DEVELOPMENT OF NOVEL SYNTHETIC ROUTES TO THE EPOXYKETOOCTADECANOIC ACIDS (EKODES) AND THEIR BIOLOGICAL EVALUATION AS ACTIVATORS OF THE PPAR FAMILY OF NUCLEAR RECEPTORS By ROOZBEH ESKANDARI Submitted in partial fulfillment of the requirements for The Degree of Doctor of Philosophy Thesis Advisor: Gregory P. Tochtrop, Ph.D. Department of Chemistry CASE WESTERN RESERVE UNIVERSITY January, 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ROOZBEH ESKANDARI Candidate for the Ph.D degree *. (signed) Anthony J. Pearson, PhD (Chair of the committee) Gregory P. Tochtrop, PhD (Advisor) Michael G. Zagorski, PhD Blanton S. Tolbert, PhD Witold K. Surewicz, PhD (Department of Physiology and Biophysics) (date) 14th July, 2015 *We also certify that written approval has been obtained for any proprietary material contained therein. I dedicate this work to my sister Table of Contents Table of Contents ........................................................................................................................ i List of Tables .............................................................................................................................. vi List of Figures ........................................................................................................................... vii List of Schemes .......................................................................................................................... ix Acknowledgements ................................................................................................................... xi List or Abbreviations ............................................................................................................ xiii Abstract ................................................................................................................................... xxiii CHAPTER 1 General Introduction ........................................................................................ 1 1.1 Discovery of prostaglandins .............................................................................................. 1 1.2 The role of synthesis in the advancement of prostaglandins research ...................3 1.3 Discovery of leukotrienes ...........................................................................................6 1.4 The role of synthesis in the advancement of leukotrienes research .......................6 1.5 Non-enzymatic oxidation of PUFAs ..........................................................................8 1.6 The role of synthesis in the advancement of LPO products ..................................10 1.7 Formation of LPO products .....................................................................................12 1.8 Oxidative stress (OS) ................................................................................................16 1.8.1 Formation of reactive oxygen species/reactive nitrogen species (ROS/RNS) ............................................................................................................................................17 1.8.2 Reactive oxygen species (ROS) ............................................................................. 17 i 1.8.2.1 Singlet oxygen .................................................................................... 17 1.8.2.2 Superoxide radical ............................................................................. 18 1.8.2.3 Hydrogen peroxide ............................................................................ 19 1.8.2.4 Hydroxyl radical................................................................................. 19 1.8.3 Reactive nitrogen species (RNS) .................................................................... 19 1.8.3.1 Nitric oxide (NO.) .............................................................................. 19 1.8.3.2 Peroxynitrite (ONOO-) .................................................................... 19 1.8.4 Reactivity of ROS/RNS. ................................................................................... 20 1.9. Biological roles of oxidized lipids .................................................................................. 22 1.9.1 Oxidized lipid and G protein-coupled receptors (GPCRs) ...................... 23 1.9.2 Oxidized lipid and peroxisome-proliferator-activated receptors (PPARs) ...................................................................................................................................... 24 1.10 References ......................................................................................................................... 26 CHAPTER 2 Development of novel synthetic routes to the epoxyketooctadecenoic acids (EKODEs) ........................................................................................................................ 31 2.1 Introduction ........................................................................................................................ 31 2.2 EKODE structure and nomenclature ............................................................................ 34 2.3 EKODE-(E)-I family ......................................................................................................... 35 2.3.1 Retrosynthetic analysis of trans-EKODE-(E)- I ......................................... 36 2.3.2 Synthesis of trans-EKODE-(E)- Ia ................................................................ 37 ii 2.3.3 Synthesis of trans-EKODE-(E)- Ib ................................................... 38 2.3.4 Retrosynthetic analysis of cis-EKODE-(E)- Ib .............................. 40 2.3.5 Synthesis of cis-EKODE-(E)- Ib ....................................................... 40 2.4 EKODE-(E)-II family .......................................................................................... 42 2.4.1 Retrosynthetic analysis of trans-EKODE-(E)-II ........................... 44 2.4.2 Synthesis of bifunctional conjunctive ylide .................................... 45 2.4.3 Synthesis of trans-EKODE-(E)-II ..................................................... 47 2.4.4 Divergent synthesis for diverse library of trans-EKODE-(E)-II .......................................................................................................................................... 49 2.4.4.1 Synthesis of alkyne-end aldehyde ..................................... 49 2.4.4.2 Synthesis of deuterated labeled aldehyde ....................... 49 2.5 Experimental part ................................................................................................ 52 2.5.1 General experimental details ............................................................. 52 2.5.2 Syntheses ................................................................................................ 54 2.6 References ............................................................................................................... 88 Chapter 3. Biological evaluation of EKODEs as potential activators of the PPAR family of nuclear receptors .................................................................................................... 91 3.1 Peroxisome proliferator-activated receptors (PPARs) structure and activity ..... 91 3.2. The physiological functions of the PPARs .................................................................. 95 3.2.1 Functional role of PPARα ............................................................................... 95 iii 3.2.1.1 Exogenous ligands (synthetic Xenobiotics) of PPARα .............. 95 3.2.1.2 Endogenous ligands (biological molecules) of PPARα ............. 96 3.2.2 Functional role of PPARɣ ................................................................................ 97 3.2.2.1 Exogenous ligands of PPARɣ ......................................................... 98 3.2.2.2 Endogenous ligands of PPARɣ ....................................................... 99 3.2.3. Functional role of PPARβ/δ ......................................................................... 101 3.2.3.1 Exogenous ligands of PPARβ/δ .................................................... 102 3.2.3.2 Endogenous ligands of PPARβ/δ ................................................. 102 3.3 EKODEs as potential endogenous metabolites ......................................................... 103 3.4 Target reporter assay ..................................................................................................... 105 3.4.1 Bioluminescence ............................................................................................... 105 3.4.2 Cell-based reporter assay .............................................................................. 105 3.4.3 Single luciferase reporter assay ................................................................... 106 3.4.4 Luciferase enzyme mechanism ..................................................................... 107 3.4.5 Colorimetric β-galactosidase assay ............................................................. 108 3.5 Experimental .................................................................................................................... 110 3.5.1 Materials ............................................................................................................ 110 3.5.2 Cell transient transfection assay .................................................................. 110 3.5.3 Methods ............................................................................................................