5 '-modified monomer, oligonucleotide and double-stranded RNA

Abstract

The technology described herein relates to 5 '-modified nucleosides, nucleotides, oligonucleotides and double stranded RNAs, such as siRNAs, as well as kits comprising them and their use for inhibition of target genes.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Application No. 63 / 521,256, filed June 15, 2023, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The technology described herein generally relates to 5'-modified nucleosides, nucleotides, oligonucleotides, and double-stranded RNA (e.g., siRNA), compositions and kits comprising them, and methods of using them to inhibit target genes. BACKGROUND

[0004] There remains a need in the art for oligonucleotides and siRNAs with improved activity and / or pharmacokinetics. The present disclosure addresses some of these needs. SUMMARY

[0005] In one aspect, provided herein is a compound of Formula I (Formula I), or a salt thereof, wherein: B is an optionally modified base (e.g., uracil or 5-methyluracil); X is O or S; each R V is independently hydrogen or a hydroxyl protecting group (e.g., ethyl or pivaloyloxymethyl ((CH3)3CC(O)OCH2-, POM); one of R2and R3is hydrogen, halogen, or -OR 20 , wherein: R 20 is hydrogen, a hydroxyl protecting group, an optionally substituted C 1-6 alkyl (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxypropan-2-yl, 2-(N- methylamino)-2-oxoethyl), an optionally substituted C 2-6 alkenyl, or an optionally substituted C 2-6 alkynyl (e.g., propargyl); R 2 and the other of R 3 is -OR 30 , wherein: R 30 is hydrogen, a hydroxyl protecting group, a reactive phosphorus group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.

[0006] In some embodiments, X is O. In some other embodiments, X is S.

[0007] In the compounds of Formula I, each R Vindependently hydrogen or a hydroxyl protecting group. In some embodiments, each R V is hydrogen. In some embodiments, each R V is a hydroxyl protecting group, such as pivaloyloxymethyl ((CH3)3CC(O)OCH2-, POM) or ethyl (CH3CH2-).

[0008] In some compounds described herein, R 3 is -OR 30 . For example, R 3 is -OR 30 , and R 30 is a reactive phosphorus group. Exemplary reactive phosphorus groups are described below, including but not limited to phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphotriesters, and phosphorus-containing chiral auxiliaries. Thus, in some embodiments, R 3 is -OR 30 , and R 30 is a reactive phosphorus group selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, and phosphotriesters, optionally, R 30 is a phosphoramidite. Accordingly, in some compounds, R 30 is -P(OR P1 )N(R P2 )2, -P(SR P1 )N(R P2 )2, -P(O)(OR P1 )N(R P2 )2, -P(S)(OR P1 )N(R P2 )2, -P(R P3 )N(R P2 )2, -P(O)(SR P1 )N(R P2 )2, -P(O)(OR P1 )H, -P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , wherein: each R P3 is independently optionally substituted C1-C 30 alkyl, optionally substituted C2-C 30 alkenyl, or optionally substituted C2-C 30 alkynyl (e.g., optionally substituted C1-C 10alkyl, optionally substituted C2-C 10 alkenyl or optionally substituted C2-C 10 alkynyl); each R P1 is independently optionally substituted C 1-6 alkyl; and each R P2 is independently optionally substituted C 1-6 alkyl (e.g., methyl, ethyl, propyl, or isopropyl, e.g., isopropyl), or two R P2 together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; or R P1 and one R P2 together with the atom to which they are attached form an optionally substituted 4-8 membered heterocyclyl.

[0009] In some embodiments, R 3 is -OR 30 , and R 30 is -P(OR P1 )N(R P2 )2. For example, R3is -OR 30 , and R 30 is -P(OR P1 )N(R P2 )2, wherein R P1 is C 1-6 alkyl substituted with cyano or -SC(O)Ph. In some embodiments, R 3 is -OR 30 , and R 30 is -P(OR P1 )N(R P2 )2, wherein R P1 is -CH2CH2CN.

[0010] In some embodiments, R 3 is -OR 30 , and R 30 is -P(OR P1 )N(R P2 )2, and each R P2 is independently methyl, ethyl, propyl, or isopropyl. For example, R 3 is -OR 30 , and R 30 is -P(OR P1 )N(R P2 )2, wherein each R P2 is isopropyl.

[0011] In some preferred embodiments, R3 For -OR 30 And R 30 -P(OR) P1 )N(R P2 )2, where R P1 C substituted with cyano or -SC(O)Ph 1-6 Alkyl groups, and each R P2 It can be methyl, ethyl, propyl, or isopropyl independently. For example, R3 is -OR. 30 And R 30 -P(OR) P1 )N(R P2 )2, where R P1 For –CH2CH2CN, and each R P2 It is isopropyl.

[0012] In some implementation schemes, R 3 For -OR 30 And R 30 It is a phosphoramidite group, such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or 3'-[(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite.

[0013] In another instance, R 3 For -OR 30 And R 30 It is a hydrogen or hydroxyl protecting group (e.g., a silyl-based hydroxyl protecting group). Used for R 3 R 30 Some exemplary hydroxyl protecting groups include, but are not limited to, tert-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylhexylsilyl, tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethoxysilyl (DPMS), or tert-butylmethoxyphenylsilyl (TBMPS), wherein the hydroxyl protecting group is TBDMS.

[0014] In another example, R 3 For -OR 30 And R 30 This is a linkage bond with a nucleoside or nucleotide, or with an oligonucleotide. For example, R 30 For the linkage with oligonucleotides. When R 30 When it is a linker bond with a nucleoside or nucleotide, or a linker bond with an oligonucleotide, R 3to the 5'-hydroxyl of the nucleoside or nucleotide or the 5'-hydroxyl of the 5' end of the oligonucleotide. It should be noted that the internucleotide linkage between a compound or nucleoside of Formula I and the nucleoside, nucleotide, or oligonucleotide to which it is attached can be an unmodified internucleotide linkage (i.e., a phosphodiester linkage) or a modified internucleotide linkage (e.g., phosphorothioate, MMI, or imidp), preferably the modified internucleotide linkage is a phosphorothioate. Exemplary modified internucleotide linkages are described below.

[0015] In some embodiments, R 3 is hydrogen or halogen (e.g., F, Br, CI, or I). For example, R 3 is H or F.

[0016] In some compounds of Formula I, R 3 is -OR 20 wherein R 20 is optionally substituted C 1-6 alkyl. For example, R 3 is -OR 20 wherein R 20 is methyl, ethyl, or propyl. In some embodiments, R 3 is -OR 20 wherein R 20 is methyl.

[0017] In some compounds of Formula I, R 3 is -OR 20 wherein R 20 is optionally substituted C 2-6 alkenyl. For example, R 3 is -OR 20 wherein R 20 is ethenyl or allyl. In some compounds of Formula I, R 3 is -OR 20 wherein R 20 is optionally substituted C 2-6 alkynyl. For example, R 3 is -OR 20 wherein R 20 is ethynyl, propargyl, or 5-hexyn-1-yl.

[0018] In some compounds of Formula I, R 3 is -OR 20 wherein R 20 is C 1-6 alkoxy C 1-6 alkyl. For example, R 3 is -OR 20 wherein R 20 is 2-methoxyethyl.

[0019] R 3 is -OR 30 , and R 2 is hydrogen, halogen, or -OR 20 . For example, R 3 is -OR 30 , and R 2 is hydrogen, halogen, or -OR 20 , wherein R 30 is a reactive phosphorus group, a hydroxyl protecting group, a linkage to a nucleoside or nucleotide, or a linkage to an oligonucleotide.

[0020] In some embodiments, R 3 is -OR 30 , R 2 is hydrogen or halogen (e.g., F, Br, CI, or I), and R 30 is a reactive phosphorus group, a hydroxyl protecting group, a linkage to a nucleoside or nucleotide, or a linkage to an oligonucleotide. For example, R 2 is H or F.

[0021] In some compounds of Formula I, R 2 is -OR 20 , wherein R 20 is optionally substituted C 1-6 alkyl. In one example, R 20 is C 1-6 alkyl substituted with one, two, or three substituents independently selected from the group consisting of halogen, -OR 22 , -N(R 22 )2, -SR 22 , -C(O)OR 22 , -C(O)N(R 22 )2, wherein R 22 is hydrogen or C 1-3 alkyl (e.g., 2,2,2-trifluoroethyl, 1,3-dimethoxypropan-2-yl). In another example, R 20 is C 1-6 alkyl substituted with one or two substituents independently selected from the group consisting of halogen, -OR 22 , -N(R 22 )2, -SR 22 , -C(O)OR 22 , -C(O)N(R 22 )2, wherein R 22 is hydrogen or C 1-3 alkyl (e.g., 2,2,2-trifluoroethyl, 1,3-dimethoxypropan-2-yl).

[0022] In another example, R 2 For -OR 20 , where R 20 It is methyl, ethyl, or propyl. In some embodiments, R 2 For -OR 20 , where R 20 It is a methyl group.

[0023] In some compounds of formula I, R 2 For -OR 20 , where R 20 C 1-6 Alkoxy C 1-6 Alkyl group. For example, R 2 For -OR 20 , where R 20 It is 2-methoxyethyl.

[0024] In some compounds of formula I, R 2 For -OR 20 , where R 20 For N-(C 1-6 alkyl)carbamoyl C 1-6 Alkyl group. For example, R 2 For -OR 20 , where R 20 It is 2-(N-methylamino)-2-oxoethyl.

[0025] In another example, R 2 For -OR 30 And R 30 It is a hydrogen or hydroxyl protecting group (e.g., a silyl-based hydroxyl protecting group). Used for R 2 R 30 Some exemplary hydroxyl protecting groups include, but are not limited to, tert-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylhexylsilyl, tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethoxysilyl (DPMS), or tert-butylmethoxyphenylsilyl (TBMPS), wherein the hydroxyl protecting group is TBDMS.

[0026] In some of the compounds described in this article, R 2 For -OR 30 For example, R 2 For -OR 30 And R 30is a reactive phosphorus group. Exemplary reactive phosphorus groups are described below, including but not limited to phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphotriesters, and phosphorus-containing chiral auxiliaries. Thus, in some embodiments, R 2 is -OR 30 , and R 30 is a reactive phosphorus group selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, and phosphotriesters, optionally R 30 is a phosphoramidite. Accordingly, in some compounds, R 30 is -P(OR P1 )N(R P2 )2, -P(SR P1 )N(R P2 )2, -P(O)(OR P1 )N(R P2 )2, -P(S)(OR P1 )N(R P2 )2, -P(R P3 )N(R P2 )2, -P(O)(SR P1 )N(R P2 )2, -P(O)(OR P1 )H, -P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , wherein: each R P3 is independently optionally substituted C1-C 30 alkyl, optionally substituted C2-C 30 alkenyl, or optionally substituted C2-C 30 alkynyl (e.g., optionally substituted C1-C 10 alkyl, optionally substituted C2-C 10 alkenyl, or optionally substituted C2-C 10 alkynyl); each R P1 is independently optionally substituted C 1-6 alkyl; and each R P2 is independently optionally substituted C 1-6 alkyl (e.g., methyl, ethyl, propyl, or isopropyl, e.g., isopropyl), or two R P2together with the atom to which they are attached form an optionally substituted 4-8 membered heterocyclyl. or R P1 and one R P2 together with the atom to which they are attached form an optionally substituted 4-8 membered heterocyclyl.

[0027] In some embodiments, R 2 is -OR 30 , and R 30 is -P(OR P1 )N(R P2 )2. For example, R 2 is -OR 30 , and R 30 is -P(OR P1 )N(R P2 )2, wherein R P1 is C 1-6 alkyl substituted with cyano or -SC(O)Ph. In some embodiments, R 2 is -OR 30 , and R 30 is -P(OR P1 )N(R P2 )2, wherein R P1 is -CH2CH2CN.

[0028] In some embodiments, R 2 is -OR 30 , and R 30 is -P(OR P1 )N(R P2 )2, and each R P2 is independently methyl, ethyl, propyl, or isopropyl. For example, R 2 is -OR 30 , and R 30 is -P(OR P1 )N(R P2 )2, wherein each R P2 is isopropyl.

[0029] In some preferred embodiments, R 2 is -OR 30 , and R 30 is -P(OR P1 )N(R P2 )2, wherein R P1 is C 1-6 alkyl substituted with cyano or -SC(O)Ph, and each R P2 is independently methyl, ethyl, propyl, or isopropyl. For example, R 2 is -OR 30 , and R 30-P(OR) P1 )N(R P2 )2, where R P1 It is -CH2CH2CN, and each R P2 It is isopropyl.

[0030] In some implementation schemes, R 2 For -OR 30 And R 30 It is a phosphoramid group, such as 2'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid or 2'-[(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramid.

[0031] In yet another example, R 2 For -OR 30 And R 30 This is a linkage bond with a nucleoside or nucleotide, or with an oligonucleotide. For example, R 30 For the linkage with oligonucleotides. When R 30 When it is a linker bond with a nucleoside or nucleotide, or a linker bond with an oligonucleotide, R 2 It can be linked to the 5'-hydroxyl group of the nucleoside or nucleotide or the 5'-terminus of the oligonucleotide. It should be noted that the internucleotide linking bond between the compound of Formula I or the nucleoside and the linked nucleoside, nucleotide, or oligonucleotide can be an unmodified internucleotide linking bond (i.e., a phosphodiester bond) or a modified internucleotide linking bond (e.g., phosphate thioester, MMI, or imidp), preferably a phosphate thioester. Exemplary modified internucleotide links are described below.

[0032] In compounds of Formula I, B is an optionally modified natural or non-natural base. For example, B is uracil, adenine, cytosine, 5-methylcytosine, guanine, or thymine (i.e., 5-methyluracil). In some embodiments, B is a modified or protected base. For example, B is a protected base containing at least one amine or hydroxyl protecting group. In some embodiments, B is adenine, cytosine, 5-methylcytosine, or guanine containing at least one amine protecting group. Exemplary modified and unmodified natural and non-natural bases are described below.

[0033] In some compounds of formula I, X is O; R 2 For hydrogen, F or -OR 20 , where R 20 Hydrogen, or optionally substituted C 1-6 Alkyl groups (e.g., methyl, ethyl, or propyl), such as C 1-6 Alkoxy C 1-6 Alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C1-6 alkyl)aminocarbonyl C 1-6 Alkyl groups (e.g., 2-(N-methylamino)-2-oxoethyl); and R 3 For -OR 30 , where R 30 It is a hydrogen or hydroxyl protecting group or a reactive phosphorus group.

[0034] In some compounds of formula I, X is O; R 2 For hydrogen, F or -OR 20 , where R 20 Hydrogen, or optionally substituted C 1-6 Alkyl groups (e.g., methyl, ethyl, or propyl), such as C 1-6 Alkoxy C 1-6 Alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C 1-6 alkyl)aminocarbonyl C 1-6 Alkyl groups (e.g., 2-(N-methylamino)-2-oxoethyl); and R 3 For -OR 30 , where R 30 The reactive phosphorus group is (e.g., phosphorous amide, H-phosphonate, alkyl-phosphonate, or triphosphate). In a further embodiment of this embodiment, the reactive phosphorus group is: -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 (For example, -P(OR) P1 )N(R P2 )2), where: each R P3 C1-C independently of optional substitution 30 Alkyl, optionally substituted C2-C30 alkenyl or optionally substituted C2-C 30 Alkyne groups (e.g., optionally substituted C1-C) 10 Alkyl, optionally substituted C2-C 10 alkenyl or optionally substituted C2-C 10 alkynyl group); each R P1 C can be substituted independently. 1-6 Alkyl groups (e.g., C groups substituted with cyano or -SC(O)Ph) 1-6 Alkyl groups, such as 2-cyanoethyl); and each R P2 C can be substituted independently. 1-6 Alkyl (e.g., methyl, ethyl, propyl, or isopropyl, preferably isopropyl), or two R P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3-8 membered heterocyclic group; or R P1 And an R P2 Together with the atoms to which they are attached, they form optional 4-8 membered heterocyclic groups.

[0035] In some compounds of formula I, X is O; R 2 For hydrogen, F or -OR 20 , where R 20 Hydrogen, or optionally substituted C 1-6 Alkyl groups (e.g., methyl, ethyl, or propyl, preferably methyl), such as C 1-6 Alkoxy C 1-6 Alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C 1-6 alkyl)aminocarbonyl C 1-6 Alkyl groups (e.g., 2-(N-methylamino)-2-oxoethyl); and R 3 For -OR 30 , where R 30 -P(OR) P1 )N(R P2 )2, where R P1 It is -CH2CH2CN, and each R P2 It is isopropyl.

[0036] In some preferred embodiments, X is O; R 2 For hydrogen, F or -OR 20 , where R 20 It is hydrogen, methyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl or 2-(N-methylamino)-2-oxoethyl; and R 3 For -OR 30 , where R 30 -P(OR) P1 )N(R P2 )2, where R P1It is -CH2CH2CN, and each R P2 It is isopropyl.

[0037] In some embodiments, the compound has the following formula: or

[0038] Or its salt, wherein: B is an optional modified base (e.g., uracil); X is O or S; each R V Independently protected by a hydrogen or hydroxyl group (e.g., ethyl or neopentanoyloxymethyl); R 2 and R 3 One of them is hydrogen, halogen, or -OR 20 ,in: R 20 Protected by hydrogen, hydroxyl group, or optionally substituted C 1-6 Alkyl groups (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl, 2-(N-methylamino)-2-oxoethyl), optionally substituted C 2-6 alkenyl or optionally substituted C 2-6 Alkyne group (e.g., propargyl); and R 2 and R 3 The other one is -OR 30 , where: R 30 It can be a hydrogen or hydroxyl protecting group, a reactive phosphorus group (e.g., phosphoramide), a linking bond with a nucleoside or nucleotide, or a linking bond with an oligonucleotide.

[0039] In some embodiments, the compound has the following formula: or

[0040] Or its salt, wherein: B is an optional modified base (e.g., uracil). X is O or S; Each R V Independently protected by a hydrogen or hydroxyl group (e.g., ethyl or neopentyloxymethyl); R 2 and R 3 One of them is hydrogen, halogen, or -OR 20 ,in: R 20 It is a hydrogen, hydroxyl protecting group, or optionally substituted C 1-6 Alkyl groups (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl, 2-(N-methylamino)-2-oxoethyl), optionally substituted C2-6 alkenyl groups, or optionally substituted C2-6 alkenyl groups. 3-6alkynyl (e.g., propynyl); and R 2 and R 3 The other one is -OR 30 ,in: R 30 It is a hydrogen, hydroxyl protecting group, reactive phosphorus group (e.g., phosphoramide), linking bond with nucleosides or nucleotides, or linking bond with oligonucleotides.

[0041] In some embodiments, the compound is of the formula... Or its salt.

[0042] In some embodiments, the compound is of the formula... Or its salt.

[0043] In some embodiments, the compound is of the formula... Or its salt.

[0044] In some implementation schemes, R 3 Yes - OR 30 For example, the compound has the formula, , , , , , or , or its salt, in: R 3 Yes - OR 30 ,in: R 30 These are hydrogen, hydroxyl protecting groups, reactive phosphorus groups (e.g., phosphorous amide), linkages with nucleosides or nucleotides, or linkages with oligonucleotides; B is an optional modified base (e.g., uracil). X is O or S; Each R P Independently protected by a hydrogen or hydroxyl group (e.g., ethyl or neopentanoyloxymethyl); and R 2 Is it hydrogen, halogen, or -OR? 20 ,in: R 20 It is a hydrogen, hydroxyl protecting group, or optionally substituted C 1-6 Alkyl groups (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl, 2-(N-methylamino)-2-oxoethyl), optionally substituted C 2-6 alkenyl or optionally substituted C 3-6 Alkyne group (e.g., propargyl);

[0045] In some embodiments, B is uracil or thymine. In some embodiments, R... V It is hydrogen. In some implementations, R V It is a hydroxyl protecting group (e.g., ethyl or neopentyloxymethyl).

[0046] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2, X is O. For example, the compound has the formula, , , , , , , or

[0047] or its salt, in: R 3’ Yes - OR 30 ,in: R 30 It is -P(OR) P1 )N(R P2 )2, in: Each R P1 C is an optional substitute 1-6 Alkyl groups (e.g., -CH2CH2CN); Each R P2 C can be substituted independently. 1-6 Alkyl groups (e.g., isopropyl); X is O; B is an optional modified base (e.g., uracil). Each R P Independently protected by a hydrogen or hydroxyl group (e.g., ethyl or neopentanoyloxymethyl); and R 2’ Is it hydrogen, halogen, or -OR? 20 ,in: R 20 It is a hydrogen, hydroxyl protecting group, or optionally substituted C 1-6 Alkyl groups (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl, 2-(N-methylamino)-2-oxoethyl), optionally substituted C 2-6 alkenyl or optionally substituted C 3-6 Alkyne (e.g., propyne).

[0048] In some embodiments, B is uracil or thymine. In some embodiments, R... V It is hydrogen. In some implementations, R V It is a hydroxyl protecting group (e.g., ethyl or neopentyloxymethyl).

[0049] In some compounds of formula (I), the compound's Partially The substituted compound, i.e., the compound is of formula [formula missing] Among them, B, X, R V R 2 and R 3 As defined in formula (Ⅰ) herein. For example, the structure of the compound is: .

[0050] In some embodiments, the compound is , , or

[0051] Or its salt, wherein: B is an optional modified base (e.g., uracil). X is O or S; Each R V Independently protected by hydrogen or hydroxyl groups (e.g., ethyl or neopentyloxymethyl); R 2 Is it hydrogen, halogen, or -OR? 20 ,in: R 20 It is a hydrogen, hydroxyl protecting group, or optionally substituted C 1-6 Alkyl groups (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl, 2-(N-methylamino)-2-oxoethyl, optionally substituted C) 2-6 alkenyl or optionally substituted C 3-6 alkynyl (e.g., propynyl); and Represents the remaining portion of an oligonucleotide (e.g., the antisense strand of a double-stranded RNA).

[0052] Previously, the oxygen atom that connects the 5' end of the oligonucleotide to the phosphorus atom is the 5' oxygen atom of the 5' terminal nucleotide of the oligonucleotide.

[0053] In some implementations, Y is S.

[0054] In this article, Used to represent oligonucleotides; such oligonucleotides can be RNA, DNA, single-stranded RNA (such as antisense oligonucleotides (ASO)), antisense strands of double-stranded RNA (such as siRNA), and oligonucleotide derivatives (such as phosphoridamide morpholino oligomers (PMO)).

[0055] In some implementation schemes, ,

[0056] or , Or its salt, wherein: R V It is a hydrogen or hydroxyl protecting group (e.g., ethyl or neopentanoyloxymethyl). In some embodiments, B is uracil or thymine. In some embodiments, R V It is hydrogen. In some implementations, R V It is a hydroxyl protecting group (e.g., neopentanoyloxymethyl). In some embodiments, Y is S. In other embodiments, Y is O.

[0057] In some embodiments, the compound is selected from the compounds shown in Table A: Table A: Some Exemplary Compounds

[0058]

[0059] In another aspect, this paper provides an oligonucleotide with a 5' end modification, the structure of which is as follows: , in: X is O or S; and Each R V It is independently protected by hydrogen or hydroxyl groups.

[0060] In some implementations, the 5' end modification includes the following structure: or .

[0061] In some implementations, X is 0. In some implementations, X is 0, and each R... V It is hydrogen. In some implementations, X is O, and each R V It is ethyl. In some embodiments, X is O, and each R V It is neopentyloxymethyl.

[0062] It is worth noting that, including (For example, or The glycosyl moiety of a nucleoside modified with the above-mentioned modifications, namely the nucleotide at the 5' end of the oligonucleotide (5' terminal nucleotide), may contain a 5- or 6-membered ring. For example, the glycosyl moiety in a nucleoside containing the above-mentioned modifications may be a furanose (e.g., ribofuranose, arabinofuranose, xyfuranose, ribonefuranose, or xynefuranose, including their α- and β-, D- and L-, deoxy-, and modified derivatives thereof), or a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, and talopyranose, including their α- and β-, D- and L-, deoxy-, and modified derivatives thereof).

[0063] generally, (For example, or This modification replaces the CH2OH group on the 5'-terminal nucleotide sugar moiety of the oligonucleotide. For example, (For example, or The modification replaces the 4'-CH2OH group on the furanose ring (e.g., ribofuranose, arabinofuranose, xylofuranose, ribonefuranose, or xylofuranose, preferably ribofuranose) or the 5'-CH2OH group on the pyranose ring (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, choropyranose, gulopyranose, idupyranose, and taropyranose, preferably glucopyranose, galactopyranose, or mannopyranose) of the 5'-terminal nucleotide of the oligonucleotide.

[0064] In some embodiments, the oligonucleotide comprises a compound of formula (I) described herein at its 5' end. For example, the 5' nucleotide of the oligonucleotide has the following structure: , in: Each R V Independently protected by hydrogen or hydroxyl groups (e.g., ethyl or neopentyloxymethyl ((CH3)3CC(O)OCH2-, POM); B is an optional modified base (e.g., uracil). R 2 and R 3 One of them is hydrogen, halogen, or -OR 20 ,in: R 20 It is a hydrogen, hydroxyl protecting group, or optionally substituted C 1-6 Alkyl groups (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl, 2-(N-methylamino)-2-oxoethyl), optionally substituted C2-6 alkenyl or optionally substituted C 3-6 alkynyl (e.g., propynyl); and R 2 and R 3 The other one is -OR 30 ,in: R 30 It is a linking bond with an oligonucleotide (e.g., an internucleotide linking bond that links the oligonucleotide to a subsequent nucleotide).

[0065] In some implementations, the 5' terminal nucleotide of the oligonucleotide has the following structure: , where R 2 and R 3 One of them is -OR 30 (For example, R) 3 For -OR 30 ), where: R 30 It is a linking bond with an oligonucleotide (e.g., an internucleotide linking bond that connects to a subsequent nucleotide of an oligonucleotide).

[0066] In some other embodiments, the 5' terminal nucleotide of the oligonucleotide has the following structure: Where R 2 and R 3 One of them is -OR 30 (For example, R) 3 For -OR 30 ), where: R 30 It is a linking bond with an oligonucleotide (e.g., an internucleotide linking bond that connects to a subsequent nucleotide of an oligonucleotide).

[0067] In some other embodiments, the 5' terminal nucleotide of the oligonucleotide has the following structure: , where R 2 and R 3 One of them is -OR 30 (For example, R) 3 For -OR 30 ), where: R 30 It is a linking bond with an oligonucleotide (e.g., an internucleotide linking bond that connects to a subsequent nucleotide of an oligonucleotide).

[0068] In some other embodiments, the 5' terminal nucleotide of the oligonucleotide has the following structure: , where R 2 and R 3 One of them is -OR 30 (For example, R) 3 For -OR 30 ), where: R 30It is a linking bond with an oligonucleotide (e.g., an internucleotide linking bond that connects to a subsequent nucleotide of an oligonucleotide).

[0069] In some other embodiments, the 5' terminal nucleotide of the oligonucleotide has the following structure: Where R 2 and R 3 One of them is -OR 30 (For example, R) 3 For -OR 30 ), where: R 30 It is a linking bond with an oligonucleotide (e.g., an internucleotide linking bond that connects to a subsequent nucleotide of an oligonucleotide).

[0070] Typically, the oligonucleotides described herein contain at least three nucleotides. For example, an oligonucleotide may contain 5 to 100, such as 10 to 50 nucleotides. In some embodiments, the oligonucleotide may contain 15 to 40 nucleotides. For example, the length of the oligonucleotide may be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the oligonucleotide may be 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides. For example, the length of the oligonucleotide may be 19, 20, 21, 22, or 23 nucleotides. It is worth noting that compounds of Formula I are counted as one nucleotide.

[0071] The oligonucleotides described herein may contain at least one nucleic acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more independently selected modifications). Exemplary nucleic acid modifications are described below, including but not limited to base modifications, sugar modifications, internucleotide linking modifications, conjugates (e.g., ligands), and combinations thereof.

[0072] In some embodiments, the oligonucleotide contains at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-OMe nucleotides.

[0073] In some embodiments, the oligonucleotide contains at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) thermally unstable modification to the duplex. For example, the oligonucleotide contains a thermally unstable modification at at least one of positions 4, 5, 6, 7 or 8, counting from the 5' end of the oligonucleotide, wherein the compound of formula I is located at position 1 at the 5' end of the oligonucleotide; optionally, the thermally unstable modification is located at position 6, 7 or 8, counting from the 5' end of the oligonucleotide, preferably, the thermally unstable modification is located at position 7, counting from the 5' end of the oligonucleotide.

[0074] In some embodiments, the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-F nucleotide. For example, the oligonucleotide comprises 2, 3, 4, 5 or 6 2'-F nucleotides, optionally, the oligonucleotide comprises 3, 4 or 6 2'-F nucleotides. In some embodiments, counting from the 5' end of the oligonucleotide, the oligonucleotide contains 2'-F nucleotides at least at positions 2, 14 and 16, wherein the compound of formula I is located at position 1 of the 5' end of the oligonucleotide. For example, counting from the 5' end of the oligonucleotide, the oligonucleotide contains 2'-F nucleotides at least at positions 2, 6, 14 and 16, optionally, the oligonucleotide contains 2'-F nucleotides at least at positions 2, 6, 9, 14 and 16, preferably, counting from the 5' end of the oligonucleotide, the oligonucleotide contains 2'-F nucleotides at least at positions 2, 6, 8, 9, 14 and 16. It is worth noting that when there is more than one 2'-F nucleotide in an oligonucleotide, each 2'-F nucleotide is an independently selected nucleotide.

[0075] The oligonucleotide may also contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy (2'-H) nucleotides. For example, the oligonucleotide contains 2, 3, 4, 5, 6 or 7 2'-deoxy nucleotides, optionally, the oligonucleotide contains 3, 4, 5 or 6 2'-deoxy nucleotides. Starting from the 5' end of the oligonucleotide, the oligonucleotide may contain a 2'-deoxy nucleotide at any of the 2, 5, 7, 12, 14 and 16 positions, wherein the compound of formula I is located at the first position of the 5' end count of the oligonucleotide. For example, starting from the 5' end of the oligonucleotide, the oligonucleotide contains at least one 2'-deoxy nucleotide at the 5th position. In some embodiments, starting from the 5' end of the oligonucleotide, the oligonucleotide contains a 2'-deoxy nucleotide at at least the 2, 5 and 9 positions. For example, starting from the 5' end of the oligonucleotide, the oligonucleotide contains a 2'-deoxy nucleotide at at least the 2, 5, 7 and 12 positions. For example, starting from the 5' end of the oligonucleotide, the oligonucleotide contains 2'-deoxynucleotides at positions 2, 5, 7, 12, 14, and 16. It is noteworthy that when multiple 2'-deoxynucleotides are present in an oligonucleotide, each 2'-deoxynucleotide is an independently chosen nucleotide.

[0076] One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) bases in an oligonucleotide may be non-natural or modified bases. For example, an oligonucleotide may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified or protected bases.

[0077] The internucleotide linkages in an oligonucleotide can be independently unmodified (e.g., phosphodiester bonds) or modified (e.g., thiophosphate bonds). Therefore, in some embodiments, the oligonucleotide contains at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified internucleotide linkages. Typically, the oligonucleotide contains at least one (e.g., 1, 2, 4 or 5) modified internucleotide linkages (e.g., thiophosphate bonds) at positions 1-5 at one or both ends of the oligonucleotide. For example, the oligonucleotide contains modified oligonucleotide linkages (e.g., thiophosphate bonds) between the first and second nucleotides counting from the 5' end of the oligonucleotide and between the second and third nucleotides; and the oligonucleotide contains modified oligonucleotide linkages (e.g., thiophosphate bonds) between the first and second nucleotides counting from the 3' end of the oligonucleotide and between the second and third nucleotides.

[0078] In some implementations, oligonucleotides are covalently linked to a vector, such as a solid-phase vector.

