ISOLATION, STRUCTURE DETERMINATION, AND BIOSYNTHETIC STUDIES OF SECONDARY METABOLITES FROM DORID NUDLBRANCHS by EDMUND IDRIS GRAZIANI B.Sc, Trinity College, University of Toronto, 1991 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1996 © Edmund I. Graziani 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Investigations of the skin extracts from a number of dorid nudibranchs have led to the isolation of two novel compounds, lovenone (2) and limaciamine (8). Lovenone (2), isolated from the North Sea dorid Adalaria loveni, represents the first triterpenoid isolated from a nudibranch, and is only the second triterpenoid ever isolated from a marine mollusc. The structure of lovenone (2) was solved using a number of two-dimensional NMR techniques. Similarly, the isolation of limaciamine (8) from the North Sea dorid Limacia clavigera, represents the only naturally occurring analogue reported to date of triophamine (9). Triophamine (9) was originally isolated from the British Columbia dorids, Triopha catalinae and Polycera tricolor. (2) (9) The isolation and structure determination of these novel compounds led directly to an investigation into the biosynthesis of secondary metabolites by dorid nudibranchs. By taking advantage of the unique biology of dorid nudibranchs, a protocol has been developed whereby 13 multiple injections over time of [1,2- C2] acetate has afforded irrefutable proof for de novo biosynthesis in a number of dorid nudibranchs. Terpenoic acid glycerides have been isolated from skin extracts of a number of dorid nudibranchs collected worldwide. Herein is reported the first unambiguous proof for the de novo synthesis of terpenoic acid glycerides 28 and 29, isolated from Archidoris odhneri and A. 13 13 montereyensis, respectively, by NMR analysis of C- C coupling arising from incorporation of intact doubly labeled acetate. Stable isotope incorporation studies with [1,2-^C2] acetate have been used to investigate the biosynthesis of the sesquiterpenoids nanaimoal (38), acanthodoral (39), and isoacanthodoral (40) by the dorid nudibranch Acanthodoris nanaimoensis. The results have shown that: i) the sesquiterpenoids are synthesized de novo by A. nanaimoensis and ii) that a previously proposed biogenetic pathway to the isoacanthodoral skeleton was not tenable and required modification. The use of stable isotope methodology has been extended to probe polyketide biosynthesis by the dorid nudibranch, Triopha catalinae. Incorporation of [ 1,2-* ^C2]acetate into triophamine (9) has clearly shown the biogenesis of (9) from two units of butyrate and one unit of acetate. This work represents the first experimental evidence for de novo polyketide biosynthesis by a dorid nudibranch; moreover, the use of doubly-labeled [1,2-13C2] acetate has provided clear evidence in support of one pathway where a number of biosynthetic pathways were possible. iv TABLE OF CONTENTS Page Abstract ii Table of Contents iv List of Tables vi List of Figures vii List of Schemes " x List of Abbreviations xi Acknowledgments xiii Dedication xiv I. General Introduction 1 LA. Introduction to Marine Natural Products 1 I.B. Introduction to Dorid Nudibranchs and Their Secondary Metabolism 3 I.C. Research Summary 6 I. D. Endnotes: Chapter I: General Introduction 6 II. Isolation and Structure Determination of Lovenone, A Cytotoxic Degraded Triterpenoid from the North Sea Dorid Nudibranch, Adalaria loveni 8 II. A. Taxonomy 9 II.B. Collection and Isolation 10 II.C. Structure Determination 10 U.D. Biological Activity 37 II. E. Origin and Proposed Biogenesis 37 H.F. Endnotes: Chapter II: A. loveni 40 III. Isolation and Structure Determination of Limaciamine, A Diacylguanidine from the North Sea Dorid Nudibranch, Limacia clavigera 41 m.A. Taxonomy 42 III. B. Collection and Isolation 43 ITJ.C. Structure Determination 52 m.D. Endnotes: Chapter ITJ: Limacia clavigera 56 v IV. Biosynthetic Studies of Isoprenoid Secondary Metabolites from Dorid Nudibranchs Using Stable Isotopes 57 IV.I.A. Introduction 57 IV.I.B. Biosynthetic Studies with Marine Invertebrates: Practical Considerations 59 IV.I.C. Introduction to Isoprenoid Biosynthesis 63 rV.I.D. Previous Studies into the Biosynthesis of Terpenoids by Marine Invertebrates 67 IV.II. Biosynthesis of Terpenoic Acid Glycerides by the Dorid Nudibranchs Archidoris odhneri and A. montereyensis 75 IV.II.A. Preamble 76 IV.II.B. Preliminary