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Glycosylation is a fundamental co-translational and/or post-translational modification process where an attachment of sugars onto either proteins or lipids can alter their biological function, subcellular location and modulate the development and physiology of an organism. Glycosylation is not a template driven process and as such produces a vastly larger array of glycan structures through combinatorial use of enzymes and of repeated common scaffolds and as a consequence it provides a huge expansion of both the proteome and lipidome. While the essential role of N- and O-glycan modifications on mammalian glycoproteins is already well documented, we are just starting to decode their biological functions in plants. Although significant advances have been made in plant glycobiology in the last decades, there are still key challenges impeding progress in the field and, as such, holistic modern high throughput approaches may help to address these conceptual gaps. In this snapshot, we present an update of the most common O- and N-glycan structures present on plant glycoproteins as well as (1) the plant glycosyltransferases (GTs) and glycosyl hydrolases (GHs) responsible for their biosynthesis; (2) a summary of microorganism-derived GHs characterized to cleave specific glycosidic linkages; (3) a summary of the available tools ranging from monoclonal antibodies (mAbs), lectins to chemical probes for the detection of specific sugar moieties within these complex macromolecules; (4) selected examples of N- and O-glycoproteins as well as in their related GTs to illustrate the complexity on their mode of action in plant cell growth and stress responses processes, and finally (5) we present the carbohydrate microarray approach that could revolutionize the way in which unknown plant GTs and GHs are identified and their specificities characterized. Fil: Strasser, Richard. University of Natural Resources and Life Sciences; Suiza Fil: Seifert, Georg. University of Natural Resources and Life Sciences; Suiza Fil: Doblin, Monika S.. La Trobe University; Australia Fil: Johnson, Kim L.. La Trobe University; Australia Fil: Ruprecht, Colin. University of Natural Resources and Life Sciences; Suiza Fil: Pfrengle, Fabian. University of Natural Resources and Life Sciences; Suiza Fil: Bacic, Antony. La Trobe University; Australia Fil: Estevez, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

The heterogeneous nature of non-cellulosic polysaccharides, such as arabinoxylan, makes it difficult to correlate molecular structure with macroscopic properties. To study the impact of specific structural features of the polysaccharides on crystallinity or affinity to other cell wall components, collections of polysaccharides with defined repeating units are required. Herein, a chemoenzymatic approach to artificial arabinoxylan polysaccharides with systematically altered branching patterns is described. The polysaccharides were obtained by glycosynthase-catalyzed polymerization of glycosyl fluorides derived from arabinoxylan oligosaccharides. X-ray diffraction and adsorption experiments on cellulosic surfaces revealed that the physicochemical properties of the synthetic polysaccharides strongly depend on the specific nature of their substitution patterns. The artificial polysaccharides allow structure-property relationship studies that are not accessible by other means.

Colloidal gold particles with functionalized organic shells were applied as novel selectin binders. The ligand shell was terminated with different monocyclic carbohydrate mimetics as simplified analogs of the sLe(x) unit found in biological selectin ligands. The multivalent presentation of the sulfated selectin binding epitopes on the gold particles led to extremely high binding affinities towards L- and P-selectin and IC(50) values in the subnanomolar range. Depending on the ring size of the sulfated carbohydrate mimetic, its substitution pattern and its configuration, different selectivities for either L-selectin or P-selectin were obtained. These selectivities were not found for gold particles with simple acyclic sulfated alcohols, diols and triols in the ligand shell. In addition, the influence of the particle size and the thickness of the hydrophobic organic shell were systematically investigated.

