Native chemical ligation

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Native chemical ligation is the most widely used form of chemical ligation, a technique for constructing a large polypeptide from two or more unprotected 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 added thiol catalyst. In a freely reversible first step, a transthioesterification occurs to yield a thioester-linked intermediate; this intermediate rearranges irreversibly under the usual reaction conditions to form a native amide ('peptide') bond at the ligation site. Native chemical ligation of unprotected peptide segments was developed in the laboratory of Stephen Kent at The Scripps Research Institute in 1994. [The transthioesterification/amide-forming intramolecular rearrangement reaction had been reported in 1953 by Theodor Wieland, who was studying possible chemistries for amino acid addition to a polypeptide chain in protein biosynthesis; Wieland's work led to the 'active ester' method for making protected peptide segments in conventional solution synthesis in organic solvents.]

Native chemical ligation is carried out in aqueous solution and frequently gives near-quantitative yields of the desired ligation product. The challenge is the preparation of the necessary unprotected peptide-thioester building blocks. Peptide-thioesters are usually prepared by Boc chemistry SPPS; a thioester-containing peptide cannot be synthesized using a nucleophilic base, thus disfavoring Fmoc chemistry. Fmoc chemistry solid phase peptide synthesis techniques for generating peptide-thioesters are known; they make use of modifications of the Kenner 'safety catch' linker. In making peptide segments for use in native chemical ligation, protecting groups that release aldehydes or ketones should be avoided since these may cap the N-terminal cysteine. For the same reason, the use of acetone should be avoided, particularly prior to lyophilization and in washing glassware.

A feature of the native chemical ligation technique is that the product polypeptide chain contains cysteine at the site of ligation. For some proteins homocysteine can be used and methylated after ligation to form methionine, although side reactions can occur in this alkylation step. The cysteine at the ligation site can also be desulfurized to alanine; more recently, other beta-thiol containing amino acids have been used for native chemical ligation, followed by desulfurization. Alternately, thiol-containing ligation auxiliaries can be used that mimic an N-terminal cysteine for the ligation reaction, but which can be removed after synthesis. The use of thiol-containing auxiliaries is not as effective as ligation at a Cys residue.

The payoff in the native chemical ligation method is that coupling long peptides by this technique is in many cases nearly quantitative and provides synthetic access to large peptides and proteins otherwise impossible to make, due to length or decoration by posttranslational modification. Native chemical ligation forms the basis of modern chemical protein synthesis, and has been used to prepare numerous proteins and enzymes by total chemical 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 semi-synthetic proteins. Native chemical ligation of this kind using a recombinant polypeptide segment is known as Expressed Protein Ligation. Similarly, a recombinant protein containing an N-terminal Cys can be reacted with a synthetic polypeptide thioester. Thus, native chemical ligation can be used to introduce chemically synthesized segments into recombinant proteins, regardless of size.

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Wieland T, Bokelmann E, Bauer L, Lang HU, Lau H. 1953. “Liebigs Ann. Chem.” 583:129-149.

Dawson PE, Muir TW, Clark-Lewis I, Kent, SBH. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779.

Muir TW, Sondhi D, Cole PA. 1998. Expressed Protein Ligation: A General Method for Protein Engineering. Proc. Natl. Acad. Sci. USA 95:6705-6710.

Nilsson BL, Soellner MB, Raines RT. 2005. Chemical Synthesis of Proteins. Annu. Rev. Biophys. Biomol. Struct. 34:91-118