ORTHOGONALLY LINKED MULTIMERIC OLIGONUCLEOTIDES

Abstract

Orthogonally linked multi-conjugates (such as multimeric oligonucleotides) are disclosed, along with methods of synthesizing them using orthogonal linking strategies to join together subunits that are biological moieties.

Claims

1. A multimeric oligonucleotide comprising a plurality of subunits ******** and a protected sulfur-containing end group, wherein: each of the subunits ******** is independently a single or double stranded oligonucleotide and is joined to another subunit by a covalent linker ●; at least one of the covalent linker ● is a sulfur-containing covalent linker 0, optionally the sulfur-containing covalent linker comprises a sulfur-containing cleavable group; the sulfur-containing end group is deprotectable under a deprotection condition; and each sulfur-containing covalent linker 0 is stable under the deprotection condition.

2. The multimeric oligonucleotide of claim 1, wherein at least one sulfur-containing covalent linker 0 comprises a cleavable group, optionally a sulfur-containing cleavable group, that is cleavable under a cleavage condition that is not the deprotection condition.

3. The multimeric oligonucleotide of claim 1, wherein the protected sulfur-containing end group comprises a protected thiol group.

4. The multimeric oligonucleotide of claim 1 comprising the following Structure 1: ##STR00081## wherein: L is a bioactive moiety that may be present or absent; each R is individually a spacer group that may be present or absent; each ******** is independently a single or double stranded oligonucleotide subunit; each ● is a covalent linker joining adjacent oligonucleotide subunits; n is an integer in the range of 1 to 9; S-PG is a protected sulfur-containing end group, optionally a protected thiol group, that is deprotectable under a deprotection condition; and at least one ● is a sulfur-containing covalent linker 0, optionally the sulfur-containing covalent linker comprises a sulfur-containing cleavable group, that is stable under the deprotection condition.

5. The multimeric oligonucleotide of claim 4, wherein at least one of the spacer groups R that is present in the multimeric oligonucleotide comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl; optionally wherein every spacer group R that is present in the multimeric oligonucleotide comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl.

6. The multimeric oligonucleotide of claim 5, wherein at least one of the spacer groups R that is present in the multimeric oligonucleotide comprises C.sub.1-10 alkyl, C.sub.1-10 alkyl ether, C.sub.1-10 alkyl ester, 6-10 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-10 alkyl)-(6-10 membered aryl), (C.sub.1-10 alkyl)-(5-10 membered heteroaryl), or (C.sub.1-10 alkyl)-(5-10 membered heterocyclyl); optionally wherein every spacer group R that is present in the multimeric oligonucleotide comprises C.sub.1-10 alkyl, C.sub.1-10 alkyl ether, C.sub.1-10 alkyl ester, 6-10 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-10 alkyl)-(6-10 membered aryl), (C.sub.1-10 alkyl)-(5-10 membered heteroaryl), or (C.sub.1-10 alkyl)-(5-10 membered heterocyclyl).

7-10. (canceled)

11. The multimeric oligonucleotide of claim 1, wherein the sulfur-containing covalent linker 0 comprises a linkage represented by -R.sup.1-R.sup.2-R.sup.1-, wherein: each R.sup.1 is individually absent or a spacer group; and R.sup.2 is a thiopropionate or disulfide group.

12. (canceled)

13. The multimeric oligonucleotide of claim 11, wherein at least one of the spacer groups R.sup.1 that is present in the linkage comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl; optionally wherein every spacer group R.sup.1 that is present in the linkage comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl.

14. The multimeric oligonucleotide of claim 13, wherein at least one of the spacer groups R.sup.1 that is present in the linkage comprises C.sub.1-10 alkyl, C.sub.1-10 alkyl ether, C.sub.1-10 alkyl ester, 6-10 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-10 alkyl)-(6-10 membered aryl), (C.sub.1-10 alkyl)-(5-10 membered heteroaryl), or (C.sub.1-10 alkyl)-(5-10 membered heterocyclyl); optionally wherein every spacer group R.sub.1 that is present in the linkage comprises C.sub.1-10 alkyl, C.sub.1-10 alkyl ether, C.sub.1-10 alkyl ester, 6-10 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-10 alkyl)-(6-10 membered aryl), (C.sub.1-10 alkyl)-(5-10 membered heteroaryl), or (C.sub.1-10 alkyl)-(5-10 membered heterocyclyl).

15-18. (canceled)

19. The multimeric oligonucleotide of claim 11, wherein at least one of the spacer groups R.sup.1 that is present in the linkage comprises a phosphate linking group, a phosphorothioate linking group, a phosphonate linking group, or a dithiophosphate linking group; optionally wherein every spacer group R.sup.1 that is present in the linkage comprises a phosphate linking group, a phosphorothioate linking group, a phosphonate linking group, or a dithiophosphate linking group.

20-29. (canceled)

30. The multimeric oligonucleotide of claim 1, wherein the protected sulfur-containing end group comprises a protecting group selected from optionally substituted alkyl, optionally substituted alkoxyalkyl, optionally substituted trialkylsilyl, optionally substituted arylalkylsilyl, optionally substituted aryl, optionally substituted benzyl, optionally substituted acyl and optionally substituted benzoyl.

31. The multimeric oligonucleotide of claim 1, wherein the protected sulfur-containing end group comprises a protecting group PG selected from trityl, methoxytrityl, dimethoxytrityl, methylmethoxy, triisopropylsilyl, dinitrophenyl, nitrophenyl, acetyl and benzoyl.

