• Acm;
  • disulfide formation;
  • EGF domain;
  • Factor VII;
  • peptide synthesis;
  • regioselective

Abstract: The study of EGF-like domains is of great general interest in protein science because of their participation in a multitude of protein–protein interactions. A common structural feature of EGF-like modules is the presence of three disulfide bonds, the regioselective formation of which still remains a challenge to peptide chemists. We report here on a method for the one-pot regioselective synthesis of an analogue of the EGF-1 domain of human coagulation Factor VII (residues 45–83) comprising an Asn57β–Asp substitution. The cysteine protecting groups trityl, t-butyl and acetamidomethyl were chosen for the three disulfide bond pairings. All three disulfide bridges were prepared directly from the crude starting product, obviating the need for the timely and costly purification of the intermediate folded products. The fully folded product was purified by preparative high-pressure liquid chromatography prior to evaluation of its biological activity in an assay to detect inhibition of FVII/TF complex formation. In addition circular dichroism spectroscopy was employed to elucidate the main structural similarities between this peptide analogue and the native human Factor VII EGF-1 domain.