A photochemical approach to C-terminal A-amidation

Abstract

The present invention relates to a photochemical process for the production of C-terminal α-amides, a class of compounds that represent up to half of all biologically relevant peptide hormones. The invention provides a simple, versatile and economically efficient process suitable for large-scale production.

Description

[Technical field]

[0001] The present invention relates to a photochemical process for producing C-terminal α-amides, and more particularly to a method for producing peptides or proteins containing C-terminal α-amides using a photochemical process. [Background technology]

[0002] C-terminal α-amidation is a common post-translational modification that occurs in over half of all biologically active peptide hormones and neuropeptides and is often required for full biological activity.

[0003] Chemical C-terminal α-amidation requires selective functionalization of the C-terminus. Amidated peptides are produced by a fully synthetic approach (Non-Patent Document 1) and by in vitro enzymatic oxidation of recombinantly expressed C-terminal glycine-extended precursors (Non-Patent Document 2). However, these approaches are complex and expensive. For example, peptidylglycine α-amidating monooxygenase (PAM) and related amidating enzymes are not only substrate specific but must also be expressed in mammalian culture, which adds cost to the manufacturing process.

[0004] Baker et al. provide a method for the selective modification of cysteines with bromomaleimides and purport to disclose a method for the photolytic modification of cysteine ​​maleimide conjugates (Non-Patent Document 3 and Non-Patent Document 4).

[0005] An enzymatic approach to selective C-terminal functionalization and α-amidation, including a photolysis step, is further presented in US Pat. No. 5,393,411 and Non-Patent Document 5. Malins et al. purportedly disclose an electrochemical approach to selective C-terminal modification and α-amidation (Non-Patent Document 6).

[0006] Nevertheless, providing a low-cost, selective, and scalable process compatible with the manufacturing-scale preparation of peptides containing C-terminal α-amides remains a challenge. There is always room for improvement. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US5580751 [Non-patent literature]

[0008] [Non-Patent Document 1] Enzyme Microb. Technol. 16, 450-456 (1994) [Non-Patent Document 2] Nat. Biotechnol. 11, 64-70 (1993) [Non-Patent Document 3] Baker et al.,Chem.Commun.6583-6585(2009) [Non-Patent Document 4] Baker et al.,Org.Biomol.Chem.14,455-459(2016) [Non-Patent Document 5] Int.J.Peptide Protein Res.41,169-180(1993) [Non-Patent Document 6] Malins et al.,J.Am.Chem.Soc.143,11811-11819(2021) Summary of the Invention

[0009] One objective of this work is to provide a photochemical process for the generation of peptides or proteins containing C-terminal α-amides. The objective is to provide a selective, low-cost C-terminal peptide and protein amidation process that can be applied to a variety of peptides while overcoming at least some of the shortcomings of conventional amidation methods.

[0010] The overall photochemical reaction is shown in Scheme 1 below and further illustrated in Figure 1. According to Scheme 1, a method for producing a peptide or protein comprising a C-terminal α-amide of formula IV is provided, comprising: [ka] wherein R2 is a polypeptide; R1-X is a photolabeling agent, R1 is a photolabel and X is a leaving group; R3 is selected from the group consisting of hydrogen, methyl, and ethyl. Step (a): coupling a peptide or protein containing a C-terminal cysteine ​​amidation tag (Formula I) with a photolabel (R1) to obtain a peptide-photolabel conjugate (Formula II); Step (b): Irradiating the peptide-photolabeled conjugate (Formula II) to obtain a C-terminal enamide (Formula III) by photochemical conversion; and step (c). cleaving the C-terminal enamide (Formula III) to obtain the C-terminal α-amide (Formula IV).

[0011] In a first aspect of the invention, a method according to Scheme 1 is provided for producing a peptide or protein comprising a C-terminal α-amide of formula IV. This is accomplished by starting with a peptide or protein comprising a C-terminal cysteine ​​amidation tag (shown as formula I) and reacting it with a photolabeling agent (shown as R1-X) to obtain a peptide-photolabeled conjugate (formula II). The peptide-photolabeled conjugate is then exposed to light and undergoes photochemical conversion to obtain a peptide or protein enamide (formula III) which is then converted to the resulting C-terminal α-amide (formula IV).

[0012] The method for generating peptides or proteins containing C-terminal α-amides may include a photolabeling agent (R1-X) selected from the group consisting of 3-bromo-1H-pyrrole-2,5-dione, 4-chloro-7-nitrobenzofurazan, 2-bromo-1,4-naphthoquinone, 1-fluoro-2,4-dinitrobenzene, and 4-fluoro-7-sulfamoylbenzofurazan. The method for generating peptides or proteins containing C-terminal α-amides may include a photolabeling agent (R1-X) selected from the group consisting of trifluoroacetic acid (TFA), hydrochloric acid (HCl), sulfuric acid (H 2 SO 4 ), tosylate (TsOH), phosphate (H 3 PO 4 ), oxalic acid, 3,6-diphenyl-1,2,4,5-tetrazine, and 6,6'-(1,2,4,5-tetrazine-3,6-diyl)dinicotinic acid.

[0013] Methods for producing a peptide or protein comprising a C-terminal α-amide may include a polypeptide R2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, or 17.

[0014] 2. A method for producing a peptide or protein comprising a C-terminal α-amide according to Scheme 2, which is seen below and further illustrated in FIG. [ka] wherein R2 is a polypeptide; R3 is hydrogen. Step (a): coupling a peptide or protein containing a C-terminal cysteine ​​amidation tag (Formula I) with 4-chloro-7-nitrobenzofurazan to obtain a peptide-photolabeled conjugate (Formula II-a); Step (b): Irradiating the peptide-photolabeled conjugate (Formula II-a) to obtain a C-terminal enamide (Formula III) by photochemical conversion; Step (c): cleaving the C-terminal enamide (Formula III) to obtain the C-terminal α-amide (Formula IV).

[0015] The present invention according to a second aspect is carried out by reacting a peptide or protein containing a C-terminal cysteine ​​of formula I with 4-chloro-7-nitrobenzofurazan as a photolabeling agent to obtain a peptide-photolabel conjugate of formula II-a, which is subsequently converted by photochemical reaction to an enamide of formula III, which is then converted by cleavage to the desired C-terminal α-amide of formula IV.

[0016] The method for generating a peptide or protein comprising a C-terminal α-amide may further comprise coupling a cysteine ​​of peptide R2 with photolabel R1 to form a photolabel-protected cysteine ​​prior to irradiation (step (b)), and releasing the photolabel-protected cysteine ​​from peptide R2 after irradiation (step (b)). The method may further comprise adding a nucleophilic sulfide to release the photolabel-protected cysteine ​​from peptide R2.

[0017] The present photochemical amidation method is simple, broad in scope, economically efficient, and suitable for large-scale production.The present invention may also solve further problems that will become apparent from the disclosure of the exemplary embodiments.

[0018] Sequence Listing SEQ ID NO:1 represents a non-limiting exemplary peptide amino acid sequence that may be provided as R2 (Scheme 1 or 2) immediately preceding the C-terminal cysteine, and the penultimate amino acid of the peptide (from the N-terminus to the C-terminus of the polypeptide) prior to the photoamidation process (i.e., Formula I in Schemes 1 and 2) may vary: SEQ ID NO:2 represents the amino acid sequence of an exemplary embodiment of a peptide capable of forming R2 (Scheme 1 or 2), SEQ ID NO:3 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing the glucagon-like peptide-1 (GLP-1) peptide; SEQ ID NO: 4 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing a pancreastatin (PST) inhibitor, more specifically, the pancreastatin inhibitor peptide-8 (PSTi8) peptide; SEQ ID NO:5 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing the pancreatic pYY(3-36) peptide; SEQ ID NO:6 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing a luteinizing hormone releasing hormone (LHRH) agonist; SEQ ID NO: 7 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing the gastrin releasing peptide (GRP) peptide; SEQ ID NO: 8 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing adrenocorticotropic hormone (ACTH), more specifically cosyntropin; SEQ ID NO:9 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing the QRF-amide peptide; SEQ ID NO: 10 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing the GLP-1 receptor-neuropeptide Y receptor 2 coagonist EP45; SEQ ID NO: 11 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing an entry inhibitor, more specifically brevirtide; SEQ ID NO: 12 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing osteoclin (OSTN) peptide; SEQ ID NO: 13 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing an antiretroviral drug, more specifically, enfuvirtide; SEQ ID NO: 14 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing an amylin receptor agonist, more specifically, pramlintide; SEQ ID NO:15 represents the amino acid sequence of a non-limiting exemplary peptide that may be provided as R2 (Scheme 1 or 2), in which a disulfide bond is desired therein: SEQ ID NO: 16 represents the amino acid sequence of the peptide portion (R2) of the starting material for producing a GLP-1 receptor-amylin receptor coagonist; SEQ ID NO: 17 represents the amino acid sequence of the peptide portion (R2) of the starting material consisting of a peptide for amidation and N-terminal extension to generate a GLP-1 receptor-amylin receptor coagonist; SEQ ID NO: 18 represents the amino acid sequence of an exemplary N-terminal extension which may form part of the peptide moiety (R2). [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the overall reaction scheme (Scheme 1) of a photochemical process for producing a peptide or protein containing a C-terminal α-amide. [Diagram 2] FIG. 2 shows an embodiment of a reaction (Scheme 2) of a photochemical process for producing a peptide or protein containing a C-terminal α-amide, in which chloro-7-nitrobenzofurazan is used as the photolabeling agent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Structure-activity studies of therapeutic products, such as amylin peptides (Cooper et al., GJS Molecular and functional characterization of amylin, a peptide associated with type 2 diabetes mellitus. Proc. Natl. Acad. Sci. USA 86, 9662-9666 (1989)), NPY (Rivier et al., Synthesis and hypertensive activity of neuropeptide Y fragments and analogues with modified N- or C-termini or D-substitutions. J. Med. Chem. 32, 597-601 (1989)), and others (Nuss et al., The current state of peptide drug discovery: back to the future? J. Med. Chem. 61, 1382-1414 (2018)), have revealed that C-terminal amidation is often required for full biological activity. However, manufacturing-scale preparation of peptides containing C-terminal α-amides is particularly challenging. Thus, there is a need for selective, low-cost, and scalable semi-recombinant technologies for producing peptides containing C-terminal amides.

[0021] The present invention relates to a photochemical process for producing peptides or proteins comprising C-terminal α-amides. The method offers the advantage of being broad in scope, such that it can be utilized to generate a wide variety of biologically active peptides with C-terminal α-amides. As will become apparent, the process for producing peptides or proteins comprising C-terminal α-amides disclosed herein is not tied to a particular peptide (R2) of the protein, but provides a method for the amidation of a wide variety of peptides or proteins via a C-terminal cysteine, which may be referred to herein as a C-terminal cysteine ​​amidation tag, given its purpose in the photochemical process. The method is specific enough to allow for C-terminal amidation of peptides and proteins even when the peptides and proteins contain more than one cysteine ​​therein (i.e., cysteines in peptide R2 in addition to the C-terminal cysteine ​​amidation tag used to produce the peptide or protein comprising a C-terminal α-amide). Similarly, or alternatively, another advantage of the present invention is that the method is economically efficient due to readily and commercially available starting materials and simple reaction set-up, making the process suitable for batch or distribution manufacturing and amenable to large-scale manufacturing.

[0022] The present invention is carried out as shown in Scheme 1 (Figure 1) by starting with a peptide or protein containing a C-terminal cysteine ​​residue (Formula I), reacting it with a photolabeling agent (R1-X) to obtain a peptide-photolabeled conjugate (Formula II), followed by exposing the peptide-photolabeled conjugate to light to obtain a peptide or protein enamide (Formula III), and then converting the resulting enamide to a C-terminal α-amide (Formula IV).

[0023] The photochemical amidation reaction described above consists of three general reaction steps: Step (a): coupling a peptide or protein containing a C-terminal cysteine ​​residue (Formula I) with a photolabeling agent (R1-X) to obtain a peptide-photolabeled conjugate (Formula II); Step (b): photochemically converting the resulting peptide-photolabeled conjugate (Formula II) to obtain a C-terminal enamide (Formula III); Step (c): cleavage of the C-terminal enamide (Formula III) to give the C-terminal α-amide (Formula IV).

[0024] These steps (a), (b), and (c) are also generally shown in Scheme 1, and in an exemplary embodiment in Scheme 2. Each step may be characterized by its own reactants and conditions, as defined below.

[0025] It is understood that the process for producing peptides or proteins comprising C-terminal α-amides disclosed herein can be carried out at various strategic times in the synthesis of biologically active peptides. In certain embodiments, the process for producing peptides or proteins comprising C-terminal α-amides disclosed herein can be used on intermediate products, including but not limited to precursors, before a filtration step, such as high performance liquid chromatography (HPLC) filtration, before an additional ligation step, and / or before cleavage of the peptide extension. The term extension here refers to at least one amino acid extending from the N-terminus of the desired peptide, such as may be the case for the precursor. Such peptide R2 may have the following structure:

[0026] (extension)-(desired peptide) In such embodiments, the combined extension and desired peptide may form part of peptide R2 during the photochemical process of the present disclosure, and the extension may be meant to be cleaved from peptide R2 in a subsequent step.

[0027] It may be considered advantageous to proceed with the photochemical amidation process early in the synthesis of biologically active peptides. Large scale C-terminal amidation by the photochemical methods described herein can be performed well before the completion of the process steps required to arrive at the final active pharmaceutical ingredient is required, minimizing the loss of downstream intermediate products that may be more costly in terms of materials used and / or labor invested in the synthesis process.

