<p>Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose.</p><p>Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns.</p><p>Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.</p>

La fonctionnalisation de liaisons C-H non-activées est un défi pour la chimie organique. Dans cette thèse, l'amination intramoléculaire, via l'insertion catalytique de nitrènes sur ces positions réputées inertes, a été effectuée sur des glycomimétiques de type C-glycoside. A l'inverse de leurs analogues 1- carbamoyloxyméthyle, cette réaction s'est montrée fortement stéréo-dépendante de la configuration du carbone pseudo-anomérique dans le cas des dérivés 1-sulfamoyloxyméthyle. L'insertion régiosélective en position pseudo-anomérique conduit à des composés spiraniques dont la fonction N,O-acétal peut être fonctionnalisée pour aboutir à des glycomimétiques originaux. Une étude sur des dérivés pipéridinique a permis d'évaluer l'influence de l'hétéroatome endocyclique sur la réaction d'insertion. L'amination intramoléculaire de ces dérivés nous a permis d'observer une régiosélectivité complètement différente de celle observée précédemment avec la formation inédite d'un cycle à sept membres. La combinaison d'effets stéréoélectroniques et conformationnels décisifs nous a permis de rationaliser ce résultat inattendu. Nous avons exploité ce résultat par la mise au point d'une stratégie de synthèse générale de 4-amino-imino-Cglycosides. En nous appuyant sur l'utilisation séquentielle du groupe 1-sulfamoyloxymethyle comme "bras moléculaire activateur", nous sommes parvenus à fonctionnaliser toutes les positions d'une pipéridine monosubstituée. La synthèse totale de divers iminosucres originaux a permis d'illustrer l'intérêt de cette stratégie et ouvre ainsi l'accès à la découverte de nouveaux composés d'intérêt thérapeutique.

Les glycosides sont reconnus pour leur potentiel pharmaceutique tels que les antibiotiques, les agents anticancéreux et antiviraux. Ils sont impliqués dans plusieurs processus biologiques entre autres la reconnaissance cellulaire, l’inflammation, la réponse immunitaire, la croissance, le transport cellulaire, l’adhésion cellulaire et les groupes sanguin. Notre groupe excelle dans la glycosidation stéréocontrôlée avec un minimum de protection suivant le concept d’activation à distance d’aglycones hétérocycliques anomériques. La présence d’une quantité sous stoechiométrique d’acide de Lewis, les (2-pyridyl)-β-D-glycosides déprotégés sont d’excellents donneurs permettant de haute sélectivité pour l’anomère- α-D de glycosides simples et complexes. Inversement, (2-pyridyl)-α-D-glycosides donnent les β-D-glycosides avec de bonne sélectivité. Des exemples de formation stéréocontrôlée de glycosides sont présentés dans cette thèse avec des accepteurs tels que les phénols, les stéroïdes, les terpènes et les acides hydroxyaminés. Cette méthodologie de glycosidation a été appliquée sur support solide. / Glycosides are well-known to be components of important antibiotics, anticancer agents and antiviral drugs. They are also involved in many biologically relevant processes such as cell-cell recognition, inflammation, immunity, cell transport, cell adhesion, and as determinants of blood group types. Our group has championed stereocontolled methods of glycoside synthesis with minimal donor protection using the concept of remote activation of anomeric heterocyclic aglycones. In the presence of sub-stoichiometric amounts of Lewis acids, unsubstituted beta-D-pyridyl glycosides are excellent donors affording simple and complex glycosides usually with high alpha-anomeric selectivity. Conversely, alpha-D-pyridyl glycosides give the inverted beta-glycosides with equally good selectivity. Examples of stereocontrolled glycoside synthesis with acceptor molecules include among others, phenols, steroids, terpenes, and hydroxyamino acids. Applications to glycoside synthesis on solid support have been pursued with promising results.

