Glycopeptide Mimetics Recapitulate High‐Mannose‐Type Oligosaccharide Binding and Function

High‐mannose‐type glycans (HMTGs) decorating viral spike proteins are targets for virus neutralization. For carbohydrate‐binding proteins, multivalency is important for high avidity binding and potent inhibition. To define the chemical determinants controlling multivalent interactions we designed glycopeptide HMTG mimetics with systematically varied mannose valency and spacing. Using the potent antiviral lectin griffithsin (GRFT) as a model, we identified by NMR spectroscopy, SPR, analytical ultracentrifugation, and microcalorimetry glycopeptides that fully recapitulate the specificity and kinetics of binding to Man₉GlcNAc₂Asn and a synthetic nonamannoside. We find that mannose spacing and valency dictate whether glycopeptides engage GRFT in a face‐to‐face or an intermolecular binding mode. Surprisingly, although face‐to‐face interactions are of higher affinity, intermolecular interactions are longer lived. These findings yield key insights into mechanisms involved in glycan‐mediated viral inhibition.     

A Spray‐Filming Self‐Healing Hydrogel Fabricated from Modified Sodium Alginate and Gelatin as a Bacterial Barrier

Self‐healing hydrogels as wound dressings still face challenges in infection prevention, especially in the dressing of mass wounds, due to their inflexibility and the slow formation of the protective film on the wound. Therefore, designing a spray‐filming (rapid‐forming) hydrogel that can serve as a bacterial barrier is of particular significance in the development of wound dressings. Here, a self‐healing hydrogel based on adipic acid dihydrazide‐modified gelatin (Gel‐ADH) and monoaldehyde‐modified sodium alginate(SA‐mCHO) is prepared. Using dynamic, Schiff base bonds, the hydrogels exhibit excellent self‐healing properties. Moreover, the gelation time of SA‐mCHO/Gel‐ADH (SG) hydrogels is shortened to 2–21 s, resulting in rapid filming by spraying the two precursor solutions. In addition, the rapid spray‐filming ability might offer sufficient flexibility and rapidity for dealing with mass and irregular wounds. Notably, the bacterial barrier experiments show that the SG hydrogel films could form an effective barrier to Staphylococcus aureus and Candida albicans for 12 h. Therefore, SG hydrogels could be used in wound dressings and they show great promise in applications associated with mass and irregular traumas.     

Synthesis and rheological investigation of self‐healable deferoxamine grafted alginate hydrogel

Deferoxamine grafted alginate (SA‐DFA) was successfully synthesized via amidation of sodium alginate with deferoxamine mesylate as determined by H‐NMR and elemental analysis. SA‐DFA with different graft yield was obtained by adjusting the ratio of sodium alginate and deferoxamine mesylate. It was found that aqueous solution of SA‐DFA could form hydrogel spontaneously due to hydrogen bonding interactions, which also endowed the SA‐DFA hydrogel with self‐healing capability. The healing efficiency of SA‐DFA hydrogels ranged from 53.64 to 90.16%. In addition, surface morphologies of SA‐DFA hydrogels before/after self‐healing process were demonstrated by SEM images. We anticipated that such self‐healable alginate hydrogel would be applied in the field of wound healing. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2017 , 55, 856–865     

Clustering of Escherichia coli type-1 fimbrial adhesins by using multimeric heptyl α-D-mannoside probes with a carbohydrate core

Heptyl α‐D ‐mannoside (HM) is a strong inhibitor of the FimH lectin that mediates the initial adhesion of the uropathogenic Escherichia coli (E. coli) to the bladder cells. We designed a set of multivalent HM ligands based on carbohydrate cores with structural valencies that range from 1 to 7. The chemical strategy used to construct the regular hydrophilic structures consisted of the repetition of a critical glucoside fragment. A primary amino group was grafted at the sugar reducing end to couple the multimers to a fluorescent label. A one‐pot synthetic approach was developed to tether the ligands and the fluorescein isothiocyanate (FITC) probe to the scaffold simultaneously. Isothermal calorimetry with the monomeric FimH lectin revealed nanomolar affinities and saturation of all structurally available binding sites on the multivalent HM ligands. Direct titrations domain showed almost strict correlation of enthalpy–entropy compensation with increasing valency of the ligand, whereas reverse titration calorimetry demonstrated negative cooperativity between the first and the second binding site of the divalent heptyl mannoside. A multivalency effect was nevertheless observed by inhibiting the haemagglutination of type‐1 piliated UTI89 E. coli, with a titer as low as 60 nM for the heptavalent HM ligand. An FITC‐labeled HM trimer showed capture and cross‐linking of living bacteria in solution, a phenomenon not previously described with low‐valency ligands.     

