No description available.

Fluorescent labeling can improve the detection sensitivity in capillary electrophoretic (CE) separations down to attomolar concentrations. However, most fluorescent labels are not compatible with CE because their fluorescence properties and charge states are pH-dependent, they are often hydrophobic and they have a tendency to significantly change the properties of the analytes after labeling. A group of fluorescent labeling reagents have been prepared whose fluorophores have properties that are optimized for CE separations. These fluorophores have fluorescence properties and charge states that are independent of pH in the 2 < pH < 11 range. Their excitation maxima are also compatible with the 488 nm line of the Argon ion laser. A mono-cationic acridine-based fluorescent label was prepared and was found to not shift the pI of a labeled model protein in capillary isoelectric focusing separation (cIEF). Lower loading, due to increased sensitivity, led to better resolution of closely spaced isoform peaks having a pI = 0.05. A tri-anionic pyrene-based fluorescent labeling reagent was also synthesized and was used in the sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) separation of proteins. The fluorophore led to an LOQ in the nM range, and did not alter the migration behavior of proteins in the sieving matrix. A third fluorescent labeling reagent was developed as a solid phase reagent (SPR) where the fluorophore was immobilized on a solid surface through a cleavable anchor. The fluorophore is di-anionic and is based on pyrene. The SPR was designed to allow the simultaneous capture and labeling of an analyte and the efficient release of the label-analyte conjugate under mild acidic conditions. The use of the SPR allowed the labeling of a diamine whose concentration was in the low nanomolar range. The SPR opens up the possibility for mono-labeling and proportional multiple labeling of proteins.

This work seeks to develop new solvents for environmentally benign chemical synthesis. Switchable solvents are a new class of compounds that change properties upon the application of some stimulus such as heat, UV light, or pH. We have developed the use of a new solvent, thiirane oxide, that has chemical properties similar to DMSO. Thiirane oxide, however, undergoes facile decomposition to two gases at temperatures above 100 C, which is much lower than the temperature required for removing DMSO. Thus we have a solvent with excellent solvation properties, but with a built-in switch for easier removal. However, thiirane oxide leaves behind sulfurous products which make the reverse reaction to reform the solvent unfeasible. We are also developing the use of another solvent, piperylene sulfone, which is expected to have good solvent properties, yet with decomposition products that can be reacted to reform the solvent. This thesis also details the work to date on piperylene sulfone. Gas-expanded liquids (GXLs) also show promise as a new reaction medium. In order to design solvent systems that take full advantage of this medium, we desire to understand the microstructure of these fluids. To that end, we are using cage reactions to probe solute-solvent and solvent-solvent interactions at the molecular level. This thesis discusses the current research on using cage reactions to probe the structure of GXLs.

Homogenous acid catalysts are prevalent throughout the chemical industry but all have the drawback of requiring post reaction neutralization and subsequent downstream removal of the product salt. The use of a base to neutralize the acid and the processing of the salt are ancillary to the process and the disposal of the salt is an environmental concern. The work presented here shows the use of alkylcarbonic acids, which form in situ with CO₂ pressure and neutralize on loss of CO₂ pressure rather than requiring a base. Thus CO₂ can be used to "switch" the acid on and off. The properties of alkylcarbonic acids are explored to gain understanding of the mechanisms by which they act. The acids are also used to catalyze the synthesis of α-pinene, methyl yellow, and benzyl iodide. These reactions are examples of common acid catalyzed reactions where this technology could be implemented. The second half of the work explores two other "switches". The first is using temperature to break an emulsion with a novel thermally cleavable surfactant. This technology has potential applications in a wide range of fields where surfactants are used including polymerization, oil recovery, and biosynthesis. The second is using CO₂ to liquefy a solid ionic compound to allow its use as a solvent. This would greatly increase the number of ionic species available for use in ionic liquid-CO₂ biphasic systems.

