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Title Page

Abstract

Contents

List of Abbreviations 14

1. GENERAL INTRODUCTION 16

1.1. REACTIVE OXYGEN SPECIES (ROS) 16

1.1.1. The introduction of ROS 17

1.1.2. Various applications of ROS 19

1.2. PHOTOSENSITIZERS (PS) 20

1.2.1. Introduction to photosensitizers 21

1.2.2. Design strategies for efficient photosensitizers and limitations 24

1.3. PROTEIN MODIFICATIONS 29

1.3.1 A series of protein modifications and their utilization 29

1.3.2. Protein oxidative modifications: oxidation and crosslinking 30

1.3.3. The utilization of protein oxidative modifications as chemical handle 31

1.4. OUTLINE OF THIS DISSERTATION 33

1.5. REFERENCES 33

2. NEW TYPE OF MECHANISM FOR SINGLET OXYGEN GENERATION: 'SPIN FLIP ELECTRON TRANSFER' 38

2.1. INTRODUCTION 38

2.2. EXPERIMENTAL METHODS 39

2.2.1. Synthesis of polymer and molecular weight (Mn) calculation[이미지참조] 39

2.2.2. Characterization of photophysical properties of polymer 41

2.2.3. Evaluation of type I ROS generation (O₂·-, H₂O₂ and OH·)[이미지참조] 41

2.2.4. Evaluation of type II ROS generation (¹O₂) 42

2.2.5. Cyclic voltammetry (CV) for investigation of electron transfer 42

2.2.6. Time dependent-density functional theory (DFT) calculation 43

2.2.7. DCFH-DA and superoxide radical assay for intracellular ROS detection 43

2.2.8. Live or dead assay for investigating photodynamic effect on cancer cells 43

2.2.9. Cell viability of polymer with photoirradiation 44

2.2.10. Phototoxicity index calculation 44

2.3. RESULTS AND DISCUSSION 44

2.3.1. Synthesis of polymer for electron transfer mediated ¹O₂ generation and characterization 45

2.3.2. Electrochemical analysis to investigate electron transfer to molecular oxygen (O₂). 50

2.3.3. Computational analyses clarifying reasonable ¹O₂ generation mechanism. 52

2.3.4. Application of electron transfer mediated ¹O₂ generation 55

2.4. CONCLUSION 57

2.5. REFERENCES 58

3. CELL ORGANELLE DEPENDENT PHOTODYNAMIC THERAPY (PDT) MECHANISMS 60

3.1. INTRODUCTION 60

3.2. EXPERIMENTAL METHODS 62

3.2.1. Materials and Methods 62

3.2.2. Synthesis of TIr1, TIr2, TIr3 TIr4 63

3.2.3. Absorbance, emission and phosphorescence quantum yield (Φp).[이미지참조] 65

3.2.4. Cyclic voltammetry (CV) analysis 65

3.2.5. Spectral measurements of phosphorescence emitted by two-photon absorption. 66

3.2.6. Time-correlated single photon counting (TCSPC) for lifetime characterization. 66

3.2.7. Cell culturing, transfection and cell imaging (One-/two-photon imaging & FLIM analysis). 66

3.2.8. Two-photon real-time scanning of SK-OV-3 67

3.2.9. Cytotoxicity studies. 67

3.2.10. ROS assay: PL quenching, ABDA assay (¹O₂), dihydrorhodamine assay (O₂·-)[이미지참조] 68

3.2.11. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) for in-vitro assay of biotin-phenol (BP) crosslinking 69