[0079] In another aspect, this paper provides a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are substantially complementary, and wherein one of the sense strand or the antisense strand is an oligonucleotide as described herein, namely an oligonucleotide with a 5' end modification, comprising the following structure: (For example or ), in: X is O or S; and Each R V It is independently protected by hydrogen or hydroxyl groups.

[0080] Preferably, the antisense chain includes the aforementioned 5' end modification.

[0081] In another aspect, this article provides a method for reducing target gene expression in a subject. The method includes administering to the subject: (i) a double-stranded RNA as described herein, wherein the antisense strand is substantially complementary to the target gene; or (ii) an oligonucleotide as described herein, wherein the oligonucleotide is substantially complementary to the target gene.

[0082] In another aspect, this article provides a pharmaceutical composition comprising, alone or in combination with a pharmaceutically acceptable carrier or excipient, the oligonucleotide or dsRNA molecule described herein.

[0083] In another aspect, this article provides a cell containing the oligonucleotide or dsRNA molecule described herein.

[0084] In another aspect, this article provides a gene silencing kit containing oligonucleotides or dsRNA molecules as described herein.

[0085] Furthermore, this article provides a method for silencing a target gene in a cell. The method includes the following steps: (i) introducing a dsRNA molecule as described herein into the cell, wherein one strand of the dsRNA, such as an antisense strand, contains a nucleotide sequence substantially complementary to the nucleotide sequence of the target gene; and / or (ii) an oligonucleotide as described herein, wherein the oligonucleotide contains a nucleotide sequence substantially complementary to the nucleotide sequence of the target gene.

[0086] In another aspect, this article provides a method for inhibiting or reducing the expression of a target gene in a subject. The method comprises administering to the subject: (i) a dsRNA molecule as described herein, wherein one strand of the dsRNA, such as an antisense strand, contains a nucleotide sequence substantially complementary to the nucleotide sequence of the target gene; and / or (ii) an oligonucleotide as described herein, wherein the oligonucleotide contains a nucleotide sequence substantially complementary to the nucleotide sequence of the target gene. Attached Figure Description

[0087] Figure 1A -1F describes synthetic schemes for some exemplary compounds of this disclosure.

[0088] Figure 2 RNAi activity of some exemplary dsRNAs targeting mTTR and comprising exemplary compounds of this disclosure is shown.

[0089] Figure 3 SOD1 evaluation of exemplary dsRNAs containing exemplary compounds in rats was described.

[0090] Figures 4-7 It describes molecular modeling research. Figure 4 In the case of 6'-E-VP-RNA, it is located in a crowded space, occupying the position of water molecules in the parent structure. Figure 5 The spacing is very tight, and the water in the 4F3T (PDB) miR-20a-Ago2 complex is displaced. Otherwise, there would be no space at all between the phosphate groups and various basic side chains now inserted deeper into the pocket. The reference structure has been superimposed on all other structures and is shown in gray. Figure 6 The 6ʹ-Z-VP modification fits well; it appears to fit better than the 6-E-VP-isomer, which has spatial conflicts with Gln-545 and Tyr-529; the stacking interaction of 6ʹ-Z-VP with Tyr-529 is better than that of 5'-Z-VP. Figure 7The 6'-Z-VP-U modification at AS1 fits well into the MID binding pocket - it looks better than the case of 6'-E-VP, which pushes the terminal phosphate group too close to multiple basic residues as well as Gln-545 and Tyr-529. Detailed Implementation

[0091] It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative only, and are not intended to limit the scope of the claimed invention. Herein, the singular is used to include the plural unless otherwise specified. Unless otherwise specified, “or” as used herein means “and / or”. Furthermore, the use of the word “including” and other forms such as “includes” and “included” is not restrictive. Moreover, unless otherwise specified, terms such as “element” or “component” include both elements and components constituting a single unit and elements or components constituting multiple sub-units.

[0092] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter. All documents or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, and papers, are hereby expressly incorporated herein by reference in their entirety for any purpose.

[0093] R 3

[0094] In all the aspects discussed in this article, R 3 It can be hydrogen, halogen, or -OR 20 or -OR 30 .

[0095] In some implementation schemes, R 3 Yes - OR 30 , where R 30 It is a hydrogen, hydroxyl protecting group, reactive phosphorus group (e.g., phosphorous acid), bond with a nucleoside or nucleotide, or bond with an oligonucleotide. For example, R 3 Yes - OR 30 R 30 It is a hydrogen or hydroxyl protecting group.

[0096] In some implementation schemes, R 3 Yes - OR 30 R 30 It is a reactive phosphorus group. For example, R 30 It is a phosphorus amide, H-phosphonate, alkylphosphonate, or triphosphate. In some embodiments, R 3 Yes - OR 30 R 30 It is -P(OR)P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 ,in: Each R P1 It is C 1-6 Alkyl group, optionally substituted with 1, 2, 3, 4 or 5 substituents, said substituents being 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6; Each R P2 Independently for C 1-6Alkyl group, optionally substituted with 1, 2, 3, 4 or 5 substituents, said substituents being 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6, optionally, each R P2 Each of the following is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl, preferably each R P2 It is isopropyl; Or two Rs P2 Together with the nitrogen atom to which it is attached, it forms an optional substituted 3-8 membered heterocyclic group; Or R P1 and R P2 One of them, together with the atoms it is attached to, forms an optionally substituted 4-8 membered heterocyclic group; and Each R P3 Independently for C 1-30 Alkyl, C2-C 30 alkenyl or C2-C 30The alkynyl group is optionally and independently substituted by 1, 2, 3, 4, or 5 substituents, said substituents being 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6, optionally each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy groups.

[0097] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )RP3 or -P(O)(SR P1 )R P3 , where: each R P1 It is C 1-6 Alkyl groups, optionally substituted with CN or –SC(O)Ph; each R P2 C can be substituted independently. 1-6 Alkyl groups; and each R P3 C can be substituted independently. 1-6 alkyl.

[0098] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is C 1-6 Alkyl groups, optionally substituted with CN or –SC(O)Ph; each R P2 Independently, each of the following is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy.

[0099] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2)2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is 2-cyanoethyl (-CH2CH2CN); each R P2 C can be substituted independently. 1-6 Alkyl groups; and each R P3 C can be substituted independently. 1-6 alkyl.

[0100] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )RP3 , where: each R P1 It is 2-cyanoethyl (-CH2CH2CN); each R P2 Independently, each of the following is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy.

[0101] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is 2-cyanoethyl (-CH2CH2CN); each R P2 Independently isopropyl; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy.

[0102] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2, where: R P1It is 2-cyanoethyl (-CH2CH2CN); each R P2 It is independently isopropyl.

[0103] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is C 1-6 Alkyl groups, optionally substituted with CN or –SC(O)Ph; R P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3-8 membered heterocyclic group; and each R P3 C can be substituted independently. 1-6 alkyl.

[0104] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2)2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is C 1-6 Alkyl groups, optionally substituted with CN or –SC(O)Ph; R P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3-8 membered heterocyclic group; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy.

[0105] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is C 1-6 Alkyl groups, optionally substituted with CN or –SC(O)Ph; R P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3-8 membered heterocyclic group; and each R P3 C can be substituted independently.1-6 alkyl.

[0106] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is 2-cyanoethyl (-CH2CH2CN); R P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3-8 membered heterocyclic group; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy.

[0107] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2)2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: R P1 and R P2 One of them, together with the atom it is attached to, forms an optionally substituted 4-8 membered heterocyclic group; the other R P2 C can be substituted independently. 1-6 Alkyl groups; and each R P3 C can be substituted independently. 1-6 alkyl.

[0108] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: R P1 and R P2 One of them, together with the atom it is attached to, forms an optionally substituted 4-8 membered heterocyclic group; the other R P2 Independently, each of the following is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl; and each R P3 C can be substituted independently. 1-6 alkyl.

[0109] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: R P1 and R P2 One of them, together with the atom it is attached to, forms an optionally substituted 4-8 membered heterocyclic group; the other R P2 C can be substituted independently. 1-6 Alkyl groups; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy.

[0110] In some implementation schemes, R 3 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1)N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: R P1 and R P2 One of them, together with the atom it is attached to, forms an optionally substituted 4-8 membered heterocyclic group; the other R P2 Independently, each of the following is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy.

[0111] In some implementation schemes, R 3 Yes - OR 30 R 30 It is a linkage bond with a nucleoside, nucleotide, or oligonucleotide. When R... 30 When the linkage between a compound of Formula I and a nucleoside, nucleotide, or oligonucleotide is a linking bond, the nucleotide linking between the compound and the nucleoside, nucleotide, or oligonucleotide can be an unmodified (e.g., phosphodiester bond) nucleotide linking bond or a modified (e.g., thiophosphate bond) nucleotide linking bond.

[0112] In some implementation schemes, R 3 Yes - OR 30 R 30 Linked to the 5' position of a nucleoside, nucleotide, or oligonucleotide via an unmodified (e.g., phosphodiester bond) or modified (e.g., thiophosphate bond) internucleotide linker. For example, R 30 It is linked to the 5'-terminus (e.g., 5'-OH) of the oligonucleotide. In some embodiments, R 30 Linked to the 5'-terminus (e.g., 5'-OH) of the oligonucleotide via an unmodified (e.g., phosphodiester bond) internucleotide linker. In some other embodiments, R 30 The oligonucleotide is linked to the 5'-terminus (e.g., 5'-OH) via a modified (e.g., thiophosphate) internucleotide linker.

[0113] In some implementation schemes, R 3’ Yes - OR 30 R30 It is a hydroxyl protecting group. For example, R 3’ Yes - OR 30 R 30It is a hydroxyl protecting group, selected from BOC or Boc, MOM, MTM, tert-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, tert-butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxymethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiaranyl, 1-methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiaranyl, 4-methoxytetrahydrothiaranyl-S,S-dioxide, CTMP, 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrothiaranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trihydroxythiaranyl Methyl-4,7-methoxybenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-phenoxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenoyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-formylbenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-pyridinecarboxyl, 4-pyridinecarboxyl, 3-methyl-2-pyridinyl N-oxide, diphenylmethyl, p,p′-dinitrophenylmethyl, 5-dibenzoyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)tonyl, 9-(9-phenyl-10-oxo)anthrayl, 1,3-benzodithiacyclopentadien-2-yl, benzisothiazolyl-S,S-dioxy, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthiahexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, TBMPS, formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate Methoxyacetate, Triphenylmethoxyacetate, Phenoxyacetate, p-Chlorophenoxyacetate, 3-Phenylacetate, 4-Oxovalerate (Aleuprolate), 4,4-(Ethylene dithio)valerate (Aleuproyl dithioacetal), Adamantate, Crotonate, 4-Methoxycrotonate, Benzoate, Paraben, 2,4,6-Trimethylbenzoate (Trimethylbenzyl ester), Alkylmethyl Carbonate, Fmoc, Alkylethyl Carbonate, Troc, TMSEC, Psec, 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-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro- 4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid ester, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid ester, chlorodiphenylacetic acid ester, isobutyrate, (E)-2-methyl-2-butenoate, o-(methoxyyl)benzoate, α-naphthylcarbamate, nitrate esters, alkyl N,N,N',N'-tetramethyldiamide phosphate, alkyl N-phenylcarbamate, borate esters, dimethylphosphonothioyl, alkyl 2,4-dinitrophenylsulfite, sulfate esters, methanesulfonates, benzyl sulfonates, and p-toluenesulfonates.

[0114] In some implementation schemes, R 3’ Yes - OR 30 R 30 It is TBDMS, TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TMPS, DPMS, dimethylxylsilyl, tribenzylsilyl, tri-p-xylsilyl or triphenylsilyl, optionally, R 30 It's TBCMS.

[0115] In some implementation schemes, R 3 It is hydrogen or halogen. For example, R 3 It is H or F.

[0116] In some implementation schemes, R 3 Yes - OR 20 , where R 20 It is a hydrogen, hydroxyl protecting group, or optionally substituted C 1-6 Alkyl groups, such as C 1-6 Alkoxy C 1-6 Alkyl (e.g., 2-methoxyethyl) or N-(C 1-6 alkyl)aminocarbonyl C 1-6 Alkyl groups (such as 2-(N-methylamino)-2-oxoethyl). For example, R 3 Yes - OR 20 R 20 It is a hydrogen or hydroxyl protecting group.

[0117] In some implementation schemes, R 3 Yes - OR 20 R20 It is C 1-6 Alkyl group, optionally substituted with 1, 2, 3, 4 or 5 substituents, said substituents being independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C 1-4 )alkyl, SO2NH(C 1-4 Alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C 1-4 [alkyl]2, C(O)NH2, C(O)NH(C 1-6 Alkyl), C(O)N(C 1-6 Alkyl)2, COOH, COO(C 1-6 Alkyl groups (e.g., COOMe), C 2-6 Acyl (e.g., acetyl), (C 1-8 )alkyl, O(C 1-8 )alkyl (i.e., C 1-8 alkoxy), O(C) 1-8 ) Haloalkyl, (C 2-8 )alkenyl, (C 2-8 Alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaryl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2—[CH(OH)] m —(CH2) p —OH, CH2—[CH(OH)] m —(CH2) p —NH2 or CH2-arylalkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5, or 6. For example, R 3 Yes - OR 20 R 20 It is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl, optionally, R 20 It is a methyl group.

[0118] In some implementation schemes, R 3 Yes - OR 20 R 20 It is C 1-6 Alkoxy C 1-6 Alkyl group. For example, R 3 Yes - OR 20 R 20 It is 2-methoxyethyl.

[0119] In some implementation schemes, R 3’ For -OR 20 R 20It is a hydroxyl protecting group. For example, R 3’ For -OR 20 R 20It is a hydroxyl protecting group, selected from BOC or Boc, MOM, MTM, tert-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, tert-butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxymethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiaranyl, 1-methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiaranyl, 4-methoxytetrahydrothiaranyl-S,S-dioxide, CTMP, 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrothiaranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trihydroxythiaranyl Methyl-4,7-methoxybenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-phenoxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenoyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-formylbenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-pyridinecarboxyl, 4-pyridinecarboxyl, 3-methyl-2-pyridinyl N-oxide, diphenylmethyl, p,p′-dinitrophenylmethyl, 5-dibenzoyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)tonyl, 9-(9-phenyl-10-oxo)anthrayl, 1,3-benzodithiacyclopentadien-2-yl, benzisothiazolyl-S,S-dioxy, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthiahexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, TBMPS, formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate Methoxyacetate, Triphenylmethoxyacetate, Phenoxyacetate, p-Chlorophenoxyacetate, 3-Phenylacetate, 4-Oxovalerate (Acetopropionate), 4,4-(Ethylene dithio)valerate (Acetopropionyl dithioacetal), Adamantate, Crotonate, 4-Methoxycrotonate, Benzoate, Paraben, 2,4,6-Trimethylbenzoate (Trimethylbenzyl ester), Alkylmethyl Carbonate, Fmoc, Alkylethyl Carbonate, Troc, TMSEC, Psec, 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-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro- 4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid ester, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid ester, chlorodiphenylacetic acid ester, isobutyrate, (E)-2-methyl-2-butenoate, o-(methoxyyl)benzoate, α-naphthylcarbamate, nitrate esters, alkyl N,N,N',N'-tetramethyldiamide phosphate, alkyl N-phenylcarbamate, borate esters, dimethylphosphonothioyl, alkyl 2,4-dinitrophenylsulfite, sulfate esters, methanesulfonates, benzyl sulfonates, and p-toluenesulfonates.

[0120] In some implementation schemes, R 3’ For -OR 20 R 20 It is TBDMS (or TBS), TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TMPS, DPMS, dimethylxylsilyl, tribenzylsilyl, tri-p-xylylsilyl or triphenylsilyl.

[0121] R 2

[0122] In all the aspects discussed in this article, R 2 It can be hydrogen, halogen, or -OR 20 or -OR 30 .

[0123] In some implementation schemes, R 2 It is hydrogen or halogen. For example, R 2 It is H or F.

[0124] In some implementation schemes, R 2 Yes - OR 20 , where R 20 It is a hydrogen, hydroxyl protecting group, or optionally substituted C 1-6 Alkyl groups, such as C 1-6 Alkoxy C 1-6 Alkyl (e.g., 2-methoxyethyl) or N-(C 1-6 alkyl)aminocarbonyl C 1-6Alkyl groups (such as 2-(N-methylamino)-2-oxoethyl). For example, R 2 Yes - OR 20 R 20 It is a hydrogen or hydroxyl protecting group. In other examples, R 2 Yes - OR 20 , where R 20 C is an optional substitute 1-6 alkyl.

[0125] In some implementation schemes, R 2 Yes - OR 20 R 20 It is C 1-6 Alkyl group, optionally substituted with 1, 2, 3, 4 or 5 substituents, said substituents being independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C 1-4 )alkyl, SO2NH(C 1-4 Alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C 1-4 [alkyl]2, C(O)NH2, C(O)NH(C 1-6 Alkyl), C(O)N(C 1-6 Alkyl)2, COOH, COO(C 1-6 Alkyl groups (e.g., COOMe), C 2-6 Acyl (e.g., acetyl), (C 1-8 )alkyl, O(C 1-8 )alkyl (i.e., C 1-8 alkoxy), O(C) 1-8 ) Haloalkyl, (C 2-8 )alkenyl, (C 2-8 Alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaryl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2—[CH(OH)] m —(CH2) p —OH, CH2—[CH(OH)] m —(CH2) p —NH2 or CH2-arylalkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5, or 6. For example, R 2 Yes - OR 20 R 20 It is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl, optionally, R 20 It is a methyl group.

[0126] In some implementation schemes, R 2 Yes - OR 20 R 20 It is C 1-6 Alkyl group, optionally substituted with 1, 2, 3, 4 or 5 substituents, said substituents being independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C 1-4 )alkyl, SO2NH(C 1-4 Alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C 1-4 [alkyl]2, C(O)NH2, C(O)NH(C 1-6 Alkyl), C(O)N(C 1-6 Alkyl)2, COOH, COO(C 1-6 Alkyl groups (e.g., COOMe), C 2-6 Acyl (e.g., acetyl), (C 1-8 )alkyl, O(C 1-8 )alkyl (i.e., C 1-8 Alkoxy), O (C) 1-8 ) Haloalkyl, (C 2-8 )alkenyl, (C 2-8 Alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaryl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2—[CH(OH)] m —(CH2) p —OH, CH2—[CH(OH)] m —(CH2) p —NH2 or CH2-arylalkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5, or 6. For example, R 2 Yes - OR 20 R 20 It is 2-methoxyethyl.

[0127] In some implementation schemes, R 2’ For -OR 20 R 20 It is a hydroxyl protecting group. For example, R 2’ For -OR 20 R 20It is a hydroxyl protecting group, selected from BOC or Boc, MOM, MTM, tert-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, tert-butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxymethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiaranyl, 1-methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiaranyl, 4-methoxytetrahydrothiaranyl-S,S-dioxide, CTMP, 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrothiaranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trihydroxythiaranyl Methyl-4,7-methoxybenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-phenoxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenoyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-formylbenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-pyridinecarboxyl, 4-pyridinecarboxyl, 3-methyl-2-pyridinyl N-oxide, diphenylmethyl, p,p′-dinitrophenylmethyl, 5-dibenzoyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)tonyl, 9-(9-phenyl-10-oxo)anthrayl, 1,3-benzodithiacyclopentadien-2-yl, benzisothiazolyl-S,S-dioxy, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthiahexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, TBMPS, formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate Methoxyacetate, Triphenylmethoxyacetate, Phenoxyacetate, p-Chlorophenoxyacetate, 3-Phenylacetate, 4-Oxovalerate (Aleuprolate), 4,4-(Ethylene dithio)valerate (Aleuproyl dithioacetal), Adamantate, Crotonate, 4-Methoxycrotonate, Benzoate, Paraben, 2,4,6-Trimethylbenzoate (Trimethylbenzyl ester), Alkylmethyl Carbonate, Fmoc, Alkylethyl Carbonate, Troc, TMSEC, Psec, 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-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro- 4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid ester, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid ester, chlorodiphenylacetic acid ester, isobutyrate, (E)-2-methyl-2-butenoate, o-(methoxyyl)benzoate, α-naphthylcarbamate, nitrate esters, alkyl N,N,N',N'-tetramethyldiamide phosphate, alkyl N-phenylcarbamate, borate esters, dimethyl thiophosphonoyl, alkyl 2,4-dinitrophenyl sulfite, sulfate esters, methanesulfonates, benzyl sulfonates, and p-toluenesulfonates.

[0128] In some implementation schemes, R 2’ For -OR 20 R 20 It is TBDMS (or TBS), TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TMPS, DPMS, dimethylxylsilyl, tribenzylsilyl, tri-p-xylsilyl or triphenylsilyl, optionally, R 20 It's TBCMS.

[0129] In some implementation schemes, R 2 Yes - OR 30 , where R 30 It is a hydrogen, hydroxyl protecting group, reactive phosphorus group (e.g., phosphorous acid), bond with a nucleoside or nucleotide, or bond with an oligonucleotide. For example, R 2 Yes - OR 30 R 30 It is a hydrogen or hydroxyl protecting group.

[0130] In some implementation schemes, R 2 Yes - OR 30 R 30 It is a reactive phosphorus group. For example, R 30 It is a phosphorus amide, H-phosphonate, alkylphosphonate, or triphosphate. In some embodiments, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SRP1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 ,in: Each R P1 It is C 1-6 Alkyl group, optionally substituted with 1, 2, 3, 4 or 5 substituents, said substituents being 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6; Each R P2 Independently for C 1-6Alkyl group, optionally substituted with 1, 2, 3, 4 or 5 substituents, said substituents being 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6, optionally, each R P2 Each of the following is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl, preferably each R P2 It is isopropyl; Or two Rs P2 Together with the nitrogen atom to which it is attached, it forms an optional substituted 3-8 membered heterocyclic group; Or R P1 and R P2 One of them, together with the atoms it is attached to, forms an optionally substituted 4-8 membered heterocyclic group; and Each R P3 Independently for C 1-30 Alkyl, C2-C 30 alkenyl or C2-C 30The alkynyl group is optionally and independently substituted by 1, 2, 3, 4, or 5 substituents, said substituents being 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6, optionally each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy groups.

[0131] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )RP3 or -P(O)(SR P1 )R P3 , where: each R P1 It is C 1-6 Alkyl groups, optionally substituted with CN or –SC(O)Ph; each R P2 C can be substituted independently. 1-6 Alkyl groups; and each R P3 C can be substituted independently. 1-6 alkyl.

[0132] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is C 1-6 Alkyl groups, optionally substituted with CN or –SC(O)Ph; each R P2 Independently, it is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy groups.

[0133] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2)2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is 2-cyanoethyl (-CH2CH2CN); each R P2 C can be substituted independently. 1-6 Alkyl groups; and each R P3 C can be substituted independently. 1-6 alkyl.

[0134] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )RP3 , where: each R P1 It is 2-cyanoethyl (-CH2CH2CN); each R P2 Independently, each of the following is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy groups.

[0135] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is 2-cyanoethyl (-CH2CH2CN); each R P2 Independently isopropyl; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy groups.

[0136] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2, where: R P1It is 2-cyanoethyl (-CH2CH2CN); each R P2 It is independently isopropyl.

[0137] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is C 1-6 Alkyl groups, optionally substituted with CN or –SC(O)Ph; R P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3-8 membered heterocyclic group; and each R P3 C can be substituted independently. 1-6 alkyl.

[0138] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2)2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is C 1-6 Alkyl groups, optionally substituted with CN or –SC(O)Ph; R P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3-8 membered heterocyclic group; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy.

[0139] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is 2-cyanoethyl (-CH2CH2CN); R P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3-8 membered heterocyclic group; and each R P3 C can be substituted independently. 1-6 alkyl.

[0140] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: each R P1 It is 2-cyanoethyl (-CH2CH2CN); R P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3-8 membered heterocyclic group; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy.

[0141] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(ORP1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: R P1 and R P2 One of them, together with the atom it is attached to, forms an optionally substituted 4-8 membered heterocyclic group; the other R P2 C can be substituted independently. 1-6 Alkyl groups; and each R P3 C can be substituted independently. 1-6 alkyl.

[0142] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: R P1 and R P2 One of them, together with the atom it is attached to, forms an optionally substituted 4-8 membered heterocyclic group; the other R P2 Independently, each of the following is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl; and each R P3 C can be substituted independently. 1-6 alkyl.

[0143] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: R P1 and R P2 One of them, together with the atom it is attached to, forms an optionally substituted 4-8 membered heterocyclic group; the other R P2 C can be substituted independently. 1-6 Alkyl groups; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy.

[0144] In some implementation schemes, R 2 Yes - OR 30 R 30 It is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(ORP1 H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 , where: R P1 and R P2 One of them, together with the atom it is attached to, forms an optionally substituted 4-8 membered heterocyclic group; the other R P2 Independently, each of the following is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl; and each R P3 It is independently methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which may optionally be substituted with NH2, OH, C(O)NH2, COOH, halogen, SH or C1-C6 alkoxy.

[0145] In some implementation schemes, R 2 Yes - OR 30 R 30 It is a linkage bond with a nucleoside, nucleotide, or oligonucleotide. When R... 30 When the linkage between the compound of formula I-II and the nucleoside, nucleotide or oligonucleotide is a linking bond, the linkage between the compound and the nucleoside, nucleotide or oligonucleotide can be an unmodified (e.g., phosphodiester bond) linkage or a modified (e.g., thiophosphate bond) linkage.

[0146] In some implementation schemes, R 2 Yes - OR 30 R 30 Linked to the 5' position of a nucleoside, nucleotide, or oligonucleotide via an unmodified (e.g., phosphodiester bond) or modified (e.g., thiophosphate bond) internucleotide linker. For example, R 30 It is linked to the 5'-terminus (e.g., 5'-OH) of the oligonucleotide. In some embodiments, R 30 Linked to the 5'-terminus (e.g., 5'-OH) of the oligonucleotide via an unmodified (e.g., phosphodiester bond) internucleotide linker. In some other embodiments, R 30 The oligonucleotide is linked to the 5'-terminus (e.g., 5'-OH) via a modified (e.g., thiophosphate) internucleotide linker.

[0147] In some implementation schemes, R 2’ Yes - OR 30 R 30 It is a hydroxyl protecting group. For example, R2’ is - OR 30 , R 30It is a hydroxyl protecting group, wherein the hydroxyl protecting group is selected from BOC or Boc, MOM, MTM, tert-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, tert-butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxymethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiaranyl, 1-methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiapyranyl, 4-methoxytetrahydrothiaranyl-S,S-dioxide, CTMP, 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrothiaranyl, 2,3,3a,4,5,6,7,7a-octahydro-7, 8,8-Trimethyl-4,7-methoxybenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-phenoxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenoyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-formylbenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-pyridinecarboxyl, 4-pyridinecarboxyl, 3-methyl-2-pyridinyl N-oxide, di Benzyl, p,p′-dinitrobenzyl, 5-dibenzoyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)tonyl, 9-(9-phenyl-10-oxo)anthrayl, 1,3-benzodithiacyclopentadien-2-yl, benzisothiazolyl-S,S-dioxy, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthiahexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, TBMPS, formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate Ester, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxovalerate (acetylpropionate), 4,4-(ethylidene dithio)valerate (acetylpropionyl dithioacetal), adamantinate, crotonate, 4-methoxycrotonate, benzoate, p-benzoate, 2,4,6-trimethylbenzoate (trimethylbenzene ester), alkyl methyl carbonate, Fmoc, alkyl ethyl carbonate, Troc, TMSEC, Psec, Peoc, alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzene 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-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro- 4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid ester, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid ester, chlorodiphenylacetic acid ester, isobutyrate, (E)-2-methyl-2-butenoate, o-(methoxyyl)benzoate, α-naphthylcarbamate, nitrate esters, alkyl N,N,N',N'-tetramethyldiamide phosphate, alkyl N-phenylcarbamate, borate esters, dimethylphosphonothioyl, alkyl 2,4-dinitrophenylsulfite, sulfate esters, methanesulfonates, benzyl sulfonates, and p-toluenesulfonates.

[0148] In some implementation schemes, R 2’ Yes - OR 30 R 30 It is TBDMS, TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TMPS, DPMS, dimethylxylmethylsilyl, tribenzylsilyl, tri-p-xylmethylsilyl or triphenylsilyl, optionally, R 30 It's TBCMS.

[0149] R V

[0150] In some implementations, at least one R V Independently protected by a hydroxyl group. For example, at least one R V For example, two Rs VIndependently, a hydroxyl protecting group is selected from ethyl, neopentanoyloxymethyl (POM), BOC or Boc, MOM, MTM, tert-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, tert-butoxymethyl, POM, silyloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxymethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiaranyl, 1-methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiaranyl, 4-methoxytetrahydrothiaranyl-S,S-dioxide, CTMP, 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrothiaranyl, 2,3,3a,4 5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methoxybenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-phenoxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenoyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-formylbenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-pyridinecarboxyl, 4-pyridinecarboxyl, 3-methyl-2-pyridinecarboxyl Pyridyl N-oxide, diphenylmethyl, p,p′-dinitrophenylmethyl, 5-dibenzoyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)tonyl, 9-(9-phenyl-10-oxo)anthrayl, 1,3-benzodithiacyclopentadien-2-yl, benzisothiazolyl-S,S-dioxy, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthiahexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, TBMPS, formate, acetate, chloroacetate, dichloroacetate, trichloroacetate Trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxovalerate (acetylpropionate), 4,4-(ethylidene dithio)valerate (acetylpropionyl dithioacetal), adamantinate, crotonate, 4-methoxycrotonate, benzoate, p-benzoate, 2,4,6-trimethylbenzoate (trimethylbenzyl ester), alkyl methyl carbonate, Fmoc, alkyl ethyl carbonate, Troc, TMSEC, Psec, 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-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro-4-(1,1, 3,3-Tetramethylbutyl)phenoxyacetic acid ester, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid ester, chlorodiphenylacetic acid ester, isobutyrate, (E)-2-methyl-2-butenoate, o-(methoxyyl)benzoate, α-naphthylcarbamate, nitrate ester, alkyl N,N,N',N'-tetramethyldiamide phosphate, alkyl N-phenylcarbamate, borate ester, dimethylphosphonothioyl, alkyl 2,4-dinitrophenylsulfite, sulfate ester, methanesulfonate (methanesulfonate), benzyl sulfonate and p-toluenesulfonate, preferably with a POM or ethyl protecting group. Therefore, in some embodiments, at least one R, V For example, two Rs V Independently, it is a neopentanoyloxymethyl (POM). In some other embodiments, at least one R V For example, two Rs V It is independent.

[0151] In some implementations, at least one R V For example, two Rs V It is hydrogen independently.

[0152] B (base)

[0153] In some embodiments of the various aspects described herein, B is an optionally modified base. It is noteworthy that the base can be either natural or non-natural. "Non-natural base" refers to a base other than adenine, guanine, cytosine, uracil, or thymine. Exemplary non-natural bases include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanine, tuberculin, and substituted or modified analogues of adenine, guanine, cytosine, and uracil, such as 2-aminoadenine and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halogenated uracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine, and thymine. Adenine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 8-halo, amino, mercapto, thioalkyl, hydroxyl and other 8-substituted adenine and guanine, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyl... Acetyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deazo-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkylcytosine, 7-deazoadenine, N6,N6 dimethyladenine, 2,6-diaminopurine, 5-aminoallyluracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 5-methoxy Uracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N4-acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentenyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine, or O-alkylated bases. Other purines and pyrimidines include those disclosed in U.S. Patent 3,687,808, those disclosed in *Concise Encyclopedia of Polymer Science and Engineering*, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, and those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613. All of these contents are incorporated herein by reference.

