# Patent

## Title
Tetragalnac and peptide containing conjugates and methods for delivery of oligonucleotides

## Abstract

Disclosed herein is a modular composition comprising 1) an oligonucleotide; 2) one or more tetraGalNAc ligands of Formula (I), which may be the same or different; optionally, 3) one or more linkers, which may be the same or different; 4) one or more peptides independently selected from Table 3, which may be the same or different; and optionally, 5) one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation application of U.S. patent application Ser. No. 14/398,369, filed on Oct. 31, 2014, which is a national-stage application of PCT Application No. PCT/US2013/039072, filed on May 1, 2013, which claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 61/641,741, filed May 2, 2012; all of which are incorporated by reference herein in their entirety.


BACKGROUND OF THE INVENTION
Scientific efforts focused on the delivery of oligonucleotides systemically for therapeutic purposes are ongoing. Three highlighted approaches to oligonucleotide delivery include 1) lipid nanoparticle (LNP) encapsulation, 2) polymer conjugation and 3) single chemical conjugation. Single chemical conjugation typically employs a targeting ligand or a lipid or a solubilizing group or an endosomolytic peptide or a cell penetrating peptide and/or a combination of two or all four attached to an oligonucleotide. Linkers may be present in the conjugate as well as other functionalities. Single chemical conjugates are known and attachment of the oligonucleotide occurs either at the 5′- or 3′-end of the oligonucleotide, at both ends, or internally. See WO2005/041859, WO2008/036825, and WO2009/126933.
Considerable amount of literature evidence supports the hypothesis that the major hurdles for oligonucleotide delivery are cell uptake and endosomal escape. There remains a need for additional single chemical conjugates that can provide effective delivery efficiency, cell uptake and/or endosomal escape.
SUMMARY OF THE INVENTION
Single chemical conjugates comprising tetraGalNAc and peptides disclosed herein have surprising properties of effective delivery efficiency, cell uptake and/or endosomal escape.
In one embodiment, a modular composition disclosed herein comprises: 1) a single stranded or double stranded oligonucleotide; 2) one or more tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different:




wherein X is —O—, —S—, —CR1R2— or —NR1—, wherein R1 and R2 are each independently selected from the group consisting of hydrogen and C1-C6alkyl; n is 1, 2, 3, or 4; and the bond with “” indicates point of attachment; optionally, 3) one or more linkers, which may be the same or different; 4) one or more peptides independently selected from Table 3, which may be the same or different; and optionally, 5) one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents. In one embodiment, R1 and R2 are each independently selected from the group consisting of hydrogen, methyl and ethyl. In another embodiment, R1 and R2 are each hydrogen.

In one embodiment, the tetraGalNAc ligand has Formula (II) wherein X, R1, R2 and n are as defined above. In another embodiment, the tetraGalNAc ligand has Formula (III) wherein X, R1, R2 and n are as defined above:




In another embodiment, a modular composition comprises: 1) a single stranded or double stranded oligonucleotide; 2) 1-8 tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different, wherein X is —O—, —S—, —CH2— or —NH—; and n is 1, 2, 3, or 4; 3) 1-24 linkers, which may be the same or different; 4) 1-8 peptides independently selected from Table 3, which may be the same or different; and optionally, 5) 1-8 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents. the tetraGalNAc ligand has Formula (II) wherein X, R1, R2 and n are as defined above.
In another embodiment, a modular composition comprises: 1) a single stranded or double stranded siRNA; 2) 1-8 tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different, wherein X is —O—, —S—, —CH2— or —NH—; and n is 1, 2, 3, or 4; 3) 1-24 linkers, which may be the same or different; 4) 1-8 peptides independently selected from Table 3, which may be the same or different; and optionally, 5) 1-8 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.
In another subset of the above embodiments, the linkers are attached to the oligonucleotide or siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the oligonucleotide or siRNA.
In another subset of the above embodiments, the tetraGalNAc ligands and/or the peptides are attached to the oligonucleotide or siRNA optionally via linkers.
In another subset of the above embodiments, the tetraGalNAc ligands and/or the peptides are attached to the oligonucleotide or siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the oligonucleotide or siRNA; and the tetraGalNAc ligands and/or the peptides are attached to the oligonucleotide or siRNA optionally via linkers.
In another subset of the above embodiments, X of Formula (I), (II) or (III) is —O—, —S—, or —CH2—; and n is 1, 2 or 3.
In another subset of the above embodiments, X of Formula (I), (II) or (III) is —O— or —CH2— and n is 1 or 2.
In another subset of the above embodiments, X of Formula (I), (II) or (III) is —O— and n is 1 or 2.
In another subset of the above embodiments, X of Formula (I), (II) or (III) is —CH2— and n is 1 or 2.
In another subset of the above embodiments, the composition comprises 1-6 tetraGalNAc ligands, or more specifically, 1-4 tetraGalNAc ligands, which may be the same or different.
In another subset of the above embodiments, the composition comprises 1-6, peptides, or more specifically, 1-4 peptides, which may be the same or different.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded; and the tetraGalNAc ligands are attached to the guide strand or the passenger strand of the oligonucleotide or siRNA at different 2′-positions of the ribose rings.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded; and the tetraGalNAc ligands are attached to the guide strand or the passenger strand of the oligonucleotide or siRNA at different terminal 3′ and/or 5′-positions.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded; and the tetraGalNAc ligands are attached to both the guide strand and the passenger strand of the oligonucleotide or siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded; and the peptides are attached to the guide strand or the passenger strand of the oligonucleotide or siRNA at different 2′-positions of the ribose rings of the siRNA.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded; and the peptides are attached to the guide strand or the passenger strand of the oligonucleotide or siRNA at different terminal 3′ and/or 5′-positions.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded; and the peptides are attached to both the guide strand and the passenger strand of the oligonucleotide or siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions.
In another subset of the above embodiments, the tetraGalNAc ligands and the peptides are attached to the same strand of the oligonucleotide or siRNA.
In another subset of the above embodiments, the tetraGalNAc ligands and the peptides are attached to different strands of the oligonucleotide or siRNA.
In another subset of the above embodiments, the tetraGalNAc ligands and the peptides are attached to the same or different strands of the oligonucleotide or siRNA via linkers.
In another subset of the above embodiments, each linker is independently selected from Table 1.
In another subset of the above embodiments, each linker is independently selected from Table 2.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded; and the optional targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents are attached to the same or different strands of the oligonucleotide or siRNA.
In one embodiment, a modular composition comprises 1) a double stranded siRNA; 2) 1-8 tetraGalNAc ligands of Formula (IV), (V) or (VI), which may be the same or different:







3) 1-24 linkers independently selected from Table 1, which may be the same or different; 4) 1-8 peptides independently selected from Table 3, which may be the same or different; and, optionally, 5) 1-8 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.

In another embodiment, a modular composition comprises 1) a double stranded siRNA; 2) 1-4 tetraGalNAc ligands of Formula (IV), (V) or (VI), which may be the same or different; 3) 1-12 linkers independently selected from Table 1, which may be the same or different; 4) 1-4 peptides independently selected from Table 3, which may be the same or different; and, optionally, 5) 1-4 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents; wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the siRNA; and wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA optionally via linkers.
In one subset of the above embodiments, the tetraGalNAc ligands and the peptides are attached to the same strand of the siRNA via linkers.
In another subset of the above embodiments, the tetraGalNAc ligands and the peptides are attached to different strands of the siRNA via linkers.
In one embodiment, a modular composition comprises 1) a double stranded siRNA; 2) 1-4 tetraGalNAc ligands of Formula (IV), (V) or (VI), which may be the same or different; 3) 1-12 linkers independently selected from Table 2, which may be the same or different; 4) 1-4 peptides independently selected from Table 4, which may be the same or different; and, optionally, 5) 1-4 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents; wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the siRNA; and wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA via linkers.
In one subset of the above embodiment, the tetraGalNAc ligands and the peptides are attached to the same strand of the siRNA via linkers.
In one subset of the above embodiment, the tetraGalNAc ligands and the peptides are attached to different strands of the siRNA via linkers.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Non-limiting examples of modular compositions comprising double stranded oligonucleotides with terminal conjugations.
FIG. 2. Non-limiting examples of modular compositions comprising double stranded oligonucleotides with terminal conjugations.
FIG. 3. Non-limiting examples of modular compositions comprising double stranded oligonucleotides with internal and/or terminal conjugations are shown in FIG. 3A to FIG. 3B.
FIG. 4. Generic structures of each nucleotide [On] or [On′] that contain a linker (L-P and/or L-G).
FIG. 5. Scheme 2 as shown in FIG. 5A-1 to FIG. 5D for preparing B Conjugates (Ex. 3-6).
FIG. 6. Scheme 3 as shown as FIG. 6A to FIG. 6B for preparing Conjugates B6-P32 and B8-seq32 (Ex. 7-8). The figures disclose SEQ ID NO: 32.
FIG. 7. Scheme 4 as shown in FIG. 7A, FIG. 7B and FIG. 7C for preparing B9, B10-seq32 and B11-seq32. The figures disclose SEQ ID NO: 32.
Scheme 5 as shown in FIG. 7D-1 and FIG. 7D-2, FIG. 7E and FIG. 7F for preparing B-13-seq13-b compound. The figures disclose SEQ ID NO: 13.
Scheme 6 as shown in FIG. 7G-1 to FIG. 7G-2 for preparing B16-seq32 and B17-seq32-b compound. FIG. 7H-1, FIG. 7H-2, and FIG. 7I show the preparation of B15-seq32 and B16-seq32-b. FIGS. 7H-1 to 7I disclose SEQ ID NO: 32.
FIG. 8. Scheme 7 as shown in FIG. 8A to FIG. 8D for preparing C1 to C3, C4-seq32 and C6-seq32 compound. The figures disclose SEQ ID NO: 32.
FIG. 9. Scheme 8 as shown in FIG. 9A to FIG. 9E for preparing C7 to C10, C11-seq32 and C12-seq32 compound. The figures disclose SEQ ID NO: 32.
FIG. 10. Scheme 9 shown in FIG. 10 A to FIG. 10D for preparing C13, C14-seq32 and C15-seq32-a compound. The figures disclose SEQ ID NO: 32.
FIG. 11. Scheme 10 as shown in FIG. 11A to FIG. 11 D for preparing D1, D3 and D4.
FIG. 12. Scheme 11 as shown in FIG. 12A-1 to FIG. 12B-2 for preparing D5-seq32 and D7-seq32 compound. The figures disclose SEQ ID NO: 32.
FIG. 13. Scheme 12 as shown in FIG. 13A to FIG. 13H-2 for preparing E compounds.
FIG. 14. Scheme 13 as shown in FIG. 14A-1 to FIG. 14B-2 for preparing E8-seq 137 and E10-seq137e compounds. The figures disclose SEQ ID NO: 137.
FIG. 15. Scheme 14 as shown in FIG. 15A to FIG. 15E-2 for preparing F compounds. The figures disclose SEQ ID NO: 463.
FIG. 16. Scheme 15 as shown in FIG. 16A-1 to FIG. 16B-2 for preparing F6seq 463-f compound. The figures disclose SEQ ID NO: 463.
FIG. 17. Scheme 16 as shown in FIG. 17A-1 to FIG. 17D-2 for preparing G compounds. The figures disclose SEQ ID NO: 489.
FIG. 18. Scheme 17 as shown in FIG. 18A-1 to FIG. 18B-2 for preparing G compounds. The figures disclose SEQ ID NO: 489.
FIG. 19. Scheme 19 as shown in FIG. 19A to FIG. 19I-2 for preparing H10-seq32-h compound. The figures disclose SEQ ID NO: 32.
FIG. 20. Scheme 20 as shown in FIG. 20A-1 to FIG. 20E-2 for preparing I10-seq1681-f compound. The figures disclose SEQ ID NOS 1737, 1737-1739, 1737, 1737, and 1737, respectively, in order of appearance.
FIG. 21. Scheme 21 as shown in FIG. 21A to FIG. 21H-2 for preparing J9-seq26-i compound.
The figures disclose SEQ ID NO: 26.
FIG. 22. Scheme 22 as shown in FIG. 22A-1 to FIG. 22D-2 for preparing K6 seq 74-b compound.
The figures disclose SEQ ID NO: 74.
FIG. 23. Scheme 23 as shown in FIG. 23A to FIG. 23C-2 for preparing L11-seq 463-j compound. The figures disclose SEQ ID NO: 463.
FIG. 24. Scheme 24 as shown in FIG. 24A-1 to FIG. 24B-2 for preparing M4-seq-j compound. The figures disclose SEQ ID NO: 463.
FIG. 25. Scheme 25 as shown in FIG. 25A to FIG. 25B-2 for preparing N4-seq 283-k compound. The figures disclose SEQ ID NO: 283.
FIG. 26. Scheme 26 as shown in FIG. 26A-1 to FIG. 26B-2 for preparing O3-seq 463-k compound. The figures disclose SEQ ID NO: 463.
FIG. 27. Scheme 27 as shown in FIG. 27A-1 to FIG. 27B-2 for preparing P2-seq-32-k compound. The figures disclose SEQ ID NO: 13.
FIG. 28. Scheme 28 as shown in FIG. 28-1 to FIG. 28-2 for preparing P2-seq 32-m compound. The figures disclose SEQ ID NO: 74.
FIG. 29. Scheme 29 as shown in FIG. 29A-1 to FIG. 29C-2 used to prepare Q3-seq74-b compound. The figures disclose SEQ ID NO: 74.
FIG. 30. Scheme 30 as shown in FIG. 30A to FIG. 30E-3 for preparing R4-seq 27-I compound. The figures disclose SEQ ID NO: 27.
FIG. 31. Scheme 32 as shown in FIG. 31A and FIG. 31B for preparing tetraGalNAc-siRNA conjugates.
FIG. 32. Scheme 33 as shown in FIG. 32A and FIG. 32B for preparing TetraGalNAc-siRNA Conjugate 19-1.
FIG. 33. Scheme 35 as shown in FIG. 33A and FIG. 33B for preparing Compound 26.
FIG. 34. Scheme 36 as shown in FIG. 34A to FIG. 34C for preparing Compounds 27 and 28.
FIG. 35. Scheme 38 as shown in FIG. 35A and FIG. 35B for preparing Conjugates 35-37.
FIG. 36. Scheme 39 as shown in FIG. 36A to FIG. 36C for preparing Conjugates 38-44.
FIG. 37. Scheme 40 as shown in FIG. 37 showing examples of different linkers from Table 2, for conjugating tetraGalNAc to siRNA.
FIG. 38. Scheme 41 as shown in FIG. 38A to FIG. 38E for preparing Compounds and/or Conjugates 46-48.
FIG. 39. Scheme 42 as shown in FIG. 39A to FIG. 39C for preparing Compounds and/or Conjugates 49-51.
FIG. 40. Scheme 43 as shown in FIG. 40 showing a general description for illustrative purposes of nomenclature used in Table 6.



DETAILED DESCRIPTION OF THE INVENTION
Disclosed herein are single chemical conjugates comprising a single stranded or double stranded oligonucleotide; one or more tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different;







wherein X is —O—, —S—, —CR1R2— or —NR1—, wherein R1 and R2 are each independently selected from the group consisting of hydrogen and C1-C6alkyl; n is 1, 2, 3, or 4; and the bond with “” indicates the point of attachment; and one or more peptides, which may be the same or different. Other functionalities, such as targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents are optionally present. In one embodiment, R1 and R2 are each independently selected from the group consisting of hydrogen, methyl and ethyl. In another embodiment, R1 and R2 are each hydrogen.

In one embodiment, the oligonucleotide is a short interfering RNA (siRNA). In another embodiment, the siRNA is a single stranded siRNA. In another embodiment, the siRNA is a double stranded siRNA.
The use of the tetraGalNAc disclosed herein provides effective delivery of the oligonuleotide or siRNA by directing the modular composition to a particular cell. For example, the targeting ligand may specifically or non-specifically bind with a molecule on the surface of a target cell and facilitate internalization of the ligand-siRNA conjugate.
The peptides may function as endosomolytic, cell penetrating and/or fusogenic agents. In addition, the peptide may have cationic, zwitterionic, neutral, anionic character. Incorporation of both the tetraGalNAc and the peptide in the modular composition may further improve the delivery efficiency of the oligonuleotide or siRNA.
A linker may be present between each peptide and the oligonucleotide or between each tetraGalNAc and the oligonucleotide. The linkers are attached to the oligonucleotide at different 2′-positions of the ribose rings and/or the terminal 3′ and/or 5′-positions of the oligonucleotide.
In one embodiment, a modular composition comprises 1) a single stranded or double stranded oligonucleotide; 2) one or more tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different, wherein X is —O—, —S—, —CH2— or —NH—; n is 1, 2, 3, or 4; and the bond with “” indicates the point of attachment; optionally, 3) one or more linkers, which may be the same or different; 4) one or more peptides independently selected from Table 3, which may be the same or different; and optionally, 5) one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.
In another embodiment, a modular composition comprises 1) a single stranded or double stranded oligonucleotide; 2) 1-8 tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different, wherein X is —O—, —S—, —CH2— or —NH—; n is 1, 2, 3, or 4; 3) 1-24 linkers, which may be the same or different; 4) 1-8 peptides independently selected from Table 3, which may be the same or different; and optionally, 5) 1-8 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.
In another embodiment, a modular composition comprises 1) a single stranded or double stranded siRNA; 2) 1-8 tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different, wherein X is —O—, —S—, —CH2— or —NH—; n is 1, 2, 3, or 4; 3) 1-24 linkers, which may be the same or different; 4) 1-8 peptides independently selected from Table 3, which may be the same or different; and optionally, 5) 1-8 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.
In one subset of the above embodiments, the tetraGalNAc ligands and/or the peptides are attached to the oligonucleotide or siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the oligonucleotide or siRNA.
In another subset of the above embodiments, the tetraGalNAc ligands and/or the peptides are attached to the oligonucleotide or siRNA optionally via linkers. In one embodiment, the linkers are present.
In another subset of the above embodiments, the tetraGalNAc ligands and/or the peptides are attached to the oligonucleotide or siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the oligonucleotide or siRNA; and the tetraGalNAc ligands and/or the peptides are attached to the oligonucleotide or siRNA via linkers.
In another subset of the above embodiments, the tetraGalNAc ligands are attached to the oligonucleotide or siRNA via linkers and the linkers are attached to the oligonucleotide or siRNA at different 2′-positions of the ribose rings.
In another subset of the above embodiments, the tetraGalNAc ligands are attached to the oligonucleotide or siRNA via linkers and the linkers are attached to the oligonucleotide or siRNA at different terminal 3′ and/or 5′-positions of the oligonucleotide.
In another subset of the above embodiments, X is —O—, —S—, or —CH2—. In another embodiment, X is —O— or —CH2—. In another embodiment, n is 1, 2 or 3. In another embodiment, X is —O— and n is 1 or 2. In another embodiment, X is —CH2— and n is 1 or 2. In another embodiment, X is —O— and n is 1. In yet another embodiment, X is —CH2— and n is 1.
In another subset of the above embodiments, the oligonucleotide or siRNA is single stranded. In another embodiment, the oligonucleotide or siRNA is double stranded.
In another subset of the above embodiments, the composition comprises 1-6 tetraGalNAc ligands. In another embodiment, the composition comprises 1-4 tetraGalNAc ligands. In another embodiment, the composition comprises 1-2 tetraGalNAc ligands. In yet another embodiment, the composition comprises 1 tetraGalNAc ligand.
In another subset of the above embodiments, the composition comprises 1-6 peptides. In another embodiment, the composition comprises 1-4 peptides. In another embodiment, the composition comprises 1-2 peptides. In yet another embodiment, the composition comprises 1 peptide.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the tetraGalNAc ligands are attached to the guide strand at different 2′-positions of the ribose rings.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded the tetraGalNAc ligands are attached to the guide strand at different terminal 3′ and/or 5′-positions.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the tetraGalNAc ligands are attached to the passenger strand at different 2′-positions of the ribose rings.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the tetraGalNAc ligands are attached to the passenger strand at different terminal 3′ and/or 5′-positions.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the tetraGalNAc ligands are attached to both the guide strand and the passenger strand at different 2′-positions of the ribose rings and/or different terminal 3′ and/or 5′-positions.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the peptides are attached to the guide strand at different 2′-positions of the ribose rings.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the peptides are attached to the guide strand at different terminal 3′ and/or 5′-positions.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the peptides are attached to the passenger strand at different 2′-positions of the ribose rings.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the peptides are attached to the passenger strand at different terminal 3′ and/or 5′-positions.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the peptides are attached to both the guide strand and the passenger strand at different 2′-positions of the ribose rings and/or different terminal 3′ and/or 5′-positions.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the tetraGalNAc ligands and the peptides are attached to the same or different strands via linkers. In one embodiment, each linker is independently selected Table 1. In another embodiment, each linker is independently selected Table 2.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the tetraGalNAc ligands and the peptides are attached to the same strand.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the tetraGalNAc ligands and the peptides are attached to different strands.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the optional targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents are attached to the same or different strands.
In another subset of the above embodiments, the oligonucleotide or siRNA is double stranded and the optional targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents are attached to the same or different strands via linkers. In one embodiment, each linker is independently selected from Table 1. In another embodiment, each linker is independently selected from Table 2.
In one embodiment, a modular composition comprises 1) a single stranded or double stranded siRNA; 2) 1-8 tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different; wherein X is —O—, —S—, —CH2— or —NH—; and n is 1, 2, 3, or 4; 3) 1-24 linkers, which may be the same or different; 4) 1-8 peptides independently selected from Table 3, which may be the same or different; and optionally, 5) 1-8 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents; wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the siRNA; and wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA optionally via linkers. In one embodiment, the linkers are present. In another embodiment, X is —O—, —S—, or —CH2—, and n is 1, 2 or 3. In another embodiment, X is —O— or —CH2—, and n is 1 or 2.
In another embodiment, a modular composition comprises 1) a double stranded siRNA; 2) 1-6 tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different; wherein X is —O—, —S—, or —CH2—; and n is 1, 2 or 3; 3) 1-18 linkers, which may be the same or different; 4) 1-6 peptides independently selected from Table 3, which may be the same or different; and optionally, 5) 1-6 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents; wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the siRNA; and wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA optionally via linkers. In one embodiment, the linkers are present. In another embodiment, X is —O—, —S—, or —CH2— and n is 1 or 2. In another embodiment, the linkers are independently selected from Table 1. In another embodiment, the linkers are independently selected from Table 2. In another embodiment, the peptides of 4) are independently selected from Table 4.
In another embodiment, a modular composition comprises 1) a double stranded siRNA; 2) 1-4 tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different; wherein X is —O—, —S—, or —CH2—; and n is 1 or 2; 3) 1-12 linkers, which may be the same or different; 4) 1-4 peptides independently selected from Table 3, which may be the same or different; and optionally, 5) 1-4 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents; wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the siRNA; and wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA via linkers. In one embodiment, X is —O— or —CH2— and n is 1 or 2. In another embodiment, the linkers are independently selected from Table 1. In another embodiment, the linkers are independently selected from Table 2. In another embodiment, the peptides are independently selected from Table 4.
In another embodiment, a modular composition comprises 1) a double stranded siRNA; 2) 1-4 tetraGalNAc ligands of Formula (IV), (V) or (VI), which may be the same or different:







3) 1-12 linkers independently selected from Table 1, which may be the same or different; 4) 1-4 peptides independently selected from Table 3, which may be the same or different; and optionally, 5) 1-4 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents; wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the siRNA; and wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA via linkers.

In another embodiment, a modular composition comprises 1) a double stranded siRNA; 2) 1-4 tetraGalNAc ligands of Formula (IV), (V) or (VI); 3) 1-12 linkers independently selected from Table 2, which may be the same or different; 4) 1-4 peptides independently selected from Table 4, which may be the same or different; and optionally, 5) 1-4 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents; wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the siRNA; and wherein the tetraGalNAc ligands and/or the peptides are attached to the siRNA via linkers.
In one subset of the above embodiments, the tetraGalNAc ligands and/or the peptides are attached to the siRNA via linkers; and wherein the tetraGalNAc ligands and/or the peptides are attached to the same strand.
In another subset of the above embodiments, the tetraGalNAc ligands and/or the peptides are attached to the siRNA via linkers; and wherein the tetraGalNAc ligands and the peptides are attached to different strands.
To illustrate the invention via cartoon, the invention features a modular composition, comprising an oligonucleotide ([O1][O2][O3] . . . [On]), one or more tetraGalNAc(s) ligands (G), one or more linker(s) (L), one or more peptide(s) (P), and one or more optional lipid(s) (X), one or more targeting ligand(s) (X), and/or one or more solubilizing group(s) (X).
In an embodiment, the modular composition may have the formula:

G-L-[O1][O2][O3] . . . [On]-L-P.

In another embodiment, the modular composition may have the formula:

P-L-[O1][O2][O3] . . . [On]-L-G.

Non-limiting examples of modular compositions comprising double stranded oligonucleotides with terminal conjugations are shown in FIG. 1.
Non-limiting examples of modular compositions comprising double stranded oligonucleotides with terminal conjugations are shown in FIG. 2.
Non-limiting examples of modular compositions comprising double stranded oligonucleotides with internal and/or terminal conjugations are shown in FIG. 3A and FIG. 3B.
These examples are used as illustration only. One skilled in the art will recognize that a variety of permutations for placing the desired components on the passenger and guide strand exist.
Any number of linkers, and therefore any number of peptides, can be attached to the oligonucleotide. The range of numbers of linkers is from 1-16. A more preferred range of numbers of linkers is from 1-12, or more specifically, 1-8, or even more specifically, 1-4.
The range of numbers of tetraGalNAc ligands is from 1-8. A more preferred range of numbers of tetraGalNAc ligands is from 1-6, or more specifically, 1-4, or even more specifically, 1-2.
The range of numbers of peptides is from 1-8. A more preferred range of numbers of peptides is from 1-6, or more specifically, 1-4, or even more specifically, 1-2.
The two strands contain n and n′ nucleotides respectively. The numbers n and n′ can be equal or different. The numbers are integers ranging from 8 to 50. Preferably, the numbers are integers ranging from 12-28. More preferably, the numbers are integers ranging from 19-21.
As an example, each nucleotide [On] or [On′], that contains a linker (L-P and/or L-G) has generic structures as shown in FIG. 4.
For each nucleotide, 1) E=oxygen (O) or sulfur (S); 2) Base=A, U, G or C, which can be modified or unmodified; 3) D is the connection point between ribose ring and linker L, D=oxygen (O), sulfur (S, S(O) or S(O)2), nitrogen (N—R, wherein R=H, alkyl, L-P or L-X), carbon (CH—R, wherein R=H, alkyl, L-P, or L-X), or phosphorus (P(O)R or P(O)(OR), wherein R=alkyl, L-P, or L-X). Preferably, D=oxygen (O).
The two nucleotides [On-1] and [On] or [On′-1] and [On′] are connected via phosphodiester or thio-phosphodiester bonds.
When the oligonucleotide is a double-stranded oligonucleotide, the “G-L”, “P-L” and the lipid, targeting ligand, and/or solubilizing group may be located on the same strand or on different strands.
In some embodiments, the “G-L” and “P-L” are on the same strand.
In some embodiments, the “G-L” and “P-L” are on the passenger strand.
In some embodiments, the “G-L” and “P-L” are on the guide strand.
In some embodiments, the “G-L” and “P-L” are located on different strands.
In some embodiments, the “G-L” is on the passenger strand while the “P-L” is on the guide strand.
In some embodiments, the “G-L” and “P-L” are on different strands but on the same terminal end of the double-stranded oligonucleotide.
In some embodiments, the “G-L” and “P-L” are on different strands and on the opposite terminal ends of the double-stranded oligonucleotide.
In some embodiments, the “G-L” can be located on multiple terminal ends of either the passenger or guide strand and “P-L” can be located on the remaining terminal ends of the passenger and guide strands.
In some embodiments, one “G-L” and two or more “P-L” are present in the oligonucleotide.
In some embodiments, two or more “G-L” and two or more “P-L” are present in the oligonucleotide.
In some embodiments, when the oligonucleotide is a double-stranded oligonucleotide and multiple “G-L” and/or “P-L” are present, such multiple “G-L” components and/or “P-L” may all be present in one strand or both strands of the double stranded oligonucleotide.
When multiple “G-L” components and/or “P-L” are present, they may all be the same or different.
In some embodiments, the “G-L” and/or “P-L” are on internal nucleotides only (i.e. excluding the 3′- and 5′-terminal ends of the oligonucleotide).
In another aspect, the invention includes a method of delivering an oligonucleotide or siRNA to a cell. The method includes (a) providing or obtaining a modular composition disclosed herein; (b) contacting a cell with the modular composition; and (c) allowing the cell to internalize the modular composition.
The method can be performed in vitro, ex vivo or in vivo, e.g., to treat a subject identified as being in need of an oligonucleotide or siRNA. A subject in need of said oligonucleotide is a subject, e.g., a human, in need of having the expression of a gene or genes, e.g., a gene related to a disorder, downregulated or silenced.
In one aspect, the invention provides a method for inhibiting the expression of one or more genes. The method comprising contacting one or more cells with an effective amount of an oligonucleotide of the invention, wherein the effective amount is an amount that suppresses the expression of the one or more genes. The method can be performed in vitro, ex vivo or in vivo.
The methods and compositions of the invention, e.g., the modular composition described herein, can be used with any oligonucleotides or siRNAs known in the art. In addition, the methods and compositions of the invention can be used for the treatment of any disease or disorder known in the art, and for the treatment of any subject, e.g., any animal, any mammal, such as any human. One of ordinary skill in the art will also recognize that the methods and compositions of the invention may be used for the treatment of any disease that would benefit from downregulating or silencing a gene or genes.
The methods and compositions of the invention, e.g., the modular composition described herein, may be used with any dosage and/or formulation described herein, or any dosage or formulation known in the art. In addition to the routes of administration described herein, a person skilled in the art will also appreciate that other routes of administration may be used to administer the modular composition of the invention.
Oligonucleotide
An “oligonucleotide” as used herein, is a double stranded or single stranded, unmodified or modified RNA or DNA. Examples of modified RNAs include those which have greater resistance to nuclease degradation than do unmodified RNAs. Further examples include those which have a 2′ sugar modification, a base modification, a modification in a single strand overhang, for example a 3′ single strand overhang, or, particularly if single stranded, a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group. Examples and a further description of oligonucleotides can be found in WO2009/126933, which is hereby incorporated by reference.
In an embodiment, an oligonucleotide is an antisense, miRNA, peptide nucleic acid (PNA), poly-morpholino (PMO) or siRNA. The preferred oligonucleotide is an siRNA.
Another preferred oligonuleotide is the passenger strand of an siRNA. Another preferred oligonucleotide is the guide strand of an siRNA.
siRNA
siRNA directs the sequence-specific silencing of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates. Methods for preparing and administering siRNA and their use for specifically inactivating gene function are known. siRNA includes modified and unmodified siRNA. Examples and a further description of siRNA can be found in WO2009/126933, which is hereby incorporated by reference.
A number of exemplary routes of delivery are known that can be used to administer siRNA to a subject. In addition, the siRNA can be formulated according to any exemplary method known in the art. Examples and a further description of siRNA formulation and administration can be found in WO2009/126933, which is hereby incorporated by reference.
The phrases “short interfering nucleic acid”, “siNA”, “short interfering RNA”, “siRNA”, “short interfering nucleic acid molecule”, “oligonucleotide”, “short interfering oligonucleotide molecule”, or “chemically modified short interfering nucleic acid molecule” refer to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication by mediating RNA interference (“RNAi”) or gene silencing in a sequence-specific manner. These terms can refer to both individual nucleic acid molecules, a plurality of such nucleic acid molecules, or pools of such nucleic acid molecules. The siNA can be a double-stranded nucleic acid molecule comprising self-complementary sense and antisense strands, wherein the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single-stranded polynucleotide having a nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single-stranded polynucleotide can further comprise a terminal phosphate group, such as a 5′-phosphate (see for example, Martinez et al., 2002, Cell, 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate.
siRNA directs the sequence-specific silencing of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates. Methods for preparing and administering siRNA and their use for specifically inactivating gene function are known. As used herein, siRNA includes chemically modified and unmodified nucleic acid molecules capable of inhibiting or down regulating gene expressions. Examples and a further description of siRNA can be found in WO2009/126933, which is hereby incorporated by reference.
A number of exemplary routes of delivery are known that can be used to administer siRNA to a subject. In addition, the siRNA can be formulated according to any exemplary method known in the art. Examples and a further description of siRNA formulation and administration can be found in WO2009/126933, which is hereby incorporated by reference.
Linkers
The covalent linkages between the tetraGalNAc and the oligonucleotide or siRNA of the modular composition and/or between the peptide and the oligonucleotide or siRNA may be mediated by a linker. This linker may be cleavable or non-cleavable, depending on the application. In certain embodiments, a cleavable linker may be used to release the oligonucleotide after transport from the endosome to the cytoplasm. The intended nature of the conjugation or coupling interaction, or the desired biological effect, will determine the choice of linker group. Linker groups may be combined or branched to provide more complex architectures. Suitable linkers include those as described in WO2009/126933, which is hereby incorporated by reference.
In one embodiment, the linkers of the instant invention are shown in Table 1:








TABLE 1

















 












 













 








 








 








 








 








 








 








 








 








 





















R = H, Boc, Cbz, Ac, PEG, lipid, targeting ligand, linker(s) and/or peptide(s).


n = 0 to 750.


“nucleotide” can be substituted with non-nucleotide moiety such as abasic or linkers as are generally known in the art.


enzymatically cleavable linker = linker cleaved by enzyme; e.g., protease or glycosidase






In another embodiment, the preferred linkers are shown in Table 2.








TABLE 2

















 












 













 








 








 








 








 








 








 











R = H, Boc, Cbz, Ac, PEG, lipid, targeting ligand, linker(s) and/or peptide(s).


n = 0 to 750.


“nucleotide” can be substituted with non-nucleotide moiety such as abasic or linkers as are generally known in the art.


enzymatically cleavable linker = linker cleaved by enzyme; e.g., protease or glycosidase






Commercial linkers are available from various suppliers such as Pierce or Quanta Biodesign including combinations of said linkers. In addition, commercial linkers attached via phosphate bonds can be used independently as linkers or in combination with said linkers. The linkers may also be combined to produce more complex branched architectures accommodating from 1 to 8 peptides as illustrated in one such example below:




Peptides

For macromolecular drugs and hydrophilic drug molecules, which cannot easily cross bilayer membranes, entrapment in endosomal/lysosomal compartments of the cell is thought to be the biggest hurdle for effective delivery to their site of action. Without wishing to be bound by theory, it is believed that the use of peptides will facilitate oligonucleotide escape from these endosomal/lysosomal compartments or oligonucleotide translocation across a cellular membrane and release into the cytosolic compartment. In certain embodiments, the peptides of the present invention may be polycationic or amphiphilic or polyanionic or zwitterionic or lipophilic or neutral peptides or peptidomimetics which can show pH-dependent membrane activity and/or fusogenicity. A peptidomimetic may be a small protein-like chain designed to mimic a peptide.
In some embodiments, the peptide is a cell-permeation agent, preferably a helical cell-permeation agent. These peptides are commonly referred to as Cell Penetrating Peptides. See, for example, “Handbook of Cell Penetrating Peptides” Ed. Langel, U.; 2007, CRC Press, Boca Raton, Fla. Preferably, the component is amphipathic. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase. A cell-permeation agent can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide or hydrophobic peptide, e.g. consisting primarily of Tyr, Trp and Phe, dendrimer peptide, constrained peptide or crosslinked peptide. Examples of cell penetrating peptides include Tat, Penetratin, and MPG. For the present invention, it is believed that the cell penetrating peptides can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and proteins across cell membranes. Cell permeation peptides can be linear or cyclic, and include D-amino acids, “retro-inverso” sequences, nonpeptide or pseudo-peptide linkages, peptidyl mimics. In addition the peptide and peptide mimics can be modified, e.g. glycosylated, pegylated, or methylated. Examples and a further description of peptides can be found in WO2009/126933, which is hereby incorporated by reference. Synthesis of peptides is well known in the art.
The peptides may be conjugated at either end or both ends by addition of a cysteine or other thiol containing moiety to the C- or N-terminus. When not functionalized on the N-terminus, peptides may be capped by an acetyl group, or may be capped with a lipid, a PEG, or a targeting moiety. When the C-terminus of the peptides is unconjugated or unfunctionalized, it may be capped as an amide, or may be capped with a lipid, a PEG, or a targeting moiety.
Suitable peptides that can be used in the conjugates disclosed herein are listed in Table 3 below:







