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.

Des études phytochimiques dediverses espèces du genre Macaranga ontconduit à la découverte d’une familleintéressante de stilbènes prénylés nommésschweinfurthines (SWs). Le NCI (NationalCancer Institute) a évalué l’activité cytotoxiquede ces composés sur un panel de 60 lignéescellulaires cancéreuses : ces composésprésentent de très bonnes activités cytotoxiquessur certaines de ces lignées. De plus, leur profild’activité original suggère que ces composésagissent selon un nouveau mécanisme d’action,encore inconnu. Ainsi, ce projet de thèse a pourbut d’éclaircir ce mécanisme par identificationdes protéines cibles ainsi que des voies designalisations impliquées. Pour cela l’obtentiond’une grande quantité de SWs était nécessaire.Dans un premier temps, l'espèce vietnamienneMacaranga tanarius a été sélectionnée par uneapproche utilisant les réseaux moléculaires. Sonétude phytochimique a conduit à l’isolement denouveaux analogues et de SWs d’intérêt àl'échelle du gramme, permettant de réaliser dansun second temps des modifications chimiquesciblées. Une fonction alcyne a, par exemple, étéintroduite sur la SW G sans altérer son profild’activité. La présence de cette fonction aensuite permis le couplage de la SW avec unfluorophore et une biotine par chimie click incellulo. Des études de localisation intracellulaireet d’identification d’un partenaire protéique ontensuite été réalisées. En parallèle de ces travaux,l’interaction entre les SWs et la protéine OSBP(Oxysterol-binding protein), décrite commeétant une cible potentielle des SWs a égalementété investiguée. / Phytochemical studies fromMacaranga species led to the discovery of aninteresting family of prenylated stilbenesnamed schweinfurthins (SWs). The NCI(National Cancer Institute) evaluated thesemolecules on a panel of 60 human cancer cells:studying these molecules is of great interestbecause they display promisingantiproliferative activities for specific tumorderivedcell lines. Furthermore, theiruncommon activity profile suggests that itimplements a new mechanism of action. Ourproject aims to decipher this mechanism byfinding the targeted protein(s) and thesignalling pathway involved. For that purpose,a large amount of these natural stilbenes wasrequired. First, a vietnamese plant has beenselected using molecular networking approachto ensure the presence of these molecules.Then, a phytochemical study on a large scaleled to the isolation of the compounds of interest(several grams) and new analogues. An alkynemoiety has been introduced on one of thesemolecules by a selective chemical modificationwithout any loss of the specific cytotoxicprofile. This alkyne function allowed the incellulo click reaction between a fluorescentprobe or a biotin. Thus, studies of intracellularlocalization and protein identification havebeen achieved. Concurrently, interactionbetween SWs and Oxysterol-binding protein(OSBP), a previously described target of thesecompounds, has been studied.

Natural compounds have been the primary resource used to produce polymeric materials by humankind since the mid-1900s. Yet, progress in bio-based polymers from renewable feedstock has encountered some obstacles, mainly due to the low prices of petroleum-based monomers, compared to natural and sustainable materials. However, most commodity plastics are non-degradable materials, and solid plastic waste accumulation adversely affects the environment. As the world population is growing and demanding chemicals, energy, and plastics materials, polymer research is focusing on synthesizing bio-based and degradable polymers. Thus, biomass, a sustainable and inexpensive feedstock, is highly appropriate for designing alternative thermoplastics that are degradable to reduce the current environmental issues. In this dissertation, three different approaches were used to afford alternative thermoplastics to petroleum‐based commodities: bio-based poly(ether-amide)s, random aromatic copolyesters, and copoly(acetal triazole)s. In our first approach, two new lignin‐derived poly(ether‐amide)s (PEA)s were prepared. Their thermal properties showed high degradation temperature (Td) ranging from 330 °C to 380 °C, and glass transition temperature (Tg) between 100 °C and 120 °C. The chemical degradation studies revealed that the PEAs were degradable in 4 M H2SO4, HNO3, and TFA in 3 days. The second polymer group synthesized were semicrystalline bio-based aromatic copolyesters with tunable thermal properties. The thermal analysis of these copolyesters revealed high Td (413 °C to 446 °C) and Tg and Tm ranging from –36 °C to 67 °C and 60 °C to 267 °C, respectively. Their crystallization behavior showed a dependence on the comonomer composition, exhibiting a pseudo-eutectic region. Finally, furfural- and benzaldehyde-based copoly(acetal triazole)s (Td range 280–340 °C) were prepared by click polymerization at room temperature. Preliminary results showed that furfural-based copoly(acetal triazole)s were susceptible to hydrolytic degradation under neutral conditions after only 8 days at 40 °C. Overall, degradable and bio-based polymers were successfully synthesized as a potential thermoplastic alternative for packaging applications.

