Implementation of anion-receptor macrocycles in supramolecular tandem assays for enzymes involving nucleotides as substrates, products, and cofactors

Abstract

A supramolecular tandem assay for direct continuous monitoring of nucleotide triphosphate-dependent enzymes such as potato apyrase is described. The underlying principle of the assay relies on the use of anion-receptor macrocycles in combination with fluorescent dyes as reporter pairs. A combinatorial approach was used to identify two complementary reporter pairs, i.e. an amino-gamma-cyclodextrin with 2-anilinonaphtalene-6-sulfonate (ANS) as dye (fluorescence enhancement factor of 17 upon complexation) and a polycationic cyclophane with 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS) as dye (fluorescence decrease by a factor of more than 2000), which allow the kinetic monitoring of potato apyrase activity at different ATP concentration ranges (microM and mM) with different types of photophysical responses (switch-ON and switch-OFF). Competitive fluorescence titrations revealed a differential binding of ATP (strongest competitor) versus ADP and AMP, which constitutes the prerequisite for monitoring enzymatic conversions (dephosphorylation or phosphorylation) involving nucleotides. The assay was tested for different enzyme and substrate concentrations and exploited for the screening of activating additives, namely divalent transition metal ions (Ni(2+), Mg(2+), Mn(2+), and Ca(2+)). The transferability of the assay could be demonstrated by monitoring the dephosphorylation of other nucleotide triphosphates (GTP, TTP, and CTP).

Determining Protease Substrate Selectivity and Inhibition by Label-Free Supramolecular Tandem Enzyme Assays

“…Cucurbit [7]uril (CB7, Figure 1) is a water-soluble macrocycle that has been investigated extensively in biological applications including drug delivery, [29][30][31][32][33] interactions with enzymes, 34,35 plasma membrane protein fishing, 36 and label-free enzyme assays. [22][23][24][25] The repeating glycoluril units produce a barrel-shaped container that has a hydrophobic cavity and negatively charged portals. 37 The latter are capable, not only for CB7 but also for its homologues, of binding inorganic cations as well as the cationic sites of organic guests, mostly ammonium groups; nonpolar groups are preferentially immersed in the inner cavity.…”

“…For this purpose, the observed fluorescence decay needed to be related to changes in absolute concentration. 25 This relationship was achieved by recording the fluorescence response obtained by addition of a known quantity of an authentic sample of reaction product (see Supporting Information). Analysis of the initial reaction rates at varying substrate concentrations (Figure 3b) yielded the characteristic proteolytic constants (kcat/KM) for the different peptide sequences (inset of Figure 3b and Table 1).…”

“…[22][23][24][25] Until now, the technique was limited to substrates and products which, owing to their low molecular weight, could essentially be fully immersed in the macrocyclic host cavity, such that the entire analyte, e.g. arginine or cadaverine, served as recognition motif.…”

Cucurbiturils: from synthesis to high-affinity binding and catalysis

“…191 Symmetrical and unsymmetrical bispyridyl ethylenes, as well as stilbazole derivatives were employed in related photocycloaddition reactions; through a combination of best fit and a minimization of electrostatic repulsion within the CB8 cavity, new reactions could be designed. 192 Ramamurthy further extended this chemistry to include the photodimerization of neutral guests, such as trans-cinnamic acid (20). 189,190 Upon irradiation of 20 encapsulated by CB8 (in aqueous solution as well as in the solid state), the reaction afforded the syn head-to-head dimer (21), along with the corresponding cis isomer (22).…”

Dynamic Octopus Amphiphiles as Powerful Activators of DNA Transporters: Differential Fragrance Sensing and Beyond

“…Compared with the many elegant, often much more practical differential chemosensors reported over the past two decades, [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] sensing systems that work in lipid bilayers are of interest because subtle differences in structure are magnified by covalent capture of several analytes by the same Figure 5. Dose-response curves for DNA activation by guanidinium hydrazides G1H4 (a) and G1H3 (c) after incubation with o-anisaldehyde O11 (*), m-anisaldehyde O12 (*), and p-anisaldehyde O13 (&).…”