BIOSYNTHETIC STUDIES OF NATURAL PLANT PRODUCTS The Biosynthesis of Petasin in Petasites hybridus (L) Compositae A Thesis submitted to the University of Glasgow for the Degree of Ph.D. by Robert Anthony Butler Keates September 1970 ProQuest Number: 11011945 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 11011945 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ACKNOWLEDGEMENTS I must express my sincere thanks to Dr. C.J.W. Brook for his guidance and interest at all times, and to Professor R.A. Raphael, F.R.S., for providing the opportunity to carry out this research. Thanks are also due to my many colleagues for useful discussions, and in particular to Dr. J.A. Zabkiewicz, who was associated with me in the early part of this work, and to Dr. A.M.K. Berrie, of the Botany Department, for his advice on botanical aspects of the work. I must also gratefully acknowledge the assistance of Dr. J. Sjovall and Dr. E. Nystrom, of the Karolinska Institutet, Stockholm, for the valuable information and inspiration that permitted the chromatographic studies described in this thesis, and for the hospitality extended in the course of a visit to their laboratory during April 1969. Finally, I thank the technical staff of this Department for excellent service at all times. The work was supported in the first year by a grant, to Dr. Brooks, from Arthur Guinness, Son & Company. The remaining two years have been completed during tenure of a Demonstratorship. Department of Chemistry, University of Glasgow, September 1970 CONTENTS Conventions and Nomenclature INTRODUCTION 1 CHEMICAL STUDIES OF P. HYBRIDUS CONSTITUENTS Introduction and Historical Background 7 Chemotaxonomic Aspects 8 Results and Discussion Preliminary Examination of Samples from Different Localities 11 G-LC Examination of Leaf Extract 12 Identification by G-C-MS of General Leaf Constituents 16 Further Subdivision of the Extract into Groups 17 Characterisation and Identification of Major and Minor Sesquiterpenoids 19 Quantitative Estimation of Leaf Sesauiterpenoids 41 Comparison of Flower, Rhizome and Leaf Sesquiterpenoids 42 Experimental General Chromatographic and Spectroscopic Procedures 47 Plant Material 51 Extraction of Plant Tissues 52 Separation Procedures 54 Chemical Techniques 57 Quantitative Estimation of Sesquiterpenoids 59 RADIOACTIVE TRACER LABELLING- STUDIES IN THE INVESTIGATION OF PETASIN BIOSYNTHESIS Introduction and Historical Background Common Pathways of Terpenoid Biosynthesis 61 Farnesyl Pyrophosphate as the Common Precursor of Cyclic Sesquiterpenoids 65 Results and Discussion General Plan of Labelling Experiments 68 Feeding of Labelled Mevalonolactone to P. hybridus Plants 72 Location of Label from (2-^C) Mevalonic Acid in the Molecule of Petasin 76 Isolation of C-5 of Desisopropylideneisopetasol 79 Isolation of C-9 of Desisopropylideneisopetasol 85 Distribution of Radioactivity in Isopetasol 87 Proposed Isolation of Tritium Label in Petasin from (2-^C-5R, 4R-4-^H) Mevalonic Acid 89 Experimental Procedures Feeding of Mevalonolactone 92 / 14 x Isolation of Isopetasol from (2- C) MVAL Feeding 95 Retro-Aldol Reaction and Isolation of Iodoform and Desisopropylideneisopetasol 97 Isolation of C-5 of Isopetasol 101 Isolation of C-9 of Isopetasol 110 Radioassay of Degradation Products 119 4 BIOCHEMISTRY AND METABOLISM OF MEVALONIC ACID IN P. HYBRIDUS 4.1.1 Introduction: The Metabolic Status of Mevalonic Acid in Higher Plants 120 4.2 Results and Discussion 4.2.1 Preliminary Feedings and Plan of Experiments 127 4.2.2 Distribution of (2-^C) MVAL and Effect on Petasin Biosynthesis 128 4.2.3 Utilisation of Mevalonic Acid in P. hybridus 133 4.2.4 Effect of Synthetic Inhibitors on Sesauiterpenoid and Sterol Production 137 14 4.2.5 Incorporation of COg in Sesquiterpenoids and Sterols 141 4.2.6 An Approach to In Vitro Methods of Study 148 4.3 Experimental Procedures 4.3.1 General Techniques 154 4.3.2 Distribution Experiments 154 4.3.3 Utilisation of Labelled Precursor 156 4.3.4 Inhibitor Feedings 158 4.3.5 ^OOg Feedings 159 4.3.6 Cell-Free System from P. hybridus Leaves 162 5 THE BIOSYNTHESIS OF PETASIN: GENERAL CONCLUSIONS 5.1 A Proposal for a Biosynthetic Sequence Leading to Petasin 164 5.2 Compartmentation of Sesquiterpenoid Biosynthesis 173 PART II STUDIES IN THE APPLICATION OF GEL CHROMATOGRAPHY 6 STUDIES OF GEL CHROMATOGRAPHIC SYSTEMS USING HYDROXYALKOXYPROPYL DERIVATIVES OF SEPHADEX 6.1.1 Introduction 178 6.2 Results and Discussion 6.2.1 General Principles 185 6.2.2 Examination of a Straight-Phase Partition System 187 6.2.3 Examination of Some Gel Filtration Systems 190 6.2.4 Examination of Reversed-Phase Partition Systems 192 6.2.5 Performance of Gel Chromatographic Systems 194 6.2.6 Applications of Gel Chromatography 199 6.3 Experimental Procedures 6.3.1 Preparation of Gels 201 6.3.2 Preparation of Columns 206 6.3.3 