Bioorthogonal reactions, due to its excellent selectivity and time-efficiency, have emerged as a popular tool for protein and cell probing. Among all the bioorthogonal reactions, the inverse electron-demand Diels-Alder reaction (IEDDA) reaction has its advantage of bearing the fastest kinetics. Although the IEDDA reaction drew considerable attention in chemical biology in the last decade, challenges lie in finding the suitable dienophiles. Strained dienophiles, for example, trans-cyclooctene derivatives, can undergo ultrafast IEDDA reactions and therefore have been extensively developed. Unstrained alkenes and alkynes, however, have not been well investigated as IEDDA handles. In general, unstrained dienophiles are more straightforward to synthesise compared with strained dienophiles, therefore they are more accessible to researchers. In addition, the absence of a highly reactive bond makes unstrained dienophiles inert to biological nucleophiles, which allows effectively cellular labelling. In this dissertation, I described three different unstrained dienophiles for different biological purposes. Allyl handle is thiol-stable and non-toxic, which was utilised to label apoptotic cells in a pre-targeting manner. Enol ethers can react with tetrazines to decage protected amino acids and prodrugs. Potassium arylethynyltrifluoroborate, as a novel dienophile, was shown to react fast with pyridyl tetrazines controllably and this new IEDDA was applied to label proteins site-selectively and to fluorescently label two proteins orthogonally. In addition to IEDDA reactions, other bioorthogonal reactions were also developed using these versatile unstrained handles. Allyl-bearing amino acids and proteins can undergo an acetophenone-mediated hetero-[2+2] photocycloaddition with maleimide derivatives, expanding the toolbox of photo-triggered chemistry for protein modification. The potassium arylethynyltrifluoroborate handle was also found reactive in copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) and showcased the huge potential for protein labelling and multicolour cellular labelling.

Bioorthogonal chemistry is a rapidly growing field that enables spatiotemporal monitoring of biomolecules using targeted probes. The development of bioorthogonal reactions therefore requires several criteria to be met. Reactions need to be selective, and fast enough so lower concentrations of reagents are used to mitigate toxicity, and they need to be stable in biological environments. 3-oxidopyridiniums are a class of water stable six-membered heteroaromatic latent dipoles, and have been previously reported to undergo [3+2] cycloadditions with electron deficient dipolarophiles, but have yet to be investigated for bioorthogonal use. To test their applicability as a bioorthogonal reagent, a series of N-methyl-3-oxidopyridiniums were synthesized with varying substituents on the 5 position and were reacted with DIBO (dibenzocyclooctyne). Electron donating 5-substituents have been shown to significantly increase the rate of the reaction, with bimolecular rate constants ranging from 3.31 x 10⁻⁴ with 5-trifluoromethyl-N-methyl-3-oxidopyridinium to 1.07 M⁻¹ s⁻¹ with 5-amino-N-methyl-3-oxidopyridinium, putting the faster reactions on par with commonly used bioorthogonal reactions for cell labelling. Strain-promoted alkyne-nitrone cycloadditions (SPANC) are a class of [3+2] cycloadditions that are commonly used for bioorthogonal reactions. In comparison to their predecessor SPAAC (strain promoted azide alkyne cycloadditions), SPANC offers much better reaction tunability. icSHAPE (in vivo click selective hydroxyl acylation analyzed by primer extension) is a labelling technique used to determine RNA structure. This is done by selectively targeting the 2' hydroxyl on the ribose sugar of RNA that is structurally available in regions of RNA that are single stranded, and the use of SPAAC allows for an improved signal to noise ratio. Herein, DMImO (1-[(p-methoxycarbonylphenyl)methyl]-2,2-dimethyl-5-oxo-3-imidazolin-3-ium-3-olate), a previously used nitrone in SPANC reactions, has been modified to include an electron deficient carbonyl imidazole to allow for a nucleophilic attack from the 2' hydroxyl of the RNA. Hydrolysis of the nitrone probe is on par with previous SHAPE reagents that are used for in vivo labelling and is able to label 5S rRNA for structure determination as effectively as previously used SHAPE reagents.

