UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Topics in natural product synthesis and biosynthesis Forrester, James McLeod 1972

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1972_A1 F67.pdf [ 6.52MB ]
Metadata
JSON: 831-1.0060119.json
JSON-LD: 831-1.0060119-ld.json
RDF/XML (Pretty): 831-1.0060119-rdf.xml
RDF/JSON: 831-1.0060119-rdf.json
Turtle: 831-1.0060119-turtle.txt
N-Triples: 831-1.0060119-rdf-ntriples.txt
Original Record: 831-1.0060119-source.json
Full Text
831-1.0060119-fulltext.txt
Citation
831-1.0060119.ris

Full Text

TOPICS IN NATURAL PRODUCT SYNTHESIS AND BIOSYNTHESIS by JAMES McLEOD FORRESTER B . S c , U n i v e r s i t y o f E d i n b u r g h , 1967 M . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the•Department o f C h e m i s t r y We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 1972 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Br i t ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Cheml8try  The University of Brit ish Columbia Vancouver 8, Canada Date September 30 . 1972 i i ABSTRACT In Part I of t h i s t h e s i s i s described the syntheses of r a d i o l a b e l l e d -bisabolene, monocyclofarnesol and farnesol-10 ,11-epoxide, and the e v a l u a t i o n of t h e i r r o l e as precursors of the mould metabolite t r i c h o t h e c i n . 15-Bisabolene was prepared by dehydration of r a d i o l a b e l l e d C<-bisabolol, which i t s e l f was prepared by the a c t i o n of "^C-methyl magnesium i o d i d e on 2-methyl-6-keto-6-(4'-methylcyclohex-3'-enyl)-hex-2-ene. This ketone was prepared i n a modified Grignard r e a c t i o n from 5-bromo-2-methyl-pent-2-ene, (obtained from c y c l o p r o p y l methyl ketone by treatment w i t h methyl magnesium io d i d e followed by r e a c t i o n of the i n t e r -mediate 2-cyclopropylpropan-2-ol w i t h 48% hydrogen bromide) and 4-methylcyclohex-3-ene c a r b o x y l i c a c i d (prepared i n a D i e l s A l d e r r e a c t i o n between a c r y l i c a c i d , or i t s methyl e s t e r , and isop r e n e ) . Monocyclofarnesol was prepared from ^§-ionone by re d u c t i o n of the c e n t r a l double bond to dihydro-^S -ionone followed by a modified W i t t i g r e a c t i o n w i t h r a d i o l a b e l l e d t r i m e t h y l phosphono-14 acetate, prepared from t r i m e t h y l phosphite and 1- C-methyl bromoacetate. Farnesol-10,11-epoxide was prepared from g e r a n i o l by formation of geranyl bromide which was condensed w i t h e t h y l acetoacetate. H y d r o l y s i s and dec a r b o x y l a t i o n then gave geranyl acetone which was tr e a t e d i n the same way as dihydro- -ionone to give 1 ^ C - l a b e l l e d f a r n e s o l . Treatment of f a r n e s o l w i t h N-bromosuccinimide i n aqueous glyme followed by base c a t a l y s e d 14 r i n g c l o s u r e gave C - l a b e l l e d farnesol-10,11-epoxide. i i i The mechanisms whereby these compounds c o u l d be c o n v e r t e d t o t r i c h o t h e c i n are d e s c r i b e d and the r e l e v a n c e , t o contemporary r e p o r t s , o f t h e i r n o n - i n c o r p o r a t i o n i n t o t r i c h o t h e c i n i s d i s c u s s e d . I n P a r t I I i s d e s c r i b e d an attempted s y n t h e s i s o f <K-cuparenone from 5 - a c e t o x y - 6 - m e t h y l - 2 - p - t o l y l h e p t a - l , 5 - d i e n e . R e a c t i o n o f p - t o l u a l d e h y d e w i t h 5-bromo-2-methylpent-2-ene gave 6-keto-2-m e t h y l - 6 - p _ - t o l y l h e x - 2 - e n e . P r o t e c t i o n o f the ketone as a k e t a l f o l l o w e d by h y d r o b o r a t i o n of the double bond and a c e t y l a t i o n o f the r e s u l t i n g a l c o h o l , gave, a f t e r d e k e t a l i s a t i o n and W i t t i g r e a c t i o n , 5 - a c e t o x y - 6 - m e t h y l - 2 - p - t o l y l h e p t -1-ene. H y d r o l y s i s o f the a c e t a t e and o x i d a t i o n gave 6 - m e t h y l - 2 - p _ - t o l y l h e p t - l - e n e 5-one which gave 5 - a c e t o x y - 6 - m e t h y l - 2 - p _ - t o l y l h e p t a - l ,5-diene on t r e a t m e n t w i t h i s o p r o p e n y l a c e t a t e and p _ - t o l u e n e s u l p f i o n i c a c i d . T h i s e n o l a c e t a t e c o u l d not be c o n v e r t e d to c<^cuparenone. I n P a r t I I I i s d e s c r i b e d the p h o t o c h e m i s t r y o f t r i a c e t i c a c i d l a c t o n e m e t h y l e t h e r . P h o t o l y s i s o f t h i s compound i n methanol s o l u t i o n gave 6,6-dimethoxy-4-methyl-2-pyrone w h i c h on t r e a t m e n t w i t h water gave c i s -|3 - c a r b o m e t h y o x y m e t h y l c r o t o n i c a c i d . The mechanism f o r t h i s p r o c e s s i s d i s c u s s e d . i v ABSTRACT TABLE OF CONTENTS LIST OF FIGURES ACKNOWLEDGEMENTS INTRODUCTION PART I INTRODUCTION DISCUSSION EXPERIMENTAL BIBLIOGRAPHY PART I I INTRODUCTION DISCUSSION EXPERIMENTAL BIBLIOGRAPHY PART I I I INTRODUCTION DISCUSSION EXPERIMENTAL BIBLIOGRAPHY TABLE OF CONTENTS Page i i i v v v i i 1 21 34 76 95 100 105 124 142 144 153 157 160 V LIST OF FIGURES F i g u r e Page INTRODUCTION I I I I I I IV V VI V I I V I I I IX XI X I I X I I I XIV XV XVI XVII Secondary M e t a b o l i s m 4 The i m p o r t a n t r o l e o f a c e t y l co-enzyme A 5 B i o s y n t h e t i c r o u t e s t o m e v a l o n i c a c i d 7 F o r m a t i o n o f 3-methyl 2 - b u t e n y l 10 pyrophosphate F o r m a t i o n o f g e r a n y l and f a r n e s y l p y r o - 10 phosphate S t e r e o c h e m i s t r y o f the c o n d e n s a t i o n o f Ct. u n i t s 13 S h i k i m i c a c i d pathway t o a r o m a t i c 1.7 compounds A c y l - p o l y m a l o n a t e r o u t e to a r o m a t i c 18 compounds PART I L o c a t i o n o f l a b e l s i n "o*-bisabolene from 2 - 1 4 c - m e v a l o n i c a c i d 25 C h a i r type f o l d i n g o f o'-bisabolene 25 a c c o r d i n g to R u z i c k a Boat type f o l d i n g of V j-bisabolene 26 A l t e r n a t i v e c h a i r type f o l d i n g o f 27 X-bisabolene L a b e l l i n g p a t t e r n i n t r i c h o t h e c i n from 28 isomer (45a) o f Y - b i s a b o l e n e L a b e l l i n g p a t t e r n i n t r i c h o t h e c i n from 28 isomer (45b) o f ^ - b i s a b o l e n e P o s i t i o n o f l a b e l i n t r i c h o t h e c i n 32 a c c o r d i n g t o Hanson and co-workers A l t e r n a t i v e s y n t h e s i s o f ketone (54) 39 C o n f i r m a t i o n o f s t r u c t u r e o f a c i d (55) 43 v i X v T I I NMR v a l u e s f o r g e r a n i o l and n e r o l 67 XIX I n f r a r e d spectrum of t r i c h o t h e c i n 72 XX N u c l e a r magnetic resonance spectrum o f t r i c h o t h e c i n 73 XXI I n f r a r e d spectrum o f t r i c h o t h e c o l o n e 74 XXII N u c l e a r magnetic resonance spectrum 75 of t r i c h o t h e c o l o n e PART I I I P r e v i o u s s y n t h e s i s o f ( X -cuparenone 104 PART I I I I A c y l - p o l y m a l o n a t e r o u t e t o a r o m a t i c 146 compounds I I P h o t o c h e m i s t r y o f t r i a c e t i c a c i d l a c t o n e 151 I I I P h o t o c h e m i s t r y of methyl t r i a c e t i c 156 a c i d l a c t o n e v i i ACKNOWLEDGEMENTS I would l i k e to e x p r e s s my g r a t i t u d e t o Dr. T. Money f o r h i s i n s p i r a t i o n , guidance and many h e l p f u l s u g g e s t i o n s made d u r i n g the c o u r s e o f t h i s work and d u r i n g the p r e p a r a t i o n o f the t h e s i s . I would a l s o l i k e t o thank Dr. D. F. MacSweeney and Mr. G. L. Hodgson f o r many i n t e r e s t i n g d i s c u s s i o n s and h e l p f u l s u g g e s t i o n s . Thanks are a l s o due t o Dr. P. S a l i s b u r y f o r the c a p a b l e c u l t i v a t i o n o f the c u l t u r e s o f T r i c h o t h e c i u m roseum. I am g r a t e f u l t o the U n i v e r s i t y of B r i t i s h Columbia f o r f i n a n c i a l a s s i s t a n c e d u r i n g the c o u r s e o f t h i s work. INTRODUCTION O r g a n i c c h e m i s t s have always been i n t e r e s t e d i n n a t u r a l l y o c c u r r i n g compounds, w h i c h may be c l a s s i f i e d i n t o two b r o a d c a t e g o r i e s p r i m a r y and secondary m e t a b o l i t e s . P r i m a r y m e t a b o l i t e s are compounds which are found u n i v e r s a l l y i n almost a l l l i v i n g organisms and p e r f o r m e s s e n t i a l l y i d e n t i c a l f u n c t i o n s i n those organisms. Examples are p r o t e i n s and n u c l e i c a c i d s . These compounds have p r i m a r i l y been the p r e s e r v e o f b i o c h e m i s t s . Secondary m e t a b o l i t e s are more the p r e s e r v e o f the o r g a n i c c h e m i s t who gave them the name " n a t u r a l p r o d u c t s " . They do not have the same b r o a d spectrum o f o c c u r r e n c e as the p r i m a r y m e t a b o l i t e s , nor do the m a j o r i t y o f t h e s e compounds have any known b i o l o g i c a l f u n c t i o n , though some have b i o l o g i c a l p r o p e r t i e s w h i c h may be e x h i b i t e d e n t i r e l y w i t h i n one i n d i v i d u a l o r ganism, (e.g. hormones), e x h i b i t e d between two o r more i n d i v i d -u a l s o f the same s p e c i e s , (e.g. pheromones), o r e x h i b i t e d between two e n t i r e l y d i f f e r e n t o r g a n i s m s , (e.g. a n t i b i o t i c s , p o i s o n s , e t c . ) . Three f a c e t s o f n a t u r a l p r o d u c t c h e m i s t r y have p a r t i c u l a r l y f a s c i n a t e d o r g a n i c c h e m i s t s , namely the s t r u c t u r e s o f the compounds, s y n t h e s e s o f the compounds, and how the compounds are b u i l t up i n the l i v i n g c e l l . A f o u r t h , and more r e c e n t f a c e t o f n a t u r a l p r o d u c t c h e m i s t r y i s s o - c a l l e d b i o g e n e t i c type s y n t h e s i s , though . the a c c u r a c y o f the term " r e c e n t " may be judged by a q u o t a t i o n from a paper p u b l i s h e d by J..N. C o l l i e i n 1893: "The attempt to a r t i f i c i a l l y produce n a t u r a l l y o c c u r r i n g s u b s t a n c e s , and t o i m i t a t e i n the l a b o r a t o r y some o f the many p r o c e s s e s w h i c h are p e r p e t u a l l y b e i n g c a r r i e d on around us i n n a t u r e , has always been - 2 -1 one o f the c h i e f aims o f the o r g a n i c c h e m i s t " . The e l u c i d a t i o n o f the s t r u c t u r e o f a n a t u r a l p r o d u c t i s the problem f i r s t posed when a new compound i s i s o l a t e d . T h i s f a l l s i n t o f o u r p a r t s : d e t e r m i n a t i o n o f the f u n c t i o n a l groups p r e s e n t ; d e t e r m i n a t i o n o f the ca r b o n s k e l e t o n and the p o s i t i o n s o f the f u n c t i o n a l groups; d e t e r m i n a t i o n o f the s t e r e o c h e m i s t r y ; and f i n a l l y a t o t a l s y n t h e s i s to c o n f i r m the s t r u c t u r e . The c l a s s i c a l method o f p e r f o r m i n g the s t r u c t u r a l e l u c i d a t i o n was by c h e m i c a l d e g r a d a t i o n , f o l l o w e d by s y n t h e s i s o f the s m a l l e r fragments so o b t a i n e d . The modern method i s t o use p h y s i c a l methods such as i n f r a r e d , u l t r a v i o l e t , n u c l e a r m agnetic r e s o n a n c e , and mass s p e c t r o s c o p y , though even those modern methods are b e i n g r e n d e r e d o b s o l e t e by the more w i d e s p r e a d use o f X-ray c r y s t a l l o g r a p h y . Once the s t r u c t u r e s o f a l a r g e number o f n a t u r a l p r o d u c t s had been e s t a b l i s h e d i t became apparent t h a t n a t u r a l p r o d u c t s c o u l d be c l a s s i f i e d a c c o r d i n g t o s t r u c t u r a l s i m i l a r i t i e s w h i c h were thought t o be a consequence o f a s i m i l a r b i o s y n t h e t i c o r i g i n . 1,2 Thus C o l l i e f i r s t s u g g e s t e d t h a t c e r t a i n p h e n o l i c compounds c o u l d be d e r i v e d from h e a d - t o - t a i l c o n d e n s a t i o n s o f a c e t i c a c i d u n i t s , t o g i v e l i n e a r poly-^S-diketones w h i c h c o u l d c y c l i s e t o g i v e p h e n o l i c compounds. T h i s h y p o t h e s i s has s i n c e been extended by 3 B i r c h and Donovan. 4 I n the i s o p r e n o i d f i e l d s t r u c t u r a l s i m i l a r i t i e s l e d R u z i c k a to p o s t u l a t e h i s famous " I s o p r e n e R u l e " w h i c h s t a t e s t h a t i s o -p r e n o i d s are d e r i v e d by h e a d - t o - t a i l c o n d e n s a t i o n o f i s o p r e n e (1) u n i t s . - 3 -C D I n the a l k a l o i d f i e l d i t was r e a l i s e d e a r l y i n t h i s c e n t u r y t h a t , f o r example, d i h y d r o x y p h e n y l a l a n i n e (2) was p r o b a b l y i n v o l v e d i n the b i o s y n t h e s i s o f c e r t a i n i s o q u i n o l i n e a l k a l o i d s , e.g. l a u d a n o s i n e ( 3 ) . (3) (2) P r a c t i c a l l y the whole v a s t a r r a y o f n a t u r a l p r o d u c t s a r i s e s from r e l a t i v e l y s i m p l e s t a r t i n g m a t e r i a l s by way o f a few r e a c t i o n t y p e s . N a t u r a l p r o d u c t s are the p r o d u c t s o f secondary m e t a b o l i s m w h i c h may be shown s c h e m a t i c a l l y i n F i g u r e I . The c a r b o h y d r a t e s w h i c h are i m p o r t a n t i n t h i s pathway are monosaccharides which are - 4 -f i r s t converted to p y r u v i c a c i d (4) and then to a c e t y l co-enzyme A ( 5 ) . C 0 2 + H 2 0 h-o c h l o r o p h y l l Carbohydrates v i a e n z y m a t i c a l l y Complex n a t u r a l c o n t r o l l e d products of ^r -every type - re a c t i o n s Carbohydrate Metabolism V Pool of simple organic compounds Figure I: Secondary Metabolism CH. 0 II C C 0 2 H (4) 6,7 A c e t y l co-enzyme A i s probably the most important i n t e r -mediate i n the b i o s y n t h e s i s of n a t u r a l products as i t i s a common intermediate on the pathway to a l l three of the main groups of n a t u r a l products, the i s o p r e n o i d s , the a l k a l o i d s and the aromatic compounds. The important r o l e of a c e t y l co-enzyme A (5 ) i s shown s c h e m a t i c a l l y i n Figure I I . The b i o s y n t h e s i s of terpenes has r e c e n t l y been reviewed and at present i s thought to proceed as f o l l o w s . The i n i t i a l b iochemical r e a c t i o n i n terpenoid b i o s y n t h e s i s i s the enzymic re d u c t i o n of (S)-3-hydroxy-3-methylg]utaryl co-enzyme A (6) to produce (R)-mevalonic a c i d (7). the reduction o c c u r r i n g by two hydrogen t r a n s f e r s from reduced nicotinamide-adenine d i n u c l e o t i d e phosphate (8a) (NADPH). The o x i d i s e d form of NADPH i s NADP+ C8b) - 5 -L i n e a r c o n d e n s a t i o n F a t t y a c i d s and A r o m a t i c compounds, e.g. A n t h r a q u i n o n e s , T e t r a c y c l i n e s e t c . A c e t y l Co-Enzyme A Amino A c i d s Krebs c y c l e A l k a l o i d s Branched c o n d e n s a t i o n M e v a l o n i c A c i d I s o p r e n o i d s F i g u r e I I : The i m p o r t a n t r o l e o£ a c e t y l co-enzyme A fj OH CH, 0 0 W I I 3 II U CH 3C-S-CH 2CH 2NHCO(CH 2) NHCOCH-C-CH2-0-P-0-P-OH CH. OH (5) HO — P As f a r as i s known at p r e s e n t , t h i s i s the o n l y method by whic h m e v a l o n i c a c i d i s produced and m e v a l o n i c a c i d i s used o n l y i n t e r p e n o i d b i o s y n t h e s i s . E s t e r ( 6 ) , however, i s a p r o d u c t o f a number o f pathways. I t can be produced by enzymic h y d r a t i o n o f g l u t a c o n i c e s t e r (9) which i s a p r o d u c t o f b i o t i n dependent - 6 -H H c a r b o x y l a t i o n o f 3 - m e t h y l c r o t o n y l co-enzyme A (.10) > a d e g r a d a t i o n p r o d u c t o f l e u c i n e . A b i o l o g i c a l l y more i m p o r t a n t pathway t o e s t e r (6) i s the c o n d e n s a t i o n of a c e t y l co-enzyme A (5) w i t h g a c e t o a c e t y l co-enzyme A ( 1 1 ) . A c e t o a c e t y l co-enzyme A (11) can be s y n t h e s i s e d e i t h e r by c o n d e n s a t i o n o f two m o l e c u l e s of a c e t y l co-enzyme A (5) o r by d e g r a d a t i o n o f f a t t y a c i d s . F i g u r e I I I summarises the s y n t h e s i s of m e v a l o n i c a c i d ( 7 ) . Thus m e v a l o n i c a c i d can be s y n t h e s i s e d from t h r e e m o l e c u l e s o f a c e t i c a c i d s i n c e t h e r e e x i s t s an enzyme f o r e s t e r i f y i n g a c e t i c - 7 -C a r b o h y d r a t e F a t • P r o t e i n (7) F i g u r e I I I : B i o s y n t h e t i c r o u t e s t o m e v a l o n i c a c i d a c i d w i t h co-enzyme A. M e v a l o n i c a c i d has been used more than any o t h e r p r e c u r s o r i n t e r p e n e b i o s y n t h e t i c s t u d i e s because i t i s c h e m i c a l l y s t a b l e , can be s y n t h e s i s e d i n a t l e a s t e l e v e n d i f f e r e n t ways depending on the l a b e l l i n g r e q u i r e m e n t s , and most i m p o r t a n t l y , i s n o t known to be u t i l i s e d f o r a n y t h i n g o t h e r t h a n t e r p e n e b i o s y n t h e s i s . Only the (R)-£orm of m e v a l o n i c a c i d i s b i o l o g i c a l l y 9 a c t i v e , the ( S ) - f o r m b e i n g m e t a b o l i c a l l y i n e r t . T h i s i s f o r t u n a t e s i n c e s y n t h e t i c m e v a l o n i c a c i d i s u s u a l l y r a c e m i c and i s d i f f i c u l t to r e s o l v e . - 8 -The n e x t two s t e p s i n the b i o s y n t h e s i s of t e r p e n o i d s are b o t h p h o s p h o r y l a t i o n s , w h i c h produce m e v a l o n i c a c i d - 5 - p h o s p h a t e (12) and m e v a l o n i c a c i d - 5 - p y r o p h o s p h a t e ( 1 3 ) . The phosphate group donated i n each r e a c t i o n i s the t e r m i n a l phosphate group o f adenosine t r i p h o s p h a t e (14) (ATP). The enzymes r e s p o n s i b l e , e s p e c i a l l y the one p r o d u c i n g the monophosphate, which i s the one w h i c h o n l y u t i l i s e s ( R ) - m e v a l o n i c a c i d , " ^ ' " ^ have been p u r i f i e d 10,12 , . 13,14 from y e a s t and from l i v e r . (7) (12) ATP ADP CH (13) NH (14) OH OH - 9 -The next s t e p i n t e r p e n o i d b i o s y n t h e s i s i s a c t u a l l y a d e g r a d a t i o n . M e v a l o n i c a c i d - 5 - p y r o p h o s p h a t e (13) r e a c t s ' enzymat-i c a l l y w i t h adenosine t r i p h o s p h a t e (14) to g i v e adenosine d i p h o s p h a t e (ADP), i n o r g a n i c phosphate, carbon d i o x i d e and 3-methyl-12 3 - b u t e n y l p y r o p h o s p h a t e (15) (synonym i s o p e n t e n y l pyrophosphate) . The oxygen o f the t e r t i a r y h y d r o x y l group ends up i n the i n o r g a n i c p h o s p h a t e , 1 5 s u g g e s t i n g t h a t t h i s h y d r o x y l group i s f i r s t p h o s p h o r y l a t e d , but no i n t e r m e d i a t e phosphate has been i s o l a t e d . The double bond must be formed by a c o n c e r t e d e l i m i n a t i o n , and n o t by a d e h y d r a t i o n f o l l o w e d by d e c a r b o x y l a t i o n , because no hydrogen from the aqueous medium appears i n the p r o d u c t . The p r o c e s s i s a t r a n s e l i m i n a t i o n " ^ and t h i s type o f r e a c t i o n has no t been found e l s e w h e r e i n enzyme c h e m i s t r y , though i t i s analogous t o some o r g a n i c r e a c t i o n s , e.g. the e l i m i n a t i o n o f carb o n d i o x i d e and h a l i d e i o n from the s a l t s of 3 - h a l o g e n o p r o p i o n i c a c i d s . 3 - M e t h y l - 3 - b u t e n y l pyrophosphate (15) i s n e x t c o n v e r t e d i n t o 3 - m e t h y l - 2 - b u t e n y l pyrophosphate (16) (synonym 3 , 3 - d i m e t h y l a l l y l p y r ophosphate) v i a a p r o t o t r o p i c s h i f t . T h i s i s one o f the few r e v e r s i b l e r e a c t i o n s i n t e r p e n o i d b i o s y n t h e s i s and s t u d i e s w i t h 17 18 p a r t i a l l y p u r i f i e d enzyme from y e a s t or l i v e r show ' t h a t the e q u i l i b r i u m l i e s r a t h e r h e a v i l y towards ( 1 6 ) . The e l i m i n a t i o n o f the p r o t o n on g o i n g from (15) t o (16) i s s t e r e o s p e c i f i c , the one e l i m i n a t e d b e i n g Ha as shown i n F i g u r e IV. The c h e m i c a l s i g n i f i c a n c e o f the change (15)-*(16) i s t h a t a s u b s t a n c e w i t h a r e l a t i v e l y u n r e a c t i v e p h o s p h o r y l group and a n u c l e o p h i l i c double bond, i s c o n v e r t e d i n t o a h i g h l y r e a c t i v e - 10 -ATP ADP CH-OH HO CH2OP206H3 He He Hd Ha Hb (13) P i CO. CH OP n H, 2 2 6 3 Ha Hb (15) H H; CH. CH.OP.OJ-L 2 2 o 3 Hb (16) F i g u r e IV: F o r m a t i o n o f 3-methyl-2-butenyl Pyrophosphate OP 0 H 2 6 3 (16) H+ OP?0,H 2U6N3 (15) O.P206H3 cm H+^SJ OP^O.H H H 2 6 3 P2°6H3 (18) F i g u r e V: F o r m a t i o n of g e r a n y l and f a r n e s y l pyrophosphates - 11 -e l e c t r o p h i l i c a l l y l p y r o p h o s p h a t e . The i n t e r m e d i a t e s (15) and (16) are n e x t j o i n e d t o g e t h e r , as shown i n F i g u r e V, by an enzyme c a l l e d a p r e n y l t r a n s f e r a s e . T h i s r e a c t i o n resembles a p o l y m e r i s a t i o n i n t h a t (16) can be c o n s i d e r e d as the i n i t i a t i n g s p e c i e s and (15) as the p r o p a g a t i n g s p e c i e s . The two m o l e c u l e s are combined w i t h l o s s o f pyrophosphate i o n from one and a hydrogen i o n from the o t h e r to form g e r a n y l p yrophosphate ( 1 7 ) . T h i s can now r e p l a c e (16) i n a f u r t h e r , a n a l a g o u s , r e a c t i o n w i t h (15) to g i v e f a r n e s y l pyrophosphate ( 1 8 ) . 1 9 I t i s now thought t h a t most monoterpenes a r i s e from g e r a n y l p yrophosphate (17) ( o r i t s c i s - d o u b l e bond isomer n e r y l pyrophos-phate) and t h a t most s e s q u i t e r p e n e s a r i s e from f a r n e s y l p y r o -phosphate (18) (or i t s c i s - d o u b l e bond i s o m e r ) . T h i s i s a s t a t e -20 21 ment o f the " B i o g e n e t i c I s o p r e n e R u l e " . ' 19 22 23 The t r a n s f e r a s e from l i v e r has been p a r t i a l l y p u r i f i e d ' ' and t h e r e i s no i n d i c a t i o n t h a t two d i f f e r e n t enzymes c a t a l y s e the two s t e p s l e a d i n g t o (17) and ( 1 8 ) . However t h i s p r e p a r a t i o n was i n c a p a b l e o f u s i n g (18) as a s u b s t r a t e f o r f u r t h e r a d d i t i o n o f u n i t s , thus presumably the s t r u c t u r e o f the enzyme can accommodate (16) and (17) but does not have space t o accommodate the l a r g e r (18) i n a c o n f i g u r a t i o n r e q u i r e d f o r r e a c t i o n w i t h ( 1 5 ) . T h i s a s s o c i a t i o n of u n i t s i s an u n u s u a l r e a c t i o n even f o r an enzymic p r o c e s s , as most c o n d e n s a t i o n s t o form c a r b o n - c a r b o n bonds i n n a t u r e are o f the a l d o l or C l a i s e n t y p e . T h i s r e a c t i o n i s i n e f f e c t an a l k y l a t i o n o f an o l e f i n and i s analogous t o the f o r m a t i o n o f " d i - i s o b u t e n e " (19) from i s o b u t e n e : -12 -CH •> ( C H 3 ) 3 C + CH H CH / CH + -H > (CH 3) 3C-CH 2-C (19) T h i s enzymic r e a c t i o n was a t f i r s t c o n s i d e r e d to be a carbonium i o n r e a c t i o n o f s i m i l a r t y p e , b u t e x p e r i m e n t s u s i n g asymmetric l a b e l l i n g w i t h hydrogen i s o t o p e s have shown t h a t f o r m a t i o n o f the c a r b o n - c a r b o n bond i s accompanied by complete i n v e r s i o n o f c o n f i g u r a t i o n at the a l l y l i c carbon atom. T h i s i s c h a r a c t e r i s t i c o f a b i m o l e c u l a r n u c l e o p h i l i c s u b s t i t u t i o n r e a c t i o n r a t h e r than a carbonium i o n r e a c t i o n . The a d d i t i o n o f 16 24 the a l l y l i c C^ u n i t and e l i m i n a t i o n o f the hydrogen i o n o c c u r i n two s t a g e s : f i r s t l y a t r a n s a d d i t i o n o f the a l l y l i c u n i t and o f an e l e c t r o n d o n a t i n g group X, f o l l o w e d by subsequent t r a n s e l i m i n a t i o n o f X and o f hydrogen i o n (H ) as shown i n F i g u r e V I . I f Hd were e l i m i n a t e d , the c o r r e s p o n d i n g c i s isomer would be o b t a i n e d and t h i s a l t e r n a t i v e s t e r e o c h e m i c a l pathway f o r the c o n d e n s a t i o n of C$ u n i t s i s i n v o l v e d i n the b i o s y n t h e s i s o f the p o l y t e r p e n e r u b b e r . The s t e r e o c h e m i c a l f a t e o f the hydrogen e l i m i n a t i o n s can be f o l l c i v e d by f e e d i n g 2R- 2D, -mevalonate and - 13 -H F i g u r e V I : S t e r e o c h e m i s t r y o f the C o n d e n s a t i o n o f C 5 u n i t s 2S-2Dj,-mevalonate f o l l o w e d by d e t e r m i n a t i o n of the s t e r i c p o s i t i o n s o f hydrogen and d e u t e r i u m i n i s o p e n t e n o l o b t a i n e d from the i s o p e n t e n y l p y r o p h o s p h a t e . 