[0028] The present photochemical amidation reaction may also solve additional problems that become apparent from the disclosure of the exemplary embodiments.

[0029] In the following, the Greek letters may be represented by their symbols or by the corresponding descriptive names, e.g., α is alpha, β is beta, ε is epsilon, γ is gamma, ω is omega, etc. The Greek letter μ may also be represented by "u", e.g., μL is uL or μM is uM.

[0030] General reaction process Step (a) - Coupling a peptide or protein containing a C-terminal cysteine ​​residue (Formula I) with a photolabeling agent (R1-X) to obtain a peptide-photolabeling conjugate (Formula II) The first step (step (a)) of the photochemical amidation reaction may be referred to as coupling a peptide or protein containing a C-terminal cysteine ​​residue (also referred to as Formula I) with a photolabel (R1) to obtain a peptide-photolabel conjugate (also referred to as Formula II).

[0031] C-Terminal Cysteine ​​Residue (Formula I) Formula I is a peptide or protein comprising a polypeptide moiety (R2) and a C-terminal cysteine ​​(Cys) residue. The polypeptide moiety R2 may also be referred to as a protein or peptide. The compound of formula I may also be referred to as a peptide having a C-terminal cysteine ​​(Cys) amidation tag. The compound of formula I may also be referred to as a polypeptide or a polypeptide comprising a C-terminal cysteine ​​(Cys) residue. The C-terminal cysteine ​​residue may be a residue of the amino acid cysteine ​​substituted at the C-terminus of the corresponding peptide or protein.

[0032] When referring to R2, this is understood as the polypeptide portion of the peptide or protein of formula I that does not include the C-terminal cysteine ​​amidation tag and that will ultimately be provided with a C-terminal amide according to formula IV by this method.

[0033] The photochemical reactions disclosed herein have been tested on peptides differing in the penultimate amino acid (Example 3, Table 4, entries 1-20). To this end, an exemplary peptide R2 (when R2 of SEQ ID NO:1 forms part of formula I of Scheme 1, the C-terminus is a cysteine ​​amidation tag) was provided with the sequence IWTKDHEEVYEX (SEQ ID NO:1) differing in the penultimate amino acid. In one embodiment, X is Ala. In one embodiment, X is Asp. In one embodiment, X is Glu. In one embodiment, X is Phe. In one embodiment, X is Gly. In one embodiment, X is His. In one embodiment, X is Ile. In one embodiment, X is Lys. In one embodiment, X is Leu. In one embodiment, X is Met. In one embodiment, X is Asn. In one embodiment, X is Pro. In one embodiment, X is Gln. In one embodiment, X is Arg. In one embodiment, X is Ser. In one embodiment, X is Thr. In one embodiment, X is Val. In one embodiment, X is Trp. In one embodiment, X is Tyr. For example, when X in IWTKDHEEVYEX (SEQ ID NO: 1) is alanine (Ala), the resulting peptide R2 is IWTKDHEEVYEA (SEQ ID NO: 2), which forms a peptide having the same amino acid sequence as the amide after undergoing the photochemical amidation reaction of the present application.

[0034] Photochemical amidation reactions have been further used to prepare different intermediates of biologically active peptides (as shown in Example 5, Table 5, entries 1-12), more specifically, amidated precursors of a given biologically active peptide. In some embodiments, R2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 3-14 and 16-17. In some embodiments, R2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, and 17. In some embodiments, R2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 3, 4, 5, 16, and 17. In some embodiments, R2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 3, 4, 5, 16, and 17. In some embodiments, R2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 3, 4, 5, 16, and 17. In some embodiments, R2 may comprise the amino acid sequence shown in SEQ ID NO: 16.

[0035] In some embodiments, the photochemical amidation reaction may be used to prepare a glucagon-like peptide-1 (GLP-1) peptide or precursor thereof. The photochemical amidation reaction may be used to prepare an amylin receptor agonist or precursor thereof, such as pramlintide (the active pharmaceutical ingredient in Symlin™). The photochemical amidation reaction may be used to prepare a glucose-dependent insulinotropic polypeptide (GIP) peptide or precursor thereof. The photochemical amidation reaction may be used to prepare a GIP and GLP-1 receptor coagonist or precursor thereof, such as tirzepatide. The photochemical amidation reaction may be used to prepare a corticotropin releasing factor (CFR) peptide or precursor thereof, such as urocortin-2 (UCN2) peptide. The photochemical amidation reaction may be used to prepare a pancreatic pYY(3-36) peptide or precursor thereof. The photochemical amidation reaction may be used to prepare a pancreastatin (PST) inhibitor or precursor thereof, such as pancreastatin inhibitor peptide-8 (PSTi8) peptide. The photochemical amidation reaction may be used to prepare a luteinizing hormone releasing hormone (LHRH) agonist or precursor thereof. The photochemical amidation reaction may be used to prepare a gastrin releasing peptide (GRP) peptide or precursor thereof. The photochemical amidation reaction may be used to prepare an adrenocorticotropic hormone (ACTH) or precursor thereof, such as cosyntropin (the active pharmaceutical ingredient of Cortrosyn™). The photochemical amidation reaction may be used to prepare a QRF-amide peptide or precursor thereof. The photochemical amidation reaction may be used to prepare a GLP-1 receptor-neuropeptide Y receptor 2 coagonist or precursor thereof, such as EP45. The photochemical amidation reaction may be used to prepare an entry inhibitor or precursor thereof, such as brevirtide (the active pharmaceutical ingredient of Hepcludex™). The photochemical amidation reaction may be used to prepare a natriuretic peptide or precursor thereof, such as osteocrinin (OSTN). The photochemical amidation reaction may be used to prepare an antiretroviral drug or precursor thereof, such as enfuvirtide.This photochemical amidation reaction may be used to prepare GLP-1 receptor-amylin receptor coagonists or precursors thereof, such as those represented by SEQ ID NO:16 or 17.

[0036] The present photochemical amidation reaction can be used to produce a wide variety of therapeutically effective drugs, including glucagon-like peptide-1 (GLP-1) peptides, amylin receptor agonists such as pramlintide (the active pharmaceutical ingredient in Symlin™), glucose-dependent insulinotropic polypeptide (GIP) peptides, GLP-1 receptor coagonists such as tirzepatide, corticotropin releasing factor (CFR) peptides such as urocortin-2 (UCN2) peptide, pancreatic pYY(3-36) peptide, pancreastatin (PST) inhibitors such as pancreatic pYY(3-36) peptide, pancreastatin inhibitor peptide-8 (PSTi8) peptide, luteinizing hormone releasing hormone (LHRH) agonists, and the like. , gastrin releasing peptide (GRP) peptides, adrenocorticotropic hormones (ACTH) such as cosyntropin (the active pharmaceutical ingredient of Cortrosyn™), QRF-amide peptides, GLP-1 receptor-neuropeptide Y receptor 2 coagonists such as EP45, entry inhibitors such as brevirtide (the active pharmaceutical ingredient of Hepcludex™), natriuretic peptides such as osteocrine (OSTN) peptides, antiretroviral agents such as enfuvirtide, and GLP-1 receptor-amylin receptor coagonists such as those comprising the amino acid sequence of SEQ ID NO: 16, or precursors thereof, may be prepared.

[0037] The photochemical amidation reaction may be used to prepare glucagon-like peptide-1 (GLP-1) peptides, amylin receptor agonists such as pramlintide (the active pharmaceutical ingredient of Symlin™), pancreatic pYY(3-36) peptides, pancreastatin (PST) inhibitors such as pancreastatin inhibitor peptide-8 (PSTi8) peptide, luteinizing hormone releasing hormone (LHRH) agonists and antagonists, gastrin releasing peptide (GRP) peptides, adrenocorticotropic hormones (ACTH) such as cosyntropin (the active pharmaceutical ingredient of Cortrosyn™), QRF-amide peptides, GLP-1 receptor-neuropeptide Y receptor 2 coagonists such as EP45, entry inhibitors such as brevirtide (the active pharmaceutical ingredient of Hepcludex™), osteocrinin, antiretroviral agents such as enfuvirtide, and GLP-1 receptor-amylin receptor coagonists such as those comprising the amino acid sequence of SEQ ID NO: 16, or precursors thereof.

[0038] The present photochemical amidation reaction may be used to prepare glucagon-like peptide-1 (GLP-1) peptides, pancreatic pYY(3-36) peptides, pancreastatin inhibitor (PST) peptides, such as pancreastatin inhibitor peptide-8 (PSTi8) peptides, GLP-1 receptor-amylin receptor coagonists, such as those comprising the amino acid sequence of SEQ ID NO: 16, or precursors thereof.

[0039] The present photochemical amidation reaction may be used to prepare glucagon-like peptide-1 (GLP-1) peptides, pancreatic pYY(3-36) peptides, and GLP-1 receptor-amylin receptor coagonists, such as those comprising the amino acid sequence of SEQ ID NO: 16, or precursors thereof.

[0040] The preparation of GLP-1 peptides disclosed herein includes natural human GLP-1(7-36)-amide (e.g., the starting peptide R2 can be GLP-1(7-36), SEQ ID NO:3) and analogs thereof (GLP-1 analogs). The present photochemical amidation reaction may be used to prepare GLP-1(7-36)-amide. The present photochemical amidation reaction may be used to prepare GLP-1 receptor-amylin receptor coagonists. The preparation of GLP-1 receptor-amylin receptor coagonists disclosed herein includes the preparation of coagonists comprising the amino acid sequence shown in SEQ ID NO:16.

[0041] In some embodiments of the present photochemical amidation reaction, R2 may comprise a sequence corresponding to a precursor of a biologically active peptide product, such as a GLP-1 receptor-amylin receptor coagonist or an amylin receptor agonist. The present photochemical amidation reaction may be used to prepare a GLP-1 receptor-amylin receptor coagonist precursor comprising the amino acid sequence set forth in SEQ ID NO: 16. The present photochemical amidation reaction may be used to prepare a GLP-1 receptor-amylin receptor coagonist precursor consisting of the amino acid sequence set forth in SEQ ID NO: 16. The present photochemical amidation reaction may be used to prepare a GLP-1 receptor-amylin receptor coagonist precursor consisting of the amino acid sequence set forth in SEQ ID NO: 17.

[0042] In some embodiments of the present photochemical amidation reaction, R2 may be a peptide having an N-terminal extension. The extension may be any combination of amino acids. The extension may be 1-60 amino acids. The extension may be 1-50 amino acids. The extension may be 1-40 amino acids. The extension may be 1-20 amino acids. The extension may be 1-15 amino acids. The extension may be 5-15 amino acids. The extension may be 14 amino acids.

[0043] In some embodiments, a photochemical amidation reaction may be used to prepare a GLP-1 receptor-amylin receptor coagonist precursor with an N-terminal extension. In some embodiments, R2 may be a peptide with an amino acid sequence as shown in SEQ ID NO: 16 and any type of N-terminal extension. R2 may be a peptide with an amino acid sequence as shown in SEQ ID NO: 18 and any type of N-terminal extension. R2 may be a peptide with an amino acid sequence as shown in SEQ ID NO: 16 and an N-terminal extension with an amino acid sequence as shown in SEQ ID NO: 18. R2 may be a peptide with an amino acid sequence as shown in SEQ ID NO: 17.

[0044] In some embodiments of the photochemical amidation reaction, R2 may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 3-14, 16-17. R2 may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 3-14, 16 and an N-terminal extension. R2 may be an amino acid sequence set forth in any one of SEQ ID NOs: 3-14, 16-17. R2 may be an amino acid sequence set forth in any one of SEQ ID NOs: 3-14, 16 and an N-terminal extension.

[0045] In some embodiments of the photochemical amidation reaction, a peptide or protein containing a C-terminal cysteine ​​residue (Formula I) may be reacted with a photolabeling agent (R1-X). In another embodiment, a compound of Formula I may be coupled to a photolabel (R1).

[0046] Photolabeling Agent (R1-X) A photolabeling agent (also identified herein as R1-X) may be defined as a compound that contains a photoexcitable moiety and can be coupled to a peptide. A photolabeling agent (R1-X) may be defined as a compound that can undergo a photochemical reaction.

[0047] Photolabeling agent (R1-X) comprises a photolabel, defined herein as R1, and a leaving group, defined herein as X. As used herein, photolabel (R1) may be coupled to a peptide or protein that contains a C-terminal cysteine ​​(Formula I) by reacting the peptide or protein with photolabeling agent (R1-X) under conditions described herein.

[0048] In one embodiment of the photochemical amidation reaction, the photolabeling agent (R1-X) may be selected from the group consisting of 3-bromo-1H-pyrrole-2,5-dione, 4-chloro-7-nitrobenzofurazan, 2-bromo-1,4-naphthoquinone, 1-fluoro-2,4-dinitrobenzene, and 4-fluoro-7-sulfamoylbenzofurazan.

[0049] 3-Bromo-1H-pyrrole-2,5-dione may also be called 2-bromomaleimide and may be defined as formula 1:

[0050] Chemical formula 1: [ka] 4-Chloro-7-nitrobenzofurazan may also be referred to as NBD-Cl and may be defined as formula 2.

[0051] Chemical formula 2: [ka] In some embodiments, the photolabeling agent (R1-X) may be 3-bromo-1H-pyrrole-2,5-dione (Formula 1) or 4-chloro-7-nitrobenzofurazan (Formula 2). The photolabeling agent (R1-X) may be 3-bromo-1H-pyrrole-2,5-dione (Formula 1). The photolabeling agent (R1-X) may be 4-chloro-7-nitrobenzofurazan (Formula 2).

