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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 o f E x e t e r , 1976.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department of Chemistry.  We a c c e p t 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 p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by  the head o f  representatives.  my  It i s  understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  Department of  OH Q^NA i  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6  (3/81)  Starvd  r^Sib  written  - i iABSTRACT.  This t h e s i s camphor.  d e s c r i b e s an i n v e s t i g a t i o n  o f the b i o s y n t h e s i s o f  Camphor, a b i c y c l i c monoterpene, has been shown t o be b i o -  synthesised  f r o m . g e r a n y l pyrophosphate o r i t s isomers ( n e r y l pyro-  phosphate o r 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 p r o c e s s .  g e r a n y l pyrophosphate  camphor  I n an attempt t o d i f f e r e n t i a t e the o r i g i n o f t h e C(8) and  C(9)  m e t h y l groups,, f e e d i n g experiments w i t h a p p r o p r i a t e l y l a b e l l e d p r e c u r s o r s were a t t e m p t e d .  The two p r e c u r s o r s  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 t h a t the expected p r o d u c t s , 2 2 2 [8- H^]- and [9- H^]-camphor, c o u l d be d i f f e r e n t i a t e d by H-n.m.r.  u s i n g the s h i f t r e a g e n t E u ( t h d ) ^ 3, 5-heptanedionato.)europium) .  (Eu(thd)^ = tris(2,2,6,6-tetramethyl-  Feeding^ experiments w i t h  Rosemarinus  o f f i c a n a l i s u s i n g the c u t stem method showed no i n c o r p o r a t i o n o f deuterium l a b e l i n t o camphor e x t r a c t e d 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  iii TABLE OF CONTENTS.  Abstract  i i  Table of contents  i i i  L i s t of figures  v  Abbreviations  x  Acknowledgements I  .  Introduction  xii 1  I - ( i ) The terpenoids  1  I - ( i i ) Early studies on the terpenoids  2  I - (iii)  4  Early studies on terpenoid biosynthesis  I - ( i v ) The i d e n t i f i c a t i o n of intermediates (a)  Mevalonic acid  7  (b)  From acetate to mevalonic acid  9  (c)  From mevalonic acid to the 'active  (d) I - (v)  7  isoprene u n i t '  13  IPP to other terpenyl pyrophosphates  15  Stereochemical aspects of terpenoid formation  16  (a)  IPP isomerisation  16  (b)  Condensation of DMAPP with IPP  19  I - ( v i ) Mechanistic studies  29  I - ( v i i ) From geranyl pyrophosphate to the monoterpenes  31  (a)  Introduction  31  (b)  Regular a c y c l i c monoterpenes  35  (c)  Monocyclic monoterpenes (p_-menthanes)  38  (d)  B i c y c l i c monoterpenes  48  - iv (i)  pinanes  (ii)  bornanes  (iii)  thujanes  (iv)  II  caranes  50 55 57  (e)  Cyclopentanoid monoterpenes (iridanes)  60  (f)  Irregular monoterpenes  64  Discussion  II - ( i )  48  70  The problem  70  II - ( i i ) The method of l a b e l l i n g  76  II - ( i i i )  77  II - (iv) II - (v)  The choice of precursors The plant system Synthesis of the biosynthetic precursors  87  (i)  87  (ii) II - (vi)  83  [ 2- H ] -Mevalonic, acid (6l_) 2  2  [ 8- E^ 2  - L i n a l o o l (62)  Results  91 101  II - ( v i i ) Suggestions f o r further work  103  III  105  Experimental  References  123  LIST OF FIGURES.  Figure. 1  Page. The c o u p l i n g o f i s o p r e n e u n i t s i n a "headto- t a i l " fashion  2  3  4  3  Bonner and A r r e g u i n ' s p r o p o s a l f o r t h e earlysteps i n rubber b i o s y n t h e s i s .  6  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 14 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  The c o n v e r s i o n o f a c e t a t e t o (3R)-mevalonic a c i d (8)  5  6  7  11  The v a r i o u s pathways t o HMGCoA (7a) from g e n e r a l metabolism  12  C o n c e r t e d e l i m i n a t i o n o f C0„ and P. t o form 2 l mevalonic a c i d  14  A n t i e l i m i n a t i o n o f CO^ and P_^ from 3-phospho5-pyrophospho-MVA t o form I P P  15-  8  The i s o m e r i s a t i o n o f I P P t o DMAPP (13)  15  9  S t e r e o c h e m i s t r y o f I P P i s o m e r i s a t i o n t o 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 t h e presence o f H^O  18  11  The d e m o n s t r a t i o n o f r e f a c e a t t a c k o f H i n +  t h e i s o m e r i s a t i o n o f IPP t o DMAPP  20  12  R e g i o c h e m i s t r y o f attachment o f p r e n y l u n i t s  21  13  1'-4 condensation...  21  14  R e g i o c h e m i s t r y o f c o n d e n s a t i o n t o form p r e squalene o r prephytoene  pyrophosphates  22  15  Squalene b i o s y n t h e s i s  23  16  Phytoene  24  17  The c o n d e n s a t i o n o f t h e a l l y l i c and I P P  biosynthesis pyrophosphates  25  Inversion of configuration at  of the  a l l y l i c pyrophosphate i n t h e 1 '-4- condens a t i o n shown by P o p j a k and C o r n f o r t h S t e r e o c h e m i c a l r e q u i r e m e n t s f o r t h e forma t i o n o f p r e s q u a l e n e pyrophosphate ( o r prephytoene pyrophosphate) The r e g i o c h e m i s t r y o f a t t a c k o f t h e a l l y l i c substrate of IPP 1'-4 c o n d e n s a t i o n P o s t u l a t e d mechanism o f t h e 1'-4 c o n d e n s a t i o n . , Examples  o f t h e b a s i c s k e l e t a o f t h e mono-  terpenes R u z i c k a ' s scheme- f o r t h e f o r m a t i o n o f t h e monoterpenes The proposed b i o s y n t h e s i s o f 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 t r a n s f o r m a t i o n s o f g e r 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 t h e f o r m a t i o n o f nerol directly L a b e l l i n g p a t t e r n o b t a i n e d when r o s e p e t a l s were f e d w i t h In  [2- C,4- H.]-MVA 1/i  3  v i t r o studies of the hydrolysis of the  phosphate e s t e r s o f l i n a l o o l , g e r a n i o l , and nerol C o n v e r s i o n of.GPP t o NPP v i a LPP, w i t h o u t l o s s of  hydrogen a t C(1)  C o r i ' s proposed mechanism f o r t h e d i r e c t f o r m a t i o n o f NPP from t h e c o n d e n s a t i o n o f IPP and DMAPP  ,  Some n a t u r a l l y o c c u r r i n g m o n o c y c l i c monoterpenes Some a r o m a t i c  monoterpenes and t h e i r  r e l a t i o n s h i p w i t h t h e n o n - a r o m a t i c monoterpenes P r o p o s e d b i o s y n t h e s i s o f y - t e r p i n e n e and a-thujene  ,  Scheme t o p r o v i d e e v i d e n c e f o r t h e e x i s t e n c e o f 1,2-hydride s h i f t s i n monoterpene biosynthesis Examples o f t h e pinanes.. The bornanes P r o d u c t i o n o f t h e bornanes from t h e s t e r e o chemically appropriate a-terpinyl cation Synchronous mechanism f o r NPP t o BPP Biosynthesis o f borneol Revised  proposal f o r the biosynthesis 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 t h e thujanes The  carane monoterpenes  P r o p o s e d method o f f o r m a t i o n  o f car-3-ene.....  B i o s y n t h e s i s o f car-3-ene i n P i n u s  sylvestris.  The b i o s y n t h e s i s o f c a r - 3 - e n e , i n v o l v i n g a 1,2-proton s h i f t Examples o f t h e i r i d a n e monoterpenes Biosynthesis of the i r i d o i d s The  proposed b i o s y n t h e s i s o f t h e i r i d o i d s  ,  Figure.  Page.  52  I r r e g u l a r roonoterpene b i o s y n t h e s i s  53  Postulated  64  d i r e c t f o r m a t i o n o f f e n c h o l from  GPP  66  54  Major s k e l e t a o f t h e 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 o f 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 o f t h e i r r e g u l a r monot e r p e n e s i n v o l v i n g DMVC  57  69  I n c o r p o r a t i o n o f [ 2 - C ] - M V A (8) and U  [ 2 - ^ ^ C ] - g e r a n i o l {V^) i n t o camphor (2) 58  C o n v e r s i o n o f g e r a n y l pyrophosphate (14) t o b o r n y l pyrophosphate (37) by an  enzyme  p r e p a r a t i o n from sage 59  Numbering  71  o f t h e bornane s k e l e t o n i n b o r n e o l  (37) and camphor (2) 60  71  72  Postulated cyclisation of a  intermediate  t o b o r n y l pyrophosphate v i a and enzymebound a - t e r p i n y l s t r u c t u r e .  73  61  MVA t o camphor (2)  74  62  The f a t e s o f t h e a l l y l i c m e t h y l groups on c y c l i s a t i o n t o t h e 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 t o campherenone (59)  76  64  A s y m m e t r i c a l l a b e l l i n g found i n monoterpenoid biosynthesis  65  Precursors  77  of choice f o r the b i o s y n t h e t i c  study  78  66  Preparation of [8-  67  E f f e c t o f added s h i f t r e a g e n t (S.R.) on t h e H-n.m.r. spectrum o f [8- H^]-camphor  80  E f f e c t o f added s h i f t r e a g e n t (S.R.) on t h e 2 2 H-n.m.r. spectrum o f [9- H ]-camphor  81  68  - and [9- H ^ - camphor  1  79  viii  - ix Figure.  Page, o  69  H-N.m.r. spectrum o f a 1:1 mixture o f 2 2 [ 8 - H^]- and [ 9 - H-]]- camphor i n t h e presence o f excess s h i f t r e a g e n t  82  70  Preparation  o f [ 2 - H ] - m e v a l o n i c a c i d (61.)  88  71  Preparation  o f 4-acetoxy-2-butanone  88  72  A side r e a c t i o n i n the condensation o f  2  4-acteoxy-2-butanone (64) w i t h e t h y l a c e t a t e anion  90  73  Proposed s y n t h e t i c r o u t e  74  Mechanism o f selenium d i o x i d e o x i d a t i o n  75  Selenium d i o x i d e o x i d a t i o n o f l i n a l y l acetate  to [8-^H^]-linalool  91 92  (68)  94  76  R e d u c t i o n o f aldehyde (71.) t o a l c o h o l (69)  95  77  Mass spectrum f r a g m e n t a t i o n  96  78  Proposed f o r m a t i o n  o f aldehyde (71.)  o f minor product (72) on  selenium d i o x i d e o x i d a t i o n 79  C o n v e r s i o n o f a l c o h o l (69)  80  Attempted c o n v e r s i o n  97 t o [ 8 - H^] - l i n a l o o l . . . .  99  of geraniol to  g e r a n y l bromide  99  2 81  H-N.m.r. s p e c t r a o b t a i n e d  from camphor  e x t r a c t e d from Rosemarinus o f f i c i a n a l i s feeding with  (a) [2- H ]-MVA, and (b) 2  2  [S- ^] -linalool 2  82  after  102  Scheme f o r e s t a b l i s h i n g t h e s t e r e o chemistry  of ring closure  104  -  ABBREVIATIONS.  The following l i s t of abbreviations, most of which are commonly adopted i n chemical and biochemical l i t e r a t u r e , w i l l be employed in this thesis:  Bu  n  b u t y l , CH CH CH CH - . 3  2  2  2  CoA  coenzyme A.  DMAPP  dimethylallyl pyrophosphate.  Et-  ethyl, CH CH - .  FPP  farnesyl  g.l.c.  gas l i q u i d chromatography. •  Glu  glucoside.  GPP  geranyl pyrophosphate.  HMG  3-hydroxy-3-methyl  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 a c i d .  n.m.r.  nuclear magnetic resonance.  NPP  neryl pyrophosphate.  P  phosphate  P. 1 PP  inorganic  3  2  pyrophosphate.  g l u t a r i c acid.  phosphate.  pyrophosphate.  X -  - xi t.l.c.  t h i n layer chromatography.  TsCl  ^-toluene sulphonyl chloride,  V  frequency, cm  (^-CH^C^SC^Cl.  ACKNOWLEDGEMENTS.  I would l i k e t o thank D r . R.. T a y l o r and Mr. J . M c P h a i l l o f 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 t o "prune" t h e p l a n t s mentioned i n t h i s t h e s i s , and D r . P. S a l i s b u r y and Mr. G. H e w i t t f o r advice- on t h e f e e d i n g work.  I would a l s o l i k e t o thank t h e members  of t h e e l e c t r i c a l , , mechanical.,, and. g l a s s b l o w i n g  workshops f o r t h e i r  e x p e r t i s e i n k e e p i n g t h e lab., equipment f u n c t i o n a l , and t h e many people who have made my s t a y a t U.B.C.  enjoyable.  F i n a l l y , I would l i k e t o express my g r a t i t u d e t o P r o f e s s o r Thomas Money f o r h i s guidance,, encouragement, and anecdotes t h a t have h e l p e d t o make l i f e i n 34-8 e n j o y a b l e get t o see him i n h i s k i l t !  - even though I d i d n ' t  - 1 I.  I - (i)  INTRODUCTION..  The  terpenoids.  The group o f n a t u r a l p r o d u c t s known as the t e r p e n o i d s i s p r o b a b l y the most widespread  and c h e m i c a l l y i n t e r e s t i n g group o f n a t u r a l p r o d u c t s .  They have been known from a n t i q u i t y , h a v i n g 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 o f purposes,  a l t h o u g h 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 term " e s s e n t i a l o i l " .  o f p l a n t t i s s u e s , l e a d i n g t o the  Many p l a n t essences  are known and are used i n  perfumery, medicine,, and f o o d f l a v o u r i n g and c o l o u r i n g . Due  t o 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 t e r p e n o i d s were f a v o u r i t e o b j e c t s o f study by i n the l a t e 1800's and e a r l y 1900's.  chemists  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 t o the c h e m i s t r y of the t e r p e n o i d s , and amongst them were W a l l a c h , P e r k i n , B r e d t \ and i n more r e c e n t t i m e s , R u z i c k a 2  3  Banthorpe . 6  5  and  By the e a r l y 1900's, the g r o s s s t r u c t u r e o f most o f the  common t e r p e n o i d s 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 a r e s t i l l c o n t i n u i n g have added many d e t a i l s t o t h e s t e r e o c h e m i s t r y , r e a c t i o n s , and b i o s y n t h e s i s o f these compounds.  Many t e r p e n o i d s 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 , o r g a n i c a c i d e s t e r s , and even i n c o m b i n a t i o n w i t h p r o t e i n s . 1  The lower, members of the  class,  h a v i n g 10 o r 15 carbon atoms, can. be. r e a d i l y o b t a i n e d from p l a n t t i s s u e s by steam  distillation.  The t e r p e n o i d s are r e l a t e d by a common o r i g i n and a common structural relationship.  They a r e composed o f m u l t i p l e s o f a 5 carbon  u n i t , and are s u b d i v i d e d i n t o groups depending on the number o f  carbon  atoms i n t h e i r s k e l e t o n as f o l l o w s : *• Monoterpenoids Sesquiterpenoids C^Q  Diterpenoids Triterpenoids Carotenoids  C^Q  Rubber  (C_) 5 n  Few compounds  c o n t a i n i n g o n l y one 5 carbon atom u n i t , the  hemiterpenoid  are found t o e x i s t i n n a t u r e as s t a b l e , i s o l a b l e , m e t a b o l i c end  product  However, they a r e found t o 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 biosynthesis. I - ( i i ) E a r l y s t u d i e s on the t e r p e n o i d s .  C h e m i c a l i n v e s t i g a t i o n o f the t e r p e n o i d s began i n the e a r l y 1800' E m p i r i c a l formulae determinations  and c h e m i c a l d e g r a d a t i o n  t o the concept t h a t t h i s group o f compounds 5 carbon atom u n i t .  I n 1860, W i l l i a m s ^  a  studies l e d  c o u l d be d e r i v e d from a  heated rubber t o 300°C and  o b t a i n e d a product w i t h the c h e m i c a l f o r m u l a C^Hg which he c a l l e d "isoprene". molecular  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 o f  f o r m u l a C„_H„/ which he c a l l e d " c a t o u c h i n e " 10 16  and which was  7b  polymeric with isoprene.  Bouchardat  produced a compound o f m o l e c u l a r Williams. terpenes  showed t h a t h e a t i n g  formula C ^ H ^ ,  S t a r t i n g i n 1884., W a l l a c h  isoprene  c o n f i r m i n g the i d e a . o f  began a s y s t e m a t i c  study o f the  t h a t l e d t o the f i r s t s t r u c t u r a l formulae o f these  compounds.  2e  I n 1887  he proposed t h e " i s o p r e n e r u l e " w h i c h d i d n o t seem t o have  much e f f e c t on h i s eoritemporarias.  . I t -was o n l y -in the 1920's w i t h t h e  - 3 work o f R u z i c k a t h a t t h i s r u l e became e s t a b l i s h e d , i n t h e 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 " b i o g e n e t i c isoprene r u l e " . 5  When  W a l l a c h proposed h i s h y p o t h e s i s , t h e s t r u c t u r e o f i s o p r e n e was n o t known, a l t h o u g h he b e l i e v e d i t t o be as shown ( l _ ) .  T h i s was shown t o  be t h e c o r r e c t s t r u c t u r e f o r i s o p r e n e by Impatiew and W i t t o r f i n 1897. 8  isoprene  (D The d i m e t h y l b r a n c h o f t h i s m o l e c u l e i s commonly c a l l e d t h e " t a i l " and the u n s u b s t i t u t e d end t h e "head".  C o u p l i n g o f t h i s p r e c u r s o r t o form  the t e r p e n o i d s i s g e n e r a l l y seen t o be i n a " h e a d - t o - t a i l " f a s h i o n ( F i g u r e 1) and t h e t e r p e n o i d compounds can g e n e r a l l y • b e f o r m a l l y d i s s e c t e d i n t o C ^ - u n i t s p o s s e s s i n g t h i s arrangement, f o r example, camphor (2) and limonene ( 3 ) . These two compounds a r e monoterpenoids t h a t " f o l l o w " t h e i s o p r e n e r u l e , t h a t i s , t h e y can be seen t o be composed o f two i s o p r e n e u n i t s combined h e a d - t o - t a i l .  F i g u r e 1.  When t h e i s o p r e n e r u l e was f i r s t i n t r o d u c e d  The c o u p l i n g o f i s o p r e n e 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)  by W a l l a c h  (3)  i t was e x e m p l i f i e d by compounds t h a t were r e g u l a r .  As  time went on, compounds were d i s c o v e r e d which were t e r p e n o i d i n n a t u r e and y e t d i d n o t show t h i s l i n e a r a r r a y o f i s o p r e n e u n i t s . t h i s type o f compound, R u z i c k a f o r m u l a t e d  To encompass  the biogenetic isoprene  rule  which takes i n t o account t h e f a c t t h a t t h e compound t h a t i s f i n a l l y formed i n t h e p l a n t , w h i l s t h a v i n g 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 p r e c u r s o r , 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 d u r i n g i t s e l a b o r a t i o n i n t o t h e end p r o d u c t t h a t i s f i n a l l y  isolated.  I - ( i i i ) E a r l y s t u d i e s on t e r p e n o i d b i o s y n t h e s i s .  Numerous hypotheses f o r t e r p e n o i d b i o s y n t h e s i s were put forwardbased 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 t o be a member o f the t e r p e n o i d 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 n o t easy, as the compound i t s e l f does n o t have a c h e m i c a l f o r m u l a of C^Hg.  I n 1916, T s u j i m o t o  9  o i l and i t s m o l e c u l a r -formula\ determined.  i s o l a t e d squalene (4.) C^QH^Q,  that i s a multiple from shark l i v e r  and some o f i t s p r o p e r t i e s were  Ten y e a r s l a t e r , H e i l b r o n and c o w o r k e r s  squalene may be a p r e c u r s o r o f c h o l e s t e r o l (5),  1 0  suggested t h a t  and e l u c i d a t i o n o f t h e  s t r u c t u r e s o f these two compounds gave some s t r e n g t h t o t h i s  proposal.  - 5 However, i t was not u n t i l s t u d i e s on the b i o s y n t h e s i s o f c h o l e s t e r o l u s i n g l a b e l l e d p r e c u r s o r s were c a r r i e d out t h a t the f i n a l p r o o f of t h i s r e l a t i o n s h i p was  found.  squalene  cholesterol  U)  (5)  The f i r s t b i o c h e m i c a l use o f i s o t o p e s was i n 1923, who  c a r r i e d out by H e v e s y  s t u d i e d the uptake o f l e a d i n p l a n t s .  of deuterium i n 1932  11  W i t h the d i s c o v e r y  by U r e y , experiments i n b i o s y n t h e s i s began i n 1 2  13  earnest.  Hevesy and Hofer  biochemical  experiment.  were the f i r s t workers t o use deuterium i n a 13 14 The d i s c o v e r y o f G and G enabled a l a r g e 14  range o f s t u d i e s t o be c a r r i e d o u t .  L i t t l e and B l o c h  used 13  l a b e l l e d i n e i t h e r the c a r b o x y l or m e t h y l group w i t h  C or  acetate 14. C to  determine the r e l a t i v e u t i l i z a t i o n o f the 2 carbon atoms from a c e t a t e i n the b i o s y n t h e s i s o f c h o l e s t e r o l (5).  They determined t h a t 15 o f the  27 carbon atoms o f c h o l e s t e r o l arose from the methyl group o f a c e t a t e and 12, presumably the r e m a i n i n g 12, t o be from the c a r b o x y l Bonner and A r r e g u i n  1 5  group.  proposed t h a t acetate.was a p r e c u r s o r o f rubber  and c a r r i e d out experiments t h a t showed t h a t a c e t a t e , a c e t o a c e t a t e ,  and  3,3-dimethylacrylic  a c i d (6)  were a c t i v e i n s u p p o r t i n g r u b b e r  formation.  T h i s l e d them t o propose t h e f o l l o w i n g s y n t h e t i c sequence f o r t h e e a r l y steps i n t h e b i o s y n t h e s i s o f r u b b e r ( F i g u r e 2 ) .  A l t h o u g h t h i s i s now  known t o 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 t o note t h a t i t p r e d i c t s t h e correct l a b e l l i n g pattern 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 o f r u b b e r was r e v i e w e d i n 1954- . A t t h a t  time,  16  a modified-version  o f rubber b i o s y n t h e s i s was proposed t h a t i n c l u d e d the 3-hydroxy-3-methyl g l u t a r i c a  f o r m a t i o n o f a 6-carbori i n t e r m e d i a t e , a c i d (HMG, 7 ) .  