NOVEL NUCLEOTIDES AND OLIGONUCLEOTIDES AND RNAi AGENTS COMPRISING THE SAME

Abstract

The present disclosure relates to novel destabilizing molecules, such as a butadiol moiety, modified nucleosides incorporating the butadiol moiety, modified nucleotides incorporating the butadiol moiety, and oligonucleotides and RNAi agents comprising such modified nucleotides that improve selectivity and / or reduce off target effects while maintaining or improving knockdown of on target gene expression.

Description

NOVEL NUCLEOTIDES AND OLIGONUCLEOTIDES AND RNAi AGENTS COMPRISING THE SAMESEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled “30407_WO” created 13-February- 2025 and is 1,351 kilobytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention is directed to novel nucleotides comprising a butadiol moiety. The present invention is also directed to novel nucleosides comprising a butadiol moiety. The present invention is also directed to oligonucleotides comprising the novel nucleoside or the novel nucleotide. The present invention is also directed to oligonucleotides, such as RNAi agents, comprising a novel destabilizing nucleotide, the destabilizing nucleotide comprising a butadiol moiety. The present invention is also directed to methods for increasing the selectivity' of RNAi agents and / or oligonucleotides through the introduction of a destabilizing nucleotide comprising a butadiol moiety'.BACKGROUND OF THE INVENTION

[0003] Molecules comprising oligonucleotides that modulate mRNA levels of genes are important for the study of molecular biology' and increasingly showing promise as therapies.

[0004] RNA interference (RNAi) is a highly conserved regulatory mechanism in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA molecules.

[0005] As RNA is relatively unstable, much effort has been placed on balancing the stability of the RNA molecules and the sequence-specific knockdown capability with any cellular or organ toxicity. Nucleotides of double stranded RNA have been modified to improve their knockdown selectivity by reducing hybridization to off target RNAsequences. However, nucleotide modifications to reduce off-target hybridization, such as nucleotides comprising glycol (glycol nucleic acid, GNA), have been shown to reduce off- target and on-target binding. Thus, there remains a need for such destabilizing molecules that are more selective in reducing off target binding.SUMMARY OF THE INVENTION

[0006] Disclosed herein is an oligonucleotide comprising a compound of the formula:wherein Xi is a 5’ portion of the oligonucleotide, X2 and X3 are one or more independently selected nucleotides, X4 is a 3’ portion of the oligonucleotide, m and n are independently selected from any whole number from 0 to 40, and XB is a butadiol modified nucleotide.

[0007] Disclosed herein is an oligonucleotide comprising a compound of the formula:wherein Xi is a 5’ portion of the oligonucleotide, X2 and X3 are nucleotides, X4 is a 3’ portion of the oligonucleotide, B is a nucleobase, and m and n are independently selected from any whole number from 0 to 40.

[0008] Also disclosed herein is an oligonucleotide comprising a compound of the formula:wherein Xi is a 5’ portion of the oligonucleotide, X2 and X3 are nucleotides, X4 is a 3‘ portion of the oligonucleotide, B is a nucleobase, and m and n are independently selected from any whole number from 0 to 40.

[0009] Also disclosed herein is an oligonucleotide comprising a compound of the formula:wherein Xi is a 5’ portion of the oligonucleotide, X2 and X3 are nucleotides, X4 is a 3’ portion of the oligonucleotide, B is a nucleobase, and m and n are independently selected from any whole number from 0 to 40.

[0010] Disclosed herein is an oligonucleotide of the formula:wherein, each B is independently a nucleobase, m and n are independently selected from any whole number from 0 to 20, X is O or S, R is independently selected from H, O-Ri, F, Cl, and Ri is H, C1to C20 alkyl, or C1to C20 alkyl-O-C1to C20 alkyl.

[0011] Also disclosed herein is an oligonucleotide of the formula:wherein, each B is independently a nucleobase, m and n are independently selected from any whole number from 0 to 20, X is O or S, R is independently selected from H, O-Ri, F, Cl, and Ri is H, C1to C20 alkyl, or C1to C20 alkyl-O- C1to C20 alkyl.

[0012] Also disclosed herein is an oligonucleotide of the formula:wherein, each B is independently a nucleobase, m and n are independently selected from any whole number from 0 to 20, X is O or S, R is independently selected from H, O-R1, F, Cl, and Ri is H, C1to C20 alkyl, or C1to C20 alkyl-O- C1to C20 alkyl.

[0013] Also disclosed herein is a compound of the formula:wherein B is a nucleobase and Y is an organic protecting group.

[0014] Also disclosed herein is a compound of the formula:wherein B is a nucleobase. Y is an organic protecting group, and Z is phosphate or is a phosphoramidite represented by a compound of the formula:

[0015] In some embodiments, the oligonucleotide has a butadiol modified nucleotide at position 3, 4, 5, 6, 7, or 8 starting from the 5’ end. In other aspects, the butadiol modified nucleotide is at position 5, 6, 7, or 8 starting from the 5’ end. In other aspects, the butadiol modified nucleotide is at position 5. 6, or 8 starting from the 5’ end. In other aspects, the butadiol modified nucleotide is at position 5 starting from the 5’ end. In other aspects, the butadiol modified nucleotide is at position 8 starting from the 5’ end.

[0016] In some embodiments, the oligonucleotide has a butadiol modified nucleotide at position 22 starting from the 5’ end. In other aspects, the oligonucleotide has a butadiol modified nucleotide at position 23 starting from the 5’ end. In other aspects, the oligonucleotide has a butadiol modified nucleotide at both positions 22 and 23 starting from the 5' end.

[0017] In some embodiments, the oligonucleotide of Formula I is 15 to 30 nucleotides in length. In some aspects, the oligonucleotide is 18 to 30 nucleotides in length. In other aspects, the oligonucleotide is 23 nucleotides in length.

[0018] In some embodiments, the R of Formula I of the oligonucleotide is 2‘fluoro at one of the following positions:e. 2, 14, and 16 from the 5’ end; or f. 2, 6, 14, and 16 from the 5’ end of the oligonucleotide, and the R is 2’0-methyl at all the other positions of the oligonucleotide.

[0019] Also disclosed herein is an RNA agent comprising a sense strand oligonucleotide and an antisense strand oligonucleotide each independently 15 to 30 nucleotides in length, wherein the sense strand oligonucleotide and the antisense strand oligonucleotide form a duplex, wherein optionally one or more nucleotides of the sense strand and the nucleotides of the antisense strand are independently modified nucleotides, and wherein optionally one or more intemucleotide linkages of the sense strand oligonucleotide and the antisense strand oligonucleotide are modified intemucleotide linkages, and the antisense oligonucleotide is the oligonucleotide provided herein.

[0020] Also disclosed herein is a RNAi agent comprising a sense strand that is 21 nucleotides in length and wherein each nucleotide of the sense strand is a modified nucleotide, and wherein the positions of the modified nucleotides comprising a 2’ fluoro modification are present in a group of positions selected from the following: a. Positions 9, 10, and 1 1 from the 5 ’end; b. Positions 7, 9, and 11 from the 5 ’end; c. Positions 7, 9, and 10 from the 5 'end; and d. Positions 7, 10, and 11 from the 5 ’end, and wherein 2’O-methyl modifications are at all the other positions of the sense strand oligonucleotide.

[0021] Also disclosed herein is a method of increasing the selectivity of an RNAi agent, the method comprising replacing a sugar moiety with a butadiol moiety. An RNAi agent of the present disclosure may include a delivery moiety conjugated to an RNA duplex, optionally via a linker. In some embodiments, the delivery moiety may be a GalNAc moiety. In some embodiments, the delivery moiety is conjugated to the sense strand of the RNA duplex.DETAILED DESCRIPTION

[0022] Accordingly, disclosed herein are novel destabilizing molecules, modified nucleotides incorporating such destabilizing molecules, and oligonucleotides and RNAiagents comprising such modified nucleotides that improve selectivity of knockdown of target gene expression or reduce off target effects while maintaining or improving knockdown of on target gene expression.

[0023] It has surprisingly been found that by incorporating the disclosed destabilizing molecules into a nucleotide, oligonucleotide, and / or RNAi agent that the selectivity of knockdown of target gene expression can be improved relative to an unmodified nucleotide, oligonucleotide, and / or RNAi agent and relative to other modified RNAi agents, such as GNA.DEFINITIONS

[0024] The RNAi agents herein compnse a sense strand and an antisense strand, wherein each strand is an oligonucleotide.

[0025] As used herein, “antisense strand” means a single-stranded oligonucleotide that is complementary to a region of a target sequence. Likewise, and as used herein, “sense strand” means a single-stranded oligonucleotide that is complementary to a region of an antisense strand.

[0026] As used herein, “oligonucleotide” means a polymer of linked nucleotides, each of which can be modified or unmodified. A typical oligonucleotide is less than about 100 nucleotides in length. An oligonucleotide may be single-stranded (ss) or double stranded (ds). An oligonucleotide may or may not have duplex regions. As a set of non-limiting examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (DsiRNA), or antisense oligonucleotide (ASO).

[0027] As used herein, “nucleotide” means an organic compound having a nucleoside (a nucleobase such as, for example, adenine, cytosine, guanine, thymine, or uracil); and a pentose sugar such as, for example, ribose or 2'-deoxyribose; and a phosphate group. A “nucleotide” can serve as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0028] As used herein, “modified intemucleotide linkage” means an intemucleotide linkage having one or more chemical modifications when compared with a reference intemucleotide linkage having a phosphodiester bond. A modified intemucleotide linkagecan be a non-naturally occurring linkage. In some embodiments, the modified intemucleotide linkage is a phosphorothioate linkage.

[0029] As used herein, ‘"modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide can be a non-naturally occurring nucleotide. A modified nucleotide can have, for example, one or more chemical modification in its sugar, nucleobase, and / or phosphate group. Additionally, or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to a corresponding reference nucleotide.

