Redox-Controlled Chemical Protein Synthesis: Sundry Shades of Latency.

The last two decades have witnessed the rise in power of chemical protein synthesis to the point where it now constitutes an established corpus of synthetic methods efficiently complementing biological approaches. One factor explaining this spectacular evolution is the emergence of a new class of chemoselective reactions enabling the formation of native peptide bonds between two unprotected peptidic segments, also known as native ligation reactions. In recent years, their application has fueled the production of homogeneous batches of large and highly decorated protein targets with a control of their composition at the atomic level. In doing so, native ligation reactions have provided the means for successful applications in chemical biology, medicinal chemistry, materials science, and nanotechnology research.The native chemical ligation (NCL) reaction has had a major impact on the field by enabling the chemoselective formation of a native peptide bond between a C-terminal peptidyl thioester and an N-terminal cysteinyl peptide. Since its introduction in 1994, the NCL reaction has been made the object of significant improvements and its scope and limitations have been thoroughly investigated. Furthermore, the diversification of peptide segment assembly strategies has been essential to access proteins of increasing complexity and has had to overcome the challenge of controlling the reactivity of ligation partners.One hallmark of NCL is its dependency on thiol reactivity, including for its catalysis. While Nature constantly plays with the redox properties of biological thiols for the regulation of numerous biochemical pathways, such a control of reactivity is challenging to achieve in synthetic organic chemistry and, in particular, for those methods used for assembling peptide segments by chemical ligation. This Account covers the studies conducted by our group in this area. A leading theme of our research has been the conception of controllable acyl donors and cysteine surrogates that place the chemoselective formation of amide bonds by NCL-like reactions under the control of dichalcogenide-based redox systems. The dependency of the redox potential of dichalcogenide bonds on the nature of the chalcogenides involved (S, Se) has appeared as a powerful means for diversifying the systems, while allowing their sequential activation for protein synthesis. Such a control of reactivity mediated by the addition of harmless redox additives has greatly facilitated the modular and efficient preparation of multiple targets of biological relevance. Taken together, these endeavors provide a practical and robust set of methods to address synthetic challenges in chemical protein synthesis.

Thiol Catalysis of Selenosulfide Bond Cleavage by a Triarylphosphine.

The arylthiol 4-mercaptophenylacetic acid (MPAA) is a powerful catalyst of selenosulfide bond reduction by the triarylphosphine 3,3',3″-phosphanetriyltris(benzenesulfonic acid) trisodium salt (TPPTS). Both reagents are water-soluble at neutral pH and are particularly adapted for working with unprotected peptidic substrates. Contrary to trialkylphosphines such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP), TPPTS has the advantage of not inducing deselenization reactions. We believe that the work reported here will be of value for those manipulating selenosulfide bonds in peptidic or protein molecules.

A Selenium-based Cysteine Surrogate for Protein Chemical Synthesis.

N-selenoethyl cysteine (SetCys) in the form of its cyclic selenosulfide is a cysteine surrogate, whose reactivity depends on the reducing power of the medium. SetCys does not interfere with the native chemical ligation reaction under mild reducing conditions, that is in the absence of tris(2-carboxyethyl)phosphine (TCEP). In contrast, subjecting SetCys to TCEP results in the spontaneous loss of its N-selenoethyl appendage and thus to its conversion into a Cys residue. Therefore, SetCys can be used for the redox-controlled assembly of peptide segments using NCL. We provide in this protocol detailed procedures for the synthesis of Fmoc-protected SetCys residue and for its incorporation into peptides using standard solid-phase peptide synthesis protocols. We also describe its use for the chemical synthesis of proteins through the redox-controlled assembly of three peptide segments in one-pot.

Pedal to the Metal: The Homogeneous Catalysis of the Native Chemical Ligation Reaction.

The native chemical ligation reaction of peptide thioesters with cysteinyl peptides is a pivotal chemical process in the production of native or modified peptides and proteins, and well beyond in the preparation of various biomolecule analogs and materials. To benefit from this reaction at its fullest and to access all the possible applications, the experimentalist needs to know the factors affecting its rate and how to control it. This concept article presents the fundamental principles underlying the rate of the native chemical ligation and its homogeneous catalysis by nucleophiles. It has been prepared to serve as a quick guide in the search for an appropriate catalyst.

A cysteine selenosulfide redox switch for protein chemical synthesis.

