Photoinduced bidirectional mesophase transition in vesicles containing azobenzene amphiphiles.

The functionality and efficiency of proteins within a biological membrane are highly dependent on both the membrane lipid composition and the physiochemical properties of the solution. Lipid mesophases are directly influenced by changes in temperature, pH, water content or due to individual properties of single lipids such as photoswitchability. In this work, we were able to induce light- and temperature-driven mesophase transitions in a model membrane system containing a mixture of 1,2-dipalmitoyl-phosphatidylcholine phospholipids and azobenzene amphiphiles. We observed reversible and reproducible transitions between the lamellar and Pn3m cubic phase after illuminating the sample for 5 min with light of 365 and 455 nm wavelengths, respectively, to switch between the cis and trans states of the azobenzene N=N double bond. These light-controlled mesophase transitions were found for mixed complexes with up to 20% content of the photosensitive molecule and at temperatures below the gel-to-liquid crystalline phase transition temperature of 33°C. Our results demonstrate the potential to design bespoke model systems to study the response of membrane lipids and proteins upon changes in mesophase without altering the environment and thus provide a possible basis for drug delivery systems.

Selective Quantification of Bacteria in Mixtures by Using Glycosylated Polypyrrole/Hydrogel Nanolayers.

Here, we present a covalent nanolayer system that consists of a conductive and biorepulsive base layer topped by a layer carrying biorecognition sites. The layers are built up by electropolymerization of pyrrole derivatives that either carry polyglycerol brushes (for biorepulsivity) or glycoside moieties (as biorecognition sites). The polypyrrole backbone makes the resulting nanolayer systems conductive, opening the opportunity for constructing an electrochemistry-based sensor system. The basic concept of the sensor exploits the highly selective binding of carbohydrates by certain harmful bacteria, as bacterial adhesion and infection are a major threat to human health, and thus, a sensitive and selective detection of the respective bacteria by portable devices is highly desirable. To demonstrate the selectivity, two strains of Escherichia coli were selected. The first strain carries type 1 fimbriae, terminated by a lectin called FimH, which recognizes α-d-mannopyranosides, which is a carbohydrate that is commonly found on endothelial cells. The otherE. coli strain was of a strain that lacked this particular lectin. It could be demonstrated that hybrid nanolayer systems containing a very thin carbohydrate top layer (2 nm) show the highest discrimination (factor 80) between the different strains. Using electrochemical impedance spectroscopy, it was possible to quantify in vivo the type 1-fimbriated E. coli down to an optical density of OD600 = 0.0004 with a theoretical limit of 0.00005. Surprisingly, the selectivity and sensitivity of the sensing remained the same even in the presence of a large excess of nonbinding bacteria, making the system useful for the rapid and selective detection of pathogens in complex matrices. As the presented covalent nanolayer system is modularly built, it opens the opportunity to develop a broad band of mobile sensing devices suitable for various field applications such as bedside diagnostics or monitoring for bacterial contamination, e.g., in bioreactors.

Photoinduced bidirectional switching in lipid membranes containing azobenzene glycolipids.

Following the reaction of biological membranes to external stimuli reveals fundamental insights into cellular function. Here, self-assembled lipid monolayers act as model membranes containing photoswitchable azobenzene glycolipids for investigating structural response during isomerization by combining Langmuir isotherms with X-ray scattering. Controlled in-situ trans/cis photoswitching of the azobenzene N = N double bond alters the DPPC monolayer structure, causing reproducible changes in surface pressure and layer thickness, indicating monolayer reorientation. Interestingly, for monolayers containing azobenzene glycolipids, along with the expected DPPC phase transitions an additional discontinuity is observed. The associated reorintation represents a crossover point, with the surface pressure and layer thickness changing in opposite directions above and below. This is evidence that the azobenzene glycolipids themselves change orientation within the monolayer. Such behaviour suggests that azobenzene glycolipids can act as a bidirectional switch in DPPC monolayers providing a tool to investigate membrane structure-function relationships in depth.

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.

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.

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.

A compatibility study on the glycosylation of 4,4'-dihydroxyazobenzene.

Photoresponsive glycoconjugates based on the azobenzene photoswitch are valuable molecules which can be used as tools for the investigation of carbohydrate-protein interactions or as precursors of shape-switchable molecular architectures, for example. To access such compounds, glycosylation of 4,4'-dihydroxyazobenzene (DHAB) is a critical step, frequently giving heterogeneous results because DHAB is a challenging glycosyl acceptor. Therefore, DHAB glucosylation was studied using nine different glycosyl donors, and reaction conditions were systematically varied in order to find a reliable procedure, especially towards the preparation of azobenzene bis-glucosides. Particular emphasis was put on glucosyl donors which were differentiated at the primary 6-position (N3, OAc) for further functionalisation. The present study allowed us to identify suitable glycosyl donors and reaction conditions matching with DHAB, affording the bis-glycosylated products in fair yields and good stereocontrol.

