A STUDY ON THE BIOSYNTHESIS OF CAMPHOR By THERESE MARY ATLAY B.Sc.(Hons.) University of Exeter, 1976. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Chemistry. We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1983. © Therese Mary Atlay, 1983. 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 of OH Q^NA i The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date Starvd r^Sib DE-6 (3/81) - ii - ABSTRACT. This thesis describes an investigation of the biosynthesis of camphor. Camphor, a bicyclic monoterpene, has been shown to be bio- synthesised from.geranyl pyrophosphate or its isomers (neryl pyro• phosphate or linaloyl pyrophosphate) and several mechanisms have been proposed for this cyclisation process. geranyl pyrophosphate camphor In an attempt to differentiate the origin of the C(8) and C(9) methyl groups,, feeding experiments with appropriately labelled pre- cursors were attempted. The two precursors used were [2- H^J-mevalonic r 2 i acid and [8- H^J-linalool. It was shown that the expected products, 2 2 2 [8- H^]- and [9- H^]-camphor, could be differentiated by H-n.m.r. using the shift reagent Eu(thd)^ (Eu(thd)^ = tris(2,2,6,6-tetramethyl- 3, 5-heptanedionato.)europium) . Feeding^ experiments with Rosemarinus officanalis using the cut stem method showed no incorporation of deuterium label into camphor extracted from the plant's essential oil. [2- H ]-mevalonic acid [8- H..] -linalool iii - TABLE OF CONTENTS. Abstract ii Table of contents iii List of figures v Abbreviations x Acknowledgements . xii I Introduction 1 I - (i) The terpenoids 1 I - (ii) Early studies on the terpenoids 2 I - (iii) Early studies on terpenoid biosynthesis 4 I - (iv) The identification of intermediates 7 (a) Mevalonic acid 7 (b) From acetate to mevalonic acid 9 (c) From mevalonic acid to the 'active isoprene unit' 13 (d) IPP to other terpenyl pyrophosphates 15 I - (v) Stereochemical aspects of terpenoid formation 16 (a) IPP isomerisation 16 (b) Condensation of DMAPP with IPP 19 I - (vi) Mechanistic studies 29 I - (vii) From geranyl pyrophosphate to the monoterpenes 31 (a) Introduction 31 (b) Regular acyclic monoterpenes 35 (c) Monocyclic monoterpenes (p_-menthanes) 38 (d) Bicyclic monoterpenes 48 - iv - (i) pinanes 48 (ii) bornanes 50 (iii) thujanes 55 (iv) caranes 57 (e) Cyclopentanoid monoterpenes (iridanes) 60 (f) Irregular monoterpenes 64 II Discussion 70 II - (i) The problem 70 II - (ii) The method of labelling 76 II - (iii) The choice of precursors 77 II - (iv) The plant system 83 II - (v) Synthesis of the biosynthetic precursors 87 2 (i) [ 2- H2] -Mevalonic, acid (6l_) 87 (ii) [ 8-2E^ -Linalool (62) 91 II - (vi) Results 101 II - (vii) Suggestions for further work 103 III Experimental 105 References 123 LIST OF FIGURES. Figure. Page. 1 The coupling of isoprene units in a "head- to- tail" fashion 3 2 Bonner and Arreguin's proposal for the early- steps in rubber biosynthesis. 6 3 The labelling pattern in squalene (4) biosyn- 1 4 thesised from [2- ^-mevalonic acid (MVA) 8 4 The conversion of acetate to (3R)-mevalonic acid (8) 11 5 The various pathways to HMGCoA (7a) from general metabolism 12 6 Concerted elimination of C0„ and P. to form 2 l mevalonic acid 14 7 Anti elimination of CO^ and P_^ from 3-phospho- 5-pyrophospho-MVA to form IPP 15- 8 The isomerisation of IPP to DMAPP (13) 15 9 Stereochemistry of IPP isomerisation to DMAPP 17 10 Squalene biosynthesised in a rat liver 2 homogenate in the presence of H^O 18 11 The demonstration of re face attack of H+ in the isomerisation of IPP to DMAPP 20 12 Regiochemistry of attachment of prenyl units 21 13 1'-4 condensation... 21 14 Regiochemistry of condensation to form pre- squalene or prephytoene pyrophosphates 22 15 Squalene biosynthesis 23 16 Phytoene biosynthesis 24 17 The condensation of the allylic pyrophosphates and IPP 25 Inversion of configuration at of the allylic pyrophosphate in the 1 '-4- conden• sation shown by Popjak and Cornforth Stereochemical requirements for the form• ation of presqualene pyrophosphate (or prephytoene pyrophosphate) The regiochemistry of attack of the allylic substrate of IPP 1'-4 condensation Postulated mechanism of the 1'-4 condensation., Examples of the basic skeleta of the mono• terpenes Ruzicka's scheme- for the formation of the monoterpenes The proposed biosynthesis of DMAPP (13) from leucine (1_8) Some naturally occurring acyclic monoterpenes.. The in vitro transformations of geranyl, neryl, and linaloyl phosphates Postulated mechanism for the formation of nerol directly Labelling pattern obtained when rose petals were fed with [2-1/iC,4-3H.]