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

Metal-free photoanodes for C–H functionalization

Organic semiconductors, such as carbon nitride, when employed as powders, show attractive photocatalytic properties, but their photoelectrochemical performance suffers from low charge transport capability, charge carrier recombination, and self-oxidation. High film-substrate affinity and well-designed heterojunction structures may address these issues, achieved through advanced film generation techniques. Here, we introduce a spin coating pretreatment of a conductive substrate with a multipurpose polymer and a supramolecular precursor, followed by chemical vapor deposition for the synthesis of dual-layer carbon nitride photoelectrodes. These photoelectrodes are composed of a porous microtubular top layer and an interlayer between the porous film and the conductive substrate. The polymer improves the polymerization degree of carbon nitride and introduces C-C bonds to increase its electrical conductivity. These carbon nitride photoelectrodes exhibit state-of-the-art photoelectrochemical performance and achieve high yield in C-H functionalization. This carbon nitride photoelectrode synthesis strategy may be readily adapted to other reported processes to optimize their performance

VaporSPOT: Parallel Synthesis of Oligosaccharides on Membranes

Automated chemical synthesis has revolutionized synthetic access to biopolymers in terms of simplicity and speed. While automated oligosaccharide synthesis has become faster and more versatile, the parallel synthesis of oligosaccharides is not yet possible. Here, a chemical vapor glycosylation strategy (VaporSPOT) is described that enables the simultaneous synthesis of oligosaccharides on a cellulose membrane solid support. Different linkers allow for flexible and straightforward cleavage, purification, and characterization of the target oligosaccharides. This method is the basis for the development of parallel automated glycan synthesis platforms

Automated Laser‐Transfer Synthesis of High‐Density Microarrays for Infectious Disease Screening

Laser-induced forward transfer (LIFT) is a rapid laser-patterning technique for high-throughput combinatorial synthesis directly on glass slides. A lack of automation and precision limits LIFT applications to simple proof-of-concept syntheses of fewer than 100 compounds. Here, an automated synthesis instrument is reported that combines laser transfer and robotics for parallel synthesis in a microarray format with up to 10 000 individual reactions cm−2. An optimized pipeline for amide bond formation is the basis for preparing complex peptide microarrays with thousands of different sequences in high yield with high reproducibility. The resulting peptide arrays are of higher quality than commercial peptide arrays. More than 4800 15-residue peptides resembling the entire Ebola virus proteome on a microarray are synthesized to study the antibody response of an Ebola virus infection survivor. Known and unknown epitopes that serve now as a basis for Ebola diagnostic development are identified. The versatility and precision of the synthesizer is demonstrated by in situ synthesis of fluorescent molecules via Schiff base reaction and multi-step patterning of precisely definable amounts of fluorophores. This automated laser transfer synthesis approach opens new avenues for high-throughput chemical synthesis and biological screening

Automatisierte Synthese von Hemizellulose-Oligosacchariden aus der pflanzlichen Zellwand

