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Polysaccharides are complex biopolymers that play essential roles in the biological systems including energy storage, structural support, lubrication, and signal transduction. Despite their importance, the synthesis of polysaccharides has proven to be very challenging due to the presence of multiple hydroxyl groups and difficulty in controlling the stereochemical outcome of glycosylation reactions. As a conventional chemical method to synthesize polysaccharides, ring-opening polymerization of anhydrosugars enables the synthesis of stereoregular α-(1→6)-linked polysaccharides, but is less effective in preparing polysaccharides with other linkages. Enzymatic polymerizations have also been explored, however, these methods typically require expensive monomers, and suffer from a narrow scope of enzymes and small scale of reactions. The limited approaches to polysaccharides have inspired chemists to synthesize polysaccharide mimetics with achiral linkages that can be constructed efficiently. Poly-amido-saccharides (PASs) are a new type of saccharide polymers in which the O-glycosidic linkages in natural polysaccharides are replaced with (1→2)-amide linkages. With saccharide moieties inter-connected by amide bonds, PASs exhibit characteristics of both polysaccharides and polypeptides, such as possessing pyranose-backbones and lots of hydroxyl groups, and adopting a left-handed helical conformation. However, due to lack of sufficient terminal saccharide residues, previously synthesized glucose and galactose PASs display weak interactions with carbohydrate binding lectins and receptors, limiting their applications in biomedical and pharmaceutical fields. Herein, the design and synthesis of PASs with novel structures and properties is described. By pre-installing the stereochemistry in the monomer, Altrose PASs (Alt-PASs) with β-(1→2)-amide linkages are prepared via ring-opening polymerization of an altrose-based β-lactam followed by debenzylation. Circular dichroism shows that Alt-PASs adopt a right-handed helical conformation in aqueous solution. Via the polymerization of disaccharide-based β-lactams, two PASs with either 4-O-α-D-glucose branches (Mal-PASs) or 6-O-β-D-glucose branches (Gen-PASs) are obtained. Biological studies reveal that Mal-PASs are multivalent ligands to lectin Concanavalin A, while Gen-PASs activate RAW 264.7 macrophage cells by enhancing the secretion of TNF-α and NO. The anionic ring-opening polymerization of sugar-based β-lactams is a useful method to synthesize well-defined polysaccharide mimetics, and this method expands the current repertory of approaches available to complex saccharide polymers with biological activities. / 2021-02-15T00:00:00Z

In this thesis entitled “Saccharide Recognition - Boronic acids as Receptors in Polymeric Networks” different aspects of boronic acid synthesis, their analysis and incorporation or attachment to different polymeric networks and characterisation thereof were investigated. The following key aspects were considered: • Provision of a variety of different characterised arylboronic acids and benzoboroxoles • Attachment of certain derivatives to nanoparticles and the characterisation of saccharide binding by means of isothermal titration calorimetry and displacement assay (ARS) to enhance the association constant to saccharides at pH 7.4 • Enhancement of selectivity in polymeric systems by means of molecular imprinting using fructose as template and a polymerisable benzoboroxole as functional monomer for the recognition at pH 7.4 (Joined by a diploma thesis of F. Grüneberger) • Development of biomimetic saccharide structures and the development of saccharide (especially glucose and fructose) binding MIPs by using these structures as template molecules. In the first part of the thesis different arylboronic acid derivatives were synthesised and their binding to glucose or fructose was investigated by means of isothermal titration calorimetry (ITC). It could be derived, which is in parallel to the literature, that derivatives bearing a methylhydroxyl-group in ortho-position to the boron (benzoboroxole) exhibit in most cases a two-fold higher association constant compared to the corresponding phenylboronic acid derivative. To gain a deeper understanding NMR spectroscopy and mass spectrometry with the benzoboroxole and glucose or fructose was performed. It could be shown that the exchange rate in terms of NMR time scale is quite slow since in titration experiments new peaks appeared. Via mass spectrometry of a mixture between benzoboroxole and glucose or fructose, different binding stoichiometries could be detected showing that the binding of saccharides is comparable with their binding to phenylboronic acid. In addition, the use of Alizarin Red S as an electrochemical reporter was described for the first time to monitor the saccharide binding to arylboronic acids not only with spectroscopy. Here, the redox behaviour and the displacement were recorded by cyclic voltammograms. In the second part different applications of boronic acids in polymeric networks were investigated. The attachment of benzoboroxoles to nanoparticles was investigated and monitored by means of isothermal titration calorimetry and a colourimetric assay with Alizarin Red S as the report dye. The investigations by isothermal titration calorimetry compared the fructose binding of arylboronic acids and benzoboroxoles coupled to these nanoparticles and “free” in solution. It could be shown that the attached derivatives showed a higher binding constant due to an increasing entropy term. This states for possible multivalent binding combined with a higher water release. Since ITC could not characterise the binding of glucose to these