Protocol for protein modification using oxalyl thioester-mediated chemoselective ligation.

The development of fast ligation chemistries for the site-specific modification of proteins has become a major focus in chemical biology. We describe steps for preparing an oxalyl thioester precursor in the form of an N-oxalyl perhydro-1,2,5-dithiazepine handle, i.e.

Protein desulfurization and deselenization.

Methods enabling the dechalcogenation of thiols or selenols have been investigated and developed for a long time in fields of research as diverse as the study of prebiotic chemistry, the engineering of fuel processing techniques, the study of biomolecule structures and function or the chemical synthesis of biomolecules. The dechalcogenation of thiol or selenol amino acids is nowadays a particularly flourishing area of research for being a pillar of modern chemical protein synthesis, when used in combination with thiol or selenol-based chemoselective peptide ligation chemistries. This review offers a comprehensive and scholarly overview of the field, emphasizing emerging trends and providing a detailed and critical mechanistic discussion of the dechalcogenation methods developed so far.

Incorporation of a Highly Reactive Oxalyl Thioester-Based Interacting Handle into Proteins.

Providing biomolecules with extended physicochemical, biochemical, or biological properties is a contemporary challenge motivated by impactful benefits in life or materials sciences. In this study, we show that a latent and highly reactive oxalyl thioester precursor can be efficiently introduced as a pending functionality into a fully synthetic protein domain following a protection/late-stage deprotection strategy and can serve as an on-demand reactive handle. The approach is illustrated with the production of a 10 kDa ubiquitin Lys48 conjugate.

Electrostatic Assistance of 4-Mercaptophenylacetic Acid-Catalyzed Native Chemical Ligation.

4-Mercaptophenylacetic acid (MPAA) is a popular catalyst of the native chemical ligation (NCL) but has to be used in large excess for achieving practically useful rates (up to 50-100 equiv). We report here that the catalytic potency of MPAA can be boosted by introducing a stretch of arginines in the departing thiol from the thioester. By doing so, the electrostatically assisted NCL reaction proceeds rapidly by using substoichiometric concentrations of MPAA, an advantage that enables useful synthetic applications.

An Iron-Catalyzed Protein Desulfurization Method Reminiscent of Aquatic Chemistry.

One pillar of protein chemical synthesis based on the application of ligation chemistries to cysteine is the group of reactions enabling the selective desulfurization of cysteine residues into alanines. Modern desulfurization reactions use a phosphine as a sink for sulfur under activation conditions involving the generation of sulfur-centered radicals. Here we show that cysteine desulfurization by a phosphine can be effected efficiently by micromolar concentrations of iron under aerobic conditions in hydrogen carbonate buffer, that is using conditions that are reminiscent of iron-catalyzed oxidation phenomena occurring in natural waters.

A biomimetic electrostatic assistance for guiding and promoting N-terminal protein chemical modification.

The modification of protein electrostatics by phosphorylation is a mechanism used by cells to promote the association of proteins with other biomolecules. In this work, we show that introducing negatively charged phosphoserines in a reactant is a powerful means for directing and accelerating the chemical modification of proteins equipped with oppositely charged arginines. While the extra charged amino acid residues induce no detectable affinity between the reactants, they bring site-selectivity to a reaction that is otherwise devoid of such a property.

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.

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.

Fast Protein Modification in the Nanomolar Concentration Range Using an Oxalyl Amide as Latent Thioester.

We show that latent oxalyl thioester surrogates are a powerful means to modify peptides and proteins in highly dilute conditions in purified aqueous media or in mixtures as complex as cell lysates. Designed to be shelf-stable reagents, they can be activated on demand to enable ligation reactions with peptide concentrations as low as a few hundred nM at rates approaching 30 M  s .

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.

Insights into the Mechanism and Catalysis of Peptide Thioester Synthesis by Alkylselenols Provide a New Tool for Chemical Protein Synthesis.

While thiol-based catalysts are widely employed for chemical protein synthesis relying on peptide thioester chemistry, this is less true for selenol-based catalysts whose development is in its infancy. In this study, we compared different selenols derived from the selenocysteamine scaffold for their capacity to promote thiol-thioester exchanges in water at mildly acidic pH and the production of peptide thioesters from (2-sulfanylethyl)amido (SEA) peptides. The usefulness of a selected selenol compound is illustrated by the total synthesis of a biologically active human chemotactic protein, which plays an important role in innate and adaptive immunity.

