Detection of 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) carbamoyl amino acids generated by post-column desulfuration in the simultaneous determination of the sequence and D/L-configuration of peptides using a fluorogenic Edman reagent, 7-N

摘要

A modified Edman sequencing method using 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) for amino acid sequence and D/L-configuration determination has been developed. DBD-thiazolinone (TZ)-amino acids generated by Edman sequencing method were hydrolyzed and then oxidized to DBD-carbamoyl (CA)-amino acids, instead of the conversion reaction to thiohydantoin (TH)-amino acid in the conventional Edman method. The oxidative desulfuration step operated manually with formation of DBD-CA-amino acids was difficult to control because of the production of by-products by the side-chain oxidation of amino acid and inapplicability to the conventional automated protein sequencer. Therefore, post-column desulfuration method in which the reaction was carried out after the separation of analyte on a column was designed. DBD-thiocarbamoyl (TC)-amino acids generated by the modified sequencing method were separated on the reversed phase or Pirkle type chiral stationary phase column and then oxidized to DBD-CA-amino acids with hydrogen peroxide as a post-column reagent and detected fluorimetrically. Using the method, the determination of the amino acid sequence and D/L-configuration of a D-amino acid containing peptide was achieved and gave us the recognition to the utility of the method, (C) 2000 Elsevier Science B.V. All rights reserved. [References: 32]