nanoparticles due to experimental restrictions the glucose binding at pH 7.4 was shown with ARS. Here, the displacement of ARS by fructose and also glucose could be followed and consequently these nanoparticles can be used for saccharide determination. Within this investigation also the temperature stability of these nanoparticles was examined and after normal sterilisation procedures (121°C, 20 min.) the binding behaviour was still unchanged. To target the selectivity of the used polymeric networks, molecular imprinting was used as a technique for creating artificial binding pockets on a molecular scale. As functional monomer 3-methacrylamidobenzoboroxole was introduced for the recognition of fructose. In comparison to polymers prepared with vinylphenylboronic acid the benzoboroxole containing polymer had a stronger binding at pH 7.4 which was shown for the first time. In addition, another imprinted polymer was synthesised especially for the recognition of glucose and fructose employing biomimetic saccharide analogues as template molecule. The advantage to use the saccharide analogues is the defined template-functional monomer complex during the polymerisation which is not the case, for example, for glucose-boronic acid interaction. The biomimetic character was proven through structural superimposition of crystal structures of the analogues with already described crystal structures of boronic acid esters of glucose and fructose. A molecularly imprinted polymer was synthesised with vinylphenylboronic acid as the functional monomer to show that both glucose and fructose are able to bind to the polymer which was predicted by the structural similarity of the analogues. The major scientific contributions of this thesis are • the determination of binding constants for some, not yet reported saccharide – boronic acid / benzoboroxole pairs, • the use of ARS as electrochemical reporter for saccharide detection, • the thermodynamic characterisation of a saccharide binding nanoparticle system containing benzoboroxole and functioning at pH 7.4, • the use of a polymerisable benzoboroxole as functional monomer for saccharide recognition in neutral, aqueous environments • and the synthesis and utilisation of biomimetic saccharide analogues as template molecules especially for the development of a glucose binding MIP. / Die vorliegende Arbeit beschäftigte sich mit der Synthese verschiedener Boronsäurederivate und deren Charakterisierung und Anbindung oder Einbau in polymeren Netzwerken. Die synthetisierten Polymere werden charakterisiert bezüglich ihrer Morphologie und besonders hinsichtlich ihrer Saccharidbindung. Im Wesentlichen wurden die folgenden Aspekte bearbeitet: • Bereitstellung einer Reihe von verschiedenen Arylboronsäuren und Benzoboroxolderivaten • Modifizierung von Nanopartikeln mit verschiedenen Derivaten aus der Bibliothek mit dem Ziel die Bindingkonstanten zu Monosacchariden bei pH 7.4. zu erhöhen. Dabei werden isothermale Titrationskalorimetrie und ein spektroskopischer Verdrängungsassay (Alizarin Rot S) zum Bindungsnachweis verwendet. • Erhöhung der Selektivität von Polymeren mittels molekularen Prägens von Fruktose als Matrizenmolekül. Durch den Einsatz eines polymerisierbaren Benzoboroxolderivatives konnte die Anbindung bei pH 7.4 stattfinden (Dieser Teil der Arbeit wurde durch eine Diplomarbeit von F. Grüneberger unterstützt). • Entwicklung von biomimetischen saccharidanalogen Strukturen, die als Matrizenmoleküle während des molekularen Prägens eingesetzt werden, um ein Polymer für die Anbindung von Monosacchariden wie etwa Fruktose oder Glukose zu generieren. Im ersten Teil der Arbeit wurde die Synthese und Charakterisierung von verschiedenen Boronsäurederivaten beschrieben. Die Charakterisierung der Bindungskonstanten wurde mittels isothermaler Titrationskalorimetrie durchgeführt. Dabei konnte im Einklang mit der Literatur gezeigt werden, dass Derivative, die zusätzlich mit einer Methylhydroxygruppe in ortho-Position zum Bor (Benzoboroxol) derivatisiert sind, eine meist zweifach höhere Bindungskonstante als gleich substituierte Arylboronsäurederivate aufweisen. Durch 1H-NMR-Spektroskopie und Massenspektrometrie mit Benzoboroxol und Glukose oder Fruktose konnten die Bindung weiter charakterisiert werden. Dabei zeigten die Ergebnisse der 1H-NMR-Spektroskopie, dass die Bindung bezogen auf NMR-Zeitskalen recht langsam stattfindet, da es zu einer Verbreiterung der Peaks kommt. Die Ergebnisse der Massenspektrometrie zeigen, dass hinsichtlich der Stöchiometrie der Binding verschiedene Komplexe möglich sind, was in ähnlicher Weise für Phenylboronsäure schon gezeigt wurde. Zusätzlich wurde in diesem Teil der Arbeit wurde die Elektrochemie von Alizarin Rot S untersucht, um den Farbstoff, der spektroskopisch schon für den Bindungsnachweis zwischen einem Boronsäurederivat und verschiedenen Zuckern beschrieben ist, auch elektrochemisch zu nutzen. Im zweiten Teil der Arbeit wurden verschiedene Anwendungen von den charakterisierten Boronsäuren in polymeren Netzwerken beschrieben. Dabei wurde zunächst ein Polystyrollatex mit Benzoboroxol modifiziert und die Bindung von Glukose oder Fruktose mittels isothermaler Titrationskalorimetrie und einem spektroskopischen Farbassay untersucht. Die Untersuchungen mittels isothermaler Titrationskalorimetrie (ITC) wurden vergleichend zu frei in Lösung befindlichem Benzoboroxol durchgeführt. Es wurde eine höhere Bindungskonstante für die modifizierten Nanopartikel ermittelt, was auf die Erhöhung der Entropie im System zurückzuführen ist. Dies kann durch eine mutivalente Bindung und einer einhergehenden erhöhten Wasserfreisetzung erklärt werden. Da es nicht möglich war, die Glukosebindung mittels ITC zu verfolgen, wurde zusätzlich ein spektroskopischer Assay bei pH 7.4. durchgeführt. Bei