Synthesis of chloroacetyl and bromoacetyl modified peptides for the preparation of synthetic peptide polymers, conjugated peptides, and cyclic peptides

• US 5,066,716 A
• Filed: 12/13/1988
• Issued: 11/19/1991
• Est. Priority Date: 12/13/1988
• Status: Expired due to Term

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Fields, Raymond L., Lindner, Wolfgang, Robey, Frank A.
• Primary Examiner(s): Nutter, Nathan M.
• Application Number:
  US07/283,849
• Time in Patent Office: 1,071 Days
• Field of Search: 530/335, 530/336, 530/337, 530/345, 530/317, 528/288, 525/54.1, 525/54.11
• US Class Current: 525/54.1
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids

Abstract

A method to incorporate bromoacetyl and chloroacetyl moieties on amino groups of synthetic peptides using a standard program with an automated peptide synthesizer has been developed. The bromoacetyl and chloroacetyl-derivatized peptides react well with sulfhydryl-containing proteins and with peptides containing cysteine residues. Autopolymerization or cyclization occurs by reaction of the free sulfhydryl of cysteine in a peptide with the bromoacetyl group (or chloroacetyl group) and reactions can generally be controlled by controlling the concentrations of starting peptide in neutral pH buffers. Analytical methods for evaluating the polymers or cyclized peptides include gel filtration chromatography, reverse phase HPLC, SDS-PAGE and amino acid analysis where the degree of reaction can be evaluated by quantifying the amount of S-carboxymethylcysteine formed after HCl hydrolysis. N-bromoacetyl-derivatized peptides are useful as reagents for potential peptide immunogens, vaccines and therapeutics, and for substances such as peptides linked to polymers, plastics, enamels, and ceramics.

Claims

1. A method for the preparation of bromoacetyl or chloroacetyl derivatized peptides, which comprises the steps of:

coupling a bromoacetyl or chloroacetyl group onto a fully protected peptide to form an amide linkage between said bromoacetyl or chloroacetyl group and said fully protected peptide; and

deprotecting said fully protected peptide while still preserving the presence of said bromoacetyl or chloroacetyl group on said peptide.

2. The method according to claim 1, wherein said derivatized peptide is an N-bromoacetyl derivatized peptide.

3. The method according to claim 1, wherein said derivatized peptide is an N-chloroacetyl derivatized peptide.

4. The method according to claim 1, wherein said fully protected peptide contains cysteine, a group with an SH group, or a reactive amino group in the peptide chain.

5. The method according to claim 1, which comprises forming a symmetric anhydride of bromoacetic acid or chloroacetic acid;

reacting said bromoacetic anhydride or chloroacetic acid anhydride with an N-terminus of a protected peptide to form said amide linkage; and

deprotecting said peptide by treatment with an acid.

6. The method according to claim 5, wherein the formation of said symmetric anhydride of bromoacetic acid or chloroacetic acid occurs at about 0°

C. to about 30°

C.

7. The method according to claim 5, wherein the reaction of the bromoacetic acid and anhydride or chloroacetic acid anhydride with the protected peptide is conducted at about 0°

C. to about 30°

C.

8. The method according to claim 5, wherein the treatment with the acid is conducted at about -5°

C. to about 5°

C.

9. The method according to claim 8, wherein the treatment with the acid is conducted for about 10 minutes to about 3 hours.

10. A method according to claim 4, which further comprises polymerizing or cyclizing said bromoacetyl or chloroacetyl derivatized peptide by treating said derivatized peptide in a solution having a pH of about 5 to about 11.

11. The method according to claim 10, wherein said polymerization or cyclization occurs in a solution of NaHCO₃ or NaHPO₄ having a pH of about 8.

12. The method according to claim 11, which is conducted at about 15°

C. to about 30°

C.

13. The method according to claim 12, which is conducted for about 0.5 to about 48 hours.

14. The method according to claim 1, which further comprises reacting the bromoacetyl or chloroacetyl derivatized peptide with a carrier protein containing cysteine, a group having an SH group, or a reactive amine to form a conjugate.

15. The method according to claim 14, wherein the reaction is conducted for about 0.5 hours to about 48 hours at a temperature of about 15°

C. to about 30°

C.

16. The method according to claim 14, wherein said carrier protein is a member selected from the group consisting of bovine serum albumin, chicken serum albumin, and keyhole lymphet hemocyanin.

17. A bromoacetyl or chloroacetyl derivatized peptide in which the bromoacetyl group or chloroacetyl group is bonded to the peptide via an amide linkage.

18. A polymer of the derivatized peptide of claim 17.

19. A cyclized form of the derivatized peptide of claim 18.

20. A conjugate of a carrier protein and the derivatized peptide of claim 17.

22. A peptide carrier conjugate produced by a process which comprises reacting the derivatized peptide of claim 17 with a carrier selected from the group consisting of polymers, enamels and ceramics.

21. A method for the preparation of bromoacetyl or chloroacetyl derivatized proteins, which comprises:

coupling a bromoacetyl or chloroacetyl group onto a fully protected peptide at a pH of about 5 to about 11 for about 10 minutes to about 48 hours at about -5°

C. to 30°

C. in an aprotic polar solvent to form an amide linkage between said bromoacetyl or chloroacetyl group and said fully protected peptide, and

deprotecting said fully protected peptide while still preserving the presence of said bromoacetyl or chloroacetyl group on said peptide at a temperature of about -5°

C. to about 5°

C. for about 10 minutes to about 3 hours in trifluoroacetic acid or hydrogen fluoride.