Depuis quelques décennies, l’étude de systèmes biologiques complexes est un domaine en plein essor. Ainsi, des outils de ligation des biomolécules avec des reporters chimiques ont été mis en place afin d’avoir une compréhension toujours fine du vivant. Les ligations sont des réactions chimiques biocompatibles permettant de lier deux entités synthétiques ou biologiques entre elles. On regroupe généralement ces ligations en deux catégories, les réactions de bioconjuguaison, qui font intervenir des fonctions chimiques naturellement présentes dans les biomolécules, et les réactions bio-orthogonales qui n’interfèrent pas avec les fonctions chimiques présentes dans ces milieux, mais nécessitent en amont une modification des partenaires de réaction. Cependant, il convient de faire la distinction avec une troisième catégorie, les réactions de conjugaison chimiosélectives, qui mettent en oeuvre des fonctions non naturellement présentes sur les biomolécules. En ce sens, elles se rapprochent donc des réactions bio-orthogonales, mais les fonctions ou conditions mises en jeu ne sont pas suffisamment bio-orthogonales ou les réactions ne sont pas suffisamment rapides pour pouvoir être réalisées dans les systèmes biologiques. Ces ligations sont toutefois très utilisées pour de la construction biomoléculaire allant de la petite molécule (par exemple oligopeptide modifié) à la biomacromolécule (type protéine modifiée) et se distinguent par une facilité de mise en oeuvre et purification des conjugués, ce qui n’est pas toujours réa lisable avec l’arsenal des réactions bio-orthogonales qui conduisent à la formation de multiple isomères. Ainsi, mes travaux de thèse se sont orientés vers la découverte et/ou l’étude de ligations chimiosélectives ainsi qu’à leur utilisation dans la préparation de sondes fluorescentes voire fluorogéniques. L’étude de la ligation Kondrat’eva préalablement développée au sein du laboratoire, a permis de mettre en évidence son caractère fluorogénique, et a été exploitée pour le marquage fluorescent de molécules via une étape unique de ligation fluorogénique. Puis, le développement d’une ligation utilisant le système tétrazine/pyrazolone a été développée afin de pallier le manque de sélectivité des réactions basées sur le motif tétrazine proposées jusqu’alors, qui conduisent aux bioconjugués sous la forme d’un mélange de produits. Cette approche a été illustrée par le marquage fluorescent d’une protéine humaine. Enfin, le développement d’une nouvelle voie d’accès aux quinoxalinones a permis leur étude photophysique et la mise en évidence de propriétés fluorogéniques utilisées notamment pour la synthèse d’une biosonde. / In recent decades, the study of complex biological systems has been a growing field. Thus, biomolecules ligation tools with chemical reporters were set up in order to have a better and fine understanding of the living. Ligations are biocompatible chemical reactions that link two synthetic or biological entities one antother. These ligations are generally gathered into two categories, bioconjugation reactions, using chemical functions naturally present in the biomolecules, and bio-orthogonal reactions which does not interfere with these function, but require a prior engineering of the biological partner. However, it is necessary to distinguish a third category, the chemoselective conjugation reactions, which implement functions not naturally present on biomolecules. In this sense, they are therefore closer to bio-orthogonal reactions, but the functions or conditions involved are not sufficiently bioorthogonal or the reactions are not fast enough to be carried out in any biological systems. These ligations are, however, widely used for biomolecular constructions ranging from the small molecule (for example modified oligopeptides) to the biomacromolecule (protein modification) and are distinguished by their ease of implementation and purification of the conjugates, which is not always feasible with the arsenal of bio-orthogonal reactions that leads to the formation of multiple isomers. Thus, my PhD work focused on the discovery and / or the study of chemoselective ligations as well as their use in the preparation of fluorescent or fluorogenic probes. The study of the Kondrat'eva ligation previously developed within the laboratory, highlighted its fluorogenic behaviour, and was exploited for the fluorescent labelling of molecules through a single fluorescence-ligation step. Then, the development of a ligation using the tetrazine / pyrazolone system was developed in order to overcome the lack of selectivity of the reactions based on the tetrazine scaffold which often lead to the formation of bioconjugates as a mixture of isomers. This approach has been illustrated by the fluorescent labelling of a human protein. Finally, the development of a new access route to quinoxalinones allowed to study their photophysical properties and to highlight their fluorogenic properties which were leveraged in particular for the synthesi s of a bioprobe.