diesem wird die Bindung von Fruktose oder Glukose durch Freisetzung von Alizarin Rot S verfolgt. In diesem Zusammenhang konnte auch gezeigt werden, dass die Nanopartikel nach Sterilisation bei 121°C für 20 Minuten eine vergleichbare Bindung des Farbstoffes zeigen, was die Temperaturstabilität der Partikel unterstreicht. Da in diesem System die Bindung von Zuckern abhängig von der Affinität der Zucker zu den Boronsäuren bestimmt wird, wurden molekular geprägte Polymere synthetisiert, die eine Selektivität für bestimmte Zucker zeigen. Als Funktionsmonomer wurde 3-Methacrylamidobenzboroxol verwendet. Im Vergleich zu Polymeren, die mit 3-Vinylphenylboronsäure als literaturbekanntes Funktionsmonomer synthetisiert worden sind, zeigt sich bei pH 7.4 eine höhere Bindung von Fruktose an Benzoboroxolpolymere. In einem anderen geprägten Polymer wurden biomimetische (zuckeranaloge) Substanzen als Matrizenmolekül verwendet. Der Vorteil dieser Substanz ist die Bildung eines definierten Boronsäureesters, der als Templat-Funktionsmonomerkomplexes einpolymerisiert wird. Im Gegensatz dazu weist beispielsweise ein Boronsäureester der Glukose keine definierte Struktur auf. Der biomimetische Charakter wurde durch strukturelle Überlagerung mit schon publizierten Kristallstrukturen von Boronsäureestern von Glukose und Fruktose gezeigt. Das zugehörige molekular geprägte Polymer wurde mit Vinylphenylboronsäure synthetisiert und zeigt eine Bindung von Glukose und Fruktose, was durch die Strukturüberlagerungen vorhergesagt wurde. Zusammenfassend sind die wesentlichen wissenschaftlichen Ergebnisse: • Bindungskostanten von neuen Zucker-Benzoboroxolpaaren, • Eignung von ARS als elektrochemische Nachweissubstanz der Saccharidbindung • Die thermodynamische Charakterisierung der Bindung von Fruktose oder Glukose an Nanopartikel, die Benzoboroxol - modifziert sind und so bei pH 7.4 Zucker binden können • Einsatz von Benzoboroxol als Funktionsmonomer • die Synthese und Nutzung von biomimetischen zuckeranalogen Matrizenmolekülen für die Entwicklung von molekular geprägten Polymeren für die Glukosebindung.

Soil structural stability plays a pivotal role in landscape preservation when a protective vegetation cover is lacking. For example, under semiarid climates seasonal rainfall cannot sustain a full vegetation cover, but still causes soil erosion. With the loss of (fertile) soil material, ecosystem productivity reduces and less C can be stored. In natural semiarid systems, soil erosion is a spatially heterogeneous process, whereby local highly erodible spots are alternated by improved soil structure under the sparse canopy cover, creating a very heterogeneous landscape. Although the physical protection by the plant canopy is well understood, the potential influence of soil archaea and bacteria on soil structural stability in relation to plants and parent material is less well known. Mainly from studies under controlled conditions, we know that certain archaeal and bacterial species have the ability to produce extracellular polymeric substances (EPS), forming an extracellular matrix. As the formed matrix connects soil particles, EPS seem to have the potential of playing a substantial role in soil aggregation, thereby controlling soil erodibility. Little is known of this gluing process by EPS and its importance under natural conditions as most evidence is derived from controlled conditions in the laboratory. This dissertation aims to unravel the role of EPS from soil archaea and bacteria in soil aggregate stabilization in semiarid grasslands by considering the potential role of plant species and parent material in this process. The sparse vegetation in semiarid grasslands provide a useful gradient in soil organic C contents to study these processes. Improved conditions for soil microbes producing EPS can be found at the root surface, while the bare canopy interspaces lack in C/resources. Two sites were selected in southeast Spain, mainly differing in graphitic C, inorganic C and nitrogen contents. On both sites, soil adjacent to the widely occurring Anthyllis cytisoides legumes shrubs and Macrochloa tenacissima grass tussocks were sampled during two campaigns. The first sampling campaign in April 2017 focused on the top soil, whereby a distance gradient from the plant stem to the bare intercanopy area was sampled. The second sampling campaign in April 2018 focused more on the effect of plant roots on soil archaeal and bacterial communities by including the rhizosphere. As the parent material of the Rambla Honda site, i.e. one of the study sites, contains a substantial amount of graphitic C, several methods were tested to quantify the different types of C in these soils to understand their role in shaping EPS contents. Furthermore, the quantification of graphitic C contents opened the possibility to study a potential interaction between graphite minerals and microbes. Although graphitic C contents explained part of the variances in microbial community, no direct link with EPS-saccharide contents was found. EPS contents were relative high in the rhizosphere, most notable at the legumes shrub Anthyllis cytisoides, and were linked to the enrichment of N-fixing bacteria. However, outside the root influenced soil, EPS contents were still substantially high, whereby the abundance of microbial species, previously associated to biofilm formation in other environments, indicated that EPS synthesis is not only restricted to the rhizosphere. Soil aggregation was linked to EPS-saccharide contents, whereby two mechanisms were hypothesized. Firstly, the strong link between soil wettability and EPS-saccharide content in the soil of the carbonate poor Rambla Honda site, indicated that aggregates become stabilized by hydrophobic bonds created by the