Photo-labile BODIPY protecting groups for glycan synthesis

Protective groups that can be selectively removed under mild conditions are an essential aspect of carbohydrate chemistry. Groups that can be selectively removed by visible light are particularly attractive because carbohydrates are transparent to visible light. Here, different BODIPY protecting groups were explored for their utility during glycan synthesis. A BODIPY group bearing a boron difluoride unit is stable during glycosylations but can be cleaved with green light as illustrated by the assembly of a trisaccharide

The Influence of the Electron Density in Acyl Protecting Groups on the Selectivity of Galactose Formation

The stereoselective formation of 1,2-cis-glycosidic bonds is a major bottleneck in the synthesis of carbohydrates. We here investigate how the electron density in acyl protecting groups influences the stereoselectivity by fine-tuning the efficiency of remote participation. Electron-rich C4-pivaloylated galactose building blocks show an unprecedented α-selectivity. The trifluoroacetylated counterpart with electron-withdrawing groups, on the other hand, exhibits a lower selectivity. Cryogenic infrared spectroscopy in helium nanodroplets and density functional theory calculations revealed the existence of dioxolenium-type intermediates for this reaction, which suggests that remote participation of the pivaloyl protecting group is the origin of the high α-selectivity of the pivaloylated building blocks. According to these findings, an α-selective galactose building block for glycosynthesis is developed based on rational considerations and is subsequently employed in automated glycan assembly exhibiting complete stereoselectivity. Based on the obtained selectivities in the glycosylation reactions and the results from infrared spectroscopy and density functional theory, we suggest a mechanism by which these reactions could proceed

Thiol-Mediated Uptake of a Cysteine-Containing Nanobody for Anticancer Drug Delivery

The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that specifically recognizes B cell lymphoma and breast cancer in a thiol-dependent manner. CB2 binding strictly requires the presence of a nonconserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 via thiol-mediated endocytosis which can be exploited to deliver cytotoxic agents. CB2 internalization combined with functionalization forms the basis for a wide range of diagnostic and therapeutic applications, rendering thiol-reactive nanobodies promising tools for targeting cancer

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

Neue Hilfsmittel für Stereoselektivität und Orthogonalität in der Automatischen Glykan-Festphasensynthese für die Konjugat-Impfstoffentwicklung

Carbohydrates represent the most diverse and prevalent class of biomolecules in nature and are essential in a wide variety of biological processes. Antigenic glycans at the surface of microbes have the potential to be developed into glycoconjugate vaccines. The Automated Glycan Assembly (AGA) approach can provide those glycans rapidly in a solid-supported synthesis. Challenges in the synthesis of glycans by AGA arise from their complex structures, which require both strict regio- and stereocontrol, but also from technical limitations of the current AGA synthesizer. The goal of my research was to expand capabilities of AGA by introducing new orthogonal protecting groups and developing new strategies for 1,2-cis stereoselective glycosylations to give access to materials for research on infectious diseases. The first part of this dissertation focuses on orthogonality and the expansion of the protecting group portfolio in AGA (Chapter 3). The introduction of microwave-assistance to the AGA synthesizer enabled a larger temperature range (-40 to 100 °C) and the development of a mannose building block with four orthogonal protecting groups. When utilized in the microwave-assisted platform, this building block allowed 1) on-resin global deprotection and 2) the synthesis of glycan structures with up to four branches. In parallel, photo-labile 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) protecting groups were developed. A BODIPY protecting group bearing a boron difluoride unit provided the optimal compromise between glycosylation-stability and photo-lability by green light irradiation. This protecting group enabled a solution-phase consecutive glycan assembly without intermediate purification up to a trisaccharide. The second part of this dissertation describes studies on factors that influence the stereoselectivity for 1,2-cis glycosylations (Chapter 4). The influence of positional and electronic effects in acyl and ether groups on the efficacy for remote participation were investigated. An array of uniquely substituted building blocks was synthesized followed by the evaluation in model glycosylations and cold-ion IR spectroscopy. The obtained mechanistic insights helped to design building blocks for the formation of α-(1→3)-galactosidic linkages. In Chapter 5, the new knowledge and developed methods were utilized to synthesize a conjugation-ready glycan library of the Porphorymonas gingivalis lipopolysaccharide (LPS). P. gingivalis has an immense social and medical impact as it is the major cause of chronical periodontitis and is associated with several systemic diseases. The improvement of our basic understanding of interactions between human immune system and LPS may allow for treatment and prevention strategies. Twelve LPS fragments of P. gingivalis were screened for IgG and IgA binding in human saliva and serum using glycan microarray studies, which enabled the identification of 5-amino-pentyl α-D-Galp-(1→6)-α-D-Glcp-(1→4)-α-L-Rhap-(1→3)-2-β-D-GalNAc as a potential glycoconjugate vaccine candidate against P. gingivalis.Kohlenhydrate sind die vielfältigsten und verbreitetsten Biomoleküle in der Natur und sind essentiell für eine Vielzahl an Abläufen in Organsimen. Oberflächen-Glykane von Pathogenen können als Antigene dienen und haben Potential für die Entwicklung von Glykokonjugatimpfstoffen. Die automatische Festphasensynthese (engl. Automated Glycan Assembly, AGA) kann diese Glykane schnell bereitstellen. Syntheseschwierigkeiten von Glykanen im AGA kommen vor allem durch ihre Komplexität, die strenge Einhaltung von Regio- und Stereokontrolle voraussetzt, aber auch durch technische Grenzen der AGA-Synthesemaschinen zustande. Das allgemeine Ziel meiner Forschung war die Verbesserung des AGA-Repertoire durch die Einführung von orthogonalen Schutzgruppen und die Entwicklung von neuen Strategien für 1,2-cis-stereoselektive Glykosylierungen. Der erste Teil dieser Dissertation fokussiert sich auf die Orthogonalität und die Erweiterung des Schutzgruppen-Portfolios (3. Kapitel). Die Einführung von Mikrowellen-assistenz in die AGA-Synthesemaschine ermöglichte eine größere Temperaturspanne (-40 bis 100 °C) und die Entwicklung eines komplett orthogonalen Mannose-Bausteines. Dieser Baustein erlaubte zum einen die globale Entschützung am Harz und zum anderen den Aufbau von Strukturen mit bis zu vier Verzweigungen. Außerdem wurden photo-labile BODIPY-Schutzgruppen entwickelt. Eine BODIPY-Schutzgruppe mit einer Bordifluorid-Einheit bot einen guten Kompromiss zwischen Glykosylierungs-Stabilität und Photoreaktivität bei grünem Licht. Dies ermöglichte eine fortlaufende Glykan-Herstellung ohne intermediäre Aufreinigungen bis zum Trisaccharid. Der zweite Teil der Dissertation beschreibt Studien zu Einflussfaktoren auf die Stereoselektivität von 1,2-cis-Glykosylierungen (4. Kapitel). Es wurden elektronische Effekte von Acyl- und Ether-Gruppen und der Effekt ihrer Position auf die Effizienz von Fern-Beteiligung untersucht. Dies wurde durch die Synthese einer Vielzahl an substituierten Bausteinen, ihrer Evaluation in Test-Glykosylierungen und Kalt-Ionen-Spektroskopie verwirklicht. Die so erhaltenen mechanistischen Einblicke halfen dabei, Bausteine für die Bildung von α-(1→3)-galactosidischen Bindungen zu entwickeln. Das erlernte Wissen und die entwickelten Methoden wurden im 5. Kapitel für die Synthese einer Glykan-Bibliothek aus konjugations-bereiten Teilen der LPS-Wiederholungseinheit von Porphyromonas gingivalis verwendet. P. gingivalis hat einen großen sozialen und medizinischen Impakt, da es der Hauptauslöser von chronischer Parodontitis ist und mit verschiedenen systemischen Krankheiten in Verbindung steht. Mikroarray-Studien zur Überprüfung von IgG- und IgA-Bindung aus Speichel und Serum ermöglichten die Identifizierung eines potentiellen Glykokonjugat-Impfstoff-Kandidaten (-D-Galp-(1→6)-α-D-Glcp-(1→4)-α-L-Rhap-(1→3)-2-β-D-GalNAc) gegen P. gingivalis