[0154] In some embodiments, the non-natural base may be selected from inosine, xanthine, hypoxanthine, nubularine, isoguanine, tuberculin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(meththio)-N6-(isopentenyl)adenine, 7-(denitro)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, and 8-(hydroxy)adenine. 8-(thioalkyl)adenine, 8-(mercapto)adenine, N6-(isopentyl)adenine, N6-(methyl)adenine, N6,N6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(denitro)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxy)guanine, 8-(thioalkyl)guanine, 8 -(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(denitro)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halogenated)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)cytosine 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-(guanidinylalkyl)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-(methoxycarbonylmethyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)uracil 5-(methyl)-2-(thio)-2-(di ... Vinyl)-4-(thio)-pseudouracil, 1-(aminocarbonylvinyl)-2,4-(dithio)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-2-(thio)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-4-(thio)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-2,4-(dithio)-pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazine-1-yl, 1-(aza)- 2-(thio)-3-(aza)-phenoxazine-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazine-1-yl, 7-(aminoalkylhydroxy)-1-(za)-2-(thio)-3-(za)-phenothiazine-1-yl, 7-(guanidinylalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazine-1-yl, 7-(guanidinylalkylhydroxy)-1-(za)-2-(thio)-3-(za)-phenothiazine-1-yl, 7-(guanidinylalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazine-1-yl 7-(guanidinoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazine-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, neopamine, tuberculin, isoguanine, inosine, 2-aza-inosine, 7-deazo-inosine, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbazyl-stiolizyl, 5-(methyl)iso Carbasteilyl, 3-(methyl)-7-(propynyl)isocarbasteilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidazopyridyl, 9-(methyl)-imidazopyridyl, pyrrolopyrazinyl, isocarbasteilyl, 7-(propynyl)isocarbasteilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthyl, anthraceneyl, phenanthryl, pyrene, stilbene alkyl, tetraphenyl, pentaphenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymidine, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N2-substituted purine, N6-substituted purine, O6-substituted purine, substituted 1,2,4-triazole, and any O-alkylated or N-alkylated derivatives thereof.

[0155] In some embodiments, the non-natural base is a modified base, meaning the base includes base modifications as described herein, such as substitutions or modifications of any natural base. Examples of base modifications include, but are not limited to: pyrimidines having an alkyl or aminoalkyl group and other cationic groups (such as guanidinyl and amidine functional groups) at the C-5 position; guanidinyl and amidine functional groups of purines known in the art, having alkyl or aminoalkyl groups at the N2- and N6- positions; and other cationic groups such as G-clamps, guanidinyl G-clamps, and pseudouridines.

[0156] In some embodiments of any aspect, the non-natural base is a universal base. As used herein, a universal base is any modified or unmodified natural or non-natural base that can pair with all bases of adenine, cytosine, guanine, and uracil without substantially affecting the melting behavior, intracellular enzyme recognition, or activity of the oligonucleotide containing the universal base. Some exemplary universal bases include, but are not limited to, 2,4-difluorotoluene, nitropyrrole, nitroindolyl, 8-aza-7-deadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbapenyl, 5-methylisocarbapenyl, 3-methyl-7-propynylisocarbapenyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridyl, 9-methyl-imidazopyridyl, pyrrolopyrazinyl, isocarbapenyl, 7-propynylisocarbapenyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methanolyl, 4,6-dimethylindolyl, phenyl, naphthylphenyl, anthraceneyl, phenanthrophenyl, pyreneyl, stilbeneyl, tetraphenyl, pentaphenyl and their structural derivatives.

[0157] In some implementations, the natural or non-natural base is a protected base. As used herein, a "protected base" means a base that contains a nitrogen protecting group, an oxygen protecting group, and a sulfur protecting group.

[0158] For example, the base is a pyrimidine modified at the C4 position. In another non-limiting example, the base is a pyrimidine modified at the C5 position.

[0159] In some embodiments, the base is a purine modified at the N2 position. In some embodiments, the base is a purine modified at the N6 position. For example, the base is a purine modified at the C6 position. In some non-limiting examples, the base is an N-7 denitropurine optionally modified at the N7 position.

[0160] In some embodiments, the base is an analogue of a modified, protected, or substituted base selected from adenine, cytosine, guanine, thymine, and uracil. For example, the base is uracil, adenine, guanine, or cytosine, optionally each independently containing a hydroxyl or amine protecting group.

[0161] Double-stranded RNA

[0162] Those skilled in the art will readily recognize that double-stranded RNAs containing 19 to 24 (particularly 21) base pairs are considered particularly effective in inducing RNA interference (RNAi). However, others have found that shorter or longer double-stranded oligonucleotides may also be effective. Therefore, in some embodiments, the longer double-stranded oligonucleotides described herein are capable of inducing RNA interference. In other words, the longer double-stranded oligonucleotides described herein can mediate RNA interference. As used herein, the phrase "mediates RNAi" refers to the ability to inhibit or reduce the expression of a target nucleic acid (e.g., target RNA, such as mRNA) in a sequence-specific manner.

[0163] Therefore, another aspect provided herein is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand substantially or 100% (e.g., completely) complementary to the sense strand, wherein one of the sense or antisense strands has a 5' end modification, comprising the following structure: , in: X is O or S; and Each R V It is independently protected by hydrogen or hydroxyl groups.

[0164] In some implementations, the 5' end modification of the justice chain or antisense chain (e.g., the antisense chain) includes the following structure: or .

[0165] In some implementations, X is 0. In some implementations, X is 0, and each R... V It is hydrogen. In some implementations, X is O, and each R V It is ethyl. In some embodiments, X is O, and each R V It is neopentyloxymethyl.

[0166] It is worth noting that, including (For example, or The glycosyl group of a nucleotide modified with the above-mentioned modification, i.e., the glycosyl group of the nucleotide at the 5' end of the sense or antisense strand (5'-terminal nucleotide), may contain a 5- or 6-membered ring. For example, the glycosyl group in a nucleotide containing the above-mentioned modification may be a furanose (e.g., ribofuranose, arabinofuranose, xyfuranose, ribonefuranose, or xynefuranose, including α and β, D and L, deoxy, and their modified derivatives), or a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, adropyranose, gulopyranose, idupyranose, and taropyranose, including α and β, D and L, deoxy, and their modified derivatives).

[0167] Typically, the (For example, or The modification replaces the CH2OH group on the 5'-terminal nucleotide sugar moiety of the sense or antisense strand. For example, (For example, or )or The modification replaces the 4'-CH2OH group on the furanose ring (e.g., ribofuranose, arabinofuranose, xylofuranose, ribonefuranose, or xylofuranose, preferably ribofuranose) or the 5'-CH2OH group on the pyranose ring (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, choropyranose, gulopyranose, idupyranose, and taropyranose, preferably glucopyranose, galactopyranose, or mannopyranose) of the 5'-terminal nucleotide of the oligonucleotide.

[0168] In some embodiments, the sense strand or antisense strand (e.g., the antisense strand) comprises a compound of formula (I) described herein at its 5' end. For example, the 5' nucleotide of the sense strand or antisense strand has the following structure: , in: Each R V Independently protected by hydrogen or hydroxyl groups (e.g., ethyl or neopentyloxymethyl ((CH3)3CC(O)OCH2-, POM); B is an optional modified base (e.g., uracil). R 2 and R 3 One of them is hydrogen, halogen, or -OR 20 ,in: R 20 It is a hydrogen, hydroxyl protecting group, or optionally substituted C 1-6 Alkyl groups (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl, 2-(N-methylamino)-2-oxoethyl), optionally substituted C 2-6 alkenyl or optionally substituted C 3-6 alkynyl (e.g., propynyl); and R 2 and R 3 The other one is -OR 30 ,in: R 30 It is a linking bond with an oligonucleotide (e.g., an internucleotide linking bond that links the oligonucleotide to a subsequent nucleotide).

[0169] In some implementations, the 5' terminal nucleotide of the sense strand or antisense strand (e.g., the antisense strand) has the following structure: , where R 2 and R3 One of them is -OR 30 (For example, R) 3 For -OR 30 ), where: R 30 It is a linking bond with an oligonucleotide (e.g., an internucleotide linking bond that connects to a subsequent nucleotide of an oligonucleotide).

[0170] In some other embodiments, the 5' terminal nucleotide of the positive or antisense strand (e.g., the antisense strand) has the following structure: , where R 2 and R 3 One of them is -OR 30 (For example, R) 3 For -OR 30 ), where: R 30 It is a linking bond with an oligonucleotide (e.g., an internucleotide linking bond that connects to a subsequent nucleotide of an oligonucleotide).

[0171] In some other embodiments, the 5' terminal nucleotide of the positive or antisense strand (e.g., the antisense strand) has the following structure: , where R 2 and R 3 One of them is -OR 30 (For example, R) 3 For -OR 30 ), where: R 30 It is a linking bond with an oligonucleotide (e.g., an internucleotide linking bond that connects to a subsequent nucleotide of an oligonucleotide).

[0172] In some other embodiments, the 5' terminal nucleotide of the sense strand or antisense strand (e.g., the antisense strand) has the following structure: , where R 2 and R 3 One of them is -OR 30 (For example, R) 3 For -OR 30 ), where: R 30 It is a linking bond with an oligonucleotide (e.g., an internucleotide linking bond that connects to a subsequent nucleotide of an oligonucleotide).

[0173] Preferably, the antisense strand of the dsRNA is an oligonucleotide as described herein.

[0174] As used herein, the term “antisense strand” refers to an oligonucleotide that is substantially or 100% (e.g., completely) complementary to the target nucleic acid. For example, an antisense strand may be wholly or partially complementary to the target nucleic acid, such as messenger RNA, non-mRNA RNA sequences (e.g., microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or coding or non-coding DNA sequences.

[0175] It is worth noting that the length of each strand of dsRNA can be between 12 and 40 nucleotides. For example, the length of each strand can independently be 14-40 nucleotides, 17-37 nucleotides, 25-37 nucleotides, 27-35 nucleotides, 17-23 nucleotides, 17-21 nucleotides, 17-19 nucleotides, 19-25 nucleotides, 19-23 nucleotides, 19-21 nucleotides, 21-25 nucleotides, 21-23 nucleotides, 25-35 nucleotides, 26-35 nucleotides, 27-34 nucleotides, 28-32 nucleotides, or 29-31 nucleotides. The sense and antisense strands can be of equal or unequal length, without restriction. In some embodiments, the antisense strand is longer than the sense strand, for example, by 1, 2, 3, 4, or 5 nucleotides.

[0176] In some embodiments, the length of each positive and negative strand is independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. For example, the length of each positive and negative strand is independently 18, 19, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the length of each strand is independently 19, 20, 21, 22, or 23 nucleotides. In some embodiments, one strand (e.g., the positive strand) is 18, 19, 20, 21, or 22 nucleotides long, and the length of the other strand (e.g., the negative strand) is 21, 22, 23, 24, or 25 nucleotides.

[0177] The sense and antisense strands of a dsRNA molecule are complementary and can hybridize to form a double-stranded region. Therefore, a dsRNA molecule has a double-stranded region. The length of the double-stranded region can be 17-25 nucleotide base pairs. For example, dsRNA can have a double-stranded region of 17-24 nucleotide base pairs. In some embodiments, dsRNA has a double-stranded region of 18, 19, 20, 21, 22, 22, 23, 24, or 25 nucleotide base pairs. In some embodiments, dsRNA has a double-stranded region of 19, 20, 21, or 22 nucleotide base pairs.

[0178] dsRNA molecules may have one or more sticky ends (i.e., single-stranded regions) and / or capping groups at the 3', 5', or both ends of the strand. Without restriction, the sticky ends can be 1-3 nucleotides long, for example, 1, 2, or 3 nucleotides in length. Sticky ends can be formed by one strand being longer than the other, or by two strands of equal length being interleaved. Sticky ends can form mismatches with the target sequence, be complementary to the target sequence, or be other sequences. The sense and antisense strands can also be linked, for example, by forming a hairpin structure with an additional base, or by other non-base linkers. Without restriction, sticky ends may be present only at the 3' end of one or both strands.

[0179] In some embodiments, the dsRNA molecule includes a sticky end. For example, the dsRNA molecule has a sticky end that is no more than one, two, or three nucleotides long. Preferably, the sticky end is two nucleotides long. In some embodiments, the sticky end is located at the 3' end of the strand (e.g., the antisense strand). In some embodiments, the dsRNA includes a two-nucleotide sticky end at the 3' end of the strand (e.g., the antisense strand). For example, the sticky end is located at the 3' end of the antisense strand. For example, the antisense nucleic acid includes one or two nucleotide sticky ends at its 3' end.

[0180] dsRNA can also have a blunt end. For example, one end of dsRNA is blunt, and the other end has a sticky end. The blunt end can be located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa; there are no restrictions. Generally, the antisense strand of double-stranded ribonucleic acid has a sticky nucleotide end at the 3' end and a blunt end at the 5' end. Although not theoretically constrained, an asymmetric blunt end at the 5' end of the antisense strand and a sticky end at the 3' end of the antisense strand are beneficial for guiding the strand loading into the RISC process. In some implementations, dsRNA has a 2-nucleotide sticky end at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0181] In some other embodiments, the dsRNA molecule has two blunt ends, i.e., at both ends of the dsRNA. For example, the two strands of the dsRNA are of equal length. In some embodiments, the antisense strand is 18 to 25 nucleotides long. In some embodiments, the antisense strand is 21-25, 19-25, 19-21, or 21-23 nucleotides long. In some specific embodiments, the antisense strand is 23 nucleotides long.

[0182] Similar to the antisense strand, in some embodiments, the length of the sense strand can be 18-25 nucleotides. In some embodiments, the length of the sense strand is 21-25, 19-25, 19-21, or 21-23 nucleotides. In some embodiments, the length of the sense strand is 21 nucleotides.

[0183] In some implementations, the length of the sense strand is 21 nucleotides and the length of the antisense strand is 23 nucleotides.

[0184] Nucleic acid modification

[0185] The longer double-stranded and single-stranded oligonucleotides described herein may contain one or more nucleic acid modifications. Exemplary nucleic acid modifications include, but are not limited to, base modifications, sugar modifications, interglycosylation modifications, conjugates (e.g., ligands), and any combination thereof. Notably, nucleic acid modifications may be present at any position on the longer double-stranded and single-stranded oligonucleotides. Nucleic acid modifications may be present only in one or both strands of the dsRNA. In some embodiments, only the antisense strand contains at least one, such as two, three, four, five, or more nucleic acid modifications. In some embodiments, only the antisense strand contains at least one, such as two, three, four, five, or more nucleic acid modifications. In some embodiments, both strands independently contain at least one, such as two, three, four, five, or more nucleic acid modifications.

[0186] The embodiments described herein list specific positions on the chain, counted starting from the end of the chain. When the chain is single-stranded, such as a long-chain oligonucleotide, the position count begins with the first nucleotide at the specified end. When the chain is part of a double-stranded molecule, such as a long double-stranded oligonucleotide, the position count may begin with the first nucleotide at the specified end of the chain, or with the first base pair nucleotide at the specified end of the chain. Preferably, the position count begins with the first nucleotide at the specified end of the chain.

[0187] Thermally unstable modification

[0188] In some embodiments of any aspect described herein, the dsRNA includes a thermally unstable modification. A “thermally unstable modification” is a modification that results in the dsRNA having a lower total melting temperature (Tm), preferably 1, 2, 3, or 4 degrees lower than the Tm of unmodified dsRNA. Exemplary thermally unstable modifications are described below and may include, but are not limited to, baseless modifications; relative nucleotide mismatches in the opposing strand; and sugar modifications such as 2'-deoxy (i.e., 2'-H) modifications, acyclic nucleotides (such as unlocked nucleic acids (UNA) or glycol nucleic acids (GNA)), threonine nucleic acids (TNA), nucleotides linked by their 2'-position (i.e., nucleotides linked to the 5' position of subsequent nucleotides by their 2'-OH group (2'-5' RNA modification)); Hyp spacer modifications; modified internucleotide linkages that reduce the thermal stability of the dsRNA double strand; or bases with impaired WC hydrogen bonds to complementary bases on the opposing strand.

[0189] In some embodiments, the dsRNA contains at least one, such as one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more independently selected thermally unstable modifications. Thermally unstable modifications can be present at any location on the dsRNA. Furthermore, thermally unstable modifications can be present on one or both strands of the dsRNA. In some embodiments, only the antisense strand contains at least one, such as two, three, four, or more thermally unstable modifications. In some embodiments, only the sense strand contains at least one, such as two, three, four, or more thermally unstable modifications. In some embodiments, both the sense and antisense strands contain at least one, such as two, three, four, or more thermally unstable modifications.

[0190] Thermally unstable modifications can occur on any nucleotide in either the sense or antisense strand. For example, a thermally unstable modification can occur on every nucleotide in both the sense and / or antisense strands; each thermally unstable modification can occur in an alternating pattern in either the sense or antisense strand; or both the sense and antisense strands may contain thermally unstable modifications in an alternating pattern. The alternating pattern of thermally unstable modifications on the sense strand can be the same as or different from that on the antisense strand, and the alternating pattern of thermally unstable modifications on the sense strand may be shifted relative to the alternating pattern of thermally unstable modifications on the antisense strand.

[0191] In some embodiments, counting from the 5' end of the antisense chain, the thermally unstable modification is located at positions 2, 3, 4, 5, 6, 7, 8, or 9, or preferably at positions 4, 5, 6, 7, or 8. In some embodiments, the thermally unstable modification is located at positions 2, 3, 4, 5, or 9 of the 5' end of the antisense chain. In some other embodiments, the thermally unstable modification is located at positions 6, 7, or 8 of the 5' end of the antisense chain. In some specific embodiments, the thermally unstable modification is located at position 7 of the 5' end of the antisense chain.

[0192] In some embodiments, only the antisense chain contains the thermally unstable modification. For example, only the antisense chain contains the thermally unstable modification, and the thermally unstable modification is located at position 4, 5, 6, 7, or 8 counting from the 5' end of the antisense chain; preferably, the thermally unstable modification is located at position 5, 6, 7, or 8; more preferably, the thermally unstable modification is located at position 6, 7, or 8. In some embodiments, only the antisense chain contains the thermally unstable modification, and the thermally unstable modification is located at position 7 counting from the 5' end of the antisense chain.

[0193] Similar to the antisense chain, the thermally unstable modification can be located at one of the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, or 9th positions counting from the 5' end of the longer ssNA, or preferably at the 4th, 5th, 6th, 7th, or 8th position. In some embodiments, the thermally unstable modification is located at the 2nd, 3rd, 4th, 5th, or 9th position counting from the 5' end of the longer ssNA. In some other embodiments, the thermally unstable modification is located at the 6th, 7th, or 8th position counting from the 5' end of the longer ssNA. In some specific embodiments, the thermally unstable modification is located at the 7th position counting from the 5' end of the longer ssNA.

[0194] Thermally stable modification

[0195] In some embodiments, the dsRNA includes a thermostable modification. A “thermally stable modification” is a modification that results in a higher overall melting temperature (Tm) of the dsRNA than that of dsRNA without such modification, preferably 1, 2, 3, or 4 degrees higher. Exemplary thermostable modifications are described below and may include, but are not limited to, 2'-fluoronucleotides (2'-F modifications), bridging nucleic acids (BNAs), such as locked nucleic acids (LNAs) and cyclohexene nucleic acids (CeNAs). In some preferred embodiments, the thermostable modification is a 2'-fluoronucleotide. Exemplary thermostable modifications are described below. Other exemplary baseless nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011 / 133876 and WO2019222479, the entire contents of which are incorporated herein by reference.

[0196] In some embodiments, the dsRNA may contain at least two, such as three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more thermostable (e.g., 2'-F) modifications. Without limitation, thermostable (e.g., 2'-F) modifications may be present in one or both strands of the dsRNA. In some embodiments, the sense strand contains at least one, such as two, three, four, or more thermostable (e.g., 2'-F) modifications. In some embodiments, the antisense strand contains at least one, such as two, three, four, or more thermostable (e.g., 2'-F) modification. In some embodiments, both the sense and antisense strands contain at least one, such as two, three, four, or more thermostable (e.g., 2'-F) modification. Thermostable (e.g., 2'-F) modifications may occur on any nucleotide of either the sense or antisense strand. For example, a thermostable (e.g., 2'-F) modification can occur on each nucleotide of the sense and / or antisense strand; each thermostable (e.g., 2'-F) modification can occur in an alternating pattern on either the sense or antisense strand; or both the sense and antisense strands contain alternating patterns of thermostable (e.g., 2'-F) modifications. The alternating pattern of a thermostable (e.g., 2'-F) modification on the sense strand can be the same as or different from that on the antisense strand, and the alternating pattern of a thermostable (e.g., 2'-F) modification on the sense strand can be shifted relative to the alternating pattern of a thermostable (e.g., 2'-F) modification on the antisense strand.

[0197] In some embodiments, the positive strand of the dsRNA contains at least one, such as two, three, four, five, six, seven, eight, nine, ten, or more thermostable (e.g., 2'-F) modifications. In some embodiments, the positive strand includes two, three, four, or five thermostable (e.g., 2'-F) modifications. For example, the positive strand includes three or four thermostable (e.g., 2'-F) modifications. Without limitation, thermostable (e.g., 2'-F) modifications in the positive strand can be present at any position. In some embodiments, the positive strand includes at least three thermostable (e.g., 2'-F) modifications. For example, the positive strand includes thermostable (e.g., 2'-F) modifications counted at least at positions 7, 10, and 11, starting from the 5' end of the positive strand. In some other embodiments, the positive strand includes at least four thermostable (e.g., 2'-F) modifications. For example, the positive strand includes thermostable (e.g., 2'-F) modifications counted at least at positions 7, 9, 10, and 11, starting from the 5' end of the positive strand.

[0198] In some embodiments, counting from the 5' end of the antisense chain, the positive chain includes thermally stable (e.g., 2'-F) modifications at positions opposite to or complementary to positions 11, 12, and 15 of the antisense chain. In some other embodiments, counting from the 5' end of the antisense chain, the positive chain includes thermally stable (e.g., 2'-F) modifications at positions opposite to or complementary to positions 11, 12, 13, and 15 of the antisense chain. In some embodiments, the positive chain includes two, three, or four segments with thermally stable (e.g., 2'-F) modifications.

[0199] In some embodiments, counting from the 5' end, the positive chain includes a thermally stable (e.g., 2'-F) modification at least in positions 7, 9, and 11, and the negative chain includes a thermally stable (e.g., 2'-F) modification at least in positions 2, 14, and 16, counting from the 5' end. In some other embodiments, the positive chain includes a thermally stable (e.g., 2'-F) modification at least in positions 7, 9, and 11, counting from the 5' end, and the negative chain includes a thermally stable (e.g., 2'-F) modification at least in positions 2, 6, 9, 14, and 16, counting from the 5' end. In still other embodiments, the positive chain includes a thermally stable (e.g., 2'-F) modification at least in positions 7, 9, and 11, counting from the 5' end, and the negative chain includes a thermally stable (e.g., 2'-F) modification at least in positions 2, 6, 8, 9, 14, and 16, counting from the 5' end.

[0200] In some embodiments, the positive chain includes a thermally stable (e.g., 2'-F) modification at least in positions 7, 9, 10, and 11, starting from the 5' end, and at least in positions 2, 14, and 16, starting from the 5' end. In some other embodiments, the positive chain includes a thermally stable (e.g., 2'-F) modification at least in positions 7, 9, 10, and 11, starting from the 5' end, and the negative chain includes a thermally stable (e.g., 2'-F) modification at least in positions 2, 6, 9, 14, and 16. In still other embodiments, the positive chain includes a thermally stable (e.g., 2'-F) modification at least in positions 7, 9, 10, and 11, starting from the 5' end, and the negative chain includes a thermally stable (e.g., 2'-F) modification at least in positions 2, 6, 8, 9, 14, and 16, starting from the 5' end.

[0201] In some implementations, the sense chain does not contain thermally stable (e.g., 2'-F) modifications at positions opposite to or complementary to the thermally unstable modifications of the bistrand in the antisense chain.

[0202] The antisense strand of the dsRNA molecule may contain at least one, such as two, three, four, five, six, seven, eight, nine, ten, or more thermostable (e.g., 2'-F) modifications. In some embodiments, the antisense strand contains two, three, four, five, or six thermostable (e.g., 2'-F) modifications. Without limitation, thermostable (e.g., 2'-F) modifications in the antisense strand can be present at any position. In some embodiments, the antisense strand contains at least three thermostable (e.g., 2'-F) modifications. For example, counting from the 5' end, the antisense strand contains at least the 2nd, 14th, and 16th positions of the thermostable (e.g., 2'-F) modification. In some other embodiments, the antisense strand contains at least four thermostable (e.g., 2'-F) modifications. For example, counting from the 5' end, the antisense strand includes at least the 2nd, 6th, 14th, and 16th positions of the thermostable (e.g., 2'-F) modification. In some further embodiments, the antisense strand contains at least five thermostable (e.g., 2'-F) modifications. For example, the antisense chain includes thermally stable (e.g., 2'-F) modifications at least at positions 2, 6, 9, 14, and 16, starting from the 5' end. In other embodiments, the antisense chain contains at least six thermally stable (e.g., 2'-F) modifications. For example, the antisense chain includes thermally stable (e.g., 2'-F) modifications at least at positions 2, 6, 8, 9, 14, and 16, starting from the 5' end.

[0203] In some embodiments, the antisense strand includes at least one thermostable (e.g., 2'-F) modification adjacent to the thermostable modification. For example, the thermostable (e.g., 2'-F) modification may be a nucleotide at the 5' or 3' end of the thermostable modification, i.e., at position -1 or +1 of the thermostable modification. In some embodiments, the antisense strand includes thermostable (e.g., 2'-F) modifications at the 5' and 3' ends of the thermostable modification, i.e., at positions -1 and +1 of the unstable modification, respectively.

[0204] In some implementations, the antisense chain includes at least two stable modifications at the 3'-end of the unstable modification, namely at positions +1 and +2 away from the unstable modification position.

[0205] In some implementations, the sense chain does not contain thermally stable (e.g., 2'-F) modifications at positions opposite to or complementary to the thermally unstable modifications of the bistrand in the antisense chain.

[0206] 2'-OMe nucleotide

[0207] In some embodiments, the dsRNA described herein may contain at least one, such as one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more 2'-OMe nucleotides. Without limitation, the 2'-OMe nucleotides may be present in one or both strands of the dsRNA. In some embodiments, both the sense and antisense strands contain at least one 2'-OMe nucleotide. 2'-OMe modification may occur on any nucleotide of the sense or antisense strand. For example, 2'-OMe modification may occur on every nucleotide of the sense and / or antisense strands; each 2'-OMe modification may occur in an alternating pattern on the sense or antisense strands; or both the sense and antisense strands may contain 2'-OMe modifications in an alternating pattern. The alternation pattern modified by 2'-OMe on the justice chain can be the same as or different from that on the antisense chain, and the alternation pattern modified by 2'-OMe on the justice chain can be shifted relative to the alternation pattern modified by 2'-OMe on the antisense chain.

[0208] The antisense strand of the dsRNA molecule may contain at least one, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or more 2'-OMe modifications. Without limitation, the 2'-OMe modification in the antisense strand can be present at any position. In some embodiments, each nucleotide of the antisense strand is independently a 2'-O-methyl nucleotide, except for any other specific modifications (e.g., thermally unstable modifications, thermally stable modifications, and / or 2'-deoxy (2'-H) modifications).

[0209] Similar to the antisense strand, the sense strand of a dsRNA molecule may contain at least one, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or more 2'-OMe modifications. Without limitation, the 2'-OMe modification in the sense strand can be present at any position. In some embodiments, each nucleotide of the sense strand is independently a 2'-O-methyl nucleotide, except for any other specific modifications (e.g., thermostable modifications, lipophilic modifications, inverted nucleotides, thermostable modifications, and / or 2'-deoxy (2'-H) modifications).

[0210] 2'-deoxy (2'-H) nucleotide

[0211] In some embodiments, the dsRNA described herein may contain 2'-deoxy, i.e., 2'-H nucleotides. For example, the longer double-stranded and single-stranded oligonucleotides described herein may contain at least one (e.g., 1, 2, 3, 4, 5 or more) 2'-deoxy nucleotides.

[0212] 2'-Deoxynucleotides can be present at any position on either the sense or antisense strand. Furthermore, 2'-Deoxynucleotides can be present on one or both strands of the dsRNA.

[0213] In some embodiments, the positive strand contains 1, 2, 3, 4, 5 or more 2'-deoxynucleotides. For example, the positive strand contains 2'-deoxynucleotides at positions 7, 9, and 11, starting from the 5' end. In some embodiments, the positive strand contains 2'-deoxynucleotides at least at position 9, starting from the 5' end. For example, the positive strand contains 2'-deoxynucleotides at positions 7 and 9, starting from the 5' end. In another non-limiting example, the positive strand contains 2'-deoxynucleotides at positions 9 and 11, starting from the 5' end.

[0214] In some embodiments, the antisense strand contains 1, 2, 3, 4, 5, 6, 7, 8 or more 2'-deoxynucleotides. For example, the antisense strand contains 2'-deoxynucleotides at any of the 2nd, 5th, 7th, 12th, 14th, and 16th positions, starting from the 5' end. In some embodiments, the antisense strand contains 2'-deoxynucleotides at least at the 5th position, starting from the 5' end. For example, the antisense strand contains 2'-deoxynucleotides at least at the 2nd, 5th, and 9th positions, starting from the 5' end. In another non-limiting example, the antisense strand contains 2'-deoxynucleotides at least at the 2nd, 5th, 7th, and 12th positions, starting from the 5' end. In yet another non-limiting example, the antisense strand contains 2'-deoxynucleotides at least at the 2nd, 5th, 7th, 12th, 14th, and 16th positions, starting from the 5' end.

[0215] Lipophilic modification

[0216] In some embodiments, the dsRNA described herein may contain lipophilic modifications. For example, the longer double-stranded and single-stranded oligonucleotides described herein may contain at least one (e.g., 1, 2, 3, 4, 5 or more) lipophilic modification. Exemplary lipophilic modifications include nucleotides modified with lipophilic groups, for example, containing a lipophilic group (e.g., C2) at its 2'-position. 10 -C 30 Alkyl or C 10 -C 30 alkenyl groups, such as C 16 Alkyl, C 16 alkenyl, C 18 Alkyl, C 18 alkenyl, C 20 Alkyl, C 20 alkenyl, C 22 Alkyl, C 22 alkenyl, C 24 Alkyl, C24 alkenyl; C 15 Alkyl, C 15 alkenyl, C 17 Alkyl, C 17 alkenyl, C 19 Alkyl, C 19 alkenyl, C 21 Alkyl, C 21 alkenyl, C 33 Alkyl or C 23 Alkenyl) nucleotides. Some exemplary lipophilic nucleotides include, but are not limited to, 2'-O-hexadecyl modified nucleotides (Nhd), 2'-O-docosahexadecyl modified nucleotides (Nda), 2'-O-(ω-hydroxyhexadecyl) modified nucleotides (NhdOH) and 2'-O-(ω-hydroxydocosahexadecyl) modified nucleotides (NdaOH).

[0217] Lipophilic modifications can be present at any position on either the sense or antisense strand. Furthermore, lipophilic modifications can be present on one or both strands of the dsRNA. In some embodiments, only the sense strand contains the lipophilic modification. For example, the sense strand contains the lipophilic modification at any of the following positions, counting from the 5' end: 1, 2, 3, 4, 5, 6, 7, 8, 13, 14, 15, 16, 17, or 18. In some embodiments, the sense strand contains the lipophilic modification at any of the following positions, counting from the 5' end: 4, 5, 6, 7, 8, 13, 14, 15, 16, 17, or 18.

[0218] In some embodiments, each residue of the sense and antisense strands is independently modified with 2'-O-methyl, 2'-fluorine, 2'-deoxy, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, or 2'-C-allyl, 2'-deoxy. The strands may contain multiple modifications. In some embodiments, each residue of the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluorine. It should be understood that these modifications are complementary to any other specific modification of the dsRNA molecule (e.g., at least one thermally unstable modification of the double strand present in the antisense strand).

