Synthetic Glycolipid Research Articles

Pyroelectricity in Synthetic Amphitropic Glycolipid for Potential Application of IR Sensor Device

Synthetic pure β-anomer glycolipid with large pyroelectric coefficient (∼80 μC/m2K under zero applied bias field) is reported. The pyroelectric and dielectric properties of glycolipid prepared in thin films, annealed at various temperatures have been investigated. The induced polarization in the glycolipid thin films systems remains stable up to 100°C, thus this material is appropriate for sensor materials for pyroelectric application. The real part of the dielectric constant is also low as compared to that of the other ferroelectric ceramic materials used in pyroelectric devices; therefore these new materials have significant potential application for infrared sensing devices.

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.

Synthetic glycolipids for glioma growth inhibition developed from neurostatin and NF115 compound

Neurostatin, a natural glycosphingolipid, and NF115, a synthetic glycolipid, are inhibitors of glioma growth. While neurostatin shows high inhibitory activity on gliomas its abundance is low in mammalian brain. On the contrary NF115 exhibits less inhibitory activity on gliomas, but could be prepared by chemical synthesis. In this study we describe synthetic compounds, structurally related to NF115, capable of inhibiting glioma growth at low micromolar range. We used DNA microarray technology to compare the genes inhibited in U373-MG human glioma cells after treatment with the natural or synthetic inhibitor. New synthetic compounds were developed to interact with the product of Rho GDP dissociation inhibitor alpha gene, which was repressed in both treatments. Compounds that were inhibitors of glioma cell growth in assays for [3H]-thymidine incorporation were then injected in C6 tumor bearing rats and the tumor size in each animal group were measured. The GC-17, GC-4 and IG-5 are new compounds derived from NF115 and showed high antiproliferative activity on tumor cell lines. The GC-17 compound inhibited U373-MG glioblastoma cells (3.2μM), the effects was fifty times more potent than NF115, and caused a significant reduction of tumor volume (P<0.05) when tested in Wistar rats allotransplanted with C6 glioma cells.

Molecular Dynamics Study of Anhydrous Lamellar Structures of Synthetic Glycolipids: Effects of Chain Branching and Disaccharide Headgroup

Glycolipids form materials of considerable potential for a wide range of surfactant and thin film applications. Understanding the effect of glycolipid covalent structure on the properties of their thermotropic and lyotropic assemblies is a key step toward rational design of new glycolipid-based materials. Here, we perform molecular dynamics simulations of anhydrous bilayers of dodecyl β-maltoside, dodecyl β-cellobioside, dodecyl β-isomaltoside, and a C(12)C(10) branched β-maltoside. Specifically, we examine the consequences of chain branching and headgroup identity on the structure and dynamics of the lamellar assemblies. Chain branching of the glycolipid leads to measurable differences in the dimensions and interactions of the lamellar assembly, as well as a more fluid-like hydrophobic chain region. Substitution of the maltosyl headgroup of βMal-C(12) by an isomaltosyl moiety leads to a significant decrease in bilayer spacing as well as a markedly altered pattern of inter-headgroup hydrogen bonding. The distinctive simulated structures of the two regioisomers provide insight into the difference of ~90 °C in their observed clearing temperatures. For all four simulated glycolipid systems, with the exception of the sn-2 chain of the branched maltoside, the alkyl chains are ordered and exhibit a distinct tilt, consistent with recent crystallographic analysis of a branched chain Guerbet glycoside. These insights into structure-property relationships from simulation provide an important molecular basis for future design of synthetic glycolipid materials.

