Nitrones are alternatives to azides in rapid strain-promoted 1,3-dipolar cycloadditions with cyclooctynes. To evaluate the differences between nitrones and azides we have performed kinetic studies of strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions of biarylazacyclooctynone (BARAC) with various acyclic and cyclic nitrones. The reactions were conducted under pseudo first-order reaction conditions using UV-visible spectroscopy. The reactivity of the acyclic nitrones was evaluated by varying the stereoelectronic and steric character of substituents at both the α-aryl and nitrogen positions. Cyclic nitrone reactivity was assessed according to the size of the ring and additional steric and strain effects. The obtained second-order rate constants for reactions of BARAC with cyclic nitrones were found to be greater than those for acyclic nitrones. However, all nitrones employed in the kinetic studies herein displayed significantly greater reactivity than azides in the analogous cycloadditions with BARAC. It is of particular note that the five-membered cyclic nitrones showed exceptional reactivity and, if used as rapid alternatives to azides in reactions with BARAC, can increase the reaction rates by up to 50 fold. An attempt to synthesize an allylated BARAC analogue is also described; the rearrangement reaction leading to the unexpected products is reported. The reaction rate for the novel rearrangement under both neutral and acidic conditions was obtained and plausible mechanisms for formation of products are proposed. Based on the results reported herein we anticipate that development of a labelling probe based on BARAC and a five-membered cyclic nitrone would allow for significant decrease of the concentrations of labelling reagents, thereby minimizing reaction time and reagent usage in life sciences applications. Nevertheless, a possible labelling decrease due to side reactions should be given consideration for prolonged labelling.

La découverte et l’exploration des réactions bio-orthogonales pour le marquage spécifique d’entités biologiques est un défi majeur à portée de main depuis une dizaine d’années. Une variété de réactions bio-orthogonales a récemment été décrite, parmi lesquelles : les réactions de Diels-Alder entre des alcynes ou alcènes tendus et des tétrazines ou encore les cycloadditions entre alcynes tendus et azotures (SPAAC). Ces réactions biocompatibles sont aujourd’hui parmi les plus utilisées pour les applications de marquages in vivo ou in vitro. Récemment notre groupe et le groupe du Pr. Chin ont identifié une nouvelle réaction bio-orthogonale impliquant une sydnone et un alcyne tendu et conduisant à la formation d’un adduit pyrazole. Cette réaction a été nommée SPSAC par analogie à la réaction SPAAC.Le but de ces travaux de thèse a été, dans un premier temps, d’améliorer la cinétique de la réaction de SPSAC en incorporant différents substituants sur le noyau sydnone, de façon a montrer l’intérêt de cette réaction pour des applications en bioconjugaison en comparaison avec la réaction de SPAAC.Dans une deuxième partie et avec l’objectif de pousser plus loin le développement de cette réaction pour des applications en bioconjugaison, nous avons synthétisé des sondes sydnones pro-fluorescentes i.e. qui deviennent fluorescentes suite la réaction de SPSAC avec un alcyne tendu. La sonde la plus prometteuse a été utilisée pour effectuer le marquage sur gel d’une protéine modèle dans des milieux biologiques.Enfin les derniers travaux de cette thèse ont permis d’étendre les applications de la SPSAC à la chimie des matériaux. Une méthodologie de synthèse de sydnones tricycliques hautement conjuguées a été développée. Ces sydnones conduisent par réaction avec des diynes ou des arynes à des structures chirales complexes aux propriétés optiques intéressantes. / The discovery and exploration of bio-orthogonal reactions for the specific labeling of biological entities is a major challenge. Up to now, a variety of bio-orthogonal reactions have been described, including the Diels-Alder reaction between strained alkynes or alkenes and tetrazines or the Strain Promoted Azide-Alkyne Cycloaddition (SPAAC). These “click reactions” are today the most popular tools for in vivo or in vitro chemical modifications of complex biomolecules.Recently, our group and Pr. Chin’s group have identified a new bio-orthogonal reaction involving sydnones and strained alkynes and leading to the formation of pyrazole adducts. This reaction, very similar to the SPAAC, was coined SPSAC for Strain Promoted Sydnone-Alkyne Cycloaddition.The aim of this PhD thesis was first to improve the kinetic properties of the SPSAC by incorporating various substituents on the sydnone ring in order to demonstrate the interest of using this reaction for bioconjugation applications.To extend the potential of this reaction for bio-labelling applications, we then investigated the synthesis of fluorogenic sydnone probes, i.e. sydnones that would emit fluorescence upon reaction with a strain alkyne. The most promising probe was involved in the fast fluorogenic labelling of a protein in a biological medium. This work is described in the second part of the manuscript.Finally, during the last part of my PhD, we extended the application of SPSAC to the field of material science. We developed a methodology enabling a straightforward access to highly conjugated tricycle sydnones. These sydnones, lead to complex chiral structures with interesting optical properties upon reaction with diynes or arynes.