32-99. (canceled)

100. A multi-conjugate comprising a plurality of subunits ******** joined to one another by one or more covalent linkers ●, wherein the multi-conjugate comprises Structure 3: ##STR00082## wherein: each of the subunits ******** is independently a bioactive moiety; at least one covalent linker • is a sulfur-containing covalent linker 0, optionally the sulfur-containing covalent linker 0 comprises a sulfur-containing cleavable group ; each of ▲.sub.1, ▲.sub.2, ▲.sub.3, and ▲.sub.4 is a group that is independently absent or comprises a functional moiety joined to a subunit and, optionally, a spacer group joining the functional moiety to the subunit; Q is a group that comprises a sulfur-containing end group, and optionally a spacer group joining Q to the subunit; and n is an integer greater than or equal to zero; optionally n is an integer in the range of 0 to 10; optionally n is an integer in the range of 0 to 10; optionally n is an integer in the range of 1 to 4; optionally, n is 1, 2, 3 or 4.

101. The multi-conjugate of claim 100, wherein at least one of the subunits ******** present in Structure 3 is not an oligonucleotide.

102. The multi-conjugate of claim 100 or 101, wherein at least one of the subunits ******** present in Structure 3 comprises an oligopeptide or a protein.

103. The multi-conjugate of claim 100, wherein at least one functional moiety is present.

104. The multi-conjugate of claim 103, wherein the at least one functional moiety that is present is a targeting ligand.

105-116. (canceled)

117. A composition comprising a multimeric oligonucleotide of claim 1 and a pharmaceutically acceptable excipient.

118-126. (canceled)

Description

DRAWING

[0103] FIG. 1 illustrates reaction Scheme 1 for making a multimeric oligonucleotide. “Lig” indicates a ligand, e.g., as described elsewhere herein with respect to L in Structure 1, such as triantennary GalNAc as described in the Example below. “-S-CL-S-” represents a covalent linker such as an internal DTME linkage as described in the Example below. “Tr” indicates a trityl group and “DTME” indicates a terminal dithiobismaleimidoethane group that reacts with a thiol group to form the -S-CL-S- linker. Solid lines represent single stranded oligonucleotides and dotted lines represent oligonucleotides that are annealed to the multimeric oligonucleotide as described in the Example below.

DETAILED DESCRIPTION

[0104] The disclosures of any patents, patent applications, and publications referred to herein are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art known to those skilled herein as of the date of the disclosure described and claimed herein.

Definitions

[0105] As used herein, the term “biological moiety” or “functional moiety” are interchangeable and have ordinary meaning as understood by those skilled in the art. The terms refers to chemical entities that are biologically active or inert when delivered into a cell or organism.

[0106] A biological moiety that can produce a biological effect, affinity, or activity within the cell or organism to which it is delivered is referred to as a “bioactive moiety.” In some embodiments the biological effect, affinity, or activity is detectable or measurable. In other instances, a bioactive moiety may be selected to augment or enhance the biological effect, affinity, or activity of another biological moiety with which it is delivered. In still other instances, a bioactive moiety may be selected for use in a method for synthesizing a synthetic intermediate or multi-conjugate (as described below).

[0107] Examples of bioactive moieties include but are not limited to nucleic acids, amino acids, peptides, proteins, lipids, carbohydrates, carboxylic acids, vitamins, steroids, lignins, small molecules, organometallic compounds, or derivatives of any of the foregoing.

[0108] In some aspects of the disclosure, a “non-nucleic acid biological moiety” refers to any biological moiety other than a nucleic acid. Non-nucleic acid biological moieties include but are not limited to amino acids, peptides, proteins, lipids, carbohydrates, carboxylic acids, vitamins, steroids, lignins, small molecules (e.g., a small molecule therapeutic or drug molecule), organometallic compounds, or derivatives of any of the foregoing. A non-nucleic acid biological moiety that can produce a biological effect or activity within the cell or organism to which it is delivered is referred to as a “non-nucleic acid bioactive moiety.”

[0109] “Alkyl” refers to a straight or branched, saturated, aliphatic radical. The number of carbon atoms present in the alkyl group may be specified by number (e.g., C.sub.3 alkyl contains three carbon atoms). The size range of an alkyl group can be specified by indicating a range of the numbers of carbon atoms (e.g., C.sub.1-C.sub.3 alkyl for a one to three carbon atom containing alkyl group). For example, C.sub.1-C.sub.6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl (i.e., 2-pentyl), 1-ethylpropyl (i.e., 3-pentyl), 3-methylpentyl, and the like. Alkyl can include any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6 carbons. The alkyl group is typically monovalent, but can be divalent, such as when the alkyl group links two moieties together, and it is understood that “alkyl” includes alkylene when two functionalities are appended.

[0110] “Alkyl ether” refers to a straight or branched chain saturated hydrocarbon containing 1-12 carbon atoms and 1-12 oxygen atoms in the chain. Examples of alkyl ethers include those represented by -((alkyl)—O—)— or —((CH.sub.2).sub.n—O—).sub.m— where n is an integer in the range of 1 to 6 and m is an integer in the range of 1 to 1 2. A polyethylene glycol (PEG) group or linker is an example of an alkyl ether that may be represented by —((CH.sub.2).sub.2—O—).sub.m—. An “alkoxy” is an example of an alkyl ether that contains a single oxygen atom attached to an end of the alkyl group e.g., —O—(alkyl). Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, butoxy, t-butoxy, or pentoxy groups.

[0111] “Aryl” refers to a monocyclic or fused bicyclic, tricyclic or greater, aromatic ring assembly containing 6 to 16 ring carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, phenanthrenyl, naphthacenyl, fluorenyl, pyrenyl, and the like. “Arylene” means a divalent radical derived from an aryl group. Aryl groups can be mono-, di- or tri-substituted by one, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy and oxy-C2-C3-alkylene; all of which are optionally further substituted, for instance as hereinbefore defined; or 1- or 2-naphthyl; or 1- or 2-phenanthrenyl.