Chez les mammifères, la réparation des CDBs par la voie NHEJ (Non Homologous End Joining) implique plusieurs complexes multi-protéines : (i) de reconnaissance (ADN-Ku70/Ku80), (ii) de maturation et (iii) de ligation comprenant XRCC4, XLF et ligase IV. Si les protéines impliquées dans le NHEJ sont connues, leurs propriétés structurales et fonctionnelles le sont moins. Au cours de ma thèse, j’ai combiné des approches biochimiques, de Microscopies Electronique et à Force Atomique, pour caractériser les propriétés de XRCC4, de XLF et leurs interactions avec le complexe de reconnaissance en particulier Ku. J’ai montré que la protéine complète XRCC4 est capable de polymériser et former des filaments alors qu’elle ne peut pas en faire en absence de la région C-terminale. Par Microscopies Electronique et à Force Atomique; nous avons montré que le filament XRCC4 forme une structure hélicoidale de chiralité gauche. XLF seule ne forme pas de filament mais peut être incorporé dans le filament XRCC4, ce que nous avons montré par immunomarquage. L’analyse d’images réalisé en collaboration et avec l’algorithme d’Edward Egelman (Université de Virginie-USA) a permis d’obtenir une reconstruction 3 D du filament XRCC4. Il est composé de 2 filaments enroulés l’un autour de l'autre avec un pas de 54 nm. Des Etudes sont en cours afin d’obtenir une structure 3D en CryoEM à haute résolution. L’étude des propriétés physicochimiques de l’assemblage du filament en fonction de la concentration, la température et le temps d’incubation a permis de montrer la dynamique du filament avec une stabilisation à basse température, et une concentration située entre 50 et 250 nM. L’ADN avec ou sans extrémités interagit avec les filaments. Cette interaction stabilise et promeut l’extension du filament. L’incorporation de XLF stabilise aussi le filament XRCC4. L’analyse des complexes formés entre l’ADN et XRCC4 ou XLF à l’état oligomérique montre des événements de pontage intra ou intermoléculaires. Parallèlement, nous avons étudié les propriétés de reconnaissance de l’ADN par l’hétérodimère Ku70/Ku80 et avons montré que le domaine KBM de XLF interagit avec Ku80 au sein de l’hétérodimère. En conclusion, nous montrons que le filament XRCC4 pourrait jouer un rôle d’architecture en maintenant les extrémités physiquement proches. Le recrutement de XLF dans le filament XRCC4 permettrait d’assembler le complexe de ligation avec le complexe de reconnaissance grâce aux interactions entre Ku et XLF. / In mammals, DSBs repair by the Non Homologous End Joining (NHEJ) pathway involves several multi-protein complexes : (i) the recognition complex (the Ku70 / Ku80 heterodimer), (ii) the maturation complex and (iii) the ligation complex comprising XRCC4, XLF, PAXX and ligase IV. If the proteins involved in NHEJ are identified and characterized, their structural and functional properties are often poorly understood. During my thesis I combined biochemical approaches, and molecular microscopies (Electron Microscopy and Atomic Force Microscopy), to characterize the properties of XRCC4, XLF and their interactions with the recognition complex (Ku-DNA). I have shown that the full length XRCC4 forms oligomers in solution (dimers and tetramers) and it polymerize into filaments whereas it can’t do it when the C-terminal region is absent. We initially characterized the structure of this filament in Electron Microscopy and Atomic Force Microscopy. XRCC4 filament forms a helicoidal structure of left chirality. XLF alone does not forms a filament but can be incorporated into the XRCC4 filament, which we have shown by immunostaining with gold beads. In collaboration with Edward Egelman (University of Virginia-USA), the image analysis performed using his algorithm allowed us to obtain a 3D reconstruction of the XRCC4 filament. It consists of 2 filaments wound around each other in a helical manner with a pitch of 54 nm. Studies in CryoEM are in progress to obtain a 3D high resolution structure. The study of the physicochemical properties of the filament assembly as a function of the concentration, the temperature and the incubation time allowed to show the dynamics of the filament with stabilization at low temperature, and a concentration between 50 and 250 nM. DNA with or without ends interacts with the filaments. This interaction stabilizes and promotes the extension of the filament. Similarly, incorporation of XLF stabilizes the Xrcc4 filament. Analysis of complexes formed between DNA and XRCC4 or XLF in the oligomeric state shows intra- or intermolecular bridging events. In parallel, we have studied the DNA recognition properties of the Ku70/Ku80 heterodimer and we have shown that KBM domain of XLF interacts with Ku80 within the heterodimer. In conclusion, we show that the XRCC4 filament could play an architectural role favoring repair events by keeping the ends physically close. The recruitment of XLF into XRCC4 filaments ans its interaction with Ku allow the link between ligation and recognition complexes.

