Pseudomonas aeruginosa (PA) est l’un des principaux germes impliqués dans les maladies nosocomiales et est aussi la principale cause de mortalité et morbidité des patients atteints de la mucoviscidose malgré l’utilisation massive d’antibiotiques. Dans la lutte contre PA, une alternative aux antibiotiques est l’inhibition de ses facteurs de virulence notamment ceux impliqués dans l’adhésion et la formation du biofilm via des interactions de type sucres/protéines. Ces protéines sont appelées lectines (PA-IL, PA-IIL, FliD). L’objectif de ce travail est la recherche de molécules inhibitrices (glycoclusters) de ces lectines impliquées dans la virulence de PA. Compte tenu du grand nombre de glycoclusters à tester et des faibles quantités de matériels biologiques disponibles, un outil de criblage innovant a été développé (glycoarray) à partir d’une lame de verre microstructurée et fonctionnalisée chimiquement afin d’immobiliser de manière organisée et ordonnée les glycoclusters. La méthode d’immobilisation choisie est la méthode d’immobilisation spécifique par hybridation de l’ADN appelée DDI : DNA Directed Immobilization. Sur ces glycoarrays, 3 méthodes indépendantes (lecture de fluorescence directe, IC50 et Kd) de mesure des interactions glycoclusters/lectines ont été mises au point et validées par une étude comparative donnant un classement similaire des glycoclusters pour leur affinité vis-à-vis des lectines Il faut noter que ces mesures faites sur glycoarrays ne consomment que quelques picomoles de glycoclusters comparées aux méthodes classiques (ITC, ELLA, RMN, …) qui nécessitent des micromoles de produits. A l’aide de ces glycoarrays, un criblage d’une bibliothèque d’une centaine de glycoclusters multivalents, de différentes topologies, charges et linkers a permis d’identifier deux structures montrant une très forte affinité vis-à-vis des lectines de PA. Ces glycoclusters sont actuellement en test in vitro et in vivo. Ces études d’interactions sur DDI-glycoarray ont été étendues à d’autres agents pathogènes tels que les bactéries Burkholderia ambifaria, Viscum album ou contre le virus de la grippe. Dans le futur, pour mieux appréhender les mécanismes d’interactions sucres/protéines, il serait intéressant de pouvoir suivre en temps réel ces interactions en utilisant des systèmes de détection sans marquage tel que, par exemple, la résonance plasmonique de surface. Aussi, le dernier chapitre donne les prémices d’une adaptation de la méthode DDI sur glycoarray sur surface d’or. / Pseudomonas aeruginosa (PA) is one of the predominant bacterium encountered in nosocomial infections. PA infections often lead to chronic inflammation and eventually to death despite aggressive antibiotic therapy. A promising approach is to inhibit the virulence factors of PA such as PA-IL, PA-IIL, FliD (lectins). Therefore, there is a great interest for studying carbohydrate/lectin interactions in order to design new treatments. The goal of this work is the research for inhibitory molecules (glycoclusters ) of these lectins involved in the virulence of PA. An innovative screening tool for studying carbohydrate/lectin interactions has been developed (glycoarray). Glycoarray are microstructured glass-slides, chemically functionalized in order to immobilize, organized and orderly, glycoclusters at the surface. The immobilization method is the specific immobilization method based on DNA hybridization called DDI (DNA Directed Immobilization). This miniaturized analytical biosystem allows multiplex test performed in one single microwell. Moreover, three independent methods of affinity measurement (direct fluorescence read-out, IC50 and Kd) have been developed and validated by a comparative study giving a similar ranking of glycoclusters for their affinity towards PA-IL. These measurements on glycoarrays consume only a few picomoles glycoclusters compared to conventional methods (ITC, ELLA...) that require micromoles of products. Using these glycoarrays, the screening of a library of hundreds of glycoclusters presenting different topologies, multivalencies, charges and linkers led to the identification of two structures showing a very strong affinity for PA lectins. These glycoclusters are currently in vitro assay and in vivo. These interaction studies on DDI-glycoarray were extended to other pathogens such as Burkholderia ambifaria bacteria, Viscum album or against the influenza virus. In the future, to better understand the mechanisms of sugar / protein interactions, it would be interesting to monitor in real time the interactions using label-free detection systems such as, for example, the surface plasmon resonance (SPR). Also, the last chapter gives the beginnings of an adaptation of the method of DDI glycoarray on gold surface

Les interactions entre les sucres et les lectines sont généralement l'étape clé dans de nombreux phénomènes biologiques et pathologiques. Malgré leu r importance cruciale, ces interactions sont paradoxalement caractérisées par des constantes d'affinité faibles et nécessite une multiprésentation des motifs saccharidiques pour être significatives. Cette augmentation est appelée "effet cluster". En outre, les techniques d'analyse actuellement utilisées en laboratoire nécessitent des quantités importantes de produits, ce qui est difficilement compatible avec les méthodes de synthèse actuelle. Pour pallier ces difficultés, une approche originale basée sur l'utilisation conjointe de glycooligonucléotide et de puces à ADN a été proposée. Les glycoconjugués basés sur des squelettes phosphodiesters et couplés à des séquences d'ADN ont été synthétisés en utilisant la chimie des oligonucléotides, couplée