[0112] “Heteroaryl” refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 4 of the ring atoms are each a heteroatom independently selected from N, O and S. Non-limiting examples of heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radicals substituted, especially mono- or di-substituted, by e.g. alkyl, nitro or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinyl represents preferably 2-, 3- or 4-quinolinyl. Isoquinolinyl represents preferably 1-, 3- or 4-isoquinolinyl. Benzopyranyl, benzothiopyranyl represents preferably 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl represents preferably 2- or 4-thiazolyl, and most preferred, 4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

[0113] “Heterocyclyl” refers to a ring system having from 3 ring members to about 20 ring members and from 1 to about 5 heteroatoms independently selected from N, O and S. For example, heterocyclyl includes, but is not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, morpholino, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, piperidinyl, indolinyl, quinuclidinyl and 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl.

[0114] The term “detectable label” as used herein has its ordinary meaning as understood by those skilled in the art. It refers to a chemical group that is attachable to a multi-conjugate and detectable by an imaging technique, such as fluorescence spectroscopy. For example, the detectable label may be a dye that comprises a fluorophore, which, after absorption of energy, emits radiation at a defined wavelength. Many suitable fluorescent labels or dyes are known. For example, Welch et al. (Chem. Eur. J. 5(3):951-960, 1999) discloses dansyl-functionalised fluorescent moieties and Zhu et al. (Cytometry 28:206-211, 1997) describes the use of the fluorescent labels Cy3 and Cy5. Other labels are described in Prober et al. (Science 238:336-341, 1987); Connell et al. (BioTechniques 5(4):342-384, 1987), Ansorge et al. (Nucl. Acids Res. 15(11):4593-4602, 1987) and Smith et al. (Nature 321 :674, 1986). Examples of commercially available fluorescent labels include, but are not limited to, fluorescein, rhodamine (such as TMR, texas red or Rox), alexa, bodipy, acridine, coumarin, pyrene, benzanthracene and cyanine (such as Cy2 or Cy4). Other forms of detectable labels include microparticles, including quantum dots (Empodocles, et al., Nature 399: 126-130, 1999), gold nanoparticles (Reichert et al., Anal. Chem. 72:6025-6029, 2000), microbeads (Lacoste et al., Proc. Natl. Acad. Sci USA 97(17):9461 -9466, 2000), and tags detectable by mass spectrometry. The detectable label may be a multi-component label that is dependent on an interaction with another compound for detection, such as the biotin-streptavidin system.

Multimeric Oligonucleotides With a Protected Sulfur-Containing End Group

[0115] The present disclosure provides a multimeric oligonucleotide comprising a plurality of subunits ******** and a protected sulfur-containing end group. Each of the subunit ******** is independently a single or a double stranded oligonucleotide and is joined to another subunit by a covalent linker .circle-solid., and at least one of the covalent linker .circle-solid. is a sulfur-containing covalent linker ◇. In an embodiment, the sulfer-containing end group comprises a protected thiol group. In some embodiments, the protected sulfur-containing end group comprises a protecting group PG selected from optionally substituted alkyl, optionally substituted alkoxyalkyl, optionally substituted trialkylsilyl, optionally substituted arylalkylsilyl, optionally substituted aryl, optionally substituted benzyl, optionally substituted acyl and optionally substituted benzoyl. In some embodiments, the protected sulfur-containing end group comprises a protecting group PG selected from trityl, methoxytrityl, dimethoxytrityl, methylmethoxy, triisopropylsilyl, dinitrophenyl, nitrophenyl, acetyl, and benzoyl. In some embodiments, the protected thiol is trityl thiol. In some embodiments, the protected sulfur-containing end group does not comprise a thiopropionate group or a disulfide group. The protected sulfur-containing end group is deprotectable under a deprotection condition known to a person of ordinary skill in the art. Each sulfur-containing covalent linker ◇ is stable under the deprotection condition.

[0116] In some embodiments, at least one sulfur-containing covalent linker ◇ comprises a cleavable group that is cleavable under an intracellular cleavage condition. Examples of the cleavable group include, but are not limited to, disulfide and thiopropionate. The cleavage condition is known to a person of ordinary skill in the art, and is not the same as the deprotection condition.

[0117] In some embodiments, the multimeric oligonucleotide disclosed herein comprises the following structure:

##STR00031##

wherein L is a bioacitve moiety that may be present or absent and has biological activity or affinity; each R is individually a spacer group that may be present or absent; each ******** is independently a single or double stranded oligonucleotide subunit; each .circle-solid. is a covalent linker joining adjacent oligonucleotide subunits; n is an integer in the range of 1 to 9; S-PG is a protected sulfur-containing end group that is deprotectable under a deprotection condition, optionally, S-PG is a protected thiol group; and at least one .circle-solid. is a sulfur-containing covalent linker ◇ that is stable under the deprotection condition. In some embodiments, the protected sulfur-containing end group does not comprise a thiopropionate group or a disulfide group.

[0118] In some embodiments, n is an integer in the range of 2 to 6.

[0119] In some embodiments, at least two subunits ******** are substantially different. In some embodiments, the multimeric oligonucleotide comprises two, three, four, five, or six subunits ******** In some embodiments, each nucleic acid strand within a subunit ******** is 5-30, 15-30, 17-27, 19-26, or 20-25 nucleotides in length.

