Synthèse biomimétique et automatisée de peptides crypto-thioester pour la ligation chimique native : application à des peptides naturels riches en cystéine

Abstract : Peptide Cα-thioesters play a prominent role in the chemical synthesis of proteins: they are key partners in native chemical ligation (NCL), a reaction that has revolutionized the field. Nonetheless, access to such peptides via the widely used Fmoc-SPPS is still limited to experts. This limitation is currently an obstacle to further popularization of NCL-based protein synthesis. The first part of this thesis describes the design and optimization of a new bio-inspired methodology for the synthesis of peptide thioester precursors, based on an N-(2-hydroxy-5-nitrobenzyl)-cysteine intramolecular thioesterification device (N-Hnb-Cys). Synthesis of peptides bearing this device was fully automated, starting from inexpensive materials. Importantly, no post-SPPS steps are required prior to NCL. The biomimetic design of the device –that operates through an N→S acyl shift–, results in fast NCL kinetics at neutral pH. A broad range of thioester precursors has been synthesized; this allowed us to explore the scope and limitation of the methodology, while stressing its efficiency even for demanding sequences and long peptides. This approach has been applied to the ligation-based synthesis of a representative variety of naturally occurring disulfide-rich peptides (DRP) sequences, from snake venom, molluscs, primates and plants. In particular, we have described the first synthesis of a Big-defensin (93 amino acids), discovered in oyster and whose biological activity is currently under evaluation. Furthermore, an original method for the synthesis of C-terminal cysteine-containing DRP has been proposed. It is based on the introduction of this amino acid through NCL, avoiding side reactions inherent to existing approaches. We have also applied our approach to the intramolecular NCL-based synthesis of the cyclic backbone of several DRP, with remarkable yields. Altogether, our results are extremely encouraging for the generalization of this methodology.
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Victor Terrier. Synthèse biomimétique et automatisée de peptides crypto-thioester pour la ligation chimique native : application à des peptides naturels riches en cystéine. Sciences agricoles. Université d'Orléans, 2016. Français. ⟨NNT : 2016ORLE2016⟩. ⟨tel-01431163⟩

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