Use of metabolic glycoengineering and pharmacological inhibitors to assess lipid and protein sialylation on cells
2023, Kranaster, Petra, Blum, Jonathan, Dold, Jeremias E.G.A., Wittmann, Valentin, Leist, Marcel
Metabolic glycoengineering (MGE) has been developed to visualize carbohydrates on live cells. The method allows the fluorescent labeling of sialic acid (Sia) sugar residues on neuronal plasma membranes. For instance, the efficiency of glycosylation along neurite membranes has been characterized as cell health measure in neurotoxicology. Using human dopaminergic neurons as model system, we asked here, whether it was possible to separately label diverse classes of biomolecules and to visualize them selectively on cells. Several approaches suggest that a large proportion of Sia rather incorporated in non-protein components of cell membranes than into glycoproteins. We made use here of deoxymannojirimycin (dMM), a non-toxic inhibitor of protein glycosylation, and of N-butyl-deoxynojirimycin (NBdNM) a well-tolerated inhibitor of lipid glycosylation, to develop a method of differential labeling of sialylated membrane lipids (lipid-Sia) or sialylated N-glycosylated proteins (protein-Sia) on live neurons. The time resolution at which Sia modification of lipids/proteins was observable was in the range of few hours. The approach was then extended to several other cell types. Using this technique of 'target-specific MGE', we found that in dopaminergic or sensory neurons > 60% of Sia is lipid bound, and thus polysialic acid-neural cell adhesion molecule (PSA-NCAM) cannot be considered the major sialylated membrane component. Different from neurons, most Sia was bound to protein in HepG2 hepatoma cells or in neural crest cells. Thus, our method allows visualization of cell-specific sialylation processes for separate classes of membrane constituents.
Photoswitching Affinity and Mechanism of Multivalent Lectin Ligands
2022-05-11, Osswald, Uwe, Boneberg, Johannes, Wittmann, Valentin
Multivalent receptor-ligand binding is a key principle in a plethora of biological recognition processes. Immense binding affinities can be achieved with the correct spatial orientation of the ligands. Accordingly, the incorporation of photoswitches, that can be used to reversibly change the spatial orientation of molecules, into multivalent ligands is a means to alter the binding affinity and possibly also the binding mode of such ligands. We report a divalent ligand for the model lectin wheat germ agglutinin (WGA) containing an arylazopyrazole photoswitch. This switch, that has been recently introduced as an alternative to the more commonly used azobenzene moiety, is characterized by almost quantitative E / Z photoswitching in both directions, high quantum yields, and high thermal stability of the Z isomer. The ligand was designed in a way that only one of the isomers is able to bridge adjacent binding sites of WGA leading to a chelating binding mode. Photoswitching induces an unprecedentedly high change in lectin binding affinity as determined by isothermal titration calorimetry (ITC). Furthermore, additional dynamic light scattering (DLS) data suggest that the binding mode of the ligand changes from chelating binding of the E isomer to crosslinking binding of the Z isomer.
In situ EPR spectroscopy of a bacterial membrane transporter using an expanded genetic code
2021-11-18, Kugele, Anandi, Ketter, Sophie, Silkenath, Bjarne, Wittmann, Valentin, Joseph, Benesh, Drescher, Malte
The membrane transporter BtuB is site-directedly spin labelled on the surface of living Escherichia coli via Diels–Alder click chemistry of the genetically encoded amino acid SCO-L-lysine. The previously introduced photoactivatable nitroxide PaNDA prevents off-target labelling, is used for distance measurements, and the temporally shifted activation of the nitroxide allows for advanced experimental setups. This study describes significant evolution of Diels–Alder-mediated spin labelling on cellular surfaces and opens up new vistas for the the study of membrane proteins.
Metabolic Glycoengineering with Azide- and Alkene-Modified Hexosamines : Quantification of Sialic Acid Levels
2021-04-06, Dold, Jeremias E.G.A., Wittmann, Valentin
Metabolic glycoengineering (MGE) is an established method to incorporate chemical reporter groups into cellular glycans for subsequent bioorthogonal labeling. The method has found broad application for the visualization and isolation of glycans allowing to probe their biological roles. Furthermore, targeting of drugs to cancer cells, that present high concentrations of sialic acids on their surface, is an attractive approach. We report the application of a labeling reaction using 1,2-diamino-4,5-methylenedioxybenzene for the quantification of sialic acid derivates after MGE with various azide- and alkene-modified ManNAc, GlcNAc, and GalNAc derivatives. We followed the time course of sialic acid production and were able to detect sialic acids modified with the chemical reporter group not only after addition of ManNAc derivatives to the cell culture. A cyclopropane-modified ManNAc derivative, being a model for the corresponding cyclopropene analog that undergoes fast inverse-electron-demand Diels-Alder reactions with 1,2,4,5-tetrazines, resulted in the highest incorporation efficiency. Furthermore, we investigated whether feeding of the cells with natural and unnatural ManNAc derivative results in increased levels of sialic acids and found that this is strongly dependent on the investigated cell type and cell fraction. For HEK 293T cells, a strong increase of free sialic acids in the cell interior was found whereas cell-surface sialic acid levels are only moderately increased.
