Small molecule fluorescent probes for alcohol dehydrogenase, catecholamines and copper(I) ion

Abstract

Due to the non-destructive nature and high spatiotemporal resolution in fluorescence imaging, small molecule fluorescent probes are powerful bioanalytical tools for studying biologically important species in living systems such as live-cell fluorescence imaging. In this thesis, the development of small molecule fluorescent probes for alcohol dehydrogenase, catecholamines and copper(I) ion detection is described.

In Chapter 1, fundamental principles of fluorescence emission is described. Applications of small molecule fluorescent probes in biological species sensing is also discussed with a number of recently published examples.

In Chapter 2, the development of alcohol dehydrogenase (ADH) fluorescent probes is described. Five ADH probes have been synthesized by covalently linking different fluorophores with a 3-hydroxypropyl group for ADH activity targeting. Fluorescence response has been tested with yeast ADH, and the triggered-release of fluorophore has been studied by LC-MS analysis. Selectivity of ADH-cyan was evaluated by testing the probe against common oxidizing agents, sugars, vitamins, amino acids, metal ions and other biologically-relevant species. Kinetics of the ADH oxidation and fluorophore release has been studied to obtain Michaelis-Menten parameters and catalytic efficiency. Molecular docking has been performed to study the binding interactions between ADH-cyan and human ADH. ADH-cyan showed a good sensitivity, selectivity, low cytotoxicity and its applicability in cellular ADH activity imaging has been studied in live human HepG2, A549 and HEK293T cells.

Chapter 3 describes chemical modifications of reaction-based catecholamines probes. Specifically, effects of electron-donating and electron-withdrawing substituents on the pyridine ligand of a catecholamine-responsive Cu(II) complex on the reactivity and selectivity towards catecholamines were studied. The derivative featuring an electron-donating methyl group was found to enhance the catecholamines response with a strong fluorescence turn-on. A good selectivity of the probe was maintained, and further modification with a NHS ester allows extracellular labelling of the probe on cell surface for extracellular catecholamines detection.

In Chapter 4, the development of an irreversible binding-based fluorescent probe CuP486 for Cu+ sensing is described. CuP486 is a [2]catenane consists of two mechanically interlocked macrocyclic ligands functionalized by a pyrene for optical output. Fluorescence response has been tested by treating CuP486 with Cu+ in PBS buffer, and selectivity against biologically relevant metal ions has been investigated. Fluorescence reversibility has been evaluated by treating Cu(I)-quenched CuP486 with strong copper binding agents. UV-Vis absorption of the copper complex was measured and relationship between light absorption and fluorescence quenching was observed. Fluorescence response has also been tested in cell lysate, and intracellular Cu+ imaging is testing in live HeLa cells.