1 ^ - 14 -G e r a n y l g e r a n y l pyrophosphate ( 2 0 ) , the p r o g e n i t o r o f the d i t e r p e n e s , i s formed by a n o t h e r a d d i t i o n o f (15) t o f a r n e s y l p yrophosphate ( 1 8 ) , and the f r e e hydroxy compound has r e c e n t l y 2 5 been shown t o be a p r e c u r s o r o f the d i t e r p e n e r o s e n o n o l a c t o n e ( 2 1 ) . A d d i t i o n o f y e t an o t h e r u n i t g i v e s g e r a n y l f a r n e s y 1 26 ' pyrophosphate (22) w h i c h has been shown to be i m p l i c a t e d i n the b i o s y n t h e s i s of the s e s t e r p e n e o p h i o b o l i n F (23) by c e l l f r e e e x t r a c t s o f C h o c h l i o b o l u s h e t e r o s t r o p h u s . G e r a n y l f a r n e s o l (22, OH i n s t e a d o f 0?2^^^) has r e c e n t l y been found i n i n s e c t 27 wax. (20) 0P o0,H. 2 6 j O P 2 ° 6 H 3 C22) - 15 -T r i t e r p e n e s ( C 3 0 ) are not b i o s y n t h e s i s e d from the C ^ Q analogue of (22) but are i n s t e a d formed from the s y m m e t r i c a l h y d r o c a r b o n 28 sq u a l e n e ( 2 4 ) . T h i s compound i s d e r i v e d from " t a i l - t o - t a i l " c o n d e n s a t i o n o f two m o l e c u l e s o f f a r n e s y l p yrophosphate (18) and i t s b i o s y n t h e s i s and c o n v e r s i o n t o t r i t e r p e n e s and s t e r o i d s have been the s u b j e c t o f c o n s i d e r a b l e i n v e s t i g a t i o n , the r e s u l t s o f 6,24,29 whi c h have been d e s c r i b e d i n s e v e r a l e x c e l l e n t r e v i e w s . (24) He - 16 -J u s t as the r e a l i s a t i o n o f the importance o f i s o p r e n e u n i t s i n the b i o s y n t h e s i s o f t e r p e n o i d s has p r o v e d i n v a l u a b l e i n the e l u c i d a t i o n and c o r r e l a t i o n o f the l a r g e v a r i e t y o f s t r u c t u r a l t y p e s c h a r a c t e r i s t i c o f t h i s c l a s s o f compound, so the r e a l i s a t i o n o f the im p o r t a n c e o f o t h e r compounds i n the b i o s y n t h e s i s o f non-i s o p r e n o i d compounds has l e d to g r e a t e r u n d e r s t a n d i n g o f the c h e m i s t r y o f the s e compounds. One such group o f compounds i s the c l a s s r e f e r r e d t o c o l l e c t i v e l y as a r o m a t i c compounds. These compounds can a r i s e i n n a t u r e i n two d i s t i n c t ways. One r o u t e i s termed the s h i k i m i c a c i d (25) r o u t e , w h i c h i s an o f f - s h o o t o f g l u c o s e m e t a b o l i s m , compounds formed by t h i s method b e i n g i m p o r t a n t i n the b i o s y n t h e s i s o f a l k a l o i d s . The s h i k i m i c a c i d pathway i s b r i e f l y summarised i n F i g u r e V I I . The o t h e r r o u t e to a r o m a t i c compounds i s the a c e t a t e 1 2 3 5 r o u t e , ' ' ' now c a l l e d the a c y l - p o l y m a l o n a t e r o u t e . As o r i g i n a l l y 1,2 e n v i s a g e d by C o l l i e , t h i s r o u t e i n v o l v e d c o n d e n s a t i o n s o f a c e t i c a c i d u n i t s , but more r e c e n t work^O has shown the importance o f m a l o n y l co-enzyme A (26) as a p r e c u r s o r o f the c h a i n p r o p a g a t i n g u n i t . As u n d e r s t o o d at p r e s e n t the f e a t u r e s o f t h i s r o u t e are as f o l l o w s . A c e t y l co-enzyme A (5) ( t h e c h a i n s t a r t e r u n i t ) condenses w i t h enzyme bound m a l o n i c a c i d (27) ( t h e c h a i n p r o p a g a t i n g u n i t ) , formed from m a l o n y l co-enzyme A (26) by e s t e r exchange. M a l o n y l co-enzyme A (26) i t s e l f i s d e r i v e d from a c e t y l co-enzyme A (5) by b i o t i n dependent c a r b o x y l a t i o n . The p r o d u c t from the conden-s a t i o n can now r e a c t s e q u e n t i a l l y w i t h more enzyme bound m a l o n i c a c i d (27) to g i v e an enzyme bound poly-yg-diketone c h a i n (28) as shown i n F i g u r e V I I I . - 1 7 -Phosphoenol P y r u v a t e S - d e h y d r o q u i n i c a c i d 5 - d e h y d r o s h i k i m i c a c i d S h i k i m i c a c i d £25) HO-C OPOJ P h e n y l a l a n i n e T y r o s i n e F i g u r e V I I : S h i k i m i c a c i d pathway t o a r o m a t i c compounds - 18 -CH COSCoA 3 (5) B i o t i n / A T P / M g + + / H C 0 3 CH 2COSCoA C0 2H (26) Enzyme CH -CO-S-Enzyme + CH 3CO SCoA C0 2H (27) 1 CHCOCH CO-S-Enzyme CH^CO-S-Enzyme ( r e p e a t e d n t i m e s ) C0 2H CH CO(CH 2CO) nCH 2CO-S-Enzyme (28) (n = 1-8) F i g u r e V I I I : A c y l - p o l y m a l o n a t e r o u t e t o a r o m a t i c compounds - 19 -The poly-^S-diketone c h a i n can now undergo i n t e r n a l c y c l i s a t i o n w h i c h can be o f two t y p e s , namely a l d o l c o n d e n s a t i o n t o g i v e o r s e l l i n i c a c i d (29) type compounds, or C l a i s e n c o n d e n s a t i o n to g i v e a c y l p h l o r o g l u c i n o l s , e.g. a c e t y l p h l o r o g l u c i n o l ( 3 0 ) . Enzyme-S-OC 1 1—»6 0 (28,n=l) A l d o l 2—»7 C l a i s e n N H0 20 OH OH (29) (30) A number o f secondary m o d i f i c a t i o n s can o c c u r , e i t h e r p r i o r to c y c l i s a t i o n or a f t e r c y c l i s a t i o n . These i n c l u d e a l k y l a t i o n s , 31 32 33 e.g. m y c o p h e n o l i c a c i d ( 3 1 ) , ' ' o x i d a t i o n , e.g. f u m i g a t i n ( 3 2 ) . r e d u c t i o n , e.g. 6-methyl s a l i c y l i c a c i d ( 3 3 ) , 30,34,35,36,37 h a l o g e n a t i o n , i n t r o d u c t i o n of n i t r o g e n , and c l e a v a g e o f the a r o m a t i c 38 39,40 r i n g , e.g. p e n i c i l l i c a c i d (34) and p a t u l i n ( 3 5 ) . F u r t h e r work on t h i s r o u t e to a r o m a t i c compounds has r e v e a l e d t h a t a c e t i c a c i d i s not the o n l y a c i d i n v o l v e d as a s t a r t e r u n i t . Other a c i d s a l s o found as s t a r t e r u n i t s i n c l u d e c i n n a m i c a c i d s . 20 -0 H 0 2C (31) 0 (32) OH OCH OH (33) OCH. ° ^ H (35) b e n z o i c a c i d s , n i c o t i n i c a c i d and a n t h r a n i l i c a c i d . These a c i d s are p r o d u c t s o f the s h i k i m i c a c i d (25) r o u t e to a r o m a t i c compounds so compounds formed by u t i l i s a t i o n o f one o f those a c i d s r e p r e s e n t a c o n f l u e n c e o f the two r o u t e s . R e p r e s e n t a t i v e examples o f 41 compounds h a v i n g such a mixed b i o g e n e s i s are y a n g o n i n (36) and p i n o s y l v i n ( 3 7 ) . 42 OCH. (36) (37) PART I INVESTIGATIONS OF TRICHOTHECIN BIOSYNTHESIS - 21 -INTRODUCTION S t r u c t u r a l E l u c i d a t i o n o f T r i c h o t h e c i n (38) The b i o l o g i c a l a c t i v i t y o f T r i c h o t h e c i u m roseum L i n k has been 43 r e p o r t e d s e v e r a l times s i n c e the b e g i n n i n g o f t h i s c e n t u r y . 44 In 1949 Freeman and M o r r i s o n r e p o r t e d the i s o l a t i o n and p r o p e r t i e s o f the compound r e s p o n s i b l e f o r the b i o l o g i c a l a c t i v i t y , and named i t t r i c h o t h e c i n . T h e i r r e s u l t s i n d i c a t e d t h a t t r i c h o t h e c i n : (a) has a m o l e c u l a r f o r m u l a o f C-| qH-j, gO^ o r C ^ ^ H ^ q O ^ ; (b) i s a n e u t r a l , u n s a t u r a t e d , c o n j u g a t e d k e t o n e , con-t a i n i n g no h y d r o x y l o r a l k o x y l group and, (c) has t h r e e C-methyl groups. L a t e r w o r k ^ 5 showed t h a t the m o l e c u l a r f o r m u l a was ^19^24* J5 a n c l t^ i a^ t r i c h o t h e c i n was an e s t e r , the components of w h i c h were t r i c h o t h e c o l o n e (39) and i s o c r o t o n i c a c i d . 0 - 22 -46 In 1959 s t r u c t u r e (40) was p r o posed f o r t r i c h o t h e c i n on the b a s i s o f a s y s t e m a t i c d e g r a d a t i o n , w h i c h , however, d i d not e x c l u d e the p o s s i b i l i t y o f a l t e r n a t i v e s t r u c t u r e ( 4 1 ) . F u r t h e r e v i d e n c e f o r s t r u c t u r e (40) was p r o v i d e d by Jones and h i s c o - w o r l c e r s , 4 7 who 48 were a l s o the f i r s t t o s t u d y the b i o s y n t h e s i s o f t r i c h o t h e c i n . In 1964 a new compound, wh i c h was named t r i c h o d e r m i n , was 49 -i s o l a t e d from a s t r a i n of T r i c h o d e r m a . The c h e m i c a l and s p e c t r a l p r o p e r t i e s of t r i c h o d e r m i n s u g g e s t e d a r e l a t i o n s h i p t o t r i c h o t h e c i n , and t h i s was c o n f i r m e d by o x i d a t i o n o f t r i c h o d e r m i n t o t r i c h o t h e c o l o n e a c e t a t e , s u g g e s t i n g s t r u c t u r e (42) f o r t r i c h o d e r m i n . However t h i s s t r u c t u r e was i n c o m p a t i b l e w i t h some of the r e a c t i o n s of t r i c h o d e r m i n , r e a c t i o n s w h i c h s u g g e s t e d the p r e s e n c e o f an e poxide r i n g i n t r i c h o d e r m i n . To r e s o l v e t h i s p roblem an X-ray a n a l y s i s o f t r i c h o d e r m o l p_-bromobenzoate (43,p_-bromobenzoate i n s t e a d of a c e t y l ) was u n d e r t a k e n , ^ w h i c h i n d i c a t e d t h a t s t r u c t u r e (43) i s the s t r u c t u r e o f t r i c h o d e r m i n and hence (3S) i s the c o r r e c t s t r u c t u r e o f t r i c h o t h e c i n . 23 -(42) B i o s y n t h e t i c P r o p o s a l s f o r T r i c h o t h e c i n 48 Jones and Low were the f i r s t t o study the b i o s y n t h e s i s of t r i c h o t h e c i n ( 3 8 ) . In d e r i v i n g the t r i c h o t h e c i n carbon s k e l e t o n from f a r n e s y l p y rophosphate (18) (as r e q u i r e d by the " B i o g e n e t i c 20 21 Isop r e n e R u l e " ' ' ) i t i s n e c e s s a r y to in v o k e e i t h e r a 1,3-methyl m i g r a t i o n o r two 1,2-methyl m i g r a t i o n s . The r e s u l t s o f Jones and 4 8 Lowe showed t h a t two 1,2-methyl m i g r a t i o n s o c c u r . U s i n g erroneous s t r u c t u r e (40.) R u z i c k a d i s c u s s e d , the b i o -s y n t h e s i s of t r i c h o t h e c i n . He proposed that f a r n e s y l pyrophosphate - 24 -(18) c y c l i s e s t o g i v e carbonium i o n (44) which, t h e n l o s e s a p r o t o n t o form ' o'-bisabolene (45) . I t s h o u l d be n o t e d t h a t t h e r e are two isomers o f 'b'-bisabolene (45a) and (45b). R u z i c k a has s t a t e d ^ 1 t h a t s t a r t i n g w i t h ( 2 - ^ C ) - m e v a l o n i c a c i d the l a b e l l i n g p a t t e r n s h o u l d be as shown i n F i g u r e IX and t h a t b o t h isomers o f 'o'-bisabolene can be d e r i v e d from f a r n e s y l pyrophosphate (18) , whether (18) (44) (45) - 25 -or not one s t a r t s with, a c i s o r a t r a n s c e n t r a l double bond. R u z i c k a has a l s o stated 5''" t h a t b o t h ^ - b i s a b o l e n e s c o u l d c y c l i s e and undergo two 1,2-methyl m i g r a t i o n s t o g i v e the s t r u c t u r e and s t e r e o c h e m i s t r y o f t r i c h o t h e c i n (as assumed at t h a t time) as shown i n F i g u r e X. A c c o r d i n g t o the l a b e l l i n g s t u d i e s o f Jones and A O Lowe i t i s the t r a n s m e t h y l group i n the i s o p r o p y l i d e n e group w h i c h m i g r a t e s . 14. (45a) (45b) F i g u r e IX: L o c a t i o n o f l a b e l i n ^ - b i s a b o l e n e from 2- 1 4C m e v a l o n i c a c i d _ . C45 a or b) 4 _ <-F i g u r e X: (40) OR C h a i r type f o l d i n g o f X - b i s a b o l e n e a c c o r d i n g t o R u z i c k a - 2 6 -However, when the c o r r e c t s t r u c t u r e C38) i s t a k e n i n t o account t h i s mechanism g i v e s the wrong s t e r e o c h e m i s t r y i n the 5-membered 49b r i n g . Two a l t e r n a t i v e mechanisms are p o s s i b l e , the f i r s t i n v o k i n g a b o a t - t y p e f o l d i n g o f the s i d e c h a i n CFigure X I ) . T h i s . scheme can be c r i t i c i s e d on a t l e a s t two c o u n t s . F i r s t l y the b o a t - t y p e f o l d i n g i s more e n e r g e t i c a l l y u n f a v o u r a b l e t h a n a c h a i r -type f o l d i n g , and s e c o n d l y the m e t h y l s h i f t s must be n o n - c o n c e r t e d to agree w i t h the l a b e l l i n g p a t t e r n . (45a or b) H < <r (38) F i g u r e X I : Boat type f o l d i n g o f X - b i s a b o l e n e A much more e l e g a n t and e n e r g e t i c a l l y more f a v o u r a b l e method of f o l d i n g the s i d e c h a i n i s to u t i l i s e a c h a i r - t y p e f o l d i n g but to f o l d the s i d e c h a i n i n f r o n t o f the p l a n e o f the c y c l o h e x a n e r i n g as i n F i g u r e X I I r a t h e r than b e h i n d i t as i n F i g u r e X. - 27 -(45a o r b) F i g u r e X I I : A l t e r n a t i v e c h a i r type f o l d i n g o f X - b i s a b o l e n e H a v i n g shown how the s i d e c h a i n may be f o l d e d , t h e next a s p e c t of the b i o s y n t h e s i s o f t r i c h o t h e c i n t o d i s c u s s i s w h i c h isomer o f "o'-bisabolene, (45a) or (45 b ) , may be i n v o l v e d . U n f o r -t u n a t e l y the r e s u l t s o b t a i n e d by two d i f f e r e n t r e s e a r c h groups are i n d i r e c t c o n t r a d i c t i o n . 14 4 S t a r t i n g from ( 2 - 1 H C ) - m e v a l o n i c a c i d l a c t o n e , Jones and Lowe have shown t h a t the l a b e l l i n g p a t t e r n i n t r i c h o t h e c i n i s as f o l l o w s : H 28 S t a r t i n g w i t h . Y - b i s a b o l e n e two a l t e r n a t e schemes can be e n v i s a g e d f o r the b i o s y n t h e s i s of t r i c h o t h e c i n C F i g u r e s X I I I and XIV) . (45a) H 0''> <r~ <— (38) F i g u r e X I I I : L a b e l l i n g p a t t e r n i n t r i c h o t h e c i n from isomer (45a) of "tf-bisabolene (38) F i g u r e XIV: L a b e l l i n g p a t t e r n i n t r i c h o t h e c i n from isomer (45b) of Y - b i s a b o l e n e - 2 9 -t h u s i t can be seen t h a t o n l y isomer C45b) o f ) f - b i s a b o l e n e g i v e s a r e s u l t w h i c h i s c o n s i s t e n t w i t h the work o f Jones and Lowe. However, t h i s i s o n l y t r u e i f t h e r e i s no r o t a t i o n about the bond j o i n i n g the 6- to the 5- membered r i n g . I f t h e r e i s r o t a t i o n about t h i s bond'then F i g u r e s X I I I and XIV become e q u i v a l e n t a f t e r the c y c l i s a t i o n s t e p . S i n c e i t i s v e r y u n l i k e l y t h a t the i n t e r m e d i a t e i s a f r e e carbonium i o n , b u t has some group Z a t t a c h e d a t t h a t p o i n t w h i c h must s u b s e q u e n t l y be e l i m i n a t e d i n a normal t r a n s -a n t i - p a r a l l e l manner, then the s t e r e o s p e c i f i c i t y d e p i c t e d i n F i g u r e s X I I I and XIV s t i l l h o l d s : -- 30 -More r e c e n t work done by Hanson and h i s c o l l a b o r a t o r s , u s i n g 3 14 doubly l a b e l l e d (2- H,2- C) f a r n e s y l p y rophosphate (18) i n d i c a t e d t h a t the o t h e r isomer (45a) o f 1 ) - b i s a b o l e n e i s i n v o l v e d i n the b i o s y n t h e s i s o f t r i c h o t h e c i n ( 3 8 ) . ( P r e v i o u s work from the same 53 group had a l r e a d y i m p l i c a t e d f a r n e s y l p y r o p h o s p h a t e (18) i n •7 14 the • b i o s y n t h e s i s o f t r i c h o t h e c i n ) . S t a r t i n g from (2-°H, 2- C) f a r n e s y l p y r o p h o s p h a t e ( 1 8 ) , the two isomers o f ^ - b i s a b o l e n e have the f o l l o w i n g l a b e l l i n g p a t t e r n s : -(18) (45a) (45b) - 31 -F i g u r e XV t r a c e s the f a t e o f the l a b e l s i n each o f these isomers as they are c o n v e r t e d to t r i c h o t h e c i n ( 3 8 ) . E x p e r i m e n t a l l y i t was found t h a t the t r i t i u m was r e t a i n e d w h i c h i s c o n s i s t e n t w i t h . i s o m e r (45a) b e i n g the one i n v o l v e d i n the b i o s y n t h e s i s o f t r i c h o t h e c i n ( 3 8 ) . The same argument a p p l i e s c o n c e r n i n g a Z group mechanism w h i c h e f f e c t i v e l y p r e v e n t s r o t a t i o n about the i n t e r - a n n u l a r bond. The f a c t t h a t the r e t e n t i o n of 52 t r i t i u m i s almost 1001 i n d i c a t e s t h a t t h e r e can be no r o t a t i o n about t h i s bond. 48 48 As i t had been shown t h a t a c e t a t e , m evalonate, and f a r n e s y l 53 pyrophosphate are i n v o l v e d i n the b i o s y n t h e s i s o f t r i c h o t h e c i n , the n e x t l o g i c a l s t e p was to determine w h i c h m o n o c y c l i c s p e c i e s ( i f any) are i n v o l v e d . P r e l i m i n a r y i n v e s t i g a t i o n s i n t h i s 54 l a b o r a t o r y have shown t h a t «K-bisabolol (46) i s not a p r e c u r s o r i n the b i o s y n t h e s i s o f t r i c h o t h e c i n ( 3 8 ) . I t was thought t h a t c < - b i s a b o l o l (46) may have been c a p a b l e o f d e h y d r a t i o n i n v i v o t o g i v e " ^ - b i s a b o l e n e (45) o r t h a t i t may be a t r u e p r e c u r s o r o f ^ - b i s a b o l e n e . OH (46) (45) - 32 -F i g u r e XV: P o s i t i o n o f l a b e l i n t r i c h o t h e c i n a c c o r d i n g t o Hanson and co-workers - 33 -The work d e s c r i b e d i n t h i s p a r t o f the t h e s i s i n v o l v e s a s y n t h e s i s o f ^ - b i s a b o l e n e (45) and the f e e d i n g of t h i s compound ( i n r a d i o - a c t i v e form) t o c u l t u r e s o f T r i c h o t h e e i u m roseuni. The s i g n i f i c a n c e o f the r e s t i l t s o b t a i n e d and t h e i r r e l e v a n c e t o work p u b l i s h e d 5 5 ' 5 ^ ' 5 ^ ' 5 ^ d u r i n g the course o f t h i s work i s th e n d i s c u s s e d . A l s o i n t h i s p a r t o f the t h e s i s are d e s c r i b e d s y n t h e s e s of r a d i o - a c t i v e m o n o c y c l o f a r n e s o l (47) and f a r n e s o l epoxide ( 4 8 ) , the p o s s i b l e mechanisms of t h e i r c o n v e r s i o n t o t r i c h o t h e c i n , and the r e s u l t s o f f e e d i n g them to T r i c h o t h e c i u m roseum. OH (47) (48) - 34 DISCUSSION P r e v i o u s work on the b i o s y n t h e s i s o f t r i c h o t h e c i n (38) has 48 48 53 shown t h a t a c e t a t e , mevalonate, and f a r n e s y l p yrophosphate 54 are p r e c u r s o r s o f t r i c h o t h e c i n ( 3 8 ) . P r e l i m i n a r y i n v e s t i g a t i o n s t o determine w h i c h m o n o c y c l i c compounds may be i n v o l v e d have shown t h a t o c - b i s a b o l o l (46) i s not a p r e c u r s o r . As the r e a s o n f o r assuming the o < - b i s a b o l o l (46) may have been a p r e c u r s o r was the f a c t t h a t i t might have been c a p a b l e o f d e h y d r a t i o n i n v i v o t o ^ - b i s a b o l e n e ( 4 5 ) , w h i c h has l o n g been c o n s i d e r e d ^ * 3 * 5 1 ' ' ^  ' ^ to be a p r e c u r s o r i n t h i s p r o c e s s , the n e x t o b v i o u s s t e p was t o t e s t V - b i s a b o l e n e (45) f o r p r e c u r s o r a c t i v i t y . Y - B i s a b o l e n e (45) was p r e p a r e d by the a c t i o n o f phosphorus o x y c h l o r i d e , i n p y r i d i n e , on o < - b i s a b o l o l ( 4 6 ) . P O C l 3 / P y r i d i n e > (46) (45) °<-Bisabolol (46) has been p r e p a r e d a number of times b e f o r e . I t 62 was f i r s t s y n t h e s i s e d by R u z i c k a and L i g u o r i i n 1932. They p r e p a r e d i t by c o n d e n s i n g the G r i g n a r d complex of bromide (49) w i t h ketone ( 5 0 ) . Bromide (49) was p r e p a r e d from d i o l ( 5 1 ) , and ketone (50) was p r e p a r e d by o z o n o l y s i s and d e h y d r a t i o n of ^-t e r p i n e o l ( 5 2 ) . 35 -(51) ( 4 9 ) 0' MgBr (50) ( 4 6 ) A s y n t h e s i s o f p - b i s a b o l e n e (53) was r e p o r t e d by M a n j a r r e z and Guzman i n 1966. The key s t e p i n t h e i r scheme was the r e a c t i o n between ketone (54) and m e t h y l t r i p h e n y I p h o s p h o n i u m bromide. Ketone (54) was p r e p a r e d by c o n d e n s a t i o n of the G r i g n a r d complex o f bromide - 36 -(49) with, the a c i d c h l o r i d e o f a c i d (55) • Bromide (49) ( t h e same 6 2 one as used by R u z i c k a and L i g u o r i ) was p r e p a r e d a f t e r the method o f J u l i a and c o - w o r k e r s * ^ from c y c l o p r o p y l m e t h y l ketone ( 5 6 ) , w h i c h was r e a c t e d w i t h m e t h y l magnesium bromide to g i v e c a r b i n o l ( 5 7 ) . Treatment o f c a r b i n o l (57) w i t h 48% hydrogen bromide gave bromide ( 4 9 ) . A c i d (55) was p r e p a r e d by h y d r o l y s i s o f e s t e r ( 5 8 ) , o b t a i n e d by D i e l s - A l d e r r e a c t i o n between i s o p r e n e (1) and m e t h y l a c r y l a t e ( 5 9 ) . (59) (1) (58) (55) - 37 -0 CH P h 3 P C H 3 B r + X (54) (53) These s y n t h e s e s have been c r i t i c i s e d by Gutsche and h i s co-w o r k e r s ^ 5 who o f f e r an a l t e r n a t i v e scheme, though u t i l i s i n g the 6 7 same key r e a c t i o n as R u z i c k a and L i g u o r i . " Their f i r s t c r i t i c i s m i s t h a t i t i s t e d i o u s to p r e p a r e ketone (50) by the method o f 62 R u z i c k a and L i g u o r i because o f the d i f f i c u l t y i n s e p a r a t i n g pure | S - t e r p i n e o l from the c o m m e r c i a l l y a v a i l a b l e m i x t u r e . They then t r i e d t o p r e p a r e ketone (50) by a D i e l s - A l d e r r e a c t i o n between m e t h y l v i n y l ketone and i s o p r e n e but had d i f f i c u l t y i n s e p a r a t i n g the i s o m e r i c m i x t u r e o b t a i n e d , w h i c h c o n t a i n e d 25% o f unwanted isomer ( 6 0 ) . They e v e n t u a l l y o b t a i n e d ketone (50) i n low y i e l d by t r e a t m e n t of 4 - k e t o - c y c l o h e x a n e c a r b o x y l i c a c i d (61) w i t h m e t h y l l i t h i u m , f o l l o w e d by d e h y d r a t i o n . S e c o n d l y they c l a i m t h a t the bromide (49) p r e p a r e d by R u z i c k a 6 2 and L i g u o r i c o n t a i n e d s i g n i f i c a n t amounts o f the i s o p r o p e n y l isomer (6 2 ) . - 38 -0 (60) 0 0 (61) OH (50) C62) Gutsche and h i s co-workers tlxen p r e p a r e d a pure sample of o f - b i s a b o l o l (.46) by c o n d e n s i n g the G r i g n a r d complex o f pure bromide ( 4 9 ) , p r e p a r e d by the meth_od of J u l i a and co-workers,^* 4 w i t h pure ketone ( 5 0 ) , p r e p a r e d by t h e i r own method. A n o t h e r s y n t h e s i s o f ketone (54) has been r e p o r t e d , ^ b u t as i t i s much l e n g t h i e r than the o t h e r s i t was c o n s i d e r e d u n s u i t a b l e . I t i s shown i n F i g u r e XVI. For a s y n t h e s i s o f r a d i o - a c t i v e c < ~ b i s a b o l o l (46) i t was c o n s i d e r e d t h a t the b e s t method was to i n t r o d u c e the r a d i o - l a b e l i n C 0 2 E t (a) Na d u s t ; H c l Cc) MeMgl CO Acetone/HCl ^ (g) B u V / B i ^ O H / ^ (O I b l 0 (b) KOH/MeOH/H20; H ; A/Cu (d) p-TSA/benzene/A (£) NaH/CO(OEt) 2/DMF/A F i g u r e XVI: A l t e r n a t i v e s y n t h e s i s o f ketone (54) - 40 -the l a s t s t e p o f t h e r e a c t i o n sequence by a l l o w i n g ketone (54) t o r e a c t w i t h r a d i o - a c t i v e m e t h y l magnesium i o d i d e . The f i r s t o b j e c t i v e was thus a c o n v e n i e n t - s y n t h e s i s o f ketone ( 5 4 ) . (54) (46) The method f i n a l l y adopted, a f t e r an i n v e s t i g a t i o n o f an u n s u c c e s s f u l a l t e r n a t i v e , was a m o d i f i c a t i o n 5 4 o f the r e a c t i o n 6 3 scheme o f M a n j a r r e z and Guzman. Thus c o n d e n s a t i o n of i s o p r e n e (1) w i t h m e t h y l a c r y l a t e (59) gave, i n good y i e l d , a m i x t u r e of e s t e r s (58) and ( 6 3 ) . [ P r e v i o u s work by Hennis has shown t h a t the r a t i o o f p r o d u c t s i n t h i s r e a c t i o n i s a p p r o x i m a t e l y 70% d e s i r e d e s t e r (58) and 30% unwanted e s t e r ( 6 3 ) ] . The m i x t u r e of e s t e r s was h y d r o l y s e d i n aqueous sodium h y d r o x i d e s o l u t i o n t o g i v e a gummy m i x t u r e of a c i d s (55) and ( 6 4 ) , m.p. 50-100°C, wh i c h was r e p e a t e d l y c r y s t a l l i s e d from p e t r o l e u m e t h e r (60-80°C) u n t i l the m e l t i n g p o i n t was c o n s t a n t at 99-100°C. E s s e n t i a l l y s i m i l a r r e s u l t s c o u l d be o b t a i n e d u s i n g a c r y l i c a c i d i n s t e a d o f m e t h y l a c r y l a t e and o m i t t i n g the h y d r o l y s i s s t e p . - 41 -MeO (59) (1) (58) (63) ~ 70% ^ 30% C o n f i r m a t i o n o f the s t r u c t u r e of a c i d (55) was o b t a i n e d by l a c t o n i s i n g the p u r i f i e d p r o d u c t w i t h 98% f o r m i c a c i d . T h i s p r o c e d u r e gave a w h i t e , c r y s t a l l i n e compound, m.p. 68-69°C, w i t h i n f r a - r e d ( i r ) a b s o r p t i o n at 1760 cm * i n carbon t e t r a c h l o r i d e s o l u t i o n and 1740 cm i n c h l o r o f o r m s o l u t i o n . T h i s i n d i c a t e d t h a t . t h e compound p o s s e s s e d a ^ - l a c t o n e r i n g , w h i c h i n c o n j u n c t i o n w i t h the n u c l e a r m agnetic resonance (nmr) spectrum ( s i n g l e t a t 8.65 T , m e t h y l group a t t a c h e d t o carbon b e a r i n g oxygen) c o n f i r m e d the s t r u c t u r e as l a c t o n e ( 6 5 ) . (65) - 42 -There i s some c o n f u s i o n i n the l i t e r a t u r e r e g a r d i n g the s t r u c t u r e o f t h i s l a c t o n e as a group of F r e n c h workers^** a s c r i b e d s t r u c t u r e (66) t o t h i s l a c t o n e on the b a s i s o f the i r a b s o r p t i o n a t 5.68yu (1760 cm \ presumably r e c o r d e d i n carbon t e t r a c h l o r i d e ) . F u r t h e r p r o o f t h a t s t r u c t u r e (66) i s not the l a c t o n e formed by l a c t o n i s i n g a c i d (55) i s the f a c t t h a t b o t h isomers of l a c t o n e (66) have been p r e p a r e d ^ 9 and n e i t h e r m e l t s a t 69°C. One o t h e r p o s s i b i l i t y i s t h a t the a c i d l a c t o n i s e d i n t h i s work may not have been a c i d (55) b u t i n s t e a d was a c i d (64) ( i . e . the D i e l s - A l d e r r e a c t i o n had g i v e n a preponderance of the wrong i s o m e r ) . T h i s 70 would l e a d t o l a c t o n e (67) w h i c h has p r e v i o u s l y been s y n t h e s i s e d and i t s m e l t i n g p o i n t i s 45°C. F i n a l l y l a c t o n e (65) has been 71 s y n t h e s i s e d by an a l t e r n a t i v e r o u t e and i t s m e l t i n g p o i n t i s 69°C and i t has an i r a b s o r p t i o n o f 1740 cm""'" (presumably r e c o r d e d i n c h l o r o f o r m s o l u t i o n ) . These r e s u l t s are summarised i n F i g u r e X V I I . • Bromide (49) was p r e p a r e d i n the same way as i n the s y n t h e s i s 6 3 r e p o r t e d by M a n j a r r e z and Guzman. Thus t r e a t m e n t o f c y c l o p r o p y l m e t h y l ketone (56) w i t h m e t h y l magnesium i o d i d e gave d i m e t h y l c a r b i n o l (57) w h i c h was c o n v e r t e d i n t o bromide (49) by the a c t i o n o f 48% hydrogen b r o m i d e . ^ 54 P r e v i o u s work has shown t h a t when the G r i g n a r d complex o f bromide (49) i s a l l o w e d to r e a c t w i t h the a c i d c h l o r i d e o f a c i d (55) i n the normal manner, i . e . a d d i n g the a c i d c h l o r i d e to the G r i g n a r d complex, the o n l y p r o d u c t o b t a i n e d i s e s t e r ( 6 8 ) . E s t e r (6 8) i s presumably formed by p r i o r f o r m a t i o n o f ketone (54) w h i c h , - 4 3 -m.p. 4S°C F i g u r e X V I I : C o n f i r m a t i o n o f s t r u c t u r e o f a c i d (55) - 44 -(49) i n the p r e s e n c e o f exc e s s G r i g n a r d r e a g e n t i s reduced t o magnesium a l c o h o l a t e (69, R = MgBr). T h e . r e d u c i n g p r o p e r t i e s o f G r i g n a r d 72 complexes are w e l l known. A l c o h o l a t e (69, R = MgBr) can now compete w i t h the G r i g n a r d r e a g e n t f o r the a c i d c h l o r i d e , to g i v e e s t e r ( 6 8 ) . 