title: Synthesis and cystine/cysteine-catalyzed oxidative folding of the amaranth alpha-amylase inhibitor.
abstract: We report here the total synthesis of the alpha-amylase inhibitor (AAI), a 32-residue-long peptide with three disulfide bridges, isolated from amaranth seeds (Chagolla-Lopez, A., Blanco-Labra, A., Patthy, A., Sanchez, R. & Pongor S. (1994) J. Biol. Chem. 269, 23675-23680). The synthesis was carried out using a stepwise solid-phase approach based on the Fmoc/t-Bu chemistry, combined with the S-acetamidomethyl protection for cysteines. The linear, reduced peptide was obtained after two reduction steps, using 1,4-dithio-DL-threitol and tri(2-carboxyethyl)phosphine hydrochloride in basic and acidic conditions, respectively. Disulfide bridges were formed by oxidative folding in a cystine/cysteine redox buffer, these conditions were found superior to air oxidation and to glutathione-catalyzed oxidative folding. The physiochemical and enzyme inhibitory properties of synthetic AAI were found identical with those of natural product. Several orthogonal protection schemes proved unsuccessful in obtaining a biologically active product.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Lozanov
authors.forename: C
authors.initials: C
authors.lastname: Guarnaccia
authors.forename: A
authors.initials: A
authors.lastname: Patthy
authors.forename: S
authors.initials: S
authors.lastname: Foti
authors.forename: S
authors.initials: S
authors.lastname: Pongor
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D003553:Cystine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry; D005449:Fluorenes / Q000737:chemistry; D007700:Kinetics; D019684:Magnoliopsida / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D017510:Protein Folding; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014361:Trypsin Inhibitors; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
chemical_list: D000596:Amino Acids; D004220:Disulfides; D004791:Enzyme Inhibitors; D005449:Fluorenes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D010940:Plant Proteins; D013438:Sulfhydryl Compounds; D014361:Trypsin Inhibitors; D003553:Cystine; D000516:alpha-Amylases; D003545:Cysteine