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.

L’adénosine est une molécule dotée d’un fort potentiel thérapeutique, mais présentant néanmoins un temps de demi-vie plasmatique extrêmement court qui limite de manière sérieuse son efficacité. Comme présenté dans l’introduction bibliographique de cette thèse, cette difficulté peut être surmontée grâce à l’utilisation de systèmes de délivrance de médicaments à base de lipides. L’adénosine peut en effet être soit encapsulée dans des liposomes, soit simplement couplée à un lipide. Parmi les « lipidizations » de l’adénosine, la « squalénisation », notamment, a favorablement modifié la biodistribution de cette substance active. Cette technique consiste à coupler l’adénosine à une molécule lipophile dérivée du squalène, générant ainsi des bioconjugués ayant la capacité de s’auto-assembler spontanément en milieu aqueux sous forme de nanoparticules d’une centaine de nanomètres de diamètre. L’injection de ces nanoparticules d’adénosine-squalène (AdSQ) par voie intraveineuse a donné des résultats très prometteurs pour le traitement de l’ischémie cérébrale et du traumatisme de la moëlle épinière. Ainsi, l’objectif de cette thèse a consisté à faire progresser le développement pré-clinique de ces nanomédicaments suivant deux axes principaux: l’étude du mécanisme d’action et l’amélioration de la formulation.De ce fait, le premier chapitre de cette thèse présente les résultats obtenus lors de l’étude in vitro du mécanisme d’action des nanoparticules d’AdSQ. Les travaux ont montré que ces nanoparticules d’AdSQ n’interagissaient pas directement avec les récepteurs à l’adénosine, mais formaient un réservoir intracellulaire d’adénosine. En effet, après internalisation, le bioconjugué d’AdSQ est clivé pour libérer l’adénosine. Celle-ci finit par être effluée par les cellules vers le milieu extracellulaire, où elle peut ainsi activer les récepteurs spécifiques situés au niveau des membranes des cellules avoisinantes. Après étude du mécanisme, l’amélioration de la formulation de ces nanoparticules a été explorée et décrite dans le deuxième chapitre. Les efforts ont été principalement concentrés sur la lyophilisation de la suspension nanoparticulaire, afin de proposer une formulation stable dans le temps et facile d’utilisation dans le cadre médical. Les conditions utilisées ont abouti au bon maintien des propriétés physico-chimiques des nanoparticules et l’obtention de solutions injectables sans risque chez l’animal. Dans son ensemble, ce travail de thèse a permis d’élargir les perspectives d’application des nanoparticules d’AdSQ grâce à une meilleure compréhension de leur mécanisme d’action ainsi que la mise au point d’une formulation plus adaptée aux besoins cliniques. / Adenosine has a high therapeutic potential but its extremely short half-life in blood seriously impairs its efficacy. As presented in the literature review, this difficulty can be overcome by using lipid-based drug delivery systems. Indeed, adenosine can be encapsulated into liposomes or conjugated to a lipid. In particular, among adenosine « lipidizations », the so-called « squalenoylation » has been shown to enhance adenosine biodistribution. This technique consists in coupling adenosine to a lipophilic squalene derivative, thus generating bioconjugates which are able to spontaneously self-assemble as nanoparticles of 100 nm of diameter in aqueous solution. Intravenous injection of these squalene-adenosine (SQAd) nanoparticles led to highly promising results for the treatment of cerebral ischemia and spinal cord injury. Thus, the aim of this thesis was to push forward the pre-clinical development of these nanomedicines following two main directions: unveiling their mechanism of action and enhancing their formulation.Thereby, the first chapter of this thesis presents the results from in vitro study on SQAd nanoparticles mechanism of action. This work has shown that SQAd nanoparticles did not interact directly with adenosine receptors, but formed an intracellular reservoir of adenosine. Indeed, after internalisation, SQAd bioconjugates acted as prodrugs by releasing free adenosine. This molecule was then efflued out of the cells into the extracellular medium, where it could activate specific membrane receptors on neighbouring cells. After studying the mechanism of action, we explored how to optimize the formulation. Results are described in the second chapter. We focus our efforts on freeze-drying the nanoparticles suspension, in order to offer a stable and easy-to-use formulation. Pre-formulation studies were conducted in order to define the optimal conditions for the preservation of nanoparticles physico-chemical properties and for an easy reconstitution of these nanoparticles suspension which can thus be safely injected intravenously. Overall, this work has widen the field of applications for SQAd nanoparticles thanks to a better understanding of their mechanism of action and the development of a formulation which is more suited to clinical needs.

