Sulfur and Azobenzenes, a Profitable Liaison: Straightforward Synthesis of Photoswitchable Thioglycosides with Tunable Properties.

Azobenzene photoswitches are valuable tools for controlling properties of molecular systems with light. We have been investigating azobenzene glycoconjugates to probe carbohydrate-protein interactions and to design glycoazobenzene macrocycles with chiroptical and physicochemical properties modulated by light irradiation. To date, direct conjugation of glycosides to azobenzenes was performed by reactions providing target compounds in limited yields. We therefore sought a more effective and reliable coupling method. In this paper, we report on a straightforward thioarylation of azobenzene derivatives with glycosyl thiols as well as other thiols, thereby increasing the scope of azobenzene conjugation. Even challenging unsymmetrical conjugates can be achieved in good yields via sequential or one-pot procedures. Importantly, red-shifted azoswitches, which are addressed with visible light, were easily functionalized. Additionally, by oxidation of the sulfide bridge to the respective sulfones, both the photochromic and the thermal relaxation properties of the core azobenzene can be tuned. Utilizing this option, we realized orthogonal three-state photoswitching in mixtures containing two distinct azobenzene thioglycosides.

Between Two Chairs: Combination of Theory and Experiment for the Determination of the Conformational Dynamics of Xylosides.

The conformational properties of monosaccharides constitute fundamental features of oligosaccharides. While the energy landscape of monosaccharides can be altered by a specific biochemical environment or by chemical modifications, the analysis of resulting dynamic conformational equilibria is not feasible by experimental means alone. In this work, a series of ß-d-xylopyranosides is used to outline how a combination of experimental NMR parameters and computed molecular properties can be used to determine conformers and quantify the composition of conformational equilibria. We demonstrate that identifying the most stable conformers using energy calculations is challenging and computing of NMR shieldings is typically not sensitive enough. On the other hand, computed spin-spin coupling constants for the xyloside ring can be used to unambiguously assign experimental NMR data of dynamic conformational equilibria and quantify the ratio of different conformers in the mixture. As a proof of principle, this procedure allowed to analyze a hitherto unknown dynamic equilibrium of a diamino-xyloside as a precursor of a molecular switch.

Synthesis of regioisomeric maltose-based Man/Glc glycoclusters to control glycoligand presentation in 3D space.

The investigation of carbohydrate recognition in a natural environment suffers from the complexity of overlapping functional effects such as multivalency and heteromultivalency effects. Another key factor in carbohydrate recognition is the presentation mode of glycoligands in three-dimensional (3D) space. In order to trace out the effect of 3D ligand presentation, we utilized an oligosaccharide model to precisely control the spatial relation between a mannose ligand (Man) and a glucose moiety (Glc). A disaccharide (maltose) served as a scaffold to alternately conjugate Man and Glc at position 6 and 6' of a synthetic maltoside, resulting in a pair of regioisomeric heterobivalent glycoclusters. The biological effect of this specific structural tuning was tested in a native system employing mannose-specific adhesion of live E. coli cells. Indeed, the variable 3D presentation of the Man ligand resulted in a 2-fold difference between the regioisomeric heterobivalent glycoclusters as inhibitors of bacterial adhesion. This can be considered a remarkable effect, which could be interpreted by computer-aided modelling of the complexes between the bacterial lectin and the synthetic regioisomeric glycoligands.

Improving the Switching Capacity of Glyco-Self-Assembled Monolayers on Au(111).

Self-assembled monolayers (SAMs) decorated with photoisomerizable azobenzene glycosides are useful tools for investigating the effect of ligand orientation on carbohydrate recognition. However, photoswitching of SAMs between two specific states is characterized by a limited capacity. The goal of this study is the improvement of photoswitchable azobenzene glyco-SAMs. Different concepts, in particular self-dilution and rigid biaryl backbones, have been investigated. The required SH-functionalized azobenzene glycoconjugates were synthesized through a modular approach, and the respective glyco-SAMs were fabricated on Au(111). Their photoswitching properties have been extensively investigated by applying a powerful set of methods (IRRAS, XPS, and NEXAFS). Indeed, the combination of tailor-made biaryl-azobenzene glycosides and suitable diluent molecules led to photoswitchable glyco-SAMs with a significantly enhanced and unprecedented switching capacity.