3.2.12. Western blot for evaluating protein photo-crosslinking in living cells 69

3.2.13. Oxidation analysis 71

3.3. RESULTS AND DISCUSSION 72

3.3.1. Ir(III) complexes as photodynamic therapy (PDT) agents. 72

3.3.2. Cytotoxicity evaluation of light activated Ir(III) complexes 76

3.3.3. Characterization of ROS generation by Ir(III) complexes 78

3.3.4. Characterization of protein crosslinking by Ir(III) complexes 82

3.3.5. Identification of oxidative modifications of endogenous proteins by TIr3 84

3.3.6. Primary subcellular localization of TIr3 in living cells 86

3.3.7. Oxidation of mitochondrial proteins related to mitochondrial physiology by TIr3 90

3.3.8. Proposed mode of action of Ir(III) complexes as PDT agents 91

3.3.9. Oxidative protein modification by mitochondria targeting Ir(III) complex (Ir-OA) 93

3.3.10. Mitochondrial photo-oxidation induced cell death mechanism 95

3.3.11. Oxidized proteome of lysosome targeting Ir(III) complex (B2) 95

3.3.12. Lysosomal photo-oxidation induced cell death mechanism 96

3.3.13. Cell organelle dependent barcode of oxidized proteome 98

3.4. CONCLUSION 100

3.5. REFERENCES 100

4. PHOTOINDUCED OXIDATION OF AMYLOID BETA FOR MODULATING ITS SELF-AGGREGATION PATHWAY 105

4.1. INTRODUCTION 105

4.2. EXPERIMENTAL METHODS 107

4.2.1. Materials and Methods 107

4.2.2. Synthesis of Ir(III) Complexes 108

4.2.3. Photophysical Properties of Ir(III) Complexes 112

4.2.4. Histidine Binding Affinities of Ir(III) Complexes 112

4.2.5. Singlet Oxygen (¹O₂) Generation by Ir(III) Complexes. 113

4.2.6. 2D NMR Spectroscopy. 113

4.2.7. Aβ Aggregation Experiments 113

4.2.8. Gel electrophoresis with Western blotting for monitoring Aβ modulation 114

4.2.9. Transmission Electron Microscopy (TEM) to visualize Aβ 114

4.2.10. Electrospray Ionization-Ion Mobility-Mass Spectrometry (ESI-IM-MS) 115

4.2.11. MTT-assay for cell viability analysis of Ir(III) Complexes 115

4.3. RESULTS AND DISCUSSION 116

4.3.1. Rational design of Ir-1 for oxidation of Aβ peptide 116

4.3.2. Oxidative modifications of Aβ peptide by Ir-1 116

4.3.3. Morphological alteration of Aβ 40 by photoactivated Ir-1 117

4.3.4. Advanced design strategies for peptide modification using Ir(III) complexes 118

4.3.5. Optical properties of Ir(III) complexes with or without histidine. 119

4.3.6. Coordination binding affinity and following enhancement of ROS generation. 120

4.3.7. Modifications of Aβ40 peptide by Ir-F[이미지참조] 123

4.3.8. Cell viability enhancement with treatment of Ir(III) complex and photo-irradiation 125

4.4. CONCLUSION 126

4.5. REFERENCES 126

5. NOVEL PROTEOMIC METHOD BASED ON PROTEIN PHOTO-CROSSLINKING 130

5.1. INTRODUCTION 130

5.2. EXPERIMENTAL METHODS 132

5.2.1. Materials and equipment 132

5.2.2. Photophysical properties of Ir(III) complexes 133

5.2.3. HaloTag protein titration and binding kinetics of Ir(III) complexes 133

5.2.4. Evaluation of electron transfer capability through type I ROS generation assay 134

5.2.5. Competition assay for identifying intracellular binding of Ir-HTL 134

5.2.6. Photo-crosslinking in FRB-FKBP25 model system 135

5.2.7. Western blot to identify photo-crosslinking 135

5.2.8. Line-cut analysis for identifying photo-crosslinking efficiency 136

5.2.9. Confocal imaging to verify GFP-GBP system 136

5.2.10. LC-MS² analysis for identifying interactome of protein of interest (POI) 137

5.2.11. Synthesis of Ir-HTL and sIr-HTL 139

5.3. R ESULTS AND DISCUSSION 144

5.3.1. Characterization of binding with HaloTag protein and optical property change 144

5.3.2. Spatially resolved localization and photo-crosslinking in live cells 149

5.3.3. Protein-protein interaction network in specific nucleus suborganelle. 153

5.4. CONCLUSION 156

5.5. REFERENCES 156

CURRICULUM VITAE 162

List of Tables

Table 1.1. Photodynamic action of photosensitizer with molecular oxygen. 22

Table 1.2. Respective cancer cell lines according to over-expressed enzymes. 28

Table 2.1. Atomic composition of PG and PAG in full XPS spectrum. 47

Table 2.2. Peak position of deconvoluted high resolution XPS spectrum (C1s, N1s, and O1s) Note that A indicates... 48