Ce manuscrit débute par un panorama général de la chimie des carbènes vinyliques. Nous nous sommes ensuite intéressés à la génération de tels carbènes à partir de cyanoazétidines, qui conduisent à la formation d’amines homopropargyliques. L’extension de cette réactivité aux cyanoépoxydes nous a menés à la découverte d’une voie de synthèse d’ α-hydroxy-β-azidotétrazoles (AHBATs), qui ont fait l’objet d’une application originale en chimie de ligation. Ainsi, ces AHBATs permettent de réaliser des réactions click de CuAAC de façon orthogonale et itérative. Enfin, la dernière partie de ce manuscrit est consacré à l’étude mécanistique et à l’optimisation de la décomposition d’ α-hydroxytétrazoles en alcynes vrais. / This manuscript begins with a general description of the chemistry of alkylidene carbenes. We then studied the generation of such carbenes from 2-cyanoazetidines, leading to the formation of homopropargylamines. The extension of this reactivity to cyanoepoxides lead us to discover a way to synthesize α-hydroxy-β-azidotetrazoles (AHBATs), that appeared to have an interesting application in ligation chemistry. These AHBATs allow to realize sequential and iterative CuAAC reactions in an orthogonal manner. Finally, the last part of this manuscript describes the mechanistic and optimization studies of the decomposition of α-hydroxytetrazoles into alkynes.

No description available.

Le principal objectif de cette thèse a consisté au développement d’une méthode de screenning pour la découverte de nouvelles réactions de ligations bioorthogonales ainsi que son application sur une bibliothèque développée pour cette étude. Par conséquent, un système de screening a été conçu en trois étapes consistant au départ en une analyse HPLC, puis une évaluation basée sur la fluorescence de haute résolution et finalement un test de microscopie confocal in cellulo. Puis, nous avons standardisé toutes les analyses avec les réactions CuAAC et SpAAC. En outre, nous avons synthétisés 18 réactifs d’intérêts et effectué un screening de 58 expériences de ligation avec une évaluation par méthode HPLC. Parmi les 9 réponses positives obtenues figure 6 réactions impliquant de nouveaux réactifs et les analyses LC‐MS ont pu tous les valider comme des réactions de cycloaddition directe à l’exception d’une réaction. Finalement, nous avons pu appliquer la méthode in cellulo développée, afin d’évaluer la pertinence des réactions de chélation CuAAC pour une application sur cellules. / The main goal of this thesis was the development of a screening method for the discovery of new bioorthogonal ligation reactions as well as its application on a self‐designed library. Therefore we designed a three step screening system consisting of a preliminary HPLC assay, a high resolution fluorescence based assay and a final in cellulo confocal microscopy assay.Subsequently we standardized all assays with the highly established CuAAC and SpAAC. Furthermore, we successfully synthesized 18 reagents of interest and screened 58 ligation experiments with the help of the HPLC setup. The 9 positive hits from this screening contained 6 reactions involving novel reagents and LCMS analysis was able to validate all but one as straight forward cycloaddition reaction. Finally we were able to apply the newly developed in cellulo assay to assess the suitability of chelating CuAAC for in cell application.

