Chemical Modification of Proteins

Abstract

The invention relates to methods for selectively converting a cysteine residue in a peptide or protein to the dehydroalanine (Dha) residue. The method also works on selenocysteine and substituted cysteine and selenocysteine residues, resulting in the Dha residue which may be converted to any natural or unnatural amino acid residue desired without the alteration of the remainder of the peptide or protein. The invention also allows ligation of a desired peptide at any point rather than at a point where there should be a naturally occurring cysteine, thereby allowing native chemical ligation to be used in the synthesis of peptides that do not contain cysteine. The methodology allows for the synthesis of very large peptides.

1 . A method for selectively converting a cysteine residue in a peptide or protein to a dehydroalanine residue comprising contacting the peptide or protein with a sulfonylhydroxylamine. 
     
     
         2 . The method of  claim 1  wherein the sulfonylhydroxylamine is O-mesitylenesulfonylhydroxylamine. 
     
     
         3 . The method of  claim 1  wherein the contacting the cysteine residue with the sulfonylhydroxylamine takes place in a solution or suspension in a polar aprotic solvent. 
     
     
         4 . The method of  claim 3  wherein the polar aprotic solvent is dimethylformamide (DMF). 
     
     
         5 . The method of  claim 1  wherein the cysteine residue is at the surface of the peptide or protein. 
     
     
         6 . The method of  claim 1  wherein the step of contacting the cysteine residue with the sulfonylhydroxylamine takes place in the presence of a base. 
     
     
         7 . The method of  claim 6  wherein the base comprises a potassium carbonate buffer or a phosphate buffer. 
     
     
         8 . The method of  claim 1  comprising a subsequent step of reacting the carbon-carbon double bond of the dehydroalanine residue. 
     
     
         9 . The method of  claim 8  wherein the step of reacting the carbon-carbon double bond is stereoselective. 
     
     
         10 . The method of  claim 8  wherein the carbon-carbon double bond of the dehydroalanine residue is reacted with a thiol to form a thioether derivatized peptide or protein. 
     
     
         11 . The method of  claim 8  wherein the carbon-carbon double bond of the dehydroalanine residue is reacted with an organoborate. 
     
     
         12 . The method of  claim 8  wherein the carbon-carbon double bond of the dehydroalanine residue is reacted with an organohalide in the presence of elemental zinc. 
     
     
         13 . A method for producing a peptide or protein comprising the steps of:
 a) joining two polypeptides by native chemical ligation; and   b) contacting the polypeptides with a sulfonylhydroxylamine to convert a cysteine reside to a dehydroalanine residue.   
     
     
         14 . The method of  claim 13  wherein the cysteine residue is located at the site of native chemical ligation. 
     
     
         15 . A peptide or protein modified by contacting the peptide or protein with a sulfonylhydroxylamine to convert a cysteine reside to a dehydroalanine residue