Chemical recognition of terminally amidated peptides
Abstract
Peptides play important regulatory roles in many organ and cell systems. Development of novel methods for the identification and purification of bioactive peptides remains as an exciting area of interest. Traditionally the discovery of peptide hormones has relied on an assay based on their biological activities. It would be useful to have a method for recognizing peptide hormones solely by their chemical properties without any specific knowledge of their biological effects. One such chemical characteristic of many of the peptide hormones is amidation at the carboxyl terminus. This C-terminal amino acid $\alpha$-amide occurs in hormones of great diversity. The biosynthetic mechanism involved with carboxyamidation has been elucidated. Thus if a naturally occurring peptide is terminally amidated, its biological activity is strongly implied. We have developed a chemical method designed to determine whether a sample peptide is terminally amidated. The reactions utilized take advantage of the reagent I,I-bis(trifluoroacetoxy)iodobenzene (PIFA). The peptide C-terminal amide is rearranged with PIFA to a geminal aminoamide derivative. This is allowed to hydrolyze in the presence of cyanide, so that the generated imine intermediate can be trapped with cyanide to form a cyanoamine derivative, which can be hydrolyzed to an amino acid. This regenerated C-terminal amino acid is identified through amino acid analysis. In the development of the proposed method, first the PIFA rearrangement was studied using N-acetylamino acid amides. Since peptides contain various side chain functional groups, this reaction was characterized for each amino acid, and side reactions, if any, were investigated. Next the cyanide trapping step was optimized under mild conditions, in which peptide side chains remain unharmed. Finally, this proposed method was applied to bioactive terminally amidated peptides at submicromolar levels, and we have validated the proposed chemistry.
Degree
Ph.D.
Advisors
Loudon, Purdue University.
Subject Area
Organic chemistry|Biochemistry
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