Die native chemische Ligation (NCL) ist eine der effizientesten Methoden, um Peptidfragmente zu verknüpfen. Diese Methode jedoch auf das Vorhandensein von Cystein angewiesen. Um diese Beschränkung zu überwinden, wurden Ligationsauxiliare entwickelt, die auch cysteinfreie Peptide zur Ligation befähigen können. Auxiliare ermöglichen zwar die Ligation, diese Ligationen sind jedoch weniger effizient als die ursprüngliche NCL. Der durch das Auxiliar eingeführte sterische Ballast an der Ligationsschnittstelle behindert die Anwendung dieser Hilfsmoleküle. Das bisher erfolgreichste schwefelhaltige Ligationsauxiliar ist das 2-Mercapto-2-phenethyl-(MPE)-Auxiliar, das als erstes Auxiliar die Durchführung von Ligationen an glycinfreien Schnittstellen ermöglichte. Allerdings ist auch das MPE-Auxiliar für die Ligation an anspruchsvolleren Schnittstellen nicht geeignet, da der S→N Acyltransfer unter diesen Umständen stark gehemmt wird. In dieser Arbeit wurde nach effizienteren Auxiliar-Designs gesucht, die Ligationen auch an stark sterisch belasteten Schnittstellen ermöglichen. Die Entwicklung neuer Auxiliare ging dabei vom MPE-Auxiliar aus und es wurden drei unterschiedliche Ansätze für die Verbesserung der Ligationseigenschaften untersucht: Die Einführung von Substituenten in para Position am Phenylring des MPE-Auxiliars, die Untersuchung des Einflusses des Dialkyl-Effektes auf die Auxiliareffektivität und die Einführung einer Base in das Auxiliargerüst in Form eines Pyridins. Das aus der dritten Variante resultierende 2-Mercapto-2(pyridin-2-yl)ethyl-(MPyE)-Auxiliar zeigte dabei als einziges neu entwickelte Auxiliar einen deutlich verbesserten Einfluss auf die Ligationseigenschaften. In verschiedenen Experimenten und durch quantenchemische Rechnungen konnte gezeigt werden, dass der Pyridin-Stickstoff als interne Base ligationsunterstützend fungiert. In dieser Arbeit wurde somit das erste Ligationsauxiliar entdeckt, in dem das Auxiliargerüst aktiv an der Ligation teilnimmt. / The Native chemical ligation (NCL) is one of the most efficient methods for linking peptide fragments, but it is dependent on the presence of cysteine. To overcome this limitation, ligation auxiliaries have been developed that can also enable cysteine-free peptide fragments to be ligated. Although auxiliaries enable ligation, these ligation reactions are less efficient than the original NCL. The steric ballast introduced by the auxiliary at the ligation junction hinders the use of these auxiliaries at most junctions. The most successful sulfur-containing ligation auxiliary to date is the 2-mercapto-2-phenethyl (MPE) auxiliary, which was the first auxiliary to allow ligations to be carried out at glycine-free junction. However, the MPE auxiliary is not suitable for ligation at more sterically demanding junctions, as the S→N acyl transfer is strongly inhibited under these circumstances. This work sought more efficient auxiliary designs that enable ligation reactions even at highly sterically loaded ligation junctions. The development of new auxiliary designs was based on the MPE auxiliary and three different approaches for improving the ligation properties were investigated: The introduction of substituents in para-position on the phenyl ring of the MPE auxiliary, the investigation of the influence of the dialkyl effect on the auxiliary efficiency and the introduction of a base into the auxiliary scaffold in the form of a pyridine. The 2-mercapto-2(pyridin-2-yl)ethyl (MPyE) auxiliary resulting from the third variant was the only newly developed auxiliary to show a significantly improved influence on its ligation properties. Various experiments and quantum chemical calculations showed that the pyridine nitrogen acts as an internal base to support ligation. In this work, the first ligation auxiliary was discovered in which the auxiliary scaffold actively participates in the ligation reaction.

