Synthetic Process for Production of Modified GCC Receptor Agonists
AssigneeIronwood Pharmaceuticals, Inc.
InventorsChristopher LEITHEISER, Gunnar STAERKAER, Ulf ANNBY
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Abstract
The present invention relates to methods of producing a synthetic peptide or pharmaceutically acceptable salts thereof of SEQ ID NO: 1.
Claims33
A method of producing a synthetic peptide, or a pharmaceutically acceptable salt thereof, the method comprising:
- (i) chemically synthesizing a linear peptide having its C-terminal bound to a solid phase support and a protected amine group at its N-terminal using a plurality of amino acids and at least one polyamino acid synthon, the linear peptide having protecting groups in one or more amino acids and/or the at least one polyamino acid synthon;
- wherein the synthon has at least one amine group acetylated and at least one carboxylic acid protecting group;
- (ii) removing the carboxylic acid protecting group of the synthon and the protecting group from the amine group at the N-terminal of the linear peptide to form a partially unprotected solid phase support-bound peptide having an unprotected amine group and an unprotected carboxylic acid group;
- (iii) coupling the unprotected amine group and the unprotected carboxylic acid group to form a cyclized solid phase support-bound peptide;
- (iv) cleaving the cyclized solid phase support-bound peptide from the solid phase support to generate a cyclized protected peptide;
- (v) globally deprotecting the cyclized protected peptide to obtain a globally deprotected peptide;
- (vi) folding the globally deprotected peptide to form one or more additional crosslinks to obtain the synthetic peptide;
- and
- (vii) purifying the synthetic peptide;
- wherein the synthetic peptide comprises the amino acid sequence:
- Ac-Cys1 Cth2 Glu3 Leu4 Cys5 Cys6 Asn7 Val8 Ala9 Cys10 Tyr11 Gly12 Cys13 (SEQ ID NO:
- 1);
- wherein the synthetic peptide contains a covalent bond between the following amino acid residues:
- a) Cys1 and Cys6,
- b) Cth2 and Cys10, and
- c) Cys5 and Cys13.
The method of claim 5, wherein the polyamino acid synthon is a compound of the formula:
- wherein:
- P2 is an amine protecting group;
- P3 is a carboxylic acid protecting group;
- and
- P4 is a thiol protecting group.
The method of claim 14 or 15, wherein the carbodiimide is selected from the group consisting of diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC) and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
The method of 15, wherein the carbodiimide is DIC.
The method of any one of claims 15-18, wherein the carbodiimide-mediated reaction further comprises an antioxidant.
The method of any one of the preceding claims, wherein the cyclization coupling reaction is mediated by a non-carbodiimide coupling agent.
A method of preparing a synthetic peptide of Formula I:
- comprising:
- (i) coupling a C-terminal resin bound Tyr-Gly-Cys peptide having protected amino acid side chains to a polyamino acid synthon of Formula II:
- wherein:
- P2 is an amine protecting group;
- P3 is a carboxylic acid protecting group;
- and
- P4 is a thiol protecting group;
- to form a resin bound peptide of Formula III:
- (ii) removing the P2 protecting group of Formula III to obtain a resin bound peptide of Formula IV having an unprotected amine group:
- (iii) coupling a P2-alanine to the resin bound peptide of Formula IV via the free amine group of Formula IV to form a resin bound peptide of Formula V:
- (iv) removing the P2 protecting group of Formula V to obtain a free amine group, followed by coupling the free amine group to P2-amino acid,
- wherein the side chain of the P2 amino acid may be protected;
- (v) repeating step (iv) for five more times to form a resin bound peptide of Formula VI:
- wherein at least one amino acid side chain is protected;
- (vi) removing the P2 protecting group and the P3 protecting group to obtain a free amine group and a free carboxylic acid group;
- (vii) coupling the free amine group and the free carboxylic acid group, to obtain a cyclized peptide of Formula VII:
- (viii) cleaving the peptide of Formula VII from the resin to obtain a cyclized peptide;
- (ix) globally deprotecting the cyclized peptide to obtain a globally deprotected peptide;
- and
- (x) folding the globally deprotected peptide by forming two disulfide bonds to obtain the synthetic peptide of Formula I.
Dependencies are inferred from the claim text.