Dissertations / Theses on the topic 'Enzyme mimics'

Les nanomatériaux à base de CuS ont suscité une attention considérable en raison de leurs propriétés intrinsèques exceptionnelles qui ont justifié leur utilisation dans diverses applications. Les nanomatériaux à base de CuS présentent une activité semblable à celle d'une enzyme, appelée «nanozymes». Ils sont considérés comme des alternatives aux enzymes peroxydases naturelles dans leur comportement catalytique vis-à-vis de l'oxydation de la réaction du substrat de 3,3 ', 5,5′-tétraméthylbenzidine (TMB) en présence de H2O2. Ce mérite en fait des candidats idéaux pour le développement de plates-formes de détection colorimétriques extrêmement sensibles et sélectives pour l'identification quantitative et qualitative de différentes espèces chimiques et biologiques.Encouragés par les propriétés optiques prometteuses des nanomatériaux à base de CuS, leurs applications à des fins biomédicales ont également été démontrées dans cette thèse. Ainsi, nous avons étudié leur utilisation en tant que candidats photothermiques et transporteurs de médicaments pour la libération à la demande d'antibiotique du lysozyme en tant que modèle de médicament antibactérien pour l'élimination bactérienne sous un laser à onde NIR à 980 nm
CuS-based nanomaterials have gained enormous attention due to their intrinsic outstanding properties that warranted their use in various applications. CuS-based nanomaterials exhibit enzyme-like activity, referred to as “nanozymes”. They are considered as alternatives to natural peroxidase enzymes in their catalytic behavior toward oxidizing the reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate in the presence of H2O2. This merit makes them ideal candidates for the development of extremely sensitive and selective colorimetric sensing platforms for the quantitative and qualitative identification of various chemical and biological species. Encouraged by the promising optical properties of CuS-based nanomaterials, their applications for biomedical purposes have been also demonstrated in this thesis. Thus, we investigated their use as photothermal candidates and drug carriers for the on-demand release of lysozyme antibiotic as an antibacterial drug model for bacterial elimination under a 980 nm NIR wave laser

Engineering artificial cells is currently an emerging area of research that involves constructing mimics of biological cells. These biomimetic cellular systems hold tremendous promise for the different biomedical applications (diagnostics, therapy, tissue engineering, gene transfection, bioactive coatings) as well as aspects of synthetic biology. A key architectural principle of the cell is a multicompartmentalized assembly, which is one of the features of biological cells that enable the performance of multiple complex biochemical reactions within confined environments. For this purpose, this study demonstrates novel artificial cells, not only presenting organelle mimics but also incorporating various stimuli for regulating enzymatic cascade reactions within the artificial cell and for controlled simultaneous and/or subsequent release of the encapsulated (therapeutic) molecules. To successfully fabricate the multifunctional polymeric multicompartmentalized systems as artificial cells aimed for, in the first step a hollow capsule as biomimetic cellular membrane was developed to simulate a key characteristic of functional artificial cells for the selective uptake and release of (bio)molecules and particles for intra- and intercellular signaling processes. Herein using LbL technique which involved alternate deposition of oppositely charged polyelectrolytes on silica template via electrostatic interaction, the pH and temperature dual-responsive and photo-crosslinked hollow capsule was fabricated and they can be used for the subsequent post-encapsulation process of protein-like macromolecules (≤ 11 nm) and their controllable release triggered by external stimuli for mimicking the controllable bio-inspired functions of cell membranes. The reversible temperature and pH dual-response ability of the hollow capsules has been analyzed. The uptake and release properties of the resulting hollow capsules with different degree of photo-crosslinking for cargos have been further investigated at various temperatures (25, 37 or 45°C) and pH (5.5 or 7.4) of the solution. Next, the design of the polymersomal subcompartmens as organelle mimics, which divide the interior of the multicompartmentalized systems into subcompartments and can stably encapsulate fragile hydrophobic and hydrophilic cargo, e.g., enzymes in order to conduct encapsulated catalysis-resembling cell organelles, was also an important subject. The fabrication of these subcompartments was starting with the synthesis of suitably end-group block copolymers to realize the enzyme-loaded, multifunctional, pH-responsive, photo-crosslinked and post-labelled polymersomes decorated with adamantane groups. The pH sensitivity and various enzymatic reactions of the established multifunctional Ada-polymersomes have been investigated. Based on the above concepts, a bottom-up approach was developed to assemble a structural and functional eukaryotic cell mimics, including “membrane-associated” multicompartmentalized system (MS1) and “free-floating” multicompartmentalized system (MS2), by loading pH-sensitive Ada-polymersomes inside the multifunctional cell membrane. The creation of these multicompartmentalized systems was based on the assembly of enzyme-loaded Ada-polymersomes as organelle mimics onto sacrificial particle templates by host-guest interaction, followed by the LbL deposition of temperature-responsive and photo-crosslinkable PMA(β-CD)/[PAH/PNMD]3 multilayers and outer protective capping PAH/PMA(PEG) bilayer as biomimetic cellular membrane. Upon photo-crosslinking the polymer biomimetic membrane and dissolution of the particle templates, multicompartmentalized systems were obtained. Spatial position of the subcompartments can be controlled using non-covalent host-guest concept, which yielded multicompartmentalized systems containing “membrane-associated” and “free-floating” subunits. Moreover, the metabolism mimicry of multicompartmentalized systems by performing multiple successive two-enzyme cascade reactions in the cells and the multiple parallel reactions by using a third enzyme for deactivating the reaction product and interfering the cascade reaction have been investigated. In conclusion, these multicompartmentalized systems, combining the advantages of both pH-responsive Ada-polymersomes as organelle mimics and multifunctional hollow capsule as biomimetic cellular membrane, present new opportunities for the development of functional cell mimics. The presented studies highlight crucial aspects for the successful applications of such cell mimics for diagnostics, tissue engineering, as nanoreactors, as carriers for multiple drug delivery with controlled release profiles, or as therapeutic artificial cells.

This thesis investigates the functional consequences of neutralising the negative charges on the bacteriophage T7 antirestriction protein ocr. The ocr molecule is a small highly negatively charged, protein homodimer that mimics a short DNA duplex upon binding to the Type I Restriction Modification (RM) system. Thus, ocr facilitates phage infection by binding to and inactivating the host RM system. The aim of this study was to analyse the effect of reducing the negative charge on the ocr molecule by mutating the acidic residues of the protein. The ocr molecule (117 residues) is replete with Asp and Glu residues; each monomer of the homodimer contains 34 acidic residues. Our strategy was to begin with a synthetic gene in which all the acidic residues of ocr had been neutralised. This so called ‘positive ocr’ (or pocr) was used as a template to gradually reintroduce codons for acidic residues by adapting the ISOR strategy proposed by D.S.Tawfik. After each round of mutagenesis an average of 4-6 acidic residues were incorporated into pocr. In this fashion a series of mutant libraries in which acidic residues were progressively introduced into pocr was generated. A high-throughput in vivo selection assay was developed and validated by assessing the antirestriction behaviour of a number of mutants of the DNA mimic proteins wtOcr and Orf18 ArdA. Further to this, selective screening of the libraries allowed us to select clones that displayed antirestriction activity. These mutants were purified and in vitro characterisation confirmed these mutants as displaying the minimum number of acidic residues deemed critical for the activity of ocr. This in vitro process effectively simulated the evolution of the charge mimicry of ocr. Moreover, we were able to tune the high-throughput assay to different selection criteria in order to elucidate various levels of functionality and unexpected changes in phenotype. This approach enables us to map the “in vitro” evolution of ocr to identify acidic residues that are required for protein expression, solubility and function proceeding to a fully functional antirestriction protein.

