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UBC Theses and Dissertations

Studies on the biosynthesis of trichothecin Forrester, James McLeod 1970

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S T U D I E S ON THE BIOSYNTHESIS OF TRIGHOTHECIN by J A M E S MCLEOD F O R R E S T E R ' B . S c , U n i v e r s i t y of E d i n b u r g h , 196? A THESIS SUBMITTED IN PARTIAL FULFILMENT OF .THE REQUIREMENTS FOR THE DEGREE O P ':'; " • ' MASTER OF SCIENCE i n the Department of 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 t o the r e q u i r e d standard. THE UNIVERSITY OF BRITISH COLUMBIA . , ' September 1 9 7 0 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D a t e IL^O <MU* *<WO ( i i ) ABSTRACT . ' The d e r i v a t i o n of trichothecin (28) from -bisabolene ( 3 ^ ) i s discussed and the contradictory r e s u l t s of two research groups noted. Radioactive <X-bisabolol ( 3 5 ) was prepared by reaction of radioactive methyl magnesium Iodide on ketone (42) which was prepared by a modification of a l i t e r a t u r e procedure. Thus Diels-Alder addition of isoprene (1) and a c r y l i c acid gave acid ( 4 l ) . The structure of the lactone prepared by treatment J of t h i s acid with 98?6 formic acid i s corrected to lactone (64) and not lactone (62). Bromide ( 3 8 ) was prepared by a l i t e r a t u r e procedure from methyl cyclopropyl ketone ( 4 4 ) . Addition of the acid chloride of acid (4l) to the Grignard complex of bromide ( 3 8 ) gave ester ( 7 0 ) which on treatment with ethereal methyl lithium s o l u t i o n followed by Jones oxidation gave ketone (42) , Radioactive o<-bisabolol (3*0 was then prepared from ketone (42) by reaction with radioactive methyl magnesium iodide, and fed to Trichothecium roseum. I s o l a t i o n of t r i c h o t h e c i n (28) showed an incorporation of 0.014$. Degradation of trichothecin (28) to trichothecolone (29) showed a s i g n i f i c a n t decrease i n r a d i o a c t i v i t y , the s i g n i f i c a n c e of which i s then discussed. ( i i i ) TABLE OP CONTENTS I n t r o d u c t i o n • • 1 D i s c u s s i o n . . « . • • - . . . . . . . . . . 27 E x p e r i m e n t a l . . . » e . . . . . « . . . » . » . . . » 50 ( i v ) ACKNOWLEDGMENT I would l i k e to express my g r a t i t u d e to Dr. T. Money f o r h i s c o n s t a n t guidance and many h e l p f u l suggestions d u r i n g the course of t h i s work and the p r e p a r a t i o n of the t h e s i s , I would a l s o l i k e to thank Dr. P. S a l i s b u r y who grew the c u l t u r e s of Trichotheciura roseura. it INTRODUCTION N a t u r a l products have f o r many years h e l d a f a s c i n a t i o n f o r o r g a n i c chemists, The problem f i r s t posed by a n a t u r a l p r o d u c t i s the e l u c i d a t i o n of i t s s t r u c t u r e , which c l a s s i c a l l y can be d i v i d e d 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 of the f u n c t i o n a l groups presents d e t e r m i n a t i o n of the carbon s k e l e t o n and the p o s i t i o n s of 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 of the s t e r e o -chemistry, and f i n a l l y a 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 o l d e r method of p e r f o r m i n g t h i s s t r u c t u r a l e l u c i d a t i o n was by chemical d e g r a d a t i o n and s y n t h e s i s of the s m a l l e r p a r t s so o b t a i n e d . The modern method i s to 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 magnetic resonance and mass sp e c t r o s c o p y . I t soon became apparent t h a t n a t u r a l products 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 to s t r u c t u r a l s i m i l a r i t i e s which were thought to be a consequence of 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 suggested ' 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 condensations of a c e t a t e u n i t s , to g i v e l i n e a r poly - ^ 8-diketones which c o u l d c y c l i s e to 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 was l a t e r extended by 3 B i r c h . I n the f i e l d of i s o p r e n o i d s 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 "Isoprene Rule" which s t a t e s t h a t i s o p r e n o i d s a r e d e r i v e d by h e a d - t o - t a i l condensation of i s o p r e n e u n i t s ( 1 ) . (1) - 2 -5 \ In 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 early i n t h i s century that, f o r example, dihydroxy phenylalanine was probably involved i n the biosynthesis of ce r t a i n isoquinollne a l k a l o i d s . P r a c t i c a l l y a l l natural products a r i s e from r e l a t i v e l y simple s t a r t i n g materials by way of a few reac t i o n types. The natural products of the organic chemist are more c o r r e c t l y c a l l e d secondary metabolites and are products of secondary metabolism which may be schematically shown as i n Figure I: C 0 2 + H 2 ° h\> chlorophyll Carbohydrates V Carbohydrate Metabolism .Complex natural products of / every type v i a enzymatically c o n t r o l l e d reactions Pool of simple organic compounds Figure I .'• The carbohydrates are monosaccharides which are converted to pyruvic a c i d (2) and then to ac e t y l co-enzyme A (3)° 0 II CH3-C-CO2H (2) A c e t y l co-enzyme A i s probably the most important intermediate i n the biosynthesis of natural products as i t i s a common intermediate on the pathway to a l l three of the main groups of natural products, the isoprenoids, the a l k a l o i d s , and the aromatic compounds. - 3 -o II CH^C-S-CHgCHgNHCO(CH2)gNHCOCH-OH CHo I i 3 C I CH CH2-0-P-0-P-OH' OH 0 (3) The important r o l e of a c e t y l co-enzyme A 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 I . L i n e a r c o n d e n s a t i o n is* F a t t y a c i d s and Aromatic compounds compounds e.g. Anthraquinones 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 Krebs ^ c y c l e Amino A c i d s V A l k a l o i d s Branched condensation Mevalonic A c i d 1 V I s o p r e n o i d s F i g u r e 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 a t pr e s e n t i s thought to proceed as f o l l o w s . The i n i t i a l b i o c h e m i c a l r e a c t i o n i n t e r p e n o i d b i o s y n t h e s i s Is the enzymic r e d u c t i o n of (S ) - 3-hydro3ry - 3-methylglutaryl co-enzyme A (k) to produce (R)-mevalonic a c i d (5)» the r e d u c t i o n proceeding by two hydrogen t r a n s f e r s from reduced n i c o t i n a m i d e - a d e n i n e 6,7 - i+ -d i n u c l e o t i d e phosphate (6a) o r NADPH .for s h o r t . The o x i d i s e d form i s NADP + (6b) . H H OH 0 R I H O — P = 0 (6b) (6a) CH T h i s Is the o n l y method, so f a r known, by which mevalonic a c i d i s produced and mevalonic a c i d i s used only f o r t e r p e n o i d b i o s y n t h e s i s o E s t e r (k), however, can be produced by a number of pathways. I t can be b l o s y n t h e s i s e d by enzymic h y d r a t i o n of the g l u t a c o n i c e s t e r (7) which comes from b i o t i n dependent c a r b o x y l a t i o n of 3 - m e t h y l c r o t o n y l co-enzyme A (8), a d e g r a d a t i o n product of l e u c i n e . A b i o l o g i c a l l y more important pathway to (k) i s the condensation of a c e t y l co-enzyme A (3) w i t h a c e t o -8 a c e t y l co-enzyme A (9) . E s t e r (9) can be s y n t h e s l s e d e i t h e r by d e g r a d a t i o n of f a t t y a c i d s or by condensation of two molecules of a c e t y l co-enzyme A, T h i s i s summarised i n F i g u r e I I I . Carbohydrate F a t P r o t e i n (11) Figure III - 6 -Thus mevalonic a c i d can be s y n t h e s i s e d from three molecules of a c e t i c a c i d s i n c e there 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 a c i d w i t h co-enzyme A. . ' Mevalonic a c i d has been used more than any other p r e c u r s o r i n terpene b i o s y n t h e t i c s t u d i e s . T h i s i 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 eleven ways, depending on the l a b e l l i n g requirements, and most i m p o r t a n t l y , i t i s not known to be u t i l i s e d f o r a n y t h i n g other than terpene b i o s y n t h e s i s . Only the (R)-form of mevalonic a c i d 9 i s b i o l o g i c a l l y a c t i v e , the (S)-form b e i n g raetabolically 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 mevalonic a c i d i s u s u a l l y racemlc and i s d i f f i c u l t to r e s o l v e . The next two enzymic processes on the pathway to t e r p e n o i d s a r e both p h o s p h o r y l a t i o n s which produce mevalonic acid~5"*Phosphate (10) and mevalonic acId - 5~PJTophosphate (11); 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 of adenosine t r i p h o s p h a t e (ATF) (12). NH 2 The enzymes r e s p o n s i b l e have been p u r i f i e d from y e a s t ' and 12 13 from l i v e r ' e s p e c i a l l y the one producing the monophosphate, , v 10,14 which Is the one which only u t i l i s e s (R)-mevalonic a c i d The next stage of 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 degradation,, Mevalonic acld-5-pyrophosphate (11) r e a c t s e n z y m a t l c a l l y w i t h adenosine t r i p h o s p h a t e to g i v e adenosine diphosphate, i n o r g a n i c phosphate, carbon d i o x i d e and 3-methyl-3-butenyl pyrophsphate (13)"^ ( a l s o c a l l e d l s o p e n t e n y l pyrophosphate). The oxygen of 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 phosphate,"1"-' 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 intermediate, phosphate has been i s o l a t e d . The double bond must be formed by a concerted e l i m i n a t i o n and not 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 f o r no hydrogen from the aqueous medium appears i n the product. 