Convergent peptide synthesis methods

Abstract

Convergent pathways for the preparation of a peptide by solid phase peptide synthesis or liquid phase peptide synthesis, as well as peptide fragments suitable for use in such pathways, are provided. The convergent pathways may be utilized for the synthesis of agonists of the glucagon-like peptide 1 receptor (GLP-1R) such as the peptide component of maridebart cafraglutide.

Description

10696-WC01-SECCONVERGENT PEPTIDE SYNTHESIS METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 739,787, filed December 30, 2024, and U.S. Provisional Patent Application No. 63 / 906,108, filed October 27, 2025.SUBMISSION OF SEQUENCE LISTING

[0002] The content of the following Sequence Listing XML is incorporated herein by reference in its entirety: file name: 10696-WC01-SEC_Seqlisting, date created: December 22, 2025; size: 772,232 bytes.FIELD

[0003] The present disclosure describes convergent pathways for the preparation of peptides on solid supports or in solution, as well as peptide intermediate fragments useful in such convergent synthesis pathways.BACKGROUND

[0004] Therapeutic peptides are commonly synthesized by either solid-phase peptide synthesis (SPPS) or liquid-phase peptide synthesis (LPPS) methods, both of which rely on amide-bond forming reactions to assemble peptides (Merrifield, J. Am. Chem. Soc., 1963; El-Faham & Albericio, Chem. Rev., 2011; Behrendt et al., J. Pept. Sci., 2016). SPPS immobilizes the growing peptide chain on a solid support, facilitating stepwise addition of amino acids and simplified purification, while LPPS methods proceed without such support and typically require intermediate purification steps between coupling reactions (El-Faham & Albericio, Chem. Rev., 2011). Due to its scalability, flexibility, and compatibility with automation techniques, SPPS is the most commonly used peptide synthesis approach at commercial scale, especially for short to medium length peptides. However, LPPS and hybrid LPPS / SPPS strategies are increasingly being explored for the production of large and complex peptides.

[0005] One intrinsic drawback of traditional stepwise peptide synthesis methods— whether performed on solid support or in solution— is the cumulative loss of yield and purity as peptides become longer and more complex (El-Faham & Albericio, Chem. Rev., 2011). Issues such as incomplete coupling, side reactions (e.g., racemization or aspartimide formation), aggregation, and poor solubility make purification increasingly difficult as peptide length increases, potentially reducing overall yield and purity (Behrendt et al., J. Pept. Sci., 2016). Convergent assembly, which involves preparing shorter peptide fragments and subsequently coupling these fragments to produce a longer peptide, offers a potential solution to these challenges for some peptides (Raibaut et al., Chem. Soc. Rev., 2012; Agouridas et al., Chem. Rev., 2019). By minimizing the number of reactions per fragment and isolating problematic regions into shorter amino acid sequences, convergent synthesis approaches can improve yield, reduce side reactions, and enhance synthetic efficiency (Kent, Chem. Soc. Rev., 2009; Agouridas et al., Chem. Rev., 2019).10696-W001-SEC

[0006] The utility of convergent peptide synthesis depends on how the desired peptide is divided into shorter sequences. Optimal fragment boundaries vary based on factors such as solubility, aggregation tendency, reactivity at coupling junctions, and susceptibility to side reactions like epimerization, making reliable in silico predictions challenging (Behrendt et al., J. Pept. Sci., 2016; Raibaut et al., Chem. Soc. Rev., 2012). Additionally, coupling efficiency is sensitive to the local amino acid composition near the coupling site, often requiring customized strategies— such as modified reaction conditions or specific protecting groups— tailored to each individual sequence (Agouridas et al., Chem. Rev., 2019; Malins & Payne, Curr. Opin. Chem. Biol., 2014; Haase et al., Angew. Chem. Int. Ed., 2008; Chen et al., Angew. Chem. Int. Ed., 2008).

[0007] Thus, a need exists in the art for new methods of preparing peptides (e.g., glucagon-like peptide 1 (GLP-1 ) receptor agonist peptides) using convergent approaches, including solid phase and liquid phase convergent peptide synthesis strategies for preparing the peptide component of maridebart cafraglutide, as well as peptide intermediate fragments useful in such approaches.SUMMARY

[0008] Solid phase and liquid phase convergent peptide synthesis processes for the preparation of the peptide component of maridebart cafraglutide (also called MariTide or AMG 133), an agonist of the glucagon-like peptide 1 receptor (GLP-1 R) and an antagonist of the glucose-dependent insulinotropic polypeptide receptor (GIPR) that is currently under investigation for the treatment of obesity and related conditions (Jastreboff, N Engl J Med, 2025), as well as peptide fragments thereof, are generally described herein. The convergent synthesis processes described herein offer several advantages over non-convergent processes for preparing a peptide component of maridebart cafraglutide, such as higher yield, reduced processing time, reduced solvent and / or reagent usage, and the ability to run reactions without the need for specialized equipment.

[0009] A peptide component of maridebart cafraglutide comprises the amino acid sequence of SEQ ID NO: 7, which is alternatively referred to as "Peptide A” herein.

[0010] One aspect of the present disclosure provides peptide fragments useful in the convergent preparation of Peptide A. In some embodiments, the present disclosure provides a peptide having a sequence of His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ser (SEQ ID NO: 1) ("Fragment 1”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ser-Tyr (SEQ ID NO: 11) ("Fragment 1 A”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of Ty r-Leu-Glu-Glu-GIn-Ala-Ala-Lys-GI u-Phe-l le-Ala (SEQ ID NO: 2) ("Fragment 2”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of Leu-Glu-Glu-GIn-Ala-Ala-Lys-Glu-Phe-lle (SEQ ID NO: 12) ("Fragment 2A”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of Tyr-Leu-Glu-Glu-GIn-Ala-Ala-Lys-Glu-Phe-lle (SEQ ID NO: 16) ("Fragment 2B") or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of Leu-Glu-Glu-GIn-Ala-Ala-Lys-Glu-Phe-lle-Ala (SEQ ID NO: 17) ("Fragment 2C”) or a protected variant thereof. In some10696-WQ01-SECembodiments, the present disclosure provides a peptide having a sequence of Trp-Leu-Val-Lys-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 3) ("Fragment 3”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 13) ("Fragment 3A”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Lys (SEQ ID NO: 4) ("Fragment 4”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly (SEQ ID NO: 5) ("Fragment 5”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Lys (SEQ ID NO: 6) ("Fragment 6”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Lys (SEQ ID NO: 9) ("Peptide B”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of Leu-Glu-Glu-GIn-Ala-Ala-Lys-Glu-Phe-lle-Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Lys (SEQ ID NO: 10) ("Peptide C”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ser-Tyr-Leu-Glu-Glu-GIn-Ala-Ala-Lys-Glu-Phe-lle (SEQ ID NO: 15) ("Peptide D”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of Trp-Leu-Val-Lys-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Lys (SEQ ID NO: 8) ("Peptide E”) or a protected variant thereof. In some embodiments, the present disclosure provides a peptide having a sequence of Tyr-Leu-Glu-Glu-GIn-Ala-Ala-Lys-Glu-Phe-lle-Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Lys (SEQ ID NO: 14) ("Peptide F”) or a protected variant thereof. In some embodiments, a protected variant described herein has a protected (such as, e.g., an Fmoc-, Boc-, or Trt-protected) N-terminus. In some embodiments, a protected variant of Fragment 4, Fragment 6, Peptide B, Peptide C, Peptide E, or Peptide F described herein comprises a C-terminal lysine residue with a side chain protecting group that can be selectively removed (i.e., is orthogonal to any other side chain or termini protecting group(s), such as, e.g., a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (IvDde), allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), or chlorobenzyloxycarbonyl (2-Cl-Z) protecting group) to enable selective modification of the s-amino group of the C-terminal Lys residue.

[0011] In certain embodiments, "having a sequence” may be replaced by "consisting of a sequence.” In addition, in some embodiments, when a peptide or protected variant thereof is described as "comprising a sequence” (or its cognates), the peptide or protected variant thereof can alternatively be described as "having a sequence” (or its cognates). Additionally, in some embodiments, any of the peptides and protected variants described herein may be isolated peptides or protected variants, respectively. In some embodiments, a peptide or protected variant described herein (e.g., an isolated or protected variant described herein) is in the form of a salt. In some embodiments, a peptide or protected variant described herein (e.g., an isolated or protected variant described herein) is in the form of an acid-addition salt (e.g., an HCI, acetate, or TFA salt). In some10696-WQ01-SECembodiments, a peptide or protected variant described herein (e.g., an isolated or protected variant described herein) is in the form of a carboxylate salt (e.g., a sodium or triethylammonium salt).

[0012] Another aspect of the present disclosure provides a method of preparing Peptide B or a protected variant thereof, comprising coupling a protected variant of Fragment 5 with a protected variant of Fragment 6 to form a protected variant of Peptide B, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9, Fragment 5 comprises the amino acid sequence of SEQ ID NO: 5, and Fragment 6 comprises the amino acid sequence of SEQ ID NO: 6.

[0013] Still another aspect of the present disclosure provides a method of preparing Peptide B or a protected variant thereof, comprising coupling a protected variant of Fragment 3A with a protected variant of Fragment 4 to form a protected variant of Peptide B, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9, Fragment 3A comprises the amino acid sequence of SEQ ID NO: 13, and Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4.

[0014] Another aspect of the present disclosure provides a method of preparing Peptide E or a protected variant thereof, comprising coupling a protected variant of Fragment 3 with a protected variant of Fragment 4 to form a protected variant of Peptide E, wherein Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of SEQ ID NO: 8.

[0015] Still another aspect of the present disclosure provides a method of preparing Peptide B or a protected variant thereof, comprising coupling a protected variant of Peptide E with an N-protected alanine to form a protected variant of Peptide B, wherein Peptide E comprises the amino acid sequence of SEQ ID NO: 8 and Peptide B comprises the amino acid sequence of SEQ ID NO: 9.

[0016] Yet another aspect of the present disclosure provides a method of preparing Peptide B or a protected variant thereof, comprising:coupling or having coupled a protected variant of Fragment 3 with a protected variant of Fragment 4 to form a first protected variant of Peptide E, wherein the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Fragment 4 has a free amino group at the N-terminus, Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of SEQ ID NO: 8;selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide E to form a second protected variant of Peptide E with a free amino group at the N-terminus; and coupling or having coupled the second protected variant of Peptide E with an N-protected alanine to form a protected variant of Peptide B, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9.10696-WQ01-SEC

[0017] Another aspect of the present disclosure provides a method of preparing Peptide D or a protected variant thereof, comprising coupling a protected variant of Fragment 1 with a protected variant of Fragment 2B in to form a protected variant of Peptide D, wherein Peptide D comprises the amino acid sequence of SEQ ID NO: 15, Fragment 1 comprises the amino acid sequence of SEQ ID NO: 1, and Fragment 2B comprises the amino acid sequence of SEQ ID NO: 16.

[0018] Still another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising coupling a protected variant of Peptide B with a protected variant of Peptide D to form a protected variant of Peptide A, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9, Peptide D comprises the amino acid sequence of SEQ ID NO: 15, and Peptide A comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the method further comprises deprotecting the protected variant of Peptide A to isolate Peptide A. In some embodiments, the method further comprises adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0019] Yet another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising:(A)(i) (a) coupling or having coupled a protected variant of Fragment 3 with a protected variant of Fragment 4 to form a first protected variant of Peptide E, wherein the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Fragment 4 has a free amino group at the N-terminus, Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of SEQ ID NO: 8; (b) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide E from step (A)(i)(a) to form a second protected variant of Peptide E with a free amino group at the N-terminus; (c) coupling or having coupled the second protected variant of Peptide E from step (A)(i)(b) with an N-protected alanine to form a first protected variant of Peptide B, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9; and (d) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide B from step (A)(i)(c) to form a second protected variant of Peptide B with a free amino group at the N-terminus; OR(ii) (a) coupling or having coupled a protected variant of Fragment 3A with a protected variant of Fragment 4 to form a first protected variant of Peptide B, wherein the protected variant of Fragment 3A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Fragment 4 has a free amino group at the N-terminus, Fragment 3A comprises the amino acid sequence of SEQ ID NO: 13, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide B comprises the amino acid sequence of SEQ ID NO: 9; and (b) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide B from step (A)(ii)(a) to form a second protected variant of Peptide B with a free amino group at the N-terminus; OR10696-WQ01-SEC(iii) (a) coupling or having coupled a protected variant of Fragment 5 with a protected variant of Fragment 6 to form a first protected variant of Peptide B, wherein the protected variant of Fragment 5 has a free carboxyl group at the C-terminus and a protected (preferably Trt- or Fmoc-protected, more preferably Trt-protected) N-terminus, the protected variant of Fragment 6 has a free amino group at the N-terminus, Fragment 5 comprises the amino acid sequence of SEQ ID NO: 5, Fragment 6 comprises the amino acid sequence of SEQ ID NO: 6, and Peptide B comprises the amino acid sequence of SEQ ID NO: 9; and (b) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide B from step (A)(iii)(a) to form a second protected variant of Peptide B with a free amino group at the N-terminus;(B) coupling or having coupled a protected variant of Fragment 1 with a protected variant of Fragment 2B to form a protected variant of Peptide D, wherein the protected variant of Fragment 1 has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus, the protected variant of Fragment 2B has a free amino group at the N-terminus, Fragment 1 comprises the amino acid sequence of SEQ ID NO: 1, Fragment 2B comprises the amino acid sequence of SEQ ID NO: 16, and Peptide D comprises the amino acid sequence of SEQ ID NO: 15; and(C) coupling or having coupled the second protected variant of Peptide B from step (A) with the protected variant of Peptide D from step (B) to form a protected variant of Peptide A, wherein Peptide A comprises the amino acid sequence of SEQ ID NO: 7.

[0020] In some embodiments, the method further comprises deprotecting or having deprotected the protected variant of Peptide A to isolate Peptide A.

[0021] In some embodiments, the method further comprises adding or having added a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0022] Another aspect of the present disclosure provides a method of preparing Peptide C or a protected variant thereof, comprising coupling a protected variant of Fragment 2A with a protected variant of Peptide B to form a protected variant of Peptide C, wherein Fragment 2A comprises the amino acid sequence of SEQ ID NO: 12, Peptide B comprises the amino acid sequence of SEQ ID NO: 9, and Peptide C comprises the amino acid sequence of SEQ ID NO: 10.

[0023] Still another aspect of the present disclosure provides a method of preparing Peptide C or a protected variant thereof, comprising coupling a protected variant of Fragment 2C with a protected variant of Peptide E to form a protected variant of Peptide C, wherein Fragment 2C comprises the amino acid sequence of SEQ ID NO: 17, Peptide E comprises the amino acid sequence of SEQ ID NO: 8, and Peptide C comprises the amino acid sequence of SEQ ID NO: 10.

[0024] Yet another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising coupling a protected variant of Fragment 1 A with a protected variant of Peptide C to form a protected variant of Peptide A, wherein Fragment 1 A comprises the amino acid sequence of SEQ ID10696-WQ01-SECNO: 11, Peptide C comprises the amino acid sequence of SEQ ID NO: 10, and Peptide A comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the method further comprises deprotecting the protected variant of Peptide A to isolate Peptide A. In some embodiments, the method further comprises adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0025] Another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising:coupling or having coupled a protected variant of Fragment 2A with a protected variant of Peptide B to form a first protected variant of Peptide C, wherein the protected variant of Fragment 2A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Peptide B has a free amino group at the N-terminus, Fragment 2A comprises the amino acid sequence of SEQ ID NO: 12, Peptide B comprises the amino acid sequence of SEQ ID NO: 9, and Peptide C comprises the amino acid sequence of SEQ ID NO: 10;selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide C to form a second protected variant of Peptide C with a free amino group at the N-terminus; and coupling or having coupled a protected variant of Fragment 1 A with the second protected variant of Peptide C to form a protected variant of Peptide A, wherein the protected variant of Fragment 1 A has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus, Fragment 1 A comprises the amino acid sequence of SEQ ID NO: 11 , and Peptide A comprises the amino acid sequence of SEQ ID NO: 7.

[0026] In some embodiments, the method further comprises deprotecting or having deprotected the protected variant of Peptide A to isolate Peptide A.

[0027] In some embodiments, the method further comprises adding or having added a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0028] Still another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising:(A)(I) (a) coupling or having coupled a protected variant of Fragment 3 with a protected variant of Fragment 4 to form a first protected variant of Peptide E, wherein the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Fragment 4 has a free amino group at the N-terminus, Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of SEQ ID NO: 8; (b) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide E from step (A)(1)(a) to form a second protected variant of Peptide E with a free amino group at the N-terminus; (c) coupling or having coupled the second protected variant of Peptide E10696-WQ01-SECfrom step (A)(i)(b) with an N-protected alanine to form a first protected variant of Peptide B, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9; and (d) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide B from step (A)(1)(c) to form a second protected variant of Peptide B with a free amino group at the N-terminus; OR(ii) (a) coupling or having coupled a protected variant of Fragment 3A with a protected variant of Fragment 4 to form a first protected variant of Peptide B, wherein the protected variant of Fragment 3A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Fragment 4 has a free amino group at the N-terminus, Fragment 3A comprises the amino acid sequence of SEQ ID NO: 13, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide B comprises the amino acid sequence of SEQ ID NO: 9; and (b) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide B from step (A)(ii)(a) to form a second protected variant of Peptide B with a free amino group at the N-terminus; OR(ill) (a) coupling or having coupled a protected variant of Fragment 5 with a protected variant of Fragment 6 to form a first protected variant of Peptide B, wherein the protected variant of Fragment 5 has a free carboxyl group at the C-terminus and a protected (preferably Trt- or Fmoc-protected, more preferably Trt-protected) N-terminus, the protected variant of Fragment 6 has a free amino group at the N-terminus, Fragment 5 comprises the amino acid sequence of SEQ ID NO: 5, Fragment 6 comprises the amino acid sequence of SEQ ID NO: 6, and Peptide B comprises the amino acid sequence of SEQ ID NO: 9; and (b) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide B from step (A)(iii)(a) to form a second protected variant of Peptide B with a free amino group at the N-terminus;(B) (I) coupling or having coupled a protected variant of Fragment 2A with the second protected variant of Peptide B from step (A) to form a first protected variant of Peptide C, wherein the protected variant of Fragment 2A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, Fragment 2A comprises the amino acid sequence of SEQ ID NO: 12, and Peptide C comprises the amino acid sequence of SEQ ID NO: 10; and (ii) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide C from step (B)(i) to form a second protected variant of Peptide C with a free amino group at the N-terminus; and(C) coupling or having coupled a protected variant of Fragment 1 A with the second protected variant of Peptide C from step (B)(ii) to form a protected variant of Peptide A, wherein the protected variant of Fragment 1 A has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus, Fragment 1 A comprises the amino acid sequence of SEQ ID NO: 11, and Peptide A comprises the amino acid sequence of SEQ ID NO: 7.

[0029] In some embodiments, the method further comprises deprotecting or having deprotected the protected variant of Peptide A to isolate Peptide A.10696-WQ01-SEC

[0030] In some embodiments, the method further comprises adding or having added a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0031] Yet another aspect of the present disclosure provides a method of preparing Peptide F or a protected variant thereof, comprising coupling a protected variant of Fragment 2 with a protected variant of Peptide E to form a protected variant of Peptide F, wherein Fragment 2 comprises the amino acid sequence of SEQ ID NO: 2, Peptide E comprises the amino acid sequence of SEQ ID NO: 8, and Peptide F comprises the amino acid sequence of SEQ ID NO: 14.

[0032] Still another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising coupling a protected variant of Fragment 1 with a protected variant of Peptide F to form a protected variant of Peptide A, wherein Fragment 1 comprises the amino acid sequence of SEQ ID NO: 1, Peptide F comprises the amino acid sequence of SEQ ID NO: 14, and Peptide A comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the method further comprises deprotecting the protected variant of Peptide A to isolate Peptide A. In some embodiments, the method further comprises adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0033] Another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising:coupling or having coupled a protected variant of Fragment 2 with a protected variant of Peptide E to form a first protected variant of Peptide F, wherein the protected variant of Fragment 2 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Peptide E has a free amino group at the N-terminus, Fragment 2 comprises the amino acid sequence of SEQ ID NO: 2, Peptide E comprises the amino acid sequence of SEQ ID NO: 8, and Peptide F comprises the amino acid sequence of SEQ ID NO: 14;selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide F to form a second protected variant of Peptide F with a free amino group at the N-terminus;coupling or having coupled a protected variant of Fragment 1 with the second protected variant of Peptide F to form a protected variant of Peptide A, wherein the protected variant of Fragment 1 has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus, Fragment 1 comprises the amino acid sequence of SEQ ID NO: 1, and Peptide A comprises the amino acid sequence of SEQ ID NO: 7.

[0034] In some embodiments, the method further comprises deprotecting or having deprotected the protected variant of Peptide A to isolate Peptide A.

[0035] In some embodiments, the method further comprises adding or having added a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0036] Another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising:10696-WQ01-SECcoupling or having coupled a protected variant of Fragment 3 with a protected variant of Fragment 4 to form a first protected variant of Peptide E, wherein the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Fragment 4 has a free amino group at the N-terminus, Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of SEQ ID NO: 8;selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide E to form a second protected variant of Peptide E with a free amino group at the N-terminus;coupling or having coupled a protected variant of Fragment 2 with the second protected variant of Peptide E to form a first protected variant of Peptide F, wherein the protected variant of Fragment 2 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, Fragment 2 comprises the amino acid sequence of SEQ ID NO: 2, and Peptide F comprises the amino acid sequence of SEQ ID NO: 14;selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide F to form a second protected variant of Peptide F with a free amino group at the N-terminus; and coupling or having coupled a protected variant of Fragment 1 with the second protected variant of Peptide F to form a protected variant of Peptide A, wherein the protected variant of Fragment 1 has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus, Fragment 1 comprises the amino acid sequence of SEQ ID NO: 1, and Peptide A comprises the amino acid sequence of SEQ ID NO: 7.

[0037] In some embodiments, the method further comprises deprotecting or having deprotected the protected variant of Peptide A to isolate Peptide A.

[0038] In some embodiments, the method further comprises adding or having added a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0039] Still another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising:coupling or having coupled a protected variant of Fragment 3 with a protected variant of Fragment 4 to form a first protected variant of Peptide E, wherein the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Fragment 4 has a free amino group at the N-terminus, Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of SEQ ID NO: 8;selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide E to form a second protected variant of Peptide E with a free amino group at the N-terminus;10696-WQ01-SECcoupling or having coupled a protected variant of Fragment 2C with the second protected variant of Peptide E to form a first protected variant of Peptide C, wherein the protected variant of Fragment 2C has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, Fragment 2C comprises the amino acid sequence of SEQ ID NO: 17, and Peptide C comprises the amino acid sequence of SEQ ID NO: 10;selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide C to form a second protected variant of Peptide C with a free amino group at the N-terminus; and coupling or having coupled a protected variant of Fragment 1 A with the second protected variant of Peptide C to form a protected variant of Peptide A, wherein the protected variant of Fragment 1 A has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus, Fragment 1 A comprises the amino acid sequence of SEQ ID NO: 11 , and Peptide A comprises the amino acid sequence of SEQ ID NO: 7.

[0040] In some embodiments, the method further comprises deprotecting or having deprotected the protected variant of Peptide A to isolate Peptide A.

[0041] In some embodiments, the method further comprises adding or having added a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0042] Yet another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising:coupling or having coupled a protected variant of Fragment 3A with a protected variant of Fragment 4 to form a first protected variant of Peptide B, wherein the protected variant of Fragment 3A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Fragment 4 has a free amino group at the N-terminus, Fragment 3A comprises the amino acid sequence of SEQ ID NO: 13, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide B comprises the amino acid sequence of SEQ ID NO: 9;selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide B to form a second protected variant of Peptide B with a free amino group at the N-terminus;coupling or having coupled a protected variant of Fragment 2A with the second protected variant of Peptide B to form a first protected variant of Peptide C, wherein the protected variant of Fragment 2A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, Fragment 2A comprises the amino acid sequence of SEQ ID NO: 12, and Peptide C comprises the amino acid sequence of SEQ ID NO: 10;selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide C to form a second protected variant of Peptide C with a free amino group at the N-terminus; and10696-W001-SECcoupling or having coupled a protected variant of Fragment 1 A with the second protected variant of Peptide C to form a protected variant of Peptide A, wherein the protected variant of Fragment 1 A has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus, Fragment 1 A comprises the amino acid sequence of SEQ ID NO: 11 , and Peptide A comprises the amino acid sequence of SEQ ID NO: 7.

[0043] In some embodiments, the method further comprises deprotecting or having deprotected the protected variant of Peptide A to isolate Peptide A.

[0044] In some embodiments, the method further comprises adding or having added a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0045] Another aspect of the present disclosure provides a method of preparing Peptide A having a sequence of His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ser-Tyr-Leu-Glu-Glu-GIn-Ala-Ala-Lys-Glu-Phe-lle-Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Lys (SEQ ID NO: 7) ("Peptide A”), or a protected variant thereof, comprising:(A)(I) (a) reacting a protected variant of Fragment 3 with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide E (SEQ ID NO: 8), wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide E is on solid support after the reacting; and(b) reacting a protected variant of Peptide E with a protected alanine under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Peptide B is on solid support; OR(ii) reacting a protected variant of Fragment 3A with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide B is on solid support after the reacting; OR(ill) reacting a protected variant of Fragment 5 with a protected variant of Fragment 6 under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Fragment 6 is on solid support prior to the reacting and the protected variant of Peptide B is on solid support after the reacting;(B) reacting a protected variant of Fragment 2A (SEQ ID NO: 12) with the protected variant of Peptide B from step (A) under solid phase peptide coupling conditions to form a protected variant of Peptide C (SEQ ID NO: 10), wherein the protected variant of Peptide C is on solid support after the reacting;10696-WQ01-SEC(C) reacting a protected variant of Fragment 1 A (SEQ ID NO: 11) with the protected variant of Peptide C from step (B) under solid phase peptide coupling conditions to form a protected variant of Peptide A, wherein the protected variant of Peptide A is on solid support after the reacting;(D) optionally deprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A; and(E) optionally adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0046] In some embodiments, the present disclosure describes a method of preparing Peptide A or a protected variant thereof, comprising:reacting a protected variant of Fragment 3 with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide E, wherein Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of 8, and further wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide E is on solid support after the reacting;reacting the protected variant of Peptide E with a protected alanine under solid phase peptide coupling conditions to form a protected variant of Peptide B, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9, and further wherein the protected variant of Peptide B is on solid support after the reacting; reacting a protected variant of Fragment 2A with the protected variant of Peptide B under solid phase peptide coupling conditions to form a protected variant of Peptide C, wherein Fragment 2A comprises the amino acid sequence of SEQ ID NO: 12 and Peptide C comprises the amino acid sequence of SEQ ID NO: 10, and further wherein the protected variant of Peptide C is on solid support after the reacting;reacting a protected variant of Fragment 1 A with the protected variant of Peptide C under solid phase peptide coupling conditions to form a protected variant of Peptide A, wherein Fragment 1 A comprises the amino acid sequence of SEQ ID NO: 11 and Peptide A comprises the amino acid sequence of SEQ ID NO: 7, and further wherein the protected variant of Peptide A is on solid support after the reacting; anddeprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A.

[0047] In some embodiments, the method further comprises adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0048] In some embodiments, the present disclosure describes a method of preparing Peptide A or a protected variant thereof, comprising:reacting a protected variant of Fragment 3A with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide B, wherein Fragment 3A comprises the amino acid sequence of SEQ ID NO: 13, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide B comprises the amino acid sequence of SEQ ID NO:9,and further wherein the protected variant of10696-WQ01-SECFragment 4 is on solid support prior to the reacting and the protected variant of Peptide B is on solid support after the reacting;reacting a protected variant of Fragment 2A with the protected variant of Peptide B under solid phase peptide coupling conditions to form a protected variant of Peptide C, wherein Fragment 2A comprises the amino acid sequence of SEQ ID NO: 12 and Peptide C comprises the amino acid sequence of SEQ ID NO: 10, and further wherein the protected variant of Peptide C is on solid support after the reacting;reacting a protected variant of Fragment 1 A with the protected variant of Peptide C under solid phase peptide coupling conditions to form a protected variant of Peptide A, wherein Fragment 1 A comprises the amino acid sequence of SEQ ID NO: 11 and Peptide A comprises the amino acid sequence of SEQ ID NO: 7, and further wherein the protected variant of Peptide A is on solid support after the reacting; anddeprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A.

