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A study on the biosynthesis of camphor Atlay, Thérèse Mary 1983

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A STUDY ON THE BIOSYNTHESIS OF CAMPHOR By THERESE MARY ATLAY B.Sc.(Hons.) U n i v e r s i t y of Exeter, 1976. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Chemistry. We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May 1983. © Therese Mary A t l a y , 1983. In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of OH Q^NA i The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date Starvd r^Sib DE-6 (3/81) - i i -ABSTRACT. This t h e s i s describes an i n v e s t i g a t i o n of the b i o s y n t h e s i s of camphor. Camphor, a b i c y c l i c monoterpene, has been shown to be b i o -synthesised from.geranyl pyrophosphate or i t s isomers ( n e r y l pyro-phosphate or l i n a l o y l pyrophosphate) and s e v e r a l mechanisms have been proposed f o r t h i s c y c l i s a t i o n process. geranyl pyrophosphate camphor In an attempt to d i f f e r e n t i a t e the o r i g i n of the C(8) and C(9) methyl groups,, feeding experiments with a p p r o p r i a t e l y l a b e l l e d pre-cursors were attempted. The two precursors used were [2- H^ J-mevalonic r 2 i a c i d and [8- H ^ J - l i n a l o o l . I t was shown that the expected products, 2 2 2 [8- H^]- and [9- H^]-camphor, could be d i f f e r e n t i a t e d by H-n.m.r. using the s h i f t reagent Eu(thd)^ (Eu(thd)^ = t r i s ( 2 , 2 , 6 , 6 - t e t r a m e t h y l -3, 5-heptanedionato.)europium) . Feeding^ experiments w i t h Rosemarinus o f f i c a n a l i s using the cut stem method showed no i n c o r p o r a t i o n of deuterium l a b e l i n t o camphor extracted from the p l a n t ' s e s s e n t i a l o i l . [2- H ]-mevalonic a c i d [8- H..] - l i n a l o o l i i i -TABLE OF CONTENTS. Abstract i i Table of contents i i i Li s t of figures v Abbreviations x Acknowledgements . x i i I Introduction 1 I - (i) The terpenoids 1 I - ( i i ) Early studies on the terpenoids 2 I - ( i i i ) 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 - ( v i i ) 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 ( i i ) bornanes 50 ( i i i ) thujanes 55 (iv) caranes 57 (e) Cyclopentanoid monoterpenes (iridanes) 60 (f) Irregular monoterpenes 64 II Discussion 70 II - (i) The problem 70 II - ( i i ) The method of labelling 76 II - ( i i i ) The choice of precursors 77 II - (iv) The plant system 83 II - (v) Synthesis of the biosynthetic precursors 87 (i) [ 2-2H2] -Mevalonic, acid (6l_) 87 ( i i ) [ 8-2E^ -Linalool (62) 91 II - (vi) Results 101 II - (vii) Suggestions for further work 103 III Experimental 105 References 123 LIST OF FIGURES. F i g u r e . Page. 1 The coupling of isoprene u n i t s i n a "head-t o - t a i l " f a s h i o n 3 2 Bonner and Arreguin's proposal f o r the early-steps i n rubber b i o s y n t h e s i s . 6 3 The l a b e l l i n g p a t t e r n i n squalene (4) biosyn-1 4 t h e s i s e d from [2- ^ - m e v a l o n i c a c i d (MVA) 8 4 The conversion of acetate to (3R)-mevalonic a c i d (8) 11 5 The va r i o u s pathways to HMGCoA (7a) from general metabolism 12 6 Concerted e l i m i n a t i o n of C0„ and P. to form 2 l mevalonic a c i d 14 7 A n t i e l i m i n a t i o n of CO^ and P_^  from 3-phospho-5-pyrophospho-MVA to form IPP 15-8 The i s o m e r i s a t i o n of IPP to DMAPP (13) 15 9 Stereochemistry of IPP i s o m e r i s a t i o n to DMAPP 17 10 Squalene b i o s y n t h e s i s e d i n a r a t l i v e r 2 homogenate i n the presence of H^ O 18 11 The demonstration of re face a t t a c k of H + i n the i s o m e r i s a t i o n of IPP to DMAPP 20 12 Regiochemistry of attachment of p r e n y l u n i t s 21 13 1'-4 condensation... 21 14 Regiochemistry of condensation t o form pre-squalene or prephytoene pyrophosphates 22 15 Squalene b i o s y n t h e s i s 23 16 Phytoene b i o s y n t h e s i s 24 17 The condensation of the a l l y l i c pyrophosphates and IPP 25 I n v e r s i o n of c o n f i g u r a t i o n a t of the a l l y l i c pyrophosphate i n the 1 '-4- conden-s a t i o n shown by Popjak and Cornforth Stereochemical requirements f o r the form-a t i o n of presqualene pyrophosphate (or prephytoene pyrophosphate) The regiochemistry of a t t a c k of the a l l y l i c s u b s t r a t e of IPP 1'-4 condensation P o s t u l a t e d mechanism of the 1'-4 condensation., Examples of the b a s i c s k e l e t a of the mono-terpenes Ruzicka's scheme- f o r the formation of the monoterpenes The proposed b i o s y n t h e s i s of DMAPP (13) from l e u c i n e (1_8) Some n a t u r a l l y o c c u r r i n g a c y c l i c monoterpenes.. The i n v i t r o transformations of ger a n y l , n e r y l , and l i n a l o y l phosphates P o s t u l a t e d mechanism f o r the formation of n e r o l d i r e c t l y L a b e l l i n g p a t t e r n obtained when rose p e t a l s were f e d with [2-1/iC,4-3H.]-MVA In v i t r o s t u d i e s of the h y d r o l y s i s of the phosphate e s t e r s of l i n a l o o l , g e r a n i o l , and n e r o l Conversion of.GPP t o NPP v i a LPP, without l o s s of hydrogen a t C(1) , Co r i ' s proposed mechanism f o r the d i r e c t formation of NPP from the condensation of IPP and DMAPP Some n a t u r a l l y o c c u r r i n g monocyclic monoterpenes Some aromatic monoterpenes and t h e i r r e l a t i o n s h i p w i t h the non-aromatic mono-terpenes Proposed b i o s y n t h e s i s of y-terpinene and a-thujene , Scheme to provide evidence f o r the exis t e n c e of 1,2-hydride s h i f t s i n monoterpene b i o s y n t h e s i s Examples of the pinanes.. The bornanes Production of the bornanes from the stereo-c h e m i c a l l y appropriate a - t e r p i n y l c a t i o n Synchronous mechanism f o r NPP to BPP Bi o s y n t h e s i s of borneol Revised proposal f o r the b i o s y n t h e s i s of the bornanes . The thujane monoterpenes P o s t u l a t e d b i o s y n t h e t i c scheme f o r the thujanes The carane monoterpenes Proposed method of formation of car-3-ene..... B i o s y n t h e s i s of car-3-ene i n Pinus s y l v e s t r i s . The b i o s y n t h e s i s of car-3-ene, i n v o l v i n g a 1,2-proton s h i f t Examples of the i r i d a n e monoterpenes B i o s y n t h e s i s of the i r i d o i d s , The proposed b i o s y n t h e s i s of the i r i d o i d s - v i i i F i g u r e . Page. 52 I r r e g u l a r roonoterpene b i o s y n t h e s i s 64 53 P o s t u l a t e d d i r e c t formation of fe n c h o l from GPP 66 54 Major s k e l e t a of the i r r e g u l a r monoterpenes 67 55 Proposed b i o s y n t h e s i s of i r r e g u l a r monoterpenes 68 56 The b i o s y n t h e s i s of the i r r e g u l a r mono-terpenes i n v o l v i n g DMVC 69 57 I n c o r p o r a t i o n of [2- UC]-MVA (8) and [2 - ^ ^ C ] - g e r a n i o l {V^) i n t o camphor (2) 71 58 Conversion of geranyl pyrophosphate (14) to b o r n y l pyrophosphate (37) by an enzyme pre p a r a t i o n from sage 71 59 Numbering of the bornane sk e l e t o n i n borneol (37) and camphor (2) 72 60 P o s t u l a t e d c y c l i s a t i o n of a intermediate to b o r n y l pyrophosphate v i a and enzyme-bound a - t e r p i n y l s t r u c t u r e . 73 61 MVA to camphor (2) 74 62 The f a t e s of the a l l y l i c methyl groups on c y c l i s a t i o n t o the b i c y c l o - [ 2 . 2 . l ] - system 75 63 FPP (1_2) c y c l i s a t i o n to campherenone (59) 76 64 Asymmetrical l a b e l l i n g found i n monoterpenoid b i o s y n t h e s i s 77 65 Precursors of choice f o r the b i o s y n t h e t i c study 78 66 P r e p a r a t i o n of [ 8 - - and [9- H^- camphor 79 67 E f f e c t of added s h i f t reagent (S.R.) on the H-n.m.r. spectrum of [8- H^]-camphor 80 68 E f f e c t of added s h i f t reagent (S.R.) on the 2 2 H-n.m.r. spectrum of [9- H 1]-camphor 81 - i x -Figure. Page, o 69 H-N.m.r. spectrum of a 1:1 mixture of 2 2 [ 8 - H^]- and [9- H-]]- camphor i n the presence of excess s h i f t reagent 82 70 Preparation of [2- H 2]-mevalonic acid (61.) 88 71 Preparation of 4-acetoxy-2-butanone 88 72 A side reaction i n the condensation of 4-acteoxy-2-butanone (64) with e t h y l acetate anion 90 73 Proposed synthetic route to [ 8 - ^ H ^ ] - l i n a l o o l 91 74 Mechanism of selenium dioxide oxidation 92 75 Selenium dioxide oxidation of l i n a l y l 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^ ] - l i n a l o o l . . . . 99 80 Attempted conversion of geraniol to geranyl bromide 99 2 81 H-N.m.r. spectra obtained from camphor extracted from Rosemarinus o f f i c i a n a l i s a f t e r feeding with (a) [2- 2H 2]-MVA, and (b) [ S - 2 ^ ] - l i n a l o o l 102 82 Scheme f o r e s t a b l i s h i n g the stereo-chemistry of r i n g closure 104 - X -ABBREVIATIONS. The following l i s t of abbreviations, most of which are commonly adopted in chemical and biochemical literature, w i l l be employed in this thesis: Bu nbutyl, 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 l i n a l y l 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. 1 inorganic phosphate. PP pyrophosphate. - xi -t . l . c . thin layer chromatography. TsCl ^-toluene sulphonyl chloride, (^-CH^C^SC^Cl. V frequency, cm ACKNOWLEDGEMENTS. I would l i k e to thank Dr. R.. Taylor and Mr. J . M c P h a i l l of the U.B.C. B o t a n i c a l Cardens f o r . a l l o w i n g me to "prune" the p l a n t s mentioned i n t h i s t h e s i s , and Dr. P. S a l i s b u r y and Mr. G. Hewitt f o r advice- on the feeding work. I would a l s o l i k e to thank the members of the e l e c t r i c a l , , mechanical.,, and. glassblowing workshops f o r t h e i r e x p e r t i s e i n keeping the lab., equipment f u n c t i o n a l , and the many people who have made my stay at U.B.C. enjoyable. F i n a l l y , I would l i k e to express my g r a t i t u d e to Pro f e s s o r Thomas Money f o r h i s guidance,, encouragement, and anecdotes t h a t have helped to make l i f e i n 34-8 enjoyable - even though I didn't get to see him i n h i s k i l t ! - 1 -I. INTRODUCTION.. I - ( i ) The terpenoids. The group of n a t u r a l products known as the terpenoids i s probably the most widespread and chemically i n t e r e s t i n g group of n a t u r a l products. They have been known from a n t i q u i t y , having been i s o l a t e d from p l a n t s and used f o r a v a r i e t y of purposes, although seldom as the pure compound 1. The group i n c l u d e s many v o l a t i l e compounds which made them r e a d i l y a v a i l a b l e by the simple d i s t i l l a t i o n of p l a n t t i s s u e s , l e a d i n g to the term " e s s e n t i a l o i l " . Many p l a n t essences are known and are used i n perfumery, medicine,, and food f l a v o u r i n g and c o l o u r i n g . Due to t h e i r abundance, a c c e s s i b i l i t y , and r e l a t i v e l y simple c o n s t i t u t i o n , the terpenoids were f a v o u r i t e objects of study by chemists i n the l a t e 1800's and e a r l y 1900's. Many d i s t i n g u i s h e d chemists were a t t r a c t e d to the chemistry of the terpenoids, and amongst them were W a l l a c h 2 , P e r k i n 3 , B r e d t \ and i n more recent times, R u z i c k a 5 and Banthorpe 6. By the e a r l y 1900's, the gross s t r u c t u r e of most of the common terpenoids had been e s t a b l i s h e d , and i n v e s t i g a t i o n s which are s t i l l c o ntinuing have added many d e t a i l s to the stereochemistry, r e a c t i o n s , and b i o s y n t h e s i s of these compounds. Many terpenoids occur f r e e i n p l a n t t i s s u e s , but some have been found as g l y c o s i d e s , organic a c i d e s t e r s , and even i n combination w i t h p r o t e i n s 1 . The lower, members of the c l a s s , having 10 or 15 carbon atoms, can. be. r e a d i l y obtained from p l a n t t i s s u e s by steam d i s t i l l a t i o n . The terpenoids are r e l a t e d by a common o r i g i n and a common s t r u c t u r a l r e l a t i o n s h i p . They are composed of m u l t i p l e s of a 5 carbon u n i t , and are subdivided i n t o groups depending on the number of carbon atoms i n t h e i r s keleton as f o l l o w s : *• Monoterpenoids Sesquiterpenoids Diterpenoids C ^ Q T r i t e r p e n o i d s Carotenoids C ^ Q Rubber ( C _ ) 5 n Few compounds co n t a i n i n g only one 5 carbon atom u n i t , the hemiterpenoid are found to e x i s t i n nature as s t a b l e , i s o l a b l e , metabolic end product However, they are found to e x i s t i n l i v i n g c e l l s as h i g h l y r e a c t i v e intermediates i n terpenoid b i o s y n t h e s i s . I - ( i i ) E a r l y s t u d i e s on the terpenoids. Chemical i n v e s t i g a t i o n of the terpenoids began i n the e a r l y 1800' E m p i r i c a l formulae determinations and chemical degradation s t u d i e s l e d to the concept t h a t t h i s group of compounds could be derived from a 5 carbon atom u n i t . In 1860, W i l l i a m s ^ a heated rubber to 300°C and obtained a product w i t h the chemical formula C^Hg which he c a l l e d "isoprene". I n t h i s experiment, W i l l i a m s a l s o i s o l a t e d a compound of molecular formula C„_H„/ which he c a l l e d "catouchine" and which was 10 16 7b polymeric w i t h isoprene. Bouchardat showed that heating isoprene produced a compound of molecular formula C ^ H ^ , confirming the idea.of W i l l i a m s . S t a r t i n g i n 1884., Wallach began a systematic study of the terpenes that l e d to the f i r s t s t r u c t u r a l formulae of these compounds. 2 e In 1887 he proposed the "isoprene r u l e " which d i d not seem to have much e f f e c t on h i s eoritemporarias. .It -was only -in the 1920's with the - 3 -work of Ruzicka that t h i s r u l e became e s t a b l i s h e d , i n the m o d i f i c a t i o n of Wallach's o r i g i n a l proposal, the "biogenetic isoprene r u l e " 5 . When Wallach proposed h i s hypothesis, the s t r u c t u r e of isoprene was not known, although he b e l i e v e d i t to be as shown ( l _ ) . This was shown to be the c o r r e c t s t r u c t u r e f o r isoprene by Impatiew and W i t t o r f 8 i n 1897. isoprene (D The dimethyl branch of t h i s molecule i s commonly c a l l e d the " t a i l " and the u n s u b s t i t u t e d end the "head". Coupling of t h i s precursor to form the terpenoids i s g e n e r a l l y seen to be i n a " h e a d - t o - t a i l " f a s h i o n (Figure 1) and the terpenoid compounds can generally•be f o r m a l l y d i s s e c t e d i n t o C^-units possessing t h i s arrangement, f o r example, camphor (2) and limonene ( 3 ) . These two compounds are monoterpenoids that " f o l l o w " the isoprene r u l e , t h a t i s , they can be seen to be composed of two isoprene u n i t s combined h e a d - t o - t a i l . When the isoprene r u l e was f i r s t introduced Figure 1. The coupling of isoprene u n i t s i n a " h e a d - t o - t a i l " f a s h i o n . camphor limonene (2) (3) by Wallach i t was exem p l i f i e d by compounds that were r e g u l a r . As time went on, compounds were discovered which were terpenoid i n nature and yet d i d not show t h i s l i n e a r array of isoprene u n i t s . To encompass t h i s type of compound, Ruzicka formulated the bi o g e n e t i c isoprene r u l e which takes i n t o account the f a c t t h a t the compound th a t i s f i n a l l y formed i n the p l a n t , w h i l s t having i t s o r i g i n i n a r e g u l a r i s o p r e n o i d precursor, may have undergone l a t e r a l t e r a t i o n i n s t r u c t u r e during i t s e l a b o r a t i o n i n t o the end product t h a t i s f i n a l l y i s o l a t e d . I - ( i i i ) E a r l y s t u d i e s on terpenoid b i o s y n t h e s i s . Numerous hypotheses f o r terpenoid b i o s y n t h e s i s were put forward-based on s t r u c t u r a l s t u d i e s . - C h o l e s t e r o l was thought to be a member of the terpenoid f a m i l y but e s t a b l i s h i n g t h i s r e l a t i o n s h i p was not easy, as the compound i t s e l f does not have a chemical formula that i s a m u l t i p l e of C^Hg. In 1916, Tsujimoto 9 i s o l a t e d squalene (4.) from shark l i v e r o i l and i t s molecular -formula \ C ^ Q H ^ Q , and some of i t s p r o p e r t i e s were determined. Ten years l a t e r , H e i l b r o n and coworkers 1 0 suggested t h a t squalene may be a precursor of c h o l e s t e r o l (5), and e l u c i d a t i o n of the s t r u c t u r e s of these two compounds gave some strength to t h i s proposal. - 5 -However, i t was not u n t i l s tudies on the b i o s y n t h e s i s of c h o l e s t e r o l using l a b e l l e d precursors were c a r r i e d out that the f i n a l proof of t h i s r e l a t i o n s h i p was found. squalene c h o l e s t e r o l U ) (5) The f i r s t biochemical use of isotopes was c a r r i e d out by Hevesy 1 1 i n 1923, who s t u d i e d the uptake of l e a d i n p l a n t s . With the disc o v e r y of deuterium i n 1932 by U r e y 1 2 , experiments i n b i o s y n t h e s i s began i n 1 3 earnest. Hevesy and Hofer were the f i r s t workers to use deuterium i n a 13 14 biochemical experiment. The discovery of G and G enabled a l a r g e 14 range of s t u d i e s to be c a r r i e d out. L i t t l e and Bloch used acetate 13 14. l a b e l l e d i n e i t h e r the carboxyl or methyl group with C or C to determine the r e l a t i v e u t i l i z a t i o n of the 2 carbon atoms from acetate i n the b i o s y n t h e s i s of c h o l e s t e r o l (5). They determined t h a t 15 of the 27 carbon atoms of c h o l e s t e r o l arose from the methyl group of acetate and 12, presumably the remaining 12, to be from the carboxyl group. Bonner and A r r e g u i n 1 5 proposed that acetate.was a precursor of rubber and c a r r i e d out experiments t h a t showed th a t acetate, acetoacetate, and 3 , 3-dimethylacrylic a c i d (6) were a c t i v e i n supporting rubber formation. This l e d them to propose the f o l l o w i n g s y n t h e t i c sequence f o r the e a r l y steps i n the b i o s y n t h e s i s of rubber (Figure 2). Although t h i s i s now known to be i n c o r r e c t , i t i s i n t e r e s t i n g to note that i t p r e d i c t s the co r r e c t l a b e l l i n g p a t t e r n f o r the isoprene residue (Figure 2). The e a r l y work on the b i o s y n t h e s i s of rubber was reviewed i n 1954-16. At that time, a modified-version of rubber b i o s y n t h e s i s was proposed that i n c l u d e d the formation of a 6-carbori intermediate, 3-hydroxy-3-methyl g l u t a r i c a a c i d (HMG, 7). (a) 2 CH3C0.0H H2° CH3C0CH2C00H CH 3C0CH 3 + CO 2 (b) CH3COCH3 + CH3C00H CH, C = C CH 3 ^GOOH 3 , 3-dimethylacrylic a c i d (6) Figure 2. (a) Bonner and A r r e g u i n ' s 1 5 proposal f o r the e a r l y steps i n rubber b i o s y n t h e s i s , (b) P a t t e r n of l a b e l l i n g of isoprene r e s i d u e , p r e d i c t e d by Bonner and Arreguin's scheme, m = methyl carbon.; c = carboxyl carbon. - 7 -3-hydroxy-3-methyl g l u t a r i c a c i d (HMG) (7) I - ( i v ) The i d e n t i f i c a t i o n of interm e d i a t e s . (a) Mevalonic a c i d . The nature of the C ^ .-starter u n i t was f i r s t discovered i n 1956 by Wolf et a l . 