STUDIES IN TERPENOID BIOSYNTHESIS A thesis submitted by JOHN ANTONY WHITTLE for the degree of Doctor of Philosophy in the University of London. Imperial College of Science and Technology, South Kensington, London S.W.7. September, 1967. 2 ABSTRACT, The various types of sesquiterpenoids are discussed in terms of their structural and biogenetic relationships. Possible biosynthetic pathways are proposed for all types, and some existing proposals are modified or extended. Emphasis is placed on the small number of different initial cyclisations of farnesyl pyrophosphate required to explain the biogenesis of the various skeletal types, and the key role of intermediates of the bisabolene and germacrane types is stressed. The published experimental studies on sesquiterpenoid biogenesis are reviewed and discussed. Experimental work on the biosynthesis in Artemisia maritima of the sesquiterpenoid bitter principle, santonin, is described. On the basis of the incorporations obtained, it is necessary to postulate two pathways for the later oxidative steps in its biosynthesis. These differ at the stage at which the first hydroxylation occurs. The incorporation of the ten-membered ring compound, 11,12-dihydrocostunolide suggests that this lactone is cyclised to a decalin system in the biosynthetic pathway. The incorporation of mevalonic acid lactone into santonin is shown to have occurred with scrambling of 3 the label. Farnesyl pyrophosphate was not incorporated, while a very low incorporation of farnesol is recorded. The occurrence of 1,2—dihydrosantonin in Artemisia stellariana is proved. The isolation of tl.e triterpenoid bitter principle, limonin,from the roots of Dictamnus albus is described. A ;ow incorporation of me7alonic acid lactone into limonin is recorded. CONTENTS ACKNOWLEDGEMENTS ••a 2•• •• ••• ••• SECTION 1 - REVIEW OF SESQUITERPENDID STRUCTURAL TYPES AND THEIR PROBABLE BIOGENESIS INTRODUCTION ••• ••• ••• ••• ••• ••• ••• 9 OCCURRENCE AND FUNCTION OF SESQUITERPENOIDS 10 IMPORTANT BIOSYNTHETIC MECHANISMS Eliminations 11 Cyclisations ••• ••• ••• ••• ••• ••• 11 Reactions of carboxyl groups 13 Oxidation and reduction ••• ••• 13 Hydroxylation and dehydration 14 FAR NESYL PYROPHOSPHATE ••• ••• •• • ••• 16 ACYCLIC SESQUITERPENOIDS ••• ••• ••• ••• 17 TEN-MEMBERED RING SESQUITERPENOIDS - GERMACRANES Introduction ••• ••• ••• ••• ••• ••• 20 Lactonic germacranes Germacranolides 25 Linderane group ... ••• ••• ••• 30 Miscellaneous ••• ••• ••• ••• 31 EUDESMANE AND RELATED SES'QUITERPENOIDS Introduction 9041 ••• 32 Eudesmanes without lactone, furan or 6,7,11-cyclopropane rings ... *00 • •• 37 Lactonic eudesmanes - Eudesmanolides ••• 45 Furanoeudesmanes ••• 49 Ag arofuran group •• • a•• ••• ••• • •• 51 Eudesmanes with 6,7,11-cyclopropane rings ••• 53 Eremophilane group ••• ..• ••• ••• ••• 54 V aleranone type 800 4,4.0 000 000 000 0 •• 59 Agarospirol type ••• ••• ••• ••• •• • 60 HYDROAZULENIC SESQUITERPENOIDS Introduction .. ... 63 Guaianes without lactone, 6,7,11-cyclopropane or oxide rings ••. ••• ••• ••• ••• 66 Lactonic guaianes - Guaianolides ... ... 68 Pseudoguaianolides ••• ••• ••• ••• ..• 70 Kessane group ••• ••• ••• ••• ••• ••• 7/4 Valerene type ••• ••• ••• • •• • 75 Guaianes with 6,7,11-cyclopropane rings ... 