Science, Faculty of
Chemistry, Department of
DSpace
UBCV
Hirkala-Schaefer, Truman
Master of Science - MSc
University of British Columbia
The prehistoric Australian conifer Wollemia nobilis was thought to be extinct until the discovery of living specimens in 1992. Due to its threatened status and restricted distribution, investigations into its metabolic constituents have been limited. Chapter 2 details the phytochemical investigation of Wollemia nobilis. Crude organic extracts from the leaves Wollemia nobilis collected on UBC campus were screened for bioactivity against a series of pathogens. Bioassay guided fractionation led to the isolation and structural elucidation of the novel compound 2.12, which upon examination of the experimental data was found to be the true structure of the previously reported compound wollemolide (2.11). Additionally, two new endoperoxide diterpenoids (2.14 & 2.15), several known compounds new to the Wollemia genus (2.13 & 2.16-2.18), as well as sandaracopimaric acid (2.4) previously reported from Wollemia nobilis were isolated and structurally elucidated. The pure compounds demonstrated no in vitro activity against the screened pathogens Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans and methicillin resistant Staphylococcus aureus (MRSA). The Indonesian sponge Petrosia contignata has been the source of several novel bioactive metabolites. Though previously examined, the crude sponge extracts of Petrosia contignata were investigated for less abundant metabolites. Chapter 3 describes the isolation and structural elucidation of the new tetrahydroxylated steroid petrosiasterol (3.9). as well as three known steroids new to the Petrosia genus (3.7, 3.8, & 3.11) and one previously reported from the Petrosia genus (3.10).
Terpenoids from Terrestrial and Marine Sources by Truman Hirkala-Schaefer B.Sc., University of Victoria, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2020 © Truman Hirkala-Schaefer, 2020 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Terpenoids from Terrestrial and Marine Sources submitted by Truman Hirkala-Schaefer in partial fulfillment of the requirements for the degree of Master of Science in Chemistry Examining Committee: Dr. Raymond J. Andersen, Professor, Chemistry, UBC Supervisor Dr. Katherine Ryan, Associate Professor, Chemistry, UBC Supervisory Committee Member Dr. Stephen Withers, Professor, Chemistry, UBC Supervisory Committee Member iii Abstract The prehistoric Australian conifer Wollemia nobilis was thought to be extinct until the discovery of living specimens in 1992. Due to its threatened status and restricted distribution, investigations into its metabolic constituents have been limited. Chapter 2 details the phytochemical investigation of Wollemia nobilis. Crude organic extracts from the leaves Wollemia nobilis collected on UBC campus were screened for bioactivity against a series of pathogens. Bioassay guided fractionation led to the isolation and structural elucidation of the novel compound 2.12, which upon examination of the experimental data was found to be the true structure of the previously reported compound wollemolide (2.11). Additionally, two new endoperoxide diterpenoids (2.14 & 2.15), several known compounds new to the Wollemia genus (2.13 & 2.16-2.18), as well as sandaracopimaric acid (2.4) previously reported from Wollemia nobilis were isolated and structurally elucidated. The pure compounds demonstrated no in vitro activity against the screened pathogens Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans and methicillin resistant Staphylococcus aureus (MRSA). The Indonesian sponge Petrosia contignata has been the source of several novel bioactive metabolites. Though previously examined, the crude sponge extracts of Petrosia contignata were investigated for less abundant metabolites. Chapter 3 describes the isolation and structural elucidation of the new tetrahydroxylated steroid petrosiasterol (3.9). as well as three known steroids new to the Petrosia genus (3.7, 3.8, & 3.11) and one previously reported from the Petrosia genus (3.10). iv Lay Summary Natural products are chemical compounds produced by organisms such as plants and marine invertebrates, and can serve as a source of inspiration for drug development. Immobile organisms such as marine invertebrates are vulnerable to predators and often produce chemical defense compounds that can be of medicinal interest. Crude mixtures of the compounds produced by these organisms can be extracted and tested for activity against pathogens and diseases, and if the mixture of chemicals shows efficacy against a particular disease or pathogen, it is implied that some compound therein is providing this activity. Through this process, medicinally relevant compounds, including antibiotics and chemotherapeutics, as well as new chemical structures that provide biological information on their source organism can be discovered. Here, I present work towards the discovery of such compounds, which led to the discovery of several new chemical structures. v Preface Chapter 2 was based on work conducted by myself at UBC, after the discovery of a Wollemia nobilis specimen growing on the UBC campus. Previous phytochemical studies of W. nobilis were reported in the literature, but only two involved NMR-based structural elucidation and both utilized 400 MHz NMR instruments, rather than the 600 MHz instrument at my disposal. All isolation and structural elucidations were carried out by myself. Jianxong Yang from Dr. Alex Wang’s group performed circular dichroism (CD) calculations, and Virayu Suthiphasilp of the Andersen group performed CD experiments. Michael LeBlanc performed the bioassays. Chapter 3 is based on work conducted in the Andersen Lab by Dr. David Borgoyne, who first investigated compounds from Petrosia contignata in 1992. My original goal was to isolate sufficient quantities of the secondary metabolite contignasterol for structure-activity relationship studies. In the process, I discovered several sterols that had not previously been reported in studies of Petrosia contignata. All isolations and structural elucidations were performed by myself. vi Table of Contents Abstract ................................................................................................................................. iii Lay Summary .......................................................................................................................... iv Preface .................................................................................................................................... v Table of Contents ................................................................................................................... vi List of Tables ........................................................................................................................... x List of Figures ......................................................................................................................... xi List of Abbreviations ............................................................................................................ xxii List of Symbols .................................................................................................................... xxv Acknowledgements ............................................................................................................ xxvi Dedication ......................................................................................................................... xxvii Chapter 1: Introduction .......................................................................................................... 1 1.1 Introduction to Natural Product Chemistry .......................................................................... 1 1.2 Sources of Natural Products & Conservation ........................................................................ 3 1.2.1 Marine Natural Products ........................................................................................................ 5 1.3 Natural Products in Drug Discovery ...................................................................................... 9 1.4 Techniques in Natural Products Isolation & Elucidation ...................................................... 12 1.5 Classes of Natural Products and Biosynthetic Origins ......................................................... 14 1.5.1 Terpenoids ............................................................................................................................ 17 vii 1.5.2 Steroids ................................................................................................................................. 19 1.6 Scope of Thesis .................................................................................................................. 22 Chapter 2: Terpenoids and Other Compounds from Wollemia nobilis .................................... 23 2.1 Introduction to Wollemia nobilis ........................................................................................ 23 2.2 Previously Isolated Secondary Metabolites from Wollemia nobilis ..................................... 24 2.3 Terpenoids from Wollemia nobilis ...................................................................................... 26 2.3.1 Isolation and Structural Elucidation of Compound 2.12 and Agathic Acid Methyl Ester (2.13) ………………………………………………………………………………………………………………………………………….27 2.3.1.1 Structural Correction of Wollemolide (2.11) ............................................................................. 37 2.3.2 Isolation and Structural Elucidation of 15-Hydroperoxy-8β,12β-epidioxy-13-abieten-19-oic acid (2.14) and 15-Hydroxy-8β,12β-epidioxy-13-abieten-19-oic acid (2.15) ..................................... 42 2.3.3 Isolation & Structural Elucidation of Abiesanordine E (2.16) and Kaur-15-en-17-al (2.17) .. 64 2.3.4 Isolation and Structural Elucidation of Isocommunic Acid (2.18) and Sandaracopimaric Acid (2.4) …………………………………………………………………………………………………………………………………………69 2.3.5 Chemotaxonomy of Wollemia nobilis ................................................................................... 74 2.3.6 Proposed Biogenetic Relationship of Compounds Isolated from Wollemia nobilis .............. 77 2.3.7 Biological Data ...................................................................................................................... 82 2.4 Conclusion ......................................................................................................................... 82 Chapter 3: Sterols from Petrosia contignata .......................................................................... 83 3.1 Sea Sponges ....................................................................................................................... 83 3.1.1 The Petrosia Genus and Marine Natural Products ............................................................... 83 3.2 Steroids from Petrosia contignata ...................................................................................... 85 viii 3.2.1 Isolation and Structural Elucidation of 24-Ethylcholest-7-ene-3b,5a,6b-triol (3.7) and 24-Ethylcholest-7-ene-3b,5a,6b,9a-tetraol (3.8) ................................................................................... 86 3.2.2 Structural Elucidation of Petrosiasterol (3.9) ....................................................................... 92 3.2.3 Isolation and Structural Elucidation of 24-Ethylcholest-6-en-5a,8a-epidioxy-3b-ol (3.10) and 3b-Hydroxy-24-ethylcholesta-5,8-dien-7-one (3.11) ................................................................ 102 3.3 Biogenetic Relationship of Steroids Isolated from Petrosia contignata ............................. 107 3.4 Conclusion ....................................................................................................................... 107 Chapter 4: Experimental ...................................................................................................... 109 4.1 General Experimental Protocol ........................................................................................ 109 4.2 Sample Collection, Extraction and Isolation of Compounds from Wollemia nobilis ........... 110 4.3 Sample Collection, Extraction and Isolation of Compounds from Petrosia contignata ...... 114 4.4 Bioassays ......................................................................................................................... 116 References .......................................................................................................................... 117 Appendices ......................................................................................................................... 126 Appendix A 2D-NMR Spectra for Compound 2.12 and 2.13 Recorded in DMSO-d6 at 600 MHz ...... 126 Appendix B Calculated & Experimental CD Spectra for Selected Compounds ................................ 131 Appendix C NMR Spectra for Compound 2.12 Recorded in CDCl3 at 600 MHz ............................... 133 Appendix D Thin Film FT-IR Spectrum of Compound 2.12 ............................................................. 135 Appendix E NMR Spectra for 15-Hydroperoxy-8β,12β-epidioxy-13-abieten-19-oic acid (2.14) and 15-Hydroxy-8b,12b-epidioxy-13-abieten-19-oic acid (2.15) Recorded in DMSO-d6 and CDCl3 at 600 MHz .................................................................................................................................................... 136 ix Appendix F NMR Spectra for Abiesanordine E (2.16) and Kaur-15-en-17-al (2.17) Recorded in DMSO-d6 at 600 MHz ............................................................................................................................... 146 Appendix G NMR Spectra for Isocommunic Acid (2.18) and Sandaracopimaric Acid (2.4) Recorded in DMSO-d6 at 600 MHz .................................................................................................................... 151 Appendix H NMR Spectra for 24-Ethylcholest-7-ene-3b,5a,6b-triol (3.7) and 24-Ethylcholest-7-ene-3b,5a,6b,9a-tetraol (3.8) Recorded in DMSO-d6 at 600 MHz ........................................................ 157 Appendix I NMR Spectra for 3.9 Recorded in DMSO-d6 at 600 MHz .............................................. 164 Appendix J NMR Spectra for 24-Ethylcholest-6-en-5a,8a-epidioxy-3b-ol (3.10) and 3b-Hydroxy-24-ethylcholesta-5,8-dien-7-one (3.11) Recorded in DMSO-d6 at 600 MHz ......................................... 166 x List of Tables Table 2.1 NMR Data for 2.12 and Agathic Acid Methyl Ester (2.13) Recorded at 600 MHz in DMSO-d6 ...................................................................................................................................... 28 Table 2.2 Comparison of Experimental and Literature NMR Data for Wollemolide (2.11) & Compound 2.12 Recorded at 600 MHz in CDCl3 ......................................................................... 39 Table 2.3 NMR Data for 2.14 and 2.15 Recorded at 600 MHz in DMSO-d6 ............................... 43 Table 2.4 NMR Data for 2.14 & 2.15 Recorded at 600 MHz in CDCl3 and Comparison to Literature Data for their 8α,12α-Epidioxy Analogues 2.14ʹ & 2.15ʹ ............................................ 61 Table 2.5 NMR Data for Abiesanordine E (2.16) and Kaur-15-en-17-al (2.17) Recorded at 600 MHz in DMSO-d6 ......................................................................................................................... 65 Table 2.6 NMR Data for Isocommunic Acid (2.18) and Sandaracopimaric Acid (2.4) Recorded at 600 MHz in DMSO-d6 .................................................................................................................. 70 Table 3.1 NMR Data for 3.7 and 3.8 Recorded in DMSO-d6 at 600 MHz ................................... 87 xi List of Figures Figure 1.1 Azadirachtin (1.1) from Azadirachta indica ................................................................. 2 Figure 1.2 Mescaline (1.2) from Lophophora williamsii ................................................................ 3 Figure 1.3 Structures of Spongothymidine (1.3), Spongouridine (1.4) and Spongosine (1.5), and their Chemical Derivatives Vidarabine (1.6) and Cytarabine (1.7) ................................................ 6 Figure 1.4 Structure of Brevetoxin A (1.8) ..................................................................................... 7 Figure 1.5 Marine Derived Natural Products Dolastatin-10 (1.9) and Halichondrin B (1.12) and their Synthetically Derived Drug Analogues Eribulin (1.11) and MMAE (1.10) .......................... 8 Figure 1.6 Historically Significant Natural Product Drugs ............................................................ 9 Figure 1.7 Camptothecin (1.14) and its Synthetic Analogues (1.15-1.17) ................................... 11 Figure 1.8 Structures of the Polyketides Discodermolide (1.23) and Tacrolimus (1.24) ............. 14 Figure 1.9 Structure and Biogenetic Origins of the Ribosomal Peptide a-Aminitin and Structure of Echinomycin ............................................................................................................................. 15 Figure 1.10 Biosynthetic Origins of the Ergot Alkaloid Lysergic Acid ....................................... 16 Figure 1.11 Biosynthesis of Terpenes ........................................................................................... 17 Figure 1.12 Several Common Diterpene Skeletons ...................................................................... 18 Figure 1.13 Biosynthesis of the Diterpenoid Abiatic Acid ........................................................... 19 Figure 1.14 Biosynthetic Origins of Lanosterol and 24-Ethyl Sterols .......................................... 20 Figure 1.15 Steroid Numbering Scheme and Several Common Skeletons .................................. 21 Figure 2.1 Wollemia nobilis Specimen Planted at UBC ............................................................... 23 Figure 2.2 Previously Reported Compounds from Wollemia nobilis ........................................... 25 Figure 2.3 Wollemia nobilis Material: Before and After Extraction with MeOH ........................ 26 xii Figure 2.4 Terpenoids Isolated from Wollemia nobilis in this Study ........................................... 26 Figure 2.5 Structure of Compound 2.12 and Agathic Acid Methyl Ester (2.13) .......................... 27 Figure 2.6 1H NMR Spectrum of Compound 2.12 Recorded at 600 MHz in DMSO-d6 .............. 29 Figure 2.7 13C NMR Spectrum of Compound 2.12 Recorded at 150 MHz in DMSO-d6 ........... 29 Figure 2.8 gradHSQC for Compound 2.12 Collected in DMSO-d6 at 600 MHz with Relevant Annotations ................................................................................................................................... 30 Figure 2.9 Expansion of gradHSQC for Compound 2.12 Collected in DMSO-d6 at 600 MHz Showing Relevant Annotations ..................................................................................................... 30 Figure 2.10 Selected HMBC and TOCSY/COSY Correlations for Compound 2.12 ................... 31 Figure 2.11 Expansion of gradHSQCTOCSY for Compound 2.12 Collected in DMSO-d6 at 600 MHz Showing Color Coded Contiguous Spin Systems ............................................................... 