REACTION MECHANISMS AND SYNTHESIS OF OXIDIZED LIPIDS by WENYUAN YU Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: Dr. Robert G. Salomon Department of Chemistry CASE WESTERN RESERVE UNIVERSITY January 2014 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Wenyuan Yu candidate for the Doctor of Philosophy degree *. (signed) Dr. Michael G. Zagorski . (Chair of the committee) Dr. James D. Burgess . Dr. Thomas G. Gray . Dr. Yanming Wang . Dr. Robert G. Salomon . (Date) 08 /05 /2013 *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents Table of Contents ii List of Schemes v List of Figures viii List of Tables x Appendix xi Acknowledgements xvi Abbreviations and Acronyms xvii Abstract xx REACTION MECHANISMS AND SYNTHESIS OF OXIDIZED LIPIDS Chapter 1 Introduction 1 1.1 Lipid Oxidation and Peroxidation Processes. 2 1.2 Mechanism Studies of Lipid Peroxidative Production of HNE 5 1.3 Oxidation Products of Levuglandin E2 (LGE2) and LGD2. 8 1.4 Effects of Fe2+ on Lipid Oxidation 10 1.5 References 12 Chapter 2 Fe2+ Catalyzed Fragmentation of 2-(3-Pentyloxiran-2-yl)vinyl 17 Hexanoate to Generate 4-Hydroxynonenal (HNE) 2.1 Background 18 2.2 Results and Discussion. 21 iii Preparation of 3,4-Epoxy-1-nonen Hexanoate (2.13) 21 Fragmentation of 3,4-Epoxy-1-nonen Hexanoate (2.13) 22 Proposed Fragmentation Mechanism for the Formation of HNE 27 2.3 Conclusions 32 2.4 Experimental Procedures 33 2.5 References 37 Chapter 3 Synthesis of Unsymmetrically Disubstituted Maleic Anhydrides and 40 3,4-Disubstituted 5-Methylhydroxyfuran-2(5H)-ones 3.1 Background 41 3.2 Results and Discussion 44 A Feasibility Study for Pathway 1 44 A Feasibility Study for Pathway 2 45 A Feasibility Study for Pathway 3 48 3.3 Conclusions 57 3.4 Experimental Procedures 58 3.5 References 69 Chapter 4 Total Synthesis of Oxidized Levuglandin D (ox-LGD ) 2 2 72 4.1 Background 73 4.2 Results and Discussion 74 Synthesis of Two Side Chains 74 iv Synthesis of ox-LGD2 75 Possible Mechanisms of ox-LGD Generation from the Oxidation of 2 86 LGD2 4.3 Conclusions 88 4.4 Experimental Procedures 89 4.5 References 103 Chapter 5 Pilot Studies 105 Part A: Model Study for the Synthesis Putative β-Alkylperoxy Hydroperoxide 106 5.1.1 Background 106 5.1.2 Result and Discussion 109 5.1.3 Conclusions 112 5.1.3 Experimental Procedures 113 5.1.4 References 116 Part B: A Novel Method for the Selective Cleavage of DTBMS Esters 117 5.2.1 Background 117 5.2.2 Result and Discussion 119 5.2.3 Conclusions 122 5.2.4 Experimental Procedures 123 5.2.5 References 126 Appendix 127 Bibliography 170 v List of Schemes Chapter 1 Scheme 1.1 The formation of CEP from DHA-PC 3 Scheme 1.2 Suggested mechanism for the generation of HNE from linoleate or 6 arachidonate hydroperoxides Scheme 1.3 Fragmentation of dimer putative β-alkylperoxy hydroperoxide to form 7 aldehyde and radicals. Scheme 1.4 Reaction of LGs with proteins. Lactams and hydroxylactams are stable 8 end products formed from protein-bound levuglandin or isolevuglandin derived pyrroles Chapter 2 Scheme 2.1 Suggested mechanisms for the generation of HNE and ONE from 19 linoleate or arachidonate hydroperoxides Scheme 2.2 Proposed multiple fragmentation of epoxy vinyl hydroperoxide 2.9 20 Scheme 2.3 Synthesis of 3,4-epoxy-1-nonen hexanoate (2.13 (2.13-E and 2.13-Z). 