[0049] In some embodiments, the method further comprises adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0050] In some embodiments, the present disclosure describes a method of preparing Peptide A or a protected variant thereof, comprising:reacting a protected variant of Fragment 5 with a protected variant of Fragment 6 under solid phase peptide coupling conditions to form a protected variant of Peptide B, wherein Fragment 5 comprises the amino acid sequence of SEQ ID NO: 5, Fragment 6 comprises the amino acid sequence of SEQ ID NO: 6, and Peptide B comprises the amino acid sequence of SEQ ID NO: 9, and further wherein the protected variant of Fragment 6 is on solid support prior to the reacting and the protected variant of Peptide B is on solid support after the reacting;reacting a protected variant of Fragment 2A with the protected variant of Peptide B under solid phase peptide coupling conditions to form a protected variant of Peptide C, wherein Fragment 2A comprises the amino acid sequence of SEQ ID NO: 12 and Peptide C comprises the amino acid sequence of SEQ ID NO: 10, and further wherein the protected variant of Peptide C is on solid support after the reacting;reacting a protected variant of Fragment 1 A with the protected variant of Peptide C under solid phase peptide coupling conditions to form a protected variant of Peptide A, wherein Fragment 1 A comprises the amino acid sequence of SEQ ID NO: 11 and Peptide A comprises the amino acid sequence of SEQ ID NO: 7, and further wherein the protected variant of Peptide A is on solid support after the reacting; anddeprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A.

[0051] In some embodiments, the method further comprises adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.10696-WQ01-SEC

[0052] Another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising:reacting a protected variant of Fragment 3 with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide E, wherein Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of 8, and further wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide E is on solid support after the reacting;reacting the protected variant of Peptide E with a protected variant of Fragment 2 under solid phase peptide coupling conditions to form a protected variant of Peptide F, wherein Peptide F comprises the amino acid sequence of SEQ ID NO: 14, and further wherein the protected variant of Peptide F is on solid support after the reacting;reacting a protected variant of Fragment 1 with the protected variant of Peptide F under solid phase peptide coupling conditions to form a protected variant of Peptide A, wherein Fragment 1 comprises the amino acid sequence of SEQ ID NO: 1 and Peptide A comprises the amino acid sequence of SEQ ID NO: 7, and further wherein the protected variant of Peptide A is on solid support after the reacting; anddeprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A.

[0053] In some embodiments, the method further comprises adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0054] Still another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising:reacting a protected variant of Fragment 3 with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide E, wherein Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of SEQ ID NO: 8; and further wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide E is on solid support after the reacting;reacting a protected variant of Fragment 2C with the protected variant of Peptide E on solid support under solid phase peptide coupling conditions to form a protected variant of Peptide C, wherein Fragment 2C comprises the amino acid sequence of SEQ ID NO: 17 and Peptide C comprises the amino acid sequence of SEQ ID NO: 10; and further wherein the protected variant of Peptide C is on solid support after the reacting; and reacting a protected variant of Fragment 1 A with the protected variant of Peptide C on solid support under solid phase peptide coupling conditions to form a protected variant of Peptide A, wherein Fragment 1 A comprises the amino acid sequence of SEQ ID NO: 11 and Peptide A comprises the amino acid sequence of10696-W001-SECSEQ ID NO: 7 and further wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide A is on solid support after the reacting; anddeprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A.

[0055] In some embodiments, the method further comprises adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0056] Another aspect of the present disclosure provides a method of preparing Peptide A or a protected variant thereof, comprising:(A)(i) (a) reacting a protected variant of Fragment 3 with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide E (SEQ ID NO: 8), wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide E is on solid support after the reacting; and(b) reacting a protected variant of Peptide E with a protected alanine under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Peptide B is on solid support; OR(ii) reacting a protected variant of Fragment 3A with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide B is on solid support after the reacting; OR(iii) reacting a protected variant of Fragment 5 with a protected variant of Fragment 6 under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Fragment 6 is on solid support prior to the reacting and the protected variant of Peptide B is on solid support after the reacting;(B) reacting a protected variant of Peptide D (SEQ ID NO: 15) with the protected variant of Peptide B from step (A) under solid phase peptide coupling conditions to form a protected variant of Peptide A (SEQ ID NO: 7), wherein the protected variant of Peptide A is on solid support after the reacting;(C) optionally deprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A; and(D) optionally adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0057] Yet another aspect of the present disclosure provides a method of preparing Peptide A or a variant thereof, comprising:(A) coupling a protected variant of Fragment 5 with a protected variant of Fragment 6 to form a protected variant of Peptide B; and / or10696-WQ01-SEC(B) coupling a protected variant of Fragment 1 with a protected variant of Fragment 2B to form a protected variant of Peptide D; and / or(C) coupling a protected variant of Peptide B with a protected variant of Peptide D to form a protected variant of Peptide A,wherein Peptide A has the amino acid sequence of SEQ ID NO: 7, Peptide B has the amino acid sequence of SEQ ID NO: 9, Peptide D has the amino acid sequence of SEQ ID NO: 15, Fragment 1 has the amino acid sequence of SEQ ID NO: 1, Fragment 2B has the amino acid sequence of SEQ ID NO: 16, Fragment 5 has the amino acid sequence of SEQ ID NO: 5, and Fragment 6 has the amino acid sequence of SEQ ID NO: 6.

[0058] Still another aspect of the present disclosure provides a molecule selected from:Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 166);H-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 30);Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 169);Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59);H-Gly-Gly-Gly-Gly-Ser(Psi(Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130);Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 170);H-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 147); andBoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 171),or a salt thereof.

[0059] Another aspect of the disclosure provides a method of preparing Peptide B or a protected variant thereof, the method comprising coupling a protected variant of Fragment 5 with a protected variant of Fragment 6 in solution to form a protected variant of Peptide B, wherein Peptide B has the amino acid sequence of SEQ ID10696-WQ01-SECNO: 9, Fragment 5 has the amino acid sequence of SEQ ID NO: 5, and Fragment 6 has the amino acid sequence of SEQ ID NO: 6, further wherein:the N-terminus, the side chain of the Trp residue at position 2, and the side chain of the Lys residue at position 5 of the protected variant of Fragment 5 are protected and the protected variant of Fragment 5 has a free carboxyl group at the C-terminus; andthe side chain of the Ser residue at position 5, the side chain of the Ser residue at position 10, the side chain of the Ser residue at position 15, and the side chain of the Lys residue at position 16 of the protected variant of Fragment 6 are protected, the protected variant of Fragment 6 has a free amino group at the N-terminus, and the protected variant of Fragment 6 does not have a free carboxyl group at the C-terminus.

[0060] In some embodiments, the side chain of the Trp residue at position 2 and the side chain of the Lys residue at position 5 of the protected variant of Fragment 5 are each independently protected with an acid-labile protecting group; the side chain of the Ser residue at position 5, the side chain of the Ser residue at position 10, and the side chain of the Ser residue at position 15 of the protected variant of Fragment 6 are each independently protected with an acid-labile protecting group; and the side chain of the Lys residue at position 16 of the protected variant of Fragment 6 is orthogonally protected relative to the side chain protecting groups of the protected variant of Fragment 5 and the protected variant of Fragment 6 and the N-terminal protecting group of the protected variant of Fragment 5. Additionally, in some embodiments, the N-terminal protecting group of the protected variant of Fragment 5 can be removed without removing the side chain protecting groups of the protected variant of Fragment 5 and the protected variant of Fragment 6. Furthermore, in some embodiments, the C-terminus of the protected variant of Fragment 6 is amidated as a C-terminal primary amide.

[0061] Still another aspect of the present disclosure provides a method of preparing a Peptide D or a protected variant thereof, the method comprising coupling a protected variant of Fragment 1 with a protected variant of Fragment 2B in solution to form a protected variant of Peptide D, wherein Peptide D has the amino acid sequence of SEQ ID NO: 15, Fragment 1 has the amino acid sequence of SEQ ID NO: 1, and Fragment 2B has the amino acid sequence of SEQ ID NO: 16, further wherein:the N-terminus, the side chain of the His residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Thr residue at position 5, the side chain of the Thr residue at position 7, the side chain of the Ser residue at position 8, the side chain of the Asp residue at position 9, the side chain of the Tyr residue at position 10, the side chain of the Ser residue at position 11 , and the side chain of the Ser residue at position 12 of the protected variant of Fragment 1 are protected and the protected variant of Fragment 1 has a free carboxyl group at the C-terminus; andthe side chain of the Tyr residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Glu residue at position 4, the side chain of the Gin residue at position 5, the side chain of the Lys residue at position 8, and the side chain of the Glu residue at position 9 of the protected variant of Fragment 2B are protected and the protected variant of Fragment 2B has a free amino group at the N-terminus.10696-WQ01-SEC

[0062] In some embodiments, the side chain of the His residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Thr residue at position 5, the side chain of the Thr residue at position 7, the side chain of the Ser residue at position 8, the side chain of the Asp residue at position 9, the side chain of the Tyr residue at position 10, the side chain of the Ser residue at position 11, and the side chain of the Ser residue at position 12 of the protected variant of Fragment 1 are each independently protected with an acid-labile protecting group; and the side chain of the Tyr residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Glu residue at position 4, the side chain of the Gin residue at position 5, the side chain of the Lys residue at position 8, and the side chain of the Glu residue at position 9 of the protected variant of Fragment 2B are each independently protected with an acid-labile protecting group. Additionally, in some embodiments, the N-terminus of the protected variant of Fragment 1 is an acid-labile protecting group.

[0063] Still another aspect of the disclosure provides a method of preparing Peptide A or a variant thereof, the method comprising coupling a protected variant of Peptide B with a protected variant of Peptide D to form a protected variant of Peptide A, wherein Peptide A has the amino acid sequence of SEQ ID NO: 7, Peptide B has the amino acid sequence of SEQ ID NO: 9, and Peptide D has the amino acid sequence of SEQ ID NO: 15, further wherein:the side chain of the Trp residue at position 2, the side chain of the Lys residue at position 5, the side chain of the Ser residue at position 13, the side chain of the Ser residue at position 18, the side chain of the Ser residue at position 23, and the side chain of the Lys residue at position 24 of the protected variant of Peptide B are protected, the protected variant of Peptide B has a free amino group at the N-terminus, and the protected variant of Peptide B does not have a free carboxyl group at the C-terminus; andthe N-terminus, the side chain of the His residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Thr residue at position 5, the side chain of the Thr residue at position 7, the side chain of the Ser residue at position 8, the side chain of the Asp residue at position 9, the side chain of the Tyr residue at position 10, the side chain of the Ser residue at position 11, the side chain of the Ser residue at position 12, the side chain of the Tyr residue at position 13, the side chain of the Glu residue at position 15, the side chain of the Glu residue at position 16, the side chain of the Gin residue at position 17, the side chain of the Lys residue at position 20, and the side chain of the Glu residue at position 21 of the protected variant of Peptide D are protected and the protected variant of Peptide D has a free carboxyl group at the C-terminus.

[0064] In some embodiments, the side chain of the Trp residue at position 2, the side chain of the Lys residue at position 5, the side chain of the Ser residue at position 13, the side chain of the Ser residue at position 18, and the side chain of the Ser residue at position 23 of the protected variant of Peptide B are each independently protected with an acid-labile protecting group; the N-terminus, the side chain of the His residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Thr residue at position 5, the side chain of the Thr residue at position 7, the side chain of the Ser residue at position 8, the side chain of the Asp residue at position 9, the side chain of the Tyr residue at position 10, the side chain of the Ser residue at position 11, the side chain of the Ser residue at position 12, the side chain of the Tyr residue at position 13, the side chain10696-WC01-SECof the Glu residue at position 15, the side chain of the Glu residue at position 16, the side chain of the Gin residue at position 17, the side chain of the Lys residue at position 20, and the side chain of the Glu residue at position 21 of the protected variant of Peptide D are each independently protected with an acid-labile protecting group; and the side chain of the Lys residue at position 24 of the protected variant of Peptide B is orthogonally protected relative to the side chain protecting groups of the protected variant of Peptide B and the protected variant of Peptide D and the N-terminal protecting group of the protected variant of Peptide D. Furthermore, in some embodiments, the C-terminus of the protected variant of Peptide B is amidated as a C-terminal primary amide.

[0065] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIGs. 1 A-1 D show schematic representations of a convergent solid phase synthesis route for a protected variant of Peptide A (SEQ ID NO: 179) from protected variants of Fragment 1A (SEQ ID NO: 95), Fragment 2A (SEQ ID NO: 53), Fragment 3 (SEQ ID NO: 56), alanine, and Fragment 4 (SEQ ID NO: 117) in accordance with certain embodiments of the present disclosure.

[0067] FIG. 2A depicts a reaction scheme for coupling a protected variant of Fragment 6 with an amidated C-terminus, where Fragment 6 has the amino acid sequence of SEQ ID NO: 6, and a protected variant of Fragment 5, where Fragment 5 has the amino acid sequence of SEQ ID NO: 5, to form a protected variant of Peptide B with an amidated C-terminus, where Peptide B has the amino acid sequence of SEQ ID NO: 9, in accordance with certain disclosed embodiments.

[0068] FIG. 2B provides a reaction scheme for coupling a protected variant of Fragment 6 with an amidated C-terminus, where Fragment 6 has the amino acid sequence of SEQ ID NO: 6, and a protected variant of Fragment 5, where Fragment 5 has the amino acid sequence of SEQ ID NO: 5, to form a protected variant of Peptide B with an amidated C-terminus, where Peptide B has the amino acid sequence of SEQ ID NO: 9, in accordance with certain disclosed embodiments.

[0069] FIG. 2C depicts another protected variant of Peptide B with an amidated C-terminus (SEQ ID NO: 147) synthesized in accordance with certain disclosed embodiments.

[0070] FIG. 2D provides another reaction scheme for coupling a protected variant of Fragment 6 with an amidated C-terminus (SEQ ID NO: 130) and a protected variant of Fragment 5 (SEQ ID NO: 59) to form a protected variant of Peptide B with an amidated C-terminus (SEQ ID NO: 147) in accordance with certain disclosed embodiments.10696-WQ01-SEC

[0071] FIG. 3A shows a reaction scheme for coupling a protected variant of Fragment 1, where Fragment 1 has the amino acid sequence of SEQ ID NO: 1, and a protected variant of Fragment 2B, where Fragment 2B has the amino acid sequence of SEQ ID NO: 16, to form a protected variant of Peptide D, where Peptide D has the amino acid sequence of SEQ ID NO: 15, in accordance with certain disclosed embodiments.

[0072] FIG. 3B depicts another protected variant of Peptide D (SEQ ID NO: 157) synthesized in accordance with certain disclosed embodiments.

[0073] FIG. 3C shows another reaction scheme for coupling a protected variant of Fragment 1 (SEQ ID NO: 166) and a protected variant of Fragment 2B (SEQ ID NO: 30) to form a protected variant of Peptide D (SEQ ID NO: 169) in accordance with certain disclosed embodiments.

[0074] FIGs. 4A and 4B show a reaction scheme for coupling a protected variant of Peptide B with an amidated C-terminus (SEQ ID NO: 147) to a protected variant of Peptide D (SEQ ID NO: 169) to form a protected variant of Peptide A with an amidated C-terminus (SEQ ID NO: 171) in accordance with certain disclosed embodiments. FIGs.4C and 4D provide a reaction scheme for coupling a protected variant of Peptide B with an amidated C-terminus (SEQ ID NO: 147) to a protected variant of Peptide D (SEQ ID NO: 168) to form a protected variant of Peptide A with an amidated C-terminus in accordance with other disclosed embodiments.

[0075] FIGs. 5A and 5B depict IvDde deprotection of a protected variant of Peptide A with an amidated C-terminus in accordance with certain disclosed embodiments.

[0076] FIG. 5C shows a partially deprotected variant of Peptide A in which the s-amino group of the C-terminal lysine residue is deprotected and the C-terminus is amidated (SEQ ID NO: 191). This molecule can be synthesized in accordance with certain embodiments of the present disclosure.

[0077] FIGs. 5D and 5E depict IvDde deprotection, bromoacetylation, and global deprotection of a protected variant of Peptide A with an amidated C-terminus in accordance with certain disclosed embodiments.

[0078] FIG. 6A depicts bromoacetylation of a partially deprotected variant of Peptide A with a C-terminal amide group in accordance with certain disclosed embodiments.

[0079] FIG. 6B shows a bromoacetylated and protected variant of Peptide A with an amidated C-terminus synthesized in accordance with certain disclosed embodiments.DETAILED DESCRIPTION

[0080] Provided herein are methods of synthesizing a peptide component of maridebart cafraglutide (Peptide A) and fragments thereof using a convergent peptide synthesis approach (e.g., a convergent solid phase peptide synthesis approach, a convergent liquid phase peptide synthesis approach, or a hybrid approach in which one or more couplings are performed on solid support and one or more couplings are performed in solution to obtain Peptide A or a fragment thereof). To develop efficient and scalable convergent approaches for the preparation of10696-WQ01-SECPeptide A, various peptide fragments and associated protecting groups, as well as various coupling conditions, were investigated.

[0081] Illustratively, it was determined that, in some embodiments, Peptide A can be efficiently prepared on solid support by first coupling (I) a protected variant of Fragment 3 and a protected variant of Fragment 4 (which is on solid support) then adding a protected Ala to the N-terminus to form a protected variant of Peptide B (which is on solid support), a synthetic intermediate as shown in FIG. 1A, or (ii) a protected variant of Fragment 3A and a protected variant of Fragment 4 (which is on solid support) to form a protected variant of Peptide B (which is on solid support), a synthetic intermediate, or (ill) a protected variant of Fragment 5 and a protected variant of Fragment 6 (which is on solid support) to form a protected variant of Peptide B on solid support. Then, a protected variant of Fragment 2A can be coupled to the protected variant of Peptide B (on solid support) to form a protected variant of Peptide C on solid support, as shown in FIG. 1B. Finally, a protected variant of Fragment 1 A can be coupled to the protected variant of Peptide C (on solid support) to form a protected variant of Peptide A on solid support, as shown in FIG. 1C. Additional deprotection and cleavage steps can be performed to isolate Peptide A from the solid support. Optionally, Peptide A can also be reacted to add a bromoacetyl moiety at its C-terminal Lys residue.

[0082] Solid phase synthesis conditions for each coupling step can be selected to provide desired yield and regioselectivity. Protecting groups for the protected peptide variants (i.e., peptides with one or more side-chain protecting groups to facilitate peptide synthesis) can be selected to provide reactive inertness during peptide coupling while minimizing impediments to such peptide couplings. These selections can be informed by the discussion and examples provided below. Additionally, in some embodiments of SPPS methods described herein, the N-terminus of a protected amino acid or peptide on solid support can initially be present as a free o-amino group, or the protected amino acid or peptide can initially be present in the form of an acid-addition salt (such as, e.g., an HCI or TFA salt following acidolytic deprotection). In certain embodiments in which the protected amino acid or peptide is initially in the form of an acid-addition salt, the protected amino acid or peptide on solid support can be contacted with a base (such as, e.g., a non-nucleophilic tertiary base, e.g., DIPEA, NMM, or collidine) to convert the acid-addition salt to a free base, thereby liberating an o-amino group for reaction with a coupling partner with an activated C-terminal carboxyl group.

[0083] Provided herein are methods of preparing Peptide A having a sequence of His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ser-Tyr-Leu-Glu-Glu-GIn-Ala-Ala-Lys-Glu-Phe-lle-Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Lys (SEQ ID NO: 7) ("Peptide A”), or a protected variant thereof, comprising:(A)(I) (a) reacting a protected variant of Fragment 3 with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide E (SEQ ID NO: 8),10696-WQ01-SECwherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide E is on solid support after the reacting; and(b) reacting a protected variant of Peptide E with a protected alanine under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Peptide B is on solid support; OR(ii) reacting a protected variant of Fragment 3A with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide B is on solid support after the reacting; OR(ill) reacting a protected variant of Fragment 5 with a protected variant of Fragment 6 under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Fragment 6 is on solid support prior to the reacting and the protected variant of Peptide B is on solid support after the reacting;(B) reacting a protected variant of Fragment 2A (SEQ ID NO: 12) with the protected variant of Peptide B from step (A) under solid phase peptide coupling conditions to form a protected variant of Peptide C (SEQ ID NO: 10), wherein the protected variant of Peptide C is on solid support after the reacting;(C) reacting a protected variant of Fragment 1 A (SEQ ID NO: 11) with the protected variant of Peptide C from step (B) under solid phase peptide coupling conditions to form a protected variant of Peptide A, wherein the protected variant of Peptide A is on solid support after the reacting;(D) optionally deprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A; and(E) optionally adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0084] In some embodiments, the method comprises:(A)(I) reacting a protected variant of Fragment 3 of any one of the aspects of the present disclosure with a protected variant of Fragment 4 of any one of the aspects of the present disclosure under solid phase peptide coupling conditions to form a protected variant of Peptide E (SEQ ID NO: 8), wherein the protected variant of Fragment 4 is on solid support before the reacting and the protected variant of Peptide E is on solid support after the reacting; and(ii) reacting the protected variant of Peptide E on solid support from step (A)(i) with a protected alanine under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Peptide B is on solid support after the reacting;10696-WQ01-SEC(B) reacting a protected variant of Fragment 2A of any one of the aspects of the present disclosure with the protected variant of Peptide B on solid support from step (A) under solid phase peptide coupling conditions to form a protected variant of Peptide C (SEQ ID NO: 10), wherein the protected variant of Peptide C is on solid support after the reacting;(C) reacting a protected variant of Fragment 1 A of any one of the aspects of the present disclosure with the protected variant of Peptide C on solid support from step (B) under solid phase peptide coupling conditions to form a protected variant of Peptide A, wherein the protected variant of Peptide A is on solid support after the reacting;(D) optionally deprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A; and(E) optionally adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0085] In some embodiments, the method comprises:(A) reacting a protected variant of Fragment 3A of any one of the aspects of the present disclosure with a protected variant of Fragment 4 of any one of the aspects of the present disclosure under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Fragment 4 is on solid support before the reacting and the protected variant of Peptide B is on solid support after the reacting;(B) reacting a protected variant of Fragment 2A of any one of the aspects of the present disclosure with the protected variant of Peptide B on solid support from step (A) under solid phase peptide coupling conditions to form a protected variant of Peptide C (SEQ ID NO: 10), wherein the protected variant of Peptide C is on solid support after the reacting;(C) reacting a protected variant of Fragment 1 A of any one of the aspects of the present disclosure with the protected variant of Peptide C on solid support from step (B) under solid phase peptide coupling conditions to form a protected variant of Peptide A, wherein the protected variant of Peptide A is on solid support after the reacting;(D) optionally deprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A; and(E) optionally adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0086] In some embodiments, the method comprises:(A) reacting a protected variant of Fragment 5 of any one of the aspects of the present disclosure with a protected variant of Fragment 6 of any one of the aspects of the present disclosure under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the10696-WQ01-SECprotected variant of Fragment 6 is on solid support before the reacting and the protected variant of Peptide B is on solid support after the reacting;(B) reacting a protected variant of Fragment 2A of any one of the aspects of the present disclosure with the protected variant of Peptide B on solid support from step (A) under solid phase peptide coupling conditions to form a protected variant of Peptide C (SEQ ID NO: 10), wherein the protected variant of Peptide C is on solid support after the reacting;(C) reacting a protected variant of Fragment 1 A of any one of the aspects of the present disclosure with the protected variant of Peptide C on solid support from step (B) under solid phase peptide coupling conditions to form a protected variant of Peptide A, wherein the protected variant of Peptide A is on solid support after the reacting;(D) optionally deprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A; and(E) optionally adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0087] Also provided herein is a method of preparing Peptide A or a protected variant thereof, comprising:reacting a protected variant of Fragment 3 with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide E, wherein Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of 8, and further wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide E is on solid support after the reacting;reacting the protected variant of Peptide E with a protected variant of Fragment 2 under solid phase peptide coupling conditions to form a protected variant of Peptide F, wherein Peptide F comprises the amino acid sequence of SEQ ID NO: 14, and further wherein the protected variant of Peptide F is on solid support after the reacting;reacting a protected variant of Fragment 1 with the protected variant of Peptide F under solid phase peptide coupling conditions to form a protected variant of Peptide A, wherein Fragment 1 comprises the amino acid sequence of SEQ ID NO: 1 and Peptide A comprises the amino acid sequence of SEQ ID NO: 7, and further wherein the protected variant of Peptide A is on solid support after the reacting; anddeprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A.

[0088] In some embodiments, the method further comprises adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0089] Also provided herein is a method of preparing Peptide A or a protected variant thereof, comprising:reacting a protected variant of Fragment 3 with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide E, wherein Fragment 3 comprises the amino10696-WQ01-SECacid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of SEQ ID NO: 8; and further wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide E is on solid support after the reacting;reacting a protected variant of Fragment 20 with the protected variant of Peptide E on solid support under solid phase peptide coupling conditions to form a protected variant of Peptide 0, wherein Fragment 20 comprises the amino acid sequence of SEQ ID NO: 17 and Peptide 0 comprises the amino acid sequence of SEQ ID NO: 10; and further wherein the protected variant of Peptide 0 is on solid support after the reacting; and reacting a protected variant of Fragment 1 A with the protected variant of Peptide 0 on solid support under solid phase peptide coupling conditions to form a protected variant of Peptide A, wherein Fragment 1 A comprises the amino acid sequence of SEQ ID NO: 11 and Peptide A comprises the amino acid sequence of SEQ ID NO: 7 and further wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide A is on solid support after the reacting; anddeprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A.

[0090] In some embodiments, the method further comprises adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0091] Also provided herein a method of preparing Peptide A or a protected variant thereof, comprising:(A)(I) (a) reacting a protected variant of Fragment 3 with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide E (SEQ ID NO: 8), wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide E is on solid support after the reacting; and(b) reacting a protected variant of Peptide E with a protected alanine under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Peptide B is on solid support; OR(ii) reacting a protected variant of Fragment 3A with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide B is on solid support after the reacting; OR(ill) reacting a protected variant of Fragment 5 with a protected variant of Fragment 6 under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Fragment 6 is on solid support prior to the reacting and the protected variant of Peptide B is on solid support after the reacting;10696-WQ01-SEC(B) reacting a protected variant of Peptide D (SEQ ID NO: 15) with the protected variant of Peptide B from step (A) under solid phase peptide coupling conditions to form a protected variant of Peptide A (SEQ ID NO: 7), wherein the protected variant of Peptide A is on solid support after the reacting;(C) optionally deprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A; and(D) optionally adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0092] In some embodiments, the method comprises:(A) reacting a protected variant of Fragment 3 with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide E (SEQ ID NO: 8), wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide E is on solid support after the reacting; and reacting a protected variant of Peptide E with a protected alanine under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Peptide B is on solid support;(B) reacting a protected variant of Peptide D (SEQ ID NO: 15) with the protected variant of Peptide B from step (A) under solid phase peptide coupling conditions to form a protected variant of Peptide A (SEQ ID NO: 7), wherein the protected variant of Peptide A is on solid support after the reacting; (C) optionally deprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A; and(D) optionally adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0093] In some embodiments, the method comprises:(A) reacting a protected variant of Fragment 3A with a protected variant of Fragment 4 under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Fragment 4 is on solid support prior to the reacting and the protected variant of Peptide B is on solid support after the reacting;(B) reacting a protected variant of Peptide D (SEQ ID NO: 15) with the protected variant of Peptide B from step (A) under solid phase peptide coupling conditions to form a protected variant of Peptide A (SEQ ID NO: 7), wherein the protected variant of Peptide A is on solid support after the reacting; (C) optionally deprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A; and(D) optionally adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.10696-WQ01-SEC

[0094] In some embodiments, the method comprises:(A) reacting a protected variant of Fragment 5 with a protected variant of Fragment 6 under solid phase peptide coupling conditions to form a protected variant of Peptide B (SEQ ID NO: 9), wherein the protected variant of Fragment 6 is on solid support prior to the reacting and the protected variant of Peptide B is on solid support after the reacting;(B) reacting a protected variant of Peptide D (SEQ ID NO: 15) with the protected variant of Peptide B from step (A) under solid phase peptide coupling conditions to form a protected variant of Peptide A (SEQ ID NO: 7), wherein the protected variant of Peptide A is on solid support after the reacting; (C) optionally deprotecting and cleaving the protected variant of Peptide A from the solid support to isolate Peptide A; and(D) optionally adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A.