[0030] As used herein, “phosphate analog” means a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5' terminal nucleotide of an oligonucleotide in place of a 5'- phosphate. A 5' phosphate analog can include a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). An oligonucleotide can have a phosphate analog at a 4'-carbon position of the sugar (referred to as a “4'-phosphate analog”) at a 5'-terminal nucleotide. An example of a 4'-phosphate analog is oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (<?.g., at its 4'- carbon) or analog thereof. In one embodiment of a butadiol -modified RNAi agent described herein, the 5’ terminal nucleotide of the antisense strand of the RNA duplex may comprise an OH group, a phosphate group or a phosphate analog. In one embodiment, the 5’ terminal nucleotide of the antisense strand comprises a vinyl phosphonate, a phosphate, or a hydroxyl group.

[0031] As used herein, “duplex,” in reference to nucleic acids or oligonucleotides, such as a sense strand or an antisense strand means a structure formed through hydrogen bonds of complementary base pairing of two antiparallel sequences of nucleotides under suitable conditions to promote such a structure. A duplex may form despite not having full complementarity7between the two strands, or when an abasic nucleotide is present.

[0032] As used herein, “abasic,” means an apurinic or apyrimidinic nucleotide wherein the N-glycosyl bonds are cleaved between the nitrogenous base and the deoxyribose sugar, leaving an intact phosphodiester backbone.

[0033] As used herein, “iRNA,’' “iRNA agent,” “RNAi,” "RNAi agent” and “RNA interference agent” means an agent that contains RNA and mediates the targeted cleavage of a RNA transcript via RNA interference, e.g., through a RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi agent has a sense strand oligonucleotide and an antisense strand oligonucleotide, and the sense strand oligonucleotide and the antisense strand oligonucleotide form a duplex. In some embodiments, the sense and antisense strand oligonucleotides of RNAi agent are 21-23 nucleotides in length. In other embodiments, the sense and antisense strand oligonucleotides can be longer, for example 25-30 nucleotides in length, in which case the longer RNAi sequences are first processed by the Dicer enzy me. In some embodiments, the sense or antisense strand oligonucleotide is conjugated to a delivery moiety to facilitate entry into the cells of various tissues. In some embodiments, the sense or antisense strand oligonucleotide is conjugated directly to the delivery moiety. In other embodiments, the sense or the antisense strand is conjugated indirectly via a linker to the delivery moiety.

[0034] As used herein, “reduced expression,” or “reducing expression”, such as with respect to a gene, means a decrease in the amount or level of RNA transcript or protein encoded by the gene and / or a decrease in the amount or level of activity of the gene in a cell, a population of cells, a sample, or a subject, when compared to an appropriate reference (e.g., a reference cell, population of cells, sample, or subject).

[0035] As used herein, “strand” refers to a single, contiguous sequence of nucleotides linked together through intemucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). A strand can have two free ends (e.g., a 5' end and a 3' end).

[0036] As used herein, “HPRT” refers to a hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT) mRNA, protein, or polypeptide. The nucleic acid sequence of a mouse HPRT mRNA transcript can be found at NM 013556.2:

[0038] As used herein, “Fas” refers to a Fas cell surface death receptor (Fas) mRNA, protein, or polypeptide. The nucleic acid sequence of a human Fas mRNA transcript can be found at NM_000043.6:

[0039] The amino acid sequence of a human Fas protein can be found at NP_000034.1:

[0040] As used herein, “Navi.8” refers to a sodium voltage-gated channel alpha subunit 10 (SCN10A) (Navi.8) mRNA. protein, or polypeptide. The nucleic acid sequence of a human Navi.8 mRNA transcript can be found at NM_001293306.2:

[0041] The amino acid sequence of a human Navi.8 protein can be found atNP_001280235.2:

[0042] As used herein, “Ang8” refers to an angiopoietin like 8 (Ang8) mRNA, protein, or polypeptide. The nucleic acid sequence of a human Ang8 mRNA transcript can be found at NM_018687.7:

[0043] The amino acid sequence of a human Ang8 protein can be found at NP_061157.3:

[0044] As used herein, “HMGCR” refers to 3-hydroxy-3-methylglutaryl-CoA reductase mRNA, protein, or polypeptide. The nucleic acid sequence of a human HMGCR mRNA transcript can be found at NM_000859.3:

[0045] The amino acid sequence of a human HMGCR protein can be found at NP_000850.1:Butadiol

[0046] Disclosed herein is a novel butadiol moiety that can be incorporated into one or more nucleosides and / or nucleotides by replacing one or more sugar moieties. The modified nucleotides can be incorporated into an oligonucleotide, such as an RNAi agent, as a destabilizing agent in the RNAi agent. The butadiol moiety can be represented by the compounds of Formula A, A’, and / or A”.Formula A. Butadiol, wherein B is a nucleobase.Formula A’. (R,S) - Butadiol, wherein B is a nucleobase.Formula A”. (S.S)-Butadiol, wherein B is a nucleobase.

[0047] The butadiol moiety’ can comprise a mixture of the (R,S) and (S,S) diastereomers (Formula A), the (R,S) diastereomer (Formula A’), or the (S,S) diastereomer (Formula A’"). In some embodiments, the butadiol moiety can comprise the (R,R), (R,S), (S,S). and / or the (S,R) diastereomers. It has been surprisingly found that the introduction of (S,S)-butadiol into a nucleotide at position 8 in an oligonucleotide can improve the targeting selectivity of the oligonucleotide.

[0048] In Formula A, A’, and A”, the nucleobase, B, can be any nucleobase. The nucleobase can be a naturally occurring nucleobase, a non-naturally occurring, artificial, or modified nucleobase, or a combination thereof. Suitable modified nucleobases can include hypoxanthine, xanthine, 7-methylguanine, inosine, xanthosine, 7-methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydoxymethylcytosine, dihydrouridine, 5- methyl cytidine, and / or psuedouridine. Suitable naturally occurring nucleobases can include adenine, guanine, cytosine, thymine, and / or uracil. In one aspect, the nucleobase is selected from the group consisting of adenine, cytosine, thymine, uracil, and guanine. In one aspect, the nucleobase is selected from the group consisting of adenine, cytosine, and uracil. In another aspect, the nucleobase is selected from the group consisting of cytosine and uracil. In another aspect, the nucleobase is uracil.Nucleoside

[0049] Disclosed herein is a novel nucleoside comprising the butadiol moiety. As disclosed herein, a butadiol moiety can be incorporated into one or more nucleosides by replacing a sugar moiety in the nucleoside. The novel nucleoside can be represented by a compound of Formula B, B’, and / or B”.Formula B. Novel Nucleoside, wherein A is independently selected from H. a phosphate, C1to C6alkyl, and an organic protecting group, and B is a nucleobase.Formula B’. Novel Nucleoside, wherein A is independently selected from H, a phosphate. C1to C6alkyl, and an organic protecting group, and B is a nucleobase.Formula B”. Novel Nucleoside, wherein A is independently selected from H, a phosphate. C1to C6alkyl, and an organic protecting group, and B is a nucleobase.

[0050] In Formula B, B', and B" , A can be independently selected from H, a phosphate, C1to C6alkyl, and an organic protecting group. Suitable organic protecting groups can include methoxylmethyl ether (MOM), Methoxyethoxymethyl ether (MEM), tert-butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acety l, benzy l (Bn), dimethoxytrityl (DMT), methoxytrityl (MMT), p-Methoxybenzyl ether (PMB), p- Methoxyphenyl ether (PMP). pivaloyl (Piv). tetrahydropyranyl (THP), tetrahydrofuran (THF), trity l (Tr), and / or trimethyl silyl (TMS). The organic protecting group can comprise dimethoxytrityl (DMT).

[0051] In Formula B, B’, and B”. the nucleobase, B, the nucleobase, B, can be any nucleobase. The nucleobase can be a naturally occurring nucleobase, a non-naturally occurring, artificial, or modified nucleobase, or a combination thereof. Suitable modified nucleobases can include hypoxanthine, xanthine, 7-methylguanine, inosine, xanthosine, 7- methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydoxymethylcytosine, dihydrouridine, 5-methylcytidine. and / or psuedouridine. Suitable naturally occurring nucleobases can include adenine, guanine, cytosine, thymine, and / or uracil. In one aspect, the nucleobase is selected from the group consisting of adenine, cytosine, thymine, uracil, and guanine. In one aspect, the nucleobase is selected from the group consisting of adenine, cytosine, and uracil. In another aspect, the nucleobase is selected from the group consisting of cytosine and uracil. In another aspect, the nucleobase is uracil.

[0052] The novel nucleoside can also be represented by a compound of Formula C, C’, and / or C”.Formula C. Novel Nucleoside, wherein B is a nucleobase and Y is an organic protecting group.Formula C’. Novel Nucleoside, wherein B is a nucleobase and Y is an organic protecting group.Formula C”. Novel Nucleoside, wherein B is a nucleobase and Y is an organic protecting group.

[0053] The novel nucleoside can also be represented by a compound of Formula D, D', and / or D”.Formula D. Novel Nucleoside, wherein B is a nucleobase.Formula D’. Novel Nucleoside, wherein B is a nucleobase.Formula D”. Novel Nucleoside, wherein B is a nucleobase.

[0054] In Formula C, C’, and C, the organic protecting group can include methoxylmethyl ether (MOM). Methoxyethoxymethyl ether (MEM), lert- butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn),dimethoxytrityl (DMT), methoxytrityl (MMT), p-Methoxybenzyl ether (PMB), p- Methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), and / or trimethyl silyl (TMS). The organic protecting group can comprise dimethoxy trityl (DMT).

