Disulfide bridges are significant "markers" for the folding of peptides and proteins, since they covalently cross-link portions of the polypeptide chain which are apart in the linear sequence but come together in three dimensions. In the present application, we seek to build on the twin expertises of this laboratory in mild chemical methods for solid-phase peptide synthesis and in sulfur chemistry to develop general new methods for the creation of sulfur-sulfur bonds in peptides. The steps for sulfur-sulfur bond formation will be carried out while a peptide remains anchored to a polymeric support, thereby taking advantage of the pseudo-dilution phenomenon which favors intramolecular cyclization. This approach requires a good repertoire of orthogonally removable cysteine protecting groups, and the capability to release unprotected monomeric peptide products from the support without breaking or scrambling the disulfides. Our multi-faceted approach will apply the best recent innovations in anchoring linkages, mild deprotection/cleavage conditions, and polymeric supports to this challenging problem. Two predetermined residues will be selectively deblocked, followed either by careful co-oxidation or by "directed" techniques to join them as a disulfide. We will assess the relative influence of reaction conditions, resin substitution level, and support characteristics on yield and purity of monomeric material. The new methods will be applied to biologically active target molecules with one to three disulfides, including oxytocin, bactenecin, apamin, and neutrophil defensins. If our syntheses of the parent structures are successful, analogies will be made where one or more disulfide is removed, ring size is decreased or increased, more conformational rigidity is introduced, and disulfides are intentionally mispaired. The secondary and tertiary structure of the analogues will be characterized by biophysical techniques, and the biological activities will be determined. Ultimately, the chemical synthesis of such rationally-designed peptide analogues may provide more potent, selective, and longer-lasting drugs.