Tze Chieh Shiao

Mannosylated G(0) Dendrimers with Nanomolar Affinities to Escherichia coli FimH

Pentaerythritol and bis‐pentaerythritol scaffolds were used for the preparation of first generation glycodendrimers bearing aryl α‐D‐mannopyranoside residues assembled using single‐step Sonogashira and click chemistry. The carbohydrate precursors were built with either para‐iodophenyl, propargyl, or 2‐azidoethyl aglycones whereas the pentaerythritol moieties were built with terminal azide or propargyl groups, respectively. Cross‐linking abilities of this series of glycodendrimers were first evaluated with the lectin from Canavalia ensiformis (Concanavalin A). Surface plasmon resonance measurements showed these two families of mannosylated clusters as the best ligands known to date toward Escherichia coli K12 FimH with subnanomolar affinities. Tetramer 4 had a Kd of 0.45 nM. These clusters were 1000 times more potent than mannose for their capacity to inhibit the binding of E. coli to erythrocytes in vitro.

Modular synthesis of amphiphilic Janus glycodendrimers and their self-assembly into glycodendrimersomes and other complex architectures with bioactivity to biomedically relevant lectins

The modular synthesis of 7 libraries containing 51 self-assembling amphiphilic Janus dendrimers with the monosaccharides D-mannose and D-galactose and the disaccharide D-lactose in their hydrophilic part is reported. These unprecedented sugar-containing dendrimers are named amphiphilic Janus glycodendrimers. Their self-assembly by simple injection of THF or ethanol solution into water or buffer and by hydration was analyzed by a combination of methods including dynamic light scattering, confocal microscopy, cryogenic transmission electron microscopy, Fourier transform analysis, and micropipet-aspiration experiments to assess mechanical properties. These libraries revealed a diversity of hard and soft assemblies, including unilamellar spherical, polygonal, and tubular vesicles denoted glycodendrimersomes, aggregates of Janus glycodendrimers and rodlike micelles named glycodendrimer aggregates and glycodendrimermicelles, cubosomes denoted glycodendrimercubosomes, and solid lamellae. These assemblies are stable over time in water and in buffer, exhibit narrow molecular-weight distribution, and display dimensions that are programmable by the concentration of the solution from which they are injected. This study elaborated the molecular principles leading to single-type soft glycodendrimersomes assembled from amphiphilic Janus glycodendrimers. The multivalency of glycodendrimersomes with different sizes and their ligand bioactivity were demonstrated by selective agglutination with a diversity of sugar-binding protein receptors such as the plant lectins concanavalin A and the highly toxic mistletoe Viscum album L. agglutinin, the bacterial lectin PA-IL from Pseudomonas aeruginosa, and, of special biomedical relevance, human adhesion/growth-regulatory galectin-3 and galectin-4. These results demonstrated the candidacy of glycodendrimersomes as new mimics of biological membranes with programmable glycan ligand presentations, as supramolecular lectin blockers, vaccines, and targeted delivery devices.

Expeditive synthesis of glycodendrimer scaffolds based on versatile TRIS and mannoside derivatives.

A new family of glycodendrimer scaffolds containing 12 and 18 peripheral alpha-d-mannopyranosidic units has been synthesized by Cu(I)-catalyzed [1,3]-dipolar cycloadditions using sulfurated dendritic scaffolds bearing alkyne functionalities and novel TRIS derivatives.

Glycodendrimers as functional antigens and antitumor vaccines

Glycodendrimers have advantageous characteristics of providing potent immunostimulating and adjuvant properties in vaccine preparation due to their molecularly defined multivalent scaffolds together with their abundant architectural variations. Their versatile syntheses allow the production of highly defined conjugates with small antigens. An overview of the use of dendrimers as carriers of carbohydrate antigens including constructs that have built-in adjuvant properties and as stand-alone adjuvants that can be mixed with carbohydrate antigens to provide efficient vaccine formulations is provided. A brief description of the innate and adaptive immune responses toward glycoconjugates is provided to allow better understanding and basic constituent requirements for future design. The necessary distinction between antigens and immunogens (vaccines) is also discussed. To counterbalance the poor immunogenicity and T-cell independent characteristics of carbohydrate antigens, chemists have developed original hybrid molecules aimed at targeting specific pattern-recognition receptors to trigger competent immune cell proliferations and protective antibody production. Although early glycodendrimers were found to lack immunogenicity, these architecturally impressive nanomolecules were shown to bind avidly to various carbohydrate binding proteins, including antibodies. More recently, the hitherto non-immunogenic dendritic components have been successfully used as adjuvants and carriers to direct other molecules to immunocompetent cells. Commendable examples against tumor associated carbohydrate antigens will illustrate the immunochemical strategies engaged in the development of potent and exclusively synthetic carbohydrate-based anticancer vaccines.