This thesis showcases a work that focused on developing processes with improved economic and environmental signatures. It illustrates the strengths of chemists and chemical engineers working together towards sustainable solutions. The joint collaboration between Drs. Liotta and Eckert allows the combination of disciplines to overcome economic and environment obstacles. This thesis depicts the application of chemical engineering and chemistry for industrial processes towards reducing cost and environmental impact. In chapter 2, a synthetic sequence yielding a pharmaceutical precursor was optimized for continuous processing. The precursor was for the pharmaceutical drug Ro 31-8959, which acts as a human immunodeficiency virus (HIV) protease inhibitor. A continuous flow reactor was designed, built and utilized successfully for the two-step reaction of the diazoketone pharmaceutical precursor, (1-benzyl-3-chloro-2-hydroxy-propyl)-carbamic acid tert-butyl ester. The best configuration for the continuous flow reactor involved a single and double coiled stainless steel reactor packed with glass beads. The yield obtained for the diazoketone was quantitative. In chapter 3, the cleavable surfactant (cleavable surfactants decompose in non-surface active ingredients upon stimulus), n-octyl thiirane oxide was synthesized, characterized and its surface activity and loss of surface activity upon heating was demonstrated. The n-octyl thiirane oxide surfactant activity was measured using a dye, Suddan III, and compared to a commercially available surfactant sodium dodecyl sulfate. In chapter 4, 5-amino-1H-tetrazole was synthesized using two novel synthetic routes starting from benign chemicals. Both routes involved Sharpless click chemistry in the first step to form the tetrazole ring. Both routes also used hydrogen transfer as the last step for the formation of the 5-amino-1H-tetrazole. These syntheses eliminated the use of highly toxic and/or explosive chemicals such as cyanamide, hydrazoic acid, and hydrazine. Finally in chapter 5, phase transfer catalysis was used as a means to improve reaction rates and yields between a siloxylated reagent (in the liquid phase) and insoluble ionic reagents (in the solid phase). The activity of commercial phase transfer catalysts like tetra-n-butylammonium bromide was compared to the activity of two novel custom-made siloxylated phase transfer catalysts. Surprisingly, the tetra-n-butylammonium resulted in superior rate constants to the custom made siloxylated phase transfer catalysts.

No description available.

Le développement de réactions chimiques compatibles avec les milieux biologiques est un défi scientifique de taille. Les outils dont actuellement disponibles pour le marquage spécifique d’entités biologiques se sont multipliés pendant cette dernière décennie. Parmi les réactions vedettes du domaine, on trouve les réactions de Diels-Alder entre alcènes tendus et tétrazines ou encore les cycloadditions entre les cycloalcynes et les azotures (SPAAC). En revanche, les réactions de clivage bioorthogonal ont retenu une attention plus faible de la part de la communauté scientifique. Récemment notre groupe a identifié une réaction bioorthogonale entre une iminosydnone et un cycloalcyne permettant d’effectuer simultanément le clivage d’un composé et la ligation de deux entités. Ces travaux de thèse ont été entièrement dirigés vers cette nouvelle réactivité.En premier lieu, nous avons étudié la synthèse et la fonctionnalisation du partenaire iminosydnone dans le but d’obtenir une librairie de composés pour l’étude de la cinétique de la réaction de click & release avec les cycloalcynes. Dans un second temps, nous avons utilisé les outils synthétiques développés pour concevoir des sondes iminosydnones à double exaltation de fluorescence. Cela a marqué l’entrée de ces composés dans le contexte de la chimie bioorthogonale puisque la sonde la plus prometteuse a été utilisée sur des cellules vivantes.Enfin, la capacité des iminosydnones à être clivées par les cycloalcynes a été exploitée sous la forme d’espaceurs clivables dans deux applications. Nous avons tout d’abord conçu des outils pour le target-fishing. Les sondes obtenues ont permis l’immobilisation de protéines sur un support solide. La libération du support des protéines a pu être effectuée de manière quantitative tout en leur apposant une étiquette fluorescente. Le second objet exploitant les propriétés d’espaceurs clivables des iminosydnones est un ADC. Celui en développement dans cette thèse est destiné à traiter les infections bactériennes, cible atteignable uniquement en employant un espaceur clivable par un agent exogène. / The development of chemical reactions compatible with the biological medium is a major challenge. The availability of chemical tools for the specific labeling of biological entities increased dramatically during the last decade. The Diels-Alder reactions between tetrazines and strained alkenes or the Strained Promoted Azide Alkyne Cycloaddition (SPAAC) are among the most popular reactions for bioorthogonal ligations. On the other hand, moderate attention was given to bioorthogonal cleavage reactions. Recently our group identified a new bioorthogonal reaction between an iminosydnone and a cycloalkyne, enabling a cleavage and the ligation of two partners at the same time.The first aim of this PhD thesis was to study the synthesis and functionalisation of the iminosydnone partner. With the large library of compounds in our hands, we could study the kinetic properties of the click & release reaction.Next, using this transformation, we synthesized double fluorogenic iminosydnone probes. The most promising probe was then used on living cells, underlining the bioorthogonality of the reaction.Finally, we took advantage of the iminosydnone properties to use them as cleavable linkers in two applications. The first field we explored was target-fishing. The tools we developped allowed protein immobilization on a solid support. The quantitative release from the support of labeled proteins was achieved using the click & release reaction. The second object using iminosydnones as cleavable linkers we considered was an ADC. The one developped in this PhD thesis is aimed at bacteria. For treatment with ADCs, this target can only be reached using a linker cleavable with an external agent.