32 Figure 2.12 Expansion 1 of gradHMBC NMR Spectrum of Compound 2.12 Collected in DMSO-d6 at 600 MHz with Relevant Annotations ................................................................................... 32 Figure 2.13 Expansion 2 of gradHMBC NMR Spectrum of Compound 2.12 Collected in DMSO-d6 at 600 MHz with Relevant Annotations ................................................................................... 33 Figure 2.14 Selected trROESY Correlations for Compound 2.12 ................................................ 34 Figure 2.15 Expansion 1 of trROESY NMR Spectrum for Compound 2.12 Collected in DMSO-d6 at 600 MHz with Relevant Annotations ................................................................................... 35 Figure 2.16 Expansion 2 of trROESY NMR Spectrum for Compound 2.12 Collected in DMSO-d6 at 600 MHz with Relevant Annotations ................................................................................... 35 Figure 2.17 Selected HMBC & COSY/TOCSY Correlations for Agathic Acid Methyl Ester (2.13) ............................................................................................................................................. 36 Figure 2.18 Selected trROESY Correlations for Agathic Acid Methyl Ester (2.13) .................... 37 xiii Figure 2.19 Comparison of Reported Structure for Wollemolide (2.11) & Compound 2.12 ....... 38 Figure 2.20 1H NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 600 MHz with Relevant Annotations .................................................................................................................... 40 Figure 2.21 13C NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 150 MHz with Relevant Annotations .................................................................................................................... 40 Figure 2.22 Expansion of gradHMBC NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 600 MHz Showing Color Coded Contiguous Spin Systems ........................................................ 41 Figure 2.23 Expansion of trROESY NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 600 MHz with Relevant Annotations ........................................................................................... 41 Figure 2.24 Structures of 15-Hydroperoxy-8β,12β-epidioxy-13-abieten-19-oic acid (2.14) and 15-Hydroxy-8b,12b-epidioxy-13-abieten-19-oic acid (2.15) ....................................................... 42 Figure 2.25 1H NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz .............................. 44 Figure 2.26 13C NMR Spectrum of 2.14 Recorded in DMSO-d6 at 150 MHz ............................. 44 Figure 2.27 gradHSQC NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz ................. 45 Figure 2.28 Expansion of gradHSQC NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations ............................................................................................................ 45 Figure 2.29 Selected HMBC & COSY/TOCSY Correlations for 2.14 ........................................ 46 Figure 2.30 Expansion of gradHSQCTOCSY NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations ........................................................................................... 47 Figure 2.31 Expansion 1 of gradHMBC NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................................................. 47 Figure 2.32 Expansion 2 of gradHMBC NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................................................. 48 xiv Figure 2.33 Expansion 3 of gradHMBC NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................................................. 48 Figure 2.34 Selected trROESY Correlations for 2.14 ................................................................... 49 Figure 2.35 Expansion 1 of trROESY NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................................................. 50 Figure 2.36 Expansion 2 of trROESY NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................................................. 50 Figure 2.37 Expansion 1 of trROESY NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz with Relevant Annotations ............................................................................................................ 51 Figure 2.38 1H NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz .............................. 52 Figure 2.39 13C NMR Spectrum of 2.15 Recorded in DMSO-d6 at 150 MHz ............................. 53 Figure 2.40 gradHSQC NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................................................................... 53 Figure 2.41 Expansion of gradHSQC NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations ............................................................................................................ 54 Figure 2.42 Selected HMBC & COSY/TOCSY Correlations for 2.15 ........................................ 55 Figure 2.43 Expansion of gradHSQCTOCSY NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz Showing Color Coded Contiguous Spin Systems ........................................................ 55 Figure 2.44 Expansion 1 of gradHMBC NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................................................. 56 Figure 2.45 Expansion 2 of gradHMBC NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................................................. 56 xv Figure 2.46 Expansion 3 of gradHMBC NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................................................. 57 Figure 2.47 Selected trROESY Correlations for 2.15 ................................................................... 58 Figure 2.48 Expansion 1 of trROESY NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................................................. 58 Figure 2.49 Expansion 2 of trROESY NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................................................. 59 Figure 2.50 Expansion of trROESY NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz with Relevant Annotations ............................................................................................................ 59 Figure 2.51 Diagnostic NOE Cited for the Assignment of 8α,12α-Epidoxy Abietanes ............... 60 Figure 2.52 Structures of 2.14 & 2.15 and their 8α,12α-Epidioxy Analogues 2.14ʹ & 2.15ʹ ....... 61 Figure 2.53 1H NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz .................................... 62 Figure 2.54 13C NMR Spectrum of 2.14 Recorded in CDCl3 at 150 MHz ................................... 62 Figure 2.55 1H NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz .................................... 63 Figure 2.56 gradHSQC NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz ...................... 63 Figure 2.57 Structures of Abiesanordine E (2.16) and Kaur-15-en-17-al (2.17) .......................... 64 Figure 2.58 Selected HMBC and COSY/TOCSY Correlations for Abiesanordine E (2.16) ....... 66 Figure 2.59 Selected trROESY Correlations for Abiesanordine E (2.16) .................................... 67 Figure 2.60 Selected HMBC and COSY/TOCSY Correlations for 2.17 ...................................... 68 Figure 2.61 Selected trROESY Correlations for 2.17 ................................................................... 68 Figure 2.62 Structures of Isocommunic Acid (2.18) and Sandaracopimaric Acid (2.4) .............. 69 Figure 2.63 Selected HMBC & COSY/TOCSY Correlations for Isocommunic Acid (2.18) ...... 71 Figure 2.64 Selected trROESY Correlations for Isocommunic Acid (2.18) ................................ 72 xvi Figure 2.65 Selected HMBC & COSY/TOCSY Correlations for Sandaracopimaric Acid (2.4) . 73 Figure 2.66 Selected trROESY Correlations for Sandaracopimaric Acid (2.4) ........................... 73 Figure 2.67 Phylogeny of Wollemia nobilis and its Relatives Within the Pinophyta Division .... 74 Figure 2.68 Proposed Biogenetic Relationship of Compounds Isolated from Wollemia nobilis . 78 Figure 2.69 Detailed Steps for the Proposed Biogenesis of Abiesanordine E (3.14) from Intermediate ii ............................................................................................................................... 79 Figure 2.70 Detailed Steps for the Proposed Biogenesis of 2.14 and 2.15 from Levopimaradiene....................................................................................................................................................... 80 Figure 3.1 Compounds Previously Isolated from the Petrosia Genus .......................................... 84 Figure 3.2 Petrosia contignata in Methanol ................................................................................. 85 Figure 3.3 Steroids Isolated from Petrosia contigata in this Study .............................................. 85 Figure 3.4 Structures of 24-Ethylcholest-7-ene-3b,5a,6b-triol (3.7) and 24-Ethylcholest-7-ene-3b,5a,6b,9a-tetraol (3.8) .............................................................................................................. 86 Figure 3.5 Selected HMBC and COSY/TOCSY Correlations for 3.7 .......................................... 88 Figure 3.6 Selected trROESY Correlations for 3.7 ....................................................................... 90 Figure 3.7 Selected HMBC and COSY/TOCSY Correlations for 3.8 .......................................... 91 Figure 3.8 Selected trROESY Correlations for 3.8 ....................................................................... 91 Figure 3.9 Structure of Petrosiasterol (3.9) ................................................................................... 92 Figure 3.10 1H NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz ....... 94 Figure 3.11 13C NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 150 MHz ...... 95 Figure 3.12 gradHSQC NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................................................. 95 xvii Figure 3.13 Expansion of gradHSQC NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations ................................................................................... 96 Figure 3.14 Selected HMBC and COSY/TOCSY Correlations for Petrosiasterol (3.9) .............. 97 Figure 3.15 Expansion of HSQCTOCSY NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations ...................................................................... 97 Figure 3.16 Expansion 1 of gradHMBC NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations ...................................................................... 98 Figure 3.17 Expansion 2 of gradHMBC NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations ...................................................................... 99 Figure 3.18 Selected trROESY correlations for 3.9 ................................................................... 100 Figure 3.19 Expansion 1 of trROESY NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................... 100 Figure 3.20 Expansion 2 of trROESY NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations .................................................................... 101 Figure 3.21 Structure of Homaxisterol C .................................................................................... 101 Figure 3.23 Selected HMBC and COSY/TOCSY Correlations for 3.10 .................................... 104 Figure 3.24 Selected trROESY Correlations for 3.10 ................................................................. 105 Figure 3.25 Selected HMBC and COSY/TOCSY Correlations for 3.11 .................................... 106 Figure 3.26 Selected trROESY correlations for 3.11 ................................................................. 107 Figure A.1 gradCOSY60 NMR Spectrum of Compound 2.12 Recorded at 600 MHz in DMSO-d6 ……………………………………………………………………………………………......124 xviii Figure A.2 gradHSQC NMR Spectrum of Compound 2.12 Recorded at 600 MHz in DMSO-d6..................................................................................................................................................... 126 Figure A.3 gradHSQCTOCSY Spectrum of Compound 2.12 Recorded at 600 MHz in DMSO-d6..................................................................................................................................................... 127 Figure A.4 gradHMBC NMR Spectrum of Compound 2.12 Recorded at 600 MHz in DMSO-d6..................................................................................................................................................... 127 Figure A.5 trROESY Spectrum of Compound 2.12 Recorded at 600 MHz in DMSO-d6 ......... 128 Figure A.6 1H NMR Spectrum of Agathic Acid Methyl Ester (2.13) Recorded in DMSO-d6 at 600 MHz ..................................................................................................................................... 128 Figure A.7 gradHSQC NMR Spectrum of Agathic Acid Methyl Ester (2.13) Recorded in DMSO-d6 at 600 MHz ................................................................................................................ 129 Figure A.8 gradCOSY60 NMR Spectrum of Agathic Acid Methyl Ester (2.13) Recorded in DMSO-d6 at 600 MHz ................................................................................................................ 129 Figure A.9 gradHMBC NMR Spectrum of Agathic Acid Methyl Ester (2.13) Recorded in DMSO-d6 at 600 MHz ................................................................................................................ 130 Figure A.10 trROESY NMR Spectrum of Agathic Acid Methyl Ester (2.13) Recorded in DMSO-d6 at 600 MHz ............................................................................................................................. 130 Figure B.1 Figure 4.11 Calculated CD Spectra for (4R,5S,9S,10R) Configuration of Compound 2.12 (Blue) & its Enantiomer (Red) ............................................................................................ 131 Figure B.2 Calculated CD Spectrum for (4S,5R,9S,10S) Configuration of Compound 2.12 .... 131 Figure B.3 Experimental CD Spectrum for Compound 2.12 Recorded in MeOH ..................... 132 Figure C.1 COSY60 NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 600 MHz ...... 133 Figure C.2 gradHSQC NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 600 MHz ... 133 xix Figure C.3 gradHMBC NMR Spectrum for Compound 2.12 Recorded in CDCl3 at 600 MHz 134 Figure C.4 trROESY NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 600 MHz ..... 134 Figure D.1 Thin Film FT-IR Spectrum of Compound 2.12 ........................................................ 135 Figure E.11 1H NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz ................................. 141 Figure E.12 gradCOSY60 NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz ................ 141 Figure E.13 gradHSQC NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz .................... 142 Figure E.14 gradHMBC NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz ................... 142 Figure E.15 trROESY NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz ...................... 143 Figure E.16 1H NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz ................................. 143 Figure E.17 gradCOSY60 NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz ................ 144 Figure E.18 gradHSQC NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz .................... 144 Figure E.19 gradHMBC NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz ................... 145 Figure E.20 trROESY NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz ...................... 145 Figure H.1 1H NMR Spectrum of 3.7 Recorded in DMSO-d6 at 600 MHz ............................... 157 Figure H.2 13C NMR Spectrum of 3.7 Recorded in DMSO-d6 at 150 MHz .............................. 157 Figure H.3 gradCOSY60 NMR Spectrum of 3.7 Recorded in DMSO-d6 at 600 MHz .............. 158 Figure H.4 gradHSQCTOCSY NMR Spectrum of 3.7 Recorded in DMSO-d6 at 600 MHz ..... 158 Figure H.5 gradHSQC NMR Spectrum of 3.7 Recorded in DMSO-d6 at 600 MHz .................. 159 Figure H.6 gradHMBC NMR Spectrum of 3.7 Recorded in DMSO-d6 at 600 MHz ................. 159 Figure H.7 trROESY NMR Spectrum of 3.7 Recorded in DMSO-d6 at 600 MHz .................... 160 Figure H.8 1H NMR Spectrum of 3.8 Recorded in DMSO-d6 at 600 MHz ............................... 160 Figure H.9 13C NMR Spectrum of 3.8 Recorded in DMSO-d6 at 150 MHz .............................. 161 Figure H.10 gradCOSY60 NMR Spectrum of 3.8 Recorded in DMSO-d6 at 600 MHz ............ 161 xx Figure H.11 gradHSQCTOCSY NMR Spectrum of 3.8 Recorded in DMSO-d6 at 600 MHz ... 162 Figure H.12 gradHSQC NMR Spectrum of 3.8 Recorded in DMSO-d6 at 600 MHz ................ 162 Figure H.13 gradHMBC NMR Spectrum of 3.8 Recorded in DMSO-d6 at 600 MHz ............... 163 Figure H.14 trROESY NMR Spectrum of 3.8 Recorded in DMSO-d6 at 600 MHz .................. 163 Figure I.1 gradCOSY60 NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz ............................................................................................................................................ 164 Figure I.2 gradHSQC NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz..................................................................................................................................................... 164 Figure I.3 gradHSQCTOCSY NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz ..................................................................................................................................... 165 Figure I.4 gradHMBC NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz..................................................................................................................................................... 165 Figure I.5 trROESY NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz..................................................................................................................................................... 166 Figure J.1 1H NMR Spectrum of 3.10 Recorded in DMSO-d6 at 600 MHz ............................... 166 Figure J.2 gradHSQC NMR Spectrum of 3.10 Recorded in DMSO-d6 at 600 MHz ................. 167 Figure J.3 gradCOSY60 NMR Spectrum of 3.10 Recorded in DMSO-d6 at 600 MHz ............. 167 Figure J.4 gradHSQCTOCSY NMR Spectrum of 3.10 Recorded in DMSO-d6 at 600 MHz .... 168 Figure J.5 gradHMBC NMR Spectrum of 3.10 Recorded in DMSO-d6 at 600 MHz ................ 168 Figure J.6 trROESY NMR Spectrum of 3.10 Recorded in DMSO-d6 at 600 MHz ................... 169 Figure J.7 1H NMR Spectrum of 3.11 Recorded in DMSO-d6 at 600 MHz ............................... 169 Figure J.8 gradHSQC NMR Spectrum of 3.11 Recorded in DMSO-d6 at 600 MHz ................. 170 Figure J.9 gradCOSY60 NMR Spectrum of 3.11 Recorded in DMSO-d6 at 600 MHz ............. 170 xxi Figure J.10 gradHMBC NMR Spectrum of 3.11 Recorded in DMSO-d6 at 600 MHz .............. 171 Figure J.11 trROESY NMR Spectrum of 3.11 Recorded in DMSO-d6 at 600 MHz ................. 