NPR-c mediated delivery to adipose tissue

Abstract

Provided herein are conjugates for selective delivery of a therapeutic molecule, such as an oligonucleotide, to adipose tissue. In particular, the conjugate includes a NPR-C binding peptide, a therapeutic agent (e.g., an oligonucleotide), and optionally a linker conjugating the peptide to the therapeutic agent. The conjugates can also include one or more fatty acids. Also provided herein are methods and uses of treating diseases and conditions associated with adipose tissue (e.g., obesity or obesity-related comorbidity) by the conjugates described herein.

Description

NPR-C MEDIATED DELIVERY TO ADIPOSE TISSUESEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled “30829_WO” created October 03, 2025 and is 267 kilobytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety.FIELD OF INVENTION

[0002] The present disclosure relates to the delivery of therapeutics, in some instances oligonucleotides, to adipose tissue, and more particularly, to peptide conjugates of such therapeutics that are targeted to adipose tissue to modulate genes expression.BACKGROUND

[0003] Adipose tissue is a connective tissue and an endocrine organ, participating in various physiological processes, including energy homeostasis, glucose metabolism, and inflammation. Dysregulation of adipose tissue function, including accumulation of excess adipose, can lead to metabolic disorders, such as obesity, diabetes, and cardiovascular diseases. Therefore, modulation of adipose tissue gene expression can be a potential strategy for the treatment of these disorders.

[0004] Natriuretic peptides (NPs) are a class of endogenous hormones which confer cardiovascular protection through regulation of body fluid homeostasis. They include several structurally related peptide hormones: Atrial Natriuretic Peptide (ANP) and variants such as Urodilatin and mANP, Brain Natriuretic Peptide (BNP), C-type Natriuretic Peptide (CNP) and Dendroaspis Natriuretic Peptide (DNP). Three subtypes of natriuretic peptide receptors (NPR) have been described and include NPR- A, NPR-B and NPR-C. Of these, NPR-C is enriched in adipose tissue, where it acts in the natriuretic peptide system to bind and clear NPs, removing them from circulating blood (Maack et al., Science 238:675-678 (1987)).

[0005] Nucleic acid therapeutics, including RNA interference (RNAi) agents such as small interfering RNA (siRNA) and antisense oligonucleotides (ASO) have the potential to selectively target and modulate individual genes related to a disease state. The effectiveness of suchtreatment can be related to how much of the therapeutic is delivered to the organ, tissue, or even cell of interest.

[0006] Attempts to selectively target adipose tissue generally involve delivery with at least one lipid component. In WO2018191719, a lipid nanoparticle for intraperitoneal delivery of a nucleic acid (a messenger RNA (mRNA)) is disclosed. WO2024148329 discloses conjugation of lipids to RNAi agents for subcutaneous or intravenous delivery of the resulting conjugates to adipose tissue.

[0007] There remains a need for technologies to selectively and effectively deliver a therapeutic agent, such as a nucleic acid, to adipose tissue, particularly technologies utilizing delivery moieties other than, or in conjunction with, lipids.SUMMARY OF INVENTION

[0008] Provided herein are conjugates comprising an NPR-C binding peptide and a therapeutic agent, such as an oligonucleotide. The NPR-C binding peptide can deliver the therapeutic agent such as an oligonucleotide to adipose tissue with good selectivity. The conjugates optionally include a linker between the NPR-C binding peptide and the therapeutic agent (e.g., an oligonucleotide). The conjugates can also include one or more fatty acids. The present disclosure also includes methods and uses of treating diseases and conditions associated with adipose tissue (e.g., obesity or obesity-related comorbidity) by the conjugates or pharmaceutical composition described herein.

[0009] In one aspect, provided herein are conjugates comprising Formula (I): O-(L-P)n, wherein O comprises an oligonucleotide; wherein L is a linker or a bond; wherein P is an NPR-C-binding peptide, and wherein n is an integer of 1 to 4. In some embodiments, O is an antisense oligonucleotide (ASO), a double stranded RNA (dsRNA), or a guide RNA. In some embodiments, P is an NPR-C-binding peptide described herein, e.g., an NPR-C-binding peptide in Tables 1-3. In some embodiments, L comprises a linker core and one or more spacers.

[0010] In some embodiments, provided herein are conjugates comprising Formula (I): O- (L-P) n, wherein O comprises an oligonucleotide; wherein L is a linker or a bond; wherein P is an NPR-C-binding peptide comprising SEQ ID NO: 1 (GX11IDX14I), wherein XI 1 is arginine, proline, or hydroxyproline, and X14 is arginine or N-methylarginine, and wherein n is an integer of 1 to 4. In some embodiments, XI 1 is arginine, e.g., L-arginine (R). In some embodiments,X14 is arginine, e.g., L-arginine (R). In some embodiments, P comprises SEQ ID NO: 2 (GRIDRI). In some embodiments, P comprises SEQ ID NO: 3 (SX7X8X9GX11IDX14I), wherein X7 is glycine, alanine, proline, hydroxyproline, serine, or cysteine; X8 is phenylalanine, or cyclohexylalanine, and X9 is glycine, alanine, or serine. In some embodiments, X7 is cysteine, e.g., L-cysteine (C). In some embodiments, X9 is glycine (G). In some embodiments, P comprises SEQ ID NO: 4 (SCFGGRIDRI). In some embodiments, P comprises SEQ ID NO: 5 (FGGRIDRIGA). In some embodiments, P comprises SEQ ID NO: 6 (RSSX7FGGRIDRI), wherein X7 is serine or cysteine. In some embodiments, P comprises SEQ ID NO: 7 (RSSX7FGGRIDRIGA). In some embodiments, X7 is cysteine. In some embodiments, X7 is serine. In some embodiments, P comprises SEQ ID NO: 8 (X7-Cha-X9GX11IDX14I), wherein X7 is proline, hydroxyproline, glycine, cysteine, or alanine; X9 is alanine, serine, or glycine;XI 1 is proline, hydroxyproline, or arginine; and X14 is arginine or N-methylarginine. In some embodiments, P comprises SEQ ID NO: 9 (fSp-Cha-aGPIDRI). In some embodiments, P comprises a sequence selected from any one of SEQ ID NO: 11-57.

[0011] In some embodiments, P is a linear peptide. In some embodiments, P is a cyclic peptide. In some embodiments, P is cyclized by covalent attachment of a side chain of a first cysteine residue to a side chain of a second cysteine residue. In some embodiments, the covalent attachment comprises a disulfide bond. In some embodiments, the covalent attachment comprises a thioacetal moiety. In some embodiments, P comprises a C-terminal hydroxyl. In some embodiments, P comprises a C-terminal amide.

[0012] In some embodiments, O is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand. In some embodiments, at least one nucleotide of the sense strand is a modified nucleotide. In some embodiments, at least one nucleotide of the antisense strand is a modified nucleotide. In some embodiments, at least one internucleotide linkage of the sense strand is a modified internucleotide linkage. In some embodiments, at least one internucleotide linkage of the antisense strand is a modified intemucleotide linkage.

[0013] In some embodiments, L comprises a linker core and one or more spacers. In some embodiments, L comprises Spacer 1 -Linker Core-Spacer2. In some embodiments, the Linker Core is selected from Table 9. In some embodiments, Spacerl and Spacer 2 are selected from Table 10. In some embodiments, L is selected from Table 11.

[0014] In some embodiments, L is attached to the 5’ end of the sense strand and to the N- tcrminal end of the peptide. In some embodiments, L is attached to the 5’ end of the sense strand and to the C-terminal end of the peptide. In some embodiments, L is attached to the 3’ end of the sense strand and to the N-terminal end of the peptide. In some embodiments, L is attached to the 3’ end of the sense strand and to the C-terminal end of the peptide. In some embodiments, L is attached to the 3’ end of the antisense strand and to the N-terminal end of the peptide. In some embodiments, L is attached to the 3’ end of the antisense strand and to the C-terminal end of the peptide.

[0015] In some embodiments, the conjugate further comprises a fatty acid (FA). In some embodiments, FA is attached to O. In some embodiments, FA is attached to P. In some embodiments, FA is attached to L. In some embodiments, the fatty acid is selected from FA1- FA27 of Table 12 herein.

[0016] In some embodiments, the conjugate comprises (FA)m-O-L-P or O-L-P-(FA)m, wherein m is an integer of 1 to 4. In some embodiments, m is 1 or 2.

[0017] In some embodiments, the conjugate further comprises a Spacer3. In some embodiments, the conjugate comprises (FA-Spacer3)m-O-L-P or O-L-P-(Spacer3-FA)m, wherein m is an integer of 1 to 4. In some embodiments, m is 1 or 2. In some embodiments, the conjugate comprises FA-Spacer3-O-L-P or O-L-P-Spacer3-FA. In some embodiments, the conjugate comprises O-L-(FA-Spacer3)m-P or O-(Spacer3-FA)m-L-P, wherein m is an integer of 1 to 4. In some embodiments, m is 1 or 2. In some embodiments, the conjugate comprises O-L- FA-Spacer3-P or O-Spacer3-FA-L-P.

[0018] In another aspect, provided herein are pharmaceutical compositions comprising a conjugate described herein and a pharmaceutically acceptable carrier.

[0019] In another aspect, provided herein are methods of treating a disease or condition of adipose tissue, e.g., obesity or obesity-related comorbidity, in a patient in need thereof, comprising administering to the patient an effective amount of a conjugate or pharmaceutical composition described herein. The conjugate or pharmaceutical composition can be administered to the patient intravenously or subcutaneously.

[0020] In another aspect, provided herein are conjugates or pharmaceutical compositions described herein for use in a therapy. In some embodiments, provided herein are conjugates orpharmaceutical compositions described herein for use in the treatment of a disease or condition of adipose tissue, e.g., obesity or obesity-related comorbidity.

[0021] Also provided herein are uses of conjugates or pharmaceutical compositions described herein in the manufacture of a medicament for treating a disease or condition of adipose tissue, e.g., obesity or obesity-related comorbidity.

[0022] Also provided herein are methods of delivering an oligonucleotide to an adipose tissue, comprising administering to a subject a conjugate or pharmaceutical composition described herein.DETAILED DESCRIPTION

[0023] Provided herein are conjugates comprising an NPR-C binding peptide and a therapeutic agent, such as an oligonucleotide. The NPR-C binding peptide can deliver the therapeutic agent such as an oligonucleotide to adipose tissue with good selectivity. The conjugates optionally include a linker between the NPR-C binding peptide and the therapeutic agent (e.g., an oligonucleotide). The conjugates can also include one or more fatty acids. The present disclosure also includes methods and uses of treating diseases and conditions associated with adipose tissue (e.g., obesity or obesity-related comorbidity) by the conjugates or pharmaceutical composition described herein.

[0024] In one aspect, provided herein are conjugates comprising Formula (I): O-(L-P)U, wherein O comprises an oligonucleotide; wherein L is a linker or a bond; wherein P is an NPR- C-binding peptide, wherein n is an integer of 1 to 4. In some embodiments, O is an antisense oligonucleotide (ASO), a double stranded RNA (dsRNA), or a guide RNA. In some embodiments, P is an NPR-C-binding peptide, e.g., an NPR-C-binding peptide in Tables 1-3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, L comprises a linker core and one or more spacers. In some embodiments, L comprises Spacerl -Linker Core- Spaced.

[0025] In some embodiments, the conjugate further comprises a fatty acid (FA) or lipid. In some embodiments, FA is attached to O. In some embodiments, FA is attached to P. In some embodiments, FA is attached to L.

[0026] In some embodiments, the conjugate comprises (FA)m-O-L-P or O-L-P-(FA)m, wherein m is an integer of 1 to 4. In some embodiments, m is 1 or 2.

[0027] In some embodiments, the conjugate further comprises a Spacer3. In some embodiments, the conjugate comprises (FA-Spaccr3)m-O-L-P or O-L-P-(Spaccr3-FA)m, wherein m is an integer of 1 to 4. In some embodiments, m is 1 or 2. In some embodiments, the conjugate comprises FA-Spacer3-O-L-P or O-L-P-Spacer3-FA. In some embodiments, the conjugate comprises O-L-(FA-Spacer3)m-P or O-(Spacer3-FA)m-L-P, wherein m is an integer of I to 4. In some embodiments, m is 1 or 2. In some embodiments, the conjugate comprises O- L-FA-Spacer3-P or O-Spacer3-FA-L-P.NPR-C binding peptide or protein (P)

[0028] The conjugates provided herein include a peptide that binds a human NPR-C receptor (“NPR-C binding peptide”) or a protein that binds human NPR-C receptor. In some embodiments, the protein or peptide binds NPR-C receptor with good affinity and selectivity.

[0029] In some embodiments, the NPR-C binding peptide is derived from, or similar to, ANP. Wild-type human ANP is a 28 amino acid peptide having a 17 amino acid loop formed by an intramolecular disulfide linkage between two cysteine residues present at positions 7 and 23 (SEQ ID NO: 10). It is a cardiac hormone that generally acts to maintain the cardiovascular’ system. It is part of the body's natural defense against hypoxia and pathological cardiac wall stress. Wild type ANP exhibits similar’ binding affinity for the receptors NPR-A and NPR-C, but a synthetic peptide C-ANP4-23 has high selectivity for NPR-C, not binding to NPR-A at detectable levels. Both linear and cyclic C-ANP4-23 bind NPR-C with high potency.

[0030] In some embodiments, the NPR-C binding peptide comprises a consensus sequence GX11IDX14I (SEQ ID NO: 1), wherein Xu is arginine, proline, or hydroxyproline, and X14 is arginine or N-methylarginine. In some embodiments, Xu is R. In some embodiments, X14 is R.

[0031] In some embodiments, the NPR-C binding peptide comprises GRIDRI (SEQ ID NO: 2).

[0032] In some embodiments, the NPR-C binding peptide comprises SX7X8X9GX11IDX14I (SEQ ID NO: 3), wherein:X7 is glycine, alanine, proline, hydroxyproline, serine, or cysteine;Xs is phenylalanine, or cyclohexylalanine, andX9 is glycine, alanine, or serine.

[0033] In some embodiments, X7 is C. In some embodiments, X9 is G.

[0034] In some embodiments, the NPR-C binding peptide comprises SCFGGRIDRI(SEQ ID NO: 4).

[0035] In other embodiments, the NPR-C binding peptide comprises FGGRIDRIGA (SEQ ID NO: 5).

[0036] In some embodiments, the NPR-C binding peptide comprises RSSX7FGGRIDRI (SEQ ID NO: 6), wherein X7 is serine or cysteine.

[0037] In some embodiments, the NPR-C binding peptide comprises SEQ ID NO: 7 (RSSX7FGGRIDRIGA). In certain embodiments, X7 is cysteine. In other embodiments, X7 is serine.

[0038] In some embodiments, the NPR-C binding peptide comprises SEQ ID NO: 8 (X7- Cha-X9GXiiIDXi4I), wherein:X7 is cysteine, proline, hydroxyproline, glycine, or alanine;X9 is alanine, serine, or glycine;Xu is proline, hydroxyproline, or arginine; and X14 is arginine or N-methylarginine.

[0039] In certain embodiments, the NPR-C binding peptide comprises SEQ ID NO: 9 (fSp-Cha-aGPIDRI).

[0040] Exemplary NPR-C binding peptides that can be used in conjugates described herein are provided in Tables 1-3. In some embodiments, the NPR-C binding peptide comprises a sequence selected from any one of SEQ ID NO: 11-57.Table 1. Exemplary NPR-C binding peptidesTable 2. Exemplary NPR-C binding peptidesTable 3. Exemplary NPR-C binding peptides

[0041] Tables 1-3 do not represent an exhaustive listing of all NPR-C binding peptides which may be utilized in a conjugate disclosed herein. In some instances, one or more amino acids may be substituted for an equivalent number of amino acids representing conservative mutations to the sequence, as are known in the art. For example, in some instances an isoleucine may be substituted where a leucine is indicated, and vice versa.

[0042] Suitable NPR-C binding peptides may be of a variety of lengths. In one aspect, the length of the peptide may be from 6 to 30 amino acids. In another aspect, the length of the peptide may be from 7 to 28 amino acids. In another aspect, the length of the peptide may be from 8 to 26 amino acids. In another aspect, the length of the peptide may be from 9 to 24 amino acids. In another aspect, the length of the peptide may be from 10 to 22 amino acids. The peptide may be 6 amino acids long, or 7 amino acids, or 8 amino acids, or 9 amino acids, or 10 amino acids, or 11 amino acids, or 12 amino acids, or 13 amino acids, or 14 amino acids, or 15 amino acids, or 16 amino acids, or 17 amino acids, or 18 amino acids, or 19 amino acids, or 20 amino acids, or 21 amino acids, or 22 amino acids, or 23 amino acids, or 24 amino acids, or 25 amino acids, or 26 amino acids, or 27 amino acids, or 28 amino acids, or 29 amino acids, or 30 amino acids in length.

[0043] As mentioned above, the NPR-C binding peptide may be modified. It may be modified at the N-terminal end, at the C-terminal end, at an internal position, or any combination of these. For example, the C-terminal carboxylic acid moiety of a peptide may be converted to an amide to generate a different peptide having the same amino acid sequence (see for example SEQ ID NO: 16 and SEQ ID NO: 17). In some embodiments, NPR-C binding peptide comprises a C-terminal hydroxyl. In some embodiments, NPR-C binding peptide comprises a C-terminal amide.

[0044] In some instances, the NPR-C binding peptide may be a linear peptide; that is, one in which there is no intramolecular bond made between any of the N-tcrminal end, the C- terminal end, and any of the side chains of the amino acids which constitute the peptide.

[0045] In other instances, the NPR-C binding peptide may be a cyclic peptide, in which there is exactly one or at least one intramolecular bond made between any of the N-terminal end, the C-terminal end, and any of the side chains of the amino acids which constitute the peptide. The bond which cyclizes the peptide may in one embodiment include atoms only derived from the parent peptide itself, for example a disulfide bond between cysteine side chains, as shown in Formula II below. As illustrated, Formula II shows the cysteine residues separated by 9 amino acids, with each X representing an amino acid residue. It will be appreciated that the amino acid chain may be of any length:

[0046] In another embodiment, the peptide may be cyclized using additional atoms in the intramolecular bond, such as an intervening alkyl group between the side chains of two cysteine residues. As illustrated, Formula III shows the cysteine residues separated by 9 amino acids, with each X representing an amino acid residue. It will be appreciated that the amino acid chain may be of any length. The value of z can be any integer from 1 to 20 inclusive.

[0047] In the formula above, when z = 1, the peptide contains a thioacetal moiety, defined by the side chains of the cysteine residues and the methylene group bridging the same.

[0048] In some embodiments, the NPR-C binding peptide is cyclized by covalent attachment of a side chain of a first cysteine residue to a side chain of a second cysteine residue. In some embodiments, the covalent attachment comprises a disulfide bond. In some embodiments, the covalent attachment comprises a thioacetal moiety.

[0049] In some instances, the NPR-C binding peptide may be a single copy of one of the sequences specified herein. Also envisaged are delivery moieties containing multiple copies of peptides disclosed herein, such as duplications of certain sequences, the use of multiple distinct peptides to generate a single construct, and so forth.Oligonucleotide

[0050] The conjugates described herein comprise an oligonucleotide. In some embodiments, O is an antisense oligonucleotide, a double stranded RNA (dsRNA), or a guide RNA.

[0051] In some embodiments, O is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand. In some embodiments, at least one nucleotide of the sense strand is a modified nucleotide. In some embodiments, at least one nucleotide of the antisense strand is a modified nucleotide. In some embodiments, at least one internucleotide linkage of the sense strand is a modified internucleotide linkage. In some embodiments, at least one internucleotide linkage of the antisense strand is a modified internucleotide linkage. In some embodiments, the dsRNA comprises a sense strand and an antisense stand, wherein the antisense strand is complementary to a target mRNA selected from SOD1, PLIN1, ALK7 / ACVR1C, PDE3B or HPRT.

[0052] Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting SOD1 mRNA are provided in Table 4.Table 4. Unmodified Nucleic Acid Sequences of dsRNA targeting mouse SOD1 mRNA (SOD1 siRNA)

[0053] Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting HPRT mRNA are provided in Table 5.Table 5. Unmodified Nucleic Acid Sequences of dsRNA targeting human HPRT mRNA (HPRT siRNA)

[0054] The dsRNA can include modifications. The modifications can be made to one or more nucleotides of the sense and / or antisense strand or to the internucleotide linkages, which are the bonds between two nucleotides in the sense or antisense strand. For example, some 2’- modifications of ribose or deoxyribose can increase RNA or DNA stability and half-life. Such 2’ -modifications can be 2’-fluoro, 2’-O-methyl (i.e., 2’-methoxy), or 2'-O-alkyl (e.g., 2’-O-Ci6 alkyl).

[0055] In some embodiments, one or more nucleotides of the sense strand and / or the antisense strand are independently modified nucleotides, which means the sense strand and the antisense strand can have different modified nucleotides. In some embodiments, each nucleotide of the sense strand is a modified nucleotide. In some embodiments, each nucleotide of the antisense strand is a modified nucleotide. In some embodiments, the modified nucleotide is a 2'- fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-Ci6 alkyl) modified nucleotide. In some embodiments, each nucleotide of the sense strand and the antisense strand is independently a modified nucleotide, e.g., a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-Ci6 alkyl) modified nucleotide.

[0056] In some embodiments, the sense strand has four 2'-fluoro modified nucleotides, e.g., at positions 7, 9, 10, 11 from the 5’ end of the sense strand. In some embodiments, the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand has four 2'-fluoro modified nucleotides, e.g., at positions 2, 6, 14, 16 from the 5’end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand arc 2'-O-mcthyl modified nucleotides.

[0057] In some embodiments, the sense strand has three 2'-fluoro modified nucleotides, e.g., at positions 9, 10, 11 from the 5’ end of the sense strand. In some embodiments, the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 7, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 8, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 3, 7, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the antisense strand has three 2'-fluoro modified nucleotides, e.g., at positions 2, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O- methyl modified nucleotides.

[0058] In some embodiments, the 5’ end of the antisense strand has a phosphate analog, e.g., 5’-vinylphosphonate (5’-VP).

[0059] In some embodiments, the sense strand or the antisense strand comprises an abasic moiety or inverted abasic moiety, e.g., a moiety shown in Table 6.Table 6. Abasic or inverted abasic (iAb) moieties“5”’ and “3”’ indicate the 5’ to 3’ direction of the sequences.

[0060] In some embodiments, the abasic moiety or inverted abasic moiety may be attached to the sense strand or the antisense strand via a phosphorothioate moiety. In oneexample, an inverted abasic moiety attached via a phosphorothioate internucleotide linkage of a sense strand has a formula as follows, where 3’ indicates the direction of the sequence:

[0061] In some embodiments, the sense strand and the antisense strand have one or more modified intemucleotide linkages. In some embodiments, the modified internucleotide linkage is phosphorothioate linkage. In some embodiments, the sense strand has four or five phosphorothioate linkages. In some embodiments, the antisense strand has four or five phosphorothioate linkages. In some embodiments, the sense strand and the antisense strand each has four or five phosphorothioate linkages. In some embodiments, the sense strand has four phosphorothioate linkages and the antisense strand has five phosphorothioate linkages.

[0062] In some embodiments, the sense strand or antisense strand comprises a modified nucleotide with a lipid moiety at the 2’ position of a ribose (e.g., 2’-O-Ci6 alkyl). In some embodiments, the modified nucleic acid is at position 6 from the 5’ end of the sense strand.

[0063] Exemplary modified sense strand and antisense strand sequences of dsRNA targeting SOD1 mRNA arc provided in Table 7.Table 7: Modified Nucleic Acid Sequences of dsRNA targeting human SOD1 mRNA (SOD1 siRNA)Abbreviations - “m” indicates 2’-0Me; “f’ indicated 2’-fluoro; indicates phosphorothioate linkage; “Uhd” indicates 2’-0-hexadecyl uridine; “Upa” indicates 2'-0-propylamino uridine; “Upi” indicates 2'-O- icosanamidopropyl uridine; “VP” indicates 5’-vinylphosphonate; “S” means the sense strand; “AS” means the antisense strand.

[0064] Exemplary modified sense strand and antisense strand sequences of dsRNA targeting human HPRT mRNA are provided in Table 8.Table 8: Modified Nucleic Acid Sequences of dsRNA targeting human HPRT mRNA (HPRT siRNA)Abbreviations - “m” indicates 2’-0Me; “f’ indicated 2’-fluoro; indicates phosphorothioate linkage; “VP” indicates 5’-vinylphosphonate; “Ahd” indicates 2’-O-hexadecyl adenosine; “S” means the sense strand; “AS” means the antisense strand.

[0065] The sense strand and antisense strand of dsRNA can be synthesized using any nucleic acid polymerization methods known in the art, for example, solid-phase synthesis by employing phosphoramidite chemistry methodology (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA), H- phosphonate, phosphortriester chemistry, or enzymatic synthesis. Automated commercial synthesizers can be used, for example, MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from Bio Automation or Applied Biosystems. Phosphorothioate linkages can be introduced using a sulfurizing reagent such as phenylacetyl disulfide or DDTT(((dimethylaminomethylidene) amino)-3H-l,2,4-dithiazaoline-3-thione). It is well known to use similar techniques and commercially available modified amidites and controllcd-porc glass (CPG) products to synthesize modified oligonucleotides or conjugated oligonucleotides.

