Reaction Mechanisms and Synthesis of Oxidized Lipids

Home , Butyl group, Vinyl group

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

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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

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

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.

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.

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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

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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

+ 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

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

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 di-t-butyl 2-allyl-3-butylmaleate (3.15) 134

Figure 3.9S 1H NMR spectrum of 3-allyl-4-butylfuran-2,5-dione (3.16) 135

Figure 3.10S H-H COSY spectrum of 3-allyl-4-butylfuran-2,5-dione (3.16) 135

Figure 3.11S 1H NMR spectrum of 4-allyl-3-butyl-5-hydroxy-5-methylfuran-2(5H)- 136 one (3.17a) and 3-allyl-4-butyl-5-hydroxy-5-methylfuran-2(5H)-one (3.17b)

Figure 3.12S 13C NMR spectrum of 4-allyl-3-butyl-5-hydroxy-5-methylfuran-2(5H)- 136

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one (3.17a) and 3-allyl-4-butyl-5-hydroxy-5-methylfuran-2(5H)-one (3.17b)

Figure 3.13S H-H COSY spectrum of 4-allyl-3-butyl-5-hydroxy-5-methylfuran- 137 2(5H)-one (3.17a) and 3-allyl-4-butyl-5-hydroxy-5-methylfuran- 2(5H)-one (3.17b)

Figure 3.14S 1H NMR spectrum of di-tert-butyl 2-allyl-3-((E)-3-((tert- 137 butyldimethylsilyl)oxy)oct-1-en-1-yl)maleate (3.18)

Figure 3.15S 13C NMR spectrum of di-tert-butyl 2-allyl-3-((E)-3-((tert- 138 butyldimethylsilyl)oxy)oct-1-en-1-yl)maleate (3.18)

Figure 3.16S 1H NMR spectrum of (E)-3-allyl-4-(3-hydroxyoct-1-en-1-yl)furan-2,5- 138 dione (3.20)

Figure 3.17S H-H COSY spectrum of (E)-3-allyl-4-(3-hydroxyoct-1-en-1-yl)furan- 139 2,5-dione (3.20)

Figure 3.18S 1H NMR spectrum of 2-allyl-3-((E)-3-((tert- 139 butyldimethylsilyl)oxy)oct-1-en-1-yl)maleic acid (3.22)

Figure 3.19S H-H COSY spectrum of 2-allyl-3-((E)-3-((tert- 140 butyldimethylsilyl)oxy)oct-1-en-1-yl)maleic acid (3.22)

Figure 3.20S 1H NMR spectrum of (E)-3-allyl-4-(3-hydroxyoct-1-en-1-yl)furan-2,5- 140 dione (3.19)

Figure 3.21S 13C NMR spectrum of (E)-3-allyl-4-(3-hydroxyoct-1-en-1-yl)furan- 141 2,5-dione (3.19)

Figure 3.22S H-H COSY spectrum of (E)-3-allyl-4-(3-hydroxyoct-1-en-1-yl)furan- 141 2,5-dione (3.19)

Figure 3.23S 1H NMR spectrum of 2-allyl-3-butylmaleic acid 142

Figure 3.24S 1H NMR spectrum of (Z)-tert-butyl 2-(2-oxo-6-pentyl-2H-pyran- 142 3(6H)-ylidene)pent-4-enoate (3.21)

Figure 3.25S 13C NMR spectrum of (Z)-tert-butyl 2-(2-oxo-6-pentyl-2H-pyran- 143 3(6H)-ylidene)pent-4-enoate (3.21)

Figure 3.26S H-H COSY spectrum of (Z)-tert-butyl 2-(2-oxo-6-pentyl-2H-pyran- 143 3(6H)-ylidene)pent-4-enoate (3.21)

Figure 4.1S 1H NMR spectrum of 7-((tert-butyldimethylsilyl)oxy)hept-2-yn-1-ol 144

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(4.6)

Figure 4.2S 13C NMR spectrum of 7-((tert-butyldimethylsilyl)oxy)hept-2-yn-1-ol 144 (4.6)

Figure 4.3S H-H COSY spectrum of 7-((tert-butyldimethylsilyl)oxy)hept-2-yn-1-ol 145 (4.6)

Figure 4.4S 1H NMR 7-((tert-butyldimethylsilyl)oxy)hept-2-yn-1-yl acetate (4.7) 145

Figure 4.5S 13C NMR 7-((tert-butyldimethylsilyl)oxy)hept-2-yn-1-yl acetate (4.7) 146

Figure 4.6S 1H NMR spectrum of 7-acetoxyhept-5-ynoic acid (4.8) and 7- 146 Hydroxyhept-5-ynoic acid (4.9)

Figure 4.7S 13C NMR spectrum of 7-acetoxyhept-5-ynoic acid (4.8) and 7- 147 Hydroxyhept-5-ynoic acid (4.9)

Figure 4.8S 1H NMR spectrum of methyl 7-hydroxyhept-5-ynoate (4.10) 147

Figure 4.9S 1H NMR spectrum of (Z)-methyl-7-hydroxyhept-5-enoate (4.11) 148

Figure 4.10S 13C NMR spectrum of (Z)-methyl-7-hydroxyhept-5-enoate (4.11) 148

Figure 4.11S 1H NMR spectrum of (Z)-methyl 7-bromohept-5-enoate (4.12) 149

Figure 4.12S 1H NMR spectrum of 4.13 149

Figure 4.13S 13C NMR spectrum of 4.13 150

Figure 4.14S H-H COSY spectrum of 4.13 150

Figure 4.15S 1H NMR spectrum of 4.14 151

Figure 4.16S 13C NMR spectrum of 4.14 151

Figure 4.17S H-H COSY spectrum of 4.14 152

Figure 4.18S 1H NMR spectrum of methyl 7,8-(di-tert-butoxycarbonyl)-11-((tert- 152 butyldimethylsilyl)oxy)-(4Z,7Z,9E)-heptadecatrieneoate (4.15)

Figure 4.19S 13C NMR spectrum of methyl 7,8-(di-tert-butoxycarbonyl)-11-((tert- 153 butyldimethylsilyl)oxy)-(4Z,7Z,9E)-heptadecatrieneoate (4.15)

Figure 4.20S Mass spectrometry 4.15 at full scan mode. M/z: 608+107,109[Ag+] 153 =715, 717

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Figure 4.21S 1H NMR spectrum of 2-((E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en- 154 1-yl)-3-((Z)-7-methoxy-7-oxohept-2-en-1-yl)maleic acid

Figure 4.22S 1H NMR spectrum of methyl (Z)-(7-(4-((E)-3-((tert- 154 butyldimethylsilyl) oxy)oct-1-en-1-yl)-2,5-dioxo-2,5-dihydrofuran-3- yl)hept-5-enoate) (4.16)

Figure 4.23S H-H COSY spectrum of methyl (Z)-(7-(4-((E)-3-((tert- 155 butyldimethylsilyl) oxy)oct-1-en-1-yl)-2,5-dioxo-2,5-dihydrofuran-3- yl)hept-5-enoate) (4.16)

Figure 4.24S 1H NMR spectrum of di-tert-butyl(methyl)silyl 7-hydroxyhept-5- 155 ynoate (4.18)

Figure 4.25S 13C NMR spectrum of di-tert-butyl(methyl)silyl 7-hydroxyhept-5- 156 ynoate (4.18)

Figure 4.26S 1H NMR spectrum of (Z)-di-tert-butyl(methyl)silyl 7-hydroxyhept-5- 156 enoate (4.19)

Figure 4.27S 1H NMR spectrum of di-tert-butyl(methyl)silyl (Z)-7-bromohept-5- 157 enoate (4.20)

Figure 4.28S 13C NMR spectrum of di-tert-butyl(methyl)silyl (Z)-7-bromohept-5- 157 enoate (4.20)

Figure 4.29S 1H NMR spectrum of (Z)-7-((tert-butyldimethylsilyl)oxy)hept-2-en-1- 158 yl acetate (4.23)

Figure 4.30S 1H NMR spectrum of (Z)-7-acetoxyhept-5-enoic acid (4.24) and (Z)-7- 158 hydroxyhept-5-enoic acid (4.20)

Figure 4.31S 1H NMR spectrum of di-tert-butylmethylsilyl 7,8-(di-tert- 159 butoxycarbonyl)-11-((tert-butyldimethylsilyl)oxy)-(4Z,7Z,9E)- heptadecatrieneoate (4.25)

Figure 4.32S 1H NMR spectrum of di-tert-butylmethylsilyl 7,8-(di-tert- 159 butoxycarbonyl)-11-((tert-butyldimethylsilyl)oxy)-(4Z,7Z,9E)- heptadecatrieneoate (4.25)

Figure 4.33S 1H NMR spectrum of 4.31 160

Figure 4.34S H-H COSY spectrum of 4.31 160

Figure 4.35S Mass spectrometry 4.25 at full scan mode. M/z: 750+107,109[Ag+] = 161

857, 859

Figure 5.1S 1H NMR spectrum of (9Z,11E)-13-((2-methoxypropan-2-yl) peroxy) 161 octadeca-9,11-dienoic acid (5.2)

Figure 5.2S 1H NMR spectrum of (9Z,11E)-(perfluorophenyl)methyl-13-((2- 162 methoxypropan-2-yl)peroxy)octadeca-9,11-dienoate (5.3)

Figure 5.3S 1H NMR spectrum of 2-((2E,4E)-hexa-2,4-dien-1-yloxy)tetrahydro- 162 2H-pyran (5.8)

Figure 5.4S 13C NMR spectrum of 2-((2E,4E)-hexa-2,4-dien-1-yloxy)tetrahydro- 163 2H-pyran (5.8)

Figure 5.5S 1H NMR spectrum of (E)-2-((3-bromo-2-(tert-butylperoxy)hex-4-en-1- 163 yl)oxy)tetrahydro-2H-pyran (5.11)

Figure 5.6S 13C NMR spectrum of (E)-2-((3-bromo-2-(tert-butylperoxy)hex-4-en- 164 1-yl)oxy)tetrahydro-2H-pyran (5.11)

Figure 5.7S H-H COSY of (E)-2-((3-bromo-2-(tert-butylperoxy)hex-4-en-1- 164 yl)oxy)tetrahydro-2H-pyran (5.11)

Figure 5.8S HMQC spectrum of (E)-2-((3-bromo-2-(tert-butylperoxy)hex-4-en-1- 165 yl)oxy)tetrahydro-2H-pyran (5.11)

Figure 5.9S 1H NMR spectrum of p-anisic acid, DTBMS ester (5.2.1) 165 Figure 5.10S 13C NMR spectrum of p-anisic acid, DTBMS ester (5.2.1) 166

Figure 5.11S 1H NMR spectrum of furylacrylic acid, DTBMS ester (5.2.2) 166

Figure 5.12S 13C NMR spectrum of furylacrylic acid, DTBMS ester (5.2.2) 167

Figure 5.13S 1H NMR spectrum of diphenylacetic acid, DTBMS ester (5.2.3) 167

Figure 5.14S 13C NMR spectrum of diphenylacetic acid, DTBMS ester (5.2.3) 168

Figure 5.15S 1H NMR spectrum of 8-hydroxy-5-octynoic acid, DTBMS ester (5.2.4) 168

Figure 5.16S 13C NMR spectrum of 8-hydroxy-5-octynoic acid, DTBMS ester 169 (5.2.4)

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Acknowledgments

First and foremost, I would like to give my deepest appreciation to my research advisor Dr. Robert G. Salomon. He spent a lot of time in my research projects and my thesis.

He taught me how to do research independently and how to work as a scientist. Without his valuable guidance, suggestions and inspiration, I could not have accomplished all of these dissertation works.

I would also like to thank my committee members, Dr. Michael Zagorski, Dr.

Thomas Gray, Dr. James Burgess, and Dr. Yanming Wang for their precious time and input on my thesis.

I would like to thank my colleagues Dr. Linetsky, Hua, Yu, Yalun, Wenzhao, Dawit,

Junhong, Guangying, Nicholas, Yu-Shiuan and my former colleague Dr. Yunfeng Xu, Dr.

Jaewoo Choi, Dr. Li Hong, Dr. Xiaoxia Zhang, Dr. Detao Gao, Liang Xin for their discussions and friendship. I have had a pleasant time here and enjoyed working in such a great group. Special thanks to Dr. James Laird and Dr. Xiaodong Gu for their helps and friendship. Dr. Laird taught me a lot about organic synthesis and provided me valuable compounds for my synthesis. Thanks to Dr. Gu for giving me valuable suggestion and ideas.

I also want to express my thanks to Dr. James Faulk for his training and doing the

HRMS, and Dr. Dale Ray for the help on the NMR, and other staff members in Department of Chemistry in Case Western Reserve University.

Last, but not least, I want to thank my parents for their unconditional love and support all these years. I especially would like to thank my wife, Jingjing, for her endless support during my Ph.D. study.

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Abbreviations and Acronyms Abbreviations & Acronyms Definition 4-HPNE 4-hydroperoxy-nonenal 9-HPODE 9-hydroperoxy-10,12-octadecadienoic acid 13 -HPODE 13-hydropeoxy-9,11-octadecadienoic acid AA arachidonic acid AMD age-related macular degeneration BDE bond dissociation energy BHT 2,6-di-tert-butyl-4-methylphenol BIBS di-tert-butylisobutyl silyl CEP ω-carboxyethylpyrrole CID collision induced dissociation COSY correlation spectroscopy COX cyclooxygenase DCC dicyclohexylcarbodiimide DHA docosahexaenoic acid DTBMS di-tert-butylmethylsilyl DTBMSH di-tert-butylmethylsilane ELSD evaporative light scattering DMAP 4-dimethylaminopyridine DMDO dimethyldioxirane DMP Dess-Martin periodinane EOD 4-epxy-12-oxododecanoyl EOH 4-epoxy-7-oxoheptanoyl HNE 4-hydroxynonenal HMQC heteronuclear multiple-quantum correlation HMPA hexamethylphosphorictriamide HODA 9-hydroxy-12-oxo-10(E)-dodecenoic acid

HODA-PC 1-hexadecanoyl -2-(9-hydroxy-12-oxo-10(E)- dodecenoyl)-3-phosphatidylcholine HOHA 4-hydroxy-7-oxohept-5-enoic acid

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HPLC high performance liquid chromatography HRMS high resolution mass spectrometry LA linoleic acid LC/MS liquid chromatography/mass spectrometry LDL low-density lipoprotein

LGD2 levuglandin D2

LGE2 levuglandin E2 LO· lipid alkoxy radicals LOO· lipid perxoyl radicals LOOHs lipid hydroperoxides LPO lipid peroxidation m/z mass-to-charge ratio NBS N-bromosuccinimide NMR nuclear magnetic resonance NOE nuclear magnetic resonance ON ω-oxononanoyl ONE 4-oxo-2-nonenal ox-LGD2 oxidized levuglandin D2 PC phosphatidylcholine PCC pyridinium chlorochromate

Pd(PPh3)4 tetrakis(triphenylphosphine)palladium (0) PFB pentaflurobenzyl PFB-Br pentafluorobenzyl bromide PhLi phenyllithium

PGH2 prostaglandin H2 PPTS pyridinium p-toluene sulfonate PUFAs polyunsaturated fatty acyls

Rf retention factor ROS rod outer segment RPE retinal pigment epithelium SUVs small unilamellar vesicles

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TBDMS tert-butyldimethylsilyl TBDMSOTf tert-butyldimethylsilyl triflate TES triethylsilyl TFA trifluoroacetic acid THF tetrahydrofuran TIC total ion current TIPS triisopropylsilyl TLC thin layer chromatography TMS trimethlysilyl TsOH p-toluenesulfonic acid monohydrate UV ultraviolet

Vit C vitamin C Vit E vitamin E

REACTION MECHANISMS AND

SYNTHESIS OF OXIDIZED LIPIDS

Abstract

WENYUAN YU

4-Hydroxy-2-nonenal (HNE) is a well-known abundant aldehyde that is generated through lipid autoxidation in vivo. Epoxy vinyl hydroperoxide is the precursor for the formation of both HNE and ONE. To further understand the mechanism of formation of

HNE in vivo, a simple model compound 3,4-epoxy-1-nonen hexanoate, a possible oxidation intermediate generated from elimination of water from epoxy hydroperoxide, was synthesized. 3,4-Epoxy-1-nonen hexanoate was incubated in physiomimetic mixed solvents

2+ 2+ 2+ CD3CN and D2O in the absence or presence of Fe , Ca or Zn as well as in the presence or absence or vitamin E. 3,4-Epoxy-1-nonen hexanoate incubated in the presence of a catalytic amount of Fe2+ delivered HNE in quantitative yield in less than one hour, while fragmentation of 3,4-epoxy-1-nonen hexanoate stopped or dramatically slowed with the addition of one equivalent of the antioxidant vitamin E depending on the amount of added

Fe2+. A free radical pathway was proposed for the rearrangement of 3,4-epoxy-1-nonen hexanoate in the presence of Fe2+ that delivers HNE. In contrast, the slow fragmentation of

3,4-epoxy-1-nonen hexanoate to form HNE in the presence of Ca2+ or Zn2+ apparently involves a Lewis acid catalytic mechanism.

xxi

Three different possible pathways that were explored to synthesize unsymmetrically disubstituted maleic anhydrides and 3,4-disubstituted 5-methyl-5-hydroxyfuran-2(5H)-ones were discussed. The reaction of 2,3-dichloromaleic anhydride with two equivalents of butyl cuprates afforded 3,4-dibutylfuran-2,5-dione. This method can be applied to the one-step synthesis of symmetrical dialkyl maleic anhydrides (3,4-dialkyl-furan-2,5-diones) in high yields. Unsymmetrical mono and disubstituted maleic anhydrides are accessible through the reaction of di-t-butyl acetylenedicarboxylate with organocopper reagents RCu(Me2S)·MgBr2 followed in situ by addition of various electrophiles that delivers mono and disubstituted di-t- butyl maleates that can be converted by Lewis acid catalysis into maleic anhydrides. The maleic anhydrides give hydroxyl lactones by reaction with methylating reagents such as methyl lithium or methyl magnesium bromide. This provided precedent for a possible route for the total synthesis of ox-LGD2, which is a new oxidized derivative of LGD2 isolated in the red algae, Gracilariaedulis in 2011.

Di-tert-butylmethylsilyl (DTBMS) is a novel reagent to protect carboxylic acid. We discovered that ethylenediamine can cleave the silyl ester of a DTBMS protected carboxylic acid in high yields. The milder base ethanolamine can also remove the DTBMS group from alkyl silyl esters. To the best of our knowledge, this is the first example of the use of ethylenediamine and ethanolamine for the cleavage of a silyl ester. Therefore, this novel method should find applications in organic synthesis. Potassium carbonate is also effective for deprotecting both aryl and alkyl DTBMS esters.

xxii

Chapter 1

Introduction

1.1 Lipid Oxidation and Peroxidation Processes.

The main biological functions of lipids include energy storage, acting as structural components of cell membranes, and participating as important signaling molecules. Normal lipid oxidation for energy production is accomplished in peroxisomes and mitochondria,1 both of which are specialized organelles that are capable of controlling the reactive compounds normally generated by lipid oxidation.2 Autoxidation of lipids plays a key role in human health. All lipids that contain unsaturated bonds have the potential for spontaneous oxidation and the production of toxic metabolites such as hydroperoxides, epoxides, aldehydes, and free radicals. It contributes to changes associated with aging, and may be recruited in the immune response (oxidative burst) or programmed cell death (apoptosis). It also is involved in pathological processes such as the ischemia-reperfusion injury associated with heart attacks and stroke, and may contribute to Alzheimer’s disease, Parkinson’s diseases3, amyotrophic lateral sclerosis,4 and contributes in several ways to pathogenesis of age-related macular degeneration.5

Docosahexaenoic acid (DHA) is the most abundant essential polyunsaturated fatty acyl (PUFA) in the retina. It comprises 60% of the PUFAs in the retina.6 It is concentrated in the light-sensitive photoreceptor rod outer segment membranes and in the retinal pigment epithelium (RPE).7,8 The DHA ester of 2-lysophosphatidylcholine (DHA-PC), gives rise to carboxyethylpyrroles (CEP) through oxidative cleavage to the 4-hydroxy-7-oxo-2-octenoate

HOHA-PC (Scheme 1.1). DHA deficiency is associated with cognitive decline.9 Retinal neovascularization resulting from diabetic retinopathy is the most common cause of new blindness in young patients. In addition, choroidal neovascularization resulting from AMD

(age-related macular degeneration) is the chief cause of severe and irreversible loss of vision in elderly patients. CEPs promote neovascularization by inducing sprouting of new vessels.10

Scheme 1.1 The formation of CEP from DHA-PC.

Reactive oxygen species are highly unstable molecules having unpaired electrons with a strong proclivity toward reaction with other substrates. They are formed during a variety of biochemical reactions and cellular activities such as mitochondrial metabolism.11

PUFAs are highly susceptible to free radical-induced oxidation owing to the presence of doubly allylic methylene groups. Hydrogen atoms on these methylene carbons can be abstracted, forming free radicals that then trigger self-propagating chain reactions with oxygen and additional PUFAs in cell membranes, leading to production of new pentadienyl radicals and hydroperoxides.2 Oxidative degradation of cellular lipids, particularly their component PUFAs, is called lipid peroxidation or lipoxidation (LPO).12

Lipoxygenases can transform PUFAs to lipid hydroperoxides (LOOHs) which can react with iron ion to generate LO· and LOO· radicals.13-16 In turn, these active radicals will induce autocatalytic lipid peroxidation to regenerate L· from unsaturated acyls (LH) by abstracting allylic and especially doubly allylic hydrogen atoms. This cycle repeats as the new fatty acid radical (L·) reacts with an oxygen molecule to give a dienyl peroxyl radical

(LOO·).17 On the other hand, LOOH is not stable toward oxidative degradation through various pathways. Several reactive aldehydes such as malondialdehyde (MDA), acrolein, 2,4-

3 decadienal (DDE), 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE) were generated through oxidative fragmentation involving allylic hydroperoxide intermediates.18-21

Figure 1.1 Lipid oxidation and the formation of aldehyde modified proteins.22

These compounds can cause molecular cross-linking enzyme inhibition, and produce insoluble lipofuscin deposits from proteins, thus destroying macromolecules and interfering with cell functioning. They also directly damage DNA, and so are mutagenic and carcinogenic. Cellular aging, arteriosclerosis, malignant transformation, immune dysfunction, or cell death may be the ultimate result of extensive autoxidation.

1.2 Mechanism Studies of Lipid Peroxidative Production of HNE.

The peroxidation of lipids generates reactive aldehydes, that may act as mutagens23 or inactivate enzymes,24,25 or react with proteins and nucleic acids to form heterogeneous crosslinks.26 4-Hydroxy-2-nonenal (HNE) is a well-known aldehyde generated upon lipid peroxide reactions in vivo.27,28 HNE is an oxidative product of fragmentation of arachidonic acid (AA) or linoleic acid (LA) groups, e.g., in phospholipids.29 HNE reacts with nucleophilic residues of proteins such as lysine, histidine and cysteine and generates Michael and Schiff base adducts as well as a pyrrole adduct.30,31 HNE can also mediate the formation of lysine-lysine crosslinks.32,33 It is highly cytotoxic to Ehrlich ascites tumor cells. It can cause lysis of erythrocytes and arouse chemiluminescence and pentane production in isolated hepatocytes.33,34 HNE and its protein adducts are therefore considered as established markers for oxidative stress.35 This reactive cytotoxic agent covalently adducts with various proteins or DNA, and thereby modifies proteins and DNA to form biologically relevant adducts.36,37 HNE has been linked to the pathology of several diseases such as neurodegenerative diseases, Alzheimer’s disease and Parkinson’s disease; cataract, atherosclerosis, and cancer, and it can induce cell apoptosis.38,39 However, the mechanisms of its formation have proven to be quite elusive. One of the possible mechanisms suggested by

Pryor and Porter for the generation of the unsaturated aldehyde HNE from linoleate or arachidonate hydroperoxides is shown in Scheme 1.2.40

Homolytic cleavage reduction of hydroperoxide in the presence of metal ions (e.g.,

Fe(II)) gives an alkoxyl radical which can cyclize to give hydroperoxy epoxide. The resulting alkoxyl radical intermediate can cyclize to produce a diepoxycarbinyl radical that gives an epoxyhydroperoxide by reaction with oxygen and hydrogen atom abstraction. Conversion of

5 epoxy hydroperoxide to a 3,4-epoxy aldehyde is suggested to occur by Lewis or protic acid catalysis.41 The epoxy aldehyde is known to undergo ring opening and rearrangement to

HNE under very mild conditions.42,43 This mechanism postulates a pseudosymmetric diepoxycarbinyl radical intermediate in the fragmentation, and also involves the conversion of an intermediate epoxy hydroperoxide to an epoxy aldehyde precursor, assumed to be unstable with respect to ring opening catalyzed by acid or base to deliver HNE.40

Scheme 1.2 Suggested mechanism for the generation of HNE from linoleate or arachidonate hydroperoxides.

Alternatively, a mechanism involved the fragmentation of β-alkylperoxy hydroperoxide provides another pathway to deliver truncated aldehydes as well as hydroxyl and alkoxyl radicals (Scheme 1.3). The oxidation of PUFAs or their esters can generate relatively stable pentadienyl radicals upon hydrogen atom abstraction,44, 45 which can be captured by oxygen to deliver conjugated dienyl perxoy radicals that can abstract hydrogen atoms from additional polyunsaturated fatty acids or their esters to generate new hydroperoxide, such as 13-HPODE.46 These hydroperoxides can further oxidized to HNE, hexanal and its “mirror image” 9-hydroxy-12-oxododec-10-enoic acid (HODA).47-52 It was suggested that intermediates containing a peroxide linked dimer with a hydroperoxy group

6 readily generate hydroxyl and alkoxyl radicals under mild conditions (Scheme 1.3).44 It is the generation of these radicals during the fragmentation reactions, driven by the formation of two carbonyl groups, that results in their ability to be autocatalytic intermediates in lipid oxidation reactions. The putative β-alkylperoxy hydroperoxide intermediate is shown in scheme 1.3.

