Des peptides antigéniques sont présentés à la surface des cellules cancéreuses ou infectées par un virus pour être reconnus par des cellules du système immunitaire. Cette reconnaissance déclenche alors une réponse immunitaire spécifique contre les cellules présentatrices ciblées. L'objectif de ce projet est de stimuler cette réponse immunitaire afin qu'elle soit un moyen thérapeutique pour détruire spécifiquement les cellules cancéreuses ou infectées. Toutefois, les peptides antigéniques ne peuvent pas être administrés tels quels, du fait de leur pauvre stabilité en milieu biologique. Il est donc nécessaire de les modifier afin qu’ils soient plus résistants. Le défi de ce projet est de synthétiser des peptidomimes bio-résistants, capables de reproduire la même réponse immunitaire que celle du peptide antigénique naturel. / Abstract

Catalysis plays a key role in chemical conversions by making them faster and more selective. Despite its widespread use and decades of academic and industrial research, limited catalyst selectivity and stability still call for major improvements in catalyst performance to meet the demands of a sustainable society. Phosphine ligands are ubiquitous in transition metal chemistry and lead to extremely reactive and versatile homogeneous catalysts. Fast development of tailor-made catalysts and catalyst recovery are key issues in (asymmetric) homogeneous catalysis. Therefore libraries of ligands have to be synthesised and screened in an efficient way, which could be facilitated by Solid Phase Synthesis (SPS). Currently, most polymer bound ligands are anchored to the support after the synthesis in solution. However, the main advantages of synthesising the ligands directly on the polymeric support are not only easy catalyst recycling and product separation, but also the ease of purification during the synthesis steps, namely by simple washing and filtration. The use of SPS is very efficient for high throughput synthesis and screening of ligand libraries, however applications of SPS towards libraries of phosphorus ligands are rare, because the synthetic methodologies are still lacking. Here we present the development of methodologies towards novel immobilised bis(phosphine) ligands synthesised on polystyrene and JandaJel™ resin. By performing the synthesis steps on a solid support, the advantages of SPS are fully utilised. Successful routes have been developed towards immobilised secondary phosphine-boranes, which were versatile synthons to prepare a variety of new polymer-supported (C-chiral) bis(phosphine) ligands. These ligands were then tested for their catalytic activity in rhodium catalysed hydrogenation reactions.

Chemical biology can be defined as the area of science where chemical tools are used to study biological systems. The simplest way this can be achieved is in the identification of compounds which inhibit or modulate a biological pathway and the consequences studied. However, novel tools are required to enable, for example, the development of assays to allow simpler screening of difficult targets such as membrane proteins and protein-protein interactions. A series of kisspeptin analogues were synthesised for the development of a screening platform compatible with G-protein coupled receptors and tagged one bead one compound (OBOC) combinatorial libraries. Fluorescently labelled kisspeptin showed good affinity for GPCR54 and an on-bead version of the peptide, with the required C-terminal amide presented away from the bead was prepared and used for testing possible screening methods. GPCR54 was expressed in a number of formats and a kisspeptin based OBOC library designed and synthesised. Investigation into the C-terminal RF-amide motif of Kisspeptin was also carried out in order to assess the importance of the carbonyl moiety. The corresponding peptide amine was synthesised and the compound biologically assessed. This led to the development of a novel acid labile benzofuranone (ALBA) linker for anchoring amines to a solid support. For the preparation of fluorescent kisspeptin ligands, a novel general synthetic route which gives direct access to single isomer functionalised rhodamine dyes from phthalides has been developed. This circumvents the arduous task of isomer separation usually associated with the synthesis of functionalised rhodamines. The route has been demonstrated with a range of linkage groups and rhodamine types. This rhodamine material was used as a reporter group in various multifunctional reagents synthesised using a trifunctional orthogonally protected backbone (TOBa), which was prepared on a solid support and enables rapid synthesis of trifunctional reagents. This resin takes advantage of protecting group orthogonality and the high yields of peptide bond formation. A series of trifunctional reagents for screening use were prepared using this resin. A proof of concept study was carried out involving the simultaneous labelling and immobilisation of a protein for applications in probing protein-protein interactions. Development of a trifunctional hydroxamic acid containing cross-linker was carried out which takes advantage of its reaction with boronic acids to enable reversible capture on solid support for enrichment of cross-linked peptides. A new benzophenone based heterobifunctional reagent was prepared for protein cross-linking and mass spectrometry analysis. This was shown to give complimentary reactivity to existing cross-linkers, allowing more structural information to be extracted from protein samples.