Results Using Liposomes 78 IV. II.C. Successful Incorporation Studies 82 IV.III. Stable Isotope Incorporation Studies on Sesquiterpenoids from the Dorid Nudibranch Acanthodoris nanaimoensis 99 IV. IV. Endnotes: Chapter IV: Biosynthesis of Isoprenoids 130 V. Stable Isotope Investigations on the Biosynthesis of Triophamine by Triopha catalinae 134 V. A. Introduction to Polyketide Biosynthesis 135 V.B. Precedents for a Polyketide Origin of Triophamine 137 V.C. Feeding Experiments with Triopha catalinae 149 V.D. Endnotes: Chapter V: Biosynthesis of Triophamine 160 VI. Concluding Remarks 162 VLB. Endnotes: Chapter VI: Concluding Remarks 171 VII. Experimental 172 VII.B. Endnotes: Chapter VU: Experimental 181 VIII: Appendix A: Nuclear Magnetic Resonance Techniques 182 IX. Appendix B: Isolation of Known Compounds from New Sources 190 vi LIST OF TABLES Page Table 1: lH, 13C, COSY, HMBC, and nOe NMR Data for lovenone (2) 19 Table 2: 1H,13C, COSY, and HMBC Data for limaciamine (8) 53 Table 3: Specific Incorporation Data for the [1,2-13C2] acetate Feeding Experiments with Archidoris odhneri and A. montereyensis 95 Table 4: NMR Data for nanaimool (41) and isoacanthodorol (43) 121 Table 5: NMR Incorporation Data for Labeled nanaimool (41) and isoacanthodorol (43) 127 Table 6: Specific Incorporation Data for triophamine (9) 159 vii LIST OF FIGURES Page Figure 1: Anatomy of a Typical Dorid Nudibranch 5 Figure 2: Color Plate of Adalaria loveni 8 Figure 3: 1H NMR Spectrum of lovenone (2) [500 MHz, C^] 12 Figure 4: 13C Spectrum of lovenone (2) [ 125 MHz, C&>6\ 13 Figure 5: APT Spectrum of lovenone (2) [125 MHz, C^Dd 14 Figure 6: HMQC Spectrum of lovenone (2) [500 MHz, QDg] 15 Figure 7: Expanded Upfield Region of HMQC Spectrum of lovenone (2) [500 MHz, C6D6] 16 Figure 8: Electron Impact Mass Spectrum of lovenone (2) 17 Figure 9: Fourier Transform Infrared (FT-IR) Spectrum of lovenone (2) 18 Figure 10: Fragment Describing Ring C of lovenone (2) 21 Figure 11: COSY Spectrum of lovenone (2) [500 MHz, C6D6] 22 Figure 12: Expanded Upfield Region of COSY Spectrum of lovenone (2) [500 MHz, C6D6] 23 Figure 13: Expanded Methyl Region of HMBC Spectrum of lovenone (2) [500 MHz, C6D6] 24 Figure 14: Fragment Describing Ring B of lovenone (2) 25 Figure 15: Expanded Region of HMBC Spectrum of lovenone (2) [500 MHz, C6D6] Showing Correlations from the lH at 5 3.67 26 Figure 16: Expanded Region of HMBC Spectrum of lovenone (2) [500 MHz, C6D6] Showing Correlations from the *H at 8 2.05 27 Figure 17: Expanded Downfield Region of HMBC Spectrum of lovenone (2) [500 MHz, C6D6] 28 Figure 18: Fragment Describing Ring D of lovenone (2) 29 Figure 19: Downfield Region of ]H Spectrum of lovenone (2) Recorded in d6-DMSO [500 MHz] 30 Figure 20: Downfield Region of !H Spectrum of lovenone (2) Recorded in d6-DMSO + D20 [500 MHz] 30 Figure 21: Expanded Region of COSY Spectrum of lovenone (2) in d6-DMSO 500 MHz] 31 Figure 22: Fragment Describing Side-Chain of lovenone (2) 33 Figure 23: Expanded Region (*H 8 1.5 to 1.8) of HMBC Spectrum of lovenone (2) [500 MHz, C^] 34 Figure 24: Proposed Conformation of lovenone (2) 35 Figure 25: Selected Difference nOe Spectra of lovenone (2) 36 Figure 26: Color Plate of Limacia clavigera 41 Figure 27: !H Spectrum of limaciamine (8) [500 MHz, CDCI3] 44 Figure 28: FT-IR Spectrum of limaciamine (8) [NaCl, thin film, neat] 45 Vlll Figure 29: Electron Impact Mass Spectrum of limaciamine (8) 46 Figure 30: 13C Spectrum of limaciamine (8) [125 MHz, CDCI3] 47 Figure 31: HMQC Spectrum of limaciamine (8) [500 MHz, CDCI3] 48 Figure 32: COSY Spectrum of limaciamine (8) [500 MHz, CDCI3] 49 Figure 33: Selected Region of HMBC Spectrum of limaciamine (8) [500 MHz, CDCI3] 50 Figure 34: Methyl Region of HMBC Spectrum of limaciamine (8) [500 MHz, CDCI3] 51 Figure 35: Color Plates of Archidoris odhneri (top) and A. montereyensis (bottom) 75 Figure 36: 1H Spectrum of farnesic acid glyceride (28) [500 MHz, CDCI3] 83 Figure 37: 13C Spectrum of farnesic acid glyceride (28) [125 MHz, CDCI3] 84 Figure 38: HMQC Spectrum of farnesic acid glyceride (28) [500 MHz, CDCI3] 85 Figure 39: Expanded Upfield Region of HMQC Spectrum of farnesic acid glyceride (28) [500 MHz, CDCI3] 86 Figure 40: HMBC Spectrum of farnesic acid glyceride (28) [500 MHz, CDCI3] 87 Figure 41: Expanded Upfield Region of HMBC Spectrum of farnesic acid glyceride (28) [500 MHz, CDCI3] 88 Figure 42: Expanded Region (Upfield