Carbohydrate-Functionalized Anthracene Carboximides as Multivalent Ligands and Bio-Imaging Agents.

Anthracene carboximides (ACIs) conjugated with gluco-, galacto- and mannopyranosides are synthesized, by glycosylation of N-hydroxyethylanthracene carboximide acceptor with glycosyl donors. Glycoconjugation of anthracene carboximide increases the aq. solubility by more than 3-fold. The glycoconjugates display red-shifted absorption and emission, as compared to anthracene. Large Stokes shift (λabs/λem=445/525 nm) and high fluorescence quantum yields (Φ) of 0.86 and 0.5 occur in THF and water, respectively. The ACI-glycosides undergo facile photodimerization in aqueous solutions, leading to the formation of the head-to-tail dimer, as a mixture of syn and anti-isomers. Solution phase and solid-state characterizations by dynamic light scattering (DLS), microscopic imaging by atomic force (AFM) and transmission electron (TEM) microscopies reveal self-assembled vesicle structures of ACI glycosides. These self-assembled structures act as multivalent glycoclusters for ligand-specific lectin binding, as evidenced by the binding of Man-ACI to Con A, by fluorescence and turbidity assays. The conjugates do not show cellular cytotoxicity (IC50) till concentrations of 50 µM with HeLa and HepG2 cell lines and are cell-permeable, showing strong fluorescence inside the cells. These properties enable the glycoconjugates to be used in cell imaging. The non-selective cellular uptake of the glycoconjugates suggests a passive diffusion through the membrane.

Trivalent dialkylaminopyridine-catalyzed site-selective mono-O-acylation of partially-protected pyranosides.

This work demonstrates trivalent tris-(3-N-methyl-N-pyridyl propyl)amine (1) catalyzing the site-selective mono-O-acylation of glycopyranosides. Different acid anhydrides were used for the acylation of monosaccharides, mediated by catalyst 1, at a loading of 1.5 mol%; the extent of site-selectivity and the yields of mono-O-acylation products were assessed. The reactions were performed between 2 and 10 h, depending on the nature of the acid anhydride, where the bulkier pivalic anhydride required a longer duration for acylation. The glycopyranosides are maintained as diols and triols, and from a set of experiments, the site-selectivity of acylations was observed to follow the intrinsic reactivities and stereochemistry of hydroxy functionalities. The trivalent catalyst 1 mediates the reactions with excellent site-selectivities for mono-O-acylation product formation in the studied glycopyranosides, in comparison to the monovalent N,N-dimethylamino pyridine (DMAP) catalyst. This study illustrates the benefits of the multivalency of catalytic moieties in catalysis.

Cyclic Disaccharide Formation Enforced by a Ring Contraction: 2,3-Dideoxy Pyranoside Glycoside Donor to a Furanoside Macrocycle.

The synthesis of a disaccharide macrocycle through 2,3-dideoxy glucopyranosyl monosaccharide is reported. 2,3-Dideoxy-erythro-hexopyranosyl thioglycoside possessing a free hydroxy functionality at the C-4 carbon is prepared, and cycloglycosylation is conducted. In the event, the cycloglycosylation occurs with a ring contraction of the monosaccharide moiety and affords the cyclic furanoside disaccharide. Solution-phase and single-crystal X-ray diffraction structural characterizations permit the features of the macrocycle to be uncovered. The solubilization and encapsulation properties of the macrocycle are studied in aqueous solutions with 1-aminoadamantane.

The barley lectin, horcolin, binds high-mannose glycans in a multivalent fashion, enabling high-affinity, specific inhibition of cellular HIV infection.