The ability to safely delivery efficacious amounts of nucleic acids to cells and tissues remains an important goal for the gene therapy field. Viruses are very efficient at delivering DNA, but safety concerns limit their clinical use. Nonviral vectors are not as efficient at DNA delivery, but have a better safety profile. Limiting the efficaciousness of nonviral vectors are the numerous extra and intracellular barriers that must be overcome for successful DNA delivery in vivo. While single polymers can successfully transfect immortalized cell lines in vitro, multicomponent gene delivery systems are required for delivery in vivo. Key in the development of multicomponent systems is their syntheses. Optimization of a nonviral gene delivery system requires the development of methodologies that incorporate the different components in a controlled fashion, generating homogeneous gene delivery vectors. Such syntheses ensure every polymer has the different components required for successful delivery. The amount of each component and location within the gene delivery system can also be varied systemically, allowing optimization of the vector. The overall scope of this thesis is to develop a chemical method to iteratively couple gene delivery peptides through reducible disulfide bonds. The synthesis of such polypeptides allows the triggered disassembly of a polypeptide polyplexed with DNA upon cellular uptake. To synthesize homogeneous gene delivery polypeptides, a novel iterative reducible ligation strategy was developed, based upon the use of a thiazolidine masked cysteine. Initial studies demonstrated that a thiazolidine could be unmasked to a cysteine in the presence of a disulfide bond without side reaction, though the reported thiazolidine hydrolysis conditions of aqueous methoxyamine were insufficiently robust for high yielding ligations. Discovery of a novel silver trifluoromethanesulfonate hydrolysis led to an efficient process for generating reducible polypeptides, as evidenced in the synthesis of a 4 component polypeptide. Due to the success of the thiazolidine mediated iterative ligation strategy, cysteines were replaced by penicillamines to produce more stable disulfide bonds. The mild thiazolidine hydrolysis and subsequent peptide conjugation reactions led to attempt the iterative ligation strategy on a solid support, eliminating purification steps that lowered the yields in the solution phase methodology. Initial progress at generating gene delivery peptides that could be incorporated into the synthetic strategy included the generation of a tri-orthogonal cysteine protecting scheme that allowed a third cysteine to be derivatized with a targeting ligand or stealthing polymer. Due to the use of terminal cysteines in the iterative ligation strategy, a PEG stealthing polymer could be placed in the center of a polyacridine gene delivery peptide with only a small decrease in the ability to condense and protect DNA during systemic circulation. A convergent synthesis was also developed that was able to synthesize large polypeptides in fewer linear steps. The synthetic methodology of thiazolidine mediated iterative reducible ligation developed in this thesis is important in the gene therapy field as it allows the construction of polypeptides that can be systemically optimized, potentially resulting in highly efficacious nonviral gene delivery.

<p>After the completion of the human genome project the human genome was annotated with the surprisingly small amount of 24 000 (www.ensemble.com) genes. This has focused research on the contribution of splice variants, posttranslational modifications and interactions of proteins at the proteome level and other regulatory elements in the cell to fully understand the complexity of functions in a higher organism. Proteomic oriented projects are currently aiming to investigate all the splice variants and posttranslational modifications of all the proteins present in an organism or cell type and annotate their function and interaction partners. Projects on this scale are at the moment difficult to achieve and new methodologies are needed. </p><p>Proximity ligation assays (PLAs) are based on a novel protein detection strategy that converts the presence of a target molecule in a unique DNA tag through ligation reactions. PLA detection of proteins requires several independent recognition events by affinity reagents that have been converted into proximity probes. Different formats of the proximity ligation strategy have been developed in both heterogeneous and homogeneous format[1-4]. This thesis presents the development of an antibody based proximity ligation approach and the development of a novel proximity ligation based detection strategy named triple specific proximity ligation (3PLA). To extend the range of target molecules we adapted the proximity ligation assay for the use with antibodies by converting matched monoclonal antibody pairs and polyclonal antibody batches into proximity probes and used them for the detection of several cytokines in complex biological fluids. The novel 3PLA requires the simultaneous detection by three independent affinity binders to create one specific DNA based signal. This requirement for triple recognition extends the biological specificity of immunoassays and allows a proximity ligation design