-MVA In vitro studies of the hydrolysis of the phosphate esters of linalool, geraniol, and nerol Conversion of.GPP to NPP via LPP, without loss of hydrogen at C(1) , Cori's proposed mechanism for the direct formation of NPP from the condensation of IPP and DMAPP Some naturally occurring monocyclic monoterpenes Some aromatic monoterpenes and their relationship with the non-aromatic mono• terpenes Proposed biosynthesis of y-terpinene and a-thujene , Scheme to provide evidence for the existence of 1,2-hydride shifts in monoterpene biosynthesis Examples of the pinanes.. The bornanes Production of the bornanes from the stereo- chemically appropriate a-terpinyl cation Synchronous mechanism for NPP to BPP Biosynthesis of borneol Revised proposal for the biosynthesis of the bornanes . The thujane monoterpenes Postulated biosynthetic scheme for the thujanes The carane monoterpenes Proposed method of formation of car-3-ene..... Biosynthesis of car-3-ene in Pinus sylvestris. The biosynthesis of car-3-ene, involving a 1,2-proton shift Examples of the iridane monoterpenes Biosynthesis of the iridoids , The proposed biosynthesis of the iridoids - viii Figure. Page. 52 Irregular roonoterpene biosynthesis 64 53 Postulated direct formation of fenchol from GPP 66 54 Major skeleta of the irregular monoterpenes 67 55 Proposed biosynthesis of irregular monoterpenes 68 56 The biosynthesis of the irregular mono• terpenes involving DMVC 69 57 Incorporation of [2-UC]-MVA (8) and [2-^^C]-geraniol {V^) into camphor (2) 71 58 Conversion of geranyl pyrophosphate (14) to bornyl pyrophosphate (37) by an enzyme preparation from sage 71 59 Numbering of the bornane skeleton in borneol (37) and camphor (2) 72 60 Postulated cyclisation of a intermediate to bornyl pyrophosphate via and enzyme- bound a-terpinyl structure. 73 61 MVA to camphor (2) 74 62 The fates of the allylic methyl groups on cyclisation to the bicyclo-[2.2.l]- system 75 63 FPP (1_2) cyclisation to campherenone (59) 76 64 Asymmetrical labelling found in monoterpenoid biosynthesis 77 65 Precursors of choice for the biosynthetic study 78 66 Preparation of [8- - and [9- H^- camphor 79 67 Effect of added shift reagent (S.R.) on the H-n.m.r. spectrum of [8- H^]-camphor 80 68 Effect of added shift reagent (S.R.) on the 2 2 H-n.m.r. spectrum of [9- H1]-camphor 81 - ix - Figure. Page, o 69 H-N.m.r. spectrum of a 1:1 mixture of 2 2 [8- H^]- and [9- H-]]- camphor in the presence of excess shift reagent 82 70 Preparation of [2- H2]-mevalonic acid (61.) 88 71 Preparation of 4-acetoxy-2-butanone 88 72 A side reaction in the condensation of 4-acteoxy-2-butanone (64) with ethyl acetate anion 90 73 Proposed synthetic route to [8-^H^]-linalool 91 74 Mechanism of selenium dioxide oxidation 92 75 Selenium dioxide oxidation of linalyl acetate (68) 94 76 Reduction of aldehyde (71.) to alcohol (69) 95 77 Mass spectrum fragmentation of aldehyde (71.) 96 78 Proposed formation of minor product (72) on selenium dioxide oxidation 97 79 Conversion of alcohol (69) to [8- H^] -linalool.... 99 80 Attempted conversion of geraniol to geranyl bromide 99 2 81 H-N.m.r. spectra obtained from camphor extracted from Rosemarinus officianalis after 2 feeding with (a) [2- H2]-MVA, and (b) [S-2^] -linalool 102 82 Scheme for establishing the stereo• chemistry of ring closure 104 - X - ABBREVIATIONS. The following list of abbreviations, most of which are commonly adopted in chemical and biochemical literature, will be employed in this thesis: n Bu butyl, CH3CH2CH2CH2- . CoA coenzyme A. DMAPP dimethylallyl pyrophosphate. Et- ethyl, CH3CH2- . FPP farnesyl pyrophosphate. g.l.c. gas liquid chromatography. • Glu glucoside. GPP geranyl pyrophosphate. HMG 3-hydroxy-3-methyl glutaric acid. IPP isopentenyl pyrophosphate. J coupling constant, Hz. LDA lithium diisopropylamide. LPP linalyl pyrophosphate. MeOH methanol, CH^OH. MsCl methane sulphonyl chloride, CH^SOgCl. MVA mevalonic acid. n.m.r. nuclear magnetic resonance. NPP neryl pyrophosphate. P phosphate P. inorganic phosphate. 1 PP pyrophosphate. - xi - t.l.c. thin layer chromatography. TsCl ^-toluene sulphonyl chloride, (^-CH^C^SC^Cl. V frequency, cm ACKNOWLEDGEMENTS. I would like to thank Dr. R.. Taylor and Mr. J. McPhaill of the U.B.C. Botanical Cardens for.allowing me to "prune" the plants mentioned in this thesis, and Dr. P. Salisbury and Mr. G. Hewitt for advice- on the feeding work. I would also like to thank the members of the electrical,, mechanical.,, and.