Modern Extensions of Native Chemical Ligation for Chemical Protein Synthesis

Abstract

Over the past 20 years, native chemical ligation has facilitated the synthesis of numerous complex peptide and protein targets, with and without post-translational modifications, as well as the design and construction of a variety of engineered protein variants. This powerful methodology has also served as a platform for the development of related chemoselective ligation technologies which have greatly expanded the scope and flexibility of ligation chemistry. This chapter details a number of important extensions of the original native chemical ligation manifold, with particular focus on the application of new methods in the total chemical synthesis of proteins. Topics covered include the development of auxiliary-based ligation methods, the post-ligation manipulation of Cys residues, and the synthesis and utility of unnatural amino acid building blocks (bearing reactive thiol or selenol functionalities) in chemoselective ligation chemistry. Contemporary applications of these techniques to the total chemical synthesis of peptides and proteins are described.

Amide-forming chemical ligation via O -acyl hydroxamic acids

“…Due to the facility of the original native chemical ligation chemistry, many methods have been developed to extend its applicability beyond Xaa-Cys ligation sites, by the use of various cysteine surrogates in place of an N-terminal cysteine (17)(18)(19). The abilities of certain nonthiol side-chain functionalities-including imidazoles (20) and carboxylates (21,22)-to facilitate the aminolysis of a peptide-α thioester by N-terminal histidine or Asp/Glu-peptides have been documented.…”

A molecular engineering toolbox for the structural biologist

“…2014; Malins & Payne, 2015). As a consequence of this massive effort, the technique has become a central tool in protein science, having been applied to literally hundreds of protein targets.…”

“…The preceding discussion highlights just a few of the many refinements and extensions the NCL strategy has undergone since its introduction over 20 years ago (Harmand et al . 2014; Malins & Payne, 2015). As a consequence of this massive effort, the technique has become a central tool in protein science, having been applied to literally hundreds of protein targets.…”

Internal Activation of Peptidyl Prolyl Thioesters in Native Chemical Ligation

“…As a milestone advance, in conjunction with the broadly scoped metal-free desulfurization (MFD), NCL is not only one of the most powerful methods in the field of protein chemical synthesis, but it has inspired chemists seeking new chemical methods and tools to study biological problems. A number of ligation methods were thus invented, including auxiliary-based ligation methods, Staudinger ligation, α-ketoacid-hydroxylamine (KAHA) ligation, seleno-amino-acids based ligation, serine/threonine ligation (STL), and peptide hydrazides-based ligation, among others. However, nature always poses new challenges to chemists.…”