EPS. Secondly, results from the carbonate rich Alboloduy site indicates that EPS has a facilitating role in creating stable aggregates by precipitating carbonates on the EPS structure. This likely lead to a higher soil structural stability, as carbonate bindings are more stable when prolonged drought reduces soil biological activity and thereby EPS contents. Overall, EPS play a substantial role in soil aggregate stabilization in semiarid grasslands, whereby EPS contents were increased by legume plants, by means of enriching EPS producing bacteria. / Die Stabilität der Bodenstruktur spielt eine entscheidende Rolle in der Erhaltung der Landschaft, insbesondere wenn keine schützende Vegetationsbedeckung vorhanden ist. So ist beispielsweise unter semiariden Klimabedingungen wegen der Saisonalität der Niederschläge keine vollständige Vegetationsbedeckung vorhanden, was Bodenerosion verursacht. Durch den Verlust von (fruchtbarem) Bodenmaterial verringert sich die Produktivität des Ökosystems. Dadurch kann weniger Kohlenstoff (C) im Boden gespeichert werden. In natürlichen semiariden Systemen ist die Bodenerosion ein räumlich heterogener Prozess, bei dem sich stark erosionsanfällige Stellen mit solchen Bereichen abwechseln, welche durch günstige Bodenstruktur unter der spärlichen Pflanzendecke gekennzeichnet sind. Hierdurch entsteht eine sehr heterogene Landschaft. Während zum physikalischen Schutz durch Vegetationsüberschirmung viele Erkenntnisse vorliegen, ist über den möglichen Einfluss von Archaeen und Bakterien auf die strukturelle Stabilität des Bodens in Bezug auf Pflanzen und Ausgangsmaterial weit weniger bekannt. Hauptsächlich aus Studien unter kontrollierten Bedingungen wissen wir, dass bestimmte Archaen- und Bakterienarten die Fähigkeit besitzen, extrazelluläre polymere Substanzen (EPS) zu produzieren, die eine extrazelluläre Matrix bilden. Da die gebildete Matrix Bodenpartikel verbindet, scheint EPS das Potenzial für eine maßgebliche Beeinflussung der Bodenaggregation zu haben und dadurch die Erosionsanfälligkeit zu steuern. Über solche Klebemechanismen von EPS und deren Bedeutung unter natürlichen Bedingungen ist aber wenig bekannt; die meisten Hinweise stammen aus kontrollierten Bedingungen im Labor. Diese Dissertation zielt darauf ab, die Bedeutung von EPS von Archaeen und Bakterien hinsichtlich der Stabilisierung von Bodenaggregaten in semiariden Graslandschaften unter Berücksichtigung der möglichen Rolle von Pflanzenarten und Ausgangsmaterial in diesem Prozess aufzuklären. Zur Untersuchung solcher Prozesse bietet die spärliche Vegetation in semiariden Graslandschaften einen zweckdienlichen Gradienten bezüglich des Gehalt an organischem C im Boden. Günstige Bedingungen für EPS-produzierende Bodenmikroorganiosmen sind an der Wurzeloberfläche zu finden, während dem unbedeckten Boden zwischen Stellen ohne Pflanzenbedeckung C / Ressourcen fehlen. Es wurden zwei Standorte in Südostspanien ausgewählt, die sich hauptsächlich in den Gehalten an graphitischem C, anorganischem C und Stickstoff unterscheiden. An beiden Standorten wurden im Rahmen von zwei Feldkampagnen Böden in unmittelbarer Nähe zu der weit verbreiteten Leguminosenart Anthyllis cytisoides-Hülsenfrüchten und Grasbüscheln von Macrochloa tenacissima beprobt. Die erste Probenahmekampagne im April 2017 konzentrierte sich auf den obersten Boden, wobei ein Abstandsgradient vom Pflanzenspross zum unbedeckten Boden zwischen der Pflanzendecke beprobt wurde. Die zweite Probenahmekampagne im April 2018 konzentrierte sich mehr auf die Wirkung von Pflanzenwurzeln auf Archaeen- und Bakteriengemeinschaften durch Beprobung der Rhizosphäre. Am Rambla Honda-Standort enthält das Ausgangsmaterial eine erhebliche Menge an graphitischem C. Deshalb wurden verschiedene Methoden getestet, um die verschiedenen Arten von C in diesen Böden zu quantifizieren und ihre Rolle bei der Gestaltung des EPS-Gehalts zu verstehen. Darüber hinaus eröffnete die Quantifizierung des graphitischen C-Gehalts die Möglichkeit, die Wechselwirkung zwischen Graphitmineralen und Mikroorganismen zu untersuchen. Obwohl der Gehalt an graphitischem C einen Teil der Varianzen in der mikrobiellen Gemeinschaft erklärte, wurde kein direkter Zusammenhang mit dem EPS-Saccharidgehalt gefunden. Die EPS-Gehalte waren in der Rhizosphäre relativ hoch - am deutlichsten bei der Leguminosenart Anthyllis cytisoides - und mit der Anreicherung von N-fixierenden Bakterien verbunden. Außerhalb des von der Wurzel beeinflussten Bodens war der EPS-Gehalt jedoch immer noch deutlich erhöht. Dabei wies die Häufigkeit von Mikroorganismenarten, die zuvor mit der Bildung von Biofilmen in anderen Umgebungen in Verbindung gebracht wurden, darauf hin, dass die EPS-Synthese nicht nur auf die Rhizosphäre beschränkt ist. Die Bodenaggregation zeigte eine Verbindung mit dem EPS-Saccharidgehalt auf, wobei zwei Mechanismen angenommen wurden: Erstens wies der starke Zusammenhang zwischen der Bodenbenetzbarkeit und dem EPS-Saccharidgehalt im Boden des karbonatarmen Rambla Honda-Standorts auf eine Aggregatstabilisierung durch EPS-erzeugte hydrophobe Bindungen hin. Zweitens zeigen die Ergebnisse des Standorts Alboloduy-Standorts mit karbonatreichem Boden, dass EPS eine unterstützende Funktion bei der Erzeugung stabiler Aggregate besitzt, indem Karbonate auf der EPS-Struktur ausgefällt werden. Dies führt wahrscheinlich zu einer höheren Stabilität der Bodenstruktur, da Karbonatbindungen stabiler sind, wenn eine längere Trockenheit zu einer Verringerung der biologischen Aktivität im Boden und damit des EPS-Gehalts führt. Insgesamt spielt EPS eine wesentliche Rolle bei der Stabilisierung von Bodenaggregaten in semiariden Graslandschaften, wobei der EPS-Gehalt durch Leguminsosen, mittels Anreicherung von EPS-produzierenden Bakterien, erhöht wurde.