[0219] In some embodiments, at least two different modifications are typically present on the sense and antisense strands. These two modifications may be 2'-deoxy, 2'-O-methyl, or 2'-fluorine modifications, which are thermally unstable. In some embodiments, the sense and antisense strands each contain two different modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue of the sense and antisense strands is independently modified with a 2'-O-methyl nucleotide, a 2'-deoxy nucleotide, a 2'-deoxy-2'-fluorine nucleotide, a 2'-ON-methylacetamido(2'-O-NMA) nucleotide, a 2'-O-dimethylaminoethoxyethyl(2'-O-DMAEOE) nucleotide, a 2'-O-aminopropyl(2'-O-AP) nucleotide, or a 2'-ara-F nucleotide. For example, each residue of the sense and antisense strands is independently modified with a 2'-O-methyl nucleotide, a 2'-deoxy nucleotide, or a 2'-deoxy-2'-fluorine nucleotide. Similarly, it should be understood that these modifications are supplementary to any thermally unstable modifications of the bistrand present in the antisense chain.

[0220] In some embodiments, the antisense strand comprises at least one thermally unstable modification, and the remaining nucleotides are independently 2'-O-methylnucleotides, 2'-deoxynucleotides, 2'-deoxy-2'-fluoronucleotides, 2'-ON-methylacetamido(2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl(2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl(2'-O-AP) nucleotides, or 2'-ara-F nucleotides. For example, the antisense strand comprises a thermally unstable modification, and the remaining nucleotides are independently 2'-O-methylnucleotides, 2'-deoxynucleotides, or 2'-deoxy-2'-fluoronucleotides. In some embodiments, the antisense strand comprises: (i) thermally unstable modifications counting at positions 5, 6, 7, or 8 starting from the 5' end of the antisense strand; (ii) at least two, for example, 3, 4, 5, or 6, 2'-fluoronucleotides; and (iii) the remaining nucleotides are independently 2'-O-methylnucleotides or 2'-deoxynucleotides.

[0221] In some embodiments, each nucleotide of the positive strand is independently a 2'-O-methylnucleotide, a 2'-deoxynucleotide, a 2'-deoxy-2'-fluoro(2'-F)nucleotide, a 2'-ON-methylacetamido(2'-O-NMA)nucleotide, a 2'-O-dimethylaminoethoxyethyl(2'-O-DMAEOE) nucleotide, a 2'-O-aminopropyl(2'-O-AP) nucleotide, or a 2'-ara-F nucleotide. For example, each nucleotide of the positive strand is independently a 2'-O-methylnucleotide, a 2'-deoxynucleotide, or a 2'-deoxy-2'-fluoronucleotide. In some embodiments, the positive strand contains at least two, for example, three, four, five, or six 2'-fluoronucleotides, with the remaining nucleotides being independently 2'-O-methylnucleotides or 2'-deoxynucleotides.

[0222] In some embodiments, at least one of the first 1, 2, 3, 4, or 5 base pairs in the 5' end double-stranded region of the dsRNA antisense strand can be independently selected from: A:U, G:U, I:C, and mispairings, such as non-standard or non-standard pairings, or pairings containing universal bases, to facilitate dissociation of the antisense strand at the 5' end of the double-stranded region. In some embodiments, the first nucleotide in the 5' end double-stranded region of the antisense strand is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the 5' end double-stranded region of the antisense strand is an AU base pair. For example, the first base pair in the 5' end double-stranded region of the antisense strand is an A:U base pair. Notably, both the sense and antisense strands can contain adenosine (A) nucleotides.

[0223] Modified internucleotide linkage

[0224] The dsRNA described herein may contain at least one, such as two, three, four, five, six, seven, eight, nine, ten or more modified internucleotide bonds. As used herein, “internucleotide link” refers to a covalent link between adjacent nucleotides. Exemplary modified internucleotide bonds include, but are not limited to, phosphodiester bonds, thiophosphate bonds (R, S or racemic), dithiophosphate bonds, methylene methylimine bonds (MMI, 3'-CH2-N(CH3)-O-5'), phosphotriester bonds, alkylphosphonate bonds (e.g., methylphosphonate), phosphoramide ester bonds, methylene methylimine bonds (—CH2-N(CH3)-O—CH2-), thiodiester bonds (—O—C(O)—S—), thiocarbamate bonds (—O—C(O)(NH)—S—), and siloxane bonds (—O—S). i(H)2-O—and dialkylsiloxane), N,N′-dimethylhydrazine bond (—CH2-N(CH3)-N(CH3)-), amide-3 bond (3'-CH2-C(=O)-N(H)-5'), amide-4 bond (3'-CH2-N(H)-C(=O)-5'), hydroxylamine bond, siloxane bond (dialkylsiloxane), formamide bond, carbonate bond, carboxymethyl bond, carbamate bond, carboxylic acid ester bond, thioether bond, ethylene oxide linker, sulfide bond, sulfonate bond, sulfonamide bond, sulfonate bond, thioacetal bond ( ), acetal bond ( ), oxime bond, methylene imine bond, methylene carbonyl amino bond, methylene hydrazine bond, methylene dimethyl hydrazine bond, methylene oxymethyl imine bond, ether bond (C3'-O-C5'), thioether bond (C3'-S-C5'), thioacetamide bond (C3'-N(H)-C(=O)-CH2-S-C5', C3'-OP(O)-O-SS-C5'), C3'-CH2-NH-NH-C5', 3'-NHP(O)(OC H3)-O-5', 3'-NHP(O)(OCH3)-O-5', phosphoramide bond (“imidp”), 2'->5' inter-nucleoside bond, 2'->3 inter-nucleoside bond, 3'->3 inter-nucleoside bond and 5'->5' inter-nucleoside bond; optionally, the modified inter-nucleoside bond is a thiophosphate bond, a methylphosphonate bond, an imidp or MMI bond, more preferably, the modified inter-nucleoside bond is a thiophosphate (PS) bond.

[0225] Modified internucleotide linker bonds can occur on any nucleotide of the sense or antisense strand, and at any position on the strand. For example, internucleotide linker bond modifications can occur on every nucleotide of the sense and / or antisense strand; each internucleotide linker bond modification can occur in an alternating pattern on the sense or antisense strand; or the sense or antisense strand may contain internucleotide linker bond modifications in an alternating pattern. The alternating pattern of internucleotide linker bond modifications on the sense strand can be the same as or different from that on the antisense strand, and the alternating pattern of internucleotide linker bond modifications in the sense strand can be shifted relative to the alternating pattern of internucleotide linker bond modifications in the antisense strand.

[0226] In some embodiments, the dsRNA contains modified internucleotide linkers (e.g., phosphate thioester bonds or methylphosphonic acid nucleotide linkers) in the sticky-terminal region. For example, the sticky-terminal region contains two nucleotides with a modified internucleotide linker (e.g., phosphate thioester or methylphosphonic acid nucleotide linker) between them. Internucleotide linker modifications can also be made to link the sticky-terminal nucleotide to terminal paired nucleotides within the double-stranded region. For example, at least two, three, four, or all of the sticky-terminal nucleotides can be linked by modified internucleotide linkers (e.g., phosphate thioester bonds or methylphosphonic acid nucleotide linkers), and optionally, additional modified intranucleotide linkers (e.g., phosphate thioester bonds or methylphosphonic acid nucleotide linkers) can connect the sticky-terminal nucleotide to adjacent paired nucleotides. For example, at least one modified internucleotide linker (e.g., phosphate thioester or methylphosphonic acid nucleotide linker) may exist between the three terminal nucleotides, wherein two of the three nucleotides are sticky-terminal nucleotides and the third is a paired nucleotide adjacent to the sticky-terminal nucleotide. Preferably, these terminal three nucleotides can be located at the 3'-end of the antisense strand.

[0227] Regarding the position of the nucleotide linker, the indicated position refers to the nucleotide linker that connects the nucleotide at that position to the nucleotide one position downstream of that position. In other words, the nucleotide linker at position N means that it is located between nucleotides N and N+1. Therefore, counting from the 5' end, the nucleotide linker at position 1 means that the linker is located between the nucleotides at positions 1 and 2, counting from the 5' end.

[0228] In some embodiments, the positive chain includes 1-5 (e.g., 1, 2, 3, 4, or 5) modified nucleoside bonds (e.g., phosphate thioester bonds or methylphosphonic acid nucleotide bonds) within positions 1-5, counted from the 5' end of the positive chain, and 1-5 (e.g., 1, 2, 3, 4, or 5) modified nucleoside bonds (e.g., phosphate thioester bonds or methylphosphonic acid nucleotide bonds) within positions 1-5, counted from the 5' end of the positive chain. For example, the positive chain includes modified nucleoside bonds (e.g., phosphate thioester bonds or methylphosphonic acid nucleotide bonds) between nucleotides 1 and 2 and between nucleotides 2 and 3, counted from the 5' end of the positive chain, and the positive chain also includes modified nucleoside bonds between nucleotides 1 and 2 and between nucleotides 2 and 3, counted from the 3' end of the positive chain.

[0229] In some embodiments, the antisense strand includes 1-5 (e.g., 1, 2, 3, 4, or 5) modified nucleoside bonds (e.g., phosphate thioester bonds or methylphosphonate nucleotide bonds) within positions 1-5, counted from the 5' end of the antisense strand, and 1-5 (e.g., 1, 2, 3, 4, or 5) modified nucleoside bonds (e.g., phosphate thioester bonds or methylphosphonate nucleotide bonds) within positions 1-5, counted from the 5' end of the antisense strand. For example, the antisense strand includes modified nucleoside bonds (e.g., phosphate thioester bonds or methylphosphonate nucleotide bonds) between nucleotides 1 and 2 and between nucleotides 2 and 3, counted from the 5' end of the antisense strand, and the antisense strand also includes modified nucleoside bonds between nucleotides 1 and 2 and between nucleotides 2 and 3, counted from the 3' end of the antisense strand.

[0230] In some embodiments, the sense strand includes modified internucleotide bonds (e.g., phosphate thioester or methylphosphonate nucleotide bonds) at positions 1 and 2, counting from the 5' end of the sense strand, and modified intranucleotide bonds (e.g., phosphate thioester or methylphosphonate nucleotide bonds) at positions 1 and 2, counting from the 3' end of the sense strand; and the antisense strand includes modified internucleotide bonds (e.g., phosphate thioester or methylphosphonate nucleotide bonds) at positions 1 and 2, counting from the 5' end of the antisense strand, and modified internucleotide bonds (e.g., phosphate thioester or methylphosphonate nucleotide bonds) at positions 1 and 2, counting from the 3' end of the antisense strand.

[0231] Sticky end modification

[0232] The nucleotides in the sticky-terminal region of the dsRNA molecule can be independently modified or unmodified nucleotides, including but not limited to 2'-sugar modifications such as 2'-fluoro, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine, 2'-O-methoxyethyl adenosine, 2'-O-methoxyethyl-5-methylcytidine, GNA (ethylene glycol nucleic acid), SNA (serine nucleic acid), TNA (threonine nucleic acid), and any combination thereof. For example, TT (or UU) can be a sticky-terminal sequence at either end. The 5' or 3' sticky ends of the sense strand, antisense strand, or both strands of the dsRNA molecule can be phosphorylated. In some embodiments, the sticky-terminal region contains two nucleotides linked by a phosphate-thionucleotide bond, wherein the two nucleotides in the sticky-terminal region can be the same or different.

[0233] 5'-modification

[0234] lack (For example, or The 5' end of the modified dsRNA strand can also be modified. Exemplary modifications to the 5' end include, but are not limited to, 5'-morpholinonucleotides (e.g., nucleotides in which the 5'-OH group is replaced by a morpholino group), 5'-dimethylaminonucleotides (e.g., nucleotides in which the 5'-OH group is replaced by a dimethylamino group), 5'-deoxynucleotides, reverse nucleotides (i.e., nucleotides linked to the rest of the strand via a 5'->5' link), reverse abase-free nucleotides (e.g., abase-free nucleotide linked via a 5'->5' link), or reverse abase-free locked nucleic acid modifications located at the 5' end (i.e., LNAs lacking a base and linked via a 5'->5' link).

[0235] In some implementations, the sense strand of the dsRNA contains a 5'-morpholinonucleotide, 5'-dimethylaminonucleotide, 5'-deoxynucleotide, reverse nucleotide, reverse abase-free nucleotide, or reverse abase-free locked nucleic acid modification at the 5' end. For example, the sense strand contains a reverse nucleotide, reverse abase-free nucleotide, or reverse abase-free locked nucleic acid modification at the 5' end. For example, the sense strand of the double-stranded ribonucleic acid contains a reverse nucleotide, reverse abase-free nucleotide, or reverse abase-free locked nucleic acid modification at the 3' end.

[0236] In some implementations, the justice chain contains a ligand at its 3'-terminus.

[0237] Ligand

[0238] In some implementations, longer double-stranded and single-stranded oligonucleotides may contain ligands. Where one does not wish to be bound by theory, the ligand may alter one or more properties of the linked molecule (e.g., dsRNA as described herein), including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake (cellular targeting), charge, and clearance.

[0239] In some implementations, the ligand is a targeting ligand. As used herein, the term "targeting ligand" refers to any molecule that provides enhanced affinity to a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, e.g., a cell, tissue, or organ compartment. Some exemplary targeting ligands include, but are not limited to, 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. Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, polygalactose, N-acetyl-D-galactosamine (GalNAc), polyvalent GalNAc (e.g., GalNAc2 and GalNAc3), D-mannose, polymannose, polylactose, N-acetylglucosamine, polyfusose, glycosylated polyamino acids, and lectins. The term "polyvalent" indicates the presence of multiple monosaccharide units. These monosaccharide subunits can be linked to each other via glycosidic bonds or to scaffold molecules.

[0240] In some embodiments, the ligand is a desialylate glycoprotein receptor (ASGPR) ligand. An ASGPR ligand refers to a ligand that binds to an ASGPR. In some embodiments, the ASGPR ligand comprises one or more (e.g., 1, 2, 3, or more) GalNAc or GalNAc derivatives linked by divalent or trivalent branched linkers. Exemplary ASGPR ligands are: or , or Where n = 0-10 (e.g., 1 or 4); or ,or An oligonucleotide that forms a cyclic structure in which 3 or 4 consecutive nucleosides are modified by a ligand containing GalNAc; for example, oligonucleotides containing a cyclic structure. (SEQ ID NO:1), where each G* and A* is replaced in bits 2'-0 with .

[0241] Exemplary carbohydrate-based targeting ligands are also described in U.S. Patents 5,994,517 and 6,906,182, and PCT application PCT / US2022 / 047102, the entire contents of which are incorporated herein by reference. Exemplary folic acid and folic acid analogues of targeting ligands are described in U.S. Patents 2,816,110; 5,552,545; 6,335,434 and 7,128,893, the entire contents of which are incorporated herein by reference.

[0242] It is worth noting that when two or more ligands are present, the ligands may all have the same properties, or they may all have different properties, or some ligands may have the same properties while others have different properties. In a preferred embodiment, all ligands have different properties.

[0243] It is worth noting that the ligand can be attached to any position on either strand of the dsRNA. For example, the ligand can be located at the 5' end, 3' end, or internal position of the strand, such as the sense or antisense strand of a double-stranded RNA.

[0244] In some implementations, the justice chain includes a ligand, meaning the ligand is coupled to the justice chain. For example, the ligand is coupled to the 3′ end of the justice chain.

[0245] Exemplary sense strand

[0246] In some implementations, the sense strand of dsRNA has one of the following modification patterns:

[0247]

[0248] in n is a 2'-O-methyl nucleotide; (dN) is a 2'-deoxynucleotide; Nf is a 2'-fluorinated nucleotide (e.g., a 2'-deoxy-2'-fluorinated nucleotide); and The chain of justice may optionally include: (a) Choose either the 3'-end or 5'-end modification from the following options: (i) 5'-(L1)-, optionally linked to the 5'-terminal nucleotide via a divalent linker, such as a phosphodiester bond or a thiophosphate bond (e.g., via the 5'-O of the terminal nucleoside). (ii)-(L2)-3', optionally linked to the 3'-terminal nucleotide (e.g., via the 3'-O of the terminal nucleoside) via a divalent linker, such as a phosphodiester bond or a thiophosphate bond. (iii) 5'-(L1)(I)-, optionally linked to the 5'-terminal nucleotide (5'-5') via a divalent linker, such as a phosphodiester bond or a thiophosphate bond; or (iv)-(I)(L2)-3', optionally linked to the 3'-terminal nucleotide (3'-3') via a divalent linker, such as a phosphodiester bond or a thiophosphate bond. in Each (I) is a reverse nucleotide (e.g., a reverse abase nucleotide, such as a reverse abase ribonucleotide, such as a reverse abase deoxyribonucleotide). (L1) and (L2) are independently hydrogen or groups containing ligand (L), wherein the ligand is selected from: (i) Lipophilic groups; examples include those containing C 10 -C 30 Alkyl or C 10 -C 30 alkenyl groups, such as C 10 Alkyl, C 10 alkenyl, C 12 Alkyl, C 12 alkenyl, C 14 Alkyl, C 14 alkenyl, C 15 Alkyl, C 15 alkenyl, C 16 Alkyl, C 16 alkenyl, C 18 Alkyl, C 18 alkenyl, C 20 Alkyl, C 20 alkenyl, C 22 Alkyl, C 22 alkenyl, C 24 Alkyl, C 24 alkenyl; C 15 Alkyl, C 15 alkenyl, C 17 Alkyl, C 17 alkenyl, C 19 Alkyl, C 19 alkenyl, C 21 Alkyl, C 21 alkenyl, C 23 Alkyl or C 23 Alkenyl; examples include, but are not limited to, hexadecyl, docosyl, ω-hydroxyhexadecyl, and ω-hydroxydocosyl; or (ii) Receptor-targeting ligands, such as groups containing ASGPR ligands; or (iii) a precursor functional group, wherein the precursor functional group is suitable for post-synthetic functionalization with a ligand containing or coupled to a complementary reactive functional group (e.g., as described in (i) or (ii)); examples of the precursor functional group include, but are not limited to, amino, carboxyl, primary amide (-C(O)NH2), N-succinimide, azide (-N3), mercapto (-SH), and active esters (e.g., N-hydroxysuccinimide ester (NHS ester) or pentafluorobenzene). 1,2,4,5-tetraazinyl (e.g., 3-methyl-1,2,4,5-tetraazinyl, 3-(pyridin-2-yl)-1,2,4,5-tetraazinyl or 3-(pyrimidin-2-yl)1,2,4,5-tetraazinyl); cyclooctynyl (e.g., bicyclo[6.1.0]nonynyl (BCN) or dibenzocyclooctynyl alcohol (DBCO)), transcyclooctenyl, 2-methylsulfonylpyrimidinyl, 4-vinylpyridinyl and their protected forms; or (b) As described above, the ligand-modified nucleotides that replace the aforementioned nucleotides; examples of lipophilic modified nucleotides include (Nhd)-2'-O-hexadecyl modified nucleotides; (Nda)-a 2'-O-docosahexadecyl modified nucleotides; (NhdOH)-2'-O-(ω-hydroxyhexadecyl) modified nucleotides; or (NdaOH)-a 2'-O-(ω-hydroxydocosahexadecyl) modified nucleotides.

[0249] In each of the above positive strands, each nucleotide is tandemly linked by an optionally modified nucleotide linker (i.e., in a 3'->5' configuration). For example, each nucleotide is linked by a phosphodiester or thiophosphate nucleotide linker.

[0250] In some embodiments, counting from the 5' end of the oligonucleotide, the nucleotides at positions 1 and 2 are linked by a phosphate thioester nucleoside bond; the nucleotides at positions 2 and 3 are linked by a phosphate thioester nucleoside bond; and the remaining nucleotides are linked by a phosphodiester bond.

[0251] In some implementations, counting from the 5' end of the oligonucleotide, the nucleotides at positions 1 and 2 are linked by a phosphate-thionucleotide bond; the nucleotides at positions 2 and 3 are linked by a phosphate-thionucleotide bond; the nucleotides at positions 3 and 4 are linked by a phosphate-thionucleotide bond; and the remaining nucleotides are linked by a phosphodiester bond.

[0252] In some embodiments, counting begins from the 5' end of the oligonucleotide, where for nucleotides of length m, the m-1 and m positions are linked by a phosphate thioester nucleoside bond. That is, for a nucleotide of length 23 nucleotides, counting begins from the 5' end of the oligonucleotide, with the nucleotides at positions 22 and 23 linked by a phosphate thioester nucleoside bond; for a nucleotide of length 21 nucleotides, counting begins from the 5' end of the oligonucleotide, with the nucleotides at positions 20 and 21 linked by a phosphate thioester nucleoside bond.

[0253]

[0254]

[0255]

[0256]

[0257] in n is a 2'-O-methyl nucleotide; (dN) is a 2'-deoxynucleotide; Nf is a 2'-fluorinated nucleotide; (L) is a ligand-modified nucleotide, for example, containing a lipophilic group (e.g., C). 16 Or C 22 (Modified) or ASGPR ligands; and s is an intermolecular bond between phosphate-thionucleotides. (s) is a thiophosphate bond or a phosphodiester nucleotide linking bond (e.g., in some embodiments, each (s) is a thiophosphate bond). (I) is a reverse abase-free nucleotide (e.g., a reverse abase-free ribonucleotide or a reverse abase-free deoxyribonucleotide). (L1) and (L2) are independently hydrogen or groups containing ligands, and the positive strand optionally contains 5'-morpholinonucleotide, 5'-dimethylaminonucleotide, 5'-deoxynucleotide, reverse nucleotide, reverse abase-free nucleotide or reverse abase-free locked nucleic acid modification at its 5'-terminus.

[0258] In some implementations, the antisense chain of dsNA has one of the following modification patterns:

[0259]

[0260] in: n is a 2'-O-methyl modified nucleotide; s is a phosphate thionucleotide inter-bond (3'-5'). (dN) is a 2'-deoxynucleotide; Nf is a 2'-fluorinated nucleotide; Z is (For example, or ),in: X is O or S; and Each R V Independently protected by hydrogen or hydroxyl groups; and The above structure replaces the 4'-CH2OH group on furanose (such as ribose) or the 5'-CH2OH group on the 5'-terminal nucleotide pyranose ring.

[0261] In each of the preceding antisense strands, each nucleotide is linked in tandem by a phosphodiester or thiophosphate nucleotide bond (i.e., in a 3'->5' manner).

[0262] In one embodiment of any of AS1-AS39, one nucleotide from position 5 to 8, counting from the 5' end of the antisense strand, is replaced by a thermally unstable modification (G), for example: (Ngn)-ethylene glycol nucleic acid, S-isomer; (N2p)-2′-phosphate nucleotide (i.e., 3'-RNA linked by 3'-5' and 2'-5' nucleotide bonds in the 5' and 3' directions, respectively); (Tna) - a threonucleotide (linked by 3'-3' and 2'-5' nucleotide bonds in the 5' and 3' directions, respectively); (MM) base mismatch with the positive chain; or (Nul) A type of unlocked nucleic acid.

[0263] In one embodiment of any of AS1-AS39, nucleotide position 5, counting from the 5' end of the antisense strand, is replaced by a heat-labile modification (G). In one embodiment of any of AS1-AS39, nucleotide position 6, counting from the 5' end of the antisense strand, is replaced by a heat-labile modification (G). In one embodiment of any of AS1-AS39, nucleotide position 7, counting from the 5' end of the antisense strand, is replaced by a heat-labile modification (G). In one embodiment of any of AS1-AS39, nucleotide position 8, counting from the 5' end of the antisense strand, is replaced by a heat-labile modification (G).

[0264] For example, the antisense chain can be selected from any of AS40-AS58 as described in Table E:

[0265] In some embodiments, counting begins from the 5' end of the oligonucleotide, with the nucleotides at positions 1 and 2 linked by a phosphate-thionucleotide bond; the nucleotides at positions 2 and 3 linked by a phosphate-thionucleotide bond; and the remaining nucleotides linked by a phosphodiester bond.

[0266] In some embodiments, nucleotides of length m are counted starting from the 5' end of the oligonucleotide, with the m-1 and m-position nucleotides linked by a phosphate-thionucleotide bond. That is, for a nucleotide of length 23 nucleotides, counting starts from the 5' end of the oligonucleotide, with the 22nd and 23rd nucleotides linked by a phosphate-thionucleotide bond; for a nucleotide of length 21 nucleotides, counting starts from the 5' end of the oligonucleotide, with the 20th and 21st nucleotides linked by a phosphate-thionucleotide bond.

[0267] In some embodiments, nucleotides of length m are counted starting from the 5' end of the oligonucleotide, with the m-2 and m-1 nucleotides linked by a phosphate-thionucleotide bond, and the m-1 and m nucleotides linked by a phosphate-thionucleotide bond. That is, for a nucleotide of length 23 nucleotides, counting starting from the 5' end of the oligonucleotide, the 21st and 22nd nucleotides are linked by a phosphate-thionucleotide bond; the 22nd and 23rd nucleotides are linked by a phosphate-thionucleotide bond; for a nucleotide of length 21 nucleotides, counting starting from the 5' end of the oligonucleotide, the 20th and 21st nucleotides are linked by a phosphate-thionucleotide bond.

[0268] In some implementations, the nucleotides of length m are counted starting from the 5' end of the oligonucleotide: (a) The nucleotides at positions 1 and 2 are linked by a phosphate-thionucleotide bond; (b) The nucleotides at positions 2 and 3 are linked by a phosphate-thionucleotide bond; (c) The nucleotides at positions m-2 and m-1 are linked by a phosphorothioate bond; and (d) The nucleotides at positions m-1 and m are linked by a phosphorothioate bond. The remaining nucleotides are linked by phosphodiester bonds.

[0269] In other words, for a nucleotide with a length of 23 nucleotides, counting from the 5' end of the oligonucleotide, positions 1 and 2; 2 and 3; 21 and 22; and 22 and 23 are linked by phosphate-thionucleotide bonds, while the remaining nucleotides are linked by phosphodiester bonds. For a nucleotide with a length of 21 nucleotides, counting from the 5' end of the oligonucleotide, positions 1 and 2; 2 and 3; 19 and 20; and 20 and 21 are linked by phosphate-thionucleotide bonds, while the remaining nucleotides are linked by phosphodiester bonds.

[0270] In some implementations, the nucleotides of length m are counted starting from the 5' end of the oligonucleotide: (a) The nucleotides at positions 1 and 2 are linked by a phosphate-thionucleotide bond; (b) The nucleotides at positions 2 and 3 are linked by a phosphate-thionucleotide bond; (c) The nucleotides at positions 3 and 4 are linked by a phosphorothioate bond; and (d) The nucleotides at positions m-1 and m are linked by a phosphorothioate bond. The remaining nucleotides are linked by phosphodiester bonds.

[0271] In other words, for a nucleotide with a length of 23 nucleotides, counting from the 5' end of the oligonucleotide, the nucleotides at positions 1 and 2; 2 and 3; 3 and 4; and 22 and 23 are linked by phosphate-thionucleotide bonds, while the remaining nucleotides are linked by phosphodiester bonds. For a nucleotide with a length of 21 nucleotides, counting from the 5' end of the oligonucleotide, the nucleotides at positions 1 and 2; 2 and 3; 3 and 4; and 20 and 21 are linked by phosphate-thionucleotide bonds, while the remaining nucleotides are linked by phosphodiester bonds.

[0272] For example, in some implementations, the antisense strand of the dsRNA has one of the following modification patterns in Table F.

[0273]

[0274]

[0275]

[0276] In one embodiment of any of AS58-AS135, one of the nucleotides at positions 5-8, counting from the 5' end of the antisense strand, is replaced by a heat-labile modification (G), for example: (Ngn)-ethylene glycol nucleic acid, S-isomer; (N2p)-2'-phosphate nucleotides (i.e., 3'-RNA linked by 3'-5' and 2'-5' nucleotide bonds in the 5' and 3' directions, respectively); (Tna) - a threonucleotide (linked by 3'-3' and 2'-5' nucleotide bonds in the 5' and 3' directions, respectively); (MM) base mismatch with the positive chain; or (Nul) A type of unlocked nucleic acid.

[0277] In one embodiment of any of AS58-AS135, the 5th nucleotide, counting from the 5' end of the antisense strand, is replaced by a heat-labile modification (G). In one embodiment of any of AS58-AS135, the 6th nucleotide, counting from the 5' end of the antisense strand, is replaced by a heat-labile modification (G). In one embodiment of any of AS58-AS135, the 7th nucleotide, counting from the 5' end of the antisense strand, is replaced by a heat-labile modification (G). In one embodiment of any of AS58-AS135, the 8th nucleotide, counting from the 5' end of the antisense strand, is replaced by a heat-labile modification (G).

[0278] For example, the antisense chain can be selected from any of AS136-AS171 described in Table G.

[0279]

[0280]

[0281] In another embodiment of the foregoing exemplary positive and negative strands, each of the positive strands S1 to S123 can hybridize or form a double strand with any of the negative strands AS1 to AS171. In other words, the longer double-stranded oligonucleotide product described herein includes the product formed by hybridization / double-stranding of any of the positive strands S1 to S123 with any of the negative strands AS1 to AS171. In some embodiments, the positive strand is selected from embodiments having 21 nucleotides, and the negative strand is selected from embodiments containing 23 nucleotides. In some embodiments, the positive strand is selected from embodiments having 21 nucleotides, and the negative strand is selected from embodiments containing 21 nucleotides. In some embodiments, the positive strand is selected from embodiments having 19 nucleotides, and the negative strand is selected from embodiments containing 21 nucleotides. In some embodiments, the positive strand is selected from embodiments having 19 nucleotides, and the negative strand is selected from embodiments containing 19 nucleotides.

[0282] In some implementations, the longer single-stranded oligonucleotides described herein are any one of the sense strands S1 to S123 or any one of the antisense strands AS1 to AS171.

[0283] Composition

[0284] The dsRNA or oligonucleotides described herein can be formulated into compositions. For example, the dsRNA or oligonucleotides described herein can be formulated into pharmaceutical compositions for therapeutic use. Therefore, in another aspect, this document provides a pharmaceutical composition comprising an effective therapeutic amount of one or more dsRNAs or oligonucleotides described herein, for administration alone or in combination with one or more pharmaceutically acceptable carriers (additives), excipients, and / or diluents.

[0285] Pharmaceutical compositions can be specifically formulated for administration in solid or liquid form, including pharmaceutical compositions suitable for: (1) oral administration, e.g., oral enemas (aqueous or non-aqueous solutions or suspensions), tablets (e.g., tablets for oral, sublingual and systemic absorption), pills, powders, granules, pastes (for the tongue); (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous or epidural injection, e.g., sterile solutions or suspensions, or sustained-release formulations; (3) topical administration, e.g., as creams, ointments or controlled-release patches or sprays applied to the skin; (4) vaginal or rectal administration, e.g., as suppositories, creams or foams; (5) sublingual; (6) ocular; (7) percutaneous; or (8) nasal. Delivery by subcutaneous or intravenous injection may be particularly advantageous.

[0286] As used herein, the term “effective therapeutic amount” refers to an amount of compound, material, or composition comprising the conjugate described herein, which is capable of effectively producing some desired therapeutic effect in at least one cell subpopulation of animals with a reasonable benefit / risk ratio suitable for any medical treatment.

[0287] As used in this article, "pharmaceutically acceptable" means compounds, materials, compositions, and / or dosage forms that are suitable for use in human and animal tissues within the bounds of reasonable medical judgment without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0288] As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. The use of such media and reagents for pharmaceutically active substances is well known in the art. Use in a composition may be considered unless any conventional media or reagent is incompatible with the active compound. Additional active compounds may also be incorporated into the composition. Drug carriers include sterile aqueous solutions or dispersions, as well as sterile powders for the provisional preparation of sterile injectable solutions or dispersions. The use of such media and reagents for pharmaceutically active substances is known in the art.

[0289] dsRNA or oligonucleotides can be formulated for parenteral administration via injection, such as by bolus or continuous infusion. Injectable formulations can be presented in unit dosage forms, such as ampoules or multi-dose containers, and may include preservatives. The composition can be in the form of a suspension, solution, or emulsion in an oily or aqueous carrier and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the active ingredient can be in powder form for reconstitution with a suitable carrier (e.g., sterile, pyrogen-free water) prior to use.