0 ( 5 4 ) - 45 -I t was thought t h a t v e r y slow i n v e r s e a d d i t i o n , i . e . adding the G r i g n a r d r e a g e n t t o the a c i d c h l o r i d e would g i v e the d e s i r e d ketone (54) d i r e c t l y , and t h i s p r o v e d to be the case. However, ketone (54) p r e p a r e d i n t h i s manner always c o n t a i n e d an i m p u r i t y , w i t h an i r a b s o r p t i o n a t 1730 cm 1 , which was v e r y d i f f i c u l t t o remove. The i m p u r i t y proved to be e s t e r (70) presumably formed by a i r o x i d a t i o n of the G r i g n a r d complex f o l l o w e d by r e a c t i o n o f the r e s u l t i n g magnesium a l c o h o l a t e w i t h the a c i d c h l o r i d e . ( 7 0 ) Thus the b e s t method f o r p r e p a r a t i o n o f ketone ( 5 4 ) was by a normal G r i g n a r d r e a c t i o n t o g i v e e s t e r ( 6 8 ) which on t r e a t m e n t w i t h e i t h e r l i t h i u m aluminium h y d r i d e o r m e t h y l l i t h i u m gave a l c o h o l ( 6 9 , R = H) w h i c h c o u l d then be o x i d i s e d u s i n g Jones 7 3 Reagent. T h i s method was used i n t h i s s y n t h e s i s of ketone ( 5 4 ) , w h i c h was then c o n v e r t e d , i n almost q u a n ' t i t i v e y i e l d , t o © C-bisabolol ( 4 6 ) by the a c t i o n of met h y l magnesium i o d i d e . A s i m i l a r s y n t h e s i s of c x - b i s a b o l o l ( 4 6 ) has r e c e n t l y been 74 p u b l i s h e d by some R u s s i a n w o r k e r s , u s i n g n i t r i l e ( 7 1 ) and the G r i g n a r d complex o f bromide ( 4 9 ) to g i v e ketone ( 5 4 ) which was then c o n v e r t e d t o o ( - b i s a b o i o l ( 4 6 ) w i t h m e t h y l magnesium i o d i d e . - 47 -0 ( 4 6 ) - 48 -0 . (71) (54) In the same paper i s d e s c r i b e d an a l t e r n a t i v e s y n t h e s i s o f <X-b i s a b o l o l u s i n g the same r e a g e n t s but r e a c t i n g them i n a d i f f e r e n t o r d e r . Thus n i t r i l e ( 7 1 ) , on t r e a t m e n t w i t h m e t h y l magnesium i o d i d e , gave ketone (50) which, was c o n v e r t e d i n t o c < - b i s a b o l o l by the a c t i o n o f the G r i g n a r d complex of bromide ( 4 9 ) , as i n the o r i g i n a l s y n t h e s i s o f R u z i c k a and L i g u o r i . (46) - 49 -D e h y d r a t i o n o f 0 < - b i s a b o l o l C46) w i t h phosphorus o x y c h l o r i d e i n . p y r i d i n e gave a m i x t u r e o f X - b i s a b o l e n e (45) and o t h e r i s o m e r i c b i s a b o l e n e s such as ^ - b i s a b o l e n e ( 5 3 ) . S e p a r a t i o n o f t h i s m i x t u r e by p r e p a r a t i v e gas l i q u i d chromatography ( g l c ) (30% Carbowax, 180°C) gave pure X - b i s a b o l e n e (45) (^50% o f the m i x t u r e ) w h i c h was c h a r a c t e r i s e d by a n a l y s i s o f i t s i r and nmr s p e c t r a . P r e p a r a t i o n o f r a d i o - l a b e l l e d ^ - b i s a b o l e n e was a c h i e v e d i n an i d e n t i c a l manner from k e t o n e (54) u s i n g r a d i o - a c t i v e m e t h y l i o d i d e i n the i n i t i a l G r i g n a r d r e a c t i o n . (46) (45) (53) + o t h e r i s o m e r ( s ) A s a t i s f a c t o r y s y n t h e s i s o f ^ - b i s a b o l e n e (45) h a v i n g been a c h i e v e d , i t s p r e c u r s o r a c t i v i t y i n the b i o s y n t h e s i s o f t r i c h o t h e c i n (38) c o u l d now be i n v e s t i g a t e d . To t h i s end an aqueous s o l u t i o n o f r a d i o - l a b e l l e d $ - b i s a b o l e n e (due t o the d i f f i c u l t i e s i n p u r i f y i n g s m a l l amounts of r a d i o - a c t i v e m a t e r i a l the m i x t u r e o f isomers was u s e d ) , s o l u b i l i s e d w i t h Tween 20, was added t o one - 50 -week o l d c u l t u r e s o f T r 1 chothiecium r o s e u m , ^ and the c u l t u r e s a l l o w e d t o grow f o r a f u r t h e r t h r e e weeks. The b r o t h was then d e c a n t e d and the m y c e l i a washed w i t h a l i t t l e w a t e r . The combined b r o t h and washings were e x t r a c t e d w i t h carbon t e t r a c h l o r i d e , and e v a p o r a t i o n o f the s o l v e n t gave a crude gum w h i c h was p u r i f i e d by t h i n l a y e r chromatography on s i l i c a g e l . The t r i c h o t h e c i n (38) so o b t a i n e d was then r e c r y s t a l l i s e d from p e t r o l e u m e t h e r (60-80°C) to c o n s t a n t r a d i o - a c t i v i t y , w h i c h r e p r e s e n t e d a s p e c i f i c i n c o r p o r a t i o n o f 0.005%. H y d r o l y s i s o f t r i c h o t h e c i n (38) t o t r i c h o t h e c o l o n e (39) w i t h m e t h a n o l i c p o t a s s i u m h y d r o x i d e s o l u t i o n , f o l l o w e d by r e c r y s t a l l i s a t i o n o f the t r i c h o t h e c o l o n e t o c o n s t a n t r a d i o - a c t i v i t y showed t h a t ~ 4 0 % o f the r a d i o - a c t i v i t y had been l o s t , i n d i c a t i n g t h a t d e g r a d a t i o n o f the 'o'-bisabolene had o c c u r r e d p r i o r t o i n c o r p o r a t i o n . These r e s u l t s i n d i c a t e t h a t fl'-bisabolene (45) i s not a p r e c u r s o r o f t r i c h o t h e c i n . D u r i n g the c o u r s e o f t h i s work two groups o f w o r k e r s have r e p o r t e d r e s u l t s 5 5 ' 5 ^ ' 5 7 ' 5 ^ w h i c h agree w i t h t h i s c o n c l u s i o n . The n e g a t i v e i n c o r p o r a t i o n o f ^ - b i s a b o l e n e (45) prompted c o n s i d e r a t i o n o f an a l t e r n a t i v e b i o s y n t h e t i c r o u t e t o t r i c h o t h e c i n (38) from f a r n e s o l pyrophosphate ( 1 8 ) . I t has been s u g g e s t e d ' * t h a t c a t i o n (72) might be i n v o l v e d i n the b i o s y n t h e s i s o f s e v e r a l 75.76 ? r types o f s e s q u i t e r p e n e s s uch as ot-chamigrene ( 7 3 ) , p - chamigrene ( 7 4 ) / J 7 5 76 t h u j o p s e n e (75) and cuparene ( 7 6 ) . A l t h o u g h c a t i o n (72) may be f o r m a l l y d e r i v e d by c y c l i s a t i o n of ^ - b i s a b o l e n e (45) i t has been 7 (\ su g g e s t e d t h a t i t may a r i s e by c y c l i s a t i o n of m o n o c y c l o f a r n e s o l C47). (73) - 52 -Thus i t i s p o s s i b l e t o c o n s t r u c t a mechanisja wLereby mono-c y c l o f a r n e s o l (47) c o u l d be c o n v e r t e d t o the t r i c h . o t h . e c i n s k e l e t o n . C y c l i s a t i o n o f m o n o c y l o f a r n e s o l (47) c o u l d p r o v i d e s p i r o - b i c y c l i c c a t i o n (72) w h i c h , a f t e r r i n g c o n t r a c t i o n , would p r o v i d e the cuparane type c a t i o n (77) i d e n t i c a l to t h a t o b t a i n e d by c y c l i s i n g & - bisabolene (45) . (45) - 53 -To t e s t t h i s p r o p o s a l a r a d i o - l a b e l l e d sample o f monocyclo-f a r n e s o l (47} was p r e p a r e d as f o l l o w s . The n a t u r a l l y o c c u r r i n g C k e t o n e , |3-ionone (78) was r e d u c e d , u s i n g Raney n i c k e l , a t the s t e r i c a l l y l e s s h i n d e r e d d i - s u b s t i t u t e d double bond t o g i v e d i h y d r o - ^ - i o n o n e ( 7 9 ) . I t was found t h a t d u r i n g the r e a c t i o n a p p r o x i m a t e l y 10% of tetrahydro-y5-ionone (80) was formed. R e a c t i o n o f dihydro - jS - ionone (79) i n a m o d i f i e d W i t t i g r e a c t i o n w i t h t r i m e t h y l phosphonoacetate (81) gave a m i x t u r e o f c i s - and t r a n s - m e t h y l m o n o c y c l o f a r n e s a t e (82) i n a r a t i o o f a p p r o x i m a t e l y 60:40. R e d u c t i o n o f m e t h y l m o n o c y c l o f a r n e s a t e (82) w i t h l i t h i u m aluminium h y d r i d e then gave m o n o c y c l o f a r n e s o l ( 4 7 ) . - 54 -(MeO) 2-P-CH -CO"2Me ^ (MeO) 2-P-CH-C0 2Me (82) (47) A sample o f r a d i o - a c t i v e l y l a b e l l e d m o n o c y c l o f a r n e s o l was p r e p a r e d i n an e x a c t l y s i m i l a r manner u s i n g r a d i o - l a b e l l e d t r i m e t h y l p hosphonoacetate p r e p a r e d by h e a t i n g t r i m e t h y l p h o s p h i t e and 1 - ^ C - m e t h y l bromoacetate ( 8 3 ) . A d m i n i s t r a t i o n o f a sample o f r a d i o - l a b e l l e d m o n o c y c l o f a r n e s o l (4 7) t o T r i c h o t h e c i u m roj^cum, f o l l o w e d by i s o l a t i o n o f the t r i c h o t h e c i n (38) i n the manner d e s c r i b e d above gave a s p e c i f i c - 55 -0 (MeO) 3P + Br-CH 2-C0 2Me (83) (MeO) 2-P-CH 2C0 2Me (81) (79) OH LAH (47) *C0 2Me (82) i n c o r p o r a t i o n o f 0.003%. H y d r o l y s i s t o t r i c h o t h e c o l o n e (39) w i t h l o s s o f 70% o f the r a d i o a c t i v i t y i n d i c a t e d t h a t s u b s t a n t i a l d e g r a d a t i o n had t a k e n p l a c e p r i o r t o i n c o r p o r a t i o n . I n d i r e c t s u p p o r t f o r th e s e r e s u l t s w i t h ^ - b i s a b o l e n e (45) and m o n c c y c l o f a r n e s o l (47) has been p r o v i d e d d u r i n g the co u r s e 55-58 of t h i s work by two groups o f workers who have r e p o r t e d r e s u l t s w h i c h are c o n s i s t e n t w i t h our c o n c l u s i o n s . 5 5 N The f i r s t o f t h o s e concerns the b i o s y n t h e s i s o f h e l i c o b a s i d i n ( 8 4 ) , a cuparane type o f s e s q u i t e r p e n e . OH H0 2C CH 20H 4R-(4- 3H, 2- 1 4C) m e v a l o n i c a c i d 5 6 -When 4R-(4-^H, 2- 1 4C) m e v a l o n i c a c i d l a c t o n e was f e d t o the organi s m w h i c h produced h e l i c o b a s i d i n (84) i t was found t h a t 2 o f the pro -R-hydrogen atoms from the mevalonic a c i d l a c t o n e ( t r i t i u m i n t h i s case) were r e t a i n e d i n the h e l i c o b a s i d i n . S i n c e t h e s e are the hydrogen atoms w h i c h are known to be l o c a t e d on the double 2 4 bonds o f f a r n e s y l pyrophosphate t h i s seems to p r e c l u d e the i n t e r m e d i a c y o f ^ - b i s a b o l e n e i n the b i o s y n t h e s i s o f h e l i c o b a s i d i n ( 8 4 ) , and hence p r o b a b l y a l l cuparane type s e s q u i t e r p e n e s , a t l e a s t i n m i c r o - o r g a n i s m s . As the t r i c h o t h e c a n e s k e l e t o n i s a r e a r r a n g e d cuparane s k e l e t o n t h i s r e s u l t a l s o seems t o p r e c l u d e the i n t e r m e d i a c y o f ^ - b i s a b o l e n e i n the b i o s y n t h e s i s o f t r i c h o t h e c i n . The o t h e r r e s u l t s 5 ^ 5 ^ are from Hanson's l a b o r a t o r y and con c e r n h e l i c o b a s i d i n ( 8 4 ) , t r i c h o t h e c i n (38) and t r i c h o d e r m i n ( 4 3 ) . T h i s group has s t u d i e d the f a t e o f m e v a l o n o i d hydrogen atoms and from these s t u d i e s has o b t a i n e d i n f o r m a t i o n on the b i o s y n t h e s i s 3 14 of t h e s e t h r e e compounds. Thus when (2- H 2, 2- C) m e v a l o n i c a c i d was f e d i t was f o u n d 5 ^ t h a t 5 t r i t i u m atoms had been i n c o r p o r -a t e d i n t o t r i c h o d e r m i n ( 4 3 ) . H y d r o l y s i s o f the a c e t a t e group and o x i d a t i o n of the secondary a l c o h o l so formed r e s u l t e d i n l o s s o f 1 - - 3 14 t r i t i u m atom. When 2R-(2- H, 2- C) m e v a l o n i c a c i d was f e d i t was found t h a t t h e r e were 2 t r i t i u m atoms i n c o r p o r a t e d b e f o r e and a f t e r o x i d a t i o n . T h i s d e f i n e s w h i c h hydrogen i s l o s t i n i n t r o d u c i n g the a c e t o x y group. A s i m i l a r experiment w i t h t r i c h o t h e c i n (38) showed the i n c o r p o r a t i o n o f 4 t r i t i u m atoms b e f o r e o x i d a t i o n and 2 t r i t i u m atoms a f t e r o x i d a t i o n when (2- 3H , 2- 1 4C) m e v a l o n i c acid, was f e d as a p r e c u r s o r . The c o n c l u s i o n s drawn from th e s e e x p e r i m e n t s are t h a t the h y d r o x y l a t i o n o f the 5-membered r i n g o c c u r s w i t h o v e r a l l r e t e n t i o n o f c o n f i g u r a t i o n at C^, and t h a t i n the b i o s y n t h e s i s o f t r i c h o t h e c i n (38) a t r i t i u m atom m i g r a t e s from C p to Cr/ (where i t - 57 -( 4 3 ) 4 T r e t a i n e d 5 T r e t a i n e d 2T r e t a i n e d 2T r e t a i n e d - 58 -4T r e t a i n e d 2T r e t a i n e d R = i s o c r o t o n y l ( 3 8 ) 4T r e t a i n e d i s e a s i l y exchanged, hence the l o s s o f 2 t r i t i u m atoms a f t e r o x i d a t i o n ) . F u r t h e r e v i d e n c e f o r t h i s m i g r a t i o n was p r o v i d e d 3 14 by f e e d i n g ( 5 - H^, 2 - C) m e v a l o n i c a c i d when o n l y 4 o f the e x p e c t e d 5 tritium„atoms are r e t a i n e d . The a u t h o r s suggest t h a t the epoxide c r o t o c i n ( 8 5 ) may be i n v o l v e d i n the b i o s y n t h . e s i s o f t r i c h o t h e c i n and t h a t i t may be r e a r r a n g e d t o t r i c h o t h e c i n ( 3 8 ) . - 59 -57 In the. second paper o f t h i s s e r i e s Hanson and h i s group r e p e a t the work o f the Japanese w o r k e r s 5 5 on h e l i c o b a s i d i n (84) 3 14 g e t t i n g the same r e s u l t . I n a d d i t i o n , by f e e d i n g (2- H, 2~ C) g e r a n y l pyrophosphate (17) they show t h a t one o f the t r i t i u m 3 14 atoms r e t a i n e d when 4R-(4- H, 2- C) m e v a l o n i c a c i d i s f e d must be from the c e n t r a l m e v a l o n o i d u n i t , thus c l e a r l y showing t h a t ^ - b i s a b o l e n e (45) cannot be a p r e c u r s o r as t h i s hydrogen i s l o s t 5 2 i n f o r m i n g " f t - b i s a b o l e n e . In a p r e v i o u s paper t h i s group had shown t h a t 2 t r i t i u m atoms are r e t a i n e d i n t r i c h o t h e c i n when 3 14 4R-(4- H, 2- C) mevalonate was f e d t o the organi s m p r o d u c i n g t r i c h o t h e c i n . One o f t h e s e t r i t i u m atoms, l o c a t e d at C ^ Q , was 52 shown to come from the t e r m i n a l m e v a l o i d u n i t of f a r n e s y l p y r ophosphate ( 1 8 ) , and the o t h e r t r i t i u m atom was l o c a t e d at C^, but i t s o r i g i n was not d e f i n e d . By f e e d i n g ( 2 , H, 2- C) 57 g e r a n y l p y rophosphate (17) they now showed t h a t the hydrogen l o c a t e d at C 0 comes from the c e n t r a l m e v a l o n o i d u n i t o f f a r n e s y l p yrophosphate ( 1 8 ) . T h i s a l s o c l e a r l y p r e c l u d e s Y - b i s a b o l e n e as a p r e c u r s o r o f t r i c h o t h e c i n . Hanson and h i s c o l l a b o r a t o r s 5 7 suggest a p o s s i b l e c o n c e r t e d mechanism' f o r the b i o s y n t h e s i s - 60 -- 61 -o f t r i c h o t h e c i n , a mechanism i n w h i c h a hydrogen s h i f t o c c u r s i n an enzyme d i s p l a c e m e n t s t e p . 5 8 In the t h i r d paper t h i s group d e s c r i b e s some f u r t h e r e x p e r i m e n t s on the b i o s y n t h e s i s o f h e l i c o b a s i d i n (84) w h i c h add c o n f i r m a t i o n t o t h e i r s u g g e s t e d mechanism f o r the b i o s y n t h e s i s of cuparane type s e s q u i t e r p e n e s . I n an a l t e r n a t i v e mechanism f o r the b i o s y n t h e s i s o f t r i c h o t h e c i n (38) i t was c o n s i d e r e d t h a t the oxygen f u n c t i o n which appears at °f t r i c h o t h e c i n (38) may be i n t r o d u c e d v e r y e a r l y from a p r e c u r s o r such as f a r n e s o l e p o x i d e ( 4 8 ) , w h i c h c o u l d e x p l a i n the f a i l u r e t o i n c o r p o r a t e the p r e v i o u s l y t e s t e d compounds. OH (48) as f o r mono-c y c l o f a r n e s o l H = .0 OR (38) 62 -Although, f a r n e s o l e p o x i d e (48) has not been found i n n a t u r e 79 one o f i t s e n a n t i o m e r i c forms has been found t o be m e t a b o l i s e d by c e r t a i n f u n g i , and t h i s f a c t has been used i n a r e s o l u t i o n o f ra c e m i c f a r n e s o l e poxide ( 4 8 ) . F a r n e s o l epoxide has a l s o been p o s t u l a t e d as a p r e c u r s o r i n the b i o s y n t h e s i s o f the s e s q u i t e r p e n o i d 80 ' , 81 compounds i r e s i n (86) and f a r n e s i f e r o l - A ( 8 7 ) . (86) (87) G e r a n y l acetone. (88) was c o n s i d e r e d t o be a s u i t a b l e i n t e r -mediate i n a s y n t h e s i s o f r a d i o - l a b e l l e d f a r n e s o l e poxide ( 4 8 ) . 8 2 G e r a n y l acetone (88) has been prepared, b e f o r e u by c o n d e n s i n g g e r a n y l bromide (89) w i t h the a n i o n o f e t h y l a c e t o a c e t a t e (90) , f o l l o w e d by h y d r o l y s i s and d e c a r b o x y l a t i o n o f the i n t e r m e d i a t e , g - k e t o - e s t e r ( 9 1 ) . (89) (90) , (91) - 63 -l)NaOH/H 20 2)H 0 ^ (88) G e r a n y l bromide (89) was p r e p a r e d by the a c t i o n o f t r i p h e n y l phosphine and carbon t e t r a b r o m i d e on g e r a n i o l ( 9 2 ) . The r e a c t i o n + C B r 4 + PPh,. — > (92) (89) 8 3 i s thought to p r o c e e d as f o l l o w s : + Br-CBr. Ph 3P:' + P h 3 P - B r Ph,?0-R ~ + 3 CBr. P h 3 P - B r + ROH - , P h 3 P CBr Ph^-O-R + 3 Br CHBr. 0 RBr From g e r a n y l acetone (88) two r o u t e s t o f a r n e s o l e poxide are p o s s i b l e , namely p r i o r f o r m a t i o n of the e p o x i d e f u n c t i o n f o l l o w e d by subsequent e l a b o r a t i o n o f the a l l y l i c a l c o h o l m o i e t y , or the r e v e r s e sequence. For reasons o u t l i n e d below the f i r s t o f t h e s e methods was chosen f o r the s y n t h e s i s o f f a r n e s o l e p o x i d e . Thus i t was c o n s i d e r e d t h a t (a) t h i s r o u t e e n t a i l e d i n s e r t i o n o f the - 64 -r a d i o - l a b e l a t a l a t e r s t a g e t h a n the a l t e r n a t e r o u t e , and (b) i t i n v o l v e d e p o x i d a t i o n o f one double bond s e l e c t i v e l y from o n l y two double bonds r a t h e r than from t h r e e . Model s t u d i e s were p e r f o r m e d r e a c t i n g v a r i o u s d e r i v a t i v e s o f 2-bromo-ethanol i n W i t t i g r e a c t i o n s w i t h ketone (93) to g i v e d e r i v a t i v e s o f g e r a n i o l (92) but the r e s u l t s were d i s a p p o i n t i n g w i t h no e v i d e n c e f o r the f o r m a t i o n o f g e r a n i o l d e r i v a t i v e s b e i n g o b t a i n e d . B r C H 2 C H 2 O R ' ' \ •> Ph^CH^CHAOR - Br In a n o t h e r attempt to produce g e r a n i o l (92) from ketone (93) 84 a m o d i f i c a t i o n o f a r e a c t i o n used by Corey and Yamamoto i n a s y n t h e s i s o f °<-santalol (94) was t r i e d . In t h e i r s y n t h e s i s o f c * - s a n t a l o l (94) Corey and Yamamoto r e a c t e d aldehyde (95) w i t h e t h y l i d i n e t r i p h e n y l phosphorane a t -78°C to g i v e the normal W i t t i g type i n t e r m e d i a t e ( 9 6 ) . At t h i s low tem p e r a t u r e i n t e r -mediate (96) i s s t a b l e and a d d i t i o n o f one e q u i v a l e n t o f base gave 6 -oxidephosphonium y l i d e (97) w h i c h was a l l o w e d t o r e a c t w i t h - 65 -p a r a f o r m a l d e h y d e a t Q°C. Aqueous work up then gave fc<-santalol C94). The mechanism and s t e r e o c h e m i s t r y of t h i s i n t e r e s t i n g H 0 (94) r e a c t i o n have been r e v i e w e d . I t was thought t h a t by u s i n g m e t h y l ketone (9 3) i n s t e a d o f aldehyde ( 9 5 ) , and u s i n g methylene t r i p h e n y l phosphorane i n s t e a d o f e t h y l i d i n e t r i p h e n y l phosphorane, g e r a n i o l (92) might be p r o d u c e d ; When the r e a c t i o n was c a r r i e d out u s i n g t h o s e r e a g e n t s a compound - 66 -was produced i n about 25% y i e l d ( p u r i f i e d by g l c ) which, e x h i b i t e d s p e c t r a l c h a r a c t e r i s t i c s very s i m i l a r to g e r a n i o l (92). However c l o s e r examination of the nmr s p e c t r u m ^ (Figure X V I I I ) , and comparison w i t h an a u t h e n t i c sample showed that the compound was the cis-double bond isomer, n e r o l (98). (98) - 67 -g e r a n i o l n e r o l P r e d i c t e d 8 . 34 C.2) ., 8 . 40 (1) 8 . 27 (1) , 8 . 34 U ) , 8 . 40 (1) y a l u e A u t h e n t i c -8 . 33(2) , 8 .41 (1) 8 . 27 C I ) , 8 . 31 QL) , 8 . 39 CI ) sample S y n t h e t i c sample 8.27 C I ) ,8.32 CI) ,8.38(1) Cvalues are i n % u n i t s , f i g u r e s i n parentheses are no. of methyl groups) F i g u r e X V I I I : nmr values f o r g e r a n i o l and n e r o l As the y i e l d o f t h i s r e a c t i o n was r a t h e r low, and the p u r i f i c a t i o n o f a s m a l l r a d i o - a c t i v e sample would be d i f f i c u l t , t h i s r o u t e was abandoned. The b e s t r o u t e t o f a r n e s o l e poxide C48) thus appeared to be p r i o r f o r m a t i o n o f f a r n e s o l C99) , p r e p a r e d from g e r a n y l acetone C88) v i a m e t h y l f a r n e s a t e (10 0) i n a manner analogous to the p r e p a r a t i o n o f m o n o c y c l o f a r n e s o l C47) from dihydro-jS-ionone ( 7 9 ) , f o l l o w e d by s e l e c t i v e e p o x i d a t i o n o f the 10,11-double bond. 0 CMeO) 2-P-CH 2C0 2Me (83) > (88) (100) - 68 -LAH (48) R e a c t i o n o f g e r a n y l acetone (88) w i t h the a n i o n formed by the a c t i o n o f sodium h y d r i d e on t r i m e t h y l p h o s p h o n o a c e t a t e (81) gave m e t h y l f a r n e s a t e (100) i n good y i e l d . Subsequent r e d u c t i o n e s t e r (100) w i t h l i t h i u m aluminium h d y r i d e gave f a r n e s o l (99) i d e n t i c a l i n a l l r e s p e c t s w i t h an a u t h e n t i c sample o f f a r n e s o l . 8 7 88 I t has a l r e a d y been shown by van Tamelen and co-workers ' t h a t polyenes such as f a r n e s o l (99) and squalene (24) may be s e l e c t i v e l y e p o x i d i s e d a t the t e r m i n a l double bond by t r e a t m e n t w i t h N-bromosuccinimide i n aqueous glyme (dimethoxy e t h a n e ) , f o l l o w e d by base c a t a l y s e d r i n g c l o s u r e o f the i n t e r m e d i a t e bromo h y d r i n . The s e l e c t i v i t y i n the s e r e a c t i o n s i s p r o b a b l y due to the f a c t t h a t i n t h i s s o l v e n t system the f a r n e s o l c h a i n i s c o i l e d up on i t s e l f e x p o s i n g o n l y the ends t o the r e a g e n t . Treatment o f f a r n e s o l (99) w i t h one e q u i v a l e n t o f N-bromo-s u c c i n i m i d e i n 70% aqueous glyme, f o l l o w e d by r e a c t i o n w i t h m e t h a n o l i c p o t a s s i u m h y d r o x i d e s o l u t i o n gave a p r o d u c t whose s p e c t r a l c h a r a c t e r i s t i c s i n d i c a t e d t h a t i t was f a r n e s o l - 1 0 , 1 1 -e p o x i d e ( 4 8 ) . - 69 -NBS aqueous glyme (99) KOH/MeOH (48) P r e p a r a t i o n o f a r a d i o - l a b e l l e d sample o f f a r n e s o l e p o x i d e (48) was a c h i e v e d i n an e x a c t l y analogous manner s t a r t i n g w i t h 14 1- C-methyl bromoacetate (83) and t r i m e t h y l p h o s p h i t e . (MeO) 3P + Br-CH 2-C0 2Me (83) 0 II ^ (MeO) 2P -CH 2C0 2Me (81) C791 A d m i n i s t r a t i o n o f a sample o f r a d i o - l a b e l l e d f a r n e s o l epoxide (48) t o T r i c h o t h e c i u m roseum f o l l o w e d by i s o l a t i o n o f the t r i c h o t h e c i n (38) i n the u s u a l manner gave a s p e c i f i c i n c o r p o r a t i o n o f 0.007%. H y d r o l y s i s to t r i c h o t h e c o l o n e (39) w i t h l o s s of over 501 o f the r a d i o a c t i v i t y i n d i c a t e d t h a t f a r n e s o l e p o x i d e i s not a p r e c u r s o r o f t r i c h o t h e c i n . As Y _ b i s a b o l e n e ( 4 5 ) , m o n o c y c l o f a r n e s o l (47) and f a r n e s o l e p oxide (48) have a l l been shown not to e x h i b i t p r e c u r s o r a c t i v i t y i n the b i o s y n t h e s i s of t r i c h o t h e c i n ( 3 8 ) , i t would seem t h a t the d i r e c t c y c l i s a t i o n o f f a r n e s y l pyrophosphate (18) t o a cuparane s k e l e t o n , as p r o p o s e d 5 ^ 5 ^ by Hanson and h i s c o l l a b o r a t o r s i s p r o b a b l y c o r r e c t . H o w e v e r i t i s p o s s i b l e t h a t the h y d r i d e m i g r a t i o n i s more complex t h a t as i t i s p o s t u l a t e d i n t h e i r mechanism. 8 E x p e r i m e n t a l d e t a i l s o f t h e i r work have v e r y r e c e n t l y been p u b l i s h e d . 90 A v e r y r e c e n t p u b l i c a t i o n by Machida and Nozoe d e s c r i b e s the i n c o r p o r a t i o n of t r i c h o d i e n e (101) i n t o t r i c h o t h e c i n ( 3 8 ) . T r i c h o d i e n e i s the compound o b t a i n e d a f t e r the m i g r a t i o n o f the m e t h y l groups i n the p o s t u l a t e d b i o s y n t h e t i c schemes (see F i g u r e s X I I I and X V I ) . - 71 -(101) F i g u r e XX: N u c l e a r magnetic resonance spectrum o f T r i c h o t h e c i n 3000 2000 1500 CM-l 1000 900 800 700 j tit i l i . t t - , i i I i i I 1 i I i t t t i t , I I , I , U ,f, i.(, i ,1, „ i , ?, w , ,1 7 8 9 10 WAVELENGTH (MICRONS) F i g u r e XXI: I n f r a r e d spectrum o f T r i c h o t h e c o l o n e T ~ T 1 1 1 1 1 1 1 1 I I I . ! .1 1 1 1 1 | 1 1 j 1 1 1 1 1 1 1 1 1 1 1 ' 1 * r • 5 0 0 > 1 I i 1 I 1 I 1 1 4 0 0 ! • 1 . 3 0 0 1 1 | | | 1 1 1 I 1 ! 1 1200 •! : 1 I 1 1 1 100 I )_ j i ! ( ) t F i g u r e X X I I : N u c l e a r magnetic resonance spectrum o f T r i c h o t h e c o l o n e - 76 -EXPERIMENTAL A l l m e l t i n g p o i n t s were d e t e r m i n e d on a K o f l e r b l o c k and are u n c o r r e c t e d . I n f r a r e d s p e c t r a CO max) were r e c o r d e d on a P e r k i n - E l m e r I n f r a c o r d model 137 s p e c t r o p h o t o m e t e r . N u c l e a r magnetic resonance s p e c t r a (f) were d e t e r m i n e d In ca r b o n t e t r a -c h l o r i d e or d e u t e r i o c h l o r o f o r m s o l u t i o n and r e c o r d e d on a J e o l c o C-60H s p e c t r o m e t e r o r on V a r i a n A s s o c i a t e s s p e c t r o m e t e r s , model A-60 o r model T-60. S i g n a l p o s i t i o n s are g i v e n i n the T i e r s 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 as an i n t e r n a l s t a n d a r d ; the m u l t i p l i c i t y , c o u p l i n g c o n s t a n t s (where a p p r o p r i a t e ) and i n t e g r a t e d peak areas are i n d i c a t e d i n p a r e n t h e s e s ; s = s i n g l e t , b r . s = b r o a d s i n g l e t , d = d o u b l e t , t = t r i p l e t , q = q u a r t e t , m = m u l t i p l e t . Mass s p e c t r a (M +) were d e t e r m i n e d w i t h an AEI model MS 9 s p e c t r o m e t e r . M i c r o a n a l y s e s were p e r f o r m e d by Mr. 