Silicone polymers and network-materials have proven extremely useful in a variety of applications owing to their superb properties when compared to carbon-based polymers. Polysiloxanes containing functional groups other than simple alkyl moieties have allowed for further manipulations of pendant groups along the polymer backbone leading to a greater range of possible chemical transformations, as well as changes in physical/interfacial properties. One aspect of functional polymers that has yet to be explored with respect to primarily silicone-based systems is that of stimuli-responsive materials. In order for this unique application to work, silicones must be functionalized with a group or groups that can influence the polymer’s properties based on that group’s response to specific external stimuli. Boronic acids represent one such group, wherein the most common stimuli used to affect changes in ionization state and solubility are pH and diol-binding. Boronic acids are also capable of forming weak hydrogen-bonded dimers with other boronic acids, and dynamic covalent bonds with Lewis bases. It is proposed that the covalent attachment of boronic acids and their derivatives onto silicones could lead to stimuli-responsive silicone materials. Herein, the synthesis of silicone-boronic acids and their protected boronic esters is described. The simple two-step method involving boronic acid protection followed by hydrosilylation has led to a variety of molecules differing in molecular weight and three-dimensional geometry through the use of commercially available hydride-functional silicones. Initial results regarding saccharide binding selectivity and the impacts on silicone solubility are provided. The unique interfacial behaviour of silicone-boronic esters and their propensity to form self-assembled, crosslinked films at an air/water interface are also reported. Using several different diol protecting groups and a variety of aqueous sub-phases, the mechanism for changes in physical properties as well as crosslinking were revealed. Finally, the production of new thermoplastic silicone elastomers from silicone-boronic esters and amine-containing molecules is discussed. The Lewis acid/Lewis base complexation that occurs between nitrogen and boron can provide enough strength to produce robust, yet recyclable, silicone elastomers without the use of catalyst or solvent. Elastomers can be easily dissolved and reformed through the introduction and removal of a mono-functional Lewis base. The impact of crosslink density, controlled by the quantities and molecular weights of each polymer component used, on physical characteristics is reported. / Thesis / Doctor of Philosophy (PhD)

Interactions between proteins and saccharide moieties play an indispensable role in mammalian reproduction as they stand behind of such processes as maturation and mutual recognition of gametes and sperm oviductal reservoir formation. In my dissertation thesis I focused on activities of glycosidases from bovine and porcine follicular fluids and their changes connected with follicle development. Activities of five glycosidases were detected in tertiary and preovulatory follicles in both species. The most active enzymes were α-L-fucosidase in cow and α-D-mannosidase in sow and both enzymes also demonstrated the most pronounced increase in their activities during follicle maturation. Interestingly, both α-L-fucose in cow and α-D-mannose in sow were described as saccharides responsible for the formation of the sperm oviductal reservoir and we offered a hypothetical mechanism of synchronisation between sperm release from their reservoir with the time of ovulation based on a surge of activities of corresponding follicular glycosidases through the oviduct. Subsequently, it was demonstrated that β-D-galactosidase and α-D-mannosidase affect sperm-zona pellucida binding in pig, as they both decrease interaction between sperm receptors for zona pellucida and zona pellucida. This may explain the observation...

