Peptide synthesis reagents and method of use

• US 4,507,230 A
• Filed: 05/12/1982
• Issued: 03/26/1985
• Est. Priority Date: 05/12/1982
• Status: Expired due to Fees

• Current Assignee: Research Corporation
• Original Assignee: Research Corporation
• Inventors: Tam, James P., Merrifield, Robert B., Heath, William F. Jr.
• Primary Examiner(s): Phillips, Delbert R.
• Assistant Examiner(s): Moezie, F.
• Application Number:
  US06/377,443
• Time in Patent Office: 1,049 Days
• Field of Search: 424/177, 260/112.5 R, 525/54.11
• US Class Current: 530/334
• CPC Class Codes: C07K 1/042 characterised by the nature of the carrier
• CPC Class Codes: C07K 1/12 by hydrolysis , i.e. solvolysis in general
• CPC Class Codes: C07K 14/605 Glucagons

Abstract

A method of releasing a functional group present in an amino acid or amino acyl residue from a resin or protecting residue which is bonded to the functional group by a linkage having proton affinity, which comprises: reacting the functional group bonded to the organic residue, with a mixture of HF and a base for a time and under conditions effective to produce the release; wherein the amounts of HF and base in the mixture are adjusted so that said release occurs substantially by an S_N 2 mechanism.

Claims

1. In a method of synthesizing peptides by the solid phase methodology, wherein the growing peptide chain is covalently attached by a functional group through a linkage having proton affinity, to the organic residue of a resin, the improvement wherein said peptide is detached from said resin using a mixture of HF and a base selected from the group consisting of di-C₁ -C_1O -alkyl sulfides and alkyl phenyl sulfides, wherein the amounts of HF and base in said mixture are adjusted so that said detachment occurs predominantly by an S_N 2 mechanism, said HF being present in said mixture in an amount between 0.1 and 60% by volume.

2. The method of claim 1 wherein said base also serves as diluent in said mixture.

3. The method of claim 1 wherein said base has a pKa of between -2 and -11.

4. The method of claim 1 wherein said dialkylsulfide is dimethyl sulfide.

5. The method of claim 1 wherein said linkage having proton affinity is an ester, ether or carbamate linkage.

6. The method of claim 1 wherein said HF is present in said mixture in an amount sufficient to protonate at least 1% of said linkage.

7. The method of claim 1 wherein said HF is present in said mixture in an amount such that detectable amounts of unprotonated base remain in the solution.

8. The method of claim 7 wherein said base is protonated no more than 99%.

9. The method of claim 1 wherein the mixture further comprises about 1 to about 20 percent by volume of scavengers for carbocations including phenols, indoles, ethers, thioethers, thiophenols and sulfides.

10. The method of claim 1 wherein said mixture also contains 1-50% by volume of a compound capable of swelling a polymeric resin.

11. The method of claim 1 wherein said functional group is --COOH, --NH₂, --SH, --OH, >

NH, --S-- or ##STR9##

12. The method of claim 1 wherein said mixture also comprises a polar solvent.

13. The method of claim 1 wherein the organic residue of said resin is a benzyl residue.

14. The method of claim 1 wherein said resin is a polystyrene resin.

15. The method of claim 1 wherein said mixture is a binary mixture comprising HF and dimethylsulfide, wherein the HF is present in said mixture in 0.1-60% by volume.

16. The method of claim 1 wherein said mixture also contains between 1 and 10% by volume of a C₁ -C₁₀ alkyl thiol or any aryl thiol.

17. The method of claim 14 wherein said mixture contains 1-20% by volume of a phenol.

18. The method of claim 1 which, after said reaction of said mixture with said peptide, comprises the additional step of reacting said peptide with HF at a concentration of greater than 60% by volume.

19. In the method of claim 1 wherein said peptide is selected from the group consisting of C-terminal-pentapeptide amide of gastrin, cecropin A (1-33), glucagon, Tyr²² -glucagon, and human gastrin (1-17).