Ce travail a consisté en la synthèse d’outils chimiques et au développement de leurs applications biologiques. Dans un premier temps, des sondes pour l’étude de la Topoisomérase IIA humaine ont été synthétisées. Ces sondes ont alors été testées sur lysat cellulaire pour la capture des protéines présentant une affinité pour ces médicaments. Dans un second temps, un nouveau lien clivable en conditions non dénaturantes pour des applications en protéomique chimique a été developpé. Ainsi, après optimisation de la structure, il a été intégré au sein d’une sonde d’affinité pour évaluer sa capacité de capture et libération de la PARP 1. Enfin, la réaction de click entre un azoture et un cyclooctyne a été appliquée à l’élimination d’une drogue circulante dans le sang.Après l’étude cinétique de la réaction, l’activité biologique et la pharmacocinétique des différents composés ont été évaluées pour optimiser la réaction de click in vivo. / The main goal of this work was to synthesize chemical tools and to developp their biological applications. In the first part, probes for the study of Topoisomerase II via chemical proteomic were synthesized. They were then used for pulldown experiments on cell lysats. In a second part, a new cleavable linker in non denaturing conditions was developped for chemical proteomic applications. After optimisation of the structure, it was incorporated in an affinity probe and tested for the pulldown of PARP 1. Finally, a click chemistry reaction, the SPAAC, was used to provok the elimination of a circulating drug. After the study of the kinetic of the reaction, the biological activity and the pharmacokinetic of the different compounds were evaluated to optimise the click reaction in vivo.