H  (a)  2 CH C0.0H  2°  CH C0CH C00H  3  3  CH C0CH  2  3  + CO  CH COCH 3  3  +  CH C00H  3  2  CH,  3  C = C CH  ^GOOH  3  3,3-dimethylacrylic  acid  (6)  (b)  F i g u r e 2.  (a) Bonner and A r r e g u i n ' s  1 5  p r o p o s a l f o r t h e 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 o f l a b e l l i n g o f i s o p r e n e r e s i d u e , p r e d i c t e d by Bonner and A r r e g u i n ' s  scheme,  m = methyl carbon.; c = c a r b o x y l 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 o f  (a)  intermediates.  Mevalonic a c i d .  The n a t u r e o f the C ^ . - s t a r t e r u n i t was f i r s t d i s c o v e r e d Wolf e t a l .  1 7  ,  when they d i s c o v e r e d  certain l a c t o b a c i l l i .  1 8  an a c e t a t e - r e p l a c i n g f a c t o r .for  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, and c o w o r k e r s  i n 1956 by  o r mevalonolactone  (8).  Tavormina  showed t h a t t h i s compound, as the a c i d - a l c o h o l form,  c o u l d suppress the i n c o r p o r a t i o n o f a c e l l - f r e e r a t l i v e r homogenate.  -acetate i n t o c h o l e s t e r o l i n U s i n g s y n t h e t i c , l a b e l l e d (3RS)-  m e v a l o n i c a c i d , t h e y 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 o n l y 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  s t e p i n the i n c o r p o r a t i o n o f  m e v a l o n i c a c i d i n t o the s t e r o l s would be the l o s s o f the c a r b o x y l 14 group.  To prove t h i s they f e d [1-  C]-mevalonic a c i d and found no  i n c o r p o r a t i o n o f i s o t o p e i n the c h o l e s t e r o l , u n l i k e w i t h a c i d , i n d i c a t i n g t h a t the c a r b o x y l carbon i s i n d e e d l o s t .  [2-^^c]-mevalonic Various  groups  mevalonolactone.  (3R)-mevalonic a c i d .  (8)  (8)  of r e s e a r c h e r s  0  f e d [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 F i g u r e 3.  Amdur,. e t a l . ° 2  C  a l s o f e d [2-^ ^G, 5-^H]-mevalonic  t h a t both hydrogens a t G(5) of MVA  a c i d (MVA)  and  are-retained.--' They a l s o showed t h a t  f o r a y e a s t enzyme system t o c o n v e r t MVA  t o s q u a l e n e , the a d d i t i o n o f  2+ Mn  , ATP, and a p y r i d i n e n u c l e o t i d e was  F i g u r e 3.  required.  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  [2- V| 1  found  -mevalonic a c i d (MVA).  ( * = C U  ).  from  By 1960,  MVA  had been shown t o be i n c o r p o r a t e d i n t o s e v e r a l  t e r p e n o i d compounds, i n c l u d i n g a monoterpene, a-pinene (9) p o s i t i o n as the p r e c u r s o r of the. t e r p e n o i d s was  set.  and i t s  1 9  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 p r e c u r s o r i n t e r p e n o i d 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 enabling l a b e l l i n g with and C(3 ), and u s i n g ;l  13  C  2 2  or  H of  of t h i s molecule are a v a i l a b l e a t p o s i t i o n s C ( 2 ) , C(3>,  CU),  H at. the p r o ' c h i r a l ' p o s i t i o n s ' 2,4,  C(5),  and;  5,  see ( 8 ) .  a-pmene  (9)  (b)  From a c e t a t e t o m e v a l o n i c a c i d .  For the b i o s y n t h e s i s of m e v a l o n i c a c i d , i t was  reasonable  to  suggest t h a t i t i s formed" i n the organism from a c e t a t e , as a c e t a t e i s seen t o be i n c o r p o r a t e d i n t o the t e r p e n o i d s , 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 a c e t a t e began. and A r r e g u i n ' , 1 5  1 6  a c e t a t e was  produce a c e t o a c e t a t e .  proposed by Bonner  thought t o undergo a s e l f - c o n d e n s a t i o n t o  The enzymic c o n v e r s i o n of a c e t a t e t o  i n p i g e o n l i v e r e x t r a c t s was ATP  As was  shown by Soodak and L i p m a n n  2 3 a  acetoacetate , who  found  and coenzyme A t o be r e q u i r e d e o f a c t o r s o f 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 t h a t both a c e t a t e  m o l e c u l e s needed to be a c t i v a t e d f o r c o n v e r s i o n t o a c e t o a c e t a t e ;  and  they  proposed the f i r s t s t e p t o be the c o n v e r s i o n of a c e t a t e t o i t s t h i o l e s t e  10 acetylcoenzyme A (acetylGoA,  1_0). T h i s was soon v e r i f i e d by t h e i s o l a t i o n 2 4-27  of a c e t y l C o A . condensation  V a r i o u s groups t o acetoacetate  proposed t h e n e x t s t e p a f t e r t h e t o be t h e f o r m a t i o n of• '3-hydroxy-3-methyl  g l u t a r i c a c i d (HMG, 7) o r i t s t h i o l e s t e r , HMGGoA - see F i g u r e 4.  HMG  was i s o l a t e d from v a r i o u s s o u r c e s , i n c l u d i n g by h y d r o l y s i s o f the 28  alkaloid dicrotaline  ,. and i t s e x i s t e n c e i n p l a n t s strengthened  this  p o s t u l a t e d r o u t e . ' Enzymatic r e d u c t i o n o f HMG produces (3R)-mevalonic 0 0 0 0 0 NH, I CH CSCH CH NHC'(CH ) NHGGHCH -0-P-0-|-0-CH o  o  o  2  2  2  OH  OH  OH  = phosphate 0 II = -P-OH I OH ;  = CH^SCoA Acetylcoenzyme A ( a c e t y l C o A ) . (10)  a c i d ( F i g u r e 4-)» which was shown t o occur i n a y e a s t e x t r a c t s y s t e i and i n a mammalian enzyme s y s t e m  3 0  29  i n t h e presence o f NADPH, ATP, CoA,  2+ g l u c a t h i o n e , and Mg  as c o f a c t o r s .  I n t h i s s t e p , 2 hydrogens a r e  t r a n s f e r r e d from t h e reduced n i c o t i n a m i d e - a d e n i n e (NADPH).  d i n u c l e o t i d e phosphate  The r e 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  o n l y (3R)-MVA.  W i t h o u t NADPH i n the r e d u c t i o n s y s t e m  shown to,be c o n v e r t e d  3 0 1 3  a c e t a t e was  t o HMGCoA and no f u r t h e r , showing the r e d u c t i o n  step' t o r e q u i r e NADPH.  I n t h e r e d u c t i o n , t h e 5-pro-S hydrogen o f MVA 3 0C  i s i n t r o d u c e d from NADPH  - 11 2 CH^COSCoA  COSCoA  acetylcoenzyme A (10)  •A  H,  -COSCoA  H,  . HO,  J3H,H "H*  NADP+  NADPH*  HOOCXZXOH (3R)-mevalonic  acid  H C H00C  OH COSCoA  H ^^'H 1  2  (3S)-3-hydroxy-3-methyl-  glutarylCoA (8)  F i g u r e 4.  (7a)  The c o n v e r s i o n o f a c e t a t e t o ( 3 R ) - m e v a l o n i c a c i d ( 8 ) .  As f a r as can be a s c e r t a i n e d , MVA i s n o t produced by any o t h e r pathway, n o r i s i t used f o r a n y t h i n g o t h e r t h a n t h e s y n t h e s i s o f t h e terpenoids.  The t h i o l e s t e r , HMGCoA, on t h e o t h e r hand, l i e s on more t h a n  one b i o s y n t h e t i c r o u t e , and i t s proposed methods o f f o r m a t i o n a r e o u t l i n e d i n F i g u r e 5. HMGCoA i s p r o b a b l y t h e o n l y c o n n e c t i o n , and a one way one a t t h a t , l e a d i n g from g e n e r a l metabolism t o m e v a l o n i c a c i d , t h e key i n t e r m e d i a t e i n t e r p e n o i d b i o s y n t h e s i s .  Only t h e ( 3 R ) - form o f  m e v a l o n i c a c i d i s used i n metabolism, t h e ( 3 S ) - form i s m e t a b o l i c a l l y inert.  F i g u r e 5.  The v a r i o u s pathways t o HMGCoA (7a) from g e n e r a l m e t a b o l i s m .  - 13 (c)  From m e v a l o n i c a c i d t o t h e ' a c t i v e i s o p r e n e u n i t ' .  The n e x t q u e s t i o n i n t h e b i o s y n t h e s i s  o f t h e t e r p e n o i d s was t h e  conversion o f mevalonic a c i d i n t o the 'active isoprene u n i t ' .  Studies  on t h e metabolism o f m e v a l o n i c a c i d l e d t o t h e i d e n t i f i c a t i o n o f 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 e t a l . i d e n t i f i e d 3  OH  OH  5-pyrophosphomevalonic mevalonic a c i d .  1  OH  a c i d as a r i s i n g from t h e metabolism o f 5-phospho-  These workers a l s o found a new compound w h i c h , when  1L.  -MVA was used, was n o t l a b e l l e d , t h a t i s , t h e c a r b o x y l group o f 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 - i s o p e n t e n y l pyrophosphate ( l _ t ) .  Lynen and c o w o r k e r s  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 - i s o p e n t e n y l pyrophosphate ( I P P , 1_1_) and examined i t s f o r m a t i o n and use i n e x t r a c t s from y e a s t c e l l s .  They found  0-P-0-P-0H OH  OH  A - i s o p e n t e n y l pyrophosphate  (IPP).  (11:)  t h a t y e a s t c e l l s , i n t h e absence o f NADPH, d i d n o t c o n v e r t MVA o r 5-pyrophosphomevalonic a c i d (5-pyrophospho-MVA) t o s q u a l e n e , b u t t o a ^-compound, f a r n e s y l pyrophosphate (FPP, 1_2). e x t r a c t s w i t h 5-pyrophospho-['2A  4  Incubation o f the yeast  C]-MVA i n t h e presence o f i o d o a c e t a m i d e  an enzyme i n h i b i t o r , gave n o t FPP b u t l a b e l l e d I P P .  Synthetic,  l a b e l l e d IPP i n c u b a t e d i n t h e y e a s t enzyme system w i t h NADPH and M g gave r a d i o a c t i v e  s q u a l e n e , and w i t h o u t NADPH gave r a d i o a c t i v e FPP,  2 +  -u -  OPP  F a r n e s y l pyrophosphate (12)  PP = pyrophosphate 0 0 II II =-P-0-P*0H 1 I OH OH  (FPP).  .  i n d i c a t i n g IPP t o be the ' a c t i v e i s o p r e n e u n i t ' . mechanism.of IPP f o r m a t i o n  was  3 3  Examination  c a r r i e d out by c o n v e r t i n g MVA  to  »  2 squalene i n the presence of  H^O.  t h a t l e s s than one deuterium  atom per m o l e c u l e of MVA  T h i s v e r y p r e l i m i n a r y study showed was  incorporated  i n t o squalene i n d i c a t i n g t h a t d e c a r b o x y l a t i o n o c c u r s w i t h o u t of the earbon c h a i n .  of the  protonation  K i n e t i c s t u d i e s r e v e a l e d 00^ f o r m a t i o n and  ADP  f o r m a t i o n t o occur a t 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 t o be a c o n c e r t e d e l i m i n a t i o n of i n o r g a n i c phosphate C0_, F i g u r e 6.  F i g u r e 6.  I n t h i s r e a c t i o n , 5-pyrophospho-MVA undergoes a  Concerted  e l i m i n a t i o n of C0„  and P. t o form IPP.  and  phosphorylation  t o g i v e the 5-pyrophospho-3-monophospho- d e r i v a t i v e .  T h i s compound then e l i m i n a t e s CO^ c o n c e r t e d p r o c e s s t o y i e l d IPP. t o be a n t i - ( F i g u r e 7 ) . 3  and i n o r g a n i c phosphate (P^) i n a 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  L a b e l l i n g s t u d i e s have shown the oxygen from  the t e r t i a r y a l c o h o l t o be i n the i n o r g a n i c phosphate a f t e r t h i s reaction . 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-pyrophosphoMVA  (d)  t o form  IPP.  IPP t o o t h e r t e r p e n y l pyrophosphates.  E x a m i n a t i o n of the c o n v e r s i o n f i r s t s t e p t o be the c o n v e r s i o n  of IPP  ( i t ) t o FPP  of IPP t o i t s a l l y l i c i s o m e r ,  a l l y l pyrophosphate (DMAPP, 13), F i g u r e  8  3 6  8.  The  synthetic [1-  (13)  i s o m e r i s a t i o n o f IPP t o  An enzyme p r e p a r a t i o n  dimethyl-  .  (II) Figure  (1_2) showed the  DMAPP.(t3)  from b a k e r ' s y e a s t was  1h C]-IPP t o r a d i o a c t i v e DMAPP.  shown t o c o n v e r t  At about the same t i m e a s :  - 16 DMAPP was i d e n t i f i e d and shown t o a r i s e by. a l l y l i c rearrangement o f I P P , 3 7  o t h e r a l l y l i c pyrophosphates were found  38  '  / 38  .  Goodman and Popjak  i d e n t i f i e d g e r a n y l ( 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 i n t e r m e d i a t e s i n t h e c o n v e r s i o n o f MVA t o squalene, and  OPP  Geranyl- pyrophosphate  (GPP).  (u) i t was p o s s i b l e t o summarise t h e c o n v e r s i o n o f MVA t o squalene as follows : MVA  -IPP  IPP  -DMAPP  IPP. + DMAPP  —GPP  . GPP + IPP FPP + FPP  I - (v)„  (a)  ••FPP N  A  D  P  H  - squalene  Stereochemical aspects of terpenoid formation,  IPP i s o m e r i s a t i o n .  The  s t e r e o c h e m i s t r y o f t h e i s o m e r i s a t i o n o f IPP t o DMAPP was 39—4 1  examined by many groups  .  I n examining  t h i s r e a c t i o n there are 3  q u e s t i o n s which must be answered :(1) Which p r o t o n i s removed from 0(2) o f IPP i n f o r m i n g DMAPP?  - 17 (2)  What i s t h e s t e r e o c h e m i s t r y o f t h e new m e t h y l group i n DMAPP? .  (3)  I s t h e new m e t h y l group formed by r e o r s i f a c e p r o t o n a t i o n o f t h e double bond i n IPP?  The  f i r s t o f these q u e s t i o n s was answered b y G o r n f o r t h  et a l .  3 9  w h i l s t w o r k i n g on t h e b i o s y n t h e s i s o f squalene w i t h p a r t i c u l a r  interest  i n the stereochemistry of the condensation  In this  o f IPP w i t h DMAPP.  study, C o r n f o r t h f e d (3R.4R)- and ( 3 R . 4 S ) - .[ 2- ^C,4- H] -MVA t o an 1  3  enzyme p r e p 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 t h e squalene produced. 3  He found t h a t t h e ( 4 S ) - i s o m e r gave no  H i n t h e squalene produced, 3 14  whereas t h e ( 4 R ) - isomer had an i d e n t i c a l starting material.  T h i s he concluded  H/  G r a t i o as i n t h e  t o show t h a t t h e ( 4 S ) - hydrogen o f  MVA (,(2R)-hydrogen o f IPP)' i s - l o s t i n t h e i s o m e r i s a t i o n o f I P P t o DMAPP a n d ' i n t h e subsequent c o u p l i n g r e a c t i o n , i . e . , t h e s t e r e o c h e m i s t r y o f i s o m e r i s a t i o n i s i d e n t i c a l t o t h a t o f bond f o r m a t i o n , and i s as/shown,; -.(Figure 9) .  "  HOOC  F i g u r e 9.  Stereochemistry  o f I P P • i s o m e r i s a t i o n t o DMAPP.  From C o r n f o r t h . 3 9  - 18 F o r the second of these problems, van T a m e l e n 1  40  examined the  H-n.m.r. spectrum o f 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 1.67  ppm,  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 a t  d e s i g n a t e d as the oo-(E)-methyl group.  This,' he suggested,  2 i n d i c a t e d the m e t h y l group was l a b e l l e d w i t h  H, and i s thus the m e t h y l  group produced by the i s o m e r i s a t i o n , see F i g u r e 10. 1.57  1.61  1.61  1.61  F i g u r e 10.  1.61  1.57  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 - h o m o g e n a t e i n the 2 presence of  H^O-.  Numbers i n d i c a t e c h e m i c a l s h i f t ( i n ppm)  o f the methyl groups.  The f i n a l q u e s t i o n i n the s t e r e o c h e m i s t r y 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 s o l v e d u n t i l i d e n t i f i c a t i o n o f the c h i r a l i t y of a  -  m e t h y l group l a b e l l e d w i t h a l l t h r e e hydrogen i s o t o p e s was a c h i e v e d . F o r t h i s problem, C o r n f o r t h  4 1  used two enzyme systems o f known- e n a n t i o -  s e l e c t i v i t y t o 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. C o r n f o r t h i n c u b a t e d ( 2 R , 3 R ) - [ 2 - H ] - and ( 2 S . 3 R ) - [ 2 - H ] - MVA w i t h s o l u b l e 3  3  2 enzymes from p i g l i v e r i n (FPP) produced.  T h i s was  subjected to ozonolysis.  H^O  and i s o l a t e d the f arnesylpy'rophosphate  ..hydrolysed t o the f r e e a l c o h o l and then 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 t o i o d o f o r m and a c e t a t e w i t h o u t the o p p o r t u n i t y f o r 'exchange o f the methyl hydrogens w i t h t h e  - 19 -  medium, and t h e a c e t a t e was c o n v e r t e d  t o acetylCoA.  two e n z y m a t i c r e a c t i o n s , t h i s a c e t y l C o A form ( S ) - m a l a t e  (see F i g u r e 1 1 ) .  I n the f i r s t of the  was condensed w i t h g l y o x a l a t e t o  Incubation of t h i s  (S)-malate  with  fumarase a l l o w e d f o r e q u i l i b r a t i o n between ( S ) - m a l a t e and t h e c o n j u g a t e d 3 a l k e n e - d i a c i d 1_5. The percentage i s o m e r i s a t i o n was- measured.  H remaining  bound t o carbon a f t e r  this  Due t o t h e s t e r e o c h e m i s t r y o f t h e fumarase,  ( R ) - [ H , H ] - a c e t a t e s h o u l d r e t a i n most o f i t s t r i t i u m , whereas ( S ) - [ H , H ] 2  3  2  a c e t a t e s h o u l d l o s e most o f i t s t r i t i u m .  3  I n t h i s experiment, acetate  o b t a i n e d from FPP produced when ( 2 R , 3 R ) - [ 2 - H ] -MVA was f e d gave a malate J  which r e t a i n e d 63. k% o f i t s . t r i t i u m , whereas a c e t a t e produced from FPP a f t e r f e e d i n g w i t h ( 2 S , 3 R ) - [ 2 - H ] - M V A gave a malate which l o s t 2 3  of i t s t r i t i u m , thus i n d i c a t i n g t h a t  63.5%  H i s added t o t h e r e f a c e o f t h e  double bond i n t h e i s o m e r i s a t i o n o f I P P t o - DMAPP ( F i g u r e 1 1 ) . (b)  C o n d e n s a t i o n o f DMAPP w i t h  IPP.  To form t h e t e r p e n o i d s , IPP and DMAPP condense t o form g e r a n y l pyrophosphate (GPP,1U),  t h e C^Q p r e c u r s o r o f t h e monoterpenoids.  GPP,  b e i n g an a l l y l i c pyrophosphate, can condense w i t h a second m o l e c u l e of IPP t o form f a r n e s y l pyrophosphate (FPP, .12), t h e C ^ p r e c u r s o r o f the s e s q u i t e r p e n o i d s .  The r e g i o c h e m i s t r y o f t h i s attachment can be  d e s c r i b e d as h e a d - t o - t a i l , o r as a 1 -A l  o u t l i n e d by P o u l t e r  et a l . * . 1  2  condensation  This condensation  ( F i g u r e 12) as  reorganises  three  bonds: • 1. C l e a v e s  t h e C-0 bond i n t h e a l l y l i c pyrophosphate ( p r e n y l  donor). 2. Forms a bond between t h e p r e n y l donor and t h e p r e n y l (IPP).  acceptor  - 20 -  F i g u r e 11.  The  d e m o n s t r a t i o n of r e f a c e a t t a c k of H  of IPP t o DMAPP.  From C o r n f o r t h  i n the i s o m e r i s a t i o n  et a l . * 1  1  - 21 -  l  1'-4  1 '-1  (head-to-tail)  (head-to-head)  F i g u r e 12.  R e g i o c h e m i s t r y of attachment et  al.  3. Loses H e.g., f o r a 1'-4  4 2  of prenyl u n i t s . ( c f . P o u l t e r  ).  from e i t h e r the p r e n y l a c c e p t o r or the p r e n y l donor, condensation (Figure  13)»  OPP  F i g u r e 13.  1'-4 c o n d e n s a t i o n .  C o n d e n s a t i o n o f IPP w i t h an a l l y l i c pyrophosphate g i v e s the C -homologue, so g e r a n y l pyrophosphate i s produced from DMAPP and I P P . 5 GPP, condenses w i t h I P P . t o form f a r n e s y l pyrophosphate (FPP), and FPP can condense w i t h IPP t o produce g'eranylgeranylr-pyrophosph'ate (GGPP, 16) the C  ? n  p r e c u r s o r o f the d i t e r p e n e s .  To form the p r e c u r s o r o f the  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 m o l e c u l e s o f f a r n e s y l pyrophosphate (FPP) condense t o g e t h e r i n an o v e r a l l  o r head-to-  head, f a s h i o n - ( F i g u r e 12) and s i m i l a r l y f o r phytoene, t h e c a r o t e n o i d p r e c u r s o r , two m o l e c u l e s o f g e r a n y l g e r a n y l pyrophosphate condense 'head-to-head'. condensation  (GGPP, 16)  T h i s c o n d e n s a t i o n . i s thought t o be a 1'-2,3  ( F i g u r e 14-) •' t o form a c y c l o p r o p a n e r i n g ,  giving  p r e s q u a l e n e pyrophosphate o r prephytoene pyrophosphate, f o l l o w e d by opening o f t h e c y c l o p r o p a n e r i n g t o g i v e . a c a r b o c a t i o n which i s quenched by h y d r i d e from NADPH (see F i g u r e s 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 c o n d e n s a t i o n s o f t h e a l l y l i c p r e c u r s o r s  and I P P i a r e summarised i n F i g u r e 17.  1'-2,3 c o n d e n s a t i o n . F i g u r e 14-. R e g i o c h e m i s t r y o f c o n d e n s a t i o n t o form p r e s q u a l e n e o r prephytoene- pyrophosphates'* . 2  The  s t e r e o c h e m i s t r y o f t h e 1'-4 c o n d e n s a t i o n was s t u d i e d by Popjak 3  and C o r n f o r t h biosynthesis.  9 , 4 3 -  during- work on t h e s t e r e o c h e m i s t r y o f squalene These workers found t h e same s t e r e o c h e m i s t r y o f  c o n d e n s a t i o n i n two enzyme systems from w i d e l y d i f f e r i n g s o u r c e s and c o n c l u d e d t h a t t h e s t e r e o c h e m i s t r y i s a "highly- conserved p r o p e r t y o f prenyl transferases.  T h e i r work answered t h r e e major q u e s t i o n s about  OPP  presqualene pyrophosphate  F i g u r e 15.  Squalene  biosynthesis  /OPP  - 25 -  Monoterpenes + IPP DMAPP  + nIPP G e r a n y l pyrophosphate  Rubbers  + IPP  Sesquiterpenes diniGri ss  F a r n e s y l pyrophosphate  «- Squalene 1 '-1  Phytosterols  Triterpenoids  + IPP Steroids G e r a n y l g e r a n y l pyrophosphate  • Diterpenes  d i m e r i s e 1 -1 1  Phytoene  F i g u r e 17.  this  %*• C a r o t e n o i d s .  