Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/13—Labelling of peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/003—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by transforming the C-terminal amino acid to amides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a photochemical process for the manufacturing of a C-terminal a-amide, and more specifically to a method of producing a peptide or protein comprising a C-terminal a-amide with a photochemical process.
• C-terminal a-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.
• Baker et al. provides a method for the selective modification of cysteines with bromomaleimides and purports to disclose a method for photolytic modification of cysteine maleimide conjugates (Chem. Commun. 6583-6585 (2009) and Org. Biomol. Chem. 14, 455- 459 (2016)).
• One objective of the present work is to provide a photochemical process for the production of a peptide or protein comprising a C-terminal a-amide. It is an objective to provide a selective and low-cost C-terminal peptide and protein amidation process capable of being applied to a variety of peptides while overcoming at least some of the drawbacks of traditional amidation methods.
• R1-X is a photolabeling agent, wherein R1 is a photolabel and X is a leaving group,
• R3 is selected from the group consisting of hydrogen, methyl and ethyl, comprising the steps of
• Step (a) coupling a peptide or protein comprising a C-terminal cysteine amidation tag (formula I) with the photolabel (R1) to obtain a peptide-photolabel conjugate (formula II);
• Step (b) irradiating the resulting peptide-photolabel conjugate (formula II) to obtain a C- terminal enamide (formula III) via a photochemical conversion;
• Step (c) cleaving the C-terminal enamide (formula III) to obtain the C-terminal a-amide (formula IV).
• a method following Scheme 1 for producing a peptide or protein comprising a C-terminal a-amide of formula IV is provided. This is achieved by starting with a peptide or protein comprising a C-terminal cysteine amidation tag (depicted as formula I) and reacting it with a photolabeling agent (depicted as R1-X) to obtain a peptide- photolabel conjugate (formula II). Subsequently, the peptide-photolabel conjugate is exposed to light, and undergoes a photochemical conversion, to obtain a peptide or protein enamide (formula III), which is subsequently converted to the resulting C-terminal a-amide (formula IV).
• the method for producing a peptide or protein comprising a C-terminal a-amide may comprise a photolabeling agent (R1-X) selected from the group consisting of 3-Bromo-1 H- pyrrole-2, 5-dione, 4-chloro-7-nitrobenzofurazan, 2-bromo-1 ,4-naphthoquinone, 1-fluoro-2,4- dinitrobenzene and 4-fluoro-7-sulfamoylbenzofurazan.
• a photolabeling agent R1-X
• the method for producing a peptide or protein comprising a C-terminal a-amide may comprise the use of cleavage reagent selected from the group consisting of trifluoroacetic acid (TFA), hydrochloric acid (HCI), Sulfuric acid (H2SO4), tosylic acid (TsOH), phosphoric acid (H3PO4), oxalic acid, 3,6-diphenyl-1 ,2,4,5- tetrazine, and 6,6'-(1 ,2,4,5-tetrazine-3,6-diyl)dinicotinic acid.
• TFA trifluoroacetic acid
• HCI hydrochloric acid
• Sulfuric acid H2SO4
• TsOH tosylic acid
• H3PO4 phosphoric acid
• oxalic acid 3,6-diphenyl-1 ,2,4,5- tetrazine, and 6,6'-(1 ,2,4,5-tetrazine-3,6-d
• the method for producing a peptide or protein comprising a C-terminal a-amide may comprise a polypeptide R2 comprising an amino acid sequence as set out in any one of SEQ. ID No.: 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 16 or 17.
• R3 is hydrogen, comprising the steps of
• Step (a) coupling a peptide or protein comprising a C-terminal cysteine amidation tag (formula I) with 4-chloro-7-nitrobenzofurazan to obtain a peptide-photolabel conjugate (formula ll-a);
• Step (b) irradiating the peptide-photolabel conjugate (formula ll-a) to obtain a C-terminal enamide (formula III) via photochemical conversion;
• Step (c) cleaving the C-terminal enamide (formula III) to obtain the C-terminal a-amide (formula IV).
• the invention according to the second aspect is carried out by reacting a peptide or protein comprising a C-terminal cysteine of formula I with 4-chloro-7-nitrobenzofurazan as the photolabeling agent, obtaining a peptide-photolabel conjugate of formula ll-a.
• the conjugate is subsequently converted by a photochemical reaction to the enamide of formula III, which is converted by cleavage to the desired C-terminal a-amide of formula IV.
• the method for producing a peptide or protein comprising a C-terminal a-amide may further comprise the steps of coupling a cysteine of the peptide R2 with the photolabel R1 forming a photolabel-protected cysteine prior to irradiation (step (b)); and releasing the photolabel-protected cysteine from the peptide R2 after irradiation (step (b)).
• the method may further comprise the step of adding a nucleophilic sulfide for releasing the photolabel-protected cysteine from the peptide R2.
• the photochemical amidation method is mild, broad in scope, economically efficient, and suitable for large scale manufacturing.
• the invention may also solve further problems that will be apparent from the disclosure of the exemplary embodiments.
• SEQ ID No: 1 represents the amino acid sequence of a non-limiting example peptide that may be provided as R2 (Scheme 1 or 2) immediately preceding a C-terminal cysteine, wherein the penultimate amino acid of the peptide (from N-terminal to C-terminal of the polypeptide) prior to the photoamidation process (ie. Formula I in scheme 1 and 2) can be varied;
• SEQ ID No: 2 represents the amino acid sequence of an example embodiment of the peptide that can form R2 (Scheme 1 or 2);
• SEQ ID No: 3 represents the amino acid sequence of the peptide part (R2) of the starting material for producing glucagon-like peptide-1 (GLP-1) peptides;
• SEQ ID No: 4 represents the amino acid sequence of the peptide part (R2) of the starting material for producing a pancreastatin (PST) inhibitor, more specifically pancreastatin inhibitor peptide-8 (PSTi8) peptide;
• SEQ ID No: 5 represents the amino acid sequence of the peptide part (R2) of the starting material for producing pancreatic pYY(3-36) peptide;
• SEQ ID No: 6 represents the amino acid sequence of the peptide part (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 part (R2) of the starting material for producing a gastrin releasing peptide (GRP) peptide;
• SEQ ID No: 8 represents the amino acid sequence of the peptide part (R2) of the starting material for producing an adrenocorticotropic hormone (ACTH), more specifically Cosyntropin;
• SEQ ID No: 9 represents the amino acid sequence of the peptide part (R2) of the starting material for producing a QRF-amide peptide
• SEQ ID No: 10 represents the amino acid sequence of the peptide part (R2) of the starting material for producing a GLP-1 receptor-neuropeptide Y receptor 2 co-agonist EP45;
• SEQ ID No: 11 represents the amino acid sequence of the peptide part (R2) of the starting material for producing an entry inhibitor, more specifically Bulevirtide;
• SEQ ID No: 12 represents the amino acid sequence of the peptide part (R2) of the starting material for producing osteocrin (OSTN) peptide
• SEQ ID No: 13 represents the amino acid sequence of the peptide part (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 part (R2) of the starting material for producing a amylin receptor agonist, more specifically Pramlintide;
• SEQ ID No: 15 represents the amino acid sequence of a non-limiting example peptide that may be provided as R2 (Scheme 1 or 2), for which a disulfide bond is desired therein;
• SEQ ID No: 16 represents the amino acid sequence of the peptide part (R2) of the starting material for producing a GLP-1 receptor-amylin receptor co-agonist
• SEQ ID No: 17 represents the amino acid sequence of the peptide part (R2) of the starting material, consisting of the peptide for amidation and an N-terminal extension, for producing an a GLP-1 receptor-amylin receptor co-agonist;
• SEQ ID No: 18 represent the amino acid sequence of an example N-terminal extension which may form part the peptide part (R2).
• Fig. 1 depicts the overall reaction (Scheme 1) of the photochemical process for the manufacturing of a peptide or protein comprising a C-terminal a-amide;
• Fig. 2 depicts an embodiment of the reaction (Scheme 2) of the photochemical process for the manufacturing of a peptide or protein comprising a C-terminal a-amide, wherein chloro-7-nitrobenzofurazan is used as the photolabeling agent.
