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-1 s-1 .

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 bis(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.

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.

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.

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. They also enable rate accelerations of four orders of magnitude in some cases, thereby permitting chemical processes to proceed at the protein level in the low micromolar range, using reactions that are normally too slow to be useful in such dilute conditions.

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/CO2 buffer. In addition to acting as a catalyst, arginine prevents the aggregation of proteins during ligation. With its dual properties as a nucleophilic catalyst and a protein aggregation inhibitor, arginine hydrochloride is a useful addition to the hydrazone/oxime ligation toolbox.

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.