Multivalent Glycoclusters Research Articles

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.

The disulfide bond as a key motif for the construction of multivalent glycoclusters

S-Glycosylated dendrons having a thioacetate group in their focal points led to multivalent glycoclusters by spontaneous O2-oxidation of sulfides.

Synthesis of dimeric and tetrameric trithiomannoside clusters through convenient photoinitiated thiol-ene click protocol for efficient inhibition of gram-negative bacteria

Glycoclusters with high glycan density accessible through grouping of monosaccharide units in multimeric forms are important due to their high affinity and capability in recognition with protein receptors as a result of multivalent effect. In this report, we have achieved the assembly of glycoclusters in dimeric to tetrameric forms using efficient light initiated free radical mediated thiol-ene click reaction. Bacterium (UTI89) inhibition study by yeast agglutination assay demonstrated that these multivalent glycoclusters showed more efficient inhibition of bacteria when compared to monovalent sugar units.

Multivalent assembly of a pyrene functionalized thio-N-acetylglucosamine: Synthesis, spectroscopic and WGA binding studies

We introduce here a new fluorescent derivative of 1-thio-β-N-acetylglucosamine linked to a pyrene system through a triazolylpentyl spacer, designed to self-assemble into a multivalent glycocluster. The synthesis was achieved by efficient CuAAC click reaction between a pyrene functionalized with an azide group and a suitable alkynyl thiomonosaccharide. Spectroscopic studies by fluorometry indicated that the self-assembly in aqueous medium is modulated by concentration and pH changes, the latter due to the presence of the amino group close to the π system. Circular dichroism experiments revealed a moderate positive signal, suggesting that the pyrene-thioGlcNAc conjugate can aggregate into a chiral supramolecular assembly. The sugar moiety showed to specifically and reversibly interact with the wheat germ agglutinin, a fact that was demonstrated by turbidity assay. SEM microscopy of a lyophilized solution at pH 10 revealed a fibrillar morphology compatible with the presence of tubular micelles, whereas crystalline and amorphous solids are formed at lower pHs.

Direct and Regioselective Di-α-fucosylation on the Secondary Rim of β-Cyclodextrin.

A straightforward glycosylation method is described to regio‐ and stereoselectively introduce two α‐l‐fucose moieties directly to the secondary rim of β‐cyclodextrin. Using NMR and MS fragmentation studies, the nonasaccharide structure was determined, which was also visualized using molecular dynamics simulations. The reported glycosylation method proved to be robust on gram‐scale, and may be generally applied to directly glycosylate β‐cyclodextrins to make well‐defined multivalent glycoclusters.

Development of a Microwave-assisted Chemoselective Synthesis of Oxime-linked Sugar Linkers and Trivalent Glycoclusters.

A rapid, high-yielding microwave-mediated synthetic procedure was developed and optimized using a model system of monovalent sugar linkers, with the ultimate goal of using this method for the synthesis of multivalent glycoclusters. The reaction occurs between the aldehyde/ketone on the sugars and an aminooxy moiety on the linker/trivalent core molecules used in this study, yielding acid-stable oxime linkages in the products and was carried out using equimolar quantities of reactants under mild aqueous conditions. Because the reaction is chemoselective, sugars can be incorporated without the use of protecting groups and the reactions can be completed in as little as 30 min in the microwave. As an added advantage, in the synthesis of the trivalent glycoclusters, the fully substituted trivalent molecules were the major products produced in excellent yields. These results illustrate the potential of this rapid oxime-forming microwave-mediated reaction in the synthesis of larger, more complex glycoconjugates and glycoclusters for use in a wide variety of biomedical applications.

The uncommon strong inhibition of α-glucosidase by multivalent glycoclusters based on cyclodextrin scaffolds.

The homeostasis disruption of d-glucose causes diabetes, a dramatic chronic disease worldwide. Type 1 diabetes is a successfully treatable form, where blood d-glucose is regulated by insulin treatment. In contrast type 2 diabetes, the non-insulin dependent kind, is problematic. The control of the d-glucose blood level via intestinal α-d-glucosidase inactivation can be achieved by using competitive inhibitors, such as iminosugars (e.g. acarbose) or sulfonium sugar derivatives (e.g. salacinol). Recently, an unprecedented result showed that multivalent diamond nanoparticles grafted with unmodified sugars displayed α-glucosidase inhibition at low micromolar concentrations. Herein we describe the synthesis of multivalent glycoclusters using cyclodextrins (CDs) as scaffolds and an assessment of their role as inhibitors of α-d-glucosidase. The glycoclusters were efficiently obtained from per-azido α, β and γ-CD derivatives and propargyl glycosides using click-chemistry under microwave irradiation. The methodology was successfully applied to various protected and non-protected propargylated monosaccharides, including both O- and S-glycosides, giving clear evidence of its versatility. The targeted 6-per-glycosylated CDs were isolated in moderate to excellent yields (30-90%) by silica gel chromatography. The results showed inhibition of α-glucosidase from Saccharomyces cerevisiae with IC50 values in the 32-132 μM range, lower than that of acarbose (IC50 = ∼250 μM), a well-known competitive inhibitor used in the clinical treatment of type 2 diabetes. Preliminary experiments suggest a mixed-type non-competitive inhibition mode for these new glycoclusters.