• amylin peptide a peptide associated with type 2 diabetes mellitus. Proc. Natl. Acad. Sci. USA 86, 9662-9666 (1989)
• NPY Rudet 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 a/., The current state of peptide drug discovery: back to the future? J. Med. Chem.
• the present invention relates to a photochemical process for the manufacturing of a peptide or protein comprising a C-terminal a-amide.
• the instant method provides the advantage of being broad in scope, such that it may be utilised to produce a great variety of biologically active peptides with a C-terminal a-amide.
• the process for the manufacturing of a peptide or protein comprising a C-terminal a-amide disclosed herein is not bound to a particular peptide of protein (R2) and provides a method for the amidation of a broad range of peptides or proteins via a C-terminal cysteine, which may be referred to herein as a C-terminal cysteine amidation tag in view of its purpose in the photochemical process.
• the method is sufficiently specific to permit the C-terminal amidation of peptides and proteins even when it contains more than one cysteine therein (ie.
• cysteines within the peptide R2 in addition to the C-terminal cysteine amidation tag of use for manufacturing of the peptide or protein comprising a C-terminal a-amide).
• another advantage of the invention is that the method is economically efficient due to the readily and commercially available starting materials and a simple reaction setup, which makes the process suitable for batch or in flow manufacturing, and permits the process to be of use in large scale manufacturing.
• the invention is carried out as depicted in Scheme 1 (Fig. 1) by starting with a peptide or protein comprising a C-terminal cysteine residue (formula I) and reacting it with a photolabeling agent (R1-X) to obtain a peptide-photolabel conjugate (formula II), followed by exposing the peptide-photolabel conjugate to light to obtain a peptide or protein enamide (formula III), and subsequently converting the resulting enamide to the C-terminal a-amide (formula IV).
• the process for the manufacturing of a peptide or protein comprising a C-terminal a-amide disclosed herein may be carried at varying strategic moments of the synthesis of biologically active peptides.
• the process for the manufacturing of a peptide or protein comprising a C-terminal a-amide disclosed herein can be used on intermediate products, including but not limited to precursors, prior to filtration steps such as high-performance liquid chromatography (HPLC) filtration for instance, prior to additional ligation steps and/or prior to cleavage of a peptide extension.
• HPLC high-performance liquid chromatography
• extension here refers to at least one amino acid extending from the N-terminal of a desired peptide, such as may be the case for instance for a precursor.
• Such a peptide R2 may have the following structure:
• the combined extension and desired peptide form part of the peptide R2 during the photochemical process of the present disclosure, where the extension may be meant to be cleaved from peptide R2 in a subsequent step.
• the photochemical amidation reaction may also solve further problems that will be apparent from the disclosure of the exemplary embodiments.
• Step (a) Coupling of peptide or protein comprising C-terminal cysteine residue (formula I) with a photolabeling agent (R1-X) to obtain a peptide-photolabel conjugate (formula II)
• the first step (step (a)) of the photochemical amidation reaction may be referred to as the coupling of a peptide or protein comprising 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).
• Formula I is a peptide or protein, comprising a polypeptide part (R2) and a C-terminal cysteine (Cys) residue.
• the polypeptide part R2 may also be referred to as a protein or peptide.
• the compound of formula I may be referred to as a peptide with 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.
• X is Ala.
• X is Asp.
• X is Glu.
• X is Phe.
• X is Gly.
• X is His. In one embodiment, X is lie. 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 Gin. In one embodiment, X is Arg. In one embodiment, X is Ser. In one embodiment, X is Thr. In one embodiment, X is Vai. In one embodiment, X is Trp. In one embodiment, X is Tyr. For example, in the case where X of
• IWTKDHEEVYEX (SEQ. ID NO 1) is an Alanine (Ala), the resulting peptide R2 is
• IWTKDHEEVYEA (SEQ. ID NO 2), forming an peptide having the same amino acid sequence with an amide once having undergone the photochemical amidation reaction of the present application.
• R2 may comprise an amino acid sequence as set out in any one of SEQ ID No. 3-14 and 16-17.
• R2 may be an amido acid sequence as set out in any one of SEQ. ID NO.: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16 and 17.
• R2 may comprise an amino acid sequence as set out in any one of SEQ. ID NO. 3, 4, 5, 16 and 17. In some embodiments, R2 may be an amino acid sequence as set out in any one of SEQ. ID NO. 3, 4, 5, 16 and 17. In some embodiments, R2 may comprise an amino acid sequence as set out in SEQ ID No. 16.
• the photochemical amidation reaction may be used to prepare glucagon-like peptide-1 (GLP-1) peptides or precursors thereof.
• the photochemical amidation reaction may be used to prepare amylin receptor agonists such as Pramlintide (the active pharmaceutical ingredient in SymlinTM), or precursor thereof.
• the photochemical amidation reaction may be used to prepare glucose-dependent insulinotropic polypeptide (GIP) peptides or precursors thereof.
• GIP glucose-dependent insulinotropic polypeptide
• the photochemical amidation reaction may be used to prepare GIP and GLP-1 receptor co-agonists such as Tirzepatide, or precursors thereof.
• the photochemical amidation reaction may be used to prepare corticotropin-releasing factor (CFR) peptides such as urocortin-2 (UCN2) peptide, or precursors thereof.
• CFR corticotropin-releasing factor
• the photochemical amidation reaction may be used to prepare pancreatic pYY(3-36) peptide or precursors thereof.
• the photochemical amidation reaction may be used to prepare pancreastatin (PST) inhibitors, such as pancreastatin inhibitor peptide-8 (PSTi8) peptide, or precursors thereof.
• PST pancreastatin
• the photochemical amidation reaction may be used to prepare luteinizing hormone-releasing hormone (LHRH) agonist or precursors thereof.
• LHRH luteinizing hormone-releasing hormone
• the photochemical amidation reaction may be used to prepare gastrin releasing peptide (GRP) peptides or precursors thereof.
• the photochemical amidation reaction may be used to prepare adrenocorticotropic hormones (ACTH) such as Cosyntropin (active pharmaceutical ingredient of CortrosynTM), or precursors thereof.
• the photochemical amidation reaction may be used to prepare QRF-amide peptides or precursors thereof.
• the photochemical amidation reaction may be used to prepare GLP-1 receptor-neuropeptide Y receptor 2 co-agonist, such as EP45, or precursors thereof.
• the photochemical amidation reaction may be used to prepare entry inhibitors, such as Bulevirtide (active pharmaceutical ingredient of HepcludexTM), or precursors thereof.
• the photochemical amidation reaction may be used to prepare natriuretic peptides such as osteocrin (OSTN) peptide or precursors thereof.
• the photochemical amidation reaction may be used to prepare antiretroviral drugs such as Enfuvirtide or precursors thereof.
• the photochemical amidation reaction may be used to prepare GLP-1 receptor-amylin receptor co-agonists or precursors thereof, such as that represented by SEQ ID NO. 16 or 17.
• 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 in SymlinTM), glucose-dependent insulinotropic polypeptide (GIP) peptides, GLP-1 receptor co-agonists such as Tirzepatide, corticotropin-releasing factor (CFR) peptides such as Urocortin-2 (UCN2) peptide, pancreatic pYY(3-36) peptide, pancreastatin (PST) inhibitors such as pancreastatin inhibitor peptide-8 (PSTi8) peptide, luteinizing hormone-releasing hormone (LHRH) agonist, Gastrin releasing peptide (GRP) peptides, adrenocorticotropic hormones (ACTH) such as Cosyntropin (active pharmaceutical ingredient of CortrosynTM), QRF-amide peptides, GLP-1 receptor
• 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 in SymlinTM), pancreatic pYY(3-36) peptide, 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 (active pharmaceutical ingredient of CortrosynTM), QRF-amide peptides, GLP-1 receptor-neuropeptide Y receptor 2 co-agonist such as EP45, entry inhibitors, such as Bulevirtide (active pharmaceutical ingredient of HepcludexTM), Osteocrin, antiretroviral drugs such as Enfuvirtide
• the photochemical amidation reaction may be used to prepare glucagon-like peptide- 1 (GLP-1) peptides, pancreatic pYY(3-36) peptide, pancreastatin (PST) inhibitors such as pancreastatin inhibitor peptide-8 (PSTi8) peptide, GLP-1 receptor-amylin receptor co-agonists such as those comprising the amino acid sequence of SEQ ID No. 16, or precursors thereof.