[0095] Also provided herein is a method of preparing Peptide B or a protected variant thereof, comprising coupling a protected variant of Fragment 5 with a protected variant of Fragment 6 to form a protected variant of Peptide B, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9, Fragment 5 comprises the amino acid sequence of SEQ ID NO: 5, and Fragment 6 comprises the amino acid sequence of SEQ ID NO: 6.

[0096] In some embodiments, the protected variant of Fragment 5 is coupled with the protected variant of Fragment 6 in solution. In some embodiments, the protected variant of Fragment 5 is coupled with the protected variant of Fragment 6 under liquid phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 5 is coupled with the protected variant of Fragment 6 under classical solution phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 5 is coupled with the protected variant of Fragment 6 under tag-assisted liquid phase peptide coupling conditions.

[0097] In other embodiments, the protected variant of Fragment 5 is coupled with the protected variant of Fragment 6 on solid support. In other embodiments, the protected variant of Fragment 5 is coupled with the protected variant of Fragment 6 under solid phase peptide coupling conditions.

[0098] In some embodiments, a C-terminal carboxyl group of the protected variant of Fragment 5 is preactivated. In other embodiments, a C-terminal carboxyl group of the protected variant of Fragment 5 is activated in situ.

[0099] In some embodiments, the method comprises:contacting the protected variant of Fragment 5 with a coupling composition to form a protected variant of Fragment 5 with an activated C-terminal carboxyl group ("activated Fragment 5"); andcontacting the protected variant of Fragment 6 with the activated Fragment 5 to form the protected variant of Peptide B.10696-WC01-SEC

[0100] In some embodiments, the method comprises:contacting the protected variant of Fragment 5 with a coupling composition on solid support to form a protected variant of Fragment 5 with an activated C-terminal carboxyl group ("activated Fragment 5"); and contacting the protected variant of Fragment 6 with the activated Fragment 5 on solid support to form the protected variant of Peptide B.

[0101] In some embodiments, the method comprises:contacting the protected variant of Fragment 5 with a coupling composition in solution to form a protected variant of Fragment 5 with an activated C-terminal carboxyl group ("activated Fragment 5"); and contacting the protected variant of Fragment 6 with the activated Fragment 5 in solution to form the protected variant of Peptide B.

[0102] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 5 is pre-activated), the method comprises:contacting the protected variant of Fragment 5 with a coupling composition to form a protected variant of Fragment 5 with an activated C-terminal carboxyl group ("activated Fragment 5"); andcontacting the protected variant of Fragment 6 with the activated Fragment 5 to form the protected variant of Peptide B, wherein the contacting does not occur in the presence of a coupling reagent.

[0103] In some embodiments, the protected variant of Fragment 5 is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 5 and the coupling composition.

[0104] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 5 is pre-activated), the method comprises:contacting the protected variant of Fragment 5 with a coupling composition on solid support to form a protected variant of Fragment 5 with an activated C-terminal carboxyl group ("activated Fragment 5"); and contacting the protected variant of Fragment 6 with the activated Fragment 5 on solid support to form the protected variant of Peptide B, wherein the contacting does not occur in the presence of a coupling reagent.

[0105] In some embodiments, the protected variant of Fragment 5 is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 5 and the coupling composition.

[0106] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 5 is pre-activated), the method comprises:contacting the protected variant of Fragment 5 with a coupling composition in solution to form a protected variant of Fragment 5 with an activated C-terminal carboxyl group ("activated Fragment 5"); and10696-WC01-SECcontacting the protected variant of Fragment 6 with the activated Fragment 5 in solution to form the protected variant of Peptide B, wherein the contacting does not occur in the presence of a coupling reagent.

[0107] In some embodiments, the protected variant of Fragment 5 is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 5 and the coupling composition.

[0108] In some embodiments, the contacting of the protected variant of Fragment 5 with the coupling composition and the contacting of the activated Fragment 5 with the protected variant of Fragment 6 occur in the same reaction vessel. In other embodiments, the contacting of the protected variant of Fragment 5 with the coupling composition and the contacting of the activated Fragment 5 with the protected variant of Fragment 6 occur in different reaction vessels.

[0109] In some embodiments, the activated Fragment 5 is contacted with the protected variant of Fragment 6 in the presence of a base and / or a coupling additive but not a coupling reagent (such as, e.g., in certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 5 is pre-activated and the protected variant of Fragment 5 is present in molar excess relative to the coupling reagent in the coupling composition during the contacting to form activated Fragment 5).

[0110] In some embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 5 is pre-activated, the protected variant of Fragment 6 has a free N-terminal amino group and a free C-terminal carboxyl group.

[0111] In some embodiments, the coupling composition comprises a coupling reagent and a base. In some embodiments, the coupling composition comprises a coupling reagent (e.g., a carbodiimide, e.g., DIG, DCC, EDC) and a coupling additive (e.g., HOBt, 5-CI-HOBt, Oxyma).

[0112] In some embodiments, the coupling composition comprises a coupling reagent, a coupling additive, and a base.

[0113] In some embodiments, the coupling reagent is a carbodiimide, such as, e.g., DIG, DCC, or EDC. In other embodiments, the coupling reagent is BOP-CI.

[0114] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 5 is activated in situ), the method comprises coupling the protected variant of Fragment 5 with the protected variant of Fragment 6 in the presence of a coupling composition. In some embodiments, the method comprises coupling the protected variant of Fragment 5 with the protected variant of Fragment 6 in solution in the presence of a coupling composition.

[0115] In some embodiments, the protected variant of Fragment 6 does not have a free carboxyl group at the C-terminus. In some embodiments, the C-terminus of the protected variant of Fragment 6 is protected. In some embodiments, the C-terminus of the protected variant of Fragment 6 is amidated. In some embodiments, the protected variant of Fragment 6 has a free amino group at the N-terminus. In preferred embodiments, the10696-WC01-SECprotected variant of Fragment 6 has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus. In particularly preferred embodiments, the protected variant of Fragment 6 has a free amino group at the N-terminus and an amidated C-terminus.

[0116] In some embodiments, the protected variant of Fragment 5 does not have a free amino group at the N-terminus. In some embodiments, the N-terminus of the protected variant of Fragment 5 is protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected). In some embodiments, the protected variant of Fragment 5 has a free carboxyl group at the C-terminus. In preferred embodiments, the protected variant of Fragment 5 has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Fragment 5 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected) N-terminus.

[0117] In some embodiments, the protected variant of Fragment 6 has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 5 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected) N-terminus.

[0118] In some embodiments, the C-terminus of the protected variant of Fragment 6 is amidated, and the N-terminus of the protected variant of Fragment 5 is protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected).

[0119] In preferred embodiments, the protected variant of Fragment 6 has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 5 has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Fragment 6 has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 5 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected) N-terminus.

[0120] In some embodiments, the protected variant of Fragment 6 has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 5 has a free carboxyl group at the C-terminus and an Fmoc-protected N-terminus. In some embodiments, the protected variant of Fragment 6 has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 5 has a free carboxyl group at the C-terminus and a Trt-protected N-terminus.

[0121] In some embodiments, the N-terminus, the side chain of the Trp residue at position 2, and the side chain of the Lys residue at position 5 of the protected variant of Fragment 5 are protected and the protected variant of Fragment 5 has a free carboxyl group at the C-terminus.

[0122] In some embodiments, the side chain of the Ser residue at position 5, the side chain of the Ser residue at position 10, the side chain of the Ser residue at position 15, and the side chain of the Lys residue at position 16 of the protected variant of Fragment 6 are protected, the protected variant of Fragment 6 has a free10696-WQ01-SECamino group at the N-terminus, and the protected variant of Fragment 6 does not have a free carboxyl group at the C-terminus.

[0123] In some embodiments, the N-terminus, the side chain of the Trp residue at position 2, and the side chain of the Lys residue at position 5 of the protected variant of Fragment 5 are protected, the protected variant of Fragment 5 has a free carboxyl group at the C-terminus, the side chain of the Ser residue at position 5, the side chain of the Ser residue at position 10, the side chain of the Ser residue at position 15, and the side chain of the Lys residue at position 16 of the protected variant of Fragment 6 are protected, the protected variant of Fragment 6 has a free amino group at the N-terminus, and the protected variant of Fragment 6 does not have a free carboxyl group at the C-terminus.

[0124] In some embodiments, the side chain of the Trp residue at position 2 and the side chain of the Lys residue at position 5 of the protected variant of Fragment 5 are each protected with a tert-butyloxycarbonyl (Boc) protecting group.

[0125] In some embodiments, the Ser residues at position 5, position 10, and position 15 of the protected variant of Fragment 6 are each protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser and the side chain of the Lys residue at position 16 of the protected variant of Fragment 6 is protected with an ivDde (1 -(4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl) protecting group.

[0126] In some embodiments, the side chain of the Trp residue at position 2 and the side chain of the Lys residue at position 5 of the protected variant of Fragment 5 are each protected with a tert-butyloxycarbonyl (Boc) protecting group, the Ser residues at position 5, position 10, and position 15 of the protected variant of Fragment 6 are each protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser, and the side chain of the Lys residue at position 16 of the protected variant of Fragment 6 is protected with an ivDde (1 -(4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl) protecting group.

[0127] In some embodiments, the protected variant of Fragment 5 is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59).

[0128] In some embodiments, the protected variant of Fragment 6 is Gly-Gly-Gly-Gly-Ser(Psi (Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130) or Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 121). In some embodiments, the protected variant of Fragment 6 is Gly-Gly-Gly-Gly-Ser(Psi(Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130). In some embodiments, the protected variant of Fragment 6 is Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 121).

[0129] In some embodiments, the protected variant of Fragment 5 is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59), and the protected variant of Fragments is Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-10696-W001-SECGly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130) or Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 121).

[0130] In some embodiments, the protected variant of Fragment 5 is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59), and the protected variant of Fragment 6 is Gly-Gly-Gly-Gly-Ser(Psi (Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130).

[0131] In some embodiments, the protected variant of Fragment 5 is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59), and the protected variant of Fragment 6 is Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 121).

[0132] In some embodiments, a protected variant of Peptide B can be efficiently prepared in solution by coupling a protected variant of Fragment 6 with an amidated C-terminus and a protected variant of Fragment 5 to form a protected variant of Peptide B with an amidated C-terminus as shown in FIG. 2A. Alternatively, a protected variant of Peptide B can be efficiently prepared in solution by coupling a protected variant of Fragment 6 with an amidated C-terminus and another protected variant of Fragment 5 to form a protected variant of Peptide B with an amidated C-terminus as shown in FIG. 2B. FIG. 2C depicts another protected variant of Peptide B with an amidated C-terminus, which can be prepared by an analogous process to that shown in FIG.2B. Alternatively, a protected variant of Peptide B can be efficiently prepared in solution by coupling a protected variant of Fragment 6 with an amidated C-terminus and another protected variant of Fragment 5 to form a protected variant of Peptide B with an amidated C-terminus as shown in FIG. 2D.

[0133] In some embodiments, the coupling composition comprises 1 to 10 equivalents of a coupling reagent, optionally 1 to 10 equivalents of a base, and optionally 1 to 10 equivalents of a coupling additive, wherein equivalents are relative to the protected variant of Fragment 5. In some embodiments, the coupling composition comprises 2 to 10 equivalents of a coupling reagent, optionally 2 to 10 equivalents of a base, and optionally 2 to 10 equivalents of a coupling additive, wherein equivalents are relative to the protected variant of Fragment 5.

[0134] In some embodiments, the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0135] In some embodiments, the coupling composition comprises PyBOP and TEA. In some embodiments, the coupling composition comprises PyBOP and TEA in DMSO. In some embodiments, the coupling composition comprises PyBOP and TEA in DMSO and MeOH. In some embodiments, the coupling composition comprises PyBOP and TEA in 1-5:1 DMSO:MeOH. In some embodiments, the coupling composition comprises PyBOP and TEA in 1:1 DMSO:MeOH.

[0136] In some embodiments, the coupling composition comprises TBTU and collidine. In some embodiments, the coupling composition comprises TBTU and collidine in a solvent selected from DMSO, acetonitrile, and combinations thereof. In some embodiments, the coupling composition comprises TBTU and10696-W001-SECcollidine in DMSO. In some embodiments, the coupling composition comprises TBTU and collidine in acetonitrile. In some embodiments, the coupling composition comprises TBTU and collidine in DMSO and acetonitrile.

[0137] In some embodiments, the coupling composition comprises TBTU and TMEDA. In some embodiments, the coupling composition comprises TBTU and TMEDA in acetonitrile.

[0138] In some embodiments, the protected variant of Fragment 5 is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59), the protected variant of Fragment 6 is Gly-Gly-Gly-Gly-Ser(Psi(Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me, Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130) or Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 121), and the coupling composition comprises TBTU / collidine, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0139] In some embodiments, the protected variant of Fragment 5 is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59), the protected variant of Fragment 6 is Gly-Gly-Gly-Gly-Ser(Psi(Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130), and the coupling composition comprises PyBOP and TEA. In some embodiments, the coupling composition comprises PyBOP and TEA in DMSO. In some embodiments, the coupling composition comprises PyBOP and TEA in 1-5:1 DMSO:MeOH. In some embodiments, the coupling composition comprises PyBOP and TEA in 1:1 DMSO:MeOH.

[0140] In some embodiments, the protected variant of Fragment 5 is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59), the protected variant of Fragment 6 is Gly-Gly-Gly-Gly-Ser(Psi (Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130), and the coupling composition comprises TBTU and collidine. In some embodiments, the coupling composition comprises TBTU and collidine in a solvent selected from DMSO, acetonitrile, and combinations thereof. In some embodiments, the protected variant of Fragment 5 is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59), the protected variant of Fragment 6 is Gly-Gly-Gly-Gly-Ser(Psi(Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130), and the coupling composition comprises TBTU and TMEDA. In some embodiments, the coupling composition comprises TBTU and TMEDA in acetonitrile.

[0141] In some embodiments, the protected variant of Fragment 5 is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59), the protected variant of Fragment 6 is Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 121), and the coupling composition comprises TBTU and collidine. In some embodiments, the coupling composition comprises TBTU and collidine in DMSO and acetonitrile.

[0142] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C. In some embodiments, the10696-W001-SECcoupling occurs at a temperature in the range of 20°C to 30°C. In some embodiments, the coupling occurs at a temperature in the range of 30°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 35°C to 45°C.

[0143] In some embodiments, the method comprises coupling the protected variant of Fragment 5 and the protected variant of Fragment 6 for a time in the range of 1 hour to 72 hours. In some embodiments, the time is in the range of 6 hours to 24 hours. In other embodiments, the time is in the range of 1 hour to 2 hours.

[0144] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 6 hours to 24 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 6 hours to 24 hours.

[0145] In some embodiments, the coupling occurs at a temperature in the range of 20°C to 40°C for a time in the range of 1 hour to 2 hours.

[0146] In some embodiments, the protected variant of Fragment 5 is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59), the protected variant of Fragment 6 is Gly-Gly-Gly-Gly-Ser(Psi(Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me, Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130) or Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 121), the coupling composition comprises TBTU / collidine, PyBOP / TEA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine (preferably TBTU / collidine or PyBOP / TEA), and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours).

[0147] In some embodiments, the method further comprises selectively deprotecting the N-terminus of the protected variant of Peptide B (e.g., using a Trt, Boc, or Fmoc deprotection method described herein).

[0148] In some embodiments, the N-terminus of the protected variant of Peptide B formed by coupling the protected variant of Fragment 5 with the protected variant of Fragment 6 is Trt-protected, and the method further comprises contacting the protected variant of Peptide B with a Bronsted acid or a Lewis acid (preferably TFA or HOI), optionally in the presence of a scavenger (preferably TIS), to selectively remove the trityl protecting group at the N-terminus. In some embodiments, the protected variant of Peptide B is contacted with a Bronsted acid (preferably a dilute Bronsted acid). In some embodiments, the Bronsted acid is selected from TFA, HOI, and dichloroacetic acid (preferably dilute TFA, HOI, or dichloroacetic acid). In other embodiments, the protected variant of Peptide B is contacted with a Lewis acid (preferably a dilute Lewis acid).10696-WC01-SEC

[0149] In some embodiments, the scavenger is selected from hydrosilanes (e.g., TIS, triethylsilane (TES)), phenols (e.g., phenol, anisole, m-cresol), thioethers (e.g., thioanisole, dimethyl sulfide), thiols / dithiols (e.g., EDT, DTT, 1 -dodecanethiol), water, indole, and combinations of any of the foregoing.

[0150] In some embodiments, the method further comprises contacting the protected variant of Peptide B with HCI. In some embodiments, the method further comprises contacting the protected variant of Peptide B with dilute HCI. In some embodiments, the method further comprises contacting the protected variant of Peptide B with 1 eq. to 10 eq. HCI. In some embodiments, the method further comprises contacting the protected variant of Peptide B with 1 eq. to 5 eq. HCI. In some embodiments, the method further comprises contacting the protected variant of Peptide B with 2 eq. to 5 eq. HCI.

[0151] In some embodiments, the method further comprises contacting the protected variant of Peptide B with TFA in the presence of TIS. In some embodiments, the method further comprises contacting the protected variant of Peptide B with dilute TFA in the presence of TIS.

[0152] In some embodiments, the contacting occurs at a temperature in the range of 0°C to 60°C for a time in the range of 1 hour to 72 hours.

[0153] In some embodiments, the N-terminus of the protected variant of Peptide B formed by coupling the protected variant of Fragment 5 with the protected variant of Fragment 6 is Trt-protected, and the method further comprises contacting the protected variant of Peptide B with a Bronsted acid or a Lewis acid (preferably TFA or HCI), optionally in the presence of a scavenger selected from hydrosilanes, phenols, thioethers, thiols / dithiols, water, indole, and combinations of any of the foregoing (preferably TIS), to selectively remove the trityl protecting group at the N-terminus, wherein the contacting occurs at a temperature in the range of 0°C to 60°C for a time in the range of 1 hour to 72 hours. In some embodiments, the contacting occurs at a temperature in the range of 30°C to 50°C for a time in the range of 6 hour to 24 hours.

[0154] In some embodiments, the N-terminus of the protected variant of Peptide B formed by coupling the protected variant of Fragment 5 with the protected variant of Fragment 6 is Trt-protected, and the method further comprises contacting the protected variant of Peptide B with a dilute Bronsted acid or a dilute Lewis acid (preferably dilute TFA or HCI), optionally in the presence of a scavenger selected from hydrosilanes, phenols, thioethers, thiols / dithiols, water, indole, and combinations of any of the foregoing (preferably TIS), to selectively remove the trityl protecting group at the N-terminus, wherein the contacting occurs at a temperature in the range of 0°C to 60°C for a time in the range of 1 hour to 72 hours. In some embodiments, the contacting occurs at a temperature in the range of 30°C to 50°C for a time in the range of 6 hour to 24 hours.

[0155] In some embodiments, the N-terminus of the protected variant of Peptide B formed by coupling the protected variant of Fragment 5 with the protected variant of Fragment 6 is Trt-protected, and the method further comprises contacting the protected variant of Peptide B with 1 to 10 eq. HCI to selectively remove the trityl protecting group at the N-terminus, wherein the contacting occurs at a temperature in the range of 25°C to 50°C (e.g., 25°C to 35°C, e.g., 30°C) for a time in the range of 6 hour to 36 hours (e.g., 12 hours to 24 hours). In some10696-WQ01-SECembodiments, the N-terminus of the protected variant of Peptide B formed by coupling the protected variant of Fragment 5 with the protected variant of Fragment 6 is Trt-protected, and the method further comprises contacting the protected variant of Peptide B with 2 eq. to 5 eq. HCI to selectively remove the trityl protecting group at the N-terminus, wherein the contacting occurs at a temperature in the range of 25°C to 35°C for a time in the range of 12 hours to 24 hours.

[0156] Also provided herein is a method of preparing Peptide B or a protected variant thereof, comprising coupling a protected variant of Fragment 3A with a protected variant of Fragment 4 to form a protected variant of Peptide B, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9, Fragment 3A comprises the amino acid sequence of SEQ ID NO: 13, and Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4.

[0157] In some embodiments, the protected variant of Fragment 3A is coupled with the protected variant of Fragment 4 in solution. In some embodiments, the protected variant of Fragment 3A is coupled with the protected variant of Fragment 4 under liquid phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 3A is coupled with the protected variant of Fragment 4 under classical solution phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 3A is coupled with the protected variant of Fragment 4 under tag-assisted liquid phase peptide coupling conditions.

[0158] In other embodiments, the protected variant of Fragment 3A is coupled with the protected variant of Fragment 4 on solid support. In some embodiments, the protected variant of Fragment 3A is coupled with the protected variant of Fragment 4 under solid phase peptide coupling conditions.

[0159] In some embodiments, a C-terminal carboxyl group of the protected variant of Fragment 3A is preactivated. In other embodiments, a C-terminal carboxyl group of the protected variant of Fragment 3A is activated in situ.

[0160] In some embodiments, the method comprises:contacting the protected variant of Fragment 3A with a coupling composition to form a protected variant of Fragment 3A with an activated C-terminal carboxyl group ("activated Fragment 3A"); andcontacting the protected variant of Fragment 4 with the activated Fragment 3A to form the protected variant of Peptide B.

[0161] In some embodiments, the method comprises:contacting the protected variant of Fragment 5 with a coupling composition on solid support to form a protected variant of Fragment 5 with an activated C-terminal carboxyl group ("activated Fragment 5"); and contacting the protected variant of Fragment 6 with the activated Fragment 5 on solid support to form the protected variant of Peptide B.10696-WC01-SEC

[0162] In some embodiments, the method comprises:contacting the protected variant of Fragment 3A with a coupling composition in solution to form a protected variant of Fragment 3A with an activated C-terminal carboxyl group ("activated Fragment 3A"); and contacting the protected variant of Fragment 4 with the activated Fragment 3A in solution to form the protected variant of Peptide B.

[0163] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 3A is pre-activated), the method comprises:contacting the protected variant of Fragment 3A with a coupling composition to form a protected variant of Fragment 3A with an activated C-terminal carboxyl group ("activated Fragment 3A"); andcontacting the protected variant of Fragment 4 with the activated Fragment 3A to form the protected variant of Peptide B, wherein the contacting does not occur in the presence of a coupling reagent.

[0164] In some embodiments, the protected variant of Fragment 3A is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 3A and the coupling composition.

[0165] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 3A is pre-activated), the method comprises:contacting the protected variant of Fragment 3A with a coupling composition on solid support to form a protected variant of Fragment 3A with an activated C-terminal carboxyl group ("activated Fragment 3A"); and contacting the protected variant of Fragment 4 with the activated Fragment 3A on solid support to form the protected variant of Peptide B, wherein the contacting does not occur in the presence of a coupling reagent.

[0166] In some embodiments, the protected variant of Fragment 3A is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 3A and the coupling composition.

[0167] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 3A is pre-activated), the method comprises:contacting the protected variant of Fragment 3A with a coupling composition in solution to form a protected variant of Fragment 3A with an activated C-terminal carboxyl group ("activated Fragment 3A"); and contacting the protected variant of Fragment 4 with the activated Fragment 3A in solution to form the protected variant of Peptide B, wherein the contacting does not occur in the presence of a coupling reagent.10696-WC01-SEC

[0168] In some embodiments, the protected variant of Fragment 3A is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 3A and the coupling composition.

[0169] In some embodiments, the contacting of the protected variant of Fragment 3A with the coupling composition and the contacting of the activated Fragment 3A with the protected variant of Fragment 4 occur in the same reaction vessel. In other embodiments, the contacting of the protected variant of Fragment 3A with the coupling composition and the contacting of the activated Fragment 3A with the protected variant of Fragment 4 occur in different reaction vessels.

[0170] In some embodiments, the activated Fragment 3A is contacted with the protected variant of Fragment 4 in the presence of a base and / or a coupling additive but not a coupling reagent (such as, e.g., in certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 3A is pre-activated and the protected variant of Fragment 3A is present in molar excess relative to the coupling reagent in the coupling composition during the contacting to form activated Fragment 3A).

[0171] In some embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 3A is pre-activated, the protected variant of Fragment 4 has a free N-terminal amino group and a free C-terminal carboxyl group.

[0172] In some embodiments, the coupling composition comprises a coupling reagent and a base. In some embodiments, the coupling composition comprises a coupling reagent (e.g., a carbodiimide, e.g., DIG, DCC, EDC) and a coupling additive (e.g., HOBt, 5-CI-HOBt, Oxyma).

[0173] In some embodiments, the coupling composition comprises a coupling reagent, a coupling additive, and a base.

[0174] In some embodiments, the coupling reagent is a carbodiimide, such as, e.g., DIG, DCC, or EDC. In other embodiments, the coupling reagent is BOP-CI.

[0175] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 3A is activated in situ), the method comprises coupling the protected variant of Fragment 3A with the protected variant of Fragment 4 in the presence of a coupling composition. In some embodiments, the method comprises coupling the protected variant of Fragment 3A with the protected variant of Fragment 4 in solution in the presence of a coupling composition.

[0176] In some embodiments, the protected variant of Fragment 4 does not have a free carboxyl group at the C-terminus. In some embodiments, the C-terminus of the protected variant of Fragment 4 is protected. In some embodiments, the C-terminus of the protected variant of Fragment 4 is amidated. In some embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus. In preferred embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and does not have a free carboxyl10696-W001-SECgroup at the C-terminus. In particularly preferred embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and an amidated C-terminus.

[0177] In some embodiments, the protected variant of Fragment 3A does not have a free amino group at the N-terminus. In some embodiments, the N-terminus of the protected variant of Fragment 3A is protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected). In some embodiments, the protected variant of Fragment 3A has a free carboxyl group at the C-terminus. In preferred embodiments, the protected variant of Fragment 3A has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Fragment 3A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected) N-terminus.

[0178] In some embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 3A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected) N-terminus.

[0179] In some embodiments, the C-terminus of the protected variant of Fragment 4 is amidated, and the N-terminus of the protected variant of Fragment 3A is protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected).

[0180] In preferred embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 3A has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 3A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected) N-terminus.

[0181] In some embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 3A has a free carboxyl group at the C-terminus and an Fmoc-protected N-terminus. In some embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 3A has a free carboxyl group at the C-terminus and a Trt-protected N-terminus.

[0182] In some embodiments, the protected variant of Fragment 3A is Fmoc-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 57).

[0183] In some embodiments, the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 109).

[0184] In some embodiments, the protected variant of Fragment 3A is Fmoc-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 57), and the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 109).10696-WQ01-SEC

[0185] In some embodiments, the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0186] In some embodiments, the coupling composition comprises DIC / Oxyma, Py BOP / collidine, PyOxim / collidine, or TBTU / collidine. In some embodiments, the coupling composition comprises DIC / Oxyma, Py BOP / collidine, PyOxim / collidine, or TBTU / collidine in DSMO.

[0187] In some embodiments, the coupling composition comprises DIO and Oxyma. In some embodiments, the coupling composition comprises DIO and Oxyma in DMSO.

[0188] In some embodiments, the coupling composition comprises TBTU and collidine. In some embodiments, the coupling composition comprises TBTU and collidine in DMSO.