[0066] Purification methods can be used to exclude the unwanted impurities from the final oligonucleotide product. Commonly used purification techniques for single stranded oligonucleotides include reverse-phase ion pair high performance liquid chromatography (RP-IP- HPLC), capillary gel electrophoresis (CGE), anion exchange HPLC (AX-HPLC), and size exclusion chromatography (SEC). After purification, oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nm. The sense strand and antisense strand can then be annealed to form a dsRNA.Linker

[0067] The conjugates described herein can include a linker (L).

[0068] In some embodiments, L comprises a linker core (LC) and one or more spacers. Exemplary LC structures are provided in Table 9. The spacers may be small organic groups or polymer chains or the like. In some embodiments, L comprises Spacer 1 -Linker Core-Spacer2. In some embodiments, Spacerl and Spacer 2 are selected from Table 10. Exemplary linkers are provided in Table 11.Table 9. Exemplary Linker Core (LC) Structures

[0069] It will be appreciated that in some instances, the linker structures above represent final structural configurations of the linkers where precursor molecules on different portions of the conjugate have been reacted to yield such structures. That is, a first precursor to the linker may be covalently attached to the oligonucleotide, and a second precursor to the linker may be covalently attached to the peptide. Then the two precursors are brought into proximity with one another under proper conditions and form a covalent linkage yielding one of the structures shown herein.Table 10: Exemplary Linker Spacers (SL, SP)Table 11: Exemplary Linkers (L)

[0070] Various linkers and spacers may be combined to yield an overall linker structure.For instance, in one embodiment, the linker is of formula:wherein X represents a position to which the 3’ end of the sense strand of O is conjugated, and Y represents a position to which an N-terminal end of P is conjugated.Fatty Acids

[0071] In some embodiments, the conjugates further comprise a fatty acid (FA) or lipid. The FA can be saturated or unsaturated. In some embodiments, the FA is a C12-C22 fatty acid. Exemplary fatty acids are provided in Table 12.

[0072] In some embodiments, FA is attached to O. In some embodiments, FA is attached to P. In some embodiments, FA is attached to L.

[0073] In some embodiments, the conjugate comprises (FA)m-O-L-P or O-L-P-(FA)m, wherein m is an integer of 1 to 4. In some embodiments, m is 1 or 2.

[0074] In some embodiments, the conjugate further comprises a Spacer3. Exemplary fatty acid spacers (Spacer3) are provided in Table 13.

[0075] Exemplary FA-Spacer3 pairs are provided in Table 14.

[0076] In some embodiments, the conjugate comprises (FA-Spacer3)m-O-L-P or O-L-P- (Spacer3-FA)m, wherein m is an integer of 1 to 4. In some embodiments, m is 1 or 2. In some embodiments, the conjugate comprises FA-Spacer3-O-L-P or O-L-P-Spacer3-FA. In some embodiments, the conjugate comprises FA-Spacer3-O-Spacerl-LinkerCore-Spacer2-P or O- Spaccrl-LinkcrCorc-Spaccr2-P-Spaccr3-FA.

[0077] In some embodiments, the conjugate comprises O-L-(FA-Spacer3)m-P or O- (Spacer3-FA)m-L-P, wherein m is an integer of 1 to 4. In some embodiments, m is 1 or 2. In some embodiments, the conjugate comprises O-L-FA-Spacer3-P or O-Spacer3-FA-L-P. In some embodiments, the conjugate comprises O-Spacerl-LinkerCore-Spacer2- FA-Spacer3-P or O- Spacer3-FA-Spacerl-LinkerCore-Spacer2-P.

[0078] The fatty acids FA of Table 12 may be incorporated into conjugates of the present disclosure by several different methods. For example, these lipids may be conjugated to an RNA molecule after synthesis is complete, or they may be incorporated at an internal position of the RNA by being provided as part of an amidite, or by any other conventional method.Table 12: Exemplary Fatty Acids (FA)Table 13: Exemplary Spacers for Fatty Acids (Spacer3)* For SF1, “(2’-OH)-C3-NH-“ the aminopropyl moiety extends from the position where the 2’- hydroxyl hydrogen of an RNA would otherwise exist, with reference to the Upa 2'-O-propylamino uridine modified base.Table 14. Exemplary FA-Spacer3 PairsConjugates

[0079] Certain exemplary conjugates of the present disclosure are provided in Table 15:Table 15: Exemplary conjugates comprising NPR-C binding peptide and dsRNAFor C22 and C23, parentheses around (FA2) represent the connectivity of the enclosed fatty acid carbonyl FA2, which attaches via amide bond to the amine of the 2'-0-propylamino uridine (Upa) modified base of R (SEQ ID NO 5).AFor C26, parentheses around (LC3-SP1-P) represent the connectivity of the enclosed linker- spacer-peptide moiety LC3-SP1-P, which attaches via amide bond between the cyclohexylcarbonyl carbon of LC3 and the amine of the 2'-O-propylamino uridine (Upa) modified base of R (SEQ ID NO 5). iFor C72, parentheses around (FA27) represent the connectivity of the enclosed fatty acid carbonyl FA27 which attaches via amide bond to the amine within the Upi 2'-O-icosanamidopropyl uridine modified base of R (SEQ ID NO 8).LOOO8OJ The conjugates described herein can be made by a variety of procedures known to one of ordinary skill in the art, some of which are illustrated in the preparations and examplesbelow. One of ordinary skill in the art recognizes that the specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare conjugates. The product of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. The reagents and starling materials are readily available to one of ordinary skill in the art.Pharmaceutical Composition

[0081] In another aspect, provided herein are pharmaceutical compositions comprising any of the conjugates described herein, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can also comprise one or more pharmaceutically acceptable excipient, diluent, or carrier. Pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 23rd edition (2020), A. Loyd et al., Academic Press). In certain embodiments, the pharmaceutical composition may be an aqueous formulation. In some embodiments, a pharmaceutically active salt may comprise at least one of sodium, calcium, magnesium, potassium, or coformulations of the same.Method of Treatment and Therapeutic Use

[0082] In another aspect, provided herein are methods of treating a disease or condition of adipose tissue, e.g., obesity or obesity-related comorbidity, in a patient in need thereof, and such the method comprises administering to the patient an effective amount of a conjugate or pharmaceutical composition described herein. In one aspect, the method or use includes a method to reduce excess body weight and maintain weight reduction long term in adults with obesity or adults with overweight in the presence of at least one weight-related comorbid condition. In one embodiment, the method or use includes treating moderate to severe obstructive sleep apnea (OSA) in adults with obesity. The conjugate or pharmaceutical composition can be administered to the patient intravenously or subcutaneously.

[0083] Also provided herein are methods of delivering an oligonucleotide to an adipose tissue, comprising administering to a subject a conjugate or pharmaceutical composition described herein.

[0084] Dosage regimens for the conjugates described herein may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0085] Dosage values may vary with the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

[0086] In some embodiments, methods of treatment with a conjugate of the present disclosure comprise administering a conjugate and an incretin to the patient in simultaneous, separate, and sequential combinations. Examples of incretin include glucagon like peptide- 1 (GLP- 1) or GLP-1 analogs, glucose-dependent insulinotropic polypeptide (GIP) or GIP analogs, oxyntomodulin or oxyntomodulin analogs; dual GIP and GLP-1 receptor agonists; GCG, and GIP receptor agonist and GLP-1 receptor tri-agonists. In certain embodiments, the incretin may be selected from semaglutide, dulaglutide, tirzepatide, exenatide, liraglutide, albiglutide, lixisenatide, or retatrutide. In one embodiment, the patient to be treated is being treated with an incretin. In one embodiment, the patient to be treated has been treated with an incretin.

[0087] In another aspect, provided herein are conjugates or pharmaceutical compositions described herein for use in a therapy. In some embodiments, provided herein are conjugates or pharmaceutical compositions described herein for use in the treatment of a disease or condition of adipose tissue, e.g., obesity or obesity-related comorbidity.

[0088] Also provided herein are uses of conjugates or pharmaceutical compositions described herein in the manufacture of a medicament for treating a disease or condition of adipose tissue, e.g., obesity or obesity-related comorbidity.

[0089] Also provided herein are methods of delivering an oligonucleotide to an adipose tissue, comprising administering to a subject a conjugate or pharmaceutical composition described herein.

[0090] As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

[0091] As used herein, the term “alkyl” means saturated linear or branched-chain monovalent hydrocarbon radical, containing the indicated number of carbon atoms. For example, “C1-C22 alkyl” means a radical having 1-22 carbon atoms in a linear- or branched arrangement.

[0092] As used herein, “antisense strand” means a single-stranded oligonucleotide that is complementary to a region of a target sequence. Likewise, and as used herein, “sense strand” means a single-stranded oligonucleotide that is complementary to a region of an antisense strand.

[0093] The terms “bind” and “binds” as used herein are intended to mean, unless indicated otherwise, the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which results in proximity of the two proteins or molecules as determined by common methods known in the art.

[0094] As used herein, “complementary” means a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand, e.g., a hairpin) that permits the two nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. Complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. Likewise, two nucleic acids may have regions of multiple nucleotides that are complementary with each other to form regions of complementarity, as described herein.

[0095] As used herein, “duplex,” in reference to nucleic acids or oligonucleotides, means a structure formed through complementary base pairing of two antiparallel sequences of nucleotides (i.e., in opposite directions), whether formed by two separate nucleic acid strands or by a single, folded strand (e.g., via a hairpin).

[0096] An “effective amount” refers to an amount necessary (for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount of a protein or conjugate may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein or conjugate to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the protein or conjugate are outweighed by the therapeutically beneficial effects.

[0097] The term “knockdown” or “expression knockdown” refers to reduced mRNA or protein expression of a gene after treatment of a reagent.

[0098] As used herein, “modified internucleotide linkage” means an internucleotide linkage having one or more chemical modifications when compared with a reference intemucleotide linkage having a phosphodiester bond. A modified intemucleotide linkage can be a non-naturally occurring linkage. In some embodiments, the modified intemucleotide linkage is phosphorothioate linkage.

