Stable and sensitive probes for lysosomes: cell-penetrating fluorescent calix[4]arenes accumulate in acidic vesicles.

The uptake of a fluorescently labeled cationic calix[4] (NBDCalAm) in live, nonfixed cells has been investigated. The compound is taken into the cells rapidly and shows distinct endosomal distribution after 2 hours. This distribution pattern shows colocalization with lysosomal staining. The uptake is not altered by inhibition of clathrin or caveolae dependent pathways nor by depletion of the cellular ATP-pool. Immediately after uptake the probe is localized in the Golgi and brefeldin A treatment prevents transport to lysosomes. Pulse chase experiments with bafilomycin A1, monensin, and sodium azide showed that accumulation and retention of the probe in lysosomes is primarily driven by the activity of vacuolar ATPases. The NBD labeled calix[4]arene provides a very stable and sensitive marker for lysosomes, and has a considerable advantage over some commercially available lysosomal markers in so far that the fluorescent signal is stable even when the cells are incubated in dye-free medium after staining.

Taking multicalixarenes into the nanoworld: first third-generation calixarene dendrimer.

We report the first synthesis of a dendritic multicalixarene, featuring twenty-one calixarene units, which when adsorbed onto mica, forms regular assemblies which can then further aggregate to form larger clusters.

Achieving high affinity towards a bacterial lectin through multivalent topological isomers of calix[4]arene glycoconjugates.

A family of seven topologically isomeric calix[4]arene glycoconjugates was prepared through the synthesis of a series of alkyne-derivatised calix[4]arene precursors that are suitable for the attachment of sugar moieties by microwave-assisted copper(I)-catalysed azide-alkyne cycloaddition (CuAAC). The glycoconjugates thus synthesised comprised one mono-functionalised derivative, two 1,2- or 1,3-divalent regioisomers, one trivalent and three tetravalent topoisomers in the cone, partial cone or 1,3-alternate conformations. The designed glycoconjugates were evaluated as ligands for the galactose-binding lectin PA-IL from the opportunistic bacterium Pseudomonas aeruginosa, a major causative agent of lung infections in cystic fibrosis patients. Binding affinities were determined by isothermal titration calorimetry (ITC), and the interaction with the lectin was shown to be strongly dependant on both the valence and the topology. Whereas the trivalent conjugate displayed enhanced affinity when compared to a monosaccharide model, the tetravalent conjugates are to-date the highest-affinity ligands measured by ITC. The topologies presenting carbohydrates on both faces of calixarene are the most potent ones with dissociation constants of approximately 200 nM. Molecular modelling suggests that such a multivalent molecule can efficiently chelate two of the binding sites of the tetrameric lectin; this explains the 800-fold increase of affinity achieved by the tetravalent molecule. Surface plasmon resonance (SPR) experiments confirmed that this glycoconjugate is the strongest inhibitor for binding of PA-IL to galactosylated surfaces for potential applications as an anti-adhesive agent.

Efficient gene transfection with functionalised multicalixarenes.

Novel amino-functionalised multicalixarenes have been synthesised which show low cellular toxicity, effective DNA binding and, when featuring aliphatic amines, are efficient gene transfection agents.