13-PODE 13-ODE HO C H (CH ) COOH 4 9 2 6 C4H9 (CH2)6COOH Lenoleic acid (LA) 13-HPODE 13-HODE HOH LA

O 2 C5H11 (CH2)7COOH LA C5H11 (CH2)7COOH C H (CH ) COOH Fe2+ 5 11 2 7

OO LA HOO O 13-ODE 13-PODE 13-HPODE OH C5H11 HOOC(H2C)7 H OH

HOOC(H2C)7 ON O LA C5H11 (CH2)7COOH

C H HO O O OH 5 11 LA O O OH O2 13-HODE O C5H11 (CH2)7COOH

C5H11 (CH2)7COOH O HNE alkylperoxy hydroperoxide

Scheme 1.3 Fragmentation of dimer putative β-alkylperoxy hydroperoxide to form aldehyde and radicals.

1.3 Oxidation Products of Levuglandin E2 (LGE2) and LGD2.

Autoxidation of arachidonic phospholipids can produce prostaglandin H2 (PGH2) by cyclooxygenase (COX) or H2-isoprostanes by non-enzymatic peroxidation, which can

53,54 generate reactive levuglandin E2 (LGE2) and LGD2, or their isomers isoLGs. LGs and isoLGs can react spontaneously with free primary amino groups in proteins and DNA to form covalent adducts and cross-link proteins and nucleic acids.55-57

Scheme 1.4 Reaction of LGs with proteins. Lactams and hydroxylactams are stable end products formed from protein-bound levuglandin- or isolevuglandin-derived pyrroles.

The reaction of free amino groups of proteins with LGs or isoLGs through a Schiff base and its enamine tautomer leads to pyrrole adducts. Oxidation of the pyrroles can eventually yield lactams and hydroxylactams.59-60 Thus, LGs or isoLGs interfere with protein function and are among the most potent neurotoxic products of lipid oxidation. Because they can accumulate over the lifetimes of proteins, iso[n]LG-protein adducts represent a convenient dosimeter of oxidative stress. 61-63

In 2011, Yotsu-Yamashita and co-workers discovered that a new oxidation derivative of LGD2, e.g. ox-LGD2, in the red algae, Gracilaria edulis. It was also identified in mouse tissues and the lysate of PMA-treated THP-1 cells incubated with arachidonic acid. These

8 results suggest that ox-LGD2 is a common oxidized metabolite of lipid oxidation

64 intermediate LGD2.

Figure 1.2 Oxidation of arachidonic phospholipids.

1.4 Effects of Fe2+ on Lipid Oxidation.

An iron ion is contained in the active center of lipoxygenases. Lipid hydroperoxides of unsaturated fatty acids (LOOH) can be decomposed to alkoxy radicals (LO·) in the presence of iron or copper ions in a Fenton-like reaction.65 Cu2+ ions correspond to Fe3+ in their oxidation state. LO· can be further cleaved to aldehyde. The generated Fe3+ may also induce the generation of lipid perxoyl radicals (LOO·). 66

LOOH + Fe2+ LO·+ Fe3+ + OH-

LOOH + Fe3+ LOO· + Fe2+ + H+

For example, linoleic acid can be induced to form alkoxy radicals by catalytic amounts of either Fe2+ or Fe3+ ions since the conversion between these two ions are always in equilibrium. The generation of lipid peroxidation (LPO) products in diseases is mainly assumed to be caused by a nonenzymatic LPO. The dysfunction of mitochondria can be explained by this. In mitochondria, oxygen is transformed to water to produce hydroxyl radical in four steps.67

2+ 3+ · - H2O2 + Fe Fe + OH + OH First, a superoxide anion is generated by transferring one electron to oxygen.68 The anion can react with H2O to produce a radical, the combination of which will generate H2O2

69 70 and O2. H2O2 can react with iron ion in a Fenton reaction to give OH radical. OH· is a very active species which can abstract a hydrogen from allylic methylene groups in PUFAs to give allylic radicals and then perxoyl radicals by reacting with oxygen. Perxoyl radicals not only attack unsaturated double bonds, but also proteins and DNA as discussed in chapter

1.1. The ability of peroxyl radicals to react with molecules in biological systems serve as the primary reason that nonenzymatic lipid peroxidation is a deleterious process.67

1.5 References

1. Shils, M. E.; Shike, M.; Caballero, B.; Cousins, B. J. Modern Nutrition in Health and

Disease: assessment and requirements. 10th ed. Philadelphia: Lippincott Williams & Wilkins,

2006

2. Fahy, E.; Subramaniam, S.; Brown, H.A. J Lipid Res 2005, 46, 839-861.

3. Clausen, J. Acta Neurol Scand 1984, 70, 345-355.

4. Davies, K. J. Biochem Soc Symp 1995, 61, 1-31.

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6. Meharban, S. Indian J Pediatrics 2005, 72, 239-42.

7. Wang, N.; Anderson, R.E. Curr Eye Res 1992, 11, 783-791.

8. Alvarez, R. A.; Aguirre, G. D.; Acland, G. M.; Anderson, R.E. Invest Ophthalmol Vis Sci

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9. Lukiw, W. J.; Cui, J. G.; Marcheselli, V. L.; Bodker, M.; Botkjaer, A.; Gotlinger, K.;

Serhan, C. N.; Bazan, N. G. J Clin Invest 2005, 115, 2774-2783.

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Crabb, J.; Anand-Apte, B. PNAS. 2006, 103, 13480–13484.

11. Simonian, N. A.; Coyle, J. T. Annu Rev Pharmacol Toxicol 1996, 36, 83-106.

12. Shewfelt, R. L.; Purvis, A. C. HortScience 1995, 30, 213-218.

13. Yamamoto, S. Biochim Biophys Acta 1992, 117-131.

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16. Halliwell, B. S. Afr J Sci 1991, 87, 576-584.

17. Bradley, D. E.; Min, D. B., Cat Rev Food Sci Nutri 1992, 31, 211-236.

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20. Lee, S. H.; Blair, I. A., Chem Res Toxicol 2000, 13, 698-702.

21. Rosahl, S. Z. Naturforsch 1996, 51c, 123-138.

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Mutat Res 1985, 148, 25-34.

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25. Szweda, L. I.; Uchida, K.; Tsai, L.; Stadtman, E. R., J Biol Chem 1993, 268, 3342-3347.

26. Chio, K. S.; Tappel, A. L. Biochemistry 1969, 8, 2821-2826.

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30. Cadenas, E.; Muller, A.; Brigelius, R.; Esterbauer, H.; Sies, H., Biochem J 1983, 214,

479-487.

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32. Sayre, L. M.; Sha, W.; Xu, G.; Kaur, K.; Nadkarni, D.; Subbanagounder, G.; Salomon, R.

G., Chem Res Toxicol 1996, 9, 194-201.

33. Xu, G.; Sayre, L. M., Chem Res Toxicol 1998, 11, 247-251.

34. Salomon, R. G.; Kaur, K.; Podrez, E.; Hoff, H. F.; Krushinsky, A. V.; Sayre, L. M. Chem

Res Toxicol 2000, 13, 557-564.

35. Zarkovic, N., Mol Aspects Med 2003, 24, 281-291.

36. Esterbauer, H.; Schaur, R. J.; Zollner, H. Free Radic Biol Med 1991, 11, 81-128.

37. Nair, J.; Barbin, A.; Velic, I.; Bartsch, H. Mutat Res 1999, 424, 59-69.

38. Fazio, V. M.; Rinaldi, M.; Ciafre, S.; Barrera, G.; and Farace, M. G. Mol. Aspects Med

1993, 14, 217-228.

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40. Pryor, W. A.; Porter, N. A. Free Radic Biol Med 1990, 8, 541-543.

41. Gardner, H. W.; Selke, E. Lipids 1984, 19, 375-380.

42. Yadagiri, P.; Lumin, S.; Mosset, P.; Capdevila, J.; Falck, J. R. Tetrahedron Lett 1986, 27,

6039-6040.

43. Gardner, H. W.; Bartelt, R. J.; Weisleder, D. Lipids 1992, 27, 686-689.

44. Moritaa, M.; Tokita, M. Lipids 2006, 41, 91-95.

45. Nicholls, S. J.; Hazen, S. L. J Lipid Res 2009, 50 Suppl, S346-351.

46. Min, D. B.; Boff, J. M. Comprehensive Rev Food Sci Food Safety (CRFSFS) 2002, 1, 58-

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1986, 27, 6039-6040.

52. Gardner, H. W.; Bartelt, R. J.; Weisleder, D. Lipids 1992, 27, 686-689.

53. Salomon , R. G. , Miller, D. B.; Zagorski, M. G.; Coughlin, D. J. J Am Chem Soc 1984 ,

106, 6049- 6060 .

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323-326.

55. Salomon , R. G.; Sha, W.; Brame, C.; Kaur, K.; Subbanagounder, G.; O’Neil, J.; Hoff, H.

F.; Roberts II, L. J. J Biol Chem 1999, 274, 20271-20280.

56. Salomon, R. G.; Subbanagounder, G.; Singh, U.; O’Neil, J.; Hoff. H. F. Chem Res

Toxicol 1997, 10, 750-759.

57. Brame, C. J.; Salomon , R. G.; Morrow , J. D. ; Roberts II L. J. J Biol Chem 1999, 274,

13139-13146.

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60. IYER, R.S., M.E. KOBIERSKI & R.G. SALOMON. J Org Chem 1994, 59, 6038-6043.

61. Salomon. R. G. Antioxid Redox Signal 2005, 7, 185-201.

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Boutaud , O.; Oates, J. A. J Neurochem 2005, 94, 1140-1145.

63. Subbanagounder, G.; Salomon, R. G.; Murthi, K.; Brame, C.; Roberts, L. J. J Org Chem

1997, 62, 7658–7666.

64. Kanai, Y.; Hiroki, S.; Koshino, H.; Konoki, K.; Cho, Y.; Cayme, M.; Fukuyo, Y.;

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65. Gardner, H.W. Biol Med 1989, 7, 65–86.

66. Esterbauer, H., Rotheneder, M., Striegl, G., Waeg, G., Ashy, A., Sattler, W., Jurgens, G.,

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67. Spiteller, G. Med Hypoth 2003, 60, 69–83.

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Chapter 2

Fe2+ Catalyzed Fragmentation of

2-(3-Pentyloxiran-2-yl)vinyl Hexanoate to

Generate 4-Hydroxynonenal (HNE)

2.1 Background

4-Hydroxy-2-nonenal (HNE) is a well-known abundant aldehyde that is generated through lipid autoxidation in vivo.1,2 This reactive cytotoxic agent reacts with and covalently modifies proteins and DNA to form biologically relevant adducts.3,4 HNE has been linked with the pathology of several diseases such as neurodegenerative diseases,

Alzheimer’s and Parkinson’s disease, cataract, atherosclerosis, and cancer, and it can induce cell apoptosis.5,6 However, understanding the mechanisms of its formation has proven to be elusive. Scheme 2.1 shows a mechanism suggested by Pryor and Porter for the generation of the hydroxyl unsaturated aldehyde HNE from linoleate or arachidonate hydroperoxides.7

Homolytic reductive cleavage of hydroperoxide 2.1 in the presence of metal ions, e.g., Fe (II), gives an alkoxyl radical 2.2 that can cyclize to give hydroperoxy epoxide 2.3. Homolytic cleavage of 2.3 then gives alkoxyl radical 2.4, which can cyclize to produce diepoxycarbinyl radical 2.5. Epoxy hydroperoxide 2.7 is then produced by the cleavage of 2.5 and oxidation of 2.6 in the presence of oxygen. Conversion of 2.7 to 3,4-epoxy aldehyde 2.8 was suggested to occur by Lewis or protic acid catalysis.8 Epoxy aldehyde 2.8 is known to be unstable and undergoes ring opening rearrangement to HNE under mild conditions.9,10

This mechanism postulates a pseudosymmetrical diepoxycarbinyl radical intermediate 2.5 in the fragmentation, and also involves the conversion of immediate epoxy hydroperoxide 2.7 to epoxy aldehyde precursor 2.8, and assumes that 2.8 is unstable with respect to acid or base catalyzed ring opening to deliver HNE.7

Instead of HNE, 4-oxo-2-nonenal (ONE) was found to be a principal decomposition product of linoleic acid autoxidation based on LC/MS analysis of adducts generated from 13- hydroperoxylinoleic acid that react with DNA bases.11 It is conceivable that ONE is simply

18 the oxidation product of initial intermediate HNE. However, since products that would be formed by reaction of HNE or its epoxide with dGuo could not be detected, instead of fragmenting from 2.8, Blair proposed that ONE is the direct product from a fragmentation of intermediate 2.7 that loses one molecule of water with concomitant loss of a long chain fatty acid aldehyde.

Scheme 2.1 Suggested mechanisms for the generation of HNE and ONE from linoleate or arachidonate hydroperoxides.

Hydroperoxy epoxide 2.3 can thus serve as a common intermediate to give both γ- hydroxyalkenals and γ-oxoalkenals, and epoxy hydroperoxide 2.7 is the precursor for the formation of both HNE and ONE. By comparing these two mechanisms (Scheme 2.1), despite the fact that the ring opening of epoxide is expected to be catalyzed by acid or base, it remained to be determined whether or not the epoxy aldehyde 2.8 can serve as a precursor of

HNE under mild neutral biological reaction conditions.

Scheme 2.2 Proposed multiple fragmentation of epoxy vinyl hydroperoxide 2.9.

To further understand the mechanism of formation of HNE in vivo, we synthesized a simple model compound 3,4-epoxy-1-nonen hexanoate (2.13) that contains both epoxy and vinyl group, and is a possible oxidation intermediate generated from elimination of water from epoxy hydroperoxide (2.12) (Scheme 2.2). Owing to symmetry, radical 2.10, which is functionally and structurally similar to the suggested diepoxycarbinyl radical intermediate

2.5, was expected to give a single aldehyde product upon decomposition, and this would facilitate quantitative evaluation of reaction progress. After treating with Fe(II), this epoxy vinyl compound would undergo a series of radical rearrangements that could generate HNE.

2.2 Results and Discussion

Preparation of 3,4-Epoxy-1-nonen Hexanoate (2.13).

Partial oxidation of (Z)-non-3-en-1-ol to aldehyde 2.14 was carried out in CH2Cl2 solution by treatment with the Dess-Martin periodinane (DMP). Preparation of the enol ester

2.15 was accomplished by the reaction of the sodium enolate of 2.14, which was prepared by adding 2.14 to sodium bis(trimethylsilyl)amide in THF at -78 ºC, with hexanoyl chloride.

The ratio of the two isomers of 2.15 (2.15E and 2.15Z) is E : Z = 1 : 0.64 according to the 1H

NMR peak areas of vinyl hydrogens. The yield of 2.15 in this reaction is improved (72.1%), compared with the yield (22%) of an enol ester reported in the reference paper using the same reaction strategy.12 Epoxidation of 2.15 can be performed using commercially available oxone or H2O2, but the yield is only about 50% and the product is difficult to recover. By comparison, when reacting with dimethyldioxirane (DMDO) that was freshly prepared in acetone, the reaction was carried out in acetone solution and was completed in about 20 minutes at room temperature to give pure epoxides 2.13E and 2.13Z.13 It is of interest to note that the epoxidation reaction occurred specifically at the double bond between carbons 3 and

4 of 2.15.

NaHMDS, THF, -78 oC HO DMP, CH2Cl2 OHC C H hexanoyl chloride C H 4 9 4 9 2.14

C H O 4 9 DMDO C4H9 O O O Acetone C5H11 2.15E C5H11 2.15Z O

O C5H11

C4H9 O O

O C4H9 O 2.13E C5H11 2.13Z

Scheme 2.3 Synthesis of 3,4-epoxy-1-nonen hexanoate (2.13 (2.13E and 2.13Z)).

The use of various amounts of DMDO was tested to epoxidize 2.15. When the ratio of alkene ester 2.15 to DMDO is 1 : 1.5, the epoxy ester 2.13 was formed, however it was unstable and further decomposed to HNE and hexanoic acid that were separated by flash chromatograph and their identities were confirmed by 1H NMR (Scheme 2.4). When a ratio of 2.15 to DMDO of 1 : 2.5 was employed, HNE and hexanoic acid were the only final products. No trace of epoxy ester 2.13 could be detected in the 1H NMR spectrum of the reaction product mixture. If the epoxy compound 2.13 formed during the reaction, it was rapidly transformed into HNE. In order to get pure epoxy ester 2.13, the ratio of 2.15 to

DMDO must be maintained at 1 : 1.05-1.1. Compound 2.13 must be stored in diethyl ether solution at -20 ºC. It is stable for several months under this condition.

Scheme 2.4 Epoxidation of 2.15 with 1.5 equivalent of DMDO.

Fragmentation of 3,4-Epoxy-1-nonen Hexanoate (2.13).

It is well known that hydroperoxides can undergo transition metal ion-mediated decomposition through single electron transfer to form alkoxy radicals. For example, iron has the capacity to interconvert between ferrous (Fe2+) and ferric (Fe3+), donating and accepting electrons readily. One type of reaction promoted by iron is the formation of lipid radicals, that is, ferrous (Fe2+) iron can react with lipid hydroperoxides (LOOH) to form alkoxyl radicals (LO·).14-16 To model the reaction conditions that promote decomposition of lipid

22 hydroperoxides that may involve the intermediacy of 2.13, this vinyl ester was exposed to

Fe(II) choloride in physiomimetic mixed solvent (D2O/CD3CN = 1 : 1) to assess the possibility that it would promote fragmentation of 2.13 at 37 ºC. The ratio of 2.13 and HNE

1 in D2O and CD3CN was determined by the area of vinyl hydrogens in H NMR.

Chelex 100 ion chelating resin is a chelating material used to purify chemical compounds via ion exchange. It is noteworthy for its ability to bind transition metal ions,17 such as Fe, Cu. In our experiment, Chelex 100 was used to purify D2O and CD3CN before or after the incubation depending on the experimental reaction conditions. Vitamin E (α- tocopherol) is a biological lipid-soluble antioxidant. It performs its functions as antioxidant in the glutathione peroxidase pathway,18 and it protects cell membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction.19,20 By removing free radical intermediates, it prevents the oxidation reaction from continuing. We also examined the effect of α-tocopherol on the Fe2+ promoted decomposition of 2.13.

Fragmentation of epoxy vinyl ester 2.13 generated HNE and hexanoic acid (Scheme

2.5). First, we examined fragmentation of 2.13 in a physiomimetic mixed solvent (D2O and

CD3CN). When the solvents were used as received and not filtered through a pad of Chelex

100, slow decomposition of 2.13 was observed. In contrast, no decomposition occurred if the solvent mixture was filtered through Chelex 100 beforehand, indicating that traces of transition metal ions in the unfiltered solvent promoted the fragmentation of 2.13 (Figure 2.1,

Table 2.1).

Scheme 2.5 Fe2+ Catalyzed fragmentation of 3,4-epoxy-1-nonen hexanoate to generate HNE.

y= -7.24*exp(-x/104)+7.27 r2 = 0.9903 88.3%

76.0%

73.6%

70.3%

55.6%

19.7% HNE / (reactant + HNE))HNE (reactant /

5.3%

0 100 200 300 400 Time (min)

Figure 2.1 HNE generated in the reaction of 2.13 in the putative presence of traces of transition metal ions in unfiltered solvents (D2O and CD3CN) at 37 ºC.

Table 2.1 HNE generated upon the incubation of 2.13 in unfiltered solvents (D2O and

CD3CN).

Solvents filtered Reaction time Unreacted HNE through Chelex 100 (min) (%) (%) Yes 400 100% 0% No 15 94.7% 5.3% No 40 80.3% 19.7% No 60 44.4% 55.6% No 90 29.7% 70.3% No 120 26.4% 73.6% No 165 24% 76.0% No 400 11.7% 88.3%

Subsequently, the effects of exposure of 2.13 to various mole percentage of Fe2+

(0.5%, 1%, 5%, 100%) were examined. Fragmentation 2.13 generated HNE that decomposed completely in 20 min or 1 hour in the presence of one equivalent or 5 mole percent of Fe2+ respectively.

HNE (n=3) 1.1

1.0

0.9

0.8

0.7

0.6

0.5

0.4

HNE/(HNE+starting material) 0.3

0.2

20 30 40 50 60 Time (min)

2+ Figure 2.2 Generation of HNE upon incubation of 2.13 with 0.5% Fe in D2O and CD3CN at 37 ºC.

Table 2.2 HNE generated upon incubation of 2.13 with Fe (II) in D2O and CD3CN at 37 ºC.

Epoxide Additive mol % Reaction time Unreacted (%) HNE (%)

2+ 2.13 Fe 100% 20 min 0 100 2+ 2.13 Fe 5% 60 min 0 100 2+ 2.13 Fe 1% 10 min 29.6% 70.4%

2+ 2.13 Fe 0.5% 20 min 76.3% 23.7% 2+ 2.13 Fe 0.5% 30 min 63.4% 36.6% 2+ 2.13 Fe 0.5% 40 min 39.9% 60.1% 2+ 2.13 Fe 0.5% 60 min 0 100%

Fragmentation of 2.13 in the presence of a catalytic amount of Fe2+ (0.5 mol %) is shown in Table 2.2 and Figure 2.2. All the starting material 2.13 was decomposed to HNE in

60 min. Thus, fragmentation of epoxy vinyl ester 2.13 is much faster when it is treated with even catalytic amount of Fe2+ (0.5 mol %) (Figure 2.2) compared with treatment with unfiltered blank solvents that putatively contain traces of redox active metal ions (Table 2.1).

In a third experiment, 2.13 was found to be quite stable when treated with one equivalent of Vit E in CD3CN and D2O (Table 2.3). After treatment of 2.13 with one equivalent of Fe2+ and Vit E for 1 hour, 20% of 2.13 remained unreacted. The yield of HNE is 53% from decomposition of 2.13 in the presence of one equivalent vitamin E and 10 mol % of Fe2+. However, no HNE was detected upon treatment with a catalytic amount (0.01 equiv.) of Fe2+ and one equivalent in the presence of the antioxidant vitamin E (Table 2.3).

Table 2.3 HNE generated in the incubation of 2.13 with Fe (II) and Vit E at 37 ºC.

Epoxide Additives mol% Time Unreacted (%) HNE 2.13 Vit E 100% 25 h 100% 0 2+ 2.13 Vit E, Fe 100% : 100% 60 min 20% 80% 2+ 2.13 Vit E, Fe 100% : 1% 60 min 100% 0 2+ 2.13 Vit E, Fe 200% : 0.5% 60 min 100% 0 2.13 Vit E, Fe2+ 100% : 10% 60 min 47.3% 52.7% 2.13 Vit E, Fe2+ 200% : 10% 60 min 38.7% 61.3% 2+ 2.13 Fe 0.5% 40 min 39.9% 60.1% 2+ 2.13 Fe 5.0% 60 min 0% 100%

In the previously proposed mechanism for the Lewis or protic acid catalyzed formation of HNE (Scheme 2.1), epoxy aldehyde 2.8, is suggested as a precursor to HNE. In our experiment, 2.8 was incubated with various amounts of Fe2+ at 37 °C for 1 hour in

CD3CN and D2O (Scheme 2.6). In contrast, 2.8 that is believed to be unstable with respect to ring opening catalyzed by acid or base to deliver HNE, decomposed much more slowly than

2.13 in the presence of Fe2+ (Table 2.4). Thus, 2.8 remains unreacted when exposed to 5 mole % of Fe2+ at 37 °C for one hour.

O OH O CH C H O HC C H C H O ? O HNE ? 2.13 (2.13E and 2.13Z) O C H 2.8

Scheme 2.6 Fe2+ catalyzed fragmentation of 2.13 and 2.8 to generate HNE.

Table 2.4 HNE generated in the reaction of 2.8 and 2.13 with Fe (II) and Vit C at 37 ºC.

Epoxide Additive mol% Reaction time (min) Unreacted (%) HNE 2+ 2.8 Fe 1% 60 100% 0 2+ 2.8 Fe 5% 60 100% 0 2+ 2.13 Fe 5% 60 0 100 2+ 2.13 Fe 0.5% 40 39.9% 60.1% 2.8 vit C 5% 20 100% 0 2.13, vit C 5% 20 28% 72%

Proposed Fragmentation Mechanism for the Formation of HNE

Bond energy is a measure of the strength of a chemical bond. A general trend is that the shorter the bond length, the higher the bond energy. The larger the bond energy, the stronger the bond.21, 22 The average bond energy is calculated as the average of the bond energies of a specific bond type in different molecules. There are several contributing factors but usually the most important is the difference in the electronegativity of the two atoms bonding together.23, 24

For example, the average bond energy of C-H, C-C, O-O, C-O is 413, 348, 145 and

360 kJ/mol respectively.25 The same bond can appear in different molecules, but it will have different bond energies in each molecule because the other bonds in the molecule will affect the bond energy of the specific bond. To be more specific, the energy required to break one

C–O bond in ethanol and dimethyl ether is 405.85 kJ/mol (based on electron impact) and

334.72 kJ/mol (calculated using enthalpies of radicals formation) respectively.25 Angle strain is one of the factors that affect bond energy. Angle strain occurs in cyclic molecules whereas non-cyclic molecules will thermodynamically conform to the most favorable stable bond angle state. According to the Baeyer strain theory, bond angles deviate from the ideal bond angle to achieve maximum bond strength. A specific conformation will change the bond length, which consequently will further change the bond energy. For instance, the epoxy ring of ethylene oxide is an almost regular triangle with bond angles of 61.62° which is much less than the normal angle of 104.4° for bonds between atoms with sp3 hybridized orbitals of oxygen.26,27 This significant angular stress (strain energy) corresponding to the energy of 105 kJ/mol, reduces the compound’s carbon-oxygen bond energy, making it more reactive than normal C-O bonds.28

Due to this strain energy, the energy required to break two C–O bonds in ethylene oxide is significantly reduced to 354.38 kJ/mol (177 kJ/mol for one C-O bond on average), which is calculated based on atomic enthalpies.25 The ring strain in epoxides makes them much more reactive than other ethers towards breaking the carbon-oxygen bond.