• GLP-1 receptor-amylin receptor co-agonists such as those comprising the amino acid sequence of SEQ ID No. 16, or precursors thereof.
• the photochemical amidation reaction may be used to prepare glucagon-like peptide- 1 (GLP-1) peptides, pancreatic pYY(3-36) peptide, and GLP-1 receptor-amylin receptor co- agonists such as those comprising the amino acid sequence of SEQ ID No. 16, or precursors thereof.
• GLP-1 glucagon-like peptide- 1
• pancreatic pYY(3-36) peptide pancreatic pYY(3-36) peptide
• GLP-1 receptor-amylin receptor co- agonists such as those comprising the amino acid sequence of SEQ ID No. 16, or precursors thereof.
• the preparation of GLP-1 peptides as disclosed herein includes native human GLP- 1 (7-36)-amide (such as starting peptide R2 may be GLP-1(7-36), SEQ ID No. 3) as well as analogues thereof (GLP-1 analogues)).
• the photochemical amidation reaction may be used to prepare GLP-1 (7-36)-amide.
• the photochemical amidation reaction may be used to prepare a GLP-1 receptor-amylin receptor co-agonists.
• the preparation of GLP-1 receptor-amylin receptor co-agonists disclosed herein includes the preparation of co-agonists comprising an amino acid sequence as set out in SEQ ID No. 16.
• R2 may comprise a sequence corresponding to a precursor of a bioactive peptide product, such as a GLP-1 receptor-amylin receptor co-agonist or amylin receptor agonists.
• the photochemical amidation reaction may be used to prepare a GLP-1 receptor-amylin receptor co-agonist precursor comprising an amino acid sequence as set out in SEQ ID No. 16.
• the photochemical amidation reaction may be used to prepare a GLP-1 receptor-amylin receptor co-agonist precursor consisting of an amino acid sequence as set out in SEQ ID No. 16.
• the photochemical amidation reaction may be used to prepare a GLP-1 receptor-amylin receptor co-agonist precursor consisting of an amino acid sequence as set out in SEQ ID No. 17.
• R2 may be a peptide with an N-terminal extension.
• the extension may be any combination of amino acids.
• the extension may be between 1-60 amino acids.
• the extension may be between 1-50 amino acids.
• the extension may be between 1-40 amino acids.
• the extension may be between 1-20 amino acids.
• the extension may be between 1-15 amino acids.
• the extension may be between 5-15 amino acids.
• the extension may be 14 amino acids.
• the photochemical amidation reaction may be used to prepare a GLP-1 receptor-amylin receptor co-agonist precursor with an N-terminus extension.
• R2 may be a peptide with an amino acid sequence as set out in SEQ ID No. 16 plus any type of N-terminal extension.
• R2 may be a peptide with an N-terminal extension with an amino acid sequence as set out in SEQ ID No. 18.
• R2 may be a peptide with an amino acid sequence as set out in SEQ ID No. 16 plus an N-terminal extension with an amino acid sequence as set out in SEQ ID No. 18.
• R2 may be a peptide with an amino acid sequence as set out in SEQ ID No. 17.
• R2 may comprise an amino acid sequence as set out in any one of any one of SEQ ID Nos. 3-14, 16-17.
• R2 may comprise an amino acid sequence as set out in any one of SEQ ID Nos. 3-14, 16 plus an N- terminal extension.
• R2 may be an amino acid sequence as set out in any one of SEQ ID Nos: 3-14, 16-17.
• R2 may be an amino acid sequence as set out in any one of SEQ ID NOs: 3-14, 16 plus an N-terminal extension.
• the peptide or protein comprising a C-terminal cysteine residue may be reacted with a photolabeling agent (R1-X).
• a compound of formula I may be coupled to a photolabel (R1).
• a photolabeling agent (herein also identified as R1-X) may be defined as a chemical compound that contains a photo-excitable moiety and that is capable of coupling to a peptide.
• the photolabeling agent (R1-X) may be defined as a compound capable of undergoing a photochemical reaction.
• the photolabeling agent (R1-X) comprises a photolabel defined herein as R1 and a leaving group defined herein as X.
• the photolabel (R1) may be coupled to a peptide or protein comprising a C-terminal cysteine (formula I) by reacting the peptide or protein with the photolabeling agent (R1-X) under the conditions described herein.
• the photolabeling agent (R1-X) may be selected from the group consisting of 3-bromo-1 H-pyrrole-2, 5-dione, 4-chloro- 7-nitrobenzofurazan, 2-bromo-1 ,4-naphthoquinone, 1-fluoro-2,4-dinitrobenzene and 4-fluoro- 7-sulfamoylbenzofurazan.
• 3-Bromo-1 H-pyrrole-2, 5-dione may also be referred to as 2-bromomaleimide and may be defined as Chem 1 :
• the photolabeling agent (R1-X) may be 3-bromo-1 H-pyrrole- 2, 5-dione (Chem. 1) or 4-chloro-7-nitrobenzofurazan (Chem. 2).
• the photolabeling agent (R1- X) may be 3-bromo-1 H-pyrrole-2, 5-dione (Chem. 1).
• the photolabeling agent (R1-X) may be 4-chloro-7-nitrobenzofurazan (Chem. 2).
• between 1 and 5 equivalents of the photolabeling agent (R1-X) relative to the peptide (formula I) may be provided. In some embodiments, between 1 and 3 equivalents of the photolabeling agent (R1-X) relative to the peptide (formula I) may be provided. In some embodiments, between 1.5 and 2.5 equivalents of the photolabeling agent (R1-X) relative to the peptide (formula I) may be provided. In some embodiments, between 1.8 and 2.2 equivalents of the photolabeling agent (R1-X) relative to the peptide (formula I) may be provided. In some embodiments, about 2 equivalents of the photolabeling agent (R1-X) relative to the peptide (formula I) may be provided. Reaction conditions of peptide or protein comprising C-terminal cysteine residue with photolabeling agent
• Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer.
• the photochemical amidation reaction may take place in an aqueous reaction buffer, such as an aqueous buffer selected from the group consisting of bis-tris methane (may be referred to as bis-tris), tris, triethanolamine and phosphate.
• Steps (a) of the photochemical amidation reaction may take place in an aqueous buffer at pH 4-9.
• Steps (a) of the photochemical amidation reaction may take place in an aqueous buffer comprising bis-tris methane at pH 4-9.
• Steps (a) of the photochemical amidation reaction may take place in an aqueous buffer at pH 6-8.
• Steps (a) of the photochemical amidation reaction may take place in an aqueous buffer comprising bis-tris methane at pH 6-8. Steps (a) of the photochemical amidation reaction may take place in an aqueous buffer at pH 6-7. Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer comprising bis-tris methane at pH 6-7. Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer at pH 6.3-7. Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer comprising bis-tris methane at about pH 6.3-7.
• Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer at about pH 6.4.
• Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer comprising bis-tris methane at about pH 6.4.
• Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer comprising bis-tris methane at about pH 6.3.
• Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer comprising bis-tris methane at about pH 7.
• Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer comprising about 25mM bis-tris methane at about pH 6.4.
• Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer comprising 25mM bis-tris methane at about pH 6.3.
• Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer with glycine.
• Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer with about 50 mM of glycine.
• step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer comprising about 25 mM bis-tris methane at about pH 6.4 with about 50 mM glycine.
• Step (a) of the photochemical amidation reaction may take place in an aqueous reaction buffer comprising about 25 mM bis-tris methane at about pH 6.3 with about 50 mM glycine.
• step (a) of the photochemical amidation reaction may take place at room temperature, which defined herein as being between 15-25 °C.
• Step (a) of the photochemical amidation reaction may take place at a temperature between 4-80 °C.
• Step (a) of the photochemical amidation reaction may take place at a temperature between 10-60 °C.
• Step (a) of the photochemical amidation reaction may take place at a temperature between 10-40 °C.