16 The process 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 of r e a c t i o n has not been found elsewhere i n enzyme chemistry but i s analogous to 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 of carbon 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-halogeno-proplonic a c i d s . A p r o t o t r o p i c s h i f t next c o n v e r t s 3~methyl - 3-butenyl-pyrophosphate (13) i n t o 3-methyl - 2-butenyl pyrophosphate (1*0 ( a l s o c a l l e d 3 , 3-dimethyl a l l y l pyrophosphate). T h i s i s one of the few r e v e r s i b l e r e a c t i o n s i n terpene b i o s y n t h e s i s and s t u d i e s 17 18 w i t h 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 ' 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*0 • The e l i m i n a t i o n of the p r o t o n on going from (13) to (1*0 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 He, 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 of the change (13)-$(1*0 i s t h a t a substance w i t h a r e l a t i v e l y u n r e a c t l v e phosphoryl group - 8 -and a n u c l e o p h l l i c double bond i s converted into a highly-reactive e l e c t r o p h i l i c a l l y l pyrophosphate. The intermediates (13) and (14) are now joined together, as shown In Figure V, by an enzyme c a l l e d a prenyl transferase. ATP • ADP ' . -.. Hd Figure IV (14) This reaction resembles a polymerisation i n that (14) can be considered as the i n i t i a t i n g species and (13) as the propagating species, The two molecules are combined with loss of pyrophosphate ion from one and a hydrogen ion from the other to form geranyl pyrophosphate '(1.5) • This can now replace (14) i n a further, analogous reaction with (13) to give farnesyl 19 pyrophosphate (16) . I t i s now thought that most monoterpenes a r i s e from g e r a n y l pyrophosphate (or i t s c i s - d o u b l e bond isomer) and 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 pyrophosphate (or i t s c i s -double bond i s o m e r ) . T h i s i s a statement of the " B i o g e n e t i c 20,21 Isoprene Rule" 0 O P 2 ° 6 H 3 0 P 2 0 6 H 3 T O P OgH O P o O 2°6 H3 (13) O P 2 0 6 H 3 Figure V OP 2°6* 3 (16) The transferase from l i v e r has been p a r t i a l l y 19 22 23 p u r i f i e d ' ' and there i s no i n d i c a t i o n that two d i f f e r e n t enzymes catalyse the two steps leading to (15) and (16). However, t h i s preparation i s incapable of using (16) as a substrate f o r further additions of u n i t s , thus presumably the structure of the enzyme can accomodate (1*0 and (15) but has not space to accomodate the bulkier (16) i n a configuration required f o r - 10 -r e a c t i o n w i t h (13)« T h i s a s s o c i a t i o n of u n i t s i s an unusual r e a c t i o n , even f o r an enzymic process as most condensations i n nature to form carbon-carbon bonds are of the a l d o l or C l a i s e n type. 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 of an o l e f i n and i s analogous to the f o r m a t i o n of " d i i s o b u t e n e " (1?) from isobutenes ^y=CEz — > ( C H 3 ) 3 C + CH 3 H \ CH —1 > (CH3) 3C-CH 2 — C ( C H 3) 3C-CH 2- C \ .CH (17) ' At f i r s t t h i s enzymic r e a c t i o n was c o n s i d e r e d as a carbonium i o n r e a c t i o n of s i m i l a r type. However experiments 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 of the carbon-carbon bond i s accompanied by complete i n v e r s i o n of c o n f i g u r a t i o n a t the a l l y l i c carbon atom, which i s c h a r a c t e r i s t i c of a b i m o l e c u l a r n u c l e o p h l 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 of the a l l y l i c C. unit" 1"^ and e l i m i n a t i o n of hydrogen i o n proceed i n a s t e r e o s p e c i f i c manner which suggests t h a t the p r o c e s s occurs i n two stages; f i r s t a tran s a d d i t i o n of t h e . a l l y l i c u n i t and of an electron donating group X, followed by trans elimination of X and of hydrogen ion as shown i n Figure VI „ H Figure VI A l t e r n a t i v e l y i f Hd were eliminated the corresponding els isomer would be obtained and t h i s stereochemical pathway f o r the condensation of units i s involved i n the biosynthesis of the polyterpene rubber. The stereochemical fate of the hydrogen eliminations can be followed by feeding 2R-2D1 -mev-alonate and 2S-2D -mevalonate followed by determining the s t e r l c positions of hydrogen and deuterium i n isopentenol obtained from the 16 isopentenyl pyrophosphate Geranylgeranyl pyrophosphate (18), the postulated progenitor of the diterpenes, i s formed by another addition of (13) to farnesyl pyrophosphate „ Ad-dition of yet another u n i t gives geranylfarnesyl pyrophosphate (19) which has very 25 recently been shown to be implicated In the biosynthesis of the sesterpene ophiobolin P (20.) by c e l l free extracts of Chochllobolus heterostrophus. HO" (20) The C ^ Q terpenoids (triterpenes) are not biosynthesised from the C ^ Q analogue of (19) but instead are formed from the 26 symmetrical hydrocarbon squalene (21) " » The l a t t e r compound i s derived by " t a i l - t o - t a i l " condensation of two molecules of far n e s y l pyrophosphate. The biosynthesis of squalene and i t s conversion to triterpenes and steroids have been the subject of considerable i n v e s t i g a t i o n and the r e s u l t s have been described 6,24,27 i n several excellent reviews . OPP 2PPi + Hb + Ha Kb OPP NADPH ** NADP"1 Ha H (21) - Ik -As mentioned above, the key Intermediate i n the b i o s y n t h e s i s of s e s q u i t e r p e n e s i s f a r n e s y l pyrophosphate (16). Almost a l l s e s q u i t e r p e n e s , comprising 50-60 s k e l e t a l types, can be f o r m a l l y d e r i v e d from f a r n e s y l pyrophosphate by a s e r i e s of i o n i c c y c l i s a t i o n s (see F i g u r e V I I ) . The o r i g i n a l t h e o r e t i c a l s u g g e s t i o n s have r e c e n t l y been reviewed and extended by Parker, 28 Roberts and Ramage . (2?) S t r u c t u r a l E l u c i d a t i o n of T r i c h o t h e c i n T r i c h o t b e c i n (28) Is a metab o l i t e of Trichotheclum roseum L i n k which e x h i b i t s a n t i - f u n g a l a c t i v i t y . The b i o l o g i c a l a c t i v i t y of the organism, as a whole, has been reported s e v e r a l 29 times s i n c e the beginning of t h i s century . I n 19^9 Freeman 30 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 of 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 c a l l e d i t t r i c h o t h e c i n o They concluded that t r i c h o t h e c i n r had a formula of C^H^gO^ or C1^H2o°Z|» w a s a n e u t r a l , unsaturated, conjugated ketone, contained no hydroxyl or a l k o x y l groups and 31 had three C-methyl groups. L a t e r work showed th a t the molecular formula was C10H24O5 and tha t t r i c h o t h e c i n was an e s t e r , the components of which were t r i c h o t h e c o l o n e (29) and i s o c r o t o n i c a c i d . 0 H (29) - 16 -32 In 1959 Freeman and his coworkers proposed structure (30) f o r t r i c h o t h e c i n on the basis of a systematic degradation, which however did not exclude the p o s s i b i l i t y of (3D-OR (30) 33 Further evidence f o r (30) was provided by Jones and his 34 .collaborators, who also studied some aspects of the bio-synthesis of t r i c h o t h e c i n . In deriving the t r i c h o t h e c i n skeleton from fa r n e s y l pyrophosphate i t i s necessary to invoke either a 1,3-methyl migration or two 1,2-methyl migrations. The r e s u l t s of Jones indicate that two 1,2-methyl migrations occur. In 1964 a new compound, which was named trichodermln 35 (32) was i s o l a t e d J from a s t r a i n of Trichoderma. The chemical and s p e c t r a l properties of trichodermln suggested a r e l a t i o n s h i p to t r i c h o t h e c i n , and t h i s was confirmed by oxidation of trichodermln to trichothecolone acetate, suggesting structure (33) f o r trichodermln. - 17 -H ( 3 3 ) • • 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 l n , which suggested the presence of an epoxide r i n g . To r e s o l v e t h i s problem an X-ray a n a l y s i s of t r i c h o d e r m o l p-bromobenzoate ( 3 2 , p-bromo b e n z o y l i n s t e a d of a c e t y l ) was undertaken-^ which i n d i c a t e d t h a t ( 3 2 ) i s the s t r u c t u r e of t r i c h o d e r m i n and hence (28) i s the s t r u c t u r e of t r i c h o t h e c i n . - 18 -Proposed 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 7 U s i n g s t r u c t u r e ( 3 0 ) Ruzicka has 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 t h a t f a r n e s y l pyrophosphate ( 1 6 ) c y c l l s e s to g i v e carbonium i o n ( 2 2 ) , (16) ( 2 2 ) T h i s then l o s e s a p r o t o n to form ^ - b i s a b o l e n e ( 3 ^ ) . I t should be noted t h a t t h e r e are two forms of ft-bisabolene ( 3 ^ a ) and ( 3 4 b ) (3La) (34b) R u z i c k a has s t a t e d t h a t s t a r t i n g w i t h ( 2 - C ) -mevalonic a c i d the l a b e l l i n g should be as shown and t h a t both - b i s a b o l e n e s can be d e r i v e d from f a r n e s y l pyrophosphate, whether or not one s t a r t s w i t h a e l s or t r a n s c e n t r a l double bond. . 