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.
A rapid synthetic route to a nontoxic fluorescently labeled water-soluble calixarene has been developed. Investigation of the cellular uptake of the labeled calixarene, via confocal microscopy, through coincubation with uptake inhibitors demonstrates that uptake is not through the common clathrin coated pits or caveolae (lipid raft) endocytic pathways and that the calixarene derivative localizes within the cytoplasm and does not enter the nucleus. The study demonstrates the power of fluorescent labeling for investigation of interactions between calixarenes and biological systems and the potential for calixarene based intracellular imaging agents
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.
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. ' 2010 International Society for Advancement of Cytometry Key termslysosome; calixarene; cell-penetration; immunofluorescence CALIXARENES (1) are a family of readily synthesized and functionalized macrocycles which have found applications in a number of research areas. In particular the ability of derivatives to complex anions (2,3) and cations (4,5) has been exploited for the development of sensors. Recently, interest has focused on the use of calixarene derivatives in biological systems be it as drugs, drug delivery systems, or imaging agents. In the design of drug molecules, particular attention has been paid to calixarenes as antimicrobials (6-8), as vaccines (9) and as anticancer agents (10). In the field of drug delivery, agents for nucleic acids based on single calixarenes (11-13) have shown promise and we have recently shown that larger arrays of cationic calix[4]arene, so-called multicalixarenes, are able to transfect DNA effectively (14). In addition a number of studies have combined the ability of calixarene derivatives to complex cations with their low toxicity in the development of noncovalently labeled protein MRI imaging agents (15,16).While the nontoxic (17) and non-immunogenicity (18,19) status of calixarenes has been established, less information is available on how calixarenes are processed within cells and their cellular fate, both of which are important features which must be characterized before such derivatives are taken further for clinical development. We recently demonstrated that fluorescently labeled calix [4]arene have the potential to track progress of the macrocycle in cells, as an NBD labeled cationic derivative was readily and speedily taken up into cells and provided a very stable, nontoxic read-out system to investigate cellular localization (20). The absorbance and emission spectra for the NBDCalAm showed that it can easily be visualized using a GFP filter (20).
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