TABLE 3







Peptide Sequence Listing and ID








Sequence
SEQ ID





CGLFEAIEEFIENLWELLIDGWYGYGRKKRRQRR
SEQ ID NO: 1


CGLFEAIEGFIENGWEGMIDGWYGYGHKKHHQHH
SEQ ID NO: 2


C-bAla-LFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 3


CGLFEAIEGFIENGLKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 4


CGLFEAIEGFIEWGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 5


CRRQRRKKRGYGYWGDIMGEWGNEIFGEIAEFLG
SEQ ID NO: 6


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQR
SEQ ID NO: 7


CYGRKKRRQRRGLFEAIEGFIENGWEGMIDGWYG
SEQ ID NO: 8


CIFGAIAGFIKNILKGLIDG
SEQ ID NO: 9


CIFGAIAGFIRNIW
SEQ ID NO: 10


CGLFHALLHLLHSLWHGLLHAWYGYGHKKHHQHR
SEQ ID NO: 11


CGLFEAIEGLIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 12


CGLFELIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 13


CGLFEAIEGFIENGWEGLIDGWYGYGOOOOOQRR (O = ornithine)
SEQ ID NO: 14


CGLFGAIEGFIENGWEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 15


CGLFEAIEGFLENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 16


CGLFEAIEGFIENGLEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 17


CGLFGAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 18


CGLFEAIEGFIENGWEG-Nle-IDGWYGYGRKKRRQRR
SEQ ID NO: 19


CGIFGAIAGFIKNIWKGLIDW
SEQ ID NO: 20


CYGRKKRRQRRGLFEAIEGFIENGWKGLIDAWYG
SEQ ID NO: 21


CGLLEALEGLLESLWEGLLEAWYGYGRKKRRQRR
SEQ ID NO: 22


CGLFEAIEGFIENGWEGMIDNWYGYGRKKRRQRR
SEQ ID NO: 23


CIFGAIAGFIKNIWEGLIEAWYGLHLLHHLLHHLHHLLHHLLHL
SEQ ID NO: 24


CIFGAIAGFIKNIWEGLIDAF
SEQ ID NO: 25


CIFGAIAGFIKNIWEGLI
SEQ ID NO: 26


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRK(stearyl)
SEQ ID NO: 27


CGLFEAIAGFIEGGWPGLINGWYGYGRKKRRQRRLHLLHHLLHHLH
SEQ ID NO: 28


HLLHHLLHLLHHLLHHL



CGLFEAIEGFIENGWEGMIDGWYGGGGLHLLHHLLHHLHHLLHHL
SEQ ID NO: 29


LHLLHHLLHHL



CGLFEAIEGFIENGWEGMIDGWYGLHLLHHLLHHLHHLLHHLLHL
SEQ ID NO: 30


CGLFEALLELLESLWELLLEAYGRKKRRQRR
SEQ ID NO: 31


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 32


CGLFEAIEGFIENGWEGMADGWYGYGRKKRRQRR
SEQ ID NO: 33


CGIFGAIAGFIKNIWEGLIDWWYGYGRKKRRQRR
SEQ ID NO: 34


CGFLPAIAGILSQLFEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 35


CFFGAIWGFIKSIL
SEQ ID NO: 36


CIFGAIAGFIKNIWKGLIDWWYG
SEQ ID NO: 37


CGLFEAIEGFIWNGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 38


CGLFEAIAEFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 39


CYGRKKRRQRRGLFEAIEGFIENGWKGLIDWWYG
SEQ ID NO: 40


CGLFEAIEGFIEEGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 41


CGLFEAIEGFIENAWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 42


CGLFEAIEGFIENGWEGMIDLWYGYGRKKRRQRR
SEQ ID NO: 43


CRLLRLLLRLWRRLLRLLR
SEQ ID NO: 44


CGGFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 45


CGLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 46


CGLFEAIEGFIENGWENMIDGWYGYGRKKRRQRR
SEQ ID NO: 47


CIFGAIAGFIKNILKGL
SEQ ID NO: 48


CIFGAIAGFIKNILKGLIDGWYG
SEQ ID NO: 49


CGLFEAIEGFIENGWEGMIDGWYG-(PEG)3-YGRKKRRQRR
SEQ ID NO: 50


CGLFEALLELLESLWELLLEAYGRKKRRQRRLHLLHHLLHHLH
SEQ ID NO: 51


HLLHHLLHL



CYGRKKRRQRRWEAALAEALAEALAEHLAEALAEALEALAA
SEQ ID NO: 52


CIFGAIAGFIKNIWEGLIDGWYGKLALKLALKALKAALKLA
SEQ ID NO: 53


CFFGAIWEFIRSILEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 54


CGLFHALLHLLHSLWHLLLHAWYGYGRKKRRQRR
SEQ ID NO: 55


CGLFHALLHLLHSLWHLLLHAWYGYGHKKHHQHR
SEQ ID NO: 56


CGLFGALLELLESLWKGLLEWYGRKKRRQRR
SEQ ID NO: 57


CRRQRRKKRGYGYWGDILGEWGNEIFGEIAEFLG
SEQ ID NO: 58


CGLFEALEGFLENGWEGLLDGWYGYGROORRQRR (O = ornithine)
SEQ ID NO: 59


CGLFGEIEELIENGLKNLIDWWYGYGRKKRRQRR
SEQ ID NO: 60


CRRQRRKKRGYGYWWDILGKWGNEIFGEIAEFLG all (D) aminos
SEQ ID NO: 61


CGIFGAIAGFIKNIL
SEQ ID NO: 62


CGIFGAIAGLLKNIFK
SEQ ID NO: 63


CIFGAIAGFIKNIWKGLIDW
SEQ ID NO: 64


CIFGAIAGFIKNIWK
SEQ ID NO: 65


CGLFEEIEGFIENGWEGLIDWWYGYGHKKHHQHR
SEQ ID NO: 66


CGLFGEIEELIENGLKNLIDWWYGYGHKKHHQHR
SEQ ID NO: 67


CGLFEEIEEFIENGWEGLIDWWYGYGHKKHHQHR
SEQ ID NO: 68


stearyl-WEAALAEALAEALAEHLAEALAEALEALAAYGRKKRRQRRC
SEQ ID NO: 69


CGLFEAIEGFIENGWKGLIDGWYGGLFEAIEGFIENGWKGLIDWWYG
SEQ ID NO: 70


CGFFHAFFHFFHSFWHGFFEA
SEQ ID NO: 71


CGNFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 72


CFFGAIWEFIRNILEGF
SEQ ID NO: 73


CFFGAIWEFIHSIL
SEQ ID NO: 74


CGLFHALLHLLHSLWHGLLEA
SEQ ID NO: 75


CIFGAIAGFIKNIWEGL
SEQ ID NO: 76


CIFGAIAGLLKNIFEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 77


CGFIGAIANLLSKIFEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 78


CGLFEAIEELIENLWKGLIDAWYGYGRKKRRQRR
SEQ ID NO: 79


CGIFGAIAGLLKNIFKGLIDA
SEQ ID NO: 80


CGIFGAIAGLLKNIFKGLIDW
SEQ ID NO: 81


CGIFEAIAGLLKNIFK
SEQ ID NO: 82


CGIFEEIAGLLKNIFK
SEQ ID NO: 83


CGLFEAIAGFIEGGWPGLINGWYGYGRKKRRQRRLHLLHHLLH
SEQ ID NO: 84


HLHHLLHHLLHL



CGLFEAIEGFIENGWKGMIDWWYGYGRKKRRQRRK(stearyl)
SEQ ID NO: 85


CGLFGEIEEFIENGWKGLIDWWYG
SEQ ID NO: 86


CIFGAIAGFIKNIWLHLLHHLLHHLHHLLHHLLHL
SEQ ID NO: 87


CGIFGAIEGFIENGWKGLIDAWYGYRKKRRQRR
SEQ ID NO: 88


CELFGAIEGFIENGWKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 89


CIFGIDDLIIGLLFVAIVEAGIGGYLLGSYGRKKRRQRR
SEQ ID NO: 90


GLFGALAEALAEALAEHLAEALAEALEALAAGGSC
SEQ ID NO: 91


CGFIGAIANLLSKIFEGLIDGWYGYGRKKRRQRR all (D)
SEQ ID NO: 92


CFFGAIWEFIRSILKGLI
SEQ ID NO: 93


CFFGAIWEFIRSILK
SEQ ID NO: 94


CFFGAIWEFIRSILE
SEQ ID NO: 95


CIFGAIAGFIKNIWE
SEQ ID NO: 96


CIFGAIAGFIKNIWKGLIDA
SEQ ID NO: 97


CFFEAIEEFIKNILK
SEQ ID NO: 98


CIFGAIAGLLRNIF
SEQ ID NO: 99


CGIFGAIAGLLKNIW
SEQ ID NO: 100


CLFGAIWEFIKSIL
SEQ ID NO: 101


CFWGAIWEFIKSIL
SEQ ID NO: 102


CFGGAIWEFIKSIL
SEQ ID NO: 103


CFAGAIWEFIKSIL
SEQ ID NO: 104


CGLFEAIEGFIENGWEGM(SO2)IDGWYGYGRKKRRQRR
SEQ ID NO: 105


CGLFEAIEGFIENGWEGMIDWWYGYGRKKRRQRR
SEQ ID NO: 106


CFFGAIWEFIKSIG
SEQ ID NO: 107


CFFGAIWEFIKSIA
SEQ ID NO: 108


CFFGAIWEFIKSIN
SEQ ID NO: 109


CFFGAIWEFIKSIW
SEQ ID NO: 110


CFFGAIWEFIKSILEGLIDWWYGYGHKKHHQHR
SEQ ID NO: 111


Ac-CLHLLHHLLHHLHHLLHHLLHLLHHLLHHL-NH2
SEQ ID NO: 112


Ac-LHLLHHLLHHLHHLLHHLLHLLHHLLHHLGGGRKKRRQRRR
SEQ ID NO: 113


PPQC-NH2



CRKKRRQRRRPPQGGGLHLLHHLLHHLHHLLHHLLHLLHHLLHHL
SEQ ID NO: 114


CLHLLHHLLHHLHHLLHHLLHLLHHLLHHLGGGRKKRRQRRRPPQ
SEQ ID NO: 115


CGLFHAIAHFIHGGWHGLIHGWYGYGRKKRRQRR
SEQ ID NO: 116


CGLFKAIAKFIKGGWKGLIKGWYGYGRKKRRQRR
SEQ ID NO: 117


CGLFEAIAGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 118


CWEAALAEALAEALAEHLAEALAEALEALAAYGRKKRRQRR
SEQ ID NO: 119


CGLFEAIEGFIENGWEGMIDGWYGRKKRRQRRRPPQ
SEQ ID NO: 120


GLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRC
SEQ ID NO: 121


Ac-LHLLHHLLHHLHHLLHHLLHLLHHLLHHLRKKRRQRRRPPQ-NH2
SEQ ID NO: 122


Ac-LHLLHHLLHHLHHLLHHLLHLLHHLLHHLGPGRKKRRQRRR
SEQ ID NO: 123


PPQ-NH2



Ac-LIRLWSHLIHIWFQNRRLKWKKK-NH2
SEQ ID NO: 124


Ac-RKKRRQRRRPPQQQQQQ-NH2
SEQ ID NO: 125


Ac-GLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR-NH2
SEQ ID NO: 126


Ac-LHLLHHLLHHLHHLLHHLLHLLHHLLHHLGGGRRRRRRRRR-NH2
SEQ ID NO: 127


Ac-LHLLHHLLHHLHHLLHHLLHLLHHLLHHL-(Peg)12-RKKRRQRRR
SEQ ID NO: 128


PPQ-NH2



Ac-GLFGAIAGFIENGWEGMIDGWYGLIRLWSHLIWFQNRRLKWL
SEQ ID NO: 129


LL-NH2



Ac-HHHHHRKKRRQRRRPPQGGGLHLLHHLLHHLHHLLHHLLHLLHH
SEQ ID NO: 130


LLHHL-NH2



Ac-LHLLHHLLHHLHHLLHHLLHLLHHLLHHL-(Peg)2-RKKRRQRRR
SEQ ID NO: 131


PPQ-NH2



Ac-LHLLHHLLHHLHHLLHHLLLLHHLLHHLGGGRQIKIWFQNRRM
SEQ ID NO: 132


KWKKGG-NH2



Ac-KLLKLLLKLWLKLLKLLLKLLGGGRKKRRQRRRPPQ-NH2
SEQ ID NO: 133


Ac-LHHLLHHLLHLLHHLLHHLHHLLHHLLHLC-NH2 all (D)
SEQ ID NO: 134


Ac-LHLLHHLLHHLHHLLHHLLHLLHHLLHHL-PEG6-RKKRRQRRR
SEQ ID NO: 135


PPQC-NH2



Ac-GLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRC-NH2
SEQ ID NO: 136


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR all (D)
SEQ ID NO: 137


CGLFEAIEGFIENGWEGMIDGWYGYGRRRRRRRRR-NH2
SEQ ID NO: 138


YGRKKRRQRRGLFEAIEGFIENGWEGMIDGWYGC-NH2
SEQ ID NO: 139


CGVFVLGFLGFLATAGSYGRKKRRQRR-NH2
SEQ ID NO: 140


CGLFKAIAKFIKGGWKGLIKGWYG-NH2
SEQ ID NO: 141


CGLFEAIEGFIENGWEGMIDGWYGYGRKKR
SEQ ID NO: 142


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRYGRKKRRQRR
SEQ ID NO: 143


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRYGRKKRRQRR
SEQ ID NO: 144


CGLFEAIKGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 145


CGLFEAIHGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 146


CGLFEAIRGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 147


CGLFEAIDGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 148


CRLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 149


CGGGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 150


CGLFEAIEGFIENGWEGMIDGWYGGGGYGRKKRRQRR
SEQ ID NO: 151


CGLFEAIEGFIENGWEGMIDGWYG-(PEG)11-YGRKKRRQRR
SEQ ID NO: 152


CFLGFLLGVGSAIASGIAVSKVLHL
SEQ ID NO: 153


CGVFVLGFLGFLATAGSAMGARSLTLSAYGRKKRRQRR
SEQ ID NO: 154


Ac-GLWRALWRLLRSLWRLLWRA-mercaptoethylamide
SEQ ID NO: 155


C-Nle-LFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 156


CELFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 157


CGFFGAIAGFLEGGWEGMIAGWHGYGRKKRRQRR
SEQ ID NO: 158


CFLGFLLGVGSAIASGIAVSKVLHLYGRKKRRQRR
SEQ ID NO: 159


GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC
SEQ ID NO: 160


CGLFEAIEGFIENGWEGMIDGWYGLHLLHHLLHHLHHLLHHLLH
SEQ ID NO: 161


LLHHLLHHL



CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRLHLLHHLL
SEQ ID NO: 162


HHLHHLLHHLLHLLHHLLHHL



CGLFGAIAGFIEGGWTGMIDGWYGYGRKKRRQRR
SEQ ID NO: 163


CGLFGAIAGFIEGGWQGMVDGWYGYGRKKRRQRR
SEQ ID NO: 164


CGLFGAIAGFIENGWQGLIDGWYGYGRKKRRQRR
SEQ ID NO: 165


CGLFGAIAGFIENGWEGLVDGWYGYGRKKRRQRR
SEQ ID NO: 166


CGLFGAIAGFIEGGWSGMIDGWYGYGRKKRRQRR
SEQ ID NO: 167


CGLFGAIAGFIEGGWPGLVAGWYGYGRKKRRQRR
SEQ ID NO: 168


CGLFGAIAGFIENGWEGMVDGWYGYGRKKRRQRR
SEQ ID NO: 169


CGLFGAIAGFIEGGWPGLINGWYGYGRKKRRQRR
SEQ ID NO: 170


CGLFGAIAGFIENGWEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 171


CGLFGAIAGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 172


CGLFGAIAGFIENGWEGMIDGWYGSSKKKK
SEQ ID NO: 173


CGLFGAIAGFIENGWEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 174


CGLFEAIEGFIENGWEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 175


CGLFGAIAGFIENGWEGLIEGWYGGGRKKRRQRR
SEQ ID NO: 176


CGLFEAIEGFIENGWEGMIDGWYGGGRKKRRQRR
SEQ ID NO: 177


CGLFEAIAGFIENGWEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 178


CGLFEAIAEFIENGWEGLIEGWYGGRKKRRQRR
SEQ ID NO: 179


CGLFEAIEGFIENGWEGMIDGWYGRKKRRQRRR
SEQ ID NO: 180


CKLLKLLLKLWLKLLKLLLKLL
SEQ ID NO: 181


CKLLKLLLKLWLKLLKLLLKLLYGRKKRRQRR
SEQ ID NO: 182


GLFEAIEGFIENGWEGMIDGWYGC
SEQ ID NO: 183


CVLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 184


CSLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 185


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQ
SEQ ID NO: 186


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRR
SEQ ID NO: 187


CGLFEAIEGFIENGWEGMIDGWYGYGKKKKKQKK
SEQ ID NO: 188


CGLFEAIEGFIENGWEGMIDGWYGGLFEAIEGFIENGWEGMIDGWYG
SEQ ID NO: 189


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRGLFEAIEG
SEQ ID NO: 190


FIENGWEGMIDGWYGYGRKKRRQRR



RRQRRKKRGYGYWGDIMGEWGNEIFGEIAEFLGC
SEQ ID NO: 191


CRRQRRKKRGYGYWGDIMGEWGNEIFGEIAEFLG
SEQ ID NO: 192


GLFEAIEGFIENGWEGMIDGWYGYGRK-K(D)-RRQRR
SEQ ID NO: 193


GLFEAIEGFIENGWEGMIDGWYGYGRKK-R(D)-RQRR
SEQ ID NO: 194


GL-F(D)-EAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 195


GLF-E(D)-AIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 196


CGLFEAIEGFIENGWEGMIDGWYG
SEQ ID NO: 197


CYGRKKRRQRR
SEQ ID NO: 198


YGRKKRRQRRC
SEQ ID NO: 199


RRQRRKKRGYGYWGDIMGEWGNEIFGEIAEFLGC all(D)
SEQ ID NO: 200


CRRQRRKKRGYGYWGDIMGEWGNEIFGEIAEFLG all(D)
SEQ ID NO: 201


CGLFEAIEGFIENGWEGMIDGAYGYGRKKRRQRR
SEQ ID NO: 202


CGLFEALLELLESLWELLLEAWYGYGRKKRRQRR
SEQ ID NO: 203


CGLFEAIEGFNENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 204


CGLFEAIEGFIENEWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 205


K(stearoyl)GLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRC
SEQ ID NO: 206


CGLFEAIK(stearoyl)GFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 207


CGLFEAIKGFIENGWEGMIDGWYGYGRK(stearoyl)KRRQRR
SEQ ID NO: 208


CGLFEAIEGFIENPWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 209


(stearyl)GLFEAIEGFIENPWEGMIDGWYGYGRKKRRQRRC
SEQ ID NO: 210


CGLFGAIAGFIEGGWPGLINGWYGYGRKKRRQRRLHLLHHLLH
SEQ ID NO: 211


HLHHLLHHLLHLLHHLLHHL



CGLFGAIAGFIEGGWPGLINGWYGYGRKKRRQRRLHLLHHLLH
SEQ ID NO: 212


HLHHLLHHLLHL



CGLFEAIAGFIEGGWPGLINGWYGYGRKKRRQRR
SEQ ID NO: 213


CGLEEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 214


CGLFNAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 215


CGLFAAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 216


CGLFEAIENFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 217


CGLFEAIEKFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 218


CGLFEAIEGFAENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 219


CGLFEAIEGFIENWWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 220


CGLFEAIEGFIENNWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 221


CGLFEAIEGFIENGEEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 222


CGLFEAIEGFIENGWAGMIDGWYGYGRKKRRQRR
SEQ ID NO: 223


CGLFEAIEGFIENGWNGMIDGWYGYGRKKRRQRR
SEQ ID NO: 224


CGLFEAIEGFIENGWGGMIDGWYGYGRKKRRQRR
SEQ ID NO: 225


CGLFEAIEGFIENGWEGMIDAWYGYGRKKRRQRR
SEQ ID NO: 226


CGLFEAIEGFIENGWLGMIDGWYGYGRKKRRQRR
SEQ ID NO: 227


CGLFEAIEGFIENGWKGMIDGWYGYGRKKRRQRR
SEQ ID NO: 228


CGLFEAIEGFIENGWEGMIDKWYGYGRKKRRQRR
SEQ ID NO: 229


CGLFEAIEGFIENGWEGMIDEWYGYGRKKRRQRR
SEQ ID NO: 230


CGLFEAIEGFIENGWEGMIDGLYGYGRKKRRQRR
SEQ ID NO: 231


CGLFEAIEGFIENGWEGMIDGNYGYGRKKRRQRR
SEQ ID NO: 232


CGLFEAIEGFIENGWEGMIDGKYGYGRKKRRQRR
SEQ ID NO: 233


CGLFEAIEGFIENGWEGMIDGEYGYGRKKRRQRR
SEQ ID NO: 234


CGLFEALEELLEGGWEGLIEAWYGYGRKKRRQRR
SEQ ID NO: 235


CELFGAIWEFIEGGWEGLIEAWYGYGRKKRRQRR
SEQ ID NO: 236


CGLFEALEEFIEGGWEGLLEAWYGYGRKKRRQRR
SEQ ID NO: 237


CGLFEALEEFIENGWEGLLEAWYGYGRKKRRQRR
SEQ ID NO: 238


CGLFEAIEGFIESGWEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 239


CGLFEAIEEFIEGGWEGLIEAWYGYGRKKRRQRR
SEQ ID NO: 240


CGLFEAIEGFIENGWEGLIDAWYGYGRKKRRQRR
SEQ ID NO: 241


CGLFEAIEGFILNGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 242


CGLFEAIEGFIKNGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 243


CGLFEAIEGFIGNGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 244


CGLFEAIEGFIELGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 245


CGLFEAIEGFIEKGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 246


CGLFEAIAEFIEGGWEGLIEGWYGYGRKKRRQRR
SEQ ID NO: 247


CRGWEVLKYWWNLLQY
SEQ ID NO: 248


CRGWEVLKYWWNLLQYYGRKKRRQRR
SEQ ID NO: 249


CGLFGAIAGFIENGWEGMIDGWYGFRYGRKKRRQRR
SEQ ID NO: 250


Ac-CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR-CO2H
SEQ ID NO: 251


CGLLEALEGLLENGWEGLLEAWYGYGRKKRRQRR
SEQ ID NO: 252


CLRHLLRHLLRHLRHLLRHLRHLLRHLLRH
SEQ ID NO: 253


CGIFEAIEGFIENGWEGIIDGWYGYGROORRQRR (O = ornithine)
SEQ ID NO: 254


CGIGAVLKVLTTGLPALISWIKRKRQQ
SEQ ID NO: 255


CGIGAVLKVLTTGLPALISWIHHHHQQ
SEQ ID NO: 256


CGAFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 257


Ac-LHLLHHLLHHLHHLLHHLLHLLHHLLHHLRRRRR
SEQ ID NO: 258


CGLFGAIWGFIENWWKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 259


CGLFGAIEGFIENGWKGLIDAWYGYGRKKRRQRR
SEQ ID NO: 260


CGLFEAIAGFIENGWKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 261


GLFEAIEGFIENGWKGLIDWWYGYGRKKRRQRRC
SEQ ID NO: 262


YGRKKRRQRRGLFEAIEGFIENGWKGLIDAWYGC
SEQ ID NO: 263


YGRKKRRQRRGLFEAIEGFIENGWKGLIDWWYGC
SEQ ID NO: 264


CGLFHAIHGFIENGWHGLIDWWYGYGRKKRRQRR
SEQ ID NO: 265


CGLFEAIEGFIENGWKGLIDWWYGYGRKKRRQRRK(stearyl)
SEQ ID NO: 266


CGLFKALLKLLKSLWKLLLKAWYGYGHKKHHQHR
SEQ ID NO: 267


CGLFKALLKLLKSLWKGLLKAWYGYGHKKHHQHR
SEQ ID NO: 268


CGLAKALLKLLKSLWKGLIEAWYGYGRKKRRQRR
SEQ ID NO: 269


CGIFGAIAGFIKNIW
SEQ ID NO: 270


CIFGAIAGFIKNIWEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 271


CGIFGAIAGFIKNIWEGLIDGYGRKKRRQRR
SEQ ID NO: 272


CGIFGAIAGFIKNIWKGLIDAWYGYGRKKRRQRR
SEQ ID NO: 273


CIFGAIAGFIKNIWKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 274


CLFGAIAGFIKNIW
SEQ ID NO: 275


CGL(R5)EAIEGF(S8)ENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 276


CGLFEA(S5)EGF(S5)ENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 277


CGLFEAIEGFIENGWEGAIDGWYGYGRKKRRQRR
SEQ ID NO: 278


CGLFEAIEGFIENGWEGEIDGWYGYGRKKRRQRR
SEQ ID NO: 279


CGIFGAIAGFIKNGWEGMVDWYGYGRKKRRQRR
SEQ ID NO: 280


CGLFEAIAGFIENGWEGMIDGWYGFYGRKKRRQRR
SEQ ID NO: 281


CGIFGAIAGFIKNGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 282


CIFGAIAGFIKNIW
SEQ ID NO: 283


CIFGAIAGFIKNIWYGRKKRRQRR
SEQ ID NO: 284


CGIFGAIAGFIKNIWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 285


CGLFEAIEGFIENGWEGLIEAYGRKKRRQRR
SEQ ID NO: 286


CGLFEALLGFIENGWEGLIDGYGRKKRRQRR
SEQ ID NO: 287


CGLFGAIEGFIENGWEGLIDGWYGYGRKKRRQRRR
SEQ ID NO: 288


CELFGAIEGFIENGWEGMIDGWYGYGRKKRRQRRR
SEQ ID NO: 289


CGLFEAIEGFIENGWEGMIDGWYGYGHKKHHQHR
SEQ ID NO: 290


CGLFGAIEGFIEGGWPGLINGWYGYGRKKRRQRRR
SEQ ID NO: 291


CGLFKALLKLLKSLWKLLLKAYGRKKRRQRR
SEQ ID NO: 292


CGLFKALLKLLKSLWKLLLKAWYGYGRKKRRQRR
SEQ ID NO: 293


CGLFRALLRLLRSLWRLLLRAYGRKKRRQRR
SEQ ID NO: 294


CGLFEAILGFIENGWEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 295


CGLFEAIWEFIENGWEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 296


CGLFEAIEGFIENGWEGMIDGWYGGGGLHLLHHLLHHLHHLLHHLLHL
SEQ ID NO: 297


CGPVEDAITAAIGRVADTVGTYGRKKRRQRR
SEQ ID NO: 298


CMDGTLFPGDDDLAIPATEFFSTKA
SEQ ID NO: 299


CGLFEALEEFIEGGWEGLLEAWYGYGRKKRRQRR
SEQ ID NO: 300


CGLFEALEEFIENGWEGLLEAWYGYGRKKRRQRR
SEQ ID NO: 301


CELFGAIWEFIEGGWEGLIEAYGRKKRRQRR
SEQ ID NO: 302


CGLFEAIEGFIEEGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 303


CGLFEAIAEFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 304


CGLFEAIAEFIEGLWEGLIEGWYGYGRKKRRQRR
SEQ ID NO: 305


CGLLEALEGLLESLWEGLLEAWYGYGRKKRRQRR
SEQ ID NO: 306


CGLFEAIEGFIENGWEGMIDIWYGYGRKKRRQRR
SEQ ID NO: 307


CGLFEAIEGFIENGWRGMIDGWYGYGRKKRRQRR
SEQ ID NO: 308


CGLFEAIEGFIENGWDGMIDGWYGYGRKKRRQRR
SEQ ID NO: 309


CGLFEAIEGFIENHWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 310


CGLFEAIEGFIENWWKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 311


GLFEAIEGFIENGWKGLIDAWYGYGRKKRRQRRC
SEQ ID NO: 312


CGLFEAIEGFIENGWKGMIDAWYGYGRKKRRQRR
SEQ ID NO: 313


CGLFEAIEGFIENGWKGMIDWWYGYGRKKRRQRR
SEQ ID NO: 314


CGLAEAIEGFIENGLKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 315


RRQRRKKRGYGYVVGDILGEWGNEIFGEIAEFLGC all(D)
SEQ ID NO: 316


CRRQRRKKRGYGYWGDILGEWGNEIFGEIAEFLG all(D)
SEQ ID NO: 317


CGLFEAIEGFIENGWKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 318


CGFFEAIEGFIENGLKGLIDAWYGYGRKKRRQRR
SEQ ID NO: 319


CGLFEAIEGFIENGLKGLIDAWYGYGRKKRRQRR
SEQ ID NO: 320


CELFGAIEGFIENGWKGLIDAWYGYGRKKRRQRR
SEQ ID NO: 321


CGLFKAIKGFIKNGWKGLIKAWYGYGRKKRRQRR
SEQ ID NO: 322


CGLAEALLELLESLWKGLIEAYGRKKRRQRR
SEQ ID NO: 323


CGIFGAIEGFIENGWKGLIDAWYGYGRKKRRQRR
SEQ ID NO: 324


CGIAGAIAGFIKNIWEGLIDWWYGYGRKKRRQRR
SEQ ID NO: 325


CGIAGAIAGFIKNIWKGLIDAWYGYGRKKRRQRR
SEQ ID NO: 326


CGIFGAIAGFIKNIWEGLIDGWYGKKKKKKKKK
SEQ ID NO: 327


CG(R5)FEAIEG(S8)IENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 328


CGLFEAIEGF(R5)ENGWEG(S8)IDGWYGYGRKKRRQRR
SEQ ID NO: 329


GLFEAIEGFIENGWEGMIDGWYGCYGRKKRRQRR
SEQ ID NO: 330


GLFEAIEGFIENGWEGMIDGWYGGCGYGRKKRRQRR
SEQ ID NO: 331


GLLEALEGLLENGWEGLLDGWYGYGRKKRRQRR
SEQ ID NO: 332


CFFGAIWEFIRNIL
SEQ ID NO: 333


CIFGAIAGFIRSIL
SEQ ID NO: 334


CGLFEEIEEFIENGWEGLIDWWYGYGRKKRRQRR
SEQ ID NO: 335


CGFFGAIWEFIKSIL
SEQ ID NO: 336


GFFGAIWEFIKSILC
SEQ ID NO: 337


CGLFEALEGFIENGWEGLLDGWYGYGROORRQRR (O = ornithine)
SEQ ID NO: 338


CGLFEALLELLENGWELLLEAWYGYGRKKRRQRR
SEQ ID NO: 339


CGLFEALLELLENGWELLLDGWYGYGRKKRRQRR
SEQ ID NO: 340


CALFEAIEAFIENGWEAMIDAWYGYGRKKRRQRR
SEQ ID NO: 341


CGLFGAIWGFIENGWEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 342


CGLFEAIEELIENLWKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 343


CGLFEEIEGFIENGWKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 344


CGLFEEIEGFIENGWKGLIDWWYGYGHKKHHQHR
SEQ ID NO: 345


CFFGAIWEFIKNILKGLIDGWYG
SEQ ID NO: 346


CGIFGAIAGFIRSIL
SEQ ID NO: 347


CGLFEEIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 348


CGLFEAIEGFIENGWEGMIDGWNGYGRKKRRQRR
SEQ ID NO: 349


AGYLLGKINLKALAALAKKILHHHHHHKKKKKKC
SEQ ID NO: 350


Bis CGLFEAIEGFIENGWEGMIDWWYGYGRKKRRQRR
SEQ ID NO: 351


CGLFEAIEGFIENGWEGMIDGWYG-(PEG)6-YGRKKRRQRR
SEQ ID NO: 352


CGIFGAIWNGIKSLFEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 353


CGIFGAIEGFIENGWEGLIDWWYGYGRKKRRQRR
SEQ ID NO: 354


CIFGAIAGFIKNIWEGLIDWWYGYGRKKRRQRR
SEQ ID NO: 355


CGLFEAIEGFIENGWKGLIDGWYGGLFEAIEGFIENGWKGLIDWWYG
SEQ ID NO: 356


CWEAALAEALAEALAEHLAEALAEALEALAAYGRKKRRQRRK(stearyl)
SEQ ID NO: 357


CGLFEAIEGFIENGWKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 358


CGLFEELEELLEEGWEGLLEAYGRKKRRQRR
SEQ ID NO: 359


CGNFEEIEEFIEEGLRNFIDWWYGYGHKKHHQHR
SEQ ID NO: 360


CFFGAIWEFIRNILEGLIDWWYGYGRKKRRQRR
SEQ ID NO: 361


CFFGAIWEFIKNILLHLLHHLLHHLHHLLHHLLHL
SEQ ID NO: 362


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR all(D)
SEQ ID NO: 363


CGFFHAFFHFFHSFWHGFFEA
SEQ ID NO: 364


CGLFHALLHLLHSLWHGLLHWWYGYGHKKHHQHR
SEQ ID NO: 365


CGLFGALLELLESLWEGLLEWYGRKKRRQRR
SEQ ID NO: 366


CGLFGALLELLESLWEGLLEWYGHKKHHQHR
SEQ ID NO: 367


CGLFHALLHLLHSLWKGLLEWWYGF
SEQ ID NO: 368


CIFGAIAGFIRSILEGF
SEQ ID NO: 369


CGIFGAIAGFIKNIWKGLIDA
SEQ ID NO: 370


CFFEAIEEFIKNIWK
SEQ ID NO: 371


CGLFEAIEGFIENGWKGLIDWLAEALAEALEALAA
SEQ ID NO: 372


GCGIFGAIAEFIKNIW
SEQ ID NO: 373


CIFGAIAEFIKNIWKGLIDW
SEQ ID NO: 374


CFFGAIWEFIKSILELLLEAYGHKKHHQHRR
SEQ ID NO: 375


CWFGAIWEFIKSIL
SEQ ID NO: 376


CAFGAIWEFIKSIL
SEQ ID NO: 377


CFLGAIWEFIKSIL
SEQ ID NO: 378


CFFGAIWEFIKSIK
SEQ ID NO: 379


CGFIGAIANLLSKIFEGLIDGWYGYGRKKRRQRR all(D)
SEQ ID NO: 380


CFFGAIWEFIKSIL
SEQ ID NO: 381


CIFGAIAGFIKNIWLHLLHHLLHHLHHLLHHLLHL all(D)
SEQ ID NO: 382


CFFGAIAEFIKNIW
SEQ ID NO: 383


CIFEAIWGFIKNIW
SEQ ID NO: 384


stearyl-AGYLLGKINLKALAALAKKILHHHHHHKKKKKKC
SEQ ID NO: 385


CIFEAIAGFIKNIWKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 386


CGLFEAIEGFIENGWKGLIDWWYGGRPRESGKKRKRKRLKP
SEQ ID NO: 387


C(b-Ala)GFGEIEEFIENGLKNLIDWWYGYGHKKHHQHR
SEQ ID NO: 388


C(b-Ala)GFEFIEEFIENGLKNLIDWWYGYGRKKRRQRR
SEQ ID NO: 389


C(b-Ala)GFEFIEEFIENGLKNLIDWWYGYGHKKHHQHR
SEQ ID NO: 390


CGGIEEIAGLLSKILKGLIDWWYGYGHKKHHQHR
SEQ ID NO: 391


CGFIGAIANLLSKIFEGLIDWWYGYGRKKRRQRR
SEQ ID NO: 392


CGFIGAIAELLEKIFEGLIDWWYGYGRKKRRQRR
SEQ ID NO: 393


CGFIGAIAELLEKIFEGLIDWWYGYGHKKHHQHR
SEQ ID NO: 394


CFFGAIWEFIRNILEGLIDWWYGYGHKKHHQHR
SEQ ID NO: 395


CFFGAIWEFIKSILLHLLHHLLHHLHHLLHHLLHL
SEQ ID NO: 396


CFFGAIWEFIRSILLHLLHHLLHHLHHLLHHLLHL
SEQ ID NO: 397


CGFFGAIWEFIRSILEGFIDWWYGYGYGHKKHHQHR
SEQ ID NO: 398


CGLFEAIWEFIKSILEGLLEAYGHKKHHQHR
SEQ ID NO: 399


CGLFEAIWEFIKSILEGLLEAWYGYGHKKHHQHR
SEQ ID NO: 400


CGIFGAIAGFIKNIWKYGRKKRRQRR
SEQ ID NO: 401


CGLFEALLELLESLWELLLEAWYGYGHKKHHQHR
SEQ ID NO: 402


CIFGAIAGFIRNIWKGLIDGWYG
SEQ ID NO: 403


CGIFGAIAGFIRNIWKGLIDGWYG
SEQ ID NO: 404


CFFGAIWEFIKNILKLHLLHHLLHHLHHLLHHLLHL
SEQ ID NO: 405


CFFGAIWEFIRNILLHLLHHLLHHLHHLLHHLLHL
SEQ ID NO: 406


CFFGKIWEFIKSIL
SEQ ID NO: 407


CYGRKKRRQRRGLFEALLELLESLWELLLEA
SEQ ID NO: 408


FFGAIWEFIKSILC
SEQ ID NO: 409


CWWGAIEGFIKSIL
SEQ ID NO: 410


CFFGAIWEWIKSIL
SEQ ID NO: 411


CFFGAIWEFWKSIL
SEQ ID NO: 412


CFFGAIWEFIKFIL
SEQ ID NO: 413


CFFGAIWEFIKKIL
SEQ ID NO: 414


CFFGAIWEFIKGIL
SEQ ID NO: 415


CFFGAIWEFIKLIL
SEQ ID NO: 416


CFFGAIWEFIKWIL
SEQ ID NO: 417


CFFGAIWEFIKSFL
SEQ ID NO: 418


CFFGAIWEFIKSKL
SEQ ID NO: 419


CFFGFIWEFIKSIL
SEQ ID NO: 420


CIFGAIAGFIKNILKGLIDAF
SEQ ID NO: 421


CFFGKIWELWEWIL
SEQ ID NO: 422


CFFGAIWEFAKSIL
SEQ ID NO: 423


CFFGAIWEFIKSAL
SEQ ID NO: 424


CFFGAIWEFIKSWL
SEQ ID NO: 425


CFFGAIWEFIKSILK
SEQ ID NO: 426


CFFGAIWEFIKSILE
SEQ ID NO: 427


CFFKAIWEFIKSIL
SEQ ID NO: 428


CFFNAIWEFIKSIL
SEQ ID NO: 429


CFFGGIWEFIKSIL
SEQ ID NO: 430


CFFGNIWEFIKSIL
SEQ ID NO: 431


CFFGALWEFIKSIL
SEQ ID NO: 432


CFFGAAWEFIKSIL
SEQ ID NO: 433


CGLFHALLHLLHSLWHGLLDG
SEQ ID NO: 434


CGLFHALLHLLHSLWHGLLEW
SEQ ID NO: 435


CGLFHALLHLLHSLWHLLLEA
SEQ ID NO: 436


CGLFHALLHLLHSLWKLLLEW
SEQ ID NO: 437


CKFGAIWEFIKSIL
SEQ ID NO: 438


CFKGAIWEFIKSIL
SEQ ID NO: 439


CFFGAIWKFIKSIL
SEQ ID NO: 440


CFFGAIWAFIKSIL
SEQ ID NO: 441


CFFGAIWLFIKSIL
SEQ ID NO: 442


CFFGAIWFFIKSIL
SEQ ID NO: 443


CFFGAIWNFIKSIL
SEQ ID NO: 444


CFFGAIWELIKSIL
SEQ ID NO: 445


CFFGAIWEAIKSIL
SEQ ID NO: 446


CGLFEAIEGFIENGWEGLAEALAEALEALAAYGRKKRRQRR
SEQ ID NO: 447


CIFGAIAGFIKNIWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 448


CIFGAIAGFIKNIWEGLIDAWYGYGRKKRRQRR
SEQ ID NO: 449


CIFGAIAGFIKNIWKGLIDAWYGYGRKKRRQRR
SEQ ID NO: 450


CIFGAIAGFIKNIWIFGAIAGFIKNIWWYGYGRKKRRQRR
SEQ ID NO: 451


CGLFGAIAGFIENGWEGLIEGWYG
SEQ ID NO: 452


CGLFEAIEGFIENGWEGLIDGWYGYGOOOOOQRR (O = ornithine)
SEQ ID NO: 453


CGLFEAIEGFIENGWKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 454


CGLFEAIEGFIENGWEGLIDGWYGYGRKKRRQRRK(stearyl)
SEQ ID NO: 455


CYGHKKHHQHRGLFEAIEGFIENGWKGLIDWWYG
SEQ ID NO: 456


CYGHKKHHQHRGLFEAIEEFIENGWEGLIDGWYG
SEQ ID NO: 457


CGLFEAIEGFIENGWKGLIDGWYGYGRKKRRQRRK(stearyl)
SEQ ID NO: 458


CGLFEAIEGFIENGWHGMIDGWYGYGRKKRRQRR
SEQ ID NO: 459


IFGIDDLIIGLLFVAIVEAGIGGYLLGSYGRKKRRQRRC
SEQ ID NO: 460


CGFFGEIAELIEEGLKGLIDWWNG
SEQ ID NO: 461


CGLFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 462


CFFGAIWEFIHSIL all (D)
SEQ ID NO: 463


CFFGAIWEFIHNIL
SEQ ID NO: 464


CFFGAIWEFIHSIFK
SEQ ID NO: 465


CGIFEAIAGLLKWIFK
SEQ ID NO: 466


CGIFELIAGLLKNIFK
SEQ ID NO: 467


CGIFEAIAGLLKSILKK(stearyl)
SEQ ID NO: 468


CGIFGAIAGLLKSILKK(stearyl)
SEQ ID NO: 469


CIFGAIAGFIKNILKGL all (D)
SEQ ID NO: 470


CIFGAIAGFIKNILKGLIDGWWYG
SEQ ID NO: 471


CIFGAIAGFIKNIWHGLI
SEQ ID NO: 472


CIFGAIAGFIKNILKGLK(stearyl)
SEQ ID NO: 473


GLGKLINKIFGAIAGFIC all (D)
SEQ ID NO: 474


CGIFEAIAGLLKNIFD
SEQ ID NO: 475


CGIFEAIAGLLKNIFE
SEQ ID NO: 476


CGIFEAIAGLLKNIFR
SEQ ID NO: 477


CGIFEAIAGLLKNIFH
SEQ ID NO: 478


CGIFEAIAGLLKNIFO (O = ORNITHINE)
SEQ ID NO: 479


CGIFEAIAGLLKNIFN
SEQ ID NO: 480


CGIFEAIAGLLKNIFCit (Cit = citrulline)
SEQ ID NO: 481


CGIFEAIWGLLKNIFK
SEQ ID NO: 482


CGIFGAIWGLLKNIFK
SEQ ID NO: 483


CIFGAIAGLLKNIFK
SEQ ID NO: 484


CIFEAIAGLLKNIFK
SEQ ID NO: 485


CFFGAIAGLLKNIFK
SEQ ID NO: 486


CFFEAIAGLLKNIFK
SEQ ID NO: 487


CGFFEAIAGLLKNIFK
SEQ ID NO: 488


CIFGAIAGFIKNIWEGLI all (D)
SEQ ID NO: 489


CIFGAIAGLLKNIFK all(D)
SEQ ID NO: 490


CGLFGEIEELIEEGLENLIDWWNG all(D)
SEQ ID NO: 491


CGNFGEIEELIEEGLENLIDWWNG all(D)
SEQ ID NO: 492


CGFFGEIAELIEEGLKGLIDWWNG all(D)
SEQ ID NO: 493


CGLFGEIEELIEEGLENLIDWWNE
SEQ ID NO: 494


CGFFGAIAGLLKNIFK
SEQ ID NO: 495


CGLFELIEGFIENGWEGMIDGWYGYGRKKRRQRRK(stearyl)
SEQ ID NO: 496


CGLFELIEGFIEWGWEGMIDGWYGYGRKKRRQRRK(stearyl)
SEQ ID NO: 497


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRK(2H,2H,3H,3H-
SEQ ID NO: 498


perfluorononanoyl)



CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRK(2H,2H,3H,
SEQ ID NO: 499


3H-perfluoro-10 methylundecanoyl)



CIFGAIAGFIKNIWEGLIK(2H,2H,3H,3H-perfluorononanoyl)
SEQ ID NO: 500


CIFGAIAGFIKNIWEGLIK(2H,2H,3H,3H-perfluoro-10 methylundecanoyl)
SEQ ID NO: 501


CGLFEAIEGFIEWGWEGMIDGWYGYGRKKRRQRRK(2H,2H,3H,3H-
SEQ ID NO: 502


perfluorononanoyl)



CGLFEAIEGFIEWGWEGMIDGWYGYGRKKRRQRRK(2H,2H,3H,3H-
SEQ ID NO: 503


perfluoro-10 methylundecanoyl)



CGLFELIEGFIENGWEGMIDGWYGYGRKKRRQRRK(2H,2H,3H,3H-
SEQ ID NO: 504


perfluorononanoyl)



CGLFELIEGFIENGWEGMIDGVVYGYGRKKRRQRRK(2H,2H,3H,3H-
SEQ ID NO: 505


perfluoro-10 methylundecanoyl)



