Development of fluorescent probes for detection of hypochlorous acid, hydroxyl radical and mitochondrial pH

Abstract

Abstract of thesis entitled Development of Fluorescent Probes for Detection of Hypochlorous Acid, Hydroxyl Radical and Mitochondrial pH submitted by Xiaoyu Bai for the degree of Doctor of Philosophy at The University of Hong Kong in August 2017

In order to investigate the biological roles of reactive oxygen species and mitochondrial pH, sensitive and selective detection methods are highly needed. Small-molecule based fluorescent probes are indispensable tools because of their rapid response, high sensitivity, extraordinary temporal and spatial resolution both in vitro and in vivo. This thesis summarizes the designs, syntheses and applications of small-molecule based fluorescent probes for detection of hypochlorous acid, hydroxyl radical and mitochondrial pH. Rhodol based yellow fluorescent probe HKOCl-4 was designed for the detection of HOCl (Figure 1). HKOCl-4, which is composed of a dichlorophenol as a reaction site for HOCl and a rhodol as the fluorophore, exhibited excellent sensitivity and selectivity towards HOCl. Obvious increase in fluorescence intensity was observed of testing solution of HKOCl-4 upon treatment with HOCl, while negligible fluorescence responses was recorded towards other reactive oxygen and nitrogen species. HKOCl-4 was successfully applied in detecting HOCl in PMA stimulated RAW264.7 macrophage. Figure 1

For the detection of hydroxyl radical, green fluorescent probes Compound A and HKOH-1 were designed on the basis of the electron transfer reaction between hydroxyl radical and a bulky diiodophenol, which was coupled to fluorescein and 2′,7′-dichlorofluorescein, respectively (Figure 2). Both probes showed strong turn-on response towards hydroxyl radical generated from Fenton reagent or TCBQ/H2O2. Moreover, these probes did not respond to other ROS/RNS, which indicates that Compound A and HKOH-1 could specific distinguish hydroxyl radical from other ROS/RNS. Probe HKOH-1r, which is a derivative of HKOH-1 for better cellular uptake and retention, have also been proved to be effective in monitoring intracellular hydroxyl radical in PMA stimulated RAW264.7 cells and UVA-irradiated Hela cells by both confocal imaging and flow cytometry analysis. Figure 2

Inspired by the success of HKOH-1, red rhodol based fluorescent probe HKOH-2 was designed and synthesized (Figure 3). By using a julolidine containing rhodol as the fluorophore, HKOH-2 was developed with excellent sensitivity and selectivity towards hydroxyl radical. HKOH-2 was employed in detection of intracellular •OH in PMA stimulated RAW264.7 cells. Two specific organelle target probes HKOH-2m and HKOH-2L, were also developed for targeting mitochondria and lysosomes, respectively (Figure 3). While HKOH-2m proved to be suitable for detecting mitochondrial hydroxyl radical in PMA stimulated RAW264.7 cells, HKOH-2L was applied successfully in detecting lysosomal hydroxyl radical in PMA stimulated and glucose deprivated RAW264.7 cells.

Figure 3

In the development of fluorescent probes for mitochondrial pH, HKpH-1 was designed by introducing two methyl groups into a commercial probe carboxyl SNARF-1 (Figure 4). The electron donating effect of the two methyl groups was expected to increase the pKa of the phenol (Figure 4). HKpH-1 showed a dual emission at 580 nm and 670 nm with an excitation at 560 nm, and the ratio of fluorescence intensity at 580 nm and 670 nm (F580/F670) was found to have a linear relationship with pH in the range of 7.0–9.0. HKpH-1m was designed for specific mitochondrial targeting and was successfully applied in monitoring mitochondrial pH in live cells by confocal imaging and flow cytometry analysis.

Figure 4