• Step (a) of the photochemical amidation reaction may take place at a temperature between 10-30 °C.
• Step (a) of the photochemical amidation reaction may take place at a temperature between 15-30 °C.
• Step (a) of the photochemical amidation reaction may take place at a temperature of about 20 °C.
• a surfactant may be added to the reaction buffer prior to photochemical conversion (described in step (b) below). This surfactant may be used to improve the solubility of the photolabel intermediates. For example, and as will be discussed below regarding the reaction with disulfide containing peptides, the surfactants may provide improved solubility of the 4- chloro-7-nitrobenzofurazan (NBD-CI, Chem. 2) alkylated intermediates when the NBD-CI photolabeling agent is used to globally alkylate all the cysteines within a peptide (R2).
• NBD-CI 4- chloro-7-nitrobenzofurazan
• a surfactant selected from the group consisting of sodium dodecyl sulfate (SDS), sodium desoxycholate (SDC), 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), octyl phenol ethoxylate (TritonTM X-100, ThermoFisher Scientific Cat. No. 28314, 85111 , 85112), polysorbate 20 (Tween-20, ThermoFisher Scientific Cat. No. 28320, 85113, 85115), Tetrapropylammonium hydroxide (TAPH-40) and sodium octanoate may be added to the reaction buffer.
• SDS sodium dodecyl sulfate
• SDC sodium desoxycholate
• CHAPS 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate
• octyl phenol ethoxylate Tri
• sodium dodecyl sulfate may be added to the reaction buffer.
• sodium desoxycholate SDC
• 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate CHAPS
• sodium octanoate may be added to the reaction buffer.
• octyl phenol ethoxylate TritonTM X-100, ThermoFisher Scientific Cat. No. 28314, 85111 , 85112
• polysorbate 20 (Tween-20, ThermoFisher Scientific Cat. No. 28320, 85113, 85115) may be added to the reaction buffer.
• Tetrapropylammonium hydroxide (TAPH-40) may be added to the reaction buffer.
• the surfactant may be added to the reaction buffer in a concentration between 40-80 mM.
• the surfactant may be added to the reaction buffer in a concentration between 50-70 mM.
• the surfactant may be added to the reaction buffer in a concentration between 55-65 mM.
• the surfactant may be added to the reaction buffer in a concentration of about 60 mM. In some embodiments, no surfactant may be added to the reaction buffer.
• the second step (step (b)) of the reaction may be referred to as the photochemical conversion of conjugate (also referred to as formula II) to C-terminal enamide (also referred to as formula III).
• Step (b) may also be referred to as the conversion of the peptide-photolabel conjugate to obtain the C-terminal enamide.
• the peptide-photolabel conjugate may be referred to as a conjugate.
• the photochemical conversion of step (b) may be obtained by subjecting the peptide- photolabel conjugate of formula II to light.
• the photochemical conversion of step (b) may be obtained by irradiating the peptide-photolabel conjugate.
• the light shined on the conjugate of formula II may be absorbed by the photolabel, leading to a light promoted chemical reaction, which may be referred to herein generally as a photochemical conversion, to ultimately form the C-terminal enamide of formula III.
• the photochemical conversion of step (b) may be referred to as a photolytic cleavage by radical decarboxylation of the C-terminal.
• the enamide of formula III may be said to be monosubstituted.
• the effective wavelength for this photochemical conversion may vary based on the photolabel R1 coupled to the C-terminal cysteine residue (formula I) in part a.
• a variety of light sources may be used to generate the light spectrum desired for the photochemical reaction to occur.
• the light sources may be selected from fluorescent lamps, light-emitting diodes, mercury lamps, lasers, etc.
• the light source may be ambient light, such as light provided by the sun.
• 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.
• more than one light source may be used for irradiating the conjugate of formula II to light uniformly.
• the light source may be more than one CLF.
• the light source may be more than one LED.
• the light source may be a series of LEDs uniformly arranged to irradiate the conjugate.
• the light source may be arrays of LEDs, which can be referred to as strips of LEDs when arranged in a row.
• 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 between 300- 700 nm.
• the wavelength of the light provided may be between 365-550 nm.
• the wavelength of the light provided may be between 365-500 nm.
• the wavelength of the light provided may be between 365-450 nm.
• the wavelength of the light provided may be about 365 nm.
• the wavelength of the light provided may be between 400-500 nm.
• the wavelength of light provided may be between 400-470 nm.
• the wavelength of light provided may be between 400- 450 nm.
• the wavelength of light provided may be between 405-430 nm.
• the wavelength provided may be of 365nm.
• the wavelength provided may be of 405nm.
• the wavelength provided may be of 430nm.
• the wavelength provided may be of 450nm.
• the source of light may be LEDs providing light having a wavelength of 365 nm.
• the source of light may be a CFL providing white light.
• the source of light may be LEDs providing light having a wavelength of 405 nm.
• the source of light may be LEDs providing light having a wavelength of 430 nm.
• the source of light may be LEDs providing light having a wavelength of 450 nm.
• the source of light may be an array of monochromatic LEDs providing light having a wavelength of 365 nm.
• the source of light may be an array of monochromatic LEDs providing light having a wavelength of 405 nm.
• the source of light may be an array of monochromatic LEDs providing light having a wavelength of 430 nm.
• the source of light may be an array of monochromatic LEDs providing light having a wavelength of 450 nm.
• 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 and may further restrict the spectral range irradiating the conjugate.
• 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 and may further restrict the spectral range irradiating the conjugate.
• 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 20nm.
• the spectral width of the light may have a FWHM of 10nm.
• the reaction condition for photochemical conversion of the peptide-photolabel conjugate (formula II) to obtain a C-terminal enamide (formula III) may correspond to that provided for the coupling of peptide or protein comprising C-terminal cysteine residue (formula I) with a photolabeling agent (R1-X) to obtain a peptide-photolabel conjugate (formula II) in step (a) above.
• step (b) may take place in the same aqueous reaction buffer for step (a).
• the step (b) may take place at a temperature corresponding to that of step (a).
• the third and last step of the reaction may be referred to as the cleavage of the resulting C-terminal enamide (also referred to as formula III) to obtain the C-terminal a- amide (also referred to as formula IV).
• the cleavage may be referred to as the conversion of the C-terminal enamide.
• the C-terminal enamide may be referred to as enamide.
• the C-terminal a-amide may be referred to as a C-terminal amide.
• the cleavage of the enamide to a C-terminal a-amide may be carried out using different cleavage methods.
• the cleavage may be carried out using acidolysis.
• the cleavage may be carried out using acidolysis with an acid that may be categorized as being a strong acid, where strong acid here is defined as an acid with a pKa below or equal to 4.
• the cleavage reagent may have a pKa below or equal to 4.
• the cleavage reagent may have a pKa below or equal to 3.
• the cleavage may be carried out using acidolysis with a weak acid (ie. an acid with a pKa above 4) with the addition of Lewis acids to accelerate the acidolysis.
• the cleavage may be carried out using inverse-electron demand Diels-Alder (I EDDA).
• the enamide may be cleaved by acid mediated hydrolysis.
• the enamide may be hydrogenated.
• the enamide may be cleaved by acid catalysed, oxidizing agent mediated hydrolysis or by an Inverse Electron Demand Diels-Alder reaction.
• the enamide may be cleaved by acid catalysed or by oxidizing agent mediated hydrolysis.
• the enamide may be cleaved by acid catalysed hydrolysis.
• the enamide may be cleaved by oxidizing agent mediated hydrolysis.
• the enamide may be cleaved by an Inverse Electron Demand Diels-Alder reaction.
• the vinyl amide may be reduced to N-ethyl amide. In some embodiments, the vinyl amide may be used for conjugation, such as a thiol-ene reaction.
• an additive may be added to the step.
• the additives may be selected from, but not limited to, the group consisting of methionine, indole or caffeic acid.
• the different cleavage methods may require different reaction conditions, such as different cleavage reagents and temperature.
• the cleavage reagent is added to the reaction with the intermediate of formula III therein.
• the photochemical amidation reaction takes place at a constant temperature.
• the temperature may be change between the steps (a), (b) and/or (c).