[0120] In some embodiments, one or more subunits ******** are a double-stranded RNA. In some embodiments, one or more subunits ******** are a double-stranded RNA. In some embodiments, one or more subunits ******** are a single-stranded RNA. In some embodiments, the subunits ******** comprises a combination of single-stranded and double-stranded oligonucleotides.

[0121] In some embodiments, each subunit ******** is an RNA, a DNA, or an artificial or non-natural nucleic acid analog thereof. In some embodiments, each subunit ******** is an siRNA, an saRNA, or a miRNA. In some embodiments, each subunit ******** is a double-stranded siRNA.

[0122] In some embodiments, at least one of the covalent linkers .circle-solid. is a cleavable covalent linker CL, the cleavable covalent linker CL being different from the sulfur-containing covalent linker ◇. In some embodiments, the cleavable covalent linker CL comprises an acid cleavable bond, a reductant cleavable bond, a bio-cleavable bond, or an enzyme cleavable bond. In some embodiments, the cleavable covalent linker CL is cleavable under intracellular conditions.

[0123] In some embodiments, at least one of the spacer groups R that is present in the multimeric oligonucleotide of Structure 1 comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl. In some embodiments, every spacer group R that is present in the multimeric oligonucleotide of Structure 1 comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl. In some embodiments, at least one of the spacer groups R that is present in the multimeric oligonucleotide of Structure 1 comprises C.sub.1-.sub.10 alkyl, C.sub.1-.sub.10 alkyl ether, C.sub.1-10 alkyl ester, 6-10 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-.sub.10 alkyl)-(6-10 membered aryl), (C.sub.1-.sub.10 alkyl)-(5-10 membered heteroaryl), or (C.sub.1-.sub.10 alkyl)-(5-1 0 membered heterocyclyl). In some embodiments, every spacer group R that is present in the multimeric oligonucleotide comprises C.sub.1-10 alkyl, C.sub.1-.sub.10 alkyl ether, C.sub.1-.sub.10 alkyl ester, 6-10 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-10-alkyl)-(6-10 membered aryl), (C.sub.1-.sub.10 alkyl)-(5-10 membered heteroaryl), or (C.sub.1-10alkyl)-(5-10 membered heterocyclyl).

[0124] In some embodiments, at least one of the spacer groups R that is present in the multimeric oligonucleotide of Structure 1 comprises C.sub.2-C.sub.10 alkyl, C.sub.2-C.sub.10 alkyl ether, C.sub.2-C.sub.10 alkyl ester, or C.sub.6-C.sub.10 aryl. In some embodiments, every spacer group R that is present in the multimeric oligonucleotide comprises C.sub.2-C.sub.10 alkyl, C.sub.2-C.sub.10 alkyl ether, C.sub.2-C.sub.10 alkyl ester, or C.sub.6-C.sub.10 aryl.

[0125] In some embodiments, at least one of the spacer groups R that is present in the multimeric oligonucleotide of Structure 1 comprises C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl. In some embodiments, every spacer group R that is present in the multimeric oligonucleotide comprises C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl.

[0126] In some embodiments, at least one of the spacer groups R that is present in the multimeric oligonucleotide of Structure 1 comprises C.sub.6 alkyl. In some embodiments, every spacer group R that is present in the multimeric oligonucleotide comprises C.sub.6 alkyl.

[0127] In some embodiments, at least one of the spacer groups R that is present in the multimeric oligonucleotide of Structure 1 comprises 1,4-phenylene. In some embodiments, every spacer group R that is present in the multimeric oligonucleotide comprises 1,4-phenylene.

[0128] In some embodiments, the sulfur-containing covalent linker 0 comprises a linkage represented by -R.sup.1-R.sup.2-R.sup.1-, wherein each R.sup.1 is individually absent or a spacer group, and R.sup.2 is a thiopropionate or disulfide group. In some embodiments, the protected thiol group does not comprise a thiopropionate group or a disulfide group.

[0129] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl.

[0130] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises C1-.sub.10 alkyl, C1-.sub.10 alkyl ether, C1-.sub.10 alkyl ester, 6-1 0 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-.sub.10 alkyl)-(6-10 membered aryl), (C.sub.1-.sub.10 alkyl)-(5-10 membered heteroaryl), or (C.sub.1-.sub.10 alkyl)-(5-10 membered heterocyclyl). In some embodiments, every spacer group .sub.R1 that is present in the linkage comprises C.sub.1-.sub.10 alkyl, C1-.sub.10 alkyl ether, C.sub.1-.sub.10alkyl ester, 6-10 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-.sub.10alkyl)-(6-10 membered aryl), (C.sub.1-.sub.10alkyl)-(5-10 membered heteroaryl), or (C.sub.1-10alkyl)-(5-10 membered heterocyclyl).

[0131] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises C.sub.2-C.sub.10 alkyl, C.sub.2-C.sub.10 alkyl ether, C.sub.2-C.sub.10 alkyl ester, or C.sub.6-C.sub.10 aryl; optionally wherein every spacer group R1 that is present in the multimeric oligonucleotide comprises C.sub.2-C.sub.10 alkyl, C.sub.2-C.sub.10 alkyl ether, C.sub.2-C.sub.10 alkyl ester, or C.sub.6-C.sub.1O aryl.

[0132] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises C.sub.2. C.sub.3, C.sub.4, C5, or C.sub.6 alkyl. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl.

[0133] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises C.sub.6 alkyl. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises C.sub.6 alkyl.

[0134] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises 1,4-phenylene. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises 1,4-phenylene.

[0135] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises a phosphate linking group, a phosphorothioate linking group, a phosphonate linking group, or a dithiophosphate linking group. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises a phospliate linking group, a phosphorothioate linking group, a phosphonate linking group, or a dithiophosphate linking group.