The plant cell wall is the outer-layer of the cell and controls its volume and shape. It plays an important structural role and is involved in the plant’s immune response against pathogens. Plant cell walls are composed of a mixture of simple and complex biopolymers that are mainly comprised of highly complex polysaccharides. Well-defined oligosaccharides related to plant cell wall polysaccharides are fundamental molecular tools for studying cell wall structure and biosynthesis, but are difficult to obtain from natural sources. The chemical synthesis of plant oligosaccharide libraries is thus a promising approach for advancing cell wall research. Automated glycan assembly is a powerful emerging technique for the solid-phase synthesis of oligosaccharides. Through the iterative addition of different building blocks (BB) to a functionalized solid support, glycans of different lengths and branching patterns can be produced. In this work, automated glycan assembly has been used to obtain well-defined oligosaccharide fragments of the cell wall polysaccharides xyloglucan (XG) and mixed-linkage glucan (MLG). These compounds were applied for the characterization of cell wall glycan-directed monoclonal antibodies and cell wall-modifying enzymes. In Chapter 2, which constitutes the main part of this thesis, the synthesis of XG oligosaccharides is described. XGs are the main hemicellulosic polysaccharides in higher plants. They are made of a cellulosic backbone highly decorated with xylose residues that can be further substituted with galactose and fucose. Initially, we tested the possibility to use exclusively monosaccharide BBs for the assembly of the target oligosaccharides. However, this approach did not provide good -stereoselectivity when glycosylating backbone glucose residues with xylose. Therefore, a disaccharide BB carrying a pre-installed -glycosidic linkage between glucose and xylose was synthesized. Using this disaccharide BB a library of XG oligosaccharides was successful prepared. To synthesize galactosylated XG oligosaccharides p-methoxybenzyl was used at the C2-position of the xylose. This represents the first example for the use of this temporary protecting group in automated glycan assembly. In collaboration with Dr. Ruprecht, the synthetic XG oligosaccharides were used for the characterization of monoclonal antibodies (mAbs) that recognize cell wall polysaccharides and for determining the binding specificities of xyloglucan endotransglycosylases (XETs), important enzymes involved in remodeling the cell wall during cell growth. In chapter 3, a library of MLG oligosaccharides was synthesized by automated glycan assembly. MLGs are an important class of hemicellulose polymers present in grass and cereals. They are constructed from a backbone of cellulosic oligosaccharide stretches randomly connected by -1,3-glycosidic linkages. Using two differently protected glucose building blocks, a library of MLG oligosaccharides up to an octasaccharide was synthesized. MLGs are degraded by an enzyme named lichenase, which can be used to characterize the composition of MLG polysaccharides and is used in industrial processes such as brewery and animal feed production. The synthetic MLG oligosaccharides represented ideal substrates to investigate the substrate specificity of lichenase, and recent reconsiderations on the specificity of lichenase were confirmed.Die Pflanzenzellwand ist die äußere Zellschicht und bestimmt ihr Volumen und ihre Form. Sie hat eine wichtige strukturgebende Rolle und ist in die Immunreaktion gegen Krankheitserreger involviert. Pflanzenzellwände bestehen aus einer Vielzahl einfacher und komplexer Biopolymere, die hauptsächlich hochkomplexe Polysaccharide beinhalten. Kurze Oligosaccharid-Fragmente von Polysacchariden aus der Pflanzenzellwand dienen als fundamentale molekulare Hilfsmittel, um die Struktur und Biosynthese von Pflanzenzellwänden zu untersuchen, wobei strukturell definierte und reine Oligosaccharide nur schwierig aus natürlichen Quellen zu gewinnen sind. Die chemische Synthese von Pflanzenoligosaccharid-Bibliotheken ist deshalb ein vielverspechender Ansatz, um die Forschung an der Pflanzenzellwand voranzubringen. Automatische Oligosaccharidsynthese ist eine leistungsfähige Technik, um Oligosaccharide an der Festphase zu synthetisieren. Durch die iterative Zugabe von verschiedenen Bausteinen zu einem funktionalisierten festen Trägermaterial können Glykane mit verschiedenen Längen und Verzweigungen synthetisiert werden. In dieser Arbeit wurde automatische Festphasensynthese benutzt, um definierte Oliogosaccharid-Fragmente der Zellwandpolysaccharide Xyloglukan (XG) und Glukane mit gemischten Verknüpfungen (MLG) zu erhalten. Diese Verbindungen fanden Anwendung in der Charakterisierung von monoklonalen Antikörpern, die Zellwandpolysaccharide erkennen, und Enzymen, die die Zellwand modifizieren. Kapitel 2, welches den Hauptteil dieser Arbeit umfasst, beschreibt die Synthese von XG-Oligosacchariden. XG sind die am häufigsten vorkommenden hemicellulosischen Polysaccharide in vaskulären Pflanzen. Sie bestehen aus einem Celluloserückgrat, welches mit Xyloseeinheiten dekoriert ist, die wiederum mit Galaktose und Fukose substituiert sein können. Zuerst wurde die Möglichkeit getestet, die Zielstrukturen ausschließlich aus Monosaccharid- Bausteinen zu synthetisieren. Bei diesem Ansatz wurde allerdings keine gute -Stereoselektivität in der Glykosylierung von Glukoseeinheiten des Rückgrats mit Xylose erzielt. Deshalb wurde ein Disaccharid-Baustein genutzt, in dem die -glykosidische Bindung zwischen Glukose und Xylose bereits vorinstalliert war. Durch die Verwendung dieses Disaccharid-Bausteins wurde eine Bibliothek von XG-Oligosacchariden erfolgreich synthetisiert. Um Galaktose-substituierten XG Oligosaccharide zu synthetisieren, wurde p-Methoxybenzyl in der C2-Position von Xylose genutzt. Dies ist das erste Beispiel für die Verwendung dieser temporären Schutzgruppe in der automatischen Oligosaccharid- Festphasensynthese. In einer Kollaboration mit Dr. Ruprecht wurden die synthetisierten XG-Oligosaccharide für die Charakterisierung von monoklonalen Antikörpern (mAbs), die Zellwandpolysaccharide erkennen, genutzt. Außerdem die wurden die Bindungsspezifitäten von Xyloglucan-Endotransglycosylasen (XET) bestimmt, die wichtige Enzyme, der Zellwand während des Zellwachstums sind in der Reorganisation. In Kapitel 3 wird die Festphasensynthese einer Bibliothek von MLG-Oligosacchariden beschrieben. MLG sind eine wichtige Klasse von Hemicellulosepolymeren in Gräsern und Getreide. Sie bestehen aus einem Rückgrat von Cellulose-Oligosaccharidabschnitten, die unregelmäßig durch -1,3-glykosidische Bindungen verknüpft sind. Indem zwei unterschiedlich geschützte Glukosebausteine verwendet wurden, konnte eine Bibliothek von MLG- Oligosacchariden bis zur Länge eines Oktasaccharids synthetisiert werden. MLG werden durch das Enzym Lichenase abgebaut, welches für die Charakterisierung der strukturellen Zusammensetzung von MLG-Polysacchariden genutzt werden kann, und in industriellen Prozessen wie dem Brauwesen und in der Futtermittelproduktion genutzt wird. Die synthetischen MLG-Oligosaccharide stellten ideale Substrate dar, um die Substratspezifitäten von Lichenase zu untersuchen und um jüngste Neubewertungen der Spezifität von Lichenase zu bestätigen