The c o n d e n s a t i o n s of the a l l y l i c pyrophosphates and IPP.  stereochemistry: a.  I s t h e r e r e t e n t i o n o r i n v e r s i o n o f c o n f i g u r a t i o n a t C^ o f the allylic  pyrophosphate?  b.  Which s i d e of the double bond i n IPP i s a t t a c k e d ?  c.  Which p r o t o n i s l o s t i n f o r m i n g the new double bond?  a.  Squalene was b i o s y n t h e s i s e d from (3R,5R)-[5- H]-MVA u s i n g an  enzyme p r e p 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  s u b j e c t e d t o o z o n o l y s i s f o l l o w e d by c l e a v a g e of the k e t o - a c i d produced from carbon atoms 3 t o 6 t o 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 c o n c l u d e d 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 t h a t o f 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 o f c o n f i g u r a t i o n had o c c u r r e d a t C_(1) of the a l l y l i c moiety i n the 1'-4  condensation (Figure 18).  1'-1  dimerisation  V  (R)-succinic acid. F i g u r e 18.  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 i n the 1'-4  of the a l l y l i c  c o n d e n s a t i o n shown by Popjak and  pyrophosphate  Cornforth ' . 3 9  4 3  - 27 As has been mentioned e a r l i e r , t h e b i o s y n t h e s i s phytoene) i n v o l v e s a "head-to-head" of FPP ( o r GGPP).  or  o f squalene ( o r  condensation of 2 molecules  This r e s u l t s i n i n v e r s i o n of configuration a t the  t e r m i n a l carbon atom o f one molecule i n t h e r e a c t i n g s p e c i e s 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 t h e t e r m i n a l carbon atom i n t h e o t h e r m o l e c u l e .  At  the carbon atom which shows r e t e n t i o n o f c o n f i g u r a t i o n , one o f t h e hydrogen atoms i s seen t o exchange.  T h i s i s t h e 5-pro-S hydrogen o f  m e v a l o n i c a c i d and t h e proposed b i o s y n t h e s i s  o f squalene (and phytoene)  t a k e s t h i s i n t o account by p r o p o s i n g t h e i n t e r m e d i a c y o f p r e s q u a l e n e pyrophosphate  ( o r prephytoene pyrophosphate; both o f t h e s e compounds  have been i s o l a t e d from n a t u r a l s o u r c e s , F i g u r e s 15, 16, and 1 9 ) .  HOOC OPP  i \ 1  H"  H  H*  H  »  H'  i n v e r s i o n of configuration.  H* H * HT  t H*  IH  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 NADPH . A  F i g u r e 19. S t e r e o c h e m i c a l r e q u i r e m e n t s f o r t h e f o r m a t i o n o f presqualene pyrophosphate  ( o r prephytoene  pyrophosphate).  - 28 b.  The i n s i t u b i o s y n t h e s i s o f  H ] - I P P from  (Z)-[A-  (2R.3R)-  2 [2-^H] -MVA showed t h a t a d d i t i o n o f t h e a l l y l i c moiety t o I P P o c c u r s t o / \ •-/ \ the s ± f a c e o f t h e 0 ( 3 ) - 0 ( 4 ) double bond i n IPP  4 3  b  from (2R.,3R)-[2- H]-MVA was i s o l a t e d and o z o n i s e d a c t i v e s u c c i n i c a c i d , found t o be ( R ) .  .  The f a r n e s o l produced  t o g i v e an o p t i c a l l y  This i n d i c a t e s the a t t a c k t o  be on t h e .si f a c e ( F i g u r e 20.),  y/+  DMAPP  Topology o f s u b s t r a t e s d u r i n g 1'-4- c o n d e n s a t i o n .  Figure 20.  The r e g i o c h e m i s t r y  o f a t t a c k o f the a l l y l i c  s u b s t r a t e on I P P .  - 29 c.  T h i s q u e s t i o n 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 o f IPP t o DMAPP ( F i g u r e 9, S e c t i o n I - ( v ) - ( a ) ). I t i s r e a s o n a b l e  t o assume t h e s t e r e o c h e m i s t r y  of t h e 1'-4- c o u p l i n g - i s t h e same as t h a t ' o f t h e ' i s o m e r i s a t i o n o f IPP ;  :  t o DMAPPas t h e l a t t e r can be e n v i s i o n e d as a 1'-4- c o n d e n s a t i o n w i t h H  +  t a k i n g t h e p l a c e o f t h e a l l y l i c p y r o p h o s p h a t e . ( F i g u r e 21).'...  F i g u r e 21.  1'-4-  condensation.  I - ( v i ) . Mechanistic  studies.  The mechanism o f t h e 1'-4- c o n d e n s a t i o n has been e x a m i n e d the r e s u l t s r e v i e w e d by C o r n f o r t h . 4 6  45:  and  The 1'-4- c o n d e n s a t i o n r e a c t i o n  shows two major p o i n t s : ' 1 .  There i s a c l e a n i n v e r s i o n o f c o n f i g u r a t i o n a t C(1) o f t h e a l l y l i c pyrophosphate - c o n s i s t e n t w i t h a c o n c e r t e d  process i n  which C-0 bond c l e a v a g e i s accompanied by C-C bond  formation.  2 . F o r m a t i o n o f t h e C-C bond i s n o t c o n c e r t e d e l i m i n a t i o n of H concerted  from &(-2)-of  w i t h the  IPP.,- •since t h i s would i n v o k e a  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 o f t h i s t y p e  are thought t o be s t e r e o c h e m i c a l l y To e x p l a i n t h e s e two  unfavourable.  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 t o the double bond of IPP f o l l o w e d by a t r a n s - e l i m i n a t i o n of 2-pro-R hydrogen of IPP and group X t o produce a new (Figure 22)^  double bond '  T h i s mechanism i s , so f a r , based s o l e l y on  c o n s i d e r a t i o n s and a l t h o u g h  i t i s a l o g i c a l mechanism,  stereochemical stereochemical  grounds a l o n e are i n s u f f i c i e n t p r o o f t h a t i t i s c o r r e c t .  F i g u r e 22.  P o s t u l a t e d mechanism of the 1'-4  the  condensation.  - 31 I - (vii).  (a)  From, geranyl-pyrophosphate" t o . t h e monoterpenes«  Introduction.  The uses o f monoterpenes i n p l a n t s a r e 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 t o f u n c t i o n as defence substances o r i n c h e m i c a l communication, the v o l a t i l i t y and c h e m i c a l c o m p l e x i t y (and hence p o t e n t i a l i n f o r m a t i o n c o n t e n t ) making them i d e a l f o r such purposes. Monoterpenes i n p l a n t s have been shown t o a c t 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 o r seed d i s p e r s i n g i n s e c t s o r a n i m a l s  , t o r e p e l browsing  a n i m a l s and i n s e c t p e s t s ' * ^ , 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 * . 8  1  S e v e r a l hundred monoterpenes a r e k n o w n  49  8 0  and can be a s s i g n e d t o one o f  f o u r broad c a t e g o r i e s : 1. ' R e g u l a r 2.  acyclic.  Gyclohexanoid; i ) monocyclic. ii)  3.  Cyclopentanoid.  4.  Irregular.  Category 2  bicyclic.  ( c y c l o h e x a n o i d ) encompasses the l a r g e s t number o f monoterpenes.  Examples o f t h e b a s i c s k e l e t a f o r the monoterpenes a r e shown i n F i g u r e 23. the  S e v e r a l r e v i e w s o f monoterpene f o r m a t i o n have appeared, o f w h i c h r e v i e w s by Banthorpe e t a l .  6  i n . 1972 and C o r n f o r t h  5 0  i n 1968 deserve  mention. I n 1953, R u z i c k a the  5  proposed a g e n e r a l scheme f o r the f o r m a t i o n o f  r e g u l a r monoterpenes, p l u s some o f the i r r e g u l a r , which t o t h i s  date has n o t been shown t o be i n c o r r e c t . c y c l i s a t i o n of a C  i n  The scheme i n v o l v e s the  d e r i v a t i v e t o g i v e the a - t e r p i n y l c a t i o n (l_7) which  Regular.  dlmethyloctane  bornane  p_-menthane  t h u j ane  pinane  carane  Irregular.  iridane  artemisane  F i g u r e 23.  fenchane  santolinane  isocamphane  chrysanthemane  Examples o f t h e b a s i c s k e l e t a o f t h e monoterpenes.  - 33 -  fenchane  F i g u r e 2k.  bornane  isocamphane  R u z i c k a ' s scheme f o r t h e f o r m a t i o n  carane  o f t h e monoterpenes.  can undergo f u r t h e r r e a c t i o n s t o y i e l d t h e v a r i o u s monoterpenes (Figure  21,). The study o f monoterpene b i o s y n t h e s i s , a l t h o u g h o u t w a r d l y a p p e a r i n g  s i m p l y an e x t e n s i o n  of the b i o s y n t h e t i c studies of the higher  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 o f a t r a c e r i n t o p l a n t systems i s  f r a u g h t 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 a r e u s u a l . and h i g h e r t e r p e n e s ,  terpenes,  For sesqui-  t h e problem o f i n c o r p o r a t i o n does n o t a r i s e , these  t e r p e n e s a r e a l s o found as f u n g a l m e t a b o l i t e s  or occurring 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 h i g h e r p l a n t s .  The  system, i n these c a s e s ,  can' be m a i n t a i n e d on a n u t r i e n t b r o t h c o n t a i n i n g the l a b e l l e d p r e c u r s o r and h i g h i n c o r p o r a t i o n l e v e l s a c h i e v e d .  W i t h the e x c e p t i o n  of  the  i r i d o i d s , monoterpenes occur c h i e f l y i n h i g h e r 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 f e e d i n g t o an i n t a c t p l a n t or shoots.  The  monoterpenes i n t h e s e 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 g l a n d s 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 m e v a l o n i c a c i d or o t h e r i s seen t o be v e r y low  (=0.1  - 0.01  %) .  The  51  precursors  high incorporation rate  seen f o r s e s q u i - and h i g h e r t e r p e n e s of m e v a l o n i c a c i d a l l o w s  the  a c c e p t a n c e - o f m e v a l o n i c a c i d as the terpene " s t a r t e r u n i t " , and i t i s r e a s o n a b l e t o assume t h a t m e v a l o n i c a c i d i s i n d e e d 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  examined.  being  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 a c c e p t e d b e i n g the compartmentation of monoterpene b i 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 t o exogenous, stopping  the i n c o r p o r a t i o n .  precursors  Compartmentation i s w e l l known and  regarded  5 lb  as an i m p o r t a n t f e a t u r e 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 c o u l d o c c u r 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 c o u l d 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  of g e r a n i o l i n r o s e p e t a l s and v a r i o u s o t h e r p l a n t s . 5 2  of MVA  incorporation  i n t o - g e r a n i o l i n - v a r i o u s p l a n t s p e c i e s has been shown t o  52b  low  The  biosynthesis  be  53  whereas when u s i n g f l o w e r p e t a l s .  g l a n d s and  can t h u s be assumed t o be d e v o i d  incorporation is- high.  In conjunction  monoterpene b i o s y n t h e s i s , an  which do not c o n t a i n o i l of compartmentation e f f e c t s ,  t o - t h i s low l a b e l l i n g seen i n  u n s y m m e t r i c a l d i s t r i b u t i o n of the l a b e l  i s seen, t h a t i s , t h a t p a r t of the m o l e c u l e d e r i v e d from IPP i s found t o 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 p a r t d e r i v e d from DMAPP  - 35 51  54.57  c a r r i e s l i t t l e o r no d e t e c t a b l e 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 t o c o n d e n s a t i o n o f a s m a l l amount o f l a b e l l e d I P P d e r i v e d from  exogenous  l a b e l l e d MVA w i t h DMAPP p r e s e n t  b e f o r e i s o m e r i s a t i o n o f I P P t o DMAPP can o c c u r .  i n a metabolic  pool  I t i s possible that  such p o o l s p a r t i c i p a t e i n t h e b i o s y n t h e s i s o f o t h e r t e r p e n e s b u t such p a r t i c i p a t i o n i s o n l y •.noticeable when i n c o r p o r a t i o n o f 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 o f t h e 5 *• carbon atom u n i t s  reasons f o r t h e u n s y m m e t r i c a l l a b e l l i n g  5 5  5 3  .  Other  include the p o s s i b i l i t y  DMAPP i s n o t o f d i r e c t mevalonoid o r i g i n , t h a t -.ex'ogenously  that  administered  MVA may i n h i b i t I P P - i s o m e r a s e , o r a c o m b i n a t i o n o f both - t h 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 origin.  E x p e r i m e n t s u s i n g l a b e l l e d leucine'-  56  have g i v e n some i n d i c a t i o n  t h a t DMAPP may be d e r i v e d from l e u c i n e (_1_8) as shown i n F i g u r e 25. However, c o n 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 n o t y e t been shown.  The development o f c e l l - f r e e systems f o r t h e e x a m i n a t i o n o f  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 r e c e n t l y ' b e e n ^ e x a m i n e d  (b)  R e g u l a r a c y c l i c monoterpenes.  C o n v e r s i o n o f GPP t o t h e v a r i o u s members o f t h e a c y c l i c monoterpenes i s r e l a t i v e l y easy t o envisage..  H y d r o l y s i s o f GPP g i v e s g e r a n i o l , and  GPP o r g e r a n i o l can be t r a n s f o r m e d  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 , e t c . ( s e e F i g u r e 2 6 ) . studies using  Labelling  exogenous « MVA have shown l a b e l l i n g p a t t e r n s i n t h e a c y c l i c  monoterpenes t o be c o n s i s t e n t w i t h t h i s s u g g e s t i o n .  In vitro  studies  of t h e h y d r o l y s i s of . g e r a n y l , n e r y l , and 'Iinalpyl.'phosphates have shown  - 36 -  NH, SCoA  C0 H 2  (18) leucine  isovaleric acid-CoA  OH  SCoA  OPP (13) DMAPP F i g u r e 25. The proposed b i o s y n t h e s i s  g e r a n i o l (1 4-)  o f DMAPP {Y3) from l e u c i n e (18)  n e r o l (19)  l i n a l o o l (20)  *^CH0  c i t r o n e l l o l (21)  c i t r a l (22)  F i g u r e 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 f o r m a t i o n  o f 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 m o n o c y c l i c ) mono-  terpene a l c o h o l s and h y d r o c a r b o n s . 6 2  e s t e r s can be c o n s i d e r e d  The h y d r o l y s i s o f these phosphate  as a model r e a c t i o n f o r the t r a n s f o r m a t i o n s  of GPP i n v i v o ( F i g u r e 2 7 ) .  (24.)  (23).  myrcene  F i g u r e 27.  (25)  cis-ocimene  The i n v i t r o t r a n s f o r m a t i o n s l i n a l o y l phosphates.  trans-ocimene  o f g e r a n y l , n e r y l , and  - 38 -  Monocyclic  (c)  monoterpenes (p_-menthanes) .  Formation o f the monocyclic (GPP,  monoterpenes from g e r a n y l •''pyrophosphate'  1_4) does n o t seem f e a s a b l e due t o t h e t r a n s - 6 , 7 - d o u b l e  Conversion  bond.  o f GPP t o n e r y l pyrophosphate (NPP, 1_9)., w i t h t h e c i _ s - s t e r e o -  chemistry,., o r l i n a l o y l - ' p y r o p h o s p h a t e (LPP, 2 0 ) , t h e a l l y l i c i s o m e r , 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 t o occur. 2  2  as  seems  C y c l i s a t i o n can then proceed by  1  (nerol) or  ( l i n a l o o l ) mechanism t o g i v e t h e b a s i c s k e l e t o n .  R u z i c k a . p o s t u l a t e d a n e r o l - t y p e d e r i v a t i v e ( F i g u r e 24)  and t h e  5  f o r m a t i o n o f NPP d i r e c t l y from DMAPP and I P P can be e n v i s i o n e d i f , before e l i m i n a t i o n of H rotation  +  and X  i n t h e o u t l i n e d mechanism ( s e c t i o n I - ( v i ) )  o c c u r s about t h e C ( 2 ) - C ( 3 ) bond o f I P P , g i v i n g on e l i m i n a t i o n  the c i s - d o u b l e bond d i r e c t l y ( F i g u r e 2 8 ) .  I fthis  o c c u r r e d , then t h e  4-pro-R hydrogen o f MVA would be l o s t , as i s found i n t h e b i o s y n t h e s i s of r u b b e r ( a l l c i s - ) .  Work by B a n t h o r p e  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 - C , 4 - H ] - and ( 4 S ) - [ 2 - C , 4 - H ] - MVA 3  U  a t C ( 3 ) ) t o rose p e t a l s . for was  3  H/  U  3  (racemic  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  1/.  14  14  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 o f C. L a b e l l i n g w i t h C 3 14 as expected ( F i g u r e 2 9 ) . The H/ C r a t i o was m a i n t a i n e d when t h e  (4-R)-isomer was f e d and H l o s t when t h e ( 4 S ) - i s o m e r was f e d f o r both the g e r a n i o l and n e r o l produced.  T h i s i n d i c a t e d t h a t r o t a t i o n about  the C ( 2 ) - G ( 3 ) bond o f I P P does n o t occur t o g i v e t h e c i s - d o u b l e bond o f nerol.  T h i s was t a k e n t o i n d i c a t e t h a t GPP i s formed from DMAPP and I P P  and then i s o m e r i s e d t o NPP o r n e r o l .  Follow-up w o r k  6 3  indicated this  c o n v e r s i o n t o occur by. a redox r e a c t i o n .  I n studying the biosynthesis  of a b i c y c l i c monoterpenoid, d - 3 - t h u j o n e ,  Banthorpe e t a l .  3  14  l o s s o f t r i t i u m when [ 1 - H, was  C ] - g e r a n i o l was used, and one t r i t i u m atom  l o s t from- [5- H ,2-^^c]-MVA. 3  9  found a  Using  [l- H,^ ^c]-nerol 3  gave no t r i t i u m  - 39 -  HOOC +H H  *HH  pyrophosphate F i g u r e 28.  P o s t u l a t e d mechanism f o r the f o r m a t i o n o f n e r o l d i r e c t l y .  - 40 -  OH 'OH  (19)  (14)  nerol  geraniol  F i g u r e 29. . L a b e l l i n g p a t t e r n o b t a i n e d when rose p e t a l s were f e d w i t h [2- C,4- H]-MVA. U  3  * = C. U  3  H  not marked.  l o s s , i n d i c a t i n g t h a t b e f o r e c y c l i s a t i o n , GPP p r o b a b l y v i a the aldehyde c i t r a l  (22).  (pro-1S) hydrogen o f g e r a n i o l was  was  isomerised to  NPP,  S t e r e o s p e c i f i c l o s s o f the  shown t o o c c u r .  However, Suga and  c o w o r k e r s ^ examined the c o n v e r s i o n o f [5- H,2-^^C]-MVA t o monoterpenes 5  3  i n s e v e r a l p l a n t s p e c i e s and found no l o s s o f t r i t i u m i n the o f GPP  t o NPP.  Earlier, Attaway  precursor of.the monocyclic  6 5  conversion  proposed LPP' as the i n t e r m e d i a t e  monoterpenes, and the c o n v e r s i o n o f GPP  LPP w i t h o u t l o s s o f hydrogen a t C(1)  can be e n v i s i o n e d - ( F i g u r e 30)  H 0  (22) citral  to based  - 41 -  F i g u r e 30.  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 e s t e r s of l i n a l o o l , g e r a n i o l , and  nerol.  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 e s t e r s of g e r a n i o l and n e r o l LPP  over GPP  6 6  .  or NPP,  Further evidence supporting and f o r the c o n v e r s i o n  of C(1) hydrogen, was  shown by S u g a  5 8  of GPP  the p r e f e r e n c e  The  same  H/  u s i n g a c e l l - f r e e system from  C r a t i o s were found i n the i s o l a t e d  compared t o t h a t i n the p r e c u r s o r incorporated  f e d , and LPP was  t o the g r e a t e r amount..  c o n v e r t e d t o NPP  v i a LPP,  of  t o LPP w i t h o u t l o s s  14 3 Mentha s p i c a t a L. t o produce a - t e r p i n e o l from [ l - C,1- HJ- NPP, 3 14 and GPP.  linalool,  found t o  I t i s p o s s i b l e t h a t GPP  w i t h o u t l o s s of H a t C'(1)  LPP,  a-terpineol be is  ( F i g u r e 31).  As  - 42 -  0PP  (u)  T  (20)  (20)  (19) neryl  geranyl pyrophosphate  pyrophosphate  F i g u r e 31. C o n v e r s i o n of GPP at  t o NPP  pyrophosphate  v i a LPP,  w i t h o u t l o s s of hydrogen  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 a t  C('1).  S e v e r a l examples of the i s o m e r i s a t i o n of a t r a n s - t o a c i s - double bond w i t h o u t l o s s of hydrogen a t C(1) number of s e s q u i t e r p e n e s  5 7  have been demonstrated f o r a  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  this  mechanism proposes had not been examined. Shine and L o o m i s GPP  t o NPP  6 8  i s o l a t e d an enzyme capable of  w i t h o u t the d e t e c t a b l e  T h i s c i s - t r a n s i s o m e r i s a t i o n was  intermediacy-of  to g e r a n i o l i s observed.  DMAPP and I P P , was from P i n u s and was  t o be d i r e c t , i . e . no  LPP.  (cf. a l l hydrolysis  .. . i n v o k i n g the d i r e c t f o r m a t i o n  put f o r w a r d by C o r i  Citrus species.  No  6 9  , who  used enzyme  e v i d e n c e of a GPP  found i n these systems but; b o t h GPP  l o s s of the  c i t r a l (22) or  found t o be p h o t o i n d u c i b l e  t r a n s - r e t i n o l t o 1 3 - c i s - r e t i n o l ) and  An a l t e r n a t e p r o p o s a l ,  isomerising  (• 4-pro-S)-hydrogen of MVA.  and NPP  t o NPP  of NPP  preparations isomerase  were formed w i t h  They proposed two  from  the  alternatives  for this 1.  observation:  The enzyme p r e p a r a t i o n c o n t a i n s two p r e n y l t r a n s f e r a s e s ( t r a n s - and  c i s - ) which b o t h have e q u i v a l e n t l y l o c a t e d b i n d i n g s i t e s f o r t h e pyrophosphate group and t h e 2-pro-R hydrogen o f IPP (4-pro-S hydrogen o f MVA),  b u t b i n d t h e t e r m i n a l methylene o f I P P and t h e h y d r o p h o b i c and  pyrophosphate groups o f DMAPP w i t h d i f f e r e n t arrangements  (Figure 32).  