Liposomal glyco-microarray for studying glycolipid–protein interactions

A microarray enables high-throughput interaction screening of numerous biomolecules; however, fabrication of a microarray composed of cellular membrane components has proven difficult. We report fabrication of a liposomal glyco-microarray by using an azide-reactive liposome that carries synthetic and natural glycolipids via chemically selective and biocompatible liposome immobilization chemistry. Briefly, liposomes carrying anchor lipid dipalmitoylphosphatidylethanolamine (DPPE)-PEG(2000)-triphenylphosphine and ganglioside (GM1 or GM3) were prepared first and were then printed onto an azide-modified glass slide so as to afford a liposomal glyco-microarray via Staudinger ligation. Fluorescent dye release kinetics and fluorescence imaging confirmed successful liposome immobilization and specific protein binding to the intact arrayed glycoliposomes. The liposomal glyco-microarray with different gangliosides showed their specific lectin and toxin binding with different binding affinity. The azide-reactive liposome provides a facile strategy for fabrication of either a natural or a synthetic glycolipid-based membrane-mimetic glycoarray. This liposomal glyco-microarray is simple and broadly applicable and thus will find important biomedical applications, such as studying glycolipid-protein interactions and toxin screening applications.

ChemInform Abstract: Biological Activities of Synthetic Oligosaccharides and Glycolipids from Mycobacteria

AbstractReview: 111 refs.

Physicochemical Characterization of Natural-like Branched-Chain Glycosides toward Formation of Hexosomes and Vesicles

Synthetic branched-chain glycolipids have become of great interest in biomimicking research, since they provide a suitable alternative for natural glycolipids, which are difficult to extract from natural resources. Therefore, branched-chain glycolipids obtained by direct syntheses are of utmost interest. In this work, two new branched-chain glycolipids are presented, namely, 2-hexyldecyl β(α)-D-glucoside (2-HDG) and 2-hexyldecyl β(α)-D-maltoside (2-HDM) based on glucose and maltose, respectively. The self-assembly properties of these glycolipids have been studied, observing the phase behavior under thermotropic and lyotropic conditions. Due to their amphiphilic characteristics, 2-HDG and 2-HDM possess rich phase behavior in dry form and in aqueous dispersions. In the thermotropic study, 2-HDG formed a columnar hexagonal liquid crystalline phase, whereas in a binary aqueous system, 2-HDG formed an inverted hexagonal liquid crystalline phase in equilibrium with excess aqueous solution. Furthermore, aqueous dispersions of the hexagonal liquid crystal could be obtained, dispersions known as hexosomes. On the other hand, 2-HDM formed a lamellar liquid crystalline phase (smectic A) in thermotropic conditions, whereas multilamellar vesicles have been observed in equilibrium with aqueous media. Surprisingly, 2-HDM mixed with sodium dodecyl sulfate or aerosol OT induced the formation of more stable unilamellar vesicles. Thus, the branched-chain glycolipids 2-HDG and 2-HDM not only provided alternative nonionic surfactants with rich phase behavior and versatile nanostructures, but also could be used as new drug carrier systems in the future.

Synthesis of β-arabinofuranoside glycolipids, studies of their binding to surfactant protein-A and effect on sliding motilities of M. smegmatis

Surfactant protein A (SP-A), which is a lung innate immune system component, is known to bind glycolipids present at the cell surface of a mycobacterial pathogen. Lipoarabinomannan (LAM), a component of mycobacterial thick, waxy cell wall, is one of the glycolipid ligands for SP-A. In order to assess binding of synthetic glycolipids with SP-A and the glycosidic linkage preferences for the interaction, β-arabinofuranoside trisaccharide glycolipids constituted with β-(1→2), β-(1→3) and β-(1→2), β-(1→5) linkages relevant to LAM were synthesized through chemical glycosylations. The efficacies of synthetic glycolipids to interact with SP-A were assessed by using the surface plasmon resonance (SPR) technique, from which association-dissociation rate constants and equilibrium binding constants were derived. The equilibrium binding constants of the interaction of two constitutionally varying β-arabinofuranoside glycolipids with SP-A were found to be in the millimolar range. A comparison of the results with few α-anomeric arabinofuranoside glycolipids showed that glycolipids with β-anomeric linkages were having relatively lower equilibrium binding constants than those with α-anomeric linkages in binding to the protein, whereas oligosaccharides alone, without lipidic chains, exhibited higher equilibrium binding constants. Further, the synthetic compounds inhibited the growth of mycobacteria and affected sliding motilities of the bacteria, although to an extent relatively lesser than that of synthetic compounds constituted with α-anomeric linkages.