[0055] In Formula C, C’, C”, D, D’, and / or D ', the nucleobase, B, can be any nucleobase. The nucleobase can be a naturally occurring nucleobase. a non-naturally occurring, artificial, or modified nucleobase, or a combination thereof. Suitable modified nucleobases can include hypoxanthine, xanthine, 7-methylguanine, inosine, xanthosine, 7- methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydoxymethylcytosine, dihydrouridine, 5-methylcytidine. and / or psuedouridine. Suitable naturally occurring nucleobases can include adenine, guanine, cytosine, thymine, and / or uracil. In one aspect, the nucleobase is selected from the group consisting of adenine, cytosine, thymine, uracil, and guanine. In one aspect, the nucleobase is selected from the group consisting of adenine, cytosine, and uracil. In another aspect, the nucleobase is selected from the group consisting of cytosine and uracil. In another aspect, the nucleobase is uracil.Nucleotide

[0056] Disclosed herein is a novel nucleotide comprising the butadiol moiety, a nucleobase, and a phosphate or a phosphoramidite to allow for selective incorporation into an RNAi agent, such as an oligonucleotide. As disclosed herein, a butadiol moiety can be incorporated into one or more nucleotides by replacing a sugar moiety in the nucleotide. The novel nucleotide can be represented by a compound of Formula E, E’, and / or E”.Formula E. Novel Nucleotide, wherein B is a nucleobase, Y is an organic protecting group, and Z is phosphate or is a phosphoramidite represented by Formula F.Formula E’. Novel Nucleotide, wherein B is a nucleobase. Y is an organic protecting group, and Z is phosphate or is a phosphoramidite represented by Formula F.Formula E”. Novel Nucleotide, wherein B is a nucleobase, Y is an organic protecting group, and Z is phosphate or is a phosphoramidite represented by Formula F.Formula F. Phosphoramidite

[0057] In Formula E, E’, and E”, the organic protecting group can include methoxylmethyl ether (MOM), methoxyethoxymethyl ether (MEM), tert-butyldimethylsilane (TBDMS), tert-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxy trityl (DMT), methoxytrityl (MMT). -Methoxybenzyl ether (PMB), p-Methoxyphenyl ether (PMP), pivaloyl (Piv). tetrahydropyranyl (THP), tetrahydrofuran (THF). trityl (Tr), and / or trimethyl silyl (TMS). The organic protecting group can comprise dimethoxytrityl (DMT).

[0058] The novel nucleotide can also be represented by a compound of Formula G, G’, and / or G ’.Formula G. Modified Nucleotide, wherein B is a nucleobase.Formula G’. Modified Nucleotide, wherein B is a nucleobase.Formula G”. Modified Nucleotide, wherein B is a nucleobase.

[0059] In Formula E, E', E’’, G, G’, and / or G ', the nucleobase, B, can be any nucleobase. The nucleobase can be a naturally occurring nucleobase, a non-naturally occurring, artificial, or modified nucleobase, or a combination thereof. Suitable modified nucleobases can include hypoxanthine, xanthine, 7-methylguanine, inosine, xanthosine, 7- methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydoxymethylcytosine, dihydrouridine, 5-methylcytidine. and / or psuedouridine. Suitable naturally occurring nucleobases can include adenine, guanine, cytosine, thymine, and / or uracil. In one aspect, the nucleobase is selected from the group consisting of adenine, cytosine, thymine, uracil, and guanine. In one aspect, the nucleobase is selected from the group consisting of adenine, cytosine, and uracil. In another aspect, the nucleobase is selected from the group consisting of cytosine and uracil. In another aspect, the nucleobase is uracil.Oligonucleotide

[0060] Disclosed herein is a novel oligonucleotide comprising the butadiol moiety. As disclosed herein, a butadiol moiety can be incorporated into one or more nucleotides by replacing a sugar moiety in the nucleotide.

[0061] The novel oligonucleotide can be represented by a compound of Formula 1.Formula H. Oligonucleotide, wherein Xi is a 5’ portion of the oligonucleotide, X2 and X3are one or more independently selected nucleotides, X4 is a 3’ portion of the oligonucleotide, m and n are independently selected from any whole number from 0 to 40, and XB is a butadiol modified nucleotide.

[0062] In Formula H, m and n can be independently selected from any whole number from 0 to 20. In one embodiment, m and n can be independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17. 18. 19. or 20. In one embodiment, m can be from 0 to 7, n can be from 0 to 20, and m+n can be from 0 to 40.

[0063] The novel oligonucleotide can also be represented by a compound of Formula I, I’, and / or T‘.Formula I. Oligonucleotide, wherein Xi is a 5’ portion of the oligonucleotide, X2 and X3 are nucleotides, X4 is a 3’ portion of the oligonucleotide, and m and n are independently selected from any whole number from 0 to 40.Formula I’. Oligonucleotide, wherein Xi is a 5’ portion of the oligonucleotide. X2 and X3 are nucleotides, X4 is a 3’ portion of the oligonucleotide, and m and n are independently selected from any whole number from 0 to 40.Formula I”. Oligonucleotide, wherein Xi is a 5’ portion of the oligonucleotide, X2 and X3 are nucleotides, X4 is a 3’ portion of the oligonucleotide, and m and n are independently selected from any whole number from 0 to 40.

[0064] The 5’ portion of the oligonucleotide, such as Xi in Formula I, I’, and / or I”, is the 5’ end of the oligonucleotide. The 5’ portion of the oligonucleotide can include a nucleotide, a nucleoside, a nucleobase, a pentose sugar, a phosphate, a vinyl phosphonate, ahalogen, an alcohol, C1to C20 alkyl. C1to C20 alkyl-O- C1to C20 alkyl and / or combinations thereof. The 5’ end of the oligonucleotide can be easily modified using methods known to a person of ordinary skill in the art.

[0065] The 3’ portion of the oligonucleotide, such as X4 in Formula I, I’, and / or I”, is the 3’ end of the oligonucleotide. The 5’ portion of the oligonucleotide can include a nucleotide, a nucleoside, a nucleobase, a pentose sugar, a phosphate, a vinyl phosphonate, ahalogen, an alcohol, C1to C20 alkyl, C1to C20 alkyl-O- C1to C20 alkyl and / or combinations thereof. The 3 ‘ end of the oligonucleotide can be easily modified using methods know n to a person of ordinary skill in the art.

[0066] The oligonucleotide can also include one or more nucleotides, which do not contain the butadiol moiety, such as X2 and X3 in Formula I, I’, and / or I”. Suitable nucleotides can include a pentose sugar, a phosphate, a nucleobase, and / or an other phosphate linker.

[0067] In Formula I, I’, and / or I”, m and n can be independently selected from any whole number from 0 to 20. In one embodiment, m can be any whole number from 0 to 7, n can be any whole number from 0 to 40, and m+n can be any whole number from 0 to 40.

[0068] The novel oligonucleotide can be represented by a compound of Formula J.Formula J. Oligonucleotide and / or RNAi agent, wherein B is a nucleobase, m and n are independently selected from any whole number from 0 to 20, X is O or S, R is independently selected from H, O-R1, F, Cl, and Ri is H, C1to C20 alkyl, or C1to C20 alkyl-O- C1to C20 alkyl.

[0069] In Formula J, m and n can be independently selected from any whole number from 0 to 20. In one embodiment, m and n can be independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0070] In Formula J, the nucleobase can be selected from the group consisting of adenine, cytosine, thymine, uracil, and guanine. In an embodiment, the nucleobase is selected from the group consisting of adenine, cytosine, and uracil. In a furtherembodiment, the nucleobase is selected from the group consisting of cytosine and uracil. In a further embodiment, the nucleobase is uracil. In one embodiment, the modified nucleotide is at position 3, 4, 5, 6, 7, or 8 starting from the 5’ end. In one embodiment, the modified nucleotide is at position 5, 6, 7, or 8 starting from the 5 ’ end. In one embodiment, the modified nucleotide is at position 5, 6, or 8 starting from the 5' end. In one embodiment, the modified nucleotide is at position 5 starting from the 5’ end.

[0071] The modified nucleotide can be present in the antisense strand and / or the sense strand. Preferably, the modified nucleotide is present in the antisense strand.

[0072] The novel oligonucleotide can be represented by a compound of Formula K.Formula K. Oligonucleotide and / or RNAi agent, wherein B is a nucleobase, m and n are independently selected from any whole number from 0 to 20, X is O or S, R is independently selected from H, O-R1, F, Cl, and Ri is H, C1to C20 alkyl, or C1to C20 alkyl-O- C1to C20 alkyl.

[0073] In Formula K, m and n can be independently selected from any whole number from 0 to 20. In one embodiment, m and n can be independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0074] In Formula K, the nucleobase can be selected from the group consisting of adenine, cytosine, thymine, uracil, and guanine. In an embodiment, the nucleobase is selected from the group consisting of adenine, cytosine, and uracil. In a further embodiment, the nucleobase is selected from the group consisting of cytosine and uracil. In a further embodiment, the nucleobase is uracil. In a further embodiment, the modified nucleotide is at position 3, 4, 5. 6, 7, or 8 starting from the 5 ' end. In a further embodiment, the modified nucleotide is at position 5. 6, 7. or 8 starting from the 5’ end. In a further embodiment, the modified nucleotide is at position 5, 6, or 8 starting from the 5’ end. In a further embodiment, the modified nucleotide is at position 5 starting from the 5 ’ end.

[0075] The novel oligonucleotide can be represented by a compound of Formula L.Formula L. Oligonucleotide and / or RNAi agent, wherein B is a nucleobase. m and n are independently selected from any whole number from 0 to 20, X is O or S, R is independently selected from H, O-R1, F, Cl, and Ri is H, C1to C20 alkyl, or C1to C20 alkyl-O- C1to C20 alkyl.

[0076] In Formula L, m and n can be independently selected from any whole number from 0 to 20. In one embodiment, m and n can be independently selected from 0, 1, 2, 3, 4, 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, or 20.