1 7 , when they discovered an a c e t a t e - r e p l a c i n g f a c t o r .for c e r t a i n l a c t o b a c i l l i . The compound was i s o l a t e d and i d e n t i f i e d as 3-hydroxy - 3-methyl-6-valerolactone, or mevalonolactone ( 8 ) . Tavormina and coworkers 1 8 showed that t h i s compound, as the a c i d - a l c o h o l form, could suppress the i n c o r p o r a t i o n of -acetate i n t o c h o l e s t e r o l i n a c e l l - f r e e r a t l i v e r homogenate. Using s y n t h e t i c , l a b e l l e d (3RS)-mevalonic a c i d , they found 43*4% i n c o r p o r a t i o n i n t o the s t e r o l . Assuming only one enantiomer was used, t h i s i s v i r t u a l l y 100% i n c o r p o r a t i o n . Tavormina and G i b b s 1 9 p o s t u l a t e d the f i r s t step i n the i n c o r p o r a t i o n of mevalonic a c i d i n t o the s t e r o l s would be the l o s s of the carboxyl 14 group. To prove t h i s they fed [1- C]-mevalonic a c i d and found no i n c o r p o r a t i o n of isotope i n the c h o l e s t e r o l , u n l i k e w i t h [2-^^ c ]-mevalonic a c i d , i n d i c a t i n g t h a t the carboxyl carbon i s indeed l o s t . Various groups mevalonolactone. (3R)-mevalonic a c i d . (8) (8) of researchers 0 fed [2- C] -mevalonic a c i d (MVA) and examined the l a b e l l i n g p a t t e r n i n squalene, a l l f i n d i n g the r e s u l t s shown i n Figure 3. Amdur,. et al. 2° C a l s o fed [2-^ ^ G, 5-^H]-mevalonic a c i d (MVA) and found t h a t both hydrogens at G(5) of MVA are-retained.--' They al s o showed th a t f o r a yeast enzyme system to convert MVA to squalene, the a d d i t i o n of 2+ Mn , ATP, and a p y r i d i n e n u c l e o t i d e was r e q u i r e d . Figure 3. The l a b e l l i n g p a t t e r n i n squalene (4.) b i o s y n t h e s i s e d from [ 2 - 1 V | -mevalonic a c i d (MVA). ( * = U C ). By 1960, MVA had been shown to be i n c o r p o r a t e d i n t o s e v e r a l terpenoid compounds, i n c l u d i n g a monoterpene, a-pinene (9) 1 9 and i t s p o s i t i o n as the precursor of the. terpenoids was s e t . I t i s now the most g e n e r a l l y used b i o s y n t h e t i c precursor i n terpenoid b i o s y n t h e s i s and a wide v a r i e t y of s y n t h e s e s 2 2 of t h i s molecule are a v a i l a b l e enabling l a b e l l i n g w i t h 1 3 C or at p o s i t i o n s C(2), C(3>, C U ) , C(5), and C(3 ; l), and using H of H at. the pro'chiral'positions' 2,4, and; 5, see ( 8 ) . For the b i o s y n t h e s i s of mevalonic a c i d , i t was reasonable to suggest t h a t i t i s formed" i n the organism from acetate, as acetate i s seen to be i n c o r p o r a t e d i n t o the terpenoids, and so i n v e s t i g a t i o n s i n t o the b i o s y n t h e s i s of MVA.from acetate began. As was proposed by Bonner and A r r e g u i n 1 5 ' 1 6 , acetate was thought to undergo a self-condensation to produce acetoacetate. The enzymic conversion of acetate to acetoacetate i n pigeon l i v e r e x t r a c t s was shown by Soodak and L i p m a n n 2 3 a , who found ATP and coenzyme A to be r e q u i r e d eofactors of t h i s r e a c t i o n . Lipmann 23b and a s s o c i a t e s a l s o found evidence i n d i c a t i n g that both acetate molecules needed ;to be a c t i v a t e d f o r conversion to acetoacetate and they proposed the f i r s t step to be the conversion of acetate to i t s t h i o l e s t e a-pmene (9) (b) From acetate to mevalonic a c i d . 10 -acetylcoenzyme A (acetylGoA, 1_0). This was soon v e r i f i e d by the i s o l a t i o n 2 4-27 of acetylCoA. Various groups proposed the next step a f t e r the condensation to acetoacetate to be the formation of• '3-hydroxy-3-methyl g l u t a r i c a c i d (HMG, 7) or i t s t h i o l e s t e r , HMGGoA - see Figure 4. HMG was i s o l a t e d from various sources, i n c l u d i n g by h y d r o l y s i s of the 2 8 a l k a l o i d d i c r o t a l i n e ,. and i t s existence i n pl a n t s strengthened t h i s p ostulated route. ' Enzymatic r e d u c t i o n of HMG produces (3R)-mevalonic NH, 0 0 0 I  0 0 CHoCSCHoCHoNHC'(CH2)2NHGGHCH2-0-P-0-|-0-CH OH OH OH = CH^SCoA Acetylcoenzyme A (acetylCoA). (10) = phosphate 0 ; II = -P-OH I OH a c i d (Figure 4-)» which was shown to occur i n a yeast e x t r a c t s y s t e i 29 and i n a mammalian enzyme system 3 0 i n the presence of NADPH, ATP, CoA, 2+ glucathione, and Mg as c o f a c t o r s . In t h i s step, 2 hydrogens are t r a n s f e r r e d from the reduced nicotinamide-adenine d i n u c l e o t i d e phosphate (NADPH). The re d u c t i o n i s r e g i o s p e c i f i c , i r r e v e r s i b l e , and "produces only (3R)-MVA. Without NADPH i n the red u c t i o n s y s t e m 3 0 1 3 acetate was shown to,be converted to HMGCoA and no f u r t h e r , showing the re d u c t i o n step' to r e q u i r e NADPH. In the r e d u c t i o n , the 5-pro-S hydrogen of MVA 3 0C i s introduced from NADPH - 11 -2 CH^COSCoA acetylcoenzyme A (10) COSCoA H, •A H, -COSCoA . HO, J3H,H "H* H O O C X Z X O H (3R)-mevalonic a c i d NADP+ (8) NADPH* H C H00C OH COSCoA H1^^'H2 ( 3 S ) - 3 - h y d r o x y - 3 - m e t h y l -glutarylCoA (7a) Figure 4. The conversion of acetate to (3R)-mevalonic a c i d ( 8 ) . As f a r as can be as c e r t a i n e d , MVA i s not produced by any other pathway, nor i s i t used f o r anything other than the synth e s i s of the terpenoids. The t h i o l e s t e r , HMGCoA, on the other hand, l i e s on more than one b i o s y n t h e t i c r o u t e , and i t s proposed methods of formation are o u t l i n e d i n Figure 5. HMGCoA i s probably the only connection, and a one way one at t h a t , l e a d i n g from general metabolism to mevalonic a c i d , the key intermediate i n terpenoid b i o s y n t h e s i s . Only the ( 3 R ) - form of mevalonic a c i d i s used i n metabolism, the ( 3 S ) - form i s m e t a b o l i c a l l y i n e r t . Figure 5. The various pathways to HMGCoA (7a) from general metabolism. - 13 -(c) From mevalonic a c i d to the 'active isoprene u n i t ' . The next question i n the b i o s y n t h e s i s of the terpenoids was the conversion of mevalonic a c i d i n t o the 'ac t i v e isoprene u n i t ' . Studies on the metabolism of mevalonic a c i d l e d to the i d e n t i f i c a t i o n of 5-phosphomevalonic a c i d and 5-pyrophosphomevalonic a c i d (phospho :-0 0 0 -0-P-0H ; pyrophospho :- -0-P-0^P-0H ). Chaykin et a l . 3 1 i d e n t i f i e d OH OH OH 5-pyrophosphomevalonic a c i d as a r i s i n g from the metabolism of 5-phospho-mevalonic a c i d . These workers a l s o found a new compound which, when 1L. -MVA was used, was not l a b e l l e d , t h a t i s , the carboxyl group of MVA had been l o s t . They t e n t a t i v e l y i d e n t i f i e d t h i s compound as A-iso p e n t e n y l pyrophosphate ( l _ t ) . Lynen and coworkers 3 2 i s o l a t e d and i d e n t i f i e d t h i s ' compound as A-isopentenyl pyrophosphate (IPP, 1_1_) and examined i t s formation and use i n e x t r a c t s from yeast c e l l s . They found 0-P-0-P-0H OH OH A-isopentenyl pyrophosphate (IPP). (11:) th a t yeast c e l l s , i n the absence of NADPH, d i d not convert MVA or 5-pyrophosphomevalonic a c i d (5-pyrophospho-MVA) to squalene, but to a ^-compound, f a r n e s y l pyrophosphate (FPP, 1_2). Incubation of the yeast e x t r a c t s w i t h 5-pyrophospho-['2A 4C]-MVA i n the presence of iodoacetamide an enzyme i n h i b i t o r , gave not FPP but l a b e l l e d IPP. S y n t h e t i c , l a b e l l e d IPP incubated i n the yeast enzyme system with NADPH and Mg 2 + gave r a d i o a c t i v e squalene, and without NADPH gave r a d i o a c t i v e FPP, - u -OPP PP = pyrophosphate 0 0 II II =-P-0-P*0H 1 I OH OH F a r n e s y l pyrophosphate (FPP). (12) . i n d i c a t i n g IPP to be the ' a c t i v e isoprene u n i t ' . Examination of the mechanism.of IPP f o r m a t i o n 3 3 was c a r r i e d out by converting MVA to » 2 squalene i n the presence of H^O. This very p r e l i m i n a r y study showed that l e s s than one deuterium atom per molecule of MVA was incorporated i n t o squalene i n d i c a t i n g that decarboxylation occurs without protonation of the earbon chain. K i n e t i c studies revealed 00^ formation and ADP formation to occur at i d e n t i c a l r a t e s , w i t h no l a g period,' i n d i c a t i n g the r e a c t i o n to be a concerted e l i m i n a t i o n of i n o r g a n i c phosphate and C0_, Figure 6. I n t h i s r e a c t i o n , 5-pyrophospho-MVA undergoes a Figure 6. Concerted e l i m i n a t i o n of C0„ and P. to form IPP. phosphorylation to give the 5-pyrophospho-3-monophospho- d e r i v a t i v e . This compound then e l i m i n a t e s CO^ and i n o r g a n i c phosphate (P^) i n a concerted process to y i e l d IPP. The e l i m i n a t i o n has been e s t a b l i s h e d to be a n t i 3 - ( F i g u r e 7). L a b e l l i n g studies have shown the oxygen from the t e r t i a r y a l c o h o l to be i n the i n o r g a n i c phosphate a f t e r t h i s r e a c t i o n 3 5 . Figure 7. A n t i e l i m i n a t i o n of CO^ - and P^ from 3-phospho-5-pyrophospho-MVA to form IPP. (d) IPP to other t e r p e n y l pyrophosphates. Examination of the conversion of IPP ( i t ) to FPP (1_2) showed the f i r s t step to be the conversion of IPP to i t s a l l y l i c isomer, dimethyl-a l l y l pyrophosphate (DMAPP, 13), Figure 8 3 6 . (II) (13) Figure 8. The i s o m e r i s a t i o n of IPP to DMAPP.(t3) An enzyme prep a r a t i o n from baker's yeast was shown to convert 1 h s y n t h e t i c [1- C]-IPP to r a d i o a c t i v e DMAPP. At about the same time :as - 16 -DMAPP was i d e n t i f i e d and shown to a r i s e by. a l l y l i c rearrangement of IPP, 3 7 3 8 / 3 8 other a l l y l i c pyrophosphates were found ' . Goodman and Popjak i d e n t i f i e d geranyl ( H ) , f a r n e s y l (l_2), and d i m e t h y l a l l y l (13) pyro-phosphates as intermediates i n the conversion of MVA to squalene, and OPP Geranyl- pyrophosphate (GPP). (u) i t was p o s s i b l e to summarise the conversion of MVA to squalene as f o l l o w s : MVA -IPP IPP -DMAPP IPP. + DMAPP —GPP . GPP + IPP ••FPP FPP + FPP N A D P H - squalene I - (v)„ Stereochemical aspects of terpenoid formation, (a) IPP i s o m e r i s a t i o n . The stereochemistry of the i s o m e r i s a t i o n of IPP to DMAPP was 3 9 — 4 1 examined by many groups . I n examining t h i s r e a c t i o n there are 3 questions which must be answered :-(1) Which proton i s removed from 0(2) of IPP i n forming DMAPP? - 17 -(2) What i s the stereochemistry of the new methyl group i n DMAPP? . (3) I s the new methyl group formed by re or s i face protonation of the double bond i n IPP? The f i r s t of these questions was answered by Gornforth et a l . 3 9 w h i l s t working on the b i o s y n t h e s i s of squalene with p a r t i c u l a r i n t e r e s t i n the stereochemistry of the condensation of IPP with DMAPP. I n t h i s study, Cornforth f e d (3R.4R)- and (3R.4S)- .[ 2- 1 ^ C,4-3H] -MVA to an enzyme prep a r a t i o n from r a t l i v e r and i s o l a t e d the squalene produced. 3 He found that the ( 4 S ) - isomer gave no H i n the squalene produced, 3 14 whereas the ( 4 R ) - isomer had an i d e n t i c a l H/ G r a t i o as i n the s t a r t i n g m a t e r i a l . This he concluded to show that the ( 4 S ) - hydrogen of MVA (,(2R)-hydrogen of IPP)' i s - l o s t i n the i s o m e r i s a t i o n of IPP to DMAPP and'in the subsequent coupling r e a c t i o n , i . e . , the stereochemistry of i s o m e r i s a t i o n i s i d e n t i c a l to that of bond formation, and i s as/shown,; -.(Figure 9) . " HOOC Figure 9. Stereochemistry of I P P • i s o m e r i s a t i o n to DMAPP. From C o r n f o r t h 3 9 . - 18 -For the second of these problems, van Tamelen 4 0 examined the 1 H-n.m.r. spectrum of squalene b i o s y h t h e s i s e d i n a r a t l i v e r homogenate 2 prepared- i n H^O, and found a decrease i n i n t e n s i t y of the s i g n a l at 1.67 ppm, designated as the oo-(E)-methyl group. This,' he suggested, 2 i n d i c a t e d the methyl group was l a b e l l e d w i t h H, and i s thus the methyl group produced by the i s o m e r i s a t i o n , see Figure 10. 1.57 1.61 1.61 1.61 1.61 1.57 Figure 10. Squalene bio s y n t h e s i s e d i n a r a t liver-homogenate i n the 2 presence of H^ O-. Numbers i n d i c a t e chemical s h i f t ( i n ppm) of the methyl groups. The f i n a l question i n the stereochemistry of t h i s i s o m e r i s a t i o n r e a c t i o n was not solved u n t i l i d e n t i f i c a t i o n of the c h i r a l i t y of a -methyl group l a b e l l e d w i t h a l l three hydrogen isotopes was achieved. For t h i s problem, C o r n f o r t h 4 1 used two enzyme systems of known- enantio-s e l e c t i v i t y to s p e c i f i c a l l y remove: a c e r t a i n p r o c h i r a l hydrogen. Cornforth incubated (2R,3R)-[2- 3H]- and (2S.3R)-[2- 3H]- MVA w i t h s o l u b l e 2 enzymes from p i g l i v e r i n H^O and i s o l a t e d the f arnesylpy'rophosphate (FPP) produced. This was ..hydrolysed to the f r e e a l c o h o l and then subjected to o z o n o l y s i s . The acetone produced by the t e r m i n a l i s o -p r o p y l i d i n e group was c o l l e c t e d and degraded to iodoform and acetate without the opportunity f o r 'exchange of the methyl hydrogens with the - 19 -medium, and the acetate was converted to acetylCoA. In the f i r s t of the two enzymatic r e a c t i o n s , t h i s acetylCoA was condensed with g l y o x a l a t e to form (S)-malate (see Figure 1 1 ) . Incubation of t h i s (S)-malate w i t h fumarase allowed f o r e q u i l i b r a t i o n between (S)-malate and the conjugated 3 a l k e n e - d i a c i d 1_5. The percentage H remaining bound to carbon a f t e r t h i s i s o m e r i s a t i o n was- measured. Due to the stereochemistry of the fumarase, ( R ) - [ 2 H , 3 H ] -acetate should r e t a i n most of i t s t r i t i u m , whereas ( S ) - [ 2 H , 3 H ] -acetate should l o s e most of i t s t r i t i u m . I n t h i s experiment, acetate obtained from FPP produced when (2R,3R)-[ 2- JH] -MVA was fed gave a malate which r e t a i n e d 63. k% of i t s . t r i t i u m , whereas acetate produced from FPP a f t e r feeding w i t h (2S,3R)-[2- 3H]-MVA gave a malate which l o s t 63.5% 2 of i t s t r i t i u m , thus i n d i c a t i n g that H i s added to the re face of the double bond i n the i s o m e r i s a t i o n of I P P t o - DMAPP (Figure 1 1 ) . (b) Condensation of DMAPP with IPP. To form the terpenoids, IPP and DMAPP condense to form geranyl pyrophosphate (GPP,1U), the C^Q precursor of the monoterpenoids. GPP, being an a l l y l i c pyrophosphate, can condense with a second molecule of IPP to form f a r n e s y l pyrophosphate (FPP, .12), the C ^ precursor of the sesquiterpenoids. The regiochemistry of t h i s attachment can be described as h e a d - t o - t a i l , or as a 1l-A condensation (Figure 12) as o u t l i n e d by P o u l t e r et a l . 1 * 2 . This condensation reorganises three bonds: • 1. Cleaves the C-0 bond i n the a l l y l i c pyrophosphate (prenyl donor). 2. Forms a bond between the pr e n y l donor and the pren y l acceptor (IPP). - 20 -Figure 11. The demonstration of re face attack of H i n the i s o m e r i s a t i o n of IPP to DMAPP. From Cornforth et a l . 1 * 1 - 21 -l 1'-4 1 '-1 ( h e a d - t o - t a i l ) (head-to-head) Figure 12. Regiochemistry of attachment of pr e n y l u n i t s . ( c f . P o u l t e r 3. Loses H from e i t h e r the pr e n y l acceptor or the pr e n y l donor, e.g., f o r a 1'-4 condensation (Figure 13)» Figure 13. 1'-4 condensation. Condensation of IPP with an a l l y l i c pyrophosphate gives the C -homologue, so geranyl pyrophosphate i s produced from DMAPP and IPP. 5 GPP, condenses w i t h IPP.to form f a r n e s y l pyrophosphate (FPP), and FPP can condense w i t h IPP to produce g'eranylgeranylr-pyrophosph'ate (GGPP, 16) the C ? n precursor of the diterpenes. To form the precursor of the et a l . 4 2 ) . OPP t r i t e r p e n o i d s and s t e r o i d s , squalene (4) > two molecules of f a r n e s y l pyrophosphate (FPP) condense together i n an o v e r a l l or head-to-head, f a s h i o n -(Figure 12) and s i m i l a r l y f o r phytoene, the carotenoid precursor, two molecules of geranylgeranyl pyrophosphate (GGPP, 16) condense 'head-to-head'. This condensation.is thought to be a 1'-2,3 condensation (Figure 14-) •' to form a cyclopropane r i n g , g i v i n g presqualene pyrophosphate or prephytoene pyrophosphate, followed by opening of the cyclopropane r i n g to give.a carbocation which i s quenched by hydride from NADPH (see Figures 15 (squalene b i o s y n t h e s i s ) and 16 (phytoene b i o s y n t h e s i s ) ) . The condensations of the a l l y l i c precursors and IPP iare summarised i n Figure 17. 1'-2,3 condensation. Figure 14-. Regiochemistry of condensation to form presqualene or prephytoene- pyrophosphates'* 2. The stereochemistry of the 1'-4 condensation was studied by Popjak 3 9 , 4 3 -and Cornforth during- work on the stereochemistryof squalene b i o s y n t h e s i s . These workers found the same stereochemistry of condensation i n two enzyme systems from widely d i f f e r i n g sources and concluded t h a t the stereochemistry i s a "highly- conserved property of pr e n y l t r a n s f e r a s e s . Their work answered three major questions about OPP presqualene pyrophosphate Figure 15. Squalene bi o s y n t h e s i s /OPP - 25 -Monoterpenes DMAPP + IPP + nIPP Geranyl pyrophosphate Rubbers + IPP Sesquiterpenes d i n i G r i s s F a r n e s y l pyrophosphate «- Squalene 1 '-1 P h y t o s t e r o l s T r i t e r p e n o i d s + IPP S t e r o i d s Geranylgeranyl pyrophosphate • Diterpenes dimerise 11-1 Phytoene %*• Carotenoids. Figure 17. The condensations of the a l l y l i c pyrophosphates and IPP. t h i s stereochemistry: a. I s there r e t e n t i o n or i n v e r s i o n of c o n f i g u r a t i o n at C^  of the a l l y l i c pyrophosphate? b. Which side of the double bond i n IPP i s attacked? c. Which proton i s l o s t i n forming the new double bond? a. Squalene was bio s y n t h e s i s e d from (3R,5R)-[5- H]-MVA using an enzyme prep a r a t i o n from r a t l i v e r . The l a b e l l e d squalene produced was subjected to o z o n o l y s i s followed by cleavage of the k e t o - a c i d produced from carbon atoms 3 to 6 to an o p t i c a l l y a c t i v e s u c c i n i c a c i d . This s u c c i n i c a c i d was concluded to. have the ( R ) - c o n f i g u r a t i o n as i t s ORD - 26 -curve matched th a t of s y n t h e t i c (R)-[ H ] - s u c c i n i c a c i d . I t was thus concluded t h a t i n v e r s i o n of c o n f i g u r a t i o n had occurred at C_(1) of the a l l y l i c moiety i n the 1'-4 condensation (Figure 18). 