Table 2.3. Dominant transition for charge transfer excited states of PG/PAG-O₂. 54

Table 2.4. Viability of HeLa cells under dark and light (60 J/cm²) condition. 57

Table 3.1. Detail information of prepared plasmids 70

Table 3.2. All information of the photophysical and electrochemical properties for TIr1-4 and [Ru(bpy)3]2+.[이미지참조] 74

Table 3.3. Decreasing ratio between N₂ and O₂ bubbling, calculated with maximum point or area... 79

Table 3.4. All elements for calculation of ¹O₂ quantum yield. 80

Table 4.1. Photophysical properties of Ir-1. 116

Table 4.2. Photophysical properties of four Ir(III) complexes 120

Table 5.1. Plasmid construct information. 151

List of Figures

Figure 1.1. Origin of molecular oxygen (O₂) on earth. 17

Figure 1.2. Involvement of ROS in our life. 17

Figure 1.3. Physiological response depending on intracellular ROS level. 18

Figure 1.4. Two different types of ROS generation by electron transfer (type I) or energy transfer (type II)... 19

Figure 1.5. Various applications to utilize ROS. 20

Figure 1.6. Jablonski diagram to generate ROS from photoactivation of PS. 21

Figure 1.7. Milestone for development of photosensitizers. 22

Figure 1.8. 1st and 2nd generation photosensitizer based on porphyrin, chlorin moiety.[이미지참조] 23

Figure 1.9. Xanthene derivatives as photosensitizer. 23

Figure 1.10. BODIPY derivatives as photosensitizer. 24

Figure 1.11. Rationale for developing NIR absorbing photosensitizer. 25

Figure 1.12. Molecular design strategies for NIR absorption of photosensitizer. 26

Figure 1.13. Two representative design strategies for enhancing ROS generation. 27

Figure 1.14. Tumor specific enzyme reactivity of photosensitizer with functionalization. 28

Figure 1.15. Enzyme mediated-labeling tool utilizing biotinylation. 30

Figure 1.16. Oxidative modifications of amino acids. 31

Figure 1.17. Utilization of protein oxidative modifications. 32

Figure 2.1. Design strategy and working mechanism of photosensitizers for electron transfer mediated ¹O₂ generation. 39

Figure 2.2. Preparation of biocompatible PG and PAG polymer. 45

Figure 2.3. Material characterization through ¹H and ¹³C NMR spectroscopy. 46

Figure 2.4. Optical properties of the polymers and chemical binding modes of PG and PAG. 47

Figure 2.5. Investigation of Type I ROS generation by photoactivation of polymers. 49

Figure 2.6. Identifying ¹O₂ generation of polymer upon photo-irradiation. 50

Figure 2.7. Evidence of electron transfer from the polymer to oxygen. 51

Figure 2.8. Density functional theory (DFT) calculations for analyzing ¹O₂ generation properties of PG and... 53

Figure 2.9. Density functional theory (DFT) to evaluate Gibbs free energy of ground and oxidized state for... 54

Figure 2.10. Plausible mechanism of ¹O₂ generation via electron transfer cascade in the network between... 55

Figure 2.11. Application of ROS generated by direct electron transfer upon photo-irradiation of polymer... 56

Figure 2.12. Additional application of ROS generated by photo-irradiation of polymer in cells. 57

Figure 3.1. Protein modification pathways generated by Ir(III) complexes in cellular regions and optical... 61

Figure 3.2. Two Ir(III) complexes targeting mitochondria (Ir-OA) and lysosome (B2) to investigate cell... 62