[0077] In Formula L, the nucleobase can be selected from the group consisting of adenine, cytosine, thymine, uracil, and guanine. In an embodiment, the nucleobase is selected from the group consisting of adenine, cytosine, and uracil. In a further embodiment, the nucleobase is selected from the group consisting of cytosine and uracil. In a further embodiment, the nucleobase is uracil. In a further embodiment, the modified nucleotide is at position 3, 4. 5, 6, 7, or 8 starting from the 5’ end. In a further embodiment, the modified nucleotide is at position 5, 6, 7, or 8 starting from the 5’ end. In a further embodiment, the modified nucleotide is at position 5, 6, or 8 starting from the 5’ end. In a further embodiment, the modified nucleotide is at position 5 starting from the 5’ end.

[0078] One or more butadiol moi eties may be incorporated into any position in the RNAi agent. For instance, a butadiol moiety may be incorporated at position 22, or at position 23, or at both positions 22 and 23, to realize a potential increase in durability, particularly for the antisense strand of the RNAi agent. The butadiol may also be incorporated at position 1, or 2, or 9, or 10, or 11. or 12, or 13, or 14, or 15, or 16. or 17, or 18, or 19, or 20. or 21 of an oligonucleotide.

[0079] The oligonucleotide can comprise a sense strand and / or an antisense strand. In further embodiments, the oligonucleotide is an antisense oligonucleotide. In further embodiment, the antisense oligonucleotide is 15 to 30 nucleotides in length. In still further embodiments, the antisense oligonucleotide is 18 to 30 nucleotides in length. In certain embodiments, the antisense oligonucleotide is 23 nucleotides in length.RNAi Agent

[0080] Disclosed herein is an RNAi agent comprising the butadiol moiety. As disclosed herein, a butadiol moiety can be incorporated into one or more nucleotides by replacing asugar moiety in the nucleotide. The modified nucleotide can be incorporated into an RNAi agent.

[0081] The RNAi agent can comprise a sense strand oligonucleotide and an antisense strand oligonucleotide described herein. In a further embodiment, the RNAi agent is a siRNA.

[0082] In another embodiment described herein is an RNAi agent comprising a sense strand oligonucleotide and an antisense strand oligonucleotide each independently 15 to 30 nucleotides in length, wherein the sense strand oligonucleotide and the antisense strand oligonucleotide form a duplex, wherein optionally one or more nucleotides of the sense strand and the nucleotides of the antisense strand are independently modified nucleotides, and wherein optionally one or more intemucleotide linkages of the sense strand oligonucleotide and the antisense strand oligonucleotide are modified intemucleotide linkages, and wherein the antisense strand oligonucleotide is represented as Formula I, II, or III.

[0083] In yet a further embodiment of the RNAi agents disclosed herein, the antisense strand oligonucleotide of Formula J, Formula K, or Formula L, comprises a 3' overhang of two nucleotides. In a further embodiment, the antisense strand oligonucleotide of Formula J, Formula K, or Formula L is 23 nucleotides in length. In another further embodiment, the sense strand oligonucleotide is 21 nucleotides in length.

[0084] In other embodiments, each nucleotide of the sense strand oligonucleotide and antisense strand oligonucleotide of Formula J, Formula K, or Formula L are modified or further modified and independently comprise a 2-fluoro modified nucleotide or a 2’0- methyl nucleotide.

[0085] In one embodiment of the RNAi agents herein, the antisense strand oligonucleotide of Formula J, Formula K, or Formula L is 23 nucleotides in length and wherein each nucleotide of the antisense strand is a modified nucleotide, and wherein the position of the 2’ -fluoro modified nucleotides is modified to be present at a group of positions selected from the following: a. Positions 2, 3, 7, 14, and 16 from the 5’ end of the antisense strand; b. Positions 2, 5, 7, 14, and 16 from the 5’ end of the antisense strand; c. Positions 2, 3, 8, 14, and 16 from the 5’ end of the antisense strand; d. Positions 2. 5, 8, 14, and 16 from the 5’ end of the antisense strand;e. Positions 2. 6, 14, and 16 from the 5’ end of the antisense strand; and f. Positions 2, 14, and 16 from the 5’ end of the antisense strand.

[0086] In another embodiment of the RNAi agents herein, the sense strand oligonucleotide is 21 nucleotides in length and wherein each nucleotide of the sense strand is a modified nucleotide, and wherein one or more of the modified nucleotides comprises a 2’ fluoro modification that is present at a group of positions selected from the following: a. positions 9, 10, and 11 from the 5 ’-end of the sense strand; b. positions 7, 9, 10, and 11 from the 5’-end of the sense strand; c. positions 7, 9, and 10 from the 5’-end of the sense strand; or d. positions 7, 10, and 11 from the 5 '-end of the sense strand.

[0087] In a further embodiment, of the RNAi agents herein, the modified nucleotides comprise a 2’fluoro modification that is present in the antisense strand oligonucleotide of Formula J, Formula K, or Formula L in a group of positions selected from the following: a. Positions 2, 3. 7, 14, and 16 from the 5’ end of the antisense strand; b. Positions 2, 5. 7, 14, and 16 from the 5’ end of the antisense strand; c. Positions 2, 3, 8, 14, and 16 from the 5’ end of the antisense strand; d. Positions 2, 5, 8, 14, and 16 from the 5’ end of the antisense strand; e. Positions 2, 6, 14, and 16 from the 5’ end of the antisense strand; and the modified nucleotides comprise a 2’fluoro modification that is present in the sense strand in a group of positions selected from the following: a. positions 9, 10, and 11 from the 5 ’-end of the sense strand; b. positions 7, 9, 10, and 11 from the 5’-end of the sense strand; c. positions 7, 9, and 10 from the 5 ’-end of the sense strand; or d. positions 7, 10, and 11 from the 5 ’-end of the sense strand.

[0088] In further embodiments, of the RNAi agents herein, the nucleotides comprise a modification or a further modification that is a modified intemucleotide or intemucleoside linkage. In one embodiment, the sense strand oligonucleotide has four or five phosphorothioate linkages. In another embodiment, the antisense strand oligonucleotide of Formula J, Formula K, or Formula L has four or five phosphorothioate linkages.

[0089] In a further embodiment, the RNAi agents herein comprise at least four intemucleotide linkages that are phosphorothioate linkages that are present at each of the two terminal nucleotides of each of the 5’ and 3’ ends of each of the sense strand and antisense strand.

[0090] In some embodiments, the sense strand or the antisense strand further comprises an abasic moiety or an inverted abasic moiety, e.g, a moiety in Table A. In some embodiments, the sense strand comprises an inverted abasic moiety.Table A. Abasic and inverted abasic (iAb) moieties“5”’ and “3”’ indicate the 5’ to 3’ direction of the sequences.

[0091] In other embodiments, the RNAi agents herein comprise a modified nucleotide at the 5’ end of the antisense strand oligonucleotide of Formula J, Formula K, or Formula L that comprises a further modification of the phosphate group to a phosphate analog. In a further embodiment, the further modification is a phosphate analog that is 5 ’-(E)- vinylphosphonate.

[0092] In other embodiments of the RNAi agent disclosed herein, the sense strand or the antisense strand has a further modification that is an abasic moiety.

[0093] The RNAi agent can further comprise a delivery moiety which targets the RNAi agent to a particular cell, tissue, and / or organ of the patient or subject. By delivering oligonucleotides to a desired cell or tissue of the patient, gene expression can be regulated in the location where it is most beneficial. In one aspect, the delivery moiety can be a lipophilic delivery moiety.

[0094] In one aspect, the delivery moiety may be a GalNAc delivery moiety; that is, a moiety comprising at least one N-acetylgalactosamine (GalNAc). Such a moiety can target the asialoglycoprotein receptor on hepatic cells, thereby increasing delivery to the liver. In one embodiment, the delivery' moiety' can include one, two, three, or four GalNAc moieties. Suitable delivery moieties are described in WO 2022 / 271806.

[0095] In a particular, embodiment, the delivery moiety can also be described by a compound of Formula M.Formula M. Delivery Moiety

[0096] In a moiety of Formula M, L is the delivery moiety linker or a bond (that is, where an additional linker is not present), and Z is the RNA molecule.

[0097] In another aspect, the delivery moiety can be described by a compound of Formula M’:Formula M’. Delivery moiety

[0098] For Formula M?, L may be a linker or a bond, and Z is the RNA molecule.

[0099] The delivery moiety can be connected to the RNAi agent at the 3’ or 5’ end. The delivery moiety can be attached to the RNAi agent through a delivery moiety linker. The delivery' moiety linker can be any suitable organic linker. The delivery moiety linker can be described by a compound of Formula N.Formula N. Deliver}' moiety linker

[0100] In another embodiment, the linker may be described by Formula N’:Formula N’

[0101] In another embodiment, the linker may be described by Formula N”:Formula N”, which in certain embodiments may be of Formula N’ ‘-a or of Formula N”-b:Formula N”-aFormula N”-b.

[0102] In an embodiment, an RNAi agent as described herein may have the structure:wherein Z is the RNA molecule.

[0103] In another embodiment, an RNAi agent as described herein may have the structure:wherein Z is the RNA molecule.

[0104] In some embodiments, the RNAi agent comprises any one of the dsRNAs of Tables la-2d conjugated to the delivery moiety, optionally at 3’ or 5’. In some embodiments the delivery moiety is GalNAc of Preparation 29. In some embodiments, GalNAc is ofFormula M. In some embodiments the dsRNA and GalNAc are conjugated via a linker, such as the delivery moiety linker of Formula N.