19 -R u z i c k a has a l s o stated' 5'' 7 t h a t both ^ - 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 to 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 of t r i c h o t h e c i n (as assumed a t t h a t time) (see F i g u r e V I I I ) . However 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 f i v e membered r i n g when the c o r r e c t 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 a r e taken i n t o account, 35b Two a l t e r n a t i v e mechanisms a r e p o s s i b l e . The f i r s t invokes a boat type f o l d i n g of the s i d e c h a i n ( F i g u r e IX).. A c c o r d i n g 3^  to the l a b e l l i n g s t u d i e s of Jones and h i s coworkers^ i t i s the t r a n s methyl group i n the i s o p r o p y l i d e n e group which m i g r a t e s . (3^a) or (3^b) < < OR (30) F i g u r e V I I 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 counts. F i r s t l y the boat type 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 than a c h a i r type f o l d i n g . S e c o n d l y the methyl s h i f t s must be non 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 . - 2 0 -(3^a) or (34b) V H ( 2 8 ) Figure IX A much more elegant and energetically more favourable method of f o l d i n g the side chain i s to again u t i l i s e a.chair type f o l d i n g but to f o l d the side chain i n front of the plane of the cyclohexene r i n g , as i n Figure X, rather than behind the plane of the cyclohexene r i n g as In Figure VIII,, Having shown how the side chain may be folded i t only remains to discuss which of the isomers of o'-bisabolene, (34a) or (3^b), may be involved i n the biosynthesis of t r i c h o t h e c i n . Unfortunately r e s u l t s obtained by two d i f f e r e n t research groups are i n d i r e c t contradiction. (3^a) or (34b) H (28) F i g u r e X 14 S t a r t i n g from (2- C)-mevalonic a c i d l a c t o n e , Jones and 34 h i s group 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 t-H • 0 = S t a r t i n g from V - b i s a b o l e n e , the f o l l o w i n g schemes can be envisaged 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 ( F i g u r e s XI and X I I ) . - 23 -Thus i t can be seen that only isomer (3^b) of It-bisabolene gives a r e s u l t consistent with the work of Jones' group . However, t h i s i s only true If there i s no r o t a t i o n about the bond joining the 6- to the 5-membered r i n g 0 I f there i s r o t a t i o n about t h i s bond then Figures XI and XII become equivalent a f t e r the c y c l i s a t i o n 8 Since i t i s very u n l i k e l y that the intermediate i s a free carbonium ion, but has some group Z attached at that point which must subsequently be eliminated i n a normal trans-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 depicted In Figures XI and XII holds:-- 2 4 -Recent work by Hanson and his collaborators using doubly l a b e l l e d 4(R)-[4-^H,2 - X V] mevalonic a c i d and r- 3 14 1 |2- Hj,2- Cj farnesyl pyrophosphate indicates that the other isomer (34a) i s involved i n the biosynthesis of t r i c h o t h e c i n (Figure XIII),, Previous work from the same group has 39 implicated^ farnesyl pyrophosphate i n the biosynthesis of tr i c h o t h e c i n . Experimentally Hanson found that the t r i t i u m was retained which i s consistent with isomer (3^a) being the one involved i n the biosynthesis of t r i c h o t h e c i n . T (3^a) (34b) - 2 5 -Loss of T F i g u r e X I I I - 26 -The same argument applies concerning a Z group which e f f e c t i v e l y prevents r o t a t i o n about the inter-annular bond. 34 As i t has been shown that acetate and mevalonate 39 and fa r n e s y l pyrophosphate are involved i n the biosynthesis of trichothecin,, the next l o g i c a l step i s to determine what monocyclic species are involved,, and s p e c i f i c a l l y to te s t Ruzicka's proposals concerning ^-bisabolene. - 2? ~ DISCUSSION In addition to tes t i n g ^-bisabolene for precursor a c t i v i t y i t was decided, f o r several reasons, also to test cK-bisabolol (35) as a precursor because (a) the b i o l o g i c a l system may be able to dehydrate cK-bisabolol to S-bisabolene. (b) <*-bisabolol may be i n f a c t a genuine precursor, as shown i n Figure XIV 0 (c) the synthesis envisaged f o r ^-blsabolene involved the intermediacy of c<~bisabolol anyway. (3^a) (34b) Figure XIV - 28 -ex-Biasbolol ( 3 5 ) was f i r s t synthesised i n 1 9 3 2 by 4 0 Ruzicka and L i g u o r i , The key reaction i n t h e i r synthesis was .the r e a c t i o n between i r-acetyl-l-methyl cyclohexene ( 3 6 ) , prepared by ozonolysis and dehydration of y5-terpineol {37), and the Grignard of 2-methyl-5-bromo-2-pentene ( 3 8 ) prepared from 2~methyl-2,5~pentadiol ( 3 9 ) « ( 3 7 ) ' ( 3 6 ) ( 3 9 ) ( 3 8 ) ( 3 5 ) - 29 In 1966 Manjarrez and Guzman synthesised ^3-bisabolene (40) , The key step i n t h e i r synthesis was the reaction between the a c i d chloride of 4-methyl-3-cyclohexene carboxylic acid 39 (41) and the same Grignard compound used by Ruzicka , to give ketone (42). The a c i d (4l) was prepared by hydrolysis of the ester obtained by Diels-Alder condensation of Isoprene (1) and methyl acrylate (43), and the bromide (38) was prepared from 42 methyl cyclopropyl ketone (44) a f t e r the method of J u l i a , MeOgC (43) MeGgC (l) H02C (41) + MeMgBr OH - 30 -48# HBr y Br MgBr C10C (38) : (42) Ketone (42) was then converted to ^ S-bisabolene (40) by treatment w i t h triphenylmethylphosphonium bromide. PhopCHo Br (42) >3 (40) I n a rec e n t . p a p e r , Gutsche and h i s coworkers have c r i t i c i s e d these p r e v i o u s two syntheses o f c x - b i s a b o l o l and ^3-bisabolene, and o f f e r an a l t e r n a t i v e r o u t e , though u t i l i s i n g the same key r e a c t i o n as R u z i c k a and L I g u 0 r i ^ ° - 31 -T h e i r 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 prepare 4 0 ketone ( 3 6 ) "by the method of R u z i c k a and L i g u o r i „ because of d i f f i c u l t y i n s e p a r a t i n g pure ^ - t e r p i n e o l from the commercially a v a i l a b l e m ixture, They then t r i e d to prepare ketone ( 3 6 ) by a D i e l s - A l d e r r e a c t i o n between methyl v i n y l ketone and i s o p r e n e but found i t d i f f i c u l t to separate the i s o m e r i c mixture o b t a i n e d , which c o n t a i n e d 25% of unwanted isomer ( 4 5 ) , They e v e n t u a l l y p repared ketone ( 3 6 ) In low y i e l d by treatment of 4-keto-cyclohexane c a r b o x y l i c a c i d (46) w i t h methyl 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 , - 0 H0 2C (46) . ( 3 6 ) Secondly they c l a i m t h a t the bromide (3.8) prepared by R u z i c k a 40 and L i g u o r i isomer ( 4 7 ) c o n t a i n s s i g n i f i c a n t amounts of the i s o p r o p e n y l - 32 -Br (4?) T h i r d l y they c l a i m the product of Manjarrez and Guzman i s contaminated by s t r u c t u r a l isomers as the 6-membered r i n g was c o n s t r u c t e d v i a a D i e l s - A l d e r r e a c t i o n and no s e p a r a t i o n of Isomers was I n d i c a t e d , Gutsche and h i s co-workers then ' prepared J a pure sample of c x - b i s a b o l o l by condensing the Gri g n a r d 42 compound of pure bromide (38), prepared by the method of J u l i a , wi t h pure ketone (36) prepared by t h e i r own method d e s c r i b e d above, An a l t e r n a t i v e s y n t h e s i s of ketone (42) has been 44 r e p o r t e d , but i t i s r a t h e r l e n g t h y and f o r t h i s reason we c o n s i d e r e d i t u n s u i t a b l e . I t i s shown i n F i g u r e XV. F o r our s y n t h e s i s of r a d i o a c t i v e c x - b i s a b o l o l (35) 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 l a b e l i n the l a s t step of the s y n t h e s i s by r e a c t i n g ketone (42) w i t h r a d i o a c t i v e methyl magnesium i o d i d e or methyl l i t h i u m . - 33 -o o (a) Na d u s t ; H C l (b) KOH/MeOH/K20; H +{ A/Cu (c) MeMgl (d) PTSA/benzene/A (e) a c e t o n e / H C l ( f ) NaH/C0(0Et) 2/D.M.F./A (g) Bu t0"/Bu tOH/(38) F i g u r e XV (^2) (35) Our f i r s t objective therefore was to chose or devise a convenient synthesis of ketone (42) „ The synthesis reported by Manjarrez and Guzman involves synthesis of acid ( 4 l ) and i s subject to the c r i t i c i s m outlined above. We decided therefore to investigate an a l t e r n a t i v e synthetic route to ketone (42). The key reaction envisioned was the reaction between the Grignard of 4 - c h l o r o - l -methyl-cyclohexene (48) and the acid chloride of 5-Taethyl-4-hexenoic a c i d ( 4 9 ) . , • :' / C 0 2 H (48) (49) The a c i d (49) was prepared by a l i t e r a t u r e procedure by condensing l-chloro -3-methyl~2-butene (50) with the sodium s a l t of d i e t h y l malonate