1'-1 d i m e r i s a t i o n V ( R ) - s u c c i n i c a c i d . Figure 18. I n v e r s i o n of c o n f i g u r a t i o n at of the a l l y l i c pyrophosphate i n the 1'-4 condensation shown by Popjak and C o r n f o r t h 3 9 ' 4 3 . - 27 -As has been mentioned e a r l i e r , the b i o s y n t h e s i s of squalene (or phytoene) i n v o l v e s a "head-to-head" or condensation of 2 molecules of FPP (or GGPP). This r e s u l t s i n i n v e r s i o n of c o n f i g u r a t i o n at the te r m i n a l carbon atom of one molecule i n the r e a c t i n g species and r e t e n t i o n of c o n f i g u r a t i o n a t the t e r m i n a l carbon atom i n the other molecule. At the carbon atom which shows r e t e n t i o n of c o n f i g u r a t i o n , one of the hydrogen atoms i s seen to exchange. This i s the 5-pro-S hydrogen of mevalonic a c i d and the proposed b i o s y n t h e s i s of squalene (and phytoene) takes t h i s i n t o account by proposing the intermediacy of presqualene pyrophosphate (or prephytoene pyrophosphate; both of these compounds have been i s o l a t e d from n a t u r a l sources, Figures 15, 16, and 19). HOOC O P P i \ 1 i n v e r s i o n of c o n f i g u r a t i o n . H" H H* H » H ' H* H * HT t H * I H H I H ' H net r e t e n t i o n of c o n f i g u r a t i o n . H A from NADPHA. Figure 19. Stereochemical requirements f o r the formation of presqualene pyrophosphate (or prephytoene pyrophosphate). - 28 -b. The i n s i t u b i o s y n t h e s i s of (Z)-[A- H ] - I P P from (2R.3R)-2 [2-^H] -MVA showed that a d d i t i o n of the a l l y l i c moiety to I P P occurs to / \ •-/ \ 4 3 b the s ± face of the 0(3)-0(4) double bond i n IPP . The f a r n e s o l produced from (2R.,3R)-[2- H]-MVA was i s o l a t e d and ozonised to give an o p t i c a l l y a c t i v e s u c c i n i c a c i d , found to be ( R ) . This i n d i c a t e s the attack to be on the .si face (Figure 20.), y/+ DMAPP Topology of substrates during 1'-4- condensation. Figure 20. The regiochemistry of attack of the a l l y l i c substrate on I P P . - 29 -c. This question has been answered by C o r n f o r t h 3 9 when a s c e r t a i n i n g which hydrogen i s l o s t i n the i s o m e r i s a t i o n of IPP to DMAPP (Figure 9, Sec t i o n I - (v)-(a) ). I t i s reasonable to assume the stereochemistry of the 1'-4- c o u p l i n g - i s the same as t h a t ' o f ;the : 'isomerisation of IPP to DMAPPas the l a t t e r can be envisioned as a 1'-4- condensation with H + t a k i n g the place of the a l l y l i c pyrophosphate. (Figure 21).'... Figure 21. 1'-4- condensation. I - ( v i ) . Mechanistic s t u d i e s . The mechanism of the 1'-4- condensation has been examined 4 5 :and the r e s u l t s reviewed by C o r n f o r t h 4 6 . The 1'-4- condensation r e a c t i o n shows two major points:' 1 . There i s a clean i n v e r s i o n of c o n f i g u r a t i o n at C(1) of the a l l y l i c pyrophosphate - c o n s i s t e n t w i t h a concerted process i n which C-0 bond cleavage i s accompanied by C-C bond formation. 2 . Formation of the C-C bond i s not concerted w i t h the e l i m i n a t i o n of H from &(-2)-of IPP.,- •since t h i s would invoke a concerted s u p r a f a c i a l r e a c t i o n , and r e a c t i o n s of t h i s type are thought to be stereochemically unfavourable. To e x p l a i n these two p o i n t s , a 2-step mechanism has been p o s t u l a t e d , i n v o l v i n g the t r a n s - a d d i t i o n of the a l l y l i c group and an unknown.group X to the double bond of IPP followed by a t r a n s - e l i m i n a t i o n of the 2-pro-R hydrogen of IPP and group X to produce a new double bond ' (Figure 22)^ This mechanism i s , so f a r , based s o l e l y on stereochemical c o n s i d e r a t i o n s and although i t i s a l o g i c a l mechanism, stereochemical grounds alone are i n s u f f i c i e n t proof that i t i s c o r r e c t . Figure 22. P o s t u l a t e d mechanism of the 1'-4 condensation. I - ( v i i ) . From, geranyl-pyrophosphate" t o . the monoterpenes« - 31 -(a) I n t r o d u c t i o n . The uses of monoterpenes i n p l a n t s are v a r i e d , and have been demonstrated by many workers ' . Monoterpenes o c c u r r i n g i n i n s e c t s .•-.' have been seen to f u n c t i o n as defence substances or i n chemical communication, the v o l a t i l i t y and chemical complexity (and hence p o t e n t i a l i n f o r m a t i o n content) making them i d e a l f o r such purposes. Monoterpenes i n p l a n t s have been shown to act as a t t r a c t a n t s f o r 4 8a p o l l i n a t i n g or seed d i s p e r s i n g i n s e c t s or animals , to r e p e l browsing animals and i n s e c t pests'* 8^, and i n r e s i s t i n g m i c r o b i a l a t t a c k 1 * 8 0 . Several hundred monoterpenes are known 4 9 and can be assigned to one of four broad c a t e g o r i e s : 1. ' Regular a c y c l i c . 2. Gyclohexanoid; i ) monocyclic. i i ) b i c y c l i c . 3. Cyclopentanoid. 4. I r r e g u l a r . Category 2 (cyclohexanoid) encompasses the l a r g e s t number of monoterpenes. Examples of the b a s i c s k e l e t a f o r the monoterpenes are shown i n Figure 23. Several reviews of monoterpene formation have appeared, of which the reviews by Banthorpe et a l . 6 in. 1972 and C o r n f o r t h 5 0 i n 1968 deserve mention. In 1953, R u z i c k a 5 proposed a general scheme f o r the formation of the r e g u l a r monoterpenes, plus some of the i r r e g u l a r , which to t h i s date has not been shown to be i n c o r r e c t . The scheme i n v o l v e s the c y c l i s a t i o n of a C i n d e r i v a t i v e to give the a - t e r p i n y l c a t i o n (l_7) which Regular. dlmethyloctane p_-menthane pinane bornane i r i d a n e thuj ane I r r e g u l a r . carane fenchane isocamphane artemisane santolinane chrysanthemane Figure 23. Examples of the b a s i c s k e l e t a of the monoterpenes. - 33 -fenchane bornane isocamphane carane Figure 2k. Ruzicka's scheme f o r the formation of the monoterpenes. can undergo f u r t h e r r e a c t i o n s to y i e l d the various monoterpenes (Figure 21,). The study of monoterpene b i o s y n t h e s i s , although outwardly appearing simply an extension of the b i o s y n t h e t i c studies of the higher terpenes, i s much more d i f f i c u l t . I n c o r p o r a t i o n of a t r a c e r i n t o p l a n t systems i s fraught w i t h problems, and low i n c o r p o r a t i o n r a t e s are us u a l . For sesqui-and higher terpenes, the problem of i n c o r p o r a t i o n does not a r i s e , these terpenes are a l s o found as fun g a l metabolites or o c c u r r i n g i n animal - 34 -t i s s u e s from which c e l l - f r e e enzyme systems can be r e l a t i v e l y e a s i l y obtained, as w e l l as i n higher p l a n t s . The system, i n these cases, can' be maintained on a n u t r i e n t broth containing the l a b e l l e d precursor and high i n c o r p o r a t i o n l e v e l s achieved. With the exception of the i r i d o i d s , monoterpenes occur c h i e f l y i n higher p l a n t s and u n t i l r e c e n t l y s t u d i e s have i n v o l v e d feeding to an i n t a c t p l a n t or shoots. The monoterpenes i n these p l a n t s are c o l l e c t e d i n s p e c i a l i s e d o i l glands i n the l e a f t i s s u e , and i n c o r p o r a t i o n of mevalonic a c i d or other precursors i s seen to be very low (=0.1 - 0.01 %)51. The high i n c o r p o r a t i o n r a t e seen f o r sesqui- and higher terpenes of mevalonic a c i d allows the acceptance-of mevalonic a c i d as the terpene " s t a r t e r u n i t " , and i t i s reasonable to assume that mevalonic a c i d i s indeed the monoterpene precursor and i t s low i n c o r p o r a t i o n r a t e i s a r e s u l t of the system being examined. Many r a t i o n a l e s f o r t h i s low i n c o r p o r a t i o n have been put forward the most g e n e r a l l y accepted being the compartmentation of monoterpene bi o s y n t h e s i s i n t o s i t e s not r e a d i l y a c c e s s i b l e to exogenous, precursors stopping the i n c o r p o r a t i o n . Compartmentation i s w e l l known and regarded 5 l b as an important feature i n r e g u l a t i o n . Production of mevalonic a c i d i n the p l a n t could occur where i t i s r e q u i r e d , and t r a n s l o c a t i o n could be d i f f i c u l t . Evidence i n support- of t h i s can be seen i n the b i o s y n t h e s i s of g e r a n i o l i n rose p e t a l s and various other p l a n t s 5 2 . The i n c o r p o r a t i o n of MVA into- g e r a n i o l i n - v a r i o u s p l a n t species has been shown to be 52b 53 low whereas when using flower petals. which do not contain o i l glands and can thus be assumed to be devoid of compartmentation e f f e c t s , i n c o r p o r a t i o n i s - high. In conjunction t o - t h i s low l a b e l l i n g seen i n monoterpene b i o s y n t h e s i s , an unsymmetrical d i s t r i b u t i o n of the l a b e l i s seen, that i s , that part of the molecule derived from IPP i s found to c a r r y m a j o r i t y of the l a b e l , w h i l s t the part derived from DMAPP - 35 -51 54.57 c a r r i e s l i t t l e or no detectable l a b e l . This i s g e n e r a l l y a t t r i b u t e d to condensation of a small amount of l a b e l l e d IPP derived from exogenous l a b e l l e d MVA wi t h DMAPP present i n a metabolic pool before i s o m e r i s a t i o n of IPP to DMAPP can occur. I t i s p o s s i b l e t h a t such pools p a r t i c i p a t e i n the b i o s y n t h e s i s of other terpenes but such p a r t i c i p a t i o n i s only •.noticeable when i n c o r p o r a t i o n of exogenous • precursor i s low. With rose p e t a l s , having a high i n c o r p o r a t i o n r a t e , there i s an•equivalent l a b e l l i n g of the 5 *• carbon atom u n i t s 5 3 . Other reasons f o r the unsymmetrical l a b e l l i n g 5 5 i n c l u d e the p o s s i b i l i t y that DMAPP i s not of d i r e c t mevalonoid o r i g i n , that -.ex'ogenously administered MVA may i n h i b i t IPP-isomerase, or a combination of both - th a t when IPP-isomerase i s i n h i b i t e d (by MVA), DMAPP i s formed from a non-mevalonoid o r i g i n . Experiments using l a b e l l e d leucine'- 5 6 have given some i n d i c a t i o n t h a t DMAPP may be derived from l e u c i n e (_1_8) as shown i n Figure 25. However, con c l u s i v e evidence f o r t h i s r e a c t i o n scheme has not yet been shown. The development of c e l l - f r e e systems f o r the examination of monoterpene b i o s y n t h e s i s w i l l ease.these problems and many r e l a t i v e l y * 5 9—61 crude' c e l l - f r e e systems have recently'been^examined (b) Regular a c y c l i c monoterpenes. Conversion of GPP to the various members of the a c y c l i c monoterpenes i s r e l a t i v e l y easy to envisage.. H y d r o l y s i s of GPP gives g e r a n i o l , and GPP or g e r a n i o l can be transformed i n t o the v a r i e t y of a c y c l i c monoterpenes by o x i d a t i o n , r e d u c t i o n , etc.(see Figure 26). L a b e l l i n g studies using exogenous « MVA have shown l a b e l l i n g patterns i n the a c y c l i c monoterpenes to be con s i s t e n t w i t h t h i s suggestion. I n v i t r o s tudies of the h y d r o l y s i s of .geranyl, n e r y l , and 'Iinalpyl.'phosphates have shown - 36 -NH, C0 2H (18) l e u c i n e OH SCoA i s o v a l e r i c acid-CoA SCoA OPP (13) DMAPP Figure 25. The proposed b i o s y n t h e s i s of DMAPP {Y3) from l e u c i n e (18) g e r a n i o l (1 4-) n e r o l (19) l i n a l o o l (20) * ^ C H 0 c i t r o n e l l o l (21) c i t r a l (22) Figure 26. Some n a t u r a l l y o c c u r r i n g a c y c l i c monoterpenes. the formation of n a t u r a l l y o c c u r r i n g a c y c l i c (and monocyclic) mono-terpene a l c o h o l s and hydrocarbons 6 2. The h y d r o l y s i s of these phosphate este r s can be considered as a model r e a c t i o n f o r the transformations of GPP i n v i v o (Figure 27). (23). (24.) (25) myrcene cis-ocimene trans-ocimene Figure 27. The i n v i t r o transformations of geranyl, n e r y l , and l i n a l o y l phosphates. (c) Monocyclic monoterpenes (p_-menthanes) . - 38 -Formation of the monocyclic monoterpenes from geranyl •''pyrophosphate' (GPP, 1_4) does not seem feasable due to the trans-6,7-double bond. Conversion of GPP to n e r y l pyrophosphate (NPP, 1_9)., w i t h the ci_s-stereo-chemistry,., or linaloyl-'pyrophosphate (LPP, 2 0 ) , the a l l y l i c isomer, seems p r e r e q u i s i t e f o r c y c l i s a t i o n to occur. C y c l i s a t i o n can then proceed by 2 2 1 as (nerol) or ( l i n a l o o l ) mechanism to give the bas i c skeleton. Ruzicka 5. p o s t u l a t e d a nerol-type d e r i v a t i v e (Figure 24) and the formation of NPP d i r e c t l y from DMAPP and IPP can be envisioned i f , before e l i m i n a t i o n of H + and X i n the o u t l i n e d mechanism ( s e c t i o n I - ( v i ) ) r o t a t i o n occurs about the C(2)-C(3) bond of IPP, g i v i n g on e l i m i n a t i o n the cis-double bond d i r e c t l y (Figure 2 8 ) . I f t h i s occurred, then the 4-pro-R hydrogen of MVA would be l o s t , as i s found i n the b i o s y n t h e s i s of rubber ( a l l c i s - ) . Work by Banthorpe 5 3 on t h i s problem was c a r r i e d out b y f f e e d i n g (4R)-[ 2 - U C , 4 - 3 H ] - and ( 4 S ) - [ 2 - U C , 4 - 3 H ] - MVA (racemic at C ( 3 ) ) to rose p e t a l s . The g e r a n i o l and n e r o l i s o l a t e d were examined 3 1/. 14 14 f o r H/ C r a t i o and l a b e l l i n g p o s i t i o n s of C. L a b e l l i n g w i t h C 3 14 -was as expected (Figure 2 9 ) . The H/ C r a t i o was maintained when the (4-R)-isomer was fed and H l o s t when the (4S)-isomer was fed f o r both the g e r a n i o l and n e r o l produced. This i n d i c a t e d that r o t a t i o n about the C(2)-G(3) bond of IPP does not occur to give the cis-double bond of n e r o l . This was taken to i n d i c a t e t h a t GPP i s formed from DMAPP and IPP and then isomerised to NPP or n e r o l . Follow-up work 6 3 i n d i c a t e d t h i s conversion to occur by. a redox r e a c t i o n . In studying the b i o s y n t h e s i s of a b i c y c l i c monoterpenoid, d-3-thujone, Banthorpe et a l . found a 3 14 l o s s of t r i t i u m when [ 1 - H, C ] - g e r a n i o l was used, and one t r i t i u m atom was l o s t from- [5- 3H 9,2-^^c]-MVA. Using [ l - 3 H , ^ ^ c ] - n e r o l gave no t r i t i u m - 39 -HOOC +H H *HH pyrophosphate Figure 28. P o s t u l a t e d mechanism f o r the formation of n e r o l d i r e c t l y . - 40 -OH 'OH (14) (19) g e r a n i o l n e r o l Figure 29. . L a b e l l i n g p a t t e r n obtained when rose p e t a l s were fed w i t h [2- UC,4- 3H]-MVA. * = U C . 3H not marked. l o s s , i n d i c a t i n g t h a t before c y c l i s a t i o n , GPP was isomerised to NPP, probably v i a the aldehyde c i t r a l (22). S t e r e o s p e c i f i c l o s s of the (pro-1S) hydrogen of g e r a n i o l was shown to occur. However, Suga and c o w o r k e r s 5 ^ examined the conversion of [5-3H,2-^^C]-MVA to monoterpenes i n s e v e r a l p l a n t species and found no l o s s of t r i t i u m i n the conversion of GPP to NPP. E a r l i e r , A t t a w a y 6 5 proposed LPP' as the intermediate precursor of.the monocyclic monoterpenes, and the conversion of GPP to LPP without l o s s of hydrogen at C(1) can be envisioned -(Figure 30) based H 0 (22) c i t r a l - 41 -Figure 30. In v i t r o s tudies of the h y d r o l y s i s of the phosphate e s t e r s of l i n a l o o l , g e r a n i o l , and n e r o l . on i n v i t r o s t u d i e s of the h y d r o l y s i s of the phosphate est e r s of l i n a l o o l , g e r a n i o l and n e r o l 6 6 . Further evidence supporting the preference of LPP over GPP or NPP, and f o r the conversion of GPP to LPP without l o s s of C(1) hydrogen, was shown by S u g a 5 8 using a c e l l - f r e e system from 14 3 Mentha s p i c a t a L. to produce a - t e r p i n e o l from [ l - C,1- HJ- NPP, LPP, 3 14 and GPP. The same H/ C r a t i o s were found i n the i s o l a t e d a - t e r p i n e o l compared to t h a t i n the precursor f e d , and LPP was found to be incorporated to the greater amount.. I t i s p o s s i b l e t h a t GPP i s converted to NPP v i a LPP, without l o s s of H at C'(1) (Figure 31). As - 42 -0PPT (u) geranyl pyrophosphate (20) pyrophosphate (20) (19) n e r y l pyrophosphate Figure 31. Conversion of GPP to NPP v i a LPP, without l o s s of hydrogen at G(1). i s shown, t h i s would i n v o l v e an i n v e r s i o n of c o n f i g u r a t i o n at C('1). Several examples of the i s o m e r i s a t i o n of a t r a n s - to a c i s - double bond without l o s s of hydrogen at C(1) have been demonstrated f o r a number of s e s q u i t e r p e n e s 5 7 but the i n v e r s i o n of c o n f i g u r a t i o n t h i s mechanism proposes had not been examined. Shine and L o o m i s 6 8 i s o l a t e d an enzyme capable of i s o m e r i s i n g GPP to NPP without the detectable intermediacy-of c i t r a l (22) or LPP. This c i s - t r a n s i s o m e r i s a t i o n was found to be photoinducible ( c f . a l l -t r a n s - r e t i n o l to 1 3 - c i s - r e t i n o l ) and to be d i r e c t , i . e . no h y d r o l y s i s to g e r a n i o l i s observed. .. . An a l t e r n a t e proposal, i n v o k i n g the d i r e c t formation of NPP from DMAPP and IPP, was put forward by C o r i 6 9 , who used enzyme preparations from Pinus and C i t r u s species. No evidence of a GPP to NPP isomerase was found i n these systems but; both GPP and NPP were formed with the l o s s of the (• 4-pro-S)-hydrogen of MVA. They proposed two a l t e r n a t i v e s f o r t h i s observation: 1. The enzyme preparation contains two pren y l t r a n s f e r a s e s ( t r a n s - and c i s - ) which both have e q u i v a l e n t l y l o c a t e d binding s i t e s f o r the pyro-phosphate group and the 2-pro-R hydrogen of IPP (4-pro-S hydrogen of MVA), but bind the te r m i n a l methylene of IPP and the hydrophobic and pyrophosphate groups of DMAPP with d i f f e r e n t arrangements (Figure 32). On coup l i n g , the same proton of IPP i s eli m i n a t e d i n both cases, but g i v i n g a t r a n s - double bond f o r one enzyme and a c i s - double bond f o r the other, or; 2. There i s only one pren y l t r a n s f e r a s e , but a f t e r i n i t i a l coupling to form the c a t i o n ( H I ^ i n Figure 32). r o t a t i o n , of the C(2)-C(3) bond of the bound DMAPP (implying the hydrophobic group not to be r i g i d l y held) and then e l i m i n a t i o n of the 4-pro-S hydrogen of MVA w i l l give NPP. Both of these hypotheses, however, would o v e r a l l r e s u l t i n att a c k of the s i face of the double bond of IPP to .form NPP ra t h e r than the re face as i n forming GPP (proposal 1 involves, d i r e c t s i face a t t a c k , proposal 2 would appear to give such a t t a c k a f t e r r o t a t i o n of the IPP moiety). No work on t h i s problem has been reported to date, and the enzyme(-s) r e s p o n s i b l e f o r t h i s coupling have not been s u f f i c i e n t l y i s o l a t e d and p u r i f i e d to answer t h i s problem. Ruzicka's proposed b i o s y n t h e t i c scheme i n v o l v e s the c y c l i s a t i o n of GPP, NPP, or LPP to the a - t e r p i n y l c a t i o n , from which the other many monocyclic compounds can be formed. In support of t h i s proposal, crude e x t r a c t s from "Mentha p i p e r i t a L. (peppermint) have been shown to convert NPP to a - t e r p i n e o l (26) as the major c y c l i c p r o d u c t 5 9 and the same e x t r a c t has been shown to convert substrate concentration of 5 9 0 a - t e r p i n e o l to limonene (27) and a-terpinolene (28) (Figure 33) - u -Figure 32. C o r i ' s proposed mechanism f o r the d i r e c t formation of NPP from the condensation of IPP and DMAPP. - 45 -(26) (3) (27) (28) 7 0 a a - t e r p i n e o l limonene te r p i n o l e n e 3-phellandrene (29) (30) (31) 7 0 c 70(J 7 0 e 1,8-cineole carvone pulegone Figure 33. Some n a t u r a l l y o c c u r r i n g monocyclic monoterpenes. g i v i n g r i s e to the theory that the diene monoterpenes a r i s e by dehydration of a - t e r p i n e o l . The co-occurence .of these c y c l i c mono-terpenes, coupled with t h e i r . a p p a r e n t ease of i n t e r c o n v e r s i o n , seems to i n d i c a t e t h a t they are formed by s e q u e n t i a l m o d i f i c a t i o n of a s i n g l e monocyclic intermediate such as a - t e r p i n e o l r a t h e r than by formation d i f f e r e n t l y from an a c y c l i c precursor. However, l a t e r work by Croteau and K a r p 6 0 ' 6 1 using a soluble enzyme preparation from S a l v i a o f f i c i a n a l i s leaves shown to cat a l y s e the conversion of NPP to a number of mono-c y c l i c monoterpenes, i n d i c a t e s that 1,8-cineole (30), a - t e r p i n e o l (26), and the c y c l i c dienes limonene' (27) and a-terpinolene (28) are derived independently from the a c y c l i c precursor, r a t h e r than as f r e e i n t e r -mediates of a common r e a c t i o n sequence. The synthetases r e q u i r e d f o r each d i f f e r e n t monoterpene have been separated. . - 46 -L a b e l l i n g patterns examined i n various monocyclic monoterpenes derived from [^c]-MVA are co n s i s t e n t w i t h Ruzicka's h y p o t h e t i c a l scheme f o r b i o s y n t h e s i s i n v o l v i n g the i n i t i a l c y c l i s a t i o n of a NPP-type a c y c l i c precursor to the a - t e r p i n y l c a t i o n (l_7, Figure 24.) followed by o x i d a t i o n , r e d u c t i o n , e t c . (Figure 33) 7 0 . Various aromatic monoterpenes are seen to co-occur with v a r y i n g . amounts of a-terpinene, y-terpinene, and t e r p i n e n - 4 - o l , suggesting they may be b i o g e n e t i c a l l y r e l a t e d 7 1 . In v i v o and i n v i t r o s tudies have shown a r e l a t i o n s h i p between these compounds (Figure 34)• * postu l a t e d Figure 34. Some aromatic monoterpenes and t h e i r r e l a t i o n s h i p w i t h the non-aromatic monoterpenes. An enzyme preparation has been shown to convert [ l - H.]