Chemical ligation

Chemical ligation is a set of techniques used for creating long peptide or protein chains. It is the second step of a convergent approach. First smaller peptides containing 30-50 amino acids are prepared by conventional chemical peptide synthesis. They are then completely deprotected. Chemical ligation is the technique of coupling these peptides by chemoselective reaction to give a unique reaction product, usually in aqueous solution. With several coupling steps, proteins of up to 200-300 amino acids can be produced.

Methods of chemical ligation
There are various techniques described in literature. The most practical and robust method for the chemoselective reaction of unprotected peptides is native chemical ligation. Native chemical ligation has overcome the limitations of the classical synthetic organic chemistry approach to the total synthesis of proteins, and enables the routine total or semi- synthesis of protein molecules. The original chemical ligation methods involved the formation of a non-native bond at the ligation site. Subsequently, native chemical ligation was developed. Based on chemistry first reported in 1953 by Theodor Weiland, an unprotected peptide-thioester reacts with a Cys-peptide to give a ligation product with a peptide bond at the ligation site. In this method, the initial thioester-linked ligation product intermediate rearranges to form an amide bond.

Native chemical ligation relies on the presence of a cysteine residue at the ligation site, the second least common amino acid found in proteins. Methods using removable auxiliary groups can overcome this limitation in some instances, as can the use of desulfurization subsequent to the ligation (e.g. converting a Cys to an Ala).

Expressed protein ligation
By exploiting naturally occurring inteins it is possible to prepare a recombinant polypeptide C-terminal thioester. This removes the size restriction of the C-terminal thioester fragment used in native chemical ligation. The recombinant thioester can be ligated to a synthetic peptide bearing an N-terminal cysteine. Native Chemical Ligation of this kind using recombinant C-terminal thioesters is known as Expressed Protein Ligation. Recombinant expression can also be used to give a Cys-polypeptide for use in native chemical ligation.

Staudinger ligation
The Staudinger ligation, first reported in 2000, enables the ligation of peptide segments independent of the terminal amino acids. The method is based on the Staudinger reaction. The Staudinger ligation continues to be developed and has not yet found widespread use.