It is well known that hydroperoxides (LOOH) are cleaved in the presence of Fe2+ in a

Fenton-like reaction to give alkoxy radicals (LO·), which can further decompose to generate aldehydes.29,30 The energy required to break one O–O bond is 145 kJ/mol. This relatively low bond dissociation energy (BDE) facilitates the Fe2+ induced radical reaction, e.g. the formation of alkoxy radical (LO·) from hydroperoxide (LO-OH). As mentioned above, the

BDE of the C-O bond is 360 kJ/mol. Due to this high bond energy, it is difficult for Fe2+ to break normal C-O bonds. However, it is a different situation when dealing with epoxides.

The energy required to break two C–O bonds in the ethylene oxide is 354.38 kJ/mol (177 kJ/mol for one C-O bond in average). This bond energy is close that required to break one O–

O bond (145 kJ/mol). So it is possible that Fe2+ might be able to induce free radical reaction by homolysis of C-O bond of epoxy compound.

Based on analysis of the C-O bond energy of epoxides, the C-O bonds of epoxide

2.13 are considerably weaker than unstrained carbon-oxygen bonds. Apparently the fragmentation of 2.13 to HNE occurs through a mechanism not involving intermediate 2.8 depicted in Scheme 2.6. An alternative mechanism for the fragmentation of 2.13 is proposed involving reductive homolysis of the epoxide C-O bond (Scheme 2.7).

Scheme 2.7 Proposed mechanism for fragmentation of epoxy vinyl ester 2.13 to form HNE.

Fe2+ initiates the homolysis of 2.13 resulting in breaking of the allylic C-O bond by single electron transfer to generate an alkoxide and an allylic radical in 2.16. This fragmentation is also driven by the generation of an allylic radical 2.16, a relatively stable radical, which also has the resonance form 2.17. Oxidation of 2.17 by single electron transfer

3+ 2+ to Fe regenerates Fe and an allylic cation 2.18. Lone pair electrons on H2O attack the cation 2.18 and give 2.19, which will fragment to afford HNE.

Cleavage of vinyloxiranes through single electron transfer is precedented by reactions

32 with samarium diiodide (SmI2) that produce allylic alcohols (Scheme 2.8 and Table 2.5). In

29 some cases, e.g., where the substituent -Y can stabilize a radical anion intermediate, e.g.,

COSEt, SO2Ph or PO(OEt)2, this process may involve stepwise generation of an anion radical intermediate 2.21 that undergoes C-O bond cleavage in a subsequent step to generate

2.23. However, for substituents –Y such as –H, –Me, or –SPh, an alternative mechanism seems likely in which the release of ring strain resulting from partial cleavage of the oxirane facilitates generation of a transition state 2.22 in a concerted electron transfer and ring cleavage that produces 2.23 directly. In the reductive cleavage of Scheme 2.8 the allylic radical intermediate 2.23 undergoes further reduction to 2.24 and protonation to deliver 2.25.

In the proposed mechanism of Scheme 2.8, the corresponding allylic radical intermediate

2.16 undergoes oxidation to an allylic cation that can be stabilized by conjugation with an electron pair from the hexanoyl substituent.

Scheme 2.8. Reductive cleavage of vinyloxiranes 2.20 with SmI2.

Table 2.5 Reductive cleavage of vinyloxiranes with SmI2

Starting material -Y Product % yield 2.20a COSEt 2.25a 80 2.20b SO2Ph 2.25b 82 2.20c PO(OEt)2 2.25c 84 2.20d H 2.25d 69 2.20e Me 2.25e 42 2.20f SPh 2.25f 54

To test the proposition that Fe2+ merely acts as a Lewis acid catalyst, we also examined the possibility of catalyzing fragmentation of 2.13 by other divalent metal ions, e.g.

Ca2+, Zn2+ (Table 2.5). Epoxy vinyl ester 2.13 was incubated for 72 hours at 37 ºC in

1 CD3CN and D2O filtered through Chelex 100. The filtrate was detected by H NMR. No

HNE could be detected. Thus, trace amounts of Fe2+ or other redox active metal ions in the solvents catalyze the rearrangement of 2.13. Notably, 2.13 is quite sensitive to even 0.5 mol% of redox active metal ions. By contrast, 2.13 decomposed very slowly in the presence

2+ 2+ of 5 mol % of Ca and Zn in CD3CN and D2O to form HNE (Table 2.6). The very slow formation of HNE in the presence of Ca2+ or Zn2+ is apparently catalyzed by their Lewis acidity (Scheme 2.8) instead of through a free radical pathway as we found for Fe2+ (Scheme

2.7).

Table 2.6 HNE generated in the reaction of 2.13 promoted by Ca2+or Zn2+ at 37 ºC.

Epoxide Additive mol% Solvents filtered Reaction time Unreacted (%) HNE with Chelex 100 2.13 No 1 h 44.4% 55.6% 2.13 Yes 72 h 100% 0 2.13 Ca2+ 5% Yes 2 h 95% 5% 2.13 Ca2+ 5% Yes 46 h 49.1% 40.9% 2.13 Ca2+ 5% Yes 100 h 43.6% 56.4% 2.13 Zn2+ 5% Yes 2 h 95% 5% 2.13 Zn2+ 5% Yes 46 h 27.2% 72.8% 2.13 Zn2+ 5% Yes 100 h 25.9% 74.1%

Scheme 2.8 Potential mechanism for Lewis acid (Zn2+, Ca2+) catalysis of the formation of

HNE from 2.13.

2.3 Conclusions.

In this study, 3,4-epoxy-1-nonen hexanoate 2.13 was incubated in physiomimetic

2+ 2+ 2+ mixed solvents CD3CN and D2O in the absence or presence of Fe , Ca or Zn as well as in the presence or absence or vitamin E. 3,4-Epoxy-1-nonen hexanoate 2.13 incubated in the presence of a catalytic amount of Fe2+ delivered HNE in quantitative yield in less than one hour, while fragmentation of 2.13 stopped or dramatically slowed with the addition of one equivalent of the antioxidant vitamin E depending on the amount of added Fe2+. A free radical pathway was proposed for the rearrangement of 2.13 in the presence of Fe2+ that delivers HNE. In contrast, the slow fragmentation of 2.13 to form HNE in the presence of

Ca2+ or Zn2+ apparently involves a Lewis acid catalytic mechanism.

2.4 Experimental Procedures

General Methods. Proton magnetic resonance (1H NMR) spectra and carbon magnetic resonance (13C NMR) spectra were recorded on a Varian Inova AS400 spectrometer operating at 400 MHz for 1H and 100 MHz for 13C. Proton chemical shifts are reported as parts per million (ppm) on the δ scale relative to CDCl3 (δ 7.24), CD3CN (δ 1.94) or D2O (δ

4.80). 1H NMR spectral data are tabulated in terms of multiplicity of proton absorption (s, singlet; d, doublet; t, triplet; m, multiplet; br, broad), coupling constants (Hz), number of protons. All solvents for syntheses were distilled under a nitrogen atmosphere prior to use, and all materials were obtained from Aldrich unless specified. Chromatography was performed with ACS grade solvent. Rf values are quoted for plates of thickness 0.25 mm.

The plates were visualized with iodine. Flash column chromatography was performed on

230-400 mesh silica gel supplied by E. Merck. High performance liquid chromatography

(HPLC) purification was performed with HPLC solvent gradients using a Waters M600A solvent delivery system and a Waters U6K injector or a Waters 717 autosampler. The eluents were monitored using an ISCO V4 UV-vis detector or a Waters 2996 photodiode array detector. All high resolution mass spectra were recorded on a Kratos AEI MS25 RF high resolution mass spectrometer at 20 eV.

Cis-Non-3-enal (2.14). cis-3-Nonen-1-ol (50 mg, 0.35 mmol) was added to a suspension of

DMP (200 mg, 0.47 mmol, 1.3 eq) in dichloromethane (2.0 mL). The white suspension was stirred for 2 h at room temperature. After removal of CH2Cl2, the reaction mixture was filtered and triturated with pentane. The pentane solution of product was concentrated by rotary evaporation and the crude product was chromatographed on silica gel by elution with

33 petroleum ether-diethyl ether (20 : 1) to afford 2.14 (41.2 mg, 0.29 mmol, yield = 82%, Rf =

1 31 1 0.3). The H NMR spectrum was the same as reported. H NMR (400 MHz, CDCl3) 9.59 (t,

1H), 5.63 (dt, 1H), 5.48 (dt, 1H), 3.12 (d, 2H), 1.96 (m, 2H), 1.21 (m, 4H), 0.88 (t, 2H).

(1E, 3Z)-Nona-1,3-dien-1-yl hexanoate (2.15E), (1Z,3Z)-nona-1,3-dien-1-yl hexanoate

(2.15Z). Aldehyde 2.14 (70 mg, 0.50 mmol) in THF (2.0 mL) was added dropwise to a solution of sodium bis(trimethylsilyl)amide (0.55 mmol) in THF (20 mL, -78 °C) over 10 min. The solution was stirred at -78 °C for 30 min. Then this solution was added rapidly to acetyl chloride dissolved in 3 mL of THF. After the addition was completed, the solution was allowed to come to room temperature and stirred overnight. The solution was concentrated to

1/3 volume, and ether was added to extract the organic product. After evaporation, a red oil remained. The mixture was separated by flash chromatography (petroleum ether : ether =

20:1, Rf = 0.2) to give the product, a mixture of stereoisomers 2.15E and 2.15Z (yield 72.1%).

1H NMR showed that the product is a mixture of E and Z isomers. The ratio is E : Z = 1.6:1

3 1 according to areas of peaks and J values. 2.15E H NMR (400 MHz, CDCl3) 7.34 (d,

J=12.25 Hz, 1H), 6.27 (dd, J=12.25 Hz, J=11.6Hz, 1H), 5.89 (dd, J=11.6 Hz, J= 4.6 Hz, 1H),

5.46 (dt, 1H), 2.40 (t, 2H), 2.15 (dt, 2H), 1.66 (m, 2H), 1.30 (m, 10H), 0.88 (t, 6H); 2.15Z 1H

NMR (400 MHz, CDCl3) 7.06 (d, J=6.4 Hz, 1H), 6.32 (dd, J=11.2 Hz, 1H), 5.71 (ddd, J=6.4

Hz, J=11.2 Hz, J=0.8 Hz, 1H), 5.48 (m, 1H), 2.40 (t, 2H), 2.15 (dt, 2H), 1.66 (m, 2H), 1.30

+ (m, 10H), 0.88 (t, 6H). HRMS (EI): m/z calcd for C15H26O2 (M ), 238.1933, found 238.1939.

3,4-Epoxy-1(E)-nonen hexanoate (2.13E) and 3,4-epoxy-1(Z)-nonen hexanoate (2.13Z).

Dimethyldioxirane (2.5 mL, 0.07 mol/L, 1.8 mmol) in acetone was added to a mixture of

2.14E and 2.14Z (40 mg, 0.16 mmol) in 3 mL of acetone. The solution was stirred for 30 min.

After the reaction was complete, the solution was dried, and solvents were evaporated (yield

1 = 95%), Rf = 0.22 (hexanes/ether = 20 : 1). 2.13E H NMR (400 MHz, CDCl3) 7.50 (d,

J=12.52, 1H), 5.29 (dd, J=12.52Hz, J=8.55 Hz, 1H), 3.48 (dd, J=8.55 Hz, J= 4.0 Hz, 1H),

3.10 (1H), 2.38 (t, 2H), 2.15 (dt, 2H), 1.66 (m, 2H), 1.30 (m, 10H), 0.88 (t, 6H); 2.13Z 1H

NMR (400 MHz, CDCl3) 7.37 (d, J=8.75, 1H), 4.75 (dd, J=8.75Hz, J=6.65 Hz, 1H), 3.43 (dd,

J=6.65 Hz, J=4.0 Hz, 1H), 3.10 (1H), 2.38 (t, 2H), 2.15 (dt, 2H), 1.66 (m, 2H), 1.30 (m, 10H),

+ 0.88 (t, 6H). HRMS (EI): m/z calcd for C15H27O3 (MH ) 255.1960, found 255.1955.

General procedure for iron-catalyzed fragmentation of hydroperoxide 2.13

Various amounts of FeSO4·7H2O (and Vitamin E if applicable) in D2O (100 µL) were added to a CD3CN (200 µL) solution of allylic epoxide 2.13 (2.0 mg). The resulting mixture was incubated at 37 °C. Then the solution was filtered through a pad of Chelex 100 ion chelating resin. The solution was directly analyzed by 1H NMR for quantitative analysis measuring

35 various peak areas in the 1H NMR spectrum of the reaction product mixture: 4-Hydroxy-2-

1 nonenal (HNE): H NMR (400 MHz, D2O): 9.58 (d, J=7.9 Hz, 1H), 6.84 (dd, J=15.7 Hz, J=

7.9Hz, 1H), 6.31 (d, J=15.7 Hz, 1H), 4.43 (m, 1H), 1.56 (m, 2H), 1.44 (m, 6H), 0.89 (t, 3H).

2.5 References

1. Carmen Vigo-Pelfrey, E. Membrane Lipid Oxidation 1990, Vol 1.

2. Esterbauer, H. Aldehydic products of lipid peroxidation; Academic Press: London, 1982.

3. Esterbauer, H.; Schaur, R. J.; Zollner, H. Free Radic Biol Med 1991, 11, 81-128.

4. Nair, J.; Barbin, A.; Velic, I.; Bartsch, H. Mutat Res 1999, 424, 59-69.

5. Fazio, V. M.; Rinaldi, M.; Ciafre, S.; Barrera, G.; and Farace, M. G. Mol Aspects Med

1993, 14, 217-228.

6. Markesbery, W. R.; Carney, J. M. Brain Pathol 1999, 9, 133-146

7. Pryor, W. A.; Porter, N. A. Free Radic Biol Med 1990, 8, 541-543.

8. Gardner, H. W.; Selke, E. Lipids 1984, 19, 375-380.

9. Yadagiri, P.; Lumin, S.; Mosset, P.; Capdevila, J.; Falck, J. R. Tetrahedron Lett 1986, 27,

6039-6040.

10. Gardner, H. W.; Bartelt, R. J.; Weisleder, D. Lipids 1992, 27, 686-689.

10. Chabert T, P.; Ousset, J B; Mioskowsik, C. Tetrahedron Lett, 1989, 30, 179-182,

11. Rindgen, D.; Nakajima, M.; Wehrli, S.; Xu, K.; Blair, I. A. Chem Res Toxicol 1999, 12,

1195-1204.

12. Corey E. J.; Wright, S. W. J Org Chem 1990, 55, 1670-1673.

13. Murray, R. W.; Singh, M Org. Syn. 1998, 9, 288-292.

14. Halliwell, B.; Gutteridge JMC. Free Radicals in Biology and Medicine 3rd ed. Oxford:

Oxford University Press; 1999.

15. Lee, S. H.; Blair, I. A. Chem Res Toxicol 2000, 13, 698-702.

16. Posner, G. H.; O'Neill, P. M. Acc Chem Res 2004, 37, 397-404.

17. Ceo R. N.; Kazerouni, M. R.; Rengan K. J Radioanal Nucl Chem 1993, 172, 43–48.

18. Wefers, H; Sics Euro J Biochem 1988, 174, 353–357.

19. Traber; Atkinson, J Free Radic Biol Med 2007, 43, 4–15.

20. Herrera; Barbas, C. J Physiol Biochem 2001, 57, 43–56.

21. Petrucci, Ralph H., Harwood, William S., Herring, F. G., Madura Jeffrey D.

General Chemistry: Principles and Modern Applications. 9th ed. Upper Saddle River:

Pearson Education, Inc., 2007.

22. Carruth, Gorton, Ehrlich, Eugene. "Bond Energies." Volume Library. Ed. Carruth,

Gorton. Vol 1. Tennessee: Southwestern, 2002.

23. Petrucci, Ralph H., Bissonnette, Carey, Herring, F.G., and Madura Jeffrey D. General

Chemistry: Principles and Modern Applications. 10th ed. Upper Saddle River: Pearson

Education, Inc., 2010.

24. "Bond Lengths and Energies." U Waterloo, n.d. Web. 21 Nov 2010.

25. Kondrat'ev, V.N. Energy of chemical bonds. Ionization potentials and electron affinity.

Nauka. 1974, 77–78.

26. Knunyants, I.L.; "Voltage molecules". Chemical Encyclopedia. 3. "Soviet encyclopedia".

1988, 330–334.

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"Academkniga". 2004, 102–106. ISBN 5-94628-172-0.

28. The dipole moments of certain substances. ChemAnalitica.com. 1 April 2009.

29. Gardner, H.W. Free Radic Biol Med 1989, 7, 65–86.

30. Spiteller, P.; Spiteller, G. Chem Phys of Lipids 1997, 89, 131–139.

31. Wavrin, L.; Viala, J. Synthesis 2002, 326-330.

32. Molander, G.A.; La Belle, B.; Hahn, G. J Org Chem 1986, 51, 5259-5264.

Chapter 3

Synthesis of Unsymmetrically Disubstituted

Maleic Anhydrides and 3,4-Disubstituted

5-Methylhydroxyfuran-2(5H)-ones

3.1 Background

Autoxidation of arachidonic phospholipids can produce H2-isoprostanes by nonenzymatic peroxidation while cyclooxygenase (COX)-promoted oxidation generates prostaglandin H2 (PGH2). Both pathways can generate reactive levuglandin E2 (LGE2) and

1,2 levuglandin D2 (LGD2), or their stereoisomers (Figure 3.1). LGs and isoLGs react spontaneously with primary amines in proteins and aromatic amines in DNA to form covalent adducts and to cross-link proteins and nucleic acids (Figure 3.2).3-5 Such covalent adduction and cross-linking can interfere with protein function. Consequently, LGs and isoLGs are among the most potent neurotoxic products of lipid oxidation. Because they can accumulate over the lifetimes of proteins, isoLG-protein adducts represent a convenient dosimeter of oxidative stress.6-8

Figure 3.1 Oxidation products of arachidonic phospholipids.

1 1 2 1 2 Protein 2 2 Protein 2 2 Schiff Base enamine

R 3 1 N 2

R 2

Figure 3.2 Reaction of LGs with proteins. Lactams and hydroxylactams are stable end products formed from protein- bound pyrroles.

In 2011, Yotsu-Yamashita and co-workers reported the isolation of a new oxidized

9 derivative of LGD2, i.e., ox-LGD2, in the red algae, Gracilaria edulis. Ox-LGD2 was also identified in mouse tissues and the lysate of PMA-treated THP-1 cells incubated with arachidonic acid. These results suggest that ox-LGD2 is a common oxidized metabolite of the lipid oxidation product LGD2. To further examine the physiological function of ox-LGD2 and enzymatic pathways of oxidation in vitro and in vivo, it is desirable to synthesize ox-LGD2 as authentic internal standard with unambiguous structures, that can be used to confirm and

9 quantify its formation in animal tissues and cells. Ox-LGD2 is a 5-methylhydroxyfuran-

2(5H)-one with two different side chains on the 3 and 4 positions. In this chapter, three possible pathways to synthesize unsymmetrically disubstituted maleic anhydrides and 5- methylhydroxyfuran-2(5H)-ones (Scheme 3.1) were pursued, the feasibility of each pathway was investigated,9-13 and a general method to synthesize unsymmetrically disubstituted maleic anhydrides and 3,4-disubstituted 5-methylhydroxyfuran-2(5H)-ones is described.

O OH O O O O t-BuOOC COOt-Bu Pathway 3 Pathway 1 Cl Cl Cl Cl t-BuOOC COOt-Bu

Pathway 2 R1 R2 O OTBDMS O O O O OTBDMS

R1 = Cl Cl R1 R2 R2 = COOMe

O OTBDMS O OTBDMS O OH O OH O O O O

R R R R R R R R 1 2 2 1 1 2 2 1

Scheme 3.1 Three possible pathways to construct unsymmetrically disubstituted maleic anhydrides and 3,4-disubstituted 5-methylhydroxyfuran-2(5H)-ones.

3.2 Results and Discussion

A Feasibility Study for Pathway 1

The reaction of 2,3-dimethylmaleic anhydride (3.1a) with MeLi in dry THF at -78 °C for 15 min gives mainly 5-hydroxyl-3,4,5-trimethyl-2(5H)-furanone (Scheme 3.2).14 We used a similar method in our synthesis of 3.2. Thus, addition of MeLi or Grignard reagents, e.g.,

MeMgBr, to 2,3-dichloromaleic anhydride 3.1b gives the hemiactal 3,4-dichloro-5-hydroxy-

5-methyl-furanone 3.2.14,15 To avoid the formation of the dimethylation product 3,4-dichloro-

5-dimethyl-furanone, the reaction must be kept at low temperature. However, despite slow addition of MeLi to a dilute THF solution of the anhydride, minor amounts of dimethylation product were still produced. The product mixture can be easily separated by flash silica gel chromatography due to the difference of polarity.

Scheme 3.2 Feasibility study for pathway 1.

TBDMS chloride has been much used in the protection of primary hydroxyl groups with either imidazole or 4-dimethylaminopyridine as catalyst. However, difficulty is encountered in the silylation of hindered secondary or tertiary alcohols. For instance,

TBDMS chloride was ineffective for silylation of the tertiary alcohol 3.2. TBDMS triflate is a simple and useful reagent in the protection of hindered alcohols.16 The reaction of MeMgBr with 3.1b followed by protection of resulting hemiacetal 3.2 with TBDMS triflate delivered the TBDMS protected hemiacetal 3.3 in 95% yield. In a previous study,18 reaction of a 2,3- dichloromaleimide 3.1c (Scheme 3.3) with an n-butyl organocuprate complex followed by adding an allylic bromide gave a disubstituted maleimide with one chloro group replaced by the nucleophilic butyl group and the other by an electrophilic allyl group. This unprecedented transformation apparently involves a 1,4-addition followed by elimination of CuCl and subsequent metal halogen exchange to convert a vinyl chloride into a vinyl cuprate.

Accordingly, in our synthesis in Scheme 3.2, the alkyl cuprate R2CuLi was expected to react with dichloro lactone 3.3 resulting in a vinyl organocuprate intermediate 3.4, which could by quenched by electrophiles (E+), e.g. H+, allyl or vinyl, forming unsymmetrically disubstitued compounds 3.5. The THF-soluble CuCl·dimethyl sulfide complex was chosen to facilitate organocuprate generation.10, 17-20

A Feasibility Study for Pathway 2

Copper bromide-dimethyl sulfide complex (CuBr·Me2S) is a source of copper(I) that is soluble in THF that has been used to prepare lithium dialkyl cuprates.19 Therefore, dimethyl sulfide organocuprate complex was used in this study to substitute chloro group of

3.3 with alkyl cuprate. Commercial copper bromide or its dimethyl sulfide complex contains impurities that are deleterious to the reaction. Therefore, the copper(I) bromide-dimethyl sulfide complex was purified according to the method of House.19

Scheme 3.3 Feasibility study for pathway 2

Various copper (I) salts have been used in organocuprate reactions. When di-n-butyl cuprate Bu2CuLi·LiBr·Me2S derived from BuLi (2.0 equiv.) and CuBr·Me2S (1.0 equiv.), was treated with 3.3 at -78 °C, no reaction occured. All the starting material was recovered.

Even with a longer reaction time (3 hours in -78 °C) was applied, all the starting material was recovered. Since increasing the reaction temperature might enhance the speed of the reaction, the reaction mixture was gradually warmed from -78 °C to -30 °C and kept for one hour and then warmed to room temperature., 1H NMR and 13C NMR results showed that 3,4-dichloro-

5-hydroxy-5-methyl-furanone 3.2 was obtained because the TBDMS protecting group was removed during the reaction, but no butyl substituted product was produced during this reaction. Perhaps the bulky TBDMS group creates steric hindrance that prevents the substitution of the 2,3-dichloro group of 3.3.

In analogy with the reaction of 3.1c in Scheme 3.3, the reaction of dichloromaleic anhydride (3.1b) with alkyl cuprates was expected to give an alkylcopper complex intermediate 3.7, which can react with various electrophiles (E+, e.g. H+, allyl and vinyl) to provide disubstituted maleic anhydrides 3.8 (Scheme 3.3). In this study, we attempted to achieve this transformation using various cuprates.

However, in our initial efforts to employ one butyl group to substitute the chloro group of 3.1b, we found that di-butyl substituted compound 3.9 was the only product

17-20 formed. When di-n-butyl cuprate Bu2CuLi·LiBr·Me2S (one equiv.), derived from BuLi

(2 equiv.) and CuBr·Me2S (1 equiv.), reacted with 3.1b (one equiv.) at -78 °C, 3,4- dibutylfuran-2,5-dione 3.9 and some starting material 3.1b were obtained (Scheme 3.4). No mono-butyl substituted compound 3.11 was found. When the ratio of Bu2CuLi·LiBr·Me2S and 3.1b is changed to 0.4 : 1, disubstituted product 3.9 and starting material 3.1 were obtained, but no monosubstituted product 3.11 was detected.

Scheme 3.4 Reaction of cuprate with 2,3-dichloromaleic anhydride 3.1b.

