WO2025111578A1 - Linkers coupling functional ligands to macromolecules - Google Patents

Abstract

Provided herein are functional moieties including at least one beta-amino acid within an amino-acid cluster for conjugation with one or more ligands. The functional moieties are useful for linking a macromolecule with the one or more functional ligands, which may be used to facilitate delivery of the macromolecule to a site within a subject and to enhance in vitro and/or in vivo stability of the macromolecule (e.g., oligonucleotide).

Description

LINKERS COUPLING FUNCTIONAL LIGANDS TO MACROMOLECULES

RELATED APPLICATIONS

[0001] This application claims priority of U.S. provisional patent application no. 63/602,246, filed November 22, 2023, the entire contents of which are incorporated by reference herein.

SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 22, 2024, is named 0817444_00338_SL.xml and is 45,729 bytes in size.

BACKGROUND

[0003] Short-interfering RNA (siRNA)-induced RNAi responses have great potential to treat a wide variety of human diseases from cancer to pandemic viral outbreaks to Parkinson's Disease. However, following administration, bare siRNA struggles to be taken up by target cells in therapeutically relevant amounts. One solution to this problem of delivery to target cells is decorating siRNA with /V-acetylgalactosamine (GalNAc) to form siRNA conjugates for delivery to liver. Tri-GalNAc binds to the Asialoglycoprotein receptor that is highly expressed on hepatocytes, and several human carcinoma cell lines, resulting in rapid endocytosis. While the exact mechanism of escape across the endosomal lipid bilayer membrane remains unknown, sufficient amounts of siRNAs can enter the cytoplasm to induce robust, target selective RNAi responses in vivo. Multiple GalNAc-siRNA conjugate clinical trials, including two phase III trials, have been initiated to treat a wide variety of diseases, and there are three commercially available GalNAc-siRNAs. GalNAc-siRNA conjugates are one option to address the siRNA delivery problem for liver hepatocytes, and have shown the RNAi (and antisense oligonucleotide) field one path forward for targeting other tissue types.

[0004] There have been several other well-known ligands aside from GalNAc, such as Mannose/N-acetylglucosamine(GlcNAc or GluNac) delivery to macrophages through mannose receptors. Additionally, conjugation of lipophilic molecules such as cholesterol, bile acids and fatty acids increased the binding affinity of siRNA to plasma proteins, thereby improving siRNA delivery through passive targeting or through active targeting that intercepts the endogenous lipid transport pathway.

[0005] Conjugation of these ligands has been addressed previously, but in vivo stability of the conjugation linkage between ligand and siRNA remains an issue. SUMMARY

[0006] Provided herein are linkers that couple ligands to macromolecules (e.g., oligonucleotides, proteins, peptides, or the like).

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 depicts in vitro stability data of compounds described herein under conditions of rat tritosome.

[0008] FIG. 2 depicts in vitro stability data of compounds described herein under conditions of mouse liver homogenate.

[0009] FIG. 3 depicts in vitro Factor IX efficacy data for Oligonucleotide 1 with tri- GalNAc conjugates.

[0010] FIG. 4 depicts in vitro Factor VII efficacy data for Oligonucleotide 2 with tri- GalNAc conjugates.

[0011] FIG. 5 depicts in vivo Factor IX efficacy data for Oligonucleotide 1 with tri- GalNAc conjugates.

[0012] FIG. 6 depicts in vivo Factor VII efficacy data for Oligonucleotide 2 with tri- GalNAc conjugates.

[0013] FIG. 7 depicts in vitro stability data of compounds described herein under conditions of monkey liver homogenate.

[0014] FIG. 8 depicts an example synthetic scheme for preparing phosphoramidite compounds such as those of Formula (A), and the like, herein.

[0015] FIG. 9 depicts a generic synthetic scheme for preparing phosphoramidite compounds such as those of Formula (A), and the like, herein.

[0016] FIG. 10 depicts an example synthetic scheme for preparing solid-support bound compounds such as those of Formula (A), and the like, herein.

[0017] FIG. 11 depicts a generic synthetic scheme for preparing solid-support bound compounds such as those of Formula (A), and the like, herein.

[0018] FIG. 12 depicts Oligonucleotide 1 and Oligonucleotide 2 of Table 4 as doublestranded pairings of SEQ ID NO: 1 with SEQ ID N0:2 and SEQ ID N0:3 with SEQ ID N0:4.

DETAILED DESCRIPTION

Definitions

[0019] Certain terms, whether used alone or as part of a phrase or another term, are defined below.

[0020] The articles "a" and "an" refer to one or to more than one of the grammatical object of the article. [0021] Numerical values relating to measurements are subject to measurement errors that place limits on their accuracy. For this reason, all numerical values provided herein, unless otherwise indicated, are to be understood as being modified by the term "about." Accordingly, the last decimal place of a numerical value provided herein indicates its degree of accuracy. Where no other error margins are given, the maximum margin is ascertained by applying the rounding-off convention to the last decimal place or last significant digit when a decimal is not present in the given numerical value.

[0022] The term "amelioration" means a lessening of severity of at least one indicator of a condition or disease, such as a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.

[0023] The term "composition" refers to a mixture of at least two or more components.

[0024] The terms "effective amount" and "therapeutically effective amount" refer to an amount of therapeutic compound, combination of compounds, or composition, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect. In general, the therapeutically effective amount can be estimated initially either in cell culture assays or in mammalian animal models, for example, in non-human primates, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in non-human subjects and human subjects.

[0025] The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid filler, solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent, or encapsulating material, involved in carrying or transporting at least one compound described herein within or to the patient such that the compound may perform its intended function. A given carrier must be "acceptable" in the sense of being compatible with the other ingredients of a particular formulation, including the compounds described herein, and not injurious to the patient. Other ingredients that may be included in the pharmaceutical compositions described herein are known in the art and described, for example, in "Remington's Pharmaceutical Sciences" (Genaro (Ed.), Mack Publishing Co., 1985), the entire content of which is incorporated herein by reference.

[0026] The term "pharmaceutical composition" refers to a mixture of at least one compound described herein with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound, or combination thereof, to a patient or subject. Multiple techniques of administering a compound, combination, or composition, exist including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration. For example, administration of therapeutic proteins, peptides, oligosacharrides, or oligonucleotides is, in some instances, via oral, inhalational, or injected routes of administration.

[0027] The terms "treatment" or "treating" refer to the application of one or more specific procedures used for the amelioration of a disease. A "prophylactic" treatment, refers to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset.

[0028] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Accordingly, for the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0029] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the described subject matter and does not pose a limitation on the scope of the subject matter otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to practicing the described subject matter.

[0030] Groupings of alternative elements or embodiments of this disclosure are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. Furthermore, a recited member of a group may be included in, or excluded from, another recited group for reasons of convenience or patentability. When any such inclusion or exclusion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0031] References have been made to patents and printed publications throughout this specification, each of which are individually incorporated herein by reference in their entirety. [0032] It is to be understood that the embodiments of this disclosure are illustrative. Accordingly, the present disclosure is not limited to that precisely as shown and described.

Compounds/Conjugates [0033] It has been discovered that an oligonucleotide decorated with three GalNAc moieties as in Formula (A) (e.g., Formula (II)), which comprises at least one beta-amino acid (e.g., D- or L- or racemate), such as beta-lysine or beta-glutamate, is imbued with more in vitro and in vivo stability (e.g., resistance to enzymatic degradation) than the corresponding moiety having L- or D-alpha-amino acid instead of the at least one beta-amino acid (e.g., L- or D-alpha-lysine instead of beta-lysine, and L- or D-alpha-glutamate instead of betaglutamate), and it has been discovered that Formula (II) (e.g., where R² is an oligonucleotide) is at least as stable as Formula (V). Put another way, it has been discovered that compounds of Formula (A) herein improve commercial solid support synthesis or improve in vitro/vivo stability and target site delivery (e.g., cellular uptake by target cells). This is important because preparation of Formula (A) (e.g., Formula (II)) as compared to Formula (V) requires fewer synthetic steps and is more atom efficient, and thereby more cost efficient, and also would result in less foreign material delivered to a subject (e.g., a lower dosage amount as compared to a therapeutic siRNA conjugated to tris-GalNAc as in Formula (V)). Compounds of Formula (I) would similarly benefit from such efficiencies of synthesis. Corresponding phosphoramidite compounds as described herein are useful in preparing such conjugates by solid phase synthesis. Thus, compounds such as Formula (A) are found to be useful as described herein, e.g., regarding medical use they are found to be useful for improved stability or delivery to a target site in a subject (e.g., uptake by target cells), whether in the form of a phosphoramidite for atom efficient solid support facilitated synthesis of macromolecules conjugate to a ligand (e.g., GalNAC, GIcNAC, a fatty acid, or the like), or in the form of the intermediates or products of such synthesis. Intermediates described herein in the preparation of such phosphoramidites are also provided herein.

