A selective and efficient method for disulfide bond formation in peptides by dimethyl sulfoxide (DMSO) is described. Facile disulfide bond formation by DMSO in aqueous buffered solutions was found to proceed in a wide range of pH. More importantly, it overcame the limitation of the conventional oxidation method with air or mixed disulfide that was applicable only at a narrow basic pH range. The sulfur-sulfur bond reaction by DMSO was selective, and no side reactions were observed with nucleophilic amino acids such as Met, Trp, or Tyr. Because of its widely applicable pH range, the DMSO oxidation method was particularly suitable for basic and hydrophobic peptides. Monocyclic disulfide formation by 20% DMSO was observed to be completed in 0.5-4 h in a series of basic and hydrophobic peptides with ring sizes varying from 6 to 11 amino acids, while similar experiments by air oxidation at basic pH required longer duration and produced incomplete reactions. A detailed kinetic study on ten peptides showed that the DMSO oxidation method was pH-independent between pH 3 and 8 with pseudo-first-order rates ranging from 0.012 to 0. 14 min-1. However, with peptides containing a cysteine at the amino terminus, the rates became pH-dependent with an optimal pH near neutrality. Equally facile oxidations by DMSO were observed with the basic and hydrophobic, tricyclic 29-residue human defensin. In contrast, air oxidation at basic pH of human defensin led to extensive precipitation and low yield. Our results show that DMSO is a versatile and useful oxidizing agent for peptides at a wide range of pH and may bc particularly suitable for renaturation and oxidation of proteins at controlled pH.