to give dies t e r (51). ^5 - 3 5 -cl ( 5 0 ) COoEt 'CH Na"1 C 0 2 E t EtOgC COgEt ( 5 D B a s i c h y d r o l y s i s of d i e s t e r ( 5 1 ) w i t h aqueous sodium hydroxide gave a c r y s t a l l i n e d i a c i d ( 5 2 ) which was t h e r m a l l y d e c a r b o x y l a t e d "by m e l t i n g f o l l o w e d by d i s t i l l a t i o n to g i v e a c i d (49). Normally the d i a c i d was not I s o l a t e d but some was 46 r e c r y s t a l l i s e d from benzene f o r a n a l y t i c a l purposes. E t 0 2 C .C0 2Et NaOH/H20 (5D 47 ( 5 2 ) (49) «+/. B i r c h r e d u c t i o n of p-methyl a n i s o l e (53) w i t h sodium i n l i q u i d ammonia and e t h a n o l gave an almost q u a n t i t a t i v e y i e l d o f d i h y d r o compound (5*0 • C o n v e r s i o n of eno l e t h e r (54) to the b i s u l p h i t e compound f o l l o w e d by h y d r o l y s i s r e s u l t e d i n very poor y i e l d s - 36 -of d e s i r e d ^ ^ - u n s a t u r a t e d ketone (55)» However h y d r o l y s i s of enol ether (5*0 w i t h o x a l i c a c i d i n aqueous methanol gave the desiredy^X-unsaturated ketone (55) i n reasonable y i e l d {60%) OHe OMe Na/NH-^/StOH (53) (COpH)2/H20/MeOH (5*0 (55) Ketone (55) was then reduced w i t h sodium borohydride i n e t h a n o l to g i v e u n s a t u r a t e d a l c o h o l (56), NaBHjL-EtCK (55) (56) PCl5/CHGl ? or CCl^/PhoP - 3 ? -The c h l o r i d e (48) was prepared from the a l c o h o l (56) i n two ways. The f i r s t method i n v o l v e d r e a c t i o n of a l c o h o l (56) w i t h phosphorus p e n t a c h l o r i d e i n c h l o r o f o r m . The product of t h i s r e a c t i o n was a mixture of d e s i r e d c h l o r i d e ( 48 ) , a c y c l i c diene and t o l u e n e , i n the r a t i o s of 7 0 : l ? s l 3 (as determined by n.m,r,). C a r e f u l f r a c t i o n a l d i s t i l l a t i o n e v e n t u a l l y gave a sample of c h l o r i d e (48) which was r e a s o n a b l y pure but the y i e l d was o n l y approximately J0% from a l c o h o l ( 5 6 ) . The second method gave a c l e a n e r product but the y i e l d was a g a i n d i s a p p o i n t i n g l y low. T h i s method Involved r e a c t i o n of a l c o h o l (56) w i t h c a r b o n t e t r a c h l o r i d e i n the presence of 49 5 0 t r i p h e n y l phosphine . The r e a c t i o n i s a t p r e s e n t thought to proceed as f o l l o w s : -Ph~P-Cl + C C l yr'-Ul + U U . L 3 Ph^P-Cl + ° C C 1 3 + ROH > Ph^-O-R +• C l + CHCl^ P h ^ P - O j R ^ ' C l > Pl^P+O + RC1 • One c o m p l i c a t i o n w i t h t h i s r e a c t i o n i s the removal of the t r i p h e n y l phosphine oxide by-product*, T h i s was done by r e p e a t e d t r i t u r a t i o n of the s e m i - s o l i d o i l obtained on e v a p o r a t i o n of the excess carbon t e t r a c h l o r i d e , (a p r e l i m i n a r y f i l t r a t i o n p r i o r to e v a p o r a t i o n removes a good d e a l of the t r i p h e n y l phosphine o x i d e ) , u n t i l a l l the t r i p h e n y l phosphine oxide which remained was d i s s o l v e d i n the c h l o r i d e , and f i n a l l y by d i s t i l l a t i o n . I t was found t h a t a p u r e r sample r e s u l t e d i f the d i s t i l l a t i o n was c a r r i e d out at room temperature and 0,1 mm Hg pressure trapping the d i s t i l l a t e i n a dry ice/acetone bath, than by d i s t i l l i n g at 90°C at 70 mm Hg pressure. . A" more convenient method of removing the by-product i s 50 described" i n a recent communication where phosphorous trisdimethyiamide (57) i s used as reagent, giving the water-soluble phosphoric trisdimethyiamide (58) as the by-product (also known as hexamethyl phosphoraraide) (Me 2N) 3P . • (Me 2N) 3P ~ 0 (57) (58) The next step In the synthesis was the preparation of the key intermediate (42) by reacting the Grignard complex -of c h l o r i d (48) w i t h the a c i d chloride of acid (49), (42) Unfortunately the Grignard. reagent of chloride (48) formed very slowly and with great d i f f i c u l t y , and reacted with the acid chloride of a c i d (49) to give a complex mixture of products, of which the major one (~50#, p a r t i a l l y p u r i f i e d by d i s t i l l a t i o n , then p u r i f i e d further by preparative t . l . c . ) was not desired - 39 -ketone (42). Further work on the nature of thi s product i s in progress, In an a l t e r n a t i v e synthesis of ketone (42) the reaction 4 l scheme of Marjarrez and Guzman was followed. Thus condensation of ispprene (1) and methyl aerylate (43) gave, i n good y i e l d , 51 a mixture of esters (59) and (60). Previous work by Hennis has shown that the r a t i o of products was approximately 70% desired ester (59) and J0% unwanted, ester (60). Me02C, (*3) (1) Me02C Me02C (59) (60) This mixture of esters was hydrolysed i n aqueous sodium hydroxide s o l u t i o n to give a gummy mixture of acids, (41) and (61), m.p. 50-100°C. MeO C MeO„C 2 (59) H02C (60) (41) (61) This mixture of acids was repeatedly c r y s t a l l i s e d from petroleum ether (60-80°) u n t i l the m.p. was constant at 99-•100°C. E s s e n t i a l l y I d e n t i c a l r e s u l t s could be obtained by condensing isoprene and a c r y l i c a c i d . Confirmation that t h i s p u r i f i e d product was Indeed the desired a c i d (41) was obtained by l a c t o n i s i n g the p u r i f i e d - 4o -product 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 f which has been 5 2 r e p o r t e d i n the l i t e r a t u r e by some French workers, gave a white c r y s t a l l i n e compound,-m.p. 68~69°CP w i t h an I . r . a b s o r p t i o n ~1 S 2 a t 1760 cm ( i n C C l ^ ) . The F r e n c h workers a s c r i b e d s t r u c t u r e (62) to t h i s compound on the b a s i s of i t s i . ' r . spectrum - l a c t o n e ) . However, we o b t a i n e d the n.m.r. spectrum of t h i s compound and i t was i n c o m p a t i b l e w i t h s t r u c t u r e (62) as i t c l e a r l y showed t h a t the methyl group was a t t a c h e d to carbon b e a r i n g oxygen and not to carbon b e a r i n g hydrogen ( s i n g l e t a t 8.65 T', 53 no s i g n a l around 9 rC)« A l s o a more r e c e n t paper r e p o r t s the p r e p a r a t i o n of both isomers of (62) and r e p o r t s t h a t n e i t h e r melts a t 6 9°C At f i r s t we thought t h a t we had not a c i d (4l) but i t s isomer (61) which would l a c t o n i s e to g i v e l a c t o n e (63). However t h i s l a c t o n e (63) had been p r e v i o u s l y prepared by Boorman 5 4 o and L i n s t e a d and I t s m.p. i s 4-5 C. F i n a l l y l a c t o n e (64) has been prepared by Noyce-'-' and h i s coworkers and i t has a m.p. of o , -1 69 C and an i . r . a b s o r p t i o n a t 1740 cm . Thus we were sure we had l a c t o n e (64) and hence the c o r r e c t a c i d (41) but c o u l d not e x p l a i n the d i f f e r e n c e s i n the i . r , s p e c t r a . T h i s problem was r e s o l v e d by r e r u n n i n g the i . r . spectrum of our l a c t o n e In c h l o r o f o r m s o l u t i o n when i t -1 showed an a b s o r p t i o n a t 1740 cm These r e s u l t s are summarised i n F i g u r e XVI. - 4 1 -F i g u r e XVI - 42 -The a l i p h a t i c s l d e c h a i n of ketone (42) was s y n t h e s i s e d In the same way as i n the s y n t h e s i s r e p o r t e d "by Manjarrez and 40 Guzman . Treatment of c y c l o p r o p y l methyl ketone (44) w i t h e t h e r e a l methyl l i t h i u m s o l u t i o n gave a l c o h o l (65) [_plus a second component i d e n t i f i e d as d i o l (66)1 which c o u l d be converted w i t h 42 48$ hydrobromic a c i d i n t o bromide (38) . (66) A d d i t i o n of the a c i d c h l o r i d e of a c i d (41) to the G r i g n a r d r e a g e n t prepared from bromide (38) gave a compound w i t h an I . r . a b s o r p t i o n a t 173° cm" 1 and a pa r e n t peak i n the mass spectrum a t 330- E l e m e n t a l a n a l y s i s i n d i c a t e d a m o l e c u l a r f o r m u l a of C22p-3k®2* R e a c t i o n of t h i s compound w i t h e t h e r e a l - 4 3 -methyl l i t h i u m s o l u t i o n gave two products which were separated o by p r e p a r a t i v e g . l . c . on a 3 0 > Se 3 0 column a t 1 7 5 C. H 2OC ( 3 8 ) (41) MgBr > C 2 2 H 3 4 ° 2 i . r . x 7 3 0 cm mass spec. 3 3 0 '; C 2 2 H 3 4 ° 2 i . r . 