Ce travail est composé de trois projets. Le premier projet a pour objectif de découvrir de nouveaux métabolites de la vitamine A. Il a consisté en la synthèse d’un analogue du rétinaldéhyde, portant une fonction azoture et permettant de suivre son devenir in vivo. Le second projet a consisté en l’élaboration de la sonde ATPP permettant l’analyse de l’ensemble des métabolites aldéhydiques d’un échantillon. La sonde permet un gain de sensibilité en LS-MS². Une analyse de sa biodistribution a été faite, et montre que la sonde ATPP, après injection intrapéritonéale, est distribuée in vivo. Concernant le troisième projet, un réactif de couplage homobifonctionnel « thiol-thiol » a été élaboré. Les produits du couplage ont montré une excellente stabilité plasmatique. Le réactif a d’abord été appliqué avec succès au couplage de petites molécules, puis au couplage d’un oligonucléotide modifié et d’un peptide. / This work consists of three projects. The first project aims to discover new metabolites of vitamin A. An analog of retinaldehyde, carrying an azide function was synthesized. It would allow to follow its fate in vivo. The second project consisted in the development of a probe allowing the analysis of all the aldehyde metabolites in a sample. The probe provides sensitivity gain in LS-MS². An analysis of its biodistribution has been done, and showed the ATPP probe is distributed after an intraperitoneal injection. Concerning the third project, a homobifunctional coupling reagent "thiol-to-thiol" has been developed. The coupling products showed excellent plasma stability. The reagent was first successfully applied to the coupling of small molecules and then to the coupling of a modified oligonucleotide and a peptide.

Gas filled microbubbles (MBs) stabilized by a shell (e.g. lipids) are commonly used as ultrasound (US) contrast agents. Attaching biomolecules to the surface of MBs allows for molecular US imaging of various diseases. With the increased interest in targeted US imaging, new platforms to prepare disease-targeted MBs are necessary. Furthermore, attaching signaling agents to MBs creates multi-modal imaging opportunities, enhancing visualization and quantification of disease biomarkers. In this thesis, MBs labeled with 99mTc and/or rhodamine dye by taking advantage of the strong interaction between biotin and streptavidin are reported. Radiolabeling of MBs was achieved in good radiochemical yield (~ 30%). 99mTc-labeled MBs were targeted to vascular endothelial growth factor receptor 2 (VEGFR2) using an anti-VEGFR2 antibody and to prostate specific membrane antigen (PSMA) using small-molecule based PSMA inhibitors. In vitro evaluations showed successful binding of MBs to the target while in vivo targeting assessments were unsuccessful. New strategies to target MBs to the site of interest were then developed through the use of the bioorthogonal reaction between tetrazine (Tz) and trans-cyclooctene (TCO). A biotinylated derivative of Tz was loaded on streptavidin coated MBs to create a Tz-derivatized MB (MBTz). Targeting MBTz to extracellular markers of cancer such as VEGFR2, PSMA and urokinase plasminogen activator receptor (uPAR) in vitro was achieved using TCO-conjugated antibodies. In vivo targeting was successful for VEGFR2 and PSMA, but not uPAR. Translating the new strategy to other US contrast agents was then investigated. Gas vesicles (GVs) produced in halobacteria were conjugated with TCO using amide-coupling chemistry. A 99mTc-labeled derivative of Tz was loaded on TCO-GVs (RCY= 59%) and their distribution assessed by SPECT/CT imaging and ex vivo tissue counting. Having established a convenient platform to conjugate molecules to GVs and MBs, future work focuses on developing a new generation of human compatible molecular US imaging probes. / Dissertation / Doctor of Philosophy (PhD)

Glycans are macromolecules that contain several classes. Glycans can play an important role in biological activities. Studying the cell surface glycans can provide a very powerful way to understand the fundamental process. Also it could help to regulate expected cell response. Thus it is very necessary to have a method to detect cell- surface glycans efficiently. An efficient method for glycan detection is necessary. Metabolic glycan labeling and chemoenzymatic glycan labeling are most commonly used. Chemoenzymatic glycan labeling is a rapid and sensitive method which also has high specificity. This method can be applied in both vitro and vivo. However the availability of unnatural sugar nucleotides functioned by bioorthogonal groups is the main limitation for chemoenzymatic labeling. In this thesis, UDP-GlcNAc and UDP-GalNAc derivatives were prepared for further chemoenzymatic labeling by using chemoenzymatic synthesis method.