• the conditions of the photochemical reaction at steps (a), (b) and/or (c) may be different to one another.
• the cleavage reagent may be selected from the group consisting of trifluoroacetic acid (TFA), hydrochloric acid (HCI), Sulfuric acid (H2SO4), tosylic acid (TsOH), phosphoric acid (H3PO4), oxalic acid, 3,6-diphenyl-1 ,2,4,5-tetrazine, and 6,6'- (1 ,2,4,5-tetrazine-3,6-diyl)dinicotinic acid.
• TFA trifluoroacetic acid
• HCI hydrochloric acid
• Sulfuric acid H2SO4
• TsOH tosylic acid
• H3PO4 phosphoric acid
• 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 (HCI), Sulfuric acid (H2SO4), tosylic acid (TsOH), phosphoric acid (H3PO4), oxalic acid and 6,6'-(1 ,2,4,5-tetrazine-3,6- diyl)dinicotinic acid.
• TFA trifluoroacetic acid
• HCI hydrochloric acid
• Sulfuric acid H2SO4
• TsOH tosylic acid
• H3PO4 phosphoric acid
• oxalic acid 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 (HCI), Sulfuric acid (H2SO4), 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 (HCI).
• the cleavage reagent may be sulfuric acid (H2SO4).
• the cleavage reagent may be phosphoric acid (H3PO4).
• 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.
• the cleavage reagent may be between 2-8% v/v trifluoroacetic acid (TFA).
• the cleavage reagent may be between 4-6% v/v trifluoroacetic acid (TFA).
• the cleavage reagent may be 5% v/v trifluoroacetic acid (TFA).
• the cleavage reagent may be 0.5-2 M phosphoric acid (H3PO4).
• the cleavage reagent may be 0.5-1.5 M phosphoric acid (H3PO4).
• the cleavage reagent may be 0.7-1.3 M phosphoric acid (H3PO4).
• the cleavage reagent may be 0.8-1.2 M phosphoric acid (H3PO4).
• the cleavage reagent may be about 1 M phosphoric acid (H3PO4).
• the enamide cleavage may be performed at a temperature between 0 and 80 °C.
• the enamide cleavage may be performed at a temperature between 20 and 60 °C.
• the enamide cleavage may be performed at a temperature between 20 and 40 °C.
• the enamide cleavage may be performed at a temperature between 30 and 40 °C.
• the enamide cleavage may be performed at a temperature of 37 °C.
• the enamide cleavage may be performed at a temperature between 20 and 26 °C.
• the enamide cleavage may be performed at a temperature between 20 and 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.
• the reaction conditions may vary depending on the procedure of which the reaction is carried out.
• the photochemical amidation reaction described herein may be performed in a batch procedure.
• the batch procedure may also be referred to as batch scale reaction.
• the photochemical amidation process may be performed in in the same reaction vessel, which may be referred to as a “single pot” reaction.
• steps (a) and (b) of the photochemical amidation reaction described herein may take place at room temperature in a reaction vessel, while the temperature may be adjusted to, for instance about 37 °C, for step (c) while nevertheless taking place in the same reaction vessel.
• steps (a), (b) and/or (c) may be performed in different reaction vessels.
• the reaction conditions may vary depending on the photolabeling agent (R1-X) and the cleavage reagents used, as described above.
• the photochemical amidation reaction may be performed in a photo-flow procedure, wherein the solution is pumped through a circuit while being irradiated by a light source.
• the reaction may be performed in a flow reactor providing these conditions.
• the flow reactor may be referred to as a photo-flow reactor, photochemical reactor or more generally as a reactor.
• the reaction performed in a flow reactor may also be referred to as photo-flow process or reaction. In some embodiments, the reaction performed in a flow reactor may also be referred to as a photo-flow procedure.
• the flow reactor may operate at a temperature between 10 to 100 °C.
• the flow reactor may operate at a temperature between 20 to 60 °C.
• the flow reactor may operate at a temperature between 20 to 40 °C.
• the flow reactor may operate at a temperature between 20 to 30 °C.
• the flow reactor may operate at a temperature between 25 to 30 °C.
• the flow reactor may operate at a temperature of about 26 °C.
• the photochemical amidation reaction may be performed at different flow rates.
• the flow rate here refers to the volumetric flow rate which may be defined as the volume of fluid which passes through the system per unit of time.
• the flow rate may be between 0.200 and 50.00 mL/min.
• the flow rate may be between 1.00 and 25.00 mL/min.
• the flow rate may be 5.00 and 10.00 mL/min.
• the flow rate may be between 6.00 and 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.
• the flow reactor volume may be between 1 to 10 mL.
• the flow reactor volume may be between 2 and 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 here may be regarded as the volume that may be subject to irradiation at any given time by the light source of the flow reactor.
• the reaction may also be carried out using different residence times.
• the residence time here may be defined as a measure of how long a fluid stays in the flow reactor. Written otherwise, it is a measurement of time between the moment the solution enters the flow reactor, forming part of the flow reactor volume and capable of being irradiated by the flow reactor, and the moment the solution exits the flow reactor, no longer forming part of the flow reactor volume and no longer being irradiated by the flow reactor. It may also be given by the ratio of flow reactor volume to the overall flow rate.
• the residence time in the flow reactor may be between 0.04 and 50 min.
• the residence time in the flow reactor may be between 0.1 and 10 min.
• the residence time in the flow reactor may be between 0.1 and 1 min.
• the residence time in the flow reactor may be between 0.1 and 0.5 min.
• the residence time in the flow reactor may be about 0.25 min.
• the flow reactor may be a Vapourtec UV-150 photochemical reactor.
• the flow reactor may be a Corning® Lab Photo Reactor. It will be understood that any equivalent photochemical reactor which permits the irradiation of a circulating solution for the purposes of photochemical conversion as described in step (b) may be used without departing from the present disclosure.
• the flow reactor light source may irradiate at a wavelength between 300nm and 700nm.
• 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 450nm.
• the flow reactor light source may be a series of monochromatic LEDs irradiating at a wavelength of 430nm.
• 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 about 405 nm.
• the flow reactor light source may irradiate at a radiant power between 3 and 150 watts.
• the flow reactor light source may irradiate at a radiant power between 5 and 100 watts.
• the flow reactor light source may irradiate at a radiant power between 10 and 80 watts.
• the reactor light source may irradiate at a radiant power between 9 and 24 watts.
• the photochemical amidation reaction may be performed in a batch - photo-flow hybrid procedure, in which only a portion of the photochemical amidation reaction may be completed via a photo-flow procedure.
• only some of the steps of the reaction occur in a flow reactor, where the remaining steps may occur in a batch procedure, in a reaction vessel for instance.
• the reaction may generally be referred to being completed in a hybrid procedure.
• the photochemical conversion of the peptide-photolabel conjugate to obtain the C-terminal enamide of the reaction may be performed in a flow reactor.
• the coupling of the peptide comprising a C-terminal cysteine residue with a photolabel to obtain a peptide-photolabel conjugate and the photochemical conversion of the peptide-photolabel conjugate to obtain the C-terminal enamide of the reaction may be performed in a flow reactor.
• the photochemical conversion of the peptide-photolabel conjugate to obtain the C-terminal enamide and the cleavage of the C- terminal enamide to obtain the C-terminal a-amide of the reaction may be performed in a flow reactor.
• only the photochemical conversion of the peptide-photolabel conjugate to obtain the C-terminal enamide of the reaction may be performed in a flow reactor.
• the photochemical amidation reaction steps which are not performed in a flow reactor may be performed via batch procedures.
• the photochemical amidation reaction steps which are not performed in a flow reactor may be performed via batch procedures in reaction vessels.
• the photochemical amidation reaction of the present application may take place with peptides R2 that contain cysteines residues therein.
• formula I of Scheme 1 or 2 contains a peptide which comprises at least one cysteine (Cys) residue in addition to the C- terminal cysteine (Cys) residue for the photochemical amidation reaction.
• the cysteines within the peptide R2 may form disulfide bonds.
• the photochemical amidation process may proceed as generally described above while further being subject to the below.