Automated glycan assembly of peptidoglycan backbone fragments

We report the automated glycan assembly (AGA) of different oligosaccharide fragments of the bacterial peptidoglycan (PGN) backbone. Iterative addition on a solid support of an acetyl glucosamine and a new muramic acid building block is followed by cleavage from the solid support and final deprotection providing 10 oligosaccharides up to six units

Automated Laser-Transfer Synthesis of High-Density Microarrays for Infectious Disease Screening

Laser-induced forward transfer (LIFT) is a rapid laser-patterning technique for high-throughput combinatorial synthesis directly on glass slides. A lack of automation and precision limits LIFT applications to simple proof-of-concept syntheses of fewer than 100 compounds. Here, an automated synthesis instrument is reported that combines laser transfer and robotics for parallel synthesis in a microarray format with up to 10 000 individual reactions cm−2. An optimized pipeline for amide bond formation is the basis for preparing complex peptide microarrays with thousands of different sequences in high yield with high reproducibility. The resulting peptide arrays are of higher quality than commercial peptide arrays. More than 4800 15-residue peptides resembling the entire Ebola virus proteome on a microarray are synthesized to study the antibody response of an Ebola virus infection survivor. Known and unknown epitopes that serve now as a basis for Ebola diagnostic development are identified. The versatility and precision of the synthesizer is demonstrated by in situ synthesis of fluorescent molecules via Schiff base reaction and multi-step patterning of precisely definable amounts of fluorophores. This automated laser transfer synthesis approach opens new avenues for high-throughput chemical synthesis and biological screening

Metal-free photoanodes for C–H functionalization

Abstract Organic semiconductors, such as carbon nitride, when employed as powders, show attractive photocatalytic properties, but their photoelectrochemical performance suffers from low charge transport capability, charge carrier recombination, and self-oxidation. High film-substrate affinity and well-designed heterojunction structures may address these issues, achieved through advanced film generation techniques. Here, we introduce a spin coating pretreatment of a conductive substrate with a multipurpose polymer and a supramolecular precursor, followed by chemical vapor deposition for the synthesis of dual-layer carbon nitride photoelectrodes. These photoelectrodes are composed of a porous microtubular top layer and an interlayer between the porous film and the conductive substrate. The polymer improves the polymerization degree of carbon nitride and introduces C-C bonds to increase its electrical conductivity. These carbon nitride photoelectrodes exhibit state-of-the-art photoelectrochemical performance and achieve high yield in C-H functionalization. This carbon nitride photoelectrode synthesis strategy may be readily adapted to other reported processes to optimize their performance