Mara Florea

• The present doctoral thesis describes the development of novel fluorescence-based sensors for real-time monitoring of analyte concentration changes. Water-soluble macrocycles, such as cyclodextrins, calixarenes, cyclophanes, or cucurbiturils, and their ability to encapsulate biologically and environmentally relevant analytes, as well as fluorescent dyes are the basis of such sensing systems. The first part of the thesis focuses on cucurbiturils, highly symmetric and rigid pumpkin-shaped macrocycles, comprised of two carbonyl-lined portals and a hydrophobic cavity. Herein, the reversible encapsulation of neutral guests, such as volatile hydrocarbons by cucurbiturils has been investigated using a fluorescent indicator displacement approach. Measurements in salt-free aqueous solution have disclosed a surprisingly strong (binding constants up to 107 M-1) and highly selective binding towards the investigated hydrocarbons (differentiation of alkanes from alkenes, isoalkanes from n-alkanes, and cis- from trans-alkenes), contrasting the common conception of cucurbiturils as cation receptors. The second part of the thesis relates to the area of enzyme assay design, which allows real-time monitoring of analyte changes during biochemical transformations. The first introduced assay exploits the use of anion-receptor macrocycles, for following ATP dephosphorylation via a fluorescence-based supramolecular tandem assay. The methodology has also been exploited to screen for activators of the model enzyme employed, potato apyrase, as well as to monitor the dephosphorylation of other nucleotides. The second assay, also performed in accordance with the supramolecular tandem strategy, enables a label-free, continuous monitoring of histone tail peptide methylation in homogeneous solution. The third project employs nuclear magnetic resonance spectroscopy for experimental testing of molecules with inhibitory potential against L-aspartate-alpha-decarboxylase, an enzyme known for its critical role in the growth of microorganisms, such as Mycobacterium tuberculosis.