CFFGAIWEFIHSILK(2H,2H,3H,3H-perfluorononanoyl)
SEQ ID NO: 506


CFFGAIWEFIHSILK(2H,2H,3H,3H-perfluoro-10 methylundecanoyl)
SEQ ID NO: 507


CIFGAIAGFIKNILKGLK(2H,2H,3H,3H-perfluorononanoyl)
SEQ ID NO: 508


CIFGAIAGFIKNILKGLK(2H,2H,3H,3H-perfluoro-10 methylundecanoyl)
SEQ ID NO: 509


CFFGAIWEFIRNILEGFK(2H,2H,3H,3H-perfluorononanoyl)
SEQ ID NO: 510


CFFGAIWEFIRNILEGFK(2H,2H,3H,3H-perfluoro-10 methylundecanoyl)
SEQ ID NO: 511


CGLFGEIEELIEEGLENLIDWWNQ
SEQ ID NO: 512


CGIFGAIAGLLKSALK
SEQ ID NO: 513


CGIFEAIAGLLKSIWK
SEQ ID NO: 514


CGIFEAIAGLLKSILK
SEQ ID NO: 515


CGIFEAIAGLLONIFK (O = Ornithine)
SEQ ID NO: 516


CGIFEAIAGLLKNILKGLIDGWYG
SEQ ID NO: 517


CGIFGAIAGLLKNILKGLIDGWYG
SEQ ID NO: 518


CGIFGAIAGLLKNIFKGLIDGWYG
SEQ ID NO: 519


CGIFGAIWELWEWILK
SEQ ID NO: 520


CGIFEAIWELWEWILK
SEQ ID NO: 521


CGLFEAIEGFIENGWEGMIDGWYGK(stearyl)
SEQ ID NO: 522


(stearyl)GLFEAIEGFIENGWEGMIDGWYGC
SEQ ID NO: 523


CFLE-Aib-LWKLLEHLL
SEQ ID NO: 524


CFLE-Aib-LWELLEHLL
SEQ ID NO: 525


CFLEALWE-Aib-LEHLL
SEQ ID NO: 526


CFLE-Aib-LWE-Aib-LEHLL
SEQ ID NO: 527


CFLE-Aib-LWEALEKLF
SEQ ID NO: 528


(stearyl)IFGAIAGFIKNIWEGLIC
SEQ ID NO: 529


CIFGAIAGFIKNIWEGLIK(stearyl)
SEQ ID NO: 530


(stearyl)FFGAIWEFIKSILC
SEQ ID NO: 531


CFFGAIWEFIKSILK(stearyl)
SEQ ID NO: 532


(stearyl)FFGAIWEFIHSILC
SEQ ID NO: 533


CFFGAIWEFIHSILK(stearyl)
SEQ ID NO: 534


(stearyl)GIFEAIAGLLKNIFKC
SEQ ID NO: 535


CGIFEAIAGLLKNIFK(stearyl)
SEQ ID NO: 536


CGIFEAIAGLLKNIFKK(stearyl)
SEQ ID NO: 537


(stearyl)IFGAIAGFIKNILKGLC
SEQ ID NO: 538


CIFGAIAGFIKNILKGLK(stearyl)
SEQ ID NO: 539


CIFGAIAGFIKNILKGL
SEQ ID NO: 540


CGLFGEIEELIEEGLENLIDWWNS
SEQ ID NO: 541


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 542


CGFFGEIAELIEEGLKNLIDWWNG
SEQ ID NO: 543


CGLFEAIEGFIENGWKGMIDGWYGYGRKKRRQRR
SEQ ID NO: 544


CGLFEAIEGFIEWGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 545


CGLFELIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 546


CIFGAIAGFIKNIWEGLI
SEQ ID NO: 547


CGLFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 548


CGLFEEIEGFIENGWEGLIDWWYGYGHKKGGQHR
SEQ ID NO: 549


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRK(stearyl)
SEQ ID NO: 550


CGLFEALLELLESLWELLEAYGRKKRRQRR
SEQ ID NO: 551


CGLFEALLELLESLWELLEAYGRKKRRQRR
SEQ ID NO: 552


CFFGAIWEFIRNILEGF
SEQ ID NO: 553


CFFGAIWEFIRNILEGFK(stearyl)
SEQ ID NO: 554


CIFGAIAGFIKNIWEGLIK(lauryl)
SEQ ID NO: 555


(lauryl)FFGAIWEFIKSILC
SEQ ID NO: 556


CFFGAIWEFIKSILK(lauryl)
SEQ ID NO: 557


(lauryl)FFGAIWEFIHSILC
SEQ ID NO: 558


CFFGAIWEFIHSILK(lauryl)
SEQ ID NO: 559


(lauryl)GIFEAIAGLLKNIFKC
SEQ ID NO: 560


CGIFEAIAGLLKNIFK(lauryl)
SEQ ID NO: 561


CFFGAIWEFIRNILEGFK(lauryl)
SEQ ID NO: 562


(lauryl)GLFEAIEGFIENGWEGMIDGWYGC
SEQ ID NO: 563


CGLFEAIEGFIENGWEGMIDGWYGK(lauryl)
SEQ ID NO: 564


CGKFTIVFPHNQKGNWKNVPSNYHYK(stearyl)
SEQ ID NO: 565


CMDGTLFPGDDDLAIPATEFFSTKAK(stearyl)
SEQ ID NO: 566


CNPVENYIDEVLNEVLWPNINSSNK(stearyl)
SEQ ID NO: 567


CVTPHHVLVDEYTGEWVDSQFK(stearyl)
SEQ ID NO: 568


CIFGIDDLIIGLLFVAIVEAGIGGYLLGSK(stearyl)
SEQ ID NO: 569


CGAAIGLAWIPYFGPAAEK(stearyl)
SEQ ID NO: 570


CFAGWLAGAALGVATAAQITAGIALHK(stearyl)
SEQ ID NO: 571


CFLGFLLGVGSAIASGIAVSKVLHLK(stearyl)
SEQ ID NO: 572


CFFGAVIGTIALGVATSAQITAGIALAK(stearyl)
SEQ ID NO: 573


CFFGAVIGTIALGVATAAQITAGIALAK(stearyl)
SEQ ID NO: 574


GLFEAIAGFIENGGWEGMIDGGGK(stearyl)
SEQ ID NO: 575


GLFKAIAKFIKGGWKGLIKGWYGK(stearyl)
SEQ ID NO: 576


GLFHAIAHFIHGGWHGLIHGWYGK(stearyl)
SEQ ID NO: 577


CGLFEAIAEFIENGWEGLIEGWYGK(stearyl)
SEQ ID NO: 578


CGFFGAIAGFLEGGWEGMIAGWHGK(stearyl)
SEQ ID NO: 579


CFAGVVIGLAALGVATAAQVTAAVALVKK(stearyl)
SEQ ID NO: 580


CAVGIVGAMFLGFLGAAGSTMGAVSLTLTVQAK(steary1)
SEQ ID NO: 581


CGVFVLGFLGFLATAGSAMGARSLTLSAK(stearyl)
SEQ ID NO: 582


CVPFVLGFLGFLGAAGTAMGAAATALTVK(stearyl)
SEQ ID NO: 583


CAVPVAVWLVSALAMGAGVAGGITGSMSLASGK(stearyl)
SEQ ID NO: 584


CGLASTLTRWAHYNALIRAFK(stearyl)
SEQ ID NO: 585


CGPVEDAITAAIGRVADTVGTK(stearyl)
SEQ ID NO: 586


CGLGQMLESMIDNTVREVGGAK(stearyl)
SEQ ID NO: 587


CGLFEAIEGFIENGWEGMIDGWYGFK(stearyl)
SEQ ID NO: 588


(D)-(cgl)FEAIEGFIENGWEGMIDGWYGYGRKKRR(D)-(qrr)
SEQ ID NO: 589


CGODLEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 590


CIFGIDDLIIGLLFVAIVEAGIGGYLLGS(stearyl)
SEQ ID NO: 591


CVTVLALGALAGVGVG(stearyl)
SEQ ID NO: 592


CLLGRRGWEVLKYWWNLLQYWSQEL(stearyl)
SEQ ID NO: 593


CGIFEAIAGLLKNIFD
SEQ ID NO: 594


CGIFEAIAGLLKNIFE
SEQ ID NO: 595


CGIFEAIAGLLKNIFR
SEQ ID NO: 596


CGIFEAIAGLLKNIFH
SEQ ID NO: 597


CGIFEAIAGLLKNIFO (O = ORNITHINE)
SEQ ID NO: 598


CGIFEAIAGLLKNIFN
SEQ ID NO: 599


CGIFEAIAGLLKNIFCit (Cit = citrulline)
SEQ ID NO: 600


CGIFGAIWGLLKNIFK
SEQ ID NO: 601


CIFEAIAGLLKNIFK
SEQ ID NO: 602


CFFEAIAGLLKNIFK
SEQ ID NO: 603


CGFFEAIAGLLKNIFK
SEQ ID NO: 604


CGIFEAIAGLLKNIFKG
SEQ ID NO: 605


CGIFEAIAGLLKNIFKGL
SEQ ID NO: 606


CGIFEAIAGLLKNIFKGLI
SEQ ID NO: 607


CGIFEAIAGLLKNIFKGLID
SEQ ID NO: 608


CGIFEAIAGLLKNIFKGLIDG
SEQ ID NO: 609


CGIFEAIAGLLKNIFKGLIDGF
SEQ ID NO: 610


CGIFEAIAGLLKNIFKGLIDGWYG
SEQ ID NO: 611


CGIFEAIAGLLKNIFK
SEQ ID NO: 612


CGIFEAIAGLLKSILK
SEQ ID NO: 613


CGIFEAIAGLLKNIFKA
SEQ ID NO: 614


CGIFEAIAGLLKNIFKL
SEQ ID NO: 615


CGIFEAIAGLLKNIFKW
SEQ ID NO: 616


CGIFEAIAGLLKNIFKF
SEQ ID NO: 617


CGIFEAIAGLLKNAFK
SEQ ID NO: 618


CGIFGAIAGLLKNAFK
SEQ ID NO: 619


CGIFEAIAGLLONIFO (O = Ornithine)
SEQ ID NO: 620


CGIFEAIAGLLKNIFKGIFEAIAGLLKNIFK
SEQ ID NO: 621


CGIFEAIAGLLKNIFKFFGAIWEFIHSIL
SEQ ID NO: 622


CFFGAIWEFIHSILGIFEAIAGLLKNIFK
SEQ ID NO: 623


CFFGAIWEFIHSILFFGAIWEFIHSIL
SEQ ID NO: 624


CFFGAIWEFIHSILGFFGAIWEFIHSIL
SEQ ID NO: 625


CGIFEAIAGLLKNIFKGIFEAIAGLLKNIFK
SEQ ID NO: 626


CGIFEAIAGLLKNIFKFFGAIWEFIHSIL
SEQ ID NO: 627


CFFGAIWEFIHSILGIFEAIAGLLKNIFK
SEQ ID NO: 628


CGLFHALLHLLHSLWHLLLEA
SEQ ID NO: 629


CGLFHALLHLLHSLWHLLLEAK(stearyl)
SEQ ID NO: 630


CGLFHALLHLLHSLWHLLLEAK(stearyl)
SEQ ID NO: 631


(stearyl)GLFHALLHLLHSLWHLLLEAC
SEQ ID NO: 632


CFFGNIWEFIKSIL
SEQ ID NO: 633


CFFGAIWLFIKSIL
SEQ ID NO: 634


CFFGAIWNFIKSIL
SEQ ID NO: 635


CFFGAIWGFIKSIL
SEQ ID NO: 636


CFLGALFKALSKLL
SEQ ID NO: 637


CFLGALFHALSKLL
SEQ ID NO: 638


CFLGALFKALSHLL
SEQ ID NO: 639


CFLGALFHALSHLL
SEQ ID NO: 640


FLGALFKALSKLLC
SEQ ID NO: 641


FLGALFHALSKLLC
SEQ ID NO: 642


FLGALFKALSHLLC
SEQ ID NO: 643


FLGALFHALSHLLC
SEQ ID NO: 644


CFLGALFKALKSLL
SEQ ID NO: 645


CFLGALFHALKSLL
SEQ ID NO: 646


CFLGALFKALHSLL
SEQ ID NO: 647


CFLGALFHALHSLL
SEQ ID NO: 648


FLGALFKALKSLLC
SEQ ID NO: 649


FLGALFHALKSLLC
SEQ ID NO: 650


FLGALFKALHSLLC
SEQ ID NO: 651


FLGALFHALHSLLC
SEQ ID NO: 652


CGIFGAIAGFIKNIWKGLIDW
SEQ ID NO: 653


CGLFEAIEGFIENGWEG-Nle-IDGWYGYGRKKRRQRR
SEQ ID NO: 654


CGLFEAIEGFIENGLKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 655


CGLFEAIEGFIENAWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 656


CGLFEAIEGFIENGWEGMIDLWYGYGRKKRRQRR
SEQ ID NO: 657


CRLLRLLLRLWRRLLRLLR
SEQ ID NO: 658


CGIFGAIEGFIENGWKGLIDAWYGYRKKRRQRR
SEQ ID NO: 659


CFFGAIWEFAHGIL
SEQ ID NO: 660


CFFGAIWEFARGILEGF
SEQ ID NO: 661


FFGAIWEFAHGILC
SEQ ID NO: 662


FFGAIWEFARGILEGFC
SEQ ID NO: 663


CFFGAIWEFAHSIL
SEQ ID NO: 664


FFGAIWEFAHSILC
SEQ ID NO: 665


CFFGAIWEFARSILK
SEQ ID NO: 666


FFGAIWEFARSILKC
SEQ ID NO: 667


CGIFEAIAGLAKNIFK
SEQ ID NO: 668


GIFEAIAGLAKNIFKC
SEQ ID NO: 669


CGIFEAIAGLAKNIFH
SEQ ID NO: 670


CGIFEAIAGLAHNIFH
SEQ ID NO: 671


CGIFEAIAGLAHNIFK
SEQ ID NO: 672


GIFEAIAGLAKNIFHC
SEQ ID NO: 673


GIFEAIAGLAHNIFHC
SEQ ID NO: 674


CFLGALWKALSKLL
SEQ ID NO: 675


CFLGALWHALSKLL
SEQ ID NO: 676


CFLGALWKALSHLL
SEQ ID NO: 677


CFLGALWHALSHLL
SEQ ID NO: 678


FLGALWKALSKLLC
SEQ ID NO: 679


FLGALWHALSKLLC
SEQ ID NO: 680


FLGALWKALSHLLC
SEQ ID NO: 681


FLGALWHALSHLLC
SEQ ID NO: 682


CGIFGAIAGLLKNAFK
SEQ ID NO: 683


CIFEAIAGLLKNAFK
SEQ ID NO: 684


CIFGAIAGLLKNAFK
SEQ ID NO: 685


CIFEAIWEFIKNIW
SEQ ID NO: 686


CIFEAIAEFIKNIW
SEQ ID NO: 687


CIFGAIWEFIKNIW
SEQ ID NO: 688


CIFGAIAEFIKNIW
SEQ ID NO: 689


CGIFGIAIGFKINIW
SEQ ID NO: 690


CGIFEAIAGLLHNIFK
SEQ ID NO: 691


CGIFEAIWGLLHNIFK
SEQ ID NO: 692


CGFFEAIAGLLHNIFK
SEQ ID NO: 693


CGIFEAIAALLKNIFK
SEQ ID NO: 694


CGIFEAIEGLLKNIFK
SEQ ID NO: 695


CGIFEAIAGFFKNIFK
SEQ ID NO: 696


CGIFEAIAGWWKNIFK
SEQ ID NO: 697


CGIFEAIAGLLKNIWK
SEQ ID NO: 698


CGIFEAIAELLKNIFK
SEQ ID NO: 699


CGIFGAIAGLLKSALK
SEQ ID NO: 700


CGIFEAIAGLLKSIWK
SEQ ID NO: 701


CGIFEAIAGLLKSILK
SEQ ID NO: 702


CGIFEAIAGLLKNIFKGLIDA
SEQ ID NO: 703


CGIFEAIAGLLKNIFKGLIDAF
SEQ ID NO: 704


CGIFEAIAGLLKNIFKGLIDAWYG
SEQ ID NO: 705


CGIFEAIAGLLKNIFKGLIDAWYGF
SEQ ID NO: 706


CGIFEAIAGLLKNIFKGLIDGWYGF
SEQ ID NO: 707


CGIFEAIAGLLKNIFKGLIDW
SEQ ID NO: 708


CGIFEAIAGLLKNIFKGLIDWF
SEQ ID NO: 709


CGIFEAIAGLLKNIFKGLIDWWYG
SEQ ID NO: 710


CGIFEAIAGLLKNIFKGLIDWWYGF
SEQ ID NO: 711


CGIFELIAGLLKNIFK
SEQ ID NO: 712


CGIFEAIAGLLKWIFK
SEQ ID NO: 713


CGIFELIAGLLKWIFK
SEQ ID NO: 714


CGIFELIAGLLKNIFKG
SEQ ID NO: 715


CGIFEAIAGLLKWIFKG
SEQ ID NO: 716


CGIFELIAGLLKWIFKG
SEQ ID NO: 717


CGLFEALLGLLESLWK
SEQ ID NO: 718


CGIFEAIAELLKNIFK
SEQ ID NO: 719


CGIFEALLGLLKSLWK
SEQ ID NO: 720


CGIFEALLELLKSLWK
SEQ ID NO: 721


CGIFEAIAGLLKNIFK
SEQ ID NO: 722


CEIFEAIAGLLKNIFK
SEQ ID NO: 723


CEIFGAIAGLLKNIFK
SEQ ID NO: 724


CGLFEAIAGLLKNLFK
SEQ ID NO: 725


CGIWEAIAGLLKNIWK
SEQ ID NO: 726


CGLFGAIAGLLKNLFK
SEQ ID NO: 727


CGIWGAIAGLLKNIWK
SEQ ID NO: 728


CGIFDAIAGLLKNIFK
SEQ ID NO: 729


CGIFDAIWGLLKNIFK
SEQ ID NO: 730


CGIFGGIGGLLKNIFK
SEQ ID NO: 731


CAIFAAIAALLKNIFK
SEQ ID NO: 732


CGIFEAIAGLLKNIF
SEQ ID NO: 733


CGIFEAIAGLLKNI
SEQ ID NO: 734


CGIFEAIAGLLKN
SEQ ID NO: 735


CGIFEAIAGLLK
SEQ ID NO: 736


CVIFEAIAGLLKNIFK
SEQ ID NO: 737


CSIFEAIAGLLKNIFK
SEQ ID NO: 738


CGIFEEIAGLLKNIFK
SEQ ID NO: 739


CGIFEEIWGLLKNIFK
SEQ ID NO: 740


CGIFEAIEELLKNIFK
SEQ ID NO: 741


CGIFEAIAGLWKNIFK
SEQ ID NO: 742


CGIFEAIAGLLENIFK
SEQ ID NO: 743


CGIFEAIAGLLWNIFK
SEQ ID NO: 744


CGIFEAIAGLLKEIFK
SEQ ID NO: 745


CGIFEAIAGLLKNILK
SEQ ID NO: 746


CGIFEAIAGLLRNIFK
SEQ ID NO: 747


CGIFEAIAGLLKSIFK
SEQ ID NO: 748


CGIFEAIAGLLKNILK
SEQ ID NO: 749


CGFFGAIWEFIKSILK
SEQ ID NO: 750


CGFFEAIWEFIKSILK
SEQ ID NO: 751


CGFFGAIWGLLKSILK
SEQ ID NO: 752


CGFFEAIWGLLKSILK
SEQ ID NO: 753


CGFFEAIAGLLKSILK
SEQ ID NO: 754


CGFFGAIAGLLKSILK
SEQ ID NO: 755


CGIFEAIAGLLKNIFEGLI
SEQ ID NO: 756


CGIFEAIWGLLKNIFKGLI
SEQ ID NO: 757


CGIFEAIWGLLKNIFEGLI
SEQ ID NO: 758


CGIFEAIAGLLKNILKGLIDGWYG
SEQ ID NO: 759


CGIFGAIAGLLKNILKGLIDGWYG
SEQ ID NO: 760


CGIFGAIAGLLKNIFKGLIDGWYG
SEQ ID NO: 761


CGIFGAIWELWEWILK
SEQ ID NO: 762


CGIFEAIWELWEWILK
SEQ ID NO: 763


CIFGAIWELWEWILK
SEQ ID NO: 764


CIFEAIWELWEWILK
SEQ ID NO: 765


CGIFEAIAELWKNIFK
SEQ ID NO: 766


CGIFEAIAELWENIFK
SEQ ID NO: 767


CGIFEAIAELWKWIFK
SEQ ID NO: 768


CGIFEAIAELWEWIFK
SEQ ID NO: 769


CGIFEAIAGLLKNILKGLIDWWYG
SEQ ID NO: 770


CGIFGAIAGLLKNILKGLIDWWYG
SEQ ID NO: 771


CGIFGAIAGLLKNIFKGLIDWWYG
SEQ ID NO: 772


CGIFEAIAGLLKNILKGLIDGWYGF
SEQ ID NO: 773


CGIFGAIAGLLKNILKGLIDGWYGF
SEQ ID NO: 774


CGIFGAIAGLLKNIFKGLIDGWYGF
SEQ ID NO: 775


CGIFGAIAELLEKIFE
SEQ ID NO: 776


CGIFEAIAELLEKIFE
SEQ ID NO: 777


CGFIGAIAELLEKIFE
SEQ ID NO: 778


CGIFGAIAELLEKIFK
SEQ ID NO: 779


CGIFEAIAELLEKIFK
SEQ ID NO: 780


CGFIGAIAELLEKIFK
SEQ ID NO: 781


CGLFHALLHLLHSLWHLLLEA
SEQ ID NO: 782


GLFHALLHLLHSLWHGLLEAC
SEQ ID NO: 783


GFFHAFFHFFHSFWHGFFEAC
SEQ ID NO: 784


GLFHALLHLLHSLWHLLLEAC
SEQ ID NO: 785


CGLFHALLHLLHSLWHGLLEAK(stearyl)
SEQ ID NO: 786


CGFFHAFFHFFHSFWHGFFEAK(stearyl)
SEQ ID NO: 787


CGLFHALLHLLHSLWHLLLEAK(stearyl)
SEQ ID NO: 788


(stearyl)GLFHALLHLLHSLWHGLLEAC
SEQ ID NO: 789


(stearyl)GFFHAFFHFFHSFWHGFFEAC
SEQ ID NO: 790


(stearyl)GLFHALLHLLHSLWHLLLEAC
SEQ ID NO: 791


CGFFHAFFHFFHSFWHFFFEA
SEQ ID NO: 792


CGFFHAFFHFFHSFWHLFFEA
SEQ ID NO: 793


CGLFHALLHLLHSLWHGLLEW
SEQ ID NO: 794


CGLFHALLHLLHSLWHLLLEW
SEQ ID NO: 795


CGFFHAFFHFFHSFWHGFFEW
SEQ ID NO: 796


CFFGAIWEFAKSIL
SEQ ID NO: 797


CFFGAIWEFAHSIL
SEQ ID NO: 798


CFFGAIWEFAHGIL
SEQ ID NO: 799


CFFGAIWEFIHSILK
SEQ ID NO: 800


CFFGAIWEFIHSILH
SEQ ID NO: 801


CFFGAIWEFIHSILD
SEQ ID NO: 802


CFFGAIWEFIHSILR
SEQ ID NO: 803


CFFGAIWEFIHSILO
SEQ ID NO: 804


CFFGAIAEFIHSIL
SEQ ID NO: 805


CIFGAIWEFIHSIL
SEQ ID NO: 806


CGIFGAIWEFIHSIL
SEQ ID NO: 807


CFFGAIWEFIHSILE
SEQ ID NO: 808


CFFGAIWEFIHSILEG
SEQ ID NO: 809


CFFGAIWEFIHSILEGL
SEQ ID NO: 810


CFFGAIWEFIHSILEGLI
SEQ ID NO: 811


CFFGAIWEFIHSILEGLID
SEQ ID NO: 812


CFFGAIWEFIHSILEGLIDG
SEQ ID NO: 813


CFFGAIWEFIHSILEGLIEA
SEQ ID NO: 814


CFFGAIWEFIHSILEGLIDW
SEQ ID NO: 815


CFFGAIWEFIHSILEGLIDGWYG
SEQ ID NO: 816


CFFGAIWEFIHSILEGLIDGWYGF
SEQ ID NO: 817


FFGAIWEFIHSILC
SEQ ID NO: 818


CFWGAIWEFIHSIL
SEQ ID NO: 819


CFFGAIWEFIHSILKGLIDW
SEQ ID NO: 820


CAFGKIWEFAHSIL
SEQ ID NO: 821


CAFGKIWEFIHSIL
SEQ ID NO: 822


CFFGKIWEFIHSIL
SEQ ID NO: 823


CAFGAIWEFIHSIL
SEQ ID NO: 824


CAFGAIWEFAHSIL
SEQ ID NO: 825


CGFFGAIAGLLHNIFK
SEQ ID NO: 826


CFFGAIAGLLHNIFK
SEQ ID NO: 827


CGFFEAIEGLLHNIFK
SEQ ID NO: 828


CFFEAIAGLLHNIFK
SEQ ID NO: 829


CFFEAIWGLLHNIFK
SEQ ID NO: 830


CGFFGAIAELLHNIFK
SEQ ID NO: 831


CFFGAIAELLHNIFK
SEQ ID NO: 832


CGFFEAIAELLHNIFK
SEQ ID NO: 833


CFFEAIAELLHNIFK
SEQ ID NO: 834


CFFGAIWELLHNIFK
SEQ ID NO: 835


CFFEAIWELLHNIFK
SEQ ID NO: 836


CFFGAIWEFIHSILFFGAIWEFIHSIL
SEQ ID NO: 837


CFFGAIWEFIHSILGGGFFGAIWEFIHSIL
SEQ ID NO: 838


CFFGAIWEFIHSILGFFGAIWEFIHSIL
SEQ ID NO: 839


GGLFEALLELLESLWELLLEW
SEQ ID NO: 840


GGFFEAFFEFFESFWEFFFEA
SEQ ID NO: 841


GGLFEALLELLESLWEGLLEA
SEQ ID NO: 842


CGLFHALLHLLHSLWHLLLHA
SEQ ID NO: 843


CGLFEALLHLLHSLWHLLLEA
SEQ ID NO: 844


CGLFEALLELLHSLWHLLLEA
SEQ ID NO: 845


CGLFEALLHLLESLWHLLLEA
SEQ ID NO: 846


CGLFEALLHLLHSLWELLLEA
SEQ ID NO: 847


CGLFHALLELLHSLWHLLLEA
SEQ ID NO: 848


CGLFHALLHLLESLWHLLLEA
SEQ ID NO: 849


CGLFHALLHLLHSLWELLLEA
SEQ ID NO: 850


CGLFHALLELLESLWHLLLEA
SEQ ID NO: 851


CGLFHALLELLHSLWELLLEA
SEQ ID NO: 852


CGLFHALLHLLESLWELLLEA
SEQ ID NO: 853


CGLFEALLHLLESLWELLLEA
SEQ ID NO: 854


CGLFEALLELLHSLWELLLEA
SEQ ID NO: 855


CGLEALLELLESLWHLLLEA
SEQ ID NO: 856


CGLFHALLELLESLWELLLEA
SEQ ID NO: 857


CFFGAIWEFIHSILHLLLEA
SEQ ID NO: 858


CFFGAIWEFIHSILKLLLEA
SEQ ID NO: 859


CGFFGAIWEFIHSILGFFGAIWEFIHSIL
SEQ ID NO: 860


CFFGAIWEFAHSILFFGAIWEFAHSIL
SEQ ID NO: 861


CFFGAIWEFAHSILGFFGAIWEFAHSIL
SEQ ID NO: 862


CGFFGAIWEFAHSILGFFGAIWEFAHSIL
SEQ ID NO: 863


CFFGAIWEFIHSILGLFEAIEGFIENGWEGMIDG
SEQ ID NO: 864


CFFGAIWEFIHSILGLFEAIEGFIENGWEGMIDGWYG
SEQ ID NO: 865


CFFGAIWEFIHSILGLFEAIEGFIENGWEGMIDGWYGF
SEQ ID NO: 866


CFFGALLEFIHSILELLLEA
SEQ ID NO: 867


CGLFGALLEFIHSILELLLEA
SEQ ID NO: 868


CGFFGALLEFIHSILELLLEA
SEQ ID NO: 869


CFFGALLEFIHSLWELLLEA
SEQ ID NO: 870


CGLFGALLEFIHSLWELLLEA
SEQ ID NO: 871


CGFFGALLEFIHSLWELLLEA
SEQ ID NO: 872


CIFGAIAGFIKNIWK(stearyl)
SEQ ID NO: 873


(stearyl)IFGAIAGFIKNIWC
SEQ ID NO: 874


CFFGAIWEFIKSILK(stearyl)
SEQ ID NO: 875


(stearyl)FFGAIWEFIKSILC
SEQ ID NO: 876


CFFGAIWEFIHSILK(stearyl)
SEQ ID NO: 877


(stearyl)FFGAIWEFIHSILC
SEQ ID NO: 878


CIFGAIAGFIKNIWEGLIK(stearyl)
SEQ ID NO: 879


(stearyl)IFGAIAGFIKNIWEGLIC
SEQ ID NO: 880


(stearyl)IFGAIAGFIKNILKGLC
SEQ ID NO: 881


(stearyl)GIFGAIAGFIKNILKGLC
SEQ ID NO: 882


CIFGAIAGFIKNILKGLK(stearyl)
SEQ ID NO: 883


CGLFGAIAGFIVNGWVGMIDG
SEQ ID NO: 884


CGLFGAIAGFIVNGWVGMIDGWYG
SEQ ID NO: 885


CGLFEAIEGFIVNGWVGMIDGWYG
SEQ ID NO: 886


CGLFGAIAGFIVNGWVGMIDGWYGF
SEQ ID NO: 887


CGLFEAIEAGFIVNGWVGMIDGWYGF
SEQ ID NO: 888


CGLFGAIAGFIVNGWVGMIDGWYGK(stearyl)
SEQ ID NO: 889


CGLFEAIEGFIVNGWVGMIDGWYGK(stearyl)
SEQ ID NO: 890


(stearyl)GLFGAIAGFIVNGWVGMIDGWYGC
SEQ ID NO: 891


(stearyl)GLFEAIEGFIVNGWVGMIDGWYGC
SEQ ID NO: 892


(stearyl)GLFGAIAGFIVNGWVGMIDGWYGFC
SEQ ID NO: 893


(stearyl)GLFEAIEAGFIVNGWVGMIDGWYGFC
SEQ ID NO: 894


CFFGAIWGLLHSILH
SEQ ID NO: 895


CFFGAIWELLHSIL
SEQ ID NO: 896


CFFGAIWELLHSILH
SEQ ID NO: 897


CFFGAIWGLLHSILK
SEQ ID NO: 898


CFFGAIWELLHSILK
SEQ ID NO: 899


CGLFGALLHLLHSLWELLLEA
SEQ ID NO: 900


CGLFGALLELLHSLWELLLEA
SEQ ID NO: 901


CFFGAIWEFIHSILELLLEA
SEQ ID NO: 902


CFFGAIWEFIHSILHGLLEA
SEQ ID NO: 903


CFFGAIWEFIHSILEGLLEA
SEQ ID NO: 904


CGFFGAIWEFIHSILHLLLEA
SEQ ID NO: 905


CGFFGAIWEFIHSILELLLEA
SEQ ID NO: 906


CGFFGAIWEFIHSILHGLLEA
SEQ ID NO: 907


CGFFGAIWEFIHSILEGLLEA
SEQ ID NO: 908


CGFFGAIAGLLHSIL
SEQ ID NO: 909


CGFFGAIWGLLHSIL
SEQ ID NO: 910


CGFFGALLGLLHSIL
SEQ ID NO: 911


CFFGAIWEFAKSAL
SEQ ID NO: 912


CIFGAIAGFIHNILKGL
SEQ ID NO: 913


CFFGAIAGFIKNILKGL
SEQ ID NO: 914


CIFGAIWGFIKNILKGL
SEQ ID NO: 915


CIFGAIWGFIHNILKGL
SEQ ID NO: 916


CIFGAIAGLLKNILKGL
SEQ ID NO: 917


CIFGAIAGLLHNILKGL
SEQ ID NO: 918


CIFEAIAGFIKNILKGL
SEQ ID NO: 919


CIFEAIAGFIHNILKGL
SEQ ID NO: 920


CGNFGEIAELIEEGLKNLIDWWNG
SEQ ID NO: 921


CGFFGEIAELIEEGLENLIDWWNG
SEQ ID NO: 922


CGNFGEIEELIEEGLKNLIDWWNG
SEQ ID NO: 923


CGNFGEIAELIEEGLENLIDWWNG
SEQ ID NO: 924


CGFFGEIEELIEENGENLIDWWNG
SEQ ID NO: 925


CGFFGAIEELIEEGLKNLIDWWNG
SEQ ID NO: 926


CGFFGAIAELIEEGLKNLIDWWNG
SEQ ID NO: 927


CGFFGEIAELIEEGLKNLIDWWNGF
SEQ ID NO: 928


GFFGEIAELIEEGLKNLIDWWNGC
SEQ ID NO: 929


GNWWDILNKLGEEILEAIEGFFGC
SEQ ID NO: 930


CGNWWDILNKLGEEILEAIEGFFG
SEQ ID NO: 931


CGFLGEIAELIEEGLKNLIDWWNG
SEQ ID NO: 932


CGFFGEIWELIEEGLKNLIDWWNG
SEQ ID NO: 933


CGFFGEIAELWEEGLKNLIDWWNG
SEQ ID NO: 934


CGFFGEIAELIWEGLKNLIDWWNG
SEQ ID NO: 935


CGFFGEIAELIEWGLKNLIDWWNG
SEQ ID NO: 936


CGFFGEIAELIEEGLRNLIDWWNG
SEQ ID NO: 937


CGFFGEIAELIEEGLDNLIDWWNG
SEQ ID NO: 938


CGFFGEIAELIEEGLKNLNDWWNG
SEQ ID NO: 939


CGFFGEIEELIEEGLKNLIDWWNG
SEQ ID NO: 940


CGFLGEIEELIEEGLKNLIDWWNG
SEQ ID NO: 941


CGFFGLIEELIEEGLKNLIDWWNG
SEQ ID NO: 942


CGFFGEIAELIEEGLKNLIDWWNGK(stearyl)
SEQ ID NO: 943


(stearyl)GFFGEIAELIEEGLKNLIDWWNGC
SEQ ID NO: 944


CFFGAIWEFAKSILK(stearyl)
SEQ ID NO: 945


CGFFGAIWEFAKSIL
SEQ ID NO: 946


CFFGKIWEFIKSILK(stearyl)
SEQ ID NO: 947


(stearyl)FFGKIWEFIKSILC
SEQ ID NO: 948


CFFGAIWEFIKSIAK(stearyl)
SEQ ID NO: 949


(stearyl)FFGAIWEFIKSIAC
SEQ ID NO: 950


(stearyl)FFGAIWEFAKSILC
SEQ ID NO: 951


CFFGGIWEFIKSILK(stearyl)
SEQ ID NO: 952


(stearyl)FFGGIWEFIKSILC
SEQ ID NO: 953


CFFKAIWEFIKSILK(stearyl)
SEQ ID NO: 954


(stearyl)FFKAIWEFIKSILC
SEQ ID NO: 955


CFFGAIWEAIKSILK(stearyl)
SEQ ID NO: 956


(stearyl)FFGAIWEAIKSILC
SEQ ID NO: 957


CFFKAIWEFAKSIL
SEQ ID NO: 958


CFFKAIWEFAHSIL
SEQ ID NO: 959


CFFKAIWEFAKSILK(stearyl)
SEQ ID NO: 960


(stearyl)FFKAIWEFAKSILC
SEQ ID NO: 961


CFFKAIWEFAHSILK(stearyl)
SEQ ID NO: 962


CGLFGEIAELIEEGLENLIDWWNG
SEQ ID NO: 963


CGLFGEIEELIEEGLKNLIDWWNG
SEQ ID NO: 964


CFFGAIWEFAKSILK(stearyl)
SEQ ID NO: 965


CGLFGEIEELIEEGLKGLIDWWNG
SEQ ID NO: 966


CGLFGEIAELIEEGLKNLIDWWNG
SEQ ID NO: 967


CGLFGEIAELIEEGLEGLIDWWNG
SEQ ID NO: 968


GLFGEIEELIEEGLENLIDWWNGC
SEQ ID NO: 969


(stearyl)GLFGEIEELIEEGLENLIDWWNGC
SEQ ID NO: 970


CGLFGEIEELIEEGLENLIDWWNGK(stearyl)
SEQ ID NO: 971


CGNWWDILNELGEEILEEIEGFLG
SEQ ID NO: 972


CALFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 973


CELFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 974


CSLFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 975


CNLFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 976


CVLFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 977


CGFFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 978


CGVFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 979


CGIFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 980


CGWFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 981


CGYFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 982


CGLLGEIEELIEEGLENLIDWWNG
SEQ ID NO: 983


CGLVGEIEELIEEGLENLIDWWNG
SEQ ID NO: 984


CGLIGEIEELIEEGLENLIDWWNG
SEQ ID NO: 985


CGLWGEIEELIEEGLENLIDWWNG
SEQ ID NO: 986


CGLYGEIEELIEEGLENLIDWWNG
SEQ ID NO: 987


CGLFEEIEELIEEGLENLIDWWNG
SEQ ID NO: 988


CGLFAEIEELIEEGLENLIDWWNG
SEQ ID NO: 989


CGLFNEIEELIEEGLENLIDWWNG
SEQ ID NO: 990


CGLFSEIEELIEEGLENLIDWWNG
SEQ ID NO: 991


CGLFGAIEELIEEGLENLIDWWNG
SEQ ID NO: 992


CGLFGDIEELIEEGLENLIDWWNG
SEQ ID NO: 993


CGLFGNIEELIEEGLENLIDWWNG
SEQ ID NO: 994


CGLFGSIEELIEEGLENLIDWWNG
SEQ ID NO: 995


CGLFGELEELIEEGLENLIDWWNG
SEQ ID NO: 996


CGLFGEVEELIEEGLENLIDWWNG
SEQ ID NO: 997


CGLFGEFEELIEEGLENLIDWWNG
SEQ ID NO: 998


CGLFGEWEELIEEGLENLIDWWNG
SEQ ID NO: 999


CGLFGEYEELIEEGLENLIDWWNG
SEQ ID NO: 1000


CGLFGEIAELIEEGLENLIDWWNG
SEQ ID NO: 1001


CGLFGEIGELIEEGLENLIDWWNG
SEQ ID NO: 1002


CGLFGEILELIEEGLENLIDWWNG
SEQ ID NO: 1003


CGLFGEIVELIEEGLENLIDWWNG
SEQ ID NO: 1004


CGLFGEISELIEEGLENLIDWWNG
SEQ ID NO: 1005


CGLFGEIEDLIEEGLENLIDWWNG
SEQ ID NO: 1006


CGLFGEIENLIEEGLENLIDWWNG
SEQ ID NO: 1007


CGLFGEIESLIEEGLENLIDWWNG
SEQ ID NO: 1008


CGLFGEIEALIEEGLENLIDWWNG
SEQ ID NO: 1009


CGLFGEIEGLIEEGLENLIDWWNG
SEQ ID NO: 1010


CGLFGEIEEVIEEGLENLIDWWNG
SEQ ID NO: 1011


CGLFGEIEEIIEEGLENLIDWWNG
SEQ ID NO: 1012


CGLFGEIEEFIEEGLENLIDWWNG
SEQ ID NO: 1013


CGLFGEIEEAIEEGLENLIDWWNG
SEQ ID NO: 1014


CGLFGEIEEYIEEGLENLIDWWNG
SEQ ID NO: 1015


CGLFGEIEEWIEEGLENLIDWWNG
SEQ ID NO: 1016


CGLFGEIEELVEEGLENLIDWWNG
SEQ ID NO: 1017


CGLFGEIEELLEEGLENLIDWWNG
SEQ ID NO: 1018


CGLFGEIEELFEEGLENLIDWWNG
SEQ ID NO: 1019


CGLFGEIEELAEEGLENLIDWWNG
SEQ ID NO: 1020


CGLFGEIEELYEEGLENLIDWWNG
SEQ ID NO: 1021


CGLFGEIEELWEEGLENLIDWWNG
SEQ ID NO: 1022


CGLFGEIEELIDEGLENLIDWWNG
SEQ ID NO: 1023


CGLFGEIEELINEGLENLIDWWNG
SEQ ID NO: 1024


CGLFGEIEELISEGLENLIDWWNG
