N-chlorosuccinimide, an efficient peptide disulfide bond-forming reagent in aqueous solution3

Disulfide bonds are widespread in peptides and proteins where they strengthen conformation and lead to increased rigidity and proteolytic stability. The number of disulfide-rich therapeutics currently available is increasing, such as the FDA-approved peptide drugs Prialt (ziconotide) and Linzess (linaclotide), both of which contain 3 disulfide bonds. In order to prepare disulfide-rich peptides, efficient disulfide-forming reagents must be used that are compatible with all amino acids, and also preferably with the associated amino acid protecting groups. Many oxidants are used for disulfide bond formation in peptide chemistry (e.g. air, DMSO, iodine, thallium salts, mercury salts, and carbon tetrachloride). However, given that there is no universal protocol for the formation of these bonds, this process is empirical and needs to be assessed on a case to case basis. Robust and widely applicable disulfide-forming reagents are required in order to quickly determine the conditions that will allow rapid and quantitative disulfide bond formation with minimal side-reactions. Recently, we used N-chlorosuccinimide (NCS) to form a mixed disulfide between Fmoc-Cys-OH and trimethoxythiophenol under anhydrous conditions. In comparison to most common oxidants for disulfide formation, NCS is an easy to handle, cheap, and shelfstable crystalline solid that is soluble in both organic and aqueous solvent mixtures. NCS was found to be an efficient reagent for the formation of mixed cysteine disulfides in organic solvents. Subsequently, we reported NCS as a highly efficient on-resin disulfide-forming reagent for solid-phase peptide synthesis. Quantitative disulfide formation was achieved in 15 min using 2 equiv. of NCS in DMF, which we demonstrated in the synthesis of several peptides, including an on-resin regioselective synthesis of an a-conotoxin using 2 sequential NCS oxidations (Fig. 1). Shechter and co-workers reported that NCS is compatible with all amino acids except the oxidation-sensitive Trp and Met. However, we found that under our conditions both Trp and Met are compatible with NCS.{ This finding shows that all amino acids can be used with the NCS method on the solid-phase. In addition, NCS was found to be compatible with the amino acid protecting groups Trt and Mmt, while the common disulfide-forming oxidants such as iodine and thallium salts are not. These results allowed us to conclude that NCS is the most widely applicable disulfide-forming reagent for solid-phase peptide synthesis. However, solid-phase peptide synthesis has several disadvantages