Roland J. Pieters

Wedgelike glycodendrimers as inhibitors of binding of mammalian galectins to glycoproteins, lactose maxiclusters, and cell surface glycoconjugates.

Galectins are mammalian carbohydrate‐binding proteins that are involved in cell–cell and cell–matrix adhesion, cell migration, and growth regulation with relevance to inflammation and tumor spread. These important functions account for the interest to design suitable low molecular weight inhibitors that match the distinct modes of presentation of the carbohydrate recognition domains of the different galectin subfamilies. Using 3,5‐di‐(2‐aminoethoxy)benzoic acid as the branching unit, wedgelike glycodendrimers with two, four, and eight lactose moieties (G1–G3) were synthesized. They were tested in solid‐phase competition assays with lactose maxiclusters and various N‐glycan branching profiles (miniclusters) as the matrix and also in cell assays. Prototype galectins‐1 and ‐7, chimera‐type galectin‐3, a plant (AB)2 toxin, and a lactose‐binding immunoglobulin G fraction from human serum were the carbohydrate‐binding targets. Potent inhibition and remarkable cluster effects were seen for the homodimeric galectin‐1, especially in combination with biantennary N‐glycans as the matrix. Remarkably, for the tetravalent G2 glycodendrimer, the inhibitory potency of each lactose unit reached a maximum value of 1667 relative to free lactose. In haemagglutination experiments as a model for cell adhesion, galectin‐3 was markedly sensitive to increased sugar valency and a relative potency per lactose of 150 was reached. The spatial orientation of the carbohydrate recognition domains of the endogenous lectins and the branching pattern of the carbohydrates of the glycoprotein matrices used are both important factors in the design and synthesis of glycodendrimers with galectin‐selective properties.

Homodimeric galectin-7 (p53-induced gene 1) is a negative growth regulator for human neuroblastoma cells

The extracellular functions of galectin-7 (p53-induced gene 1) are largely unknown. On the surface of neuroblastoma cells (SK-N-MC), the increased GM1 density, a result of upregulated ganglioside sialidase activity, is a key factor for the switch from proliferation to differentiation. We show by solid-phase and cell assays that the sugar chain of this ganglioside is a ligand for galectin-7. In serum-supplemented proliferation assays, galectin-7 reduced neuroblastoma cell growth without the appearance of features characteristic for classical apoptosis. The presence of galectin-3 blocked this effect, which mechanistically resembles that of galectin-1. By virtue of carbohydrate binding, galectin-7 thus exerts neuroblastoma growth control similar to galectin-1 despite their structural differences. In addition to p53-linked proapoptotic activity intracellularly, galectin-7, acting as a lectin on the cell surface, appears to be capable of reducing cancer cell proliferation in susceptible systems.

Chiral rhodium(II) carboxamides. A new class of catalysts for enantioselective cyclopropanation reactions

Abstract Chiral Rh2L4 (L = 4-substituted-oxazolidinones and methyl 2-pyrrolidone-5-carboxylate) having two nitrogen-rhodium bonds in the cis geometry at each rhodium face are effective catalysts for enantioselective intermolecular cyclopropanation reactions.

Rigidified multivalent lactose molecules and their interactions with mammalian galectins: a route to selective inhibitors

New and rigid multivalent lactose molecules were prepared. The structures contain lactose-2-aminothiazoline units at the periphery that were formed from a cyclisation of the thiourea sulphur onto the triple bond of the spacer. The lactosides were evaluated as inhibitors against lectin binding in a solid phase inhibition assay. In this assay the glycoprotein asialofetuin was immobilized onto the surface of microtiter plate wells, mimicking cell surface presentation, while mammalian galectins-1, -3 or -5 were in solution. Between the three galectins, the folding pattern and sequence are closely related but the topology of presentation of the carbohydrate recognition domains differs. Strong multivalency effects were observed for the tetravalent lactoside in the inhibition of galectin-3 binding with enhancements of almost 4300-fold compared to lactose. Remarkable selectivity was obtained in the inhibition since relative potencies of the tetravalent lactoside with the proto type galectins-1 and -5 did not exceed a factor of 143 relative to lactose. The binding of the lactosides to galectin-3 was also studied by fluorescence spectroscopy with all components in solution. These studies showed no multivalency effects in the inherent binding affinities.