Carba-Sugar Analogs of Glucosamine-6-Phosphate : New Activators for the glmS Riboswitch
2023, Stängle, David, Silkenath, Bjarne, Gehle, Paul, Esser, Anna, Mayer, Günter, Wittmann, Valentin
Riboswitches are 5'-untranslated mRNA regions mostly found in bacteria. They are promising drug targets to overcome emerging bacterial resistances against commonly used antibiotics. The glmS riboswitch is unique among the family of riboswitches as it is a ribozyme that undergoes self-cleavage upon binding to glucosamine-6-phosphate (GlcN6P). Previously, we could show that carba glucosamine-6-phosphate (carba-GlcN6P) induces self-cleavage of the riboswitch with a potency similar to GlcN6P. Here, we report a synthetic approach to a new class of carba-GlcN6P derivatives with an alkoxy substituent in the carba position. Key features of the synthesis are a ring closing metathesis reaction followed by a hydroboration. The strategy gives access to libraries of carba-GlcN6P derivatives. Ribozyme cleavage assays unraveled new activators for the glmS riboswitch from Listeria monocytogenes and Clostridium difficile.
An Advanced 'clickECM' that Can be Modified by the Inverse-Electron Demand Diels-Alder Reaction
2022-01-05, Nellinger, Svenja, Rapp, Mareike A., Southan, Alexander, Wittmann, Valentin, Kluger, Petra J.
The extracellular matrix (ECM) represents the natural environment of the cells in tissue and therefore is a promising biomaterial in a variety of applications. Depending on the purpose it is necessary to equip the ECM with specific addressable functional groups for further modification with bioactive molecules, for controllable cross linking and/or covalent binding to surfaces. Metabolic glycoengineering (MGE) enables the specific modification of the ECM with such functional groups without affecting the native structure of the ECM. In a previous approach (S. M. Ruff, S. Keller, D. E. Wieland, V. Wittmann, G. E. M. Tovar, M. Bach, P. J. Kluger, Acta Biomater. 2017 , 52 , 159-170), we demonstrated the modification of an ECM with azido groups which can be addressed by bioorthogonal copper-catalyzed azide-alkyne cycloaddition (CuAAC). Here we demonstrate the modification of an ECM with dienophiles (terminal alkenes, cyclopropene) which can be addressed by an inverse-electron-demand Diels-Alder (IEDDA) reaction. This reaction is cell friendly as there are no cytotoxic catalysts needed. We show the equipment of the ECM with a bioactive molecule (enzyme) and prove the functional groups itself not to influence cellular behavior. Thus, this new material has great potential for its use as biomaterial which can be individually modified in a wide range of applications.
Nucleophilic aromatic substitution reactions under aqueous, mild conditions using polymeric additive HPMC
2021-06-09, Borlinghaus, Niginia, Ansari, Tharique N., Braje, Leon H., Ogulu, Deborah, Handa, Sachin, Wittmann, Valentin, Braje, Wilfried M.
The use of the inexpensive, benign, and sustainable polymer, hydroxypropyl methylcellulose (HPMC), in water enables nucleophilic aromatic subsitution (SNAr) reactions between various nucleophiles and electrophiles. The mild reaction conditions facilitate a broad functional group tolerance that can be utilized for subsequent derivatization for the synthesis of pharmaceutically relevant building blocks. The use of only equimolar amounts of all reagents and water as reaction solvent reveals the greenness and sustainability of the methodology presented herein.
Metabolic glycoengineering : exploring glycosylation with bioorthogonal chemistry
2023, Kufleitner, Markus, Haiber, Lisa Maria, Wittmann, Valentin
Glycans are involved in numerous biological recognition events. Being secondary gene products, their labeling by genetic methods – comparable to GFP labeling of proteins – is not possible. To overcome this limitation, metabolic glycoengineering (MGE, also known as metabolic oligosaccharide engineering, MOE) has been developed. In this approach, cells or organisms are treated with synthetic carbohydrate derivatives that are modified with a chemical reporter group. In the cytosol, the compounds are metabolized and incorporated into newly synthesized glycoconjugates. Subsequently, the reporter groups can be further derivatized in a bioorthogonal ligation reaction. In this way, glycans can be visualized or isolated. Furthermore, diverse targeting strategies have been developed to direct drugs, nanoparticles, or whole cells to a desired location. This review summarizes research in the field of MGE carried out in recent years. After an introduction to the bioorthogonal ligation reactions that have been used in in connection with MGE, an overview on carbohydrate derivatives for MGE is given. The last part of the review focuses on the many applications of MGE starting from mammalian cells to experiments with animals and other organisms.
Using polymeric hydroxypropyl methylcellulose as an alternative to 'micellar catalysis' to enable chemical reactions in water
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.
Application of the Inverse-Electron-Demand Diels-Alder Reaction for Metabolic Glycoengineering
2021-04-13, Haiber, Lisa Maria, Kufleitner, Markus, Wittmann, Valentin
The inverse electron-demand Diels-Alder (IEDDA or DAinv) reaction is an emerging bioorthogonal ligation reaction that finds application in all areas of chemistry and chemical biology. In this review we highlight its application in metabolic glycoengineering (MGE). MGE is a versatile tool to introduce unnatural sugar derivatives that are modified with a chemical reporter group into cellular glycans. The IEDDA reaction can then be used to modify the chemical reporter group allowing, for instance, the visualization or isolation of glycoconjugates. During the last years, many different sugar derivatives as well as reporter groups have been published. These probes are summarized, and their chemical and biological properties are discussed. Furthermore, we discuss examples of MGE and subsequent IEDDA reaction that highlight its suitability for application within living systems.