POLYUNSATURATED LIPID OXIDATION PRODUCTS AND THEIR BIOLOGICAL ACTIVITIES: SYNTHESIS, GENERATION, EFFECTS AND PROTECTION by YU-SHIUAN CHENG Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisors: Dr. Robert G. Salomon Department of Chemistry CASE WESTERN RESERVE UNIVERSITY May, 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Yu-Shiuan Cheng candidate for the degree of Doctor of Philosophy *. Committee Chair Dr. Michael G. Zagorski Committee Member Dr. Blanton S. Tolbert Committee Member Dr. Fu-Sen Liang Committee Member Dr. Masaru Miyagi Date of Defense March 25th, 2019 *We also certify that written approval has been obtained for any proprietary material contained therein. This thesis is dedicated to my parents and my sister. iii TABLE OF CONTENTS Table of Contents iv List of Schemes vii List of Tables x List of Figures xi Acknowledgements xvi List of Abbreviations and Acronyms xviii Abstract xxiv Polyunsaturated Lipid Oxidation Products and Their Biological Activities: Synthesis, Generation, Effects and Protection 1. Introduction 1 1.1 Reactive oxygen species and oxidative stress 2 1.2 Free radical-induced lipid peroxidation 4 1.3 Protein adducts of oxidatively truncated lipids 7 1.4 Levuglandins and their protein adducts 9 1.5 Light-induced damage in the retina 12 1.6 Age-related macular degeneration (AMD) 16 1.7 References 22 2. Total Synthesis Confirms the Molecular Structure Proposed for Oxidized Levuglandin D2 30 2.1 Background 31 iv 2.2 Results and Discussion 33 2.3 Conclusions 56 2.4 Experimental Procedures 57 2.5 References 74 3. Light-induced Generation and Toxicity of Docosahexaenoate-derived Oxidation Products in Retinal Pigmented Epithelial Cells 80 3.1 Background 81 3.2 Results 86 3.3 Discussion 107 3.4 Conclusions 120 3.5 Experimental Procedures 123 3.5 References 138 4. 4-Hydroxy-7-oxo-5-heptenoic Acid (HOHA) Lactone-Induced Oxidative Stress and Mitochondrial Dysfunction in Retinal Pigmented Epithelial Cells: Protection by a Carnosine Analogue, L-Histidyl Hydrazide 161 4.1 Background 162 4.2 Results 169 4.3 Discussion 186 4.4 Conclusions and Future Prospects 195 4.5 Experimental Procedures 197 v 4.6 References 211 Appendix 222 Bibliography 259 vi LIST OF SCHEMES Chapter 1 Scheme 1.1. Oxidative fragmentation of PUFA phospholipids produces γ-hydroxy-α,β-unsaturated aldehydes. 7 Scheme 1.2. The cyclooxygenase (COX) and radical-induced cyclooxygenation of arachidonate produces isoLGs. 10 Scheme 1.3. Metabolism of LGs: generation of protein adducts, crosslinks and oxidized metabolites 12 Scheme 1.4. Type I and Type II photosensitized oxidation reactions 14 Scheme 1.5. Class I and Class II photochemical damage 16 Chapter 2 Scheme 2.1. Generation and proposed structure of ox-LGD2 31 Scheme 2.2. Synthetic design for ox-LGD2 33 Scheme 2.3. Synthesis of the upper side chain precursor 34 Scheme 2.4. Concise total synthesis of ox-LGD2 36 Scheme 2.5. Model study for selective removal of tert-butyl groups 39 Scheme 2.6. γ-Isomer model study 42 Scheme 2.7. Rearrangment of ox-LGE2 51 Chapter 3 Scheme 3.1. The work-flow for an ARPE-19 cell light damage model 91 vii Chapter 4 Scheme 4.1. L-Histidyl hydrazide as an inhibitor of lipid peroxidation products formation. 193 viii LIST OF TABLES Chapter 2 Table 2.1. One-pot three-component coupling of alkyne 2.6 with cuprates and allylic halides 35 Table 2.2. One-pot three-component coupling of alkyne 2.6 with various vinyl cuprates to generate maleate 2.14 37 Table 2.3. Comparison of organocopper reagents for synthesis of 2.4 37 Table 2.4. Model study for the mono methylation of a maleic anhydride 40 Table 2.5. Generation of 2.1 and 2.2 by desilylation of 2.1p and 2.2p 43 Chapter 3 Table 3.1. Optimized mass spectrometer parameters, selected ions and collision energies for quantitation of the MitoClick product in ARPE-19 mitochondria-enriched cell lysate 129 Table 3.2. Workflow for Strata X-C spin column 1st SPE enrichment of HOHA/DHHA lactone-GSH derivatives from bovine retina extracts 133 Table 3.3. Workflow for Hypercarb 2nd SPE enrichment of HOHA/DHHA lactone-GSH derivatives from bovine retina extracts 133 Table 3.4. HPLC gradient for the determination of HOHA/DHHA lactone-GSH derivatives in light exposed bovine retina extract 134 Table 3.5. Optimized mass spectrometer parameters, MRM transitions and collision energies for quantitation of HOHA/DHHA lactone-GSH derivatives in bovine retina extract 135 ix Table 3.6. Workflow for Strata X-33u spin column SPE enrichment of HOHA/DHHA lactone-GSH derivatives from ARPE-19 extracellular medium 135 Table 3.7. HPLC gradient for the determination of HOHA/DHHA lactone-GSH derivatives in extracellular medium from light exposed ARPE-19 cells 136 Table 3.8. Optimized mass spectrometer parameters, selected ions and collision energies for quantitation of HOHA/DHHA lactone-GSH derivatives in ARPE-19 extracellular medium 136 x LIST OF FIGURES Chapter 1 Figure 1.1. Main sources of ROS production in cells 3 Figure 1.2. Lipid peroxidation pathways 5 Figure 1.3. Transmission of light in the human eye 13 Figure 1.4. Outer retina (photoreceptor and RPE) is susceptible to photooxidative damage. 