Native chemical ligation

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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 developed by Phillip Dawson and 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.

Access to large proteins (in excess of 300 amino acids or so) has still not been achieved even with native chemical ligation, although 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 natures inteins it is possible to prepare a recombinant C-terminal thioester the size restriction of the reacting peptide segmentst is removed. Native Chemical Ligations of this kind using recombinant C-terminal thioesters is known as Expressed Protein Ligation.