[0189] In some embodiments, the protected variant of Fragment 3A is Fmoc-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 57), the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 109), and the coupling composition comprises TBTU / collidine, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0190] In some embodiments, the protected variant of Fragment 3A is Fmoc-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 57), the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 109), and the coupling composition comprises DIC / Oxyma, Py BOP / collidine, PyOxim / collidine, or TBTU / collidine. In some embodiments, the coupling composition comprises DIC / Oxyma, Py BOP / collidine, PyOxim / collidine, or TBTU / collidine in DMSO.

[0191] In some embodiments, the protected variant of Fragment 3A is Fmoc-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 57), the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 109), and the coupling composition comprises DIO and Oxyma. In some embodiments, the coupling composition comprises DIO and Oxyma in DMSO.

[0192] In some embodiments, the protected variant of Fragment 3A is Fmoc-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 57), the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 109), and the coupling composition comprises TBTU and collidine. In some embodiments, the coupling composition comprises TBTU and collidine in DMSO.

[0193] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 30°C. In some embodiments, the coupling occurs at a temperature in the range of 30°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 35°C to 45°C.10696-W001-SEC

[0194] In some embodiments, the method comprises coupling the protected variant of Fragment 3A and the protected variant of Fragment 4 for a time in the range of 1 hour to 72 hours. In some embodiments, the time is in the range of 6 hours to 24 hours.

[0195] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 6 hours to 24 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 6 hours to 24 hours.

[0196] In some embodiments, the protected variant of Fragment 3A is Fmoc-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 57), the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 109), the coupling composition comprises TBTU / collidine, PyBOP / TEA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine (preferably TBTU / collidine or DIC / Oxyma, most preferably DIC / Oxyma), and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours).

[0197] In some embodiments, the protected variant of Fragment 3A is Fmoc-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 57), the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 109), the coupling composition comprises DIC / Oxyma, PyBOP / collidine, PyOxim / collidine, or TBTU / collidine (preferably TBTU / collidine or DIC / Oxyma, most preferably DIC / Oxyma), and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours).

[0198] In some embodiments, the method further comprises selectively deprotecting the N-terminus of the protected variant of Peptide B (e.g., using a Trt, Boc, or Fmoc deprotection method described herein).

[0199] Also provided herein is a method of preparing Peptide E or a protected variant thereof, comprising coupling a protected variant of Fragment 3 with a protected variant of Fragment 4 to form a protected variant of Peptide E, wherein Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of SEQ ID NO: 8.

[0200] In some embodiments, the protected variant of Fragment 3 is coupled with the protected variant of Fragment 4 in solution. In some embodiments, the protected variant of Fragment 3 is coupled with the protected variant of Fragment 4 under liquid phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 3 is coupled with the protected variant of Fragment 4 under classical solution phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 3 is coupled with the protected variant of Fragment 4 under tag-assisted liquid phase peptide coupling conditions.10696-WC01-SEC

[0201] In other embodiments, the protected variant of Fragment 3 is coupled with the protected variant of Fragment 4 on solid support. In some embodiments, the protected variant of Fragment 3 is coupled with the protected variant of Fragment 4 under solid phase peptide coupling conditions.

[0202] In some embodiments, a C-terminal carboxyl group of the protected variant of Fragment 3 is preactivated. In other embodiments, a C-terminal carboxyl group of the protected variant of Fragment 3 is activated in situ.

[0203] In some embodiments, the method comprises:contacting the protected variant of Fragment 3 with a coupling composition to form a protected variant of Fragment 3 with an activated C-terminal carboxyl group ("activated Fragment 3"); andcontacting the protected variant of Fragment 4 with the activated Fragment 3 to form the protected variant of Peptide E.

[0204] In some embodiments, the method comprises:contacting the protected variant of Fragment 3 with a coupling composition on solid support to form a protected variant of Fragment 3 with an activated C-terminal carboxyl group ("activated Fragment 3"); and contacting the protected variant of Fragment 4 with the activated Fragment 3 on solid support to form the protected variant of Peptide E.

[0205] In some embodiments, the method comprises:contacting the protected variant of Fragment 3 with a coupling composition in solution to form a protected variant of Fragment 3 with an activated C-terminal carboxyl group ("activated Fragment 3"); and contacting the protected variant of Fragment 4 with the activated Fragment 3 in solution to form the protected variant of Peptide E.

[0206] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 3 is pre-activated), the method comprises:contacting the protected variant of Fragment 3 with a coupling composition to form a protected variant of Fragment 3 with an activated C-terminal carboxyl group ("activated Fragment 3"); andcontacting the protected variant of Fragment 4 with the activated Fragment 3 to form the protected variant of Peptide E, wherein the contacting does not occur in the presence of a coupling reagent.10696-WC01-SEC

[0207] In some embodiments, the protected variant of Fragment 3 is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 3 and the coupling composition.

[0208] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 3 is pre-activated), the method comprises:contacting the protected variant of Fragment 3 with a coupling composition on solid support to form a protected variant of Fragment 3 with an activated C-terminal carboxyl group ("activated Fragment 3"); and contacting the protected variant of Fragment 4 with the activated Fragment 3 on solid support to form the protected variant of Peptide E, wherein the contacting does not occur in the presence of a coupling reagent.

[0209] In some embodiments, the protected variant of Fragment 3 is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 3 and the coupling composition.

[0210] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 3 is pre-activated), the method comprises:contacting the protected variant of Fragment 3 with a coupling composition in solution to form a protected variant of Fragment 3 with an activated C-terminal carboxyl group ("activated Fragment 3"); and contacting the protected variant of Fragment 4 with the activated Fragment 3 in solution to form the protected variant of Peptide E, wherein the contacting does not occur in the presence of a coupling reagent.

[0211] In some embodiments, the protected variant of Fragment 3 is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 3 and the coupling composition.

[0212] In some embodiments, the contacting of the protected variant of Fragment 3 with the coupling composition and the contacting of the activated Fragment 3 with the protected variant of Fragment 4 occur in the same reaction vessel. In other embodiments, the contacting of the protected variant of Fragment 3 with the coupling composition and the contacting of the activated Fragment 3 with the protected variant of Fragment 4 occur in different reaction vessels.

[0213] In some embodiments, the activated Fragment 3 is contacted with the protected variant of Fragment 4 in the presence of a base and / or a coupling additive but not a coupling reagent (such as, e.g., in certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 3 is pre-activated and the protected variant of Fragment 3 is present in molar excess relative to the coupling reagent in the coupling composition during the contacting to form activated Fragment 3).10696-W001-SEC

[0214] In some embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 3 is pre-activated, the protected variant of Fragment 4 has a free N-terminal amino group and a free C-terminal carboxyl group.

[0215] In some embodiments, the coupling composition comprises a coupling reagent and a base. In some embodiments, the coupling composition comprises a coupling reagent (e.g., a carbodiimide, e.g., DIG, DOC, EDO) and a coupling additive (e.g., HOBt, 5-CI-HOBt, Oxyma).

[0216] In some embodiments, the coupling composition comprises a coupling reagent, a coupling additive, and a base.

[0217] In some embodiments, the coupling reagent is a carbodiimide, such as, e.g., DIG, DOC, or EDO. In other embodiments, the coupling reagent is BOP-CI.

[0218] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 3 is activated in situ), the method comprises coupling the protected variant of Fragment 3 with the protected variant of Fragment 4 in the presence of a coupling composition. In some embodiments, the method comprises coupling the protected variant of Fragment 3 with the protected variant of Fragment 4 in solution in the presence of a coupling composition.

[0219] In some embodiments, the protected variant of Fragment 4 does not have a free carboxyl group at the C-terminus. In some embodiments, the C-terminus of the protected variant of Fragment 4 is protected. In some embodiments, the C-terminus of the protected variant of Fragment 4 is amidated. In some embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus. In preferred embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus. In particularly preferred embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and an amidated C-terminus.

[0220] In some embodiments, the protected variant of Fragment 3 does not have a free amino group at the N-terminus. In some embodiments, the N-terminus of the protected variant of Fragment 3 is protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected). In some embodiments, the protected variant of Fragment 3 has a free carboxyl group at the C-terminus. In preferred embodiments, the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected) N-terminus.

[0221] In some embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected) N-terminus.10696-W001-SEC

[0222] In some embodiments, the C-terminus of the protected variant of Fragment 4 is amidated, and the N-terminus of the protected variant of Fragment 3 is protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected).

[0223] In preferred embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc- or Trt-protected, most preferably Trt-protected) N-terminus.

[0224] In some embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and an Fmoc-protected N-terminus. In some embodiments, the protected variant of Fragment 4 has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and a Trt-protected N-terminus.

[0225] In some embodiments, the protected variant of Fragment 3 is Fmoc-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 56).

[0226] In some embodiments, the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 109).

[0227] In some embodiments, the protected variant of Fragment 3 is Fmoc-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 56), and the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 109).

[0228] In some embodiments, the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0229] In some embodiments, the coupling composition comprises DIC / Oxyma, HCTU / DIEA, or PyOxim / DIEA. In some embodiments, the coupling composition comprises DIC / Oxyma, HCTU / DIEA, or PyOxim / DIEA in DSMO.

[0230] In some embodiments, the coupling composition comprises DIG and Oxyma. In some embodiments, the coupling composition comprises DIO and Oxyma in DMSO.

[0231] In some embodiments, the protected variant of Fragment 3 is Fmoc-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 56), the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 109), and the coupling composition comprises TBTU / collidine, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.10696-W001-SEC

[0232] In some embodiments, the protected variant of Fragment 3 is Fmoc-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 56), the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 109), and the coupling composition comprises DIC / Oxyma, HCTU / DIEA, or PyOxim / DIEA (preferably DIC / Oxyma). In some embodiments, the coupling composition comprises DIC / Oxyma, HCTU / DIEA, or PyOxim / DIEA (preferably DIC / Oxyma) in DMSO.

[0233] In some embodiments, the protected variant of Fragment 3 is Fmoc-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 56), the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 109), and the coupling composition comprises DIG and Oxyma. In some embodiments, the coupling composition comprises DIG and Oxyma in DMSO.

[0234] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 30°C. In some embodiments, the coupling occurs at a temperature in the range of 30°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 35°C to 45°C.

[0235] In some embodiments, the method comprises coupling the protected variant of Fragment 3 and the protected variant of Fragment 4 for a time in the range of 1 hour to 72 hours. In some embodiments, the time is in the range of 6 hours to 24 hours.

[0236] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 6 hours to 24 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 6 hours to 24 hours.

[0237] In some embodiments, the protected variant of Fragment 3 is Fmoc-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 56), the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 109), the coupling composition comprises TBTU / collidine, PyBOP / TEA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine (preferably DIC / Oxyma), and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours).

[0238] In some embodiments, the protected variant of Fragment 3 is Fmoc-Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-OH (SEQ ID NO: 56), the protected variant of Fragment 4 is Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 109), the coupling composition comprises DIC / Oxyma, Py BOP / collidine, PyOxim / collidine, or TBTU / collidine (preferably DIC / Oxyma), and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours).10696-WC01-SEC

[0239] In some embodiments, the method further comprises selectively deprotecting the N-terminus of the protected variant of Peptide E (e.g., using an Fmoc deprotection method described herein).

[0240] Also provided herein is a method of preparing Peptide B or a protected variant thereof, comprising coupling a protected variant of Peptide E with an N-protected alanine to form a protected variant of Peptide B, wherein Peptide E comprises the amino acid sequence of SEQ ID NO: 8 and Peptide B comprises the amino acid sequence of SEQ ID NO: 9.

[0241] In some embodiments, the N-protected alanine is coupled with the protected variant of Peptide E in solution. In some embodiments, the N-protected alanine is coupled with the protected variant of Peptide E under liquid phase peptide coupling conditions. In some embodiments, the N-protected alanine is coupled with the protected variant of Peptide E under classical solution phase peptide coupling conditions. In some embodiments, the N-protected alanine is coupled with the protected variant of Peptide E under tag-assisted liquid phase peptide coupling conditions.

[0242] In other embodiments, the N-protected alanine is coupled with the protected variant of Peptide E on solid support. In some embodiments, the N-protected alanine is coupled with the protected variant of Peptide E under solid phase peptide coupling conditions.

[0243] In some embodiments, a C-terminal carboxyl group of the N-protected alanine is pre-activated. In other embodiments, a C-terminal carboxyl group of the N-protected alanine is activated in situ.

[0244] In some embodiments, the method comprises:contacting the N-protected alanine with a coupling composition to form an N-protected alanine with an activated C-terminal carboxyl group ("activated alanine"); andcontacting the protected variant of Peptide E with the activated alanine to form the protected variant of Peptide B.

[0245] In some embodiments, the method comprises:contacting the N-protected alanine with a coupling composition on solid support to form an N-protected alanine with an activated C-terminal carboxyl group ("activated alanine"); andcontacting the protected variant of Peptide E with the activated alanine on solid support to form the protected variant of Peptide B.

[0246] In some embodiments, the method comprises:contacting the N-protected alanine with a coupling composition in solution to form an N-protected alanine with an activated C-terminal carboxyl group ("activated alanine"); andcontacting the protected variant of Peptide E with the activated alanine in solution to form the protected variant of Peptide B.10696-W001-SEC

[0247] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the N-protected alanine is pre-activated), the method comprises:contacting the N-protected alanine with a coupling composition to form an N-protected alanine with an activated C-terminal carboxyl group ("activated alanine"); andcontacting the protected variant of Peptide E with the activated alanine to form the protected variant of Peptide B, wherein the contacting does not occur in the presence of a coupling reagent.

[0248] In some embodiments, the N-protected alanine is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the N-protected alanine and the coupling composition.

[0249] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the N-protected alanine is pre-activated), the method comprises:contacting the N-protected alanine with a coupling composition on solid support to form an N-protected alanine with an activated C-terminal carboxyl group ("activated alanine"); andcontacting the protected variant of Peptide E with the activated alanine on solid support to form the protected variant of Peptide B, wherein the contacting does not occur in the presence of a coupling reagent.

[0250] In some embodiments, the N-protected alanine is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the N-protected alanine and the coupling composition.

[0251] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the N-protected alanine is pre-activated), the method comprises:contacting the N-protected alanine with a coupling composition in solution to form an N-protected alanine with an activated C-terminal carboxyl group ("activated alanine"); andcontacting the protected variant of Peptide E with the activated alanine in solution to form the protected variant of Peptide B, wherein the contacting does not occur in the presence of a coupling reagent.

[0252] In some embodiments, the N-protected alanine is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the N-protected alanine and the coupling composition.

[0253] In some embodiments, the contacting of the N-protected alanine with the coupling composition and the contacting of the activated alanine with the protected variant of Peptide E occur in the same reaction vessel. In other embodiments, the contacting of the N-protected alanine with the coupling composition and the contacting of the activated alanine with the protected variant of Peptide E occur in different reaction vessels.

[0254] In some embodiments, the activated alanine is contacted with the protected variant of Peptide E in the presence of a base and / or a coupling additive but not a coupling reagent (such as, e.g., in certain10696-WC01-SECembodiments in which a C-terminal carboxyl group of the N-protected alanine is pre-activated and the N-protected alanine is present in molar excess relative to the coupling reagent in the coupling composition during the contacting to form activated alanine).

[0255] In some embodiments in which a C-terminal carboxyl group of the N-protected alanine is preactivated, the protected variant of Peptide E has a free N-terminal amino group and a free C-terminal carboxyl group.

[0256] In some embodiments, the coupling composition comprises a coupling reagent and a base. In some embodiments, the coupling composition comprises a coupling reagent (e.g., a carbodiimide, e.g., DIG, DCC, EDC) and a coupling additive (e.g., HOBt, 5-CI-HOBt, Oxyma).

[0257] In some embodiments, the coupling composition comprises a coupling reagent, a coupling additive, and a base.

[0258] In some embodiments, the coupling reagent is a carbodiimide, such as, e.g., DIG, DCC, or EDC. In other embodiments, the coupling reagent is BOP-CI.

[0259] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the N-protected alanine is activated in situ), the method comprises coupling the N-protected alanine with the protected variant of Peptide E in the presence of a coupling composition. In some embodiments, the method comprises coupling the N-protected alanine with the protected variant of Peptide E in solution in the presence of a coupling composition.

[0260] In some embodiments, the protected variant of Peptide E does not have a free carboxyl group at the C-terminus. In some embodiments, the C-terminus of the protected variant of Peptide E is protected. In some embodiments, the C-terminus of the protected variant of Peptide E is amidated. In some embodiments, the protected variant of Peptide E has a free amino group at the N-terminus. In preferred embodiments, the protected variant of Peptide E has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus. In particularly preferred embodiments, the protected variant of Peptide E has a free amino group at the N-terminus and an amidated C-terminus.

[0261] In some embodiments, the N-protected alanine is Fmoc-protected. In some embodiments, the N-protected alanine has a free carboxyl group at the C-terminus. In preferred embodiments, the N-protected alanine has a free carboxyl group at the C-terminus and an Fmoc-protected N-terminus.

[0262] In some embodiments, the protected variant of Peptide E has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the N-protected alanine has a free carboxyl group at the C-terminus and an Fmoc-protected N-terminus.

[0263] In some embodiments, the C-terminus of the protected variant of Peptide E is amidated, and the N-terminus of the N-protected alanine is Fmoc-protected.10696-WQ01-SEC

[0264] In preferred embodiments, the protected variant of Peptide E has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the N-protected alanine has a free carboxyl group at the C-terminus and an Fmoc- or Trt-protected N-terminus. In particularly preferred embodiments, the protected variant of Peptide E has a free amino group at the N-terminus and an amidated C-terminus, and the N-protected alanine has a free carboxyl group at the C-terminus and an Fmoc-protected N-terminus.

[0265] In some embodiments, the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 160).

[0266] In some embodiments, the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0267] In some embodiments, the N-protected alanine is Fmoc- or Trt-protected, the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 160), and the coupling composition comprises TBTU / collidine, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0268] In some embodiments, the N-protected alanine is Fmoc-protected, the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 160), and the coupling composition comprises TBTU / collidine, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0269] In some embodiments, the N-protected alanine is Trt-protected, the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Glyz-Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 160), and the coupling composition comprises TBTU / collidine, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0270] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 30°C. In some embodiments, the coupling occurs at a temperature in the range of 30°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 35°C to 45°C.

[0271] In some embodiments, the method comprises coupling the N-protected alanine and the protected variant of Peptide E for a time in the range of 1 hour to 72 hours. In some embodiments, the time is in the range of 6 hours to 24 hours.10696-W001-SEC

[0272] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 6 hours to 24 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 6 hours to 24 hours.

[0273] In some embodiments, the N-protected alanine is Fmoc- or Trt-protected, the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Gly7-Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 160), the coupling composition comprises TBTU / collidine, PyBOP / TEA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine, and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours).

[0274] In some embodiments, the N-protected alanine is Fmoc-protected, the protected variant of Peptide E isTrp(Boc)-Leu-Val-Lys(Boc)-Gly7-Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 160), the coupling composition comprises TBTU / collidine, PyBOP / TEA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine, and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours).

[0275] In some embodiments, the N-protected alanine is Trt-protected, the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Gly7-Ser(tBu)-Gly4-Ser(tBu)-Gly4-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 160), the coupling composition comprises TBTU / collidine, PyBOP / TEA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine, and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours).

[0276] In some embodiments, the method further comprises selectively deprotecting the N-terminus of the protected variant of Peptide B (e.g., using an Fmoc or Trt deprotection method described herein).

[0277] Also provided herein is a method of preparing Peptide B or a protected variant thereof, comprising:coupling or having coupled a protected variant of Fragment 3 with a protected variant of Fragment 4 to form a first protected variant of Peptide E, wherein the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Fragment 4 has a free amino group at the N-terminus, Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of SEQ ID NO: 8;10696-WC01-SECselectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide E to form a second protected variant of Peptide E with a free amino group at the N-terminus; and coupling or having coupled the second protected variant of Peptide E with an N-protected alanine to form a protected variant of Peptide B, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9.

[0278] In some embodiments, the protected variant of Fragment 3 and the protected variant of Fragment 4 are coupled under conditions described herein.

[0279] In some embodiments, the second protected variant of Peptide E and the N-protected alanine are coupled under conditions described herein.

[0280] In some embodiments, the protected variant of Fragment 3 and the protected variant of Fragment 4 are coupled under conditions described above, and the second protected variant of Peptide E and the N-protected alanine are coupled under conditions described herein.

[0281] In some embodiments, the N-terminus of the first protected variant of Peptide E is Fmoc-protected, and the selective deprotection comprises contacting the first protected variant of Peptide E with an amine base to selectively remove the Fmoc protecting group at the N-terminus. In some embodiments, the amine base is selected from piperidine, piperazine, and morpholine (e.g., 5-30% (v / v) piperidine, piperazine, or morpholine). In some embodiments, the amine base is piperidine. In some embodiments, the N-terminus of the first protected variant of Peptide E is Fmoc-protected, and the selective deprotection comprises contacting the first protected variant of Peptide E with piperidine in DMF, optionally in the presence of DBU.

[0282] In some embodiments, the N-terminus of the first protected variant of Peptide E is Boc-protected, and the selective deprotection comprises contacting the first protected variant of Peptide E with an acid (preferably an acid selected from TFA, HCI, and HBr) to selectively remove the Boc protecting group at the N-terminus. In some embodiments, the first protected variant of Peptide E is contacted with TFA (e.g., neat TFA or TFA in DCM, e.g., 20-50% (v / v) TFA in DCM). In some embodiments, the first protected variant of Peptide E is contacted with HCI (e.g., HCI in DCM, DCE, toluene, or dioxane, e.g., 3-4 M HCI in DCM or dioxane).

[0283] In some embodiments, the N-terminus of the first protected variant of Peptide E is Boc-protected, and the selective deprotection comprises contacting the first protected variant of Peptide E with an acid (preferably an acid selected from TFA, HCI, and HBr) in the presence of a scavenger. In some embodiments, the scavenger is selected from hydrosilanes (e.g., TIS, triethylsilane (TES)), phenols (e.g., phenol, anisole, m-cresol), thioethers (e.g., thioanisole, dimethyl sulfide), thiols / dithiols (e.g., EDT, DTT, 1 -dodecanethiol), water, indole, and combinations of any of the foregoing. In some preferred embodiments, the scavenger is a hydrosilane. In some particularly preferred embodiments, the scavenger is TIS. In some embodiments, the first protected variant of Peptide E is contacted with TFA (e.g., neat TFA or TFA in DCM, e.g., 20-50% (v / v) TFA in DCM) in the presence of a scavenger (preferably TIS). In some embodiments, the first protected variant of Peptide E is contacted with10696-WQ01-SECHCI (e.g., HCI in DCM, DCE, toluene, or dioxane, e.g., 3-4 M HCI in DCM or dioxane) in the presence of a scavenger.

[0284] In some embodiments, the N-terminus of the first protected variant of Peptide E is Trt-protected, and the selective deprotection comprises contacting the first protected variant of Peptide E with a Bronsted acid or a Lewis acid (preferably TFA, HCI, or dichloroacetic acid, most preferably TFA), optionally in the presence of a scavenger (preferably TIS), to selectively remove the trityl protecting group at the N-terminus. In some embodiments, the first protected variant of Peptide E is contacted with a Bronsted acid (preferably a dilute Bronsted acid). In some embodiments, the Bronsted acid is selected from TFA, HCI, and dichloroacetic acid (preferably dilute TFA, HCI, or dichloroacetic acid). In other embodiments, the first protected variant of Peptide E is contacted with a Lewis acid (preferably a dilute Lewis acid).

[0285] In some embodiments, the scavenger is selected from hydrosilanes (e.g., TIS, triethylsilane (TES)), phenols (e.g., phenol, anisole, m-cresol), thioethers (e.g., thioanisole, dimethyl sulfide), thiols / dithiols (e.g., EDT, DTT, 1 -dodecanethiol), water, indole, and combinations of any of the foregoing. In some embodiments, the scavenger is a hydrosilane. In some embodiments, the scavenger is TIS.

[0286] In some embodiments, the N-terminus of the first protected variant of Peptide E is Trt-protected, and the selective deprotection comprises contacting the first protected variant of Peptide E with HCI. In some embodiments, the selective deprotection comprises contacting the first protected variant of Peptide E with dilute HCI.

[0287] In some embodiments, the N-terminus of the first protected variant of Peptide E is Trt-protected, and the selective deprotection comprises contacting the first protected variant of Peptide E with TFA in the presence of TIS. In some embodiments, the selective deprotection comprises contacting the first protected variant of Peptide E with dilute TFA in the presence of TIS.

[0288] Also provided herein is a method of preparing Peptide D or a protected variant thereof, comprising coupling a protected variant of Fragment 1 with a protected variant of Fragment 2B in to form a protected variant of Peptide D, wherein Peptide D comprises the amino acid sequence of SEQ ID NO: 15, Fragment 1 comprises the amino acid sequence of SEQ ID NO: 1, and Fragment 2B comprises the amino acid sequence of SEQ ID NO: 16.

[0289] In some embodiments, the protected variant of Fragment 1 is coupled with the protected variant of Fragment 2B in solution. In some embodiments, the protected variant of Fragment 1 is coupled with the protected variant of Fragment 2B under liquid phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 1 is coupled with the protected variant of Fragment 2B under classical solution phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 1 is coupled with the protected variant of Fragment 2B under tag-assisted liquid phase peptide coupling conditions.10696-WC01-SEC

[0290] In other embodiments, the protected variant of Fragment 1 is coupled with the protected variant of Fragment 2B on solid support. In some embodiments, the protected variant of Fragment 1 is coupled with the protected variant of Fragment 2B under solid phase peptide coupling conditions.

[0291] In some embodiments, a C-terminal carboxyl group of the protected variant of Fragment 1 is preactivated. In other embodiments, a C-terminal carboxyl group of the protected variant of Fragment 1 is activated in situ.

[0292] In some embodiments, the method comprises:contacting the protected variant of Fragment 1 with a coupling composition to form a protected variant of Fragment 1 with an activated C-terminal carboxyl group ("activated Fragment 1"); andcontacting the protected variant of Fragment 2B with the activated Fragment 1 to form the protected variant of Peptide D.

[0293] In some embodiments, the method comprises:contacting the protected variant of Fragment 1 with a coupling composition on solid support to form a protected variant of Fragment 1 with an activated C-terminal carboxyl group ("activated Fragment 1"); and contacting the protected variant of Fragment 2B with the activated Fragment 1 on solid support to form the protected variant of Peptide D.

[0294] In some embodiments, the method comprises:contacting the protected variant of Fragment 1 with a coupling composition in solution to form a protected variant of Fragment 1 with an activated C-terminal carboxyl group ("activated Fragment 1"); and contacting the protected variant of Fragment 2B with the activated Fragment 1 in solution to form the protected variant of Peptide D.

[0295] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 1 is pre-activated), the method comprises:contacting the protected variant of Fragment 1 with a coupling composition to form a protected variant of Fragment 1 with an activated C-terminal carboxyl group ("activated Fragment 1"); andcontacting the protected variant of Fragment 2B with the activated Fragment 1 to form the protected variant of Peptide D, wherein the contacting does not occur in the presence of a coupling reagent.10696-WC01-SEC

[0296] In some embodiments, the protected variant of Fragment 1 is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 1 and the coupling composition.

[0297] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 1 is pre-activated), the method comprises:contacting the protected variant of Fragment 1 with a coupling composition on solid support to form a protected variant of Fragment 1 with an activated C-terminal carboxyl group ("activated Fragment 1"); and contacting the protected variant of Fragment 2B with the activated Fragment 1 on solid support to form the protected variant of Peptide D, wherein the contacting does not occur in the presence of a coupling reagent.

[0298] In some embodiments, the protected variant of Fragment 1 is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 1 and the coupling composition.

[0299] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 1 is pre-activated), the method comprises:contacting the protected variant of Fragment 1 with a coupling composition in solution to form a protected variant of Fragment 1 with an activated C-terminal carboxyl group ("activated Fragment 1"); and contacting the protected variant of Fragment 2B with the activated Fragment 1 in solution to form the protected variant of Peptide D, wherein the contacting does not occur in the presence of a coupling reagent.

[0300] In some embodiments, the protected variant of Fragment 1 is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 1 and the coupling composition.