[0290] LNP and liposome formulations

[0291] The dsRNA or oligonucleotides described herein can be formulated with one or more lipids for delivery. For example, the dsRNA or oligonucleotides described herein can be formulated into lipid particles. As used herein, the term "lipid particle" refers to a vesicle formed from one or more lipid components. In the context of drug development, lipid particles are commonly used as carriers for nucleic acid delivery. They deliver drugs or active pharmaceutical ingredients (APIs) by fusing with the cell membrane and repositioning their lipid structure. Typically, lipid particle compositions used for such delivery consist of ionizable or cationic lipids, phospholipids (particularly compounds with phosphatidylcholine groups), cholesterol, and polyethylene glycol (PEG) lipids; however, these compositions may also include other lipids. Ionizable lipids are often used to concentrate nucleic acid cargo at low pH and drive membrane binding and fusion. Phospholipids are often used to enhance fusion. Cholesterol is often used to provide membrane integrity. PEG lipids are often used to provide steric stability. The overall composition of lipids typically determines the surface characteristics of a biological system, which in turn determines the protein (opsonization) content, thereby driving biodistribution and cellular uptake characteristics. Lipid particles can be lipid nanoparticles (LNPs).

[0292] In some embodiments, the lipid particles described herein comprise ionizable lipids. As used herein, the term "ionizable lipid" refers to a lipid having at least one protonable or deprotonable group, such that the lipid is positively charged at or below a physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably at or above a physiological pH. Typically, ionizable lipids are lipids containing at least one amino group that is positively charged or protonated under acidic conditions, such as at pH 6.5 or lower. Those skilled in the art will understand that the addition or removal of protons as a function of pH is a balanced process, and references to charged or neutral lipids refer to the nature of the dominant species and do not require all lipids to be present in a charged or neutral form. Typically, the pKa of the protonable group of ionizable lipids is in the range of about 4 to about 7. Ionizable lipids are also referred to herein as cationic lipids.

[0293] In some embodiments, the ionizable lipid is MC3(6Z,9Z,28Z,31Z)-heptatrienoic acid-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA or MC3). In some embodiments, the ionizable lipid is lipid ATX-002. In some embodiments, the ionizable lipid is (13 Z ,16 Z )- N,N -Dimethyl-3-nonyldocosa-13,16-diene-1-amine. In some embodiments, the ionizable lipid is compound 6 or compound 22 as described in WO2015 / 199952, the entire contents of which are incorporated herein by reference.

[0294] There are no restrictions; ionizable lipids can account for 20-90% (moles) of the total lipids in lipid particles.

[0295] As used herein, the term "non-cationic lipid" refers to any amphiphilic lipid as well as any other neutral or anionic lipid. Therefore, non-cationic lipids can be neutral, zwitterionic, or anionic lipids. Non-cationic lipids are commonly used to enhance fusion properties. Exemplary non-cationic lipids include, but are not limited to, distearyl-sn-glycerol-phosphoethanolamine, distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylethanolamine-4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethyl esteramine (DSPE), monomethylphosphatidylethanolamine (e.g., 16-O-monomethylPE), dimethylphosphatidylethanolamine (e.g., 16-O-dimethylPE), and 18-1-trans-phosphatidylethanolamine. E, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), lecithin choline (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearate phosphatidylglycerol (DSPG), disqualylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), ditransoleoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecosylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl group in these lipids is preferably derived from a group having a C... 10 -C 24 Acyl groups of fatty acids in the carbon chain, such as lauroyl, myristyl, palmitoyl, stearyl, or oleyl.

[0296] Other examples of non-cationic lipids suitable for lipid particles include nonphospholipids such as stearamine, dodecylamine, hexadecylamine, acetyl palmitate, glyceryl ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine dodecyl sulfate, alkylaryl sulfate polyethoxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramides, sphingomyelin, etc.

[0297] In some embodiments, the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the non-cationic lipid is DSPC.

[0298] Non-cationic lipids may comprise 0-30% (moles) of the total lipids in the lipid particles. For example, the non-cationic lipid content may be 5-20% (moles) or 10-15% (moles) of the total lipids present in the lipid particles. In various embodiments, the molar ratio of ionizable lipids to neutral lipids ranges from about 2:1 to about 8:1.

[0299] In some embodiments, the lipid particles may also contain coupled lipid molecules. As used herein, the term "coupled lipid" refers to a lipid molecule coupled to a non-lipid molecule, such as PEG, polyoxazoline, polyamide, or polymer (such as cationic polymer). Typically, these lipids are used to inhibit the aggregation of lipid particles and / or provide steric stability. Exemplary coupled lipids include, but are not limited to, PEG lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide lipid conjugates (such as ATTA lipid conjugates), cationic polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the coupled lipid molecule is a PEG lipid conjugate, such as (methoxy polyethylene glycol) coupled lipid.

[0300] Exemplary PEG lipid conjugates include, but are not limited to, PEG diacylglycerol (DAG) (e.g., 1-(monomethoxy polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG dialkoxypropyl (DAA), PEG phospholipids, PEG ceramide (Cer), polyethylene glycol-modified phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol ester (PEGS-DAG) (e.g., 4-O-(2',3'-bis(tetradecyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxy polyethylene glycol 2000)-1,2-distearate-sn-glycerol-3-phosphate ethanolamine sodium salt, or mixtures thereof).

[0301] The PEG-DAA conjugate can be, for example, PEG-dilauroyloxypropyl, PEG-dimyristoyloxypropyl, PEG-dipalmitoyloxypropyl, or PEG-distearateoxypropyl. PEG lipids can be one or more of PEG-DMG, PEG-dilauroylglycerol, PEG-dipalmitoylglycerol, PEG-distearateglycerol, PEG-dilaurylglyceramide, PEG-dimyristoylglyceramide, PEG-dipalmitoylglyceramide, PEG-distearateglyceramide, PEG-cholesterol (1-[8'-(cholest-5-en-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-bistetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some examples, the PEG lipid can be PEG-DMG, 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

[0302] PEG or conjugated lipids may comprise 0-20% (moles) of the total lipids in the lipid particles. In some embodiments, the PEG or conjugated lipid content is 0.5-10% or 2-5% (moles) of the total lipids in the lipid particles.

[0303] In some embodiments, the lipid particles may also contain a component, such as a sterol, to provide membrane integrity. An exemplary sterol that can be used in lipid particles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholesterol, 5β-codasteinol, cholesterol-(2′-hydroxy)-ethyl ether, cholesterol-(4′-hydroxy)-butyl ether, and 6-ketocholesterol; nonpolar analogs such as 5α-cholestane, cholesterolenone, 5α-cholestanone, 5β-cholestanone, and cholesterol decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog such as cholesterol-(4′-hydroxy)-butyl ether.

[0304] Components that provide membrane integrity, such as sterols, may comprise 0-50% (moles) of the total lipids in the lipid particles. In some embodiments, this component comprises 20-50% (moles) to 30-40% (moles) of the total lipid content of the lipid particles.

[0305] In one embodiment, the lipid particles comprise: ionizable lipids; non-cationic lipids; coupled lipids that inhibit particle aggregation; and sterols. The molar ratio of ionizable lipids, non-cationic lipids, sterols, and PEG / coupled lipids can be varied as needed. For example, the lipid particles may comprise 30-70% ionizable lipids, 0-60% cholesterol, 0-30% non-cationic lipids, and 1-10% coupled lipids by molar weight or total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipids, 40-50% cholesterol, and 10-20% non-cationic lipids by molar weight or total weight of the composition. In some other embodiments, the composition comprises 50-75% by molar or total weight of ionizable lipids, 20-40% by molar or total weight of cholesterol, 5-10% by molar or total weight of non-cationic lipids, and 1-10% by molar or total weight of coupled lipids. The composition may comprise 60-70% by molar or total weight of ionizable lipids, 25-35% by molar or total weight of cholesterol, and 5-10% by molar or total weight of non-cationic lipids. The composition may also comprise up to 90% by molar or total weight of ionizable lipids and 2-15% by molar or total weight of non-cationic lipids. The formulation can also be a lipid particle formulation, for example comprising 8-30% by molar or total weight of ionizable lipids, 5-30% by molar or total weight of non-cationic lipids, and 0-20% by molar or total weight of cholesterol; or 4-25% by molar or total weight of ionizable lipids, 4-25% by molar or total weight of non-cationic lipids, 2-25% by molar or total weight of cholesterol, 10-35% by molar or total weight of coupled lipids, and 5% by molar or total weight of cholesterol. The composition may contain % cholesterol; or 2-30% ionizable lipids, 2-30% non-cationic lipids, 1-15% cholesterol, 2-35% coupled lipids, and 1-20% cholesterol; or even up to 90% ionizable lipids and 2-10% non-cationic lipids, or even 100% cationic lipids. In some embodiments, the lipid particle formulation comprises ionizable lipids, phospholipids, cholesterol, and PEGylated lipids in a molar ratio of 50:10:38.5:1.5.In some other embodiments, the lipid particle formulation comprises ionizable lipids, cholesterol, and PEGylated lipids in a molar ratio of 60:38.5:1.5.

[0306] In one embodiment, the lipid particles comprise: ionizable lipids comprising about 20 mol% to about 90 mol% of the total lipids present in the particles; noncationic lipids comprising about 5 mol% to about 30 mol% of the total lipids present in the particles; coupled lipids that inhibit particle aggregation comprising about 0.5 mol% to about 20 mol% of the total lipids present in the particles; and sterols comprising about 20 mol% to about 50 mol% of the total lipids present in the particles. In some embodiments, the lipid particles comprise ionizable lipids / noncationic lipids / sterols / coupled lipids in a molar ratio of 50:10:38.5:1.5.

[0307] In one embodiment, the total lipid to nucleic acid (mass or weight) ratio is from about 10:1 to about 30:1. The amount of lipid and nucleic acid can be adjusted to provide the desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher.

[0308] Administering to a subject

[0309] The dsRNA and / or oligonucleotides described herein can be formulated and administered in any convenient manner, similar to other drugs, for medical purposes, such as in humans or veterinary medicine.

[0310] The dsRNA and / or oligonucleotides or pharmaceutical compositions comprising them described herein may be administered to subjects via various delivery routes. Exemplary delivery routes include, but are not limited to, intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, and ocular routes.

[0311] The dsRNA and / or oligonucleotides described herein can be administered in various ways, depending on whether local or systemic treatment is required and the area to be treated. Administration methods may include parenteral, local (including ophthalmic, vaginal, rectal, intranasal, transdermal), or oral administration. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intraventricular administration.

[0312] The route and site of administration can be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the target muscle is a logical choice. Lung cells can be targeted by administering the dsRNA and / or oligonucleotides described herein in aerosol form. Vascular endothelial cells can be targeted by coating a balloon catheter with the dsRNA and / or oligonucleotides described herein and mechanically introducing the dsRNA and / or oligonucleotides described herein.

[0313] In one aspect, this document provides a method for administering the dsRNA and / or oligonucleotides described herein to a subject (e.g., a human subject). In another aspect, the present invention relates to the dsRNA and / or oligonucleotides described herein for inhibiting the expression of a target gene in a subject. This method or medical use includes administering a unit dose of the dsRNA and / or oligonucleotides described herein.

[0314] The defined amount can be an amount that effectively treats or prevents a disease or condition, such as one associated with a target gene. For example, a unit dose can be administered by injection (e.g., intravenous, subcutaneous, or intramuscular), inhalation, or topical application.

[0315] In some implementations, the unit dose is administered less frequently than once daily, such as less than once every 2, 4, 8, or 30 days. In another implementation, the unit dose is not administered at a specific frequency (e.g., an irregular frequency). For example, the unit dose may be administered once.

[0316] In some implementation schemes, the effective dose is administered in conjunction with other conventional treatments.

[0317] The dsRNA and / or oligonucleotides described herein can be applied to mammals, particularly large mammals such as non-human primates or humans, in a variety of ways.

[0318] In some embodiments, the administration of the dsRNA and / or oligonucleotide composition described herein is parenteral, such as intravenous injection (e.g., as a bolus or diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intravenous, intracranial, subcutaneous, transmucosal, oral, sublingual, endoscopic, rectal, oral cavity, vagina, local, pulmonary, intranasal, urethral, ​​or ocular administration. Administration may be provided by the subject or another person, such as a healthcare provider. The drug may be administered in a measured dose or in a dispenser that delivers the measured dose.

[0319] In some implementations, the dsRNA and / or oligonucleotides described herein are administered subcutaneously or intravenously.

[0320] In some implementations, the dsRNA and / or oligonucleotides described herein are administered intrathecally.

[0321] In some implementations, the dsRNA and / or oligonucleotides described herein are administered intravitreally.

[0322] Cell

[0323] This disclosure also provides a cell comprising the compounds, dsRNA, or oligonucleotides described herein. As used herein, the term "cell" refers to a single cell as well as a group (i.e., more than one) of cells. Cells can be prokaryotic or eukaryotic. Exemplary cells include, but are not limited to, bacterial cells, yeast cells, plant cells, animal (including insect) or human cells. In some embodiments, the cell is a eukaryotic cell. For example, the cell is a mammalian cell. Notably, the cell can be in vivo, in vitro, or ex vivo.

[0324] Kit

[0325] The dsRNA or oligonucleotide described herein may be provided in the form of a kit, for example, as part of a kit. For example, a kit may include (a) the dsRNA or oligonucleotide described herein, and optionally (b) informational materials. Informational materials may be descriptive, instructive, marketing, or other materials relating to the methods described herein and / or the use of the dsRNA or oligonucleotide described herein in the methods described herein. The form of the informational materials for the kit is not limited. In some embodiments, the informational materials may include information about the production of the dsRNA or oligonucleotide, its molecular weight, concentration, expiration date, batch number, or place of production. In some embodiments, the informational materials relate to the use of dsRNA or oligonucleotides to treat, prevent, or diagnose diseases and conditions.

[0326] In some embodiments, the informational material may include instructions for administering dsRNA or oligonucleotides in an appropriate manner to perform the methods described herein, such as at an appropriate dose, dosage form, or mode of administration (e.g., the dose, dosage form, or mode of administration described herein). In another embodiment, the informational material may include instructions for administering dsRNA or oligonucleotides to a suitable subject, such as a human being, for example, a person who has or is at risk of developing a disease or condition requiring treatment.

[0327] The format of informational materials for the kit is not limited. In many cases, informational materials (such as instructions) are provided in printed form, but other formats, such as computer-readable materials, are also acceptable.

[0328] The kit components, such as dsRNA or oligonucleotides, can be provided in any form, such as liquid, dry, or lyophilized. Preferably, the dsRNA or oligonucleotides are substantially pure and / or sterile. When the dsRNA or oligonucleotides are provided as a liquid solution, the liquid solution is preferably an aqueous solution, preferably a sterile aqueous solution. When the dsRNA or oligonucleotides are provided in a dry form, they are typically reconstituted by adding a suitable solvent. Solvents, such as sterile water or buffer, may optionally be provided in the kit.

[0329] The kit may include one or more containers for containing kit components. In some embodiments, the kit includes separate containers, partitions, or compartments for different components of the kit. For example, dsRNA or oligonucleotides may be contained in vials, vials, or syringes, and informational material may be contained in association with the container. In other embodiments, individual elements of the kit are contained within a single, indivisible container. For example, dsRNA or oligonucleotides may be contained in vials, vials, or syringes with informational material attached in the form of a label. In some embodiments, the kit includes multiple (e.g., a pack) separate containers, each containing one or more unit doses of dsRNA or oligonucleotides. For example, the kit includes multiple syringes, ampoules, foil packs, or blister packs, each containing a single unit dose of dsRNA or oligonucleotides. The kit containers may be hermetically sealed, waterproof (e.g., impermeable to changes in humidity or evaporation), and / or opaque.

[0330] The kit may optionally include a device suitable for administering dsRNA or oligonucleotides, such as a syringe, inhaler, dropper (e.g., an eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In some embodiments, the device is an implantable device that dispenses a dose of dsRNA or oligonucleotides. The invention also provides a method of providing the kit, for example by combining the components described herein.

[0331] In some implementations, the kit may also contain additional ingredients and / or reagents for carrying out the methods described herein using the dsRNA or oligonucleotides described herein.

[0332] Methods of inhibiting expression of a target gene

[0333] This disclosure also relates to a method for inhibiting the expression of a target gene in a subject. The method includes administering to the subject an amount sufficient to inhibit the expression of the target gene: (i) a double-stranded RNA as described herein, wherein the antisense strand is substantially complementary to the target gene; and / or (ii) an oligonucleotide as described herein, wherein the oligonucleotide is substantially complementary to the target gene.

[0334] This disclosure also relates to the use of dsRNA and / or oligonucleotides for inhibiting the expression of target genes in target cells. The invention further relates to the use of the oligonucleotides and / or dsRNA molecules described herein for inhibiting the expression of target genes in target cells in vitro.

[0335] On the other hand, the present invention relates to a method for regulating the expression of target genes in cells, comprising administering the dsRNA and / or oligonucleotides described herein to the cells. It is noteworthy that the administration to cells can be in vitro or in vivo. Methods of administering compounds to cells are well known and available to those skilled in the art. As used herein, administering the compound to cells means contacting the cells with the compound, allowing the compound to be absorbed by the cells. Typically, cells can be contacted with the dsRNA / oligonucleotide in cell culture (e.g., in vitro or ex vivo), or the compound can be administered to a subject, e.g., in vivo. The term “contacting” or “making contact” in connection with contacting cells herein includes placing cells in a suitable culture medium containing the dsRNA and / or oligonucleotides described herein. When cells are in vivo, “contacting” or “making contact” includes administering the dsRNA and / or oligonucleotides described herein to a subject via a suitable route of administration, e.g., in a pharmaceutical composition, to contact the cells in vivo. For example, when cells are in vitro, the administration to cells may include subjecting the cells to a suitable culture medium containing dsRNA / oligonucleotides. When the cells are in vivo, the administration to the cells includes administering dsRNA / oligonucleotides to the subject via a suitable route of administration, such that the dsRNA / oligonucleotides are administered to the cells in vivo.

[0336] In some implementation schemes, the target genes are selected from Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGFβ gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepcidin, activator protein C, cyclin D gene, VEGF gene, EGFR gene, and cyclin A gene. The mutations are caused by the following genes: cyclin E gene, WNT-1 gene, β-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase IIα gene, p73 gene mutation, p21 (WAF1 / CIP1) gene mutation, p27 (KIP1) gene mutation, PPM1D gene mutation, RAS gene mutation, caveolin I gene mutation, MIBI gene mutation, MTAI gene mutation, M68 gene mutation, tumor suppressor gene mutation, and p53 tumor suppressor gene mutation.

[0337] In other embodiments, the target genes are selected from APP, ATXN2, C9orf72, TARDBP, MAPT(Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, TTR, SCN9A, LRRK2, GPR75, APOE, SCD5, ELOVL1, FLNA, ALK, CHI3L1(YKL-40), RPS25, α2-AR, and GSK3α.

[0338] In one implementation, the target genes are selected from myostatin (MSTN); cholinergic receptor α1 subunit (CHRNA1); cholinergic receptor β1 subunit (CHRNB1); cholinergic receptor δ subunit (CHRND); cholinergic receptor ε subunit (CHRNE); cholinergic receptor γ subunit (CHRNG); type XIII collagen α1 chain (COL13A1); adaptor protein 7 (DOK7); LDL receptor-associated protein 4 (LRP4); muscle-associated receptor tyrosine kinase (MUSK); synaptic receptor-associated protein (RAPSN); voltage-gated sodium channel α subunit 4 (SCN4A); dual homeobox 4 (DUX4); myotonic dystrophy protein kinase (DMPK); glycogen synthase 1 (GYS1); motor neuron survival protein 1 (SMN1); and α-glucosidase. Enzymes (GAA); Adrenergic receptor β1 (ADRB1); Voltage-gated calcium channel subunit α1C (CACNA1C); Voltage-gated calcium channel subunit α1G (CACNA1G) (T-type calcium channel); Angiotensin II receptor type 1 (AGTR1); Voltage-gated sodium channel α subunit 2 (SCN2A); Hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (HCN1); Hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4); Hyperpolarization-activated cyclic nucleotide-gated potassium channel 3 (HCN3); Voltage-gated potassium channel subfamily A member 5 (KCNA5); Inward rectifier potassium channel subfamily J member 3 (KCNJ3); Inward rectifier potassium channel subfamily J member 4 (KCNJ4); Phosphoprotein receptor (PLN); Calcium / calmodulin-dependent protein kinase IIδ (CAMK2D); or phosphodiesterase 1 (PDE1).

[0339] In one embodiment, the target genes are selected from myostatin (MSTN); cholinergic receptor α1 subunit (CHRNA1); cholinergic receptor β1 subunit (CHRNB1); cholinergic receptor δ subunit (CHRND); cholinergic receptor ε subunit (CHRNE); cholinergic receptor γ subunit (CHRNG); type XIII collagen α1 chain (COL13A1); adaptor protein 7 (DOK7); LDL receptor-associated protein 4 (LRP4); muscle-associated receptor tyrosine kinase (MUSK); synaptic receptor-associated protein (RAPSN); voltage-gated sodium channel α subunit 4 (SCN4A); dual homeobox 4 (DUX4); myotonic dystrophy protein kinase (DMPK); glycogen synthase 1 (GYS1); motor neuron survival protein 1 (SMN1); and α-glucosidase (GAA).

[0340] In one embodiment, the target gene is selected from adrenaline receptor β1 (ADRB1); voltage-gated calcium channel subunit α1C (CACNA1C); voltage-gated calcium channel subunit α1G (CACNA1G) (T-type calcium channel); angiotensin II receptor type 1 (AGTR1); voltage-gated sodium channel α subunit 2 (SCN2A); hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (HCN1); hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4); hyperpolarization-activated cyclic nucleotide-gated potassium channel 3 (HCN3); voltage-gated potassium channel subfamily A member 5 (KCNA5); inward rectifier potassium channel subfamily J member 3 (KCNJ3); inward rectifier potassium channel subfamily J member 4 (KCNJ4); phosphoprotein receptor (PLN); calcium / calmodulin-dependent protein kinase IIδ (CAMK2D); or phosphodiesterase 1 (PDE1).

[0341] In one implementation, the target genes are selected from MUC5B, TSLP, IL33, ALOX15, AGER (RAGE), MUC5AC, and STAT6.

[0342] In one embodiment, the target genes are selected from δ4-desaturase, sphingolipid 1 (DEGS1), leptin, follicle-forming protein (FLCN), zinc finger protein 423 (ZFP423), cyclin-dependent kinase 6 (CDK6), mTOR complex 1 regulatory-associated protein (RPTOR), target of rapamycin kinase (mTOR), forkhead box protein P1 (FOXP1), phosphodiesterase 3B (PDE3B), and activin A receptor type 1C (ACVR1C).

[0343] In one implementation, the target genes are selected from hATTR (central nervous system, eye and whole body) TTR, myofibrillarin (MYOC), Ras homology family member A (RhoA), SSB (small RNA-binding exonuclease protective factor La), optic nerve phosphatase, carbonic anhydrase 2 (CA2), Rho-associated coiled protein kinase 1 (ROCK1), Rho-associated coiled protein kinase 2 (ROCK2), angiopoietin-like 7 (ANGPTL7) and cytochrome P450 1B1 (CYP1B1).

[0344] In another embodiment, the target gene is selected from PPARG, ADIPOQ, CD36, LPL, ADAMTS9, RASD1, GYS2, CAT, DPYS, MLXIPL, VEGFA, HLA-DQA1, LIPA, CTSC, FCGR2A, GBE1, SH2B3, CTSK, CDKN2B, ELN, ARG1, HHEX, TCF7L2, CYP2A6, ALDH2, ACADS, GLYCTK, LDLR, HAL, ACER3, SLC7A7, PTPN22, CDKN1C, LEPR, SNAI2, PGM1, IGF2BP2, TTPA, ATP7B, ASPA, ADRB3, MAN2B1, RCAN1, PIGL, TBX1, LMNB1, FBP1, ETFA, LMNA, LAT2, PRKAG2, SELENBP1, TKT, PCSK1, PSAP, NDN, ACY1, SATB2, CYP21A2, POMC, CDC73, CTSH, CFTR, CTSA, G6PD, EXT1, EXT2, CPT1A, SEMA5A, WFS1, KIT, ACAT1, GGCX, FKBP6, PPARGC1B, DGCR6, HMGCS2, PEPD, WRN, LCAT, KLF13, SLC16A2, DHCR7, ITPR3, CLDN4, FZD9, SLC30A2, APOA5, HADHA, CDKAL1, PTPN2, LIPC, CD226, PON1, MCCC1, EIF2AK3, GYG1, BCL7B, AGL, VKORC1, BAZ1B, NAGS, ASL, STAR, ACP2, POLG, GAA, ALDH3A2, MANBA, ARSA, AGA, CYP27B1, CPS1, DLAT, DCXR, EIF4H, DYRK1A, GTF2I, LAMP2, CTH, EPO, FLAD1, AKT2, WAC, GLB1, RFC2, BACH2, D2HGDH, GHRL, TBL2, RRM2B, PRKACA, DLD, NEU1, ADSL, SLC22A5, ADCY10, INSR, HSD17B10, DGCR8, NPAP1, OXCT1, SDC3, HMGCL, PGAP1, MCCC2, LMF1, PIGM, UCP3, PAH, VPS33A, BCS1L, PDP1, AHCY, ALDH18A1, ENO3, MTTP, MAT1A, GNPTAB, PHGDH, BCAT2, CBS, HDAC4, LIG3, PSAT1, HGD, CTNND2, PDHB, PDHA1, NADK2, UPB1, PKLRBCKDK, MEN1, GALT, LIMK1, SLC39A4, KCNJ11, PDHX, ACAD8, GSS, CHRNA7, SLC6A9, ERBB3, GLUD1, GSR, OAT, SLC6 A8, CLIP2, STX1A, CARTPT, SLC25A15, DGCR2, LIPT2, NR5A1, DNM1L, PHEX, SLC30A9, B3GAT3, SLC34A3, SLC12A3, EPX, SARS2, CAPN10, ASNS, ALDOB, AGRP, MFF, GK, APOC2, CLDN3, HPRT1, PFKM, AMACR, SNRPN, HNF1B, L2HGDH, SO RD, IDH2, TPMT, CYP2C19, TERT, MC4R, TMPRSS15, SLCO1B3, FGF23, PAX4, SLC30A8, MTNR1B, SI, SLCO1B1 and NR0B2. ,

[0345] Reactive phosphorus group

[0346] The embodiments described herein include reactive phosphorus groups. Without wishing to be bound by theory, reactive phosphorus groups can be used to form nucleoside internucleotide bonds, including, for example, phosphate diester and thiophosphate internucleotide bonds. Such reactive phosphorus groups are known in the art and contain P III or P V Phosphorus atoms in various valence states include, but are not limited to, phosphorous amides, H-phosphonates, alkylphosphonates, triphosphates, and phosphorus-containing chiral auxiliaries. Phosphorous amides as active phosphites (P...) III The reactive phosphorus group (in chemistry) is a preferred reactive phosphorus group for solid-phase oligonucleotide synthesis. Subsequently, the intermediate phosphite compound is oxidized to P using known methods. V This state allows for the formation of phosphate diester or thiophosphate nucleoside bonds.

[0347] In some implementations, the reactive phosphorus group is -P(OR) P1 )N(R P2 )2、-P(SR P1 )N(R P2 )2、-P(O)(OR P1 )N(R P2 )2、-P(S)(OR P1 )N(R P2 )2、-P(R P3 )N(R P2 )2、-P(O)(SR P1 )N(R P2 )2、-P(O)(OR P1H、-P(S)(OR P1 )H、-P(O)(SR P1 H、-P(O)(OR) P1 )R P3 -P(S)(OR) P1 )R P3 or -P(O)(SR P1 )R P3 .

[0348] In some implementation schemes, R P1 C is an optional substitute 1-6 Alkyl group. For example, R P1 It is C 1-6 Alkyl group, optionally substituted with 1, 2, 3, 4 or 5 substituents, said substituents being 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6, optionally each R P3 Independently, it 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, halogen, SH, or C1-C6 alkoxy groups. In some embodiments, R P1 It is C 1-6 Alkyl groups, optionally substituted with CN or –SC(O)Ph. For example, R P1 It is cyanoethyl (-CH2CH2CN).

[0349] In some implementations, each R P2 Independently select the C that is replaced 1-6 Alkyl groups. For example, each R P2It 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 groups are present in the reactive phosphorus group... P2 When there are two or more R groups, they can be the same or different. Therefore, in some non-limiting examples, when there are two or more R groups... P2 When grouping, R P2 The groups are distinct. In some other unrestricted examples, when there are two or more R groups... P2 When grouping, R P2 The groups are identical. In some implementations, each R... P2 It is isopropyl.

[0350] In some implementation schemes, R P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3- to 8-membered heterocyclic group. Exemplary heterocyclic groups include, but are not limited to, pyrrolidinyl, piperazinyl, dioxolaneyl, morpholinyl, tetrahydrofuranyl, piperidinyl, 4-morpholinyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolidinyl, 1,4-diazaperhydroheptanyl, 1,3-dioxolaneyl and 1,4-dioxanecyclohexaneyl, each of which may optionally be substituted by 1, 2 or 3 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0351] In some implementation schemes, R P1 and R P2One of the atoms, together with the atom it is attached to, forms an optionally substituted 4-8 membered heterocyclic group. Exemplary heterocyclic groups include, but are not limited to, pyrrolidinyl, piperazinyl, dioxolaneyl, morpholinyl, tetrahydrofuranyl, piperidinyl, 4-morpholinyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolidinyl, 1,4-diazaperhydroheptanyl, 1,3-dioxolaneyl and 1,4-dioxanecyclohexaneyl, each of which may optionally be substituted by 1, 2 or 3 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0352] In the reactive phosphorus group, each R P3 C1-C independently of optional substitution 30 Alkyl-substituted C1-C 30 Alkyl, optionally substituted C2-C 30 alkenyl or optionally substituted C2-C 30 Alkyne groups (e.g., optionally substituted C1-C) 10 Alkyl, optionally substituted C2-C 10 alkenyl or optionally substituted C2-C 10 (Alkyne group). For example, R P3 It can be C 1-6Alkyl group, optionally substituted with 1, 2, 3, 4 or 5 substituents, said substituents being 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R P3 It 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, halogen, SH or C1-C6 alkoxy.

[0353] In some implementations, the reactive phosphorus group is -P(OR) P1 )(N(R P2 )2). For example, the reactive phosphorus group is -P(OR) P1 )(N(R P2 )2), where R P1 It is 2-cyanoethyl (-CH2CH2CN), each R P2 It is isopropyl.