• the photolabeling agent (R1-X) may couple to the C-terminal cysteine residue, as described in step (a) above, as well as to the additional cysteine residues present in the peptide R2. This may be referred to as global coupling of the cysteines.
• the cysteines forming part of the peptide R2 having been coupled with the photolabel R1 may generally be referred to as photolabel-protected cysteines.
• the photolabeling agent may be added in a manner proportional to the quantity of cysteines to be coupled with the photolabeling agent.
• the equivalents of photolabeling agent may increase proportional to the number of disulfide bonds to be formed within the amidated peptide.
• the photolabeling agent (R1-X) relative to the peptide (formula I) may be provided for each disulfide bond to be formed in the peptide (R2).
• Between 1 and 2.5 additional equivalents of the photolabeling agent (R1-X) relative to the peptide (formula I) may be provided for each disulfide bond to be formed in the peptide (R2).
• Between 1.5 and 2.5 additional equivalents of the photolabeling agent (R1- X) relative to the peptide (formula I) may be provided for each disulfide bond to be formed in the peptide (R2).
• about 2 additional equivalents of the photolabeling agent (R1-X) relative to the peptide (formula I) may be provided for each disulfide bond to be formed in the peptide (R2).
• additional here refers to photolabeling agent equivalents which are to be summed to the photolabeling agent equivalents one would provide in the general reaction at step (a) should the peptide R2 have no disulfide bonds to be formed.
• between 3 and 5 total equivalents of the photolabeling agent (R1-X) relative to the peptide (formula I) may be provided for step a. Between 3.5 and 4.5 total equivalents of the photolabeling agent (R1-X) relative to the peptide (formula I) may be provided for step a. Between 3.8 and 4.2 total equivalents of the photolabeling agent (R1-X) relative to the peptide (formula I) may be provided for step a. In some embodiments, about 4 total equivalents of the 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.
• NBD-CI 4-chloro-7-nitrobenzofurazan
• the peptide-photolabel conjugate may be irradiated in the manner generally disclosed in step b.
• the photolabel found at the C-terminal of the conjugate may go through the photochemical conversion such as to obtain the C-terminal enamide, while the remaining cysteine coupled photolabels may remain unaffected.
• the photochemical conversion step (b) disclosed herein may be said to be selective to the C-terminal of the peptide-photolabel conjugate.
• the photochemical conversion step (b) disclosed herein may be said to be selective to the C-terminal cysteine of the peptide coupled with the photolabel.
• the cleavage of the C-terminal enamide to obtain the C-terminal a-amide may be carried out as described in step (c).
• the release of the photolabel in the photolabel-protected cysteines of the peptide R2 may be carried out.
• the release of the photolabel-protected cysteines in the peptide R2 may be carried out before the cleavage of the C-terminal enamide to obtain the C-terminal a-amide of step (c).
• the release of the photolabel-protected cysteines in the peptide R2 may be carried out simultaneously to the cleavage of the C-terminal enamide to obtain the C- terminal a-amide of step (c).
• a nucleophilic sulfide may be provided, leading to the release of the photolabels from photolabel-protected peptide cysteines.
• a nucleophilic sulfide selected from the group consisting of cysteamine, cysteine, dithiothreitol (DTT), 3,6-dioxa-1 ,8- octanedithiol (DODT), 2-mercaptoethanol, glutathione (GSH) and acetylcysteine may be provided.
• cysteamine may be provided as a nucleophilic sulfide.
• an oxidation partner may further be provided, leading to the disulfide oxidation.
• An oxidation partner selected from the group consisting of cystamine glutathione disulfide (GSSG) and cystine may be provided.
• cystamine glutathione disulfide (GSSG) may be provided as an oxidation partner.
• R1-X is a photolabeling agent, wherein R1 is a photolabel and X is a leaving group,
• R3 is selected from the group consisting of hydrogen, methyl and ethyl, comprising the steps of
• Step (a) coupling a peptide or protein comprising a C-terminal cysteine of formula I with a photolabel (R1) to obtain a peptide-photolabel conjugate of formula II, and
• Step (b) photochemical conversion of the resulting peptide-photolabel conjugate of formula II to obtain a C-terminal enamide of formula III, and
• Step (c) cleavage of the resulting C-terminal enamide of formula III to obtain the C- terminal a-amide of formula IV, may be provided.
• R3 may be selected from the group consisting of hydrogen and ethyl. In another embodiment, R3 may be hydrogen.
• R3 is hydrogen, comprising the steps of
• Step (a) coupling a peptide or protein comprising a C-terminal cysteine of formula I with 4-chloro-7-nitrobenzofurazan (Chem. 2) to obtain a peptide-photolabel conjugate of formula ll-a, and
• Step (b) photochemical conversion of the resulting peptide-photolabel conjugate of formula ll-a to obtain a C-terminal enamide of formula III, and
• Step (c) cleavage of the resulting C-terminal enamide of formula III to obtain the C- terminal a-amide of formula IV, may be provided.
• Step (a) coupling a peptide or protein comprising a C-terminal cysteine amidation tag (formula I) with 4-chloro-7-nitrobenzofurazan to obtain a peptide-photolabel conjugate (formula I l-a);
• Step (b) irradiating the peptide-photolabel conjugate (formula I l-a) with light having a wavelength between 400-450nm to obtain a C-terminal enamide (formula III) via photochemical conversion;
• Step (c) cleaving the C-terminal enamide (formula III) to obtain the C-terminal a-amide (formula IV).
• the purification of the C-terminal a-amide may be performed in a batch procedure.
• the purification may be performed by continuous precipitation.
• the reaction may be monitored by Liquid Chromatography Mass Spectrometry (LC- MS).
• the purification of the C-terminal a-amide may be performed via High Performance Liquid Chromatography (HPLC) or Ultra Filtration Diafiltration (UFDF).
• HPLC High Performance Liquid Chromatography
• UFDF Ultra Filtration Diafiltration
• HPLC High Performance Liquid Chromatography
• UFDF Ultra Filtration Diafiltration
• Reaction samples may be analysed by UPLC-MS analysis.
• the reaction samples may be analysed using extracted ion chromatography.
• the reaction may be monitored by LC-MS.
• R1-X is a photolabeling agent, wherein R1 is a photolabel and X is a leaving group,
• R3 is selected from the group consisting of hydrogen, methyl and ethyl, comprising the steps of
• Step (a) coupling a peptide or protein comprising a C-terminal cysteine amidation tag of formula I with the photolabel (R1) to obtain a peptide-photolabel conjugate of formula II;
• Step (b) irradiating the peptide-photolabel conjugate of formula II to obtain a C- terminal enamide of formula III via a photochemical conversion
• Step (c) cleaving the C-terminal enamide of formula III to obtain the C-terminal a- amide of formula IV.
• R3 is selected from the group consisting of hydrogen and ethyl. 3. The method according to any of the preceding embodiments, wherein R3 is hydrogen.
• R2 comprises an amino acid sequence as set out in any one of SEQ. ID NO.: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17.
• R2 consists of an amino acid sequence as set out in any one of SEQ. ID NO.: 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 16, 17.
• R2 is an amino acid sequence as set out in any one of SEQ. ID NO. 3-14 and 16-17.
• R2 is an amino acid sequence as set out in any one of SEQ. ID NO.: 3, 4, 5, 16, 17.
• R2 comprises an amino acid sequence as set out in SEQ ID No. 16.
• R2 further comprises a N-terminal extension.
• R2 further comprises a N-terminal extension having between 1-60 amino acids.
• R2 further comprises a N-terminal extension having between 1-50 amino acids.
• R2 further comprises a N-terminal extension having between 1-40 amino acids.
• R2 further comprises a N-terminal extension having between 1-20 amino acids.
• R2 further comprises a N-terminal extension having between 1-15 amino acids.
• R2 further comprises a N-terminal extension having between 5-15 amino acids.
• R2 further comprises a N-terminal extension having between 14 amino acids.
• R2 further comprises a N-terminal extension according to SEQ ID No. 18.
• R2 is an amino acid sequence as set out in SEQ ID No. 17.
• glucagon-like peptide-1 GLP-1
• the method is used in the preparation of an amylin receptor agonist.
