH2CH2-C(O)N(R N' )(R N'' ).

[0029] In some embodiments of any one of the aspects described herein, R 3 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkoxy, a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support. For example, herein, R 3 is hydrogen, hydroxyl, protected hydroxyl, halogen, a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support. In some embodiments of any one of the aspects described herein, R 3 is hydrogen, hydroxyl, protected hydroxyl, a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support. For example, R 3 is a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support. In some embodiments of any one of the aspects described herein, R 3 is a reactive phosphorus(III), or a linker covalently attached to a solid support. For example, R 3 is a reactive phosphorus(III) group (e.g., a phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite).

[0030] In the compound of formula (I), one of R 2 and R 3 should be noted that only one of them is a reactive phosphorus (III) group, a solid support, or a linker covalently attached to the solid support.

[0031] Optionally, only one of R 2 and R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ). Thus, in some embodiments, one of R 2 and R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ), and the other of R 2 and R 3 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkoxy, a reactive phosphorus (III) group, a solid support, a linker, or a linker covalently attached to the solid support. For example, one of R 2 and R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ), and R 2 and R 3The other of them is hydrogen, hydroxyl, protected hydroxyl, halogen, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to the solid support.

[0032] In some embodiments of any one described herein, R 2 and R 3 One of them is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and the other of R 2 and R 3 is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to the solid support. For example, one of R 2 and R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and the other of R 2 and R 3 is reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to the solid support. In some embodiments of any one described herein, one of R 2 and R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(RN'' ) and R 2 and R 3 the other of which is a reactive phosphorus(III) or a linker covalently attached to a solid support. For example, one of R 2 and R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and the other of R 2 and R 3 is a reactive phosphorus(III) group (e.g., phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite).

[0033] In some embodiments of any one of the aspects described herein, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 3 is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to a solid support. For example, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R3 is a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to the solid support. In some embodiments of any one of the aspects described herein, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ), and R 3 is a reactive phosphorus(III), or a linker covalently attached to the solid support. For example, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ), and R 3 is a reactive phosphorus(III) group (e.g., a phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite).

[0034] In some embodiments of any one of the aspects described herein, R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ), and R 2 is hydrogen, hydroxyl, protected hydroxyl, a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to the solid support. For example, R3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 2 is a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support. In some embodiments of any one of the aspects described herein, R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 2 is a reactive phosphorus(III), or a linker covalently attached to a solid support. For example, R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 2 is a reactive phosphorus(III) group (e.g., a phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite).

[0035] In some embodiments of the various aspects described herein, R 4 is H. In some other embodiments of the various aspects described herein, R 4 is R MA and For example, R4 is -O(CH2) m1 -X M' -R M' (for example, -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ).

[0036] In the compound of formula (I), R 5 is R MA , hydrogen, hydroxyl, protected hydroxyl, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy, optionally substituted 3- to 8-membered heterocyclyl (e.g., morpholin-1-yl, piperidin-1-yl, or pyrrolidin-1-yl), halogen, alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxalate, amino, alkylamino, dialkylamino, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), vinylphosphonate (VP) group (e.g., =CH-X P , X P is a phosphate group), C 3~6Cycloalkyl phosphonates (e.g., cyclopropyl phosphonate), monophosphates ((HO)2(O)P - O - 5'), diphosphates ((HO)2(O)P - O - P(HO)(O) - O - 5'), triphosphates ((HO)2(O)P - O - (HO)(O)P - O - P(HO)(O) - O - 5'); monothiophosphates (phosphorothioates, (HO)2(S)P - O - 5'), monodithiophosphates (phosphorodithioates; (HO)(HS)(S)P - O - 5'), phosphorothiolates ((HO)2(O)P - S - 5'); alpha - thiotriphosphates; beta - thiotriphosphates; gamma - thiotriphosphates; phosphoramidates ((HO)2(O)P - NH - 5', (HO)(NH2)(O)P - O - 5'), alkyl phosphonates [(R P )(OH)(O)P - O - 5', R P is an optionally substituted C 1~30 alkyl, e.g., methyl, ethyl, isopropyl, or propyl)], alkyl ether phosphonates [(R P1 )(OH)(O)P - O - 5', R P1 is an alkoxyalkyl, e.g., methoxymethyl (CH2OMe) or ethoxymethyl], (HO)2(X)P - O[-(CH2) a - O - P(X)(OH) - O] b - 5' or (HO)2(X)P - O[-(CH2) a - P(X)(OH) - O] b - 5' or (HO)2(X)P - [-(CH2) a - O - P(X)(OH) - O] b - 5', or an optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimetics (e.g., HO[-(CH2) a - O - P(X)(OH) - O] b - 5', H2N[-(CH2) a - O - P(X)(OH) - O] b - 5', H[-(CH2) a - O - P(X)(OH) - O] b - 5', Me2N[-(CH2) a - O - P(X)(OH) - O] b-5', HO[-(CH2) a -P(X)(OH)-O] b -5', H2N[-(CH2) a -P(X)(OH)-O] b -5', H[-(CH2) a -P(X)(OH)-O] b -5', Me2N[-(CH2) a -P(X)(OH)-O] b -5' can be, where X is O or S, and a and b are each independently 1 to 10, and each R 8 and R 9 are independently H, a targeting ligand (e.g., GalNac), a pharmacokinetic modifier, optionally substituted C 1~30 alkyl, optionally substituted C 1~30 alkenyl, or optionally substituted C 1~30 alkynyl.

[0037] In some embodiments of any one of the aspects described herein, R 5 is -O(CH2) m1 -X M' -R M' For example, R 5 is -O(CH2) m1 -O-R M' Another non-limiting example, R 5 is -O(CH2) m1 -S-R M' is.

[0038] In some embodiments of any one of the aspects described herein, R 5 is -OCH2CH2-X M' -R M' For example, R 5 is -OCH2CH2-O-R M' Another non-limiting example, R 5 is -OCH2CH2-S-R M' is.

[0039] In some embodiments of any one of the aspects described herein, R 5is -O(CH2) n1 -C(Y M )N(R N' )(R N'' ). For example, -O(CH2) n1 -C(Y M )N(R N' )(R N'' ). In some embodiments, R 5 is -OCH2-C(O)N(R N' )(R N'' ). In some other embodiments, R 5 is -OCH2CH2-C(O)N(R N' )(R N'' ).

[0040] In some embodiments of any one of the aspects described herein, R 5 is hydroxyl, protected hydroxyl, optionally substituted C 1~30 alkoxy, vinyl phosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha - thiotriphosphate, beta - thiotriphosphate, gamma - thiotriphosphate, phosphoramidate, alkyl phosphonate, alkyl ether phosphonate, dialkyl terminal phosphate or phosphate mimetic. For example, R 5 is hydroxyl, protected hydroxyl, vinyl phosphonate (VP) group, cyclopropyl phosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha - thiotriphosphate, beta - thiotriphosphate, gamma - thiotriphosphate, phosphoramidate, alkyl phosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, or phosphate mimetic. In some embodiments of any one of the aspects described herein, R 5 is hydroxyl or protected hydroxyl. In some other embodiments of any one of the aspects described herein, R 5is a vinylphosphonate (VP) group.

[0041] In some embodiments of any one of the aspects described herein, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 3 is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus (III) group, solid support, linker, or a linker covalently attached to a solid support, and R 5 is hydroxyl, protected hydroxyl, optionally substituted C 1~30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate or phosphate mimetic. For example, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 3 is a reactive phosphorus (III) group, solid support, linker, or a linker covalently attached to a solid support, and R 5is hydroxyl, protected hydroxyl, vinylphosphonate (VP) group, cyclopropylphosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, or a phosphate mimetic. In some embodiments of any one of the aspects described herein, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 3 is a reactive phosphorus(III), or a linker covalently attached to a solid support, and R 5 is hydroxyl or protected hydroxyl. For example, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 3 is a reactive phosphorus(III) group (e.g., a phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiol phosphoramidite), and R 5 is hydroxyl or protected hydroxyl. In another non-limiting example, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M'or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 3 is a reactive phosphorus(III) group (e.g., phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite), and R 5 is a vinylphosphonate (VP) group. In yet another non-limiting example, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 3 is a linker covalently attached to the solid support, and R 5 is hydroxyl or protected hydroxyl. In yet another non-limiting example, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' (R N'' ) and R 3 is a linker covalently attached to the solid support, and R 5 is a vinylphosphonate (VP) group.

[0042] In some embodiments of any one of the aspects described herein, R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' (R N'' ) and R2 is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to a solid support, and R 5 is hydroxyl, protected hydroxyl, optionally substituted C 1~30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate or phosphate mimetic. For example, R 3 is -O(CH2) m1 -X M' -R M' (e.g. -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 2 is a reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to a solid support, and R 5 is hydroxyl, protected hydroxyl, vinylphosphonate (VP) group, cyclopropylphosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, or phosphate mimetic.

[0043] In some embodiments of any one of the aspects described herein, R 3 is -O(CH2) m1 -X M' -R M' (e.g. -OCH2CH2-X M'-R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 2 is a reactive phosphorus(III) or a linker covalently attached to a solid support, and R 5 is hydroxyl or protected hydroxyl. For example, R 3 is -O(CH2) m1 -X M' -R M' (such as -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 2 is a reactive phosphorus(III) group (such as phosphoramidite like [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite), and R 5 is hydroxyl or protected hydroxyl. In another non-limiting example, R 3 is -O(CH2) m1 -X M' -R M' (such as -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 2 is a reactive phosphorus(III) group (such as phosphoramidite like [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite), and R 5 is a vinylphosphonate (VP) group. In yet another non-limiting example, R 3 is -O(CH2) m1 -X M' -R M' (such as -OCH2CH2-X M' -R M' ) or -O(CH2)n1 -C(O)N(R N' )(R N'' ) and R 2 is a linker covalently attached to the solid support, and R 5 is hydroxyl or protected hydroxyl. In yet another non-limiting example, R 3 is -O(CH2) m1 -X M' -R M' (for example -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 2 is a linker covalently attached to the solid support, and R 5 is a vinylphosphonate (VP) group.

[0044] In some compounds of formula (I), R 2 is -O(CH2) m1 -X M' -R M' (for example -OCH2CH2-X M' -R M' ) and R 5 is hydroxyl or protected hydroxyl, R 4 is H, and when R 3 is hydroxyl, protected hydroxyl, phosphate group or reactive phosphorus (III) group, R M' is neither unsubstituted C 6~21 alkyl, nor unsubstituted C 6~21 alkenyl, nor unsubstituted C 6~21 alkynyl.

[0045] In some compounds of formula (I), R 2 is -O(CH2) n1 -C(O)N(R N' )(R N'' ) and n1 is 1, R 5 is hydroxyl or protected hydroxyl, R 4 is H, R 3is hydroxyl, protected hydroxyl, phosphate group or reactive phosphorus (III) group, and R N' and R N'' if one of them is H, then R N' and R N'' the other one is not substituted or unsubstituted C 5~8 alkyl. For example, R 2 is -O(CH2) n1 -C(O)N(R N' )(R N'' ), n1 is 1, R 5 is hydroxyl or protected hydroxyl, R 4 is H, R 3 is hydroxyl, protected hydroxyl, phosphate group or reactive phosphorus (III) group, and R N' and R N'' if one of them is H, then R N' and R N'' the other one is not -(CH2)6CH3, not -(CH2)7CH3, not -(CH2)8CH3, not -(CH2)5NHCOCF3, not -(CH2)6NHCOCF3, not -(CH2)7NHCOCF3, not -(CH2)5N(CH3)2, not -(CH2)6N(CH3)2, not -(CH2)7N(CH3)2.

[0046] Certain ones of the compounds of formula (I) are useful for preparing oligonucleotides. Accordingly, in another aspect, the present specification provides oligonucleotides prepared using the compounds of formula (I). For example, oligonucleotides containing at least one nucleotide of formula (II). TIFF2025522811000003.tif33128

[0047] In the nucleotide of formula (II), one of R 22 , R 23 , R 4 and R 25 is R MA . Optionally, R 22 , R 23 , R4 and R 25 Only one of them is R MA is. Therefore, in some aspects of the various aspects described in this specification, R 22 , R 23 , R 4 and R 25 Only one and only one of them is R MA is.

[0048] In the nucleotide of formula (II), B is an optionally modified nucleobase. For example, B can be a natural or unnatural nucleobase, and each of these nucleobases can optionally be modified with one or more of a functional group, a ligand, a protecting group, etc. In some aspects, B is an unmodified nucleobase. In some other aspects, B is a modified nucleobase.

[0049] In the nucleotide of formula (II), R 22 is R MA , a bond to the internucleotide linker for the subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), a ligand, a linker covalently bonded to one or more ligands, a solid support, a linker, or a linker covalently bonded to a solid support.

[0050] In some embodiments of any one of the aspects described herein, R 22 is -O(CH2) m1 -X M' -R M' For example, R 22 is -O(CH2) m1 -O-R M' In another non-limiting example, R 22 is -O(CH2) m1 -S-R M'

[0051] In some embodiments of any one of the aspects described herein, R 22 is -OCH2CH2-X M' -R M' For example, R 22 is -OCH2CH2-O-R M' In another non-limiting example, R 22 is -OCH2CH2-S-R M'

[0052] In some embodiments of any one of the aspects described herein, R 22 is -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) For example, R 22 is -O(CH2) n1 -C(O)N(R N' )(R N'' ) In some embodiments, R 22 is -OCH2-C(O)N(R N' )(R N'' ) In some other embodiments, R 22 is -OCH2CH2-C(O)N(R N' )(R N'' )

[0053] In some embodiments of any one of the aspects described herein, R 22 is a bond to a internucleotide linker for a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C​​1~30 is an alkoxy, a solid support, a linker, or a linker covalently attached to a solid support. For example, R 22 is a bond to a nucleotide internucleoside linkage for a subsequent nucleotide, a hydroxyl, a protected hydroxyl, a solid support, a linker, or a linker covalently attached to a solid support. In some embodiments of any one of the aspects described herein, R 22 is a bond to a nucleotide internucleoside linkage for a subsequent nucleotide. In some embodiments of any one of the aspects described herein, R 22 is a linker covalently attached to a solid support. In some embodiments of any one of the aspects described herein, R 22 is a hydroxyl or a protected hydroxyl.

[0054] In the nucleotide of formula (II), R 23 is R MA , a bond to a nucleotide internucleoside linkage for a subsequent nucleotide, hydrogen, a hydroxyl, a protected hydroxyl, a halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, a 5- to 8-membered heterocyclyl, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support.

[0055] In some embodiments of any one of the aspects described herein, R23 is -O(CH2) m1 -X M' -R M' as follows. For example, R 23 is -O(CH2) m1 -O-R M' as follows. In another non-limiting example, R 23 is -O(CH2) m1 -S-R M' as follows.

[0056] In some embodiments of any one of the aspects described herein, R 23 is -OCH2CH2-X M' -R M' as follows. For example, R 23 is -OCH2CH2-O-R M' as follows. In another non-limiting example, R 23 is -OCH2CH2-S-R M' as follows.

[0057] In some embodiments of any one of the aspects described herein, R 23 is -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) as follows. For example, R 23 is -O(CH2) n1 -C(O)N(R N' )(R N'' ) as follows. In some embodiments, R 23 is -OCH2-C(O)N(R N' )(R N'' ) as follows. In some other embodiments, R 23 is -OCH2CH2-C(O)N(R N' )(R N'' ) as follows.

[0058] In some embodiments of any one of the aspects described herein, R 23 is a bond to the internucleotide linker for the subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30It is an alkoxy, a solid support, a linker, or a linker covalently attached to the solid support. For example, R 23 is a bond to the internucleotide linker for the subsequent nucleotide, a hydroxyl, a protected hydroxyl, a solid support, a linker, or a linker covalently attached to the solid support. In some embodiments of any one of the aspects described herein, R 23 is a bond to the internucleotide linker for the subsequent nucleotide. In some embodiments of any one of the aspects described herein, R 23 is a linker covalently attached to the solid support. In some embodiments of any one of the aspects described herein, R 23 is a hydroxyl or a protected hydroxyl.

[0059] In the nucleotide of formula (II), note that only one of R 22 and R 23 is a bond to the internucleotide linker for the subsequent nucleotide, a solid support, or a linker covalently attached to the solid support.

[0060] Thus, in some embodiments of any one of the aspects described herein, one of R 22 and R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ), and the other of R 22 and R 23 is a bond to the internucleotide linker for the subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkoxy, a solid support, a linker, or a linker covalently attached to the solid support. For example, R 22 and R23 One of them is -O(CH2) m1 -X M' -R M' (for example, -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and the other of R 22 and R 23 is a linkage to the internucleotide linker for the subsequent nucleotide, a hydroxyl, a protected hydroxyl, or a linker covalently attached to a solid support. In some embodiments, one of R 22 and R 23 is -O(CH2) m1 -X M' -R M' (for example, -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and the other of R 22 and R 23 is a linkage to the internucleotide linker for the subsequent nucleotide. In some other embodiments, one of R 22 and R 23 is -O(CH2) m1 -X M' -R M' (for example, -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and the other of R 22 and R 23 is a hydroxyl, a protected hydroxyl, or a linker covalently attached to a solid support.

[0061] In some embodiments of any of the aspects described herein, R 22 is -O(CH2)m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 23 is a bond to a nucleotide internucleoside linker for a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkoxy, solid support, linker, or a linker covalently attached to a solid support. For example, R 22 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 23 is a bond to a nucleotide internucleoside linker for a subsequent nucleotide, hydroxyl, protected hydroxyl, or a linker covalently attached to a solid support. In some embodiments, R 22 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 23 is a bond to a nucleotide internucleoside linker for a subsequent nucleotide. In some other embodiments, R 22 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(RN' )(R N'' ) and R 23 is hydroxyl, protected hydroxyl, or a linker covalently attached to a solid support.

[0062] In some embodiments of any of the aspects described herein, R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 22 is a bond to a nucleotide internucleoside linker for a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkoxy, solid support, linker, or a linker covalently attached to a solid support. For example, R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 22 is a bond to a nucleotide internucleoside linker for a subsequent nucleotide, hydroxyl, protected hydroxyl, or a linker covalently attached to a solid support. In some embodiments, R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 22is a bond to the internucleotide linkage for the subsequent nucleotide. In some alternative embodiments, R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ), and R 22 is a hydroxyl, a protected hydroxyl, or a linker covalently attached to a solid support.

[0063] In the nucleotide of formula (II), R 25 is a bond to the internucleotide linkage for the preceding nucleotide, a hydroxyl, a protected hydroxyl, an optionally substituted C 1~30 alkoxy, a vinyl phosphonate (VP) group, a monophosphate, a diphosphate, a triphosphate, a monothiophosphate (phosphorothioate), a monodithiophosphate, a phosphorothiolate, an alpha-thiotriphosphate, a beta-thiotriphosphate, a gamma-thiotriphosphate, a phosphoramidate, an alkyl phosphonate, an alkyl ether phosphonate, a dialkyl terminal phosphate or a phosphate mimetic. For example, R 25 is a bond to the internucleotide linkage for the preceding nucleotide, a hydroxyl, a protected hydroxyl, a vinyl phosphonate (VP) group, a cyclopropyl phosphonate, a monophosphate, a diphosphate, a triphosphate, a monothiophosphate (phosphorothioate), a monodithiophosphate, a phosphorothiolate, an alpha-thiotriphosphate, a beta-thiotriphosphate, a gamma-thiotriphosphate, a phosphoramidate, an alkyl phosphonate, an alkyl ether phosphonate, a dialkyl terminal phosphate, or a phosphate mimetic.

[0064] In some embodiments of any one of the aspects described herein, R25 is a bond to the internucleotide linkage relative to the preceding nucleotide.

[0065] In some embodiments of any one of the aspects described herein, R 25 is hydroxyl or protected hydroxyl.

[0066] In some embodiments of any one of the aspects described herein, R 25 is a vinylphosphonate (VP) group, cyclopropylphosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, or a phosphate mimetic. For example, R 25 is a vinylphosphonate (VP) group.

[0067] R 22 and R 23 If neither is a bond to the internucleotide linkage relative to the subsequent nucleotide, note that R 25 is a bond to the internucleotide linkage relative to the preceding nucleotide.

[0068] In some embodiments of any of the aspects described herein, R 22 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 23is a bond to an internucleotide linkage to a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, solid support, linker, or a linker covalently attached to a solid support, R 25 is a bond to an internucleotide linkage to a preceding nucleotide, hydroxyl, protected hydroxyl, vinylphosphonate (VP) group, cyclopropylphosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, or a phosphate mimetic, provided that R 23 is a bond to an internucleotide linkage to a subsequent nucleotide or R 25 is a bond to an internucleotide linkage to a preceding nucleotide.

[0069] In some embodiments, R 22 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 23 is a bond to an internucleotide linkage to a subsequent nucleotide, R 25is a hydroxyl, protected hydroxyl, vinyl phosphonate (VP) group, cyclopropyl phosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkyl phosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, or phosphate mimetic. For example, R 22 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) ' or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 23 is a bond to the internucleotide linker to the subsequent nucleotide, and R 25 is a hydroxyl, protected hydroxyl or vinyl phosphonate (VP) group.

[0070] In some embodiments, R 22 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 23 is a bond to the internucleotide linker to the subsequent nucleotide, and R 25 is a bond to the internucleotide linker to the preceding nucleotide.

[0071] In some embodiments of any of the aspects described herein, R 22 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-XM' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 23 is hydroxyl, protected hydroxyl, solid support, linker, or a linker covalently attached to a solid support, and R 25 is a bond to a nucleotide internucleoside linker relative to the preceding nucleotide. For example, R 22 is -OCH2CH2-X M' -R M' or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 23 is hydroxyl, protected hydroxyl, or a linker covalently attached to a solid support.

[0072] In some embodiments of any of the aspects described herein, R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 22 is a bond to a nucleotide internucleoside linker relative to the subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, solid support, linker, or a linker covalently attached to a solid support, and R 25is a linkage to an internucleotide linkage to a preceding nucleotide, a hydroxyl, a protected hydroxyl, a vinylphosphonate (VP) group, a cyclopropylphosphonate, a monophosphate, a diphosphate, a triphosphate, a monothiophosphate (phosphorothioate), a monodithiophosphate, a phosphorothiolate, an alpha-thiotriphosphate, a beta-thiotriphosphate, a gamma-thiotriphosphate, a phosphoramidate, an alkylphosphonate, an alkyl ether phosphonate, a dialkyl terminal phosphate, or a phosphate mimetic, provided that R 22 is a linkage to an internucleotide linkage to a subsequent nucleotide, or R 25 is assumed to be a linkage to an internucleotide linkage to a preceding nucleotide.

[0073] In some embodiments, R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ), where R 22 is a linkage to an internucleotide linkage to a subsequent nucleotide and R 25 is a hydroxyl, a protected hydroxyl, a vinylphosphonate (VP) group, a cyclopropylphosphonate, a monophosphate, a diphosphate, a triphosphate, a monothiophosphate (phosphorothioate), a monodithiophosphate, a phosphorothiolate, an alpha-thiotriphosphate, a beta-thiotriphosphate, a gamma-thiotriphosphate, a phosphoramidate, an alkylphosphonate, an alkyl ether phosphonate, a dialkyl terminal phosphate, or a phosphate mimetic. For example, R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -RM' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) and R 22 is a bond to the internucleotide linker for the subsequent nucleotide, and R 25 is a hydroxyl, protected hydroxyl or vinylphosphonate (VP) group.

[0074] In some embodiments, R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 22 is a bond to the internucleotide linker for the subsequent nucleotide, and R 25 is a bond to the internucleotide linker for the preceding nucleotide.

[0075] In some embodiments of any of the aspects described herein, R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 22 is a hydroxyl, protected hydroxyl, solid support, linker, or a linker covalently attached to a solid support, and R 25 is a bond to the internucleotide linker for the preceding nucleotide. For example, R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2)n1 -C(O)N(R N' )(R N'' ) is such that R 22 is hydroxyl, protected hydroxyl, or a linker covalently attached to a solid support.

[0076] In some embodiments of any of the aspects described herein, R 25 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) is such that one of R 22 and R 23 is a bond to a nucleotide internucleoside linker for a subsequent nucleotide. For example, R 25 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) is such that R 23 is a bond to a nucleotide internucleoside linker for a subsequent nucleotide. In another non-limiting example, R 25 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(Y M )N(R N' )(R N'' ) is such that R 22 is a bond to a nucleotide internucleoside linker for a subsequent nucleotide.

[0077] Optionally, in the nucleotide of formula (II), R 22is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ), and R 25 is hydroxyl, protected hydroxyl, or a bond to an internucleotide linker for a preceding nucleotide, and R 4 is H, and R 23 is hydroxyl, protected hydroxyl, a bond to an internucleotide linker for a subsequent nucleotide, a solid support, a linker, or a linker covalently attached to a solid support, and when at least one of R 23 and R 25 is a bond to an internucleotide linker, R M' is neither unsubstituted C 5~21 alkyl, nor unsubstituted C 5~21 alkenyl, nor unsubstituted C 5~21 alkynyl.

[0078] Optionally, in the nucleotide of formula (II), R 22 is -O(CH2) n1 -C(O)N(R N' )(R N'' ), n1 is 1, and R 5 is hydroxyl, protected hydroxyl or a bond to an internucleotide linker for a preceding nucleotide, and R 4 is H, and R 3 is hydroxyl, protected hydroxyl, a bond to an internucleotide linker for a subsequent nucleotide, a solid support, a linker, or a linker covalently attached to a solid support, and at least one of R 23 and R 25 is a bond to an internucleotide linker, and one of R N' and R N'' is H, and when at least one of R 23 and R 25 is a bond to an internucleotide linker, R N' and R N''The other one of them is not -(CH2)6CH3, -(CH2)7CH3, -(CH2)8CH3, -(CH2)5NHCOCF3, -(CH2)6NHCOCF3, -(CH2)7NHCOCF3, -(CH2)5N(CH3)2, -(CH2)6N(CH3)2, or -(CH2)7N(CH3)2.

[0079] In another aspect, as used herein, R 2 , R 3 , R 4 and R 5 wherein one of them is -O(CH2) n1 -C(Y M )N(R N' )(R N'' ), a method for preparing an oligonucleotide comprising at least one nucleotide of formula (II) is provided. The method comprises reacting an oligonucleotide comprising a nucleotide of formula (II'): TIFF2025522811000004.tif33128 with an amine of formula HN(R N' )(R N'' ).

[0080] In the nucleotide of formula (II'), B is an optionally modified nucleobase, and R 22' is -O(CH2) n1 -C(Y M )OR LV , a bond to the internucleotide linker for the subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9)、 -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 )、 a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support, where R 23' is -O(CH2) n1 -C(Y M )OR LV 、 a bond to a nucleotide internucleoside linker for a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 )、 -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 )、 a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support, where R 25 ' is -O(CH2) n1 -C(O)OR LV 、 a bond to a nucleotide internucleoside linker for a preceding nucleotide, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30Alkoxy, optionally substituted 3- to 8-membered heterocyclyl (e.g., morpholin-1-yl, piperidin-1-yl, or pyrrolidin-1-yl), halogen, alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxalate, amino, alkylamino, dialkylamino, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 )、-O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 )、vinylphosphonate (VP) group (e.g., =CH-X P 、X P is a phosphate group), C 3~6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), phosphorothiolate ((HO)2(O)P-S-5'), alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), alkylphosphonate [(R P )(OH)(O)P-O-5', R P is optionally substituted C 1~30 alkyl, e.g., methyl, ethyl, isopropyl, or propyl)], alkyl ether phosphonate [(R P1 )(OH)(O)P-O-5', R P1 is alkoxyalkyl, e.g., methoxymethyl (CH2OMe) or ethoxymethyl], (HO)2(X)P-O[-(CH2) a -O-P(X)(OH)-O] b -5', or (HO)2(X)P-O[-(CH2) a-P(X)(OH)-O] b -5', or (HO)2(X)P - [-(CH2) a -O-P(X)(OH)-O] b -5', or optionally substituted alkyl, and dialkyl terminal phosphate and phosphate mimics (e.g., HO[-(CH2) a -O-P(X)(OH)-O] b -5', H2N[-(CH2) a -O-P(X)(OH)-O] b -5', H[-(CH2) a -O-P(X)(OH)-O] b -5', Me2N[-(CH2) a -O-P(X)(OH)-O] b -5', HO[-(CH2) a -P(X)(OH)-O] b -5', H2N[-(CH2) a -P(X)(OH)-O] b -5', H[-(CH2) a -P(X)(OH)-O] b -5', Me2N[-(CH2) a -P(X)(OH)-O] b -5' and (where X is O or S, and a and b are each independently 1 to 10), R 4 is hydrogen, optionally substituted C 1~6 alkyl, optionally substituted C 2~6 alkenyl, optionally substituted C 2~6 alkynyl, or optionally substituted C 1~6 alkoxy, and R LV is C1 - C6 alkyl (e.g., ethyl), each R 8 and R 9 are independently H, a targeting ligand (e.g., GalNac), a pharmacokinetic modulating substance, optionally substituted C 1~30 alkyl, optionally substituted C 1~30 alkenyl, or optionally substituted C 1~30 alkynyl.

[0081] In some embodiments of any one of the aspects described herein, R 22' is -O-R LV wherein.

[0082] In some embodiments of any one of the aspects described herein, R 23' is -O-R LV wherein.

[0083] In some embodiments of any one of the aspects described herein, R 24' is -O-R LV wherein.

[0084] In some embodiments of any one of the aspects described herein, R 25' is -O-R LV wherein.

[0085] In some embodiments of any one of the aspects described herein, the oligonucleotide comprising the nucleoside of formula (II') is linked to a solid support. For example, the oligonucleotide comprising the nucleoside of formula (II') is reacted with an amine while the oligonucleotide is still attached to the solid support.

[0086] In some embodiments of any one of the aspects described herein, the oligonucleotide comprising the nucleoside of formula (II') is linked to a solid support. For example, the method includes the step of cleaving the oligonucleotide from the solid support prior to reaction with an amine.

[0087] In some embodiments of any one of the aspects described herein, the oligonucleotide comprising the nucleoside of formula (II') comprises at least one modified internucleoside linkage.

[0088] In some embodiments of any one of the aspects described herein, the oligonucleotide comprising a nucleotide of formula (II') comprises at least one hydroxyl protecting group, phosphate protecting group or amino protecting group. For example, the oligonucleotide comprising a nucleoside of formula (II') is reacted with an amine while the oligonucleotide comprises at least one hydroxyl protecting group, phosphate protecting group or amino protecting group.

[0089] In some embodiments of any one of the aspects described herein, the oligonucleotide comprising a nucleotide of formula (II') comprises at least one hydroxyl protecting group, phosphate protecting group or amino protecting group, and the method comprises the step of cleaving the at least one hydroxyl protecting group, phosphate protecting group or amino protecting group before reacting the oligonucleotide with an amine.

[0090] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein comprise 3 to 50 nucleotides.

[0091] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein comprise at least one ribonucleotide (e.g., 2'-OH).

[0092] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein comprise at least one 2'-deoxyribonucleotide.

[0093] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein comprise at least one nucleotide having a modified nucleobase or unnatural nucleobase.

[0094] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein comprise at least one nucleotide having a modified ribose sugar.

[0095] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein include at least one nucleotide containing a group that is neither H nor OH at the 2'-position of the ribose sugar.

[0096] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein include at least one nucleotide having 2'-F ribose.

[0097] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein include at least one nucleotide having 2'-OMe ribose.

[0098] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein include at least one nucleotide containing a moiety other than ribose sugar.

[0099] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein include at least one modified nucleotide internucleoside linkage.

[0100] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein include nucleosides containing at least one hydroxyl protecting group, phosphate protecting group or amino protecting group.

[0101] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein include at least one ligand.

[0102] Also provided herein are double-stranded nucleic acids, such as double-stranded RNA, comprising a first strand and a second strand, wherein at least one of the first strand or the second strand is an oligonucleotide described herein. For example, a double-stranded RNA comprising a first strand and a second strand, wherein the first strand and / or the second strand is an oligonucleotide comprising a nucleotide of formula (II).

[0103] In another aspect, the present specification provides a method for inhibiting or reducing the expression of a target gene in a subject. The method includes the step of administering to the subject (i) a double-stranded RNA described herein, wherein one of the strands of the dsRNA is complementary to the target gene, and / or (ii) an oligonucleotide described herein that is complementary to the target gene.

Brief Description of the Drawings

[0104]

Figure 1-1

Figure 1-2

Figure 2-1

Figure 2-2

Figure 3-1

Figure 3-2

Figure 4-1

Figure 4-2

Figure 4-3

Figure 4-4

Figure 5-1

Figure 5-2

Figure 6

Figure 7-1

Figure 7-2

Figure 7-3

Figure 7-4

Figure 7-5

Figure 8

Figure 9A

Figure 9B

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

MODE FOR CARRYING OUT THE INVENTION

[0105] Detailed Description It should be understood that the above summary and the following detailed description are merely illustrative and explanatory and are not limitations of the invention described in the claims. In this specification, the use of the singular form includes the plural unless otherwise specifically stated. The use of "or" in this specification means "and / or" unless otherwise stated. Further, the use of the term "including" and other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" include both elements and components that contain one unit and those that contain two or more subunits, unless otherwise specifically stated.

[0106] The section headings used in this specification are for structural purposes only and should not be construed as limiting the subject matter described. Any document or part of a document referred to in this application, including, for example, patents, patent applications, papers, books, and treatises, is hereby incorporated by reference in its entirety for all purposes.

[0107] X M' One aspect of any of the aspects described herein is X M' including. In some aspects of any of the aspects described herein, X M' is O. In some other aspects of any of the aspects described herein, X M' is S. In some further other aspects of any of the aspects described herein, X M' is N(R MX ), where R MX is hydrogen or R M' .

[0108] Y M One aspect of any of the aspects described herein is Y Mincludes. In some aspects of any one described herein, Y M' is O. In some other aspects of any one of the aspects described herein, Y M is S.

[0109] R M' One aspect of any one of the aspects described herein is R M' includes. In some aspects, R M' is optionally substituted C 6~30 alkyl, optionally substituted C 6~30 alkenyl, optionally substituted C 6~30 alkynyl, or optionally substituted C 3~30 cycloalkyl, optionally substituted 3- to 8-membered heterocyclyl C 3~30 alkyl, optionally substituted C 3~10 cycloalkyl C 3~30 alkyl, optionally substituted aryl C 3~30 alkyl, optionally substituted heteroaryl C 3~30 alkyl, optionally substituted C 1~30 alkoxy C 1~30 alkyl, -(CH2CH2O) mq -R MQ , a lipid, a ligand, a linker, or a linker for one or more ligands, where mq is an integer selected from 1 to 10, and R MQ is hydrogen or C 1~6 alkyl.