[0052] In some embodiments, 1 to 5 equivalents of the photolabeling agent (R1-X) may be provided relative to the peptide (Formula I). ​​In some embodiments, 1 to 3 equivalents of the photolabeling agent (R1-X) may be provided relative to the peptide (Formula I). ​​In some embodiments, 1.5 to 2.5 equivalents of the photolabeling agent (R1-X) may be provided relative to the peptide (Formula I). ​​In some embodiments, 1.8 to 2.2 equivalents of the photolabeling agent (R1-X) may be provided relative to the peptide (Formula I). ​​In some embodiments, about 2 equivalents of the photolabeling agent (R1-X) may be provided relative to the peptide (Formula I).

[0053] Reaction conditions for photolabeling of peptides or proteins containing C-terminal cysteine ​​residues Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer. The photochemical amidation reaction may be carried out in an aqueous reaction buffer, such as an aqueous buffer selected from the group consisting of bis-trismethane (which may also be referred to as bis-tris), tris, triethanolamine, and phosphate. Step (a) of the photochemical amidation reaction may be carried out in an aqueous buffer having a pH of 4-9. Step (a) of the photochemical amidation reaction may be carried out in an aqueous buffer having a pH of 4-9. Step (a) of the photochemical amidation reaction may be carried out in an aqueous buffer having a pH of 6-8. Step (a) of the photochemical amidation reaction may be carried out in an aqueous buffer comprising bis-trismethane having a pH of 6-8. Step (a) of the photochemical amidation reaction may be carried out in an aqueous buffer having a pH of 6-7. Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer comprising bis-trismethane having a pH of 6-7. Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer of pH 6.3-7. Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer containing bis-trismethane of about pH 6.3-7. Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer of about pH 6.4. Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer containing bis-trismethane of about pH 6.4. Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer containing bis-trismethane of about pH 6.3. Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer containing bis-trismethane of about pH 7. Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer containing about 25 mM bis-trismethane of about pH 6.4. Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer containing 25 mM bis-trismethane at about pH 6.3.

[0054] Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer having glycine. Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer having about 50 mM glycine.

[0055] In another embodiment, step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer comprising about 25 mM bis-trismethane at about pH 6.4 with about 50 mM glycine. Step (a) of the photochemical amidation reaction may be carried out in an aqueous reaction buffer comprising about 25 mM bis-trismethane at about pH 6.3 with about 50 mM glycine.

[0056] In some embodiments, step (a) of the photochemical amidation reaction may be carried out at room temperature, defined herein as 15-25°C. Step (a) of the photochemical amidation reaction may be carried out at a temperature of 4-80°C. Step (a) of the photochemical amidation reaction may be carried out at a temperature of 10-60°C. Step (a) of the photochemical amidation reaction may be carried out at a temperature of 10-40°C. Step (a) of the photochemical amidation reaction may be carried out at a temperature of 10-30°C. Step (a) of the photochemical amidation reaction may be carried out at a temperature of 15-30°C. Step (a) of the photochemical amidation reaction may be carried out at a temperature of about 20°C.

[0057] A detergent may be added to the reaction buffer prior to the photochemical conversion (described in step (b) below). The detergent may be used to improve the solubility of the photolabeled intermediate. For example, as discussed below for reactions with disulfide-containing peptides, detergents may improve the solubility of the 4-chloro-7-nitrobenzofurazan (NBD-Cl, Formula 2) alkylated intermediate when the NBD-Cl photolabeling agent is used to globally alkylate all cysteines in the peptide (R2).

[0058] In some embodiments, a surfactant selected from the group consisting of sodium dodecyl sulfate (SDS), sodium desoxycholate (SDC), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), octylphenol ethoxylate (Triton™ X-100, ThermoFisher Scientific Catalog Nos. 28314, 85111, 85112), polysorbate 20 (Tween-20, ThermoFisher Scientific Catalog Nos. 28320, 85113, 85115), tetrapropylammonium hydroxide (TAPH-40), and sodium octanoate may be added to the reaction buffer. In some embodiments, sodium dodecyl sulfate (SDS) may be added to the reaction buffer. In some embodiments, sodium desoxycholate (SDC) may be added to the reaction buffer. In some embodiments, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) may be added to the reaction buffer. In some embodiments, sodium octanoate may be added to the reaction buffer. In some embodiments, octylphenol ethoxylate (Triton™ X-100, ThermoFisher Scientific Catalog Nos. 28314, 85111, 85112) may be added to the reaction buffer. In some embodiments, polysorbate 20 (Tween-20, ThermoFisher Scientific Catalog Nos. 28320, 85113, 85115) may be added to the reaction buffer. In some embodiments, tetrapropylammonium hydroxide (TAPH-40) may be added to the reaction buffer.

[0059] The surfactant may be added to the reaction buffer at a concentration of 40-80 mM. The surfactant may be added to the reaction buffer at a concentration of 50-70 mM. The surfactant may be added to the reaction buffer at a concentration of 55-65 mM. The surfactant may be added to the reaction buffer at a concentration of about 60 mM. In some embodiments, no surfactant may be added to the reaction buffer.

[0060] Step (b) - Photochemically converting the resulting peptide-photolabeled conjugate (Formula II) to give the C-terminal enamide (Formula III). The second step of the reaction (step (b)) may be referred to as the photochemical conversion of the conjugate (also referred to as formula II) to the C-terminal enamide (also referred to as formula III). Step (b) may also be referred to as the conversion of the peptide-photolabeled conjugate to obtain the C-terminal enamide. In some embodiments, the peptide-photolabeled conjugate may be referred to as the conjugate.

[0061] The photochemical conversion of step (b) may be obtained by subjecting the peptide-photolabel conjugate of formula II to light. In another way, the photochemical conversion of step (b) may be obtained by irradiating the peptide-photolabel conjugate. The light shone on the conjugate of formula II may be absorbed by the photolabel, resulting in a light-promoted chemical reaction, which may generally be referred to herein as photochemical conversion, ultimately forming the C-terminal enamide of formula III. The photochemical conversion of step (b) may also be referred to as photolytic cleavage by radical decarboxylation of the C-terminus. The enamide of formula III may be said to be monosubstituted.

[0062] It is understood that the effective wavelength of this photochemical conversion can vary based on the photolabel R1 coupled to the C-terminal cysteine ​​residue (Formula I) in part a. It is further understood that a variety of light sources can be used to generate the desired light spectrum for the photochemical reaction to occur. The light source may be selected from a fluorescent lamp, a light emitting diode, a mercury lamp, a laser, and the like. In some embodiments, the light source may be ambient light, such as light provided by sunlight. In some embodiments, the light source may be a compact fluorescent lamp (CFL). The light source may be a light emitting diode (LED). The LED may be a monochromatic LED. In some embodiments, two or more light sources may be used to uniformly illuminate the conjugate of formula II with light. The light source may be two or more CFLs. The light source may be two or more LEDs. The light source may be a series of LEDs uniformly arranged to illuminate the conjugate. The light source may be an array of LEDs, which may be referred to as a strip of LEDs when arranged in a row.

[0063] The light provided may be white light. The wavelength of the light provided may be within the visible light spectrum. The wavelength of the light provided may be 300-700 nm. The wavelength of the light provided may be 365-550 nm. The wavelength of the light provided may be 365-500 nm. The wavelength of the light provided may be 365-450 nm. The wavelength of the light provided may be about 365 nm. The wavelength of the light provided may be 400-500 nm. The wavelength of the light provided may be 400-470 nm. The wavelength of the light provided may be 400-450 nm. The wavelength of the light provided may be 405-430 nm. The wavelength of the light provided may be 365 nm. The wavelength of the light provided may be 405 nm. The wavelength of the light provided may be 430 nm. The wavelength of the light provided may be 450 nm. The light source may be an LED providing light having a wavelength of 365 nm. The light source may be a CFL providing white light. The light source may be an LED providing light having a wavelength of 405 nm. The light source may be an LED providing light having a wavelength of 430 nm. The light source may be an LED providing light having a wavelength of 450 nm. The light source may be an array of monochromatic LEDs providing light having a wavelength of 365 nm. The light source may be an array of monochromatic LEDs providing light having a wavelength of 405 nm. The light source may be an array of monochromatic LEDs providing light having a wavelength of 430 nm. The light source may be an array of monochromatic LEDs providing light having a wavelength of 450 nm.

[0064] Of course, when a single wavelength is mentioned herein, reference is made to a spectrum of light having a spectral width generally centered on that wavelength.For example, if the light provided by the light source is 405 nm in wavelength, it is understood that this should not be interpreted as being strictly limited to light of 405 nm in wavelength, but rather to a spectrum having a given width that encompasses the wavelength of 405 nm.For light having a wavelength of 405 nm, and for example, a full width at half maximum (FWHM) of 50 nm, it is understood that the effective wavelength for irradiating the conjugate extends from 380 to 430 nm.

[0065] The light source may be combined with spectral filters, such as high-pass filters, low-pass filters, and / or bandgap filters, which may be considered to form part of the light source, to further restrict the spectral range in which the conjugate is irradiated.

[0066] In some embodiments, the spectral width of the light may have a FWHM of 50 nm. The spectral width of the light may have a FWHM of 40 nm. The spectral width of the light may have a FWHM of 30 nm. The spectral width of the light may have a FWHM of 20 nm. The spectral width of the light may have a FWHM of 10 nm.

[0067] The reaction conditions for photochemically converting the peptide-photolabeled conjugate (Formula II) to obtain the C-terminal enamide (Formula III) may correspond to the conditions provided in step (a) above for coupling a peptide or protein containing a C-terminal cysteine ​​residue (Formula I) with a photolabeling agent (R1-X) to obtain the peptide-photolabeled conjugate (Formula II). For example, step (b) may be carried out in the same aqueous reaction buffer as step (a). Similarly, step (b) may be carried out at a temperature corresponding to the temperature of step (a).

[0068] Step (c) - Cleavage of the C-terminal enamide (Formula III) to give the C-terminal α-amide (Formula IV) The third and final step of the reaction (step (c)) may be referred to as cleavage of the resulting C-terminal enamide (also referred to as formula III) to obtain the C-terminal α-amide (also referred to as formula IV). In some embodiments, the cleavage may be referred to as conversion of the C-terminal enamide. The C-terminal enamide may be referred to as an enamide. The C-terminal α-amide may be referred to as a C-terminal amide.

[0069] Cleavage of the enamide to the C-terminal α-amide may be performed using different cleavage methods. Cleavage may be performed using acidolysis. In some embodiments, cleavage may be performed using acidolysis with an acid that can be classified as a strong acid, where a strong acid is defined as an acid with a pKa of 4 or less. In some embodiments, the cleavage reagent may have a pKa of 4 or less. In some embodiments, the cleavage reagent may have a pKa of 3 or less. In some embodiments, cleavage may be performed using acidolysis with a weak acid (i.e., an acid with a pKa greater than 4) with the addition of a Lewis acid to accelerate the acidolysis.

[0070] Cleavage may be performed using inverse electron demand Diels-Alder (IEDDA). In some embodiments, the enamide may be cleaved by acid-mediated hydrolysis. The enamide may be hydrogenated. The enamide may be cleaved by acid-catalyzed hydrolysis, oxidant-mediated hydrolysis, or by an inverse electron demand Diels-Alder reaction. The enamide may be cleaved by acid-catalyzed hydrolysis, or by oxidant-mediated hydrolysis. The enamide may be cleaved by acid-catalyzed hydrolysis. The enamide may be cleaved by oxidant-mediated hydrolysis. The enamide may be cleaved by an inverse electron demand Diels-Alder reaction.

[0071] In some embodiments, vinyl amides may be reduced to N-ethyl amides, hi some embodiments, vinyl amides may be used in conjugations such as thiol-ene reactions.

[0072] In some embodiments, additives may be added to the process. The additives may be selected from the group consisting of, but are not limited to, methionine, indole, or caffeic acid.

[0073] Reaction conditions for enamide cleavage As noted above, different cleavage methods may require different cleavage reagents and different reaction conditions such as temperature, etc. In some embodiments, a cleavage reagent is added to the reaction with the intermediate of formula III.

[0074] It is understood that some variables of reaction conditions may be kept constant throughout steps (a), (b), and (c). For example, in some embodiments, the photochemical amidation reaction is carried out at a constant temperature. In other embodiments, the temperature may be varied between steps (a), (b), and / or (c). In other embodiments, the photochemical reaction conditions in steps (a), (b), and / or (c) may differ from one another.

[0075] In some embodiments, the cleavage reagent is trifluoroacetic acid (TFA), hydrochloric acid (HCl), sulfuric acid (H 2 SO 4 ), tosylate (TsOH), phosphate (H 3 PO 4 ), oxalic acid, 3,6-diphenyl-1,2,4,5-tetrazine, and 6,6'-(1,2,4,5-tetrazine-3,6-diyl)dinicotinic acid. The cleavage reagent may be selected from the group consisting of trifluoroacetic acid (TFA), hydrochloric acid (HCl), sulfuric acid (H 2 SO 4 ), tosylate (TsOH), phosphate (H 3 PO 4 ), oxalic acid, and 6,6'-(1,2,4,5-tetrazine-3,6-diyl)dinicotinic acid. The cleavage reagent may be selected from the group consisting of trifluoroacetic acid (TFA), hydrochloric acid (HCl), sulfuric acid (H 2 SO 4), tosylic acid (TsOH), and 6,6'-(1,2,4,5-tetrazine-3,6-diyl)dinicotinic acid. The cleavage reagent may be trifluoroacetic acid (TFA). The cleavage reagent may be hydrochloric acid (HCl). The cleavage reagent may be sulfuric acid (H 2 SO 4 The cleavage reagent may be phosphate (H 3 PO 4 The cleavage reagent may be tosylic acid (TsOH). The cleavage reagent may be 6,6'-(1,2,4,5-tetrazine-3,6-diyl)dinicotinic acid.

