coworkers had reported the S-to-N acyl shift as early as 1953, when the reaction of valine-thioester and cysteine amino acid in aqueous buffer was shown to yield the dipeptide valine-cysteine. The reaction proceeded through the intermediacy of a thioester containing the sulfur of the cysteine residue. However, Wieland's work did NOT lead to the development of the native chemical ligation reaction. Rather, the study of amino acid thioester reactions led Wieland and others to develop the 'active ester' method for the synthesis of protected peptide segments by conventional chemical methods carried out in organic solvents. Features Native chemical ligation forms the basis of modern chemical protein synthesis, and has been used to prepare numerous proteins and enzymes by total chemical synthesis. The payoff in the native chemical ligation method is that coupling long peptides by this technique is typically near quantitative and provides synthetic access to large peptides and proteins otherwise impossible to make, due to their large size, decoration by post-translational modification, and containing non-coded amino acid or other chemical building blocks. Native chemical ligation is inherently 'Green' in its atom economy and its use of benign solvents. It involves the reaction of an unprotected peptide thioester with a second, unprotected peptide that has an N-terminal cysteine residue. It is carried out in aqueous solution at neutral pH, usually in 6 M guanidine.hydrochloride, in the presence of an arylthiol catalyst and typically gives near-quantitative yields of the desired ligation product. Peptide-thioesters can be directly prepared by Boc chemistry SPPS; however, thioester-containing peptides are not stable to treatment with a nucleophilic base, thus preventing direct synthesis of peptide thioesters by Fmoc chemistry SPPS. Fmoc chemistry solid phase peptide synthesis techniques for generating peptide-thioesters are based on the synthesis of peptide hydrazides that are converted to peptide thioesters post-synthetically. Polypeptide C-terminal thioesters can also be produced in situ, using so-called N,S-acyl shift systems. Bis(2-sulfanylethyl)amido group, also called SEA group, belongs to this family. Polypeptide C-terminal bis(2-sulfanylethyl)amides (SEA peptide segments) react with Cys peptide to give a native peptide bond as in NCL. This reaction, which is called SEA Native Peptide Ligation, is a useful variant of native chemical ligation. In making peptide segments that contain an N-terminal cysteine residue, exposure to ketones should be avoided since these may cap the N-terminal cysteine. Do not use protecting groups that release aldehydes or ketones. For the same reason, the use of acetone should