SEQ ID NO: 1025


CGLFGEIEELIEDGLENLIDWWNG
SEQ ID NO: 1026


CGLFGEIEELIEYGLENLIDWWNG
SEQ ID NO: 1027


CGLFGEIEELIESGLENLIDWWNG
SEQ ID NO: 1028


CGLFGEIEELIEQGLENLIDWWNG
SEQ ID NO: 1029


CGLFGEIEELIENGLENLIDWWNG
SEQ ID NO: 1030


CGLFGEIEELIEEALENLIDWWNG
SEQ ID NO: 1031


CGLFGEIEELIEENLENLIDWWNG
SEQ ID NO: 1032


CGLFGEIEELIEESLENLIDWWNG
SEQ ID NO: 1033


CGLFGEIEELIEEQLENLIDWWNG
SEQ ID NO: 1034


CGLFGEIEELIEEGWENLIDWWNG
SEQ ID NO: 1035


CGLFGEIEELIEEGVENLIDWWNG
SEQ ID NO: 1036


CGLFGEIEELIEEGIENLIDWWNG
SEQ ID NO: 1037


CGLFGEIEELIEEGFENLIDWWNG
SEQ ID NO: 1038


CGLFGEIEELIEEGAENLIDWWNG
SEQ ID NO: 1039


CGLFGEIEELIEEGYENLIDWWNG
SEQ ID NO: 1040


CGLFGEIEELIEEGLRNLIDWWNG
SEQ ID NO: 1041


CGLFGEIEELIEEGLHNLIDWWNG
SEQ ID NO: 1042


CGLFGEIEELIEEGLONLIDWWNG
SEQ ID NO: 1043


CGLFGEIEELIEEGLDNLIDWWNG
SEQ ID NO: 1044


CGLFGEIEELIEEGLKNLIDWWNG
SEQ ID NO: 1045


CGLFGEIEELIEEGLEGLIDWWNG
SEQ ID NO: 1046


CGLFGEIEELIEEGLEYLIDWWNG
SEQ ID NO: 1047


CGLFGEIEELIEEGLEQLIDWWNG
SEQ ID NO: 1048


CGLFGEIEELIEEGLESLIDWWNG
SEQ ID NO: 1049


CGLFGEIEELIEEGLEALIDWWNG
SEQ ID NO: 1050


CGLFGEIEELIEEGLE(Cit)LIDWWNG
SEQ ID NO: 1051


CGLFGEIEELIEEGLENMIDWWNG
SEQ ID NO: 1052


CGLFGEIEELIEEGLENFIDWWNG
SEQ ID NO: 1053


CGLFGEIEELIEEGLENIIDWWNG
SEQ ID NO: 1054


CGLFGEIEELIEEGLENWIDWWNG
SEQ ID NO: 1055


CGLFGEIEELIEEGLENVIDWWNG
SEQ ID NO: 1056


CGLFGEIEELIEEGLENYIDWWNG
SEQ ID NO: 1057


CGLFGEIEELIEEGLEN(Nle)IDWWNG
SEQ ID NO: 1058


CGLFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 1059


CGLFGEIEELIEEGLENLVDWWNG
SEQ ID NO: 1060


CGLFGEIEELIEEGLENLFDWWNG
SEQ ID NO: 1061


CGLFGEIEELIEEGLENLWDWWNG
SEQ ID NO: 1062


CGLFGEIEELIEEGLENLYDWWNG
SEQ ID NO: 1063


CGLFGEIEELIEEGLENLIEWWNG
SEQ ID NO: 1064


CGLFGEIEELIEEGLENLINWWNG
SEQ ID NO: 1065


CGLFGEIEELIEEGLENLISWWNG
SEQ ID NO: 1066


CGLFGEIEELIEEGLENLIQWWNG
SEQ ID NO: 1067


CGLFGEIEELIEEGLENLIDGWNG
SEQ ID NO: 1068


CGLFGEIEELIEEGLENLIDAWNG
SEQ ID NO: 1069


CGLFGEIEELIEEGLENLIDFWNG
SEQ ID NO: 1070


CGLFGEIEELIEEGLENLIDLWNG
SEQ ID NO: 1071


CGLFGEIEELIEEGLENLIDIWNG
SEQ ID NO: 1072


CGLFGEIEELIEEGLENLIDVWNG
SEQ ID NO: 1073


CGLFGEIEELIEEGLENLIDWGNG all (D)
SEQ ID NO: 1074


CGLFGEIEELIEEGLENLIDWANG
SEQ ID NO: 1075


CGLFGEIEELIEEGLENLIDWFNG
SEQ ID NO: 1076


CGLFGEIEELIEEGLENLIDWING
SEQ ID NO: 1077


CGLFGEIEELIEEGLENLIDWVNG
SEQ ID NO: 1078


CGLFGEIEELIEEGLENLIDWYNG
SEQ ID NO: 1079


CGLFGEIEELIEEGLENLIDWWQG
SEQ ID NO: 1080


CGLFGEIEELIEEGLENLIDWWTG
SEQ ID NO: 1081


CGLFGEIEELIEEGLENLIDWWSG
SEQ ID NO: 1082


CGLFGEIEELIEEGLENLIDWWEG
SEQ ID NO: 1083


CGLFGEIEELIEEGLENLIDWW(Cit)G
SEQ ID NO: 1084


CGLFGEIEELIEEGLENLIDWWNA
SEQ ID NO: 1085


CGLFGEIEELIEEGLENLIDWWNN
SEQ ID NO: 1086


CGLFGEIEELIEEGLENLIDWWNS
SEQ ID NO: 1087


CGLFGEIEELIEEGLENLIDWWNY
SEQ ID NO: 1088


CGLFGEIEELIEEGLENLIDWWNW
SEQ ID NO: 1089


CFFGAIWGLLHSIL
SEQ ID NO: 1090


CFFGK(stearyI)IWEFIKSIL
SEQ ID NO: 1091


CFFGK(stearyl)IWEFIHSIL
SEQ ID NO: 1092


CFFK(stearyl)AIWEFIKSIL
SEQ ID NO: 1093


CGFFGAIWGLLHSILK
SEQ ID NO: 1094


CGFFEAIWGLLHSIL
SEQ ID NO: 1095


CFFGAIWGLLKSIL
SEQ ID NO: 1096


CGFFGAIWGLLKSIL
SEQ ID NO: 1097


CFFEAIWGLLKSIL
SEQ ID NO: 1098


CGFFEAIWGLLKSIL
SEQ ID NO: 1099


CFFGAIWGLLHSILKGLIDWWNG
SEQ ID NO: 1100


CFFGAIWGLLHSILKGLIDGWYG
SEQ ID NO: 1101


CGIFGAIAGLLKNIFKG
SEQ ID NO: 1102


CGIFGAIAGLLKNIFKA
SEQ ID NO: 1103


CGIFGAIAGLLKNIFKL
SEQ ID NO: 1104


CGIFGAIAGLLKNIFKW
SEQ ID NO: 1105


CGIFGAIAGLLKNIFKF
SEQ ID NO: 1106


CGIFGAIAGLLKNIFKN
SEQ ID NO: 1107


CGIFGAIAGLLKNIFKE
SEQ ID NO: 1108


CGIFGAIAGLLKNIFKS
SEQ ID NO: 1109


CGIFGAIAGLLKNIFK(stearyl)
SEQ ID NO: 1110


CGIFGAIAGLLKNIFKK(stearyl)
SEQ ID NO: 1111


(stearyl)GIFGAIAGLLKNIFKC
SEQ ID NO: 1112


CGIFGAIAGLLKNIFK(lauryl)
SEQ ID NO: 1113


CGIFGAIAGLLKNIFKK(lauryl)
SEQ ID NO: 1114


(lauryl)GIFGAIAGLLKNIFKC
SEQ ID NO: 1115


CGIFGAIAGLLHNIFK
SEQ IDNO: 1116


CGIFGAIAGLLONIFK
SEQ IDNO: 1117


CGIFGAIAGLLRNIFK
SEQ IDNO: 1118


CGIFGAIAGLLENIFK
SEQ IDNO: 1119


CGIFGAIAGLLDNIFK
SEQ ID NO: 1120


CGIFGAIAGLLKNIFH
SEQ IDNO: 1121


CGIFGAIAGLLKNIFO
SEQ IDNO: 1122


CGIFGAIAGLLKINFE
SEQ IDNO: 1123


CGIFGAIAGLLKNIFD
SEQ IDNO: 1124


CGIFGAIAGLLKNIFN
SEQ IDNO: 1125


CGIFGAIAGLLNNIFK
SEQ IDNO: 1126


CGIFGIAIGLLKNIFKGIFGAIAGLLKNIFK
SEQ ID NO: 1127


CGIFGAIWGLLKNIFKG
SEQ IDNO: 1128


CGIFGAIWGLLKNIFKA
SEQ IDNO: 1129


CGIFGAIWGLLKNIFKL
SEQ ID NO: 1130


CGIFGAIWGLLKNIFKW
SEQ IDNO: 1131


CGIFGAIWGLLKNIFKF
SEQ IDNO: 1132


CGIFGAIWGLLKNIFKN
SEQ IDNO: 1133


CGIFGAIWGLLKNIFKE
SEQ IDNO: 1134


CGIFGAIWGLLKNIFKS
SEQ IDNO: 1135


CGIFGAIWGLLKNIFK(stearyl)
SEQ ID NO: 1136


CGIFGAIWGLLKNIFKK(stearyl)
SEQ ID NO: 1137


(stearyl)GIFGAIWGLLKNIFKC
SEQ ID NO: 1138


CGIFGAIWGLLKNIFK(lauryl)
SEQ ID NO: 1139


CGIFGAIWGLLKNIFKK(lauryl)
SEQ ID NO: 1140


(lauryl)GIFGAIWGLLKNIFKC
SEQ ID NO: 1141


CGIFGAIWGLLHNIFK
SEQ IDNO: 1142


CGIFGAIWGLLONIFK
SEQ IDNO: 1143


CGIFGAIWGLLRNIFK
SEQ IDNO: 1144


CGIFGAIWGLLENIFK
SEQ IDNO: 1145


CGIFGAIWGLLDNIFK
SEQ IDNO: 1146


CGIFGAIWGLLKNIFH
SEQ IDNO: 1147


CGIFGAIWGLLKNIFO
SEQ IDNO: 1148


CGIFGAIWGLLKINFE
SEQ IDNO: 1149


CGIFGAIWGLLKNIFD
SEQ ID NO: 1150


CGIFGAIWGLLKNIFN
SEQ IDNO: 1151


CGIFGAIWGLLNNIFK
SEQ IDNO: 1152


CFFGAIWGLLKNIFK
SEQ IDNO: 1153


CGFFGAIWGLLKNIFK
SEQ ID NO: 1154


CIFGAIWGLLKNIFK
SEQ ID NO: 1155


CGIFGAIWIGLLKNIFKGIFGAIWGLLKNIFK
SEQ ID NO: 1156


CGIFGAIWGLLHNIFH
SEQ ID NO: 1157


CGIFGAIWGLLONIFO
SEQ ID NO: 1158


CGIFGAIAGLLHSILK
SEQ ID NO: 1159


CGIFGAIWGLLHSILK
SEQ ID NO: 1160


CGIFGAIAGLLHSIL
SEQ ID NO: 1161


CGIFGAIWGLLHSIL
SEQ ID NO: 1162


CGIFGAIWELLKNIFK
SEQ ID NO: 1163


CGIFGAIWGLLHNIFHGIFGAIWGLLHNIFK
SEQ ID NO: 1164


CGIFEAIWGLLHNIFHGIFEAIWGLLHNIFH
SEQ ID NO: 1165


CGIFEAIWGLLKNIFHGIFEAIWGLLHNIFH
SEQ ID NO: 1166


CGIFEAIWGLLKNIFKGIFEAIWELLKNIFH
SEQ ID NO: 1167


CGIFEAIWGLLKNIFHGIFEAIWGLLKNIFH
SEQ ID NO: 1168


CGLFEALLELLESLWELLLEAWNG
SEQ ID NO: 1169


CGLFEALLELLESLWELLLEWWNG
SEQ ID NO: 1170


CGLFGELEELLEEGLENLLDWWNG
SEQ ID NO: 1171


CGLFGELEELLEEGLENLLEWWNG
SEQ ID NO: 1172


CGLFGELEELLEEGWELLLEAWNG
SEQ ID NO: 1173


CGLFGELEELLEEGWELLLEWWNG
SEQ ID NO: 1174


CGLFGELEELLEEGWELLLDWWNG
SEQ ID NO: 1175


CGLFGALLELLEEGLENLIDWWNG
SEQ ID NO: 1176


CGLFEALLELLEEGLENLIDWWNG
SEQ ID NO: 1177


CGLFEALLELLESLLENLIDWWNG
SEQ ID NO: 1178


CGLFGELAELLEEGLENLLDWWNG
SEQ ID NO: 1179


GLFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 1180


CFFGNIWEFIHSIL
SEQ ID NO: 1181


CFFGAIWNFIHSIL
SEQ ID NO: 1182


CFFGNIWNFIHSIL
SEQ ID NO: 1183


CGIFGNIWNFIKNIFK
SEQ ID NO: 1184


CGIFGNIWNLLKNIFK
SEQ ID NO: 1185


CGIFGNIWGLLKNIFK
SEQ ID NO: 1186


CGIFGNIWNFIKNIFH
SEQ ID NO: 1187


CGIFGNIWNLLKNIFH
SEQ ID NO: 1188


CGIFGNIWGLLKNIFH
SEQ ID NO: 1189


CGIFENIWNFIKNIFK
SEQ ID NO: 1190


CGIFENIWNFIKNIFH
SEQ ID NO: 1191


CGIFENIWGLLKNIFK
SEQ ID NO: 1192


CGIFENIWGLLKNIFH
SEQ ID NO: 1193


CGIFENIWNLLKNIFK
SEQ ID NO: 1194


CGIFENIWNLLKNIFH
SEQ ID NO: 1195


CGLFGAIAGLLENIFENLIDWWNG
SEQ ID NO: 1196


CGLFGAIAGLLNKIFKNLIDWWNG
SEQ ID NO: 1197


CGLFGAIAGLLENIFKNLIDWWNG
SEQ ID NO: 1198


CGLFGAIAGLLKNIFENLIDWWNG
SEQ ID NO: 1199


CGLFGAIAGLLKNIFHNLIDWWNG
SEQ ID NO: 1200


CLIGAILKVLATGLPTLISWIKNKRKQ
SEQ ID NO: 1201


CGLLEEIEELLEEGLENLIDWWNG
SEQ ID NO: 1202


CGLFEELEELLEEGLENLIDWWNG
SEQ ID NO: 1203


CGLFEELEELLEEGLENLIEA
SEQ ID NO: 1204


CGLFEELEELLEEGLENLIEAWNG
SEQ ID NO: 1205


CGLFEELEELLEEGLENLIEW
SEQ ID NO: 1206


CGLFEELEELLEEGLENLIEWWNG
SEQ ID NO: 1207


CGLFEELEELLEEGLENLIDA
SEQ ID NO: 1208


CGLFEELEELLEEGLENLIDAWNG
SEQ ID NO: 1209


CGLFEELEELLEEGLENLIDW
SEQ ID NO: 1210


CFLGALKFALKSLL
SEQ ID NO: 1211


CFLGALHFALKSLL
SEQ ID NO: 1212


CFLGALKFALHSLL
SEQ ID NO: 1213


CFLGALHFALHSLL
SEQ ID NO: 1214


FLGALKFALKSLLC
SEQ ID NO: 1215


GFLGALKFALKSLLC
SEQ ID NO: 1216


CGLFGELEELIEEGLENLLDWWNG
SEQ ID NO: 1217


CGLFGEIEELLEEGLENLLDWWNG
SEQ ID NO: 1218


CGLFGELEELLEEGLENLIDWWNG
SEQ ID NO: 1219


CGLFGEIEELIEEGLENLMDWWNG
SEQ ID NO: 1220


CGLFGEIEELIEEGLENLEDWWNG
SEQ ID NO: 1221


CGLFGEIEELIEEGLENLDDWWNG
SEQ ID NO: 1222


CGLFGEIEELIEEGLENLNDWWNG
SEQ ID NO: 1223


CGLFGEIEELIEEGLENLSDWWNG
SEQ ID NO: 1224


CGLFGEIEELIEEGLENLQDWWNG
SEQ ID NO: 1225


CGLFGEIEELIEEGLENL-CIT-DWWNG
SEQ ID NO: 1226


CGLFGEIEELIEELLENLIDWWNG
SEQ ID NO: 1227


CGLFGEIEELIEEILENLIDWWNG
SEQ ID NO: 1228


CGLFGEIEELIEEVLENLIDWWNG
SEQ ID NO: 1229


CFLGALWKLLSHLL
SEQ ID NO: 1230


CFLGALWKILSHLL
SEQ ID NO: 1231


CFLGALWVKVLSHLL
SEQ ID NO: 1232


CFLGALWKFLSHLL
SEQ ID NO: 1233


CFLEALWKALSHLL
SEQ ID NO: 1234


CFLHALWKALSHLL
SEQ ID NO: 1235


CFLKALWKALSHLL
SEQ ID NO: 1236


CFLNALWKALSHLL
SEQ ID NO: 1237


CFLSALWKALSHLL
SEQ ID NO: 1238


CFLQALWKALSHLL
SEQ ID NO: 1239


CFLEALWEALSHLL
SEQ ID NO: 1240


CFLGALWEALSHLL
SEQ ID NO: 1241


CFLEALWKLLSHLL
SEQ ID NO: 1242


CFLEALWEALEELL
SEQ ID NO: 1243


CFLEELWEALEELL
SEQ ID NO: 1244


CFLEALWEALEHLL
SEQ ID NO: 1245


CFLEELWEALEHLL
SEQ ID NO: 1246


CFLEELWELLEELL
SEQ ID NO: 1247


CFLEELWELLEHLL
SEQ ID NO: 1248


CGLFGEIEELLEEGLE-CIT-LIDWWNG
SEQ ID NO: 1249


CGLFEEIEELLEEGLE-CIT-LIDWWNG
SEQ ID NO: 1250


CGLFGEIAELLEEGLE-CIT-LlDWWNG
SEQ ID NO: 1251


CGLFEEIAELLEEGLE-CIT-LIDWWNG
SEQ ID NO: 1252


CGLFGEIEELLEEGLE-CIT-LVDWWNG
SEQ ID NO: 1253


CGLFEEIEELLEEGLE-CIT-LVDWWNG
SEQ ID NO: 1254


CGLFGEIAELLEEGLE-CIT-LVDWWNG
SEQ ID NO: 1255


CGLFEEIAELLEEGLE-CIT-LVDWWNG
SEQ ID NO: 1256


CGLFGEIEELLEEGLE-CIT-LIDWWNE
SEQ ID NO: 1257


CGLFEEIEELLEEGLE-CIT-LIDWWNE
SEQ ID NO: 1258


CGLFGEIAELLEEGLE-CIT-LIDWWNE
SEQ ID NO: 1259


CGLFEEIAELLEEGLE-CIT-LIDWWNE
SEQ ID NO: 1260


CGLFGEIEELLEEGLH-CIT-LIDWWNG
SEQ ID NO: 1261


CGLFEEIEELLEEGLH-CIT-LIDWWNG
SEQ ID NO: 1262


CGLFGEIAELLEEGLH-CIT-LIDWWNG
SEQ ID NO: 1263


CGLFEEIAELLEEGLH-CIT-LIDWWNG
SEQ ID NO: 1264


CGLFGEIEELLEEGLE-CIT-LVDWWNE
SEQ ID NO: 1265


CGLFEEIEELLEEGLE-CIT-LVDWWNE
SEQ ID NO: 1266


CGLFGEIAELLEEGLE-CIT-LVDWWNE
SEQ ID NO: 1267


CGLFEEIAELLEEGLE-CIT-LVDWWNE
SEQ ID NO: 1268


CFFKNIWEFIKSIL
SEQ ID NO: 1269


CFFKNIWNFIKSIL
SEQ ID NO: 1270


CFFKAIWEFIKSILE
SEQ ID NO: 1271


CFFKAIWEFIKNIFK
SEQ ID NO: 1272


CFFKAIWEFIKNIFKE
SEQ ID NO: 1273


CFFKAIWELLKSIL
SEQ ID NO: 1274


CFFKAIWGLLKSIL
SEQ ID NO: 1275


CFFKAIWEFIKSILK
SEQ ID NO: 1276


CFFKNIWGLLKSIL
SEQ ID NO: 1277


CFFKAIWGLLKNIFK
SEQ ID NO: 1278


CFFKAIWELLKNIFK
SEQ ID NO: 1279


CFFKNIWGLLKNIFK
SEQ ID NO: 1280


CFFKNIWELLKNIFK
SEQ ID NO: 1281


CFFKAIWEFIRSIL
SEQ ID NO: 1282


CFFKAIWEFIKSLL
SEQ ID NO: 1283


CFFKAIWEFIKSAL
SEQ ID NO: 1284


CFFKAIWEFIKSIF
SEQ ID NO: 1285


CFFKALWEFLKSLL
SEQ ID NO: 1286


CIFKAIWEFIKSIL
SEQ ID NO: 1287


CFFKAIWEFIKSIW
SEQ ID NO: 1288


CFFHAIWEFIKSIL
SEQ ID NO: 1289


CFFEAIWEFIKSIL
SEQ ID NO: 1290


CFFKAIAEFIKSIL
SEQ ID NO: 1291


CFFKAIEEFIKSIL
SEQ ID NO: 1292


CFFKAILEFIKSIL
SEQ ID NO: 1293


CFFKAIFEFIKSIL
SEQ ID NO: 1294


CFFKAIWGFIKSIL
SEQ ID NO: 1295


CFFKAIWHFIKSIL
SEQ ID NO: 1296


CFFKAIWKFIKSIL
SEQ ID NO: 1297


CFFEAIWKFIKSIL
SEQ ID NO: 1298


CFFKAIWELIKSIL
SEQ ID NO: 1299


CFFKALWELLKSLL
SEQ ID NO: 1300


CFFKAIWEAIKSIL
SEQ ID NO: 1301


CFFKAIWEFLKSIL
SEQ ID NO: 1302


CFFKAIWEFIHSIL
SEQ ID NO: 1303


CFFKAIWEFIESIL
SEQ ID NO: 1304


CFFKAIWEFIKNIL
SEQ ID NO: 1305


CFFKAIWEFIKWIL
SEQ ID NO: 1306


CFFKAIWEFIKEIL
SEQ ID NO: 1307


CFFKAIWEFIKGIL
SEQ ID NO: 1308


CFFKAIWEFIKSGL
SEQ ID NO: 1309


CFFKAIWEFIKSII
SEQ ID NO: 1310


CFFKAIWEFIK-CIT-IL
SEQ ID NO: 1311


CFFKAIWEFIKSIA
SEQ ID NO: 1312


CFFKAIWEFIKQIL
SEQ ID NO: 1313


CGFFKAIWEFIKSIL
SEQ ID NO: 1314


CFFKAIWEFIKSILKGLIDG
SEQ ID NO: 1315


CFFKAIWEFIKSILKGLIDGWYG
SEQ ID NO: 1316


CFFKAIWEFIKSILEGLIDG
SEQ ID NO: 1317


CFFKAIWEFIKSILEGLIDGWYG
SEQ ID NO: 1318


CFFKAIWEFIKNIFKGLIDG
SEQ ID NO: 1319


CFFKAIWEFIKNIFKGLIDGWYG
SEQ ID NO: 1320


CFFGNIWEFIKSILKGLIDG
SEQ ID NO: 1321


CFFGNIWEFIKSILKGLIDGWYG
SEQ ID NO: 1322


CFFGNIWEFIKSILEGLIDG
SEQ ID NO: 1323


CFFGNIWEFIKSILEGLIDGWYG
SEQ ID NO: 1324


CFFGNIWEFIKNIFKGLIDG
SEQ ID NO: 1325


CFFGNIWEFIKNIFKGLIDGEYG
SEQ ID NO: 1326


CFFKAIWGLLKSILKGLIDG
SEQ ID NO: 1327


CFFKAIWGLLKSILKGLIDGWYG
SEQ ID NO: 1328


CFFKAIWGLLKSILEGLIDG
SEQ ID NO: 1329


CFFKAIWGLLKSILEGLIDGWYG
SEQ ID NO: 1330


CFFKAIWGLLKNIFKGLIDG
SEQ ID NO: 1331


CFFKAIWGLLKNIFKGLIDGWYG
SEQ ID NO: 1332


CFFKAIWGLLKNIFEGLIDG
SEQ ID NO: 1333


CFFKAIWGLLKNIFEGLIDGWYG
SEQ ID NO: 1334


CFFKAIWEFIKSILKGLIDGWNG
SEQ ID NO: 1335


CFFKAIWEFIKNIFKGLIDGWNG
SEQ ID NO: 1336


CIFGAIAGLLKNILKGLIDG
SEQ ID NO: 1337


CIFGAIAGLLKNILKGLIDGWYG
SEQ ID NO: 1338


CFLEALWKALEHLL
SEQ ID NO: 1339


CFLEALWEALSKLL
SEQ ID NO: 1340


CFLEALWEALEKLL
SEQ ID NO: 1341


CFLEALWEALEHLLK(stearyl)
SEQ ID NO: 1342


(stearyl)FLEALWEALEHLLC
SEQ ID NO: 1343


(stearyl)GFLEALWEALEHLLC
SEQ ID NO: 1344


CFLEALWKALSKLL
SEQ ID NO: 1345


CFLEALWEALDHLL
SEQ ID NO: 1346


CFLEALWEALTHLL
SEQ ID NO: 1347


CFLEALWEALNHLL
SEQ ID NO: 1348


CFLEALWEALQHLL
SEQ ID NO: 1349


CFLEALWEALEHLLH
SEQ ID NO: 1350


CFLEALWEALEHLLK
SEQ ID NO: 1351


CFLEALWEALEHLLE
SEQ ID NO: 1352


CWLEALEALEHLL
SEQ ID NO: 1353


CLLEALWEALEHLL
SEQ ID NO: 1354


CFFEALWEALEHLL
SEQ ID NO: 1355


CFLEALEEALEHLL
SEQ ID NO: 1356


CFLEALAEALEHLL
SEQ ID NO: 1357


CFLEALFEALEHLL
SEQ ID NO: 1358


CLFEALWEALHHLL
SEQ ID NO: 1359


CLFEALWEALKHLL
SEQ ID NO: 1360


CFLEALWEALEHGL
SEQ ID NO: 1361


CLFEALWEALEHLF
SEQ ID NO: 1362


CLFEALWEALEHFL
SEQ ID NO: 1363


CLFEALWEALEHLLEGLIDWWYG
SEQ ID NO: 1364


CLFEALWEALEHLLEGLIDWWNG
SEQ ID NO: 1365


CLFEALWEALEHLLENLIDWWNG
SEQ ID NO: 1366


CFLEELWELLEKLL
SEQ ID NO: 1367


CFLEELWELLEELLE
SEQ ID NO: 1368


CFLEELWELLEELLELLE
SEQ ID NO: 1369


CFLEELWELLEHLLELLD
SEQ ID NO: 1370


CFLEELWELLEELLELID
SEQ ID NO: 1371


CFLEELWELLEELLELLD
SEQ ID NO: 1372


CFLEELWELLEHLLEGLE
SEQ ID NO: 1373


CFLEELWELLEHLLEGLD
SEQ ID NO: 1374


CFLEELWELLEHLLEEGLI
SEQ ID NO: 1375


CFLEELWELLEHLLEGLIDWWYG
SEQ ID NO: 1376


CFLEELWELLEHLLENLIDWWNG
SEQ ID NO: 1377


CFLEALWEALEHLLELLD
SEQ ID NO: 1378


CGLFGELEELLEEGLENLTDWWNG
SEQ ID NO: 1379


CGLFGELEELLEEGLENL-(ALLO-I)-DWWNG
SEQ ID NO: 1380


CFLEALWEALEHLLELID
SEQ ID NO: 1381


CELFEELEELLEEGLENLIDWWNG
SEQ ID NO: 1382


CGLFEELEELLEEGLELLIDWWNG
SEQ ID NO: 1383


CGLFEELEELLEEGLELLIDWWNK
SEQ ID NO: 1384


CGLFEELEELLEEGLENLIDWWNK
SEQ ID NO: 1385


CGLFGELEELLEEGLENLIDWWNQ
SEQ ID NO: 1386


CGLFGELEELLEEGLENLIDWWNE
SEQ ID NO: 1387


CGLFGELEELLEEGLENLIDWWNN
SEQ ID NO: 1388


CGLFGELEELLEEGLENLIDWWNS
SEQ ID NO: 1389


CGLFEELEELLEEGLENLIDWWNQ
SEQ ID NO: 1390


AC-CFLEELWELLEHLL
SEQ ID NO: 1391


AC-CFLEELWELLEELL
SEQ ID NO: 1392


CGLLGEIEELLEEGLENLIDWWNG
SEQ ID NO: 1393


CGLLAEIEELLEEGLENLIDWWNG
SEQ ID NO: 1394


CGLLGEIEELLEEGLENLIDWWNQ
SEQ ID NO: 1395


CGLLAEIEELLEEGLENLIDWWNQ
SEQ ID NO: 1396


CGLLEEIEELLEEGLENLIDWWNQ
SEQ ID NO: 1397


CGLLGEIEELLEEGLENLIDWWNE
SEQ ID NO: 1398


CGLLAEIEELLEEGLENLIDWWNE
SEQ ID NO: 1399


CGLLEEIEELLEEGLENLIDWWNE
SEQ ID NO: 1400


CGLLGEIEELLEEGLENLIDWWNS
SEQ ID NO: 1401


CGLLAEIEELLEEGLENLIDWWNS
SEQ ID NO: 1402


CGLLEEIEELLEEGLENLIDWWNS
SEQ ID NO: 1403


CGLFAELEELLEEGLENLLEWWNG
SEQ ID NO: 1404


CGLFEELEELLEEGLENLLEWWNG
SEQ ID NO: 1405


CGLFGELEELLEEGLENLLEWWNE
SEQ ID NO: 1406


CGLFAELEELLEEGLENLLEWWNE
SEQ ID NO: 1407


CGLFEELEELLEEGLENLLEWWNE
SEQ ID NO: 1408


CGLLGELEELLEEGLENLLEWWNG
SEQ ID NO: 1409


CGLLGELEELLEEGLENLLEWWNE
SEQ ID NO: 1410


CGILGEIEELLEEGLENLIDWWNG
SEQ ID NO: 1411


CGILGEIEELLEEGLENLIDWWNE
SEQ ID NO: 1412


CGILGEIEELLEEGLENLIDWWNS
SEQ ID NO: 1413


CGILAEIEELLEEGLENLIDWWNG
SEQ ID NO: 1414


CGILEEIEELLEEGLENLIDWWNG
SEQ ID NO: 1415


CIFGAIAELLKNIFK
SEQ ID NO: 1416


CIFGAIAELLENIFK
SEQ ID NO: 1417


CIFGAIAGLLENIFK
SEQ ID NO: 1418


CFLEELWGLLEHLL
SEQ ID NO: 1419


CGILAEIEELLEEGLENLIDWWNQ
SEQ ID NO: 1420


CGILAEIEELLEEGLENLIDWWNE
SEQ ID NO: 1421


CGLFAEIEELLEEGLENLIDWWNQ
SEQ ID NO: 1422


CGLFAEIEELLEEGLENLIDWWNE
SEQ ID NO: 1423


CGLFGELEELLEEGLENLLEWWNQ
SEQ ID NO: 1424


CGLFAEIAELLEEGLE-CIT-LIDWWNE
SEQ ID NO: 1425


CGILAEIEELLEEGLENLLEWWNG
SEQ ID NO: 1426


CGILEEIEELLEEGLENLIDWWNE
SEQ ID NO: 1427


CGILEEIEELLEEGLENLIDWWNQ
SEQ ID NO: 1428


CGLFGEIEELIWEGLENLIDWWNG
SEQ ID NO: 1429


CGLFGEIAELIWEGLENLIDWWNG
SEQ ID NO: 1430


CGLFEEIAELIEEGLENLIDWWNG
SEQ ID NO: 1431


CGLFEEIAELIWEGLENLIDWWNG
SEQ ID NO: 1432


CELFEEIAELIWEGLENLIDWWNG
SEQ ID NO: 1433


CELFEEIAELLWEGLENLIDWWNG
SEQ ID NO: 1434


CGLFEEIAELLWEGLENLIDWWNG
SEQ ID NO: 1435


CGLFEELAELLWEGLENLIDWWNG
SEQ ID NO: 1436


CELFEELAELLWEGLENLIDWWNG
SEQ ID NO: 1437


CELFEELAELLWEGLENLIDWWNS
SEQ ID NO: 1438


CGLFEELAELLWEGLENLIDWWNS
SEQ ID NO: 1439


CGIFEELAELLWEGLENLIDWWNG
SEQ ID NO: 1440


CGIFEELAELLWEGLENLIDWWNS
SEQ ID NO: 1441


CGLFEELEELLEELLENLIDWWNS
SEQ ID NO: 1442


CELFEELEELLEELLENLIDWWNS
SEQ ID NO: 1443


CELFEELEELLEELLELLIDWWNS
SEQ ID NO: 1444


CEFLEELEELLEELLENLIDWWNS
SEQ ID NO: 1445


CELFEELEELLEHLLENLIDWWNS
SEQ ID NO: 1446


CELFEELEELLHELLENLIDWWNS
SEQ ID NO: 1447


CGLFGELEELLWEGLENLIDWWNG
SEQ ID NO: 1448


CGLFGELEELLWEGLHNLIDWWNG
SEQ ID NO: 1449


CGLFGELWELLEHGLENLIDWWNG
SEQ ID NO: 1450


CGL-R6H-GELEEL-S7H-EEGLENLIDWWNG
SEQ ID NO: 1451


CGLFEAIEGFIENGWEGMIDGWNG
SEQ ID NO: 1452


CGLFEAIEGFIENGWEGMIDWWNG
SEQ ID NO: 1453


CGLFGAIEGFIENGWEGMIDWWNG
SEQ ID NO: 1454


CGLFAEIEELLEEGLENLLEWWNG
SEQ ID NO: 1455


CGLFAELEELLEEGLENLIDWWNG
SEQ ID NO: 1456


CGIFAEIEELLEEGLENLIDWWNG
SEQ ID NO: 1457


CGLFAEIEELLEEGLENLIDWWNGF
SEQ ID NO: 1458


CGLFAEIEELLEEGLENLIDWWNA
SEQ ID NO: 1459


CGLFAEIEELLEEGLENLIDWWNS
SEQ ID NO: 1460


CGLFAEIEELLEEGLENLIDWWN-CIT
SEQ ID NO: 1461


CGLFGEIAGLLEEGLHNLIDWWNG
SEQ ID NO: 1462


CGLFGEIAGLLEQGLHNLIDWWNG
SEQ ID NO: 1463


CGLFGEIAGLLESGLHNLIDWWNG
SEQ ID NO: 1464


CGLFAEIAGLLEQGLHNLIDWWNG
SEQ ID NO: 1465


CGLFAEIAGLLEEGLHNLIDWWNG
SEQ ID NO: 1466


CGLFAEIAGLLESGLHNLIDWWNG
SEQ ID NO: 1467


CGIFEAIAGLLEQGLHNLIDWWNG
SEQ ID NO: 1468


CGLFGAIAELLEEGLHNLIDWWNG
SEQ ID NO: 1469


CGLFAAIAELLEEGLHNLIDWWNG
SEQ ID NO: 1470


CGIFEAIAGLLKNIFKNLIDWWNG
SEQ ID NO: 1471


CGIFGAIWELLEQGLHNLIDWWNG
SEQ ID NO: 1472


CGLFAELAGLLEQGLHNLIDWWNG
SEQ ID NO: 1473


CGILAELAGLLEQGLHNLIDWWNG
SEQ ID NO: 1474


CGLFGEIEELLEHLL
SEQ ID NO: 1475


CGLFGEIEELLEELL
SEQ ID NO: 1476


CGLFGEIEELLEEGL
SEQ ID NO: 1477


CGLFGEIEELLEHGL
SEQ ID NO: 1478


CGLFHEIEELLEHLL
SEQ ID NO: 1479


CFLGALWKALSELLE
SEQ ID NO: 1480


CGLFGEIWELLEEGL
SEQ ID NO: 1481


CGLFGEIWELLEEGLI
SEQ ID NO: 1482


CGLFGEIWELLEELL
SEQ ID NO: 1483


CGLFEEIEELLEELLE
SEQ ID NO: 1484


CGLFELIEGFIEWGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 1485


CIFGAIAGFIKNIWLHLLHHLLHHLHHLLHHLLHL
SEQ ID NO: 1486


CEALFGKINAIFIGKL
SEQ ID NO: 1487


CEENWIGLFGGGNIWEEEEILDLL
SEQ ID NO: 1488


CLELWLEHLFLELE
SEQ ID NO: 1489


CGNFEEIEGFIENGWEGLIDGWYGYGRKKRRQRR
SEQ ID NO: 1490


CRGKWYMGFGEIKRQGEGRRYGLFEDWIAENRGI
SEQ ID NO: 1491


GLFEAIEGFIENGWEGLAELAEALEALAAGGSC
SEQ ID NO: 1492


GLFGALAEALAEALAEHLAEALAEALEALAAGGSC
SEQ ID NO: 1493


CGFFGEIAGLLENGLHNLIDWWNG
SEQ ID NO: 1494


CGFFGEIAALLENGLENLIDWWNG
SEQ ID NO: 1495


CGFFGEIAEFIHSGLKNLIDWWNG
SEQ ID NO: 1496


CGFFGEIAGLLKNGLKNLIDWWNG
SEQ ID NO: 1497


CGFFGEIAGFIKNGLKNLIDWWNG
SEQ ID NO: 1498


CGFFGEIAEFIHSILKNLIDWWNG
SEQ ID NO: 1499


CGFFGEIAGLLKNILKNLIDWWNG
SEQ ID NO: 1500


CGFFGEIAGFIKNILKNLIDWWNG
SEQ ID NO: 1501


CFLGALFHALSELL
SEQ ID NO: 1502


CFLGALWHALSELL
SEQ ID NO: 1503


CFLGALWHALSHLL
SEQ ID NO: 1504


CFLGALWELLSHLL
SEQ ID NO: 1505


CFLGALWKALSHLL
SEQ ID NO: 1506


CFLGALWHALSKLL
SEQ ID NO: 1507


CFLGALFHLLSHLL
SEQ ID NO: 1508


CFLGALFHLLSELL
SEQ ID NO: 1509


CFLGALWHLLSHLL
SEQ ID NO: 1510


CFLGALWHLLSELL
SEQ ID NO: 1511


CFLGALFHALSHLLE
SEQ ID NO: 1512


CFLGALFHLLSHLLE
SEQ ID NO: 1513


CGLFGALFHALSHLLE
SEQ ID NO: 1514


CFLGALWKALSHLL
SEQ ID NO: 1515


CGLFAEIEELLEEGLENLIDWWNG
SEQ ID NO: 1516


CGLFGEIEELIEEGLE-Cit-LIDWWNG
SEQ ID NO: 1517


CGLFGEIEELIEEGLENLIDWWNE
SEQ ID NO: 1518


CFFGAIWEFIHSILK(stearyl)
SEQ ID NO: 1519


CIFGAIAGFIKNIWEGLIK(stearyl)
SEQ ID NO: 1520


CGIFEAIAGLLKNIFK(stearyl)
SEQ ID NO: 1521


CGIFEAIAGLLKNIFKK(stearyl)
SEQ ID NO: 1522


CFLGALFHALSHLL
SEQ ID NO: 1523


Ac-CIFGAIAGFIKNILKGLIDG
SEQ ID NO: 1524


CIFGAIAGFIKNILKGLK(stearylL)
SEQ ID NO: 1525


Ac-CIFGAIAGFIKNILKGLK(stearyl)
SEQ ID NO: 1526


CGLFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 1527


CFLGALWKALSELLKNLIDWWNG
SEQ ID NO: 1528


CGFLGALWKALSELLKNLIDWWNG
SEQ ID NO: 1529


CFLGALFHALSHLLENLIDWWNG
SEQ ID NO: 1530


CGFLGALFHALSHLLENLIDWWNG
SEQ ID NO: 1531


CGLFGELEGFIENGLKNLIDWWNG
SEQ ID NO: 1532


CGLFGELEGLLWHGLKNLIDWWNG
SEQ ID NO: 1533


CGLFGELAELLWHGLKNLIDWWNG
SEQ ID NO: 1534


CGLFGELAELLWQGLKNLIDWWNG
SEQ ID NO: 1535


CGLFGELWELLWHGLKNLIDWWNG
SEQ ID NO: 1536


CGLFGELWELLWQGLKNLIDWWNG
SEQ ID NO: 1537


CGLFEELAGLLWHGLKNLIDWWNG
SEQ ID NO: 1538


CGLFEELWGLLWHGLKNLIDWWNG
SEQ ID NO: 1539


CGLFEELAGLLWQGLKNLIDWWNG
SEQ ID NO: 1540


CGLFEELWGLLWQGLKNLIDWWNG
SEQ ID NO: 1541


CGLFGELAELLWHGLKNLIDWWNK
SEQ ID NO: 1542


CGLFEELAELLWHGLKNLIDWWNK
SEQ ID NO: 1543


CGLFGELAELLWHGLKNLIDWWNH
SEQ ID NO: 1544


CGLFEELAELLWHGLKNLIDWWNH
SEQ ID NO: 1545


CGLFAELWGLLWQGLKNLIDWWNG
SEQ ID NO: 1546


CGLFAELWGLLWHGLKNLIDWWNG
SEQ ID NO: 1547


CGLFAELWGLLWHGLHNLLDWWNG
SEQ ID NO: 1548


CGLFAELAELLWEGLKNLIDWWNG
SEQ ID NO: 1549


CGLFAELAELLWHGLKNLIDWWNG
SEQ ID NO: 1550


CGLFAELELLWQGLKNLIDWWNG
SEQ ID NO: 1551


CELFGELAGLLWHGLKNLIDWWNG
SEQ ID NO: 1552


CLFEALWE-Aib-LEKLF
SEQ ID NO: 1553


CFLEALWELLEHLL
SEQ ID NO: 1554


CFLEALWKALEKLL
SEQ ID NO: 1555


CGLF-Aib-EIAGLLEEGLHNLIDWWNG
SEQ ID NO: 1556