The production of reactive oxygen species by mitochondria is implicated in the mitochondrial dysfunction associated with a range of diseases and ageing. In contrast, reactive oxygen species produced by mitochondria are involved in redox signalling pathways necessary for modulating a number of cell processes. Mitochondrially targeted antioxidants comprised of an antioxidant moiety linked to a lipophilic triphenylphosphonium cation have recently been used to decrease reactive oxygen species-mediated oxidative damage to mitochondria and to investigate the role of mitochondrial reactive oxygen species in redox signalling. These lipophilic cations are selectively accumulated by mitochondria within cells due to the mitochondrial membrane potential. This thesis presents the synthesis and characterization of mitochondrially targeted antioxidant superoxide dismutase and thiol peroxidase mimetics. A mitochondrially targeted derivative of the Mn(II) macrocycle SOD mimetic M40403 (MitoSOD) was synthesized by Mn(II) template synthesis of a chiral tetraamine component and a triphenylphosphonium derivative of 2,6-pyridinedialdehyde. Racemic tetraamine was synthesized by mono-protection of racemic diamine followed by reductive amination of glyoxal and deprotection of di-protected tetraamine but overall this was found to be less efficient than a reported method based on trityl protection. The synthesis of the triphenylphosphonium derivative of 2,6-pyridinedialdehyde involved substitution of protected 4-bromo-2,6-pyridinedialdehyde by the thiolate of 3-mercaptoproanol followed by simultaneous deprotection and alkyl bromide formation, and triphenylphosphine substitution of the thioalkyl bromide substituent. MitoSOD was found to be more lipophilic than M40403 and was kinetically stable to dissociation to Mn(II) and macrocyclic ligand at physiological pH. Pulse radiolysis kinetic studies indicated both MitoSOD and M40403 catalyse the dismutation of superoxide. Fast conductivity and spectrophotometric measurements indicated the mechanism of catalysis involved reaction of the Mn(II) centre with superoxide to give a Mn(III)-peroxide intermediate which reacted with further superoxide to give the parent Mn(II) macrocycle. MitoSOD was significantly accumulated by mitochondria and this was dependent to some extent on the mitochondrial membrane potential. In addition, MitoSOD appeared to react with a product of mitochondrial succinate respiration. A mitochondrially targeted derivative of the organoselenium thiol peroxidase mimetic ebselen (Mitoebselen) was synthesized by O-alkylation of a phenolic ebselen derivative with a triphenylphosphonium derivative of an alkyl iodide. Reaction of excess triphenylphosphine with an ebselen derivative containing an alkyl iodide substituent resulted in substitution of iodide and, unexpectedly, reduction of the isoselenazole moiety to the diselenide redox form. Mitoebselen and its diselenide were both readily reduced to a selenol by an excess of the physiological thiol glutathione. Reaction of the selenol with excess peroxide generated the diselenide, possibly via reaction of unreacted selenol with Mitoebselen formed from a selenenic acid intermediate or with selenenic acid directly. Mitoebselen and its diselenide were both oxidized by excess peroxide to a selenoxide but these reactions were much slower than those between selenol and peroxides, and those between Mitoebselen or its diselenide with glutathione. Together these studies suggested cyclic pathways other than a selenolisoselenazole-selenol cycle could be involved in Mitoebselen or ebselen-catalysed thiol peroxidation.

Ce mémoire présente une exploration des polymères à empreintes moléculaires (MIP) comme outils d’une libération contrôlée de bioactifs olfactifs ou pour le criblage/préselection de composés à activité antivirales ou anti-tumorales sur le site actif d’une enzyme. La première partie est une étude de la complexation de la vanilline sur des billes polymériques sphériques en vue d’une libération contrôlée (pH, salinité, …). Ces études portent sur les caractéristiques de l'absorption et la libération de la molécule d'intérêt dans le milieu aqueux sur les microsphères fonctionnalisées fourni par Merck ESTAPOR® Microsphères. Nous avons ensuite synthétisé divers MIP de vanilline au format monolithique. Plusieurs stratégies d’impression ont été étudiées: non covalente, covalente et semi-covalente. La composition du MIP préparé dans chaque approche a été optimisée pour obtenir les meilleures propriétés et performances. L'affinité, la sélectivité et la capacité du MIP ont été déterminées. Les MIPs ont été évalués par extraction en phase solide (SPE) d'analogues structuraux de la vanilline dans des échantillons naturels (extrait de vanille, vin). La deuxième partie de ce mémoire concerne l’évaluation de MIPs de l’adénosine 5’-monophosphate (AMP) Le polymère a été préparé par une approche non-covalente et son efficacité de recapture a été caractérisée par analyse frontale (FA). L’analyse frontale est une technique qui permet de discriminer des interactions spécifiques des non spécifiques et de comprendre les mécanismes de liaison dans des cavités spécifiques
This thesis report presents the exploration of molecularly imprinted polymers (MIP) for the application in controlled release and targeting antivirus and anticancer drugs. The first part of this study describes the imprinting of vanillin as a monolith. Several strategies were studied: non-covalent, covalent and semi-covalent. The composition of the MIP prepared in each approach was optimized to obtain the best properties and performance. The affinity, selectivity and capacity of MIP were determined. MIPs were evaluated in solid-phase extraction (SPE) of structural analogues in natural samples (vanilla extract, wine). We also present the study of the exploration of spherical beads as potential tools for the controlled release of vanillin. These studies concern the characteristics of uptake and release of the molecule of interest in the aqueous medium on functionalised microspheres supplied by Merck ESTAPOR Microspheres®. The second part of this thesis is devoted to studies on the evaluation of MIP of adenosine 5'-monophosphate (AMP). The polymer was prepared in non-covalent approach and efficiency of binding was characterised using frontal analysis (FA). FA is a useful technique that allows discriminate specific and nonspecific interactions and to understand the binding mechanisms in specific cavities

As a first step to producing a shape selective catalysts or enzyme mimic, two preorganized host molecules were synthesized. Binding studies of the two hosts with a variety of guests in three solvents demonstrated that an important driving force in the association was the formation of C-H???X-R hydrogen bonds (X = halogen). A deuterated host was utilized to further examine the formation of the C-H???X-R hydrogen bonds. In an effort to place functionality in the hydrophobic pocket of these hosts, two methods were developed. The first utilized directed ortho metallation to place electrophiles above and/or directed into the cavity. Perlithiation of the host could lead to sixty-nine products but reaction conditions and host rigidity limited product formation. This reaction technique led to the placement of carboxylic acid groups onto the host and the isolation of twelve products. Two different positions of the carboxylic acids (endoand exo-) direct the orientation of the guest. 1D- and 2D-NMR were utilized to examine how the was orientated inside the host. The second method employed to place functionality on the host, sited a tripodal zinc binding ligand on the side of the hydrophobic pocket of the host. The synthesized host was able to bind zinc strongly and in a 1:1 manner.

I. Synthesis and inhibitory activity of sialic acid derivatives targeting viral sialate-O-acetylesterases and sialate-O-acetyltransferases Sialate-O-acetylation is a common structural modification of sialic acid, which has been associated with many human disease states (including cancer and autoimmune disease). This highly regulated and tissue-specific modification is carried out by sialate- O-acetylesterase (SOAE) and sialate-O-acetyltransferase (SOAT) enzymes. The availability of these enzymes make inhibition studies a viable endeavour, considering that SOAT/SOAE inhibitors may provide interesting tools/drug leads for the development of antiviral compounds or treatments for various disease states. A synthesis of suitable 4-O- and 9-O-functionalised sialic acid derivatives has been established which enabled the investigation of 4- and 9-sialate-O-acetylesterase enzymes. Sialic acid derivatives were screened for the inhibition of a set of viral SOAEs and while no inhibition of 4-SOAE could be detected, a 9-O-methyl derivative showed inhibition of the recombinant influenza C virus SOAE. The functionalised sialic acid motif thus serves as an initial template for the design and synthesis of future sialic acid derivatives towards SOAT/SOAT inhibition. II. New tools for the characterization and investigation of influenza virus neuraminidases: towards novel influenza virus sensors Tamiflu™ (Oseltamivir), has been employed as a mimetic of the sialic acid “oxocarbenium” intermediate formed during enzymatic hydrolysis, leading to inhibition of virus-bound neuraminidase (NA) enzyme. Phospha-isosteres of oseltamivir provide access to monoesters which retain the efficacy of the pharmacophore and allow the synthesis of novel influenza neuraminidase-specific materials. Phospha-oseltamivir-stabilised gold nanoparticles (“TamiGold”) have been synthesised and NA inhibition studies with “small TamiGold” show activity against influenza virus strains investigated compared to control gold nanoparticles. The binding interactions displayed by “large TamiGold” may provide the basis for a colorimetric method of influenza detection and as such a novel prototype influenza sensor. To the best of our knowledge this is the first example of a multivalent approach to influenza virus binding utilising sialylmimetic scaffolds immobilised on a nanoparticle platform which specifically target the NA (instead of the hemagglutinin, HA). The synthesis of phospha-oseltamivir conjugates and their ligation to biological reporter groups afford small molecule tools with high affinity and selectivity towards influenza NA. These derivatives can be applied towards novel multivalent phospha-oseltamivir materials and used as novel diagnostics, independent of existing methods.