Positron Emission Tomography (PET) has great value in research and clinical applications from oncology to neurodegenerative disorders. However, there is a barrier in translating biological knowledge into new PET applications due in part to the lack of efficient, widely applicable methods for labelling compounds with PET radioisotopes. Herein, a generic approach to radiolabelling is presented which is direct, broadly applicable and potentially adaptable to either of the two most commonly used PET radioisotopes, 11C and 18F. This approach employs the advantages of solid phase synthesis to achieve selective release of only the desired radiolabelled product from a solid support in a single step, simplifying purification and hence improving synthetic efficiency. Polystyrene resin was functionalised with a 1,2-diol group; this allowed the covalent attachment of compounds bearing boronic acid groups via formation of a boronate ester linkage. A Suzuki-Miyaura reaction with methyl iodide was used to cleave a model compound from the resin in 61% conversion after five minutes. This reaction was adapted to develop a fully automated radiosynthesis with [11C]- methyl iodide which generated a radiolabelled model compound in 2 – 7% non-decay-corrected radiochemical yield. This provided proof of concept for the simultaneous cleavage of compounds from the resin and radiolabelling with 11C. A boronic acid precursor of the known radiotracer [11C]-M-MTEB was attached to the resin and successfully radiolabelled with 11C in 2.4% non-decay-corrected radiochemical yield and 96 – 100% radiochemical purity under the same conditions. Furthermore, the potential adaptability of this solid phase approach to 18F radiolabelling was demonstrated by treatment of the resin-bound small molecules and peptides with potassium bifluoride, which released the compounds rapidly as trifluoroborate salts.

<p>This thesis investigates three different surface modifications, and the route to design and synthesize them. The thesis is therefore divided into three sub- projects. (i.) Design and synthesis of a peptide which secondary structure could be controlled by a negatively charged surface. (ii.) Design and synthesis of a cyclic peptide, that would self-organize prior to surface interaction, using the type I anti-freeze protein of a winter flounder as template. (iii.) The use of solid-phase synthesis to make the synthesis of SAM-molecules easier.</p>

This thesis investigates three different surface modifications, and the route to design and synthesize them. The thesis is therefore divided into three sub- projects. (i.) Design and synthesis of a peptide which secondary structure could be controlled by a negatively charged surface. (ii.) Design and synthesis of a cyclic peptide, that would self-organize prior to surface interaction, using the type I anti-freeze protein of a winter flounder as template. (iii.) The use of solid-phase synthesis to make the synthesis of SAM-molecules easier.

Thesis (Ph.D.)--University of Western Sydney, 2008. / A thesis presented to the University of Western Sydney, College of Health and Science, School of Biomedical and Health Sciences, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographies.

The detection and quantification of biomolecules, and proteins in particular, are of great interest since these molecules are of fundamental importance to our well-being. Body fluids, as for instance human blood, are well suited for sampling of protein levels. However, the complexity of the fluids and the low abundance of many of the interesting biomolecules makes detection and quantification difficult. This has spurred an interest into the development of many protein detection methods, and of these, ligand assays have proven particularly suitable. In this thesis, different types of ligand assays for protein detection have been developed using affibody molecules as ligands. In a first study, a homogeneous competitive detection assay was investigated, based on antiidiotypic affibody molecule pairs and fluorescence resonance energy transfer (FRET) as reporting system. The individual members of two anti-idiotypic affibody pairs, each consisting of a target binding (idiotypic) and an anti-idiotypic affibody ligand, were labeled with a donor fluorophore and an acceptor fluorophore, respectively. Incubation with the two target proteins IgA and Taq DNA polymerase resulted in a concentration dependent decrease in the FRET signal, allowing for target protein detection and quantification. For Taq DNA polymerase, detection in 25% human plasma was also possible in the same concentration span as in buffer. In a second study, a homogeneous, non-competitive detection system was described. Affibody molecules of 58 amino acids directed against IgA and IgG were produced with chemical synthesis, and two fluorophores capable of FRET were site-specifically introduced. Binding of target protein induced a concentration-dependent change in the relative emission of the two fluorophores, which formed the basis for the detection system. In two studies, affibody molecules were evaluated and shown to function well as capture ligands on microarrays. Synthetic affibody molecules directed against Taq DNA polymerase and IgA were modified by the introduction of immobilization tags. Specific immobilization via a C-terminal cysteine or a biotin moiety, or random immobilization via amino groups, were studied in protein microarray experiments and SPR-based biosensor studies. The experiments showed that all immobilization chemistries resulted in functional capture molecules. A short spacer was also introduced, situated between the affibody and the cysteine and biotin moieties, which was shown to improve binding for all constructs. Multidomain affibody constructs of up to four N- to C-terminally linked domains were shown to increase the amount of bound target, compared to monomeric affibody ligands. Six dimeric affibody constructs directed against IgA, IgG, IgE, Taq DNA polymerase, TNF-α and insulin, respectively, showed low limits of detections for their targets and little or no cross-reactivity with the other target proteins. Dimeric affibody molecules directed against IgA and TNF-α were also shown to function in a sandwich format with antibodies for detection of targets in buffer and in human serum and plasma. Successful discrimination between normal and IgA-deficient sera showed that affibody molecules could be used for specific detection of protein in highly complex backgrounds on microarrays. / QC 20100916

Thesis (M.S.)--State University of New York at Binghamton, Department of Chemistry, 2006. / Includes bibliographical references.

