Synthesis of Proteins by Native Chemical Ligation

Abstract

A simple technique has been devised that allows the direct synthesis of native backbone proteins of moderate size. Chemoselective reaction of two unprotected peptide segments gives an initial thioester-linked species. Spontaneous rearrangement of this transient intermediate yields a full-length product with a native peptide bond at the ligation site. The utility of native chemical ligation was demonstrated by the one-step preparation of a cytokine containing multiple disulfides. The polypeptide ligation product was folded and oxidized to form the native disulfide-containing protein molecule. Native chemical ligation is an important step toward the general application of chemistry to proteins.

Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase

“…8 Here, we report the first use of this technique to localize dynamic effects in hydrogen transfer reactions. EcDHFR derivatives that were isotopically labeled in the N‐terminal (residues 1–28; NT‐EcDHFR) and C‐terminal (residues 29–159; CT‐EcDHFR) segments were constructed.…”

Total synthesis by modern chemical ligation methods and high resolution (1.1 Å) X‐ray structure of ribonuclease A

“…The protein has 124 amino acid polypeptide chain ( Figure 1) that contains eight cysteine residues which form four disulfide bonds, providing ample options for thioestermediated native chemical ligation at cysteine residues. 11 In exploratory syntheses we evaluated several possible synthetic schemes using a variety of these potential ligation sites.…”

“…Chemical ligation involves the chemoselective joining, by means of unique mutually reactive moieties-one on each peptide-of two unprotected peptide segments. Native chemical ligation 11 has proven to be the most practical of the ligation chemistries and has found the most use in the total chemical synthesis of proteins. In native chemical ligation a native peptide bond is formed between an unprotected peptide and a peptide-a thioester, both prepared by solid phase peptide synthesis.…”

From Chemical Mutagenesis to Post‐Expression Mutagenesis: A 50 Year Odyssey

“…Creative application of such an approach, aided by the rapid maturation of ligation technologies, has significant potential for new approaches in chemical mutagenesis. Native‐chemical ligation (NCL; Scheme 3 b)8c is perhaps the seminal example of the utility of chemical approaches to protein synthesis (and thus the incorporation of modifications). Although our focus is on varied methods, some key features that lie behind the success of the reaction are worth considering as potential guidelines for the design of future reactions that rely on other disconnections.…”