Magister Pharmaceuticae - MPharm / Sutherlandia frutescens (tribe Galegeae, Fabaceae), is a popular medicinal plant traditionally used in South Africa. In 2000, a company called Phyto Nova (Pty) Ltd. initiated large-scale cultivation and contract manufacturing of tablets, made from the powdered herb (i.e. thin stems and leaves). Most of these commercial Sutherlandia solid dosage forms are made from the dried leaf powder but recently a new product, viz. Promune™ capsules, made from a freeze-dried aqueous extract, came on the market and was claimed to be “better” as it mimics the traditional tea. However, the pharmaceutical quality and stability of these preparations have not yet been investigated. The objectives of this study were firstly, to develop a validated stability-indicating HPLC assay for sutherlandioside B (SU-B); secondly, to compare the SU-B levels in the two commercially available Sutherlandia products viz, the Phyto Nova Sutherlandia SU1™ tablet and the Promune™ capsule, and, thirdly, to determine the effect of elevated temperature and humidity as well as acid hydrolysis on the SU-B levels in these two products. / South Africa

<p>This thesis describes stereoselective syntheses of four amino acids, three of which are C-glycosidic analogues of glycosylated amino acids. The overall goal of the project was to probe the interactions between MHC molecules, glycopeptide antigens and T cell receptors, that are essential for development of collagen induced arthritis. Collagen induced arthritis is a frequently used mouse model for rheumatoid arthritis, an autoimmune disease that attacks joint cartilage and leads to a painful and eventually crippling condition.</p><p>The thesis is based on four studies. The first study describes the synthesis of hydroxylysine, an amino acid that is found in collagen and is an important constituent of the glycopeptide proposed as an antigen in collagen induced arthritis. During the synthesis of hydroxylysine some new insight into the mechanism of the reductive opening of <i>p</i>-methoxybenzylidene acetals was obtained.</p><p>The remaining three studies deals with the synthesis of C-glycosidic analogues of glycosylated amino acids, hydroxy norvaline, threonine and hydroxylysine.The synthesis of each amino acid required control of several stereogenic centra and utilizes a variety of approaches such as use of stereoselective reactions, chiral auxilaries, chiral templates and asymmetric catalysis.</p><p>The C-glycosidic analogues of galactosylated hydroxynorvaline and hydroxylysine were incorporated in glycopeptides from type II collagen and evaluated in T cell response assays. It was found that the T cells were stimulated by the C-glycopeptides, but that higher concentrations were required than for the native O-glycopeptide</p>

This thesis describes stereoselective syntheses of four amino acids, three of which are C-glycosidic analogues of glycosylated amino acids. The overall goal of the project was to probe the interactions between MHC molecules, glycopeptide antigens and T cell receptors, that are essential for development of collagen induced arthritis. Collagen induced arthritis is a frequently used mouse model for rheumatoid arthritis, an autoimmune disease that attacks joint cartilage and leads to a painful and eventually crippling condition. The thesis is based on four studies. The first study describes the synthesis of hydroxylysine, an amino acid that is found in collagen and is an important constituent of the glycopeptide proposed as an antigen in collagen induced arthritis. During the synthesis of hydroxylysine some new insight into the mechanism of the reductive opening of p-methoxybenzylidene acetals was obtained. The remaining three studies deals with the synthesis of C-glycosidic analogues of glycosylated amino acids, hydroxy norvaline, threonine and hydroxylysine.The synthesis of each amino acid required control of several stereogenic centra and utilizes a variety of approaches such as use of stereoselective reactions, chiral auxilaries, chiral templates and asymmetric catalysis. The C-glycosidic analogues of galactosylated hydroxynorvaline and hydroxylysine were incorporated in glycopeptides from type II collagen and evaluated in T cell response assays. It was found that the T cells were stimulated by the C-glycopeptides, but that higher concentrations were required than for the native O-glycopeptide

