A chemical method for the sequential degradation of peptides from the carboxy terminus
Abstract
A chemical method is described that will determine sequentially from the carboxy terminus the amino acid sequence of a protein or peptide fragment. The procedure involves attachment of peptide to a rigid glass support specifically at the amino terminus. Once the peptide is immobilized on the support, organic reagents and solvents may be used without difficulty. The free carboxy-terminal amino acid anion is converted to an acyl azide when allowed to react with bis(p-nitrophenyl)phosphorylazide in dimethylformamide. This acyl azide is then themolyzed to an isocyanate via the Curtius rearrangement in the presence of p-methoxybenzyl alcohol (pMBA), ultimately leading to an N-(1-aminoalkyl)peptidyl amide. This compound as the free base is coupled directly to ethyl chlorooxalate. Employing basic conditions, the N-(1-)O-ethyloxalyl)aminoalkyl)amide intermediate is cyclized internally via the amide nitrogen to yield an N-acetylated-2-alkyl-4,5-imidazolidinedione intermediate that is cleaved from the solid support most efficiently employing strong basic conditions. This procedure leaves the remaining peptide attached to the glass support, and the degraded amino acid derivatized as a 2-alkyl-4,5-imidazolidinedione (2I45D). The identity of the amino acid-2I45Ds are evaluated along with the cleavage efficiency using reverse-phase high-performance liquid chromatography. The exact conditions necessary to obtain maximum yields in all these transformations are discussed and described. Problems associated with cleavage efficiency are also discussed, in addition to experiments proposed to deal with these problems. The new carboxy-terminal amino acid of the adhered peptide continues the stepwise sequential degradation using this procedure.
Degree
Ph.D.
Advisors
Loudon, Purdue University.
Subject Area
Pharmacology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.