UBC Theses and Dissertations

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UBC Theses and Dissertations

Novel metabolites from British Columbia nudibranchs Gustafson, Kirk 1984

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NOVEL METABOLITES FROM BRITISH COLUMBIA NUDIBRANCHS BY KIRK GUSTAFSON B . S c , The U n i v e r s i t y o f D e l a w a r e , 1979 THESIS SUBMITTED IN PARTIAL FULFILLMENT THE. REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Depa r t m e n t 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 t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 1984 ©Kirk G u s t a f s o n , 1984 I n 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 a n 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 m a y b e g r a n t e d b y t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r 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 b e 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 C\r>i» rv\vS"V r ^ 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 1956 M a i n M a l l V a n c o u v e r , C a n a d a V6T 1Y3 D a t e ^ a ^ 3 ^ B \ (3/81) ABSTRACT S k i n e x t r a c t s f r o m a number o f B r i t i s h C o l u m b i a n u d i b r a n c h s h a v e p r o v e n t o be a r i c h s o u r c e o f n o v e l m e t a b o l i t e s . T h i s t h e s i s d e s c r i b e s t h e s t r u c t u r a l i n v e s t i g a t i o n o f s e v e r a l o f t h e s e compounds and i n c l u d e s some p r e l i m i n a r y s t u d i e s o f t h e i r b i o s y n t h e t i c o r i g i n and p h y s i o l o g i c a l r o l e . The u n i q u e d i a c y l g u a n i d i n e t r i o p h a m i n e (7_5) h a s b e en i s o l a t e d f r o m T r i o p h a c a t a l i n a e and P o l y c e r a t r i c o l o r . I t s s t r u c t u r e was d e d u c e d by c l a s s i c a l c h e m i c a l methods and c o n f i r m e d by an unambiguous s y n t h e s i s . A n i s o d o r i s n o b i l i s p r o v i d e d t h e o d o r i f e r o u s d e g r a d e d s e s q u i t e r p e n e d i h y d r o a p o f a r n e s a l (1_5) . The s t r u c t u r e o f a l d e h y d e jL_5 was a s s i g n e d by i n t e r p r e t a t i o n o f s p e c t r a l d a t a . E x t r a c t s o f A r c h i d o r i s m o h t e r e y e n s i s c o n t a i n t h e d i t e r p e n o i c a c i d g l y c e r i d e 3/7, i t s two m o n o a c e t a t e s _18 and 1J3, t h e d r i m a n e s e s q u i t e r p e n o i c a c i d g l y c e r i d e 2fJ and i t s m o n o a c e t a t e 21, t h e m o n o c y c l o f a r n e s i c a c i d g l y c e r i d e 2_2 and t h e g l y c e r y l e t h e r 23^. T h e s e compounds were e s t a b l i s h e d by a c o m b i n a t i o n o f s p e c t r a l a n a l y s i s , c h e m i c a l i n t e r c o n v e r s i o n a n d / o r s i n g l e c r y s t a l X - r a y d i f f r a c t i o n a n a l y s i s . The s t r u c t u r e o f a f r e e d i t e r p e n o i c a c i d f o u n d i n o n l y one c o l l e c t i o n o f A. m o n t e r e y e n s i s , was a l s o t e n t a t i v e l y i d e n t i f i e d . T h e i s o m e r i c f a r n e s i c a c i d g l y c e r i d e 2_7 i n a d d i t i o n t o i i i g l y c e r i d e s 1Q_, 20, 22_ and e the r 22_ have a l s o been i s o l a t e d from A r c h i d o r i s o d h n e r i . The d r imeno i c a c i d g l y c e r i d e 20. an<^ t n e g l y c e r y l e the r 23^  e x h i b i t a n t i f e e d a n t a c t i v i t y a g a i n s t f i s h . B i o s y n t h e t i c s t u d i e s showed t h a t l t + C l a b e l e d meva lon ic a c i d i s i n c o r p o r a t e d i n t o the t e r p e n o i d p o r t i o n o f g l y c e r i d e s 1_7 and 20_ by A. montereyens is and i n t o the f a r n e s i c a c i d p o r t i o n o f g l y c e r i d e 24 by A r c h i d o r i s  o d h n e r i . P r e l i m i n a r y r e s u l t s suggest t ha t t r i ophamine (75) i s s y n t h e s i z e d i n s i t u by T . c a t a l i n a e and the drimane s k e l e t o n o f a l b i c a n y l a ce t a t e (3_6) i s b i o s y n t h e s i z e d by C a d l i n a l u t e o m a r g i n a t a . i v TABLE OF CONTENTS A b s t r a c t i i Table o f Contents i v L i s t o f F i g u r e s v i L i s t of Schemes v i i i L i s t s o f Tables v i i i L i s t of P l a t e s v i i i Acknowledgments i x A b b r e v i a t i o n s x INTRODUCTION I. Nudibranch Defenses 1 I I . Chemistry of Other Opisthobranchs 5 I I I . Nudibranch Chemistry 9 CHEMICAL STUDIES OF TRIOPHA CATALINAE I. I s o l a t i o n and S t r u c t u r a l E l u c i d a t i o n of Triophamine (7^5) 3 5 I I . B i o l o g i c a l C o n s i d e r a t i o n s 53 I I I . S y n t h e s i s of (± )-Triophamine (75a) 55 CHEMICAL STUDIES OF ANISODORIS NOBILIS I. D i h y d r o a p o f a r n e s a l (1_5) 65 C H E M I C A L S T U D I E S OF A R C H I D O R I S M O N T E R E Y E N S I S A N D A . O D H N E R I I. A r c h i d o r i s m o n t e r e y e n s i s 72 I I . A r c h i d o r i s o d h n e r i 1 1 6 I I I . A n t i f e e d a n t B i o a s s a y 1 1 8 I V . B i o s y n t h e s i s o f A r c h i d o r i s M e t a b o l i t e s 1 2 0 V . A d d i t i o n a l B i o s y n t h e t i c S t u d i e s 1 2 3 SUMMARY A N D D I S C U S S I O N 1 2 4 E X P E R I M E N T A L i 1 3 2 B I B L I O G R A P H Y 1 5 2 v i L I S T OF FIGURES 1. P h y l o g e n e t i c c l a s s i f i c a t i o n o f n u d i b r a n c h s 2 2. T y p i c a l d o r i d n u d i b r a n c h ' 4 3. A c y l i m i n e t a u t o m e r s o f t r i o p h a m i n e (7j>) 42 4. I n t e r p r e t a t i o n o f t h e HRMS o f 75. 4 5 5. 400 MHz 'H NMR s p e c t r u m o f 75 46 6. 100 MHz 1 3 C NMR s p e c t r u m o f 75 47 7. 100 MHz SFORD 1 3 C NMR s p e c t r u m o f 75 48 8. IR s p e c t r u m o f 75 49 9. 400 MHz 'H NMR d i f f e r e n c e NOE s p e c t r u m o f 75 50 10. 270 MHz 'H NMR s p e c t r u m o f 78 51 11. 400 MHz 'H NMR s p e c t r u m o f 83 52 12. C o m p a r i s o n o f 400 MHz 'H NMR r e s o n a n c e s o f n a t u r a l t r i o p h a m i n e (7j5) and s y n t h e t i c d i a s t e r e o m e r s 75b . 63 13. I n t e r p r e t a t i o n o f t h e HRMS o f 15 69 14. 400 MHz *H NMR s p e c t r u m o f 15 70 15. Computer g e n e r a t e d s t e r e o c h e m i c a l r e p r e s e n t a t i o n o f r7 76 16. I n t e r p r e t a t i o n o f t h e HRMS o f 17 77 17. 400 MHz 'H NMR s p e c t r u m o f 17 78 18. 100 MHz 1 3 C NMR s p e c t r u m o f 17 79 19. 100 MHz SFORD 1 3 C NMR s p e c t r u m o f 17 80 20. IR s p e c t r u m o f _17 81 21. 400 MHz 'H NMR s p e c t r u m o f 97 82 22. 100 MHz 1 3 C NMR s p e c t r u m o f 9_7 83 v i i 23. IR spectrum of 97 84 24. 400 MHz 'H NMR spectrum o f 1J3 87 25. IR spectrum o f 18 88 26. 400 MHz 'H NMR spectrum of 19 89 27. IR spectrum of 19 90 28. I n t e r p r e t a t i o n of the HRMS of 20 94 29. 400 MHz 1H NMR spectrum of 20 95 30. 100 MHz 1 3 C NMR spectrum of 20 96 31. IR spectrum of 20 97 32. 400 MHz 'H NMR d i f f e r e n c e NOE spectrum of 20 98 33. 270 MHz 'H NMR spectrum of 98 99 34. 400 MHz 'H NMR spectrum of 21 100 35. IR spectrum o f _21 101 36. I n t e r p r e t a t i o n of the HRMS of 22 104 37. 400 MHz 'H NMR spectrum of .22 105 38. IR spectrum o f 22_ 106 39. P o s s i b l e mass s p e c t r a l fragmentation of 100 108 40. P o s s i b l e mass s p e c t r a l fragmentation of 101 109 41. 400 MHz 'H NMR spectrum of the d i t e r p e n o i c a c i d ( s ) 100 and/or 101 I l l 42. IR spectrum of 100 and/or 101 112 43. 270 MHz 'H NMR spectrum of 23_ 114 44. IR spectrum of _23 . 115 45. 270 MHz 'H NMR spectrum of 21_ 117 46. C, n-hydrocarbon p o r t i o n of triophamine (7_5) 125 v i i i L I S T OF SCHEMES 1. S y n t h e t i c s e q u e n c e f o r p r o d u c t i o n o f t h e i s o m e r i c a c i d s 94 and 83a 56 2. F i n a l s t e p s i n t h e s y n t h e s i s o f ( ± ) - t r i o p h a m i n e (75a) and t h e d i a s t e r e o m e r s 75b 59 L I S T OF TABLES 1. N u d i b r a n c h M e t a b o l i t e s 10 2. 'H and 1 3 C NMR D a t a f o r T r i o p h a m i n e (25) 38 3. 'H NMR D e c o u p l i n g S t u d i e s o f T r i o p h a m i n e (7j>) .... 39 4. F i s h A n t i f e e d a n t R e s u l t s 119 5. R e s u l t s o f 1 4 C M e v a l o n i c A c i d I n c o r p o r a t i o n E x p e r i m e n t s 122 L I S T OF PLATES 1. T r i o p h a c a t a l i n a e 34 2. A n i s o d o r i s n o b i l i s 64 3. A r c h i d o r i s m o n t e r e y e n s i s 71 i x ACKNOWLEGEMENTS I am indebted to numerous people who have p r o v i d e d me with a s s i s t a n c e and encouragement d u r i n g the course of t h i s work. In p a r t i c u l a r , I would l i k e t o thank Dr. Raymond Andersen f o r i n t r o d u c i n g me t o the f i e l d of n a t u r a l products c h e m i s t r y . H i s u n f a i l i n g e n t h u s i a s m has i n d e e d been i n s p i r a t i o n a l . The r e l i a b l e and f r i e n d l y support o f Mike LeBlanc and Steven Ayer i s g r e a t l y a p p r e c i a t e d . Warm thanks are extended to a l l of the d i v e r s who aided i n the c o l l e c t i o n of the marine organisms t h a t were e s s e n t i a l t o t h i s r e s e a r c h . I am g r a t e f u l t o Rosemary Armstrong, who k i n d l y s u p p l i e d a sample of a u t h e n t i c drimenol. The p a t i e n t and understanding s t a f f of the departmental NMR and MS l a b o r a t o r i e s are a l s o acknowledged. X L I S T OF ABBREVIATIONS DCC = D i c y c l o h e x y l c a r b o d i i m i d e DIBAL = D i i s o b u t y a l u m i n u m h y d r i d e DMSO = D i m e t h y l s u l f o x i d e DNPH = 2 , 4 - D i n i t r o p h e n y l h y d r a z i n e E = E l e c t r o n i c n o i s e EtOH = E t h a n o l GC = Gas c h r o m a t o g r a p h y HPLC = H i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y HRMS = H i g h r e s o l u t i o n mass s p e c t r o s c o p y IR = I n f r a r e d LC = L i q u i d c h r o m a t o g r a p h y MeOH = M e t h a n o l NEN = New E n g l a n d N u c l e a r 'H NMR = P r o t o n n u c l e a r m a g n e t i c r e s o n a n c e 1 3 C NMR = Carbon-13 n u c l e a r m a g n e t i c r e s o n a n c e NOE = N u c l e a r O v e r h a u s e r enhancement RT = Room t e m p e r a t u r e S = S o l v e n t s i g n a l SFORD = S i n g l e f r e q u e n c y o f f - r e s o n a n c e d e c o u p l e d THF = T e t r a h y d r o f u r a n TLC = T h i n - l a y e r c h r o m a t o g r a p h y UV = U l t r a v i o l e t W = Water s i g n a l 1. I N T R O D U C T I O N I. N U D I B R A N C H D E F E N S E S M o l l u s c s o f t h e c l a s s G a s t r o p o d a , s u b c l a s s O p i s t h o b r a n c h i a h a v e b e e n t h e s u b j e c t o f i n t e n s e s c r u t i n y b y n a t u r a l p r o d u c t c h e m i s t s i n r e c e n t y e a r s . A d u l t o p i s t h o b r a n c h s a r e s l o w m o v i n g a n i m a l s w h o s e s y n o n y m s i n c l u d e s e a s l u g a n d n a k e d s n a i l . W h i l e m o s t o p i s t h o b r a n c h s a r e s h e l l - l e s s , s o m e f o r m s h a v e i n t e r n a l o r g r e a t l y r e d u c e d s h e l l s t h a t p r o v i d e v e r y l i t t l e p r o t e c t i o n . E v e n t h o u g h t h e y h a v e e x p o s e d s o f t t i s s u e a n d a r e o f t e n b r i g h t l y c o l o r e d , t h e s e a n i m a l s h a v e f e w k n o w n p r e d a t o r s 1 . T h i s r e l a t i v e i m p u n i t y f r o m p r e d a t i o n h a s l e d t o s p e c u l a t i o n t h a t o p i s t h o b r a n c h s u t i l i z e s o m e f o r m o f c h e m i c a l d e f e n s e . N u d i b r a n c h s c o m p r i s e t h e l a r g e s t o f s e v e n o r d e r s i n t h e s u b c l a s s O p i s t h o b r a n c h i a ( F i g u r e 1 ) . T h e y a r e c h a r a c t e r i z e d b y t h e c o m p l e t e a b s e n c e o f a s h e l l a n d , a s t h e i r n a m e i m p l i e s , t h e y p o s s e s s e x t e r n a l r e s p i r a t o r y a p p e n d a g e s . A d u l t n u d i b r a n c h s r a n g e i n s i z e f r o m 3 - 3 0 0 mm a n d t h e i r d i e t s i n c l u d e s p o n g e s , b r y o z o a n s a n d c o e l e n t e r a t e s 2 . C o l o r a t i o n r a n g e s f r o m c a m o u f l a g e d c o l o r s c h e m e s t h a t m i m i c t h e p r e f e r r e d s u b s t r a t e o f a s p e c i e s t o v e r y b r i g h t , c o n s p i c u o u s c o l o r s t h a t s e e m t o b l a t a n t l y a d v e r t i s e t h e p r e s e n c e o f t h e o r g a n i s m . S e l e c t e d m e m b e r s o f f o u r g r o u p s o f a n i m a l s , o p i s t h o b r a n c h s , c r u s t a c e a n s , a s t e r o i d s a n d f i s h e s , a r e k n o w n t o p r e y o n n u d i b r a n c h s 3 , b u t t h e l e v e l o f MOLLUSCA PHYLUM GASTROPODA CLASS OPISTHOBRANCHIA SUBCLASS 1 BULLOMORPHA 1 APLYSIAMORPHA 1 PLEUOBRANCHOMORPHA 1 PTEROPODA ORDER SACOGLASSA NUDIBRANCHIA PYRAMIDELLA AEOLIDACEA ARMINACEA DENDRONOTACEA DORIDACEA SUBORDER N.B. Organisms c l a s s i f i e d a c co rd ing to B e h r e n s 2 . to F igu re 1. Phy logene t i c c l a s s i f i c a t i o n o f nud ibranchs 3. p r e d a t i o n i s v e r y low. T h e r e f o r e , w h i l e t h e i r s o f t body f o r m and l i m i t e d m o b i l i t y m i g h t s u g g e s t t h a t n u d i b r a n c h s a r e p a r t i c u l a r l y v u l n e r a b l e t o a t t a c k , t h i s i s c l e a r l y n o t t h e c a s e . I t i s b e l i e v e d t h a t t h e y employ a v a r i e t y o f s o p h i s t i c a t e d d e f e n s i v e s t r a t e g i e s t o d e t e r p o t e n t i a l p r e d a t o r s . C r y p t i c c o l o r a t i o n i n c o n c e r t w i t h c o m p l e m e n t a r y h a b i t a t s e l e c t i o n r e d u c e s t h e c o n s p i c u o u s n e s s o f c e r t a i n n u d i b r a n c h s . S e v e r a l s p e c i e s d i r e c t l y u t i l i z e p i g m e n t s a c q u i r e d f r o m t h e i r prey 1*, w h i l e o t h e r s use p i g m e n t s w h i c h t h e y p r o d u c e t h e m s e l v e s t o match t h e c o l o r scheme o f t h e i r s u r r o u n d i n g s . Such c o l o r a t i o n may r e d u c e h a r a s s m e n t by p o t e n t i a l p r e d a t o r s , b u t Thompson 1 f o u n d t h a t n u d i b r a n c h s were s t i l l n o t a c c e p t a b l e t o f i s h when p l a c e d i n a s e t t i n g where c o l o r o f f e r e d no c o n c e a l m e n t . Once d i s c o v e r e d , c r y p t i c n u d i b r a n c h s a p p a r e n t l y r e l y on a s e c o n d l e v e l o f d e f e n s e . The o u t e r c o v e r i n g o f a n u d i b r a n c h i s t e r m e d t h e m a n t l e o r dorsum and i t c o m p r i s e s t h e b u l k o f t h e o r g a n i s m ' s e x p o s e d t i s s u e ( F i g u r e 2 ) . O t h e r e x t e r n a l f e a t u r e s i n c l u d e t h e r h i n o p h o r e s , b r a n c h i a l plume and f o o t . R h i n o p h o r e s a r e p a i r e d s e n s o r y a p p a r a t u s f o u n d a r o u n d a n u d i b r a n c h ' s h e a d , w h i l e t h e b r a n c h i a