Novel Solid-Phase Reagents for Facile Formation of Intramolecular Disulfide Bridges in Peptides under Mild Conditions1,2

Abstract

The controlled formation of intramolecular disulfide bridges in peptides, while avoiding unwanted oligomerization, is a significant challenge. Ellman's reagent, 5,5‘-dithiobis(2-nitrobenzoic acid), was developed originally in the context of an assay for measuring free thiol concentration under physiological conditions. The present studies demonstrate that this reagent, when bound through two sites to a suitable solid support (PEG-PS, modified Sephadex, or controlled-pore glass), is an effective mild oxidizing reagent that promotes the formation of disulfide bridges. Rates and yields of the reactions were determined as a function of pH, excess of oxidizing reagent, resin loading, and parent support, for the preparation of oxytocin and deamino-oxytocin (9 residues, disulfide bridge between residues 1 and 6), somatostatin (14 residues, disulfide bridge between residues 3 and 14), α-conotoxin SI (13 residues, disulfide bridges between residues 2 and 7; 3 and 13), and apamin (18 residues, disulfide bridges bet...