171 xxii List of Abbreviations 1D- one dimensional- 2D- two dimensional- ax axial ACN acetonitrile br broad CBD Convention on Biological Diversity CD circular dichroism CDCl3 deuterated chloroform COSY60 correlation spectroscopy with 60o pulse field gradient d doublet DCM dichloromethane dd doublet of doublets DMSO-d6 deuterated dimethyl sulfoxide EtOAc ethyl acetate eq equatorial ECD electronic circular dichroism FDA food and drug administration g gram(s) grad gradient HMBC heteronuclear multiple bond multiple quantum coherence HPLC high performance liquid chromatography xxiii HRESIMS high-resolution electrospray ionization mass spectrometry HSQC heteronuclear single quantum coherence hz Hertz LRESIMS low-resolution electrospray ionization mass spectrometry m multiplet Me MET MHz MVA methyl methylerythritol phosphate megahertz mevalonic acid MeOH methanol MRSA methicillin resistant Staphylococcus aureus mg milligram(s) m/z mass-to-charge ratio NAD(P) nicotinamide adenine dinucleotide (phosphate) NMR nuclear magnetic resonance nOe nuclear Overhauser effect PKS polyketide synthase ppm parts per million ROESY rotating frame Overhauser effect spectroscopy Rt chromatographic retention time s singlet SCUBA self-contained underwater breathing apparatus xxiv SAM s-adenosyl methionine TFA trifluoroacetic acid TLC thin-layer chromatography tr transverse TOCSY total correlation spectroscopy t triplet UBC University of British Columbia UV-VIS ultraviolent/visible light xxv List of Symbols % per cent 1H proton 13C carbon-13 δ chemical shift in parts per million lmax local UV-VIS absorbance maxima µg microgram xxvi Acknowledgements I would first and foremost like to thank my supervisor Dr. Raymond Andersen for providing me with a supportive environment in which to foster my understanding of natural products chemistry and Science at large. Your guidance will always be appreciated. I would also like to extend gratitude to David Williams for showing me the ropes of natural products chemistry, and also to Michael LeBlanc for being the anchor of the Andersen Lab. My thanks also go out to all of my Andersen Group lab mates, both past and present. I would also like to thank the UBC Faculty of Chemistry for providing an excellent learning environment. I owe particular gratitude to Dr. Glenn Sammis and Dr. Gregory Dake, who helped me achieve a more complete understanding of organic chemistry. Most importantly, I would like to extend gratitude to my partner Jenn, who supported me through all of my ups and downs and made my home life so much better when things were particularly challenging in other areas of life. Lastly, I would like to express my gratitude to Greg D, for pointing out the wollemi pine specimen on campus. xxvii Dedication To my partner, Jenn1 Chapter 1: Introduction 1.1 Introduction to Natural Product Chemistry Throughout human history, plant and animal products have played a crucial role in society and medicine. The organic chemical compounds derived from these living organisms are generally referred to as primary or secondary metabolites. A primary metabolite is a molecule produced by an organism that is directly related to a fundamental biochemical pathway intrinsic to an organism’s survival, such as the lactic acid pathway. By contrast, the term ‘secondary metabolite’ is used interchangeably with ‘natural product’ and broadly refers to biologically active compounds produced by organisms that are not inextricably linked to an organism’s existence. Furthermore, the term ‘specialized metabolite’ is sometimes used in place of ‘secondary metabolite’. Though it is not fully understood why organisms produce secondary metabolites, it has been hypothesized that these compounds typically bestow some evolutionary advantage.1 This is believed to be the case for the apparent chemical warfare compound azadirachtin (1.1) from Azadirachta indica, which has potent insecticide properties and has served as a key ingredient in non-synthetic pesticides2,3 (Figure 1.1, photo by Raimunda C. dos Santos). 2 Figure 1.1 Azadirachtin (1.1) from Azadirachta indica However, some secondary metabolites do not present clear biological utility, and it has also been postulated that some these metabolites are simply waste byproducts or carry some vestigial role in the organism’s survival.4,5 An example of this is mescaline (1.2) from Lophophora williamsii (Figure 1.2), which is potent hallucinogen with a rich history of use among North American First Nations and has been used to study human neurochemistry.6 Whatever the case, natural products remain a vitally important source of novel chemistry, and as illustrated by mescaline (1.2), a lack of apparent evolutionary purpose does not preclude meaningful biological activity in humans. 3 Figure 1.2 Mescaline (1.2) from Lophophora williamsii 1.2 Sources of Natural Products & Conservation Natural products can be isolated from a diverse array of microorganisms, plants and animals. However, one thing that should be highlighted is the value of these natural products sources as a resource. Nearly 70% of all known plant and animal species are distributed within 12 countries: Brazil, Ecuador, Columbia, Peru, Mexico, Madagascar, Zaire, China, India, Indonesia and Australia.7 If we accept the notion that a greater degree of biodiversity affords a greater degree of chemodiversity,8 then extreme care should be taken to preserve these resources. In order to place value on these resources, the Convention on Biological Diversity (CBD) in 1992 established rules protecting genetic resources from outside nations, and mandating that the transfer of genetic resources should benefit the nation of origin.9 The Nagoya Protocol of 2014 further established the agency of countries over their natural product resources by introducing language that specifically protected natural products and traditional knowledge.10 As of 2019, 114 nations have ratified the Nagoya Protocol.11 4 With this in mind, an issue of great ecological importance surrounds how we approach and value natural products chemistry. Ecosystems of immense biodiversity should be protected as rich sources of useful medicines and new scientific discoveries. A salient example lies in the Amazon Rainforest, which contains an estimated one third of all known species, including upwards of 40,000 species of plants, and 100,000 species of invertebrates.12,13 This does not count the great microbial biodiversity within the Amazon, estimates for which seem near impossible to quantify.14 Seemingly inconsistent with its immense biodiversity, few drug leads have emerged from the Amazon.15 This is considered the result of scarce studies with respect to the opportunity presented by the abundance of the Amazon, due in part to the difficulty of accessing the remote geography of the Amazon,16 but also the strict governmental regulations surrounding foreign access of these natural resources.17 Despite this apparent value, habitat loss in the Amazon and within tropical forests across the globe continues at untenable rates,18,19 and it has been hypothesized that extinction rates within these ecosystems are comparable to those observed during prehistoric mass extinction events.20 Biodiverse ecosystems such as the Amazon Rainforest clearly have potential as medicinal treasure troves, and serve as salient examples of the importance of biological conservation within the context of natural products chemistry.21,22 By more closely examining the genetic resources of such abundant ecosystems, a greater perception of value could be placed on the organisms within, and one beyond their utility as physical resources such as lumber. If such value is associated with these organisms in terms of their potential role in drug development, it is possible that conservation could be incentivized through economic means resulting in legislative intervention. As evidenced by efforts to interface with indigenous cultures and government while 5 investigating natural products sources in countries such as Madagascar and Suriname, commodifying ecosystems as abundant sources of drugs has the potential to foster an imperative of ecological conservation.23 If executed effectively, the resulting economic benefit could extend to both local communities and environmental conservation while facilitating advancements in both medicine and natural products chemistry. 1.2.1 Marine Natural Products Marine organisms are of particular interest in the context of natural products chemistry, as the harsh conditions of the marine environment are thought to have given rise to an especially diverse array of bioactive chemical defense compounds.24 Studies on the isolation of marine natural products date back as far as 1951, when the Bergman Group at Yale isolated three nucleosides of unknown identity from the Caribbean sponge Cryptothethia crypta and subsequently structurally elucidated the nucleoside spongothymidine (1.3).25 In the following years, the structural elucidations of the other two nucleosides spongouridine (1.4)26 and spongosine (1.5)27 were reported, which in conjunction with spongothymidine led to the eventual development of the first marine derived drugs: the anti-viral vidarabine (1.6) and the chemotherapeutic cytarabine (1.7)28 (Figure 1.3). 6 Figure 1.3 Structures of Spongothymidine (1.3), Spongouridine (1.4) and Spongosine (1.5), and their Chemical Derivatives Vidarabine (1.6) and Cytarabine (1.7) As advances in self-contained underwater breathing apparatus (SCUBA) continued, so did the opportunity to collect marine specimens and by 1975, three main subfields had risen from marine natural products chemistry: marine toxins, biomedicinal chemistry and chemical ecology.29 The study of structurally complex marine toxins such as brevetoxin A from the dinoflagellate Kareni brevis were crucial in the development of modern structural elucidation methods,30,31 but it would take until the isolation of the conotoxins in 1978 by Olivera et al.32 for the first pharmaceutical from the sea to be FDA approved. Two decades later, these marine snail-derived toxins served as the lead for the potent analgesic ziconotide (trade name Prialt®), which was given FDA approval in 2004.33 7 Figure 1.4 Structure of Brevetoxin A (1.8) Currently, marine-derived secondary metabolites are a source of several commercially important drugs and the source of many more in clinical development.34,35 Dolastatin-10 (1.9) isolated from a sea hare is a tubulin polymerization inhibitor with picomolar potency.36 It has been developed into the synthetic analogue monomethyl auristatin E (MMAE; 1.10) by Seattle Genetics37 for use in their antibody-drug conjugate Brentuximab vedotin® (Figure 1.5; common pharmacophores in red). Eribulin (trade name Halaven®; 1.11) is a synthetic derivative of the anti-mitotic agent halichondrin B (1.12) isolated from the marine sponge Halichondra okadai and it has been approved as a chemotherapeutic agent38,39 (Figure 1.5; common pharmacophores shown in red and blue). 8 Figure 1.5 Marine Derived Natural Products Dolastatin-10 (1.9) and Halichondrin B (1.12) and their Synthetically Derived Drug Analogues Eribulin (1.11) and MMAE (1.10) Despite this abundance of novel bioactive compounds, marine natural products are still a somewhat untapped resource. As of 2017, more than 25,000 marine natural products have been discovered, yet only 7 marine derived drugs are currently on the market; however, this number increased from 4 in 2010 to 7 in 2014, with some 262 more in some stage clinical trials.35,40 As such, it seems clear that marine natural products will continue to present an intriguing and invaluable resource for drug development. 9 1.3 Natural Products in Drug Discovery Figure 1.6 Historically Significant Natural Product Drugs The history of pharmaceuticals and natural products chemistry both have roots in the crystallization of morphine (1.13; Figure 1.6) in 1804, which is generally considered the first pure natural product isolated41. Later in the 1870s, crude morphine isolated from the opium poppy Papaver somniferum was treated with acetic anhydride to yield heroin (1.14), ostensibly the first semisynthetic drug. However, there is a rich history of traditional medicines that has served as the compass for many natural product discoveries. Foxglove has documented historical significance tracing back to 10th century Europe and in the 1700s its active metabolite digitoxin (1.15) was found to enhance heart function.42 Quinine (1.16), isolated from Cinchona officianilis, has centuries of documented use for the treatment of malaria and fever and was approved as an anti-malarial drug by the FDA in 2004.43 Pilocarpine (1.17) from Pilocarpus sp. has been used treat glaucoma and dry mouth for over a century.44 Artemisia annua (sweet wormwood) has 10 documented use in traditional Chinese medicine as an antimalarial treatment leading to the isolation of the pure active natural product artemisinin (1.18). The 2015 Nobel prize in medicine was awarded to Tu Yu for the discovery of atemisinin.45 Though natural products and their derivatives represent 49% of small-molecule drugs approved for clinical use from the 1940s until end of 2014,46 they are often not suitable for direct use as drugs. Between 1981 and 2014, 67 or 6% of all FDA approved small molecule-drugs were unmodified natural products as opposed to 482 natural product derivatives or synthetic analogues of natural products.46 In general, bioactive metabolites serve as the inspiration or lead for drug development, and require optimization of pharmacokinetic and bioavailability properties.47 For example, chemical optimization the topoisomerase inhibitor camptothecin (1.19; Figure 1.7) and several analogues lead to the development of the synthetic drugs exatecan (1.20), topotecan (1.21) and irinotecan (1.22).48 11 Figure 1.7 Camptothecin (1.14) and its Synthetic Analogues (1.15-1.17) Notwithstanding the seemingly undeniable significance of natural products discovery in drug development, most pharmaceutical companies have moved away from in-house discovery programs. The uncertainty of these programs producing viable drug leads towards a specific target may be present, but leaving natural products research purely to the realm of academia deprives research programs of industrially backed financial resources and is to the detriment of drug-development in general. Areas where innovation will soon be essential such as the antibiotic pipeline will necessitate a diverse array of approaches to problem solving, and it is crucial that natural products research programs be equipped with sufficient funding and resources to properly approach this subject.49,50 As illustrated by the past, natural products discovery will be an invaluable resource, whether in untying or cutting the Gordian knots of medicinal chemistry. 12 1.4 Techniques in Natural Products Isolation & Elucidation The general process of natural product discovery has taken on many forms. However, ‘bioassay guided fractionation’ is generally considered the classical approach, in which crude extracts of all metabolites from a source organism are screened for in vitro bioactivity against a particular pathogen or disease model. Next, these extracts are subjected to a series of chromatographic steps including size exclusion (LH-20), reverse-phase, and normal phase chromatographies to crudely fractionate the metabolites. At each step, bioactivity screening is performed to determine which fraction contains the bioactive metabolite, and after semi-pure fractions of interest are obtained, the compounds are purified by high-performance liquid chromatography (HPLC) to isolate pure compounds. Upon obtaining pure compounds, their chemical structures are most often elucidated by detailed analyses of mass spectrometric (LR/HRESIMS), 1D-NMR (1H and 13C) and 2D-NMR data. Several 2D NMR experiments are of particular importance. HSQC provides single bond proton-carbon chemical shift correlations, allowing for the identification of which protons are attached to which carbons. TOCSY in conjunction with COSY identifies contiguous spin systems of coupled protons. HSQCTOCSY can further isolate these spin systems by distributing them on the basis on carbon shifts, which are frequently better separated on the 2D spectrum than in the case of aliphatic methylene signals in the proton NMR spectrum. HMBC provides 2 and 3 bond correlations between proton and carbon chemical shifts. trROESY experiments show nuclear Overhauser (nOe) interactions which can be used to assign relative configurations of 13 substituents. Absolute configurations can then be determined by methods such as X-ray crystallography, circular dichroism (CD) and various methods involving chiral auxiliaries. During the process of natural products discovery, the efficiency with which extracts can be screened against disease models or pathogens is of extreme importance with respect to the output of results. As such, high-throughput screening (HTS) methods have become invaluable in obtaining novel bioactive molecules.51 Furthermore, the process of early stage identification of known molecules if of great importance. This process is typically referred to as ‘dereplication’ and if effectively integrated into a natural products discovery program, can maximize efficiency by eliminating unnecessary efforts into the isolation and purification of known molecules.52 Methods including genome mining, computational chemistry, and screening against mass spectrometric databases have been implemented in dereplication efforts.53-55 Later stage dereplication consists of searching solved structures, or even molecular formulas against databases such as Scifinder® to determine if a structure of interest is novel, or if a structure that matches unsolved data can be observed. With sufficient experience, recognition of semi-pure compounds via their NMR spectra is in itself a tenable method of dereplication. Furthermore, an interesting NMR spectrum, whether or not from a bioactive compound, can be worth investigating. Identification of already known molecules is nevertheless important, as they can be used for high throughput screening against pathogens and disease models not screened in the initial study. Additionally, these known compounds can still be sources of chemically interesting scaffolds and can aid in the characterization of biosynthetic pathways within the organism of provenance. 14 1.5 Classes of Natural Products and Biosynthetic Origins Natural products are generally categorized as a function of their biosynthetic origin. For example, polyketides such as the microtubule stabilizing agent (+)-discodermolide (1.23; Figure 1.8) are produced from protein anchored acetyl groups via an iterative series of enzymatically mediated aldol-type reactions to yield their core scaffolds.56,57 This class encompasses macrolides, which are macrocyclic lactone polyketides and have been the source of many important antibiotic compounds such as tacrolimus (1.24; Figure 1.8).58 Figure 1.8 Structures of the Polyketides Discodermolide (1.23) and Tacrolimus (1.24) Peptides are produced by enzymatically mediated peptide linkage and can be synthesized both ribosomally, as is the case for the potent RNA polymerize inhibitor α-aminitin (1.25; Figure 1.9),59 or non-ribosomally as in the case of the bis-intercalater echinomycin (1.26; Figure 1.9).60 15 Figure 1.9 Structure and Biogenetic Origins of the Ribosomal Peptide a-Aminitin and Structure of Echinomycin Alkaloids such as the oxytocic lysergic acid (1.27) are typically produced from building blocks including nicotinic acid, amino acids, and anthranilic acid (Figure 1.10).61 However, the alkaloid class is not based on biosynthetic origins, but rather on the presence of a basic nitrogen moiety. This classification is somewhat ambiguous and is based on the traditional extraction process for alkaloids. Historically, this process involves an acidification step to protonate these basic nitrogens in crude extracts, allowing for aqueous extraction, subsequent basification and organic extraction to isolate the alkaloidal components of crude natural product extracts. Some biosynthetic transformations are observed across all classes of natural products. For example, s-adenosylmethionine (SAM) is a cosubstrate involved in the methylation of various 16 natural products.62 Additionally, reductions and oxidations involving a variety of enzymes and cosubstrates are frequently observed. Figure 1.10 Biosynthetic Origins of the Ergot Alkaloid Lysergic Acid 17 1.5.1 Terpenoids Figure 1.11 Biosynthesis of Terpenes Terpenes are a class of hydrocarbon natural products derived from C5 isoprene units formed through either the mevalonic acid (MVA) or methylerithritol (MEP) pathways (Figure 1.11). As they are based on C5 units, terpenes are named based on the number of carbons contained in their skeleton and broadly classified as monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20; Figure 1.12), sesterterpenes (C25), triterpenes (C30) and tetraterpenes (C40). 