Chemical Protein Synthesis in Medicinal Chemistry.

Although the majority of proteins used for biomedical research are produced using living systems such as bacteria, biological means for producing proteins can be advantageously complemented by protein semisynthesis or total chemical synthesis. The latter approach is particularly useful when the proteins to be produced are toxic for the expression system or show unusual features that cannot be easily programmed in living organisms. The aim of this review is to provide a wide overview of the use of chemical protein synthesis in medicinal chemistry with a special focus on the production of post-translationally modified proteins and backbone cyclized proteins.

Catalysis of Hydrazone and Oxime Peptide Ligation by Arginine.

Hydrazone and oxime peptide ligations are catalyzed by arginine. The catalysis is assisted intramolecularly by the side-chain guanidinium group. Hydrazone ligation in the presence of arginine proceeds efficiently in phosphate buffer at neutral pH but is particularly powerful in bicarbonate/CO buffer.

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.

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.

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.

A statistical view of protein chemical synthesis using NCL and extended methodologies.

Native chemical ligation and extended methodologies are the most popular chemoselective reactions for protein chemical synthesis. Their combination with desulfurization techniques can give access to small or challenging proteins that are exploited in a large variety of research areas. In this report, we have conducted a statistical review of their use for protein chemical synthesis in order to provide a flavor of the recent trends and identify the most popular chemical tools used by protein chemists.

From protein total synthesis to peptide transamidation and metathesis: playing with the reversibility of N,S-acyl or N,Se-acyl migration reactions.

Amide forming reactions are central to the field of peptide and protein synthesis and are considered to be poorly reversible reactions owing to the high stability of peptide bonds. One amide-forming reaction is native chemical ligation (NCL) which is driven by a sulfur to nitrogen acyl migration process from a transient thioester intermediate. However, recent studies have revealed the reversibility of the S,N-acyl shift reaction or of the related Se,N-acyl shift process using mild aqueous conditions.

Cyclic sulfamidates as vehicles for the synthesis of poly- and diversely substituted benzosultams via unusual S(O)2-O bond cleavage.

1,2-Cyclic sulfamidates undergo novel, efficient, and regiospecific intramolecular nucleophilic cleavage with aryllithiated species to provide an entry to poly-, diversely, and enantiopure N-substituted benzosultams.

Effect of fluorination on the pharmacological profile of 11beta isomers of fulvestrant in breast carcinoma cells.

We describe the synthesis of an 11beta isomer 3 of the steroidal antiestrogen fulvestrant 2. Partial fluorination of the 11beta side chain in 3 leads to 4, which still shows strong antiproliferative activity on MCF-7 cells. However, unlike 2 and 3, compound 4 fails to down-regulate estrogen receptor alpha (ERalpha).

Fluorous tolerance of the estrogen receptor alpha as probed by 11-polyfluoroalkylestradiol derivatives.

The concern of this work was to try to delineate factors, inherent to fluorination, susceptible to influence estradiol binding to the estrogen receptor alpha (ERalpha). For this purpose, fluorinated chains were linked at 11beta position of the steroid (i.e.

Loss of antagonistic activity of tamoxifen by replacement of one N-methyl of its side chain by fluorinated residues.

Efforts to limit the metabolic alteration of the aminoalkyl side chain of tamoxifen by fluorination largely decrease its ER-mediated antagonistic properties in MCF-7 cells (i.e., ability to inhibit growth, to stabilize ER, and to modulate ERE and AP-1 transcriptional activity).

Synthesis of 11beta-perfluorohexylestradiol.

[reaction: see text] We report the synthesis of 11beta-perfluorohexylestradiol 1e using a perfluoroorganometallic reagent for the introduction of the fluorous part. This compound is useful for biological studies and for imaging the ERalpha estradiol receptor distribution in the whole cell by secondary ion mass spectrometry (SIMS). The key step of this synthesis involves the radical reduction of an 11beta-oxalate derivative.