Method of acylating peptides and novel acylating agents

• US 6,451,974 B1
• Filed: 03/13/2000
• Issued: 09/17/2002
• Est. Priority Date: 03/17/1999
• Status: Expired due to Term

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Novo Nordisk A/S
• Inventors: Hansen, Louis Brammer
• Primary Examiner(s): Low, Christopher S. F.
• Assistant Examiner(s): Mohamed, Abdel A.
• Application Number:
  US09/523,783
• Time in Patent Office: 918 Days
• Field of Search: 530/345, 530/308, 530/333, 530/402, 436/86, 436/90
• US Class Current: 530/345
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 14/605 Glucagons

Abstract

Provided is a method for acylating one or more amino groups of a peptide or protein such as GLP-1. This method includes (a) reacting a peptide or protein having at least one free amino group with an acylating agent of formula I

wherein n is 0-8; R¹ is COOR⁴; R² is a lipophilic moiety; R³ and its attached carboxyl group designate a reactive ester or a reactive N-hydroxy imide ester; and R⁴ is selected from hydrogen, C_1-12-alkyl and benzyl, under basic conditions in a mixture of an aprotic polar solvent and water; and (b) if R⁴ is not hydrogen, saponifying the acylated peptide or protein ester group under basic conditions in order to obtain an N-acylated peptide or an N-acylated protein.

Claims

1. A method for acylating an amino group of a peptide or a protein, the method comprising:

(a) reacting a peptide or protein having at least one free amino group with an acylating agent of formula I

 

wherein

n is 0-8;

R¹ is COOR⁴;

R² is a lipophilic moiety;

R³ and its attached carboxyl group designate a reactive ester or a reactive N-hydroxy imide ester; and

R⁴ is selected from the group consisting of hydrogen, C_1-12-alkyl and benzyl, under basic conditions in a mixture of an aprotic polar solvent and water; and

(b) if R⁴ is not hydrogen, saponifying the acylated peptide or protein ester group (COOR⁴) under basic conditions;

in order to obtain an N-acylated peptide or an N-acylated protein.

2. The method of claim 1, wherein R⁴ is hydrogen.

3. The method of claim 1, wherein R⁴ is C_1-8-alkyl or benzyl.

4. The method of claim 1, wherein R³ and its attached carboxyl group designate a reactive N-hydroxy imide ester.

5. The method of claim 1, wherein the mixture of the aprotic solvent and water is a 1:

5 to 5;

1 mixture of N-methyl-2-pyrrolidon and water.

6. The method of claim 1, wherein the reaction between the peptide or protein and the acylating agent in step (a) is conducted at a pH range of 9-13.

7. The method of claim 1, wherein the reaction between the peptide or protein and the acylating agent in step (a) is conducted at a temperature range of 0-5°

C.

8. The method of claim 1, wherein the acylated peptide ester is saponified at a pH value in the range of 10-14.

9. The method of claim 1, wherein R² is selected from the group consisting of C_3-39-alkyl, C_3-39-alkenyl, C_3-39-alkadienyl and steroidal residues.

10. The method of claim 1, wherein R² is C_7-25-alkyl.

11. The method of claim 1, wherein the peptide is selected from the group consisting of GLP-1(7-37) and analogues thereof, exendin and analogues thereof, and GLP-2(1-34) and analogues thereof.

12. The method of claim 1, wherein the peptide is selected from the group consisting of exendin-3, exendin-4, Arg²⁶-GLP-1(7-37), Arg³⁴-GLP-1(7-37), Val⁸GLP-1(7-37), Thr⁸GLP-1(7-37), Met⁸GLP-1(7-37), Gly⁸GLP-1(7-37), Val⁸GLP-1(7-36) amide, Thr⁸GLP-1(7-36) amide, Met⁸GLP-1(7-36) amide, and Gly⁸GLP-1(7-36) amide.

17. The method of claim 1, wherein the mixture of the aprotic solvent and water is a 1:

10 to 10;

1 mixture.

13. A compound of formula I

wherein

n is 0-8;

R¹ is COOR⁴;

R² is a lipophilic moiety;

R³ and its attached carboxyl group designate a reactive ester or a reactive N-hydroxy imide ester; and

R⁴ is selected from the group consisting of hydrogen, C₁-C₁₂ alkyl and benzyl.

14. The compound of claim 13, wherein n is 0 or 1 and R³ and its attached carboxyl group designate a reactive N-hydroxy imide ester.

15. The compound of claim 13, wherein R² is selected from the group consisting of C_3-39-alkyl, C_3-39-alkenyl, C_3-39-alkadienyl and steroidal residues.

16. The compound of claim 13, wherein R⁴ is hydrogen.