18 While the unmodified skeletons in this class are referred to as terpenes, their chemically modified derivatives are commonly referred to as terpenoids. Additionally, terpenes bearing chemical formulas representing losses of methyl groups such as podocarpane (1.33) are referred to as norterpenes. . Figure 1.12 Several Common Diterpene Skeletons The biosynthesis of terpenoids involves the head-to-tail linking of isoprene units to establish hydrocarbon backbones, followed by a series of cyclizations and cationic rearrangements to produce the diverse array of chemical scaffolds that are characteristic to this class. The biosynthesis of (-)-abietic acid (1.34) demonstrates several canonical transformations observed in terpenoid biosynthesis (Figure 1.13). Upon forming the characteristic skeletons, final chemical modifications such as oxidations and methyl transfer steps involving SAM can occur to produce further structural diversity. With over 50 000 terpenoids isolated to date,63 there are more of these compounds than any other class of natural product.64 19 Figure 1.13 Biosynthesis of the Diterpenoid Abiatic Acid 1.5.2 Steroids Though generally considered a subclass of terpenoids, steroids are frequently referred to as a separate class of natural products and are based on the tetracyclic core of the triterpenoid lanosterol (1.35). However, they lack the methyl groups found at C-4 and C-14 within lanosterol. Steroids are frequently biologically active and have found use in medicine as anti-inflammatories and immunomodulators.65 20 Figure 1.14 Biosynthetic Origins of Lanosterol and 24-Ethyl Sterols Steroid biosynthesis is well illustrated by the production of the 24-ethyl sterols (Figure 1.14), beginning with the oxidation and subsequent cyclization of squalene. Following this 21 cyclization, a variety of cationic arrangements can give rise to the differences in configurations between steroids. Next, demethylations at C-4 and C-14 can occur to give rise to the characteristic steroid skeleton. Finally, reductive and oxidative steps, as well as further cationic rearrangements and demethylations give rise to the structural diversity and canonical chemical scaffolds and sidechains found in steroids (Figure 1.15). Figure 1.15 Steroid Numbering Scheme and Several Common Skeletons 22 1.6 Scope of Thesis The goal of my research was to isolate and structurally elucidate novel natural products by examining natural product extracts from the prehistoric coniferous tree Wollemia nobilis and the sponge Petrosia contignata. Though bioactive compounds were not discovered, several novel compounds were isolated and the identification of known secondary metabolites new to their source organisms provides pertinent chemotaxonomic and biogenetic insight. Chapter 2 will describe the isolation and elucidation of metabolites produced by Wollemia nobilis, including the isolation of two new endoperoxide diterpenoids and the structural correction of an erroneously assigned labdane diterpenoid. Chapter 3 will detail investigations into the metabolites produced by Petrosia contiganata, as well as metabolites previously isolated from the Petrosia genus. Purification and structural elucidation protocols will be described for each all compounds isolated and biogenetic relationships between compounds will be proposed where relevant. 23 Chapter 2: Terpenoids and Other Compounds from Wollemia nobilis 2.1 Introduction to Wollemia nobilis Wollemia nobilis (Figure 2.1) is an Australian coniferous tree belonging to the family Araucariaceae.66 It is the only known member of its genus and is considered a living fossil, as its pollen specimens are near identical to Jurassic era specimens collected in New South Wales.67 These pollen fossils were not found to belong to any extant species, and as such they were initially assigned to a new species: Dilwynites granulatus. However, in 1994 living specimens of Wollemia nobilis were discovered in a remote temperate rainforest in Wollemi National Park, New South Wales and renamed after its discoverer David Noble.68 As fewer than 100 individuals were discovered in the wild, the species is considered critically endangered.66 However, cultivation efforts have led to its sale in suitable climates around the world, including British Columbia, and recovery programs are in place to preserve the species. Figure 2.1 Wollemia nobilis Specimen Planted at UBC 24 2.2 Previously Isolated Secondary Metabolites from Wollemia nobilis Previous studies on the chemical constituents of the wollemi pine have been limited, partly due to the limited availability of specimens. However, 6 chemical isolation studies have been performed as of 2019, and various natural products have been reported (Figure 2.2), such as several common diterpenoids including isocupressic acid (2.1), acetyl-isocupressic acid (2.2), agathic acid (2.3) and sandaracopimaric acid (2.4).69 Caffeic acid (2.5), shikimic acid (2.6) and pheophorbide A (2.7), as well as diflavenones such as 4ʹ-O-methyl-scutellarein (2.8) have also been reported.70,71 Furthermore, three novel compounds have been reported from W. nobilis: a bicyclic monoterpene 6ʹ-O-acetyl-pina-2-ene-4,10-diol-10-O-D-glucopyranoside (2.9),72 and the modified labdane wollemol (2.10),70 and most recently, a labdane derivative wollemolide (2.11).73 25 Figure 2.2 Previously Reported Compounds from Wollemia nobilis All previous phytochemical investigations of W. nobilis used lower field NMR instruments than those available at UBC. As such, it was hypothesized that studies using higher field NMR instruments with greater sensitivity and resolution, as well as more advanced HPLC systems could reveal new chemistry and provide higher quality data for previously isolated compounds. 26 2.3 Terpenoids from Wollemia nobilis Figure 2.3 Wollemia nobilis Material: Before and After Extraction with MeOH The novel labdane diterpenoid 2.12 (Figure 2.4) was isolated from methanolic extracts of Wollemia nobilis along with two new endoperoxide abietane diterpenoids (2.14 and 2.15), four known terpenoids new to the Wollemia genus (2.13 & 2.16-2.18), and a compound previously reported from Wollemia nobilis (2.4). Along with the new structures discovered, elucidation of the metabolites produced by W. nobilis provides both biogenetic and chemotaxonomic insight on a sparsely studied prehistoric species. Figure 2.4 Terpenoids Isolated from Wollemia nobilis in this Study 27 2.3.1 Isolation and Structural Elucidation of Compound 2.12 and Agathic Acid Methyl Ester (2.13) Figure 2.5 Structure of Compound 2.12 and Agathic Acid Methyl Ester (2.13) Compound 2.12 was obtained as a colorless oil via reverse-phase HPLC purification. HRESIMS analysis showed a [M + Na]+ ion at m/z 369.2046 (calculated for C21H30NaO4, 369.2042), requiring 7 degrees of unsaturation. Analysis of the HRESIMS, 1D and 2D NMR data (Table 2.1; Figures 2.6 & 2.7; Appendix A) indicated that 2.12 was a diterpenoid, and a comparison of this data to that reported for previously isolated compounds from W. nobilis suggested it to be a labdane terpenoid, bearing a resemblance to agathic acid (2.3).69 Analysis of the 13C and gradHSQC NMR spectra revealed 13C resonances at δ 50.6 (OCH3), 72.3 (OCH2), 108.9 (CH), 114.8 (CH2), 146.3 (C), 160.1 (C), 166.2 (C) and 175.2 (C), indicating the presence of a methoxy group, an oxymethylene, 2 olefins, and 2 carboxylic acid derivatives, respectively, as well as 2 methyls, 7 methylenes and 2 methines (Figures 2.8 & 2.9). Furthermore, as the δ 166.2 resonance was more deshielded than a typical carboxylic acid derivative, this group was suspected to be conjugated to one of the olefins. 28 Table Error! Style not defined..1 NMR Data for 2.12 and Agathic Acid Methyl Ester (2.13) Recorded at 600 MHz in DMSO-d6 No. 2.12 2.13 δC δH (J in Hz) δC δH (J in Hz) 1eq ax 36.7 2.22 1.26 39.0 1.98 0.78 2eq ax 20.9 1.72 1.46 20.2 1.99 1.30 3eq ax 39.3 1.64 1.53 39.3 1.71 0.95 4 43.0 ¾ 43.6 ¾ 5 47.9 1.60 (dd, 13.0, 3.8) 56.1 1.08 6ax eq 26.9 1.96 0.97 (dq, 13.0, 4.4) 26.2 1.99 1.84 7eq ax 36.1 2.40 1.97 38.5 2.30 1.78 8 146.3 ¾ 148.1 ¾ 9 49.5 1.83 (d, 11.1) 54.7 1.47 10 38.2 ¾ 40.2 ¾ 11 72.3 4.12 (dd, 11.9) 4.18 (dd, 11.9, 2.1) 12.9 0.64 12 21.1 1.79 1.37 20.9 1.64 1.41 13 39.2 2.27 2.06 38.7 2.22 1.92 14 160.1 ¾ 160.6 ¾ 15 18.4 2.13 (d, 0.8) 18.2 2.08 16 114.8 5.64 (d, 0.8) 114.0 5.61 17 166.2 ¾ 166.0 ¾ 18 108.9 5.05 (s) 4.64 (s) 104.9 4.78 4.41 19 23.2 1.05 30.1 0.96 20 175.2 ¾ 178.1 ¾ OMe 50.6 3.59 (s) 50.4 3.58 *See Appendix A for full NMR data. 29 Figure 2.6 1H NMR Spectrum of Compound 2.12 Recorded at 600 MHz in DMSO-d6 Figure 2.7 13C NMR Spectrum of Compound 2.12 Recorded at 150 MHz in DMSO-d6 0.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.8f1 (ppm)0.950.950.970.970.991.001.011.021.071.231.231.241.241.251.251.261.261.271.281.281.281.331.341.351.361.361.371.371.381.381.391.461.471.471.481.491.501.501.511.511.531.541.551.561.581.591.601.611.641.651.651.661.671.671.711.711.721.721.731.731.741.741.741.761.761.761.771.771.781.781.791.801.821.841.931.941.951.951.961.971.981.992.002.002.012.112.202.212.212.222.222.232.242.242.252.262.272.282.282.392.392.392.402.402.412.422.433.604.114.134.174.174.194.194.655.035.65102030405060708090100110120130140150160170180f1 (ppm)18.4320.8621.0623.1626.8836.1436.7238.2039.10 DMSO39.24 DMSO39.37 DMSO39.51 DMSO39.65 DMSO39.79 DMSO39.9139.93 DMSO40.0543.0247.8849.4650.6472.28108.89114.81146.34160.12166.23175.1930 Figure 2.8 gradHSQC for Compound 2.12 Collected in DMSO-d6 at 600 MHz with Relevant Annotations Figure 2.9 Expansion of gradHSQC for Compound 2.12 Collected in DMSO-d6 at 600 MHz Showing Relevant Annotations 31 Analysis of the HSQCTOCSY data allowed for the identification of three contiguous spin systems: H-1/H-1ʹ/H-2/H-2ʹ/H-3/H-3ʹ, H-5/H-6/H-6ʹ/H-7/H-7ʹ and H-12/H-12ʹ/H-13/H-13ʹ (Figures 2.10 & 2.11). Furthermore, the most notable different from agathic acid (2.3) was the presence of the deshielded oxymethylene 1H resonances at δ 4.12 and 4.18 (H-11/H-11ʹ), as well as HMBC correlations from this group to C-1, C-5, C-9, C-10 and C-20 (Figures 2.12 & 2.13). Accounting for this and the remaining degree of unsaturation, this group (C-11) was determined to be a part of a lactone moiety within the structure of 2.12. The connectivity of the remaining fragments was facilitated by HMBC correlations observed from Me-15 to C-13, C-14 and C-16, and Me-19 to C-3, C-4, C-5 and C-20, as well as correlations from the methoxy group to C-17 and from the olefinic protons H-18/H-18ʹ to C-7 and C-9 (Figures 2.12 & 2.13). Figure 2.10 Selected HMBC and TOCSY/COSY Correlations for Compound 2.12 32 Figure 2.11 Expansion of gradHSQCTOCSY for Compound 2.12 Collected in DMSO-d6 at 600 MHz Showing Color Coded Contiguous Spin Systems Figure 2.12 Expansion 1 of gradHMBC NMR Spectrum of Compound 2.12 Collected in DMSO-d6 at 600 MHz with Relevant Annotations 0.80.91.01.11.21.31.41.51.61.71.81.92.02.12.22.32.42.52.6f2 (ppm)36373839404142434445464748495051f1 (ppm)H -7 H -7 ' H -6 H -5 H -6 'H -1 3 H -1 3 ' H -9H -1 2 'H -1 H -2 H -3 H -3 ' H -2 ' H -1 'H -1 2C-9C-5C-1H-1H-7H-13H-7'H-9H-13' H-6H-12H-2 H-3 H-5 H-3'/H-2' H-12' H-1' H-6'33 Figure 2.13 Expansion 2 of gradHMBC NMR Spectrum of Compound 2.12 Collected in DMSO-d6 at 600 MHz with Relevant Annotations trROESY experiments provided relative configuration of 2.12 (Figures 2.14-2.16). Correlations from the olefinic proton H-16 to H-13/H-13ʹ coupled with the absence of a correlation to Me-15 provided the E configuration of the C-14/C-16 alkene (Figures 2.15 & 2.16). Furthermore, correlations from H-11/H-11ʹ to H-12/H-12ʹ indicated the lactone moiety and C-9 substituent to be of the same orientation. Moreover, a system of correlations including H-1ax/H-5, H-1ax/H-9, and H-5/H-9 confirmed these substituents to be opposite the orientation of the lactone and C-9 substituent and established a trans configuration for the ring junction (Figure 2.13). Additionally, Me-19 was suspected to be of an equatorial configuration based on the tricyclic ring structure of 2.12, and this hypothesis was confirmed by trROESY correlations of Me-19/H-6ax and Me-19/H-5 (Figure 2.13). The absolute configuration of 2.12 was determined by electronic circular dichroism (ECD) calculations and subsequent comparison to experimental 34 ECD spectra (Appendix B). Lastly, to ensure methylation of the ester moiety was not an artifact of using methanol as the extraction solvent, the extraction and isolation were in the absence of methanol and the methyl ester was still observed, thus confirming it to be the product of a biosynthetic transformation. No previous reports of 2.12 exist in the literature and these results represent the structural elucidation of a novel natural product. Figure 2.14 Selected trROESY Correlations for Compound 2.12 35 Figure 2.15 Expansion 1 of trROESY NMR Spectrum for Compound 2.12 Collected in DMSO-d6 at 600 MHz with Relevant Annotations Figure 2.16 Expansion 2 of trROESY NMR Spectrum for Compound 2.12 Collected in DMSO-d6 at 600 MHz with Relevant Annotations 36 Agathic acid methyl ester (2.13) was obtained as a colorless oil via reverse-phase HPLC. HRESIMS analysis afforded a [M + H]+ ion at m/z 349.2307 (calculated for C21H33O4, 349.2381), requiring 6 degrees of unsaturation. Analysis of the HRESIMS, 1D and 2D NMR data collected for 2.13 (Table 2.1; Appendix A) indicated it to be a diterpenoid, with significant structural similarities to 2.12, as well as and only superficial differences from the previously isolated agathic acid (2.3).69 In fact, the only apparent difference from agathic acid was the presence of a methoxy group showing an HMBC correlation to C-17, thus suggesting 2.13 to be the methyl ester of agathic acid. An analysis of 1D and 2D NMR data was consistent with this hypothesis (Figure 2.17), and this structural assignment was further supported by the observation that 2.13 is ostensibly a non-lactonized analogue of 2.12. Figure 2.17 Selected HMBC & COSY/TOCSY Correlations for Agathic Acid Methyl Ester (2.13) The relative configuration of 2.13 was assumed to be analogous to that determined for 2.12 and confirmed by trROESY analysis. Correlations were observed from Me-11 to H-2ax, H-37 6ax, and H-12, as were the system of correlations including H-1ax, H-3ax, H-5, H-7ax and H-9 (Figure 2.18). Furthermore, a trROESY correlation from H-16 to H-15 was observed, indicating the same E configuration for the C-14/C-16 olefin as was observed for 2.12. The absolute configuration of 2.13 was assumed based on its apparent biogenetic relationship to 2.12. Though the occurrence of agathic acid methyl ester (2.13) has previously been reported,74 these results represent its first isolation from Wollemia nobilis. Figure 2.18 Selected trROESY Correlations for Agathic Acid Methyl Ester (2.13) 2.3.1.1 Structural Correction of Wollemolide (2.11) During the writing of this dissertation, the structure of a labdane terpenoid wollemolide (2.11; Figure 2.19) bearing the same molecular formula and demonstrating numerous structural similarities to 2.12 was reported as isolated from Wollemia nobilis by Venditti et al.73 However, based on the data collected and several errors and shortcomings in the authors structural assignment, it appears that this compound was an erroneously assigned sample of 2.12. 38 First, a NMR data set for compound 2.12 collected in CDCl3 (Appendix C), the same solvent used in this recent study of 2.11 reported by Venditti et al.73 revealed identical spectra and chemical shifts to those provided for 2.11 (Table 2.2; Figures 2.20 & 2.21) confirming that compounds 2.11 and 2.12 were of the same identity. Additionally, a diagnostic HMBC correlation from the bridgehead methyl (Me-19) is observed to the lactone carbon C-20, both in the collected HMBC spectrum (Figure 2.22) as well as that reported in the literature,73 which would not be observed based on the authors proposed structure (Figure 2.19). Furthermore, several of the observed trROESY correlations would be absent based on structure 2.11, particularly from H-11/H-11ʹ to H-12/H-12ʹ (Figure 2.23). Moreover, a thin film IR spectrum collected on 2.12 was missing a characteristic beta-lactone absorption at 1820 cm-1 +/- 20 cm-1 and instead displayed an absorption at 1725 cm-1 typical of a delta lactone (Appendix D). Thus, it can be concluded that the reported structure of 2.11 was incorrect and the compound reported as wollemolide has the structure 2.12. Lastly, no trROESY or CD data was provided to support the configuration reported by Venditti et al.,73 which was assigned based on a comparison to previously observed chemical shifts for other labdane (1.28) and sclarene (1.32) derivatives, and it is likely that these configurations were also misassigned. Figure 2.19 Comparison of Reported Structure for Wollemolide (2.11) & Compound 2.12 39 Table Error! Style not defined..2 Comparison of Experimental and Literature NMR Data for Wollemolide (2.11) & Compound 2.12 Recorded at 600 MHz in CDCl3 No. Experimental Literature73 δC δH (J in Hz) δC δH (J in Hz)a 1 40.0 1.88 1.53 40.0 1.88-1.84 2 27.3 2.02 1.18 27.3 2.01-1.96 3 38.0 2.21 1.28 38.0 2.19-2.14 4 43.8 ¾ 44.3 ¾ 5 49.5 1.50 49.5 1.54-1.48 6 21.3 1.84 1.64 21.3 1.80-1.76 7 36.9 2.46 (dd, 13.0, 2.5) 1.97 36.9 2.46 (dd, 12.9, 2.6) 2.01-1.96 8 146.0 ¾ 146.0 ¾ 9 50.3 1.83 50.3 1.82-1.79 10 38.9 ¾ 38.9 ¾ 11 73.1 4.38 (dd, 12.1, 2.3) 4.02 (d, 12.1) 73.1 4.37 (dd, 12.0, 2.2) 4.01 (d, 12.0) 12 21.6 1.80 1.40 21.6 1.69-1.68 13 39.8 2.33 2.06 39.8 2.01-1.96 1.88-1.84 14 159.7 ¾ 158.7 ¾ 15 19.0 2.16 (d, 0.9) 18.9 2.15 (d, 0.9) 16 115.7 5.64 (d, 0.9) 115.7 5.63 (d, 0.9) 17 167.2 ¾ 167.2 ¾ 18 109.5 5.04 (s) 4.64 (s) 109.4 5.04 (s) 4.64 (s) 19 23.5 1.19 (s) 23.5 1.18 (s) 20 176.4 ¾ 176.4 ¾ OMe 51.1 3.69 (s) 51.0 3.69 (s) a). The 1H resonances in the aliphatic region reported by Venditti et al.73 are poorly resolved from one another and their assignments are questionable. b). See Appendix C for full NMR data. 40 Figure 2.20 1H NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 600 MHz with Relevant Annotations Figure 2.21 13C NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 150 MHz with Relevant Annotations 41 Figure 2.22 Expansion of gradHMBC NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 600 MHz Showing Color Coded Contiguous Spin Systems Figure 2.23 Expansion of trROESY NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 600 MHz with Relevant Annotations 42 2.3.2 Isolation and Structural Elucidation of 15-Hydroperoxy-8β,12β-epidioxy-13-abieten-19-oic acid (2.14) and 15-Hydroxy-8β,12β-epidioxy-13-abieten-19-oic acid (2.15) Figure 2.24 Structures of 15-Hydroperoxy-8β,12β-epidioxy-13-abieten-19-oic acid (2.14) and 15-Hydroxy-8b,12b-epidioxy-13-abieten-19-oic acid (2.15) 15-hydroperoxy-8β,12β-epidioxy-13-abieten-19-oic acid (2.14) was obtained as a colorless oil via reverse-phase HPLC. HRESIMS analysis afforded a [M + Na]+ ion at m/z 389.1941 (calculated for C20H30NaO6, 389.1940) requiring 6 degrees of unsaturation. Analysis of the HRESIMS, 1D and 2D NMR data collected for 2.14 (Table 2.3; Figures 2.25 & 2.26; Appendix D) indicated it to be a diterpenoid with structural similarities to abietic acid (1.34). Analysis of the 13C and gradHSQC NMR spectra revealed resonances at δ 80.3 (C), 71.3 (CH), 76.0 (C), 127.3 (CH), 146.6 (C) and 179.9 (C) indicating the presence of three oxygenated carbons, an olefin, and a carboxylic acid derivative, respectively, as well as 2 quaternary carbons, 4 methyl groups, 6 methylene groups and 2 methines (Figures 2.27 & 2.28). Furthermore, a sharp 1H resonance bearing no HSQC correlation was observed at δ 11.1, which along with the highly deshielded 13C resonance at δ 80.3 indicated the presence of a hydroperoxy group. 