[0099] As used herein, “modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide can have, for example, one or more chemical modification in its sugar, nucleobase, and / or phosphate group. Additionally, or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to a corresponding reference nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl (e.g., 2’-O-Ci6 alkyl) modified nucleotide. In some embodiments, the modified nucleotide has a phosphate analog, e.g., 5’-vinylphosphonate. In some embodiments, the modified nucleotide has an abasic moiety or inverted abasic moiety, e.g., a moiety shown in Table 6.[000100] As used herein, “nucleotide” means an organic compound having a nucleoside (a nucleobase, e.g., adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar, e.g., ribose or 2'-deoxyribose) linked to a phosphate group. A “nucleotide” can serve as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).[000101] As used herein, “oligonucleotide” means a polymer of linked nucleotides, each of which can be modified or unmodified. An oligonucleotide is typically less than about 100 nucleotides in length.[000102] The term “patient”, as used herein, refers to a human patient.[000103] As used herein, “phosphate analog” means a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5’ terminal nucleotide of an oligonucleotide in place of a 5’- phosphate, which is often susceptible to enzymatic removal. A 5’ phosphate analog can include aphosphatase-resistant linkage. Examples of phosphate analogs include 5’ methylene phosphonate (5’-MP) and 5’-(E)-vinylphosphonatc (5’-VP). In some embodiments, the phosphate analog is 5’-VP.[000104] As used herein, “polypeptide” or “peptide” means a polymer of amino acid residues comprising two (2) or more amino acids and / or amino acid derivatives which, in general, are linked via peptide bonds. The term applies to polymers comprising naturally occurring amino acids and polymers comprising one or more non-naturally occurring amino acids. Embodiments may include modifications or amino acid derivatives, including synthetic modifications, some of which may resemble post-translational modifications such as, phosphorylation, hydroxylation, sulfonation, acylation, glycosylation and disulfide formation. [000105] As used herein, the term “protein” refers to at least one polypeptide. A protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Proteins may include moieties other than amino acids (e.g., glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. [000106] In some embodiments, the peptides described herein include a fatty acid conjugated, for example, by way of a direct bond or linker to a natural or non-natural amino acid with a functional group available for conjugation. Such a conjugation is sometimes referred to as acylation. In certain instances, the amino acid with a functional group available for conjugation can be K, C, E, and D. In particular instances, the amino acid with a functional group available for conjugation is K, where the conjugation is to an 8-amino group of a K side-chain. In some embodiments, the peptides described herein are amidated. In some embodiments, the peptides described herein have a modification of the C-terminal group, wherein the modification is NH2. In some embodiments, the peptides described herein have a modification of the C-terminal group where the modification is absent.[000107] Amino acids which may be incorporated into the peptides of the present disclosure include the twenty standard or canonical amino acids, and a number of other nonstandard amino acids. Some of the nonstandard amino acids are shown below in Table 16.Table 16. Selected nonstandard amino acids[000108] As used herein, where the full name of an amino acid is spelled out (“arginine,” etc.), all stereoisomers of that amino acid is encompassed (e.g., L-arginine and D-arginine.) Where a single-letter abbreviation is used, an uppercase letter denotes the L isomer (that is, the isomer incorporated into polypeptides in nature), and a lowercase letter denotes the D isomer. Noncanonical amino acids are generally denoted by a three-letter abbreviation rather than a single letter, or as specified in the table above.[000109] The peptides described herein may react with any number of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts.Pharmaceutically acceptable salts and common techniques for preparing them are well known in the art (see, e.g, Stahl et. al, Handbook of Pharmaceutical Salts: Properties, Selection, and Use, 2ndRevised Edition (Wilcy-VCH, 2011)). Pharmaceutically acceptable salts for use herein include sodium, potassium, trifluoroacetate, hydrochloride and / or acetate salts. The disclosure also provides and therefore encompasses novel intermediates and methods of synthesizing the polypeptides described herein, or pharmaceutically acceptable salts thereof. The intermediates and polypeptides described herein can be prepared by a variety of techniques known in the art. For example, a method using chemical synthesis is illustrated in the Examples below.[000110] The specific synthetic steps for each of the routes described may be combined in different ways to prepare the polypeptides described herein. The reagents and starting materials are readily available to one of skill in the art.[000111] As used herein, “strand” refers to a single, contiguous sequence of nucleotides linked together through intemucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). A strand can have two free ends (e.g., a 5’ end and a 3’ end).[000112] As used herein, “treatment” or “treating” refers to all processes wherein there may be a slowing, controlling, delaying, or stopping of the progression of the disorders or disease disclosed herein, or ameliorating disorder or disease symptoms, but does not necessarily indicatea total elimination of all disorder or disease symptoms. Treatment includes administration of a protein or nucleic acid or vector or composition for treatment of a disease or condition in a patient, particularly in a human.[000113] As used herein, “NPR-C” refers to natriuretic receptor C protein or polypeptide.NPR-C is also known as NPR3 (natriuretic peptide receptor 3). NPR-C and NPR3 are used interchangeably throughout this disclosure. Several human NPR-C isoforms exist.[000114] The amino acid sequence of the longest human NPR-C isoform (isoform 1) can be found at NP_001191304.1:1 MPSLLVLTFS PCVLLGWALL AGGTGGGGVG GGGGGAGIGG GRQEREALPP QKIEVLVLLP61 QDDSYLFSLT RVRPAIEYAL RSVEGNGTGR RLLPPGTRFQ VAYEDSDCGN RALFSLVDRV121 AAARGAKPDL ILGPVCEYAA APVARLASHW DLPMLSAGAL AAGFQHKDSE YSHLTRVAPA181 YAKMGEMMLA LFRHHHWSRA ALVYSDDKLE RNCYFTLEGV HEVFQEEGLH TSIYSFDETK241 DLDLEDIVRN IQASERWIM CASSDTIRSI MLVAHRHGMT SGDYAFFNIE LFNSSSYGDG301 SWKRGDKHDF EAKQAYSSLQ TVTLLRTVKP EFEKFSMEVK SSVEKQGLNM EDYVNMFVEG361 FHDAILLYVL ALHEVLRAGY SKKDGGKI IQ QTWNRTFEGI AGQVSIDANG DRYGDFSVIA421 MTDVEAGTQE VIGDYFGKEG RFEMRPNVKY PWGPLKLRID ENRIVEHTNS SPCKSSGGLE481 ESAVTGIWG ALLGAGLLMA FYFFRKKYRI TIERRTQQEE SNLGKHRELR EDSIRSHFSV541 A(SEQ ID NO: 88).[000115] The amino acid sequence of NPR-C isoform 2 can be found at NP_000899.1 :1 MPSLLVLTFS PCVLLGWALL AGGTGGGGVG GGGGGAGIGG GRQEREALPP QKIEVLVLLP 61 QDDSYLFSLT RVRPAIEYAL RSVEGNGTGR RLLPPGTRFQ VAYEDSDCGN RALFSLVDRV 121 AAARGAKPDL ILGPVCEYAA APVARLASHW DLPMLSAGAL AAGFQHKDSE YSHLTRVAPA181 YAKMGEMMLA LFRHHHWSRA ALVYSDDKLE RNCYFTLEGV HEVFQEEGLH TSIYSFDETK241 DLDLEDIVRN IQASERWIM CASSDTIRSI MLVAHRHGMT SGDYAFFNIE LFNSSSYGDG301 SWKRGDKHDF EAKQAYSSLQ TVTLLRTVKP EFEKFSMEVK SSVEKQGLNM EDYVNMFVEG361 FHDAILLYVL ALHEVLRAGY SKKDGGKI IQ QTWNRTFEGI AGQVSIDANG DRYGDFSVIA421 MTDVEAGTQE VIGDYFGKEG RFEMRPNVKY PWGPLKLRID ENRIVEHTNS SPCKSCGLEE481 SAVTGIWGA LLGAGLLMAF YFFRKKYRIT IERRTQQEES NLGKHRELRE DSIRSHFSVA(SEQ ID NO: 89)[000116] Other NPR-C isoforms include isoform 3 (NP_001191305.1), isoform 4(NP_OO135O581.1), isoform 5 (NP_001351387.1), isoform 6 (NP_001351389.1).[000117] The following examples are offered to illustrate, but not to limit, the claimed inventions.EXAMPLESExample 1: General strategy for synthesis of dsRNA-peptide conjugatesAs depicted in Routel, NPR-C binding peptides (Route 1A), RNA sense strands (Route IB) andRNA antisense strands (Route 1C) were all synthesized using standard solid phase peptide oroligonucleotide synthesis techniques. Further functionalization steps to incorporate optional spacers (SL, Sp, SF), fatty acids (FA), and linker fragments activated for subsequent conjugation (LF), were performed either directly on solid support, or in solution following cleavage from the solid support, depending on the chemistry involved.Route 1:Asteps as needed p pF- Optional Fatty Acid (FA) vatedsteps as neededF- Optional Spacer (SF)- Optional Fatty Acid (FA)As depicted in Route 2, functionalized RNA sense strand intermediates were conjugated in solution to functionalized NPR-C binding peptides using appropriate conditions. The resulting RNA sense strand-peptide conjugate intermediates were then annealed to the corresponding RNA antisense strand, to provide the dsRNA-peptide conjugate, with appropriate purification steps.Route 2RNA Sense Strand - Peptide Conjugate Lc= Linker CoredsRNA - Peptide Conjugate- Optional Spacers (SLand SF)- Optional Fatty Acid (FA)As depicted in Route 3, an alternative synthetic approach entails annealing the functionalized RNA sense strand to the corresponding RNA antisense strand, prior to conjugation. Using appropriate conditions and purification steps, the functionalized NPR-C binding peptide can then be conjugated to the functionalized dsRNA in solution, to provide the dsRNA-peptide conjugate.Route 3dsRNA - Peptide Conjugate- Optional Spacers (SLand SF)- Optional Fatty Acid (FA) Lc= Linker CoreExample 2: Preparation of functionalized peptide intermediates for use in synthesis of dsRNA-NPR-C-binding peptide conjugatesExamples 2A-2EExample 2A: General Procedure for solid phase synthesis of NPR-C binding peptides [000118] Peptide synthesis was carried out using standard 9-fluorenyl-methyloxycarbonyl (Fmoc) tert-Butyl (t-Bu) solid phase peptide chemistry protocols on either a Symphony 12- channel multiplex peptide synthesizer or a Symphony -X 24-channel multiplex peptide synthesizer (Gyros Protein Technologies, Inc.), at a 0.1 mmol scale.[000119] The solid support used was pre-loaded H-Cys(Trt)-2-CTC resin (S-trityLL- cysteine-2-Chlorotrityl Resin, Peptides International), (100-200 mesh) with a 1% DVB crosslinked polystyrene core and a substitution of ~ 0.50 meq / g for the generation of peptide acids, or Low Loading 4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucyL4- Methylbenzhydrylamine resin (Fmoc-Rink-MBHA Low Loading resin, EMD Millipore), (100- 200 mesh) with a 1% DVB cross-linked polystyrene core and a substitution range of 0.3-0.4 meq / g for peptide amides. Standard sidechain protecting groups were used for all Fmoc-L- Amino Acids utilized. Non-standard amino acids used in the syntheses herein can be found in Table 17 or 18.[000120] Fmoc deprotection prior to each coupling step was accomplished by treatments with 20% piperidine (PIP; Sigma Aldrich) in DMF (Fisher Chemicals), 2 x 7 minutes with nitrogen mixing, followed by 8 x DMF wash cycles. For the synthesis of peptide acids on 2- chlorotrityl resin, typical unhindered couplings were performed for 1 hour using the Fmoc amino acid (0.3 M in DMF), N,N,N',N'-Tetramethyl-O-(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, Ambeed Inc.; 0.9 M in DMF ) and N,N-diisopropylethylamine (DIPEA, Sigma Aldrich; 1.2 M in DMF), at a 9-fold molar excess of AA / HBTU and a 12-fold molar excess of DIPEA over the reported resin loading level.[000121] For the coupling of hindered building blocks such as Fmoc-Arg(Pbf)-OH, Fmoc- N-Methyl-AAs or Fmoc-aMeAAs, a double coupling protocol was used, and the coupling time was extended to 2 hours each. For the synthesis of peptide amides on Fmoc-Rink-MBHA resin, typical unhindered couplings were performed for 1 hour using the Fmoc amino acid (0.3 M, Advanced ChemTech, in DMF), N,N'-diisopropylcarbodiimide (DIC, Chemlmpex, 1.2 M in DCM) and ethyl cyanohydroxyiminoacetate (Oxyma Pure, Chemlmpex; 0.9 M in DMF), at a 9- fold molar excess of AA / Oxyma and a 12-fold molar excess of DIC over the reported resinloading level. For hindered couplings such as Fmoc-Arg(Pbf)-OH, Fmoc-N-Methyl-AAs or Fmoc-aMcAA, the coupling time was extended to 6 hours.[000122] For the solid phase coupling of active esters, such as MSPT-PEG2-NHS, a manual addition of a 3x excess of the ester along with a lOx excess of DIPEA was used after the final Fmoc was removed from the peptidyl resin. After the primary sequence of the desired peptide was completed, the peptidyl resin was transferred as a DCM slurry to disposable fritted plastic syringe fitted with Teflon stopcock. To prepare the resin for cleavage, further washes with DCM were done, and the resin was thoroughly dried in vacuo.[000123] TFA cleavage: The dry peptidyl resins were treated with 10 mL of cleavage cocktail consisting of trifluoroacetic acid (TFA, Acros Organics), Water, Thioanisole (Sigma- Aldrich) and Triisopropylsilane (TIPS; Acros Organics), (TFA:Water:Thioanisole:TIPS;90.5:2.5:2.5 v / v) or TFA, water, 3,6-dioxa-l,8-octanedithiol (DODT; Sigma Aldrich), triisopropylsilane, (TFA:Water:DODT:TIPS; 90:5:2.5:2.5 v / v) for 2 hours at room temperature. After the 2 hour cleavage incubation, the resin was filtered off, washed twice with 2 mL of neat TFA, and the combined filtrates / washes were collected in a 50 ml conical disposable tube. The cleavage solution was then treated with 35 mL of cold diethyl ether (Fisher Chemicals) (-20°C) to precipitate the crude peptide. The peptide / ether suspension was then centrifuged at 4000 rpm for 2 min to form a solid pellet, the supernatant was decanted, and the solid pellet was triturated with fresh ether and the process was repeated two additional times, finally drying the peptide pellet in vacuo.Table 17: Nonstandard amino acids and other peptide componentsExample 2B: General procedure for disulfide bridge formation in NPR-C binding peptides [000124] Crude peptides were solubilized and diluted, in a suitable glass vessel, with 25% aqueous acetic acid to relatively low concentration (0.2-0.5 mg / ml crude peptide). The solution was then placed on magnetic stirrer with a spin vane, mixed vigorously and titrated with a few drops a saturated Iodine in methanol solution until a faint yellow endpoint was achieved. After reaching the yellow endpoint, the reaction was incubated at RT for 15 min, at which point the excess Iodine was quenched by the addition of a few drops of 0.1 M aqueous ascorbic acid (Sigma Aldrich).Example 2C: General procedure for thioacetal bridge formation in NPR-C binding peptides[000125] Peptides were solubilized in 3 mL of water; the solution was then diluted with water and AcCN to achieve about a 30 / 70 mixture of Water / AcCN (-400 mL total volume) witha low concentration of peptide (-0.2-0.5 mg / ml). The solution was then adjusted to pH 8 with tricthylaminc (TEA, Acros Organics) - 10 equivalents, reducing conditions to prevent disulfide bridge formation were achieved by the addition of 2-4 equivalents of Tris(2-carboxyethyl) phosphine hydrochloride (TCEP-HC1, Sigma Aldrich) reducing agent. The thioacetal bridge was formed by the addition of 7-10 equivalents of Diiodomethane (CH2I2, Alfa Aesar). The thioacetal formation reaction was carried out by incubating the solution for 18 hours at RT with magnetic stirring. Progress of the reaction was monitored using analytical LC-MS and by observing the change in mass of +12 Daltons from the starting reduced peptide molecular weight and an accompanying shift in retention of the starting material HPLC peak.Example 2D: General procedure for solution-phase acylation of NPR-C Peptide N- Terminus with linker precursors: Mal-Dap(Boc)-OPfp, MSPT-PEG2-NHS, Mpa-NHS, and Fmoc-Cys(Trt)-Opfp[000126] For cyclic peptides containing a Thioacetal bridge, a solution phase acylation step was necessary to introduce the requisite reactive handle for conjugation. In a 15 mL falcon tube equipped with a spin vane, the purified, lyophilized precursor peptide was dissolved in 1000 pL of DMSO (Acros Organics) to it were added 75-100 pL of DIPEA (TCI America) to adjust the pH to -8-9. The reaction was magnetically stirred at RT. The active ester of the desired residue / moiety was dissolved in DMSO (-200-300 uL), 50 pL aliquots were added one at time while monitoring the progression of the reaction by LC-MS. Once the starting material was consumed, the reaction was stopped by the addition of -10 mL cold diethyl ether (Fisher Chemicals) (-20°C). The tube was mixed vigorously and centrifuged to force the peptide into a pellet or oil phase. Another fresh volume of cold diethyl ether was added, and the process was repeated, this time a white, tacky precipitate was formed as the final crude product.Additional steps following solution phase coupling of Fmoc-Cys(Trt)-OPfp:[000127] After the acylation and ether precipitation step, the pellet was further treated with 1 ml of 20 % PIP in DMF (for -15 min) to remove the Fmoc protecting group. Another round of precipitation was carried out using cold diethyl ether, washed and additional time with the cold ether. The pellet was finally treated with TFA (with 2%TIPS) to remove the Trityl protecting group from the Cys residue. Cold diethyl ether precipitation was once again used to isolate the final crude product.Additional steps following solution phase coupling of Mal-Dap(Boc)-OPfp:[000128] After the acylation and ether precipitation step, the pellet was further treated with neat TFA to remove the Boe protecting group from the Dap residue. Cold diethyl ether precipitation was once again used to isolate the final crude product.Example 2E: General procedure for Reverse-Phase HPLC purification of peptides [000129] Preparative HPLC was carried out using either a Waters 2545 Binary HPLC Systems or a Shimadzu LC-8A Binary HPLC Systems, both equipped with a column heater; using a Luna Phenyl-Hexyl RP-HPLC column (Phenomenex Inc.; 5pm, 100A; 250 x 21.2 mm). The running buffers used were either Formic acid; A: 0.1% Formic Acid / H2O and B: 0.1% Formic / Acetonitrile (AcCN, Fisher Chemicals) or TFA; A: 0.1% TFA / H2O and B: 0.1% TFA / Acetonitrile (AcCN, Fisher Chemicals). The initial loadings of the peptides were typically done either at 0%, 5% B, or 10% depending on the hydrophobicity of the peptide, with a 10 min isocratic step after loading at the respective starting conditions for column equilibration. Linear gradients of 0-60% B, or 5-65% B or 10-70% B over 60 min, at a flow of 15, 20 or 25 mL / min, with column heating set at 60 °C were used. UV monitoring was done at either 214 or 220nm. Fractions that were determined to contain the desired product, as confirmed by LC-MS analysis (Agilent LC / MSD XT), were pooled together. Typically, the solutions were then frozen and lyophilized to give a white amorphous solid product, as the TFA salts of the peptides (TFA was added to the pooled fraction isolated with Formic acid buffers). The purity was assessed by RP- HPLC (Agilent Infinity II), and MW was confirmed by LC-MS analysis (Agilent LC / MSD XT).Example 3: Representative examples of NPRC-binding peptide synthesis and functionalizationExamples 3A-3GExample 3A: Synthesis of Azidoacetyl-(SP1)-(SEQ ID: 11)[000130] Below is a depiction of the structure of the title compound using the standaid single letter code for L- Amino Acids except for the 2-(2-(2-Aminoethoxy)-ethoxy) acetic acid spacer (AEEA), L-Cysteine (Cys) residues at positions 7 and 23, and Azidoacetic Acid at the N- Terminus, where the structures of the residues have been expanded.[000131] The primary peptide sequence of the title compound was synthesized using standard 9- Fluorenyl-methyloxycarbonyl (Fmoc) tert-Butyl (t-Bu) solid phase peptide chemistry protocols on a Symphony, 12-channel multiplex peptide synthesizer (Gyros Protein Technologies, Inc.), at a 0.1 mmol scale. The solid support used consisted of pre-loaded H-Cys(Trt)-2-CTC resin (S- trityl-L-cysteine-2-Chlorotrityl Resin, Peptides International), (100-200 mesh) with a 1% DVB cross-linked polystyrene core and a substitution of ~ 0.50 meq / g. Standard sidechain protecting groups were used for all Fmoc-L- Amino Acids utilized. The non-standard amino acid used in the synthesis of the title compound was 2-[2-[2-(Fmoc-amino) ethoxy] ethoxy] acetic acid (Fmoc- AEEA-OH, AappTec Peptides). Azidoacetic Acid was used to cap the N-terminus of the sequence; the residue provides a reactive handle for conjugation chemistries. Fmoc deprotection prior to each coupling step was accomplished by treatments with 20% Piperidine (PIP; Sigma Aldrich) in Dimethylformamide (DMF; Fisher Chemicals), 2 x 7 minutes with nitrogen mixing, followed by 8 x DMF wash cycles. All amino acid couplings were performed for 1 hour using the Fmoc Amino Acid (0.3 M in DMF), N, N, N',N'-Tetramethyl-O-(lH-benzotriazol-l- yl)uronium hexafluorophosphate (HBTU, Ambeed Inc.; 0.9 M in DMF ) and N,N- Diisopropylethylamine (DIPEA, Sigma Aldrich; 1.2 M in DMF), at a 9-fold molar- excess of AA / HBTU and a 12-fold molar excess of DIPEA over the reported resin loading level. After the primary sequence of the peptide was synthesized up the third Fmoc-AEEA-OH residue, the final N-Terminal Fmoc was removed with PIP, and the DMF washes were carried out, the peptidyl- resin was capped with a 9-fold excess of Azidoacetic Acid. Coupling was done using DIC / Oxyma (1.2M / 0.9M); the coupling time was extended to 5 hrs. After coupling, 3x DMF washes were done, then the peptidyl resin was transferred as a DCM slurry to disposable fritted plastic syringe fitted with Teflon stopcock. Further washes with DCM were done, and finally, the resin was thoroughly dried in vacuo. The dry resin was then treated with 10 mL of cleavage cocktail consisting of trifluoroacetic acid (TFA, Acros Organics), water, and Triisopropylsilane (TIPS; Acros Organics), (TFA:Water:TIPS; 92.5:5:2.5 v / v) for 2 hours at room temperature. After the 2 hr cleavage incubation, the resin was filtered off, washed twice with 2 mL of neatTFA, and the combined filtrates / washes were collected in a 50 ml conical disposable tube. The cleavage solution was then treated with 35 mL of cold diethyl ether (Fisher Chemicals) (-20°C) to precipitate the crude peptide. The peptide / ether suspension was then centrifuged at 4000 rpm for 2 min to form a solid pellet, the supernatant was decanted, and the solid pellet was triturated with fresh ether and the process was repeated two additional times, finally drying the peptide pellet in vacuo.Disulfide Bridge Formation[000132] The crude peptide was solubilized, in a suitable glass vessel, with 25% aqueous Acetic Acid to a relatively low concentration (0.2-0.5 mg / ml crude peptide). The solution was then placed on magnetic stirrer with the necessary spin vane, mixed vigorously and titrated with a few drops a saturated Iodine in methanol solution until a faint yellow endpoint was achieved. After reaching the yellow endpoint, the reaction was incubated at RT for 15 min, at which point the excess Iodine was quenched by the addition of a few drops of 0.1 M aqueous ascorbic acid (Sigma Aldrich).HPLC Purification[000133] The reaction solution containing the crude oxidized peptide was then loaded, via injection valve, onto a preparative HPLC system (Shimadzu LC-8A Binary Preparative HPLC Systems) using a Luna Phenyl-Hexyl RP-HPLC column (Phenomenex Inc.; 5pm, 100A; 250 x 21.2 mm). The running buffers used were A: 0.1% TFA / H2O and B: 0.1% TFA / Acetonitrile (ACCN, Fisher Chemicals). The initial loading was done at 0% B, with 10 min isocratic equilibration after loading. The sample was eluted using a linear’ 0-60 % B gradient over 60 min, at a flow of 25 mL / min, with column heating set at 60 °C. Fractions containing the desired product (analysis by Agilent LC-MS) were pooled, frozen and lyophilized to give a white amorphous solid product, as the TFA salt of the title compound. The purity assessed by RP- HPLC 1 (Agilent HPLC System) was found to be >95%, with the observed LC-MS molecular weight of 2556.20 Dalton; matching the theoretical calculated molecular weight of 2556.82 Dalton.Example 3B: Synthesis of Cys-(SP1)-(SEQ ID: 11)[000134] Below is a depiction of the structure of the title compound using the standard single letter code for L-Amino Acids except for the 2-(2-(2-Aminoethoxy)-ethoxy) acetic acid spacer (AEEA) and L-Cysteine (Cys) residues at the N-Terminus and at positions 7 and 23 where the structures of the residues have been expanded.[000135] The primary peptide sequence of the title compound was synthesized using standard 9- Fluorenyl-methyloxycarbonyl (Fmoc) tert-Butyl (t-Bu) solid phase peptide chemistry protocols on a Symphony-X, 24-channel multiplex peptide synthesizer (Gyros Protein Technologies, Inc.), at a 0.1 mmol scale. The N-terminal Cysteine residue is omitted from the solid phase peptide synthesis protocol and was added in solution after the isolation of the thioacetal bridged precursor peptide: H-AEEA-AEEA-AEEA-RSS[CFGGRIDRIGAQSGLGC]-OH (Thioacetal Bridge Cys7-Cys23). The solid support used consisted of pre-loaded Fmoc-Cys(Trt)-2-CTC resin (Fmoc-S-trityl-L-cysteine-2-Chlorotrityl Resin, Chemlmpex), (200-400 mesh) with a 1% DVB cross-linked polystyrene core and a substitution range of 0.3-0.8 meq / g. Standard sidechain protecting groups were used for all Fmoc-L- Amino Acids used. The non-standard amino acid used in the synthesis of the title compound was 2-[2-[2-(Fmoc-amino) ethoxy]ethoxy]acetic acid (Fmoc-AEEA-OH, AappTec Peptides). Fmoc deprotection prior to each coupling step was accomplished by treatments with 20% Piperidine (PIP; Sigma Aldrich) in Dimethylformamide (DMF; Fisher Chemicals), 2 x 7 minutes with nitrogen mixing, followed by 8 x DMF washing cycles. All amino acid couplings were performed for 1 hour using the Fmoc Amino Acid (0.3 M in DMF), N, N, N',N'-Tetramethyl-O-(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, Ambccd Inc.; 0.9 M in DMF ) and N,N-Diisopropylcthylaminc (DIPEA, Sigma Aldrich; 1.2 M in DMF), at a 9-fold molar excess of AA / HBTU and a 12-fold molar excess of DIPEA over the reported resin loading level. After the primary sequence of the peptide was synthesized up the third Fmoc-AEEA-OH residue, the final N-Terminal Fmoc was removed, the required DMF washes were completed, the peptidyl resin was transferred as a DCM slurry to disposable fritted plastic syringe fitted with Teflon stopcock. Further washes with DCM weredone, and finally, the resin was thoroughly dried in vacuo. The dry resin was then treated with 10 mL of cleavage cocktail consisting of trifluoroacctic acid (TFA, Acros Organics), water, 3,6- dioxa-l,8-octanedithiol (DODT; Sigma Aldrich), triisopropylsilane (TIPS; Acros Organics), (TFA:Water:DODT:TIPS; 90:5:2.5:2.5 v / v) for 2 hours at room temperature. After the 2 hr cleavage incubation, the resin was filtered off, washed twice with 2 mL of neat T FA, and the combined filtrates / washes were collected in a 50 ml conical disposable tube, the solution was then treated with 35 mL of cold diethyl ether (Fisher Chemicals) (-20°C) to precipitate the crude peptide. The peptide / ether suspension was then centrifuged at 4000 rpm for 2 min to form a solid pellet, the supernatant was decanted, and the solid pellet was triturated with fresh ether and the process was repeated two additional times, finally drying the peptide pellet in vacuo.Disulfide Bridge Formation[000136] The crude peptide was solubilized, in a suitable glass vessel, with 25% aqueous Acetic Acid to relatively low concentration (0.2-0.5 mg / ml crude peptide). The solution was then placed on magnetic stirrer with the requisite spin vane, mixed vigorously and titrated with a few drops a saturated Iodine in methanol solution until a faint yellow endpoint was achieved. After reaching the yellow endpoint, the reaction was incubated at RT for 15 min, at which point the excess Iodine was quenched by the addition of a few drops of 0.1 M aqueous ascorbic acid (Sigma Aldrich).HPLC Purification[000137] The oxidation solution containing the crude peptide was loaded directly onto a preparative HPLC system (Waters 2545 Binary Systems) equipped with a column heater and using a Luna Phenyl-Hexyl RP-HPLC column (Phenomenex Inc.; 5pm, 100A; 250 x 21.2 mm). The running buffers used were A: 0.1% TFA / H2O and B: 0.1% TFA / Acetonitrile (AcCN, Fisher Chemicals). The initial loading was done at 5% B, with 10 min isocratic wash after loading for column equilibration. The sample was eluted using a linear 5 - 65 % B gradient over 60 min, at a flow of 20 mL / min, with column heating set at 60 °C. Fractions that were determined to contain the desired product (analysis by LC-MS) were pooled, frozen and lyophilized to give a white amorphous solid product, as the TFA salt of the title compound. The purity assessed by RP-HPLC was found to be >95%, with the observed LC-MS molecular weight of 2473.73 Dalton, matching the theoretical calculated molecular weight of 2473.77 Dalton.Thioacetal Bridge Formation[000138] After the purification of the disulfide bridged peptide, the lyophilized disulfide material was solubilized in 3 mL of water, the solution was then diluted with water and AcCN to achieve about a 30 / 70 mixture of Water / AcCN (-200-400 mL total volume) with a low concentration of peptide (-0.2-0.5 mg / ml). The solution was then adjusted to pH 8 with Triethylamine (TEA, Acros Organics) - 10 equivalents, and the peptide’s disulfide bridge was reduced with the addition of 2-4 equivalents of Tris(2-carboxyethyl) phosphine hydrochloride (TCEP-HC1, Sigma Aldrich) reducing agent. After the disulfide bridge reduction, the thioacetal bridge was formed by the addition of 7-10 equivalents of Diiodomethane (CH2I2, Alfa Aesar). The Thioacetal formation reaction was carried out by incubating the solution for 18 hours at RT with magnetic stirring. Progress of the reaction was monitored using analytical LC-MS and by observing the change in mass of +12 Daltons from the starting reduced peptide molecular weight and an accompanying shift in retention of the starting material HPLC peak.HPLC Purification[000139] The thioacetal reaction solution was loaded directly onto a preparative HPLC system (Waters 2545 Binary Systems) equipped with a column heater and using a Luna Phenyl-Hexyl RP-HPLC column (Phenomenex Inc.; 5pm, 100A; 250 x 21.2 mm). The running buffers used were A: 0.1% Formic Acid / ILO and B: 0.1% Formic / Acetonitrile (AcCN, Fisher Chemicals). The initial loading was done at 5% B, with 10 min isocratic wash after loading equilibration. The sample was eluted using a linear 5 - 65 % B gradient over 60 min, at a flow of 15 mL / min, with column heating set at 60 °C. Fractions that were determined to contain the desired product (analysis by EC-MS) were pooled, 0.05 mF of TFA were added to convert the final product to a TFA salt, the solution was then frozen and lyophilized to give a white amorphous solid product, as the TFA salt of the title compound. The purity assessed by RP-HPLC was found to be >95%, with the observed LC-MS molecular weight of 2487.77 Dalton, matching the theoretical calculated molecular weight of 2487.79 Dalton.