Development of Imidazoline-2-Thiones Based Two-Photon Fluorescence Probes for Imaging Hypochlorite Generation in a Co-Culture System
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by Prof. Juyoung Yoon (jyoon@ewha.ac.kr)

Department of Chemistry and Nanoscience

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Hypochlorite (OCl⁻) is an important antimicrobial ion in the immune system and it is believed to be involved in some diseases such as rheumatoid arthritis and atherosclerotic lesions. Methods to detect OCl⁻ generation in live cell are important in order to enable the study of its role in living systems. As a result, the design of new chemo-recognition processes that are selective for OCl⁻ is required. In this effort, we designed, prepared and successfully tested the new imidazoline-2-thione containing OCl⁻ probes, PIS and NIS, which operate through specific reactions with OCl⁻ that yield corresponding fluorescent imidazolium ions (Scheme). Importantly, we demonstrated that PIS can be employed to image OCl⁻ generation in macrophages in a co-culture system (Figure). We have also employed two-photon microscopy and PIS to image OCl⁻ in live cells and tissues, a finding that shows that this probe could have wide biological applications.

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Scheme. Synthesis of imidazoline-2-thiones (a) and the proposed reaction mechanism with OCl⁻ (b). Reagents and conditions: (i) CH₃I, NaH/THF, R. T. (ii) CH₃I, reflux. (iii) Potassium tert-butoxide, S/CH₃OH, 65^oC. (iv) NaOCl. (v) CH₃I/CH₃CN, reflux.

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Figure. Fluorescence (upper) and brightfield (middle) and merge (lower) images of RAW 264.7 macrophages and HeLa cell treated with various stimulants and incubated with PIS. PIS, 20 min (a), LPS (100 ng/ml) for 16 h, IFN-γ (50 ng/ml) for 4 h, PMA (10 nM), 30 min and then PIS (10 μM) for 20 min (b), LPS (100 ng/ml) for 16 h, IFN-γ (50 ng/ml) for 4 h, PMA (10 nM) + 4-ABAH (50 uM) for 16h and PIS (10 μM) for 20 min (c) and LPS (100 ng/ml), IFN-γ (50 ng/ml) for 4 h, PMA (10 nM) + FFA (50 uM) for 16 h and PIS (10 μM) for 20 min (d). scale bar: 10 μM. RAW 264.7 macrophages: small round cell (blue arrow), HeLa cell: large spindle cell (red arrow). Excitation source: 405 nm diode laser and emission: 490 – 590 nm with confocal microscopy.

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* Related Article
“Development of Imidazoline-2-Thiones Based Two-Photon Fluorescence Probes for Imaging Hypochlorite Generation in a Co-Culture System” Angewandte Chemie, April 13, 2015, 54(16), 4890–4894

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​“A Highly Specific Fluorescent Probe for Hypochlorous Acid and Its Application in Imaging Microbe-Induced HOCl Production”  J. Am. Chem. Soc. 2013, 135(26), 9944-9949.