Native chemical ligation

Native chemical ligation is a common form of chemical ligation, a technique for constructing a large peptide from two or more smaller peptides. In native chemical ligation a peptide containing a C-terminal thioester reacts with another peptide containing an N-terminal cysteine, in the presence of an exogenous thiol catalyst. In a thermodynamically-controlled, freely reversible first step a transthioesterification occurs. The product rearranges irreversibly under the usual reaction conditions to form the desired amide bond. The process was based off of a reaction reported in 1953 by Theodor Wieland and further developed in the laboratory of Stephen Kent at The Scripps Research Institute in 1994.



Peptide-thioesters to be used in native chemical ligation are usually prepared by Boc chemistry SPPS; the thioester piece cannot be synthesized with a nucleophilic base, thus disfavoring Fmoc chemistry. Fmoc techniques for generating thioesters involving the modifications of the Kenner 'safety catch' linker are known. Protecting groups on the N-terminal piece cannot have constituents which release as aldehydes or ketones since these will cap the N-terminal cysteine. For the same reason, acetone should be avoided in general use, particularly prior to lyophilization and in washing glassware.

The payoff is that coupling long peptides by this technique is in many cases nearly quantitative and provides synthetic access to proteins otherwise impossible to make, due to length or decoration by posttranslational modification.

A limitation of this technique is that cysteine has to be part of the produced protein at a suitable place. For some proteins homocysteine can be used and methylated after coupling to form methionine. Cysteine can also be desulfurized to alanine. Alternately, ligation auxiliaries can be used that mimic an N-terminal cysteine for the ligation reaction, but which can be removed after synthesis.

Polypeptide C-terminal thioesters produced by recombinant DNA techniques can be reacted with an N-terminal Cys containing polypeptide by the same native ligation chemistry to provide very large semisynthesized proteins. Thus, by exploiting nature's inteins to prepare a recombinant C-terminal thioester, the size restriction of the reacting peptide segment is removed. Similarly, a recombinant protein containing an N-terminal Cys can be reacted with a synthetic polypeptide thioester. Native Chemical Ligation of this kind using a recombinant polypeptide segment is known as Expressed Protein Ligation.