On c o u p l i n g , t h e same p r o t o n o f IPP i s e l i m i n a t e d i n b o t h cases, b u t 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 o n l y one p r e n y l t r a n s f e r a s e , b u t a f t e r i n i t i a l  coupling  t o form t h e c a t i o n ( H I ^ i n F i g u r e 32). r o t a t i o n , o f t h e C(2)-C(3) bond of t h e bound DMAPP ( i m p l y i n g t h e hydrophobic group n o t t o be r i g i d l y h e l d ) and t h e n e l i m i n a t i o n o f t h e 4-pro-S hydrogen o f MVA w i l l g i v e NPP. Both o f t h e s e h y p o t h e s e s , however, would o v e r a l l r e s u l t i n a t t a c k of t h e s i f a c e o f t h e double bond o f IPP t o .form NPP r a t h e r than t h e r e f a c e as i n forming  GPP ( p r o p o s a l 1 i n v o l v e s , d i r e c t s i f a c e a t t a c k ,  p r o p o s a l 2 would appear t o g i v e such a t t a c k a f t e r r o t a t i o n o f t h e IPP moiety).  No work on t h i s problem has been r e p o r t e d t o d a t e , and t h e  enzyme(-s) r e s p o n s i b l e f o r t h i s c o u p l i n g have n o t 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 t o answer t h i s Ruzicka's  problem.  proposed b i o s y n t h e t i c scheme i n v o l v e s t h e c y c l i s a t i o n  of GPP, NPP, o r LPP t o t h e a - t e r p i n y l c a t i o n , from which t h e o t h e r many m o n o c y c l i c compounds can be formed.  I n 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 t o c o n v e r t NPP t o a - t e r p i n e o l (26) as t h e major c y c l i c p r o d u c t  5 9  and t h e  same e x t r a c t has been shown t o c o n v e r t s u b s t r a t e c o n c e n t r a t i o n o f 59 0  a - t e r p i n e o l t o limonene (27) and a - t e r p i n o l e n e  (28)  ( F i g u r e 33)  -u -  F i g u r e 32.  C o r i ' s proposed mechanism f o r the d i r e c t f o r m a t i o n of NPP  from the c o n d e n s a t i o n of IPP and DMAPP.  - 45 -  (3)  (26) a-terpineol  terpinolene  70  70(J  c  3-phellandrene  (31)  (30)  1,8-cineole  (28)  a  limonene  (29)  F i g u r e 33.  (27) 70  carvone  70  e  pulegone  Some n a t u r a l l y o c c u r r i n g m o n o c y c l i c monoterpenes.  g i v i n g r i s e t o t h e t h e o r y t h a t t h e diene monoterpenes a r i s e by dehydration terpenes,  of a-terpineol.  The co-occurence .of these c y c l i c mono-  coupled w i t h t h e i r . a p p a r e n t ease o f i n t e r c o n v e r s i o n , seems  t o i n d i c a t e t h a t they a r e formed by s e q u e n t i a l m o d i f i c a t i o n o f 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 f o r m a t i o n  d i f f e r e n t l y from an a c y c l i c p r e c u r s o r . and K a r p leaves  6 0  '  6 1  However, l a t e r work by Croteau  u s i n g a s o l u b l e enzyme p r e p a r a t i o n from S a l v i a o f f i c i a n a l i s  shown t o c a t a l y s e t h e c o n v e r s i o n  o f NPP t o a number o f mono-  c y c l i c monoterpenes, i n d i c a t e s t h a t 1 , 8 - c i n e o l e and  (30),  t h e c y c l i c d i e n e s limonene' (27) and a - t e r p i n o l e n e  i n d e p e n d e n t l y from t h e a c y c l i c p r e c u r s o r , mediates o f a common r e a c t i o n sequence.  a-terpineol  (26),  (28) a r e d e r i v e d  r a t h e r than as f r e e i n t e r The s y n t h e t a s e s r e q u i r e d f o r  each d i f f e r e n t monoterpene have been s e p a r a t e d . .  - 46 L a b e l l i n g p a t t e r n s examined i n v a r i o u s m o n o c y c l i c  monoterpenes  d e r i v e d from [^c]-MVA a r e c o n s i s t e n t w i t h R u z i c k a ' 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 t h e i n i t i a l c y c l i s a t i o n o f a NPP-type a c y c l i c p r e c u r s o r t o t h e a - t e r p i n y l c a t i o n (l_7, F i g u r e 24.)  f o l l o w e d by o x i d a t i o n ,  reduction, e t c . (Figure 33) . 70  V a r i o u s a r o m a t i c monoterpenes a r e seen t o c o - o c c u r w i t h v a r y i n g . amounts o f a - t e r p i n e n e , y - t e r p i n e n e , and t e r p i n e n - 4 - o l , 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  .  suggesting  I n v i v o and i n v i t r o  have shown a r e l a t i o n s h i p between these compounds ( F i g u r e  studies 34)•  * postulated F i g u r e 34.  Some a r o m a t i c  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 p r e p a r a t i o n has been shown t o c o n v e r t [ l - H.]-GPP or  3  3  [1- H]-NPP t o y-[3-  H]-terpinene with approximately equal e f f i c i e n c y . 7 2  T h i s system shows no evidence o f c o n v e r s i o n of GPP  t o 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 p r o d u c t , the f o l l o w i n g mechanism has  been proposed ( F i g u r e 35).  F i g u r e 35.  T h i s mechanism -proposes a 1,2-hydride s h i f t  Proposed b i o s y n t h e s i s o f y - t e r p i n e n e and  a-thujene.  from C(J+) t o C(8) t o produce Y " t -  e r  pi  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 o f a p r o t o n from C(6) and f o r m a t i o n o f a c y c l o p r o p y l r i n g , encompasses t h e f o r m a t i o n o f a - t h u j e n e , a minor p r o d u c t from t h i s preparation.  The n a t u r e o f t h e i n t e r m e d i a t e s 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 , b u t l i m i t e d s t u d i e s u s i n g LPP and a - t e r p i n y l p y r o phosphate as s u b s t r a t e s s u p p o r t t h e p r o p o s a l .  The 1,2-hydride  shifts  proposed i n t h i s mechanism have a l s o been p o s t u l a t e d f o r r e l a t e d m o n o c y c l i c monoterpenes b u t never demonstrated.  1,3-hydride s h i f t s i n 72b  r e l a t e d analogous s e s q u i t e r p e n e cases have been demonstrated V a r i o u s schemes have been proposed t o show the''existence o f 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 ( F i g u r e 3 6 ) (d)  Bicyclic  (i)  Pinanes.  7 3  .  monoterpenes.  The major pinane monoterpenes- are a- and 3- pinene ( F i g u r e 3 7 ) , and t h e s e compounds a r e t h e most common n a t u r a l l y o c c u r r i n g monoterpenes V a r i o u s oxygenated d e r i v a t i v e s a l s o o c c u r n a t u r a l l y b u t 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 r o u t e s t o t h e pinanes have been deduced on chemotaxanomic g r o u n d s ? a-Pinene i s found t o o c c u r i n an o p t i c a l l y 7 4  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 t o e o - o c c u r w i t h 3-pinene o f t h e o p p o s i t e a b s o l u t e c o n f i g u r a t i o n .  The  s t r u c t u r e o f t h e pinenes suggests t h e i r f o r m a t i o n by M a r k o w n i k o f f 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 1L  exogenous-  [ 2 - GJ-MVA a r e 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 . A c e l l 7 5  f r e e p r e p a r a t i o n from P i n u s r a d i a t a has been shown t e n t a t i v e l y t o  - 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 o f p_-cymene produced  from o i - t e r p i n e n e , t o b e n z o i c 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  F i g u r e 36.  shift  7 3  .  Scheme t o p r o v i d e e v i d e n c e f o r t h e e x i s t e n c e o f 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)  a-pinene  (3£)  (35)  (36)  3-pinene  myrtenol  verbenone  F i g u r e 37.  Examples of the p i n a n e s .  c y c l i s e NPP  t o a- and 3-  pinenes .  T h i s system does n o t seem t o  7 6  s y n t h e s i s e the a c y c l i c monoterpenes, and t h e r e f o r e does not s u p p o r t the t h e o r y t h a t eis-ocimene  (24.)  and myrcene (23)  are i n t e r m e d i a t e s  i n pinane b i o s y n t h e s i s . The o r i g i n o f the oxygenated  pinanes has not been examined i n  d e t a i l , but i t seems f e a s a b l e t h a t 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 o f oxygenated  (ii)  compounds.  Bornanes.  B o r n e o l (38)  and.camphor (2)  ( F i g u r e 38)  are the most i m p o r t a n t  members o f t h i s c l a s s o f compounds,.and t h e s e compounds commonly cooccur i n a number o f p l a n t s p e c i e s .  Both enantiomers  occur i n n a t u r e ,  e.g., d-camphor and d - b o r n e o l 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)  borneol  F i g u r e 38.  The  camphor  bornanes.  L a b e l l i n g p a t t e r n s i n b o r n e o l and exogenous  (2)  camphor b i o s y n t h e s i s e d from  [ 2 - ^ G ] - M V A 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  R u z i c k a ' s h y p o t h e s i s f o r - t h e c y c l i s a t i o n o f the a - t e r p i n y l c a t i o n i n an a n t i - M a r k o w n i k o f f  sense.  The  e q u i v a l e n t l a b e l l i n g seen i n o p p o s i t e  enantiomers i m p l i e s c y c l i s a t i o n of the s t e r e o c h e m i c a l l y a p p r o p r i a t e 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 t o g i v e the enantiomer ( F i g u r e 3 9 ) .  As b o r n e o l and  camphor co-occur  a b s o l u t e c o n f i g u r a t i o n i t seems- r e a s o n a b l e interconverted.  that borneol i s f i r s t  78  t o c a t a l y s e the c a t i o n -  H]-NPP i n t o d-[3- H ] - b o r n e o l  and the  dependent o x i d a t i o n o f t h i s b o r n e o l i n t o d- [3- H]-camphor. e x p e r i m e n t , a water s o l u b l e , d i a l y z a b l e i n t e r m e d i a t e was and  be  An enzyme p r e p 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 ) l e a v e s has been s h o w n dependent c y c l i s a t i o n o f [ l -  w i t h the. same  t o assume t h a t they can  E v i d e n c e c o l l e c t e d suggests  formed and then o x i d i s e d t o camphor.  opposite  shown t o be i n v o l v e d i n the r e a c t i o n .  NAD-  In t h i s  also isolated  T h i s compound was  identified  - 52 -  F i g u r e 39.  Production  of the bornanes from the  stereochemical^  appropriate a - t e r p i n y l cation.  as b o r n y l pyrophosphate, and gave some s u p p o r t t o the t h e o r y b i c y c l i s a t i o n occurs as one  that  s t e p , r a t h e r t h a n v i a the f o r m a t i o n  of a  3  monocyclic intermediate. converted  S y n t h e t i c d l - [ 3 - H ] - a - t e r p i n e o l was  t o b o r n e o l 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 t h a t a - t e r p i n e o l  i s not a f r e e i n t e r m e d i a t e f o r NPP  i n the b i o s y n t h e s i s .  t o b o r n y l pyrophosphate (BPP)  The  not  was  A synchronous mechanism  thus proposed ( F i g u r e  4-0).  i s o m e r i c a l c o h o l , i s o b o r n e o l (38) i s seen t o o c c u r i n n a t u r e  t o a l i m i t e d e x t e n t , and may  be p o s t u l a t e d t o a r i s e from the  (38) isoborneol  reduction  - 53 -  F i g u r e 4-0.  Synchronous mechanism  f o r NPP t o BPP.  of camphor r a t h e r t h a n from the c y c l i s a t i o n o f NPP, GPP or the aterpinyl cation.  The r e a s o n f o r t h i s p o s t u l a t i o n can be seen when  b o r n e o l b i o s y n t h e s i s i s examined.  I n 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 d e v e l o p i n g  c a t i o n a t the  C(2)  p o s i t i o n i n t h e bornane s k e l e t o n i s r e a d i l y quenched by pyrophosphate i o n ( i n a synchronous mechanism) or by water ( F i g u r e 4-1) from the l e s s hindered,  endo f a c e o f the m o l e c u l e .  Figure 41.  Biosynthesis of borneol.  This i s a l s o the p r e f e r r e d trans--  - 54 a d d i t i o n o f s u b s t i t u e n t s a c r o s s a double bond, and generates endo a l c o h o l .  o n l y the  R e d u c t i o n o f camphor would proceed by h y d r i d e a d d i t i o n .  t o the l e s s h i n d e r e d 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 p r e p a r a t i o n from sage m e n t i o n e d c o n v e r t GPP t o BPP, but l e s s e f f i c i e n t l y .  78  was a l s o found t o  Examination  o f the t e r p e n y l  pyrophosphate h y d r o l a s e s p r e s e n t i n t h i s r e l a t i v e l y crude p r e p a r a t i o n revealed that they 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 h y d r o l y s e d 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 p r e p a r a t i o n removed these competing phosphatases,  and the  p r e p a r a t i o n then showed a p r e f e r e n c e f o r GPP over NPP, and no i n t e r c o n v e r s i o n was seen.  T h i s f i n d i n g made the p r e v i o u s p o s t u l a t e d mechanism  untenable and a m u l t i s t e p p r o c e s s i n v o l v i n g GPP was proposed ( F i g u r e 42) 79  PPO? PPO  OPP  F i g u r e 42.  R e v i s e d p r o p o s a l f o r the b i o s y n t h e s i s o f the bornanes.  -  I t i s suggested t h a t the pyrophosphate i o n i s the o n l y  55  nucleophile  p r e s e n t i n a h y d r o p h o b i c pocket of the enzyme i n which the m o l e c u l e i s s i t t i n g , and t h u s the d e v e l o p i n g  c a t i o n i s quenched w i t h pyro-  phosphate i o n (PPO—) r a t h e r than w a t e r .  (iii)  Thujanes.  The (4-0)  t h u j a n e monoterpenes, of which t h u j o n e (39)  ( F i g u r e 4-3)  and  isothujone  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 o f  a - t e r p i n y l c a t i o n -as i n R u z i c k a ' s s c h e m e . 80  the  Time course s t u d i e s  and  8 1  14 the i n c o r p o r a t i o n of a-[ vulgare  are c o n s i d e r e d  a c y c l i c precursor  e]-terpineol  8 2  i n t o 3-thujone i n Tanecetum  t o 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  t o 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 u s i n g s p e c i f i c a l l y l a b e l l e d - a - [ H, MVA,  Banthorpe e t a l . -  8 3  obtained  (39) d - 3 - t h u j one  The  C ] - t e r p i n e o l and  e v i d e n c e f o r two h y d r i d e  p o s t u l a t e d the' f o l l o w i n g b i o s y n t h e t i c . p a t h w a y  F i g u r e 43.  an  thujane- monoterpenes.  (Figure  shifts  44).  (40) 1 - 3 - i s o t h u j one  [ H, and  C]-  -  - 56 -  d-3-thuj anol  F i g u r e 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 (iv)  Carane.  Car-3-ene (_4J_) i s t h e o n l y commonly o c c u r r i n g monoterpene w i t h the carane s k e l e t o n ( F i g u r e 45),  car-2-ene (43)  i s thought t o be an  a r t i f a c t o f t h e method o f e x t r a c t i o n .  (41)  (42) d-car-2-ene  d-car-3-ene F i g u r e 4-5.  The carane monoterpenes.  R u z i c k a proposed the f o r m a t i o n o f car-3-ene from the a - t e r p i n y l c a t i o n by a 1 , 3 - e l i m i n a t i o n , and t h e c o - o c c u r r e n c e o f t e r p i n o l e n e (28) w i t h car-3-ene was c o n s i d e r e d p r o o f o f the t h e o r y ( F i g u r e 46).  Formation  14 of car-3-ene from- [ 2 -  CJ-MVA * shows the l a b e l t o be p r e d o m i n a n t l y a t 81  C(4) o f the carane s k e l e t o n , 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 t h e a - t e r p i n y l cation-, f o r m a t i o n o f the c y c l o p r o p y l r i n g must be accompanied by double bond m i g r a t i o n ( F i g u r e Using- P i n u s s y l v e s t r i s , Banthorpe and A k h i l a  8 5  46).  have'" examined the  b i o s y n t h e s i s o f car-3-ene and have shown f o r m a t i o n o f t h e double bond t o be accompanied by an u n u s u a l 1,2-proton s h i f t . double bond,-the (2-pro-S)-hydrogen of. MVA. i s lost..  I n f o r m i n g the After feeding with  [ ^ ^"C, 1 -H,-,] - 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 t h e 3  double bond o z o n i s e d ( F i g u r e 47).  L o s s o f 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 c o n v e r s i o n t o n e r o l by a redox mechanism.  Nerol  F i g u r e 47.  B i o s y n t h e s i s o f car-3-ene i n P i n u s  sylvestris.  -  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 i t i u m when f o r m i n g the c y c l o p r o p y l r i n g . p r o d u c t (A, F i g u r e  47)  t r a n s f e r of  E n o l i s a t i o n of the  ozonolysis  showed the compound d e r i v e d from n e r o l t o l o s e  h a l f i t s t r i t i u m , whereas t h a t from g e r a n i o l e x h i b i t e d no such l o s s . e x p l a i n these r e s u l t s , B a n t h o r p e of g e r a n i o l was  proposed t h a t the  8 5  l o s t on c o n v e r s i o n  t o n e r o l , and  p r o t o n remains on the c y c l o p r o p y l r i n g . syn i n t e r a c t i o n s would be proposed u n l e s s  enzyme bound, and  the r e m a i n i n g l a b e l l e d  F o r ' c y c l i s a t i o n , "forbidden" an "X-group" mechanism i s  48.  The  This  of the a - t e r p i n y l c a t i o n t o remain  t h i s i s supported by the o b s e r v a t i o n  i s not i n c o r p o r a t e d  Figure  equivalent  To  (1-pro-S)-hydrogen  i n v o k e d , 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 ( F i g u r e 4-8) . i m p l i e s the b i o g e n e t i c  59  that  a-terpineol  i n t o car-3-ene.  biosynthesis  of car-3-ene, i n v o l v i n g a 1,2-proton s h i f t .  -  - 60 (e)  C y c l o p e n t a n o i d monoterpenes ( i r i d a n e s ) .  The i r i d a n e group of monoterpenes g a i n e d t h e i r name from I r i d o myrmex, the s p e c i e s of a n t from which they were f i r s t i s o l a t e d .  Several  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 t h e y o f t e n occur as- the 3-D-glueosides.  Rates o f i n c o r p o r a t i o n o f exogenous  p r e c u r s o r s are h i g h and n o t plagued w i t h a s y m m e t r i c a l l a b e l l i n g  problems  as t h e s e compounds are not i s o l a t e d t o o i l g l a n d s but occur throughout the p l a n t .  A few examples o f the i r i d a n e monoterpenes are shown i n . - .u.  F i g u r e 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;  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  The s e c o i r i d o i d s are  b e l i e v e d t o a r i s e by cleavage o f - t h e C(7)-C(8) bond- of the c y c l o p e n t a n e r i n g i n loganin- (43)  f o l l o w e d by. numerous secondary t r a n s f o r m a t i o n s .  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 r e v i e w s i n t h i s a r e a have  appeared . 8 6  (43)  (44)  loganin  nepetalactone  G l u = 3-D-glucose  F i g u r e 49.  Examples of the i r i d a n e monoterpenes.  (45) sweroside  - 61 L a b e l l i n g s t u d i e s with. [ 2- ^c]-MVA and v a r i o u s [• H ] - l a b e l l e d MVA's 1  3  have been c o n s i s t e n t w i t h the o r i g i n o f l o g a n i n t h r o u g h t h e a c c e p t e d pathway o f 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 o r GPP ( F i g u r e S7_89  50)  90  .  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 t o t h e -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 p r e c u r s o r s ,  and. o n l y t h e 1 0 - h y d r o x y - g e r a n i o l  and  1 0 - h y d r o x y - n e r o l were found t o g i v e good i n c o r p o r a t i o n .  t i s s u e c u l t u r e has-been shown...to t r a n s f o r m  l i n a l o o l s e l e c t i v e l y t o the  trans-hydroxy-compound - . • I n some c a s e s , c o n s i d e r a b l e 9  1  9 2  A tobacco  randomisation  of l a b e l from 0(9) and C(10) were found when l o g a n i n was b i o s y n t h e s i s e d  alkaloids  F i g u r e 50.  Biosynthesis of the i r i d o i d s .  compound demonstrated t o be an i n t e r m e d i a t e between t h e 10-hydroxy(46)  compounds and l o g a n i n i s d e o x y l o g a n i n  (43) . 9k  (47)  93  as w e l l as l o g a n i c a c i d  I n the conversion of loganin t o the a l k a l o i d s ,  secologanin  i s shown t o be an i n t e r m e d i a t e . 9 5  On t h e b a s i s o f t h e f i n d i n g s so f a r , t h e 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 ( F i g u r e 51 ) . 9 6  OGlu  OGlu  -0  0  HO  C00H  COOMe  (46)  (43)  deoxyloganin  loganic acid  CHO  COOMe  (47) secologanin  - 63 -  H, Me  COOR  GOOR  R = H, Me  F i g u r e 51.  The proposed b i o s y n t h e s i s o f the  R = H, Me  iridoids.  - 64 (f)  I r r e g u l a r monoterpenes.  The i r r e g u l a r monoterpenes a r e o f two t y p e s ; 1) those t h a t c o n t a i n l e s s t h a n 10 carbon atoms ;  and 2) those which c o n t a i n 10 carbon atoms  but n o t 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 c o n t a i n i n g l e s s t h a t 10 carbon atoms can  be assumed t o a r i s e by d e g r a d a t i o n o f a r e g u l a r monoterpene  (Figure  52);. The two compounds shown, 3 - p h e l l a n d r e n e and c r y p t o n e , a r e found t o c o - o c c u r . The b i o s y n t h e s i s o f t h i s type o f i r r e g u l a r monoterpene i s the same as f o r t h e i r p a r e n t compound f o l l o w e d by o x i d a t i v e c l e a v a g e , e t c .  (28)  (49)  3-phellandrene  F i g u r e 52.  2)  cryptone  I r r e g u l a r monoterpene b i o s y n t h e s i s .  