43 Table Error! Style not defined..3 NMR Data for 2.14 and 2.15 Recorded at 600 MHz in DMSO-d6 No. 2.14 2.15 δC δH (J in Hz) δC δH (J in Hz) 1eq ax 37.2 1.24 0.87 36.4 1.29 0.94 2ax eq 17.4 1.38 1.28 16.6 1.48 1.40 3ax eq 37.3 1.65 1.24 36.3 1.64 1.45 4 46.1 ¾ 45.8 ¾ 5 49.3 1.84 (dd, 11.8, 2.4) 48.8 1.75 6eq ax 20.9 1.42 1.31 20.9 1.51 1.30 7eq ax 32.0 1.68 1.20 31.9 1.76 1.23 8 76.2 ¾ 76.0 ¾ 9 50.2 1.69 50.1 1.73 10 35.7 ¾ 35.6 ¾ 11eq ax 24.2 2.03 1.27 24.7 2.06 1.08 12 71.3 4.79 71.6 4.86 13 146.6 ¾ 149.5 ¾ 14 127.3 6.07 (d, 4.1) 123.5 5.98 (d, 4.1) 15 80.3 ¾ 68.9 ¾ 16 22.2 1.35 (s) 28.3 1.22 (s) 17 24.2 1.21 (s) 27.9 1.24 (s) 18 18.0 0.91 (s) 16.0 1.02 (s) 19 179.9 ¾ 178.8 ¾ 20 14.5 0.48 (s) 14.7 0.50 (s) OH/OOH ¾ 11.1 (s) ¾ 4.77 (br s) a). C-17 and C-18 are interchangeable. b). See Appendix E for full NMR data. 44 Figure 2.25 1H NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz Figure 2.26 13C NMR Spectrum of 2.14 Recorded in DMSO-d6 at 150 MHz45 Figure 2.27 gradHSQC NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz Figure 2.28 Expansion of gradHSQC NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations 46 Analysis of the HSQCTOCSY data allowed for the identification of three contiguous spin systems: H-1/H-1ʹ/H-2/H-2ʹ/H-3/H-3ʹ, H-5/H-6/H-6ʹ/H-7/H-7ʹ and H-9/H-11/H-11ʹ/H-12 (Figures 2.29 & 2.30). The connectivity of these fragments and assignment to the abietane skeleton was facilitated by HMBC correlations observed from Me-20 to C-1, C-5, C-9 and C-10 and Me-18 to C-3, C-4, C-5 and C-19, as well as correlations from Me-16/17 to C-13 and C-15 (Figures 2.31 & 2.32). Furthermore, HMBC correlations from the olefinic proton H-14 to C-8, C-9, C-12, C-13 and C-15 confirmed the assignments in the C ring (Figure 2.33). As the alcohol, carboxylic acid, tricyclic abietane skeleton and olefin account for 5 of the 6 degrees of saturation and 3 of the 5 oxygens within the molecular formula of 2.14, the presence of an endoperoxide moiety was suspected. Based on the remaining oxygenated 13C resonances at δ 71.3 and 76.2, the endoperoxide in 2.14 was determined to be between the oxygens at C-8 and C-12. Figure 2.29 Selected HMBC & COSY/TOCSY Correlations for 2.14 47 Figure 2.30 Expansion of gradHSQCTOCSY NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz Showing Color Coded Contiguous Spin Systems Figure 2.31 Expansion 1 of gradHMBC NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations 1.01.52.02.53.03.54.04.5f2 (ppm)363738394041424344454647484950f1 (ppm)H -1H -3H -3 'H -2 'H -2H -1 'C-1C-3H -7 H -7 'H -5 H -6H -6 'H -1 1H -1 2 H -1 1 'H -9C-9C-5H-12 H-11 H-5H-3H-7H-9H-2H-6H-6' H-1'H-1H-3H-2'' H-7'48 Figure 2.32 Expansion 2 of gradHMBC NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations Figure 2.33 Expansion 3 of gradHMBC NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations 49 The relative configuration of 2.14 was determined via trROESY analysis. Sets of correlations between H-2ax, H-6ax, Me-20 and Me-18, as well as H-1ax, H-5, and H-9 allowed for assignment of the relative configurations shown in the A and B rings (Figures 2.34 & 2.35). The configuration of the endoperoxide was determined through the observation of correlations from the olefinic proton H-14 to both H-7ax and H-7eq (Figure 2.36). Finally, in order to confirm the identity of the H-14 correlations involving overlapping signals, NMR data was obtained in CDCl3 (Table 2.4; Figure 2.37; Appendix D) and the same H-14/H-7ax and H-14/H-7eq signals were observed. The absolute configuration of 2.14 was assumed based on its proposed biogenetic relationship to compound 2.12, but not unambiguously determined. Figure 2.34 Selected trROESY Correlations for 2.14 50 Figure 2.35 Expansion 1 of trROESY NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations Figure 2.36 Expansion 2 of trROESY NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations 51 Figure 2.37 Expansion 1 of trROESY NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz with Relevant Annotations 15-hydroxy-8β,12β-epidioxy-13-abieten-19-oic acid (2.15) was obtained as a colorless oil via reverse-phase HPLC. HRESIMS analysis afforded a [M + Na]+ ion at m/z 373.2001 (calculated for C20H30NaO5, 373.1991) requiring 6 degrees of unsaturation. Analysis of the HRESIMS, 1D and 2D NMR data collected for 2.15 (Table 2.3; Figures 2.38 & 2.39; Appendix D) indicated it to be a diterpenoid with only minor differences from 2.14 in the aliphatic region, suggesting a similar oxidized abietane skeleton. Indeed, as with 2.14 13C resonances were observed at δ 68.9 (C), 71.6 (CH), 76.0 (C), 123.5 (CH), 149.5 (C) and 178.8 (C) indicating the presence of three oxygenated carbons, an olefin, and a carboxylic acid derivative, respectively, as well as the 2 quaternary carbons, 4 methyl groups, 6 methylene 52 groups and 2 methines (Figures 2.40 & 2.41). However, a key difference was observed in the absence of the sharp downfield 1H singlet at δ 11.1 observed for 2.14 and corresponding upfield 13C shift for C-16 to δ 68.9 (vs δ 80.3 for 2.14), as well as the presence of a new broad 1H singlet at δ 4.77 showing no HSQC correlation. Accounting for the one less oxygen present in the molecular formula of 2.15 as compared to 2.14, as well as the comparatively deshielded 13C resonances for Me-16/17, 2.15 was strongly suspected to be the 15-hydroxy analogue of 2.14. Figure 2.38 1H NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz 0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.5f1 (ppm)0.510.850.860.860.910.910.930.940.950.960.980.991.021.051.061.071.081.091.101.121.131.141.161.171.181.201.211.231.241.281.281.291.311.371.381.391.401.401.451.471.491.501.511.521.551.561.621.621.641.651.661.671.721.731.741.751.771.941.951.982.032.042.052.052.062.072.074.774.864.865.9912.2353 Figure 2.39 13C NMR Spectrum of 2.15 Recorded in DMSO-d6 at 150 MHz Figure 2.40 gradHSQC NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations 54 Figure 2.41 Expansion of gradHSQC NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations Analysis of the HSQCTOCSY for 2.15 revealed three contiguous spin systems: H-1/H-1ʹ/H-2/H-2ʹ/H-3/H-3ʹ, H-5/H-6/H-6ʹ/H-7/H-7ʹ and H-9/H-11/H-11ʹ/H-12 (Figures 2.42 & 2.43). As with 2.14, the connectivity of these fragments was determined through the observation of HMBC correlations from Me-20 to C-1, C-5, C-9 and C-10 and Me-18 to C-3, C-4, C-5 and C-19, as well as correlations from Me-16/17 to C-13 and C-15 (Figures 2.43 & 2.44). Furthermore, HMBC correlations were observed from the OH resonance to C-13 and C-15, indicating the OH group to be bound to C-15 (Figure 2.45). Finally, HMBC correlations from the olefinic proton H-14 to C-8, C-9, C-12, C-13 and C-15 confirmed the assigned connectivity in the C ring (Figure 2.46). As the alcohol, carboxylic acid, tricyclic abietane skeleton and olefin accounted for 5 of the 6 degrees of saturation and 3 of the 5 oxygens within the molecular formula of 2.15, the 55 presence of an endoperoxide moiety was suspected. Based on the remaining oxygenated 13C resonances at δ 71.6 and 76.0, the endoperoxide in 2.15 was determined to be between the oxygens at C-8 and C-12, thus confirming its hypothesized identity as the 15-hydroxy analogue of 2.14. Figure 2.42 Selected HMBC & COSY/TOCSY Correlations for 2.15 Figure 2.43 Expansion of gradHSQCTOCSY NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz Showing Color Coded Contiguous Spin Systems 1.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.8f2 (ppm)36373839404142434445464748495051f1 (ppm)H -7H -7 'H -6H -5H -6 'H -1 1H -1 2 H -1 1 'H -9H -1H -3H -3 'H -2H -2 'H -1 'C-1C-3C-9C-5H-12 H-11 H-3H-5H-7H-9H-1'H-3'H-2H-2'H-6H-6'H-7'H-1 H-11'56 Figure 2.44 Expansion 1 of gradHMBC NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations Figure 2.45 Expansion 2 of gradHMBC NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations 57 Figure 2.46 Expansion 3 of gradHMBC NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations The relative configuration of 2.15 was suspected to be the same as that determined for 2.14, and confirmed via trROESY analysis. The same sets of correlations between H-2ax, H-6ax, Me-20 and Me-18, as well as between H-1ax, H-5, and H-9 observed for 2.14 were likewise present in the trROESY data for 2.15 (Figures 2.47 & 2.48). Additionally, correlations from the olefinic proton H-14 to both H-7ax and H-7eq were present supporting the assignment of the 8β-12β configuration of the endoperoxide moiety (Figure 2.49). Furthermore, as with 2.14, NMR data was obtained in CDCl3 (Table 2.4; Figure 2.50; Appendix D) and confirmed that trROESY correlations from H-14 involving overlapping signals were correctly interpreted. The absolute configuration of 2.15 was inferred to be the same as that of 2.14 as assumed based on its proposed biogenetic relationship to compound 2.12 but not unambiguously determined. 58 Figure 2.47 Selected trROESY Correlations for 2.15 Figure 2.48 Expansion 1 of trROESY NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations 0.50.60.70.80.91.01.11.21.31.41.51.61.71.81.9f2 (ppm)0.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.92.02.1f1 (ppm)H - '1 /H -5 H -1 '/H -9H -1 '/H -3M e -2 0 /H -2H -1 '/H -1 M e -2 0 /H -6 'M e -2 0 /H -1M e -2 0 /H -1 1 'M e -2 0 /M e -1 8H -2 /M e -1 8H -1 /H -1 'H -5 /H -1 ' H -9 /H -1 'H -6 '/M e -2 0 H -1 1 '/M e -2 0 M e -1 8 /M e -2 0H -1 /M e -2 0H -2 /M e -2 0H-5 H-9 H-6' H-1M-18 Me-20H-11' H-2Me-20M-18H-11' H-1H-1' H-1' H-6'H-2H-9H-559 Figure 2.49 Expansion 2 of trROESY NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz with Relevant Annotations Figure 2.50 Expansion of trROESY NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz with Relevant Annotations 60 The 8β,12β-epidioxy configurations observed for 2.14 and 2.15 represent the first isolation of such compounds from natural product extracts. However, these compounds represent endoperoxide stereoisomers of previously isolated 8α,12α-epidioxy abietane terpenoids (2.14ʹ & 2.15ʹ; Figure 2.52).75,76 Indeed, NMR data for 2.14/2.15 collected in analogous solvent conditions to those reported for 2.14ʹ/2.15ʹ (Table 2.4; Figures 2.53-2.56; Appendix E) showed differences in both 13C and 1H chemical shifts, most notably at H-12 and H-14 (Table 2.3). Furthermore, another notable difference existed in the absence of trROESY correlations of Me-20/H-14, which Martínez-Casares et al.75 cited as the primary evidence for the assignment of the 8α,12α configuration for the endoperoxides in 2.14ʹ and 2.15ʹ (Figure 2.51). Figure 2.51 Diagnostic NOE Cited for the Assignment of 8α,12α-Epidoxy Abietanes 61 Figure 2.52 Structures of 2.14 & 2.15 and their 8α,12α-Epidioxy Analogues 2.14ʹ & 2.15ʹ Table Error! Style not defined..4 NMR Data for 2.14 & 2.15 Recorded at 600 MHz in CDCl3 and Comparison to Literature Data for their 8α,12α-Epidioxy Analogues 2.14ʹ & 2.15ʹ No. 2.14 2.14ʹ 2.15 2.15ʹ δ13C (ppm) δ1H (ppm) δ13C (ppm)75 δ1H (ppm)75 δ13C (ppm) δ1H (ppm) δ13C (ppm)75 δ1H (ppm)75 1eq ax 36.0 1.30 0.92 36.5 1.40 1.03 35.8 1.29 0.92 36.0 1.34 0.99 2ax eq 16.4 1.48 1.40 16.9 1.52 16.0 1.45 1.06 16.9 1.47 3ax eq 36.2 1.69 1.51 36.8 1.76 1.54 36.0 1.66 1.48 36.6 1.71 1.58 4 45.8 ¾ 46.7 ¾ 45.5 ¾ 46.6 ¾ 5 48.3 1.81 48.8 1.90 48.5 1.78 48.8 1.84 6eq ax 20.9 1.47 1.40 21.6 1.55 20.9 1.46 1.37 21.4 1.42 7eq ax 31.4 1.76 1.42 31.9 1.88 1.54 31.0 1.72 1.41 31.8 1.83 1.46 8 76.1 ¾ 77.2 ¾ 76.6 ¾ 76.6 ¾ 9 49.5 1.85 49.8 1.98 49.0 1.80 49.7 1.92 10 35.6 ¾ 36.2 ¾ 35.6 ¾ 36.4 ¾ 11eq ax 21.8 2.11 1.33 24.5 2.27 1.26 24.5 2.11 1.10 24.9 2.22 1.12 12 71.7 4.85 72.0 4.91 71.4 4.82 72.3 4.86 13 146.9 ¾ 146.0 ¾ 148.4 ¾ 149.9 ¾ 14 126.9 5.96 129.2 6.13 123.3 5.91 124.7 6.00 15 80.5 ¾ 81.8 ¾ 69.1 ¾ 70.5 ¾ 16 23.7 1.36 (s) 22.5 1.44 28.1 1.27 (s) 27.9 1.37 17 23.9 1.28 (s) 22.9 1.35 27.5 1.27 (s) 27.7 1.33 18 15.8 1.03 (s) 16.2 1.14 15.5 1.02 (s) 16.1 1.09 19 180.2 ¾ 183.4 1.44 180.0 ¾ 183.4 ¾ 20 14.3 0.51 (s) 15.0 0.56 14.2 0.49 (s) 15.0 0.51 OH/OOH ¾ 10.3 (s) ¾ ¾ ¾ ¾ ¾ * See Appendix E for full NMR data. 62 Figure 2.53 1H NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz Figure 2.54 13C NMR Spectrum of 2.14 Recorded in CDCl3 at 150 MHz 0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.5f1 (ppm)0.510.900.900.910.910.920.930.940.951.031.041.281.281.301.301.311.311.321.331.341.341.351.361.371.381.391.391.401.411.411.421.431.431.441.441.451.461.461.481.481.491.491.501.511.511.521.681.691.711.711.751.751.761.761.771.771.801.801.801.821.821.821.831.841.851.862.092.102.102.112.122.132.134.854.864.865.965.9610.2710.2711.5463 Figure 2.55 1H NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz Figure 2.56 gradHSQC NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz 64 2.3.3 Isolation & Structural Elucidation of Abiesanordine E (2.16) and Kaur-15-en-17-al (2.17) Figure 2.57 Structures of Abiesanordine E (2.16) and Kaur-15-en-17-al (2.17) Abiesanordine E (2.16) was obtained as a colorless oil via reverse-phase HPLC. HRESIMS analysis afforded a [M + H]+ ion at m/z 277.1809 (calculated for C17H25O3, 277.1805), requiring 6 degrees of unsaturation. Analysis of the HRESIMS, 1D and 2D NMR data collected for 2.16 (Table 2.5; Appendix F) indicated it to be a norditerpenoid. 13C resonances extracted from the HSQC and HMBC spectra were observed at δ 197.8 (C), 165.9 (C) and 124.9 (CH), indicating the presence of an a,b-unsaturated ketone. Additionally, a 13C resonance at δ 179.9 (C) was observed, suggesting the presence of a carboxylic acid, thus accounting for all oxygens and 3 of the 6 degrees of unsaturation. Taking this and the 1H NMR data into account, 2.16 was determined to be a tricyclic oxygenated terpenoid with similarities to the podocarpane skeleton (1.33), as well as the abietane terpenoids previously reported in extracts of W. nobilis. 65 Table Error! Style not defined..5 NMR Data for Abiesanordine E (2.16) and Kaur-15-en-17-al (2.17) Recorded at 600 MHz in DMSO-d6 2.16 2.17 δC δH (J in Hz) δC δH (J in Hz) 1 38.0 1.65 1.10 39.2 1.77 0.73 (ddd, 13.0, 3.9) 2 18.1 1.40 0.89 17.5 1.60 1.37 3 36.9 1.65 1.27 40.6 1.36 1.12 4 46.0 ¾ 32.1 ¾ 5 48.1 2.12 54.5 0.83 6 23.3 1.44 1.31 17.8 1.55 1.24 7 36.1 2.41 2.18 37.4 1.76 1.62 8 165.9 ¾ 49.9 ¾ 9 50.8 2.14 45.2 1.08 10 37.5 ¾ 38.9 ¾ 11 14.6 0.73 (s) 17.0 1.03 (s) 12 19.5 1.92 1.62 17.4 1.61 1.39 13 34.7 2.40 2.18 (ddd, 12.3, 2.6) 24.3 1.52 1.37 14 198.8 ¾ 36.6 2.88 15 124.9 5.73 147.3 ¾ 16 18.4 0.98 (s) 161.3 6.80 (s) 17 179.9 ¾ 189.0 9.70 (s) 18 ¾ ¾ 42.0 2.21 (d, 10.7) 1.46 19 ¾ ¾ 20.7 0.79 (s) 20 ¾ ¾ 32.8 0.85 (s) * See Appendix F for full NMR data. HMBC correlations were observed from Me-11 to C-1, C-5, C-9 and C-10 and Me-16 to C-3, C-4, C-5 and C-17, allowing for assignment of these methyls on a podocarpane skeleton (Figure 2.58). Furthermore, analysis of COSY data allowed for the identification of three contiguous spin systems: H-1/H-1ʹ/H-2/H-2ʹ/H-3/H-3ʹ, H-5/H-6/H-6ʹ/H-7/H-7ʹ and H-9/H-12/H-12ʹ/H-13/H-13ʹ. The connectivity of these fragments was facilitated by the methyl HMBC 66 correlations, along with HMBC correlations from the olefinic proton H-16 to C-8/C-9 and from H-12 to C-13 and C-14 (Figure 2.58). Comparison of experimental data to that reported in the literature was consistent with this structural assignment.77 Figure 2.58 Selected HMBC and COSY/TOCSY Correlations for Abiesanordine E (2.16) The relative configuration of 2.16 was assigned on the basis trROESY data (Figure 2.59). Key correlations from Me-11 to H-2ax, H-6ax and Me-16, as well as from H-5 to H-1ax, H-3ax, H-7ax and H-9 allowed for assignment of the relative configurations at the ring junctions as shown (Figure 2.59). The absolute configuration of 2.16 was assumed based on its proposed biogenetic relationship to compound 2.12, but not unambiguously determined and these results represent the first isolation of 2.16 from Wollemia nobilis. 67 Figure 2.59 Selected trROESY Correlations for Abiesanordine E (2.16) Kaur-15-en-17-al (2.17) was obtained as an off yellow oil via reverse-phase HPLC. HREIMS analysis afforded a [M]+ ion at m/z 286.2287 (calculated for C20H30O, 286.2297), requiring 6 degrees of unsaturation. Analysis of the HRESIMS, 1D and 2D NMR data (Table 2.5; Appendix F) indicated that 2.17 was a diterpenoid with structural relationship to the kaurene (1.29) skeleton. Furthermore, the presence of 13C resonances at δ 189.0 (CH), 161.3 (C) and 147.3 (CH) as well as the extremely downfield proton resonance for H-17 at δ 9.70 suggested the presence of an a,b-unsaturated aldehyde, thus accounting for the additional degree of unsaturation as compared to kaurene (1.29). Additionally, 3 contiguous spin systems were observed in the gradHSQCTOCSY and COSY spectra: H-1/H-1ʹ/H-2/H-2ʹ/H-3/H-3ʹ, H-5/H-6/H-6ʹ/H-7/H-7ʹ, and H-9/H-12/H-12ʹ/H-13/H-13ʹ/H-14/H-18/H-18ʹ (Figure 2.60). HMBC correlations observed from Me-11 to C-1, C-5, C-9 and C-10 and Me-19/20 to C-3, C-4, C-5 and C-20/19, as well as correlations from the aldehyde proton H-17 to C-14 and from the olefinic proton to C-7, C-8, C-17, and C-18 (Figure 2.36) established the connectivity of these fragments. As such, 2.17 was determined to be 68 a C-17 oxygenated analogue of kaurene (1.19), and a comparison of the experimental NMR data with previous reports in the literature supported this structural assignment.78 Figure 2.60 Selected HMBC and COSY/TOCSY Correlations for 2.17 The relative configuration of 2.17 was determined on the basis of trROESY data (Figure 2.61). Correlations from Me-11 to H-2ax, H-6ax, H-18 and Me-19, as well as from H-5 to H-1ax, H-3ax, and H-9 allowed for assignment of the relative configurations shown (Figure 2.61). As with the previous terpenoids, the absolute configuration of 2.14 was assumed based on its proposed biogenetic relationship to compound 2.12, but not unambiguously determined and these results represent the first reported isolation of 2.16 from Wollemia nobilis. Figure 2.61 Selected trROESY Correlations for 2.17 69 2.3.4 Isolation and Structural Elucidation of Isocommunic Acid (2.18) and Sandaracopimaric Acid (2.4) Figure 2.62 Structures of Isocommunic Acid (2.18) and Sandaracopimaric Acid (2.4) Isocommunic acid (2.18) was obtained as a colorless oil via reverse-phase HPLC. HRESIMS analysis afforded a [M + H]+ ion at m/z 303.2331 (calculated for C20H31O2, 303.2324), requiring 6 degrees of unsaturation. Analysis of the HRESIMS, 1D and 2D NMR data collected for 2.18 (Table 2.6; Appendix G) indicated it to be a diterpenoid, with significant structural similarities to 2.13, as well as the previously reported labdane terpenoids isolated from W. nobilis. 