[0301] In some embodiments, the contacting of the protected variant of Fragment 1 with the coupling composition and the contacting of the activated Fragment 1 with the protected variant of Fragment 2B occur in the same reaction vessel. In other embodiments, the contacting of the protected variant of Fragment 1 with the coupling composition and the contacting of the activated Fragment 1 with the protected variant of Fragment 2B occur in different reaction vessels.

[0302] In some embodiments, the activated Fragment 1 is contacted with the protected variant of Fragment 2B in the presence of a base and / or a coupling additive but not a coupling reagent (such as, e.g., in certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 1 is pre-activated and the protected variant of Fragment 1 is present in molar excess relative to the coupling reagent in the coupling composition during the contacting to form activated Fragment 1).10696-W001-SEC

[0303] In some embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 1 is pre-activated, the protected variant of Fragment 2B has a free N-terminal amino group and a free C-terminal carboxyl group.

[0304] In some embodiments, the coupling composition comprises a coupling reagent and a base. In some embodiments, the coupling composition comprises a coupling reagent (e.g., a carbodiimide, e.g., DIG, DOC, EDO) and a coupling additive (e.g., HOBt, 5-CI-HOBt, Oxyma).

[0305] In some embodiments, the coupling composition comprises a coupling reagent, a coupling additive, and a base.

[0306] In some embodiments, the coupling reagent is a carbodiimide, such as, e.g., DIG, DOC, or EDO. In other embodiments, the coupling reagent is BOP-CI.

[0307] In some embodiments, a protected variant of Peptide D can be prepared in solution by coupling a protected variant of Fragment 2B and a protected variant of Fragment 1 as shown in FIG. 3A. FIG. 3B depicts another protected variant of Peptide D which can be prepared by an analogous process to that shown in FIG.3A. Alternatively, a protected variant of Peptide D can be efficiently prepared in solution by coupling a protected variant of Fragment 2B and a protected variant of Fragment 1 as shown in FIG. 3C.

[0308] In some embodiments, the protected variant of Fragment 1 is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 166) or Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 167), the protected variant of Fragment 2B is Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 30), the coupling composition comprises Py BOP / collidine, DIC / Oxyma, DIC / Oxyma / collidine, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HCTU / 6-CI-HOBt / collidine, EDC / HOBt, EDC / HOBt / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine (preferably Py BOP / collidine), and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C, more preferably a temperature in the range of 20°C to 30°C) for a time in the range of 30 minutes to 72 hours (preferably a time in the range of 30 minutes to 24 hours).

[0309] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 1 is activated in situ), the method comprises coupling the protected variant of Fragment 1 with the protected variant of Fragment 2B in the presence of a coupling composition. In some embodiments, the method comprises coupling the protected variant of Fragment 1 with the protected variant of Fragment 2B in solution in the presence of a coupling composition.

[0310] In some embodiments, the protected variant of Fragment 2B does not have a free carboxyl group at the C-terminus. In some embodiments, the C-terminus of the protected variant of Fragment 2B is protected. In some embodiments, the protected variant of Fragment 2B has a free amino group at the N-terminus. In preferred10696-WC01-SECembodiments, the protected variant of Fragment 2B has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus. In particularly preferred embodiments, the protected variant of Fragment 2B has a free amino group at the N-terminus and a protected C-terminus.

[0311] In some embodiments, the protected variant of Fragment 1 does not have a free amino group at the N-terminus. In some embodiments, the N-terminus of the protected variant of Fragment 1 is protected (preferably Boc-protected). In some embodiments, the protected variant of Fragment 1 has a free carboxyl group at the C-terminus. In preferred embodiments, the protected variant of Fragment 1 has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Fragment 1 has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus.

[0312] In some embodiments, the protected variant of Fragment 2B has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 1 has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus.

[0313] In some embodiments, the C-terminus of the protected variant of Fragment 2B is protected, and the N-terminus of the protected variant of Fragment 1 is protected (preferably Boc-protected).

[0314] In preferred embodiments, the protected variant of Fragment 2B has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 1 has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Fragment 2B has a free amino group at the N-terminus and a protected C-terminus, and the protected variant of Fragment 1 has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus.

[0315] In some embodiments, the N-terminus, the side chain of the His residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Thr residue at position 5, the side chain of the Thr residue at position 7, the side chain of the Ser residue at position 8, the side chain of the Asp residue at position 9, the side chain of the Tyr residue at position 10, the side chain of the Ser residue at position 11, and the side chain of the Ser residue at position 12 of the protected variant of Fragment 1 are protected and the protected variant of Fragment 1 has a free carboxyl group at the C-terminus.

[0316] In some embodiments, the side chain of the Tyr residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Glu residue at position 4, the side chain of the Gin residue at position 5, the side chain of the Lys residue at position 8, and the side chain of the Glu residue at position 9 of the protected variant of Fragment 2B are protected and the protected variant of Fragment 2B has a free amino group at the N-terminus.

[0317] In some embodiments, the N-terminus, the side chain of the His residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Thr residue at position 5, the side chain of the Thr residue10696-W001-SECat position 7, the side chain of the Ser residue at position 8, the side chain of the Asp residue at position 9, the side chain of the Tyr residue at position 10, the side chain of the Ser residue at position 11, and the side chain of the Ser residue at position 12 of the protected variant of Fragment 1 are protected, the protected variant of Fragment 1 has a free carboxyl group at the C-terminus, the side chain of the Tyr residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Glu residue at position 4, the side chain of the Gin residue at position 5, the side chain of the Lys residue at position 8, and the side chain of the Glu residue at position 9 of the protected variant of Fragment 2B are protected, and the protected variant of Fragment 2B has a free amino group at the N-terminus.

[0318] In some embodiments, the side chain of the His residue at position 1 of the protected variant of Fragment 1 is protected with a trityl (Trt) protecting group; the side chain of the Glu residue at position 3 of the protected variant of Fragment 1 is protected with a tert-butyl ester (OtBu) protecting group; the side chains of the Thr residues at position 5 and position 7 of the protected variant of Fragment 1 are each protected with a tertbutyl (tBu) protecting group; the side chains of the Ser residues at position 8 and position 11 of the protected variant of Fragment 1 are each protected with a tBu protecting group; the side chain of the Asp residue at position 9 of the protected variant of Fragment 1 is protected with an OtBu protecting group; the side chain of the Tyr residue at position 10 of the protected variant of Fragment 1 is protected with a tBu protecting group; and the Ser residue at position 12 of the protected variant of Fragment 1 is protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser.

[0319] In some embodiments, the side chain of the Tyr residue at position 1 of the protected variant of Fragment 2B is protected with a tBu protecting group; the side chains of the Glu residues at position 3, position 4, and position 9 of the protected variant of Fragment 2B are each protected with a OtBu protecting group; the side chain of the Gin residue at position 5 of the protected variant of Fragment 2B is protected with a Trt protecting group; and the side chain of the Lys residue at position 8 of the protected variant of Fragment 2B is protected with a tert-butyloxycarbonyl (Boc) protecting group.

[0320] In some embodiments, the side chain of the His residue at position 1 of the protected variant of Fragment 1 is protected with a trityl (Trt) protecting group; the side chain of the Glu residue at position 3 of the protected variant of Fragment 1 is protected with a tert-butyl ester (OtBu) protecting group; the side chains of the Thr residues at position 5 and position 7 of the protected variant of Fragment 1 are each protected with a tertbutyl (tBu) protecting group; the side chains of the Ser residues at position 8 and position 11 of the protected variant of Fragment 1 are each protected with a tBu protecting group; the side chain of the Asp residue at position 9 of the protected variant of Fragment 1 is protected with an OtBu protecting group; the side chain of the Tyr residue at position 10 of the protected variant of Fragment 1 is protected with a tBu protecting group; the Ser residue at position 12 of the protected variant of Fragment 1 is protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser; the side chain of the Tyr residue at position 1 of the protected variant of Fragment 2B is protected with a tBu protecting group; the side chains of the Glu residues at position 3, position 4, and position 9 of the protected variant of Fragment 2B are each protected with10696-WQ01-SECa OtBu protecting group; the side chain of the Gin residue at position 5 of the protected variant of Fragment 2B is protected with a Trt protecting group; and the side chain of the Lys residue at position 8 of the protected variant of Fragment 2B is protected with a tert-butyloxycarbonyl (Boc) protecting group.

[0321] In some embodiments, the protected variant of Fragment 1 is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 166) or Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 167). In some embodiments, the protected variant of Fragment 1 is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 166). In some embodiments, the protected variant of Fragment 1 is Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 167).

[0322] In some embodiments, the protected variant of Fragment 2B is Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-l le-X, wherein X is a C-terminal protecting group (SEQ ID NO: 46).

[0323] In some embodiments, the protected variant of Fragment 1 is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 166) or Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 167), and the protected variant of Fragment 2B is Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-X, wherein X is a C-terminal protecting group (SEQ ID NO: 46). In some embodiments, the protected variant of Fragment 1 is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 166), and the protected variant of Fragment 2B is Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-X, wherein X is a C-terminal protecting group (SEQ ID NO: 46). In some embodiments, the protected variant of Fragment 1 is Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 167), and the protected variant of Fragment 2B is Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-X, wherein X is a C-terminal protecting group (SEQ ID NO: 46).

[0324] In some embodiments, the coupling composition comprises 1 to 10 equivalents of a coupling reagent, optionally 1 to 10 equivalents of a base, and optionally 1 to 10 equivalents of a coupling additive, wherein equivalents are relative to the protected variant of Fragment 2B. In some embodiments, the coupling composition comprises 1 to 5 equivalents of a coupling reagent, optionally 1 to 5 equivalents of a base, and optionally 1 to 5 equivalents of a coupling additive, wherein equivalents are relative to the protected variant of Fragment 2B. In some embodiments, the coupling composition comprises 2 to 10 equivalents of a coupling reagent, optionally 2 to 10 equivalents of a base, and optionally 2 to 10 equivalents of a coupling additive, wherein equivalents are relative to the protected variant of Fragment 2B.

[0325] In some embodiments, the coupling composition comprises Py BOP / collidine, DIC / Oxyma, DIC / Oxyma / collidine, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HCTU / 6-CI-HOBt / collidine, EDC / HOBt,10696-W001-SECEDC / HOBt / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine. In some embodiments, the coupling composition comprises Py BOP / collidine, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0326] In some embodiments, the coupling composition comprises PyBOP and collidine. In some embodiments, the coupling composition comprises PyBOP and collidine in THF and DMSO.

[0327] In some embodiments, the coupling composition comprises EDO and HOBt. In some embodiments, the coupling composition comprises EDO, HOBt, and collidine.

[0328] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 30°C. In some embodiments, the coupling occurs at a temperature in the range of 30°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 35°C to 45°C.

[0329] In some embodiments, the method comprises coupling the protected variant of Fragment 1 and the protected variant of Fragment 2B for a time in the range of 30 minutes to 72 hours. In some embodiments, the time is in the range of 30 minutes to 24 hours.

[0330] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 30 minutes to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 30 minutes to 24 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 30 minutes to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 30 minutes to 24 hours.

[0331] In some embodiments, the protected variant of Fragment 1 is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 166) or Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 167), the protected variant of Fragment 2B is Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-X, wherein X is a C-terminal protecting group (SEQ ID NO: 46), the coupling composition comprises Py BOP / collidine, DIC / Oxyma, DIC / Oxyma / collidine, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HCTU / 6-CI-HOBt / collidine, EDC / HOBt, EDC / HOBt / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine (preferably Py BOP / coll idine), and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C, more preferably a temperature in the range of 20°C to 30°C) for a time in the range of 30 minutes to 72 hours (preferably a time in the range of 30 minutes to 24 hours).10696-WQ01-SEC

[0332] In some embodiments, the method further comprises selectively deprotecting the N-terminus of the protected variant of Peptide D (e.g., using a Boc or Fmoc deprotection method described herein).

[0333] Also provided herein is a method of preparing Peptide A or a protected variant thereof, comprising coupling a protected variant of Peptide B with a protected variant of Peptide D to form a protected variant of Peptide A, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9, Peptide D comprises the amino acid sequence of SEQ ID NO: 15, and Peptide A comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the method further comprises deprotecting the protected variant of Peptide A to isolate Peptide A. In some embodiments, the method further comprises adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0334] In some embodiments, the protected variant of Peptide B is coupled with the protected variant of Peptide D in solution. In some embodiments, the protected variant of Peptide B is coupled with the protected variant of Peptide D under liquid phase peptide coupling conditions. In some embodiments, the protected variant of Peptide B is coupled with the protected variant of Peptide D under classical solution phase peptide coupling conditions. In some embodiments, the protected variant of Peptide B is coupled with the protected variant of Peptide D under tag-assisted liquid phase peptide coupling conditions.

[0335] In other embodiments, the protected variant of Peptide B is coupled with the protected variant of Peptide D on solid support. In some embodiments, the protected variant of Peptide B is coupled with the protected variant of Peptide D under solid phase peptide coupling conditions.

[0336] In some embodiments, a C-terminal carboxyl group of the protected variant of Peptide D is preactivated. In other embodiments, a C-terminal carboxyl group of the protected variant of Peptide D is activated in situ.

[0337] In some embodiments, the method comprises:contacting the protected variant of Peptide D with a coupling composition to form a protected variant of Peptide D with an activated C-terminal carboxyl group ("activated Peptide D"); andcontacting the protected variant of Peptide B with the activated Peptide D to form the protected variant of Peptide A.

[0338] In some embodiments, the method comprises:contacting the protected variant of Peptide D with a coupling composition on solid support to form a protected variant of Peptide D with an activated C-terminal carboxyl group ("activated Peptide D"); and contacting the protected variant of Peptide B with the activated Peptide D on solid support to form the protected variant of Peptide A.10696-WC01-SEC

[0339] In some embodiments, the method comprises:contacting the protected variant of Peptide D with a coupling composition in solution to form a protected variant of Peptide D with an activated C-terminal carboxyl group ("activated Peptide D"); andcontacting the protected variant of Peptide B with the activated Peptide D in solution to form the protected variant of Peptide A.

[0340] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Peptide D is pre-activated), the method comprises:contacting the protected variant of Peptide D with a coupling composition to form a protected variant of Peptide D with an activated C-terminal carboxyl group ("activated Peptide D"); andcontacting the protected variant of Peptide B with the activated Peptide D to form the protected variant of Peptide A, wherein the contacting does not occur in the presence of a coupling reagent.

[0341] In some embodiments, the protected variant of Peptide D is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Peptide D and the coupling composition.

[0342] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Peptide D is pre-activated), the method comprises:contacting the protected variant of Peptide D with a coupling composition on solid support to form a protected variant of Peptide D with an activated C-terminal carboxyl group ("activated Peptide D"); and contacting the protected variant of Peptide B with the activated Peptide D on solid support to form the protected variant of Peptide A, wherein the contacting does not occur in the presence of a coupling reagent.

[0343] In some embodiments, the protected variant of Peptide D is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Peptide D and the coupling composition.

[0344] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Peptide D is pre-activated), the method comprises:contacting the protected variant of Peptide D with a coupling composition in solution to form a protected variant of Peptide D with an activated C-terminal carboxyl group ("activated Peptide D"); andcontacting the protected variant of Peptide B with the activated Peptide D in solution to form the protected variant of Peptide A, wherein the contacting does not occur in the presence of a coupling reagent.10696-WC01-SEC

[0345] In some embodiments, the protected variant of Peptide D is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Peptide D and the coupling composition.

[0346] In some embodiments, the contacting of the protected variant of Peptide D with the coupling composition and the contacting of the activated Peptide D with the protected variant of Peptide B occur in the same reaction vessel. In other embodiments, the contacting of the protected variant of Peptide D with the coupling composition and the contacting of the activated Peptide D with the protected variant of Peptide B occur in different reaction vessels.

[0347] In some embodiments, the activated Peptide D is contacted with the protected variant of Peptide B in the presence of a base and / or a coupling additive but not a coupling reagent (such as, e.g., in certain embodiments in which a C-terminal carboxyl group of the protected variant of Peptide D is pre-activated and the protected variant of Peptide D is present in molar excess relative to the coupling reagent in the coupling composition during the contacting to form activated Peptide D).

[0348] In some embodiments in which a C-terminal carboxyl group of the protected variant of Peptide D is pre-activated, the protected variant of Peptide B has a free N-terminal amino group and a free C-terminal carboxyl group.

[0349] In some embodiments, the coupling composition comprises a coupling reagent and a base. In some embodiments, the coupling composition comprises a coupling reagent (e.g., a carbodiimide, e.g., DIG, DCC, EDC) and a coupling additive (e.g., HOBt, 5-CI-HOBt, Oxyma).

[0350] In some embodiments, the coupling composition comprises a coupling reagent, a coupling additive, and a base.

[0351] In some embodiments, the coupling reagent is a carbodiimide, such as, e.g., DIG, DCC, or EDC. In other embodiments, the coupling reagent is BOP-CI.

[0352] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Peptide D is activated in situ), the method comprises coupling the protected variant of Peptide D with the protected variant of Peptide B in the presence of a coupling composition. In some embodiments, the method comprises coupling the protected variant of Peptide D with the protected variant of Peptide B in solution in the presence of a coupling composition.

[0353] In some embodiments, the protected variant of Peptide B does not have a free carboxyl group at the C-terminus. In some embodiments, the C-terminus of the protected variant of Peptide B is protected. In some embodiments, the C-terminus of the protected variant of Peptide B is amidated. In some embodiments, the protected variant of Peptide B has a free amino group at the N-terminus. In preferred embodiments, the protected variant of Peptide B has a free amino group at the N-terminus and does not have a free carboxyl group10696-WC01-SECat the C-terminus. In particularly preferred embodiments, the protected variant of Peptide B has a free amino group at the N-terminus and an amidated C-terminus.

[0354] In some embodiments, the protected variant of Peptide D does not have a free amino group at the N-terminus. In some embodiments, the N-terminus of the protected variant of Peptide D is protected (preferably Boc-protected). In some embodiments, the protected variant of Peptide D has a free carboxyl group at the C-terminus. In preferred embodiments, the protected variant of Peptide D has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Peptide D has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus.

[0355] In some embodiments, the protected variant of Peptide B has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Peptide D has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus.

[0356] In some embodiments, the C-terminus of the protected variant of Peptide B is amidated, and the N-terminus of the protected variant of Peptide D is protected (preferably Boc-protected).

[0357] In preferred embodiments, the protected variant of Peptide B has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Peptide D has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Peptide B has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Peptide D has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus.

[0358] In some embodiments, the side chain of the Trp residue at position 2, the side chain of the Lys residue at position 5, the side chain of the Ser residue at position 13, the side chain of the Ser residue at position 18, the side chain of the Ser residue at position 23, and the side chain of the Lys residue at position 24 of the protected variant of Peptide B are protected, the protected variant of Peptide B has a free amino group at the N-terminus, and the protected variant of Peptide B does not have a free carboxyl group at the C-terminus.

[0359] In some embodiments, the N-terminus, the side chain of the His residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Thr residue at position 5, the side chain of the Thr residue at position 7, the side chain of the Ser residue at position 8, the side chain of the Asp residue at position 9, the side chain of the Tyr residue at position 10, the side chain of the Ser residue at position 11, the side chain of the Ser residue at position 12, the side chain of the Tyr residue at position 13, the side chain of the Glu residue at position 15, the side chain of the Glu residue at position 16, the side chain of the Gin residue at position 17, the side chain of the Lys residue at position 20, and the side chain of the Glu residue at position 21 of the protected variant of Peptide D are protected and the protected variant of Peptide D has a free carboxyl group at the C-terminus.10696-W001-SEC

[0360] In some embodiments, the side chain of the Trp residue at position 2, the side chain of the Lys residue at position 5, the side chain of the Ser residue at position 13, the side chain of the Ser residue at position 18, the side chain of the Ser residue at position 23, and the side chain of the Lys residue at position 24 of the protected variant of Peptide B are protected, the protected variant of Peptide B has a free amino group at the N-terminus, the protected variant of Peptide B does not have a free carboxyl group at the C-terminus, the N-terminus, the side chain of the His residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Thr residue at position 5, the side chain of the Thr residue at position 7, the side chain of the Ser residue at position 8, the side chain of the Asp residue at position 9, the side chain of the Tyr residue at position 10, the side chain of the Ser residue at position 11, the side chain of the Ser residue at position 12, the side chain of the Tyr residue at position 13, the side chain of the Glu residue at position 15, the side chain of the Glu residue at position 16, the side chain of the Gin residue at position 17, the side chain of the Lys residue at position 20, and the side chain of the Glu residue at position 21 of the protected variant of Peptide D are protected, and the protected variant of Peptide D has a free carboxyl group at the C-terminus.

[0361] In some embodiments, the side chain of the Trp residue at position 2 and the side chain of the Lys residue at position 5 of the protected variant of Peptide B are each protected with a tert-butyloxycarbonyl (Boc) protecting group, the Ser residues at position 13, position 18, and position 23 of the protected variant of Peptide B are each protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser, and the side chain of the Lys residue at position 24 of the protected variant of Peptide B is protected with an ivDde (1 -(4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl) protecting group.

[0362] In some embodiments, the side chain of the His residue at position 1 of Peptide D is protected with a trityl (Trt) protecting group; the side chains of the Glu residues at position 3, position 15, position 16, and position 21 of Peptide D are each protected with a tert-butyl ester (OtBu) protecting group; the side chains of the Thr residues at position 5 and position 7 of Peptide D are each protected with a tert-butyl (tBu) protecting group; the side chains of the Ser residues at position 8 and position 11 of Peptide D are each protected with a tBu protecting group; the side chain of the Asp residue at position 9 of Peptide D is protected with an OtBu protecting group; the side chains of the Tyr residues at positions 10 and 13 of Peptide D are each protected with a tBu protecting group; the Ser residue at position 12 of Peptide D is protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser; the side chain of the Gin residue at position 17 of Peptide D is protected with a Trt protecting group; and the side chain of the Lys residue at position 20 of Peptide D is protected with a tert-butyloxycarbonyl (Boc) protecting group.

[0363] In some embodiments, the side chain of the Trp residue at position 2 and the side chain of the Lys residue at position 5 of the protected variant of Peptide B are each protected with a tert-butyloxycarbonyl (Boc) protecting group, the Ser residues at position 13, position 18, and position 23 of the protected variant of Peptide B are each protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser, the side chain of the Lys residue at position 24 of the protected variant of Peptide B is protected with an ivDde (1 -(4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl) protecting group, the side10696-WQ01-SECchain of the His residue at position 1 of Peptide D is protected with a trityl (Trt) protecting group; the side chains of the Glu residues at position 3, position 15, position 16, and position 21 of Peptide D are each protected with a tert-butyl ester (OtBu) protecting group; the side chains of the Thr residues at position 5 and position 7 of Peptide D are each protected with a tert-butyl (tBu) protecting group; the side chains of the Ser residues at position 8 and position 11 of Peptide D are each protected with a tBu protecting group; the side chain of the Asp residue at position 9 of Peptide D is protected with an OtBu protecting group; the side chains of the Tyr residues at positions 10 and 13 of Peptide D are each protected with a tBu protecting group; the Ser residue at position 12 of Peptide D is protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser; the side chain of the Gin residue at position 17 of Peptide D is protected with a Trt protecting group; and the side chain of the Lys residue at position 20 of Peptide D is protected with a tert-butyloxycarbonyl (Boc) protecting group.

[0364] In some embodiments, the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 147).

[0365] In some embodiments, the protected variant of Peptide D is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 157) or Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-l le-OH (SEQ ID NO: 168). In some embodiments, the protected variant of Peptide D is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 157). In other embodiments, the protected variant of Peptide D is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 168).

[0366] In some embodiments, the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2 (SEQ ID NO: 147), and the protected variant of Peptide D is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 157) or Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 168).

[0367] In some embodiments, the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2 (SEQ ID NO: 147), and the protected variant of Peptide D is Boc-His(Boc)-10696-W001-SECAib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 157).

[0368] In some embodiments, the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2 (SEQ ID NO: 147), and the protected variant of Peptide D is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 168).

[0369] In some embodiments, a protected variant of Peptide B with an amidated C-terminus and a protected variant of Peptide D can be coupled in solution to form a protected variant of Peptide A with an amidated C-terminus as shown in FIGs. 4A and 4B.

[0370] In some embodiments, the coupling composition comprises 1 to 10 equivalents of a coupling reagent, optionally 1 to 10 equivalents of a base, and optionally 1 to 10 equivalents of a coupling additive, wherein equivalents are relative to the protected variant of Peptide D. In some embodiments, the coupling composition comprises 1 to 5 equivalents of a coupling reagent, optionally 1 to 10 equivalents of a base, and optionally 1 to 5 equivalents of a coupling additive, wherein equivalents are relative to the protected variant of Peptide D.

[0371] In some embodiments, the coupling composition comprises DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, EDC / HOBt / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, Oxyma / PyOxim / collidine, Py BOP / HOBt / collidine, or HCTU / 6-CI-HOBt / collidine.

[0372] In some embodiments, the coupling composition comprises HCTU and collidine.

[0373] In some embodiments, the coupling composition comprises HATU and collidine.

[0374] In some embodiments, the coupling composition comprises EDC, HOBt, and collidine. In some embodiments, the coupling composition comprises EDC, HOBt, and collidine in THF and water. In some embodiments, the coupling composition comprises EDC, HOBt, and collidine in 4:1 THF:water.

[0375] In some embodiments, the coupling composition comprises PyBOP, HOBt, and collidine. In some embodiments, the coupling composition comprises PyBOP, HOBt, and collidine in THF and acetonitrile. In some embodiments, the coupling composition comprises PyBOP, HOBt, and collidine in 1:1 THF:acetonitrile.

[0376] In some embodiments, the coupling composition comprises PyOxim, Oxyma, and collidine. In some embodiments, the coupling composition comprises PyOxim, Oxyma, and collidine in THF and acetonitrile. In some embodiments, the coupling composition comprises PyOxim, Oxyma, and collidine in 1:1 THF:acetonitrile.

[0377] In some embodiments, the coupling composition comprises HCTU, 6-CI-HOBt, and collidine. In some embodiments, the coupling composition comprises HCTU, 6-CI-HOBt, and collidine in a solvent selected from DMF, acetonitrile, THF, 2-MeTHF, and combinations of any of the foregoing. In some embodiments, the coupling composition comprises HCTU, 6-CI-HOBt, and collidine in acetonitrile.10696-W001-SEC

[0378] In some embodiments, the coupling composition comprises 1 to 10 equivalents of EDC, 1 to 10 equivalents of collidine, and 1 to 10 equivalents of HOBt in THF and water, wherein equivalents are relative to the protected variant of Peptide D. In some embodiments, the coupling composition comprises 1 to 5 equivalents of EDC, 1 to 10 equivalents of collidine, and 1 to 5 equivalents of HOBt in THF and water, wherein equivalents are relative to the protected variant of Peptide D.

[0379] In some embodiments, the coupling composition comprises 1 to 10 equivalents of EDC, 1 to 10 equivalents of collidine, and 1 to 10 equivalents of HOBt in 3-5:1 THF:water (e.g., 4:1 THF:water), wherein equivalents are relative to the protected variant of Peptide D. In some embodiments, the coupling composition comprises 1 to 5 equivalents of EDO, 1 to 10 equivalents of collidine, and 1 to 5 equivalents of HOBt in 3-5:1 THF:water (e.g., 4:1 THF:water), wherein equivalents are relative to the protected variant of Peptide D.

[0380] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 30°C. In some embodiments, the coupling occurs at a temperature in the range of 30°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 35°C to 45°C.

[0381] In some embodiments, the method comprises coupling the protected variant of Peptide B and the protected variant of Peptide D for a time in the range of 1 hour to 72 hours. In some embodiments, the time is in the range of 6 hours to 24 hours.

[0382] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 6 hours to 24 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 6 hours to 24 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 30°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 30°C for a time in the range of 6 hours to 24 hours.