CGLFGEI-Aib-GLLEEGLHNLIDWWNG
SEQ ID NO: 1557


CGFFGEIAGLLEE-Aib-LHNLIDWWNG
SEQ ID NO: 1558


CGLFGEIAGLLEEGLHNLIDWWN-Aib
SEQ ID NO: 1559


CGLF-Aib-EIAGLLEE-Aib-LHNLIDWWNG
SEQ ID NO: 1560


CGFFGEI-Aib-GLLEE-Aib-LHNLIDWWNG
SEQ ID NO: 1561


CGFFGEI-Aib-ELIWEGLKNLIDWWNG
SEQ ID NO: 1562


CGFFGEIAELIWELKNLIDWWN-Aib
SEQ ID NO: 1563


CGFFAib-EIAELIWE-Aib-LKNLIDWWNG
SEQ ID NO: 1564


AC-CFLGALWKALSHLL
SEQ ID NO: 1565


AC-CFLEELWELLEELLE
SEQ ID NO: 1566


AC-CLFGALWKALSELL
SEQ ID NO: 1567


AC-CGIGAVLKVLTTGLPALISWIKRKRQQ
SEQ ID NO: 1568


AC-CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 1569


AC-CGLFEAIEGFIENGWEGMIDGVVWYGYGRKKRRQRRK(stearyl)
SEQ ID NO: 1570


Ac-CFLGALWKALSHLL
SEQ ID NO: 1571


Ac-CFLGALWKALSELL
SEQ ID NO: 1572


CELFEEIAELLWEGLENLIDWWNG
SEQ ID NO: 1573


CGLFGEIAELIWEGLENLIDWWNG
SEQ ID NO: 1574


CGLFGEIEELLEEGLENLIDWWNG
SEQ ID NO: 1575


CGLFAELAELLWEGLENLIDWWNG
SEQ ID NO: 1576


CGLFAELAELLEEGLENLIDWWNG
SEQ ID NO: 1577


CGLFAELAELLWEGLENLIDWWNS
SEQ ID NO: 1578


CGLFAELAELLEEGLENLIDWWNS
SEQ ID NO: 1579


CGLFAELAELLWEGLENLIDWWNQ
SEQ ID NO: 1580


CGLFAELAELLEEGLENLIDWWNQ
SEQ ID NO: 1581


CGLFAELAELLWEGLENLIDWWNE
SEQ ID NO: 1582


CGLFAELAELLEEGLENLIDWWNE
SEQ ID NO: 1583


CELFEELAELLWEGLENLIDWWNQ
SEQ ID NO: 1584


CELFEELAELLWEGLENLIDWWNE
SEQ ID NO: 1585


CELFEELAELLEEGLENLIDWWNG
SEQ ID NO: 1586


CELFAELAELLWEGLENLIDWWNG
SEQ ID NO: 1587


CELFAELAELLEEGLENLIDWWNG
SEQ ID NO: 1588


CELFAELAELLWEGLENLIDWWNS
SEQ ID NO: 1589


CELFAELAELLEEGLENLIDWWNS
SEQ ID NO: 1590


CELFAELAELLWEGLENLIDWWNQ
SEQ ID NO: 1591


CELFAELAELLEEGLENLIDWWNQ
SEQ ID NO: 1592


CELFAELAELLWEGLENLIDWWNE
SEQ ID NO: 1593


CELFAELAELLEEGLENLIDWWNE
SEQ ID NO: 1594


CELFEELAELLWEGLHNLIDWWNG
SEQ ID NO: 1595


CELFEELAELLWEGLHNLIDWWNS
SEQ ID NO: 1596


CELFEELAELLWEGLHNLIDWWNQ
SEQ ID NO: 1597


CELFEELAELLWEGLHNLIDWWNE
SEQ ID NO: 1598


CELFGELEGFIENGLENLIDWWNG
SEQ ID NO: 1599


CGLFEELEGFIENGLENLIDWWNG
SEQ ID NO: 1600


CGLFAELAGFIENGLENLIDWWNG
SEQ ID NO: 1601


CGLFAELEGFIENGLENLIDWWNG
SEQ ID NO: 1602


CGLFGELAGFIENGLENLIDWWNG
SEQ ID NO: 1603


CELFEELEGFIENGLENLIDWWNG
SEQ ID NO: 1604


CELFAELAGFIENGLENLIDWWNG
SEQ ID NO: 1605


CGLFGELEGFIWNGLENLIDWWNG
SEQ ID NO: 1606


CGLFGELEGFIENGLENLIDWWNG
SEQ ID NO: 1607


CGLFGELEGFIENGLENLIDWWNQ
SEQ ID NO: 1608


CGLFGELEGFIENGLENLIDWWNE
SEQ ID NO: 1609


CELFEELEGFIENGLENLIDWWNE
SEQ ID NO: 1610


CGLLEEIAELLEEGLENLIDWWNS
SEQ ID NO: 1611


CGLLEEIEELLWEGLENLIDWWNS
SEQ ID NO: 1612


CELLEEIEELLEEGLENLIDWWNS
SEQ ID NO: 1613


CGLLEEIAELLWEGLENLIDWWNS
SEQ ID NO: 1614


CELLEEIAELLWEGLENLIDWWNS
SEQ ID NO: 1615


CELLEEIEELLEEGLENLIDWWNE
SEQ ID NO: 1616


CGLLEELEELLEEGLENLIDWWNS
SEQ ID NO: 1617


CGLLEELEELLEEGLENLLEWWNS
SEQ ID NO: 1618


CGLLEEIAELLEEGLENLIDWWNG
SEQ ID NO: 1619


CGLLAEIAELLEEGLENLIDWWNS
SEQ ID NO: 1620


CGLLAEIAELLWEGLENLIDWWNS
SEQ ID NO: 1621


CGLLEEIEGFIENGLENLIDWWNS
SEQ ID NO: 1622


CGLLEEIEGFIENGLENLIDWWNG
SEQ ID NO: 1623


CGLLEEIEELLEEGLE-Cit-LIDWWNS
SEQ ID NO: 1624


CGLLEEIEELLEQGLENLIDWWNS
SEQ ID NO: 1625


CGLLAELAELLEEGLENLIDWWNS
SEQ ID NO: 1626


CGLLEEIEELLEEGLENLIDWWNA
SEQ ID NO: 1627


CGLL-Aib-EIEELLEEGLENLIDWWNS
SEQ ID NO: 1628


CGLLEEIEELLEEGLENLIDWWN-Aib
SEQ ID NO: 1629


CGLLEEIEELLEE-Aib-LENLIDWWNG
SEQ ID NO: 1630


CGLFGHIHHLIHHGLHNLIDWWNG
SEQ ID NO: 1631


CGLFGEIHHLIHHGLHNLIDWWNG
SEQ ID NO: 1632


CGLFGEIHHLIHHGLENLIDWWNG
SEQ ID NO: 1633


CGLFGEIHELIHHGLENLIDWWNG
SEQ ID NO: 1634


CELLEEIEELLEEGLENLIDWWNS
SEQ ID NO: 1635


CGLFGELEELIEEGLENLIDWWNG
SEQ ID NO: 1636


CGLLAEIEELLWEGLENLIDWWNS
SEQ ID NO: 1637


CGLLEEIEELLEEGLENLLEWWNS
SEQ ID NO: 1638


C(b-ALA)LLEEIEELLEEGLENLIDWWNS
SEQ ID NO: 1639


CGLLEEIEELLEEGLENLIDLWNS
SEQ ID NO: 1640


CGLLEEIEELLEWGLENLIDWWNS
SEQ ID NO: 1641


CGLFGEIEELIEEGLENLIDWGNG
SEQ ID NO: 1642


CGFFGEIAELIEEGLKNLIDWGNG
SEQ ID NO: 1643


CGLFGEIEELIEEGLENLIDWANG
SEQ ID NO: 1644


CGLFGEIEELIEEGLENLIDWSNG
SEQ ID NO: 1645


CGLFGEIEELIEEGLENLIDW-(Aib)-NG
SEQ ID NO: 1646


CGLFGEIEELIEEGLENLIDWPNG
SEQ ID NO: 1647


CGLFGEIEELIEEGLENLIDWHNG
SEQ ID NO: 1648


CGLFGEIEELIEEGLENLIDWQNG
SEQ ID NO: 1649


CGLFGEIEELIEEGLENLIDWENG
SEQ ID NO: 1650


CGLFEEIAELIEEGLENLIDWGNG
SEQ ID NO: 1651


CELFEELAELLWEGLENLIDWGNS
SEQ ID NO: 1652


CGLFGEIAELIWEGLENLIDWGNG
SEQ ID NO: 1653


CGLLEEIEELLEEGLENLIDWGNS
SEQ ID NO: 1654


CGLFAEIEELLEEGLENLIDWGNG
SEQ ID NO: 1655


CGLL-(Aib)-EIEELLEEGLENLIDWWNS
SEQ ID NO: 1656


CGLFGEIEELIEEGLENLIDWNNG
SEQ ID NO: 1657


CGLFGEIEELIEEGLENLIDWDNG
SEQ ID NO: 1658


CGLFGEIEELIEEGLENLIDWONG
SEQ ID NO: 1659


CGLFAEIEELLEEGLENLIDWGNG
SEQ ID NO: 1660


CGLL-Aib-EIEELLEEGLENLIDWGNS
SEQ ID NO: 1661


CGLFGEIEELIEEGLENLIDGWNG
SEQ ID NO: 1662


CGLFGEIEELIEEGLENLIDLWNG
SEQ ID NO: 1663


CGWFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 1664


CGLFGEVEELIEEGLENLIDWWNG
SEQ ID NO: 1665


CGLFGEIEEVIEEGLENLIDWWNG
SEQ ID NO: 1666


CGLFGEIEELVEEGLENLIDWWNG
SEQ ID NO: 1667


CGLFGEIEELAEEGLENLIDWWNG
SEQ ID NO: 1668


CGLFGEIEELIDEGLENLIDWWNG
SEQ ID NO: 1669


CGLFGEIEELIEDGLENLIDWWNG
SEQ ID NO: 1670


CGLFGEIEELIEEGLEALIDWWNG
SEQ ID NO: 1671


CGLFGEIEELIEEGLENIIDWWNG
SEQ ID NO: 1672


CGLFGEIEELIEEGLEN-(Nle)-IDWWNG
SEQ ID NO: 1673


CGLFGEIEELIEEGLENLIGWWNG
SEQ ID NO: 1674


CGLFGEIEELIEEGLENLIDAWNG
SEQ ID NO: 1675


CGLLEEIEELLEEGLENLIDWWNE
SEQ ID NO: 1676


CELFEELAELLWEGLENLIDWWNE
SEQ ID NO: 1677


CGLFGEIEELIEEGLENLIGWWNG
SEQ ID NO: 1678


CGLFELIEGFIENGWEGMIDGWYGYGRKKRRQRR all (D)
SEQ ID NO: 1679


CGLFEAIEGFIENGWEGMIDGWYG all (D)
SEQ ID NO: 1680


CGLFGEIEELIENGLKNLIDWWYGYGRKKRRQRR all (D)
SEQ ID NO: 1681


CGLFEALLELLESLWELLLEAYGRKKRRQRR all (D)
SEQ ID NO: 1682


CGLFEEIEGFIENGWEGLIDWWYGYGHKKHHQHR all (D)
SEQ ID NO: 1683


CGLFGEIEELIEEGLENLIDWWNE all (D)
SEQ ID NO: 1684


CGLFGEIEELIEEGLENLIDWWNS all (D)
SEQ ID NO: 1685


CGLFGEIEELIEEGLENLIDWWNQ all (D)
SEQ ID NO: 1686


CYGRKKRRQRRLIRLWSHLIHIWFQNRRLKWKKK
SEQ ID NO: 1687


CGLFEAIEEFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 1688


CGLFFAIEGFIENGWEGMIDWWYGYGRKKRRQRR ALL (D)
SEQ ID NO: 1689


CGLFELIEGFIENGWEGMIDGWYGYGRKKRRQRRK(STEARYL) 
SEQ ID NO: 1690


ALL (D)



(STEARYL)GLFELIEGFIENGWEGMIDGWYGYGRKKRRQRRC 
SEQ ID NO: 1691


ALL (D)



CFFGAIWEFIKSILK(STEARYL) ALL(D)
SEQ ID NO: 1692


CGIFEAIAGLLKNIFKGIFEAIAGLLKNIFK ALL (D)
SEQ ID NO: 1693


CIFGAIAGFIKNILKGLIDG ALL (D)
SEQ ID NO: 1694


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRK(STEARYL) 
SEQ ID NO: 1695


ALL(D)



(LAURYL)FFGAIWEFIKSILC ALL (D)
SEQ ID NO: 1696









The D-amino acid, retro-inverso, and cysteine conjugation point variants of the peptides shown in Table 3 are also suitable.
The preferred peptides are listed in Table 4 below:







TABLE 4







Peptide Listing and ID








Sequence
SEQ ID





CGLFEAIEGFIENGWEGMIDGWYGYGHKKHHQHH
SEQ ID NO: 2


C-bAla-LFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 3


CGLFEAIEGFIEWGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 5


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQR
SEQ ID NO: 7


CGLFHALLHLLHSLWHGLLHAWYGYGHKKHHQHR
SEQ ID NO: 11


CGLFELIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 13


CGLFEAIEGFIENGWEG-Nle-IDGWYGYGRKKRRQRR
SEQ ID NO: 19


CGLLEALEGLLESLWEGLLEAWYGYGRKKRRQRR
SEQ ID NO: 22


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRK(stearyl)
SEQ ID NO: 27


CGLFEAIAGFIEGGWPGLINGWYGYGRKKRRQRRLHLLHHLLHHLHHL
SEQ ID NO: 28


LHHLLHLLHHLLHHL



CGLFEAIEGFIENGWEGMIDGWYGGGGLHLLHHLLHHLHHLLHHLLHL
SEQ ID NO: 29


LHHLLHHL



CGLFEAIEGFIENGWEGMIDGWYGLHLLHHLLHHLHHLLHHLLHL
SEQ ID NO: 30


CGLFEALLELLESLWELLLEAYGRKKRRQRR
SEQ ID NO: 31


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR
SEQ ID NO: 32


CGLFHALLHLLHSLWHLLLHAWYGYGRKKRRQRR
SEQ ID NO: 55


CGLFHALLHLLHSLWHLLLHAWYGYGHKKHHQHR
SEQ ID NO: 56


CGIFGAIAGLLKNIFK
SEQ ID NO: 63


CIFGAIAGFIKNIWKGLIDW
SEQ ID NO: 64


stearyl-WEAALAEALAEALAEHLAEALAEALEALAAYGRKKRRQRRC
SEQ ID NO: 69


CGFFHAFFHFFHSFWHGFFEA
SEQ ID NO: 71


CGNFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 72


CFFGAIWEFIRNILEGF
SEQ ID NO: 73


CFFGAIWEFIHSIL
SEQ ID NO: 74


CGLFGEIEEFIENGWKGLIDWWYG
SEQ ID NO: 86


CIFGIDDLIIGLLFVAIVEAGIGGYLLGSYGRKKRRQRR
SEQ ID NO: 90


CFFGAIWEFIRSILK
SEQ ID NO: 94


CFFGAIWEFIRSILE
SEQ ID NO: 95


CGLFEAIEGFIENGWEGMIDWWYGYGRKKRRQRR
SEQ ID NO: 106


CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR all (D)
SEQ ID NO: 137


CRRQRRKKRGYGYWGDIMGEWGNEIFGEIAEFLG
SEQ ID NO: 192


RRQRRKKRGYGYWGDIMGEWGNEIFGEIAEFLGC all(D)
SEQ ID NO: 200


CRRQRRKKRGYGYWGDIMGEWGNEIFGEIAEFLG all(D)
SEQ ID NO: 201


CGLFEAIEGFIENGWKGMIDGWYGYGRKKRRQRR
SEQ ID NO: 228


CGLFEAIEGFIENGWKGMIDGWYGYGRKKRRQRR
SEQ ID NO: 228


CGLFEAIEGFIENGWKGLIDWWYGYGRKKRRQRR
SEQ ID NO: 266


CIFGAIAGFIKNIW
SEQ ID NO: 283


CFFGAIWEFIRNIL
SEQ ID NO: 333


FFGAIWEFIKSILC
SEQ ID NO: 409


CFFGKIWEFIKSIL
SEQ ID NO: 407


CFFGAIWEFAKSIL
SEQ ID NO: 423


CGLFHALLHLLHSLWHLLLEA
SEQ ID NO: 436


CGLFHALLHLLHSLWKLLLEW
SEQ ID NO: 437


CGFFGEIAELIEEGLKGLIDWWNG
SEQ ID NO: 461


CGLFGEIEELIEEGLENLIDWWNG
SEQ ID NO: 462


CFFGAIWEFIHSIL all (D)
SEQ ID NO: 463


CGIFEAIAGLLKSILKK(stearyl)
SEQ ID NO: 468


CGIFGAIAGLLKSILKK(stearyl)
SEQ ID NO: 469


CIFGAIAGFIKNILKGL all (D)
SEQ ID NO: 470


CIFGAIAGFIKNILKGLK(stearyl)
SEQ ID NO: 473


GLGKLINKIFGAIAGFIC all (D)
SEQ ID NO: 474


CGLFGEIEELIEEGLENLIDWWNG all(D)
SEQ ID NO: 491


CGNFGEIEELIEEGLENLIDWWNG all(D)
SEQ ID NO: 492


CGFFGEIAELIEEGLKGLIDWWNG all(D)
SEQ ID NO: 493


CGIFEAIAGLLKNIFK all(D)
SEQ ID NO: 612


CIFGAIAGFIKNIWEGLI all (D)
SEQ ID NO: 489


CGLFGEIEELIEEGLENLIDWGNG all (D)
SEQ ID NO: 1074


CGLFGEIEELIEEGLENLIDWGNG
SEQ ID NO: 1642


CGLFELIEGFIENGWEGMIDGWYGYGRKKRRQRR all (D)
SEQ ID NO: 1679


CGLFEAIEGFIENGWEGMIDGVVYG all (D) 
SEQ ID NO: 1680


CGLFGEIEELIENGLKNLIDWWYGYGRKKRRQRR all (D)
SEQ ID NO: 1681


CGLFEALLELLESLWELLLEAYGRKKRRQRR all (D)
SEQ ID NO: 1682


CGLFEEIEGFIENGWEGLIDWWYGYGHKKHHQHR all (D)
SEQ ID NO: 1683


CGLFGEIEELIEEGLENLIDWWNE all (D)
SEQ ID NO: 1684


CGLFGEIEELIEEGLENLIDWWNS all (D)
SEQ ID NO: 1685


CGLFGEIEELIEEGLENLIDWWNQ all (D)
SEQ ID NO: 1686


GFFGAIWEFIKSILC
SEQ ID NO: 337









The D-amino acid, retro-inverso, and cysteine conjugation point variants of the peptides shown in Table 4 are also preferred.
Targeting Ligands
The modular compositions of the present invention may comprise a targeting ligand. In some embodiments, this targeting ligand may direct the modular composition to a particular cell. For example, the targeting ligand may specifically or non-specifically bind with a molecule on the surface of a target cell. The targeting moiety can be a molecule with a specific affinity for a target cell. Targeting moieties can include antibodies directed against a protein found on the surface of a target cell, or the ligand or a receptor-binding portion of a ligand for a molecule found on the surface of a target cell. Examples and a further description of targeting ligands can be found in WO2009/126933, which is hereby incorporated by reference.
The targeting ligands are selected from the group consisting of an antibody, a ligand-binding portion of a receptor, a ligand for a receptor, an aptamer, D-galactose, N-acetyl-D-galactose (GalNAc), multivalent N-acytyl-D-galactose, D-mannose, cholesterol, a fatty acid, a lipoprotein, folate, thyrotropin, melanotropin, surfactant protein A, mucin, carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-gal actosamine, N-acetyl-glucosamine, multivalent mannose, multivalent fructose, glycosylated polyaminoacids, transferin, bisphosphonate, polyglutamate, polyaspartate, a lipophilic moiety that enhances plasma protein binding, a steroid, bile acid, vitamin B12, biotin, an RGD peptide, an RGD peptide mimic, ibuprofen, naproxen, aspirin, folate, and analogs and derivatives thereof.
The preferred targeting ligands are selected from the group consisting of D-galactose, N-acetyl-D-galactose (GalNAc), GalNAc2, and GalNAc3, cholesterol, folate, and analogs and derivatives thereof.
Lipids
Lipophilic moieties, such as cholesterol or fatty acids, when attached to highly hydrophilic molecules such as nucleic acids can substantially enhance plasma protein binding and consequently circulation half life. In addition, lipophilic groups can increase cellular uptake. For example, lipids can bind to certain plasma proteins, such as lipoproteins, which have consequently been shown to increase uptake in specific tissues expressing the corresponding lipoprotein receptors (e.g., LDL-receptor or the scavenger receptor SR-B1). Lipophilic conjugates can also be considered as a targeted delivery approach and their intracellular trafficing could potentially be further improved by the combination with endosomolytic agents.
Exemplary lipophilic moieties that enhance plasma protein binding include, but are not limited to, sterols, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, phenoxazine, aspirin, naproxen, ibuprofen, vitamin E and biotin etc. Examples and a further description of lipids can be found in WO2009/126933, which is hereby incorporated by reference.
The preferred lipid is cholesterol.
Solubilizing Agents
The modular composition may comprise one or more other moieties/ligands that may enhance aqueous solubility, circulation half life and/or cellular uptake. These can include naturally occurring substances, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); or a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid). These moieties may also be a recombinant or synthetic molecule, such as a synthetic polymer or synthetic polyamino acids. Examples include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-0.5K, PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), methyl-PEG (mPEG), [mPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2 ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Examples and a further description of solubilizing agents can be found in WO2009/126933, which is hereby incorporated by reference.
The preferred solubilizing group is PEG 0.5K to 30K.
Method of Treatment
In one aspect, the invention features, a method of treating a subject at risk for or afflicted with a disease that may benefit from the administration of the modular composition of the invention. The method comprises administering the modular composition of the invention to a subject in need thereof, thereby treating the subject. The oligonucleotide that is administered will depend on the disease being treated. See WO2009/126933 for additional details regarding methods of treatments for specific indications.
Formulation
There are numerous methods for preparing conjugates of oligonucleotide compounds. The techniques should be familiar to those skilled in the art. A useful reference for such reactions is Bioconjugate Techniques, Hermanson, G. T., Academic Press, San Diego, Calif., 1996. Other references include WO2005/041859; WO2008/036825 and WO2009/126933.
EXAMPLES
The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. The siRNAs described herein were designed to target the ubiquitously expressed gene SSB (Sjogren syndrome antigen B; NM_009278.4).
Linker groups may be connected to the oligonucleotide or siRNA strand(s) at a linkage attachment point (LAP) and may include any carbon-containing moiety, in some embodiments having at least one oxygen atom, at least one phosphorous atom, and/or at least one nitrogen atom. In some embodiments, the phosphorous atom forms part of a terminal phosphate, or phosphorothioate, group on the linker group, which may serve as a connection point for the oligonucleotide strand. In certain embodiments, the nitrogen atom forms part of a terminal ether, ester, amino or amido (NHC(O)—) group on the linker group, which may serve as a connection point for the linkers of interest, endosomolytic unit, cell penetrating peptide, solubilizing group, lipid, targeting group, or additional linkers of interest. These terminal linker groups include, but are not limited to, a C6 hexyl, C5 secondary-hydroxy, C3 thiol or C6 thiol moiety. An example from the RNA sequences described below is C6 hexyl: [(CH2)6NH2].
The siRNA sequences described in the Examples herein are shown in Table 5.












TABLE 5






Sequence



SEQ ID


Entry
Code
Compound
strand
Sequence
NO:




















1
b
CTNNB1
passenger
[6amiL][iB][omeC][omeU][clickG][omeU][omeU][fluG][fluG]
1697






[fluA][omeU][omeU][fluG][fluA][omeU][omeU][omeC][fluG]







[clickA][fluA][fluA][omeUs][omeU][iB][C3SH]





CTNNB1
guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU][omeC]
1698






[fluA][omeA][fluU][omeC][fluC][omeA][fluA][omeC][fluA]







[omeG][omeUs][omeU]



2
c
ApoB
passenger
[C6SH][iB][omeC][omeU][omeU][omeU][fluA][fluA][omeC]
1699






[fluA][fluA][omeU][omeU][omeC][omeC][omeU][fluG][fluA]







[fluA][fluA][omeU][dTs]dT[iB][6amiL]





ApoB
guide
[rAs][rUs][rUs][omeU][omeC][fluA][fluG][fluG][fluA][fluA][omeU]
1700






[omeU][fluG][fluU][omeU][fluA][fluA][fluA][fluG][omeUs]







[omeU]



3
d
CTNNB1
passenger
[6amiL][iB][omeC][omeU][clickG][omeU][omeU][fluG][fluG]
1701






[fluA][omeU][omeU][fluG][fluA][omeU][omeU][omeC][fluG]







[clickA][fluA][fluA][omeUs][omeU][iB][C3SH]





CTNNB1
guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU]
1702






[omeC][fluA][omeA][fluU][omeC][fluC][omeA][fluA][omeC]







[fluA][omeG][omeUs][omeU]



4
e
CTNNB1
passenger
[6amiL][iB][omeC][omeU][fluG][omeU][omeU][fluG][fluG]
1703






[fluA][omeU][omeU][fluG][fluA][omeU][omeU][omeC][fluG]







[clickA][fluA][fluA][omeUs][omeU][iB][C3SH]





CTNNB1
guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU]
1704






[omeC][fluA][omeA][fluU][omeC][fluC][omeA][fluA][omeC]







[fluA][omeG][omeUs][omeU]



5
f
CTNNB1
passenger
[6amiL][iB][omeC][omeU][clickG][omeU][omeU][fluG][fluG]
1705






[fluA][omeU][omeU][fluG][fluA][clickU][omeU][omeC]







[fluG][fluA][clickA][fluA][omeUs][omeU][iB][C3SHSup]





CTNNB1
guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU]
1706






[omeC][fluA][omeA][fluU][omeC][fluC][omeA][fluA][omeC]







[fluA][omeG][omeUs][omeU]



6
g
CTNNB1
passenger
[6amiL][iB][omeC][omeU][clickG][omeU][omeU][fluG]
1707






[fluG][clickA][omeU][omeU][fluG][fluA][clickU][omeU][omeC]







[fluG][fluA][clickA][fluA][omeUs][omeU][iB][C3SHSup]





CTNNB1
guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU][omeC]
1708






[fluA][omeA][fluU][omeC][fluC][omeA][fluA][omeC][fluA]







[omeG][omeUs][omeU]



7
h
CTNNB1
passenger
[LiCholinker][iB][omeC][omeU][fluG][omeU][omeU][fluG]
1709






[fluG][fluA][omeU][omeU][fluG][fluA][omeU][omeU][omeC]







[fluG][fluA][fluA][fluA][omeUs][omeUl[iBl[6amiL]





CTNNB1
guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU]
1710






[omeC][fluA][omeA][fluU][omeC][fluC][omeA][fluA][omeC]







[fluA][omeG][omeUs][omeU]



8
i
CTNNB1
passenger
[amino modifier C2dT]
1711






[iB][omeC][omeU][clickG][omeU][omeU][fluG][fluG][fluA]







[omeU][omeU][fluG][fluA][omeU][omeU][omeC][fluG][clickA]







[fluA][fluA][omeUs][omeU][iB][C3SSC3OH]





CTNNB1
guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU][omeC]
1712






[fluA][omeA][fluU][omeC][fluC][omeA][fluA][omeC][fluA]







[omeG][omeUs][omeU]



9
j
CTNNB1
passenger
[6amiL][iB][omeC][omeU][clickG][omeU][omeU][fluG][fluG]
1713






[fluA][omeU][omeU][fluG][fluA][omeU][omeU][omeC][fluG]







[fluA][fluA][fluA][omeUs][omeU][iB][C3SH]





CTNNB1
guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU][omeC]
1714






[clickA][omeA][fluU][omeC][fluC][clickA][fluA][omeC]







[fluA][omeG][omeUs][omeUSup]



10
k
CTNNB1
passenger
[6amiL][iB][omeC][omeU][clickG][omeU][omeU][fluG][fluG]
1715






[fluA][omeU][omeU][fluG][fluA][omeU][omeU][omeC][fluG]







[fluA][fluA][fluA][omeUs][omeU][iB][C3SH]





CTNNB1
guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU][omeC]
1716






[fluA][omeA][fluU][omeC][fluC][clickA][fluA][omeC][fluA]







[omeG][omeUs][omeU]



11
l
CTNNB1
passenger
[6amiL][iB][omeC][omeU][fluG][omeU][omeU][fluG][fluG]
1717






[fluA][omeU][omeU][fluG][fluA][omeU][omeU][omeC][fluG]







[fluA][fluA][fluA][omeUs][omeU][iB][6amiL]





CTNNB1
guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU]
1718






[omeC][fluA][omeA][fluU][omeC][fluC][clickA][fluA][omeC]







[fluA][omeG][omeUs][omeU]



12
m
CTNNB1
passenger
[6amiL][iB][omeC][omeU][clickG][omeU][omeU][fluG][fluG]
1719






[fluA][omeU][omeU][fluG][fluA][clickU][omeU][omeC][fluG]







[fluA][clickA][fluA][omeUs][omeU][iB][C3SHSup]





CTNNB1
guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU]
1720






[omeC][fluA][omeA][fluU][omeC][fluC][clickA][fluA][omeC][fluA]







[omeG][omeUs][omeU]





As used herein, ome = 2′ methoxy; flu = 2′ fluoro; click = 2′ propagyl; iB = inverted abasic; “s” subscript = phosphorothioate; and r = 2′ ribo; 6amil = n-hexylamino; C3SH = n-propylthiol; and C6SH = n-hexylthiol.






Preparations of tetraGalNAc ligands and tetraGalNAc-siRNA conjugates are described below in the examples and synthetic schemes. Note that the siRNA depictions below are for illustrative purposes. Specific sequence information can be found in Table 5.
Section A
Examples 1-2
Synthesis of TetraGalNAc Ligand Compounds A9 and A10
The following Scheme 1 was used to prepare TetraGalNAc Compounds 9 and 10.