13C NMR resonances were observed at δ 104.4 (CH2), 113.2 (CH2), 115.4 (CH2), 138.4 (CH), 147.0 (C), 149.3 (C) and 178.9 (C) indicating the presence of three olefins, and a carboxylic acid derivative, respectively. Furthermore, based on the HSQC data for the olefins, it was observed that two geminal olefins and one monosubstituted olefin were present in 2.18. 70 Table Error! Style not defined..6 NMR Data for Isocommunic Acid (2.18) and Sandaracopimaric Acid (2.4) Recorded at 600 MHz in DMSO-d6 No. 2.18 2.4 δC δH (J in Hz) δC δH (J in Hz) 1 39.2 1.66 0.94 38.1 1.65 1.04 2 19.8 2.01 1.24 17.8 1.55 1.45 3 39.3 2.00 0.73 36.8 1.66 1.34 4 43.7 ¾ 46.4 ¾ 5 56.0 1.00 48.6 1.89 6 25.9 2.04 1.83 24.3 1.25 1.23 7 38.7 2.29 1.79 35.2 2.14 1.94 8 149.3 ¾ 137.0 ¾ 9 55.1 1.56 50.0 1.71 10 39.9 ¾ 37.4 ¾ 11 12.7 0.59 (s) 14.7 0.75 (s) 12 21.3 1.63 1.40 18.2 1.42 1.03 13 29.9 2.28 1.94 33.20 1.41 1.31 14 147.0 ¾ 36.7 ¾ 15 115.4 5.01 (s) 4.97 (s) 25.6 1.00 (s) 16 138.4 6.35 (dd, 17.6, 10.8) 148.4 5.76 17 113.2 5.20 (dd, 17.6, 1.2) 5.06 (d, 10.8) 110.3 4.90 4.87 18 104.4 4.79 (s) 4.44 (s) 128.0 5.17 19 0.94 (s) 30.0 179.8 ¾ 20 178.9 ¾ 17.6 1.02 (s) * See Appendix G for full NMR data. Analysis of the COSY and HSQCTOCSY data for 2.18 allowed for the identification of three contiguous spin systems: H-1/H-1ʹ/H-2/H-2ʹ/H-3/H-3ʹ, H-5/H-6/H-6ʹ/H-7/H-7ʹ and H-12/H-12ʹ/H-13/H-13ʹ (Figure 2.63). The connectivity of these fragments was facilitated by HMBC correlations observed from Me-11 to C-1, C-5, C-9 and C-10 and Me-19 to C-3, C4, C-5 71 and C-20, as well as correlations from the olefinic protons H-18/H-18ʹ to C-7, C-8 and C-9, from H-15 to C-14 and C-16, from H-16 to C-13 and C-14 and from H-17/H-17ʹ to C-14 and C-16 (Figure 2.63). This structural assignment was confirmed by comparison of experimental data with that reported in the literature.79 Figure 2.63 Selected HMBC & COSY/TOCSY Correlations for Isocommunic Acid (2.18) Relative configurations for 2.18 were determined via trROESY analysis. Correlations were observed from Me-11 to H-2ax, H-6ax, and H-12, as were the system of correlations including H-1ax, H-3ax, H-5, H-7ax and H-9, indicating an analogous relative configuration to 3.13 (Figure 2.64). The absolute configuration of 2.18 was assumed based on its proposed biogenetic relationship to compound 2.12, but not unambiguously determined. The occurrence of 2.18 has previously been reported,79 but these results represent its first isolation from Wollemia nobilis. 72 Figure 2.64 Selected trROESY Correlations for Isocommunic Acid (2.18) Sandaracopimaric Acid (2.4) was obtained as a colorless oil via reverse-phase HPLC. HRESIMS analysis afforded a [M + H]+ ion at m/z 303.2329 (calculated for C20H31O2, 303.2324), requiring 6 degrees of unsaturation. Analysis of the HRESIMS, 1D and 2D NMR data collected for 2.4 (Table 2.6; Appendix G) indicated it to be a diterpenoid, and upon first inspection appeared to be sandaracopimaric acid (2.4) previously reported from W. nobilis.69 Comparison of experimental data to that reported in the literature were consistent with this hypothesis and 2.4 was determined to be a sample of sandaracopimaric acid. Experimental HMBC, COSY and HSQCTOCSY data were also consistent with this structure assignment (Figure 2.65). 73 Figure 2.65 Selected HMBC & COSY/TOCSY Correlations for Sandaracopimaric Acid (2.4) The relative configuration of 2.4 was determined through trROESY analysis, and key correlations from Me-11 to H-2ax, H-6ax, H-12 and Me-15, as well as from H-5 to H-1ax, H-3ax, and H-12 allowed for assignment of the relative configurations shown (Figure 2.66). The absolute configuration of 2.4 was assumed based on its proposed biogenetic relationship to compound 2.12, but not unambiguously determined. Figure 2.66 Selected trROESY Correlations for Sandaracopimaric Acid (2.4) 74 2.3.5 Chemotaxonomy of Wollemia nobilis Phylogenetically, Wollemia nobilis is a member of the Araucariaceae within the Pinophyta division (Figure 2.67), a family containing 40 species including the common landscaping plant Araucaria araucana or monkey puzzle tree.80 This placement was established by chloroplast genome sequencing and comparison with other members of the Pinophyta through which Wollemia was found to be monophyletic with Agathis and Araucaria, with particular genetic similarity to the Filipino conifer Agathis Damarra.69,81 Figure 2.67 Phylogeny of Wollemia nobilis and its Relatives Within the Pinophyta Division Phytochemical studies on the Pinophyta have yielded several observations on the chemotaxonomic affinities of various terpenoids common to each genus within this division. Common to all Pinophyta is the occurrence of various orders of terpenoids, and in general the Araucariaceae are characterized by the production of bi-, tri, and tetracyclic diterpenoids.82 Specifically, the Araucariaceae are frequently observed to contain a prevalence of kauranes and 75 agathic acid derivatives, named for the genus Agathis within this family, and a study on Cretaceous French ambers found kauranes and agathic acid as the primary biomarkers of the Araucariaceae.83 Conversely, labdanes, pimaranes and abietanes are considered biomarkers of the Pinaceae.83 However, as the Pinaceae are genetically considered a sister genus to all of the Pinophyta,81 the presence of abietane terpenoids across these genera is expected. Consistent with its phylogenetic placement, all of the compounds isolated from Wollemia nobilis in this study have been connected to the Araucariaceae or its related families. Compound 2.12, agathic acid methyl ester (2.13), and isocommunic acid (2.18) are labdane terpenoids and typical biomarkers of the Araucariaceae. Furthermore, sandaracopimaric acid (2.4) is a pimarane which are considered biomarkers of Pinaceae, and kaur-15-en-17-al (2.17) is a kaurene, which are closely linked to the Araucariaceae. Moreover, abiesanordine E (2.16) is a podocarpane which are considered biomarkers and named for the Podocarpaceae, a sister family to Araucariaceae. 2.14 and 2.15 are abietanes, which are considered biomarkers of all Pinophyta. Furthermore, previous isolations of the 8α,12α-epidioxy configurations of 2.14 and 2.15 were reported from Pinus heldreichii,75 a sister genus to all Pinophyta and similar 11β,13β-epidioxy abietane terpenoids were reported from Juniperus phoenicia, a member of Araucariaceae’s sister family Cupressaceae.84 The previously reported compounds isolated from Wollemia nobilis in this study are also consistent with its phylogenetic placement, as previous phytochemical studies on Wollemia nobilis also observed an abundance of labdane, abietane and kaurene diterpenoids. 76 Additionally, a steam volatile oil study of Wollemia nobilis found 16-kaurene to be the primary terpenoid within its essential oil, comprising 60% of these extracts.85 The chemotaxonomic placement of the compounds isolated in this study are consistent with the phylogenetic placement of Wollemia within the Araucariaceae. Furthermore, identification of the abundant metabolites within Wollemia aids in taxonomically linking it to other species both genetically and biochemically in the future. The co-occurrence of specific biomarker compounds across members of Pinophyta also implies a biogenetic relationship between these compounds, as is known to be the case for the abietanes and labdanes. Furthermore, despite a prevalence of one of these classes in a given genera, it should be expected that these compounds would frequently be isolated from genetically related species. Identification of the compounds produced by Wollemia nobilis provides chemotaxonomic insight and further support for its phylogenetic placement. 77 2.3.6 Proposed Biogenetic Relationship of Compounds Isolated from Wollemia nobilis The diterpenoids isolated from Wollemia nobilis in this study appear to demonstrate a clear biogenetic relationship, and a proposed biogenesis for these diterpenoids deriving from a common labdadienyl pyrophosphate intermediate (Figure 1.13) via several divergent pathways is shown in Figure 2.68. One pathway begins with the elimination of the pyrophosphate group to generate sclarene, followed by an oxidation at C-20 to yield isocommunic acid (2.18). Conversely, agathic acid methyl ester (2.13) is formed from labdadienyl pyrophosphate by an oxidative elimination of the pyrophosphate group and SAM mediated methylation of the resulting C-17 carboxylic acid, followed by an isomerization of the olefin and C-20 oxidation. Oxidation of the ring junction methyl on 2.13 and subsequent lactone formation with the carboxylic acid at C-20 then yields compound 2.12. The abietanes and structurally related terpenoids are formed through a separate series of transformations starting from labdadienyl pyrophosphate. First, a sandaracopimarenyl cation is formed by a cyclization and elimination of the pyrophosphate group, and then goes on to yield several compounds. Sandaracopimaric acid (2.4) is produced via an elimination and subsequent oxidation of C-19. Alternatively, intermediate i is produced through a cation rearrangement. Finally, a 1,2-alkyl shift and cation rearrangement of the sandaracopimarenyl cation to yield the kaurenyl cation, followed by an elimination and oxidation yields 2.17. 78 Figure 2.68 Proposed Biogenetic Relationship of Compounds Isolated from Wollemia nobilis 79 The remaining compounds are formed from intermediate i, which is either quenched by water to form intermediate ii or rearranged via a 1,2-methy shift to yield the abietenyl cation. Intermediate ii then yields abiesanordine E via the elimination of CO2 and acetic acid, followed by a subsequent oxidation (Figure 2.69). A precedent for the oxidation and subsequent loss of acetic acid is observed in Baeyer-Villiger monooxygenases.86 Alternatively, the abietenyl cation gives 2.14 by 2 peroxidations, including a [4+2] cycloaddition to yield the endoperoxide, followed by a C-19 oxidation (Figure 2.70), after which 2.15 is formed via a reduction of 2.14. Figure 2.69 Detailed Steps for the Proposed Biogenesis of Abiesanordine E (3.14) from Intermediate ii 80 Figure 2.70 Detailed Steps for the Proposed Biogenesis of 2.14 and 2.15 from Levopimaradiene A general mechanism for endoperoxide and hydroperoxide biosynthesis can be derived from a model hypothesized and later confirmed for the production of the marine norditerpenoid sigmosceptrellin D.87 This mechanism demonstrates the addition of a hydroperoxide to an olefin and subsequent oxidation and has since been applied to the biosynthesis of other endoperoxide metabolites.88,89 Another mechanism of endoperoxide generation is derived from the biosynthesis of ergosterols and involves the cycloaddition of oxygen to 1,3 dienes.90 With this in mind, the formation of 2.14 and 2.15 raises questions related to the biosynthesis of peroxides. Numerous noteworthy endoperoxide metabolites have been reported including the anti-malarial artemisinin and the hormone prostaglandin.91,92 However, the role of enzymes in their biosynthesis has been the subject of some debate, and some doubt remains as to whether or not these reactions are truly enzymatic in nature. 81 1,3 and 1,4-dienes are somewhat reactive to photoperoxidation and autoperoxidation reactions involving these substrates have been reported.93,94 However, accounts of [4+2] cycloaddition reactions involving levopimaradiene derivatives with oxygen cite fairly harsh synthetic conditions including 1.0 M O2, and as such it is unlikely that compounds such as 2.14 and 2.15 could form entirely by autoperoxidation under ambient conditions.94 Additionally, endoperoxides have been isolated from organisms deriving from anoxic environments and persisted even when extractions were done in the absence of air and light.95,96 Furthermore, there are reports endoperoxide containing natural products extracts doped with the 1,3-diene bearing ergosterol showed no ergosterol peroxide, suggesting such compounds to be true metabolites.97 Taking this into account, the artefactual origin of 2.14 and 2.15 endoperoxides is further unlikely due to the isolation of only the 8β,12β-epidioxy analogue rather than a mixture including the 8α,12α-epidioxy analogues. Controversy pertaining to the biogenetic origins of certain chemical moieties is not unique to endoperoxides. Recently, there has been debate regarding the enzymatic origins of [4+2] cycloadditions in secondary metabolism, and the purported ‘Diels-Alderases’ remain elusive.98,99 The isolation of endoperoxides that are ostensibly the result of a biosynthetic transformation lends credence to the enzymatic hypothesis for their biosynthesis. As with Diels-Alderases, the role of endoperoxidases in natural product biosynthesis is still a matter of scientific discourse, but at present it appears that at least some peroxide metabolites including 2.14 and 2.15 are the result of an enzymatic inclusion of oxygen. 82 2.3.7 Biological Data Disc diffusion assays of 2.4 & 2.12-2.18 revealed no apparent biological activity against MRSA, Escherichia coli, Candida albicans, Bacillus subtilis, Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. 2.4 Conclusion Bioassay guided fractionation of methanolic extracts from the prehistoric Australian conifer Wollemia nobilis led to the isolation and structural elucidation of 2.12 and subsequent correction of the previously reported structure for the labdadiene diterpenoid wollemolide (2.11). The same extracts yielded the isolation and structural elucidation of two new 8β,12β-epidioxy abietane diterpenoids (2.14 and 2.15), four terpenoids new to the Wollemia genus (2.13 & 2.16-2.18) and the reisolation of the previously reported sandaracopimaric acid (2.4). Though the crude extracts initially demonstrated activity against MRSA, the pure compounds showed no activity against a series of bacterial pathogens. Identification of the phytochemical constituents of Wollemia nobilis provides both biogenetic and chemotaxonomic insight on a sparsely studied prehistoric species. Furthermore, the isolation of 2.14 and 2.15 provides support for the hypothesized enzymatic origins of endoperoxide biosynthesis. Future work could involve the determination of absolute configurations for the compounds isolated, studies on the alkaloids and more polar chemical constituents of Wollemia nobilis, and further biological screening. 83 Chapter 3: Sterols from Petrosia contignata 3.1 Sea Sponges Sea sponges are filter-feeding aquatic invertebrates belonging to the phylum Porifera. They are soft and sessile organisms lacking physical defense mechanisms such as shells or spines. Consequently, it is necessary for sea sponges to produce chemical defense compounds to facilitate their survival in an environment as hostile as the ocean, and a diverse array of novel bioactive metabolites have been isolated from these organisms. In fact, Porifera are considered the most abundant resource for novel marine secondary metabolites, with over 4851 compounds isolated,100 representing roughly 30% of all marine secondary metabolites isolated as of 2017.101 3.1.1 The Petrosia Genus and Marine Natural Products Investigations of extracts from Petrosia genus sponges have led to the isolation of numerous natural products (Figure 3.1). For example, petrosin A (3.1) is an anti-HIV alkaloid isolated from P. similis.102 Polyoxygenated steroids such as the anti-inflammatory drug lead contignasterol (3.2) and the sulfonlyated weinbersterol A (3.3) were isolated from P. contignata and P. weinbergi, respectively.103,104 Halogenated acyclic ene-yenes such as (5E, 9Z)-6-bromo-heptacosadienoic acid (3.4) have likewise been reported.105 Furthermore, terpenoids such as the meroditerpenoid strongylophorine 26 (3.5) from P. corticata and the mitochondrial respiratory chain inhibitor terretonin E (3.6) from P. ficiformis have also been reported.106,107 84 Figure 3.1 Compounds Previously Isolated from the Petrosia Genus 85 3.2 Steroids from Petrosia contignata Figure 3.2 Petrosia contignata in Methanol The novel steroid petrosiasterol (3.9) was isolated from methanolic extracts of Petrosia contignata, along with four other known steroids, three of which are new to the Petrosia genus (3.7, 3.8, and 3.11; Figure 3.3). Figure 3.3 Steroids Isolated from Petrosia contigata in this Study 86 3.2.1 Isolation and Structural Elucidation of 24-Ethylcholest-7-ene-3b,5a,6b-triol (3.7) and 24-Ethylcholest-7-ene-3b,5a,6b,9a-tetraol (3.8) Figure 3.4 Structures of 24-Ethylcholest-7-ene-3b,5a,6b-triol (3.7) and 24-Ethylcholest-7-ene-3b,5a,6b,9a-tetraol (3.8) 24-Ethylcholest-7-ene-3b,5a,6b-triol (3.7) was obtained as a pale-yellow oil via reverse-phase HPLC. Low-resolution electrospray ionization mass spectrometry (LRESIMS) gave a [M + Na]+ ion at m/z 469.4 and a [M - H]- ion at m/z 445.3, which in conjunction with the observed NMR data was in agreement with a molecular formula for 2.7 as C29H50O3, requiring 5 degrees of unsaturation. Analysis of ESI-MS, 1H and 13C NMR data (Table 3.1; Appendix H) indicated 3.7 to be a steroid. Analysis of the 1H, 13C and gradHSQC NMR spectra revealed the presence of 3 downfield signals corresponding to 3 oxygenated carbons (C-3, C-5 and C-6) and an alkene (C-7 and C-8), as well as 3 hydroxyl groups, 10 methylene groups, 6 methyl groups and 6 methine groups. 