<p>Sutherlandia frutescens (tribe Galegeae, Fabaceae), is a popular medicinal plant traditionally used in South Africa. In 2000, a company called Phyto Nova (Pty) Ltd. initiated large-scale cultivation and contract manufacturing of tablets, made from the powdered herb (i.e. thin stems and leaves). Most of these commercial Sutherlandia solid dosage forms are made from the dried leaf powder but recently a new product, viz. Promune&trade / capsules, made from a freeze-dried aqueous extract, came on the market and was claimed to be &ldquo / better&rdquo / as it mimics the traditional tea. However, the pharmaceutical quality and stability of these preparations have not yet been investigated. The objectives of this study were firstly, to develop a validated stability-indicating HPLC assay for sutherlandioside B (SU-B) / secondly, to compare the SU-B levels in the two commercially available Sutherlandia products viz, the Phyto Nova Sutherlandia SU1&trade / tablet and the Promune&trade / capsule, and, thirdly, to determine the effect of elevated temperature and humidity as well as acid hydrolysis on the SU-B levels in these two products.</p>

Wood formation or xylogenesis is a fundamental process for so diverse issues as industry, shelter and a sustainable environment. Wood is comprised of secondary xylem, rigid large cells with thick cell walls that are lignified. The basis for the sturdy cells is an advanced composite made up of cellulose fibers cross-linked by hemicelluloses and finally embedded in lignin. This fiber-composite is the secondary cell walls of woody plants. Cell division and differentiation is regulated by switching on and off genes. Proteins encoded by these genes execute the major functions in the cells. They steer the entire machinery operating the structure and function of the cells, maintaining growth and synthesising essential products such as the cell wall carbohydrates.   Here we describe the investigation of genes and proteins involved in xylan formation as well as the development of a model system that will aid the functional analysis of wood formation. Xylan is the main hemicellulose or cross linking glycan in dicot wood and thereby one of the most abundant carbohydrates on earth. We demonstrate that hybrid aspen cell suspension cultures can be used as a model system for secondary cell wall formation. We have also examined glycosyltransferases from CAZy family 43 that play a part in secondary cell wall formation. We have focused on one of these, Pt×tGT43A, a likely ortholog of Arabidopsis IRX9, which plays a crucial role in xylan formation. The protein was transiently expressed in Nicotiana benthamiana and its function and localization is described. Also, we investigate a glycoside hydrolase, Pt×tXyn10A, involved in wood formation. Its role is not clear but it most likely modifies xylan as it gets incorporated into the secondary cell wall after secretion from the Golgi. This influences the interaction between cellulose, xylan and lignin in the finished wood cell. We have also cloned a transcription factor, Pt×tMYB021, a likely ortholog of Arabidopsis MYB46 and we show that it activates GT43A, GT43B and Xyn10A. By analysis of the promoter sequences we identify a CA-rich motif putatively important for xylem-specific genes.   By mastering proteins involved in xylogenesis we will acquire the tools to improve and develop the wood product market. Xylan is an immense unexploited source of renewable carbohydrate. New products envisioned include e.g. faster growing trees, changed fiber characteristics, optimised utilization of wood carbohydrates for biofuels and biomaterials as well as invention of intelligent materials by biomimetic engineering. / Vedbildning, eller xylogenes, är en grundläggande mekanism för så skilda områden som industri, boende och en hållbar miljö. Ved består av sekundärt xylem som är starka, stora celler med tjocka cellväggar som är lignifierade. Grunden för de starka cellerna är en avancerad komposit bestående av cellulosafibrer tvärbundna av hemicellulosa och slutligen ingjutet i lignin. Denna fiberkomposit är den sekundära cellväggen i vedartade växter. Celldelning och differentiering regleras genom att sätta igång och stänga av gener. Proteiner som kodas av dessa gener utför de viktigaste funktionerna i cellerna. De styr hela maskineriet som upprätthåller cellernas struktur och funktion, underhåller tillväxt samt tillverkar nödvändiga produkter såsom cellväggskolhydraterna. Här beskriver vi utforskningen av gener och proteiner som är inblandade i xylanbildning liksom utvecklandet av ett modellsystem som kommer vara en hjälp i den funktionella analysen av vedbildning. Xylan är den vanligaste hemicellulosan, eller korsbindande glykanen, i lövträd och därför en av de vanligaste kolhydraterna på jorden. Vi demonstrerar att hybridaspcellkulturer i suspension kan användas som ett modellsystem för sekundär cellväggsbildning. Vi har också undersökt glykosyltransferaser från CAZy-familj 43 som tycks spela en viktig roll i bildandet av sekundär cellvägg. Vi har fokuserat på en av dessa, Pt×tGT43A, en trolig ortolog till Arabidopsis IRX9 som spelar en viktig roll i xylanbildning. Proteinet har uttryckts övergående i Nicotiana benthamiana och dess funktion och lokalisering beskrivs. Dessutom undersöker vi ett glykosidhydrolas, Pt×tXyn10A, involverad i vedbildning. Dess roll är oklar men högst sannolikt modifierar det xylan medan det inkorporeras i sekundära cellväggen efter sekretion från Golgi. Detta influerar interaktionen mellan cellulosa, hemicellulosa och lignin i den slutliga vedcellen. Vi har också klonat en transkriptionsfaktor, Pt×tMYB021, en trolig ortolog till Arabidopsis MYB46 och vi visar att den aktiverar GT43A, GT43B och Xyn10A. Genom analys av promotorsekvenserna har vi identifierat ett CA-rikt motiv förmodat viktigt för xylemspecifika gener.Genom att bemästra proteinerna som är ansvariga för vedbildning får vi verktyg att utveckla skogsproduktsmarknaden. Xylan är en ofantligt stor outnyttjad källa till förnyelsebara kolhydrater. En vision är nya produkter som till exempel snabbväxande träd, ändrade fiberegenskaper, optimerat användande av vedkolhydrater för biobränsle och biomaterial såväl som utvecklandet av intelligenta material genom biomimetisk ingenjörskonst. / QC20100730