[0354] Some exemplary aspects of this disclosure are described by one or more of the following numbered embodiments:

[0355] Implementation Scheme 1: An oligonucleotide comprising at least one nucleoside of formula (A): (Formula A) in: B is an optional modified base; X S It is O, CH2, S, or NH; R 22 It is a hydroxyl group, a hydroxyl protecting group, a halogen, or an optional substituted C. 1-30Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optional substituted C 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 Alkyne group, alkoxyalkylamine, alkoxyoxycarboxylic acid ester, amino group, alkylamino group, dialkylamino group, 5-8 membered heterocyclic group, -OC group 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 -OC 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 ), ligands, linkers covalently linked to one or more ligands, or nucleoside-to-nucleoside bonds to subsequent nucleosides; R 23 It is the nucleoside linking bond with the subsequent nucleoside, hydroxyl group, hydroxyl protecting group, halogen, optional substituted C. 2-30 alkynyl group, optionally substituted C 1-30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optional substituted C 1-30 Alkyl, optionally substituted C 2-30 Alkenyl, alkoxyalkylamine, alkoxyoxycarboxylic acid ester, amino, alkylamino, dialkylamino, 5-8 membered heterocyclic group, -OC 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 -OC 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 ), phosphate groups, ligands, or linkers covalently linked to one or more ligands; R 24 It is hydrogen, and the C is optionally substituted. 1-6 Alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl or optionally substituted C 1-6 Alkoxy; Or R 22 and R 24 Together they form 4'-C(R) 10 R 11 ) v -Y-2' or 4'-YC(R) 10 R 11 ) v -2'; Y represents -O-, -CH2-, -CH(Me)-, -C(CH3)2-, -S-, -N(R) 12)-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -OC(O)-, -C(O)O-, -N(R 12 C(O)- or -C(O)N(R) 12 )-; R 10 and R 11 Independently H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 ynyl; R 12 It is hydrogen, and the C is optionally substituted. 1-30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1-4 Halogenated alkyl groups, optionally substituted C 2-4 alkenyl, optionally substituted C 2-4 -Alynyl group, optionally substituted C1-C 30 Alkyl-CO2H or nitrogen-protected groups; v is 1, 2, or 3; and R 5 -L 5 -CH=CH-X P , Among them, L 5 It is a key or C 1-30 Alkylene (e.g., L) 5 It is a key, namely R 5 It is -CH=CH-X P ),as well as X P It is a phosphate group (e.g., a protected phosphate group). The prerequisite is R 22 and R 23 One of them is the bond between nucleotides that are subsequently linked to a nucleoside, and R 22 and R 23 Only one of them is a bond that connects to the nucleic acid of the subsequent nucleoside.

[0356] Implementation Scheme 2: The oligonucleotide of Implementation Scheme 1, wherein X S It can be O or CH2.

[0357] Implementation Scheme 3: Oligonucleotides from any one of Implementation Schemes 1-2, wherein X S It is O.

[0358] Implementation Scheme 4: Oligonucleotides from any one of Implementation Schemes 1-3, wherein R 23 It is a bond that links nucleotides to subsequent nucleosides.

[0359] Implementation Scheme 5: The oligonucleotide of Implementation Scheme 4, wherein R 22It is a hydroxyl group, a hydroxyl protecting group, a halogen, or an optional substituted C. 1-30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optional substituted C 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 Alkyne group, alkoxyalkylamine, alkoxyoxycarboxylic acid ester, amino group, alkylamino group, dialkylamino group, 5-8 membered heterocyclic group, -OC group 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 -OC 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 ); or R 24 and R 25 Together they form 4'-C(R) 10 R 11 ) v -Y-2' or 4'-YC(R) 10 R 11 ) v -2'.

[0360] Implementation Scheme 6: An oligonucleotide from any one of Implementation Schemes 4-5, wherein R 22 It is a hydroxyl group, a hydroxyl protecting group, a halogen, or an optional substituted C. 1-30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, amino, alkylamino, or dialkylamino; or R 22 and R 24 Together they form 4'-C(R) 10 R 11 ) v -Y-2' or 4'-YC(R) 10 R 11 ) v -2'.

[0361] Implementation Scheme 7: Oligonucleotides from any one of Implementation Schemes 4-6, wherein R 22 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine, methoxy, ethoxy, or 2-methoxyethoxy; or R 22 and R 24 Together they form 4'-C(R) 10 R 11 ) v -Y-2' or 4'-YC(R) 10 R 11 ) v -2'.

[0362] Implementation Scheme 8: Oligonucleotides from any one of Implementation Schemes 4-7. Wherein R 22 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy.

[0363] Implementation Scheme 9: Oligonucleotides from any one of Implementation Schemes 4-7, wherein R 22 and R 24 Together they form 4'-C(R) 10 R 11 ) v -Y-2' or 4'-YC(R) 10 R 11 ) v -2'.

[0364] Implementation Scheme 10: The oligonucleotide of Implementation Scheme 9, wherein R 2 and R 4 Together they form 4'-C(R) 10 R 11 ) v -Y-2', where v is 1 or 2.

[0365] Implementation Scheme 11: Oligonucleotides of Implementation Scheme 9 or 10, wherein R 10 and R 11 One of them is H, and the other is H or an optional substituted C1-C6 alkyl group.

[0366] Implementation Scheme 12: The oligonucleotide of Implementation Scheme 11, wherein R 22 and R 24 Together they form 4'-CH2-O-2'.

[0367] Implementation Scheme 13: Oligonucleotides from any one of Implementation Schemes 4-8, wherein R 24 It is H.

[0368] Implementation Scheme 14: Oligonucleotides from any one of Implementation Schemes 1-3, wherein R 22 It is a bond that links nucleotides to subsequent nucleosides.

[0369] Implementation Scheme 15: The oligonucleotide of Implementation Scheme 14, wherein R 23 It is a hydroxyl group, a hydroxyl protecting group, a halogen, or an optional substituted C. 1-30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optional substituted C 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 Alkyne group, alkoxyalkylamine, alkoxyoxycarboxylic acid ester, amino group, alkylamino group, dialkylamino group, 5-8 membered heterocyclic group, -OC group 4-30 Alkyl-ON(CH2R)8 (CH2R) 9 ) or -OC 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 ).

[0370] Implementation Scheme 16: An oligonucleotide from any one of Implementation Schemes 14-15, wherein R 23 It is a hydroxyl group, a hydroxyl protecting group, a halogen, or an optional substituted C. 1-30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, amino, alkylamino or dialkylamino.

[0371] Implementation Scheme 17: An oligonucleotide from any one of Implementation Schemes 14-16, wherein R 23 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine, methoxy, ethoxy, or 2-methoxyethoxy; or R 2 and R 4 Together they form 4'-C(R) 10 R 11 ) v -Y-2' or 4'-YC(R) 10 R 11 ) v -2'.

[0372] Implementation Scheme 18: An oligonucleotide from any one of Implementation Schemes 14-17, wherein R 23 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy.

[0373] Implementation Scheme 19: An oligonucleotide of any one of Implementation Schemes 14-18, wherein R 24 It is H.

[0374] Implementation Scheme 20: An oligonucleotide of any one of Implementation Schemes 1-18, wherein the nucleotide of formula (A) is of formula (A-VP) or (A-VP'): (Formula A-VP) or (Formula A-VP').

[0375] Implementation Scheme 21: The oligonucleotide of Implementation Scheme 20, wherein R 23 It is a bond that links nucleotides to subsequent nucleosides.

[0376] Implementation Scheme 22: The oligonucleotide of Implementation Scheme 21, wherein R 22 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy.

[0377] Implementation Scheme 23: An oligonucleotide from any one of Implementation Schemes 20-22, wherein X P -P(O)(OR)V )2, where each R V It is an independent H or oxygen protecting group.

[0378] Implementation Scheme 24: Oligonucleotides of Implementation Scheme 23, wherein each R V H stands for H independently.

[0379] Implementation Scheme 25: The oligonucleotide of Implementation Scheme 20, wherein X P It is -P(O)(OR) V )2, R 23 It is the bond between nucleotides that connects to subsequent nucleotides, R 22 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy, and each of the R groups is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy. V It is independently a H or oxygen protecting group.

[0380] Implementation Scheme 26: Oligonucleotides of Implementation Scheme 25, wherein each R V H stands for H independently.

[0381] Implementation Scheme 27: Oligonucleotides of Implementation Scheme 20, wherein R 22 It is a nucleotide-to-nucleotide bond that links to subsequent nucleotides.

[0382] Implementation Scheme 28: The oligonucleotide of Implementation Scheme 27, wherein R 23 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy.

[0383] Implementation Scheme 29: Oligonucleotides of Implementation Scheme 27 or 28, wherein X P -P(O)(OR) V )2, where each R V It is an independent H or oxygen protecting group.

[0384] Implementation Scheme 30: Oligonucleotides of Implementation Scheme 29, wherein each R V H stands for H independently.

[0385] Implementation Scheme 31: Oligonucleotides of Implementation Scheme 20, wherein X P It is -P(O)(OR) V )2, R 22 It is the bond between nucleotides that connects to subsequent nucleotides, R 23 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy, and each of the R groups is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy. V It is independently a H or oxygen protecting group.

[0386] Implementation Scheme 32: Oligonucleotides of Implementation Scheme 31, wherein each R V H stands for H independently.

[0387] Implementation Scheme 33: An oligonucleotide of any one of Implementation Schemes 1-32, wherein the oligonucleotide comprises 3 to 50 nucleotides.

[0388] Implementation Scheme 34: An oligonucleotide of any one of Implementation Schemes 1-33, wherein the oligonucleotide comprises at least one ribonucleotide.

[0389] Implementation Scheme 35: An oligonucleotide of any one of Implementation Schemes 1-34, wherein the oligonucleotide comprises at least one 2'-deoxyribonucleotide.

[0390] Implementation Scheme 36: An oligonucleotide of any one of Implementation Schemes 1-35, wherein the oligonucleotide comprises, in addition to the nucleoside of Formula (A), at least one nucleoside having a modified or non-natural base.

[0391] Implementation Scheme 37: An oligonucleotide of any one of Implementation Schemes 1-36, wherein the oligonucleotide comprises, in addition to the nucleoside of Formula (A), at least one nucleoside having a modified ribose.

[0392] Implementation Scheme 38: An oligonucleotide of any one of Implementation Schemes 1-37, wherein the oligonucleotide, in addition to the nucleoside of Formula (A), comprises at least one nucleoside containing a group other than H or OH at the 2'-position of the ribose.

[0393] Implementation Scheme 39: An oligonucleotide of any one of Implementation Schemes 1-38, wherein the oligonucleotide comprises, in addition to the nucleoside of Formula (A), at least one nucleoside having a 2'-F ribose.

[0394] Implementation Scheme 40: An oligonucleotide of any one of Implementation Schemes 1-39, wherein the oligonucleotide comprises, in addition to the nucleoside of Formula (A), at least one nucleoside having a 2'-OMe ribose.

[0395] Implementation Scheme 41: An oligonucleotide of any one of Implementation Schemes 1-40, wherein the oligonucleotide comprises, in addition to the nucleoside of Formula (A), at least one nucleoside containing a group other than ribose.

[0396] Implementation Scheme 42: An oligonucleotide of any one of Implementation Schemes 1-41, wherein the oligonucleotide comprises at least one modified nucleoside linker.

[0397] Implementation Scheme 43: An oligonucleotide of any one of Implementation Schemes 1-42, wherein the nucleotide linking bond with the subsequent nucleotide is a modified nucleotide linking bond.

[0398] Implementation Scheme 44: The oligonucleotide of Implementation Scheme 43, wherein the modified nucleotide linking bond is a phosphate thioester linking bond.

[0399] Implementation Scheme 45: An oligonucleotide of any one of Implementation Schemes 1-44, wherein the oligonucleotide is attached to a solid support.

[0400] Implementation Scheme 46: An oligonucleotide of any one of Implementation Schemes 1-45, wherein the oligonucleotide contains at least one ligand.

[0401] Implementation Scheme 47: An oligonucleotide of any one of Implementation Schemes 1-46, wherein the oligonucleotide comprises at least one hydroxyl, phosphate, or amino protecting group.

[0402] Implementation Scheme 48: A double-stranded nucleic acid comprising a first oligonucleotide chain and a second oligonucleotide chain substantially complementary to the first chain, wherein the first or second chain is an oligonucleotide of any one of Implementation Schemes 1-47.

[0403] Implementation Scheme 49: The double-stranded nucleic acid of Implementation Scheme 48, wherein one of the first and second strands is an oligonucleotide of any one of Implementation Schemes 1-47.

[0404] Implementation scheme 50: The double-stranded nucleic acid of implementation scheme 48 or 50, wherein the first and second strands are independently 15 to 25 nucleotides in length.

[0405] Implementation scheme 51: A double-stranded nucleic acid from any of Implementation schemes 48-50, wherein the double-stranded nucleic acid is capable of inducing RNA interference.

[0406] Implementation Scheme 52: The double-stranded nucleic acid of Implementation Scheme 51, wherein the double-stranded nucleic acid comprises an antisense strand and a sense strand, and wherein the antisense strand is an oligonucleotide of any one of Implementation Schemes 1-47.

[0407] Implementation Scheme 53: A double-stranded nucleic acid of any one of Implementation Schemes 48-52, wherein one or both strands have 1-5 nucleotide sticky ends at their respective 5' or 3' ends.

[0408] Implementation scheme 54: A double-stranded nucleic acid of any one of implementation schemes 48-53, wherein only one strand has a sticky end of 2 nucleotides at its 5' or 3' end.

[0409] Implementation scheme 55: A double-stranded nucleic acid of any one of implementation schemes 48-54, wherein only one strand has a sticky end of 2 nucleotides at its 3' end.

[0410] Implementation Scheme 56: A pharmaceutical composition comprising an oligonucleotide of any one of Implementation Schemes 1-47, alone or in combination with a pharmaceutically acceptable carrier or excipient, or a dsRNA molecule of any one of Implementation Schemes 48-55.

[0411] Implementation Scheme 57: A gene silencing kit comprising an oligonucleotide of any one of Implementation Schemes 1-47 or a dsRNA molecule of any one of Implementation Schemes 48-55.

[0412] Implementation Scheme 58: A method for silencing a target gene in cells, the method comprising the step of introducing the following substance into the cell: (i) a double-stranded RNA according to any one of Implementation Schemes 48-55, wherein the antisense strand comprises a nucleotide sequence substantially complementary to the target gene; or (ii) an oligonucleotide according to any one of Implementation Schemes 1-47, wherein the oligonucleotide comprises a nucleotide sequence substantially complementary to the target gene.

[0413] Implementation Scheme 59: A method for reducing the expression of a target gene in a subject, comprising administering to the subject: (i) a double-stranded RNA according to any one of Implementation Schemes 48-55, wherein the antisense strand comprises a nucleotide sequence substantially complementary to the target gene; or (ii) an oligonucleotide according to any one of Implementation Schemes 1-47, wherein the oligonucleotide comprises a nucleotide sequence substantially complementary to the target gene.

[0414] Implementation Scheme 60: The method of Implementation Scheme 59, wherein the administration is subcutaneous or intravenous.

[0415] Implementation Scheme 61: Compound of Formula (B): (Formula B) in: B is an optional modified base; X S It is O, CH2, S, or NH; R 2 It is a hydroxyl group, a hydroxyl protecting group, a halogen, or an optional substituted C. 1-30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optional substituted C 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 Alkyne group, alkoxyalkylamine, alkoxyoxycarboxylic acid ester, amino group, alkylamino group, dialkylamino group, 5-8 membered heterocyclic group, -OC group 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 -OC 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 ), phosphate groups, ligands, or linkers covalently linked to one or more ligands; R 3 It is a reactive phosphorus group, hydroxyl group, hydroxyl protecting group, halogen, or optionally substituted C.2-30 alkynyl group, optionally substituted C 1-30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optional substituted C 1-30 Alkyl, optionally substituted C 2-30 Alkenyl, alkoxyalkylamine, alkoxyoxycarboxylic acid ester, amino, alkylamino, dialkylamino, 5-8 membered heterocyclic group, -OC 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 -OC 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 ), phosphate groups, ligands, or linkers covalently linked to one or more ligands; R 4 It is hydrogen, and the C is optionally substituted. 1-6 Alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl or optionally substituted C 1-6 Alkoxy; Or R 4 and R 2 Together they form 4'-C(R) 10 R 11 ) v -Y-2' or 4'-YC(R) 10 R 11 ) v -2'; Y represents -O-, -CH2-, -CH(Me)-, -C(CH3)2-, -S-, -N(R) 12 )-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -OC(O)-, -C(O)O-, -N(R 12 C(O)- or -C(O)N(R) 12 )-; R 10 and R 11 Independently H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 ynyl; R 12 It is hydrogen, and the C is optionally substituted. 1-30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1-4 Halogenated alkyl groups, optionally substituted C 2-4 alkenyl, optionally substituted C 2-4 -Alynyl group, optionally substituted C1-C 30 Alkyl-CO2H or nitrogen-protected groups; v is 1, 2, or 3; and R 5 -L 5 -CH=CH-X P , Among them, L 5 It is a key or C 1-30 Alkylene (e.g., L) 5 It is a key, namely R 5 It is -CH=CH-X P ),as well as X P It is a phosphate group (e.g., a protected phosphate group). The prerequisite is that when R 2 and R 3 When one of them is a reactive phosphorus group, R 2 and R 3 Only one of them is a reactive phosphorus group.

[0416] Implementation Scheme 62: A compound of any one of Implementation Scheme 61, wherein X S It can be O or CH2.

[0417] Implementation Scheme 63: A compound of any one of Implementation Schemes 61-62, wherein X S It is O.

[0418] Implementation Scheme 64: A compound of any one of Implementation Schemes 61-63, wherein R 3 It is a reactive phosphorus group, hydroxyl group, or hydroxyl protecting group.

[0419] Implementation Scheme 65: The compound of Implementation Scheme 64, wherein R 3 It is a reactive phosphorus group.

[0420] Implementation Scheme 66: A compound of any one of Implementation Schemes 64-65, wherein R 2 It is a hydroxyl group, a hydroxyl protecting group, a halogen, or an optional substituted C. 1-30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optional substituted C 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 Alkyne group, alkoxyalkylamine, alkoxyoxycarboxylic acid ester, amino group, alkylamino group, dialkylamino group, 5-8 membered heterocyclic group, -OC group 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 -OC 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9); or R 24 and R 25 Together they form 4'-C(R) 10 R 11 ) v -Y-2' or 4'-YC(R) 10 R 11 ) v -2'.

[0421] Implementation Scheme 67: A compound of any one of Implementation Schemes 64-66, wherein R 2 It is a hydroxyl group, a hydroxyl protecting group, a halogen, or an optional substituted C. 1-30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, amino, alkylamino, or dialkylamino; or R 2 and R 4 Together they form 4'-C(R) 10 R 11 ) v -Y-2' or 4'-YC(R) 10 R 11 ) v -2'.

[0422] Implementation Scheme 68: A compound of any one of Implementation Schemes 64-67, wherein R 2 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine, methoxy, ethoxy, or 2-methoxyethoxy; or R 2 and R 4 Together they form 4'-C(R) 10 R 11 ) v -Y-2' or 4'-YC(R) 10 R 11 ) v -2'.

[0423] Implementation Scheme 69: A compound of any one of Implementation Schemes 64-68, wherein R 2 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy.

[0424] Implementation Scheme 70: A compound of any one of Implementation Schemes 64-69, wherein R 2 and R 4 Together they form 4'-C(R) 10 R 11 ) v -Y-2' or 4'-YC(R) 10 R 11 ) v -2'.

[0425] Implementation Scheme 71: The compound of Implementation Scheme 70, wherein R 2 and R 4 Together they form 4'-C(R) 10 R 11 ) v -Y-2', where v is 1 or 2.

[0426] Implementation Scheme 72: The compound of Implementation Scheme 70 or 71, wherein R 10 and R 11 One of them is H, and the other is H or an optional substituted C1-C6 alkyl group.

[0427] Implementation Scheme 73: The compound of Implementation Scheme 72, wherein R 2 and R 4 Together they form 4'-CH2-O-2'.

[0428] Implementation Scheme 74: A compound of any one of Implementation Schemes 64-69, wherein R 4 It is H.

[0429] Implementation Scheme 75: A compound of any one of Implementation Schemes 61-63, wherein R 2 It is a reactive phosphorus group, hydroxyl group, or hydroxyl protecting group.

[0430] Implementation Scheme 76: The compound of Implementation Scheme 75, wherein R 2 It is a reactive phosphorus group.

[0431] Implementation Scheme 77: A compound of any one of Implementation Schemes 75-76, wherein R 3 It is a hydroxyl group, a hydroxyl protecting group, a halogen, or an optional substituted C. 1-30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optional substituted C 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 Alkyne group, alkoxyalkylamine, alkoxyoxycarboxylic acid ester, amino group, alkylamino group, dialkylamino group, 5-8 membered heterocyclic group, -OC group 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 ) or -OC 4-30 Alkyl-ON(CH2R) 8 (CH2R) 9 ).

[0432] Implementation Scheme 78: A compound of any one of Implementation Schemes 75-77, wherein R 3 It is a hydroxyl group, a hydroxyl protecting group, a halogen, or an optional substituted C. 1-30Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, amino, alkylamino or dialkylamino.

[0433] Implementation Scheme 79: A compound of any one of Implementation Schemes 75-78, wherein R 3 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine, methoxy, ethoxy, or 2-methoxyethoxy; or R 2 and R 4 Together they form 4'-C(R) 10 R 11 ) v -Y-2' or 4'-YC(R) 10 R 11 ) v -2'.

[0434] Implementation Scheme 80: A compound of any one of Implementation Schemes 75-79, wherein R 3 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy.

[0435] Implementation Scheme 81: A compound of any one of Implementation Schemes 75-80, wherein R 4 It is H.

[0436] Implementation Scheme 82: The compound of Implementation Scheme 61, wherein the compound is of formula (B-VP) or (B-VP'): (Formula B-VP) or (Formula B-VP').

[0437] Implementation Scheme 83: The compound of Implementation Scheme 82, wherein R 3 It is a reactive phosphorus group (e.g., phosphoramide, such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide or 3'-[(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramide).

[0438] Implementation Scheme 84: The compound of Implementation Scheme 83, wherein R 2 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy.

[0439] Implementation Scheme 85: A compound of any one of Implementation Schemes 82-84, wherein X P -P(O)(OR) V )2, where each R V It is an independent H or oxygen protecting group.

[0440] Implementation Scheme 86: Compounds of Implementation Scheme 85, wherein each R V H stands for H independently.

[0441] Implementation Scheme 87: The compound of Implementation Scheme 82, wherein XP is X P It is -P(O)(OR) V )2;R 3 It is a reactive phosphorus group (e.g., phosphoramide, such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide or 3'-[(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramide); R 2 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy.

[0442] Implementation Scheme 88: Compounds of Implementation Scheme 87, wherein each R V It is an oxygen protecting group independently.

[0443] Implementation Scheme 89: The compound of Implementation Scheme 82, wherein R 2 It is a reactive phosphorus group (e.g., phosphoramide, such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide or 3'-[(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramide).

[0444] Implementation Scheme 90: The compound of Implementation Scheme 89, wherein R 3 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy.

[0445] Implementation Scheme 91: The compound of Implementation Scheme 89 or 90, wherein X P -P(O)(OR) V )2, where each R V It is an independent H or oxygen protecting group.

[0446] Implementation Scheme 92: The compounds of Implementation Scheme 91, wherein each R V H stands for H independently.

[0447] Implementation Scheme 93: The compound of Implementation Scheme 82, wherein X P It is -P(O)(OR) V )2;R 2 It is a reactive phosphorus group (e.g., phosphoramide, such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide or 3'-[(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramide); R 3 It is hydrogen, hydroxyl, hydroxyl protecting group, fluorine or methoxy.

[0448] Implementation Scheme 94: Compounds of Implementation Scheme 93, wherein each R V It is an oxygen protecting group independently.

[0449] Implementation Scheme 95: The compound of Implementation Scheme 1, wherein the compound is , , , , or .

[0450] In some nucleotides of formula (A) or compounds of formula (B), R 5 -CH=CH-X P It is worth noting that when R 5 -CH=CH-X P At this time, the double bond can be in either a cis or trans configuration. Therefore, in some embodiments of any aspect described herein, R 5 -CH=CH-X P The double bond is in cis configuration. In some other embodiments of any aspect described herein, R 5 -CH=CH-X P The double bond is in cis configuration.

[0451] Hydroxyl protecting group

[0452] Some embodiments of the various aspects described herein include hydroxyl protecting groups. Hydroxyl protecting groups include, but are not limited to, -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、-CH2OC(=O)R OP1 -S(=O)R OP1 -SO2R OP1 、-Si(R OP1 )3、-P(R OP3 )2、-P(R OP3 ) + 3X - -P(OR) OP3 2. -P(OR) OP3 )3 X- -P(=O)(R OP1 )2、-P(=O)(OR OP3 )2 and -P(=O)(N(R OP2 )2)2; where each X - It is a counterion; each R OP1 Independently for C 1-10 Alkyl, C 1-10 All-halogenated alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 Heteroalkyl, C 2-10 Heterene, C 2-10 Neyne group, C 3-10 Carbocyclic groups, 3-14 membered heterocyclic groups, C 6-14 aryl or 5-14 heteroaryl, or two R OP1 Group linkages form 3-14 membered reactive heteroaryl or 5-14 membered heteroaryl rings; each R OP2 It 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 All-halogenated alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 Heteroalkyl, C 2-10 Heterene, C 2-10 Neyne group, C 3-10 Carbocyclic groups, 3-14 membered heterocyclic groups, C 6-14 aryl or 5-14 heteroaryl, or two R OP2Groups are linked to form 3-14 membered heterocyclic groups or 5-14 membered heteroaryl rings; and each R OP3 Independently hydrogen, C 1-10 Alkyl, C 1-10 All-halogenated alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 Heteroalkyl, C 2-10 Heterene, C 2-10 Neyne group, C 3-10 Carbocyclic groups, 3-14 membered heterocyclic groups, C 6-14 aryl or 5-14 heteroaryl, or two R OP3 Groups are linked to form 3-14 membered heterocyclic groups or 5-14 membered heteroaryl rings; and R OP1 R OP2 and R OP3 Each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aralkyl, aryl, and heteroaryl group may optionally be substituted by 1, 2, 3, 4, or 5 substituents, said substituents being 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0453] Hydroxyl protecting groups are well known in the art, including Greene's *Protecting Groups in Organic Synthesis*, PGM Wuts, 5 th The hydroxyl protecting groups described in detail in Edition, John Wiley & Sons, 2014, are incorporated herein by reference.

[0454] Exemplary hydroxyl protecting groups include, but are not limited to, methyl, tert-butoxycarbonyl (BOC or Boc), methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyl dimethylsilyl)methoxymethyl ester (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacol methyl (GUM), tert-butoxymethyl, neopentyloxymethyl (POM), acetoxymethyl (AM), silyloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, and 2-(trimethylsilyl) Ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiaranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiaranyl, 4-methoxytetrahydrothiaranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methylenebenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methyl oxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenoyl)ethyl, tert-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-pyridinemethyl, 4-pyridinemethyl, 3-methyl-2-pyridinemethyl N-oxide, diphenylmethyl, p,p′-dinitrodiphenylmethyl, 5-dibenzo[a]benzoyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methyl 4-(4′-bromophenylacetoxyphenyl)diphenylmethyl, di(p-methoxyphenyl)benzyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenylacetoxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidephenyl)methyl, 4,4′,4″-tris(pentanoyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)di(4′,4″-dimethoxyphenyl)methyl, 1,1-di(4-methoxyphenyl)-1′-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthrayl, 1,3-benzodithiacyclopenten-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylhexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl, methoxyacetyl, triphenylmethoxyacetyl, phenoxyacetyl, p-chloro Phenoxyacetyl, 3-phenylpropionyl, 4-oxopentanoyl (pentanoyl), 4,4-(ethylenedithio)pentanoate (pentanoyl dithioacetal), adamantyl ester, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (trimethylbenzene ester), alkylmethyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkylethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonium)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-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoic acid Esters, 2,6-dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid ester, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid ester, chlorodiphenylacetic acid ester, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxyyl)benzoate, α-naphthylcarbamate, nitrates, alkyl N,N,N',N'-tetramethyldiamide phosphate, alkyl N-phenylcarbamate, borates, dimethyl thiophosphonoyl, alkyl 2,4-dinitrophenyl sulfite, sulfates, methanesulfonates, benzyl sulfonates, and p-toluenesulfonates (Ts).

[0455] In some embodiments, the hydroxyl protecting group is benzyl, benzoyl, 2,6-dichlorobenzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, methanesulfonate, toluenesulfonate, 4,4′-dimethoxytriphenylmethyl (DMT), 9-phenylxanth-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanth-9-yl (MOX). In some embodiments, the hydroxyl protecting group is selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and dimethoxytriphenylmethyl, with 4,4′-dimethoxytriphenylmethyl being a more preferred hydroxyl protecting group. In some embodiments, the hydroxyl protecting group is neopentyloxymethyl.

[0456] The terms "hydroxyl protecting group" and "protected hydroxyl group" used in this article refer to the formula -OR Pro The group, wherein R Pro It is the oxygen protecting group defined in this article.

[0457] Amine protecting group

[0458] Some embodiments of the various aspects described herein include amine protecting groups (also referred to herein as amino protecting groups). Amine protecting groups include, but are not limited to, -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 (e.g., aralkyl, heteroaralkyl), C 2-10 alkenyl, C 2-10 alkynyl group, C 3-10 Carbocyclic groups, 3-14 membered heterocyclic groups, C 6-14 aryl or 5-14 heteroaryl, wherein each R NP1 Independently for C 1-10 Alkyl, C 1-10 All-halogenated alkyl, C2-10 alkenyl, C 2-10 alkynyl group, C 1-10 Heteroalkyl, C 2-10 Heterene, C 2-10 Neyne group, C 3-10 Carbocyclic groups, 3-14 membered heterocyclic groups, C 6-14 aryl or 5-14 heteroaryl; and each R NP2 Independently hydrogen, C 1-10 Alkyl, C 1-10 All-halogenated alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 Heteroalkyl, C 2-10 Heterene, C 2-10 Neyne group, C 3-10 Carbocyclic groups, 3-14 membered heterocyclic groups, C 6-14 aryl or 5-14 heteroaryl, or two R SP3 Groups are linked to form 3-14 membered heterocyclic groups or 5-14 membered heteroaryl rings; and R NP1 and R NP2 Each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aralkyl, aryl, and heteroaryl group may optionally be substituted by 1, 2, 3, 4, or 5 substituents, said substituents being 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0459] Amine protecting groups are well known in the art, including Greene's *Protecting Groups in Organic Synthesis*, PGM Wuts, 5th The amine protecting groups described in detail in Edition, John Wiley & Sons, 2014, are incorporated herein by reference.

[0460] Exemplary amide-based (e.g., -C(=O)R) NP1 The amine protecting groups of (e.g., formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, pyridinecarboxamide, 3-pyridinecarboxamide, N-benzoylphenylpropionyl derivatives, benzamide, p-benzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetylacetamide, (N′-dithiobenzyloxyacetamide)acetamide, 3-(p-hydroxyphenyl)propionamide, 3-(o-nitrophenyl)propionamide, 2-methyl-2-(o-nitrophenoxy)propionamide, 2-methyl-2-(o-phenylazophenoxy)propionamide, 4-chlorobutyramide, 3-methyl-3-nitrobutyramide, o-nitrocinnamamide, N-acetylated methionine derivatives, o-nitrobenzamide, and o-(benzyloxymethyl)benzamide.

[0461] Exemplary carbamate-based (e.g., -C(=O)OR) NP1The amine protecting groups of ) include, but are not limited to, methyl carbamates, ethyl carbamates, 9-fluorenemethyl carbamate (Fmoc), 9-(2-sulfonic acid)fluorenemethyl carbamate, 9-(2,7-dibromo)fluorenemethyl carbamate, 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothiopheneyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenylacetyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), and 1-(1-adamantyl)-1-methylethyl carbamate. (Adpoc), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethylcarbamate (TCBOC), 1-methyl-1-(4-biphenyl)ethylcarbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethylcarbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethylcarbamate (Pyoc), 2-(N,N-dicyclohexylcarbamate)ethylcarbamate, tert-butylcarbamate (BOC or Boc), 1-adamantylcarbamate (Adoc), vinylamino Formate (Voc), Allyl Carbamate (Alloc), 1-Isopropyl Allyl Carbamate (Ipaoc), Cinnamyl Carbamate (Coc), 4-Nitrocinnamyl Carbamate (Noc), 8-Quinolinyl Carbamate, N-Hydroxypiperidinyl Carbamate, Alkyl Dithiocarbamate, Benzyl Carbamate (Cbz), p-Methoxybenzyl Carbamate (Moz), p-Nitrobenzyl Carbamate, p-Bromobenzyl Carbamate, p-Chlorobenzyl Carbamate, 2,4-Dichlorobenzyl Carbamate, 4-Methylsulfonylmethanebenzyl Carbamate (Msz), 9-Anthracenemethyl Carbamate, Diphenylmethyl Carbamate, 2-Methylthioethyl Carbamate, 2-Methylsulfonyl 2-(p-Toluenesulfonyl)ethylcarbamate, [2-(1,3-dithiaalkyl)]methylcarbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthiophenylcarbamate (Bmpc), 2-phosphorylethylcarbamate (Peoc), 2-triphenylphosphorylisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethylcarbamate, m-chloro-p-acyloxybenzylcarbamate, p-(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chromonelmethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3,5-Dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl)methyl carbamate, tert-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decoxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethoxycarbonylamino)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethoxycarbonylamino)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanmethyl carbamate, 2-iodobenzyl Ethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicoyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-tert-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

[0462] Exemplary sulfonamide-based (e.g., -S(=O)2R) NP1 The amine protecting group of the amine includes, but is not limited to, 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-trimethylsilyl ethyl sulfonamide (SES), 9-anthracitesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenylacetylsulfonamide.