1 7 3 0 cm-mass spec. 3 3 0 Me'Li OH ( 6 7 ) H OH ( 6 8 ) _ L L _ The more v o l a t i l e component was shown by comparison of i . r . and n.m.r. spectra and g , l , c . retention time to be c<-terpineol ( 6 7 ) » and the less v o l a t i l e component assigned structure (68) on the basis of i . r . and n.m.r. spectra. Reaction of the product from the Grignard reaction with lithium aluminium hydride i n ether again gave two products separable by preparative g . l . c . under the same conditions as before. The less v o l a t i l e component was i d e n t i c a l with compound ( 6 8 ) and the more v o l a t i l e component was assigned structure ( 6 9 ) on the basis of i . r . and n.m.r. spectra. The sequence of reactions i s shown i n Figure X V I I . On the basis of the evidence presented above we have assigned structure ( 7 0 ) to the Grignard product. The mechanism f o r t h i s i n t r i g u i n g r eaction presumably involves p r i o r formation of ketone (42) which, i n the presence of excess Grignard reagent, i s reduced to alcohol ( 6 8 ) , The 5 6 reducing properties of Grignard complexes are well known , The alcohol ( 6 8 ) then competes with the Grignard reagent f o r the a c i d chloride of acid (41) to give ester ( 7 0 ) . I f t h i s explanation i s correct, then very slow Inverse addition, i . e . • adding the Grignard reagent to the a c i d chloride, should give the desired ketone (42). This was tested and did indeed give - 45 -a product having an i . r . a b s o r p t i o n a t 1710 cm \ However th e r e was a l s o a s l i g h t a b s o r p t i o n a t 1730 cm~\ P r e p a r a t i v e g . l . c under the p r e v i o u s c o n d i t i o n s gave one main component which s t i l l showed two c a r b o n y l a b s o r p t i o n s . Thus e s t e r (70) was excluded as the i m p u r i t y as i t has a much l a r g e r r e t e n t i o n time. The n.m.r. spectrum of t h i s mixture showed a t r i p l e t a t 6.0 °f so i t was thought t h a t the i m p u r i t y might be e s t e r (71) formed by o x i d a t i o n of the G r i g n a r d complex, by mol e c u l a r oxygen, and r e a c t i o n of 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 . • (71) T h i s was shown to be tr u e by r e d u c i n g the mixture w i t h sodium b o r o h y d r i d e and by p u r i f y i n g the products by chromatography on alumina. E l u t i o n w i t h p e t . ether ( 6 0 - 8 0 ° ) gave a compound w i t h i . r . and n.m.r. s p e c t r a c o n s i s t e n t w i t h s t r u c t u r e (71) whereas a l c o h o l (68) from r e d u c t i o n of ketone (42) remained on the column. - 46 -. . D i r e c t comparison of ketone (42), p u r i f i e d by d i s t i l l a t i o n , with the compound obtained by Jones oxidation of alcohol (68), obtained from ester (70) by reaction with either methyl lithium or l i t h i u m aluminium hydride, showed them to be i d e n t i c a l i n a l l respects. Ketone (42) was further characterised by reduction to alcohol (68) with sodium borohydride. Treatment of ketone (42) with excess of either ethereal methyl lithium s o l u t i o n or ethereal methyl magnesium iodide solution gave c<-bisabolol ( 35 ) i n almost quantitative y i e l d . (42) (35 ) An i d e n t i c a l synthesis using radioactive methyl magnesium iodide yielded radioactive cx-bisabolol l a b e l l e d on the t e r t i a r y methyl group. (^2) , ( 35 ) - 4? -During the course of t h i s work an a l t e r n a t i v e , but 57 e s s e n t i a l l y s i m i l a r synthesis of ketone (42) was published by some Russian workers, u t i l i s i n g n i t r i l e (72), Instead of aci d (4l) and the Grignard reagent prepared from bromide (38)0 0 . ... . m ) The same authors then prepared o<-bisabolol using methyl magnesium iodide on ketone (42). .In the same paper they also give an a l t e r n a t i v e synthesis of «c-bisabolol, using the same reagents but reac t i n g them i n a d i f f e r e n t order. Thus n i t r i l e (72), on treatment with methyl magnesium iodide, gave ketone (36), which could be converted to <K-bisabolol by reaction with the Grignard of bromide (38) as i n the o r i g i n a l synthesis of Ruzlcka and 40 '. L i g u o r i . • • . . . . V : (35) - 48 -Having accomplished a s a t i s f a c t o r y synthesis of radioactive «x-bisabolol (35) w © could now test i t s precursor a c t i v i t y i n the biosynthesis of t r l c o t h e c i n , Por that purpose an aqueous so l u t i o n of tx-bisabolol ( s o l u b i l i s e d by Tween 20) 30 was added to five-day-old cultures of Trlcotheclum roseum and the cultures allowed to grow for a further three weeks. The broth was then decanted and the raycelia washed with a l i t t l e water. The combined broth and washings were extracted with carbon t e t r a c h l o r i d e . Evaporation of the carbon t e t r a c h l o r i d e gave a gum which was p u r i f i e d by preparative thin layer chromatography on s i l i c a g e l . The t r i c h o t h e c i n so obtained was then r e c r y s t a l l i s e d from pet. ether (60-80°) to constant r a d i o a c t i v i t y , which represented an incorporation of 0.014,^. A preliminary, degradation to determine i f the incorporation was s p e c i f i c was c a r r i e d out by hydrolysing the t r i c h o t h e c i n to trichothecolone (29) with methanolic potassium hydroxide 32 34 s o l u t i o n . Jones and his co-workers have shown that i f acetate i s used as the precursor, then 95% of the r a d i o a c t i v i t y resides i n the isocrotonyl side chain. R e c r y s t a l l i s a t l o n of the trichothecolone to constant r a d i o a c t i v i t y showed that - 4 9 -over 60% of the r a d i o a c t i v i t y of the trichothecin resided i n the isoprenoid portion, as compared with 5% when acetate i s used as the precursor. This shows that there has been a s i g n i f i c a n t amount of degradation of the ©<-bisabolol, presumably to acetate, but that approximately 60$ of the <X - b i s a b o l o l has been incorporated i n t a c t . However a systematic degradation of the trichothecolone w i l l be required before a d e f i n i t i v e answer can be given, and t h i s w i l l form the basis of further work. The rather low incorporation (0.014^) can be explained i n two ways. F i r s t l y the c*>bisabolol may not be getting to the s i t e of the synthesis of trichothecin. <X -Bisabolol i s water-insoluble so there may be transportation problems. Secondly, and more probably, what i s being seen Is an aberrant biosynthetlc pathway, i . e . c^-bisabolol i s not d i r e c t l y on the main bio-synthetic route to tri c h o t h e c i n , but i s being converted to a compound which i s on the pathway. This compound i s most probably ^-bisabolene (34) and work i s i n progress to prepared ""^"c-labelled. .&-bisabolene and feed i t to Trlchotheclum roseum, when a marked increase i n incorporation should be seen. EXPERIMENTAL A l l melting points v^ere determined on a K o f l e r block and are uncorrected. Infrared spectra (\)max) were recorded on a Perkin-Elmer Infracord model 137 spectrophotometer. Nuclear magnetic resonance spectra CX) were determined i n carbon t e t r a c h l o r i d e , deuteriochloroform or neat and recorded on a Jeolco C-60H spectrometer or on VarIan Associates spectrometers, model A-60 or model T-60. Signal positions are given i n the T i e r s f scale, with tetramethylsllane as an i n t e r n a l standard; the m u l t i p l i c i t y , coupling constants (where appropriate) and integrated peak areas are indicated i n parentheses; s = s i n g l e t , br.s = broad s i n g l e t , d = doublet, t = t r i p l e t , q = quartet, quin «= quintet, d.t = double t r i p l e t , t, quin = t r i p l e quintet, m = m u l t i p l e t . Mass spectra (M +) were determined with an MS 9 spectrometer. Microanalyses were performed by Mr. P. Borda, M i c r o a n a l y t i c a l Laboratory, Uni v e r s i t y of B r i t i s h Columbia, Thin la y e r chromatographic r e s u l t s were obtained using s i l i c a gel H F 2 ^ of 0.5 mm thickness. Preparation of l-methoxy-4-methyl-cyclohexa-l,4-diene (54) p-Methyl anisole (53) (24.4 g, 0.20 mole) and ethanol (75 ml) were added to l i q u i d ammonia (approx. 450 ml) contained i n a 21 f l a s k , equipped with a mechanical s t i r r e r , and immersed i n a dry ice-acetone bath. Sodium (28.0 g) was added, with s t i r r i n g , i n small pieces as quickly as possible, yet not so f a s t as to cause f r o t h i n g (ca. 1 hour), to give a pasty mass from which a l l the blue colour had disappeared a f t e r s t i r r i n g f o r about 2 hours. Most of the ammonia was allowed to evaporate - 51 -and water (300 ml) added cautiously. The r e s u l t i n g s o l u t i o n was extracted 3 times with ether p the ethereal layer washed 2 times with brine, dried (NagSO^) and the ether was removed at reduced pressure. D i s t i l l a t i o n gave l-methoxy~4-methyl-cyclohexa-1 ,4-diene (54) (22 .9 g, 93/0 as a colourless o i l , b.p. 70-73°C (25 mm) [ l i t . ' 4 ' 7 , b.p. 167-170° (760 mm)"]. \> m o r r ( l i q u i d f i l m ) , 2900, 2830, 1700, 1660, 1210, H 6 5 , 776 cm"1? T (CCl^) 4.62 (m, I H ) , 5.51 (br.s, I H ) , 6.58 ( S , 3H), 7.36 (br.s, 4 H ) , 8.37 (S, 3H). Preparation of 4-methyl-cyclohex-3-enone (55) l-Methoxy -4-methyl-cyclohex-l ,4-diene (54) (22 ,9 g, O . I 8 5 mole) dissolved i n methanol (50 ml) was added to oxalic a c i d ( 6 . 9 g) i n water (90 ml) (ca. 0..1M s o l u t i o n ) . This two phase system was magnetically s t i r r e d f o r 40 minutes, by which time the s o l u t i o n was homogeneous and t . l . c . analysis showed no st a r t i n g material present. Ether and water were added, the aqueous layer extracted 2 times with ether, the combined ether extracts washed with 2% sodium bicarbonate sol u t i o n then brine, dried (Na 2 S0j i | ) and the ether removed. D i s t i l l a t i o n gave 4-methyl-cyclohex-3-enone (55) (12 .5 S» 6 1 . 5 $ ) as a colourless o i l , b.p. •76-77°C (26 mm) [ l i t . ^ 7 , 74°C (17 mm) or 169-172°C (755 mm)]. •\} m a x ( l i q u i d f i l m ) , 2970, 1730, 1196, 780 cm"1? T (neat) 4.60 (m, I K ) , 7.29 (m, 2 H ) , 7.6l (S, 4 H ) , 8 . 