N-Linked glycans are critical to the infection cycle of HIV, and most neutralizing antibodies target the high-mannose glycans found on the surface envelope glycoprotein-120 (gp120). Carbohydrate-binding proteins, particularly mannose-binding lectins, have also been shown to bind these glycans. Despite their therapeutic potency, their ability to cause lymphocyte proliferation limits their application. In this study, we report one such lectin named horcolin (Hordeum vulgare lectin), seen to lack mitogenicity owing to the divergence in the residues at its carbohydrate-binding sites, which makes it a promising candidate for exploration as an anti-HIV agent. Extensive isothermal titration calorimetry experiments reveal that the lectin was sensitive to the length and branching of mannooligosaccharides and thereby the total valency. Modeling and simulation studies demonstrate two distinct modes of binding, a monovalent binding to shorter saccharides and a bivalent mode for higher glycans, involving simultaneous interactions of multiple glycan arms with the primary carbohydrate-binding sites. This multivalent mode of binding was further strengthened by interactions of core mannosyl residues with a secondary conserved site on the protein, leading to an exponential increase in affinity. Finally, we confirmed the interaction of horcolin with recombinant gp120 and gp140 with high affinity and inhibition of HIV infection at nanomolar concentrations without mitogenicity.

Surface Ligand Density Switches Glycovesicles between Monomeric and Multimeric Lectin Recognition.

Carbohydrate-protein interactions define a multitude of cellular recognition events. We present herein synthetic glycovesicles as cell-surface mimics in order to switch the nature of lectin recognition. The covalent glycovesicles, constituted with diacetylene monomers of various ligand densities at their surfaces, are prepared through photo-polymerization. Vesicles with sparsely imbedded ligands engage in a lectin interaction leading to the formation of a dense, crosslinked multimeric complex. On the other hand, vesicles with many ligands, or completely covered with them, switch the lectin interaction to form a fully soluble monomeric complex, without crosslinking. Nanomolar dissociation constants govern these interactions, as assessed by a ligand-displacement assay. The study demonstrates the switching nature - between monomeric and multimeric - of the interaction as a function of ligand density in the vesicles; the results are directly relevant to understanding such a phenomenon occurring at cell surfaces.

Surface Density of Ligands Controls In-Plane and Aggregative Modes of Multivalent Glycovesicle-Lectin Recognitions.

Glycovesicles are ideal tools to delineate finer mechanisms of the interactions at the biological cell membranes. Multivalency forms the basis which, in turn, should surpass more than one mechanism in order to maintain multiple roles that the ligand-lectin interactions encounter. Ligand densities hold a prime control to attenuate the interactions. In the present study, mannose trisaccharide interacting with a cognate receptor, namely, Con A, is assessed at the vesicle surface. Synthetic (1→3)(1→6)-branched mannose trisaccharides tethered with a diacetylene monomer and glycovesicles of varying sugar densities were prepared. The polydiacetylene vesicles were prepared by maintaining uniform lipid concentrations. The interactions of the glycovesicles with the lectin were probed through dynamic light scattering and UV-Vis spectroscopy techniques. Binding efficacies were assessed by surface plasmon resonance. Aggregative and in-plane modes of interactions show ligand-density dependence at the vesicle surface. Vesicles with sparsely populated ligands engage lectin in an aggregative mode (trans-), leading to a cross-linked complex formation. Whereas glycovesicles embedded with dense ligands engage lectin interaction in an in-plane mode intramolecularly (cis-). Sub-nanomolar dissociation constants govern the intramolecular interaction occurring within the plane of the vesicle, and are more efficacious than the aggregative intermolecular interactions.

Display of Rich Reactivities of Endo- and Exocyclic Unsaturated Sugars that Parallel the Native Sugars.

Unsaturated monosaccharides expand the scope of reactivities in a sugar, directly leading to the development of newer methodologies, molecular structures and functional entities. The unsaturation as a reactive moiety can either be within the molecule, namely, endocyclic, or as a pendant moiety around the molecule, namely, exocyclic. One carbon homologations aided by reactions at the unsaturated moiety expand the molecular structures in both endo- and exocyclic sugars and lead to structures that are largely hitherto unknown. Molecular shifts and rearrangements permit interchanging the reactivities from one carbon to the other in unsaturated sugars. Activations of exocyclic unsaturated sugars also find newer possibilities to reactions central to the sugar chemistry, namely, the glycosylations. The personal reflections result from a couple of decades of explorations that traverse through the unsaturated sugars from different vantage points.

Carbon tetrachloride-free allylic halogenation-mediated glycosylations of allyl glycosides.