21 Scheme 2.4 Epoxidation of 2.15 with 1.5 equivalent of DMDO 22 Scheme 2.5 Fe2+ Catalyzed fragmentation of 3,4-epoxy-1-nonen hexanoate to 23 generate HNE Scheme 2.6 Fe2+ catalyzed fragmentation of 2.13 and 2.8 to generate HNE 27 Scheme 2.7 Proposed mechanism for fragmentation of epoxy vinyl ester 2.13 to 29 form HNE Scheme 2.8 Reductive cleavage of vinyloxiranes 2.20 with SmI2. 30 Scheme 2.9 Suggested mechanism for the formation of HNE in Lewis acid (Zn2+, 31 Ca2+) Chapter 3 Scheme 3.1 Three possible pathways to construct unsymmetrically disubstituted 43 maleic anhydrides and 3,4-disubstituted 5-methylhydroxyfuran-2(5H)- ones. vi Scheme 3.2 Feasibility study for pathway 1 44 Scheme 3.3 Feasibility study for pathway 2 46 Scheme 3.4 Reaction of cuprate with 2, 3-dichloromaleic anhydride 3.1b. 47 Scheme 3.5 A simple model study to achieve a three-component coupling 49 construction of an unsymmetrically disubstituted maleic anhydride and it’s conversion to hydroxy lactones. Scheme 3.6 Synthesis of hydroxyl lactone 3.23 51 Scheme 3.7 Formation of diene through the reaction of (Z)-vinylstannanes with 52 BuLi. Scheme 3.8 Three different pathways for the cleavage of di-t-butyl ester 3.15 52 Scheme 3.9 Three different pathways for the cleavage of di-t-butyl carbonate 3.18 53 Scheme 3.10 Two different pathways for the formation of maleic anhydride 3.19 54 Chapter 4 Scheme 4.1 Synthesis of ox-LGD2 74 Scheme 4.2 Synthesis of top sidechain(Z)-methyl 7-bromohept-5-enoate 4.3 75 Scheme 4.3 Two confirmed byproducts 4.13 and 4.14 formed during the reaction 76 Scheme 4.4 Synthesis of DTBMS triflate 78 Scheme 4.5 Synthesis of DTBMS protected ester 4.21 79 Scheme 4.6 An alternative synthetic route to 4.21 79 Scheme 4.7 Another synthetic route of ox-LGD2. 80 Scheme 4.8 Byproduct formed in the cuprate reaction of ox-LGD2. 81 Scheme 4.9 Possible mechanisms (pathway 1 and pathway 2) to form ox-LGD2 87 from LGD2 Chapter 5 Scheme 5.1.1 Fragmentation of putative β-alkylperoxy hydroperoxide to form 106 aldehyde and radicals. vii Scheme 5.1.2 Proposed synthetic method of β-alkylperoxy hydroperoxide. 107 Scheme 5.1.3 Model study for the synthesis of β-alkylperoxy hydroperoxide 108 Scheme 5.1.4 Four possible intermediates in the reaction of 5.8 with NBS and t- 109 BuOOH. Scheme 5.2.1 Reduction of 4.18 to 4.19 by H2 and ethylenediaimine. 118 Scheme 5.2.2 Two synthetic methods to prepare DTBMSH and DTBMS triflate 119 viii List of Figures Chapter 1 Figure 1.1 Lipid oxidation and the formation of aldehyde modified proteins 4 Figure 1.2 Oxidation of arachidonic phospholipids 9 Chapter 2 Figure 2.1 HNE generated in the reaction of 2.13 in the putative presence of 24 traces of transition metal ions in unfiltered solvents (D2O and CD3CN) at 37 ºC 2+ Figure 2.2 Generation of HNE upon incubation of 2.13 with 0.5% Fe in D2O 25 and CD3CN at 37 ºC. Chapter 3 Figure 3.1 Oxidation products of arachidonic phospholipids. 