Nature-like synthetic alkyl branched-chain glycolipids: a review on chemical structure and self-assembly properties

This review presents the structure/property relationship of two classes of synthetic branched-chain glycolipids, namely the Guerbet and isoprenoid glycosides, in particular highlighting the importance of chain branching to their self-assembly properties. Alkyl chain branching in glycolipids tends to stabilise the reversed type phases as well as a lamellar (Lα) phase. These glycolipids exhibit several reversed bicontinuous cubic phases (), the reversed hexagonal phase (H II) and the lamellar phase. The detailed structures of the phases are governed by the balance between the hydrophobic chain bulkiness and the headgroup interaction, including hydrogen bonding. Among the isoprenoid glycolipids, an aqueous isoprenoid xyloside exhibits a bicontinuous cubic phase with a diamond (Pn3m) space group at full hydration and that of a glucoside adopts the stable reversed micellar cubic phase of an Fd3m space group. In the series of Guerbet glycolipids, the ones with longer chains may form stable bicontinuous cubic phases (diamond and Schwarz primitive) in dry condition, as well as the gyroid (Ia3d) in excess water. The phase behaviours of these synthetic glycolipids are comparable to those observed in other natural glycolipids, thus making them potentially useful for applications in both nano-electronics and biomedicine, as therapeutic delivery systems.

In Vitro and in Vivo Anticancer Activity of a Synthetic Glycolipid as Toll-like Receptor 4 (TLR4) Activator

Activation of Toll-like receptor 4 (TLR4) triggers the innate immune response and leads to the induction of adaptive immunity. TLR4 agonists are known to function as immunostimulants and exhibit promising therapeutic potential for cancer immunotherapy. We have previously developed a synthetic serine-based glycolipid (designated as CCL-34) that can activate TLR4-dependent signaling pathways. In this study, the anticancer immunity of CCL-34 was further demonstrated. CCL-34-activated macrophages induced cancer cell death via the apoptotic pathway, and this cytotoxicity was significantly inhibited by NG-monomethyl-L-arginine (an inducible NOS inhibitor). Notably, conditioned medium collected from CCL-34-treated splenocytes also induced cytotoxicity toward cancer cells. Furthermore, CCL-34 treatment suppressed tumor growth and increased the survival rate in TLR4-functional C3H/HeN mice but not in TLR4-defective C3H/HeJ mice. Increased apoptosis, the induction of cytokines (IFN-γ and IL-12) and chemokines (CXCL9 and CXCL10), and the elevation of leukocyte markers (CD11b, CD11c, CD4, and CD8) were detected at tumor sites in C3H/HeN mice but not in C3H/HeJ mice. Structure-and-activity relationship analysis of CCL-34 and its structural analogs revealed that a sugar moiety is essential for its activity. However, the substitution of the galactose in CCL-34 with glucose or fucose did not reduce its activity. Altogether, this study reveals the anticancer activity of a new synthetic TLR4 agonist and broadens the molecular basis of TLR4-activating glycolipids.

Abstract C172: A synthetic glycolipid induces macrophage-mediated apoptosis of cancer cells via nitric oxide-dependent downregulation of thoc1 gene.