The action of the DNA repair enzyme alkyladenine DNA glycosylase (AAG), as part of the Base Excision Repair pathway, on alkylation-induced DNA damage has been shown in mice to lead to cell death in the retina, spleen, thymus and cerebellum. The action of AAG has also been linked to damage caused by ischaemia/reperfusion (I/R) events in liver, brain and kidney. As a result, small molecule inhibitors of AAG are required for ongoing studies into the biological mechanism of this cellular damage, as well as to become potential drug leads for some types of retinal degeneration, I/R-related tissue damage, or as protective agents for patients undergoing alkylative chemotherapy and showing an increased AAG activity. They could also serve the opposite effect, acting as an alkylating agent (TMZ) sensitiser in paediatric glioblastoma (GBM). Two DNA oligomers, containing etheno-cytidine or an abasic pyrrolidine, are reported in the literature to show potent AAG inhibition in vitro. Unfortunately, their size and the charged nature of DNA chains makes them unsuitable for use as potential drug leads in vivo, as they would show low membrane permeability and face degradation by nucleases. However, the motifs present in these oligomers, together with examination of the enzyme active site, led to the conception of two types of small drug-like pyrrolidine-based inhibitor candidates termed 2-(hydroxymethyl)pyrrolidines and 4-(hydroxymethyl)pyrrolidines. The synthetic routes to these inhibitor candidates have been studied and optimised. That to the 2-(hydroxymethyl)pyrrolidines failed at the final step of attachment of DNA base-mimicking aryl groups. However, five 4-(hydroxymethyl)pyrrolidines nucleoside mimetics were successfully synthesised, bearing imidazole and pyridine groups to represent a DNA base. These were subsequently tested in vitro against AAG in a surface-bound hairpin loop colorimetric DNA oligomer assay. The most promising candidate, (+)-395, showed an IC50 of 157 μM corresponding to a ligand efficiency of 0.37 kcal·mol-1·heavy atom-1. Due to its low molecular weight (197 g·mol-1), this inhibitor constitutes a viable starting point for a future lead optimisation programme.

Glycosaminoglycans (GAGs) are linear polysaccharides whose disaccharide building blocks consist of an amino sugar and either uronic acid or galactose. They are expressed on virtually all mammalian cells, usually covalently attached to proteins, forming proteoglycans. GAGs are highly negatively charged due to an abundance of sulfate and carboxylic acid groups, and are structurally very diverse, with differences arising from chain length, the type of monomeric units, the linkages between each monomeric unit, the position of sulfate groups, and the degree of sulfation. GAGs are known to interact with a multitude of proteins, impacting diverse physiological and pathological processes. In addition, most of the biological interactions mediated by proteoglycans are believed to be primarily because of the GAG chains present on their surface. Considering the involvement of GAGs in multiple diseases, their use in the development of drugs has been of significant interest in the pharmaceutical field. Heparin, the first GAG-based drug developed in 1935, is still the most widely used anticoagulant in the world. The therapeutic potential of GAGs for the treatment of many other disease states, including cancer, inflammation, infection, wound healing, lung diseases, and Alzheimer’s disease, is being actively studied with many GAGs currently in clinical trials. However, challenges associated with the heterogeneous and complex structure of GAGs, limit their successful development. To combat such issues, our lab has focused on developing Non- Saccharide GAG Mimetics (NSGMs) as structural mimics of GAGs. NSGMs, being synthetic molecules, offer multiple advantages over GAGs. The studies mentioned here describe our efforts in the development of NSGMs as potential therapeutics for cancer, and cystic fibrosis.

Les espèces réactives de l'oxygène (ROS) sont produites en continu dans tous les organismes aérobies et sont impliquées dans la signalisation cellulaire, les défenses contre les pathogènes, mais aussi le stress oxydant. Ce dernier correspond à un déséquilibre entre la production des ROS et leur prise en charge par les défenses anti-oxydantes de la cellule. Le stress oxydant est associé à de nombreuses pathologies, notamment les maladies inflammatoires chroniques de l'intestin (MICI). Parmi les métallo-enzymes qui contrôlent la concentration en ROS, les superoxide dismutases (SOD) jouent un rôle essentiel. Ces enzymes sont responsables de la régulation du superoxyde le premier ROS produit lors de la réduction du dioxygène. Dans ces travaux, des complexes de Mn(II) mimant l'activité de la Mn-SOD (SODm) ont été conçus en utilisant une approche biomimétique. Leur intérêt pour limiter le stress oxydant et l'inflammation dans un modèle cellulaire des MICI a été examiné. En particulier, leur activité biologique a été étudiée au vu de leurs propriétés physico-chimiques et de leur biodisponibilité. Les résultats obtenus avec un complexe parent ont mené à la conception d'une deuxième génération de SODm couplés à une sonde multimodale, à des peptides pénétrants, ou à des peptides adressant aux mitochondries. L'étude du complexe parent fonctionnalisé par des peptides polyarginines a démontré l'influence de charges positives portées par le ligand sur la constante de vitesse. Dans la continuité de l'approche biomimétique développée ici, la conception de SODm de novo est présentée et constitue un premier pas vers l'imitation de l'influence de la seconde sphère de coordination
Reactive oxygen species (ROS) are produced continuously in all aerobic organisms and are involved in cell signaling, defenses against pathogens, but also oxidative stress. This latter corresponds to an imbalance between ROS production and their consumption by the antioxidant defenses of the cell. Oxidative stress is associated with numerous pathologies, such as inflammatory bowel diseases (IBD). Among the metalloenzymes controlling the concentration of ROS, superoxide dismutases (SOD) play a crucial role. These enzymes are responsibles for the regulation of superoxide, the first ROS produced by the reduction of oxygen. In this work, Mn(II) complexes mimicking the activity of the Mn-SOD (SODm) were designed using a biomimetic approach. Their relevance to limit oxidative stress and inflammation in a cellular model of IBD was investigated. In particular, their biological activity was studied in light of their physico-chemical properties and of their bioavailability. The results obtained with a parent complex led to the design of a second generation of SOD mimics conjugated with a single core multimodal probe, cell-penetrating peptides, or mitochondria-penetrating peptides. An effect of electrostatic interactions on the catalytic rate constant of the parent complex functionalized with polyarginines peptides was demonstrated, similarly to what is observed for the enzyme. In the continuity of the biomimetic approach envisioned here, the design of de novo SOD mimics is presented and constitutes a first step toward the mimicry of second sphere influence

Within a short timeframe, the CuI-catalysed 1,3-dipolar cycloaddition (1,3-DCR) of an organic azide to a terminal acetylene to form a 1,4-disubstituted-1,2,3-triazole, has emerged as a powerful synthetic transformation in combinatorial chemistry, organic synthesis and bioconjugation research. This synthetic methodology, now known as click chemistry, has had an appreciable impact in the drug discovery and biotechnology sectors and has shown broad scope and compatibility with small molecule and polymeric substrates. The application of this powerful synthetic transformation, specifically in carbohydrate based drug discovery and glycobiology is a recent and emerging trend. Chapter one of this thesis is a review of the current literature concerning the use of click chemistry in carbohydrate based drug discovery and glycobiology. Several examples have appeared within the literature highlighting the potential of click chemistry for rapidly generating structurally diverse neoglycoconjugates, ranging from small molecule drug leads to multivalent constructs, as well as a bioconjugation strategy for labelling cell-surface glycoconjugates. The review aims to be exhaustive in its coverage, with emphasis on future perspective. This thesis presents the investigation of click chemistry as a synthetic tool in carbohydrate chemistry, and its application for generating novel carbohydrate based enzyme inhibitor libraries for lead discovery and optimisation purposes. Chapter two describes the utility of click chemistry and the glycosyl triazole moiety in synthetic carbohydrate chemistry. The reaction is well suited to the synthesis of mimetics of complex oligosaccharides and glycoconjugates, owing to the mild ambient nature and remarkable regio- and stereo- selectivity. The transformation was therefore interrogated under conditions typically encountered in carbohydrate chemistry, including glycosylation reactions and protective group manipulations. The study represents the first exhaustive investigation into the stability of the triazole moiety under these conditions as well as the synthetic utility of the CuI-catalysed 1,3-DCR as a potential orthogonal transformation in carbohydrate chemistry and an adjunct to existing methods. The first aspect of the study aimed to examine the stability of the glycosyl triazole moiety under conditions employed in protective group chemistry and the compatibility of the transformation with pre-installed functional groups. Using click chemistry, the triazole moiety could be introduced onto the carbohydrate scaffold in the presence of a wide range of protective functional groups. In addition, the 1,2,3-triazole moiety was indeed shown to be a robust entity that is compatible with essential protecting group manipulations and glycosylation chemistry - an important outcome with respect to its potential utility as an additional tool for the synthesis of oligosaccharide/glycoconjugate mimetics, which are often heavily reliant on orthogonal reaction sequences. Next, the utility of the reaction with respect to solvent and catalyst conditions was examined. The reaction was performed in different organic and aqueous solvents in the presence of two different CuI-catalyst systems. It was shown that the reaction is reasonably insensitive to the nature of the solvent or aqueous co-solvent and the catalyst system. Reaction times and yields displayed little variation with respect to the solvent and catalyst system. In all cases, the 1,4-disubstituted 1,2,3-glycosyl triazole model compound was isolated in high yields and required minimal purification. The work also amply demonstrated, in a proof-of-concept manner, the powerful scope of the reaction for preparing structurally diverse neoglycoconjugates in high yield and purity. Several artificial glycomimetics were prepared using a suite of glycosyl azides through the facile 1,3-DCR to a series of acetylenes. Chapter three presents an extensive study into the preparation and biological activity of glycoconjugate benzene sulfonamides as a novel class of carbonic anhydrase (CA) inhibitor. The conjugation of carbohydrate “tails” to a benzene sulfonamide pharmacophore provides access to CA inhibitors which are neutral, water-soluble and features high chiral density and polyfunctionality that may be exploited for tissue delivery applications and to survey active site architectures in order to impart isozyme selectivity. Glycoconjugate benzene sulfonamides could also display compromised plasma membrane permeability allowing for the selective targeting of tumour associated isozymes with extracellular catalytic domains. Glycoconjugate benzene sulfonamides have received little attention as CA inhibitors, and this work represents the first comprehensive study in the area. By utilising a novel “click-tailing” strategy developed in our laboratory, a panel of structurally diverse carbohydrate “tails” were appended to the primary arylsulfonamide (ArSO2NH2) pharmacophore. A panel of azido sugars and propargyl glycosides were reacted with acetylene- and azide-functionalised benzene sulfonamide scaffolds, respectively, and subsequently evaluated for their inhibition of human carbonic anhydrase (hCA) isozymes hCA I, II, IX, XII and XIV in vitro. In this manner, a total of 50 glycoconjugate benzene sulfonamides belonging to three libraries were prepared and assessed for their inhibition of human cystolic isozymes hCA I, II and transmembrane isozymes hCA IX, XII and XIV. Selective inhibition among CA isozymes is challenging owing to conservation of active site topology within this enzyme family, yet the design of selective CA inhibitors is necessary for the development of efficacious and safe CA-based therapeutics which are void of side effects arising from systemic CA inhibition. Many of the glycoconjugate benzene sulfonamides exhibited a non-clustered in vitro inhibition profile, demonstrating that the carbohydrate tail was a powerful structural element able to distinguish isozyme selectivity. A significant outcome of this study was the discovery of several potent and selective CA inhibitors of the tumour-associated transmembrane isozyme, hCA IX, and the physiologically dominant cytosolic isozyme, hCA II. Chapter four of this thesis explores the synthetic utility of click chemistry for the solution-phase synthesis of N-alkylated azasugar libraries. To date, click chemistry has seen limited application for the synthesis and screening of natural product-based libraries. To the best of my understanding, this work represents the first example of the use of click chemistry for the generation of azasugars containing structurally diverse N-alkyl substituents as potential glycosidase and glycosyltransferase inhibitors. By employing the click chemistry methodology, various synthetically accessible aliphatic and aromatic azides were conjugated to the acetylene-functionalised 6- and 7-membered ring N-propynyl azasugar scaffolds using click chemistry, thus providing expedient access to N-methylene triazole-substituted azasugars in a single, high yielding step. The work demonstrates the applicability of the reaction for generating not only the structural diversity deemed necessary for distinguishing inhibitory potency and selectivity, but also a powerful means of tuning the physicochemical properties of the azasugar for in vivo targeting and lead optimisation purposes.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Full Text