Fullerene‐sp2‐Iminosugar Balls as Multimodal Ligands for Lectins and Glycosidases: A Mechanistic Hypothesis for the Inhibitory Multivalent Effect

Concerted functioning of lectins and carbohydrate‐processing enzymes, mainly glycosidases, is essential in maintaining life. It was commonly assumed that the mechanisms by which each class of protein recognizes their cognate sugar partners are intrinsically different: multivalency is a characteristic feature of carbohydrate–lectin interactions, whereas glycosidases bind to their substrates or substrate‐analogue inhibitors in monovalent form. Recent observations on the glycosidase inhibitory potential of multivalent glycomimetics have questioned this paradigm and led to postulate an inhibitory multivalent effect. Here the mechanisms at the origin of this phenomenon have been investigated. A D ‐gluco‐configured sp²‐iminosugar glycomimetic motif, namely 1‐amino‐5N,6O‐oxomethylydenenojirimycin (1N‐ONJ), behaving, simultaneously, as a ligand of peanut agglutinin (PNA) lectin and as an inhibitor of several glycosidases, has been identified. Both the 1N‐ONJ–lectin‐ and 1N‐ONJ–glycosidase‐recognition processes have been found to be sensitive to multivalency, which has been exploited in the design of a lectin–glycosidase competitive assay to explore the implication of catalytic and non‐glycone sites in enzyme binding. A set of isotropic dodecavalent C₆₀‐fullerene–sp²‐iminosugar balls incorporating matching or mismatching motifs towards several glycosidases (inhitopes) was synthesized for that purpose, thereby preventing differences in binding modes arising from orientational preferences. The data supports that: 1) multivalency allows modulating the affinity and selectivity of a given inhitope towards glycosidases; 2) multivalent presentation can switch on the inhibitory capacity for some inhitope–glycosidase pairs, and 3) interactions of the multivalent inhibitors with non‐glycone sites is critical for glycosidase recognition. The ensemble of results point to a shift in the binding mode on going from monovalent to multivalent systems: in the first case a typical ′′key–lock′′ model involving, essentially, the high‐affinity active site can be assumed, whereas in the second, a lectin‐like behavior implying low‐affinity non‐glycone sites probably operates. The differences in responsiveness to multivalency for different glycosidases can then be rationalized in terms of the structure and accessibility of the corresponding carbohydrate‐binding regions.     

Dual‐Functional Dextran‐PEG Hydrogel as an Antimicrobial Biomedical Material

Microbial infections continually present a major worldwide public healthcare threat, particularly in instances of impaired wound healing and biomedical implant fouling. The development of new materials with the desired antimicrobial property to avoid and treat wound infection is urgently needed in wound care management. This study reports a novel dual‐functional biodegradable dextran‐poly(ethylene glycol) (PEG) hydrogel covalently conjugated with antibacterial Polymyxin B and Vancomycin (Vanco). The hydrogel is designed as a specialized wound dressing that eradicates existing bacteria and inhibits further bacteria growth, while, ameliorating the side effects of antibiotics and accelerating tissue repair and regeneration. The hydrogel exhibits potent antibacterial activities against both gram‐negative bacteria Escherichia coli (E. coli) and gram‐positive bacteria Staphylococcus aureus (S. aureus) with no observable toxicity to mouse fibroblast cell line NIH 3T3. These results demonstrate the immense potential of dextran‐PEG hydrogel as a wound dressing healthcare material in efficiently controlling bacteria growth in complex biological systems.     

Multivalent Gold Glycoclusters: High Affinity Molecular Recognition by Bacterial Lectin PA‐IL

Multivalent protein‐carbohydrate interactions are involved in the initial stages of many fundamental biological and pathological processes through lectin–carbohydrate binding. The design of high affinity ligands is therefore necessary to study, inhibit and control the processes governed through carbohydrate recognition by their lectin receptors. Carbohydrate‐functionalised gold nanoclusters (glyconanoparticles, GNPs) show promising potential as multivalent tools for studies in fundamental glycobiology research as well as biomedical applications. Here we present the synthesis and characterisation of galactose functionalised GNPs and their effectiveness as binding partners for PA‐IL lectin from Pseudomonas aeruginosa. Interactions were evaluated by hemagglutination inhibition (HIA), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) assays. Results show that the gold nanoparticle platform displays a significant cluster glycoside effect for presenting carbohydrate ligands with almost a 3000‐fold increase in binding compared with a monovalent reference probe in free solution. The most effective GNP exhibited a dissociation constant (K_d) of 50 nM per monosaccharide, the most effective ligand of PA‐IL measured to date; another demonstration of the potential of glyco‐nanotechnology towards multivalent tools and potent anti‐adhesives for the prevention of pathogen invasion. The influence of ligand presentation density on their recognition by protein receptors is also demonstrated.     