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 , U n i v e r s i t y o f B r i t i s h C o l umbia. T h i n l a y e r c h r o m a t o g r a p h i c r e s u l t s were o b t a i n e d 254 u s i n g s i l i c a g e l HF o f 0.5 mm t h i c k n e s s . R a d i o a c t i v i t y was measured w i t h a N u c l e a r - G h i c a g o Mark I Model 6860 L i q u i d S c i n t i l l a t i o n c o u n t e r . The r a d i o a c t i v i t y o f a sample was c a l c u l a t e d t a k i n g i n t o account the c o u n t i n g e f f i c i e n c y w h i c h was d e t e r m i n e d f o r each sample by the e x t e r n a l s t a n d a r d t e c h n i q u e (see i n s t r u m e n t manual) u t i l i s i n g the b u i l t - i n barium-133 gamma s o u r c e . The l i q u i d s c i n t i l l a t i o n s o l u t i o n used w i t h the c o u n t e r was made up from L i q u i f l u o r (New E n g l a n d N u c l e a r ) (42 ml) and t o l u e n e (1 1 ) , g i v i n g the f o l l o w i n g c o n c e n t r a t i o n s of r e a g e n t s : t o l u e n e (1 1 ) , 2 , 5 - d i p h e n y l o x a z o l e (4 g) and 1,4-bis [ 2 - ( 5 - p h e n y l o x a z o l y l ) ] benzene (0.05 g ) . - 77 -In p r a c t i c e a weighed sample was d i s s o l v e d i n benzene (1 ml) i n a c o u n t i n g v i a l and made up to 15 ml w i t h s c i n t i l l a t i o n s o l u t i o n . M e t h y l ( 4 - m e t h y l c y c l o h e x -3-ene) c a r b o x y l a t e (58) Iso p r e n e (1) (9.0 g, 0.13 m o l e ) , m e t h y l a c r y l a t e (59) (11.3 g, 0.13 mole) and hydroquinone (0.5 g) were s e a l e d , under vacuum, i n a g l a s s tube and h e a t e d a t 80°C o v e r n i g h t . The r e s u l t i n g c o l o u r l e s s o i l was d i s t i l l e d t o g i v e m e t h y l ( 4 - m e t h y l -c y c l o h e x - 3-ene) c a r b o x y l a t e (58) [plus a l i t t l e o f the 3-methyl isomer (63)] (14.7 g, 73.5%) as a c o l o u r l e s s o i l , b.p. 78-80°C (11 mm) [ l i t . 6 7 85-86° (15 mm)]. X ) ( l i q u i d f i l m ) 2925, ITlclX 1735, 1430 , 1170 cm' 1; T ( C C 1 4 ) 4.63 ( s , 1H) , 6.36 ( s , 3H) , 7.20-8.20 (m, 7H), 8.33 ( s , 3H). 4-M e t h y l c y c l o h e x - 3 - e h e c a r b o x y l i c a c i d (5 5) (a) M e t h y l ( 4 - m e t h y l c y c l o h e x - 3 - e n e ) c a r b o x y l a t e (58) (14.7 g, 0.09 mole) was added t o a s o l u t i o n of sodium h y d r o x i d e (11.5 g) i n w a t e r (60 m l ) . The m i x t u r e was s t i r r e d a t 65°C u n t i l i t was homogeneous («~-^ 2 h r ) , when i t was c o o l e d and e x t r a c t e d w i t h e t h e r t o remove unchanged s t a r t i n g m a t e r i a l . The b a s i c s o l u t i o n was then a c i d i f i e d ( l i t m u s ) w i t h c o n c e n t r a t e d s u l p h u r i c a c i d and e x t r a c t e d t h r e e times w i t h benzene. The combined benzene e x t r a c t s were washed t h o r o u g h l y w i t h b r i n e and e v a p o r a t e d i n vacuo to g i v e a gummy w h i t e s o l i d w h i c h was r e p e a t e d l y c r y s t a l l i s e d from p e t r o l e u m e t h e r (60-80°C) to g i v e 4-methylcyclohex-3-ene c a r b o x y l i c a c i d (55) (2.5 g, 19%) as w h i t e , g l i s t e n i n g n e e d l e s , m.p. 99-100°C ( l i t . 6 7 98.5-99°C). "O ( C H C l j ) 2905, 2655, 1705 c m - 1 ; T ( C D C 1 3 ) 1.13 ( s , 1H) , 4.36 ( s , 1H) , 7.4-8.2 (m, 7H) , - 78 -8.37 ( s , 3H) . (b) A c r y l i c a c i d (45.2 g, 0.63 m o l e ) , i s o p r e n e (42.8 g, 0.63 mole) and hydroquinone (0.8 g) were s e a l e d , under vacuum, i n 4 g l a s s tubes and h e a t e d a t 95°C f o r 16 h r . The r e s u l t i n g gummy s o l i d was r e p e a t e d l y c r y s t a l l i s e d from p e t r o l e u m e t h e r (60-80°C) t o g i v e 4 -methylcyclohex-3-ene c a r b o x y l i c a c i d (55) (26.6 g, 301) i d e n t i c a l i n a l l r e s p e c t s t o t h a t p r e p a r e d under (a) above. 4-Hydroxy-4-methylcyclohexane c a r b o x y l i c a c i d l a c t o n e (65) 4-M e t h y l c y c l o h e x - 3 - e n e c a r b o x y l i c a c i d (55) (100 mg, 0.72 mmole) and 98% f o r m i c a c i d (2 ml) were h e a t e d o v e r n i g h t a t 80°C. The r e a c t i o n m i x t u r e was worked up by a d d i n g e t h e r and wa t e r . The aqueous l a y e r was e x t r a c t e d t w i c e w i t h e t h e r and the combined e t h e r e x t r a c t s washed t w i c e w i t h b r i n e , t w i c e w i t h s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n , t w i c e w i t h b r i n e and d r i e d (Na2SO^). E v a p o r a t i o n o f the e t h e r a t reduced p r e s s u r e gave s l i g h t l y y e l l o w c r y s t a l s w h i c h were r e c r y s t a l l i s e d from p e t r o l e u m e t h e r (60-80°C) to g i v e 4 - h y d r o x y - 4 - m e t h y l c y c l o h e x a n e c a r b o x y l i c a c i d l a c t o n e (65) (52.0 mg, 52.0%) as w h i t e p l a t e s , m.p. 68-69°C ( l i t . 7 1 m.p. 68.0-68.7°C). " O m a x (CC1 4) 2950, 1760, 1230, 1070, 960 c m _ 1 ; ^ 0 ( C H C 1 J 2965,.1740, 1230, 1070, 960 II13.X o cm" 1 ; T ( C C 1 4 ) 7.50 ( s , 1H) , 8.20 ( s , 8H) , 8.65 ( s , 3H) . 2 - c y c l o p r o p y l p r o p a n - 2 - o l (57) M e t h y l i o d i d e (186.0 g, 1.31 mole) i n e t h e r (350 ml) was added t o magnesium t u r n i n g s (35.0 g, 1.46 mole) a t such a r a t e so as to m a i n t a i n a v i g o r o u s r e f l u x . A f t e r the r e a c t i o n s u b s i d e d the r e a c t i o n m i x t u r e was h e a t e d on a hot wa t e r b a t h f o r 0.5 h r . - 79 -On c o o l i n g t o 0°C, c y c l o p r o p y l m e t h y l ketone (56) (100.0 g, 1.19 mole) i n e t h e r (350 ml) was added w i t h s t i r r i n g , and the r e a c t i o n m i x t u r e s t i r r e d a t room t e m p e r a t u r e f o r 2 h r , when i t was a g a i n c o o l e d to 0°C and s a t u r a t e d aqueous ammonium a c e t a t e s o l u t i o n added. The m i x t u r e was s t i r r e d u n t i l two c l e a r l a y e r s r e s u l t e d . They were then s e p a r a t e d and the aqueous l a y e r was e x t r a c t e d 3 times w i t h e t h e r . The combined o r g a n i c l a y e r s were washed w i t h b r i n e , d r i e d (MgSO^) and the e t h e r e v a p o r a t e d i n vacuo to g i v e a y e l l o w o i l which was d i s t i l l e d t o g i v e . 2 - c y c l o p r o p y l p r o p a n - 2 - o l (57) (86.5 g, 73%) as a c o l o u r l e s s o i l , b.p. 124-128°C (760 mm) ( l i t . 6 4 b.p. 121-122°C). X ) 0 ( l i q u i d f i l m ) 3400, 3100, 2950, 111 3. JC 1460, 1150, 1020 , 960 , 920, 845, 825 cm" 1 ; T ( C C 1 4 ) , 7.66 ( s , 1H) , 8.86 ( s , 6H), 9.00-9.40 (m, 4H), 9.73 ( d , J = 6 Hz, 6H). 5-bromo-2-methylpent-2-ene (49) To 2 - c y c l o p r o p y l p r o p a n - 2 - o l (57) (86.5 g, 0.865 mole) was added, w i t h c o o l i n g and s t i r r i n g , 48% HBr (410 ml) over a p e r i o d o f 0.3 h r . A f t e r s t i r r i n g f o r 0.5 h r w i t h c o o l i n g and 0.5 h r a t room temperature the r e a c t i o n m i x t u r e was e x t r a c t e d 3 times w i t h e t h e r , the combined e t h e r e x t r a c t s were washed t h o r o u g h l y w i t h s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n , washed w i t h b r i n e , d r i e d (Na2S0 4) and the e t h e r e v a p o r a t e d a t reduced p r e s s u r e to g i v e an o i l w h i c h was d i s t i l l e d t o g i v e 5-bromo-2-methylpent-2-ene (49) 102.7 g, 72.5%) as a c o l o u r l e s s o i l , b.p. 77-82°C (60 mm) [ l i t . 6 4 b.p. 84-85°C (84 mm)]. \) ( l i q u i d f i l m ) 2960, in 3 .x 2925, 1670, 1445, 1375, 1265, 1205, 1095, 835 c m " 1 ; T ( C C 1 4 ) 4.83 ( t , J = 7Hz, 1H), 6.77 ( t , J = 7Hz, 2H), 7.53 ( t , J = 7Hz, 2H), 8.33 ( d , J = 4Hz, 6H) . - 80 -1~C4 ' -^Methylcyclohex-3 ' - e n y l ) - 1 - (4 ' - m e t h y l c y c l o h e x - 5 1 - e n y l carboxy) -5-methylhex-4-ene (68) To the G r i g n a r d complex, p r e p a r e d from 5-bromo-2-methylpent-2-ene (49) (34.2 g, 0.21 mole) and magnesium t u r n i n g s (5.5 g, 0.23 mole) i n dry e t h e r (100 m l ) , was added cuprous c h l o r i d e (200 mg) and the a c i d c h l o r i d e o f 4-methylcyclohex-3-ene c a r b o x y l i c a c i d (55) i n e t h e r (25 ml) p r e p a r e d from 4-methylcyclohex-3-ene-= c a r b o x y l i c a c i d (55) (26.6 g, 0.19 mole) and o x a l y l c h l o r i d e (26.7 g, 0.21 mole). Excess o x a l y l c h l o r i d e was removed by add i n g d r y benzene and e v a p o r a t i n g in. vacuo t o g i v e a y e l l o w o i l . A f t e r s t i r r i n g a t room temp e r a t u r e o v e r n i g h t the r e a c t i o n m i x t u r e was worked up by a d d i n g s a t u r a t e d aqueous ammonium a c e t a t e s o l u t i o n . The e t h e r l a y e r was s e p a r a t e d and the aqueous phase e x t r a c t e d t w i c e w i t h e t h e r . The combined e t h e r l a y e r s were washed t w i c e w i t h 2% aqueous sodium b i c a r b o n a t e s o l u t i o n , once w i t h b r i n e and d r i e d ( N a 2 S 0 4 ) . E v a p o r a t i o n o f the e t h e r at reduced p r e s s u r e gave a y e l l o w o i l which was d i s t i l l e d t o g i v e 1 - ( 4 - m e t h y l c y c l o h e x -3 ' - e n y l ) - 1 - ( 4 ' - m e t h y l c y c l o h e x - 3 ' - e n y l c a r b o x y ) - 5-methylhex-4-ene (68) (11.4 g, 35.01) as a c o l o u r l e s s o i l , b.p. 157-166°C ' (0.1 mm). OmSLX ( l i q u i d f i l m ) 2915 , 1730 , 1440 , 1170 , 920 , 835 , 805 , 770 , cm" 1; T ( C C 1 4 ) 4.68 ( s , 2H) , 4.80- 5.40 (m, 2H) , 7.20- 8.27 (m, 18H) , 8.33 (m,12H); M + M/e 330; C, 80.23; H, 10.33 ( c a l c . f o r C22 H34°2' C ' 8 0 - 0 0 ' H,10,30). 2 - M e t h y l - 6 - k e t o - 6 - ( 4 ' - m e t h y l c y c l o h e x - 5 ' - e n y l ) - h e x - 2 - e n e (54) (a) E t h e r e a l m e t h y l l i t h i u m s o l u t i o n (21.3 ml o f 2.4 M s o l u t i o n , 5.12 mmole) was added d r o p w i s e , w i t h s t i r r i n g and c o o l i n g t o a s o l u t i o n o f 1 - ( 4 ' m e t h y l c y c l o h e x - 3 ' - e n y l ) - 1 - ( 4 ' -m e t h y l c y c l o h e x - 3 ' - e n y l carboxy)-5-methylhex-4-ene (68) - 81 (4.2 g, 12.8 mmole) i n e t h e r (15 m l ) . A f t e r s t i r r i n g . a t room tem p e r a t u r e f o r 0.5 h r w a t e r (50 ml) was added. A f t e r s e p a r a t i o n o f the e t h e r l a y e r the aqueous phase was e x t r a c t e d t w i c e w i t h e t h e r and the combined e t h e r l a y e r s washed w i t h b r i n e and d r i e d (Na2S0^). A f t e r e v a p o r a t i o n o f the e t h e r i n vacuo the crude 7 3 p r o d u c t was d i s s o l v e d i n acetone (10 ml) and Jones r e a g e n t (1.65 ml) added dropwise w i t h s t i r r i n g and c o o l i n g . A f t e r s t i r r i n g a t room temperature f o r 0.25 h r the r e a c t i o n m i x t u r e was worked up by a d d i n g e t h e r and w a t e r . A f t e r s e p a r a t i o n o f the two l a y e r s the aqueous phase was e x t r a c t e d t w i c e w i t h e t h e r and the combined e t h e r l a y e r s washed w i t h b r i n e , d r i e d (Na2SO^) and the e t h e r e v a p o r a t e d a t reduced p r e s s u r e . D i s t i l l a t i o n o f the r e s u l t i n g y e l l o w o i l gave 2 - m e t h y l - 6 - k e t o - 6 - ( 4 ' - m e t h y l c y c l o h e x - 3 ' - e n y l ) -hex-2-ene (54) as a c o l o u r l e s s o i l , b.p. 80-84°C (0.25 mm) [ l i t . 6 3 b.p. 123-125°C (2 mm)]. \) ( l i q u i d f i l m ) 2905, 1710, in 3.x 1445, 1375, 920, 835, 805 cm" 1 ; T ( C C 1 4 ) 4.63 ( s , 1H), 4.95 Ct, J = 7Hz, 1H),7.20-8.20 (m, 11H), 8.33 ( s , 9H); M + m/e 206. (b) To the a c i d c h l o r i d e o f a c i d (55) p r e p a r e d as above from 4-methylcyclohex-3-ene c a r b o x y l i c a c i d (55) (0.9 g, 6.4 mmole) and o x a l y l c h l o r i d e (0.9 g, 7.0 mmole) was s l o w l y added the G r i g n a r d complex of bromide (49) p r e p a r e d as above from 5-bromo-2-methylpent-2-ene (49) (1.05 g, 6.4 mmole) and magnesium (0.17 g, 7.0 mmole). The s o l u t i o n was a l l o w e d t o s t i r a t room te m p e r a t u r e f o r 2 h r then worked up by add i n g s a t u r a t e d aqueous ammonium a c e t a t e and then e x t r a c t e d t h r e e times w i t h e t h e r . The e t h e r was e x t r a c t e d once w i t h s a t u r a t e d aqiieous sodium b i c a r b o n a t e s o l u t i o n to remove unchanged a c i d (55) , t h e n washed w i t h b r i n e - 82 -and d r i e d ( N a 2 S 0 4 ) . Removal o f the e t h e r i n vacuo gave a y e l l o w o i l CO.97 g, 75%) w h i c h was t r e a t e d i n two ways:-Ci) d i s t i l l a t i o n gave 2 - m e t h y l - 6 - k e t o - 6 - ( 4 ' - m e t h y l c y c l o h e x - 3 1 -en y l ) - h e x - 2 - e n e (54) i d e n t i c a l i n a l l r e s p e c t s t o t h a t o b t a i n e d i n (a) above. Cii) R e d u c t i o n o f a p o r t i o n o f the crude o i l w i t h sodium b o r o h y d r i d e i n m e t h a n o l , f o l l o w e d by chromatography on a l u m i n a gave a p r o d u c t t e n t a t i v e l y a s s i g n e d as 4 - m e t h y l p e n t - 3 - e n y l C 4 1 - m e t h y l c y c l o h e x - 3 1 - e n e ) c a r b o x y l a t e (70) as a c o l o u r l e s s o i l . -tt Y ( l i q u i d f i l m ) 2915 , 1735 , 1445, 1165 , 830 cm'1; T C C C 1 . ) 4.67 ( s , 1H), 4.90 ( t , J = 7Hz, 1H), 6.00 ( t , J = 7Hz, 2H), 7.33-8.20 Cm, 9H), 8.33 ( s , 9H). c < - B i s a b o l o l (46) To a s o l u t i o n o f m e t h y l magnesium i o d i d e p r e p a r e d from met h y l i o d i d e (106.5 mg, 0.75 mmole) and magnesium powder (19.8 mg, 0.83 mmole) i n e t h e r (5 m l ) , was added 2-methyl-6-keto-6-C4'-methyl-c y c l o h e x - 3 ' - e n y l ) -hex- 2 -ene (54) (51.5 mg, 0.25 mmole) i n e t h e r (2 m l ) . The m i x t u r e was a l l o w e d t o s t i r a t room te m p e r a t u r e f o r two hours then worked up by a d d i n g s a t u r a t e d aqueous ammonium a c e t a t e s o l u t i o n . The e t h e r l a y e r was s e p a r a t e d and the aqueous phase e x t r a c t e d t w i c e w i t h e t h e r . The combined e t h e r l a y e r s were washed w i t h b r i n e and d r i e d C N ^ ^ 0 ^ . E v a p o r a t i o n o f the e t h e r i n vacuo gave 0 < - b i s a b o l o l (46) (55 mg, 100%) as a c o l o u r l e s s o i l . \i ^ ( l i q u i d f i l m ) 3435 , 2915, 1440 , 1370, 1105, 83*0 , 800 cm" 1; t C C C l 4 ) 4.67 ( s , 1H) , 4.90 ( t , J = 7Hz, 1H) 7.60-8.23 (m, 7H) , 8.37 C s , 9H),8.50-8.87 (m, 5H), 8.93 ( d , J = 2Hz, 3H); M + m/e 222. - 83 -^ - B i s a b o l e n e (4 5) To ©(-bisabolol (46) (75 mg, 0.3 mmole) d i s s o l v e d i n p y r i d i n e (1 m l ) , was added a s o l u t i o n of phosphorus o x y c h l o r i d e (0.5 ml) i n p y r i d i n e (2 m l ) . The r e a c t i o n m i x t u r e was a l l o w e d t o s t i r o v e r n i g h t and then worked up by p o u r i n g onto i c e / w a t e r , f o l l o w e d by e t h e r e x t r a c t i o n . The combined e t h e r e x t r a c t s were washed w i t h b r i n e , d r i e d (MgSO^) and the e t h e r e v a p o r a t e d i n vacuo to g i v e a y e l l o w o i l (45.6 mg, 7 5 % ) . P u r i f i c a t i o n o f t h i s o i l by p r e p a r a t i v e g l c (180°C, 30% Carbowax) gave two components w i t h v e r y s i m i l a r r e t e n t i o n t i m e s . The l e s s v o l a t i l e peak (~"50%) was shown to be ^ - b i s a b o l e n e (45) ; " > 0 m a - j C ( l i q u i d f i l m ) 2935, 1440 , 1370 , 1145, 1100 , 910 , 825, 800 cm" 1; (CC1 4) 4.67 ( b r . s , 1H), 4.93 ( t , J - 7 Hz, 1H), 7.37 (m, 2H), 8.07 (m, 8H), 8.38 (m, 12H) . The more v o l a t i l e peak (~50%) was shown to be - b i s a b o l e n e (53) p l u s o t h e r u n i d e n t i f i e d i s o m e r ( s ) ,"\) m„ v ( l i q u i d Iii ci X. f i l m ) 2940, 1650, 1445, 1375, 920, 890, 830, 800 cm" 1; T (CC1 4) v e r y s i m i l a r to the nmr spectrum o f "tf-bisabolene (45) b u t showing f e a t u r e s [e.g. 5.30 ( s ) j a t t r i b u t a b l e to double bond isomers o f ^ - b i s a b o l e n e (45) . 1 4 C - L a b e l l e d Q ( - b i s a b o l o l (46) 14 c < - B i s a b o l o l (46) was p r e p a r e d as above u s i n g C-methyl i o d i d e (213.0 mg, 1.5 mmole, lmC, 0.667 mC/mmole) magnesium powder (39.6 mg , 1.65 mmole) and 2-methyl-6-keto-6 -(4'-methy1-c y c l o h e x - 3 1 - e n y l ) - h e x - 2 - e n e (54) (103 mg, 0.5 mmole). A f t e r work up t i c a n a l y s i s showed a t r a c e of s t a r t i n g ketone (54) l e f t 14 so i n a c t i v e m e t h y l l i t h i u m was added. The y i e l d o f C-o<-bisabolol (46) was 110.0 mg ( 1 0 0 % ) , and the s p e c i f i c a c t i v i t y was det e r m i n e d - 84 -to be 0.37 mC/mmole. 14 v C - L a b e l l e d p - b i s a b o l e n e (45) 14 C - L a b e l l e d o t - b i s a b o l o l (55 mg) was de h y d r a t e d as above and the crude p r o d u c t p u r i f i e d by chromatography on alu m i n a t o 14 v y i e l d C - l a b e l l e d o - b i s a b o l e n e (45) ( p l u s i s o m e r s ) (13 mg) as a c o l o u r l e s s o i l . I s o l a t i o n o f T r i c h o t h e c i n (38) from T r i c h o t h e c i u m roseum T r i c h o t h e c i u m roseum was grown as d e s c r i b e d i n the l i t -44 91 e r a t u r e ' ex c e p t t h a t the pH was not a d j u s t e d . T r i c h o t h e c i n Q 1 was i s o l a t e d as d e s c r i b e d i n the l i t e r a t u r e e x c e p t t h a t p r e n a r a t i v e t h i n l a y e r chromatography on s i l i c a g e l was used i n t p u r i f i c a t i o n r a t h e r than column chromatography on a l u m i n a . I n a t y p i c a l e x periment 10 f l a s k s o f T. roseum were a l l o w e d t o grow f o r t h r e e weeks. The b r o t h was then f i l t e r e d , the m y c e l i a washed w i t h a l i t t l e w a t e r and the combined washings and b r o t h e x t r a c t e d t w i c e , i n 1000 ml p o r t i o n s , w i t h carbon t e t r a c h l o r i d e (200 m l ) . E v a p o r a t i o n o f the carbon t e t r a c h l o r i d e gave a gum (148.6 mg) wh i c h was p u r i f i e d by t h i n l a y e r chromatography on s i l i c a g e l , d e v e l o p i n g w i t h p e t r o l e u m e t h e r : e t h y l a c e t a t e ( 1 : 1 ) . Under uv r a d i a t i o n the t r i c h o t h e c i n showed up as a dark band a p p r o x i m a t e l y h a l f way up the p l a t e . These bands were s c r a p e d o f f and the t r i c h o t h e c i n (38) l e a c h e d w i t h e t h y l a c e t a t e . E v a p o r a t i o n o f the e t h y l a c e t a t e gave t r i c h o t h e c i n (38) (94.3 mg) which was r e c r y s t a l l i s e d from p e t r o l e u m e t h e r (60-80°C) to g i v e pure t r i c h o t h e c i n (38) as w h i t e n e e d l e s , m.p. 116-118°C ( l i t . ^ m.p. 1 1 8 ° C ) ; ^ (CHCU) 1715 , 1685 cm" 1, max j - 85 T r i c h o t h e c o l o n e " (39) T r i c h o t h e c i n (38) (50 mg, 0.15 mmole) was s t i r r e d o v e r n i g h t a t room temperature i n IN m e t h a n o l l c p o t a s s i u m h y d r o x i d e s o l u t i o n (2 m l ) . Water (1.5 ml) was the n added and the methanol e v a p o r a t e d i n vacuo. The aqueous l a y e r was then e x t r a c t e d w i t h c h l o r o f o r m , removal o f w h i c h a t reduced p r e s s u r e gave a c o l o u r l e s s gum w h i c h s l o w l y c r y s t a l l i s e d . T h i s was r e c r y s t a l l i s e d from benzene-p e t r o l e u m e t h e r (60-80°C) (2:1) t o g i v e t r i c h o t h e c o l o n e (39) (39.8 mg, 100%) as w h i t e n e e d l e s , m.p. 182-184°C ( l i t . 4 6 183-184°C). • v m a x (CHC1 3), 3560, 1680 cm" 1. 14 v F e e d i n g of C - l a b e l l e d o - b i s a b o l e n e (45) t o T r i c h o t h e c i u m roseum A w e l l shaken m i x t u r e o f 1 4 C - l a b e l l e d ^ - b i s a b o l e n e (45) (13 mg, 0.024 mC) wat e r (5ml) and Tween 20 (0.5 ml) was e v e n l y d i s t r i b u t e d among 5 f l a s k s o f 1 week o l d c u l t u r e s o f T. roseum. A f t e r 3 weeks t r i c h o t h e c i n (38) was i s o l a t e d as b e f o r e and r e c r y s -t a l l i z e d t o c o n s t a n t s p e c i f i c a c t i v i t y (1.88 x 10 ^ mC/mmole) r e p r e s e n t i n g a s p e c i f i c i n c o r p o r a t i o n o f 0.005%. The r e m a i n i n g t r i c h o t h e c i n (38) was h y d r o l y z e d as above to g i v e t r i c h o t h e c o l o n e (39) w i t h s p e c i f i c a c t i v i t y o f 1.15 x 10 5 mC/mmole r e p r e s e n t i n g l o s s o f 40% o f the r a d i o a c t i v i t y . D i h y d r o - f t - i o n o n e (79) 3^ -Ionone (78) (19.2 g, 0.1 mole) was hydrog e n a t e d i n e t h a n o l (50 ml) u s i n g Raney n i c k e l (^4 g) as c a t a l y s t . A f t e r uptake o f the t h e o r e t i c a l volume o f hydrogen the c a t a l y s t was removed by f i l t r a t i o n , the e t h a n o l e v a p o r a t e d i n vacuo and the r e s u l t i n g o i l d i s t i l l e d t o g i v e a p r o d u c t (14.0 g, 72%) w h i c h was homogeneous - 86 -on one g l c column (175°C, 101 Carbowax) b u t was shown to c o n s i s t o f 2 p r o d u c t s i n the r a t i o o f <^9:1 on a second column (150°C, 5% Q F I ) . S e p a r a t i o n o f these two compounds (200°C, 3 0 % QFI) gave dihydro-/3-ionone (79) as a c o l o u r l e s s o i l , b.p. 115-116°C (9 mm) [ l i t . 7 8 b.p. 122-122.5°C (12 mm)];\) ( l i q u i d f i l m ) 2940, 1725, 1360, 1155 c m - 1 ; (CC1 4) 7.68 (m, 4H), 7.94 ( s , 3H), 8.43 ( s , 3H), 9.00 ( s , 6H); M + m/e 194; C,80.59; H, 11.48 ( c a l c . f o r C 1 5 H 2 2 0 : C, 80.35 ; H, 11.41; and t e t r a h y d r o - p - i o n o n e ( 8 0 ) ; ^ m a x ( l i q u i d f i l m ) v e r y s i m i l a r to t h a t o f d i h y d r o - ^ - i o n e ( 7 9 ) ; *£ (CC1 4) 7.70 (m, 2H), 7.95 ( s , 3H), 8.33-8.93 (m, 10H), 9.07 ( d , J = 5Hz, 6H), 9.13 ( d , J = 7 Hz, 3H); M + m/e 196. M e t h y l monocycTofarnesate (8 2) E t h e r washed 501 sodium h y d r i d e / o i l d i s p e r s i o n (.0.48 g, 0.01 mole NaH) was added t o dry benzene (50 m l ) , and t r i m e t h y l p h o s p h o n o -a c e t a t e (81) (1.82 g, 0.01 m o l e ) , i n benzene (5 m l ) , added a t room t e m p e r a t u r e . A f t e r s t i r r i n g a t room te m p e r a t u r e f o r 0.5 h r , dihydro-/S-ionone (79) (1.35 g, 0.007 m o l e ) , i n benzene (5 ml) was added dropwise and the s o l u t i o n r e f l u x e d f o r 15 h r . On c o o l i n g , w a t e r was added and the r e s u l t i n g o i l e x t r a c t e d 3 times w i t h e t h e r , the e t h e r was d r i e d (MgS0 4) and e v a p o r a t e d i n vacuo. D i s t i l l a t i o n gave methyl m o n o c y c l o f a r n e s a t e (82) (1.0 g, 571) as a c o l o u r l e s s o i l , b.p. 81-83°C (0.02 mm) ; ""O ( l i q u i d f i l m ) 2935, 1720, 1645, 1430, 1355, 1220, 1140 cm" 1; T ( C C 1 4 ) 4.37 ( b r . s , 1H), 6.37 ( s , 3H), 7.80 ( s , 2H), 7.84 ( s , 3H), 8.38 ( s , 3H), 8.97 ( s , 6H); M + m/e 250; C, 77.16; H, 10.59 ( c a l c . f o r C 1 6 H 2 6 ° 2 : C> 7 6 ' 7 6 > H> 10-47). - 87 -M o n o c y c l o f a r n c s o l (47) L i t h i u m aluminium h y d r i d e (30 mg, 0.79 mmole) was added t o a s o l u t i o n o f me t h y l m o n o c y c l o f a r n e s a t e (82) (300 mg, 1.2 mmole) i n e t h e r (10 m l ) . The r e s u l t i n g s o l u t i o n was s t i r r e d o v e r n i g h t and worked up by p o u r i n g onto i c e / d i l u t e HC1. The e t h e r l a y e r was s e p a r a t e d and the aqueous l a y e r was e x t r a c t e d 3 times w i t h e t h e r . The combined e t h e r e x t r a c t s were washed t h o r o u g h l y w i t h b r i n e , d r i e d (MgSO^) and the e t h e r e v a p o r a t e d in vacuo t o g i v e monocyclo-f a r n e s o l ( 4 7 ) , 252 mg, 95%) as a c o l o u r l e s s o i l , \ ) ( l i q u i d f i l m ) 3350, 2940, 1670, 1460, 1380, 1110, 1000 cm" 1; ~£ (CC1 4) 4.64 ( t , J = 7 Hz, 1H), 5.98 ( d , J = 7 Hz, 2H), 8.99 ( s , 6H); M + m/e 222. 1 4 C - T a b e l l e d m o n o c y c l o f a r n e s o l (47) l - 1 4 C - M e t h y l bromoacetate (83) (306 mg, 2 mmole, 1 mC, 0.5 mC/ mmole), t r i m e t h y l p h o s p h i t e (248 mg, 2 mmole) and benzene (1 ml) were r e f l u x e d f o r 5 h r . On c o o l i n g , benzene (4 ml) and pentane washed sodium h y d r i d e (501 o i l d i s p e r s i o n ) (240 mg, 5 mmole) were added and the s o l u t i o n a l l o w e d t o s t i r a t room temperature f o r 1.5 h r . D i h y d r o - ^ - i o n o n e (79) 388 mg, 2 mmole) i n benzene (5 ml) was th e n added and the s o l u t i o n a l l o w e d t o r e f l u x o v e r n i g h t . The r e a c t i o n m i x t u r e was worked up by adding w a t e r f o l l o w e d by e t h e r e x t r a c t i o n t o g i v e a y e l l o w o i l w h i c h was p u r i f i e d by chromato-graphy on s i l i c a . The f r a c t i o n e l u t i n g w i t h p e t r o l e u m e t h e r (40-60 u C ) / b enzene (3:1) c o n s i s t e d of m e t h y l m o n o c y c l o f a r n e s a t e (82) (171 mg). M e t h y l m o n o c y c l o f a r n e s a t e (82) (171 mg, 0.68 mmole) was s t i r r e d i n e t h e r (10 ml) w i t h l i t h i u m aluminium h y d r i d e (17.1 ^ 88 -mg, 0.45 mmole) f o r 10 h r . The r e a c t i o n m i x t u r e was worked up by-ad d i n g d i l u t e HC1 f o l l o w e d by e t h e r e x t r a c t i o n and e v a p o r a t i o n o f the e t h e r -to g i v e 1 4 C - l a b e l l e d m o n o c y c l o f a r n e s o l (47) (110 mg). The s p e c i f i c a c t i v i t y was d e t e r m i n e d t o be 0.29 mC/mmole. 14 F e e d i n g o f C - l a b e l l e d m o n o c y c l o f a r n e s o l (47) to T r i c h o t h e c i u m  roseum A w e l l shaken m i x t u r e o f 1 4 C - l a b e l l e d m o n o c y c l o f a r n e s o l (47) (55.0 mg, 0.071 mC), water (18 ml) and Tween 20 (2 ml) was e v e n l y d i s t r i b u t e d among 10 f l a s k s o f 7 day o l d c u l t u r e s o f T. roseum. A f t e r a f u r t h e r 24 days the t r i c h o t h e c i n (38) was i s o l a t e d i n the u s u a l manner and r e c r y s t a l l i z e d t o c o n s t a n t s p e c i f i c a c t i v i t y (0.867 x 10 5 mC/mmole), r e p r e s e n t i n g an i n c o r p o r a t i o n o f 0.003%. The r e m a i n i n g t r i c h o t h e c i n (38) was h y d r o l y z e d as above to g i v e t r i c h o t h e c o l o n e (39) w i t h s p e c i f i c a c t i v i t y o f 0.237 x 10 5 mC/ mmole r e p r e s e n t i n g l o s s o f 70% o f the r a d i o a c t i v i t y . G e r a n y l bromide (89) To a s t i r r e d s o l u t i o n o f g e r a n i o l (92) (30.8 g, 0.2 mole) and c a r b o n t e t r a b r o m i d e (99.6 g, 0.3 mole) i n e t h e r (200 ml) was added, w i t h s t i r r i n g , a s o l u t i o n o f t r i p h e n y l phosphine (57.6 g, 0.22 mole) i n e t h e r (200 m l ) , over a p e r i o d o f 0.5 h r . The r e a c t i o n m i x t u r e was a l l o w e d t o s t i r o v e r n i g h t then the e t h e r was e v a p o r a t e d a t reduced p r e s s u r e . P e t r o l e u m e t h e r (60-80°C) was added t o p r e c i p i t a t e t r i p h e n y l phosphine o x i d e w h i c h was f i l t e r e d o f f . The p e t r o l e u m e t h e r was e v a p o r a t e d i n vacuo t o g i v e a y e l l o w o i l which was d i s t i l l e d to g i v e g e r a n y l bromide (89) (27.1 g , 62.5%) as a c o l o u r l e s s o i l , b.p. 100-108°C (11 mm). - 89 --\) v ( l i q u i d f i l m ) 2940, 1660, 1440 , 1375, 120.0, 840,. 