Thrombin is the key protease that regulates hemostasis; the delicate balance between procoagulation and anticoagulation of blood. In clotting disorders, like deep vein thrombosis or pulmonary embolism, procoagulation is up-regulated, but propagation of clotting can be inhibited with drugs targeting the proteases involved, like thrombin. Such drugs however, have serious side effects (e.g., excessive bleeding) and some require monitoring during the course of treatment. The reason for these side effects is the mechanism by which the drugs’ act. The two major mechanisms are direct orthosteric and indirect allosteric inhibition, which will completely abolish the protease’s activity. Herein we sought an alternative mechanism called allosteric, partial inhibition, that has shown promise to truly regulate coagulation. Partial inhibition through allosteric mechanisms are well described for membrane-bound and oligomeric proteins. However proteases, specifically monomeric proteases (i.e., thrombin), have not shown this phenomenon until now. A small library of coumarin-based sulfated allosteric modulators (CSAMs) was synthesized to target a surface region called exosite 2; mainly composed of highly positively charged residues surrounded by hydrophobic patches. Studies revealed a non-competitive mechanism of binding with a range of IC50s between 0.2-58 µM combined with inhibitory efficacies (ΔY) between 22-73%; indicative of allosteric, partial inhibition. The KD was determined for the most potent compound (3g; IC50 = 0.2 µM, ΔY = 47%) at 0.15 µM. 3g was observed to bind at exosite 2 through unfractionated heparin competition and thrombin mutant studies. Additional computational studies were in agreement with the mutant and competition studies, showing the sulfate of 3g binding within a pocket containing R126 and R233. Fluorescence quenching and antithrombin inactivation rate studies described a conformational change to thrombin’s active site in the presence of 3g, supporting reduction of thrombin’s catalytic efficiency, without complete inhibition of thrombin’s proteolytic activities. Coupled enzyme assays and gel electrophoresis showed that in the presence of 3g, hydrolysis of fibrinogen (IC50 = 0.51 µM, ΔY = 94%) and protein C activation (IC50 = 1.7 µM, ΔY = 91%) is fully inhibited. Alternatively, FXIII activation was shown to be only partially inhibited by the presence of 3g, and FXI activation did not show any significant activation or inhibition. 3g was also shown to be active in human plasma and whole blood, but requiring much higher concentrations to induce an anticoagulant effect. Mice studies looking at the effects of 3g in vivo showed that even at high concentrations, showed no abnormal bleeding or any other irregularities. This work highlights a novel occurrence regarding thrombin’s allosteric functionality against multiple endogenous substrates. This library of compounds may be useful in the future development of allosteric inhibitors and probes that pose little to no risk of bleeding events by inducing partial inhibition.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1997.
Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Protein and peptide scaffolds are of interest for many bioengineering applications. The incorporation of catalytic activity in such structures is of major importance, although it represents a challenge. Several strategies are being used to obtain peptide catalysts and protein/peptide-based hydrogels, but the merging of the two fields is still in infancy. This work aims to study a new peptide scaffold, based on the small domain peptide, which is part of PTEN, a tumor suppressor protein. To fully characterize this peptide scaffold, the peptide was produced by two strategies; (i) small domain fused with GFP (SD-GFP) and; (ii) the small domain without fusion partner (SD). Both strategies were developed by using bacterial cells as hosts and purified by chromatographic-based techniques. The soluble SD-GFP scaffold was obtained with 93% purity, and showed catalytic activity towards pNPP, presenting a rate of 5x10-4 s-1, which is an order of magnitude lower than the kcat of PTEN towards pNPP. The production of SD-GFP also led to the formation of inclusion bodies, which were solubilized and matrix-assisted refolded on-column with 69% purity, leading to a self-supporting hydrogel at 4 ºC. The gel was washed by using three cycles of PBS and distilled water, which allowed to increase hydrogel purity. This corroborates the fact that hydrogel network is being formed by SD-GFP. The SD peptide was produced in the soluble form in bacterial cells and was purified yielding 90% of purity. The presence of paired cysteines in the SD peptide sequence lead to the formation of disulfide bridges forming tetrameric assemblies. These assemblies present an α-helical structure but are not catalytically active. In the future, the peptide should be in its reduced form (in monomer), so that cysteines can act as a nucleophile, catalyzing the dephosphorylation of pNPP.

This thesis presents research in the field of transition metal complex chemistry. The major project, reported in Part I, is focussed on the mimicking of enzymes in order to produce simple catalyst molecules that enhance the rate of ester hydrolysis. This work involved a systematic characterisation of a suite of potential enzyme mimics which were based on the simple template molecules tris(2-aminoethyl)amine (tren) and 1,4,7-triazacyclononane (tacn). The effects of substituting tren and tacn with methyl groups and β-cyclodextrin and then complexing these with Zn²⁺, Cu²⁺, Cd²⁺ and Ni²⁺ were examined. The aim was to produce stable complexes with lowered pKₐs for their associated aqua ligands (‘pKₐʜ₂ᴏ’), such that the catalytically active hydroxo ligands (responsible for the ester hydrolysis) were readily available over a broad pH range. The thirty two resulting transition metal complex systems were characterised by potentiometric titration under identical experimental conditions (aqueous NaClO₄ solution, I = 0.10 mol dm⁻³, 298.2 K). For selected systems which were characterised by large stability constants and relatively low aqua ligand pKₐʜ₂ᴏs, their ability to enhance the rate of hydrolysis of 4-nitrophenyl acetate was measured using UV-Visible spectroscopy. A modified Michaelis- Menten enzyme kinetics analysis was developed specifically for this study. Information about the possible mechanism of catalysis was also obtained by a qualitative investigation of the UV-Vis spectra of several systems over the course of the reaction. The chapters comprising Part I therefore describe the synthesis of the ligands (including two new compounds; 6ᴬ-{2-[bis(2-dimethylamino)ethyl]amino}-6ᴬ-deoxy-β-cyclodextrin (βCDMe₅tren) and 6ᴬ-(1,4,7-trimethyl-1,4,7-triazacyclononan-1-yl)-6ᴬ-deoxy-β-cyclodextrin (βCDMe₂tacn)), potentiometric titrations to establish speciation of the systems and the subsequent monitoring of the hydrolysis of 4-nitrophenyl acetate using UV-Vis spectroscopy. The secondary project, reported in Part II, is focussed on the detection of physiological Zn²⁺ using a newly characterised ligand; 2-((E)-2-phenyl)ethenyl-8-(N-4- methylbenzenesulfonyl)aminoquinol-6-yloxyacetic acid. A styryl functional group was added to the commercially available Zn²⁺-selective fluorophore “Zinquin” which detects physiological Zn²⁺ by complexing it and producing a fluorescent signal exclusively on Zn²⁺ binding. The intention of the styryl addition was to enhance the bulk of the Zinquin analogue and thereby hope to improve the selectivity of this fluorophore for free, intracellular Zn²⁺ over the Zn²⁺ found in the structures and catalytic centres of proteins and enzymes. This styryl Zinquin derivative was characterised using potentiometric titrimetry, UV-Vis spectroscopy and fluorimetry. This includes an analysis of the photoisomerism introduced by the styryl functionality. The absorbance and fluorescence characteristics of the free and Zn²⁺-complexed ligand were measured and stability constants for the Zn²⁺ complexes were determined. The results were compared to Zinquin under the same experimental conditions. Like Zinquin, the new styryl analogue was found to be Zn²⁺ selective.
Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2015