[0110] In some aspects, R M' is C 6~30 alkyl, C 6~30 alkenyl or C 6~30 alkynyl, where C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl may be optionally substituted with at least one substituent. For example, R M' is C 6~30 alkyl, C 6~30 alkenyl or C6~30 is alkynyl, where C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl may be substituted with at least one substituent selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclil, heterocyclil, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, acyloxy, cyano or ureido. For example, C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl is OH, CN, -SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) pOptionally substituted with 1, 2, 3, 4 or 5 groups selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0111] In some embodiments, R M' is C 6~30 alkyl, C 6~30 alkenyl or C 6~30 alkynyl, where C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl may be optionally substituted at the terminus with at least one substituent. "alyl

[0112] In some embodiments, R M' is C 6~30 alkyl, C 6~30 alkenyl or C 6~30 alkynyl, where C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl are optionally substituted at the terminus with at least one substituent selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclil, heterocyclil, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino, alkylcarbonoyl, arylcarbonoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, acyloxy, cyano or ureido. For example, R M' is R M' is C 6~30 alkyl, C 6~30 alkenyl or C 6~30 alkynyl, where C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl are R M'At the end away from the point where it is attached to the remaining part of the molecule, OH, CN, -SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p It may be substituted with at least one substituent selected from the group consisting of -NH2 or CH2-aryl-alkoxy, and "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0113] In some embodiments of any one of the aspects described herein, R M' is C 3~30 alkyl, C 3~30 alkenyl or C 3~30 alkynyl, where C 3~30 alkyl, C 3~30 alkenyl and C 3~30 alkynyl are optionally substituted 3- to 8-membered heterocyclyl, optionally substituted C 3~10 cycloalkyl, optionally substituted aryl or optionally substituted heteroaryl, and the terminus is substituted. For example, R M' is C 3~30 alkyl, C 3~30 alkenyl or C 3~30 alkynyl, where C 3~30 alkyl, C 3~30 alkenyl and C 3~30Alkynyl is R M' At the terminal away from the point where it is attached to the rest of the molecule, it is substituted with an optionally substituted 3- to 8-membered heterocyclyl, an optionally substituted C 3~10 Cycloalkyl, an optionally substituted aryl or an optionally substituted heteroaryl. C 3~30 Alkyl, C 3~30 Alkenyl and C 3~30 Note that alkynyl may optionally be substituted with 1, 2, 3 or 4 or more additional independently selected substituents.

[0114] In some embodiments of any one of the aspects described herein, R M' is -(CH2) mc -R MC wherein mc is an integer from 3 to 30 and R MC is an optionally substituted 3- to 8-membered heterocyclyl, an optionally substituted C 3~10 Cycloalkyl, an optionally substituted aryl or an optionally substituted heteroaryl.

[0115] In some embodiments of any one of the aspects described herein, R M' is C 1~30 Alkyl, C 2~30 Alkenyl or C 2~30 Alkynyl, wherein C 1~30 Alkyl, C 2~30 Alkenyl and C 2~30 Alkynyl is substituted at the terminal with an optionally substituted C 1~30 Alkoxy. For example, R M' is C 1~30 Alkyl, C 2~30 Alkenyl or C 2~30 Alkynyl, wherein C 1~30 Alkyl, C 2~30 Alkenyl and C 2~30 Alkynyl is at the terminal away from the point where R M' is attached to the rest of the molecule, an optionally substituted C 1~30is substituted with an alkoxy group. C 1~30 alkyl, C 2~30 alkenyl and C 2~30 It should be noted that the alkynyl may optionally be substituted with one, two, three or four or more additional independently selected substituents.

[0116] In some embodiments of any one of the aspects described herein, R M' is -(CH2) md -R MD wherein md is an integer from 1 to 30 and R MD is an optionally substituted alkoxy group. 1~30 C

[0117] In some embodiments of any one of the aspects described herein, R M'' is -(CH2CH2O) mq -R MQ wherein mq is an integer selected from 1 to 10 and R MQ I R M' at the end remote from the point where it is attached to the remainder of the molecule is hydrogen or C 1~6 alkyl. For example, R M'' is -(CH2CH2O) mq -R MQ wherein mq is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and R MQ is H. In another non-limiting example, R M'' is -(CH2CH2O) mq -R MQ wherein mq is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and R MQ is C 1~6 alkyl such as methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, or hexyl. It should be noted that the C MQ alkyl group of R 1~6 can optionally be substituted with one, two, three or four or more independently selected substituents. In some embodiments of any one of the aspects described herein, RM' is C 6~30 alkyl, C 6~30 alkenyl or C 6~30 alkynyl, where C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl are substituted with an anionic or cationic group at the terminal remote from the point where R M' is attached to the remainder of the molecule.

[0118] In some embodiments of any one of the aspects described herein, R M' is -(CH2) mm -R ME where mm is an integer from 6 to 29 and R ME is methyl, CO2H, CO2Me, NH2, SH, OH, CH=CH2 or C≡CH.

[0119] In some embodiments of any one of the above aspects, R M' is -(CH2) mm -R ME where mm is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. For example, mm is 13, 14, 15, 16, 17, 18, 19 or 20. In another non-limiting example, mm is 13, 15, 17 or 19. In yet another non-limiting example, mm is 14, 16, 18 or 20.

[0120] In some embodiments of any one of the above aspects, R ME is methyl, CO2H, CO2Me or NH2. For example, R ME is CO2H, CO2Me or NH2.

[0121] In some embodiments of any one of the above aspects, mm is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and R ME is methyl, CO2H, CO2Me or NH2. For example, mm is 14, 16, 18 or 20 and RME is CO2H, CO2Me or NH2. In another non-limiting example, mm is 13, 15, 17 or 19, and R ME is methyl.

[0122] In some embodiments of any one of the aspects described herein, R M' is hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, or icosadecyl. For example, R M' is tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, or icosadecyl. In some embodiments, R M' is tetradecyl, hexadecyl, octadecyl, or icosadecyl.

[0123] In some embodiments of any one of the aspects described herein, R M' is hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, or icosadecyl, each substituted with at least one substituent selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclil, heterocyclil, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino, alkylcarbonoyl, arylcarbonoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, acyloxy, cyano or ureido. For example, R M'where each is OH, CN, -SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, or icosadecyl substituted with at least one substituent selected from the group consisting of -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6.

[0124] In some embodiments, R M' are each R M'At the terminal end away from the point where it is attached to the rest of the molecule, OH, CN, -SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p Hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, or icosadecyl substituted with at least one substituent selected from the group consisting of -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R M'' Each is such that R M' At the terminal end away from the point where it is attached to the rest of the molecule, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, or icosadecyl substituted with OH, oxo (=O), SH, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, or COOMe.

[0125] In some embodiments, R M' Each is such that R M'At the terminal end, which is away from the point where it is attached to the rest of the molecule, it is tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, or icosadecyl substituted with NH2, CO2H, or CO2Me.

[0126] In some embodiments of any one of the aspects described herein, R M' is (9Z)-tetradec-9-enyl, (6Z)-hexadec-6-enyl, (9Z)-hexadec-9-enyl, (9Z)-octadec-9-enyl, (9E)-octadec-9-enyl, (11E)-octadec-11-enyl, (9Z,12Z)-octadeca-9,12-dienyl, (9E,12E)-octadeca-9,12-dienyl, (9Z,12Z,15Z)-octadeca-9,12,15-trienyl, (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenyl, (5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenyl, or (13Z)-docosa-13-enyl. For example, R M' is (9Z)-octadec-9-enyl, (9E)-octadec-9-enyl, (11E)-octadec-11-enyl, (9Z,12Z)-octadeca-9,12-dienyl or (9E,12E)-octadeca-9,12-dienyl. In some embodiments, R M' is (9Z)-octadec-9-enyl or (9Z,12Z)-octadeca-9,12-dienyl or (9E,12E)-octadeca-9,12-dienyl.

[0127] In some embodiments of any one of the aspects described herein, R 2 is -OCH2CH2-O-R M' where R 5 is hydroxyl or protected hydroxyl, R 4 is H, and when R 3 is hydroxyl, protected hydroxyl, phosphate group or reactive phosphorus(III) group, R M' is unsubstituted C 6~21 alkyl or unsubstituted C6~21 It is neither alkenyl nor unsubstituted C 6~21 nor alkynyl.

[0128] In some embodiments of any one of the aspects described herein, R M' is a ligand, a linker, or a linker for one or more ligands. For example, R M' is L-R L where L is a linker and R L is a ligand.

[0129] R N' and R N'' One aspect of any one of the aspects described herein includes R N' and R N'' In some embodiments of any one of the aspects described herein, R N' and R N'' are independently hydrogen, optionally substituted C 6~30 alkyl, optionally substituted C 6~30 alkenyl, optionally substituted C 6~30 alkynyl, or optionally substituted C 3~30 cycloalkyl, lipid, ligand, linker, or a linker for one or more ligands. Optionally, at least one of R N' and R N'' is not hydrogen.

[0130] In some embodiments of any one of the aspects described herein, one of R N' and R N'' is hydrogen.

[0131] In some embodiments of any one of the aspects described herein, R N' and R N'' are both not hydrogen. When both R N' and R N'' are not hydrogen, R N' and R N''Note that they can be the same or different. Thus, in some embodiments of any one of the aspects described herein, R N' and R N'' are both not hydrogen, and R N' and R N'' are the same. In some other embodiments of any one of the aspects described herein, R N' and R N'' are both not hydrogen, and R N' and R N'' are different.

[0132] In some embodiments, at least one of R N' and R N'' is C 6~30 alkyl, C 6~30 alkenyl or C 6~30 alkynyl, where C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl may be substituted with at least one substituent. For example, at least one of R N' and R N'' is C 6~30 alkyl, C 6~30 alkenyl or C 6~30 alkynyl, where C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl are substituted with at least one substituent selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, aralkyl, aryl, heteroaryl, cyclil, heterocyclil, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino, alkylcarbonoyl, arylcarbonoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, acyloxy, cyano or ureido. For example, C 6~30Alkyl, C 6~30 Alkenyl and C 6~30 Alkynyl is OH, CN, -SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p Optionally substituted with 1, 2, 3, 4 or 5 groups selected from -NH2 or CH2-aryl-alkoxy, and "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0133] In some embodiments, R N' and R N'' At least one of which is C 6~30 Alkyl, C 6~30 Alkenyl or C 6~30 Alkynyl, where C 6~30 Alkyl, C 6~30 Alkenyl and C 6~30 Alkynyl is optionally substituted with at least one substituent at the terminus. For example, at least one of R N' and R N'' is C 6~30 Alkyl, C 6~30 Alkenyl or C 6~30 Alkynyl, where C 6~30 Alkyl, C 6~30 Alkenyl and C 6~30 Alkynyl is R N'and R N'' At least one of them may be substituted with at least one substituent at a terminal remote from the point where it is attached to the remainder of the molecule.

[0134] In some embodiments, R N' and R N'' At least one of them is C 6~30 alkyl, C 6~30 alkenyl or C 6~30 alkynyl, where C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl are each optionally substituted at the terminal with at least one substituent selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino, alkylcarbonoyl, arylcarbonoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, acyloxy, cyano or ureido. For example, at least one of R N' and R N'' At least one of them is R N' and R N'' At least one of them is C 6~30 alkyl, C 6~30 alkenyl or C 6~30 alkynyl, where C 6~30 alkyl, C 6~30 alkenyl and C 6~30 alkynyl are R N' and R N''At least one of which is attached to the terminal away from the part where it is attached to the rest of the molecule, OH, CN, -SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p It may be substituted with at least one substituent selected from the group consisting of -NH2 or CH2-aryl-alkoxy, and "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0135] In some embodiments of any one of the aspects described herein, R N' and R N'' At least one of which is -(CH2) mn -R NE wherein mn is an integer from 6 to 29, and R NE is methyl, CO2H, CO2Me, NH2, SH, OH, CH=CH2 or C≡CH.

[0136] In some embodiments of any one of the above aspects, R N' and R N'' At least one of which is -(CH2) mn -R NEand where mn is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. For example, mn is 10, 11, 12, 13, 14, 15, 16, or 17. In another non-limiting example, mn is 11, 13, 15 or 17. In yet another non-limiting example, mn is 10, 12, 14, 16 or 18.

[0137] In some embodiments of any one of the above aspects, R NE is methyl, CO2H, CO2Me, NH2, CH=CH2 or C≡CH. For example, R NE is CO2H, CO2Me, NH2, CH=CH2 or C≡CH.

[0138] In some embodiments of any one of the above aspects, mn is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and R NE is methyl, CO2H, CO2Me, NH2, CH=CH2 or C≡CH. For example, mn is 9, 11, 13, 15 or 17 and R NE is CO2H, CO2Me, NH2, CH=CH2 or C≡CH. In another non-limiting example, mn is 10, 12, 14, 16 or 18 and R NE is CO2H, CO2Me, NH2, CH=CH2 or C≡CH. In yet another non-limiting example, mn is 9, 11, 13, 15, 17 or 19 and R NE is methyl. In yet another non-limiting example, mn is 10, 12, 14, 16 or 18 and R NE is methyl.

[0139] In some embodiments of any one of the above aspects, R N' and R N'' are independently selected from C 6~10 alkyl, C 6~10 alkenyl or C 6~10 alkynyl, where C 6~10 alkyl, C 6~10 alkenyl and C6~10 The alkynyl may be substituted with at least one substituent.

[0140] In some embodiments of any one of the aspects described herein, R N' and R N'' at least one of which is hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, or icosadecyl.

[0141] In some embodiments of any one of the aspects described herein, R N' and R N'' at least one of which is (9Z)-tetradeca-9-enyl, (6Z)-hexadeca-6-enyl, (9Z)-hexadeca-9-enyl, (9Z)-octadeca-9-enyl, (9E)-octadeca-9-enyl, (11E)-octadeca-11-enyl, (9Z,12Z)-octadeca-9,12-dienyl, (9E,12E)-octadeca-9,12-dienyl, (9Z,12Z,15Z)-octadeca-9,12,15-trienyl, (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenyl, (5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenyl, or (13Z)-docosa-13-enyl. For example, R N' and R N'' at least one of which is (9Z)-octadeca-9-enyl, (9E)-octadeca-9-enyl, (11E)-octadeca-11-enyl, (9Z,12Z)-octadeca-9,12-dienyl or (9E,12E)-octadeca-9,12-dienyl. In some embodiments, R N' and R N'' at least one of which is (9Z)-octadeca-9-enyl or (9Z,12Z)-octadeca-9,12-dienyl or (9E,12E)-octadeca-9,12-dienyl.

[0142] In some embodiments of any one of the aspects described herein, R N' and R N'' at least one of which is optionally substituted C 3~30 cycloalkyl. Note that the cycloalkyl can be partially unsaturated, i.e., the cycloalkyl can contain one or more double bonds and / or triple bonds. For example, R N' and R N'' at least one of which is cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl or cyclododecyl. In some embodiments, R N' and R N'' at least one of which is cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl. For example, R N' and R N'' at least one of which is cyclooctyl.

[0143] In some embodiments of any one of the aspects described herein, R N' and R N'' at least one of which is a ligand, a linker, or a linker to one or more ligands. For example, R N' and R N'' at least one of which is -L-R L where L is a linker and R L is a ligand. In some embodiments, one of R N' and R N'' is hydrogen and the other of R N' and R N'' is -L-R L Examples of exemplary ligands for R N' and R N'' include, but are not limited to, triGalNAc, monoGalNac, cyclic RGD and other peptide targeting ligands, folic acid, DUPA, biotin, carboxyfluorescein, lipoic acid and mannose ligands.

[0144] In some embodiments, R N' and R N'' at least one of which is TIFF2025522811000005.tif101146TIFF2025522811000006.tif179138, where R is TIFF2025522811000007.tif21128.

[0145] For example, one of R N' and R N'' is hydrogen and the other is the structure shown in the above paragraph.

[0146] R LV Any one embodiment of the aspects described herein includes R LV . In some embodiments, R LV is C 1~6 alkyl. For example, R LV is methyl, ethyl, propyl, butyl, isobutyl, pentyl, or hexyl. In some embodiments of any one of the aspects described herein, R LV is ethyl.

[0147] R 2 In some embodiments of any one of the aspects described herein, R 2 is -O(CH2) m1 -X'-R M' , -O(CH2) n1 -C(O)N(R N' )(R N'' ), hydrogen, hydroxyl, protected hydroxyl, phosphate group, reactive phosphorus(III) group, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30Alkoxy (e.g., methoxy, 2 - methoxyethoxy), alkoxyalkyl (e.g., methoxyethyl such as 2 - methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, protected aminoalkyl, -O - N - methylacetamide, -O - C 4~30 alkyl - ON(CH2R 8 )(CH2R 9 ), or -O - C 4~30 alkyl - ON(CH2R 8 )(CH2R 9 ), a solid support, a linker or a linker covalently attached to a solid support. For example, R 2 is -O(CH2) m1 -X'-R M' , -O(CH2) n1 -C(O)N(R N' )(R N'' ), hydrogen, hydroxyl, protected hydroxyl, phosphate group, reactive phosphorus(III) group, halogen, optionally substituted C 1~30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, -O - N - methylacetamide, or C 6~24 alkyl (e.g., n - C 6~24 alkyl).

[0148] In some embodiments of any one of the aspects described herein, R 2 is -O(CH2) m1 -X M' -R M' . For example, R 2 is -O(CH2) m1 -O - R M' . In another non - limiting example, R 2 is -O(CH2) m1 -S - R M' .

[0149] In some embodiments of any one of the above aspects, R 2 is -OCH2CH2 - X'-RM' is. For example, R 2 is -OCH2CH2-O-R M' is. In another non-limiting example, R 2 is -OCH2CH2-S-R M' is.

[0150] In some embodiments of any one of the aspects described herein, R 2 is -O(CH2) n1 -C(O)N(R N' )(R N'' ). Note that when R 2 is -O(CH2) n1 -C(O)N(R N' )(R N'' ), n1 can be 1 or 2. Thus, in some embodiments of any one of the aspects described herein, R 2 is -OCH2-C(O)N(R N' )(R N'' ). In some other embodiments of any one of the aspects described herein, R 2 is -OCH2CH2-C(O)N(R N' )(R N'' ).

[0151] In some embodiments of any one of the above aspects, R 2 is hydrogen, hydroxyl, halogen, protected hydroxyl, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., methoxyethyl such as 2-methoxyethyl), alkoxyalkylamine, alkoxyoxalate, amino, alkylamino, dialkylamino, -O-N-methylacetamide, C 6~24 alkyl (e.g., n-C 6~24 alkyl), or -O-C 4~30Alkyl-ON(CH2R8)(CH2R9), or -O-C 4~30 is Alkyl-ON(CH2R8)(CH2R9). For example, R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkoxy, optionally substituted C 1~30 alkyl or alkoxyalkyl (e.g., methoxyethyl).

[0152] In some embodiments, R 2 is hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, ethoxy, 2-methoxyethoxy, or -O-N-methylacetamide. For example, R 2 is hydrogen, hydroxyl, protected hydroxyl, fluoro or methoxy.

[0153] In some embodiments, R 2 is -OR 222 wherein R 222 is hydrogen, an oxygen protecting group, optionally substituted C 1~30 alkyl, C 1~30 haloalkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, or optionally substituted C 1~30 alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl.

[0154] In some embodiments, R 222 is hydrogen, i.e., R 2 is OH.

[0155] In some embodiments, R 222 is an oxygen protecting group, i.e., R 2 is -OR Pro wherein R Prois selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl. In some embodiments, R 2 is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl.

[0156] In some embodiments of any one of the aspects described herein, R 2 is a reactive phosphorus group. Optionally, only one of R 2 and R 3 is a reactive phosphorus(III) group.

[0157] Without wishing to be bound by theory, the reactive phosphorus group serves to form, for example, an internucleoside linkage that includes a phosphodiester and phosphorothioate nucleoside internucleoside linkage. Such reactive phosphorus groups are known in the art and contain a phosphorus atom in a P III or P V valence state, such as phosphoramidites, H-phosphonates, phosphate triesters, and phosphorus-containing chiral auxiliaries. The reactive phosphorus(III) group in the form of a phosphoramidite (P III chemistry) is a preferred reactive phosphorus(III) group for solid-phase oligonucleotide synthesis. Subsequently, the intermediate phosphite compound is oxidized to the Pv state using known methods to obtain a phosphodiester or phosphorothioate nucleoside internucleoside linkage.

[0158] In some embodiments of any one of the aspects described herein, the reactive phosphorus(III) group is -OP(OR P )(N(R P2 )2), -OP(SR P )(N(R P2 )2), -OP(O)(OR P )(N(R P2)2), -OP(S)(OR P )(N(R P2 )2), -OP(O)(SR P )(N(R P2 )2), -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 . For example, the reactive phosphorus(III) group is -OP(OR P )(N(R P2 )2).

[0159] In some embodiments of any one of the above situations, R P is optionally substituted C 1~6 alkyl. For example, R P is OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-aryl-alkoxy, and is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from C 1~6is alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6. In some embodiments, R p may be substituted with CN or -SC(O)Ph, C 1~6 alkyl. For example, R p is cyanoethyl (-CH2CH2CN).

[0160] In the reactive phosphorus(III) group, each R P2 is independently optionally substituted C 1~6 alkyl. For example, each R P2 can be independently selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl. It should be noted that when two or more R P2 groups are present in the reactive phosphorus(III) group, they can be the same or different. Thus, in some non-limiting examples, when two or more R P2 groups are present, those R P2 groups are different. In some other non-limiting examples, when two or more R P2 groups are present, those R P2 groups are the same. In some embodiments of any one of the above aspects, each R P2 is isopropyl.

[0161] In some embodiments of any one of the above aspects, both R P2together with the nitrogen atom to which they are attached form an optionally substituted 3- to 8-membered heterocyclyl. Exemplary heterocyclyls include, but are not limited to, pyrrolidinyl, piperazinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholinyl, 4-piperazinyl, pyrrolidinyl, perhydro-pyrrolidinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl, 1,4-dioxanyl, etc., each of which is optionally OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p can be substituted with 1, 2 or 3 substituents independently selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0162] In some embodiments of any one of the above aspects, R P and R P2One of them, together with the atom to which they are attached, forms an optionally substituted 4- to 8-membered heterocyclyl. Exemplary heterocyclyls include, but are not limited to, pyrrolidinyl, piperazinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydro-pyrrolidinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl, 1,4-dioxanyl, etc., each of which is optionally OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p can be substituted with one, two or three substituents independently selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0163] In the reactive phosphorus(III) group, each R P3 is independently optionally substituted C 1~6 alkyl. For example, R P3is OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from -NH2 or CH2-aryl-alkoxy 1~6 can be alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6. For example, R P3 is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy.

[0164] In some embodiments of any one of the above aspects, the reactive phosphorus(III) group is -OP(OR P )(N(R P2 )2). For example, the reactive phosphorus(III) group is -OP(OR P )(N(R P2 )2), where R P is cyanoethyl (-CH2CH2CN) and each R P2 is isopropyl.

[0165] In some embodiments of any one of the aspects described herein, R 2 is -OP(OR P )(N(R P2 )2), -OP(SR P )(N(R P2 )2), -OP(O)(OR P )(N(R P2 )2), -OP(S)(OR P )(N(R P2 )2), -OP(O)(SR P )(N(R P2 )2), -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 .

[0166] In some embodiments of any one of the above aspects, R 2 is -OP(OR P )(N(R P2 )2), -OP(SR P )(N(R P2 )2), -OP(O)(OR P )(N(R P2 )2), -OP(S)(OR P )(N(R P2 )2), -OP(O)(SR P )(N(R P2 )2), -OP(O)(OR P )H, -OP(S)(OR P ), optionally substituted C 1~6 alkyl, each R P2 is independently optionally substituted C 1~6 alkyl, and each R P3 is independently optionally substituted C 1~6 alkyl.

[0167] In some embodiments, R 2is [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite.

[0168] In some embodiments of any one of the above aspects, R 2 is -OP(OR P )(N(R P2 ))2). For example, R 2 is -OP(OR P )(N(R P2 ))2), where R P is cyanoethyl (-CH2CH2CN) and each R P2 is isopropyl.

[0169] In some embodiments, R 2 is -OP(SR P )(N(R P2 ))2). For example, R 2 is -OP(SR P )(N(R P2 ))2), where R P is β-thiobenzoylethyl and the two Rs P2 , together with the nitrogen to which they are attached, form pyrrolidine.

[0170] In some embodiments of any one of the aspects described herein, R 2 is a solid support or a linker covalently attached to a solid support. For example, R 2 is -OC(O)CH2CH2C(O)NH-Z, where Z is a solid support. In some embodiments, R 2 is -OC(O)CH2CH2CO2H.

[0171] R 2 and R 3 Note that only one of can be a linker covalently attached to a solid support.

[0172] R 3 In some embodiments of any one of the aspects described herein, R 3 is -O(CH2) m1 -X'-R M' 、-O(CH2) n1 -C(O)N(R N' )(R N'' ), hydrogen, hydroxyl, protected hydroxyl, phosphate group, reactive phosphorus(III) group, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., methoxyethyl such as 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, protected aminoalkyl, -O-N-methylacetamide, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), or -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), a solid support, a linker or a linker covalently attached to a solid support. For example, R 3 is -O(CH2) m1 -X'-R M' 、-O(CH2) n1 -C(O)N(R N' )(R N'' ), hydrogen, hydroxyl, protected hydroxyl, phosphate group, reactive phosphorus(III) group, halogen, optionally substituted C 1~30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-N-methylacetamide, or C 6~24 alkyl (e.g., n-C 6~24 alkyl).

[0173] In some embodiments of any one of the aspects described herein, R 3 is -O(CH2) m1 -X M' -R M' For example, R 3 is -O(CH2) m1 -O-R M' In another non-limiting example, R 3 is -O(CH2) m1 -S-R M'

[0174] In some embodiments of any one of the above aspects, R 3 is -OCH2CH2-X'-R M' For example, R 3 is -OCH2CH2-O-R M' In another non-limiting example, R 3 is -OCH2CH2-S-R M'

[0175] In some embodiments of any one of the aspects described herein, R 3 is -O(CH2) n1 -C(O)N(R N' )(R N'' ) Note that when R 3 is -O(CH2) n1 -C(O)N(R N' )(R N'' ), n1 can be 1 or 2. Therefore, in some embodiments of any one of the aspects described herein, R 3 is -OCH2-C(O)N(R N' )(R N'' ). In some other embodiments of any one of the aspects described herein, R 3 is -OCH2CH2-C(O)N(R N' )(R N'' )

[0176] In some embodiments of any one of the above aspects, R 3 ​​is hydrogen, hydroxyl, halogen, protected hydroxyl, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., methoxyethyl such as 2-methoxyethyl), alkoxyalkylamine, alkoxyoxocarboxylate, amino, alkylamino, dialkylamino, -O-N-methylacetamide, C 6~24 alkyl (e.g., n-C 6~24 alkyl), or -O-C 4~30 alkyl-ON(CH2R8)(CH2R9), or -O-C 4~30 alkyl-ON(CH2R8)(CH2R9). For example, R 3 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkoxy, optionally substituted C 1~30 alkyl or alkoxyalkyl (e.g., methoxyethyl).

[0177] In some embodiments, R 3 is hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, ethoxy, 2-methoxyethoxy, or -O-N-methylacetamide. For example, R 2 is hydrogen, hydroxyl, protected hydroxyl, fluoro or methoxy.

[0178] In some embodiments, R 3 is -OR 222 wherein R 222 is hydrogen, an oxygen protecting group, optionally substituted C 1~30 alkyl, C 1~30 haloalkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, or optionally substituted C 1~30It is alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

[0179] In some embodiments, R 222 is hydrogen, i.e., R 3 is OH.

[0180] In some embodiments, R 222 is an oxygen protecting group, i.e., R 3 is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl. In some embodiments, R 3 is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl.

[0181] In some embodiments of any one of the aspects described herein, R 3 is a reactive phosphorus group. Optionally, only one of R 2 and R 3 is a reactive phosphorus(III) group.

[0182] In some embodiments, R 3 is [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite.

[0183] In some embodiments of any one of the above aspects, R 3 is -OP(OR P )(N(R P2 )2). For example, R 3 is -OP(OR P )(N(R P2 )2) wherein R Pis cyanoethyl (-CH2CH2CN), each R P2 is isopropyl.

[0184] In some embodiments, R 3 is -OP(SR P )(N(R P2 )2). For example, R 3 is -OP(SR P )(N(R P2 )2), where R P is β-thiobenzoylethyl, and the two Rs P2 form pyrrolidine together with the nitrogen to which they are attached.

[0185] In some embodiments of any one of the aspects described herein, R 3 is a solid support or a linker covalently attached to a solid support. For example, R 3 is -OC(O)CH2CH2C(O)NH-Z, where Z is a solid support. In some embodiments, R 3 is -OC(O)CH2CH2CO2H. Note that only one of R 2 and R 3 can be a linker covalently attached to a solid support.

[0186] In some embodiments, one of R 2 and R 3 is -OCH2CH2-X M' -R M' or -O(CH2) n1 -C(O)N(R N' )(R N'' ), and the other of R 2 and R 3 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkoxy, a reactive phosphorus (III) group, a solid support, a linker, or a linker covalently attached to a solid support. For example, R 2 and R 3One of them is -OCH2CH2-X M' -R M' Or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and the other of R 2 and R 3 is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus(III) group, linker, or a linker covalently attached to a solid support.

[0187] In some embodiments, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 3 is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus(III) group, linker, or a linker covalently attached to a solid support.

[0188] In some other embodiments, R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 2 is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus(III) group, linker, or a linker covalently attached to a solid support.

[0189] R 5 In some embodiments of any one of the various aspects described herein, R 25 is -O(CH2) m1 -X M' -R M', -O(CH2) n1 -C(O)N(R N' )(R N'' ), hydroxyl, protected hydroxyl, optionally substituted C 1~30 alkoxy, vinyl phosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkyl phosphonate, alkyl ether phosphonate, dialkyl terminal phosphate or phosphate mimetic. For example, R 25 is -O(CH2) m1 -X M' -R M' , -O(CH2) n1 -C(O)N(R N' )(R N'' ), hydroxyl, protected hydroxyl, vinyl phosphonate (VP) group, cyclopropyl phosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkyl phosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, or phosphate mimetic. In some embodiments of any one of the various aspects described herein, R 25 is a vinyl phosphonate group, cyclopropyl phosphonate. In some other embodiments, R 25 is hydroxyl or protected hydroxyl.

[0190] In some embodiments of any one of the aspects described herein, R 5 is -O(CH2) m1 -X M' -R M' . For example, R 5 is -O(CH2) m1 -O-R M'is. In another non-limiting example, R 5 is -O(CH2) m1 -S-R M' is.

[0191] In some embodiments of any one of the above situations, R 5 is -OCH2CH2-X'-R M' is. For example, R 5 is -OCH2CH2-O-R M' is. In another non-limiting example, R 5 is -OCH2CH2-S-R M' is.

[0192] In some embodiments of any one of the situations described herein, R 5 is -O(CH2) n1 -C(O)N(R N' )(R N'' ) is. Note that when R 5 is -O(CH2) n1 -C(O)N(R N' )(R N'' ), n1 can be 1 or 2. Thus, in some embodiments of any one of the situations described herein, R 5 is -OCH2-C(O)N(R N' )(R N'' ). In some other embodiments of any one of the situations described herein, R 3 is -OCH2CH2-C(O)N(R N' )(R N'' ).

[0193] In some embodiments of the various situations described herein, R 5 is R 551 , optionally substituted C 1~6 alkyl-R 551 , optionally substituted -C 2~6 alkenyl-R 551 , or optionally substituted -C 2~6 alkynyl-R 551 , where R 551 is -OR552 , -SR 553 can be hydrogen, a phosphorus(III) group, a solid support, or a linker to a solid support. R 551 is -OR 552 When R 552 is, R 1~30 can be hydrogen, an oxygen protecting group, optionally substituted C 1~30 alkyl, C 2~30 haloalkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 1~30 alkynyl, or optionally substituted C 551 When R 553 is -SR 553 is, R 1~30 can be hydrogen, a sulfur protecting group, optionally substituted C 1~30 alkyl, C 2~30 haloalkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 1~30 alkynyl, or optionally substituted C

[0194] In some embodiments of any one of the aspects described herein, R 5 is -OR 552 where is hydrogen or an oxygen protecting group. Exemplary hydroxyl protecting groups for R 552 include, but are not limited to, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX).

[0195] In some embodiments of any one of the various aspects described herein, R 552 is hydrogen, i.e., R 5 is OH.

[0196] In some embodiments of any one of the various aspects described herein, R 552 is an oxygen protecting group, i.e., R 5 is -OR Pro wherein R Pro is an oxygen protecting group. For example, R 5 is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl. In some embodiments of any one of the various aspects described herein, R 5 is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl.

[0197] In some embodiments of any one of the various aspects described herein, R 5 is -OR Pro wherein R Pro is 4,4'-dimethoxytrityl (DMT).

[0198] In some embodiments of any one of the various aspects described herein, one of R 2 and R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and the other of R 2 and R 3 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30An alkoxy, a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support, R 5 is hydroxyl or -OR Pro and optionally, R Pro is DMT. For example, one of R 2 and R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and the other of R 2 and R 3 is hydrogen, hydroxyl, protected hydroxyl, a reactive phosphorus(III) group, a linker, or a linker covalently attached to a solid support, R 5 is hydroxyl or -OR Pro and optionally, R Pro is DMT.

[0199] In some embodiments, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 3 is hydrogen, hydroxyl, protected hydroxyl, a reactive phosphorus(III) group, a linker, or a linker covalently attached to a solid support, R 5 is hydroxyl or -OR Pro and optionally, R Pro is DMT.

[0200] In some other embodiments, R 3 is -O(CH2) m1 -X M' -R M'(e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ), where R 3 is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus(III) group, linker, or a linker covalently attached to a solid support, and R 5 is hydroxyl or -OR Pro and optionally, R Pro is DMT.