It was concluded that the reaction of 2,3-dichloromaleic anhydride 3.1b with cuprate afforded 3,4-dibutylfuran-2,5-dione 3.9, and not the potential alternative product (Z)-6,7- dichlorododec-6-ene-5,8-dione 3.10, since the chemical shift of carbonyl carbon of 3.10 should be above 190 ppm at 13C NMR, while the actual chemical shift of this compound is

166 ppm. In addition, another cuprate BuCuLiCN reacted with 3.1b (1:1) to give the same product 3.9 and starting material 3.1b. Generation of the disubstituted product 3.9 is the

47 expected result of two consecutive 1,4-addition-elimination reactions. This result further emphasizes the novelty of the reaction of dichloromaleic anhydride 3.1c with organocuprates.

Although we did not successfully obtain unsymmetrical substituted maleic anhydride

3.8, this method provides an efficient synthesis of symmetrical dialkyl maleic anhydrides

(3,4-dialkyl-furan-2,5-diones). This is the first synthesis of 3,4-dibutylfuran-2,5-dione (3.9).

A Feasibility Study for Pathway 3

Normant reported a method to prepare disubstituted alkenylcopper complexes through addition of Grignard reagents to acetylenes to give disubstituted alkenylcopper complexes.22 The subsequent reactions of this intermediate with various electrophiles gives a variety of substituted alkenes. Furthermore, the reaction of di-t-butyl acetylenedicarboxylate with organocopper reagents RCu(Me2S)·MgBr2, followed by reaction of the intermediate vinyl cuprate with various electrophiles afforded mono and disubstituted di-t-butyl maleates.23

It appeared to us that this three component coupling would be a very promising approach to disubstituted maleates and maleic anhydrides. In our initial study (Scheme 3.5), di-t-butyl acetylenedicarboxylate 3.13 was prepared by adding tert-butanol and acetylenedicarboxylic acid 3.12 to the solution of anhydrous magnesium sulfate in concentrated sulfuric acid.24 Initial studies linked the acetylene, butyl lithium and allyl bromide. A Grignard reagent BuMgBr was generated by treatment of butyl lithium with magnesium bromide-etherate complex (MgBr2·Et2O). The Grignard reagent was added to

CuBr·SMe2 to give an organocopper complex BuCu(Me2S)·MgBr2 that reacted with 3.13 to produce a vinyl cuprate complex 3.14 at -78 °C.

COOH COOt-Bu t-BuOOC Bu MgBr OEt ,BuLi t-BuOH, MgSO4 2 2 Pd(PPh3)4,HMPA cont. H SO O allyl bromide 2 4 CuBr SMe2,THF,-78C t-BuOOC Cu(SMe2)MgBr2 COOH COOt-Bu 3.12 3.13 3.14

O HO O t-BuOOC Formic acid O MeMgBr, THF O O -78O C t-BuOOC Bu Bu Bu Bu O O HO 3.15 3.16 3.17a 1:1 3.17b

Scheme 3.5 A simple model study to achieve a three-component coupling construction of an unsymmetrically disubstituted maleic anhydride and it’s conversion to hydroxy lactones.

The reaction of cuprate 3.14 with a variety of electrophilic reagents (e.g. ally bromide) in the presence of hexamethylphosphorictriamide (HPMA) produced disubstituted maleate 3.15. A catalytic amount of tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) was used to favor retention of configuration. From H-H COSY of 3.15 (Figure 3.3), the vinyl hydrogen resonance at 5.70 ppm (H2) showed coupling with the hydrogen signals at 5.00 ppm (H1) and 2.99 ppm (H3), while the hydrogen at 2.23 ppm (H6) showed a coupling with the hydrogen signal at 1.31 ppm (H7). The hydrogen at 1.31 ppm (H8) also coupled with H7 and hydrogen signal at 0.88 ppm (H9). This indicated that allylic group and butyl group were the two side chains of 3.15. To determine the configuration of 3.15, a 1D NOE difference experiment was carried out (Figure 3.3, Figure 3.7S (control)). An allylic hydrogen was selectively preirradiated until saturation was achieved. The NOE peak at the other allylic hydrogen was 3% as intense as the irradiated peak, which indicated that 3.15 has the cis (Z) configuration.

Figure 3.3 H-H COSY (CDCl3, 400 MHz) and 1D NOE difference spectra (DMSO-d6, 600

MHz) of 3.15.

The configuration of 3.15 was further confirmed by the formation of maleic anhydride 3.16 upon treating with formic acid. Reaction of 3.16 with methyl lithium at -

78 °C in anhydrous THF produced 3.17a and 3.17b. TLC showed only one spot, however, 1H

NMR and 13C NMR showed that this mixture is actually two products, 3.17a and 3.17b. The polarities of these two compounds are almost the same. The ratio of hydroxy lactones 3.17a and 3.17b is 1:1 according to 1H NMR. HPLC with a methanol/water gradient – which started with 15 % methanol for 10 min and rose to 100% methanol in 10 min, then was held at 100% methanol for another 10 min, and then was reversed to 15% methanol in 0.5 min, and held at 15% methanol for 10 min before the next run – showed two peaks in the UV and

ELSD chromatographs. Subsequently, this method was pursued for the synthesis of more complex unsymmetrically disubstituted maleic anhydrides and hydroxy lactones (Scheme

3.6).

Scheme 3.6 Synthesis of hydroxyl lactone 3.23.

The required lower chain vinyllithium synthon, was prepared by treatment of a (Z)- vinylstannane with n-BuLi in THF at -78 °C.25 Then the vinyl lithium was reacted with magnesium bromide-etherate complex (MgBr2·Et2O), followed by solid CuBr·SMe2 to give an organocopper complex RCu(Me2S) ·MgBr2. The temperature must be kept below -45 °C.

Otherwise the vinyl intermediate will either isomerize to the corresponding allenolate or give diene by thermal decomposition.26 Actually, some diene is formed even at -78 °C (Scheme

3.7). In addition, 1,4 O- to C-silyl migration may occur at higher temperature.27 The organocopper regaent was then added to 3.13 followed by the addition of allyl bromide in the presence of HMPA and Pd(PPh3)4 to give 3.18 in 10% yield. Compared with the yield of 3.15

(75%), the yield of 3.18 is much lower owing to the formation of diene and other unidentified byproducts.

Scheme 3.7 Formation of diene through the reaction of (Z)-vinylstannanes with BuLi.

Scheme 3.8 Three different pathways for the cleavage of di-t-butyl ester 3.15.

The TBDMS protecting group is stable during the reaction. It was desired to deprotect the di-tert-butyl carbonate of 3.18 without affecting the TBDMS group to provide 3.19. Di-t-

52 butyl esters are stable to mild basic hydrolysis, to hydrazine, and to ammonia; and they are cleaved by moderately acidic hydrolysis. Various reagents were tested to hydrolyze the di- tert-butyl esters 3.15 (Scheme 3.8) and 3.18 (Scheme 3.9).

Scheme 3.9 Three different pathways for the cleavage of di-t-butyl carbonate 3.18

Previously, a TBDMS protected prostaglandin E2 was converted to prostaglandin

28 E2 by exposure to acetic acid-THF-H2O with ratio of 3:1:1 at 25 °C for 20 hours.

However, we found that neither the OTBDMS group nor the t-butyl ester of 3.18 was affected by acetic acid. Although di-t-butylmaleates 3.15 and 3.18 are stable in the presence of acetic acid, maleic anhydride 3.16 was generated upon treatment of 3.15 with formic acid, and formic acid removed both the t-butyl and TBDMS groups of 3.18 to give anhydride 3.20. Zinc bromide catalyzed conversion of 3.15 to the corresponding dicarboxylic acid. And 3.18 underwent transesterification to deliver lactone 3.21 under catalysis by ZnBr2. Refluxing 3.15 in a toluene suspension of SiO2 generated maleic anhydride 3.16. Although the OTBDMS group was not affected, SiO2 selectively catalyzed conversion of the di-t-butyl ester 3.18 into dicarboxylic acid 3.22 but not the

53 desired anhydride product 3.19. From H-H COSY of 3.19 (Figure 3.4), the vinyl hydrogen resonance at 5.77 ppm (H2) showed coupling with the hydrogen signals at 5.14 ppm (H1) and 3.24 ppm (H3). The hydrogen at 6.52 ppm (H6) showed a coupling with the hydrogen signal at 7.24 ppm (H7). The hydrogen at 4.34 ppm (H8) also couples with hydrogen signal at 1.52 ppm (H9). This indicated that allylic group and butyldimethylsilyloxyoctenyl group were the two side chains of 3.19.

Scheme 3.10 Two different pathways for the formation of maleic anhydride 3.19.

Since the OTBDMS group of 3.18 is stable in acetic acid, and formic acid catalyzes formation of anhydrides 3.16 and 3.20 from presumed dicarboxylic acid precursors, acetic acid was used to catalyze the formation of anhydride 3.19 from dicarboxylic acid 3.22 (Scheme 3.10). 3.19 could also be obtained by reprotecting the

OH group of 3.20 with TBDMS-OTf. The 1H NMR spectrum of the samples of 3.19 generated from the two methods was the same.

Organic compounds containing hard or soft Lewis base sites can be ionized by

+ + + 29 cations such as Li , Na and Ag . This technique known as coordination ion spray mass spectrometry (CIS-MS) can be used to detect analyte that can form a positively charged complex adduct, such as [M + Ag+], and be detected by mass spectrometry in the positive

+ mode. The mass spectrum of 3.19 in the presence of silver ions (AgBF4) exhibited Ag adducts of 3.19 with m/z = 485 and 487 ([M + Ag+] = 378 + 107 or + 109).30

Figure 3.4 H-H COSY of 3.19 (CDCl3, 400 MHz).

The task was to methylate 3.19 to give hydroxy lactone 3.23. First, MeMgBr was added to a solution of 3.19 in THF at -78 °C and quenched with saturated aqueous NH4Cl solution. No reaction occurred and starting material was recovered. Still no reaction occurred when the temperature was raised to -20 °C before quenching. Both MeMgBr and MeLi reacted with 3.19 to give the methylation product hydroxy lactone 3.23 at 0 °C.

By comparison, maleic anhydride 3.16 readily reacts with methyl lithium at -78 °C in

THF to give hydroxy lactone 3.17. The protecting TBDMS group of 3.23 was cleaved by

55 formic acid to form 3.24a and 3.24b. The mass spectra of the products in the presence of

+ silver ions exhibited Ag complex adducts of 3.24a and 3.24b (with AgBF4) is m/z = 387 and 389 ([M + Ag+] = 280.2 +107 or + 109).

3.3 Conclusions

In this chapter, we have discussed three different possible pathways that were explored to synthesize unsymmetrically disubstituted maleic anhydrides and 3,4-disubstituted 5-methyl-

5-hydroxyfuran-2(5H)-ones. The reaction of 2,3-dichloromaleic anhydride with two equivalents of butyl cuprates afforded 3,4-dibutylfuran-2,5-dione 3.9. This method can be applied to the one-step synthesis of symmetrical dialkyl maleic anhydrides (3,4-dialkyl- furan-2,5-diones) in high yields. Unsymmetrical mono and disubstituted maleic anhydrides are accessible through the reaction of di-t-butyl acetylenedicarboxylate with organocopper reagents RCu(Me2S)·MgBr2 followed in situ by addition of various electrophiles that delivers mono and disubstituted di-t-butyl maleates that can be converted by Lewis acid catalysis into maleic anhydrides. The maleic anhydrides give hydroxyl lactones by reaction with methylating reagents such as methyl lithium or methyl magnesium bromide.

3.4 Experimental Procedures General Methods. Proton magnetic resonance (1H NMR) spectra and carbon magnetic resonance (13C NMR) spectra were recorded on a Varian Inova AS400 spectrometer operating at 400 MHz for 1H and 100 MHz for 13C or Varian Inova 600. Proton chemical shifts are reported as parts per million (ppm) on the δ scale relative to CDCl3 (δ

7.24). 1H NMR spectral data are tabulated in terms of multiplicity of proton absorption (s, singlet; d, doublet; t, triplet; m, multiplet; br, broad), coupling constants (Hz), number of protons. All solvents were distilled under a nitrogen atmosphere prior to use, and all materials were obtained from Aldrich unless specified. Chromatography was performed with

ACS grade solvent. Rf values are quoted for plates of thickness 0.25 mm. The plates were visualized with iodine. Flash column chromatography was performed on 230-400 mesh silica gel supplied by E. Merck. High performance liquid chromatography (HPLC) purification was performed with HPLC solvent gradients using a Waters M600A solvent delivery system and a Waters U6K injector or a Waters 717 autosampler. The eluents were monitored using an

ISCO V4 UV-vis detector, a Waters 2996 photodiode array detector, and/or a Sedex evaporative light scattering detector (ELSD). All high resolution mass spectra were recorded on a Kratos AEI MS25 RF high resolution mass spectrometer at 20 eV.

3,4-Dichloro-5-hydroxy-5-methyl-furanone (3.2) To a dry THF (10.0 mL) solution of 2,3- dichloromaleic anhydride (100 mg, 0.62 mmol) at -78 C, methylmagnesium bromide (1.2 eq,

0.73 mmol, 3.0 M in ether) was added dropwise. The mixture was further stirred for 30 min.

The reaction was then quenched by addition of saturated NH4Cl aqueous solution (2 mL) and

58 extracted with diethyl ether. The product was purified by flash chromatography

(ether/petroleum ether = 3 : 2, Rf = 0.25) to separate the product from starting material and

1 other impurities impurity (yield 55%). H NMR (400 MHz, CDCl3): δ 4.63 (1H), 1.72 (s,

13 3H); C NMR (100 MHz, CDCl3): δ 163.97, 152.73, 122.30, 105.00, 23.67; HRMS (EI):

+ m/z calcd for C5H4Cl2O3 (M ), 181.9537, 183.9508, found 181.9561,183.9516.

5-((tert-Butyldimethylsilyl)oxy)-3,4-dichloro-5-methylfuran-2(5H)-one (3.3) To a solution of 3.2 (130 mg, 0.71 mmol) and 2,6-lutidine (456 mg, 6.0 equiv.) in CH2Cl2 at 0 °C,

TBMDS triflate (750 mg, 4 equiv. ) was added. TBDMS-OTf was freshly prepared from

TBDMSCl and triflate acid at room temperature, followed by distillation. The mixture was warned to room temperature and stirred for 2 hours and then diluted by addition of saturated aqueous NH4Cl solution. The aqueous layer was extracted with CH2Cl2. The combined organic phases were dried over Na2SO4. The residue was purified flash chromatography

(petroleum ether/ ether = 40:1 to 20:1) (yield 95%). Rf = 0.3 (petroleum ether/ ether = 20:1).

1 13 H NMR (400 MHz, CDCl3): δ 1.76 (s, 3H), 0.91 (s, 9H), 0.2 (3H), 0.1 (3H); C NMR (100

MHz, CDCl3): δ 163.27, 154.17, 121.44, 105.26, 17.99, 1.25, -3.21, -3.58; HRMS (EI): m/z

+ calcd for C7H9Cl2O3Si (M -C4H9) 238.9698, 240.9669, found 238.9688, 240.9661.

3,4-Dibutylfuran-2,5-dione (3.9) Butyllithium (0.412 mL of 1.6 M in hexane, 2.2 eq, 0.66 mmol) was added dropwise to a suspension of CuBr·Me2S (68 mg, 0.33 mmol) in THF (3

59 mL) in -78 °C under argon. The reaction mixture was stirred for about 20 min until a clear solution of cuprate was formed. Then the cuprate solution was added via a needle to a solution of 3.1b (50.0 mg, 0.3 mmol) in THF (2 mL). A dark brown color developed immediately. The reaction mixture was stirred for 2 hours at -78 °C, and then warmed to room temperature after being quenched by addition of an aqueous solution of NH4Cl. The mixture was then extracted with ethyl acetate. The organic solvent was washed with water and brine, dried and concentrated. The residue was purified by flash chromatography with

1 petroleum ether/ether = 40/1 (Rf = 0.20) to give pure product 3.9 (yield 40%). H-NMR (400

13 MHz, CDCl3): δ 2.45 (2H), 1.58 (2H), 1.40 (2H), 0.93 (3H); C-NMR (100 MHz, CDCl3): δ

+ 166, 144, 31, 25, 23, 15; HRMS (EI): m/z calcd for: C12H19NO3 (MH ), 210.1256, found

210.1258.

3, 4-Dibutylfuran-2,5-dione (3.9) Butyllithium (0.468 µL of 1.6 M in hexane, 2.5 eq, 0.75 mmol) was added dropwise to a suspension of CuCN (687mg, 0.75 mmol) in THF (3 mL) in

-78 °C under agron. The reaction mixture was stirred for about 20 min until a clear solution of cuprate was formed. Then the cuprate solution was added via a needle to a solution of 2,3- dichloromaleic anhydride (50.0 mg, 0.3 mmol) in THF (2 mL). A dark brown color developed immediately. The reaction mixture was stirred for 2 hours at -78 °C. After being quenched by addition of an aqueous solution of NH4Cl, the mixture was then extracted with ethyl acetate. The organic solvent was washed with water and brine, dried and concentrated.

The residue was purified by flash chromatography with petroleum ether/ether = 40/1 (Rf =

1 0.20) to give product 3.9 (yield 40%). H-NMR (400 MHz, CDCl3): δ 2.45 (2H), 1.58 (2H),

13 1.40 (2H), 0.93 (3H); C-NMR (100 MHz, CDCl3): δ 166, 144, 31, 25, 23, 15; HRMS (EI):

+ m/z calcd for: C12H19NO3 (MH ), 210.1256, found 210.1258.

Di-t-butyl maleate (3.13) Concentrated sulfuric acid (200 mg, 2 mmol) was added to a vigorously stirred suspension of magnesium sulfate (0.95 g, 8.0 mmol) in dichloromethane

(10 mL).30 The suspension was stirred for 15 min, after that the acetylenedicarboxylic acid

(228 mg, 2 mmol) was added, followed by tert-butanol (10 mmol, 1 mL). The mixture was then kept at room temperature and stirred overnight. After the reaction was completed, the mixture was extracted with diethyl ether and washed with aqueous sodium bicarbonate

(NaHCO3). The extract was dried, filtered, and concentrated under reduced pressure to afford a colorless oil. The oil was flash chromatographed with diethyl ether/petroleum ether (1: 25,

1 Rf = 0.22) to afford di-t-butyl acetylenedicarboxylate (86%). H NMR δ 1.35 (s, 3H) is the same as reported.35

Di-t-butyl 2-allyl-3-butylmaleate (3.15) To a cold (-78 °C) solution of butyl lithium (0.80 mL, 1.6 M in hexane, 1.28 mmol) in anhydrous THF (10 mL) was added MgBr2·OEt2 (330 mg, 1.28 mmol) under Argon. A white suspension formed and the mixture was stirred for another 20 min. Then the above suspension was transferred dropwise via a cannula to a solution of cuprous bromide-dimethyl sulfide (263 mg, 1.28 mmol) in anhydrous THF (2.0 mL) under -78 °C. The resulting yellow solid was stirred at -78 °C for 2 hours.

A solution of di-t-butyl acetylenedicarboxylate (250 mg, 1.10 mmol) in anhydrous

THF (2 mL) at -78 °C was slowly added via cannula to the cuprate solution (-78 °C). The mixture was stirred at -78°C for 45 min. Freshly distilled hexamethylphophorous triamide

(HMPA) (2.82 mmol, 0.6 mL) was added. After about 15 min, tetrakis(triphenylphosphine) palladium (Pd(PPh3)4) (0.07 mmol, 20 mg) in 1 mL of THF was added by syringe, followed by the addition of allyl bromide (210 mg, 1.8 mmol) in THF (1 mL) at -78 °C. Stirring was continued for 12 hours at -78 °C and was then allowed to stir at -45 °C for one hour. And the reaction mixture was quenched with saturated aqueous ammonium chloride (2 mL), and then slowly warned to 0 °C. Diethyl ether was used to extract the mixture. The crude product was separated by flash chromatography with petroleum ether/Et2O (20:1 to 10:1). Rf = 0.15

1 13 (petroleum ether/Et2O = 20/1). H NMR, C NMR and H-H COSY showed that the product

1 is diethyl 2-allyl-3-butylmaleate (yield 75%). H NMR (400 MHz, CDCl3): δ 5.70 (m, 1H),

5.00 (2H), 2.99 (d, 2H), 2.23 (t, 2H), 1.40 (s, 18H), 1.31 (m, 4H), 0.88 (t, 3H); 13C NMR (100

MHz, CDCl3): δ 168.66, 167.58, 140.72, 134.29, 134.03, 116.64, 81.34, 81.27, 33.78, 30.39,

+ 30.01, 28.18, 28.17, 28.11, 22.78, 22.78, 14.06; HRMS (EI): m/z calcd for C19H33O4 (MH ),

325.2379, found 325.2383.

3-Allyl-4-butylfuran-2,5-dione (3.16) 25 mg of dibutyl 2-allyl-3-butylmaleate was added to formic acid (0.5 mL, 99%), and the mixture was stirred at room temperature for about 2 hours until all the starting material disappeared. The mixture was concentrated mechanical high vacuum pump, trapping volatiles in a dry ice-cooled trap. Then the residue was flash

62 chromatographed with petroleum ether/ether (20:1, Rf = 0.2) to provide 3-allyl-4-butylfuran-

2,5-dione as a colorless oil (10 mg, 72.8%). 1H-NMR and 13C NMR showed that the

1 dicarboxylic acid had dehydrated to form the anhydride. H NMR (400 MHz, CDCl3): δ 5.77

(m, 1H), 5.11 (m, 2H), 3.16 (m, 2H), 2.41 (t, 2H), 1.50 (2H), 1.33 (m, 2H), 0.87 (t, 3H); 13C

NMR (100 MHz, CDCl3): δ 166.1, 165.9, 146.3, 140.0, 118.9, 30.1, 28.6, 24.4, 22.9, 13.9;

+ HRMS (EI): m/z calcd for C11H14O3 (M ), 194.0943, found 194.0945.

4-Allyl-3-butyl-5-hydroxy-5-methylfuran-2(5H)-one (3.17a) and 3-allyl-4-butyl-5- hydroxy-5-methylfuran-2(5H)-one (3.17b) To a solution of 3-allyl-4-butylfuran-2,5-dione

(40 mg, 0.21 mmol) in dry THF (1 mL) at -78 °C was added 100 µL of MeMgBr (3M in hexane). The solution was stirred for about 1 hour at -78 °C, and then was quenched with saturated aqueous NH4Cl (0.5 mL). The mixture was then extracted with ethyl acetate. The organic extract was washed with water and then brine, dried and concentrated. The residue was purified by flash chromatography with petroleum ether/ether (5:1, Rf = 0.25) to give

1 3.17a and 3.17b (31 mg, yield = 69%). 3.17a H NMR (400 MHz, CDCl3): δ 5.77 (m, 1H),

5.11 (m, 2H), 3.10 (m, 1H), 2.18 (t, 2H), 1.60 (s, 3H), 1.40 (m, 4H), 0.87 (t, 3H); 3.17b 1H

NMR (400 MHz, CDCl3): δ 5.77 (m, 1H), 5.11 (m, 2H), 2.95 (m, 1H), 2.30 (t, 2H), 1.60 (s,

13 3H), 1.40 (m, 4H), 0.87 (t, 3H); C NMR (100 MHz, CDCl3): δ 172.0, 163.6, 159.1, 133.5,

132.8, 130.3, 120.4, 118.1, 116.9, 105.9, 105.61, 30.2, 30.1, 27.9, 25.8, 24.5, 24.3, 23.5, 22.8,

+ 14.0; HRMS (EI): m/z calcd for C12H17O2 (M -OH), 193.1229, found 193.1244.

Di-tert-butyl 2-allyl-3-((E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)maleate (3.18) A solution of tri-n-butyl-1-(3-t-butyldimethylsiloxy)octenyltin34 (555 mg, 0.886 mmol) in dry

THF (-78 °C, 5 mL) was treated with n-BuLi (1.6 M in hexanes, 0.53 mL, 0.83 mmol). After

20 min, MgBr2·OEt2 (227 mg, 0.886 mmol) was added to the mixture under argon. A white suspension formed and the mixture was stirred for another 20 min. Then the above suspension was transferred dropwise via a cannula to a solution of cuprous bromide-dimethyl sulfide (180.9 mg, 0.886 mmol) in anhydrous THF (2 mL) under -78 °C. The resulting suspension of a yellow solid was stirred at -78 °C for 2 hours.

A solution of di-t-butyl acetylenedicarboxylate (200 mg, 1.10 mmol) in anhydrous

THF (2 mL) at -78 °C was slowly added via cannula to the cuprate (at -78 °C). The mixture was stirred at -78 °C for 45 min. Freshly distilled HMPA (2.0 mmol, 0.5 mL) was added.

After about 15 min, Pd(PPh3)4 (0.05 mmol, 15 mg) in 1 mL of THF was added by syringe, followed by the addition of allyl bromide (140 mg, 1.1 mmol) in 1 mL THF. Stirring was continued overnight at -78 °C and then the mixture was allowed warm to -45 °C and then stirred for one more hour. Then the reaction mixture was quenched with aqueous ammonium chloride (2.0 mL), and slowly warned to 0 °C. Diethyl ether was used to extract the mixture.

The crude product was separated by flash chromatography with petroleum ether/Et2O (40:1

1 13 to 20:1). Rf = 0.16 (petroleum ether/Et2O = 40/1). H NMR, C NMR showed that the

1 product is 3.18 (41 mg, yield 10%). H NMR (400 MHz, CDCl3): δ 6.40 (d, 2H), 6.01 (q, 1H),

5.75 (m, 1H), 5.02 (q, 2H), 4.22 (m, 1H), 3.10 (d, 2H), 1.4-1.6 (21H), 1.3 (6H), 0.91 (s, 9H),

13 0.88 (t, 3H), 0.06 (6H); C NMR (100 MHz, CDCl3): δ 167.78, 166.29, 142.15, 140.92,

134.59, 129.11, 122.09, 116.38, 81.84, 81.26, 72.98, 38.16, 32.40, 32.04, 28.26, 28.23, 26.07,

+ 24.87, 22.82, 18.42, 14.15, -4.19, -4.60; HRMS (EI): m/z calcd for C28H49O5Si (M -CH3),

+ 493.3349, found 493.3350; calcd for C25H43O5Si (M -C4H9), 451.2880, found 451.2889.