[0034] Thus, in some embodiments, provided herein are compounds comprising Formula (A):

or a salt (e.g., a pharmaceutically acceptable salt) thereof, wherein

R¹ is hydrogen (H), -R⁷-(solid support) (e.g., wherein the solid support is selected from, but not limited to, a silica gel, a controlled pore glass (CPG) (e.g., long chain alkylamine CPG), or a resin (e.g., a polystyrene)), or a phosphoramidite (e.g., 3- ((diisopropylamino)phosphaneyl)oxy)propanenitrile moiety (abbreviated as DIPA CEP):

R² comprises, or is, H, an oligonucleotide (e.g., with or without protecting groups from standard cyanoethyl phosphoramidate oligonucleotide (e.g., DNA or RNA, optionally single stranded or double stranded, including optional nucleotide analogs therein) solid support synthesis), a peptide (i.e., 2 to 49 amino acid residues with or without protecting groups from standard FMOC amide solid support synthesis), a protein (i.e., 50 or more amino acid residues with or without protecting groups from standard FMOC amide solid support synthesis), or an acid labile protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltri phenylmethyl, dimethyltriphenyl methyl, trimethyltriphenylmethyl, monochlorotriphenyl methyl, dichlorotri phenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl); or R¹ is an acid labile protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltri phenylmethyl, dimethyltriphenyl methyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenyl methyl, monomethoxymethylsulfonyltriphenylmethyl, di methoxymethylsulfonyltri phenyl methyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl), and R² comprises, or is, an oligonucleotide (e.g., with or without protecting groups from standard cyanoethyl phosphoramidate oligonucleotide (e.g., DNA or RNA, optionally single stranded or double stranded) solid support synthesis), a peptide (i.e., 2 to 49 amino acid residues with or without protecting groups from standard FMOC amide solid support synthesis), a protein (i.e., 50 or more amino acid residues with or without protecting groups from standard FMOC amide solid support synthesis), the oligonucleotide, peptide, or protein covalently linked to a solid support (e.g., directly or by a linker as shown in R¹, above, e.g., -R⁷-(solid support));

R³, R⁴, and R⁵ are, independently, selected from hydrogen (H),

each R⁶ is, independently, selected from H, an acyl group (including, but not limited to, acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4- isopropylphenoxyacetyl, or pivaloyl), silyl groups (including, but not limited to, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyld imethylsilyl (TBDMS), triphenylsilyl (TPS), or tert-butyld iphenylsi lyl (TBDPS)), carbonate groups (including, but not limited to, 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p- nitrophenyl)ethoxycarbonyl, or fluorenylmethoxycarbonyl), or benzyl groups (including, but not limited to, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl);

R⁸ is O or NH (i.e., R7 and R8, together with the atoms to which they are attached, form a moiety that includes an ester and an ester on each end of the moiety or an ester and an amide, one on each end of the moiety); and n is 2, 3, 4, 5, or 6, or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0035] In some embodiments, the oligonucleotides referred to herein comprise one or more nucleotide analogs. In some embodiments, the nucleotide analogs are selected, independently from a 2'-O-methyl nucleotide (e.g., mA, mC, mG, mil, mT), a 2'-deoxy-2'- fluoro nucleotide (e.g., fA, fC, fG, fU, f ), or a vinylphosphonate nucleotide (e.g., 5'-(E)- vinylphosphonate dA, dC, dG, dll, dT, mA, mC, mG, mil, mT, fA, fC, fG, fU, or fT). In some embodiments, the oligonucleotides referred to herein comprise a single stranded or a double stranded oligonucleotide. In some embodiments, the oligonucleotide is a double stranded oligonucleotide, each strand having a length, independently, of about 15-30 nucleotides in length. In some embodiments, each oligonucleotide has the same nucleotide length, or a different nucleotide length. In some embodiments, the difference in nucleotide length is 1, 2, 3, 4, or 5 nucleotides. In some embodiments, the oligonucleotide(s) is, independently, about 15-30 nucleotides in length, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the oligonucleotide(s) is, independently, about 18-25 nucleotides in length. In some embodiments, the oligonucleotide(s) is, independently, about 20-25 nucleotides in length. In some embodiments, the oligonucleotide(s) is, independently, about 21 nucleotides in length.

[0036] In some embodiments, of the formulae herein, R¹ is -R⁷-(solid support) (e.g., a solid support selected from, but not limited to, a silica gel, a controlled pore glass (CPG), or a resin (e.g., a polystyrene)), R² is an acid labile protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltri phenylmethyl, dimethyltriphenyl methyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenyl methyl, monomethoxymethylsulfonyltriphenylmethyl, di methoxymethylsulfonyltri phenyl methyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl), and each R⁶ is, independently, selected from an acyl group (including, but not limited to, acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4- isopropylphenoxyacetyl, or pivaloyl), silyl groups (including, but not limited to, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-buty Id imethy Isilyl (TBDMS), triphenylsilyl (TPS), or tert-butyld iphenylsilyl (TBDPS)), carbonate groups (including, but not limited to, 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p- nitrophenyl)ethoxycarbonyl, or fluorenylmethoxycarbonyl), or benzyl groups (including, but not limited to, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl). In some embodiments, of the formulae herein, R¹ is -R⁷-(sol id support) (e.g., a solid support selected from, but not limited to, a silica gel, a controlled pore glass (CPG), or a resin (e.g., a polystyrene)), R² is DMTr, and R⁶ is acetyl.

[0037] In some embodiments, of the formulae herein, R¹ is an acid labile protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl), and R² comprises, or is, an oligonucleotide (e.g., with or without protecting groups from standard cyanoethyl phosphoramidate oligonucleotide (e.g., DNA or RNA, optionally single stranded or double stranded) solid support synthesis), a peptide (i.e., 2 to 49 amino acid residues with or without protecting groups from standard FMOC amide solid support synthesis), a protein (i.e., 50 or more amino acid residues with or without protecting groups from standard FMOC amide solid support synthesis), the oligonucleotide, peptide, or protein covalently linked to a solid support, and each R⁶ is, independently, selected from an acyl group (including, but not limited to, acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4- isopropylphenoxyacetyl, or pivaloyl), silyl groups (including, but not limited to, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-buty Id imethylsilyl (TBDMS), triphenylsilyl (TPS), or tert-butyld iphenylsilyl (TBDPS)), carbonate groups (including, but not limited to, 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p- nitrophenyl)ethoxycarbonyl, or fluorenylmethoxycarbonyl), or benzyl groups (including, but not limited to, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl). In some embodiments, of the formulae herein, R¹ is DMTr, R² is an oligonucleotide, peptide, or protein covalently linked to a solid support (e.g., -R⁷-(solid support)), and R⁶ is acetyl. In some embodiments, of the formulae herein, R¹ is DMTr, R² is an oligonucleotide, peptide, or protein covalently linked to a solid support (e.g., -R⁷-(solid support)), and R⁶ is H. In some embodiments, of the formulae herein, R¹ is H, R² is an oligonucleotide, peptide, or protein covalently linked to a solid support (e.g., -R⁷-(solid support)), and R⁶ is acetyl. In some embodiments, of the formulae herein, R¹ is H, R² is an oligonucleotide, peptide, or protein covalently linked to a solid support (e.g., -R⁷- (solid support)), and R⁶ is H. [0038] In some embodiments, of the formulae herein, R¹ is H, R² is H, and R⁶ is H.

[0039] In some embodiments, of the formulae herein, R¹ is H, R² is H, and R⁶ is acetyl.

[0040] In some embodiments, of the formulae herein, R¹ is H, R² is H, and each R⁶ is, independently, selected from an acyl group (including, but not limited to, acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, or pivaloyl), silyl groups (including, but not limited to, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), or tertbutyldiphenylsilyl (TBDPS)), carbonate groups (including, but not limited to, 2- cya noethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, or fluorenylmethoxycarbonyl), or benzyl groups (including, but not limited to, benzyl, 2- nitrobenzyl, or 4-nitrobenzyl).