[0076] In some embodiments, the cleavage reagent may be 2-8% v / v trifluoroacetic acid (TFA). The cleavage reagent may be 4-6% v / v trifluoroacetic acid (TFA). The cleavage reagent may be 5% v / v trifluoroacetic acid (TFA).

[0077] In some embodiments, the cleavage reagent is 0.5-2M phosphoric acid (H 3 PO 4 The cleavage reagent may be 0.5 to 1.5 M phosphoric acid (H 3 PO 4 The cleavage reagent may be 0.7 to 1.3 M phosphoric acid (H 3 PO 4 The cleavage reagent may be 0.8 to 1.2 M phosphoric acid (H 3 PO 4 The cleavage reagent may be about 1M phosphoric acid (H 3 PO 4 ) may also be used.

[0078] In some embodiments, the enamide cleavage may be performed at a temperature of 0-80° C. The enamide cleavage may be performed at a temperature of 20-60° C. The enamide cleavage may be performed at a temperature of 20-40° C. The enamide cleavage may be performed at a temperature of 30-40° C. The enamide cleavage may be performed at a temperature of 37° C. The enamide cleavage may be performed at a temperature of 20-26° C. The enamide cleavage may be performed at a temperature of 20-23° C. The enamide cleavage may be performed at a temperature of about 20° C. The enamide cleavage may be performed at a temperature of about 23° C. The enamide cleavage may be performed at room temperature, which may also be referred to as ambient temperature.

[0079] The reaction conditions may vary depending on the procedure in which the reaction is carried out.

[0080] Batch Procedure The photochemical amidation reaction described herein may be carried out in a batch procedure. A batch procedure may also be referred to as a batch-scale reaction. In some embodiments, the photochemical amidation process may be carried out in the same reaction vessel, which may be referred to as a "single-pot" reaction. Of course, the reaction may occur in a single reaction vessel, but this should not be construed as saying that the reaction conditions therein should be kept constant. For example, steps (a) and (b) of the photochemical amidation reaction described herein may occur at room temperature in a reaction vessel, while for step (c), the temperature may be adjusted, for example, to about 37°C, and still occur in the same reaction vessel.

[0081] In other embodiments, steps (a), (b), and / or (c) may be carried out in different reaction vessels. The reaction conditions may vary depending on the photolabeling agent (R1-X) and the cleavage reagent used, as described above.

[0082] Photo Flow Procedure The photochemical amidation reaction may be carried out in a photoflow procedure, in which a solution is pumped through a circuit while being irradiated by a light source. The reaction may be carried out in a flow reactor that provides these conditions. The flow reactor may be called a photoflow reactor, a photochemical reactor, or more generally a reactor.

[0083] In some embodiments, reactions carried out in a flow reactor may also be referred to as photoflow processes or reactions. In some embodiments, reactions carried out in a flow reactor may also be referred to as photoflow procedures.

[0084] The flow reactor may be operated at a temperature of 10 to 100°C. The flow reactor may be operated at a temperature of 20 to 60°C. The flow reactor may be operated at a temperature of 20 to 40°C. The flow reactor may be operated at a temperature of 20 to 30°C. The flow reactor may be operated at a temperature of 25 to 30°C. The flow reactor may be operated at a temperature of about 26°C.

[0085] The photochemical amidation reaction may be carried out at different flow rates, where flow rate refers to the volumetric flow rate, which may be defined as the volume of fluid passing through the system per unit of time.

[0086] The flow rate may be 0.200 to 50.00 mL / min. The flow rate may be 1.00 to 25.00 mL / min. The flow rate may be 5.00 to 10.00 mL / min. The flow rate may be 6.00 to 9.00 mL / min. The flow rate may be about 8.00 mL / min. The flow rate may be about 10 mL / min. The flow rate may be about 20 mL / min.

[0087] The flow reactor volume may be 1-10 mL. The flow reactor volume may be 2-5 mL. The flow reactor volume may be about 2 mL. The flow reactor volume may be about 2.7 mL. The flow reactor volume herein may be considered as the volume of the flow reactor available for irradiation by the light source at any given time.

[0088] The reaction may also be carried out using different residence times. Residence time here may be defined as a measure of how long the fluid remains in the flow reactor. Stated differently, it is a measurement of the time between the moment the solution enters the flow reactor, forms part of the flow reactor volume and can be irradiated by the flow reactor, and the moment the solution leaves the flow reactor, no longer forms part of the flow reactor volume and is no longer irradiated by the flow reactor. It may also be given by the ratio of the flow reactor volume to the total flow rate.

[0089] The residence time in the flow reactor may be 0.04 to 50 minutes. The residence time in the flow reactor may be 0.1 to 10 minutes. The residence time in the flow reactor may be 0.1 to 1 minute. The residence time in the flow reactor may be 0.1 to 0.5 minutes. The residence time in the flow reactor may be about 0.25 minutes.

[0090] The flow reactor may be a Vapourtec UV-150 photochemical reactor. The flow reactor may be a Corning® Lab Photo Reactor. Of course, any equivalent photochemical reactor that allows for irradiation of the circulating solution for the purpose of photochemical conversion, as described in step (b), may be used without departing from the present disclosure.

[0091] In some embodiments, the flow reactor light source may irradiate at a wavelength between 300 nm and 700 nm. The flow reactor light source may be a series of LEDs irradiating at a wavelength between 365 and 525 nm. The flow reactor light source may be a series of monochromatic LEDs irradiating at a wavelength between 400 and 450 nm. The flow reactor light source may be a series of monochromatic LEDs irradiating at a wavelength of 430 nm. The flow reactor light source may be a series of monochromatic LEDs irradiating at a wavelength between 390 and 420 nm. The flow reactor light source may be a series of monochromatic LEDs irradiating at a wavelength of 405 nm.

[0092] In some embodiments, the flow reactor light source may irradiate with a radiant power of 3 to 150 Watts. The flow reactor light source may irradiate with a radiant power of 5 to 100 Watts. The flow reactor light source may irradiate with a radiant power of 10 to 80 Watts. The reactor light source may irradiate with a radiant power of 9 to 24 Watts.

[0093] Batch-photoflow hybrid procedure The photochemical amidation reaction may be carried out in a batch-photoflow hybrid procedure, in which only a portion of the photochemical amidation reaction may be completed via the photoflow procedure. Stated differently, in some embodiments, only a portion of the steps of the reaction may occur in a flow reactor, with the remaining steps occurring in a batch procedure, for example in a reaction vessel. When only a single step or only a portion of the steps of the photochemical amidation reaction occur in a photoflow reactor, the reaction may generally be referred to as being completed in a hybrid procedure.

[0094] In some embodiments, the photochemical conversion of the peptide-photolabeled conjugate to obtain the C-terminal enamide of the reaction may be carried out in a flow-through reactor. In some embodiments, the coupling of a peptide containing a C-terminal cysteine ​​residue with a photolabel to obtain a peptide-photolabeled conjugate and the photochemical conversion of the peptide-photolabeled conjugate to obtain the C-terminal enamide of the reaction may be carried out in a flow-through reactor. In some embodiments, the photochemical conversion of the peptide-photolabeled conjugate to obtain the C-terminal enamide and the cleavage of the C-terminal enamide to obtain the C-terminal α-amide of the reaction may be carried out in a flow-through reactor. In some embodiments, only the photochemical conversion of the peptide-photolabeled conjugate to obtain the C-terminal enamide of the reaction may be carried out in a flow-through reactor. In some embodiments, the photochemical amidation reaction step that is not carried out in a flow-through reactor may be carried out via a batch procedure. In some embodiments, the photochemical amidation reaction step that is not carried out in a flow-through reactor may be carried out via a batch procedure in a reactor.

[0095] Photochemical reactions of disulfide bond-containing peptides. The photochemical amidation reaction of the present application may be carried out using peptide R2 containing a cysteine ​​residue therein. In other words, formula I of scheme 1 or 2 contains a peptide containing at least one cysteine ​​(Cys) residue in addition to the C-terminal cysteine ​​(Cys) residue for photochemical amidation reaction. The cysteine ​​in peptide R2 may form a disulfide bond.

[0096] In such embodiments, the photochemical amidation process generally proceeds as described above and may simultaneously be further subjected to the following:

[0097] Global coupling of photolabeling agents The photolabeling agent (R1-X) may be coupled to the C-terminal cysteine ​​residue and to any additional cysteine ​​residues present in peptide R2, as described in step (a) above. This may be referred to as global coupling of cysteines. The cysteine ​​that forms part of peptide R2 coupled with photolabel R1 may generally be referred to as a photolabel-protected cysteine.

[0098] The photolabeling agent may be added in a manner proportional to the amount of cysteine ​​that is coupled to the photolabeling agent. The amount of photolabeling agent may be increased proportionally to the number of disulfide bonds formed in the amidated peptide.

[0099] In some embodiments, 0.5 to 3 additional equivalents of photolabeling agent (R1-X) relative to the peptide (Formula I) may be provided for each disulfide bond formed in the peptide (R2). 1 to 2.5 additional equivalents of photolabeling agent (R1-X) relative to the peptide (Formula I) may be provided for each disulfide bond formed in the peptide (R2). 1.5 to 2.5 additional equivalents of photolabeling agent (R1-X) relative to the peptide (Formula I) may be provided for each disulfide bond formed in the peptide (R2). In some embodiments, about 2 additional equivalents of photolabeling agent (R1-X) relative to the peptide (Formula I) may be provided for each disulfide bond formed in the peptide (R2). The term additional herein refers to a photolabeling agent equivalent that is added to the photolabeling agent equivalent that may be provided in the general reaction of step (a) when the peptide R2 does not have a disulfide bond formed.

[0100] In some embodiments, 3 to 5 total equivalents of photolabeling agent (R1-X) relative to the peptide (Formula I) may be provided for step a. 3.5 to 4.5 total equivalents of photolabeling agent (R1-X) relative to the peptide (Formula I) may be provided for step a. 3.8 to 4.2 total equivalents of photolabeling agent (R1-X) relative to the peptide (Formula I) may be provided for step a. In some embodiments, about 4 total equivalents of photolabeling agent (R1-X) relative to the peptide (Formula I) may be provided for step a. The term total refers to the resulting amount of photolabeling equivalents provided in the reaction during step (a) of the photochemical amidation reaction.

[0101] In some embodiments, about 4 total equivalents of 4-chloro-7-nitrobenzofurazan (NBD-Cl) relative to the peptide may be provided for step a.

[0102] Selective C-terminal photochemical transformation The peptide-photolabel conjugate may be irradiated in the manner generally disclosed in step b. The photolabel found at the C-terminus of the conjugate may undergo photochemical conversion to yield a C-terminal enamide, while the remaining cysteine-coupled photolabels may remain unaffected.

[0103] In some embodiments, the photochemical conversion step (b) disclosed herein can be said to be selective for the C-terminus of the peptide-photolabel conjugate. In some embodiments, the photochemical conversion step (b) disclosed herein can be said to be selective for the C-terminal cysteine ​​of the peptide coupled with the photolabel.

[0104] Release and oxidation Cleavage of the C-terminal enamide to give the C-terminal α-amide may be carried out as described in step (c).

[0105] In addition, release of the photolabel at the cysteine ​​protected by the photolabel of peptide R2 may be performed. Release of the cysteine ​​protected by the photolabel in peptide R2 may be performed before cleaving the C-terminal enamide to obtain the C-terminal α-amide in step (c). Release of the cysteine ​​protected by the photolabel in peptide R2 may be performed simultaneously with cleaving the C-terminal enamide to obtain the C-terminal α-amide in step (c).

[0106] A nucleophilic sulfide may be provided to release the photolabel from the photolabel-protected peptide cysteine. In some embodiments, the nucleophilic sulfide may be provided selected from the group consisting of cysteamine, cysteine, dithiothreitol (DTT), 3,6-dioxa-1,8-octanedithiol (DODT), 2-mercaptoethanol, glutathione (GSH), and acetylcysteine. In some embodiments, cysteamine may be provided as the nucleophilic sulfide.

[0107] In some embodiments, an oxidation partner may be further provided to effect disulfide oxidation. An oxidation partner selected from the group consisting of cystamine glutathione disulfide (GSSG) and cystine may be provided. In some embodiments, cystamine glutathione disulfide (GSSG) may be provided as the oxidation partner.

[0108] Overall response In a first aspect of the invention, there is provided a method for producing a peptide or protein comprising a C-terminal α-amide of formula IV according to scheme 1, as defined above, comprising: [ka] wherein R2 is a polypeptide; R1-X is a photolabeling agent, R1 is a photolabel and X is a leaving group; R3 is selected from the group consisting of hydrogen, methyl, and ethyl. Step (a): coupling a peptide or protein containing a C-terminal cysteine ​​of formula I with a photolabel (R1) to obtain a peptide-photolabel conjugate of formula II; Step (b): photochemically converting the resulting peptide-photolabeled conjugate of formula II to obtain a C-terminal enamide of formula III; Step (c). cleaving the resulting C-terminal enamide of formula III to obtain the C-terminal α-amide of formula IV; A method may be provided that includes:

[0109] In some embodiments, R3 may be selected from the group consisting of hydrogen and ethyl. In other embodiments, R3 may be hydrogen.