-GPP or 3 3 [1- H]-NPP to y-[3- H]-terpinene with approximately equal e f f i c i e n c y 7 2 . This system shows no evidence of conversion of GPP to NPP. Based on 3 s p e c i f i c l o c a t i o n of H i n the product, the f o l l o w i n g mechanism has been proposed (Figure 35). This mechanism -proposes a 1,2-hydride s h i f t Figure 35. Proposed b i o s y n t h e s i s of y-terpinene and a-thujene. from C(J+) to C(8) to produce Y - " t e r p i n e n e • A s i m i l a r mechanism i n v o l v i n g l o s s of a proton from C(6) and formation of a c y c l o p r o p y l r i n g , encompasses the formation of a-thujene, a minor product from t h i s p r e p a r a t i o n . The nature of the intermediates i n t h i s mechanism i s h i g h l y s p e c u l a t i v e , but l i m i t e d s tudies using LPP and a - t e r p i n y l pyro-phosphate as substrates support the proposal. The 1,2-hydride s h i f t s proposed i n t h i s mechanism have a l s o been postulated f o r r e l a t e d monocyclic monoterpenes but never demonstrated. 1,3-hydride s h i f t s i n 72b r e l a t e d analogous sesquiterpene cases have been demonstrated Various schemes have been proposed to show the''existence of 1,2-hydride s h i f t s , which would u t i l i z e s o l u b l e enzyme systems (Figure 3 6 ) 7 3 . (d) B i c y c l i c monoterpenes. ( i ) Pinanes. The major pinane monoterpenes- are a- and 3- pinene (Figure 37), and these compounds are the most common n a t u r a l l y o c c u r r i n g monoterpenes Various oxygenated d e r i v a t i v e s a l s o occur n a t u r a l l y but t h e i r d i s t r i b u t i o n i s l i m i t e d (Figure 37). Two enzymic routes to the pinanes have been deduced on chemo-taxanomic grounds 7 4? a-Pinene i s found to occur i n an o p t i c a l l y impure form whereas 3-pinene i s u s u a l l y o p t i c a l l y pure, and a-pinene i s found to eo-occurwith 3-pinene of the opposite absolute c o n f i g u r a t i o n . The s t r u c t u r e of the pinenes suggests t h e i r formation by Markownikoff a d d i t i o n w i t h i n the - a - t e r p i n y l c a t i o n , and l a b e l l i n g s t u d i e s from 1 L exogenous- [2- GJ-MVA are c o n s i s t e n t w i t h t h i s h y p o t h e s i s 7 5 . A c e l l -f r e e p r e p a r a t i o n from Pinus r a d i a t a has been shown t e n t a t i v e l y to - 49 -A 50% decrease i n s p e c i f i c a c t i v i t y on o x i d a t i o n of p_-cymene produced from oi-terpinene, to benzoic a c i d would i n d i c a t e a 1,3-hydride s h i f t . No change would i n d i c a t e a 1,2-hydride s h i f t 7 3 . Figure 36. Scheme to provide evidence f o r the existence of 1,2-hydride s h i f t s i n monoterpene b i o s y n t h e s i s . - 50 -(9) (3£) (35) (36) a-pinene 3-pinene myrtenol verbenone Figure 37. Examples of the pinanes. c y c l i s e NPP to a- and 3- p i n e n e s 7 6 . This system does not seem to synthesise the a c y c l i c monoterpenes, and therefore does not support the theory t h a t eis-ocimene (24.) and myrcene (23) are intermediates i n pinane b i o s y n t h e s i s . The o r i g i n of the oxygenated pinanes has not been examined i n d e t a i l , but i t seems feasable that these compounds a r i s e by o x i d a t i o n of the pinenes r a t h e r than d i r e c t c y c l i s a t i o n of oxygenated compounds. ( i i ) Bornanes. Borneol (38) and.camphor (2) (Figure 38) are the most important members of t h i s c l a s s of compounds,.and these compounds commonly co-occur i n a number of p l a n t species. Both enantiomers occur i n nature, e.g., d-camphor and d-borneol i n S a l v i a o f f i c i a n a l i s and 1-camphor and 1-borneol i n Rosemarinus o f f i c i a n a l i s . - 51 -(37) (37) (2) (2) borneol camphor Figure 38. The bornanes. L a b e l l i n g patterns i n borneol and camphor bios y n t h e s i s e d from exogenous [2-^G]-MVA and [ 2 - ^ ^ c ] - g e r a n i o l 7 7 are c o n s i s t e n t w i t h Ruzicka's hypothesis f o r - t h e c y c l i s a t i o n of the a - t e r p i n y l c a t i o n i n an anti-Markownikoff sense. The equivalent l a b e l l i n g seen i n opposite enantiomers i m p l i e s c y c l i s a t i o n of the stereochemically appropriate a - t e r p i n y l c a t i o n , r a t h e r than a 1,3-hydride s h i f t to give the opposite enantiomer (Figure 39). As borneol and camphor co-occur w i t h the. same absolute c o n f i g u r a t i o n i t seems- reasonable to assume th a t they can be i n t e r c o n v e r t e d . Evidence c o l l e c t e d suggests that borneol i s f i r s t formed and then o x i d i s e d to camphor. An enzyme prep a r a t i o n from sage ( S a l v i a o f f i c i a n a l i s ) leaves has been shown 7 8 to c a t a l y s e the c a t i o n -dependent c y c l i s a t i o n o f [ l - H]-NPP i n t o d-[3- H]-borneol and the NAD-dependent o x i d a t i o n of t h i s borneol i n t o d- [3- H]-camphor. In t h i s experiment, a water s o l u b l e , d i a l y z a b l e intermediate was a l s o i s o l a t e d and shown to be i n v o l v e d i n the r e a c t i o n . This compound was i d e n t i f i e d - 52 -Figure 39. Production of the bornanes from the s t e r e o c h e m i c a l ^ appropriate a - t e r p i n y l c a t i o n . as b o r n y l pyrophosphate, and gave some support to the theory that b i c y c l i s a t i o n occurs as one step, r a t h e r than v i a the formation of a 3 monocyclic intermediate. S y n t h e t i c d l - [ 3 - H ] - a - t e r p i n e o l was not converted to borneol by t h i s p r e p a r a t i o n , i n d i c a t i n g that a - t e r p i n e o l i s not a f r e e intermediate i n the b i o s y n t h e s i s . A synchronous mechanism f o r NPP to b o r n y l pyrophosphate (BPP) was thus proposed (Figure 4-0). The isomeric a l c o h o l , i s o b o r n e o l (38) i s seen to occur i n nature to a l i m i t e d extent, and may be p o s t u l a t e d to a r i s e from the r e d u c t i o n (38) i s o b o r n e o l - 53 -Figure 4-0. Synchronous mechanism f o r NPP to BPP. of camphor r a t h e r than from the c y c l i s a t i o n of NPP, GPP or the a-t e r p i n y l c a t i o n . The reason f o r t h i s p o s t u l a t i o n can be seen when borneol b i o s y n t h e s i s i s examined. In the example above, or even i f the a - t e r p i n y l c a t i o n . i s i n v o l v e d , the developing c a t i o n at the C(2) p o s i t i o n i n the bornane skeleton i s r e a d i l y quenched by pyrophosphate i o n ( i n a synchronous mechanism) or by water (Figure 4-1) from the l e s s hindered, endo face of the molecule. This i s a l s o the p r e f e r r e d trans--Figure 41. B i o s y n t h e s i s of borneol. - 54 -a d d i t i o n of s u b s t i t u e n t s across a double bond, and generates only the endo a l c o h o l . Reduction of camphor would proceed by hydride a d d i t i o n . to the l e s s hindered endo f a c e , r e s u l t i n g i n the exo a l c o h o l . The enzyme preparation from sage mentioned 7 8 was a l s o found to convert GPP to BPP, but l e s s e f f i c i e n t l y . Examination of the t e r p e n y l pyrophosphate hydrolases present i n t h i s r e l a t i v e l y crude preparation revealed t h a t t h e y contained high l e v e l s of a hydrolase that p r e f e r e n t i a l l y hydrolysed GPP'to the i n a c t i v e monophosphate. P a r t i a l p u r i f i c a t i o n of the enzyme pre p a r a t i o n removed these competing phosphatases, and the preparation then showed a preference f o r GPP over NPP, and no i n t e r -conversion was seen. This f i n d i n g made the previous p o s t u l a t e d mechanism untenable and a m u l t i s t e p process i n v o l v i n g GPP was proposed (Figure 42) 79 PPO? PPO OPP Figure 42. Revised proposal f o r the b i o s y n t h e s i s of the bornanes. - 55 -I t i s suggested that the pyrophosphate i o n i s the only n u c l e o p h i l e present i n a hydrophobic pocket of the enzyme i n which the molecule i s s i t t i n g , and thus the developing c a t i o n i s quenched with pyro-phosphate i o n (PPO—) r a t h e r than water. ( i i i ) Thujanes. The thujane monoterpenes, of which thujone (39) and isothujone (4-0) (Figure 4-3) are the most common members, show l a b e l l i n g patterns 14 from exogenous • [ 2 - C]-MVA c o n s i s t e n t w i t h the c y c l i s a t i o n of the a - t e r p i n y l c a t i o n -as i n Ruzicka's scheme 8 0. Time course s t u d i e s 8 1 and 14 the i n c o r p o r a t i o n of a-[ e ] - t e r p i n e o l 8 2 i n t o 3-thujone i n Tanecetum  vulgare are considered to e l i m i n a t e the d i r e c t b i c y c l i s a t i o n of an a c y c l i c precursor to the thujane- s k e l e t o n , and the- a - t e r p i n y l c a t i o n , or i t s b i o s y n t h e t i c e q u i v a l e n t , i s i n v o l v e d . From the r e s u l t s of experiments using s p e c i f i c a l l y l a b e l l e d - a - [ H, C ] - t e r p i n e o l and [ H, C]-MVA, Banthorpe et a l . - 8 3 obtained evidence f o r two hydride s h i f t s and p o s t u l a t e d the' f o l l o w i n g biosynthetic.pathway (Figure 4 4 ) . (40) 1 - 3 - i s o t h u j one (39) d - 3-thuj one Figure 4 3 . The thujane- monoterpenes. - 56 -d-3-thuj anol Figure 44. P o s t u l a t e d b i o s y n t h e t i c scheme f o r the thujanes. - 57 -( i v ) Carane. Car-3-ene (_4J_) i s the only commonly oc c u r r i n g monoterpene with the carane skeleton (Figure 45), car-2-ene (43) i s thought to be an a r t i f a c t of the method of e x t r a c t i o n . (41) d-car-3-ene Figure 4-5. The carane monoterpenes. (42) d-car-2-ene Ruzicka proposed the formation of car-3-ene from the a - t e r p i n y l c a t i o n by a 1 , 3-elimination, and the co-occurrence of te r p i n o l e n e (28) with car-3-ene was considered proof of the theory (Figure 46). Formation 14 of car-3-ene from- [2- CJ-MVA81* shows the l a b e l to be predominantly at C(4) of the carane skeleton, r a t h e r than C(2), i n d i c a t i n g i f car-3-ene does a r i s e from the a - t e r p i n y l cation-, formation of the c y c l o p r o p y l r i n g must be accompanied by double bond migration (Figure 46). Using- Pinus s y l v e s t r i s , Banthorpe and A k h i l a 8 5 have'" examined the b i o s y n t h e s i s of car-3-ene and have shown formation of the double bond to be accompanied by an unusual 1,2-proton s h i f t . In forming the double bond,-the (2-pro-S)-hydrogen of. MVA. i s lost.. A f t e r feeding w i t h [ ^  ^ "C, 1 - 3H,-,] - g e r a n i o l and n e r o l , the car-3-ene was i s o l a t e d and the double bond ozonised (Figure 47). Loss of one H atom from g e r a n i o l i n d i c a t e d p r i o r conversion to n e r o l by a redox mechanism. N e r o l Figure 47. B i o s y n t h e s i s of car-3-ene i n Pinus s y l v e s t r i s . - 59 -e x h i b i t e d no l o s s of t r i t i u m , i n d i c a t i n g an i n t r a m o l e c u l a r t r a n s f e r of t r i t i u m when forming the c y c l o p r o p y l r i n g . E n o l i s a t i o n of the o z o n o l y s i s product (A, Figure 47) showed the compound derived from n e r o l to l o s e h a l f i t s t r i t i u m , whereas that from g e r a n i o l e x h i b i t e d no such l o s s . To e x p l a i n these r e s u l t s , Banthorpe 8 5 proposed that the (1-pro-S)-hydrogen of g e r a n i o l was l o s t on conversion to n e r o l , and the remaining l a b e l l e d proton remains on the c y c l o p r o p y l r i n g . F o r ' c y c l i s a t i o n , "forbidden" syn i n t e r a c t i o n s would be proposed unless an "X-group" mechanism i s invoked, a l l o w i n g these i n t e r a c t i o n s to- be a n t i (Figure 4-8) . This i m p l i e s the b i o g e n e t i c equivalent of the a - t e r p i n y l c a t i o n to remain enzyme bound, and t h i s i s supported by the observation that a - t e r p i n e o l i s not i n c o r p o r a t e d i n t o car-3-ene. Figure 48. The b i o s y n t h e s i s of car-3-ene, i n v o l v i n g a 1,2-proton s h i f t . - 60 -(e) Cyclopentanoid monoterpenes ( i r i d a n e s ) . The i r i d a n e group of monoterpenes gained t h e i r name from I r i d o -myrmex, the species of ant from which they were f i r s t i s o l a t e d . S everal hundred i r i d a n e s have been i s o l a t e d from i n s e c t s and p l a n t s , and they often occur as- the 3-D-glueosides. Rates of i n c o r p o r a t i o n of exogenous precursors are high and not plagued w i t h asymmetrical l a b e l l i n g problems as these compounds are not i s o l a t e d to o i l glands but occur throughout the p l a n t . A few examples of the i r i d a n e monoterpenes are shown i n . - .u. Figure 49. The i r i d a n e s can be s p l i t f u r t h e r i n t o two groups; the i r i d o i d s and the s e e o i r i d o i d s (e.g. swero.side). The s e c o i r i d o i d s are b e l i e v e d to a r i s e by cleavage of-the C(7)-C(8) bond- of the cyclopentane r i n g i n loganin- (43) followed by. numerous secondary transformations. Much i n t e r e s t i n the i r i d a n e s stems from t h e i r r o l e i n i n d o l e - and i s o q u i n o l i n e - a l k a l o i d b i o s y n t h e s i s , and s e v e r a l reviews i n t h i s area have app e a r e d 8 6 . (43) (44) (45) l o g a n i n nepetalactone sweroside Glu = 3-D-glucose Figure 49. Examples of the i r i d a n e monoterpenes. - 61 -L a b e l l i n g s t u d i e s with. [ 2-1 ^c]-MVA and various [• 3 H ] - l a b e l l e d MVA's have been c o n s i s t e n t w i t h the o r i g i n of lo g a n i n through the accepted pathway of monoterpene b i o s y n t h e s i s , i . e . , v i a g e r a n i o l or GPP (Figure S7_89 90 50) . Tracer experiments'with Vinca rosea i n d i c a t e the f i r s t step i n the i n c o r p o r a t i o n of g e r a n i o l or n e r o l i n t o the i r i d o i d s i s o x i d a t i o n to the -10-hydroxy-compound and 10-hydroxy-compounds were i s o l a t e d from p l a n t s r i c h i n l o g a n i n . 10-hydroxy- g e r a n i o l , n e r o l , and c i t r o n e l l o l were t e s t e d as precursors, and. only the 10-hydroxy-geraniol and 10-hydroxy-nerol were found to give good i n c o r p o r a t i o n . A tobacco t i s s u e c u l t u r e has-been shown...to transform l i n a l o o l s e l e c t i v e l y to the trans-hydroxy-compound 9- 1. • I n some c a s e s 9 2 , considerable randomisation of l a b e l from 0(9) and C(10) were found when log a n i n was bio s y n t h e s i s e d a l k a l o i d s Figure 50. Bi o s y n t h e s i s of the i r i d o i d s . compound demonstrated to be an intermediate between the 10-hydroxy-compounds and log a n i n i s deoxyloganin (46)93 as w e l l as l o g a n i c a c i d (43) 9 k . In the conversion of loganin to the a l k a l o i d s , secologanin (47) i s shown to be an i n t e r m e d i a t e 9 5 . On the b a s i s of the f i n d i n g s so f a r , the f o l l o w i n g scheme f o r i r i d o i d b i o s y n t h e s i s has been proposed (Figure 51 ) 9 6 . OGlu -0 COOMe HO OGlu 0 C00H (46) deoxyloganin (43) l o g a n i c a c i d CHO COOMe (47) secologanin - 63 -H, Me COOR GOOR R = H, Me R = H, Me Figure 51. The proposed b i o s y n t h e s i s of the i r i d o i d s . - 64 -( f ) I r r e g u l a r monoterpenes. The i r r e g u l a r monoterpenes are of two types; 1) those that contain l e s s than 10 carbon atoms ; and 2) those which contain 10 carbon atoms but not i n a r e g u l a r " h e a d - t o - t a i l " f a s h i o n . 1.) The i r r e g u l a r monoterpenes containing l e s s that 10 carbon atoms can be assumed to a r i s e by degradation of a r e g u l a r monoterpene (Figure 52);. The two compounds shown, 3-phellandrene and cryptone, are found to co-occur. The b i o s y n t h e s i s of t h i s type of i r r e g u l a r monoterpene i s the same as f o r t h e i r parent compound followed by o x i d a t i v e cleavage, e t c . (28) (49) 3-phellandrene cryptone Figure 52. I r r e g u l a r monoterpene b i o s y n t h e s i s . 