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Oligosaccharides play very crucial biological roles in the human body. They are structurally diverse and very challenging to analyze. In an attempt to contribute towards the analysis of oligosaccharides in the growing filed of 'Glycomics', we explored different ways to make novel efficient oligosaccharide tags to label N -linked oligosaccharides from glycoproteins. We synthesized and characterized various oligosaccharide tags which are useful to analyze the oligosaccharide by mass spectrometry, HPLC, and bioaffinity. In these tags we incorporated ar UV/fluorescent core, a biotin moiety and an amino/azido/alkyne terminus. We varied the structures of the tags for improved solubility in common organic solvents. These tags were used to label standard oligosaccharides, and the labeling efficiency was evaluated.

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In the scope of our BACE1 inhibitor project we used an originally designed microtiter plate-based screening to discover 4 triazole-linked reduced amide isosteres that showed modest (single digit micromolar) BACE1 inhibition. Our ligands were designed based on a very potent (single digit nanomolar) isopththalamide ligand from Merck. We supplanted one of the amide linkages in order to incorporate our triazole and saw a 1000-fold decrease in potency. We then enlisted Molsoft, L.L.C. to compare our ligand to Merck's in silico to account for this discrepancy. They found that the triazole linkage gives rise to a significantly different docking pose in the active site of the BACE1 enzyme, therefore diminishing its potency relative to the Merck ligand. The ability to control the regio- and stereochemical outcome of organic reactions is an ongoing interest and challenge to synthetic chemists. The pre-association of reacting partners through hydrogen bonding (H-bonding) can often to yield products with extremely high stereoselectivity. We were able to show that anilines, due to their enhanced acidity relative to amines, can serve as substrate directing moieties in the opening of cyclohexene oxides. We observed that by judicious choice of conditions we could control the regiochemical outcome of the reaction. These studies demonstrate that an intramolecular anilino-NH hydrogen bond donor can direct Fürst-Plattner epoxide opening. A unified mechanism for this phenomenon has been proposed in this work which consists of a novel mechanistic route we call "NH-directed Fürst-Plattner." We further studied the opening of cyclohexene oxides by incorporating amide and amide derivative substituents in both the allylic and homoallylic position relative to the epoxide moiety. Our attempts to control regioselectivity in the allylic systems were unsuccessful; however when the directing substituent was in the homoallylic position, we could demonstrate some degree of regioselectivity. An additional project that the author worked on for approximately one year during his graduate student tenure is not described within this work. In February of 2009 AstraZeneca, Mayo Clinic, and Virginia Tech Intellectual Properties Inc. concomitantly announced that AstraZeneca licensed a portfolio of preclinical Triple Reuptake Inhibitor (TRI) compounds for depression. The lead compound, PRC200, was discovered by a collaborative effort between the Carlier and Richelson (Mayo Clinic Jacksonville) research groups in 1998. The author was tasked to develop backup candidates of PRC200 in order to improve the pharmacokinetics of the lead compound. Due to confidentiality agreements, this work is not reported herein. / Ph. D.