Protein-protein interactions (PPIs) are responsible for the regulation of a variety of important functions within living organisms. Compounds which can selectively modulate aberrant PPIs are novel therapeutic candidates for treating human diseases. Whilst PPIs have traditionally been considered as "undruggable", research in this area has led to the emergence of several effective methodologies for targeting PPIs. One such methodology is peptide stapling, which involves constraining a short peptide into its native alpha-helical form by forming a covalent link between two of its amino acid side-chains. The Sondheimer dialkyne reagent has previously been used in strain-promoted double-click cycloadditions with diazidopeptides to generate stapled peptides that are capable of inhibiting PPIs. However, the Sondheimer dialkyne suffers from poor water-solubility; it decomposes rapidly in aqueous solutions which limits its application in biological systems. This dissertation describes the design and synthesis of new substituted variants of the Sondheimer dialkyne with increased solubility and stability, that are suitable for application in strain promoted double click peptide stapling. In total, ten different derivatives were generated; of these, a meta-trimethylammonium substituted variant was found to have particularly high water-solubility and aqueous stability, as well as high azide reactivity. The substituted Sondheimer dialkynes were applied to the strain promoted double click stapling of p53-based diazido peptides in an effort to generate stapled peptide-based inhibitors of the oncogenic p53 MDM2 PPI, a validated target for anticancer therapeutics. Three stapled peptides were found to have inhibitory activity, thus demonstrating the utility of the novel dialkynes in the preparation of PPI inhibitors. The functionalised stapled peptide formed from a meta-fluoro substituted Sondheimer dialkyne was found to be the most potent inhibitor. All ortho-substituted Sondheimer dialkynes were found to be unreactive, whereas those with a meta-trimethylammonium substituent were highly reactive when compared to other meta-substituted dialkynes. These patterns in azide reactivity could be explained through X-ray crystallographic studies and density functional theory calculations.

As new therapeutic targets and drugs are discovered for B-cell lymphoma and other cancers, companion diagnostics are also needed to determine target engagement, therapeutic efficacy, and patient segmentation for clinical trials. We first employed synthetic chemistry to build a platform for modifying small molecule drugs into imaging probes, using the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor AZD2281 (Olaparib) as a model for technology development. Our results showed that small-molecule companion imaging drugs can be used for fluorescence imaging in cells, as well as for pharmacokinetic studies and positron emission tomography (PET) imaging in vivo, without significantly perturbing their target binding properties or cellular uptake. To apply this approach to B-cell lymphoma drugs currently in clinical trials, we modified an irreversible inhibitor of Bruton's Tyrosine Kinase (BTK), PCI-32765 (Ibrutinib), with the fluorophore Bodipy FL (BFL), and used it for imaging in cells and in a mouse window-chamber xenograft model. The excellent co-localization of our probe (Ibrutinib-BFL) with BTK demonstrated its utility for studying additional BTK inhibitors and as a companion imaging probe. In parallel, we hypothesized that central nervous system (CNS) lymphoma diagnosis from paucicellular cerebrospinal fluid (CSF) samples could be improved with molecular profiling of putative lymphoma cells trapped in a customized microfluidic chip. Following fabrication and characterization of a polydimethylsiloxane (PDMS) diagnostic device containing an array of affinity-free single-cell capture sites, we were able to efficiently recover >90% of lymphocytes, perform immunostaining on chip, and apply an image-processing algorithm to group cells based on their molecular marker expression, such as kappa/lambda light chain restriction. Additionally, in combination with Ibrutinib-BFL or other imaging drugs, we demonstrated the potential for on-chip drug imaging for use in conjunction with drug development. Finally, we applied bioorthogonal conjugation chemistries on cellulose paper for potential applications in lowering the cost of drug screening. We anticipate that these approaches will enable direct, molecular information for personalized treatment decisions in B-cell lymphomas, as well as provide a roadmap for the development of companion diagnostic probes and devices for additional indications.

This study is focused on changes in protein expression in a glioblastoma cell line during infection with tick-borne encephalitis virus. Newly synthesized proteins were distinguished from previously synthesized proteins using bioorthogonal chemistry (BONCAT method) to observe changes in protein synthesis. Labelled proteins were visualized using two-dimensional PAGE and western blotting followed by Click reaction on membrane. Differences in protein pattern between control and infected cells were observed.