Can Heterocyclic γ-Peptides Provide Polyfunctional Platforms for Synthetic Glycocluster Construction?

Sugars play key roles in many molecular and cellular communication processes involving a family of proteins named lectins. The low affinity associated with sugar recognition is generally counterbalanced by the multivalent nature of the interaction. While many polyglycosylated architectures have been described, only a few studies focused on the impact of topology variations of the multivalent structures on the interaction with lectin proteins. One major interest of our group concerns the design of new highly predictable and stable molecular pseudo-peptide architectures for therapeutic applications. In such a context, we described a class of constrained heterocyclic γ-amino acids built around a thiazole ring, named ATCs. ATC oligomers are helical molecules resulting from the formation of a highly stable C9 hydrogen-bonding pattern. Following our program, we herein address the potential of ATC oligomers as tunable scaffolds for the development of original multivalent glycoclusters.

Supramolecular nanofiber of pyrene-lactose conjugates and its two-photon fluorescence imaging

A lactose modified pyrene derivative (Py-Lac) was synthesized, with which novel twisted supramolecular nanofibers in diameter about 20 nm were constructed by self-assembly. The nanofibers showed solid-state fluorescence between 400 nm and 650 nm with the maximum emission at 495 nm. Furthermore, its recognition reaction with PNA lectin was investigated by fluorescence spectra and turbidity assays. It is interesting found that the supramolecular assembly as multivalent glycocluster exhibited unique and selectively binding interactions with PNA lectin with the binding constant of 5.74 × 106 M−1. Moreover, compound Py-Lac showed two-photon fluorescence imaging with Hep G2 cells.

Mechanistic Insight into Heptosyltransferase Inhibition by using Kdo Multivalent Glycoclusters.

The synthesis of unprecedented multimeric Kdo glycoclusters based on fullerene and calix[4]arene central scaffolds is reported. The compounds were used to study the mechanism and scope of multivalent glycosyltransferase inhibition. Multimeric mannosides based on porphyrin and pillar[5]arenes were also generated in a controlled manner. Twelve glycoclusters and their monomeric ligands were thus assayed against heptosyltransferase WaaC, which is an important bacterial glycosyltransferase that is involved in lipopolysaccharide biosynthesis. It was first found that all the multimers interact solely with the acceptor binding site of the enzyme even when the multimeric ligands mimic the heptose donor. Second, the novel Kdo glycofullerenes displayed very potent inhibition (Ki =0.14 μm for the best inhibitor); an inhibition level rarely observed with glycosyltransferases. Although the observed "multivalent effects" (i.e., the enhancement of affinity of a ligand when presented in a multimeric fashion) were in general modest, a dramatic effect of the central scaffold on the inhibition level was evidenced: the fullerene and the porphyrin scaffolds being by far superior to the calix- and pillar-arenes. We could also show, by dynamic light scattering analysis, that the best inhibitor had the propensity to form aggregates with the heptosyltransferase. This aggregative property may contribute to the global multivalent enzyme inhibition, but probably do not constitute the main origin of inhibition.

Synthesis of multivalent difluorinated zanamivir analogs as potent antiviral inhibitors

An efficient synthesis of mono-, di-, tetra-, and octa-valent difluorinated zanamivir analogs as potent inhibitors of influenza virus is reported. The mono difluorinated zanamivir with an azide linker attached at the C-7 position was synthesized in good yield from sialic acid using Selectfluor® and (diethylaminosulfur trifluoride) DAST as the fluorination reagent. This key intermediate was attached on various alkynlated scaffolds via “Click Chemistry” to afford multivalent glycoclusters. A neuraminidase inhibition assay was used to evaluate the compounds for their inhibitory activity using H7N9 virus like particle. These multivalent sialosides show enhanced inhibition with IC50 values ∼100-fold better than the monomer indicative of the strength of using a multivalent approach.