l plume i s a r e s p i r a t o r y s t r u c t u r e ( g i l l ) u s u a l l y l o c a t e d n e a r t h e anus. The f o o t a n c h o r s t h e a n i m a l t o i t s s u b s t r a t e and i s i n v o l v e d i n l o c o m o t i o n . When n u d i b r a n c h s a r e d i s t u r b e d , most s p e c i e s r e t r a c t t h e s e e x t e r n a l s t r u c t u r e s l e a v i n g o n l y t h e d o r s u m e x p o s e d t o w o u l d b e a t t a c k e r s . T h e p r e s e n c e o f s p i c u l e s i n t h e m a n t l e o f d o r i d ( s u b o r d e r D o r i d a c e a ) n u d i b r a n c h s h a s b e e n p o s t u l a t e d a s a p o s s i b l e d e t e r e n t t o p r e d a t i o n 5 . S p i c u l e s a r e c a l c a r e o u s s p i n e s a c q u i r e d f r o m d i e t a r y s p o n g e s . H o w e v e r , t h e r e l a t i v e i m p o r t a n c e o f s p i c u l e s i n t h w a r t i n g p r e d a t o r s i s b e l i e v e d t o b e q u i t e l o w 3 . B r a n c h i a l Plume F i g u r e 2. T y p i c a l d o r i d n u d i b r a n c h N e m a t o c y s t s , w h i c h a r e s t i n g i n g o r g a n e l l e s p r o d u c e d b y c o e l e n t e r a t e s , p r o v i d e a d e t e r e n t t o p o t e n t i a l p r e d a t o r s f o r c e r t a i n a e o l i d , ( s u b o r d e r A e o l i d a c e a ) , n u d i b r a n c h s 1 . U n f i r e d n e m a t o c y s t s o b t a i n e d f r o m d i e t a r y o r g a n i s m s t r a v e l t h r o u g h t h e d i g e s t i v e g l a n d o f t h e n u d i b r a n c h a n d a r e s t o r e d i n a f u n c t i o n a l s t a t e i n s p e c i a l i z e d c y n i d o c y s t c e l l s 3 . T h e s e s t o r a g e c e l l s a r e l o c a l i z e d i n p r o j e c t i o n s f r o m t h e n u d i b r a n c h s s k i n s u r f a c e . T h u s s i t u a t e d , t h e y a r e r e a d i l y 5. e n c o u n t e r e d d u r i n g t h e i n i t i a l i n s p e c t i o n by an i n q u i s i t i v e p r e d a t o r . When an a e o l i d i s d i s t u r b e d , t h e c y n i d o c y s t c e l l s r e l e a s e c l o u d s o f s t i n g i n g n e m a t o c y s t s as a d e f e n s i v e r e s p o n s e . N e m a t o c y s t u t i l i z a t i o n by n u d i b r a n c h s i l l u s t r a t e s t h e r e m a r k a b l e a b i l i t y o f an a n i m a l t o s e l e c t i v e l y s e q u e s t e r and u t i l i z e f o r i t s own d e f e n s e an i n t a c t c e l l o r g a n e l l e f r o m a d i e t a r y a n i m a l o f a d i f f e r e n t phylum. I t h as become i n c r e a s i n g l y a p p a r e n t t h a t a l a r g e number o f n u d i b r a n c h s employ c h e m i c a l s e c r e t i o n s as p a r t o f t h e i r d e f e n s i v e a r s e n a l . Most s p e c i e s a r e known t o p o s s e s s an abundance o f non-mucous s k i n g l a n d s whose f u n c t i o n s a r e s p e c u l a t e d t o be d e f e n s i v e 1 . S e v e r a l n u d i b r a n c h s c a n s e c r e t e h i g h l y a c i d i c (H2SO^, pH = 1) s o l u t i o n s a l o n g t h e i r s k i n s u r f a c e when m o l e s t e d 6 . W h i l e t h e r e l e a s e o f s t r o n g a c i d p r o v i d e s p r o t e c t i o n f o r some s p e c i e s , t h e s k i n s e c r e t i o n o f most n u d i b r a n c h s i s n o n - a c i d i c 7 . The c h e m i c a l n a t u r e o f t h e s e s e c r e t i o n s and t h e i r p o s s i b l e d e f e n s i v e r o l e h a s p r ompted much o f t h e i n t e r e s t by n a t u r a l p r o d u c t c h e m i s t s . I I . CHEMISTRY OF OTHER OPISTHOBRANCHS P r i o r t o d i s c u s s i n g t h e c h e m i c a l d e f e n s e o f n u d i b r a n c h s , a n o v e r v i e w o f t h e c h e m i s t r y o f o t h e r o p i s t h o b r a n c h s i s i n o r d e r . An e x h a u s t i v e r e v i e w o f o p i s t h o b r a n c h c h e m i s t r y i s b e y o n d t h e s c o p e o f t h i s t h e s i s . R a t h e r , t h e h i g h l i g h t s and g e n e r a l t r e n d s a r e p r e s e n t e d 6. a l o n g w i t h f i n d i n g s t h a t h a v e been m i r r o r e d i n n u d i b r a n c h work. I n i t i a l o p i s t h o b r a n c h s t u d i e s f o c u s e d o n A p l y s i a s p e c i e s b e c a u s e t h e y a r e l a r g e ( a v e r a g e l e n g t h s e v e r a l h u n d r e d mm) and o f t e n c o n t a i n h i g h c o n c e n t r a t i o n s o f n o v e l compounds. P r o d u c t s i s o l a t e d f r o m A p l y s i a s p p . i n c l u d e m o n o t e r p e n e s 8 , s e s q u i t e r p e n e s 9 , d i t e r p e n e s 1 0 a n d C-15 a c e t a t e d e r i v a t i v e s 1 1 . Many o f t h e s e c o n s t i t u e n t s c o n t a i n one o r more h a l o g e n atoms. S t r o n g c i r c u m s t a n t i a l e v i d e n c e i n d i c a t e s t h a t A p l y s i a a c c u m u l a t e s compounds f r o m d i e t a r y s o u r c e s , p r i m a r i l y a l g a e . I n v a r i a b l y t h e h i g h e s t m e t a b o l i t e c o n c e n t r a t i o n i s f o u n d i n t h e d i g e s t i v e g l a n d o f t h e s e h e r b i v o r s . The c o m p o s i t i o n o f a s p e c i e s ' e x t r a c t s c a n v a r y w i t h t h e l o c a t i o n and t i m e o f y e a r t h a t t h e a n i m a l s were c o l l e c t e d , due t o g e o g r a p h i c a l and s e a s o n a l d i f f e r e n c e s i n t h e a v a i l a b i l i t y o f d i e t a r y a l g a e . Numerous p r o d u c t s i s o l a t e d f r o m A p l y s i a h a v e a l s o b e e n f o u n d i n o n e o r m o r e s p e c i e s o f a 1 g a e 1 0 a ' 1 0 c ' 1 2 . S t a l l a r d e_t a _ l . 8 b c l e a r l y d e m o n s t r a t e d t h a t A. c a l i f o r n i c a c o n c e n t r a t e s and s t o r e s i n i t s d i g e s t i v e g l a n d h a l o g e n a t e d m o n o t e r p e n e s o b t a i n e d f r o m t h e a l g a P l o c a m i u m c o c c i n e u m v a r . p a c i f i c u m . I n some i n s t a n c e s , m e t a b o l i t e s c o n c e n t r a t e d f r o m t h e d i e t a r e c h e m i c a l l y m o d i f i e d f o l l o w i n g i n g e s t i o n . The a l g a l p r o d u c t s l a u r i n t e r o l (JL) and p a c i f e n o l (2) a r e c o n v e r t e d t o a p l y s i n (3) and p a c i f i d i e n e (4) r e s p e c t i v e l y , w i t h i n t h e 7. d i g e s t i v e g l a n d o f A . c a l i f o r n i c a 1 3 . Br Br 2 1 M a n y o f t h e k n o w n A p l y s i a c o m p o u n d s p o s s e s s s o m e t y p e o f b i o l o g i c a l a c t i v i t y 9 a ' 9 b | 1 1 1 a n d a p p a r e n t l y a r e a c t i v e p r i n c i p l e s o f t h e a n i m a l s ' d e f e n s i v e s e c r e t i o n s . B r a s i l e n y n e (5_) a n d c i s - d i h y d r o r h o d o p h y t i n (6)11, w h i c h a r e f o u n d b o t h i n t h e b o d y w a l l a n d d i g e s t i v e g l a n d o f A . b r a s i l i a n a , h a v e d e m o n s t r a t e d a n t i f e e d a n t a c t i v i t y t o w a r d f i s h . S o f t - b o d i e d m a r i n e m o l l u s c s o t h e r t h a n A p l y s i a a l s o e m p l o y c h e m i c a l d e f e n s i v e s u b s t a n c e s t o t h w a r t p r e d a t o r s . T h e o p i s t h o b r a n c h O n c h i d e l l a b i n n e y i p r o d u c e s a r e p u g n a n t e x u d a t e c o n t a i n i n g t h e s e s q u i t e r p e n e o n c h i d a l (2)1 5 a r i d a p o l y p r o p i o n a t e d e r i v a t i v e , p e c t i n a t o n e (8_) 1 6 , h a s b e e n i m p l i c a t e d a s t h e d e t e r r e n t i n a m u c o u s s e c r e t i o n . f r o m t h e s i p h o n a r i d S i p h o n a r i a p e c t i n a t a . 8. The a b i l i t y to a c q u i r e f u n c t i o n a l o r g a n e l l e s from d i e t a r y organisms i s not unique t o a e o l i d n u d i b r a n c h s . S e v e r a l op i s thob ranchs o f the o rde r Sacog lossa are known to a s s i m i l a t e p h o t o s y n t h e t i c a l l y a c t i v e c h l o r o p l a s t s from t h e i r a l g a l d i e t . I r e l a n d et a l . 1 7 showed tha t p o l y p r o p i o n a t e m e t a b o l i t e s such as 9 , 1 O - d e o x y t r i d a c h i o n e (.9)/ f rom P lacobranchus o c e l l a t u s , are b i o s y n t h e s i z e d s i t u by the m o l l u s c - c h l o r o p l a s t p a i r . T h i s was the f i r s t demons t ra t ion t h a t an o p i s t h o b r a n c h a c t i v e l y p a r t i c i p a t e s i n t he b i o s y n t h e s i s of secondary m e t a b o l i t e s . 9 . I t i s c l e a r t h a t o p i s t h o b r a n c h m o l l u s c s h a v e d e v e l o p e d m a n y s o p h i s t i c a t e d s y s t e m s o f c h e m i c a l d e f e n s e . U s i n g a v a r i e t y o f p o t e n t i a l l y n o x i o u s c h e m i c a l s t h e y s u c c e s s f u l l y r e s i s t p r e d a t i o n . T h e s o u r c e o f t h e s e c o m p o u n d s i s o f t e n d i r e c t l y f r o m t h e d i e t , a l t h o u g h s o m e d i e t a r y c o n s t i t u e n t s a r e c h e m i c a l l y t r a n s f o r m e d f o l l o w i n g i n g e s t i o n . I n a d d i t i o n , s e v e r a l s p e c i e s a r e c a p a b l e o f in s i t u p r o d u c t i o n o f u n i q u e m e t a b o l i t e s v i a a s y m b i o t i c a s s o c i a t i o n w i t h a l g a l c h l o r o p l a s t s . I l l . N U D I B R A N C H C H E M I S T R Y N u d i b r a n c h s h a v e b e e n t h e f o c u s o f n u m e r o u s c h e m i c a l i n v e s t i g a t i o n s i n r e c e n t y e a r s . R e v i e w s b y T h o m p s o n e t a _ l . 2 5 a n d S c h u l t e e t a _ l . 2 9 n i c e l y s u m m a r i z e t h e w o r k u p t o 1 9 8 2 . T o d a t e , n a t u r a l p r o d u c t s h a v e b e e n r e p o r t e d f r o m o v e r f o r t y s p e c i e s o f n u d i b r a n c h s . T h e s e c o m p o u n d s e n c o m p a s s a n a m a z i n g d i v e r s i t y o f m o l e c u l a r s i z e , f u n c t i o n a l i t y a n d s t r u c t u r a l t y p e . F o r a c o m p i l a t i o n o f a l l k n o w n n u d i b r a n c h m e t a b o l i t e s w i t h c o m m e n t s a b o u t t h e i r s o u r c e a n d b i o l o g i c a l a c t i v i t y s e e T a b l e I. I t c a n b e s e e n t h a t t h e m a j o r i t y o f n u d i b r a n c h s k i n c h e m i c a l s a r e t e r p e n o i d i n n a t u r e . S e s q u i t e r p e n e s c o m p r i s e t h e l a r g e s t s t r u c t u r a l c l a s s a n d e x h i b i t a n u m b e r o f r e c u r r i n g s t r u c t u r a l f e a t u r e s . I n a d d i t i o n , t h e y h a v e p r o v i d e d m u c h o f o u r i n s i g h t i n t o t h e c h e m i c a l e c o l o g y o f n u d i b r a n c h s . TABLE I: NUDIBRANCH METABOLITES SPECIES METABOLITES COMMENTS Acanthodoris nanaimoensis (Ref. 18) c PC ^-CHO \ These isomeric aldehydes represent three new carbon skeletons. A biogenetic proposal suggests that acanthodoral (11) i s the pre-cursor of nanaimoal (10) and isoacanthodoral 02). nanaimoal CHO ^CHO acanthodoral <J2) isoacanthodoral (12) Adalaria sp. (Ref. 19) Compound (13) i s the major component of a mixture of s t e r o i d a l peroxides. J l TABLE I: continued SPECIES METABOLITES Aldisa sanguinea  cooperi (Ref. 20, 21) Anisodoris n o b i l i s (Ref. 21, 22) dihydroapofarnesal (15) NH cHriV> H H0CH2 ft 0 0 H H doridosine (16) COMMENTS In addition to s t e r o i d a l ketone J^4, i t s analog with an a8-unsaturated acid was also present. Acid _1_4, the major s t e r o i d , has antifeedant properties toward goldfish. I t i s suggested that cholestenone obtained from the sponge Anthroarcuata graceae may be converted by the nudibranch to f i s h -antifeedant J4^. Glyceryl ether 23>, reported for Archidoris montereyensis i s also found in A. sanguinea cooperi. The degraded sesquiterpene aldehyde dihydroapof arnesal (JJ^) i s the p r i n c i p l e odoriferous constituent of the nudibranch. Doridosine (^6) which was i s o l a t e d from the nudibranch's digestive gland, i s water soluble and exhibits a variety of pharmacological a c t i v i t i e s . I t has also been i s o l a t e d from the Australian sponge Tedania d i g i t a t a . TABLE I: continued SPECIES METABOLITES COMMENTS Archidoris montereyensis (Ref. 21, 23) ( ^ r ^ OR 2 / \ H V 7 R 1 = H R 2 = H J 8 Rx = Ac R2 = H J 9 Rx = H R 2 = Ac OH [ I J ) 20 R = H 21 R = AC OH 22 OH 23 Compound 17 and i t s 1 ° and 2° monoacetates are the major metabolites. Glyceride 2£ and glyceryl ether 23 act as feeding deterents to the tide pool sculpin Oligocottus maculosus while 17 i s inactive. Compound 23 has strong a n t i b i o t i c a c t i v i t y and i s also found i n the dietary sponge Halichondria panicea. l l +C-labeled mevalonic acid fed to A. montereyensis i s incorporated into the terpenoid portions of compounds V7 and 20. Thus the nudibranch seems to acquire glycerol derivatives from both dietary sources and de novo biosynthesis. Compounds 1 7 , 1 8 , 20, 22 and 23^  have also been is o l a t e d from the nudibranch Archidoris odhneri while compound 23 has been found i n Aldisa sanguinea cooperi. TABLE I: continued SPECIES METABOLITES COMMENTS Archidoris 0 0 R 2 24 Rx = H R2 = H 25 . R1 = Ac R 2 = H 26 R1 = H R 2 = Ac o H 0 27 H O Farnesic acid glyceride 2A and i t s 1° and 2° monoacetates are the p r i n c i p l e metabolites. The isomeric glyceride 27 i s found at a much lower abundance. Compounds \T_, ^8, 2:0, 22^  and 23, reported for A. montereyensis, are also present i n trace amounts. labeled mevalonic acid fed to the nudibranch i s incorporated into the farnesic acid portion of 24. Glyceride 2A shows a n t i b i o t i c a c t i v i t y against gram p o s i t i v e bacteria but no antifeedant a c t i v i t y towards f i s h . odhneri (Ref. 24) TABLE I: continued SPECIES METABOLITES COMMENTS Cadlina dendrolasin (28) p l e r a p l y s i l l i n - 1 (29) 3_0 microcionin-2 (31) The compounds present i n C. luteomarginata luteomarginata (Ref. 25, 26, 27) extracts show wide v a r i a b i l i t y depending on the location and time of year of c o l l e c t i o n . Many of these metabolites can be traced to dietary sponge sources. Furodysinin (33), pallescensin-A (34) and possibly the other furanosesquiterpenes are obtained from Dysidea amblia, idiadione (37) comes from L e i o s e l l a i d i a and compounds 39 and 40 have been traced to Axin e l l a sp. Albicanol (35) i s known from the liverwort Diplophyllum ablicans and i s present i n the nudibranch primarily as al b i c a n y l acetate (_36) • The source of the degraded terpenoid luteone (38) i s not clea r . While several of these metabolites have been detected i n the gut contents of C. luteomarginata, they are a l l stored i n the dorsum of the nudibranch. Furodysinin, pallescensin-A, idiadione and compounds 39 and 40^  are toxic to goldfis h . They also show varying degrees of antifeedant a c t i v i t y toward goldfish and the sculpin Clinocottus a n a l i s . Albicanyl acetate i s also an antifeedant toward gold-f i s h . The nudibranch Hypselodoris g h i s e l i n i i s known to contain