Figure 3.3. Synthetic pathway of four cyclometalated Ir III complexes (TIr1, TIr2, TIr3, and TIr4).[이미지참조] 63

Figure 3.4. Optical and electrochemical properties of TIr1-4 and correlation of their energy levels with the... 73

Figure 3.5. Characterization of non-linear two-photon absorption properties. 74

Figure 3.6. Optical imaging for provided four Ir(III) complexes. 75

Figure 3.7. Cell death triggered by Ir(III) complexes and real time tracking of cell morphological alteration... 77

Figure 3.8. Cell death induced by Ir(III) complexes upon light activation in MCF-7 cells. 78

Figure 3.9. Triplet state quenching by O₂ bubbling to figure out their ability for one electron process to... 79

Figure 3.10. Absorbance attenuation of ABDA by ¹O₂ generation from photosensitizers. 80

Figure 3.11. Absorbance change of ABDA for neutral and anionic Ir(III) complexes according to bandgap narrowing. 81

Figure 3.12. DHR123 assay for identifying O₂·- production under photo-irradiation.[이미지참조] 82

Figure 3.13. MALDI-MS analysis for BP crosslinking with TIr3 and [Ru(bpy)3]2+ as reference.[이미지참조] 83

Figure 3.14. Analysis of photo-crosslinking in HEK293T cells by TIr1-4 and [Ru(bpy)3]2+.[이미지참조] 84

Figure 3.15. Identification of photo-crosslinking in HEK293T cells for Neutral-3 and Anionic-3 compared... 84

Figure 3.16. Western blot analysis with methionine sulfoxide antibody in HEK293T cell 85

Figure 3.17. Identification of the oxidative damage to the proteome induced by TIr3 upon photo-activation... 88

Figure 3.18. Confocal laser scanning microscope (CLSM) images for HeLa cells. 88

Figure 3.19. Confocal laser scanning microscope (CLSM) images for HEK293T cells. 89

Figure 3.20. Mitochondrial morphological change by photoactivation of TIr3. 90

Figure 3.21. Oxidized proteome related to protein quality control in ER. 91

Figure 3.22. Proposed mode of action of Ir(III) complexes for photodynamic therapy (PDT). 92

Figure 3.23. Plausible oxidation induced cell death mechanism focusing on the perturbation of ER-... 92

Figure 3.24. Investigation of photo-crosslinking as oxidative protein modification by Ir-OA. 93

Figure 3.25. Proteomic analysis for figuring out oxidized proteome with mitochondria-localized Ir-OA. 94

Figure 3.26. Overview of photo-induced cell death pathway in mitochondria. 95

Figure 3.27. Characterization of lysosome targeting Ir(III) complexes. 96

Figure 3.28. Proteomic analysis for figuring out oxidized proteome with lysosome-localized B2 97

Figure 3.29. Overview of photo-oxidation induced cell death pathway in lysosome. 98

Figure 3.30. Heat map diagram for cell-organelle dependent oxidized proteome distribution. 99

Figure 3.31. Oxidized proteome groups sorted by specific protein function depending on cell organelles. 99

Figure 4.1. Ir(III) complexes for modulating Aβ aggregation pathway. 106

Figure 4.2. Synthetic routes to IrIII complexes for photoinduced modulation of amyloid beta self-...[이미지참조] 108

Figure 4.3. Mass spectrometric analyses to identify Aβ modification with Ir-1 and photo-irradiation. 117

Figure 4.4. TEM images to observe morphological change of Aβ 40 aggregates with Ir-1. 118

Figure 4.5. Characterization of Ir(III) complexes to induce Aβ peptide modification. 119

Figure 4.6. Investigation of binding ability of Ir(III) complexes to histidine, Aβ monomer, oligomers, and fibrils. 121

Figure 4.7. Analysis of the amount of singlet oxygen (¹O₂) generated by Ir(III) complexes with or without... 122

Figure 4.8. DHR123 assay to detect O₂·- produced by Ir(III) complexes with or without histidine (His).[이미지참조] 123