2) Those i r r e g u l a r monoterpenes co n t a i n i n g 10.carbon atoms can be assumed to a r i s e by one of two methods: ( i ) by rearrangement of r e g u l a r monoterpenes, ther e f o r e being u l t i m a t e l y derived from GPP. Examples of t h i s group are: fenchane and camphane: Wagner-Meerwein s h i f t •OH pmane fenchane fenchol (50) - 65 -bornane . isocamphane camphene (51) s e c o i r i d o i d s : COOMe CHO COOMe loga n i n (43) secologanin (47) These i r r e g u l a r monoterpenes are b e l i e v e d to be produced i n v i v o , presumably enzymatieally, and although few i n . v i v o s t u d i e s have been c a r r i e d out, chemically reasonable routes have been post u l a t e d having many i n v i t r o a n a l o g i e s . I t has been p o s t u l a t e d 9 7 that 1-endo-fenchol'(50) could a r i s e from GPP d i r e c t l y via-an enzyme-bound l i n a l o y l and p i n y l c a t i o n (Figure 53) . i i ) : . a s primary b i o s y n t h e t i c products, and not derived from GPP or any other r e g u l a r monoterpene'precursor. These are by f a r the most i n t e r -e s t i n g i r r e g u l a r monoterpenes. Their b i o s y n t h e s i s i s l a r g e l y a mystery, t h e i r r e l a t i v e l y low n a t u r a l abundance posing the major problem i n studying-them. However, some studies,- both i n v i v o and i n v i t r o , have l e d to a general proposal- f o r t h e i r b i o s y n t h e s i s . The major- s k e l e t a of t h i s • c l a s s of monoterpenes are the a r t e m i s y l ('52), chrysanthemyl (53) , l a v a n d u l y l (54) , and s a n t o l i n y l (55) s k e l e t a , gure 5 3 . , P o s t u l a t e d d i r e c t formation of fenchol from GPP. - 67 -(540 (55). (53) l a v a n d u l y l s a n t o l i n y l chrysanthemyl (52) (56) a r t e m i s y l a r t e m i s i a ketone Figure 54-. Major s k e l e t a of the i r r e g u l a r monoterpenes. shown i n Figure 54-, and the most widespread i r r e g u l a r monoterpene i s a r t e m i s i a ketone (56). L a b e l l i n g s t u d i e s i n A r t e m i s i a annua have shown MVA, DMAPP, and IPP to be incorpo r a t e d i n t o a r t e m i s i a ketone,.and g e r a n i o l i s not 9 8 a incorporated without p r i o r degradation. . High i n c o r p o r a t i o n of MVA 9 8 b i n t o chrysanthamic a c i d has a l s o been reported . Various b i o s y n t h e t i c 9 9 1 0 0 pathways have been proposed ' and these g e n e r a l l y propose the formation of the chrysanthemyl skeleton i n the f i r s t i n s t a n c e . Subsequent rearrangements of t h i s skeleton can lead to the s a n t o l i n y l and a r t e m i s y l s k e l e t a (Figure 55). Feeding and tra p p i n g e x p e r i m e n t s 1 0 1 have shown chrysanthemyl a l c o h o l to be an.intermediate i n . t h e b i o s y n t h e s i s of chrysanthamic a c i d , whereas l a v a n d u l o l and a r t e m i s i a a l c o h o l are not. A c e l l - f r e e e x t r a c t was shown to convert MVA,. IPP, DMAPP, and d i m e t h y l v i n y l c a r b i n o l (DMVC) (57) to a r t e m i s i a ketone and a l c o h o l , - 68 -Figure 55. Proposed b i o s y n t h e s i s of i r r e g u l a r monoterpenes. - 69 -l a v a n d u l o l , and chrysanthemyl a l c o h o l 1 0 2 . G e r a n i o l , n e r o l , l i n a l o o l and t h e i r pyrophosphates were not seen to be inc o r p o r a t e d . From these and other s t u d i e s 1 0 3 , the f o l l o w i n g scheme f o r the b i o s y n t h e s i s of these i r r e g u l a r monoterpenes has been proposed (Figure 56). Figure 56. The b i o s y n t h e s i s of the i r r e g u l a r monoterpenes i n v o l v i n g DMVC. - 70 -I I •DISCUSSION. I I - ( i ) The problem. The b i o s y n t h e s i s of camphor ( 2 ) , as o u t l i n e d i n Secti o n I - ( v i i ) - d ( i i ) , has been shown to be c o n s i s t e n t w i t h Ruzicka's scheme f o r the c y c l i s a t i o n of the a - t e r p i n y l c a t i o n 7 7 , w i t h l a b e l from exogeneous [2- C]- MVA (8) and [ 2 - ' V ) - g e r a n i o l (1J.) being incorporated s p e c i f i c a l l y i n accordance with t h i s scheme (Figure 57). Work w i t h enzyme preparations from sage 7 8 7 9 ( S a l v i a o f f i c i a n a l i s ) ' has shown th a t geranyl ( H ) , n e r y l (l_9), and l i h a l o y l (20) pyrophosphates can be r e a d i l y converted i n t o borneol (37) and camphor (2) without the detectable intermediacy of an a - t e r p i n y l s t r u c t u r e . To account f o r the ready conversion of geranyl pyrophosphate to b o r n y l pyrophosphate, the f i r s t formed b i c y c l i c product, a m u l t i s t e p process has been proposed 7 9 (Figure 58). Geranyl pyrophosphate, having t r a n s - stereochemistry at. the. 2,3-double bond, i s of an i n c o r r e c t stereo-chemistry f o r c y c l i s a t i o n . The mechanism.proposed 7 9 ( F i g u r e 58) 1A 19 20 geranyl n e r y l l i n a l o y l • pyrophosphate pyrophosphate pyrophosphate - 71 -OPP (37) Figure 58. Conversion of geranyl .pyrophosphate. (^ .) to bo r n y l pyro-phosphate (37) by an enzyme preparation from s a g e 7 9 . - 72 -borneol camphor (37) (2) Figure 59. Numbering of the bornane skeleton i n borneol (37) and camphor (2) postulates the i s o m e r i s a t i o n . t o a l i n a l o y l - t y p e enzyme bound intermediate w i t h the developing c a t i o n at C(2) of the bornane skeleton (see Figure 59 f o r numbering of the bornane skeleton) being quenched by pyrophosphate i o n h e l d i n a hydrophobic pocket of the enzyme.. This enzyme preparation d i d not convert s y n t h e t i c , l a b e l l e d a - t e r p i n e o l . i n t o borneol and was taken as evidence that an a - t e r p i n y l s t r u c t u r e i s not i n v o l v e d . A l t e r n a t i v e l y , the a - t e r p i n y l s t r u c t u r e may be present as an enzyme bound intermediate which c y c l i s e s to the bornane skeleton (Figure 60). Thus exogeneous a - t e r p i n e o l would not be.accepted by t h i s enzyme. C y c l i s a t i o n of the a - t e r p i n y l , skeleton has been proposed 5 to occur- d i r e c t l y by anti-Markovnikov a d d i t i o n to the 'double bond, or by c y c l i s a t i o n to the pinane skeleton (Markovnikov a d d i t i o n ) followed by a 1,2 s h i f t of the bridge carbon r a t h e r than proton l o s s to give the double bond found i n the pinenes. f 1 (2) ' (37) Figure 60. P o s t u l a t e d c y c l i s a t i o n of a C^Q intermediate to b o r n y l pyrophosphate v i a an enzyme-bound a - t e r p i n y l s t r u c t u r e . To provide f u r t h e r evidence f o r t h i s mode of c y c l i s a t i o n of the C^Q precursor,' we decided to examine the f a t e of the a l l y l i c methyl groups i n the conversion of the a c y c l i c -C^Q precursor (_5_8) to camphor (2) ( F i g u r e 61)'. In. the precursor, these two methyl groups have d i f f e r e n t i d e n t i t i e s , being, c i s - or t r a n s - on the double bond, and i f they can be s e p a r a t e l y l a b e l l e d , then t h e i r eventual p o s i t i o n s a f t e r c y c l i s a t i o n - can-be examined. I f the b i c y c l i s a t i o n was d i r e c t or v i a an enzyme-bound a - t e r p i n y l s t r u c t u r e , i t would be expected t h a t one of these two methyl groups would become, the C(8)-methyl group i n camphor w h i l s t the other became the C(9)-methyl group (Figure 62). I f a f r e e c a t i o n i c intermediate (1_7) i s i n v o l v e d , f r e e r o t a t i o n about the now s i n g l e bond would r e s u l t - i n a l o s s of i d e n t i t y and the two methyl groups - 1U -MVA camphor (58) ( 2 ) Figure 61. MVA to camphor ( 2 ) . would be randomly d i s t r i b u t e d between the C ( 8 ) - a n d G(9)- p o s i t i o n s . Thus l a b e l l i n g one of these two methyl groups i n the a c y c l i c precursor and examining-the camphor bi o s y n t h e s i s e d from t h i s compound w i l l give a greater i n s i g h t i n t o the mechanism..of formation of the b i c y c l o - [ 2.2.1]-compounds. By analogy to the sesquiterpenoids, - i t would be expected th a t the two groups would r e t a i n , t h e i r i n t e g r i t y . Campherenone (_5_9), the C.| -analogue of camphor- (Z) , i s found i n . t h e e s s e n t i a l o i l of Cinnammomum- camphora Sieb. while the isomer epi-campherenone (60) has not yet been reported as a n a t u r a l product. The c y c l i s a t i o n of f a r n e s y l pyrophosphate (l_2) to campherenone (59) i s analogous to the c y c l i s a t i o n of GPP (l_£) to camphor (2).and occurs i n a s t e r e o s e l e c t i v e fashion-(Figure 63). Figure 62. The f a t e s of the a l l y l i c methyl groups on c y c l i s a t i o n to the b i c y c l o - [ 2 . 2 . l ] - system. - 76 -epi-campherenone Figure 63. FPP (l_2) c y c l i s a t i o n to campherenone (59). I I — ( i i ) The method of. l a b e l l i n g . L a b e l l i n g of one of the a l l y l i c methyl groups i n the Q-acyclic precursor (58, Figure 61) could be c a r r i e d out i n the two f o l l o w i n g ways; (a) by using mevalonic a c i d . l a b e l l e d at 0(2) or C(3')> (b) by using a l a b e l l e d C^Q intermediate, e.g., l i n a l o o l (20), g e r a n i o l (14.K or n e r o l (l_9). The a c t u a l method of l a b e l l i n g should be detectable by a non-degradative means (e.g., nuclear magnetic resonance (n.m.r.)) as•degradative studies on camphor to d i s t i n g u i s h between the 8- or 9- methyl groups r e q u i r e a ioi+a considerable amount of m a t e r i a l . Even mass spectrometry cannot d i s t i n g u i s h between-these g r o u p s 1 0 ^ but the use of a non-radioactive - 77 -n..m..r.. detectable l a b e l would solve t h i s problem. 13 C has a n a t u r a l abundance of approximately 1.1$, and n a t u r a l abundance IJ>C-n.m.r. are r e l a t i v e l y easy to o b t a i n . I n c o r p o r a t i o n of l a b e l would r e s u l t i n enhancement of peak s i z e and consequently a r e l a t i v e l y l a r g e i n c o r p o r a t i o n would be r e q u i r e d f o r an unambiguous assignment. 'As i n c o r p o r a t i o n l e v e l s f o r monoterpene b i o s y n t h e s i s - a r e n o t o r i o u s l y low, t h i s i s not 2 the-method of choice. I n c o n t r a s t , H has a low n a t u r a l abundance (0.016$) and thus any i n c o r p o r a t i o n would r e s u l t i n a s i g n i f i c a n t 2 2 increase i n H present and thus i n s i g n a l s i z e . I n c o r p o r a t i o n of H 13 i n t o a precursor would be e a s i e r to detect than i n c o r p o r a t i o n of C, 2 and t h e r e f o r e H was chosen as the l a b e l . I l l - ( i i i ) . The choice of precursors. The precursor of choice f o r terpenoid b i o s y n t h e s i s i s mevalonic a c i d (MVA, 8). However, with monoterpenoids, there are d i f f i c u l t i e s w i t h t h i s precursor i n that asymmetric l a b e l l i n g of the f i n a l mono-terpene o f t e n o c c u r s 5 2 . This r e s u l t s in-approximately 90% of the l a b e l being found i n that' part of the s t r u c t u r e derived' from IPP, whereas that p a r t derived from DMAPP i s v i r t u a l l y u n l a b e l l e d (Figure 64., see a l s o S e c t i o n I - ( v i i ) - ( a ) ) . As i t i s that part, of the molecule derived from > 90% of l a b e l here < 10$ of l a b e l here Figure 64.. Asymmetrical l a b e l l i n g found i n monoterpenoid b i o s y n t h e s i s . DMAPP" which i s r e q u i r e d to be l a b e l l e d , use of a l a b e l l e d MVA alone could be i n c o n c l u s i v e . Using a l a b e l l e d C^Q d e r i v a t i v e (e.g., l i n a l o o l (20), g e r a n i o l (j_4_), or n e r o l (1^))- would overcome t h i s problem. The choice of G^Q d e r i v a t i v e i s a r b i t r a r y as a l l three compounds have been shown to be incorpo r a t e d i n t o the monqterpenoids but recent work has given an i n d i c a t i o n that l i n a l o o l (20) can be incorporated w i t h greater e a s e 5 8 . Examination of the l i t e r a t u r e showed that r e g i o s p e c i f i c f u n c t i o n a l i s a t i o n of the t r a n s - a l l y l i c methyl group i n l i n a l o o l can be accomplished and th a t various s y n t h e t i c procedures are a v a i l a b l e f o r the synthesis of mevalonic a c i d l a b e l l e d w i t h deuterium at 0(2), C(3'), 0(4), and 0(5). Thus we decided to prepare two precursors, mevalonic a c i d and l i n a l o o l , l a b e l l e d w i t h H at the C(2) and C(8) p o s i t i o n s r e s p e c t i v e l y (Figure 65). D (61) (62) 2 2 [2- H^]-mevalonic a c i d . [8- ] - l i n a l o o l Figure 65. Precursors of choice f o r the b i o s y n t h e t i c study. 2 Examination of the H-n.m.r. of camphor bios y n t h e s i s e d from 2 2 [2- H^]-mevalonic a c i d (6l_) or [8- H ^ ] - l i n a l o o l (62) would show i f the C(8) and C(9) methyl groups had been derived from a s p e c i f i c methyl group i n the a c y c l i c precursor (58). Samples of 8-deuterio- and - 79 -9-bromocamphor [9- H.]-camphor Figure 66. P r e p a r a t i o n of [8- H^-and [9- H^]- camphor. 9-deuterio- camphor, obtained from the corresponding bromo-compounds by r e d u c t i o n w i t h t r i - n - b u t y l t i n deutride (Figure 6 6 ) , were examined 2 by H-n.m.r. spectroscopy and i t was noted that the chemical s h i f t 2 d i f f e r e n c e between the deuterium s i g n a l s was very small (8- H- at I.OOOppm, 2 and 9- H- at 1.159 ppm.). An obvious method of enhancing t h i s s h i f t 2 d i f f e r e n c e i s the use of a lanthanide s h i f t reagent, and the H-n.m.r. i n the presence of added increments of Eu(thd)^ s h i f t reagent were taken. (Eu(thd)' 3 = Resolve-Al ( A l d r i c h ) = t r i s ( 2 , 2 , 6 , 6 - t e t r a m e t h y l -3,5-heptanedionato)europium.) The, s h i f t reagent binds to the carbonyl 2 group of camphor and i t would be expected f o r the [8- H^]- s i g n a l to be a f f e c t e d to a greater extent than the '[9- H^]- s i g n a l . This was indeed found and the r e s u l t i n g chemical s h i f t w i t h respect to the amount of s h i f t reagent added noted (Figures.67, 68, and 6 9 ) . This gave a - 80 -Figure 67. E f f e c t of added s h i f t reagent (S.R.) on the H-n.m.r. 2 spectrum of [8- H ]-camphor. - 8 1 -CDC1 Figure 68.. E f f e c t of added s h i f t reagent (S.R.) on the H-n.m.r. 2 spectrum 'of '[9- H-.] -camphor. Figure 69. H-N .m.r. spectrum of a mixture of [8- H^ ] - and [9- H^ ] - camphor i n the presence to i 'of excess s h i f t reagent. - 83 -convenient, non-destructive method f o r the a n a l y s i s of the r e s u l t s obtained. I I - ( i v ) The p l a n t system. According' to the l i t e r a t u r e 1 0 5 , three plants a v a i l a b l e on the U.B.C. campus are known to contain- a workable q u a n t i t y of camphor i n t h e i r e s s e n t i a l o i l s . These were Cinnamomum camphora Sieb. (the camphor t r e e ) , R o s e m a r i n u s • o f f i c i a n a l i s . ( r o s e m a r y ) , .and S a n t o l i r i a chamaecyparissus (cotton l a v e n d e r ) . The camphor tree r e q u i r e s a hot climate f o r optimum growth and was maintained i n the U.B.C. . h o r t i c u l t u r e b u i l d i n g . The pl a n t i t s e l f was not very robust and - experimentation'using i t would no doubt have k i l l e d the t r e e . So the -alternatives-were examined. Steam d i s t i l l a t i o n of the ground p l a n t s . f o l l o w e d by petroleum ether (30°-60°C) e x t r a c t i o n of the d i s t i l l a t e , provided -the e s s e n t i a l o i l s of R. o f f i c i a n a l i s and S. chamae c y p a r i s sus, two hardy, shrubs, grown in. the U.B.C. B o t a n i c a l gardens. G.l.c. examination, showed t h a t the e s s e n t i a l o i l from R. o f f i c i a n a l i s contained approximately 20% camphor•whilst that obtained from S. chamae c y p a r i s sus contained l e s s than.-5% camphor. Chromatography of the e s s e n t i a l o i l from rosemary on s i l i c a . g e l gave camphor (90$ g . l . c . p u r i t y ) which could be r e a d i l y sublimed to higher p u r i t y (>99%>). The s p e c t r a l d a t a ' f o r • t h i s sample of camphor was i d e n t i c a l to that of an a u t h e n t i c sample. Thus Rosemarinus o f f i c i a n a l i s was chosen f o r the b i o s y n t h e t i c s t u d i e s . For b i o s y n t h e t i c s t u d i e s , there are a v a r i e t y of methods a v a i l a b l e f o r f o r c i n g a p l a n t system to incorporate ..a l a b e l l e d precursor. Much of the work to date on monoterpenoid b i o s y n t h e s i s has been c a r r i e d out on the i n t a c t p l a n t or c u t t i n g s , although some workers are now succeeding i n separating the reasonably .pure enzyme preparations r e s p o n s i b l e f o r 7 8 7 9 the d e s i r e d transformations (e.g. -R.iCroteau ' ). Another method used i n b i o s y n t h e t i c ' s t u d i e s i s that of p l a n t t i s s u e c u l t u r e s , u n d i f f e r e n t i a t e d c e l l s grown i n or on a n u t r i e n t medium. - However, the production of secondary metabolites, by. c e l l cultures- has- been fraught w i t h p r o b l e m s 1 0 6 . Some plan t t i s s u e c u l t u r e s have been used f o r the production of various secondary me t a b o l i t e s , e.g., alk a l o i d s , - - p h e n o l i c , and f l a v o n o i d s 1 0 7 , with l e v e l s close to or exceeding those-found i n the i n t a c t p l a n t s . In some cases, the c u l t u r e .has produced compounds which are not present i n the i n t a c t plant.- I t has been p o s t u l a t e d 1 0 7 -that r e a c t i o n s that l e a d to secondary metabolites do not have a b s o l u t e . s p e c i f i c i t y f o r s u b s t r a t e s , and i f s u i t a b l e analogues are a v a i l a b l e , analogues of the secondary metabolites w i l l be produced. .;I-n - the i n t a c t plant-, : compartmentation e f f e c t s would r e s t r i c t access to these-•alternative• s u b s t r a t e s . For c e r t a i n secondary products (e.g.. e s s e n t i a l , o i l s ) - i t i s thought that accumulation of product only occurs i n s p e c i f i c morphological s t r u c t u r e s (e.g. o i l glands)- and accumulation can only occur when these .structures are present. In-the u n d i f f e r e n t i a t e d c e l l s , t h e r e f o r e , these products are not accumulated to.any great extent, and t h e i r synthesis v i a pla n t t i s s u e c u l t u r e s i s l i m i t e d . . . I t , has been r e p o r t e d 1 0 8 , t h a t i n c u l t u r e s of pepper and spearmint, mint o i l . o r menthane d e r i v a t i v e s cannot be detected i f "the c e l l - c u l t u r e does not. .contain - the biochemical and s t r u c t u r a l features. 1 of o i l glands.. . In a d d i t i o n , v o l a t i l e o i l s of Ruta  g r a v e o ^ ^ 1 -and • Pimpi-nella anisum have been detected i n c e l l aggregates where s p e c i a l i s e d - c e l l s have formed. . The formation-of c e l l aggregates i s regarded as a p r e r e q u i s i t e f o r - o i l formation and before the i n v i t r o b i o s y n t h e t i c • study.. of. terpenoids using p l a n t t i s s u e • c u l t u r e s can be. c a r r i e d out, there must be. progress .towards inducing c e r t a i n l e v e l s of - 85 -c y t o d i f f e r e n t i a t i o n i h these c u l t u r e s . Both of these newer methods of b i o s y n t h e t i c study, c e l l c u l t u r e and enzyme preparations,•are i n . v i t r o .methods of studying b i o s y n t h e s i s and although they are good i n d i c a t i o n s of the mode of operation o c c u r r i n g i n a p l a n t , i t must be'remembered th a t they are s t i l l i n v i t r o s t u d i e s . In v i v o s t u d i e s , using the i n t a c t p l a n t system, obviously give a c l o s e r view of the exact mode of operation occurring- and thus i n v i t r o s t u d i e s should be backed up by i n v i v o s t u d i e s . '• As has been p o s t u l a t e d 1 0 7 , the s p e c i f i c i t y of the r e a c t i o n s y i e l d i n g - secondary•metabolites i s thought to be r e l a t i v e l y low, and In v i t r o s tudies may show a suspected precursor to be incorpo r a t e d i n t o a secondary.product which i n v i v o i s not a true precursor, being denied access to the-, s i t e of- synthesis '.by compartmentation e f f e c t s . Secondly, a metabolite-may be produced which i s . not u s u a l l y present i n the system, -possibly-duetto the lack-of- s p e c i f i c i t y of the r e a c t i o n pathway. A l t e r n a t i v e l y , - a compound produced i n the pl a n t by a c e r t a i n route may, i n v i t r o , be produced-by.an. a l t e r n a t i v e route. Whether any or a l l of t h e s e - a l t e r n a t i v e s occur i s unknown, but the p o s s i b i l i t y can only be reduced to .a.minimum by .using a system as clos e to the i n t a c t p l a n t as p o s s i b l e . • Even i n these !cases when an i n t a c t p l a n t i s used, the effect..of. increased, l e v e l s - o f • the suspected precursor i s unknown, and may r e s u l t i n a l t e r n a t i v e , u s u a l l y unused, pathways being brought i n t o operation (e.g., the reported production of DMAPP from l e u c i n e , see s e c t i o n I - ( v i i ) - (a).)