Synthèse Totale de Protéines : Développement d'Outils Innovants pour la Ligation Native de Fragments Peptidiques. – SynProt

Thanks to the development of native chemical ligation (NCL) in early 90's by Kent and co-workers, chemical protein synthesis has shifted into the realms of the achievable. The basis of this methodology is the chemo- and regioselective reaction of a peptide thioester and a N-terminal cysteinyl peptide, and produces a 'natural' peptide bond. This allowed the production of site-specific modified proteins (glycoproteins, phosphorylation) as well as cytotoxic or hydrophobic proteins previously unattainable by molecular biology. Contrasting with the major scientific breakthrough, the applicability of NCL is restricted to proteins containing a cysteine residue which participates in the reaction as nucleophilic reaction partner in inducing a preliminary dynamic thioester exchange step : this dramatically reduces the usefulness of this reaction to a handful of proteins, as cysteine residues are seldom conveniently distributed throughout a peptide sequence so as to facilitate NCL. To remove this restriction, a lot of strategies are currently being actively explored by a few world-leading groups, mostly based in the USA but also in UK and Germany (French teams being almost virtually absent from this emerging discipline). The goal of my project resides in overtaking one the main drawback of classic NCL, the first step based on a slow, reversible, thioester exchange. To explore innovative native ligation procedures where the initial reaction that link the two unprotected peptide fragment would be a fast and irreversible process, two dramatically different concepts will be investigated: the triazole-capture ligation (TCL) and a more universal 'DMAP-handle Native chemical Ligation' (DMAP-NL). If the former implies the exceptional chemo- and regioselectivities of the recently-discovered Copper(I)-catalyzed Alkyne-Azide Cycloaddition to assist the native ligation process, the latter is based on a totally new concept of DMAP-organocatalyzed native ligation. The breakthrough provided by the exploration of the TCL and DMAP-NL will be extended to the multi-ligation strategies which are required for the synthesis of proteins bigger than 60 to 100 amino acids. The final biological application of this chemical project concerns the production of MccJ25, a new anti-microbial peptide whose production is extremely challenging due to its original [1]rotaxane peptidic topology. In a long-term course, this project will output on the synthesis of modified microcins J25 lasso-peptides, crucial chemical tools to clarify the molecular bases of the biochemical pathway to this naturally-occurring, mechanically-interlocked peptide.