dendrolasin while H. porterae contains furodysinin and H. c a l i f o r n i e n s i s contains dendrolasin, furodysinin and pallescensin-A. TABLE I: continued SPECIES METABOLITES COMMENTS Cadlina luteomarginata (continued) furodysin (32) furodysinin (33) pallescensin-A (34) 35 R = H 36 R = Ac • TABLE I: continued SPECIES METABOLITES COMMENTS Cadlina luteomarginata (continued) 0 0 idiadione (37) O H C jT X A H luteone (38) 39 40 TABLE I: continued COMMENTS A t o t a l of four acetylated derivatives of 4 1 and 4 2 were also reported. These furanoditerpenes are i d e n t i c a l to, or are s t r u c t u r a l l y related to, compounds previously i s o l a t e d from an Australian Spongia sp. Pu'ulenal (43) i s r e a d i l y hydrolyzed to polygodial (55) which i s known to deter predation by f i s h . TABLE I: continued SPECIES METABOLITES Chromodoris maridadilus (Ref. 30) Chromodoris marislae (Ref. 31) nakafuran-8 (44) nakafuran-9 (45) marislin (46) aR= ^0 b R COMMENTS Nakafuran-8 (44) and nakafuran-9 (45) were isolated from the nudibranchs C. maridadilus and Hypselodoris godeffroyana and traced to the dietary sponge Dysidea f r a g i l i s . Both compounds possess antifeedant properties against the common reef fishes Chaetodon spp. The nudibranch Hypselodoris g h i s e l i n i also contains nakafuran-9 and a related methoxy butenolide 67 while Hypselodoris c a l i f o r n i e n s i s contains nakafuran-8. Marislin (4f5) i s the p r i n c i p a l constituent and i t readily rearranges to the sponge metabolite p l e r a p l y s i l l i n - 2 (formally a [3,3] sigmatrophic rearrangement). This close s t r u c t u r a l r e l a t i o n s h i p indicates that there may be chemical a l t e r a t i o n by the nudibranch of a metabolite that i t obtains from i t s d i e t . TABLE I: continued COMMENTS The rearranged diterpene n o r r i s o l i d e (49) was also found i n the south P a c i f i c sponge Dendrilla sp. A biosynthetic proposal for the formation of the norrisane skeleton from the spongian skeleton was presented. A t o t a l of f i v e s c a l a r i n type t e t r a c y c l i c terpenes were i s o l a t e d . Sednolxde (50) i n h i b i t s the growth of the marine bacterium V i b r i o anguillarum. An Australian dorid nudibranch, Chromodoris splendida was also found to contain a mixture of 24-methylscalarins. Similar Cgg s c a l a r i n derivatives have been isolated from sponges of the genus Phyllospongia. TABLE I: continued SPECIES METABOLITES Chromodoris see Chromodoris sedna splendida (Ref. 33) Coryphella OH lineata (Ref. 34, 35) Dendrodoris  grandiflora (Ref. 35) Dendrodoris  k r e b s i i (Ref. 36) polygodial (55) olepupuane (56) COMMENTS A number of polyhydroxylated steriods were isolated from C. li n e a t a and from the hydroid Eudendrium sp. upon which they prey. The major steroid i s compound J53. A similar mixture of polyhydroxylated steroids was also found in the nudibranchs F l a b e l l i n a a f f i n i s and Hervia peregrina. Fa s c i c u l a t i n (54) i s a furanosesterterpene isolated from the digestive gland of D. grandiflora. I t i s known to be a metabolite of the sponge I r c i n i a f a s c i c u l a t a . The nudibranchs D. k r e b s i i , I), nigra and D. tuberculosa were found to contain polygodial (55) and olepupuane (56). Polygodial i s a known f i s h and insect antifeedant while olepupuane was found to i n h i b i t feeding of the P a c i f i c damsel f i s h Dascyllus aruanus. Polygodial has also been isolated from the nudibranch Dendrodoris limbata while olepupuane has been found in D o r i o p s i l l a  albopunctata and D o r i o p s i l l a janaina. TABLE I: continued SPECIES METABOLITES COMMENTS Dendrodoris limbata (Ref. 37, 38) 0 II R-CH-CO /\ H 57. A sesquiterpene with the drimane skeleton was found e s t e r i f i e d to a series of f a t t y acids with varying degrees of unsaturation. These esters were l o c a l i z e d i n the digestive gland while polygodial (55) was found i n extracts of the mantle. Polygodial i s a potent antifeedant against f i s h but the sesquiterpene esters are i n a c t i v e . l^C-labeled mevalonic acid fed to D. limbata is incorporated into polygodial and the sesquiterpene portion of the esters. It i s suggested that the sesquiterpene esters a r i s e from the de t o x i f i c a t i o n of polygodial. Similar sesquiterpene esters have also been isolated from the nudibranchs D o r i o p s i l l a albopunctata and D o r i o p s i l l a janaina. Dendrodoris see Dendrodoris k r e b s i i nigra (Ref. 36) Dendrodoris see Dendrodoris k r e b s i i tuberculosa (Ref. 36) TABLE I: continued SPECIES METABOLITES Diaulula sandiegensis (Ref. 39, 40) NH 2 H H0CH 2 isoguanosine (58) ft 0 0 H H 59 D o r i o p s i l l a see Dendrodoris limbata albopunctata Dendrodoris k r e b s i i (Ref. 36) Do r i o p s i l l a see Dendrodoris limbata janaina (Ref. 36) Dendrodoris kr e b s i i F l a b e l l i n a see Coryphella lineata a f f i n i a (Ref. 34, 35) Glossodoris g r a c i l i s (Ref. 35) COMMENTS Isoguanosine (58) was iso l a t e d from a water extract of the nudibranch's digestive gland. It i s a purine riboside and exhibits a variety of pharmacological a c t i v i t i e s . A series of nine s t r a i g h t chain chlorinated acetylenes of general structure 59 were also isolated from skin extracts. Compound 59 shows antimicrobial a c t i v i t y against several bacteria and a yeast. Longifolin (60) i s a known metabolite from the sponge P l e r a p l y s i l l a s p i n i f e r a . It i s a deterent to feeding by the marine f i s h Chromis chromis and has also been i s o l a t e d from the mantle and digestive gland of the nudibranch Glossodoris valenciennesi. TABLE I: continued SPECIES METABOLITES COMMENTS Glossodoris t r i c o l o r (Ref. 41) AcO 0-A Furoscalarol (61) and deoxoscalarin (62) were also i s o l a t e d from the dietary sponge Cacospongia m o l l i o r . Both compounds show antifeedant properties toward the marine f i s h Chromis chromis. AH furoscalarol (61) HO Aco "y-o T J AH deoxoscalarin (62) Glossodoris see Glossodoris g r a c i l i s valenciennesi (Ref. 41) Hervia see Coryphella lineata peregrina (Ref. 34, 35) Hypselodoris agassizi (Ref. 42) Agassizin (63) i s s t r u c t u r a l l y related to the sponge metabolite pallescensin-G. agassizin (63) TABLE I: continued SPECIES METABOLITES COMMENTS Hypselodoris Euryfuran (64) is a metabolite of the sponge californiensis Euryspongia sp. Four other sponge (Ref. 42) Cu metabolites, dendrolasin (28), nakafuran-8 (44), furodysinin (33) and pallescensin-A euryfuran (64) (34) were also isolated from H. californiensis. Hypselodoris porterae is also known to contain euryfuran. Hypselodoris Spiniferin-2 (65) is a known metabolite of danielae the sponge Pleraplysilla spinifera. (Ref. 29) spiniferin-2 (65) Hypselodoris o In addition to ghiselinin (66), H. ghiselini ghiselini contains dendrolasin (28), nakafuran-9 (45) (Ref. 42) and a methoxy butenolide 6>7 related to f i Y nakafuran-9. /\ H ghiselinin (66) X H > ° ' ^ M e O 67 TABLE I: continued SPECIES METABOLITES COMMENTS Hypselodoris see Chromodoris maridadilus godeffroyana (Ref. 42) Hypselodoris see Hypselodoris c a l i f o r n i e n s i s and porterae Cadlina luteomarginata (Ref. 42) Melibe Compound 68 i s the p r i n c i p a l odoriferous leonina constituent of M. leonina. This degraded (Ref. 43) monoterpene i s also present as a carboxylic ea 0 acid. Petodoris HCiC-CH-CH = CH(CH.) CH = CH"C = C-CH-C5C(CH.) -CH=CH-CH-C = CH A series of high molecular weight straight atromaculata OH I I OH OH chain polyacetylenes (C^g, C^g, C 5 2 a n d C5g) (Ref. 44) of general structure 69 were is o l a t e d from 69 the nudibranch's digestive gland and from the dietary sponge Petrosia f i c i f o r m i s . P h y l l i d i a w u Axisonitrile-1 (70) originates i n the dietary p u l i t z e r i T I , sponge Axinella cannabina. I t i s inactive as (Ref. 41) a f i s h antifeedant but i s toxic to f i s h at NC I concentrations as low as 8 ppm. axi s o n i t r i l e - 1 (70) TABLE I: continued SPECIES METABOLITES P h y l l i d i a  varicosai (Ref. 45, 46) Phyllodesmium l o n g i c i r r a (Ref. 47) isocyanopupukeanane socyanopupukeanane ( < trocheliophorol (73) COMMENTS The isocyanosesquiterpenes _71 and 72 are sequestered from the dietary sponge Hymeniacidon sp. They are the active constituents of the nudibranch's mucous skin secretion that i s l e t h a l to f i s h and crustaceans. Trocheliophorol (7_3) i s obtained from the dietary soft coral Sarcophyton trocheliophorium and i s concentrated i n the cerata of the nudibranch. Two other cembranoid diterpenes related to trocheliophoral were also found i n both the nudibranch and the soft c o r a l . TABLE I: continued SPECIES METABOLITES Polycera  t r i c o l o r (Ref. 21) Roboastra t i g r i s (Ref. 48) Tamb j e  abdere (Ref. 48) Tambj e  e l i o r a (Ref. 48) Triopha catalinae (Ref. 49) see Triopha catalinae see Tambje abdere OMe tambjamine-A (74) see Tambje abdere 0 NHL 0 1 2 H triophamine (75) COMMENTS Tambjamine-A (74) and three related bipyrroles were traced to the dietary bryozoan Sessibugula translucens. Tambjamines B, C and D d i f f e r from Tambjamine A by the presence of bromine and/or an isobutylamine f u n c t i o n a l i t y . These compounds shown a n t i b i o t i c a c t i v i t y and i n h i b i t c e l l d i v i s i o n of f e r t i l i z e d sea urchin eggs. They have also been found in the nudibranchs Tambje e l i o r a and Roboastra t i g r i s . Triophamine (75) i s present primarily i n the mucous secretion of T. catalinae but does not deter feeding by g o l d f i s h . I t has also been isolated from the nudibranch Polycera t r i c o l o r . 2 8 . T h e f i r s t n a t u r a l p r o d u c t s r e p o r t e d f r o m a n u d i b r a n c h w e r e t h e r e a r r a n g e d s e s q u i t e r p e n e 9 - i s o c y a n o p u p u k e a n a n e ( 7 ^ i ) 4 5 a n ( j i t s 2 - i s o c y a n o i s o m e r 7 2 ^ 6 • T h e s e t w o i s o n i t r i l e s a r e t h e a c t i v e c o n s t i t u e n t s o f a t o x i c m u c o u s s e c r e t i o n p r o d u c e d b y P h y l l i d i a v a r i c o s a a n d w e r e b o t h f o u n d i n t h e d i e t a r y s p o n g e H y m e n i a c i d o n s p . T h i s c l e a r l y e s t a b l i s h e d t h a t n u d i b r a n c h s a r e c a p a b l e o f s e q u e s t e r i n g a n d s t o r i n g s p o n g e m e t a b o l i t e s f o r u s e a s d e f e n s i v e a l l o m o n e s . B i o l o g i c a l l y a c t i v e i s o c y a n o s e s q u i t e r p e n e s o f s p o n g e o r i g i n a r e a l s o k n o w n f r o m P h y l l i d i a p u l i t z e r i 1 * 1 a n d C a d l i n a  l u t e o m a r g i n a t a 2 5 . T h e s e c o m p o u n d s h a v e r e g u l a r i s o p r e n o i d s k e l e t o n s a n d a r e t o x i c t o f i s h . I n m a r i n e s p o n g e s , i s o c y a n o c o m p o u n d s o f t e n o c c u r a l o n g w i t h t h e i r i s o t h i o c y a n a t e a n d f o r m a m i d e d e r i v a t i v e s . E x c e p t f o r t h e i s o t h i o c y a n a t e a n a l o g 4 0 f r o m C . l u t e o m a r g i n a t a , s u c h d e r i v a t i e s h a v e n o t b e e n r e p o r t e d f r o m n u d i b r a n c h s . A n o t h e r c o m m o n f u n c t i o n a l i t y i n n u d i b r a n c h s e q u i t e r p e n e s i s t h e f u r a n r i n g . A s e r i e s o f s e v e n r e l a t e d f u r a n o s e s q u i t e r p e n e s h a s b e e n i s o l a t e d f r o m C . l u t e o m a r g i n a t a 2 5 . T h e s e c o n s t i t u e n t s a r e l o c a l i z e d i n t h e n u d i b r a n c h 1 s m a n t l e a n d s e v e r a l o f t h e m h a v e d e m o n s t r a t e d t o x i c i t y o r a n t i f e e d a n t a c t i v i t y t o w a r d f i s h . S e s q u i t e r p e n e s , o r t h e i r d e r i v a t i v e s , w h i c h p o s s e s s a f u r a n r e s i d u e a r e a l s o k n o w n f r o m C h r o m o d o r i s m a r i s l a e 3 1 , G l o s s o d o r i s g r a c i l i s 3 5 a n d a n u m b e r o f H y p s e l o d o r i s s p e c i e s 2 9 ' 1 * 2 . A l l o f t h e s e f u r a n - c o n t a i n i n g c o m p o u n d s c a n b e t r a c e d t o a s p o n g e s o u r c e o r a r e s t r u c t u r a l l y r e l a t e d t o k n o w n s p o n g e p r o d u c t s . C i r c u m s t a n t i a l e v i d e n c e s u g g e s t s t h a t C . m a r i s l a e c a n c h e m i c a l l y t r a n s f o r m t h e s p o n g e m e t a b o l i t e p l e r a p l y s i l l i n - 2 (7[6) 5 0 i n t o m a r i s l i n (46) . T h e s e t w o f u r a n c o m p o u n d s a r e f o r m a l l y r e l a t e d b y a [3,3] s i g m a t r o p i c r e a r r a n g e m e n t a n d t h e t r a n s f o r m a t i o n o f 46 t o J76 o c c u r s s p o n t a n e o u s l y d u r i n g r o u t i n e h a n d l i n g 3 1 . W h e n a c o n c l u s i v e p r e d a t o r - p r e y r e l a t i o n s h i p h a s b e e n e s t a b l i s h e d , t h e e x t r a c t s o f a n u d i b r a n c h a n d i t s s p o n g e p r e y c a n s t i l l s h o w s u b s t a n t i a l v a r i a n c e . S o m e m e t a b o l i t e s a r e r e t a i n e d b y t h e n u d i b r a n c h w h i l e o t h e r s a r e a b s e n t o r i n r e d u c e d c o n c e n t r a t i o n . C h r o m o d o r i s m a r i d a d i l u s a n d H y p s e l o d o r i s g o d e f f r a y a n a a r e k n o w n t o o b t a i n n a k a f u r a n - 8 (44) a n d n a k a f u r a n - 9 (4_5) f r o m t h e s p o n g e D y s i d e a  f r a g i l i s 3 2 . T h e s e t w o f u r a n o s e s q u i t e r p e n e s s h o w a n t i f e e d a n t a c t i v i t y a g a i n s t f i s h . A n o t h e r D y s i d e a s e s q u i t e r p e n e , u p i a l (77) h a s n o r e p e l l e n t p r o p e r t i e s a n d i s n o t p r e s e n t i n t h e n u d i b r a n c h s . T h e n u d i b r a n c h s a p p a r e n t l y r e t a i n s p o n g e m e t a b o l i t e s f r o m w h i c h t h e y d e r i v e b e n e f i t a n d r e j e c t t h o s e t h a t a r e i n a c t i v e . 30. 77 Carbon ske l e t ons observed i n the ma jo r i t y o f nud ibranch s e s q u i t e r p e n e s are w e l l known from o the r s o u r c e s . Compounds p r e v i o u s l y d i s c u s s e d from P_. v a r i c o s a , H. gode f f r oyana and C. m a r i d a d i l u s , i n a d d i t i o n to the i s o m e r i c a ldehydes nanaimoal (_10) , a c an thodo ra l (1_1) and i s o a c a n t h o d o r a l (12) f rom A c a n t h o d o r i s n a n a i m o e n s i s 1 8 , r e p r e s e n t n o t a b l e e x c e p t i o n s . The d r imane c a r b o n s k e l e t o n o c c u r s i n t e r r e s t r i a l and m a r i n e n a t u r a l p r o d u c t s , i n c l u d i n g nud ibranch c o n s t i t u e n t s such as eu r y fu ran olepupuane (56) 3 6 , p u ' u l e n a l (£3) 2 9 and a l b i c a n o l (_35) 2 6 . Whi le the s i g n i f i c a n c e o f the carbon s k e l e t o n i s not c l e a r , i t i s i n t e r e s t i n g t o note t h a t , many drimane d e r i v a t i v e s e x h i b i t a n t i f e e d a n t or o the r b i o l o g i c a l a c t i v i t i e s . The a c q u i s i t i o n o f secondary m e t a b o l i t e s from d i e t a r y sources i s f i r m l y e s t a b l i s h e d f o r nud ib r anchs , however Cimino et a l . 