[0041] In some embodiments, of the formulae herein, R¹ is H and R² is an acid labile protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltri phenylmethyl, dimethyltriphenyl methyl, trimethyltriphenylmethyl, monochlorotriphenyl methyl, dichlorotri phenylmethyl, trichlorotriphenylmethyl, methylsulfonyltri phenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydi methylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl). In some embodiments, of the formulae herein, R¹ is H and R² is DMTr. In some embodiments, of the formulae herein, R¹ is H, R² is DMTr, and each R⁶ is, independently, selected from an acyl group (including, but not limited to, acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4- isopropylphenoxyacetyl, or pivaloyl), silyl groups (including, but not limited to, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), or tert-butyld iphenylsilyl (TBDPS)), carbonate groups (including, but not limited to, 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p- nitrophenyl)ethoxycarbonyl, or fluorenylmethoxycarbonyl), or benzyl groups (including, but not limited to, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl). In some embodiments, of the formulae herein, R¹ is H, R² is DMTr, and R⁶ is acetyl.

[0042] In some embodiments, of the formulae herein, R¹ is DIPA CEP, R² is an acid labile protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltri phenylmethyl, dimethyltriphenyl methyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl), and each R⁶ is, independently, selected from an acyl group (including, but not limited to, acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, or pivaloyl), silyl groups (including, but not limited to, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tertbutyldimethylsilyl (TBDMS), triphenylsilyl (TPS), or tert-butyldiphenylsilyl (TBDPS)), carbonate groups (including, but not limited to, 2-cyanoethylcarbonyl, 2-(2,4- dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, or fluorenylmethoxycarbonyl), or benzyl groups (including, but not limited to, benzyl, 2- nitrobenzyl, or 4-nitrobenzyl). In some embodiments, of the formulae herein, R¹ is DIPA CEP, R² is DMTr, and R⁵ is acetyl.

[0043] In some embodiments, provided herein are compounds comprising Formula (I) :

or a pharmaceutically acceptable salt thereof, wherein

R¹ is hydrogen (H);

R² comprises, or is, an oligonucleotide or a peptide (i.e., 2 to 49 amino acid residues) or a protein (i.e., 50 or more amino acid residues); and

R³, R⁴, and R⁵ are, independently, ligands selected from, but not limited to, hydrogen (H), ETA, CPA, GalNAc, GluNAc, PGA, CA, UDA, DDA, DDA 12-OH, TDA, MA, PDA, PA, HDA, SA, SA 18-OH, SA 12-OH, SA 2-OH, ACA, BA, DHA, ARA, EPA, ALA, GLA, RA, OA, EA, or LA. The abbreviated compounds above are depicted below in Table 1.

[0044] In some embodiments, R³, R⁴, and R⁵ are, independently, linked to ligands through a bond, selected from, but not limited to, a direct bond, -0-, -NH-, -N(CH3)-, -S-S-, -C(0)-0-, -O-C(O)-, -C(O)-S-, -S-C(O)-, -C(O)-N(H)-, -N(H)-C(O)-, -C(O)-N(CH₃)-, -N(CH₃)- C(0)-, -0-C(0)-0-, -0-C(0)-0-, -O-C(O)-N(H)-, -N(H)-C(O)-O-, -O-C(O)-N(CH₃)-, -N(CH₃)- C(0)-0-, -N(H)-C(O)-N(H)-, and N(CH₃)-C(O)-N(CH₃)-.

[0045] In some embodiments, R³, R⁴, and R⁵ are the same.

[0046] In some embodiments, R³, R⁴, and R⁵ are GalNAc.

[0047] In some embodiments, R³, R⁴, and R⁵ are GluNAc.

Table 1. Chemical names of some abbreviations used herein

[0048] In some embodiments, R² is an oligonucleotide, and R³, R⁴, and R⁵ are GalNAc:

[0049] In some embodiments, R² is an oligonucleotide, and R³, R⁴, and R⁵ are GluNAc:

[0050] In some embodiments, the present invention provides compounds of Table 2.

Table 2. Compounds related to Formula (A) (e.g., Formula (I)) (H = hydrogen)

[0051] In some embodiments, Formula (I) is further conjugated to an oligonucleotide or a peptide. In some embodiments, Formula (I) is further conjugated to a ribonucleic acid, e.g., Formula (I) includes a dsRNA.

[0052] In some embodiments, provided herein are compounds of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein

R¹ is hydrogen (H), and

R² comprises, or is, an oligonucleotide or a peptide (i.e., 2 to 49 amino acid residues) or a protein (i.e., 50 or more amino acid residues). [0053] In some embodiments, R² is an oligonucleotide. In some embodiments, the oligonucleotide is an siRNA.

[0054] Also provided herein are compounds comprising Formula (III) :

(HI), wherein

R¹ is -R⁷-(solid support) as defined above, the solid support, selected from, but not limited to, a silica gel, a controlled pore glass (CPG), or a resin, for example, a polystyrene (PS), or R¹ is a phosphoramidite such as 3- ((diisopropylamino)phosphaneyl)oxy)propanenitrile moiety (abbreviated as DIPA CEP):

R² is triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenyl methyl, trichlorotri phenylmethyl, methylsulfonyltriphenyl methyl, monomethoxymethylsulfonyltriphenylmethyl, di methoxymethylsulfonyltri phenyl methyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl; and

R³, R⁴, and R⁵ are, independently, ligands selected from, but not limited to, hydrogen (H), ETA, CPA, GalNAc, GluNAc, PGA, CA, UDA, DDA, DDA 12-OH, TDA, MA, PDA, PA, HDA, SA, SA 18-OH, SA 12-OH, SA 2-OH, ACA, BA, DHA, ARA, EPA, ALA, GLA, RA, OA, EA, or LA (see Table 1), wherein each hydroxyl moiety of GalNAc, GluNAc, DDA 12-OH, SA 18-OH, SA 12-OH, and SA 2-OH is acetylated. In some embodiments, R³, R⁴, and R⁵ are, independently, linked to ligands through a bond, selected from, but not limited to, -0-, -NH-, -N(CH₃)-, -S- S-, -C(0)-0-, -O-C(O)-, -C(O)-S-, -S-C(O)-, -C(O)-N(H)-, -N(H)-C(O)-, -C(O)-N(CH₃)-, - N(CH₃)-C(O)-, -0-C(0)-0-, -0-C(0)-0-, -O-C(O)-N(H)-, -N(H)-C(O)-O-, -O-C(O)-N(CH₃)-, - N(CH₃)-C(O)-O-, -N(H)-C(O)-N(H)-, and N(CH₃)-C(O)-N(CH₃)-. In some embodiments, R³, R⁴, and R⁵ are, independently, a GalNAc moiety or a GluNAc moiety linked through an oxygen bond at the anomeric carbon of the galactosamine or glucosamine ring. In some embodiments, R³, R⁴, and R⁵ are the same. In some embodiments, R³, R⁴, and R⁵ are GalNAc, wherein each hydroxyl moiety of GalNAc is protected with a base-labile protecting group, e.g., acetylated, e.g., GalNAc(OAc)₃ or, pictorially,

[0055] In some embodiments, R³, R⁴, and R⁵ are the same. In some embodiments, R³, R⁴, and R⁵ are GluNAc, wherein each hydroxyl moiety of GluNAc is protected with a base- labile protecting group, e.g., acetylated, e.g., GluNAc(OAc)₃ or, pictorially,

[0056] In some embodiments, R³, R⁴, and R⁵ are the same. In some embodiments, R³, R⁴, and R⁵ are GluNAc, GalNAc, DDA 12-OH, SA 18-OH, SA 12-OH, or SA 2-OH. In some embodiments the free hydroxyl groups of each GluNAc, GalNAc, DDA 12-OH, SA 18-OH, SA 12-OH, or SA 2-OH are protected with base sensitive protecting groups (e.g., R⁶, which may be indicated as -OR⁶). Any suitable base sensitive protecting group may be selected. In some embodiments, the base sensitive protecting group is an acetyl moiety as described above. In other embodiments, the base sensitive protecting group can be another acyl group (including, but not limited to, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4- isopropylphenoxyacetyl, and pivaloyl), silyl groups (including, but not limited to, TMS, TES, TIPS, TBDMS, TPS, and TBDPS), carbonate groups (including, but not limited to, 2- cya noethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, aand fluorenylmethoxycarbonyl), and benzyl groups (including, but not limited to, benzyl, 2- nitrobenzyl, and 4-nitrobenzyl).