[0105] The RNAi agent can also comprise other suitable delivery moi eties. Other suitable delivery' moieties can comprise cholesterol, such as 5' or 3' attached cholesteroltetraethylene CE-Phosphoramidite (CholTEG). Suitable phosphoramidites that can be attached to a cholesterol molecule can include the phosphoramidites listed in Table B.Table B: phosphoramiditesEXAMPLES

[0106] Certain abbreviations are defined as follows: ’'AS" refers to antisense strand; BD " and “bd” are used interchangeably to refer to butadiol; ’t-BuOGH" refers to tert-butyl hydroperoxide; “CT” refers to cycle threshold; “DCM” refers to dichloromethane; “DIPT” refers to diisopropyl tartrate; “DMF” refers to dimethylformamide; “DMT” refers to dimethoxytrityl; “dsRNA” refers to double stranded ribonucleic acid; “EtOAc” refers to ethyl acetate; “GNA” refers to glycol nucleic acid; “hiPSC” refers to human induced pluripotent stem cell; “MeCN” refers to acetonitrile; “MeOH” refers to methanol and methyl alcohol; “PBS” phosphate-buffered saline; “PCR” refers to polymerase chain reaction; “RBF” refers to round bottom flask; “RT-PCR” refers to reverse transcription polymerase chain reaction; “siRNA” refers to small interfering RNA; “SS” refers to sense strand; “TEA” refers to triethylamine; and “Ti(Oi-Pr)4” refers to titanium isopropoxideStep Step

[0107] Scheme 1, step A depicts the synthesis of the compound (2) using the Sharpless asymmetric epoxidation protocol, the conditions of which will be known by one skilled in the art. Step B shows the conversion of compound (2) to compound (3) using Mitsunobu reaction conditions with 4-nitrobenzoic acid, the conditions of which will be known by one skilled in the art. Step C shows hydrolysis of compound (3) in a solvent such as dichloromethane using a base such as cesium carbonate to afford compound (4) which was then protected with DMT to afford compound (6) in step E. Step D was performed in a manner essentially analogous to the method of step E to provide compound (5).Scheme 2

[0108] Scheme 2, step A, compound (5) was treated with compound (7) and using a base such as NaH in a suitable solvent such as DMF to provide compound (8). In step B, the secondary alcohol of compound (8) was phosphitylated with 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile in an appropriate solvent such as DCM to provide compound (9).

[0109] The scheme was repeated starting with compound (6) to provide the other diastereomer.Scheme 3

[0110] Scheme 3, step A was performed in a manner essentially analogous to the method in step A of Scheme 2 to give compound (11). Step B was performed in a manner essentially analogous to the method in step B of Scheme 2 to give compound (12). The scheme was repeated starting with compound (6) to provide the other diastereomer.Scheme 416

[0111] Scheme 4, step A was performed in a manner essentially analogous to the method in step A of Scheme 2 to give compound (14). For Step B. compound (14) was treated with 1,1 -dimethoxy -N,N-dimethylmethanamine in an appropriate solvent such as DMF and heated to provide compound (15). Step C was performed in a manner essentially analogous to the method in step B of Scheme 2 to give compound (16). The scheme was repeated starting with compound (6) to provide the other diastereomer.

[0112] Scheme 5, step A was performed in a manner essentially analogous to the method in step A of Scheme 2 to give compound (18). In step B the benzyl protecting group was removed using catalytic hydrogenation with Pd / C in an appropriate solvent such as EtOAc to provide compound (19). Step C was performed in a manner essentially analogous to the method in step B of Scheme 4 to give compound (20). Step D was performed in a manner essentially analogous to the method in step B of Scheme 2 to give compound (21). The scheme was repeated starting with compound (6) to provide the other diastereomer.Preparation 1But-3 -en-2-ol

[0113] Starting material was purchased from Combi-Blocks, CAS number 598-32-3.Preparation 2(R)- 1 -((S)-Oxiran-2-yl)ethan- 1 -ol

[0114] 4A molecular sieves (20 g) were added to a dry three-neck RBF under nitrogen. DCM (160 mL) was added and the suspension was cooled to -23 °C. (-)-DIPT (1.00 mL, 4.88 mmol) was added, followed by Ti(Oz-Pr)4 (1.20 mL, 4.2 mmol). t-BuOOH (18.2 mL, 100 mmol) was added slowly. Using a mechanical stirrer, the reaction was stirred for 30 minutes. 3-Buten-2-ol (6.00 g, 83.2 mmol) was added dropwise via addition funnel as a solution in DCM (20 mL). The reaction was allowed to stir for 64 hours at -23 °C. The reaction was quenched by the addition of dimethyl sulfide (7 mL). The reaction flask was removed from the cold bath and allowed to warm to ambient temperature. A solution of acetone / water (100 mL / 40 mL) was added, and the suspension was stirred for 1 hour. The reaction was filtered through a pad of diatomaceous earth, washing with DCM. The filtrate was dried over Mg2SO4 and filtered. The solution of crude product was carefully concentrated in vacuo. The resulting solution was purified via silica gel flash chromatography eluting with 0-10% MeOH in DCM to give the title compound as a colorless oil (3.15 g, 43%). 'H NMR (CDCh) 5 4.01(m, 1H), 3.02 (m. 1H), 2.79 (dd, J= 5.0, 3.0 Hz, 1H), 2.73 (dd, J = 4.8, 4.4 Hz, 1H), 1.85 (m, 1H), 1.26 (d, J= 6.4 Hz, 3H).Preparation 3(S)-l -((S)-Oxiran-2-yl)ethyl 4-nitrobenzoate

[0115] To a dry RBF was added (R)-l-((S)-oxiran-2-yl)ethan-l-ol (3.00 g, 34 mmol) under nitrogen. DCM was added (60 mL), followed by 4-nitrobenzoic acid (2.85 g, 17.0 mmol) and triphenylphosphine (4.47 g, 17.0 mmol). The solution was cooled to 0 °C, and DIAD (2.65 mL, 13.6 mmol) was added slowly as a solution in DCM (5 mL). After stirring for 10 minutes, the cold bath was removed and the reaction was stirred at ambient temperature for 2 hours. The reaction was concentrated in vacuo and the resulting solution was purified via silica gel flash chromatography eluting with 0-30% EtOAc in hexanes to give the title compound as a white solid (1.72 g, 64%). 'H NMR (CDCb) 5 8.30 (d, J = 9.2 Hz. 2H), 8.23 (d, J = 9.2 Hz, 2H), 5.02 (quintet, J = 6.4 Hz. 1H), 3.25 (m, 1H). 2.91 (dd, J= 4.4, 4.4 Hz, 1H), 2.72 (dd, J= 4.8, 2.8 Hz, 1H), 1.48 (d, J= 6.4 Hz, 3H).Preparation 4(S)- 1 -((S)-Oxiran-2-y l)ethan-l -ol

[0116] To a dry RBF was added (S)-l-((S)-oxiran-2-yl)propyl 4-nitrobenzoate (1.72 g, 7.25 mmol) under nitrogen. DCM was added (10 mL). followed by cesium carbonate (2.60 g, 7.98 mmol). MeOH (1.5 mL, 36.3 mmol) was added and the reaction was stirred for 2 hours. The suspension was filtered and carefully concentrated in vacuo. The resulting residue was purified via silica gel flash chromatography eluting with 0-10% MeOH in DCM in hexanes to give the title compound as a colorless oil (0.614 g. 96%). 'H NMR (CDCh) 6 3.63 (m. 1H), 2.97 (m, 1H). 2.82 (dd. J= 4.8. 4.0 Hz. 1H), 2.71 (dd, J= 4.8. 2.4 Hz, 1H), 1.88 (br s, 1H), 1.35 (d, .7= 6.4 Hz, 3H).Preparation 5(S)-2-((S)-l-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)oxirane

[0117] To a dry RBF was added (S)-l-((S)-oxiran-2-yl)ethan-l-ol (0.614 g, 6.97 mmol) under nitrogen. DCM was added (35 mL) and the solution was cooled to 0°C. 2,4,6- trimethylpyridine (5.53 mL, 41.8 mmol) was added, followed by 4,4'- (chloro(phenyl)methylene)bis(methoxybenzene) (3.31 g, 9.76 mmol). AgNOs (1.54 g, 9.06 mmol) was added slowly in 5 portions over 10 minutes. The cold bath was removed and the reaction was allowed to stir at ambient temperature for 3 hours. The reaction was diluted with EtOAc (15 mL) and quenched with water (3 mL). The suspension was stirred for 10 minutes and then fdtered through a pad of diatomaceous earth, washing with DCM. The organic layer was separated and dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting solution was purified via silica gel flash chromatography eluting with0-30% EtOAc in hexanes (eluants contain 1% TEA) to give the title compound as a colorless oil (2.40 g, 88%). 'H NMR (CDCh) 5 7.52 (d, J = 8.0 Hz, 2H), 7.41 (m, 4H), 7.30-7.19 (m, 3H), 6.82 (d, J = 8.8 Hz, 4H), 3.79 (s, 6H), 3.62 (quintet, J = 6.4 Hz, 1H), 2.85 (m, 1H), 2.61 (dd, J= 4.8, 4.4 Hz, 1H), 2.51 (dd, J = 4.8, 2.8 Hz, 1H), 0.72 (d, J= 6A Hz, 3H).Table 1: Preparation 5 synthesized in a manner essentially analogous to that of Preparation 6Preparation 7 l-((2S,3R)-3-(Bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)pyrimidine-

[0118] To a dry RBF was added pyrimidine-2,4(lH,3H)-dione (0.517 g, 4.61 mmol) under nitrogen. DMF was added (5 mL), followed by NaH (46 mg, 1. 15mmol, 60% dispersion in mineral oil). The suspension was stirred for 1.5 hours. (S)-2-((R)-l-(bis(4- methoxyphenyl)(phenyl)methoxy)ethyl)oxirane (1.50 g, 3.84 mmol) was added as a solution in DMF (5 mL). The reaction was heated to 110 °C and stirred for 48 hours. Thereaction was cooled to ambient temperature and diluted with EtOAc (20 mL) and washed with a solution of 5% NaCl in water (2 x 50 mL). The organic layer was separated and concentrated in vacuo. The resulting solution was purified via silica gel flash chromatography eluting with 0-100% EtOAc in hexanes (eluants contain 1% TEA) to give the title compound as a white foam (1.32 g, 68%). ES / MS (m / z): 501.3 (M-H).Table 2: Preparations below synthesized in a manner essentially analogous to that ofPreparation 7.Preparation 15(2S,3R)-3-(Bis(4-methoxyphenyl)(phenyl)methoxy)-l-(2,4-dioxo-3,4-dihydropyrimidin- l(2H)-yl)butan-2-yl (2-cy anoethyl) diisopropylphosphoramidite