A Photoswitchable Trivalent Cluster Mannoside to Probe the Effects of Ligand Orientation in Bacterial Adhesion.

We have recently demonstrated, by employing azobenzene glycosides, that bacterial adhesion to surfaces can be switched through reversible reorientation of the carbohydrate ligands. To investigate this phenomenon further, we have turned here to more complex-that is, multivalent-azobenzene glycoclusters. We report on the synthesis of a photosensitive trivalent cluster mannoside conjugated to an azobenzene hinge at the focal point. Molecular dynamics studies suggested that this cluster mannoside, despite the conformational flexibility of the azobenzene-glycocluster linkage, offers the potential for reversibly changing the glycocluster's orientation on a surface. Next, the photoswitchable glycocluster was attached to human cells, and adhesion assays with type 1 fimbriated Escherichia coli bacteria were performed. They showed marked differences in bacterial adhesion, dependent on the light-induced reorientation of the glycocluster moiety. These results further underline the importance of orientational effects in carbohydrate recognition and likewise the value of photoswitchable glycoconjugates for their study.

Pseudoenantiomeric glycoclusters: synthesis and testing of heterobivalency in carbohydrate-protein interactions.

Multivalent carbohydrate-protein interactions are key events in cell recognition processes and have been extensively studied by means of synthetic glycomimetics. To date, frequently the valency, i.e. the multiplicity of the ligand attached to a polyvalent scaffold, has been considered in the design of multivalent structures but these studies have not led to a conclusive understanding of glycan recognition at the molecular level. In this work, we add a new aspect to carbohydrate-lectin recognition studies by designing the first heterobivalent diastereomeric glycoclusters in order to investigate the influence of both heteromultivalency and relative ligand orientation. Two enantiomeric scaffolds, derived from d- and l-serine, respectively, were glycosylated with two distinct carbohydrate ligands to obtain a library of pseudoenantiomeric glycoclusters. They all have an α-d-mannosyl residue in common as a specific ligand for lectins FimH and ConA, while they differ in the second carbohydrate portion, consisting of a ß-d-glucosyl, a ß-d-galactosyl or a ß-d-glucosaminyl residue as unspecific ligands. The synthesised heteroclusters were tested in standard binding-inhibition assays investigating FimH-mediated bacterial adhesion and ConA binding to mannosylated surfaces. A striking difference was observed between the potencies of the two pseudoenantiomeric glucose-containing glycoclusters as inhibitors of FimH-mediated bacterial adhesion. For the other diastereomeric glycocluster pairs smaller inhibitory potency differences were detected in the bacterial adhesion assay. In contrast, the assays with ConA showed no significant variation for all tested cluster pairs. The results obtained with the diastereomeric glucose-mannose glycocluster pair were rationalised by molecular docking. Binding energies for the FimH carbohydrate recognition domain were calculated for both diastereomers and are in agreement with experimental data obtained in the bacterial adhesion assays.

Lectins of Mycobacterium tuberculosis - rarely studied proteins.

The importance of bacterial lectins for adhesion, pathogenicity, and biofilm formation is well established for many Gram-positive and Gram-negative bacteria. However, there is very little information available about lectins of the tuberculosis-causing bacterium, Mycobacterium tuberculosis (Mtb). In this paper we review previous studies on the carbohydrate-binding characteristics of mycobacteria and related Mtb proteins, discussing their potential relevance to Mtb infection and pathogenesis.

Photoswitchable Glycolipid Mimetics: Synthesis and Photochromic Properties of Glycoazobenzene Amphiphiles.

Glycolipids as constituents of cell membranes play an important role in cell membrane functioning. To enable the structural modification of membranes on demand, embedding of photosensitive glycolipid mimetics was envisioned and novel amphiphilic glycolipid mimetics comprising a photoswitchable azobenzene unit were synthesized. In this study, the photochromic properties of these glycolipid mimetics were analyzed by means of UV/Vis spectroscopy and reversible photoswitching. The glycolipids were based on a racemic glycerolipid derivative to be comparable in DPPC (dipalmitoylphosphatidylcholine) phospholipid membrane monolayers. Carbohydrate head groups were altered between a ß-glucoside and a ß-lactosyl unit, as well as acyl chain lengths between C12 and C16, resulting in altered photoswitching. Langmuir isotherms showed that photoswitching of Langmuir films comprising the synthetic photosensitive glycoamphiphiles was successful.

