2022, Borlinghaus, Niginia, Wittmann, Valentin, Braje, Wilfried M.

Synthetic chemistry relies heavily on organic solvents which are often toxic and hazardous as well as expensive in purchase and disposal. The application of water as an alternative reaction medium is not only advantageous from an environmental and safety perspective but can also offer improved selectivity and reaction rates. The exploration of this new, sustainable approach comes along with an ever-growing spectrum of reaction procedures. In this current opinion, we provide an overview of a new methodology that uses a polymeric cellulose derivative, hydroxypropyl methylcellulose, to enable chemical reactions in water.

2018-10-18, Weickert, Sabrina, Seitz, Torben, Myers, William K., Timmel, Christiane R., Drescher, Malte, Wittmann, Valentin

Multivalent carbohydrate–lectin interactions are a key concept in biological processes mediating, for example, signaling and adhesion. Binding affinities of multivalent ligands often increase by orders of magnitude compared to a monovalent binding situation. Thus, the design of multivalent ligands as potent inhibitors is a highly active field of research, where knowledge about the binding site topology is crucial. Here, we report a general strategy for precise distance measurements between the binding sites of multivalent target proteins using monovalent ligands. We designed and synthesized Monovalent, conformationally Unambiguously Spin-labeled LIgands (MUeSLI). Distances between the binding sites of the multivalent model lectin wheat germ agglutinin in complex with a GlcNAc-derived MUeSLI were determined using pulsed electron paramagnetic resonance spectroscopy. This approach is an efficient method for exploring multivalent systems with monovalent ligands, and it is readily transferable to other target proteins, allowing the targeted design of multivalent ligands without structural information available.

2017-07-04, Dold, Jeremias E.G.A., Pfotzer, Jessica, Späte, Anne-Katrin, Wittmann, Valentin

Sialic acids play an important role in numerous cell adhesion processes, and sialylation levels are known to be altered under certain pathogenic conditions, such as cancer. Metabolic glycoengineering with mannosamine derivatives is a convenient way to introduce non-natural chemical reporter groups into sialylated glycoconjugates, offering the opportunity to label sialic acids by using bioorthogonal ligation chemistry. The labeling intensity depends not only on the rate of the ligation reaction but also on the extent to which the natural sialic acids are replaced by the modified ones; that is, the incorporation efficiency. Here, we present a comparative study of eight mannosamine derivatives featuring terminal alkenes as chemical reporter groups that can be labeled by an inverse-electron-demand Diels-Alder (DAinv) reaction. The derivatives differed in chain length as well as the type of linkage (carbamates, amides, and a urea) that connects the terminal alkene to the sugar. As a general trend, increasing chain lengths resulted in higher DAinv reactivity and, at the same time, reduced incorporation efficiency. Carbamates were better accepted than amides with the same chain length; nevertheless, the latter resulted in more intense cell-surface staining, visible by live-cell fluorescence microscopy. A urea derivative was also shown to be accepted.

2016-07-20, Dauner, Martin, Batroff, Ellen, Bachmann, Verena, Hauck, Christof R., Wittmann, Valentin

Glycosphingolipids are an important component of cell membranes that are involved in many biological processes. Fluorescently labeled glycosphingolipids are frequently used to gain insight into their localization. However, the attachment of a fluorophore to the glycan part or-more commonly-to the lipid part of glycosphingolipids is known to alter the biophysical properties and can perturb the biological function of the probe. Presented here is the synthesis of novel glycosphingolipid probes with mono- and disaccharide head groups and ceramide moieties containing fatty acids of varying chain length (C4 to C20). These glycosphingolipids bear an azide or an alkyne group as chemical reporter to which a fluorophore can be attached through a bioorthogonal ligation reaction. The fluorescent tag and any linker connected to it can be chosen in a flexible manner. We demonstrate the suitability of the probes by selective visualization of the plasma membrane of living cells by confocal microscopy techniques. Whereas the derivatives with the shorter fatty acids can be directly applied to HEK 293T cells, the hydrophobic glycosphingolipids with longer fatty acids can be delivered to cells using fusogenic liposomes.

2019-09-25, Kaur, Gurjot, Fahrner, Raphael, Wittmann, Valentin, Stieger, Bruno, Dietrich, Daniel R.

Water contamination by cyanobacterial blooms is a worldwide health hazard to humans as well as livestock. Exposure to Microcystins (MCs), toxins produced by various cyanobacterial or blue green algae found in poorly treated drinking water or contaminated seafood such as fish or prawns are associated with hepatotoxicity, nephropathy and neurotoxicity and in extreme cases, death in humans. MC congeners, currently >240 known, differ dramatically in their uptake kinetics, i.e. their uptake via OATP1B1 and OATP1B3, in OATP overexpressing human HEK293 cells and primary human hepatocytes. It is thus likely that MC congeners will also differ with respect to the cellular efflux of the parent and conjugated congeners, e.g. via MRPs, MDRs, BCRP or BSEP. Consequently, the role and kinetics of different human efflux transporters - MRP, MDR, BCRP and BSEP in MC efflux was studied using insect membrane vesicles overexpressing the human transporters of interest. Of the efflux transporters investigated, MRP2 displayed MC transport. Michaelis-Menten kinetics displayed mild co-operativity and thus allosteric behavior of MRP2. MC transport by MRP2 was MC congener-specific, whereby MC-LF was transported more rapidly than MC-LR and -RR. Other human transporters (BCRP, BSEP, MRP1,3,5, MDR1) tested in this study did not exhibit interaction with MC. Although MRP2 showed specific MC transport, the MC-LR-GSH conjugate, was not transported suggesting the involvement of other transporters than MRP2 for the conjugate efflux.