[0057] In some embodiments, the present invention provides compounds of Table 3.

Table 3. Compounds related to Formula (A) (e.g., Formula (III)).

(IV).

[0059] In some embodiments, provided herein are compounds of Formula (V):

or a pharmaceutically acceptable salt thereof, wherein

R¹ is hydrogen (H); and

R² is an oligonucleotide. [0060] In some embodiments, provided herein are compounds of Formula (VI) :

or a pharmaceutically acceptable salt thereof, wherein

R¹ is hydrogen (H); and

R² is an oligonucleotide.

[0061] Also provided herein are compositions, comprising a compound herein and a carrier. In some embodiments, the composition is a pharmaceutical composition, e.g., including a pharmaceutically acceptable carrier.

[0062] Abbreviations used herein include those of Table 1. In context, use of abbreviations may refer to an "yl" or "di-yl" or corresponding "ate" or "amide" or "amidyl" of the reference compound. For example, GalNAc, which refers to 2-(acetylamino)-2-deoxy-D- galactose parent compound, may also refer to 2-(acetylamino)-2-deoxy-D-galactosyl moiety, and CA, which refers to decanoic acid, may also refer to decanoyl or decanoate or decanamide. [0063] In some embodiments, provided herein are compositions, comprising one or more compounds provided herein. The compositions may include one or more carriers, including, without limitation, one or more solvents. In some embodiments, provided herein are pharmaceutical compositions comprising one or more of the compounds provided herein, and at least one pharmaceutically acceptable carrier. In some embodiments, the composition is a solid composition. In some embodiments, the composition is an implantable composition. In some embodiments, the composition is an inhalable composition. In some embodiments, the composition is an orally ingestible composition. In some embodiments, the composition is an injectable composition. In some embodiments, the composition is a flowable powder composition. In some embodiments, the composition is a liquid composition, including, without limitation, a suspension or emulsion of the compound therein. In some embodiments, the composition is a gel, cream, or ointment comprising the compound.

Methods

[0064] The amino-acid clusters herein, except the corresponding phosphoramidite compounds, may be useful as components of therapeutic applications. Thus, it is understood that such compounds are administrable in conjunction with methods of treatment in a subject in need thereof. Thus, provided herein are, at least, methods, comprising administering the compound to a subject. Routes of administration may be via any route suitable for delivery of the compounds herein to a subject, including those described herein.

Kits

[0065] In some embodiments, provided herein are packaged forms of a compound provided herein, packaged compositions, or packaged pharmaceutical compositions comprising a container holding a therapeutically effective amount of a compound described herein, and instructions for using the compound in accordance with one or more of the methods provided herein.

[0066] The present compounds and associated materials can be finished as a commercial product by the usual steps performed in the present field, for example by appropriate sterilization and packaging steps. For example, at doses of 25-35 kGy, both e- beams and gamma radiation may effectively sterilize pharmaceuticals. Alternatively, the material can be treated by UV/vis irradiation (200-500 nm), for example using photoinitiators with different absorption wavelengths (e.g., Irgacure 184, 2959), preferably water- soluble initiators (e.g., Irgacure 2959). Such irradiation is usually performed for an irradiation time of 1-60 min, but longer irradiation times may be applied, depending on the specific method. The material according to the present disclosure can be finally sterile-wrapped so as to retain sterility until use and packaged (e.g. by the addition of specific product information leaflets) into suitable containers (boxes, etc.). The compounds may also be packaged under inert conditions (e.g., de-oxygenated or dehydrated atmosphere, e.g., nitrogen or argon atmosphere), to preserve the compound from degradation.

[0067] According to further embodiments, the present compounds can also be provided in kit form combined with other components, including without limitation, those necessary for use of the material for synthetic methods or administration of the material to the patient. For example, disclosed kits, such as for use in treatments, can further comprise, for example, administration materials.

[0068] The compounds or compositions provided herein may be prepared and placed in a container for storage at ambient or elevated temperature. When the compound or composition is stored in a polyolefin plastic container as compared to, for example, a polyvinyl chloride plastic container, discoloration of the compound or composition may be reduced, whether suspended in a liquid composition (e.g., an aqueous or organic liquid solution), or as a solid. Without wishing to be bound by theory, the container may reduce exposure of the container's contents to electromagnetic radiation, whether visible light (e.g., having a wavelength of about 380-780 nm) or ultraviolet (UV) light (e.g., having a wavelength of about 190-320 nm (UV B light) or about 320-380 nm (UV A light)). Some containers also include the capacity to reduce exposure of the container's contents to infrared light, or a second component with such a capacity. Some containers further include the capacity to reduce the exposure of the container's contents to heat or humidity. The containers that may be used include those made from a polyolefin such as polyethylene, polypropylene, polyethylene terephthalate, polycarbonate, polymethylpentene, polybutene, or a combination thereof, especially polyethylene, polypropylene, or a combination thereof. In some embodiments, the container is a glass container, including without limitation an amber colored glass container. The container may further be disposed within a second container, for example, a paper container, cardboard container, paperboard container, metallic film container, or foil container, or a combination thereof, to further reduce exposure of the container's contents to UV, visible, or infrared light. Articles of manufacture benefiting from reduced discoloration, decomposition, or both during storage, include phosphoramidites described herein or dosage forms that include a form of the compounds or compositions described herein. The compounds or compositions provided herein may need storage lasting up to, or longer than, three months; in some cases up to, or longer than one year. The containers may be in any form suitable to contain the contents— for example, a bag, a bottle, or a box, or any combination thereof.

EMBODIMENTS

[0069] Embodiment 1. A compound, having a formula of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein R¹ is hydrogen (H); R² comprises, or is, an oligonucleotide or a peptide (i.e., 2 to 49 amino acid residues) or a protein (i.e., 50 or more amino acid residues); and R³, R⁴, and R⁵ are, independently, ligands, selected from, but not limited to, hydrogen (H), ETA, CPA, GalNAc, GluNAc, PGA, CA, UDA, DDA, DDA 12-OH, TDA, MA, PDA, PA, HDA, SA, SA 18-OH, SA 12-OH, SA 2-OH, ACA, BA, DHA, ARA, EPA, ALA, GLA, RA, OA, EA, or LA (see Table 1).

[0070] R³, R⁴, and R⁵ are, independently, linked to ligands through a bond, selected from, but not limited to, , -O-, -NH-, -N(CH3)-, -S-S-, -C(O)-O-, -O-C(O)-, -C(O)-S-, -S-C(O)-, -C(O)-N(H)-, -N(H)-C(O)-, -C(O)-N(CH₃)-, -N(CH₃)-C(O)-, -O-C(O)-O-, -O-C(O)-O-, -O-C(O)- N(H)-, -N(H)-C(O)-O-, -O-C(O)-N(CH₃)-, -N(CH₃)-C(O)-O-, -N(H)-C(O)-N(H)-, and N(CH₃)- C(O)-N(CH₃)-.

[0071] Embodiment 2. The compound of embodiment 1, wherein R³, R⁴, and R⁵ are the same.

[0072] Embodiment 3. The compound of embodiment 1, wherein R³, R⁴, and R⁵ are different.

[0073] Embodiment 4. The compound of embodiment 1, wherein R³, R⁴, and R⁵ are GalNAc, linked through -0-.

[0074] Embodiment 5. The compound of embodiment 1, wherein R² is an oligonucleotide, and R³, R⁴, and R⁵ are GalNAc, linked through -0-.

[0075] Embodiment 6. The compound of embodiment 5, wherein the oligonucleotide is a ribonucleic acid (e.g., an siRNA).

