We introduce a new economic, convenient and general assay principle based on the reversible interaction of water-soluble macrocycles and fluorescent dyes. We show that amino acid decarboxylase activity can be continuously monitored by measuring changes in fluorescence, which result from the competition of the enzymatic product and the dye for forming a complex with a cucurbituril or calixarene macrocycle. The new assay provides a complementary method to the use of antibodies, radioactive markers and labeled substrates.

Attractive in theory and confirmed to exist, anion-pi interactions have never really been seen at work. To catch them in action, we prepared a collection of monomeric, cyclic and rod-shaped naphthalenediimide transporters. Their ability to exert anion-pi interactions was demonstrated by electrospray tandem mass spectrometry in combination with theoretical calculations. To relate this structural evidence to transport activity in bilayer membranes, affinity and selectivity sequences were recorded. pi-acidification and active-site decrowding increased binding, transport and chloride > bromide > iodide selectivity, and supramolecular organization inverted acetate > nitrate to nitrate > acetate selectivity. We conclude that anion-pi interactions on monomeric surfaces are ideal for chloride recognition, whereas their supramolecular enhancement by pi,pi-interactions appears perfect to target nitrate. Chloride transporters are relevant to treat channelopathies, and nitrate sensors to monitor cellular signaling and cardiovascular diseases. A big impact on organocatalysis can be expected from the stabilization of anionic transition states on chiral pi-acidic surfaces.

More than 100 bacterial isolates from various marine habitats were screened for AHL production by using gfp reporter constructs based on the lasR system of Pseudomonas aeruginosa and the luxR system of Vibrio fischeri. Of the 67 Alphaproteobacteria tested, most of which belonged into the so-called Roseobacter clade, 39 induced fluorescence in either one or both sensor strains up to 103-fold compared to controls. Acylated homoserine lactones were identified by GC-MS analysis and shown to have chain lengths of C8, C10, C13-C16, and C18. One or two double bonds were often present, while a keto or hydroxyl group occurred only rarely in the side chain. Most strains produced several different AHLs. C18-en-HSL and C18-dien-HSL were produced by Dinoroseobacter shibae, an aerobic anoxygenic phototrophic bacterium isolated from dinoflagellates, and are among the longest AHLs found to date. Z7-C14-en-HSL, which has previously been detected in Rhodobacter sphaeroides, was produced by Roseovarius tolerans and Jannaschia helgolandensis. This signal molecule was synthesised and shown to induce a similar response to the culture supernatant in the respective sensor strain. The widespread occurrence of quorum-sensing compounds in marine Alphaproteobacteria, both free-living strains and those associated to eukaryotic algae, points to a great importance of this signalling mechanism for the adaptation of the organisms to their widely different ecological niches.

A combination of moderately selective host-guest binding with the impressive specificity of enzymatic transformations allows the real-time monitoring of enzymatic reactions in a homogeneous solution. The resulting enzyme assays ("supramolecular tandem assays") exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate and product. Two examples of enzymatic reactions were investigated: the hydrolysis of arginine to ornithine catalyzed by arginase and the oxidation of cadaverine to 5-aminopentanal by diamine oxidase, in which the substrates have a higher affinity to the macrocycle than the products ("substrate-selective assays"). The depletion of the substrate allows the fluorescent dye to enter the macrocycle in the course of the enzymatic reaction, which leads to the desired fluorescence response. For arginase, p-sulfonatocalix[4]arene was used as the macrocycle, which displayed binding constants of 6400 M(-1) with arginine, 550 M(-1) with ornithine, and 60,000 M(-1) with the selected fluorescent dye (1-aminomethyl-2,3-diazabicyclo[2.2.2]oct-2-ene); the dye shows a weaker fluorescence in its complexed state, which leads to a switch-off fluorescence response in the course of the enzymatic reaction. For diamine oxidase, cucurbit[7]uril (CB7) was used as the macrocycle, which showed binding constants of 4.5 x 10(6) M(-1) with cadaverine, 1.1 x 10(5) M(-1) with 1-aminopentane (as a model for the thermally unstable 1-aminopentanal), and 2.9 x 10(5) M(-1) with the selected fluorescent dye (acridine orange, AO); AO shows a stronger fluorescence in its complexed state, which leads to a switch-on fluorescence response upon enzymatic oxidation. It is demonstrated that tandem assays can be successfully used to probe the inhibition of enzymes. Inhibition constants were estimated for the addition of known inhibitors, i.e., S-(2-boronoethyl)-L-cysteine and 2(S)-amino-6-boronohexanoic acid for arginase and potassium cyanide for diamine oxidase. Through the sequential coupling of a "product-selective" with a "substrate-selective" assay it was furthermore possible to monitor a multistep biochemical pathway, namely the decarboxylation of lysine to cadaverine by lysine decarboxylase followed by the oxidation of cadaverine by diamine oxidase. This "domino tandem assay" was performed in the same solution with a single reporter pair (CB7/AO).

The coupling of an enzymatic transformation with dynamic host-guest exchange allows the unselective binding of macrocycles to be used for highly selective analyte sensing. The resulting supramolecular tandem enzyme assays require the enzymatic substrate and its corresponding product to differ significantly in their affinity for macrocycles, for example, cation receptors, and to show a differential propensity to displace a fluorescent dye from its host-guest complex. The enzymatic transformation results in a concomitant dye displacement that can be accurately followed by optical spectroscopy, specifically fluorescence. By exploiting this label-free continuous enzyme assay principle with the fluorescent dye Dapoxyl and the macrocyclic host cucurbit[7]uril, a multiparameter sensor array has been designed, which is capable of detecting the presence of amino acids (e.g. histidine, arginine, lysine, and tyrosine) and their decarboxylases. Only in the presence of both, the particular amino acid and the corresponding decarboxylase, is the amine or diamine product formed. These products are more highly positively charged than the substrate, have a higher affinity for the macrocycle and, therefore, displace the dye from the complex. The extension of the high selectivity and muM sensitivity of the tandem assay principle has also allowed for the accurate measurement of D-lysine enantiomeric excesses of up to 99.98 %, as only the L-enantiomer is accepted by the enzyme as a substrate and is converted to the product that is responsible for the observed fluorescence signal.