Inhibition of Streptococcus suis Adhesion by Dendritic Galabiose Compounds at Low Nanomolar Concentration

A series of mono-, di-, and tetravalent galabiose (Galalpha1-4Gal) compounds were synthesized in good yields by coupling of a general carboxylic acid-bearing sugar building block to dendritic scaffolds based on the 3,5-di-(2-aminoethoxy)benzoic acid branching unit. Furthermore, a poly(amidoamine)- (PAMAM-) based dendritic galabioside was synthesized containing eight galabiose units. All galabiosides were tested in a hemagglutination assay and a surface plasmon resonance (SPR) competition assay in order to establish their potency in the binding to the bacterial Gram-positive pathogen Streptococcus suis. A monovalent galabioside containing a short spacer was used as a reference compound in all the assays. Variations in the scaffold as well as in the spacer arms were introduced to determine their influence on the inhibition. The best inhibitor of hemagglutination was an octavalent galabioside with a minimal inhibitory concentration (MIC) of 0.3 nM, to the best of our knowledge the first example of inhibition of bacterial binding by a soluble carbohydrate at a subnanomolar concentration.

Multivalent Carbohydrate Recognition on a Glycodendrimer-Functionalized Flow-Through Chip

Dendrimers were fitted out with up to eight mannose moieties by “click” chemistry. They were subsequently attached to aluminum oxide chips via a spacer that was linked to the dendrimer core; this resulted in a microarray of glycodendrimers. Binding of the glycodendrimers to the fluorescent lectins ConA and GNA was observable in real time. In a single experiment it was possible to observe the multivalency enhancement or cluster effect in the binding event. This effect was small for ConA, in agreement with its widely spaced binding sites, whereas it was large for GNA, with its twelve much more closely spaced binding sites. The dendrimer‐fitted chip represents a valuable screening tool for multivalency effects. Furthermore kinetic and thermodynamic data on binding events can be deduced. Inhibition experiments are also possible with the system as was shown for ConA with α‐methyl mannose as the inhibitor.

Inhibition and Detection of Galectins

More and more studies report on the roles that galectins play in numerous types of cancer. These roles can be varied, as has been shown particularly for galectin‐3. These studies have created the need for inhibitors that can block unwanted effects, and the need to detect galectins in tissues, in order to better understand their role, and aid in diagnosis and prognosis. Since galectins bind β‐galactosides, monovalent galactose‐derived inhibitors have been prepared but also peptidic ones have appeared. Since galectins often induce crosslinking and partake in aggregation phenomena, multivalency has been a successful design element in inhibitor development. Currently, there are no cheap and convenient solutions available for the detection of, ideally multiple, galectins in tissue samples, although antibody‐based methods such as ELISA and Western blot analysis are being used. Besides these, a chemical probe‐based method also shows potential as an alternative.

A new chemical probe for proteomics of carbohydrate-binding proteins.

Selective capture of galectins, while leaving other proteins untouched, was achieved by activating photoaffinity labels that were precisely positioned on noncovalently bound carbohydrate ligands. The labelled proteins were visualised in-gel by “clicking-on” a rhodamine moiety afterwards.

Efficient microwave-assisted synthesis of multivalent dendrimeric peptides using cycloaddition reaction (click) chemistry

Multivalent dendrimeric peptides were synthesized via a microwave-assisted Huisgen 1,3-dipolar cycloaddition between azido peptides and dendrimeric alkynes in yields ranging from 46 to 96%.

The influence of ligand valency on aggregation mechanisms for inhibiting bacterial toxins.

Divalent and tetravalent analogues of ganglioside GM1 are potent inhibitors of cholera toxin and Escherichia coli heat‐labile toxin. However, they show little increase in inherent affinity when compared to the corresponding monovalent carbohydrate ligand. Analytical ultracentrifugation and dynamic light scattering have been used to demonstrate that the multivalent inhibitors induce protein aggregation and the formation of space‐filling networks. This aggregation process appears to arise when using ligands that do not match the valency of the protein receptor. While it is generally accepted that multivalency is an effective strategy for increasing the activity of inhibitors, here we show that the valency of the inhibitor also has a dramatic effect on the kinetics of aggregation and the stability of intermediate protein complexes. Structural studies employing atomic force microscopy have revealed that a divalent inhibitor induces head‐to‐head dimerization of the protein toxin en route to higher aggregates.