Automated glycan assembly of xyloglucan oligosaccharides

We report the automated glycan assembly of oligosaccharide fragments related to the hemicellulose xyloglucan (XG). Iterative addition of monosaccharide and disaccharide building blocks to a solid support provided seven cellulose and xyloglucan fragments including XXGG- and XXXG-type oligosaccharides

Multivalent interaction and selectivities in selectin binding of functionalized gold colloids decorated with carbohydrate mimetics

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

Active Site Mapping of Xylan-Deconstructing Enzymes with Arabinoxylan Oligosaccharides Produced by Automated Glycan Assembly

Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as the production of biofuel. To determine the substrate specificities of these enzymes, we prepared a collection of complex xylan oligosaccharides by automated glycan assembly. Seven differentially protected building blocks provided the basis for the modular assembly of 2-substituted, 3-substituted, and 2-/3-substituted arabino- and glucuronoxylan oligosaccharides. Elongation of the xylan backbone relied on iterative additions of C4-fluorenylmethoxylcarbonyl (Fmoc) protected xylose building blocks to a linker-functionalized resin. Arabinofuranose and glucuronic acid residues have been selectively attached to the backbone using fully orthogonal 2-(methyl)naphthyl (Nap) and 2-(azidomethyl)benzoyl (Azmb) protecting groups at the C2 and C3 hydroxyls of the xylose building blocks. The arabinoxylan oligosaccharides are excellent tools to map the active site of glycosyl hydrolases involved in xylan deconstruction. The substrate specificities of several xylanases and arabinofuranosidases were determined by analyzing the digestion products after incubation of the oligosaccharides with glycosyl hydrolases.Fil: Senf, Deborah. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; AlemaniaFil: Ruprecht, Colin. Max Planck Institut für Kolloid und Grenzflächenforschung; AlemaniaFil: de Kruijff, Goswinus H. M.. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; Alemania. University Mainz. Institute of Institute of Organic Chemistry, Johannes Gutenberg; AlemaniaFil: Simonetti, Sebastián Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. Max Planck Institut für Kolloid und Grenzflächenforschung; AlemaniaFil: Schuhmacher, Frank. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; AlemaniaFil: Seeberger, Peter H.. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; AlemaniaFil: Pfrengle, Fabian. Max Planck Institut für Kolloid und Grenzflächenforschung; Alemania. Freie Universität; Alemani

Cracking the “Sugar Code”: A Snapshot of N- and O-Glycosylation Pathways and Functions in Plants Cells

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; SuizaFil: Seifert, Georg. University of Natural Resources and Life Sciences; SuizaFil: Doblin, Monika S.. La Trobe University; AustraliaFil: Johnson, Kim L.. La Trobe University; AustraliaFil: Ruprecht, Colin. University of Natural Resources and Life Sciences; SuizaFil: Pfrengle, Fabian. University of Natural Resources and Life Sciences; SuizaFil: Bacic, Antony. La Trobe University; AustraliaFil: 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; Argentin

Arabinoxylan-Oligosaccharides Act as Damage Associated Molecular Patterns in Plants Regulating Disease Resistance

[EN] Immune responses in plants can be triggered by damage/microbe-associated molecular patterns (DAMPs/MAMPs) upon recognition by plant pattern recognition receptors (PRRs). DAMPs are signaling molecules synthesized by plants or released from host cellular structures (e.g., plant cell walls) upon pathogen infection or wounding. Despite the hypothesized important role of plant cell wall-derived DAMPs in plant-pathogen interactions, a very limited number of these DAMPs are well characterized. Recent work demonstrated that pectin-enriched cell wall fractions extracted from the cell wall mutant impaired in Arabidopsis Response Regulator 6 (arr6), that showed altered disease resistance to several pathogens, triggered more intense immune responses than those activated by similar cell wall fractions from wild-type plants. It was hypothesized that arr6 cell wall fractions could be differentially enriched in DAMPs. In this work, we describe the characterization of the previous immune-active fractions of arr6 showing the highest triggering capacities upon further fractionation by chromatographic means. These analyses pointed to a role of pentose-based oligosaccharides triggering plant immune responses. The characterization of several pentose-based oligosaccharide structures revealed that b-1,4-xylooligosaccharides of specific degrees of polymerization and carrying arabinose decorations are sensed as DAMPs by plants. Moreover, the pentasaccharide 33-a-L-arabinofuranosyl-xylotetraose (XA3XX) was found as a highly active DAMP structure triggering strong immune responses in Arabidopsis thaliana and enhancing crop disease resistance.SIThis work was supported by grants IND2017/BIO-7800 of the Comunidad de Madrid Regional Government.This work has been also financially supported by the “Severo Ochoa Programme for Centres of Excellence in R&D” from the Agencia Estatal de Investigación of Spain (grant SEV-2016- 0672 (2017-2021) to the CBGP). In the frame of this program HM was supported with a postdoctoral fellow. DR was the recipient of an Industrial PhD Fellow (IND2017/BIO-7800) and IH was the recipient of an PhD FPU fellow from the Spanish Ministry of Education (FPU16/07118). FP thanks the Max Planck Society and the German Research Foundation (DFG, Emmy Noether program PF850/1-1 to FP) for financial support

Synthesis and application of branched type II arabinogalactans

The synthesis of linear and (1 → 6)-branched β-(1 → 3)-d-galactans, structures found in plant arabinogalactan proteins (AGPs), is described. The synthetic strategy relies on iterative couplings of monosaccharide and disaccharide thioglycoside donors, followed by a late-stage glycosylation of heptagalactan backbone acceptors to introduce branching. A key finding from the synthetic study was the need to match protective groups in order to tune reactivity and ensure selectivity during the assembly. Carbohydrate microarrays were generated to enable the detailed epitope mapping of two monoclonal antibodies known to recognize AGPs: JIM16 and JIM133

Synthesis of C-branched amino sugars and related carbohydrate mimetics as potential ligands of multivalent conjugates

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

Addition of lithiated enol ethers to nitrones and subsequent Lewis acid induced cyclizations to enantiopure 3,6-dihydro-2H-pyrans – an approach to carbohydrate mimetics

A stereodivergent synthesis of enantiopure 3,6-dihydro-2H-pyrans is presented. The addition of lithiated enol ethers to carbohydrate-derived nitrones afforded syn- or anti-configured hydroxylamine derivatives 4a–d that were cyclized under Lewis acidic conditions to yield functionalized dihydropyrans cis- or trans-5a–d containing an enol ether moiety. This functional group was employed for a variety of subsequent reactions such as dihydroxylation or bromination. Bicyclic enol ether 19 was oxidatively cleaved to provide the highly functionalized ten-membered ring lactone 20. The synthesized enantiopure aminopyrans 24, 26, 28 and 30 can be regarded as carbohydrate mimetics. Trimeric versions of 24 and 28 were constructed via their attachment to a tricarboxylic acid core

Determining Substrate Specificities of β1,4-Endogalactanases Using Plant Arabinogalactan Oligosaccharides Synthesized by Automated Glycan Assembly

Pectin is a structurally complex plant polysaccharide with many industrial applications in food products. The structural elucidation of pectin is aided by digestion assays with glycosyl hydrolases. We report the automated glycan assembly of oligosaccharides related to the arabinogalactan side chains of pectin as novel biochemical tools to determine the substrate specificities of endogalactanases. Analysis of the digestion products revealed different requirements for the lengths and arabinose substitution pattern of the oligosaccharides to be recognized and hydrolyzed by the galactanases