[0463] Other exemplary amine protecting groups include, but are not limited to, phenthiazinyl-(10)-acyl derivatives, N′-p-toluenesulfonamide acyl derivatives, N′-phenylaminothioacyl derivatives, N-benzoylphenylalanine derivatives, N-acetylated methionine derivatives, 4,5-diphenyl-3-oxazoline-2-one, N-phthalimide, N-dithiosuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethylsilylazacyclopentane adduct (STABASE), and 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexane. Alkyl-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexane-2-one, 1-substituted 3,5-dinitro-4-pyridinone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrololin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzopyridine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluoreneamine (PhF) N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenemethylamino (Fcm), N-2-pyridinemethylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylamine, N-p-methoxybenzylamine, N-diphenylmethyleneamine, N-[(2-pyridyl)trimethylmethyl]methyleneamine, N-(N′,N′-diaminomethylene)amine, N,N′-isopropylidenediamine, N-p-nitrobenzylamine, N-salicylaldehyde imine, N-5-chlorosalicylaldehyde imine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylimine, N-(5,5-dimethyl-3- Oxo-1-cyclohexenyl)amine, N-borane and N-diphenylboronic acid derivatives, N-[phenyl(pentylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitrosamine, N-nitrosamine, amine N-oxide, diphenylphosphamide (Dpp), dimethylthiophosphamide (Mpt), diphenylthiophosphamide (Ppt), dialkylphosphamide ester, dibenzylphosphamide ester, diphenylphosphamide ester, benzenesulfonamide, o-nitrobenzenesulfonamide (Nps), 2,4-dinitrobenzenesulfonamide, pentachlorobenzenesulfonamide, 2-nitro-4-methoxybenzenesulfonamide, triphenylmethanesulfonamide and 3-nitropyridinesulfonamide (Npys).

[0464] Thiol protecting group

[0465] Some embodiments of the various aspects described herein include thiol protecting groups. Thiol protecting groups include, but are not limited to, -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, where: X- is the counter ion; each R SP1 Independently for C 1-10 Alkyl, C 1-10 All-halogenated alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 Heteroalkyl, C 2-10 Heterene, C 2-10 Neyne group, C 3-10 Carbocyclic groups, 3-14 membered heterocyclic groups, C 6-14 aryl or 5-14 heteroaryl, or two R SP1 Group linkages form 3-14 membered heterocycles or 5-14 membered heteroaryl rings; each R SP2 They are all 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 -SO2ORSP3 -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 All-halogenated alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 Heteroalkyl, C 2-10 Heterene, C 2-10 Neyne group, C 3-10 Carbocyclic groups, 3-14 membered heterocyclic groups, C 6-14 aryl or 5-14 heteroaryl, or two R SP2 Groups are linked to form 3-14 membered heterocyclic groups or 5-14 membered heteroaryl rings; and each R SP3 Independently hydrogen, C 1-10 Alkyl, C 1-10 All-halogenated alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 Heteroalkyl, C 2-10 Heterene, C 2-10 Neyne group, C 3-10 Carbocyclic groups, 3-14 membered heterocyclic groups, C 6-14 aryl or 5-14 heteroaryl, or two R SP3 Groups are linked to form 3-14 membered heterocyclic groups or 5-14 membered heteroaryl rings; and R SP1 R SP2 and R SP3Each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aralkyl, aryl, and heteroaryl group may optionally be substituted by 1, 2, 3, 4, or 5 substituents, said substituents being 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, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylamino, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p -NH2 or CH2-arylalkoxy, wherein "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0466] Sulfur protecting groups are well known in the art, including Greene's Protecting Groups in Organic Synthesis, PGM Wuts, 5 th The sulfur protecting groups described in detail in Edition, John Wiley & Sons, 2014, are incorporated herein by reference.

[0467] Definitions

[0468] For convenience, certain terms used herein, in the specification, embodiments, and appended claims are summarized. Unless otherwise stated or implied from the context, the following terms and phrases have the following meanings. Unless otherwise expressly stated or obvious from the context, the following terms and phrases do not exclude the meaning that a term or phrase has in the art to which it pertains. These definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Furthermore, unless the context requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms.

[0469] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention pertains. Although any known methods, apparatus, and materials may be used in the practice or testing of this invention, methods, apparatus, and materials in this regard are described herein. Definitions of commonly used terms in immunology and molecular biology can be found in: The Merck Manual of Diagnosis and Therapy, 20th Edition, 2018, Merck Sharp & Dohme Corp. (ISBN 0911910190, 978-0911910421); The Encyclopedia of Molecular Cell Biology and Molecular Medicine, edited by Robert S. Porter et al., 1999–2012, Blackwell Science Ltd. (ISBN 9783527600908); and Molecular Biology and Biotechnology: a Comprehensive Desk Reference, edited by Robert A. Meyers, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), WW Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, Jones & Bartlett, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (editor), Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (Editor), John Wiley and Sons, 2014 (ISBN 047150338X,9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (Editor), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 003 (ISBN 0471142735, 9780471142737), the entire contents of which are incorporated herein by reference.

[0470] Furthermore, unless otherwise stated, the present invention can be practiced using conventional techniques within the scope of the art, including those in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology. These techniques are well explained in the literature, such as *Molecular Cloning: A Laboratory Manual, 2nd Edition* (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by MJ Gait, 1984); *Animal Cell Culture* (edited by RI Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Current Protocols in Molecular Biology* (edited by FM Ausubel et al., 1987 and regularly updated); *PCR: The Polymerase Chain Reaction* (edited by Mullis et al., 1994); *A Practical Guide to Molecular Cloning* (Perbal Bernard V.., 1988); *Phage Display: A Laboratory Manual* (Barbas et al., 2001).

[0471] If a range of values ​​is provided, it is understood that, unless the context explicitly specifies otherwise, every intermediate value between the upper and lower limits of the range and any other stated or intermediate value within that range, up to one-tenth of the lower limit unit, is included in this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also within this invention, but are subject to any specific exclusions from the range. Where the range includes one or two limits, the range excluding one or both of those included limits is also included in this invention.

[0472] Unless otherwise stated in the operational examples or elsewhere, all figures used herein to indicate quantities of ingredients or reaction conditions should be understood to be modified by the word “about” in all cases. When used in conjunction with percentages, “about” can mean ±1%. In some embodiments of the various aspects described herein, when used in conjunction with percentages, the term “about” can mean ±5%. The term “about” is used herein to provide textual support for the exact number preceding it and for numbers that are close to or approximate to the number preceding it. In determining whether a number is close to or approximate to a specifically cited number, a close to or approximate uncited number may be a number that provides a substantially equivalent quantity to the specifically cited quantity in its presented context.

[0473] As used herein, the term "comprising" or "including" refers to compositions, methods and their respective components that are essential to the invention, but may also include unspecified elements, whether or not they are essential.

[0474] The term "composed of" refers to the compositions, methods and their corresponding components described herein, excluding any elements not mentioned in the description of this embodiment.

[0475] As used herein, the term "consistent primarily of" refers to those elements required for a given embodiment. This term allows for the presence of additional elements that do not materially affect the basic, novel, or functional features of an embodiment of the invention.

[0476] Unless the context clearly indicates otherwise, the singular terms “a,” “an,” and “the” include plural references. Similarly, unless the context clearly indicates otherwise, the word “or” is intended to include “and.” It should also be noted that a claim can be drafted to exclude any optional elements. Therefore, the claim is intended to provide a prior basis for the use of exclusive terms such as “solely,” “only,” or the use of negative limiting terms to state claim elements.

[0477] The abbreviation “eg” comes from the Latin word *exempli gratia*, used here to indicate a non-restrictive example. Therefore, the abbreviation “eg” is synonymous with the term “for example”.

[0478] As used herein, the terms “siRNA” and “iRNA reagent” are used interchangeably to refer to a reagent that can mediate the silencing of a target RNA, such as mRNA, or the transcript of a gene encoding a protein. For convenience, this mRNA is also referred to herein as the silenced mRNA. The gene is also referred to as the target gene. Generally, the RNA to be silenced is an endogenous gene, an exogenous gene, or a pathogen gene. In addition, RNA other than mRNA can also be targeted, such as tRNA and viral RNA.

[0479] As used in this article, the phrase “mediated RNAi” refers to the ability to silence target genes (such as mRNA) in a sequence-specific manner. While not wishing to be bound by theory, it is believed that silencing is achieved using RNAi mechanisms or processes and guide RNAs, such as the antisense strand of dsRNA, which is 21 to 23 nucleotides long.

[0480] As used herein, those skilled in the art will understand that, unless otherwise stated, when used to describe a first nucleotide sequence associated with a second nucleotide sequence, the term "complementarity" refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions and form a double-stranded structure. For example, these conditions may be stringent conditions, which might include: 400 mM NaCl, 40 mM PIPES at pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12–16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered in vivo, may also apply. Those skilled in the art will be able to determine the most suitable combination of conditions for testing the complementarity of the two sequences based on the final application of the hybridized nucleotides.

[0481] As used herein, the term "fundamentally complementary" nucleotide sequence relative to a reference nucleotide sequence refers to a nucleotide sequence whose percentage of identity with the precisely complementary reference nucleotide sequence is at least 80%. For example, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (i.e., perfectly complementary). Preferably, identity is assessed at a length of at least 15, for example, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides.

[0482] The term "off-target" and the phrase "off-target effect" refer to any situation in which an effector molecule targeting a given target causes an unintended effect by interacting directly or indirectly with another target sequence, DNA sequence, or cellular protein or other part. For example, an "off-target effect" can occur when other transcripts are simultaneously degraded due to partial homology or complementarity between other transcripts and the sense and / or antisense strands of siRNA.

[0483] The terms “reduce,” “reduction,” “lower,” or “inhibit” are used herein to refer to a statistically significant reduction. In some implementations, “reduce,” “reduction,” or “lower” or “inhibit” generally means a reduction of at least 10% compared to a reference level (e.g., in the absence of a specific treatment or drug), and may include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduce” or “inhibit” does not include complete inhibition or reduction compared to a reference level. “Complete inhibition” means 100% inhibition compared to a reference level. For individuals without a specific disease, the reduction is preferably reduced to an acceptable level within the normal range.

[0484] The terms “increased,” “enhanced,” “strengthened,” or “activated” all refer herein to an increase in a statically significant amount. In some implementations, the terms “increased,” “enhanced,” or “activated” may refer to an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at most about 90%, or up to and including 100% or any increase between 10% and 100% compared to a reference level, or at least about 2 times, or at least 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 times and 10 times or more compared to a reference level. In the context of a marker or symptom, “increased” means that the level is statistically significant.

[0485] As used in this article, the “end” of a chain refers to position 1, which is the position from which the counting begins at the nearest end of the chain. For example, the 5’ end refers to position 1, which is the position from the 5’ end of the chain. Similarly, the 3’ end refers to position 1, which is the position from the 3’ end of the chain.

[0486] As used herein, the “end region” of a chain refers to positions 1-4, counting from the nearest end of the chain, such as positions 1, 2, 3, and 4. For example, the 5’ end region refers to positions 1-4, such as positions 1, 2, 3, and 4, counting from the 5’ end of the chain. Similarly, the 3’ end region refers to positions 1-4, such as positions 1, 2, 3, and 4, counting from the 3’ end of the chain.

[0487] For example, the 5' end region of the antisense chain is bits 1, 2, 3, and 4, counted starting from the 5' end of the antisense chain. Preferably, the 5' end region of the antisense chain is bits 1, 2, and 3, counted starting from the 5' end of the antisense chain. The 3' end region of the antisense chain can be bits 1, 2, 3, and 4, counted starting from the 3'-end of the chain. Preferably, the 3'-end region of the antisense chain is bits 1, 2, and 3, counted starting from the 3'-end of the antisense chain.

[0488] Similarly, the 5' end region of the justice chain is the first, second, third, and fourth bits counted from the 5' end of the justice chain. The preferred 5' end region of the justice chain is the first, second, and third bits counted from the 5' end of the justice chain. The 3' end region of the justice chain can be the first, second, third, and fourth bits counted from the 3' end of the chain. The preferred 3' end region of the justice chain is the first, second, and third bits counted from the 3' end of the justice chain.

[0489] As used herein, the “center region” of a chain refers to positions 5-17, counting from the 5' end of the chain. Examples include positions 6-16, 6-15, 6-14, 6-13, 6-12, 7-15, 7-14, 7-13, 7-12, 8-16, 8-15, 8-14, 8-13, 8-12, 9-16, 9-15, 9-14, 9-13, 9-12, 10-16, 10-15, 10-14, 10-13, or 10-12. For example, the center region of a line refers to positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the line. The preferred central region of the justice chain is bits 6, 7, 8, 9, 10, 11, 12, 13, and 14, counting from the 5' end of the justice chain. A more preferred central region of the justice chain is bits 7, 8, 9, 10, 11, 12, and 13, counting from the 5' end of the justice chain. The preferred central region of the antisense chain is bits 9, 10, 11, 12, 13, 14, 15, 16, and 17, counting from the 5' end of the antisense chain. A more preferred central region of the antisense chain is bits 10, 11, 12, 13, 14, 15, and 16, counting from the 5' end of the antisense chain.

[0490] As used herein, the term “in vitro” refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, cell culture, etc., rather than an event that occurs within an organism (such as an animal or plant). As used herein, the term “ex vivo” refers to cells that have been removed from a living organism and cultured outside the organism (e.g., in a test tube). As used herein, the term “in vivo” refers to an event that occurs within an organism (e.g., an animal, plant, and / or microorganism).

[0491] As used herein, the terms “subject” or “patient” refer to any organism to which the compositions disclosed herein may be administered, for example, for experimental, diagnostic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Animals are typically vertebrates such as primates, rodents, livestock, or prey. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Livestock and prey include cows, horses, pigs, deer, bison, buffalo, felines (such as domestic cats), canines (such as dogs, foxes, and wolves), birds (such as chickens, emus, and ostriches), and fish (such as trout, catfish, and salmon). Patients or subjects include any subset of the foregoing, such as all of the foregoing, but do not include one or more groups or species such as humans, primates, or rodents. In some embodiments of the aspects described herein, the subject is a mammal, such as a primate, for example, a human. The terms "patient" and "subject" are used interchangeably herein. The subject can be male or female.

[0492] Preferably, the subject is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cattle, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects in animal models representing human diseases and disorders. Furthermore, the compounds, compositions, and methods described herein can be used with domestic animals and / or pets.

[0493] Subjects can be individuals who have been previously diagnosed with or identified as having a disease requiring treatment. Alternatively, subjects can be individuals who have not been previously diagnosed. A "subject" requiring testing for a specific disease can be someone who has the disease, has been diagnosed with the disease, or is at risk of developing the disease.

[0494] In some embodiments, the subject is a human. In another embodiment, the subject is an experimental animal or animal substitute used as a disease model. The term does not indicate a specific age or sex. Therefore, adult and neonatal subjects, as well as fetuses, whether male or female, will be included. Examples of subjects include humans, dogs, cats, cattle, goats, and mice. The term "subject" is also intended to include transgenic species. In some embodiments, the subject may be of European descent. In some embodiments, the subject may be of African American descent. In some embodiments, the subject may be of Asian descent.

[0495] In jurisdictions where patentability is prohibited for methods performed on humans, the meaning of "administering" a composition to a human subject should be limited to specifying a controlled substance that the subject would administer spontaneously by any technique (e.g., oral, inhalation, topical application, injection, insertion, etc.). This is intended to be the broadest reasonable interpretation consistent with the laws or regulations defining patentable subject matter. In jurisdictions where patentability is not prohibited for methods performed on humans, "administering" a composition includes both the method performed on the human body and the activities described above.

[0496] As used herein, the term "parenteral administration" refers to administration by injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous, intravenous, or intramuscular administration.

[0497] As used in this article, the term "subcutaneous administration" means administration just beneath the skin. "Intravenous administration" means administration into a vein.

[0498] As used herein, the term "dosage" refers to a specified amount of pharmaceutical preparation provided in a single administration. In some embodiments, the dosage may be administered in the form of two or more pills, tablets, or injections. For example, in some embodiments, when subcutaneous administration is required, the desired dosage necessitates a volume that is not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dosage. In some embodiments, the dosage may be administered in two or more injections to minimize individual injection site reactions.

[0499] As used herein, the term "dosage unit" refers to the form in which a pharmaceutical preparation is provided. In some embodiments, a dosage unit is a vial containing lyophilized antisense oligonucleotides. In some embodiments, a dosage unit is a vial containing recombinant antisense oligonucleotides.

[0500] The terms “treat,” “treating,” or “treatment of” (and their grammatical variations) refer to a reduction, at least partial improvement, or stabilization of the severity of a subject’s condition and / or a degree of relief, reduction, decrease, or stabilization of at least one clinical symptom, and / or a delay in the progression of the disease or disorder.

[0501] The terms “prevent,” “preventing,” and “prevention” (and their grammatical variations) refer to preventing and / or delaying the onset of a subject’s disease, disorder, and / or clinical symptoms, and / or reducing the severity of the onset of a disease, disorder, or clinical symptom compared to the absence of the method of the present invention. Prevention can be complete, for example, the complete absence of disease, disorder, and / or clinical symptoms. Prevention can also be partial, such that the occurrence and / or severity of disease, disorder, and / or clinical symptoms in the subject is less than in the absence of the present invention.

[0502] The terms “statistically significant” or “significantly” refer to statistical significance, which usually means a difference of two standard deviations (2SD) or greater.

[0503] Glycolic acid nucleic acid (GNA) is Where B is a modified or unmodified base, and * is... R , S Or a racemic mixture.

[0504] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose, for example, in which any bonds between the 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 oxygen (e.g., C1', C2', C3', C4', or O4') is not present in the nucleotide, either independently or in combination. In some embodiments, the acyclic nucleotide is: , , , or Where B is a modified or unmodified base, and R 1 and R 2 It is independently H, halogen, OR3 or alkyl; R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar.

[0505] Unlocking nucleic acid (UNA) modifications involves removing the monomeric bond between C1' and C4' (i.e., the covalent carbon-oxygen-carbon bond between C1' and C4' carbons). In another example, the C2'-C3 bond of the sugar (i.e., the covalent carbon-carbon bond between C2 and C3 carbons) is removed (see Mikhailov et al.). Tetrahedron Letters , 26 (17): 2059 (1985); Fluiter et al., Mol. Biosyst (The entire contents of this paper are incorporated herein by reference in ., 10: 1039 (2009). Acyclic nucleotides can be linked via 2'-5' or 3'-5'. In some embodiments, UNA has the following structure: In this embodiment, B is a modified or unmodified base; R, R', R”, R”', and R”” are independently H, OH, CH3, CH2CH3, O-alkyl, NH2, NHMe, or NMe2; and each* is independently R, S, or racemic. In some embodiments, the UNA modification is, where B is a modified or unmodified base and R is H, OH, or O-alkyl.

[0506] Modified unlocking nucleic acid ( m UNA modifications include, but are not limited to, the following: , Where the bases are modified or unmodified, * is R , S Or a racemic mixture.

[0507] In some implementations, the UNA modification is selected from the following group: .

[0508] The term "base-free modification" refers to a nucleotide or its analogue that contains no bases. Some exemplary base-free modifications include, but are not limited to, the following:

[0509] Where R is H, Me, Et, or OMe; R' is H, Me, Et, or OMe; R” is H, Me, Et, or OMe; * indicates R , S Or a racemic mixture.

[0510] In some implementations, the thermally unstable modification is selected from the group consisting of: .

[0511] Where B is a modified or unmodified base, and * is... R , S Or a racemic mixture.

[0512] As used in this article, 2'-5' RNA is The bases are modified or unmodified nucleotides.

[0513] As used herein, TNA is a nucleotide containing threonine rather than ribose, wherein its 3' position is linked to the 3' position of its upstream nucleotide, and its 2' position is linked to the 5' position of its downstream nucleotide. TNA has the following structure. Some exemplary TNA modifications include, but are not limited to, the following: , where B is a modified or unmodified nucleobase.

[0514] As used herein, hydroxyproline spacer modifications include hydroxyproline monomers, for example, R is either H or a modified or unmodified nucleobase.

[0515] Other exemplary sugar modifications include, but are not limited to, the following: and Where B is a modified or unmodified nucleobase, and R is H or a C1-C6 alkyl group (e.g., methyl or ethyl).

[0516] Exemplary nucleotides with impaired WC hydrogen bonds to complementary bases on the opposite strand include, but are not limited to, nucleotides containing bases independently selected from: .

[0517] Exemplary noncanonical bases whose ability to form hydrogen bonds with bases in the opposite chain is impaired or completely eliminated include, but are not limited to, inosine, nebularine, 2-aminopurine, 2,4-difluorotoluene, 5-nitroindole, 3-nitropyrrole, 4-fluoro-6-methylbenzimidazole, and 4-methylbenzimidazole.

[0518] Exemplary α-nucleotides include, but are not limited to, B is a modified or unmodified nucleobase, and R is H, OH, OCH3, F, NH2, NHMe, NMe2, or O-alkyl.

[0519] Exemplary phosphate modifications known to reduce the thermal stability of dsNA double chains compared to natural phosphodiester bonds include, but are not limited to, the following: , The alkyl group R can be a C1-C6 alkyl group. Specific alkyl groups of R include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0520] As used herein, the term "bridging nucleic acid" includes, but is not limited to, nucleotides containing a five- or six-membered bridging structure with a fixed 3′-endo conformation, also known as the north conformation. The bridging structure links the 2′-carbon (e.g., 2′-oxygen) of the ribose to the 4′-carbon. Various different bridging structures containing carbon, oxygen, nitrogen, and hydrogen atoms are possible.

[0521] In some implementations, BNA is locked nucleic acid (LNA). As used herein, the term "locked nucleic acid" (LNA) generally refers to a class of BNA in which the ribose ring is "locked" by a methylene bridge connecting the 2′ oxygen of the ribose and the 4′ carbon of the ribose.

[0522] As used herein, the term "aliphatic" refers to a saturated or unsaturated straight-chain, branched, and / or cyclic hydrocarbon with a defined number of carbon atoms. Examples include alkyl, alkenyl, ynyl, cycloalkyl, cycloalkylalkanyl, cycloalkenyl, and cycloalkynyl groups with a defined number of carbon atoms.

[0523] As used herein, the term "alkyl" refers to a straight-chain or branched aliphatic hydrocarbon group having 1 to 60 carbon atoms in the chain, preferably having about 6 to 50 carbon atoms in the chain. "Lower alkyl" refers to an alkyl group having 1 to 8 carbon atoms. "Higher alkyl" refers to an alkyl group having about 10 to 20 carbon atoms. An alkyl group may optionally be substituted with one or more identical or different alkyl substituents, wherein "alkyl substituent" includes halogen, amino, aryl, hydroxyl, alkoxy, aryloxy, alkylthio, arylthio, arylalkoxy, arylalkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. "Branched" refers to a lower alkyl group, such as methyl, ethyl, or propyl, attached to a straight-chain alkyl chain. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl, and hexadecyl. Useful alkyl groups include branched or straight-chain alkyl groups with 6 to 50 carbons, as well as lower alkyl groups with 1 to 4 carbons and higher alkyl groups with about 12 to 16 carbons.

[0524] A "heteroalkyl" group is any carbon atom of an alkyl group that is replaced by a heteroatom (e.g., a CH2 group connected to an NH or O group) having an appropriate number of hydrogen atoms attached. The term "heteroalkyl" includes optionally substituted alkyl, alkenyl, and ynyl groups having one or more skeletal chain atoms selected from atoms other than carbon, such as oxygen, nitrogen, sulfur, phosphorus, silicon, or combinations thereof. In some embodiments, the heteroatom is located at any internal position of the heteroalkyl group. Examples include, but are not limited to, -CH2-O-CH3, -CH2-CH2-O-CH3, -CH2-NH-CH3, -CH2-CH2-NH-CH3, -CH2-N(CH3)-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and –CH=CH-N(CH3)-CH3. In some embodiments, up to two heteroatoms are consecutive, such as -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0525] As used herein, the term "alkenyl" refers to an alkyl group containing at least one carbon-carbon double bond. Alkenyl groups may optionally be substituted with one or more "alkyl substituents". Exemplary alkenyl groups include vinyl, allyl, n-pentenyl, decenyl, dodecenyl, tetradecadienyl, heptadecan-8-en-1-yl, and heptadecan-8,11-dien-1-yl.

[0526] As used herein, the term "alkynyl" refers to an alkyl group containing a carbon-carbon triple bond. An alkynyl group may optionally be substituted with one or more "alkyl substituents". Exemplary alkynyl groups include ethynyl, propynyl, n-pentynyl, decanynyl, and dodecanynyl. Useful alkynyl groups include lower alkynyl groups.

[0527] As used herein, the term "cycloalkyl" refers to a non-aromatic monocyclic or polycyclic cyclic system of about 3 to about 12 carbon atoms. Cycloalkyl groups may optionally be partially unsaturated. Cycloalkyl groups may also optionally be substituted with aryl substituents, oxo groups, and / or alkylene groups. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl. Useful polycyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decahydronaphthyl, camphor, camphene, and noradamantyl.

[0528] "Heterocyclic group" refers to a non-aromatic 3-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system. If it is a monocyclic ring, it has 1-3 heteroatoms; if it is a bicyclic ring, it has 1-6 heteroatoms; and if it is a tricyclic ring, it has 1-9 heteroatoms. The heteroatoms are selected from O, N, or S (e.g., for monocyclic, bicyclic, or tricyclic rings, this means carbon atoms and 1-3, 1-6, or 1-9 N, O, or S heteroatoms, respectively). Cx heterocyclic groups and Cx cyclic heterocyclic groups are commonly used, where X and Y represent the number of carbon atoms in the ring system. In some embodiments, 1, 2, or 3 hydrogen atoms in each ring may be substituted with substituents. Exemplary heterocyclic groups include, but are not limited to, piperazinyl, pyrrolyl, dioxyl, morpholinyl, tetrahydrofuranyl, piperidinyl, 4-morpholinyl, 4-piperazinyl, pyrrolyl, perhydropyrroloazinyl, 1,4-diazadihydroepenyl, 1,3-dioxyl and 1,4-dioxyl.

[0529] "Aryl" refers to an aromatic carbocyclic group containing about 3 to about 13 carbon atoms. An aryl group may optionally be substituted with one or more aryl substituents, which may be the same or different, wherein "aryl substituents" may include alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxyl, alkoxy, aryloxy, carboxyl, arylacyl, halogen, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, arylalkoxycarbonyl, acyloxy, amide, arylacylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NRR', wherein R and R' are each independently hydrogen, alkyl, aryl, and arylalkyl. Exemplary aryl groups include substituted or unsubstituted phenyl and substituted or unsubstituted naphthyl groups.

[0530] "Heteroaryl" refers to aromatic 3-8 membered monocyclic, 8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ring systems. If it is a monocyclic ring, it has 1-3 heteroatoms; if it is a bicyclic ring, it has 1-6 heteroatoms; and if it is a tricyclic ring, it has 1-9 heteroatoms. The heteroatoms are selected from O, N, or S (for example, if they are monocyclic, bicyclic, or tricyclic, they are carbon atoms and 1-3, 1-6, or 1-9 N, O, or S heteroatoms, respectively).

[0531] Exemplary aryl and heteroaryl groups include, but are not limited to, phenyl, pyridyl, pyrimidinyl, furanyl, thiopheneyl, imidazolyl, thiazolyl, pyrazolyl, pyrazinyl, triazinyl, tetrazolyl, indoleyl, benzyl, naphthyl, anthraceneyl, azulel, fluorenyl, indanyl, indanyl, naphthyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiopheneyl, benzooxazolyl, benzooxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazoleyl, benzoisooxazolyl, benzoisothiazolyl, benzimidazolinyl, carbazoleyl, 4aH-carbazoleyl, carbolinyl, chromanyl, Chromenyl, cenolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofurano[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazoalkyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolyl, 3H-indolinyl, isatinoyl, isobenzofuranyl, isochromyl, isoindolinyl, isoindolinyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholine Naphthyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolylalkyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridine, phenanthroline, phenazinyl, phenothiazinyl, phenothiazinyl, phthalazinyl, piperazine, piperidinyl, piperidinoneyl, 4-piperidinoneyl, piperinyl, pteridinyl, purine, pyranyl, pyrazinyl, pyrazolylalkyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridinyl, pyridoxazole, pyridinium, pyridoxazole, pyridoxazole, pyridinyl(pyr) The compounds include idinyl, pyridyl, pyrimidinyl, pyrrolylyl group, pyrrolinyl group, 2H-pyrroleyl, pyrroleyl, quinazolinyl, quinolinyl, 4H-quinazinyl, quinoxalinyl, quininecycloyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, phenylthio, and xanthonyl, etc. In some embodiments, one, two, three, or four hydrogen atoms in each ring may be substituted with substituents.

[0532] As used herein, the term "halogen" or "halogenated" refers to an atom selected from fluorine, chlorine, bromine, and iodine. The term "halogenated radioisotope" or "halogenated isotope" refers to a radionuclide selected from atoms of fluorine, chlorine, bromine, and iodine.

[0533] "Halogen-substituted portion" or "halogenated portion", as an independent group or part of a larger group, refers to the aliphatic, alicyclic, or aromatic portion described herein, which is substituted by one or more "halogenated" atoms as defined in this application.

[0534] As used herein, the term "haloalkyl" refers to an alkyl and alkoxy structure having at least one substituent of fluorine, chlorine, bromine, or iodine, or a combination thereof. In embodiments, when the group contains more than one halogen, the halogens may be the same or different. The terms "fluoroalkyl" and "fluoroalkoxy" respectively include haloalkyl and haloalkoxy, wherein the halogen is fluorine. Exemplary haloalkyls include haloalkyl, dihaloalkyl, trihaloalkyl, and perhaloalkyl, etc. (e.g., halo(C1-C3)alkyls include chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF3), perfluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-1,1-dichloroethyl, etc.).

[0535] As used herein, the term "amino" refers to -NH2. The term "alkylamino" refers to a nitrogen moiety having a single, straight-chain or branched, unsaturated aliphatic, cyclic, or heterocyclic group attached to a nitrogen atom, such as -NH(alkyl). The term "dialkylamino" refers to a nitrogen moiety having two single-chain or branched, unsaturated aliphatic, cyclic, or heterocyclic groups attached to a nitrogen atom, such as -N(alkyl)(alkyl). The term "alkylamino" includes "alkenylamino," "alkynylamino," "cycloamino," and "heterocyclicamino." The term "arylamino" refers to a nitrogen moiety having at least one aryl group attached to a nitrogen atom. For example, -NHaryl and -N(aryl)2. The term "heteroarylamino" refers to a nitrogen moiety having at least one heteroaryl group attached to a nitrogen atom. For example, -NHhearyl and -N(heteroaryl)2. Optionally, the two substituents may also form a ring together with the nitrogen atom. Unless otherwise stated, compounds containing an amino moiety described herein may include their protected derivatives. Protecting groups suitable for the amino moiety include acetyl, tert-butoxycarbonyl, benzyloxycarbonyl, etc. Exemplary alkylamino groups include, but are not limited to, NH (C1-C2). 10 Alkyl groups, such as -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, and -NHCH(CH3)2. Exemplary dialkylamino groups include, but are not limited to, -N(C1-C2). 10 Alkyl)2, such as N(CH3)2, -N(CH2CH3)2, -N(CH2CH2CH3)3 and -N(CH(CH3)3)2.