Figure 4.9. Mass spectrometric analysis of Aβ 40 species generated upon treatment with Ir-F. 124

Figure 4.10. Collision-induced dissociation (CID) spectrum of the singly oxidized Aβ40.[이미지참조] 124

Figure 4.11. TEM images to observe morphological change of Aβ 40 aggregates with Ir(III) complexes. 125

Figure 4.12. MTT-assay to confirm the alleviation of toxicity from Aβ species in N2a cells. 125

Figure 5.1. Previous methods for labeling interactome; immunoprecipitation, enzyme-mediated labeling,... 131

Figure 5.2. Schematic description of photo-crosslinking-based interactome analysis. 132

Figure 5.3. Synthetic pathway of Ir-HTL and sIr-HTL. 139

Figure 5.4. Characterization of photophysical properties of two photo-crosslinking reagents. 145

Figure 5.5. Investigation for binding between Ir(III) complexes and HaloTag protein. 146

Figure 5.6. Identification of ROS generation by Ir-HTL and sIr-HTL. 147

Figure 5.7. Effect of triplet excited state lifetime toward photo-crosslinking efficiency. 147

Figure 5.8. In-vitro photo-crosslinking in chemically induced dimerization system with FRB-FKBP25. 148

Figure 5.9. Live cell photo-crosslinking in FRB-FKBP25 model system with Ir-HTL. 149

Figure 5.10. Schematic illustration of GFP-GBP binding system and protein of interest (PTBP1; splicing... 150

Figure 5.11. Identification of specific localization of Ir-HTL led by GBP-GFP interaction in nucleus. 152

Figure 5.12. Photo-crosslinking of POI by Ir-HTL depending on its location. 152

Figure 5.13. Identification interactome of three POIs through developed photo-crosslinking method. 153

Figure 5.15. Distribution of subcellular localization of enriched interactomes visualized by pie chart. 155

Figure 5.16. Validation for interactome of each POIs by Western blotting and co-localization. 155

초록보기

Reactive oxygen species (ROS) are continually produced under normal conditions, following aerobic energy metabolism. Because ROS are remarkably reactive chemical species to induce severe impairment through oxidative modifications of many biomolecules, it leads to various diseases like cancer or neurodegenerative diseases if the ROS could not be properly handled by redox metabolism in our body. Otherwise, ROS also give an opportunity to treat disease by exogenously generated excessive ROS, leading to oxidative stress on the specific lesion of disease not on the normal. In particular, photodynamic therapy (PDT), which is invasive cancer therapy by only light and photoactivatable materials, is one of therapeutics with ROS.

In this point, various chemicals have been emerged to provide exogenous ROS, which could be utilized to oxidation organic reactions, antimicrobial effect, water waste purification, and PDT. Among chemicals, in particular, photosensitizer (PS) that can generate ROS through photoexcitation by irradiation source was highlighted because ROS generation was elaborately controlled owing to the advantage from usage of photon (spatiotemporal resolution). Although a variety of PSs have been developed, lots of issues still remain of which breakthrough leads to optimal advancement of PS. Thereby, endeavors for various molecular design strategies to provide the optimal PSs have to be dedicated.

In this thesis, four molecular design strategies for advancement of PSs are introduced to efficiently generate ¹O₂ according to (i) distance between PS and O₂ (chapter 2), (ii) optical bandgap of PS (chapter 3), (iii) on-target toxicity (chapter 4) and (iii) triplet excited-state lifetime (chapter 5). Each different molecular engineering was successfully applied to respective application (oxidation-induced cell death by PDT; strategy (i-ii), modulation of amyloidogenic peptide (Aβ) aggregation through oxidation-induced structural change; strategy (iii), and method for proteomics via photo-crosslinking - one of oxidative protein modifications; strategy (iv)). Furthermore, chemical and biological mode of action of oxidation-induced cell death depending on cell organelles in PDT are elucidated by oxidized proteome analysis. These insights would offer pertinent PS in accordance with specific situation and based on detail mechanism for cell organelle dependent oxidation-induced cell death.

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