이광자 흡수 벤조쿠마린 형광체 및 감마-글루타밀 전이효소 프로브의 개발

다국어 초록 (Multilingual Abstract)

Section I. Development of in cellulo super-bright benzocoumarin dyes
Fluorescent methods are important in studying biochemical phenomena in cells and tissues through microscopic imaging analysis. Researchers usually assume that the fluorescent properties of a fluorophore in solutions is simiar to be in cells. However, as the fluorescence intensity and wavelength are highly environment-sensitive, there is a gap between the “in solution” and “in cellulo” fluorescence properties. Herein, I developed benzoumarin dyes which are super bright in cells compared to commonly-used dyes. In addition, I compared their distinctive emission properties in each of organic, aqueous solvents and cells in aspect of the maximum emission wavelength. Thus, I found one of the new fluorophores, exceptionally bright inside cells and relatively environment-insensitive through comparing process and proved that it is able to provide reliable quantification data.

Section II. Development of ratiometric imaging probes for ϒ-glutamyl transferase in cells and tissues
Ratiometric imaging of ϒ-glutamyl transferase unperturbed by pH, polarity, and viscosity changes
ϒ-Glutamyltransferase (GGT) is involved in maintaining the intracellular glutathione levels and, at its elevated levels, is associated with various diseases including cancer and myocardial infarction. To study this enzyme in biological systems, fluorescent probes have received significant attention recently. As fluorescence signal is sensitive to environmental fluctuations, however, it is challenging to address the signal fluctuation issue. Disclosed is the benzocoumarin-based probe that enables ratiometric imaging of GGT activity levels in cells as well as in tissues, essentially unperturbed by medium pH, viscosity, and polarity changes. Validity of the probe is demonstrated by determining the GGT activity level in HeLa cells directly through ratiometric imaging. Furthermore, the probe and its enzymatic product are two-photon absorbing, extending its applicability to tissue: an 8.5-fold higher level of GGT in cancerous tissue over the normal tissue is determined, and the GGT activity lev-els between different mouse organ tissues are quantitatively compared. The probe with practicality holds great promise for studying GGT associated biological processes directly through ratiometric imaging by two-photon microscopy.
 
Cellular-membrane-localizing ϒ-glutamyl transpeptidase probe for ratiometric imaging
ϒ-Glutamyltransferase (GGT) localizes on the exterior surface of cell membrane, Therefore, it is crucial to develop a fluorescence probe that localizes in the cellular membrane; however, non of membrane-targeting GGT probes are known. Moreover, only a few membrane localizing probes for other analytes have been developed so far. I developed the membrane-localizing GGT probe by introducing a zwitterionic and a hydrophobic group for the first time. Using the probe, the GGT activity level in three normal cell lines (HEK-293T, HUVEC, and FHC) and three cancer cell lines (A549, HeLa, and HCT116) were deterimed directly through ratiometric imaging. Furthermore, using probe’s two-photon absorbing property, the relative GGT levels of eight different organs (muscle (normal/cancer), colon, lung, liver, spleen, stomach, pancreas, and kidney) in a xenograft cancer mouse (by subcutaneous injection of CT-26 cells) were determined and compared with those of the normal mouse: The GGT activity levels have been elevated in the tissues in the order of lung, colon and liver. Thus, the probe has a great potential for monitoring GGT levels in cells as well as in tissues under given biological processes.

목차 (Table of Contents)

• Contents
• Abstract 5
• Contents 8
• Abbreviations 11
• I. General features 12
• I-1. Fluorescence method 12
• I-2. Two-photon Microscopy 12
• I-3. Ratiometric probes 13
• II. References 13
• Section I. Development of in cellulo super-bright benzocoumarin dyes
• I. Introduction 16
• II. Results and discussion 17
• II-1. Importance of brightness in cellulo 17
• II-2. Photophysical properties of benzocoumarin dyes 18
• II-3. Selection of commonly used dyes and benzocoumarin dyes 19
• II-4. Evaluation of in cellulo and in solution fluorescence brightness of fluorophores 20
• II-5. Evaluation of in cellulo emission wavelengths of fluorophores 22
• II-6. Effect of the environmental sensitivity of fluorophores on ratiometric quantification 23
• III. Conclusion 25
• IV. Experimental section 26
• IV-1. General information 26
• IV-2. Synthesis 27
• IV-3. Fluorescence assays 34
• IV-4. Cell culturing and imaging 35
• IV-5. Tissue preparation and imaging 35
• IV-6. Additional data for other benzocoumarin dyes 36
• V. References 44
• Section II. Development of Ratiometric Imaging Probes for ϒ-Glutamyl Transferase in Cells and Tissues
• Section II-A. Ratiometric Imaging of γ-Glutamyl Transpeptidase Unperturbed by pH, Polarity, and Viscosity Changes
• I. Introduction 48
• II. Results and discussion 49
• II-1. Design of pH insensitive platform 49
• II-2. Synthesis 50
• II-3. Photophysical properties of Py+BC_Glu 51
• II-4. pH, viscosity and polarity test 53
• II-5. Two-photon action cross section (TPACS) 55
• II-6. Cellular imaging 55
• II-7. Quantification of GGT in HeLa cells 56
• II-8. Tissue imaging 59
• II-9. The relative GGT activity levels in several organs 60
• III. Conclusion 63
• IV. Experimental section 64
• IV-1. General information 64
• IV-2. Synthesis 64
• IV-3. Fluorescence assays 67
• IV-4. Cell culturing and imaging 67
• IV-5. Tissue preparation and imaging 68
• V. References 69
• Section II-B. Cellular-Membrane-Localizing, Ratiometric ϒ-Glutamyl Transpeptidase Probe
• I. Introduction 72
• II. Results and discussion 73
• II-1. Design of membrane targeting platform 73
• II-2. Photophysical properties of mNVP_Glu 77
• II-3. Colocalization 78
• II-4. Two-photon action cross section (TPACS) 79
• II-5. Cellular imaging and tissue imaging 80
• III. Conclusion 84
• IV. Experimental section 85
• IV-1. General information 85
• IV-2. Synthesis 86
• IV-3. Fluorescence assays 95
• IV-4. Cell culturing and imaging 96
• IV-5. Tissue preparation and imaging 97
• V. References 98
• Summary (in Korean) 100
• Curriculum vitae 101