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Synthèse chimio-enzymatique de sondes moléculaire pour la caractérisation de protéines affines des chitinoligosaccharides

Abstract : Chitinoligosaccharides (COs) play major roles in plants. While long COs (6-8 saccharide units) are elicitors activating plant defense mechanisms against pathogenic microorganisms, short COs (4-5 saccharide units) would participate in the establishment of symbioses with beneficial microorganisms allowing better assimilation of soil nutrients. Identifying the receptors involved in these processes to understand how plants discriminate these signal molecules requires having access to pure molecules with well-defined degrees of polymerization. In this thesis, we focused on the synthesis of well-defined COs and their modification to obtain new affinity probes. For this purpose, a design of experiments was developed in order to optimize the production of COs and more particularly of long ones by enzymatic hydrolysis of chitin with a commercial enzyme, hen egg-white lysozyme. Subsequently, a new type of affinity-based probe allowing the specific labeling of CO-binding proteins has been developed. We have shown for the first time that triazinyl glycosides can be effectively used to introduce a fluorescent group on an oligosaccharide-binding protein without any external chemical or physical activation. After demonstrating the proof of concept with lectins, a fluorescent activity-based probe allowing continuous assay of chitinases and their labeling at the same time was synthesized. These new tools offer exciting perspectives for the characterization of CO receptors in plants as well as for the discovery of new lectins and carbohydrate-active enzymes.
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Arnaud Masselin. Synthèse chimio-enzymatique de sondes moléculaire pour la caractérisation de protéines affines des chitinoligosaccharides. Chimie organique. Université Grenoble Alpes, 2018. Français. ⟨NNT : 2018GREAV054⟩. ⟨tel-02076347⟩

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