3 8 r e c e n t l y demonstrated t h a t Dendrodor i s  l imba t a i s capab le of d_e novo s y n t h e s i s of a drimane s e s q u i t e r p e n e . l l * C - l a b e l e d meva lon ic a c i d fed t o the n u d i b r a n c h was i n c o r p o r a t e d i n t o t h e d i a l d e h y d e p o l y g o d i a l (5_5) . P o l y g o d i a l , l o c a l i z e d i n t h e n u d i b r a n c h 1 s m a n t l e , i s a p o t e n t a n t i f e e d a n t a g a i n s t f i s h . I t has p r e v i o u s l y b een i s o l a t e d f r o m a number o f h i g h e r p l a n t s where i t s e r v e s as an i n s e c t a n t i f e e d a n t 5 1 ; The m i x e d e s t e r s 5_7, i n w h i c h a d r i m a n e s e s q u i t e r p e n e i s e s t e r i f i e d t o a s e r i e s o f f a t t y a c i d s , were a l s o f o u n d t o c o n t a i n t h e l i +C l a b e l . I t was s u g g e s t e d t h e y a r i s e f r o m t h e d e t o x i f i c a t i o n o f p o l y g o d i a l 2 8 , b u t t h i s r e l a t i o n s h i p r e m a i n s ambiguous. P o l y g o d i a l h a s b e e n i s o l a t e d f r o m t h r e e o t h e r s p e c i e s o f D e n d r o d o r i s w h i c h d i d n o t c o n t a i n t h e m i x e d e s t e r s and two s p e c i e s o f D o r i o p s i 1 1 a c o n t a i n t h e e s t e r s b u t n o p o l y g o d i a l 3 6 . W h i l e t h e y a r e n o t d i r e c t l y r e l a t e d t o t h e e s t e r s 57_, f a r n e s i c a c i d g l y c e r i d e s f r o m A r c h i d o r i s  odhneri 2*» a r e a n o t h e r example o f n u d i b r a n c h s e s q u i t e r p e n e s o f mixed b i o g e n e s i s . D i t e r p e n e s f o u n d i n n u d i b r a n c h s i n c l u d e f u r a n o d i t e r p e n e s o f s p o n g e o r i g i n f r o m C a s e l l a  a t r o m a r g i n a t a 2 8 , C a d l i n a l u t e o m a r g i n a t a 2 5 and H y p s e l o d o r i s  g h i s e l i n i 1 * 2 . N o r r i s o l i d e (4^), a r e a r r a n g e d d i t e r p e n e f r o m C h r o m o d o r i s n o r r i s i 3 2 , i s a l s o o b t a i n e d f r o m a sponge w h i l e a number o f c e m b r a n o i d s f r o m P h y l l o d e s m i u m l o n g i c i r r a * * 7 were t r a c e d t o a d i e t a r y s o f t c o r a l . S e s t e r t e r p e n e s f r o m n u d i b r a n c h s f a l l i n t o two g r o u p s ; s c a l a r i n d e r i v a t i v e s and s t r a i g h t - c h a i n f u r a n o compounds. T h e s e m e t a b o l i t e s a p p a r e n t l y o r i g i n a t e i n d i e t a r y s p o n g e s o r 32 . r e s u l t from t r a n s f o r m a t i o n of sponge products. A s e r i e s of C 2 6 methylated s c a l a r i n s , i n c l u d i n g the novel d e r i v a t i v e s e d n o l i d e (5_0), was f o u n d i n C h r o m o d o r i s s e d n a 3 3 . B i o l o g i c a l a c t i v i t y of s e s t e r t e r p e n e s i s evidenced by the i c h t h y o t o x i c i t y o f i d i a d i o n e ( 3 _ 7 ) 2 5 and t h e f e e d i n g d e t e r r e n c e of two s c a l a r i n s from G l o s s o d o r i s t r i c o l o r 1 * 1 . Degraded terpenes are represented by the C 9 aldehyde 68 from Melibe l e o n i n a 1 * 3 and C 2 3 luteone (3_8) from C a d l i n a  l u t e o m a r g i n a t a 2 7 . Nudibranchs are a l s o the source of i n t e r e s t i n g s t e r o i d a l p e r o x i d e s 1 9 and p o l y h y d r o x y l a t e d s t e r o i d s 3 1 * . B i l e a c i d d e r i v a t i v e s from A l d i s a sanguinea  c o o p e r i 2 0 p o s s i b l y a r i s e from chemical m o d i f i c a t i o n of a d i e t a r y compound. I t i s suggested t h a t A 4-cholesta - 3-one, obtained from the sponge Anthroarcuata graceae, may be converted by the nudibranch to the observed b i l e a c i d s . A growing number of non-terpenoid c o n s t i t u e n t s have r e c e n t l y b e e n r e p o r t e d . A s e r i e s o f c h l o r i n a t e d p o l y a c e t y l e n e s were obtained from D i a u l u l a s a n d i e g e n s i s 1 * 0 , and P e l t o d o r i s atromaculata 1* ** p r o v i d e d another s e r i e s of a c e t y l e n i c a l c o h o l s . Tambjamine-A (7_4) 8 and three r e l a t e d b i p y r r o l e s occur i n s e v e r a l nudibranch s p e c i e s and o r i g i n a t e i n a d i e t a r y bryozoan. Other nitrogenous m e t a b o l i t e s i n c l u d e novel p u r i n e r i b o s i d e s from A n i s o d o r i s n o b i l i s 2 2 and D i a l u l a s a n d i e g e n s i s 3 9 . The a r r a y o f n a t u r a l p r o d u c t s i d e n t i f i e d from n u d i b r a n c h s i s t r u e l y r e m a r k a b l e . L i k e t h e o t h e r 33. o p i s t h o b r a n c h s , nud ibranchs have deve loped the a b i l i t y t o o b t a i n complex, mo lecu les from t h e i r d i e t and may be ab le to c h e m i c a l l y modify them f o l l o w i n g i n g e s t i o n . These organisms are s e l e c t i v e i n the d i e t a r y compounds they concen t r a t e and s t o r e . In some cases nud ibranchs are d i r e c t l y r e s p o n s i b l e f o r the b i o s y n t h e s i s o f secondary m e t a b o l i t e s . A n t i f e e d a n t a c t i v i t y a s s o c i a t e d w i th nud ibranch c o n s t i t u e n t s suppor t s t h e i r a s c r i b e d f u n c t i o n as defense a l l omones . A d a p t a t i o n o f a chemica l defense has apparenty d e t e r r e d p r e d a t i o n and a l l owed nud ibranchs to e l i m i n a t e the me t abo l i c l oad r e q u i r e d t o produce and ma in t a i n a p r o t e c t i v e s h e l l . P l a t e 1. T r i o p h a c a t a l i n a e . P h o t o g r a p h e r : S i m o n F r a s e r U n i v e r s i t y . Ron L o n g , C H E M I C A L S T U D I E S OF T R I O P H A C A T A L I N A E I . I S O L A T I O N A N D 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 O P H A M I N E ( 7 5 ) T r i o p h a c a t a l i n a e ( r e c e n t l y T r i o p h a c a r p e n t e r i ) 2 i s a n u d i b r a n c h o f t h e s u b o r d e r d o r i d a c e a . I t i s b e a u t i f u l l y d e c o r a t e d w i t h o r a n g e t i p p e d r h i n o p h o r e s , g i l l s a n d p a p i l l a e ( f i n g e r l i k e p r o c e s s e s e x t e n d i n g f r o m t h e d o r s u m ) p r o j e c t i n g f r o m a w h i t e b o d y s u r f a c e . W i t h a n a v e r a g e l e n g t h o f a p p r o x i m a t e l y 7 0 mm, T . c a t a l i n a e ' s w h i t e , o r a n g e - s p o t t e d b o d y i s q u i t e c o n s p i c u o u s i n t h e m a r i n e e n v i r o n m e n t . T h i s s t r i k i n g n u d i b r a n c h f e e d s o n b r y o z o a n s 1 * a n d i s f o u n d a l o n g t h e P a c i f i c c o a s t o f N o r t h A m e r i c a f r o m V a n c o u v e r I s l a n d , B r i t i s h C o l u m b i a t o S a n D i e g o , C a l i f o r n i a 2 . D u e t o i t s r e l a t i v e a b u n d a n c e , c o n s p i c u o u s c o l o r a t i o n a n d a p p a r e n t l a c k o f p r e d a t i o n , a n i n v e s t i g a t i o n o f t h e n a t u r a l p r o d u c t s f r o m T r i o p h a c a t a l i n a e w a s i n i t i a t e d i n 1 9 8 0 . T h e s p e c i m e n s u s e d i n t h i s s t u d y w e r e c o l l e c t e d i n B a r k l e y S o u n d , a l o n g t h e w e s t c o a s t o f V a n c o u v e r I s l a n d . A l t h o u g h f o u n d i n a n a s s o r t m e n t o f h a b i t a t s , T . c a t a l i n a e w e r e m o s t a b u n d a n t o n e x p o s e d r o c k y o u t c r o p p i n g s w h i c h s u p p o r t l a r g e b r y o z o a n c o m m u n i t i e s . T h i s d o r i d n u d i b r a n c h w a s c o l l e c t e d t h r o u g h o u t t h e y e a r f r o m d e p t h s o f 1 - 1 3 m e t e r s . F r e s h l y c o l l e c t e d s p e c i m e n s o f T r i o p h a c a t a l i n a e w e r e r a p i d l y i m m e r s e d i n m e t h a n o l a n d s t o r e d a t r o o m t e m p e r a t u r e . 36. The s o l v e n t was decanted and the nudibranchs washed with a d d i t i o n a l methanol. E v a p o r a t i o n _in vacuo of the combined e x t r a c t s produced an aqueous suspension t h a t was p a r t i t i o n e d between c h l o r o f o r m and water. The c h l o r o f o r m s o l u b l e m a t e r i a l was c o n c e n t r a t e d t o a b r o w n i s h o i l composed p r i m a r i l y of f a t s , pigments, s t e r o i d s and the compound of i n t e r e s t , triophamine (75)1*9. The o i l was f r a c t i o n a t e d by s i l i c a g e l f l a s h chromatography u s i n g step g r a d i e n t s of e t h y l a c e t a t e i n hexane. N o n - p o l a r l i p i d s were e l u t e d w i t h 5% e t h y l acetate/hexane. Triophamine (7j>) and s e v e r a l b r i g h t orange pigments were e l u t e d with 15% e t h y l acetate/hexane while the s t e r o i d f r a c t i o n was e l u t e d w i t h more p o l a r s o l v e n t mixtures. A t t e n t i o n was drawn to triophamine by i t s s t r o n g UV absorbance and the presence of i n t e r e s t i n g s i g n a l s i n the 'H NMR o f t h e c r u d e f r a c t i o n from t h e f l a s h column. Triophamine was f u r t h e r p u r i f i e d t o a l i g h t y e l l o w o i l by p r e p a r a t i v e t h i n - l a y e r chromatography (1:1 h e x a n e / d i e t h y l ether, Rf = 0.5). Triophamine (T5) i s o p t i c a l l y a c t i v e (COCDD -7.0° (C 37. 1.7, MeOH)) a n d i t c o n t a i n s a UV c h r o m o p h o r e w i t h a ^max = 2 5 0 n m ( £ 12000, MeOH). T r e a t m e n t w i t h a c i d c a u s e s a h y p s o c h r o m i c s h i f t i n t h e X m a x ^o 2-*-4 n m ( £ 14500, MeOH/HCl), w h i l e t r e a t m e n t w i t h b a s e (MeOH/NaOH) c a u s e s t r i o p h a m i n e t o decompose t o a new s u b s t a n c e w i t h a \ m a x = 232 nm ( T 1 / 2 = 30 min, e 10000). T h e m o l e c u l a r f o r m u l a o f t r i o p h a m i n e {75^) was e s t a b l i s h e d as C 2 1 H 3 7 N 3 0 2 b y e l e c t r o n - i m p a c t h i g h r e s o l u t i o n mass s p e c t r o m e t r y (M +, m/z 363.2885, c a l c ' d 3 63.2885). M a j o r f r a g m e n t i o n s r e s u l t i n g f r o m l o s s o f CH 3 (m/z 348), C 2 H 5 (m/z 334), C 6 H 1 Q (m/z 281) and C g H 1 7 (m/z 238) r e s i d u e s were a l s o o b s e r v e d . E x a m i n a t i o n o f t h e 'H a n d 1 3 C NMR s p e c t r a o f t r i o p h a m i n e (7_5) l e d t o t h e c o n c l u s i o n t h a t i n t h e NMR t i m e s c a l e t h e m o l e c u l e must e x h i b i t an e l e m e n t o f symmetry. O n l y 11 s i g n a l s were o b s e r v e d i n t h e 1 3 C NMR s p e c t r u m and t h e 'H NMR s p e c t r u m showed w e l l r e s o l v e d r e s o n a n c e s f o r o n l y 17 n o n e x c h a n g e a b l e p r o t o n s ( T a b l e I I ) . When t h e 'H NMR s p e c t r a were o b t a i n e d w i t h DMSO-d g as s o l v e n t one a d d i t i o n a l e x c h a n g e a b l e p r o t o n a t 6 9 . 4 and a v e r y b r o a d e x c h a n g e a b l e s i g n a l t h a t i n t e g r a t e d f o r l e s s t h a n one p r o t o n a t 11 ppm was o b s e r v e d . I t was a p p a r e n t t h a t e a c h 'H r e s o n a n c e must a c c o u n t f o r d o u b l e t h e number o f p r o t o n s i n d i c a t e d by i n t e g r a t i o n . T h u s , t r i o p h a m i n e (7_5) c o n t a i n s 3 e x c h a n g e a b l e and 34 38. TABLE I I . *H and 1 3 C NMR d a t a f o r T r i o p h a m i n e (75) C a r b o n *H NMR,a 6 1 3 C N M R , b 6 1,1* 185.6 ( s ) 2,2' 2.30-2.48 (m, IH) 50.3 (d) 3,3' 2.12 (dd, J = 6, 13 Hz, IH) 40.1 ( t ) 2.30-2.42 (m, IH) 4,4' 140.4 (s ) 5,5' 5.21 ( q , J = 7 Hz, IH) 120.5 (d) 6,6' 1.56 (d, J = 7 Hz, 3H) 12.1 ( q ) d 7,7' 2.01 (q, J = 7 Hz, 2H) 23.2 ( t ) e 8,8' 0.95 ( t , J = 7 Hz, 3H) 12.9 ( q ) d 9,9' 1.46-1.67 (m, 2H) 26.1 ( t ) e 10,10' 0.91 ( t , J = 7 Hz, 3H) 13.1 ( q ) d 11 158.9 ( s ) NH^ 9.4 ( b r , IH) NH C 11.0 ( b r , 0.5H) a 400 MHz, CDC1 3 D 100 MHz, a c e t o n e - d 6 c 100 MHz, DMS0-d 6 d May be r e v e r s e d . e May be r e v e r s e d . 3 9 . n o n e x c h a n g e a b l e p r o t o n s . I n a d d i t i o n , 1 0 o f t h e 1 1 r e s o n a n c e s o b s e r v e d i n t h e 1 3 C NMR s p e c t r u m m u s t e a c h a c c o u n t f o r 2 c a r b o n s . A 1 3 C S F O R D NMR e x p e r i m e n t ( F i g u r e 7 ) c l e a r l y s h o w e d s i g n a l s f o r 3 m e t h y l g r o u p s , 3 m e t h y l e n e c a r b o n s , a m e t h i n e c a r b o n a n d a t r i s u b s t i t u t e d o l e f i n . T h e s e o b s e r v a t i o n s a n d a f a c i l e l o s s o f C g H 1 7 i n t h e m a s s s p e c t r u m d e m o n s t r a t e d t h a t t r i o p h a m i n e c o n t a i n s t w o i d e n t i c a l 9 - c a r b o n s e g m e n t s , e a c h w i t h 1 7 n o n e x c h a n g e a b l e p r o t o n s . T h e s t r u c t u r e o f t h e C g H 1 7 r e s i d u e w a s a s s i g n e d b y a n a l y s i s o f ' H NMR s p e c t r a a n d a s e r i e s o f p r o t o n d e c o u p l i n g e x p e r i m e n t s . T A B L E I I I . ' H N M R a D e c o u p l i n g S t u d i e s o f T r i o p h a m i n e ( 7 5 ) R e s o n a n c e s I r r a d i a t e d O b s e r v e d S p e c t r a l C h a n g e s 6 1 . 4 6 - 1 . 6 7 6 0 . 9 1 ( t ) s i n g l e t 5 . 2 1 ( q ) -> s i n g l e t 2 . 0 1 0 . 9 5 ( t ) -> s i n g l e t 2 . 1 2 2 . 3 0 - 2 . 4 8 -y s h a r p e n u p 2 . 3 0 - 2 . 4 8 2 . 1 2 ( d , d ) -»• s i n g l e t 2 . 4 3 2 . 1 2 ( d , d ) -> ( d , J = 1 3 H z ) a 4 0 0 M H z , C D C 1 3 T h e 1 3 C S F O R D NMR e x p e r i m e n t h a d a l r e a d y e s t a b l i s h e d t h e p r e s e n c e o f a t r i s u b s t i t u t e d d o u b l e b o n d . A n o l e f i n i c m e t h y l s i g n a l a t 6 1 . 