Abstract : Adverse drug reactions (ADRs) are undesirable effects caused after administration of a single dose or prolonged administration of drug or result from the combination of two or more drugs. Idiosyncratic drug reaction (IDR) is an adverse reaction that does not occur in most patients treated with a drug and does not involve the therapeutic effect of the drug. IDRs are unpredictable and often life-threatening. Idiosyncratic reaction is dependent on drug chemical characteristics or individual immunological response. IDRs are a major problem for drug development because they are usually not detected during clinical trials. In this study we focused on IDRs of Nevirapine (NVP), which is a non-nucleoside reverse transcriptase inhibitor used for the treatment of Human Immunodeficiency Virus (HIV) infections. The use of NVP is limited by a relatively high incidence of skin rash. NVP also causes a rash in female Brown Norway (BN) rats, which we use as animal model for this study. Our hypothesis is that idiosyncratic skin reactions associated with NVP treatment are due to post-translational modifications of proteins (e.g., glutathionylation) detectable by MS. The main objective of this study was to identify the proteins that are targeted by a reactive metabolite of Nevirapine in the skin. The specific objectives derived from the general objective were as follow: 1) To implement the click chemistry approach to detect proteins modified by a reactive NVP-Alkyne (NVP-ALK) metabolite. The purpose of using NVP-ALK was to couple it with Biotin using cycloaddition Click Chemistry reaction. 2) To detect protein modification using Western blotting and Mass Spectrometry techniques, which is important to understand the mechanism of NVP induced toxicity. 3) To identify the proteins using MASCOT search engine for protein identification, by comparing obtained spectrum from Mass Spectrometry with theoretical spectrum to find a matching peptide sequence. 4) To test if the drug or drug metabolites can cause harmful effects, as the induction of oxidative stress in cells (via protein glutathionylation). Oxidative stress causes cell damage that mediates signals, which likely induces the immune response. The results showed that Nevirapine is metabolized to a reactive metabolite, which causes protein modification. The extracted protein from the treated BN rats matched 10% of keratin, which implies that keratin was the protein targeted by the NVP-ALK. / Résumé : Les effets indésirables (EI) sont les effets indésirables causés après l'administration d'une dose unique ou une administration prolongée du médicament ou le résultat de la combinaison de deux médicaments ou plus. La Réaction idiosyncratique (IDR) est une réaction indésirable qui ne se produit pas dans la plupart des patients traités avec un médicament et qui ne comporte pas l'effet thérapeutique du médicament. IDR sont imprévisibles et peuvent mettre la vie du malade en danger. Cette réaction dépend des caractéristiques chimiques du médicaments et/ou de la réponse immunitaire individuelle du patient. IDR est un problème majeur pour le développement de médicaments car ils ne sont généralement pas détectés au cours des essais cliniques. Dans cette étude, nous nous sommes concentrés sur la Réaction idiosyncratique de névirapine (NVP) qui est un inhibiteur de transcriptase inverse non nucléosidique utilisé pour le traitement du virus d'immunodéficience humaine (VIH). L'utilisation de NVP est limitée par une incidence relativement élevée d'éruption cutanée. NVP provoque également une éruption cutanée chez les rats femelles de souche Brown Norway. Notre étude vise à mieux comprendre les IDRs induites par l'administration de NVP chez l'animal. La présente étude vise à vérifier l'hypothèse que les problèmes cutanés associés à la prise de NVP soient attribuables à la modification post-traductionnelle de protéines détactable par spectrométrie de masse. Les principaux objectifs de ce projet étaient : 1) Déterminer si la Nevirapine alcynes (NVP-ALK), un analogue de la NVP peut développer la même éruption cutanée que la NVP. La NVP-ALK a été couplé avec de la biotine en utilisant la réaction chimique (click chemistry). 2) Détecter les modifications post-traductionelles des proteines par Western blot et des techniques de spectrométrie de masse, pour comprendre le mécanisme de la toxicité induite par la NVP. 3) Identifier les protéines modifiées en utilisant le moteur de recherche MASCOT pour l'identification des protéines, en comparant le les spectres de masse obtenus avec les spectres théoriques pour trouver une séquence correspondante de peptide. 4) Tester si la NVP et ses métabolites peuvent provoquer des effets nocifs, comme l'induction d'un stress oxydatif dans les cellules (par la mesure de la glutathionylation des protéines). Les résultats ont montré que la névirapine est métabolisé en métabolite réactif ce qui provoque une modification de la kératine. Ainsi nos résultats suggèrent que la kératine est la cible des métabolites de la NVP-ALK.