Structural Insight into Multivalent Galactoside Binding to Pseudomonas aeruginosa Lectin LecA

Multivalent galactosides inhibiting Pseudomonas aeruginosa biofilms may help control this problematic pathogen. To understand the binding mode of tetravalent glycopeptide dendrimer GalAG2 [(Gal-β-OC6H4CO-Lys-Pro-Leu)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2] to its target lectin LecA, crystal structures of LecA complexes with divalent analog GalAG1 [(Gal-β-OC6H4CO-Lys-Pro-Leu)2Lys-Phe-Lys-Ile-NH2] and related glucose-triazole linked bis-galactosides 3u3 [Gal-β-O(CH2)n-(C2HN3)-4-Glc-β-(C2HN3)-[β-Glc-4-(N3HC2)]2-(CH2)n-O-β-Gal (n = 1)] and 5u3 (n = 3) were obtained, revealing a chelate bound 3u3, cross-linked 5u3, and monovalently bound GalAG1. Nevertheless, a chelate bound model better explaining their strong LecA binding and the absence of lectin aggregation was obtained by modeling for all three ligands. A model of the chelate bound GalAG2·LecA complex was also obtained rationalizing its unusually tight LecA binding (KD = 2.5 nM) and aggregation by lectin cross-linking. The very weak biofilm inhibition with divalent LecA inhibitors suggests that lectin aggregation is necessary for biofilm inhibition by GalAG2, pointing to multivalent glycoclusters as a unique opportunity to control P. aeruginosa biofilms.

Functionalized halloysite multivalent glycocluster as a new drug delivery system.

A new design for halloysite nanotube materials was obtained by grafting chemically modified cyclodextrin units onto the nanotube surface. In particular, grafted cyclodextrins were decorated with thiosaccharide pendants, in order to mimic the well-known binding of sugars to proteins and the glyco-cluster effect occurring during cellular recognition events. The obtained materials were characterized by using a combination of varied techniques (FT-IR spectroscopy, thermogravimetric analysis, scanning electron microscopy, dynamic light scattering, turbidimetry), and their potential drug-delivery abilities were tested by studying their interactions with the common naturally occurring anticancer agent curcumin. A suitable model describing the interaction between our materials and curcumin is proposed.

Molecular arrangement between multivalent glycocluster and Pseudomonas aeruginosa LecA (PA‐IL) by atomic force microscopy: influence of the glycocluster concentration

New therapeutics strategy against cystic fibrosis seeks to prevent the adhesion of the bacterium Pseudomonas aeruginosa (PA) on the epithelial cells in the lungs. One of the factors that induces the adhesion is the interaction between natural glycocluster present on the cells and lectins such as the PA lectin LecA (PA-IL) present on the bacterium. By introducing synthetic glycoclusters with a great affinity with the lectin PA-IL, the adhesion can be prevented. In this study, we characterized, by atomic force microscopy, the interaction between a tetra-galactosylated glycocluster and the PA-IL lectin for high concentration of lectins (2.5 μM).We showed that the strong lectin/lectin interaction is reduced even for low concentration of glycoclusters (1 for 20 000 lectins). We assumed that it is due to the tensioactive behavior of the glycoclusters. It was shown that the arrangement of the created complexes induced different structures evolving from one-dimensional elongated aggregates to two-dimensional compact islands when increasing the glycocluster concentration. This evolution can be interpreted as the predominance of the glycocluster/lectin interaction.

Synthesis of Octopus Type Trimethylene Glycol β-D-glucopyranosides

ABSTRACT The octopus type oligo-hydroxypropylene glycol β-D-glucopyranosides were successfully synthesized in this studies: the sterospecific compound of allyl 2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosides (1) was deacetylated with NaOMe in MeOH lead to allyl-β-D-glucopyranoside (2) which was perallylated and followed by hydroboration and subsequent oxidation afforded (3-hydroxy-propyl) 2, 3, 4, 6-tetraO(3-hydroxy-propyl)- β-D-glucopyranoside (4). As a result, not only allylpropylene glycol β-D-glucopyranosides (5, 7, and 9) but also hydroxypropylene glycol β-D-glucopyranosides (6, 8, and 10) were synthesized by repeated sequential perallylation followed by hydroboration/oxidation. Keywords : hydroboration/oxidation, perallylation, oligo-hydroxypropylene glycol β-D-glucopyranosides 1. INTRODUCTION Carbohydrates are naturally occurring abund-ant compounds that have been widely studied for a plethora of applications (Hanessian, 1983; Scott, 1994; Middleton et al., 1993). Of parti-cular interest has been their use as polyfunctional molecules for the design and/or synthesis of car-bohydrate-centered multivalent glycoclusters and glycodendrimers (Lindhorst et al., 1998; Lind-horst et al., 2001; Lindhorst et al., 2006). Most of them are based on a multi-step synthesis con-sisting of oligo-glycosylation in order to achieve a uniform O-hydroxy-spacer, such as ethylene or propylene glycol not only in the 2, 3, 4, 6-tetra hydroxy groups but also at the α-anomeric po-sition, turning them into carbohydrate-centered cluster glycosides (Lindhorst et al., 1998; Lind-horst et al., 2001; Lindhorst et al., 2006).Thus, it became our goal to attempt the syn-thesis of the exhaustive glycosylation of octopus type β-D-glucopyranosides. This could provide us with a great potential opportunity of a novel class of high-molecular weight monosaccharide mimetics, which could possibly be adapted for the generation of various spacers with different