[0536] The term "aminoalkyl" refers to alkyl, alkenyl, and alkynyl groups as defined above, unless one or more substituted or unsubstituted nitrogen atoms (-N-) are located between the carbon atoms of the alkyl, alkenyl, or alkynyl group. For example, (C2-C6)aminoalkyl refers to a chain containing 2 to 6 carbons and one or more nitrogen atoms located between the carbon atoms.

[0537] The terms "hydroxyl" and "hydroxyl radical" refer to the free radical -OH.

[0538] As used herein, the term "alkoxyl" or "alkoxy" refers to an alkyl group as described above, which is attached to an oxygen group and can be represented by one of -O-alkyl, -O-alkenyl, and -O-ynyl. An aryloxy group can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein. Alkoxy and aryloxy groups can substitute for alkyl groups as described above. Exemplary alkoxy groups include, but are not limited to, O-methyl, O-ethyl, O-n-propyl, O-isopropyl, O-n-butyl, O-isobutyl, O-sec-butyl, O-tert-butyl, O-pentyl, O-hexyl, O-cyclopropyl, O-cyclobutyl, O-cyclopentyl, O-cyclohexyl, etc.

[0539] As used herein, the term "carbonyl" refers to the group -C(O)-. It is worth noting that the carbonyl group can be further substituted by various substituents to form different carbonyl groups, including acids, acid halides, amides, esters, ketones, etc.

[0540] As used in this article, the term "oxo" refers to the double oxygen bond, i.e., =O.

[0541] The term "carboxyl group" refers to the group -C(O)O-. It should be noted that compounds containing a carboxyl moiety described herein may include their protected derivatives, where the oxygen atom is replaced by a protecting group. Protecting groups suitable for the carboxyl moiety include benzyl, tert-butyl, etc. As used herein, the carboxyl group includes -COOH, i.e., the carboxyl radical group.

[0542] The term "ester" refers to the chemical part of the formula -C(=O)OR, where R is selected from alkyl, cycloalkyl, aryl, heteroaryl, and heterocycloalkyl.

[0543] The term "cyano" refers to the free radical - CN.

[0544] The term "nitro" refers to the free radical -NO2.

[0545] The term "heteroatom" refers to an atom that is not a carbon atom. Specific examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and halogens. A "heteroatomic moiety" includes a portion to which the atom connecting the moiety is not carbon. Examples of heteroatomic moiety include -N=, -NRN-, -N+(O-)=, -O-, -S- or -S(O)2-, -OS(O)2-, and -SS-, where RN is H or another substituent.

[0546] The terms "alkathio" and "thioalkoxy" refer to alkoxy groups as described above, wherein the oxygen atom is replaced by a sulfur atom. In a preferred embodiment, the "alkathio" moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-ynyl. Representative alkathio groups include methylthio, ethylthio, etc. The term "alkathio" also includes cycloalkyl, olefin, and cycloolefin groups, as well as alkynyl groups. "Aryl" refers to aryl or heteroaryl groups.

[0547] The term "sulfinyl group" refers to the -SO- group. It is worth noting that the sulfinyl group can be further substituted by various substituents to form different sulfinyl groups, including sulfinic acids, sulfinamides, sulfinic esters, sulfoxides, etc.

[0548] The term "sulfonyl" refers to the free radical -SO2-. It is worth noting that the sulfonyl group can be further replaced by various substituents to form different sulfonyl groups, including sulfonic acids (-SO3H), sulfonamides, sulfonates, sulfones, etc.

[0549] The term "thiocarbonyl" refers to the group -C(S)-. It is worth noting that thiocarbonyl can be further substituted by various substituents to form different thiocarbonyl groups, including thioacids, thioamides, thioesters, thioketones, etc.

[0550] "Acyl" refers to an alkyl-CO- group, wherein the alkyl group is as previously described. Exemplary acyl groups include alkyl groups having 1 to 30 carbon atoms. Exemplary acyl groups also include acetyl, propionyl, 2-methylpropionyl, butyryl, and palmitoyl.

[0551] "Aromatic acyl" refers to an aryl-CO- group, wherein the aryl group is as described above. Exemplary aromatic acyl groups include benzoyl and 1- and 2-naphthoyl.

[0552] "Aryl thio" refers to an aryl-S-group, wherein the aryl group is as described above. Exemplary aryl thio groups include phenylthio and naphthio.

[0553] "Arylalkyl" refers to an arylalkyl group, wherein the aryl group and alkyl group are as described above. Exemplary arylalkyl groups include benzyl, phenethyl, and naphthylmethyl.

[0554] "Arylalkoxy" refers to an aralkyl-O- group, wherein the aralkyl group is as described above. An exemplary arylalkoxy is a benzyloxy group.

[0555] "Arylthio" refers to an aralkyl-S-group, wherein the aralkyl group is as described above. An exemplary arylthio group is benzylthio.

[0556] "Alkoxycarbonyl" refers to an alkyl-O-CO- group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butoxycarbonyl, and tert-butoxycarbonyl.

[0557] "Aryloxycarbonyl" refers to an aryl -O-CO- group. Exemplary aryloxycarbonyl groups include phenoxycarbonyl and naphthoxycarbonyl.

[0558] "Aryl alkoxycarbonyl" refers to an aralkyl-O-CO- group. An exemplary aryl alkoxycarbonyl is benzyloxycarbonyl.

[0559] "Carbamoyl" refers to the H2N-CO- group.

[0560] "alkylcarbamoyl" refers to the R'RN-CO- group, where one of R and R' is hydrogen and the other is an alkyl group as described above.

[0561] "Dialkylcarbamoyl" refers to the R'RN-CO- group, where R and R' are each independently an alkyl group as described above.

[0562] "Acyloxy group" refers to an acyl-O- group, wherein the acyl group is as described above. "Acylamino group" refers to an acyl-NH- group, wherein the acyl group is as described above. "Aromatic acylamino group" refers to an aromatic acyl-NH- group, wherein the aromatic acyl group is as described above.

[0563] The term "optionally substituted" means that the specified group or portion is unsubstituted, or substituted by one or more (typically 1, 2, 3, 4, 5, or 6 substituents) selected independently from the group of substituents listed in the following definition of "substituent" or otherwise specified. The term "substituent" means a group that is substituted at any atom of the substituent. Suitable substituents include, but are not limited to, halogens, hydroxyl groups, carboxyl groups, oxo groups, nitro groups, haloalkyl groups, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, cycloyl groups, heterocyclic groups, aralkyl groups, alkoxy groups, aryloxy groups, amino groups, amide groups, alkylcarbamoyl groups, arylcarbamoyl groups, aminoalkyl groups, alkoxycarbonyl groups, carboxyl groups, hydroxyalkyl groups, alkylsulfonyl groups, arylsulfonyl groups, alkylsulfonamide groups, arylsulfonamide groups, arylalkylsulfonamide groups, alkylcarbonyl groups, acyloxy groups, cyano groups, or ureyl groups. In some cases, two substituents together with the carbon atom to which they are attached can form a ring.

[0564] For example, any alkyl, alkenyl, cycloalkyl, heterocyclic, heteroaryl, or aryl group may optionally be substituted with 1, 2, 3, 4, or 5 substituents, said substituents being 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, O(C Substituents of 1-C8) haloalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylamino, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)alkyl, C(O)-alkyl, alkylcarbonylamino, CH2—[CH(OH)]m—(CH2)p—OH, CH2—[CH(OH)]m—(CH2)p—NH2 or CH2-aryl-alkoxy; “m” and “p” are independently 1, 2, 3, 4, 5 or 6.

[0565] In some embodiments, the optionally substituted group is replaced by one substituent. In some other embodiments, the optionally substituted group is replaced by two independently selected substituents, which may be the same or different. In some other embodiments, the optionally substituted group is replaced by three independently selected substituents, which may be the same, different, or any combination of the same and different. In still other embodiments, the optionally substituted group is replaced by four independently selected substituents, which may be the same, different, or any combination of the same and different. In still other embodiments, the optionally substituted group is replaced by five independently selected substituents, which may be the same, different, or any combination of the same and different.

[0566] In some embodiments, any alkyl, alkenyl, cycloalkyl, heterocyclic, heteroaryl, or aryl (e.g., alkyl) group is optionally substituted with one, two, or three substituents, said substituents being independently selected from halogens, -OR 22 -N(R) 22 )2、-SR 22 -C(O)OR 22 -C(O)N(R) 22 In )2, where R 22 Is it hydrogen or C? 1-3 Alkyl (e.g., 2,2,2-trifluoroethyl, 1,3-dimethoxypropyl-2-yl). For example, any alkyl, alkenyl, cycloalkyl, heterocyclic, heteroaryl, or aryl (e.g., alkyl) may optionally be selected independently by one or two halogens, -OR 22 -N(R)22 )2、-SR 22 -C(O)OR 22 -C(O)N(R) 22 Substituents in )2, where R 22 Is it hydrogen or C? 1-3 Alkyl groups (e.g., 2,2,2-trifluoroethyl, 1,3-dimethoxypropyl-2-yl).

[0567] The "isocyanate" group refers to the NCO group.

[0568] The "thiocyanate" group refers to the CNS group.

[0569] The "isothiocyanate" group refers to the NCS group.

[0570] "Alkoxy" refers to the RC(=O)O- group.

[0571] "alkyl" refers to the RC (=O)- group.

[0572] It should be understood that this disclosure is not limited to the specific methods, schemes, and reagents provided herein, and therefore may vary. The terminology used herein is for describing specific embodiments only and is not intended to limit the scope of this disclosure, which is defined solely by the claims. The invention is further illustrated by the following examples, which should not be construed as further limitations.

[0573] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each component may be cited and claimed individually, or in any combination with other components of that group or other elements herein. For convenience and / or patentability reasons, one or more components of a group may be included in or removed from the group. When any such inclusion or removal occurs, this specification is hereby deemed to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

[0574] The description of embodiments of this disclosure is not intended to be exhaustive or to limit this disclosure to the precise form disclosed. While specific embodiments and examples of this disclosure are described herein for illustrative purposes, those skilled in the art will recognize that various equivalent modifications can be made within the scope of this disclosure. For example, although method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or may perform functions substantially simultaneously. The teachings of the disclosure provided herein can be suitably applied to other procedures or methods. The various embodiments described herein can be combined to provide further embodiments. If desired, aspects of this disclosure can be modified to incorporate the compositions, functions, and concepts of the foregoing references and applications to provide yet another embodiment of this disclosure. Furthermore, due to considerations of biological functional equivalence, some changes can be made to the protein structure without affecting the type or amount of biological or chemical action. These and other changes can be made to this disclosure based on the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0575] Any particular element in any of the above embodiments can be combined with or substituted for elements in other embodiments. Furthermore, while advantages relating to certain embodiments of this disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit these advantages to fall within the scope of this disclosure.

[0576] Example

[0577] The techniques described herein are further illustrated by the following embodiments, which should not be construed as further limitations.

[0578] Example 1: Synthesis of an exemplary compound Synthesis of Compound 21

[0579] Compound 21 was synthesized according to scheme 1.

[0580]

[0581] Option 1

[0582] N-[9-[(2R,5S)-4-[tert-butyldimethyl)silyl]oxy-5-formyl-3-methoxy-tetrahydrofuran-2-yl]purin-6-yl]benzamide (Compound 10) N-[9-[(2R,5R)-4-[tert-butyldimethyl)silyl]oxy-3-methoxy-5-vi...

Claims

1. A compound of formula (I), (Formula I), or its salt, in: B is an optional modified base (e.g., uracil or 5-methyluracil); X is O or S; Each R V Independently protected by hydrogen or hydroxyl groups (e.g., ethyl or neopentyloxymethyl ((CH3)3CC(O)OCH2-, POM); One of R2 and R3 is hydrogen, halogen, or -OR 20 ,in: R 20 Protected by hydrogen, hydroxyl group, or optionally substituted C 1-6 Alkyl groups (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl, 2-(N-methylamino)-2-oxoethyl), optionally substituted C 2-6 alkenyl or optionally substituted C 2-6 Alkynyl (e.g., propargyl); R 2 and R 3 The other one is -OR 30 ,in: R 30 It is a hydrogen, hydroxyl protecting group, reactive phosphorus group (e.g., phosphoramide), bond with nucleoside or nucleotide, or bond with oligonucleotide.

2. The compound according to claim 1, wherein the compound has the following structure: Or its salt.

3. The compound according to claim 1, wherein the compound has the following structure: Or its salt.

4. The compound according to any one of claims 1-3, wherein each R V Independently for hydrogen, optionally, each R V It is hydrogen on its own.

5. The compound according to any one of claims 1-3, wherein at least one R V For hydroxyl protecting groups (e.g., ethyl or POM), optionally, each R V It is a hydroxyl protecting group.

6. The compound according to any one of claims 1-3 or 5, wherein each R V It is -CH2CH3.

7. The compound according to any one of claims 1-6, wherein X is O.

8. The compound according to any one of claims 1-7, wherein Selected from *-P(O)(OH)2, *-P(O)(OCH2CH3)2 or *-P(O)(OCH2OC(O)C(CH3)3)2, where * indicates a bond with the rest of the compound.

9. The compound according to any one of claims 1-6, wherein X is S.

10. The compound according to claim 1, wherein the compound has the following structure: Or its salt.

11. The compound according to claim 10, wherein the compound has the following structure: Or its salt.

12. The compound according to claim 10, wherein the compound has the following structure: Or its salt.

13. The compound according to any one of claims 1-12, wherein B is a modified or protected nucleobase.

14. The compound of claim 13, wherein B is a protected nucleobase containing at least one amino or hydroxyl protecting group.

15. The compound according to any one of claims 1-14, wherein R 2 It is hydrogen or halogen (e.g., fluorine).

16. The compound according to any one of claims 1-14, wherein R 2 Yes - OR 20 .

17. The compound according to claim 16, wherein R 20 C is an optional substitute 1-6 alkyl.

18. The compound according to claim 17, wherein R 20 It is methyl, ethyl, propyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl or 2-(N-methylamino)-2-oxoethyl, optionally, R 20 It is methyl or 2-(N-methylamino)-2-oxoethyl.

19. The compound according to claim 16, wherein R 20 C is an optional substitute 2-6 Alkenyl group.

20. The compound according to claim 16, wherein R 20 C is an optional substitute 2-6 Alkyne group (e.g., propynyl group).

21. The compound according to any one of claims 15-20, wherein R 3 Yes - OR 30 , where R 30 It is a hydrogen or hydroxyl protecting group or a reactive phosphorus group.

22. The compound according to claim 21, wherein R 30 It is a hydrogen or hydroxyl protecting group.

23. The compound according to claim 22, wherein R 30 It is hydrogen.

24. The compound according to claim 22, R 30 It is a hydroxyl protecting group.

25. The compound of claim 24, wherein the hydroxyl protecting group is tert-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylhexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylsilyl (DPMS), or tert-butylmethoxyphenylsilyl (TBMPS), optionally, the hydroxyl protecting group is TBDMS.

26. The compound according to claim 21, wherein R 30 It is a reactive phosphorus group.

27. The compound of claim 26, wherein the reactive phosphorus group is a phosphoramide, H-phosphonate, alkyl-phosphonate, or triphosphate.

28. The compound according to claim 27, wherein the reactive phosphorus group is -P(OR P )N(R P2 )2, -P(SR P )N(R P2 )2, -P(O)(OR P )N(R P2 )2, -P(S)(OR P )N(R P2 )2, -P(R P3 )N(R P2 )2, -P(O)(SR P )N(R P2 )2, -P(O)(OR P )H, -P(S)(OR P )H, -P(O)(SR P )H, -P(O)(OR P )R P3 , -P(S)(OR P )R P3 or -P(O)(SR P )R P3 , wherein: Each R P3 C1-C independently of optional substitution 30 Alkyl, optionally substituted C2-C 30 alkenyl or optionally substituted C2-C 30 Alkyne groups (e.g., optionally substituted C1-C) 10 Alkyl, optionally substituted C2-C 10 alkenyl or optionally substituted C2-C 10 (alkynyl group); Each R P C can be substituted independently. 1-6 Alkyl; and Each R P2 C can be substituted independently. 1-6 Alkyl (e.g., methyl, ethyl, propyl, or isopropyl, such as isopropyl). Or two Rs P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3-8 membered heterocyclic group; Or R P And an R P2 Together with the atoms to which they are attached, they form optional 4-8 membered heterocyclic groups.

29. The compound according to claim 28, wherein the reactive phosphorus group is -P(OR) P )N(R P2 )2.

30. The compound according to claim 28 or 29, wherein R P It is a C that is substituted with a cyano group or -SC(O)Ph. 1-6 alkyl.

31. The compound according to any one of claims 28-30, wherein R P It is –CH2CH2CN.

32. The compound according to any one of claims 28-31, wherein each R P2 It can be methyl, ethyl, propyl or isopropyl independently.

33. The compound according to any one of claims 28-32, wherein each R P2 It is isopropyl.

34. The compound according to any one of claims 28-33, wherein R P3 It is an optional substituted C1-C6 alkyl group (e.g., methyl).

35. The compound according to any one of claims 15-20, wherein R 3 Yes - OR 30 , where R 30 It is a bond with a nucleoside or nucleotide, or a bond with an oligonucleotide.

36. The compound of claim 35, wherein the R 30 It is a bond with oligonucleotides.

37. The compound according to claim 36, wherein R 3 Linked to oligonucleotides via phosphodiester bonds or modified internucleotide linkages (e.g., thiophosphate bonds).

38. The compound according to claim 36 or 37, wherein R 3 Linked to the 5'-end of the oligonucleotide (e.g., the 5'-hydroxyl group at the 5'-end of the oligonucleotide).

39. The compound according to any one of claims 1-14, wherein R 3 It is hydrogen or halogen (e.g., F).

40. The compound according to any one of claims 1-14, wherein R 3 Yes - OR 20 .

41. The compound according to claim 40, wherein R 20 C is an optional substitute 1-6 alkyl.

42. The compound according to claim 41, wherein R 20 It is methyl, ethyl, propyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl or 2-(N-methylamino)-2-oxoethyl, optionally, R 20 It is methyl or 2-(N-methylamino)-2-oxoethyl.

43. The compound according to claim 40, wherein R 20 C is an optional substitute 2-6 Alkenyl group.

44. The compound according to claim 40, wherein R 20 C is an optional substitute 2-6 Alkenyl (e.g., propargyl).

45. The compound according to any one of claims 39-44, wherein R 2 Yes - OR 30 , where R 30 It is a hydrogen or hydroxyl protecting group or a reactive phosphorus group.

46. ​​The compound according to claim 45, wherein R 30 It is a hydrogen or hydroxyl protecting group.

47. The compound according to claim 46, wherein R 30 It is hydrogen.

48. The compound according to claim 47, wherein R 30 It is a hydroxyl protecting group.

49. The compound according to claim 48, wherein the hydroxyl protecting group is tert-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylhexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylsilyl (DPMS), or tert-butylmethoxyphenylsilyl (TBMPS), optionally, the hydroxyl protecting group is TBDMS.

50. The compound according to claim 45, wherein R 30 It is a reactive phosphorus group.

51. The compound of claim 50, wherein the reactive phosphorus group is a phosphoramide, H-phosphonate, alkylphosphonate, or triphosphate.

52. The compound according to claim 51, wherein the reactive phosphorus group is -P(OR P )N(R P2 )2, -P(SR P )N(R P2 )2, -P(O)(OR P )N(R P2 )2, -P(S)(OR P )N(R P2 )2, -P(R P3 )N(R P2 )2, -P(O)(SR P )N(R P2 )2, -P(O)(OR P )H, -P(S)(OR P )H, -P(O)(SR P )H, -P(O)(OR P )R P3 , -P(S)(OR P )R P3 or -P(O)(SR P )R P3 , wherein: Each R P3 C1-C independently of optional substitution 30 Alkyl, optionally substituted C2-C 30 alkenyl or optionally substituted C2-C 30 Alkyne groups (e.g., optionally substituted C1-C) 10 Alkyl, optionally substituted C2-C 10 alkenyl or optionally substituted C2-C 10 (alkynyl group); Each R P C can be substituted independently. 1-6 Alkyl; and Each R P2 C can be substituted independently. 1-6 alkyl, Or two Rs P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3-8 membered heterocyclic group; Or R P And an R P2 Together with the atoms to which they are attached, they form optional 4-8 membered heterocyclic groups.

53. The compound according to claim 52, wherein the reactive phosphorus group is -P(OR) P )N(R P2 )2.

54. The compound according to any one of claims 52 or 53, wherein R P It is a C that is substituted with a cyano group or -SC(O)Ph. 1-6 alkyl.

55. The compound according to any one of claims 52-54, wherein R P It is –CH2CH2CN.

56. The compound according to any one of claims 52-55, wherein each R P2 It can be methyl, ethyl, propyl or isopropyl independently.

57. The compound according to any one of claims 52-56, wherein each R P2 It is isopropyl.

58. The compound according to any one of claims 52-57, wherein R P3 It is an optional substituted C1-C6 alkyl group (e.g., methyl).

59. The compound according to any one of claims 39-44, wherein R 2 Yes - OR 30 , where R 30 It is a bond with a nucleoside or nucleotide, or a bond with an oligonucleotide.

60. The compound according to claim 59, wherein R 30 It is a bond with oligonucleotides.

61. The compound according to claim 60, wherein R 2 Linked to oligonucleotides via phosphodiester bonds or modified internucleotide linkages (e.g., thiophosphate bonds).

62. The compound according to claim 60 or 61, wherein R 2 Linked to the 5'-end of the oligonucleotide (e.g., the 5'-hydroxyl group at the 5'-end of the oligonucleotide).

63. The compound according to any one of claims 1-6, wherein: R 2 Is it hydrogen, F, or -OR? 20 , Where R 20 It is hydrogen, and the C atoms can be substituted. 1-6 Alkyl groups (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl, 2-(N-methylamino)-2-oxoethyl), optionally substituted C 2-6 Alkenyl or optionally substituted C 2-6 Alkyne (e.g., propynyl); and R 3 is - OR 30 , in R30 It is a hydrogen or hydroxyl protecting group or a reactive phosphorus group.

64. The compound according to claim 63, wherein R 30 It is a hydrogen or hydroxyl protecting group.

65. The compound according to claim 63, wherein R 30 It is a reactive phosphorus group.

66. The compound of claim 65, wherein the reactive phosphorus group is a phosphoramide, H-phosphonate, alkylphosphonate, or triphosphate.

67. The compound according to claim 66, wherein the reactive phosphorus group is -P(OR P1) N(R P2 )2, -P(SR P1) N(R P2 )2, -P(O)(OR P1) N(R P2 )2, -P(S)(OR P1) N(R P2 )2, -P(R P3 )N(R P2 )2, -P(O)(SR P1) N(R P2 )2, -P(O)(OR P1) H, -P(S)(OR P1) H, -P(O)(SR P1) H, -P(O)(OR P1) R P3 , -P(S)(OR P1) R P3 or -P(O)(SR P1) R P3 where: Each R P3 C1-C independently of optional substitution 30 Alkyl, optionally substituted C2-C 30 alkenyl or optionally substituted C2-C 30 Alkyne groups (e.g., optionally substituted C1-C) 10 Alkyl, optionally substituted C2-C 10 alkenyl or optionally substituted C2-C 10 (alkynyl group); Each R P1 C can be substituted independently. 1-6 Alkyl; and Each R P2 C can be substituted independently. 1-6 alkyl, Or two Rs P2 Together with the nitrogen atom to which it is attached, it forms an optionally substituted 3-8 membered heterocyclic group; Or R P1 And an R P2 Together with the atoms to which they are attached, they form optional 4-8 membered heterocyclic groups.

68. The compound according to claim 67, wherein the reactive phosphorus group is -P(OR) P1 )N(R P2 )2.

69. The compound according to claim 67 or 68, wherein R P1 It is a C that is substituted with a cyano group or -SC(O)Ph. 1-6 alkyl.

70. The compound according to any one of claims 67-69, wherein R P1 It is -CH2CH2CN.

71. The compound according to any one of claims 67-70, wherein each R P2 It can be methyl, ethyl, propyl or isopropyl independently.

72. The compound according to any one of claims 67-71, wherein each R P2 It is isopropyl.

73. The compound according to any one of claims 66-72, wherein the reactive phosphorus group is -P(OR) P1 )N(R P2 )2, where R P1 It is –CH2CH2CN, and each R P2 It is isopropyl.

74. The compound according to any one of claims 63-73, wherein R 2 Is it hydrogen, F, or -OR? 20 , where R 20 It is methyl or 2-methoxyethyl.

75. The compound according to any one of claims 63-74, wherein each R V Independently for hydrogen (e.g., It is *-P(O)(OH)2).

76. The compound according to any one of claims 64-74, wherein each R V Independently protected by a hydroxyl group (e.g., It is *-P(O)(OCH2CH3)2 or *-P(O)(OCH2OC(O)C(CH3)3)2).

77. The compound according to claim 1, wherein the compound is selected from: , , , , , , or .

78. An oligonucleotide with a 5' end modification, the structure of which is as follows: , in: X is O or S; and Each R V It is independently protected by hydrogen or hydroxyl groups.

79. The oligonucleotide of claim 78, wherein the 5' end modification comprises the following structure: 。 80. The oligonucleotide of claim 78, wherein the 5' end modification comprises the following structure: 。 81. An oligonucleotide wherein the 5' terminal nucleotide has the following structure: , in: B is an optional modified base (e.g., uracil). Each R V Independently protected by hydrogen or hydroxyl groups (e.g., ethyl or neopentyloxymethyl(CH3)3CC(O)OCH2-, POM); R 2 and R 3 One of them is hydrogen, halogen, or -OR 20 ,in: R 20 It is a hydrogen, hydroxyl protecting group, or optionally substituted C 1-6 Alkyl groups (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxypropyl-2-yl, 2-(N-methylamino)-2-oxoethyl), optionally substituted C2-6 alkenyl groups, or optionally substituted C2-6 alkenyl groups. 3-6 alkynyl (e.g., propynyl); and R 2 and R 3 The other one is -OR 30 ,in: R 30 It is a bond with an oligonucleotide (e.g., an internucleotide linking the oligonucleotide to a subsequent nucleotide).

82. The oligonucleotide of claim 81, wherein the 5' terminal nucleotide has the following structure: 。 83. The compound according to claim 81, wherein the compound has the following structure: 。 84. The oligonucleotide of claim 81, wherein the 5' terminal nucleotide has the following structure: 。 85. The oligonucleotide of claim 84, wherein the 5' terminal nucleotide has the following structure: 。 86. The oligonucleotide of claim 84, wherein the 5' terminal nucleotide has the following structure: 。 87. The oligonucleotide according to any one of claims 79-86, wherein R 3 Yes - OR 30 .

88. An oligonucleotide, wherein the oligonucleotide is a compound according to any one of claims 35-38 or 59-62.

89. The oligonucleotide according to any one of claims 78-88, wherein the length of the oligonucleotide is 10 to 50 nucleotides (e.g., 15 to 40 nucleotides), wherein the compound of formula (I) is a single nucleotide.

90. The oligonucleotide of claim 89, wherein the oligonucleotide is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, optionally, the oligonucleotide is 17, 18, 19, 21, 22, 23, 24 or 25 nucleotides in length.

91. The oligonucleotide according to any one of claims 78-90, wherein the oligonucleotide comprises at least one nucleic acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more independently selected modifications).

92. The oligonucleotide of claim 91, wherein the oligonucleotide comprises at least one nucleic acid modification selected from nucleobase modifications, sugar modifications, internucleotide linkage modifications, conjugates (e.g., ligands), and any combination thereof.

93. The oligonucleotide according to any one of claims 78-92, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-OMe nucleotide.

94. The oligonucleotide according to any one of claims 78-93, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the thermally unstable modification of the double strand.

95. The oligonucleotide according to claim 94, wherein the double-stranded thermally unstable modification is located at position 4, 5, 6, 7 or 8 counting from the 5' end of the oligonucleotide, wherein the compound of formula (I) is located at position 1 counting from the 5' end of the oligonucleotide, optionally, the double-stranded thermally unstable modification is located at position 6, 7 or 8 counting from the 5' end of the oligonucleotide, preferably, the double-stranded thermally unstable modification is located at position 7 counting from the 5' end of the oligonucleotide.

96. The oligonucleotide according to any one of claims 78-95, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-F nucleotide.

97. The oligonucleotide of claim 96, wherein the oligonucleotide contains a 2'-F nucleotide at least at positions 2, 14, and 16, counting from the 5' end of the oligonucleotide, wherein the compound of formula (I) is located at position 1 at the 5' end of the oligonucleotide, optionally, the oligonucleotide contains a 2'-F nucleotide at least at positions 2, 6, 14, and 16, counting from the 5' end of the oligonucleotide, preferably, the oligonucleotide contains a 2'-F nucleotide at least at positions 2, 6, 9, 14, and 16 or at least at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the oligonucleotide.

98. The compound according to any one of claims 78-97, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy(2'-H) nucleotide.

99. The oligonucleotide of claim 98, wherein the oligonucleotide comprises at least 2'-deoxynucleotides at positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the oligonucleotide, wherein the compound of formula (I) is at position 1, counting from the 5' end of the oligonucleotide; optionally, the oligonucleotide comprises at least 2'-deoxynucleotides at positions 5, counting from the 5' end of the oligonucleotide; preferably, the oligonucleotide comprises at least one 2'-deoxynucleotide at positions 2, 5, and 9, or at least at positions 2, 5, 7, and 12, or at least at positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the oligonucleotide.

100. The oligonucleotide according to any one of claims 78-99, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) non-natural or modified nucleobases.

101. The oligonucleotide according to any one of claims 78-100, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified internucleotide linker.

102. The oligonucleotide according to claims 78-101, wherein the oligonucleotide comprises, starting from the 5' end of the oligonucleotide, phosphate thioester linkages between nucleotides at positions 1 and 2 and between nucleotides at positions 2 and 3, wherein the compound of formula (I) is located at nucleotide position 1 at the 5' end of the oligonucleotide; and the oligonucleotide comprises, starting from the 3' end of the oligonucleotide, phosphate thioester linkages between nucleotides at positions 1 and 2 and between nucleotides at positions 2 and 3.

103. The oligonucleotide according to any one of claims 78-102, wherein the oligonucleotide is covalently linked to a carrier, such as a solid carrier.

104. A double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are substantially complementary, and wherein one of the sense strand or the antisense strand is an oligonucleotide according to any one of claims 78-103.

105. The dsRNA of claim 104, wherein the antisense strand is an oligonucleotide of any one of claims 78-103.

106. The dsRNA according to claim 104 or 105, wherein the dsRNA is capable of inducing RNA interference.

107. A method for reducing the expression of a target gene in a subject, comprising administering to the subject: (i) a double-stranded RNA according to any one of claims 104-106, wherein the antisense strand is substantially complementary to the target gene; or (ii) an oligonucleotide according to any one of claims 78-103, wherein the oligonucleotide is substantially complementary to the target gene.

108. A composition comprising a compound according to any one of claims 1-77, an oligonucleotide according to any one of claims 78-103, or a dsRNA according to any one of claims 104-106.

109. A kit comprising the compound of any one of claims 1-77, the oligonucleotide of any one of claims 78-103, or the dsRNA of any one of claims 104-106.

110. A cell comprising the compound of any one of claims 1-77, the oligonucleotide of any one of claims 78-103, or the dsRNA of any one of claims 104-106.

111. The cell of claim 110, wherein the cell is in vivo.

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