Synthesis of (2S)-2, 6-bis[bis (prop-2-yn-1-yl)amino]hexanoic acid (Compound A1)
Into a 2000-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed a solution of (2S)-2,6-diaminohexanoic acid (50 g, 342.03 mmol, 1.00 equiv) in acetonitrile (1000 mL) and heated to 50° C. To this was added potassium hydroxide (22.6 g, 0.4025 mol, 1.00 equiv, 85%). The resulting solution was stirred for 30 min. Then 3-bromoprop-1-yne (29.5 mL, 1.00 equiv) was added. The resulting solution was stirred for 1 hour at 50° C. additional potassium hydroxide (22.6 g, 0.4025 mol, 1.00 equiv) was added to the solution and stirred for 30 min at 50° C. To this was added 3-bromoprop-1-yne (29.5 mL, 1.00 equiv). The resulting solution was stirred for 1 hour. To this was added potassium hydroxide (22.6 g, 0.4025 mol, 1.00 equiv) again. The resulting solution was stirred for 30 min at 50° C., followed by addition of more 3-bromoprop-1-yne (29.5 mL, 1.00 equiv). The resulting solution was stirred for 1 hour. To this was added potassium hydroxide (22.6 g, 0.4025 mol, 1.00 equiv). The resulting solution was stirred for 30 min. To this was added 3-bromoprop-1-yne (29.5 mL, 1.00 equiv). The resulting solution was stirred for 3 hours. The reaction mixture was cooled to 25° C. with a water/ice bath. The solid was filtered out. The filtrate was adjusted to pH 4 with HCl (6M). The solid was filtered out. The filtrate was concentrated under vacuum. The residue was applied onto a silica gel column and eluted with dichloromethane/methanol (100:1-25:1). This resulted in (2S)-2, 6-bis[bis (prop-2-yn-1-yl)amino]hexanoic acid (Compound A1) as a light yellow oil.
MS (ES, m/z): 297.2, [M−H]−1HNMR(CDCl3, 500 MHz, ppm): 3.62 (d, J=2.0 Hz, 4H), 3.52-3.49 (m, 1H), 3.50 (d, J=2.4 Hz, 4H), 2.62 (t, J=7.1 Hz, 2H), 2.30 (t, J=2.4 Hz, 2H), 2.27 (t, J=2.4 Hz, 2H), 1.88-1.79 (m, 2H), 1.60-1.53 (m, 2H), 1.52-1.43 (m, 2H).
Synthesis of 2-(2-hydroxyethoxy)ethyl 4-methylbenzenesulfonate (Compound A3)
Into a 2000-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed a solution of 2-(2-hydroxyethoxy)ethan-1-ol (A2, 42.4 g, 399.55 mmol, 1.00 equiv) in dichloromethane (1000 mL) and triethylamine (27.9 g, 275.72 mmol, 0.25 equiv). To the above was added p-toluenesulfonyl chloride (19.1 g, 100.18 mmol, 0.50 equiv). After stirred for 1 h at 25° C., the resulting mixture was washed with 1×500 mL of aq. potassium hydrosulfate (1M) and 1×500 mL of aq. sodium bicarbonate (5%) respectively. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column and eluted with dichloromethane/methanol (100:1). This resulted in 2-(2-hydroxyethoxy)ethyl 4-methylbenzenesulfonate (Compound A3) as a colorless oil.
Synthesis of 2-(2-azidoethoxy)ethan-1-ol (Compound A4)
Into a 500-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed a solution of 2-(2-[[(4-2-(2-hydroxyethoxy)ethyl 4-methylbenzenesulfonate (A3, 50 g, 192.08 mmol, 1.00 equiv) in N,N-dimethylformamide (250 mL). This was followed by the addition of sodium azide (18.79 g, 289.03 mmol, 1.50 equiv) at 25° C. The resulting solution was stirred for 5 h at 100° C. in an oil bath. The reaction mixture was cooled and filtered. The filtrate was concentrated under vacuum. The residual solution was diluted with 1000 mL of dichloromethane and washed with 1×500 mL of water. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column and eluted with dichloromethane/methanol (80:1). This resulted in 2-(2-azidoethoxy)ethan-1-ol (Compound A4) as a colorless oil.
1HNMR (CDCl3, 400 MHz, ppm): 3.42-3.45 (t, J=4.8 Hz, 2H), 3.63-3.65 (t, J=4.8 Hz, 2H), 3.71-3.74 (t, J=4.8 Hz, 2H), 3.71-3.79 (m, 2H).
Synthesis of (3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (Compound A6)
Into a 2000-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed a solution of (3R,4R,5R,6R)-3-amino-6-(hydroxymethyl)tetrahydro-2H-pyran-2,4,5-triol hydrochloride (A5, 120 g, 556.50 mmol, 1.00 equiv) in pyridine (1200 mL). This was followed by the addition of acetic anhydride (341.6 g, 3.35 mol, 6.00 equiv) dropwise with stirring at 0° C. The resulting solution was stirred overnight at 25° C. The reaction was then quenched by the addition of 8000 mL of water/ice. The solid was collected by filtration. This resulted in (3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (Compound A6) as a white solid.
Synthesis of (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazole-6,7-diyl diacetate (Compound A7)
Into a 2000-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed a solution of (3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (A6, 30 g, 77.05 mmol, 1.00 equiv) in dichloromethane (1500 mL), then added iron (III) chloride (30 g, 184.95 mmol, 2.40 equiv). The resulting mixture was stirred for 2 h at 25° C. The reaction was then quenched by the addition of 1000 mL of water/ice. The organic layer was washed with 1×1000 mL of sodium aq. bicarbonate and 1×1000 mL of water, dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazole-6,7-diyl diacetate (Compound A7) as yellow oil. 1HNMR(CDCl3, 300 MHz, ppm): 2.03 (s, 9H), 2.12 (s, 3H), 3.97-4.27 (m, 4H), 4.90-4.93 (m, J=3.3 Hz, 1H), 5.45-5.47 (t, J=3.0 Hz, 1H), 5.98-6.00 (d, J=6.6 Hz, 1H).
Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-[2-(2-azidoethoxy)ethoxy]tetrahydro-2H-pyran-3,4-diyl diacetate (Compound A8)
Into a 500-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed a solution of (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazole-6,7-diyl diacetate (A7, 40 g, 121.47 mmol, 1.00 equiv) in 1,2-dichloroethane (200 mL), 2-(2-azidoethoxy)ethan-1-ol (A4, 23.89 g, 182.18 mmol, 1.50 equiv). To the above several 4A zeolite was added. The resulting mixture was stirred for 1 h at 25° C. Then trimethylsilyl trifluoromethanesulfonate (10.8 mL, 0.50 equiv) was added. After stirred overnight at 25° C., the reaction mixture was diluted with 500 mL of dichloromethane and washed with 1×500 mL of water, 1×500 mL of aq. sodium bicarbonate and 1×500 mL of water. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column and eluted with dichloromethane/methanol (100:1). This resulted in (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-[2-(2-azidoethoxy)ethoxy]tetrahydro-2H-pyran-3,4-diyl diacetate (A8) as a colorless oil.
MS (m/z): 461.1, [M+H]+
1HNMR(CDCl3, 500 MHz, ppm) 5.78 (d, J=8.90 Hz, 1H), 5.36 (d, J=2.9 Hz, 1H), 5.22 (dd, J=11.2, 3.6 Hz, 1H), 4.77 (d, J=8.3 Hz, 1H), 4.19-4.12 (m, 2H), 4.11-4.05 (m, 1H), 3.98-3.92 (m, 2H), 3.82-3.78 (m, 1H), 3.71-3.63 (m, 4H), 3.49-3.38 (m, 2H), 2.16 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H), 1.97 (s, 3H).
Synthesis of (S)-2,6-bis(bis((1-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)hexanoic acid (Compound A9, tetraGalNAc Acetate) (A9) (Ex. 1)
Into a 250-mL round bottom flask purged and maintained with an inert atmosphere of nitrogen was charged (2S)-2, 6-bis [bis (prop-2-yn-1-yl) amino]hexanoic acid (A1, 1.0 g, 1.0 equiv), (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-[2-(2-azidoethoxy)ethoxy]tetrahydro-2H-pyran-3,4-diyl diacetate (A8, 9.26 g, 6.0 equiv), anhydrous THF 50 mL, CuBrSMe2 (0.138 g, 0.20 equiv), and anhydrous DBU (1.5 ml, 3.0 equiv) in respective order. The resulting solution was stirred for 16 h at room temperature, quenched with acetic acid (0.75 mL, 4.0 equiv), treated with MP-TMT resin (Part No: 801472, from Biotage) (9 g), aged at room temperature for 16 h, filtered, and concentrated the filtrate to a foam solid. The solid was then dissolved in CH2Cl2 (140 mL), and washed with AcOH/NaCl solution (140 mL). The AcOH/NaCl solution was prepared with 1 mL AcOH and 100 mL 20% NaCl solution. The bottom organic layer was concentrated, and purified on a SiO2 column (220 g), eluting with CH2Cl2/MeOH. This resulted in (S)-2,6-bis(bis((1-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)hexanoic acid (Compound A9) as a white solid.
MS (m/z): 2139.5, [M+H]+
Synthesis of (S)-2,6-bis(bis((1-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)hexanoic acid (Compound A10, TetraGalNAc) (A10) (Ex. 2)
Into a 250-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen was charged (S)-2,6-bis(bis((1-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)hexanoic acid (A9, 6.9 g, 1.0 equiv), Na2CO3 (6.83 g, 20 eq), water (56 mL), and MeOH (32 mL) in respective order. The reaction was aged at room temperature for 16 h, concentrated to residue, redissolved in water (50 mL), and purified on Combiflash C18 gold reverse column (415 g), eluting with water/MeCN. After concentration under vacuum, the product was dissolved in minimum amount of water, and lyophilized to obtain (S)-2,6-bis(bis((1-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4, 5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)hexanoic acid (Compound A10) as a white solid.
MS (m/z): 1657 [M+Na]+
1HNMR (D2O, 500 MHz, ppm): 8.05 (s, 2H), 7.91 (s, 2H), 4.62 (t, J=5.0 Hz, 4H), 4.57 (t, J=5.0 Hz, 4H), 4.45-4.41 (d, J=8.6 Hz, 4H), 3.99-3.82 (m, 28H), 3.80-3.61 (m, 28H), 3.14 (t, J=7.1 Hz, 1H), 2.52 (broad s, 2H), 1.99 (s, 6H), 1.98 (s, 6H), 1.73 (m, 2H), 1.60 (m, 2H), 1.29 (m, 2H).
Section B
Preparation of B2 to B5
Examples 3-6
Scheme 2 as shown in FIG. 5A-1 to FIG. 5D, was used to prepare B Conjugates (Ex. 3-6).
Synthesis of B2 (Ex. 3)
A10 (86 mg, 0.053 mmol) and DIEA (57.6 μL, 0.330 mmol) were dissolved in DMSO (500 μL), then added to a solution of HATU (301 μL, 0.079 mmol) and stirred for 15 min. Starting material passenger strand B1 (101 mg, 0.013 mmol) was dissolved in water (168 μL) and DMSO (1.5 mL). The HATU solution was added to the RNA solution and aged for 15 min. The reaction mixture was diluted with water (50 mL) and centrifugal dialyzed three times against water over a 3 k membrane. The concentrate was loaded onto an HPLC fitted with a Dionix ProPac SAX 22×250 mm column. The product was gradient eluted starting at 95% A (2:3 H2O:2,2,2-trifluoroethanol, 20 mM TEA) up to 40% solvent B (2:3 H2O:2,2,2-trifluoroethanol, 20 mM TEA, 1M CsCl). The fractions were diluted with water to reduce the 2,2,2-trifluoroethanol content to 25% and centrifugal dialyzed three times against water over a 3 k membrane. The concentrate was freeze dried to afford the product as a white amorphous solid. Expected mass: 9267.5, found mass: 9267.0
Synthesis of B3 (Ex. 4)
To a solution of B2 (606 mg, 0.065 mmol) in water (32 mL) was added TEAA (1.64 mL, 2M), aqueous DTT (0.65 mL, 1M), and TEA (0.65 mL, 4.69 mmol). The reaction mixture was aged for 10 min. The reaction mixture was then diluted with water and centrifugal dialyzed three times against water over a 3 k membrane. The concentrate was taken forward without further isolation. Expected mass: 9177.4, found mass: 9179.0
Synthesis of B4 (Ex. 5)
To a solution of B3 (350 mg, 0.038 mmol) in water (3 mL) was added N-(2-aminoethyl)-maleimide trifluoroacetate salt (194 mg, 0.763 mmol). The reaction mixture was aged for 30 min, after which it was purified by RP-HPLC (95:5-5:95% A:B linear gradient (A=100 mM aqueous TEAA; B=100 mM TEAA in acetonitrile) Waters Phenyl xbridge Column. Fractions containing B4 were centrifugal dialyzed three times against water over a 3 k membrane and the concentrate was lyophilized to give product as a white amorphous solid.
Synthesis of B5 (Ex. 6)
To a solution of B4 (286 mg, 0.031 mmol) in aqueous sodium bicarbonate (3.0 mL, 200 mM) was added a solution of NHS-dPEG12-SPDP (280 mg, 0.307 mmol) in acetonitrile (0.5 mL). The reaction mixture was aged for 30 min, after which it was treated with aqueous TEAA (1.0 mL, 2M) and purified by RP-HPLC (95:5-5:95% A:B linear gradient (A=100 mM aqueous TEAA; B=100 mM TEAA in acetonitrile) Waters Phenyl xbridge Column. Fractions containing B5 were centrifugal dialyzed three times against water over a 3K membrane and the concentrate was lyophilized to give product as a white amorphous solid. Measured mass=10117
Examples 7-8
Preparation of B6-Seq32
Scheme 3 as shown in FIG. 6A to FIG. 6B was used to prepare Conjugates B6-P32 and B8-seq32 (Ex. 7-8).
Synthesis of Conjugate B6-Seq32 (Ex. 7)
B5 (50 mg, 5 umol, 1 eq.) was dissolved in 50 mM AcOH in 2,2,2-trifluoroethanol (5 mL). Peptide Seq32 (51 mg, 13 umol, 2.5 eq.) was dissolved in guanidine-HCl (8M, 500 uL), diluted with 50 mM AcOH in 2,2,2-trifluoroethanol (5 mL). The peptide solution was added dropwise to the stirring RNA solution over 5 min, and the reaction was left at room temperature for 1 hour. The reaction was diluted with formamide (10 mL), and 1.5 mL aliquots of the reaction mixture were loaded onto an HPLC fitted with a Dionex ProPac SAX-10 22×250 mm column. The product was gradient eluted starting at 98% solvent A (2:3 H2O:2,2,2-trifluoroethanol, 40 mM TEA) up to 35% solvent B (2:3 H2O:2,2,2-trifluoroethanol, 40 mM TEA, 1M guanidine-HCl) over 10 min at 20 mL/min. The fractions were diluted with water to reduce the 2,2,2-trifluoroethanol content to 25% and centrifugal dialyzed three times against water over a 10 k membrane. The concentrate was freeze dried to afford the product as a white amorphous solid. Expected mass: 13961.9, found mass: 13962.0
Synthesis of Conjugate B8-Seq32-b (Ex. 8)
Guide strand (B7, 17.7 mg) was dissolved in water (5 mL) and added to a vial containing B6-seq 42 (36.2 mg). The solution was thoroughly mixed and left at room temperature for 2 hours. The solution was freeze dried to afford the duplex as a white amorphous solid.
Synthesis of Additional B8-Peptide Conjugates
Additional conjugates of B8 and Peptide Sequence and duplexes were prepared in a manner analogous to that used for B8-seq32-b.
Examples 9-11
Preparation of B9 and B10-Seq32 and 11-Seq32
Scheme 4 as shown in FIG. 7A, FIG. 7B and FIG. 7C was used to prepare B9, B10-seq32 and B11-seq32.
Synthesis of B9 (Ex. 9)
Compound B3 (120 mg, 0.0132 mmol) in water (5 mL) was added dropwise to a stirring solution of 2,2′-dipyridyldisulfide (29 mg, 0.132 mmol, 10 eq.) dissolved in methanol (5 mL). The solution was diluted with water to bring the methanol content to 20% and centrifugal dialyzed three times against water over a 3K membrane. The concentrate was freeze dried to afford the product as an amorphous white solid. Expected mass: 9166.5, found mass: 9165.5
Synthesis of B10-Seq32 (Ex. 10)
B9 (15 mg, 1.615 umol) was dissolved in water (150 uL) and was diluted with 50 mM AcOH in TFE (1.5 mL). In a separate vial, P32 (8.79 mg, 2.155 umol) was dissolved in 8 M guanidine HCl (60 uL) and diluted with 50 mM AcOH in TFE (1.5 mL), then added to the RNA solution. The reaction mixture was aged for 15 min, then was diluted with formamide and purified by AEX (95:5-55:45 A:B linear gradient (A=20 mM TEA in 60% aqueous TFE; B=1M CsCl and 20 mM TEA in 60% aqueous TFE), Dionix Propac column. Fractions containing B10-Seq 32 were centrifugal dialyzed three times against water over a 10K membrane and the concentrate was lyophilized to give product as a white amorphous solid.
Synthesis of B11-Seq32-b (Ex. 11)
B10-seq 32 (9.68 mg, 0.730 umol) was treated with a solution of B7 (5.00 mg, 0.730 umol) dissolved in PBS (500 uL) and aged for 30 min. Excess guide strand was removed by AEX purification (95:5-55:45 A:B linear gradient (A=20 mM TEA in 60% aqueous TFE; B=1M CsCl and 20 mM TEA in 60% aqueous TFE), Dionix Propac column. Fractions containing B11-seq 32 were centrifugal dialyzed three times against water over a 10K membrane and the concentrate was lyophilized to give product as a white amorphous solid.
Examples 12-14
Additional Synthesis of B1-Peptide Conjugates
Additional conjugates of B11 and peptide sequences and corresponding duplexes were prepared in a manner analogous to that used for B11-seq32-b.
Scheme 5 is shown in FIG. 7D, FIG. 7E and FIG. 7F.
Synthesis of B12 (Ex. 12)
B3 (50 mg, 5.4 μmol) was dissolved in water (3 mL,  ̃17 mg/mL) and Compound 1, 1,1′-(ethane-1,2-diyl)bis(1H-pyrrole-2,5-dione), (16 mg, 0.073 mmol) was dissolved in DMF (1.2 mL) in separate vials. The B3 solution was added to Compound 1 solution and stirred for 10 min. The reaction was diluted with water to 15 mL and then dialyzed 4 times on 3 K MWCO membrane against water. The reaction was then filtered (0.22 μm syringe filter) and lyophilized to afford a white solid, B12. Expected mass: 9397.535. Observed mass: 9400.0.
Synthesis of B12-Seq13 (Ex. 13)
See Synthesis of B10-seq32 for reaction procedure. B12-seq13. Expected mass: 13518.215
Synthesis of B13-Seq 13-b (Ex. 14)
See Synthesis of B11-seq32 for reaction procedure. B13-seq13-b. Expected mass: 20370.215
Additional Synthesis of B13-Peptide Conjugates
Additional conjugates of B13 and peptide sequences were prepared in a manner analogous to that used for B13-seq13.
Examples 15-16
Preparation of B15-Seq32 and B16-Seq32-b
Scheme 6 as shown in FIG. 7G-1 to FIG. 7G-2 was used to prepare B16-seq32 and B17-seq32-b.
Synthesis of B14
B3 (100 mg, 10.9 μmol) was dissolved in water (10 mL) and dioxane (20 mL) was treated with bis maleimide dissolved in dioxane (3.8 mL) to give a cloudy mixture. The reaction was stirred for 1.5 hours, after which it was quenched with N-methylmaleimide (36.3 mg, 0.327 mmol). The reaction mixture was diluted with water and centrifugal dialyzed once against water over a 3 k membrane. The concentrate was filtered and purified by RP-HPLC (95:5-5:95% A:B linear gradient (A=100 mM aqueous TEAA; B=100 mM TEAA in acetonitrile) Waters Phenyl xbridge Column). Fractions containing product were dialyzed and lyophilized to give B14 as an amorphous white powder. Measured mass=9531
Synthesis of B15-Seq 32 (Ex. 15)
B14 (5 mg, 0.524 μmol) was dissolved in formamide solution (2M thiourea, 50 mM MES buffer at pH 6.5, 500 μL). In a separate vial, peptide sequence 32 (4.28 mg, 1.048 μmol) was dissolved in formamide solution (2M thiourea, 50 mM MES buffer at pH 6.5, 500 μL), then was added to the RNA solution. After aging one hour at room temperature, the reaction mixture was loaded onto an HPLC fitted with a Dionex ProPac SAX-10 22×250 mm column. The product was gradient eluted starting at 98% solvent A (2:3 H2O:2,2,2-trifluoroethanol, 40 mM TEA) up to 35% solvent B (2:3 H2O:2,2,2-trifluoroethanol, 40 mM TEA, 1M guanidine-HCl) over 10 min at 20 mL/min. The fractions were diluted with water to reduce the 2,2,2-trifluoroethanol content to 25% and centrifugal dialyzed three times against water over a 10 k membrane. The concentrate was freeze dried to afford the product as a white amorphous solid.
Synthesis of B16-Seq32-b (Ex. 16)
B15-seq 32 (2.11 mg, 0.155 μmol) was treated with a solution of B7 (1.062 mg, 0.155 μmol) in water (212 μL) and aged at room temperature for 2 hours. The solution was lyophilized to give the product as a white amorphous solid.
Section C
Examples 17-21
Preparation of C1 to C3, C4-seq32 and C6-seq32
Scheme 7 as shown in FIG. 8A to FIG. 8D was used to prepare C1 to C3, C4-seq32 and C6-seq32.
Synthesis of C1 (Ex. 17)
1,2-Diaminododecane (100 mg, 0.499 mmol) was dissolved in chloroform (3.3 mL) and cooled to 0° C., then treated with N-methoxycarbonyl-maleimide (234 mg, 1.50 mmol) and tetrabutylammonium hydrogen sulfate (170 mg, 0.499 mmol). DIPEA (209 uL, 1.20 mmol) was slowly added and the reaction aged for 10 minutes at 0° C. The ice bath was removed and the reaction was treated with aqueous saturated sodium bicarbonate solution (6.6 mL). After aging 3.5 hours at room temperature, the reaction mixture was extracted with ethyl acetate (3×15 mL). The combined organic layers were dried with sodium sulfate and then solvent removed in vacuo. The crude product was purified by flash chromatography with a 100:0-0:100% A:B linear gradient (A=hexanes; B=ethyl acetate). Fractions containing product were pooled and concentrated to give C1 as a fine white powder. 1H NMR (CDCl3): 1.24-1.28 (m, 12H), 1.55-1.61 (m, 4H), 3.50 (t, 4H J=7.4 Hz), 6.68 (s, 4H). Measured mass=361.
Synthesis of C2 (Ex. 18)
Step 1.
3′ Hamino 5′ C6 disulfide siRNA (46.9 mg, 6.16 μmol) was dissolved in 9:1 DMSO/water (782 μl). TetraGalNAc (40.0 mg, 0.025 mmol) and DIEA (26.9 μl, 0.154 mmol) were dissolved in DMSO (200 μl), then added solution of HATU (14.0 mg, 0.037 mmol) in DMSO (141 μL) and stirred at RT for 15 minutes. This solution was added to the RNA solution and aged for 30 minutes. The reaction was diluted with DI water and dialyzed once to remove DMSO and purified by AEX (95:5-65:35 A:B linear gradient (A=20 mM TEA in 60% aqueous TFE; B=1M CsCl and 20 mM TEA in 60% aqueous TFE), Dionix Propac column). Fractions containing product were pooled, dialyzed, and lyophilized. Measured mass=9233.
Step 2.
To this solid (30.8 mg, 3.34 μmol) was added TCEP (19.13 mg, 0.067 mmol) and DI water (2 mL). The reaction was stirred at RT for 1 hour, then aged overnight at 5° C. The reaction was diluted with DI water and dialyzed twice against DI water to give a solution of C2 that was used in further reactions without isolation.
Synthesis of C3 (Ex. 19)
C2 (60.1 mg, 6.60 umol, prepared in a manner analogous to B3) dissolved in DI water (37 mL) was treated with C1 (23.8 mg, 66.0 umol) dissolved in DMF (7 mL) to give a cloudy solution. The reaction was aged overnight, at which point dioxane (18 mL) was added to solubilize the reaction mixture. After aging for 30 additional minutes, the reaction was diluted with DI water. It was then dialyzed once against DI water, filtered, and purified by RP-HPLC (95:5-5:95% A:B linear gradient (A=100 mM aqueous TEAA; B=100 mM TEAA in acetonitrile) Waters Phenyl xbridge Column). Fractions containing product were dialyzed and lyophilized to give C3 as an amorphous white powder. Measured mass=9458.
Synthesis of C4-seq32 (Ex. 20)
C3 (10 mg, 1.057 umol) was dissolved in formamide modified with 20 mM MES buffer and 2 M thiourea (1 mL) and was added to P32 (8.62 mg, 2.11 umol). After 20 mins, LC-MS indicated good conversion to desired product. Reaction was purified by AEX (95:5-55:45 A:B linear gradient (A=20 mM TEA in 60% aqueous TFE; B=1M CsCl and 20 mM TEA in 60% aqueous TFE), Dionix Propac column). Fractions containing product were dialyzed to give C4-P32.
Synthesis of C6-seq32-(Ex. 21)
C4 (6.78 mg, 0.501 μmol) dissolved in DI water (3.40 mL) was treated with guide strand C5 (3.44 mg, 0.501 μmol) dissolved in DI water (530 μL). Analytical SAX indicated good duplex purity with some excess guide strand observed. Solution was lyophilized to give C6 as an amorphous white powder. Measured mass=passenger strand: 13539, guide strand: 6869.
Additional Synthesis of C6-peptide Conjugates
Additional conjugates of C6 and Peptide Sequence were prepared in a manner analogous to that used for C6-seq32-c.
Examples 22-27
Preparation of C7 to C10, C11-P32 and C12-seq32-a
Scheme 8 as shown in FIG. 9A to FIG. 9E was used to prepare C7 to C10, C11-seq32 and C12-seq32.
Synthesis of C7 (Ex. 22)
Icosanedioic acid (600 mg, 1.752 mmol) was suspended in toluene (11 mL) and treated with DIEA (673 μL, 3.85 mmol) and DPPA (793 uL, 3.68 mmol). After stirring at room temp for 30 minutes, the reaction was slowly heated to 80° C., then to gentle reflux for two hours. Reaction was cooled and treated with tBuOH (1.675 mL, 17.52 mmol) and copper iodide (200 mg, 1.051 mmol) and heated back to reflux for 2 additional hours. Reaction was cooled (precipitation observed), diluted with DCM, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography with a 100:0-0:50% A:B linear gradient (A=hexanes; B=ethyl acetate). Fractions containing product were pooled and concentrated to give C7. Measured mass=486.
Synthesis of C8 (Ex. 23)
C7 (101 mg, 0.208 mmol) was dissolved in DCM (20 mL) and treated with TFA (20 mL). The reaction was aged for five minutes, after which solvent and TFA were removed in vacuo to give C8 as a colorless oily solid that was used without further purification. Measured mass=286.
Synthesis of C9 (Ex. 24)
C8 (100.0 mg, 0.209 mmol) was suspended in chloroform (28 mL) and treated with tetrabutylammonium hydrogen sulfate (70.9 mg, 0.209 mmol), N-methoxy carbonyl maleimide (98.0 mg, 0.631 mmol), and DIEA (88.0 μL, 0.502 mmol). Saturated sodium bicarbonate (28 mL) was added. The reaction was stirred vigorously for 25 hours, after which it was extracted 3×50 mL DCM. The combined organic layers were dried with sodium sulfate, then evaporated to dryness. The crude product was purified by flash chromatography with a 100:0-0:50% A:B linear gradient (A=hexanes; B=ethyl acetate). Fractions containing the desired product were combined and evaporated to give C9. 1H NMR (CDCl3): 1.24-1.26 (m, 28H), 1.55-1.59 (m, 4H), 3.50 (t, 4H J=7.4 Hz), 6.68 (s, 4H). Measured mass=445.
Synthesis of C10 (Ex. 25)
C2 (12.0 mg, 1.31 μmol) was dissolved in 1:3 water:dioxane (14.4 mL) and was treated with C9 (5.8 mg, 13.1 μmol) dissolved in 1.4 mL dioxane. After aging overnight, the reaction was quenched with N-methyl maleimide (4.38 mg, 39.4 μmol) and was diluted with DI Water. The crude reaction was dialyzed once against DI water, filtered, and purified by RP-HPLC (95:5-5:95% A:B linear gradient (A=100 mM aqueous TEAA; B=100 mM TEAA in acetonitrile) Waters Phenyl xbridge Column). Fractions containing product were dialyzed against DI water and lyophilized to give C10. Measured mass: 9546.
Synthesis of C11-Seq32 and C12-Seq32-c (Ex. 26 and Ex. 27)
Conjugates C11-seq32 and C12-seq32-c were prepared in a manner analogous to that used for C4-seq32 and C6-seq32.
Additional Synthesis of C12-Peptide Conjugates
Additional conjugates of C12 and peptide sequence were prepared in a manner analogous to that used for C12-seq32.
Section D
Examples 28-30
Preparation of C13, C14-Seq32 and C15-Seq32
Scheme 9 shown in FIG. 10 A to FIG. 10D was used to prepare C13, C14-seq32 and C15-seq32-a.
Synthesis of C13 (Ex. 28)
C2 (11 mg, 1.22 μmol) dissolved in DI water (3.5 mL) was treated with C2 bismaleimide (2.69 mg, 12.20 umol) dissolved in DMF (270 μL). After one hour, LC-MS indicated good conversion to desired product. Reaction was dialyzed 3 times against DI water and lyophilized to give C13. Measured mass: 9317.
Synthesis of C14-Seq32 (Ex. 29)
C13 (10.53 mg, 1.13 μmol) was dissolved in DI water (50 μL) and diluted with TFE modified with 50 mM AcOH (2.0 mL), then was added to seq32 (9.22 mg, 2.26 μmol) dissolved in 8M guanidine hydrochloride (60 μL). The reaction was aged for 10 minutes. Reaction was purified by AEX (95:5-55:45 A:B linear gradient (A=20 mM TEA in 60% aqueous TFE; B=1M CsCl and 20 mM TEA in 60% aqueous TFE), Dionix Propac column). Fractions containing product were dialyzed to give C14-seq32.
Synthesis of C15-Seq32-c (Ex. 30)
C14-seq32 (9.81 mg, 0.738 μmol) dissolved in DI water (2.6 mL) was treated with guide strand C5 (7.76 mg, 0.738 μmol) dissolved in DI water (751 μL). Solution was lyophilized to give the desired product C15-seq32-c. Measured mass=passenger strand: 13396, guide strand: 6868
Additional Synthesis of C15-Peptide Conjugates
Additional conjugates of C15 and peptide sequence were prepared in a manner analogous to that used for C15-seq32.
Examples 31-33
Preparation of D1, D3 and D4
Scheme 10 as shown in FIG. 11A to FIG. 11 D was used to prepare D1, D3 and D4.
Synthesis of D1 (Ex. 31)
To a solution of NHS ester (100.0 mg, 0.320 mmol) in 0.5 mL anhydrous DCE were added azido amine (253.0 mg, 0.480 mmol) in 0.5 mL anhydrous DCE and 1.5 eq. triethylamine. The resulting solution was stirred for 1 h at room temperature, and the reaction mixture was loaded on a silica column, eluding with MeOH/DCM=0/100 to 10/90 over 25 min. The collected fraction was subject to LC-MS analysis and the result indicated >95% purity.
Synthesis of D3 (Ex. 32)
Oligonucleotide D2 (10 mg, 1.3 μmol) and azide linker D1 (5.6 mg, 7.8 μmol) were dissolved in degassed 3:1 DMA/water (1000 μL) in an Eppendorf tube, then a solution of copper(I) bromide-dimethyl sulfide (0.05 mg, 0.26 μmol) in degassed MeCN (100 μL) was added to the reaction mixture. After 60 min at 40° C., D2 was completely consumed monitored by LC-MS. The reaction mixture was diluted with 0.4 M EDTA (5 mL) and stirred for additional 15 min, then dialyzed against water using a Millipore 3K membrane and purified by RP HPLC (5%-60% A in B, A: 100 mM TEAA in MeCN, B: 100 mM TEAA in water). The product fractions were dialyzed against water and lyophilized to afford D3 as a white powder.
Synthesis of D4 (Ex. 33)
TetraGalNAc A10 (5.7 mg, 3.5 μmol), HATU (2.0 mg, 5.2 μmol), N,N-diisopropylethylamine (1.8 mg, 14 μmol) were dissolved in DMSO (100 μL). After 10 min, the activated ester was added to oligonucleotide D3 (6.4 mg, 0.70 μmol) in DMF (350 μL) and water (50 μL). The resulting reaction mixture was stirred for 15 min and quenched by addition of water, then purified by RP HPLC (5%-60% A in B, A: 100 mM TEAA in MeCN, B: 100 mM TEAA in water). The product fractions were dialyzed against water and lyophilized to afford R3 as a whiter powder.
Examples 34-35
Preparation of D5-Seq32 and D7-Seq32
Scheme 11 as shown in FIG. 12A-1 to FIG. 12B-2 was used to prepare D5-seq32 and D7-seq32.
Synthesis of D5-Seq32 (Ex. 34)
Oligonucleotide D4 (6.5 mg, 0.60 μmol) in 200 μL formamide/pH=6.8 Tris buffer=3/1 was treated with peptide seq32 (9.8 mg, 2.4 μmol) in 200 μL of the same buffer and the resulting reaction mixture was stirred for 1 h. The reaction was diluted by addition of formamide 2.5 mL and purified by strong anion exchange chromatography on a Sepax Proteomix SAX NP10, 21.2×50 mm column (2%-30% B in A over 8 min, A: 60:40 trifluoroethanol:water, 40 mM triethylamine, B: 60:40 trifluoroethanol:water, 40 mM triethylamine, 1 M guanidine-HCl, 20 mL/min) to afford D5-seq32 as a white powder.
Synthesis of D7-Seq32 (Ex. 35)
Oligonucleotide D5-seq32 (5.7 mg, 0.304 μmol) and the corresponding antisense strand D6 (2.0 mg, 0.29 μmol) were mixed in RNase free water for 1 h. The reaction mixture was lyophilized and the product D7-seq32-d was submitted for in vivo evaluation.
Synthesis of Additional D7-Peptide Conjugates
Additional conjugates of D7 and peptide sequence were prepared in a manner analogous to that used for D7-seq32.
Section E. Synthesis of Hybrid of Lipid and Peptide Conjugates
Examples 36-42
Scheme 12 is shown in FIG. 13A to FIG. 13H-2.
Synthesis of E2 (Ex. 36)
Oligonucleotide E1 (300 mg, 39 μmol) and the PEG9 azide linker (58.5 mg, 78 mol) were dissolved in degassed 3:1 DMA/water (10 mL) in a glass vial, then a solution of copper(I) bromide-dimethyl sulfide (20.06 mg, 98 μmol) in degassed DMSO (699 μL) was added to the reaction mixture. After 40 min at 45° C., E1 was completely consumed monitored by LC-MS. The reaction mixture was diluted with 0.4 M EDTA (20 mL) and stirred for additional 15 min, then dialyzed against water using a Millipore 3K membrane and lyophilized to afford E2 as a white powder.
Synthesis of E3 (Ex. 37)
TetraGalNAc A10 (237 mg, 145 μmol), HATU (55.2 mg, 145 μmol), N,N-diisopropylethylamine (94 mg, 726 μmol) were dissolved in DMSO (700 μL). After 10 min, the activated ester was added to oligonucleotide E2 (306 mg, 36 μmol) in DMA (7.5 mL) and water (2.5 mL). The resulting reaction mixture was stirred for 15 min and quenched by addition of water, then purified by RP HPLC (5%-60% A in B, A: 100 mM TEAA in MeCN, B: 100 mM TEAA in water). The product fractions were dialyzed against water and lyophilized to afford E3 as a whiter powder.
Synthesis of E4 (Ex. 38)
To a solution of E3 (246 mg, 24 μmol, 1 eq.) in water (8000 μL) was added TCEP-HCl (70 mg, 244 μmol, 10 eq.). The reaction mixture was mixed until TCEP-HCl fully dissolved. The solution was left at room temperature for 2 hours. The solution was centrifugal dialyzed two times against water over a 3K membrane to afford crude E4 which was directly used in the next step.
Synthesis of E5 (Ex. 39)
To a solution of E4 (244 mg, 24 μmol) in water (12 mL) was added N-(2-aminoethyl)maleimide trifluoroacetate salt (62.2 mg, 0.245 mmol, 10 eq.) dissolved in MeCN (0.5 mL). The solution was left at room temperature for 1 hour. LCMS indicated complete conversion. The solution was centrifugal dialyzed twice against water over a 3K membrane and lyophilized to afford E5 as a white powder.
Synthesis of E6 (Ex. 40)
E5 (40 mg, 3.95 μmol, 1 eq.) was dissolved in 4:1 DMA/water (500 μL). DIPEA (10.2 mg, 79 μmol, 20 eq.) was added to the above solution. Cholesterol chloroformate (18 mg, 40 μmol, 10 eq.) was dissolved in THF (500 μL). The two solutions were mixed together, and the reaction mixture was left at room temperature for 1 hour. LCMS indicated that the reaction was done. The reaction mixture was purified by RP HPLC (5%-95% B in A, A: 100 mM TEAA in water, B: 100 mM TEAA in MeCN). The product fractions were dialyzed against water and lyophilized to afford E6 as a whiter powder.
Synthesis of E7 (Ex. 41)
To a solution of E6 (24.5 mg, 2.3 μmol, 1 eq.) in water (1000 μL) was added piperidine in DMF (200 μL, 20% by volume, 200 eq.). The reaction mixture was left at room temperature for 1 hour. LCMS indicated that the reaction was done. The reaction mixture was filtered (0.2 uM), dialyzed against water, and lyophilized to give E7 as a whiter powder.
Synthesis of E8 (Ex. 42)
E7 (16 mg, 1.55 μmol, 1 eq.) was dissolved in freshly prepared aqueous sodium bicarbonate (0.1M, 400 μL). SPDP (4.85 mg, 0.016 mmol, 10 eq.) was dissolved in acetonitrile (400 uL). The two solutions were mixed together, and the reaction mixture was left at room temperature for 1 hour. The reaction mixture was purified by RP HPLC (5%-95% B in A, A: 100 mM TEAA in water, B: 100 mM TEAA in MeCN). The product fractions were dialyzed against water and lyophilized to afford E8 as a whiter powder.
Examples 43-44
Preparation of E8-Seq137 and E9-Seq 137
Scheme 13 is shown in FIG. 14A-1 to FIG. 14B-2.
Synthesis of E9-Seq137 (Ex. 43)
Oligonucleotide E8 (3.0 mg, 0.286 μmol) in 100 μL of 2 M Thiourea/20 mM MES in Formamide pH 6.5 was treated with peptide seq 137 (2.33 mg, 0.572 μmol) in 100 μL of the same buffer and the resulting reaction mixture was left at RT for 30 min. The reaction was diluted by addition of formamide 1 mL and purified by strong anion exchange chromatography on a Propac SAX 22×250 mm column (5%-45% B in A over 15 min, A: 60:40 trifluoroethanol:water, 20 mM triethylamine, B: 60:40 trifluoroethanol:water, 20 mM triethylamine, 1 M guanidine-HCl, 20 mL/min) to afford E9-seq-137 as a white powder.
Synthesis of E10-Seq137-e (Ex. 44)
Passenger strand E9-seq137 (1.30 mg, 0.077 μmol) and the corresponding guide strand B7 (0.561 mg, 0.077 μmol) were mixed in RNase free water and heated to 90° C. for 1 min, then left at RT for 10 min. The duplex was lyophilized and the resulting product isolated as an amorphous white powder.
Synthesis of Additional E10-Peptide Conjugates
Additional conjugates of E10 and peptide sequence were prepared in a manner analogous to that used for E10-Seq137-e.
Section F. Preparation of 3, 13, 18 Tripeptide Conjugates
Examples 45-49
Scheme 14 is shown in FIG. 15A to FIG. 15E-2.
Synthesis of Compound F2 (Ex. 45)
Compound A10 (210 mg, 0.129 mmol) was dissolved in dry N-methyl-2-pyrrolidinone (3 ml). HATU (48.9 mg, 0.129 mmol) and dry diisopropylethylamine (0.046 ml, 0.257 mmol) were added, and the mixture was sonicated until the solid was fully dissolved. The reaction was left at RT for 5 min. In a separate vial, compound F1 (500 mg, 0.0646 mmol) was dissolved in water (2 ml) and N-methyl-2-pyrrolidinone (5 ml). The A10 solution was added to the F1 solution, and the reaction was left at RT for 5 min. The reaction mixture was loaded on to an HPLC fitted with an Agilent PL-SAX 8 um 50×150 mm column heated to 60° C. The product was gradient eluted by starting at 100% solvent A (4:1 H2O:ethanol, 20 mM triethylammonium acetate pH 7.0) and increasing to 80% solvent B (4:1 H2O:ethanol, 20 mM triethylammonium acetate pH 7.0, 1M guanidinium hydrochloride) over 30 min at 100 ml/min. The fractions were combined, and the ethanol content was reduced to 5% by diluting with water. The solution was pump loaded onto a Waters XBridge 5 um 50×50 mm column at 50 ml/min, and the product was washed with water at 100 ml/min for 5 min. The desalted product was eluted by reversing the column and flowing 2:3 H2O:acetonitrile at 50 ml/min through the column. The fraction was freeze dried to afford F2 as a white amorphous solid. Expected mass: 9363.6, found mass: 9363.5.
Synthesis of Compound F3 (Ex. 46)
F2 (500 mg, 0.0534 mmol) and azido-peg9-amine (253 mg, 0.481 mmol) were dissolved in 2,2,2-trifluoroethanol (5 ml) and water (5 ml). Nitrogen was bubbled through the solution for 1 min. In a separate vial, copper(I) bromide dimethyl sulfide (43.9 mg, 0.214 mmol) was dissolved in acetonitrile (2.5 ml). Nitrogen was bubbled through the solution for 1 min. The two solutions were mixed together, and nitrogen was bubbled through the reaction mixture for 1 min. The vial was sealed and left at RT for 1 hour. The reaction mixture was quenched with EDTA solution (0.5M, pH 8.0, 1 mL) and loaded onto an HPLC fitted with a Waters XBridge 5 um 50×250 mm column. The product was gradient eluted by starting at 100% solvent A (H2O, 0.1M triethylammonium acetate pH 7.0) and increasing to 40% solvent B (acetonitrile) at 100 ml/min over 30 minutes. The fractions were combined, and the acetonitrile content was reduced to 5% by diluting with water. The solution was pump loaded onto a Waters XBridge 5 um 50×50 mm column at 50 ml/min, and the product was washed with water at 100 ml/min for 5 min. The desalted product was eluted by reversing the column and flowing 2:3 H2O:acetonitrile at 50 ml/min through the column. The fraction was freeze dried to afford F3 as a white amorphous solid. Expected mass: 10943.5, found mass: 10943.2.
Synthesis of Compound F4 (Ex. 47)
F3 (467 mg, 0.0427 mmol) was dissolved in sodium bicarbonate solution (0.1M, 4.5 mL). NHS-SPDP (120 mg, 0.384 mmol) was dissolved in acetonitrile (1 mL). The solutions were mixed together, and the reaction was left at RT for 15 min. The reaction mixture was loaded onto an HPLC fitted with a Waters XBridge 5 um 50×250 mm column. The product was gradient eluted by starting at 100% solvent A (H2O, 0.1M triethylammonium acetate pH 7.0) and increasing to 40% solvent B (acetonitrile) at 100 ml/min over 30 min. The fractions were combined, and the acetonitrile content was reduced to 5% by diluting with water. The solution was pump loaded onto a Waters XBridge 5 um 50×50 mm column at 50 ml/min, and the product was washed with water at 100 ml/min for 5 min. The desalted product was eluted by reversing the column and flowing 2:3 H2O:acetonitrile at 50 ml/min through the column. The fraction was freeze dried to afford F4 as a white amorphous solid. Expected mass: 11535.3, found mass: 11535.1.
Synthesis of F5-Seq 463 (Ex. 48)
Peptide Seq. 612 (8.75 mg, 0.00520 mmol) was dissolved in DMSO (1 mL) containing 20 mM acetic acid. In a separate vial, F4 (10 mg, 0.000867 mmol) was dissolved in DMSO (1 ml) containing 20 mM acetic acid. The two solutions were mixed together and left at RT for 1 hour. The reaction was quenched with N-methylmaleimide (5.78 mg, 0.0520 mmol) and loaded onto an HPLC fitted with an Agilent PL-SAX 10 um 25×50 mm column. The product was gradient eluted by starting at 100% solvent A (2:3 H2O:2,2,2-trifluoroethanol, 20 mM triethylamine) and increasing to 70% solvent B (2:3 H2O:2,2,2-trifluoroethanol, 20 mM triethylamine, 0.5M guanidinium hydrochloride) at 30 ml/min over 20 min. The fractions were combined and loaded onto an HPLC fitted with a Waters XBridge 5 um 19×250 mm column. The product was gradient eluted by starting at 85% solvent A (H2O, 0.1M hexylammonium acetate pH 7.0) and increasing to 65% solvent B (tetrahydrofuran) at 20 ml/min over 30 min. The fractions were combined, and the tetrahydrofuran content was reduced to less than 5% under vacuum. The solution was centrifugal dialyzed over a 10 k membrane once against water, once against 4:1 H2O:ethanol containing 0.1M sodium chloride, and two more times against water. The concentrate was freeze dried to afford F5-Seq 463 as a white amorphous solid. Expected mass: 16247.8, found mass: 16247.9.
Example 49
Scheme 15 is shown in FIG. 16A-1 to FIG. 16B-2.
Synthesis of F6 Seq 463-f (Ex. 49)
F5-Seq 463 (7.75 mg, 0.000477 mmol) and Guide B7 (3.27 mg, 0.000477 mmol) were dissolved in H2O (0.5 mL). The solution was left at RT for 1 hour and then freeze dried to afford the duplex of F6 Seq 463-f as a white amorphous solid (11 mg, quantitative). Expected mass of passenger strand: 16247.8, found mass: 16247.9. Expected mass of guide strand: 6852.5, found mass: 6852.7.
Synthesis of Additional F10-Peptide Conjugates an Duplexes
Additional conjugates of F10 and peptide sequences and their duplexes were prepared in a manner analogous to that used for F6-Seq 463-f.
Section G Preparation of 3,8,13,18 Tetrapeptides
Examples 50-53
Scheme 16 is shown in FIG. 17A-1 to FIG. 17D-2.
Synthesis of G2 (Ex. 50)
A10 (210 mg, 0.129 mmol) was dissolved in dry N-methyl-2-pyrrolidinone (3 ml). HATU (48.9 mg, 0.129 mmol) and dry diisopropylethylamine (0.046 ml, 0.257 mmol) were added, and the mixture was sonicated until the solid was fully dissolved. The reaction was left at RT for 5 min. In a separate vial, G1 (500 mg, 0.0643 mmol) was dissolved in water (2 ml) and N-methyl-2-pyrrolidinone (5 ml). The A10 solution was added to the G1 solution, and the reaction was left at RT for 5 min. The reaction mixture was loaded on to an HPLC fitted with an Agilent PL-SAX 8 um 50×150 mm column heated to 60° C. The product was gradient eluted by starting at 100% solvent A (4:1 H2O:ethanol, 20 mM triethylammonium acetate pH 7.0) and increasing to 80% solvent B (4:1 H2O:ethanol, 20 mM triethylammonium acetate pH 7.0, 1M guanidinium hydrochloride) over 30 minutes at 100 ml/min. The fractions were combined, and the ethanol content was reduced to 5% by diluting with water. The solution was pump loaded onto a Waters XBridge 5 um 50×50 mm column at 50 ml/min, and the product was washed with water at 100 ml/min for 5 min. The desalted product was eluted by reversing the column and flowing 2:3 H2O:acetonitrile at 50 ml/min through the column. The fraction was freeze dried to afford the G2 as a white amorphous solid. Expected mass: 9399.7, found mass: 9399.5.
Synthesis of G3 (Ex. 51)
G2 (483 mg, 0.0514 mmol) and azido-peg9-amine (324 mg, 0.617 mmol) were dissolved in 2,2,2-trifluoroethanol (5 ml) and water (5 ml). Nitrogen was bubbled through the solution for 1 min. In a separate vial, copper(I) bromide dimethyl sulfide (50 mg, 0.244 mmol) was dissolved in acetonitrile (2.5 ml). Nitrogen was bubbled through the solution for 1 min. The two solutions were mixed together, and nitrogen was bubbled through the reaction mixture for 1 min. The vial was sealed and left at RT for 1 hour. The reaction mixture was quenched with EDTA solution (0.5M, pH 8.0, 1 mL) and loaded onto an HPLC fitted with a Waters XBridge 5 um 50×250 mm column. The product was gradient eluted by starting at 100% solvent A (H2O, 0.1M triethylammonium acetate pH 7.0) and increasing to 40% solvent B (acetonitrile) at 100 ml/min over 30 min. The fractions were combined, and the acetonitrile content was reduced to 5% by diluting with water. The solution was pump loaded onto a Waters XBridge 5 um 50×50 mm column at 50 ml/min, and the product was washed with water at 100 ml/min for 5 min. The desalted product was eluted by reversing the column and flowing 2:3 H2O:acetonitrile at 50 ml/min through the column. The fraction was freeze dried to afford G3 as a white amorphous solid. Expected mass: 11506.2, found mass: 11506.0.
Synthesis of G4 (Ex. 52)
G3 (455 mg, 0.0396 mmol) was dissolved in sodium bicarbonate solution (0.1M, 5 mL). NHS-SPDP (160 mg, 0.512 mmol) was dissolved in acetonitrile (1.5 mL). The solutions were mixed together, and the reaction was left at RT for 15 min. The reaction mixture was loaded onto an HPLC fitted with a Waters XBridge 5 um 50×250 mm column. The product was gradient eluted by starting at 100% solvent A (H2O, 0.1M triethylammonium acetate pH 7.0) and increasing to 40% solvent B (acetonitrile) at 100 ml/min over 30 min. The fractions were combined, and the acetonitrile content was reduced to 5% by diluting with water. The solution was pump loaded onto a Waters XBridge 5 um 50×50 mm column at 50 ml/min, and the product was washed with water at 100 ml/min for 5 min. The desalted product was eluted by reversing the column and flowing 2:3 H2O:acetonitrile at 50 ml/min through the column. The fraction was freeze dried to afford G4 as a white amorphous solid. Expected mass: 12295.3, found mass: 12295.1.
Synthesis of G5-Seq 489 (Ex. 53)
Peptide SEQ ID NO: 489 (CIFGAIAGFIKNIWEGLI all (D)) (13.6 mg, 0.00694 mmol) was dissolved in DMSO (1 mL) containing 20 mM acetic acid. In a separate vial, G4 (10 mg, 0.000867 mmol) was dissolved in DMSO (1 ml) containing 20 mM acetic acid. The two solutions were mixed together and left at RT for 1 hour. The reaction was quenched with N-methylmaleimide (7.71 mg, 0.0694 mmol) and loaded onto an HPLC fitted with an Agilent PL-SAX 10 um 25×50 mm column. The product was gradient eluted by starting at 100% solvent A (2:3 H2O:2,2,2-trifluoroethanol, 20 mM triethylamine) and increasing to 70% solvent B (2:3 H2O:2,2,2-trifluoroethanol, 20 mM triethylamine, 0.5M guanidinium hydrochloride) at 30 ml/min over 20 min. The fractions were combined and loaded onto an HPLC fitted with a Waters XBridge 5 um 19×250 mm column. The product was gradient eluted by starting at 85% solvent A (H2O, 0.1M hexylammonium acetate pH 7.0) and increasing to 65% solvent B (tetrahydrofuran) at 20 ml/min over 30 min. The fractions were combined, and the tetrahydrofuran content was reduced to less than 5% under vacuum. The solution was centrifugal dialyzed over a 10 k membrane once against water, once against 4:1 H2O:ethanol containing 0.1M sodium chloride, and two more times against water. The concentrate was freeze dried to afford G5-Seq 489 as a white amorphous solid. Expected mass: 19708.1, found mass: 19708.0.
Example 54
Scheme 17 is shown in FIG. 18A-1 to FIG. 18B-2.
Synthesis of G6-Seq 489-g (Ex. 54)
G5-Seq 489 (8.5 mg, 0.000434 mmol) and B7 (2.98 mg, 0.000434 mmol) were dissolved in H2O (0.5 mL). The solution was left at RT for 1 hour and then freeze dried to afford the duplex G6-Seq 489-g as a white amorphous solid. Expected mass of passenger strand: 19708.1, found mass: 19708.3. Expected mass of guide strand: 6852.5, found mass: 6852.6.
Synthesis of Additional G6-Peptide Conjugates and Duplexes
Additional conjugates of G6 and peptide sequences and their duplexes were prepared in a manner analogous to that used for G6-Seq 489-g.
Section H. Preparation of 3,8,13,18 Tetrapeptide
Examples 55-58
Scheme 18 below was used to prepare H1 to H5.