76 Tricyclic guaianes ••• ••• ••• ••• ••• 77 5 SESQUITERPENOIDS REGARDED AS DERIVED FROM BISABOLENE TYPE INTERMEDIATES Introduction • • • • • • • • ••• ••• ••. 80 Bi sabolene type • • • • • • • • • ••• 000 80 Cadinene type • • . • • • • • • • • • • • . • • • 82 Helminthosporal group • • • • • • . • • • .• 86 Acorone and cedrene groups et. 000 0.0 88 Trichothecin group • • • • • • • • • . • . • • • 88 ELEVEN-MEMBERED RING SESQUITERPENOIDS AND OTHERS REGARDED AS DERIVED THEREFROM Humulene group 91 Illudins and marasmic acid 00. 92 SESQUITERPENOIDS FOR WHICH SEVERAL POSSIBLE BIO- SYNTHETIC ROUTES ARE AVAILABLE Copaene, longifolene., santalene and bergamotene groups ... 080 94 Himachalene group and allohimachalol 100 Widdrol type including thujopsene and other cyclopropanes 101 Chamigrene, cuprene, laurene and aplysin types 102 BICYCLOFARNESOL GROUP ... • • • • •0 ••• ••• ••• 104 MISCELLANEOUS SMALL GROUPS Maturin and cacalol group • • • • • • • • • • • • 106 Elemene group e.. • • • • 4. • • • • • 0 • 107 Carotane type • • • • • 0 • • • • • • 108 Tutin group O.& 000 • • • • • • • • • • • • 109 CONCLUSIONS • • • • • • • • • • • • • • • 110 SECTION 2 - REVIEW OF THE PUBLISHED EXPERIMENTAL INVESTIGATIONS INTO SESQUITERPENOID BIOSYNTHESIS FARNESYL PYROPHOSPHATE • • • • • • • • • •• • • • • 112 IPOMEAMRONE 600 • • • • • 0 • • • • • • • • • 112 HELMINTHOSPORAL • • • • • • • • • •• • • • • 115 CARYOPHYLLENE 0.0 .00 • e J 0 0 • • • •• • • • • 117 ILLUDINS AND MARASMIC ACID • • • • • 0 • • 0 ••• •O• 117 LONGIFOLENE 00. 000 • • • • • • • • • ••• ••• 11, 9 CAROTOL .0. Os. *Ile • • • • • • • • • ••• ••• 1.20 GOSSYPOL os. 000 000 • • 0 • • • • • • •• • • 0 0 1.20 SECTION 3 - BRIEF REVIEW OF THE ISOLATION AND DETERMINATION OF STRUCTURE OF SANTONIN EXTRACTION AND ISOLATION OF SANTONIN O ,04. • • • .0. 123 GROSS STRUCTURE 000 .0. 000 0.0 0 • 0 0 • 0 1.24 STEREOCHEMISTRY 00. .00 0.0 ... ... 1.26 TOTAL SYNTHESIS O 0. 060 00. 127 CONCLUSION ... 000 000 00. e iro 000 000 129 6 SECTION 4 - DISCUSSION OF EXPERIMENTAL WORK CARRIED OUT ON THE BIOSYNTHESIS OF SANTONIN PRELIMINARYWORK • • • • • • • • • • • • • . • • • • •• • 131 FIRST SEASON Introd=tion • • • • • • • .• • • .. • • • •.. 132 Prepation of the lactonic possible precursors 134 Preption of the acidic possible precursors 139 Raditivity determinations 004 000 000 140 Synthis of the labelled compounds for feeding experiments • • • • • • • • • • • • 140 FeediLg Experiments • • • • • • •• • •• • •• • 147 SECOND SEAS7 11,12-Dihydrocostunolide • • • •• • . • • •• • 153 Further eudesmanolide possible precursors 00. 156 Labelling of precursors for second season's feedings ... ... 0.. .06 4.0 161 Feeding experiments • • • • • • •• • •• • •• • 162 Degradation of the santonin from the mevalonate feeding ... 000 040 000 167 THIRD SEASON Preliminary considerations • • • • • • • • • • • • 171 Farnesyl pyrophosphate ... ... ... ... 172 Attempted syntheses of hydroxyketo lactone (401) 172 Douglanine ... • • • • • • • • • •• • •• • •• • 175 Germacrene 41410 .00 000 000 00. 