Photo-labile BODIPY protecting groups for glycan synthesis

Protective groups that can be selectively removed under mild conditions are an essential aspect of carbohydrate chemistry. Groups that can be selectively removed by visible light are particularly attractive because carbohydrates are transparent to visible light. Here, different BODIPY protecting groups were explored for their utility during glycan synthesis. A BODIPY group bearing a boron difluoride unit is stable during glycosylations but can be cleaved with green light as illustrated by the assembly of a trisaccharide.This article is published as Leichnitz, Sabrina, Komadhie C. Dissanayake, Arthur H. Winter, and Peter H. Seeberger. "Photo-labile BODIPY protecting groups for glycan synthesis." Chemical Communications (2022). DOI: 10.1039/D2CC03851J. Copyright 2022 The Royal Society of Chemistry. Creative Commons Attribution 3.0 Unported Licence. Posted with permission

Microwave-assisted Automated Glycan Assembly

Automated synthesis of DNA, RNA, and peptides provides quickly and reliably important tools for biomedical research. Automated glycan assembly (AGA) is significantly more challenging as highly branched carbohydrates require strict regio- and stereocontrol during synthesis. A new AGA synthesizer enables rapid temperature adjustment from -40 °C to +100 °C to control glycosylations at low temperature and accelerates capping, protecting group removal, and glycan modifications by using elevated temperatures. Thereby, the temporary protecting group portfolio is extended from two to four orthogonal groups that give rise to oligosaccharides with up to four branches. In addition, sulfated glycans and unprotected glycans can be prepared. The new design reduces the typical coupling cycles from 100 min to 60 min while expanding the range of accessible glycans. The instrument drastically shorten and generalizes the synthesis of carbohydrates for use in biomedical and material science.<br /

Microwave-Assisted Automated Glycan Assembly

Automated synthesis of DNA, RNA, and peptides provides quickly and reliably important tools for biomedical research. Automated glycan assembly (AGA) is significantly more challenging, as highly branched carbohydrates require strict regio- and stereocontrol during synthesis. A new AGA synthesizer enables rapid temperature adjustment from −40 to +100 °C to control glycosylations at low temperature and accelerates capping, protecting group removal, and glycan modifications using elevated temperatures. Thereby, the temporary protecting group portfolio is extended from two to four orthogonal groups that give rise to oligosaccharides with up to four branches. In addition, sulfated glycans and unprotected glycans can be prepared. The new design reduces the typical coupling cycles from 100 to 60 min while expanding the range of accessible glycans. The instrument drastically shortens and generalizes the synthesis of carbohydrates for use in biomedical and material science