2 6 (m, 3K). - 5 2 -Preparation of 4-methylcyIohex-3-enol ( 5 6 ) 4-Methylcyclohex~3~enone ( 5 5 ) ( 3 8 . 1 g, 0.35 mole) dissolved i n ethanol (90.ml) was slowly added to a solution of sodium borohydride (4.0 g, 0.11 mole) dissolved i n ethanol ( 1 5 0 ml) contained i n a 5 0 0 ml f l a s k equipped with a reflux, condenser. The rate of addi t i o n was adjusted so as to maintain a gentle r e f l u x . The s o l u t i o n was allowed to r e f l u x f o r 3 0 minutes a f t e r the addition was completed and then water (100 ml) added. The ethanol was removed at reduced pressure and the aqueous s o l u t i o n extracted 3 times with ether. The ether extracts were washed with brine, dried U^SO^) and the ether removed at reduced pressure. D i s t i l l a t i o n gave 4-methylcyclohex-3-enol ( 5 6 ) (26.9 g. 69.9/0 as a colourless o i l , b.p. 70-74°C ( 1 5 mm) [ l i t , 5 8 b.p. 83°C (I8mm)]\) Q V ( l i q u i d f i l m ) , 3 3 1 0 , .2920, 1060, 798 cm"1; T ( C C l ^ ) 4,?7 (m, IH), 5.60 (S, IH), 6 . 3 0 (br.m, IH), 7.96-8.22 (m, 6H), 8 . 3 8 (S, 3 H ) . Preparation of 4-chIoro-l-methylcyclohexene (48) (a) 4-Methylcyclohex-3-enol ( 5 6 ) (26.9 g, 0.24 mole), potassium carbonate (170 g, 1.23 mole) and chloroform (1100 ml) were cooled, with mechanical s t i r r i n g , i n a 31 f l a s k immersed i n an ice-water bath. Phosphorus pentachloride (110 g, 0.53 mole) was added, with s t i r r i n g and cooling, over a period of 4 5 minutes. The reaction mixture was s t i r r e d , with cooling, f o r 3 hours, then s t i r r e d at room temperature f o r 18 hours. The reaction mixture was cooled i n an ice-water bath, water (400 ml) added and the s o l u t i o n s t i r r e d , with cooling u n t i l the s o l u t i o n - 53 -was c l e a r ( c a , 5 h o u r s ) , then s t i r r e d a t room temperature f o r 18 hours. The c h l o r o f o r m l a y e r was separated and the aqueous l a y e r . e x t r a c t e d 2 times w i t h c h l o r o f o r m . The combined c h l o r o f o r m 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 (NagSO^) and the c h l o r o f o r m evaporated a t reduced p r e s s u r e . D i s t i l l a t i o n gave 4 - c h l o r o - l - m e t h y l c y c l o h e x e n e (48) (10.0 g, 31.8$) as a c o l o u r l e s s o i l , b.p. 89-94°C (82 mm). \) m a x 2910, 1440, 860, 800, 7 7 0 , 745 cm" 1; T (CCl^) 4 .71 (br.m, IH), 5 .91 ( q u i n . , J = 5 .5 Hz, IH), 7.5-8.1 (m, 6H), 8 .33 (S, 3H). (b) T r i p h e n y l phosphine (30.0 g, 0.11 mole) was added s l o w l y , w i t h s t i r r i n g , to 4-methyl-cyclohex-4-enol ( 56 ) (9.2 g, 0.08 mole) i n C C l ^ (75 m l ) . The r e a c t i o n mixture was r e f l u x e d o v e r n i g h t , then c o o l e d and most of the t r i p h e n y l phosphine oxide f i l t e r e d o f f . The f i l t r a t e was evaporated, a l i t t l e C C l ^ added, the mixture f i l t e r e d and the f i l t r a t e evaporated. T h i s was repeated u n t i l no t r i p h e n y l phosphine oxide remained as a s o l i d i n the evaporated f i l t r a t e , (Some however remained d i s s o l v e d i n the crude c h l o r i d e ) . T h i s o i l was d i s t i l l e d to g i v e 4 - c h l o r o - l -methyl-cyclohexene (48) (2 . 3 g» 22%) as a c o l o u r l e s s o i l , b.p. 2 5 ° C ( 0 . 1 mm) and i d e n t i c a l s p e c t r o s c o p i c p r o p e r t i e s to t h a t prepared i n (a) above. P r e p a r a t i o n of D i e t h y l (3-methyl-but-2-enyI)-maIonate (51) D i e t h y l malonate (80.0 g, 0 .50 mole) was added to a s o l u t i o n of sodium ethoxide i n e t h a n o l , prepared by adding sodium (11.5 g, 0 . 5 0 mole) to a b s o l u t e e t h a n o l (150 ml) ( d r i e d - 54 -by formation of magnesium ethoxide) i n a 500 ml f l a s k equipped with magnetic s t i r r e r , r e f l u x condenser, dropping funnel and nitrogen i n l e t tube. The mixture was then heated with s t i r r i n g i n an o i l bath at a bath temperature of 100°C f o r 1 hour, a f t e r which the mixture was cooled s l i g h t l y and l-chloro - 3-methyl-but-2-ene ( 50 ) ( 5 . 2 3 g, 0 .50 mole) added dropwise with s t i r r i n g . O , V The mixture was then heated at 100 C (bath temperature), with s t i r r i n g , u n t i l the basic reaction to litmus had disappeared (ca. 1 hour). After cooling the reaction mixture, the ethanol was removed under reduced pressure and the pasty residue dissolved i n the minimum amount of water. The o i l y layer was separated and the aqueous layer extracted 3 times with ether. The combined o i l and ether extracts were washed with brine, dried (NagSO^) and the ether evaporated at reduced pressure. D i s t i l l a t i o n gave d i e t h y l (3-methyl-but-2-ehyl)-malonate ( 51 ) (77.6 g, 68.0$) as a colourless o i l , b.p. 127-130°C (13 mm) j U l t ^ 5 b.p. 249-275°C )]• "^max d i c l u l d f i l m ) , 2995, 1730, 1440, 1360, 1040, 863 cm"1; (neat) 4.91 ( t , J = 7Hz, IH), 5.88 (q, J = 7Hz, 4H), 6 . 7 3 ( t , J = 7.5Hz, IH), 7 .50 ( t , J = 7.5Hz, 2H), 8 . 39 (br.s, 6H), 8.80 ( t , J = 7Hz, 6H). Preparation of 3-methyl-but-2~enyl malonic acid ( 52 ) To a solution of sodium hydroxide (50.0 g) i n water (110 ml) i n a 500 ml f l a s k equipped with magnetic s t i r r e r , r e f l u x condenser and dropping funnel, d i e t h y l (3-methyl-but-2-enyl)-malonate ( 51 ) (100.1 g, 0 .44 mole) was added dropwise. The s o l u t i o n was then heated at a bath temperature of 90-100°C f o r 5a hours. S u f f i c i e n t water was added to dissolve some of the sodium s a l t which had p r e c i p i t a t e d out, and the reaction mixture was extracted twice with'ether to remove unreacted s t a r t i n g material. The reaction mixture was a c i d i f i e d with 6N HC1 and the o i l y layer separated. The aqueous layer was extracted 3 times with ether and the combined o i l and ether extracts were vrashed thoroughly with brine, dried (W^SOjj,) , and the ether evaporated to give a yellow o i l which c r y s t a l l i s e d on scratching and cooling. This was r e c r y s t a l l i s e d from benzene to give 3-methyl-but-2-enyl malonic a c i d ( 52 ) ( 5 3 » 7 g, 71.0$) as white c r y s t a l s , m.p. 95 -97°C ( l i t . ^ 6 m.p. 95.5-96°C ). X > m a z (nujol mull) 2600, 1700, 826, ?80 cm"1; T ( C D C l 3 ) -1 ,30 (S, 2H) , 4.28 (t, J = 7.5Hz, IH), 6 .50 ( t , J = 7.5Hz, IH), 7 . 3 2 ( t , J = 7.'5Hz, 211), . 8 .30 (d, J = 3Kz, 6H), Preparation of 5-methyl-hex-4-enoic acid ( 4 9 ) 3-Methyl-but-2-enyl malonic a c i d (52) ( 5 3 . 7 g, 0.31 mole) was placed in a 25p ml f l a s k and heated u n t i l i t melted. The o i l was then d i s t i l l e d at reduced pressure, care being taken to avoid f r o t h i n g , to give 5-methyl-hex-4-enoic acid ( 49 ) ( 3 ^ . 2 g, 85«5%) as a colourless o i l , b.p. 110 - l l 4°C (15 mm) ( l i t . ^ 5 , b.p. 230-237°C). "X) m a x ( l i q u i d f i l m ) , 2900, 1700, 826 cm"1; T (neat) 1 .33 (S, IH), 4 . 9 0 (br.s, IH), 7.69 (d, J = 3Hz, 4H), 8 .34 (d, J = 311 z, 6H). - 5 6 - . •' Attempted P r e p a r a t i o n of 2-methyl~6-keto-6--(4 / -methyl-cyclohex- 3 / - e n y l ) - h e x - 2 - e n e (42) To the G r i g n a r d complex prepared from 4 - c h l o r o - l - m e t h y l -cyclohexene ( 48) (1.0 g, 7.7 mmole) and magnesium t u r n i n g s ( 0 . 2 1 g, 8.4 mmole) i n dry ether ( 1 0 ml) and cuprous c h l o r i d e ( 5 0 mg) was added, i n ether (10 ml ) , the a c i d c h l o r i d e of 5-methyl-hex-4-enoic a c i d (49) prepared from 5-methyl-hex-4-enoic a c i d (49) (0.87 go 7.7 mmole) and o x a l y l c h l o r i d e ( 1 . 0 1 g, 8.4 mmole). Excess o x a l y l c h l o r i d e was removed by adding dry benzene and e v a p o r a t i n g to 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 temperature o v e r n i g h t , the r e a c t i o n mixture 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 ether l a y e r was s e p a r a t e d and t h e aqueous phase e x t r a c t e d twice w i t h e t h e r . The combined e t h e r l a y e r s were washed twice 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 (NagSO^). E v a p o r a t i o n of the ether gave a red o i l ( 0 . 9 2 g) which was shown by t . l . c . to be a complex mixture of p r o d u c t s . D i s t i l l a t i o n ( 8 0 - 9 0 ° C , 0.3 mm) gave a predominance of one prod u c t which was f u r t h e r p u r i f i e d by p r e p a r a t i v e t . l . c . on s i l i c a g e l to g i v e a produ c t which was shown, by comparison of i . r . and -n.m.r. s p e c t r a not t o be the d e s i r e d 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 7 -enyl)-hex-2-ene ( 4 2 ) . P r e p a r a t i o n of 2 - c y c l o p r o p y l - p r o p a n - 2 - o l ( 6 5 ) To methyl c y c l o p r o p y l ketone (44) ( 5 0 g, 0.6 mole) d i s s o l v e d i n ether (400 ml) was added, dropwise w i t h c o o l i n g and s t i r r i n g , a s o l u t i o n of methyl l i t h i u m i n ether ( 3 0 0 ml of 2.4 M s o l u t i o n , 0.72 mole). A f t e r s t i r r i n g a t room temperature - 57 -f o r 2 hours the reaction mixture was worked up by adding Ice cold water to the cooled s o l u t i o n . After separation of the ether layer the aqueous phase was extracted twice with ether, the combined ether layers were washed once with brine, dried (Na^ SOjj,.) and the ether evaporated to give a pale yellow o i l which on d i s t i l l a t i o n gave 2 f r a c t i o n s : -v (a) 2-cyclo-propyl-propan-2-ol (65) (32,8 g, 53$) as a colourless o i l , b ep„ 120-124°C ( 7 6 0 mm) ( l l t , ^ 2 b . p „ 1 2 1 - 1 2 2 ° C ) , N) m a x ( l i q u i d f i l m ) , 3^ -00, 3 1 0 0 , 2 9 5 0 , 1 4 6 0 , 1 1 5 0 , 1 0 2 0 , 9 6 0 , 9 2 0 , 845, 8 2 5 cm"1; T (CCl^) 7 .66 (S, IH), 8,86 (S, 6 H ) , 9.00-9.40 (m, 4 H ) , 9.73 (d, J = 6Hz, 6 H ) . (b) 2,4-dicyclopropyl-pentan-2,4-diol ( 6 6 ) ( 9 , 3 g, .15.7&) as a colourless o i l , b.p. 128-132°C ( 1 0 mm), \) x - ( l i q u i d film) 3 3 5 0 , 3 0 9 0 , 2 9 9 5 , 1 0 5 0 , ' 1 0 2 0 , 9 1 0 , 8 3 0 cm"1 i t (CCl^) 6.26 (S, 2 H ) , 8.20 (d, J =4Hz, 2H), 8 .72 (S, 6 H ) , 8 . 