Synthesis, optical properties and binding interactions of a multivalent glycocluster based on a fluorescent perylene bisimide derivative

A water-soluble fluorescent perylene bisimide derivative, PBI-Cl-Man, grafted with six mannose groups was synthesized. Its optical properties were investigated by UV-vis and fluorescence spectra, which indicated that compound PBI-Cl-Man exists in the monomer state at low concentrations and exhibits high fluorescence with a fluorescence quantum yield of 59.9% in water. Furthermore, the binding interactions of PBI-Cl-Man with Con A were studied by fluorescence spectra, CD spectra, turbidity assays and ELLA experiments. The almost complete quenching of the fluorescence upon binding with Con A was observed, with a binding constant of 2.4 × 107 M−1 (4.0 × 106 M−1 for monomeric mannose, valency corrected), nearly 3 orders of magnitude higher than that of monovalent mannose ligands (α-MMP). The ELLA experiments indicated that PBI-Cl-Man, as a multivalent glycocluster, exhibits a strong binding interaction with Con A with an IC50 value of 34 μM (204 μM, valency corrected), 7 times stronger than that of the reference compound (α-MMP).

Efficient Synthesis of Thiolactoside Glycoclusters by Ruthenium‐Catalyzed Cycloaddition Reaction of Disubstituted Alkynes on Carbohydrate Scaffolds

AbstractLectin‐carbohydrate interactions are responsible for several cellular processes involved in the immune system and the development of certain types of cancer. To further understanding of the cellular responses triggered by these interactions, complex carbohydrates are designed and prepared. Here, we describe the synthesis of a family of multivalent glycoclusters based on carbohydrate cores bearing thiolactosides or thiogalactosides as recognition elements with structural valencies ranging from 2 to 8. The synthetic strategy involves a key ruthenium‐catalyzed cycloaddition reaction between symmetric disubstituted alkynes bearing two thiosugar units and azide‐containing carbohydrate scaffolds. This methodology afforded high‐valency glycoconjugates in good to excellent yields. Binding affinities of the synthetic β‐thiolactosides for peanut lectin were measured by isothermal titration calorimetry. These titrations revealed micromolar affinities as well as a multivalent effect. A tetravalent glycoconjugate based on a trehalose scaffold displayed the highest binding affinity.

Multivalent glycoclusters constructed by chiral self-assembly of mannose functionalized perylene bisimide

Water-soluble perylene bisimide derivative 7 modified with six mannoses was synthesized and its self-assembled properties were studied by UV-Vis and CD spectroscopy, which revealed an interesting self-assembly with a solvent-tuning chiral conformation in H(2)O-DMSO solution. As H(2)O was added to the DMSO solution until a 60% (or 70%) v/v proportion was achieved, the self-assembly of the mannose functionalized compound 7 exhibited a left-handed helical conformation. More interestingly, when the volume of H(2)O constituted beyond 85% of the solution, the conformation of the self-assembly turned out to be a right-handed helical conformation. Furthermore, the binding interactions between the self-assembly of compound 7 and Con A were investigated by turbidity assay, CD spectra, TEM and SEM images, and ELLA experiment, which indicated that the self-assembly of compound 7 as multivalent glycoclusters exhibited specific binding to Con A with an IC(50) value of 24 μM (144 μM, valency corrected), 10 times stronger than the reference compound (α-MMP).

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.

Chiral self-assembly of lactose functionalized perylene bisimides as multivalent glycoclusters

A chiral self-assembly has been constructed from a d-lactose functionalized perylene bisimide derivative, showing right-handed supramolecular stacking induced by the chiral d-lactose moieties. Benefiting from the grafting of D-lactose, the self-assembled multivalent glycoclusters exhibited specific binding with PNA lectin.