[0383] In some embodiments, the coupling composition comprises 1 to 10 equivalents of EDC, 1 to 10 equivalents of collidine, and 1 to 10 equivalents of HOBt in 3-5:1 THF:water (e.g., 4:1 THF:water), wherein equivalents are relative to the protected variant of Peptide D, and the coupling occurs at a temperature in the range of 20°C to 30°C for a time in the range of 6 hours to 24 hours. In some embodiments, the coupling composition comprises 1 to 5 equivalents of EDO, 1 to 10 equivalents of collidine, and 1 to 5 equivalents of HOBt in 3-5:1 THF:water (e.g., 4:1 THF:water), wherein equivalents are relative to the protected variant of Peptide D, and the coupling occurs at a temperature in the range of 20°C to 30°C for a time in the range of 6 hours to 24 hours.10696-W001-SEC

[0384] In some embodiments, the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2 (SEQ ID NO: 147), the protected variant of Peptide D is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 157) or Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 168), the coupling composition comprises DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, EDC / HOBt / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, Oxyma / PyOxim / collidine, Py BOP / HOBt / collidine, or HCTU / 6-CI-HOBt / collidine (preferably HCTU / 6-CI-HOBt / collidine or EDC / HOBt / collidine), and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C, more preferably a temperature in the range of 20°C to 30°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours).

[0385] In some embodiments, the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 140), the protected variant of Peptide D is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 168), and the coupling composition comprises HCTU, 6-CI-HOBt, and collidine. In some embodiments, the protected variant of Peptide B and the protected variant of Peptide D are contacted at room temperature. In some embodiments, the protected variant of Peptide B and the protected variant of Peptide D are contacted at room temperature for a time in the range of 6 hours to 24 hours.

[0386] In some embodiments, the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2 (SEQ ID NO: 147), the protected variant of Peptide D is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 157), and the coupling composition comprises EDO, HOBt, and collidine. In some embodiments, the protected variant of Peptide B and the protected variant of Peptide D are contacted at room temperature for a time in the range of 6 hours to 24 hours.

[0387] In some embodiments, the protected variant of Peptide B and / or the protected variant of Peptide D is prepared by a method provided herein.

[0388] Additionally, in certain embodiments, a protected variant of Peptide A can be partially deprotected as illustrated in FIG. 5A to form a different protected variant of Peptide A (FIG. 5B) in which the protecting group on the s-amino group of the lysine residue at the C-terminus has been removed. FIG. 5C depicts another partially deprotected variant of Peptide A which can be prepared by an analogous process to that shown in FIG. 5A.10696-WC01-SEC

[0389] In some embodiments, the side chain of the Lys residue at the C-terminus of the protected variant of Peptide A formed by coupling the protected variant of Peptide B with the protected variant of Peptide D is protected with a protecting group that can be selectively removed (such as, e.g., a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde), allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), or chlorobenzyloxycarbonyl (2-CI-Z) protecting group) and the method further comprises contacting the protected variant of Peptide A with a selective deprotection reagent to remove the protecting group and form a partially deprotected variant of Peptide A.

[0390] In some embodiments, the side chain of the Lys residue at the C-terminus of the protected variant of Peptide A formed by coupling the protected variant of Peptide B with the protected variant of Peptide D is protected with a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) protecting group, and the method further comprises contacting the protected variant of Peptide A with an o-nucleophile to remove the ivDde protecting group and form a partially deprotected variant of Peptide A. In some embodiments, the o-nucleophile is hydrazine or hydroxylamine, preferably hydrazine. In some embodiments, the o-nucleophile is hydrazine, and the contacting occurs in a solvent selected from DMF, DMSO, DMI, acetonitrile, methanol, THF, and combinations of any of the foregoing. In some embodiments, the o-nucleophile is hydroxylamine, and the contacting occurs in a solvent selected from acetonitrile, DMF, THF, DMSO, NMP, DMI, methanol, water, and combinations of any of the foregoing.

[0391] In some embodiments, the contacting occurs at a temperature in the range of 0°C to 60°C. In some embodiments, the contacting occurs at a temperature in the range of 20°C to 50°C. In some embodiments, the contacting occurs at a temperature in the range of 20°C to 30°C.

[0392] In some embodiments, the side chain of the Lys residue at the C-terminus of the protected variant of Peptide A formed by coupling the protected variant of Peptide B with the protected variant of Peptide D is protected with a 1 -(4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl (ivDde) protecting group, and the method further comprises contacting the protected variant of Peptide A with hydrazine (e.g., hydrazine in DMI) at a temperature in the range of 20°C to 30°C to remove the ivDde protecting group and form a partially deprotected variant of Peptide A.

[0393] In some embodiments, the side chain of the Lys residue at the C-terminus of the protected variant of Peptide A formed by coupling the protected variant of Peptide B with the protected variant of Peptide D is protected with a 1 -(4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl (ivDde) protecting group, and the method further comprises contacting the protected variant of Peptide A with hydroxylamine (e.g., hydroxylamine in DMSO and THF) at a temperature in the range of 20°C to 30°C to remove the ivDde protecting group and form a partially deprotected variant of Peptide A.

[0394] In some embodiments, the method further comprises contacting the partially deprotected variant of Peptide A with bromoacetic acid or bromoacetic anhydride in the presence of a second coupling composition to10696-WG01-SECform a bromoacetylated protected variant of Peptide A. In some embodiments, the second coupling composition comprises a coupling reagent, a base, and optionally a coupling additive.

[0395] In some embodiments, the method further comprises contacting the partially deprotected variant of Peptide A with bromoacetic acid in the presence of a second coupling composition to form a bromoacetylated protected variant of Peptide A, wherein the second coupling composition comprises TBTU and collidine. In some embodiments, the second coupling composition further comprises N-hydroxysuccinimide ester (NHS).

[0396] In some embodiments, the method further comprises contacting the partially deprotected variant of Peptide A with bromoacetic acid in the presence of a second coupling composition to form a bromoacetylated protected variant of Peptide A, wherein the second coupling composition comprises DIG and 2,6-lutidine. In some embodiments, the second coupling composition comprises DIG and 2,6-lutidine in heptane and MTBE.

[0397] In some embodiments, the method further comprises contacting the partially deprotected variant of Peptide A with bromoacetic anhydride in the presence of a second coupling composition to form a bromoacetylated protected variant of Peptide A, wherein the second coupling composition comprises lithium bromide and N-methylmorpholine. In some embodiments, the second coupling composition comprises lithium bromide and N-methylmorpholine in THF.

[0398] In some embodiments, the method further comprises deprotecting the bromoacetylated protected variant of Peptide A by contacting the bromoacetylated protected variant of Peptide A with a Bronsted acid or a Lewis acid (preferably trifluoroacetic acid (TFA)), optionally in the presence of a scavenger (preferably triisopropylsilane (TIS)). In some embodiments, the scavenger is selected from hydrosilanes (e.g., TIS, triethylsilane (TES)), phenols (e.g., phenol, anisole, m-cresol), thioethers (e.g., thioanisole, dimethyl sulfide), thiols / dithiols (e.g., EDT, DTT, 1 -dodecanethiol), water, indole, and combinations of any of the foregoing. In some embodiments, the method further comprises deprotecting the bromoacetylated protected variant of Peptide A by contacting the bromoacetylated protected variant of Peptide A with an aqueous solution comprising TFA and TIS.

[0399] In some embodiments, the method further comprises globally deprotecting the bromoacetylated protected variant of Peptide A.

[0400] In some embodiments in which the N-terminus of the bromoacetylated protected variant of Peptide A is Boc-protected, the method further comprises deprotecting the bromoacetylated protected variant of Peptide A by contacting the bromoacetylated protected variant of Peptide A with a Bronsted acid or a Lewis acid (preferably trifluoroacetic acid (TFA)), optionally in the presence of a scavenger (preferably triisopropylsilane (TIS)). In some embodiments, the scavenger is selected from hydrosilanes (e.g., TIS, triethylsilane (TES)), phenols (e.g., phenol, anisole, m-cresol), thioethers (e.g., thioanisole, dimethyl sulfide), thiols / dithiols (e.g., EDT, DTT, 1 -dodecanethiol), water, indole, and combinations of any of the foregoing. In some embodiments in which the N-terminus of the bromoacetylated protected variant of Peptide A is Boc-protected, the method further10696-WQ01-SECcomprises deprotecting the bromoacetylated protected variant of Peptide A by contacting the bromoacetylated protected variant of Peptide A with an aqueous solution comprising TFA and TIS.

[0401] Alternatively, a protected variant of Peptide A can be partially deprotected to form a protected variant of Peptide A in which the protecting group on the s-amino group of the lysine residue at the C-terminus has been removed; the s-amino group of the lysine residue at the C-terminus can then be bromoacetylated and the protected peptide globally deprotected as shown in FIG. 5D to form the product shown in FIG. 5E.

[0402] In alternative embodiments, the C-terminal lysine residue of a protected variant of Peptide A can be bromoacetylated as illustrated in FIG. 6A. FIG. 6B depicts a bromoacetylated and protected variant of Peptide A with an amidated C-terminus which can be prepared by the process shown in FIG. 6A.

[0403] Also provided herein is a method of preparing Peptide A or a protected variant thereof, comprising:(A)(I) (a) coupling or having coupled a protected variant of Fragment 3 with a protected variant of Fragment 4 to form a first protected variant of Peptide E, wherein the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Fragment 4 has a free amino group at the N-terminus, Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of SEQ ID NO: 8; (b) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide E from step (A)(1)(a) to form a second protected variant of Peptide E with a free amino group at the N-terminus; (c) coupling or having coupled the second protected variant of Peptide E from step (A)(1)(b) with an N-protected alanine to form a first protected variant of Peptide B, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9; and (d) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide B from step (A)(1)(c) to form a second protected variant of Peptide B with a free amino group at the N-terminus; OR(ii) (a) coupling or having coupled a protected variant of Fragment 3A with a protected variant of Fragment 4 to form a first protected variant of Peptide B, wherein the protected variant of Fragment 3A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Fragment 4 has a free amino group at the N-terminus, Fragment 3A comprises the amino acid sequence of SEQ ID NO: 13, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide B comprises the amino acid sequence of SEQ ID NO: 9; and (b) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide B from step (A)(ii)(a) to form a second protected variant of Peptide B with a free amino group at the N-terminus; OR(ill) (a) coupling or having coupled a protected variant of Fragment 5 with a protected variant of Fragment 6 to form a first protected variant of Peptide B, wherein the protected variant of Fragment 5 has a free carboxyl group at the C-terminus and a protected (preferably Trt- or Fmoc-protected, more preferably Trt-10696-WQ01-SECprotected) N-terminus, the protected variant of Fragment 6 has a free amino group at the N-terminus, Fragment 5 comprises the amino acid sequence of SEQ ID NO: 5, Fragment 6 comprises the amino acid sequence of SEQ ID NO: 6, and Peptide B comprises the amino acid sequence of SEQ ID NO: 9; and (b) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide B from step (A)(iii)(a) to form a second protected variant of Peptide B with a free amino group at the N-terminus;(B) coupling or having coupled a protected variant of Fragment 1 with a protected variant of Fragment 2B to form a protected variant of Peptide D, wherein the protected variant of Fragment 1 has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus, the protected variant of Fragment 2B has a free amino group at the N-terminus, Fragment 1 comprises the amino acid sequence of SEQ ID NO: 1, Fragment 2B comprises the amino acid sequence of SEQ ID NO: 16, and Peptide D comprises the amino acid sequence of SEQ ID NO: 15; and(C) coupling or having coupled the second protected variant of Peptide B from step (A) with the protected variant of Peptide D from step (B) to form a protected variant of Peptide A, wherein Peptide A comprises the amino acid sequence of SEQ ID NO: 7.

[0404] In some embodiments, the method further comprises deprotecting or having deprotected the protected variant of Peptide A to isolate Peptide A.

[0405] In some embodiments, the method further comprises adding or having added a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0406] In some embodiments, the method comprises step (A)(i), step (B), and step (C). In other embodiments, the method comprises step (A)(ii), step (B), and step (C). In other preferred embodiments, the method comprises step (A)(iii), step (B), and step (C).

[0407] In some embodiments, the method comprises step (A)(i), step (B), and step (C), wherein each coupling is performed under liquid phase peptide coupling conditions. In other embodiments, the method comprises step (A)(ii), step (B), and step (C), wherein each coupling is performed under liquid phase peptide coupling conditions. In other preferred embodiments, the method comprises step (A)(iii), step (B), and step (C), wherein each coupling is performed under liquid phase peptide coupling conditions.

[0408] In some embodiments, the method comprises step (A)(i), step (B), and step (C), wherein each coupling is performed under solid phase peptide coupling conditions. In other embodiments, the method comprises step (A)(ii), step (B), and step (C), wherein each coupling is performed under solid phase peptide coupling conditions. In other embodiments, the method comprises step (A)(iii), step (B), and step (C), wherein each coupling is performed under solid phase peptide coupling conditions.

[0409] In some embodiments, the protected variant of Fragment 3 and the protected variant of Fragment 4 are coupled in step (A)(1)(a) under conditions described herein.10696-WC01-SEC

[0410] In some embodiments, the second protected variant of Peptide E and the N-protected alanine are coupled in step (A)(i)(c) under conditions described herein.

[0411] In some embodiments, the protected variant of Fragment 3A and the protected variant of Fragment 4 are coupled in step (A)(ii)(a) are coupled under conditions described herein.

[0412] In some embodiments, the protected variant of Fragment 5 and the protected variant of Fragment 6 are coupled in step (A)(iii)(a) are coupled under conditions described herein.

[0413] In some embodiments, the protected variant of Fragment 1 and the protected variant of Fragment 2B are coupled in step (B) are coupled under conditions described herein.

[0414] In some embodiments, the second protected variant of Peptide B and the protected variant of Peptide D are coupled in step (C) are coupled under conditions described herein.

[0415] In some embodiments, the method comprises step (A)(i), the N-terminus of the first protected variant of Peptide E is Fmoc-protected, and the selective deprotection of step (A)(i)(b) comprises contacting the first protected variant of Peptide E with an amine base to selectively remove the Fmoc protecting group at the N-terminus. In some embodiments, the amine base is selected from piperidine, piperazine, and morpholine (e.g., 5-30% (v / v) piperidine, piperazine, or morpholine). In some embodiments, the amine base is piperidine. In some embodiments, the method comprises step (A)(i), the N-terminus of the first protected variant of Peptide E is Fmoc-protected, and the selective deprotection of step (A)(i)(b) comprises contacting the first protected variant of Peptide E with piperidine in DMF, optionally in the presence of DBU.

[0416] In some embodiments, the method comprises step (A)(i), the N-terminus of the first protected variant of Peptide E is Boc-protected, and the selective deprotection of step (A)(i)(b) comprises contacting the first protected variant of Peptide E with an acid (preferably an acid selected from TFA, HCI, and HBr) to selectively remove the Boc protecting group at the N-terminus. In some embodiments, the first protected variant of Peptide E is contacted with TFA (e.g., neat TFA or TFA in DCM, e.g., 20-50% (v / v) TFA in DCM). In some embodiments, the first protected variant of Peptide E is contacted with HCI (e.g., HCI in DCM, DCE, toluene, or dioxane, e.g., 3-4 M HCI in DCM or dioxane).

[0417] In some embodiments, the method comprises step (A)(i), the N-terminus of the first protected variant of Peptide E is Boc-protected, and the selective deprotection of step (A)(i)(b) comprises contacting the first protected variant of Peptide E with an acid (preferably an acid selected from TFA, HCI, and HBr) in the presence of a scavenger. In some embodiments, the scavenger is selected from hydrosilanes (e.g., TIS, triethylsilane (TES)), phenols (e.g., phenol, anisole, m-cresol), thioethers (e.g., thioanisole, dimethyl sulfide), thiols / dithiols (e.g., EDT, DTT, 1 -dodecanethiol), water, indole, and combinations of any of the foregoing. In some preferred embodiments, the scavenger is a hydrosilane. In some particularly preferred embodiments, the scavenger is TIS. In some embodiments, the first protected variant of Peptide E is contacted with TFA (e.g., neat TFA or TFA in DCM, e.g., 20-50% (v / v) TFA in DCM) in the presence of a scavenger (preferably TIS). In some embodiments,10696-WC01-SECthe first protected variant of Peptide E is contacted with HCI (e.g., HCI in DCM, DCE, toluene, or dioxane, e.g., 3-4 M HCI in DCM or dioxane) in the presence of a scavenger.

[0418] In some embodiments, the method comprises step (A)(i), the N-terminus of the first protected variant of Peptide E is Trt-protected, and the selective deprotection of step (A)(1)(b) comprises contacting the first protected variant of Peptide E with a Bronsted acid or a Lewis acid (preferably TFA, HCI, or dichloroacetic acid, most preferably TFA), optionally in the presence of a scavenger (preferably TIS), to selectively remove the trityl protecting group at the N-terminus. In some embodiments, the first protected variant of Peptide E is contacted with a Bronsted acid (preferably a dilute Bronsted acid). In some embodiments, the Bronsted acid is selected from TFA, HCI, and dichloroacetic acid (preferably dilute TFA, HCI, or dichloroacetic acid). In other embodiments, the first protected variant of Peptide E is contacted with a Lewis acid (preferably a dilute Lewis acid).

[0419] In some embodiments, the method comprises step (A)(i), the scavenger is selected from hydrosilanes (e.g., TIS, triethylsilane (TES)), phenols (e.g., phenol, anisole, m-cresol), thioethers (e.g., thioanisole, dimethyl sulfide), thiols / dithiols (e.g., EDT, DTT, 1 -dodecanethiol), water, indole, and combinations of any of the foregoing. In some embodiments, the scavenger is a hydrosilane. In some embodiments, the scavenger is TIS.

[0420] In some embodiments, the method comprises step (A)(i), the N-terminus of the first protected variant of Peptide E is Trt-protected, and the selective deprotection of step (A)(1)(b) comprises contacting the first protected variant of Peptide E with HCI. In some embodiments, the selective deprotection of step (A)(1)(b) comprises contacting the first protected variant of Peptide E with dilute HCI.

[0421] In some embodiments, the method comprises step (A)(i), the N-terminus of the first protected variant of Peptide E is Trt-protected, and the selective deprotection of step (A)(1)(b) comprises contacting the first protected variant of Peptide E with TFA in the presence of TIS. In some embodiments, the selective deprotection of step (A)(1)(b) comprises contacting the first protected variant of Peptide E with dilute TFA in the presence of TIS.

[0422] In some embodiments, the N-terminus of the first protected variant of Peptide B is Fmoc-protected, and the selective deprotection of step (A)(1)(d), step (A)(ii)(b), or step (A)(iii)(b) comprises contacting the first protected variant of Peptide B with an amine base to selectively remove the Fmoc protecting group at the N-terminus. In some embodiments, the amine base is selected from piperidine, piperazine, and morpholine (e.g., 5-30% (v / v) piperidine, piperazine, or morpholine). In some embodiments, the amine base is piperidine. In some embodiments, the N-terminus of the first protected variant of Peptide B is Fmoc-protected, and the selective deprotection of step (A)(1)(d), step (A)(ii)(b), or step (A)(iii)(b) comprises contacting the first protected variant of Peptide B with piperidine in DMF, optionally in the presence of DBU.

[0423] In some embodiments, the N-terminus of the first protected variant of Peptide B is Boc-protected, and the selective deprotection of step (A)(1)(d), step (A)(ii)(b), or step (A)(iii)(b) comprises contacting the first protected variant of Peptide B with an acid (preferably an acid selected from TFA, HCI, and HBr) to selectively remove the Boc protecting group at the N-terminus. In some embodiments, the first protected variant of Peptide10696-WC01-SECB is contacted with TFA (e.g., neat TFA or TFA in DCM, e.g., 20-50% (v / v) TFA in DCM). In some embodiments, the first protected variant of Peptide B is contacted with HCI (e.g., HCI in DCM, DCE, toluene, or dioxane, e.g., 3-4 M HCI in DCM or dioxane).

[0424] In some embodiments, the N-terminus of the first protected variant of Peptide B is Boc-protected, and the selective deprotection of step (A)(1)(d), step (A)(ii)(b), or step (A)(iii)(b) comprises contacting the first protected variant of Peptide B with an acid (preferably an acid selected from TFA, HCI, and HBr) in the presence of a scavenger. In some embodiments, the scavenger is selected from hydrosilanes (e.g., TIS, triethylsilane (TES)), phenols (e.g., phenol, anisole, m-cresol), thioethers (e.g., thioanisole, dimethyl sulfide), thiols / dithiols (e.g., EDT, DTT, 1 -dodecanethiol), water, indole, and combinations of any of the foregoing. In some preferred embodiments, the scavenger is a hydrosilane. In some particularly preferred embodiments, the scavenger is TIS. In some embodiments, the first protected variant of Peptide B is contacted with TFA (e.g., neat TFA or TFA in DCM, e.g., 20-50% (v / v) TFA in DCM) in the presence of a scavenger (preferably TIS). In some embodiments, the first protected variant of Peptide B is contacted with HCI (e.g., HCI in DCM, DCE, toluene, or dioxane, e.g., 3-4 M HCI in DCM or dioxane) in the presence of a scavenger.

[0425] In some embodiments, the N-terminus of the first protected variant of Peptide B is Trt-protected, and the selective deprotection of step (A)(i)(d), step (A)(ii)(b), or step (A)(iii)(b) comprises contacting the first protected variant of Peptide B with a Bronsted acid or a Lewis acid (preferably TFA, HCI, or dichloroacetic acid, most preferably TFA), optionally in the presence of a scavenger (preferably TIS), to selectively remove the trityl protecting group at the N-terminus. In some embodiments, the first protected variant of Peptide B is contacted with a Bronsted acid (preferably a dilute Bronsted acid). In some embodiments, the Bronsted acid is selected from TFA, HCI, and dichloroacetic acid (preferably dilute TFA, HCI, or dichloroacetic acid). In other embodiments, the first protected variant of Peptide B is contacted with a Lewis acid (preferably a dilute Lewis acid).

[0426] In some embodiments, the scavenger is selected from hydrosilanes (e.g., TIS, triethylsilane (TES)), phenols (e.g., phenol, anisole, m-cresol), thioethers (e.g., thioanisole, dimethyl sulfide), thiols / dithiols (e.g., EDT, DTT, 1 -dodecanethiol), water, indole, and combinations of any of the foregoing. In some embodiments, the scavenger is a hydrosilane. In some embodiments, the scavenger is TIS.

[0427] In some embodiments, the N-terminus of the first protected variant of Peptide B is Trt-protected, and the selective deprotection of step (A)(i)(d), step (A)(ii)(b), or step (A)(iii)(b) comprises contacting the first protected variant of Peptide B with HCI. In some embodiments, the selective deprotection of step (A)(i)(d), step (A)(ii)(b), or step (A)(iii)(b) comprises contacting the first protected variant of Peptide B with dilute HCI.

[0428] In some embodiments, the N-terminus of the first protected variant of Peptide B is Trt-protected, and the selective deprotection of step (A)(i)(d), step (A)(ii)(b), or step (A)(iii)(b) comprises contacting the first protected variant of Peptide B with TFA in the presence of TIS. In some embodiments, the selective deprotection of step (A)(i)(d), step (A)(ii)(b), or step (A)(iii)(b) comprises contacting the first protected variant of Peptide B with dilute TFA in the presence of TIS.10696-WQ01-SEC

[0429] Also provided herein is a method of preparing Peptide C or a protected variant thereof, comprising coupling a protected variant of Fragment 2A with a protected variant of Peptide B to form a protected variant of Peptide C, wherein Fragment 2A comprises the amino acid sequence of SEQ ID NO: 12, Peptide B comprises the amino acid sequence of SEQ ID NO: 9, and Peptide C comprises the amino acid sequence of SEQ ID NO: 10.

[0430] In some embodiments, the protected variant of Fragment 2A is coupled with the protected variant of Peptide B in solution. In some embodiments, the protected variant of Fragment 2A is coupled with the protected variant of Peptide B under liquid phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 2A is coupled with the protected variant of Peptide B under classical solution phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 2A is coupled with the protected variant of Peptide B under tag-assisted liquid phase peptide coupling conditions.

[0431] In other embodiments, the protected variant of Fragment 2A is coupled with the protected variant of Peptide B on solid support. In some embodiments, the protected variant of Fragment 2A is coupled with the protected variant of Peptide B under solid phase peptide coupling conditions.

[0432] In some embodiments, a C-terminal carboxyl group of the protected variant of Fragment 2A is preactivated. In other embodiments, a C-terminal carboxyl group of the protected variant of Fragment 2A is activated in situ.

[0433] In some embodiments, the method comprises:contacting the protected variant of Fragment 2A with a coupling composition to form a protected variant of Fragment 2A with an activated C-terminal carboxyl group ("activated Fragment 2A"); andcontacting the protected variant of Peptide B with the activated Fragment 2A to form the protected variant of Peptide C.

[0434] In some embodiments, the method comprises:contacting the protected variant of Fragment 2A with a coupling composition on solid support to form a protected variant of Fragment 2A with an activated C-terminal carboxyl group ("activated Fragment 2A"); and contacting the protected variant of Peptide B with the activated Fragment 2A on solid support to form the protected variant of Peptide C.

[0435] In some embodiments, the method comprises:contacting the protected variant of Fragment 2A with a coupling composition in solution to form a protected variant of Fragment 2A with an activated C-terminal carboxyl group ("activated Fragment 2A"); and contacting the protected variant of Peptide B with the activated Fragment 2A in solution to form the protected variant of Peptide C.10696-W001-SEC

[0436] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 2A is pre-activated), the method comprises:contacting the protected variant of Fragment 2A with a coupling composition to form a protected variant of Fragment 2A with an activated C-terminal carboxyl group ("activated Fragment 2A"); andcontacting the protected variant of Peptide B with the activated Fragment 2A to form the protected variant of Peptide C, wherein the contacting does not occur in the presence of a coupling reagent.

[0437] In some embodiments, the protected variant of Fragment 2A is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 2A and the coupling composition.

[0438] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 2A is pre-activated), the method comprises:contacting the protected variant of Fragment 2A with a coupling composition on solid support to form a protected variant of Fragment 2A with an activated C-terminal carboxyl group ("activated Fragment 2A"); and contacting the protected variant of Peptide B with the activated Fragment 2A on solid support to form the protected variant of Peptide C, wherein the contacting does not occur in the presence of a coupling reagent.

[0439] In some embodiments, the protected variant of Fragment 2A is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 2A and the coupling composition.

[0440] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 2A is pre-activated), the method comprises:contacting the protected variant of Fragment 2A with a coupling composition in solution to form a protected variant of Fragment 2A with an activated C-terminal carboxyl group ("activated Fragment 2A"); and contacting the protected variant of Peptide B with the activated Fragment 2A in solution to form the protected variant of Peptide C, wherein the contacting does not occur in the presence of a coupling reagent.

[0441] In some embodiments, the protected variant of Fragment 2A is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 2A and the coupling composition.

[0442] In some embodiments, the contacting of the protected variant of Fragment 2A with the coupling composition and the contacting of the activated Fragment 2A with the protected variant of Peptide B occur in the same reaction vessel. In other embodiments, the contacting of the protected variant of Fragment 2A with the coupling composition and the contacting of the activated Fragment 2A with the protected variant of Peptide B occur in different reaction vessels.10696-WC01-SEC

[0443] In some embodiments, the activated Fragment 2A is contacted with the protected variant of Peptide B in the presence of a base and / or a coupling additive but not a coupling reagent (such as, e.g., in certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 2A is pre-activated and the protected variant of Fragment 2A is present in molar excess relative to the coupling reagent in the coupling composition during the contacting to form activated Fragment 2A).

[0444] In some embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 2A is pre-activated, the protected variant of Peptide B has a free N-terminal amino group and a free C-terminal carboxyl group.

[0445] In some embodiments, the coupling composition comprises a coupling reagent and a base. In some embodiments, the coupling composition comprises a coupling reagent (e.g., a carbodiimide, e.g., DIG, DCC, EDC) and a coupling additive (e.g., HOBt, 5-CI-HOBt, Oxyma).

[0446] In some embodiments, the coupling composition comprises a coupling reagent, a coupling additive, and a base.

[0447] In some embodiments, the coupling reagent is a carbodiimide, such as, e.g., DIG, DCC, or EDC. In other embodiments, the coupling reagent is BOP-CI.