5 6 ( 3 H , d , J = 7 H z ) c o u p l e d t o a 40. o n e - p r o t o n q u a r t e t a t 5.21 (J = 7Hz) ppm i n d i c a t e d a m e t h y l g r o u p and a p r o t o n were a t t a c h e d t o t h e same c a r b o n o f t h e o l e f i n . A m e t h y l e n e r e s o n a n c e a t 62.01 (2H, q, J = 7Hz) c o u p l e d t o a t h r e e - p r o t o n t r i p l e t a t 0.95 (J = 7Hz) ppm r e v e a l e d t h a t t h e t h i r d o l e f i n i c s u b s t i t u e n t was an e t h y l g r o u p . An a l i p h a t i c e t h y l r e s i d u e on t h e h y d r o c a r b o n c h a i n was e v i d e n c e d by a m e t h y l t r i p l e t a t 60.91 (J = 7Hz) c o u p l e d t o a t w o - p r o t o n m u l t i p l e t a t 1.46-1.67 ppm. C a t a l y t i c h y d r o g e n a t i o n (Pd/C, EtOH) o f t r i o p h a m i n e (75) gave t e t r a h y d r o t r i o p h a m i n e (7JB) . The 'H NMR s p e c t r u m o f 78 ( F i g u r e 10) r e v e a l e d t h a t r e s o n a n c e s f o r a p a i r o f g e m i n a l m e t h y l e n e p r o t o n s o b s e r v e d a t 62.12 (dd, J = 6, 13Hz, IH) and 2.30-2.42 (m, IH) ppm i n t r i o p h a m i n e h a d u n d e r g o n e an u p f i e l d s h i f t . T h i s e s t a b l i s h e d t h a t a m e t h y l e n e g r o u p was a t t a c h e d t o t h e e t h y l s u b s t i t u e d c a r b o n o f t h e o l e f i n . A p r o m i n a n t peak i n t h e mass s p e c t r u m o f _78 a t m/z 283, r e s u l t i n g f r o m a M c L a f f e r t y r e a r r a n g e m e n t , s u p p o r t e d t h i s a s s i g n m e n t . A o n e - p r o t o n m u l t i p l e t d e s h i e l d e d t o 62.37 ppm i n t e t r a h y d r o t r i o p h a m i n e i n d i c a t e d t h a t t h e h y d r o c a r b o n c h a i n was a t t a c h e d t o t h e r e m a i n d e r o f t h e m o l e c u l e v i a a m e t h i n e m o i e t y . D e c o u p l i n g e x p e r i m e n t s w i t h n a t u r a l t r i o p h a m i n e showed t h a t t h e m e t h i n e p r o t o n was c o u p l e d t o b o t h t h e a l l y l i c m e t h y l e n e p r o t o n s a t 62.12 and 2.30-2.42 ppm and t h e m e t h y l e n e p r o t o n s o f t h e a l i p h a t i c e t h y l g r o u p . T h i s l e d t o t h e c o n c l u s i o n t h a t t r i o p h a m i n e c o n t a i n s two 9 - c a r b o n r e s i d u e s o f g e n e r a l s t r u c t u r e 79, 41. w h i c h was c o n s i s t e n t w i t h t h e o b s e r v e d mass s p e c t r a l f r a g m e n t a t i o n p a t t e r n ( s e e F i g u r e 4 ) . The g e o m e t r y a b o u t t h e d o u b l e bond was e l u c i d a t e d by means o f a d i f f e r e n c e N u c l e a r O v e r h a u s e r Enhancement ( N O E ) 5 2 e x p e r i m e n t ( F i g u r e 9 ) . I r r a d i a t i o n o f t h e o l e f i n p r o t o n a t 65.21 c a u s e d a p o s i t i v e e n h a n c e m e n t o f t h e a l l y l i c m e t h y l e n e p r o t o n a t 2.12 and a n e g a t i v e enhancement o f t h e p r o t o n a t 2.30-2.42 ppm. N e g a t i v e h o m o n u c l e a r N O E 1s o f p r o t o n s i g n a l s a r e n o t uncommon i n s m a l l m o l e c u l e s . C e r t a i n g e o m e t r i c a l a r r a n g e m e n t s o f s p i n s c a n l e a d t o d i r e c t p o s i t i v e N OE 1s and i n d i r e c t n e g a t i v e N OE 1s i n t h e same m o l e c u l e 5 2 * 3 . No enhancement was o b s e r v e d i n t h e o l e f i n i c e t h y l r e s o n a n c e s . T h i s d e m o n s t r a t e d t h a t t h e o l e f i n p r o t o n c o n t r i b u t e s s i g n i f i c a n t l y t o t h e r e l a x a t i o n o f t h e m e t h y l e n e i n t h e main h y d r o c a r b o n s u b s t i t u e n t . S i n c e t h e m a g n i t u d e o f t h i s e f f e c t i s i n v e r s e l y p r o p o r t i o n a l t o t h e s i x t h power o f t h e i n t e r n u c l e a r d i s t a n c e 5 3 , t h e o l e f i n p r o t o n and t h e main c h a i n m e t h y l e n e g r o u p must be c i s . The o l e f i n t h e r e f o r e h a s 0 N H 2 0 78 79 4 2 . a n ( E ) c o n f i g u r a t i o n . T h e r e m a i n i n g p o r t i o n o f t r i o p h a m i n e (7_5) c o n s i s t e d o f C 3 H 3 N 3 0 2 a n d i t h a d t o i n c o r p o r a t e t h r e e u n i t s o f u n s a t u r a t i o n . T h e 1 3 C NMR r e s o n a n c e o b s e r v e d a t 6 1 5 8 . 9 p p m w a s h i g h l y s u g g e s t i v e o f a g u a n i d i n e s u b s t i t u e n t . A n i n f r a r e d a b s o r p t i o n a t 1 7 0 0 c m - 1 ( F i g u r e 8 ) a n d t h e r e m a i n i n g 1 3 C r e s o n a n c e a t 6 1 8 5 . 6 p p m s u g g e s t e d t h e p r e s e n c e o f t w o c a r b o n y l c a r b o n s . L o w t e m p e r a t u r e ' H NMR s t u d i e s s h o w e d s u b s t a n t i a l s i g n a l b r o a d e n i n g a t - 4 0 ° C w h i c h i n c r e a s e d a t - 6 0 ° C . T h e e l e m e n t o f s y m m e t r y o b s e r v e d o n t h e NMR t i m e s c a l e a t r o o m t e m p e r a t u r e w a s l o s t a s t h e t e m p e r a t u r e w a s l o w e r e d . F r o m t h e s e o b s e r v a t i o n s i t w a s c l e a r t h a t t r i o p h a m i n e c o n s i s t e d o f a d i a c y l a t e d g u a n i d i n e m o i e t y . M o n o a c y l g u a n i d i n e s a r e k n o w n t o e x i s t a l m o s t e x c l u s i v e l y a s t h e i r a c y l i m i n e t a u t o r a e r 5 " * . T h e s y m m e t r y o b s e r v e d i n t h e NMR s p e c t r a o f t r i o p h a m i n e r e s u l t s f r o m r a p i d e q u i l i b r i u m b e t w e e n i t s t w o a c y l i m i n e t a u t o m e r s . R 0 N H 2 0 H N H 2 0 R N N R H F i g u r e 3 . A c y l i m i n e t a u t o m e r s o f t r i o p h a m i n e ( 7 5 ) . 4 3 . T o t e s t t h i s c o n c l u s i o n d i a c e t y l g u a n i d i n e 8_0 w a s p r e p a r e d b y r e a c t i n g g u a n i d i n e c a r b o n a t e w i t h a c e t i c a n h y d r i d e . T h e UV c h a r a c t e r i s t i c s o f c o m p o u n d 8 0 [ \ m a x = 2 4 8 nm (e 1 6 8 0 0 , MeOH) a n d \ m a x = 2 1 2 nm U 1 9 9 0 0 , M e O H / H C l ) ] w e r e c o m p a r a b l e t o t h o s e o b s e r v e d f o r t r i o p h a m i n e (7_5) . I t u n d e r w e n t r a p i d b a s e - c a t a l y z e d h y d r o l y s i s ( M e O H / N a O H , r o o m t e m p e r a t u r e ) t o g i v e m o n o a c e t y l g u a n i d i n e w i t h a X m a x = 2 3 0 n m ( E 1 4 3 0 0 , M e O H ) . U n d e r s i m i l a r r e a c t i o n c o n d i t i o n s t r i o p h a m i n e w a s c o n v e r t e d t o i t s m o n o a c y l d e r i v a t i v e 81 w h o s e UV a b s o r b a n c e C^max = 2 3 2 n m ( £ 1 0 0 0 0 , M e O H ) ] c o r r e l a t e s w e l l w i t h m o n o a c e t y l g u a n i d i n e . T h e 1 3 C N M R r e s o n a n c e s o f d i a c e t y l g u a n i d i n e a t 6 1 5 9 . 0 a n d 1 8 0 . 1 p p m a r e i n g o o d a g r e e m e n t w i t h t h o s e o f t r i o p h a m i n e . T h e d e s h i e l d i n g o f t h e c a r b o n y l c a r b o n s i n t r i o p h a m i n e r e l a t i v e t o d i a c e t y l g u a n i d i n e i s a l m o s t e q u a l i n m a g n i t u d e t o t h e d i f f e r e n c e b e t w e e n p r o p i o n a m i d e ( 6 1 7 7 . 2 ) a n d a c e t a m i d e ( 6 1 7 2 . 7 ) r e p o r t e d b y L e v y e t a l . 5 5 . 0 N H 2 0 H N H 2 0 H 2 N ^ N 44. F i n a l p r o o f o f t h e s t r u c t u r e was o b t a i n e d by b a s e -c a t a l y z e d h y d r o l y s i s (MeOH/NaOH, 48h) o f t r i o p h a m i n e (75) . U s i n g t h e FCNP s p r a y r e a g e n t 5 6 ( f e r r i c y a n a t e - s o d i u m n i t r o p r u s s i d e ) , g u a n i d i n e c o u l d be d e t e c t e d i n t h e r e a c t i o n m i x t u r e by TLC [ C H C 1 3 / M e O H / a c e t i c a c i d ( 5 0 : 3 5 : 5 ) , R f = 0 . 3 ] . T r e a t m e n t w i t h a c e t y l a c e t o n e a l l o w e d t h e i s o l a t i o n o f g u a n i d i n e as i t s 4 , 6 - d i m e t h y l p y r i m i d i n e d e r i v a t i v e 82. A f t e r r e p e a t e d a t t e m p t s , t h e 1 0 - c a r b o n c a r b o x y l i c a c i d 83_ was a l s o i s o l a t e d i n low y i e l d as a p r o d u c t o f h y d r o l y s i s . 82 83 - C H - - C2H1 F i g u r e 4. I n t e r p r e t a t i o n o f the HRMS o f 75. PPM 16) F i g u r e 6 . 1 0 0 MHz b r o a d b a n d d e c o u p l e d 1 3 C NMR s p e c t r u m o f 75 i n a c e t o n e - d 6 * F i g u r e 7 . 1 0 0 MHz S F O R D 1 3 C NMR s p e c t r u m o f 75. i n C D C 1 3 . M I C R O M E T E R S ( M m ) 4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400 F R E Q U E N C Y ( C M 1 ) vo F igu re 8. IR spectrum o f 75 i n CHC lg . F i g u r e 9 . 4 0 0 MHz ' H NMR d i f f e r e n c e NOE s p e c t r u m o f 75 w h e n t h e o l e f i n i c p r o t o n a t 6 5 . 2 1 p p m w a s i r r a d i a t e d : a ) e n h a n c e d s i g n a l s o f t h e m a i n c h a i n m e t h y l e n e p r o t o n s ; b ) e n h a n c e d s i g n a l o f t h e v i n y l m e t h y l g r o u p c ) a l i p h a t i c m e t h y l s . Figure 10. 270 MHz 'H NMR spectrum of 78 i n CDC1 3. 5 3 . I I . B I O L O G I C A L C O N S I D E R A T I O N S A n u m b e r o f i n t e r e s t i n g a n d u n i q u e g u a n i d i n e c o n t a i n i n g c o m p o u n d s h a v e b e e n r e p o r t e d f r o m m a r i n e o r g a n i s m s . A t h o r -o u g h r e v i e w o f t h e s e c o m p o u n d s w a s p r o v i d e d b y C h e v o l o t 5 7 i n 1 9 8 1 . S i n c e t h e n s e v e r a l o t h e r g u a n i d i n e d e r i v a t i v e s o f m a r i n e o r i g i n h a v e a l s o b e e n d e s c r i b e d 5 8 ' 5 9 . H o w e v e r , t o t h e b e s t o f o u r k n o w l e d g e , t r i o p h a m i n e (7_5) i s t h e f i r s t n a t u r a l l y o c c u r r i n g d i a c y l a g u a n i d i n e . T h e i m p e t u s f o r s t u d y i n g T r i o p h a c a t a l i n a e w a s t h e i m p l i c i t a s s u m p t i o n t h a t t h e n u d i b r a n c h e m p l o y e d a c h e m i c a l d e f e n s e s y s t e m . D i s s e c t i n g t h e o r g a n i s m p r i o r t o e x t r a c t i o n s h o w e d t h a t t r i o p h a m i n e (7_5) w a s l o c a l i z e d e x c l u s i v e l y i n t h e m a n t l e . W h e n p r o d d e d r e p e a t e d l y , l i v e s p e c i m e n s p r o d u c e d a m u c o u s e x u d a t e r i c h i n 7_5. T r i o p h a m i n e s e e m s t o b e s t r a t e g i c a l l y s t o r e d n e a r t h e o u t e r s u r f a c e o f t h e n u d i b r a n c h w h e r e i t c a n b e r a p i d l y m o b i l i z e d i n a m u c o u s s e c r e t i o n . S u c h l o g i s t i c s a r e i d e a l l y s u i t e d f o r a d e f e n s i v e s u b s t a n c e , b u t i n a f i s h a n t i f e e d a n t b i o a s s a y u s i n g g o l d f i s h , t r i o p h a m i n e d i d n o t d e t e r f e e d i n g . I n a d d i t i o n , i n a s t a n d a r d a s s a y a g a i n s t g r a m p o s i t i v e b a c t e r i a t r i o p h a m i n e f a i l e d t o i n h i b i t g r o w t h . W h i l e a l a r g e n u m b e r o f c o m p o u n d s c o n t a i n g t h e g u a n i d i n e u n i t p o s s e s s p o w e r f u l b i o l o g i c a l a c t i v i t y , i n t h e s e t w o t e s t s t r i o p h a m i n e p r o v e d t o b e i n a c t i v e . O n t h e b a s i s o f t h e s e n e g a t i v e r e s u l t s , i t s h o u l d n o t b e c o n c l u d e d t h a t t r i o p h a m i n e h a s n o v a l u e a s a c h e m i c a l d e t e r e n t . F r e s h w a t e r g o l d f i s h m i g h t r e s p o n d 54. d i f f e r e n t l y t h a n t h e i r m a r i n e c o u n t e r p a r t s o r i t may be t h a t t r i o p h a m i n e d e t e r s p r e d a t o r s o t h e r t h a n f i s h . The egg masses o f numerous n u d i b r a n c h s p e c i e s c o n s i s t o f c o n c e n t r i c r i n g s o f s o f t , spongy m a t e r i a l . T h e s e s t r u c t u r e s a r e o f t e n b r i g h t l y c o l o r e d and d e p o s i t e d on u n o b s t r u c t e d s u b s t r a t e s . N u d i b r a n c h eggs w o u l d a p p e a r t o be a s u i t a b l e f o o d s o u r c e f o r many a n i m a l s b u t p r e d a t i o n i s r a r e l y o b s e r v e d . The c o n s p i c u o u s n a t u r e o f T. c a t a l i n a e egg m a s s e s p r o m p t e d a s e a r c h f o r c h e m i c a l a n t i f e e d a n t s . M e t h a n o l e x t r a c t s o f T r i o p h a egg masses p r o d u c e d l i p i d s and s e v e r a l s t e r o i d s b u t no t r i o p h a m i n e (T5) o r o t h e r u n u s u a l m e t a b o l i t e s . The a p p a r e n t immunity t o p r e d a t i o n e n j o y e d by t h e s e egg masses r e m a i n s an unanswered p u z z l e . T r i o p h a m i n e (T5) was s u b s e q u e n t l y f o u n d i n e x t r a c t s o f t h e v i v i d l y c o l o r e d d o r i d n u d i b r a n c h P o l y c e r a t r i c o l o r . P. t r i c o l o r a l s o f e e d s e x c l u s i v e l y on b r y o z o a n s , a g r o u p o f s e s s i l e m a r i n e a n i m a l s f r o m w h i c h n i t r o g e n o u s compounds h a v e p r e v i o u s l y b e en i s o l a t e d 6 0 . S i n c e many n u d i b r a n c h s h a v e t h e a b i l i t y t o s e q u e s t e r m e t a b o l i t e s o b t a i n e d t h r o u g h t h e i r d i e t , a s e a r c h was u n d e r t a k e n t o f i n d a d i e t a r y s o u r c e f o r t r i o p h a m i n e . C h e m i c a l a n a l y s i s o f b r y o z o a n s p e c i e s e n c o u n t e r e d a t t h e c o l l e c t i o n s i t e s f a i l e d t o r e v e a l t h e p r e s e n c e o f t r i o p h a m i n e . W h i l e i t i s p o s s i b l e t h a t t r i o p h a m i n e o r i g i n a t e s i n an u n d e t e c t e d d i e t a r y s o u r c e , a p r e l i m i n a r y 1 4 C i n c o r p o r a t i o n s t u d y s u g g e s t s i t i s p r o d u c e d w i t h i n t h e n u d i b r a n c h ( s e e C h a p t e r 4, V ) . 55. I I I . S Y N T H E S I S OF ( ± ) - T R I O P H A M I N E ( 7 5 a ) A s y n t h e s i s o f t r i o p h a m i n e (7_5) w a s u n d e r t a k e n t o p r o v i d e d e f i n a t i v e p r o o f o f t h e p r o p o s e d s t r u c t u r e . T h e s y n t h e t i c s c h e m e i n v o l v e d p r o d u c t i o n o f t h e C 1 Q - a c i d 83_ a n d c o u p l i n g t w o o f t h e s e m o i e t i e s t o g u a n i d i n e . T h i s p r o j e c t w a s p e r f o r m e d i n c o n j u n c t i o n w i t h P i e r s a n d C h o n g 6 1 w h o s y n t h e s i z e d t h e h y d r o c a r b o n p o r t i o n o f t h e m o l e c u l e . A t t h e t i m e t h e s y n t h e s i s w a s i n i t i a t e d t h e g e o m e t r y a b o u t t h e o l e f i n i c b o n d s i n t r i o p h a m i n e h a d n o t b e e n e s t a b l i s h e d . P i e r s a n d C h o n g a p p l i e d a r e c e n t l y d e v e l o p e d m e t h o d 6 2 e m p l o y i n g o r g a n o c o p p e r r e a g e n t s t o s t e r o s p e c i f i c a l l y p r o d u c e C 1 Q - a c i d s w i t h ( E ) a n d (Z^) o l e f i n s . T h e i r s y n t h e s e s o f t h e CyQ-acids a r e s u m m a r i z e d b e l o w ( s e e S c h e m e 1 ) . R e a c t i o n o f t h e a , 8 - a c e t y l e n i c e s t e r 8 4 w i t h l i t h i u m ( p h e n y l t h i o ) ( t r i - n - b u t y l s t a n n y l ) c u p r a t e J35 i n T H F a t - 4 8 ° C a f f o r d e d u p o n w o r k u p , e t h y l ( Z ) - 3 - ( t r i - n - b u t y l s t a n n y l ) -2 - p e n t e n o a t e 86_. A l t e r n a t e l y , w h e n 8 4 w a s r e a c t e d i n T H F a t - 7 8 ° C w i t h t h e ( t r i - n - b u t y l s t a n n y l ) c o p p e r r e a g e n t 87_ t h e c o r r e s p o n d i n g g e o m e t r i c i s o m e r 8 8 w a s p r o d u c e d . C o m p l e t e r e d u c t i o n o f t h e e s t e r m o i e t i e s t o m e t h y l g r o u p s i n 8_6 a n d 8 8 f o l l o w e d b y t r a n s m e t a l a t i o n y i e l d e d Q9_ a n d 9_0 r e s p e c t i v e l y . C o n j u g a t e a d d i t i o n o f t h e v i n y l i t h i u m s p e c i e s 8 9 t o t h e N , N , N ' - t r i m e t h y l h y d r a z i d e d e r i v a t i v e o f 2 - e t h y l p r o p e n o i c a c i d 9_1 p r o v i d e d t h e t r i m e t h y l h y d r a z i d e 9 2 . I n a s i m i l a r f a s h i o n 9_3 w a s p r o d u c e d v i a 1 , 4 - a d d i t i o n o f 9 0 t o 9 1 . T h e t r i m e t h y l h y d r a z i d e r e s i d u e s w e r e r e m o v e d C H , C H o C = C - C 0 9 E t Scheme 1. S y n t h e t i c sequence f o r p r o d u c t i o n of the i s o m e r i c a c i d s 94 and 83a. Note : the " a " de s i gna t e s a r a c e m i c s y n t h e t i c p r o d u c t t h a t s t r u c t u r a l l y co r responds w i th m a t e r i a l d e r i v e d from a n a t u r a l s o u r c e . 