PROCESS FOR CONTROLLED STEPWISE SYNTHESIS OF POLYPEPTIDES

• US 3,846,399 A
• Filed: 03/01/1971
• Issued: 11/05/1974
• Est. Priority Date: 04/10/1969
• Status: Expired due to Term

• Current Assignee: Ralph F Hirschmann, Robert G. Denkewalter
• Original Assignee: Ralph F Hirschmann, Robert G. Denkewalter
• Inventors: Hirschmann, Ralph F., Denkewalter, Robert G.
• Primary Examiner(s): Roberts, Elbert L.
• Application Number:
  US05/120,063
• Time in Patent Office: 1,345 Days
• Field of Search: 260/112.5
• US Class Current: 530/342
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 1/02 in solution C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C07K 1/08 using activating agents C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C07K 14/695 Corticotropin [ACTH]
• CPC Class Codes: C07K 5/0812 and aromatic or cycloaliphatic
• CPC Class Codes: C07K 5/101 the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
• CPC Class Codes: C07K 5/1013 the side chain containing O or S as heteroatoms, e.g. Cys, Ser
• CPC Class Codes: C07K 5/1019 with the first amino acid being basic
• CPC Class Codes: C07K 7/18 Kallidins; Bradykinins; Related peptides
• CPC Class Codes: Y10S 930/26 Containing cys-cys disulfide bridge between nonadjacent cysteine residues

Abstract

The invention disclosed herein relates to a novel process for the controlled, stepwise synthesis of polypeptides and proteins. More particularly, it is concerned with a process for the rapid and efficient preparation of polypeptides in which multiple sequential steps are carried out without isolation of intermediate peptides. This process for the controlled stepwise synthesis of polypeptides involves reacting a starting amino acid or peptide (or derivative thereof) with an N-carboxy amino acid anhydride (or derivative thereof) in an aqueous medium under conditions of controlled pH such that the only amino group present in appreciable concentration in reactive form during the course of the reaction is the amino group in the starting amino acid or peptide.

Claims

1. THE PROCESS FOR THE CONTROLLED STEPWISE SYNTHESIS OF PEPTIDES AND PROTECTED DERIVATIVES THERE OF WHICH COMPRISES REACTING A STARTING AMINO COMPOUND SELECTED FROM THE GROUP CONSISTING OF AMINO ACIDS, PEPTIDES, AND DERIVATIVES THEREOF WHEREIN ADDITIONAL FUNCTIONA;

NCA REAGENT SELECTED FROM THE GROUP CONSISTING OF N-CARBOXY AMINO ACID ANHYDRIDES, AND DERIVATIVES THEREOF WHEREIN ADDITIONAL FUNCTIONAL GROUPS ARE PROTECTED SAID REACTION BEING CONDUCTED BY BRINGING TOGETHER SAID AMINO COMPOUND AND SAID NCA REAGENT IN AN AQUEOUS MEDIUM WHILE MAINTAINING THE PH AT THE PROTECTING PH (WHEREBY THE ONLY AMINO GROUP PRESENT IN APPRECIABLE CONCENTRATION IN REACTIVE FORM DURING THE COURSE OF THE REACTION IS THE AMINO GROUP OF THE STARTING AMINO COMPOUND WHICH IS TO PARTICIPATE IN THE REACTION WITH THE SAID NCA REAGENT) THEREBY FORMING THE CORRESPONDING N-CARBOXY PEPTIDE AND, DECARBOXYLATING BY ACIDIFICATION, STANDING, HEATING OR FREEZE-DRYING SAID N-CARBOXY PEPTIDE.

2. The process, as defined in claim 1, for the controlled stepwise synthesis of peptides and protected derivatives thereof having four amide linkages which comprises reacting a starting material selected from the group consisting of tetrapeptides, and derivatives thereof wherein additional functional groups are protected, with an NCA reagent selected from the group consisting of N-carboxy amino acid anhydrides, and derivatives thereof wherein additional functional groups are protected, said reaction being conducted by bringing together said starting material and said NCA reagent in an aqueous medium under conditions of controlled pH (such that the only amino group present in appreciable concentration in reactive form during the course of the reaction is the amino group of the starting material which is to participate in the reaction with the said NCA reagent) thereby forming the corresponding N-carboxy peptide, and decarboxylating by acidification, standing, heating or freeze-drying said N-carboxy peptide.

3. The process as defined in claim 1 wherein the synthesized peptide or protected derivative thereof contains an aspartic acid or glutamic acid segment, and the NCA reagent is N-carboxy aspartic acid anhydride or N-carboxy glutamic acid anhydride.

4. The process as defined in claim 1 wherein the reaction is conducted at a protecting pH of from about 4 to about 11.

5. The process for the controlled stepwise synthesis of peptides and protected derivatives thereof which comprises adding an NCA reagent selected from the group consisting of N-carboxy amino acid anhydrides, and derivatives thereof wherein additional functional groups are protected, to an aqueous medium containing a pH controlling buffer and a starting amino compound selected from the group consisting of amino acids, peptides, anD derivatives thereof wherein additional functional groups are protected, under conditions of intimate mixing at a protecting pH of from 4 to 11 (such that the only amino group present in appreciable concentration in reactive form during the course of the reaction is the amino group of the starting amino compound which is to participate in the reaction with the said NCA reagent) thereby forming the corresponding N-carboxy peptide, and reacting resulting peptide (following decarboxylation by acidification, standing, heating or freeze-drying) with NCA reagent as defined hereinabove under reaction conditions as aforesaid.

6. The process as defined in claim 5 wherein the pH controlling buffer is a borate buffer, and wherein the protecting pH, designed for stabilization of the carbamate protecting group, is from about 8 to about 10.5.

7. The process as defined in claim 5 for the controlled stepwise synthesis of peptides and protected derivatives thereof which comprises adding an NCA reagent selected from the group consisting of N-carboxy amino acid anhydrides, and derivatives thereof wherein additional functional groups are protected, to an aqueous medium containing a starting amino compound selected from the group consisting of amino acids, peptides, and derivatives thereof wherein additional functional groups are protected, under conditions of intimate mixing at a protecting pH of from 4 to 6 (such that, using protonation to protect the amino group, the only amino group present in appreciable concentration in reactive form during the course of the reaction is the amino group of the starting amino compound which is to participate in the reaction with the said NCA reagent) thereby forming the corresponding N-carboxy peptide, and reacting resulting peptide (following decarboxylation by acidification, standing, heating or freeze-drying) with NCA reagent as defined hereinabove under reaction conditions as aforesaid.

8. The process as defined in claim 5 for the controlled stepwise synthesis of peptides and protected derivatives thereof which comprises adding an NCA reagent selected from the group consisting of N-carboxy amino acid anhydrides, and derivatives thereof wherein additional functional groups are protected, to an aqueous medium containing a starting amino compound selected from the group consisting of amino acids, peptides, and derivatives thereof wherein additional functional groups are protected, under conditions of intimate mixing while concurrently adding sufficient alkaline reagent to said medium to maintain a protecting pH (so that the only amino group present in appreciable concentration in reactive form during the course of the reaction is the amino group of the starting amino compound which is to participate in the reaction with the said NCA reagent) thereby forming the corresponding N-carboxy peptide, and reacting resulting peptide (following decarboxylation by acidification, standing, heating or freeze-drying) with NCA reagent as defined hereinabove under reaction conditions as aforesaid.

9. The process as defined in claim 1 wherein the reaction is conducted by adding a mixture of said NCA reagent together with said starting amino compound to said aqueous medium under conditions of intimate mixing and at the protecting pH.