Abstract Activation of Toll-like receptor 4 (TLR4) triggers both innate and adaptive immunity. We previously developed a synthetic TLR4 agonist, CCL-34, which can induce anticancer immunity in vitro and in vivo. The CCL-34-activated macrophages were shown to induce nitric oxide (NO)-dependent apoptosis of cancer cells. In addition, the THO complex 1 (thoc1) gene was suppressed in cancer cells co-cultured with CCL-34-activated macrophages and in tumors of CCL-34-treated mice, suggesting the involvement of Thoc1 in the CCL-34-induced anticancer activity. It has been shown that Thoc1 promoted cell survival and was overexpressed in many cancer cells. However, regulation of thoc1 and its downstream effectors in Thoc1-mediated cell survival remains unknown. In this study, we investigated the role of Thoc1 in the cancer cell apoptosis induced by CCL-34-activated macrophages and the underlying molecular mechanisms. Our data showed that the suppressed Thoc1 in bladder cancer cells (MBT-2) co-cultured with CCL-34-activated macrophages was restored by iNOS inhibitor (L-NMMA), and direct treatment of MBT-2 cells with NO donor repressed Thoc1 expression. Moreover, treatment of either CCL-34 or NO donor resulted in the suppression of thoc1 promoter activity in MBT-2 cells, whereas mutations in certain region of this promoter region abolished such repression. Notably, MBT-2 cells overexpressing thoc1 (MBT/thoc1) exhibit higher resistance to the cytotoxicity induced by treatment of NO donor or CCL-34-activated macrophages than MBT-2 cells, while both treatments suppressed Bcl-2 expression in MBT-2 cells but not in MBT-2/thoc1 cells. Together, our results demonstrate that NO-mediated thoc1 downregulation is a key step in the cancer cell apoptosis induced by CCL-34- treated macrophages. In addition, the decreased expression of thoc1 could lead to downregulation of Bcl-2 and subsequent induction of cancer cell apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C172.

Intranasal but not intravenous delivery of the adjuvant α‐galactosylceramide permits repeated stimulation of natural killer T cells in the lung

Efficient induction of antigen-specific immunity is achieved by delivering multiple doses of vaccine formulated with appropriate adjuvants that can harness the benefits of innate immune mediators. The synthetic glycolipid α-galactosylceramide (α-GalCer) is a potent activator of NKT cells, a major innate immune mediator cell type effective in inducing maturation of DCs for efficient presentation of co-administered antigens. However, systemic administration of α-GalCer results in NKT cell anergy in which the cells are unresponsive to subsequent doses of α-GalCer. We show here that α-GalCer delivered as an adjuvant by the intranasal route, as opposed to the intravenous route, enables repeated activation of NKT cells and DCs, resulting in efficient induction of cellular immune responses to co-administered antigens. We show evidence that after intranasal delivery,α-GalCer is selectively presented by DCs for the activation of NKT cells, not B cells. Furthermore, higher levels of PD-1 expression, a potential marker for functional exhaustion of the NKT cells when α-GalCer is delivered by the intravenous route, are not observed after intranasal delivery. These results support a mucosal route of delivery for the utility of α-GalCer as an adjuvant for vaccines, which often requires repeated dosing to achieve durable protective immunity.

Biological Activities of Synthetic Oligosaccharides and Glycolipids from Mycobacteria

Glycolipids are essential components of mycobacterial cell envelopes. They are highly bioactive and involved in many aspects of virulence and persistence of mycobacteria. Many glycolipids have been isolated from mycobacterial surfaces. Some mycobacterial glycans and glycolipids, as well as their analogs, have been chemically synthesized. This report focuses on recent contributions in the synthesis of mycobacterial glycans and glycolipids to the study of their biological mechanisms and to the optimization of their activities, with highlights on the lipid and saccharide mimetics.