[000140] Below is a depiction of the structure of intermediate product H-AEEA-AEEA-AEEA- RSS[CFGGRIDRIGAQSGLGC]-OH (Thioacctal Bridge Cys7-Cys23), using the standard single letter code for L-Amino Acids except for the 2-(2-(2-Aminoethoxy)-ethoxy) acetic acid spacer (AEEA) where the structures of the residues have been expanded.Chemical Formula: C1OI H171N33036S2 Exact Mass: 2486.20 Molecular Weight: 2487.79Acylation of intermediate thioacetal peptide with Fmoc-Cys(Trt)-OPfp[000141] In a 15 mL falcon tube equipped with a spin vane, the purified, lyophilized precursor peptide was dissolved in 1000 pL of DMSO (Acros Organics) to it were added 75 pL of DIPEA (TCI America) to adjust the pH to -8-9, the reaction was magnetically stirred. Fmoc-Cys(Trt)- OPfp (-200 mg, Chemlmpex) was dissolved in DMSO, 50 pL aliquots were added one at time while monitoring the progression of the reaction by LC-MS. Once the starting material was consumed, the reaction was stopped by the addition of -10 mL cold diethyl ether (Fisher Chemicals) (-20°C). The tube was mixed vigorously and centrifuged to force the peptide into a pellet (oil phase). Another fresh volume of cold diethyl ether was added, and the process was repeated, this time a white tacky precipitate was formed. The pellet was treated with 1 ml of 20 % PIP in DMF (for -15 min). Another round of precipitation was carried out using cold diethyl ether, washed and additional time with the cold ether. The pellet was finally treated with TFA (2%TIPS) to remove the Trityl protecting group from the Cys residue. The cold diethyl ether precipitation was once again used to isolate the final crude product.HPLC Purification[000142] The crude peptide was dissolved in 15 mL of water and then loaded, via injection valve onto a preparative HPLC system (Shimadzu LC-8A Binary Systems) using a Luna Phenyl- Hexyl RP-HPLC column (Phenomenex Inc.; 5pm, 100A; 250 x 21 .2 mm). The running buffers used were A: 0.1% TFA / H2O and B: 0.1% TFA / Acctonitrilc (AcCN, Fisher Chemicals). Theloading was done at 0% B, with 5 min isocratic wash at 0% B for column equilibration. The sample was eluted using a linear 0 - 60 % B gradient over 60 min, at a flow of 25 mL / min, with column heating set at 60 °C. Fractions that were determined to contain the desired product (analysis by LC-MS) were pooled, frozen and lyophilized to give a white amorphous solid product, as the TFA salt of the title compound. The purity assessed by RP-HPLC 1 was found to be >95%, with the observed molecular weight of 2590.40 Dalton; matching the theoretical calculated molecular weight of 2590.93 Dalton.Example 3C: Synthesis of Cys-(SP1)-(SEQ ID: 17)[000143] Below is a depiction of the structure of the title compound using the standard single letter code for L-Amino Acids except for the 2-(2-(2-Aminoethoxy)-ethoxy) acetic acid spacer (AEEA) and L-Cysteine (Cys) at the N-Terminus where the structures of the residues have been expanded.[000144] The primary peptide sequence of the title compound is essentially similar to Example 3B. For this iteration, the disulfide bridge was removed by replacing Cys7 and Cys23 with Ser7 and Ser23 residues resulting in a lineal' analog without a connecting disulfide or thioacetal bridge. The synthesis of the title compound was carried out in a similar manner as Example 3B, with the noted Cys->Ser replacements, and was made as a C-Terminal Amide instead of a C- Terminal acid. The solid support resin used consists of low loading 4-(2',4'-Dimethoxyphenyl- Fmoc-aminomethyl)-phenoxyacetamido-norleucyl-4-Methylbenzhydrylamine resin (Fmoc-Rink- MBHA Low Loading resin, EMD Millipore), (100-200 mesh) with a 1% DVB cross-linked polystyrene core and a substitution range of 0.3-0.4 meq / g. Amide couplings were performed for 1 hour using the Fmoc Amino Acid (0.3 M, Advanced ChemTech, in DMF), N,N'- Diisopropylcarbodiimide (DIC, Chemlmpex, 1.2 M in DCM) and Ethyl Cyanohydroxyiminoacetate (Oxyma Pure, Chemlmpex; 0.9 M in DMF), at a 9-fold molar excess of AA / Oxyma and a 12-fold molar excess of DIC over the reported resin loading level. After theSPPS was complete, the peptidyl resin was transferred as a DCM slurry to disposable fritted plastic syringe fitted with Teflon stopcock. Further washes with DCM were done, and finally, the resin was thoroughly dried in vacuo. The dry resin was then treated with 10 mL of cleavage cocktail consisting of trifluoroacetic acid (TFA, Acros Organics), water, 3,6-dioxa-l,8- octanedithiol (DODT; Sigma Aldrich), triisopropylsilane (TIPS; Acros Organics), (TFA:Water:DODT:TIPS; 90:5:2.5:2.5 v / v) for 2 hours at room temperature. After the 2 hr cleavage incubation, the resin was filtered off, washed twice with 2 mL of neat TFA, and the combined filtrates / washes were collected in a 50 ml conical disposable tube, the solution was then treated with 35 mL of cold diethyl ether (Fisher Chemicals) (-20°C) to precipitate the crude peptide. The peptide / ether suspension was then centrifuged at 4000 rpm for 2 min to form a solid pellet, the supernatant was decanted, and the solid pellet was triturated with fresh ether and the process was repeated two additional times, finally drying the peptide pellet in vacuo.HPLC Purification[000145] The crude peptide was dissolved in 15 mL of water and then loaded, via injection valve onto a preparative HPLC system (Shimadzu LC-8A Binary Systems) using a Luna Phenyl- Hexyl RP-HPLC column (Phenomenex Inc.; 5pm, 100A; 250 x 21.2 mm). The running buffers used were A: 0.1% TFA / H2O and B: 0.1% TFA / Acetonitrile (AcCN, Fisher Chemicals). The loading was done at 0% B, with 5 min isocratic wash at 0% B for equilibration. The sample was eluted using a linear 0 - 60 % B gradient over 60 min, at a flow of 25 mL / min, with column heating set at 60 °C. Fractions that were determined to contain the desired product (analysis by LC-MS) were pooled, frozen and lyophilized to give a white amorphous solid product, as the TFA salt of the title compound. The purity assessed by RP-HPLC 1 was found to be >95%, with the observed molecular weight of 2545.60 Dalton; matching the theoretical calculated molecular weight of 2545.82 Dalton.Example 3D: Synthesis of Mpa-(SPl)-( SEQ ID: 22)[000146] Below is a depiction of the structure of the title compound with all residues expanded.[0001 7] The protocol used for the synthesis of the title compound is essentially similar as that used in Example 3C. In this iteration, the starting resin used was 2 [3-((Mcthyl-Fmoc- amino)-methyl)indol-l-yl]acetyl AM resin (Methyl Indole AM Resin), (100-200 mesh) with a 1% DVB cross-linked polystyrene core with a substitution range of 0.3-0.4 meq / g. This solid support generates an N-methyl substituted carboxamide containing peptide (-NH-CH3). The previously outlined cleavage and purification protocols were used for the workup of the material.Example 3E: Synthesis of MalDap-(SP12)-(SEQ ID: 21)[000148] Below is a depiction of the structure of the title compound using the standard single letter code for L-Amino Acids, except for the 2-(2-(2-Aminoethoxy)-ethoxy) acetic acid spacer (AEEA), D-Phe, D-Pro, D-Ala, p-cyclohexyl-L-alanine (Cha), and Mal-Dap-at the N-Terminus where the structures of the residues have been expanded.Wteewter Weight 32§3.3 i[000149] The synthesis of the title compound was carried out in a similar manner as previous preparations. The solid support resin used Fmoc-Rink-MBHA Low Loading resin(EMD Millipore), (100-200 mesh) with a 1% DVB cross-linked polystyrene core and a substitution range of 0.3-0.4 meq / g. Standard 1 hour couplings were performed using Fmoc Amino Acid (0.3 M, Advanced ChemTech, in DMF), N,N '-Diisopropylcarbodiimide (DIC, Chemlmpex, 1.2 M in DCM) and Ethyl Cyanohydroxyiminoacetate (Oxyma Pure, Chemlmpex; 0.9 M in DMF), at a 9- fold molar excess of AA / Oxyma and a 12-fold molar excess of DIC over the reported resin loading level. Of note, Fmoc-Gly-Gly-OH dipeptide (Chemlmpex) was used to build the (GGGGS)6 (SEQ ID: 79) repeat, coupling time was extended to 2 hrs for the dipeptide. MaL Dap(Boc)-OH (Chemlmpex) was used to cap the N-terminus of the peptide using standard coupling conditions. The previously outlined cleavage and purification protocols were used for the workup of the material.Example 3F: Synthesis of Mpa-(SP1)-(SEQ ID: 21)-(SF4)-(FA4)[000150] Below is a depiction of the structure of the title compound using the standard single letter code for L-Amino Acids, except for the 2-(2-(2-Aminoethoxy)-ethoxy) acetic acid spacer (AEEA), y-Glutamic Acid, D-Phe, D-Pro, D-Ala, P-cyclohexyLL- alanine (Cha), C-Terminal Lys, and 3-Mercaptopropionic Acid at the N-Terminus where the structures of the residues have been expanded.[000151] The synthesis of the title compound was carried out in a similar manner as described previously. The solid support resin used Fmoc-Rink-MBHA Low Loading resin (EMD Millipore), (100-200 mesh) with a 1% DVB cross-linked polystyrene core and a substitution range of 0.3-0.4 meq / g. Standard 1 hour couplings were performed using Fmoc Amino Acid (0.3 M, Advanced ChemTech, in DMF), N,N'-Diisopropylcarbodiimide (DIC, Chemlmpex, 1.2 M in DCM) and Ethyl Cyanohydroxyiminoacetate (Oxyma Pure, Chemlmpex; 0.9 M in DMF), at a 9- fold molar excess of AA / Oxyma and a 12-fold molar excess of DIC over the reported resin loading level. The first amino acid coupled to the resin was Fmoc-Lys(Mtt)-OH (Advanced ChemTech) and was done using standard coupling conditions. The use of orthogonal Mtt protection on Lysine residues allows for selective on-resin side chain deprotection (s-amino group) and directed lipidation of the peptides. For the Mtt deprotection, the resin is thoroughly washed with DCM after the last coupling step of Trt-3-Mpa-OH and then treated 4 times with a sufficient volume of 30% 1,1,1,3,3,3-Hexafluoroisopropanol (HF1P, Chemlpex) in DCM. Each HFIP treatment is done for 30 minutes with nitrogen mixing. After Mtt removal the resin was thoroughly washed with DCM (8x), and subsequently DMF (8x). Acylation with OtBu-C20- (yGlu-OtBu)-AEEA-AEEA-OH afforded the side chain linker fatty diacid, coupling was done using DIC / Oxyma at 3x excess for ~18hrs. The previously outlined cleavage and purification protocols were used for the workup of the material.Example 3G: Synthesis of FA1[000152] Below is a depiction of the structure of the title compound:[000153] The title compound was synthesized manually in a fritted glass reaction vessel (50 mL) using standard 9-Fluorcnyl-mcthyloxycarbonyl (Fmoc) tcrt-Butyl (t-Bu) solid phase peptide chemistry protocols at a 1.0 mmol scale. The solid support used consisted of 4-(2z,4Z- Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin (Rink-Resin LS, CreoSalus), (100-200 mesh) with a 1% DVB cross-linked polystyrene core and a substitution of ~ 0.29 meq / g. The building blocks used for the synthesis were: Fmoc-L-Lys(Mtt)-OH (AappTec Peptides), Fmoc-3- (2-cyano-4-pyridyl)alanine (Fmoc-Cpa-OH, WuXi), Fmoc-Glu-OtBu (Advanced ChemTech), 1- Acetylimidazole (Acros Organics) Fmoc-AEEA-OH, and 20-(tert-Butoxy)-20-oxoicosanoic acid (HO-C20-OtBu, WuXi). Fmoc deprotection prior to each coupling step was accomplished by treatments with 20% Piperidine (PIP; Sigma Aldrich) in Dimethylformamide (DMF; Fisher Chemicals), 2 x 10 minutes with nitrogen mixing, followed by 8 x DMF wash cycles. Fmoc- Lys(Mtt)-OH and Fmoc-Cpa-OH were performed for 3 hours using a 5x excess of the Fmoc Amino Acids, DIC and Oxyma. Capping or acetylation of the N-terminus was done using Acetyl-imidazole in DCM (6x excess) reaction time 2 hrs. After the capping step, the resin was washed with DCM 8x. The Mtt side chain deprotection of Lysine was done with using 20% HFIP in DCM (3x 20min), the resin washed 3x DCM, 4x DMF. Coupling of Fmoc-AEEA-OH residues and Fmoc-Glu-OtBu were performed for 5 hours using a 5x excess of the Fmoc Amino Acids, DIC and Oxyma. Coupling of OtBu-C20-OH was done using PyBop / DIPEA 3x excess (overnight). After the coupling of the fatty diacid, the resin washed 3x DMF, 3x DCM, 2 Ether and dried in vacuo. The dry resin was then treated with 100 mL of cleavage cocktail consisting of trifluoroacetic acid (TFA, Acros Organics), water, triisopropylsilane (TIPS; Acros Organics),(TFA:Water:TIPS; 90:5:5 v / v) for 2 hours at room temperature. After the 2 hr cleavage incubation, the resin was filtered off, washed 00 mL of DCM T FA, and the combined filtrates / washes were collected in six 50 ml conical disposable tubes, the solutions were then treated with 35 mL of cold diethyl ether (Fisher Chemicals) (-20°C) each to precipitate the crude product. The ether suspensions were then centrifuged at 4000 rpm for 2 min to form solid pellets, the supernatant was decanted, and the solid pellets were triturated with fresh ether and the process was repeated two additional times, finally drying the pellets in vacuo. The crude material was used without further purification.Example 4: Synthesis of oligonucleotides for targeting HPRT and SOD1 (e.g., siRNA) and reagents for oligonucleotide functionalizationExamples 4A-4EE[000154] Single strands (sense and antisense) were typically synthesized on solid support via a MerMade 12 (LGC Biosearch Technologies), K&A H-8 SE (K&A Labs GmbH) oligonucleotide synthesizer. The sequences of the sense and antisense strands were shown in Table 7 and 8. In addition to the strand sequence, appropriate amidites and CPGs were utilized to append appropriate linkers to enable the conjugates in table 15. The sense strands were synthesized using an appropriate CPG such as “3'-Amino Modifier C-3 Icaa CPG 500A” (part number N-8271-05, Chemgenes) or “3’ Thiol Modifier C6 SS” (part number ON-526-UC, Hongene - C6SSC6-CPG) whereas the antisense strands used standard 2’-O-Methyl supports (LGC Biosearch Technologies). The oligonucleotides were synthesized via phosphoramidite chemistry at an appropriate scale for in-vitro or in-vivo experimentation.[000155] Standard reagents were used in the oligo synthesis (Table 18), where 0.1M xanthane hydride in pyridine was used as the sulfurization reagent and 20% DEA in ACN was used as an auxiliary wash post synthesis. For antisense strands, the 20% DEA wash was typically omitted and ACN wash was used instead. All monomers (Table 19, phosphoramidites) were made at 0.1M in ACN and contained a molecular sieves trap bag. When necessary, inclusion of 10% volume equivalents of DMF or 50% volume equivalents of DCM were utilized to maintain amidite solubility (i.e. mU amidite). For lipid-containing amidites such as those which were used to synthesize sense strands bearing 5 ’-terminal FA5 through FA9, FA11-FA18 and internal Upa-conjugated FA27, the appropriate monomers were typically dissolved at 0.1 or 0.2M in 50% volume equivalents of DCM:ACN or 100% DCM and used under standard phosphoramiditc coupling conditions. For lipid-containing amidites bearing carboxylic acid esters such as those which were used to synthesize sense strands bearing 5’-terminal FA10-SF8, FA26-SF8, or FA19-FA25, the appropriate monomers were typically dissolved at 0.1 or 0.2M in 50% volume equivalents of DCM:ACN or 100% DCM.[000156] The sense strands made with 3 ’-phthalimide C6 amino CPG were typically cleaved and deprotected from the CPG using 50% (methylamine / ammonia hydroxide 28-30%) at ambient temperature (about 25 °C) for 2-3 hrs. For lipid-containing sense strands bearing carboxylic acid such as those bearing FA10-SF8, FA26-SF8, or FA19-FA25, cleavage and deprotection (C / D) typically was achieved using 0.4 M NaOH in 4: 1 Me0H / H20 at ambient temperature (about 25 °C) for 24 hours. The antisense strands were typically cleaved and deprotected (C / D) at 45 °C for 17 hours using a solution of 3% DEA in ammonia hydroxide (28- 30%, cold). C / D was determined complete by IP-RP LCMS when the resulting mass data confirmed the identity of sequence. Dependent on scale, the CPG was filtered via 0.45 um PVDF syringeless filter, 0.22 pm PVDF Steriflip® vacuum filtration or 0.22 pm PVDF Stericup® Quick release.[000157] The CPG was typically back washed / rinsed with RNAse free water or 30% EtOH / RNAse free water, then filtered through the same filtering device and combined with the first filtrate. This was repeated twice. The material was then divided evenly into conical centrifuge tubes to remove organics via Genevac™. After concentration, the crude oligonucleotides were diluted back to synthesized scale with RNAse free water and filtered either by 0.45 pm PVDF syringeless filter, 0.22 pm PVDF Steriflip® vacuum filtration or 0.22 pm PVDF Stericup® Quick release.[000158] Sense strands containing C6SSC6 protected thiol moiety installed on solid phase either originating from a 3 ’-modification via C6SSC6-CPG or 5 ’-modification via C6SSC6-PA (Table 19) were typically treated with an excess (tris(2-carboxyethyl)phosphine)-HCl to reduce the dithio bond after removal of volatiles and prior to purification, unless functionalization of an amino moiety is necessary prior to fiunctionalization of the liberated thiol moiety. In this case, purification as below may take place and disulfide cleavage may be similarly employed following amino functionalization.[000159] The crude oligonucleotides were purified via AKTA™ Pure purification system using anion-exchange (AEX) or reverse-phase (RP) chromatography utilizing a gradient of Mobile Phase A (MPA) to Mobile Phase B as appropriate for the product. For AEX, an ES Industry Source™ 15Q column with MPA: 20mM NaFhPC , 15% ACN, pH 7.4 and MPB: 20 mM NaH2PO4, IM NaBr, 15% ACN, pH 7.4. For RP, an ES Industry Source™ 15RPC with MPA: lOmM NaOAc 2% Acetonitrile and MPB: 80% Acetonitrile in water. Fractions were analyzed by IP-RP LCMS and those which contained a mass purity greater than 85% without impurities >5% were combined.[000160] The purified oligonucleotides were typically desalted using 15 mL 3K MWCO centrifugal spin tubes at 3500xg for ~30 min. The oligonucleotides were rinsed with RNAse free water until the eluent conductivity reached < 100 usemi / cm. After desalting was complete, 2-3 mL of RNAse free water was added then aspirated lOx, the retainment was transferred to an appropriately sized conical tube, this was repeated until complete transfer of oligo by measuring concentration of compound on filter via nanodrop. The final oligonucleotide was then typically nano filtered via 15 mL 100K MWCO centrifugal spin tubes at 3500xg for 2 min. The final desalted oligonucleotides were analyzed for concentration (nanodrop at A260). The molecular weight of the product was confirmed by LC-MS analysis using a Linear Ion Trap Mass Spectrometer (LTQ XL, Thermo Fisher), and purity was confirmed by UPLC analysis.[000161] Oligonucleotide UPLC analysis was typically conducted under Ion-Pairing Reversed-Phase Ultra Performance Liquid Chromatography (IP-RP UPLC) conditions using a Waters™ ACQUITY™ UPLC Oligonucleotide BEH C18 Column 2.1x50 mm, 1.7 pm column. The gradient used Mobile Phase A (MPA) of 7 mM triethylamine with 100 mM hexafluoroisopropanol, and a Mobile Phase B (MPB) of 7:3 Methanol to Acetonitrile.Oligonucleotides with low lipophilicity were typically analyzed over a gradient of 0 to 25% MPB over 10 minutes while oligonucleotides with high lipophilicity were analyzed over a gradient of 5 to 90% MPB over 10 minutes.Table 18: Oligonucleotide Synthesis ReagentsTable 19: PhosphoramiditesN / A denotes not applicable.Example 4A: Hexadecyl A phosphoroamiditeN-[9-[(2R,3R,4R,5R)-5-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxy-3-hexadecoxy-tetrahydrofuran-2-yl]purin-6-yl]benzamide[000162] Prepared the title compound, referred to herein as Hcxadccyl A phosphoroamidite, according to the protocols described in WO2019217459.1H-NMR (CD3CN) 8 9.37 (s, 1H), 8.57 (d, J = 9.4 Hz, 1H), 8.27 (d, J = 10.3 Hz, 1H), 7.99 (d, J = 7.6 Hz, 2H), 7.61 (d, J = 7.4 Hz, 1H), 7.52 (t, J = 7.6 Hz, 2H), 7.42 (t, J = 7.3 Hz, 2H), 7.34 - 7.16 (m, 7H), 6.85 -6.77 (m, 4H), 6.1 1 (dd, J = 5.0, 2.5 Hz, 1H), 4.80 (m, 1H), 4.69 (m, 1H), 4.32 (m, 1H), 3.97 -3.78 (m, 1H), 3.74 (d, J - 3.1 Hz, 7H), 3.64 (m, 4H), 3.56 - 3.40 (m, 2H), 3.33 (m, 1H), 2.73 - 2.59 (m, 1H), 2.50 (t, J = 6.0 Hz, 1H), 1.52 - 1.45 (m, 2H), 1.33 - 1.12 (m, 37H), 1.09 (d, J = 6.8 Hz, 3H), 0.87 (t, J = 6.8 Hz, 3H).31P NMR (CD3CN) 8 151.19, 150.78.Example 4B: Hexadecyl U phosphoroamidite3-[[(2R,3R,4R,5R)-2-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hexadecoxy-5-(2- hydroxy-4-oxo-pyrimidin-l-yl)THF-3-yl]oxy-(diisopropylamino)phosphanyl]oxypropanenitrile[000163] Prepared the title compound, referred to herein as Hexadecyl U phosphoramidite, according to the protocols described in WO2019217459. 1H NMR (CD3CN): 7.86-7.73 (m, 1H), 7.51-7.43 (m, 2H), 7.40-7.23 (m, 7H), 6.95-6.87 (m, 4H), 5.90-5.84 (m, 1H), 5.29-5.21 (m, 1H), 4.54-4.40 (m, 1H), 4.21-4.13 (m, 1H), 4.10-3.56 (m, 13H), 3.50-3.34 (m, 2H), 2.75-2.62 (m, 1H), 2.55 (t, J= 6.0 Hz, 1H), 1.66-1.51 (m, 2H), 1.40-1.14 (m, 35H), 1.08 (d, J= 6.8 Hz, 3H), 0.91 (t, J= 6.8 Hz, 3H). 