(E)-3-Allyl-4-(3-hydroxyoct-1-en-1-yl)furan-2,5-dione (3.20) Diester 3.18 (20 mg) was stirred in 99% of formic acid (0.5 mL) for about 2 hours. Then volatiles were evaporated under a stream of dry argon gas to deliver 3.20 (7.9 mg, yield = 81%). 1H NMR (400 MHz,

CDCl3): δ 8.02 (s, 1H), 7.01 (q, 1H), 6.40 (d, 1H), 5.78 (m, 1H), 5.50 (q, 1H), 5.10 (m, 2H),

13 3.23 (d, 2H), 1.70 (m, 2H), 1.25 (m, 6H), 0.90 (t, 3H); C NMR (100 MHz, CDCl3): δ 166.0,

160.3, 143.09, 119.2, 117.6, 136.5, 73.8, 34.2, 31.6, 28.2, 24.8, 22.7, 14.2; HRMS (EI): m/z

+ calcd for: C15H18O3 (M -H2O), 246.1256, found 246.1258.

(E)-3-Allyl-4-(3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)furan-2,5-dione (3.19) To a

CH2Cl2 solution of 3.20 (5 mg, 0.019 mmol) at 0 °C was added 2,6-lutidine (5 mg, 0.05 mmol) and TBDMS triflate (8.0 mg, 0.03 mmol). The solution was stirred for 30 min.

Diethyl ether was used to extract the mixture. The crude product was separated by flash chromatography with petroleum ether/Et2O (40:1 to 20:1) to give 3.20 (5.8 mg, yield 92%);

1 Rf = 0.15 (petroleum ether/Et2O = 40/1) . H NMR (400 MHz, CDCl3): δ 7.24 (dd, 1H), 6.52

(dd, 1H), 5.77 (m, 1H), 5.14 (m, 2H), 4.34 (dt, 1H), 3.24 (dt, 2H), 1.52 (m, 2H), 1.25 (m, 6H),

13 0.92 (s, 9H), 0.87 (t, 3H), 0.03 (s, 6H); C NMR (100 MHz, CDCl3): δ 166, 164.8, 150.1,

137.6, 137.1, 131.1, 118.7, 114.8, 72.5, 37.7, 32.0, 28.1, 26.0, 24.8, 22.8, 18.5, 14.2, 1.3, -

+ 4.44, -4.58; HRMS (EI): m/z calcd for C20H31O4Si (M -CH3), 363.1992, found 363.1992;

+ calcd for C16H23O4Si (M -C5H11), 307.1366, found 307.1361.

2-Allyl-3-((E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)maleic acid (3.22) To a

32 solution of 3.18 (20 mg) in toluene (3 mL) was added SiO2 (250 mg). The solution was refluxed under argon for about 6 hours, then cooled to room temperature and diluted with 5.0 mL of 10% methanol/CH2Cl2, and filtered through a pad of Celite. The solution was

1 concentrated under vacuum to deliver crude product 3.22. H NMR (400 MHz, CDCl3): δ

7.24 (q, 1H), 6.50 (dd, 1H), 5.80 (m, 1H), 5.14 (m, 2H), 4.38 (m, 1H), 3.24 (dt, 2H), 1.52 (m,

2H), 1.25 (m, 6H), 0.90 (s, 9H), 0.87 (t, 3H), 0.03 (s, 6H) that was used without further purification for the preparation of 3.19.

(E)-3-Allyl-4-(3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)furan-2,5-dione (3.19) The crude product 3.22 was stirred in acetic acid/acetic anhydride/THF (2:1:2, 1 mL). After 8 hours, the reaction was completed. The mixture was concentrated by evaporation of volatiles into a dry ice-cooled trap with a mechanical vacuum pump, and then the residue was flash chromatographed with petroleum ether/ether (20:1, Rf = 0.20) to give 5.9 mg of 3.19 (yield =

1 40%). 3.19 H NMR (400 MHz, CDCl3): δ 7.24 (dd, 1H, J=15.6 Hz, J=4.0 Hz), 6.52 (dd, 1H,

J=15.6 Hz), 5.77 (ddt, 1H, J=17.2, 10.6, 6.4 Hz), 5.14 (m, 2H, J=17.2 Hz), 4.34 (dt, 1H,

J=4.0 Hz), 3.24 (dt, 2H, J=6.4 Hz), 1.52 (m, 2H), 1.25 (m, 6H), 0.90 (s, 9H), 0.85 (t, 3H),

13 0.03 (s, 6H); C NMR (100 MHz, CDCl3): δ 166, 164.8, 150.1, 137.6, 137.1, 131.1, 118.7,

114.8, 72.5, 37.7, 32.0, 28.1, 26.0, 24.8, 22.8, 18.5, 14.2, 1.3, -4.44, -4.58; HRMS (EI): m/z

+ + calcd for C20H31O4Si (M -CH3), 363.1992, found 363.1992; calcd for C16H23O4Si (M -

C5H11), 307.1366, found 307.1361.

Hydroxylactones 3.23a and 3.23b. Methyl lithium (8 µL, 1.6 M in diethyl ether, 1 equiv.) was added to 3.19 (5 mg) in dry THF (0.5 mL) at 0 °C. The reaction was kept at 0 °C for about 1 hour. The reaction mixture was quenched with aqueous NH4Cl. Diethyl ether was used to extract the mixture. The crude product was separated by flash chromatography with

1 petroleum ether/Et2O (10 : 1, Rf = 0.15, yield = 25%). H NMR data showed there are two peaks (δ 4.03 and δ 4.23) for the allylic proton at the lower side chain. This indicates that the

1 product is a mixture of two hydroxyl lactones. H NMR (400 MHz, CDCl3): δ 6.90 (d, 1H, J=

15.6 Hz), 6.22 (dd, 1H, J = 15.6 Hz), 5.75 (m, 1H), 5.14 (m, 2H), 4.23 (dt, 1H), 4.02 (dt, 1H),

3.10 (dt, 2H), 2.20 (s, 3H) 1.52 (m, 2H), 1.25 (m, 6H), 0.90 (s, 9H), 0.87 (t, 3H), 0.03 (s, 6H).

+ HRMS (EI): m/z Calcd for C21H35O4Si (M -CH3) 379.2305, found 379.2303.

2-Allyl-3-butylmaleic acid. To a solution of di-tert-butyl ester 3.15 (10 mg, 0.03 mmol) in

0.5 mL of dichloromethane was added zinc bromide (10 mg, 0.05 mmol) and the solution was stirred for 24 hours. Then water was added and the mixture was stirred for another 2 hours.33 Diethyl ether was used to extract the mixture. The crude product was separated by flash chromatography with petroleum ether/Et2O (1:2, Rf = 0.15) to give 2-allyl-3-

1 butylmaleic acid (4.6 mg, yield 73%); H NMR (400 MHz, CDCl3): δ 5.8 (m, 1H), 5.12 (2H),

3.18 (d, 2H), 2.4 (t, 2H), 1.3-1.5 (m, 4H), 0.88 (3H); HRMS (EI): m/z calcd for C11H14O3

(M+-H2O) 194.0943, found 194.0943.

(Z)-tert-Butyl-2-(2-oxo-6-pentyl-2H-pyran-3(6H)-ylidene)pent-4-enoate (3.21) To a solution of 3.18 (10 mg, 0.02 mmol) in CH2Cl2 (1.0 mL) was added zinc bromide (10 mg,

0.05 mmol) and the mixture was stirred overnight. Diethyl ether was used to extract the mixture. The crude product was separated by flash chromatography with petroleum

1 ether/Et2O (20 : 1, Rf = 0.2) to give 3.21 (5.7 mg, yield = 91%). H NMR (400 MHz,

CDCl3): δ 5.80 (m, 2H), 5.41 (d, 1H), 5.32 (m, 1H), 5.10 (m, 2H), 3.38 (dd, 2H), 2.05 (dt,

13 2H), 1.55 (s, 9H), 1.40 (m, 6H), 0.88 (t, 3H); C NMR (100 MHz, CDCl3): δ 163.2, 162.1,

149.7, 138.4, 136.7, 132.6, 120.3, 117.7, 83.6, 81.9, 32.4, 31.5, 29.0, 28.6, 28.3, 22.7, 14.2;

+ HRMS (EI): m/z calcd for: C19H28O4 (MH ) 320.1988, found 320.1982; calcd for C15H20O4

+ (M -C4H8), 264.1362, found 264.1351.

3.5 References

1. Salomon , R. G.; Miller, D. B.; Zagorski, M. G.; Coughlin, D. J. J Am Chem Soc 1984 ,

106, 6049- 6060.

2. Salomon, R. G.; Miller, D. B. Adv. Prostaglandin Thromboxane Leukot Res 1985, 15, 323-

326.

3. Salomon, R. G.; Sha, W.; Brame, C.; Kaur, K.; Subbanagounder, G.; O’Neil, J.; Hoff, H.

F.; Roberts II. L.J. J Biol Chem 1999, 274, 20271-20280.

4. Salomon, R. G.; Subbanagounder, G.; Singh, U.; O’Neil, J.; Hoff, H. F. Chem Res Toxicol

1997, 10, 750 -759.

5. Brame, C. J.; Salomon , R. G.; Morrow , J. D. ; Roberts II L. J. J Biol Chem 1999, 274,

13139-13146.

6. Salomon, R. G. Antioxid Redox Signal 2005, 7, 185-201.

7. Zagol-Ikapitte , I. ; Masterson, T. S.; Amarnath, V.; Montine , T. J.; Andreasson, K. I.;

Boutaud , O.; Oates, J. A. J Neurochem 2005, 94, 1140-1145.

8. Subbanagounder, G.; Salomon, R. G.; Murthi, K.; Brame, C.; Roberts, L. J. J Org Chem

1997, 62, 7658–7666.

9. Kanai, Y.; Hiroki, S.; Koshino, H.; Konoki, K.; Cho, Y.; Cayme, M.; Fukuyo, Y.;

Yotsu-Yamashita, M J Lipid Res 2011, 52, 2245-2254.

10. Johnson, C.; Penning, D. J Am Chem Soc 1988, 110, 4726-4735.

11. Marfat, A.; McGuirk, P.; Helquist, P. J Org Chem 1979, 44, 3888–3901.

12. Colette, M.; Gaudreault, R. Synth Commun 1990, 20, 2491-2500.

13. Miller D. B.; Raychaudhuri , S.; Avasthi, K.; Lal, K.; Levison, B.; Salomon, G. S. J Org

Chem 1990, 55, 3164–3175.

14. Surmont, R.; Verniest, G.; Kimpe, N.; J Org Chem 2010, 75, 5750–5753.

15. Yang, H.; Hu, G.; Chen, J.; Wang, Y.; Wang Z.H. Bioorg Medl Chem Lett 2007, 17,

5210-5213.

16. Corey, E.J.; Cho, H.; Rücker, C.; Hua, D. H. Tetrahedron Lett. 1981, 22, 3455-3458.

17. Kingsbury, C. L.; Sharp, K. S.; Robin, A., Smith, J. Tetrahedron 1999, 55, 14693-14700.

18. Mingjiang Sun, Ph.D Thesis, Case Western Reserve University.

19. Jarowicki, K.; Kocienski, P. J. Liu Q. Org Synth 2002, 79, 11-14.

20. (a) Lipshutz , B. H.; Wilhelm, R. S.; Floyd, D. M. J Am Chem Soc 1981, 103, 7672-7674.

(b) Lipshutz, B. H.; Sharma, S.; Ellsworth, E. J Am Chem Soc 1990, 112, 4032–4034.

21. House, H. O.; Chu, C.; Wilkins, J. M. Umen M. J. J Org Chem 1975, 40, 1460-1469.

22. Normant, J.F.; Bourgain, M. Tetrahedron Lett 1971, 2583-2596.

23. Colette, M.; Gaudreault, R. Synth Commun 1990, 20, 2491-2500.

24. Chandrasekaran, S.; Kluge, F.; Edwards, J. A. J Org Chem 1977, 42, 3972–3974.

25. Linderman, R.J.; Ghannam, A. J Org.Chem 1988, 53, 2878–2880.

26. (1) Alexakis, A.; Commercon, A.; Coulentianos, C.; Normant, J F Pure Appl Chem 1983,

55, 1759-1766 (2) Normant, J.F.; Cahiez, G.; Chuit, C.; Villieras, J. J Organomet Chem

1974, 77, 269-279.

27. Spinazzé, P.G.; Keay, B. A. Tetrahedron Lett 1989, 30, 1765-1768.

28. (a) Yamamoto,Y.; Yatagai, H.; Maruyama, K. J Chem Soc Chem Comm 1979,157-158.

(b) Corey, E.J.; Venkateswarlu. A. J Am Chem Soc 1972, 94, 6190-6191.

29. Havrilla, C. M.; Hachey, D. L.; Porter, N.A. J Am Chem Soc 2000, 122, 8042-8055.

30. Seal, J.; Porter, N. Anal Bioanal Chem 2004, 378, 1007-1013.

31. Wright, S. W.; Hageman, D. L.; Wright, A. S.; McClure, L. D. Tetrahedron Lett 1997, 38,

7345-7348.

32. Jackson, R.; Tetrahedron Lett. 2001, 42, 5163-5165.

33. Wu, Y.; Limburg, D. C.; Wilkinson, E.; Vaal, M.; Hamilton, S. Tetrahedron Lett. 2000,

41, 2847-2849.

34. Johnson, C. R.; Braun, M. P. J Am Chem Soc 1993, 115, 11014–11015.

35. http://www.sigmaaldrich.com/spectra/fnmr/FNMR004997.PDF.

Chapter 4

Total Synthesis of Oxidized

Levuglandin D2 (ox-LGD2)

4.1 Background.

In 2011, Yotsu-Yamashita and co-workers reported the isolation of a new oxidized derivative of LGD2, i.e., ox-LGD2, in the red algae, Gracilariaedulis (Figure 4.1). Ox-LGD2 was also identified in mouse tissues and the lysate of PMA-treated THP-1 cells incubated with arachidonic acid. These results suggest that ox-LGD2 is a common oxidized metabolite

1 of the lipid oxidation product LGD2. To further examine the physiological function of ox-

LGD2 and enzymatic pathways of oxidation in vitro and in vivo, it is desirable to synthesize ox-LGD2 to provide an authentic standard with unambiguous structure that can be used to confirm and quantify its formation in animal tissues and cells. In Chapter 3, a general method to synthesize unsymmetrically disubstituted maleic anhydrides and 3,4-disubstituted 5- methylhydroxyfuran-2(5H)-ones was discussed. This provided precedent for a possible route for the total synthesis of ox-LGD2. In this chapter, the practical total synthesis of ox-LGD2 will be discussed.

Figure 4.1 Oxidation of arachidonic phospholipids.

4.2 Results and Discussion

To synthesize ox-LGD2, two side chains 4.2 and 4.3 (Scheme 4.1) need to be prepared first. The lower side chain 4.2 was synthesized according to the reported method.2

The top side chain 4.3 was synthesized as outlined in Scheme 4.2.3

Synthesis of Two Side Chains

Scheme 4.1 Synthesis of ox-LGD2

TBDMS protected 4-iodobutyl alcohol 4.5 is readily available through ring opening of THF 4.4 by TBDMS chloride and sodium iodide. Two equivalents of butyl lithium were used to deprotonate one proton from the alcohol hydroxyl and one from the terminal alkyne of propargyl alcohol in the presence of dry HMPA. Subsequent addition of 4.5 delivered 4.6.

To prevent oxidation of the propargyl alcohol by Jones reagent in the next step, the hydroxyl group of 4.6 was acetylated with acetic anhydride in pyridine to produce 4.7. Desilylation and oxidation of 4.7 by Jones reagent afforded 4.8, the acetyl group of which was removed in 74

10% aqueous NaOH, followed by acidification with 2.0 M HCl to produce hydroxy acid 4.9.

4.9 was esterified with CH3OH in the presence of p-toluene sulfonic acid to give methyl ester

4.10. The stereo selective reduction of 4.10 to cis-alkene 4.11 was accomplished by using the

P-2 nickel boride catalyst and hydrogen. Reaction of alcohol 4.11 with phosphorus tribromide in an inert solvent such as hexanes gave allylic bromide 4.3.4,5

Scheme 4.2 Synthesis of top side chain (Z)-methyl 7-bromohept-5-enoate 4.3.

Synthesis of ox-LGD2

The required lower chain vinyllithium synthon, was prepared by treatment of a (Z)- vinylstannane 4.2 with one equivalent of n-BuLi in THF at -78 °C.6 Then the vinyl lithium was reacted with magnesium bromide-etherate complex (MgBr2·Et2O), followed by solid

CuBr·SMe2 to give an organocopper complex RCu(Me2S) ·MgBr2. Then RCu(Me2S)·MgBr2 was added to di-t-butyl acetylene dicarboxylate 4.1. The solution was kept at -78 °C for 2 hours, followed by the addition of HMPA, Pd(PPh3)4 and allyl bromide 4.3 to give 4.15. The

75 reaction temperature should be kept below -45 °C. 1,4 O- to C-silyl migration may occur at higher temperature.7

The intermediate vinyl organometallic can either isomerize to corresponding allenolate or give diene by thermal decomposition.8 Byproducts 4.13 and 4.14 were formed during the reaction (Scheme 4.3). 1,3-Diene 4.13 apparently arises by coupling of the cuprate

4.12 at -78 °C. Cuprate 4.12 can also react with allyl bromide 4.3 to give 4.14. It has been suggested that such coupling is induced by various oxidizing agents, including some transition-metal ions, or promoted by impurities (e.g. Cu2+) present in the cuprous halide.9

This three component coupling involved multistep reactions which further reduced the yield of the desired product. The yield of 4.15 was low (about 7%).

Scheme 4.3 Two confirmed byproducts 4.13 and 4.14 formed during the reaction

Previously, dry hydrocholoric acid gas was bubbled into a solution of di-tert-butyl-2- allyl-3-isobutylmaleate in nitromethane to deliver the corresponding dicarboxylic acid. Then water was removed by azeotropic distillation, and the corresponding maleic anhydride was obtained.10 This method seemed inappropriate for the conversion of 4.15 to 4.16 since hydrocholoric acid gas is expected to cleave both the t-butyl and TBDMS groups and promote dehydration or halodeoxygenation of the allylic alcohol of 4.15.

Either acid or base can hydrolyze a TBDMS protected hydroxyl group. SiO2 is an

11 excellent reagent to cleave tert-butyl esters. SiO2 selectively catalyzed cleavage of the di-t-

76 butyl ester 4.15 to give the corresponding dicarboxylic acid, and the OTBDMS survived the

SiO2-promoted cleavage process. The dicarboxylic acid was further cyclized to form maleic anhydride 4.16 by treatment with acetic acid (Scheme 4.1).

The next step was to introduce methyl group into the maleic anhydride 4.16.

Methyllithium has been used as methylation reagent.12 In the first attempt, commercially available MeLi in diethyl ether was slowly added to the maleic anhydride 4.16 in diethyl ether at -78 °C. No reaction occurred at -78 °C and starting material 4.16 was recovered.

Compounds with two or more carbon-carbon double bonds can coordinate with silver to give two positive signals with almost equal peak heights in their mass spectrum.13 When the reaction temperature was increased to 0 °C, a mixture of products are generated in the methylation reaction. The mass spectra of the methylation products in the presence of

+ silver ions exhibited Ag complex adducts (with AgBF4) that exhibited two peaks at m/z

= 601 and 603 ([M+Ag+] = 494 +107 or + 109), which indicated that hydroxy lactones

4.17a and 4.17b might be present in the reaction product mixture.

Grignard reagents MeMgBr and MeMgI are other reagents have been used in methylations.14,15 MeMgI is more reactive than MeMgBr. No reaction occurred when adding

MeMgBr or MeMgI to the maleic anhydride 4.16 in diethyl ether at -78 °C. In contrast, many products were generated when the temperature was increased 0 °C.

It seemed possible that the methyl ester group in the top side chain of 4.16 was undergoing methylation. Organometallics such as magnesium and cadmium reagents were reported to be more specific for reaction at acid anhydrides.16-17 The Grignard reagent

MeMgBr was transformed to Me2Cd by the treatment with CdCl2 in dry diethyl ether. Then

Me2Cd was added dropwise to an ether solution of 4.16 at 0 °C. No reaction occurred after

77 stirring for 30 min. When the reaction was performed at room temperature, only traces amount of methylation product were found at the m/z 601and 603 ([M + Ag+] = 494 +107 or

+ 109)in the mass spectrum of the reaction product mixture.

Presuming that the methyl ester in 4.16 was reacting with the methyl organometallics, an alternative was explored. Bulky groups such as TBDMS or DTBMS may be used as protecting groups for carboxylic acids. The reactivity toward methylating reagents of DTBMS esters is low owing to the steric hindrance of DTBMS group. So the coupling of methyl group might occur only at the carbonyl group of the maleic anhydride.

In addition, DTBMS esters are more readily hydrolyzed with weak acid catalysis compared with methyl esters. Thus, it would be easier to convert a DTBMS ester than methyl ester into a free carboxylic acid in the last step of the synthesis. Therefore, we decided to synthesize a DTBMS protected top side chain as a modification of the synthetic strategy of Scheme 4.2.

DTBMS triflate is available in two steps (Scheme 4.4),18-19 The reaction of methyldichlorosilane with tert-butyllithium delivers di-tert-butylmethylsilane in 85% yield.

The reaction of trifluoromethanesulfonic acid with di-tert-butylmethylsilane produces

DTBMS triflate that is readily isolated by distillation under reduced pressure. DTBMS triflate is a colorless non-fuming liquid.

Scheme 4.4 Synthesis of DTBMS triflate

Scheme 4.5 outlines the synthesis a DTBMS protected ester 4.21. The carboxylic acid 4.9 (Scheme 4.2) was converted to the DTBMS ester 4.18 in 95% yield by reaction

78 with DTBMS triflate and anhydrous triethylamine in THF solution. Semihydrogeneration of 4.18 with P2 Nickel in the presence of ethylenediamine furnished Z-enol 4.19 in only

10% yield. Most of product was free acid 4.20 which was converted from a DTBMS ester

4.19 by ethylenediamine at room temperature. This reaction revealed a novel reagent to remove the DTBMS protecting group. This will be discussed in Chapter 5.

DTBMS-OTf COOH Ni(OAc)2, NaBH4 HO NEt , THF HO COODTBMS 3 H , ethylenediamine 4.9 4.18 2

CH3SO2Cl COODTBMS COODTBMS HO Et3N, LiBr Br 4.19 4.21 HO COOH 4.20 Scheme 4.5 Synthesis of DTBMS protected ester 4.21

Scheme 4.6 An alternative synthetic route to 4.21

Due the low yield of 4.19, a new method to synthesize 4.21 was pursued (Scheme

4.6). Semihydrogeneration of alkyne 4.6 (from Scheme 4.5) with P2 Nickel in the presence of ethylenediamine and NaBH4 produced (Z)-allylic alcohol 4.22. The TBDMS ether is stable toward the weak base ethylenediamine. To prevent the oxidation of alcohol by Jones reagent in the next step, allylic alcohol 4.22 was acetylated with acetic anhydride to produce

4.23. Compound 4.24, the oxidation product of 4.23 by Jones reagent, was deprotected with

10% aqueous NaOH solution, followed by acidification with 2.0 M HCl to produce carboxylic acid 4.20. 4.20 was esterified by DTBMS triflate and anhydrous triethylamine to give DTBMS ester 4.19. Allylic alcohol 4.19 reacted with methanesulfonyl chloride and lithium bromide to give allylic bromide 4.21.20

Scheme 4.7 Another synthetic route of ox-LGD2.

To assemble the carbon skeleton of the target ox-LGD2, (Z)-vinylstannane 4.2 was treated with one equivalent of n-BuLi in THF at -78 °C to give a vinyllithium intermediate

(Scheme 4.7). Then vinyl lithium was then reacted with magnesium bromide-etherate complex (MgBr2·Et2O), followed by solid CuBr·SMe2 to give an organocopper complex

RCu(Me2S)·MgBr2 4.30. The organocopper reagent 4.30 was then added to di-t-butyl

80 acetylene dicarboxylate 4.1, followed by the addition of HMPA, Pd(PPh3)4 and allyl bromide

4.21 to deliver the di-tert-butyl maleate 4.25. Besides 4.25, an isomeric byproduct 4.31 was generated. Attack of the organocopper complex 4.30 on the vinyl carbon of 4.21 gave 4.31

(Scheme 4.8).

Scheme 4.8 Byproduct formed in the cuprate reaction of ox-LGD2.

The structural isomers 4.25 and 4.31 cannot be separated by flash silica gel chromatography since they have almost the same polarity. Rather, HPLC was used to separate them. With a binary solvent gradient of 2-propanol and hexanes (Table 4.1) and normal phase HPLC, both UV and ELSD showed only one peak (Figure 4.2).

Table 4.1 2-Propanol/hexanes binary gradient used to separate 4.25 and 4.31

Time Flow %A (2- %B propanol) (hexanes) 1 0.01 1.00 0.0 100.0 2 5.00 1.00 0.0 100.0 3 15.00 1.00 5 95 4 25.00 1.00 5 95 5 26.00 1.00 100.0 0.0 6 30.00 1.00 100.0 0.0 7 31.00 1.00 0.0 100.0

W2996 at 254.00 - PDA 210.0 to 400.0 nm at 1.2 nm 0.20

0.18

0.16

0.14

0.12

AU 0.10

0.08

0.06

0.04

0.02

0.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 Minutes

500.00 SATIN

450.00

400.00

350.00

300.00

250.00 mV 200.00

150.00

100.00

50.00

0.00

-50.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 Minutes

Figure 4.2 UV (above) and ELSD (below) of a mixture 4.25 and 4.31

Reverse phase HPLC with methanol and H2O as eluents still failed to separate 4.25 and 4.31. However, when the binary solvents were changed to 2-propanol and acetonitrile in reverse phase HPLC, two main peaks were detected at 21.47 min and 22.70 min (Figure 4.3).