[0119] To a dry RBF was added l-((2S,3R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2- hydroxybutyl)pyrimidine-2,4(lH,3H)-dione (1.32 g, 2.63 mmol) under nitrogen. DCM was added (13 mL), followed by 2H-tetrazole (4.09 mL, 1.84 mmol, 0.45 M in MeCN). 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (0.918 mL, 2.89 mmol) was added. The reaction was stirred for 20 hours. The reaction was concentrated in vacuo. The resulting solution was purified via silica gel flash chromatography eluting with 0-100% EtOAc in hexanes (eluants contain 1% TEA) to give the title compound as a colorless foam (1.32 g, 71%).31P NMR (CDCh) 5 149.87, 149.43; ES / MS (m / z): 701.4 (M-H).Table 3: Preparations below synthesized in a manner essentially analogous to that ofPreparation 15.Preparation 23 (E)-N'-(9-((2S,3R)-3-(Bis(4-methoxyphenyl)(phenyl)methoxy)-2 -hydroxybutyl)- 9EI- purin-6-yl)-N,N-dimethylformi midamide

[0120] To a dry RBF was added (2S,3R)-l-(6-amino-9H-purin-9-yl)-3-(bis(4- methoxyphenyl)(phenyl)methoxy)butan-2-ol (2.04 g, 3.88 mmol) under nitrogen. DMF was added (15 mL), followed by 1,1 -dimethoxy -N,N-dimethylmethanamine (2.75 mL, 20.6 mmol). The suspension was heated to 60 °C and stirred for 1.5 hours. The reaction was cooled to ambient temperature and diluted with EtOAc (20 mL) and washed with asolution of 5% NaCl in water (2 x 70 mL). The organic layer was separated and concentrated in vacuo. The resulting solution was purified via silica gel flash chromatography eluting with 0-20% MeOH in EtOAc (eluants contain 1% TEA) to give the title compound as a light-yellow foam (1.58g, 70%). ES / MS (m / z): 581.4 (M+H).Table 4: Preparations below synthesized in a manner essentially analogous to that ofPreparation 23.Preparation 272-Amino-9-((2S,3R)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxybutyl)-l,9- dihydro-6H-purin-6-one

[0121] In a dry RBF, (2S,3R)-l-(2-amino-6-(benzyloxy)-9H-purin-9-yl)-3-(bis(4- methoxyphenyl)(phenyl)methoxy)butan-2-ol (1.82 g, 2.88 mmol) and Pd / C (0.92 g, 10% on carbon) were suspended in EtOAc (72 rnL). The reaction flask was purged with nitrogen, followed by hydrogen. The reaction mixture was stirred under an atmosphere of hydrogen for 3 hours. The reaction mixture was filtered through diatomaceous earth, washing with 5: 1 DCM / MeOH. The solution was concentrated in vacuo to give the title compound as a white solid (1.32 g, 85%). ES / MS (m / z): 542.2 (M+H).Table 5: Preparations below synthesized in a manner essentially analogous to that of Preparation 27.Preparation 29Resin loaded 4-[[l-[(2S)-2-[6-[[(2S)-2-[5-[3-Acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5- diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5- oxo-pentanoyl]amino]hexanoylamino]-3-[bis(4-methoxyphenyl)-phenyl- methoxy] propanoyl]-4-piperidyl] methoxy ]-4-oxo-butanoic acid

[0122] The title compound was prepared in a manner analogous to the preparations found in WO2022 / 271806.Table 6a - HPRT unmodified sequencesTable 6b - Fas unmodified sequencesTable 6c - Navl.8 unmodified sequencesTable 7a- HPRT modified sequencesTable 7d - Angl.8 modified sequencesAbbreviations - “m” indicates 2 -OMe; “f ’ indicated 2’-fluoro; indicates phosphorothioate linkage; “sg” indicates GNA; “rsbd” indicates R,S-butadiol; and “ssbd” indicates S.S-butadiol.

[0123] The sense strand and antisense strand of dsRNA can be synthesized using any nucleic acid polymerization methods known in the art, for example, solid-phase synthesis by employing phosphoramidite chemistry methodology (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphortriester chemistry, or enzymatic synthesis. Automated commercial synthesizers can be used, for example, MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems. Phosphorothioate linkages can be introduced using a sulfurizing reagent such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene) amino)-3H-l,2,4- dithiazaoline-3-thione).

[0124] Each of the modified sequences of Tables 7a, 7b, 7c, 7d, and 7e were further modified through the introduction of a delivery moiety (see Table 7f below) at the 3’ end of the sense strand of the corresponding dsRNA. GalNAc (of Preparation 29) was added to the 3’ end of the sense strand of the dsRNA compounds of Table 7a, 7b, 7d, and 7e. It is well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products, including the resin bound GalNAc of Preparation 29, to synthesize modified oligonucleotides or conjugated oligonucleotides. CholTEG (Cholesterol triethylene glycol, ChemGenes: N-9166-05) was added to dsRNA compounds of Table 7c (see Table 7f) via conjugation methods well known to a person of ordinary skill in the art. Each of these delivery moiety-modified dsRNA compounds were used in Examples 1-5.

[0125] Purification methods can be used to exclude the unwanted impurities from the final oligonucleotide product. Commonly used purification techniques for single stranded oligonucleotides include reverse-phase ion pair high performance liquid chromatography (RP-IP-HPLC), capillary gel electrophoresis (CGE), anion exchange HPLC (AX-HPLC),and size exclusion chromatography (SEC). After purification, oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nm. The sense strand and antisense strand can then be annealed to form a dsRNA.Table 7f. Delivery MoietiesPreparation 292ChemGenes, Product number N-91 6-05EXAMPLE 1:In vitro knockdown of mouse HPRT in wildtype mouse primary hepatocytes (MPH) with Gall -conjugated HPRT siRNA with BD and GNA chemical modification at seed region

[0126] Knockdown of HPRT expression by the Gall -conjugated HPRT siRNA with BD and GNA chemical modification at seed region was assayed using the following procedure: mouse primary hepatocytes (MPH) were freshly isolated from a three-month-old wildtype mouse, and three hours after isolation, were added to Coming 96-well plates at 15,000 cells per well, and siRNA were added directly to the well. To generate concentration / dose response curves final concentrations of 1000, 333, 111, 37, 12, 4, 1.37, 0.46, 0.15, 0.05, and 0.017 nM of Gall -conjugated siRNA concentration was used.

[0127] Treated cells were lysed directly into the 96 well plate and RNA was isolated using the Quick-RNA 96 Kit (Zymo Research). The eluted RNA w as used immediately or stored frozen. cDNA was synthesized using Fast Advanced RT Master Mix (Invitrogen) and using the following steps in a thermocycler: 37 °C for 30 minutes, 95 °C for 5 minutes, and 4 °C hold. Polymerase Chain Reaction (PCR) was performed via TaqMan RT PCR (LifeTechnologies) using the following cycles temperatures and times: 50 °C for 2 minutes, 95 °C for 10 minutes, 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute.

[0128] The mouse HPRT levels were normalized to mouse RplpO (Life Technologies) and represented the relative knockdown of mouse Hprt mRNA expression as compared to vehicle-treated control cells. ICso values were calculated using a 4-parameter fit model using XLFit.

[0129] Table 8A show-s the result of ICso and percent maximum knockdown in wdldtype mouse primary hepatocytes by free uptake with the indicated Gall -conjugated HPRT siRNA with BD and GNA chemical modification at seed region. Table 8 A shows that in nearly all compounds tested, the introduction of a destabilizing nucleotide comprising a butadiol moiety maintains potency and percent knockdown relative to the parent compound without a butadiol modified nucleotide (2a).

[0130] A second study was conducted, which was run identically to the first, except that cells were isolated from a two-month-old mouse, and these were seeded immediately after isolation. The results of this study are shown in Table 8B.Table 8A:Table 8B:EXAMPLE 2:

[0131] Knockdown of FAS expression by the Gall -conjugated FAS siRNA with butadiol (BD) modification at seed region was assayed using the following procedure: Transfection reagent RNAiMAX (Life Technologies) at 0.3pl / well was mixed with siRNA in Coming plates before adding HepG2 (ATCC) cells at 15k per well. To generate concentration / dose response curves final concentrations of 200, 66.7, 22.2, 7.4, 2.5, 0.82, 0.27, 0.09, 0.03, 0.01, and 0.003nM of GalN Ac-conjugated siRNA concentration was used.

[0132] Treated cells are lysed and RNA was isolated using the Quick-RNA 96 Kit (Zymo Research) directly into the 96 well plate. The eluted RNA was used immediately or stored frozen. cDNA was synthesized using Fast Advanced RT Master Mix (Invitrogen) and using the following steps in a thermocycler: 37 °C for 30 minutes, 95 °C for 5 minutes, and 4 °C hold. Polymerase Chain Reaction (PCR) is performed via TaqMan RT PCR (Life Technologies) using the following cycles temperatures and times: 50 °C for 2 minutes, 95 °C for 10 minutes, 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute.

[0133] The human FAS levels are normalized to human RPLP0 (Life Technologies) and represent the relative knockdown of human FAS mRNA expression as compared to vehicle-treated control cells. IC50 values are calculated using a 4-parameter fit model using XLFit.

[0134] Table 9 shows that in nearly all compounds tested, the introduction of a destabilizing nucleotide comprising a butadiol moiety maintains potency and percent knockdown relative to the parent compound without a butadiol modified nucleotide (2b).Table 9 - Fas HepG2EXAMPLE 3:

[0135] DRG hiPSC-derived sensory neurons (purchased from Anatomic Inc.) were plated at 20,000 cells / well on a poly-omithine (P3655; Sigma Aldrich) and human Laminin (AMS-892-012; amsbio)-coated 96 well plates. The hiPSC sensory neurons were maintained for a week in Senso Maturation medium (Anatomic Inc.). At one week postplating. hiPSC sensory neurons were treated with siRNA at 4 pM and incubated for another week before harvest.