Metallohydrolases with dinuclear-zinc active sites perform many important biological hydrolytic reactions on a variety of substrates. In this regard, metallo-β-lactamases (mβ1, class B) represent a unique subset of zine hydrolases that hydrolyze the β-lactam ring in several antibiotics. The antibiotic resistance that results from this hydrolysis is becoming an increased threat for the clinical community. These metalloenzymes can hydrolyze a wide range of β-lactam substrates, such as cephamycins and imipenem that are generally resistant t the serine-containing β-lactamases. Therefore, the clinical application of the entire range of antibiotics is severely compromised in bacteria that produce mβls. Due to the lack of information on the mechanism of mβls, to-date, no clinically known inhibitors is there for mβls. In this present study, we synthesized several mono and dizinc complexes as models for the mβls and investigated the differences in their hydrolytic properties. This study supports the assumption that the second zinc in the dinuclear enzymes does not directly involve in the catalysis, but may orient the substrates for hydrolysis and the basic amino acid residues such as Asp and His may activate the zinc-bound water molecules, fulfilling the role of the second zinc in the mononuclear enzymes. The effect of various side chains on the hydrolysis of some commonly used cephalosporin antibiotics by mβl from B.cereus is described. It is shown that the cephalosporins having heterocyclic thiol side chains are more resistance to mβl-mediated hydrolysis than the antibiotics that do not have such side chains. This is partly due to the inhibition of enzyme activity by the thiol moieties eliminated during the hydrolysis. It is also observed that the heterocyclic side chains in pure form inhibit the lactamase activity of mβl as well as its synthetic mimics. The mode of binding of these heterocyclic side chains to the zinc has been analyzed from the crystal structure of the tetranuclear zinc complexes. The theoretical studies suggest that the eliminated heterocyclic thiols undergo a rapid tautomerism to produce the corresponding thiones. These thiones are found to irreversibly inhibit the LPO-catalyzed iodination reaction. The reaction of various thiones with I2 leads to the formation of thione-iodine complexes similar to that of the most commonly used antithyroid drug methimazole(MMI). These observations suggest that some of the latest generation of antibiotics may show negative effects on thyroid gland upon hydrolysis. Synthetic organophosphorus compounds have been used extensively as pesticides and petroleum additives. These compounds are very toxic to mammals and their widespread use in agriculture leads to serious environmental problems. Therfore, degradation of organophosphorus trimesters and remediation of associated contaminated sites are of worldwide concern. In this regards, the bacterial phsophotriesterase (PTE) enzyme plays an important role in degrading a wide range of organophosphorus esters and the active side of PTE has been shown to be very similar to that of mβl. This identification prompted us to check the hydrolysis of phosphotriesters by the mβl and its mimics. It has been observed that the dinuclear zine(II) complexes that do not allow a strong binding of phosphodiestes would be a better PTE mimics.

Metallohydrolases with dinuclear-zinc active sites perform many important biological hydrolytic reactions on a variety of substrates. In this regard, metallo-β-lactamases (mβ1, class B) represent a unique subset of zine hydrolases that hydrolyze the β-lactam ring in several antibiotics. The antibiotic resistance that results from this hydrolysis is becoming an increased threat for the clinical community. These metalloenzymes can hydrolyze a wide range of β-lactam substrates, such as cephamycins and imipenem that are generally resistant t the serine-containing β-lactamases. Therefore, the clinical application of the entire range of antibiotics is severely compromised in bacteria that produce mβls. Due to the lack of information on the mechanism of mβls, to-date, no clinically known inhibitors is there for mβls. In this present study, we synthesized several mono and dizinc complexes as models for the mβls and investigated the differences in their hydrolytic properties. This study supports the assumption that the second zinc in the dinuclear enzymes does not directly involve in the catalysis, but may orient the substrates for hydrolysis and the basic amino acid residues such as Asp and His may activate the zinc-bound water molecules, fulfilling the role of the second zinc in the mononuclear enzymes. The effect of various side chains on the hydrolysis of some commonly used cephalosporin antibiotics by mβl from B.cereus is described. It is shown that the cephalosporins having heterocyclic thiol side chains are more resistance to mβl-mediated hydrolysis than the antibiotics that do not have such side chains. This is partly due to the inhibition of enzyme activity by the thiol moieties eliminated during the hydrolysis. It is also observed that the heterocyclic side chains in pure form inhibit the lactamase activity of mβl as well as its synthetic mimics. The mode of binding of these heterocyclic side chains to the zinc has been analyzed from the crystal structure of the tetranuclear zinc complexes. The theoretical studies suggest that the eliminated heterocyclic thiols undergo a rapid tautomerism to produce the corresponding thiones. These thiones are found to irreversibly inhibit the LPO-catalyzed iodination reaction. The reaction of various thiones with I2 leads to the formation of thione-iodine complexes similar to that of the most commonly used antithyroid drug methimazole(MMI). These observations suggest that some of the latest generation of antibiotics may show negative effects on thyroid gland upon hydrolysis. Synthetic organophosphorus compounds have been used extensively as pesticides and petroleum additives. These compounds are very toxic to mammals and their widespread use in agriculture leads to serious environmental problems. Therfore, degradation of organophosphorus trimesters and remediation of associated contaminated sites are of worldwide concern. In this regards, the bacterial phsophotriesterase (PTE) enzyme plays an important role in degrading a wide range of organophosphorus esters and the active side of PTE has been shown to be very similar to that of mβl. This identification prompted us to check the hydrolysis of phosphotriesters by the mβl and its mimics. It has been observed that the dinuclear zine(II) complexes that do not allow a strong binding of phosphodiestes would be a better PTE mimics.

Nanomaterials possess a myriad of potential in various biomedical fields such as cancer diagnostics, biosensing, imaging, immunoassay, drug delivery and therapeutics etc. In particular, the ability of the nanozymes to modulate the catalytic activities and biological functions of natural enzymes attracts burgeoning interest for various biomimetic applications. There is a significant relevance of nanozymes which can regulate the reactive oxygen species (ROS) levels in cells, mimicking the cellular antioxidant enzymes due to their possible therapeutic potential in various oxidative stress related disorders. However, there are certain drawbacks associated with their selectivity, limited surface area due to functionalization and biocompatibility. Further, the mechanistic insights revealing the enzymatic role of nanozymes in a cellular environment are not well explored. Our study introduces novel enzyme-mimetics as artificial antioxidants for further development as a potential therapy against oxidative stress-mediated pathological conditions.

