Development of Novel Technologies for Parallel Synthesis of Glycans and Glycopeptides for Microarray Generation

Carbohydrates are ubiquitous biopolymers mediating fundamental biological processes such as cell–cell interaction, immune response, and host-pathogen interactions. They can selectively interact with each other (carbohydrate-carbohydrate interactions), with glycan binding proteins (carbohydrate-protein interactions), and pathogens. In order to study these interactions, glycan microarray technology has been developed, becoming nowadays a primary analytical tool. Glycan arrays enable high-throughput screening of these interactions in a fast manner, to identify new drug candidates, biomarkers, or vaccine candidates. The main strategy to generate glycan arrays is the immobilization of pure and structurally defined oligosaccharides and glycomimetics on solid surfaces. The required oligosaccharides can either be isolated from natural sources or synthesized enzymatically and/or chemically. Automated chemical synthesis of oligosaccharides has become a streamline process, revolutionizing and simplifying the synthesis of linear and branched natural and unnatural glycan collections. In contrast to other common biopolymers, there is no method available for parallel oligosaccharide synthesis. In this work, different cost- and time-efficient approaches have been developed for parallel synthesis of oligosaccharides and glycopeptides on solid supports.Kohlenhydrate sind ubiquitäre Biopolymeren, die fundamentale biologische Prozesse wie Zell-Zell-Interaktionen, Immunreaktionen und Interaktionen zwischen Wirt und Erreger vermitteln. Sie können selektiv untereinander (Kohlenhydrat-Kohlenhydrat- Wechselwirkungen) wechselwirken, mit Glykan-bindenden Proteinen (Kohlenhydrat-Protein- Wechselwirkungen) und mit Pathogenen wechselwirken. Um diese Interaktionen zu untersuchen, wurde die Glykan-Microarray-Technologie entwickelt, die heute zu einem wichtigen Analyseinstrument geworden ist. Glykan-Arrays ermöglichen ein schnelles Hochdurchsatzscreening dieser Wechselwirkungen, um neue Arzneimittelkandidaten, Biomarker oder Impfstoffkandidaten zu identifizieren. Die wichtigste Strategie zur Herstellung von Glykan-Arrays ist die Immobilisierung von reinen und strukturell definierten Oligosacchariden und Glykomimetika auf planaren Oberflächen. Die benötigten Oligosaccharide können entweder aus natürlichen Quellen isoliert oder enzymatisch und/oder chemisch synthetisiert werden. Die automatisierte chemische Synthese von Oligosacchariden hat sich zu einem Standardverfahren entwickelt, das die Synthese von linearen und verzweigten natürlichen und unnatürlichen Glykanen revolutioniert und erheblich vereinfacht hat. Im Gegensatz zu anderen gängigen Biopolymeren gibt es jedoch bisher keine Methode zur parallelen Oligosaccharidsynthese. In dieser Arbeit wurden verschiedene kosten- und zeiteffiziente Ansätze für die parallele Festphasensynthese von Oligosacchariden und Glykopeptiden entwickelt

Multivalent glycan arrays

Glycan microarrays have become a powerful technology to study biological processes, such as cell–cell interaction, inflammation, and infections. Yet, several challenges, especially in multivalent display, remain. In this introductory lecture we discuss the state-of-the-art glycan microarray technology, with emphasis on novel approaches to access collections of pure glycans and their immobilization on surfaces. Future directions to mimic the natural glycan presentation on an array format, as well as in situ generation of combinatorial glycan collections, are discussed

VaporLIFT: On-Chip Chemical Synthesis of Glycan Microarrays

Laser-induced forward transfer (LIFT) of polymers is a versatile printing method for parallel in situ synthesis of peptides on microarrays. Chemical building blocks embedded in a polymer matrix are transferred and coupled in a desired pattern to a surface, generating peptides on microarrays by repetitive in situ solid-phase synthesis steps. To date, the approach is limited to simple, heat induced chemical reactions. The VaporLIFT method, disclosed here, combines LIFT with chemical vapor glycosylation to rapidly generate glycans on microarray surfaces while maintaining inert, low temperature conditions required for glycosylations. Process design and parameter optimization enables the synthesis of a collection of glycans at defined positions on a glass surface. The synthetic structures are detected by mass spectrometry, fluorescently labeled glycan-binding proteins, and covalent staining with fluorescent dyes. VaporLIFT is ideal for parallel screening of other chemical reactions, that require inert and well-defined reaction conditions