[0076] Embodiment 7. A compound, having a formula of Formula (III) :

wherein R¹ is the solid support, selected from, but not limited to, a silica gel, a controlled pore glass (CPG), or a resin, for example, a polystyrene (PS), or the phosphoramidite such as 3- ((diisopropylamino)phosphaneyl)oxy)propanenitrile moiety (abbreviated as DIPA CEP):

R² is triphenylmethyl, monomethoxytriphenylmethyl, di methoxytri phenyl methyl, tri methoxytri phenyl methyl monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenyl methyl, trichlorotri phenylmethyl, methylsulfonyltriphenyl methyl, monomethoxymethylsulfonyltriphenylmethyl, di methoxymethylsulfonyltri phenyl methyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl; and R³, R⁴, and R⁵ are, independently, ligands, selected from, but not limited to, hydrogen (H), ETA, CPA, GalNAc, GluNAc, PGA, CA, UDA, DDA, DDA 12-OH, TDA, MA, PDA, PA, HDA, SA, SA 18-

OH, SA 12-OH, SA 2-OH, ACA, BA, DHA, ARA, EPA, ALA, GLA, RA, OA, EA, or LA (see Table 1), wherein each hydroxyl moiety of GalNAc, GluNAc, DDA 12-OH, SA 18-OH, SA 12-OH, and SA 2-OH is acetylated; R³, R⁴, and R⁵ are, independently, linked to ligands through a bond, selected from, but not limited to, , -O-, -NH-, -N(CH3)-, -S-S-, -C(O)-O-, -O-C(O)-, -C(O)-S-, -S-C(O)-, -C(O)-N(H)-, -N(H)-C(O)-, -C(O)-N(CH₃)-, -N(CH₃)-C(O)-, -O-C(O)-O-, -O-C(O)- O-, -O-C(O)-N(H)-, -N(H)-C(O)-O-, -O-C(O)-N(CH₃)-, -N(CH₃)-C(O)-O-, -N(H)-C(O)-N(H)-, and N(CH₃)-C(O)-N(CH₃)-.

[0077] Embodiment 8. The compound of embodiment 7, wherein R³, R⁴, and R⁵ are the same.

[0078] Embodiment 9. The compound of embodiment 7, wherein R³, R⁴, and R⁵ are different.

[0079] Embodiment 10. The compound of embodiment 7, wherein R³, R⁴, and R⁵ are GalNAc, linked through -O-, wherein each hydroxyl moiety of GalNAc is acetylated

[0080] Embodiment 11. A composition, comprising the compound of one of embodiments 1-10 and a carrier, optionally wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0081] Embodiment 12. A method, comprising administering the compound (e.g., in the form of a pharmaceutical compound, i.e., not a phosphoramidite) of one of embodiments 1-10, or the composition of embodiment 11, to a subject in need thereof.

[0082] Embodiment 13. An article of manufacture, comprising the compound of one of claims 1-10, or the composition of embodiment 11, and instructions for use thereof. [0083] The compounds and processes described herein will be better understood by reference to the following examples, which are intended as an illustration of and not a limitation upon the scope of the present description.

EXAMPLES

[0084] The following selected examples describe certain techniques for producing specific and general synthetic methods of pharmaceutically stability improved functional moieties and their siRNA conjugates as described herein, as well as certain analyses of stability and activities of certain compounds described herein. Syntheses and results of these examples are depicted throughout FIGs. 1-12.

Example 1. General method for the synthesis of oligonucleotide containing multivalent ligand.

A. General method for the synthesis of multivalent ligand solid supports from Fmoc or ivDde AmC7 (DMT) CPG (controlled pore glass) or PS (p oolystyrene) or CPSG (controlled porosity silica gel) or Nitto Phase.

[0085] Fmoc-Beta-homolysine(Boc)-OH was reacted with Cbz-CI under the condition of DMAP and DIPEA in DCM to obtain the Cbz-protected intermediate, and Fmoc protecting group was removed by TEA in isopropanol. Free amine of backbone was elongated with Boc- D-lysine(Boc)-OPfp under the condition of DIPEA in EtOAc. After global deprotection of Boc protecting groups with 4 M HCI in 1,4-dioxane, GalNAc-C5 acid was conjugated by utilizing the activated ester. After Cbz protecting group was removed by hydrogenation, elongation with AmC7 moiety from reaction condition of BOP and DIPEA in DCM/DMF. Free hydroxy group reacted with succinic anhydride with TEA in DMF to afford carboxylic acid form to load solid support. The resulting acid was loaded on amino-functionalized CPG with HBTU, DMPA and DIEA followed by capped with acetyl anhydride. Loading capacity is measured by DMT quantification. Additional compounds of Formula (A) are prepared analogously. See FIG. 8, FIG. 9, FIG. 10, and FIG. 11.

[0086] Alternatively, Fmoc or ivDde protected AmC7 (DMT) CPG is placed in solid phase reactor and rinsed with DCM and DMF. Fmoc protection group is removed by 20% 4- methylpiperidine in DMF and ivDde protection group is removed by 4% hydrazine in DMF. The first beta-amino acid is coupled under the condition with HATU, DIPEA in DMF. Then, the next amino acids are sequentially coupled on the backbone and/or side chain by repeating the N- terminal deprotection of Fmoc or ivDde protection group and coupling reaction under the condition with HATU, DIPEA in DMF until the targeted multivaltent ligand is obtained. Loading capacity is measured by DMT quantification.

B. General method for the synthesis of oligonucleotides with multivalent ligand solid supports. [0087] A functionalized oligonucleotide (e.g., having about 10 to 30 NT in length) is synthesized on multivalent ligand solid supports by automated oligonucleotide solid phase synthesizer. Oligonucleotides containing multivalent ligands are synthesized by standard process using phosphoramidite technology on multivalent ligand solid supports. Depending on the scale either a MerMade 12 (Bioautomation) or a Dr. Oligonucleotide48 (Biolytic) or OligoPilot 100 (Cytiva) is used. All phosphoramidites are purchased from, but not limited to, ChemGenes and Glen Research. All amidities are dissolved in anhydrous acetonitrile and/or DMF and/or DCM in adequate concentration. Deblock solution is selected from, but not limited to, acetic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, or trifluoroacetic acid in an inert solvent such as DCM or toluene. Activator solution is selected from, but not limited to, acidic azole catalysts including lH-tetrazole, 5-ethylthio-lH-tetrazole (ETT) and 2- benzylthio-lH-tetrazole (BTT) or 4,5-dicyanoimidazole (DCI) or a number of similar compounds which is dissolved in anhydrous acetonitrile in adequate concentration. Capping solution is selected from, but not limited to, a mixture of acetic anhydride and pyridine in THF and /V-methylimidazole in acetonitrile. Oxidizing solution is selected from, but not limited to iodine in water, pyridine and THF and tert-butyl hydroperoxidie, (lS)-(+)-(10- camphorsulfonyl)-oxaziridine (CSO). Sulfurization solution is selected from, but not limited to, 3-(dimethylaminomethylidene)amino-3/7-l,2,4-dithiazole-3-thione (DDTT), 3/7- 1,2- benzodithiol-3-one 1,1-dioxide (Beaucage reagent), xanthane hydride, or N,N,N',N'- tetraethylthiramdisulfide (TETD).

C. General method for the synthesis of multivalent ligand phosphoramidite.

[0088] Multivalent ligand phosphoramidite synthesis method 1, by utilizing solid phase synthesis: Fmoc or ivDde protected AmC7 (DMT) solid support is placed in solid phase reactor and rinsed with DCM and DMF. Fmoc protection group is removed by 20% 4-methylpiperidine in DMF and ivDde protection group is removed by 4% hydrazine in DMF. The first beta-amino acid is coupled under the condition with HATU, DIPEA in DMF. Then, the next amino acids are sequentially coupled on the backbone and/or side chain by repeating the /V-terminal deprotection of Fmoc or ivDde protection group and coupling reaction under the condition with HATU, DIPEA in DMF until the targeted multivaltent ligand is obtained. Then, solid support is removed by ammonium hydroxide solution, and the resulting alcohol compound is transformed into multivalent ligand phosphoramidite by phosphitylation reaction.

[0089] Multivalent ligand phosphoramidite synthesis method 2, by stepwise organic synthesis (see FIG. 8) : Fmoc-Beta-homolysine(Boc)-OH was reacted with Cbz-CI under the condition of DMAP and DIPEA in DCM to obtain the Cbz-protected intermediate, and Fmoc protecting group was removed by TEA in isopropanol. Free amine of backbone was elongated with Boc-D-lysine(Boc)-OPfp under the condition of DIPEA in EtOAc. After global deprotection of Boc protecting groups with 4 M HCI in 1,4-dioxane, GalNAc-C5 acid was conjugated by utilizing the activated ester. After Cbz protecting group was removed by hydrogenation, elongation with AmC7 moiety was performed under the condition of BOP and DIPEA in DCM/DMF to afford the substrate for phosphitylation. Tri-GalNAc phosphoramidite was finally obtained after the reaction with ETT and 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile in DCM/DMF. Later, this phosphoramidite, corresponding to Formula (IV), was used for oligonucleotide synthesis, where oligonucleotides is corresponding to Formula (II). Additional compounds of Formula (A) are similarly prepared using corresponding alternate intermediates, e.g., acetyl may be replaced with an alternate base-labile protecting group and DMTr may be replaced with an alternate acid-labile protecting group.

