Synthesis of N-substituted peptide amides

• US 4,569,967 A
• Filed: 10/24/1983
• Issued: 02/11/1986
• Est. Priority Date: 10/24/1983
• Status: Expired due to Fees

• Current Assignee: Salk Institute for Biological Studies
• Original Assignee: Salk Institute for Biological Studies
• Inventors: Rivier, Jean E. F., Porter, John S., Anderson, Harry A., Kornreich, Wayne D.
• Primary Examiner(s): Phillips, Delbert R.
• Application Number:
  US06/545,077
• Time in Patent Office: 841 Days
• Field of Search: 260/112.5 R, 525/54.11
• US Class Current: 525/54.11
• CPC Class Codes: C07K 1/04 on carriers C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C07K 5/0823 and Pro-amino acid; Derivatives thereof

Abstract

Peptide N-alkylamides, and other C-terminal N-substituted amides, can be synthesized using solid-phase synthesis on a benzene-containing resin which is suitably methylated. Reactive amino groups are attached directly or indirectly to the methyl groups, for example, such as by reacting commercially available chloromethylated polystyrene resins with an alkylamine to create a resin-amine. The C-terminal amino acid of the desired peptide is linked to the resin-amine via an amide linkage, and the peptide is thereafter built in normal fashion. Treatment of the completed peptide intermediate with HF is effective to both effect deprotection and cleave the peptide from the resin in the form of the N-substituted amide. Examples include peptide N-ethylamides, N-fluoroethylamide, N-anilides and other substituted N-benzylamides.

Claims

1. A method for synthesizing a peptide having a substitute amide at its C-terminus using solid phase synthesis, which method comprises

providing a synthetic resin having a plurality of p-benzyl moieties, the methyl groups of which are substituted only with -NHQ, wherein Q is lower alkyl, lower fluoroalkyl, phenyl or substituted phenyl,

reacting said synthetic resin with an amino acid having its alpha-amino group blocked to cause coupling of the carboxyl group thereof to said substituent by a substituted amide linkage,

deblocking said alpha-amino group,

repeating said reacting step to create a peptide and

cleaving said peptide from said resin so that the C-terminus of said peptide is --CONHQ.

2. A method in accordance with claim 1 wherein said resin is a cross-linked polystyrene resin.

3. A method in accordance with claim 1 wherein said cleaving is carried out with HF.

4. A method in accordance with claim 1 wherein Q is --C₆ H₄ NH₂.

5. A method in accordance with claim 4 wherein said peptide is cleaved as a N-aminoanilide which is subsequently oxidized to a N-nitroanilide.

6. A method in accordance with claim 5 wherein said oxidation is effected by treating with H₂ O₂ under acidic conditions.

7. A method in accordance with claim 5 wherein the C-terminus of said peptide is ##STR1## which is oxidized to create a para-nitroanilide.

8. A method in accordance with claim 6 wherein HCl is used to provide said acidic conditions and said peptide is ##STR2##

9. A method in accordance with claim 1 wherein Q is --CH₂ CH₃.

10. A method in accordance with claim 1 wherein Q is --CH₂ CF₃.

11. A method in accordance with claim 1 wherein Q is --CH₂ CHF₂.

12. A method in accordance with claim 1 wherein Q is --CH₂ CH₂ CH₃.

13. A method in accordance with claim 1 wherein Q is --CH₃.

14. A method in accordance with claim 1 wherein Q is CHCH₃ CH₃.

15. A method in accordance with claim 4 wherein a chloromethylated polystyrene resin is reacted with p-aminoaniline in DMF at about 60°

-100°

C. for at least about 16 hours to form said substituted resin.

16. A method in accordance with claim 9 wherein a chloromethylated polystyrene resin is reacted with ethylamine at about 0°

to about 5°

C. to form said substituted resin.

17. A method for synthesizing a peptide containing at least one Asp or Glu residue within its chain and having a substituted amide at its C-terminus using solid phase synthesis, which method comprises

providing a synthetic resin having a plurality of substituted p-benzl moieties of the formula --C₆ H₄ CH₂ NHQ wherein Q is lower alkyl, lower fluoroalkyl, phenyl or substituted phenyl,

reacting said synthetic resin with an amino acid having its alpha-amino group blocked to cause coupling of the carboxyl group thereof to the nitrogen of said moiety by a substituted amide linkage,

deblocking said alpha-amino group,

repeating said reacting and deblocking steps with such additional amino acids which couple sequentially to each last-coupled amino acid residue to create a peptide containing at least one Asp or Glu residue the side chain of which is protected and

simultaneously cleaving said peptide from said resin and deprotecting said side chain in a manner that the C-terminus of said peptide is --CONHQ.

18. The method in accordance with claim 17 wherein said resin is a cross-linked polystyrene resin and said cleaving and deprotecting is carried out with HF.

19. A method in accordance with claim 17 wherein the side chain of the Asp or Glu residue is protected with benzyl ester.

20. A method for synthesizing a peptide having an N-nitroanilide at its C-terminus using solid phase synthesis, which method comprises

providing a synthetic resin having a plurality of moieties of the formula --C₆ H₄ CH₂ NHC₆ H₄ NH₂,

reacting said synthetic resin with an amino acid having its alpha-amino group blocked so as to cause coupling of the carboxyl group thereof to the amino group of said moiety by an amide linkage,

deblocking said alpha-amino group,

repeating said reacting and deblocking steps with such additional amino acids which couple sequentially to each last-coupled amino acid residue to create a peptide of desired length,

cleaving said peptide from said resin in a manner such that the C-terminus of said peptide is an N-aminoanilide and

oxidizing said N-aminoanilide to an N-nitroanilide.