The allylic bromination of allyl glycosides is conducted using NBS/AIBN reagents in (EtO)2CO and PhCF3 solutions, without using CCl4 as a solvent. The activated mixed halo-allyl glycosides led to glycosylations, mediated by a triflate, in a latent-active manner, with the allyl glycosides acting as donors and acceptors. Systematic glycosylation studies are performed with different triflate promoters, non-glycosyl acceptors and various allyl glycosyl donors. One-pot allylic halogenations and subsequent glycosylations are developed in PhCF3 solutions. This newer glycosylation method is utilized to obtain xylo-pyranoside di- and trisaccharides.

Sugar Vinyl Sulfoxide Glycoconjugation of Peptides and Lysozyme: Abrogation of Proteolysis at the Lysine Sites.

We describe a glycoconjugation strategy in which a sugar vinyl sulfoxide, acting as Michael donor, reacts efficiently with amine nucleophiles arising from the lysine side chain in peptides and proteins, at physiological pH and temperature. The method permits glycoconjugation of the lysine residues present in lysozyme with the sugar vinyl sulfoxide. The glycoconjugation of the protein abrogates the trypsin-mediated proteolysis at the lysine sites. The modified protein catalyzes digestion of the Gram-negative Escherichia coli cell wall and retains the same antimicrobial property as the native lysozyme.

Mannopyranoside Glycolipids Inhibit Mycobacterial and Biofilm Growth and Potentiate Isoniazid Inhibition Activities in M.†smegmatis.

Lipomannan and lipoarabinomannan are integral components of the mycobacterial cell wall. Earlier studies demonstrated that synthetic arabinan and arabinomannan glycolipids acted as inhibitors of mycobacterial growth, in addition to exhibiting inhibitory activities of mycobacterial biofilm. Herein, it is demonstrated that synthetic mannan glycolipids are better inhibitors of mycobacterial growth, whereas lipoarabinomannan has a higher inhibition efficiency to biofilm. Syntheses of mannan glycolipids with a graded number of mannan moieties and an arabinomannan glycolipid are conducted by chemical methods and subsequent mycobacterial growth and biofilm inhibition studies are conducted on Mycobacterium smegmatis. Growth inhibition of (73±3) % is observed with a mannose trisaccharide containing a glycolipid, whereas this glycolipid did not promote biofilm inhibition activity better than that of arabinomannan glycolipid. The antibiotic supplementation activities of glycolipids on growth and biofilm inhibitions are evaluated. Increases in growth and biofilm inhibitions are observed if the antibiotic is supplemented with glycolipids, which leads to a significant reduction of inhibition concentrations of the antibiotic.

Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria.

Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of the stringent response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by RelMsm in the presence of designed inhibitors in a dose-dependent manner, which we further confirmed by monitoring the enzyme kinetics. The Rel enzyme inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long-term persistence, biofilm formation, and biofilm disruption were assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In vivo, (p)ppGpp levels were found to be downregulated in M. smegmatis treated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis The compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry.

Synthetic Arabinomannan Heptasaccharide Glycolipids Inhibit Biofilm Growth and Augment Isoniazid Effects in Mycobacterium smegmatis.

Biofilm formation, involving attachment to an adherent surface, is a critical survival strategy of mycobacterial colonies in hostile environmental conditions. Here we report the synthesis of heptasaccharide glycolipids based on mannopyranoside units anchored on to a branched arabinofuranoside core. Two types of glycolipids-2,3-branched and 2,5-branched-were synthesized and evaluated for their efficacies in inhibiting biofilm growth by the non-pathogenic mycobacterium variant Mycobacterium smegmatis. Biofilm formation was inhibited at a minimum biofilm growth inhibition concentration (MBIC) of 100 µg mL-1 in the case of the 2,5-branched heptasaccharide glycolipid. Further, we were able to ascertain that a combination of the drug isoniazid with the branched heptasaccharide glycolipid (50 µg mL-1 ) potentiates the drug, making it three times more effective, with an improved MBIC of 30 µg mL-1 . These studies establish that synthetic glycolipids not only act as inhibitors of biofilm growth, but also provide a synergistic effect when combined with significantly lowered concentrations of isoniazid to disrupt the biofilm structures of the mycobacteria.

Synthetic arabinomannan glycolipids impede mycobacterial growth, sliding motility and biofilm structure.