..- I t was the r e f o r e decided t h a t , f o r our experiments on the b i o s y n t h e s i s of- camphor,-the i n t a c t p l a n t system would be-used. -;For the -introduction of a precursor i n t o the i n t a c t p l a n t system there.are. a.number of techniques, which may be used depending on the type of plan t being, examined and the suspected d u r a t i o n of the b i o s y n t h e s i s being i n v e s t i g a t e d 1 0 9 . . The major feeding methods - 86 -used f o r p l a n t s are as f o l l o w s : 1 . In.jection; an aqueous s o l u t i o n of the l a b e l l e d m a t e r i a l i s i n j e c t e d i n t o the va s c u l a r system ( s t a l k ) . 2. Cotton-wick; f o r plants w i t h a robust but not too woody stem. One end of a cotton wick (5-10 mm.) i s i n s e r t e d through a s m a l l , l o n g t i t u d i n a l s l i t i n the p l a n t stem, and the other end immersed i n the s o l u t i o n (0.5-1.0 ml.) to be incorporated,, u s u a l l y i n a small tube attached to the stem. 3» C u t t i n g the s t a l k ; cut shoots are maintained i n an aqueous s o l u t i o n c o n t a i n i n g the l a b e l l e d m a t e r i a l . This method i s only s u i t a b l e f o r r a p i d b i o s y n t h e t i c processes as when tr e a t e d i n t h i s way, the metabolism of most p l a n t s slows down a f t e r a few hours. 4- S c a r r i n g ;•• the s o l u t i o n containing. the l a b e l l e d precursor i s added . dropwise to scars at v a r i o u s points on the stem exposing the e x t e r n a l v e i n s . 5» Brush; f o r very long term experiments. The l a b e l l e d precursor d i s p e r s e d • i n . s i l i c o n e o i l i s brushed onto the leaves of the p l a n t i n the ground or i n a vase. The p l a n t being used f o r t h i s b i o s y n t h e t i c study, Rosemarinus  o f f i c i a n a l i s , i s a hardy shrub and i s not amenable to the cotton-wick technique. The p l a n t stem was too. robust f o r the i n j e c t i o n method and, as the b i o s y n t h e s i s of monoterpenes i s -generally thought to be r a p i d , and the leaves of the p l a n t are s m a l l , the brush method was a l s o r u l e d out. Examination of the remaining two. techniques showed the c u t t i n g the s t a l k techinque to be the simp l e s t , and many, w o r k e r s 1 1 0 have used t h i s method with great success. We.decided,, t h e r e f o r e , to'use t h i s technique. - 87 -I I - (v) Synthesis of-, the, b i o s y n t h e t i c precursors of camphor. 2 As p r e v i o u s l y described,.two p r e c u r s o r s , [2- H^]-mevalonic a c i d 2 (61.) and [8- H ^ ] - l i n a l o o l . (62), were to be prepared f o r feeding to Rosemarinus o f f i c i a n a l i s . (61) (62) 2 2 [2- H_]-mevalonic a c i d [8- H ^ ] - l i n a l o o l ( i ) [2- H^]-mevalonic a c i d (61). Many methods f o r the.preparation of various l a b e l l e d mevalonic acids have been r e p o r t e d 2 2 . . The synthesis of E l l i s o n and B h a t n a g a r 1 1 1 however provides an opportunity to prepare a G.(2).-labelled d e r i v a t i v e from r e a d i l y a v a i l a b l e s t a r t i n g materials.. . The synthesis (Figure 70) y i e l d s mevalonic 'acid••• lactone -(61a) which can.be converted to mevalonic a c i d (6l_) p r i o r to the feeding experiments. 4-Acetoxy-2=butanone (64) was r e a d i l y obtained from the r e a c t i o n of methyl v i n y l . k e t o n e (66) w i t h g l a c i a l a c e t i c a c i d .(Figure 7-1). f o l l o w i n g the method'of C o r n f o r t h 1 1 2 . 2 S p e c t r a l data was c o n s i s t e n t w i t h the s t r u c t u r e given. [2- H 3 ] - E t h y l 2 acetate can be obtained, by e s t e r i f i e a t i o n of .readily a v a i l a b l e [ H^1_ a c e t i c a c i d , obtainable i n high i s o t o p i c p u r i t y as an n.m.r. s o l v e n t . - 88 -LDA = 'l i t h i u m diisopropylamide Figure 70. Pr e p a r a t i o n of [2- H„]-mevalonic a c i d (61). (66) (M) Figure 71. P r e p a r a t i o n of 4-aeetoxy-2-butanone. To prepare t h i s d e u t e r i o e t h y l acetate, an a c i d c a t a l y s e d e s t e r i f i c a t i o n of f H . l - a c e t i c a c i d w i t h ethanol was attempted. Excess ethanol was 4-employed to ensure completion of r e a c t i o n , .but i t was found d i f f i c u l t - 89 -to separate e t h y l acetate from excess solvent'. Examination of standard t a b l e s 1 1 3 showed these two compounds to form an a z e o t r o p i c mixture, and so other s o l v e n t s - ( d i e t h y l ether, benzene, toluene, and o-xylene) were t r i e d , a l l without success. I t was necessary f o r the prepared e t h y l acetate to be dry and a c i d - f r e e f o r the next step i n the r e a c t i o n sequence and preparation to such a degree of p u r i t y was d i f f i c u l t by t h i s method. Base cat a l y s e d e s t e r i f i c a t i o n would e l i m i n a t e the problem of contamination by a c i d , and carry.over of excess base would not impede the condensation. A l s o , i n b a s e . c a t a l y s i s , the. conjugate a c i d of the base can sometimes be p r e c i p i t a t e d by choice of an appropriate solvent during the course of the r e a c t i o n , and can. thus b e . e a s i l y removed by f i l t r a t i o n . P r e p a r a t i o n o f - [ 2 - H ^ ] - e t h y l acetate using base c a t a l y s i s and a dry i n e r t solvent and atmosphere would a l l o w f o r t r a n s f e r of the reagent to a s o l u t i o n o f - l i t h i u m diisopropylamide (LDA) without p r i o r p u r i f i c a t i o n . A convenient, base catalysed, e s t e r i f i c a t i o n method using 1 ,8-diazabicyclo- . [5.4.0]-undec-7-'ene .:(DBU) as the . b a s e 1 1 4 was c a r r i e d out i n dry d i e t h y l ether w i t h a c e t i c a c i d and e t h y l bromide. The s a l t DBU.HBr p r e c i p i t a t e d during the 'reaction.and-transfer of t h i s s o l u t i o n of [2- H^]-ethyl acetate i n dry d i e t h y l , ether to l i t h i u m diisopropylamide (LDA) i n dry d i e t h y l ether through, a glass, wool plug ensured no c a r r y over of the p r e c i p i t a t e . Reaction of.the..ethyl acetate anion generated i n t h i s r e a c t i o n w i t h 4.-acetox-y-2-butanone (64) gave an o i l , which was p u r i f i e d by column chromatography, to provide hydroxy-diester (65) 1 , 8-diazabicyclov .[5.4-.0] -undec-7-ene - 90 -(Figure .70) along w i t h some non-deuterated m a t e r i a l . The H-n.m.r. of hydroxy-diester (65) i n d i c a t e d that the compound was saturated w i t h deuterium a t 0(2) since the s i n g l e t a t 62.55 normally assigned to the G(2)-hydrogens i n the non-deuterated analogue could not be detected. A minor product from t h i s r e a c t i o n . e x h i b i t e d i n f r a red ( i . r . ) peaks a t -1 -1 1 3500 cm (v OH) and 1750 cm (v C=0., ester) and H-n.m.r. peaks at-6 5.2 (d.d.,1H), 5.22 (d.d.,1H), 6.0•(d.d.,1H), T.2 (t.,3H), and 4.2 (q.,2H), c h a r a c t e r i s t i c of a monosubstituted double bond and an ethoxy group. From t h i s data, s t r u c t u r e (67) was t e n t a t i v e l y assigned (Figure 72). The formation of t h i s compound can-be.- envisioned as removal by base of a proton a- to the ketone carbonyl followed by e l i m i n a t i o n of acetate anion to give m e t h y l . v i n y l ketone. .This then r e a c t s w i t h the anion [LiCD 2C0 2C 2H 5] to - give the observed- product. The base i n v o l v e d i n t h i s minor r e a c t i o n could.be the anion derived, from CD^GOOC-H^. (67) Figure 72. A side r e a c t i o n i n the-condensation o f 4-acetoxy-2-butanone (64) w i t h e t h y l acetate anion. 2 Conversion of the d i e s t e r a l c o h o l (65) to [2- H ,_J-mevalonic a c i d (61) was r e a d i l y achieved by s t i r r i n g w i t h 10% potassium hydroxide i n methanol. D i s t i l l a t i o n of the crude, o i l . o b t a i n e d on work-up gave pure 2 [2- H„]-mevalonic a c i d lactone (61a) as a c o l o u r l e s s o i l . ( i i ) [8- H ^ J - l i n a l o o l (62) OH (20) l i n a l o o l (62) [ 8 - 2 E ] - l i n a l o o l I t was considered t h a t i n t r o d u c t i o n of deuterium i n t o the t r a n methyl group of l i n a l o o l (20) could be- accomplished i n a r e l a t i v e l y simple f a s h i o n by r e g i o s e l e c t i v e o x i d a t i o n of the methyl groups (Figure 73). Selenium d i o x i d e .oxidation-of a l l y l i c methyl groups has 1 1 5 - 1 1 9 been shown'--exclusively-to occur t r a n s - to-a side chain , and n (20) R = a c t i v a t i n g group. 0G0CH, 0C0CH, (68) (62). (70) Figure 73. Proposed s y n t h e t i c route to [8- H 1 ] - l i n a l o o l . - 92 evidence f o r t h i s t r a n s - assignment had been r e p o r t e d 1 2 0 . The mechanism proposed f o r t h i s r e g i o s e l e c t i v e o x i d a t i o n i s shown i n Figure 7 4 1 1 9 . For e x p l a i n a t i o n of l e t t e r i n g (a)-(.e), see t e x t . Figure 74-. Mechanism of selenium d i o x i d e o x i d a t i o n 1 1 3 . Experimentation has .shown path (b) ([2,3]-sigmatropic rearrangement) to be dominant i n t h i s mechanism and l i t t l e or no formation of the a l l y l c a t i o n (path (a) or path (b) followed by path (c)) can be detected. The high r e g i o s e l e c t i y i t y of t h i s r e a c t i o n can be explained by t h i s [2,3]-s h i f t . ' At high temperatures .(70°-100°C) path (c) may be favoured. - 93 -A l l y l i c o x i d a t i o n of l i n a l o y l acetate (68) w i t h selenium d i o x i d e according to the method of Wakayama et a l . 1 1 5 (Figure 75) gave a low y i e l d of aldehyde (71.), pure by g . l . c . and t . l . c . examinations. However the yellow colour of t h i s product i n d i c a t e d that i t was contaminated by selenium and t h i s colour was r e t a i n e d even a f t e r d i s t i l l a t i o n and chromatography. The method of Umbreit and S h a r p l e s s 1 1 7 , using a minimum amount of selenium .dioxide and t e r t - b u t y l hydroperoxide to r e o x i d i s e the selenium formed, gave a mixture of 2:2:1 of 8 - o x o l i n a l o y l acetate (71.), 8 - h y d r o x y l i n a l o y l acetate (69) and s t a r t i n g m a t e r i a l (68) as a c o l o u r l e s s o i l . The s t a r t i n g m a t e r i a l could be separated from the product mixture by chromatography but any attempts to separate the a l c o h o l (69) from the aldehyde (71.) f a i l e d . In a d d i t i o n , t h i s method gave low y i e l d s presumably because much of the product c o n s i s t e d of organoselenium byproducts. The mixture of a l c o h o l (69) and aldehyde (7J.) was reduced w i t h sodium boro-hydride (NaBH.) i n an attempt to increase the y i e l d of a l c o h o l and make 4 the p u r i f i c a t i o n simpler. However, t h i s only r e s u l t e d i n a more complex mixture since r e d u c t i o n of the a, (3-unsaturated aldehyde to the f u l l y saturated a l c o h o l (73) a l s o occurred (Figure 76). Thus a method of producing mainly a l c o h o l or aldehyde was r e q u i r e d . Examination of the l i t e r a t u r e 1 1 9 showed th a t u s i n g one equivalent of selenium d i o x i d e produces mainly aldehyde and v i r t u a l l y no a l c o h o l , whereas usi n g 0.5 equivalents of selenium d i o x i d e gives a mixture of aldehyde and a l c o h o l . The procedure of A l t m a n 1 1 8 , us i n g 1 equivalent of selenium d i o x i d e , produced mainly aldehyde.with l i t t l e a l c o h o l i n an acceptable y i e l d . Chromatography of the r e a c t i o n product provided pure aldehyde, and the 1 s i g n a l at 69.4 i n the H-n.m.r. spectrum was assigned to the t r a n s -aldehyde group by analogy with the r e s u l t s of Chan et a l . 1 2 0 . The mass spectrum (low r e s o l u t i o n ) of t h i s compound does not e x h i b i t a M+ peak - 94 -OCOCH, OCOGH 3 (68) f *-( i i ) ( i ) Se0 2/dioxane/80 C/5h 1 1 5, (71) X OCOCH 3 -CHO ^ (71) \ . O C O C H 3 ( i i i ) [ [j CHO (71) ( i i ) Se0 2/^Bu00H/CH 2Cl 2/l0°C/4.5h 1 1 7, ( i i i ) S e 0 2/ethanol/A/24h 1 1 8. OCOCH, (69) CHO OH (72) Figure 75. Selenium dio x i d e o x i d a t i o n of l i n a l y l acetate (68). - 95 -OCOCH -CHO (71) (62)' crude mixture OCOCH, ( i ) COCH 3 : i i ) —• ( i i i ) (v) OCOCH 3 OCOGH 3 (69) ( i v ) (74) OCOCH 3 .OCOCH 3 -H _H OH (73) COCH 3 OMs (69) ( i ) NaBH /EUOH/RT ( i i ) NaBH. /E'tOH/RT ( i i i ) DIBAL/hexane/0°C 4 4 ( i v ) MsCl/Et3N/Et20/0°G (v) NaBH^/EtOH/0°C Figure 76. Reduction of aldehyde (71) to a l c o h o l (69). - 96 -but has peaks at m/e 168,. 150, and 135. The peak at m/e 168 corresponds to l o s s of ketene from the acetoxy- grouping and the peak at m/e150 corresponds to a McLafferty rearrangement of the same grouping (Figure 77). m/e.168 m/e 150 Figure 77. Mass spectrum fragmentation of aldehyde (71_). A minor product obtained from t h i s ' r e a c t i o n e x h i b i t e d i . r . peaks at 3500 cm - 1 (v OH), 1765 cm - 1 "(v 0=0 e s t e r ) , 1.700 •cm-1 (v 0=0 a, (3--1 1 unsaturated aldehyde) and I64O cm (v C=C). .The' H-n.m.r. spectrum of t h i s product e x h i b i t e d the tra n s - a l d e h y d i c proton resonance at 69.23 as w e l l as a resonance: at 64.1 which was assigned to -CH^OH. No resonances f o r the gem-dimethyl•groups, were present. Low r e s o l u t i o n mass spectrometry gave a molecular i o n of 226 mass u n i t s and s t r u c t u r e - 97 -OCOCH, SeO, " 0 ^ x OCOCH 3 (c) + H •OCOCH 3 + Se(OH), OCOCH 3 CHO OH (72) SeO, (b) OH + H20/- H OCOCH 3 Figure 78. Proposed formation of- minor' product (72) on selenium d i o x i d e o x i d a t i o n (see a l s o Figure.74). (72) was t e n t a t i v e l y assigned. I t thus appears that o x i d a t i o n has occurred c i s - to the side chain i n this-example (Figure 78). This can occur when path (c) i n the proposed mechanism i s followed (Figure 74). Reduction of aldehyde (71') to al c o h o l . (69) could be e a s i l y performed using sodium borohydride. I n i t i a l l y the..reduction was d i f f i c u l t to achieve without concomitant 1 .,4—reduction of the a,3-unsaturated system and so diisobutylaluminum hydride (DIBAL.)' was t r i e d ( F i g u r e 76). This reducing agent, which s p e c i f i c a l l y reduces the carbonyl group of-a,3-unsaturated systems, also, reduces est e r s and so the r e s u l t i n g product was 8 - h y d r o x y l i n a l o o l " (?4 ;). . I t . was.- hoped that the primary a l c o h o l i n t h i s compound could be s e l e c t i v e l y converted to a•good l e a v i n g group - 98 -( i n t h i s case the mesylate) i n the presence of the l e s s r e a c t i v e t e r t i a r y a l c o h o l . Attempts' to prepare the req u i r e d hydroxy-mesylate were un s u c c e s s f u l . Further experiments w i t h sodium borohydride showed that r e d u c t i o n of the a,3-unsaturated system i n (71_) to the saturated a l c o h o l (73) could be.: reduced and v i r t u a l l y e l i m i n a t e d by c a r r y i n g out the r e a c t i o n at- 0°C and using an a c c u r a t e l y measured 1 equivalent of sodium-borohydride. The pure a l c o h o l , obtained by chroma-1 tography of the crude r e a c t i o n product, showed i n - t h e H-n.m.r. spectrum a s i n g l e t at 64.0 corresponding to -CH^OH, and- the s i g n a l assigned to the a l l y l i c hydrogen -CH=C(CH^) (CHgOH') to-be'a broad t r i p l e t at 65.4-0, u p f i e l d from i t s p o s i t i o n i n the aldehyde (66.50). The f i n a l step i n the synthesis of 8 - d e u t e r i o l i n a l o o l (62) i n v o l v e d the deutride displacement of the C( 8 ) - a l c o h o l grouping i n 8-hydroxylina'lo-yl acetate "(69) (Figure 79) ••- I t . was i n i t i a l l y considered t h a t t h i s could be accomplished by treatment of the corresponding t o s y l a t e w i t h sodium borodeuteride•in 80% ,'h^xamethylphosphoramide (HMPA)-water. P r e p a r a t i o n of • the t o s y l a t e . i n the-usual way ( t o s y l c h l o r i d e / p y r i d i n e ) 1 2 2 gave a l o w . y i e l d . o f an o i l whose H-n.m.r. spectrum was con s i s t e n t w i t h the r e q u i r e d t o s y l a t e (75). Reduction using sodium borodeuteride in. 80% HMPA/water gave .'8 ^ d e u t e r i o l i n a l o y l acetate (77.) i n approximately 86% y i e l d . The preparation- of the t o s y l a t e was not easy to achieve i n - a r e l a t i v e l y acceptable y i e l d , and-so an a l t e r n a t i v e to t o s y l - as an a c t i v a t i n g group was sought. Sodium-borodeuteride i n 80% HMPA/water i s a l s o reported to work w e l l f o r h a l i d e s 1 2 1 and so an attempt to prepare the a l l y l i c bromide, u s i n g - g e r a n i o l (l_4.) as a model , was t r i e d (Figure 80). Using the method of Hooz and G i l a n i 1 2 3 , g e r a n i o l was t r e a t e d w i t h ear.bon tetrabromideand•tri-n-octylphosphine i n an attempt to convert the alcohol- to the bromide.. The o i l obtained i n t h i s r e a c t i o n - 99 -OCOCH 3 OCOCH, ( i ) or OCOCH ( i i ) R = M S f ( v / (15) R=Ts (76) R=Ms COCH3 (77) (62) ( i ) TsCl/py ( i i ) Msd/Et^N/Et^O ( i i i ) NaBD^/80$ HMPA/H20 ( i v ) NaOH/MeOH (v) 2.1 L i B D E t 3 / E t 2 0 ( v i ) 4.1 LiBDEtyEtgO Figure 79. Conversion of a l c o h o l (69) to 8 - d e u t e r i o l i n a l o o l (62). (U) ( i ) C B r 4 / ( n C g H 1 7 ) 3 P E t 2 0 (80). Figure 80. Attempted conversion of g e r a n i o l to geranyl bromide. - 100 -was subjected- to d i s t i l l a t i o n , and chromatography without a c h i e v i n g p u r i t y . Both of these proceedures•were seen to cause decomposition or rearrangement of the g e r a n i o l s k e l e t o n . Rearrangement to the a l l y l i c isomer (80) was thought to be o c c u r r i n g and could be seen i n the H-n.m.r. spectrum by the disappearance of a doublet (2H) at 64.0 (-CH^-X, X=Br or OH?) and•appearance of a four l i n e p a t t e r n (d.d.,1H) at 66.2 (-CH^CH^) . I n comparison, i t was : demonstrated that the conversion of 8Thydroxylinaloyl acetate (69)., to 'the corresponding mesylate (76) i n v o l v e d no i s o m e r i s a t i o n of ;the double bond, and so preparation of the mesylate looked' the most promising. As b e n z y l i c mesylates are known to be u n s t a b l e 1 2 1 * , i t was assumed that t h i s a l l y l i c mesylate would be s i m i l a r l y unstable and thus no attempts to p u r i f y t h i s 1 compound were made. Examination by H-n.m.r. spectroscopy of the crude product obtained on t r e a t i n g 8 - h y d r o x y l i n a l o y l acetate (69) w i t h methanesulphonyl c h l o r i d e and t r i e t h y l a m i n e i n dry d i e t h y l ether showed i t to contain mainly the mesylbxy - compound,, .indicated by the presence of a s i n g l e t (3H) at 62.9, assigned'to-GH^-SO^-, -and a s i n g l e t at 64.46 (2H), assigned to the 8-methylene p o s i t i o n , ; s h i f t e d from 64.0 i n the s t a r t i n g a l c o h o l . Reduction using NaBD^.in 80% HMPA/H^O gave only a 10% y i e l d of [.8- H g J - i i n a l o y l a c e t a t e ' (77), and so an a l t e r n a t i v e r e d u c t i o n was t r i e d . : The mesylate (76.)