[0136] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises a linking group represented by

##STR00032##

wherein each X independently comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises a linking group represented by

##STR00033##

wherein each X independently comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl.

[0137] In some embodiments, at least one of the spacer groups R1 that is present in the linkage comprises a pyrrolidine-2,5-dione. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises a pyrrolidine-2,-dione.

[0138] In some embodiments, the linkage represented by -R.sup.1-R.sup.2-R.sup.1- can also be represented by:

##STR00034##

wherein each R.sup.1a is independently absent,

##STR00035##

or

##STR00036##

each R.sup.1b is independently absent,

##STR00037##

or

##STR00038##

each R.sup.1c is X; and R.sup.2 is a thiopropionate or disulfide group. Each X independently comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl.

[0139] The linking group

##STR00039##

also includes

##STR00040##

and

##STR00041##

also includes

##STR00042##

X in the linking group would be the moiety that is connected to R.sup.1b.

[0140] In some embodiments, each X independently comprises C1-.sub.10 alkyl, C.sub.1-.sub.10 alkyl ether, C.sub.1-.sub.10alkyl ester, 6-10 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-.sub.10 alkyl)-(6-1 0 membered aryl), (C1-10 alkyl)-(5-10 membered heteroaryl), or (C1,10alkyl)-(5-10 membered heterocyclyl). In some embodiments, each X independently comprises C.sub.2-C.sub.10 alkyl, C.sub.2-C.sub.10 alkyl ether, C.sub.2-C.sub.10 alkyl ester, or C.sub.6-C.sub.10aryl In some embodiments, each X independently comprises C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl. In some embodiments, each X comprises C.sub.6 alkyl. In some embodiments, each X comprises 1,4-phenylene.

[0141] In some embodiments, the linkage represented by -R.sup.1-R.sup.2-R.sup.1 comprises

##STR00043##

or a ring-opened derivative thereof, such as

##STR00044##

##STR00045##

##STR00046##

[0142] In some embodiments, the linkage represented by -R.sup.1-R.sup.2_R.sup.1- is:

##STR00047##

or a ring-opened derivative thereof, wherein each X is independently as defined above, m1 are each individually an integer in the range of 1 to 10.

[0143] In some embodiments, the linkage represented by -R.sup.1-R.sup.2-R.sup.1- is:

##STR00048##

or a ring-opened derivative thereof; wherein m and m1 are each individually an integer in the range of 1 to 10.

[0144] In some embodiments, L comprises a targeting ligand. In some embodiments, the targeting ligand comprises an aptamer, N-Acetylgalactosamine (GalNAc), folate, lipid, cholesterol, or transferrin. In some embodimetns, L comprises an endosomal escape moiety. In some embodiments, the endosomal escape moiety is a membrane disrupting, altering, or destabilizing peptide, lipid, polymer, or small molecule.

[0145] In some embodiments, L comprises a detectable label. In some embodiments, the detectable label selected from fluorescein, a rhodamine (such as TMR, texas red or Rox), alexa, bodipy, acridine, coumarin, pyrene, benzanthracene and a cyanine (such as Cy2 or Cy4). For example, in an embodiment, the detectable labels are Cy2 and Cy4.into a light drug, a rhodamine (such as TMR, texas red or Rox), alexa, bodipy, acridine, coumarin, pyrene, benzanthracene and a cyanine (such as Cy2 or Cy4). For example, in an embodiment, the detectable labels are Cy2 and Cy4.

Method of Making Multimeric Oligonucleotides

[0146] The multimeric oligonucleotides described herein may be made in various ways. The disclosure provides a process for preparing a multimeric oligonucleotide as described herein. The process includes deprotecting a compound of Structure 1a to form a compound of Structure 1b; and reacting the compound of Structure 1b with a compound of Structure 1c under conditions selected to form a compound of Structure 1d, as follows:

##STR00049##

##STR00050##

##STR00051##

##STR00052##

wherein L is a bioactive moiety that may be present or absent; each R is individually a spacer group that may be present or absent; each ******** is independently a single or double stranded oligonucleotide; each .circle-solid. is a covalent linker joining adjacent oligonucleotide subunits; S-PG is a protected sulfur-containing end group, optionally a protected thiol group, that is deprotectable under a deprotection condition; Y is a reactive group selected to react with the —R—SH group of Structure 1b to form one of the covalent linkers » of Structure 1d; y is an integer in the range of 1 to 9; cr, and are each individually an integer in the range of 0 to 8, selected such that a + P + 1 = y; and at least one o is a sulfur-containing covalent linker 0 that is stable under the deprotection condition.

[0147] The disclosure provides a process for preparing another multimeric oligonucleotide as described herein. The process includes deprotecting a compound of Structure 1a to form a compound of Structure 1b; and reacting the compound of Structure lb with a compound of Structure 1e under conditions selected to form a compound of Structure 1f, as follows:

##STR00053##

##STR00054##

##STR00055##

##STR00056##

wherein L is a moiety that may be present or absent and has biological activity or affinity; each R is individually a spacer group that may be present or absent; each ******** is independently a single or double stranded oligonucleotide. Each » is a covalent linker joining adjacent oligonucleotide subunits; S-PG is a protected sulfur-containing end group, optionally a protected thiol group, that is deprotectable under a deprotection condition; Y is a reactive group selected to react with the —R—SH group of Structure 2b to form one of the covalent linkers .circle-solid. of Structure If; y is an integer in the range of 1 to 9; a and β are each individually an integer in the range of 0 to 8, selected such that a + 0 + 1 = y; at least one .circle-solid. is a sulfur-containing covalent linker 0 that is stable under the deprotection condition.