à la "click chemistry". La séquence d'ADN quant à elle a permis l'ancrage sur une puce à ADN et donc la mesure de leur affinité vis-à-vis de différentes lectines. Ce manuscrit rapporte le développement des nouvelles méthodologies de synthèse des glycooligonucléotides ainsi que la préparation de nombreux glycoconjugués originaux, dont l'affinité pour différentes lectines a été mesurée via l'utilisation de la puce à ADN. L'influence de plusieurs paramètres a été étudiée: le nombre de résidus, l'arrangement spatial, la lipophilie etc. Il s'avère que l'arrangement spatial semble être l'un des points les plus importants dans la mise au point d'un glycoconjugué. / The interactions between carbohydrates and lectins are generally the "key step" in many biological and pathological phenomena. Despite their importance, these interactions are paradoxically characterized by low affinity constants and requires multipresence of saccharide to be significant. This increase is called "cluster effect". In addition, the analysis techniques currently used in the laboratory requires large quantities of products, which is hardly compatible with the current methods of synthesis. To circumvent these difficulties, a original approach based on the combined use of glycooligonucleotides and DNA microarrays has been proposed. Glycoconjugates based on phosphodiester skeletons linked to DNA sequences have been synthesized using the chemistry of oligonucleotides, coupled with the "click chemistry". The DNA sequence has allowed the anchoring on a DNA chip and therefore the measurement of their affinity versus different lectins.This manuscript reports the development of new synthetic methodologies for the glycooligonucleotides synthesis and the preparation of many original glycoconjugates, whose affinity for various lectins was measured through the use of DNA microarray. The influence of several parameters was studied: the number of residues, the spatial arrangement, etc. lipophilicity. The spatial arrangement appears to be one of the most important parameters in the development of a glycoconjugate.

Microarrays enable high throughput analysis with minute amounts of analyte. They are widely used in the ’omics’ fields both as diagnostic and analytical tools. Their ability to dramatically impact an entire field of research has focused our attention on the development of novel methods for the formation, analysis and applications of microarrays to study carbohydrate-protein interactions and the analysis of glycosylation patterns of biomolecules. Availability of appropriately modified ligands is often a limiting factor in the preparation of microarrays. To address this issue robust routes for the synthesis of nine amino ethylglycosides were developed that can be employed for microarray formation. The syntheses of more complex ligands typically deliver small quantities of material despite the requirements for special skills, equipment and long preparation times. Considering the number of complex oligosaccharides that are necessary for systematic microarray studies, the problem of availability of these complex structures is difficult to address solely with synthetic ligands. A modified native chemical ligation (NCL) strategy, in which a surface bound oxo-ester is used instead of a thioester, was optimised and used for efficient chemoselective immobilisation of sugars and peptides carrying N-terminal cysteines. The reaction proceeds under physiological conditions and has the potential to become a valuable tool for immobilisation of N-terminal cysteine-containing molecules from biological samples. The new NCL coupling methodology was developed on gold surfaces and analysed by MALDI-ToF MS. The majority of array systems, however, rely on secondary protein interactions on glass or polystyrene surfaces. A direct, more accurate analytical tool could ease the analysis and significantly improve the quality of data read-out from glass microarrays. MALDI-ToF MS that is applicable to gold microarrays cannot be used on surfaces that do not provide the necessary electrical conductivity. The undertaken experiments indicated that application of conductive tape to the back of glass or polystyrene slides made MALDI-ToF analysis on poorly conducting surfaces possible. Furthermore, the triphenylmethyl (trityl) groups attached to the surface-molecules were shown to act as ’internal-matrix’ and enable the direct MALDI analysis. Once the new array formation and analysis techniques were developed, we turned our attention towards the application of microarrays to analyse carbohydrate-protein interactions. The tools for analysis of glycosylation of biomolecules are laborious and can only be used in specialised labs. As glycosylated biomolecules gain prominence in research, clinical and industrial settings, high throughput analysis of glycosylation patterns is becoming a requirement for quality control. A technique for screening of glycosylation patterns in glycopeptides on microarrays was developed based on biophotonic scattering. This technique enables the detection of glycosylation patterns by screening immobilised glycoproteins with a range of lectins. To study the interactions between enzymes and carbohydrates, a chemoenzymatic synthesis of a mannopeptide, which consisted of four carbohydrate units, was shown in solution and on chip. Three different glycosyl transferases were successfully employed. New methods for microarray formation and analysis were developed and applied to carbohydrate-protein interaction studies. This yielded a new technique to determine protein glycosylation patterns and to produce complex glycans by enzymatic synthesis.