87 Table Error! Style not defined..7 NMR Data for 3.7 and 3.8 Recorded in DMSO-d6 at 600 MHz 3.7 3.8 No. δC δH (J in Hz) δC δH (J in Hz) 1 32.5 1.47 1.29 26.8 2.05 (ddd J=13.7, 13.7, 3.8 Hz) 1.14 2 31.2 1.59 1.23 30.9 1.65 1.31 3 66.0 3.75 65.6 3.76 4 39.4 1.88 1.48 40.1 1.91 (dd, 13.5, 11.3) 1.47 5 74.5 ¾ 77.1 ¾ 6 72.7 3.35 71.5 3.50 7 119.5 5.12 120.1 5.13 (dd, 5.3, 2.2) 8 139.7 ¾ 141.6 ¾ 9 42.3 1.93 73.5 ¾ 10 36.6 ¾ 39.4 ¾ 11 22.6 1.51 1.38 27.4 1.68 1.45 12 38.1 1.97 1.22 34.9 1.70 1.39 13 43.1 ¾ 43.2 ¾ 14 54.1 1.78 49.9 2.38 15 22.6 1.50 1.37 27.6 1.86 1.27 16 27.5 1.83 1.24 22.5 1.49 1.31 17 55.4 1.21 55.3 1.26 18 11.9 0.52 (s) 11.3 0.54 (s) 19 17.7 0.89 (s) 21.3 0.95 (s) 20 36.1 1.31 36.0 1.33 21 18.9 0.92 (d, 6.6) 18.7 0.91(d, 6.4) 22 33.3 1.35 0.99 33.1 1.34 0.98 23 25.9 1.28 1.03 25.7 1.30 1.03 24 45.4 0.91 45.3 0.91 25 28.5 1.64 28.0 1.23 26a 18.8 0.82 (d, 6.7) 18.7 0.79 (d, 6.8) 27a 19.6 0.79 (d, 6.7) 19.4 0.82 (d, 6.8) 28 22.5 1.31 1.10 22.5 1.30 1.11 29 12.2 0.83 (t, 7.4) 12.1 0.84 (t, 7.4) 3-OH ¾ 4.24 (d, 5.6) ¾ 4.31 (d, 5.6) 5-OH ¾ 3.61 (s) ¾ 5.18 (s) 6-OH 9-OH ¾ ¾ 4.51 (d, 5.6) ¾ ¾ 4.75 (d, 5.6) 4.97 (s) 88 a). C-26 and C-27 assignments are interchangeable. b). See Appendix H for full NMR data. HMBC correlations (Figure 3.5) from the methyl groups were key in elucidating the structure, and were observed from Me-19 to C-1, C-5, and C-9 and C-10 and Me-18 to C-12, C-13, C-14 and C-17, allowing for placement of these groups on the steroid skeleton. The connectivity of the OH groups to their respective carbons was established on the basis of COSY60 data, in which correlations of 3-OH/H-3 and 6-OH/H-6 were observed. Furthermore, these OH groups each showed 3 HMBC correlations: 3-OH to C-2, C-3 and C-4 and 6-OH to C-5, C-6 and C-7. The remaining hydroxyl group showed 4 HMBC correlations (C-4, C-5, C-6 and C-7), which in conjunction with the HMBC correlation between Me-19 and C-5 suggested this hydroxyl group to be adjacent to C-19 on C-5. Figure 3.5 Selected HMBC and COSY/TOCSY Correlations for 3.7 gradHSQCTOCSY and COSY NMR data (Figure 3.5) revealed a contiguous spin system containing 3 methylene groups, 1 oxymethine and 1 hydroxyl group assigned to H-1/H-1ʹ/H-2/H-2ʹ/H-3/3-OH/H-4/H-4ʹ. Three more spin systems were observed between H-9/H-11/H-11ʹ/H-12/H-12ʹ, between H-14/H-15/H-15ʹ/H-16/H-16ʹ/H-17/H-20/Me-21/H-22/H-22ʹ/H-23/H-23ʹ/H-89 24//H-25/Me-26/Me-27/H-28/H-28ʹ/Me-29, and between 6-OH/H-6/H-7. Attachment of these spin system fragments was accomplished through the observed HMBC correlations from Me-19 to C-1, C-5 and C-9, Me-18 to C-12, C-14 and C-17, and 6-OH to C-5, C-6 and C-7, giving the core structural connectivity of 3.7. Identification of the side chain was facilitated by the observation of two doublets and a triplet assigned to Me-26/27 and Me-29, respectively, as well as the HMBC correlations from Me-26/27 to C-24 and C-25 and from Me-29 to C-24 and C-28 indicating a stigmastane (1.43) or poriferastane (1.44) type sidechain. Furthermore, an HMBC correlation from Me-21 to C-17 confirmed the attachment of this side chain at C-17. The relative configuration of 3.7 was established on the basis of trROESY data (Figure 3.6). First, a set of trROESY correlations were observed from Me-19 to 6-OH and H-11, as well as from Me-18 to H-11, indicating all were on the same face of the core ring system. Furthermore, a separate set of trROESY correlations was observed including H-3, H-9, H-14, and 5-OH, indicating all were of the opposite face relative to the methyl groups. Additionally, Me-19 showed correlations to the protons H-2ax and H-4ax. The relative configuration of the side chain was established on the basis of the NOE correlation observed between Me-18 and Me-21, as well as one between H-14 and H-17. The configuration at C-20 was not definitively assigned, but rather assumed based on the “natural” configuration observed in steroids.74 The relative configuration at C-24 was not unambiguously determinable by NMR techniques. Several isolations of 3.7 have previously been reported, including those from Spongionella gracilis and 90 the freshwater sponge Spongilla lacustris.108,109 However, these results represent the first isolation of 3.7 from the Petrosia genus. Figure 3.6 Selected trROESY Correlations for 3.7 24-Ethylcholest-7-ene-3ß,5a,6ß,9-tetraol (3.8) was obtained as a pale-yellow oil via reverse-phase HPLC. LRESIMS gave a [M + Na]+ ion at m/z 485.4 and a [M - H]- at m/z 461.4, which in conjunction with the observed NMR data was in agreement with a molecular formula for 3.8 as C29H50O3, requiring 5 degrees of unsaturation. Analysis of the ESI-MS, 1H and 13C NMR data (Table 3.1) indicated 3.8 to be steroid with an additional site of oxygenation as compared to 3.7. Furthermore, comparison of the NMR data to that of 3.7, as well as the new HMBC correlations (Figure 3.7) observed from Me-19 to δ 73.5 indicated the additional site of oxygenation to be at C-9. The rest of the NMR data were consistent with an otherwise identical structure to 3.8. The NMR data reported in the literature were consistent with those obtained experimentally.76 91 Figure 3.7 Selected HMBC and COSY/TOCSY Correlations for 3.8 The relative configuration of 3.8 was determined on the basis of trROESY NMR, and it was hypothesized that it should have analogous relative configurations to 3.7. The relative configuration at the additional site of oxygenation at C-9 was assigned on the basis of trROESY correlations from 9-OH to 5-OH and H-14, and determined to be of the a orientation. The remaining trROESY data were consistent with otherwise identical relative configurations to 3.7 (Figure 2.8). Though one previous isolation of 3.8 from Spongia officinalis was reported by Migliuolo et al. in 1990,110 these results represent its first isolation from the Petrosia genus. Figure 3.8 Selected trROESY Correlations for 3.8 92 3.2.2 Structural Elucidation of Petrosiasterol (3.9) Figure 3.9 Structure of Petrosiasterol (3.9) 24-Ethylcholest-7-ene-3ß,5a,9a-triol-6-one (3.9; given trivial name petrosiasterol) was obtained as a pale-yellow oil via reverse-phase HPLC. High-resolution electrospray ionization mass spectrometry (HRESIMS) analysis gave a [M + Na]+ ion peak at m/z 483.3433 (calculated for C29H48NaO4, 483.3450) requiring 6 sites of unsaturation and one additional site compared to 3.7 and 3.8. Analysis of the HRESIMS, 1H and 13C NMR data (Table 3.2; Figures 3.10 & 3.11; Appendix I) indicated 3.9 to be a steroid with structural similarities to 3.7 and 3.8. Analysis of the 1H, 13C and gradHSQC spectra revealed the presence of several downfield signals corresponding to 4 oxygenated carbons (C-3, C-5, C-6 & C-9) and an alkene (C-7 & C-8), as well as 3 hydroxyl groups, 10 methylene groups, 6 methyl groups and 6 methine groups (Figures 3.12 & 3.13). Furthermore, the 13C NMR data showed a resonance at δ 197.8, as well as two alkene resonances at δ 119.2 and 162.6, indicating the presence of an a,b-unsaturated ketone, thus accounting for the additional site of unsaturation with respect to 3.7 and 3.8. Comparison of the 1D and 2D NMR spectra to those collected for 3.8 suggested 3.9 to be analogous in structure 93 and implied the same sites of oxygenation (C-4, C-5, C-6 and C-9) and unsaturation (C-7 & C-8), thereby indicating the ketone to be at C-6. Table Error! Style not defined..8 NMR Data for Petrosiasterol (3.9) Recorded at 600 MHz in DMSO-d6 No. δC δH (J in Hz) 1ax eq 25.0 2.14 (dt, 13.8, 4.0) 1.35 2eq ax 30.0 1.70 1.28 3 65.2 3.72 4eq ax 35.7 1.83 1.43 5 78.1 ¾ 6 197.8 ¾ 7 119.2 5.44 (s) 8 162.6 ¾ 9 73.9 ¾ 10 40.9 ¾ 11ax eq 27.7 1.79 1.64 12eq ax 34.4 1.79 1.60 13 44.6 ¾ 14 50.7 2.62 15 21.8 1.53 1.44 16 27.2 1.91 1.31 17 55.4 1.33 18 11.6 0.55 (s) 19 19.6 0.85 (s) 20 36.2 1.33 21 18.6 0.92 (d, 5.3) 22 33.0 1.35 0.98 23 25.7 1.33 1.03 24 45.3 0.91 25 28.4 1.65 26a 19.4 0.82 (d, 6.8) 27a 18.8 0.80 (d, 6.8) 28 22.4 1.31 1.12 29 12.0 0.84 (t, 7.2) 94 a). C-26 and C-27 assignments are interchangeable. b). OH signals were not well resolved or assignable. c). See Appendix I for full NMR data. Figure 3.10 1H NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz 0.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.6f1 (ppm)0.550.750.760.770.770.790.800.810.820.840.850.850.880.910.920.930.960.970.980.991.001.021.021.031.041.041.051.071.081.101.111.121.131.141.201.231.251.271.281.291.291.301.311.311.321.321.331.331.341.341.361.361.371.411.421.431.441.451.451.461.501.511.521.531.541.551.551.561.571.591.601.621.641.651.651.661.661.671.681.711.731.761.771.781.791.801.801.801.811.831.831.841.841.851.861.861.861.881.912.112.122.132.142.152.162.622.632.632.642.642.652.653.703.713.723.723.734.505.445.4495 Figure 3.11 13C NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 150 MHz Figure 3.12 gradHSQC NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations 96 Figure 3.13 Expansion of gradHSQC NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations As with 3.7 and 3.8, gradHSQCTOCSY NMR data was key in elucidating the structure of 3.9 and revealed a contiguous spin system containing 3 methylene groups and 1 oxymethine assigned to H-1/H-1ʹ/H-2/H-2ʹ/H-3/H-4/H-4ʹ (Figures 3.14 & 3.15). Two additional spin systems were observed between H-11/H-11ʹ/H-12/H-12ʹ, and H-14/H-15/H-15ʹ/H-16/H-16ʹ/H-17/H-20/Me-21/H-22/H-22ʹ/H-23/H-23ʹ/H-24//H-25/Me-26/Me-27/H-28/H-28ʹ/Me-29 (Figure 3.15). The attachment of these spin systems was determined by HMBC correlations from Me-19 to C-1, C-5 and C-9, Me-18 to C-12, C-14 and C-17, and H-7 to C-5, C-11 and C-14 (Figures 3.16 & 3.17), providing the core structure of 3.9. Identification of the side chain was facilitated by the observation of two doublets and a triplet assigned to Me-26/27 and Me-29, respectively, as well as the HMBC correlations from Me-26/27 to C-24 and C-25 and from Me-29 to C-24 and C-28 (Figure 3.16) indicating a stigmastane (1.43) or poriferastane (1.44) type sidechain. Furthermore, 97 an HMBC correlation from Me-21 to C-17 confirmed the attachment of this side chain at C-17, confirming the structure of 3.9. Figure 3.14 Selected HMBC and COSY/TOCSY Correlations for Petrosiasterol (3.9) Figure 3.15 Expansion of gradHSQCTOCSY NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz Showing Color Coded Contiguous Spin Systems 98 Figure 3.16 Expansion 1 of gradHMBC NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations 99 Figure 3.17 Expansion 2 of gradHMBC NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations The relative configuration of 3.9 was assigned on the basis of trROESY data (Figure 3.18). First, a set of trROESY correlations were observed from Me-19 to H-1ax, H-4ax and H-11ax, as well as from Me-18 to H-11ax, indicating all were of the same orientation (Figure 3.19). Furthermore, a separate set of trROESY correlations was observed including H-3/H-1ax, indicating a β orientation for 3-OH (Figure 3.20). The relative configuration of the side chain was established on the basis of the NOE correlation observed between Me-18 and Me-21, as well as one between H-14 and H-17 (Figures 3.19 & 3.20). As with 3.7 and 3.8, the configuration at C-20 was not definitively assigned, but rather assumed based on the “natural” configuration observed in steroids.108 The relative configuration at C-24 was not unambiguously determinable by NMR techniques. 100 Figure 3.18 Selected trROESY correlations for 3.9 Figure 3.19 Expansion 1 of trROESY NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations 101 Figure 3.20 Expansion 2 of trROESY NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz with Relevant Annotations Though previous isolations of the core scaffold of 3.9 have been reported including homaxisterol c from the sponge Homaxinella sp. (Figure 3.21),111 these results represent its first isolation bearing a stigmastane or poriferastane sidechain. Figure 3.21 Structure of Homaxisterol C 102 3.2.3 Isolation and Structural Elucidation of 24-Ethylcholest-6-en-5a,8a-epidioxy-3b-ol (3.10) and 3b-Hydroxy-24-ethylcholesta-5,8-dien-7-one (3.11) Figure 3.22 Structures of 24-Ethylcholest-6-en-5a,8a-epidioxy-3b-ol (3.10) and 3b-Hydroxy-24-ethylcholesta-5,8-dien-7-one (3.11) 24-Ethylcholest-6-en-5a,8a-epidioxy-3b-ol (3.10) was obtained as a pale-yellow oil via reverse-phase HPLC. LRESIMS gave a [M + Na]+ ion at m/z 467.5 and a [M - H]- ion at m/z 443.4, which combined with analysis of the NMR data was in agreement with a molecular formula of 3.10 as C29H48O3, requiring 6 sites of unsaturation. 103 Table Error! Style not defined..9 NMR Data for 24-Ethylcholest-6-en-5a,8a-epidioxy-3b-ol (3.10) and 3b-Hydroxy-24-ethylcholesta-5,8-dien-7-one (3.11) Recorded at 600 MHz in DMSO-d6 No. 3.10 3.11 δ13C (ppm) δ1H (ppm) δ13C (ppm) δ1H (ppm) 1 34.5 1.71 1.60 33.8 2.05 1.14 2 29.6 1.63 1.37 30.0 1.79 1.58 3 64.3 3.56 70.2 3.40 4 36.2 1.82 1.78 41.6 2.48 2.40 5 81.4 ¾ 163.3 ¾ 6 134.8 6.22 (d) 125.0 5.90 7 129.8 6.43 (d) 186.3b ¾ 8 78.3 ¾ 132.5 ¾ 9 50.5 1.31 161.6 ¾ 10 36.4 ¾ 41.4 ¾ 11 22.2 1.46 1.08 23.6 2.45 1.29 12 38.2 1.88 1.16 34.6 2.03 1.44 13 44.4 ¾ 41.7 ¾ 14 51.0 1.42 47.4 2.16 15 22.5 1.40 1.16 23.6 2.45 1.29 16 27.2 1.82 1.30 28.4 1.91 1.30 17 54.8 1.17 52.4 1.20 18 12.1 0.75 (s) 11.0 0.57 19 18.4 0.79 (s) 22.8 1.27 20 34.7 1.32 35.9 1.36 21 18.2 0.89 (d, 5.2) 18.7 0.95 (d, 5.3) 22 32.7 1.32 0.95 33.3 1.38 0.98 23 25.4 1.30 1.02 25.4 1.31 1.05 24 45.3 0.90 45.0 0.92 25 28.1 1.64 28.1 1.66 26a 18.1 0.79 (d, 6.7) 18.2 0.80 (d, 6.7) 27a 18.8 0.81 (d, 6.7) 18.9 0.81 (d, 6.7) 28 22.1 1.31 1.08 22.1 1.33 1.12 29 12.1 0.83 (t, 7.2) 11.8 0.86 (t, 7.2) a). C-26 and C-27 are interchangeable. b). Value not observed, but taken from literature78 c). See Appendix J for full NMR data. 104 Analysis of the ESI-MS, 1H and 13C NMR data (Table 3.3) indicated 3.10 was a steroid bearing the same side chain and C-3 hydroxylation as 3.7-3.9, but with significant differences within the B ring. However, analysis of the 1D and 2D NMR data revealed the presence of 2 new downfield 1H resonances at δ 6.22 and 6.43, corresponding to an alkene and 2 new downfield 13C resonances at 81.4 and 78.3, corresponding to tertiary sites of oxygenation. HMBC correlations from Me-19 to the 13C resonance at δ 81.4, and from H-7 to δ 78.3, as well as the downfield 13C resonances observed for C-9 and C-14 allowed for placement of these sites of oxygenation at C-5 and C-8 (Figure 3.23). Since 5a,8a-epidioxy sterols have frequently been reported in the literature,112-114 the remaining degree of unsaturation with respect to 3.7 and 3.8 was deduced to be from such an endoperoxide moiety. The rest of the NMR data were consistent with a structure analogous to 3.7-3.9, and the assigned structure of 3.10 was confirmed by comparison with reported literature data.112-114 Figure 3.23 Selected HMBC and COSY/TOCSY Correlations for 3.10 The relative configuration of 3.10 was suspected to be analogous to those observed in 3.7-3.9, and trROESY correlations from Me-19 to H-2ax, H-4ax, and H-12ax, and from Me-18 to H-12ax and Me-21, as well as from H-14 to H-9 supported this hypothesis (Figure 3.24). The 105 5a,8a configuration of the epidioxy moiety was assigned based on comparison of the observed chemical shifts of H-5 and H-8 to those reported in the literature,112-115 while the relative configuration at C-24 was not unambiguously determined. Numerous isolations of 3.10 have been reported, including those from the Petrosia genus.112-114 However, these results represent the first isolation of 3.10 from Petrosia contignata. Figure 3.24 Selected trROESY Correlations for 3.10 3b-hydroxy-24-ethylcholesta-5,8-dien-7-one (3.11) was obtained as a colorless oil via reverse-phase HPLC. LRESIMS gave a [M + Na]+ ion at m/z 449.6 and a [M - H]- ion at m/z 425.5, which combined with analysis of NMR data was in agreement with a molecular formula for 3.11 as C29H46O2, requiring 7 sites of unsaturation. Analysis of the ESI-MS, 1H and 13C NMR data (Table 3.3) indicated 3.11 was a steroid bearing the same side chain and the C-3 hydroxylation as 3.7-3.10, but with key differences in the B ring. Analysis of the 1D and 2D NMR data revealed the presence of four olefinic carbons at 163.3, 161.6, 132.5 and 125.0, with these especially deshielded olefinic resonances suggesting the presence of a doubly a,b-unsaturated ketone. HMBC correlations were observed from Me-19 to the 13C resonances at 163.3 and 161.6, allowing for placement of this moiety within the B ring 106 (Figure 3.25). Though the ketone resonance was not directly observed, the remaining oxygen in the molecular formula of 3.11, in conjunction with the remaining degree of unsaturation and chemical shifts of the two alkenes supported the hypothesized ketone at C-6. A comparison of the collected NMR data to that reported in the literature was consistent with this structural assignment.115 Figure 3.25 Selected HMBC and COSY/TOCSY Correlations for 3.11 The relative configuration of 3.11 was suspected to be analogous to those determined for 3.7-3.10, and trROESY correlations observed Me-19 to H-2ax, H-4ax, and H-12ax, and from Me-18 to H-12ax and Me-21, as well as from H-14 to H-9 supported this hypothesis (Figure 3.26). As with 3.7-3.10, the relative configuration at C-24 was not unambiguously determined. Though one isolation of 3.11 from the sponge Polymastia tenax has been reported,116 these results represent its first isolation from the Petrosia genus. 107 Figure 3.26 Selected trROESY correlations for 3.11 3.3 Biogenetic Relationship of Steroids Isolated from Petrosia contignata As steroid biosynthesis is well characterized, it can reasonably be hypothesized that 3.7-3.11 arise from oxidative modifications of the stigmastane/poriferastane skeleton (Figure 1.14). Furthermore, there is a clear biogenetic relationship between 3.7-3.9, which differ only by sites of oxidation, with an additional hydroxyl group at C-9 for 3.8 compared to 3.7, and the formation of a ketone at C-6 for 3.9 compared to 3.8. Additionally, the co-occurrence of the core scaffolds of 3.10 and 3.11 has been well documented, and it has been hypothesized that 3.11 is a byproduct of the generation of 3.10.90,117 3.4 Conclusion Investigation of the organic extracts from Petrosia contignata led to the isolation and structural elucidation of the new polyoxygenated steroid petrosiasterol (3.9), along with three known polyoxygenated steroids (3.7, 3.8, & 3.11) new to the Petrosia genus and one steroid (3.10) previously observed in extracts from Petrosia sponges. Furthermore, the occurrence of the endoperoxide 3.10 in the extracts of Petrosia contignata, in conjunction with the isolation of 108 2.14 and 2.15 from W. nobilis further demonstrates the prevalence of endoperoxide moieties in secondary metabolites. Future work in this area could involve determination of the C-24 configurations of 3.7-3.11, as well as screening for biological activity. 109 Chapter 4: Experimental 4.1 General Experimental Protocol All solvents used extraction and chromatographic steps were HPLC grade, obtained from Sigma-Aldrich and used without further purification. NMR solvents were obtained from both Sigma Aldrich and Cambridge Isotopes. Si Gel chromatography was performed using Silicycle® G60 Si Gel. Thin-layer chromatography (TLC) was performed using plastic backed ultrapure Si Gel 250 µm plates. Low-resolution electrospray ionization mass spectrometry (LRESIMS) was recorded with Bruker HCT mass spectrometer. High-resolution electrospray ionization mass spectrometry (HRESIMS) was recorded with a Waters/Micromass LCT. 1H and 13C NMR spectra were recorded using a Bruker Advance 600 MHz spectrometer equipped with a z-gradient TCI cryoprobe. All signals NMR signals were referenced to residual undeuterated DMSO (δH=2.50 ppm and δC=39.5 ppm) or CHCl3 (δH=7.26 ppm and δC=77.2 ppm). HPLC was performed using either a Waters 2998 system equipped with a photodiode array detector (also used for UV-VIS measurements), or a Breeze system equipped with a dual wavelength detector. In both cases, an ODS IntertSustain® C18 column (10 x 250 mm) was used for reverse-phased HPLC separations. Circular dichroism (CD) experiments were performed using a Jasco J-815 spectrophotometer. Optical rotations were recorded using a Jasco P-1010 polarimeter at the sodium D line (589 nm). 