[0148] In some embodiments, the spacer groups R that is present in the multimeric oligonucleotide of Structure 1 comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl. In some embodiments, every spacer group R that is present in the multimeric oligonucleotide of Structure 1 comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl. In some embodiments, at least one of the spacer groups R that is present in the multimeric oligonucleotide of Structure 1 comprises C.sub.1-10alkyl, C.sub.1-10alkyl ether, C.sub.1-10alkyl ester, 6-10 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1 -.sub.10 al.sub.kyl)-( 6-10 membered aryl), (C.sub.1-10alky(5-.sub.10 membered heteroaryl), or (C.sub.1-10 alky10 membered heterocyclyl). In some embodiments, every spacer group R that is present in the multimeric oligonucleotide comprises C.sub.1-10alkyl, C.sub.1-10alkyl ether, C.sub.1-10alkyl ester, 6-10membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-10alkyl-(6-10 membered aryl), (C.sub.1-10 alkyl)-(5-10 membered heteroaryl), or (C.sub.1-10 alkyl(5-10membered heterocyclyl).

[0149] In some embodiments, at least one of the spacer groups R that is present in the multimeric oligonucleotide of Structure 1 comprises C.sub.2-C.sub.10 alkyl, C.sub.2-C.sub.1.sub.0 alkyl ether, C.sub.2-C.sub.10 alkyl ester, or C.sub.6-C.sub.10 aryl. In some embodiments, every spacer group R that is present in the multimeric oligonucleotide comprises C.sub.2-C.sub.10 alkyl, C.sub.2-C.sub.10 alkyl ether, C.sub.2-C.sub.10 alkyl ester, or C.sub.6-C1.sub.0 aryl.

[0150] In some embodiments, at least one of the spacer groups R that is present in the multimeric oligonucleotide of Structure 1 comprises C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl. In some embodiments, every spacer group R that is present in the multimeric oligonucleotide comprises C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl.

[0151] In some embodiments, at least one of the spacer groups R that is present in the multimeric oligonucleotide of Structure 1 comprises C.sub.6 alkyl. In some embodiments, every spacer group R that is present in the multimeric oligonucleotide comprises C.sub.6 alkyl.

[0152] In some embodiments, at least one of the spacer groups R that is present in the multimeric oligonucleotide of Structure 1 comprises 1,4-phenylene. In some embodiments, every spacer group R that is present in the multimeric oligonucleotide comprises 1,4-phenylene.

[0153] In some embodiments, the sulfur-containing covalent linker 0 comprises a linkage represented by -R.sup.1-R.sup.2-R.sup.1-, wherein each R.sup.1 is individually absent or a spacer group, and R.sup.2 is a thiopropionate or disulfide group. In some embodiments, the protected thiol group does not comprise a thiopropionate group or a disulfide group.

[0154] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl.

[0155] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises C.sub.1-10 alkyl, C.sub.1-.sub.10 alkyl ether, C.sub.1-10 alkyl ester, 6-10 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-10 alkyl)-(6-10 membered aryl), (C.sub.1-10 alkyl)-(5-10 membered heteroaryl), or (C.sub.1-.sub.10alkyl)-(5-10 membered heterocyclyl). In some embodiments, every spacer group R.sub.1 that is present in the linkage comprises C.sub.1-10 alkyl, C.sub.1-10 alkyl ether, C.sub.1-10 alkyl ester, 6-10 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-10alkyl)-(6-10 membered aryl), (C.sub.1-.sub.10alkyl)-(5-10 membered heteroaryl), or (C.sub.1-10alkyl)-(5-10 membered heterocyclyl).

[0156] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises C.sub.2-C.sub.10 alkyl, C.sub.2-C.sub.10 alkyl ether, C.sub.2-C.sub.10 alkyl ester, or C.sub.6-C.sub.10 aryl; optionally wherein every spacer group Rthat is present in the multimeric oligonucleotide comprises C.sub.2-C.sub.10 alkyl, C.sub.2-C.sub.10 alkyl ether, C.sub.2-C.sub.10 alkyl ester, or C.sub.6-C.sub.10 aryl.

[0157] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl.

[0158] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises C.sub.6 alkyl. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises C.sub.6 alkyl.

[0159] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises 1,4-phenylene. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises 1,4-phenylene.

[0160] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises a phosphate linking group, a phosphorothioate linking group, a phosphonate linking group, or a dithiophosphate linking group. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises a phosphate linking group, a phosphorothioate linking group, a phosphonate linking group, or a dithiophosphate linking group.

[0161] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises a linking group represented by

##STR00057##

, wherein each X independently comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises a linking group represented by

##STR00058##

wherein each X independently comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl. In some embodiments, R.sup.1 may comprise a linking group represented by

##STR00059##

[0162] In some embodiments, at least one of the spacer groups R.sup.1 that is present in the linkage comprises a pyrrolidine-2,5-dione. In some embodiments, every spacer group R.sup.1 that is present in the linkage comprises a pyrrolidine-2,5-dione.

[0163] In some embodiments, the linkage represented by -R.sup.1-R.sup.2-R.sup.1- can also be represented by:

##STR00060##

wherein each R.sup.1a is independently absent,

##STR00061##

or

##STR00062##

each R.sup.1b is independently absent,

##STR00063##

, or

##STR00064##

; each R.sup.1c is X; and R.sup.2 is a thiopropionate or disulfide group. Each X independently comprises alkyl, alkyl ether, ester, aryl, heteroaryl, heterocyclyl, alkyl-aryl, alkyl-heteroaryl, or alkyl-heterocyclyl.

