Thesis advisor: Jia Niu / Thesis advisor: Abhishek Chatterjee / The functions of natural nucleic acids such as DNA and RNA have transcended from serving as the primary information carrier in cells and have emerged as a new class of functional material with applications encompassing medicine, diagnosis, and research tools. While the vulnerability of natural nucleic acids to nuclease degradation as well as the lack of chemical functionality have imposed a significant constraint on their ever-expanding applications, scientists have put in the effort to develop new classes of synthetic nucleic acids (XNAs) to overcome current limitations. In this dissertation, we will describe the development of a novel XNA oligonucleotide structure, the “click handle-modified FANA” (cmFANA), as the next-generation nucleic acid-based biopolymer that is capable of post-polymerization functionalization and evolution. In this dissertation, we divide our graduate research into three chapters: the development of the essential building block for cmFANA and the synthesis of cmFANA oligonucleotide as Chapter 1; the evolution and application of cmFANA as a sugar-presenting affinity reagent that targets disease-related Carbohydrate-Binding Proteins (CBPs) as Chapter 2; and other collaboration projects as Chapter 3. Together, we described a highly potential XNA structure that goes beyond established impressions of nucleic acids and carries the ability to be a versatile platform technology. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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.

Bacterial quorum sensing is regarded as a novel target for the design of antimicrobials. Based on lead structures identified from HTS, 39 analogues have been synthesized and evaluated in Vibrio haveyi. Potent inhibitors with IC50 values at single-digit micromolar concentrations for AI-2 mediated quorum sensing have been identified. On the second project, post-synthesis modifications of DNA provide easy functionalizations for expanded applications such as aptamer selection. A CBT-modified thymidine analogue (CBT-TTP) has been synthesized and used for enzymatic incorporation into DNA. Post-synthesis modifications through condensation with 1,2-aminothiol for installation of a boronic acid moiety or a fluorophore have been achieved. On the third project, H2S has been recognized as an important gasotransmitter and its concentration is relevant to a variety of diseases. A novel fluorescent probe (DNS-Az) has been developed for quantitation of H2S in aqueous solutions. This probe has been used to measure H2S concentrations in the blood.

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.

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.

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

No description available.

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.