Those i r r e g u l a r monoterpenes c o n t a i n i n g 10.carbon atoms can be  assumed t o a r i s e by one o f two methods: (i)  by rearrangement  d e r i v e d from GPP. fenchane and  o f r e g u l a r monoterpenes, t h e r e f o r e b e i n g u l t i m a t e l y  Examples o f t h i s group a r e :  camphane: •OH  Wagner-Meerwein shift pmane  fenchane  f e n c h o l (50)  - 65 -  bornane  . isocamphane  camphene (51)  secoiridoids:  COOMe  CHO  l o g a n i n (43)  COOMe  secologanin  (47)  These i r r e g u l a r monoterpenes are b e l i e v e d t o be produced i n v i v o , presumably e n z y m a t i e a l l y , and a l t h o u g h c a r r i e d out, c h e m i c a l l y r e a s o n a b l e many i n v i t r o  few i n . v i v o s t u d i e s have been  r o u t e s have been p o s t u l a t e d  having  analogies.  I t has been p o s t u l a t e d  9 7  t h a t 1-endo-fenchol'(50) c o u l d a r i s e from  d i r e c t l y v i a - a n enzyme-bound l i n a l o y l and p i n y l c a t i o n ( F i g u r e 5 3 ) .  GPP  i i ) . a s primary b i o s y n t h e t i c products,  and not d e r i v e d from GPP o r any  o t h e r 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.  T h e i r 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 l o w n a t u r a l abundance p o s i n g the major problem i n studying-them. However, some studies,- b o t h i n v i v o and i n v i t r o , have l e d t o a g e n e r a l 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 o f t h i s • c l a s s o f monoterpenes are the a r t e m i s y l ('52), c h r y s a n t h e m y l ( 5 3 ) , l a v a n d u l y l ( 5 4 ) , 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 f o r m a t i o n o f f e n c h o l from  GPP.  - 67 -  (55).  (540  lavandulyl  (53)  santolinyl  chrysanthemyl  (56)  (52) artemisyl  Figure  54-.  artemisia  ketone  Major s k e l e t a o f t h e i r r e g u l a r monoterpenes.  54-,  shown i n F i g u r e a r t e m i s i a ketone  and t h e most widespread i r r e g u l a r monoterpene i s (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 t o be i n c o r p o r a t e d  i n t o a r t e m i s i a ketone,.and 9 8  incorporated  without p r i o r degradation.  geraniol i s not  a  High i n c o r p o r a t i o n o f MVA 9 8b i n t o c h r y s a n t h a m i c a c i d has a l s o been r e p o r t e d . Various biosynthetic 99  pathways have been proposed formation  .  100  '  and these g e n e r a l l y propose t h e  o f t h e chrysanthemyl s k e l e t o n i n t h e f i r s t i n s t a n c e .  rearrangements  Subsequent  o f t h i s s k e l e t o n can l e a d t o t h e 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 ( F i g u r e 55).  Feeding and t r a p p i n g e x p e r i m e n t s  1 0 1  have shown  c h r y s a n t h e m y l a l c o h o l t o be a n . i n t e r m e d i a t e i n . t h e b i o s y n t h e s i s o f c h r y s a n t h a m i c 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 a r e n o t . A c e l l - f r e e e x t r a c t was shown t o c o n v e r t MVA,. I P P , 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)  t o a r t e m i s i a ketone and a l c o h o l ,  - 68 -  F i g u r e 55.  Proposed b i o s y n t h e s i s o f i r r e g u l a r monoterpenes.  - 69 l a v a n d u l o l , and c h r y s a n t h e m y l a l c o h o l  1 0 2  .  Geraniol, nerol,  and  t h e i r pyrophosphates were n o t seen t o be i n c o r p o r a t e d .  and  other s t u d i e s  1 0 3  56.  From these  , 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  i r r e g u l a r monoterpenes has been proposed ( F i g u r e  Figure  linalool  The b i o s y n t h e s i s  o f these  56).  o f the i r r e g u l a r monoterpenes i n v o l v i n g DMVC.  - 70 II  •DISCUSSION.  I I - ( i ) The problem.  The b i o s y n t h e s i s o f camphor ( 2 ) , as o u t l i n e d i n S e c t i o n has been shown t o be c o n s i s t e n t w i t h R u z i c k a ' s  scheme f o r t h e c y c l i s a t i o n  of t h e a - t e r p i n y l c a t i o n , w i t h l a b e l from exogeneous 7 7  and  [ 2 - ' V ) - g e r a n i o l (1J.)  I-(vii)-d(ii),  [2- C ] - MVA  (8)  b e i n g i n c o r p o r a t e d s p e c i f i c a l l y i n accordance  w i t h t h i s scheme ( F i g u r e 5 7 ) . Work w i t h enzyme p r e p a r a t i o n s from sage 78  (Salvia officianalis)  79  '  has  shown t h a t g e r a n y l ( 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 and camphor (2) w i t h o u t t h e d e t e c t a b l e i n t e r m e d i a c y structure.  i n t o b o r n e o l (37)  o f an a - t e r p i n y l  To account f o r t h e ready c o n v e r s i o n o f g e r a n y l  pyrophosphate  t o b o r n y l pyrophosphate, t h e f i r s t formed b i c y c l i c p r o d u c t , a m u l t i s t e p p r o c e s s has been p r o p o s e d trans- stereochemistry  ( F i g u r e 5 8 ) . G e r a n y l pyrophosphate, h a v i n g  at. the. 2,3-double bond, i s o f an i n c o r r e c t s t e r e o -  chemistry f o r c y c l i s a t i o n .  1A  7 9  The mechanism.proposed  19  79  ( F i g u r e 58)  20  geranyl  neryl  linaloyl •  pyrophosphate  pyrophosphate  pyrophosphate  - 71 -  OPP (37) F i g u r e 58.  C o n v e r s i o n of g e r a n y l .pyrophosphate. (^.)  to b o r n y l pyro-  phosphate (37) by an enzyme p r e p a r a t i o n from  sage . 7 9  - 72 -  borneol  camphor  (37)  Figure  59.  (2)  Numbering of the bornane s k e l e t o n i n b o r n e o l and  (37)  camphor (2)  p o s t u l a t e s 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 w i t h the d e v e l o p i n g 59  c a t i o n a t C(2)  intermediate  of the bornane s k e l e t o n (see  Figure  f o r numbering of the bornane s k e l e t o n ) being quenched by pyrophosphate  i o n h e l d i n a h y d r o p h o b i c pocket of the enzyme.. d i d not c o n v e r t  T h i s enzyme p r e p a r a t i o n  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 b o r n e o l and  was  t a k e n as e v i d e n c e t h a t 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 p r e s e n t  as an enzyme  bound i n t e r m e d i a t e which c y c l i s e s t o the bornane s k e l e t o n ( F i g u r e  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 , s k e l e t o n has been p r o p o s e d occur- d i r e c t l y by a n t i - M a r k o v n i k o v  5  to  a d d i t i o n t o the 'double bond, or  by c y c l i s a t i o n t o the pinane s k e l e t o n (Markovnikov a d d i t i o n ) f o l l o w e d by a 1,2  s h i f t of the b r i d g e carbon r a t h e r than p r o t o n l o s s t o g i v e  double bond found i n the  pinenes.  the  1  f  (2)  F i g u r e 60.  '  P o s t u l a t e d c y c l i s a t i o n o f a C^Q  (37)  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 p r o v i d e 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  p r e c u r s o r , ' we d e c i d e d t o examine the f a t e of the a l l y l i c  methyl  groups i n the c o n v e r s i o n o f the a c y c l i c -C^Q p r e c u r s o r (_5_8) t o camphor (2) ( F i g u r e 61)'.  I n . the p r e c u r s o r , these two m e t h y l 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 t h e y 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 e v e n t u a l 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 m e t h y l groups would become, the C ( 8 ) - m e t h y l group i n camphor w h i l s t the o t h e r became the C ( 9 ) - m e t h y l  group ( F i g u r e 6 2 ) .  I f a free  c a t i o n i c i n t e r m e d i a t e (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 o f i d e n t i t y and the two m e t h y l groups  - 1U -  MVA  camphor (58)  (2)  F i g u r e 61. MVA t o camphor ( 2 ) .  would be randomly d i s t r i b u t e d between t h e 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 o f t h e s e two m e t h y l groups i n t h e a c y c l i c  precursor  and examining-the camphor b i o s y n t h e s i s e d from t h i s compound w i l l g i v e a g r e a t e r i n s i g h t i n t o t h e mechanism..of f o r m a t i o n o f t h e b i c y c l o - [ 2.2.1]compounds.  By a n a l o g y t o t h e s e s q u i t e r p e n o i d s , - i t would be expected  t h a t t h e 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 o f camphor- (Z) , i s found i n . t h e e s s e n t i a l o i l o f Cinnammomum- camphora S i e b . w h i l e t h e isomer epi-campherenone n o t y e t been r e p o r t e d as a n a t u r a l p r o d u c t . pyrophosphate  (60) has  The c y c l i s a t i o n o f f a r n e s y l  (l_2) t o campherenone (59) i s analogous t o t h e c y c l i s a t i o n  o f GPP (l_£) t o camphor (2).and o c c u r s i n a s t e r e o s e l e c t i v e f a s h i o n (Figure 63).  F i g u r e 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.  - 76 -  epi-campherenone F i g u r e 63.  II — (ii)  FPP  (l_2) c y c l i s a t i o n t o campherenone ( 5 9 ) .  The method of. l a b e l l i n g .  L a b e l l i n g o f one o f the a l l y l i c methyl groups i n the precursor  Q-acyclic  (58, F i g u r e 61) c o u l d 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 u s i n g mevalonic a c i d . l a b e l l e d a t 0(2) or C(3')>  (b)  by u s i n g a l a b e l l e d C^Q i n t e r m e d i a t e , e.g., l i n a l o o l ( 2 0 ) , g e r a n i o l (14.K  o r n e r o l (l_9).  The a c t u a l method o f l a b e l l i n g should be d e t e c t a b l e by a n o n - d e g r a d a t i v e means ( e . g . , n u c l e a r magnetic resonance (n.m.r.)) a s • d e g r a d a t i v e  studies  on camphor t o d i s t i n g u i s h between the 8- o r 9- methyl groups r e q u i r e a ioi+ c o n s i d e r a b l e amount of m a t e r i a l d i s t i n g u i s h between-these  groups  a  . 1 0  Even mass s p e c t r o m e t r y cannot  ^ but the use of a n o n - r a d i o a c t i v e  - 77 n..m..r.. d e t e c t a b l e l a b e l would s o l v e t h i s problem. abundance o f a p p r o x i m a t e l y r e l a t i v e l y easy t o o b t a i n .  1.1$,  13  C has a n a t u r a l  and n a t u r a l abundance  IJ>  C-n.m.r. a r e  I n c o r p o r a t i o n o f l a b e l would r e s u l t i n  enhancement o f peak s i z e and c o n s e q u e n t l y a r e l a t i v e l y l a r g e i n c o r p o r a t i o n would be r e q u i r e d f o r an unambiguous assignment.  'As i n c o r p o r a t i o n  l e v e l s f o r monoterpene b i o s y n t h e s i s - a r e n o t o r i o u s l y l o w , t h i s i s n o t the-method o f c h o i c e .  In contrast,  2 H has a low n a t u r a l abundance  (0.016$) 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 i n c r e a s e i n H p r e s e n t and t h u s i n s i g n a l s i z e .  Incorporation of  2 H  13 i n t o a p r e c u r s o r would be e a s i e r t o d e t e c t than i n c o r p o r a t i o n o f and t h e r e f o r e  2 H was chosen as t h e l a b e l .  Ill  The c h o i c e o f p r e c u r s o r s .  - (iii).  C,  The p r e c u r s o r o f c h o i c e f o r t e r p e n o i d b i o s y n t h e s i s i s m e v a l o n i c a c i d (MVA, 8).  However, w i t h monoterpenoids, t h e r e a r e d i f f i c u l t i e s  w i t h t h i s p r e c u r s o r i n t h a t asymmetric l a b e l l i n g o f t h e f i n a l monoterpene o f t e n o c c u r s . 5 2  This r e s u l t s in-approximately  90% o f t h e l a b e l  b e i n g found i n that' p a r t o f t h e s t r u c t u r e d e r i v e d ' from I P P , whereas t h a t p a r t d e r i v e d from DMAPP i s v i r t u a l l y u n l a b e l l e d ( F i g u r e 64., Section I - ( v i i ) - ( a ) ) .  see a l s o  As i t i s t h a t part, o f t h e molecule d e r i v e d from  > 90% o f l a b e l here  < 10$  o f l a b e l here  F i g u r e 64..  A s y m m e t r i c a l 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 t o be l a b e l l e d , use o f a l a b e l l e d MVA alone c o u l d be i n c o n c l u s i v e . (20),  U s i n g a l a b e l l e d C^Q d e r i v a t i v e (e.g.,  linalool  g e r a n i o l (j_4_), o r n e r o l (1^))- would overcome t h i s problem.  The  c h o i c e o f 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 t h r e e compounds have been shown t o be i n c o r p o r a t e d i n t o t h e monqterpenoids b u t r e c e n t work has g i v e n an i n d i c a t i o n t h a t l i n a l o o l (20) ease . 5 8  can be i n c o r p o r a t e d w i t h g r e a t e r  E x a m i n a t i o n o f t h e l i t e r a t u r e showed t h a t 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 o f t h e t r a n s - a l l y l i c m e t h y l group i n l i n a l o o l can be a c c o m p l i s h e d and t h a t v a r i o u s s y n t h e t i c p r o c e d u r e s a r e a v a i l a b l e f o r the s y n t h e s i s o f m e v a l o n i c a c i d l a b e l l e d w i t h deuterium a t 0(2), 0(4),  and 0(5).  C(3'),  Thus we d e c i d e d t o prepare two p r e c u r s o r s , m e v a l o n i c  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 r e s p e c t i v e l y (Figure  H a t t h e C(2)  and C(8) p o s i t i o n s  65).  D (62) 2 [8]-linalool  (61) 2 [2- H^]-mevalonic a c i d .  F i g u r e 65. P r e c u r s o r s o f c h o i c e f o r t h e b i o s y n t h e t i c study.  2 E x a m i n a t i o n o f t h e H-n.m.r. o f camphor b i o s y n t h e s i s e d from 2 2 [2- H^]-mevalonic a c i d (6l_) o r [8- H ^ ] - l i n a l o o l (62) would show i f t h e C(8) and C(9) m e t h y l groups had been d e r i v e d from a s p e c i f i c m e t h y l group i n t h e a c y c l i c p r e c u r s o r  (58).  Samples o f 8 - d e u t e r i o - and  -  9-bromocamphor  79 -  [ 9 - H.]-camphor  Figure 66. P r e p a r a t i o n of [8- H^-and  [ 9 - H^]- camphor.  9 - d e u t e r i o - camphor, o b t a i n e d from t h e c o r r e s p o n d i n g  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 d e u t r i d e ( F i g u r e 6 6 ) , were examined 2  by  H-n.m.r. s p e c t r o s c o p y  and i t was n o t e d t h a t t h e c h e m i c a l s h i f t 2  d i f f e r e n c e between t h e deuterium s i g n a l s was v e r y s m a l l ( 8 - H- a t I.OOOppm, 2  and 9- H- a t 1.159 ppm.).  An obvious method o f 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 o f a lanthanide s h i f t reagent, i n t h e presence o f added i n c r e m e n t s taken.  (Eu(thd)'  3  = Resolve-Al  3,5-heptanedionato)europium.)  and t h e H-n.m.r.  o f E u ( t h d ) ^ s h i f t reagent  (Aldrich) = tris(2,2,6,6-tetramethylThe, s h i f t reagent  group o f camphor and i t would be expected  binds t o the carbonyl 2  f o r t h e [ 8 - H^]- s i g n a l t o be  a f f e c t e d t o a g r e a t e r e x t e n t than t h e '[9- H^]- s i g n a l . found and t h e r e s u l t i n g c h e m i c a l s h i f t reagent  were  T h i s was i n d e e d  s h i f t w i t h r e s p e c t t o t h e amount o f  added noted ( F i g u r e s . 6 7 , 68, and 6 9 ) . T h i s gave a  - 80 -  F i g u r e 67.  E f f e c t o f added s h i f t reagent (S.R.) on t h e 2 spectrum o f [8- H ]-camphor.  H-n.m.r.  -  CDC1  F i g u r e 68..  E f f e c t o f added s h i f t reagent (S.R.) on t h e 2 spectrum 'of '[9- H-.] -camphor.  H-n.m.r.  8 1  -  F i g u r e 69.  H-N .m.r.  spectrum of a  m i x t u r e of [8- H^] - and  [9- H^] - camphor i n the presence  to i  'of excess s h i f t r e a g e n t .  - 83 convenient,  non-destructive  method f o r t h e a n a l y s i s o f t h e r e s u l t s  obtained.  II - (iv)  The p l a n t system.  According' t o the l i t e r a t u r e  1 0 5  , t h r e e p l a n t s a v a i l a b l e on t h e  U.B.C. campus a r e known t o contain- a workable q u a n t i t y o f camphor i n their essential oils.  These were Cinnamomum camphora S i e b . ( t h e 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 lavender).  The camphor t r e e r e q u i r e s a h o t c l i m a t e f o r optimum  growth and was m a i n t a i n e d  i n t h e U.B.C. . h o r t i c u l t u r e b u i l d i n g .  p l a n t i t s e l f was n o t v e r y r o b u s t and - e x p e r i m e n t a t i o n ' u s i n g doubt have k i l l e d the t r e e .  The  i t would no  So the - a l t e r n a t i v e s - w e r e examined.  Steam  d i s t i l l a t i o n o f t h e ground p l a n t s . f o l l o w e d by petroleum e t h e r (30°-60°C) e x t r a c t i o n o f the d i s t i l l a t e , p r o v i d e d -the e s s e n t i a l o i l s o f R.  officianalis  and S. chamae c y p a r i s sus, two hardy, shrubs, grown i n . the U.B.C. B o t a n i c a l gardens.  G . l . c . examination, showed t h a t t h e 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 from S. chamae c y p a r i s sus c o n t a i n e d  20% camphor•whilst t h a t l e s s than.-5% camphor.  obtained  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 c o u l d be r e a d i l y sublimed t o h i g h e r 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 o f camphor was i d e n t i c a l t o t h a t o f 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 t h e  biosynthetic studies. For b i o s y n t h e t i c s t u d i e s , t h e r e a r e a v a r i e t y o f 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 t o i n c o r p o r a t e ..a l a b e l l e d p r e c u r s o r .  Much  of the work t o 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 o r c u t t i n g s , a l t h o u g h  some workers a r e now s u c c e e d i n g  i n s e p a r a t i n g the r e a s o n a b l y .pure enzyme p r e p a r a t i o n s 78  the d e s i r e d t r a n s f o r m a t i o n s  (e.g. -R.iCroteau  responsible f o r  79  '  ).  Another method used  i n biosynthetic' studies 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 o r on a n u t r i e n t medium. - However, the p r o d u c t i o n  of  secondary m e t a b o l i t e s , by. c e l l c u l t u r e s - has- been f r a u g h t w i t h  problems  Some p l a n t t i s s u e c u l t u r e s have been used f o r the p r o d u c t i o n  of various  secondary m e t a b o l i t e s ,  e.g., a l k 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  1 0 6  ,  w i t h l e v e l s c l o s e t o o r e x c e e d i n g t h o s e - f o u n d i n the i n t a c t p l a n t s .  In  some c a s e s , t h e c u l t u r e .has produced compounds which a r e n o t p r e s e n t i n the i n t a c t p l a n t . -  I t has been p o s t u l a t e d  secondary m e t a b o l i t e s  1 0 7  -that r e a c t i o n s t h a t l e a d t o  do n o t 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 a r e a v a i l a b l e , analogues o f t h e 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 a c c e s s t o t h e s e - • a l t e r n a t i v e • s u b s t r a t e s .  For  c e r t a i n secondary p r o d u c t s (e.g.. e s s e n t i a l , o i l s ) - i t i s thought t h a t accumulation of product only occurs i n s p e c i f i c morphological  structures  (e.g. o i l glands)- and a c c u m u l a t i o n can o n l y occur when these .structures are p r e s e n t .  I n - t h e 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 p r o d u c t s  are n o t accumulated to.any g r e a t extent, and t h e i r s y n t h e s i s v i a p l a 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 ,  that i n cultures  of pepper and s p e a r m i n t , 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 b i o c h e m i c a l  and  s t r u c t u r a l f e a t u r e s . of o i l glands.. . I n a d d i t i o n , v o l a t i l e o i l s o f Ruta 1  graveo^^  1  -and • Pimpi-nella anisum have been d e t e c t e d  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 f o r m a t i o n - o f  c e l l aggregates  i s r e g a r d e d as a p r e r e q u i s i t e f o r - o i l f o r m a t i o n and b e f o r e b i o s y n t h e t i c • study.. of. t e r p e n o i d s  the i n v i t r o  u s i n g 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 o u t , t h e r e must be. p r o g r e s s .towards i n d u c i n g c e r t a i n l e v e l s o f  .  - 85 cytodifferentiation  i h these c u l t u r e s .  Both o f t h e s e newer methods o f b i o s y n t h e t i c study, and enzyme p r e p a r a t i o n s , • a r e and a l t h o u g h  c e l l culture  i n . v i t r o .methods o f s t u d y i n g  biosynthesis  t h e y a r e good i n d i c a t i o n s o f t h e mode o f o p e r a t i o n  occurring  i n a p l a n t , i t must be'remembered t h a t t h e y a r e s t i l l i n v i t r o s t u d i e s . I n v i v o s t u d i e s , u s i n g t h e i n t a c t p l a n t system, o b v i o u s l y g i v e a c l o s e r v i e w o f t h e e x a c t mode o f o p e r a t i o n occurring- and t h u s i n v i t r o  studies  s h o u l d 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 s p e c i f i c i t y o f the r e a c t i o n s y i e l d i n g - secondary•metabolites  1 0 7  , the  i s thought  t o be r e l a t i v e l y low, and I n v i t r o s t u d i e s may show a s u s p e c t e d  precursor  t o be i n c o r p o r a t e d i n t o a s e c o n d a r y . p r o d u c t which i n v i v o i s n o t a t r u e p r e c u r s o r , b e i n g denied a c c e s s t o the-, s i t e of- s y n t h e s i s '.by compartmentation effects. present  S e c o n d l y , a m e t a b o l i t e - m a y be produced which i s . not u s u a l l y i n t h e system, - p o s s i b l y - d u e t t o t h e l a c k - o f - s p e c i f i c i t y o f t h e  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 t h e p l a n t by  a c e r t a i n r o u t e may, i n v i t r o , be produced-by.an. a l t e r n a t i v e r o u t e . Whether any o r a l l o f t h e s e - a l t e r n a t i v e s o c c u r i s unknown, b u t t h e p o s s i b i l i t y can o n l y be reduced t o .a.minimum by .using a system as c l o s e t o t h e i n t a c t p l a n t as p o s s i b l e . • Even i n t h e s e c a s e s when an i n t a c t !  