Artificial Formation and Tuning of Glycoprotein Networks on Live Cell Membranes: A Single‐Molecule Tracking Study

We present a method to artificially induce network formation of membrane glycoproteins and show the precise tuning of their interconnection on living cells. For this, membrane glycans are first metabolically labeled with azido sugars and then tagged with biotin by copper‐free click chemistry. Finally, these biotin‐tagged membrane proteins are interconnected with streptavidin (SA) to form an artificial protein network in analogy to a lectin‐induced lattice. The degree of network formation can be controlled by the concentration of SA, its valency, and the concentration of biotin on membrane proteins. This was verified by investigation of the spatiotemporal dynamics of the SA‐protein networks employing single‐molecule tracking. It was also proven that this network formation strongly influences the biologically relevant process of endocytosis as it is known from natural lattices on the cell surface.     

Switching of Bacterial Adhesion to a Glycosylated Surface by Reversible Reorientation of the Carbohydrate Ligand

The surface recognition in many biological systems is guided by the interaction of carbohydrate‐specific proteins (lectins) with carbohydrate epitopes (ligands) located within the unordered glycoconjugate layer (glycocalyx) of cells. Thus, for recognition, the respective ligand has to reorient for a successful matching event. Herein, we present for the first time a model system, in which only the orientation of the ligand is altered in a controlled manner without changing the recognition quality of the ligand itself. The key for this orientational control is the embedding into an interfacial system and the use of a photoswitchable mechanical joint, such as azobenzene.     

Differential effects of oligosaccharides on the hydration of simple cations

Changed ion hydration properties near surfaces, proteins, and deoxyribose nucleic acid have been reported before in the literature. In the present work, we extend this work to carbohydrates: We have performed classical-mechanical molecular dynamics simulations to study solvation properties of simple cations of biological relevance (Na(+),K(+),Mg(2+),Ca(2+)) in explicit water, near single and multiple oligosaccharides as glycocalyx models. We find that our oligosaccharides prefer direct contact with K(+) over Na(+), but that the Na(+) contacts are longer lived. These interactions also lead to strong but short-lived changes in oligosaccharide conformations, with oligosaccharides wrapping around K(+) with multiple contacts. These findings may have implications for current hypotheses on glycocalyx functions.     

Synthesis of Bifunctional Azobenzene Glycoconjugates for Cysteine‐Based Photosensitive Cross‐Linking with Bioactive Peptides

Azobenzene linker molecules can be utilized to control peptide/protein function when they are ligated to appropriately spaced amino acid side chains of the peptide. This is because the photochemical E/Z isomerization of the azobenzene N?N double bond allows to switch peptide conformation between folded and unfolded. In this context, we have introduced carbohydrate‐functionalized azobenzene derivatives in order to advance the biocompatible properties of azobenzene peptide linkers. Chloroacetamide‐functionalized and O‐allylated carbohydrate derivatives were synthesized and conjugated with azobenzene to achieve new bifunctional cross‐linkers, in order to allow ligation to cysteine side chains by nucleophilic substitution or thiol‐ene reaction, respectively. The photochromic properties of the new linker glycoconjugates were determined and first ligation reactions performed.     

Saccharide-modified nanodiamond conjugates for the efficient detection and removal of pathogenic bacteria

The detection and removal of bacteria, such as E. coli in aqueous environments by using safe and readily available means is of high importance. Here we report on the synthesis of nanodiamonds (ND) covalently modified with specific carbohydrates (glyco-ND) for the precipitation of type 1 fimbriated uropathogenic E. coli in solution by mechanically stable agglutination. The surface of the diamond nanoparticles was modified by using a Diels-Alder reaction followed by the covalent grafting of the respective glycosides. The resulting glyco-ND samples are fully dispersible in aqueous media and show a surface loading of typically 0.1 mmol g(-1). To probe the adhesive properties of various ND samples we have developed a new sandwich assay employing layers of two bacterial strains in an array format. Agglutination experiments in solution were used to distinguish unspecific interactions of glyco-ND with bacteria from specific ones. Two types of precipitates in solution were observed and characterized in detail by light and electron microscopy. Only by specific interactions mechanically stable agglutinates were formed. Bacteria could be removed from water by filtration of these stable agglutinates through 10 μm pore-size filters and the ND conjugate could eventually be recovered by addition of the appropriate carbohydrate. The application of glycosylated ND allows versatile and facile detection of bacteria and their efficient removal by using an environmentally and biomedically benign material.     

Coloring Carbohydrates: Investigation of Azulene Derivatives as Blue Protecting Groups

Coloring carbohydrate derivatives by a chromophore tag greatly facilitates all purification steps during a synthetic sequence, according to a methodology called “chromophore-supported purification” (CSP). Herein an Fmoc-analogous blue protective group for CSP is introduced, based on guaiazulene. Following a mechanistic rational, the synthesis and introduction of this new protecting group is shown, together with its removal under variable conditions and its application for the synthesis of glycoclusters of a glycopeptide and glycopeptoid type.     

Pellet freezing of in vitro produced bovine embryos using dry ice

In vitro produced bovine embryos were frozen by pellet freezing or vitrification method. In the pellet freezing method, the embryos were cooled on the dry ice and then frozen as pellets. At warming, the pellets were immersed directly into 0.5 M sucrose. The survival rates of blastocysts frozen by the pellet freezing method were higher (P<0.01) in 40% ethylene glycol (EG) than those in the lower concentrations (20 and 30% EG). Higher survival rates of blastocysts frozen by the pellet freezing method were obtained but the development rates did not differ, as compared with those by the vitrification method. There were no significant differences between the pellet freezing and vitrification method in the frequencies of post-thaw survival of hatched blastocysts. These results demonstrate that the pellet freezing method using dry ice can be used successfully for the cryopreservation of blastocysts.