Chemical modification of proteins
View Patent ↗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 converting an unmodified cysteine residue of a peptide or protein to a dehydroalanine residue, the method comprising:
reacting an unmodified cysteine residue of the peptide or protein with a sulfonylhydroxylamine of formula:
wherein R is an electron withdrawing group, and wherein the method is performed at a pH of between 6 and 10, to convert the unmodified cysteine residue to a dehydroalanine residue.
2. The method of claim 1 wherein the sulfonylhydroxylamine is O-mesitylenesulfonylhydroxylamine.
3. The method of claim 1 wherein said reacting the unmodified 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 said reacting the unmodified cysteine residue with sulfonylhydroxylamine takes place in the presence of a base.
6. The method of claim 5 wherein the base comprises a potassium carbonate or a phosphate buffer.
7. The method of claim 1 comprising a subsequent step of reacting the carbon-carbon double bond of the dehydroalanine residue.
8. The method of claim 7 wherein the step of reacting the carbon-carbon double bond is stereoselective.
9. The method of claim 7 wherein the carbon-carbon double bond of the dehydroalanine residue is reacted with a thiol to form a thioether derivatized peptide or protein.
10. The method of claim 7 wherein the carbon-carbon double bond of the dehydroalanine residue is reacted with an organoborate.
11. The method of claim 7 wherein the carbon-carbon double bond of the dehydroalanine residue is reacted with a organohalide in the presence of elemental zinc.
12. A method for producing a peptide or protein comprising the steps of:
a) joining two polypeptides by native chemical ligation, thereby forming a joined polypeptide; and
b) contacting the joined polypeptide with a sulfonylhydroxylamine to achieve conversion of an unmodified cysteine residue of the polypeptide to a dehydroalanine residue,
wherein the sulfonylhydroxylamine has the formula:
and wherein R is an electron withdrawing group, and wherein the method is performed at a pH of between 6 and 10.
13. The method of claim 12 wherein the unmodified cysteine residue is located at the site of native chemical ligation.
14. A method of converting an unmodified cysteine residue of a peptide or protein to a dehydroalanine residue, the method comprising:
reacting an unmodified cysteine residue of the peptide or protein with O-mesitylenesulfonylhydroxylamine, wherein the step of contacting takes place in dimethylformamide, and wherein the method is performed at a pH between 6 and 10, to convert the unmodified cysteine residue to a dehydroalanine residue.
15. The method of claim 1 , further comprising reversibly denaturing the peptide or protein before said reacting.