Investigation of Highly-Reducing Polyketide Synthase Enzymes That Produce the Fungal Polyketides Lovastatin, Fumonisin Bj, and Hypothemycin

University of Alberta

Investigation of Highly-Reducing Polyketide Synthase Enzymes that Produce the Fungal Polyketides Lovastatin, Fumonisin Bj, and Hypothemycin

Jesse W.-H Li

A thesis submitted to the Faculty of Graduate Studies and Research

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Department of Chemistry

Spring, 2011

Edmonton, Alberta

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This thesis focuses on the highly-reducing polyketide synthase enzymes that biosynthesize three polyketides: lovastatin, fumonisin Bi, and hypothemycin.

Lovastatin (1), a cholesterol-lowering drug, is made by the polyketide synthase enzymes LovB and LovC in the fungus Aspergillus terreus. LovB and LovC were cloned and expressed in Saccharomyces cerevisiae (strain BJ5464-NpgA).

The catalytic activity of each domain of LovB and its role during biosynthesis was probed in vitro by incubation with a series of cofactors (malonyl-CoA, NADPH,

SAM). Full reconstitution of the biosynthetic pathway was achieved when LovB was incubated with enoyl reductase (ER) LovC, malonyl-CoA, NADPH, and

SAM to furnish dihydromonacolin L 2, an intermediate in the biosynthesis of 1.

Exogenous thioesterase (TE) domain (PKS13 or PKS4) or KOH was required to liberate 2 from LovB. Standards were synthesized to confirm the structures of proposed shunt metabolites from the in vitro assays.

The post-PKS enzyme Fum6p is a cytochrome P450 that hydroxylates the fumonisin carbon chain. Fum6p was expressed and purified from yeast, allowing in vitro experiments to probe its substrate specificity. Incubation of Fum6p with cofactors and the substrates stearic acid or sphinganine did not yield any detectable hydroxylated products. However, synthesis of fumonisin Bi mimic

(2/?,6/?)-2,6-dimethyldecanoic acid (64), and incubation with Fum6p microsomes furnished four monohydroxylated isomeric products. Synthetic standards were prepared to allow identification of the structures from the enzyme assay. Expression and purification of the hypothemycin (105) polyketide synthases Hpm8 and Hpm3 allowed for analysis of the catalytic activity of each enzyme during the biosynthesis of 105. Hpm8 was tested in a series of in vitro assays to furnish three pyrones. The structures of these pyrones were confirmed through comparison with synthetic standards. Full reconstitution of the biosynthetic pathway was achieved by incubation of Hpm3 with Hpm8 and all cofactors to produce dehydrozearalenol (107a). Acknowledgem ents

I would like to acknowledge and express my deepest gratitude to my research supervisor Professor John C. Vederas. His support and encouragement throughout my PhD has allowed me to flourish and mature as a scientist. I am thankful for the bright minds and vibrant personalities in the Vederas group, which has made my time working in the laboratory unforgettable. The rich intellectual environment they have created has prepared me well for the future. I am especially grateful to Dr. David Dietrich and Dr. Jennifer Chaytor for their assistance in proofreading my thesis. I would like to thank the dedicated support staff in the mass spectral services, NMR services, and analytical services for their invaluable assistance with my research projects.

I would like to thank all my collaborators and their gifted graduate students for their assistance and expertise with my various research projects.

I would like to thank all the friends that I have made in the Department of

Chemistry, which has made the time during my PhD truly memorable. I am thankful for the financial support by the University of Alberta. I am indebted to my family, who have supported and encouraged me throughout my life.

Finally, I would like to thank Anita who has provided endless love and support throughout this process. I am eternally grateful for her presence in my life. Table of Contents

1 Chapter 1: Introduction to Polyketides Biosynthesis, and Biosynthetic

Studies on Lovastatin 1

1.1 Introduction 1

1.2 Types of PKS enzymes 8

1.2.1 Type 1 9

1.2.2 Type II 11

1.2.3 Type III 13

1.3 The fungal polyketide lovastatin and its biological properties 15

1.3.1 Statin drugs and inhibition of cholesterol biosynthesis 15

1.3.2 Lovastatin biosynthesis 17

1.4 Results and discussion 26

1.4.1 Expression and purification of LovB and LovC 26

1.4.2 Investigation of the catalytic activity of pure LovB with malonyl-

CoA in the absence of NADPH and SAM 29

1.4.3 In vitro enzyme assay of LovB with malonyl-CoA and NADPH .... 33

1.4.4 In vitro enzyme assay of LovB with malonyl-CoA, NADPH, and

SAM 37

1.4.5 Complete reconstitution of the biosynthetic pathway to

dihydromonacolin L 39

1.4.6 Substrate specificity of LovC 42

1.4.7 Investigation of interactions between LovB and ER enzymes 43 1.4.8 Synthesis of standards to confirm structures of pyrones 4,5, and 7

produced from the various enzyme assays 47

1.4.9 Synthesis of standards to confirm tetra- and pentaketide ketones 9

and 12 generated from the enzyme assay 49

1.4.10 Synthesis of desmethyl-dihydromonacolin L (32) standard to

confirm its structure from the enzyme assay 50

1.4.11 Role of truncated NRPS section in lovastatin biosynthesis 57

1.4.12 In vitro studies with purified LovC 63

1.4.13 In vitro fluorometric enzyme assay 70

2 Chapter 2: Biosynthetic Studies on Fumonisin Bi 76

2.1 Fumonisin Biosynthesis 76

2.2 Cytochrome P450 and Fum6p 81

2.3 Results and discussion 84

2.3.1 In vitro experiments with Fum6p 84

3 Chapter 3: Biosynthetic Studies on Hypothemycin 99

3.1 Hypothemycin Biosynthesis 99

3.2 Results and discussion 103

3.2.1 In vitro experiments with Hpm8 103

3.2.2 In vitro experiments with Hpm8 and Hpm3 107

4 Experimental procedures 114

4.1 General experimental methods 114

4.1.1 Reagents, solvents, and solutions 114

4.1.2 Purification Techniques 114 4.1.3 Instrumentation for compound characterization 115

4.2 Synthesis and characterization of compounds 116

(3E, 5E, 7£)-Nona-3, 5, 7-trien-2-one (9) 116

(2E, 4E, 6£)-Ar-Methoxy-iV-methylocta-2,4, 6-trienamide (10) 117

{IE, 4E, 6E)-Octa-2,4, 6-trienal (11) 118

(3E, 5E, IE, 9£)-Undeca-3, 5, 7,9-tetraen-2-one (12) 119

2-(3-Bromopropyl)-l,3-dioxolane (14) 120

2-[(5E, 7£)-Nona-5, 7-dienyl]-l, 3-dioxolane (16) 121

(6E, 8£)-Deca-6, 8-dienal (17) 122

(2E, 8E, 10£)-Dodeca-2, 8,10-trienal (19) 123

(15, 25,4a/?, 8a5)-2-Methyl-l, 2,4a, 5, 6, 7, 8, 8a-octahydronaphthalene-l-

carbaldehyde (21) 124

Ethyl 3-[( 15,25,4a/?, 8a5)-2-methyl-l, 2, 4a, 5, 6, 7, 8, 8a-

octahydronapthalen-l-yl]acrylate (24) 126

Methyl 3-[(15, 25, 4a/?, 8a5)-2-methyl-l, 2,4a, 5, 6, 7, 8, 8a-

octahydronapthalen-l-yl]propanoate (25) 127

3-[(15,25,4a/?, 8a5)-2-Methyl-l, 2,4a, 5, 6, 7, 8, 8a-octahydronapthalen-l-

yl]propanal (27) 128

(/?)-l-[(15,25,4aR, 8a5)-2-Methyl-l, 2,4a, 5, 6, 7, 8, 8a-

octahydronapthalen-l-yl]hex-5-en-3-ol (28) 130

(/?)-l-[(15,25,4aR, 8a5)-2-Methyl-l, 2,4a, 5, 6, 7, 8, 8a-

octahydronapthalen-l-yl]hex-5-en-3-yl aery late (29) 131

(/?)-6-[2-Ethyl]-5, 6-dihydro-2//-pyran-2-one (30) 133 (1R, AR, 6/?)-4-[2-{(15,2S, 4a/?, 8aS)-2-Methyl-l, 2, 4a, 5,6, 7, 8, 8a- octahydronapthalen-1 -y1} ethyl]-3, 7-dioxabicyclo[4.1.0]heptan-2-one (31)

134

(4Z?, 6i?)-4-Hydroxy-6-[2-{(15,25,4aR, 8aS)-2-methyl-l, 2,4a, 5, 6, 7, 8,

8a-octahydronapthalen-1 -yl}ethy l]tetrahydro-2//-pyran-2-one (32) 135

(£)-5-2-Acetamidoethyl but-2-enethioate (39) 136

(2E, 4jE)-5-2-Acetamidoethyl hexa-2,4-dienethioate (40) 137

(£)-Oct-2-enoic acid (41) 138

(IE, 4E, 6£)-Ethyl 2-methylocta-2,4, 6-trienoate (43) 139

(IE, AE, 6£)-2-Methylocta-2,4, 6-trienoic acid (44) 140

(2E, AE, 6£)-5-2-Acetamidoethyl 2-methylocta-2,4, 6-trienthioate (45) ..141

(2E, AE, 6£)-Ethyl octa-2,4, 6-trienoate (47) 142

(2E, AE, 6£)-Octa-2,4,6-trienoic acid (48) 143

(2E, AE, 6£)-5-2-Acetamidoethyl octa-2,4,6-trienethioate (49) 144

Ethyl 2-[bis(2, 2, 2-trifluorethoxy)phosphoryl]propanoate (51) 145

(2Z, AE, 6£)-Ethyl 2-methylocta-2, 4, 6-trienoate (52) 146

(2Z, AE, 6£)-2-Methylocta-2,4,6-trienoic acid (53) 147

(2Z, AE, 6£)-5,-2-Acetamidoethyl 2-methylocta-2,4, 6-trienethioate (54). 148

(2Z, AE, 6^)-Methyl octa-2, 4, 6-trienoate (58) 149

(2Z, AE, 6E)- Octa-2, 4, 6-trienoic acid (59) 150

(2Z, AE, 6£)-5-2-Acetamidoethyl octa-2,4, 6-trienethioate (60) 151

(i?)-4-Benzyl-3-hexanoyloxazolidin-2-one (67) 152

(i?)-4-Benzyl-3-[(R)-2-methylhexanoyl]oxazolidin-2-one (68) 153 (i?)-2-Methy lhexan-1 -ol (69) 154

(/?)-2-Methylhexanal (70) 155

(i?)-6-Methyldec-4-enoic acid (72) 156

(i?)-6-Methyldec-4-enoyl chloride (73) 157

(/?)-4-Benzyl-3-[(i?)-6-methyldec-4-enoyl]oxazolidin-2-one (74) 157

(/?)-4-Benzyl-3-[(2/?, 6R)-2,6-dimethyldec-4-enoyl]oxazolidin-2-one (75)

159

(2R, 6R)-2, 6-Dimethyldec-4-enoic acid (76) 160

(3R, 5i?)-5-[(lj/?, 2i?)-l-Iodo-2-methylhexyl]-3-methyldihydrofuran-2(3//)- one (77) and (3R, 55)-5-[(15, 2R)-1-Iodo-2-methylhexyl]-3 - methyldihydrofuran-2(3//)-one (78) 161

(3R, 55)-3-Methyl-5-[(/?)-2-methylhexyl]dihydrofuran-2(3//)-one (79).... 163

Lithium (2R, 45, 6/?)-4-hydroxy-2, 6-dimethyldecanoate (80) 164

(3R, 55)-5-[( 1S, 2R)-1 -Hydroxy-2-methylhexyl]-3-methyldihydrofuran-

2(3//)-one (81) 164

0-(15, 2i?)-2-Methyl-l-[(25,4/?)-4-methyl-5-oxotetrahydrofuran-2-yl]hexyl

1//-imidazole-1-caarbothioate (82) 166

(3R, 5i?)-3-Methyl-5-[(^)-2-methylhexyl]dihydrofuran-2(3//)-one (83)... 167

Lithium (2R, 4R, 6/?)-4-hydroxy-2,6-dimethyldecanoate (84) 168

(3R, 5S)-5-[( 1S, 2R)-1 -Hydroxy-2-methylhexyl]-3-methyldihydrofuran-

2(3//)-one (81) and (3R, 5R)-5-[(\R, 2/?)-l-Hydroxy-2-methylhexyl]-3- methyldihydrofuran-2(3//)-one (85) 169 (3R, 55)-5-[(15, 2/?)-l-(Benzyloxy)-2-methylhexyl]-3-methyldihydrofuran-

2(3//)-one (87) and (3R, 5R)-5-[(\R, 2/?)-l-(Benzyloxy)-2-methylhexyl]-3- methyldihydrofuran-2(3//)-one (88) 170

(2R, 45, 55, 6/?)-5-(Benzyloxy)-2, 6-dimethyldecane-l, 4-diol (89) and (2R,

4R, 5R, 6/?)-5-(Benzyloxy)-2, 6-dimethyldecane-l, 4-diol (90) 172

(2R, 45, 55,6/?)-5-(Benzyloxy)-l-(/erf-butyldimethylsilyloxy)-2, 6- dimethyldecane-4-ol (91) 174

0-(2R, 45, 55, 6/?)-5-(Benzyloxy)-l-(te/*/-butyldimethylsilyloxy)-2, 6- dimethyldecane-4-yl 1//-imidazole-1-carbothioate (92) 175

(2/?, 5R, 6/?)-5-(Benzyloxy)-2,6-dimethyldecan-l-ol (93) 176

(2R, 5R, 6/?)-5-(Benzyloxy)-2, 6-dimethyldecanal (94) 177

(2R, 5R, 6/?)-5-(Benzyloxy)-2,6-dimethyldecanoic acid (95) 178

(3R, 6/?)-6-[(/?)-Hexan-2H-yl]-3-methyltetrahydro-2//-pyran-2-one (96). 179

Lithium (2R, 5R, 6/?)-5-hydroxy-2,6-dimethyldecanoate (97) 180

(2/2, 4R, 5R, 6/?)-5-(Benzyloxy)-l-(/er?-butyldimethylsilyloxy)-2, 6- dimethyldecane-4-ol (98) 181

0-(2R, 4R, 5R, 6/?)-5-(Benzyloxy)-l-(ter/-butyldimethylsilyloxy)-2, 6- dimethyldecane-4-yl 1//-imidazole-1-carbothioate (99) 182

(2R, 5R, 6/?)-5-(Benzyloxy)-2, 6-dimethyldecan-l-ol (100) 183

(2R, 55, 6/?)-5-(Benzyloxy)-2, 6-dimethyldecanal (101) 183

(2R, 55, 6/?)-5-(Benzyloxy)-2, 6-Dimethyldecanoic acid (102) 184

(3R, 65)-6-[(/?)-Hexan-2-yl]-3-Methyltetrahydro-2//-pyran-2-one (103).. 185

Lithium (2R, 5R, 6/?)-5-hydroxy-2,6-dimethyldecanoate (104) 186 4-(Benzyloxy)-6-Methyl-2//-pyran-2-one (110) 186

4-(Benzyloxy)-6-(2-oxopropyl)-2//-Pyran-2-one (111) 187

(S)- 4-(Benzyloxy)-6-(2-hydroxypropyl)-2//-Pyran-2-one (112) 188

(S)- 4-6-(2-Hydroxypropyl)-2//-pyran-2-one (113) 189

4.3 Biological procedures 190

4.3.1 Expression and purification of LovB 190

4.3.2 Mass spectrum analysis of LovB fragments 192

4.3.3 Expression and purification of LovC 192

4.3.4 In vitro enzyme assays for LovB and LovC 194

5 References 196

6 Appendix A1 - LC-MS/MS spectrum of holo LovB fragment

IDQGVDSLGAVTVGTW containing peaks corresponding to the two Ppant elimination products 219

7 Appendix A2 - Crystal Data for (li?, 4R, 6i?)-4-[2-{(15,2S, 4aR, 8aS)-2-

Methyl-1, 2, 4a, 5, 6, 7, 8, 8a-octahydronapthalen-l-yl}ethyl]-3, 7- dioxabicyclo[4.1.0]heptan-2-one (31) 220

8 Appendix A3 - Crystal Data for (4R, 61?)-4-Hydroxy-6-[2-{(lS', 2S, 4aR,

8aS)-2-methyl-l, 2, 4a, 5, 6, 7, 8, 8a-octahydronapthalen-l-yl}ethyl]tetrahydro-

2//-pyran-2-one (32) 229

9 Appendix A4 - Crystal Data for (3R, 5R)-5-[(\R, 2/?)-l-Iodo-2-methylhexyl]-

3-methyldihydrofuran-2(3//)-one (77) 238 10 Appendix A5 - Crystal Data for (1/?, 2i?)-2-methyl-l-[(2/?, 4/?)-4-methyl-5- oxotetrahydrofuran-2-yl]hexyl-4-bromobenzoate (116) 246 List of Figures

Figure 1: Examples of polyketides, displaying prominent biological activities 2

Figure 2: FAS crystal structure. (A) Ribbon structure. (B) Domain illustration

(Adapted from Maier et. al.) 8

Figure 3: Different types of PKS enzymes and their subclasses2 9

Figure 4: Lovastatin and its semi-synthetic analogues 15

Figure 5: Lovastatin biosynthetic gene cluster in A. terreus.4 19

Figure 6: Chymotrypsin digestion of LovB and LC-MS/MS analysis of fragments

Figure 7: LC-MS data for proposed pyrone 3. (A) LC chromatogram. (B) UV-

spectrum 30

Figure 8: Three factors providing superior enantiocontrol in intramolecular Diels-

Alder reaction in the presence of organocatalyst 20. (A) Reversible imine

formation, which lowers the energy of the LUMO and decreases the energy

difference between the HOMO and LUMO. (B) Controlled formation of E-

isomer due to steric hindrance. (C) Sterically favorable approach of diene

from bottom face 55

Figure 9: Proposed transition state for regioselective epoxidation of 30 using

TBHP 55 Figure 10: Proposed mechanism for regioseiective opening of epoxide 31 in the

presence of phenyl selenide 56

Figure 11: General architecture of lovastatin and equisetin domains and their

respective polyketide structure 57

Figure 12: Possible off-loading mechanism of polyketides via amide bond

formation in (A) equesetin and (B) 2 59

Figure 13: Summary of LovB-AC in vitro assays with various cofactors and their

products 62

Figure 14: Structures of coenzyme A and N-acetylcysteamine 63

Figure 15: Mechanism for HWE reaction when EWG group is attached to the

phosphonate 67

Figure 16: Substrates for LovC in vitro assay 69

Figure 17: Chemical structures of fumonisin B series 75

Figure 18: Fumonisin gene cluster in F. verticillioides 75

Figure 19: GC-MS analysis of reaction mixture. (A) Fum6p microsomes

incubated with 64 and (B) Fum6p microsomes alone [negative control] 87

Figure 20: (A) Structures and masses of 64 and four possible TMS-protected

hydroxylated isomers. Low-resolution MS of (B) the four new peaks

detected from Fum6p incubated with 64. (C) Peaks in the negative control

with Fum6p alone 88 Figure 21: Proposed mechanism of iodolactonization to form iodo-lactones 77

and 78 91

Figure 22: Resorcylic acid lactones 97

Figure 23: Hypothemycin gene cluster in H. subiculosus 100

Figure 24: Domain architecture of the HR-PKS (Hpm8) and the NR-PKS (Hpm3)

100 List of Schemes

Scheme 1: Generic steps in fatty acid biosynthesis by a fatty acid synthase

enzyme (FAS)1 3

Scheme 2: General biosynthesis of fatty acids and polyketides3 6

Scheme 3: Generic biosynthetic scheme for modular type I PKS containing a

>\ loading and three chain extension/modifying modules 11

Scheme 4: Schematic diagram of aromatic polyketide biosynthesis by type II

PKS2 12

Scheme 5: Schematic diagram of chalcone biosynthesis by type III PKS34 14

Scheme 6: Inhibition of HMG-CoA reductase by lovastatin preventing the

formation of mevalonic acid en route to the biosynthesis of cholesterol.1... 16

Scheme 7: Generic scheme for the biosynthesis of dihydromonacolin L (2) and

Lovastatin (l).1 17

Scheme 8: Isotopic labeling pattern of Lovastatin through isotopic feeding

experiments in A. terreus 17

Scheme 9: Proposed biosynthesis of 2 by LovB and LovC1 21

Scheme 10: Off-loading of incorrect hexa- and heptaketide-pyrones from LovB

in the absence of LovC4 23 Scheme 11: Proposed post-PKS steps that form intermediates monacolin L and

monacolin J through the actions of LovA, LovD, and LovF to construct 11 24

Scheme 12: Proposed mechanisms for the formation of the two Ppant eliminated

LovB products and their respective Ppant elimination product 28

Scheme 13: In vitro assay of LovB with malonyl-CoA in the absence of NADPH

and SAM. Inactive domains are shown in red and non-functional domains

are coloured white. Pyrone 3 is the product of this system 30

Scheme 14: Self-loading of malonyl-CoA onto ACP and comparison of normal

programmed steps with derailment process in the absence of NADPH and

SAM 32

Scheme 15: In vitro assay of LovB with malonyl-CoA and NADPH produces

pyrones 4-6 and hydrolysis products 9 and 12 33

Scheme 16: Comparison of normal programmed steps with derailment process in

the absence of SAM and LovC to produce heptaketide-pyrone 5 35

Scheme 17: Proposed mechanism for the formation of hydrolytic ketones 9 and 12

Scheme 18: Proposed formation of pentaketide-pyrone 4 and heptaketide-pyrone

6 when LovB is incubated with malonyl-CoA and NADPH 36

Scheme 19: In vitro assay of LovB with malonyl-CoA, NADPH, and SAM to

produce methylated pyrones 7 and 8 37 Scheme 20: In the absence of LovC, LovB produces the methylated pyrone 7

instead of biosynthesizing 2 38

Scheme 21: Proposed formation of methylated pyrone 8 in the absence of LovC

Scheme 22: Complete reconstitution of the biosynthesis of 2 requires thioester

cleavage by base hydrolysis 40

Scheme 23: Complete reconstitution of the biosynthesis of 2 requires thioester

cleavage by TE domains (PKS13 or PKS4) 41

Scheme 24: The three stages of LovC reduction: Formation of (A) tetra-, (B)

penta-, and (C) heptaketides 42

Scheme 25: In vitro assay of LovB with malonyl-CoA, NADPH, and LovC shows

the formation of shunt products 5,6, 9, and 12 instead of the predicted 32.43

Scheme 26: In vitro assay of LovB with MlcG, malonyl-CoA, and NADPH

shows the formation of 32 following base hydrolysis or treatment with the

PKS13 TE domain 44

Scheme 27: Fates of methylated and unmethylated tetraketides in the presence of

LovC or MlcG, an ER enzyme from P. citrinum 45

Scheme 28: Synthesis of (A) pentaketide pyrone 4 and (B) hexaketide pyrone 5 46

Scheme 29: Synthesis of methylated-hexaketide pyrone 7 47 Scheme 30: Synthesis of (A) tetraketide ketone 9 and (B) pentaketide ketone 12

Scheme 31: Synthesis of 21, an intermediate in the synthesis of desmethyl-

dihydromonacolin L (32) 50

Scheme 32: Synthesis of desmethyl-dihydromonacolin L (32) 52

Scheme 33: General scheme of fluorometric in vitro assay 62

Scheme 34: Synthesis of (A) diketide 39 (B) triketide 40, and (C) tetraketide

analogue 41 63

Scheme 35: Synthesis of fr-a/w-methylated tetraketide 45 64

Scheme 36: Synthesis of unmethylated tetraketide 49 65

Scheme 37: Synthesis of c/s-methylated tetraketide 54 66

Scheme 38: Synthesis of c/s-unmethylated tetraketide 60 68

Scheme 39: Proposed PLP-dependant mechanism of off-loading 18-carbon

fumonisin backbone from Fumlp 79

Scheme 40: Proposed post-PKS steps to give fiimonisins B1-B4 81

Scheme 41: Proposed mechanism for hydroxylation 82

Scheme 42: In vitro experiments with Fum6p microsomes and stearic acid or

sphinganine 85 Scheme 43: Synthesis of intermediate toward left-side portion of fumonisin B]

mimic 64 85

Scheme 44: Synthesis of left-side portion fumonisin Bi mimic 64 86

Scheme 45: The four proposed hydroxylated compounds from the Fum6p

enzyme assay 88

Scheme 46: Synthesis of left-side portion of fumonisin mimic attached to ACP

(65) 89

Scheme 47: No hydroxylated products are observed from the Fum6p enzyme

assay with 65 90

Scheme 48: Synthesis of intermediate 76 en route to predicted Fum6p

hydroxylated isomer 80 91

Scheme 49: Synthesis of predicted Fum6p hydroxylation product isomer 80.... 92

Scheme 50: Synthesis of predicted Fum6p hydroxylation product isomer 84 .... 94

Scheme 51: Synthesis of intermediate 91 en route to predicted Fum6p

hydroxylated product isomer 97 95

Scheme 52: Synthesis of predicted Fum6p hydroxylated product isomer 97 from

intermediate 91 96

Scheme 53: Synthesis of predicted Fum6p hydroxylated product isomer 104 from

diol 90 97 Scheme 54: Proposed biosynthesis of 105 102

Scheme 55: In vitro assay of Hpm8 with malonyl-CoA produces pyrone 3 103

Scheme 56: In vitro assay of Hpm8 with malonyl-CoA and NADPH to produce

tetraketide pyrone 113 and pentaketide pyrone 109 104

Scheme 57: Comparison of normal programmed steps with derailment steps

when Hpm3 is absent to produce pyrones 109 and 113 105

Scheme 58: Synthesis of predicted pyrone 113 from in vitro assay of Hpm8 with

NADPH 106

Scheme 59: Mechanism for CBS reduction of compound 111 107

Scheme 60: In vitro assay of Hpm8, Hpm3, malonyl-CoA, and NADPH to

biosynthesize dehdyrozearalenol 107a 108

Scheme 61: In vitro assay of Hpm8 with Hpm3-SAT°, malonyl-CoA, and

NADPH to furnish pyrone 3. This product was identical to those obtained

when Hpm8 is incubated with NADPH 110

Scheme 62: In vitro assay of Hpm3 or Hpm3-SAT° with malonyl-CoA and 115

to furnish 107a 111 List of Tables

Table 1: Summary of the three types of PKS enzymes and their products2

Table 2: Summary of results obtained from LovC in vitro assay LIST OF ABBREVIATIONS a" specific rotation

A adenylation domain

AcOH acetic acid

ACP acyl carrier protein

AIBN 2,2'-azobis(2-methylpropionitrile)

AT acyl transferase ap apparent

Bn benzyl br broad n-BuLi rt-butyl lithium c concentration in g mL"1 (for optical rotation)

C condensation domain

CoA coenzyme A

CBS Corey-Bakshi-Shibata

COSY correlation spectroscopy

6 chemical shift in parts per million downfield from TMS Da daltons

DBU 1,8-diazabicyclo[5.4.0]undec-7-ene

DCC 1,3-dicyclohexylcarbodiimide

DIBAL diisobutyl aluminum hydride

DIPEA N, N-Diisopropylethylamine

DMAP 4-(dimethylamino)pyridine

DMSO dimethyl sulfoxide

DMF dimethylformamide

DH dehydratase

DMP Dess-Martin periodinane d doublet (in NMR)

ER enoyl reductase eq equivalents(s)

EWG electron withdrawing group

FAD flavin adenine dinucleotide

FAS fatty acid synthase

FMN flavin mononucleotide FTIR fourier transform infrared spectroscopy

GC-MS gas chromatography coupled with mass spectrometry

HWE Horner-Wadsworth-Emmons

HMBC heteronuclear multiple bond correlation spectroscopy

HMQC heteronuclear multiple quantum coherence spectroscopy

HREI high-resolution electron impact

HRES high-resolution electrospray

HR-PKS highly-reducing polyketide synthase

HMG-CoA (35)-hydroxy-3-methylglutaryl coenzyme A

IR infrared

IPTG isopropyl thio-P-D-galactoside

J coupling constant in hertz kDa kilodaltons

KR ketoreductase

KS ketosynthase

LiHMDS lithium hexamethyldisilazane

LC-MS liquid chromatography coupled with mass spectrometry LC-MS/MS liquid chromatography coupled with tandem mass spectrometry m multiplet mlz mass to charge ratio

MAT malonyl-CoA:ACP transacylase

Me methyl

MeOH methanol

MeT methyl transferase

MS mass spectrometry

MW molecular weight

MWCO molecular weight cut off

NAC jV-acetylcysteamine

NADPH ^-nicotinamide adenine dinucleotide phosphate (reduced form)

NaHMDS sodium hexamethyldisilazane

NR-PKS non-reducing polyketide synthase

NMR nuclear magnetic resonance

NRPS non-ribosomal peptide synthetase

PR-PKS partially-reducing polyketide synthase PKS polyketide synthase ppm parts per million

Ppant phosphopantetheine

PT product template q quartet quant. quantitative yield

R reduction domain

RDS rate-determining step s singlet

SAM S-adenosyl-I-methionine

SAT starter unit:ACP transacylase

T thiolation domain

/-BuOH ter/-butanol t triplet

TBDMSC1 terf-butyldimethylsilyl chloride

TBDMS ter/-butyldimethylsilyl

TBHP tert-butyl hydroperoxide TE thioesterase

TFA trifluoroacetic acid

UV ultraviolet spectroscopy Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin 1

1 Chapter 1: Introduction to Polyketides Biosynthesis, and Biosynthetic

Studies on Lovastatin

1.1 Introduction

Polyketides1"4 are a class of natural products that have garnered substantial interest from the scientific community since their discovery in organisms such as bacteria,5 fungi,6 and plants7 all over the globe. They are structurally complex molecules that exhibit a wide range of biological activities including: antibiotic,8 anticancer,9'10 antiviral," anti-cholesterol,4 kinase inhibitory,12 and mycotoxicity

(Figure l).13 During 2005-2007, polyketides represented more than a third of naturally produced and naturally derived drugs that were approved by the FDA.14

From the roughly 7000 known polyketide structures, 20 have become commercially available drugs producing total annual revenues of over US $20 billion.15 Due to their vast array of biological activities and structural diversity, polyketides continue to be a source of interest to natural product and synthetic chemists. Synthetic organic chemists challenge themselves by designing efficient routes towards these compounds, while natural product chemists investigate nature's biosynthetic capabilities in assembling these complex molecules. The ultimate goal is harnessing the molecular machinery to biosynthesize any molecule that the chemist desires quickly and efficiently. Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin

ho2c co2h

OH OH ho2C^V^T° co2ho

Fumonlsin B, Lovastatin mycotoxin anti-cholesterol

O OH °'" -N/ H OH

Equlsetln Erythromycin A Doxorubicin HIV inhibitor antibiotic anticancer

OH O

MeHN MeO'

Neocarzlnostatln Hypothemycln anticancer kinase inhibitor

Figure 1: Examples of polyketides, displaying prominent biological activities

Polyketides are assembled from simple carboxylic acid derivatives, typically as their coenzyme A (CoA) thioesters (eg. acetyl-CoA, malonyl-CoA, and methylmalonyl-CoA) by enzymes known as polyketide synthases (PKS).'6

The process of polyketide biosynthesis is reminiscent of fatty acid synthesis as shown in Scheme 1. Fatty acids are constructed by enzymes known as fatty acid synthases (FAS) and the biosynthesis begins with the loading of the typical starting unit, acetyl-CoA, onto the active site thiol of the FAS enzyme. Malonyl-

CoA is then used as the chain extender, adding an additional two carbons to the acetyl starter unit via decarboxylation and Claisen condensation to produce the |3- Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin keto intermediate. This P-ketone can undergo reduction by NADPH to produce the alcohol, followed by dehydration to form the olefin, and finally reduction once again to furnish the saturated carbon chain. At this stage the fully saturated intermediate becomes the new starting unit and undergoes another round of chain elongation, adding an additional two carbons, where the newly formed P-ketone can be reduced to the saturated moiety once again. This process continues until the correct carbon chain length is obtained and the FAS enzyme releases the fatty acid. Scheme 1 does not specify the individual domains housed within the FAS enzyme that are responsible for catalyzing each reaction.

o CoA-S^ starting unit

H & °=< o=<"o=< o o SH 0 0 (s s CoA-S-S MOH decarboxylation

chain extender Claisen condensation I new starting unit I reduction 0 O OH sA/S. AA reduction gjjjg dehydration H repeated cycles W n = 1 to > 20

fatty acid Scheme 1: Generic steps in fatty acid biosynthesis by a fatty acid synthase enzyme (FAS) Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin 4

The catalytic domains in FAS are similar to those in PKS, and they are used to biosynthesize fatty acids and polyketides, respectively.17 The domains involved in starter unit priming and chain extension are the acyl carrier protein

(ACP), ketosynthase (KS), and malonyl-CoA:ACP acyltransferase (MAT). The domains responsible for reductions and dehydrations of the (3-keto intermediate are the ketoreductase (KR), dehydratase (DH), and the enoyl reductase (ER). The individual steps in polyketide and fatty acid biosynthesis involving these specific domains are shown in Scheme 2. The first step is the conversion of the inactive

(apo) ACP to the active (holo) form by attaching a phosphopantetheine (Ppant) moiety derived from Co A to the active site serine (1), which is a post-translational modification catalyzed by the enzyme phosphopantetheine transferase.18 The attached Ppant serves as a long flexible arm that carries the growing chain to the required domains to allow for further chain extensions and/or functional group modifications. Once the ACP is active, the starting units, in this case acetyl-CoA and malonyl-CoA, are loaded onto their respective domains; the active site cysteine of the KS domain and the phosphopantetheinyl thiol on the ACP. The transfer of malonyl-CoA to the ACP (2a) and acetyl-CoA to the KS domain (2b), is facilitated by the MAT domain which recognizes and selects the correct starter and extender units.19 During the first extension, the KS domain catalyzes decarboxylation and Claisen condensation (3) to form the (3-keto intermediate bound to the ACP.20 This extended chain can undergo two fates: the ACP-bound

|3-keto thioester can by-pass all tailoring domains and be transferred back to the

KS domain (4) to undergo further chain elongation with another malonyl unit, or Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin 5 it can continue to one or more of the reductive domains [KR (5), DH (6), and ER

(7)] before being transferred back to the KS domain for subsequent chain extension (8). FAS enzymes typically have successive cycles of chain elongation, along with use of all tailoring domains, to construct a product of required length

(usually 14, 16 or 18 carbons) that is fully reduced (9) as shown in Scheme 2.3

This is the point however, where the two enzymatic systems differ. PKS enzymes can selectively control reductive modification of the p-carbonyl to construct polyketides that are reduced (10) or unreduced (11), as shown in Scheme 2. This type of inherent biochemical "programming" is the most fascinating, and least understood, aspect of the biosynthetic machinery. The key question is how these enzymes recognize the correct starting materials and construct a polyketide, of appropriate length, while controlling reductions and dehydrations to produce the final product. Natural product chemists are striving to answer this fundamental question. Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin

0 9H H H OH © r?-°0

o o o o -M©« o o !_ @ AA,S-CoA

O O O (jl) ; ^

O OH 5AA

= Acyl carrier protein

= Ketosynthase ©I L®UJ Malonyl-CoA:acyl Dehydration "rvY transferase OH O O I o Reduced KRj = Ketoreductase SA^ Polyketide

mm m « | JDHJ = Dehydratase ©I mi mi = Enoyl reductase I Reduction HO Fatty Acid , 5 ° ;" © ©

Scheme 2: General biosynthesis of fatty acids and polyketides3

Significant advances in structural biology have allowed deconstruction of the complex biochemical "programming" of the PKS and FAS enzymes. Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin 1

Structural NMR and X-ray crystallography have been critical in providing three- dimensional structures of individual domains and complete enzyme systems.

These studies have aided our understanding of polyketide and fatty acid biosynthesis including substrate loading, chain extension, reductions, and chain termination.21"25 The FAS systems have been studied extensively, and recently a complete mammalian FAS was crystallized and analyzed by Maier et. al. (Figure

2)26 This landmark discovery has expanded our understanding of the overall architecture of these large proteins or megasynthases and has shown the structural similarities between PKS and FAS enzymes26 The ribbon structure (Figure 2A) illustrates that the mammalian FAS is a homodimer with two distinct portions; an upper modifying region (containing KR, DH, ER domains) and a lower chain condensing region (containing MAT and KS domains). The structure also has two additional domains which are non-functional: the inactive methyltransferase

(tyME) and the truncated ketoreductase domains (x^KR) which are believed to be an evolutionary relic of domains that were used in the past.26 Another key feature is the linker domains (LD), which are originally proposed to allow communication between domains and serve as a tether to hold them close together.27 These flexible linkers, as shown in Figure 2B, facilitate opening and closing of reaction chambers to allow the ACP to shuttle the bound fatty acid intermediate to the appropriate domain.26 Unfortunately due to the resolution of only 3.2 A, the flexible ACP still remains unresolved. Although this crystal structure is only a single snapshot of the entire biochemical process, it has shed Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin 8 light on many of the unsolved questions present in polyketide and fatty acid biosynthesis.

Figure 2: FAS crystal structure. (A) Ribbon structure. (B) Domain illustration

(Adapted from Maier et. al.)

1.2 Types of PKS enzymes

There are three classes of PKS enzymes, each with sub-divisions as shown in Figure 3. Each class will be discussed in more detail in the following sections. Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin 9

ivpm of pks / \

modular iterative iterative iterative

1 \ I 1 HR-PKS PR-PKS NR-PKS HR/NR- HR/NRPS- PKS hybrid hybrid

HR = Highly Reducing NR = Non-Reducing PR = Partially Reducing NRPS = Nonribosomal Peptide Synthetase

Figure 3: Different types of PKS enzymes and their subclasses2

1.2.1 Type I

Type I PKS enzymes are large multi-functional proteins with two main classes: modular (found mainly in bacteria)28 and iterative (found mainly in fungi).29 The modular PKS systems have been studied extensively. One example is the well-studied biosynthesis of the antibiotic erythromycin B (seen in Figure

1), which is produced by the bacterium Saccharopolyspora erythraea.30 Modular

PKS enzymes work in a similar fashion to an assembly line where the starting unit is first loaded onto the ACP, and subsequently the chain is extended and modified by the domains present in the module (Scheme 3). Each of these modules are Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin used only once in a non-iterative fashion, which is significantly different from the iterative Type I PKS systems. The iterative Type I PKS enzymes contain only one set of domains that are used repeatedly to construct either a reduced or unreduced polyketide (Scheme 2). It is still a mystery as to how these type I iterative PKS enzymes control the use of the modifying domains (KR, DH, ER) on the growing chain to biosynthesize the correct natural product. There are also three sub-divisions within the iterative type I PKS termed non-reducing (NR), partially-reducing (PR), and highly-reducing (HR) that reflect the level of (3- carbon processing by the KR, DH, and ER domains.29 These sub-divisions of

Type I iterative PKS enzymes produce polyketides such as: aflatoxin B],31 6-

32 13 methylsalicylic acid, and fumonisin Bt respectively. In addition to the three sub-classes, there are another two separate divisions known as the HR - nonribosomal peptide synthetase (NRPS) and the HR/NR PKS hybrid systems.

The HR-NRPS enzyme attaches an amino acid to the polyketide backbone, while the HR/NR PKS hybrid manufactures a highly reduced and a non-reduced polyketide scaffold. Two well known products of these hybrid PKS systems are equisetin,33 and hypothemycin,12 as shown in Figure 1. Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin 11

Load Module 1 Module 2 Module 3 II KR i DH

•i^rnii |nrrii|

-OH

Scheme 3: Generic biosynthetic scheme for modular type I PKS containing a loading and three chain extension/modifying modules.

1.2.2 Type II

In contrast to the type I PKS enzymes that are megasynthases, the type II

PKS enzymes are dissociable proteins that function iteratively and are found typically in bacteria.34 Similar to the iterative type I PKS enzymes, they contain one module that encodes a set of minimal PKS and tailoring domains to produce an aromatic polyketide. The minimal PKS consists of KSa, KSp, and the ACP which are required for starter unit recruitment and chain extension (Scheme 4).2 In addition, the MAT domain is used to capture malonyl-CoA, with subsequent loading onto the ACP, which is used exclusively as the extender unit in type II

PKS enzymes.35 When the starter unit is selected and loaded onto the ACP, decarboxylation and a Claisen condensation are catalyzed by the KSa domain to form the P-keto intermediate. Successive chain extensions occur in an iterative Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin manner to construct the highly reactive poly-^-ketone intermediate, which is proposed to be stabilized by the KSp domain. This reactive intermediate is immediately modified by the KR, cyclase (CYC), and aromatase (ARO) domains.

These tailoring domains can regiospecifically reduce, cyclize, and dehydrate the poly-P-ketone intermediate to afford the scaffold for aromatic polyketides. The varying assortment of aromatic structures is ultimately determined by the starter unit chosen, length of the poly-P-ketone intermediate, regioselective cyclizations,

•j and intricate control of tailoring domains. A well known iterative type II polyketide is the anti-cancer agent doxorubicin produced by the bacterium

Streptomyces peucetius (Scheme 4). 37

OH o\ o Doxorubicin 1

iMi ks. j LjcfcJ —• hmmmd KSp I f 1

poly-p-ketone t Iteration Scheme 4: Schematic diagram of aromatic polyketide biosynthesis by type II

PKS Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin

1.2.3 Type III

The type III PKS family of enzymes, known as the chalcone or stilbene synthases, produce polyketides such as flavonoids from bacteria, fungi, and plants.38 These homodimeric iterative PKS enzymes differ from all other PKS enzymes in that they do not require an ACP. Instead all decarboxylations, Claisen condensations, aromatizations, and cylization reactions occur at two independent active sites in the PKS.39 The chalcone synthase utilizes three malonyl-CoA units along with p-coumaroyl-CoA, derived from L-phenylalanine from the phenylpropanoid pathway, to biosynthesize chalcones.40 Plants biosynthesize chalcones as a natural defense mechanism since they contain antibiotic, anticancer, and antifungal activity; they are also produced because they are used as building blocks for flavonoids (Scheme 5).41 Downstream tailoring enzymes further decorate the basic aromatic scaffold providing a wide range of structures that possess various biological activities. Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin

CoA-S n times CoA-S O O CoA-S'" +

o_) OH R p-coumaroyl-CoA ¥ OH O

chalcone

Scheme 5: Schematic diagram of chalcone biosynthesis by type III PKS34

Table 1 summarizes the three broad categories of PKS enzymes along with their typical products and mode of substrate activation.

Type Mode of Producing Mode of substrate Typical of PKS operation organisms activation product I Modular bacteria ACP Reduced I Iterative fungi, bacteria ACP Aromatic and reduced II Iterative bacteria ACP Aromatic III Iterative plants, fungi, CoA Aromatic bacteria Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin

1.3 The fungal polyketide lovastatin and its biological properties

1.3.1 Statin drugs and inhibition of cholesterol biosynthesis

One polyketide that has attracted much interest in the pharmaceutical industry is lovastatin (1). Lovastatin belongs to a family of cholesterol lowering drugs, known as statins, that grossed US $14.3 billion with over 211 million prescriptions dispensed in the United States in 2009.42 These drugs include simvastatin,43 fluvastatin,44 atorvastatin,45 and rosuvastatin.46 Lovastatin (1) was the first commercially available statin in 1987, and was produced by the fungus

Aspergillus terreus.47 In the following 16 years, analogues of 1 became some of the best selling drugs in first world nations (Figure 4).