Reactive oxygen species (ROS) such as superoxide radical anion (O2•¯), hydroxylradical (OH•), hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) that are produced during the metabolism of oxygen under oxidative stress in aerobic organisms destroy several key biomolecules and lead to a number of disease states. Mammalian systems possess several effective defense mechanisms including antioxidant enzymes to detoxify these ROS. The selenocysteine-containing Glutathione peroxidase (GPx) is particularly an efficient enzyme in the detoxification of H2O2 and other hydroperoxides by using glutathione (GSH) as cofactor. The chemistry at the active siteof GPx has been extensively investigated with the help of synthetic selenium compounds. Although the anti-inflammatory compound ebselen(2-phenyl-1,2-benzoisoselenazol-3(2H)-one) is undergoing phase III clinical trial as antioxidant, the chemistry of ebselen is still not understood. The present study on a number of ebselen derivatives with various N-substitutions reveals that the substitution at the N atom is important for the antioxidant activity. This study also suggests that the nature for thiol cofactor has a dramatic effect on the GPx activity of ebselen derivatives. It has been shown that ebselen exhibits very poor catalytic activity in the presence of aromatic thiols mainly due to strong Se….O nonbonded interactions that lead to extensive thiol exchange reactions in the selenenyl sulfide intermediate. To prevent the se….O interactions, a series of tertiary amide-based diselenides have been synthesized along with their secondary amide counterparts. Detailed structure-activity correlation studies reveal that the GPx-like activity of the sec-amide-based compounds can be significantly enhanced by the substitution at the free-NH group of sec-amide functionality. The N,N-dialkylbenzylamine-based diselenides exhibit their catalytic activities via the generation of selenols which was confirmed by the reaction with anti-arthritic gold(I) compounds. Interestingly, the replacement of the hydrogen atom at the 6th position of the benzene ring of N,N-dialkylbenzylamine-based diselenides by a methoxy group prevents the thiol exchange reactions mainly be weakening the Se…N interactions and thus enhances the GPx activity. On the other hand, the catalytic activity of the tert-amine-based diselenides can also be increased by replacing the tert-amino groups with the corresponding sec-amine moieties. It has been observed that the basic amino group in the amine-based diselenides deprotonates the selenol and also the thiol cofactor, which is crucial for the higher catalytic activities of the amine-based compounds. Peroxynitrite (PN, ONOO), a strong nitrating agent, is known to inactivate a number of proteins, enzymes and other biomolecules by nitration of tyrosine residues. In this study, we have shown that the commonly used antithyroid drugs and their analogues inhibit protein tyrosine nitration. This study reveals that antithyroid agents having PN scavenging activity may be beneficial of hyperthyroidism as these compounds may protect the thyroid gland from nitrative or nitrosative stress.

Reactive oxygen species (ROS) such as superoxide radical anion (O2•¯), hydroxylradical (OH•), hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) that are produced during the metabolism of oxygen under oxidative stress in aerobic organisms destroy several key biomolecules and lead to a number of disease states. Mammalian systems possess several effective defense mechanisms including antioxidant enzymes to detoxify these ROS. The selenocysteine-containing Glutathione peroxidase (GPx) is particularly an efficient enzyme in the detoxification of H2O2 and other hydroperoxides by using glutathione (GSH) as cofactor. The chemistry at the active siteof GPx has been extensively investigated with the help of synthetic selenium compounds. Although the anti-inflammatory compound ebselen(2-phenyl-1,2-benzoisoselenazol-3(2H)-one) is undergoing phase III clinical trial as antioxidant, the chemistry of ebselen is still not understood. The present study on a number of ebselen derivatives with various N-substitutions reveals that the substitution at the N atom is important for the antioxidant activity. This study also suggests that the nature for thiol cofactor has a dramatic effect on the GPx activity of ebselen derivatives. It has been shown that ebselen exhibits very poor catalytic activity in the presence of aromatic thiols mainly due to strong Se….O nonbonded interactions that lead to extensive thiol exchange reactions in the selenenyl sulfide intermediate. To prevent the se….O interactions, a series of tertiary amide-based diselenides have been synthesized along with their secondary amide counterparts. Detailed structure-activity correlation studies reveal that the GPx-like activity of the sec-amide-based compounds can be significantly enhanced by the substitution at the free-NH group of sec-amide functionality. The N,N-dialkylbenzylamine-based diselenides exhibit their catalytic activities via the generation of selenols which was confirmed by the reaction with anti-arthritic gold(I) compounds. Interestingly, the replacement of the hydrogen atom at the 6th position of the benzene ring of N,N-dialkylbenzylamine-based diselenides by a methoxy group prevents the thiol exchange reactions mainly be weakening the Se…N interactions and thus enhances the GPx activity. On the other hand, the catalytic activity of the tert-amine-based diselenides can also be increased by replacing the tert-amino groups with the corresponding sec-amine moieties. It has been observed that the basic amino group in the amine-based diselenides deprotonates the selenol and also the thiol cofactor, which is crucial for the higher catalytic activities of the amine-based compounds. Peroxynitrite (PN, ONOO), a strong nitrating agent, is known to inactivate a number of proteins, enzymes and other biomolecules by nitration of tyrosine residues. In this study, we have shown that the commonly used antithyroid drugs and their analogues inhibit protein tyrosine nitration. This study reveals that antithyroid agents having PN scavenging activity may be beneficial of hyperthyroidism as these compounds may protect the thyroid gland from nitrative or nitrosative stress.

Thyroid hormones (THs; T4 and T3), secreted from thyroid gland, play an important role in human growth and development. T3 (3,5,3′-triiodothyronine) is the active hormone and the conversion of T4 (3,3′,5,5′-tetraiodothyronine) to T3 in cells is mediated by iodothyronine deiodinases enzymes (DIOs). DIOs are selenocysteine containing enzymes and are classified into three types (DIO1, DIO2 and DIO3). DIO1 catalyzes the outer-ring deiodination (ORD; T3 formation) and inner-ring deiodination (IRD; rT3 formation) reactions, involving in the activation (T4 to T3 conversion) and inactivation (T4 to rT3 conversion), respectively. DIO2 and DIO3 catalyse the ORD and IRD reactions, respectively. This homeostasis is regulated tightly and any deviation would lead to diseases like hyperthyroidism or hypothyroidism. Recently it is of interest to many research groups to develop iodothyronine deiodinase mimics and we have developed naphthalene-based peri-substituted thioselenol pair at 1,8-positions (1.25), which remove iodine selectively from inner-ring of T4. When selenium atom is substituted in place of sulfur (selenol-selenol pair; 1.26), the deiodination activity was ca. 90 times faster than with 1.25. This thesis deals with various aspects of the effect of substituents on the naphthalene-1,8-diselenol and solvent effect on the thyroid hormone deiodination by naphthalene-based iodothyronine deiodinase mimics. Figure 1. (A) Deiodination reactions by DIOs. (B) Chemical structure of 1.25 and 1.26. The thesis consists of five chapters. The first chapter provides a general overview about sialoproteins, thyroid hormone biosynthesis, thyroid hormone metabolism, halogen bonding, iodothyronine deiodinase mimics and proposed mechanisms for the deidoination of thyroid hormones. This chapter also introduces peri-naphthalene-1,8-diselenol (1.26), which is the key compound in this thesis and discusses about proposed mechanism for the deiodination of thyroxine involving co-operative halogen bonding and chalcogen bonding mechanism. Figure 2. (A) TH action. (B) Proposed mechanism for the deiodination of T4 by 1.26 involving cooperative halogen bonding and chalcogen bonding. Chapter 2 discusses about the synthesis, characterization and deiodination activity of a series of naphthalene-based peri-substituted-1,8-diselenols (Figure 3). These diselenols regioselectivity remove iodine from inner ring of thyroxine and other thyroid hormones, (T3 and 3, 5-T2). Substitution with different groups on the naphthalene ring did not change the regioselectivity of deiodination, indicating that the deiodination activity does not depend on the nature of substituents. Secondary or tertiary amine side chain group attached at the 2nd position of the naphthalene ring showed better activity. It is due to the secondary interaction, which facilitates the iodine removal. It was further confirmed with the substitutions at the 4th position of the ring to discriminate the possibility of electronic effect. The higher deiodination rate owing to the t-butyl group at second position of the ring also suggests that the steric effect may also play a role in the deiodination reaction (Figure 4). It is proposed that peri substituted naphthalene-1,8-diselenols remove iodine from thyroid hormones through halogen bonding-chalcogen bonding mechanism (Figure 2). The investigation of Se···Se bond distance from the crystal structures and through DFT calculation and NMR experiment showed that the stronger chalcogen bond could be the reason for the increase in the reactivity observed with substituted peri-naphthalene-1,8-diselenols. Figure 3. peri-substituted naphthalene-1,8-diselenols used for the study. Figure 4. Relative deiodinase activity of substituted-peri-naphthalene-1,8-diselenols with T4. In Chapter 3, we have discussed about the effect of chalcogen atom substitution in a series of deiodinase mimics on the deiodination of thyroid hormones. Moving from thiol-selenol pair (1.25) to selenol-selenol pair (1.26) in naphthalene based peri-substituted mimics, an increase in the activity was observed. In this chapter, we have shown that substituting with tellurium, as tellurium-thiol pair (3.3) and ditellurol (3.4) increases the reactivity of deiodination to several times and also regioselectivity of deiodination is changed from IRD in the case of 1.26 to both IRD and ORD for 3.3 and 3.4. The presence of two tellurol moieties (3.4) or a thiol-tellurol pair (3.3) can mediate sequential deiodination of T4, to produce all the possible thyroid hormone derivatives under physiologically relevant conditions (Figure 5). This study provided the first experimental evidence that the regioselectivity of the thyroid hormone deiodination is controlled by the nucleophilicity and the strength of halogen bond between the iodine and chalcogen atoms. Figure 5. (A) HPLC chromatograms of deiodination reaction of T4 with 3.3 and 3.4. (B) Chemical structure of 3.3 and 3.4. (C) Sequential deiodination reaction of T4 by 3.3 and 3.4. Chapter 4 describes the effect of alkyl conjugation at 4′-OH position of THs on the deiodination by iodothyronine mimics. In addition to the deiodination, iodothyronines undergo conjugation with sulfate and glucuronic acid group at 4′-hydroxyl position. Conjugation alters the physico-chemical properties of iodothyronines. For example, it is known that sulfate conjugation increases the rate of deiodination to a large extend. We have conjugated alkyl group at 4′-hydroxyl position of iodothyronines and investigated the deiodination reactions with reported peri-substituted naphthalene-1,8-diselenols. We observed that similar to sulfated thyroid hormones O-methylthyroxine also undergoes both phenolic and tyrosyl ring deiodination reactions and overall the rate of deiodination is increased at least by 5 times as compared with T4 under identical conditions. The phenolic iodine removal is favored by conjugation as compared to the tyrosyl ring iodine, which is similar to the observation made for T4S. Interestingly, when the acetamide group is conjugated at 4′-OH position, the regioselectivity of deiodination is changed exclusively to 5′-iodine. DFT calculations show that the positive potential on the iodine increase upon conjugation, which leads to stronger halogen bonding interaction with selenol, might be the reason for the change in the regioselectivity of deiodination. Figure 6. (A) HPLC chromatogram of deiodination reaction of T4(Me) with 1.26. (B) Initial rate comparison of T4 and T4(Me).(C) HPLC chromatogram of deiodination reaction of T4(AA) with 1.26 showing the formation of T3(AA) (ORD product). (D) Electron potential map of T4, T4(Me) and T4(AA) showing the increase in electro positive potential on 5′-iodine upon conjugation. Chapter 5 deals with the solvent effect on the deiodination reactions of THs by iodothyronine deiodinase mimics. As discussed in the earlier chapters, the deiodination reaction of thyroxine by naphthalene based-1,8-diselenols under physiological conditions produce, rT3 (IRD) as the only observable products. Surprisingly, when the deiodination reaction was performed in DMF or DMSO in the presence of 1.26, the regioselectivity of reaction was changed and the formation of both T3 (ORD) and rT3 was observed. In DMF or in DMSO, the deiodination reactivity of 1.26 was found to be 1000 fold higher than the reaction performed in phosphate buffer at pH 7.4. Figure 7. (A) HPLC chromatogram for the deiodination reaction of T4 in DMF by 1.26 showing both IRD and ORD. (B) A comparison of initial rate for the deiodination reactions of T4, T3 and 3,5-T2 in DMF and in DMSO by 1.26. (C) HPLC chromatograms for the deiodination reaction of T4 in DMF by 1.26 in the presence of TEMPO, showing the inhibition of deiodination (i) 0 mM TEMPO (ii) 10 mM of TEMPO (iii) 30 mM TEMPO. (D) HPLC chromatograms for the deiodination reaction of T4 in DMSO by 1.26 in the presence of TEMPO showing the inhibition of deiodination (i) 0 mM TEMPO (ii) 10 mM of TEMPO (iii) 30 mM TEMPO. 3,5-DIT was not denominated under physiological conditions, however, in DMF and in DMSO, 3,5-DIT was deiodinated by 2.4 to produce 3-MIT. We also observed that the control reactions in DMF or DMSO also showed a little deiodination activity. The very high reactivity observed in the presence of DMF or DMSO implied that the mechanism of denomination in these solvents may be different. It has been reported that DMSO or DMF radicals can be formed with small amounts of a base. Reaction mixture consisting of NaBH4 (for generating selenol from diselenide) and NaOH (T4 solution) may facilitate the radical formation. We also performed the reaction in the presence of TEMPO (free radical scavenger) and observed the inhibition of deiodination reaction. However, it is not clear whether the radical pathway could be one of the possible mechanisms of deiodination in these solvents by compounds 1.26 and 2.4. Further studies are required to propose a radical mechanism in different solvents such as DMF and DMSO.

