The present review offers an overview of nonclassical (e.g., with no pre- or in situ activation of a carboxylic acid partner) approaches for the construction of amide bonds. The review aims to comprehensively discuss relevant work, which was mainly done in the field in the last 20 years. Organization of the data follows a subdivision according to substrate classes: catalytic direct formation of amides from carboxylic and amines (section 2); the use of carboxylic acid surrogates (section 3); and the use of amine surrogates (section 4). The ligation strategies (NCL, Staudinger, KAHA, KATs, etc.) that could involve both carboxylic acid and amine surrogates are treated separately in section 5.

Nonclassical Routes for Amide Bond Formation

The native chemical ligation reaction (NCL) involves reacting a C-terminal peptide thioester with an N-terminal cysteinyl peptide to produce a native peptide bond between the two fragments. This reaction has considerably extended the size of polypeptides and proteins that can be produced by total synthesis and has also numerous applications in bioconjugation, polymer synthesis, material science, and micro- and nanotechnology research. The aim of the present review is to provide a thorough mechanistic overview of NCL and extended methods. The most relevant properties of peptide thioesters, Cys peptides, and common solvents, reagents, additives, and catalysts used for these ligations are presented. Mechanisms, selectivity and reactivity are, whenever possible, discussed through the insights of computational and physical chemistry studies. The inherent limitations of NCL are discussed with insights from the mechanistic standpoint. This review also presents a palette of O, S-, N, S-, or N, Se-acyl shift systems as thioester or selenoester surrogates and discusses the special molecular features that govern reactivity in each case. Finally, the various thiol-based auxiliaries and thiol or selenol amino acid surrogates that have been developed so far are discussed with a special focus on the mechanism of long-range N, S-acyl migrations and selective dechalcogenation reactions.

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations

Methods to prepare proteins that include a specific modification at a desired position are essential for understanding their cellular functions and physical properties in living systems. Chemical protein synthesis, which relies on the chemoselective ligation of unprotected peptides, enables the preparation of modified proteins that are not easily fabricated by other methods. In contrast to recombinant approaches, chemical synthesis can be used to prepare protein analogues such as D-proteins, which are useful in protein structure determination and the discovery of novel therapeutics. Post-translationally modifying proteins is another example where chemical protein synthesis proved itself as a powerful approach for preparing samples with high homogeneity and in workable quantities. In this Review, we discuss the basic principles of the field, focusing on novel chemoselective peptide ligation approaches such as native chemical ligation and the recent advances based on this method with a proven record of success in the synthesis of highly important protein targets.

Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins

Cyclic peptide-based therapeutics have a promising growth forecast that justifies the development of microfluidic systems dedicated to their production, in phase with the actual transitioning toward continuous flow and microfluidic technologies for pharmaceutical production. The application of the most popular method for peptide cyclization in water, i.e., native chemical ligation, under microfluidic conditions is still unexplored. Herein, we report a general strategy for fast and efficient peptide cyclization using native chemical ligation under homogeneous microfluidic conditions. The strategy relies on a multistep sequence that concatenates the formation of highly reactive S-(2-((2-sulfanylethyl)amino)ethyl) peptidyl thioesters from stable peptide amide precursors with an intramolecular ligation step. With very fast ligation rates (<5 min), even for the most difficult junctions (including threonine, valine, isoleucine, or proline), this technology opens the door toward the scale-independent, expedient preparation of bioactive macrocyclic peptides.

/pdf/accelerated-microfluidic-native-chemical-ligation-at-2bbjamztf9.pdf

Accelerated microfluidic native chemical ligation at difficult amino acids toward cyclic peptides

Various bioactive proteins have been synthesized by native chemical ligation (NCL) and its combination with subsequent desulfurization (e.g., conversion from Cys to Ala). In NCL, excess 4-mercaptophenylacetic acid (MPAA) is generally added to facilitate the reaction. However, co-elution of MPAA with the ligation product during preparative high-performance liquid chromatography sometimes reduces its usefulness. In addition, contamination of MPAA disturbs subsequent desulfurization. Here, we report for the first time that imidazole can be adopted as an alternative to MPAA in NCL using a peptide-alkylthioester. The efficiency of the imidazole-aided NCL (Im-NCL) is similar to that of traditional MPAA-aided NCL. As model cases, we successfully synthesized adiponectin(19-107) and [Ser(PO3H2)65]-ubiquitin using Im-NCL with a one-pot desulfurization.

Imidazole‐Aided Native Chemical Ligation: Imidazole as a One‐Pot Desulfurization‐Amenable Non‐Thiol‐Type Alternative to 4‐Mercaptophenylacetic Acid

Acceleration of thiol additive-free native chemical ligation by intramolecular S → S acyl transfer.

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Acceleration of thiol additive-free native chemical ligation by intramolecular S → S acyl transfer.