C-terminal peptide sequencing, activated support and reagent system therefor, and method of producing the activated support
AssigneeApplied Biosystems, LLC
InventorsVictoria L. Boyd, David H. Hawke
Life of this patent
Estimating the term…
Abstract
A method of C-terminal peptide sequencing. The peptide is reacted with an activated support derivatized with a mixed anhydride of isothiocyanic acid and carboxylic or carbonic acid, under basic conditions. The peptidyl thiohydantoin which forms is separated from the solid support and further reacted with a cleaving agent carried on a second solid support, to release a free C-terminal amino acyl thiohydantoin from the peptide. The free thiohydantoin is analyzed to determine the C-terminal peptide residue. The residual peptide can be recycled through the supports for successive C-terminal residue determinations.
Claims22
A method of producing a thiohydantoin (TH) of a protected amino acid having a free carboxylic acid group of a C-terminal amino acid of a peptide, comprising
- contacting the amino acid in solution with an activated support composed of a solid support derivatized with a mixed anhydride of isothiocyanic acid and carboxylic or carbonic acid, under basic conditions in which the acid group of the amino acid is deprotonated,
- by said contacting, forming the TH of the amino acid in solution phase, and
- separating the amino acid TH in solution from the solid support.
A method of sequencing a peptide from its C-terminal end, comprising
- contacting the peptide in solution with an activated support composed of a solid support derivatized with a mixed anhydride of isothiocyanic acid and carboxylic or carbonic acid, under basic conditions in which the carboxyl group of the peptide's C-terminal amino acid is deprotonated,
- by said contacting, forming in solution a peptidyl TH at the C-terminal amino acid of the peptide,
- separating the peptidyl TH in solution from the solid support,
- cleaving the peptidyl TH to release the amino acid TH from the residual peptide, and
- identifying the released amino acid TH.
An activated support comprising a solid support derivatized with a mixed anhydride of isothiocyanic acid and carboxylic or carbonic acid.
A solid-phase system for use in determining the C-terminal amino acid residue of a peptide, comprising
- an activated support composed of a solid support derivatized with a mixed anhydride of isothiocyanic acid and carboxylic or carbonic acid, and
- a second support composed of a solid support derivatized with a cleaving agent which is effective to cleave a peptidyl TH to form a free amino acid TH and a residual peptide.
A method of preparing an isothiocyanate containing solid support effective to convert a peptide, when contacted with the support, into a C-terminal peptidyl thiohydantoin (TH), comprising
- providing a solid support functionalized with carboxyl groups,
- reacting the solid support with an isoxazolium compound under basic conditions, to form activated enol esters of the carboxyl groups, and
- reacting the support with an isothiocyanate (ITC) selected from the group consisting of trialkylsilyl ITC and pyridine-ITC, to form a mixed anhydride of isothiocyanic acid and carboxylic acid on the support.
Dependencies are inferred from the claim text.