2018, Doll, Franziska, Hassenrück, Jessica, Wittmann, Valentin, Zumbusch, Andreas

Posttranslational protein glycosylation is conserved in all kingdoms of life and implicated in the regulation of protein structure, function, and localization. The visualization of glycosylation states of designated proteins within living cells is of great importance for unraveling the biological roles of intracellular protein glycosylation. Our generally applicable approach is based on the incorporation of a glucosamine analog, Ac4GlcNCyoc, into the cellular glycome via metabolic engineering. Ac4GlcNCyoc can be labeled in a second step via inverse-electron-demand Diels-Alder chemistry with fluorophores inside living cells. Additionally, target proteins can be expressed as enhanced green fluorescent protein (EGFP)-fusion proteins. To assess the proximity of the donor EGFP and the glycan-anchored acceptor fluorophore, Förster resonance energy transfer (FRET) is employed and read out with high contrast by fluorescence lifetime imaging (FLIM) microscopy. In this chapter, we present a detailed description of methods required to perform protein-specific imaging of glycosylation inside living cells. These include the complete synthesis of Ac4GlcNCyoc, immunoprecipitation of EGFP-fusion proteins to examine the Ac4GlcNCyoc modification state, and a complete section on basics, performance, as well as data analysis for FLIM-FRET microscopy. We also provide useful notes necessary for reproducibility and point out strengths and limitations of the approach.

2017-04-01, Ruff, S. M., Keller, Silke, Wieland, Daniel E., Wittmann, Valentin, Tovar, Günter, Bach, Monika, Kluger, Petra

In vitro cultured cells produce a complex extracellular matrix (ECM) that remains intact after decellularization. The biological complexity derived from the variety of distinct ECM molecules makes these matrices ideal candidates for biomaterials. Biomaterials with the ability to guide cell function are a topic of high interest in biomaterial development. However, these matrices lack specific addressable functional groups, which are often required for their use as a biomaterial. Due to the biological complexity of the cell-derived ECM, it is a challenge to incorporate such functional groups without affecting the integrity of the biomolecules within the ECM. The azide-alkyne cycloaddition (click reaction, Huisgen-reaction) is an efficient and specific ligation reaction that is known to be biocompatible when strained alkynes are used to avoid the use of copper (I) as a catalyst. In our work, the ubiquitous modification of a fibroblast cell-derived ECM with azides was achieved through metabolic oligosaccharide engineering by adding the azide-modified monosaccharide Ac4GalNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetylgalactosamine) to the cell culture medium. The resulting azide-modified network remained intact after removing the cells by lysis and the molecular structure of the ECM proteins was unimpaired after a gentle homogenization process. The biological composition was characterized in order to show that the functionalization does not impair the complexity and integrity of the ECM. The azides within this "clickECM" could be accessed by small molecules (such as an alkyne-modified fluorophore) or by surface-bound cyclooctynes to achieve a covalent coating with clickECM.

2019, Tong, Qiong, Schmidt, Magnus S., Wittmann, Valentin, Mecking, Stefan

Nanoparticles with a covalently bound shell of carbohydrate and sulfate groups, respectively, and a polyethylene core were generated by Ni(II)-catalyzed aqueous copolymerization of ethylene with comonomers undec-10-en-1-yl sulfate, undec-10-en-1-yl β-D-glucoside or undec-10-en-1-yl α-D-mannoside, respectively. Via remote substituents of the catalyst, the degree of branching and consequently degree of crystallinity of the polyethylene core could be controlled. This in turn impacts particle shapes, from spherical to anisotropic platelets, as observed by cryo-TEM. Enzyme-linked lectin assays revealed the mannose-decorated nanocrystals to be efficient multivalent ligands for Concavalin A.

2017-09-12, Matzner, Daniel, Schüller, Anna, Seitz, Torben, Wittmann, Valentin, Mayer, Günter

The glmS ribozyme is a bacterial gene-regulating riboswitch that controls cell wall synthesis, depending on glucosamine-6-phosphate as a cofactor. Due to the presence of this ribozyme in several human pathogen bacteria (e.g., MRSA, VRSA), the glmS ribozyme represents an attractive target for the development of artificial cofactors. The substitution of the ring oxygen in carbohydrates by functionalized methylene groups leads to a new generation of glycomimetics that exploits distinct interaction possibilities with their target structure in biological systems. Herein, we describe the synthesis of mono-fluoro-modified carba variants of α-d-glucosamine and β-l-idosamine. (5aR)-Fluoro-carba-α-d-glucosamine-6-phosphate is a synthetic mimic of the natural ligand of the glmS ribozyme and is capable of effectively addressing its unique self-cleavage mechanism. However, in contrast to what was expected, the activity is significantly decreased compared to its non-fluorinated analog. By combining self-cleavage assays with the Bacillus subtilis and Staphylococcus aureus glmS ribozyme and molecular docking studies, we provide a structure-activity relationship for fluorinated carba-sugars.

2017-03-02, Schüller, Anna, Matzner, Daniel, Lünse, Christina E., Wittmann, Valentin, Schumacher, Catherine, Unsleber, Sandra, Brötz-Oesterhelt, Heike, Mayer, Christoph, Bierbaum, Gabriele, Mayer, Günter

The ever-growing number of pathogenic bacteria resistant to treatment with antibiotics call for the development of novel compounds with as-yet unexplored modes of action. Here, we demonstrate the in vivo antibacterial activity of carba-α-d-glucosamine (CGlcN). In this mode of action study, we provide evidence that CGlcN-mediated growth inhibition is due to glmS ribozyme activation, and we demonstrate that CGlcN hijacks an endogenous activation pathway, hence utilizing a prodrug mechanism. This is the first report describing antibacterial activity mediated by activating the self-cleaving properties of a ribozyme. Our results open the path towards a compound class with an entirely novel and distinct molecular mechanism.