p l a n t i s used, t h e effect..of. i n c r e a s e d , 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 b e i n g brought i n t o o p e r a t i o n  (e.g., t h e r e p o r t e d p r o d u c t i o n  l e u c i n e , see s e c t i o n I - ( v i i ) - (a).)..-  o f DMAPP from  I t was t h e r e f o r e d e c i d e d  that,  f o r our e x p e r i m e n t s on t h e 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 - i n t r o d u c t i o n o f a p r e c u r s o r i n t o t h e  i n t a c t p l a n t system t h e r e . a r e . a.number o f techniques, which may be used depending on t h e type o f p l a n t being, examined and t h e s u s p e c t e d d u r a t i o n of t h e b i o s y n t h e s i s b e i n g i n v e s t i g a t e d  1 0 9  . . The major f e e d i n g methods  - 86 used f o r p l a n t s a r e as f o l l o w s : 1 . In.jection;  an aqueous s o l u t i o n o f t h e 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 t h e v a s c u l a r system  2.  Cotton-wick;  (stalk).  f o r p l a n t s w i t h a r o b u s t b u t n o t t o o woody stem. One end o f a c o t t o n w i c k (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 t h e p l a n t stem, and t h e o t h e r end immersed i n t h e s o l u t i o n (0.5-1.0 ml.) t o be i n c o r p o r a t e d , , u s u a l l y i n a s m a l l tube a t t a c h e d 3»  Cutting the s t a l k ;  t o t h e stem.  c u t shoots a r e m a i n t a i n e d i n an aqueous s o l u t i o n  containing the l a b e l l e d m a t e r i a l .  T h i s method i s o n l y  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 p r o c e s s e s as when t r e a t e d i n t h i s way, t h e metabolism o f most p l a n t s slows down a f t e r a few h o u r s . 4-  S c a r r i n g ;••  t h e 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 p r e c u r s o r  i s added  . dropwise t o s c a r s a t v a r i o u s p o i n t s on t h e stem e x p o s i n g the e x t e r n a l v e i n s . 5»  Brush;  f o r v e r y l o n g term e x p e r i m e n t s .  The l a b e l l e d p r e c u r s o r  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 t h e l e a v e s o f the p l a n t i n t h e ground o r i n a v a s e . The  p l a n t b e i n g used f o r t h i s b i o s y n t h e t i c s t u d y , Rosemarinus  o f f i c i a n a l i s , i s a hardy shrub and i s n o t amenable t o t h e c o t t o n - w i c k technique.  The p l a n t stem was too. r o b u s t  f o r the i n j e c t i o n method and,  as t h e b i o s y n t h e s i s o f monoterpenes i s - g e n e r a l l y thought t o be r a p i d , and out.  t h e l e a v e s o f t h e p l a n t a r e s m a l l , t h e b r u s h method was a l s o r u l e d E x a m i n a t i o n o f t h e r e m a i n i n g two. t e c h n i q u e s showed t h e c u t t i n g t h e  s t a l k t e c h i n q u e t o be t h e s i m p l e s t , and many, w o r k e r s method w i t h g r e a t s u c c e s s .  1 1 0  have used t h i s  We.decided,, t h e r e f o r e , to'use t h i s  technique.  - 87 I I - (v) S y n t h e s i s of-, the, b i o s y n t h e t i c p r e c u r s o r s o f camphor.  2 As p r e v i o u s l y d e s c r i b e d , . t w o 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 t o be prepared f o r f e e d i n g t o  Rosemarinus o f f i c i a n a l i s .  (61) [2-  (i)  (62)  2  2  H_]-mevalonic a c i d  [2-  H^]-mevalonic a c i d  [8- H ^ ] - l i n a l o o l  (61).  Many methods f o r t h e . p r e p a r a t i o n o f v a r i o u s l a b e l l e d m e v a l o n i c a c i d s have been r e p o r t e d . . 2 2  The s y n t h e s i s o f E l l i s o n and B h a t n a g a r  1 1 1  however p r o v i d e s an o p p o r t u n i t y t o 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 m a t e r i a l s . . . The s y n t h e s i s ( F i g u r e 70) y i e l d s m e v a l o n i c 'acid••• l a c t o n e -(61a) w h i c h can.be c o n v e r t e d a c i d (6l_)  p r i o r t o the feeding experiments.  t o mevalonic  4-Acetoxy-2=butanone  was r e a d i l y o b t a i n e d from t h e r e a c t i o n o f m e t h y l v i n y l . k e t o n e (66) 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 t h e method'of C o r n f o r t h  (64) with 1 1 2  .  2 S p e c t r a l d a t a was c o n s i s t e n t w i t h t h e s t r u c t u r e g i v e n .  [2-  H ]-Ethyl 3  2 a c e t a t e can be obtained, by e s t e r i f i e a t i o n o f . r e a d i l y a v a i l a b l e [ H^1_ a c e t i c a c i d , o b t a i n a b l e i n h i g h 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  F i g u r e 70.  diisopropylamide  P r e p a r a t i o n o f [2-  H„]-mevalonic a c i d ( 6 1 ) .  (M)  (66)  F i g u r e 71. P r e p a r a t i o n o f 4-aeetoxy-2-butanone.  To p r e p a r e t h i s d e u t e r i o e t h y l a c e t a t e , 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-acetic 4-  a c i d w i t h e t h a n o l was attempted.  Excess e t h a n o l was  employed t o ensure c o m p l e t i o n o f r e a c t i o n , .but i t was found  difficult  - 89 to  separate e t h y l a c e t a t e from excess solvent'.  tables  1 1 3  Examination  of  standard  showed these two compounds t o form an a z e o t r o p i c m i x t u r e ,  and  so o t h e r s o l v e n t s - ( d i e t h y l e t h e r , benzene, t o l u e n e , and o-xylene) were t r i e d , a l l without success.  I t was n e c e s s a r y  f o r the prepared  ethyl  a c e t a t e t o be d r y and a c i d - f r e e f o r the n e x t s t e p i n the r e a c t i o n sequence and p r e p a r a t i o n t o such a degree of p u r i t y was method.  d i f f i c u l t by  this  Base c a t 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 c a r r y . o v e r of excess base would not impede  the c o n d e n s a t i o n .  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 c h o i c e of an a p p r o p r i a t e s o l v e n t d u r i n g 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 filtration.  P r e p a r a t i o n o f - [ 2 - H ^ ] - e t h y l a c e t a t e u s i n g base c a t a l y s i s  and a d r y i n e r t s o l v e n t 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  purification.  (LDA)  without  prior  A c o n v e n i e n t , base c a t a l y s e d , e s t e r i f i c a t i o n method u s i n g  1 ,8-diazabicyclo-.[5.4.0]-undec-7-'ene .:(DBU) as the . b a s e  1 1 4  was  out i n d r y d i e t h y l e t h e r w i t h a c e t i c a c i d and e t h y l bromide.  carried The  salt  DBU.HBr p r e c i p i t a t e d d u r i n g the ' r e a c t i o n . a n d - t r a n s f e r of t h i s s o l u t i o n of [2(LDA)  H ^ ] - e t h y l acetate i n dry d i e t h y l , ether to l i t h i u m  diisopropylamide  i n d r y d i e t h y l e t h e r through, a g l a s s , wool p l u g 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  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 , w h i c h  p u r i f i e d by column chromatography, t o p r o v i d e h y d r o x y - d i e s t e r  1 , 8-diazabicyclov.[5.4-.0] -undec-7-ene  generated  (65)  was  -  - 90 ( F i g u r e .70) a l o n g w i t h some n o n - d e u t e r a t e d m a t e r i a l . hydroxy-diester  The  H-n.m.r. o f  (65) i n d i c a t e d t h a t t h e compound was s a t u r a t e d w i t h  deuterium a t 0(2) s i n c e t h e s i n g l e t a t 62.55 n o r m a l l y a s s i g n e d t o t h e G(2)-hydrogens i n t h e n o n - d e u t e r a t e d analogue c o u l d n o t be d e t e c t e d . 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 r e d ( i . r . ) peaks a t  -1 3500 cm  A  -1 (v OH) and 1750 cm  1 (v C=0., e s t e r ) 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 . , 3 H ) , and 4.2 (q.,2H), c h a r a c t e r i s t i c o f a m o n o s u b s t i t u t e d group. 72).  double bond and an ethoxy  From t h i s d a t a , s t r u c t u r e (67) was t e n t a t i v e l y a s s i g n e d  (Figure  The f o r m a t i o n o f t h i s compound can-be.- e n v i s i o n e d as removal by  base o f a p r o t o n a- t o t h e ketone c a r b o n y l f o l l o w e d by e l i m i n a t i o n o f a c e t a t e a n i o n t o g i v e m e t h y l . v i n y l k e t o n e . .This then r e a c t s w i t h t h e anion  [ L i C D C 0 C H ] t o - g i v e t h e observed- p r o d u c t . 2  2  2  5  The base i n v o l v e d  i n t h i s m i n o r r e a c t i o n could.be t h e a n i o n d e r i v e d , from CD^GOOC-H^.  (67)  F i g u r e 72. A s i d e r e a c t i o n i n t h e - c o n d e n s a t i o n (64)  Conversion  with e t h y l acetate  o f 4-acetoxy-2-butanone  anion.  2 o f t h e d i e s t e r a l c o h o l (65) t o [2- H,_J-mevalonic a c i d  (61) was r e a d i l y a c h i e v e d by s t i r r i n g w i t h 10% potassium h y d r o x i d e i n methanol. D i s t i l l a t i o n o f t h e 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 l a c t o n e (61a) as a c o l o u r l e s s o i l .  [8- H ^ J - l i n a l o o l (62)  (ii)  OH  (62)  (20)  [8- E]-linalool  linalool I t was c o n s i d e r e d  2  t h a t i n t r o d u c t i o n o f deuterium i n t o t h e t r a n  m e t h y l group o f l i n a l o o l (20) c o u l d be- a c c o m p l i s h e d 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 o f t h e m e t h y l groups  (Figure 7 3 ) . Selenium d i o x i d e . o x i d a t i o n - o f a l l y l i c m e t h y l groups has been shown'--exclusively-to  occur t r a n s - t o - a side  chain  115-119  0G0CH,  0C0CH,  (68)  (20)  R = a c t i v a t i n g group.  (62).  F i g u r e 73.  , and n  (70)  Proposed s y n t h e t i c r o u t e t o [8- H ] - l i n a l o o l . 1  - 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 F i g u r e  74  1 1 9  .  For e x p l a i n a t i o n o f l e t t e r i n g (a)-(.e), see t e x t .  F i g u r e 74-. Mechanism o f selenium  Experimentation  has .shown path  dioxide o x i d a t i o n  1 1 3  .  (b) ( [ 2 , 3 ] - s i g m a t r o p i c rearrangement) t o  be dominant i n t h i s mechanism and l i t t l e o r no f o r m a t i o n o f t h e a l l y l c a t i o n (path (a) o r path (b) f o l l o w e d by path high r e g i o s e l e c t i y i t y shift.'  ( c ) ) can be d e t e c t e d .  o f t h i s r e a c t i o n can be e x p l a i n e d by t h i s  A t h i g h temperatures .(70°-100°C) p a t h  The  [2,3]-  ( c ) may be f a v o u r e d .  - 93 Allylic  o x i d a t i o n of l i n a l o y l acetate  a c c o r d i n g t o the method of Wakayama et a l .  1  1  (68) w i t h selenium ( F i g u r e 75)  5  y i e l d of aldehyde (71.), pure by g . l . c . and t . l . c .  and t h i s c o l o u r was  chromatography.  The  gave a  low  examinations.  the y e l l o w c o l o u r of t h i s p r o d u c t i n d i c a t e d t h a t i t was selenium  dioxide  contaminated by  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  method of Umbreit and S h a r p l e s s  1 1 7  However  ,  and  u s i n g a minimum  amount of selenium .dioxide and t e r t - b u t y l h y d r o p e r o x i d e t o r e o x i d i s e the selenium  formed, gave a m i x t u r e  8-hydroxylinaloyl acetate oil.  The  of  (69) and  2:2:1  of 8 - o x o l i n a l o y l a c e t a t e  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  s t a r t i n g m a t e r i a l c o u l d be s e p a r a t e d  from the p r o d u c t  by chromatography but any a t t e m p t s t o separate aldehyde (71.) f a i l e d .  (71.),  mixture  the a l c o h o l (69) from the  I n a d d i t i o n , t h i s method gave low y i e l d s presumably  because much o f the p r o d u c t c o n s i s t e d o f organoselenium b y p r o d u c t s .  The  mixture  of a l c o h o l (69) and aldehyde (7J.) was  hydride  (NaBH.) i n an attempt t o i n c r e a s e 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 s i m p l e r . mixture  reduced w i t h sodium boro-  However, t h i s o n l y r e s u l t e d i n a more complex  s i n c e r e d u c t i o n o f the a, (3-unsaturated  s a t u r a t e d a l c o h o l (73) a l s o o c c u r r e d producing  ( F i g u r e 76).  m a i n l y a l c o h o l or aldehyde was  literature  1 1 9  showed t h a t u s i n g one  produces m a i n l y  aldehyde t o the  required.  fully  Thus a method o f E x a m i n a t i o n o f the  e q u i v a l e n t of selenium  dioxide  aldehyde and v i r t u a l l y no a l c o h o l , whereas u s i n g  e q u i v a l e n t s o f selenium  dioxide gives a mixture  The  1 1 8  procedure o f A l t m a n  0.5  o f aldehyde and a l c o h o l .  , u s i n g 1 e q u i v a l e n t of selenium  dioxide,  produced m a i n l y a l d e h y d e . w i t h l i t t l e a l c o h o l i n an a c c e p t a b l e  yield.  Chromatography of the r e a c t i o n p r o d u c t p r o v i d e d pure a l d e h y d e , and 1 s i g n a l a t 69.4  i n the  H-n.m.r. spectrum was  assigned  the  t o the t r a n s -  aldehyde group by analogy w i t h the r e s u l t s o f Chan et a l .  1 2 0  .  spectrum (low r e s o l u t i o n ) o f t h i s compound does not e x h i b i t a M  The +  mass  peak  - 94 -  OCOCH,  f  *-  (71)  OCOGH 3  X  (ii)  OCOCH 3  OCOCH,  -CHO  (68)  (71)  (69)  \.OCOCH  3  [ [j  ^  (iii)  CHO  CHO OH  (i) (ii) (iii)  Se0 /dioxane/80 2  C/5h  1 1 5  ,  (71)  (72)  Se0 /^Bu00H/CH Cl /l0°C/4.5h , 117  2  2  2  Se0 /ethanol/A/24h 2  1 1 8  .  F i g u r e 75. Selenium d i o x i d e o x i d a t i o n o f l i n a l y l a c e t a t e ( 6 8 ) .  - 95 OCOCH,  OCOCH  OCOCH  3  (i)  .OCOCH  3  -H _H  -CHO  (71)  OH  (73)  (62)'  crude m i x t u r e  COCH  OCOGH  3  :ii)  —•  3  (69) COCH  3  (iv)  (iii)  OMs (74)  OCOCH  3  (v)  (69)  (i)  NaBH /EUOH/RT ( i i ) NaBH. /E'tOH/RT (iii) 4 4 ( i v ) MsCl/Et N/Et 0/0°G ( v ) NaBH^/EtOH/0°C  DIBAL/hexane/0°C  F i g u r e 76.  (69).  3  2  R e d u c t i o n o f aldehyde  (71)  to alcohol  - 96 but has peaks a t m/e  168,. 150, and 135.  The peak a t m/e  168 corresponds  t o l o s s of ketene from the a c e t o x y - g r o u p i n g and the peak a t m/e150 corresponds t o a M c L a f f e r t y rearrangement o f the same g r o u p i n g ( F i g u r e 77).  m/e.168  m/e  F i g u r e 77.  150  Mass spectrum f r a g m e n t a t i o n o f aldehyde (71_).  A minor p r o d u c t o b t a i n e d from t h i s ' r e a c t i o n e x h i b i t e d i . r . a t 3500 cm  -1  (v OH), 1765 cm  -1  "(v 0=0 e s t e r ) , 1.700 •cm -1  u n s a t u r a t e d aldehyde) and I64O cm  -1  peaks  (v 0=0 a, (3-  1 (v C=C).  .The' H-n.m.r. spectrum  o f t h i s p r o d u c t e x h i b i t e d the t r a n s - a l d e h y d i c p r o t o n resonance a t 69.23 as w e l l as a resonance: a t 64.1 which was a s s i g n e d t o -CH^OH. resonances f o r the gem-dimethyl•groups, were p r e s e n t .  Low  No  resolution  mass s p e c t r o m e t r y gave a m o l e c u l a r i o n o f 226 mass u n i t s and  structure  - 97 -  OCOCH,  OCOCH  x  SeO,  •OCOCH  3  3  (c) + Se(OH),  + H "0^  + H 0/- H 2  OCOCH  OCOCH  3  3  SeO, (b) CHO OH  OH  (72) F i g u r e 78. Proposed f o r m a t i o n 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 F i g u r e . 7 4 ) .  (72) was t e n t a t i v e l y a s s i g n e d .  I t thus appears t h a t o x i d a t i o n has  occurred c i s - t o the side chain i n this-example can occur when p a t h Reduction  This  (c) i n the proposed mechanism i s f o l l o w e d ( F i g u r e 7 4 ) .  o f aldehyde (71') t o a l c o h o l . (69) c o u l d be e a s i l y performed  u s i n g sodium b o r o h y d r i d e . achieve without  (Figure 78).  I n i t i a l l y the..reduction was d i f f i c u l t t o  concomitant 1 .,4—reduction o f t h e a,3-unsaturated system  and so d i i s o b u t y l a l u m i n u m  hydride  (DIBAL.)' was t r i e d ( F i g u r e 7 6 ) .  T h i s r e d u c i n g agent, which s p e c i f i c a l l y reduces t h e c a r b o n y l group ofa,3-unsaturated systems, a l s o , reduces e s t e r s and so t h e r e s u l t i n g was  product  8 - h y d r o x y l i n a l o o l " (?4 ). . I t . was.- hoped t h a t t h e p r i m a r y a l c o h o l i n ;  t h i s compound c o u l d be s e l e c t i v e l y c o n v e r t e d  t o a•good l e a v i n g group  - 98 ( i n t h i s case t h e m e s y l a t e ) i n t h e presence o f t h e l e s s r e a c t i v e tertiary alcohol. were u n s u c c e s s f u l .  Attempts' t o prepare t h e r e q u i r e d hydroxy-mesylate F u r t h e r experiments w i t h sodium  borohydride  showed t h a t r e d u c t i o n o f t h e a , 3 - u n s a t u r a t e d system i n (71_) t o t h e s a t u r a t e d a l c o h o l (73) c o u l d 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 t h e r e a c t i o n at- 0°C and u s i n g an a c c u r a t e l y measured 1 e q u i v a l e n t o f sodium-borohydride.  The pure a l c o h o l , o b t a i n e d by chroma1  tography o f t h e crude r e a c t i o n p r o d u c t , a s i n g l e t a t 64.0  corresponding  showed i n - t h e  H-n.m.r. spectrum  t o -CH^OH, and- the s i g n a l a s s i g n e d t o  the a l l y l i c hydrogen -CH=C(CH^) (CHgOH') to-be'a broad t r i p l e t a t 65.4-0, u p f i e l d from i t s p o s i t i o n i n t h e aldehyde  (66.50).  The f i n a l s t e p i n t h e s y n t h e s i s o f 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 t h e d e u t r i d e displacement  o f t h e C ( 8 ) - a l c o h o l grouping i n  8-hydroxylina'lo-yl a c e t a t e "(69) ( F i g u r e 79) ••- I t . was i n i t i a l l y  considered  t h a t t h i s c o u l d be a c c o m p l i s h e d by t r e a t m e n t o f t h e c o r r e s p o n d i n g t o s y l a t e w i t h sodium b o r o d e u t e r i d e • i n 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 t h e - u s u a l way ( t o s y l c h l o r i d e /  pyridine)  1 2 2  gave a l o w . y i e l d . o f  a n o i l whose  consistent with the required t o s y l a t e (75).  H-n.m.r. spectrum was  Reduction  u s i n g sodium  b o r o d e u t e r i d e in. 80% HMPA/water gave . ' 8 ^ d e u t e r i o l i n a l o y l a c e t a t e (77.) i n approximately  86% y i e l d .  The p r e p a r a t i o n - o f the t o s y l a t e was n o t easy  t o a c h i e v e i n - a r e l a t i v e l y a c c e p t a b l e y i e l d , and-so an a l t e r n a t i v e t o 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 r e p o r t e d t o work w e l l f o r h a l i d e s  1 2 1  and so an attempt  t o prepare t h e 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 ( F i g u r e 80). was  U s i n g t h e method o f Hooz and G i l a n i  1 2 3  , geraniol  t r e a t e d w i t h ear.bon t e t r a b r o m i d e a n d • t r i - n - o c t y l p h o s p h i n e  i n an attempt  t o c o n v e r t t h e a l c o h o l - t o t h e bromide.. The o i l o b t a i n e d i n t h i s r e a c t i o n  - 99 -  OCOCH  OCOCH  OCOCH,  3 (i) or (ii)  (15) R=Ts  R = M S f ( v /  (76) R=Ms COCH  3  (62)  (77)  (i) (iv)  TsCl/py NaOH/MeOH  F i g u r e 79.  ( i i ) Msd/Et^N/Et^O  ( i i i ) NaBD^/80$ HMPA/H 0 2  (v) 2.1 L i B D E t / E t 0 3  2  ( v i ) 4.1 L i B D E t y E t g O  C o n v e r s i o n o f a l c o h o l (69) t o 8 - d e u t e r i o l i n a l o o l (62).  (i)  CBr /( C H ) P n  4  Et 0 2  (U)  (80). F i g u r e 80. Attempted c o n v e r s i o n o f g e r a n i o l t o g e r a n y l bromide.  g  1 7  3  - 100 was  subjected- t o d i s t i l l a t i o n , and chromatography w i t h o u t a c h i e v i n g  purity.  Both of these proceedures•were seen t o cause  o r rearrangement o f the g e r a n i o l s k e l e t o n . a l l y l i c isomer the  (80) was  at  66.2  (-CH^CH^) .  Rearrangement t o the  thought t o be o c c u r r i n g and c o u l d be seen i n  H-n.m.r. spectrum by the disappearance  (-CH^-X, X=Br o r OH?)  of a d o u b l e t (2H) a t  (76)  I n comparison,  i t was demonstrated t h a t the :  corresponding  i n v o l v e d no i s o m e r i s a t i o n o f ;the double bond, and  p r e p a r a t i o n o f the mesylate mesylates  64.0  and•appearance o f a f o u r l i n e p a t t e r n (d.d.,1H)  c o n v e r s i o n o f 8 T h y d r o x y l i n a l o y l a c e t a t e (69)., to 'the mesylate  decomposition  looked' the most p r o m i s i n g .  are known t o be u n s t a b l e  1 2 1  * , i t was  so  As b e n z y l i c  assumed t h a t t h i s  mesylate would be s i m i l a r l y u n s t a b l e and thus no attempts  allylic  to p u r i f y t h i s  1 compound were made.  