Mycobacterium has evolved distinct cell wall and strategies such as biofilm formation, which helps it to survive in hostile conditions. We have reported previously that arabinofuranoside containing glycolipids exhibit inhibition activities against the above functions of the mycobacterial species M. smegmatis. In search for activities mediated by oligosaccharide glycolipids, we report herein the inhibitory activities of a linear and a branched pentasaccharides having arabinan and mannan moieties. In the presence of the pentasaccharide glycolipids, a significant reduction in mycobacterial growth is observed, concomitant with reductions in sliding motility and colonization through biofilm formation, at the optimal glycolipid concentrations of 50-100 µg mL(-1). Especially the biofilm coat is ruptured by ~80-85 % in the presence of glycolipids. Pentasaccharides alone without the lipidic chain show only a weak effect. The glycolipids are non-toxic, as evaluated through their effect on RBCs. Analysis of the mycolic acid profile of glycolipid treated biofilm shows that α- and epoxy mycolic acids are downregulated significantly, in comparison to glycolipid untreated biofilms. Lipidomics profile analysis through mass spectrometry further reveals profound downregulation of phosphatidylinositol mannosides, acylatedphosphoglycerols and mycolic acid family, namely, keto-, alpha- and methoxymycolic acids.

Synthesis and Structure of Cyclic Trisaccharide with Expanded Glycosidic Linkages.

A new cyclic trisaccharide is synthesized by cycloglycosylation of a linear trisaccharide, modified with hydroxymethyl moiety at C4 of glucopyranose moiety. The cyclic trisaccharide possesses a rarely observed perfect trigonal symmetry in the P3 space group, in a narrow cone shape, and a brick-wall type arrangement of molecules in the solid state, and exhibits a significantly enhanced binding affinity to 1-aminoadamantane in aqueous solution.

Multivalent glycoliposomes and micelles to study carbohydrate-protein and carbohydrate-carbohydrate interactions.

This tutorial review describes multivalent carbohydrate-protein and carbohydrate-carbohydrate interaction studies that utilize self-assembled aggregates of thermodynamically stable liposomes and micelles. Strategies to prepare multivalent glycoliposomes and micelles include: (i) insertion of synthetic glycolipids into matrix lipids; (ii) preparation of glycolipids that aggregate to liposomes and micelles and (iii) modification of the hydrophilic surfaces with desired sugars. Several design strategies have been developed in order to obtain constituent glycolipids, having multivalent sugar moieties and their subsequent interactions with proteins were assessed in relation to the type of linkers that connect the hydrophilic and lipophilic segments. Lipophilic segments other than alkyl chains have also been developed. Polymer based glycoliposomes and micelles form an emphasis. Further, glycoliposomes facilitate studies of carbohydrate-carbohydrate interactions. An overview of the various types of glycoliposomes and micelles used to study carbohydrate-protein and carbohydrate-carbohydrate recognition phenomena is presented.

Lymph Node Targeting Mediated by Albumin Hitchhiking of Synthetic Tn Glycolipid Leads to Robust In Vivo Antibody Production.

Tn antigen is a tumor-associated carbohydrate antigen, which is present prominently on the tumor cell surfaces and attracts an interest in vaccine development. This work demonstrates that a synthetic Tn antigen carrying glycoconjugate forms a complex with circulating albumin, delivers the antigen to lymph nodes (LNs), and leads to the efficient production of antibodies against the antigen. Synthetic Tn antigen glycoconjugate, possessing DSPE-PEG2000 linker and lipophilic moieties, undergoes micellization in PBS buffer. In the presence of bovine serum albumin (BSA), demicellization of the glycolipid occurs, with a rate constant of 0.18 min-1. In vitro studies show that the glycoconjugate binds preferentially to BSA in the presence of cells. Immunological assessments in mice models reveal the albumin-enabled delivery of the Tn glycoconjugate to antigen-presenting cells in the LNs, specifically leading to a robust humoral immune response. ELISA titers show superior binding, with a saturation dilution of 1:51 200 for Tn glycoconjugate, in comparison to that mediated by the Tn-BSA covalent conjugate with a saturation dilution of 1:6400. Immunohistochemical staining shows delivery of Tn glycoconjugate at the LNs, specifically at the subcapsular sinus and interfollicular areas. The work highlights the potential of albumin-mediated target delivery strategy for cancer immunotherapies.