[0136] For gene expression analysis, first RNA was purified from siRNA treated hiPSC- sensory neurons using RNAdvance v2 for Cell kit (A47943; Beckman Coulter) according to manufacturer’s instructions. Total RNA was then used as atemplate in one-step universal probe RT-qPCR (E3006E; NEB) supplemented with Taqman assays. Taqman assays for SCN1A (Hs00374696_ml; Thermo Fisher), SCN2A (Hs01109871_ml; Thermo Fisher), SCN3A (Hs00366902_ml; Thermo Fisher), SCN4A (Hs01109480_ml; Thermo Fisher), SCN5A (Hs01668074_ml; Thermo Fisher), SCN7A (Hs00161546_ml; Thermo Fisher), SCN8A (Hs00274075_ml; Thermo Fisher), SCN9A (Hs01076699_ml; Thermo Fisher), SCN10A (Hs01045137_ml; Thermo Fisher), SCN11A (Hs00204222_ml; Thermo Fisher ), KCNT2 (Hs01591277_ml; Thermo Fisher), GTF2F1 (Hs00157845_ml; Thermo Fisher), TPCN1 (Hs00330542_ml; Thermo Fisher), NALCN (Hs00291166_ml; Thermo Fisher). TRIP1 l(Hs00188542_ml; Thermo Fisher), DCP2 (Hs00968155_ml; Thermo Fisher), NF1 (Hs01035108_ml ; Thermo Fisher) GAPDH (Hs99999905_ml; Thermo Fisher), were used to evaluate gene expression in QuanStudio6 Pro system. Quantitative data was obtained from Design & Analysis Software 2.6.0 followed by data quantification / visualization using GraphPad 9.0. All values in Table 10a below represent percent of the specified target transcript detected by qPCR, normalized to GAPDH.Table 10a - Nayl.8 gene expression assayExample 4

[0137] Knockdown of ANGPTL8 expression by the Gall -conjugated ANGPTL8 siRNA with butadiol (BD) modification at seed region was assayed using the following procedure: mouse primary’ hepatocytes (MPH) were freshly isolated from an AAV-ANGPTL8 humanized mouse, added to Coming plates at 15k per well, and siRNA were added directly to the well. For Hep3B (ATCC) cells, transfection reagent RNAiMAX (Life Technologies) at 0.3pl / well was mixed with siRNA in Coming plates before adding cells at 8k per well. To generate concentration / dose response curves final concentrations of 1000, 333, 111, 37, 12, 4. 1.37. 0.46, 0. 15, 0.05, and 0.017nM of GalNAc-conjugated siRNA concentration was used.

[0138] Treated cells were lysed and RNA was isolated using the Quick-RNA 96 Kit (Zymo Research) directly into the 96 well plate. The eluted RNA was used immediately or stored frozen. cDNA was synthesized using Fast Advanced RT Master Mix (Invitrogen) and using the following steps in a thermocycler: 37 °C for 30 minutes, 95 °C for 5 minutes, and 4 °C hold. Polymerase Chain Reaction (PCR) was performed via TaqMan RT PCR (Life Technologies) using the following cycles temperatures and times: 50 °C for 2 minutes, 95 °C for 10 minutes, 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute.

[0139] The human ANGPTL8 levels were normalized to mouse (for MPH) or human (for Hep3B) RplpO (Life Technologies) and represent the relative knockdown of human ANGPTL8 mRNA expression as compared to vehicle-treated control cells. IC50 values were calculated using a 4-parameter fit model using XLFit the results of w hich are found in Table 11.

[0140] Table 11 shows that in nearly all compounds tested, the introduction of a destabilizing nucleotide comprising a butadiol moiety maintains potency and percent knockdown relative to the parent compounds without butadiol modified nucleotides (2d- 6d).Example 5: ANGPTL8 siRNA in vivo methodsIn vivo Single dose Screen

[0141] GalNAc-siRNA (n=9) were tested in male mice (Taconic farms). The siRNAs were tested in a single dose study. Mice were dosed by retro-orbital injection with an adeno-associated virus (AAV) vector containing a plasmid with an albumin promoter and the coding sequence for human ANGPTL8 (NM_018687.7) (Vector BioLabs). Blood was collected from mice via retro-orbital puncture 14 days post AAV administration. Serum was prepared from blood and triglycerides were measured utilizing a COBAS clinical chemistry' analyzer (Roche) and ANGPTL4 / 8 was measured by an in house ELISA (Meso Scale Diagnostics). Body weights of mice were measured 22 days after AAV administration. Mice were assigned to groups with similar body weight, semm triglyceride levels, and serum ANGPTL4 / 8 (n=5). Either PBS or test article GalNac-siRNA, at a dose of 5 mg / kg, was administered subcutaneously to mice. Seven days post subcutaneous injection, blood was collected from all mice and serum was analyzed for triglycerides as before. Fourteen days post subcutaneous injection mice were euthanized under isoflurane anesthesia. Liver was collected from the mice and frozen in liquid nitrogen. Livers were homogenized in TriZol (Invitrogen) using Lysing Matrix D bead tubes on a FastPrep-24 (MP Bio). Chloroform was added and the aqueous phase was mixed with ethanol to precipitate the RNA. RNA was isolated on columns using PureLink Pro96 Total RNA purification kit (Invitrogen) according to manufacturer’s protocol and quantitated on the NanoDrop (ThermoFisher). Equal amounts (1 pg) of RNA were reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription kit (Life Technologies) on Mastercycler Nexus (Eppendorf). Thermocycler settings were 25 °C for 10 min, 37 °C for 2 hrs, then 85 °C for 5 min. Template cDNA was combined with Taqman Universal Master Mix and Assays on Demand primer / probesets and RT-PCR was performed on the QuantStudio Pro7 (ThermoFisher) with the following parameters: 50 °C for 2 min, 95 °C for 10 min then 40 cycles of 95 °C for 15 sec and 60 °C for 1 min. Fold changes (FC) were calculated as follows: the CT value of mouse RplpO was subtracted from CT value of human ANGPTL8 to obtain the delta CT value. Then the delta delta CT value was calculated by subtracting the delta CT value of the untreated sample (PBS control) from the delta CT value of each test sample. Fold change was calculated by taking the log base2 of the negative delta delta CT value. Percent knock down (%KD) was calculated by subtracting FC from one and multiplying by 100. Data was shown in Table 12.

[0142] Table 12 shows in vivo knockdown of Ang8 in an AAV mouse model. Additionally, Table 12 shows positional and stereochemical variability in knockdown depending on the location of the modified nucleotide.Table 12. ANGPTL8 mRNA reduction at 5mg / kgExample 6: In vitro knockdown of HMGCR in Hep3B cells with Butanediol- Modified HMGCR siRNA by Transfection

[0143] Knockdown of HMGCR expression by the butanediol-modified HMGCR siRNA was assayed using the following procedure: transfection reagent RNAiMAX (Life Technologies) at 0.3pl / well was mixed with siRNA in Coming plates before adding Hep3B (ATCC) cells at 20,000 per well. To generate concentration / dose response curves final concentrations of 10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.014, 0.005, 0.002, 0.0005, and 0.0002 nM of butanediol-modified siRNA concentration was used.

[0144] Treated cells were lysed and RNA was isolated using the Quick-RNA 96 Kit (Zymo Research) directly into the 96 well plate. The eluted RNA was used immediately or storedfrozen. cDNA was synthesized using Fast Advanced RT Master Mix (Invitrogen) and using the following steps in a thermocycler: 37°C for 30 minutes, 95°C for 5 minutes, and 4°C hold. Polymerase Chain Reaction (PCR) was performed via TaqMan RT PCR (Life Technologies) using the following cycles temperatures and times: 50°C for 2 minutes, 95°C for 10 minutes. 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

[0145] The human HMGCR levels were normalized to human RPLPO (Life Technologies) and represent the relative knockdow n of human HMGCR mRNA expression as compared to vehicle-treated control cells. IC50 values are calculated using a 4-parameter fit model using XLFit.

[0146] The data demonstrate strong potency of butadiol-containing RNAi agents, particularly when the butadiol is incorporated at position 6 (counting from the 5’ end of the antisense strand).

[0147] Table 13A: shows the result of IC50 with percent maximum knockdown in Hep3B cells by transfection reagent, RNAiMAX, with the indicated HMGCR siRNA. Data are expressed as percent of HMGCR message knockdown relative to untreated cells.Table 13A: HMGCR IC50 with percent maximum knockdown in Hep3B cellsExample 7HMGCR siRNA in vivo methods in mice

[0148] GalNAc-siRNAs were tested in male C57bl / 6 mice (Taconic farms) seven to ten weeks of age. Mice were weighed within approximately one week prior to dosing test article or vehicle. Body weights of mice were measured and mice were assigned to groups with similar body weight. At the beginning of study (Day 0) either PBS or test article GalNac-siRNA, at doses such as 5mg / kg were administered subcutaneously to mice. Ten animals were included in each group. At 2 weeks post siRNA administration, three mice from each group were euthanized under isoflurane anesthesia, liver was collected from the mice and frozen in liquid nitrogen. At 8 weeks post siRNA administration, the remaining seven mice were euthanized under isoflurane anesthesia. Liver was collected from mice.Livers were homogenized in TriZol (Invitrogen) using Lysing Matrix D bead tubes using a FastPrep-24 (MP Bio). Chloroform was added and the aqueous phase was mixed with ethanol to precipitate the RNA. RNA was isolated on column using PureLink Pro96 Total RNA purification kit (Invitrogen) according to manufacturer’s protocol and quantified using aNanoDrop (ThermoFisher). Equal amounts (lug) of RNA were reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription kit (Life Technologies) using Mastercycler Nexus (Eppendorf). Thermocycler settings were 25°C for 10 minutes, 37°C for 2 hours, then 85°C for 5 minutes. Template cDNA was combined with Taqman Universal Master Mix and Assays on Demand primer / probesets and RT-PCR was performed using the QuantStudio Pro7 (ThermoFisher) with the following parameters: 50°C for 2minutes, 95°C for lOminutes then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Fold changes (FC) were calculated as follows: the CT value of mouse RplpO was subtracted from CT value of mouse HMGCR to obtain the delta CT value. Then the delta delta CT value was calculated by subtracting the delta CT value of the untreated sample (average of PBS control) from the delta CT value of each test sample. Fold change was calculated by taking the log base 2 of the negative delta delta CT value. Percent mRNA remaining was calculated multiplying the fold change by 100. Data is shown in Table 14. The data demonstrate good knockdown in vivo by RNAi agents in which butadiol is incorporated at position 6 of the antisense strand.Table 14: HMGCR 2 week, mRNA reduction at 5mg / kgSEQUENCE LISTING

Claims

CLAIMSWhat is claimed is:

1. An oligonucleotide comprising a compound of the formula:Xl(X2)m—XB - (x3)n- X4wherein Xi is a 5’ portion of the oligonucleotide, X2 and X3 are one or more independently selected nucleotides, X4 is a 3' portion of the oligonucleotide, m and n are independently selected from any whole number from 0 to 40, and XB is a butadiol modified nucleotide.