Lovastatin (1) Compactin Simvastatin

O Fluvastatin Atorvastatin Rosuvastatin

Figure 4: Lovastatin and its analogues Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin

Statin drugs inhibit (3S)-hydroxy-3-methylglutaryl-coenzyme A (HMG-

CoA) reductase, an NADPH-dependant enzyme that catalyzes the reduction of

HMG-CoA to produce mevalonic acid. This acid is a key intermediate in the biosynthesis of cholesterol (Scheme 6).48 The mechanism of action of the statins involves mimicking the mevaldic acid thiohemiacetal intermediate during thioester reduction of HMG-CoA (Scheme 6). The upper portion binds in the active site and the lower hydrophobic section binds in a hydrophobic pocket in

HMG-CoA reductase.49 Modifying the hydrophobic portion can significantly increase its efficacy. For example Atorvastatin (Lipitor™)45 is the top selling statin drug grossing over US $12.4 billion in 2008.50

HMG-CoA HO HOv CO2H Reductase p^co2H

S-CoA

HMG-CoA Mevaldic acid Mevalonic acid thiohemiacetal

Mevaldic acid mimic I Hydrophobic core

HO Cholesterol

Scheme 6: Inhibition of HMG-CoA reductase by lovastatin preventing the formation of mevalonic acid en route to the biosynthesis of cholesterol.1 Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin

1.3.2 Lovastatin biosynthesis

The fungus A. terreus uses a HR-PKS pathway, incorporating units of acetyl-CoA and malonyl-CoA, to construct dihydromonacolin L (2), an intermediate in lovastatin biosynthesis. Dihydromonacolin L (2) is further decorated by post-PKS enzymes to produce lovastatin (1) (Scheme 7). The biogenic origins of all atoms in 1 have been well established through isotopic feeding experiments and NMR studies (Scheme 8).51'52 The specific labeling pattern suggests that 1 is constructed from nine acetate units, forming the core of lovastatin (2), with a methyl group derived from S-adenosyl-Z-methionine (SAM).

The methyl-butryl side chain (containing a methyl group from SAM) is attached to an alcohol on the decalin ring (originating from molecular oxygen) through an ester bond.

o -^S-CoA + o o

HC^^S-CoA H Dihydromonacolin L (2) Lovastatin (1) Scheme 7: Generic scheme for the biosynthesis of dihydromonacolin L (2) and lovastatin (l).1

o hov /v. 11 I 1 ^ONa 0 k, .>0 11 J A. terreus "rv„( H3C s fHz m HOY^^s^V°yN^^NH2 n Au. >—' CH3 Ncn^N HO OH SAM Scheme 8: Isotopic labeling pattern of lovastatin through isotopic feeding experiments in A. terreus. Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin

Molecular biology techniques have revealed that a total of 18 genes are in the lovastatin biosynthetic gene cluster in A. terreus as shown in Figure 5.4 In order to determine which genes are involved in the biosynthesis of 1, pioneering work by the Vederas and Hutchinson labs expressed certain genes from the lovastatin gene cluster in the heterologous host Aspergillus nidulans4 Expression of the genes lovB and lovC in A. nidulans allowed the isolation of the metabolite 2 from fermentation cultures, indicating that the lovastatin nonaketide synthase

(LovB) and an accessory enzyme (LovC) are involved in the biosynthesis of 1.

LovB contains all the necessary HR-PKS domains: KS, MAT, DH, MeT, ER°,

KR, and ACP. Interestingly, the inherent ER domain in LovB that has sequence homology to other ER domains is inactive (ER0).4 Therefore, an auxiliary ER enzyme (LovC) is required to complete the biosynthesis of 1. A series of post-

PKS enzymes (LovF, LovD, and LovA) were also discovered to be vital for the transformation of 2 into 1. The gene lovA encodes a cytochrome P450 that hydroxylates the lower portion of 2.4 LovF, a PKS enzyme, biosynthesizes the methyl-butyryl moiety, while LovD attaches the side chain to the hydroxyl group installed by LovA. The other genes have been found to be involved in the production of 1, however their exact roles have not been elucidated.4 Some are believed to enhance export of the metabolite and provide immunity to lovastatin for the producer organism. Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin

37 kb

ORFtf k ORF13 ORFtt^ 64 kb

Potential u..,. ,,,j„ Unknown .Transporter resistance Transesterase functjon ^ ge^es gene

Cytochrome i Regulatory i j Polyketide M P450 genes I -' genes ••• biosynthesis Figure 5: Lovastatin biosynthetic gene cluster in A. terreus.4

LovB (335 kDa) and LovC (39 kDa) work in tandem to take acetyl-CoA and eight units of malonyl-CoA through approximately thirty chemical reactions to assemble 2. Details of the proposed biosynthesis of 2 are shown in Scheme 9.

Once the initial acetyl-CoA unit is loaded onto the KS domain, malonyl-CoA is loaded onto the ACP where decarboxylation and Claisen-condensation occurs, catalyzed by the KS domain, to generate a (3-keto intermediate.3 The P-carbonyl is then reduced to the alcohol and dehydrated, by the KR and DH domains, respectively, to produce a diketide. Another round of chain extension occurs using malonyl-CoA, which includes decarboxylation and Claisen-condensation

(as illustrated by the solid grey arrow in Scheme 9). The (3-carbonyl is reduced and dehydrated to construct the triketide. To assemble the tetraketide, a methyltransferase (MeT) domain and the ER enzyme LovC are required to install the methyl group (derived from SAM) and reduce the double bond. The growing chain is then extended to the linear hexaketide, which then undergoes a stereoselective Diels-Alder reaction to generate the cyclized hexaketide.53 Chain extensions with controlled reductions and dehydrations continue to occur until the Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin nonaketide is assembled. Finally lactonization and thioester hydrolysis releases 2 from the LovB enzyme.1 What is most fascinating is that only two enzymes

(LovB and LovC) are involved in catalyzing over thirty chemical reactions on route to 2. However, how LovB and LovC decide when to perform certain chemical reactions in assembling such a complex core structure remains to be elucidated. Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin

o o X II HO"^ S-CoA "" I if KR J if + ^ Enz_SY o o . 1 o I i diketide triketide S-CoA

„ Enz KR KR KmJ ^ O^S-Enz .

o- ItoT I HHHH tetraketide pentaketide L22J

Enz-S^.0 S-Enz KR DH I ^ 0 Enz Diels-Alder r"-S mt linear hexaketide cyclized hexaketide heptaketide

O^S-Enz HO^^jj^S-Enz HO^/ > ,

H H octaketide nonaketide 2

= two carbon unit derived from malonyl-CoA

= two carbon unit derived from acetyl-CoA

Scheme 9: Proposed biosynthesis of 2 by LovB and LovC1

The role of LovC in the biosynthesis of 2 was investigated by expression of only the lovB gene in A. nidulans.4 It was found that LovB alone can self-load units of malonyl-CoA and acetyl-CoA, catalyze chain extensions, perform reductions and dehydrations, and methylate the carbon chain.4 However, in the absence of LovC, LovB diverts at the tetraketide stage. Without enoyl reduction, derailment in the normal programmed steps occurs as indicated by the isolation of Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin 22 the hexa- and heptaketide-pyrones (Scheme 10). When LovC is present, LovB can correctly convert the triketide to the tetraketide that will be further elaborated to produce 2. When LovC is absent, enoyl reduction does not occur and LovB is unable to use the unreduced tetraketide. Therefore it off-loads the incorrectly produced intermediate by catalyzing two more rounds of chain extension, without reductions or dehydrations, followed by pyrone formation to furnish the hexaketide-pyrone. The heptaketide-pyrone is formed via the addition of two more carbons to the unreduced-tetraketide along with reduction and dehydration.

Two additional rounds of chain extension occur, without reductions or dehydrations, and subsequent pyrone formation to yield the heptaketide-pyrone.

These experiments indicate that LovC plays a critical role in the biosynthesis of

2.4 Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin

HOs KR Ik OH J O^S-Enz ^ Enz-S^ ,0

triketide m tetraketide

H 2

0^,S-Enz / malonyl-CoA x 2 *

unreduced-tetraketide • malonyl-CoA x 3 hexaketide-pyrone KR II DH

heptaketide-pyrone

Scheme 10: Off-loading of incorrect hexa- and heptaketide-pyrones from LovB in the absence of LovC4

The final steps in the biosynthesis of 1 involve post-PKS enzymes that further functionalize dihydromonacolin L (2) into lovastatin (1). The first proposed step is oxidation at C-3 by the cytochrome P450 LovA, followed by spontaneous dehydration to form the diene system of monacolin L (Scheme 11).

Another oxidation step installs the hydroxyl group at C-8 to form monacolin J, Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin followed by the attachment of a butyryl side chain [(biosynthesized by the lovastatin diketide synthase (LovF)] via the transesterase LovD to construct l.54'55

Interestingly, LovF uses the same biosynthetic machinery as LovB to construct the diketide except that it contains a fully functioning ER domain, meaning it does not require an accessory ER enzyme (LovC). LovF is capable of condensing units of acetyl-CoA and malonyl-CoA to form a diketide. Subsequent reduction and dehydration to the alcohol, followed by reduction of the olefin (using its own fully functioning ER domain) and methylation produces the methyl-butyryl side chain.

-H,0

6^4a

Monacolin L

S-Enz

Monacolin J

acetyl-CoA malonyl-CoA SAM Scheme 11: Proposed post-PKS steps that form intermediates monacolin L and monacolin J through the actions of LovA, LovD, and LovF to construct 11 Chapter 1 - Introduction to Polyketides, and Biosynthetic Studies on Lovastatin

To gain a better understanding of the biosynthetic programming involved in the biosynthesis of 1, PKS genes need to be isolated and their enzymes expressed in appreciable quantities for in vitro assays. This chapter will focus on the biosynthetic steps toward 2, through studying of the purified enzymes LovB and

LovC, in order to elucidate fundamental aspects of the biosynthetic machinery.

Questions that remain to be answered are: (1) the catalytic role of each domain in

LovB, (2) substrate specificity of LovC and each domain in LovB and (3) factors that ultimately govern the order of operations of each catalytic domain. Chapter 1 - Results and discussion 26

1.4 Results and discussion

1.4.1 Expression and purification of LovB and LovC

Dihydromonacolin L (2) is assembled by the iterative HR-PKS LovB along with its accessory ER enzyme, LovC. Pioneering genetic work by the

Hutchinson and Vederas labs have identified the PKS genes that biosynthesize 1 and expressed them into the heterologous host A. nidulans.4 However, the inability to obtain pure LovB and LovC enzymes in large amounts has hampered the in-depth investigation into the biochemical programming of the megasynthase

LovB. Collaboration with Professor Yi Tang and Professor Nancy Da Silva from

UCLA and UC Irvine respectively, allowed the expression and purification of

LovB from an engineered strain of Saccharomyces cerevisiae (strain BJ5464-

NpgA). This particular strain has the majority of the protease genes knocked out and houses a copy of the Ppant transferase gene npgA, from A. nidulans,56,57 By following the Tang and Da Silva expression and purification procedures, yeast cells transformed with LovB, were grown in 1 L of YPD media with 1% dextrose for 72 hours at 28 °C. Cells were harvested by centrifugation, lysed by sonication, and cellular debris was removed through centrifugation once again. The supernatant was isolated and treated with Ni-affinity agarose resin for 2 hours to allow binding of the C-terminal hexahistadine-tagged LovB to the nickel.

Purification of LovB was done through a gravity flow column with increasing imidazole concentration. Anion-exchange chromatography was used as a final step to achieve protein homogeneity to afford a yield of 4.6 mg/L of culture. Chapter 1 - Results and discussion 27

To determine if the Ppant arm is attached to LovB, a Ppant ejection assay was conducted using LC-MS/MS. LovB was digested with chymotrypsin at 36

°C over night and the reaction was stopped upon acidification to pH > 3. The bulk of the water was removed under vacuum and the resulting mixture was desalted using a Ci8 Ziptip™. Elution with 85% MeCN, concentration under vacuum, and

MS analysis of the fragments by LC-MS/MS indicated that LovB is in the holo

(active) form (Figure 6).

335 000 Da J^Chymotrypsin

+ other fragments

LC-MS/MS I T

Ppant IDQGVDSLGAVTVGTW Q ( Expected Ppant eliminated mass: oD I 1598.6 Da o-l-0 IDQGVDSLGAVTVGTW Expected holo mass: f d>H 1957.8 Da IDQGVDSLGAVTVGTW Expected Ppant eliminated mass: 1696.5 Da

1599.737 / I 1581.697 I 1697.694 1958.680 ^1 I | | Li699.729 .

1550 1600 1750 1700 1750 1800 1850 1900 1950 Figure 6: Chymotrypsin digestion of LovB and LC-MS/MS analysis of

fragments Chapter 1 - Results and discussion 28

There are three peaks from the LC-MS/MS data that suggest that LovB is in the holo form (Figure 6). The first peak with a mass of 1958.6 Da agrees with the predicted calculated mass of the holo LovB fragment containing the Ppant arm. Also, the observed masses 1697.6 Da and 1599.7 Da agree with the calculated masses of LovB that underwent two different mechanisms of Ppant moiety elimination during the tandem MS/MS process. Scheme 12 shows the proposed mechanisms of the formation of the two LovB products following Ppant co elimination, which are consistent with the literature. The expected masses of both Ppant elimination products were found in the MS/MS data (see Appendix

Al), indicating that the expression system yielded mostly holo LovB.

°VLGAVTVGTW IDQGVD.,NU Yh 0 J o) OH H H2 HO H 0 active LovB: 1957.8 Da

• \ IDQGVDyN^A IDQGVD LGAVTVGTW LGAVTVGTW O ©O Tr'/ 0-P=0 OH Ppant eliminated LovB: 1695.5 Da Ppant eliminated LovB: 1598.6 Da + + OH OH

0=P-0 OH u i_I l I I H "2 0HlXtYN^rS~~"H O o o Ppant elimination product: 261.3 Da Ppant elimination product: 359.4 Da Scheme 12: Proposed mechanisms for the formation of the two Ppant eliminated

LovB products and their respective Ppant elimination product Chapter 1 - Results and discussion 29

The Tang lab also cloned, expressed, and purified LovC from Escherichia coli,59 We repeated the expression and purification protocols. E. coli cells were grown in 1 L of LB medium with 100 jxg/mL ampicillin at 37 °C until an OD600 of

0.4 was obtained. The cells were then incubated on ice and induced with 0.1 mM isopropyl thio-p-D-galactoside (IPTG) for 16 hours at 16 °C. The cells were harvested by centrifugation, lysed using a sonicator, and cellular debris was removed using centrifugation. The crude hexahistadine-tagged LovC protein was treated with Ni-affinity agarose resin for 2 hours and then loaded onto a gravity flow column. Elution with increasing concentration of imidazole yielded pure

LovC protein for further studies.

By systematically removing cofactors and LovC from the enzyme assay, we are able to evaluate the roles of each of the domains in LovB. First the cofactors NADPH and SAM were eliminated, thereby rendering the KR and MeT domains inactive.

1.4.2 Investigation of the catalytic activity of pure LovB with malonyl-CoA

in the absence of NADPH and SAM

To ascertain the catalytic activity of purified LovB, it was incubated with malonyl-CoA overnight at 23 °C. The reaction was quenched and extracted by the addition of ethyl acetate with 1% TFA. The combined organic layers were removed under vacuum and the residue was analyzed by LC-MS. Mass-filtering of the total ion count allowed the identification of pyrone 3, a proposed truncated product resulting from the action of LovB utilizing malonyl-CoA in the absence of certain cofactors (Scheme 13). Scheme 13 shows the seven domains within the Chapter I - Results and discussion 30

LovB enzyme that are active (black), inactive (red - because of missing cofactor), and non-functional (white). There are three key pieces of data that are obtained from the LC-MS: retention time on the LC column, UV-spectrum, and low- resolution MS (Figure 7).

Cofactors: LovB triketide

o o HO'^-^"S-CoA cr o 3 MW =126.1 g/mol

Scheme 13: In vitro assay of LovB with malonyl-CoA in the absence of NADPH and SAM. Inactive domains are shown in red and non-functional domains are coloured white. Pyrone 3 is the product of this system mAUfx1j000> A 3.0 Ex**ct-280nm.4nm (1.0 " c •» [M-H]-

2.0 . . TO. ... U5C 190 C 'TiO 2X3 1.0 mfz

A. l A 0.0

10 20 30 40 mm

B HP- 3.0-

2.0

1.0-

0.0- S S 3 S 8 200 280 300 360 400 480 500 650 -

Figure 7: LC-MS data for proposed pyrone 3. (A) LC chromatogram. (B) UV- spectrum. (C) Low-resolution mass spectrum in negative ion mode. Chapter 1 - Results and discussion 31

Cofactors NADPH and SAM are not present in this experiment, therefore both the KR and MeT domains are disabled. The ER° domain has sequence homology to other known ER enzymes, however it is non-functional in LovB.

Since LovB only produces small quantities of 3, direct characterization with NMR was difficult. Another method to confirm the structure of 3 is to compare it with a standard; commercially available 4-hydroxy-6-methyl-2//-pyran-2-one was purchased and injected into the LC-MS system, where it shows an identical retention time, UV-spectrum, and MS as the proposed pyrone 3. The formation of pyrone 3 indicates that recombinant LovB is catalytically active, can self-load malonyl-CoA, and catalyze decarboxylation to generate the acyl starting unit

(Scheme 14). This result initially appears contradictory to previous reports that the first two carbons (starter units) are derived from acetate.51 The use of malonyl-

CoA as both the starting and chain extender unit is surprising. However, with no acetyl-CoA present, LovB may have no choice but to use malonyl-CoA. Once

LovB generates the starting acyl unit, it can then be transferred to the KS domain while the ACP loads another unit of malonyl-CoA. The KS domain catalyzes decarboxylation and Claisen condensation to generate the (3-keto intermediate.

Since the KR domain is inactive, due to the absence of NADPH, LovB derails at the (3-keto intermediate. Unable to continue with the normal programmed steps, an additional round of chain extension occurs without reductions or dehydrations, to produce a triketide that cyclizes to release the pyrone 3 and free LovB (Scheme

14). Chapter 1 - Results and discussion 32

o o

SH S^OH o o i + HO^^S-CoA • 1) decarboxylation malonyl-CoA 2) acyl transfer

o o o SH S -£*7) J I malonyl-CoA i

starting acyl unit chain extender unit

1) decarboxylation 12) Claisen condensation

derailment - absence of NADPH and SAM normal programmed steps \J0 0V 0V ± A o o o KR r^-.Xl i

(3-keto intermediate diketide

1) enolization I2) pyrone formation OH SH }

o^ o 3

Scheme 14: Self-loading of malonyl-CoA onto ACP and comparison of normal programmed steps with derailment process in the absence of NADPH and SAM Chapter 1 - Results and discussion 33

1.4.3 In vitro enzyme assay of LovB with maionyl-CoA and NADPH

Next, we sought to determine the role of the MeT domain by eliminating

SAM from the assay. Malonyl-CoA can be generated in situ by the enzyme malonyl-CoA synthase (MatB) from Rhizobium trifolii, with malonate and CoA present in the reaction mixture.60 This malonyl-CoA generating enzyme was provided by the Tang lab. Addition of NADPH to the enzyme assay enables the

KR domain in LovB, and analysis of the reaction mixture by LC-MS reveals the proposed pyrones 4,5, and 6 along with hydrolytic products 9 and 12, as shown in

Scheme 15. Again, LovB is observed to be capable of self-priming, catalyzing decarboxylation, Claisen-condensations, and chain extensions to construct the p- keto intermediate.

Cofactors: LovB pentaketide-pyrone

+ + NADPH

hexaketide-pyrone heptaketlde-pyrone OH OH

Scheme 15: In vitro assay of LovB with malonyl-CoA and NADPH produces pyrones 4-6 and hydrolysis products 9 and 12 Chapter 1 - Results and discussion 34

This assay allows the (3-carbonyl in the (3-keto intermediate to be reduced and dehydrated by the KR and DH domains, respectively, since NADPH is present. This process continues to the tetraketide stage, where LovB derails since the MeT lacks substrate and ER° is non-functional. LovB then off-loads the incomplete products through different mechanisms. Chain extension without reduction, followed by enolization and cyclization afford the proposed pentaketide-pyrone 4. A similar process with extra rounds of chain extension with reduction and dehydration produce pyrones 5 and 6 (Scheme 16 and 17).

Chain extension, with thioester hydrolysis and decarboxylation generates the proposed ketones 9 and 12 as seen in Scheme 18. In both cases, LovB is able to catalyze chain extensions and reductions after failing to methylate the tetraketide, allowing the production of longer homologs such as 5,6, and 12. Chapter 1 - Results and discussion 35

o o o XJk KR 5 malonyl-CoA i }

KR DH 0-keto intermediate diketide triketide

malonyl-CoA

O 0

derailment - absence of SAM normal programmed steps and LovC

tetraketide malonyl-CoA x 2

1) enolization 2) pyrone formation

o-^o"

Scheme 16: Comparison of normal programmed steps with derailment process in the absence of SAM and LovC to produce heptaketide-pyrone 5. Chapter 1 - Results and discussion 36

0 0 0 o o OH malonyl-CoA 0^0 I

malonyl-CoA

malonyl-CoA x 2 j

Scheme 18: Proposed formation of pentaketide-pyrone 4 and heptaketide-pyrone

6 when LovB is incubated with malonyl-CoA and NADPH o , decarboxylation 9 (?) &"H thioester hydrolysis I with H20 o o

KR L DH

malonyl-CoA

o 1) thioester hydrolysis with H,0

12 2) decarboxylation

Scheme 17: Proposed mechanism for the formation of hydrolytic ketones 9 and

12 Chapter 1 - Results and discussion 37

1.4.4 In vitro enzyme assay of LovB with malonyl-CoA, NADPH, and SAM

Addition of SAM to the enzyme assay activates every domain present in

LovB allowing the construction of the natural methylated tetraketide. However, the accessory enzyme LovC is absent forcing LovB to stall at the methylated tetraketide stage. This results in the formation of methylated hexa- and heptaketide-pyrones 7 and 8 (Scheme 19). The structures of methylated pyrones 7 and 8 are consistent with results previously reported when LovB is expressed in

A. nidulans without LovC.4 LovB follows the normal programmed steps towards the tetraketide, however in the absence of LovC an unreduced tetraketide is formed. Derailment from the normal biosynthesis occurs resulting in production of methylated pyrone 7 and 8 as shown in Schemes 20 and 21 respectively.

Cofactors: LovB methylated hexaketide-pyrone o o HO'^v^S-CoA + + NADPH + SAM

methylated heptaketide-pyrone

Scheme 19: In vitro assay of LovB with malonyl-CoA, NADPH, and SAM to produce methylated pyrones 7 and 8 Chapter 1 - Results and discussion 38

o o derailment - absence of LovC normal programmed steps

Juced tetraketide tetraketide malonyl-CoA x 2 l| ooo

1) enolization I 2) pyrone formation OH

0^0

Scheme 20: In the absence of LovC, LovB produces the methylated pyrone 7 instead of biosynthesizing 2 Chapter 1 - Results and discussion 39

o o

malonyl-CoA malonyl-CoAx 2 • unreduced tetraketide

OH OOO 1) enolization 2) pyrone formation

Scheme 21: Proposed formation of methylated pyrone 8 in the absence of LovC

1.4.5 Complete reconstitution of the biosynthetic pathway to

dihydromonacolin L

The next experiment was to completely reconstitute the biosynthetic pathway to 2 by incubation of LovB, with all necessary cofactors (NADPH, malonyl-CoA, and SAM), and its partner enzyme LovC. Extraction of the reaction mixture with ethyl acetate including 1% TFA, removal of solvent, and analysis of the residue by LC-MS shows that neither 2, nor any other pyrones are produced. This is surprising since A. nidulans expressing both LovB and LovC produces substantial quantities of 2.4 Further evaluation of the results led us to believe that a key enzyme is missing from the assay, which prevents the release of

2 from LovB. Since no off-loading occurred, it seemed that a precursor of 2 was still covalently attached to LovB. By adding an excess of a 1M KOH solution to the enzyme assay, followed by acidification, extraction, removal of solvent, and Chapter 1 - Results and discussion 40 analysis of the residue by LC-MS, the formation of 2 was observed in stoichiometric amounts. The structure of 2 was confirmed by comparison with a standard produced by A. nidulans expressing LovB and LovC. Both compounds show identical LC retention times and masses. Using base hydrolysis to release 2 from LovB demonstrates that the assay mixture is lacking an enzyme or a cofactor to allow LovB to liberate 2 (Scheme 22).

Cofactors: LovB

o o Predicted HO'^v^vS-CoA rnummmt H + NADPH + SAM + LovC £ Experimental

Scheme 22: Complete reconstitution of the biosynthesis of 2 requires thioester cleavage by base hydrolysis

Thioesterase (TE) domains are present in PKS enzymes in both fungi and bacteria. Their function is to catalyze ACP-thioester cleavage releasing the PKS product. This is achieved through mainly lactone formation or hydrolysis, but other mechanisms also exist.61 Hence, fungal TE domains from Gibberella zeae Chapter 1 - Results and discussion 41

(PKS13) and Gibberella fujikuroi (PKS4) were added to the enzyme assay.

Addition of an exogenous fungal TE domain (either PKS13 or PKS4) to the mixture of LovB and LovC with all cofactors (malonyl-CoA, NADPH, and SAM) allowed release of 2 from LovB (Scheme 23). This result indicates that 2 can be liberated from LovB through fungal TE enzymes that are not initially present in the lovastatin gene cluster. Time-course experiments (performed by the Tang lab) show that both PKS13 and PKS4 are capable of producing 2 with multiple turnovers, albeit PKS13 is more efficient than PKS4. One potential reason for

PKS13's higher efficiency rate is its broad substrate specificity. ' However, addition of a TE domain from a bacterial PKS such as in erythromycin biosynthesis, does not liberate 2 from LovB. This result indicates that perhaps there are particular protein-protein interactions that are required for thioester cleavage that are absent between LovB and the bacterial TE domain from erythromycin biosynthesis.

Cofactors: LovB .0

+ NADPH + SAM + LovC H 2

Scheme 23: Complete reconstitution of the biosynthesis of 2 requires thioester cleavage by TE domains (PKS 13 or PKS4) Chapter 1 - Results and discussion 42

1.4.6 Substrate specificity of LovC

The role of LovC is to reduce the a, (3-unsaturated double bond at three stages in the biosynthesis of 2: tetraketide, pentaketide, and heptaketide (Scheme

24). Interestingly, LovC reduces an a-methylated substrate to form the tetraketide, in contrast to the penta- and heptaketides that are produced by reduction of an unmethylated substrate (Scheme 24). To determine the influence of the a-methyl on LovC reduction, the following assay was conducted.

KR DH O^S-Enz / Q^S-Enz Enz-S o hoc' triketide MtT tetraketide malonyl-CoA

Enz

malonyl-CoA h3c' h,c'xxr tetraketide pentaketide

Enz-S^O Enz-S^O

KR I^ PH j

1 malonyl-CoA 4 v H^C ' hsc ' h,c '

cyclized hexaketide heptaketide

Scheme 24: The three stages of LovC reduction: Formation of (A) tetra-, (B) penta-, and (C) heptaketides

LovB was incubated with LovC and all cofactors except SAM. An expected product from the assay would be a desmethyl version of Chapter 1 - Results and discussion 43 dihydromonacolin L (32), since the MeT domain in LovB would be inactive.

After extraction and analysis of the residual products by LC-MS, shunt products

5, 6, 9, and 12 are detected (Scheme 25). The results suggest that a-methylation of the tetraketide is crucial for LovC substrate recognition, even though the KR and DH domains function normally in the presence or absence of the a-methyl.

Cof actors: LovB Predicted

O o KOH

HO'^^S-CoA + + NADPH + LovC

Experimental

Scheme 25: In vitro assay of LovB with malonyl-CoA, NADPH, and LovC results in the formation of shunt products 5,6, 9, and 12 instead of the predicted

1.4.7 Investigation of interactions between LovB and ER enzymes

To investigate if LovB can interact with other ER enzymes than LovC, the

ER enzyme from the compactin biosynthetic pathway was chosen as a partner.

Compactin, produced by the fungus Penicillium citrinum, is an anti-cholesterol Chapter 1 - Results and discussion 44 agent that is structurally similar to 1 (Figure 4).64 The compactin nonaketide synthase MlcA works in conjunction with its ER enzyme MlcG, presumably to make desmethyl-dihydromonacolin L (32). In this case MlcA and MlcG would reduce an umethylated tetraketide, in contrast to the LovB and LovC system.

Therefore, it is interesting to examine if LovB can work in conjunction with MlcG to biosynthesize 32.

Incubation of LovB with MlcG, and all necessary cofactors except SAM, followed by base hydrolysis and extraction affords 32 with no detectable pyrone or hydrolytic products (Scheme 26). It appears that LovB, in the presence of

MlcG, can continue past the tetraketide stage without using its MeT domain to furnish 32. Compound 32 can be liberated from LovB through both base hydrolysis and action of the TE domain, PKS13.

Cofactors: LovB

O O KOH or HOM S-CoA

+ NADPH + MlcG

Scheme 26: In vitro assay of LovB with MlcG, malonyl-CoA, and NADPH shows the formation of 32 following base hydrolysis or treatment with the PKS13

TE domain

Interestingly, addition of SAM to this system produces 2. These results suggest that MlcG is tolerant of both methylated and unmethylated tetraketides to construct both 2 and 32 respectively (Scheme 27). As described earlier, LovC has Chapter 1 - Results and discussion 45 stringent requirements for an a-methylated tetraketide; otherwise pyrones and hydrolytic products (5, 6, 9, and 12) are produced. It appears that LovC possesses intricate control over the biosynthesis of 2. If LovB assembles a methylated tetraketide, LovC allows the biosynthesis of 2. However, if LovB constructs an unmethylated tetraketide, LovC does not allow LovB to biosynthesize 2 (Scheme

27). Chapter 1 - Results and discussion

5, 6, 9, and 12

Example: pyrone5

Scheme 27: Fates of methylated and unmethylated tetraketides

LovC or MlcG, an ER enzyme from P. citrinum Chapter 1 - Results and discussion 47

1.4.8 Synthesis of standards to confirm structures of pyrones 4, 5, and 7

produced from the various enzyme assays

To confirm the structure of the pyrones produced from the enzyme assays, synthetic standards were required for comparison. All pyrones are synthesized by post-doctoral fellow Dr. Viji Moorthie. Pentaketide pyrone 4 and hexaketide pyrone 5 were synthesized as shown in Scheme 28. The key step in the synthesis of pyrones 4 and 5 is the final cyclization in the presence of DBU. Analysis of these standards by LC-MS shows identical retention times, UV spectra, and low resolution MS as the corresponding pyrones obtained in the enzymatic experiments, thus confirming their structures.

o o O O OH

MOEt DBU, 70 °C t 1) NaH benzene, 21% 2) n-BuLi THF, 0 °C 7% B

O O O O O OH ^P3 EtO X AAOEt

CH2CI2, 81% 1) NaH 2) n-BuLi THF, 0 °C 9%

DBU, 70 °C | benzene, 24%

Scheme 28: Synthesis of (A) pentaketide pyrone 4 and (B) hexaketide pyrone Chapter 1 - Results and discussion 48

Heptaketide pyrone 6 could not be synthesized due to its instability.

Therefore, the Tang lab used 13C-labelled malonyl-CoA as a cofactor along with

LovB and NADPH. The resulting 26 mass unit increase in the MS as well as the red shift in the UV spectrum, when compared to 5, indicates that the proposed structure for the heptaketide 6 is correct.

To confirm the structure of proposed methylated hexaketide pyrone 7, a standard was synthesized by Dr. Viji Moorthie as shown in Scheme 29. The synthesized pyrone 7 has identical retention time, UV spectra, and low resolution

MS when compared with the pyrone produced from the enzyme assay. Identical mass and UV spectrum of methylated heptaketide pyrone 8 and a standard that was previously reported by Vederas and coworkers confirmed its structure.4

o O 0 O O OH

1) NaH 2) />BuLi THF, 0 °C 11 %

DBU, 70°C < Benzene, 27 % o

Scheme 29: Synthesis of methylated-hexaketide pyrone 7 Chapter 1 - Results and discussion 49

1.4.9 Synthesis of standards to confirm tetra- and pentaketide ketones 9

and 12 generated from the enzyme assay

The synthesis of ketone 9 proceeds by the reaction of commercially available trans, ,4-hexa-dienal with the Horner-Wadsworth-Emmons

(HWE) reagent to afford the product with a trans double bond (Scheme 30 A). To prepare ketone 12, the same aldehyde is treated with the Weinreb amide modified

HWE reagent to yield trans compound 10, which is then reduced to the aldehyde using lithium aluminum hydride to afford 11. Aldehyde 11 reacts with the HWE reagent, to give trans ketone 12 (Scheme 30 B). The rationale for the generation of trans double bonds in the HWE reactions is well precedented.65 Comparison of synthesized standards 9 and 12 to the ketones produced from the enzyme assay shows identical LC-retention times, UV-spectra, and low-resolution mass spectra on LC-MS, thus confirming their structure.

+ Om y\f NaH D / \N (EtO)2P thf, 0°c 45%

O NaH ^ ^ ^ U LiAIH ff A OMe ,OMe 4 ,P—/ N (EtO)2P N' THF,0°C Y THF, -40 °C ^ 93% 10 66%

(BO)2P- thf,o°c 11 22% 12 Scheme 30: Synthesis of (A) tetraketide ketone 9 and (B) pentaketide ketone 12 Chapter 1 - Results and discussion 50

1.4.10 Synthesis of desmethyl-dihydromonacolin L (32) standard to confirm

its structure from the enzyme assay

To confirm the structure of 32, an optically pure standard was synthesized as shown in Schemes 31 and 32. The starting ethyl ester 13 is readily reduced to the aldehyde using DIBAL, and then subsequent protection using ethylene glycol affords 14. The Grignard reagent is prepared by treatment of 14 with magnesium, and a copper-catalyzed substitution of the allylic acetate in 15 is achieved with a catalytic amount of U2CUCI4 to yield diene 16.66 Fortunately, no y-substitution product is observed. The dioxolane protecting group is removed under acidic conditions to provide 17, which is immediately condensed with the dioxolane protected Wittig reagent 18 in the presence of /-BuOK. Acidic work-up, using

10% aqueous oxalic acid, hydrolyzes the a,|3-unsaturated acetal with concomitant isomerization to the thermodynamically stable £-enal 19.67 Enal 19 undergoes an enantioselective intramolecular Diels-Alder reaction in the presence of the organocatalyst 20 and a catalytic amount of TfOH to give 21 as a mixture of AS diastereomers (4:1 endo.exo). The desired diastereomer is isolated by recrystallization through a three-step process: (1) reduction to the alcohol in the presence of NaBH4, (2) recrystallization of the white solid from hot pentane, and

(3) oxidation to the aldehyde via Dess-Martin periodinane (DMP) to afford 21 as a single diastereomer. Chapter 1 - Results and discussion 51

0 1) DIBAL, CH2CI2, 1)Mg, THF, 30 °C =a3; . Br EtO 2) ho' VDH , p-TsOH 2) AcO'a^H^^^ 13 benzene, reflux 14 15 16 Li CuCI (cat.), 74% over 2 steps 2 4 THF, -30 °C, 84%

1) f-BuOK, THF, 0 °C CH,

Br® © 0 (Ph)3P. BrfA n A THF/H20/AC0H XH (1.5:2:1), 90 °C 18 20 98% 2) 10% Oxalic acid TfOH 17 H20, 0°Cto23°C 19 MeCN, -5 °C 61% over 2 steps 69%

H ? DMP, CH2CI2 1) NaBH4, EtOH, 0°C 23 °C 2) recrystallization 73% over H > 99% ee 3 steps 22

4:1 endo/exo

Scheme 31: Synthesis of 21, an intermediate in the synthesis of desmethyl- dihydromonacolin L (32)

The aldehyde 21 is condensed with triethyl phosphonoacetate to yield 24 as a 2:3 mixture of cis/trans isomers. In this particular case a mixture of cis/trans isomers is isolated instead of solely the trans product as described in the synthesis of ketones 9 and 12 (Scheme 30). Ethyl ester 24 does not have extensive conjugation as ketones 9 or 12, therefore the thermodynamic stability of the final product may not control the formation of a cis or trans olefin. The a,(5- Chapter 1 - Results and discussion 52 unsaturated double bond is reduced with magnesium in MeOH to give transesterification product 25. Attempts to reduce methyl ester 25 in the presence of DIBAL led to a mixture of alcohol 26 and aldehyde 27. A simple oxidation of alcohol 26 yields aldehyde 27, which then undergoes an enantioselective allylation using (-)-Ipc2B(allyl)borane at -100 °C to furnish 28 as a single diastereomer. Alcohol 28 is then acylated with acryloyl chloride in the presence of DIPEA to yield ester 29, which is primed for ring closing metathesis using

Grubbs 1st generation catalyst. The cyclization smoothly generates a,|3- unsaturated lactone 30. Lactone 30 undergoes regiospecific nucleophilic epoxidation of the a,|3-unsaturated double bond to afford epoxide 31 as a single diastereomer. The stereochemistry of 31 was confirmed through X-ray crystallography (see Appendix A2). Epoxide 31 then undergoes ring opening using phenyl selenide (generated by treatment of diphenyl diselenide with NaBH4 and AcOH) to furnish 32.69 The synthesized standard has identical mass and retention time on the LC-column as the sample produced from the enzyme assay.

These results show that LovB is capable of biosynthesizing a desmethyl analogue of 2 when incubated with an ER enzyme similar to LovC, namely MlcG. Chapter 1 - Results and discussion 53

Mg, MeOH (EtO) P 2 reflux DIBAL, -78 °C

DIPEA, CH2CI2 .OH •OH o °C 1)

c Et2Q, -100 °C (I ' 2) NaOH, H 0 , 2 2 91% H H H20, 23 °C 26 27 81% | DMP, CH2CI2, 23 °C f 80% over 2 steps

C ,Ph ,Ru= cr | O-OH (Cy)3P Triton B CH2CI2 (0.005 M) reflux toluene, 0 °C 60% H 85% 30 31

NaBH4, AcOH

THF/EtzO (2:1), 0 °C 87%

Scheme 32: Synthesis of desmethyl-dihydromonacolin L (32)

In summary, there are three key steps during the synthesis of 32: (1) formation of four stereocenters and the trans-fused decalin ring via a Diels-Alder reaction (21), (2) regioselective nucleophilic epoxidation with tert-butyl hydrogen peroxide (TBHP) to afford 31, and (3) regioselective epoxide opening in the Chapter 1 - Results and discussion 54 presence of phenyl selenide to give 32. These steps will be described in greater detail below.

The first key step involves imidazolidinone 20, which is a unique organocatalyst in that it has precise enantiocontrol when establishing the four required stereocenters in the decalin ring system of 21. There are three distinct features of organocatalyst 20: The first feature involves reversible generation of an iminium ion, which lowers the LUMO of the dienophile in a Lewis acid catalyzed fashion (Figure 8 A). Decreasing the energy difference between the

HOMO (of the diene) and the LUMO (of the dienophile) increases the rate of reaction. Having both the /erf-butyl and the benzyl groups on the same face increases the rate of iminium ion formation.70 The participating nitrogen lone pair becomes more exposed since it is positioned away from the steric bulk, therefore increasing its overall nucleophilicity. This exposes a reactive enantioface in the catalyst primed for iminium formation. The second feature involves high control of the iminium geometry. The catalyst exclusively selects the £-isomer, through reduction of non-bonding interactions between the carbon-carbon double bond and the tert-butyl group (Figure 8 B).70 In addition, there are n-n interactions between the phenyl ring and the dienophile that provide further stabilization of the

£-iminium geometry. The third feature is a result of both the ter/-butyl and the benzyl groups shielding the top face of the dienophile forcing the diene to approach from only the bottom face.70 The approach of the diene from the bottom face to the construct the decalin ring system in an endo-transition state is shown in

Figure 8 C.71 Chapter 1 - Results and discussion 55

x ©

n N +. "2-fW,RN' 33 . . N' hhx

bottom

B CH3 °VyNH Bn i

O. ,CH3 ^ I E-isomer V-N + Bn1 N>^f "Ri H »

<\ ch3 Vnh Bn ^•'80mer

Ri Steric crowding

Figure 8: Three factors providing superior enantiocontrol in intramolecular

Diels-Alder reaction in the presence of organocatalyst 20. (A) Reversible imine formation, which lowers the energy of the LUMO and decreases the energy difference between the HOMO and LUMO. (B) Controlled formation of E- isomer due to steric hindrance. (C) Sterically favorable approach of diene from bottom face

The second key step is the regioselective nucleophilic epoxidation of the a,P-unsaturated lactone 30 to afford epoxide 31. The nucleophilic attack of

TBHP is under stereoelectronic control as shown in Figure 9; attack of the Chapter 1 - Results and discussion 56 nucleophile from the top face of 30 yields the favorable chair-like enolate transition state (33), whereas attack from the bottom face furnishes the unfavorable boat-like enolate transition state 34. These transition states indicate that the former pathway is favored providing 31 as a single diastereomer.72

Figure 9: Proposed transition state for regioselective epoxidation of 30 using

TBHP

The final key step involves the regioselective opening of epoxide 31 in the presence of phenyl selenide to afford 32. The regioselective opening of epoxide

31 is governed by the Furst-Plattner rule (trans-diaxial effect), which states that six member rings containing an epoxide undergo ring opening with nucleophiles

TX to furnish diaxial products. Axial attack of the a-carbon, via phenyl selenide, generates the diaxial a-phenylseleno ketone and alkoxide (35), which goes through the favorable chair-like transition state (Figure 10). If the phenyl selenide attacked the ^-carbon, the transition state would proceed through the unfavorable Chapter 1 - Results and discussion 57 twist-boat conformation (36). Intermediate 35 then undergoes proton transfers to generate 37, where another phenyl selenide attacks the selenium of the a- phenylseleno group to furnish enolate 38. The enolate is protonated to furnish the

P-hydroxy lactone 32. The stereochemistry was confirmed through X-ray crystallography (as seen in Appendix A2).

hS 31 35 J 37 . 38 cT (PhSe)2 q-n H 0 Co L 0-V- o (0-5 ?iL J °\ 0H O.x ^ ®) Se-^-H ^.Se« O- "»S "J. Ph-SeH 38

O01 I A 36 wvyv O^JL_ r H O

o ph'©h r ysST R seH C3~ 32

Figure 10: Proposed mechanism for regioselective opening of epoxide 31 in the presence of phenyl selenide

1.4.11 Role of truncated NRPS section in lovastatin biosynthesis

Lovastatin PKS (LovB) is not only a HR-PKS, it also contains a small portion of an NRPS enzyme. The truncated NRPS region is thought be an evolutionary relic of a fungal system that contained a fully functioning HR-NRPS hybrid housing condensation (C), adenylation (A), thiolation (T), and reduction

(R) domains.74 Comparison of the domain architecture of 1 with that of a Chapter 1 - Results and discussion 58 polyketide of similar structure, such as equisetin, shows that the HR-PKS portion is identical while the NRPS region is considerably different (Figure 11).

H equisetin domains equisetin

Wmt\ -" l— fti^r >Mfw fi

• . " • 1 • I lovastatin domains PKS NRPS

o V> "VV

••

1 Figure 11: General architecture of lovastatin and equisetin domains and their respective polyketide structure

The role of the C and truncated A domains in lovastatin biosynthesis is still unknown. In equisetin biosynthesis the A domain is responsible for recognizing, and activating (through the formation of mixed phosphoric anhydride using ATP) the correct amino acid, and the C domain catalyzes the formation of an amide bond between an amino acid (serine or N-Me serine) and the polyketide backbone

(Figure 12A). Another function of the complete NRPS region is off-loading of the polyketide chain through amide bond formation with an amino acid (Figure Chapter I - Results and discussion 59

12). Perhaps in the biosynthesis of 1, the C and A domains identify and catalyze off-loading of 2 from LovB through a similar reaction (Figure 12B). Could this be the role of the enigmatic C and A domains in lovastatin biosynthesis?

A - equsMtln

B - proposed dlhydromonacolin L 7

Figure 12: Possible off-loading mechanism of polyketides via amide bond formation in (A) equesetin and (B) 2

To investigate this possibility, LovB was incubated with LovC and all necessary cofactors required to produce 2 together with L-configuration common amino acids (experiments conducted in the Tang lab). These amino acids were added in groups of five. Mass-filtering was used during LC-MS analysis to find a mass that corresponds to 2 attached to an amino acid. Unfortunately, no evidence Chapter 1 - Results and discussion 60 of 2 linked to an amino acid was found in the LC-MS traces. These results suggest that the C domain is not involved in liberation of 2 from LovB via amide bond formation with an amino acid.

