Native chemical ligation

Native chemical ligation or NCL is an important extension of the chemical ligation field, a concept for constructing a large polypeptide formed by the assembling of two or more unprotected peptides segments. Especially, NCL is the most powerful ligation method for synthesizing native backbone proteins or modified proteins of moderate size (i.e., small proteins< 200 AA).

In native chemical ligation, the thiol group of an N-terminal cysteine residue of an unprotected peptide 2 attacks the C-terminal thioester of a second unprotected peptide 1 in an aqueous buffer at pH 7.0, 20 °C<T<37 °C. This reversible transthioesterification step is chemoselective and regioselective and leads to form a thioester intermediate 3. This intermediate rearranges by an intramolecular S,N-acyl shift that results in the formation of a native amide ('peptide') bond 4 at the ligation site (scheme 1).

Remarks :

NCL reaction is catalyzed by in situ transthioesterification with thiol additives. The most common thiol catalysts to date have been either a mixture of thiophenyl, 4-mercaptophenylacetic acid (MPAA), or 2-mercaptoethanesulfonate (MESNa). (ref)

The main property of the NCL method 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 exogenous thiol added as 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, under the reaction conditions used, of the second (S-to-N acyl shift) amide-forming step.

No side-products are formed from reaction with the other functional groups present in either peptide segment (acids or basics amino groups, phenolic hydroxyls, etc.).

In 1953, Theodor Wieland and coworkers discovered the chemical foundation for this reaction, when the reaction of valine-thioester and cysteine amino acid in aqueous buffer was shown to yield the dipeptide valine-cysteine. The reaction proceeded through the intermediacy of a thioester containing the sulfur of the cysteine residue. Wieland's work led to the 'active ester' method for making protected peptide segments in conventional solution synthesis in organic solvents.

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