Abstract Time-consuming diagnosis of bacterial blood stream infections and inappropriate antibiotic therapy have critical implications for patient outcome – with mortality figures rising for every hour of delayed treatment. The development of diagnostic methods that are capable of selective and rapid bacteria detection, and do not rely on preliminary blood culturing and Gram-staining procedures, is imperative in providing effective therapy and preventing multi-resistance. The aim of this dissertation was to develop a quantum dot based and Gram-specific bacteria labelling protocol. Focused on the detection of Gram-negative species, a two-step conjugation protocol was produced to functionalise quantum dots with anti-lipid A antibodies. Ionic adsorption and EDC chemistry were used to obtain oriented and covalent conjugation of antibodies to the quantum dot surface. In order to reduce non-specific binding of unreacted carboxylic groups on the conjugates to the bacterial membrane, and optimise the accuracy of detection, blocking experiments were conducted with molecules that could provide a neutral surface charge and sterically block open sites. To access lipid A on E. coli cells, three different antigen retrieval methods were tested. As a result, the developed quantum dot-anti lipid A conjugates were able to detect and specifically label Gram-negative E. coli cells after treatment with 0.6mM EDTA or acetic acid pH 3.58 at 42.5°C. 1% BSA reduced non-specific binding to untreated E. coli cells. Furthermore, in comparison to experiments performed with Tris as a blocking agent, the protein reduced non-specific binding to Gram-positive cells. The results obtained in this project are a step further in the development of a new method to rapidly detect bacteria Gram-specifically.

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No description available.

Site-selective protein modification has become an important tool to study protein functions in chemical biology. In the preliminary work, allyl sulfides were found to be reactive substrates in aqueous cross-metathesis (CM) enabling the first examples of protein modification via this approach. In order to access the enhanced CM reactivity of allyl sulfide on proteins, facile chemical methods to install S-allyl cysteine on protein surface were developed. In particular, a cysteine-specific allylating reagent – allyl selenocyanate was used on protein substrate for the first time. The substrate scope of allyl sulfide-tagged proteins and factors that affect the outcome of CM was also investigated. A range of metathesis substrates containing different olefin tether of various lengths were screened; allyl ethers were found to be most suitable as CM partners. By reducing the steric hindrance around the allyl sulfide on protein surface through a chemical spacer, the rate and conversion of metathesis reaction on proteins was greatly enhanced. Moreover, allyl selenides were found to be more reactive than allyl sulfides in CM and enabled reactions with substrates that were previously impossible for the corresponding sulfur-analogue. Through this work, substrate selection guidelines for successful metathesis reaction on proteins were established. Rapid Se-relayed CM was further investigated through biomimetic chemical access to Se-allyl selenocysteine (Seac) via dehydroalanine. On-protein reaction kinetics revealed rate constants of Seac-mediated CM to be comparable or superior to off-protein rates of many current bioconjugations. This CM strategy was applied to histone proteins to install a mimic of acetylated lysine (K9Ac, an epigenetic marker). The resulting synthetic H3 was successfully recognized by antibody that binds natural H3-K9Ac. A Cope-type selenoxide elimination subsequently allowed the removal of such modification to regenerate dehydroalanine. Finally, preliminary research efforts towards metabolic incorporation of allyl sulfide-containing amino acid into proteins, and CM on cell surfaces were discussed.