670 cm ; tT' ( C C l 4 ) 4.45 ( t , J = 8 Hz, 1H), 4.94 ( b r . s , 1H) , 6.07 ( d , J = 8 Hz, 2H), 7.92 (d, J = 3 Hz, 4H), 8.23-8.43 (m, 9H). E t h y l 2 - a c e t y l - 5 , 9 - d i m e t h y l d e c a - 4 , 8 - d i e n o a t e (91) E t h y l a c e t o a c e t a t e (90) (16.4 g, 0.126 mole) was added t o an e t h a n o l i c s o l u t i o n o f sodium e t h o x i d e p r e p a r e d from sodium (2.9 g, 0.126 mole) and e t h a n o l (55 ml) and the s o l u t i o n was a l l o w e d t o r e f l u x f o r 1 h r . On s l i g h t c o o l i n g g e r a n y l bromide (89) (27.1 g, 0.126 mole) was added and the m i x t u r e was a l l o w e d to r e f l u x a f u r t h e r 5 h r . The e t h a n o l was removed a t reduced p r e s s u r e , w a t e r added and the o i l y p r o d u c t e x t r a c t e d w i t h 3 p o r t i o n s o f e t h e r . The e t h e r s o l u t i o n was t h o r o u g h l y washed w i t h b r i n e , d r i e d (MgSO^) and the e t h e r e v a p o r a t e d i n vacuo to g i v e an o i l w h i c h was d i s t i l l e d to g i v e e t h y l 2 - a c e t y l - 5 , 9 -d i m e t h y l d e c a - 4 , 8 - d i e n o a t e (91) (12.9 g, 44%) as a c o l o u r l e s s o i l , b.p. 100-102°C (0.03 mm). ^ ( l i q u i d f i l m ) 2905, 1740, 1715, 1150 , 1030 cm" 1; 'X'(CC1 4) 4.97 ( t , J = 7 Hz, 2H), 5.85 (q, J = 7 Hz, 2H), 6.71 ( t , J = 7 Hz, 1H), 7.54 ( t , J = 7.5 Hz, 2H), 7.85 ( s , 3H), 8.02 (d, J = 3 Hz, 4H), 8.36 (m, 9H), 8.72 ( t , J = 7 Hz, 3H); M + m/e 266. 4 , 8 - D i m e t h y l h e x a - 3 , 7 - d i e n y l m e t h y l ketone ( g e r a n y l acetone) (88) E t h y l 2 - a c e t y l - 5 , 9 - d i m e t h y l d e c a - 4 , 8 - d i e n o a t e (91) (12.5 g, 0.047 m o l e ) , sodium h y d r o x i d e (lOg) and w a t e r (20 ml) were h e a t e d , w i t h s t i r r i n g , at~100°C o v e r n i g h t . The s o l u t i o n was c a r e f u l l y a c i d i f i e d ( l i t m u s ) w i t h 6N h y d r o c h l o r i c a c i d s o l u t i o n and the aqueous l a y e r e x t r a c t e d 3 times w i t h e t h e r . The combined e t h e r s o l u t i o n s were washed w i t h b r i n e , d r i e d (MgSO.) and the e t h e r - 90 -e v a p o r a t e d a t reduced p r e s s u r e to g i v e a r e d o i l w h i c h was d i s t i l l e d t o g i v e 4 , 8 - d i m e t h y l h e x a - 3 , 7 - d i e n y l m e t h y l ketone ( g e r a n y l acetone) (88) (6.5 g, -71.5%) as a c o l o u r l e s s o i l , b.p. 64-65°C (0.15 mm) [ l i t . ° 122-122.5°C(12mm)]. ^ m a x ( l i q u i d f i l m ) 2915 , 1720 , 1355, 1155, 830 cm" 1; % (CC1 4) 4.97 ( t , J = 6 Hz, 2H), 7.69 (m, 4H), 7.97 ( s , 3H), 8.04 ( s , 4H), 8.38 ( d , J = 4 Hz, 9H); M + m/e 194. N e r o l (98) To a s o l u t i o n o f m e t h y l t r i p h e n y l phosphonium bromide (25.7 g, 0.1 mole) i n t e t r a h y d r o f u r a n (100 ml) was added n - b u t y l l i t h i u m s o l u t i o n (42.0 ml of 2.4 M hexane s o l u t i o n , 0.1 m o l e ) . A f t e r f o r m a t i o n o f the r e d y l i d e the s o l u t i o n was c o o l e d t o -78°C and 5-methylhept-5-en-2-one (93) (12.6 g, 0.1 mole) added. A f t e r s t i r r i n g f o r 5 minutes n - b u t y l l i t h i u m s o l u t i o n (42.0 ml of 2.4M hexane s o l u t i o n , 0.1 mole) was added and the s o l u t i o n a l l o w e d t o warm up t o 0°C when p a r a f o r m a l d e h y d e (18.0 g, 0.2 mole) was added. The r e a c t i o n was a l l o w e d to s t i r at 0°C f o r 0.5 h r and at room temperature o v e r n i g h t . Water was added, the l a y e r s s e p a r a t e d and the aqueous l a y e r e x t r a c t e d 3 times w i t h e t h e r . The combined e t h e r s o l u t i o n s were washed w i t h b r i n e , d r i e d (MgSO^) and the e t h e r e v a p o r a t e d i n vacuo to g i v e an o i l (10 g, 65%) w h i c h was shown by g l c a n a l y s i s (130°C, 3% Se 30) t o c o n s i s t of ^ 3 0 % n e r o l ( 9 8 ) . A sample was p u r i f i e d by p r e p a r a t i v e g l c (180°C, 30% Se 30) and found to be i d e n t i c a l i n a l l r e s p e c t s w i t h n a t u r a l n e r o l , "\) ( l i q u i d f i l m ) 3345, 2925 , 1680 , 1445 , 1375, 1000 , 830 cm" 1; T?(CC1 4) 4.62 ( t , J = 6 Hz,- 1H) , 4.94 ( b r . s , 1H), 6.04 ( d , J = 7 Hz, 2H), 7.95 ( d , J = 4 Hz, 4H), - 91 -M e t h y l f a m e s ate (100) T r i m e t h y l p h o s p h i t e (248 mg, 2.0 mmole), m e t h y l bromoacetate (83)(306 mg,'2.0 mmole) and benzene (1 ml) were r e f l u x e d f o r 24 h r . Benzene (10 ml) and sodium h y d r i d e (50% o i l d i s p e r s i o n ) (144 mg, 3.0 mmole NaH) were added and the m i x t u r e s t i r r e d a t room temperature f o r 1.5 h r . 4 , 8 - D i m e t h y l h e x a - 3 , 7 - d i e n y l m e t h y l ketone (88) (388 mg, 2.0 mmole) i n benzene (3 ml) was added and the m i x t u r e r e f l u x e d o v e r n i g h t . Water was added and the aqueous l a y e r e x t r a c t e d 3 times w i t h e t h e r . The e t h e r was washed w i t h b r i n e , d r i e d (MgSO^) and e v a p o r a t e d i n vacuo t o g i v e an o i l w h i c h was d i s t i l l e d t o g i v e m e t h y l f a r n e s a t e (100) (305 mg, 61%) as a c o l o u r l e s s o i l , b.p. 55°C (0.1 mm) /\) ( l i q u i d f i l m ) 2925 , 1720 , 1650 , 1435, 1220, 1145 cm" 1 ; T (CC1 4) 4.40 ( s , 1H), 4.94 ( t , J = 6 Hz, 2H), 6.37 ( s , 3H), 7.87 ( s , 3H), 8.37 ( d , J = 4 Hz, 9H); M + m/e 250. F a r n e s o l (99) M e t h y l f a r n e s a t e (100) (430 mg, 1.74 mmole), l i t h i u m aluminium h y d r i d e (43 mg, 1.13 mmole) and e t h e r (10 ml) were s t i r r e d a t room temperature f o r 4 h r . Water was added and the aqueous l a y e r e x t r a c t e d 3 times w i t h e t h e r . The e t h e r s o l u t i o n was washed w i t h b r i n e , d r i e d (MgSO^) and the e t h e r e v a p o r a t e d to g i v e f a r n e s o l (99) (367 mg, 95%) as a c o l o u r l e s s o i l i d e n t i c a l i n a l l r e s p e c t s w i t h n a t u r a l f a r n e s o l , ~ \ 3 m a x ( l i q u i d f i l m ) 3335, 2905, 1670 , 1440 , 1370 , 995, 835 cm" 1; T ( C C 1 4 ) 4.63 ( t , J = 6 Hz, 1H), 4.92 ( b r . s , 2H), 5.98 ( d , J = 7 Hz, 2H), 7.23 ( b r . s , 1H), 7.97 ( t , J = 3 Hz, 8H), 8.37 ( d , J = 4 Hz, 12H). - 92 -F a r n e s o l - 1 0 , 1 1 - e p o x i d e (48) To f a r n e s o l (99) (300 mg, 1.35 mmole) d i s s o l v e d i n 701 aqueous glyme (13.9 ml) was added N-bromo-succinlmide (480 mg, 2.70 mmole) i n 70% aqueous glyme (3.5 m l ) . The m i x t u r e was s t i r r e d a t room temperature f o r 1 h r then w a t e r was added. The aqueous l a y e r was e x t r a c t e d 3 times w i t h methylene c h l o r i d e , the methylene c h l o r i d e was washed w i t h b r i n e , d r i e d (MgSO^) and e v a p o r a t e d a t reduced p r e s s u r e . The crude b r o m o h y d r i n was d i s s o l v e d i n methanol (9.5 ml) and p o t a s s i u m h y d r o x i d e (151 mg, 2.70 mmole) i n methanol (1 ml) added. The s o l u t i o n was s t i r r e d a t room temperature f o r 1 h r then w a t e r added. The aqueous l a y e r was e x t r a c t e d 3 times w i t h methylene c h l o r i d e w h i c h was then washed w i t h b r i n e , d r i e d (MgSO^) and e v a p o r a t e d i n vacuo to g i v e f a r n e s o l - 1 0 , 1 1 - e p o x i d e (48) (280 mg, 87%) as a c o l o u r l e s s o i l , ( l i q u i d f i l m ) 3310 , 2840 , 1640, 1425, 1360 , 1110, 1000, H i d - A . ( 870 cm" 1; T ( C C 1 4 ) 4.63 ( t , J = 6Hz, 1H) , 4.87 ( b r . s , 1H) , 5.98 (d, J = 6 Hz, 2H), 6.43 ( b r . s , 1H) 7.47 ( t , J = 6 Hz, 1H), 7.97 ( t , J = 4 Hz, 6H), 8.37 ( d , J = 4 Hz, 8H), 8.76.(d, J = 3 Hz, 6H) . 14 C - L a b e l l e d f a r n e s o l e p o x i d e (48) T r i m e t h y l p h o s p h i t e (248mg, 2 mmole), l - 1 ^ C - m e t h y l bromo-a c e t a t e (83) (306 mg, 2 mmole, 1 mC, 0.5 mC/mmole) and benzene ( 1 ml) were r e f l u x e d f o r 24 h r . The s o l u t i o n was c o o l e d and benzene (10 ml) added. Sodium h y d r i d e (50% o i l d i s p e r s i o n ) (144 mg, 3 mmole) was added and the m i x t u r e s t i r r e d a t room temperature f o r 1 h r . G e r a n y l acetone (88) (388 mg, 2 mmole) i n benzene (3 ml) was added and the s o l u t i o n r e f l u x e d f o r 24 h r . - 9 3 -E x a m i n a t i o n of the r e a c t i o n m i x t u r e by g l c i n d i c a t e d t h a t r e a c t i o n had o n l y gone a p p r o x i m a t e l y 50% so the crude r e a c t i o n p r o d u c t was added to h a l f m o lar e q u i v a l e n t s o f sodium h y d r i d e and t r i m e t h y l p h o s p h o n o a c e t a t e ( 8 1 ) . A f t e r a l l o w i n g the r e a c t i o n to r e f l u x f o r a f u r t h e r 24 h r , water was added and the aqueous l a y e r e x t r a c t e d 3 times w i t h e t h e r . The e t h e r l a y e r was washed w i t h b r i n e , d r i e d (MgSO^) and the e t h e r e v a p o r a t e d t o g i v e a p a l e y e l l o w o i l (500 mg, 100%) i d e n t i c a l i n a l l r e s p e c t s w i t h m e t h y l f a r n e s a t e (100) p r e p a r e d above. 1 4 C - l a b e l l e d m e t h y l f a r n e s a t e (500 mg, 2mmole), l i t h i u m aluminium h y d r i d e (50 mg, 1.4 mmole) and e t h e r (10 ml) were s t i r r e d a t room tem p e r a t u r e f o r 10 h r . D i l u t e HC1 was added and the aqueous l a y e r e x t r a c t e d 3 times w i t h e t h e r . The e t h e r l a y e r s were washed w i t h b r i n e , d r i e d (MgSO^) and the e t h e r e v a p o r a t e d to g i v e a c o l o u r l e s s o i l (426 mg, 96%) i d e n t i c a l i n a l l r e s p e c t s w i t h n a t u r a l f a r n e s o l ( 9 9 ) . 14 To C - l a b e l l e d f a r n e s o l (426 mg, 1.92 mmole) i n 70% aqueous glyme (19.7 ml) was added N-bromosuccinimide (682 mg, 3.84 mmole) i n 70% aqueous glyme (5.0 m l ) . The m i x t u r e was a l l o w e d to s t i r a t room t e m p e r a t u r e f o r 2 h r then water was added. The aqueous l a y e r was e x t r a c t e d 3 times w i t h methylene c h l o r i d e , the methylene c h l o r i d e washed w i t h b r i n e , d r i e d (MgSO^) and e v a p o r a t e d i n vacuo to g i v e an o i l which was d i s s o l v e d i n methanol (13.5 ml) and p o t a s s i u m h d r o x i d e (206 mg, 3.84 mmole) i n methanol (1.4 ml) added. The s o l u t i o n was s t i r r e d at room temperature f o r 2 hr then water added. The aqueous l a y e r was e x t r a c t e d 3 times w i t h methylene c h l o r i d e , the methylene c h l o r i d e washed w i t h b r i n e , d r i e d (MgSO^) and e v a p o r a t e d a t reduced p r e s s u r e t o g i v e an o i l (394.7 mg, 86.5%) i d e n t i c a l i n a l l r e s p e c t s w i t h f a r n e s o l - 1 0 , 1 1 -- 94 -ep o x i d e (48) p r e p a r e d as above. The s p e c i f i c a c t i v i t y was d e t e r m i n e d to be 0.10 mC/mmole. 14 Fe e d i n g o f C - l a b e l l e d f a r n e s o l - 1 0 , 1 1 - e p o x i d e (48) t o T r i c h o t h e c i u m  roseum A w e l l shaken m i x t u r e o f ^ C - l a b e l l e d f a r n e s o l - 1 0 , 1 1 - e p o x i d e (102.7 mg, 0.041 mC), wa t e r (18 ml) and Tween 20 (2 ml) was e v e n l y d i s t r i b u t e d among 15 f l a s k s o f 4 day o l d c u l t u r e s o f T. roseum. A f t e r a f u r t h e r 2 weeks t r i c h o t h e c i n (38) was i s o l a t e d i n the u s u a l manner and r e c r y s t a l l i s e d to c o n s t a n t s p e c i f i c a c t i v i t y (0.670 x 10 5 mC/mmole), r e p r e s e n t i n g an i n c o r p o r a t i o n o f 0.0071. The r e m a i n i n g t r i c h o t h e c i n was h y d r o l y s e d as above t o g i v e t r i c h o t h e c o l o n e (39) w i t h s p e c i f i c a c t i v i t y o f 0.317 x 1 0 ' 5 mC/ mmole r e p r e s e n t i n g l o s s o f over 50% o f the r a d i o a c t i v i t y . - 95 -BIBLIOGRAPHY 1. J . N. C o l l i e , T rans. Chem. S o c , 1893, 63, 329. 2. J . N. C o l l i e , T rans. Chem. S o c , 1907, 91, 1806. 3. A. J . B i r c h and F. W. Donovan, A u s t r a l . J . Chem., 1953, 6, 361. 4. L. R u z i c k a , E x p e r i e n t i a , 1953, 9_, 357. 5. See R o b i n s o n , "The S t r u c t u r a l R e l a t i o n s o f N a t u r a l P r o d u c t s " , 6. R. B. C l a y t o n , Q u a rt. Rev., 1965, 19, 168, 201. 7. J . W. C o r n f o r t h , Chem. i n B r t a i n , 1968, 102. 8. G. Popjak and J . W. C o r n f o r t h , Adv. Enzymol., 1960, _2_2, 281. 9. R. H. C o r n f o r t h , K. F l e t c h e r , H. H e l l i g , a n d G. P o p j a k , N a t u r e , 1960, 1_85, 923. 10. T. T. Tchen, J . B i o l . Chem., 1958, 233_, 1100. 11. R. H. C o r n f o r t h , J . W. C o r n f o r t h , a n d G. P o p j a k , T e t r a h e d r o n , 1962 , 1_8, 1351. 12. K. B l o c h , S. C h a y k i n , A. H. P h i l l i p s , a n d A de Waard, J . B i o l . Chem., 1959, 234, 2595. 13. H. R. Levy and G. P o p j a k , Biochem. J . , 1960 , 75, 417. 14. H. H e l l i g and G. P o p j a k , J . L i p i d Res.,1961, 2, 235. 15. W. L i n d b e r g , C. Yuan, A. De Waard, and K. B l o c h , B i o c h e m i s t r y , 1962, 1, 182. 16. J . W. C o r n f o r t h , R. H. C o r n f o r t h , G. P o p j a k , and L. S. Yengoyan, J . B i o l . Chem., 1966, 241, 3970. 17. B. W. A g r a n o f f , H. E g g e r e r , U. Henning, and F. Lynen, J . B i o l . Chem., 1960, 235, 326. 18. D. H. Shah, W. W. C l e l a n d , and J. W. P o r t e r , J . B i o l . Chem., 1965, ^40, 1946. 19. F. Lynen, B. W. A g r a n o f f , H. E g g e r e r , U. Henning, and E. M. M o s l e i n , Angew. Chem., 1939. 71,657. 20. L. R u z i c k a , A. Eschenmoser, 0. J e g e r , and D. A r i g o n i , H e l v . Chim. A c t a , 1955, 38, 1890. - 96 -21. L. R u z i c k a , P r o c . Chem. S o c , 1959 , 341. 22. C. R. B e n e d i c t , J . K e t t , and J . W. P o r t e r , A r c h . Biochem. B i o p h y s . , 1965, 110, 611. 23. P. W. Holl o w a y and G. P o p j a k , Biochem. J . , 1967, 104, 57. 24. J . W. C o r n f o r t h , R. H. C o r n f o r t h , C. Donninger, and G. P o p j a k , P r o c . Roy. Soc. ( B ) , 1966, 163, 492. 25. B. A c h i l l a d e l i s and J . R. Hanson, J . Chem. S o c , (C) , 1969 , 2010. 26. S. Nozoe and M. M o r i s a k i , Chem. Comm., 1969, 214. 27. T. R i o s and S. Perez C , Chem. Comm., 1969, 214. 28. F. Lynen, H. E g g e r e r , U. Henning, and I . K e s s e l , Angew. Chem., 1958, 70_, 738. 29. L. J . Mulheim and P. J . Ramm, Chem. Soc. Rev., 1972 , 1_, 259. 30. J . D. Bu'Lock, H. M. Sm a l l e y , a n d G. N. S m i t h , P r o c . Chem. Soc., 1960 , 209 . 31. C. T. B e d f o r d , J . C. F a i r l i e , P. K n i t t e l , T. Money, and . G. T. P h i l l i p s , Chem. Comm., 1971, 323. 32. L. C a n o n i c a , W. K r o s z c y z y n s k i , B. M. R a n z i o , B. R i n d o r e , and C. S c o l a s t i c o , Chem. Comm., 1970, 1357. 33. J . H. B i r k i n s h a w , H. R a i s t r i c k , and D. J . Ross, Biochem. J . , 1952, 50, 630. 34. A. J . B i r c h , A. C a s s e r a , and R. W. R i c h a r d s , Chem. and I n d . , 1961, 792. 35. S. Gatenbach and K. Mosbach, A c t a Chem. Scand., 1959 , 13, 1561. 36. E. W. B a s s e t t and S. W. T.anenb aum, B i o c h i m . B i o p h y s . A c t a . , 1960 , 4_0_, 535 . 37. F. Lynen, Fed. P r o c , 1961, 20_, 2441. 38. K. Mosbach, A c t a Chem. Scand., 1960, 14, 457. 39. J . H. B i r k i n s h a w , A. B r a c k e n , S. E. M i c h a e l , and H . R a i s t r i c k , L a n c e t , 1943, 245, 625. 40. P. I . F o r r e s t e r and G. M. Gaucher, B i o c h e m i s t r y , 1972, 1 1 , 1102. 41. T. S h i b a t a , T. S h i b u y a , and K. D o i , B u l l . Chem. Soc. Japan, 1972, 45, 930. - 97 -42. G. L i n s t e d t and A. M i s i o r n y , A c t a Chem. Scand., 1951, 5_, 121. 43. See r e f . 44 f o r l e a d i n g r e f e r e n c e s . 44. G. G. Freeman and R. I . M o r r i s o n , Biochem. J . , 1949, 4_4, 1. 45. G. G. Freeman and J . E. G i l l , N a t u r e , 1950 , 16_6, 698. 46. G. G. Freeman, J . E. G i l l , and W. S. Waring, J . Chem. S o c , 1959 / 1105. 47. J . Fishman, E. R. H. Jon e s , G. Lowe, and M. C. W h i t i n g , P r o c . Chem. S o c , 1959 , . 127; J . Chem. S o c , 1960 , 3948. 48. E. R. H. Jones and G. Lowe, J . Chem. S o c , 1960 , 3959. 49. W. 0. G o d t f r e d s e n and S. V a n g e d a l , (a) P r o c . Chem. S o c , 1964, 188; (b) A c t a Chem. Scand., 1965, 1088. 50. S. Abrahamsson and B. N i l s s o n , P r o c . Chem. S o c , 1964, 188; see a l s o A. T. M c P h a i l and G. A. Sim, J . Chem. Soc. ( C ) , 1966 1394. 51. L. R u z i c k a , Pure A p p l . Chem., 1963, 6, 512. 52. B. A c h i l l a d e l i s , P. M. Adams, and J . R. Hanson, Chem. Comm., 1970, 511. 53. J . R. Hanson and B. A c h i l l a d e l i s , Chem. and I n d . , 1967, 1643. 54. J . M. F o r r e s t e r , M.Sc T h e s i s , U n i v e r s i t y o f B r i t i s h C o lumbia, 19 70. 55. S. Nozoe, M. M o r i s a k i , and H. Matsumoto, Chem. Comm., 1970, 926. 56. P. M. Adams and J . R. Hanson, Chem. Comm., 1970, 1569. 57. P. M. Adams and J . R. Hanson, Chem. Comm., 1971, 1414. 58. P. M. Adams and J . R. Hanson, J . C. S. P e r k i n 1, 1972, 586. 59. W. Z u r c h e r , J . G u t z w i l l e r , and Ch. Tamm, H e l v . Chim. A c t a , 1965, 48, 839. 60. W. P a r k e r , J . S. R o b e r t s , and R. Ramage, Quart. Rev., 1967, 21, 331. 61. R. A c h i n i , B. M u l l e r , and Ch. Tamm, Chem. Comm., 1971, 404. 62. L. R u z i c k a and M. L i g u o r i , H e l v . Chim. A c t a , 1932, 1_5, 3. 63. A. M a n j a r r e z and A. Guzman, J . Org. Chem., 1966, 3_1, 348. 64. M. J u l i a , S. J u l i a , and R. Guegan, Compt. Rend., 1959, 248, 820. - 98 -65. C. P. Gutsche, J . R. Maycock, and C. T. Chang, T e t r a h e d r o n , 1968, 24, 859. 66. 0. P. V i g , K. L. M a t t a , G. S i n g , and I . R a j , J . I n d i a n Chem. S o c , 1966, 43, 27. 67. H. E-. H e n n i s , J . Org. Chem., 1963, 28, 2570. 68. M. Mousseron, M. Mousseron-Canet, and M. G r a n i e r , Compt. Rend., 1958, 247, 382. 69. D. S. Noyce and L. J . Dol b y , A b s t r a c t s o f papers p r e s e n t e d a t the 136th M e e t i n g o f the American Chemical S o c i e t y , A t l a n t i c C i t y , N.J., September 1959; J . Org. Chem., 1961, 2_6 , 1732 ; see a l s o r e f . 71. 70. E. J . Boorman and R. P. L i n s t e a d , J . Chem. S o c , 1935, 258. 71. D. S. Noyce, H. I . W e i n g a r t e n , and L. J . Dol b y , J . Org. Chem., 1961, 26, 2101. 72. See f o r example G. B u c h i , P. K u l s a , K. Ogasawara, and R. L. R o s a t i , J . Amer. Chem. S o c , 1970 , 92_, 999. 73. K. Bowden, I . M. H e i l b r o n , E. R. H. Jon e s , and B. C. L. Weedon. J . Chem. S o c , 1946, 39. 74. R. A. M y r s i n a and N.V. D u z n e t s o r , D o p o v i d i Akad. Nauk. U k r a i n . RS.R. S e r . B., 1969, 810. 75. T. K a t o , S. Kanno, and Y. K i t a h a r a , T e t r a h e d r o n , 1970, 2_6, 4287. 76. S. I t o , K. Endo, T. Y o s h i d a , M. Y a t a g a i , and M. Kodama, Chem. Comm., 1967, 186. 77. J . J . Sims, W. F e n i c a l , R. M. Wing, and P. R a d l i c k , J . Amer. Chem. S o c , 1971, 9_3, 3774. 78. A. C a l i e z i and H. S c h i n z , H e l v . Chim. A c t a . , 1950 , 3^ 5, 1129. 79. Y. S u z u k i and S. Marumo, Chem. Comm., 19 71, 1199. 80. C. D j e r a s s i and S. B u r s t e i n , J . Amer. Chem. S o c , 1958, 80, 2 593. 81. L. C a g l i o t i , H. Naef, D. A r i g o n i , and 0. J e g e r , H e l v . Chim. A c t a , 1958, 41_, 2278. 82. 0. I s l e r , R. Ruegg, L. C h o p a r d - d i t - J e a n , H. Wagner, and K. B e r h a r d , H e l v . Chim. A c t a , 1956 , 39_, 897. 83. I . M. Downie, J . B. Lee, and M. F. S. Matough, Chem. Comm., 1968, 1350. - 99 84. E. J . Corey and H. Yamamoto, J . Amer. Chem. S o c , 1970 , 92 , 226 . 85. J . R e u c r o f t and P. G.Sammes, Quart. Rev., 1971, 2_5, 135. 86. R. B. Bates and D. M. G a l e , J . Amer. Chem. S o c , 1960 , '82, 5749 . 87. E. E. van Tamelen and T. J . Curphey, T e t r a h e d r o n L e t t e r s , 1962,. 121. 88. E. E. van Tamelen and K. B. S h a r p l e s s , T e t r a h e d r o n L e t t e r s , 1967, 2655. 89. B. A. A c h i l l a d e l i s , P. M. Adams and J . R. Hanson, J.C.S. P e r k i n I , 1972, 1425. 90. Y. Machida and S. Nozoe, T e t r a h e d r o n L e t t e r s , 1972, 1969. 91. G. G. Freeman, J . Gen. M i c r o b i o l . , 1955, 12, 213. PART I I ATTEMPTED CYCLISATION OF ACYCLIC ENOL ACETATES - 100 -INTRODUCTION For many y e a r s c h e m i s t s have been i n t e r e s t e d i n a t t e m p t i n g t o s y n t h e s i s e n a t u r a l p r o d u c t s by r o u t e s w h i c h b e a r a resemblance t o e i t h e r a prove n o r a p o s t u l a t e d b i o s y n t h e t i c pathway. The f i r s t of t hose s y n t h e s e s was p r o b a b l y C o l l i e ' s s y n t h e s i s o f o r c i n o l (1) from d e h y d r o a c e t i c a c i d ( 2 ) . 1 OH 0 A r e v i e w o f b i o g e n e t i c type s y n t h e s e s has been p u b l i s h e d c o v e r i n g the l i t e r a t u r e t o 1961, and a f u r t h e r r e v i e w c o v e r i n g 3 4 developments i n the t e r p e n e a r e a i s i n the p r e s s . Van Tamelen has c l a s s i f i e d b i o g e n e t i c type s y n t h e s e s i n t o t h r e e c a t e g o r i e s : (a) A m phosynthesis, w h i c h i s a spontaneous, non-enzymic r e a c t i o n w h i c h proceeds by the same mechanism i n v i v o as i n v i t r o . (b) B i o - o r g a n i c s y n t h e s i s , w h i c h i s a non-enzymic t r a n s f o r m a t i o n c a r r i e d out on a compound, w h i c h i s v e r y s i m i l a r t o a n a t u r a l s u b s t r a t e , t o g i v e a compound v e r y s i m i l a r t o the n a t u r a l p r o d u c t formed by the enzymic r e a c t i o n . (c) C h e m i c a l Analogue o f a B i o s y n t h e s i s , w h i c h i s a s y n t h e t i c sequence r o u g h l y p a t t e r n e d a f t e r a b i o l o g i c a l sequence. Many r e s e a r c h groups are i n v o l v e d i n a t t e m p t i n g b i o g e n e t i c -type s y n t h e s e s , and sometimes the f a c i l i t y of a r e a c t i o n l e a d s t o - 101 -p r o p o s a l s c o n c e r n i n g b i o s y n t h e t i c pathways. For example a c o n v e n i e n t s y n t h e s i s o f camphor (3) from d i h y d r o c a r v o n e e n o l a c e t a t e (4) has prompted the s u g g e s t i o n t h a t the b i o s y n t h e s i s o f camphor (3) c o u l d o c c u r by a s i m i l a r t y pe o f r e a c t i o n , e.g. v i a an e n o l phosphate d e r i v a t i v e . 5 OAc B F ^/CH ?CI 9 (4) (3) In an attempt to expand the scope of t h i s r e a c t i o n o t h e r e n o l a c e t a t e s ( 5 ) - ( 7 ) were p r e p a r e d and s u b j e c t e d t o c y c l i s i n g 6 7 c o n d i t i o n s . ' I t was found t h a t e n o l a c e t a t e s (5) and (6) c y c l i s e d smoothly t o g i v e camphor d e r i v a t i v e s (8) and [9) but t h a t e n o l a c e t a t e ( 7 ) , w h i c h i t was hoped would g i v e the n a t u r a l l y 8 o c c u r r i n g s e s q u i t e r p e n e campherenone [10) gave a m i x t u r e o f 9 p r o d u c t s , the n a t u r e of w h i c h i s a t p r e s e n t under i n v e s t i g a t i o n . 10 Corey and co-workers have used the c y c l i s a t i o n of e n o l a c e t a t e (11) i n a s y n t h e s i s o f c e d r o l (12) w h i c h c l o s e l y f o l l o w s 11 the p r o p o s e d b i o s y n t h e s i s o f c e d r o l . In an attempt to t e s t the g e n e r a l i t y of the e n o l a c e t a t e c y c l i s a t i o n r e a c t i o n i t was d e c i d e d to p r e p a r e an a c y c l i c e n o l a c e t a t e and see i f i t c o u l d be c y c l i s e d . F u r t h e r i t was d e c i d e d t h a t the r e a c t i o n s h o u l d be p l a n n e d so t h a t i t had some f e a s i b i l i t y - 103 -as a b i o s y n t h e t i c r o u t e . The compound chosen f o r s y n t h e s i s was of-cuparenone (13) and the r e q u i r e d e n o l a c e t a t e i s compound (14) . (13) (14) ( + ) C u p a r e n o n e i s a n a t u r a l p r o d u c t w h i c h i s found i n the 12 e s s e n t i a l o i l o f "mayur p a n k h i " , and the a b s o l u t e c o n f i g u r a t i o n i s as d e p i c t e d i n ( 1 3 a ) . 1 3 However, b e f o r e CX-cuparenone (13) had been r e c o g n i s e d as a n a t u r a l p r o d u c t i t had been s y n t h e s i s e d as an i n t e r m e d i a t e i n the s y n t h e s i s o f the r e l a t e d s e s q u i t e r p e n e (+)-cuparene ( 1 4 ) . ^ I n t h i s s y n t h e s i s t o l u e n e was condensed w i t h 0 (13a) (14) 3-m.ethyl c y c l o h e x - 2 - e n o n e , i n the p r e s e n c e o f aluminium c h l o r i d e , t o g i v e ketone ( 1 5 ) , w h i c h was d i m e t h y l a t e d a t the s t e r i c a l l y h i n d e r e d 2 - p o s i t i o n by p r i o r p r o t e c t i o n o f the 6 - p o s i t i o n as the f u r f u r y l i d e n e d e r i v a t i v e (16) . O z o n o l y s i s o f d i m e t h y l ketone (17) - 104 -gave a d i p i c a c i d (18) which, was e s t e r i f i e d , s u b j e c t e d , t o Dieckmann c o n d e n s a t i o n , h y d r o l y s e d and d e c a r b o j c y l a t e d t o g i v e C~ \ cup are none C13). The o v e r a l l r e a c t i o n sequence i s shown i n F i g u r e I . 