[0448] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 2A is activated in situ), the method comprises coupling the protected variant of Fragment 2A with the protected variant of Peptide B in the presence of a coupling composition. In some embodiments, the method comprises coupling the protected variant of Fragment 2A with the protected variant of Peptide B in solution in the presence of a coupling composition.

[0449] In some embodiments, the protected variant of Peptide B does not have a free carboxyl group at the C-terminus. In some embodiments, the C-terminus of the protected variant of Peptide B is protected. In some embodiments, the C-terminus of the protected variant of Peptide B is amidated. In some embodiments, the protected variant of Peptide B has a free amino group at the N-terminus. In preferred embodiments, the protected variant of Peptide B has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus. In particularly preferred embodiments, the protected variant of Peptide B has a free amino group at the N-terminus and an amidated C-terminus.

[0450] In some embodiments, the protected variant of Fragment 2A does not have a free amino group at the N-terminus. In some embodiments, the N-terminus of the protected variant of Fragment 2A is protected (preferably Fmoc-protected). In some embodiments, the protected variant of Fragment 2A has a free carboxyl group at the C-terminus. In preferred embodiments, the protected variant of Fragment 2A has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Fragment 2A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus.10696-W001-SEC

[0451] In some embodiments, the protected variant of Peptide B has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 2A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus.

[0452] In some embodiments, the C-terminus of the protected variant of Peptide B is amidated, and the N-terminus of the protected variant of Fragment 2A is protected (preferably Fmoc-protected).

[0453] In preferred embodiments, the protected variant of Peptide B has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 2A has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Peptide B has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 2A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus.

[0454] In some embodiments, the protected variant of Peptide B has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 2A has a free carboxyl group at the C-terminus and an Fmoc-protected N-terminus.

[0455] In some embodiments, the protected variant of Fragment 2A is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 53).

[0456] In some embodiments, the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 140).

[0457] In some embodiments, the protected variant of Fragment 2A is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 53), and the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 140).

[0458] In some embodiments, the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0459] In some embodiments, the coupling composition comprises DIC / Oxyma, EDC / Oxyma, PyBOP / collidine, PyOxim / collidine, TBTU / collidine, TBTU / DIEA, HCTU / DIEA, or PyOxim / DIEA. In some embodiments, the coupling composition comprises DIC / Oxyma, EDC / Oxyma, PyBOP / collidine, PyOxim / collidine, TBTU / collidine, TBTU / DIEA, HCTU / DIEA, or PyOxim / DIEA in DSMO.

[0460] In some embodiments, the coupling composition comprises DIG and Oxyma. In some embodiments, the coupling composition comprises DIO and Oxyma in DMSO.

[0461] In some embodiments, the protected variant of Fragment 2A is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 53), the protected variant of Peptide B is Ala-10696-W001-SECTrp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 140), and the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0462] In some embodiments, the protected variant of Fragment 2A is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 53), the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 140), and the coupling composition comprises DIC / Oxyma, EDC / Oxyma, Py BOP / collidine, PyOxim / collidine, TBTU / collidine, TBTU / DIEA, HCTU / DIEA, or PyOxim / DIEA (preferably DIC / Oxyma). In some embodiments, the coupling composition comprises DIC / Oxyma, EDC / Oxyma,Py BOP / collidine, PyOxim / collidine, TBTU / collidine, TBTU / DIEA, HCTU / DIEA, or PyOxim / DIEA (preferably DIC / Oxyma) in DMSO.

[0463] In some embodiments, the protected variant of Fragment 2A is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 53), the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 140), and the coupling composition comprises DIG and Oxyma. In some embodiments, the coupling composition comprises DIG and Oxyma in DMSO.

[0464] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 30°C. In some embodiments, the coupling occurs at a temperature in the range of 30°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 35°C to 45°C.

[0465] In some embodiments, the method comprises coupling the protected variant of Fragment 2A and the protected variant of Peptide B for a time in the range of 1 hour to 72 hours. In some embodiments, the time is in the range of 6 hours to 24 hours.

[0466] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 6 hours to 24 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 6 hours to 24 hours.

[0467] In some embodiments, the protected variant of Fragment 2A is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 53), the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 140), the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine,10696-W001-SECTBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine, and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours).

[0468] In some embodiments, the protected variant of Fragment 2A is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 53), the protected variant of Peptide B is Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 140), the coupling composition comprises DIC / Oxyma, EDC / Oxyma, PyBOP / collidine, PyOxim / collidine, TBTU / collidine, TBTU / DIEA, HCTU / DIEA, or PyOxim / DIEA (preferably DIC / Oxyma), and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours). In some embodiments, the coupling composition comprises DIO and Oxyma in DMSO.

[0469] In some embodiments, the method further comprises selectively deprotecting the N-terminus of the protected variant of Peptide C (e.g., using an Fmoc deprotection method described herein).

[0470] Also provided herein is a method of preparing Peptide C or a protected variant thereof, comprising coupling a protected variant of Fragment 2C with a protected variant of Peptide E to form a protected variant of Peptide C, wherein Fragment 2C comprises the amino acid sequence of SEQ ID NO: 17, Peptide E comprises the amino acid sequence of SEQ ID NO: 8, and Peptide C comprises the amino acid sequence of SEQ ID NO: 10.

[0471] In some embodiments, the protected variant of Fragment 2C is coupled with the protected variant of Peptide E in solution. In some embodiments, the protected variant of Fragment 20 is coupled with the protected variant of Peptide E under liquid phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 20 is coupled with the protected variant of Peptide E under classical solution phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 20 is coupled with the protected variant of Peptide E under tag-assisted liquid phase peptide coupling conditions.

[0472] In other embodiments, the protected variant of Fragment 20 is coupled with the protected variant of Peptide E on solid support. In some embodiments, the protected variant of Fragment 20 is coupled with the protected variant of Peptide E under solid phase peptide coupling conditions.

[0473] In some embodiments, a C-terminal carboxyl group of the protected variant of Fragment 20 is preactivated. In other embodiments, a C-terminal carboxyl group of the protected variant of Fragment 20 is activated in situ.

[0474] In some embodiments, the method comprises:contacting the protected variant of Fragment 20 with a coupling composition to form a protected variant of Fragment 20 with an activated C-terminal carboxyl group ("activated Fragment 2C"); and10696-WC01-SECcontacting the protected variant of Peptide E with the activated Fragment 2C to form the protected variant of Peptide C.

[0475] In some embodiments, the method comprises:contacting the protected variant of Fragment 2C with a coupling composition on solid support to form a protected variant of Fragment 2C with an activated C-terminal carboxyl group ("activated Fragment 2C"); and contacting the protected variant of Peptide E with the activated Fragment 2C on solid support to form the protected variant of Peptide C.

[0476] In some embodiments, the method comprises:contacting the protected variant of Fragment 2C with a coupling composition in solution to form a protected variant of Fragment 2C with an activated C-terminal carboxyl group ("activated Fragment 2C"); and contacting the protected variant of Peptide E with the activated Fragment 2C in solution to form the protected variant of Peptide C.

[0477] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 2C is pre-activated), the method comprises:contacting the protected variant of Fragment 2C with a coupling composition to form a protected variant of Fragment 2C with an activated C-terminal carboxyl group ("activated Fragment 2C"); andcontacting the protected variant of Peptide E with the activated Fragment 2C to form the protected variant of Peptide C, wherein the contacting does not occur in the presence of a coupling reagent.

[0478] In some embodiments, the protected variant of Fragment 2C is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 2C and the coupling composition.

[0479] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 2C is pre-activated), the method comprises:contacting the protected variant of Fragment 2C with a coupling composition on solid support to form a protected variant of Fragment 2C with an activated C-terminal carboxyl group ("activated Fragment 2C"); and contacting the protected variant of Peptide E with the activated Fragment 2C on solid support to form the protected variant of Peptide C, wherein the contacting does not occur in the presence of a coupling reagent.10696-W001-SEC

[0480] In some embodiments, the protected variant of Fragment 2C is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 2C and the coupling composition.

[0481] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 2C is pre-activated), the method comprises:contacting the protected variant of Fragment 2C with a coupling composition in solution to form a protected variant of Fragment 2C with an activated C-terminal carboxyl group ("activated Fragment 2C"); and contacting the protected variant of Peptide E with the activated Fragment 2C in solution to form the protected variant of Peptide C, wherein the contacting does not occur in the presence of a coupling reagent.

[0482] In some embodiments, the protected variant of Fragment 2C is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 2C and the coupling composition.

[0483] In some embodiments, the contacting of the protected variant of Fragment 2C with the coupling composition and the contacting of the activated Fragment 2C with the protected variant of Peptide E occur in the same reaction vessel. In other embodiments, the contacting of the protected variant of Fragment 2C with the coupling composition and the contacting of the activated Fragment 2C with the protected variant of Peptide E occur in different reaction vessels.

[0484] In some embodiments, the activated Fragment 2C is contacted with the protected variant of Peptide E in the presence of a base and / or a coupling additive but not a coupling reagent (such as, e.g., in certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 2C is pre-activated and the protected variant of Fragment 2C is present in molar excess relative to the coupling reagent in the coupling composition during the contacting to form activated Fragment 2C).

[0485] In some embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 2C is pre-activated, the protected variant of Peptide E has a free N-terminal amino group and a free C-terminal carboxyl group.

[0486] In some embodiments, the coupling composition comprises a coupling reagent and a base. In some embodiments, the coupling composition comprises a coupling reagent (e.g., a carbodiimide, e.g., DIG, DCC, EDC) and a coupling additive (e.g., HOBt, 5-CI-HOBt, Oxyma).

[0487] In some embodiments, the coupling composition comprises a coupling reagent, a coupling additive, and a base.

[0488] In some embodiments, the coupling reagent is a carbodiimide, such as, e.g., DIG, DCC, or EDC. In other embodiments, the coupling reagent is BOP-CI.10696-WC01-SEC

[0489] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 2C is activated in situ), the method comprises coupling the protected variant of Fragment 2C with the protected variant of Peptide E in the presence of a coupling composition. In some embodiments, the method comprises coupling the protected variant of Fragment 2C with the protected variant of Peptide E in solution in the presence of a coupling composition.

[0490] In some embodiments, the protected variant of Peptide E does not have a free carboxyl group at the C-terminus. In some embodiments, the C-terminus of the protected variant of Peptide E is protected. In some embodiments, the C-terminus of the protected variant of Peptide E is amidated. In some embodiments, the protected variant of Peptide E has a free amino group at the N-terminus. In preferred embodiments, the protected variant of Peptide E has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus. In particularly preferred embodiments, the protected variant of Peptide E has a free amino group at the N-terminus and an amidated C-terminus.

[0491] In some embodiments, the protected variant of Fragment 2C does not have a free amino group at the N-terminus. In some embodiments, the N-terminus of the protected variant of Fragment 2C is protected (preferably Fmoc-protected). In some embodiments, the protected variant of Fragment 2C has a free carboxyl group at the C-terminus. In preferred embodiments, the protected variant of Fragment 2C has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Fragment 2C has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus.

[0492] In some embodiments, the protected variant of Peptide E has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 2C has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus.

[0493] In some embodiments, the C-terminus of the protected variant of Peptide E is amidated, and the N-terminus of the protected variant of Fragment 2C is protected (preferably Fmoc-protected).

[0494] In preferred embodiments, the protected variant of Peptide E has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 2C has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Peptide E has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 2C has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus.

[0495] In some embodiments, the protected variant of Peptide E has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 2C has a free carboxyl group at the C-terminus and an Fmoc-protected N-terminus.10696-W001-SEC

[0496] In some embodiments, the protected variant of Fragment 2C is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-OH (SEQ ID NO: 55).

[0497] In some embodiments, the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 160).

[0498] In some embodiments, the protected variant of Fragment 2C is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-OH (SEQ ID NO: 55), and the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 160).

[0499] In some embodiments, the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0500] In some embodiments, the coupling composition comprises DIC / Oxyma, EDC / Oxyma, PyBOP / collidine, PyOxim / collidine, TBTU / collidine, TBTU / DIEA, HCTU / DIEA, or PyOxim / DIEA. In some embodiments, the coupling composition comprises DIC / Oxyma, EDC / Oxyma, PyBOP / collidine, PyOxim / collidine, TBTU / collidine, TBTU / DIEA, HCTU / DIEA, or PyOxim / DIEA in DSMO.

[0501] In some embodiments, the coupling composition comprises DIG and Oxyma. In some embodiments, the coupling composition comprises DIG and Oxyma in DMSO.

[0502] In some embodiments, the protected variant of Fragment 2C is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-OH (SEQ ID NO: 55), the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 160), and the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0503] In some embodiments, the protected variant of Fragment 2C is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-OH (SEQ ID NO: 55), the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 160), and the coupling composition comprises DIC / Oxyma, EDC / Oxyma, PyBOP / collidine, PyOxim / collidine, TBTU / collidine, TBTU / DIEA, HCTU / DIEA, or PyOxim / DIEA (preferably DIC / Oxyma). In some embodiments, the coupling composition comprises DIC / Oxyma, EDC / Oxyma, PyBOP / collidine, PyOxim / collidine, TBTU / collidine, TBTU / DIEA, HCTU / DIEA, or PyOxim / DIEA (preferably DIC / Oxyma) in DMSO.

[0504] In some embodiments, the protected variant of Fragment 2C is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-OH (SEQ ID NO: 55), the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-10696-W001-SECSer(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 160), and the coupling composition comprises DIG and Oxyma. In some embodiments, the coupling composition comprises DIO and Oxyma in DMSO.

[0505] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 30°C. In some embodiments, the coupling occurs at a temperature in the range of 30°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 35°C to 45°C.

[0506] In some embodiments, the method comprises coupling the protected variant of Fragment 2C and the protected variant of Peptide E for a time in the range of 1 hour to 72 hours. In some embodiments, the time is in the range of 6 hours to 24 hours.

[0507] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 6 hours to 24 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 6 hours to 24 hours.

[0508] In some embodiments, the protected variant of Fragment 2C is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-OH (SEQ ID NO: 55), the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 160), the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine, and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours).

[0509] In some embodiments, the protected variant of Fragment 2C is Fmoc-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-OH (SEQ ID NO: 55), the protected variant of Peptide E is Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 160), the coupling composition comprises DIC / Oxyma, EDC / Oxyma, PyBOP / collidine, PyOxim / collidine, TBTU / collidine, TBTU / DIEA, HCTU / DIEA, or PyOxim / DIEA (preferably DIC / Oxyma), and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours). In some embodiments, the coupling composition comprises DIO and Oxyma in DMSO.

[0510] In some embodiments, the method further comprises selectively deprotecting the N-terminus of the protected variant of Peptide C (e.g., using an Fmoc deprotection method described herein).10696-WQ01-SEC

[0511] Also provided herein is a method of preparing Peptide A or a protected variant thereof, comprising coupling a protected variant of Fragment 1 A with a protected variant of Peptide C to form a protected variant of Peptide A, wherein Fragment 1A comprises the amino acid sequence of SEQ ID NO: 11, Peptide C comprises the amino acid sequence of SEQ ID NO: 10, and Peptide A comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the method further comprises deprotecting the protected variant of Peptide A to isolate Peptide A. In some embodiments, the method further comprises adding a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0512] In some embodiments, the protected variant of Fragment 1 A is coupled with the protected variant of Peptide C in solution. In some embodiments, the protected variant of Fragment 1 A is coupled with the protected variant of Peptide C under liquid phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 1 A is coupled with the protected variant of Peptide C under classical solution phase peptide coupling conditions. In some embodiments, the protected variant of Fragment 1 A is coupled with the protected variant of Peptide C under tag-assisted liquid phase peptide coupling conditions.

[0513] In other embodiments, the protected variant of Fragment 1A is coupled with the protected variant of Peptide C on solid support. In some embodiments, the protected variant of Fragment 1 A is coupled with the protected variant of Peptide C under solid phase peptide coupling conditions.

[0514] In some embodiments, a C-terminal carboxyl group of the protected variant of Fragment 1A is preactivated. In other embodiments, a C-terminal carboxyl group of the protected variant of Fragment 1 A is activated in situ.

[0515] In some embodiments, the method comprises:contacting the protected variant of Fragment 1 A with a coupling composition to form a protected variant of Fragment 1 A with an activated C-terminal carboxyl group ("activated Fragment 1 A"); andcontacting the protected variant of Peptide C with the activated Fragment 1 A to form the protected variant of Peptide A.

[0516] In some embodiments, the method comprises:contacting the protected variant of Fragment 1 A with a coupling composition on solid support to form a protected variant of Fragment 1 A with an activated C-terminal carboxyl group ("activated Fragment 1 A"); and contacting the protected variant of Peptide C with the activated Fragment 1 A on solid support to form the protected variant of Peptide A.

[0517] In some embodiments, the method comprises:contacting the protected variant of Fragment 1 A with a coupling composition in solution to form a protected variant of Fragment 1 A with an activated C-terminal carboxyl group ("activated Fragment 1 A"); and10696-W001-SECcontacting the protected variant of Peptide C with the activated Fragment 1 A in solution to form the protected variant of Peptide A.

[0518] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 1 A is pre-activated), the method comprises:contacting the protected variant of Fragment 1 A with a coupling composition to form a protected variant of Fragment 1 A with an activated C-terminal carboxyl group ("activated Fragment 1 A"); andcontacting the protected variant of Peptide C with the activated Fragment 1 A to form the protected variant of Peptide A, wherein the contacting does not occur in the presence of a coupling reagent.

[0519] In some embodiments, the protected variant of Fragment 1 A is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 1 A and the coupling composition.

[0520] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 1 A is pre-activated), the method comprises:contacting the protected variant of Fragment 1 A with a coupling composition on solid support to form a protected variant of Fragment 1 A with an activated C-terminal carboxyl group ("activated Fragment 1 A"); and contacting the protected variant of Peptide C with the activated Fragment 1 A on solid support to form the protected variant of Peptide A, wherein the contacting does not occur in the presence of a coupling reagent.

[0521] In some embodiments, the protected variant of Fragment 1 A is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 1 A and the coupling composition.

[0522] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 1 A is pre-activated), the method comprises:contacting the protected variant of Fragment 1 A with a coupling composition in solution to form a protected variant of Fragment 1 A with an activated C-terminal carboxyl group ("activated Fragment 1 A"); and contacting the protected variant of Peptide C with the activated Fragment 1 A in solution to form the protected variant of Peptide A, wherein the contacting does not occur in the presence of a coupling reagent.

[0523] In some embodiments, the protected variant of Fragment 1 A is present in molar excess relative to the coupling reagent in the coupling composition during the contacting of the protected variant of Fragment 1 A and the coupling composition.

[0524] In some embodiments, the contacting of the protected variant of Fragment 1 A with the coupling composition and the contacting of the activated Fragment 1 A with the protected variant of Peptide C occur in the same reaction vessel. In other embodiments, the contacting of the protected variant of Fragment 1 A with the10696-WC01-SECcoupling composition and the contacting of the activated Fragment 1 A with the protected variant of Peptide C occur in different reaction vessels.

[0525] In some embodiments, the activated Fragment 1 A is contacted with the protected variant of Peptide C in the presence of a base and / or a coupling additive but not a coupling reagent (such as, e.g., in certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 1 A is pre-activated and the protected variant of Fragment 1 A is present in molar excess relative to the coupling reagent in the coupling composition during the contacting to form activated Fragment 1 A).

[0526] In some embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 1 A is pre-activated, the protected variant of Peptide C has a free N-terminal amino group and a free C-terminal carboxyl group.

[0527] In some embodiments, the coupling composition comprises a coupling reagent and a base. In some embodiments, the coupling composition comprises a coupling reagent (e.g., a carbodiimide, e.g., DIG, DCC, EDC) and a coupling additive (e.g., HOBt, 5-CI-HOBt, Oxyma).

[0528] In some embodiments, the coupling composition comprises a coupling reagent, a coupling additive, and a base.

[0529] In some embodiments, the coupling reagent is a carbodiimide, such as, e.g., DIG, DCC, or EDC. In other embodiments, the coupling reagent is BOP-CI.

[0530] In some embodiments (e.g., certain embodiments in which a C-terminal carboxyl group of the protected variant of Fragment 1 A is activated in situ), the method comprises coupling the protected variant of Fragment 1 A with the protected variant of Peptide C in the presence of a coupling composition. In some embodiments, the method comprises coupling the protected variant of Fragment 1 A with the protected variant of Peptide C in solution in the presence of a coupling composition.

[0531] In some embodiments, the protected variant of Peptide C does not have a free carboxyl group at the C-terminus. In some embodiments, the C-terminus of the protected variant of Peptide C is protected. In some embodiments, the C-terminus of the protected variant of Peptide C is amidated. In some embodiments, the protected variant of Peptide C has a free amino group at the N-terminus. In preferred embodiments, the protected variant of Peptide C has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus. In particularly preferred embodiments, the protected variant of Peptide C has a free amino group at the N-terminus and an amidated C-terminus.

[0532] In some embodiments, the protected variant of Fragment 1 A does not have a free amino group at the N-terminus. In some embodiments, the N-terminus of the protected variant of Fragment 1 A is protected (preferably Boc-protected). In some embodiments, the protected variant of Fragment 1 A has a free carboxyl group at the C-terminus. In preferred embodiments, the protected variant of Fragment 1 A has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred10696-W001-SECembodiments, the protected variant of Fragment 1 A has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus.

[0533] In some embodiments, the protected variant of Peptide C has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 1 A has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus.

[0534] In some embodiments, the C-terminus of the protected variant of Peptide C is amidated, and the N-terminus of the protected variant of Fragment 1 A is protected (preferably Boc-protected).

[0535] In preferred embodiments, the protected variant of Peptide C has a free amino group at the N-terminus and does not have a free carboxyl group at the C-terminus, and the protected variant of Fragment 1 A has a free carboxyl group at the C-terminus and does not have a free amino group at the N-terminus. In particularly preferred embodiments, the protected variant of Peptide C has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 1 A has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus.

[0536] In some embodiments, the protected variant of Peptide C has a free amino group at the N-terminus and an amidated C-terminus, and the protected variant of Fragment 1 A has a free carboxyl group at the C-terminus and a Boc-protected N-terminus.

[0537] In some embodiments, the protected variant of Fragment 1 A is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-OH (SEQ ID NO: 95).

[0538] In some embodiments, the protected variant of Peptide C is Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 96).

[0539] In some embodiments, the protected variant of Fragment 1 A is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-OH (SEQ ID NO: 95), and the protected variant of Peptide C is Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2(SEQ ID NO: 96).

[0540] In some embodiments, the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0541] In some embodiments, the coupling composition comprises DIC / Oxyma, EDC / Oxyma,Py BOP / collidine, PyOxim / collidine, or TBTU / collidine. In some embodiments, the coupling composition comprises DIC / Oxyma, EDC / Oxyma, Py BOP / collidine, PyOxim / collidine, or TBTU / collidine.10696-W001-SEC

[0542] In some embodiments, the coupling composition comprises DIG and Oxyma. In some embodiments, the coupling composition comprises DIG and Oxyma in DMSO. In some embodiments, the coupling composition comprises DIO and Oxyma in DMF.

[0543] In some embodiments, the protected variant of Fragment 1 A is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-OH (SEQ ID NO: 95), the protected variant of Peptide C is Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 96), and the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine.

[0544] In some embodiments, the protected variant of Fragment 1 A is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-OH (SEQ ID NO: 95), the protected variant of Peptide C is Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 96), and the coupling composition comprises DIC / Oxyma, EDC / Oxyma, Py BOP / collidine, PyOxim / collidine, or TBTU / collidine (preferably DIC / Oxyma). In some embodiments, the coupling composition comprises DIC / Oxyma, EDC / Oxyma, Py BOP / collidine, PyOxim / collidine, or TBTU / collidine (preferably DIC / Oxyma) in DMSO. In some embodiments, the coupling composition comprises DIO and Oxyma in DMSO or DMF. In some embodiments, the coupling composition comprises DIO and Oxyma in DMSO. In some embodiments, the coupling composition comprises DIO and Oxyma in DMF.

[0545] In some embodiments, the protected variant of Fragment 1 A is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-OH (SEQ ID NO: 95), and the coupling composition comprises DIO and Oxyma. In some embodiments, the coupling composition comprises DIO and Oxyma in DMSO or DMF. In some embodiments, the coupling composition comprises DIO and Oxyma in DMSO. In some embodiments, the coupling composition comprises DIO and Oxyma in DMF.

[0546] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 30°C. In some embodiments, the coupling occurs at a temperature in the range of 30°C to 50°C. In some embodiments, the coupling occurs at a temperature in the range of 35°C to 45°C.

[0547] In some embodiments, the method comprises coupling the protected variant of Fragment 1A and the protected variant of Peptide C for a time in the range of 1 hour to 72 hours. In some embodiments, the time is in the range of 6 hours to 24 hours.

[0548] In some embodiments, the coupling occurs at a temperature in the range of 0°C to 60°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 0°C10696-W001-SECto 60°C for a time in the range of 6 hours to 24 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 1 hour to 72 hours. In some embodiments, the coupling occurs at a temperature in the range of 20°C to 50°C for a time in the range of 6 hours to 24 hours.

[0549] In some embodiments, the protected variant of Fragment 1 A is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-OH (SEQ ID NO: 95), the protected variant of Peptide C is Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 96), the coupling composition comprises TBTU / collidine, PyBOP / TEA, TBTU / TMEDA, DIC / Oxyma, T3P / Oxyma, T3P / Oxyma / collidine, HCTU / collidine, HATU / collidine, HBTU / collidine, TBTU / collidine, DMAP / EDC, Oxyma / PyOxim, or Oxyma / PyOxim / collidine, and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours).

[0550] In some embodiments, the protected variant of Fragment 1 A is Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Tyr(tBu)-OH (SEQ ID NO: 95), the protected variant of Peptide C is Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Gly-Gly-Gly-Gly-Ser(tBu)-Lys(ivDde)-NH2 (SEQ ID NO: 96), the coupling composition comprises DIC / Oxyma, EDC / Oxyma, Py BOP / collidine, PyOxim / collidine, or TBTU / collidine (preferably DIC / Oxyma), and the coupling occurs at a temperature in the range of 0°C to 60°C (preferably a temperature in the range of 20°C to 50°C) for a time in the range of 1 hour to 72 hours (preferably a time in the range of 6 hours to 24 hours). In some embodiments, the coupling composition comprises DIG and Oxyma in DMSO or DMF. In some embodiments, the coupling composition comprises DIG and Oxyma in DMSO. In some embodiments, the coupling composition comprises DIG and Oxyma in DMF.

[0551] Also provided herein is a method of preparing Peptide A or a protected variant thereof, comprising:coupling or having coupled a protected variant of Fragment 2A with a protected variant of Peptide B to form a first protected variant of Peptide C, wherein the protected variant of Fragment 2A has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Peptide B has a free amino group at the N-terminus, Fragment 2A comprises the amino acid sequence of SEQ ID NO: 12, Peptide B comprises the amino acid sequence of SEQ ID NO: 9, and Peptide C comprises the amino acid sequence of SEQ ID NO: 10;selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide C to form a second protected variant of Peptide C with a free amino group at the N-terminus; and coupling or having coupled a protected variant of Fragment 1 A with the second protected variant of Peptide C to form a protected variant of Peptide A, wherein the protected variant of Fragment 1 A has a free carboxyl group at the C-terminus and a protected (preferably Boc-protected) N-terminus, Fragment 1 A comprises10696-WC01-SECthe amino acid sequence of SEQ ID NO: 11 , and Peptide A comprises the amino acid sequence of SEQ ID NO: 7.

[0552] In some embodiments, the method further comprises deprotecting or having deprotected the protected variant of Peptide A to isolate Peptide A.

[0553] In some embodiments, the method further comprises adding or having added a bromoacetyl moiety to the Lys at the C-terminus of Peptide A or a protected variant thereof.

[0554] In some embodiments, the protected variant of Fragment 2A and the protected variant of Peptide B are coupled under conditions described herein.

[0555] In some embodiments, the protected variant of Fragment 1 A and the second protected variant of Peptide C are coupled under conditions described herein.

