Native chemical ligation (NCL) is a fundamental peptide chemistry method introduced by Philip Dawson and Stephen Kent in 1994 (Science 266:776), allowing assembly of two peptide fragments into a single native (unmodified) peptide bond via chemoselective reaction under mild aqueous conditions. This innovation revolutionised chemical synthesis of >50-residue proteins, previously limited by direct SPPS (yield dropping exponentially beyond 50 aa).
Chemical principle. NCL relies on the reaction between a C-terminal thioester peptide (—COSR, prepared by SPPS with Dawson/MeDbz/sulfonamide resin) and an N-terminal cysteine peptide (—H2N-Cys-peptide). The Cys thiol attacks the thioester carbonyl forming a transient thioester intermediate, which undergoes intramolecular S→N rearrangement (5-exo-trig) via thiolate cyclisation on the Cys free amine, producing a native peptide bond with Cys at the junction. The reaction is performed in aqueous buffer pH 6.5-7.5 with MPAA (4-mercaptophenylacetic acid) as catalytic thiol, TCEP as reducer, 6M guanidine as denaturant for insoluble peptides.
Extensions and variants. The Cys requirement at the junction is bypassed by various extensions: post-NCL desulfurisation (Cys → Ala, Yan & Dawson 2001), extended ligation with selenocysteine (Sec, Hondal 2001), alanine/valine/leucine/proline thiol-surrogates (β-thiol-Ala, γ-thiol-Val, Payne & Chen), expressed protein ligation (EPL, Muir 1998, biological thioester via intein), traceless Staudinger ligation (Raines 2000) or KAHA ligation (Bode).
Major applications. NCL and its extensions have enabled chemical synthesis of insulin, ribonuclease A, histones, ubiquitin, β-amyloid, HIV protease, transthyretin and other proteins up to ~300 aa. It allows site-specific incorporation of post-translational modifications (phosphorylation, glycosylation, acetylation, methylation, ubiquitination), non-natural amino acids, fluorescent tags and isotopic analogues — impossible by recombinant expression or classical linear SPPS.