The gradient is shown in Table 4.2. The mass spectra for these two peaks in the presence of

+ + silver ions exhibited Ag complex adducts (with AgBF4) is m/z = 857 and 859 ([M+Ag ] =

750 +107 or + 109). The structures of these two peaks were confirmed by 1H NMR (Figure

4.4). The peak at 22.7 min is the product 4.25, compound 4.31 showed up at 21.47 min. The

1H NMR difference between 4.25 and 4.31 were chemical shifts of protons at allylic position at 3.05 (2H, 4.25), 3.22 (1H, 4.31), and the vinylic protons at 5.27 (2H, 4.25), 5.85 (1H, 4.31) and 5.02 (2H, 4.31).

mV 2250 Detector A Ch1:254nm

2000

1750

1500

1250 21.468 22.704

1000

750

500

250 3.101 18.146 27.415 12.809 2.853 9.087 4.085 0 29.029

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min Figure 4.3 Reverse phase-HPLC of 4.25 and 4.31. There are only two main peaks at 21.47 min (4.31) and 22.70 min (4.25).

Table 4.2 Binary gradient of 2-propanol and acetonitrile used to separate 4.25 and 4.31

Time Flow (mL/min) %A (ACN) %B (2-propanol)

1 0.01 1 90 10

2 10 1 70 30

3 20 1 45 55

4 40 1 0 100

5 50 1 0 100

Figure 4.4 1H NMR of 4.15 and 4.31.

After purification by R-HPLC, di-tert-butyl ester 4.25 was cleaved by SiO2 in reflux toluene to give dicarboxylic acid 4.26, which further cyclized to maleic anhydride 4.27 in acetic acid. Maleic anhydride 4.27 was recovered when one equivalent of methyllithium was slowly added to 4.27 in dry ether at -78 °C. No reaction occurred when the reaction 84 temperature was increase to -20 °C. When the temperature was increased to 0 °C, the m/z

[636+ 107 or +109] [Ag+] = 743, 745 corresponding to the mass of Ag+ complex adducts with 4.28a and 4.28b were found in mass spectrum plus some other impurities and starting materials (Figure 4.4). However, the yield is very low. Using MeMgBr or MeMgI did not improve the yield.

02-01-2013-CH3-OH-636+107-Ag+-2_130201163826 #183-1973 RT: 4.09-42.79 AV: 597 NL: 1.08E5 T: + p ESI Full ms [200.00-1000.00] 743.80 100 519.80

50 431.60 658.00 40 Relative Abundance Relative 503.80 601.53 641.80 30 684.67 849.53 389.60 499.53 547.87 773.73 20 689.73 923.53 801.80 489.73 563.67 883.87 10 473.27

0 400 450 500 550 600 650 700 750 800 850 900 m/z Figure 4.5 Mass spectra of mixture products. M/z: m/z [636+ 107 or +109] [Ag+] = 743, 745

(hydroxyl lactone product, 4.28a and 4.28b).

The methylated sample mixtures were separated by HPLC using a water and methanol gradient. The eluents formed complex adducts with Ag+ and were detected 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 (Figure 4.5). More samples are needed to confirm the structures.

RT: 0.00 - 44.99 SM: 7G 31.58 NL: 8.39E5 100 Base Peak m/z= 742.50-743.50 MS 80 02-01-2013-CH3- 31.37 31.72 OH-636+107-Ag+ -2_130201163826 60 30.85 31.92 30.70 40 32.19 32.42 30.32

Relative Abundance Relative 20 32.67 22.98 27.47 29.55 33.45 2.094.02 5.49 7.84 10.39 16.50 18.55 21.88 37.89 39.97 42.99 0 31.37 NL: 4.91E5 100 Base Peak m/z= 31.11 31.72 744.50-745.50 MS 80 02-01-2013-CH3- OH-636+107-Ag+ 30.99 -2_130201163826 60 32.13 30.75 32.24 40 30.59 29.29 32.67 20 2.03 33.13 23.37 24.16 28.90 3.89 5.36 7.64 12.07 17.70 18.03 34.69 38.36 40.98 0 0 5 10 15 20 25 30 35 40 Time (min)

Figure 4.6 HPLC spectra of reaction mixtures of m/z = 743 (above), 745 (below). 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.

Possible Mechanisms of ox-LGD2 Generation from the Oxidation of LGD2

A possible mechanism for the formation of ox-LGD2 involving free radical reactions is proposed in Scheme 4.9. LGD2 can be oxidized by oxygen to peracid 4.32.

Homolysis of the hydroperoxy group of 4.32 breaking the O-O bond could generate a hydroxyl and a carboxyl radical 4.33 (pathway 1). Addition of the carboxyl radical of

4.33 to a C=C bond, and hydrogen atom abstraction gives ox-LGD2. Alternatively, allylic hydrogen atom abstraction from 4.32 forms allylic radical 4.34 (pathway 2). This fragmentation is driven by the generation of an allylic radical 4.34, which also has the resonance form vinoxy radical 4.35. 4.35 can cyclize to generate alkoxy radical 4.36, which abstracts one hydrogen radical to deliver ox-LGD2.

Scheme 4.9 Possible mechanisms (pathway 1 and pathway 2) to form ox-LGD2 from

LGD2.

4.3 Conclusions

In this chapter, we have discussed the practical total synthesis of ox-LGD2. The lower side chain 4.2 was synthesized according to the reported method. The top side chain 4.3 was assembled in eight steps. (Z)-vinylstannane 4.2 reacts with one equivalent of n-BuLi in THF at -78 °C to give vinyl lithium, followed by addition of MgBr2·Et2O and solid CuBr·SMe2 to develop an organocopper complex RCu(Me2S)·MgBr2. The reaction of di-t-butyl acetylenedicarboxylate with organocopper reagents RCu(Me2S)·MgBr2 followed in situ by addition of allylic bromide 4.3 in the presence of HMPA, Pd(PPh3)4 to give disubstituted

4.15. SiO2 selectively catalyzed cleavage of the di-t-butyl ester 4.15 to give the corresponding dicarboxylic acid, and the OTBDMS survived the SiO2-promoted cleavage process. The dicarboxylic acid was further cyclized to form maleic anhydride 4.16 by treatment with acetic acid. Introducing methyl group into the maleic anhydride 4.16 was not successful. It seemed possible that the methyl ester group in the top side chain of 4.16 was undergoing methylation. As a result, DTBMS protected top side chain 4.21 was synthesized in a modification of the synthetic strategy. Pure disubstituted compound 4.25 was separated in reverse phase HPLC with 2-propanol and acetonitrile. Methylation products of maleic anhydride 4.27 were separated by HPLC, and the eluents formed complex adducts with Ag+ and were detected by positive ESI/MS. Peaks m/z = 743 and 745 corresponding to Ag+ complex adducts with methylating products 4.28a and 4.28b showed up at 31.4 min. In additon, a possible mechanism for the formation of ox-LGD2 involving free radical reactions is proposed.

4.4 Experimental Procedures

1 reported as parts per million (ppm) on the δ scale relative to CDCl3 (δ 7.24). H NMR spectral data are tabulated in terms of multiplicity of proton absorption (s, singlet; d, doublet; t, triplet; m, multiplet; br, broad), coupling constants (Hz), number of protons. All solvents were distilled under a nitrogen atmosphere prior to use, and all materials were obtained from

Aldrich unless specified. Chromatography was performed with ACS grade solvent. Rf values are quoted for plates of thickness 0.25 mm. The plates were visualized with iodine. Flash column chromatograph was performed on 230-400 mesh silica gel supplied by E. Merck.

High performance liquid chromatography (HPLC) purification was performed with HPLC solvent gradients using a Waters M600A solvent delivery system and a Waters U6K injector or a Waters 717 autosampler. The eluents were monitored using an ISCO V4 UV-vis detector or a Waters 2996 photodiode array detector and a Sedex evaporative light scattering detector

(ELSD). All high resolution mass spectra were recorded on a Kratos AEI MS25 RFA high resolution mass spectrometer at 20 eV.

tert-Butyl(4-iodobutoxy)dimethylsilane (4.5) To a solution of tert-butyldimethylsilyl chloride (12.5 g, 0.083 mol) in acetonitrile (100 mL) was added NaI (18.7 g, 0.125 mol) and dry THF (25 mL). The mixture was stirred overnight at room temperature in the dark. After the reaction has completed, 100 mL of petroleum ether were added. And then the organic layer was washed with aqueous NaHCO3 and Na2S2O3 solution (15 mL x 3). The aqueous

89 and organic layers were separated and sequentially extracted with 20% ethyl acetate in hexanes (2 × 50 mL). The combined hexanes layer was washed with brine, dried with

Na2SO4. Solvent was then removed by rotary evaporation and the crude product was purified by chromatography on a silica gel column eluting with 100% of hexanes (Rf = 0.25) to give

1 4.5 (24.25 g, yield = 92.7%). H NMR (400 MHz, CDCl3): δ 3.60 (t, 2H), 3.21 (t, 2H), 1.82

+ (m, 2H), 1.60 (m, 2H), 0.82 (s, 9H), 0.02 (s, 6H). HRMS (EI): m/z calcd for C10H22IOSi (M )

313.0485, found 313.0487.

7-((tert-Butyldimethylsilyl)oxy)hept-2-yn-1-ol (4.6) Propargyl alcohol (1.787 g, 31.9 mmol), HMPA (22.8 g, 127 mmol, 4 equiv.) in 40 mL of dry THF was cooled to -40 °C in an acetonitrile/dry ice bath. Butyl lithium (1.6 M, 42.0 mL, 2.0 equiv.) was added dropwise and the mixture was stirred at -40 °C for about 1 hour. 4.5 (10.0 g, 31.9 mmol) in THF (10 mL) at

-40 °C was added via cannula to the mixture. The mixture was slowly warmed to room temperature. After 4 hours, the mixture was quenched with saturated aqueous NH4Cl solution

(20 mL), extracted with diethyl ether, washed with water, then brine, then dried over anhydrous Na2SO4 and concentrated by rotary evaporation to afford clear yellow oil. This crude product was purified by flash chromatography using 10% ethyl acetate in hexanes (Rf =

0.20) to deliver a clear light yellow oil 3.6 g (yield = 46.7%). The 1H-NMR spectra were the

21 1 same as reported. H NMR (400 MHz, CDCl3): δ 4.20 (d, 2H), 3.59 (t, 2H), 2.21 (m, 2H),

13 1.55 (m, 4H), 0.85 (s, 9H), 0.02 (s, 6H). C NMR (100 MHz, CDCl3): δ 86.47, 78.76, 62.86,

51.53, 32.11, 26.22, 26.16, 25.24, 18.75, 18.55, -5.10.

7-((tert-Butyldimethylsilyl)oxy)hept-2-yn-1-yl acetate (4.7) A solution of 4.6 (3.5 g, 12.3 mmol) in pyridine (15 mL) was cooled to 0 °C and acetic anhydride (4.5 mL, 3 eq.) was slowly added. The mixture was stirred for 40 min at 0 °C under argon, and then allowed to warm to room temperature. After 2 h the mixture (clear light yellow) was quenched by addition of ethanol (5 mL) and concentrated by rotary-evaporation to afford a clear yellow oil.

The residue was dissolved in diethyl ether and washed with 1.0 M HCl to remove pyridine and concentrated by rotary evaporation for the next step without further purification (3.6 g,

1 88%). (Rf = 0.25, ethyl acetate/hexanes = 1/20). H NMR (400 MHz, CDCl3): δ 4.61 (t, 2H),

3.58 (t, 2H), 2.20 (m, 2H), 2.04 (s, 3H), 1.54 (m, 4H), 0.85 (s, 9H), 0.00 (s, 6H). HRMS (EI):

+ m/z calcd for C13H25OSi (M -C2H3O2), 225.1675, found 225.1670

7-Acetoxyhept-5-ynoic acid (4.8) and 7-hydroxyhept-5-ynoic acid (4.9) To a solution of

4.7 (3.6 g, 12.7 mmol) in 15 mL acetone at 0 °C was added Jones reagent dropwise until the orange color persisted for 20 min. The reaction was then quenched by addition of isopropyl alcohol (10 mL), and the reaction mixture was filtered through Celite. The filtrate was concentrated by rotary evaporation, and the water residue was extracted with diethyl ether.

The ether extract was washed with water, then brine, then dried over anhydrous Na2SO4 and concentrated by rotary evaporation to afford 4.8 (Rf = 0.21, ethyl acetate : hexanes = 3:2). 4.8 was used for the next step without further purification. The crude product 4.8 was dissolved in ethyl acetate (20 mL) and extracted with 10% NaOH (4 x 20 mL). The aqueous layers 91 were collected and acidified with 2 M HCl to pH = 1, followed by extraction with ethyl acetate (5 x 20 mL). The organic layers were combined and washed with water, then brine, then dried over anhydrous magnesium sulfate, filtered and concentrated by rotary evaporation to afford a clear oil 4.9 (1.37 g, Rf = 0.3, ethyl acetate : hexanes = 5:1). The combined yield is 76% for the two steps. The NMR spectra were the same as reported.20 1H

NMR (400 MHz, CDCl3): δ 4.15 (2H), 2.39 (t, 2H, J = 8.0 Hz), 2.22 (t, 2H), 1.74 (t, J = 8.0

13 Hz). C NMR (100 MHz, CDCl3): δ 178.01, 84.88, 79.40, 50.85, 32.94, 23.67, 18.23.

Methyl 7-hydroxyhept-5-ynoate (4.10) A solution of 4.9 (1.37 g. 9.6 mmol) and p- toluenesulfonic acid hydrate in MeOH (15 mL) was boiled under reflux overnight. The reaction mixture was cooled and then concentrated by rotary evaporation. The residue was then dissolved in diethyl ether and washed with aqueous sodium bicarbonate solution.

Concentration of the extract, followed by flash chromatography with ethyl acetate and petroleum ether (1:2, Rf = 0.3) gave a colorless oil 1.12 g (yield =74.5%). The NMR spectra

22 1 were the same as reported. H NMR (400 MHz, CDCl3): δ 4.21 (d, 2H), 3.62 (s, 3H), 2.38 (t,

13 2H), 2.23 (m, 2H), 1.80 (m, 2H). C NMR (100 MHz, CDCl3): δ 174.0, 84.5, 80.0, 52.0,

50.6, 33.1, 24.3, 18.1.

(Z)-Methyl-7-hydroxyhept-5-enoate (4.11) Nickel acetate tetrahydrate (1.79 g, 7.2 mmol) in 95% of ethanol (100 mL) was placed under a balloon of H2. Sodium borohydride (0.272 g,

7.2 mmol) in ethanol (8 mL) was added at room temperature. After 20 min, ethylenediamine

(1.726 g, 28.8 mmol) was added, followed by adding 4.8 (1.12 g, 7.2 mmol) in ethanol. The

92 reaction was monitored by TLC. After about 3 hours, the solution was filtered through a pad of silica. The residue was extracted with diethyl ether, washed with water, then brine, then dried over anhydrous Na2SO4 and concentrated by rotary evaporation to afford a clear oil.

This crude product was purified by flash chromatography using 30 % ethyl acetate in hexanes (Rf = 0.4) to give 0.749 g (yield = 67%) of cis-alkene 4.11. The NMR spectra were

22 1 the same as reported. H NMR (400 MHz, CDCl3): δ 5.66 (dt, 1H), 5.49 (dt, 1H), 4.15 (d,

13 2H), 3.65 (s, 3H), 2.30 (t, 2H), 2.10 (t, 2H), 1.75 (m, 2H); C NMR (100 MHz, CDCl3): δ

174.3, 131.7, 129.9, 58.5, 51.8, 33.5, 26.8, 24.8.

(Z)-Methyl-7-bromohept-5-enoate (4.12) To a solution of alcohol 4.11 (300 mg, 1.90 mmol) and carbon tetrabromide (800 mg, 2.4 mmol) in dry CH2C12 (10 mL) was added triphenylphosphine (629 mg, 2.4 mmol) in portions. The reaction mixture was stirred for 40 min, and then MeOH (1 mL) was added. Concentration on a rotary evaporator, followed by flash chromatography with ethyl acetate/hexanes (50 : l, Rf = 0.25) gave 4.12 as a colorless oil (195 mg, yield = 46.4%). The NMR spectra were the same as reported.22 1H NMR (400

MHz, CDCl3): δ 5.70 (m, 1H), 5.53 (m, 1H), 3.94 (d, 2H), 3.61 (s, 3H), 2.30 (m, 2H), 2.10

13 (m, 2H), 1.71 (m, 2H); C NMR (100 MHz, CDCl3): δ 174.1, 134.6, 126.6, 51.8, 33.5, 27.1,

26.4, 24.4.

Methyl-7,8-(di-tert-butoxycarbonyl)-11-((tert-butyldimethylsilyl)oxy)-(4Z,7Z,9E)- heptadecatrieneoate (4.15) A solution of vinyltin reagent 4.2 (580 mg, 0.90 mmol) in dry

THF (15 mL) was treated with n-BuLi (1.6 M, 0.57 mL, 0.91 mmol) at -78 °C. After 20 min,

MgBr2·OEt2 (235 mg, 0.90 mmol) was added to the mixture under argon. A white suspension was formed and the mixture was stirred for another 20 min. Then the suspension was transferred via a cannula to a solution of cuprous bromide-dimethyl sulfide (185 mg, 0.90 mmol) in anhydrous THF (2 mL) and dimethyl sulfide (2 mL) at -78 °C. The resulting yellow solid was stirred at -78 °C for 2 hours. A solution of dibutylacetylenedicarboxylate 4.1 (215 mg, 0.95 mmol) in anhydrous THF (2 mL) at -78°C was slowly added via cannula to the cuprate solution, also cooled to -78 °C. The mixture was stirred at -78 °C for 45 min. Freshly distilled hexamethylphophoroustriamide (HMPA, 2.0 mmol, 0.5 mL) was then added. After about 15 min, tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 0.05 mmol, 15 mg) in THF

(1 mL) was added via cannula, followed by the addition of 4.3 (261 mg, 0.9 mmol) in THF (1 mL) at -78 °C. Stirring was continued overnight at -78 °C and was then warmed to -45 °C and stirred for one more hour. The reaction mixture was then quenched with aqueous NH4Cl

(2.0 mL), and slowly warned to 0 °C. Diethyl ether was used to extract the reaction mixture.

The ether extract was washed with brine, water, then dried over anhydrous Na2SO4 and

94 concentrated by rotary evaporation. The crude product was purified by flash chromatography with hexanes/Et2O (40:1 to 20:1) to give 4.15 (40 mg, yield = 7%). (Rf = 0.24, diethyl ether :

1 hexanes = 1:20). H NMR (400 MHz, CDCl3): δ 6.39 (d, 1H, J=16.0 Hz), 5.98 (dd, 1H, J =

16.0 Hz, J = 5.6 Hz), 5.37 (m, 2H), 4.20 (1H, J = 5.6 Hz), 3.65 (3H), 3.06 (dd, 2H), 2.26 (m,

2H), 2.10 (m, 2H), 1.68 (m,2H), 1.48 (20H), 1.25 (m, 6H), 0.86 (12H), 0.03 (6H); 13C NMR

(100 MHz, CDCl3): δ 174.2, 167,7, 166.5, 142.0, 141.9, 140.2, 139.8, 131.2, 127.1, 122.0,

81.9, 81.2, 73.1, 51.7, 38.3, 33.6, 32.0, 31.3, 28.3,28.2,27.0, 26.6, 26.0, 22.9, 22.8, 18.4, 14.3,

+ -4.18, -4.60; HRMS (EI): m/z calcd for C30H51SiO7 (M -C4H9), 551.3404, found 551.3406.

2-(3-tert-Butyldimethylsilyloxy-(1E)-octenyl)-3-(6-methoxycarbonyl-2(Z)-hexenyl) maleic anhydride (4.16) To a solution of 4.15 (20 mg) in toluene (3 mL) was added SiO2

(250 mg). The solution was boiled under reflux under argon for about 6 hours, then cooled to room temperature and diluted with 5 mL of 10% methanol/CH2Cl2, and filtered through a pad of Celite. The solution was concentrated under vacuum. And then the crude product was stirred with acetic acid/acetic anhydride (2:1, 1.0 mL). The reaction went to completion in 8 hours. The mixture was then concentrated on a mechanical pump and the crude product was purified by flash chromatography with hexanes/Et2O (1:5, Rf = 0.3) to

1 give 4.16 (6.3 mg, yield 40% for 2 steps). H NMR (400 MHz, CDCl3) δ 7.18 (dd, 1H, J

= 16.2 Hz) 6.50 (d, 1H, J = 16.2 Hz), 5.52 (dd, 1H), 5.37 (m, 2H), 4.38 (m, 1H), 3.62 (s,

3H), 3.22 (d, 2H), 2.26 (m, 2H), 2.10 (m, 2H), 1.68 (m,2H), 1.48 (2H), 1.30 (m, 6H), 0.88

+ (s,12H), 0.03 (s, 6H); HRMS (EI): m/z calcd for C22H33O6Si (M ), 478.2751, found

478.2777.

Di-tert-butyl(methyl)silyl-7-hydroxyhept-5-ynoate (4.18) 7-Hydroxy-5-heptynoic acid (4.9,

100 mg, 0.70 mmol) in anhydrous THF (1.0 mL) under argon was treated with dry triethylamine (1.5 eq, 107 mg) at room temperature. Then di-tert-butylmethylsulfonate (230 mg, 0.75 mmol) was added dropwise. After about 30 min, the solution was concentrated in vacuo and the residue was purified by flash chromatography with ethyl acetate and hexanes

(1 : 8, Rf = 0.3) to give 4.18 (142 mg, yield 68%). The NMR spectra were the same as

20 1 reported. H NMR (400 MHz, CDCl3): δ 4.22 (t, 2H), 2.44 (t, 2H), 2.28 (tt, 2H), 1.80 (m,

13 2H), 1.64 (s, 1H), 0.99 (18H), 0.29 (s, 3H); C NMR (100 MHz, CDCl3): δ 172.95, 85.48,

79.35, 51.59, 35.17, 27.70, 24.14, 20.49, 18.40, -7.29.

(Z)-Di-tert-butyl(methyl)silyl-7-hydroxyhept-5-enoate (4.19) To a solution of Nickel acetate tetrahydrate (83.6 mg, 0. 34 mmol) in 95% ethanol (5 mL) under a H2 atmosphere (balloon) was added NaBH4 (12.7 mg, 0.34 mmol). After about 20 min, ethylenediamine (80.5 mg, 1.31 mmol) was added, followed by addition of alkyne 4.18

(100 mg, 0.34 mmol). The mixture was stirred overnight. After the reaction was completed, the solution was filtered through a pad of silica gel. The residue was extracted with diethyl ether, washed with water, then brine, then dried over anhydrous Na2SO4 and

96 concentrated by rotary evaporation to afford a clear oil. The crude product was purified by flash chromatography using ethyl acetate/hexanes (1/8, Rf = 0.31) to give cis-alkene

1 4.19 (10 mg, yield = 10%). H NMR (400 MHz, CDCl3): δ 5. 62 (m, 1H), 5.50 (m, 1H),

4.15 (d, 2H), 2.33 (t, 2H), 2.12 (q, 2H), 1.70 (m, 2H), 0.99 (s, 18H), 0.23 (s, 2H); HRMS

+ (EI): m/z calcd for C16H31O2Si (M -OH), 283.2093, found 283.2089. It should be noted that most of 4.19 was further hydrolyzed to (Z)-7-hydroxyhept-5-enoic acid (4.20).

(Z)-7-((tert-Butyldimethylsilyl)oxy)hept-2-en-1-ol (4.22) Nickel acetate tetrahydrate

(7.14 g, 0.029 mmol) in 95% of ethanol (200 mL) was placed under a balloon of H2.

Sodium borohydride (1.09 g, 0.029 mmol) in ethanol (8 mL) was added at room temperature. After 20 min, ethylenediamine (7.0 g, 0.116 mmol) was added, followed by adding alkyne 4.6 (7.0 g, 0.029 mmol) in ethanol (10.0 mL). The reaction was monitored by TLC. After about 3 hours, the reaction was filter through a pad of silica gel. The filtrate ethanol solution was concentrated by rotary evaporation. The residue was extracted with diethyl ether, washed with water, then brine, then dried over anhydrous

Na2SO4 and concentrated by rotary evaporation to afford a clear oil. This crude product was purified by flash chromatography (hexanes : ethyl acetate = 4 :1, Rf =0 .25) gave

1 alkene 4 .22 (5.25g, 75%). H NMR (400 MHz, CDCl3): δ 5.55 (m, 2H), 4.21 (d, 2H),

3.58 (t, 2H), 2.20 (m, 2H), 1.56 (m, 4H), 0.86 (s, 9H), 0.00 (s, 6H); HRMS (EI): m/z

+ calcd for C13H27O2Si (M -H), 243.1780, found 243.1779.

(Z)-7-((tert-Butyldimethylsilyl)oxy)hept-2-en-1-yl acetate (4.23) A solution of 4.6 (3.5 g,

12.3 mmol) in pyridine (15 mL) was cooled to 0 °C and then acetic anhydride (4.5 mL, 3 eq.) was slowly added. The mixture was stirred for 40 min at that temperature under argon, and then allowed to warm to room temperature. After 2 h the mixture (clear light yellow) was quenched by addition of ethanol (5 mL) and concentrated by rotary-evaporation to afford clear yellow oil. The residue was washed with 1 M HCl and concentrated (3.6 g, 88%). The crude product, which was used for the next step without further purification, exhibited a

1 single spot by TLC Rf = 0.2 (diethyl ether : hexanes = 1:10). H NMR (400 MHz, CDCl3): δ

5.60 (dt, 1H), 5.55 (dt, 1H), 4.59 (d, 2H), 3.58 (t, 2H), 2.20 (m, 2H), 2.04 (s, 3H), 1.54 (m,

+ 4H), 0.85 (s, 9H), 0.00 (s, 6H); HRMS (EI): m/z calcd for C13H27OSi (M -C2H3O2),

227.1831, found 227.1832.

