Method for sequencing of peptides by carboxyl terminus degradation

• US 4,837,165 A
• Filed: 04/20/1988
• Issued: 06/06/1989
• Est. Priority Date: 09/26/1985
• Status: Expired due to Term

• Current Assignee: City of Hope
• Original Assignee: Beckman Research Institute of the City of Hope (City of Hope)
• Inventors: Hawke, David H.
• Primary Examiner(s): Hill, Jr., Robert J.
• Application Number:
  US07/186,667
• Time in Patent Office: 412 Days
• Field of Search: 436/89, 436/161, 530/345, 530/402, 530/427
• US Class Current: 436/89
• CPC Class Codes: C07K 1/08 using activating agents C07K1/003, C07K1/006 take precedence

Abstract

A peptide is sequenced from its carboxyl terminal by coupling with an isothiocyanate having a chemical formula R(_x)Si(NCS)_y where x=0-3 and y=1-4. R may be alkyl or aryl. The coupling reaction is preferably conducted at a temperature range of 50°-90° C. for a period to approximately sixty (60) minutes or less. A weak acid anhydride, preferably a carboxylic acid anhydride, or a weak acid chloride, preferably a carboxylic acid chloride, may be mixed with the isothiocyanate. An organic base catalyst selected from a group consisting of tertiary amines including aromatic amines such as pyridine may also be utilized.

Claims

1. In a method for sequencing a peptide by carboxyl terminal degradation which comprises coupling the carboxyl terminus of a peptide with a coupling reagent to form a peptidyl thiohydantoin derivative, cleaving the peptidyl thiohydantoin derivative to provide a thiohydantoin derivative of the amino acid previously at the carboxyl terminus of the peptide and a peptidyl residue lacking such an amino acid, and identifying the amino acid in the amino acid thiohydantoin derivative, wherein the improvement comrprises utilizing a silyl isothiocyanate as the coupling reagent.

2. The method of claim 1 in which the isothiocyanate has the formula R.sub.(x) Si(NCS)_y where x is 0 to 3, y is 1 to 4 and R is an alkyl or an aryl substituent.

3. The method of claim 2 in which the isothiocyanate is trimethylsilyl isothiocyanate.

4. The method of claim 2 in which a catalyst is included in the coupling reagent.

5. The method of claim 4 in which the catalyst is a tertiary amine.

6. The method of claim 4 in which the catalyst is pyridine.

7. The method of claim 2 in which a weak organic acid anhydride is included in the coupling reagent.

8. The method of claim 7 in which the weak organic acid anhydride is acetic anhydride.