000 000 176 Tetrahydrosantonins • • • • • • •• • •• • •• • 177 Labelling of precursors for third season's feedings • • • •• • •• • •- • 179 Feeding experiments 000 ... ... .00 000 1.79 EXAMINATION OF OTHER SPECIES OF ARTEMISIA ... 000 182 LIMONIN• • • • • • • • . • • • • • • •• • •• • •• • 183 SECTION 5 - EXPERIMENTAL DETAILS GENERALREMARKS ... • • • • • • • • . •• • •• • •.• 185 PRELIMINARY EXPERIMENTS • • 0 ••• ••• ••• ••• 188 FIRST SEASON • • • • • • • • • • • • • • • • • • • • • 189 SECOND SEASON • . • • • • • • • .. • • a • • • • • • • 204 THIRDSEASON • • • • • • . • • • • . •• • •• • •• • 222 EXTRACTIONS OF OTHER ARTEMISIA SPECIES ... 040 235 LIMONINWORK • • • • • • • • • • • • •• • •• • .• • 235 TABLES OF FEEDING EXPERIMENTS AND N.M.R. DATA ••• 237 SECTION 6 - REFERENCES REFERENCES 000 00. 000 000 000 0.0 000 247 7 ACKNOWLEDGEMENTS I wish to express my gratitude to Professor D.H.R. Barton, F.R.S., for the privilege of working under his stimulating guidance. I also wish to thank Dr. G.P. Moss for his interest, useful suggestions and helpful discussions. The assistance of the technical staffs of Imperial and Queen Mary Colleges is gratefully acknowledged. The work for this thesis was carried out during the tenure of a Science Research Council (formerly Department of Scientific and Industrial Research) Research Studentship. John A. Arhittle, September, 1967. 8 SECTION 1 ==1:MNILMI=Ir REVIEW OF sEslumpENoID STRUCTURAL TYPES AND THEIR PROBABLE BIOGENESIS 9 INTRODUCTION This section begins with a brief review of the occurrence and function of sesquiterpenoids, and discussions of certain important biosynthetic mechanisms. There follows a group by group descrip- tion of the various skeletal types, classified by structural and biosynthetic considerations. The ten- membered ring and eudesmane sesquiterpenoids are consid- ered in greater detail than other types, because of their relevance to the experimental work discussed in Section 4 of this thesis. Throughout the thesis, the term sesquiterpene is applied only to compounds of molecular formula C 15H24. All related natural products are described as sesqui- terpenoids. The numbering system used for each type is shown diagrammatically near the beginning of its section. Sesquiterpenoids have been reviewed1'2 as have some aspects of their biogenesis.3'4'5 10 OCCURRENCE AND FUNCTION OF SESQUITtRPENOIDS Sesquiterpenoids have been found in almost all forms of living matter, but occur principally in higher plants. They exhibit a rich variety of functional groups and ring systems. Apart from the 6 lactones for which the family Compositae is noted, furans, epoxides and cyclopropane rings occur frequently in the four hundred or so known sesqui- terpenoids. Less common are larger ring oxides, aldehydes, and even acetylenes. Yost sesquiterpenoids (farnesyl pyrophosphate is an important exception) are secondary metabolites, and many are produced only during certain phases of the life cycle of the organism. It is not known what function sesquiterpenoids, or indeed, any secondary metabolites fulfil, but it has been suggested7 that they are formed in order to dispose of accumulations of certain intermediates. It seems probable that, when conditions are perfectly balanced, only replication activities take place.