Dans les cellules eucaryotes, les cassures double-brin sont réparée selon deux voies principales : la recombinaison homologue et la jonction des extrémités non homologues, toutes deux bien connues dans la littérature. Cependant quelques zones d'ombres persistent quant à deux aspects singuliers de leur mise en œuvre :- Si ces deux mécanismes peuvent opérer dans les cellules, quels sont les déterminismes qui président au choix d'une voie de réparation plutôt que de l'autre ?- Dans le cas où les cassures double-brin sont induites dans l'ADN par des rayonnements ionisants – comme ceux employés en radiothérapie anticancéreuse – coment s'opère la réparation lorsque les extrémités générées ne sont pas compatibles avec une ligation immédiate ? Connaître les protéines impliquées dans ce cas permettrait d'élaborer des adjuvants à la thérapie anticancéreuse.Afin de contribuer à répondre à ces questionnements, nous avons voulu élaborer un système analytique intégré qui permît 1) le recrutement spécifique de complexes de réparation des cassures double-brin de l'ADN sur des phases chromatographiques constituées au laboration, 2) la résolution de ces complexes sur gel d'acrylamide non-dénaturants et leur visualisation et 3) la caractérisation biochimique fine des complexes séparés. La méthodologie élaborée au cours de cette thèse a concerné chacun des trois points ci-dessous : 1) nous avons conçu et mis en œuvre un système chromatographique nous permettant de distinguer les protéines recrutées spécifiquement sur des oligonucléotides duplexes d'ADN dotés d'extrémités libres de l'ADN (mimant des cassures double-brin) des autres protéines se fixant sur la séquence interne des oligonucléotides ; 2) nous avons adapté à notre problématique une méthodologie d'électrophorèse non-dénaturante permettant la résolution des complexes purifiés tout en garantissant leur intégrité au cours de la migration ; 3) grâce à la visualisation directe des complexes résolus dans le gel, nous avons pu déterminer leur composition en protéines par spectrométrie de masse.L'étude biochimique des complexes purifiés a démontré que les complexes purifiés étaient fonctionnels, c'est à dire capable de liguer deux oligonucléotides entre eux. La fouille des données de spectrométrie de masse, obtenues à partir d'un grand nombre d'expériences indépendantes, nous a permis de montrer qu'ils étaient de la physiologie de l'ADN et particulièrement représentatifs de la diversité des mécanismes de réparation.De manière intéressante, nous avons pu observer que certaines protéines recrutées spécifiquement sur les mimes de cassures double-brin de l'ADN, ne sont pourtant pas connues pour intervenir dans les processus de réponse aux dommages de l'ADN (synthèse de nucléotides, checkpoint, topologie de l'ADN, cytosquelette).Le rôle des protéines évoquées ci-dessus sera prochainement caractérisé in cellulo notamment avec des stratégies de type RNAi. D'autre part, nous utiliserons les développements méthodologiques décrits ci-dessus pour étudier les mécanismes de réparation des cassures double-brin radio-induites, tels qu'ils sont mis en jeu dans les cellules tumorales en constituants de nouvelles phases chromatographiques avec des oligonucléotides irradiés. / In eucaryotic cells, DNA double-strand breaks are repaired through two main pathways : the homologous recombination and the non homologous end joining . Altough these pathways are well characterized, two particular aspects of the repair remain poorly understood :- If two separated pathways may occur in the cells, which mechanism(s) govern the choice of the pathway that will ultimately lead to the repair ?- If the double-strand break is induced by ionizing radiations – as those employed in anti-cancerous radiotherapy – how does the repair occur if the DNA ends at the edge of the break are not compatible with a direct ligation ? A proper knowledge of the proteins involved in this repair would allow the development of additives, useful to increase the efficiency of the radiotherapy.To investigate these questions, we designed a new analytical system allowing : 1) the specific recruitment of DNA double-strand break repair complexes on home-made chromatographic phases, 2) the separation of these complexes in a non-denaturing polyacrylamide gel and their subsequent visualization and 3) their biochemical characterization.The methodology developped in this work has been focused on the following points : 1) we designed and implemented a chromatographic system allowing the distinction between proteins recruited onto duplex DNA oligonucleotide with free DNA ends (mimicking DNA double-strand breaks) and proteins fixed onto the internal sequence of the same oligonucleotides ; 2) we adapted to our problematic a methodology of non-denaturing electrophoresis thus allowing the separation of the purified complexes while guaranteeing their integrity during the migration, 3) we also determined their composition by mass spectrometry after their visualization.The biochemical study has shown that the purified complexes were still functionnal, that is they were able to efficiently ligate two oligonucleotides. The study of the data provided by the mass spectrometry analysis of independant experiences proved that the complexes belonged to the DNA physiology and were especially representative of the diversity of the DNA repair pathways.Interestingly, we observed that some of the protein specifically recruited onto the the double-strand breaks were not known to be involved in the DNA repair (nucleotide synthesis, checkpoint, DNA topology, cytoskeleton).The rôle of these proteins should be characterized in cellulo especially with siRNA. On the other hand we will also use the methodological development described above to study the repair mechanisms of radio-induced DNA double-strand breaks occuring in the irradiated tumorous cells. To achieve this study we will elaborate new chromatographic phases with pre-irradiated oligonucleotides.

Cette étude a consisté au développement et à l’utilisation de fonctions chimiques clivables en milieux biologiques. Dans le domaine de la protéomique chimique, ce travail a abouti à la conception d’une sonde d’enrichissement clivable en conditions non-dénaturantes. Appliquée à l’étude de topoisomérases, cette sonde a permis l’extraction et l’analyse de complexes fonctionnels A2B2 de gyrase. Dans un second temps, un nouveau concept de quencheur chimiquement désactivable a étéintroduit. Incorporé dans une sonde pro-fluorescente de type FRET, ce type de quencheur permet notamment de visualiser la présence de sondes non-activées dans des cellules. Enfin, une méthode a été développée pour permettre l’évaluation de la labilité d’une liaison chimique en milieux biologiques natifs. Basée sur l’utilisation de sondes pro-fluorescentes, cette méthodologie a plus particulièrement été appliquée à l’étude de la bio-labilité de groupements acido-labiles. / The general main topic of this work was the use and the development of cleavable linkers in biological systems. This study led to the design of a cleavable enrichment probe in non-denaturing conditions for chemical proteomic applications. In a topoisomerase analysis, this probe allowed the extraction and analysis of a functional DNA gyrase A2B2 complex. For fluorescence imaging, a new concept of chemically deactivatable quencher was introduced. This quencher was used to revealinactivated FRET-based probe in cell experiments. Finally, a methodology based on biolability measurements of acid-sensitive molecules was developed for the evaluation of chemical bond lability in native biological environments. This work was focused on biolability measurements of acidsensitive molecules.