Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates.

The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cell-surface glycans in the nm-range.

Biocompatible Azide-Alkyne "Click" Reactions for Surface Decoration of Glyco-Engineered Cells.

Bio-orthogonal copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) has been widely used to modify azide- or alkyne-bearing monosaccharides on metabolic glyco-engineered mammalian cells. Here, we present a systematic study to elucidate the design space for the cytotoxic effects of the copper catalyst on NIH 3T3 fibroblasts and on HEK 293-F cells. Monitoring membrane integrity by flow cytometry and RT-PCR analysis with apoptotic and anti-apoptotic markers elucidated the general feasibility of CuAAC, with exposure time of the CuAAC reaction mixture having the major influence on biocompatibility. A high labeling efficiency of HEK 293-F cells with a fluorescent alkyne dye was rapidly achieved by CuAAC in comparison to copper free strain-promoted azide-alkyne cycloaddition (SPAAC). The study details effective and biocompatible conditions for CuAAC-based modification of glyco-engineered cells in comparison to its copper free alternative.

High-affinity carbohydrate binding by trimeric benzoboroxoles measured on carbohydrate arrays.

Carbohydrates are involved in a wide range of biological processes of pharmaceutical relevance. The selective recognition of carbohydrates is therefore of great interest in biology and medicine. In this study we present the synthesis of fluorescent multimeric benzoboroxoles and the analysis of multivalent binding processes to immobilized carbohydrate arrays by fluorescence spectroscopy. We observed high binding affinities of trimeric benzoboroxoles by determination of KDsurf values for their interaction with α-Gal on glass chips. The observed KDsurf values were in the mid-nM range (49 and 104 nM) and are comparable to the KDsurf values for binding of natural lectins, such as that of ConA to immobilized α-Man (79 nM). The array technology was found to be an excellent tool for studying the binding processes of multivalent lectin mimetics with respect to profiling and quantitation.

Super-resolution imaging of plasma membrane glycans.

Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane-associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super-resolution fluorescence imaging was used to visualize plasma membrane glycans with single-molecule sensitivity. Our results demonstrate a homogeneous distribution of N-acetylmannosamine (ManNAc)-, N-acetylgalactosamine (GalNAc)-, and O-linked N-acetylglucosamine (O-GlcNAc)-modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.

Matrix-assisted laser desorption/ionization tandem mass spectrometry of N-glycans derivatized with isonicotinic hydrazide and its biotinylated form.

RATIONALE: Successful structural characterization of glycans often requires derivatization prior to mass spectrometric analysis. Here we report on a new derivatization reagent for glycans, biotinylated isonicotinic hydrazide, allowing glycan analysis by both mass spectrometry (MS) and biochemically. Fragmentation behavior in MS and its use in structural elucidation were investigated and compared with other labels. METHODS: Glycans, released from ribonuclease B and ovalbumin, were derivatized with hydrazine labels (isoniazid (INH), biotinylated isonicotinic hydrazide (BINH) and biotinamidocaproylhydrazide (BACH)). In addition, native counterparts and 2-aminobenzamide (2-AB) derivatives were prepared. Comparative matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI TOF/TOF) experiments were carried out to investigate the fragmentation pattern of the derivatives. Finally, the capability of BINH derivatives to bind lectins was explored. RESULTS: Generally, derivatization provided beneficial enhancement in the mass spectrometric signal intensity as compared to native counterparts. The mass spectrometric fragmentation varied with the kind of label used. The most significant structure-revealing ions (cross-ring cleavages) were observed in the spectra of BINH derivatives, whereas mainly glycosidic cleavages were found with native form of glycans and 2-AB derivatives. CONCLUSIONS: Hydrazine derivatization provided the means to obtain structurally informative fragment ions. Due to BINH derivatization, specific fragments of the isomers allowed the identification of diverse glycans. The derivatization reaction can be carried out without the need for purification. The biotin residue of BINH enabled for biochemical studies, i.e. protein-glycan interactions.

Synthesis of nanodiamond derivatives carrying amino functions and quantification by a modified Kaiser test.

Nanodiamonds functionalized with different organic moieties carrying terminal amino groups have been synthesized. These include conjugates generated by Diels-Alder reactions of ortho-quinodimethanes formed in situ from pyrazine and 5,6-dihydrocyclobuta[d]pyrimidine derivatives. For the quantification of primary amino groups a modified photometric assay based on the Kaiser test has been developed and validated for different types of aminated nanodiamond. The results correspond well to values obtained by thermogravimetry. The method represents an alternative wet-chemical quantification method in cases where other techniques like elemental analysis fail due to unfavourable combustion behaviour of the analyte or other impediments.

Metabolic glycoengineering of Staphylococcus aureus reduces its adherence to human T24 bladder carcinoma cells.

The Gram-positive bacterium Staphylococcus aureus is a human pathogen increasingly causing severe infections, especially in hospital environments. Moreover, strains which are resistant against various types of antibiotics are developing and spreading widely as in the case of the community-acquired MRSA (methicillin resistant S. aureus). In this study metabolic glycoengineering with N-azidoacetyl-glucosamine (GlcNAz) has been successfully applied to S. aureus for the first time. The following bioorthogonal Mendal-Sharpless-Huisgen click reaction between the azido-functionalized S. aureus cells and alkyne dyes enabled staining of these bacteria and reduced their adherence to human T24 bladder carcinoma cells by 48%. The results are of urgent interest to study S. aureus infections.

Investigating infection processes with a workflow from organic chemistry to biophysics: the combination of metabolic glycoengineering, super-resolution fluorescence imaging and proteomics.

Infectious diseases continue to be one of the major threats to public health. In the initial events of infection, glycoproteins of human cells interact with surface proteins of bacteria or viruses, the so-called environmental adhesins. In order to pinpoint the driving forces during infection, it is necessary to study the adhesive properties of human cell surface glycoproteins with regard to their primary amino acid sequence and post-translational modifications. The authors discuss how recent developments in seemingly independent fields of the natural sciences, bio-organic synthesis, biophysical visualization and bioanalysis, open the door for a promising interdisciplinary approach to study human infection processes. The use of special synthesized carbohydrate labels, in combination with new super-resolution imaging approaches, allows access to both mapping and identification of cell surface glycoproteins well below the diffraction limit. The methodology will clarify which surface molecules are involved in bacterial adherence with potential implications for bacterial and viral infection prevention.