To fiirther elucidate the role of the C domain, a truncated version of LovB was cloned and expressed right after the ACP boundary (Ser2542), thus lacking the entire C and A domains (this was done by the Tang laboratory). This variant

(LovB-AC) was incubated with LovC, PKS13, and all necessary cofactors.

Analysis of the extracted residue by LC-MS yielded no detectable amounts of 2.

In contrast, when LovB-AC was incubated with malonyl-CoA without cofactors, pyrone 3 is formed. Conducting the same enzyme assays as described in Sections

1.4.3 - 1.4.5, when LovB-AC was incubated with malonyl-CoA and NADPH, pyrones 4 to 6 and ketones 9 and 12 were detected by LC-MS. In the presence of

NADPH and SAM, LovB-AC yielded methylated pyrones 7 and 8 (Figure 13).

These results suggest that the absence of the C domain does not affect any of the main catalytic functions of the PKS. Adding the C domain as a stand-alone protein to the system along with all necessary cofactors, afforded 2 in reduced yield when a TE domain (PKS 13) was used in the assay.19 Base hydrolysis in the absence of PKS13 gave stoichiometric amounts of 2. It is possible that the C domain catalyzes the Diels-Alder reaction of the linear hexaketide to the decalin.

It has already been reported that LovB contains Diels-Alderase activity as shown through studies of cell free extracts of LovB incubated with a linear hexaketide.53

The linear hexaketide undergoes subsequent closure into the trans-fused endo product in the presence of LovB, through an unfavored endo transition-state that Chapter 1 - Results and discussion 61 cannot be accessed thermally.53 LovB is not the first example of a Diels-Alderase found in nature, but it was the first purified enzyme shown to catalyze such a reaction.53' 75, 76 These in vitro assay results support that the Diels-Alderase activity exists in LovB, potentially in the C domain. Further studies in the

Vederas group by Mr. Drew Hawranik are being conducted by incubating the stand-alone C domain with linear hexaketides to determine if the C domain is responsible for the Diels-Alder cyclization. Chapter 1 - Results and discussion 62

Malonyl-CoA, NADPH.SAII

OH Malonyt-CoA | Malonyl-CoA, ^ No detectable NADPH.8AM W amount*of2 0^0 3

Malonyl-CoA, Malonyl-CoA, NAOPH NAOPH, 8AM

4 to 6 and 7 and 8 9 and 12 OH OH

O^O' O^ O

eg. 4

Figure 13: Summary of LovB-AC in vitro assays with various cofactors and their products

In summary, these results have aided the understanding of the basic biochemical programming involved in HR-PKS systems. These in vitro experiments with purified LovB and LovC show they work in tandem to Chapter 1 - Results and discussion 63 biosynthesize 2. LovC contains the dual role of ER and gate-keeper, recognizing and reducing only an a-methylated tetraketide. Without an a-methylated substrate, pyrones and hydrolytic products are assembled. LovC can be replaced by an analogous ER enzyme, such as MlcG, to furnish 32 when incubated with

LovB and all necessary cofactors except SAM. This result shows that LovB can follow the normal programmed steps even when its natural ER enzyme, LovC, is not present. There are still, however, questions that remain unanswered. Off loading of 2 from LovB in A. nidulans and A. terreus happens efficiently.

However when using purified LovB, a key enzyme or cofactor is missing to allow

ACP-thioester cleavage and liberation of 2. Also, the enigmatic role of the truncated NRPS (C and truncated A domains) region in lovastatin biosynthesis remains to be determined. Working with purified proteins opens the door to structural analysis of these HR-PKS enzymes as well as elucidation of the putative intermediates along the biosynthetic pathway to 2.

1.4.12 In vitro studies with purified LovC

LovB requires the accessory ER enzyme LovC, because its inherent ER domain is rendered non-functional (ER°) due to deletion of key residues in the active site and cofactor binding pocket.4 It has been proposed that the ER° domain makes intimate contacts with LovC in order for the tetra-, penta-, and heptaketides to be reduced.4 However, how LovC recognizes and reduces only three out of a possible eight intermediates is not well understood. LovC belongs to a large family of fungal /ram-acting ER enzymes that also includes: MokE from Chapter 1 - Results and discussion 64

11 7R f\A Monascus pilosus, ApdC from A. nidulans, MlcG from P. citrinum, and many others.

Collaboration with Professor Sheryl Tsai from UC Irvine has allowed expression and purification of LovC from E. coli as well as development of a

NADPH fluorometric assay to explore LovC's substrate specificity. The assay consists of incubating LovC, NADPH, and a series of substrates along the biosynthetic pathway to 2, while monitoring emission at 455 nm (Scheme 33).

Loss of emission at 455 nm indicates successful reduction of the substrate by

LovC using NADPH.

NADPH

Substrate Emission at Reduced No emission at 455 nm substrate 455 nm Excitation at 340 nm Scheme 33: General scheme of fluorometric in vitro assay

Putative biosynthetic substrates were synthesized as their N- acetylcysteamine (NAC) derivatives to establish LovC's substrate specificity. It has been previously established that substrates synthesized as their NAC thioesters, instead of the natural CoA thioesters, can be recognized by PKS enzymes (Figure 14).79'80 NAC thioesters may mimic the Ppant arm on the ACP;

LovC is then able to recognize the substrate and reduce the double bond. Chapter I - Results and discussion 65

NH, O O HS^

HS_ •A W-acetylcysteamine

Figure 14: Structures of coenzyme A and JV-acetylcysteamine

yntheses of the di-, tri-, and tetraketide analogues are shown in Scheme 34.

Crotonic or sorbic acids were coupled with NAC using DCC to afford compounds

39 and 40, respectively. To examine if LovC can accept a substrate free from the

ACP, tetraketide analogue 41 was synthesized as a free acid, by the cross metathesis of 1-heptene and acrylic acid with Grubbs generation II catalyst.

H HS 'V o o DCC, DMAP

CH CI , 23 °C crotonic acid 2 2 39 85%

B H HS •v o DCC, DMAP

0H ^ CH2CI2, 23 °C s—v sorbic acid 78%

r~\ Mes"Ns^^~Mes I ,.^CI u or I Ph P(Cy) + HO 3 CH2CI2, reflux 40%

Scheme 34: Synthesis of (A) diketide 39 (B) triketide 40, and (C) tetraketide analogue 41 Chapter 1 - Results and discussion 66

As mentioned previously, LovC only recognizes an a-methylated tetraketide, whereas unmethylated tetraketides result in production of pyrones and ketones 5-8 (Scheme 27). However, it is interesting to investigate if LovC is still able to accept these substrates in the absence of LovB. The synthesis of the methylated tetraketide 45 begins with treatment of 2,4-hexadienal with the stabilized Wittig 42 to afford the ethyl ester 43 as a 10:1 mixture of E!Z isomers, as shown in Scheme 35. Ethyl ester 43 was hydrolyzed with LiOH to furnish carboxylic acid 44, which was immediately coupled with NAC in the presence of

DCC to yield 45.

42 LiOH, THF/HgO (3:2)

CH2CI2, 23 °C 70 °C, 85% 2,4-hexadienal 10:1 EIZ, 90%

hs^n^ 0 DCC. DMAP

CH2CI2, 23 °C 66%

Scheme 35: Synthesis of rram-methylated tetraketide 45

The synthesis of the unmethylated tetraketide 49 started with 2,4- hexadienal in the presence of the HWE reagent 46 to yield solely /raws-ethyl ester

47. The high yield of the /ra/w-isomer follows the same mechanism as in the synthesis of compounds 7 and 8 in Scheme 30. Again, the ethyl ester 47 was Chapter 1 - Results and discussion 67 hydrolyzed when treated with KOH to furnish carboxylic acid 48. The acid was coupled with NAC to yield unmethylated tetraketide 49 as shown in Scheme 36.

NaH

EtO-P—^i-p—' OEtr OEt OEt 46 KOH, EtOH THF, 0 °C, 85% 100 °C, quant.

HS—V O

DCC, DMAP

CH2CI2, 23 °C, 7%

Scheme 36: Synthesis of unmethylated tetraketide 49

What is also unclear is the olefin geometry of the tetraketide as it undergoes reduction by LovC. Therefore, cis versions of methylated and unmethylated tetraketides were also synthesized (Scheme 37). The modified

HWE reagent 50, was alkylated with Mel to afford 51, which was immediately reacted with 2,4-hexadienal to furnish ethyl ester 52 as a 3:2 mixture of ElZ isomers (Scheme 37). Ethyl ester 52 was hydrolyzed using KOH to yield carboxylic acid 53, which was coupled to NAC using 2-chloro-l- methylpyridinium iodide to afford c/.s-tetraketide 54. Synthesis of the cis- methylated tetraketide 54 requires using a modified HWE reagent that has high stereoselectivity for the Z-isomer. Typically the mechanism of the HWE reaction Chapter 1 - Results and discussion 68

o t ct'y follows two pathways as shown in Figure 15. ' The condensation between the phosphonate anion and an aldehyde forms transition state (TS) anti and syn at different rates. Formation of TS (syn) is slower compared to TS (anti) possibly due to steric clash. Generation of oxaphosphetanes 56a and 56b from intermediates 55a and 55b, respectively, is followed by their decomposition into their respective alkenes. Classically, HWE reagents make primarily the £-alkene since the rate of collapse of oxaphosphetane 56a is faster than the collapse of oxaphosphetane 56b. However, addition of electron withdrawing groups (EWG) on the phosphonate (R = CH2CF3) can reverse the stereoselection of the HEW reaction by altering the rate-determining step (RDS) in the mechanism. The bis(trifluoroethyl) groups facilitate collapse of the oxaphosphetanes at a faster rate than the initial condensation step. This difference in rates makes all steps essentially irreversible, and as a result the RDS resides in the generation of the least sterically hindered TS (anti) that forms the Z-alkene as the major product.

Also, the use of 18-crown-6 to prevent coordination of the metal cation further accelerates the rates of formation of the Z-alkene.83. KHMDS o 18-crown-6 (CF3CH20)2P (CF3CH20)2P THF, -78 °C 3:2 E/Z, 55%

CH2CI2, 0 °C, 9 % 52 53 54

Scheme 37: Synthesis of c/s-methylated tetraketide 54 Chapter 1 - Results and discussion 69

O o + 0 k t+ Ro..„a r Rsyn addition II . R°-^Ri ^anti addition RO"..y • •" h r2 ro 0 RO-oV"' (slower) u (slow) R = -CH2CF3 RDS H^R, TS (syn) TS (anti) steric clash

*anti (fast) (fast) "Syn

o °0 H °0 H Ro.„ a H ^trans RO<..p_vRi RO...^_>Ri k, RO'..M y R ro'»'ol no eX (fast) >r2 (fast) 0 x 0<^ H A," 0 = f„ r2 55a 56a 56b 55b

fast when R = EWG (CH2CF3)

0 q R 1 © ro-p-o + f[| + ro-p-o "X or r2 rz or

£-alkene Z-alkene

Figure 15: Mechanism for HWE reaction when EWG group is attached to the phosphonate

The same procedure was used to synthesize the unmethylated cis- tetraketide 60 (Scheme 38). First, modified HWE reagent 57 reacted with 2,4- hexadienal to furnish methyl ester 58. The methyl ester was hydrolyzed in the presence of KOH to afford acid 59, which was then coupled with NAC to yield the cw-unmethylated tetraketide 60. Chapter 1 - Results and discussion 70

KHMDS O 18-crown-6 KOH, EtOH (CF3CH20)2P- OMe ""O THF, -78 °C 100 °C, 72% 57 37%

H 58 O hs'^v-'nY^ O O DCC, DMAP O CH2CI2, 23 °C, 5%

59 60 Scheme 38: Synthesis of cw-unmethylated tetraketide 60

1.4.13 In vitro fluorometric enzyme assay

The following in vitro experiments were done in the Tsai lab. Di- and triketides 39 and 40 were incubated with LovC along with NADPH and the reaction was monitored by decrease of NADPH fluorescence. The enzymatic reaction was quenched by addition of a 1:1 mixture of Me0H/H20. The solvent was removed, and the residue was analyzed by LC-MS. A decrease in emission, was not observed, nor were substrates with an additional two mass units detected by LC-MS. These results are not surprising since LovC does not typically reduce di- and triketides during the biosynthesis of 2. In contrast, incubation of a- methylated tetraketides as trans- (45) and cw-isomers (54) are expected to result in reduction of the olefin by LovC. However, no decrease in emission or isolation of products with two additional mass units was observed under these conditions.

These results are interesting since the compounds are analogues of substrates that

LovC is proposed to reduce during the biosynthesis of 2. Incubation of LovC with unmethylated tetraketides (49 and 60) also produces similar results. A Chapter 1 - Results and discussion 71 possible explanation is that LovC does not recognize the substrate analogues as their NAC derivatives. To test this hypothesis, tetraketide analogue 41 that lacks a NAC ester was incubated with LovC along with NADPH. Again no decrease in fluorescence was observed. However, its NAC derivative (61 - Figure 16), synthesized by the Tang lab, is a substrate of LovC, and decrease in emission and isolation of a product with two additional mass units was observed. These results indicate that LovC is catalytically active and accepts the NAC moiety for recognition of some substrates. Compounds 62 and 63 (Figure 16) were proposed to resemble the three putative biosynthetic intermediates that are reduced by

LovC. LovC with NADPH reduces substrates 62 and 63. LovC accepts compounds 62 and 63, possibly through recognition of the /-butyl group by residues in the active site. But is possible that LovC needs particular protein- protein interactions with LovB in order for reduction of some putative biosynthetic intermediates as NAC esters. Without these specific contacts, perhaps only substrates with /-butyl or aryl groups interact with key residues to enable reduction.

o o o H

o CI 61 62 63 Figure 16: Substrates for LovC in vitro assay

In summary, the purified LovC, which is catalytically active, is only able to reduce compounds with certain functional group arrangements. Substrate analogues 39, 40, 41, 45, 49, 54, and 60 are not reduced, which may be due to Chapter 1 - Results and discussion 72 lack of protein-protein interactions between LovC and LovB. Table 2 summarizes the results from the LovC in vitro enzyme assay. X-ray structural studies can be done with purified LovC, to elucidate the specific interactions that need to be made between LovC and LovB. Also, a crystal structure would allow for in-depth analysis of the active site as well as identification of the key residues required for reduction.

Decrease in fluoresence Substrate Reduced Product over 5 minutes ?

no o o 39

no O o 40

o o

OH OH no 41

no Chapter I - Results and discussion 73

Decrease in fluoresence Substrate Reduced Product over 5 minutes ?

n yes

yes CI ci 62

yes

Br Br

Table 2: Summary of results obtained from LovC in vitro assay. Fluorometric assay was done over a 5 min period where the decrease in fluorescence was measured at 455 nm

In conclusion, with the help from our collaborators (Professor Yi Tang,

Professor Nancy Da Silva, and Professor Sheryl Tsai), who were able to express and purify pure LovB and LovC, we were able to study the catalytic activity of Chapter 1 - Results and discussion 74

LovB and LovC through in vitro experiments. Full reconstitution of the biosynthetic pathway to 2 was accomplished via incubation of purified LovB with

LovC and all necessary cofactors. However, 2 was only obtained when LovB was treated with KOH or an exogenous TE domain (PKS13 and PKS4). These results indicate that the enzyme system is missing either a cofactor or an enzyme that off loads 2 from the ACP of LovB. Replacement of LovC with another ER enzyme from a similar biosynthetic pathway (MlcG) in the enzyme assay with all cofactors except SAM produces desmethyl-dihydromonacolin L (32) when KOH or PKS13 is present. LovC is not only an ER enzyme; it also possesses a gate keeping function in the biosynthesis of 2. Its homolog, MlcG from the compactin pathway, is capable of reducing both methylated and unmethylated tetraketides, to produce 2 and 32, respectively. LovC is less tolerant than MlcG and requires that a-methylation of the tetraketide occur before reduction; otherwise pyrones and hydrolytic products are produced. Investigation of the truncated NRPS region in

LovB indicates that it is not involved in off-loading of 2 from LovB via amide bond formation from an amino acid. Cloning and expression of LovB lacking the truncated NRPS region (LovB-AC) does not affect its catalytic activity, as evident from production of pyrones (3 to 6), ketones (9 and 12), and methylated pyrones

(7 and 8). However, when the C domain is added to LovB-AC as a stand-alone protein, dihydromonacolin L (2) is formed. Investigation of the substrate specificity of pure LovC reveals that it does not reduce putative substrate analogues 39 to 41, 45, 49, 54, and 60. It does however reduce tetraketide NAC analogue 61 and /-butyl aryl ketones 62 and 63. This suggests that LovC requires Chapter 1 - Results and discussion 75 intimate interactions with LovB in order for reduction of actual substrates to occur.

This work has investigated the biochemical programming of HR-PKS enzymes. There are still many questions that remain to be answered in lovastatin biosynthesis. What protein-protein interactions are required for LovC to reduce substrates attached to LovB? Is the C domain responsible for catalyzing the

Diels-Alder reaction? Why does LovC reduce an a-methylated tetraketide but not unmethylated penta- and heptaketides? What enzyme or cofactor is missing that cleaves 2 or 11 from LovB? With purified LovB and LovC enzymes in hand, further structural studies can be done to obtain three-dimensional pictures of each step along the biosynthetic pathway. The information obtained from these structures may answer these difficult questions. Chapter 2 - Biosynthetic Studies on Fumonisin Bi

2 Chapter 2: Biosynthetic Studies on Fumonisin Bi

2.1 Fumonisin Biosynthesis

Fumonisins are a class of mycotoxins produced by the fungus Fusarium verticillioides, which causes ear and stalk rot in corn plants.84"86 Ingestion of infected corn plants by animals results in development of fatal diseases such as cancer in rats and mice, leukoencephalomalacia in horses, and pulmonary edema oi q«7 no in pigs. ' ' These mycotoxins have also been suspected to cause human esophageal cancer.84 Understanding how nature assembles these fumonisin mycotoxins will potentially allow development of inhibitors or fungicides to alleviate losses in revenue in the agricultural industry.

F. verticillioides houses a type I iterative HR-PKS enzyme to biosynthesize four types of fumonisins: Bt, B2, B3, and B4, where Bi comprises

70% of the total content (Figure 17).85 The biogenic origin of every atom has been well established through isotopic feeding experiments and NMR studies; the 18- carbon backbone (C3-C20), along with the hydroxyl at C-3, is derived from acetate while the amino group along with C-l and C-2 originate from alanine.89'90

The two methyl groups at C-l2 and C-l6 are derived from SAM and the hydroxyl groups at C-5, C-10, C-14, and C-15 are from molecular oxygen.91'92 The two tricarballylic acids are believed to originate from the citric acid cycle.89 Chapter 2 - Biosynthetic Studies on Fumonisin Bi

tricarballylic acid ho2c co2h

r2 oh ho2c >

6o2ho

Fumonisin bi b2 b3 b4

ri OH H OH H

r2 OH OH H H

Figure 17: Chemical structures of fumonisin B series

The 17-gene fumonisin gene cluster was recently discovered by Proctor and coworkers (Figure 18).93"95 The gene FUM1 encodes the HR-PKS enzyme,

Fumlp, that contains seven domains: KS, AT, DH, MeT, KR, ER, and ACP.94

One unit of acetyl-CoA and eight units of malonyl-CoA are used by Fumlp to construct the 18-carbon backbone of the fumonisins.96

FUM7 FUM9 FUM1 FUM6 FUM8 10 11 12 13 14 15 16 17 18 19

-| 1 1 ^ 30 40 50 60

Figure 18: Fumonisin gene cluster in F. verticillioides

HR-PKS enzymes typically do not contain a TE domain or a chain-release mechanism to off-load the polyketide product from the ACP96 During fumonisin biosynthesis, the off-loading mechanism is through a carbon nucleophile Chapter 2 - Biosynthetic Studies on Fumonisin Bi generated by the decarboxylation of alanine, catalyzed by a pyridoxal-5'- phosphate (PLP) dependant enzyme, Fum8p.97 The gene FUM8 is predicted to encode a 2-oxoamino synthetase, which consists of a group of enzymes that catalyze condensation of acyl-CoA thioesters and amino acids (Scheme 39).93 The thioester cleavage mechanism follows the sequence: deprotonation, condensation, decarboxylation, and reprotonation, to afford the fumonisin backbone with the amino group attached. The length of the polyketide chain is thought to be governed by the chain-releasing mechanism, where Fum8p has strict substrate specificity for an 18-carbon backbone.97 Chapter 2 - Biosynthetic Studies on Fumonisin B/

Transimination 0 = j-o-P-OH OH

Deprotonation

x acetyl-CoA •P* M 8 x malonyl-CoA -hf? 9 .©3 2 x SAM

Condensation

H* transfer Decartx>xylation

Transinination

Scheme 39: Proposed PLP-dependant mechanism of off-loading 18-carbon fumonisin backbone from Fumlp Chapter 2 - Biosynthetic Studies on Fumonisin Bi

Post-PKS enzymes further decorate the fumonisin skeleton with alcohols and tricarballylic acids as shown in Scheme 40. The gene FUM13 was found to encode a NADPH-dependant ketoreductase through in vitro experiments using purified Fuml3p enzyme. Fuml3p is responsible for the reduction of the C-3 carbonyl, derived from acetate, to the alcohol during fumonisin biosynthesis.98

The genes FUM3, FUM6, and FUM12 have sequence similarity to heme- dependant cytochrome P450s and are responsible for installing the hydroxyl groups at C-5, C-10, C-14, and C-15.93 It is proposed that Fum6p is the first P450 to hydroxylate the fumonisin backbone, whereas the latter P450s are proposed to be responsible for generating each individual fumonisin. Genes FUM7, FUM10,

FUMll, and FUM14 are predicted to encode fatty acid-CoA synthetases, which are responsible for esterification of the tricarballylic acids onto alcohols at C-14 and C-15.99 Disruption of these fatty acid-CoA synthetase genes did not affect fumonisin production, however, the quantities of derailment products did increase suggesting its role in biosynthesis is relevant but not essential.99 The functions of several of the FUM genes in the fumonisin gene cluster remain unknown. Chapter 2 - Biosynthetic Studies on Fumonisin Bj

NHi [.Ma?] NH2

NADPH 'OH

Furn7p i Fum14p OH NH2 WommoHUI HjirriiTttniti* OR • NH2

OH OH OR OH O COJH FutnlOp n.A-A/COjH - , .

Fumonisin B4

OR OH OH I Fumonisin B, I Fumonisin B,

OR OH OH Fumonisin B,

Scheme 40: Proposed post-PKS steps to give fiimonisins B1-B4

2.2 Cytochrome P450 and Fum6p

Cytochrome P450 enzymes are a class of monooxygenases found in mammals, plants, fungi, and other organisms, that perform key reactions in metabolism and cellular processes.100 These potent enzymes catalyze a series of transformations that include: hydroxylation of hydrocarbons, epoxidation of double bonds, and oxidation of heteroatoms.101 P450s in living organisms utilize Chapter 2 - Biosynthetic Studies on Fumonisin B/ molecular oxygen (which is typically unreactive due to high-energy barriers) and convert it into a form that is capable of performing the required oxidation reactions.102 The general mechanism of P450 oxidation begins with an iron (II) species, A, that covalently bonds with molecular oxygen to produce intermediate

B (Scheme 41). Intermediate B accepts an electron and is protonated to afford peracid C. The peracid is protonated and the loss of water generates intermediate

D. The iron-oxo species D reacts with the substrate and produces the hydroxylated product. Product release along with reequilibration with water regenerates the P450 enzyme.101

R-H R-H R-H „OH O O N- -;N 02 N I N e' N—I——n I ^-Fe'L I "" I I H+ I ^Fe'l I N— —N N— —N N— —N ABC

,, 1H2° R-H

OH2 O N- -N R-H N—j——N N^—II— |>eFe<| l>e

Scheme 41: Proposed mechanism for hydroxylation

Recent sequencing and disruption mutant studies of the FUM6 gene revealed that it was required for fumonisin biosynthesis.93 Fumonisin production was abolished when genes FUM1 or FUM8 in F. verticillioides are disrupted to generate mutants &FUM1 and AFUM8 respectively. However, when AFUM1 or Chapter 2 - Biosynthetic Studies on Fumonisin Bi

A.FUM8 mutants were co-cultured with a AFUM6 mutant, hydroxylated products were detected by LC-MS.103 The metabolites contain between one and four hydroxyl groups attached to the fumonisin backbone without the tricarballylic esters, indicating the flexibility of Fum6p. A significant drawback of these studies is the lack of full structural characterization of the intermediates obtained, and it is unclear what the exact positions are for the installed hydroxyl groups. In-depth analyses of the cytochrome P450 genes has been hampered by lack of appreciable amounts of corresponding purified proteins for in vitro experiments. This chapter will focus on studying the purified P450 enzyme Fum6p, which is proposed to be responsible for installing the vicinal diol at C-14 and C-15 on the fumonisin backbone. Specifically, the experiments include analysis of the metabolites produced from the in vitro enzyme assay and characterization of their structures through synthesis of optically pure standards. Chapter 2 - Results and discussion 84

2.3 Results and discussion

2.3.1 In vitro experiments with Fum6p

This work is a collaboration with Professor Liangcheng Du from the

University of Nebraska-Lincoln. His group did the expression and isolation of

Fum6p in yeast microsomes. The following in vitro experiments were conducted in the Du lab. To investigate the catalytic activity of Fum6p, stearic acid and sphinganine were used initially as substrates. Stearic acid is a known substrate for many P450 enzymes, and it also mimics the 18-carbon fumonisin backbone.

Sphinganine was used as a substrate since it contains the 2-amino-3-hydroxy moiety found in the fumonisin B series (Figure 17), as well as the 18-carbon backbone. To determine if these two compounds are substrates of Fum6p, they were incubated separately with necessary cofactors (NADPH, FMN, and FAD) and the yeast microsomes containing Fum6p (Scheme 42). The reaction was quenched by the addition of ethanol and the microsomes were removed by centrifugation. The solvent was removed under vacuum and the residue was treated with the silylating reagent N, 0-bis(trimethylsilyl)trifluoroacetamide before analysis by GC-MS. Stearic acid and sphinganine were readily detected by GC-

MS, however, no hydroxylated products from either substrate were detected.

These results indicate that Fum6p does not recognize these substrates even though they contain structural features similar to fumonisins. These substrates do however, lack the methyl groups at C-12 and C-16, which might be required for recognition. Chapter 2 - Results and discussion 85

no hydroxylated products stearic acid FumCp

+ cofactors no hydroxylated products OH sphinganine

Scheme 42: In vitro experiments with Fum6p microsomes and stearic acid or sphinganine

To investigate this hypothesis, a 10-carbon carboxylic acid (64) with two methyl groups in their /^-configurations were synthesized by a postdoctoral fellow, Dr. Viji Moorthie in our group (Schemes 43 and 44). This truncated chain represents the left-half portion of the fumonisin skeleton.

n-BuLi 0 0 1)NaHMDS 0 o THF, -78 °C I 2) Mel X O NH + CI O N oeo/—"96% O\_J N' THF, -78 °C \_J Bn 85% "'Bn

0 Br LiAIH4 DMP © NaHMDS , + H0A^®Ph)3 Et20, 0 °C CH2CI2, 23 °C THF, -78 °C 58% 80% 43%

Pd/C, H2 EtOH, 23 °C 96%

Scheme 43: Synthesis of intermediate toward left-side portion of fumonisin Bi mimic 64 Chapter 2 - Results and discussion 86

1) SOCI2, CH2CI2, reflux 2) n-BuLi, THF, -78 °C O x O NH 1) NaHMDS ^Bn 2) Mel 60% over 2 steps THF, -78 °C 82%

H202, LiOH

THF/H20 (3:2), 0 °C 30% 64

Scheme 44: Synthesis of left-side portion fumonisin Bi mimic 64

The Du lab then incubated 64 with the Fum6p microsomes and all necessary cofactors, followed by silylation; analysis by GC-MS resulted in detection of four new peaks with retention times: 15.97, 16.19, 16.39, and 27.74 min (highlighted in red - Figure 19A), when compared to the GC-MS trace of

Fum6p alone (Figure 19B). The negative control reactions did not contain any peaks with identical retention times to the four new peaks highlighted in red.

Low-resolution MS of these four peaks reveal identical masses of 361.1 [M+H]+, which matches the calculated mass of 360.1 corresponding to a bis-TMS protected 64 with one hydroxyl group attached (Figure 20). These results indicate that the four peaks, with identical low-resolution masses, are possibly isomers with a hydroxyl group attached at different locations along the backbone of 64

(Scheme 45). It appears that Fum6p is quite flexible at hydroxylating the vicinal diol carbons along the fumonisin backbone. Oddly enough no dihydroxylated Chapter 2 - Results and discussion 87 products were detected by GC-MS (calculated mass of 448.3 for the TMS protected product).

16.54 10CH

5.97 17.45 27.60

15.41 27.96 27.74

10u 17.46 16.55

15.42 27.61 27.97

14 16 18 20 22 24 26 28 Time (min)

Figure 19: GC-MS analysis of reaction mixture. (A) Fum6p microsomes incubated with 64 and (B) Fum6p microsomes alone [negative control]. Chapter 2 - Results and discussion 88

mass = 216.3

OTMS TMSO TMSO mass = 360.6 "TMS B Peak: 15.97 min 16.19 min 16.39 min 27.74 min

361.1 361.1 361.1 361.2

U » i*«l m Jl «. . HI H 1 L.I 1 i ...... L, III il ,1 lUilJ .I i

Peak: 15.90 min 16.01 min 16.39 min 27.74 min

359.1 355.1 359.1 359.1 , p ) if'1'! '"I1 i, 300 400 300 400" 3C3o0 400 3itoo 40C m/z

Figure 20: (A) Structures and masses of 64 and four possible TMS-protected hydroxylated isomers. (B) Low-resolution MS of the four new peaks detected from Fum6p incubated with 64. (C) Peaks in the negative control with Fum6p alone. Chapter 2 - Results and discussion 89

97 104

Scheme 45: The four proposed hydroxylated compounds from the Fum6p enzyme assay

We also wanted to investigate if Fum6p could oxidize the NAC version

(65) of 64, which mimics the polyketide attached to the ACP of Fumlp.

Compound 65 was also prepared by Dr. Viji Moorthie, and its synthesis is shown in Scheme 46.

o hs^nY HO-

64 ,0^-^01 65

CH2CI2, 23 °C 44%

Scheme 46: Synthesis of left-side portion of fumonisin mimic attached to ACP

(65)

The Du lab incubated 65 with Fum6p microsomes along with all necessary cofactors and analysis by GC-MS revealed no hydroxylated products (Scheme

47). There are two key observations: (1) Fum6p is capable of hydroxylating a carbon chain that possesses two methyl groups with (^-configuration at correct Chapter 2 - Results and discussion 90 positions along the backbone and (2) the carbon chain is probably a carboxylic acid and not attached to the ACP of the PKS enzyme Fumlp. This is consistent with previous reports that cytochrome P450s in this system are post-PKS enzymes.93 It is proposed that Fum6p is responsible for providing the dihydroxylated product. However our results, show that Fum6p is only monohydroxylating, possibly because 64 is not the true Fum6p substrate. Certain key structural moieties may be lacking that Fum6p requires for furnishing the vicinal diol.

^ h 9 I Fum«p no hydroxylated JHNHH products 65 s. >

Scheme 47: No hydroxylated products are observed from the Fum6p enzyme assay with 65

Each isomer from the Fum6p enzyme assay with 64 was chemically synthesized to confirm its structure. The synthesis of isomer 80 is shown in

Schemes 43 and 44. Hexanoyl chloride was coupled with Evans' chiral auxiliary

66 in the presence of n-BuLi to afford 67, which was immediately methylated when treated with NaHMDS followed by Mel to yield 68. The chiral auxiliary was removed by reduction using LiAlH4 to furnish alcohol 69, which was oxidized to aldehyde 70 under Swera conditions. Aldehyde 70 was condensed with unstabilized Wittig reagent 71 to produce carboxylic acid 72 as a 9:1 mixture of cis/trans isomers. The carboxylic acid was transformed to the acid chloride in Chapter 2 - Results and discussion 91 the presence of oxalyl chloride to afford 73, which was then coupled to Evan's chiral oxazolidinone 66 to furnish 74. Compound 74 is a-methylated in the presence of NaHMDS and Mel to yield 75 (Scheme 48). The chiral auxiliary was removed in the presence of LiOH and H2O2 to afford acid 76, and subsequent treatment with I2 produced iodo-lactones 77 and 78 as a 3:1 mixture via iodolactonization. Iodo-lactone 77 was isolated by recrystallization of the diastereomeric mixture. The absolute stereochemistry of 77 was confirmed through X-ray crystallography (see Appendix A3). The iodine was removed using tributyltin hydride to afford lactone 79, which was opened in the presence of LiOH to yield isomer 80 (Scheme 49).

0 n-BuLi 0 1) NaHMDS 0 n THF, -78 °C I + .A O^NH N \_y 95% °UJ ' THF, -78 °C °\J Bn Bn 92% ''Bn 66 67 68

1)(COCI)2 2) DMSO n Br§ UAIH, 3) NEt3 + HO A^^-P(Ph)3 Et20, 0 °C CH2CI2, -78 °C 82% 69 92% 70 71

NaHMDS 1)(COCI)2

THF, -78 °C CH2CI2, 23 °C 53% quant. 72 9:1 cis/trans

n-BuLi o o 1) NaHMDS THF, -78 °C A 2) Mel O O A N 81% \ , THF, -78 °C 83% "'Bn 74 75 Scheme 48: Synthesis of intermediate 75 en route to predicted Fum6p hydroxylated isomer 80 Chapter 2 - Results and discussion 92

l2, MeCN

THF/H20 (3:2), 0 °C 81%

recrystallized hot hexanes

.ojr Bu3SnH, AIBN LiOH (2 eq.) toluene, reflux MeOH, 0 °C 93% quant. 79 Scheme 49: Synthesis of predicted Fum6p hydroxylation product isomer 80

The rationale for the 3:1 ratio of 77 and 78 can be explained through the iodolactonization mechanism (Figure 21). The most stable conformation of cis- carboxylic acid 76 is shown in Figure 21. The iodine can approach the olefin from two different sides. Approach from the more sterically hindered face yields iodonium ion A. Opening of the iodonium ion from the least sterically hindered face furnishes intermediate B and subsequently iodo-lactone C. Another conformation of intermediate C is lactone D, which has the alkyl chain in the stable pseudo-equatorial position. In contrast, approach of iodine from the least sterically hindered face generates iodonium ion E. Subsequent opening of the iodonium ion from the more sterically hindered face yields iodo-lactone F with an analogous conformation G. Conformation G has the alkyl chain in the less favored pseudo-axial position. Since iodonium ion formation is reversible (I2,

MeCN), intermediates A and E can interconvert.104' 105 However, due to the Chapter 2 - Results and discussion 93 favorable conformation of iodo-lactone D over G, it is proposed that the equilibrium is shifted to intermediate A. This proposed shift in the equilibrium results in the 3:1 ration of iodo-lactone 77 to 78.

A 76 E

h3 h c °~ -ch, .xrx ch3 O.

h O- -°-nh ?

77 3:1 ratio 78

Figure 21: Proposed mechanism of iodolactonization to form iodo-lactones 77 and 78

The synthesis of the isomer 84 is shown in Scheme 50. Starting from the optically pure iodo-lactone 77, treatment with 5.3 eq. of LiOH opens the lactone and releases an alkoxide anion. This anion displaces the iodine atom in an Sn2 Chapter 2 - Results and discussion 94 fashion to afford the epoxide. The carboxylate anion immediately attacks the epoxide in an Sn2 manner to afford the 5-membered hydroxy-lactone 81. The hydroxyl group was coupled with l,r-thiocarbonyldiimidazole to furnish 82.

Thiocarbonyl 82 undergoes Barton deoxygenation in the presence of tributyltin hydride to yield lactone 83, which was subsequently opened to produce isomer

84. S OH o lAl LiOH (5.3 eq.)

THF/H20 (3:2) toluene/pyr (2:1) 0 °C, 92% 60 °C, 36% 77 81 0 O^S Bu3SnH, AlBN LiOH (2.0 eq.) .0. o. toluene, reflux THF/H20 (1:1) 64% 92% 82 83

LiO OH 84 Scheme 50: Synthesis of predicted Fum6p hydroxylation product isomer 84

Isomer 98 was synthesized from the diastereomeric iodo-lactone mixture

77 and 78. They were treated with 5.3 eq. of LiOH to afford a mixture of hydroxy-lactones 81 and 85 (Schemes 51 and 52). The absolute stereochemistry of hydroxy-lactone 85 was confirmed through X-ray crystallography of its p- bromobenzoate (as seen in Appendix A4). The hydroxyl group was protected using 86 to yield O-benzyl lactones 87 and 88. These were reduced in the Chapter 2 - Results and discussion 95 presence of LiAlHU to furnish diols 89 and 90, which were separable by column chromatography (Scheme 51). The primary alcohol in diol 89 was protected by treatment with TBDMSC1 to give 91. The secondary alcohol was acylated with l,l'-thiocarbonyldiimidazole to yield compound 92. Barton deoxygenation and deprotection under acidic conditions to produced 93. Alcohol 93 was oxidized in the presence of DMP to aldehyde 94, and further oxidized via Pinnick oxidation to yield carboxylic acid 95. The benzyl group of 95 was removed through hydrogenation. Spontaneous cyclization gave the 6-member lactone 96. Opening of the lactone with 2.0 eq. of LiOH afforded isomer 97 (Scheme 52).

+ THF/H20 (3:2) Tf0H,Et20 0 °C, 92% 23 °C, 98% OH H

78 85

OBn

OBn 91% 89 90 .0. H

Scheme 51: Synthesis of intermediate 90 en route to predicted Fum6p

hydroxylated product isomer 97 Chapter 2 - Results and discussion 96

OBn OBn Xn TBDMSCI »0 £» HO' -Si-0 DMAP, CH2CIz DMAP, CH CI , OH OH 2 2 23 °C, 80% 23 °C, 91% 89 91

1) Bu3SnH, AIBN OBn OBn toluene, 60 °C -Si-O' 2) AcOH, THF, H20 (3:3:1) 85 °C, 40% over 2 steps 93

OBn DMP

CH2CI2, 23 °C NaCI02, f-BuOH 82% H20, 23 °C 94 89% 95

OH Pd/C, H2 LiOH (2.0 eg.) LiO EtOH, 23 °C THF/H20 (1:1) 13% quant. 96 97

Scheme 52: Synthesis of predicted Fum6p hydroxylated product isomer 97 from intermediate 89

Isomer 104 was synthesized in the same fashion as 97 using diol 90 as shown in Scheme 53. Time constraints have not allowed the testing of these standards yet, but studies are underway to compare the properties of synthetic standards to those of the proposed products. Chapter 2 - Results and discussion 97

OBn OBn TBDMSCI

DMAP, CH2CI2 DMAP, CH2CI2, 23 °C, 72% 23 °C, 91%

1) Bu3SnH, AIBN OBn OBn toluene, 60 °C -Si-0 2) AcOH.THF, H20 (3:3:1) >s 85 °C, 60% over 2 steps N 100 /> C N

DMP

NaCI02, f-BuOH CH2CI2, 23 °C 72% H20, 23 °C 101 89% 102

Pd/C, Hg LiOH (2.0 eg.)

EtOH, 23 °C THF/H20 (1:1) 29% quant. 103 104

Scheme 53: Synthesis of predicted Fum6p hydroxylated product isomer 104 from diol 90

In conclusion Fum6p has been successfully expressed in yeast microsomes and shows catalytic activity. Substrates containing the 18-carbon backbone and functional groups similar to those of fumonisins, such as stearic acid and sphinganine, are not oxidized by Fum6p. However, Fum6p is capable of hydroxylating a fumonisin mimic with two methyl groups having Re configurations at C-12 and C-16. The 10-carbon carboxylic acid 64, mimicking Chapter 2 - Results and discussion 98 the left-side portion of the fumonisins, is oxidized to furnish four products with identical masses that indicate monohydroxylation. These four metabolites are believed to be isomers, and to confirm their structures, chemical syntheses of optically pure standards was completed (80, 84, 97, and 104). Experiments are being conducted in the Du lab to match these to the four isomers from the Fum6p enzyme assay. Fum6p is proposed to furnish the vicinal diol. However from our results, it is only capable of monohydroxylation of 64. A possible explanation for this observation is that 64, which is not the true fumonisin substrate, lacks structural anchors that are necessary for recognition by Fum6p. Interestingly, the

NAC derivative 65 is not oxidized by Fum6p microsomes, indicating that the cytochrome P450 most likely does not recognize substrates attached to the ACP of the PKS enzyme, Fumlp. In vitro experiments with Fum6p are laying the groundwork for understanding the post-PKS assembly steps toward fumonisin biosynthesis. Further characterization of each gene in the fumonisin gene cluster will not only provide additional knowledge in fumonisin biosynthesis, but also shed light on HR-PKS enzymes. Chapter 3 - Biosynthetic Studies on Hypothemycin

3 Chapter 3: Biosynthetic Studies on Hypothemycin

3.1 Hypothemycin Biosynthesis

The fungus Hypomyces subiculosus biosynthesizes a nanomolar inhibitor of human kinases (human ERK2 and mitogen-activated protein kinase) called hypothemycin (105).'06 It is part of a resorcylic acid lactone family that includes radicicol107'108 and zearalenone (Figure 22).109

Radicicol Zearalenone Hypothemycin (105)

Figure 22: Resorcylic acid lactones

Hypothemycin is biosynthesized by a HR/NR-PKS hybrid where the HR-

PKS first constructs the reduced portion. This is then transferred to the NR-PKS to assemble the aromatic system and complete the core structure. Post-PKS enzymes then further transform the core structure to give 105. 108 The hypothemycin gene cluster has been recently sequenced, and is shown in Figure

23. 108 Pioneering• • work by Reeves and coworkers has elucidated functions for most of the genes in the cluster through gene disruptions and in vitro experiments.108 Genes hpm8 and hpm3 show sequence similarity to HR-PKS and

NR-PKS enzymes respectively, and expression of these two genes into a Chapter 3 - Biosynthetic Studies on Hypothemycin heterologous host was crucial to support their relevance to hypothemycin biosynthesis.

hpm2 hpm6 hpml hpm3 hpm4 hpm5 hpm7 hpm8 hpm9

( -4- 1— 5K 10K 15K 20K 25K 30K 35K

P450 PolyketkJe O-methyl FMO hydroxylase biosynthesis transferase epoxidase

Glutathione __ Unknown I J MFS Alcohol S-transferase ••• function IHM transporter BHB oxidase Figure 23: Hypothemycin gene cluster in H. subiculosus

The domain architecture of the HR-PKS and NR-PKS are shown in Figure

24. The domains in Hpm8 are typical of an iterative type I HR-PKS, except for the "core" domain, which is proposed to be for structural purposes and contains non-functional KR and MeT domains from a FAS.29 Hpm3 contains domains characteristic of an iterative NR-PKS such as: KS, MAT, ACP, and TE. There are two additional domains in Hpm3 called the starter unit:ACP transacylase

(SAT) and the product template (PT). The SAT domain is thought to be involved in communication and transfer of acyl substrates between Hpm8 and Hpm3. The

PT domain is proposed to stabilize and orient the poly-p-keto intermediate before it undergoes cyclization or aromatization.110

SAT 1 KS 1 MAT i PT i ACP I TE i MtHMWMitt J L T T Hpm8 Hpm3

Figure 24: Domain architecture of the HR-PKS (Hpm8) and the NR-PKS

(Hpm3) Chapter 3 - Biosynthetic Studies on Hypothemycin

It is hypothesized that Hpm8 incorporates units of acetyl-CoA and malonyl-CoA through a series of Claisen condensations, reductions, and dehydrations to assemble a hexaketide (106). The hexaketide is then transferred downstream to Hpm3 where three more rounds of chain extension occur, using three units of malonyl-CoA (without reduction or dehydration) to afford the first product released from the PKS enzymes, dehydrozearalenol 107 (Scheme 54).