Thyroid hormones (THs; T4 and T3), secreted from thyroid gland, play an important role in human growth and development. T3 (3,5,3′-triiodothyronine) is the active hormone and the conversion of T4 (3,3′,5,5′-tetraiodothyronine) to T3 in cells is mediated by iodothyronine deiodinases enzymes (DIOs). DIOs are selenocysteine containing enzymes and are classified into three types (DIO1, DIO2 and DIO3). DIO1 catalyzes the outer-ring deiodination (ORD; T3 formation) and inner-ring deiodination (IRD; rT3 formation) reactions, involving in the activation (T4 to T3 conversion) and inactivation (T4 to rT3 conversion), respectively. DIO2 and DIO3 catalyse the ORD and IRD reactions, respectively. This homeostasis is regulated tightly and any deviation would lead to diseases like hyperthyroidism or hypothyroidism. Recently it is of interest to many research groups to develop iodothyronine deiodinase mimics and we have developed naphthalene-based peri-substituted thioselenol pair at 1,8-positions (1.25), which remove iodine selectively from inner-ring of T4. When selenium atom is substituted in place of sulfur (selenol-selenol pair; 1.26), the deiodination activity was ca. 90 times faster than with 1.25. This thesis deals with various aspects of the effect of substituents on the naphthalene-1,8-diselenol and solvent effect on the thyroid hormone deiodination by naphthalene-based iodothyronine deiodinase mimics. Figure 1. (A) Deiodination reactions by DIOs. (B) Chemical structure of 1.25 and 1.26. The thesis consists of five chapters. The first chapter provides a general overview about sialoproteins, thyroid hormone biosynthesis, thyroid hormone metabolism, halogen bonding, iodothyronine deiodinase mimics and proposed mechanisms for the deidoination of thyroid hormones. This chapter also introduces peri-naphthalene-1,8-diselenol (1.26), which is the key compound in this thesis and discusses about proposed mechanism for the deiodination of thyroxine involving co-operative halogen bonding and chalcogen bonding mechanism. Figure 2. (A) TH action. (B) Proposed mechanism for the deiodination of T4 by 1.26 involving cooperative halogen bonding and chalcogen bonding. Chapter 2 discusses about the synthesis, characterization and deiodination activity of a series of naphthalene-based peri-substituted-1,8-diselenols (Figure 3). These diselenols regioselectivity remove iodine from inner ring of thyroxine and other thyroid hormones, (T3 and 3, 5-T2). Substitution with different groups on the naphthalene ring did not change the regioselectivity of deiodination, indicating that the deiodination activity does not depend on the nature of substituents. Secondary or tertiary amine side chain group attached at the 2nd position of the naphthalene ring showed better activity. It is due to the secondary interaction, which facilitates the iodine removal. It was further confirmed with the substitutions at the 4th position of the ring to discriminate the possibility of electronic effect. The higher deiodination rate owing to the t-butyl group at second position of the ring also suggests that the steric effect may also play a role in the deiodination reaction (Figure 4). It is proposed that peri substituted naphthalene-1,8-diselenols remove iodine from thyroid hormones through halogen bonding-chalcogen bonding mechanism (Figure 2). The investigation of Se···Se bond distance from the crystal structures and through DFT calculation and NMR experiment showed that the stronger chalcogen bond could be the reason for the increase in the reactivity observed with substituted peri-naphthalene-1,8-diselenols. Figure 3. peri-substituted naphthalene-1,8-diselenols used for the study. Figure 4. Relative deiodinase activity of substituted-peri-naphthalene-1,8-diselenols with T4. In Chapter 3, we have discussed about the effect of chalcogen atom substitution in a series of deiodinase mimics on the deiodination of thyroid hormones. Moving from thiol-selenol pair (1.25) to selenol-selenol pair (1.26) in naphthalene based peri-substituted mimics, an increase in the activity was observed. In this chapter, we have shown that substituting with tellurium, as tellurium-thiol pair (3.3) and ditellurol (3.4) increases the reactivity of deiodination to several times and also regioselectivity of deiodination is changed from IRD in the case of 1.26 to both IRD and ORD for 3.3 and 3.4. The presence of two tellurol moieties (3.4) or a thiol-tellurol pair (3.3) can mediate sequential deiodination of T4, to produce all the possible thyroid hormone derivatives under physiologically relevant conditions (Figure 5). This study provided the first experimental evidence that the regioselectivity of the thyroid hormone deiodination is controlled by the nucleophilicity and the strength of halogen bond between the iodine and chalcogen atoms. Figure 5. (A) HPLC chromatograms of deiodination reaction of T4 with 3.3 and 3.4. (B) Chemical structure of 3.3 and 3.4. (C) Sequential deiodination reaction of T4 by 3.3 and 3.4. Chapter 4 describes the effect of alkyl conjugation at 4′-OH position of THs on the deiodination by iodothyronine mimics. In addition to the deiodination, iodothyronines undergo conjugation with sulfate and glucuronic acid group at 4′-hydroxyl position. Conjugation alters the physico-chemical properties of iodothyronines. For example, it is known that sulfate conjugation increases the rate of deiodination to a large extend. We have conjugated alkyl group at 4′-hydroxyl position of iodothyronines and investigated the deiodination reactions with reported peri-substituted naphthalene-1,8-diselenols. We observed that similar to sulfated thyroid hormones O-methylthyroxine also undergoes both phenolic and tyrosyl ring deiodination reactions and overall the rate of deiodination is increased at least by 5 times as compared with T4 under identical conditions. The phenolic iodine removal is favored by conjugation as compared to the tyrosyl ring iodine, which is similar to the observation made for T4S. Interestingly, when the acetamide group is conjugated at 4′-OH position, the regioselectivity of deiodination is changed exclusively to 5′-iodine. DFT calculations show that the positive potential on the iodine increase upon conjugation, which leads to stronger halogen bonding interaction with selenol, might be the reason for the change in the regioselectivity of deiodination. Figure 6. (A) HPLC chromatogram of deiodination reaction of T4(Me) with 1.26. (B) Initial rate comparison of T4 and T4(Me).(C) HPLC chromatogram of deiodination reaction of T4(AA) with 1.26 showing the formation of T3(AA) (ORD product). (D) Electron potential map of T4, T4(Me) and T4(AA) showing the increase in electro positive potential on 5′-iodine upon conjugation. Chapter 5 deals with the solvent effect on the deiodination reactions of THs by iodothyronine deiodinase mimics. As discussed in the earlier chapters, the deiodination reaction of thyroxine by naphthalene based-1,8-diselenols under physiological conditions produce, rT3 (IRD) as the only observable products. Surprisingly, when the deiodination reaction was performed in DMF or DMSO in the presence of 1.26, the regioselectivity of reaction was changed and the formation of both T3 (ORD) and rT3 was observed. In DMF or in DMSO, the deiodination reactivity of 1.26 was found to be 1000 fold higher than the reaction performed in phosphate buffer at pH 7.4. Figure 7. (A) HPLC chromatogram for the deiodination reaction of T4 in DMF by 1.26 showing both IRD and ORD. (B) A comparison of initial rate for the deiodination reactions of T4, T3 and 3,5-T2 in DMF and in DMSO by 1.26. (C) HPLC chromatograms for the deiodination reaction of T4 in DMF by 1.26 in the presence of TEMPO, showing the inhibition of deiodination (i) 0 mM TEMPO (ii) 10 mM of TEMPO (iii) 30 mM TEMPO. (D) HPLC chromatograms for the deiodination reaction of T4 in DMSO by 1.26 in the presence of TEMPO showing the inhibition of deiodination (i) 0 mM TEMPO (ii) 10 mM of TEMPO (iii) 30 mM TEMPO. 3,5-DIT was not denominated under physiological conditions, however, in DMF and in DMSO, 3,5-DIT was deiodinated by 2.4 to produce 3-MIT. We also observed that the control reactions in DMF or DMSO also showed a little deiodination activity. The very high reactivity observed in the presence of DMF or DMSO implied that the mechanism of denomination in these solvents may be different. It has been reported that DMSO or DMF radicals can be formed with small amounts of a base. Reaction mixture consisting of NaBH4 (for generating selenol from diselenide) and NaOH (T4 solution) may facilitate the radical formation. We also performed the reaction in the presence of TEMPO (free radical scavenger) and observed the inhibition of deiodination reaction. However, it is not clear whether the radical pathway could be one of the possible mechanisms of deiodination in these solvents by compounds 1.26 and 2.4. Further studies are required to propose a radical mechanism in different solvents such as DMF and DMSO.