E x a m i n a t i o n by  H-n.m.r. s p e c t r o s c o p y o f the  p r o d u c t o b t a i n e d 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 a c e t a t e (69)  crude  with  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 d r y d i e t h y l e t h e r showed i t t o c o n t a i n m a i n l y the mesylbxy - compound,, . i n d i c a t e d by the o f a s i n g l e t (3H) a t 62.9,  presence  assigned'to-GH^-SO^-, -and a s i n g l e t a t  64.46 (2H), a s s i g n e d t o the 8-methylene p o s i t i o n , ; s h i f t e d from i n the s t a r t i n g a l c o h o l .  64.0  R e d u c t i o n u s i n g NaBD^.in 80% HMPA/H^O gave  o n l y a 10% y i e l d o f [.8- H g J - i i n a l o y l a c e t a t e ' ( 7 7 ) , and so an a l t e r n a t i v e r e d u c t i o n was  tried.  :  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. H o l d e r and M a t t u r o . - U s i n g 2.1 e q u i v a l e n t s .of r e d u c i n g agent gave 2 2 [8- H ^ ] - l i n a l o y l a c e t a t e (7_7)-' w i t h some [8- H^ ] - l i n a l o o l (62) and these 1 2 5  two compounds c o u l d be e a s i l y s e p a r a t e d by:column chromatography. 2 2 [8- H^ ] - l i n a l o y l a c e t a t e (77) c o u l d be r e a d i l y c o n v e r t e d t o [8- H^] l i n a l o o l ( 6 2 ) ' u s i n g 10% sodium h y d r o x i d e i n methanol. r e d u c t i o n o f the mesylate  (76) :using 4.1  A repeat  e q u i v a l e n t s o f r e d u c i n g agent  .  -  - 101 -  gave a h i g h y i e l d o f • r e l a t i v e l y pure [ 8 - - H ^ . ] - l i n a l o o l ( 6 2 ) . Column chromatography a f f o r d e d t h e p u r e - m a t e r i a l f o r ' t h e f e e d i n g  II - (vi)  experiments.  Results.  The f e e d i n g experiments were c a r r i e d ' o u t - u s i n g t h e c u t stem method :  (see page 8 6 ) . I n t h e f i r s t f e e d i n g experiment,  t h e shoots were p l a c e d 2  i n an aqueous s o l u t i o n o f l a b e l l e d p r e c u r s o r ([-8- H ^ - l i n a l o o l (62) 2 o r [ 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 o f t h e 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 a f f 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 w i t h an a u t h e n t i c sample. The . H-F.T.-n.m-.r.. s p e c t r u m ( a p p r o x i m a t e l y 25000 t r a n s i e n t s , 24 hours a c c u m u l a t i o n •time)'-:revealed no i n c o r p o r a t i o n 2 o f l a b e l i n camphor from e i t h e r p r e c u r s o r (-[8- >H^] - l i n a l o o l (62) o r 2 [2-oH^]-mevalonic a c i d (61.)).  The-. e x p e r i m e n t s were r e p e a t e d ,  time u s i n g an aqueous s o l u t i o n o f 'ATP (0.1' m g / m l )  1 2 6  t o f e e d t h e p r e c u r s o r s and t h e shoots were m a i n t a i n e d  this  r a t h e r than water 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 b e f o r e 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 . a f t e r approximately  The' H-F.T.-n.m.r. s p e c t r a ,  20000 t r a n s i e n t s (Figure- 81) seemed t o show a peak,  but i t was assumed'that t h i s , was• an a r t i f a c t , , o n l y one p o i n t b e i n g seen on t h e peak. 4.00MHz i n s t r u m e n t  I n a d d i t i o n , -when, t h e spectrum was r e c o r d e d on a *•' 2 (approx. 60MHz f o r o b s e r v i n g  ' g l i t c h e s ' were seen.  H)'none o f these  - I t t h e r e f o r e seemed- r e a s o n a b l e  t o assume t h a t  no i n c o r p o r a t i o n o f 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 o f o t h e r s , 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 o f t h e p r e c u r s o r t o t h e s i t e o f s y n t h e s i s o r t h a t t h e p r o d u c t i o n o f camphor was a t t o o l o w a l e v e l f o r i n c o r p o r a t i o n t o be  102 -  (a)  (b).  ill. " T  4 F i g u r e 81.  H-N.m.r. s p e c t r a o b t a i n e d from camphor e x t 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 f e e d i n g w i t h ( a ) [ 2 - H~]-MVA, and ( b ) [ 8 - H ^ - l i n a l o o l .  - 103 seen.  S e a s o n a l 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  'and i n this  species,  optimum l e v e l s of camphor are seen i n O c t o b e r , w i t h a decrease i n the summer months.  I t i s r e a s o n a b l e t o assume-that the l a c k of  o f 8 - d e u t e r i o l i n a l o o l (62) i s due  or  [2-  H^]-mevalonic a c i d (6l_)  i n t o camphor  t o the f a c t t h a t the compounds were not a d m i n i s t e r e d  p l a n t a t a t i m e of r e a s o n a b l e t e r p e n o i d : b i o s y n t h e s i s precursor  b e i n g of an i n c o r r e c t n a t u r e .  has been found t o be i n c o r p o r a t e d , terpenoid  compound.  to  the  r a t h e r than  the  M e v a l o n i c a c i d , f o r example,  i n low y i e l d s , i n t o a l l t y p e s of  R e p e t i t i o n of the f e e d i n g  experiments i n the  s p r i n g , when optimum p l a n t growth g e n e r a l l y o c c u r s , may i n c o r p o r a t i o n of m e v a l o n i c a c i d and  incorporation  r e s u l t i n the  , perhaps,.linalool.  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  As has  further-work.  been suggested, r e p e t i t i o n of the f e e d i n g work d u r i n g  s p r i n g m a y - r e s u l t i n an i n c o r p o r a t i o n , o f - t h e p r e c u r s o r s . changing the mode of feeding.may a l s o b r i n g f a v o u r a b l e  In addition,  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  precursor  may  be w o r t h i n v e s t i g a t i n g .  the  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 d i m e t h y l s u l p h o x i d e (DMSO)"solution of the p r e c u r s o r be of use.  may  DMSO i s known t o p e n e t r a t e t i s s u e s , c a r r y i n g w i t h i t any  dissolved material.  T h i s may  enable the l a b e l l e d p r e c u r s o r  absorbed a t the s i t e of s y n t h e s i s and achieved.  the  to  be  thus i n c o r p o r a t i o n could  be  Once t h i s problem of i n c o r p o r a t i o n has  system i s open t o a number of f u r t h e r , s t u d i e s .  been overcome, the  The  stereochemistry  of the r i n g c l o s u r e t o 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 o f 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 a t the t e r m i n a l double bond. ( F i g u r e 82-)'. - There a r e v a r i o u s methods f o r labelling  l i n a l o o l with  H a t the 0(1) e l s - or t r a n s - p o s i t i o n s and 2  determination  o f t h e p o s i t i o n o f • H a t 0(3') i n camphor would i n d i c a t e  whether t h e r i n g c l o s u r e - 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 s t e r e o c h e m i s t r y  of r i n g c l o s u r e .  Ill  EXPERIMENTAL.  General.  Unless otherwise  stated the f o l l o w i n g are implied:  G a s - l i q u i d chromatography ( g . l . c . ) was performed on a H e w l e t t - P a c k a r d model 5831A (flame i o n i s a t i o n d e t e c t o r ) gas chromatograph u s i n g a 6' x 1/8" column w i t h 3% OV-17 as t h e s t a t i o n a r y phase supported on Chromosorb ¥ and n i t r o g e n as t h e c a r r i e r gas. C a r r i e r gas f l o w was ca 30 mL/min. were r e c o r d e d  The H 60MHz .nuclear magnetic, resonance (n.m.r.) s p e c t r a 1 on a V a r i a n A s s o c i a t e s model T60, H 80MHz F.T.-n.m.r. 1  • s p e c t r a were r e c o r d e d s p e c t r a were r e c o r d e d  on a B r u e k e r model WP80 and H 400MHz E.T.-n.m.r. on a B r u e k e r model WH4OO.  Signal positions are  g i v e n on t h e d e l t a (6) s c a l 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. s p e c t r a were  on a B r u e k e r model WP80 s p e c t r o m e t e r .  recorded  S i g n a l p o s i t i o n s a r e g i v e n 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 r e f e r e n c e  (67.51).  S i g n a l m u l t i p l i c i t y , coupling constants  integrated  ( i f observable),  a r e a and s i g n a l assignments a r e i n d i c a t e d i n p a r e n t h e s e s . spectra ( i . r . ) - w e r e recorded meter.  Infrared  on a P e r k i n Elmer 137B I n f r a c o r d s p e c t r o -  S o l u t i o n s p e c t r a were performed u s i n g a sodium c h l o r i d e c e l l  o f 0.1mm t h i c k n e s s .  Absorption ^  p o s i t i o n s (v ) a r e g i v e n i n t h e cm ^ max 6  u n i t and a r e c a l i b r a t e d by means o f t h e 1601 cm  band o f p o l y s t y r e n e .  Low r e s o l u t i o n mass s p e c t r a were determined on t h e K r a t o s AE1 model MS902 o r model MS50 i n s t r u m e n t s .  Microanalyses  P. Borda, M i c r o a n a l y t i c a l L a b o r a t o r y , Vancouver. quality.  were performed by Mr.  U n i v e r s i t y o f B r i t i s h Columbia,  A l l s o l v e n t s used f o r i . r . and n.m.r. were o f s p e c t r a l Reagents and r e a c t i o n s o l v e n t s used were o f e i t h e r Reagent  - 106 or C e r t i f i e d grade.  The term "petroleum  the low b o i l i n g f r a c t i o n o f petroleum 35 -60 C ) .  e t h e r (30°-60^C)" r e f e r s t o  d i s t i l l a t e ( b o i l i n g point ca.  Dry o r p u r i f i e d s o l v e n t s o r r e a g e n t s , 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 e t h e r . 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 c a l c i u m h y d r i d e a t atmospheric  pressure.  Diazobicycloundecene  (DBU)  by s t i r r i n g over c a l c i u m h y d r i d e and  d i s t i l l i n g a t reduced p r e s s u r e . D i e t h y l e t h e r (Et^O) o r t e t r a h y d r o f u r a n (THF)  by r e f l u x i n g over sodium  o r l i t h i u m aluminum h y d r i d e and d i s t i l l i n g . Diisopropylamine  (("""Pr^NH)  sodium h y d r o x i d e  and d i s t i l l i n g .  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  E t h y l bromide ( E t B r ) by d r y i n g w i t h c a l c i u m h y d r i d e and d i s t i l l i n g from phosphorous p e n t o x i d e (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 o n l y the 37°C b o i l i n g fraction. P y r i d i n e by s t i r r i n g w i t h potassium  h y d r o x i d e and d i s t i l l i n g .  Selenium d i o x i d e (SeO^) by s u b l i m a t i o n a t atmospheric p_-Toluenesulphonyl  pressure  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 c h l o r o f o r m ,  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 o f petroleum  e t h e r (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 c o n c e n t r a t i n g u n t i l are  (340°C).  crystals  obtained. n  B u t y l l i t h i u m used was s u p p l i e d 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 t o use. g e l f o r column chromatography w a s . S i l i c a G e l 60,  Silica  230-400 mesh,  purchased from BDH Chemicals L t d . , and was c a r r i e d out under medium pressure  (flash)  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 o u t u s i n g B a k e r - f l e x chromatography c a r d s , coated  with  s i l i c a g e l IB2F and s u p p l i e d by J.T.Baker C h e m i c a l 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 s p r a y i n g w i t h a s o l u t i o n o f dodecaphosphom o l y b d i c a c i d (5 g) i n e t h a n o l (100 mL) f o l l o w e d by h e a t i n g , o r by s p r a y i n g w i t h 5$ n i t r i c a c i d i n c o n c e n t r a t e d  sulphuric acid followed  by h e a t i n g ( s p e c i f i c spray f o r camphor).  P r e p a r a t i o n o f 1-acetoxy-3-butanone (64). M e t h y l v i n y l ketone ( ( 6 6 ) , 10 mL, 8.64  g, 0.123 mol) was  d i 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- o f water added, an the m i x t u r e heated t o 100°G f o r 24 hours.-.. The m i x t u r e 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 e t h e r , washed w i t h s a t u r a t e d sodium bicarbonate  s o l u t i o n and w a t e r , d r i e d (MgSO ) and e v a p o r a t e d t o g i v e  4 a yellow o i l .  D i s t i l l a t i o n (1.5 mm.Hg) a f f o r 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.  80 -84°C/15 mm Hg. 0  V  ( C C l . ) : 1745 ( s t r o n g , sharp, C=0 e s t e r ) , 1735 ( s t r o n g , sharp, max 4 C=0 k e t o n e ) . 6 (60MHz, C C l . ) : 1.95 ( s , 3H, CH,-C0-C-) 2.1 ( s , 3H, CH--C0-0-), 4 J i 2.7 ( t , 2H, C-C0-CH -, J=6Hz), 4-16 ( t , 2H, -C-CH -0-C0-). f  2  Model experiments f o r t h e p r e p a r a t i o n o f e t h y l a c e t a t e , (i)  Acid catalysed 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 s u 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 e t h a n o l (100 mL) and heated t o r e f l u x f o r 3  - 108 hours.  The m i x t u r e was c o o l e d and poured onto water and t h e aqueous  solution extracted  with d i e t h y l ether.  washed w i t h s a t u r a t e d  The combined o r g a n i c phases were  sodium b i c a r b o n a t e s o l u t i o n and w a t e r , d r i e d  (MgSO.), and d i s t i l l e d ( a t m o s p h e r i c p r e s s u r e ) t o g i v e a l i q u i d (3.3  4  -]  whose 60MHz  H-n.m.r. spectrum showed i t to- c o n s i s t o f a 1:1 m i x t u r e  o f e t h y l a c e t a t e and d i e t h y l e t h e r . was (ii)  g)  S e p a r a t i o n o f t h e s e two components  not achieved. Base c a t a l y s e d  esterification.  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, t o l u e n e , o r o—xylene (20 mL).  undecene (DBU, 2.54  Diazobicyclo-  g, .0.017 mol) was added dropwise and t h e m i x t u r e  s t i r r e d o v e r n i g h t a t room temperature.  The c l e a r s o l u t i o n was decanted  from t h e p r e c i p i t a t e d s a l t , washed w i t h w a t e r , and d r i e d (MgSO.). 4 D i s t i l l a t i o n afforded  e t h y l a c e t a t e (0.54 g) whose 60MHz  i n d i c a t e d t h a t i t was contaminated w i t h s o l v e n t .  H-n.m.r.  No s i g n a l s f o r  e t h y l bromide c o u l d be d e t e c t e d . Preparation of [ l - Hj,-ethyl acetate Deuterioacetic  (63).  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 d r y d i e t h y l e t h e r (5 mL) under an  atmosphere o f d r y argon.  D i a z o b i c y c l o u n d e c e n e (DBU, 11.66 mL, 0.078  mol)  was added dropwise w i t h i c e - c o o l i n g and t h e r e a c t i o n  stirred for  24 hours a t room temperature, d u r i n g which time t h e s a l t DBU.HBr precipitated.  The r e s u l t i n g d r y d i e t h y l e t h e r s o l u t i o n o f [1- H ^ ] -  e t h y l a c e t a t e was used w i t h o u t p u r i f i c a t i o n f o r t h e p r e p a r a t i o n o f e t h y l 5-acetoxy-3-hydroxy-3-methyl pentanoate  (65).  - 109 2 P r e p a r a t i o n o f e t h y l [2- H ")-5-acetoxy-3-hydroxy-3-methyl pentanoate 2  (65)•  Dry d i i s o p r o p y l a m i n e (10.9 mL, 0.078 mol) was d i s s o l v e d i n d r y d i e t h y l e t h e r (10 mL) under an atmosphere o f d r y argon, and c o o l e d t o -22°C.  " B u t y l l i t h i u m (1.6M, 47 mL, 0.078 mol) was added and t h e  m i x t u r e s t i r r e d f o r 1 hour.  The temperature  was then lowered t o -78°C  and t h e d i e t h y l e t h e r s o l u t i o n o f [ l - ^ H ^ ] - e t h y l a c e t a t e (63)  was added  through a g l a s s wool p l u g t o 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 t h e 1-acetoxy-  3-butanone ((^4), 10.14 g, 0.078 mol) added.  i n d r y d i e t h y l e t h e r (15 mL) was  S t i r r i n g was c o n t i n u e d f o r a f u r t h e r 20 minutes, water was  added t o t h e m i x t u r e and t h e s o l u t i o n a l l o w e d t o warm t o room  temperature.  The m i x t u r e was e x t r a c t e d w i t h d i e t h y l e t h e r and t h e combined o r g a n i c phases were washed w i t h water, b r i n e , and d r i e d (MgSO.). 4 o i l (7 g ) .  s a t u r a t e d sodium b i c a r b o n a t e  E v a p o r a t i o n o f t h e s o l v e n t gave a c o l o u r l e s s  Chromatography ( s i l i c a g e l , petroleum  d i e t h y l e t h e r 2:1 e l u a n t ) gave (65) (2.40 and some non-deuterated  solution,  e t h e r (30 -60 C ) :  g, 0.0109 mol, 14% y i e l d )  (65) (2.56 g, 15% y i e l d ) .  V (CC1.); 3550 (weak, broad, OH), 1745, 174.0 ( s t r o n g , sharp, C=0). max 46 (60MHz, C D C 1 ) ; 1.20 ( t , 3H, CH^CHg-O-, J=7Hz) , 1.27 ( s , 3H, t e r t i a r y 3  m e t h y l ) , 1.91 ( q , 2H, -O-CH^-CH^-C(OH)(CH^)-, J=7Hz), 2.0 ( s , 3H, CH^-CO-O), 3.61 ( b s , 1H, d i s a p p e a r s w i t h DgO, -OH), 4.1 ( q , 4H, -CH -CH -0Ac and -CO-O-CHg-CH 2  2  o v e r l a p p i n g , J=7Hz).  Mass spectrum ( l o w . r e s . ) : m / e ( r e l . i n t . ) ; 145  205 ( 1 % ) , 175 (3%),-l60 ( 1 % ) ,  ( 2 9 % ) , 133 (100%) 115 ( 2 2 % ) , 106 ( 1 6 % ) , 99 ( 7 % ) .  Analysis: For C ^ H ^ O ^  Calc:  C, 54.53;  H,  Found:  C, 54.70;  H, 8.10.  8.31. .  - 110 Preparation of [2- H l-3-hydroxy-3-methyl-5-pentanolide  (\2-^E^\-  2  2  mevalonolactone) (61 a ) . 2 E t h y l [ 2 - H^]-5-acetoxy-3-hydroxy-3-methyl pentanoate 1.45  ((65),  g, 0.0067 mol) was d i s s o l v e d i n m e t h a n o l i c potassium h y d r o x i d e  (10$ w/v,  10 mL) and s t i r r e d a t 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 o f m e t h a n o l i c hydrogen and s t i r r e d f o r 2 hours a t room t e m p e r a t u r e .  chloride  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 t h e methanol e v a p o r a t e d .  The o i l was  d i s s o l v e d i n c h l o r o f o r m , f i l t e r e d , and the c h l o r o f o r m e v a p o r a t e d . 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) a f f o r d e d mevalonolactone ((61a.), O.84 oil. V max  2  OH),  2  3450 (weak, broad, OH),  1735  C=0).  6 (80MHz, C D C l ^ ) ; L 4 O w i t h D 0,  2  g, 0.0065 mol, 97$ y i e l d ) as a c o l o u r l e s s  ( C C l , ) ; 3620 (weak, sharp, OH), 4  (strong, sharp,  [2- H ]-  1.85  ( s , 3H, t e r t i a r y m e t h y l ) , 1.69  ( d , 1H, J=5Hz) and 1.95  ( b s , 1H, d i s a p p e a r s  ( d , 1H, J=5Hz)  ( - C H - C ( 0 H ) ( C H ) - ) , 4.25-4.75 (m, 2H, -CH -0-C0-). 2  3  2  Preparation of 6-acetoxy-2,6-dimethyl-2,7-octadienal (71). (i)  A c c o r d i n g t o t h e method o f Wakayama e t L i n a l o y l a c e t a t e • ( ( 6 8 ) , 39.2  al.  1 1 5  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 t o 80°C f o r 5 h o u r s .  A f t e r c o o l i n g , the d e p o s i t e d selenium was removed  by vacuum f i l t r a t i o n t h r o u g h c e l i t e and the dioxane e v a p o r a t e d t o g i v e a red/orange o i l . A 1:1  p e t r o l e u m e t h e r (30 -60 C ) : d i e t h y l e t h e r  m i x t u r e (100 mL) was added and the m i x t u r e shaken. was decanted and t h e s o l v e n t e v a p o r a t e d .  The o r g a n i c l a y e r  The r e s u l t i n g o i l y r e s i d u e  was d i s s o l v e d i n d i e t h y l e t h e r , washed w i t h s a t u r a t e d sodium b i c a r b o n a t e  s o l u t i o n and w a t e r , d r i e d (MgSO.) and evaporated t o g i v e 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) a f 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 y e l l o w o i l , b.p. 98°-100°C/0.03 mm 1 H-n.m.r. and i . r .  Hg.  s p e c t r a 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 o f  6-acetoxy-2,6-dimethyl-2,7-octadienal.. (ii)  A c c o r d i n g t o the method o f Umbreit and S h a r p l e s s Selenium d i o x i d e (5.66  c h l o r i d e (100 mL)  g, 0.051  1 1 7  .  mol) was d i s s o l v e d i n methylene  and t - b u t y l h y d r o p e r o x i d e (20.4  mL,  0.204 mol) added.  The m i x t u r e was s t i r r e d i n the dark f o r 30 minutes, c o o l e d to and l i n a l o y l a c e t a t e ( ( 6 8 ) , 20 g, .0.102 mol) added. s t i r r e d a t 10°C  f o r 4s hours.  10°C,  The m i x t u r e was  A f u r t h e r a l i q u o t o f methylene  chloride  (75 mL) was added and the o r g a n i c phase washed w i t h s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n and water, d r i e d (MgSO. )•-and evaporated t o g i v e 4 1 a pale yellow o i l , analysis.  seen t o c o n t a i n t - b u t y l h y d r o p e r o x i d e by  H-n.m.r.  T h i s excess h y d r o p e r o x i d e was d e s t r o y e d by d i s s o l v i n g the  m i x t u r e 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 a d d i n g d i m e t h y l s u l p h i d e (30 mL) w i t h i c e c o o l i n g . -  m i x t u r e was n e u t r a l i s e d w i t h 20% w/v then e x t r a c t e d w i t h d i e t h y l e t h e r .  