Les interactions protéines-carbohydrates sont à la base de nombreux processus biologiques physiologiques aussi bien que pathologiques. Ces interactions incluent la synthèse et la dégradation enzymatique des oligosaccharides, la cohésion des tissus, l'immunité, le cancer ou encore l'infection bactérienne et virale. L'inhibition de ce type d'interaction par des molécules multivalentes synthétiques telles les glycopolymères, glycodendrimères, glycoclusters, etc. fait l'objet d'études importantes depuis plusieurs décennies. L'obtention de telles molécules pourrait permettre de développer de nouvelles thérapies qui pourraient palier notamment la multi-résistance aux antibiotiques. De plus, la détection de telles interactions par des méthodes simples et faciles à mettre en oeuvre permettrait une amélioration de la compréhension de ces phénomènes, ainsi que le diagnostic rapide de la présence de microorganismes. C'est dans ce contexte, que nous avons développé une nouvelle classe de composés glycosylés multivalents à coeur triazine. Ces glycoclusters de basse valence, ont la particularité de présenter une double fonctionnalité : l'inhibition d'interactions lectine-sucre par des effets de multivalence ainsi que la détection de ces interactions. Nous présentons dans ce manuscrit, la synthèse d'une nouvelle famille de glycoclusters polysulfurés à coeur triazine portant des épitopes saccharidiques tels que D-glucose, D-galactose, D-mannose, L-fucose, ainsi que leurs évaluations biologiques réalisées sur des lectines de Pseudomonas aeuriginosa. Nous avons ainsi mis en évidence la possibilité de reconnaître et de détecter les interactions lectine-sucre dans un premier temps par association d'un cluster mixte portant un fluorophore, et de façon plus sophistiquée, grâce à un système à géométrie variable incorporant dans le scaffold même un switch photochimique variant l'arrangement des sucres dans l'espace / Protein-carbohydrate interactions mediate a wide range of biochemical processes. Amongst these is the process of bacterial infection, which often proceeds through carbohydrate-binding lectins involved in biofilm formation. Even if the individual associations result from weak interactions, the assembly of multiple carbohydrate-protein interactions, typically more than additive, confers to the system the required specificity and avidity for their biological functions. In order to study this « glycocluster effects », a number of scaffold systems presenting multivalent carbohydrate ligands have been prepared in the literature. Dendrimers, polymers, peptides, calixarenes, to name a few, have been used as core molecules for the synthesis of multivalent glycoconjugates. The purpose of this work is to design new glycoclusters which exhibit dual functionality: the inhibition of carbohydrate-protein interactions via a multivalency effect; and detection of the interactions via fluorescence spectroscopy. A first generation of polysulfurated glycoclusters, organized around a heteroaromatic core, was synthesized using click chemistry reactions, which provided a family of highly soluble and readily accessible clusters. The glycoclusters were evaluated for their ligand-lectin interactions, multivalency effects, thermodynamic parameters, and abilty to modulate biofilm formation by Pseudomonas aeruginosa, a major causative agent of lung infections in cystic fibrosis patients. We describe a new family of ‘switchable glycoclusters’ based on photochromic behavior. They are designed to generate a modulated fluorescence signal as well as a defined change in the three-dimensional arrangement of the sugar epitopes, and may eventually provide significantly improved probes for studying the distribution, dynamics, interactions, and activities of specific lectins

Das Lektin aus <i>Pisum sativum</i>, der Gartenerbse, ist Teil der Familie der Leguminosenlektine. Diese Proteine haben untereinander eine hohe Sequenzhomologie, und die Struktur ihrer Monomere, ein all-ß-Motiv, ist hoch konserviert. Dagegen gibt es innerhalb der Familie eine große Vielfalt an unterschiedlichen Quartärstrukturen, die Gegenstand kristallographischer und theoretischer Arbeiten waren. Das Erbsenlektin ist ein dimeres Leguminosenlektin mit einer Besonderheit in seiner Struktur: Nach der Faltung in der Zelle wird aus einem Loop eine kurze Aminosäuresequenz herausgeschnitten, so dass sich in jeder Untereinheit zwei unabhängige Polypeptidketten befinden. Beide Ketten sind aber stark miteinander verschränkt und bilden eine gemeinsame strukturelle Domäne. Wie alle Lektine bindet Erbsenlektin komplexe Oligosaccharide, doch sind seine physiologische Rolle und der natürliche Ligand unbekannt. In dieser Arbeit wurden Versuche zur Entwicklung eines Funktionstests für Erbsenlektin durchgeführt und seine Faltung, Stabilität und Monomer-Dimer-Gleichgewicht charakterisiert. Um die spezifische Rolle der Prozessierung für Stabilität und Faltung zu untersuchen, wurde ein unprozessiertes Konstrukt in <i>E. coli</i> exprimiert und mit der prozessierten Form verglichen. <br /> <br /> Beide Proteine zeigen die gleiche kinetische Stabilität gegenüber chemischer Denaturierung. Sie denaturieren extrem langsam, weil nur die isolierten Untereinheiten entfalten können und das Monomer-Dimer-Gleichgewicht bei mittleren Konzentrationen an Denaturierungsmittel auf der Seite der Dimere liegt. Durch die extrem langsame Entfaltung zeigen beide Proteine eine apparente Hysterese im Gleichgewichtsübergang, und es ist nicht möglich, die thermodynamische Stabilität zu bestimmen. Die Stabilität und die Geschwindigkeit der Assoziation und Dissoziation in die prozessierten bzw. nichtprozessierten Untereinheiten sind für beide Proteine gleich. Darüber hinaus konnte gezeigt werden, dass auch unter nicht-denaturierenden Bedingungen die Untereinheiten zwischen den Dimeren ausgetauscht werden.<br /> <br /> Die Renaturierung der unprozessierten Variante ist unter stark nativen Bedingungen zu 100 % möglich. Das prozessierte Protein dagegen renaturiert nur zu etwa 50 %, und durch die Prozessierung ist die Faltung stark verlangsamt, der Faltungsprozess ist erst nach mehreren Tagen abgeschlossen. Im Laufe der Renaturierung wird ein Intermediat populiert, in dem die längere der beiden Polypeptidketten ein Homodimer mit nativähnlicher Untereinheitenkontaktfläche bildet. Der geschwindigkeitsbestimmende Schritt der Renaturierung ist die Assoziation der entfalteten kürzeren Kette mit diesem Dimer. / The lectin from <i>Pisum sativum</i> (garden pea) is a member of the family of legume lectins. These proteins share a high sequence homology, and the structure of their monomers, an all-ß-motif, is highly conserved. Their quaternary structures, however, show a great diversity which has been subject to cristallographic and theoretical studies. Pea lectin is a dimeric legume lectin with a special structural feature: After folding is completed in the cell, a short amino acid sequence is cut out of a loop, resulting in two independent polypeptide chains in each subunit. Both chains are closely intertwined and form one contiguous structural domain. Like all lectins, pea lectin binds to complex oligosaccharides, but its physiological role and its natural ligand are unknown. In this study, experiments to establish a functional assay for pea lectin have been conducted, and its folding, stability and monomer-dimer-equilibrium have been characterized. To investigate the specific role of the processing for stability and folding, an unprocessed construct was expressed in <i>E. coli</i> and compared to the processed form.<br /> <br /> Both proteins have the same kinetic stability against chemical denaturant. They denature extremely slowly, because only the isolated subunits can unfold, and the monomer-dimer-equilibrium favors the dimer at moderate concentrations of denaturant. Due to the slow unfolding, both proteins exhibit an apparent hysteresis in the denaturation transition. Therefore it has not been possible to determine their thermodynamic stability. For both proteins, the stability and the rates of association and dissociation into processed or unprocessed subunits, respectively, are equal. Furthermore it could be shown that even under non-denaturing conditions the subunits are exchanged between dimers.<br /> <br /> Renaturation of the unprocessed variants is possible under strongly native conditions with 100 % yield. The processed protein, however, can be renatured with yields of about 50 %, and its refolding is strongly decelerated. The folding process is finished only after several days. During renaturation, an intermediate is populated, in which the longer of the two polypeptide chains forms a homodimer with a native-like subunit interface. The rate limiting step of renaturation is the association of the unfolded short chain with this dimer.