This thesis presents the application of different protein engineering methods on enzymes and non-catalytic proteins that act upon xyloglucans. Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glucans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glucans such as cellulose. One important group of xyloglucan-active enzymes is encoded by the GH16 XTH gene family in plants, including xyloglucan endo-transglycosylases (XET) and xyloglucan endo-hydrolases (XEH). The molecular determinants behind the different catalytic routes of these homologous enzymes are still not fully understood. By combining structural data and molecular dynamics (MD) simulations, interesting facts were revealed about enzyme-substrate interaction. Furthermore, a pilot study was performed using structure-guided recombination to generate a restricted library of XET/XEH chimeras. Glycosynthases are hydrolytically inactive mutant glycoside hydrolases (GH) that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Different enzymes with xyloglucan hydrolase activity were engineered into glycosynthases, and characterised as tools for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns. Carbohydrate-binding modules (CBM) are non-catalytic protein domains that bind to polysaccharidic substrates. An important technical application involves their use as molecular probes to detect and localise specific carbohydrates in vivo. The three-dimensional structure of an evolved xyloglucan binding module (XGBM) was solved by X-ray diffraction. Affinity-guided directed evolution of this first generation XGBM resulted in highly specific probes that were used to localise non-fucosylated xyloglucans in plant tissue sections. / QC 20100902

In the context of ongoing efforts toward C-aryl glycoside synthesis, a recently developed approach to form C-aryl glycals from 2-deoxysugar lactones was expanded to form novel substrates. This approach has been applied to the synthesis of various furyl glycals, allowing access to C-aryl glycals via a benzyne furan (4+2) cycloaddition methodology. The hydroboration-oxidation of said C-aryl glycals has allowed access to C(2)-oxygenated C-aryl glycosides via the benzyne cycloaddition approach. An approach to the total synthesis of 5-hydoxyaloin A is detailed, in which regioselective benzyne furan (4+2) cycloadditions were achieved via the use of a silicon tether. Two approaches to the anthrone core have been applied; one in which an unsymmetrically-substituted aryl ring is first constructed by means of a silicon tether, and one in which the unsymmetrically-substituted ring is formed last, also utilizing a silicon tether. The latter approach has allowed access to the anthrone core of 5-hydroxyaloin A, and only a final desulfurization remains in order to access the natural product. / text