5 7 . v i a r e d u c t i v e c l e a v a g e . O x i d a t i o n o f t h e c r u d e r e d u c t i o n p r o d u c t s o f 9_2 a n d 93_ p r o d u c e d 9 4 a n d 8 3 a r e s p e c t i v e l y . S y n t h e t i c p r o d u c t 8 3 a p r o v i d e d I R a n d ' H NMR s p e c t r a 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 o s e o f t h e o l e f i n i c a c i d £33 i s o l a t e d f r o m n a t u r a l t r i o p h a m i n e (7_5) . S p e c t r a ( I R a n d ' H NMR) o f t h e i s o m e r i c a c i d 9 4 w e r e v e r y s i m i l a r t o , b u t d i s t i n c t l y d i f f e r e n t f r o m , t h e s p e c t r a o f t h e n a t u r a l a c i d . T h i s c o n f i r m e d t h e s t r u c t u r e o f t h e h y d r o c a r b o n p o r t i o n o f t r i o p h a m i n e w h i c h h a d p r e v i o u s l y b e e n d e d u c e d b y i n t e r p r e t a t i o n o f s p e c t r a l d a t a . T h e ( E ) c o n f i g u r a t i o n o f t h e d o u b l e b o n d i n t r i o p h a m i n e , p r e d i c t e d f r o m t h e d i f f e r e n c e NOE e x p e r i m e n t , w a s n o w d e f i n i t i v e l y e s t a b l i s h e d b y t h e u n a m b i g u o u s s y n t h e s i s o f 8 3 a . I t s h o u l d b e n o t e d t h a t a c i d - c a t a l y z e d h y d r o l y s i s o f 9 4 u s i n g H C 1 , a c e t i c a c i d , p - t o l u e n e s u l f o n i c a c i d o r o x a l i c a c i d p r o d u c e d 2 , 4 , 4 - t r i e t h y l b u t y r o l a c t o n e 9 5 a s t h e m a j o r p r o d u c t . T h i s i n d i c a t e d t h a t t h e c a r b o n - c a r b o n d o u b l e b o n d p a r t i c i p a t e d i n t h e h y d r o l y s i s p r o c e s s . W i t h t h i s o b s e r v a t i o n i n m i n d , t h e h y d r o l y s i s o f t r i o p h a m i n e (7_5) w a s r e - e x a m i n e d . W h i l e h y d r o l y s i s o f 75. i n t h e p r e s e n c e o f b a s e i s a f a c i l e p r o c e s s , t h e p e r s i s t e n t l o w y i e l d o f a c i d 8J3 f r o m t h e r e a c t i o n m i x t u r e w a s d i f f i c u l t t o e x p l a i n . I t i s n o w a p p a r e n t t h a t t h e h y d r o c a r b o n p o r t i o n o f 75_ i s p r o n e t o l a c t o n i z a t i o n i n t h e p r e s e n c e o f a c i d . A c i d i f i c a t i o n o f t h e h y d r o l y s i s m i x t u r e l e a d s t o M a r k o v n i k o v p r o t o n a t i o n o f t h e o l e f i n . T h e 3 ° c a r b o n i u m i o n w h i c h i s g e n e r a t e d c a n 58. then be a t t a cked by the c a rbony l oxygen of the c a r b o x y l i c a c i d . The Y ~ l a c t o n e 95 was subsequent l y i s o l a t e d from the a c i d i f i e d h y d r o l y s a t e o f 75. 95 The s t r a t e g y o f the remainder of the s y n t h e s i s i n v o l v e d c o n v e r t i n g a c i d 83a i n t o a s u i t a b l e d e r i v a t i v e and then c o u p l i n g i t t o g u a n i d i n e . S ince the c o u p l i n g r e a c t i o n r e q u i r e s n u c l e o p h i l i c a t t a ck o f guan id ine on the a c i d c a r b o n y l , a d e r i v a t i v e o f 83a was made which r e p l a c e d the h y d r o x y l r e s i d u e w i th a more f a v o r a b l e l e a v i n g g roup . Treatment o f the c a r b o x y l i c a c i d 83a w i th p - n i t r o p h e n o l i n the p resence of N , N ' - d i c y c l o h e x y l c a r b o d i i m i d e a f f o r d e d the p - n i t r o p h e n y l e s t e r 96 i n e x c e l l e n t y i e l d . The a b i l i t y o f the p - n i t r o p h e n y l u n i t t o s t a b i l i z e a nega t i v e change i n c r e a s e d the r e a c t i v i t y o f 9_6 t owa rd n u c l e o p h i l e s . C o u p l i n g the a c t i v a t e d e s t e r 9_6 t o guan id ine i n a 2:1 s t o i c h i o m e t r i c r a t i o would complete the s y n t h e s i s (see Scheme 2 ) . P rev ious s t u d i e s have shown t h a t wh i l e the p roduc t s o b t a i n e d on a c y l a t i o n o f guan id ine are dependent on the c o n d i t i o n s used , N, N ' - d i a c y l d e r i v a t i v e s can be produced i n F i n a l s t e p s i n t h e s y n t h e s i s o f t h e ( R , R ) a n d ( S , S ) e n a n t i o m e r s o f ( ± ) - t r i o p h a m i n e (75a) a n d t h e ( R , S ) a n d ( S , R ) d i a s t e r e o m e r s 75b. 6 0 . g o o d y i e l d 6 3 . F r o m o u r o w n s t u d i e s , i t w a s k n o w n t h a t t r e a t m e n t o f g u a n i d i n e c a r b o n a t e w i t h a c e t i c a n h y d i d e p r o d u c e d a m i x t u r e o f t h e d i a c e t y l a n d t r i a c e t y l d e r i v a t i v e s . T r i a c e t y l g u a n i d i n e c o u l d b e h y d r o l y z e d t o d i a c e t y l g u a n i d i n e i n r e f l u x i n g e t h a n o l . We s e t o u t t o f i n d s u i t a b l e c o n d i t i o n s f o r d i a c y l a t i n g g u a n i d i n e w i t h t h e p - n i t r o p h e n y l e s t e r 96^ o r i f t h e t r i a c y l d e r i v a t i v e p r e d o m i n a t e d , a c h i e v e s e l e c t i v e h y d r o l y s i s o f a s i n g l e a c y l g r o u p . I n i t i a l a t t e m p t s t o a c y l a t e t h e h y d r o c h l o r i d e o r c a r b o n a t e s a l t s o f g u a n i d i n e w e r e u n s u c c e s s f u l a s 9 6 f a i l e d t o r e a c t w i t h t h e g u a n i d i n e s a l t s . E m p l o y i n g a p r o c e d u r e d e s c r i b e d b y B a n k e r e t a l . 6 1 * , t h e f r e e b a s e o f g u a n i d i n e w a s t r e a t e d w i t h a c y l a t i n g a g e n t 9_6 i n t h e p r e s e n c e o f N , N , N ' , N ' - t e t r a m e t h y 1 g u a n i d i n e . U n d e r t h e s e c o n d i t i o n s e s t e r 9 6 r e a c t e d w i t h t h e t e t r a m e t h y l g u a n i d i n e t o y i e l d i t s m o n o a c y l d e r i v a t i v e , b u t n o r e a c t i o n o f 9 6 w i t h f r e e g u a n i d i n e w a s o b s e r v e d . B y s y s t e m a t i c a l l y v a r y i n g t h e r e a c t i o n c o n d i t i o n s , i t w a s f o u n d t h a t t h e d e s i r e d d i a c y l p r o d u c t 7 5 a c o u l d b e f o r m e d b y a d d i n g s m a l l a m o u n t s o f f r e e g u a n i d i n e t o a n e x c e s s o f 9 6 i n c h l o r o f o r m . G u a n i d i n e f r e e b a s e i s e x t r e m e l y h y g r o s c o p i c s o a n a c c u r a t e w e i g h t o f t h e t o t a l g u a n i d i n e a d d e d w a s n o t d e t e r m i n e d . R a t h e r , s m a l l p o r t i o n s w e r e a d d e d a t l h i n t e r v a l s a n d t h e c o u r s e o f t h e r e a c t i o n m o n i t o r e d b y T L C ( t h e a p p e a r a n c e o f 7 5 a a n d t h e d i s a p p e a r a n c e o f 9 6 ) . I f t h e r e a c t i o n w a s a l l o w e d t o g o t o 6 1 . c o m p l e t i o n , a s d e t e r m i n e d b y t h e d i s a p p e a r a n c e o f 9 6 , a p p r e c i a b l e q u a n t i t i e s o f s i d e p r o d u c t ( s ) f o r m e d a n d t h e y i e l d o f 7 5 a w a s v e r y l o w . T h e r e f o r e , w h e n a p p r o x i m a t e l y 5 0 - 7 5 % o f t h e s t a r t i n g m a t e r i a l 96^ h a d r e a c t e d , t h e r e a c t i o n w a s s t o p p e d b y f i l t e r i n g o f f t h e c h l o r o f o r m s o l u b l e m a t e r i a l . C h r o m a t o g r a p h y o n s i l i c a p r o v i d e d a 4 8 % y i e l d ( b a s e d o n u n r e c o v e r e d 9 6 ) o f a m i x t u r e o f ( ± ) - t r i o p h a m i n e ( 7 5 a ) a n d i t s d i a s t e r e o m e r s 7 5 b . T h e i d e n t i c a l h y d r o c a r b o n m o i e t i e s o f t r i o p h a m i n e ( 7 5 ) e a c h c o n t a i n a s i n g l e c h i r a l c e n t e r [ t h e C - 2 ( 2 ' ) m e t h i n e c a r b o n ] . A s n a t u r a l t r i o p h a m i n e i s o p t i c a l l y a c t i v e , t h e c o n f i g u r a t i o n a b o u t t h e t w o c h i r a l c e n t e r s m u s t b e ( R , R ) o r ( S , S ) . I n t h e s y n t h e s i s o f ( ± ) - t r i o p h a m i n e , t h e o l e f i n i c a c i d 8 3 a i s p r o d u c e d a s a r a c e m i c m i x t u r e . W h e n t w o a c i d r e s i d u e s a r e c o u p l e d t o g u a n i d i n e f o u r c o m b i n a t i o n s o f c o n f i g u r a t i o n s a r e f o r m e d , ( R , R ) , ( S , S ) , ( R , S ) a n d ( S , R ) . T h e m i x t u r e o f t h e ( R , R ) a n d ( S , S ) p r o d u c t s c o n s t i t u t e ( ± ) - t r i o p h a m i n e ( 7 5 a ) w h i l e t h e ( R , S ) a n d ( S , R ) p r o d u c t s r e p r e s e n t a m i x t u r e o f u n w a n t e d d i a s t e r e o m e r s 7 5 b . S e p a r a t i o n o f ( ± ) - t r i o p h a m i n e ( 7 5 a ) f r o m i t s d i a s t e r e o m e r s 7 5 b w a s a c h i e v e d b y r e p e a t e d p r e p a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y . T h e ' H NMR a n d I R s p e c t r a o f ( ± ) - t r i o p h a m i n e w e r e 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 o s e o f t h e n a t u r a l p r o d u c t . T h e s p e c t r a ( ' H NMR a n d I R ) o f t h e d i a s t e r e o m e r s s h o w e d s u b t l e b u t d i s t i n c t d i f f e r e n c e s f r o m a u t h e n t i c t r i o p h a m i n e . R e s o n a n c e s b e t w e e n 6 1 . 5 a n d 2 . 5 p p m 62. i n the 'H NMR s p e c t r a were most u s e f u l i n d i s t i n g u i s h i n g the d e s i r e d p r o d u c t ( see f i g u r e 1 2 ) . The d i a s t e r e o m e r s d i s p l a y e d a s l i g h t l y a l t e r e d c o u p l i n g p a t t e r n i n t he two-proton m u l t i p l e t a t 61.49-1.67 and a sharpened s i g n a l a t 2.12 (dd, J = 7, 14Hz, IH) ppm. The c l e a r e s t s p e c t r a l d i f f e r e n c e was the two one-proton m u l t i p l e t s a t 62 .28-2.37 and 2.37-2.47 i n the d i as te reomers t h a t were c o a l e s c e d to a t w o - p r o t o n m u l t i p l e t a t 2 . 3 0 - 2 . 4 8 ppm i n a u t h e n t i c t r i o p h a m i n e . -I 1 1 1 1 —I 1 1 1 1 2.5 2.0 1.5 PPM (6) 2.5 2.0 1.5 PPM (6) F i g u r e 1 2 . C o m p a r i s o n o f ' H NMR ( 4 0 0 M H z , C D C 1 3 ) r e s o n a n c e s CT> b e t w e e n 6 1 . 5 - 2 . 5 p p m o f : a ) n a t u r a l t r i o p h a m i n e f ( 7 5 ) a n d b ) m i x t u r e o f s y n t h e t i c d i a s t e r e o m e r s 7 5 b . P l a t e 2. A n i s o d o r i s n o b i l i s . P h o t o g r a p h e r : Simon F r a s e r U n i v e r s i t y . Ron L o n g , 6 5 . C H E M I C A L S T U D I E S OF A N I S O D O R I S N O B I L I S I. D I H Y D R O A P O F A R N E S A L ( 1 5 ) T h e d o r i d n u d i b r a n c h A n i s o d o r i s n o b i l i s i s l a r g e ( a v e r a g e s i z e 8 0 - 1 0 0 m m ) a n d f a i r l y a b u n d a n t a l o n g t h e c o a s t o f B r i t i s h C o l u m b i a . I t f e e d s o n s p o n g e s a n d i s f o u n d i n a v a r i e t y o f h a b i t a t s f r o m t h e i n t e r t i d a l z o n e t o d e p t h s o f g r e a t e r t h a n 1 0 0 m. W h e n r e m o v e d f r o m t h e w a t e r , t h e n u d i b r a n c h e m i t s a p l e a s a n t , f r u i t y a r o m a . B e c a u s e o f i t s p l e a s a n t s m e l l a n d b r i g h t y e l l o w c o l o r a t i o n , A . n o b i l i s i s c o m m o n l y r e f e r r e d t o a s t h e s p e c k l e d s e a l e m o n . A . n o b i l i s w a s t h e s u b j e c t o f a p r e v i o u s c h e m i c a l i n v e s t i g a t i o n . I n 1 9 8 0 , F u h r m a n e t a J L . 2 2 i s o l a t e d t h e u n i q u e N - m e t h y l p u r i n e r i b o s i d e d o r i d o s i n e (JL6) f r o m a q u e o u s e x t r a c t s o f t h e n u d i b r a n c h ' s d i g e s t i v e g l a n d . I t i s p h a r m a c o l o g i c a l l y a c t i v e a n d i s p r o b a b l y r e s p o n s i b l e f o r t h e t o x i c i t y t o m i c e a n d c r a b s o r i g i n a l l y o b s e r v e d i n t h e s e e x t r a c t s 6 5 . D o r i d o s i n e i s a l s o a c o n s t i t u e n t o f t h e A u s t r a l i a n s p o n g e T e d o n i a d i g i t a t a 6 6 . T h e p r e s e n t i n v e s t i g a t i o n o f A n i s o d o r i s n o b i l i s w a s p r o m p t e d b y t h e n u d i b r a n c h ' s p l e a s a n t o d o r . W h e n p e r t u r b e d , A . n o b i l i s p r o d u c e s a v o l u m i n o u s m u c o u s s e c r e t i o n w h i c h r e t a i n s t h e s m e l l o f t h e o r g a n i s m . F r e s h l y c o l l e c t e d s p e c i m e n s w e r e e x t r a c t e d w i t h c h l o r o f o r m , a s m e t h a n o l f o r m e d a n e m u l s i o n w i t h t h e m u c o u s . S u c c e s s i v e c h l o r o f o r m e x t r a c t s w e r e c o m b i n e d a n d c o n c e n t r a t e d t o a f r a g r a n t b r o w n o i l . 66. F r a c t i o n a t i o n o f t h e c r u d e e x t r a c t o n s i l i c a g e l p r o v i d e d l i p i d s , s t e r o i d s a n d a s i n g l e o d o r i f e r o u s c o m p o u n d t h a t w a s D N P H p o s i t i v e . F i n a l p u r i f i c a t i o n w a s a c h i e v e d b y p r e p a r a t i v e g a s c h r o m a t o g r a p h y t o y i e l d t h e o l e f i n i c a l d e h y d e 15^. T h e v o l a t i l e n a t u r e a n d l o w n a t u r a l a b u n d a n c e o f 1_5 r e s u l t e d i n a t o t a l y i e l d o f o n l y 2 . 8 m g ( a p p r o x i m a t e l y 0 . 1 m g / a n i m a l ) . 0 1 5 T h e m o l e c u l a r f o r m u l a C l l t H 2 4 0 w a s e s t a b l i s h e d b y e l e c t r o n i m p a c t HRMS a n d r e v e a l e d t h a t L 5 p o s s e s s e d t h r e e u n i t s o f u n s a t u r a t i o n . T h e 4 0 0 MHz ' H NMR s p e c t r u m ( F i g u r e 1 4 ) d i s p l a y e d a o n e - p r o t o n d o u b l e t a t 6 9 . 5 8 ( J = 1 H z ) p p m , i n d i c a t i v e o f a n a l d e h y d e r e s i d u e . T h r e e o l e f i n i c m e t h y l g r o u p s w e r e e v i d e n c e d b y s l i g h t l y b r o a d e n e d s i n g l e t s a t 6 1 . 6 0 , 1 . 6 1 a n d 1 . 6 9 p p m t h a t i n t e g r a t e d f o r t h r e e p r o t o n s e a c h . T w o o l e f i n i c p r o t o n s w e r e o b s e r v e d a s a m u l t i p l e t a t 6 5 . 1 2 p p m . I t w a s e v i d e n t t h e c o m p o u n d w a s a c y c l i c , a s t h e t h r e e u n i t s o f u n s a t u r a t i o n w e r e a c c o u n t e d f o r b y o n e a l d e h y d e a n d t w o o l e f i n f u n c t i o n a l i t i e s . T h e ' H NMR s p e c t r u m a l s o r e v e a l e d a m e t h y l d o u b l e t a t 6 1 . 1 0 ( J = 7 H z ) a n d s i x a l l y l i c m e t h y l e n e p r o t o n s a s a b r o a d m u l t i p l e t a t 1 . 9 6 - 2 . 1 3 p p m . O n e - p r o t o n m u l t i p l e t s a t 6 1 . 