[0201] In some embodiments of any one of the various aspects described herein, one of R 2 and R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ), and the other of R 2 and R 3 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkoxy, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to a solid support, and R 5 is hydroxyl or -OR Pro and optionally, R Pro is DMT. For example, one of R 2 and R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ), and R 2 and R 3The other of which is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus(III) group, linker, or a linker covalently attached to a solid support, and R 5 is hydroxyl or -OR Pro and optionally, R Pro is DMT.

[0202] In some embodiments, R 2 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 3 is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus(III) group, linker, or a linker covalently attached to a solid support, and R 5 is a vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate or a phosphate mimetic, and optionally, R Pro is a vinyl phosphate group.

[0203] In some other embodiments, R 3 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X M' -R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 3is a linker covalently attached to hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus(III) group, linker, or solid support, R 5 is a vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate or phosphate mimetic, optionally, R Pro is a vinyl phosphate group.

[0204] In some embodiments of any one of the aspects described herein, R 5 The methylene connecting R to the remainder of the compound of formula (I) is absent and R 5 is directly connected to the remainder of the compound of formula (I).

[0205] In some embodiments of any one of the aspects described herein, R 5 is -CH(R 554 )-R 551 wherein R 554 is hydrogen, halogen, optionally substituted C1-C 30 alkyl, optionally substituted C2-C 30 alkenyl, optionally substituted C2-C 30 alkynyl, or optionally substituted C1-C 30 alkoxy.

[0206] In some embodiments of any one of the above aspects, R 5 is -CH(R 554 )-R 551 wherein when R 554is H, or OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-aryl-alkoxy, and is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from C1-C 30 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6. For example, R 554 is H. In some other non-limiting examples, R 554 is C1-C 30 alkyl, optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy.

[0207] In some embodiments of any one of the aspects described herein, R 5 is -CH(R 554 )-O-R 552 where R 554is H, or OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-aryl-alkoxy, and is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 30 C1-C alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6. For example, R 554 is H. In some other non-limiting examples, R 554 is C1-C alkyl optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy. 30

[0208] In some embodiments of any one of the aspects described herein, R 5 is optionally substituted C 1~6 alkyl-R 551 , or optionally substituted -C 2~6 alkenyl-R 551 .

[0209] In some embodiments of any one of the aspects described herein, R 5 is -C(R 554 )=CHR 551 ​is -C(R 554 )=CHR 551 Note that the double bond in can be in the cis configuration or the trans configuration. Thus, in some embodiments of any one of the above aspects, R d is -C(R 554 )=CHR 551 wherein the double bond is in the cis configuration. In some other embodiments of any one of the above aspects, R d is -C(R 554 )=CHR 551 wherein the double bond is in the trans configuration.

[0210] In some embodiments of any one of the aspects described herein, R 5 is -CH=CHR 551 .

[0211] In some embodiments of any one of the above aspects, when R 5 is -C(R 554 )=CHR 551 , R 554 is H or OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) pC1-C which may be substituted with 1, 2, 3, 4 or 5 substituents independently selected from -NH2 or CH2-aryl-alkoxy 30 is alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6, and R 551 is a phosphorus(III) group. For example, R 5 is -CH=CHR 551 .

[0212] In some embodiments of any one of the aspects described herein, R 551 is a reactive phosphorus(III) group.

[0213] In some embodiments of any one of the above aspects, R 5 is -CH=CH-P(O)(OR 555 )2, -CH=CH-P(S)(OR 555 )2, -CH=CH-P(S)(SR 556 )(OR 555 ), -CH=CH-P(S)(SR 556 )2, -CH=CH-OP(O)(OR 555 )2, -CH=CH-OP(S)(OR 555 )2, -CH=CH-OP(S)(SR 556 )(OR 555 ), -CH=CH-OP(S)(SR 556 )2, -CH=CH-SP(O)(OR 555 )2, -CH=CH-SP(S)(OR 555 )2, -CH=CH-SP(S)(SR 556 )(OR 555 ), or -CH=CH-SP(S)(SR 556 )2, where each R 555 is independently hydrogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl, or an oxygen protecting group, and each R 556 is independently hydrogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30An alkenyl, or an optionally substituted C 2~30 alkynyl, or a sulfur protecting group.

[0214] In some embodiments of any one of the above aspects, -P(O)(OR 555 )2, -P(S)(OR 555 )2, -P(S)(SR 556 )(OR 555 )、-OP(O)(OR 555 )2、-OP(S)(OR 555 )2、-OP(S)(SR 556 )(OR 555 )、SP(O)(OR 555 )2、-SP(S)(OR 555 )2、and -SP(S)(SR 556 )(OR 555 ), at least one R 555 is hydrogen.

[0215] In some other embodiments of any one of the above aspects, -P(O)(OR 555 )2, -P(S)(OR 555 )2, -P(S)(SR 556 )(OR 555 )、-OP(O)(OR 555 )2、-OP(S)(OR 555 )2、-OP(S)(SR 556 )(OR 555 )、SP(O)(OR 555 )2、-SP(S)(OR 555 )2、or -SP(S)(SR 556 )(OR 555 ), at least one R 555 is not hydrogen. For example, P(O)(OR 555 )2, -P(S)(OR 555 )2, -P(S)(SR 556 )(OR 555 )、-OP(O)(OR 555 )2、-OP(S)(OR 555 )2、-OP(S)(SR 556 )(OR 555 )、SP(O)(OR 555)2, -SP(S)(OR 555 )2, and -SP(S)(SR 556 )(OR 555 ) at least one of at least one R 555 is optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl, or an oxygen protecting group.

[0216] In some embodiments of any one of the above aspects, -P(O)(OR 555 )2, -P(S)(OR 555 )2, -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 )2, -OP(S)(OR 555 )2, -OP(S)(SR 556 )(OR 555 ), SP(O)(OR 555 )2, -SP(S)(OR 555 )2, and -SP(S)(SR 556 )(OR 555 ), at least one R 555 is H, and at least one R 555 is other than H.

[0217] In some embodiments of any one of the above aspects, -P(O)(OR 555 )2, -P(S)(OR 555 )2, -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 )2, -OP(S)(OR 555 )2, -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 )2, -SP(O)(OR 555 )2, -SP(S)(OR 555 )2, -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 )2, in R555 are all H.

[0218] In some embodiments of any one of the above situations, -P(O)(OR 555 )2, -P(S)(OR 555 )2, -P(S)(SR 556 )(OR 555 )、-OP(O)(OR 555 )2、-OP(S)(OR 555 )2、-OP(S)(SR 556 )(OR 555 )、-OP(S)(SR 556 )2、-SP(O)(OR 555 )2、-SP(S)(OR 555 )2、-SP(S)(SR 556 )(OR 555 )、and -SP(S)(SR 556 )2, where all R 555 are other than H.

[0219] In some embodiments of any one of the above situations, -P(S)(SR 556 )(OR 555 )、-P(S)(SR 556 )2、-OP(S)(OR 555 )2、-OP(S)(SR 556 )(OR 555 )、-OP(S)(SR 556 )2、-SP(S)(SR 556 )(OR 555 )、and -SP(S)(SR 556 )2, at least one of the R 556 is H.

[0220] In some embodiments of any one of the above situations, -P(S)(SR 556 )(OR 555 )、-P(S)(SR 556 )2、-OP(S)(OR 555 )2、-OP(S)(SR 556 )(OR 555 )、-OP(S)(SR 556 )2、-SP(S)(SR 556 )(OR555 ) and -SP(S)(SR 556 )2, at least one R in 556 is other than H. For example, -P(S)(SR 556 )(OR 555 )、-P(S)(SR 556 )2、-OP(S)(OR 555 )2、-OP(S)(SR 556 )(OR 555 )、-OP(S)(SR 556 )2、-SP(S)(SR 556 )(OR 555 )、および-SP(S)(SR 556 )2, at least one R in 556 is optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl, or a sulfur protecting group.

[0221] In some embodiments of any one of the above aspects, -P(S)(SR 556 )2, -OP(S)(SR 556 )2 and -SP(S)(SR 556 )2, at least one R 556 is H, and at least one R 556 is other than H.

[0222] In some embodiments of any one of the various aspects described herein, -P(S)(SR 556 )(OR 555 )、-P(S)(SR 556 )2、-OP(S)(OR 555 )2、-OP(S)(SR 556 )(OR 555 )、-OP(S)(SR 556 )2、-SP(S)(SR 556 )(OR 555 )、および-SP(S)(SR 556 )2, R 556 are all H.

[0223] In some embodiments of any one of the various aspects described herein, -P(S)(SR 556 )(OR 555 )、-P(S)(SR 556 )2、-OP(S)(OR 555 )2、-OP(S)(SR 556 )(OR 555 )、-OP(S)(SR 556 )2、-SP(S)(SR 556 )(OR 555 )、and -SP(S)(SR 556 )2, all R 556 are other than H.

[0224] In some embodiments of any one of the above aspects, R 5 is -CH=CH-P(O)(OR 555 )2, where each R 555 is H or an oxygen protecting group.

[0225] R 22 In the nucleoside of formula (II), R 22 is -O(CH2) m1 -X'-R M' , -O(CH2) n1 -C(O)N(R N' )(R N'' ), hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxalate, amino, alkylamino, dialkylamino, protected aminoalkyl, 5- to 8-membered heterocyclyl, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), -O-N-methylacetamide, -O-C 4~30 alkyl-ON(CH2R8 )(CH2R 9 ) is a linkage to an internucleotide linkage for a subsequent nucleotide, a 3'-oligonucleotide capping group, a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support.

[0226] In some embodiments of any one of the aspects described herein, R 22 is -O(CH2) m1 -X M' -R M' For example, R 22 is -O(CH2) m1 -O-R M' In another non-limiting example, R 22 is -O(CH2) m1 -S-R M'

[0227] In some embodiments of any one of the above aspects, R 22 is -OCH2CH2-X'-R M' For example, R 22 is -OCH2CH2-O-R M' In another non-limiting example, R 22 is -OCH2CH2-S-R M'

[0228] In some embodiments of any one of the aspects described herein, R 22 is -O(CH2) n1 -C(O)N(R N' )(R N'' ) Note that when R 22 is -O(CH2) n1 -C(O)N(R N' )(R N'' ), n1 can be 1 or 2. Thus, in some embodiments of any one of the aspects described herein, R 22 is -OCH2-C(O)N(R N' )(R N'' ​​) is. In some other embodiments of any one of the aspects described herein, R 22 is -OCH2CH2-C(O)N(R N' )(R N'' ).

[0229] In some embodiments, R 22 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), amino, alkylamino, dialkylamino, protected aminoalkyl, -O-C 4~30 alkyl-ON(CH2R8)(CH2R9), -O-C 4~30 alkyl-ON(CH2R8)(CH2R9), -O-N-methylacetamide, alkoxyoxycarboxylate, solid support, linker, or a linker covalently attached to a solid support. For example, R 22 is hydrogen, hydroxyl, halogen, protected hydroxyl, optionally substituted C1-30 alkyl, optionally substituted C2-30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, protected aminoalkyl, -O-N-methylacetamide, -O-C 4~30 alkyl-ON(CH2R8)(CH2R9), or -O-C 4~30 alkyl-ON(CH2R8)(CH2R9).

[0230] In some embodiments of any one of the aspects described herein, R 22is a bond to the internucleotide linkage, a linker, or a linker covalently attached to a solid support, to a subsequent nucleotide. For example, R 22 is a bond to the internucleotide linkage to a subsequent nucleotide.

[0231] R 23 In the nucleoside of formula (II), R 23 is -O(CH2) m1 -X'-R M' -O(CH2) n1 -C(O)N(R N' )(R N'' ), hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, protected aminoalkyl, 5- to 8-membered heterocyclyl, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), -O-N-methylacetamide, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), a bond to the internucleotide linkage to a subsequent nucleotide, a 3'-oligonucleotide capping group, a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support.

[0232] In some embodiments of any one of the aspects described herein, R 23 is -O(CH2) m1 -X M' -R M' For example, R 23 is -O(CH2)m1 -O-R M' is. In another non-limiting example, R 23 is -O(CH2) m1 -S-R M' is.

[0233] In some embodiments of any one of the above aspects, R 23 is -OCH2CH2-X'-R M' is. For example, R 23 is -OCH2CH2-O-R M' is. In another non-limiting example, R 23 is -OCH2CH2-S-R M' is.

[0234] In some embodiments of any one of the aspects described herein, R 23 is -O(CH2) n1 -C(O)N(R N' )(R N'' ) is. Note that when R 23 is -O(CH2) n1 -C(O)N(R N' )(R N'' ), n1 can be 1 or 2. Thus, in some embodiments of any one of the aspects described herein, R 23 is -OCH2-C(O)N(R N' )(R N'' ). In some other embodiments of any one of the aspects described herein, R 23 is -OCH2CH2-C(O)N(R N' )(R N'' ).

[0235] In some embodiments, R 23 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), amino, alkylamino, dialkylamino, protected aminoalkyl, -O-C 4~30 alkyl-ON(CH2R8)(CH2R9), -O-C 4~30 alkyl-ON(CH2R8)(CH2R9), -O-N-methylacetamide, alkoxyoxalate, solid support, linker, or a linker covalently attached to a solid support. For example, R 23 is hydrogen, hydroxyl, halogen, protected hydroxyl, optionally substituted C1-30 alkyl, optionally substituted C2-30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxalate, amino, alkylamino, dialkylamino, protected aminoalkyl, -O-N-methylacetamide, -O-C 4~30 alkyl-ON(CH2R8)(CH2R9), or -O-C 4~30 alkyl-ON(CH2R8)(CH2R9).

[0236] In some embodiments of any one of the aspects described herein, R 23 is a bond to a nucleotide internucleoside linker, a linker, or a linker covalently attached to a solid support. For example, R 23 is a bond to a nucleotide internucleoside linker for a subsequent nucleotide.

[0237] In some embodiments, R 22 and R 23 One of them is -OCH2CH2-X'-R M' or -O(CH2) n1 -C(O)N(R N' )(R N'' ), and R 22 and R 23The other of which is hydrogen, hydroxyl, protected hydroxyl, a bond to an internucleotide linkage for a subsequent nucleotide, a 3'-oligonucleotide capping group, a solid support, a linker, or a linker covalently attached to a solid support. For example, R 22 is -OCH2CH2-X'-R M' or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 23 is hydrogen, hydroxyl, protected hydroxyl, a bond to an internucleotide linkage for a subsequent nucleotide, a 3'-oligonucleotide capping group, a solid support, a linker, or a linker covalently attached to a solid support. In another non-limiting example, R 23 is -OCH2CH2-X'-R M' or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 22 is hydrogen, hydroxyl, protected hydroxyl, a bond to an internucleotide linkage for a subsequent nucleotide, a 3'-oligonucleotide capping group, a solid support, a linker, or a linker covalently attached to a solid support.

[0238] In some embodiments, R 22 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X'-R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 23 is a bond to an internucleotide linkage for a subsequent nucleotide, a linker, or a linker covalently attached to a solid support.

[0239] In some embodiments, R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X'-RM' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ), where R 22 is a bond to an internucleotide linker, a linker, or a linker covalently attached to a solid support, for a subsequent nucleotide.

[0240] R 25 In the nucleoside of formula (II), R 25 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X'-R M' ), -O(CH2) n1 -C(O)N(R N' )(R N'' ), a bond to an internucleotide linker for a preceding nucleotide, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy, optionally substituted 3- to 8-membered heterocyclyl (e.g., morpholin-1-yl, piperidin-1-yl, or pyrrolidin-1-yl), halogen, alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxalate, amino, alkylamino, dialkylamino, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), vinylphosphonate (VP) group (e.g., =CH-X P , X P is a phosphate group), C 3~6Cycloalkyl phosphonates (such as cyclopropyl phosphonate), monophosphates ((HO)2(O)P-O-5'), diphosphates ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphates ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphates (phosphorothioates, (HO)2(S)P-O-5'), monodithiophosphates (phosphorodithioates; (HO)(HS)(S)P-O-5'), phosphorothiolates ((HO)2(O)P-S-5'); alpha-thiotriphosphates; beta-thiotriphosphates; gamma-thiotriphosphates; phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), alkyl phosphonates [(R P )(OH)(O)P-O-5', R P is optionally substituted C 1~30 alkyl, such as methyl, ethyl, isopropyl, or propyl)], alkyl ether phosphonates [(R P1 )(OH)(O)P-O-5', R P1 is alkoxyalkyl, such as methoxymethyl (CH2OMe) or ethoxymethyl], (HO)2(X)P-O[-(CH2) a -O-P(X)(OH)-O] b -5' or (HO)2(X)P-O[-(CH2) a -P(X)(OH)-O] b -5' or (HO)2(X)P-[-(CH2) a -O-P(X)(OH)-O] b -5', or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimetics (such as HO[-(CH2) a -O-P(X)(OH)-O] b -5', H2N[-(CH2) a -O-P(X)(OH)-O] b -5', H[-(CH2) a -O-P(X)(OH)-O] b -5', Me2N[-(CH2) a -O-P(X)(OH)-O] b-5', HO[-(CH2) a -P(X)(OH)-O] b -5', H2N[-(CH2) a -P(X)(OH)-O] b -5', H[-(CH2) a -P(X)(OH)-O] b -5', Me2N[-(CH2) a -P(X)(OH)-O] b represents -5', where X is O or S, a and b are each independently 1 to 10, and each R 8 and R 9 are independently H, a targeting ligand (e.g., GalNac), a pharmacokinetic modifier, optionally substituted C 1~30 alkyl, optionally substituted C 1~30 alkenyl, or optionally substituted C 1~30 alkynyl.

[0241] In some embodiments of any one of the above aspects, R 25 is -O(CH2) m1 -X'-R M' For example, R 25 is -O(CH2) m1 -O-R M' In another non-limiting example, R 25 is -O(CH2) m1 -S-R M'

[0242] In some embodiments of any one of the above aspects, R 25 is -OCH2CH2-X'-R M' For example, R 25 is -OCH2CH2-O-R M' In another non-limiting example, R 25 is -OCH2CH2-S-R M'

[0243] In some embodiments of any one of the aspects described herein, R 25 is -O(CH2) n1 -C(O)N(R​​N' )(R N'' ) is. R 25 is -O(CH2) n1 -C(O)N(R N' )(R N'' ) is the case, note that n1 can be 1 or 2. Therefore, in some embodiments of any one of the aspects described herein, R 25 is -OCH2-C(O)N(R N' )(R N'' ) is. In some other embodiments of any one of the aspects described herein, R 25 is -OCH2CH2-C(O)N(R N' )(R N'' ) is.

[0244] In some nucleosides of formula (II), R 25 is a bond to the internucleotide linker to the preceding nucleotide, a hydroxyl, a protected hydroxyl, an optionally substituted C 1~30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, phosphate mimetic, or a bond to the internucleotide linker to the preceding nucleotide. For example, R 25 is a hydroxyl, an optionally substituted C 1~30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, or gamma-thiotriphosphate.

[0245] In some embodiments of any one of the aspects described herein, R 25is a bond to the internucleotidic linkage relative to the preceding nucleotide, a hydroxyl, a protected hydroxyl, an optionally substituted C 2~30 alkenyl, an optionally substituted C 1~30 alkoxy or vinyl phosphonate (VP) group.

[0246] In some embodiments of any one of the aspects described herein, R 25 is a bond to the internucleotidic linkage relative to the preceding nucleotide.

[0247] In some embodiments of any one of the aspects described herein, R 25 is a hydroxyl or a protected hydroxyl.

[0248] In some embodiments of any one of the aspects described herein, R 25 is an optionally substituted C 2~30 alkenyl or an optionally substituted C 1~30 alkoxy.

[0249] In some embodiments of any one of the aspects described herein, R 25 is a vinyl phosphonate group.

[0250] In some embodiments of any one of the aspects described herein, R 25 is not connected to the remainder of the nucleoside of formula (II) by a methylene connecting R 25 directly to the remainder of the nucleoside of formula (II).

[0251] In some embodiments of any one of the aspects described herein, R 25 is -CH(R 51 )-X 5 -R 52 wherein X 5 is absent or is a bond or O, and R 51 is hydrogen, an optionally substituted C 1~30 alkyl, an optionally substituted -C2~30 is alkenyl, or optionally substituted -C 2~30 is alkynyl, and R 52 is a bond to the internucleoside linker to the preceding nucleotide.

[0252] In some embodiments of any one of the aspects described herein, X 5 is O or a bond. For example, X 5 is O. In some other embodiments of any one of the aspects described herein, X 5 is absent, i.e., R 25 is -CH(R 51 )R 52 is.

[0253] In some embodiments of any one of the aspects described herein, R 25 is -CH(R 51 )-R 52 or -C(R 51 )=CHR 52 wherein R 51 is hydrogen, optionally substituted C 1~30 alkyl, optionally substituted -C 2~30 alkenyl, or optionally substituted -C 2~30 alkynyl, and R 52 is a bond to the internucleoside linker to the preceding nucleotide.

[0254] In some embodiments of any one of the aspects described herein, R 25 is -CH(R 51 )-X 5 -R 52 For example, R 25 is -CH(R 51 )-X 5 -R 52 wherein R 51is H or is selected independently from OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C which may be substituted with 1, 2, 3, 4 or 5 substituents independently selected from -NH2 or CH2-aryl-alkoxy 1~30 is alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6. For example, R 51 is H. In some other non-limiting examples, R 51 is C1-C 30 alkyl which may be substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy.

[0255] In some embodiments of any one of the aspects described herein, R 25 is -CH(R 51 )-O-R 52 wherein, R 51is H or OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or may be substituted with 1, 2, 3, 4 or 5 substituents independently selected from 1, 2, 3, 4 or 5 substituents independently selected from CH2-aryl-alkoxy, C1-C 30 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6. For example, R 51 is H. In some other non-limiting examples, R 51 is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, C1-C 30 alkyl.

[0256] In some embodiments of any one of the aspects described herein, R 25 is -C(R 51 )=CHR 52 Note that the double bond in -C(R 51 )=CHR 52 can be in the cis configuration or the trans configuration. Thus, in some embodiments of any one of the above aspects, R 25 is -C(R 51 )=CHR52 and in the formula, the double bond is in the cis configuration. In some other embodiments of any one of the above aspects, R 25 is -C(R 51 )=CHR 52 and in the formula, the double bond is in the trans configuration. In some embodiments of any one of the aspects described herein, R 25 is -CH=CHR 52 .

[0257] In some embodiments of any one of the aspects described herein, R 52 is a bond to the internucleoside linker to the preceding nucleotide.

[0258] In one embodiment of any one of the aspects described herein, R 25 is optionally substituted C 1~6 alkyl-R 53 , optionally substituted -C 2~6 alkenyl-R 53 , or optionally substituted -C 2~6 alkynyl-R 53 . In one embodiment of any one of the aspects described herein, R 53 is -OR 54 , -SR 55 , -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 )2, -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 )2, -SP(O)(OR 56 )2, -SP(S)(OR 56 )2, -SP(S)(SR 57 )(OR 56 ), or -SP(S)(SR 57 )2, where R 54 is hydrogen or an oxygen protecting group, R55 is a hydrogen or sulfur protecting group, and each R 56 is independently hydrogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl, or an oxygen protecting group, and each R 57 is independently hydrogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl, or a sulfur protecting group.

[0259] In some embodiments of any one of the above aspects, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), SP(O)(OR 56 )2, -SP(S)(OR 56 )2, and -SP(S)(SR 57 )(OR 56 ), at least one of the R 56 is hydrogen.

[0260] In some other embodiments of any one of the above aspects, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), SP(O)(OR 56 )2, -SP(S)(OR 56 )2, or -SP(S)(SR 57 )(OR 56 ), at least one of the R56 is not hydrogen. For example, P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 )、-OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 )、SP(O)(OR 56 )2, -SP(S)(OR 56 )2, and -SP(S)(SR 57 )(OR 56 ) at least one of the at least one R 56 is optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl, or an oxygen protecting group.

[0261] In some embodiments of any one of the above aspects, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), SP(O)(OR 56 )2, -SP(S)(OR 56 )2, and -SP(S)(SR 57 )(OR 56 ), at least one R 56 is H, and at least one R 56 is other than H.

[0262] In some embodiments of any one of the above aspects, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56)2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 )2, -SP(O)(OR 56 )2, -SP(S)(OR 56 )2, -SP(S)(SR 57 )(OR 56 ), and -SP(S)(SR 57 )2, R 56 are all H.

[0263] In some embodiments of any one of the above situations, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 )2, -SP(O)(OR 56 )2, -SP(S)(OR 56 )2, -SP(S)(SR 57 )(OR 56 ), and -SP(S)(SR 57 )2, R 56 are all other than H.

[0264] In some embodiments of any one of the above situations, -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 )2, -SP(S)(SR 57 )(OR 56 ), and -SP(S)(SR 57 )2, at least one of the R 57 is H.

[0265] In some embodiments of any one of the above situations, -P(S)(SR 57 )(OR 56 )、-P(S)(SR 57 )2、-OP(S)(OR 56 )2、-OP(S)(SR 57 )(OR 56 )、-OP(S)(SR 57 )2、-SP(S)(SR 57 )(OR 56 )、and -SP(S)(SR 57 )2, at least one of the R 57 is other than H. For example, -P(S)(SR 57 )(OR 56 )、-P(S)(SR 57 )2、-OP(S)(OR 56 )2、-OP(S)(SR 57 )(OR 56 )、-OP(S)(SR 57 )2、-SP(S)(SR 57 )(OR 56 )、and -SP(S)(SR 57 )2, at least one of the R 57 is optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl, or a sulfur protecting group.

[0266] In some embodiments of any one of the above situations, in -P(S)(SR 57 )2、-OP(S)(SR 57 )2 and -SP(S)(SR 57 )2, at least one of the R 57 is H, and at least one of the R 57 is other than H.

[0267] In some embodiments, -P(S)(SR 57 )(OR 56 )、-P(S)(SR 57 )2、-OP(S)(OR 56 )2、-OP(S)(SR57 )(OR 56 )、 -OP(S)(SR 57 )2、 -SP(S)(SR 57 )(OR 56 )、 and -SP(S)(SR 57 )2, where R 57 is all H.

[0268] In some embodiments, -P(S)(SR 57 )(OR 56 )、 -P(S)(SR 57 )2、 -OP(S)(OR 56 )2、 -OP(S)(SR 57 )(OR 56 )、 -OP(S)(SR 57 )2、 -SP(S)(SR 57 )(OR 56 )、 and -SP(S)(SR 57 )2, where R 57 is all other than H.

[0269] In some embodiments of any one of the aspects described herein, R 25 may be optionally substituted -C 2~6 alkenyl-R 53 . For example, R 25 is -C 2~6 alkenyl-R 53 , where C 2~6The alkenyl is OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p It may be substituted with 1, 2, 3, 4 or 5 substituents independently selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6, and R 53 is -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 )、-P(S)(SR 57 )2, -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 )、-OP(S)(SR 57 )2, -SP(O)(OR 56 )2, -SP(S)(OR 56 )2, -SP(S)(SR 57 )(OR 56 )、or -SP(S)(SR 57 )2.

[0270] In some embodiments of any one of the above situations, R 25 is -CH=CHR53 is optionally substituted -C 2~6 alkenyl -R 53 Note that the double bond in can be in the cis or trans configuration. Thus, in some embodiments of any one of the above aspects, R 25 is -CH=CHR 53 wherein the double bond is in the cis configuration. In some other embodiments of any one of the above aspects, R 25 is -CH=CHR 53 wherein the double bond is in the trans configuration.

[0271] In some embodiments of any one of the above aspects, R 25 is -CH=CH-P(O)(OR 56 )2, -CH=CH-P(S)(OR 56 )2, -CH=CH-P(S)(SR 57 )(OR 56 )、-CH=CH-P(S)(SR 57 )2、-CH=CH-OP(O)(OR 56 )2、-CH=CH-OP(S)(OR 56 )2、-CH=CH-OP(S)(SR 57 )(OR 56 )、-CH=CH-OP(S)(SR 57 )2、-CH=CH-SP(O)(OR 56 )2、-CH=CH-SP(S)(OR 56 )2、-CH=CH-SP(S)(SR 57 )(OR 56 )、or -CH=CH-SP(S)(SR 57 )2. For example, R 25 is -CH=CH-P(O)(OR 56 )2.

[0272] In some embodiments of any one of the above aspects, R 54 is hydrogen or an oxygen protecting group. For example, R 54 is hydrogen or 4,4'-dimethoxytrityl (DMT). In some preferred embodiments, R 54 is H.

[0273] In some embodiments of any one of the aspects described herein, R 25 is optionally substituted -C 1~6 alkenyl-R 53 For example, R 25 is -C 1~6 alkenyl-R 53 wherein C 1~6 alkenyl is OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-aryl-alkoxy, and is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected therefrom, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6, and R 53 is -OR 54 -SR 55 -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 )2, -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56)、 -OP(S)(SR 57 )2、 -SP(O)(OR 56 )2、 -SP(S)(OR 56 )2、 -SP(S)(SR 57 )(OR 56 )、 or -SP(S)(SR 57 )2.

[0274] In some embodiments of any one of the aspects described in this specification, R 25 can be -CH(R 58 )-R 53 , where R 53 is -OR 54 , -SR 55 , -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 )2, -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 )2, -SP(O)(OR 56 )2, -SP(S)(OR 56 )2, -SP(S)(SR 57 )(OR 56 ), or -SP(S)(SR 57 )2, and R 58 is H, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl.

[0275] In some embodiments of any one of the aspects described in this specification, R 58is H or OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-aryl-alkoxy and may be substituted with 1, 2, 3, 4 or 5 substituents independently selected from C1-C 30 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6. In a non-limiting example, R 58 is H. In some other non-limiting examples, R 58 is C1-C 30 alkyl which may be substituted with a substituent selected from NH2, OH, C(O)NH2, COOH, halo, SH, and C1-C6 alkoxy.

[0276] In some embodiments of any one of the aspects described herein, R 25 is -CH(R 58 )-O-R 59 where R 59 is H, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 )2, -OP(O)(OR 56) It is 2. For example, R 25 is -CH(R 58 )-O-R 59 , where R 58 is H or optionally substituted C1-C 30 alkyl, and R 59 is H or -P(O)(OR 56 )2.

[0277] In some embodiments of any one of the aspects described herein, R 25 is -CH(R 58 )-S-R 60 , where R 60 is H, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 )、-P(S)(SR 57 )2、-OP(O)(OR 56 )2.

[0278] In the nucleoside of formula (II), among R 22 and R 23 , there is at most one bond to the internucleotide linkage for the subsequent nucleotide. It should be noted that when neither R 22 nor R 23 is a bond to the internucleotide linkage, R 25 is a bond to the internucleotide linkage for the preceding nucleotide.

[0279] In some embodiments, one of R 22 and R 23 is -O(CH2) m1 -X M' -R M' (for example, -OCH2CH2-X'-R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ), and R 22 and R 23The other of which is hydrogen, hydroxyl, protected hydroxyl, a bond to an internucleotide linker for a subsequent nucleotide, a 3'-oligonucleotide capping group, a solid support, a linker, or a linker covalently attached to a solid support, R 25 is hydroxyl, protected hydroxyl, a bond to an internucleotide linker for a preceding nucleotide or a vinyl phosphate group. For example, R 22 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X'-R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 23 is hydrogen, hydroxyl, protected hydroxyl, a bond to an internucleotide linker for a subsequent nucleotide, a 3'-oligonucleotide capping group, a solid support, a linker, or a linker covalently attached to a solid support, R 25 is hydroxyl, protected hydroxyl, a bond to an internucleotide linker for a preceding nucleotide or a vinyl phosphate group. In another non-limiting example, R 23 is -OCH2CH2-X'-R M' or -O(CH2) n1 -C(O)N(R N' )(R N'' ) and R 22 is hydrogen, hydroxyl, protected hydroxyl, a bond to an internucleotide linker for a subsequent nucleotide, a 3'-oligonucleotide capping group, a solid support, a linker, or a linker covalently attached to a solid support, R 25 is hydroxyl, protected hydroxyl, a bond to an internucleotide linker for a preceding nucleotide or a vinyl phosphate group.

[0280] In some embodiments, R 22 is -O(CH2) m1 -X M' -R M'(e.g., -OCH2CH2-X'-R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ), where R 23 is a bond to a nucleotide linker for a subsequent nucleotide, a linker, or a linker covalently attached to a solid support, and R 25 is a hydroxyl, a protected hydroxyl, a bond to a nucleotide linker for a preceding nucleotide, or a vinyl phosphate group.

[0281] In some embodiments, R 23 is -O(CH2) m1 -X M' -R M' (e.g., -OCH2CH2-X'-R M' ) or -O(CH2) n1 -C(O)N(R N' )(R N'' ), where R 22 is a bond to a nucleotide linker for a subsequent nucleotide, a linker, or a linker covalently attached to a solid support, and R 25 is a hydroxyl, a protected hydroxyl, a bond to a nucleotide linker for a preceding nucleotide, or a vinyl phosphate group.

[0282] R 22' In the nucleoside of formula (II'), R 22' is -O(CH2) n1 -C(O)OR LV , hydrogen, hydroxyl, a protected hydroxyl, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, protected aminoalkyl, 5- to 8-membered heterocyclyl, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), -O-N-methylacetamide, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), a bond to a nucleotide internucleoside linkage for a subsequent nucleotide, a 3'-oligonucleotide capping group, a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support.

[0283] In some embodiments of any one of the aspects described herein, R 22' is -O(CH2) n1 -C(O)OR LV . Note that when R 22' is -O(CH2) n1 -C(O)OR LV , n1 can be 1 or 2. Thus, in some embodiments of any one of the aspects described herein, R 22' is -OCH2-C(O)OR LV . In some other embodiments of any one of the aspects described herein, R 22' is -OCH2CH2-C(O)OR LV .

[0284] In some embodiments, R 22' is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), amino, alkylamino, dialkylamino, protected aminoalkyl, -O-C 4~30 alkyl-ON(CH2R8)(CH2R9), -O-C 4~30 alkyl-ON(CH2R8)(CH2R9), -O-N-methylacetamide, alkoxyoxalate, solid support, linker, or a linker covalently attached to a solid support. For example, R 22' is hydrogen, hydroxyl, halogen, protected hydroxyl, optionally substituted C1-30 alkyl, optionally substituted C2-30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxalate, amino, alkylamino, dialkylamino, protected aminoalkyl, -O-N-methylacetamide, -O-C 4~30 alkyl-ON(CH2R8)(CH2R9), or -O-C 4~30 alkyl-ON(CH2R8)(CH2R9).