[0556] In some embodiments, the protected variant of Fragment 2A and the protected variant of Peptide B are coupled under conditions described above, and the protected variant of Fragment 1 A and the second protected variant of Peptide C are coupled under conditions described herein.

[0557] In some embodiments, the N-terminus of the first protected variant of Peptide C is Fmoc-protected, and the selective deprotection comprises contacting the first protected variant of Peptide C with an amine base. In some embodiments, the amine base is selected from piperidine, piperazine, and morpholine (e.g., 5-30% (v / v) piperidine, piperazine, or morpholine). In some embodiments, the amine base is piperidine. In some embodiments, the N-terminus of the first protected variant of Peptide C is Fmoc-protected, and the selective deprotection comprises contacting the first protected variant of Peptide C with piperidine in DMF, optionally in the presence of DBU.

[0558] In some embodiments, the N-terminus of the first protected variant of Peptide C is Boc-protected, and the selective deprotection comprises contacting the first protected variant of Peptide C with an acid (preferably an acid selected from TFA, HCI, and HBr). In some embodiments, the first protected variant of Peptide C is contacted with TFA (e.g., neat TFA or TFA in DCM, e.g., 20-50% (v / v) TFA in DCM). In some embodiments, the first protected variant of Peptide C is contacted with HCI (e.g., HCI in DCM, DCE, toluene, or dioxane, e.g., 3-4 M HCI in DCM or dioxane).

[0559] In some embodiments, the N-terminus of the first protected variant of Peptide C is Boc-protected, and the selective deprotection comprises contacting the first protected variant of Peptide C with an acid (preferably an acid selected from TFA, HCI, and HBr) in the presence of a scavenger. In some embodiments, the scavenger is selected from hydrosilanes (e.g., TIS, triethylsilane (TES)), phenols (e.g., phenol, anisole, m-cresol), thioethers (e.g., thioanisole, dimethyl sulfide), thiols / dithiols (e.g., EDT, DTT, 1 -dodecanethiol), water, indole, and combinations of any of the foregoing. In some preferred embodiments, the scavenger is a hydrosilane. In some particularly preferred embodiments, the scavenger is TIS. In some embodiments, the first protected variant of Peptide C is contacted with TFA (e.g., neat TFA or TFA in DCM, e.g., 20-50% (v / v) TFA in DCM) in the presence10696-WQ01-SECof a scavenger (preferably TIS). In some embodiments, the first protected variant of Peptide C is contacted with HCI (e.g., HCI in DCM, DCE, toluene, or dioxane, e.g., 3-4 M HCI in DCM or dioxane) in the presence of a scavenger.

[0560] In some embodiments, the N-terminus of the first protected variant of Peptide C is Trt-protected, and the selective deprotection comprises contacting the first protected variant of Peptide C with a Bronsted acid or a Lewis acid (preferably TFA, HCI, or dichloroacetic acid, most preferably TFA), optionally in the presence of a scavenger (preferably TIS), to selectively remove the trityl protecting group at the N-terminus. In some embodiments, the first protected variant of Peptide C is contacted with a Bronsted acid (preferably a dilute Bronsted acid). In some embodiments, the Bronsted acid is selected from TFA, HCI, and dichloroacetic acid (preferably dilute TFA, HCI, or dichloroacetic acid). In other embodiments, the first protected variant of Peptide C is contacted with a Lewis acid (preferably a dilute Lewis acid).

[0561] In some embodiments, the scavenger is selected from hydrosilanes (e.g., TIS, triethylsilane (TES)), phenols (e.g., phenol, anisole, m-cresol), thioethers (e.g., thioanisole, dimethyl sulfide), thiols / dithiols (e.g., EDT, DTT, 1 -dodecanethiol), water, indole, and combinations of any of the foregoing. In some embodiments, the scavenger is a hydrosilane. In some embodiments, the scavenger is TIS.

[0562] In some embodiments, the N-terminus of the first protected variant of Peptide C is Trt-protected, and the selective deprotection comprises contacting the first protected variant of Peptide C with HCI. In some embodiments, the selective deprotection comprises contacting the first protected variant of Peptide C with dilute HCI.

[0563] In some embodiments, the N-terminus of the first protected variant of Peptide C is Trt-protected, and the selective deprotection comprises contacting the first protected variant of Peptide C with TFA in the presence of TIS. In some embodiments, the selective deprotection comprises contacting the first protected variant of Peptide C with dilute TFA in the presence of TIS.

[0564] Also provided herein is a method of preparing Peptide A or a protected variant thereof, comprising:(A)(i) (a) coupling a protected variant of Fragment 3 with a protected variant of Fragment 4 to form a first protected variant of Peptide E, wherein the protected variant of Fragment 3 has a free carboxyl group at the C-terminus and a protected (preferably Fmoc-protected) N-terminus, the protected variant of Fragment 4 has a free amino group at the N-terminus, Fragment 3 comprises the amino acid sequence of SEQ ID NO: 3, Fragment 4 comprises the amino acid sequence of SEQ ID NO: 4, and Peptide E comprises the amino acid sequence of SEQ ID NO: 8; (b) selectively deprotecting the N-terminus of the first protected variant of Peptide E from step (A)(i)(a) to form a second protected variant of Peptide E with a free amino group at the N-terminus; (c) coupling the second protected variant of Peptide E from step (A)(i)(b) with an N-protected alanine to form a first protected variant of Peptide B, wherein Peptide B comprises the amino acid sequence of SEQ ID NO: 9; and (d) selectively10696-WQ01-SECdeprotecting the N-terminus of the first protected variant of Peptide B from step (A)(i)(c) to form a second protected variant of Peptide B with a free amino group at the N-terminus; OR(ii) (a) coupling a protected variant of Fragment 3A with a protected variant of Fragment 4 to form a first protected variant of Peptide B, wherein the protected variant of Fragment 3A has a free carboxyl group at the C-terminus and a protec...

Claims

1. 10696-WQ01-SECWhat is claimed is:

1. A method of preparing Peptide A or a variant thereof, comprising:(A) coupling a protected variant of Fragment 5 with a protected variant of Fragment 6 to form a protected variant of Peptide B; and / or(B) coupling a protected variant of Fragment 1 with a protected variant of Fragment 2B to form a protected variant of Peptide D; and / or(C) coupling a protected variant of Peptide B with a protected variant of Peptide D to form a protected variant of Peptide A,wherein Peptide A has the amino acid sequence of SEQ ID NO: 7, Peptide B has the amino acid sequence of SEQ ID NO: 9, Peptide D has the amino acid sequence of SEQ ID NO: 15, Fragment 1 has the amino acid sequence of SEQ ID NO: 1, Fragment 2B has the amino acid sequence of SEQ ID NO: 16, Fragment 5 has the amino acid sequence of SEQ ID NO: 5, and Fragment 6 has the amino acid sequence of SEQ ID NO: 6.

2. The method of claim 1 , wherein the coupling of any of step (A), step (B), or step (C) is independently mediated by a coupling reagent and optionally a coupling additive and / or a base.

3. The method of claim 2, wherein each coupling reagent is independently selected from carbodiimides, aminium salts, phosphonium salts, phosphonic acid anhydrides, phosphate-type coupling reagents, and combinations of any of the foregoing.

4. The method of claim 2 or claim 3, wherein each coupling reagent is independently selected from N,N'-diisopropylcarbodiimide (DIG), N, N'-dicyclohexylcarbodiimide (DOC), 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDO), O-(1 H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(1 H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HCTU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TATU), O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU), 2-(1H-benzotriazol-1-yl)-1 ,1 ,3,3-tetramethylaminium nitrate (TNTU), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholino)]uronium hexafluorophosphate (COMU), 0-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCIQP), (benzotriazol-1 -10696-W001-SECyloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-CI), (ethyl cyano(hydroxyimino)acetato-O2)tri(1 -pyrrolidinyl)phosphonium hexafluorophosphate (PyOxim), propanephosphonic acid anhydride (T3P), 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT), and combinations of any of the foregoing.

5. The method of any one of claims 1 to 4, wherein the coupling of any of step (A), step (B), or step (C) is independently mediated by a coupling reagent, a coupling additive, and optionally a base.

6. The method of any one of claims 2 to 5, wherein each coupling additive is independently selected from 4-(dimethylamino)pyridine (DMAP), 1 -hydroxybenzotriazole (HOBt), 1-hydroxy-6-chlorobenzotriazole (6-CI-HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 2-pyridinol 1 -oxide (HOPO), ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma), and combinations of any of the foregoing.

7. The method of any one of claims 1 to 6, wherein the coupling of any of step (A), step (B), or step (0) is independently mediated by a coupling reagent, a base, and optionally a coupling additive.

8. The method of any one of claims 2 to 7, wherein each base is independently selected from N,N-diisopropylethylamine (DIPEA), N-methylmorpholine (NMM), N-ethylmaleimide (NEM), triethylamine (TEA), 2,6-lutidine, 2,4,6-collidine, 2,2,6,6-tetramethylpiperidine (TMP), and combinations of any of the foregoing.

9. The method of any one of claims 1 to 8, wherein the coupling of any of step (A), step (B), or step (0) is independently mediated by DIC / Oxyma, DIC / HOPO, EDC / DMAP, EDC / HOBt / 2,4,6-collidine, HATU / 2,4,6-collidine, HBTU / 2,4,6-collidine, HCTU / 2,4,6-collidine, Py BOP / 2,4,6-collidine, PyBOP / TEA, PyOxim / Oxyma, PyOxim / Oxyma / 2,4,6-collidine, T3P / 0xyma, T3P / Oxyma / 2,4,6-collidine, or TBTU / 2,4,6-collidine.

10. The method of any one of claims 1 to 9, wherein the coupling of any of step (A), step (B), or step (C) is conducted at a temperature in the range of 10°C to 60°C.

11. The method of any one of claims 1 to 10, wherein the coupling of any of step (A), step (B), or step (C) is conducted for a time in the range of 1 hour to 72 hours.

12. The method of any one of claims 1 to 11, wherein the coupling of any of step (A), step (B), or step (C) is conducted in a mixture comprising an organic solvent.

13. The method of any one of claims 1 to 12, wherein the coupling of any of step (A), step (B), or step (C) is conducted in a solution comprising an organic solvent.10696-W001-SEC14. The method of any one of claims 1 to 13, wherein the coupling of any of step (A), step (B), or step (C) is independently mediated by DIC / Oxyma, DIC / HOPO, EDC / DMAP, EDC / HOBt / 2,4,6-collidine, HATU / 2,4,6-collidine, HBTU / 2,4,6-collidine, HCTU / 2,4,6-collidine, Py BOP / 2,4,6-collidine, PyBOP / TEA, PyOxim / Oxyma, PyOxim / Oxyma / 2,4,6-collidine, T3P / 0xyma, T3P / Oxyma / 2,4,6-collidine, or TBTU / 2,4,6-collidine, and further wherein the coupling is conducted at a temperature in the range of 10°C to 60°C for a time in the range of 1 hour to 72 hours in a solution comprising an organic solvent.

15. The method of any one of claims 1 to 14, wherein the method comprises coupling a protected variant of Fragment 5 with a protected variant of Fragment 6 in solution to form a protected variant of Peptide B, wherein:the N-terminus, the side chain of the Trp residue at position 2, and the side chain of the Lys residue at position 5 of the protected variant of Fragment 5 are protected and the protected variant of Fragment 5 has a free carboxyl group at the C-terminus; andthe side chain of the Ser residue at position 5, the side chain of the Ser residue at position 10, the side chain of the Ser residue at position 15, and the side chain of the Lys residue at position 16 of the protected variant of Fragment 6 are protected, the protected variant of Fragment 6 has a free amino group at the N-terminus, and the protected variant of Fragment 6 does not have a free carboxyl group at the C-terminus.

16. The method of claim 15, wherein the coupling is mediated by a coupling reagent and a base.

17. The method of claim 15 or claim 16, wherein the coupling is mediated by 1 to 10 equivalents of a coupling reagent and 1 to 10 equivalents of a base, wherein equivalents are relative to the protected variant of Fragment 5.

18. The method of claim 16 or claim 17, wherein the coupling reagent comprises benzotriazol-1 -yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and the base comprises triethylamine.

19. The method of any one of claims 15 to 18, wherein the coupling is conducted at a temperature in the range of 20°C to 50°C in a solution comprising dimethyl sulfoxide (DMSO) and methanol (MeOH).

20. The method of any one of claims 15 to 19, wherein the coupling is mediated by 1 to 10 equivalents of PyBOP and 1 to 10 equivalents of triethylamine, wherein equivalents are relative to the protected variant of Fragment 5, and further wherein the coupling is conducted at a temperature in the range of 20°C to 40°C in a solution comprising DMSO and MeOH.

21. The method of claim 19 or claim 20, wherein the solution comprises 1-5:1 DMSO:MeOH.10696-WQ01-SEC22. The method of any one of claims 15 to 21, wherein:the side chain of the Trp residue at position 2 and the side chain of the Lys residue at position 5 of the protected variant of Fragment 5 are each protected with a tert-butyloxycarbonyl (Boc) protecting group; and the Ser residues at position 5, position 10, and position 15 of the protected variant of Fragment 6 are each protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser and the side chain of the Lys residue at position 16 of the protected variant of Fragment 6 is protected with an ivDde (1 -(4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl) protecting group.

23. The method of any one of claims 1 to 21, wherein the protected variant of Fragment 5 is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59) and the protected variant of Fragments is H-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130).

24. The method of any one of claims 1 to 23, wherein the method comprises coupling a protected variant of Fragment 1 with a protected variant of Fragment 2B in solution to form a protected variant of Peptide D, wherein:the N-terminus, the side chain of the His residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Thr residue at position 5, the side chain of the Thr residue at position 7, the side chain of the Ser residue at position 8, the side chain of the Asp residue at position 9, the side chain of the Tyr residue at position 10, the side chain of the Ser residue at position 11 , and the side chain of the Ser residue at position 12 of the protected variant of Fragment 1 are protected and the protected variant of Fragment 1 has a free carboxyl group at the C-terminus; andthe side chain of the Tyr residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Glu residue at position 4, the side chain of the Gin residue at position 5, the side chain of the Lys residue at position 8, and the side chain of the Glu residue at position 9 of the protected variant of Fragment 2B are protected and the protected variant of Fragment 2B has a free amino group at the N-terminus.

25. The method of claim 24, wherein the coupling is mediated by a coupling reagent, a coupling additive, and a base.

26. The method of claim 24 or claim 25, wherein the coupling is mediated by 1 to 10 equivalents of a coupling reagent, 1 to 5 equivalents of a coupling additive, and 1 to 10 equivalents of a base, wherein equivalents are relative to the protected variant of Fragment 2B.10696-WQ01-SEC27. The method of any one of claims 24 to 26, wherein the coupling is mediated by 1 to 5 equivalents of PyBOP and 1 to 5 equivalents of 2, 4,6-col lidi ne, wherein equivalents are relative to the protected variant of Fragment 2B.

28. The method of any one of claims 24 to 27, wherein the coupling is performed at a temperature in the range of 20°C to 50°C in a solution comprising tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO).

29. The method of any one of claims 24 to 28, wherein the coupling is mediated by 1 to 5 equivalents of PyBOP and 1 to 5 equivalents of 2, 4,6-col lidi ne, wherein equivalents are relative to the protected variant of Fragment 2B, and further wherein the coupling is performed at a temperature in the range of 30°C to 50°C in a solution comprising THF and DMSO.

30. The method of claim 28 or claim 29, wherein the solution comprises 5-8:1 THF:DMSO.

31. The method of any one of claims 24 to 30, wherein:the side chain of the His residue at position 1 of the protected variant of Fragment 1 is protected with a trityl (Trt) protecting group; the side chain of the Glu residue at position 3 of the protected variant of Fragment 1 is protected with a tert-butyl ester (OtBu) protecting group; the side chains of the Thr residues at position 5 and position 7 of the protected variant of Fragment 1 are each protected with a tert-butyl (tBu) protecting group; the side chains of the Ser residues at position 8 and position 11 of the protected variant of Fragment 1 are each protected with a tBu protecting group; the side chain of the Asp residue at position 9 of the protected variant of Fragment 1 is protected with an OtBu protecting group; the side chain of the Tyr residue at position 10 of the protected variant of Fragment 1 is protected with a tBu protecting group; and the Ser residue at position 12 of the protected variant of Fragment 1 is protected as a dimethylated pseudoproline (Psi(Me.Me)pro) moiety in which the oxazolidine is derived from Ser; andthe side chain of the Tyr residue at position 1 of the protected variant of Fragment 2B is protected with a tBu protecting group; the side chains of the Glu residues at position 3, position 4, and position 9 of the protected variant of Fragment 2B are each protected with a OtBu protecting group; the side chain of the Gin residue at position 5 of the protected variant of Fragment 2B is protected with a Trt protecting group; and the side chain of the Lys residue at position 8 of the protected variant of Fragment 2B is protected with a tert-butyloxycarbonyl (Boc) protecting group.

32. The method of any one of claims 1 to 31, wherein the protected variant of Fragment 1 is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 166) and the protected variant of Fragment 2B is H-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 30).10696-WC01-SEC33. The method of any one of claims 1 to 32, wherein the method comprises coupling a protected variant of Peptide B with a protected variant of Peptide D in solution to form a protected variant of Peptide A, wherein: the side chain of the Trp residue at position 2, the side chain of the Lys residue at position 5, the side chain of the Ser residue at position 13, the side chain of the Ser residue at position 18, the side chain of the Ser residue at position 23, and the side chain of the Lys residue at position 24 of the protected variant of Peptide B are protected, the protected variant of Peptide B has a free amino group at the N-terminus, and the protected variant of Peptide B does not have a free carboxyl group at the C-terminus; andthe N-terminus, the side chain of the His residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Thr residue at position 5, the side chain of the Thr residue at position 7, the side chain of the Ser residue at position 8, the side chain of the Asp residue at position 9, the side chain of the Tyr residue at position 10, the side chain of the Ser residue at position 11, the side chain of the Ser residue at position 12, the side chain of the Tyr residue at position 13, the side chain of the Glu residue at position 15, the side chain of the Glu residue at position 16, the side chain of the Gin residue at position 17, the side chain of the Lys residue at position 20, and the side chain of the Glu residue at position 21 of the protected variant of Peptide D are protected and the protected variant of Peptide D has a free carboxyl group at the C-terminus.

34. The method of claim 33, wherein the coupling is mediated by a coupling reagent, a coupling additive, and a base.

35. The method of claim 33 or claim 34, wherein the coupling is mediated by 1 to 10 equivalents of a coupling reagent, 1 to 10 equivalents of a coupling additive, and 1 to 10 equivalents of a base, wherein equivalents are relative to the protected variant of Peptide D.

36. The method of any one of claims 33 to 35, wherein the coupling is mediated by 1 to 10 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1 to 10 equivalents of 1-hydroxybenzotriazole (HOBt), and 1 to 10 equivalents of 2,4,6-collidine, wherein equivalents are relative to the protected variant of Peptide D.

37. The method of any one of claims 33 to 36, wherein the coupling is conducted at a temperature in the range of 20°C to 50°C in a solution comprising tetrahydrofuran (THF) and water (H2O).

38. The method of any one of claims 33 to 37, wherein the coupling is mediated by 1 to 10 equivalents of EDC, 1 to 10 equivalents of HOBt, and 1 to 10 equivalents of 2,4,6-collidine, wherein equivalents are relative to the protected variant of Peptide D, and further wherein the coupling is conducted at a temperature in the range of 20°C to 40°C in a solution comprising THF and H2O.10696-WQ01-SEC39. The method of claim 37 or claim 38, wherein the solution comprises 3-5:1 THF:H2O.

40. The method of any one of claims 1 to 39, wherein:the side chain of the T rp residue at position 2 and the side chain of the Lys residue at position 5 of the protected variant of Peptide B are each protected with a tert-butyloxycarbonyl (Boc) protecting group, the Ser residues at position 13, position 18, and position 23 of the protected variant of Peptide B are each protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser, and the side chain of the Lys residue at position 24 of the protected variant of Peptide B is protected with an ivDde (1 -(4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl) protecting group; andthe side chain of the His residue at position 1 of Peptide D is protected with a trityl (Trt) protecting group; the side chains of the Glu residues at position 3, position 15, position 16, and position 21 of Peptide D are each protected with a tert-butyl ester (OtBu) protecting group; the side chains of the Thr residues at position 5 and position 7 of Peptide D are each protected with a tert-butyl (tBu) protecting group; the side chains of the Ser residues at position 8 and position 11 of Peptide D are each protected with a tBu protecting group; the side chain of the Asp residue at position 9 of Peptide D is protected with an OtBu protecting group; the side chains of the Tyr residues at positions 10 and 13 of Peptide D are each protected with a tBu protecting group; the Ser residue at position 12 of Peptide D is protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser; the side chain of the Gin residue at position 17 of Peptide D is protected with a Trt protecting group; and the side chain of the Lys residue at position 20 of Peptide D is protected with a tertbutyloxycarbonyl (Boc) protecting group.

41. The method of any one of claims 1 to 40, wherein the protected variant of Peptide B and the protected variant of Peptide D that are coupled to form the protected variant of Peptide A are H-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 147) and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 169), respectively.

42. The method of claim 1 , the method comprising:(A) (i) coupling or having coupled a protected variant of Fragment 5 with a protected variant of Fragment 6 to form a first protected variant of Peptide B, wherein the protected variant of Fragment 5 has a free carboxyl group at the C-terminus and a protected N-terminus, the protected variant of Fragment 6 has a free amino group at the N-terminus, Fragment 5 has the amino acid sequence of SEQ ID NO: 5, Fragment 6 has the amino acid sequence of SEQ ID NO: 6, and Peptide B has the amino acid sequence of SEQ ID NO: 9; and (ii) selectively deprotecting or having selectively deprotected the N-terminus of the first protected variant of Peptide B from step (A)(i) to form a second protected variant of Peptide B with a free amino group at the N-terminus; and10696-WQ01-SEC(B) coupling or having coupled a protected variant of Fragment 1 with a protected variant of Fragment 2B to form a protected variant of Peptide D, wherein the protected variant of Fragment 1 has a free carboxyl group at the C-terminus and a protected N-terminus, the protected variant of Fragment 2B has a free amino group at the N-terminus, Fragment 1 has the amino acid sequence of SEQ ID NO: 1, Fragment 2B has the amino acid sequence of SEQ ID NO: 16, and Peptide D has the amino acid sequence of SEQ ID NO: 15; and (C) coupling or having coupled the second protected variant of Peptide B from step (A) with the protected variant of Peptide D from step (B) to form a protected variant of Peptide A, wherein Peptide A has the amino acid sequence of SEQ ID NO: 7.

43. The method of claim 42, wherein:the side chain of the Trp residue at position 2 and the side chain of the Lys residue at position 5 of the protected variant of Fragment 5 are protected;the side chain of the Ser residue at position 5, the side chain of the Ser residue at position 10, the side chain of the Ser residue at position 15, and the side chain of the Lys residue at position 16 of the protected variant of Fragment 6 are protected, and the protected variant of Fragment 6 does not have a free carboxyl group at the C-terminus;the side chain of the His residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Thr residue at position 5, the side chain of the Thr residue at position 7, the side chain of the Ser residue at position 8, the side chain of the Asp residue at position 9, the side chain of the Tyr residue at position 10, the side chain of the Ser residue at position 11, and the side chain of the Ser residue at position 12 of the protected variant of Fragment 1 are protected; andthe side chain of the Tyr residue at position 1, the side chain of the Glu residue at position 3, the side chain of the Glu residue at position 4, the side chain of the Gin residue at position 5, the side chain of the Lys residue at position 8, and the side chain of the Glu residue at position 9 of the protected variant of Fragment 2B are protected.

44. The method of claim 42 or claim 43, wherein:the side chain of the Trp residue at position 2 and the side chain of the Lys residue at position 5 of the protected variant of Fragment 5 are each protected with a tert-butyloxycarbonyl (Boc) protecting group;the Ser residues at position 5, position 10, and position 15 of the protected variant of Fragment 6 are each protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser and the side chain of the Lys residue at position 16 of the protected variant of Fragment 6 is protected with an ivDde (1 -(4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl) protecting group;10696-WQ01-SECthe side chain of the His residue at position 1 of the protected variant of Fragment 1 is protected with a trityl (Trt) protecting group; the side chain of the Glu residue at position 3 of the protected variant of Fragment 1 is protected with a tert-butyl ester (OtBu) protecting group; the side chains of the Thr residues at position 5 and position 7 of the protected variant of Fragment 1 are each protected with a tert-butyl (tBu) protecting group; the side chains of the Ser residues at position 8 and position 11 of the protected variant of Fragment 1 are each protected with a tBu protecting group; the side chain of the Asp residue at position 9 of the protected variant of Fragment 1 is protected with an OtBu protecting group; the side chain of the Tyr residue at position 10 of the protected variant of Fragment 1 is protected with a tBu protecting group; and the Ser residue at position 12 of the protected variant of Fragment 1 is protected as a dimethylated pseudoproline (Psi(Me,Me)pro) moiety in which the oxazolidine is derived from Ser; andthe side chain of the Tyr residue at position 1 of the protected variant of Fragment 2B is protected with a tBu protecting group; the side chains of the Glu residues at position 3, position 4, and position 9 of the protected variant of Fragment 2B are each protected with a OtBu protecting group; the side chain of the Gin residue at position 5 of the protected variant of Fragment 2B is protected with a Trt protecting group; and the side chain of the Lys residue at position 8 of the protected variant of Fragment 2B is protected with a tert-butyloxycarbonyl (Boc) protecting group.

45. The method of any one of claims 42 to 44, wherein:the protected variant of Fragment 5 is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59);the protected variant of Fragment 6 is H-Gly-Gly-Gly-Gly-Ser(Psi(Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130);the protected variant of Fragment 1 is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 166); andthe protected variant of Fragment 2B is H-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 30).

46. The method of any one of claims 42 to 45, wherein:the first protected variant of Peptide B is Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-G ly-Ser (Psi (Me, Me) Pro)-Gly-Gly-Gly-Gly-Ser(Psi (Me, M e)Pro)-G ly-G ly-G ly-G ly-Ser (Psi (Me, Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 170);the second protected variant of Peptide B is H-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 147); and10696-WQ01-SECthe protected variant of Peptide D is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 169).

47. The method of any one of claims 42 to 46, wherein the protected variant of Peptide A is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 171).

48. The method of any one of claims 1 to 47, wherein the method further comprises bromoacetylating an E-amino group of the C-terminal Lys residue of a protected variant of Peptide A to form a bromoacetylated protected variant of Peptide A, optionally wherein the bromoacetylating comprises selectively deprotecting the side chain of the C-terminal Lys residue of the protected variant of Peptide A to form a partially deprotected variant of Peptide A and then coupling the partially deprotected variant of Peptide A with bromoacetic acid or bromoacetic anhydride to form a bromoacetylated protected variant of Peptide A, wherein the coupling is mediated by a coupling reagent and optionally a coupling additive and / or a base.

49. The method of claim 48, wherein the method further comprises contacting the bromoacetylated protected variant of Peptide A with a Bronsted acid or a Lewis acid, optionally in the presence of a scavenger, to form a bromoacetylated variant of Peptide A, wherein the bromoacetylated variant of Peptide A has the amino acid sequence of SEQ ID NO: 1, the N-terminus and the side chains of the amino acids at positions 1-46 of the bromoacetylated variant of Peptide A are not protected, the C-terminal Lys residue is bromoacetylated, and the C-terminus of the bromoacetylated variant of Peptide A is amidated as a C-terminal primary amide.

50. A molecule selected from:Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (SEQ ID NO: 166);H-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 30);Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-OH (SEQ ID NO: 169);Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-OH (SEQ ID NO: 59);10696-WQ01-SECH-Gly-Gly-Gly-Gly-Ser(Psi(Me, Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 130);Trt-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 170);H-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 147); andBoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ser(Psi(Me,Me)Pro)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala-Trp(Boc)-Leu-Val-Lys(Boc)-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Gly-Gly-Gly-Gly-Ser(Psi(Me,Me)Pro)-Lys(ivDde)-NH2(SEQ ID NO: 171),or a salt thereof.

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