- was. reduced w i t h Superdeuteride ( l i t h i u m '-triethylborodeuteri-de, LiBDEt^-) • .according;...to the. method of. Holder and M a t t u r o 1 2 5 . - Using 2.1 equivalents .of reducing agent gave 2 2 [8- H ^ ] - l i n a l o y l acetate (7_7)-' w i t h some [8- H^  ] - l i n a l o o l (62) and these two compounds could be e a s i l y separated by:column chromatography. 2 2 [8- H^  ] - l i n a l o y l acetate (77) could be r e a d i l y converted to [8- H^] -l i n a l o o l (62)'using 10% sodium hydroxide i n methanol. A repeat r e d u c t i o n of the mesylate (76) :using 4.1 equivalents of reducing agent . - - 101 -gave a high y i e l d o f • r e l a t i v e l y pure [ 8--H^.]-linalool (62). Column chromatography aff o r d e d the pure-material f o r ' t h e feeding experiments. I I - ( v i ) R e s u l t s . The feeding experiments were carried' : out-using the cut stem method (see page 86). I n the f i r s t feeding experiment, the shoots were placed 2 i n an aqueous s o l u t i o n of l a b e l l e d precursor ([-8- H ^ - l i n a l o o l (62) 2 or [ 2 - H^]-mevalonic a c i d (61.)) and-maintained on-water f o r 3 days. E x t r a c t i o n of the steam d i s t i l l e d ' e s s e n t i a l o i l f o l l o w e d by column chromatography aff o r d e d camphor, whose s p e c t r a l . d a t a -was c o n s i s t e n t 2 wi t h an authe n t i c sample. The . H-F.T.-n.m-.r.. spectrum (approximately 25000 t r a n s i e n t s , 24 hours accumulation •time)'-:revealed no i n c o r p o r a t i o n 2 of l a b e l i n camphor from e i t h e r precursor (-[8- >H^ ] - l i n a l o o l (62) or 2 [2-oH^]-mevalonic a c i d (61.)). The-. experiments were repeated, t h i s time using an aqueous s o l u t i o n of 'ATP (0.1' mg/ml) 1 2 6 r a t h e r than water to feed the precursors and the shoots were maintained on t h i s ATP s o l u t i o n f o r 3 days.' E x t r a c t i o n and chromatography as before gave 2 camphor, i d e n t i f i e d by s p e c t r a l ' d a t a . The' H-F.T.-n.m.r. sp e c t r a , a f t e r approximately 20000 t r a n s i e n t s (Figure- 81) seemed to show a peak, but i t was assumed'that t h i s , was• an a r t i f a c t , , only one poi n t being seen on the peak. In a d d i t i o n , -when, the spectrum was recorded on a *•' 2 4.00MHz instrument (approx. 60MHz f o r observing H)'none of these ' g l i t c h e s ' were seen. -It t h e r e f o r e seemed- reasonable to assume th a t no i n c o r p o r a t i o n of e i t h e r precursor, was seen.- - From these r e s u l t s and the r e s u l t s of others, i t i s most l i k e l y , that, a problem arose i n e i t h e r the t r a n s p o r t of the precursor to the s i t e of s y n t h e s i s or that the production of camphor was at too low a l e v e l f o r i n c o r p o r a t i o n to be 102 -(a) (b). ill. " T 4 Figure 81. H-N.m.r. spectra obtained from camphor ext r a c t e d from Rosemarinus o f f i c i a n a l i s a f t e r feeding w i t h ( a ) [ 2 - H~]-MVA, and (b)[8- H ^ - l i n a l o o l . - 103 -seen. Seasonal v a r i a t i o n s i n the l e v e l of monoterpenes i n P i c e a  s i t c h e n s i s ( S i t k a spruce) have been, r e p o r t e d 1 2 7 ' a n d i n t h i s species, optimum l e v e l s of camphor are seen i n October, with a decrease i n the summer months. I t i s reasonable to assume-that the l a c k of i n c o r p o r a t i o n of 8 - d e u t e r i o l i n a l o o l (62) or [2- H^]-mevalonic a c i d (6l_) i n t o camphor i s due to the f a c t that the compounds were not administered to the p l a n t at a time of reasonable terpe n o i d : b i o s y n t h e s i s r a t h e r than the precursor being of an i n c o r r e c t nature. Mevalonic a c i d , f o r example, has been found to be i n c o r p o r a t e d , i n low y i e l d s , i n t o a l l types of terpenoid compound. R e p e t i t i o n of the feeding experiments i n the s p r i n g , when optimum pla n t growth g e n e r a l l y occurs, may r e s u l t i n the i n c o r p o r a t i o n of mevalonic a c i d and , p e r h a p s , . l i n a l o o l . These experiments w i l l be attempted -in the near- f u t u r e . I I - ( v i i ) Suggestions f o r further-work. As has been suggested, r e p e t i t i o n of the feeding work during the s p r i n g may-result i n an i n c o r p o r a t i o n , of-the precursors. I n a d d i t i o n , changing the mode of feeding.may a l s o b r i n g favourable r e s u l t s , to t h i s end, growing shoots complete w i t h r o o t s , i n a- s o l u t i o n of the precursor may be worth i n v e s t i g a t i n g . A l t e r n a t i v e l y , spraying the p l a n t w i t h a dimethylsulphoxide (DMSO)"solution of the precursor may be of use. DMSO i s known to penetrate t i s s u e s , c a r r y i n g w i t h i t any d i s s o l v e d m a t e r i a l . This may enable the l a b e l l e d precursor to be absorbed at the s i t e of synthesis and thus i n c o r p o r a t i o n could be achieved. Once t h i s problem of i n c o r p o r a t i o n has been overcome, the system i s open to a number of further, s t u d i e s . The stereochemistry of the r i n g c l o s u r e to the b i c y c l i c monoterpenes,-be i t syn- or a n t i - , - 104 -can be examined by the use of a l i n a l o o l d e r i v a t i v e l a b e l l e d at the t e r m i n a l double bond. (Figure 82-)'. - There are various methods f o r l a b e l l i n g l i n a l o o l w i t h H at the 0(1) e l s - or tr a n s - p o s i t i o n s and 2 determination of the p o s i t i o n of • H a t 0(3') i n camphor would i n d i c a t e whether the r i n g closure- had .-oecured•• v i a a syn-- or a n t i - mode. Figure 82. Scheme-for e s t a b l i s h i n g ' t h e stereochemistry of r i n g c l o s u r e . I l l EXPERIMENTAL. General. Unless otherwise s t a t e d the f o l l o w i n g are i m p l i e d : G a s - l i q u i d chromatography ( g . l . c . ) was performed on a Hewlett-Packard model 5831A (flame i o n i s a t i o n detector) gas chromatograph usi n g a 6' x 1/8" column w i t h 3% OV-17 as the s t a t i o n a r y phase supported on Chromosorb ¥ and n i t r o g e n as the c a r r i e r gas. C a r r i e r gas flow was ca 30 mL/min. The H 60MHz .nuclear magnetic, resonance (n.m.r.) spectra 1 were recorded on a Varian A s s o c i a t e s model T60, H 80MHz F.T.-n.m.r. 1 •spectra were recorded on a Brueker model WP80 and H 400MHz E.T.-n.m.r. spectra were recorded on a Brueker model WH4OO. S i g n a l p o s i t i o n s are given on the d e l t a (6) scal e w i t h t e t r a m e t h y l s i l a n e (TMS) as an i n t e r n a l reference (60.00). The 12.3MHz F.T.-n.m.r. spectra were recorded on a Brueker model WP80 spectrometer. S i g n a l p o s i t i o n s are given on the d e l t a (6) s c a l e w i t h CDCl^ as a n - i n t e r n a l reference (67.51). S i g n a l m u l t i p l i c i t y , c o upling constants ( i f observable), i n t e g r a t e d area and s i g n a l assignments are i n d i c a t e d i n parentheses. I n f r a r e d spectra ( i . r . ) - w e r e recorded on a P e r k i n Elmer 137B I n f r a c o r d spectro-meter. S o l u t i o n spectra were performed u s i n g a sodium c h l o r i d e c e l l of 0.1mm t h i c k n e s s . Absorption p o s i t i o n s (v ) are given i n the cm ^ ^ max 6 u n i t and are c a l i b r a t e d by means of the 1601 cm band of polystyrene. Low r e s o l u t i o n mass spectra were determined on the Kratos AE1 model MS902 or model MS50 instruments. Microanalyses were performed by Mr. P. Borda, M i c r o a n a l y t i c a l Laboratory, U n i v e r s i t y of B r i t i s h Columbia, Vancouver. A l l solvents used f o r i . r . and n.m.r. were of s p e c t r a l q u a l i t y . Reagents and r e a c t i o n solvents used were of e i t h e r Reagent - 106 -or C e r t i f i e d grade. The term "petroleum ether (30°-60^C)" r e f e r s to the low b o i l i n g f r a c t i o n of petroleum d i s t i l l a t e ( b o i l i n g p o i n t ca. 35 -60 C). Dry or p u r i f i e d solvents or reagents, where i n d i c a t e d , were prepared as f o l l o w s 1 2 8 : A z o - b i s - i s o b u t y r o n i t r i l e (AIBN) by r e c r y s t a l l i s a t i o n from d i e t h y l ether. Benzene by r e f l u x i n g w i t h and d i s t i l l i n g from calcium hydride at atmospheric pressure. Diazobicycloundecene (DBU) by s t i r r i n g over calcium hydride and d i s t i l l i n g a t reduced pressure. D i e t h y l ether (Et^O) or tetrahydrofuran (THF) by r e f l u x i n g over sodium or l i t h i u m aluminum hydride and d i s t i l l i n g . Diisopropylamine (("""Pr^NH) o r t r i e t h y l a m i n e (Et^N) by s t i r r i n g w i t h sodium hydroxide and d i s t i l l i n g . E t h y l bromide (EtBr) by d r y i n g w i t h calcium hydride and d i s t i l l i n g from phosphorous pentoxide (P,_>0^ ). Pentane by c a r e f u l d i s t i l l a t i o n c o l l e c t i n g only the 37°C b o i l i n g f r a c t i o n . P y r i d i n e by s t i r r i n g w i t h potassium hydroxide and d i s t i l l i n g . Selenium d i o x i d e (SeO^) by sublimation at atmospheric pressure (340°C). p_-Toluenesulphonyl c h l o r i d e ( t o s y l c h l o r i d e ) by d i s s o l v i n g i n chloroform, p r e c i p i t a t i n g i m p u r i t i e s by the a d d i t i o n of petroleum ether (30 -60 C), f i l t e r i n g , c l a r i f y i n g w i t h c h a r c o a l , and concentrating u n t i l c r y s t a l s are obtained. n B u t y l l i t h i u m used was supplied by A l d r i c h Co. and t i t r a t e d w i t h .s-butanol u s i n g 1 ,1O-phenanthroline as i n d i c a t o r p r i o r to use. S i l i c a g e l f o r column chromatography w a s . S i l i c a Gel 60, 230-400 mesh, purchased from BDH Chemicals L t d . , and was c a r r i e d out under medium pressure ( f l a s h ) 1 2 9 . A n a l y t i c a l t h i n l a y e r chromatography ( t . l . c . ) was c a r r i e d out usin g B a k e r - f l e x chromatography cards, coated w i t h s i l i c a g e l IB2F and supplied by J.T.Baker Chemical Co., P h i l l i p s b u r g N.J. P l a t e s were v i s u a l i s e d under f a r u l t r a v i o l e t (u.v.) r a d i a t i o n and were developed by spraying w i t h a s o l u t i o n of dodecaphospho-molybdic a c i d (5 g) i n ethanol (100 mL) followed by heating, or by spraying w i t h 5$ n i t r i c a c i d i n concentrated s u l p h u r i c a c i d followed by heating ( s p e c i f i c spray f o r camphor). P r e p a r a t i o n of 1-acetoxy-3-butanone (64). Methyl v i n y l ketone ( ( 6 6 ) , 10 mL, 8.64 g, 0.123 mol) was di s s o l v e d i n g l a c i a l a c e t i c a c i d (70 mL), 2-drops- of water added, an the mixture heated to 100°G f o r 24 hours.-.. The mixture was d i s t i l l e d (15 mm Hg) and m a t e r i a l b o i l i n g above 50°C c o l l e c t e d . This yellow l i q u i d was d i s s o l v e d i n d i e t h y l ether, washed with saturated sodium bicarbonate s o l u t i o n and water, d r i e d (MgSO ) and evaporated to give 4 a yellow o i l . D i s t i l l a t i o n (1.5 mm.Hg) affor d e d pure 1-acetoxy-3-butanone ((64K 9.14 g» 0.070 mol, 57$ y i e l d ) as a c o l o u r l e s s o i l , b.p. 800-84°C/15 mm Hg. V ( C C l . ) : 1745 (stron g , sharp, C=0 e s t e r ) , 1735 (s t r o n g , sharp, max 4 C=0 ketone). 6 (60MHz, C C l . ) : 1.95 ( s , 3H, CH,-C0-C-)f 2.1 (s , 3H, CH--C0-0-), 4 J i 2.7 ( t , 2H, C-C0-CH2-, J=6Hz), 4-16 ( t , 2H, -C-CH -0-C0-). Model experiments f o r the preparation of e t h y l acetate, ( i ) A c i d c a t a l y s e d e s t e r i f i c a t i o n . A c e t i c a c i d (5 g, 0.08 mol) and su l p h u r i c a c i d (0 .5 mL, 0.01 mol) were d i s s o l v e d . i n ethanol (100 mL) and heated to r e f l u x f o r 3 - 108 -hours. The mixture was cooled and poured onto water and the aqueous s o l u t i o n e x t r a c t e d w i t h d i e t h y l ether. The combined organic phases were washed with saturated sodium bicarbonate s o l u t i o n and water, d r i e d (MgSO.), and d i s t i l l e d (atmospheric pressure) to give a l i q u i d (3.3 g) 4 -] whose 60MHz H-n.m.r. spectrum showed i t to- c o n s i s t of a 1:1 mixture of e t h y l acetate and d i e t h y l ether. Separation of these two components was not achieved. ( i i ) Base c a t a l y s e d e s t e r i f i c a t i o n . A c e t i c a c i d (1 g, 0.017 mol) and e t h y l bromide (1.8 g, 0.017 mol) were d i s s o l v e d i n benzene, toluene, or o—xylene (20 mL). D i a z o b i c y c l o -undecene (DBU, 2.54 g, .0.017 mol) was added dropwise and the mixture s t i r r e d overnight at room temperature. The c l e a r s o l u t i o n was decanted from the p r e c i p i t a t e d s a l t , washed w i t h water, and d r i e d (MgSO.). 4 D i s t i l l a t i o n a f f o r d e d e t h y l acetate (0.54 g) whose 60MHz H-n.m.r. i n d i c a t e d t h a t i t was contaminated with solve n t . No s i g n a l s f o r e t h y l bromide could be detected. P r e p a r a t i o n of [ l - H j , - e t h y l acetate (63). D e u t e r i o a c e t i c a c i d (5 g, 0.078 mol) and e t h y l bromide (5.8 mL, 0.078 mol) were d i s s o l v e d i n dry d i e t h y l ether (5 mL) under an atmosphere of dry argon. Diazobicycloundecene (DBU, 11.66 mL, 0.078 mol) was added dropwise with i c e - c o o l i n g and the r e a c t i o n s t i r r e d f o r 24 hours at room temperature, during which time the s a l t DBU.HBr p r e c i p i t a t e d . The r e s u l t i n g dry d i e t h y l ether s o l u t i o n of [1- H^]-e t h y l acetate was used without p u r i f i c a t i o n f o r the preparation of e t h y l 5-acetoxy -3-hydroxy -3-methyl pentanoate (65). - 109 -2 Pre p a r a t i o n of e t h y l [2- H 2")-5-acetoxy-3-hydroxy-3-methyl pentanoate (65)• Dry diisopropylamine (10.9 mL, 0.078 mol) was d i s s o l v e d i n dry d i e t h y l ether (10 mL) under an atmosphere of dry argon, and cooled to -22°C. " B u t y l l i t h i u m (1.6M, 47 mL, 0.078 mol) was added and the mixture s t i r r e d f o r 1 hour. The temperature was then lowered to -78°C and the d i e t h y l ether s o l u t i o n of [ l - ^ H ^ ] - e t h y l acetate (63) was added through a glass wool plug to ensure no p r e c i p i t a t e d s a l t was c a r r i e d over. The r e a c t i o n was s t i r r e d f o r 20 minutes and the 1-acetoxy-3-butanone ((^4), 10.14 g, 0.078 mol) i n dry d i e t h y l ether (15 mL) was added. S t i r r i n g was continued f o r a f u r t h e r 20 minutes, water was added to the mixture and the s o l u t i o n allowed to warm to room temperature. The mixture was ex t r a c t e d w i t h d i e t h y l ether and the combined organic phases were washed with water, saturated sodium bicarbonate s o l u t i o n , b r i n e , and d r i e d (MgSO.). Evaporation of the solvent gave a c o l o u r l e s s 4 o i l (7 g). Chromatography ( s i l i c a g e l , petroleum ether (30 -60 C): d i e t h y l ether 2:1 eluant) gave (65) (2.40 g, 0.0109 mol, 14% y i e l d ) and some non-deuterated (65) (2.56 g, 15% y i e l d ) . V (CC1.); 3550 (weak, broad, OH), 1745, 174.0 (st r o n g , sharp, C=0). max 4-6 (60MHz, CDC1 3); 1.20 ( t , 3H, CH^CHg-O-, J=7Hz) , 1.27 ( s , 3H, t e r t i a r y methyl), 1.91 (q, 2H, -O-CH^-CH^-C(OH)(CH^)-, J=7Hz), 2.0 ( s , 3H, CH^-CO-O), 3.61 (bs, 1H, disappears w i t h DgO, -OH), 4.1 (q, 4H, -CH2-CH2-0Ac and -CO-O-CHg-CH overlapping, J=7Hz). Mass spectrum ( l o w . r e s . ) : m/e(rel. i n t . ) ; 205 ( 1 % ) , 175 (3%),-l60 ( 1 % ) , 145 (29%), 133 (100%) 115 (22%), 106 (16%), 99 ( 7 % ) . A n a l y s i s : For C ^ H ^ O ^ C a l c : C, 54.53; H, 8.31. . Found: C, 54.70; H, 8.10. - 110 -P r e p a r a t i o n of [2- 2H 2 l - 3-hydroxy - 3-methyl - 5-pentanolide (\2-^E^\-mevalonolactone) (61 a ) . 2 E t h y l [2- H^] -5-acetoxy -3-hydroxy -3-methyl pentanoate (( 6 5 ) , 1.45 g, 0.0067 mol) was d i s s o l v e d i n methanolic potassium hydroxide (10$ w/v, 10 mL) and s t i r r e d at room temperature f o r 1 hour. The r e a c t i o n was a c i d i f i e d by the a d d i t i o n of methanolic hydrogen c h l o r i d e and s t i r r e d f o r 2 hours at room temperature. The p r e c i p i t a t e d potassium c h l o r i d e was f i l t e r e d o f f and the methanol evaporated. The o i l was d i s s o l v e d i n chloroform, f i l t e r e d , and the chloroform evaporated. D i s t i l l a t i o n ( k u g e l r o h r , 0.03 mm Hg, =110°C) af f o r d e d [ 2 - 2 H 2 ] -mevalonolactone ((61a.), O.84 g, 0.0065 mol, 97$ y i e l d ) as a c o l o u r l e s s o i l . V ( C C l , ) ; 3620 (weak, sharp, OH), 3450 (weak, broad, OH), 1735 max 4 (stro n g , sharp, C=0). 6 (80MHz, CDCl^); L 4 O ( s , 3H, t e r t i a r y methyl), 1.69 (bs, 1H, disappears with D 20, OH), 1.85 (d, 1H, J=5Hz) and 1.95 (d, 1H, J=5Hz) (-CH 2-C(0H)(CH 3)-), 4.25-4.75 (m, 2H, -CH 2-0-C0-). Pr e p a r a t i o n of 6-acetoxy-2 , 6-dimethyl-2,7-octadienal (71). ( i ) According to the method of Wakayama et a l . 1 1 5 L i n a l o y l a c e t a t e • ( ( 6 8 ) , 39.2 g, 0.2 mol) and selenium d i o x i d e (22.2 g, 0.2 mol) were d i s s o l v e d i n dioxane (100 mL) and heated to 80°C f o r 5 hours. A f t e r c o o l i n g , the deposited selenium was removed by vacuum f i l t r a t i o n through c e l i t e and the dioxane evaporated to give a red/orange o i l . A 1:1 petroleum ether (30 -60 C ) : d i e t h y l ether mixture (100 mL) was added and the mixture shaken. The organic l a y e r was decanted and the solvent evaporated. The r e s u l t i n g o i l y residue was d i s s o l v e d i n d i e t h y l ether, washed w i t h saturated sodium bicarbonate s o l u t i o n and water, d r i e d (MgSO.) and evaporated to give a red/orange 4 o i l (39.11 g). D i s t i l l a t i o n (0.03 mm Hg) af f o r d e d the aldehyde (71.) (11.06 g, 0.053 mol, 26% y i e l d , 90% pure by g.l.c.) as a yellow o i l , b.p. 98°-100°C/0.03 mm Hg. 1 H-n.m.r. and i . r . spectra were c o n s i s t e n t w i t h the s t r u c t u r e of 6-acetoxy-2,6-dimethyl-2,7-octadienal.. ( i i ) According to the method of Umbreit and S h a r p l e s s 1 1 7 . Selenium d i o x i d e (5.66 g, 0.051 mol) was d i s s o l v e d i n methylene c h l o r i d e (100 mL) and t - b u t y l hydroperoxide (20 .4 mL, 0.204 mol) added. The mixture was s t i r r e d i n the dark f o r 30 minutes, cooled to 10°C, and l i n a l o y l acetate (( 6 8 ) , 20 g, .0.102 mol) added. The mixture was s t i r r e d at 10°C f o r 4s hours. A f u r t h e r a l i q u o t of methylene c h l o r i d e (75 mL) was added and the organic phase washed with saturated sodium bicarbonate s o l u t i o n and water, d r i e d (MgSO. )•-and evaporated to give 4 1 a pale yellow o i l , seen to contain t - b u t y l hydroperoxide by H-n.m.r. a n a l y s i s . This excess hydroperoxide was destroyed by d i s s o l v i n g the mixture i n c o l d a c e t i c a c i d (20 mL) and c a r e f u l l y adding dimethyl sulphide (30- mL) w i t h i c e c o o l i n g . A f t e r s t i r r i n g f o r 2 hours, the mixture was n e u t r a l i s e d w i t h 20% w/v potassium carbonate s o l u t i o n and then e x t r a c t e d w i t h d i e t h y l ether. The combined organic phases were washed with water, d r i e d (MgSO.),. and evaporated to give a c o l o u r l e s s o i l seen to be 20% s t a r t i n g m a t e r i a l , 38% aldehyde, and 40% a l c o h o l by g . l . c . Chromatography ( s i l i c a g e l , gradient e l u t i o n from petroleum ether (30°-60°C) to 30% d i e t h y l ether/petroleum ether (30°-60°C)) gave recovered s t a r t i n g m a t e r i a l (^ 68) (1 .56 g, 90% g . l . c . p u r i t y , 0.0079 mol), aldehyde (71.) (1.20 g, 90% g . l . c . p u r i t y , 0.0057 mol, 5.6% y i e l d ) , a l c o h o l (69) (1.01 g,.87% g . l . c . p u r i t y , 0.0048 mol, 4.7% y i e l d ) and a 1:1 mixture of a l c o h o l (69) and aldehyde (71.). (2.06 g, 0.010 mol, 9.8% - 112 -y i e l d ) which could not be f u r t h e r separated. A l a r g e amount of m a t e r i a l was l o s t as organoselenium byproducts i n t h i s p reparation and the y i e l d could not be r a i s e d . ( i i i ) According to the method of Altman et a l . 