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.     

Light‐Switchable Self‐Healing Hydrogel Based on Host–Guest Macro‐Crosslinking

A self‐healing hydrogel is prepared by crosslinking acrylamide with a host–guest macro‐crosslinker assembled from poly(β‐cyclodextrin) nanogel and azobenzeneacrylamide. The photoisomerizable azobenzene moiety can change its binding affinity with β‐cyclodextrin, therefore the crosslinking density and rheology property of the hydrogel can be tuned with light stimulus. The hydrogel can repair its wound autonomously through the dynamic host–guest interaction. In addition, the wounded hydrogel will lose its ability of self‐healing when exposed to ultraviolet light, and the self‐healing behavior can be recovered upon the irradiation of visible light. The utilizing of host–guest macro‐crosslinking approach manifests the as‐prepared hydrogel reversible and light‐switchable self‐healing property, which would broaden the potential applications of self‐healing polymers.

     

Antiadhesive Nanosomes Facilitate Targeting of the Lysosomal GlcNAc Salvage Pathway through Derailed Cancer Endocytosis

Activated endocytosis of extracellular macromolecules and their intracellular trafficking to lysosomes is an essential metabolic mechanism in cancer cells during their rapid proliferation. Cancer cells reuse a vast amount of N‐acetylglucosamine (GlcNAc) supplied from the GlcNAc salvage pathway for the accelerated synthesis of a pivotal uridine diphosphate (UDP)‐GlcNAc. A method to inactivate key glycosidases in lysosomes could critically contribute to the development of potent anticancer therapy. Here we demonstrate that “nanosomes” made of core metals covered by an antiadhesive mixed self‐assembled monolayer allow for avoiding nonspecific surface protein corona and targeted molecular delivery through activated endocytosis. Nanosomes carrying suicide substrates showed that lysosomal glycosidases such as β‐hexosaminidase and β‐galactosidase in cancer cells are promising targets for novel anticancer therapeutic nanomedicine that induce apoptotic cell death through lysosomal membrane permeabilization. The advantage of this method is evident because multivalent surface loading by antiadhesive nanosomes makes it possible to highlight “weak interactions” such as carbohydrate–lectin interactions independent of surface protein corona.     

A Multivalent Ligand for the Mannose‐6‐Phosphate Receptor for Endolysosomal Targeting of an Activity‐Based Probe

The ubiquitously expressed mannose‐6‐phosphate receptors (MPRs) are a promising class of receptors for targeted compound delivery into the endolysosomal compartments of a variety of cell types. The development of a synthetic, multivalent, mannose‐6‐phosphate (M6P) glycopeptide‐based MPR ligand is described. The conjugation of this ligand to fluorescent DCG‐04, an activity‐based probe for cysteine cathepsins, enabled fluorescent readout of its receptor‐targeting properties. The resulting M6P‐cluster–BODIPY–DCG‐04 probe was shown to efficiently label cathepsins in cell lysates as well as in live cells. Furthermore, the introduction of the 6‐O‐phosphates leads to a completely altered uptake profile in COS and dendritic cells compared to a mannose‐containing ligand. Competition with mannose‐6‐phosphate abolished all uptake of the probe in COS cells, and we conclude that the mannose‐6‐phosphate cluster targets the MPR and ensures the targeted delivery of cargo bound to the cluster into the endolysosomal pathway.     