Synthesis of H1 (Ex. 55)
Into a 500-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed a solution of di-tert-butyl 1-(tert-butylthio)hydrazine-1,2-dicarboxylate (15 g, 46.8 mmol, 2.00 equiv) in N,N-dimethylformamide (30 mL). A solution of 2-aminoethanethiol hydrochloride (2.66 g, 23.4 mmol, 1 eqiv) in N, N-dimethylformamide (80 ml) was added slowly into the round-bottom flask. This was followed by the addition of triethylamine (2.36 g, 23.4 mmol, 1 equiv). After stirring at RT overnight, a white solid was precipitating. Dry N, N-dimethylformamide (100 ml) was added to obtain a nearly clear solution. Triethylamine was added until a white solid was precipitating again. The reaction mixture was stirred at RT for 8 hours. The solution was filtered and evaporated under reduced pressure. Diethyl ether (200 ml) was added to the residue and filtered. The white solid was collected and dried in dessicator. Afterward, this white solid was dissolved five times in diethyl ether (5×10 ml), stirred for several minutes and filtered. The desired product was obtained as a white solid. 1HNMR (CDCl3, 500 MHz, ppm): 1.36 (s, 9H), 3.07 (t, 2H), 3.4 (t, 2H), 8.3 (s, 2H).
Synthesis of H3 (Ex. 56)
Lithocholic acid (H2) (7 gm, 18.59 mmol, 1 equiv) was dissolved in dry dicholormethane (200 ml) and then cooled to 0° C. Following this N, N-dicyclohexylcarbodiimide (4.6 g, 22.31 mmol, 1.2 equiv) was added to the solution. After stirring for 30 min at 0° C., pentafluorophenol (3.76 gm, 20.45 mmol, 1.1 equiv) in dichloromethane (13 ml) was added. Stirring was then continued at RT under argon for an additional 20 h. The precipitated N, N-dicyclohexylurea was filtered off and washed with cold dichloromethane. Combined filterates were then evaporated under reduced pressure. The oily residue obtained was then diluted with dichloromethane (50 ml) and washed with sat. aq. NaCl (60 ml) and water (80 ml). The organic phase was dried over Na2SO4, filtered and evaporated to dryness. The dried compound was purified using column chromatography (elution with CH2Cl2/CH3OH, 100/0-97/3). MS (m/z); 566 [M+Na]+
Synthesis of H4 (Ex. 57)
Compound H3 (4.5 gm, 8.29 mmol, 1 equiv) was dissolved in dry dichloromethane (15 ml) and then cooled to 0° C. A cold mixture of 2-(tert-butyldisulfanyl)ethanamine (H1) (2.057 gm, 12.44 mmol, 1.5 equiv) and triethylamine (2.56 gm, 2.52 mmol, 3 equiv) in dichloromethane (7 ml) was added to the resulting solution. The reaction mixture was stirred at RT for 2 h. TLC confirmed the formation of product. The reaction mixture was washed with sat. aq. NaCl (20 ml×2) and water (20 ml×2). The organic phase was dried over Na2SO4, filtered and dried over vacuum. The crude product was purified via silica gel column chromatography (elution with CH2Cl2/CH3OH, 100/0-95/5) yielding pure compound H4. MS (m/z); 524.35, [M+1]+
Synthesis of H5 (Ex. 58)
H4 (3 gm, 5.73 mmol, 1 equiv) was dissolved in dry dichloromethane (15 ml) and triethylamine was added (0.869 g, 8.59 mmol, 1.5 equiv). The reaction mixture was cooled to 0° C. 2-Cyanoethyl-N, N-diisopropylaminochlorophosphite (2.71 gm, 11.45 mmol, 2 equiv) in dry dichloromethane (10 ml) was added dropwise to the reaction mixture. The resulting solution was stirred for 1 h. TLC confirmed the formation of product. The reaction mixture was evaporated and purified on silica gel column (elution with hexanes/ethylacetate/triethylamine, 100/0/1.5 to 60/40/1.5). MS (m/z); 724.46 [M+1]+ 31P NMR (CDCl3, 500 MHz, ppm); 146.5
Examples 59-66
Scheme 19 as shown in FIG. 19A to FIG. 19I-2 was used to prepare Ex. 59 to Ex. 66.
Synthesis of H6 (Ex. 59)
See synthesis of B2 for reaction procedure. Expected mass: 9609.071, found mass: 9605.
Synthesis of H7 (Ex. 60)
To a solution of H6 (15 mg, 1.56 umol, 1 eq) in water (1400 ul) was added TCEP-HCl (26.8 mg, 0.094 mmol, 60 eq). The reaction mixture was mixed until TCEP-HCl fully dissolved. The solution was left at RT overnight. The solution was centrifugal dialyzed two times against water over 3K membrane. Expected mass: 9520, found mass: 9517.
Synthesis of H8 (Ex. 61)
See synthesis of B9 for reaction procedure. Expected mass: 9630, found mass: 9627.
Synthesis of H9-Seq32 (Ex. 62)
See the synthesis of B10-seq32 for reaction procedure. Expected mass: 13597, found mass: 13598.
Synthesis of H7-Seq32-h (Ex. 63)
See the synthesis of B11-seq32 for reaction procedure.
Synthesis of H8 (Ex. 64)
See the synthesis of C13 for reaction procedure. Expected mass: 9741.
Synthesis of H9-Seq32 (Ex. 65)
See the synthesis of C14 for reaction procedure. Expected mass: 13819, found mass: 13820.
Synthesis of H10-Seq32-h (Ex. 66)
See the synthesis of C15-Seq32 for reaction procedure.
Additional Synthesis of H7 and H10 Peptide Conjugates
Additional conjugates of H7 and H10 and peptide sequences and their duplexes were prepared in a manner analogous to that used for H7-Seq32-h and H10-Seq32-h.
Section I. Preparation of 3,13,18 Trienzymatic Cleavble Linker Peptide Conjugates
Examples 67-73
Scheme 20 is shown in FIG. 20A-1 to FIG. 20E-2.
Synthesis of I3 (Ex. 67)
I1 (160 mg, 0.209 mmol) and I2 (48.8 mg, 0.219 mmol) were dissolved in DMA (1 mL) and were treated with N-methylmorpholine (46 μL, 0.417 mmol). The reaction was stirred at RT for 6 hours, then purified by RP-HPLC (95:5-20:80% A:B linear gradient (A=0.1% aqueous TFA; B=0.1% TFA in acetonitrile) Waters C18 xbridge Column 19×250 mm). Fractions containing 13 were extracted with 2:1 DCM:MeOH, dried over Na2SO4, filtered, and concentrated in vacuo to give the product. Measured mass=814.3
Synthesis of I4 (Ex. 68)
I3 (88 mg, 0.108 mmol) was dissolved in DMA (1 mL) and was treated with piperidine (200 μL, 2.02 mmol) and stirred at 10° C. for 10 min. TFA (156 μL, 2.02 mmol) was added to quench the reaction. The reaction mixture was purified by RP-HPLC (95:5-60:40% A:B linear gradient (A=0.1% aqueous TFA; B=0.1% TFA in acetonitrile) Waters C18 xbridge Column 30×250 mm). Fractions containing 14 were lyophilized to give the product. Measured mass=592.3.
Synthesis of I5 (Ex. 69)
I4 (912 mg, 1.324 mmol) was dissolved in DMSO (7.7 mL) and treated with L1 (1.0 g, 1.40 mmol) and DIEA (463 μL, 2.65 mmol). The reaction mixture was stirred for 15 min and was purified by RP-HPLC (100:0-0:100% A:B linear gradient (A=0.1% aqueous TFA; B=0.1% TFA in acetonitrile) Waters C18 xbridge column. Fractions containing I5 were lyophilized to give the product. Measured mass=609.5 [M+2]
Synthesis of I7 (Ex. 70)
I6 (500 mg, 0.065 mmol) and I5 (236 mg, 0.194 mmol) were dissolved in a pH 5.5 MES buffer (51.6 ml, 500 mM) and acetonitrile (12.91 ml). The solution was degassed with nitrogen for 10 min, after which it was treated with CuBr.SMe2 (133 mg, 0.646 mmol) and degassed for an additional five minutes with nitrogen. The reaction mixture was sonicated and stirred for 30 min, then purified by RP-HPLC (95:5-5:95% A:B linear gradient (A=100 mM aqueous TEAA; B=100 mM TEAA in acetonitrile) Waters Phenyl xbridge Column). Fractions containing product were dialyzed twice against 0.32M EDTA pH 6.5 over a 3K membrane, then three times against water. The concentrate was then dialyzed twice against 200 mM TEAA and then three times against water. The concentrate was lyophilized to give the product as an amorphous white solid. Measured mass=11400
Synthesis of I8 (Ex. 71)
I7 (287 mg, 0.025 mmol) was suspended in water (100 uL) and diluted with NMP (2.0 mL), which produced a homogeneous solution upon standing. HATU (13 mg, 0.035 mmol) was dissolved in NMP (200 uL) and was added to A10 (62 mg, 0.038 mmol). The reaction mixture was diluted with NMP (200 uL) and was then treated with DIEA (13 uL, 0.076 mmol). The HATU reaction mixture was then added to the RNA solution in one portion and aged for 10 min. Reaction was diluted with DI water and purified by RP-HPLC (95:5-5:95% A:B linear gradient (A=100 mM aqueous TEAA; B=100 mM TEAA in acetonitrile) Waters Phenyl xbridge Column). Fractions containing 18 were dialyzed three times against water over a 3K membrane. The concentrate was lyophilized to give the product as an amorphous white solid. Measured mass=13027.
Synthesis of I9-Seq 1681 (Ex. 72)
I8 (20 mg, 1.537 μmol) was dissolved in TFE modified with 50 mM AcOH (2 mL). In a separate vial, Seq ID 1681 (8.63 mg, 6.15 umol) was suspended in 8M Gn.HCl (400 uL) and was diluted with 50 mM AcOH in TFE (2 mL) to form a slightly cloudy suspension, then added to the RNA solution. After 10 min, more Seq ID 1681 (8.63 mg, 1.54 umol) was added and the reaction was aged 30 min, after which AEX indicated near-complete conversion to desired product. Reaction was quenched with N-methylmaleimide (6.83 mg, 61.5 μmol) and was purified by AEX (0-40% 1M Gn.HCl in 1:1 water:TFE with 40 mM TEAA pH 7.5, Proteomix NP10 column heated to 60° C.). Material was repurified using 70:30-25:75 gradient of 200 mM HAA pH 7.5: ACN and an Agilent PLRP-S column. Pure fractions were pooled, dialyzed, and lyophilized to give 19-Seq 1681 (6.37 mg, 0.302 μmol, 19.65% yield).
Synthesis of I10-Seq 1681-f (Ex. 73)
I9-seq 1681 (3.02 mg, 0.143 μmol) was dissolved in water (950 μl) and was treated with a solution of B7 (0.980 mg, 0.143 μmol) in water (144 μl). The reaction mixture aged for 15 min and was then lyophilized to give the product as an amorphous white solid. Measured mass=21107.
Additional Synthesis of I10 Peptide Conjugates an Duplexes
Additional conjugates of I10 and peptide sequences and their duplexes were prepared in a manner analogous to that used for I10-seq-1681-f.
Section J. Preparation of Amino Modified C2 Linkers
Examples 74-82
Scheme 21 is shown in FIG. 21A to FIG. 21H-2.
Synthesis of A10B (Ex. 74)
In a test tube equipped with a stir bar, A10 (100 mg, 0.061 mmol) was dissolved in DMSO (611 μl) followed by the addition of Hunig's Base (133 μl, 0.764 mmol) and HATU (76 mg, 0.199 mmol). After 20 min, N-(2-aminoethyl)maleimide trifluoroacetate salt (12.85 mg, 0.092 mmol) dissolved in 400 μL of DMSO was added. After 20 min, the reaction was determined complete and quenched with water (1.5 mL) until yellow color almost dissipated. The reaction was purified by reverse phase chromatography (Gilson 2020, Solvent A) 0.1% TFA in water/Solvent B) 0.1% TFA in ACN, 0-50% gradient for 15 min, 40 mL/min, XBridge Prep C18 5 μm OBD 30×250 mm). The resulting fractions were lyophilized to afford a white solid, A10B. [M+1, expected]=1757.807, [M+1, observed]=1759.0.
Synthesis of J2 (Ex. 75)
See Synthesis of B3 for reaction procedure. J2 [M+1, expected]=7604.750, [M+1, observed]=7600.0.
Synthesis of J3 (Ex. 76)
A10B (10.26 mg, 5.84 μmol) was dissolved in water (700 μL) and added to a 1.8 mL solution (1 water: 1 acetate buffer: 2 formamide) of J2 (29.6 mg, 3.89 μmol). The reaction was shaken at RT for 20 min and then determined complete. The reaction mixture was purified using strong anion exchange chromatography (Gilson PLC 2020, Sepax Proteomix SAX NP10 21.2×50 mm, Buffer A: 3:2 trifluoroethanol:water, 40 mM triethylamine/Buffer B: 3:2 trifluoroethanol:water, 40 mM triethylamine, 1000 mM guanidine-HCl, 1% B hold for 3 minutes, then 5% B-45% B over 12 minutes). The fractions were dialyzed three times against water over a 3K membrane to afford a white solid, J3. [M+1, expected]=9362.556, [M+1, observed]=9359.0.
Synthesis of J4 (Ex. 77)
To an Eppendorf vial, J3 (6.34 mg, 0.678 μmol) was dissolved in water (250 μL). In a separate Eppendorf vial, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (0.831 mg, 2.035 μmol) was dissolved in DMSO (50 μL). The SPDP solution was added to the RNA solution. After 4 hours, the reaction was recharged with additional SPDP (2.77 mg, 6.78 μmol) which was dissolved in 50 μL DMSO. After 24 hr, the reaction was recharged with additional SPDP (2.77 mg, 6.78 μmol) which was dissolved in 50 μL DMSO. After 72 hr, the reaction was diluted to 3 mg/mL with the addition of 390 μL of pH 8.1 sodium bicarbonate. After 2 hr, an additional 3 eq. of SPDP in 50 μL DMSO were added. The reaction mixture was dialyzed three times against water over a 3K membrane and lyophilized to afford a white solid, J4. [M+1, expected]=9543.834, [M+1, observed]=9554.0.
Synthesis of J5-Seq26 (Ex. 78)
See Synthesis of B10-Seq32 for reaction procedure. J5-Seq26—Mass observed: 11413.
Synthesis of J6-Seq26-i (Ex. 79)
See Synthesis of B11-Seq32-b for reaction procedure. J6-Seq26-i—Mass observed: 18265.
Synthesis of J7 (Ex. 80)
To an Eppendorf vial, J3 (5.8 mg, 0.621 μmol) was dissolved in water (250 μL). In a separate Eppendorf vial, Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (0.727 mg, 1.862 μmol was dissolved in DMSO (50 μL) and the pH was adjusted to pH 5 with the addition of 1 small drop of TFA. The SMCC solution was added to the RNA solution. After several hours, the pH was titrated to pH 7 with the gradual addition of 0.1N NaOH. After 18 hr, 6 eq. of SMCC were dissolved in 50 μL DMSO and added to the reaction mixture. After 4 hr, an additional 3 eq. of SMCC in 50 μL DMSO was added to the reaction. After several hr, 300 μL of pH 8.1 sodium bicarbonate solution was added to the reaction. The reaction was dialyzed three times against water over a 3K membrane and lyophilized to afford a white solid, J7. [M+1, expected]=9543.834, [M+1, observed]=9554.0.
Synthesis of J8-Seq26 (Ex. 81)
See Synthesis of B10-Seq32 for reaction procedure. J8-Seq26—Mass observed: 11545.
Synthesis of J9-Seq26-i (Ex. 82)
See Synthesis of B11-Seq32 for reaction procedure. J9-Seq26-I—Mass expected: 18397.
Additional Synthesis of J6 & J9 Peptide Conjugates
Additional conjugates of J6 and J9 and peptide sequences and their duplexes were prepared in a manner analogous to that used for J6-Seq26, J9-Seq26 and J6-Seq26-i, J9-Seq26-i.
Section K. 3′ Bis Peptide Linkers
Examples 83-87
Scheme 22 is shown in FIG. 22A-1 to FIG. 22D-2.
Synthesis of K2 (Ex. 83)
In a 20 mL vial, 3-(tritylthio)propanoic acid (158 mg, 0.454 mmol) was dissolved in DMF (1.514 mL) followed by the addition of HATU (184 mg, 0.484 mmol) and Hunig's base (0.158 mL, 0.908 mmol). The reaction solution turned light yellow in color. After 5 min, K1 (100 mg, 0.151 mmol) was added as a solid and the reaction solution turned transparent orange in color. The reaction was stirred at RT for 15 min and then determined complete.
The reaction was purified by reverse phase chromatography (Gilson 2020, 5-95% ACN/Water with a 0.1% TFA modifier, flow rate: 20 mL/min, gradient time: 22 min, column: XBridge prep OBD 5 μm C18 19×250 nm). The resulting fractions were lyophilized to afford a white solid, K2. [M+1, expected]=877.059, [M+1, observed]=877.4
Synthesis of K3 (Ex. 84)
In an Eppendorf vial, K2 (10.07 mg, 0.011 mmol) was dissolved in formamide (0.5 mL). In a 15 mL Falcon tube, peptide Seq ID 74 (57.92 mg, 0.034 mmol) was dissolved in formamide (1 mL). The peptide/formamide solution was added to the linker/formamide solution and stirred at RT for 20 min.
The reaction was determined complete and the reaction was purified by reverse phase chromatography (Gilson 2020, 5-100% ACN/Water with a 0.1% TFA modifier, flow rate: 20 mL/min, gradient time: 30 minutes, column: XBridge prep OBD 5 μm C18 19×250 nm). The resulting fractions were lyophilized to afford a white solid, K3. [M+3, expected]=1416.03, [M+3, observed]=1415.0
Synthesis of K4 (Ex. 85)
In a 40 mL vial, a solution of TFA (1000 μL), water (96 μL), and triisopropylsilane (96 μL) in a 0.83:0.08:0.08 mixture by volume was combined and added to K3 (47 mg, 0.011 mmol) in a 20 mL vial which was stirred at RT for 10 min. An additional 500 L of TFA was added and the reaction was stirred for an additional 10 min. The reaction was determined complete, concentrated under reduced pressure, diluted with 3.5 mL of 2M thiourea pH 6.5 in FMD and MES, and purified by reverse phase chromatography (Gilson 2020, 5-80% ACN/Water with a 0.1% TFA modifier, flow rate: 20 mL/min, gradient time: 20 minutes, column: XBridge prep OBD 5 m C18 19×250 nm). The resulting fractions were lyophilized to afford a white solid, K4. [M+3, expected]=1334.34, [M+3, observed]=1334.4
Synthesis of K5-Seq 74 (Ex. 86)
See Synthesis of B10-Seq32 for reaction procedure. K5-Seq 74—Expected mass: 13178.103.
Synthesis of K6-Seq 74-b (Ex. 87)
See Synthesis of B10-Seq32 for reaction procedure. Observed mass passenger=15907; Observed mass guide=8744; duplex=24651.
Additional Synthesis of K5 Peptide Conjugates and Duplexes
Additional conjugates of K5 and peptide sequences and the corresponding duplexes were prepared in a manner analogous to that used for K5-Seq 74 and K6-Seq 74-b.
Section L. Preparation of Guide Strand Position 2′-10,15 ECL Peptide Conjugates
Examples 88-94
Scheme 23 is shown below, and in FIG. 23A to FIG. 23C-2.




Synthesis of L3 (Ex. 88)
(9H-fluoren-9-yl)methyl ((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate L1 (500 mg, 0.652 mmol), 2-(pyridin-2-yldisulfanyl)ethanamine hydrochloride (153 mg, 0.685 mmol), and N-methylmorpholine (0.143 mL, 1.30 mmol) were dissolved in N,N-Dimethylacetamide (3 mL). The reaction mixture was aged for 16 h at RT and purified by reverse phase chromatography on a Waters Xbridge C18 column (5 uM, 30×250 mm) using a gradient of 5-80% ACN/water with 0.1% TFA over 20 min at 40 mL/min. The product was lyophilized to give L3 as a solid. MS (m/z): 814 (M+1).
Synthesis of L4 (Ex. 89)
(9H-fluoren-9-yl)methyl ((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((4-((((2-(pyridin-2-yldisulfanyl)ethyl)carbamoyl)oxy)methyl)phenyl)amino)-5-ureidopentan-2-yl)amino)butan-2-yl)carbamate L3 (343 mg, 0.421 mmol) and piperidine (200 uL, 2.02 mmol) were dissolved in N,N-Dimethylacetamide (3 mL). The reaction mixture was aged for 10 min at RT, quenched with trifluoroacetic acid (156 uL, 2.02 mmol), and purified by reverse phase chromatography on a Waters Xbridge C18 column (5 uM, 30×250 mm) using a gradient of 5-40% acetonitrile/water with 0.1% trifluoroacetic acid over 20 min at 40 mL/min. The product was lyophilized to give L4 as a solid. MS (m/z): 592 (M+1).
Synthesis of L6 (Ex. 90)
To a solution of 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-(pyridin-2-yldisulfanyl)ethyl)carbamate L4 (238 mg, 0.346 mmol) in dimethylsulfoxide (1.5 mL) was added a solution of bis(2,5-dioxopyrrolidin-1-yl) octanedioate L5 (509 mg, 1.382 mmol) and triethylamine (0.096 mL, 0.691 mmol). The reaction mixture was aged for 15 min and purified on a silica gel column (80 g) using a gradient of 1-10% methanol/dichloromethane over 30 min at 60 mL/min to give L6 as a solid. MS (m/z): 845 (M+1)
Synthesis of L8 (Ex. 91)
RNA compound L7 (163 mg, 0.024 mmol) and 2-azidoethanamine hydrochloride (30 mg, 0.245 mmol) were dissolved in an argon degassed, 3:1 mixture of N,N-Dimethylacetamide:water (2 mL). An argon degassed solution of copper (I) bromide dimethyl sulfide complex (12 mg, 0.059 mmol) was added and the mixture was aged at 45° C. for 16 h. The mixture was quenched with a 0.5 M solution of EDTA (3 mL) and let stand for 15 min. The product was isolated by spin dialysis against water (3×) followed by lyophilization to give a solid. MS (m/z): 7086.
Synthesis of L9 (Ex. 92)
RNA compound L8 (46 mg, 6.49 μmol) and N-methylmorpholine (7.1 mL, 65 μmol) were dissolved in water (250 μL) and DMSO (250 μL) at 10° C. To this mixture was added a solution of 2,5-dioxopyrrolidin-1-yl 8-(((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((4-((((2-(pyridin-2-yldisulfanyl)ethyl)carbamoyl)oxy)methyl)phenyl)amino)-5-ureidopentan-2-yl)amino)butan-2-yl)amino)-8-oxooctanoate L6 (18 mg, 21 μmol) dissolved in DMSO (500 μL). The reaction mixture was aged for 16 h, diluted with water (1.5 mL) and purified by ion pairing chromatography on a Waters Xbridge phenyl column (5 μM, 19×250 mm) using a gradient of 0-55% acetonitrile/water with 100 mM triethylammonium acetate over 15 min at 20 mL/min. The product was isolated by spin dialysis against water (3×) followed by lyophilization to give a solid. MS (m/z): 8547.
Synthesis of L10-Seq 463 (Ex. 93)
RNA compound L9 (11 mg, 1.29 μmol) was dissolved in trifluoroethanol containing 50 mM acetic acid (500 μL). To this solution was added peptide Seq 463 (8.66 mg, 5.15 μmol) dissolved in trifluoroethanol containing 50 mM acetic acid (1000 μL). The mixture was aged for 10 min, quenched with N-methylmaleimide (1.9 mg, 44 μmol), and purified by ion pairing chromatography on a Waters Xbridge phenyl column (10 μM, 19×250 mm) using a gradient of 5-95% acetonitrile/water with 100 mM triethylammonium acetate over 15 min at 20 mL/min. The product was isolated by spin dialysis against water (3×) followed by lyophilization to give a solid. MS (m/z): 11687.
Synthesis of L11-Seq 463-i (Ex. 94)
A solution of L10-Seq 463 (2.46 mg, 0.27 μmol) dissolved in DI water (300 μL) was added to B2 (3.1 mg, 0.27 μmol) and heated at 90° C. for 1 min. Solution was lyophilized to give duplex as a white solid. MS (m/z) passenger strand: 9267, guide strand: 11686.
Additional Synthesis of L10 Peptide Conjugates and L11Duplexes
Additional L10 conjugates of peptide sequences and the corresponding duplexes L11 were prepared in a manner analogous to that detailed above.
Section M. Synthesis of Guide Strand Position 2′-10,15 Disulfide Peptide Conjugates
Examples 95-98
Scheme 24 is shown in FIG. 24A-1 to FIG. 24B-2.
Synthesis of M1 (Ex. 95)
3-(Pyridin-2-yldisulfanyl)propanoic acid (506 mg, 2.35 mmol), 2-azidoethanamine hydrochloride (317 mg, 2.59 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (496 mg, 2.59 mmol), 1-hydroxy-7-azabenzotriazole (199 mg, 1.46 mmol), and n-methylmorpholine (0.44 mL, 4.7 mmol) were dissolved in dichloromethane (25 mL). The mixture was aged for 1 h, diluted with saturated sodium bicarbonate solution (25 mL) and organic layer separated. Extracted aquous later with dichloromethane (2×25 mL), dried combined organics over anhydrous sodium sulfate, filtered off solids and concentrated in vacuo. The mixture was purified on a silica gel column (80 g) using a gradient of 0-50% ethyl acetate/dichloromethane over 15 min at 30 mL/min to give a clear oil of M1. MS (m/z): 284.
Synthesis of M2. (Ex. 96)
RNA compound L7 (180 mg, 26 μmol) and M1 (59 mg, 208 μmol) were dissolved in a 100 mM, pH 5.5 MES buffer (3.6 mL) and acetonitrile (0.9 mL). This mixture was degassed with argon for 15 min. To this solution was added a degassed solution of copper (I) bromide dimethyl sulfide complex (13 mg, 65 μmol) dissolved in acetonitrile (0.45 mL) and aged at RT for 28 h. The mixture was quenched with a 100 mM, pH 8 solution of EDTA (5 mL) and allowed to stand for 15 min. The mixture was purified by ion pairing chromatography on a Waters Xbridge phenyl column (5 μM, 30×150 mm) using a gradient of 0-30% acetonitrile/water with 100 mM triethylammonium acetate over 15 min at 30 mL/min. The product was isolated by spin dialysis against water (3×) followed by lyophilization to give a solid of M2. MS (m/z): 7481.
Synthesis of M3-Seq 463 (Ex. 97)
RNA compound M2 (27.3 mg, 3.65 μmol) was dissolved in trifluoroethanol containing 50 mM acetic acid (1300 μL). To this solution was added peptide Seq 463 (15.4 mg, 9.13 μmol) dissolved in trifluoroethanol containing 50 mM acetic acid (1300 μL). The mixture was aged for 10 min, quenched with N-methylmaleimide (10.1 mg, 91 μmol), and purified by ion pairing chromatography on a Waters Xbridge phenyl column (10 μM, 19×250 mm) using a gradient of 5-80% acetonitrile/water with 100 mM triethylammonium acetate over 15 min at 20 mL/min. The product was isolated by spin dialysis against water (3×) followed by lyophilization to give a solid of M3-Seq 463. MS (m/z): 10624.
Synthesis of M4-Seq 463-i (Ex. 98)
A solution of B2 (2.18 mg, 0.24 μmol) dissolved in DI water (290 μL) was added to M3-Seq 463 (2.5 mg, 0.24 μmol) and heated at 90° C. for 1 min. This solution was lyophilized to give duplex M4-Seq 463-j as a white solid. MS (m/z) passenger strand: 9267, guide strand: 10621
Additional Synthesis of M3 Peptide Conjugates and M4 Duplexes
Additional M3 conjugates of peptide sequences and the corresponding duplexes M4 were prepared in a manner analogous to that detailed above.
Section N. Synthesis of Guide Strand Position 2′-15 Disulfide Peptide Conjugates
Examples 99-100
Scheme 25 is shown in FIG. 25A to FIG. 25B-2.
Synthesis of N3-Seq 283 (Ex. 99)
RNA compound N2 (11 mg, 1.54 μmol; prepared as detailed in Section M for the di-click substrate) was dissolved in trifluoroethanol containing 50 mM acetic acid (1300 μL). To this solution was added peptide seq283 (3.57 mg, 2.31 μmol) dissolved in trifluoroethanol containing 50 mM acetic acid (1300 μL). The mixture was aged for 10 min, and purified by ion pairing chromatography on a Waters Xbridge phenyl column (10 μM, 19×250 mm) using a gradient of 5-80% acetonitrile/water with 100 mM triethylammonium acetate over 15 min at 20 mL/min. The product was isolated by spin dialysis against water (3×) followed by lyophilization to give a solid. MS (m/z): 8600.
Synthesis of N4-Seq 283-k (Ex. 100)
A solution of B2 (5.65 mg, 0.609 μmol) dissolved in DI water (423 μL) was added to N3-Seq 283 (5.24 mg, 0.609 μmol) and heated at 90° C. for 1 min. Solution was lyophilized to give duplex as a whlte solid. MS (m/z) passenger strand: 9268, guide strand: 8601.
Additional Synthesis of N3 Peptide Conjugates and N4 Duplexes
Additional N3 conjugates of peptide sequences and the corresponding duplexes N4 were prepared in a manner analogous to that detailed above.
Section O. Synthesis of Guide Strand Position 2′-15 ECL Peptide Conjugates
Examples 101-103
Scheme 26 is shown in FIG. 26A-1 to FIG. 26B-2.
Synthesis of O2 (Ex. 101)
RNA compound O1 (20.7 mg, 2.97 μmol; prepared in an anlogous manner to L8) was dissolved in 100 mM NaHCO3 (400 μL) and DMSO (300 μL). To this mixture was added a solution of 2,5-dioxopyrrolidin-1-yl 8-(((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((4-((((2-(pyridin-2-yldisulfanyl)ethyl)carbamoyl)oxy)methyl)phenyl)amino)-5-ureidopentan-2-yl)amino)butan-2-yl)amino)-8-oxooctanoate L6 (6.28 mg, 7.43 μmol) dissolved in DMSO (250 μL). The reaction mixture was aged for 1.5 h, diluted with water (1.5 mL) and purified by ion pairing chromatography on a Waters Xbridge phenyl column (5 μM, 19×250 mm) using a gradient of 0-60% acetonitrile/water with 100 mM triethylammonium acetate over 15 min at 20 mL/min. The product was isolated by spin dialysis against water (3×) followed by lyophilization to give a solid. MS (m/z): 7696.
Synthesis of O2-Seq 463 (Ex. 102)
RNA compound O2 (10 mg, 1.30 μmol) was dissolved in trifluoroethanol containing 50 mM acetic acid (1000 μL). To this solution was added peptide Seq 463 (3.28 mg, 1.95 μmol) dissolved in trifluoroethanol containing 50 mM acetic acid (500 μL). The mixture was aged for 1 hr and purified by ion pairing chromatography on a Waters Xbridge phenyl column (5 μM, 19×250 mm) using a gradient of 5-90% acetonitrile/water with 100 mM triethylammonium acetate over 15 min at 20 mL/min. The product was isolated by spin dialysis against water (3×) followed by lyophilization to give a solid. MS (m/z): 9268.
Synthesis of O3-Seq 463-k (Ex. 103)
A solution of O2-Seq 463 (3.02 mg, 0.326 μmol) dissolved in DI water (303 μL) was added to B2 (3.02 mg, 0.326 μmol) and heated at 90° C. for 1 min. Solution was lyophilized to give duplex as a whlte solid. MS (m/z) passenger strand: 9267, guide strand:9264.
Additional Synthesis of O2 Peptide Conjugates and O3 Duplexes
Additional O2 conjugates of peptide sequences and the corresponding duplexes O3 were prepared in a manner analogous to that detailed above.
Section P. Synthesis of Guide Strand Position 2′-15 Cholesterol and Peptide Conjugates
Examples 104-106
Scheme 27 is shown in FIG. 27A-1 to FIG. 27B-2.
Synthesis of P1 (Ex. 104)
RNA compound N2 (67.2 mg, 9.39 μmol) and diisopropylethylamine (13.1 μL, 75 μmol) was dissolved in water (750 μL), N,N-dimethylacetamide (750 μL), and tetrahydrofuran (1200 μL). To this mixture was added a solution of thiocholesterol (30.2 mg, 75 μmol) dissolved in tetrahydrofuran (300 μL). The mixture was aged for 30 min, diluted with 2M triethylammonium acetate (100 μL), and purified by ion pairing chromatography on a Waters Xbridge phenyl column (10 μM, 19×250 mm) using a gradient of 5-95% acetonitrile/water with 100 mM triethylammonium acetate over 15 min at 20 mL/min. The product was isolated by spin dialysis against water (3×) followed by lyophilization to give a solid. MS (m/z): 7451.
Synthesis of P2-Seq 32-k (Ex. 105)
A solution of P1 (1.0 mg, 0.134 μmol) dissolved in DI water (200 μL) was added to B10-Seq 32 (1.86 mg, 0.129 μmol) and heated at 90° C. for 1 min. Solution was lyophilized to give duplex as a whlte solid. MS (m/z) passenger strand: 13295, guide strand: 7450.
Synthesis of P2-Seq 32-m (Ex. 106)
Guide strand P1 was also duplexed with passenger strand F6-Seq 32 in a manner identical to that detailed above in Example 105 to provide duplex P2-Seq 32-m:
Scheme 28 is shown in FIG. 28-1 to FIG. 28-2.
Section Q. 3′ Enzymatically Cleaved Linker Bis Peptides
Examples 107-109
Scheme 29 is shown in FIG. 29A-1 to FIG. 29C-2.
Synthesis of Q1 (Ex. 107)
In a Falcon tube, L6 (13.82 mg, 0.016 mmol) was dissolved in DMSO (1963 μl) and cooled to 10° C. in an ice-bath. In a separate Falcon tube, B4 (76.2 mg, 8.18 μmol) was dissolved in pH 8.3 NaHCO3 200 mM (1309 μl). The RNA solution was added to the DMSO solution and the reaction was determined complete after 5 min.
The reaction was purified by ion-pairing chromatography (GX-281, XBridge Prep Phenyl 5 um, OBD, 30×150 mm, 30 mL/min, 5-45% of 100 mM TEAA in water/100 mM TEAA in ACN, 20 min gradient). The resulting fractions were dialyzed against water 3x on Millipore 3K, 15 mL tubes, (4200 rpm, 4° C.) and then lyophilized to afford a white solid. Expected mass: 10052.834. Found mass: 10051.0.
Synthesis of Q2-Seq 74 (Ex. 108)
See Synthesis of B10-Seq74 for reaction procedure. Q2-Seq 74—Found mass: 13940.012.
Synthesis of Q3-Seq 74-b (Ex. 109)
See Synthesis of B11-Seq74 for reaction procedure. Q3-Seq 74-b—Found mass: 20792.
Section R. 5′,3′ Di-Lipopeptide Conjugates
Examples 110-112
Scheme 30 is shown in FIG. 30A to FIG. 30E-3.
Synthesis of R2 (Ex. 110)
L6 (23.2 mg) was dissolved in formamide (300 μl) and DMSO (300 μl), then added R1 (50 mg) dissolved in pH 8.3 200 mM NaHCO3 aqueous solution (600 μl). After 5 min, precipitation appeared. Additional DMSO (300 μl) was added, whereupon most of solids redissolved. After a 15 min incubation, the reaction was purified using an XBridge Prep Phenyl column (5 uM, 30×150 mm) using a gradient of 5-45% CH3CN (100 mM TEAA)/water (100 mM TEAA), 20 min at 20 mL/min, collecting at 260 nm. The product fractions were diluted with water to reduce the CH3CN content to below 20% and centrifugal dialyzed four times against water over a 3K membrane. The retentate was frozen and lyophilized to a white solid.
Synthesis of R3 (Ex. 111)
Dissolved R2 in 500 μl of water, dissolved Compound 35 of SCHEME 38 separately in 500 μl of water, then added GS solution to PS solution, vortexed thoroughly at RT, then checked analytical SAX HPLC confirming the formation of duplex. The solution was freeze dried to afford the duplex as a white amorphous solid.
Synthesis of R4-Seq 27-1 (Ex. 112)
Dissolved siRNA R3 in 2,2,2-trifluoroethanol containing 50 mM acetic acid (500 uL). Dissolved peptide in 2,2,2-trifluoroethanol containing 50 mM acetic acid (500 uL), then added 8 M aqueous guanidinium hydrochloride (30 uL). The siRNA solution was added to the peptide solution to give a clear solution. After 1 h, the reaction mix was diluted with formamide (1 mL) and was purified on neutral SAX system (Buffer A: 1:1 water:TFE 20 mM MES pH 5.5 Buffer B: 1:1 water: TFE 20 mM MES pH 5.5 1M CsCl) in two runs. The product fractions were diluted with water to reduce the TFE content to below 50% and dialyzed three times against water over a 3K membrane. The retentate was frozen and lyophilized to a white solid.
Additional Synthesis of R3 Peptide Conjugates and R4 Duplexes
Additional R3 conjugates of peptide sequences and the corresponding R4 duplexes were prepared in a manner analogous to that detailed above.
Section S. Preparation of Alternative TetraGalNAc Ligands
Examples 113-115
Synthesis of TetraGalNAc Ligand Compounds 17a, 17b and 17c
The following Scheme 31 was used to prepare TetraGalNAc Compounds 17a, 17b and 17c.







Synthesis of Compound 13
To a solution of 5-chloro-1-pentanol (3.0 g, 24.47 mmol) Compound 11 in DMF (20 mL) was added sodium azide (1.909 g, 29.4 mmol) Compound 12. After being stirred at 60° C. for overnight, the reaction mixture was concentrated in vacu. The residue was purified by silica gel chromatography (EtOAc/Hexane 1:3), to give product Compound 13 as clear liquid. 1H NMR (500 MHz, CDCl3) δ 3.62 (m, 2H), 3.25 (t, J=6.9 Hz, 2H), 1.63-1.53 (m, 4H), 1.45-1.40 (m, 2H).
Synthesis of Compound 15
Compound 13 (0.796 g, 6.16 mmol) and D-galactosamine pentaacetate (2.00 g, 5.14 mmol) Compound 14 were suspended in 20 mL DCM, followed by addition of trifluoromethanesulfonic acid (0.154 g, 1.027 mmol). The resulting mixture was brought to reflux for overnight. LC-MS indicated completed conversion of SM, the reaction mixture was diluted with EtOAc and washed with sodium bicarbonate and dried over sodium sulfate. Solvent was removed and the residue was purified by ISCO DCM/MeOH from 100/0 to 90/10 over 30 min to afford Compound 15 as a white solid. 1H NMR (500 MHz, CDCl3) δ: 1.97 (6H, s), 2.02 (6H, s), 2.06 (6H, s), 2.15 (6H, s), 3.28 (6H, t, J=6.89 Hz), 3.50 (3H, dt, J=9.63, 6.66 Hz), 3.68 (1H, q, J=5.98 Hz), 3.94-3.92 (7H, m), 4.16-4.15 (5H, m), 4.73 (2H, d, J=8.34 Hz), 5.31 (2H, dd, J=11.16, 3.48 Hz), 5.40-5.38 (5H, m). Calculated mass: [M+H]+: C19H31N4O9, 459.2; observed: 459.4.
Synthesis of Compound 16
Lys-alkyne Compound A1 (130 mg, 0.436 mmol) and GalNAc Azide 6 (999 mg, 2.178 mmol) were dissolved in THF (5 mL, degassed). Copper (I) bromide-dimethyl sulfide complex (17.91 mg, 0.087 mmol) was added in one portion to the reaction mixture and the THF solution was stirred for overnight at 40° C. The reaction color changed to blue/green, indicating Cu2+, fresh sodium ascorbate 37 mg in 0.2 mL of water was added to reaction mixture and allowed to react overnight. The reaction was concentrated and purified by RP HPLC 5-60 MeCN (0.5% TFA)/Water (0.5% TFA) over 20 min. The collected fractions were combined and lyophilized to afford Compound 8 as a white solid. Calculated mass: [M+3H]3+: C94H145N18O38, 2134.0, m/z=711.3; observed: 711.9.
Synthesis of Compound 17a (Ex. 113)
To protected TetraGalNAc Compound 8 (300 mg, 0.141 mmol) in DCM/MeOH=1/1 5 mL at 0° C. was added Sodium Methoxide (91 mg, 1.688 mmol). The reaction was stirred for 1 h and quenched by addition of 2 mL of water. Volatile solvent was removed, and the reaction mixture was purified by P4 bio gel with water and the collect fractions were combined and lyophilized to afford Compound 9 as a white solid. Calculated mass: [M+3H]3+: C70H121N18O26, 1629.9, m/z=543.3; observed: 543.8; [M+2H]2+: C70H120N18O26, 1628.9, m/z=814.5; observed: 814.9.
Synthesis of Compounds 17b and 17c (Ex. 114 and Ex. 115)
Syntheses of Compounds 17b and 17c which have the following structures were accomplished in a manner similar to that used for Compound 17a using the appropriate azide source.