Synthesis, biological studies of linear and branched arabinofuranoside-containing glycolipids and their interaction with surfactant protein A

Oligoarabinofuranoside-containing glycolipids relevant to mycobacterial cell wall components were synthesized in order to understand the functional roles of such glycolipids. A series of linear tetra-, hexa-, octa- and a branched heptasaccharide oligoarabinofuranosides, with 1→2 and 1→5 α-linkages between the furanoside residues, were synthesized by chemical methods from readily available monomer building blocks. Upon the synthesis of glycolipids, constituted with a double alkyl chain-substituted sn-glycerol core and oligosaccharide fragments, biological studies were performed to identify the effect of synthetic glycolipids on the biofilm formation and sliding motilities of Mycobacterium smegmatis. Synthetic glycolipids and arabinofuranosides displayed an inhibitory effect on the growth profile, but mostly on the biofilm formation and maturation. Similarly, synthetic compounds also influenced the sliding motility of the bacteria. Further, biophysical studies were undertaken, so as to identify the interactions of the glycolipids with a pulmonary surfactant protein, namely surfactant protein A (SP-A), with the aid of the surface plasmon resonance technique. Specificities of each glycolipid interacting with SP-A were thus evaluated. From this study, glycolipids were found to exhibit higher apparent association constants than the corresponding oligosaccharide portion alone, without the double alkyl group-substituted glycerol core.

Synthesis and antigenicity of BBGL-2 glycolipids of Borrelia burgdorferi, the causative agent of Lyme disease

Borrelia burgdorferi is the etiological agent for Lyme disease (LD), the most common vector borne disease in the United States. There is no human vaccine against LD currently available. Our approach to a vaccine is based on its surface-exposed glycolipids. One group of these glycolipids termed BBGL-2 consists of 1,2-di-O-acyl-3-O-(α-d-galactopyranosyl)-sn-glycerol congeners having palmitic, oleic, stearic, linoleic, and myristic acids. In order to delineate the immunodominant region(s) of the BBGL-2 components, we embarked on a synthetic project to provide available structurally defined, homogeneous analogs of BBGL-2 that might help identify the best vaccine candidate. The antigenicity of the synthetic glycolipids was examined by dot-blot analysis using mice sera obtained by immunization with killed B. burgdorferi cells, with native BBGL-2 in complete Freund’s adjuvant, as well as sera obtained from patients with Lyme disease. We found that the presence of two acyl groups in the glycerol moiety was essential for antigenicity. At least one of these groups must be an oleoyl moiety. Neither the anomeric configuration of the galactose nor the configuration of the glycerol at C-2 was a decisive factor. Based on these findings we designed an ‘unnatural’ BBGL-2 analog having the structure 3-O-(β-d-galactopyranosyl)-1,2-di-O-oleoyl-dl-glycerol which is easier and less expensive to synthesize than the other BBGL-2 congeners prepared in this study. This substance proved to be antigenic and is considered a candidate vaccine for Lyme disease.

The NKT cell ligand αgalactosylceramide suppresses allergic airway inflammation by induction of a Th1 response

One experimental approach for the treatment of allergic reactions is the stimulation of immunoregulatory NKT cells with the synthetic glycolipid αgalactosylceramide. For a first evaluation of the immunomodulatory potential of αGalCerMPEG a human in vitro allergy model was exploited. Acting as an adjuvant, the glycolipid induced an enhanced Th1-biased allergen-specific immune response of autologous lymphocytes. In a mouse model of allergic airway inflammation, αGalCerMPEG-activated NKT cells promoted a cytokine environment in the spleen, leading to priming of Th1 cells. The shift towards a Th1-dominated allergen-specific immune response thus might mediate the abrogation of allergic airway inflammation and thereby might provide a valid option for therapeutic intervention.

Activation of natural killer T cells by α-carba-GalCer (RCAI-56), a novel synthetic glycolipid ligand, suppresses murine collagen-induced arthritis