Tri- and hexavalent mannoside clusters as potential inhibitors of type 1 fimbriated bacteria using pentaerythritol and triazole linkages

Several oligomannoside clusters having a hundred-fold increase in affinities toward E. coli were synthesized by Cu(I)-catalyzed [1,3]-dipolar cycloadditions using pentaerythritol scaffolds bearing either alkyne or azide functionalities.

Improved detection of reactive metabolites with a bromine-containing glutathione analog using mass defect and isotope pattern matching.

Drug bioactivation leading to the formation of reactive species capable of covalent binding to proteins represents an important cause of drug-induced toxicity. Reactive metabolite detection using in vitro microsomal incubations is a crucial step in assessing potential toxicity of pharmaceutical compounds. The most common method for screening the formation of these unstable, electrophilic species is by trapping them with glutathione (GSH) followed by liquid chromatography/mass spectrometry (LC/MS) analysis. The present work describes the use of a brominated analog of glutathione, N-(2-bromocarbobenzyloxy)-GSH (GSH-Br), for the in vitro screening of reactive metabolites by LC/MS. This novel trapping agent was tested with four drug compounds known to form reactive metabolites, acetaminophen, fipexide, trimethoprim and clozapine. In vitro rat microsomal incubations were performed with GSH and GSH-Br for each drug with subsequent analysis by liquid chromatography/high-resolution mass spectrometry on an electrospray time-of-flight (ESI-TOF) instrument. A generic LC/MS method was used for data acquisition, followed by drug-specific processing of accurate mass data based on mass defect filtering and isotope pattern matching. GSH and GSH-Br incubations were compared to control samples using differential analysis (Mass Profiler) software to identify adducts formed via the formation of reactive metabolites. In all four cases, GSH-Br yielded improved results, with a decreased false positive rate, increased sensitivity and new adducts being identified in contrast to GSH alone. The combination of using this novel trapping agent with powerful processing routines for filtering accurate mass data and differential analysis represents a very reliable method for the identification of reactive metabolites formed in microsomal incubations.

Structural basis of the affinity for oligomannosides and analogs displayed by BC2L-A, a Burkholderia cenocepacia soluble lectin

The opportunistic pathogen Burkholderia cenocepacia contains three soluble carbohydrate-binding proteins, related to the fucose-binding lectin PA-IIL from Pseudomonas aeruginosa. All contain a PA-IIL-like domain and two of them have an additional N-terminal domain that displays no sequence similarities with known proteins. Printed arrays screening performed on the shortest one, B. cenocepacia lectin A (BC2L-A), demonstrated the strict specificity for oligomannose-type N-glycan structures (Lameignere E, Malinovská L, Sláviková M, Duchaud E, Mitchell EP, Varrot A, Sedo O, Imberty A, Wimmerová M. 2008. Structural basis for mannose recognition by a lectin from opportunistic bacteria Burkholderia cenocepacia. Biochem J. 411:307-318.). The disaccharides alphaMan1-2Man, alphaMan1-3Man, and alphaMan1-6Man and the trisaccharide alphaMan1-3(alphaMan1-6)Man were tested by titration microcalorimetry in order to evaluate their affinity for BC2L-A in solution and to characterize the thermodynamics of the binding. Oligomannose analogs presenting two mannoside residues separated by either flexible or rigid spacer were also tested. Only the rigid one yields to high affinity binding with a fast kinetics of clustering, while the flexible analog and the trimannoside display moderate affinities and no clustering effect on short time scale. The crystal structures of BC2L-A have been obtained in complex with alphaMan1-3Man disaccharide and alphaMan1(alphaMan1-6)-3Man trisaccharide. The lengthy time required for the co-crystallization with the trisaccharide allowed for the formation of cluster since in the BC2L-A-trimannose complex solved at 1.1 A resolution, the sugar creates a bridge between two adjacent dimers, yielding to molecular strings. AFM experiments were performed in order to visualize the filaments formed in solution by this type of interaction.