• the amylin receptor agonist is Pramlintide.
• the method is used in the preparation of a glucose-dependent insulinotropic polypeptide (GIP) peptide.
• GIP glucose-dependent insulinotropic polypeptide
• the method is used in the preparation of a GIP and GLP-1 receptor co-agonist.
• glucagon-like peptide-1 GLP-1
• amylin receptor agonist such as Pramlintide
• GIP glucose-dependent insulinotropic polypeptide
• GLP- 1 receptor co-agonist such as Tirzepatide, urocortin-2 (UCN2) peptide, pancreatic pYY(3-36) peptide, pancreastatin inhibitor peptide-8 (PSTi8) peptide
• LHRH urocortin-2
• GLP pancreatic pYY(3-36) peptide
• pancreastatin inhibitor peptide-8 PSTi8) peptide
• luteinizing hormone-releasing hormone (LHRH) agonist gastrin releasing peptide (GRP) peptide
• adrenocorticotropic hormones ACTH
• GLP- 1 receptor-neuropeptide Y receptor 2 co-agonist such as EP45
• entry inhibitors such as Bulevirtide, a natriuretic peptid
• the photolabeling agent (R1-X) is selected from the group consisting of 3-Bromo-1 H-pyrrole-2, 5-dione, 4-chloro-7-nitrobenzofurazan, 2-bromo-1 ,4-naphthoquinone, 1-fluoro-2,4- dinitrobenzene and 4-fluoro-7-sulfamoylbenzofurazan.
• Chem. 1 The method according to any of the preceding embodiments, wherein the photolabeling agent is 4-chloro-7-nitrobenzofurazan (Chem. 2):
• Chem. 2 The method according to any of the preceding embodiments, wherein the method takes place in an aqueous reaction buffer.
• step (a) of the method takes place in an aqueous reaction buffer.
• step (a) of the method takes place in an aqueous reaction buffer, the aqueous buffer selected from the group consisting of bis-tris methane, tris, triethanolamine and phosphate.
• step (a) of the method takes place in an aqueous reaction buffer of bis-tris methane.
• step (a) of the method takes place in an aqueous reaction buffer of about 25mM bis-tris methane.
• step (a) of the method takes place in an aqueous reaction buffer of tris.
• step (a) of the method takes place in an aqueous reaction buffer of triethanolamine.
• step (a) of the method takes place in an aqueous reaction buffer of phosphate.
• step (a) of the method takes place in an aqueous reaction buffer at pH 4-9.
• step (a) of the method takes place in an aqueous reaction buffer at pH 6-8.
• step (a) of the method takes place in an aqueous reaction buffer at pH 6-7.
• step (a) of the method takes place in an aqueous reaction buffer at pH 6.3-7.
• step (a) of the method takes place in an aqueous reaction buffer at pH 6.3-6.4.
• step (a) of the method takes place in an aqueous reaction buffer at about pH 6.4.
• step (a) of the method takes place in an aqueous reaction buffer at about pH 6.3.
• step (a) of the method takes place in an aqueous reaction buffer at about pH 7.
• step (a) of the method takes place in an aqueous reaction buffer with glycine.
• step (a) of the steps of the method take place in an aqueous reaction buffer with about 50 mM of glycine.
• step (a) of the steps of the method take place in an aqueous reaction buffer with about 50 mM of glycine.
• a source of light for irradiating the peptide-photolabel conjugate is a compact fluorescent lamp (CFL).
• the source of light for irradiating the peptide-photolabel conjugate is a at least one light emitting diode (LED).
• the source of light for irradiating the peptide-photolabel conjugate is a plurality of light emitting diodes (LEDs).
• cleavage reagent for cleaving the enamide
• the cleavage reagent is selected from the group consisting of trifluoroacetic acid (TFA), hydrochloric acid (HCI), Sulfuric acid (H2SO4), tosylic acid (TsOH), phosphoric acid (H3PO4), oxalic acid, 6,6'-(1 ,2 ,4, 5-tetrazine-3,6-diyl)dinicotinic acid.
• cleavage reagent is selected from the group consisting of trifluoroacetic acid (TFA), hydrochloric acid (HCI), Sulfuric Acid (H2SO4), tosylic acid (TsOH), phosphoric acid (H3PO4), oxalic acid and 6,6'-(1 ,2,4,5-tetrazine-3,6-diyl)dinicotinic acid.
• TFA trifluoroacetic acid
• HCI hydrochloric acid
• H2SO4 Sulfuric Acid
• TsOH tosylic acid
• H3PO4 phosphoric acid
• oxalic acid 6,6'-(1 ,2,4,5-tetrazine-3,6-diyl)dinicotinic acid.
• cleavage reagent is selected from the group consisting of trifluoroacetic acid (TFA), hydrochloric acid (HCI), Sulfuric Acid (H2SO4), tosylic acid (TsOH), phosphoric acid (H3PO4) and 6,6'-(1 ,2 ,4, 5-tetrazine-3,6-diyl)dinicotinic acid.
• cleavage reagent is 2-8% v/v trifluoroacetic acid (TFA).
• cleavage reagent is 4-6% v/v trifluoroacetic acid (TFA).
• cleavage reagent is hydrochloric acid (HCI).
• cleavage reagent is phosphoric acid (H3PO4).
• cleavage reagent is 0.5-1.5 M phosphoric acid (H3PO4).
• cleavage reagent is 0.7-1.3 M phosphoric acid (H3PO4).
• cleavage reagent is about 1 M phosphoric acid (H3PO4).
• cleavage reagent is 6,6'-(1 ,2,4,5-tetrazine-3,6-diyl)dinicotinic acid.
• the method according to any one of embodiments 124-134 wherein the flow rate of the flow reactor is 6.00 to 9.00 mL/min. .
• the method according to any one of embodiments 124-136 wherein a reactor volume of the flow reactor is 1 to 10 mL. .
• the method according to any one of embodiments 124-138 wherein the reactor volume of the flow reactor is 1 to 3 mL. .
• a surfactant selected from the group consisting of sodium dodecyl sulfate (SDS), sodium desoxycholate (SDC), 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate (CHAPS), TritonTM X-100, Tween-20, Tetrapropylammonium hydroxide (TAPH-40) and sodium octanoate.
• SDS sodium dodecyl sulfate
• SDC sodium desoxycholate
• CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate
• TritonTM X-100 TritonTM X-100
• Tween-20 Tetrapropylammonium hydroxide
• TAPH-40 Tetrapropylammonium hydroxide
• nucleophilic sulfide for releasing the photolabel-protected cysteine from the 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.
• DTT dithiothreitol
• DODT 3,6-dioxa-1 ,8-octanedithiol
• GSH glutathione
• acetylcysteine acetylcysteine
• R3 is hydrogen, comprising the steps of
• Step (a) coupling a peptide or protein comprising a C-terminal cysteine amidation tag of formula I with 4-chloro-7-nitrobenzofurazan to obtain a peptide- photolabel conjugate of formula ll-a;
• Step (b) irradiating the peptide-photolabel conjugate of formula ll-a to obtain a C- terminal enamide of formula III via photochemical conversion;
• Step (c) cleaving the C-terminal enamide of formula III to obtain the C-terminal a- amide of formula IV.
• R3 is hydrogen, comprising the steps of Step (a), coupling a peptide or protein comprising a C-terminal cysteine amidation tag of formula I with 4-chloro-7-nitrobenzofurazan to obtain a peptide- photolabel conjugate of formula ll-a;
• Step (b) irradiating the peptide-photolabel conjugate of formula ll-a with light having a wavelength between 400-450nm to obtain a C-terminal enamide of formula III via photochemical conversion;
• Step (c) cleaving the C-terminal enamide of formula III to obtain the C-terminal a- amide of formula IV.
• a method of producing a pharmaceutical composition comprising the steps of a. producing a compound of formula IV, according to any of the embodiments 1- 181 , b. preparing said pharmaceutical composition using the compound of formula IV obtained in a.
• Peptide cleavage was conducted in a 90/2.5/2.5/2.5/2.5 solution of TFA/triisopropylsilane (TIPS)/water/thioanisole/3,6-dioxa-1 ,8-octanedithiol (DODT).