Le travail présenté dans ce manuscrit concerne le développement d’inhibiteurs sélectifs de NO Synthases, des enzymes qui catalysent la formation du monoxyde d’azote (NO) à partir de l’arginine. Trois isoformes ont été identifiées, la NOS neuronale (nNOS), la NOS endothéliale (eNOS) et la NOS inductible (iNOS). Une surproduction de NO dans l’organisme, notamment par la iNOS ou la nNOS, est impliquée dans différentes maladies, tel que le choc septique, l’arthrite, des maladies neurodégénératives ou encore certains cancers. L’inhibition des NOSs apparait donc une approche intéressante dans le traitement de ces maladies. Cependant, cette inhibition doit être sélective pour ne pas interférer avec les fonctions physiologiques des autres isoformes.Les composés synthétisés sont constitués d’une partie analogue du substrat de type S-Ethyl-Isothiocitrulline munie d’une extension sensée interagir dans le canal d’accès du substrat où la conservation entre les trois isoformes est plus faible que dans le site de liaison du substrat. Ces deux parties sont reliées par un lien qui peut être de type amide ou hétérocyclique. Ces composés sont obtenus grâce à une stratégie de synthèse en phase solide via un ancrage par la chaîne latérale, qui permet la synthèse rapide de bibliothèques de composés. Notre premier travail a été de tenter d’optimiser deux inhibiteurs sélectionnés de travaux antérieurs, l’un plutôt sélectif de la iNOS et l’autre de la nNOS. Pour cela, nous avons choisi certaines modifications selon une approche aléatoire. Mais nous avons également entamé une approche plus rationnelle en développant un protocole de docking, basé sur des structures de NOSs issues de la Protein Data Bank et destiné à prédire le mode de liaison des inhibiteurs dans le site actif des enzymes. Nous avons également développé de nouvelles séries de composés. En particulier, nous avons mis au point la synthèse sur support d’analogues où le carbone alpha de l’analogue de substrat est remplacé par un azote (composés de type aza). Nous avons aussi tenté la synthèse en solution de nouveaux analogues du substrat séléniés.Enfin, grâce au modèle de docking, un criblage virtuel de molécules issues de deux bases de données dont celle de la Chimiothèque Nationale a été effectué pour découvrir de nouvelles structures avec un potentiel pouvoir inhibiteur. Nous présentons donc dans ce document la conception et la synthèse de nouvelles séries d’inhibiteurs de NOSs, l’élaboration d’un protocole de docking pour apporter une approche plus rationnelle, et enfin l’évaluation des composés sur les trois enzymes recombinantes et, pour certains, sur deux lignées cellulaires productrices de NO. / This work is focused on the development of selective NO Synthases inhibitors, enzymes, which catalyse the production of nitric oxide (NO) from arginine. Three isoforms have been identified, the. neuronal NOS (nNOS), the endothelial NOS (eNOS) and the inducible NOS (iNOS). An overproduction of NO, in particular by iNOS or nNOS, is involved in many diseases such as septic shock, arthritis, neurodegenerative diseases or some cancers. Therefore, NOS inhibition looks very interesting to treat those diseases. However, this inhibition has to be selective of one isoform to not interfere with the physiological functions of the two others.The synthetic compounds are constituted of both an S-Ethyl-Isothiocitrulline as substrate analogue and an extension expected to interact into the substrate access channel, a less conserved region between the three isoforms compared to the substrate binding site. These two parts are joined together by a peptide bond or a heterocyclic link. These molecules have been obtained thanks to a solid-phase strategy through a side chain anchoring approach. This protocol allows the rapid synthesis of compound libraries. In a first work, we attempted to optimize two inhibitors selected from previous studies. One is rather selective of iNOS and the other to nNOS. We chose some modifications in a random fashion. But we also started a more rational approach by developing a docking protocol, based on NOS structures from the Protein Data Bank and designed to predict the binding mode of the inhibitors in the enzyme active sites.We also prepared new series of compounds. In particular, we developed the solid phase synthesis of derivatives where the alpha carbon of the substrate analogue is replaced by a nitrogen (aza-type compounds).. We also attempted the solution synthesis of new substrate analogues containing selenium.Finally, thanks to the docking model, we performed a virtual screening of molecules from two data bases including the Chimiothèque Nationale, to discover new structures with potential inhibitory activity.Therefore, we present in this manuscript the design and synthesis of new series of NOS inhibitors, the development of a docking protocol to provide a more rational approach, and finally the evaluation of compounds on the three recombinant isoforms and, for some, on two cell lines producing NO.