[0110] Similarly, or alternatively, a second aspect of the invention provides a method for producing a peptide or protein comprising a C-terminal α-amide of formula IV according to Scheme 2, comprising: [ka] wherein R2 is a polypeptide; R3 is hydrogen. Step (a): coupling a peptide or protein containing a C-terminal cysteine ​​of formula I with 4-chloro-7-nitrobenzofurazan (chemical formula 2) to obtain a peptide-photolabeled conjugate of formula II-a; Step (b): photochemically converting the resulting peptide-photolabeled conjugate of formula II-a to obtain a C-terminal enamide of formula III; Step (c). cleaving the resulting C-terminal enamide of formula III to obtain the C-terminal α-amide of formula IV; A method may be provided that includes:

[0111] In yet another embodiment, a method for producing a peptide or protein comprising a C-terminal α-amide of formula IV according to Scheme 2, comprising: [ka] wherein R2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:16; R3 is hydrogen. Step (a): coupling a peptide or protein containing a C-terminal cysteine ​​amidation tag (Formula I) with 4-chloro-7-nitrobenzofurazan to obtain a peptide-photolabeled conjugate (Formula II-a); Step (b): Irradiating the peptide-photolabeled conjugate (Formula II-a) with light having a wavelength of 400 to 450 nm to obtain a C-terminal enamide (Formula III) by photochemical conversion; Step (c): cleaving the C-terminal enamide (Formula III) to obtain the C-terminal α-amide (Formula IV).

[0112] Purification and characterization Purification of the C-terminal α-amide may be carried out in a batch procedure. Purification may be carried out by continuous precipitation.

[0113] The reaction may be monitored by liquid chromatography-mass spectrometry (LC-MS).

[0114] Purification of the C-terminal α-amide may be carried out via high performance liquid chromatography (HPLC) or ultrafiltration diafiltration (UFDF). Purification of the C-terminal α-amide may be carried out via high performance liquid chromatography (HPLC). Purification of the C-terminal α-amide may be carried out via ultrafiltration diafiltration (UFDF).

[0115] The reaction samples may be analyzed by UPLC-MS analysis. In some embodiments, the reaction samples may be analyzed using extractive ion chromatography. In some embodiments, the reaction may be monitored by LC-MS.

[0116] As used herein, terms provided in the singular also include the plural unless otherwise indicated.

[0117] The term "compound" is used herein to refer to a molecular entity, and therefore a "compound" may have different structural elements other than the minimum elements defined for each compound or group of compounds.

[0118] This specification also describes compounds and methods in which open-ended terms such as "comprises" and "comprising" and limiting terms such as "consists of," "consisting of," and the like may be used.

[0119] Where the plural is used for compounds, starting materials, intermediates, salts and the like, it is intended to mean one (preferably) or more than one single compound, salt, intermediate, etc., and where the singular or indefinite article ("a", "an") is used, this is not intended to exclude a plural, but only preferably means "one".