41 Figure 3.2 Reaction of LGs with proteins. Lactams and hydroxylactams are stable 42 end products formed from protein- bound pyrroles. Figure 3.3 H-H COSY (CDCl3, 400 MHz) and 1D NOE difference spectra 50 (DMSO-d6, 600 MHz) of 3.15. Figure 3.4 H-H COSY of 3.19 (CDCl3, 400 MHz). 55 Chapter 4 Figure 4.1 Oxidation of arachidonic phospholipids. 73 Figure 4.2 UV (above) and ELSD (below) of a mixture 4.25 and 4.31 82 Figure 4.3 Reverse phase-HPLC of 4.25 and 4.31. There are only two main peaks 83 at 21.47 min (4.31) and 22.70 min (4.25). Figure 4.4 1H NMR of compounds 4.15 and 4.31. 84 Figure 4.5 Mass spectra of mixture products. M/z: m/z [636+ 107 or +109] [Ag+] 85 = 743, 745 (hydroxyl lactone product, 4.28a and 4.28b) Figure 4.6 HPLC spectra of reaction mixtures of m/z = 743 (above), 745 (below). 86 ix + The reaction product mixtures formed complex adducts with Ag . These adducts were analyzed by positive ESI-MS. Peaks m/z=743 and 745 corresponding to Ag+ complex adducts with 4.28a and 4.28b showed up at 31.4 min at the gradients of methanol and water Chapter 5 Figure 5.1.1 Carbon chemical shifts for bromo and oxycarbon. 110 Figure 5.1.2 H-H COSY (above) and HMQC (below) of 5.11 (CDCl3, 600 MHz). 111 x List of Tables Chapter 2 Table 2.1 HNE generated upon the incubation of 2.13 in unfiltered solvents (D2O 24 and CD3CN) Table 2.2 HNE generated upon incubation of 2.13 with Fe (II) in D2O and 25 CD3CN at 37 ºC Table 2.3 HNE generated in the incubation of 2.13 with Fe (II) and Vit E at 37 ºC 26 Table 2.4 HNE generated in the reaction of 2.8 and 2.13 with Fe (II) and vitamin 27 C at 37 ºC Table 2.5 Reductive cleavage of vinyloxiranes with SmI2 30 Table 2.6 HNE generated in the reaction of 2.13 promoted by Ca2+ or Zn2+ at 37ºC 31 Chapter 4 Table 4.1 2-Propanol/hexanes binary gradient used to separate 4.25 and 4.31 81 Table 4.2 Binary gradient of 2-propanol and acetonitrile used to separate 4.25 and 83 4.31 Chapter 5 Table 5.2.1 Cleavage of DTBMS esters with various reagents in 95% ethanol 120 xi Appendix Figure 2.1S 1H NMR spectrum of cis-non-3-enal (2.14) 128 Figure 2.2S 1H NMR spectrum of (1E,3Z)-nona-1,3-dien-1-yl hexanoate (2.15E), 128 (1Z,3Z)-nona-1,3-dien-1-yl hexanoate (2.15Z) Figure 2.3S 13C NMR spectrum of (1E,3Z)-nona-1,3-dien-1-yl hexanoate (2.15E), 129 (1Z,3Z)-nona-1,3-dien-1-yl hexanoate (2.15Z) Figure 2.4S 1H NMR spectrum of 3,4-epoxy-1(E)-nonen hexanoate (2.13E), 3,4- 129 Epoxy-1(Z)-nonen hexanoate (2.13Z) Figure 2.5S 1H NMR spectrum of 4-hydroxy-2-nonenal (HNE) 130 Figure 2.6S 1H NMR spectrum of mixture of HNE and 2.13 130 Figure 3.1S 1H NMR spectrum of 3,4-dichloro-5-hydroxy-5-methyl-furanone (3.2) 131 Figure 3.2S 13C NMR spectrum of 3,4-dichloro-5-hydroxy-5-methyl-furanone (3.2) 131 Figure 3.3S 1H NMR spectrum of 5-((tert-butyldimethylsilyl)oxy)-3,4-dichloro-5- 132 methylfuran-2(5H)-one (3.3) Figure 3.4S 13C NMR spectrum of 5-((tert-butyldimethylsilyl)oxy)-3,4-dichloro-5- 132 methylfuran-2(5H)-one (3.3) Figure 3.5S 1H NMR spectrum of 3, 4-dibutylfuran-2, 5-dione (3.9) 133 Figure 3.6S 13C NMR spectrum of 3,4-dibutylfuran-2, 5-dione (3.9) 133 Figure 3.7S 1H NMR spectrum of di-t-butyl 2-allyl-3-butylmaleate (3.15) 134 Figure 3.8S 13C NMR spectrum