Modern Extensions of Native Chemical Ligation for Chemical Protein Synthesis

Malins, Lara R.; Payne, Richard J.

doi:10.1007/128_2014_584

Cited by 19 publications

(6 citation statements)

References 165 publications

(296 reference statements)

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“…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.…”

mentioning

confidence: 99%

Amide-forming chemical ligation via O -acyl hydroxamic acids

Dunkelmann

Hirata

Totaro

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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The facile rearrangement of "-acyl isopeptides" to native peptide bonds via ,-acyl shift is central to the success of native chemical ligation, the widely used approach for protein total synthesis. Proximity-driven amide bond formation via acyl transfer reactions in other contexts has proven generally less effective. Here, we show that under neutral aqueous conditions, "-acyl isopeptides" derived from hydroxy-asparagine [aspartic acid-β-hydroxamic acid; Asp(β-HA)] rearrange to form native peptide bonds via an ,-acyl shift. This process constitutes a rare example of an ,-acyl shift that proceeds rapidly across a medium-size ring (t ∼ 15 min), and takes place in water with minimal interference from hydrolysis. In contrast to serine/threonine or tyrosine, which form -acyl isopeptides only by the use of highly activated acyl donors and appropriate protecting groups in organic solvent, Asp(β-HA) is sufficiently reactive to form-acyl isopeptides by treatment with an unprotected peptide-thioester, at low mM concentration, in water. These findings were applied to an acyl transfer-based chemical ligation strategy, in which an unprotected -terminal Asp(β-HA)-peptide and peptide-thioester react under aqueous conditions to give a ligation product ultimately linked by a native peptide bond.

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mentioning

confidence: 99%

Amide-forming chemical ligation via O -acyl hydroxamic acids

Dunkelmann

Hirata

Totaro

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…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.…”

Section: Molecular Engineering Toolbox For Complex Biological Samplesmentioning

confidence: 99%

“…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.…”

Section: Molecular Engineering Toolbox For Complex Biological Samplesmentioning

confidence: 99%

A molecular engineering toolbox for the structural biologist

Debelouchina

Muir

2017

Quart. Rev. Biophys.

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Exciting new technological developments have pushed the boundaries of structural biology, and have enabled studies of biological macromolecules and assemblies that would have been unthinkable not long ago. Yet, the enhanced capabilities of structural biologists to pry into the complex molecular world have also placed new demands on the abilities of protein engineers to reproduce this complexity into the test tube. With this challenge in mind, we review the contents of the modern molecular engineering toolbox that allow the manipulation of proteins in a site-specific and chemically well-defined fashion. Thus, we cover concepts related to the modification of cysteines and other natural amino acids, native chemical ligation, intein and sortase-based approaches, amber suppression, as well as chemical and enzymatic bio-conjugation strategies. We also describe how these tools can be used to aid methodology development in X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy and in the studies of dynamic interactions. It is our hope that this monograph will inspire structural biologists and protein engineers alike to apply these tools to novel systems, and to enhance and broaden their scope to meet the outstanding challenges in understanding the molecular basis of cellular processes and disease.

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“…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.…”

Section: Introductionmentioning

confidence: 99%

Internal Activation of Peptidyl Prolyl Thioesters in Native Chemical Ligation

Gui

Qiu

et al. 2016

J. Am. Chem. Soc.

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Prolyl thioesters have shown significantly lower reactivities in native chemical ligation (NCL) in comparison to that of the alanyl thioester. This report describes a mild and efficient internal activation protocol of peptidyl prolyl thioesters in NCL without using any thiol-based additives, where the introduction of a 4-mercaptan substituent on the C-terminal proline significantly improves the reactivity of prolyl thioesters via the formation of a bicyclic thiolactone intermediate. The kinetic data indicate that the reaction rate is comparable to that of the reported data of alanyl thioesters, and the mechanistic studies suggest that the ligation of two peptide segments proceeds through an NCL-like pathway instead of a direct aminolysis, which ensures the chemoselectivity and compatibility of various amino acid side chains. This 4-mercaptoprolyl thioester-based protocol also allows an efficient one-pot ligation-desulfurization procedure. The utility of this method has been further demonstrated in the synthesis of a proline-rich region of Wilms tumor protein 1.

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Modern Extensions of Native Chemical Ligation for Chemical Protein Synthesis

Cited by 19 publications

References 165 publications

Amide-forming chemical ligation via O -acyl hydroxamic acids

Amide-forming chemical ligation via O -acyl hydroxamic acids

A molecular engineering toolbox for the structural biologist

Internal Activation of Peptidyl Prolyl Thioesters in Native Chemical Ligation

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