[0285] In some embodiments of any one of the aspects described herein, R 22' is a bond to a nucleotide internucleoside linker, a linker, or a linker covalently attached to a solid support. For example, R 22' is a bond to a nucleotide internucleoside linker for a subsequent nucleotide.

[0286] R 23' In the nucleoside of formula (II'), R 23' is -O(CH2) n1 -C(O)OR LV , hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30Alkenyl, optionally substituted C 2~30 Alkynyl, optionally substituted C 1~30 Alkoxy (e.g., methoxy, 2 - methoxyethoxy), alkoxyalkyl (e.g., 2 - methoxyethyl), alkoxyalkylamine, alkoxyoxocarboxylate, amino, alkylamino, dialkylamino, protected aminoalkyl, 5 - to 8 - membered heterocyclyl, -O - C 4~30 alkyl - ON(CH2R 8 )(CH2R 9 ), -O - N - methylacetamide, -O - C 4~30 alkyl - ON(CH2R 8 )(CH2R 9 ), a bond to a nucleotide - to - nucleotide linker for a subsequent nucleotide, a 3'-oligonucleotide capping group, a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support.

[0287] In some embodiments of any one of the aspects described herein, R 23' is -O(CH2) n1 -C(O)OR LV . Note that when R 23' is -O(CH2) n1 -C(O)OR LV , n1 can be 1 or 2. Thus, in some embodiments of any one of the aspects described herein, R 23' is -OCH2 - C(O)OR LV . In some other embodiments of any one of the aspects described herein, R 23' is -OCH2CH2 - C(O)OR LV .

[0288] In some embodiments, R 23' is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30Alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy, 2 - methoxyethoxy), alkoxyalkyl (e.g., 2 - methoxyethyl), amino, alkylamino, dialkylamino, protected aminoalkyl, -O - C 4~30 alkyl - ON(CH2R8)(CH2R9), -O - C 4~30 alkyl - ON(CH2R8)(CH2R9), -O - N - methylacetamide, alkoxyoxalate, solid support, linker, or a linker covalently attached to a solid support. For example, R 23' is hydrogen, hydroxyl, halogen, protected hydroxyl, optionally substituted C1 - 30 alkyl, optionally substituted C2 - 30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxalate, amino, alkylamino, dialkylamino, protected aminoalkyl, -O - N - methylacetamide, -O - C 4~30 alkyl - ON(CH2R8)(CH2R9), or -O - C 4~30 alkyl - ON(CH2R8)(CH2R9).

[0289] In some embodiments of any one of the aspects described herein, R 23' is a bond to a nucleotide - nucleotide linker for a subsequent nucleotide, a linker, or a linker covalently attached to a solid support. For example, R 23' is a bond to a nucleotide - nucleotide linker for a subsequent nucleotide.

[0290] In some embodiments, one of R 22' and R 23' is -O(CH2) n1 -C(O)OR LV where R 22' and R 23'The other of which is hydrogen, hydroxyl, protected hydroxyl, a linkage to an internucleotide linker for a subsequent nucleotide, a 3'-oligonucleotide capping group, a solid support, a linker, or a linker covalently attached to a solid support. For example, R 22' is -O(CH2) n1 -C(O)OR LV where R 23' is hydrogen, hydroxyl, protected hydroxyl, a linkage to an internucleotide linker for a subsequent nucleotide, a 3'-oligonucleotide capping group, a solid support, a linker, or a linker covalently attached to a solid support. In another non-limiting example, R 23' is -O(CH2) n1 -C(O)OR LV where R 22' is hydrogen, hydroxyl, protected hydroxyl, a linkage to an internucleotide linker for a subsequent nucleotide, a 3'-oligonucleotide capping group, a solid support, a linker, or a linker covalently attached to a solid support.

[0291] In some embodiments, R 22' is -O(CH2) n1 -C(O)OR LV where R 23' is a linkage to an internucleotide linker for a subsequent nucleotide, a linker, or a linker covalently attached to a solid support.

[0292] In some embodiments, R 23' is -O(CH2) n1 -C(O)OR LV where R 22' is a linkage to an internucleotide linker for a subsequent nucleotide, a linker, or a linker covalently attached to a solid support.

[0293] R 25' In the nucleoside of formula (II'), R 25' is -OCH2CH2-X'-R M' -O(CH2) n1-C(O)N(R N' )(R N'' ), a bond to an internucleotide linker relative to a preceding nucleotide, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, optionally substituted C 2~30 alkynyl, optionally substituted C 1~30 alkoxy, optionally substituted 3- to 8-membered heterocyclyl (e.g., morpholin-1-yl, piperidin-1-yl, or pyrrolidin-1-yl), halogen, alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), -O-C 4~30 alkyl-ON(CH2R 8 )(CH2R 9 ), vinylphosphonate (VP) group (e.g., =CH-X P , X P is a phosphate group), C 3~6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), alkylphosphonate [(R P )(OH)(O)P-O-5', R P is optionally substituted C 1~30(alkyl, such as methyl, ethyl, isopropyl, or propyl), alkyl ether phosphonate [(R P1 )(OH)(O)P-O-5', R P1 is alkoxyalkyl, such as methoxymethyl (CH2OMe) or ethoxymethyl], (HO)2(X)P-O[-(CH2) a -O-P(X)(OH)-O] b -5' or (HO)2(X)P-O[-(CH2) a -P(X)(OH)-O] b -5' or (HO)2(X)P-[-(CH2) a -O-P(X)(OH)-O] b -5', or optionally substituted alkyl, and dialkyl terminal phosphate and phosphate mimics (e.g., HO[-(CH2) a -O-P(X)(OH)-O] b -5', H2N[-(CH2) a -O-P(X)(OH)-O] b -5', H[-(CH2) a -O-P(X)(OH)-O] b -5', Me2N[-(CH2) a -O-P(X)(OH)-O] b -5', HO[-(CH2) a -P(X)(OH)-O] b -5', H2N[-(CH2) a -P(X)(OH)-O] b -5', H[-(CH2) a -P(X)(OH)-O] b -5', Me2N[-(CH2) a -P(X)(OH)-O] b -5' represents, where X is O or S, and a and b are each independently 1 to 10, and each R 8 and R 9 are independently H, a targeting ligand (e.g., GalNac), a pharmacokinetic modifier, optionally substituted C 1~30 alkyl, optionally substituted C 1~30 alkenyl, or optionally substituted C 1~30It is alkynyl.

[0294] In some embodiments of any one of the aspects described herein, R 25' is -O(CH2) n1 -C(O)OR LV is. R 25' is -O(CH2) n1 -C(O)OR LV is. Note that when it is, n1 can be 1 or 2. Thus, in some embodiments of any one of the aspects described herein, R 25' is -OCH2-C(O)OR LV is. In some other embodiments of any one of the aspects described herein, R 25' is -OCH2CH2-C(O)OR LV is.

[0295] In some nucleosides of formula (II'), R 25' is a bond to the internucleotide linker to the preceding nucleotide, hydroxyl, protected hydroxyl, optionally substituted C 1~30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, phosphate mimetic, or a bond to the internucleotide linker to the preceding nucleotide. For example, R 25' is hydroxyl, optionally substituted C 1~30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, or gamma-thiotriphosphate.

[0296] In some embodiments of any one of the aspects described herein, R 25' is a linkage to a nucleotide - nucleotide linker relative to the preceding nucleotide, a hydroxyl, a protected hydroxyl, an optionally substituted C 2~30 alkenyl, an optionally substituted C 1~30 alkoxy or a vinyl phosphonate (VP) group.

[0297] In some embodiments of any one of the aspects described herein, R 25' is a linkage to a nucleotide - nucleotide linker relative to the preceding nucleotide.

[0298] In some embodiments of any one of the aspects described herein, R 25' is a hydroxyl or a protected hydroxyl.

[0299] In some embodiments of any one of the aspects described herein, R 25' is an optionally substituted C 2~30 alkenyl or an optionally substituted C 1~30 alkoxy.

[0300] In some embodiments of any one of the aspects described herein, R 25' is a vinyl phosphonate group.

[0301] In some embodiments of any one of the aspects described herein, R 25' there is no methylene connecting R to the remainder of the nucleoside of formula (II'), and R 25' is directly connected to the remainder of the nucleoside of formula (II').

[0302] In some embodiments of any one of the aspects described herein, R 25' is - CH(R 51 ) - X 5 - R 52 wherein X 5 is absent or is a bond or O, and R 51is hydrogen, optionally substituted C 1~30 alkyl, optionally substituted -C 2~30 alkenyl, or optionally substituted -C 2~30 alkynyl, and R 52 is a bond to the internucleoside linker to the preceding nucleotide.

[0303] In some embodiments of any one of the aspects described herein, X 5 is O or a bond. For example, X 5 is O. In some other embodiments of any one of the aspects described herein, X 5 is absent, i.e., R 25' is -CH(R 51 )R 52 .

[0304] In some embodiments of any one of the aspects described herein, R 25' is -CH(R 51 )-R 52 or -C(R 51 )=CHR 52 , where R 51 is hydrogen, optionally substituted C 1~30 alkyl, optionally substituted -C 2~30 alkenyl, or optionally substituted -C 2~30 alkynyl, and R 52 is a bond to the internucleoside linker to the preceding nucleotide.

[0305] In some embodiments of any one of the aspects described herein, R 25' is -CH(R 51 )-X 5 -R 52 . For example, R 25' is -CH(R 51 )-X 5 -R 52 , where R 51is H, or OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p is C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from -NH2 or CH2-aryl-alkoxy 1~30 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6. For example, R 51 is H. In some other non-limiting examples, R 51 is C1-C optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy 30 alkyl.

[0306] In some embodiments of any one of the aspects described herein, R 25' is -CH(R 51 )-O-R 52 wherein, R 51is H, or OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-aryl-alkoxy, and is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 1, 2, 3, 4 or 5 substituents independently selected from 30 C1-C alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6. For example, R 51 is H. In some other non-limiting examples, R 51 is C1-C alkyl optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy. 30

[0307] In some embodiments of any one of the aspects described herein, R 25' is -C(R 51 )=CHR 52 . Note that the double bond in -C(R 51 )=CHR 52 can be in the cis configuration or the trans configuration. Thus, in some embodiments of any one of the above aspects, R 25' is -C(R 51 )=CHR​52 and in the formula, the double bond is in the cis configuration. In some other embodiments of any one of the above aspects, R 25' is -C(R 51 )=CHR 52 and in the formula, the double bond is in the trans configuration. In some embodiments of any one of the aspects described herein, R 25' is -CH=CHR 52 .

[0308] In some embodiments of any one of the aspects described herein, R 52 is a bond to the internucleoside linker to the preceding nucleotide.

[0309] In one embodiment of any one of the aspects described herein, R 25' is optionally substituted -C 1~6 alkyl-R 53 , optionally substituted -C 2~6 alkenyl-R 53 , or optionally substituted -C 2~6 alkynyl-R 53 . In one embodiment of any one of the aspects described herein, R 53 is -OR 54 , -SR 55 , -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 )2, -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 )2, -SP(O)(OR 56 )2, -SP(S)(OR 56 )2, -SP(S)(SR 57 )(OR 56 ), or -SP(S)(SR 57 )2, where R 54 is hydrogen or an oxygen protecting group, R55 is a hydrogen or sulfur protecting group, and each R 56 is independently hydrogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl, or an oxygen protecting group, and each R 57 is independently hydrogen, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl, or a sulfur protecting group.

[0310] In some embodiments of any one of the above aspects, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), SP(O)(OR 56 )2, -SP(S)(OR 56 )2, and at least one R in -SP(S)(SR 57 )(OR 56 ) 56 is hydrogen.

[0311] In some other embodiments of any one of the above aspects, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), SP(O)(OR 56 )2, -SP(S)(OR 56 )2, or at least one R in -SP(S)(SR 57 )(OR 56 )56 is not hydrogen. For example, P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 )、-OP(O)(OR 56 )2、-OP(S)(OR 56 )2、-OP(S)(SR 57 )(OR 56 )、SP(O)(OR 56 )2、-SP(S)(OR 56 )2、and -SP(S)(SR 57 )(OR 56 ) at least one of the at least one R 56 is optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl, or an oxygen protecting group.

[0312] In some embodiments of any one of the above aspects, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), SP(O)(OR 56 )2, -SP(S)(OR 56 )2, and -SP(S)(SR 57 )(OR 56 ), at least one R 56 is H, and at least one R 56 is other than H.

[0313] In some embodiments of any one of the above aspects, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56)2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 )2, -SP(O)(OR 56 )2, -SP(S)(OR 56 )2, -SP(S)(SR 57 )(OR 56 ), and -SP(S)(SR 57 )2, where R 56 are all H.

[0314] In some embodiments of any one of the above situations, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 )2, -SP(O)(OR 56 )2, -SP(S)(OR 56 )2, -SP(S)(SR 57 )(OR 56 ), and -SP(S)(SR 57 )2, where R 56 are all other than H.

[0315] In some embodiments of any one of the above situations, -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 )2, -SP(S)(SR 57 )(OR 56 ), and -SP(S)(SR 57 )2, at least one of the R 57 is H.

[0316] In some embodiments of any one of the above situations, -P(S)(SR 57 )(OR 56 )、-P(S)(SR 57 )2、-OP(S)(OR 56 )2、-OP(S)(SR 57 )(OR 56 )、-OP(S)(SR 57 )2、-SP(S)(SR 57 )(OR 56 )、and -SP(S)(SR 57 )2, at least one R 57 is other than H. For example, -P(S)(SR 57 )(OR 56 )、-P(S)(SR 57 )2、-OP(S)(OR 56 )2、-OP(S)(SR 57 )(OR 56 )、-OP(S)(SR 57 )2、-SP(S)(SR 57 )(OR 56 )、and -SP(S)(SR 57 )2, at least one R 57 is optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl, or a sulfur protecting group.

[0317] In some embodiments of any one of the above situations, in -P(S)(SR 57 )2、-OP(S)(SR 57 )2 and -SP(S)(SR 57 )2, at least one R 57 is H, and at least one R 57 is other than H.

[0318] In some embodiments, -P(S)(SR 57 )(OR 56 )、-P(S)(SR 57 )2、-OP(S)(OR 56 )2、-OP(S)(SR57 )(OR 56 )、 -OP(S)(SR 57 )2、 -SP(S)(SR 57 )(OR 56 )、 and -SP(S)(SR 57 )2, where R 57 is all H.

[0319] In some embodiments, -P(S)(SR 57 )(OR 56 )、 -P(S)(SR 57 )2、 -OP(S)(OR 56 )2、 -OP(S)(SR 57 )(OR 56 )、 -OP(S)(SR 57 )2、 -SP(S)(SR 57 )(OR 56 )、 and -SP(S)(SR 57 )2, where R 57 is all other than H.

[0320] In some embodiments of any one of the aspects described herein, R 25' may be a substituted -C 2~6 alkenyl-R 53 . For example, R 25' is -C 2~6 alkenyl-R 53 , where C 2~6Alkenyl is OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p It may be substituted with 1, 2, 3, 4 or 5 substituents independently selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6, and R 53 is -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 )、-P(S)(SR 57 )2, -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 )、-OP(S)(SR 57 )2, -SP(O)(OR 56 )2, -SP(S)(OR 56 )2, -SP(S)(SR 57 )(OR 56 )、or -SP(S)(SR 57 )2.

[0321] In some embodiments of any one of the above situations, R 25' is -CH=CHR53 which may be substituted -C 2~6 alkenyl -R 53 Note that the double bond in can be in the cis configuration or the trans configuration. Thus, in some embodiments of any one of the above aspects, R 25' is -CH=CHR 53 wherein the double bond is in the cis configuration. In some other embodiments of any one of the above aspects, R 25' is -CH=CHR 53 wherein the double bond is in the trans configuration.

[0322] In some embodiments of any one of the above aspects, R 25' is -CH=CH-P(O)(OR 56 )2, -CH=CH-P(S)(OR 56 )2, -CH=CH-P(S)(SR 57 )(OR 56 ), -CH=CH-P(S)(SR 57 )2, -CH=CH-OP(O)(OR 56 )2, -CH=CH-OP(S)(OR 56 )2, -CH=CH-OP(S)(SR 57 )(OR 56 ), -CH=CH-OP(S)(SR 57 )2, -CH=CH-SP(O)(OR 56 )2, -CH=CH-SP(S)(OR 56 )2, -CH=CH-SP(S)(SR 57 )(OR 56 ), or -CH=CH-SP(S)(SR 57 )2. For example, R 25' is -CH=CH-P(O)(OR 56 )2.

[0323] In some embodiments of any one of the above aspects, R 54 is hydrogen or an oxygen protecting group. For example, R 54 is hydrogen or 4,4'-dimethoxytrityl (DMT). In some preferred embodiments, R 54 is H.

[0324] In some embodiments of any one of the aspects described herein, R 25' is optionally substituted -C 1~6 alkenyl-R 53 For example, R 25' is -C 1~6 alkenyl-R 53 wherein C 1~6 alkenyl is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6, and R 53 is -OR 54 -SR 55 -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 )2, -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56)、 -OP(S)(SR 57 )2、 -SP(O)(OR 56 )2、 -SP(S)(OR 56 )2、 -SP(S)(SR 57 )(OR 56 )、 or -SP(S)(SR 57 )2.

[0325] In some embodiments of any one of the aspects described herein, R 25' can be -CH(R 58 )-R 53 , where R 53 is -OR 54 , -SR 55 , -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 )2, -OP(O)(OR 56 )2, -OP(S)(OR 56 )2, -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 )2, -SP(O)(OR 56 )2, -SP(S)(OR 56 )2, -SP(S)(SR 57 )(OR 56 ), or -SP(S)(SR 57 )2, and R 58 is H, optionally substituted C 1~30 alkyl, optionally substituted C 2~30 alkenyl, or optionally substituted C 2~30 alkynyl.

[0326] In some embodiments of any one of the aspects described herein, R 58is H, or OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-aryl-alkoxy, and is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 30 C1-C 58 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6. In a non-limiting example, R 58 is H. In some other non-limiting examples, R 30 is C1-C

[0327] In some embodiments of any one of the aspects described herein, R 25' is -CH(R 58 )-O-R 59 where R 59 is H, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 )2, -OP(O)(OR 56) It is 2. For example, R 25' is -CH(R 58 )-O-R 59 where R 58 is H or optionally substituted C1-C 30 alkyl, and R 59 is H or -P(O)(OR 56 )2.

[0328] In some embodiments of any one of the aspects described herein, R 25' is -CH(R 58 )-S-R 60 where R 60 is H, -P(O)(OR 56 )2, -P(S)(OR 56 )2, -P(S)(SR 57 )(OR 56 ) -P(S)(SR 57 )2, -OP(O)(OR 56 )2.

[0329] In the nucleoside of formula (II'), at most one of R 22' and R 23' is a bond to the internucleotide linker for the subsequent nucleotide. It should be noted that when neither R 22' nor R 23' is a bond to the internucleotide linker, R 25' is a bond to the internucleotide linker for the preceding nucleotide.

[0330] In some embodiments, one of R 22' and R 23' is -O(CH2) n1 -C(O)OR LV and the other of R 22' and R 23' is hydrogen, hydroxyl, protected hydroxyl, a bond to the internucleotide linker for the subsequent nucleotide, a 3'-oligonucleotide capping group, a solid support, a linker, or a linker covalently attached to a solid support, and R 25'is a hydroxyl, protected hydroxyl, a bond to an internucleotide linkage to a preceding nucleotide or a vinyl phosphate group.

[0331] For example, R 22' is -O(CH2) n1 -C(O)OR LV wherein R 23' is hydrogen, hydroxyl, protected hydroxyl, a bond to an internucleotide linkage to a subsequent nucleotide, a 3'-oligonucleotide capping group, a solid support, a linker, or a linker covalently attached to a solid support, and R 25' is a hydroxyl, protected hydroxyl, a bond to an internucleotide linkage to a preceding nucleotide or a vinyl phosphate group. In another non-limiting example, R 23' is -O(CH2) n1 -C(O)OR LV wherein R 22' is hydrogen, hydroxyl, protected hydroxyl, a bond to an internucleotide linkage to a subsequent nucleotide, a 3'-oligonucleotide capping group, a solid support, a linker, or a linker covalently attached to a solid support, and R 25' is a hydroxyl, protected hydroxyl, a bond to an internucleotide linkage to a preceding nucleotide or a vinyl phosphate group.

[0332] In some embodiments, R 22' is -O(CH2) n1 -C(O)OR LV wherein R 23' is a bond to an internucleotide linkage to a subsequent nucleotide, a linker, or a linker covalently attached to a solid support, and R 25' is a hydroxyl, protected hydroxyl, a bond to an internucleotide linkage to a preceding nucleotide or a vinyl phosphate group.

[0333] In some embodiments, R 23' is -O(CH2) n1 -C(O)ORLV and R 22' is a bond to a internucleotide linkage to a subsequent nucleotide, a linker, or a linker covalently attached to a solid support, and R 25' is a hydroxyl, a protected hydroxyl, a bond to an internucleotide linkage to a preceding nucleotide or a vinyl phosphate group.

[0334] R 4 One aspect of any one of the aspects described herein is R 4 including. In any one of the aspects described herein, R 4 is R MA , hydrogen, optionally substituted C 1~6 alkyl, optionally substituted C 2~6 alkenyl, optionally substituted C 2~6 alkynyl, or optionally substituted C 1~6 alkoxy. For example, R 4 is R MA , hydrogen, optionally substituted C 1~6 alkyl or optionally substituted C 1~6 alkoxy.

[0335] In some aspects of any one of the aspects described herein, R 4 is H.

[0336] In some aspects of any one of the aspects described herein, R $ is -O(CH2) m1 -X M' -R M' For example, R 4 is -OCH2CH2-X M' -R M' .

[0337] R L In one aspect of any one of the aspects described herein, each R Lcan be independently selected from the group consisting of H, carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, detectable labels, diagnostic agents (e.g., bitoin), fluorescent dyes, polyethylene glycol (PEG), antibodies, antibody fragments (e.g., nanobodies).

[0338] In some embodiments of any one of the aspects described herein, R L is a ligand.

[0339] When two or more Rs L are present, note that they can be the same or different. Thus, in some embodiments of any one of the aspects described herein, all Rs L are the same. In some other embodiments of any one of the aspects described herein, Rs L are different.

[0340] Ligand One aspect of any one of the aspects described herein includes a ligand. Exemplary ligands include, but are not limited to, carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, detectable labels, diagnostic agents (e.g., bitoin), fluorescent dyes, polyethylene glycol (PEG), antibodies, antibody fragments (e.g., nanobodies).

[0341] Although not wishing to be bound by theory, a ligand modifies one or more properties of an attached molecule (such as an oligonucleotide described herein), such as, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, intracellular distribution, cellular uptake, charge, and clearance. Ligands are routinely used in the chemical arts and are attached directly to the parent compound or via an optional linker or linking group. Preferred lists of ligands include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocolesterol, cholate moieties, folic acid, lipids, phospholipids, biotin, phenazines, phenanthridines, anthraquinones, adamantanes, acridines, fluoresceins, rhodamines, coumarins, and dyes.

[0342] Preferred ligands applicable to the present invention include a lipid moiety, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), thioether, such as hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306, Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), thiocolesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphatic chain, such as dodecanediol or undecyl residue (Saison-Behmoaras et al., EMBO J., 1991, 10, 111, Kabanov et al., FEBS Lett., 1990, 259, 327, Svinarchuk et al., Biochimie, 1993, 75, 49), phospholipid, such as di-hexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651, Shea et al., Nucl. Acids Res., 1990, 18, 3777), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or octadecylamine or carbonyloxy cholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0343] The ligand can include natural molecules, recombinant molecules, or synthetic molecules. Exemplary ligands include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphazene, polyethyleneimine, cationic groups, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyamino acid, transferrin, bisphosphonate, polyglutamate, polyaspartate, aptamer, asialofetuin, hyaluronan, procollagen, immunoglobulin (e.g., antibody), insulin, transferrin, albumin, sugar-albumin conjugate, intercalator (e.g., acridines), crosslinker (e.g., psoralen, mitomycin C), porphyrin (e.g., TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbon (e.g., phenazine, dihydrophenazine), artificial endonuclease (e.g., EDTA), lipophilic molecule (e.g., steroid, bile acid, cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithocholic acid, O3-(oleoyl) cholenic acid, dimethoxytrityl, or phenoxazine), peptide (e.g., alpha helix peptide, amphiphilic peptide, RGD peptide, cell-penetrating peptide, endosome-lytic / membrane-fusing peptide), alkylating agent, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled marker, enzyme, hapten (e.g., biotin), transport / absorption promoter (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonuclease (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu3+ complex of tetraaza macrocyclic compound), dinitrophenyl, HRP, AP, antibody, hormone and hormone receptor, lectin, carbohydrate, polyvalent carbohydrate, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamin cofactor, lipopolysaccharide, activator of p38 MAP kinase, activator of NF-κB, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor alpha (TNF alpha), interleukin-1 beta, gamma interferon, natural or recombinant low density lipoprotein (LDL), natural or recombinant high density lipoprotein (HDL), and cell permeant (e.g., a. helical cell-permeation agent), but are not limited thereto.,

[0344] Peptides and peptidomimetic ligands include natural peptides or modified peptides, such as D or L peptides; α, β or γ peptides; N-methyl peptides; azapeptides; peptides in which one or more amide linkages (i.e., peptide linkages) are replaced by one or more urea, thiourea, carbamate, or sulfonylurea linkages; or those having cyclic peptides. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule that can fold into a defined three-dimensional structure similar to that of a natural peptide. The peptide or peptidomimetic ligand can be about 5 to 50 amino acids in length, for example about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in length.

[0345] Exemplary amphipathic peptides include, but are not limited to, cecropin, ricin toxin, paradaxin, buforin, CPF, bomvinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, HFIAP, magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, esculentinis-1, and caerin.

[0346] As used herein, the term "endosomolytic ligand" refers to a molecule having endosomolytic properties. An endosomolytic ligand promotes the lysis of cellular compartments such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other vesicular bodies within the cell, and / or the transport of the compositions of the invention or components thereof from there to the cytoplasm of the cell. Exemplary endosomolytic ligands include, but are not limited to, imidazole, poly- or oligoimidazole, linear or branched polyethyleneimine (PEI), linear and branched polyamines such as spermine, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers having masked or unmasked cationic or anionic charges, dendrimers having masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic membrane-fusogenic lipids, natural and synthetic cationic lipids.

[0347] Exemplary endosomolytic / membrane-fusogenic peptides include, but are not limited to, TIFF2025522811000008.tif103160.

[0348] Although not wishing to be bound by theory, membrane-fusogenic lipids fuse with membranes and as a result, destabilize the membranes. Membrane-fusogenic lipids typically have a small head and unsaturated acyl chains. Exemplary membrane-fusogenic lipids include, but are not limited to, 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA) and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (also referred to herein as XTC).

[0349] Synthetic polymers having endosomal lysis activity applicable to the present invention are described in U.S. Patent Application Publication Nos. 2009 / 0048410, 2009 / 0023890, 2008 / 0287630, 2008 / 0287628, 2008 / 0281044, 2008 / 0281041, 2008 / 0269450, 2007 / 0105804, 20070036865 and 2004 / 0198687, the contents of which are hereby incorporated by reference in their entirety.

[0350] Exemplary cell-penetrating peptides include TIFF2025522811000009.tif81159, but are not limited thereto.

[0351] Exemplary cationic groups include protonated amino groups, such as O-amines (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamine); aminoalkoxy, such as O(CH2) n amines (such as amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamine), amino (such as NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid), and NH(CH2CH2NH) n Those derived from CH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino) are included, but are not limited thereto.

[0352] As used herein, the term "targeting ligand" refers to any molecule that confers enhanced affinity for a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, such as a compartment of a cell, tissue or organ. Exemplary targeting ligands include antibodies, antigens, folic acid, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands, but are not limited thereto.

[0353] Examples of carbohydrate-based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc such as GalNAc2 and GalNAc3, D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, multivalent fucose, glycosylated polyamino acids, and lectins. The term multivalent indicates the presence of two or more monosaccharide units. Such monosaccharide subunits can be linked to each other by glycosidic bonds or to a scaffold molecule.

[0354] Folic acid and folic acid analogs applicable to the present invention are described in part in U.S. Patent Nos. 2,816,110, 5,552,545, 6,335,434, and 7,128,893, the entire contents of which are incorporated herein by reference.

[0355] As used herein, the terms "PK modulating ligand" and "PK modulating substance" refer to molecules that can modulate the pharmacokinetics of the oligonucleotides described herein. Exemplary PK modulating substances include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogs, peptides, protein binding agents, vitamins, fatty acids, phenoxazines, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEG, biotin, and transthyretia-binding ligands (e.g., tetraiodothyroacetic acid, 2,4,6-triiodophenol, and flufenamic acid). Since it is also known that oligomeric compounds containing some phosphorothioate sugar internucleotide linkages bind to serum proteins, short oligomeric compounds, such as oligonucleotides of about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) and containing a plurality of phosphorothioate linkages in their backbone, are also applicable to the present invention as ligands (e.g., PK modulating ligands). PK modulating oligonucleotides can contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate and / or phosphorodithioate linkages. In some embodiments, all of the internucleoside linkages in the PK modulating oligonucleotide are phosphorothioate and / or phosphorodithioate linkages. In addition, aptamers that bind to serum components (e.g., serum proteins) are also applicable to the present invention as PK modulating ligands. Binding to serum components (e.g., serum proteins) can be predicted from albumin binding assays, such as those described in Oravcova, et al., Journal of Chromatography B (1996), 677:1-27.

[0356] When there are two or more ligands, all of these ligands can have the same properties, all can have different properties, or some ligands can have the same properties while at the same time other ligands have different properties. For example, a ligand can have targeting properties, endosome lysis activity, or PK modulation properties. In a preferred embodiment, all ligands have different properties.

[0357] In some embodiments of any one of the above aspects, the ligand has a structure represented by any of formulas (IV) to (VII): TIFF2025522811000010.tif82149wherein, q 2A 、q 2B 、q 3A 、q 3B 、q4 A 、q 4B 、q 5A 、q 5B and q 5C each independently represents from 0 to 20 for each occurrence, and the repeating units can be the same or different, P 2A 、P 2B 、P 3A 、P 3B 、P 4A 、P 4B 、P 5A 、P 5B 、P 5C 、T 2A 、T 2B 、T 3A 、T 3B 、T 4A 、T 4B 、T 5A 、T 5B 、T 5C each independently is absent or is CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O, Q 2A 、Q 2B 、Q 3A 、Q 3B 、Q 4A 、Q 4B 、Q 5A 、Q5B and Q 5C is, independently for each occurrence, absent or is alkylene, substituted alkylene, where one or more methylenes are interrupted by or terminated with one or more of O, S, S(O), SO2, N(R N ), C(R')=C(R''), C≡C or C(O). R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C is, independently for each occurrence, absent or is NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a ), C(O)-CH(R a ), -NH-, CO, CH=N-O, TIFF2025522811000011.tif18153 or heterocyclyl. L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represent a ligand, i.e., independently for each occurrence, a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide. R a is H or an amino acid side chain.

[0358] In some embodiments of any one of the above aspects, the ligand is a ligand of formula (VII): TIFF2025522811000012.tif40128 wherein L 5A , L 5B and L 5C represent monosaccharides, such as GalNAc derivatives.

[0359] Exemplary ligands include the following ligands Examples include, but are not limited to, TIFF2025522811000013.tif58128, TIFF2025522811000014.tif210146, and TIFF2025522811000015.tif118128.

[0360] In some embodiments of any one of the aspects described herein, the ligand is the ligand described in U.S. Patent No. 5,994,517 or U.S. Patent No. 6,906,182, the entire contents of each of which are incorporated herein by reference.

[0361] In some embodiments, the ligand can be the triantenna ligand described in FIG. 3 of U.S. Patent No. 6,906,182. For example, the ligand is selected from the following triantenna ligands. TIFF2025522811000016.tif192140

[0362] Note that when two or more ligands are present, they can be the same or different. Thus, in some embodiments of any one of the aspects described herein, all ligands are the same. In some other embodiments of any one of the aspects described herein, the ligands are different.

[0363] L (linker) One aspect of any one of the aspects described herein includes a linker L. As used herein, the term "linker" means an organic moiety that connects two parts of a compound. The linker typically is a direct bond or an atom such as oxygen or sulfur, NR 1 , C(O), C(O)O, C(O)NR 1, units such as SO, SO2, SO2NH, or chains of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or more methylenes are interrupted or terminated by O, S, S(O), SO2, N(R 1 ),2, C(O), a cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic group, and R 1 is hydrogen, acyl, an aliphatic group or a substituted aliphatic group.

[0364] In some embodiments, the linker is a cleavable linker. A cleavable linker is one that releases two parts held together by the linker depending on processes within the target cell, such as reduction in the cytoplasm, exposure to acidic conditions in lysosomes or endosomes, or cleavage by a specific enzyme (e.g., protease) within the cell. Thus, a cleavable linker allows the release of the two parts in their original forms after translocation into the cell and processing within the target cell. Cleavable linkers include, but are not limited to, bonds cleavable by an enzyme (e.g., a peptide linker), bonds cleavable by reducing conditions (e.g., a disulfide linker), or bonds cleavable by acidic conditions (e.g., hydrazone and carbonate).

[0365] Generally, a cleavable linker contains at least one cleavable linking group. A cleavable linking group is a linking group that is sufficiently stable extracellularly but is cleaved upon entry into the target cell to release the two parts linked by the linker. In a preferred embodiment, the cleavable linking group is cleaved at least 10-fold, preferably at least 100-fold faster within the target cell or under a first reference condition (which can be selected to mimic or represent, for example, intracellular conditions) than in the subject's blood or serum or under a second reference condition (which can be selected to mimic or represent conditions found in blood or serum).

[0366] Cleavable linking groups are sensitive to cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, the cleavage agent is more dominant, at a higher level or activity, inside the cell than in serum or blood. Examples of such degradable agents include redox agents selected for a specific substrate or redox agents without substrate specificity, such as oxidases or reductases, or reducing agents such as mercaptans present in cells that can decompose an oxidoreductively cleavable linking group by reduction; esterases; agents that can create an endosome or acidic environment, such as those that bring about a pH of 5 or less; enzymes, peptidases (which can be substrate-specific), and phosphatases that can hydrolyze or decompose an acid-cleavable linking group by acting as a general acid.