5 F i g u r e I : P r e v i o u s s y n t h e s i s o f (X-cuparenone (13) - 105 -DISCUSSION The c y c l i s a t i o n o f e n o l a c e t a t e s has p r o v i d e d a c o n v e n i e n t method o f p r e p a r i n g some mono-and s e s q u i t e r p e n e s by r e a c t i o n s w h i c h 5 7 may have an a l o g y i n n a t u r e . ' I n o r d e r t o t e s t the g e n e r a l i t y of t h i s r e a c t i o n a s y n t h e s i s o f o(,-cuparenone (13) was d e v i s e d , the key s t e p o f w h i c h was c y c l i s a t i o n o f e n o l a c e t a t e ( 1 4 ) , i n a manner w h i c h may have s i g n i f i c a n c e i n the b i o s y n t h e s i s o f <X-cuparenone ( 1 3 ) . (14) Thus the f i r s t o b j e c t i v e i n the s y n t h e t i c scheme was an e f f i c i e n t s y n t h e s i s of ketone ( 1 5 ) . (15) - 106 -R e a c t i o n o f heptenone Cl°) w i t h the G r i g n a r d complex o f p_-bromotoluene gave t e r t i a r y a l c o h o l (.17} i n good y i e l d . A n a l y s i s o f the crude r e a c t i o n p r o d u c t by t h i n l a y e r chromatography ( t i c ) showed the p r e s e n c e o f a n o n - p o l a r i m p u r i t y , and as d i s -t i l l a t i o n l e d t o d e h y d r a t i o n o f a l c o h o l (17) , the p r o d u c t was p u r i f i e d by column chromatography on s i l i c a g e l . The n o n - p o l a r i m p u r i t y , w h i c h was e l u t e d w i t h p e t r o l e u m e t h e r (40-60°C) was found to be 4 , 4 ' - d i m e t h y l b i p h e n y l (18) by a n a l y s i s o f i t s i n f r a - r e d ( i r ) and n u c l e a r m agnetic resonance. (nmr) s p e c t r a , and i t s m e l t i n g 16 p o i n t . I t i s presumably formed by n u c l e o p h i l i c d i s p l a c e m e n t o f the bromine i n p-bromotoluene by the G r i g n a r d complex o f a n o t h e r m o l e c u l e o f p_-bromotoluene. Such G r i g n a r d c o u p l i n g s have been 1 7 n o t e d b e f o r e . E l u t i o n o f the column w i t h methanol then gave pure a l c o h o l ( 1 7 ) . OH (18) - 107 -The n e x t s t e p i n the sequence was i n t r o d u c t i o n o f the oxygen f u n c t i o n which, would u l t i m a t e l y become the k e tone group i n ketone ( 1 5 ) . T h i s was a c c o m p l i s h e d by h y d r o b o r a t i o n o f a l c o h o l (17) f o l l o w e d by an o x i d a t i v e work up u s i n g b a s i c hydrogen p e r o x i d e 18 s o l u t i o n to g i v e d i o l C19), a. c o l o u r l e s s s y r u p w h i c h s l o w l y c r y s t a l l i s e d . From d i o l (19) i t was thought t h a t two f u r t h e r r e a c t i o n s would be s u f f i c i e n t t o g i v e k e t o - o l e f i n ( 1 5 ) , namely an o x i d a t i o n o f the s e c o n d a r y h y d r o x y l f u n c t i o n , f o l l o w e d by d e h y d r a t i o n o f the t e r t i a r y one. There i s a p r e c e d e n t f o r d e h y d r a t i o n of t e r t i a r y a l c o h o l s o f t h i s type e x c l u s i v e l y t o g i v e the exomethylene double bond, as a l c o h o l (17) has been d e h y d r a t e d 19 w i t h aqueous o x a l i c a c i d t o g i v e d i e n e (20) m However i t was found t h a t d i o l (19) c o u l d not be smoothly c o n v e r t e d i n t o k e t o l ( 2 1 ) . (20) - 108 -(21) The f i r s t o x i d a t i v e c o n d i t i o n w h i c h was t r i e d was a Jones 20 o x i d a t i o n . I t appeared t h a t t h e s e c o n d i t i o n s o v e r - o x i d i s e d d i o l (19) w i t h c o n c o m i t a n t l o s s o f a t h r e e carbon fragment t o g i v e a compound t e n t a t i v e l y a s s i g n e d s t r u c t u r e (22) on the b a s i s o f a peak a t 1775 cm 1 i n the i r spectrum and no s a t u r a t e d m e t h y l s i g n a l i n the nmr spectrum. Such o x i d a t i v e c l e a v a g e s , and f o r m a t i o n o f - l a c t o n e s , have been r e p o r t e d b e f o r e d u r i r i g o x i d a t i o n o f 21 compounds c o n t a i n i n g p a r t s t r u c t u r e (23) . (23) - 109 -Other o x i d a t i v e c o n d i t i o n s which, were t r i e d were the a c t i o n o f B - b r o m o s u c c i n i m i d e CNBS) i n a s o l v e n t system c o n s i s t i n g o f 2 2 2 2 w a t e r , methanol and e t h e r , NBS i n aqueous d i o x a n and 23 manganese d i o x i d e i n . a c e t o n i t r i l e . A l l t h r e e of t h e s e r e a c t i o n s gave a complex m i x t u r e o f u n i d e n t i f i e d p r o d u c t s . A f u r t h e r method 24 w h i c h was t r i e d was the S a r e t t r e a g e n t , w h i c h i s the complex formed between p y r i d i n e and chromium t r i o x i d e . R e a c t i o n o f d i o l (19) w i t h t h i s r e a g e n t gave a m i x t u r e o f p r o d u c t s , one o f w h i c h was i d e n t i f i e d by i r and nmr s p e c t r a as k e t o n e ( 2 1 ) . However, at b e s t the r e a c t i o n was s l o w , and the e x c e s s o f r e a g e n t and r e a c t i o n temperature were found t o be v e r y c r i t i c a l : i f e i t h e r was too low, r e a c t i o n was v e r y s l o w , and i f too h i g h o v e r - o x i d a t i o n o c c u r r e d . As i t was found i m p o s s i b l e to c l e a n l y t r a n s f o r m d i o l (19) i n t o k e t o l C21) , w h i c h c o u l d t h e n be d e h y d r a t e d , i t was d e c i d e d t h a t the d e h y d r a t i o n s h o u l d be p e r f o r m e d f i r s t , f o l l o w e d by a l a t e r o x i d a t i o n . T h i s p l a n , however, n e c e s s i t a t e d the i n t r o d u c t i o n of two e x t r a s t e p s i n the sequence, namely p r o t e c t i o n o f the ; secondary a l c o h o l as an a c e t a t e and subsequent h y d r o l y s i s o f t h i s a c e t a t e a f t e r d e h y d r a t i o n . Thus t r e a t m e n t o f d i o l Q 9 ) w i t h a c e t i c a n h y d r i d e i n p y r i d i n e gave, i n h i g h y i e l d , h y d r o x y a c e t a t e ( 2 4 ) . 19 D e h y d r a t i o n o f h y d r o x y a c e t a t e C 24) w i t h aqueous o x a l i c a c i d 2 5 or w i t h t h i o n y l c h l o r i d e i n p y r i d i n e gave a m i x t u r e o f o l e f i n s , o f w h i c h o n l y a p p r o x i m a t e l y one t h i r d (as d e t e r m i n e d by a n a l y s i s o f the nmr spectrum) was the d e s i r e d a c e t o x y o l e f i n C25), the r e m a inder b e i n g c i s - and t r a n s - a c e t o x y o l e f i n C26}. - 110 -r^J 66% Because of the d i s a p p o i n t i n g r e s u l t o f the d e h y d r a t i o n r e a c t i o n i t was d e c i d e d to abandon t h i s r e a c t i o n scheme and d e v i s e an a l t e r n a t i v e r o u t e to k e t o - o l e f i n (15) i n which the exomethylene 7 0 double bond i s p r e p a r e d by a s t e r e o s e l e c t i v e W i t t i g r e a c t i o n ' on some a p p r o p r i a t e l y f u n c t i o n a l i s e d k e t o n e . - I l l -In d e v i s i n g a r e a c t i o n scheme f o r the s y n t h e s i s o f k e t o -o l e f i n (15) i n w h i c h the double bond i s p r e p a r e d by a W i t t i g r e a c t i o n , and w h i c h f o r c o n v e n i e n c e f o l l o w s i n g e n e r a l o u t l i n e the f i r s t scheme t r i e d , n o t i c e must be t a k e n o f s e v e r a l p o i n t s . (a) F o r m a t i o n o f the ketone would be by G r i g n a r d r e a c t i o n between an a p p r o p r i a t e o l e f i n i c bromide and a r o m a t i c a c i d c h l o r i d e . (b) B e f o r e h y d r o b o r a t i o n the ketone must be p r o t e c t e d as i t i s s u s c e p t i b l e to r e d u c t i o n by .diborane. (c) The a l c o h o l i n t r o d u c e d by the h y d r o b o r a t i o n r e a c t i o n must be p r o t e c t e d d u r i n g the W i t t i g r e a c t i o n . T a k i n g i n t o account those p o i n t s the f o l l o w i n g sequence o f r e a c t i o n s was attempted. Treatment o f c y c l o p r o p y l m e t h y l ketone w i t h m e t h y l magnesium i o d i d e gave c a r b i n o l (27) w h i c h was smoothly c o n v e r t e d i n t o bromide (28) w i t h 48% HBr a f t e r the method o f 27 J u l i a and c o - w o r k e r s . Br (28) 2 8 D u r i n g p r e v i o u s work i n v o l v i n g G r i g n a r d r e a c t i o n s o f bromide (28) w i t h a c i d c h l o r i d e s i t was found t h a t the magnesium complex o f bromide (28) c o u l d reduce the i n i t i a l l y formed ketone to g i v e a magnesium a l c o h o l a t e w h i c h r e a c t e d w i t h the a c i d c h l o r i d e t o g i v e an e s t e r as the main p r o d u c t . Such r e d u c t i o n s by 2 9 G r i g n a r d complexes have precedence i n the l i t e r a t u r e . T h i s r e a c t i o n c o u l d be a v o i d e d by v e r y slow i n v e r s e a d d i t i o n , i . e . a d d i t i o n o f the G r i g n a r d complex to the a c i d c h l o r i d e , but v e r y g r e a t c a r e had to be t a k e n t o e x c l u d e oxygen d u r i n g the t r a n s f e r and a d d i t i o n o f the G r i g n a r d complex, o t h e r w i s e the G r i g n a r d complex became o x i d i s e d by m o l e c u l a r oxygen t o g i v e a n o t h e r magnesium a l c o h o l a t e , w h i c h a g a i n would r e a c t w i t h the a c i d c h l o r i d e . - 113 -To a v o i d these c o m p l i c a t i o n s i t was d e c i d e d t o r e a c t the G r i g n a r d complex o f bromide (28) w i t h p_-tolualdehyde t o g i v e a l c o h o l (29) w h i c h c o u l d , i n a subsequent s t e p , be o x i d i s e d t o g i v e ketone ( 3 0 ) . However when e q u i m o l a r amounts o f bromide (28) H OH (30) and p_- t o l u a l d e h y d e were r e a c t e d t o g e t h e r the crude p r o d u c t was shown by gas l i q u i d chromatography ( g l c ) to c o n s i s t o f 3 compounds, none o f wh i c h was p_- t o l u a l d e h y d e } but wh i c h had a c a r b o n y l and a h y d r o x y l a b s o r p t i o n i n the i r spectrum. Jones o x i d a t i o n o f t h i s crude p r o d u c t gave a p r o d u c t w i t h a c a r b o n y l a b s o r p t i o n i n the i r s pectrum b u t w h i c h was shown by g l c to c o n s i s t o f 2 compounds. These were i d e n t i f i e d as p_-tolualdehyde and the r e q u i r e d ketone (3 0 ) . Thus what appeared to have happened was t h a t the f i r s t formed magnesium a l c o h o l a t e was r e d u c i n g some o f the u - t o l u a l d e h y d e t o p - t o l y l m e t h a n o l (.31) and was i t s e l f o x i d i s e d t o ketone (30) , - 114 -the 3 products i n the crude r e a c t i o n p r o d u c t b e i n g ketone (.30) and a l c o h o l s (29) and ( 3 1 ) . Such r e a c t i o n s o f G r i g n a r d complexes have been n o t e d b e f o r e . 3 ^ I t was d e c i d e d to make use o f t h i s r e a c t i o n , and so a v o i d the subsequent o x i d a t i v e s t e p , by u s i n g a 2 molar excess of p-t o l u a l d e h y d e i n the G r i g n a r d r e a c t i o n . R e a c t i o n o f the G r i g n a r d o f bromide (28) w i t h 2 moles of r j 4 o l u a l d e h y d e gave a m i x t u r e o f ketone (30) and p - t o l y l j n e t b a n o l (31) from w h i c h ketone (30) c o u l d be e a s i l y o b t a i n e d i n good y i e l d by d i s t i l l a t i o n and chroma-tograp h y on s i l i c a . - 115 -Ketone (30) was neat k e t a l i s e d u s i n g excess d i o l (32) i n r e f l u x i n g benzene w i t h l p _ - t o l u e n e s u l p h o n i c a c i d (p_-TSA) as a c i d c a t a l y s t , t o g i v e k e t a l (33) w h i c h was smoothly c o n v e r t e d i n t o h y droxy k e t a l (34) by the a c t i o n o f d i b o r a n e f o l l o w e d by b a s i c 18 hydrogen p e r o x i d e s o l u t i o n . (30) (32) (33) - 116 -Treatment o f hydroxy k e t a l C34) w i t h a c e t i c a n h y d r i d e i n p y r i d i n e gave a c e t o x y k e t a l C35) w h i c h was h y d r o l y s e d w i t h d i l u t e HC1 i n acetone t o g i v e a c e t o x y ketone C36). (36) R e a c t i o n o f a c e t o x y ketone (36) w i t h the y l i d e d e r i v e d by the a c t i o n of sodium m e t h y l s u l p h i n y l m e t h i d e on m e t h y l t r i p h e n y l phosphonium bromide gave, i n d i s a p p o i n t i n g l y low y i e l d , a c e t o x y o l e f i n ( 2 5 ) . H y d r o l y s i s o f a c e t o x y - o l e f i n (25) w i t h sodium 20 h y d r o x i d e s o l u t i o n , f o l l o w e d by Jones o x i d a t i o n o f the r e s u l t i n g a l c o h o l (37) gave k e t o o l e f i n ( 1 5 ) . 0 - + • CH 3-S-CH 2Na + + 0 3P-CH 3Br > 0 3-P-CH 2 - 117 -0 M-™2 + AcC (36) NaOH/MeOH/H20 <- Jones o x i d a t i o n (15) (37) As p r e l i m i n a r y a t t e m p t s t o c o n v e r t k e t o - o l e f i n C IS ) t o e n o l a c e t a t e ( 1 4 ) , u s i n g i s o p r o p e n y l a c e t a t e and p_-TSA, gave a m i x t u r e o f p r o d u c t s , w i t h rearrangement o f the exomethylene double bond, i t was d e c i d e d t o p e r f o r m a model s t u d y on ketone C38), e a s i l y p r e p a r e d by G r i g n a r d r e a c t i o n between i s o b u t y r a l d e h y d e and n - b u t y l 2 0 bromide, f o l l o w e d by Jones o x i d a t i o n o f the i n t e r m e d i a t e secondary a l c o h o l ( 3 9 ) . Treatment o f ketone (38) w i t h s u f f i c i e n t t r i t y l l i t h i u m C p r e p a r e d from t r i p h e n y l methane and n - b u t y l 1 1 8 -(38) l i t h i u m ) t o g i v e a p e r s i s t e n t p i n k c o l o u r , f o l l o w e d by quenching of the e n o l a t e w i t h a c e t i c a n h y d r i d e , gave a m i x t u r e o f e n o l a c e t a t e s (40) and (41) i n a r a t i o of a p p r o x i m a t e l y 9:1, as e s t i m a t e d by g l c and nmr s p e c t r o s c o p y . T h i s p r o d u c t r a t i o i s 31 c o n s i s t e n t w i t h the work o f House and Kramer. Attempts t o produce e n o l a c e t a t e s o f ketone C38) under a v a r i e t y o f m i l d a c i d c o n d i t i o n s were a l l u n s u c c e s s f u l : (a) Treatment of ketone (38) w i t h p e r c h l o r i c a c i d i n a c e t i c 3 2 a n h y d r i d e f o r 6 minutes at room temperature gave no r e a c t i o n . - 119 -OAc OAc (40) (41) (b) Treatment o f ketone (38) w i t h i s o p r o p e n y l a c e t a t e (37) and o x a l i c a c i d at 110°C f o r 15 hours gave no r e a c t i o n . 33 (c) A r e p o r t on the s y n t h e s i s o f camphor e n o l a c e t a t e (42) s u g g e s t e d t h a t t h i s compound might be an e f f i c i e n t e n o l a c e t y l a t i n g agent under m i l d c o n d i t i o n s . E n o l a c e t a t e (42) 33 was p r e p a r e d as r e p o r t e d i n the l i t e r a t u r e from camphor (4) u s i n g n - b u t y l l i t h i u m and quenching the e n o l a t e w i t h a c e t i c a n h y d r i d e . However t r e a t m e n t of ketone (38) w i t h e n o l a c e t a t e (42) i n r e f l u x i n g benzene and o x a l i c a c i d o r p_-TSA gave no i n d i c a t i o n o f f o r m a t i o n o f e n o l a c e t a t e s (40) or ( 4 1 ) . (d) Treatment of ketone (38) w i t h i s o p r o p e n y l a c e t a t e and 20% by w e i g h t of p_-TSA gave a m i x t u r e of 3 compounds s e p a r a t e d by p r e p a r a t i v e g l c and i d e n t i f i e d by i r and nmr s p e c t r a as e n o l a c e t a t e s ( 4 0 ) , (41) and ( 4 3 ) . The r e l a t i v e amounts o^ (40) , (41) and (43) , as e s t i m a t e d by g l c are 34.8%, 31.7% and 33.6% r e s p e c t i v e l y - 120 -0 (4) 1) n-BuLi 2) A c 2 0 OAc (42) w h i c h i s i n the same r e g i o n as t h a t found by House and Kramer i n a s t u d y o f s i m i l a r compounds. 31 - 121 -As a t t e m p t s t o form e n o l a c e t a t e (41) under m i l d c o n d i t i o n s were s i n g u l a r l y u n s u c c e s s f u l , i t was d e c i d e d t o t r y and i s o m e r i s e the m i x t u r e w h i c h c o n s i s t e d m o s t l y o f e n o l a c e t a t e (40) ( p r e p a r e d by q u e n c h i n g the e n o l a t e a n i o n of ketone (38) w i t h a c e t i c a n h y d r i d e ) . U n f o r t u n a t e l y a l l e f f o r t s d i r e c t e d towards t h i s g o a l . were a l s o u n s u c c e s s f u l . Thus t r e a t m e n t o f the m i x t u r e w i t h p e r c h l o r i c a c i d 32 i n a c e t i c a n h y d r i d e f o r 7 minutes at room t e m p e r a t u r e gave no i s o m e r i s a t i o n ; t r e a t m e n t o f the m i x t u r e w i t h r e f l u x i n g benzene, i n 34 w h i c h was d i s s o l v e d one c r y s t a l o f i o d i n e , gave no i s o m e r i s a t i o n ; and t r e a t m e n t o f the m i x t u r e w i t h i s o p r o p e n y l a c e t a t e (37) and 31 p_-TSA at 90°C f o r 24 hours gave no i s o m e r i s a t i o n . To summarise t h i s s e r i e s of r e a c t i o n s , f o r m a t i o n o f e n o l a c e t a t e s o f ketone (38) v i a e n o l a t e f o r m a t i o n gave d e s i r e d e n o l a c e t a t e (41) i n o n l y a p p r o x i m a t e l y 10% y i e l d , t r e a t m e n t o f k e tone (38) under a v a r i e t y of m i l d a c i d i c c o n d i t i o n s gave no e n o l a c e t a t e f o r m a t i o n , t r e a t m e n t o f ketone (38) w i t h more f o r c i n g a c i d i c c o n d i t i o n s [probably s t r o n g enough t o i s o m e r i s e k e t o - o l e f i n ( 1 5 ) ] gave a p p r o x i m a t e l y e q u a l q u a n t i t i e s o f e n o l a c e t a t e s ( 4 0 ) , (41) and ( 4 3 ) , and l a s t l y e n o l a c e t a t e (40) c o u l d not be i s o m e r i s e d under a v a r i e t y o f m i l d c o n d i t i o n s . Thus i t seemed t h a t the b e s t r o u t e t o e n o l a c e t a t e (14). was by q u e n c h i n g the m i x t u r e o f e n o l a t e anions o f ketone (15) w i t h a c e t i c a n h y d r i d e t o g i v e a m i x t u r e o f e n o l a c e t a t e s (14) and ( 4 4 ) . To t h i s end a s o l u t i o n o f t r i t y l l i t h i u m , p r e p a r e d from t r i p h e n y l methane and n - b u t y l l i t h i u m , was added t o ketone (15) u n t i l a f a i n t p i n k c o l o u r p e r s i s t e d , i n d i c a t i n g a s l i g h t excess o f base. A c e t i c a n h y d r i d e was added and a f t e r f o u r hours the - 122 -C14) . C44) r e a c t i o n was worked up to g i v e a crude p r o d u c t from which the t r i p h e n y l methane, was removed by c r y s t a l l i s a t i o n and f i l t r a t i o n . The i s o m e r i c e n o l a c e t a t e s (14) and (44) [ (14):(44) = 1:6] were s e p a r a t e d by p r e p a r a t i v e g l c and c h a r a c t e r i s e d by a n a l y s i s o f t h e i r i r and nmr s p e c t r a . E n o l a c e t a t e ( 1 4 ) , d i s s o l v e d i n methylene c h l o r i d e , was exposed to gaseous boron t r i f l u o r i d e f o r f i v e m i n u t e s . A f t e r removal o f excess boron t r i f l u o r i d e , e v a p o r a t i o n o f the s o l v e n t gave a gum which showed no a b s o r p t i o n between 1700 and 1800 cm"-* i n t h e i r spectrum, and gave a complex m i x t u r e o f peaks on g l c a n a l y s i s . Thus i t would appear t h a t e n o l a c e t a t e (14) i s not - 123 -c y c l i s e d t o o ( -cuparenone (13) under t h e s e r e a c t i o n c o n d i t i o n s . Lack o f m a t e r i a l p r e c l u d e d f u r t h e r i n v e s t i g a t i o n on the c y c l i s a t i o n o f e n o l a c e t a t e ( 1 4 ) . I t would seem t h a t , i n f u r t h e r work o f t h i s k i n d i n v o l v i n g s i m i l a r e n o l a c e t a t e s , some method s h o u l d be found which g i v e s the d e s i r e d e n o l a c e t a t e s t e r e o -s e l e c t i v i t y , f o r example by r e d u c t i o n o f s u i t a b l e enones or c ^ - c h l o r o - k e t o n e s . CI A n o t h e r method which c o u l d perhaps be t r i e d i s d e c a r b o x y l a t i o n of a s u i t a b l e /3-keto a c i d . - 124 -EXPERIMENTAL A l l m e l t i n g p o i n t s were d e t e r m i n e d on a K o f l e r b l o c k and are u n c o r r e c t e d . ' I n f r a r e d s p e c t r a (-0 ) were r e c o r d e d on a P e r k i n -Elmer I n f r a c o r d model 137 s p e c t r o p h o t o m e t e r . N u c l e a r magnetic resonance s p e c t r a (*£) were d e t e r m i n e d i n carbon t e t r a c h l o r i d e o r d e u t e r i o c h l o r o f o r m and r e c o r d e d on a J e o l c o C-60H s p e c t r o m e t e r o r on V a r i a n A s s o c i a t e s s p e c t r o m e t e r s , model A-60 o r model T-60. S i g n a l p o s i t i o n s are g i v e n i n the T i e r s % 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 as an i n t e r n a l s t a n d a r d ; the m u l t i p l i c i t y , c o u p l i n g c o n s t a n t s (where a p p r o p r i a t e ) and i n t e g r a t e d peak areas 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 ; s = s i n g l e t , b r . s = b r o a d s i n g l e t , d= d o u b l e t , t = t r i p l e t , q = q u a r t e t , m = m u l t i p l e t . Mass s p e c t r a (M +) were d e t e r m i n e d w i t h an AEI MS9 s p e c t r o m e t e r . M i c r o a n a l y s e s were performed by Mr. P. Bo r d a , 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 , U n i v e r s i t y o f B r i t i s h Columbia. T h i n l a y e r c h r o m a t o g r a p h i c ( t i c ) 254 r e s u l t s were o b t a i n e d u s i n g s i l i c a g e l HF o f 0.5 mm t h i c k n e s s ; s o l v e n t A was p e t r o l e u m e t h e r (60-80°C); s o l v e n t B was p e t r o l e u m e t h e r (60-80°C): e t h y l a c e t a t e ( 1 : 1 , v/v) . 6 - M e t h y l - 2 - p - t o l y l h e p t - 5 - e n - 2 - o l (17) To magnesium t u r n i n g s (1.7 g, 0.07 mole) i n a 3-necked f l a s k e q u i p p e d , w i t h a d r o p p i n g f u n n e l , r e f l u x condenser and n i t r o g e n i n l e t , w a s added, w i t h magnetic s t i r r i n g , p_-bromotoluene (12.1 g, 0.07 mole) i n e t h e r (80 ml) a t such a r a t e so as to m a i n t a i n a v i g o r o u s r e f l u x , a f t e r i n i t i a t i o n by g r i n d i n g the magnesium. When the r e a c t i o n s u b s i d e d the f l a s k was h e a t e d on a h o t w a t e r b a t h f o r 0.25 h r t o d r i v e the r e a c t i o n t o c o m p l e t i o n . A f t e r . - 125 -c o o l i n g the r e a c t i o n m i x t u r e i n an i c e b a t h , 6-me'thylhept-5-en-2-one (16) (8.9g, 0.07 m o l e ) , d i s s o l v e d i n e t h e r (20 ml) was s l o w l y added and the m i x t u r e then a l l o w e d to s t i r at room temperature o v e r n i g h t , when i t was worked up by adding s a t u r a t e d aqueous ammonium a c e t a t e s o l u t i o n . The l a y e r s were s e p a r a t e d and the aqueous l a y e r e x t r a c t e d 3 times w i t h e t h e r , the e t h e r washed w i t h b r i n e , d r i e d (MgSO^) and the e t h e r e v a p o r a t e d i n vacuo to g i v e crude 6 - m e t h y l - 2 - p - t o l y l h e p t - 5 - e n - 2 - o l (17) (12.9 g, 831). A n a l y s i s by t i c ( s o l v e n t A) showed the presence o f a n o n - p o l a r i m p u r i t y so the crude p r o d u c t was a p p l i e d to a s i l i c a g e l column and the column e l u t e d w i t h p e t r o l e u m e t h e r (40-60°C), e v a p o r a t i o n o f w h i c h gave 4 , 4 ! - d i m e t h y l b i p h e n y l (18) as a w h i t e s o l i d , m.p. 16 122-1240C ( l i t . m.p. 121- 122°C) . "\^ m, v ( n u j o l m u l l ) 1495, 1105, 805 cm" 1; X (CC1 4) 2.76 (q, J = 8Hz, 8H), 7.67 ( s , 6H). F u r t h e r e l u t i o n o f the column w i t h m e t h a n o l , f o l l o w e d by e v a p o r a t i o n of the m e t h a n o l , gave pure 6-me thy 1 - 2-p_- t o l y l h e p t - 5 -e n - 2 - o l (17) (10.6 g, 68.5%) as a p a l e y e l l o w o i l . " 0 m a x ( l i q u i d f i l m ) 3425, 2905, 1105, 815 cm" 1; T (CC1 4) 2.87 (q, J = 8 H z , 4H), 4.96 ( b r . s , I K ) , 7.47 ( b r . s , 1H), 7.70 ( s , 3H), 8.23 ( s , 4H), 8.38 ( s, 3H), 8.55 ( s , 6H). 6 - M e t h y l - 2 - p _ - t o l y l h e p t a- 2, 5 - d i o l (19) To a s o l u t i o n o f 6-methy 1 - 2 - p _ - t o l y l h e p t - 5 - e n - 2 - o l (17) (49.6 g, 0.23 mole) i n r e d i s t i l l e d (from l i t h i u m aluminium h y d r i d e ) t e t r a h y d r o f u r a n ( T H F ) (100 ml) c o n t a i n e d i n a 3-necked f l a s k e q u i p p e d w i t h m a g n e t i c s t i r r e r , r e f l u x c ondenser, d r o p p i n g f u n n e l and n i t r o g e n i n l e t , was added a s l u r r y o f sodium boro-h y d r i d e (4.2 g, 0,11 mole) i n THF (30 m l ) , f o l l o w e d by a s o l u t i o n - 126 -of b o r o n t r i f l u o r i d e e t h e r a t e (24.7 g, 0.17 mole) i n THF (30 m l ) , added d r o p w i s e . The r e a c t i o n m i x t u r e was s t i r r e d a t room temp e r a t u r e f o r 2 h r , then c o o l e d and aqueous 3 M sodium h y d r o x i d e s o l u t i o n (130 ml).added, f o l l o w e d by 30% hydrogen p e r o x i d e s o l u t i o n (65 m l ) . The r e a c t i o n m i x t u r e was the n a l l o w e d t o s t i r a f u r t h e r 2 h r a t room t e m p e r a t u r e when the l a y e r s were s e p a r a t e d and the aqueous phase e x t r a c t e d 3 times w i t h e t h e r . The combined o r g a n i c l a y e r s were washed w i t h b r i n e , d r i e d (MgSO^) and the s o l v e n t e v a p o r a t e d a t reduced p r e s s u r e t o g i v e crude 6 - m e t h y l - 2 - p _ - t o l y l -h e p t a - 2 , 5 - d i o l (19) (50.4 g, 94%) as a p a l e y e l l o w s y r u p w h i c h s l o w l y c r y s t a l l i s e d . R e c r y s t a l l i s a t i o n from p e t r o l e u m e t h e r (60-80°C) gave pure 6-methyl-2-p_-tolylhepta-2 , 5 - d i o l (19) as w h i t e p r i s m s , m.p. 107-109°C. *\} ( n u j o l m u l l ) 3235, 1030 , 930 , 900 , IT13.X 870, 825 cm" 1; T ( C D C 1 3 ) 2.78 (m, 4H) 6.75 (m, 1H) , 7.42 ( b r . s , 2H), 7.67 ( s, 3H) , 8.09 (m, 2H)_, 8.47 ( s , 3H) , 8.64 (m, 2H) , 9.16 (d , J = 6Hz, 6H). O x i d a t i o n of 6 - m e t h y l - 2 - p - t o l y l h e p t a - 2 , 5 - d i o l (19) (a) To 6 - m e t h y l - 2 - p _ - t o l y l h e p t a - 2 , 5 - d i o l (19) (2.0 g, 8.5 mmole) d i s s o l v e d i n acetone (10 ml) and c o o l e d i n an i c e b a t h was 20 added Jones' r e a g e n t (1.1 m l ) . The r e a c t i o n m i x t u r e was a l l o w e d t o s t i r a t room temperature f o r 0.5 h r , then worked up by a d d i n g w a t e r , s e p a r a t i o n o f the l a y e r s and e t h e r e x t r a c t i o n o f the aqueous phase. D r y i n g (MgSO^) and e v a p o r a t i o n o f the s o l v e n t in vacuo gave a y e l l o w o i l (1.75 g) w h i c h e x h i b i t e d a s t r o n g i r a b s o r p t i o n a t 1775 cm \ and wh i c h was t e n t a t i v e l y a s s i g n e d as 4-methyl-4-p_- t o l y l b u t y r o l a c t o n e ( 2 2 ) . - 127 -(b) 6 - M e t h y l - 2 - p _ - t o l y l h e p t a - 2 , 5 - d i o l (19) (1.0 g, 4.2 mmole) was added to the complex formed between chromium t r i o x i d e 24 (0.85 g, 8.5 mmole) and p y r i d i n e (8.5 m l ) . The r e a c t i o n m i x t u r e was a l l o w e d t o s t i r a t room tem p e r a t u r e f o r 6 days and worked up by p o u r i n g onto benzene and w a t e r . The l a y e r s were s e p a r a t e d and the aqueous l a y e r e x t r a c t e d w i t h benzene. The combined o r g a n i c l a y e r s were washed w i t h b r i n e , d r i e d (MgSO^) and the s o l v e n t e v a p o r a t e d a t reduced p r e s s u r e t o g i v e a y e l l o w o i l (893 mg) which was p u r i f i e d by p r e p a r a t i v e t i c ( s o l v e n t B ) . Thus crude p r o d u c t (60 mg) gave pure 5-keto-6-methyl-2-p_- t o l y l -h e p t a n - 2 - o l (21) (30 mg, 45% o v e r a l l y i e l d ) . ^0 ( l i q u i d f i l m ) 3415, 2915, 1700, 820 cm" 1 ; T .(CDClj) 2.84 (m, 4H) , 7.70 ( s , 3H) , 8.48 ( s , 3H), 9.00 ( d , J = 7Hz, 6H). 5- A c e t o x y - 6 - m e t h y 1 - 2 - p - t o l y 1 - h e p t a n - 2 - o l (24) A c e t i c a n h y d r i d e (45.0 g, 0.44 mole) was added t o 6-methyl-2 - p _ - t o l y l h e p t a - 2 , 5 - d i o l (19) (50.4 g, 0.22 mole) i n p y r i d i n e (200 ml) and the r e a c t i o n m i x t u r e was a l l o w e d t o s t i r o v e r n i g h t at room t e m p e r a t u r e . Water was added and the p r o d u c t e x t r a c t e d 3 times w i t h e t h e r , the e t h e r was washed s u c c e s s i v e l y w i t h b r i n e , d i l u t e HC1, s a t u r a t e d aqueous 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 . A f t e r d r y i n g (MgSO^) the e t h e r was e v a p o r a t e d i n vacuo to g i v e 5 - a c e t o x y - 6 - m e t h y l - 2 - p _ - t o l y l h e p t a n - 2 - o l (24) (48.8 g, 95%) as a p a l e y e l l o w o i l . A) ( l i q u i d f i l m ) 3510 , 2950 , 1725 , HI 3.X 1235, 1015, 820 cm" 1 ; T ( C C 1 4 ) , 'l.90 (m, 4H), 5.36 (m, 1H), 7.52 ( b r . s , 1H), 7.70 ( s , 3H), 8.06 ( s , 3H), 8.55 ( s , 3H), 9.19 (d, J = 6.5 Hz, 6H). 