2. An oligonucleotide of claim 1, wherein the compound of the formula:wherein B is a nucleobase.

3. The oligonucleotide of claim 1 or 2, wherein the compound is of the formula:wherein B is a nucleobase.

4. The compound of any one of claims 1 and 2, wherein the compound is of the formula:wherein B is a nucleobase.

5. The compound of claim 1 or 2. wherein the compound is of the formula:wherein each B is an independently selected nucleobase, m and n are independently selected from any whole number from 0 to 20, X is independently O or S, R is independently selected from H, O-R1, F, Cl, and Ri is H, C1to C20 alkyl, or C1to C20 alkyl-O-C1to C20 alkyd.

6. The oligonucleotide of claim 1 or 3, wherein the compound is of the formula:wherein each B is an independently selected nucleobase, m and n are independently selected from any whole number from 0 to 20. X is independently O or S, R is independently selected from H, O-R1, F, Cl, and Ri is H, C1to C20 alkyl, or C1to C20 alkyl-O-C1to C20 alkyl.

7. The oligonucleotide of claim 1 or 4, wherein the compound is of the formula:wherein each B is an independently selected nucleobase, m and n are independently selected from any whole number from 0 to 20, X is independently O or S, R is independently selected from H, O-R1, F, Cl, and Ri is H, C1to C20 alkyl, or C1to C20 alkyl-O-C1to C20 alkyl.

8. The oligonucleotide of any one of claims 1 to 7, wherein each nucleobase is independently selected from the group consisting of hypoxanthine, xanthine, 7- methylguanine, inosine, xanthosine, 7-methylguanosine, 5,6-dihydrouracil, 5- methylcytosine, 5-hydoxymethylcytosine, dihydrouridine, 5-methylcytidine, psuedouridine, adenine, guanine, cytosine, thymine, and uracil.

9. The oligonucleotide of any one of claims 1 to 8, wherein each nucleobase is independently selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

10. The oligonucleotide of any one of claims 1 to 9, wherein the nucleobase in the butadiol modified nucleotide is independently selected from the group consisting of adenine, cytosine, and uracil.

11. The oligonucleotide of any one of claims 1 to 10, wherein the nucleobase in the butadiol modified nucleotide is independently selected from the group consisting of cytosine and uracil.

12. The oligonucleotide of any one of claims 1 to 11, wherein the nucleobase in the butadiol modified nucleotide is uracil.

13. The oligonucleotide of any one of claims 1 to 12, wherein the oligonucleotide is 15 to 30 nucleotides in total length.

14. The oligonucleotide of any one of claims 1 to 13, wherein the oligonucleotide is 18 to 30 nucleotides in total length.

15. The oligonucleotide of any one of claims 1 to 14, wherein the oligonucleotide is 23 nucleotides in total length.

16. The oligonucleotide of any one of claims 1 to 15, wherein the butadiol modified nucleotide is at position 3, 4. 5, 6, 7. or 8 starting from the 5’ end.

17. The oligonucleotide of any one of claims 1 to 16, wherein the butadiol modified nucleotide is at position 5, 6, 7, or 8 starting from the 5‘ end.

18. The oligonucleotide of any one of claims 1 to 17, wherein the butadiol modified nucleotide is at position 5, 6, or 8 starting from the 5’ end.

19. The oligonucleotide of any one of claims 1 to 18, wherein the butadiol modified nucleotide is at position 5 starting from the 5’ end.

20. The oligonucleotide of claim 19, wherein R is 2’fluoro at one of the following positions: a.

2. 3, 7, 14, and 16 from the 5’ end; b. 2, 5, 7, 14, and 16 from the 5’ end; c. 2, 3, 8, 14, and 16 from the 5’ end; d. 2, 5, 8, 14, and 16 from the 5’ end; or e.

2. 6, 14, and 16 from the 5' end of the oligonucleotide, and R is 2’0-methyl at all the other positions of the oligonucleotide.

21. The oligonucleotide of claim 19, wherein R is 2’ fluoro at positions 2, 14, and 16 from the 5‘ end of the oligonucleotide, and R is 2’ O-methyl at all other positions of the oligonucleotide.

22. The oligonucleotide of any one of claims 1-14, wherein the butadiol modified oligonucleotide is at position 22, position 23, or positions 22 and 23 starting from the 5’ end.

23. A compound of the formula:wherein B is a nucleobase and Y is an organic protecting group.

24. The compound of claim 23, wherein the compound is of the formula:

25. The compound of claim 23, wherein the compound is of the formula:

26. The compound of any one of claims 23 to 25, wherein the organic protecting group is methoxylmethyl ether (MOM), methoxyethoxymethyl ether (MEM), tertbutyldimethylsilane (TBDMS), tert-buty l (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxy trityl (DMT), methoxytrityl (MMT), -Methoxybenzyl ether (PMB), p- Methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trity l (Tr), or trimethyl silyl (TMS).

27. The compound of any one of claims 23 to 26, wherein the organic protecting group is dimethoxytrityl (DMT).

28. The compound of claim 23, wherein the compound is of the formula:

29. The compound of claim 23, wherein the compound is of the formula:

30. The compound of claim 23, wherein the compound is of the formula:

31. The compound of any one of claims 23 to 30, wherein the nucleobase is selected from the group consisting of hypoxanthine, xanthine, 7-methylguanine, inosine, xanthosine, 7- methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydoxymethylcytosine, dihydrouridine, -methylcytidine, psuedouridine, adenine, guanine, cytosine, thymine, and uracil.

32. The compound of any one of claims 23 to 31, wherein the nucleobase is selected from the group consisting of adenine, cytosine, thymine, uracil, and guanine.

33. The compound of any one of claims 23 to 32, wherein the nucleobase is selected from the group consisting of adenine, cytosine, and uracil.

34. The compound of any one of claims 23 to 33, wherein the nucleobase is selected from the group consisting of cytosine and uracil.

35. The compound of any one of claims 23 to 34, wherein the nucleobase is uracil.

36. A compound of the formula:wherein B is a nucleobase, Y is an organic protecting group, and Z is phosphate or is a phosphoramidite represented by a compound of the formula:

37. The compound of claim 36, wherein the compound is of the formula:

38. The compound of claim 36, wherein the compound is of the formula:

39. The compound of any one of claims 36 to 38, wherein the organic protecting group is methoxylmethyl ether (MOM), Methoxyethoxymethyl ether (MEM), tert- butyldimethylsilane (TBDMS), / e / 7-butyl (tBu), benzyl ether (Bz), acetyl, benzyl (Bn), dimethoxytrityl (DMT), methoxytrityl (MMT), -Methox benz l ether (PMB), p- Methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), or trimethyl silyl (TMS).

40. The compound of any one of claims 36 to 39, wherein the organic protecting group is dimethoxytrityl (DMT).

41. The compound of claim 39, wherein the compound is of the formula:

42. The compound of claim 36, wherein the compound is of the formula:

43. The compound of claim 36, wherein the compound is of the formula:

44. The compound of any one of claims 36 to 43, wherein the nucleobase is selected from the group consisting of adenine, cytosine, thymine, uracil, and guanine.

45. The compound of any one of claims 36 to 44, wherein the nucleobase is selected from the group consisting of adenine, cytosine, and uracil.

46. The compound of any one of claims 36 to 45, wherein the nucleobase is selected from the group consisting of cytosine and uracil.

47. The compound of any one of claims 36 to 46, wherein the nucleobase is uracil.

48. An RNAi agent comprising:(a) a sense strand oligonucleotide having a length of 15 to 30 nucleotides,(b) an antisense strand oligonucleotide comprising the oligonucleotide of any one of claims 1 to 22, wherein the sense strand oligonucleotide and the antisense strand oligonucleotide form a duplex.

49. An RNAi agent comprising:(a) a sense strand oligonucleotide having a length of 15 to 30 nucleotides,(b) an antisense strand oligonucleotide comprising the oligonucleotide of claim 21 or claim 22, wherein the sense strand oligonucleotide and the antisense strand oligonucleotide form a duplex.

50. The RNAi agent of claim 48 or claim 49, comprising a delivery moiety.

51. The RNAi agent of claim 50. wherein the delivery moiety comprises a GalNAc moiety.

52. The RNAi agent of claim 51, where in the GalNAc moiety has the structure:wherein L is a linker or a bond, and wherein Z is the duplex RNA.

53. The RNAi agent of claim 52, wherein L is:

54. The RNAi agent of any one of claims 50-53, wherein the delivery moiety is conjugated to the sense strand.

55. The RNAi agent of any one of claims 50-54, wherein the delivery moiety is conjugated to the 3’ end of the sense strand.