[0367] Cleavable linking groups, such as disulfide bonds, can be sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower than that, in the range of about 7.1 - 7.3. Endosomes have a pH range of 5.5 - 6.0 and are more acidic, and lysosomes have a more acidic pH of around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH to release a cationic lipid from a ligand inside the cell or into a desired compartment of the cell.

[0368] Linkers can contain cleavable linking groups that are cleavable by specific enzymes. The type of cleavable linking group incorporated into the linker can depend on the target cell. For example, a liver-targeting ligand can be linked to a cationic lipid via a linker containing an ester group. Since liver cells are rich in esterases, the linker will be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other cell types rich in esterases include cells of the lung, renal cortex, and testis. Linkers containing peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synovial cells.

[0369] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linker. It would also be similarly desirable to test the candidate cleavable linker for its ability to resist cleavage in blood or when contacted with other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between a first condition selected to show cleavage in target cells and a second condition selected to show cleavage in other tissues or biological fluids, such as blood or serum. The evaluation can be carried out in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It would be useful to perform the initial evaluation under cell-free or cell culture conditions and confirm it by further evaluation in whole animals. In a preferred embodiment, a useful candidate compound is cleaved at least 2-fold, 4-fold, 10-fold or 100-fold faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0370] One class of cleavable linking groups is redox-cleavable linking groups, which can be used in the present invention and are cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-S-S-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can rely on the methods described herein. For example, a candidate can be evaluated by incubating it with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in cells, e.g., target cells. The candidate can also be evaluated under conditions selected to mimic blood conditions or serum conditions. In a preferred embodiment, the candidate compound is cleaved by at most 10% in blood. In a preferred embodiment, a useful candidate compound is degraded at least 2-fold, 4-fold, 10-fold or 100-fold faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of a candidate compound can be determined using standard enzyme kinetics assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0371] The phosphate-based cleavable linking groups that can be used in the present invention are cleaved by agents that decompose or hydrolyze the phosphate group. Examples of agents that cleave the phosphate group in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S-, where Rk can be, independently for each occurrence, hydrogen, C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, C7-C12 aralkyl. Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-. A preferred embodiment is -O-P(O)(OH)-O-. These candidates can be evaluated using methods similar to the methods described above.

[0372] The acid-cleavable linking groups that can be used in the present invention are linking groups that are cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment where the pH is about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by an agent such as an enzyme that can act as a general acid. In cells, specific low-pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. The acid-cleavable group can have the general formula -C=NN-, C(O)O or -OC(O). A preferred embodiment is when the carbon (alkoxy group) attached to the oxygen of the ester is an aryl group, a substituted alkyl group or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0373] The ester-based cleavable linking groups that can be used in the present invention are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene groups, alkenylene groups and alkynylene groups. The ester-cleavable linking group has the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0374] The peptide-based cleavable linker that can be used in the present invention is cleaved by enzymes such as peptidases and proteases in cells. The peptide-based cleavable linker is a peptide bond formed between amino acids to give oligopeptides (such as dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not contain an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynylene. The peptide bond is a special type of amide bond formed between amino acids to give peptides and proteins. The peptide-based cleaving group is generally limited to the peptide bond (i.e., amide bond) formed between amino acids to give peptides and proteins, and does not include the entire amide functional group. The peptide-based cleavable linker has the general formula -NHCHR A C(O)NHCHR B C(O)-, wherein R A and R B are the R groups of two adjacent amino acids.

[0375] In some embodiments of any one of the aspects described herein, L is optionally substituted C1-C 20 alkylene (such as -(CH2) b -, where b is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 15, 16, 17, 18, 19 or 20), or optionally substituted C2-C 20 alkynylene, wherein the main chain of the alkylene or alkynylene is interrupted by or may end with one or more of O, S, S(O), SO2, NR N1 , NR N1 -C(O), C(O), C(O)O, a cleavable linker, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclic group, and R N1 is hydrogen, acyl, an aliphatic group or a substituted aliphatic group.

[0376] In some embodiments of any one of the above aspects, L is optionally substituted C1-C20 is alkylene, where the main chain of the alkylene is interrupted by and / or terminated with NHC(O). For example, L may be substituted C5-C 15 is alkylene, where the main chain of the alkylene is interrupted by and / or terminated with NHC(O).

[0377] In some embodiments of any one of the above aspects, L is -(CH2) LM -NHC(O)-(CH2) LN -, where LM and LN are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. LM and LN can be the same or they can be different. In some embodiments, LM is 4, 5, 6, 7 or 8. In some embodiments, LN is 9, 10, 11 or 12.

[0378] B (nucleobase) One embodiment of any one of the aspects described herein includes a modified or unmodified nucleobase B.

[0379] Note that the nucleobase can be a natural nucleobase or an unnatural nucleobase. "Unnatural nucleobase" means a nucleobase other than adenine, guanine, cytosine, uracil or thymine. Exemplary unnatural nucleobases include inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, as well as substituted or modified analogs of adenine, guanine, cytosine and uracil, such as 2-aminoadenine, as well as other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine and 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and the same 8-substituted guanines, 5-trifluoromethyl and other 5-substituted uracils and the same 5-substituted cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkylcytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazole, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3 carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N 4-acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentenyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine, or O-alkylated bases, but not limited thereto. Further purines and pyrimidines include those disclosed in U.S. Patent No. 3,687,808, those disclosed on pages 858 - 859 of Concise Encyclopedia of Polymer Science and Engineering, edited by Kroschwitz, J.I., John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, the contents of all of which are incorporated herein by reference.

[0380] In some embodiments, the unnatural nucleobases are inosine, xanthine, hypoxanthine, nebularine, isoguanosine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(isopentenyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N 6 -(isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4 -(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazol-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(Methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diazanyl)-2-oxophenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-azanyl-2-sulfanyl-3-azanylphenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-diazanyl-2-oxophenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-azanyl-2-sulfanyl-3-azanylphenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-diazanyl-2-oxophenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-azanyl-2-sulfanyl-3-azanylphenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-diazanyl-2-oxophenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-azanyl-2-sulfanyl-3-azanylphenthiazin-1-yl, 1,3,5-triazanyl-2,6-dioxonaphthalene, inosine, xanthine, hypoxanthine, tubercidin, toyocamycin, isoguanosine, inosinyl, 2-azainosinyl, 7-deazainosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridinyl, 9-methyl-imidazopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-propynylisocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthrenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 6-azathymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-azapyrimidine, 2-aminopurine, 2,6-diaminopurine, 5-substituted pyrimidine, N, 2 -substituted purine, N6 - Replacement purine, O 6 - It can be selected from the group consisting of replacement purine, replacement 1,2,4-triazole, and any O-alkylated or N-alkylated derivatives thereof.

[0381] In some embodiments, the unnatural nucleobase is a modified nucleobase. That is, the nucleobase includes the nucleobase modifications described herein. For example, the nucleobase is a substitution or modified analog of any of the natural nucleobases. Examples of nucleobase modifications include C-5 pyrimidine modifications by alkyl groups or aminoalkyl and other cationic groups, such as guanidinium and amidine functionality, alkyl groups or aminoalkyl and other cationic groups, such as guanidinium and amidine functionality, of purine N 2 - and N 6 - Modifications, G-clamps, guanidinium G-clamps, and pseudouridine are included, but are not limited thereto.

[0382] In some embodiments of any one of the above aspects, the unnatural nucleobase is a universal nucleobase. As used herein, a universal nucleobase refers to any modified or unmodified natural or unnatural nucleobase that can base pair with all of adenine, cytosine, guanine, and uracil without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of an oligonucleotide containing the universal nucleobase. Exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbolstyryl, 5-methylisocarbolstyryl, 3-methyl-7-propynylisocarbolstyryl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridinyl, 9-methylimidazopyridinyl, pyrrolopyridinyl, isocarbolstyryl, 7-propynylisocarbolstyryl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof.

[0383] In some embodiments of any one of the aspects described herein, the unnatural nucleobase is a protected nucleobase. As used herein, "protected nucleobase" refers to a nucleobase that includes a nitrogen protecting group, and / or an oxygen protecting group, and / or a sulfur protecting group.

[0384] In some embodiments of any one of the aspects described herein, the unnatural nucleobase is a modified, protected, or substituted analog of a nucleobase selected from adenine, cytosine, guanine, thymine, and uracil.

[0385] Oxygen protecting group Some aspects of any one of the aspects described in this specification include an oxygen protecting group (also referred to herein as a hydroxyl protecting group). Examples of the oxygen protecting group include -R OP1 , -N(R OP2 )2, -C(=O)SR OP1 , -C(=O)R OP1 , -CO2R OP1 , -C(=O)N(R OP2 )2, -C(=NR OP2 )R OP1 , -C(=NR OP2 )OR OP1 , -C(=NR OP2 )N(R OP2 )2, -S(=O)R OP1 , -SO + 2R OP1 , -Si(R OP1 )3, -P(R OP3 )2, -P(R OP3 ) + 3X - , -P(OR OP3 )2, -P(OR OP3 )3X - , -P(=O)(R OP1 )2, -P(=O)(OR OP3 )2, and -P(=O)(N(R OP2 )2)2, but are not limited thereto, wherein each X - is a counterion, and each R OP1 is independently C 1~10 alkyl, C 1~10 perhaloalkyl, C 2~10 alkenyl, C 2~10 alkynyl, hetero C 1~10 alkyl, hetero C 2~10 alkenyl, hetero C 2~10 alkynyl, C 3~10 carbocyclic, 3- to 14-membered heterocyclic, C 6~14 aryl, or 5- to 14-membered heteroaryl, or two R OP1 groups together form a 3- to 14-membered heterocyclic or 5- to 14-membered heteroaryl ring, and each R OP2 is hydrogen, -OH, -OR OP1 , -N(R OP3) 2, -CN, -C(=O)R OP1 , -C(=O)N(R OP3 )2, -CO2R OP1 , -SO2R OP1 , -C(=NR OP3 )OR OP1 , -C(=NR OP3 )N(R OP3 )2, -SO2N(R OP3 )2, -SO2R OP3 , -SO2OR OP3 , -SOR OP1 , -C(=S)N(R OP3 )2, -C(=O)SR OP3 , -C(=S)SR OP3 , -P(=O)(R OP1 )2, -P(=O)(OR OP3 )2, -P(=O)(N(R OP3 )2)2, C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, hetero C 1~10 Alkyl, hetero C 2~10 Alkenyl, hetero C 2~10 Alkynyl, C 3~10 Carbocyclic, 3 - 14 membered heterocyclic, C 6~14 Aryl, and 5 - 14 membered heteroaryl, or two R OP2 groups together form a 3 - 14 membered heterocyclic or 5 - 14 membered heteroaryl ring, and each R OP3 is, independently, hydrogen, C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, hetero C 1~10 Alkyl, hetero C 2~10 Alkenyl, hetero C 2~10 Alkynyl, C 3~10 Carbocyclic, 3 - 14 membered heterocyclic, C 6~14 Aryl, and 5 - 14 membered heteroaryl, or two R OP3 groups together form a 3 - 14 membered heterocyclic or 5 - 14 membered heteroaryl ring, where ROP1 , R OP2 and R OP3 Each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aralkyl, aryl, and heteroaryl of R, R, and R may optionally be substituted with one, two, three, four, or five substituents independently selected from OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6.

[0386] Oxygen protecting groups are well known in the art and include those detailed in "Greene's Protecting Groups in Organic Synthesis" P.G.M. Wuts, 5th Edition, John Wiley & Sons, 2014, which is hereby incorporated by reference in its entirety.

[0387] Exemplary oxygen protecting groups include methyl, t-butyloxycarbonyl (BOC or Boc), methoxymethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyl-oxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiol-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyltexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuSP3inoate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts) are included, but are not limited thereto.,

[0388] In some embodiments of any one of the aspects described herein, the oxygen protecting group is acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl, trimethylsilyl (TMS), triisopropylsilyl (TIPS), mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In certain embodiments, the hydroxyl protecting group is selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl (TMS), triisopropylsilyl (TIPS), and dimethoxytrityl, where the more preferred hydroxyl protecting group is 4,4'-dimethoxytrityl.

[0389] As used herein, the terms "protected hydroxyl" and "protected hydroxyl" refer to a group of the formula -OR Pro , where R Pro is an oxygen protecting group as defined herein.

[0390] Nitrogen protecting group Some embodiments of any one of the aspects described herein include a nitrogen protecting group (also referred to herein as an amino protecting group). As the nitrogen protecting group, -OH, -OR NP1 , -N(R NP2 )2, -C(=O)R NP1 , -C(=O)N(R NP2 )2, -CO2R NP1 , -SO2R NP1 , -C(=NR NP2 )R NP1 , -C(=NR NP2 )OR NP1 , -C(=NR NP2 )N(R NP2 )2, -SO2N(R NP2 )2, -SO2R NP2 , -SO2OR NP2 , -SOR NP1 , -C(=S)N(R NP2 )2, -C(=O)SR NP2, -C(=S)SR NP2 , C 1~10 alkyl (such as aralkyl, heteroaralkyl), C 2~10 alkenyl, C 2~10 alkynyl, C 3~10 carbocyclic, 3- to 14-membered heterocyclic, C 6~14 aryl, and 5- to 14-membered heteroaryl groups are included, but not limited thereto, where each R NP1 is independently C 1~10 alkyl, C 1~10 perhaloalkyl, C 2~10 alkenyl, C 2~10 alkynyl, hetero C 1~10 alkyl, hetero C 2~10 alkenyl, hetero C 2~10 alkynyl, C 3~10 carbocyclic, 3- to 14-membered heterocyclic, C 6~14 aryl, or 5- to 14-membered heteroaryl, or two R NP1 groups together form a 3- to 14-membered heterocyclic or 5- to 14-membered heteroaryl ring, and each R NP2 is independently hydrogen, C 1~10 alkyl, C 1~10 perhaloalkyl, C 2~10 alkenyl, C 2~10 alkynyl, hetero C 1~10 alkyl, hetero C 2~10 alkenyl, hetero C 2~10 alkynyl, C 3~10 carbocyclic, 3- to 14-membered heterocyclic, C 6~14 aryl, and 5- to 14-membered heteroaryl, or two R SP3 groups together form a 3- to 14-membered heterocyclic or 5- to 14-membered heteroaryl ring, and in the formula, R NP1 and R NP2Each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aralkyl, aryl, and heteroaryl may optionally be substituted with one, two, three, four, or five substituents independently selected from OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p and can be substituted with 1, 2, 3, 4, or 5 substituents independently selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6.

[0391] Nitrogen protecting groups are well known in the art and include those detailed in "Greene's Protecting Groups in Organic Synthesis" P.G.M. Wuts, 5th Edition, John Wiley & Sons, 2014, which is hereby incorporated by reference in its entirety.

[0392] Exemplary amides (e.g., -C(=O)R NP1)As nitrogen protecting groups, there may be mentioned, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyl-oxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrosinamide, N-acetylmethionine derivative, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0393] Exemplary carbamates (e.g., -C(=O)OR NP1)As nitrogen protecting groups, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)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-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)methylcarbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthiophenylcarbamate (Bmpc), 2-phosphonioethylcarbamate (Peoc), 2-triphenylphosphonioisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethylcarbamate, m-chloro-p-acetyloxybenzylcarbamate, p-(dihydroxylboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chromonylmethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzylcarbamate, o-nitrobenzylcarbamate, 3,4-dimethoxy-6-nitrobenzylcarbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amylcarbamate, S-benzylthiocarbamate, p-cyanobenzylcarbamate, cyclobutylcarbamate, cyclohexylcarbamate, cyclopentylcarbamate, cyclopropylmethylcarbamate, p-decyloxybenzylcarbamate, 2,2-dimethoxyacylvinylcarbamate, o-(N,N-dimethylcarboxamide)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamide)propylcarbamate, 1,1-dimethylpropynylcarbamate, di(2-pyridyl)methylcarbamate, 2-furanylmethylcarbamate, 2-iodoethylcarbamate, isobornylcarbamate, isobutylcarbamate, isonicotinylcarbamate, p-(p'-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1-methylcyclohexylcarbamate, 1-methyl-1-cyclopropylmethylcarbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethylcarbamate, 1-methyl-1-(p-phenylazophenyl)ethylcarbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethylcarbamate, phenylcarbamate, p-(phenylazo)benzylcarbamate, 2,4,6-tri-t-butylphenylcarbamate, 4-(trimethylammonium)benzylcarbamate, and 2,4,Examples include, but are not limited to, 6-trimethylbenzyl carbamate.,

[0394] Exemplary sulfonamides (e.g., -S(=O)2R NP1 ) As nitrogen protecting groups, for example, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide are included, but are not limited thereto.

[0395] As further exemplary nitrogen protecting groups, phenothiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuNP2inimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberilylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane and N-diphenylboric acid derivatives, N-[phenyl(pentNP1cylchromium)- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphine amide (Dpp), dimethylthiophosphine amide (Mpt), diphenylthiophosphine amide (Ppt), dialkylphosphoramidate, dibenzylphosphoramidate, diphenylphosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys), but are not limited thereto.,

[0396] Sulfur protecting group Some embodiments of any one of the aspects described herein include a sulfur protecting group (also referred to herein as a thiol protecting group). As the sulfur protecting group, -R SP1 , -N(R SP2 )2, -C(=O)SR SP1 , -C(=O)R SP1 , -CO2R SP1 , -C(=O)N(R SP2 )2, -C(=NR SP2 )R SP1 , -C(=NR SP2 )OR SP1 , -C(=NR SP2 )N(R SP2 )2, -S(=O)R SP1 , -SO2R SP1 , -Si(R SP1 )3, -P(R SP3 )2, -P(R SP3 ) + 3X - , -P(OR SP3 )2, -P(OR SP3 ) + 3X - , -P(=O)(R SP1)2, -P(=O)(OR SP3 )2, and -P(=O)(N(R SP2 )2)2 are included, but not limited thereto, wherein

[0397] X - is a counter ion, and each R SP1 is, independently, C 1~10 alkyl, C 1~10 perhaloalkyl, C 2~10 alkenyl, C 2~10 alkynyl, hetero C 1~10 alkyl, hetero C 2~10 alkenyl, hetero C 2~10 alkynyl, C 3~10 carbocyclic, 3 - to 14 - membered heterocyclic, C 6~14 aryl, or 5 - to 14 - membered heteroaryl, or two R SP1 groups together form a 3 - to 14 - membered heterocyclic or 5 - to 14 - membered heteroaryl ring, and each R SP2 is hydrogen, -OH, -OR SP1 , -N(R SP3 )2, -CN, -C(=O)R SP1 , -C(=O)N(R SP3 )2, -CO2R SP1 , -SO2R SP1 , -C(=NR SP3 )OR SP1 , -C(=NR SP3 )N(R SP3 )2, -SO2N(R SP3 )2, -SO2R SP3 , -SO2OR SP3 , -SOR SP1 , -C(=S)N(R SP3 )2, -C(=O)SR SP3 , -C(=S)SR SP3 , -P(=O)(R SP1 )2, -P(=O)(OR SP3 )2, -P(=O)(N(R SP3 )2)2, C 1~10 alkyl, C 1~10 perhaloalkyl, C 2~10 alkenyl, C 2~10 alkynyl, hetero C 1~10Alkyl, hetero C 2~10 Alkenyl, hetero C 2~10 Alkynyl, C 3~10 Carbocyclic, 3- to 14-membered heterocyclic, C 6~14 Aryl, and 5- to 14-membered heteroaryl, or two R SP2 groups together form a 3- to 14-membered heterocyclic or 5- to 14-membered heteroaryl ring, and each R SP3 is independently hydrogen, C 1~10 alkyl, C 1~10 perhaloalkyl, C 2~10 alkenyl, C 2~10 alkynyl, hetero C 1~10 alkyl, hetero C 2~10 alkenyl, hetero C 2~10 alkynyl, C 3~10 carbocyclic, 3- to 14-membered heterocyclic, C 6~14 aryl, and 5- to 14-membered heteroaryl, or two R SP3 groups together form a 3- to 14-membered heterocyclic or 5- to 14-membered heteroaryl ring, where R SP1 , R SP2 and R SP3 alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aralkyl, aryl, and heteroaryl are optionally OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p-OH, CH2-[CH(OH)] m -(CH2) p Optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from -NH2 or CH2-aryl-alkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0398] Sulfur protecting groups are well known in the art and include those detailed in "Greene's Protecting Groups in Organic Synthesis" P.G.M. Wuts, 5th Edition, John Wiley & Sons, 2014, which is incorporated herein by reference.

[0399] Internucleoside linker As used herein, the term "internucleoside linker" refers to a linker formed by a covalent bond between adjacent nucleosides. The two major classes of internucleoside linkers are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing linkers include, but are not limited to, phosphodiester (P=O), phosphorotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing linking groups include, but are not limited to, methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-), siloxane (-O-Si(H)2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleoside linkers can typically be used to alter, and in some cases increase, the nuclease resistance of oligonucleotide compounds as compared to the native phosphodiester linker. In certain embodiments, linkers having chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Representative chiral linkers include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus(III)-containing and non-phosphorus(III)-containing linkers are well known to those of skill in the art.

[0400] The phosphate group in the internucleoside linkage can be modified by replacing one of its oxygens with a different substituent. One result of this modification can be an increase in the resistance of the oligonucleotide to nucleolytic breakdown. Examples of modified phosphate groups include phosphorothioate, phosphorodithioate, phosphororoselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphodiester internucleoside linkage can be replaced by any of the following: S, Se, BR3 (where R is hydrogen, alkyl, aryl), C (i.e., an alkyl group, aryl group, etc.), H, NR2 (where R is hydrogen, optionally substituted alkyl, aryl), or OR (where R is optionally substituted alkyl or aryl). The phosphorus(III) atom in the unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the above atoms or atomic groups makes the phosphorus(III) atom chiral. In other words, the phosphorus(III) atom in the phosphate group modified in this way is an asymmetric center. The asymmetric phosphorus(III) atom can have either an "R" configuration (referred to herein as Rp) or an "S" configuration (referred to herein as Sp).

[0401] Phosphorodithioate has both non-bridging oxygens replaced by sulfur. The phosphorus center in phosphorodithioate is achiral, thereby precluding the formation of oligonucleotide diastereomers. Thus, without wishing to be bound by theory, modification of both non-bridging oxygens to remove the chiral center, such as phosphorodithioate formation, may be desirable in that it cannot give rise to a mixture of diastereomers. The non-bridging oxygen can independently be any of O, S, Se, B, C, H, N, or OR (where R is alkyl or aryl).

[0402] The phosphodiester nucleoside internucleoside linkage can also be modified by replacement of the bridging oxygen (i.e., the oxygen that links the phosphate to the sugar of the nucleoside) with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), and carbon (bridging methylene sulfonate). The replacement can be done with one or both of the linking oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, replacement with carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, replacement with nitrogen is preferred.

[0403] A modified phosphate linkage in which at least one of the oxygens linked to the phosphate is replaced or the phosphate group is replaced with a non-phosphorus(III) group is also referred to as a "non-phosphodiester sugar-sugar linkage" or a "non-phosphodiester linker".

[0404] In certain embodiments, the phosphate group can be replaced with a non-phosphorus containing connector, such as a dephospho linker. A dephospho linker is also referred to herein as a non-phosphodiester linker. Without wishing to be bound by theory, since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with a neutral structural mimic should confer enhanced nuclease stability. Again, without wishing to be bound by theory, in some embodiments, it may be desirable to introduce a modification in which the charged phosphate group is replaced with a neutral moiety.

[0405] Examples of moieties that can replace the phosphate group include amides (e.g., amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylic acid ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonic acid ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3'-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'-N(H)-C(=O)-CH2-S-C5', C3'-O-P(O)-O-SS-C5', C3'-CH2-NH-NH-C5', 3'-NHP(O)(OCH3)-O-5' and 3'-NHP(O)(OCH3)-O-5'), and nonionic linkers containing mixed N, O, S, and CH2 constituent parts, but are not limited thereto. See, for example, "Carbohydrate Modifications in Antisense Research", edited by Y.S. Sanghvi and P.D. Cook, ACS Symposium Series 580, Chapters 3 and 4 (pages 40 - 65). In preferred embodiments, methylenemethylimino (MMI), methylenecarbonylamino, amide, carbamate, and ethylene oxide linker are included.

[0406] In certain instances, it is well known to those skilled in the art that replacement of unbridged oxygen can lead to enhanced cleavage of the sugar - sugar linkage by adjacent 2'-OH. Thus, modification of the unbridged oxygen can, in many instances, require modification of the 2'-OH, such as modifications that do not participate in cleavage of the adjacent sugar - sugar linkage, such as arabinose sugar, 2'-O-alkyl, 2'-F, LNA, and ENA.

[0407] Preferred non-phosphodiester internucleoside linkages include phosphorothioate, phosphorothioate with an enantiomeric excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the Sp isomer, phosphorothioate with an enantiomeric excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the Rp isomer, phosphorodithioate, phosphotriester, aminoalkylphosphotrioester, alkylphosphonater (e.g., methyl-phosphonater), selenophosphate, phosphoramidate (e.g., N-alkylphosphoramidate), and boranophosphonate.

[0408] Further exemplary non-phosphorus-containing internucleoside linking groups are described in U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, 5,792,608, 5,646,269 and 5,677,439, the contents of each of these documents being incorporated herein by reference.

[0409] In some embodiments of any one of the aspects described herein, the oligonucleotides of the invention include one or more neutral nucleoside internucleotide linkages that are non-ionic. Suitable neutral nucleoside internucleotide linkages include phosphotriester, methylphosphonate, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), and thioformacetal (3'-S-CH2-O-5'); non-ionic linkages containing siloxane (dialkylsiloxane), carboxylic acid ester, carboxamide, sulfide, sulfonic acid ester and / or amide (see, for example, "Carbohydrate Modifications in Antisense Research", edited by Y.S. Sanghvi and P.D. Cook, ACS Symposium Series 580, Chapters 3 and 4 (pages 40-65)); and non-ionic linkages containing mixed N, O, S and CH2 constituent parts, but are not limited thereto.

[0410] In one aspect, non-phosphodiester backbone linkages include, for example, normal 3'-5' linkages, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having a backbone in which a pair of adjacent nucleoside units have a reversed polarity from a 3'-5' linkage to a 5'-3' linkage, or from a 2'-5' linkage to a 5'-2' linkage, but are not limited thereto.

[0411] Also included are various salt forms, mixed salt forms, and the free acid form. In some embodiments of the present invention, the dsRNA agent of the present invention is in the free acid form. In another embodiment of the present invention, the dsRNA agent of the present invention is in a salt form. In one embodiment, the dsRNA agent of the present invention is in the sodium salt form. In certain embodiments, when the dsRNA agent of the present invention is in the sodium salt form, sodium ions are present as counterions for substantially all of the phosphodiester groups and / or phosphorothioate groups present in the agent. The phosphodiester linkages and / or phosphorothioate linkages that do not have sodium counterions in an agent containing sodium counterions for substantially all of the phosphodiester linkages and / or phosphorothioate linkages are 5, 4, 3, 2, or 1 or less. In some embodiments, when the dsRNA agent of the present invention is in the sodium salt form, sodium ions are present as counterions for all of the phosphodiester groups and / or phosphorothioate groups present in the agent.

[0412] Representative U.S. patents that teach the preparation of the above phosphorus-containing linking group include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,316, 5,550,111, 5,563,253, 5,571,799, 5,587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209, 6,239,265, 6,277,603, 6,326,199, 6,346,614, 6,444,423, 6,531,590, 6,534,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Reissue Patent No. 39464. The entire contents of each of these patent documents are incorporated herein by reference.

[0413] In some embodiments of any one of the aspects described herein, R 23 and R 25 are both bonds to a modified nucleoside internucleoside linking group.

[0414] In some embodiments of any one of the aspects described herein, R 23 is a bond to a phosphodiester nucleoside internucleoside linking group.

[0415] In some embodiments of any one of the aspects described herein, R 25 is a bond to a phosphodiester nucleoside internucleoside linkage.

[0416] In some embodiments of any one of the aspects described herein, R 23 is a bond to a modified nucleoside internucleoside linkage, and R 25 is a bond to a phosphodiester nucleoside internucleoside linkage.

[0417] In some embodiments of any one of the aspects described herein, R 5 is a bond to a modified nucleoside internucleoside linkage, and R 23 is a bond to a phosphodiester nucleoside internucleoside linkage.

[0418] In some embodiments of any one of the above aspects, the oligonucleotide can include one or more, for example 1, 2, 3, 4, 5, 6, 7, 8 or more, modified nucleoside internucleoside linkages. For example, the oligonucleotide can include 1, 2, 3, 4, 5 or 6 (e.g., 1, 2, 3 or 4) modified nucleoside internucleoside linkages. In some embodiments, the oligonucleotide includes at least two modified nucleoside internucleoside linkages between the first five nucleotides counted from the 5' end of the oligonucleotide, and further includes at least two modified nucleoside internucleoside linkages between the first five nucleotides counted from the 3' end of the oligonucleotide. For example, the oligonucleotide includes modified nucleoside internucleoside linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 5' end of the oligonucleotide, and between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 3' end of the oligonucleotide.

[0419] In some embodiments of any one of the above aspects, the modified internucleoside linker is phosphorothioate. Thus, in some embodiments of any one of the above aspects, the oligonucleotide comprises one or more, for example 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleoside linkers. For example, the oligonucleotide comprises 1, 2, 3, 4, 5 or 6 (e.g., 1, 2, 3, or 4) phosphorothioate internucleoside linkers. In some embodiments, the oligonucleotide comprises at least two phosphorothioate internucleoside linkers between the first five nucleotides counted from the 5' end of the oligonucleotide, and further comprises at least two phosphorothioate internucleoside linkers between the first five nucleotides counted from the 3' end of the oligonucleotide. For example, the oligonucleotide comprises modified internucleoside linkers between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 5' end of the oligonucleotide, and between nucleotides 1 and 2 and between nucleotides 2 and 3 counted from the 3' end of the oligonucleotide.

[0420] In some embodiments, the oligonucleotide comprises 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate internucleoside linkers separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate internucleoside linkers. For example, the oligonucleotide comprises 2 to 9 blocks of 2 phosphorothioate or methylphosphonate internucleoside linkers separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleoside linkers.

[0421] In some embodiments of any one of the aspects described herein, the oligonucleotide comprises a pattern of backbone chiral centers. In some embodiments, the common pattern of backbone chiral centers comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 or more nucleotide linkages in the Sp configuration. In some embodiments, the number of nucleotide linkages in the Rp configuration included in the common pattern of backbone chiral centers is 8 or less, 7 or less, 6 or less, 5 or less, or 4 or less. In some embodiments, the number of achiral nucleotide linkages (such as, by way of non-limiting example, phosphodiesters) included in the common pattern of backbone chiral centers is 8 or less, 7 or less, 6 or less, 5 or less, or 4 or less. In some embodiments, the common pattern of backbone chiral centers comprises 10, 11, 12, 13, 14, or 15 or more nucleotide linkages in the Sp configuration and 8 or less, 7 or less, 6 or less, 5 or less, or 4 or less achiral nucleotide linkages. In some embodiments, the nucleotide linkages in the Sp configuration are optionally consecutive or non-consecutive. In some embodiments, the nucleotide linkages in the Rp configuration are optionally consecutive or non-consecutive. In some embodiments, the achiral nucleotide linkages are optionally consecutive or non-consecutive.

[0422] In some embodiments, the oligonucleotide comprises a block that is a stereochemical block. For example, the oligonucleotide comprises a block that is an Rp block in that each internucleotide linkage of the block is Rp. In some embodiments, the oligonucleotide comprises a block that is an Sp block in that each internucleotide linkage of the block is Sp. In some embodiments, the oligonucleotide comprises an Rp block at the 5' end. In some embodiments, the oligonucleotide comprises an Rp block at the 3' end. In some embodiments, the oligonucleotide comprises an Sp block at the 5' end. In some embodiments, the oligonucleotide comprises an Sp block at the 3' end. In some embodiments, the oligonucleotide comprises both an Rp block and an Sp block. In some embodiments, the oligonucleotide comprises one or more Rp blocks but does not comprise an Sp block. In some embodiments, the oligonucleotide comprises one or more Sp blocks but does not comprise an Rp block. In some embodiments, the oligonucleotide comprises one or more PO blocks in which each internucleotide linkage is a native phosphate linkage.

[0423] In some embodiments, Sp follows adenosine in the oligonucleotide. In some embodiments, Rp follows adenosine in the oligonucleotide. In some embodiments, a native phosphate linkage (PO) follows adenosine in the oligonucleotide. In some embodiments, Sp follows uridine in the oligonucleotide. In some embodiments, Rp follows uridine in the oligonucleotide. In some embodiments, a native phosphate linkage (PO) follows uridine in the oligonucleotide. In some embodiments, Sp follows cytidine in the oligonucleotide. In some embodiments, Rp follows cytidine in the oligonucleotide. In some embodiments, a native phosphate linkage (PO) follows cytidine in the oligonucleotide. In some embodiments, Sp follows guanosine in the oligonucleotide. In some embodiments, Rp follows guanosine in the oligonucleotide. In some embodiments, a native phosphate linkage (PO) follows guanosine in the oligonucleotide. In some embodiments, Sp follows cytidine and uridine. In some embodiments, Rp follows cytidine and uridine. In some embodiments, a native phosphate linkage (PO) follows cytidine and uridine. In some embodiments, Sp follows adenosine and guanosine. In some embodiments, Rp follows adenosine and guanosine.

[0424] Oligonucleotide Modification - Sugar In some embodiments of any one of the aspects described herein, the oligonucleotide further comprises a nucleoside having a modified sugar, i.e., in addition to the nucleoside of formula (II). "Modified sugar" means a sugar or moiety other than 2'-deoxy (i.e., 2'-H) ribose sugar or 2'-OH ribose sugar. Exemplary nucleotides containing modified sugars include 2'-F ribose, 2'-OMe ribose, 2'-O,4'-C-methylene ribose (locked nucleic acid, LNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA), cyclohexene (cyclohexene nucleic acid, CeNA), 2'-methoxyethyl ribose, 2'-O-allyl ribose, 2'-C-allyl ribose, 2'-O-N-methylacetamide (2'-O-NMA) ribose, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) ribose, 2'-O-aminopropyl (2'-O-AP) ribose, 2'-F arabinose (2'-ara-F), threose (threose nucleic acid, TNA), and 2,3-dihydroxylpropyl (glycol nucleic acid, GNA), among others. Note that nucleosides having modified sugars can be present at any position of the oligonucleotide.