Post-PKS enzymes further decorate 107 via phenol methylation (Hpm5), epoxidation (Hpm9), and hydroxylation (Hpml) to furnish aigialomycin C (108).

The particular order of these reactions is still under investigation. The next set of post-PKS enzymes further elaborate 108 through hydroxylation (Hpml), trans- to c/s-olefin isomerization (Hpm2), and oxidation (Hpm7) to afford 105. Again the exact order of these reactions is unclear. What is hindering further investigation into the biochemical programming involved in the biosynthesis of 105 is sufficient quantities of purified PKS enzymes for in vitro experiments. Therefore, the focus of this chapter will be on determining the catalytic role of each domain in Hpm8 and Hpm3, as well as reconstitution of the biosynthesis of dehydrozearalenol (107). Chapter 3 - Biosynthetic Studies on Hypothemycin

o AS-CoA

O O

S-CoA

OH O

2'\^ v "*OH (6'S, 10'S)-trans-7'-8'-dehydrozearalenol (107) HpmQ ' i'MHH f i

OH O OH o

MeO MeO OH OH OH Aigialomycin C (108) Hypothemycin (105)

Scheme 54: Proposed biosynthesis of 105 Chapter 3 - Results and discussion 103

3.2 Results and discussion

3.2.1 In vitro experiments with Hpm8

Our collaborator Professor Yi Tang from UCLA expressed and purified

Hpm8 (-1.5 mg/L) from S. cerevisiae (strain BJ5464-NpgA, the same strain of yeast used to express LovB).12 The following in vitro experiments were conducted in the Tang lab. To probe the catalytic activity of Hpm8, it was incubated with malonyl-CoA alone, and then the reaction was quenched by addition of ethyl acetate with 1% TFA. The solvent was removed under vacuum, and the residue was analyzed by LC-MS. The main product isolated has identical retention time,

UV spectrum, and low resolution MS as triketide pyrone 3 from the LovB enzyme assay (Scheme 55). Hpm8 is able to self-prime malonyl-CoA and catalyze decarboxylations and Claisen-condensations to produce the (3-keto intermediate.

However, NADPH is absent and the KR and ER domains are inactive. Therefore

Hpm8 stalls at the diketide stage, inducing its off-loading mechanism of cyclization to produce pyrone 3. The mechanism of off-loading is reminiscent of

LovB (Scheme 14).

Cofactors: Hpm8 triketide

Scheme 55: In vitro assay of Hpm8 with malonyl-CoA produces pyrone 3 Chapter 3 - Results and discussion 104

Addition of NADPH to the enzyme assay activates all domains in Hpm8, however hexaketide 106, which is proposed to be constructed, was not detected.

Instead pyrones proposed to be 109 and 113 were observed (Scheme 56). These results suggest that another enzyme is missing that is crucial for the biosynthesis of hexaketide 106. The proposed biosynthesis of derailment products 109 and

113 is shown in Scheme 57. Hpm8 is able to self-load malonyl-CoA and catalyze chain extensions and dehydrations to form the triketide. At this point a specific enzyme is absent from the enzyme assay (possibly Hpm3), which causes Hpm8 to stall and derail from the normal programmed steps. Off-loading of the shunt product occurs by the addition of another two carbon units, enolization, and pyrone formation to generate 113.

pentaketide- tetraketide Cofactors: Hpm8 pyrone pyrone

NADPH

Scheme 56: In vitro assay of Hpm8 with malonyl-CoA and NADPH to produce pentaketide pyrone 109 and tetraketide pyrone 113 Chapter 3 - Results and discussion 105

o o oh o

i KR *

p-keto intermediate diketide 1Imalonyl-CoA derailment - absence of Hpm3 oh o o normal programmed steps

malonyl-CoA malonyl-CoA x 3 r tnketide oh o o o

* \Uir*1

1) enolization I 2) pyrone formation malonyl-CoA x 2 OH O oh o oh 113

OH 1) enolization 2) pyrone formation 109

Scheme 57: Comparison of normal programmed steps with derailment steps when Hpm3 is absent to produce pyrones 109 and 113

To confirm the structure of proposed pyrone 113, a standard was synthesized as shown in Scheme 58. Starting with commercially available pyrone Chapter 3 - Results and discussion 106

3, the phenol was protected with benzyl bromide in the presence of potassium carbonate to afford 110. Compound 110 was condensed with acetyl chloride following deprotonation by LiHMDS to furnish compound 111. The ketone then underwent Corey-Bakshi-Shibata (CBS) reduction to yield 112 as a single enantiomer.111 The benzyl group was removed by hydrogenation to afford pyrone

113. Chemically synthesized pyrone 113 has an identical MS, LC-retention time, and UV spectrum as the proposed pyrone from the enzyme assay. Thus, pyrone

113 is confirmed to be the off-loaded product in Scheme 56. The pentaketide pyrone 109 was synthesized by a graduate student in our group, Mr. Zhizeng Gao, and was also found to match the corresponding shunt metabolite produced from the enzyme assay.

K2C03 LiHMDS

EtOAc, 23 °C 43% 0 85% OH 112 113 Scheme 58: Synthesis of predicted pyrone 113 from in vitro assay of Hpm8 with

NADPH

The key step in the synthesis of pyrone 113 is obtaining the desired hydroxy1 in its ^-configuration using the CBS reagent, which acts as both a Lewis acid and a chiral auxiliary (Scheme 59). The coordination of the nitrogen to the Chapter 3 - Results and discussion 107 borane makes it a stronger hydride donor, as well as increasing the Lewis acidity of the endocyclic boron. Addition of the ketone allows coordination of the boron to the least sterically crowded side of the ketone, in this case the methyl group.

Selective hydride delivery from the top face via a six-membered transition state occurs. Collapse of the intermediate and formation of the alcohol, by work-up, regenerates the CBS catalyst.82

BH3 Ph

BH3 • SM62

Scheme 59: Mechanism for CBS reduction of compound 111

3.2.2 In vitro experiments with Hpm8 and Hpm3

The Tang lab has also expressed and purified Hpm3 using S. cerevisiae to provide 2 mg/L of pure protein. To investigate its catalytic activity, Hpm8 and

Hpm3 were incubated in the Tang lab with malonyl-CoA and NADPH, and a new peak with a m/z coinciding with that of 107 was detected by LC-MS (Scheme 60).

Luckily, 107 could be isolated in appreciable quantities for NMR and X-ray crystallography analysis via S. cerevisiae transformed with expression plasmids for Hpm3 and Hpm8.108 We used NMR and X-ray crystallography to confirm the structure of 107 from the Hpm3 and Hpm8 enzyme assay. Surprisingly, the Chapter 3 - Results and discussion 108 crystal structure shows that the alcohols at C-10' and at C-6' both have identical stereochemistry (^-configuration) (107a). The structure of 107 was initially 113 proposed to be 107b. It was expected that the KR domain reduces with the same facial selectivity when biosynthesizing the hexaketide 114; this would give the C-6'R and a C-lO'S diastereomer 107b. However, the KR domain reduces the

C-6' carbonyl with opposite facial selectivity when constructing the hexaketide

106, to furnish 107a (Scheme 60). Mr. Zhizeng Gao of our group is currently investigating why the KR domain reduces the C-6' ketone with opposite facial selectivity. These results suggest that Hpm3 is catalytically active, as it can accept the biosynthesized hexaketide 106 from the HR-PKS Hpm8, and catalyze subsequent chain extension and macrolactonization to yield the nonaketide 107a.

Cofactors: Hpm8

o o ACP KR 1 Predicted HO-AAu, —-S-COA + it*KS cnEB| i Hpm3npma ij ••••••

NADPH

Experimental HO"< 6ft

HO- <10'S

HO-< 6'S OH O OH O

HO" ('OS

107b Scheme 60: In vitro assay of Hpm8, Hpm3, malonyl-CoA, and NADPH to biosynthesize dehdyrozearalenol 107a Chapter 3 - Results and discussion 109

Hpm3 and Hpm8 clearly interact to transfer the hexaketide 106 from the

HR-PKS (Hpm8) to the NR-PKS (Hpm3) to furnish 107a. It is predicted that the

SAT domain, present in Hpm3, is responsible for the crosstalk between PKS enzymes.113 To further examine the role of the SAT domain during the biosynthesis of 107a, the active site serine residue was mutated to alanine

(S121A) by the Tang lab. Incubation of Hpm8 and Hpm3 with the deactivated

SAT domain (Hpm3-SAT°), along with all necessary cofactors, abolishes production of 107a (Scheme 61). These results indicate that even though Hpm8 is proposed to be fully capable of biosynthesizing hexaketide 106, a key interaction is absent to allow transfer of 106 to Hpm3. This result is consistent with the previous experiment when Hpm8 is incubated with NADPH in the absence of

Hpm3, thereby preventing the formation of 107a. It now seems that the SAT domain is crucial for communication or transfer of biosynthesized intermediates between Hpm8 and Hpm3. Without the SAT domain, pyrones 3,109, and 113 are produced by Hpm8. Chapter 3 - Results and discussion 110

Cofactors: Hpm3-SAT°

0 o SAT0 TE OH S-CoA + O'1^0-^A NADPH 3

OH O

O^OA OH 3 109 o

OH 113 Scheme 61: In vitro assay of Hpm8 with Hpm3-SAT°, malonyl-CoA, and

NADPH to furnish pyrone 3. This product was identical to those obtained when

Hpm8 is incubated with NADPH

We next wanted to examine if Hpm3 can accept and self-prime using

NAC derivative 115. Incubation of Hpm3 with acetyl-CoA and malonyl-CoA did not produce any detectable polyketide products by LC-MS, indicating its inability to begin biosynthesis as a stand-alone protein (performed in the Tang lab).

However, incubation of Hpm3 with 115, (prepared by Mr. Zhizeng Gao) allows the assembly of 107a (Scheme 62). This result suggests that Hpm3 can self-prime a chemically synthesized hexaketide 115, and continue with the normal programmed steps toward 107a. Interestingly enough, incubation of 115 with

Hpm3-SAT° also furnishes 107a, as detected by LC-MS (Scheme 62). These results signify that although the SAT domain is critical for Hpm8 and Hpm3 to biosynthesize 107a, Hpm3-SAT° is able to capture 115, probably by direct Chapter 3 - Results and discussion 111 priming of its KS domain. The rest of its machinery is then fully functional to make 107a.

Cofactors: Hpm3 V=o SAT TE \ KS ACP

115 Scheme 62: In vitro assay of Hpm3 or Hpm3-SAT° with malonyl-CoA and

115 to furnish 107a

In conclusion, in the collaborative effort the HR/NR-PKS hybrid, Hpm8 and Hpm3 was expressed and purified. Hpm8 is catalytically active, as shown by detection of pyrones 3, 109, and 113, whose identity was confirmed by synthetic standards. Successful communication between Hpm3 and Hpm8 allows detection of 107a. Its structure was confirmed as 107a through NMR and X-ray crystallography. The protein-protein interactions between Hpm8 and Hpm3 are facilitated via the SAT domain, which is determined to be vital for the biosynthesis of 107a. Inactivation of the SAT domain in Hpm3 is not fatal for the latter biosynthetic steps toward 107a, if rescued by the chemically synthesized hexaketide 115. Chapter 3 - Results and discussion 112

Polyketides are a vast resource of structurally complex and medicinally important compounds. These factors alone have gathered substantial attention from the scientific community. They are produced by fungi, bacteria, and plants, although the focus of this thesis is fungal polyketides. Lovastatin, fumonisin Bi, and hypothemycin are produced by HR-PKS or HR/NR-PKS hybrid enzymes.

Through advances in molecular biology, we and our collaborations have been able to express and purify appreciable quantities of PKS proteins. In vitro experiments using these purified proteins have deconstructed some of the puzzling biochemical programming involved in the biosynthesis of these fungal polyketides. Detection of shunt metabolites by MS alone is not sufficient to confirm their identity. Therefore, the development of concise, efficient, and asymmetric synthetic routes is required to verify their structure. Our results demonstrate that many fungal PKS systems require two proteins. For example

LovB and LovC are necessary to complete synthesis of dihydromonacolin L (2) en route to lovastatin (1). Similarly, a highly-reducing PKS must cooperate with a non-reducing PKS to construct dehydrozearalenol (107a), the precursor to hypothemycin (105). Our studies show that if a partner protein is missing, the self loading PKS (LovB or Hpm8) uses chain-extension to enable pyrone formation with concomitant off-loading to regenerate active protein. The same process is observed if a cofactor is missing. This self-editing keeps the enzymes biosynthetically capable. Improvements in molecular biology, chemistry, mass spectrometry, and structural NMR techniques have enabled a look at the powerful biosynthetic machinery housed within living organisms. The ultimate goal is to Chapter 3 - Results and discussion 113 harness these enzymes to construct a wide range of molecular scaffolds with an array of functionality. In this way combinatorial biosynthesis can be brought to the forefront of drug discovery. Experimental procedures 114

4 Experimental procedures

4.1 General experimental methods

4.1.1 Reagents, solvents, and solutions

All reactions involving air or moisture sensitive reactants were conducted under a positive pressure of dry argon. All solvents, chemicals, biochemicals, and reagents were reagent grade and used as supplied unless otherwise stated. For anhydrous reactions, solvents were dried according to the procedures detailed in

Perrin and Armarego.114 Tetrahydrofuran and diethyl ether were distilled over sodium and benzophenone under at atmosphere of dry argon. Acetonitrile, dichloromethane, methanol, pyridine, and triethylamine were distilled over calcium hydride. Typical solvent removal was performed under reduced pressure, below 40 °C, using a Btichi rotary evaporator. Deionized water was obtained from a Milli-Q reagent water system (Millipore Co., Milford, MA). Unless otherwise specified, solutions of NH4C1, NaHCC>3, HC1, NaOH, KOH, Na2S2C>3, and LiOH refer to aqueous solutions. Brine refers to a saturated solution of NaCl.

4.1.2 Purification Techniques

All reactions and fractions from column chromatography were monitored by thin layer chromatography (TLC). Analytical TLC was done on glass plates (5 x

1.5 cm) precoated (0.25 mm) with silica gel (normal SiC>2, Merck 60 F254).

Compounds were visualized by exposure to UV light and by dipping the plates in

1% Ce(S04)2«4H20 2.5% (NH^MotC^HzO in 10% H2S04 followed by heating Experimental procedures 115 on a hot plate. Flash chromatography was performed according to the method of

Still et. al.1,5 on silica gel (EM Science, 60A, 230-400 mesh).

4.1.3 Instrumentation for compound characterization

Optical rotations were measured on Perkin Elmer 241 polarimeter with a microcell (10 cm, 1 mL) at 23°C. All specific rotations reported were referenced against air and were measured at the sodium D line and values quoted are valid within ±1°. Infrared spectra (IR) were recorded on a Nicolet Magna 750 with

Nic-Plan microscope FT-IR spectrometer. Cast refers to the evaporation of a solution on a NaCl plate. High resolution mass spectra were recorded on Krato

IMS-50 [high resolution electron impact ionization (HREI)], or Micromass

ZapSpec Hybrid Sector-TOF positive and negative mode electrospray ionization

[high resolution electrospray (HRES)] instruments. Nuclear magnetic resonance

(NMR) spectra were obtained on Varian Inova 400, Varian mercury 400, Varian

Inova 500, Varian 500 (equipped with cryo-probe), Varian 600 MHz spectrometers. 'H NMR chemical shifts are reported in parts per million (ppm) using the residual proton resonance of solvents as reference: CDCI3 8 7.24,

13 CD2CI2 5 5.32, D20 5 4.72, and CD3OD 8 3.30. C NMR chemical shifts are reported relative to CDCb 8 77.0, CD2CI2 8 53.8, and CD3OD 8 49.0. Selective homonuclear decoupling shift correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlations

(HMBC), heteronuclear single quantum coherence (HSQC), and attached proton test (APT) were used for signal assignments. Experimental procedures 116

4.2 Synthesis and characterization of compounds

(3E, 5E, 7£)-Nona-3,5,7-trien-2-one (9)

The known compound 9 was synthesized by an alternate route.116 In a flask under

Ar containing 0.25 g (6.24 mmol) of NaH (60% dispersion in oil) and 10 mL of diy THF was cooled to 0 °C. Then 1.11 g (5.72 mmol) of diethyl (2- oxopropyl)phosphonate was added dropwise to the slurry over 10 min and the mixture was allowed to stir for 15 min at 0 °C. A separate flask under Ar containing 0.50 g (5.20 mmol) of (2E, 4£)-hexa-2,4,-dienal was dissolved in 5 mL of dry THF and cooled to 0 °C. The mixture containing phosphonate was added to the aldehyde solution dropwise over 10 min, then the mixture was allowed to stir at 0 °C for 30 min and then was warmed to 23 °C overnight. The reaction was quenched with 20 mL of H2O, and the aqueous layer was extracted with hexanes

(3x15 mL). The combined organic layers were washed with 15 mL of brine, and then dried over Na2SC>4. The solvent was removed under vacuum and the residue was purified by column chromatography using a 9:1 hexane/Et20 eluent to afford

0.32 g of 9 as a yellow liquid (45%). IR (neat film) 3021, 1665, 1609 cm"1; 'H

NMR (500 MHz, CDCI3) 6 7.14 (ddd, 1H, J= 15.6, 11.1, 0.6 Hz, H-4), 6.58 (ddt,

1H, J= 14.8, 10.8, 0.6 Hz, H-5), 6.21 (ddp, 1H, J= 14.9, 11.1,0.7 Hz, H-6), 6.14

- 6.20 (m, 1H, H-7), 6.11 (d, 1H, J= 15.5 Hz, H-3), 5.97 (dq, 1H, J= 15.0, 6.9 Experimental procedures 117

Hz, H-8), 2.26 (s, 3H, H-l), 1.84 (dd, 3H, J = 6.9, 0.6 Hz, H-9); 13C NMR (125

MHz, CDClj): 5 198.4,143.6, 141.9, 135.6, 131.3, 129.5, 127.9, 27.3, 18.6; HREI m/z calcd. for C9H12O 136.0888, found 136.0886 [M]+.

(2E, 4E, 6£)-;Y-Methoxy-A-methyIocta-2,4,6-trienamide (10)

Compound 10 was synthesized using the method described by Netz, D. F. et. al."7 In a flask under Ar containing 0.58 g (14.5 mmol) of NaH (60% dispersion in oil) and 35 mL of dry THF was cooled to 0 °C. Then 2.99 g (12.4 mmol) of diethyl (N-methoxy-N-methylcarbamoylmethyl)phosphonate was added dropwise to the slurry over 10 min, the mixture was then allowed to stir at 0 °C for 30 min.

A solution of 1.00 g (10.4 mmol) of 2E, 4£)-hexa-2,4,-dienal dissolved in 3 mL of dry THF was added dropwise to the slurry over 15 min. The mixture was allowed to stir at 0 °C for 30 min and then at 23 °C overnight. The reaction was quenched by adding 50 mL of H2O and the aqueous layer was extracted with Et20 (3 x 25 mL). The combined organic layers were washed with 15 mL of brine then dried over Na2SC>4. The solvent was removed under vacuum, and the residue purified by column chromatography using a 3:1 hexane/EtOAc eluent to afford 1.76 g of

10 as a yellow liquid (93%). IR (neat film) 2966, 2936, 1650,1606 cm"1; !H NMR

(500 MHz, CDCI3) 6 7.34 (ddd, 1H, J = 15.0, 11.3, 0.6 Hz, H-3), 6.52 (ddt, 1H, J

= 14.9,10.7, 0.6 Hz, H-4), 6.43 (d, 1H, J = 15.0 Hz, H-2), 6.26 (ddt, 1H, J = 15.0,

11.4, 0.7 Hz, H-5), 6.15 (m, 1H, H-6), 5.92 (dq, 1H, J= 15.0, 7.0 Hz, H-7), 3.84 Experimental procedures 118

(s, 3H, H-10), 3.20 (s, 3H, H-9), 1.82 (dd, 3H, J= 6.9, 1.2 Hz, H-8); 13C NMR

(125 MHz, CDC13) 6 167.4, 143.5, 140.4, 134.3, 131.3, 128.2, 117.8, 61.7, 32.5,

+ 18.5; HREI m/z calcd. forCl0Hi5NO2181.1103, found 181.1098 [M] .

(2E, 4E, 6£)-Octa-2,4,6-trienal (11)

The known aldehyde ll118 was synthesized by an alternate route.119 In a flask under Ar containing 0.75 g (4.13 mmol) of 10 was dissolved in 15 mL of dry EtaO and cooled to -40 °C. Then 2.69 mL (5.38 mmol) of LiAlH4 (2M solution in

THF) was added dropwise and the reaction mixture was allowed to warm to 23 °C over 1 h. The reaction was quenched by adding 20 mL of 1M HC1 dropwise, and the aqueous layer was extracted with Et20 (3x15 mL). The combined organic layers were washed with 15 mL of brine and then dried over Na2S04. The solvent was removed under vacuum, and the crude liquid 11, was taken directly to the next step without further purification. Experimental procedures 119

(3E, 5E, IE, 9£)-Undeca-3,5,7,9-tetraen-2-one (12)

In a flask under Ar containing 0.13 g (3.28 mmol) of NaH (60% dispersion in oil) was added to 25 mL of dry THF and cooled to 0 °C. Then 0.58 g (3.00 mmol) of diethyl (2-oxopropyl)phosphonate was added dropwise to the slurry over 10 min, and the mixture was then allowed to stir for 15 min at 0 °C. In a separate flask under Ar containing 0.33 g (2.73 mmol) of aldehyde 11 was dissolved in 10 mL of dry THF and cooled to 0 °C. Then the mixture containing phosphonate was added to 11 dropwise over 10 min, and the solution was allowed to stir at 0 °C for

30 min and then warm to 23°C overnight. The reaction was quenched with 20 mL of H2O, and the aqueous layer was extracted with hexanes (3x15 mL). The combined organic layers were washed with 15 mL of brine and then dried over

Na2S(>4. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 6:1 hexane/Et20 eluent to afford 0.10 g of 12 as a yellow solid (22%). IR (microscope) 3012, 2926, 1671, 1584 cm'1; 'H NMR

(500 MHz, CDCI3) 5 7.16 (dd, 1H, J= 15.5, 11.2 Hz, H-4), 6.62 (dd, 1H,J =

14.9, 11.2 Hz), 6.40 (dd, 1H, J= 14.9, 10.7 Hz), 6.30 (dd, 1H, Hz, J= 14.8, 11.2

Hz), 6.20 (dd, 1H,J= 14.9, 11.2 Hz), 6.15 (m, 1H), 6.12 (d, 1H, J= 15.5 Hz, H-

3), 5.88 (dq, 1H, J= 14.8, 6.9 Hz, H-10), 2.30 (s, 3H, H-l), 1.83 (d, 3H, J= 6.8

Hz, H-l 1); 13C NMR (125 MHz, CDCI3) 5 198.4, 143.5, 141.9, 137.9, 133.5, Experimental procedures 120

131.6, 129.5, 129.4, 129.3, 27.3, 18.6; HREI m/z calcd. for C,iH,40 162.1045, found 162.1044 [Mf.

2-(3-BromopropyI)-l,3-dioxolane (14)

The known compound 14 was synthesized as described.120 In a flask under Ar containing 19.0 g (97.4 mmol) of ethyl 4-bromobutyrate was dissolved in 120 mL of dry CH2CI2, and then was cooled to -78 °C. Then 97.5 mL (97.4 mmol) of

DIBAL (1M in CH2CI2) was added dropwise through an addition funnel. The reaction mixture was allowed to stir for 1 h at the same temperature. The reaction was quenched by the slow addition of 1M HC1, and the mixture was stirred until 2 clear layers formed. The aqueous layer was extracted with CH2CI2 (3 x 20 mL).

The combined organic layers were washed with 25 mL of brine, and then dried over Na2SC>4. The solvent was removed under vacuum, and the residue was redissolved in 280 mL of benzene, with the addition of 40.9 mL (730 mmol) of ethylene glycol and 0.74 g (3.89 mmol) of /?-TsOH. This mixture was heated to reflux with a Dean-Stark adapter at 95 °C for 3 h. The mixture was cooled to

23°C, and 5 g of NaHCCH was added with stirring for 10 min. The organic layer was washed with 50 mL of saturated NaHCCb and dried over K2CO3. The solvent was removed under vacuum to afford 14.0 g of 14 as a colourless liquid (74%). IR

(microscope) 2955, 2875 cm-1; 'H NMR (400 MHz, CDClj) 6 4.91 (t, 1H, J- 4.6 Experimental procedures 121

Hz, H-4), 3.98 (m, 2H, H-5), 3.88 (m, 2H, H-6), 3.48 (d, 2H, J = 6.7 Hz, H-l),

13 2.02 (m, 2H, H-2), 1.34 (m, 2H, H-3); C NMR (125 MHz, CDC13) b 103.9, 65.2,

81 33.8, 32.5, 27.4; HREI m/z calcd. for C6H10 BrO2 194.9843, found 194.9842

[M]+

2-[(5£, 7£)-Nona-5,7-dienyl]-l, 3-dioxolane (16)

171 177 f\f\ The known compound 16 ' was synthesized by an alternate method. In a flask under Ar containing 0.36 g (12.6 mmol) of Mg metal and 8 mL of dry THF was added (by addition funnel) 1.64 g (8.39 mmol) of 14 dissolved in 7 mL of dry

THF. The solution of 15 was added slowly to initiate the Grignard reaction over

1.5 h at 30°C. Then the slurry was stirred for another hour at the same temperature. Another flask under Ar containing 1.04 g (6.96 mmol) of (2E, 4E)-

2,4-hexadienyl acetate, 2.23 mL (0.22 mmol) of Li2CuCl4 (0.1M in THF), and 10 mL of dry THF was cooled to -30°C. The Grignard solution was then added to the cooled solution via cannula within 5 min, and the dark purple solution was stirred at the same temperature for 3 h. It was then warmed to 23 °C overnight.

The reaction was quenched by adding 20 mL of saturated NH4CI, and the mixture was stirred until 2 clear layers formed. The layers were separated, and the aqueous layer was extracted with Et20 (3x15 mL). The combined organic extracts were washed with 15 mL of brine, and then dried over Na2SC>4. The solvent was Experimental procedures 122 removed under vacuum. The residue was dissolved in 100 mL of 3:1 MeOH/THF and 4.0 g of NaOH was added. The mixture was stirred at room temperature for 2 h to hydrolyze remaining (IE, 4£)-2,4-hexadienyl acetate. A 1:1 mixture of saturated NaHCOj/^O (50 mL) was added, and the aqueous layer was extracted with Et20 (3 x 20 mL). The combined organic extracts were washed with 25 mL brine and dried over Na2SC>4. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 95:5 hexane/Et20 eluent to afford 0.56 g of 16 as a colorless oil (84%). IR (microscope) 3015, 2927,2880,

1 2860 cm" . 'H NMR (600 MHz, CDC13) 8 6.0 (m, 2H, H-3, H-4), 5.55 (m, 2H, H-

2, H-5), 4.84 (t, 1H, J = 4.8 Hz, H-10), 3.98 (m, 2H, H-lla, H-12a), 3.85 (m, 2H,

H-l lb, H-12b), 2.05 (m, 2H, H-6), 1.73 (dd, 3H, J = 6.8, 0.7 Hz, H-l), 1.65 (m,

,3 2H, H-9), 1.43 (m, 4H, H-7, H-8); C NMR (125 MHz, CDC13) 8 132.0, 131.9,

130.7, 127.1, 104.9, 65.1, 34.0, 32.7, 29.6, 23.9, 18.2; HREI m/z calcd. for

+ C,2H2o02 196.1463, found 196.1463 [M] .

(6E, 8£)-Deca-6,8-dienal (17)

The known aldehyde 17123 was synthesized using an alternative route.124 A flask containing 75 mL of THF, 93 mL of H20, 47 mL of AcOH, and 5.20 g (26.6 mmol) of 16 was heated to reflux at 90°C for 2.5 h. The solution was cooled to 23

°C, and 150 mL of saturated NaHC03 was added. The mixture was allowed to stir Experimental procedures 123 until bubbling ceased. The aqueous layer was extracted with hexanes (4 x 25 mL), and the combined organic extracts were washed with 40 mL of brine and dried over Na2SC>4. The solvent was removed under vacuum to afford 3.98 g of

17 as a colorless oil (98%). IR (microscope) 2930, 2868, 1712 cm"1. *H NMR

(400 MHz, CDC13) 6 9.78 (t, 1H, J= 1.9 Hz, H-10), 6.01 (m, 2H, H-4, H-3), 5.59

(m, 2H, H-5, H-2), 2.42 (dt, 2H, J= 9.1, 1.9Hz, H-9), 2.10 (q, 2H,J= 7.2 Hz, H-

6), 1.78 (d, 3H, J= 6.1 Hz, H-l), 1.63 (m, 2H, H-8), 1.42 (m, 2H, H-7); 13C NMR

(100 MHz, CDCI3) 6 202.8, 131.7, 131.2, 131.1, 127.4, 43.9, 32.4, 29.1, 21.8,

+ 18.2; HREI m/z calcd. for C,oH160 152.1201, found 152.1201 [M] .

(IE, HE, 10£)-Dodeca-2,8,10-trienal (19)

8 10 12

The known aldehyde 1968 was synthesized by an alternative route.67 A flask under

Ar containing 24.4 g (56.9 mmol) of (l,3-dioxolan-2- ylmethyl)triphenylphosphonium bromide and 75 mL of dry THF was cooled to

0°C. Then 68.2 mL (68.2 mmol) of KO'Bu (1M in THF) was added slowly (upon which the slurry turns yellow). The mixture was allowed to stir at that temperature for 30 min. Then 6.56 g (43.1 mmol) of 17 diluted with 20 mL of dry

THF was added to the slurry over 20 min, at which point the solution turns orange. This mixture was stirred at 0°C for 2 h. It was then hydrolyzed by adding

400 mL of 10% oxalic acid (42 g in 400 mL of H2O), and stirring at 23 °C for 1 h. Experimental procedures 124

The mixture was extracted with Et20 (4 x 50 mL) and the combined organic layers were washed with 100 mL of saturated NaHCC>3, 50 mL of brine, then dried over Na2SC>4. The solvent was removed under vacuum and purified by column chromatography using a 97.5:2.5 hexanes/Et20 eluent to afford 4.68 g of aldehyde 19 as an oil (61%). IR (microscope) 3013, 2927, 2855, 1692 cm"1; 'H

NMR (500 MHz, CDC13) 6 9.53 (d, 1H, J= 8.2 Hz, H-12), 6.84 (dt, 1H, J= 15.6,

6.8 Hz, H-10), 6.12 (ddt, 1H, J= 15.6, 7.9, 1.5 Hz, H-ll), 6.00 (m, 2H, H-3, H-4),

5.63 - 5.48 (m, 2H, H-5, H-2) 2.35 (q, 2H, J= 6.9 Hz, H-9), 2.08 (q, 2H, 7.2

Hz, H-6), 1.73 (d, 3H, J= 6.1 Hz, H-l), 1.52 (m, 2H, H-8), 1.44 (m, 2H, H-7); 13C

NMR (100 MHz, CDCI3) 6 194.0, 158.6, 133.1, 131.5, 131.1, 130.8, 127.2, 32.6,

32.2, 28.8, 27.3, 17.9; HREI m/z calcd. for C,2Hi80 178.1357, found 178.1353

[M]+.

(15, 25, 4a/?, 8aiS)-2-Methyl-l, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-l- carbaldehyde (21)

5 H 4 4a

The known compound 21125 was synthesized by an alternate route.68 In a vial containing 0.85 g (3.45 mmol) of (2S, 5S)-5-benzyl-2-/er/-butyl-3- methylimidazolidin-4-one was added 0.31 mL (3.45 mmol) of trifluoromethane sulfonic acid and 0.83 mL of 2% v/v H2O/CH3CN. This catalyst mixture was added to a flask containing 3.07 g (17.3 mmol) of triene 19 that had been cooled to -5°C, and the solution was shaken for 48 h. The solvent was removed under Experimental procedures 125 vacuum, and the residue was purified by column chromatography using a 97.5:2.5 hexanes/Et20 eluent, to yield 2.13 g of 21 as a 4:1 mixture of endo/exo isomers as a colorless oil (69%). Diasteromeric mixture 21 was then dissolved in 25 mL of anhydrous EtOH and cooled to 0°C. Then 0.68 g (18.0 mmol) of NaBH4 was added slowly, and the mixture was stirred for 1 h at the same temperature. Once the reaction was complete, the majority of the EtOH was removed under vacuum, and 25 mL of 1M citric acid was slowly added. The mixture was diluted with 15 mL of Et20 and stirred until 2 clear layers formed. The layers were separated and the aqueous layer was extracted with Et20 (3 x 20 mL). The combined organic layers were washed with 20 mL of water, 20 mL of brine, and then dried over

Na2S04. The solvent was removed under vacuum to yield a white solid, which was then recrystallized with hot pentane. The resulting diastereomerically pure alcohol (1.0 g, 5.50 mmol), 23, was dissolved in 25 mL of CH2CI2. To this was slowly added 3.03 g (7.14 mmol) of Dess-Martin periodinane, and the mixture was stirred for 30 min at 23 °C. A 1:1 mixture of saturated NaHC03/lM Na2S203

(30 mL) was added and the mixture was stirred until 2 clear layers formed. The layers were separated and the aqueous layer was extracted with CH2CI2 (3 x 20 mL). The combined organic layers were washed with 15 mL of saturated

NaHC03, 15 mL of brine, and then dried over Na2S04. The solvent was removed under vacuum, and the residue was purified by column chromatography using a

97.5:2.5 hexanes/Et20 eluent to yield 0.92 g of 21 as a colorless oil (43% over 2

1 steps). IR (film) 2927, 1715, 1452, 1267 cm" ; 'H NMR (500 MHz, CD2C12) 5

9.18 (d, 1H, J= 4.2 Hz, H-9), 5.56 (ddd, 1H, J= 9.9, 4.5, 2.7 Hz, H-3), 5.43 (d, Experimental procedures 126

1H, J= 9.8 Hz, H-4), 2.59 (m, 1H, H-2), 2.36 (ddd, 1H, J= 11.4, 6.1, 4.2 Hz, H-

1), 1.84-1.55 (m, 6H, H-4a, H-8a, H-8, H-5), 1.42-1.20 (m, 4H, H-7, H-8), 1.04

13 (d, 3H, J = 7.1 Hz, 2-CH3); C NMR (125 MHz, CD2C12) 6 206.9, 131.7, 131.3,

55.9, 42.6, 35.9, 33.4, 32.1, 30.5, 27.0, 26.9, 17.1; HREI m/z calcd. for C,2Hi80

178.1357, found 178.1355 [M]+.

Ethyl 3-KLS1,25,4a/f, 8a5)-2-methyl-l, 2,4a, 5,6, 7,8,8a-octahydronapthalen- l-yl]acrylate (24)

n | 12 8a po

l6 I J 5 H 4 4a

A flask under Ar containing 0.08 g (1.92 mmol) of NaH (60% dispersion in oil) and 25 mL of dry THF was cooled to 0°C. Then 0.36 g (1.60 mmol) of triethyl phosphonoacetate was added slowly to the slurry (with the evolution of H2 gas).

This was stirred at 0°C for 30 min. A solution containing 0.23 g (1.28 mmol) of

21 in 5 mL of dry THF was slowly added to the mixture and allowed to stir at 0°C for 30 min, and then warmed to 23 °C overnight. The reaction was quenched by adding 10 mL of saturated NH4CI, and the aqueous layer was extracted with Et20

(3x15 mL). The combined organic layers were washed with 10 mL brine and then dried over Na2SC>4. The solvent was concentrated under vacuum, and the residue was purified by column chromatography using a 95:5 hexanes/Et20 eluent Experimental procedures 127 to afford 0.29 g of 24 as a colorless oil (91% cis/trans = 2:3). IR (Microscope)

1 2926, 2854, 1722, 1650 cm- ; 'H NMR (500 MHz, CDC13) (trans isomer) 6 6.93

(dd, 1H, J= 15.6, 10.5 Hz, H-9), 5.83 (dd, 1H, J= 15.6, 0.6 Hz, H-8), 5.57 (ddd,

1H, J = 9.9, 4.5, 2.7 Hz, H-3), 5.41 (d, 1H, J = 9.8 Hz, H-4), 4.2 (q, 2H, J = 7.1

Hz, H-12), 2.35 (m, 1H, H-2), 2.27 (m, 1H, H-l), 1.79-1.65 (m, 6H, H-4a, H-8a,

H-8, H-5), 1.29 (t, 3H, J = 7.1 Hz, H-l3), 1.30-1.23 (m, 4H, H-7, H-8), 0.98 (d,

13 3H, J = 7.1 Hz, 2-CH3); C NMR (125 MHz, CDC13) 5 166.4, 151.9, 131.8,

131.1, 121.9, 119.9, 60.1, 47.2, 42.9, 38.4, 35.7, 33.1, 30.9, 26.7, 26.6, 16.5;

+ HRES m/z calcd for Ci6H2402Na 271.1669, found 271.1667 [M+Na] .

Methyl 3-[(15, 25, 4a/?, 8a5)-2-methyl-l, 2, 4a, 5, 6, 7, 8, 8a- octahydronapthalen-l-yI]propanoate (25)

5 H 4 4a

Compound 25 was prepared following an analogous procedure of Hudlicky et. al.126 In a flask under Ar containing 0.71 g (2.87 mmol) of 24, 20 mL of dry

MeOH, and 0.70 g (28.73 mmol) of Mg was stirred until vigorous bubbling ensued. The solution was then cooled using an ice bath to control the rate of the reaction. After 3 h, the slurry was acidified with 30 mL of 1M HC1 and stirred until the Mg dissolved. The aqueous layer was extracted with Et20 (3x15 mL).

The combined organic extracts were washed with 15 mL of saturated NaHCC>3, 15 Experimental procedures 128

mL of brine, and then dried over Na2SC>4. The solvent was removed under vacuum, and the oily residue was purified by column chromatography using a

97.5:2.5 hexanes/Et20 eluent to afford 0.62 g of 25 as a colorless oil (92%). IR

(microscope) 2920, 2853, 1741 cm-1; 'H NMR (600 MHz, CDCI3) 5 5.58 (ddd,

1H, J= 9.9, 4.5, 2.7, H-3), 5.35 (d, 1H, J= 9.8 Hz, H-4), 3.68 (s, 3H, H-12), 2.39

(ddd, 1H, J = 15.6,10.4, 5.3 Hz, H-lOa), 2.22 (m, 1H, H-lOb), 2.20 (ddd, 1H, J =

15.5, 10.0, 6.3 Hz, H-4a), 1.93 (m, 1H, H-2), 1.82 (m, 2H, H-9), 1.69 (m, 3H, H-

1, H-5), 1.48 (ddt, 1H, J = 21.9, 11.1, 4.7 Hz, H-8a), 1.4-1.2 (m, 4H, H-8, H-6),

13 1.1-1.0 (m, 2H, H-7), 0.85 (d, 3H, J = 7.0 Hz, 2-CH3); C NMR (100 MHz,

CDCI3) 6 174.7, 132.5, 131.5, 51.8, 44.2, 41.5, 39.5, 33.2, 32.3, 32.1, 29.4, 27.2,

26.8, 25.3, 15.1; a" = 69.59 (c = 1.00, CHCI3); HRES m/z calcd for C,5H2402Na

259.1669, found 259.1668 [M+Na]+.

3-[(lS, 2S, 4ai?, 8aS)-2-Methyl-l, 2, 4a, 5, 6, 7, 8, 8a-octahydronapthalen-l- yl]propanal (27)

5 H 4 4a

Compound 27 was prepared using the same procedure used to synthesize compound 14. In a flask under Ar containing 0.62 g (2.63 mmol) of 25 was dissolved in 25 mL of dry CH2CI2. This was cooled to -78°C. A solution of 3.95 mL (3.95 mmol) of DIBAL (1M in CH2CI2) was added slowly to the mixture, Experimental procedures 129 which was then stirred at -78°C for 1 h. The reaction was quenched by adding 20 mL of saturated sodium potassium tartrate, and the mixture was stirred until 2 clear layers formed. The layers were separated, and the aqueous layer was extracted with Et20 (3 x 15 mL). The combined organic extracts were washed with 15 mL of brine and then dried over Na2S(>4. The solvent was removed under vacuum, and the oily residue was purified by column chromatography using a

95:5 hexanes/Et20 eluent to afford 27 as a colorless oil. Eluting the column further with 1:1 EtOAc/hexanes gave the corresponding alcohol 26. In a flask containing 0.23 g (1.10 mmol) of 26 dissolved in 20 mL of CH2CI2 was added

0.6lg (1.43 mmol) of Dess-Martin periodinane. This was stirred at 23 °C for 30 min. A 1:1 mixture of saturated NaHCCVIM Na2S203 (20 mL) was added, and the mixture was stirred until 2 clear layers formed. The layers were separated, and the aqueous layer was extracted with CH2C12 (3 x 10 mL). The combined organic layers were washed with 10 mL of saturated NaHCC>3, 10 mL of brine, and then dried over Na2S04. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 95:5 hexanes/Et20 eluent to afford 0.41 g of 27 as a colorless oil (80% over 2 steps). IR (film) 2931, 2853,

1 1727 cm" ; 'H NMR (500 MHz, CDC13) 5 9.79 (t, 1H, J = 2.1 Hz, H-ll), 5.57

(ddd, 1H, J= 9.9, 4.5, 2.7 Hz, H-3), 5.37 (d, 1H, J= 9.8 Hz, H-4), 2.5 (m, 1H, H-

10a), 2.38 (m, 1H, H-lOb), 2.22 (m, 1H, H-2), 1.94 (m, 1H, H-4a), 1.90-1.59 (m,

5H, H-l, H-5, H-9), 1.49 (ddt, 1H, J = 21.9, 11.1, 4.7 Hz, H-8a), 1.40-1.20 (m,

13 4H, H-6, H-8), 1.15-0.98 (m, 2H, H-7), 0.85 (d, 3H, J = 7.1 Hz, 2-CH3); C

NMR (100 MHz, CDCI3) 6 203.1, 132.3, 131.7, 44.2, 42.1, 41.5, 39.6, 33.6, 32.1, Experimental procedures 130

29.4, 27.2, 26.7, 21.2, 15.1; a% = 59.43 (c = 1.00, CHC13); HREI m/z calcd. for

+ C,4H220 206.1671, found 206.1676 [M] .

(/?)-l-[(lS,2S, 4a/?, 8aS)-2-Methyl-l, 2, 4a, 5, 6, 7, 8, 8a-octahydronapthalen-

1 -yljhex-5-en-3-ol (28)

12 14c ,xOH 13 11 9, I 8a ^10 H 1^8 1 (6 K 5 H 4 4a

Compound 28 was prepared following the method of Racherla.127 A flask under

Ar containing 2.52 mL (2.53 mmol) of (-)-Ipc2B(allyl)borane (1M in pentane) and

10 mL of dry Et20 was cooled to -100°C. In another flask under Ar, a mixture of

5 mL of dry Et20 and 0.44 g (2.11 mmol) of 27 was cooled to -78°C. The solution of 27 was then added to the (-)-Ipc2B(allyl)borane solution via cannula and allowed to stir at -100°C for 2 h. The mixture was then treated with 0.93 mL

(2.78 mmol) of 3M NaOH and 0.93 mL of H2O2 (30% w/w), and allowed to warm to 23 °C and stirred for 4 h. The aqueous layer was extracted with Et20 (3x10 mL), and the combined organic layers were washed with 10 mL of water, 10 mL of brine, and then dried over Na2SC>4. The solvent was concentrated under vacuum, and the oily residue was purified by column chromatography using a 9:1 hexanes/Et20 eluent to afford 0.43 g of 28 as a colorless oil (81%). IR (film)

3349, 2920, 2854, 1640 cm-'; 'H NMR (500 MHz, CDCI3) 6 5.85 (m, 1H, H-13), Experimental procedures 131

5.60 (ddd, 1H,J= 9.9, 4.5, 2.7 Hz, H-3), 5.35 (d, 1H, J= 9.8 Hz, H-4), 5.18-5.12

(m, 2H, H-14), 3.65 (m, 1H, H-ll), 2.32 (m, 1H, H-12a), 2.27 (m, 1H, H-2), 2.18

(m, 1H, H-4a), 1.82 (m, 2H, H-12b, H-l), 1.80-1.40 (m, 6H, H-10, H-8a, H-5,

OH), 1.38-1.30 (m, 1H, H-6a), 1.30-0.90 (m, 7H, H-7, H-8, H-9, H-6b), 0.85 (d,

13 3H, J = 7.1 Hz, 2-CH3); C NMR (100 MHz, CDCI3) 5 135.2, 132.8, 131.6,

118.4, 71.2, 44.3, 42.4, 41.9, 39.7, 34.5, 33.7, 32.3, 29.5, 27.2, 26.8, 24.5, 15.1; a* = 47.49 (c = 1.00, CHCI3); HREI m/z calcd. for C,7H280 248.2140, found

248.2139 [M]+.