Anomeric modification of carbohydrates using the Mitsunobu reaction.

The Mitsunobu reaction basically consists in the conversion of an alcohol into an ester under inversion of configuration, employing a carboxylic acid and a pair of two auxiliary reagents, mostly triphenylphosphine and a dialkyl azodicarboxylate. This reaction has been frequently used in carbohydrate chemistry for the modification of sugar hydroxy groups. Modification at the anomeric position, leading mainly to anomeric esters or glycosides, is of particular importance in the glycosciences. Therefore, this review focuses on the use of the Mitsunobu reaction for modifications of sugar hemiacetals. Strikingly, unprotected sugars can often be converted regioselectively at the anomeric center, whereas in other cases, the other hydroxy groups in reducing sugars have to be protected to achieve good results in the Mitsunobu procedure. We have reviewed on the one hand the literature on anomeric esterification, including glycosyl phosphates, and on the other hand glycoside synthesis, including S- and N-glycosides. The mechanistic details of the Mitsunobu reaction are discussed as well as this is important to explain and predict the stereoselectivity of anomeric modifications under Mitsunobu conditions. Though the Mitsunobu reaction is often not the first choice for the anomeric modification of carbohydrates, this review shows the high value of the reaction in many different circumstances.

Recent applications of click chemistry for the functionalization of gold nanoparticles and their conversion to glyco-gold nanoparticles.

Glycoscience, despite its myriad of challenges, promises to unravel the causes of, potential new detection methods for, and novel therapeutic strategies against, many disease states. In the last two decades, glyco-gold nanoparticles have emerged as one of several potential new tools for glycoscientists. Glyco-gold nanoparticles consist of the unique structural combination of a gold nanoparticle core and an outer-shell comprising multivalent presentation of carbohydrates. The combination of the distinctive physicochemical properties of the gold core and the biological function/activity of the carbohydrates makes glyco-gold nanoparticles a valuable tool in glycoscience. In this review we present recent advances made in the use of one type of click chemistry, namely the azide-alkyne Huisgen cycloaddition, for the functionalization of gold nanoparticles and their conversion to glyco-gold nanoparticles.

Diazirine-functionalized mannosides for photoaffinity labeling: trouble with FimH.

Photoaffinity labeling is frequently employed for the investigation of ligand-receptor interactions in solution. We have employed an interdisciplinary methodology to achieve facile photolabeling of the lectin FimH, which is a bacterial protein, crucial for adhesion, colonization and infection. Following our earlier work, we have here designed and synthesized diazirine-functionalized mannosides as high-affinity FimH ligands and performed an extensive study on photo-crosslinking of the best ligand (mannoside 3) with a series of model peptides and FimH. Notably, we have employed high-performance mass spectrometry to be able to detect radiation results with the highest possible accuracy. We are concluding from this study that photolabeling of FimH with sugar diazirines has only very limited success and cannot be regarded a facile approach for covalent modification of FimH.

En route from artificial to natural: Evaluation of inhibitors of mannose-specific adhesion of E. coli under flow.

We investigated the properties of six Escherichia coli adhesion inhibitors under static and under flow conditions. On mannan-covered model substrates and under static conditions, all inhibitors were able to almost completely abolish lectin-mediated E. coli adhesion. On a monolayer of living human microvascular endothelial cells (HMEC-1), the inhibitors reduced adhesion under static conditions as well, but a large fraction of bacteria still managed to adhere even at highest inhibitor concentrations. In contrast, under flow conditions E. coli did not exhibit any adhesion to HMEC-1 not even at inhibitor concentrations where significant adhesion was detected under static conditions. This indicates that the presence of shear stress strongly affects inhibitor properties and must be taken into account when evaluating the potency of bacterial adhesion inhibitors.

Azido Pentoses: A New Tool To Efficiently Label Mycobacterium tuberculosis Clinical Isolates.

