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. Then, they are 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.
Native chemical ligation
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. In native chemical ligation, an unprotected peptide-thioester reacts with a Cys-peptide to give a ligation product with a native amide ('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. Methods using removable auxiliary groups can in some instances extend the use of native chemical ligation to non-cysteine residues, 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 enables the use of large recombinant protein-derived thioesters 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.
The Staudinger ligation, first reported in 2000, in principle 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.
Ser/Thr ligation (STL) was introduced into protein chemical synthesis as an alternative ligation strategy. Serine/Threonine-ligation involves merger of a side-chain unprotected peptide segment containing a C-terminal salicylaldehyde (SAL) ester and another peptide segment with an N-terminal Ser or Thr residue. The chemoselective reaction between the peptide SAL ester and 1,2-hydroxylamine group of Ser or Thr leads to the formation of an N,O-benzylidene acetal linked intermediate, which undergoes acidolysis to afford a natural peptidic Xaa-Ser/Thr linkage. This method does not involve the use of unnatural amino acids, and it is simple to operate. Ser/Thr ligation provides a complementary method for protein chemical synthesis and semisynthesis.
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