A f t e r s t i r r i n g f o r 2 h o u r s , the potassium carbonate s o l u t i o n and The combined o r g a n i c phases were  washed w i t h w a t e r , d r i e d (MgSO.),. and evaporated t o g i v e a c o l o u r l e s s o i l seen t o be 20% s t a r t i n g m a t e r i a l , 38% a l d e h y d e , and 40% g.l.c.  a l c o h o l by  Chromatography ( s i l i c a g e l , g r a d i e n t e l u t i o n from p e t r o l e u m  e t h e r (30°-60°C) t o 30% d i e t h y l e t h e r / p e t r o l e u m e t h e r (30°-60°C)) gave r e c o v e r e d 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 m o l ) , aldehyde (71.) (1.20 g, 90% g . l . c . p u r i t y , 0.0057 mol, 5.6% 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%  yield),  y i e l d ) and a  1:1 m i x t u r e o f 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 c o u l d n o t be f u r t h e r s e p a r a t e d . was l o s t as organoselenium  byproducts  A l a r g e amount o f m a t e r i a l  i n t h i s p r e p a r a t i o n and t h e y i e l d  c o u l d n o t be r a i s e d . (iii)  A c c o r d i n g t o t h e method o f Altman e t a l .  1 1 8  L i n a l o y l a c e t a t e ( ( 6 8 ) , 10.8 g, 0.055 mol) was d i s s o l v e d i n e t h a n o l (95$,  75 mL), f r e s h l y sublimed selenium d i o x i d e (6.115 g,  0.055 mol) added, and t h e m i x t u r e r e f l u x e d f o r 24 hours. selenium was removed by f i l t r a t i o n evaporated  The d e p o s i t e d  through e e l i t e and t h e e t h a n o l  t o g i v e a red/orange o i l . .  The o i l was d i s s o l v e d i n d i e t h y l  e t h e r , washed w i t h s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n and b r i n e , d r i e d (MgSO.) and evaporated 4 graphy ( s i l i c a  t o g i v e a r e d o i l (13.05 g ) •  g e l , 1:1 petroleum  Chromato-'  e t h e r (30°-60°C):diethyl e t h e r e l u a n t )  gave t h e pure aldehyde (71.) (3.81 g, 0.018 mol, 33$ y i e l d , >99$ pure by g . l . c . ) as a c o l o u r l e s s , o i l and a m i x t u r e o f 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 c o u l d be c a r e f u l l y reduced by sodium b o r o h y d r i d e i n t h e next step. V (CCl,); max 4 C=0,  1735 ( s t r o n g , sharp, C=0 e s t e r ) , 1685 ( s t r o n g , sharp,  a, (3-unsaturated a l d e h y d e ) , 1655 (weak, sharp, C=C), 1255 ( s t r o n g ,  broad, C-0-C e s t e r ) . 6 (400MHz, C D C 1 ) ; 3  1.59 ( s , 3H, t e r t i a r y m e t h y l ) , 1.745 ( d , 3H,  a l l y l i c CH_, J , ,. CH -CH -CH=C, J 2  2  1 2  =1.5Hz), 2.02 (,.s, 3H, CH.C0-0-), 1.95  =7.5Hz, J  23  ( d t , 2H,  = 8 H z ) , 2.37 ( q , 2H, C ( 0 A c ) - C H - , J=8Hz), 2  5.18 (dd, 1H, -CH=CHH trans,. J . =12Hz, J =0.75Hz), 5.205 (dd, 1H, cis 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 -CH=C(CHj (CH0), J , =7.5Hz, trans cis 2 — 3 1,2 0  3, =1.5Hz), 9.40 1,3trans  ( s , 1H, -CH0). —  0  Mass spectrum ( l o w - r e s . ) : m / e ( r e l . i n t . ) ; 168 (1%), 150 ( 2 7 $ ) , 135 ( 7 . 5 $ ) , 43 ( 1 0 0 $ ) . Analysis:  ForC ^ g C ^  Calc:  C, 68.55;  H, 8.63.  Found:  C, 68.78;  H, 8.60.  Reduction o f 6-acetoxy-2,6-dimethyl-2,7octadienal (71). (i)  U s i n g d i i s o b u t y l a l u m i n u m h y d r i d e (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 - d r y hexanes under an atmosphere o f d r y argon.  The  s o l u t i o n was c o o l e d t o 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 o f methanol and water was. added-to p r e c i p i t a t e t h e aluminum s a l t s .  The mixture, was f i l t e r e d and the o r g a n i c s o l u t i o n  s e p a r a t e d , d r i e d (MgSO ) , and e v a p o r a t e d t o g i v e a c o l o u r l e s s 4 P u r i f i c a t i o n by chromatography  oil.  ( s i l i c a g e l , d i e t h y l e t h e r e l u a n t ) 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$ yield). 6 (60MHz, C C l , ) ; 4  . 1.33 ( s , 3h, t e r t i a r y m e t h y l ) , 1.66  ( s , 3H, a l l y l i c  m e t h y l ) , 1.5-2.4 (m, 4H> -CIL^-CH^-), 3.2 ( b s , 2H, d i s a p p e a r s w i t h D^O, 2 -OH), — J  gem  4.1' ( s , 2H, -CH -0H), 5.0 (dd, 1H, -CH=CHH t r a n s , J . =12Hz, —2 — cxs o  =1Hz), 5.16 (dd, 1H, -CH=CHH c i s , . J . =18Hz, J =1Hz), 5.36 trans gem  ( b t , 1H, -CH=C(:CH )(CH 0H)), 5.90 (dd, .1H, -CH=CHH, J . =12Hz, 5 2 — cis —  o  o  J, =18Hz). trans ( i i ) U s i n g sodium b o r o h y d r i d e (NaBH ) . 4 6 - A c e t o x y - 2 , 6 - d i m e t h y l - 2 , 7 - o e t a d i e n a l ((71_), 2.718 g, 0.0129 mol) was d i s s o l v e d i n e t h a n o l (95$,. 20 mL) and NaBH .:(0.536 g, 0.014 4 mol) added.  The m i x t u r e was s t i r r e d , a t i c e - b a t h temperature f o r 2  hours,-' a f t e r which time d i l u t e h y d r o c h l o r i c a c i d (20$ v/v) was added  - 1U u n t i l t h e s o l u t i o n remained a c i d i c .  The m i x t u r e was e x t r a c t e d w i t h  e t h y l a c e t a t e and t h e combined o r g a n i c phases were d r i e d (MgSO^). Evaporation  o f t h e s o l v e n t gave t h e 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 p e t r o l e u m e t h e r (30 -60 C ) : d i e t h y l e t h e r e l u a n t ) t o g i v e t h e pure a l c o h o l (2.334 g» 0.011 mol, 85% yield). V  max  (CC1,); 4  3640 (weak, sharp, OH), 3450 (weak, b r o a d , OH), 1745  ( s t r o n g , sharp, C=0 e s t e r ) , 1655 (weak, sharp, C=C), 1245 ( s t r o n g , b r o a d , C-O-C) . 6 (80MHz, C D C 1 ) ;  1.25 ( b s , 1H, d i s a p p e a r s  3  t e r t i a r y methyl),  1 .675  w i t h T)£, OH), 1.55 ( s , 3H,  ( s , 3H, a l l y l i c m e t h y l ) ,  2.025 ( 2 , 3H, CH_ -C0-0), 3  1 .75-2.25 (m, 4H, C(0Ac)-CH_ -CH -CH=), 4.O (s,. 2H, -CHg-OH) , 5.113 ( d , 2  1H,  -CH=CHH t r a n s , J . = U H z ) , 5.163 ( d , 1H, -CH=CHH c i s , J , =16HZ), cis trans  5.40 J  2  ( b t , 1H, -CH=C(CH,)(CH OH)),  6.0 (dd, 1H, -CH=CHH, J  =16HZ,  . =UHz). CIS  Mass spectrum (low r e s . ) : m/e ( r e l . i n t . ) ; 2 1 2 . ( 1 . 2 % ) , 195 ( 6 % ) , 170 ( 1 . 3 % ) , 152 ( 1 4 % ) , 134(28%), 121 ( 6 5 % ) , 71 ( 1 0 0 % ) . Analysis:  ForC  1 2  H  2 Q  0  3  Calc.r  C, 67.89;  Found:  C,. 67.64;  Preparation o f 8 - t o s y l o x y l i n a l o y l acetate  H, 9.50. H, 9.60.  (75).  8 - H y d r o x y l i n a l o y l a c e t a t e ( ( 6 9 ) , 0.05 g, 0.0024 mol) was d i s s o l v e d i n d r y p y r i d i n e (15 mL) and c o o l e d . t o  0°C. F r e s h l y p u r i f i e d  _p_- t o l u e n e s u l p h o n y l 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 m i x t u r e 24 h o u r s .  The r e a c t i o n m i x t u r e  k e p t a t o r below 0°C f o r  was poured onto i c e and e x t r a c t e d w i t h  d i e t h y l e t h e r and t h e combined o r g a n i c 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 e v a p o r a t e d  to g i v e (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. s p e c t r a showed t h e presence o f a t o s y l - grouping and was c o n s i s t e n t w i t h t h i s o i l . b e i n g f a i r l y pure 8 - t o s y l o x y l i n a l o y l a c e t a t e (75).  A t t e m p t s t o 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 t h e r e f o r e t h e  compound was reduced w i t h o u t f u r t h e r . p u r i f i c a t i o n . Reduction  o f 8 - t o s y l o x y l i n a l o y l a c e t a t e (.75):- u s i n g sodium  borohydride.  8 - T o s y l o x y l i n a l o y l a c e t a t e ((_75), 0.7 g, 0.002 mol) was d i s s o l v e d i n 80$ HMPA/H' 0 (20 mL) and sodium b o r o h y d r i d e 2  added.  (0.6g, 0.016 mol)  The m i x t u r e was heated t o 50°C f o r t h i r t y minutes, and then  c o o l e d and d i l u t e d w i t h w a t e r ,  The mixture.was e x t r a c t e d w i t h d i e t h y l  e t h e r and t h e combined o r g a n i c phases, were washed w i t h water, d r i e d (MgSO,) and evaporated 4  t o g i v e a y e l l o w o i l , . D i s t i l l a t i o n (25 mm Hg)  a f f o r d e d l i n a l o y l a c e t a t e (.(68) , . 0,15 g, 0.00076 mol, 38$ y i e l d ) as a c o l o u r l e s s - 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 a u t h e n t i c sample. •  Attempted c o n v e r s i o n , o f g e r a n i o l . ( 1 4 ) , t o g e r a n y l bromide (79)  1 23  .  G e r a n i o l ((14)» 2 g, 0.013 mol) was d i s s o l v e d i n d r y d i e t h y l e t h e r (25 mL) and c a r b o n . t e t r a b r o m i d e  (8.6 g, 0.026 .mol)  added.  The  r e a c t i o n was c o o l e d d u r i n g t h e a d d i t i o n o f 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 a t 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 o x i d e and t h e s o l i d extracted with d i e t h y l ether.  The combined o r g a n i c l a y e r s were washed  w i t h water, d r i e d (MgSO ) , and evaporated 4 Chromatography ( s i l i c a g e l , petroleum  t o give a pale yellow o i l .  e t h e r (30 -60 C) e l u a n t ) gave  an impure c o l o u r l e s s o i l whose H-n.m.r. spectrum showed t h e presence 1  o f TOP and evidence  o f decomposition  o f t h e g e r a n y l bromide formed t o  - 116 the a l l y l i c isomer (see D i s c u s s i o n , page 98, F i g u r e 6 (60MHz, CC1.);  6.2  (dd, 1H,  -CH=CH , J  8-Hydroxylinaloyl acetate i n dry d i e t h y l e t h e r (20 mL) added.  The  (0.887 mL,  s o l u t i o n was 0.0115 mol)  20 minutes, f i l t e r e d , and 96%  6 (60MHz, CC1  );  g, 0.0115 mol)  and t r i e t h y l a m i n e (2.81  mL,  was  0.02  dissolved mol)  and methane s u l p h o n y l c h l o r i d e  added d r o p w i s e .  The m i x t u r e  was  stirred for  evaporated .to g i v e a p a l e y e l l o w o i l (3.22  y i e l d ) whose s p e c t r a l d a t a was  the m e s y l a t e ( 7 6 ) .  (76).  ((69), 2.43  c o o l e d t o 0°C  was  =15Hz, J . =11Hz).  0  Preparation of 8-mesyloxylinaloyl acetate  80).  g,  c o n s i s t e n t w i t h t h a t expected f o r  The mexylate was  reduced w i t h o u t  2.9 ( s , 3H, CE^O^),  4.46 ( s , 2H,  Attempted r e d u c t i o n of 8 - m e s y l o x y l i n a l o . y l a c e t a t e  further purification. -CHg-OMs).  (76) w i t h NaBD,  4 i n - 8 0 % 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 t o t h a t  d e s c r i b e d f o r the. t o s y l a t e (75)  (page .115)  u s i n g the m e s y l a t e  (2.73  g, 0.0094.mol) i n 80%HMPA/H 0'. (20 mL)  (0.77  g, 0.0188 m o l ) .  2  d i s t i l l e d (25 mm Hg) 0.U  g, 0.0022 mol,  and  sodium  (76)  borodeuteride  Workup as d e s c r i b e d above gave an o i l which t o g i v e pure , . 8 - d e u t e r i o l i n a l o y l a c e t a t e  24% y i e l d ) .  H -n.m.r. .data was  t h a t expected f o r 8 - d e u t e r i o l i n a l o y l a c e t a t e  was  ((62),  consistent with  (62).  Preparation of 8 - d e u t e r i o l i n a l o o l (62). 8-Hydroxylinaloyl acetate i n d r y d i e t h y l e t h e r (20 mL) c o o l e d t o 0°C.  Triethylamine  ((69), 2.97  g, 0.014  mol)  was  under an atmosphere o f d r y argon (2.16  mL,. 1.57  and then methane s u l p h o n y l c h l o r i d e (1.19  mL,  g, 0.0154- mol)  was  dissolved and added  1.764- g, 0.0154- mol)  was  - 117 added d r o p w i s e . the of  The r e a c t i o n was s t i r r e d a t 0°C f o r 20 minutes and  precipitated triethylamine hydrochloride f i l t e r e d o f f . Evaporation the d i e t h y l e t h e r gave t h e crude m e s y l a t e (76)., t h e H-n.m.r.  spectrum (60MHz) o f which showed the presence o f the mesyl g r o u p i n g 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 t o 3 p r o t o n s . (2 p r o t o n s ) i n d i c a t e d the methylene  A s i n g l e t a t 64.46  g r o u p i n g t o which the m e s y l a t e  group was a t t a c h e d s i n c e i t s resonance . w a s - s h i f t e d d o w n f i e l d from i t s p o s i t i o n (64.O) i n t h e s t a r t i n g a l c o h o l . ,  T h i s crude compound was  i m m e d i a t e l y d i s s o l v e d i n d r y THF (20 mL) and a 1M s o l u t i o n o f Superd e u t e r i d e ( 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 e q u i v a l e n t s ) was added o v e r - a p e r i o d o f 15 minutes. The r e a c t i o n m i x t u r e was s t i r r e d f o r k hours a t room temperature and t h e n c o o l e d t o 0°C. Water was added,, c a r e f u l l y and t h e n 3N sodium h y d r o x i d e s o l u t i o n and 30% hydrogen p e r o x i d e s o l u t i o n t o d e s t r o y t h e organoboranes  formed i n the r e a c t i o n .  The m i x t u r e was r e f l u x e d f o r  24 hours and then e x t r a c t e d w i t h d i e t h y l , e t h e r .  The combined o r g a n i c  phases were washed w i t h water., d r i e d (MgSO.), and e v a p o r a t e d t o g i v e an 4 o i l which was seen by g . l . c . t o be mainly... the Column chromatography  d e s i r e d compound.  ( s i l i c a g e l , p e t r o l e u m e t h e r (30 -60 C ) : d i e t h y l  e t h e r 9:1 e l u a n t ) 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 max  (CC1,); 4  3600 (weak, s h a r p , OH), 3500-3300 (weak, b r o a d , OH),  2150, 2180 (weak, s h a r p , CD). 6 (60MHz, C D C 1 ) ; 3  ......  1.27 ( s , 3H, t e r t i a r y m e t h y l ) , 1.58 ( b s , 1H,  d i s a p p e a r s w i t h DgO, OH)-, 1-.-59 - (s,. 3H, a l l y l i c CH ) , 1.65 ( b s , 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). "trcLns  GXS  "Gra,ns  Mass spectrum  (low r e s . ) : m / e ( r e l . i n t . ) ;  155 ( 1 $ ) , 140 (3%), 137  ( 1 3 $ ) , 128 (2%), 122 ( 1 2 $ ) , 121 ( 8 $ ) , 110 ( 6 $ ) , 108 ( 5 $ ) , 9 6 ( 1 2 $ ) , 9 3 ( 5 6 $ ) , 84 ( 3 1 $ ) , 71 (100$). Analysis.  ForC^H  OD  Calc:  C, 77.36;  H, 11.76.  Found:  C, 77.34;  H, 11.76.  P r e p a r a t i o n o f 9-deuteriocamphor. 9-Bromocamphor (3.96 g, 0.0171 mol) and 2 , 2 ' - a z o b i s i s o b u t y r o 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 d r y benzene (20 mL) under a d r y argon atmosphere.  T r i - . n - b u t y l t i n d e u t e r i d e (5.0 g, 0.0171  mol) was added and the m i x t u r e 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 w i t h 5 $ w/v potassium h y d r o x i d e s o l u t i o n , 5$ w/v p o t a s s i u m f l u o r i d e s o l u t i o n , w a t e r , and b r i n e .  Removal o f d r i e d s o l v e n t  gave a y e l l o w s e m i - s o l i d which was shown by g . l . c . e x a m i n a t i o n t o c o n s i s t o f camphor and AIBN.  P e t r o l e u m e t h e r (30 -60 C) was added  and the m i x t u r e c h i l l e d t o p r e c i p i t a t e . AIBN.  The petroleum e t h e r  (30 -60 C) was decanted and .evaporated t o g i v e a l e s s m o b i l e o i l . U  The - p r e c i p i t a t i o n ; " of' AIBN. i n t h i s f a s h i o n was repeated- t w i c e more and t h e r e s u l t i n g s o l i d was then sublimed (60°C, a t m o s p h e r i c p r e s s u r e ) t o g i v e 9 - d e u t e r i o c a m p h o r (1.96 g, 0.0128 mol, 75$ y i e l d ) which was shown by g . l . c . e x a m i n a t i o n t o c o n t a i n 5$ AIBN.  F u r t h e r p u r i f i c a t i o n was  not attempted. V 1  max  (CCl,); 4  2180 (weak, s h a r p , CD-), • 17-4'5 ( s t r o n g , s h a r p , C=0) .  H-n.m.r., 6 (400MHz, C D C l ^ ;  10-CH  0.836 ( s , 3H, 8-CH_ ), 0.913 ( s , 3H, 3  ) , 0.942 ( t , 2H, 9-CH D, J=2Hz).  Mass spectrum  2  (low r e s . ) : m/e ( r e l . i n t . ) ;  153 ( 5 7 $ ) , 138 ( 8 $ ) , 111  ( 2 8 $ ) , 110 ( 3 6 $ ) , 109 ( 8 9 $ ) , 96 (100$), 95 ( 5 7 $ ) , 94 ( 2 3 $ ) , 84 (42$), 82 ( 1 7 $ ) , 81 (94$).  - 119 2  H-n.m.r., 6 (12.3 MHz,  CC1,);  1.159  ( t , J=2Hz).  P r e p a r a t i o n - o f 8-deuteriocamphor. 8-Deuteriocamphor was p r e p a r e d i n an analogous manner t o 9-deuteriocamphor, u s i n g 8-bromocamphor ( 3 . 9 6  g, 0.0171 m o l ) , AIBN  (2.8 g, 0.0171 mol) and t r i - n - b u t y l t i n d e u t e r i d e (5.0 g, 0.0171 m o l ) . Pure 8-deuteriocamphor was obtained, b y s u b l i m a t i o n o f t h e 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 o f AIBN, giving--8-deuteriocamphor ( 1 . 9 5 mol,. 7 5 % - y i e l d ) - .  g, 0.0128  G . l . c . examination' showed t h i s t o c o n t a i n 3% AIBN.  F u r t h e r p u r i f i c a t i o n was n o t a t t e m p t e d . V  (CC1.); ma.x  1  2180 (weak, s h a r p , CD),. 174-5 ( s t r o n g , s h a r p , C=0) .  i\,  H-n.m.r., 6 (4-OOMHz, C D C l ^ ;  0.826 ( t , 2E, 8-CH D . J=2Hz), 2  ( s , 3H, 10-CH ), 0.945 ( s , 3H,  0.912  .  3  Mass spectrum ( l o w r e s . ) : m/e  f  (rel. int.);  153 ( 5 5 % ) , 138 ( 7 % ) , 111  ( 2 8 % ) , 110 ( 3 0 % ) , 109 (74%)-, 96 (100%)-,- 95 (-32-%)-, 94 ( 1 0 % ) , 84 ( 3 7 % ) , 82 ( 1 6 % ) , 81  (50%).  ^H-n.m.r., 6 (12.3MHz, CC1.); 4  1.000  ( t , J=2Hz).  2 E f f e c t o f a s h i f t r e a g e n t on t h e -H-n,.m.r.. o f 8-. and-9- deuteriocamphor. 8-Deuteriocamphor  (50-mg) o r 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. t u b e . The 2 - H-n.m.r.. spectrum were - r e c o r d e d and t h e n a weighed a l i q u o t o f Resolve-Al (Aldrieh,'Eu(thd)y dienone) was added and the  thd = 2,2,6,6-tetramethyl-3,5-hepta-  H-n.m.r... s p e c t r a r e c o r d e d a g a i n .  The  s h i f t o f the s i g n a l w i t h r e s p e c t t o ..the amount o f s h i f t r e a g e n t added was n o t e d and the procedure r e p e a t e d .  Deuteriochloroform  (CDCl^)  was added t o the n.m.r. s o l u t i o n as an i n t e r n a l s t a n d a r d (7.510 and s i g n a l p o s i t i o n s were a d j u s t e d a c c o r d i n g l y .  ppm.)  - 120 N.m.r. s p e c t r a .  Amount o f  M o l . o f S.R.  M o l . S.R./  Signal  S..R..* added  added x 1 0  mol camphor.  position  6  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  0.00  1.159 1.317  9- Deuteriocamphor. 1  0,00  0.00  2  0.01791  25.00  0.0765  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 r e a g e n t ( R e s o l v e - A l ) .  S c r e e n i n g o f p l a n t s , f o r camphor. Samples o f 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 c u t from two p l a n t s a t t h e U.B.C. B o t a n i c a l Gardens on a sunny June a f t e r n o o n . 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 c r u s h e d . 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 a p p r o x i m a t e l y  750 mL o f d i s t i l l a t e were c o l l e c t e d .  T h i s aqueous s o l u t i o n was  e x t r a c t e d w i t h p e t r o l e u m e t h e r (30°-60°C) and then w i t h d i e t h y l e t h e r , these e x t r a c t s were examined s e p a r a t e l y .  G . l . c . examination  l e d to  the c o n c l u s i o n t h a t camphor was p r e s e n t i n t h e p e t r o l e u m e t h e r (30 e x t r a c t from b o t h plants..  F u r t h e r examination  -60  C)  showed t h e e x t r a c t from  S a n t o l i n a chamaecyparissus t o c o n t a i n l e s s than 5$ camphor whereas t h a t from Rosemarinus o f f i c i a n a l i s c o n t a i n e d a p p r o x i m a t e l y  20% camphor.  Chromatography ( s i l i c a g e l , g r a d i e n t e l u t i o n from pentane t o p e n t a n t : d i e t h y l e t h e r 9:1) a f f o r d e d a w h i t e s o l i d ( a p p r o x i m a t e l y examination  showing i t t o be 95% p u r e .  20 mg), g . l . c .  H-N.m.r. and i . r . s p e c t r a l  d a t a was c o n s i s t e n t w i t h t h i s sample b e i n g camphor. V max  (CCl,);. 1745 ( s t r o n g , sharp, C=0). 4  6 (80MHz, CDC1 ) ; O.84  ( s , 3H, 8-CH ) , 0.92 ( s , 3H, 10-CH  ) , 0.97  ( s , 3H, 9-CH ), 1.1-2.7 (m, 7H). 3  Mass spectrum ( l o w r e s . ) ; m/e ( r e l , i n t . ) ; 152 ( 3 0 $ ) , 137 (5%),  108  ( 5 0 $ ) , 95 (100$). F e e d i n g e x p e r i m e n t s u s i n g Rosemarinus o f f i c i a n a l i s . Experiment 1. The p l a n t s were c u t on J u l y 28th,., 1982 ( r a i n i n g ) from two shrubs a t t h e U n i v e r s i t y o f B r i t i s h Columbia B o t a n i c a l Gardens and s t o r e d under w a t e r .  A s o l u t i o n o f [ 2 - Hg]-mevalonic a c i d ( ( 6 1 ) ,  50 mg) was made by s t i r r i n g t h e l a c t o n e (61 a) w i t h 1 e q u i v a l e n t o f sodium b i c a r b o n a t e i n water f o r 1 hour a t room t e m p e r a t u r e .  [8- H^]2  L i n a l o o l ( ( 6 2 ) , 50 mg) was s o l u b i l i z e d u s i n g a 1$ 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 p l a c e d i n t o t h e l a b e l l e d s o l u t i o n  and m a i n t a i n e d  f o r 3 days on w a t e r .  The p l a n t m a t e r i a l was then f r o z e n  - 122 with l i q u i d  n i t r o g e n , c r u s h e d , and steam d i s t i l l e d .  Chromatography  of t h e e s s e n t i a l o i l o b t a i n e d by pentane e x t r a c t i o n o f t h e 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 d a t a were i d e n t i c a l w i t h an a u t h e n t i c by ^H-n.m.r. showed no  sample.  Analysis  t o be p r e s e n t i n t h e camphor from e i t h e r  f e e d i n g : e x p e r i m e n t and so t h e work was r e p e a t e d . Experiment 2. The p l a n t m a t e r i a l was c u t on September 7 t h . , 1982 ( o v e r c a s t ) , as f o r experiment  1. S o l u t i o n s o f l a b e l l e d m a t e r i a l were made up a s  b e f o r e , u s i n g a 0.1 mg/mL ATP s o l u t i o n i n s t e a d o f water.  The p l a n t  m a t e r i a l was p l a c e d ' i n t h e s o l u t i o n o f l a b e l l e d m a t e r i a l and m a i n t a i n e d on a 0.1 mg/mL ATP s o l u t i o n f o r 3 days..  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