[0090] D. General method for the synthesis of oligonucleotides with multivalent ligand phosphoramidite

[0091] UnyLinker CPG is placed in synthetic column and a functionalized oligonucleotide is synthesized on solid support by automated oligonucleotide solid phase synthesizer. Multivalent ligand phosphoramidite is dissolved in anhydrous acetonitrile and/or DCM and/or DMF in adequate concentration. Oligonucleotide synthesis follows the general method for the synthesis of oligonucleotide shown in B.

[0092] E. General method for the reverse synthesis of oligonucleotides followed by multivalent ligand post-synthesis

[0093] A functionalized oligonucleotide is reverse-synthesized by automated oligonucleotide solid phase synthesizer, followed by post-synthesis using step-by-step conjugation with beta-amino acid, amino acid, and ligands under the condition of HATU, DIPEA and DMF. Oligonucleotides are reverse-synthesized by standard process using phosphoramidite technology on UnyLinker solid supports. Depending on the scale either a MerMade 12 (Bioautomation) or a Dr. Oligonucleotide48 (Biolytic) or OligoPilot 100 (Cytiva) is used. All reverse-phosphoramidites are purchased from, but not limited to, ChemGenes and Glen Research. All reverse-phosphoramidities are dissolved in anhydrous acetonitrile and/or DMF and/or DCM in adequate concentration. Deblock solution is selected from, but not limited to, acetic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, or trifluoroacetic acid in an inert solvent such as DCM or toluene. Activator solution is selected from, but not limited to, acidic azole catalysts including IH-tetrazole, 5-ethylthio-lH-tetrazole (ETT) and 2- benzylthio-lH-tetrazole (BTT) or 4,5-dicyanoimidazole (DCI) or a number of similar compounds which is dissolved in anhydrous acetonitrile in adeauate concentration. Capping solution is selected from, but not limited to, a mixture of acetic anhydride and pyridine in THF and /V-methylimidazole in acetonitrile. Oxidizing solution is selected from, but not limited to iodine in water, pyridine and THF and tert-butyl hydroperoxidie, (lS)-(+)-(10- camphorsulfonyl)-oxaziridine (CSO). Sulfurization solution is selected from, but not limited to, 3-(dimethylaminomethylidene)amino-3/7-l,2,4-dithiazole-3-thione (DDTT), 3/7- 1,2- benzodithiol-3-one 1,1-dioxide (Beaucage reagent), xanthane hydride, or N,N,N',N'- tetraethylthiramdisulfide (TETD).

[0094] F. Duplexation of single strand RNAs

[0095] Sense and antisense strands are carefully mixed in equal molar amount and vortexed for at least 30 seconds. After quantification of sense and antisense strands by in process analysis, the sense or antisense strand is adjusted to make sure no residual single stranded material. The duplex solution is heated to 85 °C for 3 minutes and gradually cooled to room temperature, followed by lyophilization.

Example 2. Stability test 1 under the condition of protein digestion

[0096] The stability of oligonucleotides containing tri-GalNAc conjugate is tested under a protein digestion condition. Test materials are prepared by duplexation with sense strand and antisense. Test materials are prepared with IX PBS (Gibco, 10010-023). 10 pL of 10 pM diluted test materials are added into a mixture of lysis buffer (LGC Biosearch Technologies #MTC096H) 32.5 pL and proteinase K (50 mg/mL) 2.5 pL, and the mixtures are incubated at 37 °C for 1 hour, about 5 days, or about 7 days. After adding 2.5 pL of 3 M KCI, the sample is mixed well and vortexed, followed by incubation on ice for 10 minutes to precipitate SDS. After centrifugation for 10 minutes at 10000g at 4 °C, supernatant (40 pL) is transferred to a clean pre-chilled tube. Then, the mixture 10 pL is mixed with 6x loading dye (Promega, G190A) 2 pL. Total 12 pL was loaded on 12% Native PAGE at 120 V constant for 30 minutes, followed by staining with GelRed (Biotuum, 41003) for 15 minutes.

[0097] All oligonucleotide samples containing beta-amino acid conjugated ligands show better stability under the condition of protein digestion than oligonucleotide samples containing only D- or L-alpha-amino acid moieties (Data not shown).

[0098] The stability of oligonucleotides containing tri-GalNAc conjugate is tested under the conditions of mouse plasma, mouse serum, and rat tritosome. 6-Week C57BL/6 mouse is purchased from KOATECH (Korea, Pyeongtaek). After 4 weeks, the mouse is sacrificed and plasma and serum isolated. To prepare mouse plasma, blood was centrifuged with EDTA at 2500 g for 15 minutes at RT. To prepare mouse serum, blood was centrifuged at 2500 g for 15 minutes at RT. Each portion was carefully separated from blood centrifuge samples. Test materials are prepared by duplexation with sense strand and antisense. Test materials are prepared with IX PBS (Gibco, 10010-023). 1 pL of 10 pM diluted test materials are added into 9 pL of mouse plasma or mouse serum, and the mixture are incubated at 37 °C for 17 hours. For the rat tritosome test, 1 pL of 10 pM diluted test materials are added into a mixture of 5 pL of rat tritosome (0.5 mg/mL), 1 pL of catabolic butter (10X), and 3 pL of UPW, and the mixtures are incubated at 37 °C for 5 days. Then, the mixture 10 pL is mixed with 6x loading dye (Promega, G190A) 2 pL. Total 12 pL is loaded on 12% Native PAGE at 120 V constant for 30 minutes, followed by staining with GelRed (Biotuum, 41003) for 15 minutes. [0099] All oligonucleotide samples containing beta-amino acid conjugated ligands show better stability under the condition of mouse plasma, mouse serum, ad rat tritosome than oligonucleotide samples containing only D- or L-alpha amino acid moieties. FIG. 1 is one representative in vitro stability data under the condition of rat tritosome. Sequences with Formula (VI) which contained tri-GalNAc conjugate with alpha-lysine showed the lower stability than sequences with Formula (II) or Formula (V). Sequences with (II) and Formula (V) showed the similar in vitro stability data, where Formula (V) contained one more elongation by gamma-butyric acid (GABA) than Formula (II). Synthetically efficient Formula (II) was proven to be competitive to Formula (V).

Example 5. In vitro test 3 under the condition of mouse liver homogenate.

[O1OO] The stability of oligonucleotides containing tri-GalNAc conjugate are tested under the conditions of mouse liver homogenate. 6-Week C57BL/6 mouse is purchased from KOATECH (Korea, Pyeongtaek). After 3 weeks, the mouse is sacrificed and whole liver (about 2.5 g) is separated. To prepare liver homogenate, the whole liver is fully homogenized and placed in 50 mL polycarbonate centrifuge tubes including 10 mL of homogenization buffer (100 mM Tris, 1 mM magnesium acetate, pH 8.0). The liver homogenate is pre-incubated at 37 °C for 72 hours before adding the test materials. Test materials are prepared by duplexation with sense strand and antisense. Test materials are prepared with IX PBS (Gibco, 10010-023). Test materials are prepared by duplexation with sense strand and antisense. 1 pL of 10 pM diluted test materials are added into 9 pL of liver homogenates, and the mixtures are incubated at 37 °C for 24 hours, 48 hours, 72 hours, and 96 hours. After incubation, the homogenate samples are mixed with 6x loading dye (Promega, G190A) and heated at 65 °C for 10 minutes. 3 pL of samples are loaded on 10% Native PAGE at 100 V constant for 30 minutes, followed by staining with GelRed (Biotuum, 41003) for 5 minutes.