Le développement des nanoparticules fluorescentes, appelées quantum dots (QDs) est devenu l'un des domaines les plus prometteurs de la science des matériaux. Dans cette étude une procédure de synthèse de QDs a été mise au point, comprenant la synthèse de noyaux ultra-minces de CdSe, la purification de noyau haute performance, le revêtement central avec une coquille épitaxiale en ZnS. Cette approche a permis d’obtenir des QDs d’une taille de 3,7 nm possédant un rendement quantique supérieur à 70%. Les QDs développés ont été utilisés pour concevoir des conjugués de QDs compacts avec les nouveaux dérivés d'acridine, ayant une affinité élevée pour le G-quadruplex des télomères, ainsi que leur effet inhibiteur sur la télomérase, une cible importante du traitement du cancer. Les résultats de cette étude ouvrent la voie à l'ingénierie de nanosondes multifonctionnelles possédant une meilleure pénétration intracellulaire, une plus forte brillance et une stabilité colloïdale plus importante. / Development of the fluorescent nanoparticles referred to as quantum dots (QDs) has become one of the most promising areas of materials sciences. In this study, a procedure of synthesis of QDs, which includes the synthesis of ultrasmall CdSe cores, high-performance purification, core coating with an epitaxial ZnS shell has been developed. This approach has allowed obtaining 3.7-nm QDs with a quantum yield exceeding 70%. The QDs have been used: to engineer compact conjugates of QDs with the novel acridine derivatives, which have a high affinity for the telomere G-quadruplex; to demonstrate their inhibitory effect on telomerase, an important target of anticancer therapy; and to accelerate transmembrane penetration of ultrasmall QDs into cancer cells while retaining a high brightness and colloidal stability. The results of this study pave the way to the engineering of multifunctional nanoprobes with improved intracellular penetration, brightness, and colloidal stability.

Dynamic covalent chemistry (DCC) is currently exploited in several areas of biomedical applications such as in drug discovery, sensing, molecular separation, catalysis etc. Hydrazone and oxime chemistry have several advantages, such as mild reaction conditions, selectivity, efficiency, and biocompatibility and therefore, have the potential to be for bioconjugation applications. However, these reactions suffer from major drawbacks of slow reaction rate and poor bond stability under physiological conditions. In this regard, the work presented in this thesis focuses on designing novel bioconjugation reactions amenable under physiological conditions with tunable reaction kinetics and conjugation stability. The first part of the thesis presents different strategies of dynamic covalent reactions utilized for biomedical applications. In the next part, a detailed study related to the mechanism and catalysis of oxime chemistry was investigated in the presence of various catalysts. Aniline, carboxylate and saline were selective as target catalysts and their reaction kinetics were compared under physiological conditions (Paper I and II). Then we attempted to explore the potential of those chemistries in fabricating 3D hydrogel scaffolds for regenerative medicine application. A novel mild and regioselective method was devised to introduce an aldehyde moiety onto glycosaminoglycans structure. This involved the introduction of amino glycerol to glycosaminoglycans, followed by regioselective oxidation of tailed flexible diol without affecting the C2-C3 diol groups on the disaccharide repeating unit. The oxidation rate of the tailed flexible diol was 4-times faster than that of C2-C3 diol groups of native glycosaminoglycan. This strategy preserves the structural integrity of the glycosaminoglycans and provides a functional aldehyde moiety (Paper III). Further, different types of hydrazones were designed and their hydrolytic stability under acidic condition was carefully evaluated. The hydrazone linkage with the highest hydrolytic stability was utilized in the preparation of extracellular matrix hydrogel for delivery of bone morphogenetic proteins 2 in bone regeneration (Paper IV) and studied for controlled release of the growth factor (Paper III). In summary, this thesis presents a selection of strategies for designing bioconjugation chemistries that possess tunable stability and reaction kinetics under physiological conditions. These chemistries are powerful tools for conjugation of biomolecules for the biomedical applications.