(J?)-1-[(1S, 2S, 4aR, 8aS)-2-Methyl-l, 2, 4a, 5, 6, 7, 8,8a-octahydronapthalen- l-yl]hex-5-en-3-yl aerylate (29)

5 H 4 4a

1 7R Compound 29 was synthesized following the protocol of Inoue et. al. A flask under Ar containing 10 mL of dry CH2CI2, 0.43 g (1.72 mmol) of 28, and 0.58 mL

(3.31 mmol) of DIPEA was cooled to 0°C. Then 0.22 mL (2.78 mmol) of acryloyl chloride was added slowly, and the mixture was stirred for 2 h at 0°C.

The reaction was quenched by adding 10 mL of saturated NH4CI, and the aqueous layer was extracted with CH2CI2 (3x10 mL). The combined organic layers were Experimental procedures 132 washed with 10 mL of brine and dried over Na2SC>4. The solvent was removed under vacuum, and the yellow residue was purified by column chromatography using 9:1 hexane/Et20 eluent yield 0.47 g of 29 as a colorless liquid (91%). IR

1 (microscope) 2921, 2855, 1724, 1638 cm" ; 'H NMR (600 MHz, CDC13) 6 6.37

(dd, 1H, J = 17.4, 1.5 Hz, H-17a), 6.11 (dd, 1H, J = 17.3, 10.3 Hz, H-16), 5.85

(dd, 1H, J = 10.4, 1.5 Hz, H-17b),5.76 (m, 1H, H-13), 5.57 (ddd, 1H,J=9.9, 4.5,

2.7 Hz, H-3), 5.34 (d, 1H, J= 9.8 Hz, H-4), 5.08 (m, 2H, H-14), 4.98 (m, 1H, H-

11), 2.38 (m, 2H, H-12), 2.22 (m 1H, H-4a), 1.79 (d, 2H, J = 12.1 Hz, H-2, H-l),

1.72-1.60 (m, 4H, H-10, H-5), 1.50-1.40 (m, 2H, H-6), 1.31-1.19 (m, 3H, H-7, H-

8a), 1.13-1.05 (m, 1H, H-9a), 1.12-0.95 (m, 3H, H-8, H-9b), 0.82 (d, 3H, J = 7.0

13 Hz, 2-CH3); C NMR (100 MHz, CDC13) 6 166.1, 133.9, 132.7, 131.6, 130.6,

129.1, 117.9, 74.1, 44.2, 41.7, 39.6, 39.1, 33.7, 32.1, 31.4, 29.3, 27.2, 26.8, 24.1,

5 15.0; a* = 52.89 (c = 1.00, CHCI3); HRES m/z calcd. for C2oH3o02Na 325.2138, found 325.2140 [M+Naf. Experimental procedures 133

(/?)-6-[2-EthyI]-5,6-dihydro-2//-pyran-2-one (30)

13r^ 15f 12^- ,,0 li1 8a J10 hJ(9 2S

3 4a

Compound 30 was prepared following the protocol of Inoue et. al..m A flask under Ar containing 0.47 g (1.57 mmol) of 29 and 313 mL of dry CH2CI2

(0.005M) was heated to reflux. Then 0.13 g (0.16 mmol) of Cl2(Cy3P)2Ru=CHPh was added, and the mixture was stirred at the same temperature for 6 h. The solvent was removed under vacuum, and the dark red oil was purified using column chromatography using a 4:1 hexane/EtOAc to afford 30 as an oil (85%).

IR (film) 2924, 2854, 1719 cm'1; *H NMR (500 MHz, CDCI3) 6 6.88 (ddd, 1H, J

= 9.7, 4.6, 3.6 Hz, H-13), 6.03 (dt, 1H, J = 9.7, 1.9 Hz, H-14), 5.57 (ddd, 1H, J =

9.9, 4.5, 2.7 Hz, H-3), 5.34 (d, 1H, J= 9.8 Hz, H-4) 4.42 (dq, 1H, J = 7.6, 4.7 Hz,

H-ll), 2.38 (m, 1H, H-12a), 2.23 (m, 1H, H-12b), 1.95-1.50 (m, 10H, H-10, H-8a,

H-8, H-5, H-4a, H-2, H-l), 1.40-1.20 (m, 4H, H-9, H-7), 1.10-1.00 (m, 2H, H-6),

13 0.88 (d, 3H, J = 7.1H, 2-CH3); C NMR (100 MHz, CDCI3) 6 164.8, 145.2,

132.6, 131.7, 121.8, 78.6,44.2, 41.8, 39.6, 33.6, 32.8, 32.2, 29.8, 29.5, 27.2, 26.8,

5 23.9, 15.1; ctp = -4.86 (c = 1.00, CHC13); HRES m/z calcd. for Cig^C^Na

297.1825, found 297.1827 [M+Naf. Experimental procedures 134

(\R, 4R, 6fl)-4-[2-{(lS, 25, 4aR, 8aS>2-Methyl-l, 2, 4a, 5, 6, 7, 8, 8a- octahydronapthalen-1 -yl}ethy 1]-3, 7-dioxabicyclo[4.1.0] heptan-2-one (31)

°s 14 13P isf 12\ ,xO ll1 8a ^J 10 H f9

5 H 4 4a

Compound 31 was prepared following the protocol of Inoue et. al..m A flask under Ar at 0°C a mixture of 0.05 g (0.19 mmol) of 30, 8 mL of dry toluene, and

75.3 fiL (0.38 mmol) of TBHP was treated with 26.2 jxL (0.06 mmol) of Triton B

(40% solution in MeOH). The reaction mixture was stirred at 0°C for 2 h. The reaction was quenched by adding 10 mL of saturated NH4CI, and the aqueous layer was extracted with EtOAc (3x10 mL). The combined organic layers were washed with 10 mL of brine and then dried over Na2S04. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 4:1 hexanes/EtOAc to afford 0.030 g of 31 as a white solid (60%). IR

-1 (film) 2923, 2854, 1743 cm ; 'H NMR (600 MHz, CDC13) 6 5.57 (ddd, 1H, J =

9.9, 4.5, 2.7 Hz, H-3), 5.34 (d, 1H, J= 9.8 Hz, H-4), 4.5 (m, 1H, H-ll), 3.65 (t,

1H, J = 3.6 Hz, H-13), 3.62 (d, 1H, J = 3.9 Hz, H-14), 2.35 (dt, 1H, J = 15.1, 2.9

Hz, H-12a), 2.20 (m, 1H, H-12b), 1.95 (dd, 1H, J= 15.1, 11.9 Hz, H-4a), 1.80 (d,

2H, J= 10.6 Hz, H-2, H-l) 1.75-1.55 (m, 4H, H-10, H-5), 1.48-1.39 (m, 3H, H-6,

H-8a), 1.35-1.18 (m, 4H, H-8, H-7), 1.08-0.90 (m, 2H, H-9), 0.88 (d, 3H, J = 7.1 Experimental procedures 135

13 Hz, 2-CH3); C NMR (100 MHz, CDC13) 5 167.9, 132.5, 131.7, 74.3, 52.3, 49.4,

44.2, 41.6, 39.7, 33.6, 32.6, 32.2, 29.7, 29.4, 27.2, 26.8, 23.7, 15.1; a% = 4.03 (c

= 1.00, CHCI3); HRES m/z calcd. for C^OjNa 313.1774, found 313.1771.

(4/?, 6fl)-4-Hydroxy-6-[2-{(lS, 2S, 4aR, 8aS)-2-methyl-l, 2, 4a, 5, 6, 7, 8, 8a- octahy d ronapthalen-1-y ljethy I]tetrahydro-2//-pyran-2-one (32)

HOJ 3,

Compound 32 was prepared following the protocol of Inoue et. al..m'69 A flask under Ar at 0°C a mixture of 156 mg (0.500 mmol) of (PhSe)2 in 5 mL of anhydrous EtOH was treated with 40.0 mg (1.00 mmol) of NaBH4. The mixture was stirred for 15 min before 75.0 jj,L (1.30 mmol) of AcOH was added at 23 °C to prepare the PhSeH solution. In a separate flask under Ar containing a solution of 77.0 mg of 31 in a THF/EtOH (4 mL/ 2 mL) was treated with 4.06 mL of the

PhSeH solution at 0°C. The mixture was then stirred for 2 h. The reaction was quenched by adding 10 mL of saturated NaCl, and the aqueous layer was extracted with EtOAc (5 x 10 mL). The combined organic extracts were then dried over Na2S04. The solvent was removed under vacuum, and oily residue was purified by column chromatography using a 2:1 EtOAc/hexanes to afford Experimental procedures 136

68.3 mg of 32 as a white solid (87%). IR (microscope) 3432, 2921, 2854, 1712

1 cm" ; *H NMR (500 MHz, CDC13) 5 5.57 (ddd, 1H, J = 9.9, 4.5, 2.7 Hz, H-3),

5.38 (d, 1H, J= 9.8 Hz, H-4) 4.70 (m, 1H, H-ll), 4.40 (m, 1H, H-13), 2.72 (dd,

1H, J = 17.5, 5.0 Hz, H-14a), 2.64 (ddd, 1H, J = 17.6, 3.7, 1.7 Hz, H-14b), 2.23

(m, 1H, H-2), 2.10-1.90 (m, 2H, H-12a, H4a), 1.88-1.55 (m, 6H, H-12b, H-8, H-5,

H-l), 1.54-1.45 (m, 2H, H-6), 1.45-1.20 (m, 5H, H-9, H-7, H-8a), 1.10-0.95 (m,

13 3H, H-10, OH), 0.87 (d, 3H, J = 7.1 Hz, 2-CH3); C NMR (100 MHz, CDC13) 5

170.5, 132.5, 131.7, 76.9, 63.2,44.2, 41.8, 39.6, 38.9, 36.4, 33.6, 33.5, 32.2, 29.5,

27.2, 26.8, 23.9, 15.1; a* = -0.87 (c = 0.46, CHC13); HRES m/z calcd. for

C i gHzgOjNa 315.1931, found 315.1930 [M+Naf.

(Z^-S-l-Acetamidoethyl but-2-enethioate (39)

The known compound 39129 was synthesized by an alternate route.130 In a flask under Ar containing 0.10 g (1.16 mmol) of crotonic acid was dissolved in 5 mL of dry CH2CI2. This was cooled to 0 °C. A solution of 0.26 g (1.28 mmol) of DCC and 14.0 mg (0.12 mmol) of DMAP in 5 mL of dry CH2CI2 and 0.15 g (1.28 mmol) of iV-acetylcysteamine in 5 mL of dry CH2CI2 were added to the crotonic acid solution simultaneously with two syringes. The mixture was then stirred for

2 days at 23 °C. The slurry was filtered, and the solvent removed under vacuum.

The residue was purified by column chromatography using a 2:1 EtOAc/hexanes Experimental procedures 137 eluent to afford 0.10 g of 39 as a white solid (47% yield). IR (microscope) 3251,

-1 3073, 2944, 1662, 1638 cm ; 'H NMR (400 MHz, CDC13) 8 6.94 (dq, 1H, J =

15.5, 6.9 Hz, H-7), 6.15 (dq, 1H,J = 15.5, 1.8 Hz, H-6), 6.00 (br s, 1H, N-H),

3.45 (q, 2H, J = 6.7 Hz, H-3), 3.08 (t, 2H, J= 6.7 Hz, H-4), 1.95 (s, 3H, H-l),

13 1.89 (dd, 3H, J = 7.0, 1.8 Hz, H-8); C NMR (100 MHz, CDC13) 8 190.1, 170.2,

141.7, 129.8, 39.7, 28.1, 23.1, 17.9; HRES m/z calcd. for CgHuNC^SNa

210.0559, found 210.0561 [M+Na]+.

(2E, 4£)-S-2-Acetamidoethyl hexa-2,4-dienethioate (40)

The known compound 40m was synthesized by an alternate route.130 In a flask under Ar containing 0.10 g (0.89 mmol) of sorbic acid was dissolved in 5 mL of dry CH2C12. This was cooled to 0 °C. A solution of 0.20 g (0.98 mmol) of DCC and 12.3 mg (0.10 mmol) of DMAP in 5 mL of dry CH2CI2 and 0.12 g (0.98 mmol) of jV-acetylcysteamine in 5 mL of dry CH2CI2 were added to the sorbic acid solution simultaneously with two syringes. The mixture was then stirred for

2 days at 23 °C. The slurry was filtered, and the solvent removed under vacuum.

The residue was purified by column chromatography using a 2:1 EtOAc/hexanes eluent to afford 0.11 g of 40 as a yellow solid (56% yield). IR (microscope) 3265,

1 3077, 2931, 1670, 1635 cm' ; H NMR (500 MHz, CDC13) 5 7.24 (dd, 1H, J =

15.2, 10.7 Hz, H-7), 6.25 (dq, 1H, J = 15.1, 6.7 Hz, H-9), 6.16 (m, 1H, H-6), 6.08 Experimental procedures 138

(dd, J = 15.2, 0.6 Hz, H-8), 5.98 (br s, 1H, N-H), 3.47 (q, 2H, J= 5.9 Hz, H-3),

3.11 (t, 2H, J= 6.6 Hz, H-4), 1.96 (s, 3H, H-l), 1.88 (dd, 3H, J= 6.7, 0.7 Hz, H-

10); 13C NMR (125 MHz, CDClj) 8 190.4, 170.3, 141.9, 141.8, 129.5, 125.7,

39.9, 28.3, 23.2, 18.9; HRES m/z calcd. for C,oH15N02SNa 236.0715, found

236.0713 [M+Naf.

(£)-Oct-2-enoic acid (41)

The known compound 4159 was synthesized as follows.132 In a flask under Ar containing 0.5 g (6.93 mmol) of acrylic acid, 0.75 g (7.63 mmol) of 1-heptene,

0.29 g (0.35 mmol) of Grubbs generation II catalyst was dissolved in 25 mL of dry CH2CI2. This was heated to reflux at 40 °C overnight. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 6:1 hexane/EtOAc to afford 0.39 g of 41 as a colourless oil (40%). IR

(microscope) 3300 - 2500, 2958, 2931, 2860, 1697, 1650 cm"1; 'H NMR (500

MHz, CDCI3) 6 7.10 (dt, 1H, J= 15.8, 6.9 Hz, H-3), 5.83 (dt, 1H, J - 15.6, 2.5

Hz, H-2), 2.24 (m, 2H, H-4), 1.48 (m, 2H, H-5), 1.32 (m, 4H, H-7, H-6), 0.90 (t,

3H, J= 10.2 Hz, H-8); ,3C NMR (125 MHz, CDCI3) 8 172.0, 152.5, 120.6, 32.2,

31.3, 27.5, 22.4, 13.9; HRES m/z calcd. for C8H1402Na 165.0886, found 165.0887

[M+Na]+. Experimental procedures 139

(2E, 4E, 6£)-Ethyl 2-methylocta-2,4,6-trienoate (43)

The known compound 43133 was prepared as follows.134 A flask under Ar containing 2.07 g (5.72 mmol) of (carbethoxyethylidene)triphenylphosphorane and 50 mL of dry CH2CI2 was cooled to 0 °C. Then 0.50 g (5.20 mmol) of (2E,

4£)-hexa-2,4,-dienal was added. The reaction mixture was allowed to stir and warm to 23 °C for 2 days. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 2.5:97.5 Et20/hexanes eluent to afford 0.70 g of 43 as a colourless liquid (74%). IR (microscope) 2981,

2931, 1702, 1613 cm"1; 'H NMR (600 MHz, CDCI3) 6 7.18 (d, 1H, J= 11.5 Hz,

H-3), 6.46 (dd, 1H, J= 14.9,10.8 Hz, H-5), 6.34 (dd, 1H, J= 14.8, 11.6 Hz, H-4),

6.17 (m, 1H, H-6), 5.88 (dq, 1H, J = 13.9, 6.9 Hz, H-7), 4.19 (q, 2H, J - 7.1 Hz,

H-10), 1.93 (s, 3H, H-9), 1.81 (d, 3H, J= 6.8 Hz, H-8), 1.29 (t, 3H, J= 8.0 Hz, H-

13 11); C NMR (100 MHz, CDC13) 8 168.4, 139.5, 138.4, 133.8, 131.7, 126.1,

125.4, 60.4, 18.4, 14.2, 12.6; HREI m/z calcd. for CnH,602 180.1150, found

180.1148 [M]+. Experimental procedures 140

(2E, 4E, 6£)-2-MethyIocta-2,4,6-trienoic acid (44)

jie The known compound 44 was synthesized as follows. In a flask containing

0.50 g (2.77 mmol) of 43 and 0.31 g (7.49 mmol) of LiOH was dissolved in 60 mL of THF and 40 mL of H2O. The reaction mixture was then heated to 70 °C overnight. The mixture was acidified to pH < 1, and the aqueous layer was extracted with ether (3x15 mL). The combined organic layers were washed with

15 mL of brine then dried over Na2SC>4. The solvent was removed under vacuum, and the residue purified by recrystallization using hot hexane to afford 0.35 g of

44 as a light yellow solid (83%). IR (microscope) 3500 - 2400, 2919, 2564,

1675, 1601 cm"1; "H NMR (400 MHz, CDCI3) 5 7.32 (d, 1H, J= 11.5 Hz, H-3),

6.54 (dd, 1H, J = 14.8, 10.7 Hz, H-5), 6.38 (dd, 1H, J= 14.8, 11.4 Hz, H-4), 6.21

(ddd, 1H, J= 14.8, 10.6, 1.4 Hz, H-6), 5.96 (dq, 1H, J= 14.8, 6.9 Hz, H-7), 1.94

(s, 3H, H-9), 1.84 (d, 3H, 6.7 Hz, H-8); 13C NMR (125 MHz, CDCI3) 5 173.9,

140.9, 140.8, 134.8, 131.6, 125.2, 125.0, 18.5, 12.3; m.p. = 127.5 - 138.0 °C;

+ HREI m/z calcd. for C9Hi202 152.0837, found 152.0835 [M] . Experimental procedures 141

(2E, 4E, 6£)-S-2-Acetamidoethyl 2-methylocta-2,4,6-trienthioate (45)

O 9 2A JS, .N 12^3 S vT T

Compound 45 was synthesized using the protocol of Liu et. al. In a flask under

Ar containing 0.10 g (0.66 mmol) of 44 was dissolved in 5 mL of dry CH2CI2.

This was cooled to 0 °C. A solution of 0.15 g (0.72 mmol) of DCC and 8.00 mg

(0.07 mmol) of DMAP in 5 mL of dry CH2CI2 and 0.08 g (0.72 mmol) of N- acetylcysteamine in 5 mL of dry CH2CI2 were added to the reaction mixture simultaneously with two syringes. The mixture was then stirred for 2 days at 23

°C. The slurry was filtered, and the solvent removed under vacuum. The residue was purified by column chromatography using a 2:1 EtOAc/hexanes eluent to afford 0.11 g of 45 as a white solid (66% yield). IR (microscope) 3285, 3077,

1 2930,1651, 1608, 1552 cm" ; *H NMR (400 MHz, CDC13) 5 7.18 (d, 1H, J= 10.4

Hz, H-3), 6.56 (dd, 1H, J= 14.7, 10.6 Hz, H-5), 6.34 (dd, 1H, J= 14.2, 11.3 Hz,

H-4), 6.18 (m, 2H, H-6, NH), 5.94 (dq, 1H, J= 13.8, 6.8 Hz, H-7), 3.42 (q, 2H, J

= 6.2 Hz, H-ll), 3.06 (t, 2H, J= 6.7 Hz, H-10), 1.96 (s, 3H, H-13), 1.93 (s, 3H,

H-9), 1.81 (d, 3H, 6.2 Hz, H-8); 13C NMR (125 MHz, CDCI3) 8 193.2, 170.2,

141.5, 138.1, 135.4, 133.3, 131.6, 124.8, 39.9, 28.5, 23.2, 18.6, 12.6; HRES m/z

+ calcd. for Ci3H19N02SNa 276.1028, found 276.1027 [M+Na] . Experimental procedures 142

(2Ey 4E, 6£)-Ethyl octa-2,4,6-trienoate (47)

The known compound 47136 was prepared in the same manner as 9 with a modified procedure. A flask under Ar containing 0.58 g (14.5 mmol) of NaH

(60% dispersion in oil) and 50 mL of dry THF was cooled to 0 °C. Then 2.79 g

(12.4 mmol) of triethyl phosphonoacetate was added slowly, and the reaction mixture was allowed to stir at the same temperature for 15 min. A solution of 1.0 g (10.1 mmol) of (2E, 4£)-hexa-2,4,-dienal in 3 mL of dry THF was added, and the mixture was allowed to warm to 23 °C over night. The reaction was quenched by the addition of 20 mL of H2O, and the aqueous layer was extracted with Et20

(3x15 mL). The combined organic layers were washed with 15 mL of brine and then dried over Na2S(>4. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 8:1 hexane/Et20 eluent to afford

1.46 g of 47 as a white solid (85%). IR (cast film) 2985, 2939, 1721, 1645 cm"1;

'H NMR (400 MHz, CDCI3) 8 7.30 (dd, 1H,J = 15.3, 11.2 Hz, H-3), 6.54 (dd,

1H, J= 14.8, 10.8 Hz, H-5), 6.20 (m, 2H, H-4, H-6), 5.95 (dq, 1H, J= 15.0, 7.0

Hz, H-7), 5.85 (d, 1H, J= 15.2 Hz, H-2), 4.21 (q, 2H, J= 7.1 Hz, H-9), 1.84 (d,

3H, J= 6.8 Hz, H-8), 1.31 (t, 3H, J=7.1 Hz, H-10); ,3C NMR (100 MHz, CDCI3)

5 167.4, 145.0, 141.2, 135.2, 131.5, 127.8, 120.3, 60.4, 18.8, 14.5; HREI m/z

+ calcd. for C,oH,402 166.0993, found 166.0991 [M] . Experimental procedures 143

(2E, 4E, 6£)-Octa-2,4,6-trienoic acid (48)

The known compound 48137 was synthesized using a modified procedure. A flask containing 100 mL of 1 M KOH and 200 mL of EtOH was cooled to 0 °C. A solution of 1.35 g (8.12 mmol) of 47 in 5 mL of EtOH was added to the cooled reaction mixture and stirred at that temperature for 15 min before heating to reflux at 100 °C for 1 h. The solution was acidified with 1 M HC1 to pH = 1 and the aqueous layer was extracted with Et20 (3 x 20 mL). The combined organic layers were washed with 30 mL of brine and then dried over Na2SC>4. The solvent was removed under vacuum, and the residue was purified through recrystallization with hot hexane to afford 1.12 g of 48 as a white solid (quant.). IR (cast film)

1 3500 - 2500, 2970, 2931, 1686, 1614 cm" ; 'H NMR (500 MHz, CD3OD) 6 7.28

(dd, 1H, J= 15.2, 11.3 Hz, H-3), 6.59 (dd, 1H, J= 15.6, 10.7 Hz, H-5), 6.28 (m,

1H, H-4), 6.20 (m, 1H, H-6), 5.98 (dq, 1H,J= 15.1, 6.9 Hz, H-7), 5.81 (d, 1H, J=

13 15.3 Hz, H-2), 1.82 (d, 3H, J= 6.9 Hz, H-8); C NMR (100 MHz, CD3OD) 5

169.5, 145.6, 141.4, 134.9, 131.4, 127.6, 119.8, 17.4; m.p. = 132.2 - 133.9 °C;

+ HREI m/z calcd. for C8H10O2 138.0681, found 138.0679 [M] . Experimental procedures 144

(2E, 4E, 6£)-5-2-Acetamidoethyl octa-2,4,6-trienethioate (49)

Compound 49 was prepared using the protocol of Liu et. al.m A flask under Ar containing 0.50 g (3.62 mmol) of 48 was dissolved in 50 mL of dry DMF and cooled to 0 °C. A solution of 0.82 g (3.98 mmol) of DCC and 44.0 mg (0.36 mmol) of DMAP in 5 mL of dry DMF and 0.47 g (3.98 mmol) of N- acetylcysteamine in 5 mL of dry DMF were added to the reaction mixture simultaneously with two syringes. The mixture was then stirred overnight at 23

°C. The slurry was filtered, and the solvent removed under vacuum. The residue was purified by column chromatography using a 1:1 EtOAc/hexanes eluent to afford 0.060 g of 49 as a white solid (7% yield). IR (microscope) 3297, 3090,

1 ! 3013, 2930, 1649, 1564 cm' ; H NMR (500 MHz, CDC13) 6 7.28 (dd, 1H, J =

15.1, 11.4 Hz, H-3), 6.62 (dd, 1H, J= 14.8, 10.8 Hz, H-5), 6.20 (m, 3H, H-6, H-4,

H-2), 6.11 (dq, 1H, J= 13.8, 6.8 Hz, H-7), 5.92 (br s, 1H, N-H), 3.48 (q, 2H, J =

5.9 Hz, H-10), 3.12 (t, 2H, J= 6.6 Hz, H-9), 1.97 (s, 3H, H-12), 1.85 (d, 3H, J =

13 6.8 Hz, H-8); C NMR (125 MHz, CDC13) 5 190.3, 170.5, 143.6, 142.0, 136.6,

131.5, 127.4, 126.7, 40.2, 28.6, 23.5, 18.8; HRES m/z calcd. for CuHnNOaS

239.0980, found 239.0977 [M]+. Experimental procedures 145

Ethyl 2-[bis(2,2,2-trifluorethoxy)phosphoryl]propanoate (51)

O CI (CF3CH20)2P OEt

The known compound 51138 was synthesized following a literature procedure.83,

139 A flask under Ar containing 0.72 g (18.1 mmol) of NaH (60% dispersion in oil) and 25 mL of dry THF was cooled to 0 °C. Then 5.0 g (15.1 mmol) of ethyl

[bis(2,2,2-trifluoroethoxy)phosphinyl]acetate was added, and the reaction mixture was allowed to warm to 23 °C over 2.5 h. The solution was cooled to 0 °C, and

2.56 g (15.1 mmol) of Mel was added. The mixture was allowed to warm to 23°C over 1 h. The reaction was quenched by the addition of 10 mL of brine and 20 mL of CH2CI2. The aqueous layer was extracted with CH2CI2 (3x10 mL) and the combined organic layers were dried over Na2SC>4. The solvent was removed under vacuum to afford 4.84 g of 51 as a yellow oil, which was taken directly to the next step without further purification. Experimental procedures 146

(2Z, 4E, 6£)-Ethyl 2-methylocta-2,4,6-trienoate (52)

O 9 11

Compound 52 was synthesized following the protocol of Still et. alP In a flask under Ar containing 4.44 g (11.9 mmol) of 18-crown-6 and 3.44 g (9.94 mmol) of

51 was dissolved in 25 mL of dry THF. This was cooled to -78 °C. A solution of

18.9 mL (9.44 mmol) of KHMDS (0.5 M in THF) was added slowly, and the mixture was allowed to stir for 20 min at the same temperature. Then 0.88 g (9.24 mmol) of (2E, 4£)-hexa-2,4,-dienal was added, and the mixture was allowed to stir at -78 °C for 1 h. The reaction was quenched by adding 10 mL of saturated

NH4CI. The aqueous layer was extracted with Et20 (3 x 10 mL) and the combined organic layers were washed with 15 mL of brine and dried over

Na2SC>4. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 98:2 hexane/Et20 eluent to afford 0.92 g of 52 as a yellow oil (55%). IR (cast film) 2980, 2927, 1704, 1612 cm"1; 'H NMR (400

MHz, CDClj) 5 7.20 (dd, 1H, J= 14.9, 11.5 Hz, H-3), 6.44 (d, 1H, J= 12.4 Hz,

H-5), 6.34 (dd, 1H, J= 15.0, 10.7 Hz, H-4), 6.20 (m, 1H, H-6), 5.87 (dq, 1H, J =

14.7, 7.6 Hz, H-7), 4.25 (q, 2H, J= 5.4 Hz, H-10), 1.98 (s, 3H, H-9), 1.82 (d, 3H,

J= 7.2 Hz, H-8), 1.34 (t, 3H, J= 7.1 Hz, H-ll); 13C NMR (125 MHz, CDCI3) 5

167.8, 140.7, 138.9, 132.9, 131.9, 127.5, 125.2, 60.3, 20.9, 18.5, 14.2; HRES m/z

+ calcd. for CnHi602Na 203.1043, found 203.1043 [M+Na] . Experimental procedures 147

(2Z, 4E, 6£)-2-Methylocta-2,4,6-trienoic acid (53)

o HO 1

Compound 53 was synthesized in a similar fashion as 48. A flask containing 25 mL of 1 M KOH and 50 mL of EtOH was cooled to 0 °C. A solution of 0.92 g

(5.08 mmol) of 52 in 5 mL of EtOH was added to the cooled reaction mixture and stirred at that temperature for 15 min before heating to reflux at 100 °C for 1 h.

The solution was acidified with 1 M HC1 to pH 1, and the aqueous layer was extracted with Et20 (3x15 mL). The combined organic layers were washed with

20 mL of brine and then dried over Na2S04. The solvent was removed under vacuum, and the residue was purified through precipitation with hot hexane to afford 0.43 g of 53 as a white solid (56%). IR (solid) 3500 - 2200, 1653, 1576

-1 cm ; *H NMR (400 MHz, CDC13) 5 7.25 (dd, 1H, J= 15.0, 10.4 Hz, H-3), 6.59

(d, 1H,J = 11.6 Hz, H-5), 6.41 (dd, 1H,J=14.6, 10.8 Hz, H-4), 6.25 (m, 1H, H-

6), 5.92 (dq, 1H, J = 14.0, 6.8 Hz, H-7), 2.0 (s, 3H, H-9), 1.82 (d, 3H, J = 7.2 Hz,

13 H-8); C NMR (125 MHz, CDC13) 5 200.3 143.3, 140.2, 133.8, 131.9, 127.4,

+ 123.5,20.7, 18.5; HREI m/z calcd. for C9Hi202 152.0837, found 152.0836 [M] . Experimental procedures 148

(2Z, 4E, 6£)-S-2-Acetamidoethyl 2-methylocta-2,4,6-trienethioate (54)

Compound 54 was synthesized following the protocol of Liu et. al.m A flask under Ar containing 0.04 g (0.25 mmol) of 53, 0.08 g (0.32 mmol) of 2-chloro-l- methylpyridinium iodide, and 0.03 g (0.28 mmol) of N-acetylcysteamine was dissolved in 25 mL of dry CH2CI2. This mixture was cooled to 0 °C. Then 15.0 mg (0.13 mmol) of DMAP and 76.9 jxL (0.55 mmol) of NEt3 was added to the reaction mixture. The mixture was then stirred for 2 h at 23 °C. The mixture was diluted with 20 mL of hexanes, and the slurry was filtered through a pad of silica.

Elute with 100 mL of 40 % EtOAc/hexanes and removed solvent under vacuum.

The residue was purified by column chromatography using a 1:4 EtOAc/hexanes eluent to afford 6.02 mg of 54 as a white solid (9% yield). IR (microscope) 3285,

-1 3077, 2930,1651,1608, 1552 cm ; 'HNMR(400 MHZ, CDC13) 6 7.12 (dd, 1H, J

= 14.9, 11.6 Hz, H-3), 6.40 (m, 1H, H-5), 6.28 (d, 1H, J = 10.5 Hz, H-4), 6.20 (m,

2H, H-6, NH), 5.90 (dq, 1H, J= 14.1, 6.8 Hz, H-7), 3.48 (q, 2H,J= 6.0 Hz, H-

11), 3.11 (t, 2H, J= 6.6 Hz, H-12), 2.11 (s, 3H, H-13), 1.99 (s, 3H, H-9), 1.82 (d,

3H, J= 5.8 Hz, H-8); 13C NMR (125 MHz, CDCI3) 5 192.7, 170.5, 141.0, 138.9,

134.2, 131.7, 129.9, 126.9, 39.9, 28.3, 23.1, 20.5, 18.5; HRES m/z calcd. for

C,3Hi9N02SNa 276.1025, found 276.1026 [M+Naf. Experimental procedures 149

(2Z, 4E, 6£)-Methyl octa-2,4,6-trienoate (58)

Compound 58 was prepared in the same manner as 52. In a flask under Ar containing 3.50 g (9.39 mmol) of 18-crown-6 and 3.44 g (9.94 mmol) of methyl

P,P-bis(2,2,2-trifluoroethyl)-phosphonoacetate was dissolved in 30 mL of diy

THF. This was cooled to-78 °C. A solution of 17.9 mL (8.93 mmol) of KHMDS

(0.5 M in THF) was added slowly, and the mixture was allowed to stir for 20 min at the same temperature. Then 0.70 g (7.28 mmol) of (2E, 4£)-hexa-2,4,-dienal was added, and the mixture was allowed to stir at -78 °C for 1 h. The reaction was quenched by adding 10 mL of saturated NH4CI. The aqueous layer was extracted with Et20 (3x10 mL), and the combined organic layers were washed with 15 mL of brine and dried over Na2SC>4. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 7:1 hexane/Et20 eluent to afford 0.41 g of 58 as a yellow oil (37%). IR (solid) 2987,

2952, 1718, 1644, 1603 cm"1; *H NMR (400 MHz, CDCI3) 5 7.40 (dd, 1H, J =

15.5, 11.6 Hz, H-3), 6.59 (t, 1H, J= 11.4 Hz, H-5), 6.46 (dd, 1H, J= 15.0, 10.7

Hz, H-4), 6.23 (m, 1H, H-6), 5.94 (dq, 1H, J= 15.0,6.8 Hz, H-7), 5.62 (d, 1H, J=

11.2 Hz, H-2), 3.72 (s, 3H, H-9), 1.83 (d, 3H, J= 7.3 Hz, H-8); 13C NMR (100

MHz, CDCI3) 5 167.2,145.4, 142.3, 135.3, 131.9, 126.6, 116.0, 51.3, 18.8; HREI

+ m/z calcd. forC9H,202 152.0837, found 152.0835 [M] . Experimental procedures 150

(2Z, 4Ey 6E)- Octa-2,4,6-trienoic acid (59)

Compound 59 was prepared using the same procedure as 48. A flask containing

50 mL of 1M KOH and 100 mL of MeOH was cooled to 0 °C. A solution of 0.41 g (2.69 mmol) of 58 in 5 mL of MeOH was added to the cooled reaction mixture and stirred at that temperature for 15 min before heating to reflux at 100 °C for 1 h. The solution was acidified with 1 M HC1 to pH 1, and the aqueous layer was extracted with Et20 (3x15 mL). The combined organic layers were washed with

25 mL of brine and then dried over Na2S04. The solvent was removed under vacuum, and the residue was purified through precipitation with hot hexane to afford 0.27 g of 59 as a solid (72%). IR (microscope) 3500 - 2500, 3013, 2944,

1 1699, 1608 cm" ; *H NMR (400 MHz, CD3OD) 6 7.40 (dd, 1H,J= 15.1, 12.1 Hz,

H-3), 6.59 (t, 1H,J = 11.4 Hz, H-5), 6.46 (dd, 1H,J = 15.0, 10.8 Hz, H-4), 6.24

(m, 1H, H-6), 5.94 (dq, 1H,J= 15.0, 6.8 Hz, H-7), 5.61 (d, 1H,J = 11.2 Hz, H-2),

13 1.82 (d, 3H, J = 7.2 Hz, H-8); C NMR (100 MHz, CD3OD) 5 169.5, 145.6,

141.4, 134.9, 131.4, 127.6, 119.8, 18.7; HREI m/z calcd. for C8H10O2 138.0681, found 138.0680 [M]+. Experimental procedures 151

(2Z, 4E, 6£)-5-2-Acetamidoethyl octa-2,4,6-trienethioate (60)

O 10 23 5 J 8 9 ¥ 46 O

Compound 60 was prepared using the protocol of Liu et. al.m In a flask under Ar containing 0.20 g (1.41 mmol) of 59 was dissolved in 20 mL of dry DMF. This was cooled to 0 °C. A solution of 0.32 g (1.55 mmol) of DCC and 17.0 mg (0.14 mmol) of DMAP in 5 mL of dry DMF and 0.19 g (1.55 mmol) of N- acetylcysteamine in 5 mL of dry DMF were added to the reaction mixture simultaneously with two syringes. The mixture was then stirred for 2 days at 23

°C. The slurry was filtered, and the solvent removed under vacuum. The residue was purified by column chromatography using a 2:1 EtOAc/hexanes eluent to afford 0.02 g of 60 as a white solid (5%). IR (solid) 3303,2932,1656, 1551 cm'1;

'H NMR (400 MHz, CDC13) 6 7.28 (dd, 1H, J= 15.2, 11.1 Hz, H-3), 6.64 (dd,

1H, J = 15.1, 10.9 Hz, H-5), 6.20 (m, 3H, H-6, H-4, H-2), 6.02 (dq, 1H, J- 15.2,

6.7 Hz, H-7), 5.92 (br s, 1H, N-H), 3.50 (q, 2H, J= 5.8 Hz, H-10), 3.13 (t, 2H, J=

6.5 Hz, H-9), 2.00 (s, 3H, H-12), 1.87 (d, 3H, J = 6.7 Hz, H-8); 13C NMR (100

MHz, CDC13) 8 190.3, 170.5, 143.6, 142.1, 136.6, 131.5, 127.4, 126.7, 40.2, 28.6,

23.4,18.9; HRES m/z calcd. for C12H17N02S 239.0980, found 239.0981 [Mf. Experimental procedures 152

(/?)-4-Benzyl-3-hexanoyloxazoIidin-2-one (67)

7{JS

The known compound 67140 was prepared using a modified procedure. A flask under Ar containing 1.48 g (8.38 mmol) of /?-(+)-4-benzyl-2-oxazolidinone dissolved in 40 mL of dry THF was cooled to -78°C. Then 3.35 mL of n-BuLi

(2.5 M in hexanes, 8.38 mmol) was added dropwise over 10 min, and the solution was stirred for 15 min at -78°C. Hexanoyl chloride, 1.54 mL (11.1 mmol), was added and the solution was stirred at -78°C for 45 min and then warmed to 23°C over 30 min. Saturated NaHCC>3, 10 mL, was added slowly, and then the bulk of the THF/hexanes mixture was removed under vacuum. The aqueous layer was extracted with CH2CI2 (3x15 mL), and the combined organic layers were washed with 20 mL of 1M NaOH, 20 mL of brine, and dried over Na2SC>4. The solvent was then removed under vacuum, and the residue was purified by column chromatography using a 4:1 hexanes/EtOAc eluent to afford 2.20 g of 67 as an oil

(96%). IR (microscope): 2956, 2930, 2871, 1784, 1701 cm-1; 'H NMR (500

MHz, CDCI3) 6 7.32 (m, 2H, H-6, H-6'), 7.22 (m, 1H, H-8), 7.18 (m, 2H, H-7, H-

7'), 4.67 (m, 1H, H-3), 4.17 (m, 2H, H-2a, H-2b), 3.30 (dd, 1H, J= 13.4, 3.2 Hz,

H-4a), 2.94 (m, 2H, H-10), 2.78 (dd, 1H, 13.4, 9.6 Hz, H-4b), 1.70 (m, 2H, H-

11), 1.38 (m, 4H, H-13, H-12), 0.92 (t, 3H, 7.1 Hz, H-14); 13C NMR (125 Experimental procedures 153

MHz, CDCI3) 6 173.4, 153.4, 135.3, 129.4, 128.9, 127.3, 66.1, 55.1, 37.9, 35.5,

31.3, 23.9, 22.4, 13.9; HRES m/z calcd for Ci6H2iN03Na 298.1413, found

298.1415 [M+Na]+.

(if )-4-Benzy1-3- [(R)-2-methy lhexanoy I] oxazoIidin-2-one (68)

The known compound 68141 was prepared as follows. In a flask under Ar containing 7.40 g (26.9 mmol) of 67 was dissolved in 125 mL of dry THF. This was cooled to -78°C. Then 29.6 mL (29.6 mmol) of NaHMDS (1.0 M in THF) was added dropwise. The solution was then stirred for 15 min at -78°C. Mel, 5.03 mL (80.6 mmol), was slowly added, and the mixture was allowed to stir for 3 h at

-78°C, and then warmed to 23°C over 12 h. The reaction was quenched with 50 mL of water. The bulk of the THF/H2O was removed under vacuum. The aqueous layer was extracted with EtOAc (3 x 20 mL), and the combined organic layers were washed with 20 mL of brine and dried over Na2SC>4. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 6:1 hexane/Et20 eluent to afford 6.61 g of 68 as an oil (85%). IR (cast film) 2958, 2932, 2860, 1782, 1693 cm'1; 'H NMR (500 MHz, CDCI3) 5 7.32 (m,

2H, H-6, H-6'), 7.26 (m, 1H, H-8), 7.21 (m, 2H, H-7, H-7'), 4.65 (m, 1H, H-3),

4.18 (m, 2H, H-2a, H-2b), 3.72 (AX5, 1H, J = 6.9 Hz, H-10), 3.28 (dd, 1H, J = Experimental procedures 154

13.5, 3.3 Hz, H-4a), 2.79 (dd, 1H, J= 13.4, 9.6 Hz, H-4b), 1.75 (m, 1H, H-lla),

1.43 (m, 1H, H-llb), 1.30 (m, 4H, H-12, H-13), 1.22 (d, 3H,J= 6.9 Hz, H-15),

0.90 (t, 3H, J = 7.0 Hz, H-14); 13C NMR (125 MHz, CDClj) 6 177.3, 153.0,

135.4, 129.4, 128.9, 127.3, 65.9, 55.3, 37.9, 37.7, 33.1, 29.4, 22.7, 17.4, 13.9; a*

= -117.04 (c = 0.47, MeOH); HRES m/z calcd for CnHisNOjNa 312.1570, found

312.1574 [M+Naf.

(/?)-2-lYlethylhexan-l-ol (69)

3 5

The known alcohol 69142 was prepared by an alternate method.143 A flask under

Ar containing 3.75 g (13.1 mmol) of 68 dissolved in 75 mL of dry Et20 was cooled to 0°C. LiAlH4 (1.0 M in THF), 13.6 mL (13.6 mmol), was added over 1 h. The mixture was stirred for 3.5 h at 0°C. The reaction was quenched by the addition of 10 mL of 1 M HC1, and the aqueous layer was extracted with EtaO (3 x 10 mL). The combined organic layers were washed with 15 mL of brine and then dried over NaaSO-j. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 1:1 hexanes/EtOAc eluent to afford 0.87 g of 69 as a liquid (58%). IR (microscope) 3334, 2957, 2927, 2873

1 cm" ; 'H NMR (500 MHz, CD2C12) 6 3.46 (dd, 1H, J= 10.4, 5.7 Hz, H-la), 3.37

(dd, 1H, J= 10.4, 6.5 Hz, H-lb), 1.58 (m, 1H, H-2), 1.42-1.60 (m, 7H, H-5, H-4,

H-3, OH), 0.90 (t, 3H, J= 6.9 Hz, H-7), 0.98 (d, 3H, 6.8 Hz, H-6); 13C NMR Experimental procedures 155

(125 MHz, CD2C12) 8 68.5, 36.2, 33.2, 29.6, 23.4, 16.7, 14.2; a* = 18.36 (c =

0.31, MeOH); HREI m/z 98.1096 (30.8%), 85.1021 (80.5%).