Les aminoglycosides, des dérivés aminés de saccharides, interfèrent avec le mécanisme de synthèse des protéines chez les bactéries en se fixant au site de décodage de l'ARN de transfert aminoacylé (site A) situé en 3' de l'ARN ribosomique 16S. Au cours de ce travail de thèse, les structures de trois complexes entre des fragments d'ARN incorporant le site A et les aminoglycosides paromomycine, tobramycine et généticine, ont été résolues par cristallographie aux rayons X à 2,40-2,54 Å. L'analyse des structures montre que la reconnaissance et la fixation spécifiques des aminoglycosides au site A font intervenir de nombreuses liaisons hydrogène directes et pontées par des molécules d'eau. Dans ces structures, la partie néamine commune aux aminoglycosides (cycles I et II) s'intercale dans l'hélice de manière similaire : le cycle I (non plan) forme une pseudo paire de bases avec l'adénine 1408 ; la néamine oblige les adénines 1492 et 1493 à pointer hors de l'hélice. La comparaison des structures 3D de ces trois complexes offre des explications moléculaires aux différents résultats de biochimie et de microbiologie, ainsi qu'à certains phénomènes de résistances et de toxicités. Les conformations du site A et des aminoglycosides au sein de ces complexes sont similaires à celles du site A et de la paromomycine au sein de la sous-unité ribosomique 30S. Ainsi, la stratégie développée permet une description des interactions et des modes de fonctionnement des aminoglycosides proche du contexte naturel mais plus précise, essentielle à notre connaissance du système ARN/aminoglycoside. De ces résultats découlent des lignes directrices laissant envisager sous un jour nouveau la conception d'antibiotiques moins sujets aux résistances et moins toxiques.

Connor O’Hara July 29, 2019 Inhibition of Cancer Stem Cells by Glycosaminoglycan Mimetics In the United States cancer is the second leading cause of death, with colorectal cancer (CRC) being the third deadliest cancer and expected to cause over 51,000 fatalities in 2019 alone.1 The current standard of care for CRC depends largely on the staging, location, and presence of metastasis.2 As the tumor grows and invades nearby lymph tissue and blood vessels, CRC has the opportunity to invade not only nearby tissue but also metastasize into the liver and lung (most commonly).3 The 5-year survival rate for metastasized CRC is <15%, and standard of care chemotherapy regimens utilizing combination treatments only marginally improve survival.3-5 Additionally, patients who have gone into remission from late-stage CRC have a high risk of recurrence despite advances in treatment.6-7 The Cancer Stem-like Cell (CSC) paradigm has grown over the last 20 years to become a unifying hypothesis to support the growth and relapse of tumors previously regressed from chemotherapy (Figure 1).8 The paradigm emphasizes the heterogeneity of a tumor and its microenvironment, proposing that a small subset of cells in the tumor are the source of tumorigenesis with features akin to normal stem cells.9 The CSCs normally in a quiescent state survive this chemotherapy and “seed” tumor redevelopment.10 First observed in acute myeloid lymphoma models, CSCs have since been identified in various other cancers (to include CRC) by their cell surface antigens and unique properties characterizing them from normal cancer cells.11-12 These include tumor initiation, limitless self-renewal capacity to generate clonal daughter cells, as well as phenotypically diverse, mature, and highly differentiated progeny.13-14 Previously our lab has identified a novel molecule called G2.2 (Figure 2) from a unique library of sulfated compounds showing selective and potent inhibition of colorectal CSCs in-vitro.15 G2.2 is a mimetic of glycosaminoglycans (GAGs) and belongs to a class of molecules called non-saccharide GAG mimetics (NSGMs). Using a novel dual-screening platform, comparisons were made on the potency of G2.2 in bulk monolayer cells, primary 3D tumor spheroids of the same cell line, and subsequent generations of tumor spheroids. This work has shown in-vitro the fold-enhancement of CSCs when culturing as 3D tumor spheroids. Spheroid culture serves as a more accurate model for the physiological conditions of a tumor, as well as the functional importance of upregulating CSCs. Evaluation of G2.2 and other NSGMs was performed in only a few cell lines, developing a need to better understand the ability of G2.2 to inhibit spheroids from a more diverse panel of cancer cells to better understand G2.2’s mechanism. The last few decades have seen the advancement in fundamental biological and biochemical knowledge of tumor cell biology and genetics.16 CRC, in particular, has served as a useful preclinical model in recapitulating patient tumor heterogeneity in-vitro.17 Recent work has characterized the molecular phenotypes of CRC cell lines in a multi-omics analysis, stratifying them into 4 clinically robust and relevant consensus molecular subtypes (CMS).18-19 Our work was directed to screen a panel of cells from each of the molecular subtypes and characterize the action of G2.2 and 2nd generation lipid-modified analogs, synthesized to improve the pharmacokinetic properties of the parent compound. Four NSGMs, namely G2.2, G2C, G5C, and G8C (Figure 2) were studied for their ability to inhibit the growth of primary spheroids across a phenotypically diverse panel. Compound HT-29 IC50 (μM) Panel Average IC50 (μM) G2.2 28 ± 1 185 ± 55 G2C 5 ± 2 16 ± 15 G5C 8 ± 2 63 ± 19 G8C 0.7 ± 0.2 6 ± 3 Primary spheroid inhibition assays were performed comparing the potency of new NSGMs to G2.2. Fifteen cell lines were evaluated in a panel of colorectal adenocarcinoma cell lines with several cell lines representing each CMS. Primary spheroid inhibition assays revealed 3 distinct response with regard to G2.2’s ability to inhibit spheroid growth. Cells from CMS 3 and 4, which display poor clinical prognosis, metabolic dysregulation, and enhanced activation of CSC pathways, showed the most sensitivity to G2.2 (mean IC50 = 89 ± 55 μM). Mesenchymal CMS 4 cell lines were over 3-fold more sensitive to treatment with G2.2 when compared to CMS 1 cell lines. Resistant cell lines were composed entirely of CMS 1 and 2 (mean IC50 = 267 ± 105 μM). In contrast, all lipid-modified analogs showed greater potency than the parent NSGM in almost every CRC cell line. Of the three analogs, G8C showed the greatest potency with a mean IC50 of less than 15 μM. Of the CRC spheroids studied, HT-29 (CMS 3) was most sensitive to G8C (IC50 = 0.73 μM). To evaluate the selectivity of NSGMs for CSC spheroid inhibition, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) cytotoxicity assays were performed on monolayer cell culture, and the fold-selectivity of NSGM for spheroids was analyzed. Data shows that NSGMs preferentially target CSC-rich spheroids compared with monolayer cellular growth, with G2.2 having over 7-fold selectivity for spheroid conditions. This fold selectivity was enhanced in CMS 3/4, supporting the idea that G2.2 targets a mesenchymal and stem-like phenotype. To further validate this selectivity, limiting dilution assays were performed across the panel to determine the tumor-initiating capacity of each cell line. Cell lines which showed a sensitive response to G2.2 were over 2-fold more likely to develop into spheroids, validating the previous hypothesis. Further characterization was performed analyzing the changes G2.2 induced on CSC markers, as well as the basal expression of a unique pair of cancer cells. Western blots showed a reduction in self-renewal marker across all CMS after treatment with G2.2, and that cell lines sensitive to G2.2-treatment overexpress mesenchymal and stem-like markers. G2.2-resistant cell lines show an epithelial phenotype, lacking this expression. The positive results observed in these studies enhance the understanding of G2.2 and analogs, and further evaluation with additional cell lines of various tissues would improve the knowledge thus far gained. However, all experiments described take valuable time to perform and analyze. Thus, there became a need to develop a high-throughput screening (HTS) platform for our assays that standardized analysis and enhanced productivity. Initial development of the method for this assay are underway, and recent evidence from these evaluations of breast cancer spheroids suggests that G2.2 and analogs may be tissue-specific compounds for the treatment of cancer. Future work entails refining the application of this method for evaluation of the NCI-60 (National Cancer Institute) tumor cell panel. Overall, these results make several suggestions concerning the NSGMs evaluated against the panel. First, G2.2 selectively targets CSCs with limited toxicity to monolayer cells of the same cell line. Further, G2.2 has the greatest potency with CMS 3/4, whose mesenchymal phenotypes are associated with poor clinical prognosis and enrichment of CSCs. Supporting evidence include that sensitive cell lines are highly tumorigenic and show enhanced expression of mesenchymal/CSC markers compared to resistant cell lines. Lipid-modification of G2.2 enhances in-vitro potency against spheroid growth, with nM potency reached in the most sensitive cell lines. Evidence in the development of a HTS platform also suggests these NSGMs show tissue specificity to cancers of the intestine. Further work characterizing the mechanism of NSGMs in a broader multi-tissue panel will enhance our understanding of the compounds as a potential therapy to dramatically improve patient survival through specific targeting of tumorigenesis. References 1. Colorectal Cancer Facts & Figures 2017-2019. American Cancer Society 2017. 2. Compton, C. C.; Byrd, D. R.; Garcia-Aguilar, J.; Kurtzman, S. H.; Olawaiye, A.; Washington, M. K. Colon and rectum. In AJCC Cancer Staging Atlas, 2nd ed.; Ed. Springer Science: New York, 2012; pp 185–201. 3. 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