4 2 a n d 1 . 7 8 p p m w e r e a p p r o p r i a t e f o r a m e t h y l e n e t h a t i s 8 t o b o t h a n o l e f i n a n d a l d e h y d e s u b s t i t u e n t . T h e ' H NMR s p e c t r u m o f lj> s h o w e d g r e a t s i m i l a r i t y t o t h a t o f c o m p o u n d 6 8 , r e c e n t l y i s o l a t e d a s t h e o d o r i f e r o u s p r i n c i p l e o f t h e n u d i b r a n c h M e l i b e l e o n i n a 1 * 3 . 0 6 8 T h e f r a g r a n t c o m p o u n d f r o m A n i s o d o r i s n o b i l i s w a s e v i d e n t l y t h e ^ - a n a l o g , d i h y d r o a p o f a r n e s a l ( 2 , 6 , 1 0 -t r i m e t h y l - 5 , 9 - u n d e c a d i e n - l - a l ) . T h e p r o p o s e d s t r u c t u r e i s s u p p o r t e d b y a p r o m i n e n t m a s s s p e c t r a l f r a g m e n t i o n a t m/z 1 5 0 ( M + - C H 3 C H C H 0 + H ) r e s u l t i n g f r o m a M c L a f f e r t y r e a r r a n g e m e n t a n d t h e b a s e p e a k a t m/z 6 9 r e s u l t i n g f r o m a d o u b l y a l l y l i c c l e a v a g e w h i c h e x p e l s t h e t e r m i n a l i s o p r e n e u n i t ( F i g u r e 1 3 ) . T h e g e o m e t r y o f t h e c e n t r a l o l e f i n h a s a r b i t r a r i l y b e e n a s s i g n e d a s t r a n s . A l d e h y d e _15_ i s c l e a r l y a d e g r a d e d s e s q u i t e r p e n e . T h e m u c o u s s e c r e t i o n o f A n i s o d o r i s n o b i l i s w h i c h c o n t a i n s d i h y d r o a p o f a r n e s a l JU5 i s k n o w n t o b e r e p u g n a n t t o t h e p r e d a t o r y s e a s t a r P y c n o p o d i a h e l i a n h o i d e s 1 * . I t i s i n t e r e s t i n g t o n o t e t h a t t h e e x u d a t e f r o m M e l i b e l e o n i n a 6 8 . e l i c i t s a s i m i l a r escape response from P. h e l i a n t h o i d e s 6 7 and con t a i n s 60^ as i t s p r i n c i p a l component. The low y i e l d and v o l a t i l e na ture o f JL5 p reven ted i t s t e s t i n g i n a f i s h a n t i f e e d a n t b i o a s s a y . D i h y d r o a p o f a r n e s a l has . not been r e p o r t e d p r e v i o u s l y as a n a t u r a l p roduc t but appears to have found use i n the perfume i n d u s t r y 6 8 . m/z 1 9 0 ( 5 % ) 1 5 m/z 2 0 8 ~ ~ ( 5 % ) m/z 1 6 5 ( 5 0 % ) / m/z 1 5 0 N. A ( 2 0 % ) X 7=\ m/z 6 9 ( 1 0 0 % ) 7 = ^ -m / z 8 1 ( 7 0 % ) F i g u r e 1 3 . I n t e r p r e t a t i o n o f t h e HRMS o f 1 5 . P l a t e 3. A r c h i d o r i s L o n g , Simon m o n t e r e y e n s i s . P h o t o g r a p h e r : F r a s e r U n i v e r s i t y . Ron 72. CHEMICAL STUDIES OF ARCHIDORIS MONTEREYENSIS AND A. ODHNERI I . A r c h i d o r i s m o n t e r e y e n s i s T h e d o r i d n u d i b r a n c h A r c h i d o r i s m o n t e r e y e n s i s s u p e r f i c i a l l y r e s e m b l e s t h e s p e c k l e d s e a lemon A n i s o d o r i s  n o b i l i s . A. m o n t e r e y e n s i s r a n g e s f r o m s u b t i d a l t o 25 m d e p t h , b u t i s most common i n r e l a t i v e l y s h a l l o w , sponge d o m i n a t e d c o m m u n i t i e s . The o b s e r v a t i o n t h a t i t s c r u d e m e t h a n o l e x t r a c t s e x h i b i t e d a n t i b i o t i c a c t i v i t y a g a i n s t t h e gram p o s i t i v e b a c t e r i a S t a p h y l o c o c c u s a u r e u s and B a c i l l u s  s u b t i l i s p r o m p t e d t h e p r e s e n t c h e m i c a l i n v e s t i g a t i o n 2 1 . S p ecimens o f A r c h i d o r i s m o n t e r e y e n s i s were c o l l e c t e d i n B a r k l e y Sound, B.C. and i m m e d i a t e l y immersed i n m e t h a n o l . The m e t h a n o l was d e c a n t e d and c o n c e n t r a t e d u n d e r r e d u c e d p r e s s u r e t o an aqueous s u s p e n s i o n . P a r t i t i o n i n g between c h l o r o f o r m and w a t e r p r o v i d e d an o r g a n i c e x t r a c t w h i c h r e t a i n e d t h e a n t i b i o t i c a c t i v i t y . B i o a s s a y g u i d e d f r a c t i o n a t i o n was p e r f o r m e d by f l a s h c h r o m a t o g r a p h y on s i l i c a g e l u s i n g s t e p g r a d i e n t s o f h e x a n e - e t h y l a c e t a t e m i x t u r e s . The m a t e r i a l e l u t e d w i t h 100% e t h y l a c e t a t e was s u b j e c t e d t o r a d i a l t h i n - l a y e r c h r o m a t o g r a p h y e l u t i n g w i t h c h l o r o f o r m - m e t h a n o l ( 9 5 : 5 ) . F i n a l s e p a r a t i o n by HPLC w i t h a h e x a n e - e t h y l a c e t a t e - m e t h a n o l (50:40:2) e l u a n t p r o v i d e d p u r e samples o f compounds 1_7, 20_, 2_2 and 23. The m a j o r m e t a b o l i t e 17_, was an o p t i c a l l y a c t i v e (Calrjj = - 1 2 . 5 ° , C H C 1 3 ) , c r y s t a l i n e s o l i d . E l e c t r o n i m p a c t 7 3 . H R M S e s t a b l i s h e d i t s m o l e c u l a r f o r m u l a a s C ^ H g g O ^ . A b s o r b a n c e s i n t h e I R s p e c t r u m r e v e a l e d t h e p r e s e n c e o f h y d r o x y l ( 3 6 0 0 - 3 3 0 0 c m - 1 ) a n d e s t e r ( 1 7 3 0 c m - 1 ) f u n c t i o n a l i t i e s . A r e s o n a n c e a t 6 1 7 3 . 4 p p m i n t h e 1 3 C NMR ( 1 0 0 M H z , C D C 1 3 ) s p e c t r u m s u p p o r t e d t h e p r e s e n c e o f a n e s t e r . T h e ' H NMR ( 4 0 0 M H z , C D C 1 3 ) c o n t a i n e d r e s o n a n c e s h i g h l y s u g g e s t i v e o f a l - a c y l o x y - 2 , 3 - d i h y d r o x y p r o p a n e m o i e t y . T h e c a r b i n o l p r o t o n s o f a p r i m a r y a l c o h o l w e r e s e e n a s t w o d o u b l e d d o u b l e t s a t 6 3 . 6 3 ( J = 6 , 12 H z ) a n d 3 . 7 0 ( J = 4 , 12 H z ) p p m t h a t e a c h i n t e g r a t e d f o r o n e p r o t o n . A o n e - p r o t o n m u l t i p l e t a t 6 3 . 9 5 p p m w a s i n d i c a t i v e o f a s e c o n d a r y a l c o h o l . A b r o a d , t w o - p r o t o n s i n g l e t a t 6 2 . 4 4 p p m t h a t e x c h a n g e d w i t h D 2 0 s u p p o r t e d t h e p r e s e n c e o f t w o h y d r o x y l g r o u p s . T w o d o u b l e d d o u b l e t s d e s h i e l d e d t o 6 4 . 1 5 ( J = 7 , 12 H z , I H ) a n d 4 . 2 2 ( J = 5 , 12 H z , I H ) p p m w e r e a p p r o p r i a t e f o r a n e s t e r i f i e d p r i m a r y a l c o h o l . T h e s e d i s t i n c t i v e r e s o n a n c e s w e r e v i r t u a l l y i d e n t i c a l t o t h o s e o f t h e g l y c e r o l u n i t i n t h e f a r n e s i c a c i d d e r i v a t i v e 2 4 , p r e v i o u s l y i s o l a t e d f r o m 7 4 . A r c h i d o r i s o d h n e r i 2 * * . P r o m i n e n t p e a k s i n t h e m a s s s p e c t r u m a t m/z 2 8 6 ( M + - C 3 H 8 0 3 ) a n d 2 5 8 ( M + - C 1 + H 8 O l 4 ) c o n f i r m e d t h a t t h e a l c o h o l c o m p o n e n t o f t h e e s t e r w a s a g l y c e r o l r e s i d u e . 0 OH 2 4 T h e m o l e c u l a r f o r m u l a ( C 2 0 H 3 l O ^ ° ^ t l i e a c Y 1 p o r t i o n o f t h e e s t e r s u g g e s t e d t h a t c o m p o u n d 17_ w a s a d i t e r p e n o i c a c i d e s t e r i f i e d t o g l y c e r o l . T w o o f t h e f i v e t o t a l u n i t s o f u n s a t u r a t i o n i n t h e p a r e n t g l y c e r i d e c o u l d b e a c c o u n t e d f o r b y t h e e s t e r c a r b o n y l a n d a t r i s u b s t i t u t e d o l e f i n t h a t w a s e v i d e n c e d b y 1 3 C NMR r e s o n a n c e s a t 6 1 2 4 . 3 ( d ) a n d 1 2 8 . 6 ( s ) p p m a n d a s i n g l e o l e f i n i c p r o t o n s i g n a l a t 6 5 . 5 4 ( b s ) p p m . T h e t e r p e n o i d p o r t i o n o f I7_ c l e a r l y c o n t a i n e d t h r e e c a r b o c y c l i c r i n g s . T h e ' H NMR s p e c t r u m r e v e a l e d f o u r m e t h y l s i n g l e t s a t 6 0 . 8 2 , 0 . 8 7 , 0 . 9 2 a n d 0 . 9 6 , a v i n y l m e t h y l a t 1 . 6 1 a n d a b r o a d , o n e - p r o t o n s i n g l e t a t 2 . 9 3 p p m a p p r o p r i a t e f o r a n a l l y l i c m e t h i n e t h a t i s a l s o a t o a n e s t e r c a r b o n y l . T h e o l e f i n , e s t e r a n d m e t h y l s u b s t i t u e n t s a c c o u n t e d f o r e i g h t o f t h e t w e n t y c a r b o n s i n t h e d i t e r p e n o i d a c y l r e s i d u e . A 1 3 C S F O R D NMR e x p e r i m e n t e s t a b l i s h e d t h a t t h e r e m a i n i n g t w e l v e c o n s i s t e d o f s i x m e t h y l e n e , t h r e e m e t h i n e a n d t h r e e q u a r t e r n a r y c a r b o n s . T h i s s p e c t r a l d a t a s u g g e s t e d t h a t c o m p o u n d 17_ h a d t h e r e g u l a r i s o p r e n e s t r u c t u r e s h o w n . T h e 75. b a s e peak i n t h e mass s p e c t r u m a t m/z 192, r e s u l t i n g f r o m a r e t r o D i e l s - A l d e r c l e a v a g e o f t h e C r i n g , s u p p o r t e d t h i s h y p o t h e s i s . However, t r a c e c o n t a m i n a t i o n by compound 20 r e s u l t e d i n a p e r s i s t e n t peak a t m/z 310 w h i c h was presumed t o be a l o s s o f C 5 H g f r o m t h e p a r e n t g l y c e r i d e . A r a t i o n a l mechanism f o r s u c h a l o s s f r o m ^7 c o u l d n o t be e n v i s i o n e d . Due .to t h i s s p e c t r a l a m b i g u i t y and a l i m i t e d q u a n t i t y o f t h e n a t u r a l p r o d u c t , t h e • s t r u c t u r e was v e r i f i e d by s i n g l e c r y s t a l X - r a y d i f f r a c t i o n a n a l y s i s c o n d u c t e d by C l a r d y and C h e n 2 3 . The X - r a y e x p e r i m e n t c o n f i r m e d t h e s t r u c t u r e b u t d e f i n e d o n l y t h e r e l a t i v e s t e r e o c h e m i s t r y o f t h e m o l e c u l e . The a b s o l u t e c o n f i g u r a t i o n was d e t e r m i n e d by r e d u c i n g JL7 w i t h DIBAL t o t h e known a l c o h o l 9J_. C o m p a r i s o n o f t h e s p e c i f i c r o t a t i o n (CCXDD = - 9 ° ) o f t h e r e d u c t i o n p r o d u c t w i t h t h e l i t e r a t u r e v a l u e 6 9 (Calrj, = - 9 ° ) c o n f i r m e d t h e a b s o l u t e c o n f i g u r a t i o n as shown (5S, 8R, 9R 10S, 14R). 97 F i g u r e 15. Computer generated s t e r e o c h e m i c a l r e p r e s e n t a t i o n o f o b t a i n e d by s i n g l e c r y s t a l X - r a y d i f f r a c t i o n a n a l y s i s . O H F i g u r e 16. I n t e r p r e t a t i o n of t h e HRMS of 17. 17 160 I 120 I XX) T" 80 -r-60 40 20 PPM (6) Figure 18. 100 MHz broad band decoupled 1 3 C NMR spectrum of 17 i n CDC1 3. vo 3.5 M I C R O M E T E R S ium) 6 7 I ! i 10 1112 13 14 16 IB 20 25 4 0 0 0 3600 3200 2800 2400 2000 1800 1600 1400 F R E Q U E N C Y (CM"') 1200 1000 800 600 400 CO F igure 20. IR spectrum o f _17 i n CHC lg . T 1 1—: r 1 ' 1 ——i 1 1 r-PPM (6) F i g u r e 2 2 . 1 0 0 MHz b r o a d b a n d d e c o u p l e d 1 3 C NMR s p e c t r u m o f 9 7 i n C D C 1 , . MICROMETERS (/im) 4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400 FREQUENCY (CM"') CO F i g u r e 2 3 . I R s p e c t r u m o f 91_ i n C H C 1 3 -85. M a t e r i a l e l u t e d w i t h h e x a n e - e t h y l a c e t a t e (1:1) i n t h e i n i t i a l f r a c t i o n a t i o n o f A . m o n t e r e y e n s i s e x t r a c t s c o n t a i n e d t w o m i n o r m e t a b o l i t e s r e l a t e d t o 1T_. T h e m o l e c u l a r f o r m u l a o f JL8, e s t a b l i s h e d b y HRMS a s C 2 5 H m } ° 5 ' a n d i n t e n s e m a s s s p e c t r a l p e a k s a t m/z 286 ( M + - C 5 H x Q 0 h ) a n d 258 ( M + - C 6 H 1 Q 0 5 ) s u g g e s t e d i t d i f f e r e d f r o m _17 b y a d d i t i o n o f a c e t a t e t o t h e g l y c e r o l f r a g m e n t . T h e ' H NMR s p e c t r u m o f 18 c o n t a i n e d r e s o n a n c e s v i r t u a l l y i d e n t i c a l t o t h o s e o f t h e d i t e r p e n o i d p o r t i o n o f 1_7, i n a d d i t i o n t o a s h a r p m e t h y l s i n g l e t a t 62.11 a n d a s i n g l e e x c h a n g e a b l e p r o t o n a t 2.48 p p m . A d e s h i e l d e d f i v e - p r o t o n m u l t i p l e t a t 64.08-4.25 p p m , w h i c h c o r r e s p o n d e d w e l l w i t h t h e c h e m i c a l s h i f t s a n d c o u p l i n g p a t t e r n s p r e v i o u s l y r e p o r t e d f o r t h e g l y c e r y l a c e t a t e p r o t o n s i n 2_5, e s t a b l i s h e d t h e 1 - a c y l o x y - 2 - h y d r o x y -3 - a c e t o x y p r o p a n e r e s i d u e i n JL8. R e d u c t i o n o f a c e t a t e 1J3 w i t h D I B A L g a v e a l c o h o l 97_ w h i c h w a s 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 e r e d u c t i o n p r o d u c t o f 17. 86. A s e c o n d m o n o a c e t y l g l y c e r i d e 19 , a p p e a r e d t o be a p o s i t i o n a l isomer of 18 as i t a l s o had a mo lecu l a r fo rmula o f C 2 5 H 1 + Q 0 5 and showed mass s p e c t r a l l o s s e s o f M + - C 5 H 1 0 O 1 + and M + - C 6 H 1 Q 0 5 . The 1 H NMR spectrum con t a i ned resonances c h a r a c t e r i s t i c o f the t e r p e n o i d p o r t i o n o f JL7 and a methyl s i n g l e t at 62.09 ppm. Resonances o f the g l y c e r y l a c e t a t e r e s i d u e at 63.76 (d, J = 6 Hz, 2H), 4.26 (dd, J = 7, 12 Hz, IH) , 4.32 (dd, J = 6, 14 Hz, IH) and 5.06 (m, IH) ppm were i d e n t i c a l to those p r e v i o u s l y observed f o r 26_ and con f i rmed the g l y c e r o l moiety o f 19_ was a c e t y l a t e d at the 2 - p o s i t i o n . Reduc t ion o f 19 w i th DIBAL a l s o gave a l c o h o l 97. 90. 5 o > •J z u D o u X u c O E D u -p O d) ft w OS M CN <D U 3 •H N O I S S I W S N V b i ! N 3 0 d 3 d A t h i r d m i n o r m e t a b o l i t e w a s i s o l a t e d d u r i n g H P L C p u r i f i c a t i o n o f g l y c e r i d e C o m p o u n d 20_ w a s a n o p t i c a l l y a c t i v e s o l i d ([<X]D = + 2 3 . 1 ° ) w i t h a m o l e c u l a r f o r m u l a o f C18 H30° '+* I t s i - n f r a r e d s p e c t r u m s h o w e d h y d r o x y l ( 3 6 0 0 - 3 3 0 0 c m - 1 ) a n d e s t e r c a r b o n y l ( 1 7 3 0 c m - 1 ) a b s o r b a n c e b a n d s . F r a g m e n t s i n t h e m a s s s p e c t r u m a t m / z 2 1 8 ( M + - C 3 H g 0 3 ) a n d 1 9 0 ( M + - C [ t H 8 O l f ) r e v e a l e d t h a t _2°_ w a s a g l y c e r i d e e s t e r . OH 2 0 R = H 21 R = A c R e s o n a n c e s i n t h e ' H NMR ( 4 0 0 M H z , C D C 1 3 ) s p e c t r u m a t 6 2 . 4 8 ( 2 H , e x c h a n g e a b l e ) , 3 . 6 3 ( d d , J = 6 , 12 H z , I H ) , 3 . 7 0 ( d d , J = 4 , 12 H z , I H ) , 3 . 9 5 ( m , I H ) , 4 . 1 5 ( d d , J = 7 , 12 H z , I H ) a n d 4 . 2 2 ( d d , J = 5 , 12 H z , I H ) p p m , e s t a b l i s h e d t h a t t h e g l y c e r o l f r a g m e n t w a s a c y l a t e d o n a p r i m a r y a l c o h o l . M e t h y l s i n g l e t s a t 6 0 . 8 9 , 0 . 9 2 a n d 0 . 9 8 , a v i n y l m e t h y l a t 1 . 6 2 , a n d a s i n g l e o l e f i n i c p r o t o n a t 5 . 5 7 p p m s u g g e s t e d t h a t t h e C , _ a c y l p o r t i o n o f t h e e s t e r w a s a 9 2 . b i c y c l i c s e s q u i t e r p e n o i c a c i d . A b r o a d e n e d o n e - p r o t o n s i n g l e t d e s h i e l d e d t o 6 2 . 