[0164] In some embodiments, each R.sup.1a is independently absent, or is present and is

##STR00065##

, or

##STR00066##

, where m is an integer in the range of 1 to 10;

[0165] In some embodiments, each X independently comprises C.sub.1-.sub.10 alkyl, C.sub.1-.sub.10 alkyl ether, C.sub.1-10alkyl ester, 6-10 membered aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclyl, (C.sub.1-10alkyl)-(6-10 membered aryl), (C.sub.1-10alkyl)-(5-10 membered heteroaryl), or (C.sub.1-10 alkyl)-(5-10 membered heterocyclyl). In some embodiments, each X independently comprises C.sub.2-C.sub.10 alkyl, C.sub.2-C.sub.10 alkyl ether, C.sub.2-C.sub.10 alkyl ester, or C.sub.6-C.sub.10 aryl. In some embodiments, each X independently comprises C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl. In some embodiments, each X comprises C.sub.6 alkyl. In some embodiments, each X comprises 1,4-phenylene,

[0166] In some embodiments, the linkage represented by -R.sup.1-R.sup.2-R.sup.1- comprises

##STR00067##

or a ring-opened derivative thereof, such as

##STR00068##

##STR00069##

##STR00070##

[0167] In some embodiments, the linkage represented by -R.sup.1-R.sup.2-R.sup.1- is:

##STR00071##

or a ring-opened derivative thereof; wherein each X is independently as defined above, m1 are each individually an integer in the range of 1 to 10.

[0168] In some embodiments, the linkage represented by -R.sup.1-R.sup.2-R.sup.1- is:

##STR00072##

, or a ring-opened derivative thereof; wherein m and ml are each individually an integer in the range of 1 to 10.

[0169] In some embodiments, L comprises a targeting ligand. In some embodiments, the targeting ligand comprises an aptamer, N-Acetylgalactosamine (GalNAc), folate, lipid, cholesterol, or transferrin. In some embodiments, L comprises an endosomal escape moiety. In some embodiments, the endosomal escape moiety is a membrane disrupting, altering, or destabilizing peptide, lipid, polymer, or small molecule.

[0170] In some embodiments, L comprises a detectable label. In some embodiments, the detectable label selected from fluorescein, a rhodamine (such as TMR, texas red or Rox), alexa, bodipy, acridine, coumarin, pyrene, benzanthracene and a cyanine (such as Cy2 or Cy4). For example, in an embodiment, the detectable labels are Cy2 and Cy4.into a light drug, a rhodamine (such as TMR, texas red or Rox), alexa, bodipy, acridine, coumarin, pyrene, benzanthracene and a cyanine (such as Cy2 or Cy4). For example, in an embodiment, the detectable labels are Cy2 and Cy4.

[0171] In some embodiments, Y is a reactive group represented by

##STR00073##

or a ring-opened derivative thereof, wherein each R.sup.1c is independently C.sub.1-10 alkylene or C.sub.1-10alkyleneoxy; R.sup.2 is a thiopropionate or disulfide group; m is an integer in the range of 1 to 10; and ml is an integer in the range of 1 to 10.

[0172] In some embodiments, Y is a reactive group represented by

##STR00074##

or a ring-opened derivative thereof.

Multi-Conjugates

[0173] The disclosure also provides a multi-conjugate comprising a plurality of subunits ******** joined to one another by one or more covalent linkers .circle-solid., wherein the multi-conjugate comprises Structure 4:

##STR00075##

wherein each of the subunits ******** is independently a bioactive moiety; at least one covalent linker .circle-solid. is a sulfur-containing covalent linker ◊; at least one covalent linker .circle-solid. is a sulfur-containing covalent linker ◊; each of .triangle-solid..sub.1, .triangle-solid..sub.2, .triangle-solid..sub.3, and.triangle-solid..sub.4 is a group that is independently absent or comprises a functional moiety joined to a subunit and, optionally, a spacer group joining the functional moiety to the subunit; Q is a group that comprises a sulfur-containing end group, and optionally a spacer group joining Q to the subunit; and n is an integer greater than or equal to zero. In some emboidments, n is an integer in the range of 0 to 10. In some embodiments, n is an integer in the range of 1 to 4. In some embodiments, n is 1, 2, 3, or 4.

[0174] In some embodiments, .triangle-solid. .sub.2, .triangle-solid..sub.3, and .triangle-solid..sub.4 are absent.

[0175] In some embodiments, at least one of the subunits ******** present in Structure 4 is not an oligonucleotide. In some embodiments, at least one of the subunits ******** present in Structure 4 comprises an oligopeptide or a protein.

[0176] In some embodiments, at least one functional moiety is present. In some embodiments, at least one functional moiety that is present is a targeting ligand. In some embodiments, the at least one functional moiety that is present is a detectable label; optionally, the detectable label is a dye.

[0177] In some embodiments, the sulfur-containing end group Q comprises a protected thiol group that is deprotectable under a deprotection condition; and the sulfur-containing covalent linker ◊ is stable under the deprotection condition. In some embodiments, the sulfur-containing covalent linker ◊ comprises a sulfur-containing cleavable group, including but not limited to C.sub.2-C.sub.10 alkyldiothio, thioether, thiopropionate, or disulfide. In an embodiment, the sulfur-containing covalent linker ◊ comprises a sulfur-containing cleavable group that is cleavable under a cleavage condition that is not the deprotection condition.

[0178] In some embodiments, the sulfur-containing end group Q comprises a protected thiol group.

