PREPARATION OF FLUORESCENCE QUENCHED LIBRARIES CONTAINING INTERCHAIN DISULFIDE BONDS FOR STUDIES OF PROTEIN DISULFIDE ISOMERASES

Protein disulphide isomerase is an enzyme that catalyses disulphide re dox reactions in proteins. In this paper, fluorogenic and interchain d isulphide bond containing peptide libraries and suitable substrates, u seful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a spli t-synthesis library, two substrates containing an interchain disulphid e bond, a fluoroescent probe and a quencher were synthesized. The libr ary consists of a Cys residue flanked by randomized amino acid residue s at both sides and the fluoroescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library w as linked to PEGA-beads via methionine so that the peptides could be s electively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead-linked library and a peptide contai ning the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the inter chain disulphide bonds in the library were monitored under a fluoroesc ence microscope. Substrates to investigate the properties of protein d isulphide isomerase in solution were also synthesized. (C) 1998 Europe an Peptide Society and John Wiley & Sons, Ltd.