Activation of the Carboxy Terminus of a Peptide for Carboxy-Terminal Sequencing

Article abstract of DOI:10.1021/jo00113a042

Previously, we reported a new method of sequencing proteins from the carboxy terminus (C-terminus).The carboxyl group at the C-terminus is activated and derivatized into a thiohydantoin (TH).We reported that, by alkylating the TH formed at the C-terminus, the TH is converted into a readily displaced leaving group.Reaction with (-) under acidic conditions cleaves the alkylated thiohydantoin (ATH) and derivatizes the freshly exposed C-terminus into a new proteinyl-TH.The efficiency of the initial activation of the carboxy group at the C-terminus is critical to the initial yield of the first ATH residue.In order to directly observe the intermediates that form during activation of the C-terminus, a model tripeptide, acetyl-alanine-alanine-alanine-OH (Ac-Ala-Ala-Ala-OH) was subjected to the reagents used to form the peptidyl-TH, Ac-Ala-Ala-Ala-TH.The reaction was monitored by nuclear magnetic resonance spectroscopy.An oxazolone was observed to form immediately at the C-terminus during the reaction with diphenyl chlorophosphate (DPCP), tetraphenyl pyrophosphate (TPPP),or tetramethylchlorouronium chloride (TMU-Cl).The oxazolone was observed to react with an excess of the carboxy group-activating reagents while under basic conditions.Diketopiperazine formation at the C-terminus was also observed.These side reactions prevent or retard the reaction of (-) to form a peptidyl-TH and correlate with a reduced initial yield observed during automated C-terminal protein sequencing.The carboxylic acid-reactive reagents react with the side-chain carboxylic acid groups of aspartic and glutamic acid residues as well as the C-terminus.We found that the side-chain carboxylic acid gropus in a protein could be selectively amidated in the presence of the proteinyl-oxazolone at the C-terminus.