110 4.2 Sample Collection, Extraction and Isolation of Compounds from Wollemia nobilis 100 g of leaves and stems of Wollemia nobilis collected outside of the Earth Sciences Building on UBC campus were crushed and chopped then immersed in methanol. The methanolic extracts were periodically decanted and replenished until the color had been removed from the plant material (Figure 3.2). The combined methanolic extracts were then concentrated in vacuo to yield a green-brown suspension, which was subsequently diluted in water (200 mL). Next, this suspension was sequentially extracted with hexanes (4x200 mL), DCM (4x200 mL) and EtOAc (4x200 mL), after which in vitro bioassay fraction demonstrated slight activity by the DCM fraction against methicillin-resistant Staphylococcus aureus (MRSA). The DCM component (650 mg) was fractionated via SephadexTM LH-20 chromatography (100% MeOH as eluent) to yield fractions A through E, of which fraction C was found to contain the majority of small-molecule organics, as evidenced by 1H NMR and TLC screening. Following this, fraction C (85.1 mg) was further chromatographed by step-gradient Si Gel chromatography (eluent: 100% Hexanes to 100% EtOAc; 200% increase in EtOAc for every 50 mL eluted) to yield fractions A through G. Final purification was achieved through reverse-phase HPLC to yield pure compounds as outlined in the following: 111 Compound 2.12: colorless oil (4.8 mg) from LH-20 fraction C, Sep-Pak® fraction B via reverse-phase HPLC (H2O/ACN; linear gradient from 50% ACN to 98% ACN at 80 mins; Rt=42.5 mins); UV (H2O/ACN 1:3) lmax (log ε) 216.1 nm; [α]D22 +4.6 (c 0.005 MeOH); CD (MeOH) Δε215 +3, Δε265 0; IR (thin film) νmax 2936, 1867, 1717, 1645, 1435, 1143, 1072 cm-1; 1H NMR and 13C NMR data (600 MHz, DMSO-d6 & CDCl3) in Table 2.1 & 2.2; HRESIMS [M + Na]+ m/z 369.2046 (calculated for C21H30NaO4, 369.2042). Agathic acid methyl ester (2.13): colorless oil (3.6 mg) from LH-20 fraction C, Sep-Pak® fraction B via reverse-phase HPLC (H2O/ACN; linear gradient from 50% ACN to 98% ACN at 80 mins; Rt=48.1 mins); UV (H2O/ACN 1:4) lmax (log ε) 240.2 nm; [α]D21 +27 (c 0.005 MeOH); CD (MeOH) Δε203 -9, Δε225 +1; 1H NMR and 13C NMR data (600 MHz, DMSO-d6) in Table 2.1; HRESIMS [M + H]+ m/z 349.2380 (calculated for C21H33O4, 349.2374). 15-hydroxy-8β,12β-epidioxy-13-abieten-19-oic acid (2.14): colorless oil (1.7 mg) from LH-20 fraction C, Sep-Pak® fraction E via reverse-phase HPLC (H2O/ACN 65:35; Rt=36.9 mins); [α]D21 +8.4 (c 0.005 MeOH); UV (H2O/ACN 13:7) lmax (log ε) 197 nm; 1H NMR and 13C NMR data (600 MHz, DMSO-d6 & CDCl3) in Table 2.3 & 2.4; HRESIMS [M + Na]+ m/z 373.2001 (calculated for C20H30NaO5, 373.1991). 112 15-hydroperoxy-8β,12β-epidioxy-13-abieten-19-oic acid (2.15): colorless oil (4.3 mg) from LH-20 fraction C, Sep-Pak® fraction E via reverse-phase HPLC (H2O/ACN 65:35; Rt=58.6 mins); [α]D21 +14 (c 0.005 MeOH); UV (H2O/ACN 13:7) lmax (log ε) 288.7 nm; 1H NMR and 13C NMR data (600 MHz, DMSO-d6 & CDCl3) in Table 2.3 & 2.4; HRESIMS [M + Na]+ m/z 389.1941 (calculated for C20H30NaO6, 389.1940). Abiesanordine E (2.16): colorless oil (2.2 mg) from LH-20 fraction C, Sep-Pak® fraction E via reverse-phase HPLC (H2O/ACN 65:35; Rt=55.2 mins); UV (ACN:H2O 7:13) lmax (log ε) 246.1 nm; [α]D21 -72 (c 0.005 MeOH); 1H NMR and 13C NMR data (600 MHz, DMSO-d6) in Table 2.5; HRESIMS [M + H]+ m/z M + H]+ m/z 277.1809 (calculated for C17H25O3, 277.1805). Kaur-15-en-17-al (2.17): off-yellow oil (1.9 mg) from LH-20 fraction C, Sep-Pak® fraction A via reverse-phase HPLC (H2O/ACN; linear gradient from 50% ACN to 98% ACN at 50 mins and onward; Rt=68.9 mins); [α]D21 +73 (c 0.005 MeOH); UV (ACN) lmax (log ε) 253.2 nm; 1H NMR and 13C NMR data (600 MHz, DMSO-d6) in Table 2.5; HREIMS [M ]+ m/z 386.2287 (calculated for C20H30O, 286.2297). Isocommunic acid (2.18): colorless oil (2.7 mg) from LH-20 fraction C, Sep-Pak® fraction B via reverse-phase HPLC (H2O/ACN; linear gradient from 50% ACN to 98% ACN at 80 mins; Rt=61.2 mins); [α]D21 +20 (c 0.005 MeOH); UV (H2O/ACN 3:17) lmax (log ε) 223 nm; 1H NMR and 13C NMR data (600 MHz, DMSO-d6) in Table 2.6; HRESIMS [M + H]+ m/z 303.2331 (calculated for C20H31O2, 303.2324). 113 Sandaracopimaric Acid (2.4): colorless oil (2.1 mg) from LH-20 fraction C, Sep-Pak® fraction B via reverse-phase HPLC (H2O/ACN; linear gradient from 50% ACN to 98% ACN at 80 mins; Rt=64.2 mins); [α]D21 -12 (c 0.005 MeOH); UV (H2O/ACN 1:9) lmax (log ε) 205.0 nm; 1H NMR and 13C NMR data (600 MHz, DMSO-d6) in Table 2.6; HRESIMS [M + H]+ m/z 303.2329 (calculated for C20H31O2, 303.2324). An alternative extraction and isolation was performed to ensure that the methyl esters in 3.11 and 3.12 were not artifacts of exposure to methanol. 50 g of leaves and stems of Wollemia nobilis collected outside of the Earth Sciences Building on UBC campus were crushed and chopped then immersed in acetone. The acetone extracts were periodically decanted and replenished until colorless extracts were obtained. The combined extracts were concentrated in vacuo to yield a dark green gum, which was subsequently diluted in water (100 mL). Next, this suspension was sequentially extracted with hexanes (4x50 mL) and DCM (4x50 mL). The DCM extracts were combined and concentrated in vacuo to yield a yellow/green oil then filtered through a Si Gel plug. Following this, the resulting material was further chromatographed by step-gradient Si Gel chromatography (eluent: 100% Hexanes to 100% EtOAc; 200% increase in EtOAc for every 50 mL eluted) to yield fractions A through G. 3.11 and 3.12 were then obtained as pure compounds from fraction B via reverse-phase HPLC as per the conditions outlined above. 114 4.3 Sample Collection, Extraction and Isolation of Compounds from Petrosia contignata 1 kg of frozen Petrosia contignata collected by SCUBA in Madang, Papau New Guinea was cut into 2-3 cm pieces and submerged in 4 L of MeOH (Figure 2.2). The methanolic extracts were periodically decanted and replenished until the extracts were colorless. The combined methanolic extracts were concentrated in vacuo to yield a brown aqueous suspension. This suspension was sequentially extracted with 4x500 mL hexanes, followed by 4x 500 mL DCM. The DCM extracts were subsequently concentrated to yield 1.1 g of a red-brown gum, which was subsequently redissolved in a minimum quantity of methanol and fractionated using SephadexTM LH-20 chromatography (3:1 MeOH:H2O) to yield the 5 crude fractions A-H. These LH-20 fractions were combined on the basis of common TLC spots within the collected culture tube volumes, and then screened using 1H NMR, which revealed a series of aliphatic signals in Fraction G, as well as some broad downfield signals, suggesting the presence of oxygenated steroids. Fraction G (131 mg) was then subjected to normal-phase Sep-Pak® (solvent system hexanes/EtOAc; starting with 100% Hexanes and increasing EtOAc by 20% every 50 mL of eluent collected) yielding fractions A through F, corresponding to each 50 mL eluent collected throughout the step gradient process. Final purification was achieved through reverse-phase HPLC to yield pure compounds as outlined in the following: 115 24-Ethylcholest-7-ene-3b,5a,6b-triol (3.7): pale-yellow oil (1.6 mg) from LH-20 fraction G, Sep-Pak® fraction E via reverse-phase HPLC (H2O/ACN 4:1; Rt=70.0 mins); UV (H2O/ACN 4:1) lmax (log ε) 202.0 nm; [α]D21 -13 (c 0.005 MeOH); 1H NMR and 13C NMR data (600 MHz, DMSO-d6) in Table 3.1; LRESIMS [M + Na]+ m/z 469.4 and [M - H]- m/z 445. 24-Ethylcholest-7-ene-3ß,5a,6ß,9-tetraol (3.8): pale-yellow oil (2.3 mg) from LH-20 fraction G, Sep-Pak® fraction F via reverse-phase HPLC (H2O/ACN 4:1; Rt=38.2 mins); UV (H2O/ACN 4:1) lmax (log ε) 201.1 nm; [α]D21 -14 (c 0.005 MeOH); 1H NMR and 13C NMR data (600 MHz, DMSO-d6) in Table 3.1; LRESIMS [M + Na]+ m/z 485.4 and [M - H]- m/z 461.4. Petrosiasterol (3.9): pale-yellow oil (1.3 mg) from LH-20 fraction G, Sep-Pak® fractions C & D via reverse-phase HPLC (H2O/ACN; 20% ACN for 60 mins then a linear gradient to 65% ACN at 100 mins; Rt=67.8 mins); [α]D22 -3.6 (c 0.005 MeOH); UV (H2O/ACN 7:3) lmax (log ε) 241.6 nm; 1H NMR and 13C NMR data (600 MHz, DMSO-d6) in Table 3.2; HRESIMS [M + Na]+ m/z 483.3433 (calculated for C29H48O4Na, 483.3450). 24-Ethylcholest-6-en-5a,8a-epidioxy-3b-ol (3.10): pale-yellow oil (0.9 mg) from LH-20 fraction G, Sep-Pak® fractions A via reverse-phase HPLC (H2O/ACN; linear gradient from 80% ACN to 90% ACN at 50 mins then a linear gradient to 95% ACN at 110 mins; Rt=93.2 mins); [α]D21 +11 (c 0.005 MeOH); UV (H2O/ACN 1:19) lmax (log ε) 237.4 nm; 1H NMR and 13C NMR data (600 MHz, DMSO-d6) in Table 3.3; LRESIMS [M + Na]+ m/z 467.5 and [M - H]- m/z 443.4. 116 3b-hydroxy-24-ethylcholesta-5,8-dien-7-one (3.11): colorless oil (0.9 mg) from LH-20 fraction G, Sep-Pak® fraction C via reverse-phase HPLC (H2O/ACN; 20% ACN for 60 mins, then a linear gradient to 65% ACN at 100 mins, followed by a linear gradient to 98% ACN at 140 mins; Rt=131.2 mins); [α]D21 +4.2 (c 0.005 MeOH); UV (H2O/ACN 1:9) lmax (log ε) 246.1 nm; 1H NMR and 13C NMR data (600 MHz, DMSO-d6) in Table 3.3; LRESIMS [M + Na]+ m/z 449.6 and [M - H]- m/z 425.5. 4.4 Bioassays In vitro biological assays were done via disc diffusion assays using sterile apparatus. 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Chem. 2014, 4 (10), 709–716. 126 Appendices Appendix A 2D-NMR Spectra for Compound 2.12 and 2.13 Recorded in DMSO-d6 at 600 MHz Figure A.1 gradCOSY60 NMR Spectrum of Compound 2.12 Recorded at 600 MHz in DMSO-d6 Figure A.2 gradHSQC NMR Spectrum of Compound 2.12 Recorded at 600 MHz in DMSO-d6 1.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.0f2 (ppm)2030405060708090100110f1 (ppm)127 Figure A.3 gradHSQCTOCSY Spectrum of Compound 2.12 Recorded at 600 MHz in DMSO-d6 Figure A.4 gradHMBC NMR Spectrum of Compound 2.12 Recorded at 600 MHz in DMSO-d6 0.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.0f2 (ppm)102030405060708090100110120130140150160170180f1 (ppm)128 Figure A.5 trROESY Spectrum of Compound 2.12 Recorded at 600 MHz in DMSO-d6 Figure A.6 1H NMR Spectrum of Agathic Acid Methyl Ester (2.13) Recorded in DMSO-d6 at 600 MHz 129 Figure A.7 gradHSQC NMR Spectrum of Agathic Acid Methyl Ester (2.13) Recorded in DMSO-d6 at 600 MHz Figure A.8 gradCOSY60 NMR Spectrum of Agathic Acid Methyl Ester (2.13) Recorded in DMSO-d6 at 600 MHz 130 Figure A.9 gradHMBC NMR Spectrum of Agathic Acid Methyl Ester (2.13) Recorded in DMSO-d6 at 600 MHz Figure A.10 trROESY NMR Spectrum of Agathic Acid Methyl Ester (2.13) Recorded in DMSO-d6 at 600 MHz 131 Appendix B Calculated & Experimental CD Spectra for Selected Compounds Figure B.1 Figure 4.11 Calculated CD Spectra for (4R,5S,9S,10R) Configuration of Compound 2.12 (Blue) & its Enantiomer (Red) Figure B.2 Calculated CD Spectrum for (4S,5R,9S,10S) Configuration of Compound 2.12 132 Figure B.3 Experimental CD Spectrum for Compound 2.12 Recorded in MeOH 133 Appendix C NMR Spectra for Compound 2.12 Recorded in CDCl3 at 600 MHz Figure C.1 COSY60 NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 600 MHz Figure C.2 gradHSQC NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 600 MHz 134 Figure C.3 gradHMBC NMR Spectrum for Compound 2.12 Recorded in CDCl3 at 600 MHz Figure C.4 trROESY NMR Spectrum of Compound 2.12 Recorded in CDCl3 at 600 MHz 135 Appendix D Thin Film FT-IR Spectrum of Compound 2.12 Figure D.1 Thin Film FT-IR Spectrum of Compound 2.12 136 Appendix E NMR Spectra for 15-Hydroperoxy-8β,12β-epidioxy-13-abieten-19-oic acid (2.14) and 15-Hydroxy-8b,12b-epidioxy-13-abieten-19-oic acid (2.15) Recorded in DMSO-d6 and CDCl3 at 600 MHz Figure E.1 gradCOSY60 NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz Figure E.2 gradHSQC NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz 137 Figure E.3 gradHSQCTOCSY NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz Figure E.4 gradHMBC NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz 0.51.01.52.02.53.03.54.04.55.05.56.0f2 (ppm)2030405060708090100110120130f1 (ppm)138 Figure E.5 trROESY NMR Spectrum of 2.14 Recorded in DMSO-d6 at 600 MHz Figure E.6 gradCOSY60 NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz 0.51.01.52.02.53.03.54.04.55.05.56.0f2 (ppm)0.51.01.52.02.53.03.54.04.55.05.56.0f1 (ppm)0.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.06.2f2 (ppm)0.51.01.52.02.53.03.54.04.55.05.56.0f1 (ppm)139 Figure E.7 gradHSQC NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz Figure E.8 gradHSQCTOCSY NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz 140 Figure E.9 gradHMBC NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz Figure E.10 trROESY NMR Spectrum of 2.15 Recorded in DMSO-d6 at 600 MHz 0.51.01.52.02.53.03.54.04.55.05.56.06.5f2 (ppm)0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.0f1 (ppm)141 Figure E.11 1H NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz Figure E.12 gradCOSY60 NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz 142 Figure E.13 gradHSQC NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz Figure E.14 gradHMBC NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz 143 Figure E.15 trROESY NMR Spectrum of 2.14 Recorded in CDCl3 at 600 MHz Figure E.16 1H NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz 144 Figure E.17 gradCOSY60 NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz Figure E.18 gradHSQC NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz 145 Figure E.19 gradHMBC NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz Figure E.20 trROESY NMR Spectrum of 2.15 Recorded in CDCl3 at 600 MHz 146 Appendix F NMR Spectra for Abiesanordine E (2.16) and Kaur-15-en-17-al (2.17) Recorded in DMSO-d6 at 600 MHz Figure F.1 1H NMR Spectrum of Abiesanordine E (2.16) Recorded in DMSO-d6 at 600 MHz Figure F.2 gradHSQC NMR Spectrum of Abiesanordine E (2.16) Recorded in DMSO-d6 at 600 MHz 147 Figure F.3 gradCOSY60 NMR Spectrum of Abiesanordine E (2.16) Recorded in DMSO-d6 at 600 MHz Figure F.4 gradHMBC NMR Spectrum of Abiesanordine E (2.16) Recorded in DMSO-d6 at 600 MHz 148 Figure F.5 trROESY NMR Spectrum of Abiesanordine E (2.16) Recorded in DMSO-d6 at 600 MHz Figure F.6 1H NMR Spectrum of 2.17 Recorded in DMSO-d6 at 600 MHz 1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)0.740.740.760.770.790.800.820.830.830.840.850.850.860.860.931.041.041.091.101.111.131.131.151.161.181.241.261.261.271.281.281.301.311.351.361.361.371.371.371.381.381.391.391.401.401.411.421.431.491.501.511.551.561.571.581.591.601.611.611.621.621.631.641.641.681.741.751.761.771.781.781.781.791.802.122.142.882.896.819.70149 Figure F.7 gradHSQC NMR Spectrum of 2.17 Recorded in DMSO-d6 at 600 MHz Figure F.8 gradCOSY60 NMR Spectrum of 2.17 Recorded in DMSO-d6 at 600 MHz 150 Figure F.9 gradHSQCTOCSY NMR Spectrum of 2.17 Recorded in DMSO-d6 at 600 MHz Figure F.10 gradHMBC NMR Spectrum of 2.17 Recorded in DMSO-d6 at 600 MHz 151 Figure F.11 trROESY NMR Spectrum of 2.17 Recorded in DMSO-d6 at 600 MHz Appendix G NMR Spectra for Isocommunic Acid (2.18) and Sandaracopimaric Acid (2.4) Recorded in DMSO-d6 at 600 MHz Figure G.1 1H NMR Spectrum of Isocommunic Acid (2.18) Recorded in DMSO-d6 at 600 MHz 152 Figure G.2 gradHSQC NMR Spectrum of Isocommunic Acid (2.18) Recorded in DMSO-d6 at 600 MHz Figure G.3 gradCOSY60 NMR Spectrum of Isocommunic Acid (2.18) Recorded in DMSO-d6 at 600 MHz 153 Figure G.4 gradHSQCTOCSY NMR Spectrum of Isocommunic Acid (2.18) Recorded in DMSO-d6 at 600 MHz Figure G.5 gradHMBC NMR Spectrum of Isocommunic Acid (2.18) Recorded in DMSO-d6 at 600 MHz 154 Figure G.6 trROESY NMR Spectrum of Isocommunic Acid (2.18) Recorded in DMSO-d6 at 600 MHz Figure G.7 1H NMR Spectrum of Sandaracopimaric Acid (2.4) Recorded in DMSO-d6 at 600 MHz 155 Figure G.8 gradHSQC NMR Spectrum of Sandaracopimaric Acid (2.4) Recorded in DMSO-d6 at 600 MHz Figure G.9 gradCOSY60 NMR Spectrum of Sandaracopimaric Acid (2.4) Recorded in DMSO-d6 at 600 MHz 156 Figure G.10 gradHMBC NMR Spectrum of Sandaracopimaric Acid (2.4) Recorded in DMSO-d6 at 600 MHz Figure G.11 trROESY NMR Spectrum of Sandaracopimaric Acid (2.4) Recorded in DMSO-d6 at 600 MHz 0.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.8f2 (ppm)1.01.52.02.53.03.54.04.55.05.5f1 (ppm)157 Appendix H NMR Spectra for 24-Ethylcholest-7-ene-3b,5a,6b-triol (3.7) and 24-Ethylcholest-7-ene-3b,5a,6b,9a-tetraol (3.8) Recorded in DMSO-d6 at 600 MHz Figure H.1 1H NMR Spectrum of 3.7 Recorded in DMSO-d6 at 600 MHz Figure H.2 13C NMR Spectrum of 3.7 Recorded in DMSO-d6 at 150 MHz 158 Figure H.3 gradCOSY60 NMR Spectrum of 3.7 Recorded in DMSO-d6 at 600 MHz Figure H.4 gradHSQCTOCSY NMR Spectrum of 3.7 Recorded in DMSO-d6 at 600 MHz 0.51.01.52.02.53.03.54.04.55.0f2 (ppm)0.51.01.52.02.53.03.54.04.55.0f1 (ppm)159 Figure H.5 gradHSQC NMR Spectrum of 3.7 Recorded in DMSO-d6 at 600 MHz Figure H.6 gradHMBC NMR Spectrum of 3.7 Recorded in DMSO-d6 at 600 MHz 160 Figure H.7 trROESY NMR Spectrum of 3.7 Recorded in DMSO-d6 at 600 MHz Figure H.8 1H NMR Spectrum of 3.8 Recorded in DMSO-d6 at 600 MHz 0.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.0f2 (ppm)0.51.01.52.02.53.03.54.04.55.0f1 (ppm)161 Figure H.9 13C NMR Spectrum of 3.8 Recorded in DMSO-d6 at 150 MHz Figure H.10 gradCOSY60 NMR Spectrum of 3.8 Recorded in DMSO-d6 at 600 MHz 5101520253035404550556065707580859095100105110115120125130135140f1 (ppm)11.4112.1618.8318.9019.5221.4322.5622.5925.8126.9127.4927.7228.4930.9933.2535.0136.0840.0440.2343.3545.4050.0355.4565.7471.5773.6277.21120.31141.080.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.2f2 (ppm)0.51.01.52.02.53.03.54.04.55.0f1 (ppm)162 Figure H.11 gradHSQCTOCSY NMR Spectrum of 3.8 Recorded in DMSO-d6 at 600 MHz Figure H.12 gradHSQC NMR Spectrum of 3.8 Recorded in DMSO-d6 at 600 MHz 163 Figure H.13 gradHMBC NMR Spectrum of 3.8 Recorded in DMSO-d6 at 600 MHz Figure H.14 trROESY NMR Spectrum of 3.8 Recorded in DMSO-d6 at 600 MHz 0.51.01.52.02.53.03.54.04.55.0f2 (ppm)0.51.01.52.02.53.03.54.04.55.0f1 (ppm)164 Appendix I NMR Spectra for 3.9 Recorded in DMSO-d6 at 600 MHz Figure I.1 gradCOSY60 NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz Figure I.2 gradHSQC NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz 0.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.6f2 (ppm)102030405060708090100110120f1 (ppm)165 Figure I.3 gradHSQCTOCSY NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz Figure I.4 gradHMBC NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz 0.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.6f2 (ppm)102030405060708090100110120f1 (ppm)0.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.6f2 (ppm)102030405060708090100110120130140150160170f1 (ppm)166 Figure I.5 trROESY NMR Spectrum of Petrosiasterol (3.9) Recorded in DMSO-d6 at 600 MHz Appendix J NMR Spectra for 24-Ethylcholest-6-en-5a,8a-epidioxy-3b-ol (3.10) and 3b-Hydroxy-24-ethylcholesta-5,8-dien-7-one (3.11) Recorded in DMSO-d6 at 600 MHz Figure J.1 1H NMR Spectrum of 3.10 Recorded in DMSO-d6 at 600 MHz 0.51.01.52.02.53.03.54.04.55.05.5f2 (ppm)0.51.01.52.02.53.03.54.04.55.05.5f1 (ppm)1.01.52.02.53.03.54.04.55.05.56.06.5f1 (ppm)0.740.780.790.800.810.820.830.840.850.850.860.880.890.900.900.910.910.920.950.960.970.981.011.011.011.021.031.031.041.061.071.081.091.091.111.111.121.121.131.141.141.151.171.171.181.201.231.281.291.301.301.311.321.331.331.341.351.361.391.391.401.411.421.421.451.451.461.471.481.501.581.581.591.601.611.611.621.631.631.641.641.651.651.661.661.671.691.691.711.711.731.741.801.811.821.831.841.861.871.871.881.891.891.982.052.066.226.236.436.44167 Figure J.2 gradHSQC NMR Spectrum of 3.10 Recorded in DMSO-d6 at 600 MHz Figure J.3 gradCOSY60 NMR Spectrum of 3.10 Recorded in DMSO-d6 at 600 MHz 168 Figure J.4 gradHSQCTOCSY NMR Spectrum of 3.10 Recorded in DMSO-d6 at 600 MHz Figure J.5 gradHMBC NMR Spectrum of 3.10 Recorded in DMSO-d6 at 600 MHz 169 Figure J.6 trROESY NMR Spectrum of 3.10 Recorded in DMSO-d6 at 600 MHz Figure J.7 1H NMR Spectrum of 3.11 Recorded in DMSO-d6 at 600 MHz 170 Figure J.8 gradHSQC NMR Spectrum of 3.11 Recorded in DMSO-d6 at 600 MHz Figure J.9 gradCOSY60 NMR Spectrum of 3.11 Recorded in DMSO-d6 at 600 MHz 171 Figure J.10 gradHMBC NMR Spectrum of 3.11 Recorded in DMSO-d6 at 600 MHz Figure J.11 trROESY NMR Spectrum of 3.11 Recorded in DMSO-d6 at 600 MHz
Thesis/Dissertation
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Chemistry
Vancouver : University of British Columbia Library
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Terpenoids from terrestrial and marine sources
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