(Z)-7-Acetoxyhept-5-enoic acid (4.24) and (Z)-7-hydroxyhept-5-enoic acid (4.20) To a solution of 4.3 (3.6 g, 12.6 mmol) in acetone (15 mL) at 0 °C was added Jones reagent dropwise until the orange color persisted for 20 min. The reaction was then quenched by addition of isopropyl alcohol (10 mL), and then filtered through Celite to afford 7- acetoxyhept-5-ynoic acid 4.24 after removal of solvents by rotary evaporation. The crude product was dissolved in ethyl acetate (20 mL) and extracted into 10% NaOH (4 x 20 mL).

The aqueous layers were collected and acidified with 6 M HCl to pH = 1, followed by

98 extraction with ethyl acetate (3 x 20 mL). The organic layers were combined and washed with water, then brine, then dried over anhydrous magnesium sulfate, filtered and concentrated by rotary evaporation to afford a clear oil 4.20 (1.02 g) that exhibited a single

1 spot Rf = 0.3 by TLC with ethyl acetate. The yield is 56% for the two steps. H NMR (400

MHz, CDCl3): δ 5.63 (dt, 1H), 5.55 (dt, 1H), 4.20 (d, 2H), 2.32 (t, 2H), 2.15 (t, 2H), 1.68 (m,

+ 2H); HRMS (EI): m/z calcd for C7H10O2 (M -H2O), 126.0681, found 126.0681.

Di-tert-butyl(methyl)silyl (Z)-7-hydroxyhept-5-enoate (4.19) 4.20 (100 mg, 0.69 mol) in anhydrous THF (1.0 mL) under argon was treated with dry triethylamine (1.5 eq, 107 mg,

1.04 mmol) at room temperature. And then di-tert-butylmethyl trifluoromethanesulfonate

(230 mg, 0.72 mmol) was added dropwise. After about 30 min, the solution was concentrated in vacuo and the residue was purified by flash chromatography with ethyl acetate/hexanes

1 (1:4, Rf = 0.3) to give 4.19 (145 mg, yield 70%). H NMR (400 MHz, CDCl3): δ 5. 62 (dt,

1H), 5.50 (dt, 1H), 4.15 (d, 2H), 2.33 (t, 2H), 2.12 (q, 2H), 1.70 (m, 2H), 0.99 (s, 18H), 0.23

+ (s, 2H); HRMS (EI): m/z calcd for C16H31 O2Si(M -OH), 283.2093, found 283.2089.

Di-tert-butyl(methyl)silyl (Z)-7-bromohept-5-enoate (4.20) Methanesulfonyl chloride

(68 mg, 3.0 mmol) was added dropwise to a chilled solution of 4.19 (50 mg, 0.16 mmol), triethylamine (40 mg, 0.32 mmol) in CH2Cl2 (1.2 mL) at -50 °C. The resulting white suspension was stirred for 45 min and then treated with a solution of lithium bromide

(116 mg, 0.64 mmol) in THF (0.5 mL). The colorless mixture was then warmed slowly to

-20 °C and stirred for 1 h. Then the reaction mixture was poured over water and extracted with pentane. The pentane extracts were washed with water, then brine, then dried and concentrated by rotary evaporation to deliver crude allylic bromide. The crude product was purified by flash chromatography with ethyl acetate/hexanes (1 : 25, Rf = 0.2) to give

1 pure 4.21 (43.6 mg, 53%). H NMR (400 MHz, CDCl3): δ 5.77 (m, 1H), 5.58 (m, 1H),

3.98 (d, 2H), 2.35 (t, 2H), 2.19 (q, 2H,), 1.70 (m, 2H), 1.00 (s, 18H), 0.30 (s, 3H). 13C

NMR (100 MHz, CDCl3): δ 173.09, 134.82, 126.49, 35.72, 27.73, 27.15, 26.49, 24.71,

+ 20.49, -7.29. HRMS (EI): m/z calcd for: C16H32BrO2Si (MH ), 363.1355, 365.1334,

+ found 363.1351, 365.1335. calcd for: C12H22BrO2Si (M -C4H9), 305.0572, 307.0552,

Found 305.0575, 307.0553.

Di-tert-butylmethylsilyl 7,8-(di-tert-butoxycarbonyl)-11-((tert-butyldimethylsilyl) oxy)-(4Z,7Z,9E)-heptadecatrieneoate (4.25) A solution of vinyltin reagent 4.2 (241 mg,

0.45 mmol) in dry THF (8 mL) at -78 °C was treated with n-BuLi (1.6 M, 0.28 mL, 0.45 mmol). After 20 min, MgBr2·OEt2 (120 mg, 0.45 mmol) was added to the mixture under argon. A white suspension formed and the mixture was stirred for another 45 min. Then the suspension was transferred dropwise via a cannula to a solution of cuprous bromide- dimethyl sulfide (110 mg, 0.45 mmol) in anhydrous THF (3 mL) at -78 °C. The resulting yellow solid was stirred at -78 °C for 2 hours. Then a solution of di-t-butyl 100 acetylenedicarbonate (110 mg, 0.45mmol) in anhydrous THF (1 mL) was slowly added via cannula to the cuprate at -78 °C. The mixture was stirred at -78 °C for 45 min.

Freshly distilled HMPA (138 mg, 0.78 mmol) was then added. After about 15 min,

Pd(PPh3)4 (10 mg, 0.03 mmol) in THF (0.5 mL) was added by cannula, followed by the addition of 4.21 (161 mg, 0.45mmol) in THF (0.5 mL). Stirring was continued overnight at -78 °C and the mixture was then warmed to -45 °C and stirred for one more hour. Then the reaction mixture was quenched by addition of aqueous ammonium chloride (1 mL), and slowly warned up 0 °C. Diethyl ether was used to extract the mixture. The crude product was purified by flash chromatography with hexanes/Et2O (40 : 1 to 20 : 1) to give

1 4.25 (17 mg, yield = 5%). Rf = 0.25 (diethyl ether: hexanes = 1 : 20). H NMR (400 MHz,

CDCl3): δ 6.40 (d, 1H, J = 15.8 Hz), 5.98 (dd, 1H, J = 15.8 Hz, J = 5.2 Hz), 5.37 (m, 2H),

4.20 (d, 1H, J = 5.2 Hz), 3.05 (dd, 2H), 2.26 (t, 2H), 2.10 (m, 2H), 1.68 (m, 2H), 1.48

13 (20H), 1.25 (m, 6H), 1.06 (s, 18H) 0.86 (12H), 0.03 (6H); C NMR (100 MHz, CDCl3):

δ 173.25, 167.72, 166.50, 141.99, 139.81, 130.68, 130.35, 127.00, 122.10, 81.85, 81.38,

73.17, 38.23, 35.93, 32.03, 30.54, 29.92, 28.30, 27.74, 26.64, 25.21, 24.88, 22.81, 20.49,

18.41, 14.23, -4.15, -4.58, -7.28; HRMS (EI): m/z calcd for: C38H70O7Si2 (M-C4H8)

694.4460, found 694.4463.

101

2-(3-tert-Butyldimethylsilyloxy-(1E)-octenyl)-3-(6-di-tert-butylmethylsilyloxy carbonyl-

2(Z)-hexenyl) maleic anhydride (4.27) To a solution of 4.25 (20 mg) in toluene (3 mL) was added SiO2 (250 mg).The solution was boiled under reflux under argon for about 6 hours, then cooled to room temperature, then diluted with 5 mL of 10% methanol/CH2Cl2, and filtered through a pad of Celite. The solution was concentrated under vacuum. Then the crude product was stirred with acetic acid/acetic anhydride (2 : 1, 1 mL). After the reaction is completed, the mixture was concentrated with a mechanical pump and the crude product was purified by flash chromatography with hexanes/Et2O (1 : 5, Rf = 0.3) to give 4.27 (6.3 mg,

1 yield 42% for 2 steps). H NMR (400 MHz, CDCl3): δ 7.21 (dd, 1H, J = 16 Hz, J = 4.0 Hz),

6.52 (dt, 1H, J = 16 Hz), 5.55 (dt, 1H), 5.36 (dt, 1H), 4.35 (dd,1H, J = 4.0 Hz), 3.24 (d, 2H),

2.35 (t, 2H), 2.20 (m, 2H), 1.72 (tt, 2H) 1.53 (m, 2H), 1.28 (m, 6H), 1.01 (s, 18H), 0.91 (s,

+ 9H), 0.88 (t, 3H) 0.30 (s, 3H), 0.04 (s, 6H); HRMS (EI): m/z calcd for C30H51O6Si2 (M -

C4H9), 563.3224, found 563.3226.

102

4.5 References

1. Kanai, Y.; Hiroki, S.; Koshino, H.; Konoki, K.; Cho, Y.; Cayme, M.; Fukuyo, Y.;

Yotsu-Yamashita, M. J Lipid Res 2011, 52, 2245-2254.

2. Johnson, C. R.; Braun, M. P. J Am Chem Soc 1993, 115, 11014–11015.

3. Yunfeng Xu. Ph.D thesis, Case Western Reserve University.

4. Ganem, B.; Osby, J. O. Chem Rev 1986, 86, 763–780.

5. Brown, C. A.; Ahuja, V. K. J Org Chem 1973, 38, 2226–2230.

6. Linderman, R.J.; Ghannam, A. J Org Chem 1988, 53, 2878–2880.

7. Spinazzé, P.G.; Keay, B. A. Tetrahedron Lett 1989, 30, 1765-1768.

8. (1) Alexakis, A.; Commercon, A.; Coulentianos, C.; Normant, J F Pure ApplmChem 1983,

55, 1759-1766 (2) Normant, J.F.; Cahiez, G.; Chuit, C.; Villieras, J. J Organomet Chem 1974,

77, 269-279.

9. Marfat, A.; McGuirk, P.; Helquist, P. J Org Chem 1979, 44, 3888–3901.

10. Colette, M.; Gaudreault, R. Synth Commun 1990, 20, 2491-2500.

11. Jackson, R.; Tetrahedron Lett 2001, 42, 5163-5165.

12. Koch, H. Monatsh Chem 1962, 93, 292-295.

13. Jones, P. R.; Congdon, S. L. J Am Chem Soc 1959, 81, 4291–4294.

14. El-Shishtawy, R.; Fukunishi, K. Synthesis 1994, 1411-1412.

15. Surmont, R.; Verniest, G.; Kimpe, N.; J Org Chem 2010, 75, 5750–5753.

16. Araki, S.; Katsumura, N.; Ito, H.; Butsugan, Y. Tetrahedron Lett 1989, 30, 1581-1582.

17. Yang, H.; Hu, G.; Chen, J.; Wang, Y.; Wang Z.H. Bioorg Med Chem Lett 2007, 17,

5210-5213.

18. Barton, T. J.; TullyC. R. J Org Chem 1978, 43, 3649-3653.

103

19. Chadeayne, A.R.; Wolczanski, P. T.; Lobkovsky, E.B. Inorg Chem 2004, 43, 3421-3432.

20. Bhide, R. S.; Levison, B. S.; Sharma, R. B.; Ghosh, S.; Salomon. R. G. Tetrahedron Lett

1986, 27, 671-674.

21. Mukai, C.; Yamaguchi,S.; Sugimoto,Y.; Miyakoshi,N.; Kasamatsu, E.;Hanaoka, M. J

Org Chem 2000, 65, 6761–6765.

22. Johnson, C.; Penning, D. J Am Chem Soc 1988, 110, 4726-4735.

104

Chapter 5

Pilot Studies

Part A: Model Study for the Synthesis Putative β-

Alkylperoxy Hydroperoxide

Part B: A Novel Method for the Selective Cleavage of DTBMS Esters

105

Part A: Model Study for the Synthesis Putative β-Alkylperoxy Hydroperoxide

5.1.1 Background

Owing to the presence of a doubly allylic methylene group, polyunsaturated fatty acyls (PUFAs) are highly susceptible to free radical induced oxidation, which involves generation of relatively stable pentadienyl radicals upon hydrogen atom abstraction.1,2

Oxygen can capture these radicals to deliver conjugated dienyl peroxy radicals that can abstract hydrogen atoms from additional polyunsaturated fatty acyls to generate a hydroperoxide, such as 13-HPODE.3 The hydroperoxide can undergo further oxidation and fragmentation reactions to generate aldehyde products such as HNE, hexanal and HODA

(Scheme 5.1.1).4-9 It was suggested that intermediates containing peroxide linked dimer with a vicinal hydroperxoy group readily generate hydroxyl and alkoxyl radicals under mild conditions through a fragmentation reaction, which is driven by the formation of two carbonyl groups.1 The generation of radicals during the fragmentation makes the putative β- alkylperoxy hydroperoxides initiators of an autocatalytic lipid oxidation process.

Scheme 5.1.1 Fragmentation of putative β-alkylperoxy hydroperoxide to form aldehyde and radicals.

106

To test the hypothesis that a diperoxide β-alkylperoxy hydroperoxide intermediate can readily undergo fragmentation to deliver aldehyde and radicals, it is desirable to prepare

β-alkylperoxy hydroperoxide 5.6. As proposed in Scheme 5.1.2, 2-methoxyproene will be used to protect the hydroperoxy group of 13-HPODE 5.1 to afford 5.2, followed by reacting with pentafluorobenzyl bromide (PFB-Br) to give 5.3. The reaction of diene 5.3 with N- bromosuccinimide and t-butyl peroxide10 will produce a β-bromo dialkylperoxide 5.4.

C5H11 O (CH2)7COOH C5H11 PFB-Br 2-methoxypropene HOOC(H2C)7 O PPTS OOH OMe 5.2 5.1

C5H11 C5H11 O O NBS PFB-OOC(H2C)7 O PFB-OOC(H2C)7 O OOH OMe OMe

5.3 Br OOC(CH3)3 5.4

C5H11 O

PFB-OOC(H2C)7 O

(CH2)7COO-BFP C5H11 AgOTFA OMe O OOC(CH3)3 OOH HPODE-PFB O

(CH ) COO-PFB C5H11 2 7

C5H11 5.5 O

PFB-OOC(H2C)7 OH

AcOH/H2O O OOH O

C5H11 (CH2)7COO-PFB 5.6

Scheme 5.1.2 Proposed synthetic method of β-alkylperoxy hydroperoxide.

107

Primary, secondary, and tertiary alkyl hydroperoxides and dialkyl peroxides can be prepared from the appropriate alkyl bromide or iodide and hydrogen peroxide or alkyl hydroperoxide in the presence of AgOTFA.11 Alkylation of the alkylperoxide HPODE-PFB with alkyl bromide 5.4 promoted by AgOTFA will produce dimer 5.5. Finally, removal of the protecting group will deliver β-alkylperoxy dihydroperoxide 5.6. According to the proposed mechanism of Scheme 5.1.1, β-alkylperoxy dihydroperoxide 5.6 should fragment to deliver aldehyde, such as ON, HNE, and alkoxyl and hydroxyl radicals.

In a pilot study to test the feasibility of synthetic route envisioned in Scheme 5.1.2, the much simpler synthesis outlined in scheme 5.1.3 was attempted.

Scheme 5.1.3 Model study for the synthesis of β-alkylperoxy hydroperoxide.

108

5.1.2 Results and Discussion.

Protection of the hydroxyl group in 2,4-hexadien-1-ol 5.7 by reaction with 3,4- dihydro-2H-pyran gives tetrahydropyranyl ether 5.8. Initially, 2-methoxypropene and PPTS was used to protect the hydroxyl group of 5.7 to form 2-(methoxypropyl) ketal, in this step.

3,4-dihydro-2H-pyran gave better yield of protected derivatives and less impurity. N-

Bromosuccinimide (NBS) is a convenient source of cationic bromine and well- known in radical substitution and electrophilic addition reactions in organic synthesis. The reaction of diene 5.8 with NBS and t-butyl hydroperoxide in the presence of NaHCO3 could afford at least four regioisomeric bromide intermediates, 5.9, 5.10, 5.11, 5.12 (Scheme 5.1.4).

Scheme 5.1.4 Four possible intermediates in the reaction of 5.8 with NBS and t-BuOOH.

Only one major product was obtained (76% yield). The 1H NMR, 13C NMR, H-H

COSY and heteronuclear multiple-quantum correlation (HMQC) spectra were measured in order to elucidate the structure of product. The double bond was shown to have the E

3 geometry by the large vicinal JHH value of 16.0 Hz between it is olefinic hydrogen signals.

In the H-H COSY spectrum, the vinyl hydrogen resonance at 5.71 ppm showed a coupling with the hydrogen signal at 1.77 ppm, while the other vinyl hydrogen at 5.37 ppm showed a coupling with the hydrogen signal at 3.91 ppm, which is an oxymethine, oxymethene proton or bromomethine hydrogen. Based on the above information, we can exclude the compounds

109

5.9 and 5.13 which have allylic oxymethine, oxymethene or bromomethine hydrogens adjacent to both vinyl carbons. In the HMQC spectrum, the chemical shift at H4 (3.91 ppm) has three hydrogens, which are bound to carbons at 52.82 ppm, 69.59 ppm, 68.66 ppm. The carbon with chemical shift at 52.82 ppm has a bromo group attached, the carbon with chemical shift around 68 ppm is has an oxygen attached (Figure 5.1.1). Since C4 (52.82 ppm) is next to a vinyl carbon, Br is connected with C4. According to all the information from 1H NMR, 13C NMR, COSY and HMQC, 5.11 is the structure of the major product formed in this reaction, and not the desired product compound 5.9. Apparently, the remarkably selective addition of Br+ to the 2-3 C=C bond was unexpected. The noteworthy selectivity of this reaction of NBS with diene 5.8 may be of considerable synthetic utility.

However, it completely fails to provide any access to the synthetic target of our project.

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

5.1.3 Conclusions

In this part of Chapter 5, a simple model study to synthesize β-alkylperoxy dihydroperoxide was performed. The addition reaction of diene 5.8 with NBS and t-butyl hydroperoxide in the presence of NaHCO3 selectively gives only one product 5.11. Although the desired compound 5.9 was not successfully obtained, the noteworthy selectivity of this reaction of NBS with diene 5.8 may be of considerable synthetic utility.

112

5.1.4 Experimental Procedures

General Methods. Proton magnetic resonance (1H NMR) spectra and carbon magnetic resonance (13C NMR) spectra were recorded on a Varian Inova AS400 spectrometer operating at 400 MHz for 1H NMR and 100 MHz for 13C NMR or Varian Inova 600. Proton chemical shifts are reported as parts per million (ppm) on the δ scale relative to CDCl3 (δ

MS25 RFA high resolution mass spectrometer at 20 eV.

(9Z,11E)-13-((2-Methoxypropan-2-yl)peroxy)octadeca-9,11-dienoic acid (5.2) To a solution of hydroperoxide 13-HPODE (15 mg, 0.048 mmol) in CH2Cl2 (3 mL) was added 2- methoxypropene (5.9 mg, 0.082 mmol) and pyridinium p-toluenesulfonate (PPTS, 1 mg).

After being stirred for 1 hour, the reaction was quenched with water and extracted with

CH2Cl2. The organic layer was dried (Na2SO4) and concentrated to afford a colorless oil. The crude product was separated by flash chromatography with petroleum ether/Et2O (2:1, Rf =

1 0.15) to give 5.2 (82%). H NMR (400 MHz, CDCl3): δ 6.40 (q, 1H), 5.97 (m, 1H), 5.58 (q,

113

1H), 5.40 (q, 1H), 4.33 (m, 1H), 3.22 (s, 3H), 2.31 (m, 2H), 2.12 (m, 2H), 1.60 (2H), 1.1-1.5

+ (16H), 0.83 (3H); HRMS (EI): m/z calcd for C18H30O2 (M -C4H10O2), 278.2246, found

278.2262.

(9Z,11E)-(Perfluorophenyl)methyl-13-((2-methoxypropan-2-yl)peroxy)octadeca-9,11- dienoate (5.3) To CH2Cl2 (3 mL) was added 5.2 (15 mg, 0.039 mmol), pentafluorobenzyl bromide (10 mg, 0.039 mmol) and triethylamine (15 mg, 0.15 mmol), and the mixture was stirred until the reaction was complete to give HPODE-PFB ketal 5.3. Then the reaction mixture was extracted with diethyl ether and washed with aqueous sodium sulfate. The extract was dried, filtered, and concentrated under reduced pressure to afford a colorless oil.

The crude product was separated by flash chromatography with petroleum ether/Et2O (5:1, Rf

1 = 0.21) to give pure 5.3 (14.7 mg, 85% yield). H NMR (400 MHz, CDCl3): δ 6.43 (q, 1H),

5.97 (m, 1H), 5.58 (q, 1H), 5.41 (q, 1H), 5.16 (s, 2H), 4.35 (m, 1H), 3.22 (s, 3H), 2.31 (m,

2H), 2.12 (m, 2H), 1.60 (2H), 1.1-1.5 (16H), 0.83 (3H); HRMS (EI): m/z calcd for

+ C25H31F5O2 (M -C4H10O3), 458.2244, found 458.2248.

2-((2E, 4E)-Hexa-2,4-dien-1-yloxy)tetrahydro-2H-pyran (5.8) To a solution of 2,4-hexadien-

1-ol 5.7 (50 mg) in CH2Cl2 (5.0 mL), was added 3,4-dihydro-2H-pyran (55 mg) and amberlyst 15. The solution was stirred for about 1 hour. The resulting mixture was filtered through silica gel and extracted with CH2Cl2. The extract was dried, filtered, and

114 concentrated under reduced pressure to afford colorless oil. The crude product was separated

1 by flash chromatography with petroleum ether/Et2O (40 : 1, Rf = 0.20) (yield 86%). H NMR

3 3 (400 MHz, CDCl3): δ 6.21 (q, 1H, J = 16.0 Hz), 6.05 (q, 1H, J = 16.0 Hz), 5.7 (m, 2H), 4.62

(t, 1H), 4.25 (m, 1H), 4.0 (m, 1H), 3.91 (1H), 3.50 (1H), 1.5-1.9 (m, 9H); HRMS (EI): m/z

+ calcd for C11H18O2 (M ), 182.1307, found 182.1310.

(E)-2-((3-Bromo-2-(tert-butylperoxy)hex-4-en-1-yl)oxy)tetrahydro-2H-pyran (5.11). To a solution of 5.8 (11.3 mg, 0.062 mmol) in CH2Cl2 (5 mL) was added NBS (11.0 mg, 0.95 equivalent, 0.058 mmol) and NaHCO3 (3.0 mg, 0.036 mmol), followed 18 µL (5M, 1.5 eq) of t-butyl hydroperoxide. The mixture was stirred at RT overnight and then diluted with hexane- ether (8: 2). The solvents were removed and the crude product was flash chromatographed on silica gel eluting with petroleum ether and ether (20 : 1) (Rf = 0.25) to afford 5.11 (16.6 mg,

1 3 3 yield 76%). H NMR (400 MHz, CDCl3): δ 5.71 (m, 1H, J=16.0 Hz), 5.37 (m, 1H, J = 16.0

Hz), 5.04 (m, 1H), 4.38 (1H), 3.91 (3H), 3.62 (1H), 3.42 (m, 1H), 1.95 (1H), 1.85 (1H), 1.77

13 (3H), 1.56 (3H), 1.45 (1H), 1.24 (9H, OC(CH3)3); C NMR (100 MHz, CDCl3): δ 131.40,

128.96, 108.83, 81.46, 80. 65, 69.59, 68.66, 52.82, 28.70, 26.65, 25.27, 17.95; HRMS (EI):

+ m/z calcd for C11H18BrO2 (M -O2C4H9), 261.0490, 263.0470, found, 261.0496, 263.0462.

115

5.1.5 References

1. Moritaa, M.; Tokita, M. Lipids 2006, 41, 91-95.

2. Nicholls, S. J.; Hazen, S. L. J Lipid Res 2009, 50 Suppl, S346-351.

3. Min, D. B.; Boff, J. M. Comprehensive Rev Food Sci Food Safety (CRFSFS) 2002, 1, 58-

72.

4. Porter, N. A. Acc Chem Res 1986, 19, 262-268.

5. Greer, A. Acc Chem Res 2006, 39, 797-804.

6. Esterbauer, H. Aldehydic products of lipid peroxidation; Academic Press: London, 1982.

7. Wilcox, A. L.; Marnett, L. J. Chem Res Toxicol 1993, 6, 413-416.

8. Yadagiri, P.; Lumin, S.; Mosset, P.; Capdevila, J.; Falck, J. R. Tetrahedron Lett 1986, 27,

6039-6040.

9. Gardner, H. W.; Bartelt, R. J.; Weisleder, D. Lipids 1992, 27, 686-689.

10. Tokuyasu, T.; Masuyama, A.; Nojima, M.; McCullough, K. JOrg Chem 2000, 65, 1069-

1075.

11. Cookson P.G.; Davies, A.G.; Roberts, B.P. J Chem Soc Chem Commun. 1976, 24, 1022-

1025.

116

Part B: A Novel Method for the Selective Cleavage of DTBMS Esters

5.2.1 Background

In the past four decades, various trialkylsilyls, such as trimethlysilyl (TMS), triethylsilyl (TES ), t-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), have been among the most frequently used protecting groups for oxygen and nitrogen containing functionality. 1 Trialkylsilyl groups are widely used for the protection of alcohols and carboxylic acids due to the fact that they can be synthesized and removed very selectively under mild conditions. For example, silyl ethers can be removed by treatment with fluoride

2 3 4 5 6 + - 7,8 ion, HF, acetic acid, K2CO3, citric acid, FeCl3, and Bu4N F with the optimal choice depending on the various alkyl groups on silicon. For removal of silyl ester protecting groups many of the methods applied to alcohol deprotection are also applicable to carboxylic acids.