Alpha-carba-GalCer (RCAI-56), a novel synthetic analogue of α-galactosylceramide (α-GalCer), stimulates invariant natural killer T (NK T) cells to produce interferon (IFN)-γ. IFN-γ exhibits immunoregulatory properties in autoimmune diseases by suppressing T helper (Th)-17 cell differentiation and inducing regulatory T cells and apoptosis of autoreactive T cells. Here, we investigated the protective effects of α-carba-GalCer on collagen-induced arthritis (CIA) in mice. First, we confirmed that α-carba-GalCer selectively induced IFN-γ in CIA-susceptible DBA/1 mice in vivo. Then, DBA/1 mice were immunized with bovine type II collagen (CII) and α-carba-GalCer. The incidence and clinical score of CIA were significantly lower in α-carba-GalCer-treated mice. Anti-IFN-γ antibodies abolished the beneficial effects of α-carba-GalCer, suggesting that α-carba-GalCer ameliorated CIA in an IFN-γ-dependent manner. Treatment with α-carba-GalCer reduced anti-CII antibody production [immunoglobulin (Ig)G and IgG2a] and CII-reactive interleukin (IL)-17 production by draining lymph node (DLN) cells, did not induce apoptosis or regulatory T cells, and significantly increased the ratio of the percentage of IFN-γ-producing T cells to IL-17-producing T cells (Th1/Th17 ratio). Moreover, the gene expression levels of IL-6 and IL-23p19, Th17-related cytokines, were reduced significantly in mice treated with α-carba-GalCer. In addition, we observed higher IFN-γ production by NK T cells in α-carba-GalCer-treated mice in the initial phase of CIA. These findings indicate that α-carba-GalCer polarizes the T cell response toward Th1 and suppresses Th17 differentiation or activation, suggesting that α-carba-GalCer, a novel NK T cell ligand, can potentially provide protection against Th17-mediated autoimmune arthritis by enhancing the Th1 response.

SOCS1 regulates type I/type II NKT cell balance by regulating IFN signaling

Suppressor of cytokine signaling-1 (SOCS1) has been shown to be an essential negative regulator of cytokine responses, including those of IFNγ, IL-2, IL-4 and IL-7. SOCS1 deficiency resulted in hyperactivation not only of T cells in general but also of NKT cells specifically. Consistent with previous reports, T- and NKT-cell-specific deletion of Socs1 in mice resulted in enhanced sensitivity to ConA-induced hepatitis. Compared with wild-type (WT) NKT cells, SOCS1-deficient NKT cells produced larger quantities of IFNγ in response to ConA and proliferated faster in response to IL-2 and IL-15. To our surprise, however, SOCS1-deficient NKT cells did not respond to the synthetic glycolipid ligand alpha-galactosylceramide (α-GalCer), though they did respond to sulfatide. α-GalCer-CD1d-tetramer-positive type I NKT [invariant NKT (iNKT)] cells were marginally detected in the periphery of SOCS1-conditional knockout (cKO) mice, suggesting that most of the SOCS1-deficient NKT cells at the periphery were type II NKT cells. Consistently, invariant Vα14 expression was much lower in SOCS1-deficient NKT cells than in WT NKT cells, indicating that iNKT cell homeostasis was abnormal in SOCS1-cKO mice. This reduction in iNKT cells was not observed in mice of an IFNγ-deficient background. These results suggest that SOCS1 is an important regulator of the balance between type I and type II NKT cells at the periphery.

Stabilization of vesicular and supported membranes by glycolipid oxime polymers

We report herein new synthetic glycolipid dimers and polymers that provide unprecedented stability to both supported (SLBs) and vesicular lipid bilayers against dehydration and serum exposure. These novel physical properties will enable pharmaceutical delivery and development of SLB bioanalytical devices.

Identification of glycolipid ligands for human MINCLE. (136.22)

Abstract We are studying the role of C-type lectin-like molecules in innate immunity. We have produced recombinant human MINCLE (CLEC4E) protein and generated a specific antibody against this. We find limited expression in a range of immune cells. Human MINCLE co-immunoprecipitates with the FcR gamma chain. Recombinant MINCLE was screened for binding to a variety of glycan arrays and found to bind to several glycolipids, including synthetic glycolipids. Using a quantitative cellular reporter assay, these glycolipids were shown to cause potent cellular activation through MINCLE. Further investigation are underway to assess the relevance of the interactions of MINCLE in human disease. These ligands have great potential as adjuvants.