31P NMR (CD3CN): 149.6, 149.2.Example 4C: S-mercaptotertbutyl-L-cystine-transcyclohexylamideo phosphoramidite[000164](9H-fluoren-9-yl)methyl ((2S)-3-(tert-butyldisulfaneyl)-l-(((trans)-4-(((2- cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)cyclohexyl)amino)-l-oxopropan-2- yl)carbamate, referred to herein as S-mercaptotertbutyl-L-cystine-transcyclohexylamido phosphoramidite, was prepared according to the protocol described in Nucleosides, Nucleotides & Nucleic Acids (2000), 19(10-12), 1751-1764.Scheme 1[000165] Scheme 1, step A shows the alkylative esterification of commercially available 2,2-dimethyl-4-oxo-3,8,l l-trioxa-5-azatridecan-13-oic acid (1, CAS number 108466-89-3) which took place utilizing benzyl bromide in acetone in presence of the base potassium carbonate to give compound (2). Step B shows the acidic deprotection of compound (2) which took place by treating with hydrochloric acid in the solvent ethyl acetate to give compound (3). Step C shows the amide coupling of (3) with (1) which took place using the amide coupling reagent EDCI in presence of HOBt and the base DIEA in the solvent DCM to give compound (4). Step D shows the acidic deprotection of compound (4) which took place with hydrochloric acid in the solvent ethyl acetate to give compound (5). One skilled in the art will recognize that a variety of coupling reagents, bases, and solvents can be used to perform an amide coupling, and a variety of acids can be used to perform a BOC deprotection.Scheme 2[000166] Scheme 2, step A shows the coupling of commercially available N-Boc-L- glutamic acid 5-benzyl ester (6, CAS number 13574-13-5) with 3-hydroxypropionitrile which took place under coupling conditions utilizing DCC in presence of the base DMAP in the solvent DCM to give compound (7). Step B shows the hydrogenative debenzylation of compound (7) which took place in presence of hydrogen gas and the catalyst palladium on carbon in the solvent THF to give compound (8). One skilled in the ail will recognize that a variety coupling reagents, bases and solvents can be used to perform an ester coupling, and a variety of catalysts and hydrogen sources can be used to perform a benzyl ester removal.Scheme 3[000167] Scheme 3, step A shows the coupling of commercially available 20-(tert-Butoxy)- 20-oxoicosanoic acid (9, CAS number 683239-16-9) with 3-hydroxypropionitrile which took place under coupling conditions utilizing DCC in presence of the base DMAP in the solvent DCM to give compound (10). Step B shows the acidic deprotection of compound (10) which took place with the acid TFA in the solvent DCM to give compound (11). One skilled in the art will recognize that a variety coupling reagents, bases and solvents can be used to perform an ester coupling, and a variety of acids can be used to perform a BOC deprotection.Scheme 4[000168] Scheme 4, step A shows the amide coupling of (4) with compound (8) which took place using the amide coupling reagent EDCI in presence of HOBt and the base DIEA in the solvent DCM followed by acidic deprotection with hydrochloric acid in the solvent ethyl acetate to give compound (12). Step B shows the amide coupling of (12) with compound (11) using the amide coupling reagent EDCI in presence of HOBt and the base DIEA in the solvent DCM to give compound (13). Step C shows the hydrogenative debenzylation of compound (13) which took place in presence of hydrogen gas and the catalyst palladium on carbon in the solvent ethyl acetate to give compound (14). One skilled in the ail will recognize that a variety coupling reagents, bases and solvents can be used to perform an ester coupling, and a variety of acids can be used to perform a BOC deprotection. One skilled in the ail will also recognize a variety of catalysts and hydrogen sources can be used to perform a benzyl ester removal.Scheme 5[000169] Scheme 5, step A shows the amide coupling of (14) with 6-amino-l -hexanol which took place in presence of the amide coupling reagent EDC in the solvent DCM to give compound (15). Step B shows the conversion of alcohol (15) to a phosphoramidite which took place by treatment with the phosphoramidite precursor 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile in presence of the activator 5- (ethylthio)-lH-tetrazole in the solvent DCM to give compound (16) “C20-Diacid-CE phosphoramidite”. One skilled in the art will recognize a variety of amide coupling reagents and solvents can be used to perform an amide coupling, and a variety of phosphoramidite precursor reagents and activators can be used to form a phosphoramidite.Scheme 6[000170J Scheme 6, step A shows the amide coupling of (4) with commercially available N-Boc-L-glutamic acid 5-methyl ester (CAS number 45214-91-3) which took place using the amide coupling reagent EDCI in presence of HOBt and the base DIEA in the solvent DCM followed by acidic deprotection with hydrochloric acid in the solvent ethyl acetate to givecompound (17). Step B shows the amide coupling of (17) with commercially available 20- Mcthoxy-20-oxoicosanoic acid (CAS number 1767-98-2) which took place using the amide coupling reagent EDCI in presence of HOBt and the base DIEA in the solvent DCM to give compound (13). Step C shows the hydrogenative debenzylation of compound (13) which took place in presence of hydrogen gas and the catalyst palladium on carbon in the solvent ethyl acetate to give compound (19). One skilled in the ait will recognize that a variety coupling reagents, bases and solvents can be used to perform an ester coupling, and a variety of acids can be used to perform a BOC deprotection. One skilled in the art will also recognize a variety of catalysts and hydrogen sources can be used to perform a benzyl ester removal.Scheme 7[000171] Scheme 7, step A shows the formation of an activated N-hydroxy succinimide ester by treatment of compound (19) with 1 -hydroxypyrrolidine-2, 5-dione in presence of a coupling reagent such as EDC in a solvent such as DCM to give compound (20) referred herein as “C20-Diacid-ME NHS Ester.” One skilled in the art will recognize that a variety coupling reagents and solvents can be used to perform an activated ester formation coupling.Example 4D: Benzyl 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan- 13-oate[000172] To a mixture of 2,2-dimethyl-4-oxo-3,8,l l-trioxa-5-azatridecan- 13-oic acid (50.0 g, 99% Wt, 1 Eq, 188 mmol) in Acetone (500 mL) were added Benzyl bromide (36.1 g, 25.1 mL, 98% Wt, 1.1 Eq, 207 mmol) and Potassium carbonate (65.6 g, 99% Wt, 2.5 Eq, 470 mmol) at 0 °C, then the reaction mixture was stirred at 65 °C for 90 min under N2. This procedure was repeated twice and the reaction mixtures of were combined to work-up and purify. The reaction mixture was filtered and the filter cake was washed with EtOAc (100 mL * 3). The filtrate was concentrated under reduced pressure to give a residue. To the residue was added H2O (500 mL) and extracted with EtOAc (500 mL * 2). The combined organic layers were washed with brine (1000 mL * 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give benzyl 2,2-dimethyl-4-oxo-3,8,l l-trioxa-5-azatridecan-13-oate (128 g, 0.35 mol, 95 %, 97% Purity) as a yellow oil. LCMS m / z. = 354.1 (M+l).JH NMR (400 MHz, DMSO-tfc) 5 ppm 7.20 - 7.47 (m, 5 H) 6.73 (br t, 7=5.20 Hz, 1 H) 5.14 (s, 2 H) 4.18 (s, 2 H) 3.56 - 3.64 (m, 2 H) 3.47 - 3.54 (m, 2 H) 3.34 - 3.37 (m, 2 H) 3.05 (q, 7=6.00 Hz, 2 H) 1.36 (s, 9 H).Example 4E: Benzyl 2-(2-(2-aminoethoxy)ethoxy)acetate hydrochloride[000173] A mixture of benzyl 2,2-dimethyl-4-oxo-3,8, 1 l-trioxa-5-azatridecan-13-oate (60 g, 93% Wt, 1 Eq, 0.16 mol) in 2.0 M HC1 in EtOAc (43.8 g, 600 mL, 2 molar, 7.6 Eq, 1.20 mol) was stirred at 26 °C for 60 min. The reaction mixture was concentrated under reduced pressure to give benzyl 2-(2-(2-aminoethoxy)ethoxy)acetate hydrochloride (50 g, 0.16 mol, 99 %, 91% Purity) as a pink oil. LCMS m / z = 254.1 (M+l).Example 4F: benzyl 2,2-dimethyl-4,13-dioxo-3,8,ll,17,20-pentaoxa-5,14-diazadocosan-22- oate[000174] To a mixture of benzyl 2-(2-(2-aminoethoxy)ethoxy)acetate hydrochloride (54 g, 90% Wt, 1 Eq, 0.17 mol) in DCM (500 mL) were added boc-8-amino-3,6-dioxaoctanoic acid (46 g, 40 mL, 95% Wt, 1 Eq, 0.17 mol),l-(3-Dimethylaminopropyl)-3- ethylcarbodiimideHydrochloride(EDCI) (49 g, 99% Wt, 1.5 Eq, 0.25 mol), 1 -Hydroxy- 1H- benzotriazole (34 g, 99% Wt, 1.5 Eq, 0.25 mol) and Diisopropylethylamine (0.11 kg, 0.15 L, 99% Wt, 5 Eq, 0.84 mol) at 0 °C, then the reaction mixture was stirred at 27 °C for 16 hour. The reaction mixture was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel chromatography (Biotage®; Agela®Flash Column Silica- CS (330X2 g), Eluent of 0-50% EtOAc / petroleum ether gradient @ 100 mL / min) to give benzyl 2,2-dimethyl-4,13-dioxo-3,8,l l,17,20-pentaoxa-5,14-diazadocosan-22-oate (85 g, 0.16 mol, 97 %, 95% Purity) as a colorless gel. LCMS m / z = 499.3 (M+l).Example 4G: benzyl 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoate hydrochloride[000175] A mixture of benzyl 2,2-dimethyl-4,13-dioxo-3,8,l l,17,20-pentaoxa-5,14- diazadocosan-22-oate (60 g, 80% Wt, 1 Eq, 96 mmol) in 2.0 M HCI in EtOAc (43.8 g, 600 mL, 2 molar, 12 Eq, 1.20 mol) was stirred at 26 °C for 60 min. The reaction mixture was concentrated under reduced pressure to give benzyl 17-amino-10-oxo-3,6,12,15-tctraoxa-9-azahcptadccanoatc hydrochloride (50 g, 92 mmol, 96 %, 80% Purity) as pink gel. LCMS m / z = 399.2.1H NMR (400 MHz, DMSO-<76) 8 ppm 8.02 - 8.29 (m, 3 H) 7.75 (br t, 7=5.20 Hz, 1 H) 7.23 - 7.47 (m, 5 H) 5.14 (s, 2 H) 4.19 (s, 2 H) 3.89 (s, 2 H) 3.58 - 3.65 (m, 8 H) 3.50 - 3.54 (m, 2 H) 3.43 (t, 7=6.00 Hz, 2 H) 3.26 (q, 7=6.00 Hz, 2 H) 2.90 - 2.99 (m, 2 H).Example 4H: 5-benzyl l-(2-cyanoethyl) (tert-butoxycarbonyl)-L-glutamate[000176] To a solution of (S)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5- oxopentanoic acid (50.0 g, 1 Eq, 148 mmol) in DCM (500 mL) were added 4- Dimethylaminopyridine (3.62 g, 0.2 Eq, 29.6 mmol) and Dicyclohcxylcarbodiimidc (61.2 g, 2 Eq, 296 mmol) and Ethylene cyanohydrin (12.6 g, 12.1 mL, 1.2 Eq, 178 mmol) at 0 °C. The solution was stirred at 26 °C for 16 hour. The reaction mixture was filtered and the filter cake was washed with DCM (100 mL * 3). The filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel chromatography (Biotage®; Agela®Flash Column Silica-CS (330 g), Eluent of 0-30% EtOAc / hexanes gradient @ 100 mL / min) to give 5-benzyl l-(2-cy anoethyl) (tert-butoxycarbonyl)-L-glutamate (50 g, 0.10 mol, 69 %, 80% Purity) as a white solid. LCMS m / z - 291.1 (M+l-100). ’ H NMR (400 MHz, DMSO-d6) 8 ppm 7.30 - 7.39 (m, 5 H) 5.09 (s, 2 H) 4.14 - 4.34 (m, 2 H) 3.98 - 4.11 (m, 1 H) 2.86 (t, J=6.00 Hz, 2 H) 2.44 - 2.52 (m, 2 H) 1.95 - 2.10 (m, 1 H) 1.74 - 1.92 (m, 1 H) 1.28 - 1.46 (m, 9 H).Example 41: (S)-4-((tert-butoxycarbonyl)amino)-5-(2-cyanoethoxy)-5-oxopentanoic acid[000177] A solution of 5-benzyl l-(2-cyanoethyl) (tert-butoxycarbonyl)-L-glutamate (50 g, 80% Wt, 1 Eq, 0.10 mol) in THF (500 mL) was degassed with Argon, and Pd / C(dry basis) (25 g, 10% Wt, 0.23 Eq, 23 mmol) was added. The reaction mixture was evacuated and backfilled three times with hydrogen. The mixture was stirred at 25 °C for 2 hour under an atmosphere ofhydrogen 15 psi. Upon completion, the reaction mixture was filtered through a pad of celite and the filtrate was concentrated under reduced pressure to afford the crude product. Pd / C is recycled into an activated metal recycling bucket. The crude product was purified by flash silica gel chromatography (Biotage®; Agela®Flash Column Silica-CS (220 g), Eluent of 0-50% EtOAc / hexanes gradient @ 100 mL / min) to give (S)-4-((tert-butoxycarbonyl)amino)-5-(2-cyanoethoxy)- 5 -oxopentanoic acid (25 g, 75 mmol, 73 %, 90% Purity) as colorless oil.!H NMR (400 MHz, METHANOL-^) 5 ppm 4.26 - 4.38 (m, 2 H) 4.19 (dd, 7=9.20, 5.20 Hz, 1 H) 2.85 (t, 7-6.00 Hz, 2 H) 2.42 (t, 7=7.60 Hz, 2 H) 2.15 (m, 1 H) 1.80 - 1.98 (m, 1 H) 1.44 (s, 9 H).Example 4J: 1- (tert-butyl) 20-(2-cyanoethyl) icosanedioate[000178] To a mixture of 20-(tert-butoxy)-20-oxoicosanoic acid (25.0 g, 1 Eq, 62.7 mmol) in DCM (300 mL) were added Ethylene cyanohydrin (8.92 g, 8.56 mL, 2 Eq, 125 mmol), DCC (25.9 g, 2 Eq, 125 mmol) and DMAP (1.53 g, 0.2 Eq, 12.5 mmol) at 0 °C, then the reaction mixture was stirred at 27 °C for 16 hour under N2. The reaction mixture was filtered and the filter cake was washed with DCM (100 mL * 3). The filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel chromatography (Biotage®; Agela®Flash Column Silica-CS (220 g), Eluent of 0-10% EtOAc / hexanes gradient @ 100 mL / min) to give 1- (tert-butyl) 20-(2-cy anoethyl) icosanedioate (20 g, 40 mmol, 64 %, 90% Purity) as a white solid.1H NMR (400 MHz, CHLOROFORM-7) 6 ppm 4.27 (t, 7=6.00 Hz, 2 H) 2.70 (t, 7=6.40 Hz, 2 H) 2.34 (t, 7=7.20 Hz, 2 H) 2.18 (t, 7=7.60 Hz, 2 H) 1.60 - 1.67 (m, 2 H) 1.52 - 1.59 (m, 2 H) 1.43 (s, 9 H) 1.22 - 1.31 (m, 28 H).Example 4K: 20-(2-cyanoethoxy)-20-oxoicosanoic acid[000179] To a solution of 1 -(tert-butyl) 20-(2-cyanoethyl) icosanedioate (17 g, 90% Wt, 1 Eq, 34 mmol) in DCM (150 mL) was added TFA (3.9 g, 90 mL, 1 Eq, 34 mmol) . The reaction mixture was stirred at 26 °C for 60 min. The reaction mixture was concentrated under reduced pressure to give 20-(2-cyanoethoxy)-20-oxoicosanoic acid (15 g, 34 mmol, 100 %, 90% Purity) as a pink solid. 'H NMR (400 MHz, DMSO-tfc) 8 ppm 4.14 - 4.21 (m, 2 H) 2.86 (t, 7=6.00 Hz, 1 H) 2.32 (t, 7=7.20 Hz, 2 H) 2.17 (br t, 7=7.20 Hz, 2 H) 1.50 (td, 7=13.60, 7.20 Hz, 4 H) 1.23 (s, 28 H).Example 4L: 11-benzyl 23-(2-cyanoethyl) (S)-22-((tert-butoxycarbonyl)amino)-10,19-dioxo- 3,6,12,15-tetraoxa-9,18-diazatricosanedioate[000180] To a mixture of benzyl 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoate hydrochloride (45 g, 80% Wt, 1 Eq, 83 mmol) in DCM (500 mL) were added DIEA (32 g, 43 mL, 3 Eq, 0.25 mol),(S)-4-((tert-butoxycarbonyl)amino)-5-(2-cyanoethoxy)-5-oxopentanoic acid (25 g, 90% Wt, 0.91 Eq, 75 mmol),l-(3-Dimethylaminopropyl)-3- ethylcarbodiimideHydrochloride(EDCI) (24 g, 1.5 Eq, 0.12 mol) and 1 -Hydroxy- 1H- benzotriazole (17 g, 1.5 Eq, 0.12 mol) at 0 °C, then the reaction mixture was stirred at 26 °C for 16 hour. The reaction mixture was concentrated under reduced pressure to give the residue. The residue was added to H2O (500 mL) and extracted with EtOAc (400 mL * 2). The combined organic layers were washed with brine (400 mL * 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by flash silica gel chromatography (Biotage®; Agela®Flash Column Silica-CS (330 g), Eluent of 0-100% EtOAc I hexanes gradient @ 100 mL / min) to give 1-benzyl 23-(2-cyanoethyl) (S)-22- ((tert-butoxycarbonyl)amino)-10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosanedioate (40 g, 52 mmol, 62 %, 88% Purity) as a yellow oil. LCMS m / z =681.3 (M+l). ’H NMR (400 MHz, DMSO-rfe) 8 ppm 7.85 (br t, 7=5.20 Hz, 1 H) 7.63 (br t, 7=5.60 Hz, 1 H) 7.25 - 7.41 (m, 5 H)5.15 (s, 2 H) 4.15 - 4.30 (m, 4 H) 3.91 - 4.01 (m, 1 H) 3.87 (s, 2 H) 3.50 - 3.63 (m, 8 H) 3.42 (dt, 7=11.60, 6.00 Hz, 4 H) 3.32 (s, 2 H) 3.26 (q, 7=6.00 Hz, 2 H) 3.20 (q, 7=5.60 Hz, 2 H) 2.87 (t, 7=5.60 Hz, 2 H) 2.18 (br t, 7=7.60 Hz, 2 H) 1.87 - 1.98 (m, 1 H) 1.76 (m, 1 H) 1.38 (s, 9 H).Example 4M: 1-benzyl 23-(2-cyanoethyl) (S)-22-amino-10,19-dioxo-3,6,12,15-tetraoxa-9,18- diazatricosanedioate hydrochloride[000181] A mixture of 1-benzyl 23-(2-cy anoethyl) (S)-22-((tert-butoxycarbonyl)amino)- 10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosanedioate (38 g, 88% Wt, 1 Eq, 49 mmol) in 2.0 M HC1 in EtOAc (29.2 g, 400 mL, 2 molar, 16 Eq, 800 mmol) was stirred at 27 °C for 60 min. The reaction mixture was concentrated under reduced pressure to give 1-benzyl 23-(2- cyanoethyl) (S)-22-amino-10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosanedioate hydrochloride (34 g, 45 mmol, 92 %, 82% Purity) as a red oil. LCMS m / z =581.4 (M+l).1H NMR (400 MHz, DMSO-76) 8 ppm 8.59 - 8.85 (m, 3 H) 8.11 (br t, 7=5.60 Hz, 1 H) 7.69 (br t, 7=5.60 Hz, 1 H) 7.17 - 7.46 (m, 5 H) 5.14 (s, 2 H) 4.25 - 4.40 (m, 2 H) 4.19 (s, 2 H) 3.87 (s, 2 H) 3.49 - 3.64 (m, 9 H) 3.39 - 3.45 (m, 4 H) 3.17 - 3.29 (m, 4 H) 2.96 (t, 7=6.00 Hz, 2 H) 2.23 - 2.41 (m, 2 H) 1.99 - 2.06 (m, 2 H).Example 4N: 1-benzyl 21,41-bis(2-cyanoethyl) (S)-9, 18, 23-trioxo-2, 5,11, 14-tetraoxa-8, 17,22- triazahentetracontane-l,21,41-tricarboxylate[000182] To a mixture of 1-benzyl 23-(2-cyanoethyl) (S)-22-amino-10,19-dioxo-3,6,12,15- tetraoxa-9,18-diazatricosanedioate hydrochloride (20.5 g, 82% Wt, 1 Eq, 27.2 mmol) in DCM (300 mL) were added N-ethyl-N-isopropylpropan-2-amine (14.1 g, 4 Eq, 109 mmol),20-(2-cyanoethoxy)-20-oxoicosanoic acid (12.0 g, 90% Wt, 1 Eq, 27.2 mmol),lH- bcnzo[d][l,2,3]triazol-l-ol (5.52 g, 1.5 Eq, 40.9 mmol) and 3-(((cthylimino)mcthylcnc)amino)- N,N-dimethylpropan-l -amine hydrochloride (7.83 g, 1.5 Eq, 40.9 mmol) at 26 °C, then the reaction mixture was stirred at 26 °C for 16 hour. The reaction mixture was concentrated under reduced pressure to give the residue. The residue was added to H2O (500 mL) and extracted with EtOAc (400 mL * 2). The combined organic layers were washed with brine (400 mL * 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by flash silica gel chromatography (Biotage®; Agela®Flash Column Silica-CS (220 g), Eluent of 0-10% MeOH / DCM gradient @ 100 mL / min) to give 1-benzyl 21, 4 l-bis(2-cy anoethyl) (S)-9,18,23-trioxo-2,5,ll,14-tetraoxa- 8,17,22-triazahentetracontane-l,21,41-tricarboxylate (24 g, 18 mmol, 66 %, 72% Purity) as a white solid. LCMS m / z = 958.5 (M+l). ’H NMR (400 MHz, DMSO-6) 8 ppm 8.22 (d, 7=7.20 Hz, 1 H) 7.86 (br t, 7=5.20 Hz, 1 H) 7.64 (br t, 7=5.60 Hz, 1 H) 7.22 - 7.44 (m, 5 H) 5.14 (s, 2 H) 4.17 - 4.21 (m, 5 H) 3.86 (s, 2 H) 3.48 - 3.64 (m, 9 H) 3.40 - 3.47 (m, 4 H) 3.13 - 3.30 (m, 5 H) 2.86 (td, 7=6.00, 2.80 Hz, 4 H) 2.32 (t, J=1.60 Hz, 2 H) 2.14 - 2.20 (m, 2 H) 2.10 (t, 7=7.60 Hz, 2 H) 1.90 - 1.99 (m, 1 H) 1.74 - 1.85 (m, 1 H) 1.43 - 1.56 (m, 4 H) 1.22 (s, 28 H).Example 40: (S)-46-cyano-22-((2-cyanoethoxy)carbonyl)-10, 19,24, 43-tetraoxo-3, 6, 12, 15,44- pentaoxa-9,18,23-triazahexatetracontanoic acid[000183] A solution of 1-benzyl 21, 4 Lbis(2-cy anoethyl) (S)-9,18,23-trioxo-2,5,l l,14- tetraoxa-8,17,22-triazahentetracontane-l,21,41-tricarboxylate (12.0 g, 72% Wt, 1 Eq, 9.02 mmol) in THF (120 mL) was degassed with Argon and Pd / C(wet basis) (3.0 g, 10% Wt, 0.31 Eq, 2.8 mmol) was added. The reaction mixture was evacuated and backfilled three times with hydrogen. The mixture was stirred at 26 °C for 30 min under an atmosphere of hydrogen 15 psi. Upon completion, the reaction mixture was filtered through a pad of celite and the filtrate was concentrated under reduced pressure to afford the crude product. Pd / C is recycled into anactivated metal recycling bucket. The crude product was purified by flash silica gel chromatography (Biotagc®; Agcla®Flash Column Silica-CS (220 g), Eluent of 0-10% McOH / DCM gradient @ 60 mL / min) to give (S)-46-cyano-22-((2-cyanoethoxy)carbonyl)-10,19,24,43- tetraoxo-3,6,12,15,44-pentaoxa-9,18,23-triazahexatetracontanoic acid (11694.41 mg, 13.3 mmol, 147 %, 98.4% Purity) as a white solid. LCMS m / z = 869.0 (M+l). 'H NMR (400 MHz, METHANOL-^) 5 ppm 4.24 - 4.42 (m, 5 H) 4.12 (s, 2 H) 4.01 (s, 2 H) 3.63 - 3.72 (m, 8 H) 3.58 (dt, 7-10.40, 4.80 Hz, 4 H) 3.35 - 3.48 (m, 4 H) 2.80 - 2.88 (m, 3 H) 2.22 - 2.42 (m, 6 H) 1.92 - 2.21 (m, 2 H) 1.55 - 1.68 (m, 4 H) 1.18 - 1.41 (m, 29 H).Example 4P: 2-cyanoethyl (S)-29-((2-cyanoethoxy)carbonyl)-l-hydroxy-8,17,26,31- tetraoxo-10,13,19,22-tetraoxa-7,16,25,30-tetraazapentacontan-50-oate[000184] A solution of (S)-46-cyano-22-((2-cyanoethoxy)carbonyl)-10,19,24,43-tetraoxo- 3,6,12,15,44-pentaoxa-9,18,23-triazahexatetracontanoic acid (3.95 g, 4.55 mmol), EDC (1.05 g, 5.46 mmol), 6-amino-l -hexanol (0.59 g, 5.01 mmol), and DCM (23 mL) was stirred at ambient temperature for 18 h. The crude reaction was concentrated, the residue was dissolved in 3:1 CHCh / i-PrOH (150 mL) and washed with brine (acidified to pH 3 with 1 N HC1). The organic layer was isolated, dried (MgSCL), and concentrated to a light yellow, waxy solid (3.20 g, 73%), 2-cyanoethyl (29S)-l-(((2-cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)-29-((2- cyanoethoxy)carbonyl)-8,17,26,31-tetraoxo-10,13,19,22-tetraoxa-7,16,25,30- tetraazapentacontan-50-oate. This was used in the next preparation without further purification or characterization.Example 4Q: C20-Diacid-CE phosphoramidite2-cyanoethyl (29S)-l-(((2-cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)-29-((2- cyanoethoxy)carbonyl)-8, 17,26, 31-tetraoxo-10, 13,19, 22-tetraoxa-7, 16, 25,30- tetraazapentacontan-50-oate[000185] A solution of 2-cyanoethyl (S)-29-((2-cyanoethoxy )carbonyl)-l -hydroxy-8,17, 26, 31-tetraoxo- 10, 13, 19, 22-tetraoxa-7, 16,25, 30-tetraazapentacontan-50-oate (3.20 g, 3.31 mmol), 5-(ethylthio)-lH-tetrazole (0.25 M in MeCN; 6.6 mL, 1.65 mmol), 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (1.26 mL, 3.97 mmol), and DCM (20 mL) was stirred at ambient temperature. After 1.5 hours, the crude reaction was poured into a slurry of silica gel (15 g) in DCM (30 mL), concentrated in vacuo to a dry powder, and purified via silica gel flash chromatography eluting with 5-30% MeOH / EtOAc to give 2-cyanoethyl (29S)-l-(((2-cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)-29-((2-cyanoethoxy)carbonyl)- 8,17,26,31-tetraoxo-10,13,19,22-tetraoxa-7,16,25,30-tetraazapentacontan-50-oate as a sticky, white foam (1.40 g, 36%). ’H NMR (d6-DMSO) d 8.22 (d, 1 H), 7.88 (t, 1 H), 7.70-7.60 (m, 2 H), 4.29-4.12 (m, 5 H), 3.88 (s, 2 H), 3.85 (s, 2 H), 3.82-3.49 (m, 14 H), 3.49-3.37 (m, 4 H), 3.32-3.25 m, 2 H), 3.24-3.16 (m, 2 H), 3.13-3.04 (m, 2 H), 2.91-2.84 (m, 4 H), 2.76 (t, 2 H), 2.33 (t, 2 H), 2.18 (t, 2 H), 2.11 (t, 2 H), 1.99-1.90 (m, 1 H), 1.86-1.74 (m, 1 H), 1.59-1.04 (m, 52 H).31P NMR (d6-DMSO) d 146.3.Example 4R: 1-benzyl 23-methyl (S)-22-((tert-butoxycarbonyl)amino)-10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosanedioate[000186] To a mixture of benzyl 17-amino-10-oxo-3,6,12,15-tctraoxa-9-azahcptadccanoatc hydrochloride (35 g, 73% Wt, 1 Eq, 59 mmol) in DCM (350 mL) was added (S)-4-((tert- butoxycarbonyl)amino)-5-methoxy-5-oxopentanoic acid (16 g, 99% Wt, 1 Eq, 59 mmol, CAS number 45214-91-3), l-(3-Dimethylaminopropyl)-3-ethylcarbodiimideHydrochloride(EDCI) (17g, 99% Wt, 1.5 Eq, 88 mmol), 1 -Hydroxy- IH-benzotriazole (12 g, 99% Wt, 1.5 Eq, 88 mmol) and Diisopropylcthylaminc (31 g, 41 mL, 99% Wt, 4 Eq, 0.23 mol) at 0 °C, then the reaction mixture was stirred at 26 °C for 16 hour. The reaction mixture was concentrated under reduced pressure to give the residue. To the residue was added H2O (500 mL) and extracted with EtOAc (300 mL * 2). The combined organic layers were washed with brine (500 mL * 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by flash silica gel chromatography (Biotage®; Agela®Flash Column Silica-CS (220 g), Eluent of 0-100% EtOAc / petroleum ether gradient @ 100 mL / min) to give 1-benzyl 23-methyl (S)-22-((tert-butoxycarbonyl)amino)-10,19-dioxo-3,6,12,15-tetraoxa- 9,18-diazatricosanedioate (25.5 g, 38 mmol, 65 %, 96% Purity) as colorless gel. LCMS m / z = 642.3 (M+l) ’ H NMR (400 MHz, METHANOL-^) 5 ppm 7.24 - 7.44 (m, 5 H) 5.19 (s, 2 H) 4.21 (s, 2 H) 3.98 (s, 2 H) 3.69 - 3.73 (m, 5 H) 3.60 - 3.68 (m, 7 H) 3.55 (dt, 7=10.80, 5.20 Hz, 4 H) 3.40 - 3.46 (m, 2 H) 3.34 - 3.39 (m, 2 H) 2.30 (br t, 7=7.20 Hz, 2 H) 2.05 - 2.18 (m, 1 H) 1.79 - 1.94 (m, 1 H) 1.43 (s, 9 H).Example 4S: 1-benzyl 23-methyl (S)-22-amino-10,19-dioxo-3,6,12,15-tetraoxa-9,18- diazatricosanedioate hydrochloride[000187] To a mixture of 1-benzyl 23-methyl (S)-22-((tert-butoxycarbonyl)amino)-10,19- dioxo-3,6,12,15-tetraoxa-9,18-diazatricosanedioate (25.5 g, 96% Wt, 1 Eq, 38.1 mmol) in EtOAc (50 mL) was added to 2M Hydrogen chloride in ethyl acetate (13.9 g, 191 mL, 2 molar, 10 Eq, 381 mmol) at 0 °C. Then the reaction mixture was stirred at 26 °C for 60 min. The reaction mixture was concentrated under reduced pressure to give 1-benzyl 23-methyl (S)-22-amino- 10,19-dioxo-3,6,12,15-tetraoxa-9,18-diazatricosanedioate hydrochloride (25 g, 38 mmol, 100 %, 89% Purity) as a pink gel. LCMS m / z = 542.3(M+1). ’H NMR (400 MHz, METHANOL-74) 5 ppm 7.30 - 7.39 (m, 5 H) 4.60 (s, 2 H) 4.17 (s, 2 H) 4.05 (s, 2 H) 3.84 (s, 3 H) 3.69 - 3.71 (m, 4 H) 3.64 - 3.67 (m, 4 H) 3.56 - 3.61 (m, 6 H) 3.44 - 3.48 (m, 2 H) 3.40 (t, 7=5.60 Hz, 2 H) 2.49 - 2.54 (m, 2 H) 2.15 - 2.26 (m, 2 H).Example 4T: 1-benzyl 21, 41 -dimethyl (S)-9, 18, 23-trioxo-2, 5,11, 14-tetraoxa-8, 17,22- triazahentetracontane-l,21,41-tricarboxylate[000188] To a mixture of 1-benzyl 23-methyl (S)-22-amino-10,19-dioxo-3,6,12,15- tetraoxa-9,18-diazatricosanedioate hydrochloride (25.0 g, 89% Wt, 1 Eq, 38.5 mmol) in DCM (250 mL) were added 20-methoxy-20-oxoicosanoic acid (13.9 g, 99% Wt, 1 Eq, 38.5 mmol, CAS 1767-98-2), l-(3-Dimethylaminopropyl)-3-ethylcarbodiimideHydrochloride(EDCI) (11.2 g, 99% Wt, 1.5 Eq, 57.7 mmol), 1 -Hydroxy- IH-benzo triazole (7.88 g, 99% Wt, 1.5 Eq, 57.7 mmol) and Diisopropylethylamine (25.1 g, 33.5 mL, 99% Wt, 5 Eq, 192 mmol) at 0 °C, then the reaction mixture was stirred at 26 °C for 16 hour. The reaction mixture was concentrated under reduced pressure to give the residue. To the residue was added H2O (300 mL) and extracted with EtOAc (300 mL * 2). The combined organic layers were washed with brine (300 mL * 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by flash silica gel chromatography (Biotage®;Agela®Flash Column Silica-CS (120 g), Eluent of 0~7% MeOH / DCM gradient @ 80 mL / min) to give 1-benzyl 21,41 -dimethyl (S)-9,18,23-trioxo-2,5,l l,14-tetraoxa-8,17,22- triazahentetracontane- 1,21,41 -tricarboxylate (35 g, 39 mmol, 100 %, 97% Purity) as a white solid. LCMS m / z = 880.6 (M+l). ’H NMR (400 MHz, DMSO-ri6) 8 ppm 8.17 (d, 7=7.20 Hz, 1 H) 7.88 (br t, 7=5.20 Hz, 1 H) 7.64 (br t, 7=5.60 Hz, 1 H) 7.22 - 7.49 (m, 5 H) 5.14 (s, 2 H) 4.14 - 4.22 (m, 3 H) 3.86 (s, 2 H) 3.50 - 3.59 (m, 19 H) 3.16 - 3.29 (m, 5 H) 2.27 (t, 7=7.60 Hz, 2 H) 2.11 (m, 4 H) 1.43 - 1.54 (m, 4 H) 1.22 (s, 28 H).Example 4U: (S)-24-(methoxycarbonyl)-3, 22,27, 36-tetraoxo-2, 31, 34,40, 43-pentaoxa-23,28,37-triazapentatetracontan-45-oic add[000189] A solution of 1-benzyl 21,41 -dimethyl (S)-9,18,23-trioxo-2,5,l 1,14-tetraoxa- 8,17,22-triazahentetracontane-l,21,41-tricarboxylate (30.0 g, 97% Wt, 1 Eq, 33.1 mmol) in THF (300 mL) was degassed with Argon, and Pd / C, wet basis (14.1 g, 10% Wt, 0.4 Eq, 13.2 mmol)was added. The reaction mixture was evacuated and backfilled three times with hydrogen. The mixture was stirred at 25 °C for 120 min under an atmosphere of hydrogcnl5 psi. The reaction mixture was filtered through a pad of celite and the filtrate was concentrated under reduced pressure to afford the crude product. Pd / C is recycled into a special recycling bucket. The crude product was purified by flash silica gel chromatography (Biotage®; Agela®Flash Column Silica- CS (120 g), Eluent of 0-15% MeOH / DCM gradient @ 80 mL / min)to give (S)-24- (methoxycarbonyl)-3,22,27,36-tetraoxo-2,31,34,40,43-pentaoxa-23,28,37-triazapentatetracontan- 45-oic acid (17493.85 mg, 21.4 mmol, 64.8 %, 96.8% Purity) as a white solid. LCMS m / = 791.0 (M+l). ‘H NMR (400 MHz, METHANOL-^) 5 ppm 4.39 (dd, 7=8.80, 5.20 Hz, 1 H) 4.11 (s, 2 H) 4.01 (s, 2 H) 3.62 - 3.74 (m, 14 H) 3.57 (dt, 7=10.80, 5.20 Hz, 4 H) 3.42 - 3.48 (m, 2 H) 3.35 - 3.40 (m, 2 H) 2.31 (td, 7=7.20, 2.80 Hz, 4 H) 2.24 (br t, 7=7.60 Hz, 2 H) 2.13 (m, 1 H) 1.90 - 2.01 (m, 1 H) 1.54 - 1.68 (m, 4 H) 1.29 (br s, 28 H).Example 4V: C20-Diacid-ME NHS Ester l-(2,5-dioxopyrrolidin-l-yl) 21,41-dimethyl (S)-9, 18, 23-trioxo-2, 5,11, 14-tetraoxa-8, 17,22- triazahentetracontane-l,21,41-tricarboxylate[000190J A solution of (S)-24-(methoxycarbonyl)-3, 22, 27, 36-tetraoxo-2, 31,34,40,43- pentaoxa-23,28,37-triazapentatetracontan-45-oic acid (500 mg, 1 Eq, 633 pmol) lOmL of DCM, l-hydroxypyrrolidine-2,5-dione (109 mg, 1.5 Eq, 949 pmol) and 3- (((ethylimino)methylene)amino)-N,N-dimethylpropan-l -amine hydrochloride (121 mg, 1 Eq, 633 pmol), was stirred at room temperature for 18 hours. The crude reaction was concentrated, and loaded onto 12g silica cartridge, purified via silica gel flash chromatography eluting with 0- 30% MeOH / EtOAc to give l-(2,5-dioxopyrrolidin-l-yl) 21,41-dimethyl (S)-9,18,23-trioxo- 2,5,l l,14-tetraoxa-8,17,22-triazahentetracontane-l,21,41-tricarboxylate as a sticky, white foam (320mg, 54%). ’H NMR (d6-DMSO) 5 8.15 (d, 1 H), 7.86 (t, 1 H), 7.65 (t, 1 H), 4.61 (s, 2 H), 4.16-4.22 (m, 2 H), 4.01 (m, 1 H), 3.61 (s, 3 H), 3.58 (s, 3 H), 3.52-3.59 (m, 12 H), 3.38-3.48 (m, 4 H), 2.29 (t, 2 H), 2.07-2.18 (m, 6 H), 1.43-1.56 (m, 4 H), 1.24 (s, 28 H).Scheme 8[000191] The synthesis of MSPT linker is shown in Scheme 8. The synthesis began from commercially available intermediate 4-(5-mercapto-lH-tetrazol-l-yl)phenol (CAS# 52431-78-4). Alkylation of the free sulfide with methyliodide took place under the influence of DIPEA in the solvent THF to yield the methyl sulfide intermediate (2), Step A. As captured in Step B, Williamson ether reaction effected the coupling between the phenol and a bromo-PEG2 reactant that produced ether (4). Step C shows oxidation of the methyl sulfide to the methyl sulfone which used aqueous hydrogen peroxide and catalytic Molybdenum, and the crude sulfone intermediate was treated with trifluoroacetic acid in DCM to yield acid (5). The terminal carboxylic acid was transformed into the NHS-ester with EDCI and NHS-OH in Step D, which provided the activated ester (6) that is reactive to primary amines.Example 4W: 4-(5-(methylthio)-lH-tetrazol-l-yl)phenol:[000192] 4-(5-mercapto-lH-tetrazol- l-yl)phenol (4.00 g, 20.6 mmol) was dissolved in tetrahydrofuran (50mL), and the mixture cooled in an ice-bath, prior to the addition of N,N- diisopropylethylamine (4.31 g, 33.3 mmol). A suspension formed after stirring for 10 minutes, lodomethane (1.54 mL, 24.7 mmol) was added dropwise via syringe over 1 min. The reaction mixture was stirred for 20 minutes with cooling in an ice-bath, then at room temperature for 12 hours. The mixture was diluted with EtOAc (100 mL), and washed with saturated aqueous NH4CI (2x 50 mL). The organic layer was separated, then dried over sodium sulfate, filtered and concentrated in vacuo to provide 4-(5-(methylthio)-lH-tetrazol-l-yl)phenol (4.2 g, 93% yield) that can be used directly in the next step without further purification. LCMS - mz = 209 (M+l).Example 4X: tert-butyl 2-(2-(2-(4-(5-(methylthio)-lH-tetrazol-l- yl)phenoxy)ethoxy)ethoxy)acetate:[000193] A 200 mL pressure vessel was charged with 4-(5-(methylthio)-lH-tetrazol-l- yl)phenol (2.50 g, 11.4 mmol), tert-butyl 2-(2-(2-bromoethoxy)ethoxy)acetate (4.33 g, 14.8 mmol) and acetone (60 mL). Potassium carbonate was added (3.15 g, 22.8 mmol), and the vessel sealed and heated at 80 °C for 8 hours with vigorous stirring. The reaction mixture was cooled to room temperature and filtered to remove potassium carbonate, then washed with acetone / DCM / EtOAc (30 mL each). The filtrate was concentrated in vacuo to provide crude material, which was purified by flash chromatography (80 g, 100% DCM for 5 minutes, then gradient to 100% EtOAc over 20 minutes). The product tert-butyl 2-(2-(2-(4-(5-(methylthio)-lH- tetrazol-l-yl)phenoxy)ethoxy)ethoxy)acetate (3.98g, 85% yield) was isolated as a white powder. LCMS - mz = 411 (M+l).Example 4Y: 2-(2-(2-(4-(5-(methylsulfonyl)-lH-tetrazol-l-yl)phenoxy)ethoxy)ethoxy)acetic acid:[000194] Tert-butyl 2-(2-(2-(4-(5-(methylthio)-lH-tetrazol-l- yl)phenoxy)ethoxy)ethoxy)acetate (3.98 g, 9.21 mmol) was dissolved in ethanol (100 mL) and cooled to 5-10 °C in an ice-water bath, prior to the addition of 30% aqueous hydrogen peroxide (19.0 mL, 184 mmol), followed by ammonium molybdate (VI) tetrahydrate (1.14 g, 0.921 mmol). The reaction mixture was stirred at room temperature for 4 hours in an ice bath, then at room temperature for 12 hours. The mixture was diluted with DCM (1 0 mL) and then washed with brine. The organic phase was separated, dried over sodium sulfate and concentrated in vacuo to dryness. Purification by flash column chromatography (80g silica, 100% DCM for 5 minutes, then gradient to 100% EtOAc over 20 minutes) provided 2-(2-(2-(4-(5- (methylsulfonyl)-lH-tetrazol-l-yl)phenoxy)ethoxy)ethoxy)acetic acid (3.00g, 80% yield) as a thick oil. LCMS - mz = 385 (M-l).Example 4Z: MSPT-PEG2-NHS Ester2,5-dioxopyrrolidin-l-yl 