Vanya D. Uzunova

Supramolecular tandem enzyme assays for multiparameter sensor arrays and enantiomeric excess determination of amino acids.

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.

Operational calixarene-based fluorescent sensing systems for choline and acetylcholine and their application to enzymatic reactions

Electron-rich anionic calixarenes and resorcinarenes are known receptors for trimethylammonium-containing neurotransmitters, but the development of practical sensor applications has been impeded by the lack of suitable supramolecular sensing ensembles as well as the low selectivity and sensitivity of the macrocyclic cation-receptor hosts. The host–guest complexes between p-sulfonatocalix[n]arenes (n = 4–5) and the cationic aromatic fluorescent dye lucigenin (LCG) have been characterised by optical spectroscopic techniques, NMR, cyclic voltammetry, isothermal titration calorimetry, and X-ray crystallography. The dye is complexed with binding constants of the order of 107 M−1 and undergoes a strong static fluorescence quenching (factor 140) upon complexation as a consequence of exergonic electron transfer within the complex. LCG has been utilised in combination with p-sulfonatocalix[4]arene to set-up a refined reporter pair for label-free continuous real-time enzyme assays according to the supramolecular tandem assay principle. This affords product-selective tandem assays for amino acid decarboxylases with a one order of magnitude higher sensitivity and a 3 orders of magnitude lower host/dye concentration range, a convenient substrate-selective tandem assay for direct monitoring of choline oxidase, and a conceptually novel substrate-selective enzyme-coupled tandem assay for acetylcholinesterase. The applicability of the method to the measurement of enzyme-kinetic parameters, the screening for inhibitors of acetylcholinesterase, and the highly selective determination of absolute, low micromolar concentrations of both choline and acetylcholine by simple fluorescence measurements has been demonstrated. A domino tandem assay can be employed to measure the two analytes in the same sample. The described applications bypass problems related to the unselective binding of the macrocycle by coupling the signalling event with highly specific enzymatic transformations.

Supramolecular logic with macrocyclic input and competitive reset

The fluorescence of a designed water-soluble 4-sulfonato-1,8-naphthalimide dye can be switched on by the synchronous host-guest complexation with cucurbit[7]uril and decrease in pH (from 9 to 7). This affords a dual resettable (by addition of cadaverine as competitor or by deprotonation) logic gate based on modulation of photoinduced electron transfer.

A photoinduced pH jump applied to drug release from cucurbit[7]uril

A proof-of-principle for the application of a photoinduced pH jump for delivery of the Hoechst 33258 drug by disassembly of its host-guest complex with cucurbit[7]uril is described.

Triple Emission from p‐Dimethylaminobenzonitrile–Cucurbit[8]uril Triggers the Elusive Excimer Emission

The intriguing dual-emission behavior of p- dimethylaminobenzonitrile (DMABN) and the identity of the associated excited states is, arguably, the most extensively investigated and also controversially discussed molecule- specific phenomenon of modern photochemistry. We have now found a new, third fluorescence band when DMABN is encapsulated within the water-soluble molecular container cucurbit[8]uril (CB8). It is centered between the previously observed emissions and assigned to the elusive excimer emission from DMABN through 1:2 CB8:DMABN complex formation. Heating of the CB8⋅(DMABN)2 complex from 0 to 100 °C results in the dissociation of the ternary complex and restoration of the dual-emission properties of the monomer. Alternatively, monomer emission can be obtained by selecting cucurbit[7]uril (CB7), a host homologue that is too small to accommodate two DMABN molecules, or by introducing ethyl instead of methyl groups at the amino terminus of the aminobenzonitrile guest.

Inclusion of neutral guests by water-soluble macrocyclic hosts – a comparative thermodynamic investigation with cyclodextrins, calixarenes and cucurbiturils

Abstract The driving forces of association between three different families of macrocycles as hosts, namely cyclodextrins (α-, β-, and γ-), p-sulfonatocalix[n]arenes (n = 4–6) as well as cucurbit[n]urils (n = 6–8), and three different bicyclic azoalkane homologues as guests, namely 2,3-diazabicyclo[2.2.1]hept-2-ene (DBH), 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as well as 2,3-diazabicyclo[2.2.3]non-2-ene (DBN), were examined by means of calorimetric titrations, NMR spectroscopy and molecular dynamics simulation, all in aqueous solution. The small, spherical and uncharged guests preferably bind inside the cavities of the medium sized hosts. The inclusion complexation by β-cyclodextrin and p-sulfonatocalix[4]arene shows medium binding affinities (millimolar), while cucurbit[7]uril macrocycle shows very strong binding (micromolar). For all types of macrocycles, the complex formation is enthalpically driven (ΔH° < 0), accompanied by slightly unfavourable entropy changes (ΔS° < 0). The results are discussed in terms of the flexibility of the hosts, the hydrophobic character of their cavities and the release of high-energy water upon binding, and generalised by including two additional guests, the ketones cyclopentanone and (+)-camphor.