with reduced background signal and thus higher sensitivity. We have established proof of principle detection of the biomarkers troponin I and prostate specific antigen (PSA) alone and in complex with 1-alpha-antichymotrypsin (ACT) and detected as little as 100 molecules of vascular endothelial growth factor (VEGF). To further explore the extended biological specificity of 3PLA we adapted the assay for detection of protein complexes formed during NFκB signaling and used this system to profile the mode of action of three small molecular weight inhibitors of the IκB Kinase (IKK). The development of new protein detection methods hold promises for the investigation of complex interactions and mechanism on the proteome level which are not accessible with current technologies. We have developed tools and protocols useful for the development of new proximity ligation strategies and designs. These protocols allow the rapid and low cost custom set up of PLAs without the need for extensive conjugation protocols or purification procedures.</p>

After the completion of the human genome project the human genome was annotated with the surprisingly small amount of 24 000 (www.ensemble.com) genes. This has focused research on the contribution of splice variants, posttranslational modifications and interactions of proteins at the proteome level and other regulatory elements in the cell to fully understand the complexity of functions in a higher organism. Proteomic oriented projects are currently aiming to investigate all the splice variants and posttranslational modifications of all the proteins present in an organism or cell type and annotate their function and interaction partners. Projects on this scale are at the moment difficult to achieve and new methodologies are needed. Proximity ligation assays (PLAs) are based on a novel protein detection strategy that converts the presence of a target molecule in a unique DNA tag through ligation reactions. PLA detection of proteins requires several independent recognition events by affinity reagents that have been converted into proximity probes. Different formats of the proximity ligation strategy have been developed in both heterogeneous and homogeneous format[1-4]. This thesis presents the development of an antibody based proximity ligation approach and the development of a novel proximity ligation based detection strategy named triple specific proximity ligation (3PLA). To extend the range of target molecules we adapted the proximity ligation assay for the use with antibodies by converting matched monoclonal antibody pairs and polyclonal antibody batches into proximity probes and used them for the detection of several cytokines in complex biological fluids. The novel 3PLA requires the simultaneous detection by three independent affinity binders to create one specific DNA based signal. This requirement for triple recognition extends the biological specificity of immunoassays and allows a proximity ligation design with reduced background signal and thus higher sensitivity. We have established proof of principle detection of the biomarkers troponin I and prostate specific antigen (PSA) alone and in complex with 1-alpha-antichymotrypsin (ACT) and detected as little as 100 molecules of vascular endothelial growth factor (VEGF). To further explore the extended biological specificity of 3PLA we adapted the assay for detection of protein complexes formed during NFκB signaling and used this system to profile the mode of action of three small molecular weight inhibitors of the IκB Kinase (IKK). The development of new protein detection methods hold promises for the investigation of complex interactions and mechanism on the proteome level which are not accessible with current technologies. We have developed tools and protocols useful for the development of new proximity ligation strategies and designs. These protocols allow the rapid and low cost custom set up of PLAs without the need for extensive conjugation protocols or purification procedures.

While unprotected amino aldehydes are typically not isolable due to imine formation and consequent polymerization, stable unprotected aziridine aldehydes are useful and available reagents. Moreover, reversible hemiacetal and hemiaminal formation enable these compounds to reveal both their electrophilic and nucleophilic functional groups. This exceptional arrangement allows for aziridine aldehyde dimers to participate in and disrupt the mechanisms of an array of well-known organic reactions, including isocyanide-based multicomponent reactions. The scope and selectivity patterns of aziridine aldehyde induced amino acid or peptide macrocyclization have been investigated. A small library of constrained tri-, tetra-, and penta-peptide macrocycles – representing the most difficult cyclic peptides to synthesize – has been prepared. The scope of aziridine aldehyde participation in multicomponent reactions was also expanded to Ugi and Passerini reactions that do not employ tethered amine and acid functional groups. In order to facilitate cellular imaging of peptide macrocycles a fluorescent isocyanide reagent was prepared and applied to prepare mitochondrial targeting macrocycles. Thioester isocyanide reagents were synthesized to enable rapid assembly of cycle-tail peptides through ligation technology.