(/?)-2-Methylhexanal (70)

The known aldehyde 70144 was prepared by an alternate method.145 A flask containing 4.50 mL (51.2 mmol) of oxalyl chloride and 80 mL of dry CH2CI2 was cooled to -50 to -60 °C. DMSO (6.66 mL, 93.7 mmol) was slowly added with stirring for 2 min. Then a solution of 5.45 g (46.9 mmol) of 69 in 20 mL of dry

CH2CI2 was added within 5 min. The mixture was allowed to stir at the same temperature for 15 min. NEt3, 18.3 mL (140 mmol), was added slowly, and the mixture was allowed to stir for 5 min before being warmed to 23 °C. The reaction was quenched by adding 30 mL of H2O, and the aqueous layer was extracted with

CH2CI2 (3 x 20 mL). The combined organic layers were washed with 10 mL of brine and dried over Na2SC>4. The solvent was removed under vacuum to afford

4.94 g of 70 as an oil (92%). IR (cast film) 2959, 2930, 2861, 1708 cm'1; *H

NMR (500 MHz, CDC13) 6 9.59 (d, 1H, J= 2.0 Hz, H-l), 2.45 (AX5, 1H, J= 7.1

Hz, H-2), 1.68 (m, 1H, H-3a), 1.44 (m, 1H, H-3b), 1.32 (m, 4H, H-5, H-4), 1.07

(d, 3H,J= 7.1 Hz, H-7), 0.91 (t, 3H, J= 7.0, 13.9 Hz, H-6); 13C NMR (125 MHz,

CD2C12) 6 205.5, 46.6, 30.6, 29.5, 23.1, 14.0, 13.4; a% = -9.01 (c = 1.00,

+ CH2CI2); HREI m/z calcd. for C7H,40 113.0966, found 113.0969 [M] . Experimental procedures 156

(/?)-6-Methyldec-4-enoic acid (72)

HO 10 11

Compound 72 was prepared as follows.146 A flask under Ar containing 0.55 g

(1.27 mmol) of (3-carboxypropyl)triphenylphosphonium bromide in 20 mL of dry

THF was cooled to 0°C. Then 2.62 mL of NaHMDS (1.0 M in THF, 2.62 mmol) was added slowly, and the mixture was allowed to warm to 23°C and stirred at that temperature for 10 min. The solution was cooled to -78 °C and then 96.5 mg

(0.85 mmol) of 70 in 2 mL of dry THF was added. This was then allowed to warm to 23 °C and stirred at that temperature for 3 h. The reaction was quenched by adding 10 mL of saturated NH4CI, and the aqueous layer was extracted with

Et20 (3 x 10 mL). The combined organic layers were washed with 10 mL of brine and dried over Na2SC>4. The solvent was removed under vacuum, and the residue purified by column chromatography using a 3:1 hexanes/EtOAc eluent to afford 97.9 mg of 72 as a 9:1 mixture of cis/trans isomers (63%). IR (cast film)

3400 - 2800, 2958, 2926, 2872, 2858, 1711 cm'1; *H NMR (500 MHz, CDCI3) 6

5.28 (m, 1H, H-5), 5.20 (m, 1H, H-4), 2.40 (m, 5H, H-6, H-3, H-2) 1.38-1.25 (m,

6H, H-9, H-8, H-7), 0.91 (t, 3H, J= 6.6 Hz, H-10), 0.86 (d, 3H, J= 6.5 Hz, H-l 1);

,3C NMR (100 MHz, CDCI3) 5 179.2, 138.6, 125.6, 37.4, 34.5, 32.0, 29.9, 23.1,

23.0, 21.5, 14.3; a* = -0.13 (c = 3.72, CHCI3); HRES m/z calcd. for C,,H!0O2

183.1391, found 183.1393 [M-H]\ Experimental procedures 157

(/?)-6-Methyldec-4-enoyl chloride (73)

O CI

A flask under Ar containing 3.62 g (19.8 mmol) of 72 was cooled to 0 °C. Then

2.23 mL (25.6 mmol) of oxalyl chloride and 10 drops of DMF were added, with the evolution of CO2 gas, and the mixture was allowed to warm to 23 °C and stirred overnight. The solvent was removed under vacuum to furnish 3.99 g of 73 as a yellow oil, which was used directly in the next step without further purification.

(/?)-4-Benzyl-3-[(/?)-6-methyldec-4-cnoyl]oxazolidin-2-one (74)

Compound 74 was synthesized in the same manner as 67. In a flask under Ar containing 4.18 g (23.6 mmol) of i?-(+)-4-benzyl-2-oxazolidinone was dissolved in 100 mL of dry THF. This was cooled to -78 °C. Then 10.2 mL of n-BuLi (2.5

M in hexanes, 25.5 mmol) was added dropwise over 10 min. The solution was allowed to stir for 15 min at -78 °C. This mixture was transferred via cannula to a separate flask under Ar that was cooled to -78 °C and contained 3.99 g (19.6 mmol) of 73. This solution was stirred at -78 °C for 45 min and was then wanned to 23 °C over 30 min. The reaction was quenched by adding 25 mL of saturated Experimental procedures 158

NaHC03, and the bulk of the THF/hexanes was removed under vacuum. The aqueous layer was extracted with CH2CI2 (3 x 20 mL), and the combined organic layers were washed with 20 mL of 1 M NaOH, 20 mL of brine, and dried over

Na2S04. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 7:1 hexanes/EtOAc + 1% AcOH eluent to afford 5.37 g of 74 as an oil (79%). IR (neat film): 2956, 2926, 2858, 1784, 1702

1 cm" ; 'H NMR (600 MHz, CDC13) cis-isomer 6 7.38 (m, 2H, H6, H-6'), 7.32 (m,

1H, H-8), 7.21 (m, 2H, H-7.H-7'), 5.38 (m, 1H, H-13), 5.21 (m, 1H, H-12), 4.67

(m, 1H, H-3), 4.19 (m, 2H, H-2), 3.31 (dd, 1H, J= 13.4, 3.3 Hz, H-4a), 3.00 (m,

2H, H-10), 2.78 (dd, 1H, J= 13.4, 9.6 Hz, H-4b), 2.43 (m, 3H, H-14. H-ll), 1.38

-1.18 (m, 6H, H-17, H-16, H-15), 0.95 (d, 3H, J= 6.7 Hz, H-18), 0.88 (t, 3H,J =

6.9 Hz, H-19); 13C NMR (125 MHz, CDCI3) cis-isomer 5 172.8, 153.4, 138.1,

135.3, 129.4, 128.9, 127.3, 125.7, 66.2, 55.2, 37.9, 37.2, 35.8, 31.8, 29.7, 22.9,

22.3, 21.3, 14.1; ct% = -28.9 (c - 2.27, CHC13); HRES m/z calcd. for

C2iH29N03Na 366.2040, found 366.2041 [M+Naf. Experimental procedures 159

(/?)-4-BenzyI-3-[(2/?, 6/?)-2,6-dimethyldec-4-enoyl]oxazoIidin-2-one (75)

8 r

Compound 75 was synthesized using the same method as 68. In a flask under Ar containing 5.37 g (15.6 mmol) of 74 was dissolved in 120 mL of dry THF. This was cooled to -78 °C. Then 17.2 mL (17.2 mL) of NaHMDS (1.0 M in THF) was added slowly and the mixture was allowed to stir for 15 min at -78°C. Mel, 2.92 mL (46.9 mmol), was added slowly and the solution was allowed to stir at -78 °C for 3 h, and then warmed to 23°C overnight. The mixture was diluted with 50 mL of water, and the bulk of the THF/H2O was removed under vacuum. The aqueous layer was extracted with EtOAc (3 x 20 mL), and the combined organic layers were washed with 20 mL of brine and dried over Na2S04- The solvent was removed under vacuum, and the residue was purified by column chromatography using a 8:1 hexane/Et20 eluent to afford 4.63 g of 75 as an oil (83%). IR (neat

1 film): 2957, 2926, 2858, 1783, 1699 cm" ; 'H NMR (600 MHz, CDC13) 5 7.37

(m, 2H, H-7, H-7'), 7.32 (m, 1H, H-8), 7.21 (m, 2H, H-6. H-6'), 5.24 (m, 2H, H-

13, H-12), 4.65 (m, 1H, H-3), 4.18 (m, 2H, H-2), 3.78 (A1X5, 1H, J= 6.9 Hz, H-

10), 3.27 (dd, 1H, J= 13.4, 3.4 Hz, H-4a), 2.78 (dd, 1H, J= 13.4, 9.6 Hz, H-4b),

2.43 (m, 2H, H-ll), 2.22 (m, 1H, H-14), 1.36 - 1.17 (m, 9H, H-19, H-17, H-16,

H-15), 0.92 (d, 3H, J= 6.7 Hz, H-20), 0.88 (t, 3H,J= 7.0 Hz, H-18); 13C NMR

(100 MHz, CDCI3) 8 177.0, 153.3, 139.1, 135.6, 129.7, 129.2, 127.6, 124.7, 66.3, Experimental procedures 160

55.6, 38.2, 38.1, 37.4, 31.9, 31.6, 29.9, 23.1, 21.5, 17.4, 14.3; a* = -43.3 (c =

1.10, CHC13); HRES m/z calcd for C22H3iN03Na 380.2196, found 380.2201

[M+Naf.

(21?, 6R)-2,6-DimethyIdec-4-enoic acid (76)

11 12

Compound 76 was prepared using the protocol of Evans, et. a/.147 A flask containing 1.01 g (2.83 mmol) of 75 dissolved in a 3:1 mixture of THF/H2O (75 mL/ 25 mL) was cooled to 0 °C. Then 1.44 mL of H2O2 (30% wt in H2O, 14.1 mmol) and 0.24 g (5.7 mmol) of LiOH was added slowly to the mixture, which was allowed to stir at 4 °C for 12 h. Adding 15 mL of 1.5 N aqueous Na2S03 at

0°C quenched the reaction. The bulk of the THF was removed under vacuum, and the aqueous layer was extracted with CH2CI2 (3x15 mL). The combined organic layers were washed with brine and dried over Na2SC>4. The solvent was removed under vacuum, and the residue purified by column chromatography using a 6:1 hexane/EtOAc +1% AcOH as eluent to yield 0.45 g of 76 as a yellow oil (81%).

1 IR (neat) 3300-2750, 2958, 2927, 2873, 1708 cm' ; 'H NMR (600MHZ, CDC13) 5

5.52 (m, 2H, H-5, H-4), 2.51 (A{X5, 1H, J= 7.0 Hz, H-2), 2.43 (m, 2H, H-3), 2.23

(m, 1H, H-6), 1.32 - 1.16 (m, 9H, H-7, H-8, H-9, H-ll), 0.91 (d, 3H, J= 6.6 Hz,

H-12), 0.88 (t, 3H, J = 7.0 Hz, H-12); ,3C NMR (100 MHz, CDCI3) 6 182.6, Experimental procedures 161

139.2, 124.4, 39.9, 37.4, 31.9, 31.5, 29.9, 23.1, 21.5, 16.7, 14.3; a* = 3.60 (c =

2.93, CHCI3); HRES m/z calcd. For C,2H2i02Na 243.1331, found 243.1334

[M+Na]+.

(3R, 5R)-5-\(lR, 2/f)-l-Iodo-2-methylhexyl]-3-methyldihydrofuran-2(3//)-one

(77) and (3R, SS)-S-[(1S, 2/?)-l-Iodo-2-methylhexyl]-3-methyldihydrofuran-

2(3//)-°ne (78)

11 11 77 78

Compounds 77 and 78 were synthesized according to the protocol of Shi et. al.m

A flask under Ar containing 77.0 mg (0.39 mmol) of 76 dissolved in 10 mL of dry

MeCN was cooled to -30 °C. Then 0.31 g (1.23 mmol) of I2 was added and the mixture was allowed to stir at -30 °C for 12 h. The reaction was quenched by the addition of 10 mL of saturated Na2S203. The mixture was diluted with 10 mL of

Et20 and stirred until two clear layers had formed at 23 °C. The aqueous layer was extracted with Et20 (3 x 10 mL), and the combined organic layers were washed with 15 mL of saturated NaHC03, and then 15 mL of brine and dried over

Na2SC>4. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 9:1 hexane/EtOAc eluent to afford 0.12 g Experimental procedures 162

(94%) of a yellow solid as a 3:1 mixture of diastereomers (77:78). Recrystallizing in hexanes allowed the isolation of 72.1 mg (57%) of 77 as white crystals.

1 77: IR (cast film) 2959, 2931, 2872, 1776 cm" ; 'H NMR (600 MHz, CDC13) 6

4.15 (ddd, 1H, J- 10.6, 5.7, 4.7 Hz, H-4), 4.09 (t, 1H, J- 4.6 Hz, H-5), 2.74 (m,

1H, H-2), 2.52 (ddd, 1H, J = 12.5, 9.0, 5.8 Hz, H-3a), 1.76 (m, 1H, H-3b), 1.55

(m, 2H, H-9), 1.48 (m, 1H, H-6), 1.41-1.20 (m, 7H, H-ll, H-8, H-7), 1.08 (d,

3H, J = 6.6 Hz, H-12), 0.91 (t, 3H, J = 7.1 Hz, H-10); l3C NMR (100 MHz,

CDClj) 8 178.2, 78.8, 49.3, 38.3, 37.7, 35.7, 35.4, 29.3, 23.1, 19.8, 15.2, 14.3;

a* = -45.78 (c = 0.97, CHC13); m.p. = 99.4 - 99.8 °C; HRES m/z calcd. for

+ C,2H2iI02Na 347.0478, found 347.0479 [M+Na] .

1 78: IR (cast film) 2958, 2931, 2872, Mil cm" ; 'H NMR (600 MHz, CDC13) 8

4.39 (m, 1H, H-4), 4.25 (dt, 1H, /= 11.8 6.3Hz, H-5), 2.89 (m, 1H, H-2), 2.25 (m,

1H, H-3a), 2.06 (m, 1H, H-3b), 1.55 (m, 2H, H-9), 1.48 (m, 1H, H-6), 1.42 - 1.20

(m, 7H, H-ll, H-8, H-7), 0.99 (d, 3H, J= 6.4 Hz, H-12), 0.91 (t, 3H, J= 13 Hz,

13 H-10); C NMR (100 MHz, CDC13) 8 179.2, 80.2, 50.3, 37.9, 37.2, 36.2, 34.8,

29.3, 22.9, 18.8, 15.2, 14.3; HRES m/z calcd. for C12H2iI02Na 347.0478, found

347.0478 [M+Na]+. Experimental procedures 163

(31?, 5^)-3-Methyl-5-[(/?)-2-methyIhexyI]dihydrofuran-2(3//)-one (79)

Compound 79 was synthesized using the protocol of Hatakeyama et. al..m A flask under Ar containing 0.22 g (0.68 mmol) of 77, 0.36 mL (1.37 mmol) of

Bu3SnH, 11.16 mg (0.07 mmol) AIBN, and 15 mL of dry THF was heated to reflux for 1.5 h. The solvent was removed under vacuum, and the residue was first purified by running through a small pad of silica with 10% w/w of KF, ground with mortar and pestle, using EtOAc as an eluent. The solvent was removed, and the remaining residue was purified further using column chromatography with 10% w/w of KF added to silica gel using a 95:5 hexane/EtOAc eluent to afford 0.14 g of 79 as a colourless oil (quant.). IR (cast

1 film) 2958, 2931, 1773 cm" ; *H NMR (500 MHz, CDC13) 6 4.43 (m, 1H, H-4),

2.68 (m, 1H, H-2), 2.48 (m, 1H, H-3a), 1.77 - 1.67 (m, 2H, H-7b, H-6), 1.50 (dt,

1H, J= 10.4, 12.3 Hz , H-5a), 1.39 - 1.15 (m, 10H, H-ll, H-9, H-8, H-7, H-5b),

0.94 (d, 3H, J = 6.6 Hz, H-12), 0.88 (t, 3H, J = 6.9 Hz, H-10); 13C NMR (125

MHz, CDC13) 6 179.6, 76.9, 43.4, 38.1, 36.9, 35.9, 29.6, 29.0, 22.9, 19.3, 15.1,

14.1;

Lithium (2J?, 45,6J?)-4-hydroxy-2,6-dimethyldecanoate (80)

10 11 12

A flask containing 0.12 g (0.63 mmol) of 79 dissolved in 5 mL of MeOH was cooled to 0 °C. Then 2.5 mL (1.25 mmol) of a 0.5 M LiOH solution was added, and the mixture was allowed to warm to 23 °C overnight. The solvent was removed under vacuum, and residue was lyophilized to afford 0.12 g of 80 as a white solid containing 0.02 g of LiOH (quant.). 'H NMR (600 MHz, D2O) 5 3.73

(m, 1H, H-4), 2.39 (AB5, 1H, J= 1A Hz, H-2), 1.70 (m, 1H, H-6), 1.56 (br s, 1H,

OH), 1.43 (m, 2H, H-5), 1.30- 1.12 (m, 8H, H-9, H-8, H-7, H-3), 1.07 (d, 3H,J =

13 6.9 Hz , H-ll), 0.85 (m, 6H, H-12, H-10); C NMR (125 MHz, CDC13) 6 187.4,

68.5, 44.6, 42.8, 40.3, 37.6, 29.3,29.2, 23.2, 19.4, 18.4, 14.3; HRES m/z calcd. for

C12H23O3 215.1653, found 215.1651 [M-H]".

(3R, 5S)-5-[(lS, 2/?)-l-Hydroxy-2-methylhexvl]-3-methyldihydrofuran-2(3//)- one (81)

Compound 81 was prepared following the protocol of Itoh et. a/.150 A flask under

Ar containing 0.52 g (1.59 mmol) of 77 dissolved in 30 mL of THF and 20 mL of

H2O was cooled to 0 °C. Then 0.35 g (8.45 mmol) of LiOH was added, and the Experimental procedures 165 mixture was allowed to stir at 0 °C for 5 h. The reaction was quenched by the addition of 10 mL of 1 M HC1, and then the aqueous layer was extracted with

EtOAc (3 x 10 mL). The combined organic layers were washed with 10 mL of

H2O, 10 mL of saturated Na2S2C>3, 10 mL of brine, and then dried over Na2SC>4.

The solvent was removed under vacuum, and the crude residue was dissolved in

10 mL of CHCI3. The red solution was allowed to stand at 23 °C for 48 h. The solvent was removed, and the residue was purified by column chromatography using a 4:1 hexane/EtOAc eluent to afford 0.31 g (92%) of 81 as an oil. IR (cast

-1 film) 3446, 2958, 2931, 2873, 1759 cm ; 'H NMR (600 MHz, CDC13) 5 4.58 (m,

1H, H-4), 3.41 (t, 1H, J= 5.0 Hz, H-5), 2.80 (m, 1H, H-2), 2.31 (m, 1H, H-3a),

1.95 (m, 1H, H-3b), 1.63 (m, 1H, H-6), 1.48 (m, 1H, H-7a), 1.37 - 1.20 (m, 9H,

H-ll, H-9, H-8, H-7b, OH), 0.96 (d, 3H, J= 6.8 Hz, H-12), 0.90 (t, 3H, J= 6.9

Hz, H-10); 13C NMR (125 MHz, CDCI3) 5 180.2, 76.9, 35.2, 34.4, 33.2, 32.5,

2 5 29.3, 22.9, 16.4, 14.1, 14.0; a D = 0.48 (c = 1.00, CHC13); HRES m/z calcd. for

Ci2H22Na03 237.1461, found 237.1459 [M+Naf. Experimental procedures 166

0-(lS, 2/?)-2-Methyl-l-[(2S, 4/?)-4-methyl-5-oxotetrahydrofuran-2-yl]hexyl li/-imidazole-l-caarbothioate (82)

Compound 82 was prepared using the protocol of Ghosh et. a/.151 In a flask containing 66.6 mg (0.31 mmol) of 81 and 137 mg (0.77 mmol) of 1,1'- thiocarbonyldiimidazole was dissolved in 10 mL of dry toluene and 5 mL of dry pyridine. The solution was heated at 60 °C overnight. Then the solvent was removed under vacuum, and the residue was purified by column chromatography using a 3:1 hexane/EtOAc eluent to afford 35.7 mg (36%) of 82 as an oil. IR (cast

1 film) 2958, 2932,2872, 1778 cm' ; 'H NMR (500 MHz, CDC13) 6 8.38 (s, 1H, H-

12), 7.49 (s, 1H, H-14), 7.10 (s, 1H, H-13), 5.77 (t, 1H, J= 4.9 Hz, H-5), 4.85

(AX4, 1H, J= 4.4 Hz, H-4), 2.59 (m, 1H, H-2), 2.30 (m, 1H, H-3a), 2.09 (m, 2H,

H-6, H-3b), 1.48 (m, 1H, H-9b), 1.40 - 1.18 (m, 8H, H-ll, H-9a, H-8, H-7), 1.10

(d, 3H, J = 6.9 Hz, H-12), 0.88 (t, 3H, J = 7.0 Hz, H-10); ,3C NMR (125 MHz,

CDCI3) 6 184.4, 178.9, 131.2, 114.7, 86.7, 86.1, 75.9, 34.6, 33.7, 32.7, 32.4, 29.0,

22.7, 16.3, 15.0, 13.9; af = 3.39 (c = 0.59, CHC13), HRES m/z calcd. for

C16H25N2O3S 325.158, found 325.1582 [M+Na]+. Experimental procedures 167

(3J?, 5i?)-3-Methyl-5-[(/?)-2-methylhexyl]dihydrofuran-2(3//)-oiie (83)

Compound 83 was prepared using the protocol of Ghosh et. a/.151 In a flask containing 35.7 mg (0.11 mmol) of 82 was dissolved in 10 mL of degassed toluene. Then 58.0 JAL (0.22 mmol) of tributyltin hydride and 5.00 mg of AIBN

(0.05 mmol) were added, and the mixture was refluxed overnight. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 9.5:0.5 hexane/EtOAc eluent to afford 14.0 mg (64%) of

83 as a colourless oil. IR (cast film) 2957,2928, 2873, 2858, 1773 cm"1; 'H NMR

(500 MHz, CDC13) 5 4.09 (m, 1H, H-4), 2.68 (AX5, 1H, J= 7.3 Hz, H-2), 2.10 -

1.95 (m, 2H, H-3), 1.62-1.51 (m, 3H, H-6, H-5a, H-9a), 1.50 - 1.41 (m, 2H, H-

9b, H-5a), 1.39 - 1.10 (m, 7H, H-ll, H-8, H-7), 0.92 (d, 3H, 6.4 Hz, H-12),

13 0.88 (t, 3H, J= 7.1 Hz, H-10); C NMR (125 MHz, CDC13) 6 180.1, 76.9, 42.8,

36.2, 35.8, 34.0, 29.7, 29.0, 22.9, 19.9, 15.9, 14.1; a% = 20.19 (c = 0.52, CHC13);

HRES m/z calcd. for C i2H22Na02 221.1512, found 221.1511 [M+Naf. Experimental procedures 168

Lithium (2J?, 4R, 6/?)-4-hydroxy-2,6-dimethyldecanoate (84)

Compound 84 was prepared in a similar fashion to compound 80, using a modified procedure. In a flask containing 9.40 mg (47.4 fxmol) of 83 was dissolved in 3 mL of THF and 3 mL of H20. Then 94.8 jaL of a 1.0 M solution of

LiOH (94.8 fxmol) was added, and the mixture was stirred at 23 °C for 2 h. The solvent was removed under vacuum to afford 9.37 mg (92%) of 84 as a white solid containing 1.80 mg of LiOH. 'H NMR (600 MHz, D20) 6 3.68 (m, 1H, H-

4), 2.46 (m, 1H, H-2), 1.74 (m, 1H, H-3a), 1.52 (m, 1H, H-6), 1.48 - 1.14 (m, 9H,

H-l 1, H-9, H-8, H-7a, H-5, H-3b), 1.12-1.20 (m, 4H, H-7b, H-6, OH), 0.40 (m,

13 6H, H-l2, H-10); C NMR (125 MHz, D20) 5 187.2, 69.4, 45.2, 42.5, 40.7, 36.5,

29.6, 29.2, 23.2, 20.4, 19.2, 14.3; HRES m/z calcd. for C12H2303 215.1653, found

215.1651 [M-H]-. Experimental procedures 169

(3R, 5S)-5-[(lS, 2/f)-l-Hydroxy-2-methylhexyl]-3-methyldihydrofuran-2(3//)- one (81) and (3/?, 5/?)-5-|(l/?, 2J?)-l-Hydroxy-2-methyIhexyl]-3- methyldihydrofuran-2(3//)-one (85)

11 11

81 85

Compounds 81 and 85 were synthesized in the same fashion as 81 to afford 77.3 mg (92%) of 81 and 85 as oils in a 3:1 mixture.

85: IR (cast film) 3445, 2959, 2933, 2873, 1767 cm"1; !H NMR (500 MHz,

CDC13) 6 4.47 (ddd, 1H J= 10.1, 5.9, 4.1 Hz, H-4), 3.37 (m, 1H, H-5), 2.68 (m,

1H, H-2), 2.39 (m, 1H, H-3a), 1.83 (m, 1H, H-3b), 1.62 (m, 1H, H-6), 1.43 (m,

1H, H-7a), 1.35 - 1.13 (m, 9H, H-ll, H-9, H-8, H-7b, OH), 0.95 (d, 3H, J= 6.9

13 Hz, H-12), 0.87 (t, 3H, J= 6.2 Hz, H-10); C NMR (100 MHz, CDC13) 5 179.7,

79.1, 77.1, 36.1, 35.8, 33.1, 31.5, 29.4, 23.2; HRES m/z calcd. for C^NaOa

237.1461, found 237.1463 [M+Naf. Experimental procedures 170

(3/f, 5S)-5-[(lS, 2/?)-l-(Benzyloxy)-2-methylhexyI]-3-methyldihydrofuran-

2(3//)-one (87) and (3R, 5/?)-5-[(l/f, 2/?)-l-(Benzyloxy)-2-methylhexyl]-3- methyldihydrofuran-2(3//)-one (88) 17 16rj^|16' 15^^15'14J O

Compounds 87 amd 88 were synthesized following the protocol of Kuethe et. al.152 In a flask under Ar containing 0.52 g (2.43 mmol) of 81 and 85 was dissolved in 25 mL of dry Et20. Then 0.68 mL (3.65 mmol) of benzyl 2,2,2 - trichloroacetimidate and 3 drops of triflic acid were added, and the mixture was allowed to stir at 23 °C for 3 h. The reaction was quenched by the addition of 20 mL of 1 M HCl, and the aqueous layer was extracted with Et20 (3x15 mL). The combined organic layers were washed with 15 mL of brine and then dried over

Na2SC>4. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 7:1 hexanes/EtOAc eluent to afford 0.70 g

(95%) of 87 and 88 as a mixture of two inseparable diastereomers in a ratio of

0.7:1 as colourless oils.

87: IR (cast film) 2959, 2933, 2873, 1770, 1718 cm*1; 'H NMR (600 MHz,

CDClj) 5 4.78-4.62 (AB quartet, 1H, J= 80.2, 11.5 Hz, H-13), 4.68 (m, 1H, H- Experimental procedures 171

4), 3.30 (dd, 1H, J= 5.8, 3.9 Hz, H-5), 2.78 (m, 1H, H-2), 2.17 (m, 1H, H-3a),

1.92 (m, 1H, H-3b), 1.77 (m, 1H, H-6), 1.55 (m, 1H, H-7a), 1.36 - 1.18 (m, 8H,

H-l 1, H-9, H-8, H-7b), 0.97 (d, 3H, J= 6.8 Hz, H-12), 0.88 (t, 3H, J= 7.3 Hz, H-

13 10); C NMR (100 MHz, CDC13) 5 180.2, 138.5, 129.0, 128.4, 127.8, 127.6,

84.1, 80.2, 74.2, 34.5, 34.3, 33.3, 33.2, 22.9, 16.3, 14.6, 14.1. HRES calcd for

CioHasNaOj 327.1931, found 327.1935 [M+Na]+.

88: "H NMR (600 MHz, CDCI3) 6 4.85 - 4.60 (AB quartet, 1H, J= 80.2, 11.5 Hz,

H-l3), 4.68 (m, 1H, H-4), 3.26 (dd, 1H, J= 5.7, 3.9 Hz, H-5), 2.66 (m, 1H, H-2),

2.36 (m, 1H, H-3a), 1.92 (m, 1H, H-3b), 1.77 (m, 1H, H-6), 1.66 (m, 1H, H-7a),

1.42 - 1.12 (m, 8H, H-ll, H-9, H-8, H-7b), 1.02 (d, 3H, J = 6.7 Hz, H-12), 0.90

(t, 3H, J= 7.2 Hz, H-10); 13C NMR (100 MHz, CDCI3) 6 180.2, 138.5, 129.0,

128.4,127.8,127.6, 84.1, 80.2, 74.2,34.5, 34.3, 33.3, 33.2, 22.9,16.3,14.6,14.1. Experimental procedures 172

(2/f, 45,55, 6/?)-5-(Benzyloxy)-2,6-dimethyldecane-l, 4-diol (89) and (2if, 4R,

5Ry 6/f)-5-(Benzyloxy)-2,6-dimethyldecane-l, 4-diol (90)

17 17

Compounds 89 and 90 were synthesized using the protocol of Shi et. al.m In a flask under Ar containing 0.70 g (2.32 mmol) of 87 and 88 was dissolved in 40 mL of dry Et20. This was cooled to 0 °C. Then 0.27 g (7.25 mmol) of UAIH4 powder was slowly added, and the grey mixture was allowed to stir at 0 °C for 3 h. The reaction was quenched by the addition of 15 mL of 1M HC1. The aqueous layer was extracted with Et20 (3x15 mL). The combined organic layers were washed with brine and then dried over NaaSCV The solvent was removed under vacuum, and the two diastereomers were separated by column chromatography using a 4:1 hexane/EtOAc eluent to afford 0.64 g (0.27 g of 89 and 0.17 g of 90) as colourless oils (91%).

1 89: IR (cast film) 3373, 2956, 2928, 2872 cm" ; *H NMR (600 MHz, CDC13) 5

7.19 - 7.10 (m, 5H, H-15, H-15', H-16, H-16\ H-17), 4.74 - 4.59 (AB quartet,

2H,J= 85.2,11.3 Hz, H-13), 3.80 (m, 1H, H-4), 3.55 (m, 1H, H-la), 3.48 (m, 1H,

H-lb), 3.25 (dd, 1H, J= 6.6, 3.3 Hz, H-5), 2.80 (br s, 2H, 1-OH, 4-OH), 1.90 (m,

1H, H-2), 1.70 - 1.62 (m, 1H, H-6), 1.58 - 1.44 (m, 3H, H-7, H-3a), 1.40 - 1.20 Experimental procedures 173

(m, 5H, H-9, H-8, H-3b), 0.96 (d, 3H, J = 2.9 Hz, H-l 1), 0.95 (d, 3H, J = 2.9 Hz,

13 H-12), 0.91 (t, 3H J = 7.0 Hz, H-10); C NMR (125 MHz, CDC13) 8 138.7,

128.8, 128.1, 127.9, 86.5, 75.5, 69.6, 67.9, 38.5, 35.4, 34.5, 32.8, 30.1, 23.2, 17.7,

14.3; a* = 8.33 (c = 0.60, CHC13); HRES m/z calcd. for Ci9H32Na03 331.2244, found 331.2237 [M+Na]+.

1 90: IR (cast film) 3367, 2955, 2930, 2871 cm" ; 'H NMR (500 MHz, CDC13) 8

7.40 - 7.25 (m, 5H, H-15, H-15', H-16, H-16', H-17), 4.72 - 4.54 (AB quartet,

2H, J- 68.8, 11.2 Hz, H-13), 3.76 (ddd, 1H, J= 10.4, 4.5, 2.1 Hz, H-4), 3.54 (dd,

1H, J= 10.9,4.5 Hz, H-la), 3.40 (dd, 1H, J= 10.9, 7.2 Hz, H-lb), 3.09 (t, 1H, J =

4.7 Hz, H-5), 1.90 - 1.78 (m, 2H, H-6, H-2), 1.60 - 1.42 (m, 2H, H-7a, H-3a),

1.40 - 1.09 (m, 7H, H-9, H-8, H-7b, H-3b, 2-OH, 4-OH), 1.00 (d, 3H, J= 7.0 Hz,

H-12), 0.92 (d, 3H, J - 6.9 Hz, H-ll), 0.89 (t, 3H, J = 7.1 Hz, H-10); ,3C NMR

(125 MHz, CDC13) 8 138.3,128.5, 127.8,127.7, 87.3, 74.6, 70.3, 68.8, 40.9, 35.0,

34.5, 31.7, 29.7, 22.9, 17.9, 16.6, 14.1; a* = 9.63 (c = 0.70, CHCI3); HRES m/z

+ calcd. for C19H32Na03 331.2244, found 331.2239 [M+Na] . Experimental procedures 174

(2R, 45, 55, 6J?)-5-(Benzyloxy)-l-(/er/-butyldimethylsilyloxy)-2, 6- dimethyldecane-4-oI (91)

Compound 91 was synthesized using the protocol of Chaudhary et. al.m In a flask under Ar containing 0.26 g (0.86 mmol) of 89 was dissolved in 10 mL of dry

CH2CI2. Then 0.14 g (0.94 mmol) of ter/-butyldimethylsilyl chloride, 4.20 mg of

DMAP, and 0.14 mL of NEt3 were added, and the mixture was allowed to stir at

23 °C overnight. The reaction was quenched by the addition of 10 mL of saturated NH4CI, and the aqueous layer was extracted with CH2CI2 (3x10 mL).

The combined organic layers were washed with 15 mL of brine and dried over

Na2SC>4. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 9.5:0.5 hexane/EtOAc eluent to afford 0.29 g

(80%) of 91 as a colourless oil. IR (cast film) 2955, 2928, 2857 cm"1; !H NMR

(400 MHz, CDCI3) 8 7.43 - 7.30 (m, 5H, H-15, H-15', H-16, H-16', H-17), 4.69

(AB quartet, 2H, J= 19.5, 11.1 Hz, H-13), 3.79 (m, 1H, H-4), 3.52 (m, 2H, H-la,

H-lb), 3.16 (t, 1H, J =4.3 Hz, H-5), 2.59 (m, 1H, 4-OH), 1.90 (m, 1H, H-2), 1.78

(m, 1H, H-6), 1.62 - 1.18 (m, 8H, H-9, H-8, H-7, H-3), 0.98 (m, 18H, H-12, H-

13 11, H-10, 3 x CH3), 0.10 (s, 6H, Si-CH3); C NMR (125 MHz, CDC13) 6 138.8,

128.4, 127.7, 127.6, 86.5, 75.1, 70.1, 67.3, 38.2, 35.1, 33.9, 32.4, 29.8, 25.9, 22.9, Experimental procedures 175

17.9, 14.7, 14.1, -5.4; a* = -0.82 (c = 0.88, CHC13); HRES m/z calcd. for

+ C25H46Na03Si 445.3108, found 445.3107 [M+Naj .

0-(2R, 45, 55, 6J?)-5-(BenzyIoxy)-l-(terf-butyldimethylsilyloxy)-2, 6- dimethyldecane-4-yl l/Z-imidazole-l-carbothioate (92)

Compound 92 was synthesized using the protocol of Singh et. a/.154 In a flask under Ar containing 0.20 g (0.48 mmol) of 91 was dissolved in 5 mL of dry

CH2CI2. Then 0.42 g (2.37 mmol) of 1,1'-thiocarbonyldiimidazole and 0.12 g of

DMAP were added, and the yellow mixture was allowed to stir at 23 °C for 24 h.

The solvent was removed under vacuum, and the residue was purified by column chromatography using a 9:1 hexane/EtOAc eluent to afford 0.23 g (91%) of 92 as a colourless oil. IR (cast film) 2956, 2929, 2857 cm"1; 'H NMR (500 MHz,

CDCI3) 8 8.53 (s, 1H, H-19), 7.61 (s, 1H, H-20), 7.37 - 7.20 (m, 5H, H-15, H-15',

H-16, H-16', H-17), 7.02 (s, 1H, H-21), 5.96 (dt, 1H, J= 9.8, 4.8 Hz, H-4), 4.60

(s, 2H, H-13), 3.56 - 3.40 (ddd, 2H, J= 60.2, 9.9, 5.3 Hz, H-l), 3.48 (t, 1H, J-

5.0 Hz, H-5), 2.08 (m, 1H, H-3b), 1.80 - 1.68 (m, 2H, H-6, H-2), 1.62 - 1. 57 (m,

2H, H-3a, H-8a), 1.48 (m, 1H, H-7a), 1.38 - 1.10 (m, 4H, H-9, H-8b, H-7b), 0.99 Experimental procedures 176

(d, 3H,J= 6.8 Hz, H-12), 0.95 (d, 3H, J= 6.7 Hz, H-l 1), 0.87 (m, 12H, H-10, 3 x

13 CH3), 0.03 (s, 6H, Si-CHj); C NMR (125 MHz, CDC13) 5 183.3, 137.6, 130.1,

127.7, 127.1, 126.9, 82.8, 82.2, 73.8, 66.5, 34.3, 32.9, 32.8, 31.8, 28.9, 25.3, 22.4,

16.9, 14.5, 13.4, -5.9, -6.0; a* = -7.23 (c = 1.84, CHClj); HRES m/z calcd. for

C29H49N203NaSiS 533.3227, found 533.3218 [M+Naf

(2R, 5R, 6/?)-5-(BenzyIoxy)-2,6-dimethyldecan-l-ol (93) 17

Compound 93 was synthesized following a modified procedure from Singh et. a/.154 In a flask under Ar containing 0.23 g (0.43 mmol) of 92 was dissolved in 25 mL of dry degassed toluene. Then 0.50 g (1.72 mmol) of tributyltin hydride and

35.0 mg (0.22 mmol) of AIBN were added, and the mixture was heated to 60 °C for 4 h. The solvent was removed, and the residue was purified by column chromatography, on 10% (w/w) of KF in silica gel, using a 9.5:0.5 hexane/EtOAc eluent. The product was still contaminated with tributyltin hydride. Therefore, it was taken directly to the next step. In a flask containing crude 93 was dissolved in 6 mL of AcOH, 6 mL of H2O, and 2 mL of THF. This was heated to 85 °C for

5 h. The mixture was cooled to 23°C and then the THF was then removed under vacuum. The aqueous layer was extracted with Et20 (3x5 mL), and then the combined organic layers were washed with 10 mL of brine and dried over

Na2SC>4. The solvent was removed under vacuum, and the residue was purified Experimental procedures 177 by column chromatography 10% (w/w) of KF in silica gel, using a 8:1 hexane/EtOAc eluent to afford 0.05 g (40% over two steps) of 93 as a colourless

1 oil. IR (cast film) 3378, 2955, 2928, 2871 cm" ; 'H NMR (500 MHz, CDC13) 6

7.38 -7.22 (m, 5H, H-15, H-15', H-16, H-16', H-17), 4.52 (AB quartet, 2H, J =

20.42, 11.54 Hz, H-13), 3.43 (m, 2H, H-l), 3.22 (dt, 1H, J= 7.9,4.2 Hz, H-5), 1.8

(m, 1H, OH), 1.76 (m, 1H, H-6), 1.62 - 1.19 (m, 10H, H-9, H-8, H-7a, H-4, H-3,

H-2), 1.16 (m, 1H, H-7b), 0.90 (m, 9H, H-l2, H-l2, H-10); ,3C NMR (125 MHz,

CDC13) 5 139.2, 128.3, 127.8, 127.3, 83.5, 71.8, 68.3, 35.8, 35.5, 31.8, 29.8, 29.4,

27.6, 23.0, 16.5, 15.3, 14.1; a* = 22.60 (c = 0.46, CHCI3); HRES m/z calcd. for

+ Ci9H32Na02 315.2295, found 315.2295 [M+Na] .

(2R, 5R, 6/?)-5-(Benzvloxy)-2,6-dimethyldecanal (94)

11 12

In a flask containing 46.4 mg (0.16 mmol) of 93 and 80.7 mg (0.19 mmol) of

Dess-Martin periodinane was dissolved in 10 mL of CH2CI2. The cloudy mixture was allowed to stir at 23 °C for 2 h. The reaction was quenched by adding 10 mL of a 1:1 mixture of saturated NaHCC>3 and 1.0 M NaS2C>3. The mixture was stirred until two clear layers had formed. The aqueous layer was extracted with

CH2CI2 (3x10 mL), and the combined organic layers were washed with 10 mL of saturated NaHCC>3, 10 mL of brine and then dried over Na2SC>4. The solvent was Experimental procedures 178 removed under vacuum, and the residue was purified by column chromatography using a 9.5:0.5 hexane/EtOAc eluent to afford 37.5 mg (82%) of 94 as a colourless oil. IR (cast film) 2956, 2929, 2871, 2858, 1726 cm"1; 'H NMR (500

MHz, CDCI3) 8 9.59 (d, 1H, J= 1.9 Hz, H-l), 7.38 (m, 3H, H-16, H-16', H-17),

7.24 (m, 2H, H-15, H-15'), 4.55-4.45 (AB quartet, 2H, J= 36.9, 11.5 Hz, H-13),

3.21 (m, 1H, H-5), 2.29 (m, 1H, H-2), 1.70 (m, 2H, H-6, H-3a), 1.60 - 1.40 (m,

5H, H-8, H-4, H-3a), 1.39 - 1.18 (m, 4H, H-9, H-7), 1.08 (d, 3H, J = 6.9 Hz, H-

11), 0.89 (m, 6H, H-12, H-10); 13C NMR (125 MHz, CDCI3) 5 205.1 139.9,

128.3, 127.8, 127.4, 83.1, 71.7,46.4, 35.4, 32.7, 31.7, 29.8, 27.7, 26.9, 22.9, 15.3,

14.1, 13.4; a" = 8.81 (c = 0.64, CHC13); HRES m/z calcd. for C,9H3oNa02

313.2138, found 313.2134 [M+Na]+.

(2/f, SR, 6/?)-5-(BenzyIoxy)-2,6-dimethyldecanoic acid (95)

Compound 95 was prepared following the protocol of Hu et. al.155 The stock solution was prepared by adding 5 mL of /-butyl alcohol and 2 mL of 2-methyl-2- butene (2.0 M in THF) into a flask. Then a mixture of 86 mg of NaClCh, 57 mg of NaH2PC>4 in 2 mL of distilled H2O was added to the stock solution. This mixture was stirred vigorously at 23 °C for 10 min. A portion of this stock solution (1.6 mL) was added to a flask containing 31.1 mg (0.11 mmol) of 94, and Experimental procedures 179 the yellow mixture was stirred at 23 °C for 3 h. The reaction mixture was diluted with 5 mL of H2O, and the pH of the solution adjusted to pH 6 using 1 M HC1.

The aqueous layer was extracted with EtOAc (3x5 mL), and the combined organic layers were washed with 10 mL of brine and then dried over NaaSO,*.

The solvent was removed under vacuum, and the residue was purified by column chromatography using a 8:1 hexane/EtOAc +1% AcOH eluent to afford 32.0 mg

(quant.) of 95 as a colourless oil. IR (cast film) 3300 - 2500, 2956, 2930, 2872,

2859,1706 cm"1; 'H NMR (500 MHz, CDCI3) 5 7.37 (m, 3H, H-16, H-16', H-17),

7.26 (m, 2H, H-15, H-15'), 4.51 (AB quartet, 2H, J = 20.2, 11.5 Hz, H-13), 3.22

(m, 1H, H-5), 2.43 (m, 1H, H-2), 1.78 - 1.48 (m, 7H, H-8, H-6, H-4, H-3), 1.38 -

1.10 (m, 7H, H-l 1, H-9, H-7), 0.90 (d, 3H, 6.7 Hz, H-l 1), 0.89 (m, 3H, H-10);

13C NMR (125 MHz, CDCI3) 5 181.2,138.9, 128.3, 127.8, 127.4, 83.3, 71.8, 39.3,

35.4, 31.8, 30.2, 29.8, 28.0, 22.9, 17.0, 15.2, 14.1; a% = 6.57 (c = 0.42, CHC13);

HRES m/z calcd. for C19H29O3 305.2122, found 305.2119 [M-H]'.