9 0 - 9 . 3 6 (m, 2H), 9 . 6 0 (m, 8 H ) . Preparation of 5-3romo-2-methyl-pent-2-ene ( 3 8 ) . To 2-cyclopropyl-propan -2-ol ( 6 5 ) ( 3 2 . 8 g, O.328 mole) was added, with cooling and s t i r r i n g , 4 8 $ HBr ( 1 5 4 ml) over a period of 5 minutes. Afte r s t i r r i n g f o r 1 0 minutes with cooling and 1 0 minutes at room temperature the reaction mixture was extracted 3 times with ether, the combined ether extracts were, washed thoroughly with saturated aqueous sodium bicarbonate so l u t i o n , dried (NagSO^) and the ether evaporated. D i s t i l l a t i o n gave 5-bromo-2-methyl-pent-2-ene ( 3 8 ) ( 3 5 . 1 g, 6 5 . 5 $ ) as a colourless o i l , b.p. 72-7^°C ( 4 7 mm) [ l i t . * * 7 b.p. 84-85°C - 58 -(84 mm)]. \ ) m a x ( l i q u i d f i l m ) 2960, 2 9 2 5 , l 6 ? 0 t 1445, 1 3 7 5 , 1 2 6 5 , 1 2 0 5 , 1 0 9 5 , 835 cm"*1; T (CCl^) 4 . 8 3 ( t . q u i n , J =.7Hz, 1.5 Hz, IH), 6 . 7 7 ( d . t , J = 7Hz, 1 ,5 Hz, 2H), 7 .53 ( q u i n . J = 7Hz, 2H), 8 . 3 3 (d, J = 4Hz, 6H). P r e p a r a t i o n of methyl ( 4-methyl-cyclohex - 3-ene) c a r b o x y l a t e ( 59 ) Isoprene (1) (9.0 g, 0 .13 mole), methyl a c r y l a t e ( 43 ) (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 heated 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 methyl ( 4-methyl-cyclohex-3-ene) c a r b o x y l a t e ( 5 9 ) p l u s a l i t t l e of the 3-methyl isomer ( 6 0 ) (14 . 7 g, 7 3 . 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 . 5 1 8 5 - 8 6 ° (15 mmj] , "\3 m a „ ( l i q u i d f i l m ) 2 9 2 5 , 1 7 3 5 , 1^30, 1170 cm" 1; T ( C C l ^ ) 4 . 6 3 (S, IH), 6 .36 (S, 3H), 7 . 2 0-8 . 2 0 (m,7H), 8 . 3 3 (S, 3H). P r e p a r a t i o n of 4-methyl-cyclohex - 3-ene c a r b o x y l i c a c i d ( 4 l ) (a) Methyl ( 4-methyl-cyclohex - 3-ene) c a r b o x y l a t e (59 ) (14 . 7 g 0 . 0 9 mole) was added to a s o l u t i o n of sodium hydroxide (11 . 5 g) i n water ( 60 m l ) . The mixture was s t i r r e d a t 65°C u n t i l i t was homogeneous ( c a . 2 h o u r s ) , when i t was co o l e d and e x t r a c t e d w i t h e t h e r to 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 tho r o u g h l y w i t h b r i n e and evaporated t o g i v e a gummy white s o l i d which 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 . e t h e r ( 60-80°) to g i v e 4-methyl-cyclohex - 3-ene c a r b o x y l i c - 59 -a c i d (4l) (2.5 g» 19$) as white, g l i s t e n i n g needles, m.p, 99-lOO°c ( l i t , 5 1 93.5-99°c). ^ m a x (CHC13) 2905, 2655, 1705 cm"1? 1? (CDC1 3) 1.13 (S, IH), 4,36 (S, IK), 7.^-8,2 (m, 7H), 8.37 (S, 3H). (b) A c r y l i c a c i d (45,2 g, O.63 mole), isoprene (42.8 g, 0,63 mole) and hydroquinone (0.8 g) were sealed, under vacuum, i n 4 g l a s s tubes and heated at 95°C f o r 16 hours. The r e s u l t i n g gummy s o l i d was repeatedly c r y s t a l l i s e d from pet. ether (60-80°) to give 4-methyl-cyclohex-3-ene carboxylic a c i d (41) (26.6 g, 30$) i d e n t i c a l i n a l l respects to that prepared under (a) above. Preparation of 4-hydroxy-4-methyl-cyclohexane carboxylic a c i d  lactone (64) 4-Methyl-cyclohex-3-ene carboxylic acid (4l) (100 mg; 0.72 mmole) and 98% formic a c i d (2 ml) were-heated overnight at 80°C. The reaction mixture was worked up by adding ether and water. The aqueous layer was extracted twice with ether and the combined ether extracts washed twice with brine, twice with saturated aqueous sodium bicarbonate s o l u t i o n , twice with brine and dried (Na 2S0^). Evaporation of the ether gave s l i g h t l y yellow c r y s t a l s which were r e c r y s t a l l i s e d from pet. ether (60-80°) to give 4-hydroxy-4-methyl-cyclohexane carboxylic a c i d lactone (64) (52.0 mg, 52.0$) as white plates, m.p. 68-69°C ( l i t . 5 5 . m.p, 68.0-68.7°C). A) (CCli.) 2950, 1760, 1230, 1070, 960 cm"1? max ^ - \ ) m a x (CHCI3) 2965, 1740, 1230, 1070, 960 .cm"1;. *£ (CCli,.) 7.50 (S, IH), 8.20 (S, 8H), 8.65 (S, 3H). - 60 -P r e p a r a t i o n of e s t e r ( 7 ° ) To the G r i g n a r d complex,, prepared from 5-bromo-2-methyl-pent-2-ene (38) (34.2 g, 0.21 mole) and magnesium t u r n i n g s ( 5 . 5 g» 0,23 mole) i n dry ether (100 ml),.was added cuprous c h l o r i d e (200 mg.) and the a c i d c h l o r i d e of 4 - m e t h y l - c y c l o h e x - 3 -ene c a r b o x y l i c a c i d ( 4 l ) i n et h e r (25 ml) prepared from 4-methyl-cyclohex . - 3-ene c a r b o x y l i c a c i d (41) (26.6 g, 0.19 mole) and o x a l y l c h l o r i d e ( 2 6 . 7 g, 0.21 mole). Excess o x a l y l c h l o r i d e was removed by adding dry benzene and e v a p o r a t i n g to g i v e a y e l l o w o i l . A f t e r s t i r r i n g a t room temperature o v e r n i g h t the r e a c t i o n mixture was worked up by adding s a t u r a t e d aqueous ammonium a c e t a t e s o l u t i o n . The ether l a y e r was separated and the aqueous phase e x t r a c t e d t w i c e w i t h e t h e r . The combined ether l a y e r s were washed twice w i t h 2% aqueous sodium b i c a r b o n a t e s o l u t i o n s once w i t h b r i n e and d r i e d (NagSO^). E v a p o r a t i o n o f the et h e r gave a y e l l o w o i l which was d i s t i l l e d to g i v e e s t e r (70) (11,4 g, 35,0$) as a c o l o u r l e s s o i l , b.p. 157-l66°C. (0.1 mm). "O max ( l i q u i d f i l m ) 2915, 1730, 1440, 1 1 7 0 , 920, 835, 805, 770 cm - 1; T ( C C l ^ ) 4.68 (S, 2H), 4.80-5.40 (m, 2H), 7.20-8.27 (ra, 18H), 8 . 3 3 (m, 12H)? M + m/e 330? C, 80.23; H, 10.33 ( c a l c . f o r C22H34°2! C » 8 0 « ° ° ; H , 10..30). - 61 -R e a c t i o n s of e s t e r (70) (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 - ( 0 . 4 ml of 2.4M s o l u t i o n , 1.0 mole) was added d r o p w i s e , w i t h s t i r r i n g , t o a c o o l e d s o l u t i o n of e s t e r (70) (100 mg, 0.3 mole) I n e t h e r (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 15 minutes then worked up by ad.ding w a t e r . S e p a r a t i o n of 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 and e v a p o r a t i o n of the e t h e r gave a p a l e y e l l o w o i l (110 mg, 100%) w h i c h was shown by g . l . c . (3$ Se30, 5' x t", 150°C) t o be two p r o d u c t s . S e p a r a t i o n by p r e p a r a t i v e g . l . c . {J>0% Se30, 20' x - V 3 " , 175°C) gave, as the more v o l a t i l e component, < * - t e r p i n e o l (67), "N> m a x ( l i q u i d f i l m ) 3355, 2900, 1430, 840, 800 cm" 1; If ( C C l ^ ) 4.67 ( b r . s , I E ) , 7.93 (S, I H ) , 8.07 ( b r . s , 5H), 8.37 (S, 3K), 8.50-8.80 (m, 2H), 8.87 (S, 6H). Bo t h s p e c t r a were I d e n t i c a l w i t h those of a u t h e n t i c o < - t e r p l n e o l (67). The l e s s v o l a t i l e component was 2-methyl-6-hydroxy-6-(4"'-methyl-cyclohex-3 / - e n y l ) - h e x - 2 - e n e ( 6 8 ) ' f ^ \ Y „ ( l i q u i d f i l m ) 3365, 2900, 1435, 1370, 1075, 830, 805 cm" 1! % ( C C l ^ ) , 4.67 (S, I H ) , 4.90 ( t , J = 7Hz, I H ) , 6.67 ( b r . s , IH),,7.67-8.20 (m, 7 H ) , 8.33 (S, 9H), 8.40-8.67 (m, 4H), 8.70 ( S , I H ) . (b) To e s t e r (70) (200 mg, 0.6m mole) i n e t h e r (2 ml) was added l i t h i u m a l u m i n i u m h y d r i d e (19.0 mg, 0.5 mmole). A f t e r s t i r r i n g f o r 30 minutes t h e s o l u t i o n was worked up by a d d i n g w a t e r , 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 of t h e e t h e r t o g i v e a p a l e y e l l o w o i l (180 mg, 90f0 w h i c h was shown by g . l . c . {% Se30, 5'x |", 150°C) t o be two p r o d u c t s . S e p a r a t i o n by p r e p a r a t i v e g . l . c . (30,€ Se30, 20' x V.8"» 175°C) gave, as the more v o l a t i l e .. component, a compound 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 - c y c l o h e x -3-enyl.methanol (69), \ ) m a x ( l i q u i d f i l m ) 3320, 2905, 1440, 1050 1 0 0 5 , 800 cm ; T(CCl^) 4 .70 (m, I K ) , 6 . 5 7 (A, J = 4Hz, 2K) , 7 . 9 0-8 . 2 7 (m, 5H), 8 .33 (S, 3K), 8.50-8.80 (m, 2H), 8.90 ( S , IH). The l e s s v o l a t i l e component was 2-rnethyl-6-hydroxy-6- (4' -methyl -cycl o ~ h e x - 3 ' - e n y l } - h e x - 2 - e n e ( 6 8 ) , which showed i d e n t i c a l s p e c t r o s c o p i c p r o p e r t i e s to t h a t prepared under (a) above. P r e p a r a t i o n of 2-methyl-6-keto-6-(4^ - m e t h y l - c y c l o h e x - 3 / - e n y l ) - hex-2-ene (42) (a) E t h e r e a l methyl l i t h i u m s o l u t i o n ( 2 1 . 