2-(2-(2-(4-(5-(methylsulfonyl)-lH-tetrazol-l- yl)phenoxy)ethoxy)ethoxy)acetate:[000195] l-Hydroxypyrrolidine-2, 5-dione (1.33 g, 1.6 Eq, 11.6 mmol) was added to a solution of 2-(2-(2-(4-(5-(methyl sulfonyl)- 1 H-tetrazoL 1 -yl)phenoxy)ethoxy)ethoxy)acetic acid (2.80 g, 7.25 mmol) in DCM (50 mL) and THF (70 mL). EDCI (1.60g, 10.3 mmol) was added in one portion, upon which the solution became a cloudy mixture. Additional DCM (20 mL) was added to bring the mixture into solution again, followed by stirring at room temperature for 12hours. The solvent was removed in vacuo to provide crude material as a white foam. Purification by flash column chromatography (80 g, 100% DCM for 5 minutes, then gradient to 100% EtOAc over 20 minutes) afforded 2,5-dioxopyrrolidin-l-yl 2-(2-(2-(4-(5-(methylsulfonyl)-lH-tetrazol-l- yl)phenoxy)ethoxy)ethoxy)acetate (2.61g, 65% yield) as low melting solid. LCMS - mz = 484 (M+l).Scheme 9[000196] The synthesis of MSPOD linker is shown in Scheme 9. The synthesis began from commercially available intermediate 4-(5-mercapto-l,3,4-oxadiazol-2-yl)phenol (CAS# 69829- 90-9), and followed steps A and B as described is Scheme 8 for MPST. Oxidation with catalytic Molybdenum and aqueous hydrogen peroxide furnished the sulfone (5), Step C. Step D shows the acidic deprotection of (5) with the strong acid TFA in DCM. The terminal carboxylic acid was transformed into the NHS-ester with EDCI and NHS-OH in Step E, which provided an activated ester (7) that is reactive to primary amines.Example 4AA: 4-(5-(methylthio)-l,3,4-oxadiazol-2-yl)phenol[000197] 4-(5-Mercapto-l,3,4-oxadiazol-2-yl)phenol (3.00 g, 15.4 mmol) was dissolved in THF (50 mL), and cooled to 0 °C in an ice water bath. A,A-Diisopropylethylamine (3.46 mL, 2.60 g, 20.1 mmol) was added, resulting in a cloudy solution. The mixture was stirred for 5 minutes in the ice bath, then iodomethane (2.85 g, 20.1 mmol) was added drop-wise via syringe over a period of 1 minute. Upon addition, the mixture turned clear after 5 minutes. The cooling bath was removed and the mixture stirred at room temperature for 2 hours, after which it was diluted with dichloromethane (100 mL) and washed with saturated aqueous NH4C1 (pH was adjusted to ~5 by adding citric acid solution, 2x 50 mL). The organic layer was separated, dried over sodium sulfate, and concentrated in vacuo to dryness to afford 4-(5-methylsulfanyl-l,3,4- oxadiazol-2-yl)phenol (520 mg, 97% yield) as a pale-yellow solid that was used in the next step without further purification. LC-MS - mz = 209 (M+l).Example 4BB: Synthesis of tert-butyl 2-(2-(2-(4-(5-(methylthio)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetate[000198] 4-(5-(Methylthio)-l,3,4-oxadiazol-2-yl)phenol (3.3 g, 1 Eq, 15 mmol) and acetone (60 mL) were added to a 200 mL pressure vessel. To this solution was added tert-butyl 2-(2-(2-bromoethoxy)ethoxy)acetate (5.5 g, 20 mmol) and potassium carbonate (4.2 g, 30 mmol). The pressure vessel was sealed and heated at 70 °C for 5 hours with vigorous stirring. After cooling to room temperature, the mixture was filtered to remove solid potassium carbonate, washing with EtOAc / DCM. The filtrate was concentrated in vacuo to dryness and purified by normal phase flash column chromatography (80 g silica gold, 100% DCM for 5 minutes, then gradient to 100% EtOAc over 20 minutes). Product-containing fractions were concentrated in vacuo to afford tert-butyl 2-(2-(2-(4-(5-(methylthio)-l,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (4.8 g, 74 % yield) as a white solid. LC-MS - mz = 41 1 (M+l).Example 4CC: tert-butyl 2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetate[000199] Tert-butyl 2-(2-(2-(4-(5-(methylthio)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetate (5.20 g,12.7 mmol) was dissolved in 100 mL of ethanol, and cooled to 5-10 °C in an ice-water bath, prior to the addition of 30% hydrogen peroxide (10 mL, 97 mmol), followed by ammonium molybdate (VI) tetrahydrate (501 mg, 0.405 mmol). After two hours of vigorous stirring, an additional 15 mL of 30% hydrogen peroxide and 1 g of Ammonium molybdate (VI) tctrahydratc were added. The reaction mixture was stirred for another 6 hours, then diluted with 150 mL of DCM, and washed with brine. The organic phase was separated, dried over sodium sulfate and concentrated in vacuo to dryness. The residue was triturated with methanol to provide an initial portion of the product. The solvent was then removed from the mother liquid under reduced pressure. Purification by flash column chromatography (40g, 100% DCM for 3 minutes, then gradient to 100% EtOAc over 20 minutes) provided additional product as a white solid. Combination of both product portions yielded tertbutyl 2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (5.3g, 90% yield) as a white solid. LC-MS - mz = 387 (M - tBu).Example 4DD: 2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetic acid[000200] To a solution of tert-butyl 2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetate (5.60 g, 12.0 mmol) in DCM (60 mL) was added 2,2,2-trifluoroacetic acid (20 mL, 12.0 mmol). The reaction mixture was stirred at room temperature for 2 hours then concentrated under vacuo to afford a thick residue, which was purified by normal phase flash column chromatography (80g silica gold column, 100% DCM for 3 minutes, then gradient to 100% EtOAc over 20 minutes). Combination of product-containing fractions and concentration in vacuo yielded 2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetic acid (4.12g, 82% yield). LCMS - mz = 387 (M+l).Example 4EE: 2,5-dioxopyrrolidin-l-yl-2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetate[000201 ] 2-(2-(2-(4-(5-(Methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetic acid (3.00 g, 7.38 mmol) and l-hydroxypyrrolidine-2,5-dione (1.19 g, 10.3 mmol) were dissolved in DCM (50 mL) and THF (70 mL). To this solution was added 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-l -amine (EDO, 1.60g, 10.3 mmol). Upon addition, the solution turned cloudy and additional DCM (20 mL) was added to bring the mixture into solution again, followed by stirring at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue dissolved in DCM purified by normal phase flash column chromatography (80 g silica gold 100% DCM for 5 minutes, then gradient to 100% EtOAc over 20 minutes). Concentration of product-containing fractions afforded 2,5-dioxopyrrolidin-l-yl-2-(2-(2-(4-(5-(methylsulfonyl)-l,3,4-oxadiazol-2- yl)phenoxy)ethoxy)ethoxy)acetate (2.61g, 65% yield). LCMS - mz = 484 (M+l).Example 4FF: methyl 20-((2-hydroxyethyl)amino)-20-oxoicosanoate[000202] 20-methoxy-20-oxoicosanoic acid (3.00 g, 8.41 mmol), EDC (1 .77 g, 9.23 mmol), HOBT (1.42 g, 9.23 mmol), DIPEA (1.62 mL, 9.23 mmol), and DCM (42 mL) were stirred at ambient temperature until all solid material dissolved (~5 min). Ethanolamine (0.56 mL. 9.23 mmol) was added at which time the reaction turned to a milky white suspension. Stirring at ambient temperature continued for 18 h. The crude reaction was concentrated, the residue was suspended in water (100 mL), acidified with 5 N HC1 (2 mL), and then cooled to 0 °C for 15 min. The white solid was isolated via suction filtration, washed with water, and further dried under vacuum (3.27 g, 97%). 'H NMR (CDC13) 86.45 (br s, 1 H), 3.76 (t, 2 H), 3.69 (s, 3 H), 3.50-3.42 (m, 2 H), 2.91 (br s, 1 H), 2.37-2.24 (m, 4 H), 1.72-1.58 (m, 4 H), 1.39-1.20 (m, 28 H).Example 4GG: methyl 20-((2-(((2- cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)ethyl)amino)-20-oxoicosanoate (C20- Acid-Ethanolamide phosphoramidite)[000203] A solution of methyl 20-((2-hydroxyethyl)amino)-20-oxoicosanoate (3.25 g, 8.13 mmol), 5-(ethylthio)-lH-tetrazole (0.25 M in MeCN; 16.3 mL, 4.07 mmol), 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (3.36 mL, 10.6 mmol), and DCM (40 mL) was stirred at ambient temperature. After 3 hours, to crude reaction was added Celite (25 g) and the suspension was concentrated to a dry solid then purified via basic alumina flash chromatography eluting with 20-60% EtOAc / hexane to give the title compound as a white solid87.83 (t, 1 H), 3.81-3.67 (m, 2 H), 3.64-3.45 (m, 7 H), 3.27-3.17 (m, 2 H), 2.76 (t, 2 H), 2.29 (t, 2 H), 2.05 (t, 2 H), 1.57-1.42 (m, 4 H), 1.31-1.18 (m, 28 H), 1.14 (t, 12 H).31P NMR (d6-DMSO) 8 146.8.Example 4HH: 2-cyanoethyl (2-octyldodecyl) diisopropylphosphoramidite (C20- Octyldodecyl phosphoramidite)[000204] A solution of octyldodecanol (1.00 g, 3.35 mmol), dichloromethane (50 mL), 5- (ethylthio) tetrazole solution (7 mL, 0.25 M in MeCN, 2 mmol), and 3- ((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (1.49 mL, 4.69 mmol) was stirred at ambient temperature for 2 hours. Added Celite (12 g) and the suspension was concentrated to a dry solid then purified via 160 g neutral alumina flash chromatography eluting with 0-10% EtOAc / hexane to give the title compound as a thin oil (1.25 g, 75%). 1H NMR (CDC13) 8 3.84 (m, 2 H) 3.61 (m, 4 H) 3.49 (m, 1 H) 2.66 (t, 2 H) 1.28 (m, 32 H) 1.14 (t, 12 H) 0.90 (t,6 H). 31P NMR (CDCh) 8 147.6.Example 411: (Z)-2-cyanoethyl octadec-9-en-l-yl diisopropylphosphoramidite (Oleyl phosphoramidite)[000205][000206] A solution of oleyl alcohol (1.00 g, 3.72 mmol), dichloromethane (50 mL), 5- (ethylthio) tetrazole solution (8 mL, 0.25 M in MeCN, 2 mmol), and 3- ((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (1.68 g, 5.59 mmol) was stirred at ambient temperature for 2 hours. Added Celite (12 g) and the suspension was concentrated to a dry solid then purified via 160 g neutral alumina flash chromatography eluting with 0-7% EtOAc / hexane to give the title compound as a thin oil (1.4 g, 80%). 1H NMR (d6-DMSO) 8 5.33 (t, 2 H) 3.71 (m, 2 H) 3.57 (m, 4 H) 2.76 (t, 2 H) 1.98 (m, 4 H) 1.53 (q, 2 H) 1.25 (m, 22 H)1.14 (t, 12 H) 0.86 (t,3 H). 3 IP NMR (d6-DMSO) 8 146.49Example 4JJ: 2-cyanoethyl hexadecyl diisopropylphosphoramidite (Cetyl phosphoramidite)[000208] A solution of cetyl alcohol (2 g, 8 mmol), dichloromethane (35 mL), 2H-tetrazole (0.45 M in MeCN, 18 mL, 7 mmol), and 3- ((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (3 g, 10 mmol) was stirred at ambient temperature for 3 hours. Evaporated the solvents and redissolved in hexanes. Purified via 40 g basic alumina flash chromatography eluting with 0-7 % EtOAc / hexane to give the title compound as a thin oil (2.7 g, 70%). 1H NMR (CDC13) 83.84 (m, 2 H) 3.61 (m, 4 H) 2.66 (t, 2 H) 1.62 (m, 2 H) 1.28 (m, 24 H) 1.2 (t, 12 H) 0.90 (t, 3 H). 31P NMR (CDC13) 8 147.3.One skilled in the ail will recognize “Cetyl phosphoramidite” may be used as the next unit in standard oligonucleotide couplings after “Glycol bis(DMT)doubler phosphoramidite” with oxidizer and / or sulfurizing agent to synthesize fatty acids FA 16- 18 at the 5 ’-terminal position on a strand.Example 4KK: 2-cyanoethyl docosyl diisopropylphosphoramidite (C22-Docosyl phosphoramidite)[000209] A solution of n-docosanol (0.615 g, 1.88 mmol), dichloromethane (53 mL), 5-(ethylthio) tetrazole solution (0.25 M in MeCN, 4 mL, 1 mmol), and 3- ((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (851 mg, 2.82 mmol) was stirred at ambient temperature for 2 hours. Added Cclitc (12 g) and the suspension was concentrated to adry solid then purified via 48 g neutral alumina flash chromatography eluting with 0-8% EtOAc / hcxanc to give the title compound as a white waxy solid (0.540 g, 54%). 1H NMR (CDC13) 53.84 (m, 2 H) 3.61 (m, 4 H) 2.66 (t, 2 H) 1.62 (m, 2 H) 1.28 (m, 38 H) 1.2 (t, 12 H) 0.90 (t, 3 H). 3 IP NMR (CDC13) 5 147.1.Example 4LL: 2-((3r,5r,7r)-adamantan-l-yl)ethyl (2-cyanoethyl) diisopropylphosphoramidite (Adamantaneethanol phosphoramidite)A solution of 1-admantaneethanol (2.00 g, 11.1 mmol), dichloromethane (50 mL), 5-(ethylthio) tetrazole solution (0.25 M in MeCN, 22 mL, 5.55 mmol), and 3- ((his(diisopropylamino)phosphanyl)oxy)propanenitrile (5.02 g, 16.6 mmol) was stirred at ambient temperature for 4 hours. Added Cclitc (12 g) and the suspension was concentrated to a dry solid then purified via 160 g neutral alumina flash chromatography eluting with 0-8% EtOAc / hexane to give the title compound as a clear thin oil (0.540 g, 54%). 1H NMR (CDC13) 8 3.71 (m, 2 H) 3.57 (m, 4 H) 2.76 (t, 2 H) 1.9 (s, 6 H) 1.63 (q, 6 H) 1.5 (m, 6 H) 1.35 (t, 2 H) 1.14 (t, 12 H) 3 IP NMR (d6-DMSO) 8 146.0Example 4MM: methyl 20-(((2- cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)icosanoate (O-C20-Acid phosphoramidite)[000210] A solution of methyl 20-hydroxyicosanoate (3. 10 g, 9.05 mmol), dichloromethane (45 mL), 5-(cthylthio) tctrazolc solution (0.25 M in McCN, 18 mL, 4.5 mmol), and 3- ((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (3.55 g, 11.8 mmol) was stirred at ambient temperature for 1 hours. Added Celite (25 g) and the suspension was concentrated to a dry solid then purified via 160 g basic alumina flash chromatography eluting with 0-10% EtOAc / hexane to give the title compound as a thick oil (4.1 g, 84%). 1H NMR (d6-DMSO) 8 3.79-3.50 (m, 9 H), 2.76 (t, 2 H), 2.28 (t, 2 H), 1.58-1.46 (m, 4 H), 1.36-1.19 (m, 30 H), 1.17-1.10 (m, 12 H). 31P NMR (d6-DMSO) 8 146.3.Example 4NN: methyl 18-(((2- cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)octadecenoate (O-C18-Acid phosphoramidite)[000211] A solution of methyl 18-hydroxyoctadecanoate (3.15 g, 10.0 mmol), dichloromethane (50 mL), 5-(ethylthio) tetrazole solution (0.25 M in MeCN, 20 mL, 5 mmol), and 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (3.93 g, 13.0 mmol) was stirred at ambient temperature for 1 hours. Added Celite (25 g) and the suspension was concentrated to a dry solid then purified via 160 g basic alumina flash chromatography eluting with 0-10% EtOAc / hexane to give the title compound as a thick oil (4.0 g, 78%). 1H NMR (d6-DMSO) 8 3.79-3.50 (m, 9 H), 2.76 (t, 2 H), 2.28 (t, 2 H), 1.58-1.46 (m, 4 H), 1.36-1.19 (m, 26 H), 1.17-1.10 (m, 12 H). 3 IP NMR (d6-DMSO) 8 146.3.Example 400: methyl 16-(((2- cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)hexadecanoate (O-C16-Acid phosphoramidite)[000212] A solution of methyl 16-hydroxyhexadecanoate (3. 15 g, 11.0 mmol), 5-(ethylthio)-IH-tctrazolc (0.25 M in McCN; 22.0 mL, 5.50 mmol), 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (4.54 mL, 14.3 mmol), and DCM (55.0 mL) was stirred at ambient temperature. After 2 hours, to crude reaction was added Celite (25 g) and the suspension was concentrated to a dry solid then purified via basic alumina flash chromatography eluting with 100% hexane to give the title compound as a clear oil (5.03 g, 93% yield). 1H NMR (Acetonitrile-d3) 5 3.82-3.69 (m, 2 H), 3.66-3.54 (m, 4 H), 3.59 (s, 3 H), 2.62 (t, 2 H), 2.27 (t, 2 H), 1.60-1.52 (m, 4 H), 1.36-1.23 (m, 22 H), 1.20-1.11 (m, 12 H). 31P NMR (Acetonitrile-d3) 8 146.8.Example 4PP: methyl 16-((2-(((2- cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)ethyl)amino)-16-oxohexadecanoatev (C16-Acid Ethanolamide phosphoramidite)[000213] A solution of 16-methoxy-16-oxohexadecanoic acid (3.05 g, 10.2 mmol),DCM (50 mL), EDC (2.14 g, 11.2 mmol), HOBt (1.71 g, 11.2 mmol), and DIPEA (1.44 g, 11.2 mmol) was stirred at ambient temperature. 2-aminoethan-l-ol (682 mg, 11.2 mmol) was added resulting in a milky white suspension and stirring continued. After 18 hours, the reaction was concentrated to remove DCM, the white solid was suspended in water (100 mL) and acidified with 5 N HC1 (2 mL). The resultant aqueous suspension was cooled to 0 °C for 15 min (stirred rapidly for 5 min). White solid isolated via suction filtration, washed with 0.2 N HC1, water, then dried under vacuum to yield methyl 16-((2-hydroxyethyl)amino)-16-oxohexadecanoate (3.37 g, 97% yield).[000214] A suspension of methyl 16-((2-hydroxyethyl)amino)-16-oxohexadecanoate (3.37 g, 9.81 mmol), 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (3.84 g, 12.8 mmol), and DCM (50 mL) was stirred at ambient temperature and 5-(ethylthio)-lH-tetrazole (0.25 M in MeCN) (19.6 mL, 4.91 mmol) was added in one portion and stirring continued. The reaction turned to a clear, colorless solution after 1 hour. After 2 h, to the reaction was added Celite (-25 g), concentrated to a dry powder, and then purified via 160 g basic alumina flash chromatographyeluting with 10-60% EtOAc / hexane to give the title compound as a white solid (5.0 g, 94%). 1H NMR (d6-DMSO) 57.83 (t, 1 H), 3.81-3.67 (m, 2 H), 3.81-3.67 (m, 2 H), 2.76 (t, 2 H), 2.28 (t, 2 H), 1.58-1.46 (m, 4 H), 1 .36-1 .19 (m, 30 H), 1.17-1.10 (m, 12 H). 31P NMR (d6-DMSO) 5 146.8.Example 4QQ: methyl 16-((2-(((2- cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)ethyl)amino)-16-oxohexadecanoate (y - Glu-C16-Acid Ethanolamide phosphoramidite)[000215] Step 1: To a round bottomed flask was added: 16-methoxy-16-oxohexadecanoic acid (2.00 g, 1 Eq, 6.66 mmol, CAS 18451-85-9, CombiBlocks), DCM (33.3 mL), EDC (1.40 g, 1.1 Eq, 7.32 mmol), HOBt (1.12 g, 1.1 Eq, 7.32 mmol), and DIPEA (946 mg, 1.28 mL, 1.1 Eq, 7.32 mmol). The reaction mixture was stirred at room temperature for 30 minutes over the course of which solids slowly dissolved. Then to the reaction mixture was added 5 -(tert-butyl) 1 -methyl L-glutamate hydrochloride (1.86 g, 1.1 Eq, 7.32 mmol, CAS 6234-01-1, Alfa Aesar) and stirring at room temperature was continued. The reaction mixture was observed to be cloudy white after this addition. The reaction was allowed to proceed overnight. The next morning, reaction was still cloudy with a small amount of oily, white solid present. The reaction was diluted with DCM (50 mL) then washed with water (50 mL), again with water (50 mL) acidified with 0.5 mL 5 N HO, and then brine. The organic layer was dried (MgSO4) and concentrated to a white solid.LCMS confirmed mass of the desired product: 5 -(tert-butyl) 1-methyl (16-methoxy-16- oxohexadecanoyl)-L-glutamate. LCMS m / z = 500.2 (M+l)[000216] The product was used in subsequent reaction step without further purification.[000217] Step 2: A round bottomed flask was charged with 5 -(tert-butyl) 1-methyl (16- methoxy-16-oxohexadecanoyl)-L-glutamate (3.32 g, 1 Eq, 6.64 mmol), DCM (16.6 mL), and HC1 (2.42 g, 16.6 mL, 4 molar, 10 Eq, 66.4 mmol). Stirred at RT. The reaction mixture turned light orange (from colorless) after addition of HC1. The mixture was allowed to stir for 4 hours thenplaced in fridge (4°C) overnight. In the morning, the reaction mixture was removed from fridge and warmed to room temperature. The mixture was concentrated down under vacuum and then loaded onto a silica column and purified using silica gel chromatography on a gradient of 0-100% ethyl acetate in hexane. Product was observed to elute at -100% ethyl acetate. The fractions were concentrated down, rinsed 2x with DCM and dried to afford a white solid.LCMS confirmed mass of the desired product: (S)-5-methoxy-4-(16-methoxy-16- oxohexadecanamido)-5-oxopentanoic acid. LCMS m / z = 444.2 (M+l)[000218] Step 3: A round bottomed flask was charged with (S)-5-methoxy-4-(16-methoxy- 16-oxohexadecanamido)-5-oxopentanoic acid (1.3 g, 1 Eq, 2.9 mmol), DCM (15 mL), EDC (0.73 g, 1.3 Eq, 3.8 mmol), and DIPEA (0.45 g, 0.61 mL, 1.2 Eq, 3.5 mmol). The reaction mixture was stirred at room temperature for -5 minutes as all solid material dissolved. To the reaction mixture was then added ethanolamine (0.21 g, 0.21 mL, 1.2 Eq, 3.5 mmol) in one portion and reaction immediately turned milky white. Stirring was continued at room temperature for 24 hours.The next day, the reaction mixture was concentrated en vacuo. The white solid residue was partitioned between 3:1 CHC13 / IPA (75 mL) and water (acidified with 5 N HC1). The organic layer was then washed with brine, dried (MgSO4), and concentrated and rinsed 2x with DCM and dried to afford a white solid.[000219] LCMS confirmed mass of the desired product: methyl (S)-16-((5-((2- hydroxyethyl)amino)-l -methoxy- l,5-dioxopentan-2-yl)amino)-16-oxohexadecanoate. LCMS m / z = 487.5 (M+l)[000220] The product was used in subsequent reaction step without further purification.[000221] Step 4: A round bottomed flask was charged with methyl (S)-16-((5-((2- hydroxyethyl)amino)-l -methoxy- l,5-dioxopentan-2-yl)amino)-16-oxohexadecanoate (1.3 g, 1 Eq, 2.7 mmol), Chloroform (13 mL), 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (1.6 g, 1.7 mL, 2.0 Eq, 5.3 mmol), and 5-(ethylthio)-lH-tetrazole (0.17 g, 5.3 mL, 0.25 molar, 0.5 Eq, 1 .3 mmol). The reaction mixture was stirred at room temperature. Thin layer chromatography after 1 hr indicated full consumption of starting material. The reaction was concentrated down and loaded via liquid phase for flash chromatography using a Basic Alumina column (160 g column) with 0-100% ethyl acetate / hexane gradient. A strong signal began to elute at -90%. Fractions concentrated to a white solid.[000222] LCMS confirmed the mass of the title product: methyl 16-(((2S)-5-((2-(((2- cyanocthoxy)(diisopropylamino)phosphancyl)oxy)cthyl)amino)-l-mcthoxy-l,5-dioxopcntan-2- yl)amino)-16-oxohexadecanoate. LCMS m / z = 586.4 (M-Diisopropylamine fragmentation)Example 4RR: methyl 16-((2-(((2- cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)ethyl)amino)-16-oxohexadecanoate[000223] A solution of 16-methoxy-16-oxohexadecanoic acid (3.05 g, 10.2 mmol), DCM (50 mL), EDC (2.14 g, 11.2 mmol), HOBt (1.71 g, 11.2 mmol), and DIPEA (1.44 g, 11.2 mmol) was stirred at ambient temperature. 2-aminoethan-l-ol (682 mg, 11.2 mmol) was added resulting in a milky white suspension and stirring continued. After 18 hours, the reaction was concentrated to remove DCM, the white solid was suspended in water (100 mL) and acidified with 5 N HC1 (2 mL). The resultant aqueous suspension was cooled to 0 °C for 15 min (stirred rapidly for 5 min). White solid isolated via suction filtration, washed with 0.2 N HO, water, then dried under vacuum to yield methyl 16-((2-hydroxyethyl)amino)-16-oxohexadecanoate (3.37 g, 97% yield).[000224] A suspension of methyl 16-((2-hydroxyethyl)amino)-16-oxohexadecanoate (3.37 g, 9.81 mmol), 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (3.84 g, 12.8 mmol), and DCM (50 mL) was stirred at ambient temperature and 5-(ethylthio)-lH-tetrazole (0.25 M in MeCN) (19.6 mL, 4.91 mmol) was added in one portion and stirring continued. The reaction turned to a clear, colorless solution after 1 hour. After 2 h, to the reaction was added Celite (~25 g), concentrated to a dry powder, and flashed (160 g basic alumina column) with 10-60% EA / H to give the title compound as a white solid (5.0 g, 94%).NMR (d6-DMSO) 7.83 (t, 1 H), 3.81- 3.67 (m, 2 H), 3.63-3.45 (m, 7 H), 3.27-3.17 (m, 2 H), 2.76 (t, 2 H), 2.29 (t, 2 H), 2.05 (t, 2H), 1.57-1.42 (m, 4 H), 1.31-1.19 (m, 20 H), 1.14 (t, 12 H).31P NMR (d6-DMSO) d 146.8.Example 4SS: N-(3-(((2R,3R,4K,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-4-hydroxytetrahydrofuran-3- yl)oxy)propyl)icosanamide[000225] A solution of l-((2R,3R,4R,5R)-3-(3-aminopropoxy)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (1.00 g, 1.66 mmol), DCM (10 mL), EDC (0.35 g, 1.83 mmol), HOBt (0.28 g, 1.83 mmol), and DIPEA (0.32 mL, 1.83 mmol) was stirred at ambient temperature, icosanoic acid (0.52 g, 1.66 mmol) was added and stirring continued. After 18 hours, the reaction was diluted with DCM (30 mL) and washed with water (30 mL) acidified to pH 4 with acetic acid (95 uL). The organic layer was then washed with saturated NaHCO3, dried (MgSO4), filtered, and concentrated to a white foam to yield methyl the title compound (1.42 g, 95% yield). 'H NMR (d6-DMSO) 11.4 (br s, 1 H), 7.76-7.70 (m, 2 H), 7.42-7.21 (m, 9 H), 6.91 (d, 4 H), 5.79 (d, 1 H), 5.28 (dd, 1 H), 5.21 (d, 1 H), 4.23-4.16 (m, 1 H), 4.00-3.94 (m, 1 H), 3.93-3.88 (m, 1 H), 3.75 (s, 6 H), 3.64-3.54 (m, 2 H), 3.34-3.21 (m, 2 H), 3.20-3.03 (m, 2 H), 2.03 (t, 2 H), 1.69-1.60 (m, 2 H), 1.52-1.41 (m, 2 H), 1.32-1.16 (m, 32 H), 0.85 (t, 3 H).Example 4TT : (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2,4- dioxo-3,4-dihydropyrimidin-l(2H)-yl)-4-(3-icosanamidopropoxy)tetrahydrofuran-3-yl (2- cyanoethyl) diisopropylphosphoramidite £2'-O-icosanamidopropyl Uridine CED phosphoramidite)[000226] A solution of N-(3-(((2R,3R,4R,5R)-5-((bis(4- mcthoxyphcnyl)(phcnyl)mcthoxy)mcthyl)-2-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-4- hydroxytetrahydrofuran-3-yl)oxy)propyl)icosanamide (1.42 g, 1.58 mmol), 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (0.62 g, 2.06 mmol), and DCM (10 mL) was stirred at ambient temperature and 5-(ethylthio)-lH-tetrazole (0.25 M in MeCN) (3.15 mL, 0.79 mmol) was added in one portion and stirring continued. After 2 h, to the reaction was added Celite (~10 g), concentrated to a dry solid, and flashed (48 g basic alumina column) with 20-80% EA / H to give the title compound as a thick, colorless oil (1.58 g, 91%). ’H NMR (d6-DMSO) 11.4 (br s, 1 H), 7.84-7.76 (m, 1 H), 7.71-7.65 (m, 1 H), 7.42-7.21 (m, 9 H), 6.94-6.86 (m, 4 H), 5.83- 5.78 (m, 1 H), 5.30-5.21 (m, 1 H), 4.46-4.31 (m, 1 H), 4.15-3.96 (m, 4 H), 3.84-3.25 (m, 12 H), 3.16-3.02 (m, 2 H), 2.89 (t, 2 H), 2.05-1.98 (m, 2 H), 1.70-1.58 (m, 2 H), 1.50-1.41 (m, 2 H), 1.32- 1.06 (m, 44 H), 0.85 (t, 3 H).31P NMR (d6-DMSO) d 149.1, 148.6.Example 5: General procedure for Cpa / Cys conjugation of FAX to RNA sense strand [000227] In a suitable reaction vessel, 5’ StBu-Cys protected sense strand siRNA material was dissolved in 1 mL of lx PBS and to it was added a 6-molar excess of Ac-Cpa-Lys(AEEA2- yGlu-C20-OH)-NH2 dissolved in 50 / 50 acetonitrile / water (-200-300 uL). The pH of the solution was adjusted to -7.5 with 1 M Tris HC1, pH 8 (-100 uL). About 10 eq of 1 ,4-Dithiothreitol (DTT, Sigma Aldrich) were added to the solution and the mixture was incubated at 40 G for -18 hrs. The reaction was monitored by analytical HPLC to observe the disappearance of StBu-Cy- siRNA and the appearance of the derivatized product sense strand. After the reaction was complete, the solution was diluted to 15 ml with water and purified by RP-HPLC. The molecular weight of the product was confirmed by LC-MS analysis using a Linear Ion Trap Mass Spectrometer (LTQ XL, Thermo Fisher Scientific) equipped with a Vanquish HPLC system (Thermo Fisher Scientific).Example 6: General procedure for acylation of RNA Sense Strand 3’ or 5’ reactive amino terminus or interior reactive amino prior to peptide conjugation, with: DBCO-NHS, SPDP- NHS, MSPT-PEG2-NHS, MSPOD-PEG2-NHS[000228] In a suitable reaction vessel, the required reactive amino siRNA strand (typically 30-40 mg) was dissolved in 200-400 uL of PBS Buffer [pH 7] and 600-800 uL of DMSO alongwith 50-80 uL of DTPEA. The desired active ester (pre-dissolved in a minimum amount of DMSO) was then added in small increments, and the reaction was monitored accordingly by analytical HPLC. After the reaction was complete, the solution was diluted to 15 ml with water and purified by RP-HPLC. The molecular weight of the product was confirmed by LC-MS analysis using a Linear Ion Trap Mass Spectrometer (LTQ XL, Thermo Fisher Scientific) equipped with a Vanquish HPLC system (Thermo Fisher Scientific).Alternate procedure for acylation ofRNA Sense Strand 3 ’ or 5’ reactive amino terminus or interior nucleotide reactive amino prior to peptide conjugation, with: SMCC-NHS. Chloroacetoxy NHS or C20-Diacid-ME NHS Ester[000229] A stock solution of NHS Ester was prepared in either a minimal concentration of acetonitrile or DMSO depending on solubility. A stock solution of sodium bicarbonate was dissolved in water at 20mg / mL. In a suitable reaction vessel, the required reactive amino oligonucleotide strand (typically 30-40 mg) was solubilized with 15-30 molar equivalents of the stock solution of sodium bicarbonate. Then, 10-15 equivalents of reactive NHS ester solution was introduced to the reaction vessel with shaking at room temperature. Optionally, extra organic component of either DMSO or acetonitrile may be added to improve NHS ester solubility, typically between a 1:2 to 2:1 ratio of aqueous to organic. The reaction was monitored by LTQ-MS until the starting oligonucleotide was fully consumed. For reaction with chloroacetoxy NHS Ester, the reaction directly was diluted with water to >10% organics and subjected to 3KMWC0 spin filtration with multiple washes to remove excess reagent and side products and no further purification was employed.