The control of cysteine reactivity is of paramount importance for the synthesis of proteins using the native chemical ligation (NCL) reaction. We report that this goal can be achieved in a traceless manner during ligation by appending a simple N-selenoethyl group to cysteine. While in synthetic organic chemistry the cleavage of carbon-nitrogen bonds is notoriously difficult, we describe that N-selenoethyl cysteine (SetCys) loses its selenoethyl arm in water under mild conditions upon reduction of its selenosulfide bond. Detailed mechanistic investigations show that the cleavage of the selenoethyl arm proceeds through an anionic mechanism with assistance of the cysteine thiol group. The implementation of the SetCys unit in a process enabling the modular and straightforward assembly of linear or backbone cyclized polypeptides is illustrated by the synthesis of biologically active cyclic hepatocyte growth factor variants.

Strategies and open questions in solid-phase protein chemical synthesis.

The review gives a large overview of the strategies used for protein synthesis by chemoselective peptide segment ligation on a solid support. It discusses also important aspects that remain to be explored to further develop the technology such as the role of the solid support on reactant diffusion rates, on ligation kinetics, as well as on the folding and functionality of the proteins attached to the solid support.

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

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 at Serine Revisited.

Standard conditions for the formation of seryl-cysteinyl junctions by Native Chemical Ligation (NCL) can result in significant epimerization of the serine residue. Epimerization can be minimized to background level by adjusting peptide concentration and working at 4 °C.

Catalysis of Thiol-Thioester Exchange by Water-Soluble Alkyldiselenols Applied to the Synthesis of Peptide Thioesters and SEA-Mediated Ligation.

N-Alkyl bis(2-selanylethyl)amines catalyze the synthesis of peptide thioesters or peptide ligation from bis(2-sulfanylethyl)amido (SEA) peptides. These catalysts are generated in situ by reduction of the corresponding cyclic diselenides by tris(2-carboxyethyl)phosphine. They are particularly efficient at pH 4.0 by accelerating the thiol-thioester exchange processes, which are otherwise rate-limiting at this pH. By promoting SEA-mediated reactions at mildly acidic pH, they facilitate the synthesis of complex peptides such as cyclic O-acyl isopeptides that are otherwise hardly accessible.

Anion Recognition by Aliphatic Helical Oligoureas.

Anion binding properties of neutral helical foldamers consisting of urea type units in their backbone have been investigated. 1 H NMR titration studies in various organic solvents including DMSO suggest that the interaction between aliphatic oligoureas and anions (CH3 COO- , H2 PO4- , Cl- ) is site-specific, as it largely involves the urea NHs located at the terminal end of the helix (positive pole of the helix), which do not participate to the helical intramolecular hydrogen-bonding network. This mode of binding parallels that found in proteins in which anion-binding sites are frequently found at the N-terminus of an α-helix. 1 H NMR studies suggest that the helix of oligoureas remains largely folded upon anion binding, even in the presence of a large excess of the anion. This study points to potentially useful applications of oligourea helices for the selective recognition of small guest molecules.

Designing foldamer-foldamer interactions in solution: the roles of helix length and terminus functionality in promoting the self-association of aminoisobutyric acid oligomers.

The biological activity of antibiotic peptaibols has been linked to their ability to aggregate, but the structure-activity relationship for aggregation is not well understood. Herein, we report a systematic study of a class of synthetic helical oligomer (foldamer) composed of aminoisobutyric acid (Aib) residues, which mimic the folding behavior of peptaibols. NMR spectroscopic analysis was used to quantify the dimerization constants in solution, which showed hydrogen-bond donors at the N terminus promoted aggregation more effectively than similar modifications at the C terminus. Elongation of the peptide chain also favored aggregation. The geometry of aggregation in solution was investigated by means of titrations with [D6]DMSO and 2D NOE NMR spectroscopy, which allowed the NH protons most involved in intermolecular hydrogen bonds in solution to be identified. X-ray crystallography studies of two oligomers allowed a comparison of the inter- and intramolecular hydrogen-bonding interactions in the solid state and in solution and gave further insight into the geometry of foldamer-foldamer interactions. These solution-based and solid-state studies indicated that the preferred geometry for aggregation is through head-to-tail interactions between the N and C termini of adjacent Aib oligomers.

Stereoselective synthesis of o-bromo (or iodo)aryl P-chirogenic phosphines based on aryne chemistry.

The efficient synthesis of chiral or achiral tertiary phosphines bearing an o-bromo (or iodo)aryl substituent is described. The key step of this synthesis is based on the reaction of a secondary phosphine borane with the 1,2-dibromo (or diiodo)arene, owing to the formation in situ of an aryne species in the presence of n-butyllithium. When P-chirogenic secondary phosphine boranes were used, the corresponding o-halogeno-arylphosphine boranes were obtained without racemization in moderate to good yields and with ee up to 99%. The stereochemistry of the reaction, with complete retention of the configuration at the P atom, has been established by X-ray structures of P-chirogenic o-halogenophenyl phosphine borane complexes. The decomplexation of the borane was easily achieved without racemization using DABCO to obtain the free o-halogeno-arylphosphines in high yields.