15 Figure 1.5. Classification of AMD 17 Figure 1.6. The alternative complement pathway 20 Chapter 2 Figure 2.1. HPLC separation of three-component coupling products 2.4 and 2.4’ 38 Figure 2.2. HMBC (red) and NOESY (blue) correlations distinguishing the structures of 2.1p and 2.2p 42 1 Figure 2.3. Comparison of H NMR spectra of ox-LGD2 chemical shifts with CHD2CN resonance set at 1.90 ppm (A) at 500 MHz from total synthesis and (B) at 600 MHz from extraction of Gracilaria edulis 44 Figure 2.4. HMBC (red) and NOSEY (blue) correlations distinguishing the structures of 2.1, 2.2 and 2.2d 46 1 Figure 2.5. H NMR spectrum of the ox-LGD2 and ox-LGE2 mixture generated from the HF desilylation reaction 46 Figure 2.6. Evolution of ox-LGs in CD3CN: (Left) HPLC chromatograph; (Right) 1H NMR spectrum 48 1 6 Figure 2.7. H NMR spectrum of Δ -ox-LGE2 (2.2d) 49 xi Figure 2.8. HPLC chromatogram of ox-LGD2, two diastereomers of 6 ox-LGE2, and Δ -ox-LGE2 52 Figure 2.9. 1H NMR spectrum of peak 1 (Figure 2.8 HPLC chromatograph) collected at 44 min 53 Figure 2.10. HPLC chromatogram of ox-LGD2, minor diastereomer (b) of ox-LGE2 53 Chapter 3 Figure 3.1. Oxidative cleavage of DHA-PC delivers HOHA-PC. HOHA lactone is then released from bilayer phospholipid membranes by spontaneous intramolecular transesterifica- tion. Covalent adduction of HOHA-PC and HOHA lactone to primary amino groups of protein lysyl residues and phos- phatidyl ethanolamines (PE) produces CEP derivatives. 83 Figure 3.2. Time-course for the formation of HOHA lactone GSH derivatives 87 Figure 3.3. Photogeneration of HOHA-PC from DHA-PC is followed by release of HOHA lactone. HOHA lactone-GSH adduct is generated by glutathionylation of HOHA lactone. NADPH-dependent reduction of the HOHA lactone-GSH adduct delivers the reduced GSH adduct, DHHA lactone- GSH, that is also produced by RPE cells exposed to HOHA lactone, e.g., that may be released by oxidatively damaged photoreceptor cells. 88 Figure 3.4. Quantitative LC-MS/MS analysis of HOHA lactone GSH derivatives (HOHA lactone-GSH plus DHHA lactone-GSH) production induced in bovine retina extracts by light sources of various wavelengths 89 Figure 3.5. Internalization of A2E: representative images showing green A2E autofluorescence in ARPE-19 cells preloaded for 24 h with A2E 91 Figure 3.6. Generation of DHHA lactone-GSH upon exposure to black light (Panel A), blue light (Panel B) or white light (Panel C) in ARPE-19 cells (A2E(-)) pre-incubated with 50 μg/mL DHA for 48 h or cells (A2E(+)) pre-incubated with DHA xii for 48 h followed by 10 μM A2E for 24 h with 24 h recovery in basal medium 93 Figure 3.7. Quantitation of GSH levels remaining in DHA/A2E-laden ARPE-19 cells 10 min (Panel A) or 24 h (Panel B) after irradiation for various times from 0 to 60 min 94 Figure 3.8. Generation of CEP in ARPE-19 cells after 20 min exposure to blue light 96 Figure 3.9. Measurement of mitochondrial membrane potential in light exposed DHA-rich A2E-laden ARPE-19 cells 98 Figure 3.10. Measurement of cell viability by MTT assay 24 h after exposure of A2E-laden ARPE-19 cells to blue light (430 nm) for 0 to 60 min 99 Figure 3.11. Measurement of mitochondrial membrane potential changes in ARPE-19 cells upon exposure to HOHA lactone 100 Figure 3.12. Measurement of the cell viability of ARPE-19 cells after exposure to 0 to 40 μM HOHA lactone for 24 h assessed by MTT assay (Panel A) and by Alamar Blue assay (Panel B) 101 Figure 3.13. Senescence of ARPE-19 cells exposed to HOHA lactone 103 Figure 3.14. Lysosomal membrane perturbation in ARPE-19 cells after incubation with HOHA lactone 105 Figure 3.15. Generation of CEP in ARPE-19 cells upon exposure to HOHA lactone 106 Figure 3.16. Postulated synergistic contributions of A2E and a family of α,β-unsaturated aldehyde products of lipid oxidative cleavage to light-induced apoptosis of RPE cells 118 Chapter 4 Figure 4.1.