• TIPS triisopropylsilane
• DODT dioanisole/3,6-dioxa-1 ,8-octanedithiol
• the eluted cleavage solution was precipitated in ice cold diethyl ether.
• the peptide precipitate was pelleted using centrifugation and the pellet washed with additional ice-cold ether.
• the washed peptide pellet was re-dissolved in either 1 :1 acetonitrile/water or neat DMSO, then filtered via a 0.2 pm filter.
• the resulting solution was loaded onto a preparative RP-HPLC system, either a Waters Prep 150 LC system or an Agilent 1290 Infinity II Autoscale. Preparative LC/MSD system.
• Step (a) Coupling of peptide or protein comprising C-terminal cysteine residue with photolabel
• aqueous reaction buffer containing 25 mM bis-tris methane pH 6.4 with 50 mM glycine was degassed by bubbling nitrogen gas for 15 minutes.
• the C-terminal Cys modified peptide was dissolved in reaction buffer to generate a 1 mM stock solution.
• 25 pL of 1 mM stock was added to a well of a 96-well V-bottom assay plate, along with 65 pL of additional assay buffer.
• the plate was irradiated according to the photo aryl group utilized to generate the C-terminal enamide.
• 2-bromomaleimide a 1.5 meter strip of 365 nm LEDs served as the irradiation source, and the conversion proceeded for 4 hours. A cooling fan was used to maintain the reaction mixture at room temperature.
• NBD-chloride irradiation can be conducted with a handheld white CFL lamp or a strip of 450 nm LEDs. The conversion proceeded for 1 hour at room temperature. If desired, conversion can be monitored by LC- MS.
• Step (c) - Cleavage of the resulting C-terminal enamide to obtain the C-terminal a-amide:
• Acidolysis of the enamide proceeded using a range strong acids (Table 2).
• the acid was added to the reaction mixture from a 10X aqueous stock solution. Trifluoroacetic acid was used for the majority of testing conditions.
• TFA 10 pL of a 50/50 solution of TFA/water was added to the reaction well (5% final TFA concentration) and the plate was shaken at room temperature for up to 24 hours.
• additives such as methionine, indole or caffeic acid were utilized, the additive reagent was spiked from a 50X or 100X stock solution into the reaction prior to addition of the acid. Conversion was monitored by LC-MS.
• Solubility of the dipyridyl- tetrazine was poor in acetonitrile alone and required addition of acid to protonate pyridyl groups and improve solubility.
• 10 pL of this stock solution was added to the reaction well (1.25 mM final tetrazine cone., 5 equiv. based on peptide cone.), and the reaction was incubated at 37 °C for 24 hours. Conversion was monitored by LC-MS.
• a stock solution of (3-Bromo-1 H-pyrrole-2, 5-dione, CAS: 98026-79-0) or NBD- chloride (4-chloro-7-nitrobenzofurazan, CAS: 10199-89-0) was prepared in 1 :1 H 2 O:MeCN, and 2 equivalents relative to peptide are added to the reaction vessel for cysteine coupling. The reaction was allowed to stir for 1 hour or until the reaction was complete by LC/MS. The reaction vessel was then irradiated with 365 nm LEDs, a handheld white CFL work light, or a strip of 450 nm blue LEDs over 0.25-4 h. A cooling fan was used to maintain the reaction mixture at room temperature.
• NBD-chloride (4-chloro-7-nitrobenzofurazan, CAS: 10199-89-0) was prepared in 1 :1 H 2 O:MeCN, and 4 equivalents relative to peptide were added to the reaction vessel for global cysteine coupling.
• the reaction was allowed to stir for 2 hour or until the reaction was complete by LC/MS.
• the reaction vessel was then irradiated with a handheld white CFL work light, or a strip of 450 nm blue LEDs over 0.25-4 h. A cooling fan was used to maintain the reaction mixture at room temperature.
• a solution of nucleophilic sulfide e.g.
• cysteamine (8 equiv) and cystamine (1.2 equiv) in H2O is added and allowed to stir at ambient temperature for 2 h to liberate the backbone cysteine thiols and oxidize to the disulfide.
• Trifluoroacetic acid (TFA) was added (5% final TFA concentration) and the reaction was allowed to stir at ambient temperature for 12-24 h.
• dipyridyl-tetrazine 3,6-Di-2-pyridyl-1 ,2,4,5-tetrazine, CAS: 1671-87-0
• CAS 1671-87-0
• 5 equivalents of dipyridyl-tetrazine may be added from a stock solution to give a final concentration of 1.25 mM tetrazine (2.5 mM HCI, dissolved in 80:20 MeCN:H 2 O), and the reaction can be incubated at 37°C for 24 hours. Conversion to C- terminal a-amide was monitored by LC-MS.
• step (c) Following the general protocol for screening scale a-amidation, a broad range of acids and tetrazines were evaluated for the cleavage of the enamide to the corresponding C-terminal a-amide (step (c)). The results are presented in Table 2.
• 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 I EDDA mediated cleavage of the enamide.
• the general reaction conditions and results are presented in Tables 3 and 4 below.
• the starting material sequence of Entry 20 varies from the sequence of generic peptide and is displayed in the sequence list as SEQ. ID NO 3, and the sequence of the product of this reaction is SEQ. ID NO 4.
• the scope of the C-terminal a-amidation reaction was surveyed with respect to the penultimate amino acid position of a generic peptide. The reaction proceeds in good to excellent yields for any amino acid in the penultimate position, demonstrating a broad tolerance for side chain functionality at this position. The reaction proceeds in high efficiency when either 2-bromomaleimide or NBD-CI are utilized in the photolabeling step. High yields of the C- terminal a-amide are observed when the enamide is cleaved by acidolysis or by I EADDA reaction, allowing for choice between two complementary reactions that can be performed under acidic or neutral conditions, respectively.
• the present invention was used for the preparation of a peptide with a C-terminal a-amide which contained a disulfide bond.
• the protocol for batch scale reaction of peptides containing a disulfide bond identified above was used for this experiment following the reaction generally shown in Scheme 1 , with the exception that no surfactant was used.
• a nucleophilic sulfide of cysteamine (8 equiv.) and an oxidizing partner of cystamine (1 .2 equiv.) in H2O was used.
• the cysteine rich backbone was globally alkylated with photolabel in the first step. Selective photolysis of the C-terminal alkylated cysteine occured upon exposure to light.
• the present invention was used to synthesize biologically relevant peptides and marketed peptide therapeutics.
• the protocol outlined for screening scale a-amidation was used for these experiments, and the reaction generally followed Scheme 1 and conditions C or D as outlined in Table 3. The results are presented in Table 6.
• the C-terminal cysteine extended precursors of biologically active peptides and marketed peptide therapeutics were prepared and subjected to the C-terminal a-amidation process. Good yields of the corresponding biologically relevant C-terminal a-amides were obtained in all examples, highlighting the mild reaction conditions and broad scope of the invention.
• the C-terminal a-amidation process performed well regardless of the size and composition of the peptide, demonstrating the applicability of the chemistry to any peptide or protein.
• the present invention was applied on a larger scale using a hybrid batch - photoflow procedure described in detail below.
• the present example was done on a peptide R2 with an amino acid sequence as set out in SEQ ID No. 17, which comprises an amino acid sequence as set out in SEQ ID No. 16 plus an N-terminal extension having an amino acid sequence as set out in SEQ ID No. 18.
• the resulting solution was subjected to irradiation at 405 nm (blue LEDs, input power of 61 W) in a CorningTM Lab Photo Reactor (2.7 mL fluidic module volume, 10 mL/min flow rate, 20°C reactor heat exchange temperature), and the reactor was flushed with an additional 50 ml water.
• HPLC analysis indicated a consumption of at least 80% of the starting material.
• This example shows a larger scale production of a peptide with C-terminal amide where the respective peptide amide was produced in high yield using the photochemical C- terminal a-amidation method. Additionally, this example shows that the photochemical method can be completed in a hybrid setup, where only the photochemical conversion step (step (b) of scheme 1 or 2) was performed in a flow set-up. The example in general highlights the robustness and scalability of the method.

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