[0425] In some embodiments, the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, 2'-fluoro (2'-F) nucleotides. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-F nucleotides. Note that 2'-F nucleotides can be present at any position of the oligonucleotide.

[0426] In some embodiments, the oligonucleotide comprises (e.g., exclusively comprises) the nucleoside of formula (II) and 2'-F nucleosides.

[0427] In some embodiments, the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, 2'-OMe nucleotides. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-OMe nucleotides. Note that the 2'-OMe nucleotides can be present at any position of the oligonucleotide.

[0428] In some embodiments, the oligonucleotide comprises (e.g., exclusively comprises) the nucleosides of formula (II) and 2'-OMe nucleosides. In some other embodiments, the oligonucleotide comprises (e.g., exclusively comprises) the nucleosides of formula (II), 2'-OMe nucleosides and 2'-F nucleosides.

[0429] In some embodiments, the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, 2'-deoxy, e.g., 2'-H nucleotides. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-deoxy, e.g., 2'-H nucleotides. Note that the 2'-deoxy, e.g., 2'-H nucleotides can be present at any position of the oligonucleotide. For example, the oligonucleotide can comprise 2'-deoxy, e.g., 2'-H nucleotides at 1, 2, 3, 4, 5 or 6 of the 2nd, 5th, 7th, 12th, 14th and 16th positions counted from the 5'-end of the oligonucleotide. In some embodiments, the oligonucleotide comprises 2'-deoxy nucleotides at the 5th and 7th positions counted from the 5'-end of the oligonucleotide.

[0430] In some embodiments, the oligonucleotide comprises (e.g., consists exclusively of) nucleosides of formula (II) and 2'-deoxy(2'-H) nucleotides. In some embodiments, the oligonucleotide comprises (e.g., consists exclusively of) nucleosides of formula (I), 2'-OMe nucleosides, and 2'-deoxy(2'-H) nucleotides. In some embodiments, the oligonucleotide comprises (e.g., consists exclusively of) nucleosides of formula (I), 2'-F nucleosides, and 2'-deoxy(2'-H) nucleotides. In some embodiments, the oligonucleotide comprises (e.g., consists exclusively of) nucleosides of formula (I), 2'-OMe nucleosides, 2'-F nucleosides and 2'-deoxy(2'-H) nucleotides.

[0431] Oligonucleotide Note that the nucleosides of formula (II) can be located anywhere in the oligonucleotide. In some embodiments, the nucleosides of formula (II) are present at the 5'- or 3'-terminus of the oligonucleotide. In some embodiments, the nucleosides of formula (II) are present at an internal position of the oligonucleotide.

[0432] In some embodiments of any one of the aspects described herein, the oligonucleotide further comprises a nucleoside having a modified sugar, i.e., in addition to the nucleoside of formula (II). "Modified sugar" means a sugar or moiety other than 2'-deoxy (i.e., 2'-H) ribose sugar or 2'-OH ribose sugar. Exemplary nucleotides containing modified sugars include 2'-F ribose, 2'-OMe ribose, 2'-O,4'-C-methylene ribose (locked nucleic acid, LNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA), cyclohexene (cyclohexene nucleic acid, CeNA), 2'-methoxyethyl ribose, 2'-O-allyl ribose, 2'-C-allyl ribose, 2'-O-N-methylacetamide (2'-O-NMA) ribose, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) ribose, 2'-O-aminopropyl (2'-O-AP) ribose, 2'-F arabinose (2'-ara-F), threose (threose nucleic acid, TNA), and 2,3-dihydroxypropyl (glycol nucleic acid, GNA), etc. Note that nucleosides having modified sugars can be present at any position of the oligonucleotide.

[0433] In some embodiments, the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, 2'-fluoro (2'-F) nucleotides. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-F nucleotides. Note that 2'-F nucleotides can be present at any position of the oligonucleotide.

[0434] In some embodiments, the oligonucleotide comprises (e.g., exclusively comprises) 2'-nucleosides and 2'-F nucleosides of formula (II).

[0435] In some embodiments, the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, 2'-OMe nucleotides. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-OMe nucleotides. Note that the 2'-OMe nucleotides can be present at any position of the oligonucleotide.

[0436] In some embodiments, the oligonucleotide comprises (e.g., consists essentially of) 2'-nucleosides and 2'-OMe nucleosides of formula (II). In some other embodiments, the oligonucleotide comprises (e.g., consists essentially of) 2'-nucleosides, 2'-OMe nucleosides, and 2'-F nucleosides of formula (II).

[0437] In some embodiments, the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, 2'-deoxy, e.g., 2'-H nucleotides. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-deoxy, e.g., 2'-H nucleotides. Note that the 2'-deoxy, e.g., 2'-H nucleotides can be present at any position of the oligonucleotide. For example, the oligonucleotide can comprise 2'-deoxy, e.g., 2'-H nucleotides at 1, 2, 3, 4, 5, or 6 of the 2nd, 5th, 7th, 12th, 14th, and 16th positions counted from the 5'-end of the oligonucleotide. In some embodiments, the oligonucleotide comprises 2'-deoxynucleotides at the 5th and 7th positions counted from the 5'-end of the oligonucleotide.

[0438] In some embodiments, the oligonucleotide comprises (e.g., consists essentially of) nucleosides of formula (II) and 2'-deoxy (2'-H) nucleotides. In some embodiments, the oligonucleotide comprises (e.g., consists essentially of) nucleosides of formula (II), 2'-OMe nucleosides, and 2'-deoxy (2'-H) nucleotides. In some embodiments, the oligonucleotide comprises (e.g., consists essentially of) nucleosides of formula (II), 2'-F nucleosides, and 2'-deoxy (2'-H) nucleotides. In some embodiments, the oligonucleotide comprises (e.g., consists essentially of) nucleosides of formula (II), 2'-OMe nucleosides, 2'-F nucleosides and 2'-deoxy (2'-H) nucleotides.

[0439] In some embodiments of any one of the aspects described herein, the oligonucleotide further comprises, i.e., in addition to the nucleosides of formula (II), unnatural nucleobases. In some embodiments, the oligonucleotide can comprise nucleotides comprising one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, independently selected unnatural nucleobases. When nucleotides comprising unnatural nucleobases are present, they can be present anywhere in the oligonucleotide.

[0440] In some embodiments, the oligonucleotide further comprises a solid support attached to the oligonucleotide.

[0441] The oligonucleotides described herein can range in length from a few nucleotides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) to several hundred nucleotides. For example, the oligonucleotide can be from 5 nucleotides to 100 nucleotides in length. In some embodiments, the oligonucleotide can be from 10 nucleotides to 50 nucleotides in length. For example, the oligonucleotide is 15 - 35, more typically 18 - 25, even more typically 19 - 24, and most typically 19 - 21 base pairs in length. In some embodiments, longer oligonucleotides of 25 - 30 nucleotides in length are preferred. In some embodiments, shorter oligonucleotides of 10 - 15 nucleotides in length are preferred. In another embodiment, the oligonucleotide is at least 21 nucleotides in length.

[0442] 5'-modification In some embodiments of any one of the aspects described herein, the oligonucleotides described herein are 5'-phosphorylated or contain a phosphoryl analog at the 5'-prime end. Examples of 5'-phosphate modifications that are compatible with RISC-mediated gene silencing are provided. Suitable modifications include 5'-monophosphate ((HO)2(O)P-O-5'), 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-guanosine cap (methylated or unmethylated at the 7-position) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), 5'-monodithiophosphate (phosphorodithioate, (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); any further combination of oxygen / sulfur-substituted monophosphates, diphosphates, and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonate (e.g., RP(OH)(O)-O-5'-, R = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkenylphosphonate (i.e., vinyl, substituted vinyl, e.g., OH)2(O)P-5'-CH= or (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonate (e.g., R(OH)(O)P-O-5', R = alkyl ether, e.g., methoxymethyl (MeOCH2-), ethoxymethyl, etc.). Other exemplary 5'-modifications include those in which Z is alkyl, which may be substituted at least once, e.g., ((HO)2(X)P-O[-(CH2) a -O-P(X)(OH)-O] b-5', ((HO)2(X)P-O[-(CH2) a -P(X)(OH)-O] b -5', ((HO)2(X)P-[-(CH2) a -O-P(X)(OH)-O] b -5'; dialkyl terminal phosphate and phosphate mimics: HO[-(CH2) a -O-P(X)(OH)-O] b -5', H2N[-(CH2) a -O-P(X)(OH)-O] b -5', H[-(CH2) a -O-P(X)(OH)-O] b -5', Me2N[-(CH2) a -O-P(X)(OH)-O] b -5', HO[-(CH2) a -P(X)(OH)-O] b -5', H2N[-(CH2) a -P(X)(OH)-O] b -5', H[-(CH2) a -P(X)(OH)-O] b -5', Me2N[-(CH2) a -P(X)(OH)-O] b -5' exists, where a and b are each independently 1 to 10. Another aspect includes substitution of oxygen and / or sulfur by BH3, BH3 - and / or Se.

[0443] In some aspects of any one of the aspects described herein, the oligonucleotide contains a 5'-vinylphosphonate group. For example, the oligonucleotide contains a 5'-E-vinylphosphonate group. In some other non-limiting examples, the oligonucleotide contains a 5'-Z-vinylphosphonate group.

[0444] In some aspects of any one of the above aspects, the oligonucleotides described herein contain a 5'-morpholino, 5'-dimethylamino, 5'-deoxy, inverted abasic, or inverted abasic locked nucleic acid modification at the 5' end.

[0445] In some embodiments of any one of the above situations, the oligonucleotides described herein can include thermally destabilizing modifications. For example, the oligonucleotide can include a double-stranded thermally destabilizing modification within the first 9 nucleotide positions counted from the 5' end of the oligonucleotide. In some embodiments, the thermally destabilizing modification is located at the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, or 9th position counted from the 5' end of the antisense strand. In some embodiments, the thermally destabilizing modification is located at the 2nd to 9th, or preferably the 4th to 8th position counted from the 5' end of the oligonucleotide. In some further embodiments, the thermally destabilizing modification is located at the 5th, 6th, 7th, or 8th position counted from the 5' end of the oligonucleotide. In some further embodiments, the thermally destabilizing modification is located at the 7th position counted from the 5' end of the oligonucleotide.

[0446] The term "thermally destabilizing modification" includes modifications that result in a decrease in the overall melting temperature (Tm) of the dsRNA (preferably a Tm that is 1, 2, 3, or 4 degrees lower than the Tm of dsRNA without such modifications). In some embodiments, the thermally destabilizing modification is located at the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, or 9th position counted from the 5' end of the antisense strand.

[0447] Thermally destabilizing modifications can include, but are not limited to, abasic modifications; mismatches with opposing nucleotides in the opposing strand; and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNA) or glycol nucleic acids (GNA). For example, thermally destabilizing modifications can include, but are not limited to, the following mUNA and GNA building blocks: TIFF2025522811000017.tif106162TIFF2025522811000018.tif127164TIFF2025522811000019.tif173164

[0448] In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA.

[0449] In some embodiments, the destabilizing modification mUNA is TIFF2025522811000020.tif87151R = H, OH; OMe; Cl, F; OH; O-(CH2)2OMe; SMe, NMe2; NH2; Me; CCH (alkyne), O-nPr; O-alkyl; O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp (G-clamp); non-standard monocyclic, bicyclic and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard monocyclic, bicyclic and tricyclic heterocycles selected from the group consisting of, the stereochemistry is R or S, and for chiral centers not specified, it is a combination of R and S.

[0450] In some embodiments, the destabilizing modification mUNA is TIFF2025522811000021.tif52146R = H, OH; OMe; Cl, F; OH; O-(CH2)2OMe; SMe, NMe2; NH2; Me; CCH (alkyne), O-nPr; O-alkyl; O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard monocyclic, bicyclic and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard monocyclic, bicyclic and tricyclic heterocycles selected from the group consisting of, the stereochemistry is R or S, and for chiral centers not specified, it is a combination of R and S.

[0451] In some embodiments, the destabilizing modification mUNA is TIFF2025522811000022.tif85156R = H, OMe; F; OH; O-(CH2)2OMe; SMe, NMe2; NH2; Me; O-nPr; O-alkyl; O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard monocyclic, bicyclic and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 7-deazapurine selected from the group consisting of, the stereochemistry is R or S, and for chiral centers not specified, it is a combination of R and S.

[0452] In some embodiments, the destabilizing modification mUNA is TIFF2025522811000023.tif86151R = H, OH; OMe; Cl, F; OH; O-(CH2)2OMe; SMe, NMe2; NH2; Me; CCH (alkyne), O-nPr; O-alkyl; O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard monocyclic, bicyclic and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard monocyclic, bicyclic and tricyclic heterocycles selected from the group consisting of, the stereochemistry is R or S, and for chiral centers not specified, it is a combination of R and S.

[0453] In some embodiments, the destabilizing modification mUNA is TIFF2025522811000024.tif51146R = H, OH; OMe; Cl, F; OH; O-(CH2)2OMe; SMe, NMe2; NH2; Me; CCH (alkyne), O-nPr; O-alkyl; O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard monocyclic, bicyclic and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard monocyclic, bicyclic and tricyclic heterocycles selected from the group consisting of, the stereochemistry is R or S, and for chiral centers not specified, it is a combination of R and S.

[0454] In some embodiments, the modification mUNA is TIFF2025522811000025.tif84156R = H, OMe; F; OH; O-(CH2)2OMe; SMe, NMe2; NH2; Me; O-nPr; O-alkyl; O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard monocyclic, bicyclic and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 7-deazapurine selected from the group consisting of, the stereochemistry is R or S, and for chiral centers not specified, it is a combination of R and S.

[0455] Exemplary abasic modifications include, but are not limited to, the following modifications: TIFF2025522811000026.tif60128wherein R = H, Me, Et or OMe, R' = H, Me, Et or OMe, R'' = H, Me, Et or OMe TIFF2025522811000027.tif45132wherein B is a modified or unmodified nucleobase, and each structural asterisk represents either R, S or racemic.

[0456] Exemplary sugar modifications include, but are not limited to, the following modifications: TIFF2025522811000028.tif83132wherein B is a modified or unmodified nucleobase, and each structural asterisk represents either R, S or racemic.

[0457] In some embodiments, the double-stranded heat destabilizing modification is selected from the mUNA and GNA building blocks described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments of this, the dsRNA molecule further comprises at least one heat destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (Mod A-Mod K).

[0458] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, e.g., where any of the bonds between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') are absent and / or at least one of the ribose carbons or ribose oxygens (e.g., C1', C2', C3', C4', or O4') is independently or combinatorially deleted from the nucleotide. In some embodiments, the acyclic nucleotide is TIFF2025522811000029.tif35150, where B is a modified or unmodified nucleobase, R1 and R2 are independently H, halogen, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar). The term "UNA" refers to unlocked acyclic nucleic acids in which any of the sugar linkages have been removed to form unlocked "sugar" residues. In one example, UNA also includes monomers in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' carbon and the C4' carbon) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon covalent bond between the C2' carbon and the C3' carbon) is removed (see Mikhailov et.al., Tetrahedron Letters, 26(17):2059(1985) and Fluiter et al., Mol.Biosyst., 10:1039(2009). These references are hereby incorporated by reference in their entireties). The acyclic derivatives increase the flexibility of the backbone without affecting Watson-Crick pairing. Acyclic nucleotides can be linked by 2'-5' linkages or 3'-5' linkages.

[0459] The term "GNA" refers to glycol nucleic acid, a polymer similar to DNA or RNA but differing in that the composition of its "backbone" is composed of repeating glycerol units linked by phosphodiester bonds: Refers to TIFF2025522811000030.tif57128.

[0460] Thermal destabilizing modifications of the double strand can be mismatches (i.e., non-complementary base pairs) between a thermally destabilizing nucleotide in the dsRNA double strand and the opposing nucleotide in the reverse strand. Exemplary mismatched base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatched base pairings known in the art are also applicable to the present invention. The mismatches can occur between nucleotides that are either natural nucleotides or modified nucleotides. That is, mismatched base pairing can occur between the nucleic acid bases of each nucleotide, regardless of the modifications on the ribose sugar of the nucleotide. In certain embodiments, the dsRNA molecule contains at least one nucleic acid base that is a 2'-deoxy nucleic acid base during mismatched pairing, for example, the 2'-deoxy nucleic acid base is in the sense strand.

[0461] In some embodiments, the thermal destabilizing modification of the double strand in the seed region of the antisense strand is a nucleotide in which the W-C type H-bond to the complementary base on the target mRNA is impaired, for example including TIFF2025522811000031.tif72135.

[0462] Further examples of abasic nucleotide modifications, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are detailed in WO 2011 / 133876, which is hereby incorporated by reference in its entirety.

[0463] Thermal destabilizing modifications can also include universal bases and phosphate modifications in which the ability to hydrogen bond to the opposing base is reduced or lost.

[0464] In some embodiments, heat destabilizing modifications include nucleotides having non-standard bases, such as, without limitation, nucleobase modifications in which the ability to form hydrogen bonds with bases in the reverse strand is impaired or completely lost. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex, as described in WO 2010 / 0011895, which is hereby incorporated by reference in its entirety. Exemplary nucleobase modifications are TIFF2025522811000032.tif63150.

[0465] In some embodiments, heat destabilizing modifications of the duplex in the seed region of the antisense strand include one or more α-nucleotides complementary to bases on the target mRNA, such as TIFF2025522811000033.tif20146, where R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0466] Exemplary phosphate modifications known to decrease the thermal stability of the dsRNA duplex compared to the native phosphodiester linkage are TIFF2025522811000034.tif31139.

[0467] Alkyl as the R group can be C1-C6 alkyl. Specific alkyls as the R group include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.

[0468] In some embodiments of any one of the aspects described herein, the oligonucleotide can include one or more stabilizing modifications. For example, the oligonucleotide can include at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) stabilizing modifications.

[0469] In some embodiments, the oligonucleotide comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) stabilizing modifications. Without limitation, the stabilizing modifications in the oligonucleotide can be present at any position. In some embodiments, the oligonucleotide comprises stabilizing modifications at the 2nd, 6th, 8th, 9th, 14th, and 16th positions counting from the 5' end. In some other embodiments, the oligonucleotide comprises stabilizing modifications at the 2nd, 6th, 14th, and 16th positions counting from the 5' end. In some further other embodiments, the oligonucleotide comprises stabilizing modifications at the 2nd, 14th, and 16th positions counting from the 5' end. In some embodiments, the oligonucleotide comprises stabilizing modifications at the 7th, 10th, and 11th positions counting from the 5' end. In some other embodiments, the oligonucleotide comprises stabilizing modifications at the 7th, 9th, 10th, and 11th positions counting from the 5' end.

[0470] In some embodiments, the oligonucleotide comprises at least one stabilizing modification adjacent to a destabilizing modification. For example, the stabilizing modification can be a nucleotide at the 5' or 3' end of the destabilizing modification, i.e., at the -1 or +1 position from the position of the destabilizing modification. In some embodiments, the oligonucleotide comprises stabilizing modifications at each of the 5' and 3' ends of the destabilizing modification, i.e., at the -1 and +1 positions from the position of the destabilizing modification.

[0471] In some embodiments, the oligonucleotide comprises at least two stabilizing modifications at the 3' end of the destabilizing modification, i.e., at least two stabilizing modifications at the +1 and +2 positions from the position of the destabilizing modification.

[0472] Exemplary thermal stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermal stabilizing modifications include, but are not limited to, LNA.

[0473] double-stranded RNA It is well known to those skilled in the art that double-stranded RNAs containing a double-stranded structure of 20 to 23 base pairs, particularly 21 base pairs, are particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20: 6877-6888). However, other researchers have found that shorter or longer double-stranded oligonucleotides may be equally effective.

[0474] Thus, in one aspect, provided herein is a double-stranded RNA (dsRNA) comprising a first strand (also referred to as the antisense strand or guide strand) and a second strand (also referred to as the sense strand or passenger strand), wherein at least one of the first strand (i.e., the antisense strand) or the second strand (i.e., the sense strand) is an oligonucleotide as described herein. In other words, at least one of the first strand (i.e., the antisense strand) or the second strand (i.e., the sense strand) comprises at least one nucleotide of formula (II).

[0475] In some embodiments of any one of the aspects described herein, the antisense strand is substantially complementary to a target nucleic acid, such as a target gene or a target mRNA gene, and the dsRNA can induce target cleavage of the target nucleic acid. Without limitation, the dsRNA of the present invention can be used in place of a dsRNA molecule and can be used in gene silencing techniques based on RNA interference, including, without limitation, in vitro applications or in vivo applications.

[0476] In some embodiments of any one of the aspects described herein, the sense strand is an oligonucleotide as described herein. In other words, the sense strand comprises at least one nucleotide of formula (II).

[0477] In some embodiments of any one of the aspects described herein, the antisense strand is an oligonucleotide as described herein. In other words, the antisense strand comprises at least one nucleotide of formula (II).

[0478] As described herein, the dsRNA molecules described herein can include at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides of formula (II). Without limitation, the nucleotides of formula (II) can be present in one strand. The nucleotides of formula (II) can be present at any nucleotide at any position of the sense strand or the antisense strand, or both strands.

[0479] In some embodiments, the sense strand includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides of formula (II) described herein. The nucleotides of formula (II) described herein can be present at any position of the sense strand. For example, the nucleotides of formula (II) described herein can be present in the terminal region of the sense strand. For example, the nucleotides of formula (II) described herein can be present in one or more of the 1st, 2nd, 3rd, and 4th positions counted from the 5' end of the sense strand. In another non-limiting example, the nucleotides of formula (II) described herein can be present in one or more of the 1st, 2nd, 3rd, and 4th positions counted from the 3' end of the sense strand. In some embodiments, the nucleotides of formula (II) can be present in one or more of the 18th, 19th, 20th, and 21st positions counted from the 5' end of the sense strand. The nucleotides of formula (II) described herein can also be located in the central region of the sense strand. For example, the nucleotides of formula (II) described herein can be located in one or more of the 6th, 7th, 8th, 9th, 10th, 11th, 12th, and 13th positions counted from the 5' end of the sense strand. In some embodiments, the nucleotides of formula (II) are at the 5' end of the sense strand.

[0480] In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides of formula (II) described herein. The nucleotides of formula (II) described herein can be present at any position of the antisense strand. For example, the nucleotides of formula (II) described herein can be present in the terminal regions of the antisense strand. For example, the nucleotides of formula (II) described herein can be present in one or more of the 1st, 2nd, 3rd, and 4th positions counting from the 5' end of the antisense strand. In another non-limiting example, the nucleotides of formula (II) described herein can be present in one or more of the 1st, 2nd, 3rd, 4th, 5th, and 6th positions counting from the 3' end of the antisense strand. In some embodiments, the nucleotides of formula (II) described herein can be present in one or more of the 18th, 19th, 20th, 21st, 22nd, and 23rd positions counting from the 5' end of the antisense strand. The nucleotides of formula (II) described herein can also be located in the central region of the antisense strand. For example, the nucleotides of formula (II) described herein can be located in one or more of the 6th, 7th, 8th, 9th, 10th, 11th, 12th, and 13th positions counting from the 5' end of the antisense strand. In some embodiments, the nucleotides of formula (II) are at the 3' end of the antisense strand.

[0481] Each strand of the dsRNA molecule can be in the range of 15 to 35 nucleotides in length. For example, each strand can be 17 to 35 nucleotides in length, 17 to 30 nucleotides in length, 25 to 35 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length. Without limitation, the sense strand and the antisense strand may or may not be of equal length. For example, the sense strand and the antisense strand can independently have a length of 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0482] In some embodiments, the antisense strand is 15 to 35 nucleotides in length. In some embodiments, the antisense strand is 15 to 35, 17 to 35, 17 to 30, 25 to 35, 27 to 30, 17 to 23, 17 to 21, 17 to 19, 19 to 25, 19 to 23, 19 to 21, 21 to 25, 21 to 25 or 21 to 23 nucleotides in length. For example, the antisense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length. In some embodiments, the antisense strand is 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, the antisense strand is 21, 22, 23, 24 or 25 nucleotides in length. In some specific embodiments, the antisense strand is 22, 23 or 24 nucleotides in length. For example, the antisense strand is 23 nucleotides in length.

[0483] Similar to the antisense strand, the sense strand can also be 15 to 35 nucleotides in length in some embodiments. In some embodiments, the sense strand is 15 to 35, 17 to 35, 17 to 30, 25 to 35, 27 to 30, 17 to 23, 17 to 21, 17 to 19, 19 to 25, 19 to 23, 19 to 21, 21 to 25, 21 to 25 or 21 to 23 nucleotides in length. For example, the sense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length. In some embodiments, the sense strand is 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, the sense strand is 19, 20, 21, 22 or 23 nucleotides in length. In some specific embodiments, the sense strand is 20, 21 or 22 nucleotides in length. For example, the sense strand is 21 nucleotides in length.

[0484] In some embodiments, the sense strand can be 15 to 35 nucleotides in length, and the antisense strand can be 15 to 35 nucleotides in length independently of the sense strand. In some embodiments, the sense strand is 15 to 35, 17 to 35, 17 to 30, 25 to 35, 27 to 30, 17 to 23, 17 to 21, 17 to 19, 19 to 25, 19 to 23, 19 to 21, 21 to 25, 21 to 25 or 21 to 23 nucleotides in length, and the antisense strand is independently 15 to 35, 17 to 35, 17 to 30, 25 to 35, 27 to 30, 17 to 23, 17 to 21, 17 to 19, 19 to 25, 19 to 23, 19 to 21, 21 to 25, 21 to 25 or 21 to 23 nucleotides in length. For example, the sense strand and the antisense strand can independently be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length. In some embodiments, the sense strand and the antisense strand are independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, the sense strand is 19, 20, 21, 22 or 23 nucleotides in length, and the antisense strand is 21, 22, 23, 24 or 25 nucleotides in length. In some specific embodiments, the sense strand is 20, 21 or 22 nucleotides in length, and the antisense strand is 22, 23 or 24 nucleotides in length. For example, the sense strand is 21 nucleotides in length, and the antisense strand is 23 nucleotides in length.

[0485] The sense strand and the antisense strand typically form a double-stranded region or a duplex region. Without limitation, the double-stranded region of the dsRNA agents described herein can be 12 to 35 nucleotide (or base) pairs in length. For example, the double-stranded region can be 14 to 35 nucleotide pairs in length, 17 to 30 nucleotide pairs in length, 25 to 35 nucleotides in length, 27 to 35 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 21 nucleotide pairs in length, 17 to 19 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, 21 to 25 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length. In some embodiments, the double-stranded region is 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs in length. For example, the double-stranded region is 19, 20, 21, 22, or 23 nucleotide pairs in length. In some embodiments, the double-stranded region is 20, 21, or 22 nucleotide pairs in length. For example, the dsRNA molecule has a double-stranded region of 21 base pairs.

[0486] Use of Oligonucleotides The oligonucleotides described herein can be used for any use known in the art with respect to oligonucleotides. For example, the oligonucleotides described herein can be used in gene silencing techniques based on RNA interference. Some exemplary uses for the oligonucleotides described herein include, but are not limited to, RNA interference agents, antisense oligonucleotides, aptamers, miRNAs, ribozymes, triplex-forming oligonucleotides, and the like.

[0487] Accordingly, in another aspect, the present disclosure is directed to the use of the oligonucleotides and / or dsRNA molecules described herein for inhibiting the expression of a target gene. In some embodiments, the invention further relates to the use of the oligonucleotides and / or dsRNA molecules described herein for inhibiting the expression of a target gene in vitro.

[0488] In another aspect, the present disclosure is directed to the use of the oligonucleotides and / or dsRNA molecules described herein for use in inhibiting the expression of a target gene in a subject. The subject can be any animal, such as a mammal, such as a mouse, rat, sheep, cow, dog, cat, or human.

[0489] In some embodiments, the oligonucleotides and / or dsRNA molecules described herein are administered in a buffer.

[0490] In some embodiments, the oligonucleotides and / or dsRNA molecules described herein can be formulated for administration to a subject. The formulated oligonucleotide and / or dsRNA compositions can take various forms. In some examples, the composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% moisture). In another example, the siRNA is in an aqueous phase, such as in a solution containing water.

[0491] The aqueous or crystalline composition can be incorporated into, for example, a delivery vehicle, such as a liposome (particularly for aqueous phases) or a particle (e.g., microparticles that may be suitable for crystalline compositions). Generally, siRNA compositions are formulated in a manner compatible with the intended method of administration, as described herein. For example, in certain embodiments, the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication, freeze drying, condensation, and other self-assembly with lipids.

[0492] Preparations of oligonucleotides and / or dsRNA can be formulated in combination with another agent, such as another therapeutic agent, or an agent that stabilizes the oligonucleotide and / or dsRNA, such as a protein that complexes with the oligonucleotide and / or dsRNA. Still other agents include chelating agents, such as EDTA (e.g., to remove divalent cations such as Mg 2+ etc.), salts, RNase inhibitors (e.g., broad-specificity RNase inhibitors such as RNasin), and the like.

[0493] In some embodiments, preparations of oligonucleotides and / or dsRNA include another dsRNA compound, such as a second dsRNA capable of mediating RNAi with respect to a second gene or the same gene. Still other preparations can include at least 3, 5, 10, 20, 50, or 100 or more different siRNA species. Such dsRNAs can mediate RNAi with respect to a similar number of different genes.

[0494] In some embodiments, preparations of oligonucleotides and / or dsRNA include at least a second therapeutic agent (e.g., an agent other than RNA or DNA). For example, a composition of oligonucleotides and / or dsRNA for treating a viral disease, such as HIV, may include a known antiviral agent (e.g., a protease inhibitor or a reverse transcriptase inhibitor). In another example, a dsRNA composition for treating cancer may further include a chemotherapeutic agent.

[0495] Exemplary formulations that can be used to administer the oligonucleotides and / or dsRNA of the present invention are discussed below.

[0496] Liposomes. Preparations of oligonucleotides and / or dsRNA can be formulated into membrane molecular assemblies, such as liposomes or micelles, for delivery. As used herein, the term "liposome" refers to vesicles composed of amphiphilic lipids arranged in at least one bilayer, such as one bilayer or multiple bilayers. Liposomes include unilamellar vesicles and multilamellar vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains a composition of oligonucleotides and / or dsRNA. The lipophilic material isolates the aqueous interior from the aqueous exterior, which typically does not contain a composition of oligonucleotides and / or dsRNA, although in some instances it may contain a composition of oligonucleotides and / or dsRNA. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Since the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissues, the bilayer of the liposome fuses with the bilayer of the cell membrane. As the association between the liposome and the cell progresses, the inner aqueous contents containing oligonucleotides and / or dsRNA are delivered into the cell, where the dsRNA can specifically bind to the target RNA and mediate RNAi. In some embodiments, the liposomes are also specifically targeted, for example, to direct oligonucleotides and / or dsRNA to specific cell types.

[0497] Liposomes containing oligonucleotides and / or dsRNA can be prepared by various methods. In one example, the lipid components of the liposome are dissolved in a detergent such that micelles are formed by the lipid components. For example, the lipid components can be amphiphilic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include cholic acid, CHAPS, octyl glucoside, deoxycholic acid, and lauroyl sarcosine. Next, a dsRNA preparation is added to the micelles containing the lipid components. The cationic groups on the lipid interact with the siRNA and condense around the dsRNA to form liposomes. After condensation, the detergent is removed, such as by dialysis, to obtain a liposome preparation of the oligonucleotide and / or dsRNA.

[0498] If necessary, a carrier compound that aids in condensation can be added during the condensation reaction, such as by controlled addition. For example, the carrier compound can be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to be favorable for condensation.

[0499] Further description of methods for producing a stable polynucleotide delivery medium incorporating a polynucleotide / cationic lipid complex as a component of the delivery medium is described, for example, in WO96 / 37194. Liposome formation can also include one or more aspects of the exemplary methods described in Felgner, P.L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987, U.S. Patent No. 4,897,355, U.S. Patent No. 5,171,678, Bangham, et al. M. Mol. Biol. 23:238, 1965, Olson, et al. Biochim. Biophys. Acta 557:9, 1979, Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978, Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984, Kim, et al. Biochim. Biophys. Acta 728:339, 1983 and Fukunaga, et al. Endocrinol. 115:757, 1984, the disclosures of which are incorporated herein by reference in their entirety. Techniques commonly used to prepare lipid aggregates of a size suitable for use as a delivery medium include sonication and freeze-thawing and extrusion (see, for example, Mayer, et al. Biochim. Biophys. Acta 858:161, 1986, the disclosure of which is incorporated herein by reference in its entirety). Microfluidic techniques can be used if consistently small (50-200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984, the disclosure of which is incorporated herein by reference in its entirety). These methods can be readily adapted for the packaging of oligonucleotides and / or siRNA preparations into liposomes.

[0500] pH-sensitive or negatively charged liposomes do not form complexes with nucleic acid molecules, but rather encapsulate the nucleic acids. Since both nucleic acid molecules and lipids are similarly charged, repulsion occurs rather than complex formation. Nevertheless, some nucleic acid molecules are encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to monolayer cultures of cells. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274, which is incorporated herein by reference in its entirety).

[0501] One of the major types of liposome compositions contains phospholipids other than naturally derived phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic membrane-fusogenic liposomes are mainly formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soy PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0502] Examples of other methods for introducing liposomes into cells in vitro include U.S. Patent No. 5,283,185, U.S. Patent No. 5,171,678, WO94 / 00569, WO93 / 24640, WO 91 / 16024, Felgner, J. Biol. Chem. 269:2550, 1994, Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993, Nabel, Human Gene Ther. 3:649, 1992, Gershon, Biochem. 32:7143, 1993 and Strauss EMBO J. 11:417, 1992.

[0503] In some embodiments, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with cell membranes. Although non-cationic liposomes cannot efficiently fuse with the plasma membrane, they can be taken up by macrophages in vivo and used to deliver siRNA to macrophages.

[0504] Additional advantages of liposomes include that liposomes obtained from natural phospholipids are biocompatible and biodegradable, that liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs, and that liposomes can protect the siRNA encapsulated within their inner compartment from metabolism and degradation (Rosoff, "Pharmaceutical Dosage Forms", Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

[0505] Small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes can be formed using the positively charged synthetic cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). These liposomes have the ability to deliver siRNA as a result of fusing with the negatively charged lipids of the cell membranes of tissue culture cells (for its use with DOTMA and DNA, see, for example, Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Patent No. 4,897,355, which are incorporated herein by reference in their entirety).

