Derivatizing Reagents Based on Ferrocene for HPLC-Ecd Determination of Peptides and Proteins

Abstract

Several ferrocenic compounds for derivatization of peptides and proteins were synthesized and then tested by reaction with bovine serum albumin (BSA). The reactivity of the reagents and the electroactivity of the derivatized BSA were estimated by the height of the ECD-signal after an HPLC analysis run. The most suitable reagent was 3-ferrocenylpropionic anhydride which reacts with BSA within 15 minutes at room temperature. The anhydride presented itself as a stable compound which can be synthesized in high yields. Up to pH 9–10 it is only slowly hydrolyzed, and its derivatization products are highly electroactive. Another reagent is especially suited to derivatize those proteins whose isoelectric points are higher than 10: Ferrocenylmethyl-succinimidyl-glycine-hydrochloride. This compound develops its derivatization activity only with pH values higher than 9, but it is rather difficult to prepare.