DERIVATIZATION APPROACHES FOR A FERMENTATION DERIVED, CYCLIC PEPTIDE ANALOG

Several derivatization approaches for a cyclic, fermentation derived p eptide analog were investigated. The most viable derivatization reagen t, 5-[2-(and-3)-S-(acetylmercapto)succinoyl (SAMSA-FL), derivatized th e analyte through a Michael addition mechanism. SAMSA-FL was activated under basic conditions to produce a free thiol functionality, which i n turn added across the double bonds of available alpha,beta-unsaturat ed amide sites present on the substrate. Derivatizations performed at 50 degrees C with a 200-fold molar ratio of SAMSA-FL to peptide analog produced several fluorescently active products as seen by HPLC. These products were effectively quantitated by coeluting all of the derivat ive species into a single peak via the use of a step gradient conditio n. Quantitation of the derivatized peptide analog was achieved on actu al samples with prederivatization concentrations ranging from 25.0-500 mu M (31.1-622 ppm). The method was validated by performing an analys is on three single blind spiked samples. (C) 1998 Elsevier Science B.V . All rights reserved.