Con A lectin binding by synthetic bivalent arabinomannan tri- and pentasaccharides reveals connectivity-dependent functional valencies.

Lectin Con A, with specificity to interact with α-d-mannopyranoside, achieves tight binding affinity with the aid of optimal multivalent ligand valencies, distances and orientations between the ligands. A series of synthetic arabinomannans, possessing arabinan core and mannan at the non-reducing ends, is studied to assess the above constraints involved with lectin binding in this report. Trisaccharides, with (1 â†’ 2)(1 â†’ 3), (1 â†’ 2)(1 â†’ 5) and (1 â†’ 3)(1 â†’ 5) glycosidic bond connectivities, and a pentasaccharide with mannopyranosides at the non-reducing ends are synthesized. The binding affinities of the mannose bivalent ligands are studied with tetrameric Con A lectin by isothermal titration calorimetry (ITC). Among the derivatives, trisaccharide with (1 â†’ 2)(1 â†’ 3) glycosidic bond connectivity and the pentasaccharide undergo lectin interaction, clearly fulfilling the bivalent structural and functional valencies. Remaining oligosaccharides exhibit only a functional monovalency, defying the bivalent structural valency. The trisaccharide fulfilling the structural and functional valencies represent the smallest bivalent ligand, undergoing the lectin interaction in a trans-mode.

Linker length-dependent morphologies in self-assembled structures of anthracene glucosides.

Anthracenemethyl glucosides, that possess ethylene glycol linkers connecting the glucoside with anthracene moiety, are studied herein. Koenigs-Knorr glycosylation of ethylene glycol-tethered anthracene with acetobromo glucose, followed by removal of the protecting groups, lead to the facile formation of the target glucosides. Aq. solutions of these anthracene glucosides readily undergo self-assembly, with critical aggregation concentration varying between 0.4 and 1 mM, depending on the linker, being ethylene-, di- and tetraethylene glycol, as assessed by photophysical evaluations. Circular dichroism spectra show chiral self-assembled structures for these glucosides in solution, from which a left-handed chirality is adjudged. Morphologies of the self-assembled structures of these glucosides are controlled by the linker length. With the ethylene glycol linker, vesicles form initially, around which tendrils start to grow as the concentration of the glucoside is increased. Whereas, di- and tetraethylene glycol-spaced glucosides prefer agglomerated fractal-like structures, as assessed by microscopies. The aggregation phenomenon in the latter glucosides appears to be under the non-equilibrium-driven, dissipative control.

Anthracenemethyl Glycosides as Supramolecular Synthons for Chiral Self-Assembly and as Probes in Cell Imaging.

Chiral self-assembly of molecules warrants optimal structural features of synthons that promote formation of such self-assembled structures. A polyaromatic moiety coupled with hydrophilic, chiral-rich carbohydrates leads to segmentation of the regions and the self-assembly to supramolecular structures. Thermodynamic stability is augmented further through chiral self-assembly of the molecules, and formation of the desired chiral supramolecular structures is achieved. In the present study, we develop anthracene glycosides as efficient synthons that, in aqueous solutions, undergo facile self-assembly and lead to chiral supramolecular structures. Anthracenemethyl O-glycosides, installed with mono- and disaccharides, are studied for their self-assembly properties. Emerging chiral structures follow the configuration of the attached sugar moiety. Monosaccharide d- and l-glycopyranoside-containing derivatives alternate between left- and right-handed chiral structures, respectively. Disaccharide-containing derivatives do not exhibit chirality, even when self-assembly occurred. Photochemical [4π + 4π] cycloaddition occurs in the self-assembled structure in aqueous solution. Cell viability assay using HeLa cells shows above 80% viable cells at a concentration of 50 µM. Bioimaging assays reveal a significant imaging of HeLa cells for anthracenemethyl d-glucopyranoside; bright imaging was observed at the perinuclear region of the cells, suggestive of an active transport of the molecules through the cell membrane. d-Galactopyranoside and l-glucopyranoside-containing derivatives show weak imaging potencies.