Native chemical ligation

This transthioesterification step is reversible in the presence of an aryl thiol catalyst, rendering the reaction both chemoselective and regioselective, and leads to formation of a thioester-linked intermediate.

The intermediate rapidly and spontaneously rearranges by an intramolecular S,N-acyl shift that results in the formation of a native amide ('peptide') bond at the ligation site (scheme 1).

The key feature of native chemical ligation of unprotected peptides is the reversibility of the first step, the thiol(ate)–thioester exchange reaction.

Native chemical ligation is exquisitely regioselective because that thiol(ate)–thioester exchange step is freely reversible in the presence of an added arylthiol catalyst.

The high yields of final ligation product obtained, even in the presence of internal Cys residues in either/both segments, is the result of the irreversibility of the second (S-to-N acyl shift) amide-forming step under the reaction conditions used.

Theodor Wieland and 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 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.

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.