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.

Thermosensitive Display of Carbohydrate Ligands on Microgels for Switchable Binding of Proteins and Bacteria.

The synthesis of carbohydrate-functionalized thermosensitive poly(N-isopropylacrylamide) microgels and their ability to bind carbohydrate-binding pathogens upon temperature switch are reported. It is found that the microgels' binding affinity is increased above their lower critical solution temperature (LCST), enabling thermo-triggerable capture of pathogens. Here, a series of microgels with comparatively low mannose functionalization degrees below 1 mol % is achieved by a single polymerization step. Upon increase in mannose density, the microgel size increases, and the LCST decreases to 26 °C. Clustering with concanavalin A indicated that binding affinity is enhanced by a higher mannose content and by raising the temperature above the LCST. Binding studies with Escherichia coli confirm stronger specific interactions above the LCST and formation of mechanically stable aggregates enabling efficient separation of E. coli by filtration. For small incubation times above the LCST, the microgels' potential to release pathogens again below the LCST is confirmed also. Compared to existing switchable scaffolds, microgels nearly entirely composed of a thermosensitive material undergo a large change in volume, which allows them to drastically vary the density of ligands to switch between capture and release. This straightforward yet novel approach is likely compatible with a broad range of bioactive ligands. Therefore, thermosensitive microgels represent a promising platform for the specific capture or release of cells or pathogens.

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.

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.

New lectin ligands: Testing of Amadori rearrangement products with a series of mannoside-specific lectins.

1-(N-Phenyl)amino-1-deoxy-α-D-manno-hept-2-ulose (2) and two multivalent BSA-based structures 7 and 8, d-manno-configured C-glycosyl-type compounds derived from an Amadori rearrangement, were evaluated as ligands for mannoside-specific lectins of various sources. The determination of the concentration corresponding to 50% of inhibition (IC50) is described. Multivalency turned out to effectively influence ligand selectivity and lectin binding.

Sequence-Defined Introduction of Hydrophobic Motifs and Effects in Lectin Binding of Precision Glycomacromolecules.

This study investigates the influence of an increasingly hydrophobic backbone of multivalent glycomimetics based on sequence-defined oligo(amidoamines) on their resulting affinity toward bacterial lectins. Glycomacromolecules are obtained by stepwise assembly of tailor-made building blocks on solid support, using both hydrophobic aliphatic and aromatic building blocks to enable a gradual change in hydrophobicity of the backbone. Their binding behavior toward model lectin Concanavalin A (ConA) is evaluated using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) showing higher affinities for glycomacromolecules with higher content of hydrophobic and aromatic moieties in the backbone. Finally, glycomacromolecules are tested in a bacterial adhesion inhibition study against Escherichia coli where more hydrophobic backbones yield higher inhibitory potentials most likely due to additional secondary interactions with hydrophobic regions of the protein receptor as well as a change in conformation exposing carbohydrate ligands for increased binding. Overall, the results highlight the influence and thereby importance of the polymer backbone itself on the resulting properties of polymeric biomimetics.

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.

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.

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.

Linear Precision Glycomacromolecules with Varying Interligand Spacing and Linker Functionalities Binding to Concanavalin A and the Bacterial Lectin FimH.

A series of precision glycomacromolecules is prepared following previously established solid phase synthesis allowing for controlled variations of interligand spacing and the overall number of carbohydrate ligands. In addition, now also different linkers are installed between the carbohydrate ligand and the macromolecular scaffold. The lectin binding behavior of these glycomacromolecules is then evaluated in isothermal titration calorimetry (ITC) and kinITC experiments using the lectin Concanavalin A (Con A) in its dimeric and tetrameric form. The results indicate that both sterical and statistical effects impact lectin binding of precision glycomacromolecules. Moreover, ITC results show that highest affinity toward Con A can be achieved with an ethyl phenyl linker, which parallels earlier findings with the bacterial lectin FimH. In this way, a first set of glycomacromolecule structures is selected for testing in a bacterial adhesion-inhibition study. Here, the findings point to a one-sugar binding mode mainly affected by sterical restraints of the nonbinding parts of the respective glycomacromolecule.

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.