(3R, 6/?)-6-[(/?)-Hexan-2H-yl]-3-methyltetrahydro-2/T-pyran-2-one (96)

11 3

In a flask containing 30.4 mg (0.10 mmol) of 95 and 30.0 mg of 10% Pd/C was dissolved in 6 mL of EtOH. The black mixture was stirred vigorously under 1 atm of H2 for 24 h. The solution was filtered through a pad of celite, and the solvent was removed under vacuum. The residue was purified by column Experimental procedures 180 chromatography using a 7:1 hexane/EtOAc + 1% AcOH eluent to afford 2.60 mg

(13%) of 96 as a colourless oil. IR (cast film) 2959, 2933, 2873, 1743 cm"1; 'H

NMR (500 MHz, CDC13) 8 4.16 (ddd, 1H, J= 11.6, 4.9, 3.4 Hz, H-5), 2.60 (m,

1H, H-2), 2.09 (m, 1H, H-3a), 1.85 (m, 1H, H-4a), 1.75 - 1.64 (m, 2H, H-6, H-

4b), 1.55 - 1.46 (m, 2H, H-7a, H-3b), 1.36 - 1.14 (m, 8H, H-ll, H-9, H-8, H-7b),

0.98 (d, 3H, J = 6.8 Hz, H-12), 0.90 (t, 3H, J = 7.1 Hz, H-10); 13C NMR (125

MHz, CDClj) 8 176.7, 81.8, 37.2, 33.1, 32.0, 29.3, 25.7, 23.6, 22.9, 16.3, 14.6,

14.1; a% = -43.1 (c = 0.74, CHC13); HRES m/z calcd. for Ci2H22Na02 221.1512, found 221.1512 [M+Na]+.

Lithium (2R, 5R, 61?)-5-hydroxy-2,6-dimethyldecanoate (97)

11 12

Compound 97 was prepared in a similar fashion to 80. In a flask containing 2.7 mg (13.6 fimol) of 96, 2 mL of THF, and 2 mL of H20 was added 27.2 nL (27.2

Hmol) of a 1.0 M LiOH solution. The mixture was stirred at 23 °C for 2 h. The

THF was removed under vacuum, and the H20 was lyophilized to afford 4.96 mg

(quant.) of 97 as a white solid containing 0.93 mg of LiOH. ]H NMR (600 MHz,

D20) 8 3.44 (m, 1H, H-5), 2.22 (m, 1H, H-2), 1.60 - 1.10 (m, 10H, H-9, H-8, H-

7, H-6, H-4a, H-3), 1.09 - 1.05 (m, 1H, H-4b), 1.04 (d, 3H, J = 6.9 Hz, H-ll),

0.85 (t, 3H, J = 6.8 Hz, H-10), 0.81 (d, 3H, J = 6.8 Hz, H-12); 13C NMR (100 Experimental procedures 181

MHz, D20) 8 187.1, 75.3, 57.5,43.2, 37.1, 32.4, 31.6, 30.9, 29.0, 22.4, 17.1, 13.3;

HRES m/z calcd. for Q2H23O3 215.1653, found 215.1653 [M-H]\

(2R, 4/f, 5 R, 6i?)-5-(Benzyloxy)-l-(tert-butyldimethylsilyloxy)-2, 6- dimethyldecane-4-ol (98)

Compound 98 was synthesized in the same fashion as 91 using 90 to afford 0.17 g

(72%) of 98 as a colourless oil.

1 IR (cast film) 2955, 2929, 2857 cm" ; *H NMR (500 MHz, CDC13) 5 7.40 - 7.24

(m, 5H, H-15, H-15', H-16, H-16\ H-17), 4.70 - 4.57 (AB quartet, 2H, J= 46.5,

11.2 Hz, H-13), 3.78 (m, 1H, H-4), 3.46 (d, 2H, J= 6.1 Hz, H-l), 3.08 (t, 1H, J =

4.0 Hz, H-5), 2.50 (d, 1H, J = 6.3 Hz, OH), 1.96 - 1.80 (m, 2H, H-2, H-6), 1.65

(m, 1H, H-3a), 1.57 - 1.50 (m, 1H, H-7a), 1.42 - 1.18 (m, 6H, H-9, H-8, H-7b, H-

3b), 1.00 (d, 3H, J= 6.9 Hz, H-ll), 0.91 (d, 3H, J= 6.9 Hz, H-10), 0.91 - 0.88

,3 (m, 12H, H-l2, 3 x CH3), 0.02 (s, 6H, Si-CH3); C NMR (125 MHz, CDC13) 6

138.6, 128.4, 127.7, 127.6, 87.2, 74.4, 69.3, 69.0, 39.1, 34.9, 32.8, 31.9, 29.6,

25.9, 22.9,18.3,16.5,16.4, 14.1, -5.3, -5.4; a* = 16.21 (c = 0.75, CHC13); HRES m/z calcd. for C2sH46Na03Si 445.3108, found 445.3108 [M+Na]+.. Experimental procedures 182

0-(2R, 4R, 5R, 6/?)-5-(Benzyloxy)-l-(ter/-butyldimethylsilyloxy)-2, 6- dimethyldecane-4-yl l//-imidazole-l-carbothioate (99) 17

Compound 99 was synthesized in a similar fashion to 92 using 98 to afford 0.19 g

(91%) of 99 as a colourless oil. IR (cast film) 2955, 2929, 2857 cm"1; *H NMR

(600 MHz, CDCI3) 5 8.34 (s, 1H, H-19), 7.61 (s, 1H, H-20), 7.37 - 7.24 (m, 5H,

H-15, H-15', H-16, H-16', H-17), 7.02 (s, 1H, H-21), 6.02 (dt, 1H, J= 9.8, 3.6

Hz, H-4), 4.67 (AB quartet, 2H, J= 15.6,11.4 Hz, H-13), 3.42 (m, 3H, H-5, H-l),

2.11 (ddd, 1H, J = 14.0, 9.7, 3.9 Hz, H-3a), 1.80 (m, 1H, H-6), 1.71 (m, 1H, H-

2a), 1.59 (m, 2H, H-7a, H-3b), 1.40 - 1.18 (m, 5H, H-9, H-8, H-7b), 0.96 (d, 3H,

J= 6.8 Hz, H-l2), 0.94 (d, 3H,J= 6.6 Hz, H-ll), 0.89 (m, 12H, H-10, 3 x CH3),

0.02 (m, 6H, Si-CHj); ,3C NMR (100 MHz, CDClj) 5 184.3, 138.4, 131.0, 128.6,

128.0, 127.9, 83.5, 82.6, 74.1, 68.4, 34.7, 33.9, 32.7, 32.1, 29.4, 26.2, 23.2, 18.5,

16.9, 16.5, 14.4, 14.3, -5.2; a*5 = 7.61 (c = 1.50, CHCI3); HRES m/z calcd. for

Cz^NzOjNaSiS 533.3227, found 533.3231 [M+Na]+. Experimental procedures 183

(21?, 5R, 6/?)-5-(Benzyloxy)-2,6-dimethyldecan-l-ol (100)

O' 7 9 HO 10

11 12

Compound 100 was synthesized in a similar fashion as 93 using 99 to afford 0.06 g (60% over two steps) of 100 as a colourless oil. IR (cast film) 3403, 2956,

2929, 2872 cm"1; 'H NMR (600 MHz, CDCI3) 5 7.37 (m, 3H, H-16, H-16', H-17),

7.25 (m, 2H, H-15, H-15'), 4.54 - 4.45 (AB quartet, 2H, J= 53.9, 11.6 Hz, H-13),

3.51 (m, 1H, H-la), 3.42 (m, 1H, H-lb), 3.21 (ddd, 1H,J= 8.3, 4.7, 3.6 Hz, H-5),

1.80 (m, 1H, H-6), 1.62 -1.10 (m, 12H, H-9, H-8, H-7, H-4, H-3), 0.93 (d, 3H, J

= 6.7 Hz, H-ll), 0.90 (t, 3H, J= 7.2 Hz, H-10), 0.89 (d, 3H, J= 6.8 Hz, H-12);

13C NMR (125 MHz, CDCI3) 6 139.1, 128.3, 127.8, 127.4, 83.6, 71.5, 68.1, 35.9,

35.1, 32.6, 29.8, 29.4, 26.9, 23.0, 16.8, 14.6, 14.1; a* = -12.47 (c = 0.42,

CHCI3); HRES m/z calcd. for C^NaOz 315.2295, found 315.2294 [M+Na]+.

(2R, 55,6/?)-5-(Benzyloxy)-2,6-dimethyldecanal (101)

11 12

Compound 101 was synthesized in a similar fashion as 94 using 100 to afford

42.8 mg (72%) of 101 as a colourless oil. IR (cast film) 2957, 2926, 2856, 1726 Experimental procedures 184

1 cm" ; 'H NMR (600 MHz, CDC13) 8 9.56 (d, 1H, J= 2.1 Hz, H-l), 7.38 (m, 3H,

H-16, H-16', H-l7), 7.23 (m, 2H, H-15, H-15'), 4.57 -4.41 (AB quartet, 2H, J =

77.6, 11.5 Hz, H-l3), 3.23 (app. quartet, 1H,J= 5.1 Hz, H-5), 2.31 (m, 1H, H-2),

1.92 (m, 1H, H-3a), 1.81 (m, 1H, H-6), 1.49 (m, 2H, H-4), 1.40 - 1.12 (m, 7H, H-

9, H-8, H-7, H-3b), 1.08 (d, 3H, J = 6.9 Hz, H-l 1), 0.90 (t, 3H,J= 7.2 Hz, H-10),

13 0.88 (d, 3H, J = 6.8 Hz, H-12); C NMR (125 MHz, CDC13) 5 205.2, 138.9,

128.3, 127.8, 127.5, 82.8, 71.3, 46.4, 34.8, 32.7, 29.8, 26.9, 26.8, 22.9, 14.4, 14.1,

13.4; af = -14.5 (c = 0.52, CHC13); HRES m/z calcd. for Ci9H3oNa02 313.2138, found 313.2137 [M+Na]+.

(2R, 5S, 6/?)-5-(BenzyIoxy)-2,6-Dimethyldecanoic acid (102)

Compound 102 was synthesized in a similar fashion as 95 using 101 to afford

35.9 mg (89%) of 102 as a colourless oil. IR (cast film) 3300 - 2600, 2957, 2931,

1 2872, 1706 cm" ; 'H NMR (500 MHz, CDC13) 8 7.36 (m, 3H, H-16, H-16', H-l7),

7.26 (m, 2H, H-15, H-15'), 4.56-4.43 (AB quartet, 2H, J- 45.5, 11.6 Hz, H-13),

3.21 (m, 1H, H-5), 2.42 (m, 1H, H-2), 1.82 (m, 2H, H-6, H-3a), 1.56 - 1.10 (m,

12H, H-l 1, H-9, H-8, H-7, H-4, H-3b), 0.89 (t, 3H, J= 7.1 Hz, H-10), 0.87 (d,

13 3H, 6.9 Hz, H-12); C NMR (125 MHz, CDC13) 8 181.5, 139.0, 128.3, 127.8, Experimental procedures 185

127.4, 82.6, 71.2, 39.1, 34.9, 32.6, 29.7, 29.6, 26.9, 22.9, 17.0, 14.5, 14.1; a2* = -

19.6 (c = 0.40 CHClj); HRES m/z calcd. for C19H29O3 305.2122, found 305.2116

(3R, 65)-6-[(/f)-Hexan-2-y 1] -3-MethyItetrahydro-2//-pyran-2-one (103) 12

11 3

Compound 103 was synthesized in a similar fashion as 96 using 102 to afford

5.80 mg (29%) of 103 as a colourless oil. IR (cast film) 2958, 2931, 2873, 1731;

'HNMR (600 MHz, CDCI3) 8 4.19 (ddd, 1H,J = 11.5,5.3,3.1 Hz, H-5), 2.40 (m,

1H, H-2), 2.03 (m, 1H, H-3a), 1.83 (m, 1H, H-4a), 1.77 (m, 1H, H-6), 1.65 - 1.46

(m, 3H, H-7a, H-4b, H-3b), 1.40 - 1.13 (m, 8H, H-ll, H-9, H-8, H-7b), 0.92 (d,

3H, J = 6.8 Hz, H-12), 0.89 (t, 3H, J = 7.2 Hz, H-10); ,3C NMR (125 MHz,

CDCI3) 6 174.7, 85.6, 37.7, 36.4, 31.7, 29.4, 28.6, 24.9, 22.9, 17.5, 14.5, 14.1;

a* = 9.30 (c = 0.46, CHCI3); HRES m/z calcd. for C12H22Na02 221.1512, found

221.1512 [M+Na]+. Experimental procedures 186

Lithium (2R, 5R, 6/?)-5-hydroxy-2,6-dimethyldecanoate (104)

O OH

LiO 1 10

11 12

Compound 104 was synthesized in a similar fashion as 97 using 103 to afford

7.06 mg (quant.) of 104 as a white solid containing 1.33 mg of LiOH. 'H NMR

(600 MHz, D20) 8 3.50 (m, IH, H-5), 2.26 (m, IH, H-2), 1.62 (m, IH, H-3a),

1.56 (m, IH, H-6), 1.52 - 1.14 (m, 8H, H-9, H-8, H-7, H-4), 1.08 (m, IH, H-3b),

1.05 (d, 3H, J= 7.0 Hz, H-ll), 0.86 (t, 3H, J= 7.2 Hz, H-10), 0.83 (d, 3H,J = 7.0

13 Hz, H-12); C NMR (100 MHz, D20) 6 187.1, 76.0, 43.1, 37.8, 31.1, 30.7, 30.3,

29.1, 22.6, 18.0, 14.7, 13.6;

C12H23O3 215.1653, found 215.1649 [M-H]\

4-(Benzyloxy)-6-Methyl-2//-pyran-2-one (110)

The known compound 110 was synthesized using the protocol of Funa et. a/.156 In a flask under Ar containing 1.05 g (8.36 mmol) of 4-hydroxy-6-methyl-2-pyrone,

3.46 g (25.07 mmol) of K2CO3, and 1.48 ml (2.14 g, 12.54 mmol) of benzyl bromide was dissolved in 40 mL of dry N, iV-dimethylformamide. The slurry was Experimental procedures 187 stirred for 1 hr at 23 °C and then cooled to 0 °C. The reaction was quenched by adding 40 mL of H2O, and the aqueous layer extracted with EtOAc (3 x 20 mL).

The combined organic layers were washed with 15 mL of brine and dried over

Na2S04. The solvent was removed under vacuum, and the residue was purified by column chromatography using a 1:3 EtOAc/hexanes eluent to afford 0.75 g of

110 as a white solid (42%): IR (solid) 3086, 3035, 1738, 1651 cm'1; ]H NMR

(600 MHz, CDCb) 5 7.40 (m, 5H, H-8, H-9, H-10), 5.84 (m, 1H, H-4), 5.50 (d,

1H, J = 2.2 Hz, H-2), 5.01 (s, 2H, H-6), 2.21 (s, 3H, H-ll); 13C NMR (125 MHz,

CDCb) 5 170.2 (C-3), 164.8 (C-l), 162.2 (C-5), 134.4 (C-7), 128.8 (C-10), 128.7

(C-8), 127.8 (C-9), 100.5 (C-4), 88.5 (C-2), 70.7 (C-6), 19.9 (C-11); HRES m/z

+ calculated for Ci3Hi203Na 239.0678 found 239.0678 [M+Na] .

4-(Benzyloxy)-6-(2-oxopropyI)-2//-Pyran-2-one (111)

The known compound 111 was prepared following the protocol of Funa et. al.156

A flask under Ar containing 0.14 g (0.65 mmol) of 110 and 10 mL of dry THF was cooled to -78 °C. Then 1.09 mL (1.09 mmol) of LiHMDS (1.0 M in hexanes) was added drop wise, and the mixture was stirred at -78 °C for 1 h. Addition of

LiHMDS turns the solution from yellow to light red. After 1 h, 0.17 mL (2.33 Experimental procedures 188 mmol) of acetyl chloride was added slowly over 10 min, and the solution was allowed to stir for 3 h at -78 °C. The reaction was quenched with 10 mL of saturated NH4CI, and the layers were separated. The aqueous layer was extracted with Et20 (3x10 mL), and the combined organic layers were washed with 15 mL of brine and dried over Na2S04. The solvent was removed under vacuum, and the brown residue was purified by column chromatography using a 1:1 hexane/EtOAc eluent to provide 85.1 mg of 111 as a white solid (51%). IR (solid) 3084, 3039,

1 1730, 1715, 1654 cm" ; 'H NMR (600 MHz, CDC13) 5 7.20 (m, 5H, H-8, H-9, H-

10), 5.97 (d, 1H, J= 2.2 Hz, H-4), 5.55 (d, 1H, J= 2.2 Hz, H-2), 5.01 (s, 2H, H-

6), 3.58 (s, 2H, H-l 1), 2.27 (s, 3H, H-13); ,3C NMR (125 MHz, CDCI3) 5 201.2

(C-12), 169.7 (C-3), 164.1 (C-l), 157.7 (C-5), 134.2 (C-7), 128.9 (C-10), 128.8

(C-8), 127.8 (C-9), 103.1 (C-4), 89.6 (C-2), 70.9 (C-6), 47.8 (C-ll), 30.2 (C-13);

+ HRES m/z calculated for Ci5H,404Na 281.0784 found 281.0783 [M+Na] .

(5)- 4-(Benzyloxy)-6-(2-hydroxypropyl)-2//-Pyran-2-one (112)

Compound 112 was prepared following the protocol of Smith et. a/.157 A flask under Ar containing 0.15 g (0.59 mmol) of 111, 16.30 mg (0.06 mol) of (j*?)-(+)-2- methyl-CBS-oxazaborolidine, and 10 mL of dry CH2CI2 was cooled to -30 °C. Experimental procedures 189

Then 0.30 mL of BH3*SMe2 (0.62 mmol, 2.0 M in THF) was added over 30 min and the mixture was allowed to stir at -30 °C overnight. The reaction was quenched by the slow addition of 1 M HCI in MeOH and the solution was allowed to warm to 23 °C and stirred at that temperature for 30 min. The solvent was removed under vacuum and the residue dissolved in 10 mL of EtOAc and then washed with 10 mL of 1M HCI, 10 mL of saturated NaHCOj, 10 mL of brine, and dried over Na2SC>4. The solvent was removed under vacuum and the residue purified by column chromatography using a 2:1 EtOAc/hexanes eluent to afford

65.9 mg of 112 as a colourless oil (43%) and 71.5 mg of 111. IR (cast film) 3418,

1 2969, 2930, 1705, 1647 an ; 'HNMR (600 MHz, CDC13) 6 7.40 (m, 5H, H-8, H-

9, H-10), 5.95 (d, 1H, J = 2.2 Hz, H-4), 5.57 (d, 1H,J = 2.2 Hz, H-2), 5.01 (s, 2H,

H-6), 4.23 (m, 1H, H-12), 2.58 (m, 2H, H-ll), 1.28 (d, 3H, J= 6.3 Hz, H-13); 13C

NMR (125 MHz, CDC13) 6 170.1 (C-3), 164.8 (C-l), 162.7 (C-5), 134.3 (C-7),

128.8 (C-10), 128.7 (C- 8), 127.8 (C-9), 101.8 (C-4), 89.0 (C-2), 70.8 (C-6), 65.4

(C-12), 43.3 (C-l 1), 23.4 (C-13); a* = -18.90 (c = 0.40, MeOH); HRES m/z

+ calculated for Ci5H,604Na 283.0940 found 283.0935 [M+Na] .

(S)- 4-6-(2-Hydroxypropyl)-2//-pyran-2-one (113)

Compound 113 was prepared in a similar fashion as 96. A mixture of 29.0 mg

(0.11 mmol) of 112, 11.5 mg of Pd/C (10%), and 8 mL of EtOAc was stirred for

1.5 hr under 1 atm of H2 at 23 °C. The solution was filtered through a pad of Experimental procedures 190 celite and washed with 30 mL of EtOAc. The solvent was removed under vacuum, and the residue was purified by preparatory TLC (1000 microns) using

100:1 EtOAc/AcOH as eluent to provide 16.1 mg of 113 as an oil (85%). IR (cast

1 film) 3366, 2971, 2917, 1684, 1570 an ; *H NMR (600 MHz, CD3OD) 8 6.05 (s,

1H, H-2 or 4), 4.20 (m, 1H, H-7), 2.56 (m, 2H, H-6), 1.22 (d, 3H, J = 6.3 Hz, H-

13 8); C NMR (125 MHz, CD3OD) 6 173.6 (C-3), 168.6 (C-l), 165.6 (C-5), 103.5

(C-2), 66.2 (C-7), 44.1 (C-6), 23.4 (C-8); a*5 = -20.32 (c = 1.00, MeOH); HRES m/z calculated for CsHioC^Na 193.0471 found 193.0470 [M+Na]+.

4.3 Biological procedures

4.3.1 Expression and purification of LovB

A sterile micropipette was dipped into a glycerol stock of LovB-pADH2 in

Saccharomyces cerevisiae strain BJ5463:lNpgA and added to 5 mL of SCDt

(A,T) asceptically in growth tubes. This starter culture was shaken at 30 °C for 48 h until the culture was cloudy but not dense. For 1 L of liquid culture, a 2 L

Erlenmeyer flask with YPD media (10 g Bacto Yeast Extract, 20 g Bacto Peptone, and 900 mL of deionized water) was autoclaved and allowed to cool. A 20% dextrose solution (filtered through 0.2 [x filter and not autoclaved to prevent caramelization of the sugar) was added asceptically to the 2 L Erlenmeyer flask.

When ready to express LovB, the starter culture concentration should be at 0.1% and dextrose at 1% (ie. for a 1 L culture, add 100 mL of 20% dextrose stock and 1 mL of starter culture). The expression culture was prepared by adding 1 mL of starter culture to the YPD media, and then grown for 72 h at 30 °C. The yeast cells were harvested through centrifugation at 4 °C for 15 min at 2000 x g. The Experimental procedures 191 supernatant was removed and the cell pellets were resuspended in 20 mL of lysis buffer (50 mM NaH2P04 pH = 8.0, 0.15 M NaCl, and 10 mM imidazole). The cells were lysed by sonication for 5 min/1 L of culture with alternating 1 min sonication in an ice bath and 1 min rest. Cellular debris was removed through centrifugation at 27 000 x g for 1 h at 4 °C. The supernatant was isolated and Ni- resin (3 mL Ni-Agarose resin/1 L of cell culture) was added and stirred at 4 °C for at least 2 h to overnight. The mixture was loaded onto a gravity flow column and first eluted with Buffer A (50 mM Tris-HCl pH = 7.9, 500 mM NaCl, 2 mM DTT,

2 mM EDTA, and 5 mM imidazole). The second elution contained 40 mL of

Buffer A with 10 mM imidazole, third elution contained 30 mL of Buffer A with

20 mM of imidazole, and the final elution contained 16 mL of Buffer A with 250 mM of imidazole. A 6% SDS PAGE gel was ran on each fraction to determine which fractions contained pure LovB. Pure fractions that contained LovB were concentrated using Amicon protein concentrator (100 000 MWCO), which were pre-chilled on ice. The retentate was centrifuged at 1912 x g at 4 °C for 45 min until it was under 1 mL. The buffers were exchanged by the addition of 14 mL of

Buffer A and centrifuged at 4 °C for 45 min at 1464 x g until the retentate was less than 1 mL. The aliquots were stored at -80 °C with 10 % glycerol. Protein concentrations were determined using the Bradford assay with BSA as a standard as well as determination from protein UV absorbance at X 280 and e = 345050 AU

L mol"1 cm"1. Experimental procedures 192

4.3.2 Mass spectrum analysis of LovB fragments

In a 1.0 mL eppendorf tube was added 0.54 mg of LovB. This was diluted to 500 |xL with 100 mM NH4HCO3 and the solution was adjusted to pH 8.

Chymotrypsin 26.8 ng (sequencing grade, Roche Cat. No. 11 418 467 001) was added, and the mixture was allowed to stand at 36°C over night. The digestion was stopped by acidification to pH > 3 using 10% TFA. Water was removed under vacuum until ~50 jxL remained. The sample was then desalted using a Cig

Ziptip™ (Millipore Cat. No. ZTC8S096), and protein was eluted with 85%

CH3CN + 0.1% formic acid. This solution was concentrated under vacuum to ~ 10

(iL, and 2 was taken and diluted to 10 |xL with 0.1% formic acid. Samples were then submitted for LC-MS/MS using a Waters Q-TOF Premier instrument in positive ion mode.

4.3.3 Expression and purification of LovC

A sterile micropipette was dipped into a glycerol stock of LovC-pET in

E.coli strain BL21(DE3) and added to 5 mL of LB medium with 100 fig/mL of ampicillin in a growing test tube. This test tube was shaken at 37 °C for 12 - 14 h. For a 1 L culture, a 2 L Erlenmeyer flask containing 1 L of LB medium was autoclaved and allowed to cool. Then 100 ng/mL of ampicillin was added to the

LB media. The overnight culture was added to the sterile LB medium and grown at 37 °C until OD600 ~ 0.4 - 0.6 (approximately 3 h). The flask was then cooled on ice or in the fridge for 10 min and then induced with 110 jaL of 1 M isopropyl thio-p-D-galactoside (IPTG). The culture was allowed to grow at 16 °C for 16 -

24 h. Two 500 mL centrifuge tubes, 50 mL falcon tube, and four high-speed Experimental procedures 193 centrifuge tubes were pre-chilled in the -20 °C freezer for at least 10 min. The cell cultures were poured into the pre-chilled 500 mL centrifuge tubes and centrifuged at 2000 x g for 15 min at 4 °C. The supernatant was removed and the remaining cell culture was added to the centrifuge tubes and centrifuged again at

3500 rpm for 15 min at 4 °C. This was repeated until the entire cell culture has been spun down. The cell pellets were kept on ice and resuspended in cold lysis buffer (30 mL/lL of expression culture). Lysis buffer: 50 mM NaH2P04, 150 mM NaCl, 10 mM imidazole pH = 7-8. The cells were lysed by sonication in an ice bath in 1 min intervals with 1 min rest period for a total of 9 min. The cell lysis solution was pre-chilled in high-speed centrifuge tubes and then centrifuged at 27 000 x g for 65 min at 4 °C. The supernatant was filtered with a 0.45 ja syringe filter, work quickly so that the protein does not warm up. The sterilized supernatant was transferred to the pre-chilled 50 mL falcon tube and 1 mL of Ni-

NTA resin was added for each 1 L of expression culture and allowed to interact for 2 - 24 h. The resin mixture was loaded onto a gravity flow column in the cold room and eluted with the following buffers: (a) original flow through, (b) 40 mL

Buffer A with 10 mM imidazole and 2 mM DTT, (c) 20 mL Buffer A with 20 mM imidazole and 2 mM DTT, and (d) 15 mL Buffer A with 250 mM imidazole and 2 mM DTT. Fractions a - d were ran on an SDS-PAGE protein gel with 6% gel. Pure fractions were combined and concentrated using a pre-chilled Amicon protein concentrator (20 000 MWCO) and centrifuged at 1912 x g for 45 min at 4

°C. If the retentate was under 1 mL begin buffer exchange, otherwise it was centrifuged again at 1912 x g at 4 °C until retentate is under 1 mL. To buffer Experimental procedures 194 exchange, 14 mL of 50 mM Tris-HCl pH = 7.9, 2 mM EDTA, and 2 mM DTT was added to the upper chamber and centrifiiged at 1464 x g for 45 min at 4 °C until retentate was less than 1 mL. The protein was aliquoted into 500 |iL eppendorf tubes with 10 % glycerol and stored in -80 °C. Protein concentration was determined through the Bradford assay.

4.3.4 In vitro enzyme assays for LovB and LovC

In a 500 [iL eppendorf tube was added 25 |iM of LovB with 25 |iM MatB,

20 mM CoA, 100 mM malonate, 7 mM MgCh, and 20 mM ATP in Buffer R (100 mM Naf^PC^, pH 7.4, 10% glycerol, 2 mM DTT) at room temperature for the biosynthesis of 3. 2 mM NADPH (prepared fresh) was included to the previous reaction mixture to produce compounds 4, 5, 6, 9, and 12. In addition to NADPH,

2 mM of S-(5'-adenosyl)-L-methionine chloride (prepared fresh in 0.005 M sulfuric acid and 10 % EtOH) was added to produce compounds 7 and 8. All in vitro assays were quenched and extracted twice with an equal volume of 99 %

EtOAc/1 % TFA. The organic layers were separated, evaporated to dryness, and redissolved in 15 [iL of methanol. The organic residue was analyzed by LC-MS.

LC-MS was conducted with a Shimadzu 2010 EV liquid chromatography mass spectrometer by using both positive and negative electrospray ionization and a

Phenomenex Luna 5 [i 2.0 x 100 mm CI8 reverse-phase column. Samples were separated on a linear gradient of 5 to 95% CH3CN (v/v) in H2O supplemented with 0.05% (v/v) formic acid for 20 min at a flow rate of 0.1 mL/min at room temperature. LC retention time for (2): 33.4 min (3): 13.27 min; (4): 21.7 min; Experimental procedures 195

(5): 24.83 min; (6): 26.92 min; (9): 25.31 min; (12): 28.53 min; (7): 25.75 min;

(8): 27.9 min, (32): 32.2 min.

Compounds 2 and 32 were biosynthesized with base hydrolysis from a 250 fiL reaction containing 25 |iM of LovB with 25 |iM MatB, 20 mM CoA, 100 mM malonate, 7 mM MgCh, 2 mM NADPH and 20 mM ATP in Buffer R (100 mM

NaH2PC>4, pH 7.4,10% glycerol, 2 mM DTT) and an equal molar concentration of the corresponding dissociated enoyl reductase LovC and MlcG, respectively.

Reactions progressed for 12-24 h and were then stopped by base hydrolysis. 50

|iL of 1 N KOH was added with the overnight in vitro reaction and then heated at

65 °C for 10 minutes in an oven. The reaction was acidified by adding 100 jxL of

1 M HC1. To remove compounds 2 or 32, the reactions were extracted twice with an equal volume of 99% EtOAc/1% TFA. The organic layers were separated, evaporated to dryness, redissolved in 15 of methanol and analyzed by LC-MS.

Biosynthesis of compounds 2 and 32 using a TE domain involved the same procedure as with base hydrolysis except with the addition of an equal molar (25

HM) amount of a TE domain (PKS4 TE, PKS13 TE, or DEBS TE). After incubation for 12-24 h the reactions were quenched and extracted twice with an equal volume of 99% EtOAc/1% TFA. The organic layers were separated, evaporated to dryness, and redissolved in 15 |iL of methanol and analyzed by LC-

MS. References 196

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6 Appendix A1 - LC-MS/MS spectrum of holo LovB fragment

IDQGVDSLGAVTVGTW containing peaks corresponding to the two Ppant

elimination products.

, OH Ha

o Ppant elimination product: 261.3 Da

OH ?H H H, o o Ppant elimination product: 359.4 Da Appendix 220

7 Appendix A2 - Crystal Data for (1R, 4R, 6*)-4-[2-{(lS, 2S, 4aR, 8aS)-2-

Methyl-1, 2, 4a, 5, 6, 7, 8, 8a-octahydronapthalen-l-yl}ethyl]-3, 7-

dioxabicyclo[4.1.0]heptan-2-one (31)

The x-ray crystallography was performed by Dr. Robert McDonald at the

University of Alberta X-ray crystallography laboratory. Epoxide 31 was dissolved in a minimum amount of hot EtOAc and diluted with hot hexanes in a recrystallization tube (same diameter as NMR tube). The hot solution was allowed to cool to 23°C. However, no crystals had formed, thus the solution was placed in a -20°C freezer overnight where white crystals then formed. Due to the presence of only light atoms (no atoms heavier than oxygen) only the relative stereochemistry can be determined for 31. The relative stereochemistry is reliable based on the absolute configuration of optically pure precursor 30. Appendix 221

C18 C10 C17

C16 C11 C14

C12 C15

C13 Appendix 222

Table 1. Crystallographic Experimental Details

A. Crystal Data formula C18H26O3 formula weight 290.39 crystal dimensions (mm) 0.57 x 0.30 x 0.28 crystal system orthorhombic space group P2\2\2\ (No. 19) unit cell parameters'3

a (A) 7.1793 (4)

b(k) 9.2541 (5)

c( A) 23.6713 (13)

F(A3) 1572.67(15)

z 4

Pealed (g cm*3) 1.226

H (mm"1) 0.082

B. Data Collection and Refinement Conditions diffractometer Bruker D8/APEXII CCD'' radiation (A [A]) graphite-monochromated Mo Ka (0.71073) temperature (°C) -100 scan type co scans (0.3°) (15 s exposures) data collection 28 limit (deg) 54.96 total data collected 13852 (-9s/is 9, -12 s k s 12, -30 s Is 30) independent reflections 3585 (J?im = 0.0157) Appendix 223

2 2 number of observed reflections (NO) 3442 [Fo a 2o(F0 )] structure solution method direct methods (SHELXS-97C) refinement method full-matrix least-squares on F2 (SHELXL-97C) absorption correction method Gaussian integration (face-indexed) range of transmission factors 0.9778-0.9552

2 2 data/restraints/parameters 3585 [F0 z -3o(F0 )] / 0 /190

Flack absolute structure parameter^ -0.1(7)

e 2 2 goodness-of-fit (S) 1.057 [F0 ±-3o(F0 )] final R indices^

2 2 [F0 * 2o(F0 )] 0.0300

2 2 wR2 [F0 *-3V(F0 )] 0.0804 largest difference peak and hole 0.192 and -0.153 e A"3 aObtained from least-squares refinement of 9995 reflections with 4.72° < 26 < 54.88°.

^Programs for diffractometer operation, data collection, data reduction and absorption correction were those supplied by Bruker. cSheldrick, G. M. Acta Crystallogr. 2008, A64, 112-122.

"^Flack, H. D. Acta Crystallogr. 1983, A39, 876-881; Flack, H. D.; Bemardinelli, G. Acta Crystallogr. 1999, A55, 908-915; Flack, H. D.; Bemardinelli, G. J. Appl. Cryst. 2000, 33, 1143-1148. The Flack parameter will refine to a value near zero if the structure is in the correct configuration and will refine to a value near one for the inverted configuration. The low anomalous scattering power of the atoms in this structure (none heavier than oxygen) implies that the data can only be used for relative structure assignment. Absolute structure can be assigned based upon the established stereochemistry of the precursor compound. e 2 2 2 1 /2 S - [2w(F0 - Fc ) /(n - /?)] (n = number of data; p = number of parameters 2 2 1 2 varied; w = [cP-(F0 ) + (0.0458P) + 0.1865P]" where P = [Max(F0 , 0) + 2 2Fc ]/3).

2 2 2 2 fR\ = 2||F0| - |FC||/2|F0|; wR2 = [2w(F0 - F ) fZw(F0^ . Appendix 224

Table 2. Atomic Coordinates and Equivalent Isotropic Displacement Parameters

Atom X y z Ueq , A2

01 0.67361(11) 0.36939(8) 0.06667(3) 0.02733(17) 02 0.85062(14) 0.20141(10) 0.02955(4) 0.0401(2)* 03 0.95368(13) 0.57314(10) 0.02142(4) 0.0384(2)* CI 0.81011(17) 0.32724(12) 0.03217(4) 0.0280(2)* C2 0.90283(17) 0.43765(13) -0.00452(5) 0.0324(2)* C3 0.79787(18) 0.56959(13) -0.01683(5) 0.0334(2)* C4 0.61069(16) 0.58711(12) 0.01047(5) 0.0299(2)* C5 0.61213(15) 0.52061(11) 0.06914(4) 0.0257(2)* C6 0.42152(16) 0.51660(12) 0.09691(5) 0.0286(2)* C7 0.43277(16) 0.46370(12) 0.15803(4) 0.0279(2)* C8 0.24472(14) 0.45004(12) 0.18808(4) 0.0248(2)* C9 0.26524(15) 0.39490(12) 0.24921(4) 0.0260(2)* CIO 0.41296(17) 0.47277(13) 0.28437(5) 0.0323(2)* Cll 0.4271(2) 0.40609(15) 0.34355(5) 0.0396(3)* C12 0.2411(2) 0.40300(16) 0.37407(5) 0.0429(3)* C13 0.0905(2) 0.33216(15) 0.33829(5) 0.0397(3)* C14 0.07711(16) 0.40383(13) 0.27997(5) 0.0303(2)* C15 -0.07822(16) 0.34416(14) 0.24462(5) 0.0347(3)* C16 -0.06555(16) 0.32277(13) 0.18947(5) 0.0341(2)* C17 0.10655(15) 0.35327(13) 0.15506(4) 0.0287(2)* C18 0.19114(19) 0.20930(14) 0.13513(5) 0.0365(3)*

Anisotropically-refined atoms are marked with an asterisk (*). The form of the anisotropic displacement parameter is: exp[-2jt2(h2a*2U\ i + k2b*2U22 + 2 Pc* Ui3 + lklb*c*U23 + 2hla*c*U\3 + 2hka*b*Uu)l Appendix 225

Table 3. Selected Interatomic Distances (A)

Atoml Atom2 Distance Atoml Atom2 Distance 01 CI 1.3340(14) C8 C9 1.5414(14) 01 C5 1.4685(13) C8 C17 1.5482(15) 02 CI 1.2018(15) C9 CIO 1.5286(15) 03 C2 1.4430(15) C9 C14 1.5366(14) 03 C3 1.4395(16) CIO Cll 1.5342(16) CI C2 1.4972(16) Cll C12 1.518(2) C2 C3 1.4641(17) C12 C13 1.522(2) C3 C4 1.4999(16) C13 C14 1.5345(15) C4 C5 1.5190(14) C14 C15 1.4995(17) C5 C6 1.5187(15) C15 C16 1.3235(17) C6 C7 1.5294(15) C16 C17 1.5066(16) C7 C8 1.5313(14) C17 C18 1.5383(16)

Table 4. Selected Interatomic Angles (deg)

Atoml Atom2 Atom3 Angle Atoml Atom2 Atom3 Angle CI 01 C5 121.58(8) C7 C8 C17 112.21(8) C2 03 C3 61.05(8) C9 C8 C17 110.10(9) 01 CI 02 119.52(11) C8 C9 CIO 114.92(9) 01 CI C2 118.83(10) C8 C9 C14 110.06(8) 02 CI C2 121.58(11) CIO C9 C14 109.06(9) 03 C2 CI 117.30(9) C9 CIO Cll 110.69(10) 03 C2 C3 59.36(8) CIO Cll C12 112.58(11) CI C2 C3 117.12(10) Cll C12 C13 111.59(10) 03 C3 C2 59.59(8) C12 C13 C14 111.04(11) 03 C3 C4 115.01(9) C9 C14 C13 110.36(10) C2 C3 C4 117.74(10) C9 C14 C15 111.68(9) C3 C4 C5 110.12(9) C13 C14 C15 112.92(10) 01 C5 C4 110.60(8) C14 C15 C16 123.67(11) 01 C5 C6 105.35(8) C15 C16 C17 124.16(11) C4 C5 C6 113.55(9) C8 C17 C16 111.16(9) C5 C6 C7 111.70(9) C8 C17 C18 113.76(9) C6 C7 C8 114.79(9) C16 C17 C18 109.10(10) C7 C8 C9 112.29(8) Appendix 226

Table 5. Torsional Angles (deg)

Atoml Atom2 Atom3 Atom4 Angle Atoml Atom2 Atom3 Atom4 Angle C5 01 CI 02 -176.65(10) CI C8 C9 C14 171.94(9) C5 01 CI C2 0.36(14) CM C8 C9 CIO 174.18(9) CI 01 C5 C4 38.91(13) C17 C8 C9 C14 -62.25(11) CI 01 C5 C6 161.99(9) CI C8 C17 C16 171.23(9) C3 03 C2 CI -106.89(12) C7 C8 C17 C18 47.60(12) C2 03 C3 C4 108.77(11) C9 C8 C17 C16 45.38(12) 01 CI C2 03 45.06(15) C9 C8 C17 C18 -78.25(11) 01 CI C2 C3 -22.60(15) C8 C9 CIO Cll -177.89(9) 02 CI C2 03 -137.98(12) C14 C9 CIO Cll 58.02(12) 02 CI C2 C3 154.36(12) C8 C9 C14 C13 173.08(9) 03 C2 C3 C4 -104.19(11) C8 C9 C14 C15 46.61(12) CI C2 C3 03 107.19(11) CIO C9 C14 C13 -60.00(12) CI C2 C3 C4 2.99(15) CIO C9 C14 C15 173.52(9) 03 C3 C4 C5 -32.64(13) C9 CIO Cll C12 -54.98(13) C2 C3 C4 C5 34.67(14) CIO Cll C12 C13 52.45(15) C3 C4 C5 Ol -54.51(12) Cll C12 C13 C14 -53.75(14) C3 C4 C5 C6 -172.69(9) C12 C13 C14 C9 58.10(13) 01 C5 C6 C7 64.82(11) C12 C13 C14 C15 -176.12(10) C4 C5 C6 C7 -174.01(9) C9 C14 C15 C16 -16.74(16) C5 C6 C7 C8 -177.32(9) C13 C14 C15 C16 -141.81(12) C6 C7 C8 C9 179.42(9) C14 C15 C16 C17 0.9(2) C6 C7 C8 C17 54.77(12) C15 C16 C17 C8 -15.62(16) C7 C8 C9 CIO 48.37(12) C15 C16 C17 C18 110.62(13) nnononnoonoonnnonoooo> 8* H=r — — — — — — — — — \000>JO\^-^UN) -U> tO •— Z? 22, u> u> UJ u> 4*. 4*. u> to to U) to to to LO to u> to to s c: & la 4^ 00 00 to o On -j *-4 UJ to 4* o UJ 00 LA 00 4*> SJ 1-J »T™1 4* o NO 00 ON u> U) to u> to ON o -J NO LA to ON -o + N s «o /—•\ /—s /—'\ r—N /—s /—s /—-v /—s r—v P5 W LA ON ^w'ON 'Gi ^w' ^•w''on OS VUi LA LA 'Gi w LA LA W sLA«-»/ ''w'4*> 4* w4^ °* i K) 2 c: 3 O O o o o O o o o o o o o o o o o o o O o o b b b o b o o b o bb b b b b b b b b b w ig U) to 4* to u> to to to to to to to to to to to to to + § 4* LA o o On o u> -o On u> to U\ U) -o On 00 to U) •— ui N) 5 no 00 to LA U) ON 00 4^ /-vto NO 00 NO /-—Vo 00 u> /-~Voo y"~No O —» O LA ON ON la ON LA LA LA LA LA Ut Ui ty» s Ul 4^. S 2. •N—•n s»/ w U\ s—• N-/ w' •w

** Crtz S •' ooooooooooooooooooooo bbbbbbbbbbbbbbbbbbbboC! + ooooooooooooooooooooo^* -O O4* On O4* — o— — OO O O U> 4* — o— — U> w to OOOO^UIWOOO^^KJOOWWVONIWUOO-OO :s- /^v /—V S /—N /"~V /—V S /"—V /—S /—N /—V ^ /—N S /—»\ /—s, S /—V /-""V /—"\ /•—N 5" * o O o o o O O o o o o o O o o O o o o O o O o o b o o o o o o o b o o b o o b o bb b + o o o o o o o o o o o o o o o o o o o o to to — to o On On NO o o o o ou> o o o On ON 4^ ON 2r- On NO o NO /—vu> NO x—s 4&> 4*> to l/i LA ON L/l 4*» LA On LA /"~SUl r—s4^ /•"•v /--V /—V ^/1 •--VON u* /—V 4*. /—*s4^ 4* 4a» /—•s4>. 4a. /^VLA /—VLA 4^ 4^ u> V-/ S—• N-/ '«w' >«—• r* w s '•w' Qy * O O o o o O O o O o o o O o o o O o o o o b b b b b b b b b b b b b b b b b o o o o o o o o o o o o o o o o o o o o o o o o o to o o 4* On u> ON 4*. >—• LA u> o o — o UJ — — oo o -o oo u> o to o -o U> U) LA m-+ u> NO 4^- ON /—V /—s /•—S ZN /^SU) 'WLA 4*> LA LA •v—'LA S,^On On LA 4^ LA LA w4^ 4* sLAw' LA 4^ Appendix 228

Table 7. Derived Atomic Coordinates and Displacement Parameters for Hydrogen Atoms

Atom X y z Ueq, A2 H2 0.9867 0.4006 -0.0350 0.039 H3 0.8157 0.6143 -0.0549 0.040 H4A 0.5144 0.5394 -0.0130 0.036 H4B 0.5794 0.6911 0.0132 0.036 H5 0.6996 0.5765 0.0937 0.031 H6A 0.3388 0.4517 0.0751 0.034 H6B 0.3665 0.6147 0.0962 0.034 H7A 0.5121 0.5314 0.1797 0.033 H7B 0.4948 0.3682 0.1584 0.033 H8 0.1892 0.5490 0.1900 0.030 H9 0.3012 0.2906 0.2473 0.031 H10A 0.3802 0.5763 0.2876 0.039 H10B 0.5352 0.4657 0.2652 0.039 HI 1A 0.4752 0.3062 0.3403 0.047 HUB 0.5173 0.4625 0.3662 0.047 H12A 0.2031 0.5030 0.3834 0.051 H12B 0.2548 0.3490 0.4100 0.051 H13A -0.0309 0.3403 0.3578 0.048 H13B 0.1192 0.2282 0.3336 0.048 H14 0.0499 0.5085 0.2863 0.036 HI 5 -0.1921 0.3204 0.2628 0.042 H16 -0.1717 0.2858 0.1704 0.041 H17 0.0668 0.4079 0.1207 0.034 H18A 0.3035 0.2285 0.1128 0.044 H18B 0.1002 0.1575 0.1118 0.044 H18C 0.2236 0.1502 0.1680 0.044 Appendix 229

8 Appendix A3 - Crystal Data for (4R, 6J/?)-4-Hydroxy-6- [2-{(1 S, 2S, 4a/?,

8aS)-2-methyl-l, 2, 4a, 5, 6, 7, 8, 8a-octahydronapthalen-l-

y1} ethyl]tetrahydro-2//-pyran-2-one (32)

The x-ray crystallography was performed by Dr. Robert McDonald at the

University of Alberta X-ray crystallography laboratory. Compound 32 was dissolved in a small volume of hot Et20 and placed in a -20°C freezer overnight where a single white crystal formed. Appendix 230

Table 1. Crystallographic Experimental Details

A. Crystal Data formula C18H28O3 formula weight 292.40 crystal dimensions (mm) 0.43 x 0.28 x 0.25 crystal system monoclinic space group P2\ (No. 4) unit cell parameters0

a (A) 5.7598 (6)

b{ A) 9.5611 (10)

c(A) 15.4940(17)

P(deg) 99.2039(13)

F(A3) 842.27(15)

Z 2

Sealed (g cm"3) 1.153 fx (mm-1) 0.076

B. Data Collection and Refinement Conditions diffractometer Bruker D8/APEXII CCD^ radiation (A [A]) graphite-monochromated Mo Ka (0.71073) temperature (°C) -100 scan type a) scans (0.3°) (15 s exposures) data collection 26limit (deg) 54.80 total data collected 7413 (-7^5 7, -12 sits 12, -20 s /s20) Appendix 231

independent reflections 2024 (i?int = 0.0147)

2 2 number of observed reflections (NO) 1812 [F0 & 2o(F0 )]

structure solution method direct methods (SHELXDC)

refinement method full-matrix least-squares on F2 (SHELXL-97*)

absorption correction method Gaussian integration (face-indexed)

range of transmission factors 0.9810-0.9680

data/restraints/parameters 2024 [F02 ^ -3o(F02)] / 0 /192

extinction coefficient (x)e 0.010(4)

Flack absolute structure parameter^ 0.2(14)

2 goodness-of-fit (S)s 1.053 [Fc2a-3o(Fo )]

final R indices'1

Rx [F02 * 2o(F02)] 0.0391

2 WR2 [Fo * -3o( F02)] 0.1073

largest difference peak and hole 0.180 and-0.179 e A"3

aObtained from least-squares refinement of 5316 reflections with 5.02° <26 < 54.40°.