3 ml of 2.4 M s o l u t i o n , 5.12 mmole) was added dropwise,' w i t h s t i r r i n g and c o o l i n g to a s o l u t i o n of e s t e r (7 ) (4.2 g, 12.8 mmole) i n ether (15 m l ) . A f t e r s t i r r i n g a t room temperature f o r 30 minutes water (50 ml) was added. A f t e r s e p a r a t i o n of the ether l a y e r the aqueous phase was e x t r a c t e d twice w i t h ether and the combined ether l a y e r s washed with b r i n e and d r i e d (Na2S0^,)c A f t e r e v a p o r a t i o n of the eth e r the crude product c o n s i s t i n g mostly of a l c o h o l ( 6 8 ) and o < - t e r p i n e o l ( 6 7 ) was d i s s o l v e d i n acetone (10 ml) and Jones reagent ( I . 6 5 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 1 5 minutes the r e a c t i o n mixture was worked up by adding ether and water. A f t e r s e p a r a t i o n of the two l a y e r s the aqueous phase was e x t r a c t e d twice w i t h ether and the combined ether l a y e r s washed wi t h b r i n e , d r i e d (NagSO^.) and the ether evaporated. D i s t i l l a t i o n of the r e s u l t i n g y ellow 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 ) - h e x - 2 - e n e (42) as a c o l o u r l e s s o i l , b.p. 80-84°C ( 0 . 2 5 mm) Q i t . ^ 1 b.p. 1 2 3 -1 2 5 ° C (2 mm)]. -\3 m a x ( l i q u i d f i l m ) 2905, 1710, 1445, 1 3 7 5 , 920 >, 8 3 5 , 8 0 5 cm" 1; T(CCl^) 4.63 (S, IH), 4.95 ( t , J = 7Hz, IH), 7.20-8.20 (m, 11H), 8 . 33 (S, 9 H ) ; M + m/e 206. - 63 -(b) To the a c i d c h l o r i d e of a c i d (41) p r e p a r e d as above from 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 (41) ( 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 (38) p r e p a r e d as above from 5-bromo-2-methyl-pent-2-ene (38) (1 .05 g» 6.4 mmole) and magnesium (0.17 g, 7.0 mmole). The s o l u t i o n was allowed, t o s t i r a t room t e m p e r a t u r e f o r 2 hours t h e n 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 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 aqueous sodium b i c a r b o n a t e s o l u t i o n t o remove unchanged a c i d ( 4 l ) , t hen washed w i t h b r i n e and d r i e d (NagSC^). Removal of the e t h e r gave a y e l l o w o i l (0.97 g, 75/0 w h i c h was t r e a t e d i n two ways:-( i ) d i s t i l l a t i o n gave 2-methyl-6-keto-6 - ( 4 / - m e t h y l - c y c l o h e x -3 ' - e n y l ) - h e x - 2 - e n e (42) . 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 obtained, i n (a) above. ( i i ) R e d u c t i o n of a p o r t i o n of the crude o i l w i t h sodium borohyd.ride i n me 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-methyl-pent - 3-eny" 1 ( 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 (71) as a c o l o u r l e s s o i l . - \ ) m a x ( l i q u i d f i l m ) 2915, 1735. 1445, 1165, 830 cm" 1; T ( C C l ^ ) 4.67 (S, I H ) , 4 . 9 0 ( t , J = 7Hz, I H ) , 6.00 ( t , J = 7Hz, 2H), 7 . 3 3 -8.20 (m, 9H), 8 . 3 3 (S, 9 H ) . P r e p a r a t i o n of 2-methyl-6-h,ydroxy-6- (4 ' - m e t h y l - c y c l o h e x - 3 / - e n y l ) - hex-2-ene (68) To 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 ) - h e x - 2 -ene (42) (290 mg , 1 . 4 mmole) i n methanol (2 ml) was added sodium b o r o h y d r i d e (60 mg, 1 . 6 mmole) i n methanol ( 2 . m l ) . The m i x t u r e - 64 -was allowed to s t i r f o r 30 min.. a t room temperature, then worked up by adding water (5 ml) and e v a p o r a t i o n of the methanol. The aqueous l a y e r was e x t r a c t e d three times w i t h ether, the ether l a y e r was washed w i t h b r i n e and d r i e d (NagSO^) and the ether then evaporated. D i s t i l l a t i o n of the r e s u l t i n g o i l gave 2-methyl - 6 -h y d r o x y - 6 - ( 4 / - m e t h y l - c y c l o h e x - 3 / - e n y l ) - h e x - 2 - e n e (68) (1.50 mg,: 5 1 . 5 $ ) as a c o l o u r l e s s o i l , b.p. 92 - 9 6°C ( 0 . 2 5 mm) [ l i t . * * ~ b.p. 100-104° ( 0 . 2 5 mm]] . m a x ( l i q u i d f i l m ) 3 3 6 5 , 2 9 0 0 , 1.435, 1 3 7 0 , 1 0 7 5 , 8 3 0 , 805 cm" 1; T (CCljj,) 4,67, (S, I K ) , 4 , 9 0 ( t , J = 7Hz, I H ) , 6.67 ( b r . s , 1 H ) , 7 . 6 7-8.20 (m, ?E), 8 . 3 3 (S, 9 H ) , 8.40-8 . 6 7 (m, 4 l l ) , 8.70 (S , I K ) . P r e p a r a t i o n of frC-blsabolol (35) To a s o l u t i o n of methyl magnesium i o d i d e prepared from methyl i o d i d e (106„5 mg, 0 *75 mmole) and magnesium powder ( 1 9 . 8 mg,- O.83 mmole) i n ether ( 5 ml), was added 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 ) - h e x - 2 - e n e (42.) ( 51.5 mg, 0 . 2 5 mmole) i n ether (2 m l ) . The mixture was allowed to s t i r a t room temperature f o r two hours 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 . The ether l a y e r was separated and the aqueous phase e x t r a c t e d twice w i t h e t h e r . The combined ether l a y e r s were washed wi t h b r i n e and d r i e d (NagSO^). E v a p o r a t i o n of the ether gave o c - b i s a b o l o l (35 ) ( 5 5 mg , 100$) as a c o l o u r l e s s o i l . - \ o „ , o v ( l i q u i d f i l m ) 3^ 3 5 , 2 9 1 5 , 1 4 4 0 , 1 3 7 0 , III SIX 1 1 0 5 , 8 3 0 , 800 cm" 1; T ( C C l ^ ) 4 . 6 7 (S, 1 H ) , 4 . 9 0 ( t , J = 7Hz, IH) 7.60-8 . 2 3 (m, T H ) , 8.37 (S, 9 H ) , 8 . 5 0-8.87 (m, sH), 8 . 9 3 (d, J = 2Hz, 3H)} M + m/e 2 2 2 . P r e p a r a t i o n of C - l a b e l l e d o < - b i s a b o l o l ( 3 5 ) c< - B i s a b o l o l ( 3 5 ) was prepared as above u s i n g " C-tnethyl i o d i d e ( 2 1 3 . 0 m g » : 1 . 5 mmole, ImC, 0 . 6 6 7 mC/mmole) magnesium powder ( 3 9 . 6 mg , I . 6 5 mmole) and 2-methyl-6-keto-6-{k 1 -methyl-c y c l o h e x - 3 7 - e n y l ) - h e x - 2 - e n e ( 4 2 ) ( 1 0 3 mg., . 0 . 5 mmole). A f t e r work up t . l . 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 ( 4 2 ) 14 l e f t so i n a c t i v e methyl l i t h i u m was added. The y i e l d of C-o<-b i s a b o l o l ( 3 5 ) was 1 1 0 . 0 mg ( 1 0 0 $ ) , and the s p e c i f i c a c t i v i t y was determined to be 0 , 3 7 mC/mmole, I s o l a t i o n of T r i c h o t h e c i n ( 2 8 ) from T r l c h o t h e c l 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 • • - -• 30 ^9 l i t e r a t u r e except 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 59 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 except t h a t p r e p 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 the p u r i f i c a t i o n r a t h e r than column chromatography on alumina. I n a t y p i c a l experiment 1 0 f l a s k s of T. roseum were allowed to grow f o r three 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 water and the combined washings and b r o t h e x t r a c t e d twice, i n 1 0 0 0 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 ( 2 0 0 m l ) . E v a p o r a t i o n of the carbon t e t r a c h l o r i d e gave a gum ( 1 4 8 . 6 mg) which 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 petroleum ether: e t h y l a c e t a t e ( 1 : 1 ) . Under U.V. 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 approximately h a l f way up the p l a t e . These bands were scraped o f f and the t r i c h o t h e c i n ( 2 8 ) leached 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 of 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 ( 2 8 ) ( 9 4 . 3 mg) - 66 -w h i c h 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 ( 6 0 - 8 0 ) t o ' g i v e p u re t r i c h o t h e c i n (28) as w h i t e n e e d l e s , m.p, 116-118°C ( l i t . " * 0 m.p. 118°C ) ; - \ J (CHClo ) 1 7 1 5 , 1 6 8 5 cm" 1. max J 14 F e e d i n g of C - l a b e l l e d c x - b i s a b o l o l t o T r l c h o t h e c i u m roseum 14 To t h e C - l a b e l l e d 0 < - b i s a b o l o l ( 5 2 mg, 0.087 mC) was added w a t e r (20 ml) and Tween 20 ( 2 m l ) . T h i s w e l l shaken m i x t u r e 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 of 5 day o l d c u l t u r e s of T. roseum w h i c h were then l e f t f o r a f u r t h e r 20 d a y s . The t r i c h o t h e c i n 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 s 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 ( 5 . 3 x " 1 0 J mC/mmole) w h i c h r e p r e s e n t e d an i n c o r p o r a t i o n of 0.0143, , . P r e p a r a t i o n o f t r i c h o t h e c o l o n e ( 2 9 ) T r i c h o t h e c i n ( 5 0 mg, 0.15 mmole) was s t i r r e d over n i g h t a t room t e m p e r a t u r e i n IN 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 ( 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 v a c u o T h e 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 , r e m o v a l of w h i c h 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 b e n z e n e - p e t r o l e u m e t h e r (2:1) t o g i v e t r i c h o t h e c o l o n e (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 . 183-184°C). - O _ (CHC1 ), 3560, 1680 cm" 1. 1 ZL H y d r o l y s i s of ~ C - l a b e l l e d t r i c h o t h e c i n 14 C - l a b e l l e d t r i c h o t h e c i n (5*3 mg). was d i l u t e d w i t h i n a c t i v e t r i c h o t h e c i n (10.6 mg ) and t h i s was h y d r o l y s e d as above t o g i v e " ^ C - l a b e l l e d t r i c h o t h e c o l o n e (9.5 mg ) w h i c h was r e c r y s t a l l i s e d t o a c o n s t a n t s p e c i f i c a c t i v i t y of 1.1.x 1 0 ~ 5 mC/mmole. - 6? -BIBLIOGRAPHY 1. J . N. C o l l i e , T r a n s . Chem. S o c , 1 8 9 3 , £ 3 , 3 2 9 . 2 . J . N. C o l l i e , T r a n s , Chem. S o c , 1 9 0 7 , 9 1 , 1 8 0 6 . 3. A, J . B i r c h and F. W. Donovan, A u s t r a l . J . Chem., 1 9 5 3 . 6 , 3 6 0 . 4. L. 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