Notre laboratoire a récemment mis en évidence la réaction de cycloaddition entre les sydnones et les alcynes terminaux ou cycliques. Ces réactions sont bioorthogonales et possèdent la particularité de former deux produits : l’un provenant de la ligation (click) des deux partenaires réactionnels par cycloaddition (3+2), l’autre provenant d’un relargage (release) par réaction de rétro Diels-Alder. Le potentiel de ce type de réaction est très grand de par le nombre d’applications possibles que ce soit dans le domaine de la bioconjugaison, du marquage fluorescent ou de la libération de principes actifs.Les sydnones sont des composés hétérocycliques dipolaires qui appartiennent à la famille des mésoioniques. Cette thèse vise à découvrir de nouvelles réactions de ligation et de coupure mettant en jeu ces mésoioniques et des dipolarophiles. Vingt-cinq mésoioniques et trois familles de dipolarophiles ont été synthétisés et criblées.Lors du premier criblage, la réactivité des composés mésoioniques avec un alcyne terminal, sous une catalyse au cuivre, a été explorée. Neuf conditions de catalyse ont été testées. Ce criblage a mis en évidence la cycloaddition entre les aza-iminosydnones et les alcynes terminaux dont le produit formé dépend du système catalytique et de la nature de l’alcyne.La deuxième famille de dipolarophiles testée est celle des cycloalcynes. Nous avons découvert que, les dithiolium-olates réagissaient avec les cycloalcynes peu encombrés et que les sydnone-imines réagissent avec tous les cyloalcynes de manière bioorthogonale en libérant une molécule complexe.Enfin, les cycloalcènes ont été testés comme dipolarophiles. Dans cette partie, les résultats préliminaires du criblage nous ont amené à modifier la structure de la sydnone pour permettre la libération d’une molécule complexe. / Our laboratory has recently discovered the cycloaddition between sydnones and cyclic or terminal alkynes. These reactions are bioorthogonal and generate two products: one coming from the (3+2) cycloaddition (click) between both partners and the second from a cleavage step (release) through a retro Diels-Alder reaction. This type of new reactions would be of prime importance in the development of new reagents useful for bioconjugation and drug delivery applications.Sydnones are dipolar heterocycles belonging to the large family of compounds known as mesoionics. This work aims at discovering new chemoselective and bioorthogonal processes between mesoionic compounds and various dipolarophiles, that undergo in a same reaction both ligation and cleavage. Twenty-five mesoionics and three types of dipolarophiles were synthetized and screened.For the first screening, the reactivity between mesoionics and terminal alkyne, under a copper catalysis, was studied. Nine conditions of catalysis were tested. This screening revealed the cycloaddition between aza-iminosydnones and terminal alkynes. The structure of the formed products depends on the catalytic system and the nature of the alkyne used.The second type of dipolarophiles screened was cyclic alkynes. Dithiolium-olates were found to react with non-hindered cyclic alkynes. On the other side, imino-sydnones react with all cycloalkynes to afford a click product together with a released molecule.Finally, cycloalkenes were screened. Thanks to preliminary results from a screening, we engineered new sydnones to allow the release of a complex molecule after the cycloaddition step

Très récemment, notre équipe a mis en évidence une réaction dite de ligation et coupure entre une famille de composés mésoioniques, les sydnone-imines, et les cyclooctynes. Cette réaction bioorthogonale agit selon un processus en deux étapes, une cycloaddition [3+2] suivie d’une rétro Diels-Alder, qui génère deux nouveaux composés : un produit de ligation et un produit de coupure. L’objectif de cette thèse consiste à améliorer la cinétique de réaction entre ces deux partenaires afin de pouvoir l’utiliser en tant qu’outil pour la libération contrôlée de principes actifs in vivo.Trois stratégies ont été développées lors de cette thèse afin d’optimiser ce système réactionnel : l’étude d’une relation structure/réactivité du partenaire sydnone-imine vis-à-vis de la réaction bioorthogonale; l’utilisation de micelles constituées d’amphiphiles possédant un motif sydnone-imine en tant que lien clivable entre la partie hydrophobe et la partie hydrophile de la molécule; et enfin, l’étude de l’utilisation de la reconnaissance moléculaire entre deux brins d’acides nucléiques peptidiques (ANP) complémentaires. / Recently, our laboratory has discovered a click and release reaction involving iminosydnones, a family of mesoionic compounds, and cyclooctynes. This bioorthogonal reaction occurs via a two step process: a [3+2] cycloaddition followed by a retro Diels-Alder, to give two new compounds: a click product and a release product.The main goal of this work is to improve the kinetic of the reaction between these two partners in order to use it as a powerful tool for in vivo drug delivery. Three strategies were developed during this thesis to optimize this reaction system: the study of a structure/reactivity relationship of the iminosydnone partner regarding the bioorthogonal reaction; the development of micelles built by amphiphiles containing an iminosydnone moiety as a cleavable linker, strategically located between the hydrophobic and the hydrophilic part of the compound and finally, the use of molecular recognition between two peptide nucleic acids (PNA) complementary strands.