Example 116
Scheme of Conjugation of TetraGalNAc Ligands
Scheme 32 as shown in FIG. 31A and FIG. 31B shows a general scheme that can be used to prepare tetraGalNAc-siRNA conjugates.
Using the general scheme 32, Conjugates 10-1, 10-2, 10-3, 10a-1, 17a-1, 17b-1, 17c-1 can be obtained. The coupling procedure can be performed on a preformed siRNA duplex or on a single strand followed by annealing. Alternatively, one can utilize the protocol outlined in Bioconjug Chem. 2011, 22, pp. 1723-8.
Example 117
Synthesis of TetraGalNAc-siRNA Conjugate (A11-a) via TetraGalNAc Acetate Compound A9
To a solution of tetraGalNAc acetate (A9, 58.7 mg, 0.027 mmol) in acetonitrile (1.5 ml) was added DIPEA (2.2 mg, 0.055 mmol) and HATU (10.44 mg, 0.027 mmol). The mixture was stirred at room temperature for 30 min, transferred into a solution siRNA (0.014 mmol) in water (1.5 ml) and acetonitrile (1.5 ml) via a syringe pump over 20 min, and stirred for 30 min before it was concentrated under vacuum down to 1.5 mL. Sodium carbonate (218 mg, 2.059 mmol) was then added, followed by MeOH (0.50 ml). The resulted solution was stirred at room temperature for 16 h, concentrated, purified via dialysis, and lyophilized to yield Conjugate A11-a.
The coupling protocol described for A11-a can also be performed with A10 instead of A9.
Examples 118-119
Synthesis of Conjugates A11-b and A11-c (Ex. 118 and Ex. 119)
A similar protocol was used for Conjugates A11-b and A11-c. Duplex formation with the appropriate antisense or sense strand can be performed using the protocol described for B11.
Example 120
Synthesis of 3′5′ Bis TetraGalNAc-siRNA Conjugate Single Strand 18
To a solution of tetraGalNAc acid Compound 10 (41.2 mg, 0.025 mmol) in DMSO (200 uL) was added HATU (9.6 mg, 0.025 mmol) and DIPEA (17.6 uL, 0.126 mmol). The mixture was stirred at room temperature for 15 min, transferred into a solution of diamino-siRNA (18.8 mg, 2.52 umol) in water (40 uL) and DMSO (360 uL) and stirred for 30 min. The mixture was diluted with water (1.5 mL) and purified on a XBridge Prep Phenyl column (5 uM, 19×250 mm) using a gradient of 0-30% CH3CN/water containing 100 mM TEAA. The fractions were concentrated via dialysis and lyophilized to yield Compound 18.
Example 121
Synthesis of 3′5′ Bis TetraGalNAc-siRNA Duplex Conjugate 19-1 (Ex. 121)
Scheme 33 as shown in FIG. 32A and FIG. 32B was used to prepare TetraGalNAc-siRNA Conjugate 19-1.
A solution of 3′5′ bis tetraGalNAc-siRNA conjugate 18 (13.7 mg, 1.29 umol) in water (200 uL) was added to a solution of Guide siRNA (9.3 mg, 1.35 umol) dissolved in water (100 uL) and heated at 90 C for 1 minute. The resulting solution was cooled and lyophilized to yield duplex 19-1.
Example 122
Synthesis of TetraGalNAc Ligand Compound 24 (Ex. 122)
The following Scheme 34 was used to prepare tetraGalNAc ligand Compound 24.




Synthesis of Compound 22
To a solution of N-BOC-1,3-DIAMINOPROPANE (Compound 20, 115 mg, 0.660 mmol) in 1:1 CH2Cl2/CH3CN (1 mL) at 0° C. was added a solution of 3-maleimidopropionic acid N-hydroxysuccinimide ester (Compound 21, 185 mg, 0.695 mmol) dissolved in acetonitrile (4 mL) and CH2Cl2 (1 mL). The mixture was stirred for 1 h and concentrated in vacuo. The residue was purified by silica gel chromatography (0-5% MeOH/CH2Cl2 to give product Compound 22. Calculated mass: [M+H]+: C15H24N3O5, 326.2; observed: 326.3.
Synthesis of Compound 23
To a solution of maleimide Compound 22 (56 mg, 0.172 mmol) in CH2Cl2 (1 ml) was added a solution of 4M HCl (1 ml, 4.00 mmol) in dioxane. The mixture was stirred for 1 h and concentrated in vacuo. The residue was azeotroped with CH2Cl2 (2×) and dried under vacuum to give product Compound 23. Calculated mass: [M+H]+: C10H16N3O3, 226.1; observed: 226.3.
Synthesis of tetraGalNAc Maleimide Compound 24 (Ex. 122)
To a solution of tetraGalNAc acid Compound 10 (100 mg, 0.061 mmol) in DMF (500 uL) was added HATU (34.9 mg, 0.092 mmol), Et3N (42.6 uL, 0.306 mmol) and N-(3-aminopropyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide hydrochloride (16.0 mg, 0.061 mmol). The mixture was stirred at room temperature for 1.5 h, acidified with TFA and purified by reverse phase 0-50% CH3CN/water containing 0.1% TFA. The fractions were lyophilized to yield Compound 24. Calculated mass: [M+2H]2+: C76H125N21O32, 1843.8, m/z=921.9; observed: 922.7.
Example 123
Synthesis of Compound 26
Scheme 35 as shown in FIG. 33A and FIG. 33B was used to prepare Compound 26.
To a degassed solution of 2′-3,17 propargyl siRNA (RNA 25, 33 mg, 4.49 umol) and PEG9 SPDP azide (26 mg, 36 umol, prepared from commercial PEG-azide and pyridyl disulfide reagents) in 3:1 DMA/water (1 mL) was added a degassed solution of Copper (I) Bromide-Dimethylsulfide Complex (1.8 mg, 9.0 umol). The mixture was stirred for 72 h at room temperature, diluted with water (2 mL), filtered using a 0.45 uM syringe filter and concentrated by dialysis. The concentrated mixture was purified on a XBridge Prep Phenyl column (5 uM, 19×250 mm) using a gradient of 0-50% CH3CN/water containing 100 mM TEAA. The fractions were concentrated via dialysis and lyophilized to yield Compound 26.
Examples 124-125
Synthesis of Compounds 27 and 28 (Exs. 124-125)
Scheme 36 as shown in FIG. 34A to FIG. 34C was used to prepare Compounds 27 and 28.
Synthesis of Compound 27 (Ex. 124)
To a solution of 2′-3,17 click PEG9 SPDP Conjugate 26 (13.2 mg, 1.50 μmol) in water (1 mL) was added a solution of TCEP hydrochloride (9.15 mg, 32.2 umol) dissolved in water (0.5 mL). The mixture was stirred at RT for 30 min then purified on a XBridge Prep Phenyl column (5 uM, 19×250 mm) using a gradient of 5-40% CH3CN/water containing 100 mM TEAA. The fractions were concentrated via dialysis and lyophilized to yield Compound 27.
Synthesis of Compound 28 (Ex. 125)
To a solution of 2′-3,17-click PEG9SH 27 (3 mg, 0.35 μmol) in pH 6.0 acetate buffer (100 uL) was added a solution of tetra GalNAc maleimide (5.1 mg, 2.77 μmol) dissolved in pH 6.0 acetate buffer (100 uL). The mixture was stirred at room temperature for 30 min then purified on a XBridge Prep Phenyl column (5 uM, 19×250 mm) using a gradient of 5-40% CH3CN/water containing 100 mM TEAA. The fractions were concentrated via dialysis and lyophilized to yield Compound 28.
Example 126
Synthesis of 2′-3,17 Bis TetraGalNAc-siRNA Duplex Conjugate 29
The procedure detailed for Conjugate 19 was used to duplex 28 to make Conjugate 29.
Example 127
Synthesis of TetraGalNAc Thiol Compound 31
Scheme 37 below was used to prepare Compound 31.




To a solution of tetraGalNAc acid Compound 10 (54 mg, 0.033 mmol) in N,N-dimethylacetamide (500 μl), was added cystamine dihydrochloride 30 (14.9 mg, 0.066 mmol), EDC (12.7 mg, 0.066 mmol), HOAT (10.2 mg, 0.066 mmol) and DIPEA (57.7 μl, 0.330 mmol). The mixture was stirred at room temperature for 18 h, then added a solution of DTT (50.9 mg, 0.330 mmol) in N,N-dimethylacetamide (100 μl). The mixture was stirred at room temperature for 0.5 h, acidified with TFA and purified by reverse phase 0-30% CH3CN/water containing 0.1% TFA. The fractions were lyophilized to yield Compound 31. Calculated mass: [M+2H]2+: C68H115N19O295, 1695.8, m/z=847.9; observed: 848.0.
Examples 128-130
Synthesis of Conjugates 35-37
Scheme 38 as shown in FIG. 35A and FIG. 35B was used to prepare Conjugates 35-37.
Synthesis of Compound 33
To a degassed solution of 2′-click 15 GS Compound 32 (130 mg, 0.019 mmol) and (9H-fluoren-9-yl)methyl (2-azidoethyl)carbamate (29.1 mg, 0.095 mmol) in 3:1 DMA/water (2 mL) was added a solution of Copper (I) bromide-dimethylsulfide Complex (9.72 mg, 0.042 mmol) dissolved in degassed DMSO (0.32 mL). The mixture was stirred at 45° C. for 2 h, cooled to room temperature, and added pH 8 EDTA (0.5 M, 2 mL) to quench reaction. Stirred for 15 min and purified on a XBridge Prep Phenyl column (5 uM, 30×150 mm) using a gradient of 0-45% CH3CN/water containing 100 mM TEAA. The fractions were concentrated via dialysis. To the combined material in water (3 mL) was added a solution of piperidine (936 μL, 1.891 mmol). The mixture was stored at 4° C. for 18 h, diluted with water (10 mL) and filtered off solids through syringe filter. Added pH 8 EDTA (0.5 M, 2 mL), concentrated via dialysis and lyophilized to yield Compound 33.
Synthesis of Compound 34
To a solution of 2′-15 click C2 NH2 GS Compound 33 (43.6 mg, 6.26 μmol) in 200 mM NaHCO3 soln (2000 μl) and formamide (1000 uL) was added a solution of N-Succinimidyl-3-[2-pyridyldithio]propionate (17.9 mg, 0.057 mmol) dissolved in DMSO (298 uL). The mixture was stirred at 10° C. for 15 min, diluted with water (10 mL) and Formamide (1 mL), and concentrated by dialysis. Added 2M TEAA (200 uL) and purified on a XBridge Prep Phenyl column (5 uM, 19×250 mm) using a gradient of 5-40% CH3CN/water containing 100 mM TEAA. The fractions were concentrated via dialysis and lyophilized to yield Compound 34.
Synthesis of 2′-15 TetraGalNAc-siRNA Conjugate 35 (Ex. 128)
To a solution of 2′-15 click C2 NH2 NHS SPDP GS Compound 34 (13 mg, 1.82 μmol) in 1:1 formamide/water (200 μl) was added a solution of tetraGalNAc SH (4.62 mg, 2.72 μmol) in formamide (200 uL). The mixture was stirred at room temperature for 3.5 h, added 2M TEAA (50 uL) and purified on a XBridge Prep Phenyl column (5 uM, 19×250 mm) using a gradient of 2-35% CH3CN/water containing 100 mM TEAA. The fractions were concentrated via dialysis and lyophilized. The resulting solid was purified on a Proteomix SAX-NP10 column (22.1×50 mm) using a gradient of 2-30% (Solvent A: 60:40 TFE/water with 40 mM Et3N, Solvent B: 60:40 TFE/water with 40 mM Et3N, 1M Guanidine HCl). The fractions were concentrated via dialysis and lyophilized to yield Conjugate 35.
Synthesis of Conjugates 36 and 37 (Ex. 129 and Ex. 130)
The procedure detailed for Conjugate 19-1 was used to duplex Conjugate 35 and the appropriate passenger strand to prepare Conjugates 36 and 37, respectively.
Examples 131-139
Synthesis of Conjugates 38-45 (Exs. 131-139)
Scheme 39 as shown in FIG. 36A to FIG. 36C, was used to prepare Conjugates 38-44.
Scheme 40. Examples of different linkers from Table 2 as shown in FIG. 37, used to conjugate tetraGalNAc to siRNA.
Step 1: Passenger-RNA and Linker, Example with Proline to Illustrate Protocol
To a solution of FMOC-PRO-OH (11.11 mg, 0.033 μmol) in 120 μL DMSO were added DIPEA (43.2 μl, 0.247 μmol) followed by HATU (10.96 mg, 0.029 μmol). The mixture, slightly yellow, was stirred at room temperature for 30 min. The mixture was then added to a solution of the oligonucleotide passenger strand TEAA salt (60 mg, 8.24 μmol) in 500 μL of (10% H2O/DMSO), and the mixture continued to stir at room temperature for one hour. The reaction mixture showed desired product via LC-MS. To the reaction mixture was added diethylamine (43.0 μl, 0.412 μmol) and the mixture was stirred for one hour, confirmed desired product via LC-MS. The reaction mixture was purified by centrifugal dialysis using 3 kDa cut-off membrane. The process was repeated three times with water (14 mL each time). The resulting solution was concentrated, frozen, and lyophilized overnight to yield product as a white fluffy solid. LC/MS confirms product [7384.9].
Step 2: TetraGalNAc-Linker-Passenger RNA
To a solution of TetraGalNAc Compound 10 (53.2 mg, 0.033 μmol) in 532 μL DMSO were added DIPEA (42.6 μl, 0.244 μmol) followed by HATU (12.36 mg, 0.033 μmol). The mixture, slightly yellow, was stirred at RT for 30 min. The mixture was then added to a solution of the linker-oligonucleotide passenger strand in 500 μL of DMSO, and the mixture continued to stir at room temperature for two hours. LC/MS showed desired product. The reaction mixture was subjected to centrifugal dialysis using 3 kDa cut-off membrane. The process was repeated three times with water (14 mL each time). The resulting solution was purified by Gilson PLC 2020 using XBRIDGE PHENYL, 10-27% CH3CN with 200 μM TEAA for 35 minutes. Collection solution was concentrated via centrifugal dialysis using 3 kDa cut-off membrane. The resulting concentrated solution was treated with 1.0N NaCl and centrifugal dialysis. The process was repeated five times with water (14 mL each time). The resulting concentrated solution ( ̃1.5 mL) was frozen and lyophilized overnight to yield product as a white fluffy solid. LC/MS confirms product [9002.5].
Step 3: Duplex Formation
To a TetraGalNAc-linker-RNA (18.5 mg, 2.055 μmol) in 1.5 mL of water was duplexed with ApoB guide strand (14.12 mg, 2.055 μmol) in 1.5 mL of water. The mixture was heated at 90° C. for 5 min with stir bar. The duplex was cooled and stir bar removed. The solution was lyophilized over two days to yield desired duplex Conjugate 38 as a white fluffy solid. LC/MS confirms product [16048].
ALL the remaining conjugates were prepared using the same general procedure.
Examples 140-142
Synthesis of Compounds/Conjugates 46-48
Scheme 41 as shown in FIG. 38A to FIG. 38E was used to prepare Compounds and/or Conjugates 46-48.
Synthesis of RNA Compound 46 (Ex. 140)
SPDP Acid (2.2 mg, 10.3 μmol) was dissolved DMSO 100 μL and N,N-diisopropylethylamine (14.0 μl, 0.08 mmol), HATU (19.6 mg, 0.051 mmol) were added sequentially. RNA (15 mg, 2.06 μmol) in 200 μL of DMSO:Water (9:1) was added and the resulting reaction mixture was stirred for 1 h, reaction was quenched by addition of 3 mL water and dialyzed down to 500 μL, diluted by formamide to 3 mL and purified by SAX (Buffer A: 60% TFE in water, 20 mM TEA, Buffer B: 60% TFE in water, 20 mM TEA, 1 M CsCl, gradient A/B from 100/0 to 35/65 over 15 min). The collected fractions were combined and dialyzed against water and lyophilized to afford Compound 46 as a white solid. Calculated mass: [M−H]−: C234H300F8N72O150P23S3, 7480.1; observed: 7483.0.
Synthesis of Conjugate 47 (Ex. 141)
RNA Compound 46 (22 mg, 2.9 μmol) and tetraGalNAc Thiol Compound 31 (10.0 mg, 5.9 μmol) were dissolved in formamide:pH=6.8 Tris buffer (3:1) 400 μL and stirred for 1 h. The reaction mixture was purified by SAX (Buffer A: 60% TFE in water, 20 mM TEA, Buffer B: 60% TFE in water, 20 mM TEA, 1 M CsCl, gradient A/B from 100/0 to 35/65 over 15 min). The collected fractions were combined and dialyzed against water and lyophilized to afford Conjuate 47 as a white solid. Calculated mass: [M−H]−: C297H410F8N90O179P23S3, 9063.9; observed: 9066.2.
Synthesis of Conjugate 48 (Ex. 142)
Conjugate 47 (10.9 mg, 1.20 μmol) and guide strand (7.81 mg, 1.14 μmol) were mixed in RNAse free water 1 mL for 2 h. The reaction mixture was lyophilized to afford duplex Conjugate 48 in quantitative yield.
Examples 143-145
Synthesis of Compounds/Conjugates 49-51
Scheme 42 as shown in FIG. 39A to FIG. 39C was used to prepare Compounds and/or Conjugates 49-51.
Synthesis of RNA Compound 49 (Ex. 143)
33.3 mg of siRNA passenger strand was weighed into a 4 mL vial then 1 mL 100 mM NaHCO3 was added to dissolve. Added 0.86 uL of propionic anhydride and let stir at RT. After aging  ̃2 h, spin dialyzed 3× against water. Filtered through frit and the solution was dried via lyophilization to afford RNA Compound 49.
Synthesis of Conjugate 50 (Ex. 144)
Step 1.
Charge 2.8 mg azide, 25.7 mg siRNA, 25 ml N2 sparged DMSO and 4 ml water to 40 mL vial. Sparge with N2. Charge 2.98 mL of Cu/ligand solution (N2 sparged, 20/100umol in 10 ml DMSO). Agitate at RT under sparged N2.
Step 2.
Charge Compound 10 and 1 ml DMSO. Charge 6 uL of DIPEA and agitate for 2 min. Charge 6 mg HBTU and agitate for 2 min. Charge siRNA mixture from Step 1. The reaction was not complete so repeated with half of previous reagent charge. Evaporated the reaction mixture, dialyzed and HPLC purified (X-Bridge Phenyl, TEAA/ACN gradient). Evaporated, dialyze and lyophilized to afford Conjugate 50.
Synthesis of Conjugate 51 (Ex. 145)
Dissolve GS (Conjugate 50) 10.65 mg in 1 ml water and dissolve PS (Conjugate 49) 10.20 mg in 1.17 ml water. Added 8.7 mg of Conjugate 49 to all of Conjugate 50 to form a 1:1 duplex. Heat to 90° C. for 1 min, cool to RT over 15 min. The solution was filtered and dried via lyophilization to afford Conjugate 51 as a white solid.
RNA Silencing Activity of Compounds Transfected with Lipofectamine in Luciferase Constructs
HEK293 cells stably transfected with luciferase vector that contains target sites for siRNA in 3′UTR of renilla luciferase were generated. These cells were seeded on 96-well tissue culture plates (Corning: #3903) at a density of 7.5e3 cells per well in DMEM 10% serum media. Cellular plates were then incubated at 37° C./5% CO2 for 24 hr. After incubation, plates were treated with test compounds co-transfected with transfection reagent Lipofectamine 2000 (invitrogen: #11668-019) in Opti-MEM (Gibco: #31985) in accordance to manufacturers protocol. The treatment concentrations ranged from 10 nM to 0.03 pM. Treated plates were then incubated for 24 hr at 37° C./5% CO2. Following treatment incubation, cells were lysed and processed in accordance to Dual-GloTM Luciferase Assay (Promega: E2920) and read on a TECAN safire2 plate reader.
RNA Silencing Activity of Compounds Transfected with Lipofectamine in HepG2 Cells
HepG2 cells (ATCC: HB-8065) were seeded on collagen coated plates (BioCoat: 356649) at a density of 7.5e3 cells per well in DMEM 10% serum media. Cellular plates were then incubated at 37° C./5% CO2 for 24 hr. After incubation, plates were treated with test compounds co-transfected with transfection reagent Lipofectamine 2000 (invitrogen: 11668-019) in Opti-MEM (Gibco: 31985) in accordance to invitrogen protocol. The treatment concentrations ranged from 10 nM to 0.03 pM. Treated plates were then incubated for 24 hr at 37° C./5% CO2. Following treatment incubation, cells were lysed with PLA Buffer (AB: 4448542) in accordance to supplied protocol. Resulting cell lysate was reverse transcribed to cDNA using High Capacity cDNA Kit (AB: 4368813) and run through qPCR using Life Technology 7900.
In vivo Evaluation of RNAi Activity
CD1 female mice were dosed by subcutaneous injection in 200 μl volume. Animals were observed for behavioral or physiological changes. Animals were sacrificed 72 hrs post dose by CO2 asphyxiation followed by ex-sanguination via cardiac puncture. The liver samples were as 3 mm punches from the medial lobe and put into RNA later tubes for isolation of total RNA. The mRNA knockdown analysis was conducted by Taqman analysis using standard procedures.
Scheme 43. General Description for Illustrative Purposes of Nomenclature Used in Table 6 as shown in FIG. 40. Exact siRNA sequences used in Table 6 can be found in Table 5.
A summary of in vitro and in vivo data of selected Compounds/Conjugates is shown in Table 6 and Table 7.







TABLE 6







In vitro and In Vivo Activity for Compounds Described in Section B-D.










RBC Hemolysis Data on 




Free Peptide














EC 50 
EC 50
% KD
% KD
% KD



pH7.4
pH5.5
2.5 mpk
5 mpk
2.5 mpk


Compound #
(uM)
(uM)
(SC admin)
(iv admin)
(iv admin)















B8-seq137-b
8.3
4.3


47


B8-seq470-b
8.5
3.8


57


B8-seq1678-b
>20
5


49


B8-seq 92-b
0.3
0.3


57


B8-seq1677-b
10
0.4


57


B8-seq-463-b
18
9.8


61


B8-seq1675-b
7
4.5


47


B11-seq1-b
5.3
0.7


49


B11-seq2-b
>10
1.2


32


B11-seq3-b
>10
0.5


49


B11-seq4-b
4.3
0.2


55


B11-seq5-b
5
0.5

74



B11-seq6-b
>10
1

53



B11-seq7-b
>10
0.7

45



B11-seq8-b




22


B11-seq9-b
8.9
1.7


28


B11-seq10-b
6
1.8


35


B11-seq11-b
0.39
0.04


21


B11-seq12-b
2
0.2


45


B11-seq13-b
1.9
0.2
5

64


B11-seq14-b
2.27
1.61


26


B11-seq15-b
>10
0.4


28


B11-seq16-b
2.8
0.6


26


B11-seq17-b
4.4
0.7


34


B11-seq18-b
1
0.4

61



B11-seq19-b
>10
0.7

64



B11-seq20-b
3.7
2.05

63



B11-seq21-b
2.2
0.4

56



B11-seq22-b
6
0.5

33



B11-seq23-b
7.3
6.1

59



B11-seq24-b
>10
0.2

58



B11-seq25-b
>10
3.6

52



B11-seq26-b
4.6
1.4
38
65
57


B11-seq27-b
>10
0.4

61



B11-seq28-b
0.7
0.1


25


B11-seq29-b
>10
2


20


B11-seq30-b
>10
1.5


29


B11-seq31-b
1.5
0.3

64



B11-seq32-b
4.5
1.4

58



B11-seq33-b
0.02
0.04


35


B11-seq34-b
0.12
0.05


30


B11-seq35-b
0.03
0.03


37


B11-seq36-b
7.5
2.5


53


B11-seq37-b
6
2


22


B11-seq38-b
0.95
0.44

61



B11-seq39-b
1
0.6

58



B11-seq40-b
0.2
0.2

63



B11-seq41-b
>10
0.7
36

27


B11-seq42-b
1.3
1.9
41

57


B11-seq43-b
0.9
0.3


55


B11-seq44-b
2.1
1.4
33

56


B11-seq45-b
>10
0.07
51

53


B11-seq46-b
1.1
0.04
56

46


B11-seq47-b
>10
0.4
49

51


B11-seq48-b
3.1
1.5
47

61


B11-seq49-b
4
0.6
37

49


B11-seq50-b
>10
1.9
10

43


B11-seq51-b


11

48


B11-seq52-b
>10
6.4
14

59


B11-seq53-b
1.17
0.37


45


B11-seq54-b
0.89
0.38


49


B11-seq55-b
0.51
0.18
−7

47


B11-seq56-b
1.46
0.19
12

48


B11-seq57-b
3.5
0.59
−11




B11-seq58-b
14.47
0.31
18




B11-seq59-b
>20
0.65
7

52


B11-seq60-b
19.57
0.38


39


B11-seq61-b
1.39
0.65


55


B11-seq62-b
>20
5.86


52


B11-seq63-b
0.94
0.64


37


B11-seq64-b
>20
1.8


41


B11-seq65-b
1.38
1.87


28


B11-seq66-b
>20
0.82


54


B11-seq67-b
>20
0.87


39


B11-seq68-b
>20
5.05


56


B11-seq69-b
>20
0.91


34


B11-seq70-b
3.68
1.86


32


B11-seq71-b
>20
3.56


44


B11-seq72-b
10.63
2.54


39


B11-seq73-b
>20
4.2


38


B11-seq74-b
12.68
4.34


60


B11-seq75-b
>10
0.9


55


B11-seq76-b
6.4
1.7
3

53


B11-seq77-b
0.17
0.23


38


B11-seq78-b
0.2
0.33


47


B11-seq79-b
1.52
1.86


47


B11-seq80-b
>20
6.24


56


B11-seq81-b
>20
3.91


51


B11-seq82-b
17
1.79


40


B11-seq83-b
>20
6.19


35


B11-seq84-b
0.7
0.15


44


B11-seq85-b
>10
0.1


45


B11-seq86-b
>20
17.81


27


B11-seq87-b
>10
0.02


30


B11-seq88-b
2.35
0.07


56


B11-seq89-b
3.29
0.14


51


B11-seq90-b
>10
0.5


42


B11-seq91-b




26


B11-seq92-b




59


B11-seq93-b
>20
5.88


51


B11-seq94-b
5.2
1.61


46


B11-seq95-b
3.59
3.1


43


B11-seq96-b
16.08
4.9


55


B11-seq97-b
>20
5.56


52


B11-seq98-b
>20
3.37


40


B11-seq99-b
12.9
5.61


43


B11-seq100-b
10.24
3.45


43


B11-seq101-b
>20
4.85


46


B11-seq102-b
>20
4.87


54


B11-seq103-b
>20
3.86


43


B11-seq104-b
6.72
3.26


56


B11-seq105-b
>10
>10


30


B11-seq106-b
8.4
0.24


34


B11-seq107-b
10.41
3.52


41


B11-seq108-b
5.6
2.69


40


B11-seq109-b
>20
5.78


36


B11-seq110-b
>20
3.36


43


B11-seq111-b
>20
0.26


36


B11-seq371-b
>20
2.8


45


B11-seq-1675-b
14.2
3.5


53


B13-seq 1676-b
14.2
3.5


53


B8-seq32-c
4.5
1.4





C6-seq-31c
1.5
0.3

31



C6-seq32-c
4.5
1.4

36



C6-seq106-c
7
0.7

30



C12-seq32-c
4.5
1.4

68



C15-seq32-c
4.5
1.4

39



D7-seq32-d
4.5
1.4


52


E10-seq 137-b
>20
3.3





F6-seq26-f
>20
>20


47


F6-seq32-f
4.5
1.4


47


F6-seq463-f
18
9.8


60


F6-seq491-f
>20
3.3


72


F6-seq492-f
>20
6.3


66


F6-seq-612-f
19
6


59


F6-seq1693-f
17.1
0.6


38


F6-seq1694-f
15.6
4.4


43


G5-seq463-g
18
9.8


47


G5-seq489-g
>20
>20


48


H7-seq8-h
20
1.3
13

25


H7-seq26-h
4.6
1.4


35


H7-seq32-h
4.5
1.4
20

30


H7-seq37-h
6
2


39


H10-seq26-h
4.6
1.4


20


H10-seq32-h
4.5
1.4


33


I10-seq-1680-f
>20
1.6


67


I10-seq-1681-f
>20
1.4


66


I10-seq-1682-f
>20
1.6


66


K6-seq37-h
6
2


55


K6-seq-74-h
12.7
4.3


48


K6-seq463-h
18
9.8


55


L11-seq 463j
18
9.8


52


M4-seq463-j
18
9.8


52


N4-seq106-k
7
0.7

69



N4-seq197-k
>20
>20

63



N4-seq283-k
>20
>20

64



O3-seq-463-k
18
9.8
35

70


P2-seq32-k
4.5
1.4


61


P2-seq32-m
4.5
1.4


64


Q3-seq32-b
4.5
1.4


45


Q3-seq74-b
12.7
4.3


43


Q3-seq 1675-b
14.2
3.5


70


R4-seq1690-l
1.9
0.6


79


R4-seq1691-l
1.6
0.5


55


R4-seq1692-l
>20
>20


72


R4-seq1695-l
14.2
0.3


79


R4-seq1696-l
>20
>20


36
















TABLE 7







In vitro and In Vivo Activity for Compounds Generated in Section


E. (Starting siRNA sequence information can be found in Table 8).















Starting
Dose

IC50





siRNA
(mpk)
In
w/LF2K
ASGR




se-
Route of
vivo %
in HEK-
binding


En-
Com-
quence
Administra-
KD
Luc
IC50


try
pound
code
tion
(72 h)
[pM]
nM
















1
10a-1
51
5, 15 SC
33.6;
15.44
36.7






69.5




2
10b-1
54
SC 5, 15; IV
42, 49, 13
19.64
18.1





15





3
10-2
56
5, 50 SC
40, 56
23.4







(24 h)




4
10-3
57
1, 2.5, 5 SC
20, 45, 60
52








(HepG2)



5
17a-1
51
5 SC; 15 IV
11, 5
20.16
49.1


6
17b-1
54
5 SC; 15 IV
12, 22
43.96
33.3


7
19-1
52
5; 15 SC
32; 68
24.04
3.6


8
29
53
15 SC; 15 IV
43, 0
17.83
22


9
36
58
1, 2.5, 5 SC
16, 43, 56




10
37
58
1, 2.5, 5 SC
16, 32, 40




11
38
51
5 SC, 15 IV
36, 33
71
17


12
39
51
5 SC, 15 IV
19, 31
46.8
44


13
40
51
5, 15 SC
33, 62
76.8
77


14
41
51
5, 15 SC
28, 74
98.6
134


15
42
51
5, 15 SC
19, 73
309.7
135


16
43
51
5, 15 SC
 8, 73
64.8
45


17
44
51
5, 15 SC
31, 73
67.1
66


18
45
51
5 SC, 15 IV
20, 4
73.4
11


19
48a-1
51
5, 15 SC
10.24;
23.43







59.93




20
48b-1
53
5, 15 SC
19.87;
57.96







42.08




21
51
55
5; 15
40; 45
1838.47
94.8
















TABLE 8







Starting siRNA sequence information used to prepare conjugates from Table 7.












Entry
Gene



SEQ ID


Target
Code
Strand
Sequence
Duplex
NO.:















1
ApoB
Passenger
[6amiL][iB][omeC][omeU][omeU][omeU][fluA][fluA][om
51
1721





eC][fluA][fluA][omeU][omeU][omeC][omeC][omeU][fluG]







[fluA][fluA][fluA][omeU][dTs]dT[iB]





ApoB
Guide
[rAs][rUs][rUs][omeU][omeC][fluA][fluG][fluG][fluA][flu

1722





A][omeU][omeU][fluG][fluU][omeU][fluA][fluA][fluA][fl







uG][omeUs][omeU]




2
ApoB
Passenger
[6amiL][iB][omeC][omeU][omeU][omeU][fluA][fluA][om
52
1723





eC][fluA][fluA][omeU][omeU][omeC][omeC][omeU][fluG]







[fluA][fluA][fluA][omeU][dTs]dT[iB][6amiL]





ApoB
Guide
[rAs][rUs][rUs][omeU][omeC][fluA][fluG][fluG][fluA][flu

1724





A][omeU][omeU][fluG][fluU][omeU][fluA][fluA][fluA][fl







uG][omeUs][omeU]




3
ApoB
Passenger
[6amiL][iB][omeC][omeU][clickU][omeU][fluA][fluA][om
53
1725





eC][fluA][fluA][omeU][omeU][omeC][omeC][omeU][fluG







][fluA][clickA][fluA][omeU][dTs]dT[iB][C6SH]





ApoB
Guide
[rAs][rUs][rUs][omeU][omeC][fluA][fluG][fluG][fluA][flu

1726





A][omeU][omeU][fluG][fluU][omeU][fluA][fluA][fluA][fl







uG][omeUs][omeU]




4
ApoB
Passenger
[iB][omeC][omeU][omeU][omeU][fluA][fluA][omeC][flu
54
1727





A][fluA][omeU][omeU][omeC][omeC][omeU][fluG][fluA]







[fluA][fluA][omeU][dTs]dT[iB][6amiL]





ApoB
Guide
[rAs][rUs][rUs][omeU][omeC][fluA][fluG][fluG][fluA][flu

1728





A][omeU][omeU][fluG][fluU][omeU][fluA][fluA][fluA][fl







uG][omeUs][omeU]




5
ApoB
Passenger
[6amiL][iB][omeC][omeU][omeU][omeU][fluA][fluA][om
55
1729





eC][fluA][fluA][omeU][omeU][omeC][omeC][omeU][fluG]







[fluA][fluA][fluA][omeU][dTs]dT[iB]





ApoB
Guide
[rAs][rUs][rUs][omeU][omeC][fluA][fluG][fluG][fluA][flu

1730





A][omeU][omeU][fluG][fluU][clickU][fluA][fluA][fluA][fl







uG][omeUs][omeU]




6
SSB
Passenger
[6amiL][iB][fluA][omeC][fluA][fluA][omeC][fluA][fluG]
56
1731





[fluA][omeC][omeU][omeU][omeU][fluA][fluA][omeU][flu







G][omeU][fluA][fluA][dTs]dT[iB]





SSB
Guide
[rUs][rUs][rAs][omeC][fluA][omeU][omeU][fluA][fluA][fl

1732





uA][fluG][omeU][omeC][fluU][fluG][omeU][omeU][fluG]







[omeU][omeUs][omeU]




7
CTNNB1
Passenger
[6amiL][iB][omeC][omeU][clickG][omeU][omeU][fluG][fl
57
1733





uG][fluA][omeU][omeU][fluG][fluA][omeU][omeU][ome







C][fluG][clickA][fluA][fluA][omeUs][omeU][iB][C3SH]





CTNNB1
Guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU][

1734





omeC][fluA][omeA][fluU][omeC][fluC][omeA][fluA][ome







C][fluA][omeG][omeUs][omeU]




8
CTNNB1
Passenger
[6amiL][iB][omeC][omeU][clickG][omeU][omeU][fluG][fl
58
1735





uG][fluA][omeU][omeU][fluG][fluA][omeU][omeU][ome







C][fluG][clickA][fluA][fluA][omeUs][omeU][iB][C3SH]





CTNNB1
Guide
[omeUs][fluUs][omeUs][fluC][omeG][fluA][omeA][fluU]

1736





[omeC][fluA][omeA][fluU][omeC][fluC][clickA][fluA][om







eC][fluA][omeG][omeUs][omeU]





As used herein, ome = 2′ methoxy; flu = 2′ fluoro; click = 2′ propagyl; iB = inverted abasic; “s” subscript = phosphorothioate; and r = 2′ ribo; 6amil = n-hexylamino; C3SH = n-propylthiol; and C6SH = n-hexylthiol.






One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein, as presently representative of preferred embodiments, are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

## Claims

1. A method for preparing a tetraGalNAc compound having a formula:



wherein:
X is —O—, —S—, or —CH2—; and
n, m, and q are each 1, 2, 3, or 4,


the method comprising:
reacting an azide source having the formula of





with a compound of formula




or a compound of formula




to obtain an intermediate compound;
reacting the intermediate compound with a Lys-alkyne compound of formula





to form an acetate-protected tetraGalNAc compound; and
removing the acetate protecting groups on the acetate-protected tetraGalNAc compound, thereby forming the tetraGalNAc compound.

2. The method of claim 1, wherein the tetraGalNAc compound has the structure of

3. The method of claim 2, wherein m is 2 and q is 1.

4. A method for preparing a tetraGalNAc compound of formula (I):



wherein:
X is —O—, —S—, or —CH2—; and
n is 1, 2, 3, or 4,


the method comprising:
reacting an azide source having the formula of





with a compound of formula




or a compound of formula




to obtain an intermediate compound;
reacting the intermediate compound with a Lys-alkyne compound of formula





to form an acetate-protected tetraGalNAc compound; and
removing the acetate protecting groups on the acetate-protected tetraGalNAc compound, thereby forming the tetraGalNAc compound.

5. The method of claim 4, wherein the tetraGalNAc compound has the structure of




wherein the Lys-alkyne compound has the formula of

6. The method of claim 5, wherein X is —O— or —CH2—; and n is 1, 2, or 3.

7. The method of claim 1, wherein the reaction to obtain the intermediate compound is carried out by reacting the azide source with a compound of formula




in the presence of trifluoromethanesulfonic acid.

8. The method of claim 1, wherein the reaction to obtain the intermediate compound is carried out by reacting the azide source with a compound of formula




in the presence of trimethylsilyl trifluoromethanesulfonate.

9. The method of claim 1, wherein the reaction of the intermediate compound with the Lys-alkyne compound is carried out in the presence of CuBrSMe2.

10. The method of claim 9, wherein the reaction of the intermediate compound with the Lys-alkyne compound is carried out in the presence of CuBrSMe2 and diazabicycloundecene (DBU).

11. The method of claim 1, wherein about 4 to 6 moles of the intermediate compound are added to react with each mole of the Lys-alkyne compound.

12. The method of claim 11, wherein about 5 to 6 moles of the intermediate compound are added to react with each mole of the Lys-alkyne compound.

13. The method of claim 1, wherein the removing the acetate protecting groups on the acetate-protected tetraGalNAc compound is carried out by reacting the acetate-protected tetraGalNAc compound with sodium methoxide.

14. The method of claim 4, wherein the tetraGalNAc compound has the structure of




wherein the azide source is




and the Lys-alkyne compound has the formula of

15. The method of claim 14, wherein the tetraGalNAc compound is prepared by:
reacting





with a compound of formula




in the presence of trimethylsilyl trifluoromethanesulfonate, to obtain an intermediate compound;
reacting the intermediate compound with





in the presence of CuBrSMe2, to form an acetate-protected tetraGalNAc compound; and
reacting the acetate-protected tetraGalNAc compound with sodium methoxide to remove the acetate protecting groups on the acetate-protected tetraGalNAc compound, thereby forming the tetraGalNAc compound.

16. The method of claim 4, wherein the tetraGalNAc compound has the structure of




wherein the azide source is




and the Lys-alkyne compound has the formula of

17. The method of claim 14, wherein the tetraGalNAc compound is prepared by:
reacting





with a compound of formula




in the presence of trifluoromethanesulfonic acid, to obtain an intermediate compound of formula;
reacting the intermediate compound with





in the presence of CuBrSMe2, to form a acetate-protected tetraGalNAc compound; and
reacting the acetate-protected tetraGalNAc compound with sodium methoxide to remove the acetate protecting groups on the acetate-protected tetraGalNAc compound, thereby forming the tetraGalNAc compound.

18. The method of claim 4, wherein the tetraGalNAc compound has the structure of




wherein the azide source is




and the Lys-alkyne compound has the formula of

19. The method of claim 18, wherein the tetraGalNAc compound is prepared by:
reacting





with a compound of formula




in the presence of trifluoromethanesulfonic acid, to obtain an intermediate compound;
reacting the intermediate compound with





in the presence of CuBrSMe2, to form an acetate-protected tetraGalNAc compound; and
reacting the acetate-protected tetraGalNAc compound with sodium methoxide to remove the acetate protecting groups on the acetate-protected tetraGalNAc compound, thereby forming the tetraGalNAc compound.

20. The method of claim 4, wherein the tetraGalNAc compound has the structure of



wherein the azide source is





and the Lys-alkyne compound has the formula of

21. The method of claim 20, wherein the tetraGalNAc compound is prepared by:
reacting





with a compound of formula




in the presence of trifluoromethanesulfonic acid, to obtain an intermediate compound;
reacting the intermediate compound with





in the presence of CuBrSMe2, to form an acetate-protected tetraGalNAc compound; and
reacting the acetate-protected tetraGalNAc compound with sodium methoxide to remove the acetate protecting groups on the acetate-protected tetraGalNAc compound, thereby forming the tetraGalNAc compound.