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

Complications of chronic liver disease

Children with chronic liver disease (CLD) need a head to toe approach and an early suspicion of multi organ involvement. Nutritional assessment and management is the cornerstone of management. Consider immune dysfunction in everyday treatment decisions. Consider early heart-lung-brain involvement in transplant evaluation

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

Probing Multivalent Carbohydrate-Protein Interactions With On-Chip Synthesized Glycopeptides Using Different Functionalized Surfaces

Multivalent ligand–protein interactions are a commonly employed approach by nature in many biological processes. Single glycan–protein interactions are often weak, but their affinity and specificity can be drastically enhanced by engaging multiple binding sites. Microarray technology allows for quick, parallel screening of such interactions. Yet, current glycan microarray methodologies usually neglect defined multivalent presentation. Our laser-based array technology allows for a flexible, cost-efficient, and rapid in situ chemical synthesis of peptide scaffolds directly on functionalized glass slides. Using copper(I)-catalyzed azide–alkyne cycloaddition, different monomer sugar azides were attached to the scaffolds, resulting in spatially defined multivalent glycopeptides on the solid support. Studying their interaction with several different lectins showed that not only the spatially defined sugar presentation, but also the surface functionalization and wettability, as well as accessibility and flexibility, play an essential role in such interactions. Therefore, different commercially available functionalized glass slides were equipped with a polyethylene glycol (PEG) linker to demonstrate its effect on glycan–lectin interactions. Moreover, different monomer sugar azides with and without an additional PEG-spacer were attached to the peptide scaffold to increase flexibility and thereby improve binding affinity. A variety of fluorescently labeled lectins were probed, indicating that different lectin–glycan pairs require different surface functionalization and spacers for enhanced binding. This approach allows for rapid screening and evaluation of spacing-, density-, ligand and surface-dependent parameters, to find optimal lectin binders.BMBF, 13XP5050A, Erforschung einer neuen Methode für die Herstellung von hochdichten Molekülbibliotheken (cLIFT

On‐Chip Neo‐Glycopeptide Synthesis for Multivalent Glycan Presentation

Single glycan–protein interactions are often weak, such that glycan binding partners commonly utilize multiple, spatially defined binding sites to enhance binding avidity and specificity. Current array technologies usually neglect defined multivalent display. Laser‐based array synthesis technology allows for flexible and rapid on‐surface synthesis of different peptides. By combining this technique with click chemistry, neo‐glycopeptides were produced directly on a functionalized glass slide in the microarray format. Density and spatial distribution of carbohydrates can be tuned, resulting in well‐defined glycan structures for multivalent display. The two lectins concanavalin A and langerin were probed with different glycans on multivalent scaffolds, revealing strong spacing‐, density‐, and ligand‐dependent binding. In addition, we could also measure the surface dissociation constant. This approach allows for a rapid generation, screening, and optimization of a multitude of multivalent scaffolds for glycan binding

On-Chip Neo-Glycopeptide Synthesis for Multivalent Glycan Presentation

Single glycan-protein interactions are often weak, such that glycan binding partners commonly utilize multiple, spatially defined binding sites to enhance binding avidity and specificity. Current array technologies usually neglect defined multivalent display. Laser-based array synthesis technology allows for flexible and rapid on-surface synthesis of different peptides. By combining this technique with click chemistry, neo-glycopeptides were produced directly on a functionalized glass slide in the microarray format. Density and spatial distribution of carbohydrates can be tuned, resulting in well-defined glycan structures for multivalent display. The two lectins concanavalin A and langerin were probed with different glycans on multivalent scaffolds, revealing strong spacing-, density-, and ligand-dependent binding. In addition, we could also measure the surface dissociation constant. This approach allows for a rapid generation, screening, and optimization of a multitude of multivalent scaffolds for glycan binding