^Programs for diffractometer operation, data collection, data reduction and absorption correction were those supplied by Bruker.

cSchneider, T. R.; Sheldrick, G. M. Acta Crystallogr. 2002, D58,1772-1779.

^Sheldrick, G. M. Acta Crystallogr. 2008, A64,112-122.

e 2 3 1/4 Fc* = kFc[\ + x{0.001Fc A /sin(2#)}]- where k is the overall scale factor.

/Flack, H. D. Acta Crystallogr. 1983, A39, 876-881; Flack, H. D.; Bemardinelli, G. Acta Crystallogr. 1999, A55, 908-915; Flack, H. D.; Bemardinelli, G. J. Appl. Cryst. 2000, 33, 1143-1148. The Flack parameter will refine to a val3.8ue near zero if the structure is in the correct configuration and will refine to a value near one for the inverted configuration. The low anomalous scattering power of the atoms in this structure (none heavier than oxygen) Appendix 232

implies that the data alone cannot be used for absolute structure assignment. but should be used in conjunction with the established stereochemistry of the precursor compound. The Flack parameter is provided for informational purposes only.

2 2 2 /2 %S = [Zh>(F0 - Fc ) /(n -/>)]' (n = number of data; p = number of parameters 2 2 1 2 varied; w = [c^(F0 ) + (0.0524P) + 0.1303P]" where P = [Max(F0 , 0) +

2 2Fc ]/3).

2 2 2 = I||F0| - |FC||/2|F0|; wR2 = [2w(F0 - Fc ) /lw(F0^. Appendix 233

Table 2. Atomic Coordinates and Equivalent Isotropic Displacement Parameters

Atom X y z t/eq, A2

01 0.7262(3) -0.12783(18) -0.01322(14) 0.0671(5)* 02 0.4791(2) -0.05627(15) 0.07044(12) 0.0579(5)* 03 0.2229(2) -0.36740(16) 0.08999(13) 0.0599(5)* CI 0.6115(3) -0.1566(2) 0.04322(19) 0.0570(6)* C2 0.6261(3) -0.2985(2) 0.0862(2) 0.0627(7)* C3 0.4396(4) -0.3303(2) 0.1416(2) 0.0589(6)* C4 0.3920(5) -0.2001(2) 0.1920(2) 0.0632(7)* C5 0.3089(4) -0.0828(2) 0.12989(18) 0.0544(6)* C6 0.2788(4) 0.0585(2) 0.1708(2) 0.0592(6)* C7 0.0800(4) 0.0620(2) 0.2254(2) 0.0579(6)* C8 0.0652(4) 0.2019(2) 0.27235(17) 0.0562(6)* C9 -0.1260(4) 0.2067(2) 0.33071(15) 0.0539(5)* CIO -0.1384(6) 0.0793(3) 0.38903(18) 0.0696(7)* Cll -0.3320(6) 0.0925(3) 0.44468(18) 0.0774(9)* C12 -0.3211(8) 0.2284(4) 0.49659(18) 0.0951(11) C13 -0.3027(6) 0.3540(3) 0.43803(17) 0.0768(9)* C14 -0.0961(5) 0.3384(3) 0.38783(16) 0.0669(7)* C15 -0.0570(5) 0.4656(3) 0.3358(2) 0.0707(8)* C16 0.0036(4) 0.4612(2) 0.2570(2) 0.0628(7)* C17 0.0402(4) 0.3284(2) 0.20927(18) 0.0552(6)* C18 -0.1568(4) 0.3133(2) 0.13095(16) 0.0547(5)*

Anisotropically-refined atoms are marked with an asterisk (*). The form of the anisotropic displacement parameter is: exp[-2;r2(/?2a*2£/ii + k2b*2U22 +

2 Pc* U33 + 2klb*c*U23 + 2hla*c*U\z + 2hka*b*U\2)]. Appendix 234

Table 3. Selected Interatomic Distances (A)

Atoml Atom2 Distance Atoml Atom2 Distance 01 03fl 2.795(2)^ C8 C9 1.533(4) 01 CI 1.209(3) C8 C17 1.547(3) 01 H30a 1.96* C9 CIO 1.525(4) 02 CI 1.335(3) C9 C14 1.533(3) 02 C5 1.471(3) CIO Cll 1.520(4) 03 C3 1.415(3) Cll C12 1.524(5) CI C2 1.508(3) C12 C13 1.519(5) C2 C3 1.510(3) C13 C14 1.530(4) C3 C4 1.517(3) C14 C15 1.496(4) C4 C5 1.505(3) C15 C16 1.324(4) C5 C6 1.514(3) C16 C17 1.500(3) C6 C7 1.528(3) C17 C18 1.530(3) C7 C8 1.531(3)

Table 4. Selected Interatomic Angles (deg)

Atoml Atom2 Atom3 Angle Atoml Atom2 Atom 3 Angle CI 02 C5 123.09(17) C8 C9 CIO 115.6(2) Ol CI 02 118.1(2) C8 C9 C14 109.9(2) 01 CI C2 121.7(2) CIO C9 C14 109.0(2) 02 CI C2 120.1(2) C9 CIO Cll 112.2(2) CI C2 C3 115.99(18) CIO Cll C12 113.5(3) 03 C3 C2 111.9(2) Cll C12 C13 111.1(2) 03 C3 C4 106.31(18) C12 C13 C14 111.2(3) C2 C3 C4 109.29(18) C9 C14 C13 110.4(2) C3 C4 C5 110.4(2) C9 C14 C15 111.7(2) 02 C5 C4 110.66(17) C13 C14 C15 113.0(2) 02 C5 C6 103.94(16) C14 C15 C16 123.7(2) C4 C5 C6 116.3(2) C15 C16 C17 124.1(2) C5 C6 C7 113.42(18) C8 C17 C16 110.9(2) C6 C7 C8 112.70(19) C8 C17 C18 114.17(18) C7 C8 C9 113.97(19) C16 C17 C18 108.98(19) CI C8 C17 112.9(2) 01a H30 03 178.7* C9 C8 C17 110.24(18) "At 1-x, '/2+y, Appendix 235

TableS. Torsional Angles (deg) > > > o > 3 o 3 r-+ o o r-+ 3 Atoml 3 Atom3 Angle Atoml Atom2 Angle C5 02 CI Ol 171.6(2) C7 C8 C17 C16 174.6(2) C5 02 CI C2 -11.8(3) C7 C8 C17 C18 51.1(3) CI 02 C5 C4 31.9(3) C9 C8 C17 C16 45.9(2) CI 02 C5 C6 157.5(2) C9 C8 C17 C18 -77.7(2) 01 CI C2 C3 -167.9(2) C8 C9 CIO Cll 179.9(2) 02 CI C2 C3 15.7(3) C14 C9 CIO Cll 55.6(3) CI C2 C3 03 78.3(3) C8 C9 C14 C13 173.0(2) CI C2 C3 C4 -39.1(3) C8 C9 C14 C15 46.3(3) 03 C3 C4 C5 -61.2(2) CIO C9 C14 C13 -59.4(3) C2 C3 C4 C5 59.7(3) CIO C9 C14 C15 174.0(2) C3 C4 C5 02 -55.5(2) C9 CIO Cll C12 -52.2(3) C3 C4 C5 C6 -173.78(17) CIO Cll C12 C13 50.5(4) 02 C5 C6 C7 171.26(19) Cll C12 C13 C14 -54.0(4) C4 C5 C6 C7 -66.9(3) C12 C13 C14 C9 59.6(3) C5 C6 C7 C8 175.3(2) C12 C13 C14 C15 -174.5(2) C6 C7 C8 C9 -177.6(2) C9 C14 C15 C16 -16.0(4) C6 C7 C8 C17 55.6(3) C13 C14 C15 C16 -141.2(3) C7 C8 C9 CIO 45.3(3) C14 C15 C16 C17 0.2(4) C7 C8 C9 C14 169.2(2) C15 C16 C17 C8 -15.5(3) C17 C8 C9 CIO 173.5(2) C15 C16 C17 C18 111.0(3) C17 C8 C9 C14 -62.7(2) Appendix 236

Table 6. Anisotropic Displacement Parameters (f/jj, A2)

Atom t/ll t/22 U33 t/23 U\3 Un 01 0.0375(7) 0.0313(8) 0.1364(16) 0.0081(9) 0.0252(9) 0.0038(6) 02 0.0373(7) 0.0209(7) 0.1170(13) 0.0053(8) 0.0167(8) 0.0042(6) 03 0.0364(7) 0.0235(7) 0.1216(15) -0.0048(8) 0.0175(8) -0.0028(6) CI 0.0239(8) 0.0232(9) 0.122(2) 0.0009(11) 0.0050(10) -0.0002(7) C2 0.0309(9) 0.0241(10) 0.132(2) 0.0078(13) 0.0107(11) 0.0056(8) C3 0.0400(10) 0.0216(9) 0.1135(19) 0.0067(11) 0.0078(11) 0.0024(8) C4 0.0535(13) 0.0254(11) 0.1086(19) 0.0030(12) 0.0066(13) 0.0013(9) C5 0.0366(9) 0.0216(10) 0.1044(18) 0.0004(10) 0.0092(11) 0.0008(7) C6 0.0447(11) 0.0215(10) 0.1101(19) -0.0021(11) 0.0084(12) -0.0017(8) C7 0.0524(12) 0.0230(10) 0.0971(17) -0.0035(10) 0.0088(12) -0.0012(8) C8 0.0482(11) 0.0250(10) 0.0897(16) -0.0024(11) -0.0063(11) 0.0018(9) C9 0.0625(13) 0.0299(10) 0.0632(12) -0.0047(10) -0.0092(10) 0.0030(10) CIO 0.0852(19) 0.0437(14) 0.0746(16) 0.0087(13) -0.0037(14) 0.0092(13) Cll 0.109(2) 0.0603(17) 0.0600(14) 0.0171(13) 0.0036(15) 0.0136(17) C12 0.143(3) 0.084(3) 0.0513(13) 0.0038(15) -0.0060(16) 0.021(2) C13 0.116(2) 0.0559(17) 0.0513(13) -0.0171(12) -0.0087(14) 0.0143(17) C14 0.0816(17) 0.0439(13) 0.0635(14) -0.0129(11) -0.0239(13) 0.0063(13) C15 0.0814(18) 0.0305(12) 0.0900(19) -0.0166(12) -0.0175(15) 0.0033(12) C16 0.0645(14) 0.0244(11) 0.0938(18) -0.0069(11) -0.0047(13) -0.0001(10) C17 0.0490(11) 0.0230(9) 0.0911(16) -0.0015(10) 0.0038(11) -0.0013(8) C18 0.0517(12) 0.0348(11) 0.0778(14) -0.0007(11) 0.0105(11) 0.0014(9)

The form of the anisotropic displacement parameter is: 2 exp[-2^2(A2a*2C/n + k^b*W22 + fic* U33 + 2klb*c*U13,+ 2hla*c*U\3 + 2hka*b*Un)] Appendix 237

Table 7. Derived Atomic Coordinates and Displacement Parameters for Hydrogen Atoms

Atom X y z t/eq, A2 H30 0.2360 -0.4459 0.0669 0.090 H2A 0.7820 -0.3071 0.1234 0.075 H2B 0.6174 -0.3705 0.0399 0.075 H3 0.4942 -0.4077 0.1834 0.071 H4A 0.2707 -0.2207 0.2288 0.076 H4B 0.5375 -0.1714 0.2309 0.076 H5 0.1555 -0.1105 0.0942 0.065 H6A 0.2469 0.1294 0.1238 0.071 H6B 0.4279 0.0843 0.2085 0.071 H7A 0.1047 -0.0139 0.2693 0.069 H7B -0.0713 0.0443 0.1867 0.069 H8 0.2188 0.2139 0.3120 0.067 H9 -0.2809 0.2144 0.2914 0.065 H10A -0.1669 -0.0053 0.3520 0.084 H10B 0.0146 0.0675 0.4277 0.084 HI 1A -0.3207 0.0128 0.4859 0.093 HUB -0.4864 0.0866 0.4062 0.093 H12A -0.4643 0.2372 0.5241 0.114 H12B -0.1832 0.2263 0.5438 0.114 H13A -0.2815 0.4399 0.4741 0.092 H13B -0.4505 0.3637 0.3960 0.092 H14 0.0486 0.3249 0.4321 0.080 H15 -0.0768 0.5547 0.3608 0.085 H16 0.0252 0.5474 0.2288 0.075 H17 0.1916 0.3379 0.1859 0.066 H18A -0.1356 0.2260 0.1000 0.066 H18B -0.1521 0.3926 0.0912 0.066 H18C -0.3092 0.3116 0.1515 0.066 Appendix 238

9 Appendix A4 - Crystal Data for (3R, 5R)-5-[(\R, 27?)-1-Iodo-2-

methylhexyl]-3-methyldihydrofuran-2(3//)-one (77)

The x-ray crystallography was performed by Dr. Robert McDonald at the

University of Alberta X-ray crystallography laboratory. Compound 77 was dissolved in a small volume of hot hexane and placed in a 0°C fridge over night where a single white crystal formed. Appendix 239

Table 1. Crystallographic Experimental Details

A. Crystal Data formula C12H21IO2 formula weight 324.19 crystal dimensions (mm) 0.61 x 0.43 x 0.09 crystal system orthorhombic space group P2\2\2\ (No. 19) unit cell parameters0

a (A) 5.6404 (6)

b(A) 9.0615(9)

c(A) 27.404 (3)

F(A3) 1400.6 (2)

z 4

Pealed (g cm*3) 1.537

/1 (mm-1) 2.269

B. Data Collection and Refinement Conditions diffractometer Bruker D8/APEXII CCD* radiation (A [A]) graphite-monochromated Mo Ka (0.71073) temperature (°C) -100 scan type co scans (0.3°) (20 s exposures) data collection 28 limit (deg) 52.96 total data collected 6610(-7s As7,-ll sAs 11,-33 s 7s34) independent reflections 6610 (/fint = 0.0000) Appendix 240

2 number of observed reflections (NO) 6346 [F0 * 2o(F02)] structure solution method Patterson/structure expansion (.DIRDIF-2008C) refinement method full-matrix least-squares on F2- (SHELXL-97d) absorption correction method multi-scan (TWINABS) range of transmission factors 0.8253-0.3368 data/restraints/parameters 6610/0/137

Flack absolute structure parameter6 0.02(4) goodness-of-fit (Sf [all data] 1.075 final R indices^

R1 [F02*2o(F02)] 0.0480

w/?2 [all data] 0.1292 largest difference peak and hole 2.663 and -0.792 e A"3

^Obtained from least-squares refinement of 3404 reflections with 4.74° <26 < 43.56°.

^Programs for diffractometer operation, data collection, data reduction and absorption correction were those supplied by Bruker. The crystal used for data collection was found to display non-merohedral twinning. Both components of the twin were indexed with the program CELL NOW (Bruker AXS Inc., Madison, WI, 2004). The second twin component can be related to the first component by 176° rotation about the [1 0.04 0] axis in real space and about the [1 0.12 0.07] axis in reciprocal space. Integrated intensities for the reflections from the two components were written into a SHELXL-97 HKLF 5 reflection file with the data integration program SAINT (version 7.68A), using all reflection data (exactly overlapped, partially overlapped and non- overlapped). cBeurskens, P. T.; Beurskens, G.; de Gelder, R.; Smits, J. M. M; Garcia-Granda, S.; Gould, R. O. (2008). The DIRDIF-2008 program system. Crystallography Laboratory, Radboud University Nijmegen, The Netherlands.

^Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112-122. eFlack, H. D. Acta Crystallogr. 1983, A39, 876-881; Flack, H. D.; Bernardinelli, Appendix 241

G. Acta Crystallogr. 1999, A55, 908-915; Flack, H. D.; Bemardinelli, G. J. Appl. Cryst. 2000, 33, 1143-1148. The Flack parameter will refine to a value near zero if the structure is in the correct configuration and will refine to a value near one for the inverted configuration.

2 2 2 1/2 fS = [2w(F0 - Fc ) /(n - p)\ (n = number of data; p = number of parameters 2 2 1 2 varied; w = [o2(F0 ) + (0.0552P) + 3.5159P]* where P = [Max(F0 , 0) +

2Fc2]/3).

2 2 2 8RX = 2||F0| - |FC||/2|F0|; wR2 = [2w(F0 - Fc ) /2w(F04)]l/2. Appendix 242

Table 2. Atomic Coordinates and Equivalent Isotropic Displacement Parameters

2 Atom x y z Ueq, A

I -0.18800(5) -0.01654(4) 0.383008(12) 0.04349(11) 01 0.0645(6) 0.0593(4) 0.27976(11) 0.0332(7)* 02 0.1697(7) 0.0043(4) 0.20366(12) 0.0490(9)* CI 0.1769(9) 0.0888(5) 0.23734(15) 0.0325(9)* C2 0.3088(9) 0.2347(5) 0.24096(15) 0.0295(8)* C3 0.3193(9) 0.2581(5) 0.29603(15) 0.0305(9)* C4 0.0975(8) 0.1794(5) 0.31458(16) 0.0292(9)* C5 0.5456(10) 0.2317(7) 0.21373(19) 0.0438(12)* C6 0.1254(7) 0.1134(5) 0.36541(16) 0.0281(9)* C7 0.1837(10) 0.2277(6) 0.40496(15) 0.0356(9)* C8 -0.0053(10) 0.3493(6) 0.41032(18) 0.0403(11)* C9 0.0791(10) 0.4817(7) 0.44063(17) 0.0449(11)* CIO 0.2776(17) 0.5682(8) 0.4188(3) 0.086(3)* Cll 0.3514(18) 0.6991(9) 0.4489(3) 0.091(3)* C12 0.2375(9) 0.1520(6) 0.45381(17) 0.0415(12)*

Anisotropically-refined atoms are marked with an asterisk (*). The form of the anisotropic displacement parameter is: exp[-27t2(h2a*2U\ \ + k2b*2U22 + 2 fic* U33 + 2klb*c*U23 + 2hla*c*Un + 2hka*b*U\2)].

Table 3. Selected Interatomic Distances (A)

Atoml Atom2 Distance Atoml Atom2 Distance I C6 2.178(4) C4 C6 1.524(6) 01 CI 1.351(5) C6 C7 1.535(6) 01 C4 1.459(5) C7 C8 1.541(7) 02 CI 1.200(5) C7 C12 1.534(7) CI C2 1.521(6) C8 C9 1.535(8) C2 C3 1.525(6) C9 C10 1.492(9) C2 C5 1.530(7) C10 Cll 1.504(11) C3 C4 1.527(7) Appendix 243

Table 4. Selected Interatomic Angles (deg)

Atoml Atom2 Atom3 Angle Atoml Atom2 Atom3 Angle CI 01 C4 110.8(3) C3 C4 C6 113.7(4) 01 CI 02 121.3(4) I C6 C4 109.3(3) 01 CI C2 110.2(3) I C6 CI 112.5(3) 02 CI C2 128.4(4) C4 C6 CI 113.8(4) CI C2 C3 101.8(3) C6 C7 C8 113.7(4) CI C2 C5 112.3(4) C6 C7 C12 110.9(4) C3 C2 C5 116.8(4) C8 C7 C12 111.9(4) C2 C3 C4 103.5(4) C7 C8 C9 113.4(4) 01 C4 C3 103.6(3) C8 C9 CIO 115.2(5) 01 C4 C6 108.5(4) C9 CIO Cll 113.7(6)

Table 5. Torsional Angles (deg)

Atoml Atom2 Atom3 Atom4 Angle Atoml Atom2 Atom3 Atom4 Angle C4 01 CI 02 -179.3(4) 01 C4 C6 1 -58.5(4) C4 01 CI C2 2.4(5) 01 C4 C6 C7 174.8(4) CI Ol C4 C3 -21.2(5) C3 C4 C6 1 -173.3(3) CI Ol C4 C6 -142.4(4) C3 C4 C6 CI 60.1(5) 01 CI C2 C3 17.4(5) 1 C6 CI C8 -65.6(5) Ol CI C2 C5 143.1(4) 1 C6 CI C12 61.6(5) 02 CI C2 C3 •160.8(5) C4 C6 CI C8 59.4(5) 02 CI C2 C5 -35.1(7) C4 C6 CI C12 -173.5(4) CI C2 C3 C4 -29.0(5) C6 C7 C8 C9 -166.3(4) C5 C2 C3 C4 -151.7(4) C12 CI C8 C9 67.0(6) C2 C3 C4 Ol 31.0(4) C7 C8 C9 CIO 65.0(7) C2 C3 C4 C6 148.7(4) C8 C9 CIO Cll 178.6(7) Appendix 244

Table 6. Anisotropic Displacement Parameters (fjj, A2)

Atom C/n U22 t/33 U23 U\3 U\2 1 0.03131(15) 0.04862(18) 0.05054(18) 0.00862(15) 0.00330(12) -0.00937(14) 01 0.0281(15) 0.0335(17) 0.0381(16) -0.0015(13) -0.0032(12) -0.0071(14) 02 0.062(2) 0.044(2) 0.0410(16) -0.0115(16) -0.0002(15) -0.013(3) CI 0.034(2) 0.035(2) 0.029(2) -0.0006(17) -0.0051(19) 0.001(2) C2 0.026(2) 0.030(2) 0.033(2) -0.0003(16) 0.0012(19) -0.002(2) C3 0.028(2) 0.031(2) 0.033(2) -0.0020(16) 0.001(2) -0.008(2) C4 0.025(2) 0.032(2) 0.031(2) 0.0003(18) -0.0027(16) 0.0044(18) C5 0.035(3) 0.051(3) 0.046(3) -0.003(2) 0.008(2) -0.005(3) C6 0.019(2) 0.030(2) 0.036(2) 0.0056(17) 0.0005(15) 0.0000(16) C7 0.029(2) 0.047(3) 0.030(2) 0.0007(19) -0.001(2) 0.000(3) C8 0.040(3) 0.043(3) 0.038(2) -0.002(2) -0.001(2) 0.008(2) C9 0.052(3) 0.041(3) 0.041(2) -0.002(2) 0.010(2) 0.004(3) CIO 0.111(7) 0.060(4) 0.087(5) -0.010(4) 0.056(5) -0.028(5) Cll 0.092(7) 0.052(4) 0.129(7) -0.016(4) 0.040(6) -0.032(5) C12 0.038(3) 0.049(3) 0.037(2) 0.002(2) -0.0039(19) 0.007(2)

The form of the anisotropic displacement parameter is: 2 2 2 2 2 exp[-2j£(hla* Ui j + k b* U22 + / c» (/33 + 2klb*c*U23 + 2hla*c*U\3 + 2hka*b*U\2)]

Table 7. Derived Atomic Coordinates and Displacement Parameters for Hydrogen Atoms

Atom X y z f/eq,A2 H2 0.2074 0.3138 0.2264 0.035 H3A 0.3152 0.3644 0.3043 0.037 H3B 0.4646 0.2137 0.3100 0.037 H4 -0.0417 0.2477 0.3137 0.035 H5A 0.6229 0.3282 0.2166 0.053 H5B 0.5176 0.2093 0.1792 0.053 H5C 0.6481 0.1557 0.2279 0.053 H6 0.2622 0.0433 0.3639 0.034 H7 0.3332 0.2778 0.3945 0.043 H8A -0.0515 0.3843 0.3774 0.048 H8B -0.1480 0.3066 0.4259 0.048 H9A -0.0571 0.5489 0.4458 0.054 H9B 0.1300 0.4455 0.4731 0.054 H10A 0.2288 0.6030 0.3860 0.103 H10B 0.4160 0.5022 0.4145 0.103 HI 1A 0.4809 0.7512 0.4324 0.109 HUB 0.4053 0.6655 0.4810 0.109 HI 1C 0.2163 0.7660 0.4528 0.109 H12A 0.3594 0.0763 0.4490 0.050 H12B 0.0928 0.1058 0.4664 0.050 H12C 0.2946 0.2255 0.4773 0.050 Appendix 245

10 Appendix A5 - Crystal Data for (1R, 2/?)-2-methyl-l -[(2/?, 4Z?)-4-methyl-5-

oxotetrahydrofuran-2-yl]hexyl-4-bromobenzoate (116)

DMAP

CH2CI2, 23 °C 68%

116

A flask under Ar containing 75.0 mg (0.35 mmol) of a mixture of 85 and

86, 8 mL of dry CH2CI2,2.83 mL (350 mmol) of dry pyridine, 0.12 g (0.53 mmol) of /7-bromobenzoyl chloride, and 4.27 mg (0.04 mmol) of DMAP was allowed to stir at 23 °C overnight. The reaction was quenched by the addition of 10 mL of 1

M HCl. The layers were separated, and the organic layer was washed with 10 mL of 1 M HCl once again. The organic layer was washed with 10 mL of NaHCCb,

10 mL of H2O, 10 mL of brine, and then dried over Na2SC>4. The solvent was removed under vacuum, and the diastereomers were separated by column chromatography using a 9:1 hexanes/EtOAc eluent to afford 72.3 mg of 115 and

21.8 mg of 116 as solids (68% yield). 'H NMR (500 MHz, CDCI3) 5 7.92 (m, Appendix 246

2H), 7.61 (m, 2H), 5.07 (dd, 1H, J =7.1, 3.6 Hz), 4.72 (ddd, 1H, J= 10.1,6.2,3.7

Hz), 2.68 (m, 1H), 2.42 (m, 1H), 2.10 (m, 1H), 1.58 (m, 1H), 1.50 -1.16 (m, 8H),

13 1.04 (d, 3H, J= 6.9 Hz), 0.85 (t, 3H, J= 6.9 Hz); C NMR (125 MHz, CDC13) 8

178.7, 165.5, 131.9, 131.3, 128.6, 128.4, 77.2, 76.7, 35.2, 34.3, 33.0, 31.7, 28.8,

22.7,15.8,15.2,13.9; HRES calcd. for C^BrNaCU 419.0828, found 419.0829.

The x-ray crystallography was performed by Dr. Michael Ferguson at the

University of Alberta X-ray crystallography laboratory. Compound 116 was dissolved in a small volume of EtOAc in a one dram vial and allowed to slowly evaporate over 48 h until white crystals formed.. Appendix 247

A. Crystal Data formula Ci9H25Br04 formula weight 397.30 crystal dimensions (mm) 0.36x0.31 x 0.19 crystal system orthorhombic space group jP2 i2121 (No. 19) unit cell parameters0

a (A) 6.5512(4)

b( A) 7.9977 (5)

c(A) 36.427 (2)

F(A3) 1908.5 (2)

Z 4

Pealed (g cm"3) 1.383 fi (mm"1) 2.172

5. Data Collection and Refinement Conditions diffractometer Bruker D8/APEXII CCDft radiation (A [A]) graphite-monochromated Mo Ka (0.71073) temperature (°C) -100 scan type a> scans (0.3°) (20 s exposures) data collection 20 limit (deg) 55.36 total data collected 17005 (-8s//s 8, -10 s*s 10, -47s / £ 47) independent reflections 4463 (flint = 0.0189)

2 2 number of observed reflections (NO) 4123 [F0 a 2o(F0 )] structure solution method direct methods (SHELXS-97C) Appendix 248

refinement method full-matrix least-squares on F2 (SHELXL-97C) absorption correction method Gaussian integration (face-indexed) range of transmission factors 0.6830-0.5085 data/restraints/parameters 4463/3^/217

Flack absolute structure parameter* 0.021(12) goodness-of-fit (SF [all data] 1.061 final R indices?

*][F0222o(F02)] 0.0407

w/?2 [all data] 0.1283 largest difference peak and hole 0.767 and -0.453 e A"3

^Obtained from least-squares refinement of 9914 reflections with 5.22° <28 < 54.06°.

^Programs for diffractometer operation, data collection, data reduction and absorption correction were those supplied by Bruker. cSheldrick, G. M. Acta Crystallogr. 2008, A64,112-122.

^he C15-C16, C16-C17, and C17-C18 distances of the hexyl sidechain were restrained to be the same. eFlack, H. D. Acta Crystallogr. 1983,A39, 876-881; Flack, H. D.; Bernardinelli, G. Acta Crystallogr. 1999, A55, 908-915; Flack, H. D.; Bernardinelli, G. J. Appl. Cryst. 2000, 33, 1143-1148. The Flack parameter will refine to a value near zero if the structure is in the correct configuration and will refine to a value near one for the inverted configuration.

2 2 2 fS = [Ew(F0 - Fc ) /(n -p)]1/2 (n - number of data; p = number of parameters 2 2 1 2 varied; w = [cP-{F0 ) + (0.0764P) + 0.9153P]" where P = [Max(F0 , 0) + 2 2Fc ]/3).

2 2 2 4 ]/2 SR\ = Z||F0| - \FC\\/1\F0\- wR2 = [Zw(F0 -Fc ) flw(F0 )] .

Table 2. Atomic Coordinates and Equivalent Isotropic Displacement Parameters Appendix 249

Atom X y z U9q, A2

Br 0.66096(7) -0.01431(5) -0.018910(10) 0.06239(15) 01 -0.0634(4) 0.1561(5) 0.11276(8) 0.0688(9)* 02 0.2107(3) 0.2973(3) 0.13401(6) 0.0377(5)* 03 0.7099(3) 0.2453(4) 0.21802(7) 0.0508(6)* 04 0.4025(3) 0.3386(2) 0.20342(6) 0.0340(4)* CI 0.4914(5) 0.0551(4) 0.02053(8) 0.0421(6)* C2 0.2891(6) 0.0061(4) 0.02097(8) 0.0472(7)* C3 0.1679(5) 0.0512(4) 0.05050(9) 0.0468(7)* C4 0.2483(5) 0.1472(4) 0.07882(8) 0.0375(6)* C5 0.4533(5) 0.1951(4) 0.07777(9) 0.0415(7)* C6 0.5734(5) 0.1497(5) 0.04807(9) 0.0462(7)* C7 0.1127(5) 0.1975(4) 0.10953(9) 0.0435(7)* C8 0.0977(4) 0.3540(4) 0.16610(8) 0.0343(6)* C9 0.1954(4) 0.2740(3) 0.19948(8) 0.0310(5)* CIO 0.2195(5) 0.0836(4) 0.19805(10) 0.0420(7)* Cll 0.4222(4) 0.0520(4) 0.21698(8) 0.0361(6)* C12 0.5327(4) 0.2162(4) 0.21310(8) 0.0344(6)* C13 0.5449(6) -0.0941(5) 0.20256(12) 0.0573(9)* C14 0.1077(5) 0.5474(4) 0.16588(9) 0.0435(7)* C15 0.0132(7) 0.6289(6) 0.13225(12) 0.0717(13)* C16 -0.2119(8) 0.6041(6) 0.12736(15) 0.0844(17)* C17 -0.2789(10) 0.7080(10) 0.0945(2) 0.124(3)* C18 -0.5048(10) 0.6950(10) 0.0887(2) 0.118(3)* C19 0.0162(6) 0.6163(4) 0.20146(9) 0.0451(7)*

Anisotropically-refined atoms are marked with an asterisk (*). The form of the anisotropic displacement parameter is: exp[-2;r2(/i2a*2£/ii + k2b*2U22 + 2 Pc* U33 + 2klb*c*U23 + 2hla*c*Uu + 2hka*b*Un)l Appendix 250

Table 3. Selected Interatomic Distances (A)

Atoml Atom2 Distance Atoml Atom2 Distance Br CI 1.899(3) C5 C6 1.386(5) 01 C7 1.206(4) C8 C9 1.516(4) 02 C7 1.358(4) C8 C14 1.548(4) 02 C8 1.456(3) C9 CIO 1.532(4) 03 C12 1.197(4) CIO Cll 1.517(4) 04 C9 1.459(3) Cll C12 1.506(4) 04 C12 1.345(3) Cll C13 1.512(4) CI C2 1.382(5) C14 C15 1.519(5) CI C6 1.367(5) C14 C19 1.530(4) C2 C3 1.385(5) C15 C16 1.499(6) C3 C4 1.390(4) C16 C17 1.521(6) C4 C5 1.397(4) C17 C18 1.499(6) C4 C7 1.484(4)

Table 4. Selected Interatomic Angles (deg)

Atoml Atom2 Atom3 Angle Atoml Atom2 Atom3 Angle C7 02 C8 118.0(2) C9 C8 C14 114.1(2) C9 04 C12 110.9(2) 04 C9 C8 108.8(2) Br CI C2 119.1(2) 04 C9 CIO 105.0(2) Br CI C6 119.2(3) C8 C9 CIO 115.9(2) C2 CI C6 121.7(3) C9 CIO Cll 103.9(2) CI C2 C3 119.0(3) CIO Cll C12 103.5(2) C2 C3 C4 120.2(3) CIO Cll C13 115.8(3) C3 C4 C5 119.7(3) C12 Cll C13 112.7(3) C3 C4 C7 118.8(3) 03 C12 04 120.8(3) C5 C4 CI 121.4(3) 03 C12 Cll 128.4(3) C4 C5 C6 119.7(3) 04 C12 Cll 110.8(2) CI C6 C5 119.6(3) C8 C14 C15 114.5(3) 01 C7 02 123.3(3) C8 C14 C19 109.8(3) 01 C7 C4 124.9(3) C15 C14 C19 111.6(3) 02 C7 C4 111.8(3) C14 C15 C16 116.1(4) 02 C8 C9 107.3(2) C15 C16 C17 107.8(6) 02 C8 C14 106.6(2) C16 C17 C18 111.0(7) Appendix 251

Table 5. Torsional Angles (deg) > o bO Atoml 3 Atom3 Atom4 Angle Atoml Atom2 Atom3 Atom4 Angle C8 02 C7 01 1.3(5) C4 C5 C6 CI -1.6(5) C8 02 CI C4 -178.8(3) 02 C8 C9 04 65.2(3) C7 02 C8 C9 113.7(3) 02 C8 C9 CIO -52.8(3) CI 02 C8 C14 -123.7(3) C14 C8 C9 04 -52.7(3) C12 04 C9 C8 -139.3(2) C14 C8 C9 CIO -170.6(3) C12 04 C9 CIO -14.6(3) 02 C8 C14 C15 60.8(4) C9 04 C12 03 -179.2(3) 02 C8 C14 C19 -172.7(2) C9 04 C12 Cll -0.4(3) C9 C8 C14 C15 179.1(3) Br CI C2 C3 177.4(3) C9 C8 C14 C19 -54.4(3) C6 CI C2 C3 -1.4(5) 04 C9 CIO Cll 23.2(3) Br CI C6 C5 -177.3(3) C8 C9 CIO Cll 143.2(2) C2 CI C6 C5 1.6(5) C9 CIO Cll C12 -22.9(3) CI C2 C3 C4 1.3(5) C9 CIO Cll C13 -146.7(3) C2 C3 C4 C5 -1.3(5) CIO Cll C12 03 -166.0(3) C2 C3 C4 CI 178.3(3) CIO Cll C12 04 15.3(3) C3 C4 C5 C6 1.5(5) C13 Cll C12 03 -40.2(5) C7 C4 C5 C6 -178.1(3) C13 Cll C12 04 141.1(3) C3 C4 CI Ol 2.9(6) C8 C14 C15 C16 64.2(5) C3 C4 CI 02 -177.0(3) C19 C14 C15 C16 -61.4(5) C5 C4 CI 01 -177.5(4) C14 C15 C16 C17 174.8(5) C5 C4 CI 02 2.6(5) C15 C16 C17 C18 -177.8(6) Appendix 252

Table 6. Anisotropic Displacement Parameters (t/jj, A2)

Atom tfll U22 t/33 U23 ^13 U12 Br 0.0785(3) 0.0646(2) 0.0441(2) -0.00954(16) 0.02063(16) 0.0014(2) Ol 0.0338(12) 0.107(2) 0.0652(17) -0.0433(17) 0.0000(11) -0.0078(14) 02 0.0299(9) 0.0485(11) 0.0346(10) -0.0144(9) -0.0019(8) 0.0031(8) 03 0.0260(10) 0.0682(16) 0.0581(15) 0.0001(12) -0.0038(9) 0.0006(10) 04 0.0297(9) 0.0323(9) 0.0400(10) 0.0002(8) -0.0050(8) -0.0034(7) CI 0.0547(17) 0.0389(14) 0.0327(13) 0.0002(12) 0.0064(13) 0.0097(12) C2 0.0579(18) 0.0437(15) 0.0399(15) -0.0098(15) -0.0041(13) 0.0027(14) C3 0.0473(16) 0.0501(17) 0.0431(16) -0.0151(13) -0.0038(13) -0.0006(15) C4 0.0376(14) 0.0393(15) 0.0356(14) -0.0085(12) -0.0030(11) 0.0066(12) C5 0.0412(15) 0.0459(16) 0.0373(15) -0.0099(13) -0.0043(12) 0.0053(13) C6 0.0416(15) 0.0536(19) 0.0435(17) -0.0051(14) 0.0035(13) -0.0002(14) C7 0.0360(15) 0.0531(17) 0.0413(16) -0.0150(14) -0.0080(12) 0.0075(13) C8 0.0311(13) 0.0401(14) 0.0317(13) -0.0058(11) -0.0017(10) 0.0054(10) C9 0.0255(11) 0.0299(12) 0.0376(14) -0.0017(10) -0.0034(10) 0.0004(10) CIO 0.0381(15) 0.0277(12) 0.060(2) 0.0017(13) -0.0041(14) -0.0027(11) Cll 0.0365(13) 0.0353(14) 0.0365(14) 0.0033(10) 0.0022(11) 0.0066(11) C12 0.0290(12) 0.0445(15) 0.0297(13) -0.0017(11) -0.0006(10) 0.0042(11) C13 0.061(2) 0.0447(18) 0.066(2) -0.0024(17) 0.0053(19) 0.0195(17) C14 0.0547(18) 0.0378(15) 0.0381(15) 0.0015(12) 0.0017(13) 0.0131(13) C15 0.099(3) 0.063(2) 0.053(2) 0.0035(19) -0.007(2) 0.033(2) C16 0.110(4) 0.061(3) 0.082(3) -0.014(2) -0.045(3) 0.023(3) C17 0.120(6) 0.106(5) 0.145(7) 0.023(5) -0.058(5) 0.019(4) C18 0.140(6) 0.089(4) 0.125(6) 0.035(4) -0.044(5) 0.001(4) C19 0.0545(18) 0.0363(14) 0.0447(16) -0.0069(13) 0.0000(14) 0.0116(13)

The form of the anisotropic displacement parameter is:

2 exp[-2^2(/,2a*2t/u + ^b*W22 + /V t/33 + 2klb*c*U23 + 2hla*c*Uu + 2hka*b*U\2)] Appendix 253

Table 7. Derived Atomic Coordinates and Displacement Parameters for Hydrogen Atoms

Atom X y z t/eq,A2 H2 0.2340 -0.0576 0.0013 0.057 H3 0.0293 0.0165 0.0514 0.056 H5 0.5101 0.2585 0.0973 0.050 H6 0.7120 0.1843 0.0469 0.055 H8 -0.0477 0.3171 0.1641 0.041 H9 0.1146 0.3045 0.2218 0.037 H10A 0.1066 0.0273 0.2112 0.050 H10B 0.2225 0.0432 0.1724 0.050 HI 1 0.3951 0.0326 0.2437 0.043 H13A 0.6721 -0.1039 0.2165 0.069 H13B 0.5766 -0.0758 0.1766 0.069 H13C 0.4654 -0.1971 0.2052 0.069 H14 0.2556 0.5785 0.1659 0.052 H15A 0.0406 0.7505 0.1334 0.086 H15B 0.0834 0.5849 0.1102 0.086 H16A -0.2858 0.6405 0.1497 0.101 H16B -0.2423 0.4844 0.1230 0.101 H17A -0.2414 0.8264 0.0986 0.148 H17B -0.2069 0.6685 0.0722 0.148 H18A -0.5440 0.7617 0.0673 0.142 H18B -0.5762 0.7369 0.1105 0.142 H18C -0.5419 0.5778 0.0846 0.142 H19A 0.0228 0.7387 0.2012 0.054 H19B 0.0936 0.5736 0.2225 0.054 H19C -0.1265 0.5806 0.2035 0.054