- Activation of the Carboxy Terminus of a Peptide for Carboxy-Terminal Sequencing
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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.
- Boyd, Victoria L.,Bozzini, MeriLisa,Guga, Piotr J.,DeFranco, Robert J.,Yuan, Pau-Miau,et al.
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p. 2581 - 2587
(2007/10/02)
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- Thermitase - A Thermostable Serine Protease. II. Synthesis of Substrate Analogous Peptide Chloromethyl Ketones as Irreversibly Acting Inhibitors
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The preparation of dipeptide to pentapeptide chloromethyl ketones of alanine with variation of the amino acid in the P1 and P2 position of the peptides is described.The synthesis is performed by fragment condensation of N-acylated amino acids or peptides with the unprotected chloromethyl ketone derivatives of amino acids and peptides, respectively.Some examples of enzyme inactivation by peptide chloromethyl ketones are discussed.
- Jahreis, Guenther,Smalla, Kornelia,Fittkau, Siegfried
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