博士
國立臺灣大學
化學研究所
99
Copper zinc superoxide dismutase (CuZnSOD) is an antioxidant metalloenzyme to catalyze the dismutation of superoxide anion radical (O2－․) into hydrogen peroxide and oxygen to reduce the steady-state concentration of O2－․. A series of imidazolate-bridged homo- and hetero- dinuclear Cu(II)-Cu(II) and Cu(II)-Zn(II) model compounds were synthesized and encapsulated into various mesoporous silica/aluminosilicates (MPS/Al-MPS) to mimic the structure and functionalities of CuZnSOD enzyme. Inspired by the local environments of guanidinium group in the native CuZnSOD enzyme in facilitating the superoxide dismutation, we carefully modified the silanol surface of MPS with N-trimethoxysilylpropyl-N,N,N-trimethyl-ammonium chloride (TMAC) to prepare MPS-N+ to yield the needed local environment for the active site of model compounds and to facilitate the disproportionation of O2－․. We employed the following spectroscopic techniques: UV-Vis, EPR and EXAFS to characterize the active site-Cu(II) ion of the immobilized mimic complexes, and to obtain the structural information of Cu(II) and Zn(II) centers confined in MPSs. The O2－․ disproportionation activity of mimic complexes was measured by nitro blue tetrazolium (NBT) assay. The encapsulated complexes yielded good stability against hydrothermal treatment and enhanced the O2－․ disproportionation activity in various MPS solids. The spectroscopic studies further allow us to establish the structure-reactivity relationship between the nanostructure of MPS and the efficacy of the superoxide dismutation in MPS, and to elucidate the mechanism of the SOD-like activity of immobilized model compounds in MPS. Furthermore, we utilized the oxidative damage model of HeLa cells to study the protective effect of mimic materials in the presence of paraquat (PQ) oxidant.

This dissertation primarily focuses on biomimicry, the term coming from the Greek words bios meaning life in Greek and mimesis meaning to imitate, a field that seeks to mimic natural mechanisms, structures, and functions to exploit them into several scientific applications. We have been exploring nature-inspired catecholamine-based biopolymers to straightforwardly develop molecularly imprinted polymers (MIPs), mimetic receptors, for bioanalytical and diagnostics applications. From a more comprehensive standpoint, this dissertation addressed the broad need for simple, cost- effective, and accurate catecholamine-based assays, using animal-free reagents. The general structure of this dissertation is explained herein along with an overview of the research goals. Chapter 1 is devoted to the description of MIPs design and to how they are synthesized. The Chapter gives a brief outline of molecular imprinting technology (MIT) along with recent MIPs synthesis progresses, focusing on the selection of the template molecule, a critical factor to assemble efficient receptor mimics. Chapter 2 deals with catechol-derived biopolymers, chiefly focusing on polydopamine (PDA) and polynorepinephrine (PNE), which are becoming increasingly appreciated as soft, sustainable, versatile, and biocompatible materials able to address challenging tasks. Chapter 3 reports on the use of MIPs for the detection of the small peptide, namely gonadorelin, in biological specimens, i.e., human urines. This is the main purpose of the PhD research study which is part of a larger project entitled “New analytical approaches aimed at tackling doping in sports: development of optical biosensors for the analysis of peptide hormones through molecularly imprinted polymers” funded by the Italian Ministry of Health. First, an optical, label-free, and real time sensing strategy was developed for the detection of gonadorelin using Surface Plasmon Resonance (SPR) transduction. In addition, a portable test was settled, motivated by the lack of decentralized rapid gonadorelin assays for quick decision-making and extensive athlete monitoring before and during competitions. More in detail, a biomimetic enzyme-linked immunosorbent assay (BELISA) was developed onto disposable microplates aiming to cut testing costs and time that are usually required for gonadorelin detection (e.g., mass spectrometry). The detection of gonadorelin through a MIP-based bioanalytical approach has been a very ambitious goal, as no point-of-care devices and only a few monoclonal antibodies are commercialized targeting this analyte. The MIP based approach is able to cheaply measure the drug levels directly from human urine specimens by using a small sample volume (in order of microliters). More specifically, a polynorepinephrine (PNE) based MIP was first designed for targeting gonadorelin, and then it was employed as a receptor element in an SPR-based optical sensing platform. A competitive bioanalytical label-free assay has been built over the MIP for gonadorelin quantification in urine samples. After this, the second task of the project involved the scaling down of the competitive assay, i.e., BELISA, into a portable and simple platform to analyze human urine. The strategy developed was validated by mass spectrometric analysis. Urine samples and LC-MS/MS equipment were available at Pisa University Hospital’s Clinical Pathology Lab, partner of the project. Very soon, the journey across MIPs will continue in the direction of nano- MIPs, which we foresee could be further used as advantageous alternatives to antibodies, both in vitro diagnostics and in vivo therapeutic applications. Chapter 4 is, thus, about the preliminary development of catecholamine- based nanoparticles which will be implemented in the MIPs nanotechnological applications. Chapter 5 describes how catecholamines may be exploited in colorimetric microplate-based bioassays to screen different analytes, in addition to their use as functional monomers in mimetics (MIPs) synthesis. In this case, the redox PDA properties, and the capability to build up coating, non-imprinted material, were exploited for analytical purposes. Two colorimetric tests for molecular diagnostics were developed and applied respectively to analyze serum albumin, a biomarker for kidney function, in human fluids (urine) and to preliminarily screen hypochlorous acid, a key determinant for neurodegenerative disorders. Chapter 6 summarizes the pithiest points of the PhD research studies, discussed in the preceding chapters, and introduces considerations for future work on the topic.