1 1 8 L i n a l o y l acetate ( ( 6 8 ) , 10.8 g, 0.055 mol) was d i s s o l v e d i n ethanol (95$, 75 mL), f r e s h l y sublimed selenium d i o x i d e (6.115 g, 0.055 mol) added, and the mixture r e f l u x e d f o r 24 hours. The deposited selenium was removed by f i l t r a t i o n through e e l i t e and the ethanol evaporated to give a red/orange o i l . . The o i l was d i s s o l v e d i n d i e t h y l ether, washed with saturated sodium bicarbonate s o l u t i o n and b r i n e , d r i e d (MgSO.) and evaporated to give a red o i l (13.05 g)• Chromato-' 4 graphy ( s i l i c a g e l , 1:1 petroleum ether (30°-60°C):diethyl ether eluant) gave the pure aldehyde (71.) (3.81 g, 0.018 mol, 33$ y i e l d , >99$ pure by g.l.c.) as a colourless, o i l and a mixture of a l c o h o l (69) and aldehyde (71.) (1.449 g, 0.0069 mol, 12.55$.-yield, -80$ a l c o h o l by g.l.c.) which could be c a r e f u l l y reduced by sodium borohydride i n the next step. V ( C C l , ) ; 1735 (stron g , sharp, C=0 e s t e r ) , 1685 (s t r o n g , sharp, max 4 C=0, a, (3-unsaturated aldehyde), 1655 (weak, sharp, C=C), 1255 (strong, broad, C-0-C ester) . 6 (400MHz, CDC1 3); 1.59 ( s , 3H, t e r t i a r y methyl), 1.745 (d, 3H, a l l y l i c CH_, J , ,. =1.5Hz), 2.02 (,.s, 3H, CH.C0-0-), 1.95 ( d t , 2H, CH2-CH2-CH=C, J 1 2=7.5Hz, J 2 3=8Hz), 2.37 (q, 2H, C(0Ac)-CH 2-, J=8Hz), 5.18 (dd, 1H, -CH=CHH trans,. J . =12Hz, J =0.75Hz), 5.205 (dd, 1H, c i s gem -CH=CHH cis,. J . =16HZ, J =0,75Hz), 5.965 (.dd, 1H, -CH=CHH, — trans gem — J . =16HZ, J . =12Hz), 6.475 ( t q , 1H, CH 0-CH=C(CHj (CH0), J , 0=7.5Hz, trans c i s 2 — 3 1,2 3, =1.5Hz), 9.40 ( s , 1H, -CH0). 1,3trans — Mass spectrum ( l o w - r e s . ) : m/e(rel. i n t . ) ; 168 (1%), 150 (27$), 135 (7.5$), 43 (100$). A n a l y s i s : For C ^ g C ^ C a l c : C, 68.55; H, 8.63. Found: C, 68.78; H, 8.60. Reduction of 6-acetoxy-2 , 6-dimethyl-2,7octadienal (71). ( i ) Using diisobutylaluminum hydride (DIBAL). 6-Acetoxy-2 , 6-dimethyl-2,7-octadienal (( 7 1 ) , 1 g, 0.00476 mol) was d i s s o l v e d i n - dry hexanes under an atmosphere of dry argon. The s o l u t i o n was cooled to 0°C and DIBAL (20$ s o l u t i o n i n hexane, 13 mL, 0.019 mol) added. The r e a c t i o n was s t i r r e d - a t 0°-5°C f o r 3 hours and a 1:1 s o l u t i o n of methanol and water was. added-to p r e c i p i t a t e the aluminum s a l t s . The mixture, was f i l t e r e d and the organic s o l u t i o n separated, d r i e d (MgSO ), and evaporated to give a c o l o u r l e s s o i l . 4 P u r i f i c a t i o n by chromatography ( s i l i c a g e l , d i e t h y l ether eluant) gave 8 - h y d r o x y - l i n a l o o l (74) as a c o l o u r l e s s o i l : (0.-71. g, 0.00418 mol, 88$ y i e l d ) . . 6 (60MHz, C C l , ) ; 1.33 ( s , 3h, t e r t i a r y methyl), 1.66 ( s , 3H, a l l y l i c 4 methyl), 1.5-2.4 (m, 4H> -CIL^-CH^-), 3.2 (bs, 2H, disappears with D^O, 2 -OH), 4.1' ( s , 2H, -CH o-0H), 5.0 (dd, 1H, -CH=CHH t r a n s , J . =12Hz, — — 2 — cxs J =1Hz), 5.16 (dd, 1H, -CH=CHH c i s , . J . =18Hz, J =1Hz), 5.36 gem - tr a n s gem (b t , 1H, -CH=C(:CHo)(CHo0H)), 5.90 (dd, .1H, -CH=CHH, J . =12Hz, — 5 2 — c i s J , =18Hz). tra n s ( i i ) Using sodium borohydride (NaBH ). 4 6-Acetoxy-2 , 6-dimethyl-2,7-oetadienal ((71_), 2.718 g, 0.0129 mol) was d i s s o l v e d i n ethanol (95$,. 20 mL) and NaBH .:(0.536 g, 0.014 4 mol) added. The mixture was s t i r r e d , a t i c e - b a t h temperature f o r 2 hours,-' a f t e r which time d i l u t e h y d r o c h l o r i c a c i d (20$ v/v) was added - 1 U u n t i l the s o l u t i o n remained a c i d i c . The mixture was extr a c t e d w i t h e t h y l acetate and the combined organic phases were d r i e d (MgSO^). Evaporation of the solvent gave the crude a l c o h o l (2.8 g) which was p u r i f i e d by chromatography ( s i l i c a g e l , 1:1 petroleum ether (30 -60 C): d i e t h y l ether eluant) to give the pure a l c o h o l (2.334 g» 0.011 mol, 85% y i e l d ) . V (CC1,); 3640 (weak, sharp, OH), 3450 (weak, broad, OH), 1745 max 4 (strong , sharp, C=0 e s t e r ) , 1655 (weak, sharp, C=C), 1245 (stron g , broad, C-O-C) . 6 (80MHz, CDC1 3); 1.25 (bs, 1H, disappears with T)£, OH), 1.55 ( s , 3H, t e r t i a r y methyl), 1 .675 ( s , 3H, a l l y l i c methyl), 2.025 (2, 3H, CH_3-C0-0), 1 .75-2.25 (m, 4H, C(0Ac)-CH_2-CH2-CH=), 4.O (s,. 2H, -CHg-OH) , 5.113 (d, 1H, -CH=CHH t r a n s , J . =UHz), 5.163 (d, 1H, -CH=CHH c i s , J , =16HZ), c i s - trans 5.40 ( b t , 1H, -CH=C(CH,)(CH OH)), 6.0 (dd, 1H, -CH=CHH, J =16HZ, J . =UHz). C I S Mass spectrum (low r e s . ) : m/e ( r e l . i n t . ) ; 212.(1.2%), 195 ( 6 % ) , 170 (1.3%), 152 (14%), 134(28%), 121 (65%), 71 (100%). A n a l y s i s : For C 1 2 H 2 Q 0 3 C a l c . r C, 67.89; H, 9.50. Found: C,. 67.64; H, 9.60. Pre p a r a t i o n of 8 - t o s y l o x y l i n a l o y l acetate (75). 8 - H y d r o x y l i n a l o y l acetate ( ( 6 9 ) , 0.05 g, 0.0024 mol) was d i s s o l v e d i n dry p y r i d i n e (15 mL) and cooled.to 0°C. F r e s h l y p u r i f i e d _p_- toluene sulphonyl c h l o r i d e ( t o s y l c h l o r i d e , 0.92 g, O.OO48 mol) was added and the r e s u l t i n g homogeneous mixture kept at or below 0°C f o r 24 hours. The r e a c t i o n mixture was poured onto i c e and extracted w i t h d i e t h y l ether and the combined organic phases.were q u i c k l y washed w i t h c o l d d i l u t e h y d r o c h l o r i c a c i d and water,, d r i e d (MgSO.) and evaporated to give (75) as a c o l o u r l e s s o i l (0,124 g, 0.00034 mol, 14$ y i e l d ) . ' 1 H-n.m.r. spectra showed the presence of a t o s y l - grouping and was con s i s t e n t w i t h t h i s o i l . being f a i r l y pure 8 - t o s y l o x y l i n a l o y l acetate (75). Attempts to c r y s t a l l i s e t h i s o i l f a i l e d , and th e r e f o r e the compound was reduced without f u r t h e r . p u r i f i c a t i o n . Reduction of 8 - t o s y l o x y l i n a l o y l acetate (.75):- using sodium borohydride. 8 - T o s y l o x y l i n a l o y l acetate ((_75), 0.7 g, 0.002 mol) was d i s s o l v e d i n 80$ HMPA/H'20 (20 mL) and sodium borohydride (0.6g, 0.016 mol) added. The mixture was heated to 50°C f o r t h i r t y minutes, and then cooled and d i l u t e d w i t h water, The mixture.was e x t r a c t e d w i t h d i e t h y l ether and the combined organic phases, were washed w i t h water, d r i e d (MgSO,) and evaporated to give a yellow o i l , . D i s t i l l a t i o n (25 mm Hg) 4 afford e d l i n a l o y l acetate (.(68) , . 0,15 g, 0.00076 mol, 38$ y i e l d ) as a colourless- o i l , b.p. 124°-125°C/25 mm Hg. ^H-n.m.r. and i . r . s p e c t r a l data were c o n s i s t e n t w i t h an authe n t i c sample. • Attempted conversion, of geraniol.(14), to geranyl bromide (79) 1 2 3. Ger a n i o l ((14)» 2 g, 0.013 mol) was d i s s o l v e d i n dry d i e t h y l ether (25 mL) and carbon.tetrabromide (8.6 g, 0.026 .mol) added. The r e a c t i o n was cooled during the a d d i t i o n of t r i - n - o c t y l phosphine (TOP, 9.62 g, 0.026 mol) and s t i r r e d f o r 20 minutes at room.temperature. The s o l u t i o n was decanted from p r e c i p i t a t e d ..phosphine oxide and the s o l i d e x t r a c t e d w i t h d i e t h y l ether. The combined organic l a y e r s were washed wi t h water, d r i e d (MgSO ), and evaporated to give a pale yellow o i l . 4 Chromatography ( s i l i c a g e l , petroleum ether (30 -60 C) eluant) gave an impure c o l o u r l e s s o i l whose 1H-n.m.r. spectrum showed the presence of TOP and evidence of decomposition of the geranyl bromide formed to - 116 -the a l l y l i c isomer (see D i s c u s s i o n , page 98, Figure 80). 6 (60MHz, CC1.); 6.2 (dd, 1H, -CH=CH0, J =15Hz, J . =11Hz). Pr e p a r a t i o n of 8 - m e s y l o x y l i n a l o y l acetate (76). 8 - H y d r o x y l i n a l o y l acetate ((69), 2.43 g, 0.0115 mol) was d i s s o l v e d i n dry d i e t h y l ether (20 mL) and t r i e t h y l a m i n e (2.81 mL, 0.02 mol) added. The s o l u t i o n was cooled to 0°C and methane sulphonyl c h l o r i d e (0.887 mL, 0.0115 mol) was added dropwise. The mixture was s t i r r e d f o r 20 minutes, f i l t e r e d , and evaporated .to give a pale yellow o i l (3.22 g, 96% y i e l d ) whose s p e c t r a l data was c o n s i s t e n t w i t h that expected f o r the mesylate (76). The mexylate was reduced without f u r t h e r p u r i f i c a t i o n . 6 (60MHz, CC1 ); 2.9 ( s , 3H, CE^O^), 4.46 ( s , 2H, -CHg-OMs). Attempted r e d u c t i o n of 8-mesyloxylinalo.yl acetate (76) w i t h NaBD, 4 in-80% HMPA/XO. The r e a c t i o n was c a r r i e d out i n an analogous manner to t h a t described f o r the. t o s y l a t e (75) (page .115) using the mesylate (76) (2.73 g, 0.0094.mol) i n 80%HMPA/H20'. (20 mL) and sodium borodeuteride (0.77 g, 0.0188 mol). Workup as described above gave an o i l which was d i s t i l l e d (25 mm Hg) to give pure ,.8-deuteriolinaloyl acetate ( ( 6 2 ) , 0 . U g, 0.0022 mol, 24% y i e l d ) . H -n.m.r. .data was c o n s i s t e n t w i t h that expected f o r 8 - d e u t e r i o l i n a l o y l acetate (62). P r e p a r a t i o n of 8 - d e u t e r i o l i n a l o o l (62). 8 - H y d r o x y l i n a l o y l acetate ((69), 2.97 g, 0.014 mol) was d i s s o l v e d i n dry d i e t h y l ether (20 mL) under an atmosphere of dry argon and cooled to 0°C. Triethylamine (2.16 mL,. 1.57 g, 0.0154- mol) was added and then methane sulphonyl c h l o r i d e (1.19 mL, 1.764- g, 0.0154- mol) was - 117 added dropwise. The r e a c t i o n was s t i r r e d at 0°C f o r 20 minutes and the p r e c i p i t a t e d t r i e t h y l a m i n e hydrochloride f i l t e r e d o f f . Evaporation of the d i e t h y l ether gave the crude mesylate (76)., the H-n.m.r. spectrum (60MHz) of which showed the presence of the mesyl grouping w i t h a s i n g l e t a t 62.9 i n t e g r a t i n g to 3 protons. A s i n g l e t a t 64.46 (2 protons) i n d i c a t e d the methylene grouping to which the mesylate group was attached since i t s resonance .was-shifted downfield from i t s p o s i t i o n (64.O) i n the s t a r t i n g a l c o h o l . , This crude compound was immediately d i s s o l v e d i n dry THF (20 mL) and a 1M s o l u t i o n of Super-deuteride ( A l d r i c h Chemical Co..., l i t h i u m , t r i e t h y l b o r o d e u t e r i d e , 56 mL, 0.056 mol, 4.1 equivalents) was added over-a p e r i o d of 15 minutes. The r e a c t i o n mixture was s t i r r e d f o r k hours at room temperature and then cooled to 0°C. Water was added,, c a r e f u l l y and then 3N sodium hydroxide s o l u t i o n and 30% hydrogen peroxide s o l u t i o n to destroy the organoboranes formed i n the r e a c t i o n . The mixture was r e f l u x e d f o r 24 hours and then ex t r a c t e d w i t h d i e t h y l , ether. The combined organic phases were washed w i t h water., d r i e d (MgSO.), and evaporated to give an 4 o i l which was seen by g . l . c . to be mainly... the d e s i r e d compound. Column chromatography ( s i l i c a g e l , petroleum ether (30 -60 C ) : d i e t h y l ether 9:1 eluant) gave pure 8 - d e u t e r i o l i n a l o o l ( ( 6 2 ) , 1.19 g, 0.0077 mol, 55% y i e l d ) as a c o l o u r l e s s o i l . V (CC1,); 3600 (weak, sharp, OH), 3500-3300 (weak, broad, OH), max 4 2150, 2180 (weak, sharp, CD). ...... 6 (60MHz, CDC1 3); 1.27 ( s , 3H, t e r t i a r y methyl), 1.58 (bs, 1H, disappears w i t h DgO, OH)-, 1-.-59 - (s,. 3H, a l l y l i c CH ), 1.65 (bs, 2H, a l l y l i c CH D ) , 1.4-2.13 (m, 4H, CH^ -CH,,.); 5.05 (d, 1H, -CH=CHH t r a n s , J . =12Hz), 5.11 ( b t , 1H, -CH=C(CH.-J.(CHJD)), 5.20. (d, 1H, -CH=CHH c i s , cxs — j> <L — J , =18Hz), 5.89 (dd,.1H, -CH=CHH,. J ... =12Hz, J =18Hz). " t r c L n s GXS "Gra,ns Mass spectrum (low r e s . ) : m/e(rel. i n t . ) ; 155 (1$), 140 (3%), 137 (13$), 128 (2%), 122 (12$), 121 (8$), 110 (6$), 108 (5$), 9 6 (12$), 9 3 (56$), 84 (31$), 71 (100$). A n a l y s i s . F o r C ^ H OD C a l c : C, 77.36; H, 11.76. Found: C, 77.34; H, 11.76. Pr e p a r a t i o n of 9-deuteriocamphor. 9-Bromocamphor (3.96 g, 0.0171 mol) and 2,2'-azobisisobutyro-n i t r i l e (AIBN, 2.8 g, 0.0171 mol) were d i s s o l v e d i n dry benzene (20 mL) under a dry argon atmosphere. T r i - . n - b u t y l t i n deuteride (5.0 g, 0.0171 mol) was added and the mixture r e f l u x e d f o r 4s days. The r e s u l t i n g s o l u t i o n was washed with 5 $ w/v potassium hydroxide s o l u t i o n , 5$ w/v potassium f l u o r i d e s o l u t i o n , water, and b r i n e . Removal of d r i e d solvent gave a yellow s e m i - s o l i d which was shown by g . l . c . examination to c o n s i s t of camphor and AIBN. Petroleum ether (30 -60 C) was added and the mixture c h i l l e d to p r e c i p i t a t e . AIBN. The petroleum ether (30 -60 UC) was decanted and .evaporated to give a l e s s mobile o i l . The -precipitation;" of' AIBN. i n t h i s f a s h i o n was repeated- twice more and the r e s u l t i n g s o l i d was then sublimed (60°C, atmospheric pressure) to give 9-deuteriocamphor (1.96 g, 0.0128 mol, 75$ y i e l d ) which was shown by g . l . c . examination to contain 5$ AIBN. Further p u r i f i c a t i o n was not attempted. V ( C C l , ) ; 2180 (weak, sharp, CD-), • 17-4'5 (stro n g , sharp, C=0) . max 4 1H-n.m.r., 6 (400MHz, C D C l ^ ; 0.836 ( s , 3H, 8-CH_3), 0.913 ( s , 3H, 10-CH ), 0.942 ( t , 2H, 9-CH2D, J=2Hz). Mass spectrum (low r e s . ) : m/e ( r e l . i n t . ) ; 153 (57$), 138 (8$), 111 (28$), 110 (36$), 109 (89$), 96 (100$), 95 (57$), 94 (23$), 84 (42$), 82 (17$), 81 (94$). 2H-n.m.r., 6 (12.3 MHz, CC1,); 1.159 ( t , J=2Hz). - 119 -Preparation- of 8-deuteriocamphor. 8-Deuteriocamphor was prepared i n an analogous manner to 9-deuteriocamphor, us i n g 8-bromocamphor (3 . 9 6 g, 0.0171 mol), AIBN (2.8 g, 0.0171 mol) and t r i - n - b u t y l t i n deuteride (5.0 g, 0.0171 mol). Pure 8-deuteriocamphor was obtained, by sublimation of the s e m i - s o l i d a f t e r p r e c i p i t a t i o n of AIBN, giving--8-deuteriocamphor (1 . 95 g, 0.0128 mol,. 75%-yield)-. G.l.c. examination' showed t h i s to contain 3% AIBN. Further p u r i f i c a t i o n was not attempted. V (CC1.); 2180 (weak, sharp, CD),. 174-5 (strong, sharp, C=0) . ma.x i\, 1H-n.m.r., 6 (4-OOMHz, C D C l ^ ; 0.826 ( t , 2E, 8-CH2Df. J=2Hz), 0.912 ( s , 3H, 10-CH 3), 0.945 ( s , 3H, . Mass spectrum (low r e s . ) : m/e ( r e l . i n t . ) ; 153 (55%), 138 ( 7 % ) , 111 (28%), 110 (30%), 109 (74%)-, 96 (100%)-,- 95 (-32-%)-, 94 (10%), 84 (37%), 82 (16%), 81 (50%). ^H-n.m.r., 6 (12.3MHz, CC1.); 1.000 ( t , J=2Hz). 4 2 E f f e c t of a s h i f t reagent on the -H-n,.m.r.. of 8-. and-9- deuteriocamphor. 8-Deuteriocamphor (50-mg) or 9-deuteriocamphor (50 mg) was d i s s o l v e d i n carbon t e t r a c h l o r i d e (5 mL) i n an n.m.r. tube. The 2 - -H-n.m.r.. spectrum were - recorded and then a weighed a l i q u o t of Resolve-Al ( A l d r i e h , ' E u ( t h d ) y thd = 2,2,6,6-tetramethyl-3,5-hepta-dienone) was added and the H-n.m.r... spectra recorded again. The s h i f t of the s i g n a l w i t h respect to ..the amount of s h i f t reagent added was noted and the procedure repeated. Deuteriochloroform (CDCl^) was added to the n.m.r. s o l u t i o n as an i n t e r n a l standard (7.510 ppm.) and s i g n a l p o s i t i o n s were adjusted a c c o r d i n g l y . - 120 -N.m.r. spec t r a . Amount of Mol. of S.R. Mol. S.R./ S i g n a l S..R..* added added x 10 6 mol camphor. p o s i t i o n 6 8- Deuteriocamphor. 1 0.00 0.00 0.00 1.000 2 0.00386 5.48 0.0168 1.222 3 0.2795 39.70 0.1210 2.048 4 0.05667 80.50 0.24.60 3.000 5 0.12709 180.0 .. 0.550 4.874 6 0.28220 401.0 1.230 6.271 9- Deuteriocamphor. 1 0,00 0.00 0.00 1.159 2 0.01791 25.00 0.0765 1.317 3 0,02232 31.-70 0.0969 1 .444 4 0.1.157 144.0 0.4400 2.048 5 0.15798 224.0 0.6850 2.460 6 0.29819 424.0 1.2950 3.159 *S.R. = s h i f t reagent ( R e s o l v e - A l ) . Screening of plants, f o r camphor. Samples of Rosemarinus o f f i c i a n a l i s (approx. 70 g wet weight) and S a n t o l i n a chamaecyparissus .(approx. 70 g.wet weight) were cut from two p l a n t s a t the U.B.C. B o t a n i c a l Gardens on a sunny June afternoon. The p l a n t m a t e r i a l was frozen..in. l i q u i d n i t r o g e n and crushed. The r e s u l t i n g m a t e r i a l was c a r e f u l l y steam d i s t i l l e d and approximately 750 mL of d i s t i l l a t e were c o l l e c t e d . This aqueous s o l u t i o n was ext r a c t e d w i t h petroleum ether (30°-60°C) and then w i t h d i e t h y l ether, these e x t r a c t s were examined separately. G.l.c. examination l e d to the conclusion t h a t camphor was present i n the petroleum ether (30 -60 C) e x t r a c t from both plants.. Further examination showed the e x t r a c t from S a n t o l i n a chamaecyparissus to contain l e s s than 5$ camphor whereas tha t from Rosemarinus o f f i c i a n a l i s contained approximately 20% camphor. Chromatography ( s i l i c a g e l , gradient e l u t i o n from pentane to pentant: d i e t h y l ether 9:1) afforded a white s o l i d (approximately 20 mg), g . l . c . examination showing i t to be 95% pure. H-N.m.r. and i . r . s p e c t r a l data was c o n s i s t e n t w i t h t h i s sample being camphor. V (CCl,);. 1745 (stron g , sharp, C=0). max 4 6 (80MHz, CDC1 ); O.84 ( s , 3H, 8-CH ), 0.92 (s, 3H, 10-CH ), 0.97 (s , 3H, 9-CH 3), 1.1-2.7 (m, 7H). Mass spectrum (low r e s . ) ; m/e ( r e l , i n t . ) ; 152 (30$), 137 (5%), 108 (50$), 95 (100$). Feeding experiments using Rosemarinus o f f i c i a n a l i s . Experiment 1. The p l a n t s were cut on J u l y 28th,., 1982 ( r a i n i n g ) from two shrubs at the U n i v e r s i t y of B r i t i s h Columbia B o t a n i c a l Gardens and stored under water. A s o l u t i o n of [2- Hg]-mevalonic a c i d ( ( 6 1 ) , 50 mg) was made by s t i r r i n g the lactone (61 a) w i t h 1 equivalent of sodium bicarbonate i n water f o r 1 hour at room temperature. [8- 2H^]-L i n a l o o l ( ( 6 2 ) , 50 mg) was s o l u b i l i z e d u s i n g a 1$ T r i t o n X-100 s o l u t i o n (100 mL). The p l a n t m a t e r i a l was placed i n t o the l a b e l l e d s o l u t i o n and maintained f o r 3 days on water. The p l a n t m a t e r i a l was then frozen - 122 -wit h l i q u i d n i t r o g e n , crushed, and steam d i s t i l l e d . Chromatography of the e s s e n t i a l o i l obtained by pentane e x t r a c t i o n of the d i s t i l l a t e 1 gave -camphor, 100% by g . l . c . examination., and H-n.m.r., i . r . , and mass s p e c t r a l data were i d e n t i c a l w i t h an authentic sample. A n a l y s i s by ^H-n.m.r. showed no to be present i n the camphor from e i t h e r feeding:experiment and so the work was repeated. Experiment 2. 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