The reaction of a peptide featuring a bis(2-sulfanylethyl)amino (SEA) group on its C-terminus with a cysteinyl peptide in water at pH 7 and 37 °C leads to the chemoselective and regioselective formation of a native peptide bond. This method called SEA ligation enriches the native peptide ligation repertoire available to the peptide chemist. Preparation of an innovative solid support which allows the straightforward synthesis of peptide SEA fragments using standard Fmoc/tert-butyl solid phase peptide synthesis procedures is also described.

The design of novel methods giving access to peptide alkylthioesters, the key building blocks for protein synthesis using Native Chemical Ligation, is an important area of research. Bis(2-sulfanylethyl)amido peptides (SEA peptides) 1 equilibrate in aqueous solution with S-2-(2-mercaptoethylamino)ethyl thioester peptides 2 through an N,S-acyl shift mechanism. HPLC was used to study the rate of equilibration for different C-terminal amino acids and the position of equilibrium as a function of pH. We show also that thioester form 2 can participate efficiently in a thiol-thioester exchange reaction with 5% aqueous 3-mercaptopropionic acid. The highest reaction rate was obtained at pH 4. These experimental conditions are significantly less acidic than those reported in the past for related systems. The method was validated with the synthesis of a 24-mer peptide thioester. Consequently, SEA peptides 1 constitute a powerful platform for access to native chemical ligation methodologies.