[O1O1] All oligonucleotide samples containing beta-amino acid conjugated ligands show better stability under the condition of mouse liver homogenate than oligonucleotide samples containing only D- or L-alpha-amino acid moieties. FIG. 2 is one representative in vitro stability data under the condition of mouse liver homogenate. Sequences with Formula (VI) which contained tri-GalNAc conjugate with alpha-lysine showed the lower stability than sequences with Formula (II) or Formula (V). Sequences with Formula (II) and Formula (V) showed the similar in vitro stability data, where Formula (V) contained one more elongation by gamma-butyric acid (GABA) than Formula (II). Synthetically efficient Formula (II) was proven to be competitive to Formula (V).

Example 8. In vitro target KD efficacy test for tri-GalNAc conjugated oligonucleotide duplexes.

[0102] After treating primary human hepatocytes with each of tri-GalNAc conjugated oligonucleotide duplexes, qRT-PCR was performed thereon to measure expression levels of Factor IX mRNA or Factor VII mRNA. Specifically, the primary human hepatocytes were seeded in a 96 well plate at 3.0x l0⁴ cells/well and treated with Factor IX or Factor VII tri- GalNAc conjugated oligonucleotide duplexes at 2, 20, and 200 nM. After 24 hours of the treatment, cell lysate was prepared by using a SuperPrepTM cell lysis & RT kit for qPCR kit II (TOYOBO, SCQ-401), and cDNA was synthesized through reverse transcription using the mRNA included in the lysate as a template. Afterwards, the synthesized cDNA was used as a template for quantitative PCR performed by using a THUNDERBIRD probe qPCR MIX (TOYOBO, QPS-101), a Factor IX or Factor VII probe, and an RNA18S5 probe (Thermofisher, Hs03928985_gl). Then, the expression levels of Factor IX or Factor VII mRNA were identified by using a CFX Connect Real-Time PCR system (Bio-Rad). Meanwhile, in this example, a no treatment group (NT) was used as a control group. Data are shown in FIG. 3 and FIG. 4.

Example 6. In vivo test 1 for tri-GalNAc conjugated oligonucleotide duplexes.

[0103] 6-Week C57BL/6 Mouse is purchased from KOATECH (Korea, Pyeongtaek). Each test group is n=3. After a week of the acclimation period, oligonucleotide duplexes are injected by 3 mg/kg dose subcutaneous single injection on day 0. Oligonucleotide duplexes are prepared with IX PBS (Gibco, 10010-023). Mouse plasma is collected from the facial vein with an Animal lancet (Medipoint, GR-5). After the blood is collected, the blood is mixed with 0.109 M of trisodium citrate solution (Sigma, S1804) in a 9: 1 ratio immediately. Anticoagulated blood is centrifuged at 2,500 g, for 15 min at room temperature. Mouse plasma is collected from the supernatant, then stored at -80 °C. Mouse plasma is collected on day 0 (before oligonucleotide duplex injection), 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70 days. The Factor IX level of mouse plasma is analyzed with the Biophen Factor IX (HYPHEN BioMed, 221806-RUO) by following the manufacturer's instructions. Each mouse's Factor IX level from a different day point is normalized to day 0 Factor IX level of same individual.

[0104] All oligonucleotide samples containing beta-amino acid conjugated ligands show better stability and better efficacy than oligonucleotide samples containing only D- or L-alpha-amino acid moieties. FIG. 5 showed the in vivo Factor IX efficacy for Oligonucleotide

1 with tri-GalNAc conjugates.

Example 7. In vivo test 2 for tri-GalNAc conjugated oligonucleotide duplexes.

[0105] 6-Week C57BL/6 Mouse is purchased from KOATECH (Korea, Pyeongtaek). Each test group is n = 3. After a week of the acclimation period, oligonucleotide duplexes are injected by 2 3 mg/kg dose SC single injection on day 0. Oligonucleotide duplexes are prepared with IX PBS (Gibco, 10010-023). Mouse plasma is collected from the facial vein with an Animal lancet (Medipoint, GR-5). After the blood is collected, the blood is mixed with 0.109 M of trisodium citrate solution (Sigma, S1804) in a 9: 1 ratio immediately. Anti-coagulated blood is centrifuged at 2,500 g, for 15 min at room temperature. Mouse plasma is collected from the supernatant, then stored at -80 °C. Mouse plasma is collected on day 0 (before oligonucleotide duplex injection), 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 days. The Factor VII level of mouse plasma is analyzed with the Biophen Factor VII (HYPHEN BioMed, 221304- RUO) by following the manufacturer's instructions. Each Mouse's Factor VII level from a different day point is normalized to day 0 Factor VII level of same individual.

[0106] All oligonucleotide samples containing beta-amino acid conjugated ligands show better stability and better efficacy than oligonucleotide samples containing only D- or L-alpha-amino acid moieties. FIG. 6 showed the in vivo Factor VII efficacy for Oligonucleotide

2 with tri-GalNAc conjugates.

Example 8. In vitro test 4 under the condition of monkey liver homogenate.

[0107] The stability of oligonucleotides containing tri-GalNAc conjugate are tested under the conditions of monkey liver homogenate. 2-4 years Cynomolgus monkey (male) is purchased from KITOX (Korea, Daejeon). After 3 weeks, the monkey is sacrificed. To prepare liver homogenate, the liver left robe is fully homogenized and placed in 50 mL polycarbonate centrifuge tubes including homogenization buffer (100 mM Tris-Acetae, pH 8.0; NBB-2414, Novatein Bio. I 1 mM magnesium acetate, pH 8.0; 100 mg/mL of monkey liver homogenate). This is aliquoted to 400 uL in 1.5 mL microcentrifuge tubes. Before using it for the stability test, monkey liver homogenate is pre-incubated at 37 °C for 72 hours and the supernatant is placed into a new 1.5 mL tube before adding the test material. Test materials are prepared with IX PBS (Gibco, 10010-024). Test materials are prepared by duplexation with sense strand and antisense. 1 pL of 10 pM diluted test materials are added into 9 pL of monkey liver homogenates, and the mixtures are incubated at 37 °C for 1 day, 2 days, 3 days and 7 days. After incubation, the homogenate samples are mixed with 2 pL of 6X loading dye (Promega, G190A) to 10 pL of resuspended samples. 3 pL of samples are loaded on 12% Native PAGE at 100 V constant for 40 minutes, followed by staining with GelRed (Biotuum, 41003) for 5 minutes. Gel is imaged using ChemiDoc XRS+.

[0108] All oligonucleotide samples containing beta-amino acid conjugated ligands show better stability under the condition of monkey liver homogenate than oligonucleotide samples containing only D- or L-alpha-amino acid moieties. FIG. 7 is one representative in vitro stability data under the condition of monkey liver homogenate. Sequences with Formula (VI) which contained tri-GalNAc conjugate with alpha-lysine showed the lower stability than sequences with Formula (II) or Formula (V). Sequences with Formula (II) and Formula (V) showed the similar in vitro stability data, where Formula (V) contained one more elongation by gamma-butyric acid (GABA) than Formula (II). Synthetically efficient Formula (II) was proven to be competitive to Formula (V).

Comparative example 1. Synthesis of phosphoramidite of tri-GalNAc conjugate with beta-lysine and GABA

[0109] Phosphoramidite (VII) of tri-GalNAc conjugate with beta-lysine and GABA was synthesized by the similar synthetic pathway using beta-lysine and gamma-aminobutyric acid elongation. Later, this phosphoramidite was used for oligonucleotide synthesis, where oligonucleotides contained Formula (V).

[0110] It is found that use of the phosphoramidite compound prepared as depicted in FIG. 8 (e.g., Formula (VIII) below) in preparing an oligonucleotide conjugated thereto results in at least as stable or more stable (e.g., comparable or improved resistance to enzymatic degradation in vitro/vivo) and at least as efficacious or more efficacious (e.g., comparable or improved delivery, e.g., cellular delivery) or the oligonucleotide conjugate as compared to an oligonucleotide conjugate prepared from Formula (VII). Comparative example 2. Synthesis of phosphoramidite of tri-GalNAc conjugate with D-alpha-lysine

[0111] Phosphoramidite (VIII) of tri-GalNAc conjugate with D-alpha-lysine was synthesized by the similar synthetic pathway using D-alpha-lysine instead of beta-lysine. Later, this phosphoramidite was used for oligonucleotide synthesis, where oligonucleotides contained Formula (VI).