[0120] Specific Embodiments 1. A method for producing a peptide or protein comprising a C-terminal α-amide of formula IV according to Scheme 1, comprising: [ka] wherein R2 is a polypeptide; R1-X is a photolabeling agent, R1 is a photolabel and X is a leaving group; R3 is selected from the group consisting of hydrogen, methyl, and ethyl. Step (a): coupling a peptide or protein comprising a C-terminal cysteine ​​amidation tag of formula I with a photolabel (R1) to obtain a peptide-photolabel conjugate of formula II; Step (b). Irradiating the peptide-photolabeled conjugate of formula II to obtain the C-terminal enamide of formula III via photochemical conversion; and step (c). cleaving the C-terminal enamide of formula III to give the C-terminal α-amide of formula IV. 2. The method of embodiment 1, wherein R3 is selected from the group consisting of hydrogen and ethyl. 3. The method of any of the preceding embodiments, wherein R3 is hydrogen. 4. The method of any of the preceding embodiments, wherein R2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17. 5. The method of any of the preceding embodiments, wherein R2 consists of the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17. 6. The method of any of the preceding embodiments, wherein R2 is an amino acid sequence set forth in any one of SEQ ID NOs: 3-14 and 16-17. 7. The method of any of the preceding embodiments, wherein R2 is an amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 5, 16, 17. 8. The method of any of the preceding embodiments, wherein R2 comprises the amino acid sequence set forth in SEQ ID NO:16. 9. The method of any of the preceding embodiments, wherein R2 further comprises an N-terminal extension. 10. The method of any of the preceding embodiments, wherein R2 further comprises an N-terminal extension having 1-60 amino acids. 11. The method of any of the preceding embodiments, wherein R2 further comprises an N-terminal extension having 1-50 amino acids. 12. The method of any of the preceding embodiments, wherein R2 further comprises an N-terminal extension having 1-40 amino acids. 13. The method of any of the preceding embodiments, wherein R2 further comprises an N-terminal extension having 1-20 amino acids. 14. The method of any of the preceding embodiments, wherein R2 further comprises an N-terminal extension having 1-15 amino acids. 15. The method of any of the preceding embodiments, wherein R2 further comprises an N-terminal extension having 5-15 amino acids. 16. The method of any of the preceding embodiments, wherein R2 further comprises an N-terminal extension having 14 amino acids. 17. The method of any of the preceding embodiments, wherein R2 further comprises an N-terminal extension according to SEQ ID NO:18. 18. The method of any of the preceding embodiments, wherein R2 is the amino acid sequence set forth in SEQ ID NO:17. 19. The method of any of the previous embodiments, wherein the method is used for the preparation of a glucagon-like peptide-1 (GLP-1) peptide. 20. The method according to any one of the preceding embodiments, wherein the method is used for the preparation of an amylin receptor agonist. 21. The method of embodiment 20, wherein the amylin receptor agonist is pramlintide. 22. The method according to any one of the preceding embodiments, wherein the method is used for the preparation of a glucose-dependent insulinotropic polypeptide (GIP) peptide. 23. The method according to any one of embodiments 1 to 18, wherein the method is used for the preparation of a GIP and GLP-1 receptor coagonist. 24. The method of embodiment 23, wherein the GIP and GLP-1 receptor coagonist is tirzepatide. 25. The method according to any one of embodiments 1 to 18, wherein the method is used for the preparation of a urocortin-2 (UCN2) peptide. 26. The method according to any one of embodiments 1 to 18, wherein the method is used for the preparation of pancreatic pYY(3-36) peptide. 27. The method according to any one of embodiments 1 to 18, wherein the method is used for the preparation of a pancreastatin inhibitor peptide-8 (PSTi8) peptide. 28. The method according to any one of embodiments 1 to 18, wherein the method is used for the preparation of a luteinizing hormone releasing hormone (LHRH) agonist. 29. The method according to any one of embodiments 1 to 18, wherein the method is used for the preparation of a gastrin releasing peptide. 30. The method according to any one of the preceding embodiments, wherein the method is used for the preparation of cosyntropin. 31. The method according to any one of embodiments 1 to 18, wherein the method is used for the preparation of a QRF-amide peptide. 32. The method according to any one of embodiments 1 to 18, wherein the method is used for the preparation of a GLP-1 receptor-neuropeptide Y receptor 2 coagonist. 33. The method of embodiment 32, wherein the GLP-1 receptor-neuropeptide Y receptor 2 coagonist is EP45. 34. The method according to any one of the preceding embodiments, wherein the method is used for the preparation of brevirtide. 35. The method according to any one of embodiments 1 to 18, wherein the method is used for the preparation of osteocrine (OSTN) peptide. 36. The method according to any one of embodiments 1-18, wherein the method is used for the preparation of enfuvirtide. 37. The method according to any one of embodiments 1 to 18, wherein the method is used for the preparation of a GLP-1 receptor-amylin receptor coagonist. 38. The method of any one of embodiments 1 to 18 and 37, wherein the method is used for preparing a GLP-1 receptor-amylin receptor coagonist, and the GLP-1 receptor-amylin receptor coagonist comprises the amino acid sequence set forth in SEQ ID NO: 16. 39. The method of any one of embodiments 1-18 and 37-38, wherein the GLP-1 receptor-amylin receptor coagonist is represented by SEQ ID NO: 16. 40. The method comprises administering to a subject a therapeutically effective amount of a glucagon-like peptide-1 (GLP-1) peptide, an amylin receptor agonist such as pramlintide, a glucose-dependent insulinotropic polypeptide (GIP) peptide, a GLP-1 receptor coagonist such as tirzepatide, a urocortin-2 (UCN2) peptide, a pancreatic pYY(3-36) peptide, a pancreatic inhibitor peptide-8 (PSTi8) peptide, a luteinizing hormone-releasing hormone (LHRH) agonist, a gastrin-releasing peptide (GRP) peptide, or a cosyntropin-releasing peptide (Cosyntropin) peptide. 19. The method of any one of embodiments 1 to 18, wherein the method is used for the preparation of an adrenocorticotropic hormone (ACTH), a QRF-amide peptide, a GLP-1 receptor-neuropeptide Y receptor 2 coagonist such as EP45, an entry inhibitor such as brevirtide, a natriuretic peptide such as osteocrine (OSTN) peptide, an antiretroviral agent such as enfuvirtide, or a GLP-1 receptor-amylin receptor coagonist, such as a GLP-1 receptor-amylin receptor coagonist comprising the amino acid sequence set forth in SEQ ID NO: 16. 41. The method according to any one of embodiments 1 to 18 and 40, wherein the method is used for the preparation of a glucagon-like peptide-1 (GLP-1) peptide, a pancreatic pYY(3-36) peptide, a pancreastatin inhibitor peptide-8 (PSTi8) peptide, a luteinizing hormone releasing hormone (LHRH), a gastrin releasing peptide (GRP) peptide, a cosyntropin, a QRF-amide peptide, a GLP-1 receptor-neuropeptide Y receptor 2 coagonist EP45, brevirtide, an osteocrine (OSTN) peptide, an enfuvirtide, a GLP-1 receptor-amylin receptor coagonist comprising the amino acid sequence set forth in SEQ ID NO: 16, or an amylin receptor agonist such as pramlintide. 42. The method according to any one of embodiments 1-18 and 40-41, wherein the method is used for the preparation of a glucagon-like peptide-1 (GLP-1) peptide, a pancreatic pYY(3-36) peptide, a pancreastatin inhibitor peptide-8 (PSTi8) peptide, a GLP-1 receptor-amylin receptor coagonist comprising the amino acid sequence set forth in SEQ ID NO: 16, or an amylin receptor agonist such as pramlintide. 43. The method of any one of the preceding embodiments, wherein the method is used for the preparation of pramlintide. 44. The method according to any one of embodiments 1 to 18 and 40 to 42, wherein the method is used for the preparation of a GLP-1 receptor-amylin receptor coagonist comprising the amino acid sequence set forth in SEQ ID NO: 16. 45. The method according to any one of embodiments 1 to 18 and 40 to 42, wherein the method is used for the preparation of a precursor GLP-1 receptor-amylin receptor coagonist comprising the amino acid sequence set forth in SEQ ID NO: 16. 46. ​​The method of any of the preceding embodiments, wherein the photolabeling agent (R1-X) is selected from the group consisting of 3-bromo-1H-pyrrole-2,5-dione, 4-chloro-7-nitrobenzofurazan, 2-bromo-1,4-naphthoquinone, 1-fluoro-2,4-dinitrobenzene, and 4-fluoro-7-sulfamoylbenzofurazan. 47. The photolabeling agent (R1-X) is 3-bromo-1H-pyrrole-2,5-dione (chemical formula 1) or 4-chloro-7-nitrobenzofurazan (chemical formula 2): Chemical formula 1: [ka] The method according to any one of claims 1 to 6, wherein Chemical formula 2: [ka] 2. The method of any preceding embodiment, wherein the method is selected from the group consisting of: 48. The photolabeling agent is 3-bromo-1H-pyrrole-2,5-dione (chemical formula 1): Chemical formula 1: [ka] 3. The method of any preceding embodiment, wherein 49. The photolabeling agent is 4-chloro-7-nitrobenzofurazan (chemical formula 2): Chemical formula 2: [ka] 3. The method of any preceding embodiment, wherein 50. The method of any of the preceding embodiments, wherein the method is carried out in an aqueous reaction buffer. 51. The method of any of the previous embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer. 52. The method of any of the preceding embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer, the aqueous buffer being selected from the group consisting of bis-trismethane, tris, triethanolamine, and phosphate. 53. The method of any of the previous embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer of bis-trismethane. 54. The method of any of the previous embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer of about 25 mM bis-trismethane. 55. The method of any of the preceding embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer of Tris. 56. The method of any of the preceding embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer of triethanolamine. 57. The method of any of the preceding embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer of phosphate. 58. The method of any of the preceding embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer of pH 4-9. 59. The method of any of the preceding embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer of pH 6-8. 60. The method of any of the preceding embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer at pH 6-7. 61. The method of any of the preceding embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer of pH 6.3-7. 62. The method of any of the preceding embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer at pH 6.3-6.4. 63. The method of any of the preceding embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer at about pH 6.4. 64. The method of any one of embodiments 1-42, wherein step (a) of the method is carried out in an aqueous reaction buffer at about pH 6.3. 65. The method of any one of embodiments 1-41, wherein step (a) of the method is carried out in an aqueous reaction buffer at about pH 7. 66. The method of any of the previous embodiments, wherein step (a) of the method is carried out in an aqueous reaction buffer having glycine. 67. The method of any of the preceding embodiments, wherein the method step (a) is carried out in an aqueous reaction buffer having about 50 mM glycine. 68. The method of any of the preceding embodiments, wherein 1 to 5 equivalents of photolabel are provided to the peptide in step (a). 69. The method of any of the preceding embodiments, wherein 1 to 3 equivalents of photolabel are provided to the peptide in step (a). 70. The method of any of the preceding embodiments, wherein 1.5 to 2.5 equivalents of photolabel are provided to the peptide in step (a). 71. The method of any of the preceding embodiments, wherein 1.8 to 2.2 equivalents of photolabel are provided to the peptide in step (a). 72. The method of any of the preceding embodiments, wherein two equivalents of photolabel are provided to the peptide of step (a). 73. The method of any of the preceding embodiments, wherein the light source for illuminating the peptide-photolabeled conjugate is a compact fluorescent lamp (CFL). 74. The method of any of the previous embodiments, wherein the light source for illuminating the peptide-photolabeled conjugate is at least one light emitting diode (LED). 75. The method of any of the preceding embodiments, wherein the light source for illuminating the peptide-photolabeled conjugate is a plurality of light emitting diodes (LEDs). 76. The method of any of the preceding embodiments, wherein the light source for illuminating the peptide-photolabeled conjugate is a monochromatic LED. 77. The method of any of the preceding embodiments, wherein the light source for illuminating the peptide-photolabeled conjugate has a wavelength that provides white light. 78. The method of any of the preceding embodiments, wherein the light source for irradiating the peptide-photolabeled conjugate has a wavelength of 300 to 700 nm. 79. The method of any of the preceding embodiments, wherein the light source for irradiating the peptide-photolabeled conjugate has a wavelength of 365 to 550 nm. 80. The method of any of the preceding embodiments, wherein the light source for irradiating the peptide-photolabeled conjugate has a wavelength of 365-500 nm. 81. The method of any of the preceding embodiments, wherein the light source for irradiating the peptide-photolabeled conjugate has a wavelength of 365 to 450 nm. 82. The method of any of the preceding embodiments, wherein the light source for illuminating the peptide-photolabeled conjugate has a wavelength of 365 nm. 83. The method of any of the preceding embodiments, wherein the light source for irradiating the peptide-photolabeled conjugate has a wavelength of 400-500 nm. 84. The method of any of the preceding embodiments, wherein the light source for irradiating the peptide-photolabeled conjugate has a wavelength of 400 to 450 nm. 85. The method of any of the preceding embodiments, wherein the light source for illuminating the peptide-photolabeled conjugate has a wavelength of about 405 nm. 86. The method of any of the preceding embodiments, wherein the light source for illuminating the peptide-photolabeled conjugate has a wavelength of about 430 nm. 87. The method of any of the preceding embodiments, wherein the light source for illuminating the peptide-photolabeled conjugate has a wavelength of about 450 nm. 88. The method of any of the preceding embodiments, wherein the enamide is cleaved by acid-mediated hydrolysis. 89. The method of any of the preceding embodiments, wherein the enamide is hydrogenated. 90. The method of any of the preceding embodiments, wherein the enamide is cleaved by acid-catalyzed hydrolysis, oxidant-mediated hydrolysis, or by an inverse electron demand Diels-Alder reaction. 91. The method of any of the preceding embodiments, wherein the enamide is cleaved by acid-catalyzed hydrolysis or by oxidant-mediated hydrolysis. 92. The method of any of the preceding embodiments, wherein the enamide is cleaved by acid-catalyzed hydrolysis. 93. The method of any of the preceding embodiments, wherein the enamide is cleaved by oxidant-mediated hydrolysis. 94. The method of any of the previous embodiments, wherein the enamide is cleaved by an inverse electron demand Diels-Alder reaction. 95. The method of any of the previous embodiments, further comprising adding a cleavage reagent to cleave the enamide. 96. The method further comprising the step of adding a cleavage reagent for cleaving the enamide, the cleavage reagent being selected from the group consisting of trifluoroacetic acid (TFA), hydrochloric acid (HCl), sulfuric acid (H 2 SO 4 ), tosylate (TsOH), phosphate (H 3 PO 4 ), oxalic acid, and 6,6'-(1,2,4,5-tetrazine-3,6-diyl)dinicotinic acid. 97. The cleavage reagent is trifluoroacetic acid (TFA), hydrochloric acid (HCl), sulfuric acid (H 2 SO 4), tosylate (TsOH), phosphate (H 3 PO 4 ), oxalic acid, and 6,6'-(1,2,4,5-tetrazine-3,6-diyl)dinicotinic acid. 98. The cleavage reagent is trifluoroacetic acid (TFA), hydrochloric acid (HCl), sulfuric acid (H 2 SO 4 ), tosylate (TsOH), phosphate (H 3 PO 4 ), and 6,6'-(1,2,4,5-tetrazine-3,6-diyl)dinicotinic acid. 99. The method of any of the previous embodiments, wherein the cleavage reagent is trifluoroacetic acid (TFA). 100. The method of any of the preceding embodiments, wherein the cleavage reagent is 2-8% v / v trifluoroacetic acid (TFA). 101. The method of any of the preceding embodiments, wherein the cleavage reagent is 4-6% v / v trifluoroacetic acid (TFA). 102. The method of any of the previous embodiments, wherein the cleavage reagent is 5% v / v trifluoroacetic acid (TFA). 103. The method of any of the preceding embodiments, wherein the cleavage reagent is hydrochloric acid (HCl). 104. The cleavage reagent is sulfuric acid (H 2 SO 4 5. The method of any preceding embodiment, wherein 105. The cleavage reagent is phosphoric acid (H 3 PO 4 5. The method of any preceding embodiment, wherein 106. The cleavage reagent is 0.5 to 2 M phosphoric acid (H 3 PO 4 5. The method of any preceding embodiment, wherein 107. The cleavage reagent is 0.5 to 1.5 M phosphoric acid (H 3 PO 4 5. The method of any preceding embodiment, wherein 108. The cleavage reagent is 0.7 to 1.3 M phosphoric acid (H 3 PO 4 5. The method of any preceding embodiment, wherein 109. The cleavage reagent is 0.8 to 1.2 M phosphoric acid (H 3 PO 4 5. The method of any preceding embodiment, wherein 110. The cleavage reagent is about 1M phosphoric acid (H 3 PO 4 5. The method of any preceding embodiment, wherein 111. The method of any of the previous embodiments, wherein the cleavage reagent is tosylic acid (TsOH). 112. The method of any of the previous embodiments, wherein the cleavage reagent is 6,6'-(1,2,4,5-tetrazine-3,6-diyl)dinicotinic acid. 113. The method of any of the preceding embodiments, wherein at least a portion of the method is carried out in a batch procedure. 114. The method of any of the preceding embodiments, wherein the enamide cleavage is carried out at a temperature of 0 to 80 degrees Celsius. 115. The method of any of the preceding embodiments, wherein the enamide cleavage is carried out at a temperature of 20 to 60 degrees Celsius. 116. The method of any of the preceding embodiments, wherein the enamide cleavage is carried out at a temperature of 20-40 degrees Celsius. 117. The method of any of the preceding embodiments, wherein the enamide cleavage is carried out at a temperature of 20-26 degrees Celsius. 118. The method of any of the preceding embodiments, wherein the enamide cleavage is carried out at a temperature of 20-23 degrees Celsius. 119. The method of any of the previous embodiments, wherein the enamide cleavage is carried out at a temperature of 20 degrees Celsius. 120. The method of any of the previous embodiments, wherein the enamide cleavage is carried out at a temperature of 23 degrees Celsius. 121. The method of any of the preceding embodiments, wherein the enamide cleavage is carried out at room temperature. 122. The method of any one of embodiments 1-116, wherein the enamide cleavage is carried out at a temperature of 30-40 degrees Celsius. 123. The method of any one of embodiments 1-116 and 122, wherein the enamide cleavage is carried out at a temperature of about 37 degrees Celsius. 124. The method of any one of the preceding embodiments, wherein at least a portion of the method is carried out in a flow reactor. 125. The method of any of the preceding embodiments, wherein the irradiating step is carried out in a flow reactor. 126. The method of any of the preceding embodiments, wherein only the irradiation step is carried out in a flow reactor. 127. The method of any one of embodiments 124-126, wherein the temperature of the flow reactor is 20 to 60 degrees Celsius. 128. The method of any one of embodiments 124 to 127, wherein the temperature of the flow reactor is 20 to 40 degrees Celsius. 129. The method of any one of embodiments 124 to 128, wherein the temperature of the flow reactor is 20 to 30 degrees Celsius. 130. The method of any one of embodiments 124-129, wherein the temperature of the flow reactor is 25 to 30 degrees Celsius. 131. The method of any one of embodiments 124-130, wherein the temperature of the flow reactor is about 26 degrees Celsius. 132. The method of any one of embodiments 124 to 131, wherein the flow rate of the flow reactor is 0.200 to 50.00 mL / min. 133. The method of any one of embodiments 124 to 132, wherein the flow rate of the flow reactor is 1.00 to 25.00 mL / min. 134. The method of any one of embodiments 124 to 133, wherein the flow rate of the flow reactor is 5.00 to 10.00 mL / min. 135. The method of any one of embodiments 124 to 134, wherein the flow rate of the flow reactor is 6.00 to 9.00 mL / min. 136. The method of any one of embodiments 124-135, wherein the flow rate of the flow reactor is about 8.00 mL / min. 137. The method of any one of embodiments 124 to 136, wherein the reactor volume of the flow reactor is 1 to 10 mL. 138. The method of any one of embodiments 124 to 137, wherein the reactor volume of the flow reactor is 2 to 5 mL. 139. The method of any one of embodiments 124 to 138, wherein the reactor volume of the flow reactor is 1 to 3 mL. 140. The method of any one of embodiments 124-139, wherein the reactor volume of the flow reactor is about 2 mL. 141. The method of any one of embodiments 124-140, wherein the reactor volume of the flow reactor is about 2.7 mL. 142. The method of any one of embodiments 124 to 141, wherein the residence time in the flow reactor is 0.04 to 50 minutes. 143. The method of any one of embodiments 124 to 142, wherein the residence time in the flow reactor is 0.1 to 10 minutes. 144. The method of any one of embodiments 124 to 143, wherein the residence time in the flow reactor is 0.1 to 1 min. 145. The method of any one of embodiments 124 to 144, wherein the residence time in the flow reactor is 0.1 to 0.5 minutes. 146. The method of any one of embodiments 124-145, wherein the residence time in the flow reactor is about 0.25 minutes. 147. The method of any one of embodiments 124-146, wherein the flow reactor is a Vapourtec UV-150 photochemical reactor. 148. The method of any one of embodiments 124-146, wherein the flow reactor is a Corning® Lab Photo Reactor. 149. The method of any one of embodiments 124 to 148, wherein the light source irradiates with a radiant power of 3 to 150 watts. 150. The method of any one of embodiments 124-149, wherein the light source irradiates with a radiant power of 5-100 watts. 151. The method of any one of embodiments 124 to 150, wherein the light source irradiates with a radiant power of 10 to 80 watts. 152. The method of any one of embodiments 124-151, wherein the light source irradiates with a radiant power of 9 to 24 watts. 153. The method of any of the preceding embodiments, wherein the reaction is carried out in a single reaction vessel. 154. The method of any of the preceding embodiments, further comprising adding a surfactant. 155. The method of any of the preceding embodiments, further comprising adding a surfactant selected from the group consisting of sodium dodecyl sulfate (SDS), sodium desoxycholate (SDC), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Triton™ X-100, Tween-20, tetrapropylammonium hydroxide (TAPH-40), and sodium octanoate. 156. The method of any of the preceding embodiments, further comprising adding a surfactant, wherein the surfactant is sodium dodecyl sulfate (SDS). 157. The method of any of the preceding embodiments, further comprising adding a surfactant, wherein the surfactant is 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). 158. The method of any of the preceding embodiments, further comprising adding a surfactant, wherein the surfactant is sodium octanoate. 159. The method of any of the preceding embodiments, further comprising adding 40-80 mM of a surfactant. 160. The method of any of the preceding embodiments, further comprising adding 50-70 mM of a surfactant. 16. The method of any of the preceding embodiments, further comprising adding 161.55 to 65 mM of a surfactant. 162. The method of any of the preceding embodiments, further comprising adding about 60 mM of a surfactant. 163. Prior to step (b), coupling a cysteine ​​of peptide R2 with photolabel R1 to form a photolabel-protected cysteine; 2. The method of any of the preceding embodiments, further comprising, after step (b), releasing the photolabeled protected cysteine ​​from peptide R2. 164. The method of embodiment 163, wherein a nucleophilic sulfide and an oxidation partner are provided. 165. The method of any one of embodiments 163-164, further comprising adding a nucleophilic sulfide to release the photolabeled protected cysteine ​​from peptide R2. 166. The method of any one of embodiments 163 to 165, further comprising adding a nucleophilic sulfide to release the photolabeled protected cysteine ​​from peptide R2, wherein the nucleophilic sulfide is selected from the group consisting of cysteamine, cysteine, dithiothreitol (DTT), 3,6-dioxa-1,8-octanedithiol (DODT), 2-mercaptoethanol, glutathione (GSH), and acetylcysteine. 167. The method of any one of embodiments 163-166, further comprising adding a nucleophilic sulfide to release the photolabeled protected cysteine ​​from peptide R2, wherein the nucleophilic sulfide is cysteamine. 168. The method of any one of embodiments 163 to 167, further comprising forming a disulfide bond in peptide R2 after releasing the protected cysteine ​​with a photolabel. 169. The method of any one of embodiments 163-168, further comprising adding an oxidation partner. 170. The method of any one of embodiments 163-169, further comprising adding an oxidation partner selected from the group consisting of cystamine glutathione disulfide (GSSG) and cysteine. 171. The method of any one of embodiments 163 to 170, further comprising adding an oxidation partner selected from the group consisting of cystamine glutathione disulfide (GSSG) and cysteine. 172. The method of any one of embodiments 163-171, further comprising adding 0.5 to 3 additional equivalents of photolabel to the peptide in step (a) for each disulfide bond formed. 173. The method of any one of embodiments 163-172, further comprising adding 1 to 2.5 additional equivalents of photolabel to the peptide in step (a) for each disulfide bond formed. 174. The method of any one of embodiments 163-173, further comprising adding 1.5 to 2.5 additional equivalents of photolabel to the peptide in step (a) for each disulfide bond formed. 175. The method of any one of embodiments 163-174, further comprising adding about 2 additional equivalents of photolabel to the peptide in step (a) for each disulfide bond formed. 176. The method of any of the preceding embodiments, further comprising purifying the C-terminal α-amide, wherein purification of the C-terminal α-amide is carried out in a batch procedure or by continuous precipitation. 177. The method of any of the preceding embodiments, further comprising purifying the C-terminal α-amide, wherein purification of the C-terminal α-amide is performed via high performance liquid chromatography (HPLC) or ultrafiltration diafiltration (UFDF). 178. The method of any of the preceding embodiments, further comprising purifying the C-terminal α-amide, wherein purification of the C-terminal α-amide is performed via high performance liquid chromatography (HPLC). 179. The method of any of the preceding embodiments, further comprising purifying the C-terminal α-amide, wherein purification of the C-terminal α-amide is performed via ultrafiltration diafiltration (UFDF). 180. A method for producing a peptide or protein comprising a C-terminal α-amide of formula IV according to scheme 2, comprising: [ka] wherein R2 is a polypeptide; R3 is hydrogen. Step (a): coupling a peptide or protein containing a C-terminal cysteine ​​amidation tag of formula I with 4-chloro-7-nitrobenzofurazan to obtain a peptide-photolabel conjugate of formula II-a; Step (b): irradiating the peptide-photolabeled conjugate of formula II-a to obtain the C-terminal enamide of formula III via photochemical conversion; and step (c). cleaving the C-terminal enamide of formula III to obtain the C-terminal α-amide of formula IV. 181. A method for producing a peptide or protein comprising a C-terminal α-amide of formula IV according to scheme 2, comprising: [ka] wherein R2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:16; R3 is hydrogen. Step (a): coupling a peptide or protein containing a C-terminal cysteine ​​amidation tag of formula I with 4-chloro-7-nitrobenzofurazan to obtain a peptide-photolabel conjugate of formula II-a; Step (b): Irradiating the peptide-photolabeled conjugate of formula II-a with light having a wavelength of 400 to 450 nm to obtain the C-terminal enamide of formula III by photochemical conversion; and step (c). cleaving the C-terminal enamide of formula III to obtain the C-terminal α-amide of formula IV. 182. A method for producing a pharmaceutical composition according to any one of embodiments 1 to 181. 183. A method for producing a pharmaceutical composition, comprising: a. Producing a compound of formula IV according to any one of embodiments 1-181; b. preparing said pharmaceutical composition using the compound of formula IV obtained in a.