[0506] By using DOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) in combination with a phospholipid, DNA complexing vesicles can be formed. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Maryland) is an effective agent for delivering highly anionic nucleic acids into living tissue culture cells and contains positively charged DOTMA liposomes that interac...

Claims

1. A compound of formula (I): Wherein, B is an optionally modified nucleobase, R 2 is R MA , hydrogen, hydroxyl, protected hydroxyl, phosphate group, reactive phosphorus(III) group, halogen, optionally substituted C 1～30 alkyl, optionally substituted C 2～30 alkenyl, optionally substituted C 2～30 alkynyl, optionally substituted C 1～30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxocarboxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support, R MA is -O(CH 2 ) m1 -X M' -R M' or -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) and Y M is O or S, X M' is N(R MX ), O, or S, where R MX is hydrogen or R M' and R M' is optionally substituted C 6～30 alkyl, optionally substituted C 6～30 alkenyl, optionally substituted C 6～30 alkynyl, optionally substituted 3- to 8-membered heterocyclyl C 3～30 alkyl, optionally substituted C 3～10 cycloalkyl C 3～30 alkyl, optionally substituted aryl C 3～30 alkyl, optionally substituted heteroaryl C 3～30 alkyl, optionally substituted C 1～30 alkoxy C 1～30 alkyl, -(CH 2 CH 2 O) mq -R MQ is a lipid, ligand, linker, or a linker for one or more ligands, where mq is an integer selected from 1 to 10, and R MQ is hydrogen or C 1～6 alkyl, and Optionally, R M' is substituted at the end with an anion group or a cation group, m1 is an integer from 1 to 10, n1 is an integer from 1 to 10, R N' and R N'' each independently is hydrogen, optionally substituted C 6～30 alkyl, optionally substituted C 6～30 alkenyl, optionally substituted C 6～30 alkynyl, or optionally substituted C 3～30 cycloalkyl; a lipid, ligand, linker, or linker for one or more ligands, provided that at least one of R N' and R N'' is not hydrogen, R 3 is R MA , hydrogen, hydroxyl, protected hydroxyl, phosphate group, reactive phosphorus(III) group, halogen, optionally substituted C 1～30 alkyl, optionally substituted C 2～30 alkenyl, optionally substituted C 2～30 alkynyl, optionally substituted C 1～30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxocarboxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 )、-O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support, R 4 is R MA , hydrogen, optionally substituted C 1～6 alkyl, optionally substituted C 2～6 alkenyl, optionally substituted C 2～6 alkynyl, or optionally substituted C 1～6 alkoxy, and R 5 is R MA , hydrogen, hydroxyl, protected hydroxyl, optionally substituted C 1～30 alkyl, optionally substituted C 2～30 alkenyl, optionally substituted C 2～30 alkynyl, optionally substituted C 1～30 alkoxy, optionally substituted 3- to 8-membered heterocyclyl (e.g., morpholin-1-yl, piperidin-1-yl, or pyrrolidin-1-yl), halogen, alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxocarboxylate, amino, alkylamino, dialkylamino, -O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 )、-O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), vinylphosphonate (VP) group (e.g., =CH-X P , X P is a phosphate group), C 3～6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), monophosphate ((HO) 2 (O)P-O-5'), diphosphate ((HO) 2 (O)P-O-P(HO)(O)-O-5'), triphosphate ((HO) 2 (O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidate ((HO) 2 (O)P-NH-5', (HO)(NH 2 )(O)P-O-5'), alkylphosphonate [(R P )(OH)(O)P-O-5', R P is optionally substituted C 1～30 alkyl, such as methyl, ethyl, isopropyl, or propyl)], alkyl ether phosphonate [(R P1 )(OH)(O)P-O-5', R P1 is alkoxyalkyl, such as methoxymethyl (CH 2 OMe) or ethoxymethyl], (HO) 2 (X)P-O[-(CH 2 ) a -O-P(X)(OH)-O] b -5' or (HO) 2 (X)P-O[-(CH 2 ) a -P(X)(OH)-O] b -5' or (HO) 2 (X)P-[-(CH 2 ) a -O-P(X)(OH)-O] b -5', or optionally substituted alkyl, and dialkyl terminal phosphate and phosphate mimics (e.g., HO[-(CH 2 ) a -O-P(X)(OH)-O] b -5', H 2 N[-(CH 2 ) a -O-P(X)(OH)-O] b -5', H[-(CH 2 ) a -O-P(X)(OH)-O] b -5', Me 2 N[-(CH 2 ) a -O-P(X)(OH)-O] b -5', HO[-(CH 2 ) a -P(X)(OH)-O] b -5', H 2 N[-(CH 2 ) a -P(X)(OH)-O] b -5', H[-(CH 2 ) a -P(X)(OH)-O] b -5', Me 2 N[-(CH 2 ) a -P(X)(OH)-O] b -5' and wherein X is O or S, a and b are each independently 1 to 10, Each R 8 and R 9 are independently H, a targeting ligand (e.g., GalNac), a pharmacokinetic modifier, an optionally substituted C 1～30 alkyl, an optionally substituted C 1～30 alkenyl, or an optionally substituted C 1～30 alkynyl, Provided that, (i) R 2 , R 3 , R 4 and R 5 at least one of which is R MA and (ii) R 2 、R 3 、R 4 and R 5 only one of MA is R (iii) R 2 and R 3 only one of which is a reactive phosphorus(III) group, a solid support, or a linker covalently attached to a solid support, (iv) R 2 is -OCH 2 CH 2 -O-R M' where R 5 is hydroxyl or protected hydroxyl, R 4 is H, and R 3 is hydroxyl, protected hydroxyl, phosphate group or reactive phosphorus (III) group, R M' is neither unsubstituted C 6～21 alkyl, nor unsubstituted C 6～21 alkenyl, nor unsubstituted C 6～21 alkynyl, (v) R 2 is -O(CH 2 ) n1 -C(O)N(R N' )(R N'' ), n1 is 1, R 5 is hydroxyl or protected hydroxyl, R 4 is H, R 3 is hydroxyl, protected hydroxyl, phosphate group or reactive phosphorus (III) group, and when one of R N' and R N'' is H, the other of R N' and R N'' is not -(CH 2 ) 6 CH 3 , nor -(CH 2 ) 7 CH 3 , nor -(CH 2 ) 8 CH 3 , nor -(CH 2 ) 5 NHCOCF 3 , nor -(CH 2 ) 6 NHCOCF 3 , nor -(CH 2 ) 7 NHCOCF 3 , nor -(CH 2 ) 5 N(CH 3 ) 2 , nor -(CH 2 ) 6 N(CH 3 ) 2 , nor -(CH 2 ) 7 N(CH 3 ) 2 either.

2. R 2 is -O(CH 2 ) m1 -X M' -R M' (e.g., -OCH 2 CH 2 -X M' -R M' ) and is the compound according to claim 1.

3. X M' The compound according to claim 2, wherein X is O.

4. X M' The compound according to claim 2, wherein [is S].

5. R 2 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) and is the compound according to claim 1.

6. The compound according to claim 5, wherein n1 is 1.

7. The compound according to claim 5, wherein n1 is 2.

8. R 3 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to a solid support, a compound according to any one of claims 2 to 7.

9. R 3 The compound according to any one of claims 2 to 8, wherein R is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus (III) group, solid support, linker, or a linker covalently attached to the solid support.

10. R 3 The compound according to any one of claims 2 to 9, wherein R is a reactive phosphorus (III) group, a solid support, a linker, or a linker covalently attached to a solid support.

11. R 3 The compound according to any one of claims 2 to 10, wherein R is reactive phosphorus (III) or a linker covalently attached to a solid support.

12. R 3 wherein R is a reactive phosphorus (III) group (e.g., a phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite), a compound according to any one of claims 2 to 11.

13. R 3 The compound according to any one of claims 2 to 11, wherein R is a linker covalently attached to the solid support.

14. R 5 is hydroxyl, protected hydroxyl, optionally substituted C 1～30 alkoxy, vinyl phosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkyl phosphonate, alkyl ether phosphonate, dialkyl terminal phosphate or a phosphate mimetic, a compound according to any one of claims 2 to 13.

15. R 5 is a hydroxyl, protected hydroxyl, vinylphosphonate (VP) group, cyclopropylphosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, or phosphate mimetic, the compound according to any one of claims 2 to 14.

16. R 5 The compound according to any one of claims 2 to 15, wherein R is hydroxyl or protected hydroxyl.

17. R 3 is -O(CH 2 ) m1 -X M' -R M' (for example -OCH 2 CH 2 -X M' -R M' ) and is the compound according to claim 1.

18. X M' The compound according to claim 17, wherein X is O.

19. X M' The compound according to claim 17, wherein X is S.

20. R 3 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) as defined in claim 1, the compound according to claim 1.

21. The compound according to claim 20, wherein n1 is 1.

22. The compound according to claim 20, wherein n1 is 2.

23. R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to a solid support, a compound according to any one of claims 17 to 22.

24. R 2 The compound according to any one of claims 17 to 23, wherein R is hydrogen, hydroxyl, protected hydroxyl, halogen, reactive phosphorus (III) group, solid support, linker, or a linker covalently attached to the solid support.

25. R 2 The compound according to any one of claims 17 to 24, wherein R is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus (III) group, solid support, linker, or a linker covalently attached to the solid support.

26. R 2 The compound according to any one of claims 17 to 25, wherein R is a reactive phosphorus (III) group, a solid support, a linker, or a linker covalently attached to the solid support.

27. R 2 The compound according to any one of claims 17 to 26, wherein R is reactive phosphorus (III) or a linker covalently attached to a solid support.

28. R 2 wherein R is a reactive phosphorus(III) group such as a phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite, the compound according to any one of claims 17 to 27.

29. R 2 The compound according to any one of claims 17 to 27, wherein R is a linker covalently attached to the solid support.

30. R 5 is hydroxyl, protected hydroxyl, optionally substituted C 1～30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate or a phosphate mimetic, a compound according to any one of claims 17 to 29.

31. R 5 wherein R is hydroxyl, protected hydroxyl, vinylphosphonate (VP) group, cyclopropylphosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, or a phosphate mimetic, and the compound according to any one of claims 17 to 30.

32. R 5 The compound according to any one of claims 17 to 31, wherein R is hydroxyl or protected hydroxyl.

33. R 5 is -O(CH 2 ) m1 -X M' -R M' (for example, -OCH 2 CH 2 -X M' -R M' ) and the compound according to claim 1.

34. X M' The compound according to claim 33, wherein X is O.

35. X M' The compound according to claim 33, wherein X is S.

36. R 5 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) as described in claim 1, the compound according to claim 1.

37. The compound according to claim 36, wherein n1 is 1.

38. The compound according to claim 36, wherein n1 is 2.

39. R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to a solid support, a compound according to any one of claims 33 to 38.

40. R 2 The compound according to any one of claims 33 to 39, wherein R is hydrogen, hydroxyl, protected hydroxyl, halogen, reactive phosphorus (III) group, solid support, linker, or a linker covalently attached to a solid support.

41. R 2 The compound according to any one of claims 33 to 40, wherein R is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus (III) group, solid support, linker, or a linker covalently attached to the solid support.

42. R 2 The compound according to any one of claims 33 to 41, wherein R is a reactive phosphorus (III) group, a solid support, a linker, or a linker covalently attached to the solid support.

43. R 2 The compound according to any one of 33 to 42, wherein R is reactive phosphorus (III) or a linker covalently attached to a solid support.

44. R 2 wherein R is a reactive phosphorus(III) group, such as a phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite, a compound according to any one of claims 33 to 43.

45. R 2 The compound according to any one of claims 33 to 44, wherein R is a linker covalently attached to the solid support.

46. R 3 wherein R is hydrogen, hydroxyl, protected hydroxyl, halogen, or optionally substituted C 1～30 alkoxy, and the compound according to any one of claims 33 to 45.

47. R 3 The compound according to any one of claims 33 to 46, wherein R is hydroxyl or protected hydroxyl.

48. R 3 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to a solid support, a compound according to any one of claims 33 to 38.

49. R 3 The compound according to claim 48, wherein R is hydrogen, hydroxyl, protected hydroxyl, halogen, reactive phosphorus (III) group, solid support, linker, or a linker covalently attached to a solid support.

50. R 3 The compound according to any one of claims 48 to 49, wherein R is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus (III) group, solid support, linker, or a linker covalently attached to the solid support.

51. R 3 The compound according to any one of claims 48 to 50, wherein R is a reactive phosphorus (III) group, a solid support, a linker, or a linker covalently attached to the solid support.

52. R 3 The compound according to any one of 48 to 51, wherein R is reactive phosphorus (III) or a linker covalently attached to a solid support.

53. R 3 wherein R is a reactive phosphorus(III) group such as a phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite, a compound according to any one of claims 48 to 52.

54. R 3 The compound according to any one of claims 48 to 52, wherein R is a linker covalently attached to the solid support.

55. R 2 wherein R is hydrogen, hydroxyl, protected hydroxyl, halogen, or optionally substituted C 1～30 alkoxy, and the compound according to any one of claims 48 to 54.

56. R 2 The compound according to any one of claims 48 to 55, wherein R is hydroxyl or protected hydroxyl.

57. R 4 The compound according to any one of claims 1 to 56, wherein R is H.

58. A compound of formula (I-A): Wherein, B is an optionally modified nucleobase, R 2 is -O(CH 2 ) m1 -X M' -R M' (e.g., -OCH 2 CH 2 -X M' -R M' ), hydroxyl, protected hydroxyl, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to a solid support, R 3 is -O(CH 2 ) m1 -X M' -R M' (e.g., -OCH 2 CH 2 -X M' -R M' ), a hydroxyl, a protected hydroxyl, a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support, R 4 is -O(CH 2 ) m1 -X M' -R M' (e.g., -OCH 2 CH 2 -X M' -R M' ), hydrogen, optionally substituted C 1～6 alkyl, optionally substituted C 2～6 alkenyl, optionally substituted C 2～6 alkynyl, or optionally substituted C 1～6 alkoxy, and R 5 is -O(CH 2 ) m1 -X M' -R M' (e.g., -OCH 2 CH 2 -X M' -R M' ), hydroxyl or protected hydroxyl, R M' is optionally substituted C 6～30 alkyl, optionally substituted C 6～30 alkenyl, optionally substituted C 6～30 alkynyl, optionally substituted 3- to 8-membered heterocyclyl C 3～30 alkyl, optionally substituted C 3～10 cycloalkyl C 3～30 alkyl, optionally substituted aryl C 3～30 alkyl, optionally substituted heteroaryl C 3～30 alkyl, optionally substituted C 1～30 alkoxy C 1～30 alkyl, -(CH 2 CH 2 O) mq -R MQ , a lipid, a ligand, a linker, or a linker for one or more ligands, where mq is an integer selected from 1 to 10, and R MQ is hydrogen or C 1～6 alkyl, and Optionally, R M' is substituted at the terminus with an anion group or a cation group, X M' is N(R MX ), O or S, and Provided that, (i) R 2 , R 3 , R 4 and R 5 of which at least one is -O(CH 2 ) m1 -X M' -R M' (e.g., -OCH 2 CH 2 -X M' -R M' ) and (ii) R 2 、R 3 、R 4 and R 5 of which only one is -O(CH 2 ) m1 -X M' -R M' (e.g., -OCH 2 CH 2 -X M' -R M' ) is true, (iii) R 2 and R 3 only one of which is a reactive phosphorus(III) group, a solid support, or a linker covalently attached to a solid support, (iv) R 2 is -OCH 2 CH 2 -O-R M' and R 5 is hydroxyl or protected hydroxyl, R 4 is H, and R 3 is hydroxyl, protected hydroxyl, phosphate group or reactive phosphorus(III) group, R M' is unsubstituted C 6～21 alkyl, unsubstituted C 6～21 alkenyl, or unsubstituted C 6～21 alkynyl.

59. R 2 is -O(CH 2 ) m1 -X M' -R M' (for example, -OCH 2 CH 2 -X M' -R M' ) and is the compound according to claim 58.

60. R 3 The compound according to claim 59, wherein R is a hydroxyl group.

61. R 3 The compound according to claim 59, wherein R is a protected hydroxyl.

62. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), a compound according to claim 61.

63. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, a compound according to claim 62.

64. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, the compound according to claim 63.

65. R 3 The compound according to claim 59, wherein R is a reactive phosphorus (III) group.

66. The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 )、 -OP(SR P )(N(R P2 ) 2 )、 -OP(O)(OR P )(N(R P2 ) 2 )、 -OP(S)(OR P )(N(R P2 ) 2 )、 -OP(O)(SR P )(N(R P2 ) 2 )、 -OP(O)(OR P )H、 -OP(S)(OR P )H、 -OP(O)(SR P )H、 -OP(O)(OR P )R P3 、 -OP(S)(OR P )R P3 、 or -OP(O)(SR P )R P3 , the compound according to claim 65.

67. The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 ), the compound according to claim 66.

68. A reactive phosphorus(III) group is OP(OR P )(N(R P2 ) 2 ), where R p is cyanoethyl (-CH 2 CH 2 CN), and each R P2 is isopropyl, or both Rs P2 , together with the nitrogen atom to which they are attached, form an optionally substituted 3- to 8-membered heterocyclyl, a compound according to claim 67.

69. R 3 The compound according to claim 59, wherein R is a linker or a linker attached to a solid support.

70. R 5 The compound according to any one of claims 59 to 69, wherein R is hydroxyl.

71. R 5 The compound according to any one of claims 59 to 69, wherein R is a protected hydroxyl.

72. The protected hydroxyl is -OR Pro wherein, R Pro is an oxygen protecting group, the compound according to claim 71.

73. R Pro The compound according to claim 72, wherein R is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX).

74. R Pro The compound according to claim 73, wherein R is 4,4'-dimethoxytrityl.

75. R 3 is -O(CH 2 ) m1 -X M' -R M' (for example, -OCH 2 CH 2 -X M' -R M' ) and is a compound according to claim 58.

76. R 2 The compound according to claim 75, wherein R is a hydroxyl group.

77. R 2 The compound according to claim 75, wherein R is a protected hydroxyl.

78. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), and the compound according to claim 77

79. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, a compound according to claim 78.

80. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, the compound according to claim 79

81. R 2 The compound according to claim 75, wherein R is a reactive phosphorus (III) group.

82. The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 )、-OP(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )(N(R P2 ) 2 )、-OP(S)(OR P )(N(R P2 ) 2 )、-OP(O)(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )H、-OP(S)(OR P )H、-OP(O)(SR P )H、-OP(O)(OR P )R P3 、-OP(S)(OR P )R P3 、 or -OP(O)(SR P )R P3 , the compound according to claim 81.

83. The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 ), the compound according to claim 82.

84. The reactive phosphorus (III) group is OP(OR P )(N(R P2 ) 2 ), where R p is cyanoethyl (-CH 2 CH 2 CN), and each R P2 is isopropyl, or both R P2 , together with the nitrogen atom to which they are attached, form an optionally substituted 3- to 8-membered heterocyclyl, a compound according to claim 83.

85. R 2 The compound according to claim 84, wherein R is a linker or a linker attached to a solid support.

86. R 5 The compound according to any one of claims 75 to 85, wherein R is hydroxyl.

87. R 5 The compound according to any one of claims 75 to 85, wherein R is a protected hydroxyl.

88. The protected hydroxyl is -OR Pro wherein, R Pro is an oxygen protecting group, the compound according to claim 87.

89. R Pro The compound according to claim 88, wherein R is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX).

90. R Pro The compound according to claim 89, wherein R is 4,4'-dimethoxytrityl.

91. R 5 is -O(CH 2 ) m1 -X M' -R M' (e.g., -OCH 2 CH 2 -X M' -R M' ) and is the compound according to claim 58.

92. R 3 The compound according to claim 91, wherein R is a hydroxyl group.

93. R 3 The compound according to claim 91, wherein R is a protected hydroxyl.

94. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), a compound according to claim 93

95. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, the compound according to claim 94

96. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, a compound according to claim 95.

97. R 3 The compound according to claim 96, wherein R is a reactive phosphorus (III) group.

98. The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 )、-OP(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )(N(R P2 ) 2 )、-OP(S)(OR P )(N(R P2 ) 2 )、-OP(O)(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )H、-OP(S)(OR P )H、-OP(O)(SR P )H、-OP(O)(OR P )R P3 、-OP(S)(OR P )R P3 、or -OP(O)(SR P )R P3 , the compound according to claim 97.

99. The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 ), and the compound according to claim 98.

100. The reactive phosphorus (III) group is OP(OR P )(N(R P2 ) 2 ), where R p is cyanoethyl (-CH 2 CH 2 CN), and each R P2 is isopropyl, or both Rs P2 , together with the nitrogen atom to which they are attached, form an optionally substituted 3- to 8-membered heterocyclyl, the compound according to claim 99.

101. R 3 The compound according to claim 91, wherein R is a linker or a linker attached to a solid support.

102. R 2 The compound according to any one of claims 91 to 101, wherein R is hydroxyl or protected hydroxyl.

103. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), a compound according to claim 102.

104. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, the compound according to claim 103

105. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, the compound according to claim 104.

106. R 2 The compound according to claim 91, wherein R is a hydroxyl group. Claim 107 R 2 The compound according to claim 91, wherein R is a protected hydroxyl. Claim 108 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), the compound according to claim 107. Claim 109 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, the compound according to claim 108 Claim 110 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, the compound according to claim 109 Claim 111 R 2 The compound according to claim 110, wherein R is a reactive phosphorus (III) group. Claim 112 The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 )、-OP(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )(N(R P2 ) 2 )、-OP(S)(OR P )(N(R P2 ) 2 )、-OP(O)(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )H、-OP(S)(OR P )H、-OP(O)(SR P )H、-OP(O)(OR P )R P3 、-OP(S)(OR P )R P3 、or -OP(O)(SR P )R P3 , a compound according to claim 111. Claim 113 The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 ), the compound according to claim 112. Claim 114 The reactive phosphorus (III) group is OP(OR P )(N(R P2 ) 2 ), where R p is cyanoethyl (-CH 2 CH 2 CN), and each R P2 is isopropyl, or both R P2 , together with the nitrogen atom to which they are attached, form an optionally substituted 3- to 8-membered heterocyclyl, the compound according to claim 113. Claim 115 R 2 The compound according to claim 114, wherein R is a linker or a linker attached to a solid support. Claim 116 R 3 The compound according to any one of claims 106 to 115, wherein R is hydroxyl or protected hydroxyl. Claim 117 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), the compound according to claim 116. Claim 118 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, a compound according to claim 117 Claim 119 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, the compound according to claim 118. Claim 120 R 4 The compound according to any one of claims 58 to 119, wherein R is H. Claim 121 X M' The compound according to any one of claims 58 to 120, wherein X is O. Claim 122 Compound of formula (I-B): wherein B is an optionally modified nucleobase, R 2 is -O(CH 2 ), n1 -C(Y M )N(R N' )(R N'' ), a hydroxyl group, a protected hydroxyl group, a phosphate group, a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support, R 3 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ), a hydroxyl, a protected hydroxyl, a phosphate group, a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support, R 4 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ), hydrogen, optionally substituted C 1～6 alkyl, optionally substituted C 2～6 alkenyl, optionally substituted C 2～6 alkynyl, or optionally substituted C 1～6 alkoxy, and R 5 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ), hydroxyl or protected hydroxyl, n1 is an integer from 1 to 10, Y M is O or S, R N' and R N'' are each independently hydrogen, optionally substituted C 6～30 alkyl, optionally substituted C 6～30 alkenyl, optionally substituted C 6～30 alkynyl, or optionally substituted C 3～30 cycloalkyl, a lipid, a ligand, a linker, or a linker to one or more ligands, provided that at least one of R N' and R N'' is not hydrogen, provided that (i) R 2 、R 3 、R 4 and R 5 wherein at least one of R 2 ), n1 -C(O)N(R N' )(R N'' ) is, (ii) R 2 、R 3 、R 4 and R 5 in which only one of 2 is -O(CH n1 ) N' -C(O)N(R N'' )(R ) (iii) R 2 and R 3 only one of which is a reactive phosphorus(III) group, a solid support, or a linker covalently attached to a solid support, (iv) R 2 is -O(CH 2 ) n1 -C(O)N(R N' )(R N'' ), n1 is 1, R 35 is hydroxyl or protected hydroxyl, R 34 is H, R 33 is hydroxyl, protected hydroxyl, phosphate group, or reactive phosphorus(III) group, and when one of R N' and R N'' is H, the other of R N' and R N'' is not - (CH 2 ) 6 CH 3 , nor - (CH 2 ) 7 CH 3 , nor - (CH 2 ) 8 CH 3 , nor - (CH 2 ) 5 NHCOCF 3 , nor - (CH 2 ) 6 NHCOCF 3 , nor - (CH 2 ) 7 NHCOCF 3 , nor - (CH 2 ) 5 N(CH 3 ) 2 , nor - (CH 2 ) 6 N(CH 3 ) 2 , nor - (CH 2 ) 7 N(CH 3 ) 2 either. Claim 123 R 2 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) and optionally Y M is O, the compound according to claim 122. Claim 124 R 3 The compound according to claim 123, wherein R is a hydroxyl group. Claim 125 R 3 The compound according to claim 123, wherein R is a protected hydroxyl. Claim 126 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), a compound according to claim 125. Claim 127 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, a compound according to claim 126. Claim 128 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, a compound according to claim 127 Claim 129 R 3 The compound according to claim 128, wherein R is a reactive phosphorus (III) group. Claim 130 The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 )、 -OP(SR P )(N(R P2 ) 2 )、 -OP(O)(OR P )(N(R P2 ) 2 )、 -OP(S)(OR P )(N(R P2 ) 2 )、 -OP(O)(SR P )(N(R P2 ) 2 )、 -OP(O)(OR P )H、 -OP(S)(OR P )H、 -OP(O)(SR P )H、 -OP(O)(OR P )R P3 、 -OP(S)(OR P )R P3 、 or -OP(O)(SR P )R P3 ), the compound according to claim 129. Claim 131 The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 ), and the compound according to claim 130. Claim 132 The reactive phosphorus (III) group is OP(OR P )(N(R P2 ) 2 ), where R p is cyanoethyl (-CH 2 CH 2 CN), and each R P2 is isopropyl, or both R P2 together with the nitrogen atom to which they are attached form an optionally substituted 3- to 8-membered heterocyclyl, a compound according to claim 131. Claim 133 R 33 The compound according to claim 123, wherein R is a linker or a linker attached to a solid support. Claim 134 R 5 The compound according to any one of claims 123 to 133, wherein R is a protected hydroxyl. Claim 135 The protected hydroxyl is -OR Pro wherein R Pro is an oxygen protecting group, the compound according to claim 134. Claim 136 R Pro The compound according to claim 135, wherein R is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). Claim 137 R Pro The compound according to claim 136, wherein R is 4,4'-dimethoxytrityl. Claim 138 R 3 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) and optionally, Y M is O, the compound according to claim 122. Claim 139 R 2 The compound according to claim 123, wherein R is a hydroxyl group. Claim 140 R 2 The compound according to claim 123, wherein R is a protected hydroxyl. Claim 141 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), a compound according to claim 140. Claim 142 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, the compound according to claim 141. Claim 143 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, a compound according to claim 142. Claim 144 R 2 The compound according to claim 143, wherein R is a reactive phosphorus (III) group. Claim 145 The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 )、-OP(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )(N(R P2 ) 2 )、-OP(S)(OR P )(N(R P2 ) 2 )、-OP(O)(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )H、-OP(S)(OR P )H、-OP(O)(SR P )H、-OP(O)(OR P )R P3 、-OP(S)(OR P )R P3 、or -OP(O)(SR P )R P3 ), a compound according to claim 144. Claim 146 The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 ), the compound according to claim 145. Claim 147 The reactive phosphorus (III) group is OP(OR P )(N(R P2 ) 2 ), where R p is cyanoethyl (-CH 2 CH 2 CN), and each R P2 is isopropyl, or both Rs P2 , together with the nitrogen atom to which they are attached, form an optionally substituted 3- to 8-membered heterocyclyl, a compound according to claim 146. Claim 148 R 2 The compound according to claim 147, wherein R is a linker or a linker attached to a solid support. Claim 149 R 5 The compound according to any one of claims 138 to 148, wherein R is hydroxyl. Claim 150 R 5 The compound according to any one of claims 138 to 148, wherein R is a protected hydroxyl. Claim 151 The protected hydroxyl is -OR Pro wherein, R Pro is an oxygen protecting group, a compound according to any one of claims 150. Claim 152 R Pro The compound according to claim 151, wherein R is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). Claim 153 R Pro The compound according to claim 152, wherein R is 4,4'-dimethoxytrityl. Claim 154 R 5 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) and optionally Y M is O, the compound according to claim 122. Claim 155 R 3 The compound according to claim 154, wherein R is a hydroxyl group. Claim 156 R 3 The compound according to claim 154, wherein R is a protected hydroxyl. Claim 157 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), a compound according to claim 156 Claim 158 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, the compound according to claim 157. Claim 159 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, a compound according to claim 158. Claim 160 R 3 The compound according to claim 159, wherein R is a reactive phosphorus (III) group. Claim 161 The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 )、-OP(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )(N(R P2 ) 2 )、-OP(S)(OR P )(N(R P2 ) 2 )、-OP(O)(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )H、-OP(S)(OR P )H、-OP(O)(SR P )H、-OP(O)(OR P )R P3 、-OP(S)(OR P )R P3 、or -OP(O)(SR P )R P3 ), the compound according to claim 160. Claim 162 The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 ), and the compound according to claim 161. Claim 163 The reactive phosphorus (III) group is OP(OR P )(N(R P2 ) 2 ), where R p is cyanoethyl (-CH 2 CH 2 CN), and each R P2 is isopropyl, or both R P2 together with the nitrogen atom to which they are attached form an optionally substituted 3- to 8-membered heterocyclyl, a compound according to claim 162. Claim 164 R 3 The compound according to claim 163, wherein R is a linker or a linker attached to a solid support. Claim 165 R 2 The compound according to any one of claims 154 to 164, wherein R is hydroxyl or protected hydroxyl. Claim 166 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), a compound according to claim 165. Claim 167 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, a compound according to claim 166. Claim 168 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, a compound according to claim 167 Claim 169 R 2 The compound according to claim 154, wherein R is a hydroxyl group. Claim 170 R 2 The compound according to claim 154, wherein R is a protected hydroxyl. Claim 171 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), the compound according to claim 170. Claim 172 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, the compound according to claim 171. Claim 173 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, a compound according to claim 172. Claim 174 R 2 The compound according to claim 173, wherein R is a reactive phosphorus (III) group. Claim 175 The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 )、-OP(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )(N(R P2 ) 2 )、-OP(S)(OR P )(N(R P2 ) 2 )、-OP(O)(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )H、-OP(S)(OR P )H、-OP(O)(SR P )H、-OP(O)(OR P )R P3 、-OP(S)(OR P )R P3 、or -OP(O)(SR P )R P3 , the compound according to claim 174. Claim 176 The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 ), the compound according to claim 175. Claim 177 The reactive phosphorus (III) group is OP(OR P )(N(R P2 ) 2 ), where R p is cyanoethyl (-CH 2 CH 2 CN), and each R P2 is isopropyl, or both Rs P2 , together with the nitrogen atom to which they are attached, form an optionally substituted 3- to 8-membered heterocyclyl, a compound according to claim 176. Claim 178 R 2 The compound according to claim 177, wherein R is a linker or a linker attached to a solid support. Claim 179 R 3 The compound according to any one of claims 169 to 178, wherein R is hydroxyl or protected hydroxyl. Claim 180 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), the compound according to claim 179 Claim 181 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, the compound according to claim 180. Claim 182 The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, the compound according to claim 181. Claim 183 R 34 The compound according to any one of claims 122 to 182, wherein R is H. Claim 184 An oligonucleotide prepared using the compound according to any one of Claims 1 to 183 or 278 to 341. Claim 185 An oligonucleotide comprising at least one nucleoside of formula (II) wherein B is an optionally modified nucleobase, m1 is an integer from 1 to 10, R 22 is R MA , a bond to a internucleotide linkage to a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkyl, optionally substituted C 2～30 alkenyl, optionally substituted C 2～30 alkynyl, optionally substituted C 1～30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support, and R MA is -O(CH 2 ) m1 -X M' -R M' or -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) and Y M is O or S, X M' is N(R MX ), O, or S, where R MX is hydrogen or R M' and R M' is optionally substituted C 6～30 alkyl, optionally substituted C 6～30 alkenyl, optionally substituted C 6～30 alkynyl, optionally substituted 3- to 8-membered heterocyclyl C 3～30 alkyl, optionally substituted C 3～10 cycloalkyl C 3～30 alkyl, optionally substituted aryl C 3～30 alkyl, optionally substituted heteroaryl C 3～30 alkyl, optionally substituted C 1～30 alkoxy C 1～30 alkyl, -(CH 2 CH 2 O) mq -R MQ , a lipid, a ligand, a linker, or a linker for one or more ligands, wherein mq is an integer selected from 1 to 10, and R MQ is hydrogen or C 1～6 alkyl, and Optionally, R M' is substituted at the terminus with an anionic group or a cationic group, n1 is an integer from 1 to 10, X is O or S, R N' and R N'' each independently is hydrogen, optionally substituted C 6～30 alkyl, optionally substituted C 6～30 alkenyl, optionally substituted C 6～30 alkynyl, or optionally substituted C 3～30 cycloalkyl; a lipid, ligand, linker, or a linker for one or more ligands, provided that at least one of R N' and R N'' is not hydrogen, R 33 is R MA , a bond to an internucleotide linkage to a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkyl, optionally substituted C 2～30 alkenyl, optionally substituted C 2～30 alkynyl, optionally substituted C 1～30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support, R 4 is R MA , hydrogen, optionally substituted C 1～6 alkyl, optionally substituted C 2～6 alkenyl, optionally substituted C 2～6 alkynyl, or optionally substituted C 1～6 alkoxy, and R 25 is R MA , a bond to the internucleotide linkage to the preceding nucleotide, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C 1～30 alkyl, optionally substituted C 2～30 alkenyl, optionally substituted C 2～30 alkynyl, optionally substituted C 1～30 alkoxy, optionally substituted 3- to 8-membered heterocyclyl (e.g., morpholin-1-yl, piperidin-1-yl, or pyrrolidin-1-yl), halogen, alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxocarboxylate, amino, alkylamino, dialkylamino, -O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), vinylphosphonate (VP) group (e.g., =CH-X P , X P is a phosphate group), C 3～6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), monophosphate ((HO) 2 (O)P-O-5'), diphosphate ((HO) 2 (O)P-O-P(HO)(O)-O-5'), triphosphate ((HO) 2 (O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidate ((HO) 2 (O)P-NH-5', (HO)(NH 2 )(O)P - O - 5'), alkyl phosphonate [(R P )(OH)(O)P - O - 5', R P is optionally substituted C 1～30 alkyl, such as methyl, ethyl, isopropyl, or propyl)], alkyl ether phosphonate [(R P1 )(OH)(O)P - O - 5', R P1 is alkoxyalkyl, such as methoxymethyl (CH 2 OMe) or ethoxymethyl)], (HO) 2 (X)P - O[-(CH 2 ) a - O - P(X)(OH) - O] b - 5' or (HO) 2 (X)P - O[-(CH 2 ) a - P(X)(OH) - O] b - 5' or (HO) 2 (X)P - [-(CH 2 ) a - O - P(X)(OH) - O] b - 5', or optionally substituted alkyl, and dialkyl terminal phosphate and phosphate mimics (e.g., HO[-(CH 2 ) a - O - P(X)(OH) - O] b - 5', H 2 N[-(CH 2 ) a - O - P(X)(OH) - O] b - 5', H[-(CH 2 ) a - O - P(X)(OH) - O] b - 5', Me 2 N[-(CH 2 ) a - O - P(X)(OH) - O] b - 5', HO[-(CH 2 ) a - P(X)(OH) - O] b - 5', H 2 N[-(CH 2 ) a - P(X)(OH) - O] b - 5', H[-(CH 2 ) a -P(X)(OH)-O] b -5', Me 2 N[-(CH 2 ) a -P(X)(OH)-O] b -5' and herein a and b are each independently from 1 to 10, provided that Each R 8 and R 9 are independently H, a targeting ligand (e.g., GalNac), a pharmacokinetic modulating substance, optionally substituted C 1～30 alkyl, optionally substituted C 1～30 alkenyl, or optionally substituted C 1～30 alkynyl, the oligonucleotide. (i) R 22 , R 23 , R 24 and R 25 at least one of which is R MA and (ii)R 22 、R 23 、R 24 およびR 25 のうちの1つだけがR MA であり、 (iii) R 22 and R 23 only one of which is a bond to a solid support, a linker covalently attached to the solid support, or an internucleotide linker for a subsequent nucleotide, (iv) R 22 and R 23 are both not bonds to the internucleotide linkage to the subsequent nucleotide, R 25 is a bond to the internucleotide linkage to the preceding nucleotide, (v)R 22 is -OCH 2 CH 2 -X M' -R M' wherein R 25 is hydroxyl, protected hydroxyl or a linkage to an internucleotide linker for a preceding nucleotide, R 4 is H, R 23 is hydroxyl, protected hydroxyl, a linkage to an internucleotide linker for a subsequent nucleotide, a solid support, a linker, or a linker covalently attached to a solid support, and when at least one of R 23 and R 25 is a linkage to an internucleotide linker, R M' is neither unsubstituted C 5～21 alkyl, nor unsubstituted C 5～21 alkenyl, nor unsubstituted C 5～21 alkynyl, (vi) R 22 is -O(CH 2 ) n1 -C(O)N(R N' )(R N'' ), n1 is 1, R 5 is hydroxyl, protected hydroxyl or a bond to an internucleotide linker for a preceding nucleotide, R 4 is H, R 3 is hydroxyl, protected hydroxyl, a bond to an internucleotide linker for a subsequent nucleotide, a solid support, a linker, or a linker covalently attached to a solid support, R 23 and R 25 at least one of which is a bond to an internucleotide linker, R N' and R N'' one of which is H, and when at least one of R 23 and R 25 is a bond to an internucleotide linker, the other of R N' and R N'' is either -(CH 2 ) 6 CH 3 , or -(CH 2 ) 7 CH 3 , or -(CH 2 ) 8 CH 3 , or -(CH 2 ) 5 NHCOCF 3 , or -(CH 2 ) 6 NHCOCF 3 , or -(CH 2 ) 7 NHCOCF 3 , or -(CH 2 ) 5 N(CH 3 ) 2 , or -(CH 2 ) 6 N(CH 3 ) 2 , or -(CH 2 ) 7 N(CH 3 ) 2 Nor is it Claim 186 Claim 187 R 22 is -O(CH 2 ) m1 -X M' -R M' (for example, -OCH 2 CH 2 -X M' -R M' ) and is the oligonucleotide according to claim 185. Claim 188 X M' The oligonucleotide according to claim 186, wherein X is O. Claim 189 X M' The oligonucleotide according to claim 186, wherein Claim 190 R 22 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) as described in claim 185 of the oligonucleotide. Claim 191 The oligonucleotide according to Claim 189, wherein n1 is 1. Claim 192 The oligonucleotide according to Claim 189, wherein n1 is 2. Claim 193 R 23 is a bond to a internucleotide linkage to a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy, solid support, linker, or a linker covalently attached to a solid support, the oligonucleotide according to any one of claims 186 to 191. Claim 194 R 23 The oligonucleotide according to any one of claims 186 to 192, wherein R is a bond to an internucleotide linkage, a hydroxyl, a protected hydroxyl, a solid support, a linker, or a linker covalently attached to a solid support, for a subsequent nucleotide.