1 2 8 -D e h y d r a t i o n o f 5 - a c e t o x y - 6 - m e t h y l - 2 - p - t o l y l h e p t a n - 2 - , o l (24) (a) A s o l u t i o n o f 5 - a c e t o x y - 6 - m e t h y l - 2 - p _ - t o l y l h e p t a n - 2 - o l (24) (1.0 g, 3.6 mmole), o x a l i c a c i d (1 g) and wat e r (10 ml) was r e f l u x e d f o r 6.5 h r , then worked up by e x t r a c t i n g 3 times w i t h e t h e r . The e t h e r s o l u t i o n was t h o r o u g h l y washed w i t h b r i n e , d r i e d (MgSO^) and the e t h e r e v a p o r a t e d a t reduced p r e s s u r e t o g i v e a y e l l o w o i l (0.94 g, 100%). -» ( l i q u i d f i l m ) 2950, 1725, 1225,1025, 820 c m " 1 ; T ( C C 1 4 ) , 2.90 (m, 4H), 4.20 ( t , J = 6.5 Hz, ^ H ) , 4.,82 ( d , J = 1Hz, 1/ 3H) , 5.05 ( d , J = 1Hz, 1/ 3H) , 7.72 ( s , 3H), 8.03 ( s , 2H), 8.07 ( s , 3H), 9.12 ( t , J = 6Hz, 6H). (b) 5 - a c e t o x y - 6 - m e t h y l - 2 - p _ - t o l y l h e p t a n - 2 - o l (24) (0.5 g, 1.8 mmole) was d i s s o l v e d i n p y r i d i n e (5 ml) and c o o l e d i n a dry i c e -acetone b a t h , under a n i t r o g e n -atmosphere. F r e s h l y d i s t i l l e d t h i o n y l c h l o r i d e (0.44g, 3.6 mmole) was s l o w l y added and the m i x t u r e s t i r r e d f o r 0.25 h r , when i t was a l l o w e d t o warm up. Water was added, the p r o d u c t e x t r a c t e d w i t h e t h e r and the e t h e r washed w i t h b r i n e and d r i e d (Na^^SO^). E v a p o r a t i o n o f the e t h e r i n vacuo gave a y e l l o w o i l (0.40 g, 851) which e x h i b i t e d s p e c t r a l f e a t u r e s e s s e n t i a l l y i d e n t i c a l w i t h the s p e c t r a o f the o i l p r e p a r e d i n (a) above. 2 - C y c l o p r o p y l p r o p a n - 2 - o l (27) M e t h y l i o d i d e (186.0 g, 1.31 mole) i n e t h e r (350 ml) was added t o magnesium t u r n i n g s (35.0 g, 1.46 mole) a t such a r a t e so as t o m a i n t a i n a v i g o r o u s r e f l u x . A f t e r the r e a c t i o n s u b s i d e d the r e a c t i o n m i x t u r e was heated on a hot w a t e r b a t h f o r 0.5 h r . On c o o l i n g t o 0°C, c y c l o p r o p y l m e t h y l ketone - 129 -(100.0 g, 1.19 mole) i n e t h e r (350 ml) was added w i t h s t i r r i n g , and the r e a c t i o n m i x t u r e s t i r r e d a t room temperature f o r 2 h r , when i t was a g a i n c o o l e d t o 0°C and s a t u r a t e d aqueous ammonium a c e t a t e s o l u t i o n added. The m i x t u r e was s t i r r e d u n t i l two c l e a r l a y e r s r e s u l t e d . They were th e n s e p a r a t e d and the aqueous l a y e r was e x t r a c t e d 3 times w i t h e t h e r . The combined o r g a n i c l a y e r s were washed w i t h b r i n e , d r i e d (MgSO^) and the ether e v a p o r a t e d i n  vacuo t o g i v e a y e l l o w o i l w h i c h was d i s t i l l e d t o g i v e 2 - c y c l o p r o p y l p r o p a n - 2 - o l - (27) (86.5 g, 73%) as a c o l o u r l e s s o i l , b.p. 124-128°-C (760 mm) ( l i t . 2 7 b.p. 121-122°C). \) IIlclX ( l i q u i d f i l m ) 3400, 3100, 2950, 1460, 1150, 1020, 960, 920, 845, 825 cm" 1; T ( C C 1 4 ) , 7.66 ( s , 1H), 8 . 8 6 ( s , 6H) , 9.00-9.40 (m, 4H), 9.73 ( d , J = 6 Hz, 6H). 5-Eromo-2-methylpent-2-ene (28) To 2 - c y c l o p r o p y l p r o p a n - 2 - o l (27) (86.5 g, 0.865 mole) was added, w i t h c o o l i n g and s t i r r i n g , 48% HBr (410 ml) ov e r a p e r i o d o f 0.3 h r . A f t e r s t i r r i n g f o r 0.5 h r w i t h c o o l i n g and 0.5 h r at room t e m p e r a t u r e the r e a c t i o n m i x t u r e was e x t r a c t e d 3 times w i t h e t h e r , the combined e t h e r e x t r a c t s were washed t h o r o u g h l y w i t h s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n , washed w i t h b r i n e , d r i e d (Na2S0 4) and the e t h e r e v a p o r a t e d a t reduced p r e s s u r e t o g i v e an o i l which was d i s t i l l e d t o g i v e 5-bromo-2-methylpent-2-ene (28) (102.7 g, 72.5%) as a c o l o u r l e s s o i l , b.p. 77-82°C (60 mm) { l i t . 2 7 b.p. 84- 85°C {84 mm)]. ^  ( l i q u i d IllciX f i l m ) 2960, 2925, 1670, 1445, 1375, 1265, 1205, 1095, 835 cm" 1; T ( C C 1 4 ) 4.33 ( t , J = 7Hz, 1H) , 6.77 ( t , J = 7Hz, 2H) , 7.53 ( t , J = 7Hz, 2H), 8.33 ( d , J = 4Hz, 6H). - 130 -2 - M e t h y l - 6 - p - t o l y l h e x - 2 - e n - 6 - o l (29) To the G r i g n a r d complex p r e p a r e d from 5-bromo-2-methyl-pent-2-e i- ,e (28) (10.0 g, 0.06 mole) and magnesium (1.6 g, 0.067 mole) i n e t h e r (50 ml) was added p_-tolualdehyde (7.3 p,0.06'mole i n e t h e r (10 m l ) . The r e a c t i o n was a l l o w e d t o s t i r a t room temp-e r a t u r e o v e r n i g h t and then worked up by adding s a t u r a t e d aqueous ammonium a c e t a t e s o l u t i o n . S e p a r a t i o n o f the l a y e r s , f o l l o w e d by e t h e r e x t r a c t i o n o f the aqueous phase, b r i n e w a s h i n g , d r y i n g (MgSO^) o f the e t h e r s o l u t i o n and e v a p o r a t i o n , i n vacuo, o f the e t h e r gave a y e l l o w o i l (7.9 g, 94%) which showed 3 peaks on g l c (145°C, 3% Se30), one v o l a t i l e peak ( s i m i l a r i n r e t e n t i o n time t o , but not i d e n t i c a l w i t h p_-tolualdehyde) and two l e s s v o l a t i l e peaks. S e p a r a t i o n of the m i x t u r e by p r e p a r a t i v e g l c (200°C, 30% Se 30) gave, i n o r d e r of d e c r e a s i n g v o l a t i l i t y , p-t o l y M e t h a n o l ( 3 1 ) , m.p. 57-59°C ( l i t . 3 5 m.p. 59°C) . A) ma^ ( n u j o l ill d - A m u l l ) 3345, 1515 , 1200 , 1025, 805 cm" 1; T ( C D C 1 3 ) , 2.76, (5» 4H) , 5.37 ( s , 2H), 7.63 ( s , 3H), 8.11 ( s , 1H); 6-keto-2-methy1-6-p-t o l y l h e x ,-2-ene ( 3 0 ) , s p e c t r a l d a t a i d e n t i c a l t o t h a t g i v e n below f o r t h i s compound; and 2 - m e t h y l - 6 - p - t o l y l h e x - 2 - e n - 6 - o l £29). ~\2> m ^ x ( l i q u i d f i l m ) 3345, 2935, 1445, 1060, 815 cm" 1. 6 - K e t o - 2 - m e t h y l - 6 - p - t o l y l h e x -2-ene (30) (a) 5-Bromo-2-methylpent-2-ene (28) (37.6 g, 0.23 mole) i n e t h e r (150 ml) was added t o magnesium (6.1 g, 0.25 mole) at such a r a t e so as t o m a i n t a i n , a f t e r i n i t i a t i o n , a v i g o r o u s r e f l u x . When the r e a c t i o n slowed down the f l a s k was h e a t e d to r e f l u x on a hot water b a t h f o r 0.25 h r , then a s o l u t i o n o f p_-tolualdehyde - 131 -(61.0 g, 0.51 mole) i n e t h e r (50 ml) was added, w i t h . s t i r r i n g , at room temperature and the r e a c t i o n m i x t u r e a l l o w e d t o s t i r o v e r n i g h t a t room t e m p e r a t u r e . The r e a c t i o n was worked up by-ad d i n g s a t u r a t e d aqueous ammonium a c e t a t e s o l u t i o n . The aqueous l a y e r was s e p a r a t e d and e x t r a c t e d 3 times w i t h e t h e r . The combined o r g a n i c l a y e r s were washed w i t h b r i n e , d r i e d (MgSO^) and the e t h e r e v a p o r a t e d a t reduced p r e s s u r e t o g i v e a y e l l o w o i l w h i c h was d i s t i l l e d t o g i v e 6 - k e t o - 2 - m e t h y l - 6 - p _ - t o l y l h e x -2-ene i Q (30) (25.6 g, 55%),b.p. 103-108°C (0.2 mm) [ l i t . b.p. 93°C (0.1 mm)] which c r y s t a l l i s e d on s t a n d i n g . R e c r y s t a l l i s a t i o n from p e t r o l e u m e t h e r (60-80°C) gave an a n a l y t i c a l sample, 1 Q m.p. 45-46°C ( l i t . m.p. 47.5-48.5°C). \ ) m a x (CC1 4) 2925 , 1680 , 1610, 1170, 970, 840 c m " 1 ; T ( C C 1 4 ) 2.19 ( d , J = 4Hz, 2H), 2.81 ( d , J = 4Hz, 2H), 4.87 ( t , J = 3Hz, 1H), 7.13 ( t , J = 3.5 Hz, 2H), 7.58 ( s, 3H), 7.66 (m, 2H), 8.33 ( s , 6H); M + m/e 202; C, 82.86; H, 8.97; ( c a l c . f o r C 1 4 H l g 0 : C, 83.12; H, 8.97). (b) 2 - K e t h y l - 6 - p _ - t o l y l h e x - 2 - e n - 6 - o l (29) (1.0 g, 5.0 mmole), 20 acetone (5 ml) and Jone s ' r e a g e n t (2 ml) were s t i r r e d a t room tem p e r a t u r e f o r 0.5 h r when w a t e r was added. E t h e r e x t r a c t i o n , d r y i n g (MgS0 4) and e v a p o r a t i o n o f the e t h e r i n vacuo gave 6 - k e t o - 2 - m e t h y l - 6 - p _ - t o l y l h e x -2-ene (30) i d e n t i c a l i n a l l r e s p e c t s t o t h a t p r e p a r e d i n (a) above. 5 , 5 - D i m e t h y l - 2 - ( 4 ' - m e t h y l p e n t - 3 ' - e n y 1 ) - 2 - p - t o l y l - l , 3 - d i o x a n (33) 6-Keto-2-methyl-6-p_- t o l y l h e x -2-ene (30) (62 .7 g, 0.31 m o l e ) , 2 , 2 - d i m e t h y l p r o p a n - l , 3 - d i o l (32) (150.7 g, 1.45 m o l e ) , p_-TSA (1.86 g) and benzene (500 ml) were r e f l u x e d f o r 24 h r i n a Dean and S t a r k t r a p . On c o o l i n g the benzene was e v a p o r a t e d a t reduced - 132 -p r e s s u r e and p e t r o l e u m e t h e r (40-60°Cl added to p r e c i p i t a t e the excess d i o l w h i c h was f i l t e r e d o f f . The p e t r o l e u m e t h e r s o l u t i o n was washed t w i c e w i t h s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n , then washed t h o r o u g h l y w i t h b r i n e and d r i e d (MgSO^). Removal o f the p e t r o l e u m e t h e r a t reduced p r e s s u r e gave an o i l w hich was d i s t i l l e d t o g i v e 5 , 5 - d i m e t h y l - 2 - ( 4 1 - m e t h y l p e n t - 3 ' -e n y l ) - 2-p_- t o l y l - 1, 3-dioxan (33) (51.0 g, 57%) as a c o l o u r l e s s o i l , b.p. 110-112°C (0.03 mm), w h i c h - s l o w l y c r y s t a l l i s e d on s t a n d i n g . R e c r y s t a l l i s a t i o n from p e t r o l e u m e t h e r (60-80°C} gave an a n a l y t i c a l sample, m.p. 62-65°C). * \ ) m a x CCCl^) 2975 , 1170, 1080, 820 cm" 1; T (CC1 4) 2.81 (m, 4H), 5.00 ( t , J = 4Hz, 1H), 6.67 ( s , 4H), 7.65 ( s, 3H), 8.39 ( s , 6H), 8.45 ( s , 3H), 8.76 ( s , 3H) ; M + m/e 288; C, 79.06; H, 9.87; ( c a l c . f o r C i gH* 2 80 2: C, 79.12, H, 9.78). 5 , 5 - D i m e t h y 1 - 2 - ( 3 ' - h y d r o x y - 4 1 - m e t h y l p e n t y l ) - 2 - p - t o l y l - 1 , 3 - d i o x a n (34) To 5, 5 - d i m e t h y l - 2- (4 ' - m e t h y l p e n t - 3 ' - e n y l ) - 2 - p _ - t o l y l - l , 3-dioxan (33) (51.0 g, 0.18 mole) i n THF (100 ml) was added, w i t h s t i r r i n g , sodium b o r o h y d r i d e (3.02 g, 0.079 mole) i n THF (25 m l ) , f o l l o w e d by b oron t r i f l u o r i d e e t h e r a t e (18.9 g, 0.13 mole) i n THF (25 m l ) . The r e a c t i o n m i x t u r e was a l l o w e d to s t i r a t room temperature f o r 2 hours then 3M aqueous sodium h y d r o x i d e s o l u t i o n (100 ml) and 30% hydrogen p e r o x i d e s o l u t i o n (50 ml) added and the r e a c t i o n m i x t u r e a l l o w e d t o s t i r a f u r t h e r 2 h r a t room t e m p e r a t u r e , S e p a r a t i o n o f the l a y e r s and e t h e r e x t r a c t i o n o f the aqueous l a y e r was f o l l o w e d by b r i n e washing and d r y i n g (MgS0 4) o f the e t h e r l a y e r . E v a p o r a t i o n of the e t h e r i_n vacuo gave 5 , 5 - d i m e t h y l -2-(3' -hydroxy-4 1 - m e t h y l p e n t y l ) - 2 - p _ - t o l y l - 1 , 3 - d i o x a n (34) (53.3 g, - 133 -96.6%) as a c o l o u r l e s s , v i s c o u s o i l . " \ ) ( l i q u i d f i l m ) 3400, ' max n ' J ' 2925, 1170, 1075, 820 cm" 1; T ( C C 1 4 ) 2.82 (m, 4H), 6.64 ( s , 4H), 7.63 ( s , 3H), 8.73 ( s , 3H), 9.13 ( d , J = 5Hz, 6H), 9.41 ( s , 3H). 5,5 - D i m e t h y l - 2 - ( 5 ' - a c e t o x y - 4 ' - m e t h y l p e n t y 1 ) - 2 - p - t o l y l - l , 3 - d i o x a n (35) 5,5-Dimethy1-2-(3 '-hydroxy-4'-methylpentyl)-2-£-tolyl-l,3-d i o x a n (34) (24.0 g, 0.079 m o l e ) , a c e t i c a n h y d r i d e (15.95 g, 0.157 mole) and p y r i d i n e (50 ml) were s t i r r e d o v e r n i g h t a t room t e m p e r a t u r e , when wa t e r was added and the r e a c t i o n m i x t u r e e x t r a c t e d t h r e e times w i t h e t h e r . The e t h e r s o l u t i o n was t h o r o u g h l y washed w i t h b r i n e , d r i e d (MgS0 4) and the e t h e r e v a p o r a t e d a t reduced p r e s s u r e t o g i v e 5 , 5 - d i m e t h y l - 2 - ( 3 ' - a c e t o x y -4 ' - m e t h y l p e n t y l ) - p _ - t o l y l - l , 3 - d i o x a n (35) (26.9 g, 98.5%) as a c o l o u r l e s s o i l . ^  ( l i q u i d f i l m ) 2940, 1730, 1465, 1360, n i c i x 1235, 1170 , 1075, 1015 , 970 , 920 , 820 c m " 1 ; T ( C C l 4 ) 2.88 Cm, 4H) , 5.39 ( b r . s , 1H), 6.69 ( s , 4H), 7.64 ( s , 3H), 8.07 ( s , 3H) ; 8.78 ("s, 3H) , 9.17 ( d , J = 6Hz, 6H) , 9.44 ( s , 3H) ; M + m/e 348. 4- A c e t o x y - 5 - m e t h y l - 1 - p - t o l y l h e x a n - 1 - one (36) 5,5 - D i m e t h y l - 2 - ( 3 ' - a c e t o x y - 4 '- methylpentyl)-2-£-tolyl-1,3-d i o x a n (35) (25.0 g, 0.072 m o l e ) , acetone (300 ml) and 6N . HC1 (10 ml) were s t i r r e d o v e r n i g h t a t room t e m p e r a t u r e . Sodium b i c a r b o n a t e was added u n t i l the r e a c t i o n m i x t u r e was n e u t r a l ( l i t m u s ) and the acetone then e v a p o r a t e d i n vacuo. P e t r o l e u m e t h e r (40-40°C) was added and t h e sodium b i c a r b o n a t e removed by f i l t r a t i o n . The p e t r o l e u m e t h e r s o l u t i o n was washed w i t h b r i n e , d r i e d (MgSO^) and the s o l v e n t e v a p o r a t e d t o g i v e an o i l w h i c h was d i s t i l l e d t o g i v e 4-acetoxy-5-methyl-l-£-tolylhexan-l-one - 134 -(36) (8.78 g, 46%) a s a c o l o u r l e s s o i l , b.p. 125-128°C CO.4 mm). v> ( l i q u i d f i l m ) 2985, 1730, 1680, 1605, 1235, 1020, 820 cm" 1; ^ ( C C 1 4 X 2.18 ( d , J = 8Hz, 2H) , 2.78 ( d , J = 8Hz, 2H) , 5.25 (m, 1H) , 7.14 ( t , J = 6.5 Hz, 2H), 7.57 ( s , 3H), 8.01 ( s , 3H), 9.04 ( d , J = 7Hz, 6H); M+ m/e 262 ; C, 72.93; H, 8.53; ( c a l c . f o r C 1 6 H 2 2 0 3 : C, 73,26; H, 8.46). 5-Acetoxy-6-methy1- 2-p- t o l y h e p t -1 - ene (25) Sodium h y d r i d e (50%.. o i l d i s p e r s i o n ) (1.3 g, 0.027 mole) was washed w i t h dry e t h e r , d r i e d in vacuo, added to d i m e t h y l -s u l p h o x i d e (DMSO) (20 m l ) , and the m i x t u r e h e a t e d a f V 8 0 ° C under n i t r o g e n u n t i l e v o l u t i o n o f n i t r o g e n ceased {y0 . 5 h r ) . The m i x t u r e was c o o l e d i n an i c e / w a t e r b a t h and methyl t r i p h e n y l phosphonium bromide (9.7 g, 0.0 27 m o l e ) , d i s s o l v e d I n DMSO (40 ml) added w i t h s t i r r i n g , to g i v e a deep y e l l o w s o l u t i o n T K e s o l u t i o n was a l l o w e d t o warm t o room temperature and 4-acetoxy-5-m e t h y l - l - p _ - t o l y l h e x a n - l - o n e (36) (7.12 g, 0,027 mole) i n DMSO (10 ml) added d r o p w i s e . S t i r r i n g was c o n t i n u e d o v e r n i g h t , t h e n the r e a c t i o n m i x t u r e was e x t r a c t e d 4 times w i t h p e t r o l e u m e t h e r (40-60°C). The p e t r o l e u m e t h e r s o l u t i o n was then washed w i t h DMSO, washed w i t h b r i n e , d r i e d (MgSO^) and the s o l v e n t e v a p o r a t e d at reduced p r e s s u r e t o g i v e an o i l which was d i s t i l l e d t o g i v e 5-acetoxy-6-methy 1 - 2-p_- t o l y l h e p t - 1 - ene (25) (2.0 g, 28.5%) as a c o l o u r l e s s o i l , b.p. 96-10S°C (0.04 mm) ."O _ ( l i q u i d f i l m ) 2960 , 1735, 1370, 1235, 1025, 890, 825 c m " 1 ; T ( C C l 4 ) 2.87 (m, 4H), 4.83 ( d , J = 1.5 Hz, 1H), 5.0 5 ( d , J = 1.5 Hz, 1H), 5.29 ( q , J = 6 H z , l H ) , 7.65 ( s, 3H), 7.98 ( s, 3H), 9.14 ( d , J = 7Hz, 6H); M + m/e 260. - 135 -5- H y d r o x y - 6 - m e t h y l - 2 - p - t o l y l h e p t - l - e n e (37) 5-Acetoxy-6-methyl-2-£-tolylhept-l-ene (25) (2.0 g, 7.7 mmole) sodium h y d r o x i d e (4.0 g ) , methanol (30 ml) and w a t e r (5 ml) were a l l o w e d t o r e f l u x f o r 4 h r . The r e a c t i o n m i x t u r e was e x t r a c t e d 3 times w i t h p e t r o l e u m e t h e r (40-60°C) , the p e t r o l e u m e t h e r s o l u t i o n was washed w i t h b r i n e , d r i e d CMgSOp and the s o l v e n t removed i n vacuo to g i v e 5-hydroxy-6-methyl-2-£-tolylhept-l-ene (37) (1.34 g, 80%) as a c o l o u r l e s s o i l . "0 m a x ( l i q u i d f i l m ) 3380, 2925, 1630, 1510, 1455, 1370, 1045, 890, 825, 735 cm" 1; "C CCC1 ) 2.88 (m, 4H), 4.81 (m, 1H), 5.00 (m, 1H), 6.68 (m, 1H), 7.66 ( s , 3H), 9.15 (d , J = 6Hz, 6H). 6- Me t h y 1 - 2 - p - t o l y l h e p t - 1 - e n - 5 - o n e (15) To 5 - h y d r o x y - 6 - m e t h y l - 2 - p _ - t o l y l h e p t - l - e n e (37) (1 . 3 4 g, 6.15 mmole) i n acetone(10 ml) was added, w i t h s t i r r i n g and c o o l i n g , 20 Jones' r e a g e n t (2 m l ) . The r e a c t i o n m i x t u r e was a l l o w e d t o s t i r f o r 0.5 h r then worked up by adding w a t e r . The aqueous l a y e r was e x t r a c t e d 3 times w i t h e t h e r , the e t h e r washed w i t h b r i n e , d r i e d (MgS0 4) and the e t h e r e v a p o r a t e d a t reduced p r e s s u r e to g i v e an o i l w h i c h was d i s t i l l e d t o g i v e 6-methyl-2-£-tolylhept-l-en-5-one (15) (1.00 g, 75%) as a c o l o u r l e s s o i l , b.p. 86-88°C (0.04 mm). •"Om a v. ( l i q u i d f i l m ) 2985, 1710 , 1515, 1465, 1070 , 895, 825 cm" 1; i l l cA-A. t ( C C l 4 ) , 2.84 (m, 4H), 4.83 ( d , J = 1.5 Hz, 1H), 5.03 ( d , J = 1.5 Hz, 1H), 7.64 ( s , 3H), 8.97 ( d , J = 8Hz, 6H); M + m/e 216. 2 - M e t h y l h e p t a n - 3 - o l (59) n - B u t y l bromide (30.1 g, 0.22 mole) d i s s o l v e d i n e t h e r (100 ml) was added to magnesium t u r n i n g s (4.8 g, 0.24 mole) c o n t a i n e d i n a 3-necked f l a s k e quipped w i t h a mag n e t i c s t i r r e r , r e f l u x condenser and n i t r o g e n i n l e t . A d d i t i o n was r e g u l a t e d so as - 136 -t o m a i n t a i n a v i g o r o u s r e f l u x . When the a d d i t i o n was completed the r e a c t i o n m i x t u r e was then a l l o w e d t o s t i r f o r 2 h r a t room tem p e r a t u r e when the r e a c t i o n was worked up by c a r e f u l l y a d d i n g w a t e r u n t i l e f f e r v e s c e n c e ceased. S u f f i c i e n t 6N HC1 was then added to d i s s o l v e the e m u l s i o n formed by i n o r g a n i c s a l t s . The two l a y e r s were s e p a r a t e d and the aqueous l a y e r e x t r a c t e d 3 times w i t h e t h e r , the e t h e r washed t h o r o u g h l y w i t h b r i n e , d r i e d CMgSO^) and the e t h e r e v a p o r a t e d i n vacuo t o g i v e 2 - m e t h y l h e p t a n - 3 - o l (39) (21.4 g, 821) as a c o l o u r l e s s o i l . \ ) o v ( l i q u i d f i l m ) , 3390 , 2940 , 1460 , 990 cm" 1; f C C C l ^ , 6.73 ( b r . s , 1H) , 7.57 (s , 1H) , 8.33-8.83 Cm, 7H) , 9.12 ( d , J = 7Hz, 9H). 2-Methylheptan-3-one (38) To 2 - m e t h y l h e p t a n - 3 - o l (39) (21.4 g, 0.165 mole) d i s s o l v e d i n acetone (25 ml) was added, w i t h c o o l i n g and s t i r r i n g , J ones' r e a g e n t ^ (35 m l ) . The m i x t u r e was a l l o w e d t o s t i r a t room temp e r a t u r e f o r 4 h r then worked up by add i n g w a t e r and e x t r a c t i n g the aqueous l a y e r 3 times w i t h e t h e r . The e t h e r s o l u t i o n was washed w i t h b r i n e , d r i e d (MgSO^) and the e t h e r e v a p o r a t e d i n vacuo t o g i v e an o i l which was d i s t i l l e d t o g i v e 2-methylheptan-3-one (38) (14.3 g, 68%) as a c o l o u r l e s s o i l , b.p.82-84°C (64 mm), ^max ( l i q u i d f i l m ) 2975, 1710, 1460, 1375, 1360, 1045 c m " 1 ; T ( C C 1 4 ) 7.23-7.80 (m, 3H) , 8.30-8.83 (m, 4H), 9.00 ( t , J = 7 H z , 9 H ) . Camphor e n o l a c e t a t e (42) n - B u t y l l i t h i u m (10 ml o f 2.1 M hexane s o l u t i o n , 0.02 mole) was added t o camphor (4) (3.04 g, 0.02 mole) d i s s o l v e d i n t e t r a -h y d r o f u r a n (20 m l ) . The r e a c t i o n m i x t u r e was c o o l e d t o -78°C, a c e t i c a n h y d r i d e (4.48 g, 0.04 mole) was added and the m i x t u r e - 137 -a l l o w e d t o s t i r a t room temperature f o r 2.5 h r . Water was added and the aqueous l a y e r e x t r a c t e d 3 times w i t h e t h e r , the e t h e r washed t h o r o u g h l y w i t h b r i n e , and d r i e d (MgS0 4). E v a p o r a t i o n o f the e t h e r i n vacuo gave a r e d o i l w h i c h was p u r i f i e d by p r e p a r a t i v e g l c ( 2 00 QC, 30% Se 30) to g i v e pure camphor e n o l a c e t a t e [42) as a c o l o u r l e s s o i l . -\} ( l i q u i d f i l m ) 2940, 1760, 1360, 1205, IIlclX 1120, 1005 c m " 1 ; ^ ( C C l 4 ) 4.48 ( d , J = 4Hz, 1H) , 7.68 [ t , J = 3Hz, 1H), 7.90 ( s, 3H), 9.06 ( s , 6H), 9.24 ( s , 3H). .Enol a c e t y l a t i o n o f 2-methylheptan-5-one (38) (a) 2-Methylheptan-3-one (38) (64 mg, 0.5 mmole), camphor e n o l a c e t a t e (42) (150 g, 0.77 mmole) and o x a l i c a c i d [5 mg) were r e f l u x e d o v e r n i g h t i n benzene (2 m l ) . The benzene s o l u t i o n was 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 , d r i e d CMgSO^) and the benzene removed a t reduced p r e s s u r e . A n a l y s i s o f the p r o d u c t by g l c (100°C, 3% Se 30) i n d i c a t e d o n l y the p r e s e n c e o f the two s t a r t i n g m a t e r i a l s and camphor. ( 4 ) . (b) The above e x p e r i m e n t was r e p e a t e d , u s i n g p_-TSA. (10 mg) as c a t a l y s t , t o g i v e an e s s e n t i a l l y s i m i l a r r e s u l t . (c) 2-Methylheptan-3-one (38) [15 mg, 0.12 mmole) was s t i r r e d 32 f o r 6 minutes i n p e r c h l o r i c a c i d / a c e t i c a n h y d r i d e r e a g e n t CI•5 m l ) . The r e a c t i o n m i x t u r e was then n e u t r a l i s e d w i t h s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n and 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 . A f t e r d r y i n g (MgSO^) the e t h y l a c e t a t e was removed i n vacuo and the c r u d e " p r o d u c t i d e n t i f i e d by g l c (100°C, 3% Se 30) as 2-methylheptan-3-one [ 3 8 ) . (d) 2-Methylheptan-3-one C38) Cl•0 g> 7.8 mmole), i s o p r o p e n y l a c e t a t e (37) (20 ml) and o x a l i c a c i d (250 mg) were h e a t e d over-n i g h t at 110°C, g i v i n g a v e r y s low r a t e of d i s t i l l a t i o n . E t h e r - 138 -was added to the d i s t i l l a t i o n r e s i d u e and the e t h e r s o l u t i o n was d r i e d (MgSO^) and the e t h e r e v a p o r a t e d a t reduced p r e s s u r e t o g i v e an o i l w h i c h was shown by g l c a n a l y s i s (100°C, 3% Se 30) t o be 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 . (e) 2-Methylheptan-3-one C38) (1•0 g, 7.8 mmole), i s o p r o p e n y l a c e t a t e (37) (20 ml) and p_-TSA (200 mg) were h e a t e d a t 110°C ( g i v i n g a v e r y slow r a t e o f d i s t i l l a t i o n ) f o r 48 h r t o p p i n g up w i t h more i s o p r o p e n y l a c e t a t e (37) when r e q u i r e d . E t h e r was added to the d i s t i l l a t i o n r e s i d u e and the e t h e r s o l u t i o n washed w i t h s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n . D r y i n g (MgSO^) and e v a p o r a t i o n o f the e t h e r at reduced p r e s s u r e gave an o i l w h i c h was shown by g l c a n a l y s i s (100°C, 31 Se 30) to c o n s i s t o f 3 p r o d u c t s , none of w h i c h was s t a r t i n g m a t e r i a l . S e p a r a t i o n o f these 3 compounds by p r e p a r a t i v e g l c (160°C, 30% Se 30) gave ( i n o r d e r o f d e c r e a s i n g v o l a t i l i t y ) t r a n s - 3 - a c e t o x y - 2 - m e t h y l h e p t -3-ene (40) ( 3 4 . 8 % ) . ( l i q u i d f i l m ) 2940, 1750, 1460 , 1355, in 3.x 1205 , 1045 cm" 1; ^ ( C C l ^ , 5.10 ( t , J = 6Hz, 1H) , 7.65 ( t , J = 6Hz, 1H), 7.91 ( s , 3H), 8.24 ( t , J = 6Hz, 2H), 8.65 ( q , J = 6Hz, 2H), 8.97 ( d , J = 7Hz, 9H); c i s - 3-acetoxy-2-methylhapt-3-ene (43) ( 3 3 . 6 % ) . A) ( l i q u i d f i l m ) 2975, 1760, 1365, 1210, 1180 in 3.x 1155, 1035 cm" 1; r T j ( C C l 4 ) , 5.13 ( t , J= 8Hz, 1H) , 7.24 (m, 1H) , 7.94 ( s , 3H), 9.03 ( d , J = 7Hz, 9H); and 3 - a c e t o x y - 2 - m e t h y l h e p t - 2 -ene (41) (31.7%) ,"\) ( l i q u i d f i l m ) 2925, 1755 , 1360 , 1205 , 1135, IU3X 1060 cm" 1; ^ ( C C 1 4 ) , 7.95 ( s , 3H), 8.31 ( s , 3H), 8.52 ( s , 3H), 9.10 (m, 3H). . ( f ) To a s o l u t i o n o f 2-methylheptan-3-one (38) (1.28 g, 0.01 mmole) i n t e t r a h y d r o f u r a n (10 ml) was added, w i t h s t i r r i n g and - 139 -c o o l i n g ( i c e / w a t e r ) , s u f f i c i e n t t r i t y l l i t h i u m s o l u t i o n [ p r e p a r e d from t r i p h e n y l methane (4.88 g > n.02 mole) and n - b u t y l l i t h i u m (10 ml o f 2.1 M hexane s o l u t i o n ) ] t o - g i v e a p e r s i s t e n t p i n k c o l o u r The s o l u t i o n was then c o o l e d ( i c e / w a t e r ) , a c e t i c a n h y d r i d e (2.04 g, 0.02 mole) added, and the m i x t u r e a l l o w e d t o s t i r a t room temp e r a t u r e f o r 4 h r . Water was added and the p r o d u c t e x t r a c t e d 3 times w i t h e t h e r w h i c h was then washed t h o r o u g h l y w i t h s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n . A f t e r d r y i n g (MgSO^) the e t h e r was e v a p o r a t e d t o g i v e an o i l , w h i c h was d i s t i l l e d a t 65°C (10 mm . g) t o g i v e a c o l o u r l e s s o i l (400 mg, 23.5%) w h i c h e x h i b i t e d s p e c t r a l c h a r a c t e r i s t i c s a lmost i d e n t i c a l t o t r a n s - 3 - a c e t o x y - 2 -methylhept-3-ene (40) p r e p a r e d as i n (e) above. A n a l y s i s by g l c (100°C, 3% Se 30) showed the m i x t u r e c o n s i s t e d o f t r a n s - 3-acetoxy-2-methyl-hept-3-ene (40) (90%) and 3-acetoxy-2-methylhept-2-ene (41) ( 1 0 % ) . A ttempted i s o m e r i s a t i o n s o f t r a n s - 5 - a c e t o x y - 2 - m e t h y l h e p t - 3 - e n e (40) (a) T r a n s - 3 - a c e t o x y - 2 - m e t h y l h e p t - 3 - e n e (40) (15 mg), i n e t h y l a c e t a t e (0.75 ml) was s t i r r e d a t room tem p e r a t u r e f o r 7 minutes w i t h p e r c h l o r i c a c i d / a c e t i c a n h y d r i d e r e a g e n t . " " A f t e r washing the e t h y l a c e t a t e s o l u t i o n 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 e v a p o r a t i n g the e t h e r , g l c a n a l y s i s (100°C, 3% Se 30) i n d i c a t e d the p r e s e n c e o f o n l y t r a n s - 3-acetoxy-2-methylhept-3-ene. (b) T r a n s - 3 - a c e t o x y - 2 - m e t h y l h e p t - 3 - e n e (40) (15 mg), c y c l o h e x a n e 3 6 (1.5 ml) and i o d i n e (1 c r y s t a l ) were s t i r r e d at 90°C f o r 24 h r . On e v a p o r a t i o n o f the c y c l o h e x a n e g l c a n a l y s i s (100°C, 3% Se 30) i n d i c a t e d the p r e s e n c e of o n l y t r a n s - 3-acetoxy-2-methylhept-3-ene ( 4 0 ) . - 140 -(c) Trans - 3-acetoxy - 2-meth.ylh.ept-3-ene C4Q) (50 mg) , i s o p r o p e n y l a c e t a t e (2 ml) and p_-TS. A [10 mg) were h e a t e d at 90°C f o r 24 h r . E t h e r was added, the e t h e r s o l u t i o n washed w i t h s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n and the e t h e r e v a p o r a t e d i n vacuo t o g i v e a p r o d u c t w h i c h g l c a n a l y s i s showed t o be m a i n l y 2-methylheptan-3-one (38) and a l i t t l e t r a n s - 3-acetoxy-2-methy1-hept-3-ene ( 4 0 ) . E n o l A c e t y l a t i o n o f 6 - m e t h y l - 2 - p - t o l y l h e p t - l - e n - 5 - o n e (15) To a s o l u t i o n 0 f 6 - m e t h y l - 2 - p _ - t o l y l h e p t - l - e n - 5 - o n e (15) (1.0 g, 4.63 mmole) i n dry t e t r a h y d r o f u r a n (10 ml) was added s u f f i c i e n t t r i t y l l i t h i u m s o l u t i o n , p r e p a r e d from t r i p h e n y l methane [2.44 g, 10 mmole) i n t e t r a h y d r o f u r a n (15 ml) and n - b u t y l l i t h i u m (4.28 ml of 2.34 M hexane s o l u t i o n , 10 mmole) to g i v e a p e r s i s t e n t f a i n t p i n k c o l o u r . The s o l u t i o n was then c o o l e d i n i c e , a c e t i c a n h y d r i d e (0.945 g, 9.26 mmole) added and the m i x t u r e a l l o w e d t o s t i r a t room temperature f o r 4 h r . Water was then added, the l a y e r s s e p a r a t e d and the aqueous phase e x t r a c t e d 3 times w i t h e t h e r . The combined o r g a n i c l a y e r s were d r i e d (MgSO^) and the s o l v e n t e v a p o r a t e d i n vacuo t o g i v e a y e l l o w o i l . A l i t t l e p e t r o l e u m e t h e r (60-80°C) was added and the s o l u t i o n s t o r e d o v e r n i g h t at 0°C t o p r e c i p i t a t e t r i p h e n y l methane, w h i c h was f i l t e r e d o f f . A f t e r e v a p o r a t i o n o f the s o l v e n t the m i x t u r e was s e p a r a t e d by p r e p a r a t i v e g l c (250°C, 30% Se 30) t o g i v e i n o r d e r o f d e c r e a s i n g v o l a t i l i t y , 5-acetoxy-6-methyl-2-£-tolylhepta-l,4-diene [44) (47.3 mg), X} m a x ( l i q u i d f i l m ) 2960 , 1750, 1200 , 895, 825 c m " 1 ; ^ ( C C l 4 ) 2.85 [ q , J = 7Hz, 4H) , 4.73 ( b r . s , 1H), 5.00 ( b r . s , 1H), 5.03 ( t , J = 7Hz, 1H), - 141 -7.00 Cd, J =. 