[0121] Methods and Examples List of Abbreviations Aq., aq. Water-based Ac Acetyl CFL Compact fluorescent lamp CLND Chemiluminescent Nitrogen Detection DIC Diisopropyl-carbodiimide DODT 3,6-Dioxa-1,8-octanedithiol DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide EIC Extracted Ion Chromatogram Et Ethyl Eq. equivalent Fmoc Fluorenylmethoxycarbonyl Protecting Group h time HRMS high resolution mass spectrometry HPLC High Performance Liquid Chromatography HCl Hydrochloric acid i-Pr Isopropyl IEDDA Inverse Electron Demanding Diels-Alder LCMS Liquid Chromatography Mass Spectrometry LED Light Emitting Diode Me Methyl Min MWCO Molecular Weight Cutoff NBD-Cl 4-Chloro-7-nitrobenzofurazan NHS N-hydroxysuccinimide NMP N-Methyl-2-pyrrolidone RP-HPLC Reversed Phase High Performance Liquid Chromatography SPPS Solid Phase Peptide Synthesis TFA Trifluoroacetic acid TIPS Triisopropylsilane UPLC Ultra High Performance Liquid Chromatography UPLC-MS Ultra Performance Liquid Chromatography Mass Spectrometry Please note that in the examples, the numbering of compounds such as Compound 1 differs from the numbers representing compounds in the specification and claims, such as (I) or (II), solely for sequential clarity.

[0122] General Preparation Method The synthesis of α-amidated peptides was carried out in three different ways, either via a screening scale procedure, a batch procedure, or a continuous photoflow process.

[0123] reagent All reagents and solvents were purchased commercially and used as supplied. Orthogonally protected Fmoc-amino acids were purchased as pre-weighed cartridges from Iris Biotech and SPPS resin was purchased from NovaBioChem.

[0124] General methodology for automated SPPS Automated SPPS was performed on a Symphony X peptide synthesizer (Gyros Biotechnologies). Peptides were synthesized using standard Fmoc chemistry on preloaded Fmoc-amino acid Wang resin (approximately 0.34 mmol / g loading). Coupling was performed using 5 equivalents of Fmoc amino acid, 10 equivalents of diisopropyl-carbodiimide (DIC), 5 equivalents of 2,4,6-collidine, and 5 equivalents of ethyl cyanohydroxy-iminoacetate (Oxima) in DMF. Coupling reactions were allowed to proceed for 1.5-4 hours at room temperature with bubbling of N2 for mixing. Fmoc deprotection was performed using 20% ​​piperidine in DMF.

[0125] Common methods of cutting Peptide cleavage was carried out in a 90 / 2.5 / 2.5 / 2.5 / 2.5 / 2.5 solution of TFA / triisopropylsilane (TIPS) / water / thioanisole / 3,6-dioxa-1,8-octanedithiol (DODT). The eluted cleavage solution was precipitated in ice-cold diethyl ether. The peptide precipitate was pelleted using centrifugation, and the pellet was washed with additional ice-cold ether.

[0126] General methods for purification and quantification The washed peptide pellet was redissolved in either 1:1 acetonitrile / water or pure DMSO and then filtered through a 0.2 μm filter. The resulting solution was loaded onto either a preparative RP-HPLC system, a Waters Prep 150 LC system or an Agilent 1290 Infinity II Autoscale. Preparative LC / MSD system. Both systems were equipped with a Waters XSelect CSH C18 column (19×250 mm, 5 μm) with a flow rate of 20 mL / min (Buffer A: 89.9% water / 10% acetonitrile with 0.1% TFA, Buffer B: 89.9% acetonitrile / 10% water with 0.1% TFA). Peptides were resolved using an appropriate solvent gradient. Pure fractions were pooled, assessed for purity / identity by UPLC / LCMS and peptide content by chemiluminescence nitrogen detection (CLND), and lyophilized to powder.

[0127] Common methods of detection and characterization Analytical UPLC was performed on a Waters Acquity UPLC system equipped with a Waters BEH C18 column (2.1 x 150 mm, 1.7 μm) with a flow rate of 0.4 mL / min (Buffer A: 99.95% water with 0.05% TFA, Buffer B: 99.95% acetonitrile with 0.05% TFA, gradient: 95 / 5 A:B held for 0.5 min, 95 / 5 A:B to 5 / 95 A:B over 16 min, followed by wash and re-equilibration). UV absorbance was measured at 214 nm. MS detection was obtained using a Waters Xevo G2-XS QToF mass spectrometer in ESI+ mode for LCMS and HRMS analysis.

[0128] General reaction protocol for C-terminal α-amidation The reactions carried out in the present invention generally follow Scheme 1, shown below. [ka] The variable substituents in the above formula have the same meanings as in Scheme (1).

[0129] The α-amidation reaction of the present invention was carried out according to three different protocols: screening scale, batch scale, and flow procedure, which are described in further detail below.

[0130] General protocol for screening scale reactions: The reaction was first carried out on a screening scale according to Scheme 1. The procedures for the individual steps ((a), (b), and (c)) are described in further detail below.

[0131] Step (a) - Coupling of a peptide or protein containing a C-terminal cysteine ​​residue with a photolabel To minimize non-specific oxidation, the aqueous reaction buffer containing 25 mM bis-trismethane pH 6.4 with 50 mM glycine was degassed by bubbling nitrogen gas for 15 min. C-terminal Cys modified peptides were dissolved in the reaction buffer to generate 1 mM stock solutions. 25 μL of 1 mM stock was added to wells of a 96-well V-bottom assay plate along with 65 μL of additional assay buffer. Either 2-bromomaleimide (3-bromo-1H-pyrrole-2,5-dione, CAS: 98026-79-0) or NBD chloride (4-chloro-7-nitrobenzofurazan, CAS: 10199-89-0) was dissolved in acetonitrile to obtain a 5 mM stock solution. 10 μL of 5 mM photoconjugation reagent was added to the wells (500 μM final concentration, 2 equivalents) and allowed to incubate at room temperature for 1 h with shaking. If desired, conversion may be monitored by LC-MS.

[0132] Step (b) - The resulting peptide-photolabeled conjugate is photochemically converted to give the C-terminal enamide: After 1 hour, the plate was irradiated according to the photoaryl group utilized to generate the C-terminal enamide. For 2-bromomaleimide, a 1.5 meter strip of 365 nm LED was used as the irradiation source and the conversion was allowed to proceed for 4 hours. The reaction mixture was kept at room temperature using a cooling fan. For NBD chloride, irradiation may be performed using a handheld white CFL lamp or a strip of 450 nm LED. The conversion was allowed to proceed for 1 hour at room temperature. If desired, the conversion may be monitored by LC-MS.

[0133] Step (c) - Cleavage of the resulting C-terminal enamide to give the C-terminal α-amide: Acidolysis of enamides to C-terminal α-amide products: A variety of strong acids were used to proceed with the acidolysis of the enamides (Table 2). In general, the acid was added to the reaction mixture from a 10x aqueous stock solution. Trifluoroacetic acid was used for the majority of the conditions tested. For TFA, 10 μL of a 50 / 50 solution of TFA / water was added to the reaction well (for a final TFA concentration of 5%) and the plate was shaken at room temperature for up to 24 hours. When additives such as methionine, indole, or caffeic acid were utilized, the additive reagent was spiked into the reaction from a 50x or 100x stock solution prior to the addition of the acid. Conversion was monitored by LC-MS.

[0134] Enamide cleavage using inverse electron demand Diels-Alder (IEDDA) Enamides can also be cleaved using IEDDA chemistry with dipyridyl-tetrazine (3,6-di-2-pyridyl-1,2,4,5-tetrazine, CAS: 1671-87-0). Dipyridyl-tetrazine was dissolved in a mixture of 80% acetonitrile and 20% 125 mM aqueous HCl to give a 12.5 mM stock solution (final concentration of HCl = 25 mM, 2 equivalents based on tetrazine concentration). The solubility of dipyridyl-tetrazine was poor in acetonitrile alone, and acid had to be added to protonate the pyridyl group and improve solubility. 10 μL of this stock solution was added to the reaction well (final tetrazine concentration of 1.25 mM, 5 equivalents based on peptide concentration) and the reaction was incubated at 37 °C for 24 h. Conversion was monitored by LC-MS.

[0135] Analysis of the conversion by UPLC-MS Reaction samples were analyzed directly by UPLC-MS analysis (gradient: 95 / 5 A / B hold for 0.5 min, gradient from 95 / 5 A / B to 55 / 45 A / B over 7 min, followed by wash and re-equilibration). In some cases, it was not possible to achieve sufficient chromatographic resolution to quantify conversion using integrated UV data at 214 nm. To quantify conversion, extracted ion chromatograms (EICs) were generated by inputting the calculated masses of all peptide starting materials, intermediates, by-products, and the desired product. The integrated peaks of these EICs were summed and normalized to 100%, and the conversion to the desired product was calculated based on the normalized integral.

[0136] Protocol for batch scale reaction: In a vial equipped with a stir bar or a 12.5 mL capacity colorimeter cuvette (catalog no. 76016-356), 1.0 equivalent of protein, peptide, or polypeptide containing a C-terminal cysteine ​​residue was dissolved in bis-trismethane buffer (25 mM, pH 6.3) to a final peptide concentration of 250 uM. A stock solution of NBD (3-bromo-1H-pyrrole-2,5-dione, CAS: 98026-79-0) or NBD chloride (4-chloro-7-nitrobenzofurazan, CAS: 10199-89-0) was added in a 1:1 dilution of H 2 2 equivalents relative to peptide were added to the reaction vessel for cysteine ​​coupling. The reaction was allowed to stir for 1 hour or until reaction completion by LC / MS. The reaction vessel was then irradiated with a 365 nm LED, a handheld white CFL task light, or a strip of 450 nm blue LED for 0.25-4 hours. The reaction mixture was maintained at room temperature using a cooling fan. Once the reaction was complete by LC / MS, trifluoroacetic acid (TFA) was added (5% final TFA concentration) and the reaction was allowed to stir at ambient temperature for 12-24 hours. Alternatively, 5 equivalents of dipyridyl-tetrazine (3,6-di-2-pyridyl-1,2,4,5-tetrazine, CAS:1671-87-0) was added from a stock solution and 1.25 mM tetrazine (2.5 mM HCl, 80:20 MeCN:HCI). 2 A final concentration of 1:1 was obtained (dissolved in 0) and the reaction was incubated for 24 h at 37° C. Conversion to the C-terminal α-amide was monitored by LC-MS.