[0179] In some embodiments, at least one covalent linker .circle-solid. comprises Structure 5:

##STR00076##

wherein each R1 is independently a group comprising phosphodiester, thiophosphodiester, sulfate, amide, triazole, heteroaryl, ester, ether, thioether, disulfide, thiopropionate, acetal, glycol, or is absent; each R2 is independently a spacer group, or is absent; each A is independently the reaction product of a nucleophile and an electrophile; and R3 is a group comprising a C2-C10 alkyl, C2-C10 alkoxy, C1-C10 aryl, amide, C2-C10 alkyldithio, ether, thioether, ester, oligonucleotide, oligopeptide, thiopropionate, or disulfide. In some embodiments, each A is the same. In some embodiments, R3 of Structure 5 comprises a sulfur-containing group. In some embodiments, R3 comprises a sulfur-containing cleavable group including C.sub.2-C.sub.10 alkyldithio, thioether, thiopropionate, or disulfide.

[0180] In various embodiments, a multi-conjugate as described herein comprises one or more targeting ligands. The targeting ligand(s) may be attached to one or more of the subunits by a suitable linker. Examples of ligands that can be targeting ligands include antibody, antibody fragment, double chain Ab fragment, single chain Ab fragment; other proteins, for example, a glycoprotein (e.g., transferrin) or a growth factor; peptide (e.g., the RGD ligand or gastrin-releasing peptides); nucleic acid (e.g., an aptamer), endosomal escape moiety (e.g., peptide or nucleic acid), peptide derivative (e.g., DUPA); a natural or synthetic carbohydrate, for example, a monosaccharide (e.g., galactose, mannose, N-Acetylgalactosamine [“GalNAc”]), polysaccharide, or a cluster such as lectin binding oligo saccharide, diantennary GaiNAc, or triantennary GalNAc; a lipid, for example, a sterol (e.g., cholesterol), phospholipid (e.g., phospholipid ether, phosphatidylcholine, lecithin); a vitamin compound (e.g., tocopherol or folate); immunostimulant (e.g., a CpG oligonucleotide); an amino acid; an element (e.g., gold); or a synthetic small molecule (e.g., anisamide or polyethylene glycol). For example, in various embodiments the targeting ligand is an aptamer, N-Acetylgalactosamine (GalNAc), folate, lipid, cholesterol, or transferrin.

Method of Making Multi-Conjugates

[0181] This disclosure provides a method for making a multi-conjugate. The method includes deprotecting a compound of Structure 6a to form a compound of Structure 6b; and reacting the compound of Structure 6b with a compound of Structure 6c under conditions selected to form a compound of Structure 6d, as follows:

##STR00077##

##STR00078##

##STR00079##

##STR00080##

wherein each of the subunits ******** is independently a bioactive moiety; each .circle-solid. is a covalent linker; at least one covalent linker .circle-solid. is a sulfur-containing covalent linker ◊; each .triangle-solid. is independently a group that is absent or comprises a functional moiety joined to a subunit and, optionally, a spacer group joining the functional moiety to the subunit; Q is a group that comprises a protected sulfur-containing end group (optionally, a protected thiol) that is deprotectable under a deprotection condition and optionally a spacer group joining Q to the subunit; Y is a reactive group selected to react with the —R—SH group of Structure 6b to form one of the covalent linkers .circle-solid. of Structure 6d; γ is an integer in the range of 1 to 9; α and β are each individually an integer in the range of 0 to 8, selected such that α + β + 1 = γ; and at least one .circle-solid. is a sulfur-containing covalent linker ◊ that is stable under the deprotection condition.

Methods of Treatment

[0182] In various aspects, the disclosure provides methods for using multimeric oligonucleotides made by the process disclosed herein, for example for medical treatments, research, or for producing new or altered phenotypes in animals and plants. In some aspects, the disclosure also provides methods for using the multi-conjugates made by the process disclosed herein, for example for medical treatments, research, or for producing new or altered phenotypes in animals and plants.

[0183] In one aspect, the invention provides a method for treating a subject comprising administering an effective amount of a multimeric oligonucleotides or multi-conjugates according to the disclosure to a subject in need thereof.

[0184] In some embodiments, the multimeric oligonucleotides or multi-conjugates made by the processes disclosed herein can be administered in the form of a pharmaceutical composition.

EXAMPLE

Example 1. Synthesis of Disulfide-linked Multimeric Oligonucleotides Using Orthogonally Protected Thiols

[0185] A bis-(triantennary GalNAc) homo-hexamer of TTR siRNA (siTTR) is prepared as outlined in Scheme 1 (FIG. 1), Two monomers of siTTR sense strand are prepared on the synthesizer, one with a terminal amino group, the other with a terminal tritylated thiol. Both have a disulfide group at the other terminus. A triantennary GalNAc group is added to the terminal amino function of the first monomer and then the disulfide groups of both monomers are cleaved by DTT to the corresponding thiols.

[0186] The tritylated monomer is converted to a mono-DTME derivative by previously reported methods (see PCT Publication No. WO 2016/205410) and part of this material is reacted with the GalNAc-siTTR-thiol to yield a GalNAc-siTTR single-stranded homodimer with an internal DIME linkage (-S-CL-S-) and a terminal thiol protected by a trityl group.

[0187] The trityl group is removed from the homo-dimer by treatment with aqueous silver nitrate and after purification is treated with one molar equivalent of the tritylated mono-DTME derivate to yield a GalNAc-siTTR single-stranded homotrimer with two internal DTME linkages (-S-CL-S-) and a terminal thiol protected by a trityl group.

[0188] The trityl group is removed from the homo-trimer by treatment with aqueous silver nitrate and after purification is treated with one half-molar equivalent of DTME to yield the single stranded homo-hexamer. Annealing of six equivalents of TTR antisense siRNA yields the desired bis-(triantennary GalNAc) homo-hexamer of siTTR containing 5 disulfide linkages.