On‐Demand Dissolution of a Dendritic Hydrogel‐based Dressing for Second‐Degree Burn Wounds through Thiol–Thioester Exchange Reaction

An adhesive yet easily removable burn wound dressing represents a breakthrough in second‐degree burn wound care. Current second‐degree burn wound dressings absorb wound exudate, reduce bacterial infections, and maintain a moist environment for healing, but are surgically or mechanically debrided from the wound, causing additional trauma to the newly formed tissues. We have developed an on‐demand dissolvable dendritic thioester hydrogel burn dressing for second‐degree burn care. The hydrogel is composed of a lysine‐based dendron and a PEG‐based crosslinker, which are synthesized in high yields. The hydrogel burn dressing covers the wound and acts as a barrier to bacterial infection in an in vivo second‐degree burn wound model. A unique feature of the hydrogel is its capability to be dissolved on‐demand, via a thiol–thioester exchange reaction, allowing for a facile burn dressing removal.     

Synthesis of Oligomeric Mannosides and Their Structure‐Binding Relationship with Concanavalin A

Small glycodendrimers with α‐mannosyl ligands were synthesized by using copper‐catalyzed azide–alkyne coupling chemistry and some of these molecules were used as multivalent ligands to study the induction of concanavalin A (Con A) precipitation. The results showed that the monovalent mannose ligand could induce the precipitation of Con A. This unexpected finding initiated a series of studies to characterize the molecular basis of the ligand–lectin interaction. The atypical precipitation is found to be specific to the mannose, fluorescein moiety (FITC), and Con A. Apparently the mannose ligand binds to Con A through hydrogen‐bonding interactions, whereas the binding of FITC is mediated by hydrophobic forces.     

Heteromultivalent Glycooligomers as Mimetics of Blood Group Antigens

Precision glycomacromolecules have proven to be important tools for the investigation of multivalent carbohydrate–lectin interactions by presenting multiple glycan epitopes on a highly-defined synthetic scaffold. Herein, we present a new strategy for the versatile assembly of heteromultivalent glycomacromolecules that contain different carbohydrate motifs in proximity within the side chains. A new building block suitable for the solid-phase polymer synthesis of precision glycomacromolecules was developed with a branching point in the side chain that bears a free alkyne and a TIPS-protected alkyne moiety, which enables the subsequent attachment of different carbohydrate motifs by on-resin copper-mediated azide–alkyne cycloaddition reactions. Applying this synthetic strategy, heteromultivalent glycooligomers presenting fragments of histo-blood group antigens and human milk oligosaccharides were synthesized and tested for their binding behavior towards bacterial lectin LecB.     

Bola-Amphiphilic Glycodendrimers: New Carbohydrate-Mimicking Scaffolds to Target Carbohydrate-Binding Proteins

Dendrimers are appealing scaffolds for creating carbohydrate mimics with unique multivalent cooperativity. We report here novel bola-amphiphilic glycodendrimers bearing mannose and glucose terminals, and a hydrophobic thioacetal core responsive to reactive oxygen species. The peculiar bola-amphiphilic feature enabled stronger binding to lectin compared to conventional amphiphiles. In addition, these dendrimers are able to target mannose receptors and glucose transporters expressed at the surface of cells, thus allowing effective and specific cellular uptake. This highlights their great promise for targeted delivery.     

α‐O‐Linked Glycopeptide Mimetics: Synthesis,Conformation Analysis,and Interactions with Viscumin,a Galactoside‐Binding Model Lectin

Efficient cycloaddition of a silylidene‐protected galactal with a suitable heterodiene yielded the basis for a facile diastereoselective route to a glycopeptide‐mimetic scaffold. Its carbohydrate part was further extended by β1–3‐linked galactosylation. The pyranose rings retain their ⁴C₁ chair conformation, as shown by molecular modeling and NMR spectroscopy, and the typical exo‐anomeric geometry was observed for the disaccharide. The expected bioactivity was ascertained by saturation‐transfer‐difference NMR spectroscopy by using the galactoside‐specific plant toxin viscumin as a model lectin. The experimental part was complemented by molecular docking. The described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational properties and bioactivity establish the prepared α‐O‐linked glycopeptide mimetics as promising candidates for further exploitation of this scaffold to give O‐glycans for lectin blocking and vaccination.     

