ABSTRACT
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.
Subject(s)
Glycosphingolipids/chemistry , Glycosphingolipids/metabolism , Alkynes/chemistry , Azides/chemistry , Cell Membrane/metabolism , Cell Survival , Click Chemistry , Fluorescent Dyes/chemistry , Glycosphingolipids/chemical synthesis , HEK293 Cells , Humans , Staining and LabelingABSTRACT
Metabolic glycoengineering (MGE) allows the introduction of unnaturally modified carbohydrates into cellular glycans and their visualization through bioorthogonal ligation. Alkenes, for example, have been used as reporters that can react through inverse-electron-demand Diels-Alder cycloaddition with tetrazines. Earlier, norbornenes were shown to be suitable dienophiles; however, they had not previously been applied for MGE. We synthesized two norbornene-modified mannosamine derivatives that differ in the stereochemistry at the norbornene (exo/endo linkage). Kinetic investigations revealed that the exo derivative reacts more than twice as rapidly as the endo derivative. Through derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) we confirmed that both derivatives are accepted by cells and incorporated after conversion to a sialic acid. In further MGE experiments the incorporated sugars were ligated to a fluorophore and visualized through confocal fluorescence microscopy and flow cytometry.
Subject(s)
Bioengineering/methods , Hexosamines/chemistry , Cell Membrane Permeability , Flow Cytometry , HEK293 Cells , Hexosamines/pharmacokinetics , Humans , Kinetics , Microscopy, Confocal , N-Acetylneuraminic Acid/pharmacokinetics , Norbornanes/chemistry , Phenylenediamines/chemistry , Polysaccharides/chemistry , Polysaccharides/pharmacokinetics , StereoisomerismABSTRACT
Many applications in biotechnology and molecular biology rely on modified nucleotides. Here, we present an approach for the postsynthetic labelling of enzymatically synthesized vinyl-modified DNA by Diels-Alder reaction with inverse electron demand using a tetrazine. Labelling proceeds very efficiently and supersedes several known approaches.
Subject(s)
DNA/biosynthesis , Vinyl Compounds/chemistry , Biocatalysis , Cycloaddition Reaction , DNA/chemistry , DNA-Directed DNA Polymerase/metabolism , Deoxyadenosines/chemistry , Deoxyadenosines/metabolism , Deoxyuridine/analogs & derivatives , Deoxyuridine/chemistry , Deoxyuridine/metabolism , ElectronsABSTRACT
Controlled hydrolysis of donor-substituted titanium-salan complexes led to the formation of well-defined dinuclear complexes. Structure determination by means of X-ray and NMR-studies revealed the presence of a single µ-oxo bridge and one labile alkoxide ligand per titanium center. Concomitant cytotoxicity assays of the isolated dinuclear complexes showed cytotoxicities in the low micro-molar region, surpassing in this respect even their monomeric ancestors, thus making them possible highly active metabolites of titanium-salan anti-cancer drugs.