194. R 23 The oligonucleotide according to any one of claims 186 to 193, wherein R is a bond to an internucleotide linkage for a subsequent nucleotide.

195. R 23 The oligonucleotide according to any one of claims 186 to 194, wherein R is a linker covalently attached to a solid support.

196. R 23 is -O(CH 2 ) m1 -X M' -R M' (for example, -OCH 2 CH 2 -X M' -R M' ) and is the oligonucleotide according to claim 185.

197. X M' The oligonucleotide according to claim 196, wherein X is O.

198. X M' The oligonucleotide according to claim 196, wherein

199. R 23 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) and is the oligonucleotide according to claim 185.

200. The oligonucleotide according to claim 199, wherein n1 is 1.

201. The oligonucleotide according to claim 199, wherein n1 is 2.

202. R 22 is a bond to an internucleotide linkage, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy, solid support, linker, or a linker covalently attached to a solid support, the oligonucleotide according to any one of claims 196 to 201.

203. R 22 The oligonucleotide according to any one of claims 196 to 202, wherein R is a linkage to an internucleotide linkage, a hydroxyl, a protected hydroxyl, a solid support, a linker, or a linker covalently attached to the solid support, for a subsequent nucleotide.

204. R 22 The oligonucleotide according to any one of claims 196 to 203, wherein R is a bond to an internucleotide linkage for a subsequent nucleotide.

205. R 22 The oligonucleotide according to any one of claims 196 to 203, wherein R is a linker covalently attached to the solid support.

206. R 24 The oligonucleotide according to any one of claims 185 to 205, wherein R is H.

207. R 24 is -O(CH 2 ) m1 -X M' -R M' (e.g., -OCH 2 CH 2 -X M' -R M' ) and is the oligonucleotide according to claim 185.

208. X M' The oligonucleotide according to claim 207, wherein X is O.

209. X M' The oligonucleotide according to claim 207, wherein X is S.

210. R 24 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) as described in claim 185 of the oligonucleotide.

211. The oligonucleotide according to claim 210, wherein n1 is 1.

212. The oligonucleotide according to claim 211, wherein n1 is 2.

213. R 23 is a bond to a internucleotide linkage, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy, solid support, linker, or a linker covalently attached to a solid support, the oligonucleotide according to any one of claims 207 to 212.

214. R 23 The oligonucleotide according to any one of claims 207 to 213, wherein R is a linkage to an internucleotide linkage, a hydroxyl, a protected hydroxyl, a solid support, a linker, or a linker covalently attached to the solid support, for a subsequent nucleotide.

215. R 22 is hydrogen, hydroxyl, protected hydroxyl, halogen, or optionally substituted C 1～30 alkyl, the oligonucleotide according to any one of claims 213 to 214.

216. R 22 The oligonucleotide according to any one of claims 213 to 215, wherein R is hydrogen, hydroxyl or protected hydroxyl.

217. R 22 is a bond to a internucleotide linkage to a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy, solid support, linker, or a linker covalently attached to a solid support, the oligonucleotide according to any one of claims 207 to 212.

218. R 22 The oligonucleotide according to claim 217, wherein R is a linkage to an internucleotide linkage to a subsequent nucleotide, a hydroxyl, a protected hydroxyl, a solid support, a linker, or a linker covalently attached to the solid support.

219. R 23 is hydrogen, hydroxyl, protected hydroxyl, halogen, or optionally substituted C 1～30 alkyl, the oligonucleotide according to any one of claims 217 to 218.

220. R 23 The oligonucleotide according to any one of claims 217 to 219, wherein R is hydrogen, hydroxyl or protected hydroxyl.

221. R 25 is a bond to a internucleotide linkage to a preceding nucleotide, a hydroxyl, a protected hydroxyl, an optionally substituted C 1～30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate or phosphate mimetic, an oligonucleotide according to any one of claims 185 to 220.

222. R 25 is a nucleotide according to any one of claims 185 to 221, which is a bond to a nucleotide internucleoside linkage, a hydroxyl group, a protected hydroxyl group, a vinylphosphonate (VP) group, a cyclopropylphosphonate, a monophosphate, a diphosphate, a triphosphate, a monothiophosphate (phosphorothioate), a monodithiophosphate, a phosphorothiolate, an alpha-thiotriphosphate, a beta-thiotriphosphate, a gamma-thiotriphosphate, a phosphoramidate, an alkylphosphonate, an alkyl ether phosphonate, a dialkyl terminal phosphate, or a phosphate mimetic.

223. R 25 The oligonucleotide according to any one of claims 185 to 222, wherein R is hydroxyl or protected hydroxyl.

224. R 25 The oligonucleotide according to any one of claims 185 to 222, wherein R is a bond to a nucleotide internucleoside linkage relative to a preceding nucleotide.

225. R 25 The oligonucleotide according to any one of claims 185 to 222, wherein R is a vinylphosphonate (VP) group, cyclopropylphosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, or a phosphate mimetic.

226. R 25 is -O(CH 2 ) m1 -X M' -R M' (e.g., -OCH 2 CH 2 -X M' -R M' ) and is the oligonucleotide according to claim 185.

227. X M' The oligonucleotide according to claim 226, wherein X is O.

228. X M' The oligonucleotide according to claim 226, wherein X is S.

229. R 25 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) as described in claim 185, the oligonucleotide.

230. The oligonucleotide according to claim 229, wherein n1 is 1.

231. The oligonucleotide according to claim 229, wherein n1 is 2.

232. R 23 is a bond to an internucleotide linkage, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy, solid support, linker, or a linker covalently attached to a solid support, the oligonucleotide according to any one of claims 226 to 231.

233. R 23 The oligonucleotide according to any one of claims 226 to 232, wherein R is a bond to an internucleotide linkage, a hydroxyl, a protected hydroxyl, a solid support, a linker, or a linker covalently attached to the solid support, for a subsequent nucleotide.

234. R 22 is hydrogen, hydroxyl, protected hydroxyl, halogen, or optionally substituted C 1～30 alkyl, the oligonucleotide according to any one of claims 226 to 233.

235. R 22 The oligonucleotide according to any one of claims 226 to 234, wherein R is hydrogen, hydroxyl or protected hydroxyl.

236. R 22 is a bond to an internucleotide linkage to a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy, solid support, linker, or a linker covalently attached to a solid support, the oligonucleotide according to any one of claims 226 to 231.

237. R 22 The oligonucleotide according to claim 236, wherein R is a bond to an internucleotide linkage, a hydroxyl, a protected hydroxyl, a solid support, a linker, or a linker covalently attached to the solid support, for a subsequent nucleotide.

238. R 23 wherein R is hydrogen, hydroxyl, protected hydroxyl, halogen, or optionally substituted C 1～30 alkyl, the oligonucleotide according to claim 237.

239. R 23 The oligonucleotide according to claim 238, wherein R is hydrogen, hydroxyl or protected hydroxyl.

240. R 24 The oligonucleotide according to any one of claims 185 to 206 or 221 to 239, wherein R is H.

241. The oligonucleotide according to any one of claims 185 to 240, which is attached to a solid support.

242. The oligonucleotide according to any one of claims 185 to 241, which contains 3 to 50 nucleotides.

243. The oligonucleotide according to any one of claims 185 to 242, which contains at least one ribonucleotide.

244. The oligonucleotide according to any one of claims 185 to 243, which contains at least one 2'-deoxyribonucleotide.

245. The oligonucleotide according to any one of claims 185 to 244, which contains at least one nucleotide having a modified nucleobase or a non-natural nucleobase.

246. The oligonucleotide according to any one of claims 185 to 245, which contains at least one nucleotide having a modified ribose sugar in addition to the nucleotide of formula (II).

247. The oligonucleotide according to any one of claims 185 to 246, comprising, in addition to the nucleotide of formula (II), at least one nucleotide having a 2'-F ribose.

248. The oligonucleotide according to any one of claims 185 to 247, comprising, in addition to the nucleotide of formula (II), at least one nucleotide having a 2'-OMe ribose.

249. The oligonucleotide according to any one of claims 185 to 248, comprising, in addition to the nucleotide of formula (II), at least one nucleotide containing a moiety other than ribose sugar.

250. The oligonucleotide according to any one of claims 185 to 249, comprising at least one modified nucleotide internucleoside linkage.

251. The oligonucleotide according to any one of claims 185 to 250, comprising at least one ligand.

252. The oligonucleotide according to any one of claims 185 to 251, comprising at least one hydroxyl protecting group, phosphate protecting group or amino protecting group.

253. The oligonucleotide according to any one of claims 185 to 252, wherein m1 is 2.

254. A double-stranded nucleic acid comprising a first oligonucleotide strand and a second oligonucleotide strand substantially complementary to the first strand, wherein the first strand or the second strand is the oligonucleotide according to any one of claims 185 to 253.

255. The double-stranded nucleic acid according to claim 254, wherein the first strand and the second strand are each independently 15 to 25 nucleotides in length.

256. The double-stranded nucleic acid according to any one of claims 264 to 255, capable of inducing RNA interference.

257. The double-stranded nucleic acid according to any one of claims 264 to 256, wherein one or both strands have an overhang of 1 to 5 nucleotides at each 5' end or 3' end.

258. The double-stranded nucleic acid according to any one of claims 264 to 257, wherein only one strand has an overhang of 2 nucleotides at its 5' end or 3' end.

259. The double-stranded nucleic acid according to any one of claims 264 to 258, wherein only one strand has an overhang of 2 nucleotides at its 3' end.

260. A method for reducing the expression of a target gene in a subject, comprising (i)The double-stranded RNA according to any one of claims 254 to 259, wherein the first strand or the second strand is complementary to the target gene, or (ii)The oligonucleotide according to any one of claims 185 to 253, which is complementary to the target gene The method comprising the step of administering any one of the above.

261. A method for preparing an oligonucleotide comprising at least a nucleotide of formula (II) wherein The nucleotide of formula (II') An oligonucleotide containing is reacted with an amine of the formula HN(R N' )(R N'' ), and the method includes the step of In the formula, B is an optionally modified nucleobase, R 22 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ), a bond to an internucleotide linker for a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkyl, optionally substituted C 2～30 alkenyl, optionally substituted C 2～30 alkynyl, optionally substituted C 1～30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2 - methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, 5 - to 8 - membered heterocyclyl, -O - C 4～30 alkyl - ON(CH 2 R 8 )(CH 2 R 9 ), -O - C 4～30 alkyl - ON(CH 2 R 8 )(CH 2 R 9 ), a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support, and Y M is O or S, R N' and R N'' are independently hydrogen, optionally substituted C 6～30 alkyl, optionally substituted C 6～30 alkenyl, optionally substituted C 6～30 alkynyl, or optionally substituted C 3～30 cycloalkyl, lipid, ligand, linker, or a linker to one or more ligands, provided that at least one of R N' and R N'' is not hydrogen, n1 is an integer from 1 to 10, R 23 is -O(CH 2 ) n1 -C(O)N(R N' )(R N'' ), a bond to an internucleotide linker for a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkyl, optionally substituted C 2～30 alkenyl, optionally substituted C 2～30 alkynyl, optionally substituted C 1～30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2 - methoxyethyl), alkoxyalkylamine, alkoxyoxocarboxylate, amino, alkylamino, dialkylamino, 5 - to 8 - membered heterocyclyl, -O - C 4～30 alkyl - ON(CH 2 R 8 )(CH 2 R 9 ), -O - C 4～30 alkyl - ON(CH 2 R 8 )(CH 2 R 9 ), a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support, and R 4 is hydrogen, optionally substituted C 1～6 alkyl, optionally substituted C 2～6 alkenyl, optionally substituted C 2～6 alkynyl, or optionally substituted C 1～6 alkoxy, and R 25 is -O(CH 2 ) n1 -C(O)N(R N' )(R N'' ), a bond to an internucleotide linker relative to a preceding nucleotide, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C 1～30 alkyl, optionally substituted C 2～30 alkenyl, optionally substituted C 2～30 alkynyl, optionally substituted C 1～30 alkoxy, optionally substituted 3 - 8 membered heterocyclyl (e.g., morpholin - 1 - yl, piperidin - 1 - yl, or pyrrolidin - 1 - yl), halogen, alkoxyalkyl (e.g., 2 - methoxyethyl), alkoxyalkylamine, alkoxyoxocarboxylate, amino, alkylamino, dialkylamino, -O - C 4～30 alkyl - ON(CH 2 R 8 )(CH 2 R 9 ), -O - C 4～30 alkyl - ON(CH 2 R 8 )(CH 2 R 9 ), vinylphosphonate (VP) group (e.g., =CH - X P , X P is a phosphate group), C 3～6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), monophosphate ((HO) 2 (O)P - O - 5'), diphosphate ((HO) 2 (O)P - O - P(HO)(O) - O - 5'), triphosphate ((HO) 2 (O)P - O - (HO)(O)P - O - P(HO)(O) - O - 5'); monothiophosphate (phosphorothioate, (HO)2(S)P - O - 5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P - O - 5'), phosphorothiolate ((HO)2(O)P - S - 5'); alpha - thiotriphosphate; beta - thiotriphosphate; gamma - thiotriphosphate; phosphoramidate ((HO) 2 (O)P-NH-5', (HO)(NH 2 )(O)P-O-5'), alkyl phosphonate [(R P )(OH)(O)P-O-5', R P is optionally substituted C 1～30 alkyl, such as methyl, ethyl, isopropyl, or propyl)], alkyl ether phosphonate [(R P1 )(OH)(O)P-O-5', R P1 is alkoxyalkyl, such as methoxymethyl (CH 2 OMe) or ethoxymethyl], (HO) 2 (X)P-O[-(CH 2 ) a -O-P(X)(OH)-O] b -5' or (HO) 2 (X)P-O[-(CH 2 ) a -P(X)(OH)-O] b -5' or (HO) 2 (X)P-[-(CH 2 ) a -O-P(X)(OH)-O] b -5', or optionally substituted alkyl, and dialkyl terminal phosphate and phosphate mimics (e.g., HO[-(CH 2 ) a -O-P(X)(OH)-O] b -5', H 2 N[-(CH 2 ) a -O-P(X)(OH)-O] b -5', H[-(CH 2 ) a -O-P(X)(OH)-O] b -5', Me 2 N[-(CH 2 ) a -O-P(X)(OH)-O] b -5', HO[-(CH 2 ) a -P(X)(OH)-O] b -5', H 2 N[-(CH 2 ) a -P(X)(OH)-O] b -5', H[-(CH 2 ) a -P(X)(OH)-O] b -5', Me 2 N[-(CH 2 ) a -P(X)(OH)-O] b -5' and here X is O or S, a and b are each independently from 1 to 10, Each R 8 and R 9 are independently H, a targeting ligand (e.g., GalNac), a pharmacokinetic modifier, an optionally substituted C 1～30 alkyl, an optionally substituted C 1～30 alkenyl, or an optionally substituted C 1～30 alkynyl, R 22' is -O(CH 2 ) n1 -C(Y M )OR LV , a bond to an internucleotide linker for a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkyl, optionally substituted C 2～30 alkenyl, optionally substituted C 2～30 alkynyl, optionally substituted C 1～30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2 - methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, 5 - to 8 - membered heterocyclyl, -O - C 4～30 alkyl - ON(CH 2 R 8 )(CH 2 R 9 ), -O - C 4～30 alkyl - ON(CH 2 R 8 )(CH 2 R 9 ), a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support, and R LV is C 1 to C 6 alkyl (for example ethyl), and R 23' is -O(CH 2 ) n1 -C(Y M )OR LV , a bond to an internucleotide linker for a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkyl, optionally substituted C 2～30 alkenyl, optionally substituted C 2～30 alkynyl, optionally substituted C 1～30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2 - methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, 5 - to 8 - membered heterocyclyl, -O - C 4～30 alkyl - ON(CH 2 R 8 )(CH 2 R 9 )、-O - C 4～30 alkyl - ON(CH 2 R 8 )(CH 2 R 9 ), a ligand, a linker covalently attached to one or more ligands, a solid support, a linker, or a linker covalently attached to a solid support, and R 25 ' is -O(CH 2 ) n1 -C(Y M )OR LV , a bond to the internucleotide linker relative to the preceding nucleotide, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C 1～30 alkyl, optionally substituted C 2～30 alkenyl, optionally substituted C 2～30 alkynyl, optionally substituted C 1～30 alkoxy, optionally substituted 3- to 8-membered heterocyclyl (e.g., morpholin-1-yl, piperidin-1-yl, or pyrrolidin-1-yl), halogen, alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxylate, amino, alkylamino, dialkylamino, -O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4～30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), vinylphosphonate (VP) group (e.g., =CH-X P , X P is a phosphate group), C 3～6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), monophosphate ((HO) 2 (O)P-O-5'), diphosphate ((HO) 2 (O)P-O-P(HO)(O)-O-5'), triphosphate ((HO) 2 (O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidate ((HO) 2 (O)P-NH-5', (HO)(NH 2 )(O)P-O-5'), alkyl phosphonate [(R P )(OH)(O)P-O-5', R P is optionally substituted C 1～30 alkyl, such as methyl, ethyl, isopropyl, or propyl)], alkyl ether phosphonate [(R P1 )(OH)(O)P-O-5', R P1 is alkoxyalkyl, such as methoxymethyl (CH 2 OMe) or ethoxymethyl], (HO) 2 (X)P-O[-(CH 2 ) a -O-P(X)(OH)-O] b -5' or (HO) 2 (X)P-O[-(CH 2 ) a -P(X)(OH)-O] b -5' or (HO) 2 (X)P-[-(CH 2 ) a -O-P(X)(OH)-O] b -5', or optionally substituted alkyl, and dialkyl terminal phosphate and phosphate mimics (e.g., HO[-(CH 2 ) a -O-P(X)(OH)-O] b -5', H 2 N[-(CH 2 ) a -O-P(X)(OH)-O] b -5', H[-(CH 2 ) a -O-P(X)(OH)-O] b -5', Me 2 N[-(CH 2 ) a -O-P(X)(OH)-O] b -5', HO[-(CH 2 ) a -P(X)(OH)-O] b -5', H 2 N[-(CH 2 ) a -P(X)(OH)-O] b -5', H[-(CH 2 ) a -P(X)(OH)-O] b -5', Me 2 N[-(CH 2 ) a -P(X)(OH)-O] b -5', where X is O or S, a and b are each independently from 1 to 10, provided that (i) R 22 , R 23 , R 24 and R 25 at least one of which is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) and (ii) R 22 、R 23 、R 24 and R 25 in which only one of 2 ) n1 -C(O)N(R N' )(R N'' ) is (iii) R 22 and R 23 only one of which is a bond to the internucleotide linkage for the subsequent nucleotide, (iv) R 22 and R 23 are both not linked to the internucleotide linkage to the subsequent nucleotide, R 25 is linked to the internucleotide linkage to the preceding nucleotide, (v) R 22' , R 23' , R 24' and R 25' At least one of the -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) and (vi) R 22' , R 23' , R 24' and R 25' Only one of 2 ) n1 -C(Y M )N(R N' )(R N'' ) is (vii) R 22' and R 23' only one of which is a bond to the internucleotide linkage for a subsequent nucleotide, (viii) R 22' and R 23' are both not linked to the internucleotide linkage to the subsequent nucleotide, R 25' is linked to the internucleotide linkage to the preceding nucleotide. The above method.

262. The method according to claim 261, wherein the oligonucleotide comprising the nucleoside of formula (II') is linked to a solid support.

263. The method according to claim 261, wherein the oligonucleotide comprising the nucleoside of formula (II') is not linked to a solid support.

264. The oligonucleotide comprising the nucleoside of formula (II') is linked to a solid support, and the method comprises the step of cleaving the oligonucleotide from the solid support before reacting it with an amine of formula HN(R N' )(R N'' ), according to the method of claim 261.

265. The method according to any one of claims 261 to 264, wherein the oligonucleotide comprises 3 to 50 nucleotides.

266. The method according to any one of claims 261 to 264, wherein the oligonucleotide comprises at least one ribonucleotide (e.g., 2'-OH).

267. The method according to any one of claims 261 to 266, wherein the oligonucleotide comprises at least one 2'-deoxyribonucleotide.

268. The method according to any one of claims 261 to 267, wherein the oligonucleotide comprises at least one nucleotide having a modified nucleobase or a non-natural nucleobase.

269. The method according to any one of claims 261 to 268, wherein the oligonucleotide comprises at least one nucleotide having a modified ribose sugar.

270. The method according to any one of claims 261 to 269, wherein the oligonucleotide comprises at least one nucleotide containing a group that is neither H nor OH at the 2'-position of the ribose sugar.

271. The method according to any one of claims 261 to 270, wherein the oligonucleotide comprises at least one nucleotide having 2'-F ribose.

272. The method according to any one of claims 261 to 271, wherein the oligonucleotide contains at least one nucleotide having a 2'-OMe ribose.

273. The method according to any one of claims 260 to 272, wherein the oligonucleotide contains at least one nucleotide containing a moiety other than ribose sugar.

274. The method according to any one of claims 261 to 273, wherein the oligonucleotide containing a nucleoside contains at least one hydroxyl protecting group, phosphate protecting group or amino protecting group before the step of reacting.

275. The method according to any one of claims 261 to 274, wherein the oligonucleotide does not contain any hydroxyl protecting group, phosphate protecting group or amino protecting group before the reactive group.

276. The oligonucleotide according to any one of claims 261 to 275, wherein the oligonucleotide contains at least one ligand.

277. An oligonucleotide prepared by the method according to any one of claims 260 to 276.

278. R 4 is -O(CH 2 ) m1 -X M' -R M' (for example, -OCH 2 CH 2 -X M' -R M' ) and is the compound according to claim 1.

279. X M' The compound according to claim 278, wherein X is O.

280. X M' The compound according to claim 278, wherein X is S.

281. R 4 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) and is the compound according to claim 1.

282. The compound according to claim 281, wherein n1 is 1.

283. The compound according to claim 282, wherein n1 is 2.

284. R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to a solid support, a compound according to any one of claims 278 to 283.

285. R 2 The compound according to any one of claims 278 to 284, wherein R is hydrogen, hydroxyl, protected hydroxyl, halogen, reactive phosphorus (III) group, solid support, linker, or a linker covalently attached to the solid support.

286. R 2 The compound according to any one of claims 278 to 285, wherein R is hydrogen, hydroxyl, protected hydroxyl, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to the solid support.

287. R 2 The compound according to any one of claims 278 to 286, wherein R is a reactive phosphorus (III) group, a solid support, a linker, or a linker covalently attached to the solid support.

288. R 2 The compound according to any one of 278 to 287, wherein R is a reactive phosphorus (III) or a linker covalently attached to a solid support.

289. R 2 is a reactive phosphorus (III) group (e.g., a phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite), a compound according to any one of claims 278 to 288.

290. R 2 The compound according to any one of claims 278 to 289, wherein R is a linker covalently attached to the solid support.

291. R 3 wherein R is hydrogen, hydroxyl, protected hydroxyl, halogen, or optionally substituted C 1～30 alkoxy, and the compound according to any one of claims 278 to 290.

292. R 3 The compound according to any one of claims 278 to 291, wherein R is hydroxyl or protected hydroxyl.

293. R 3 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy, reactive phosphorus(III) group, solid support, linker, or a linker covalently attached to a solid support, a compound according to any one of claims 278 to 292.

294. R 3 The compound according to any one of claims 293, wherein R is hydrogen, hydroxyl, protected hydroxyl, halogen, a reactive phosphorus(III) group, a solid support, a linker, or a linker covalently attached to a solid support.

295. R 3 The compound according to any one of claims 293 to 294, wherein R is hydrogen, hydroxyl, protected hydroxyl, a reactive phosphorus (III) group, a solid support, a linker, or a linker covalently attached to the solid support.

296. R 3 The compound according to any one of claims 293 to 295, wherein R is a reactive phosphorus (III) group, a solid support, a linker, or a linker covalently attached to the solid support.

297. R 3 The compound according to any one of 293 to 296, wherein R is reactive phosphorus (III) or a linker covalently attached to a solid support.

298. R 3 The compound according to any one of claims 292 to 297, wherein R is a reactive phosphorus (III) group, such as a phosphoramidite such as [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or [(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite.

299. R 3 The compound according to any one of claims 293 to 298, wherein R is a linker covalently attached to a solid support.

300. R 2 wherein R is hydrogen, hydroxyl, protected hydroxyl, halogen, or optionally substituted C 1～30 alkoxy, and the compound according to any one of claims 293 to 299.

301. R 2 The compound according to any one of claims 293 to 300, wherein R is hydroxyl or protected hydroxyl.

302. R 5 is hydroxyl, protected hydroxyl, optionally substituted C 1～30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyl ether phosphonate, dialkyl terminal phosphate or a phosphate mimetic, a compound according to any one of claims 293 to 301.

303. R 5 is a hydroxyl, protected hydroxyl, vinyl phosphonate (VP) group, cyclopropyl phosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkyl phosphonate, alkyl ether phosphonate, dialkyl terminal phosphate, or phosphate mimetic, a compound according to any one of claims 278 to 302.

304. R 5 The compound according to any one of claims 278 to 303, wherein R is hydroxyl or protected hydroxyl.

305. R 4 is -O(CH 2 ) n1 -C(Y M )N(R N' )(R N'' ) as described in claim 1, the compound according to claim 1.

306. R 3 The compound according to claim 305, wherein R is a hydroxyl group.

307. R 3 The compound according to claim 306, wherein R is a protected hydroxyl.

308. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), the compound according to claim 307.

309. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, the compound of claim 308

310. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, a compound according to claim 309

311. R 3 The compound according to claim 310, wherein R is a reactive phosphorus (III) group.

312. The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 )、-OP(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )(N(R P2 ) 2 )、-OP(S)(OR P )(N(R P2 ) 2 )、-OP(O)(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )H、-OP(S)(OR P )H、-OP(O)(SR P )H、-OP(O)(OR P )R P3 、-OP(S)(OR P )R P3 、or -OP(O)(SR P )R P3 , a compound according to claim 311.

313. The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 ), the compound according to claim 312.

314. The reactive phosphorus (III) group is OP(OR P )(N(R P2 ) 2 ), where R p is cyanoethyl (-CH 2 CH 2 CN), and each R P2 is isopropyl, or both R P2 together with the nitrogen atom to which they are attached form an optionally substituted 3- to 8-membered heterocyclyl, a compound according to claim 313.

315. R 3 The compound according to claim 305, wherein R is a linker or a linker attached to a solid support.

316. R 2 The compound according to any one of claims 305 to 315, wherein R is hydroxyl or protected hydroxyl.

317. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), a compound according to claim 316.

318. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, the compound according to claim 317.

319. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, a compound according to claim 318.

320. R 2 The compound according to claim 305, wherein R is a hydroxyl group.

321. R 2 The compound according to claim 305, wherein R is a protected hydroxyl.

322. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), the compound according to claim 320.

323. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, the compound according to claim 321.

324. The protected hydroxyl is -OR Pro wherein, R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, a compound according to claim 322.

325. R 2 The compound according to claim 323, wherein R is a reactive phosphorus (III) group.

326. The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 )、-OP(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )(N(R P2 ) 2 )、-OP(S)(OR P )(N(R P2 ) 2 )、-OP(O)(SR P )(N(R P2 ) 2 )、-OP(O)(OR P )H、-OP(S)(OR P )H、-OP(O)(SR P )H、-OP(O)(OR P )R P3 、-OP(S)(OR P )R P3 、 or -OP(O)(SR P )R P3 , the compound according to claim 324.

327. The reactive phosphorus (III) group is -OP(OR P )(N(R P2 ) 2 ), and the compound according to claim 325.

328. The reactive phosphorus(III) group is -OP(OR P )(N(R P2 ) 2 ), where R p is cyanoethyl (-CH 2 CH 2 CN), and each R P2 is isopropyl, or both R P2 together with the nitrogen atom to which they are attached form an optionally substituted 3- to 8-membered heterocyclyl, a compound according to claim 326.

329. R 2 The compound according to claim 304, wherein R is a linker or a linker attached to a solid support.

330. R 3 The compound according to any one of claims 320 to 329, wherein R is hydroxyl or protected hydroxyl.

331. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), the compound according to claim 330.

332. The protected hydroxyl is -OR Pro wherein R Pro is selected from the group consisting of acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, and dimethoxytrityl, a compound according to claim 331

333. The protected hydroxyl is -OR Pro wherein, R Pro is selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl, the compound according to claim 332.

334. R 5 The compound according to any one of claims 305 to 333, wherein R is hydroxyl.

335. R 5 The compound according to any one of claims 305 to 333, wherein R is a protected hydroxyl group.

336. The protected hydroxyl is -OR Pro wherein R Pro is an oxygen protecting group, a compound according to any one of claims 335.

337. R Pro The compound according to claim 336, wherein R is selected from the group consisting of acetyl, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX).

338. R Pro The compound according to claim 337, wherein R is 4,4'-dimethoxytrityl.

339. The compound according to any one of claims 122 to 183 or 305 to 338, wherein n1 is 1.

340. The compound according to any one of claims 122 to 183 or 305 to 338, wherein n1 is 2.

341. The compound according to any one of claims 122 to 183 or 305 to 340, wherein m1 is 2.

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