[0112] Table 4 provides sequence information for certain embodiments of oligonucleotides referred to herein. FIG. 12 depicts Oligonucleotide 1 and Oligonucleotide 2 as double-stranded pairings of SEQ ID NO: 1 with SEQ ID NO:2 and SEQ ID NO:3 with SEQ ID NO:4. Abbreviations therein refer to the following:

* = phosphorothioate / null = phosphate;

(mA), (mC), (mG), (mil) = 2'-O-methyl nucleotide;

(fA), (fC), (fG), (fU) = 2'-deoxy-2'-fluoro nucleotide;

(Conjugate) = tri-GalNAc conjugate from selected formula (e.g., Formula (A), Formula (II), Formula (V), or Formula (VI), and the like); and

(EVP-mll) = 5'-(E)-vinylphosphonate 2'-0-methyluridine nucleotide of structure:

Table 4. Sequence Information.

Claims

CLAIMS What is claimed is:

1. A compound, having a formula:

or a salt thereof, wherein

R¹ is a phosphoramidite (e.g., 3-((diisopropylamino)phosphaneyl)oxy)propanenitrile moiety (abbreviated as DIPA CEP):

-R⁷-(solid support) (e.g., wherein the solid support is selected from, but not limited to, a silica gel, a controlled pore glass (CPG) (e.g., long chain alkyl-amine CPG), or a resin (e.g., a polystyrene)), or hydrogen (H);

R² comprises, or is, an acid labile protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenyl methyl, monomethoxymethylsulfonyltriphenylmethyl, di methoxymethylsulfonyltri phenyl methyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl), an oligonucleotide (e.g., with or without protecting groups from standard cyanoethyl phosphoramidate oligonucleotide (e.g., DNA or RNA, optionally single stranded or double stranded) solid support synthesis), a peptide (i.e., 2 to 49 amino acid residues with or without protecting groups from standard FMOC amide solid support synthesis), a protein (i.e., 50 or more amino acid residues with or without protecting groups from standard FMOC amide solid support synthesis), or H; or R¹ is an acid labile protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltri phenylmethyl, dimethyltriphenyl methyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenyl methyl, monomethoxymethylsulfonyltriphenylmethyl, di methoxymethylsulfonyltri phenyl methyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl), and R² comprises, or is, an oligonucleotide (e.g., with or without protecting groups from standard cyanoethyl phosphoramidate oligonucleotide (e.g., DNA or RNA, optionally single stranded or double stranded) solid support synthesis), a peptide (i.e., 2 to 49 amino acid residues with or without protecting groups from standard FMOC amide solid support synthesis), a protein (i.e., 50 or more amino acid residues with or without protecting groups from standard FMOC amide solid support synthesis), the oligonucleotide, peptide, or protein covalently linked to a solid support (e.g., directly or by a linker as shown in R¹, above, e.g., -R⁷-(solid support));

R³, R⁴, and R⁵ are, independently, selected from

each R⁶ is, independently, selected from H, an acyl group (including, but not limited to, acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4- isopropylphenoxyacetyl, or pivaloyl), silyl groups (including, but not limited to, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), or tert-butyld iphenylsi lyl (TBDPS)), carbonate groups (including, but not limited to, 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p- nitrophenyl)ethoxycarbonyl, or fluorenylmethoxycarbonyl), or benzyl groups (including, but not limited to, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl);

R⁷ is

R⁸ is O or NH (i.e., R7 and R8, together with the atoms to which they are attached, form a moiety that includes an ester and an ester on each end of the moiety or an ester and an amide, one on each end of the moiety); and n is 2, 3, 4, 5, or 6, or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

2. The compound of claim 1, wherein:

R¹ is -R⁷-(solid support) (e.g., a solid support selected from a silica gel, a controlled pore glass (CPG), or a resin);

R² is dimethoxytriphenylmethyl (DMTr), triphenylmethyl, monomethoxytriphenylmethyl, trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenyl methyl, trichlorotri phenylmethyl, methylsulfonyltriphenyl methyl, monomethoxymethylsulfonyltriphenylmethyl, di methoxymethylsulfonyltri phenyl methyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl; and each R⁶ is, independently, selected from acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, pivaloyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), tert-butyldiphenylsilyl (TBDPS), 2-cyanoethylcarbonyl, 2-(2,4- dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, fluorenylmethoxycarbonyl, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl.

3. The compound of claim 1, wherein:

R¹ is -R⁷-(solid support) (e.g., a solid support selected from a silica gel, a controlled pore glass (CPG), or a resin); R² is DMTr; and

R⁶ is acetyl.

4. The compound of claim 1, wherein:

R¹ is dimethoxytriphenylmethyl (DMTr), triphenylmethyl, monomethoxytri phenylmethyl, tri methoxytri phenyl methyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenyl methyl, trichlorotri phenylmethyl, methylsulfonyltriphenyl methyl, monomethoxymethylsulfonyltriphenylmethyl, di methoxymethylsulfonyltri phenyl methyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl; and

R² comprises, or is, an oligonucleotide, a peptide, or a protein, the oligonucleotide, peptide, or protein covalently linked to a solid support (e.g., -R⁷-(solid support)); and each R⁶ is, independently, selected from acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, pivaloyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), tert-butyldiphenylsilyl (TBDPS), 2-cyanoethylcarbonyl, 2-(2,4- dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, fluorenylmethoxycarbonyl, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl.

5. The compound of claim 1, wherein:

R¹ is DMTr;

R² is an oligonucleotide, peptide, or protein covalently linked to a solid support (e.g., -R⁷-(solid support)); and

R⁶ is acetyl.

6. The compound of claim 1, wherein:

R¹ is DMTr;

R² is an oligonucleotide, peptide, or protein covalently linked to a solid support (e.g., -R⁷-(solid support)); and

R⁶ is H.

7. The compound of claim 1, wherein:

R¹ is H;

R² is an oligonucleotide, peptide, or protein covalently linked to a solid support (e.g., -R⁷-(solid support)); and R⁶ is acetyl.

8. The compound of claim 1, wherein:

R¹ is H;

R² is an oligonucleotide, peptide, or protein covalently linked to a solid support (e.g., -R⁷-(solid support)); and

R⁶ is H.

9. The compound of claim 1, wherein:

R¹ is H;

R² is H; and

R⁶ is H.

10. The compound of claim 1, wherein:

R¹ is H;

R² is H; and

R⁶ is acetyl.

11. The compound of claim 1, wherein:

R¹ is H;

R² is H; and each R⁶ is, independently, selected from acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, pivaloyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), tert-butyldiphenylsilyl (TBDPS), 2-cyanoethylcarbonyl, 2-(2,4- dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, fluorenylmethoxycarbonyl, benzyl, 2-nitrobenzyl, or 4-nitrobenzyL

12. The compound of claim 1, wherein:

R¹ is H;

R² is dimethoxytriphenylmethyl (DMTr), triphenylmethyl, monomethoxytri phenylmethyl, tri methoxytri phenyl methyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl.

13. The compound of claim 1, wherein:

R¹ is H; and

R² is DMTr.

14. The compound of claim 1, wherein:

R¹ is H;

R² is DMTr; and each R⁶ is, independently, selected from acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, pivaloyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), tert-butyldiphenylsilyl (TBDPS), 2-cyanoethylcarbonyl, 2-(2,4- dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, fluorenylmethoxycarbonyl, benzyl, 2-nitrobenzyl, or 4-nitrobenzyL

15. The compound of claim 1, wherein:

R¹ is H;

R² is DMTr; and

R⁶ is acetyl.

16. The compound of claim 1, wherein:

R¹ is DIPA CEP;

R² is triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenyl methyl, trichlorotri phenylmethyl, methylsulfonyltriphenyl methyl, monomethoxymethylsulfonyltriphenylmethyl, di methoxymethylsulfonyltri phenyl methyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl; and each R⁶ is, independently, selected from acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, pivaloyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), tert-butyldiphenylsilyl (TBDPS), 2-cyanoethylcarbonyl, 2-(2,4- dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, fluorenylmethoxycarbonyl, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl.

17. The compound of claim 1, wherein:

R¹ is DIPA CEP;

R² is DMTr; and

R⁶ is acetyl.

18. A composition, comprising the compound of one of claims 1-17 and a carrier, optionally wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

19. An article of manufacture, comprising the compound of one of claims 1-17, or the composition of claim 18, and instructions for use thereof, optionally wherein the article of manufacture is for therapeutic use or for solid support synthesis.

20. Use of the compound, composition, or article of manufacture of one of claims 1-19, in manufacture of a medicament optionally as an oligonucleotide therapy or in solid support synthesis.