7Hz, 2H}, 7.67 Cs, 3H) , 7.88 Cs, 3H), 9.03 ( d , J = 7Hz,: 6H) ; and 5 - a c e t o x y - 6 - m e t h y l - 2 - p _ - t o l y l h e p t a - l ,5-d i e n e (14) (7.9 mg) , A) ( l i q u i d f i l m ) , 2960, 1755, 1200, 900, max 820 cm" 1; T (CC1 4) 2.87 (m, 4H) , 4.83 ( b r . s , 1H) , 5.00 ( b r . s , 1H) , 7.65 ( s , 3H), 7.92 ( s , 3H). Treatment o f 5 - a c e t o x y - 6 - m e t h y l - 2 - p - t o l y l h e p t a - l , 5 - d i e n e (14)  w i t h b oron t r i f l u o r i d e Gaseous boron t r i f l u o r i d e was p a s s e d t h r o u g h a s o l u t i o n o f 5 - a c e t o x y - 6 - m e t h y l - 2 - p _ - t o l y l h e p t a - 1 , 5-diene (14) (7.9 mg) i n methylene c h l o r i d e (25 ml) f o r 5 m i n u t e s . Excess boron t r i f l u o r i d e was removed by p a s s i n g a stream o f n i t r o g e n gas t h r o u g h the s o l u t i o n f o r 0.25 h r , a f t e r w h i c h the methylene c h l o r i d e s o l u t i o n was washed w i t h s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n , washed w i t h b r i n e , d r i e d (MgSO^) and the s o l v e n t e v a p o r a t e d at reduced p r e s s u r e t o g i v e a y e l l o w o i l w h i c h e x h i b i t e d no i n f r a r e d a b s o r p t i o n between 1700 and 1800 cm 1 and was shown by g l c a n a l y s i s (175°C, 31 Se 30) to be a complex m i x t u r e o f compounds. - 142 -BIBLIOGRAPHY 1. J . N. C o l l i e and W. S. Meyers, J . Chem. S o c , 1893, 63, 122; J . N. C o l l i e , J . Chem. S o c , 1893, 6_3, 329. 2. E. E. van Tamelen, F o r t s c h r . Chem. Org. N a t u r s t o f f e , 1961, 19, 245. 3. T. Money, P r o g . Org. Chem., 1972 , S_, i n p r e s s . 4. E. E. van Tamelen, A c c o u n t s Chem. Res., 1968, 1_, 111. 5. J V C . F a i r l i e , G. L. Hodgson, and T. Money, Chem. Comm., 1969 , 1196. 6. J . C. F a i r l i e and T. Money, u n p u b l i s h e d o b s e r v a t i o n s . 7. G. L. Hodgson, D. F. MacSweeney, and T. Money, Chem. Comm., 1971, 766. 8. H. H i k i n o , N. S u z u k i , and T. Takemoto, T e t r a h e d r o n L e t t e r s , 1967, 5069. 9. D. F. MacSweeney and T. Money, u n p u b l i s h e d o b s e r v a t i o n s . 10. E. J . Corey, N.N. G i r o t r a , and C. T. Mathew, J . Amer. Chem. Soc. , 1969 , 91, 1557. 11. W. P a r k e r , J . S. R o b e r t s , and R'. Ramage, Qu a r t . Rev., 1967 , 21, 331. 12. G. L. C h e t t y and S. Dev, T e t r a h e d r o n L e t t e r s , 1964, 73. 13. T. I r i e , T. S u z u k i , S. I t o and E. Kurosawa, T e t r a h e d r o n L e t t e r s , 1967, 3187. 14.. W. P a r k e r , R. Ramage, and R. A. R a p h a e l , P r o c . Chem. S o c , 1961, 74 ; J . Chem. S o c , 1962 , 1558. 15. A. J . B i r c h and S. M. M u k h e r j i , J . Chem. S o c , 1949 , 2531. 16. "Handbook o f C h e m i s t r y and P h y s i c s " , 49th ed., The Ch e m i c a l Rubber Company, C l e v e l a n d , 1968, p. C-209. 17. A. S. Hussey, J . Amer. Chem. S o c , 1951 , 73, 1364. 18. H. C. Brown and B. C. Subba Rao, J . Org. Chem., 1957, 22, 1136. 19. A. M a n j a r r e z and A. Guzman, J . Org. Chem., 1966, 3_1, 348. 20. K. Bowden, I . M. H e i l b r o n , E. R. H. J o n e s , and B. C. L. Weedon, J . Chem. Soc. , 1946 , 39. - 143 -21. J . S. M i l l s and A. E. A. Werner, J . Chem. S o c , 1955 , 3132 ; E. W. Warnhoff and C. M. M. H a l l s , Canad. J . Chem., 1965, 43,3311. 22. L. F. F i e s e r and S. R a j a g o p a l a n , J . Amer. Chem. S o c , 1949 , 71, 3938.. 23. I . T. H a r r i s o n , P r o c . Chem. S o c , 1964 , 110. 24. G. I . Poos, G. E. A r t h , R. E. B e y l e r , and L. H. S a r e t t , J . Amer. Chem. S o c , 1953, 75 , 422. » 25. L. F. F i e s e r and M. F i e s e r , "Reagents f o r O r g a n i c S y n t h e s i s " , John W i l e y and Sons I n c . , New Y o r k , 1967, p.1162. 26. E. J . Corey and M. Chaykovsky, J . Amer. Chem. S o c , 1962 , 84, 866; 1965, 87, 1345. 27. M. J u l i a , S. J u l i a , and R. Guegan, Compt.Rend., 1959, 24 8, 820; B u l l . Soc. Chim. F r a n c e , 1960, 1072. 28. J . M. F o r r e s t e r , M. Sc. T h e s i s , U n i v e r s i t y o f B r i t i s h C o lumbia, 1970 . 29. G. B u c h i , P. K u i s a , K. Ogasawara, and R. L. R o s a t i , J . Amer. Chem. S o c , 1970 , 9_2, 999. 30. A. S t r e i t w e i s e r , J r . , J . R. W o l f e , J r . , and W. D. S h a e f f e r , T e t r a h e d r o n , 1959, 6^ , 338; H. S. Mosher and E. Lacombe, J . Amer. Chem. S o c , 1950 , 7_2 , 3994; M. S. Kharasch and S. Weinhouse, J . Org. Chem., 1936, 1, 209; see a l s o M. S. K h a r a s c h and 0. Reinmuth " G r i g n a r d R e a c t i o n s o f Non-m e t a l l i c S u b s t a n c e s " , P r e n t i c e H a l l , New Y o r k , 1954 , p.158. 31. H. 0. House and V. Kramar, J . Org. Chem., 1963, _28, 3362. 32. B. E. Edwards and P. N. Rao, J . Org. Chem., 1966, 31_, 324. 33. G. C. J o s h i , W. D. Chambers, and E. W. Warnhoff, T e t r a h e d r o n L e t t e r s , 1967, 3613. 34. S. J . Rhoads, J . K. Chattop a d h y a y , and E. E. W a a l i , J . Org. Chem., 1970 , 3_5 , 3352. 35. "Handbook o f C h e m i s t r y and P h y s i c s " , 49th Ed. The Chemical Rubber Company, C l e v e l a n d , 1968, p.C-410. PART I I I PHOTOCHEMICAL CLEAVAGE OF PYRONES - 14.4 -INTRODUCTION 1 2 C o l l i e was the f i r s t t o su g g e s t ' t h a t some a r o m a t i c compounds c o u l d be b i o s y n t h e s i s e d from a c e t i c a c i d by way of a l i n e a r p o l y - | f i - d i k e t o n e c h a i n , a c h a r a c t e r i s t i c f e a t u r e o f compounds d e r i v e d i n t h i s manner b e i n g a 1 , 3 - r e l a t i o n s h i p between a d j a c e n t h y d r o x y l o r c a r b o n y l groups. C o l l i e e n v i s a g e d t h a t the s t a r t i n g u n i t was a c e t i c a c i d and t h a t t h i s condensed w i t h f u r t h e r m o l e c u l e s o f a c e t i c a c i d t o g i v e the l i n e a r poly-y£-d i k e t o n e c h a i n which c o u l d then undergo i n t e r n a l a l d o l or C l a i s e n c o n d e n s a t i o n s to g i v e a r o m a t i c compounds. Not o n l y d i d C o l l i e f i r s t p o s t u l a t e the p o l y a c e t a t e t h e o r y b u t he a l s o p e r f o r m e d the f i r s t b i o g e n e t i c type s y n t h e s e s o f a r o m a t i c compounds . Thus he c o n v e r t e d d e h y d r o a c e t i c a c i d ( 1 ) , a p r o t e c t e d ^ 5 - t r i k e t o n e , i n t o o r c i n o l (2) by t r e a t m e n t w i t h sodium h y d r o x i d e . He a l s o o b t a i n e d o r c i n o l [2) by a c i d c a t a l y s e d OH 0 CD (2) c y c l i s a t i o n o f h e p t a - 2 , 4 , 6 - t r i o n e (3) w h i c h i t s e l f was o b t a i n e d from d e h y d r o a c e t i c a c i d ( 1 ) . - 145 -( 3 ) ( 2 ) The p o l y a c e t a t e t h e o r y was " r e d i s c o v e r e d " and e l a b o r a t e d by B i r c h ^ and R o b i n s o n 5 . S i n c e then v a r i o u s m o d i f i c a t i o n s have been made, such as the importance o f m a l o n y l co-enzyme A as the c h a i n p r o p a g a t i n g u n i t and the use o f a c i d s o t h e r than a c e t i c a c i d as the c h a i n s t a r t e r u n i t . The t h e o r y i s now c o n s i d e r e d as the acy 1-polymalonate r o u t e t o a r o m a t i c compounds t o take i n t o a ccount th e s e more r e c e n t l y e l u c i d a t e d f e a t u r e s , and i s b r i e f l y summarised i n F i g u r e I . The secondary m o d i f i c a t i o n s i n c l u d e a l k y l a t i o n s , o x i d a t i o n s , r e d u c t i o n s , h a l o g e n a t i o n and i n t r o d u c t i o n o f n i t r o g e n . I n r e c e n t y e a r s t h e r e have been numerous r e p o r t s d e s c r i b i n g the c o n v e r s i o n of p o l y - j 3 - d i k e t o n e c h a i n s , e i t h e r p r o t e c t e d or u n p r o t e c t e d , t o a r o m a t i c compounds. One way o f p r o t e c t i n g the p o l y - ^ - d i k e t o n e c h a i n i s as a pyrone and t h i s has been used by the r e s e a r c h group a t U.B.C. The b i o g e n e t i c type s y n t h e s i s o f a r o m a t i c compounds from poly - / 5-diket.one c h a i n s has r e c e n t l y - 146 -F a t t y a c i d s P r o p i o n a t e CH 3COSCoA V + + B i o t i n / A T P / M g /HC0 3 RCOSCoA CH oC0SCoA I 2 C0 2H V Enzyme CH 2"C0S-Enzyme C0 2H c h a i n s t a r t e r u n i t A Cinnamic a c i d s B e n z o i c a c i d s N i c o t i n i c a c i d A n t h r a n i l i c a c i d A S h i k i m a t e Pathway c h a i n p r o p a g a t i n g u n i t RCOCH 2COSEnzyme c h a i n p r o p a g a t i n g u n i t RCO(CH 2CO) CH 2COSEnzyme (n = 1-8) i n t r a m o l e c u l a r c o n d e n s a t i o n s and secondary m o d i f i c a t i o n s P h e n o l i c compounds F i g u r e I : A c y 1 - p o l y m a l o n a t e r o u t e t o a r o m a t i c compounds - 147 -been r e v i e w e d by Money . As an example, b i s p y r o n e (4) can be c o n v e r t e d , under approp-r i a t e c o n d i t i o n s , to o r s e l l i n i c a c i d (5) (and d e r i v a t i v e s t h e r e o f ) 7 8 and carbomethyoxyphloracetophenone ( 6 ) . ' Other b i s p y r o n e 7a d e r i v a t i v e s gave n a t u r a l l y o c c u r r i n g p h e n o l i c compounds and (6) (5) t r i s p y r o n e (7) a l s o gave compounds of n a t u r a l type when t r e a t e d 7 under a p p r o p r i a t e c o n d i t i o n s . T e t r a p r y o n e (8) has a l s o been 7 p r e p a r e d but c o u l d not be c o n v e r t e d to a r o m a t i c compounds. Thus an a l t e r n a t i v e method of j o i n i n g the pyrone r i n g s 9 was d e v i s e d by Douglas and Money. T h i s method g i v e s r i s e t o - 148 -pyrone u n i t s which, are l i n e a r r a t h e r than condensed, .and the s t r u c t u r e s , s y n t h e s i s and r e a c t i o n s o f t h i s type o f compound, q e.g. pyrone ( 9 ) , have been f u l l y d e s c r i b e d . I t was s u b s e q u e n t l y found t h a t t r e a t m e n t o f compounds of t h i s t y p e , e.g. pyrone (10) under a v a r i e t y of b a s i c and a c i d c o n d i t i o n s d i d not l e a d to the f o r m a t i o n o f a r o m a t i c compounds. However t r e a t m e n t of the c o r r e s p o n d i n g m e t h y l e t h e r (11) under b a s i c c o n d i t i o n s gave e v e r n i n i c a c i d ( 1 2 ) . ^ A t t e m p t s t o c o n v e r t l i n e a r pyrone ( 9 ) t o a r o m a t i c compounds were o n l y p a r t i a l l y s u c c e s s f u l and a l t e r n a t i v e r i n g c l e a v a g e - a r o m a t i s a t i o n c o n d i t i o n s were - 149 -i n v e s t i g a t e d . I n p a r t i c u l a r p h o t o c h e m i c a l c o n v e r s i o n o f 0<-pyrones t o ^ - p o l y k e t o e s t e r s (and hence t o a r o m a t i c compounds) seemed to be a p o s s i b i l i t y . OMe 0 C0 2Me 0 ^ 0 (11) OMe 0 . 0 c o 2 a C0 2Me MeO CO (12) 11 P r e v i o u s s t u d i e s by de Mayo have shown t h a t 4 , 6 - d i m e t h y l e*-pyrone (13) p h o t o l y s e s i n methanol s o l u t i o n to g i v e m e t h y l p-acetonylcrotonate ( 1 4 ) . De Mayo e x p l a i n e d t h i s r e a c t i o n by h-o Me OH - o o (13) - 0 (14) C0 2Me i n v o k i n g the i n t e r m e d i a c y o f ketene (15) but Corey and S t r e i t h have s u g g e s t e d t h a t (3 - l a c t o n e (16) may be i n v o l v e d . 12 (IS) (16) - 150 -13 Thus by a n a l o g y B e d f o r d and Money c o n s i d e r e d t h a t t r i a c e t i c a c i d l a c t o n e (17) s h o u l d g i v e m e t h y l 3,5-dioxohexanoate (18) when 13 p h o t o l y s e d i n methanol s o l u t i o n . However these a u t h o r s found t h a t the major p r o d u c t formed by p h o t o l y s i s i n methanol s o l u t i o n was c i s - | 3 - c a r b o m e t h o x y m e t h y l c r o t o n i c a c i d ( 1 9 ) , w i t h o n l y a minor amount (12%) o f e s t e r ( 1 8 ) . I t was a l s o shown t h a t i r r a d i a t i o n Me0 2C C0 2H (17) (18) (19) M i n o r Major of pyrone (17) i n e t h e r or benzene gave ( S - m e t h y l g l u t a c o n i c a n h y d r i d e (20) w h i c h i n methanol was c o n v e r t e d i n t o c i s - ^ - c a r b o -m e t h o x y m e t h y l c r o t o n i c a c i d (19) . These r e s u l t s were r a t i o n a l i s e d by i n v o k i n g 0 - l a c t o n e (21) and ketene (22) and the p r o p o s e d r e a c t i o n scheme i s shown i n F i g u r e I I . The a u t h o r s s u g g e s t t h a t the 4 - h y d r o x y l group a f f e c t s the r e a c t i v i t y o f ^ - l a c t o n e (21) i n d u c i n g i r r e v e r s i b l e rearrangement to a c y c l o b u t e n o n e d e r i v a t i v e (23) w h i c h can then be c o n v e r t e d to g l u t a c o n i c a c i d d e r i v a t i v e s (19) and (20) . An a l t e r n a t i v e mechanism f o r the p h o t o c h e m i c a l c o n v e r s i o n of oc-pyrones to e s t e r s w i t h u n r e a r r a n g e d carbon s k e l e t o n s has 14 r e c e n t l y been proposed by M c i n t o s h and Chapman. They propose t h a t a d d i t i o n o f methanol to ketene (15) i s not I n v o l v e d i n t h i s p r o c e s s , b u t t h a t the p r i m a r y p h o t o r e a c t i o n i s a d d i t i o n o f - 151 -F i g u r e ! I : P h o t o c h e m i s t r y o f t r i a c e t i c a c i d l a c t o n e - . 152 -methanol t o one o f the double bonds o f the pyr o n e . Thus when 4,6-dimethyl-<X-pyrone (13) was p h o t o l y s e d i n methanol the p r i m a r y p h o t o p r o d u c t s were pyrones (24) and (25) . These compounds were s u b s e q u e n t l y opened by a c i d c a t a l y s e d m e t h a n o l y s i s t o g i v e a m i x t u r e o f i s o m e r i c m e t h y l e s t e r s such as e s t e r ( 1 4 ) . Me OH S^*0 CO 2 Me I I 0 0 + o MeO O ^ 0 H+/MeOH + o t h e r isomers C14) - 153 -DISCUSSION As an a l t e r n a t i v e method of opening pyrone r i n g s to g i v e 13 p o l y - | & - d i k e t o - e s t e r c h a i n s B e d f o r d and Money p h o t o l y s e d t r i a c e t i c a c i d l a c t o n e ( 1 7 ) , i n v a r i o u s s o l v e n t s , h o p i n g to o b t a i n e s t e r ( 1 8 ) . However p h o t o l y s i s o f t r i a c e t i c a c i d l a c t o n e (17) i n methanol gave a m i x t u r e o f p r o d u c t s c o n t a i n i n g o n l y a minor amount o f e s t e r (18) ( 1 0 - 1 5 % ) , the main p r o d u c t s b e i n g compounds w i t h r e a r r a n g e d c a r b o n s k e l e t o n s , p - c a r b o m e t h o x y m e t h y l c r o t o n i c a c i d (19) and f3 - m e t h y l g l u t a c o n i c a n h y d r i d e (20) . (17) (18) (19) (20) I n an attempt to i n c r e a s e the y i e l d o f p r o d u c t w i t h an u n r e a r r a n g e d carbon s k e l e t o n t r i a c e t i c a c i d l a c t o n e m e t h y l e t h e r (26) was p h o t o l y s e d i n methanol. P h o t o l y s i s o f a 0.2% m e t h a n o l i c s o l u t i o n of m e t h y l t r i a c e t i c a c i d l a c t o n e (26) r e s u l t e d i n an almost q u a n t i t a t i v e c o n v e r s i o n to a s i n g l e p h o t o p r o d u c t w h i c h - 154 -was i s o l a t e d as a c o l o u r l e s s o i l and had s p e c t r a l and a n a l y t i c a l p r o p e r t i e s i n agreement w i t h the a s s i g n e d s t r u c t u r e ( 2 7 ) . Thus the molecular, w e i g h t was found (by mass s p e c t r o s c o p y ) t o be 172, OMe h-0 MeOH MeO OMe (27) i n d i c a t i n g t h a t methanol had been added t o m e t h y l e t h e r (26) ; the u l t r a v i o l e t spectrum i n d i c a t e d an «<^ 3 - u n s a t u r a t e d c a r b o n y l chromophore at 220 nm (£ = 9,540) and the i n f r a r e d spectrum showed t h i s chromophore c o u l d be an ^ - u n s a t u r a t e d S - l a c t o n e . The n u c l e a r m agnetic resonance spectrum was p a r t i c u l a r l y i n f o r m a t i v e , showing a b s o r p t i o n s due t o a m e t h y l group on a double bond ( 8 . 0 2 f t ) , a p r o t o n on a double bond (4.33*£), two e q u i v a l e n t methoxyl groups (6.68 t ) and an i s o l a t e d methylene group (7.45 "£). C o n f i r m a t i o n o f s t r u c t u r e (27) was o b t a i n e d by c o n v e r s i o n , w i t h wet e t h e r , o f the p h o t o p r o d u c t t o c i s - f t - c a r b o m e t h o x y m e t h y l c r o t o n i c a c i d ( 1 9 ) . 1 3 MeO' C0 2Me C0 2H MeO (27) (19) - 155 -The c o n v e r s i o n o f methyl e t h e r (26) to dimethoxy pyrone (27) thus d i f f e r s markedly from the b e h a v i o u r o f t r a i c e t i c a c i d l a c t o n e (17) as there, i s no p r o d u c t formed w i t h an u n r e a r r a n g e d carbon s k e l e t o n . The mechanism o f t h i s r e a c t i o n seems t o i n v o l v e f o r m a t i o n o f ^ - l a c t o n e ( 2 8 ) . E v i d e n c e f o r the f o r m a t i o n o f b i c y c l i c (3 - l a c t o n e (28) d u r i n g the low t e m p e r a t u r e p h o t o l y s i s o f pyrone (26) has been o b t a i n e d by de Mayo and c o - w o r k e r s . 1 5 M e t h a n o l a d d i t i o n t o ^ - l a c t o n e (26) g i v e s r i s e to a c i d (29) w h i c h can now open t h e r m a l l y i n a c o n r o t a t o r y manner to g i v e dimethoxy a c i d (30) w h i c h then c y c l i s e s to g i v e dimethoxy pyrone ( 2 7 ) . T h i s i s shown i n F i g u r e I I I . I t i s i n t e r e s t i n g t o note t h a t t r i a c e t i c a c i d l a c t o n e (17) i s a n a t u r a l l y o c c u r r i n g p y r o n e 1 6 w h i c h i s b i o s y n t h e s i s e d by l i n e a r c o n d e n s a t i o n o f t h r e e a c e t a t e . u n i t s . The c o n v e r s i o n o f t h i s compound to a b r a n c h e d , s i x . c a r b o n compound o f the g l u t a c o n i c a c i d type might have s i g n i f i c a n c e i n t e r p e n o i d b i o s y n t h e s i s as i t i s an a l t e r n a t i v e method of d e r i v i n g m e v a l o n i c a c i d from t h r e e m o l e c u l e s o f a c e t i c a c i d . F u r t h e r work i s i n p r o g r e s s to d e v i s e a l t e r n a t i v e methods by w h i c h an e s t e r such as pyrone (10) may be p h o t o c h e m i c a l l y c o n v e r t e d i n t o a l i n e a r p o l y - | S - d i k e t o e s t e r c o n t a i n i n g 8 carbon atoms. CO-Me 1 (10). F i g u r e I I I : P h o t o c h e m i s t r y o f m e t h y l t r i a c e t i c a c i d l a c t o n e - 1 5 7 -EXPERIMENTAL I r r a d i a t i o n e x p eriments were conducted at room temperature under dry n i t r o g e n u s i n g an i n t e r n a l w a t e r - c o o l e d mercury a r c lamp ( H a n o v i a , 450W) equipped w i t h a p y r e x f i l t e r . The methanol was r i g o r o u s l y d r i e d by the f o r m a t i o n o f s m a l l amounts o f magnesium methoxide. M e l t i n g p o i n t s were d e t e r m i n e d on a K o f l e r b l o c k and are u n c o r r e c t e d . I n f r a r e d s p e c t r a (\) ) were r e c o r d e d m cix on a P e r k i n - E l m e r I n f r a c o r d model 137 s p e c t r o p h o t o m e t e r . U l t r a -v i o l e t s p e c t r a (A ) were r e c o r d e d i n methanol s o l u t i o n on a Unicam SP 800 s p e c t r o p h o t o m e t e r ; e x t i n c t i o n c o e f f i c i e n t s (£) are g i v e n i n p a r e n t h e s e s . N u c l e a r magnetic resonance s p e c t r a (X) were r e c o r d e d i n c a r b o n t e t r a c h l o r i d e o r d e u t e r i o c h l o r o f o r m on a V a r i a n A s s o c i a t e s A-60 s p e c t r o m e t e r . S i g n a l p o s i t i o n s are g i v e n i n t h e ' T i e r s ^ 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 as an i n t e r n a l s t a n d a r d ; the m u l t i p l i c i t y , c o u p l i n g c o n s t a n t s [where a p p r o p r i a t e ) and i n t e g r a t e d peak areas are i n d i c a t e d i n p a r e n t h e s e s ; s = s i n g l e t , d = d o u b l e t , b.d = b r o a d d o u b l e t , m = m u l t i p l e t . Mass s p e c t r a (M +) were d e t e r m i n e d w i t h an AEI MS 9 s p e c t r o m e t e r . M i c r o a n a l y s e s were p e r f o r m e d by Mr. 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 , U n i v e r s i t y o f B r i t i s h C olumbia. T h i n l a y e r c h r o m a t o g r a p h i c ( t i c ) ? 5 4 r e s u l t s were o b t a i n e d u s i n g s i l i c a g e l HF" J o f 0.3 mm t h i c k n e s s and e l u t i n g w i t h c h l o r o f o r m . : a c e t i c a c i d ( 9 : 1 ) . I r r a d i a t i o n o f 4-methoxy-6-methyl-2-pyrone (2,6) i n M e t h a n o l The p r o g r e s s o f the r e a c t i o n was m o n i t o r e d by the d i s a p p e a r a n c e of the l i g h t a b s o r p t i o n band at 281 nm and the appearance o f a new band at 220 nm. I t was found t h a t no t r i a c e t i c a c i d l a c t o n e m e t h y l - 158 e t h e r C26) remained a f t e r about 6 h r I r r a d i a t i o n and t i c a n a l y s i s o f the p r o d u c t i n d i c a t e d t h a t one p h o t o p r o d u c t had been formed. In a t y p i c a l .run, t r i a c e t i c a c i d l a c t o n e m e t h y l e t h e r (26) (1.00 g, 7.16 mmole) was i r r a d i a t e d i n methanol (480 ml) f o r 6 h r . E v a p o r a t i o n o f the methanol gave a p a l e y e l l o w o i l (1.07 g, 96.5%) w h i c h on d i s t i l l a t i o n y i e l d e d a c o l o u r l e s s o i l w i t h p h y s i c a l and c h e m i c a l p r o p e r t i e s c o n s i s t e n t w i t h the a s s i g n e d s t r u c t u r e ( 2 7 ) , b.p. 80°C (9 mm).-\J m a x (CC1 4) 1730, 1380 , 1360 , 850 cm" 1; ^ m a x H 2 2 0 n m & = 9 ' 5 4 0 ) ; ^ ( C C 1 4 ) > 4 ' 3 3 <>> 1 H ) > 6 - 6 8 Cs, 6 H ) > 7.45 ( s , 2H), 8.02 ( s , 3H); M + m/e 172; C, 55.62; H, 6.88 ( c a l c . C o n v e r s i o n o f p h o t o p r o d u c t (27) t o cis-/?-carbomethoxymethyl-' c r 6 t o n i c a c i d (19) (a) P h o t o p r o d u c t (27) (87.6 mg, 0.51 mmole) was d i s s o l v e d i n w a t e r s a t u r a t e d e t h e r (20 ml) and a l l o w e d t o s t a n d a t room tem p e r a t u r e f o r 24 h r . E v a p o r a t i o n to dryness f o l l o w e d by r e c r y s t a l l i s a t i o n o f the s o l i d r e s i d u e from p e t r o l e u m e t h e r (60-80°C) gave cis-|S-c a r b o m e t h y o x y m e t h y l c r o t o n i c a c i d (19) (54.2 mg, 67.2%) as w h i t e ( s , 1H), 4.16 (m, 1H), 6.30 ( b . d . , 2H), 6.34 ( s , 3H), 8.00 ( d , J = 1.4 Hz, 3H). (b) To a s o l u t i o n used t o measure the nmr spectrum o f p h o t o p r o d u c t (27) ("-30 mg i n 0.4 ml CCl^) was added w a t e r (2 drops) . P e r i o d i c nmr assay showed g r a d u a l d i s a p p e r a n c e o f peaks c o r r e s p o n d i n g to p h o t o p r o d u c t (2 7) and the appearance of peaks a t t r i b u t a b l e t o f o r C g H 1 2 0 4 : C, 55.82; H, 6.98). n e e d l e s , m.p. 80-82°C ( l i t . 77- 78°C,-\) 1650, 1270 , 1170 c m _ 1 ; > M e o H 213 nm (£ = - 159 -c i s - f t -carbometh.yoxymethylcroton.ic a c i d (19) . A f t e r 3 days r e a c t i o n was complete and a f t e r e v a p o r a t i o n to dryness the s o l i d p r o d u c t e x h i b i t e d an nmr spectrum i d e n t i c a l to that, o f pure c i s - ^ 8 - c a r b o -m e t h o x y m e t h y l c r o t o n i c a c i d (19) . - 160 -BIBLIOGRAPHY 1. J . N. C o l l i e , T r a n s . Chem. S o c , 1893, 6_3, 329. 2. J . N. C o l l i e , T r a n s . Chem. S o c , 1907, 9_1 , 1806. 3. J . N. C o l l i e and W. S. Meyers, J . Chem. S o c , 1893, 6_3, 122. 4. A. J . B i r c h and F. W. Donovan, A u s t r a l . J . Chem., 1953, 3_6, 360. 5. R. R o b i n s o n , "The S t r u c t u r a l R e l a t i o n s o f N a t u r a l P r o d u c t s " , C l a r e n d o n , O x f o r d , 1955. 6. T. Money, Chem. Rev., 1970 , 70_, 553. 7. T. Money, F. W. Comer, G. R. B. Webster, I . G. W r i g h t , and A. I . S c o t t , T e t r a h e d r o n , " 1967 , 23, 3435. 7a. J . L. Douglas and T. Money, T e t r a h e d r o n , 1967 , 2_3 , 3545. 8. J . L. Douglas and T. Money, Canad. J . Chem., 1967 , 4_5, 1990. 9. J . L. Douglas and T. Money, Canad. J . Chem., 1968 , 4_6 , 695. 10. C. T. B e d f o r d , J . L. Dougl a s , B. E. McCarry, and T. Money, Chem. Comm., 1968, 1091. 11. P. de Mayo, Adv. Org. Chem., 1960, 2^ 394. 12. E. J . Corey and J . S t r e i t h , J . Amer. Chem. S o c , 1964, 86, 950. 13. C. T. B e d f o r d and T. Money, Chem. Comm., 1969, 685. 14. C. L. M c i n t o s h and 0. L. Chapman, J . Amer. Chem. S o c , s u b m i t t e d f o r p u b l i c a t i o n . A p p r e c i a t i o n i s g r a t e f u l l y acknowledged to P r o f e s s o r Chapman f o r i n f o r m a t i o n r e g a r d i n g h i s r e s u l t s p r i o r t o p u b l i c a t i o n . 15. J . P. G u t h r i e , C. L. M c i n t o s h , and P. de Mayo, Canad. J . Chem. , 1970 , 4_8 , 237 . 16. T. M. H a r r i s , C. M. H a r r i s , and R. J . L i g h t , B i o c h i m . B i o p h y s . A c t a , 1966 , 121 , 420 . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0060119/manifest

Comment

Related Items