[0137] Protocol for batch-scale reactions of disulfide bond-containing peptides: In a vial equipped with a stir bar or a 12.5 mL capacity colorimeter cuvette (catalog no. 76016-356), 1.0 equivalent of protein, peptide, or polypeptide containing a C-terminal cysteine ​​residue was dissolved in bis-trismethane buffer (25 mM, pH 6.3) to a final peptide concentration of 250 uM. A surfactant may be added to the solution to improve the solubility of intermediates. A surfactant concentration of 60 mM may be preferred. A stock solution of NBD chloride (4-chloro-7-nitrobenzofurazan, CAS: 10199-89-0) was dissolved in 1:1 H 2 HO:MeCN and 4 equivalents relative to the peptide were added to the reaction vessel for overall cysteine ​​coupling. The reaction was allowed to stir for 2 hours or until reaction completion by LC / MS. The reaction vessel was then illuminated with a handheld white CFL task light or a strip of 450 nm blue LED for 0.25-4 hours. The reaction mixture was kept at room temperature using a cooling fan. Once the reaction was complete by LC / MS, H 2 A solution of a nucleophilic sulfide, e.g., cysteamine (8 equiv.) and cysteamine (1.2 equiv.) in O is added and allowed to stir at ambient temperature for 2 h to liberate the backbone cysteine ​​thiol and oxidize it to the disulfide. Trifluoroacetic acid (TFA) is added (5% final TFA concentration) and the reaction is allowed to stir at ambient temperature for 12-24 h. Alternatively, 5 equiv. of dipyridyl-tetrazine (3,6-di-2-pyridyl-1,2,4,5-tetrazine, CAS:1671-87-0) is added from a stock solution to give 1.25 mM tetrazine (2.5 mM HCl, 80:20 MeCN:HO). 2 A final concentration of 1,2-dichlorophenyl ether (dissolved in 200) may be obtained and the reaction may be incubated for 24 hours at 37° C. Conversion to the C-terminal α-amide was monitored by LC-MS. EXAMPLES

[0138] [Example 1: Photolabeling Experiment] Following the general procedure outlined for batch-scale α-amidation, a variety of photolabels covering a range of chemical backbones and absorption wavelengths were evaluated. Reactions were analyzed directly by UPLC-MS after photolabel conjugation and irradiation with an appropriate light source, as outlined above for conversion to the C-terminal enamide. The results are presented in Table 1. [Table 1]

[0139] Conversion was observed for photolabeling and conversion of a peptide having an R2 of the amino acid sequence shown in SEQ ID NO:2, including a C-terminal cysteine, to a C-terminal enamide. Reactions were carried out with structurally diverse photolabeling agents, and conversion of the photolabeled conjugate was achieved using visible light at 365 nm.

[0140] Example 2: Enamide cleavage experiment Following the general protocol for screening-scale α-amidation, a wide range of acids and tetrazines were evaluated for the cleavage of the enamide to the corresponding C-terminal α-amide (step (c)). The results are presented in Table 2. [Table 2]

[0141] Acidolysis of enamides to C-terminal α-amides was achieved with a variety of acids. The reaction was shown to be efficient with strong acids. Cleavage of enamides by the IEDDA reaction was even more efficient with di-2-pyridyl substituted tetrazines, providing the C-terminal α-amides in high yields.

[0142] [Example 3: Reaction performance when the penultimate AA position is varied] The performance of the reaction on the penultimate AA position of an exemplary peptide was investigated for four sets of conditions. The general protocol outlined for screening-scale α-amidation was used for these experiments.

[0143] Two photolabeling agents were selected for detailed analysis in the reactions: 3-bromo-1H-pyrrole-2,5-dione (Formula 1) and 4-chloro-7-nitrobenzofurazan (Formula 2), which typically offered the best combination of cost, high conjugation yield, and easy photolysis under UV and visible light sources, respectively. The final enamide cleavage step was performed with 5% v / v TFA for the acidolysis route and with 3,6-di-2-pyridyl-1,2,4,5-tetrazine for the IEDDA-mediated enamide cleavage. General reaction conditions and results are presented in Tables 3 and 4 below. [Table 3] [Table 4]

[0144] The scope of the C-terminal α-amidation reaction was explored for the penultimate amino acid position of generic peptides. The reaction proceeds in good to excellent yields for any amino acid in the penultimate position, indicating a broad tolerance for side chain functional groups at this position. The reaction proceeds with high efficiency when either 2-bromomaleimide or NBD-Cl is utilized in the photolabeling step. High yields of C-terminal α-amides are observed when the enamide is cleaved by acidic decomposition or by an IEADDA reaction, allowing the choice of two complementary reactions that can be carried out under acidic or neutral conditions, respectively.

[0145] Example 4: Disulfide-containing peptides The present invention was used to prepare peptides with a C-terminal α-amide containing a disulfide bond. The protocol for batch-scale reaction of peptides containing disulfide bonds specified above was used for this experiment following the reaction generally shown in Scheme 1, except that no surfactant was used. 2A nucleophilic sulfide of cysteamine (8 eq.) in O and an oxidation partner of cystamine (1.2 eq.) were used. The cysteine-rich backbone was globally alkylated with photolabeling in the first step. Selective photolysis of the C-terminal alkylated cysteine ​​occurred upon exposure to light. The remaining alkylated cysteines were removed with nucleophilic sulfide and oxidized to liberate cysteine ​​sulfides to form the desired peptides containing disulfide bonds. The reaction was then run according to the conditions outlined for enamide cleavage with TFA, yielding the results shown in Table 5 below. [Table 5]

[0146] Efficient amidation of a model peptide (IECTKSEGCEEVYEADHGEP, SEQ ID NO: 15) containing a disulfide bond in the amidated state indicates that peptides containing backbone cysteines readily undergo selective conversion to the desired C-terminal α-amide.

[0147] Example 5: Preparation of biologically active peptides The present invention was used to synthesize biologically relevant peptides and commercially available peptide therapeutics. The protocol outlined for screening scale α-amidation was used for these experiments, and the reactions generally followed Scheme 1 and conditions C or D outlined in Table 3. The results are presented in Table 6. [Table 6]

[0148] C-terminal cysteine-extended precursors of biologically active peptides and commercial peptide therapeutics were prepared and subjected to the C-terminal α-amidation process. All examples gave good yields of the corresponding biorelevant C-terminal α-amides, highlighting the facile reaction conditions and broad scope of the present invention. The C-terminal α-amidation process performed well regardless of peptide size and composition, demonstrating the applicability of the chemistry to any peptide or protein.

[0149] Example 6: Upscale preparation of GLP-1 receptor-amylin receptor coagonist intermediate The present invention was applied on a larger scale using a hybrid batch-photoflow procedure, which is described in detail below.

[0150] This example was carried out on peptide R2 having the amino acid sequence shown in SEQ ID NO:17, which comprises the amino acid sequence shown in SEQ ID NO:16 and an N-terminal extension having the amino acid sequence shown in SEQ ID NO:18.

[0151] To a 1 liter aluminum foil wrapped glass reactor under nitrogen atmosphere containing a solution of peptide R2 according to SEQ ID NO:17 with a C-terminal cysteine ​​amidated tag (12.4 g, 1.44 mmol) in 0.2-0.3 mM aqueous TCEP (780 ml, pH 7) was added a solution of NBD-Cl (519 mg, 2.6 mmol) in MeCN (5.2 ml). After stirring for 80 min at ambient temperature, HPLC analysis showed the reaction to be complete.

[0152] The resulting solution was irradiated at 405 nm (blue LED, 61 W input power) in a Corning™ Lab Photo Reactor (2.7 mL fluid module volume, 10 mL / min flow rate, reactor heat exchange temperature of 20° C.) and the reactor was flushed with an additional 50 ml of water. HPLC analysis showed at least 80% consumption of the starting material.

[0153] 6M Phosphate (H 3 PO 4, 160 mL) was added to the resulting solution. After stirring for 16 h, L-histidine (7.45 g) was added and the mixture was stirred at ambient temperature for 10 min. 6 M aqueous KOH was then carefully added until the solution reached pH 8.5 while maintaining the temperature below 40 °C. The resulting mixture was concentrated to 1.0 L by ultrafiltration (MWCO 5 kDa) and diafiltered (MWCO 5 kDa) with 3.0 L of 20 mM Tris buffer pH 8 while maintaining a constant retentate volume (continuous mode) between 0.8 and 1 L. The final mixture (800 mL) was analyzed by HPLC. The conversion results are shown below. [Table 7]

[0154] Reaction samples were diluted 10-fold with 1:1 v / v MeCN / water and analyzed by RP-HPLC analysis (Kinetex 1,7 μm C18 100 Å 100×2,1 mm, eluent A: 10% MeCN+0.1% TFA in water, eluent B: 90% MeCN+0.1% TFA in water. Gradient: 20-50% B in 12 min, UV detection at both 214 nm and 420 nm. Conversion yields were calculated using external HPLC standards.

[0155] This example shows the larger scale production of peptides with C-terminal amides, each peptide amide produced in high yield using the photochemical C-terminal α-amidation method. In addition, this example shows that this photochemical method can be completed in a hybrid setup where only the photochemical conversion step (step (b) in Scheme 1 or 2) is carried out in a flow setup. Overall, the example highlights the robustness and scalability of this method.

[0156] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will occur to those skilled in the art. It is therefore to be understood that the appended embodiments are intended to cover all such modifications and alterations that fall within the true spirit and scope of the invention.

Claims

1. 1. A method for producing a peptide or protein comprising a C-terminal α-amide of Formula IV according to Scheme 1, comprising: 【Chemistry 1】 wherein R2 is a polypeptide; R1-X is a photolabeling agent, R1 is a photolabel, and X is a leaving group; R3 is selected from the group consisting of hydrogen, methyl, and ethyl. Step (a): Coupling a peptide or protein comprising a C-terminal cysteine ​​amidated tag of Formula I with a photolabel (R1) to obtain a peptide-photolabel conjugate of Formula II; Step (b): Irradiating the peptide-photolabeled conjugate of formula II to obtain the C-terminal enamide of formula III by photochemical conversion; Step (c) cleaving the resulting C-terminal enamide of formula III to give the C-terminal α-amide of formula IV.

2. 2. The method of claim 1, wherein R3 is hydrogen.

3. 2. The method of claim 1, wherein R2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, and 17.

4. The method of claim 1, wherein R2 is an amino acid sequence shown in any one of SEQ ID NOs: 3 to 14 and 16 to 17.

5. The method of claim 1, wherein the photolabeling agent (R1-X) is selected from the group consisting of 3-bromo-1H-pyrrole-2,5-dione, 4-chloro-7-nitrobenzofurazan, 2-bromo-1,4-naphthoquinone, 1-fluoro-2,4-dinitrobenzene, and 4-fluoro-7-sulfamoylbenzofurazan.

6. The photolabeling agent (R1-X) is 3-bromo-1H-pyrrole-2,5-dione (chemical formula 1) or 4-chloro-7-nitrobenzofurazan (chemical formula 2): Chemical formula 1: 【Chemistry 2】 Chemical formula 2: 【Transformation 3】 The method of claim 1, wherein

7. The photolabeling agent is 3-bromo-1H-pyrrole-2,5-dione (chemical formula 1): Chemical formula 1: 【Chemistry 4】 The method of claim 1, wherein

8. The photolabeling agent is 4-chloro-7-nitrobenzofurazan (chemical formula 2): Chemical formula 2: 【Transformation 5】 The method of claim 1, wherein 9. The method of claim 1, wherein step (a) of the method is carried out in an aqueous reaction buffer, the aqueous buffer being selected from the group consisting of bis-trismethane, tris, triethanolamine, and phosphate.

10. The method of claim 1, wherein the light source for irradiating the peptide-photolabeled conjugate has a wavelength of 365 to 500 nm.

11. The method of claim 1, wherein at least a portion of the method is carried out in a flow reactor.

12. Before step (b), coupling a cysteine ​​of peptide R2 with photolabel R1 to form a photolabel-protected cysteine; The method of claim 1, further comprising, after step (b), releasing the photolabeled protected cysteine ​​from peptide R2.

13. The method of claim 12 wherein a nucleophilic sulfide and an oxidation partner are provided.

14. A method for producing a peptide or protein comprising a C-terminal α-amide of formula IV according to Scheme 2, comprising: 【Transformation 6】 wherein R2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16; R3 is hydrogen. Step (a): Coupling a peptide or protein containing a C-terminal cysteine ​​amidated tag of Formula I with 4-chloro-7-nitrobenzoflupazan to obtain a peptide-photolabeled conjugate of Formula II-a; Step (b): Irradiating the peptide-photolabeled conjugate of formula II-a with light having a wavelength of 400-450 nm to obtain the C-terminal enamide of formula III by photochemical conversion; Step (c): cleaving the C-terminal enamide of formula III to give the C-terminal α-amide of formula IV.

15. Step (a) producing a peptide or protein comprising a C-terminal α-amide of formula IV by the method of any one of claims 1 to 14. and step (b) producing a pharmaceutical composition comprising said peptide or protein.

Images