[000230] For reaction with SMCC-NHS Ester, the reaction was quenched to pH 5 with IN HC1 and diluted to >10% organics. The reaction mixture was filtered through a 0.2 micron filter to remove insoluble SMCC-NHS ester byproducts, then subjected to 3KMWC0 spin filtration with multiple washes to remove excess reagent and side products and no further purification was employed. The molecular weight of the product was confirmed by LC-MS analysis using a Linear Ion Trap Mass Spectrometer (LTQ XL, Thermo Fisher), and purity was typically confirmed by IP-RP UPLC. The product was quickly frozen at -20 °C or below to prevent maleimide hydrolysis and lyophilized.[000231] For reaction with C20-Diacid-ME NHS Ester, the methyl esters were hydrolyzed with the addition of 100 equivalents of LiOH (2M Li OH solution) and shaken at room temperature for 30 minutes prior to dilution to >5% organics and spin filtration (3KMWC0). The crude functionalized oligonucleotide product was loaded onto an ES Industry Source™ 15RPC with MPA: lOmM NaOAc 2% Acetonitrile and MPB: 80% Acetonitrile in water. Fractions were analyzed by IP-RP LCMS and those which contained a mass purity greater than 85% without impurities >5% were combined. The molecular weight of the product was confirmed by LC-MS analysis using a Linear- Ion Trap Mass Spectrometer (LTQ XL, Thermo Fisher), and purity was confirmed by IP-RP UPLC analysis.Example 7: General procedure for conjugation of functionalized RNA sense strands to functionalized peptides[000232] For reactive dithiol-containing siRNA strands such as those derived from SPDP- NHS or C6S-SC6 moieties, any remaining dithio bonds were reduced with a reagent such as excess (tris(2-carboxyethyl)phosphine)-HCl (TCEP) prior to conjugation. The siRNA then purified from excess reducing agent using ultrafiltration (3KMWC0 filter) or optionally chromatographic purification using similar methods as in Examples 4 and 10.[000233] The conjugation of the reactive peptides and sense strands were typically done with 20-25mg of the sense strand siRNA and a 1.5 to 2x excess of the peptide. The siRNA was dissolved in -1.5 mL of Ultra-pure DNAse / RNAse free water, the peptide was then added as a solid. The pH of the solution was raised to -7.5 with 1 M Tris HC1, pH 8 (-20-30 uL) The solution was incubated at 30 °C for -1.5 hrs, additional peptide was added as needed. Reaction was monitored by analytical HPLC to observe the disappearance of siRNA and the appearance of the conjugate. After the reaction was complete, the solution was diluted to 15 ml with water and purified by RP-HPLC. The molecular weight of the product was confirmed by LC-MS analysis using a Linear Ion Trap Mass Spectrometer (LTQ XL, Thermo Fisher).Example 8: General procedure for maleimide ring-opening of SMCC- and MalDap-linked Peptide-RNA sense strand conjugates[000234] For SMCC-linked conjugates: Following completion of the SMCC-thiol conjugation reaction, the pH of the reaction mixture was raised to -9 with IM SodiumBicarbonate buffer and incubated at RT. The ring opening reaction was monitored by LC-MS to observe the addition of 18 Daltons. Incubation times vary from a few hours to overnight, to reach complete ring opening.[000235] For MalDap-linked conjugates: Ring-opening can occur immediately in solution following coupling with no additional synthetic manipulation. To confirm that the ring opening is complete prior to purification, the pH of the solution was raised to ~8 with IM Tris HC1 and incubated at RT for 10-20 minutes.Example 9: General procedure for HPLC analysis of Peptide-RNA Sense Strand Conjugates[000236] Analytical HPLC was used to monitor the conjugation reaction of the peptide and the sense strand siRNA and to check the final purity of the purified Peptide-siRNA conjugates. Analysis was done on an analytical HPLC system (Agilent 1100) using an XBridge Protein BEH C4 analytical RP-HPLC column (Waters; 3.5pm, 300A; 4.6 x 100 mm). The running buffers used were A: 8.6 mM TEA, with 100 mM HFIP pH 8.3 and B: McOH / Acetonitrile (50 / 50 v / v, Fisher Chemicals). The gradient used was a linear 0-85 % B gradient over 15 min, at a flow of 1.5 mL / min, with column heating set at 60 °C. UV monitoring was done at 254 nm. The molecular weight of the product was confirmed by LC-MS analysis using a Linear' Ion Trap Mass Spectrometer (LTQ XL, Thermo Fisher Scientific) equipped with a Vanquish HPLC system (Thermo Fisher Scientific).Example 10: General procedure for purification of Peptide-RNA Sense Strand conjugates by RP-HPLC[000237] Solutions containing the peptide-siRNA conjugates were purified using a preparative HPLC system (Shimadzu LC-8A Binary Preparative HPLC Systems) using an XBridge Protein BEH C4 semi-preparative RP-HPLC column (Waters; 5pm, 300A; 10 x 250 mm). The running buffers used were A: 8.6 mM TEA, with 100 mM HFIP pH 8.3 and B: MeOH / Acetonitrile (50 / 50 v / v, Fisher Chemicals). The gradient used was a linear 0-90 % B gradient over 25 min, at a flow of 25 mL / min, with column heating set at 60 °C. UV monitoring was done at 254 and 220 nm. Fractions containing the desired product (HPLC analysis by Agilent HPLC) were pooled, frozen, and lyophilized to give a white amorphous solid product.The molecular weight of the product was confirmed by LC-MS analysis using a Linear Ion Trap Mass Spectrometer (LTQ XL, Thermo Fisher Scientific) equipped with a Vanquish HPLC system (Thermo Fisher Scientific).Example 11: General procedure for annealing Peptide-RNA Sense Strand conjugates to form duplexed dsRNA-Peptide Conjugates[000238] In a suitable vessel, the lyophilized ssRNA-Peptide was dissolved in 2-3 mL of RNase free water and mixed thoroughly by vortexing. The concentration of the ssRNA peptide conjugate was then determined by using a NanoPhotometer NP80 (Implen) measuring optical density at 260nm (OD260). Example calculation shown below:Where c = extinction coefficient of the ssRNA (Peptide contribution is negligible) mM = millimolar concentration (mmol / L) vol l ~ mmoles[000239] The concentration of a corresponding solution of anti-sense (asRNA), was also measured by OD260 using the same method. To anneal the strands, a molar excess of 1.5% asRNA was used with the annealing calculations shown below: mmoles ssRNA-Peptide * 1.015 x Equivalence of asRNA = mmoles asRNA required[000240] The volume of the asRNA solution required was then calculated. The required volume of the asRNA solution was combined with the ssRNA-Peptide solution. To anneal the strands, the mixture was incubated at 65°C for lOmin. using a thermomixer (Eppendorf thermomixer C), and then slowly cooled to 22 °C over 40min.[0002 1] The duplexed product was then transferred to a 100,000MWL centrifugation filter tube (Millipore) and spun at 7400 rpm for 15min. to remove any potential endotoxins. To desalt the PRC, the filtrate was then transferred into an AmiconUltra 3,000MWL centrifugation filter tube (Millipore) at 7400 rpm for 7.5min., washed twice with 2mL water each, and then twice with 2mL of IxPBS. Following endotoxin removal, water washes, and buffer exchange; theconcentrated duplex solution was then transferred to a cryogenic storage vial (Coming) of the appropriate volume. The final concentration was then determined using the NanoPhotometer with OD260 measurements. The molecular’ weight of the product was confirmed by LC-MS analysis using a Linear Ion Trap Mass Spectrometer (LTQ XL, Thermo Fisher Scientific) equipped with a Vanquish HPLC system (Thermo Fisher Scientific).Table 20: Characterization of synthesized NPR-C-binding peptide-dsRNA conjugates by mass spectrometryExample 12: In Vitro Binding of Peptide-dsRNA Conjugates to NPR-CTable 21: Summary of Reagents, Media, Reference Compounds, Supplies, and Assay Kits Used in the MethodsCell Culture[000242] Jump-in™ T-Rex™ HEK293 cells were maintained in DMEM with 4.5 g / L D- glucose culture medium supplemented with 10% FBS-HI, 20 mM HEPES, 2x GlutaMAX, lx Na pyruvate, lx P / S, 200 pg / mL hygromycin, and 5 pg / mL blasticidin in a cell culture incubator (ThermoFisher Scientific, Waltham, MA) at 37 °C in 5% CO2 and were routinely subcultured twice weekly with 0.05% trypsin-EDTA treatment.Full-Length cDNA Cloning and Generation of Cell Lines Overexpressing Natriuretic Peptide Receptors (NPRs)[000243] Vendors for the target cDNAs used to generate the NPR cell lines are listed in Table . All sequences of the cDNAs were verified by full-length sequencing performed by ACGT DNA Sequencing Services (Wheeling, IL). The target cDNA was cloned into pJTI R4 CMV-TO MCS pA vector and then co-transfected with pJTlR4 Int vector into Jump-in™ T- Rex™ HEK293 cells for mammalian inducible expression using Jump-in™ T-Rex™ HEK293 kit and Lipofectamine LTX and Plus Reagent following the manufacturer’s directions.[000244] NPRs were overexpressed in T-Rex™ HEK293 cells following the induction with 300 ng / mL tetracycline in culture medium for 48 hours. Induced cell lines in an exponential growth phase were treated with 0.05% trypsin-EDTA for a few seconds at room temperature, harvested in cell medium containing FBS to neutralize the trypsin, counted, and cryopreserved at the density of 2 million cells / mL in cell preservation solution containing FBS-HI with 5% DMSO. Cryopreserved cells were stored at -80°C for a few days prior to transferring to a liquid nitrogen tank. Induced cell lines were then used for the preparation of cell membranes to measure the binding affinity of compounds in radioligand competition binding assays, as described below.Table 22: Summary of cDNAs Used to Overexpress NPRs in T-Rex™ HEK293 CellsCell Plasma Membrane Vesicle Preparation (Cell membranes)[000245] Cell membranes were prepared from induced T-Rex™ HEK293 cells that overexpress human NPR-C as described below.[000246] Ten million T-Rex™ HEK293 cells overexpressing NPR were seeded in a Corning® cell culture flask with 225 cm2surface area in culture medium containing 300 ng / mL tetracycline and cultured for 48 h at 37°C and 5% CO2to induce the overexpression of the NPR. Cells were treated with 0.05% trypsin-EDTA for a few seconds at room temperature, harvested in culture medium containing FBS to neutralize the trypsin, and centrifuged in a Beckman GPR centrifuge (Beckman Coulter Life Sciences, Brea, CA) at 200 x g (800 rpm) for 10 minutes at room temperature. Cell pellet was weighed and stored at -80°C until it was used. Each gram of frozen cell pellet was resuspended in 10 mL of pre-cooled homogenization buffer containing 50 mM Tris-HCl, pH 7.5, and 2x protease inhibitor cocktail tablets. The cell suspension was poured into a 55 mL Wheaton glass Teflon head homogenizer and placed on ice. Cells were homogenized for 20 strokes using a Palmgren 10” Drill Press (Palmgren, Naperville, IL) at a speed set at 37. The homogenate was centrifuged in an Eppendorf Centrifuge 581 OR (Eppendorf SE, Hamburg, Germany) for 10 minutes at 1000 x g and 4°C. The supernatant was collected and the pellet was homogenized again as described above. The supernatants from the two runs of homogenization were combined and centrifuged in a Beckman Coulter Avanti J-E Centrifuge (Beckman Coulter Life Sciences, Brea, CA) using a JA-20 rotor for 1 hour at 35,000 x g and 4°C. The pellet was then resuspended in a half volume of above homogenization buffer and homogenized again as described above. The protein concentration in each membrane suspension was measured using a BCA Protein Assay kit. The membranes were aliquoted, flash frozen in liquid nitrogen, and then stored at -80°C.Assay Method: Human Natriuretic Peptide Receptor C (NPR-C) Competition Binding Assays Using125I-[Tyr ]-Human-CNP-22 as a Radioligand Probe:[000247] A SPA ligand binding assay was performed using 10 concentrations of competing test compounds (10 pM to 0.01 pM), as described below.[000248] Test compounds dissolved in DMSO (2 mM, stock solution) were first diluted 50- fold in assay buffer containing HBSS with Ca2+and Mg2+, 5 mM HEPES, 0.1% NP40, and 0.1% BSA or no BSA (pH 7.4). The 4x working stock solution was then serially diluted 1:10 in assay buffer containing BSA or no BSA to generate 10-point concentrations ranging from 40 pM to 0.04 pM.[000249] A 25 pL volume of assay buffer (normalized as 100% binding), human CNP (400 nM, normalized as 0% binding), or 4x working stock solutions of test compounds were transferred to a Costar® 3632 plate. Then, 25 pL of125I-[Tyr°]-human-CNP-22 (0.2 nM), 25 pL of cell membrane suspension (0.04 mg / mL), and 25 pL of WGA coated PVT SPA beads (4 mg / mL) were sequentially added to the plate. The final reaction volume of each well was 100 pL. The plate was sealed and shaken on a plate shaker for 1 hour at room temperature. The plate was then placed in a PerkinElmer 2450 Microplate Counter Microbeta2® and incubated for 16 hours at room temperature for beads to settle. At the end of this period, radioactivity associated with SPA beads was recorded in CPM for 2 minutes.Data Analysis[000250] The CPM were normalized to counts obtained in the presence of assay buffer as 100% binding and counts obtained in the presence of 100 nM human CNP as 0% binding. A 10- point concentration-response curve (10 pM to 0.01 pM) for each test compound was fitted to a 4- parameter model using Prism 9 (GraphPad Software, Inc., San Diego, CA) to determine the binding affinity (IC50 values). IC50 is the concentration of competing ligand which displaces 50% of the specific binding of the radioligand,125I-[Tyr°]-human-CNP-22. Data are shown in Table 23.Table 23: Binding affinity to NPR-C / 3Example 13: ANP HPRT siRNA conjugate selectively knocks down HPRT in adipose tissue [000251] On Study Day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the HPRT gene (100 nmol / kg). On day 14 post dosing, animals were sacrificed and the following tissues collected: inguinal white adipose tissue (iWAT), gonadal white adipose tissue (gWAT), interscapular brown adipose tissue (iBAT), perirenal white adipose tissue, gastrocnemius, heart, kidney and liver. Expression of HPRT was determined using qPCR, with RPLP0 used as the endogenous control gene. Average HPRT expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 24 below and demonstrate preferential knockdown in adipose tissue with the peptide siRNA conjugate.Table 24. ANP HPRT siRNA conjugate selectively knocks down HPRT in adipose tissue[000252] To understand the potential for nonselective delivery of an unconjugated siRNA to adipose tissue, in vivo testing was performed under similar- conditions to the peptide conjugated siRNA using an unconjugated RNAi designed to inhibit HPRT. On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or the unconjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBSor the unconjugated HPRT siRNA (250 nmol / kg). On day 14 post dosing, animals were sacrificed and the following tissues collected: inguinal white adipose tissue (iWAT), gonadal white adipose tissue (gWAT), interscapular brown adipose tissue (iBAT), perirenal white adipose tissue, gastrocnemius, heart, kidney and liver. Expression of HPRT was determined using qPCR, with RPLPO used as the endogenous control gene. Average HPRT expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 25 below and show lack of robust knockdown in any of the tissues tested.Table 25. Knock down of HPRT expression by unconjugated siRNAExample 14: Comparison of activity of conjugate in cells expressing NPR-C versus NPR-ATo demonstrate the activity of a peptide-RNAi conjugate mediated by NPR-C receptor, in vitro study was performed using human NPR-A or NPR-C expressing HEK cell. On study day 0, RNAi or peptide-RNAi conjugates were treated to the cell designed to inhibit the expression of the HPRT gene (0, 100, 1000 nM). On 72 hr post treatment, cells were collected and expression of HRPT was determined using qPCR, with P actin used as the endogenous control gene.Average HPRT expression for each treatment was normalized relative to the vehicle group (0 nM). Results are shown average from 2 independent experiments in Table 26 below and show NPR-C receptor dependent reductions in the expression of HPRT gene.Table 26. NPR-C receptor dependent knock down of HPRT expression by the conjugates.Example 15: ANP-SOD1 siRNA conjugate selectively knocks down SOD1 in adipose tissue [000253] To demonstrate activity against a second gene and understand the potency of a peptide-RNAi conjugate, a dose response study was performed using a peptide-conjugated to an siRNA designed to inhibit the expression of the SOD1 gene. On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide- conjugated RNAi formulated in PBS. Five (n-5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the SOD1 gene (12.5, 25, 50, 100, or 250 nmol / kg). On day 14 post dosing, animals were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of SOD1 was determined using qPCR, with RPLP0 used as the endogenous control gene. Average SOD1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 27 below and show dose dependent reductions in the expression of SOD1 in both adipose depots.Table 27. Dose dependent knock down of SOD1 expression by ANP-SOD1 siRNA conjugateExample 16: Knock down of target gene by various NPR-C binding peptide-HPRT siRNA conjugates[000254] On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the HPRT gene (100 nmol / kg). On day 14 post dosing, animals were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of HPRT was determined using qPCR, with RPLP0 used as the endogenous control gene. Average HPRT expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 28 below.Table 28. Knock down of HPRT by various NPR-C binding peptide-HPRT siRNA conjugates[000255] On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the HPRT gene (25 or 100 nmol / kg). On day 14 post dosing, animals were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT)collected. Expression of HPRT was determined using qPCR, with RPLPO used as the endogenous control gene. Average HPRT expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 29 below and show stabilized, linear NPR-C binding peptides achieve knockdown of HPRT expression in adipose tissue.Table 29. Knock down of HPRT by linear NPR-C binding peptide-HPRT siRNA conjugatesExample 17: NPR-C binding peptide-oligonucleotide conjugates knock down SOD1 in adipose tissue[000256] On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the SOD1 gene (100 nmol / kg). On day 14 post dosing, animals were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of SOD1 was determined using qPCR, with RPLPO used as the endogenous controlgene. Average S0D1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results arc shown in Table 30 below.Table 30. NPR-C binding peptide-oligonucleotide conjugates knock down SOD1 in adipose tissueEXAMPLE 18: Diacid modified NPR-C binding peptide-siRNA conjugates[000257] On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the HPRT gene (25, 50, 100, 250 or 500 nmol / kg). On day 14 post dosing, animals were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of HPRT was determined using qPCR, with RPLP0 used as the endogenous control gene. Average HPRT expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 31 below and demonstrate dose dependent knockdown of HPRT expression in adipose tissue with a C20 Diacid-modified NPR-C binding peptide conjugated siRNA.Table 31. Knock down of HPRT by C20 Diacid modified NPR-C binding peptide-siRNA conjugates[000258] An in vivo study was run to evaluate the effect of location of peptide conjugation and / or the incorporation of lipidation on activity of peptide-RNAi conjugates in adipose tissue. On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the SOD1 gene (25 or 250 nmol / kg). On day 14 post dosing, animals were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of SOD1 was determined using qPCR, with RPLP0 used as the endogenous control gene. Average SOD1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 32 below and demonstrate dose dependent reductions in SOD1 expression and highlight the ability to change the peptide location and incorporate lipidation while maintaining a similar level of knockdown in adipose tissue.Table 32. Knockdown of HPRT by lipid modified NPR-C binding peptide-siRNA conjugatesExample 19: Conjugates with different conjugation sites achieve similar knockdown[000259] On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the SOD1 gene (25 nmol / kg). On day 14 post dosing, animals were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of SOD1 was determined using qPCR, with RPLPO used as the endogenous control gene. Average SOD1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 33 below and demonstrate the ability to conjugate the peptide to several locations on the sense strand without changing activity of the molecule in adipose tissue.Table 33. Conjugates with different conjugation sites achieve similar knockdownExample 20: Comparison of peptide-siRNA conjugates to conjugates comprising lipid, peptide and siRNA[000260] An in vivo study was conducted to look at knockdown of SOD1 expression in adipose tissue when an siRNA to SOD1 is delivered using a peptide or a combination of peptide and lipid. On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the SOD1 gene (12.5, 25, 50, 100 or 250 nmol / kg). On day 14 post dosing, animals were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of SOD1 was determined using qPCR, with RPLP0 used as the endogenous control gene. Average SOD1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Tables 34A and 34B below and demonstrate greater gene knockdown at lower doses with conjugates comprising both the NPR_C binding peptide and the lipid.Table 34A: SOD1 knockdown by peptide-siRNA conjugates and conjugates comprising lipid, peptide and siRNA in Inguinal White Adipose TissueTable 34B: SOD1 knockdown by peptide-siRNA conjugates and conjugates comprising lipid, peptide and siRNA in Gonadal White Adipose TissueExample 21: Conjugates with different linkers[000261] On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of cither PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the SOD1 gene (25 nmol / kg). On day 14 post dosing, animals were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of SOD1 was determined using qPCR, with RPLPO used as the endogenous control gene. Average SOD1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 35 below.Table 35. SOD1 knockdown by conjugates with different linkersExample 22: Durability[000262] On Study day 0, female C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Animals were dosed in each group with PBS (N=6) or a peptide-conjugated RNAi (N=7) designed to inhibit the expression of the HPRT gene (25 or 254 nmol / kg). On 4 months post dosing, animals were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of HPRT was determined using qPCR, with 0 actin used as the endogenous control gene. Average HPRT expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 36 below.Table 36. Durable knockdown of HPRT by NPR-C binding peptide-siRNA conjugates[000263] Durability of gene knockdown in white adipose tissue was tested when an siRNA to SOD1 was delivered using a peptide or a combination of peptide and lipid. On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or an siRNA to SOD1 is delivered using a peptide or a combination of peptide and lipid formulated in PBS (100 nmol / kg). On days 28, 56 and 84 post dosing, animals (n=5 per group) were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of SOD1 was determined using qPCR, with RPLP0 used as the endogenous control gene. Average SOD1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Tables 37A and 37B below and demonstrate persistent reduction of SOD1 mRNA levels across the 84-day treatment period.Table 37A. Persistent reduction of SOD1 mRNA in Gonadal white adipose tissueTable 37B. Persistent reduction of SOD1 mRNA in Inguinal white adipose tissueExample 23: Constructs with various linker lengths[000264] On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the SOD1 gene (25 nmol / kg). On day 14 post dosing, animals were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of SOD1 was determined using qPCR, with RPLPO used as the endogenous control gene. Average SOD1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 38 below.Table 38. SOD1 knockdown by conjugates with different linker lengthsExample 24: Constructs with various linker lengths across different peptides[000265] On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibitthe expression of the SOD1 gene (25 nmol / kg). On day 14 post dosing, animals were sacrificed and inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of SOD1 was determined using qPCR, with RPLPO used as the endogenous control gene. Average SOD1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 39 below.Table 39. SOD1 knockdown by conjugates with different linker lengths and different peptidesExample 25: Knockdown of SOD1 by various NPR-C binding peptides conjugated to SOD1 siRNA with or without different lipids[000266] On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the SOD1 gene (100 nmol / kg). On day 14 post dosing, animals were sacrificed and the following tissues collected: inguinal white adipose tissue (iWAT), gonadal white adiposetissue (gWAT), gastrocnemius (Gast), heart, kidney and liver. Expression of SOD1 was determined using qPCR, with RPLPO used as the endogenous control gene. Average SOD1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 40 below.Table 40: Knockdown of SOD1 using NPR-C binding peptides conjugated to SOD1 siRNA with and without lipidsExample 26: Durability of knockdown of SOD1 by various NPR-C binding peptides conjugated to SOD1 siRNA with or without different lipids.[000267] On Study day 0, male C57BL / 6 mice received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the SOD1 gene (100 nmol / kg). On day 84 post dosing, animals were sacrificedand inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) collected. Expression of SOD1 was determined using qPCR, with RPLPO used as the endogenous control gene. Average SOD1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 41 below.Table 41: Durability of knockdown by NPR-C binding peptides conjugated to SOD1 siRNA with or without lipidExample 27: Knockdown of SOD1 by various NPR-C binding peptides conjugated to SOD1 siRNA with or without lipid in Sprague-Dawley rats[000268] On Study day 0, male Male Sprague Dawley Rats received a subcutaneous injection (dose volume 1 mL / kg) of either PBS or a peptide-conjugated RNAi formulated in PBS. Twelve (n=12) animals were dosed in each group with PBS or a peptide-conjugated RNAi designed to inhibit the expression of the SOD1 gene (100 nmol / kg). On days 14, 28 and 56 post dosing, four animals per group were sacrificed and the following tissues collected: inguinal white adipose tissue (iWAT), gonadal white adipose tissue (gWAT), liver, heart, kidney and gastrocnemius (Gast). Expression of SOD1 was determined using qPCR, with RPLPO used as the endogenous control gene. Average SOD1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 42 below.Table 42: Knockdown of SOD1 by NPR-C binding peptides conjugated to siRNA with or without lipid in vivoExample 28: Lipid modified NPR-C binding peptide-siRNA conjugates[000269] On Study Day 0, C57BL / 6 diet-induced obese (DIO) male mice (approx. 20 weeks old) received a subcutaneous injection (dose volume 5 mL / kg) of either PBS or a peptide- conjugated RNAi formulated in PBS. Five (n=5) animals were dosed in each group with PBS or a lipid modified NPR-C binding peptide-siRNA conjugate designed to inhibit the expression of the SOD1 gene (100 nmol / kg). On day 14 post dosing, animals were sacrificed and the followingtissues collected: inguinal white adipose tissue (iWAT), gonadal white adipose tissue (gWAT), gastrocnemius, heart, and liver. Expression of SOD1 was determined using qPCR, with RPLPO used as the endogenous control gene. Average SOD1 expression for each tissue was normalized relative to the vehicle control group (PBS). Results are shown in Table 43 below and demonstrate preferential knockdown in adipose tissue with the peptide siRNA conjugate.Table 43. Tissue Knockdown with diacid-modified NPR-C binding peptide-siRNA conjugatesSEQUENCE LISTING

