Redox-Controlled Chemical Protein Synthesis: Sundry Shades of Latency

Abstract

Conspectus 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.

An Iron‐Catalyzed Protein Desulfurization Method Reminiscent of Aquatic Chemistry

“…Designing one-pot NCL/iron-catalyzed desulfurization methods is an important goal to achieve for enabling a reduction of pot economy during chemical protein synthesis, and thus a significant gain in time and yield. [28] During the first attempts to perform an NCL/desulfurization process in one-pot, the addition of TCEP to the ligation mixture triggered the conversion of Cys residue into Ala as expected but at a rate significantly less than those measured for the desulfurization experiments performed on purified peptides. The only difference in reaction mixture composition between the one-pot NCL/desulfurization process and the desulfurization of a purified peptide is the additional presence in the former conditions of a thiol that is released from the thioester component, i.e.…”

“…Designing one‐pot NCL/iron‐catalyzed desulfurization methods is an important goal to achieve for enabling a reduction of pot economy during chemical protein synthesis, and thus a significant gain in time and yield [28] . During the first attempts to perform an NCL/desulfurization process in one‐pot, the addition of TCEP to the ligation mixture triggered the conversion of Cys residue into Ala as expected but at a rate significantly less than those measured for the desulfurization experiments performed on purified peptides.…”

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

“…The developed conditions demonstrated satisfactory effectiveness and sufficient selectivity to be applied at the level of the synthetic protein domain (Figure a). In order to ensure that the Mob protection would survive the whole assembly sequence, we engaged model thioester precursor 7a and the protected oxo SEA-containing peptide 5c in an NCL reaction. , Polypeptide 9a was isolated in 75% yield. It is worth noting that using a 3-mercaptopropionic acid-derived peptide thioester instead of SEA peptide 7a resulted in the exact same outcome, showing that different peptide acyl donors can be used for the NCL step (Supporting Information).…”