Aromatic Thioglycoside Inhibitors Against the Virulence Factor Leca from Pseudomonas Aeruginosa.

Three small families of hydrolytically stable thioaryl glycosides were prepared as inhibitors of the LecA (PA-IL) virulence factor corresponding to the carbohydrate binding lectin from the bacterial pathogen Pseudomonas aeruginosa. The monosaccharidic arylthio β-d-galactopyranosides served as a common template for the major series that was also substituted at the O-3 position. Arylthio disaccharides from lactose and from melibiose constituted the other two series members. In spite of the fact that the natural ligand for LecA is a glycolipid of the globotriaosylceramide having an α-d-galactopyranoside epitope, this study illustrated that the β-d-galactopyranoside configuration having a hydrophobic aglycon could override the requirement toward the anomeric configuration of the natural sugar. The enzyme linked lectin assay together with isothermal titration microcalorimetry established that naphthyl 1-thio-β-d-galactopyranoside () gave the best inhibition with an IC50 twenty-three times better than that of the reference methyl α-d-galactopyranoside. In addition it showed a KD of 6.3 μM which was ten times better than that of the reference compound. The X-ray crystal structure of LecA with was also obtained.

Flavonoids targeting of IκB phosphorylation abrogates carcinogen-induced MMP-9 and COX-2 expression in human brain endothelial cells.

Brain endothelial cells play an essential role as structural and functional components of the blood–brain barrier (BBB). Increased BBB breakdown and brain injury are associated with neuroinflammation and are thought to trigger mechanisms involving matrix metalloproteinase upregulation. Emerging evidence also indicates that cyclooxygenase (COX) inhibition limits BBB disruption, but the mechanisms linking metalloproteinase to COX remain unknown. In this study, we sought to investigate the nuclear factor-kappa B (NF-κB) signaling pathway, a common pathway in both the regulation of matrix metalloproteinase-9 (MMP-9) and COX-2 expression, and the inhibitory properties of several chemopreventive flavonoids. Human brain microvascular endothelial cells were treated with a combination of phorbol 12-myristate 13-acetate (PMA), a carcinogen documented to increase MMP-9 and COX-2 through NF-κB, and several naturally occurring flavonoids. Among the molecules tested, we found that fisetin, apigenin, and luteolin specifically and dose-dependently antagonized PMA-induced COX-2 and MMP-9 gene and protein expressions as assessed by qRT-PCR, immunoblotting, and zymography respectively. We further demonstrate that flavonoids impact on IκK-mediated phosphorylation activity as demonstrated by the inhibition of PMA-induced IκB phosphorylation levels. Our results suggest that BBB disruption during neuroinflammation could be pharmacologically reduced by a specific class of flavonoids acting as NF-κB signal transduction inhibitors.

Multivalent Presentation of Mannose on Hyperbranched Polyglycerol and their Interaction with Concanavalin A Lectin

We describe the synthesis of multivalent mannose derivatives by using hyperbranched polyglycerols (hPG) as a scaffold with different linker structures. Grafting of protected mannose (Man) units is achieved by using CuI‐catalyzed Huisgen click chemistry with either an anomeric azide or propargyl ether onto complementarily functionalized alkyne or azido polymer surfaces. NMR spectroscopy, dynamic light scattering (DLS), IR spectroscopy, size‐exclusion chromatography (SEC), and elemental analysis have been used to characterize the hPG–Man compounds. The surface availability and bioactivity of Man‐modified polymers were evaluated by using a competitive surface plasmon resonance (SPR)‐based binding assay by interactions of the glycopolymers with concanavalin A (Con A), a lectin that binds mannose containing molecules. The results indicated that the novel glycoarchitectures presented in this work are efficient inhibitors of Con A–mannose recognition and resulted in inhibitor concentrations (mean IC50) from the micro‐ to the nanomolar range, whereas the corresponding monovalent mannoside (methyl‐Man) requires millimolar concentrations. The results provide an interesting structure–activity relationship for libraries of materials that differ in the linkage of the sugar moiety presented on a biocompatible polyglycerol scaffold.