Claims

1. CLAIMS1. A conjugate comprising Formula:O-(L-P)n, wherein O comprises an oligonucleotide; wherein L is a linker or a bond; wherein P is an NPR-C-binding peptide comprising SEQ ID NO: 1 (GX11IDX14I), wherein Xu is arginine, proline, or hydroxyproline, and X14 is arginine or N- methylarginine; and wherein n is an integer of 1 to 4.

2. The conjugate of claim 1, wherein Xu is R.

3. The conjugate of claim 1 or claim 2, wherein X14 is R.

4. The conjugate of any one of claims 1-3, wherein P comprises SEQ ID NO: 2 (GR1DR1).

5. The conjugate of any one of claims 1-3, wherein P comprises SEQ ID NO: 3 (SX7X8X9GX11IDX14I), wherein:X7 is glycine, alanine, proline, hydroxyproline, serine, or cysteine;Xs is phenylalanine, or cyclohexylalanine, andX9 is glycine, alanine, or serine.

6. The conjugate of claim 5, wherein X7 is C.

7. The conjugate of claim 5 or claim 6, wherein X9 is G.

8. The conjugate of any one of claims 1-7, wherein P comprises SEQ ID NO: 4 (SCFGGRIDRI).

9. The conjugate of any one of claims 1-4, wherein P comprises SEQ ID NO: 5 (FGGRIDRIGA).

10. The conjugate of any one of claims 1-4, wherein P comprises SEQ ID NO: 6 (RSSX7FGGRIDRI), wherein X7 is serine or cysteine.

11. The conjugate of claim 10, wherein P comprises SEQ ID NO: 7 (RSSX7FGGRIDRIGA).

12. The conjugate of claim 10 or claim 11, wherein X7 is cysteine.

13. The conjugate of claim 10 or claim 11, wherein X7 is serine.

14. The conjugate of claim 1, wherein P comprises SEQ ID NO: 8 (X7-Cha-X9GXnIDXi4l), wherein:X7 is proline, hydroxyproline, glycine, cysteine, or alanine;X9 is alanine, serine, or glycine;Xi 1 is proline, hydroxyproline, or arginine; and X14 is arginine or N-methylarginine.

15. The conjugate of claim 14, wherein P comprises SEQ ID NO: 9 (fSp-Cha-aGPIDRI).

16. The conjugate of claim 1, wherein P comprises a sequence selected from any one of SEQ ID NOs: 11-57.

17. The conjugate of any one of claims 1-16, wherein P is a linear peptide.

18. The conjugate of any one of claims 1-16, wherein P is a cyclic peptide.

19. The conjugate of claim 18, wherein P is cyclized by covalent attachment of a side chain of a first cysteine residue to a side chain of a second cysteine residue.

20. The conjugate of claim 19, wherein the covalent attachment comprises a disulfide bond.

21. The conjugate of claim 19, wherein the covalent attachment comprises a thioacetal moiety.

22. The conjugate of any one of claims 1-18, wherein P comprises a C-terminal hydroxyl.

23. The conjugate of any one of claims 1-18, wherein P comprises a C-terminal amide.

24. The conjugate of any one of claims 1-23, wherein L comprises a linker core and one or more spacers.

25. The conjugate of any one of claims 1-24, wherein L comprises Spacer 1 -Linker Core- Spaced.

26. The conjugate of claim 24 or claim 25, wherein the Linker Core is selected from Table 9.

27. The conjugate of claim 25 or claim 26, wherein the Spacer 1 and Spacer 2 are selected from Table 10.

28. The conjugate of any one of claims 1-27, wherein L is selected from Table 11.

29. The conjugate of any one of claims 1-28, wherein n is 1 or 2.

30. The conjugate of any one of claims 1-29, wherein O is an antisense oligonucleotide, a double stranded RNA (dsRNA), or a guide RNA.

31. The conjugate of any one of claims 1-30, wherein O is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand.

32. The conjugate of claim 31 , wherein at least one nucleotide of the sense strand is a modified nucleotide.

33. The conjugate of claim 31 or 32, wherein at least one nucleotide of the antisense strand is a modified nucleotide.

34. The conjugate of any one of claims 31-33, wherein at least one intemucleotide linkage of the sense strand is a modified internucleotide linkage.

35. The conjugate of any one of claims 31-34, wherein at least one intemucleotide linkage of the antisense strand is a modified internucleotide linkage.

36. The conjugate of claim 34 or claim 35, wherein the modified intemucleotide linkage is a phosphorothioate linkage.

37. The conjugate of any one of claims 31-36, wherein the dsRNA comprises an inverted abasic moiety.

38. The conjugate of claim 37, wherein the sense strand comprises the inverted abasic moiety.

39. The conjugate of claim 37 or claim 38, wherein the inverted abasic moiety is at the 5’ end of the sense strand of the dsRNA.

40. The conjugate of any one of claims 37-39, wherein the inverted abasic moiety is covalently attached to the sense strand.

41. The conjugate of any one of claims 31-40, wherein the dsRNA comprises a vinylpho sphonate moiety.

42. The conjugate of claim 41, wherein the vinylphosphonatc moiety is at the 5’ end of the antisense strand of the dsRNA.

43. The conjugate of any one of claims 31 -42, wherein L is attached to the 5’ end of the sense strand and to the N-tcrminal end of the peptide.

44. The conjugate of any one of claims 31-42, wherein L is attached to the 5’ end of the sense strand and to the C-terminal end of the peptide.

45. The conjugate of any one of claims 31-42, wherein L is attached to the 3’ end of the sense strand and to the N-terminal end of the peptide.

46. The conjugate of any one of claims 31-42, wherein L is attached to the 3’ end of the sense strand and to the C-terminal end of the peptide.

47. The conjugate of any one of claims 31-46, wherein L comprises the formula:

48. The conjugate of any one of claims 1-47, wherein the conjugate further comprises a fatty acid (FA).

49. The conjugate of claim 48, wherein FA is attached to O.

50. The conjugate of claim 49, wherein FA is attached to P.

51. The conjugate of any one of claims 48-50, wherein the conjugate comprises (FA)m-O-L- P or 0-L-P-(FA)m, wherein m is an integer of 1 to 4.

52. The conjugate of any one of claims 48-51, wherein the conjugate further comprises a Spacer3.

53. The conjugate of claim 52, wherein the conjugate comprises (FA-Spacer3)m-0-L-P or O- L-P-(Spaccr3-FA)m, wherein m is an integer of 1 to 4.

54. The conjugate of claim 52, wherein the conjugate comprises 0-L-(FA-Spacer3)m-P or O- -(Spacer3-FA)m-L-P, wherein m is an integer of 1 to 4.

55. The conjugate of any one of claims 48-54, wherein the fatty acid is selected from the group consisting of FA1-FA27 of Table 12.

56. The conjugate of any one of claims 51, 53 and 54, wherein m is 1 or 2.

57. A conjugate comprising Formula:O-L-P, wherein O comprises a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand; wherein L is a linker comprising the formula:wherein P comprises SEQ ID NO: 90.

58. The conjugate of claim 57, wherein the linker L is attached to a 3’ end of the sense strand.

59. The conjugate of claim 57 or claim 58, wherein the linker is of formula:wherein X represents a position to which the 3’ end of the sense strand of O is conjugated, and wherein Y represents a position to which an N-terminal end of P is conjugated.

60. The conjugate of any one of claims 57-59, wherein the antisense strand is modified with 2’-fluoro at each of positions 2, 5, 7, 14, and 16.

61. The conjugate of claim 60, wherein all other positions of the antisense strand are modified with 2’-0Me.

62. The conjugate of any one of claims 57-61, wherein the antisense strand comprises phosphorothioatc linkages between positions 1 and 2, 2 and 3, 20 and 21, 21 and 22, and 22 and 23.

63. The conjugate of any one of claims 57-62, wherein the antisense comprises a vinylpho sphonate moiety conjugated to the 5’ end.

64. The conjugate of any one of claims 57-63, wherein the sense strand is modified with 2’- fluoro at each of positions 9, 10, and 11.

65. The conjugate of claim 64, wherein all other positions of the sense strand are modified with 2’-0Me.

66. The conjugate of any one of claims 57-65, wherein the sense strand comprises phosphorothioatc linkages between positions 1 and 2, 19 and 20, and 20 and 21.

67. The conjugate of claim 66, wherein the sense strand comprises the inverted abasic moiety conjugated at the 5’ end, wherein the inverted abasic moiety is conjugated to the sense strand by a phosphorothioatc linkage.

68. A pharmaceutical composition comprising a conjugate of any one of claims 1-67, and a pharmaceutically acceptable carrier.

69. A method of treating a disease or condition of adipose tissue in a patient in need thereof, comprising administering to the patient an effective amount of the conjugate of any one of claims 1-67 or the pharmaceutical composition of claim 68.

70. The method of claim 69, wherein the disease or condition is obesity or obesity-related comorbidity.

71. The method of claim 69 or 70, wherein the conjugate or pharmaceutical composition is administered intravenously or subcutaneously.

72. The method of any one of claims 69-71, wherein the conjugate or pharmaceutical composition is administered a simultaneous, sequential, or separate combination with an incretin.

73. The method of any one of claims 69-71, wherein the conjugate or pharmaceutical composition is administered to a patient who has been treated with an incretin.

74. The conjugate of any one of claims 1-67, or the pharmaceutical composition of claim 68, for use in a therapy.

75. The conjugate of any one of claims 1-67, or the pharmaceutical composition of claim 68, for use in the treatment of a disease or condition of adipose tissue.

76. The conjugate or pharmaceutical composition for use of claim 75, wherein the disease or condition is obesity or obesity -related comorbidity.

77. The conjugate or pharmaceutical composition for use of claim 75 or claim 76, which is administered in a simultaneous, sequential, or separate combination with an incretin.

78. Use of the conjugate of any one of claims 1-67 or the pharmaceutical composition of claim 68, in the manufacture of a medicament for treating a disease or condition of adipose tissue.

79. The use of claim 78, wherein the disease or condition is obesity or obesity-related comorbidity.

80. The use of claim 78 or claim 79, wherein the conjugate or pharmaceutical composition is administered in a simultaneous, sequential, or separate combination with an incretin.

81. A method of delivering an oligonucleotide to an adipose tissue, comprising administering to a subject the conjugate of any one of claims 1-67, or the pharmaceutical composition of claim 68.

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