Ziel der Dissertation war die Entwicklung neuer Synthesewege zu C2-verzweigten Aminozuckern und verwandten Kohlenhydratmimetika als potentielle Liganden multivalenter Konjugate. Im ersten Teil der Arbeit wurden, inspiriert durch eine kürzlich von Al-Harrasi beschriebene Synthese enantiomerenreiner Aminopyrane, verschieden konfigurierte C2-verzweigte 4-Aminozucker durch eine Lewis-Säure-induzierte Umlagerung von 1,2-Oxazinen hergestellt. Die benötigten 1,3-dioxolanyl-substituierten 1,2-Oxazine wurden durch eine stereodivergente Addition lithiierter Alkoxyallene an chirale Aldonitrone erhalten. Nach Einführen einer Phenylthio-Gruppe wurden die 1,2-Oxazine unter Lewis-aciden Bedingungen in bicyclische Ketone umgelagert, die unmittelbar in Glycosylierungsreaktionen eingesetzt werden konnten. Reagieren der Umlagerungsprodukte mit Methanol lieferte intern geschützte Aminozucker- Equivalente, die anschließend in Methylglycoside mit verschiedenen Konfigurationen übergeführt wurden. Diese Transformation wurde in drei einfachen reduktiven Schritten realisiert: Reduzieren der Carbonylgruppe, Debenzylierung und Spaltung der N,O-Bindung. Die Umlagerungsprodukte wurden auch als Glycosyldonor-Äquivalente eingesetzt. Glycosylierungsreaktionen mit verschiedenen Alkoholen ermöglichte die Synthese von Intermediaten, die in Di- und Trisaccharide, sowie eine glycosylierte Aminosäure und ein bivalentes Aminozuckerderivat übergeführt wurden. Im zweiten Teil der Arbeit wurden 1,3 -dioxolanyl-substituierte Hydroxylaminderivate hergestellt und in funktionalisierte Dihydropyrane cyclisiert, die anschließend in neuartige (multivalente) Kohlenhydratmimetika und andere enantiomerenreine Heterocyclen übergeführt wurden. Die linearen Vorläufer wurden in Analogie zu der Synthese der 1,2-Oxazine durch eine stereodivergente Addition lithiierter Enolether an chirale Aldonitrone erhalten. Anschließend lieferte eine Lewis-Säure- vermittelte Cyclisierung Dihydropyrane als Schlüsselintermediate für die Synthese enantiomerenreiner Aminopyrane, die als Mimetika verschieden substituierter und konfigurierter 4-Aminozucker aufgefasst werden können. Einige der in dieser Arbeit synthetisierten Verbindungen zeigten als sulfatierte Liganden von Goldnanopartikeln interessante Eigenschaften in ihrer Bindung an Selektine., Aim of the dissertation was the development of new synthetic routes to C2-branched 4-amino sugars and related carbohydrate mimetics as potential ligands for multivalent conjugates. In the first part of the thesis, inspired by a recently described synthesis of enantiopure aminopyrans by Al-Harrasi, differently configured C2-branched 4-amino sugars have been prepared by a Lewis-acid promoted rearrangement of 1,2-oxazines. The required 1,3 -dioxolanyl-substituted 1,2-oxazines have been obtained by stereodivergent addition of lithiated alkoxyallenes to a chiral aldonitrone. After introduction of a phenylthio moiety, the 1,2-oxazines were treated with Lewis acid leading to the stereoselective formation of bicyclic ketones that could directly be used in glycosidation reactions. Reaction with methanol provided internally protected amino sugar equivalents that have been transformed into methyl glycosides with different configurations. This transformation was accomplished in simple reductive steps consisting of a reduction of the carbonyl group, debenzylation and cleavage of the N,O-bond. The rearrangement products have also been used as glycosyl donor equivalents. Glycosylation reactions with different alcohols allowed the synthesis of coupled products that have been transformed into di- and trisaccharides, as well as into a glycosylated amino acid and a divalent aminosugar derivative. In the second part of the thesis, 1,3-dioxolanyl-substituted hydroxylamine derivatives have been prepared and cyclised into functionalized dihydropyrans that have been transformed into novel (multivalent) carbohydrate mimetics and other enantiomerically pure heterocycles. The required linear precursors were prepared, by analogy to the synthesis of 1,2-oxazines, via stereodivergent addition of lithiated enol ethers to carbohydrate-derived nitrones. A subsequent Lewis acid mediated cyclisation delivered dihydropyrans as crucial precursors for the synthesis of enantiopure aminopyrans that can be regarded as mimetics of differently substituted and configured 4-amino sugars. Several of the prepared compounds showed interesting properties in their binding to selectins when presented in a multivalent fashion as sulfated ligands of gold nanoparticles.