Rational Design and Synthesis of Optimized Glycoclusters for Multivalent Lectin–Carbohydrate Interactions: Influence of the Linker Arm

The design of multivalent glycoclusters requires the conjugation of biologically relevant carbohydrate epitopes functionalized with linker arms to multivalent core scaffolds. The multigram‐scale syntheses of three structurally modified triethyleneglycol analogues that incorporate amide moiety(ies) and/or a phenyl ring offer convenient access to a series of carbohydrate probes with different water solubilities and rigidities. Evaluation of flexibility and determination of preferred conformations were performed by conformational analysis. Conjugation of the azido‐functionalized carbohydrates with tetra‐propargylated core scaffolds afforded a library of 18 tetravalent glycoclusters, in high yields, by Cu^I‐catalyzed azide–alkyne cycloaddition (CuAAC). The compounds were evaluated for their ability to bind to PA‐IL (the LecA lectin from the opportunistic pathogen Pseudomonas aeruginosa). Biochemical evaluation through inhibition of hemagglutination assays (HIA), enzyme‐linked lectin assays (ELLA), surface plasmon resonance (SPR), and isothermal titration microcalorimetry (ITC) revealed improved and unprecedented affinities for one of the monovalent probes (K_d=5.8 μM ) and also for a number of the tetravalent compounds that provide several new nanomolar ligands for this tetrameric lectin.     

Synthesis of Benzaldehyde‐Functionalized Glycans: A Novel Approach Towards Glyco‐SAMs as a Tool for Surface Plasmon Resonance Studies

In recent years the interest in tools for investigating carbohydrate–protein (CPI) and carbohydrate‐carbohydrate interactions (CCI) has increased significantly. For the investigation of CPI and CCI, several techniques employing different linking methods are available. Surface plasmon resonance (SPR) imaging is a most appropriate tool for analyzing the formation of self‐assembled monolayers (SAM) of carbohydrate derivatives, which can mimic the glycocalyx. In contrast to the SPR imaging methods used previously to analyze CPI and CCI, the novel approach reported herein allows a facile and rapid synthesis of linker spacers and carbohydrate derivatives and enhances the binding event by controlling the amount and orientation of ligand. For immobilization on biorepulsive amino‐functionalized SPR chips by reductive amination, diverse aldehyde‐functionalized glycan structures (glucose, galactose, mannose, glucosamine, cellobiose, lactose, and lactosamine) have been synthesized in several facile steps that include olefin metathesis. Effective immobilization and the first binding studies are presented for the lectin concanavalin A.     

Evaluation and Comparison of a 3,5‐Dimethylphenyl Isocyanate Teicoplanin with Phenyl Isocyanate Teicoplanin Chiral Stationary Phase Using RP‐HPLC

HPLC enantiomeric separations of 8 α‐amino acids were achieved using two self‐made chiral stationary phases (CSP)–phenyl isocyanate teicoplanin (Phe‐TE) and 3,5‐dimethylphenyl isocyanate teicoplanin (DMP‐TE), using reversed phase mobile phases. The Phe‐TE or the DMP‐TE CSP was prepared from the TE using derivative agents, phenyl isocyanate or 3,5‐dimethylphenyl isocyanate, respectively. The chromatographic results were given as the retention, selectivity, resolution factor and the enantioselective free energy difference corresponding to the separation of the two enantiomers. The effect of pH, organic modifier type and amount were discussed, and the stereoselectivities for two TE‐based CSPs were compared. The chiral selectivity factor for six α‐amino acids on DMP‐TE is somewhat bigger than that on Phe‐TE CSP under reversed phase (RP) mode. Comparison of the enantiomeric separations using self‐made Phe‐TE and DMP‐TE was conducted to gain a better understanding of the chiral recognition mechanism of the macrocyclic glycopeptide CSP.     

Sugar‐Decorated Sugar Vesicles: Lectin–Carbohydrate Recognition at the Surface of Cyclodextrin Vesicles

An artificial glycocalix self‐assembles when unilamellar bilayer vesicles of amphiphilic β‐cyclodextrins are decorated with maltose and lactose by host–guest interactions. To this end, maltose and lactose were conjugated with adamantane through a tetra(ethyleneglycol) spacer. Both carbohydrate–adamantane conjugates strongly bind to β‐cyclodextrin (K_a≈4×10⁴ M ^?1). The maltose‐decorated vesicles readily agglutinate (aggregate) in the presence of the lectin concanavalin A, whereas the lactose‐decorated vesicles agglutinate in the presence of peanut agglutinin. The orthogonal multivalent interaction in the ternary system of host vesicles, guest carbohydrates, and lectins was investigated by using isothermal titration calorimetry, dynamic light scattering, UV/Vis spectroscopy, and cryogenic transmission electron microscopy. It was shown that agglutination is reversible, and the noncovalent interaction can be suppressed and eliminated by the addition of competitive inhibitors, such as D ‐glucose or β‐cyclodextrin. Also, it was shown that agglutination depends on the surface coverage of carbohydrates on the vesicles.