Sample Handling and Detection 209 APPENDIX I Mass Spectra REFERENCES CONVENTIONS AND NOMENCLATURE In the text that follows, numbers appearing as a 103)' superscript, followed by a single bracket thus denote references, while those written in line with the text, between paired brackets thus (34) indicate drawings of chemical formulae, and may be referred to in the text as ‘structure (3^)'. In drawings of structure, stereochemistry is not implied unless specifically indicated. A thickened or broken bond denotes a substituent located respectively above or below the plane of the paper. A wavy bond indicates an epimeric mixture. A structure previously drawn without stereochemical detail, e.g. (34), is written with a suffix (34A) when stereochemistry is assigned. Isotopic labelling patterns in a structure are similarly distinguished from the unlabelled compound; thus 3R-mevalonic acid (6), (2-1*C-3R) mevalonic acid (6A) and (2- C-3R,4R-4-3h ) mevalonic acid (6B). A conflicting system of identification appears, largely in the second chapter, where peaks on a chromatogram have been tabulated 1-40: these appear in the text as, for examnle ‘peak 2_8f« Following characterisation by GC-K3 and chromatographic isolation, these are then referred to as ‘compound JJ3‘, etc. 1 . 1 AL INTRODUCTION: AND SUKk AEY OF THE THESIS In recent years, the study of natural products uncoraone purely structural type of chemical investigation to consideration of the relationship o the organism of origin widespread application of modern chromatographic and spectroscopic techniques has considerably simplified the isolation and characterisation of these compounds, permitting increasing attention to turn to wider biological issues as well as purely chemical aspects. Among these are the examination of biosynthetic processes and pathways, end the solution of mechanistic 1,2 ) problems The investigation of biosynthesis has led to a greater understanding of the metabolic significance of many natural products, and to consideration of their 3) biolovical function . In addition, the application of 4) 5) chemical characters to taxonomic and phylogenetic systematics has provided a valuable new source of evidence for the relationships between taxa. This thesis describes an investigation into the biosynthesis of the sesquit^rpenoid, petasin (1), isolated from the plant Pctaitss hybridus (l ) Compositae. the structure or which war. originally determined by Aebi and ,-0 12' 0 (2) (3A) OPP OPP = Pyrophosphate HOCH, CO.H (6) (1A) 14, 2 6) Waaler . The work was initiated as part of a long-term project by Dr. Gr.H. Draff an and Dr. J.A. Zabkiewicz, formerly of this department. Their preliminary findings 7) nave already been described Petasin has been chosen for this study because it possesses the eremophilane (2) carbon skeleton, which cannot be directly derived by the Biogenetic Isoprene Rule proposed 8) ~ 9,10) by Ruzicka „ jt has been suggested that eremophilanes are derived from the eudesmane (3) skeleton by a methyl shift across the ring Junction (3A). Another factor influencing the choice of petasin has been the easy availability of P.hybridus in the wild state. Finally, the chemotaxonomic studies of 11,12) the Czech group of Sorm into various Petasites species have provided valuable background information on the chemistry of the sesauiterpenoids of this genus. • Chemical studies of P. hybridus leaves from a i, _ — number of sources thoughout Britain have demonstrated the consistent nature of the sesquiterpenoid constituents, the petasin esters being the principal components in each case. A more detailed examination, using the highly sensitive techniques of gas-liquid chromatography (G-LC) and gas chromatography coupled to mass spectrometry (G-C-K3), has revealed a number of other, non-esterified sescuiteruenoids, , ’3,14) in particular, the hydrocarbon eremophilene (4) . Many of these compounds may be significant as intermediates in the biosynthetic pathway. Using the available evidence of the sesouiterpenoid constituents, coupled with chemotaxonomic information, a rationalisation has been made of the probable biosynthetic pathway leading to petasin. This places eremophilene as the first fully cyclised component of the seouence. Stereoelectronic considerations of the cyclisation of trans,trans-farnesyl pyrophosphate (5) to eremophilene have led to a detailed proposition for the mechanism of these reactions, and for the mode of action of the enzymes responsible. This approach may also be applied to the biosynthesis of other classes of terpenoids, and is consisten with currently available experimental results. A series of conventional biochemical reactions is then postulated to derive the oxidised products, including petasin. • Radioactive tracer methods have been employed in 1 4 this investigation of petasin biosynthesis.