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (Tb), has a complex cell envelope which forms an efficient barrier to antibiotics, thus contributing to the challenges of anti-tuberculosis therapy. However, the unique Mtb cell wall can be considered an advantage and be utilized to selectively label Mtb bacteria. Here we introduce three azido pentoses as new compounds for metabolic labeling of Mtb: 3-azido arabinose (3AraAz), 3-azido ribose (3RiboAz), and 5-azido arabinofuranose (5AraAz). 5AraAz demonstrated the highest level of Mtb labeling and was efficiently incorporated into the Mtb cell wall. All three azido pentoses can be easily used to label a variety of Mtb clinical isolates without influencing Mtb-dependent phagosomal maturation arrest in infection studies with human macrophages. Thus, this metabolic labeling method offers the opportunity to attach desired molecules to the surface of Mtb bacteria in order to facilitate investigation of the varying virulence characteristics of different Mtb clinical isolates, which influence the outcome of a Tb infection.

Organizing multivalency in carbohydrate recognition.

The interactions of cell surface carbohydrates as well as of soluble glycoconjugates with their receptor proteins rule fundamental processes in cell biology. One of the supramolecular principles underlying and regulating carbohydrate recognition is multivalency. Many multivalent glycoconjugates have therefore been synthesized to study multivalency effects operative in glycobiology. This review is focused on smaller multivalent structures such as glycoclusters emphasizing carbohydrate-centered and heteromultivalent glycoconjugates. We are discussing primary, secondary and tertiary structural aspects including approaches to organize multivalency.

Chemistry Has a Commitment to Life.

"… Science that only serves its own interests, that looks away when things get uncomfortable, or that surveys favored territories rather than boldly and curiously breaking new ground will endanger society's trust in the scientific search for truth. This is not a good perspective for a learned society. As a community with responsibilities and values, the GDCh must cultivate a culture that has the well-being of the entire population and the planet in mind …" Read more in the Editorial by Thisbe K. Lindhorst.

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.

The Amadori Rearrangement for Carbohydrate Conjugation: Scope and Limitations.

The Amadori rearrangement was investigated for the synthesis of C-glycosyl-type neoglycoconjugates. Various amines including diamines, amino-functionalized glycosides, lysine derivatives, and peptides were conjugated with two different heptoses to generate non-natural C-glycosyl-type glycoconjugates of the d-gluco and d-manno series. With these studies, the scope and limitations of the Amadori rearrangement as a conjugation method have been exemplified with respect to the carbohydrate substrate, as well as the amino components.

Chemistry is a Key Science for the Future of India and the World.

The next Angewandte Symposium, which is dedicated to chemical biology and bioorganic chemistry, will take place on July 13, 2016 during the CRSI National Symposium in Chemistry. This is an occasion to reflect on the international nature and societal responsibility of science, and to place the focus on chemical research in India. The number of Communications from India submitted to Angewandte Chemie has increased by 140 % from 2010 to 2015, and the number published by as much as 325 % … Read more in the Editorial by Thisbe Lindhorst.

Microdomain Formation Controls Spatiotemporal Dynamics of Cell-Surface Glycoproteins.

The effect of galectin-mediated microdomain formation on the spatiotemporal dynamics of glycosylated membrane proteins in human microvascular endothelial cells (HMEC-1) was studied qualitatively and quantitatively by high-resolution fluorescence microscopy and artificially mimicked by metabolic glycoprotein engineering. Two types of membrane proteins, sialic acid-bearing proteins (SABPs) and mucin-type proteins (MTPs), were investigated. For visualization they were metabolically labeled with azido sugars and then coupled to a cyclooctyne-conjugated fluorescent dye by click chemistry. Both spatial (diffusion) and temporal (residence time) dynamics of SABPs and MTPs on the membrane were investigated after treatment with exogenous galectin-1 or -3. Strong effects of galectin-mediated lattice formation were observed for MTPs (decreased spatial mobility), but not for SABPs. Lattice formation also strongly decreased the turnover of MTPs (increased residence time on the cell membrane). The effects of galectin-mediated crosslinking was accurately mimicked by streptavidin-mediated crosslinking of biotin-tagged glycoproteins and verified by single-molecule tracking. This technique allows the induction of crosslinking of membrane proteins under precisely controlled conditions, thereby influencing membrane residence time and the spatial dynamics of glycans on the cell membrane in a controlled way.

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.