The various alkyl silyl protecting groups exhibit useful differences in stability of their silyl ethers. Steric hindrance around the silicon atom influences the stability and utility of various trialkylsilyl groups for the protection of hydroxyl and carboxyl groups. Their hydrolytic cleavage rates vary dramatically, as do their stability toward various reaction environments. Stability of the silyl ether increases with increasing steric bulk of R group (i.e.

Me < Et < iPr, etc.).9,10 Some larger bulky groups such as di-tert-butylmethylsilyl

(DTBMS),11 di-tert-butylisobutyl silyl (BIBS)12 and tri-tert-butylsilyl13, 14 have been developed. Tri-tert-butylsilyl is difficult to prepare, install and deprotect due to its very bulky alkyl group. The utility of BIBS for the protection of functional groups in chemical synthesis has been discussed.12

DTBMS, developed in Dr. Salomon’s group, has been successfully applied to the

11 protection of a carboxylic acid in the synthesis of levuglandin D2. In Chapter 4, DTBMS

117 triflate was used to protect a carboxylic acid. We discovered that ethylenediamine can cleave the silyl ether of a DTBMS protected carboxylic acid (Scheme 5.2.1). Thus, during the reduction of alkyne 4.18 to alkene 4.19, most of 4.19 was further hydrolyzed to carboxylic acid 4.20. To the best of our knowledge, this is the first example of the use of ethylenediamine for the cleavage of a silyl ester. In this chapter, we will discuss the synthesis of DTBMS triflate, its use to protect carboxylic acids, and the efficacy of ethylenediamine to deprotect silyl TBDMS and DTBMS esters.

Scheme 5.2.1 Reduction of 4.18 to 4.19 by H2 and ethylenediaimine.

118

5.2.2 Results and Discussion

DTBMS triflate is available in two steps from di-tert-butylchlorosilane (Scheme

5.2.2). Di-tert-butylmethylsilane (DTBMSH) can be obtained by refluxing methyllithium and di-tert-butylchlorosilane at 50 °C for 3 days. And then quenching the solution with an aqueous solution of ammonium chloride.15 Alternatively, DTBMSH can be prepared from methyldichlorosilane and tert-butyllithium in 85% yield at room temperature.16 This method is better considering the reaction temperature and time compared with using di-tert- butylchlorosilane. Reaction of trifluoromethanesulfonic acid with di-tert-butylmethylsilane at 4 °C under an atmosphere of argon followed by warming to room temperature and stirring overnight delivers DTBMS triflate that is isolated by distillation under reduced pressure.17

Bp. 63-65 °C/15.0 mmHg. DTBMS triflate was previously described as fuming liquid.11

However, I found that DTBMS triflate is a colorless non-fuming liquid.

Scheme 5.2.2 Two synthetic methods to prepare DTBMSH and DTBMS triflate

DTBMS esters were prepared in high yield (above 95%) from carboxylic acids, one equivalent of DTBMS triflate and three equivalents of anhydrous triethylamine in dry THF at room temperature. To explore the application of DTBMS esters, four representative DTBMS protected carboxylic acids were synthesized (Table 5.2.1). These DTBMS carbonates were treated with potassium carbonate, formic acid, ethylenediamine and ethanolamine to check their chemoselectivity. The result of the deprotection reactions are shown in Table 5.2.1.

119

Table 5.2.1 Cleavage of DTBMS esters with various reagents in 95% ethanol

48 h: 40% SM 24 h 12 h 24h: 50% SM 72 h: no SM 48 h: no SM

6 days: 40% SM 2 days: 50% 24 h 48h SM 5 days: no SM K2CO3 24 h: 20% SM 24h: 30% SM 24h 24h 48 h: no SM 48 h: no SM Formic No reaction. Still 4 days: 50% 24 h: 40% SM 24 h acid SM SM 48h: 10% SM SM: Starting Material

As can be seem from the data in Table 5.2.1, ethylenediamine can cleave all four substrates, albeit with different reaction rates, and the carboxylic acid is the only product. As expected, DTBMS esters of alkyl and vinyl carboxylic acids 5.2.2, 5.2.3 and 5.2.4 reacted much faster than the aryl DTBMS ester 5.2.1. Ethylenediamine can also fully hydrolyze

TBDMS heptanoate in 24 hours. These results indicate that ethylenediamine can generally be used for the deprotection of DTBMS esters.

The rate of cleavage of DTBMS esters by a less basic reagent, ethanolamine, was also examined. The reactions with ethanolamine were slower than those with ethylenediamine. It is most effective for alkyl DTBMS esters, such as 5.2.3 and 5.2.4, while for an aryl DTBMS ester 5.2.1, 40% of starting material still remained after 6 days.

DTBMS benzoate 5.2.1 was unaffected in formic acid. Formic acid can cleave only part of alkyl silyl esters 5.2.2 and 5.2.3 probably due to steric encumbrance of diphenyl group of 5.2.3 or the conjugated double bond with carbonate which decreased the sensitivity

120 of 5.2.2 toward formic acid. By contrast, acetic acid is a weaker acid than formic acid, and the TBDMS protected prostaglandin E2 carboxyl group was converted to prostaglandin E2 by

18 exposure to acetic acid-THF-H2O with ratio of 3:1:1 at 25 °C for 20 hours. As a result,

DTBMS carboxylic esters are stable derivatives which should find important applications in organic synthesis. Potassium carbonate is also an effective deprotection reagent for DTBMS esters for both aryl and alkyl carboxylic acid silyl esters as shown in Table 5.2.1.

121

5.2.3 Conclusions

In this part of Chapter 5, we have discussed the synthesis of DTBMS triflate, which can be used to protect carboxylic acid to give DTBMS carbonate. Different kinds of reagents were applied to cleave DTBMS esters. Ethylenediamine can generally be used in the deprotection of DTBMS ester in good yields. The pKa of ethylenediamine is 9.98, and the pKa of ethanolamine is 9.50.19 The milder base ethanolamine can also remove the DTBMS group from alkyl silyl esters. Therefore, this novel method should find application in organic synthesis. Potassium carbonate is also effective for deprotecting both aryl and alkyl DTBMS esters.

122

5.2.4 Experimental Procedures

General procedure to synthesize DTBMS esters.

One equivalent of carboxylic acid in anhydrous THF was treated with dry triethylamine (3.0 equiv.) at room temperature under argon. And then DTBMS triflate (1.05 eq.) was added dropwise. After about 30 min, the solution was concentrated in vacuo and the residue was extracted with diethyl ether. The organic layers were combined and washed with water, brine, dried over anhydrous magnesium sulfate, filtered and concentrated by rotary evaporation to afford crude product. The crude product was separated by flash chromatography with hexanes and diethyl ether.

p-Anisic acid, DTBMS ester (5.2.1). Yield = 95%; Rf = 0.20 (hexanes/diethyl ether = 20/1);

1 H NMR (400 MHz, CDCl3): δ 8.01 (d, 2H), 7.92 (d, 2H), 3.83 (s, 3H), 1.06 (s, 18H), 0.42 (s,

13 3H); C NMR (100 MHz, CDCl3): δ 167.1, 164.3, 133.2, 114, 55.8, 28.2, 20.5, -7.4; HRMS

+ (EI): m/z calcd for C16H25O3Si (M -CH3), 293.1573 found 293.1581

Furylacrylic acid, DTBMS ester (5.2.2). Yield = 95%; Rf = 0.22 (hexanes/diethyl ether =

1 20/1) ; H NMR (400 MHz, CDCl3): δ 7.69 (1H), 7.51 (d, 2H, J = 15.8Hz), 7.40 (1H), 6.60

13 (1H), 6.12 (d, 1H, J= 15.8 Hz), 0.96 (s, 18H), 0.36 (s, 3H); C NMR (100 MHz, CDCl3): δ

123

163.39, 144.61, 144.58, 134.95, 122.86, 120.42, 107.75, 27.74, 20.67, -7.16; HRMS (EI):

+ m/z calcd for C15H23O3Si (M -CH3), 279.1416, found 279.1428.

Diphenylacetic acid, DTBMS ester (5.2.3). Yield = 88%; Rf = 0.20 (hexanes/diethyl ether =

1 20/1); H NMR (400 MHz, CDCl3): δ 7.35 (m, 5H), 5.01 (s, 1H), 0.96 (s, 18H), 0.37 (s, 3H);

13 C NMR (100 MHz, CDCl3): δ 139.25, 129.03, 128.71, 127.32, 59.57, 27.55, 20.47, -7.40;

+ HRMS (EI): m/z calcd for C23H33O2Si (MH ), 369.2250, found 369.2255.

8-Hydroxy-5-octynoic acid, DTBMS ester (5.2.4). Yield = 68 %; Rf =0.3 (hexanes : diethyl

1 ether = 1:5) ; H NMR (400 MHz, CDCl3): δ 4.18 (d, 2H), 2.40 (t, 2H), 2.23 (t, 2H), 1.78 (m,

13 2H), 0.96 (s, 18H), 0.37 (s, 3H); C NMR (100 MHz, CDCl3): δ 173.02, 85.33, 79.41, 51.50,

35.18, 27.70, 24.14, 20.47, 18.38, -7.30; NMR spectra were the same as reported.11

General procedure for the deprotection of DTBMS esters

To one equivalent of DTBMS ester in 95% ethanol was added four to ten equivalent of ethylenediamine or ethanolamine. The mixture was stirred at room temperature. The progress of the reaction is detected by TLC exposing to UV light and TLC developing with iodine. .

The carboxylic acid is recovered by adding excessive citric acid and extracted with ethyl

124 acetate. The extract was then washed with brine, water and dried with Na2SO4 and concentrated by rotary evaporation. The structures of recovered carboxylic acids were confirmed by 1H NMR. The yields of carboxylic acids were 70-94.6%.

125

5.2.5 References

1. Nelso, T.D.; Crouch, R.D. Synthesis 1996, 1031-1069.

2. Mascarenas, J. L.； Mourino, A.; Castedo L. J Org Chem 1986, 51, 1269–1272.

3. Toshima, K.; Tatsuta, K.; Kinoshita, M. Tetrahedron Lett 1986, 27, 4741-4744.

4. Hurst, D.T.; McInnes, A. G. Can J Chem 1965, 43, 2004-2011.

5. Bundy G.L.; Peterson, D. C. Tetrahedron Lett 1978, 19, 41-44.

6. Cort, A. D. Synth Commun 1990, 20, 757-760.

7. Aizpurua, J. M.; Cossio, F. P.; Palomo, C. J Org Chem 1986, 51, 4941–4943.

8. Corey, E. J.; Snider, B. B. J Am Chem Soc 1972, 94, 2549–2550.

9. Shimizu, N.; Takesue, N.; Yasuhara, S.; Inazu, T. Chem Lett 1993, 22, 1807-1810.

10. Banfi, L.; Guanti, G.; Zannetti, M. Tetrahedron Lett 1996, 37, 521-524.

11. Bhide, R. S.; Levison, B. S.; Sharma, R. B.; Ghosh, S.; Salomon. R. G. Tetrahedron Lett

1986, 27, 671-674.

12. Liang, H.; Hu, L.; Corey E. J. Org Lett 2011, 13, 4120-4123.

13. Dexheimer, E. M.; Spialter, L. Tetrahedron Lett 1975, 16, 1771-1772.

14. Weidenbruch, M.; Peter, W. Angew Chem Int Ed Engl 1975, 14, 642-643.

15. Doyle, M. P.; West, C.T. J Am Chem Soc 1975, 97, 3777-3782.

16. Barton, T.; Tully, C. J Org Chem 1978, 43, 3649-3653.

17. Chadeayne, A. R.; Wolczanski, P. T.; Lobkovsky, E. B. Inorganic Chem 2004, 43, 3421-

3432.

18. Corey, E. J.; Venkateswarlu. A. J Am Chem Soc 1972, 94, 6190-6191.

19. Hall, H. K. J Am Chem Soc 1957, 79, 5441-5444.

126

Appendix

127

Figure 2.1S 1H NMR spectrum of cis-non-3-enal (2.14)

Figure 2.2S 1H NMR spectrum of (1E,3Z)-nona-1,3-dien-1-yl hexanoate (2.15E), (1Z,3Z)- nona-1,3-dien-1-yl hexanoate (2.15Z)

128

Figure 2.3S 13C NMR spectrum of (1E,3Z)-nona-1,3-dien-1-yl hexanoate (2.15E), (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-Epoxy- 1(Z)-nonen hexanoate (2.13Z)

129

0.93 1.00 0.95 0.97 17.40 6.28

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 f1 (ppm) Figure 2.5S 1H NMR spectrum of 4-hydroxy-2-nonenal (HNE)

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)

Figure 3.2S 13C NMR spectrum of 3,4-dichloro-5-hydroxy-5-methyl-furanone (3.2)

131

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) Figure 3.3S 1H NMR spectrum of 5-((tert-butyldimethylsilyl)oxy)-3,4-dichloro-5- methylfuran-2(5H)-one (3.3)

Figure 3.4S 13C NMR spectrum of 5-((tert-butyldimethylsilyl)oxy)-3,4-dichloro-5- methylfuran-2(5H)-one (3.3)

132

Figure 3.5S 1H NMR spectrum of 3, 4-dibutylfuran-2, 5-dione (3.9)

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)

Figure 3.8S 13C NMR spectrum di-t-butyl 2-allyl-3-butylmaleate (3.15)

134

Figure 3.9S 1H NMR spectrum of 3-allyl-4-butylfuran-2,5-dione (3.16)

Figure 3.10S H-H COSY spectrum of 3-allyl-4-butylfuran-2,5-dione (3.16)

135

1.00 2.14 0.91 1.18 1.13 1.11 1.23 3.76 5.96 4.40

Figure 3.11S 1H NMR spectrum of 4-allyl-3-butyl-5-hydroxy-5-methylfuran-2(5H)-one (3.17a) and 3-allyl-4-butyl-5-hydroxy-5-methylfuran-2(5H)-one (3.17b)

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm) Figure 3.12S 13C NMR spectrum of 4-allyl-3-butyl-5-hydroxy-5-methylfuran-2(5H)-one (3.17a) and 3-allyl-4-butyl-5-hydroxy-5-methylfuran-2(5H)-one (3.17b)

136

Figure 3.13S H-H COSY spectrum of 4-allyl-3-butyl-5-hydroxy-5-methylfuran-2(5H)-one (3.17a) and 3-allyl-4-butyl-5-hydroxy-5-methylfuran-2(5H)-one (3.17b)

Figure 3.14S 1H NMR spectrum of di-tert-butyl 2-allyl-3-((E)-3-((tert- butyldimethylsilyl)oxy)oct-1-en-1-yl)maleate (3.18)

137

Figure 3.15S 13C NMR spectrum of di-tert-butyl 2-allyl-3-((E)-3-((tert- butyldimethylsilyl)oxy)oct-1-en-1-yl)maleate (3.18)

Figure 3.16S 1H NMR spectrum of (E)-3-allyl-4-(3-hydroxyoct-1-en-1-yl)furan-2,5-dione (3.20)

138

Figure 3.17S H-H COSY spectrum of (E)-3-allyl-4-(3-hydroxyoct-1-en-1-yl)furan-2,5-dione (3.20)

Figure 3.18S 1H NMR spectrum of 2-allyl-3-((E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1- yl)maleic acid (3.22)

139

Figure 3.19S H-H COSY spectrum of 2-allyl-3-((E)-3-((tert-butyldimethylsilyl)oxy)oct-1- en-1-yl)maleic acid (3.22)

Figure 3.20S 1H NMR spectrum of (E)-3-allyl-4-(3-hydroxyoct-1-en-1-yl)furan-2,5-dione (3.19)

140

Figure 3.21S 13C NMR spectrum of (E)-3-allyl-4-(3-hydroxyoct-1-en-1-yl)furan-2,5-dione (3.19)

Figure 3.22S H-H COSY spectrum of (E)-3-allyl-4-(3-hydroxyoct-1-en-1-yl)furan-2,5-dione (3.19)

141

1.00 2.16 2.36 2.38 3.93 4.43 3.72

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) Figure 3.23S 1H NMR spectrum of 2-allyl-3-butylmaleic acid

2.14 1.00 1.03 2.14 2.22 2.23 12.48 7.98 3.75

Figure 3.24S 1H NMR spectrum of (Z)-tert-butyl 2-(2-oxo-6-pentyl-2H-pyran-3(6H)- ylidene)pent-4-enoate (3.21)

142

Figure 3.25S 13C NMR spectrum of (Z)-tert-butyl 2-(2-oxo-6-pentyl-2H-pyran-3(6H)- ylidene)pent-4-enoate (3.21)

Figure 3.26S H-H COSY spectrum of (Z)-tert-butyl 2-(2-oxo-6-pentyl-2H-pyran-3(6H)- ylidene)pent-4-enoate (3.21)

143

Figure 4.1S 1H NMR spectrum of 7-((tert-butyldimethylsilyl)oxy)hept-2-yn-1-ol (4.6)

86.46 78.75 77.67 77.25 76.82 62.85 51.52 32.10 26.22 26.16 25.24 18.76 18.55 -5.09

95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 f1 (ppm)

Figure 4.2S 13C NMR spectrum of 7-((tert-butyldimethylsilyl)oxy)hept-2-yn-1-ol (4.6)

144

Figure 4.3S H-H COSY spectrum of 7-((tert-butyldimethylsilyl)oxy)hept-2-yn-1-ol 1.00 1.01 1.02 1.63 2.04 4.53 2.88

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 (4.6) f1 (ppm) Figure 4.4S 1H NMR 7-((tert-butyldimethylsilyl)oxy)hept-2-yn-1-yl acetate (4.7)

145

Figure 4.5S 13C NMR 7-((tert-butyldimethylsilyl)oxy)hept-2-yn-1-yl acetate (4.7)

Figure 4.6S 1H NMR spectrum of 7-acetoxyhept-5-ynoic acid (4.8) and 7-hydroxyhept-5- ynoic acid (4.9)

146

Figure 4.7S 13C NMR spectrum of 7-acetoxyhept-5-ynoic acid (4.8) and 7-hydroxyhept-5- ynoic acid (4.9)

Figure 4.8S 1H NMR spectrum of methyl-7-hydroxyhept-5-ynoate (4.10)

147

Figure 4.9S 1H NMR spectrum of (Z)-methyl-7-hydroxyhept-5-enoate (4.11)

Figure 4.10S 13C NMR spectrum of (Z)-methyl-7-hydroxyhept-5-enoate (4.11)

148

1.00 0.86 1.93 3.11 2.14 1.95 2.19

Figure 4.11S 1H NMR spectrum of (Z)-methyl-7-bromohept-5-enoate (4.12)

Figure 4.12S 1H NMR spectrum of 4.13

149

Figure 4.13S 13C NMR spectrum of 4.13

Figure 4.14S H-H COSY spectrum of 4.13

150

Figure 4.15S 1H NMR spectrum of 4.14

Figure 4.16S 13C NMR spectrum of 4.14

151

Figure 4.17S H-H COSY spectrum of 4.14

Figure 4.18S 1H NMR spectrum of methyl 7,8-(di-tert-butoxycarbonyl)-11-((tert- butyldimethylsilyl)oxy)-(4Z,7Z,9E)-heptadecatrieneoate (4.15)

152

Figure 4.19S 13C NMR spectrum of methyl 7,8-(di-tert-butoxycarbonyl)-11-((tert- butyldimethylsilyl)oxy)-(4Z,7Z,9E)-heptadecatrieneoate (4.15)

05-17-2012--608-Ag+full-1 #1 RT: 0.00 AV: 1 NL: 3.90E7 T: + p ESI Full ms [150.00-1000.00] 717.00 100

40 Relative Abundance

447.47 476.80 545.07 585.93 605.00 632.27 659.20 701.13 730.93 749.13 799.07 841.93 0 450 500 550 600 650 700 750 800 m/z Figure 4.20S Mass spectrometry 4.15 at full scan mode. M/z: 608+107,109[Ag+] =715, 717

153

Figure 4.21S 1H NMR spectrum of 2-((E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)-3- ((Z)-7-methoxy-7-oxohept-2-en-1-yl)maleic acid.

Figure 4.22S 1H NMR spectrum of methyl (Z)-(7-(4-((E)-3-((tert-butyldimethylsilyl) oxy)oct-1-en-1-yl)-2,5-dioxo-2,5-dihydrofuran-3-yl)hept-5-enoate) (4.16)

154

Figure 4.23S H-H COSY spectrum of methyl (Z)-(7-(4-((E)-3-((tert-butyldimethylsilyl) oxy)oct-1-en-1-yl)-2,5-dioxo-2,5-dihydrofuran-3-yl)hept-5-enoate) (4.16)

Figure 4.24S 1H NMR spectrum of di-tert-butyl(methyl)silyl 7-hydroxyhept-5-ynoate (4.18)

155

Figure 4.25S 13C NMR spectrum of di-tert-butyl(methyl)silyl 7-hydroxyhept-5-ynoate (4.18)

Figure 4.26S 1H NMR spectrum of (Z)-di-tert-butyl(methyl)silyl 7-hydroxyhept-5-enoate (4.19)

156

Figure 4.27S 1H NMR spectrum of di-tert-butyl(methyl)silyl (Z)-7-bromohept-5-enoate (4.20)

Figure 4.28S 13C NMR spectrum of di-tert-butyl(methyl)silyl (Z)-7-bromohept-5-enoate (4.20) 157

1.00 1.02 2.12 2.45 6.08 5.40 0.26 11.05 6.69

Figure 4.29S 1H NMR spectrum of (Z)-7-((tert-butyldimethylsilyl)oxy)hept-2-en-1-yl acetate (4.23)

Figure 4.30S 1H NMR spectrum of (Z)-7-acetoxyhept-5-enoic acid (4.24) and (Z)-7- hydroxyhept-5-enoic acid (4.20)

158

Figure 4.31S 1H NMR spectrum of di-tert-butylmethylsilyl 7,8-(di-tert-butoxycarbonyl)-11- ((tert-butyldimethylsilyl)oxy)-(4Z,7Z,9E)-heptadecatrieneoate (4.25)

Figure 4.32S 1H NMR spectrum of di-tert-butylmethylsilyl 7,8-(di-tert-butoxycarbonyl)-11- ((tert-butyldimethylsilyl)oxy)-(4Z,7Z,9E)-heptadecatrieneoate (4.25)

159

Figure 4.33S 1H NMR spectrum of 4.31

Figure 4.34S H-H COSY spectrum of 4.31

160

11-30-2012-simadzu-portion-2 #1-27 RT: 0.02-0.44 AV: 27 NL: 2.20E5 T: + p ESI Full ms [200.00-1000.00] 859.27 100

40 Relative Abundance

10 813.53 519.40 641.60 669.60 713.13 763.67 449.33 472.47 553.13 613.60 911.53 939.53 967.60 0 450 500 550 600 650 700 750 800 850 900 950 1000 m/z Figure 4.35S Mass spectrometry 4.25 at full scan mode. M/z: 750+107,109[Ag+] =857, 859

Figure 5.1S 1H NMR spectrum of (9Z,11E)-13-((2-methoxypropan-2-yl) peroxy) octadeca- 9,11-dienoic acid (5.2)

161

Figure 5.2S 1H NMR spectrum of (9Z,11E)-(perfluorophenyl)methyl-13-((2- methoxypropan-2-yl)peroxy)octadeca-9,11-dienoate (5.3)

Figure 5.3S 1H NMR spectrum of 2-((2E,4E)-hexa-2,4-dien-1-yloxy)tetrahydro-2H-pyran (5.8)

162

Figure 5.4S 13C NMR spectrum of 2-((2E,4E)-hexa-2,4-dien-1-yloxy)tetrahydro-2H-pyran (5.8)

Figure 5.5S 1H NMR spectrum of (E)-2-((3-bromo-2-(tert-butylperoxy)hex-4-en-1- yl)oxy)tetrahydro-2H-pyran (5.11) 163

Figure 5.6S 13C NMR spectrum of (E)-2-((3-bromo-2-(tert-butylperoxy)hex-4-en-1- yl)oxy)tetrahydro-2H-pyran (5.11)

Figure 5.7S H-H COSY of (E)-2-((3-bromo-2-(tert-butylperoxy)hex-4-en-1- yl)oxy)tetrahydro-2H-pyran (5.11)

164

Figure 5.8S HMQC spectrum of (E)-2-((3-bromo-2-(tert-butylperoxy)hex-4-en-1- yl)oxy)tetrahydro-2H-pyran (5.11)

Figure 5.9S 1H NMR spectrum of p-anisic acid, DTBMS ester (5.2.1)

165

Figure 5.10S 13C NMR spectrum of p-Anisic acid, DTBMS ester (5.2.1).

Figure 5.11S 1H NMR spectrum of furylacrylic acid, DTBMS ester (5.2.2)

166

144.59 144.56 134.93 122.85 120.41 107.74 77.64 77.22 76.79 27.79 20.67 -7.15

Figure 5.12S 13C NMR spectrum of furylacrylic acid, DTBMS ester (5.2.2) 7.90 19.45 3.00

13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm) Figure 5.13S 1H NMR spectrum of diphenylacetic acid, DTBMS ester (5.2.3)

167

Figure 5.14S 13C NMR spectrum of diphenylacetic acid, DTBMS ester (5.2.3)

Figure 5.15S 1H NMR spectrum of 8-hydroxy-5-octynoic acid, DTBMS ester (5.2.4) 168

173.00 85.32 79.40 77.57 77.25 76.93 51.49 35.17 27.70 24.14 20.47 18.38 -7.29

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm) Figure 5.16S 13C NMR spectrum of 8-hydroxy-5-octynoic acid, DTBMS ester (5.2.4)

169

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