9 6 p p m w a s c h a r a c t e r i s t i c o f a n a l l y l i c m e t h i n e a t o a n e s t e r c a r b o n y l . A NOE d i f f e r e n c e e x p e r i m e n t s h o w e d t h a t t h i s p r o t o n p a r t i c i p a t e s s i g n i f i c a n t l y i n t h e r e l a x a t i o n o f t w o a l k y l p r o t o n s ( s e e F i g u r e 3 2 ) . T h e o b s e r v e d NOE i n d i c a t e d a 1-3 d i a x i a l o r i e n t a t i o n b e t w e e n t h e m e t h i n e p r o t o n a n d t w o o t h e r p r o t o n s i n t h e b i c y c l i c r i n g s y s t e m . T h e d r i m a n e d e r i v a t i v e 20. w a s a n o b v i o u s s t r u c t u r a l p r o p o s a l t h a t s a t i s f i e d a l l o f t h e s p e c t r a l d a t a . F u r t h e r s u p p o r t f o r t h i s h y p o t h e s i s c o u l d b e f o u n d i n p r o m i n e n t m a s s s p e c t r a l f r a g m e n t s a t m/z 1 2 4 , r e t r o D i e l s - A l d e r c l e a v a g e o f t h e B r i n g , a n d 1 0 9 , l o s s o f a n a d d i t i o n a l m e t h y l . T o c o n f i r m t h e s t r u c t u r e , was . r e d u c e d w i t h D I B A L t o t h e k n o w n s e s q u i t e r p e n e d r i m e n o l 9J3. T h e s p e c t r a l d a t a (NMR , H R M S ) a n d GC r e t e n t i o n t i m e o f a l c o h o l 9 8 w e r e 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 o s e o b t a i n e d f r o m a u t h e n t i c ( ± ) - d r i m e n o l ( k i n d l y s u p p l i e d b y R. A r m s t r o n g ) 7 0 . C o m p a r i s o n o f t h e o p t i c a l r o t a t i o n ( [ a ] r j = - 2 0 ° ) o f 9 8 w i t h t h e l i t e r a t u r e v a l u e o f d r i m e n o l ( [ O C ] D = - 2 0 ° ) 7 1 p r o v e d t h a t t h e a b s o l u t e c o n f i g u r a t i o n o f t h e s e s q u i t e r p e n o i d p o r t i o n o f 20_ i s a s s h o w n ( 5 S , 9 S , 1 0 S ) . 9 8 93. A r e l a t e d m o n o a c e t a t e 2_1, was a l s o r e c o v e r e d f r o m A. m o n t e r e y e n s i s e x t r a c t s . The m o l e c u l a r f o r m u l a < - 2 0 H 3 2 ° 5 ' d i a g n o s t i c mass s p e c t r a l f r a g m e n t s a t M + - C 5 H 1 Q 0 £ t a n d M + - C 6 H 1 0 O 5 and a r e s o n a n c e a t 62.11 ( s , 3H) ppm i n t h e 'H NMR s p e c t r u m r e v e a l e d i t t o be a m o n o a c e t y l a t e d g l y c e r y l e s t e r . A f i v e - p r o t o n m u l t i p l e t a t 6 4 . 0 5 - 4 . 2 4 ppm d e m o n s t r a t e d t h a t t h e g l y c e r o l f r a g m e n t was 1 , 3 - d i a c y l a t e d . R e s o n a n c e s c h a r a c t e r i s t i c o f t h e s e s q u i t e r p e n o i d p o r t i o n o f 20 were a l s o o b s e r v e d i n 2_1. R e d u c t i o n o f 2_1 w i t h DIBAL gave d r i m e n o l 98. m/z 295 (5%) m/z 279 (10%) C H = 0 H - C 3 H e Q 3 20 m/z 310 — (15%) Retro Diels-Alder Cleavage m/z 218 (75%) -co •CH, m/z 109 (100%) m/z 187 (40%) m/z 190 (45%) m/z 124 (70%) F i g u r e 2 8 . I n t e r p r e t a t i o n o f t h e HRMS o f 2 0 . VO Figure 30. 100 MHz broad band decoupled 1 N M R spectrum of 20 i n CDC1 3. 2.0 1.5 1.0 0.5 1 r PPM (6) F igu re 32. 400 MHz 'H NMR d i f f e r e n c e NOE spect rum o f 20 when the a l l y l i c methine p ro ton at 62.96 ppm was i r r a d i a t e d : a) enhanced s i g n a l s o f two a l k y l p r o t o n s ; b) i n c o m p l e t e l y s u b t r a c t e d m e t h y l s i g n a l s . 102. A n o t h e r t r a c e m e t a b o l i t e c o u l d o n l y be i s o l a t e d i n low y i e l d a f t e r e x t e n s i v e HPLC p u r i f i c a t i o n . I t was a n o p t i c a l l y a c t i v e o i l (CaDp = + 9 . 7 ° ) w i t h a m o l e c u l a r f o r m u l a o f C, o H o n 0 , . 22 Compound 2_2 showed i n f r a r e d a b s o r b a n c e s (3425, 1700 c m - 1 ) , a mass s p e c t r a l f r a g m e n t i o n ( M + - C 3 H g 0 3 ) and 'H NMR r e s o n a n c e s [63.62 (dd, J = 7, 12 Hz, IH) , 3.70 (dd, J = 5, 12 Hz, I H ) , 3.95 (m, 1H), 4.18 (dd, J = 7, 12 Hz, I H ) , 4.24 ( d d , J = 5, 12 H z , I H ) p p m ] d i a g n o s t i c o f a l - a c y l o x y - 2 , 3 - d i h y d r o x y p r o p a n e r e s i d u e . The p r e s e n c e o f an aB u n s a t u r a t e d e s t e r m o i e t y c o u l d be r e a d i l y d e d u c e d f r o m t h e UV a b s o r b a n c e ( X m a x = 228 nm, MeOH), IR s p e c t r u m (1700 c m - 1 ) and r e s o n a n c e s i n t h e 'H NMR f o r a d e s h i e l d e d v i n y l m e t h y l (62.16 ppm) and an o l e f i n i c p r o t o n (65.70 ppm). M e t h y l s i n g l e t s were o b s e r v e d a t 60.85 and 0.93 and an e x o c y c l i c m e t h y l e n e was e v i d e n c e d by s i n g l e t s a t 4.55 and 4.79 ppm. The aB u n s a t u r a t e d e s t e r and e x o c y c l i c o l e f i n a c c o u n t e d f o r t h r e e o f t h e f o u r t o t a l u n i t s o f u n s a t u r a t i o n i n 22, i n d i c a t i n g t h e C. a c y l p o r t i o n was a m o n o c y c l i c 103 . s e s q u i t e r p e n o i d . Resonances o f the a c y l r e s i d u e were i d e n t i c a l t o t h o s e r e p o r t e d f o r t he m e t h y l e s t e r o f t r a n s - y - m o n o c y c l o f a r n e s i c a c i d ( 9 9 ) 7 2 , thus the s t r u c t u r e of the minor m e t a b o l i t e 22^  was a s s i gned as shown. I t s mass s p e c t r a l f r agmenta t i on p a t t e r n , which i n c l u d e d prominent peaks a t m/z 176 ( C 1 3 H 2 0 ) a n d ^ 8 ^ 1 3 ^ ' w a s c o n s i s t e n t w i th t h i s p r o p o s a l (see F i g u r e 36) . 99 m/z 176 (85%) Figure 36. I n t e r p r e t a t i o n of the HRMS of 22. 107. A v e r y m i n o r c o n s t i t u e n t was f o u n d i n o n l y a s i n g l e c o l l e c t i o n o f A. m o n t e r e y e n s i s . I t h a d a m o l e c u l a r f o r m u l a o f C 2 0 H3 2°2 i n d i c a t i n g a t o t a l o f 5 u n i t s o f u n s a t u r a t i o n . I n f r a r e d a b s o r b a n c e s a t 3225-2500 and 1695 c m - 1 r e v e a l e d t h e p r e s e n c e o f an a c i d f u n c t i o n a l i t y . R e s o n a n c e s i n t h e 'H NMR s p e c t r u m o f an o l e f i n i c p r o t o n 65.70 ( b s , IH) and a v i n y l m e t h y l 2.20 ( b s , 3H) ppm w e r e a p p r o p r i a t e f o r a n aB u n s a t u r a t e d a c i d w i t h t h e m e t h y l g r o u p a t t a c h e d t o t h e B c a r b o n . A s e c o n d t r i s u b s t i t u t e d o l e f i n was e v i d e n c e d b y a o n e - p r o t o n m u l t i p l e t a t 65.07 and a v i n y l m e t h y l s i n g l e t a t 1.60 ppm. Two a l i p h a t i c m e t h y l s i n g l e t s a t 60.83 and 0.91 and r e s o n a n c e s o f an e x o c y c l i c m e t h y l e n e a t 4.53 ( b s , IH) and 4.76 ( b s , IH) ppm s u g g e s t e d t h e m e t a b o l i t e was a m o n o c y c l i c d i t e r p e n o i c a c i d . An o b v i o u s s t r u c t u r a l p r o p o s a l t h a t i s r e a s o n a b l e on b i o s y n t h e t i c g r o u n d s and c o n s i s t e n t w i t h t h e NMR d a t a i s compound 100. The mass s p e c t r u m showed i n t e n s e s i g n a l s a t m/z 289 ( M + - C H 3 ) , 124 , ( C 9 H 1 6 ) , 109 ( C g H 1 3 ) and a b a s e peak a t 81 ( C g H 9 ) w h i c h s u p p o r t t h i s h y p o t h e s i s ( F i g u r e 3 9 ) . However, a p r o m i n e n t peak o b s e r v e d a t m/z 177 ( C 1 3 H 2 1 ) i s d i f f i c u l t t o r a t i o n a l i z e f r o m f r a g m e n t a t i o n o f 100. W h i l e t h e i s o m e r i c compound 101 c o u l d be e x p e c t e d t o p r o d u c e t h e m/z 177 f r a g m e n t i o n ( F i g u r e 4 0 ) , t h i s s t r u c t u r e i s n o t e n t i r e l y c o n s i s t e n t w i t h t h e mass s p e c t r u m e i t h e r . A d o u b l y a l l y l i c c l e a v a g e o f t h e bond between C-9 and C-10 o f 101 s h o u l d r e s u l t i n a s t r o n g s i g n a l a t m/z 123 ( C - H 1 C . ) . W h i l e 108. z 304 15% F i g u r e 39. P o s s i b l e mass s p e c t r a l f r agmenta t i on of 100. 109. m/z 304 15% m/z 289 40% . m/z 177 50% F i g u r e 40 . P o s s i b l e mass s p e c t r a l f r agmenta t i on o f 101 t o produce a peak at m/z 177. 1 1 0 . a p e a k a t m / z 1 2 3 i s o b s e r v e d , t h e p e a k a t 1 2 4 i s c o n s i d e r a b l y m o r e i n t e n s e . A n a l l y l i c M c L a f f e r t y r e a r r a n g e m e n t i n c o m p o u n d 1 0 0 c o u l d r e a d i l y p r o v i d e t h e m / z 1 2 4 i o n , b u t a n a n a l o g o u s p r o t o n t r a n s f e r a n d c l e a v a g e i n c o m p o u n d 1 0 1 s e e m s l e s s p l a u s i b l e . I t i s p o s s i b l e t h a t a m i x t u r e o f t h e t w o o l e f i n i c i s o m e r s w a s i s o l a t e d , a n d t h e m a s s s p e c t r u m s h o w s f r a g m e n t i o n s f r o m b o t h 1 0 0 a n d 1 0 1 . R e s o n a n c e s i n t h e 'H NMR a r e g e n e r a l l y q u i t e s h a r p , b u t t h e a s y m m e t r i c n a t u r e o f t h e o l e f i n p r o t o n a t 6 5 . 0 7 p p m m a y s u p p o r t t h i s a r g u m e n t . D e f i n a t i v e p r o o f o f t h e s t r u c t u r e ( s ) w a s p r e v e n t e d b y a l o w y i e l d o f t h e n a t u r a l p r o d u c t a n d i t s a b s e n c e f r o m e x t r a c t s o f a l l b u t o n e c o l l e c t i o n o f A . m o n t e r e y e n s i s . F i g u r e 41. 400 MHz 'H NMR s p e c t r u m o f t h e d i t e r p e n o i c a c i d ( s ) t e n t a t i v e l y i d e n t i f i e d as 100 a n d / o r 101. Peak C i s an u n i d e n t i f i e d c o n t a m i n a n t . 113 . The f i n a l t r a c e c o n s t i t u e n t i s o l a t e d f r o m A. montereyensis had a molecular formula C i g H 4 Q 0 3 and mass s p e c t r a l fragment i o n s , m/z 255 ( M + - C 2 H 5 0 2 ) and 225 (M +-C 3H 70 3 ) sugge s t i v e of a g l y c e r y l ether moiety. The 1H NMR resonances at 63.41-3.53 (m, 4H), 3.62 (dd, J = 6, 12 Hz, IH), 3.68 (dd, J = 5, 12 Hz, IH) and 3.82 (m, IH) ppm were i n good agreement with r e p o r t e d l i t e r a t u r e values f o r g l y c e r o l e t h e r s 7 3 . A C J 6 reduced f a t t y a c i d a l k y l r e s i d u e was c l e a r l y d i s c e r n a b l e i n the 'H NMR spectrum e s t a b l i s h i n g the m e t a b o l i t e as the known compound 1-0-hexadecyl g l y c e r o l (2_3) 7 1*. Ether 23. shows potent i n - v i t r o . a n t i b i o t i c a c t i v i t y a g a i n s t Staphylcoccus aureus and B a c i l l u s s u b t i l i s , and i t alone appears t o be r e s p o n s i b l e f o r the a n t i b a c t e r i a l a c t i v i t y observed f o r crude A. montereyensis e x t r a c t s . OH n C l 6 H 3 3 23 H O O H n C l 6 H 3 3 23 1 1 6 . I I . A r c h i d o r i s o d h n e r i T h e p r i n c i p a l m e t a b o l i t e s i n e x t r a c t s o f A r c h i d o r i s  o d h n e r i w e r e p r e v i o u s l y r e p o r t e d t o b e t h e t h r e e f a r n e s i c a c i d g l y c e r i d e s 2A, 2_5 a n d J 2 6 2 4 . M e t h a n o l e x t r a c t s o f A . o d h n e r i w e r e r e - e x a m i n e d f o r t r a c e c o n s t i t u e n t s . A f t e r r i g o r o u s H P L C p u r i f i c a t i o n a p o s i t i o n a l i s o m e r o f g l y c e r i d e 2 4 w a s i s o l a t e d i n l o w y i e l d . C o m p o u n d 21_ h a d a m o l e c u l a r f o r m u l a o f C ^ H ^ O ^ a n d ' H NMR s i g n a l s c h a r a c t e r i s t i c o f a f a r n e s i c a c i d a c y l r e s i d u e . R e s o n a n c e s o f t h e g l y c e r o l f r a g m e n t a t 6 3 . 8 1 ( t , J = 6 H z , 4 H ) a n d 4 . 9 0 ( p e n t e t , J = 6 H z , I H ) d e m o n s t r a t e d i t w a s e s t e r i f i e d a t t h e s e c o n d a r y h y d r o x y l . T h e o b s e r v e d f r a g m e n t a t i o n p a t t e r n i n t h e m a s s s p e c t r u m w a s c o n s i s t e n t w i t h s t r u c t u r e 7J_. T h e d i t e r p e n o i c a c i d g l y c e r i d e s _17 a n d JL8 w e r e s u b s e q u e n t l y i s o l a t e d f r o m A . o d h n e r i e x t r a c t s i n l o w y i e l d . T h e y w e r e 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 e c o r r e s p o n d i n g m e t a b o l i t e s o f A . m o n t e r e y e n s i s . G l y c e r i d e s ^ 0 a n d _22 a n d e t h e r 2^ 3 w e r e a l s o p r e s e n t i n e x t r e m e l y t r a c e a m o u n t s . T h e y w e r e e v i d e n c e d b y c h a r a c t e r i s t i c r e t e n t i o n t i m e s i n t h e H P L C t r a c e a n d d i a g n o s t i c s i g n a l s i n t h e ' H NMR s p e c t r u m o f a p a r t i a l l y p u r i f i e d m i x t u r e o f t h e t e r p e n o i c g l y c e r i d e s . HO F i g u r e 4 5 . 2 7 0 MHz ' H NMR s p e c t r u m o f 2 7 i n C D C 1 3 . 118. I l l . A n t i f e e d a n t B i o a s s a y T h e f u n c t i o n o f n u d i b r a n c h s k i n c h e m i c a l s i s g e n e r a l l y p r e s u m e d t o i n v o l v e c h e m i c a l d e f e n s e o f t h e o r g a n i s m . T o t e s t t h i s t h e o r y , g l y c e r o l d e r i v a t i v e s f r o m A . m o n t e r e y e n s i s a n d A . o d h n e r i w e r e e v a l u a t e d f o r f i s h a n t i f e e d a n t a c t i v i t y . S i n c e t h e a s s a y s o f t e n r e q u i r e d m o r e c o m p o u n d t h a n w a s a v a i l a b l e f r o m t h e n u d i b r a n c h s , o n l y t h o s e c o n s t i t u e n t s i s o l a t e d i n s u f f i c i e n t q u a n t i t y w e r e s c r e e n e d . T h e t i d e p o o l s c u l p i n O l i g o c o t t u s m a c u l o s u s w a s c h o s e n a s a t e s t f i s h . T h e s e f i s h a r e r e g u l a r l y e n c o u n t e r e d i n a r e a s w h e r e t h e n u d i b r a n c h s w e r e c o l l e c t e d a n d t h u s r e p r e s e n t a l e g i t i m a t e p o t e n t i a l p r e d a t o r . T h e s c u l p i n s w e r e c o l l e c t e d i n t e r t i d a l l y a n d s t a r v e d f o r 2 4 h p r i o r t o t e s t i n g . C o m m e r c i a l s h r i m p p e l l e t s w e r e c o a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f n u d i b r a n c h m e t a b o l i t e s i n a c e t o n e . C o n t r o l p e l l e t s w e r e t r e a t e d o n l y w i t h a c e t o n e . T h e a s s a y i n v o l v e d a l t e r n a t e l y o f f e r i n g s c u l p i n s c o n t r o l a n d t r e a t e d p e l l e t s a n d m o n i t o r i n g t h e i r f e e d i n g r e s p o n s e d u r i n g a f i x e d p e r i o d o f t i m e . T h e f e e d i n g a b i l i t y o f t h e f i s h w a s v e r i f i e d b y o f f e r i n g u n t r e a t e d p e l l e t s b o t h b e f o r e a n d a f t e r t h e t e s t i n g o f t h e t r e a t e d p e l l e t s . F e e d i n g i n h i b i t i o n w a s a s s e s s e d a s a m a r k e d c e s s a t i o n o f f e e d i n g b e h a v i o r f o l l o w e d b y i n d i f f e r e n c e t o , o r a v o i d a n c e o f , t h e t r e a t e d p e l l e t . A l t h o u g h t h e a c t i v e p e l l e t s w e r e m o u t h e d b y t h e f i s h , t h e y w e r e q u i c k l y r e j e c t e d . I n a l l c a s e s w h e r e f e e d i n g i n h i b i t i o n w a s o b s e r v e d , s u b s e q u e n t l y o f f e r e d c o n t r o l 119. p e l l e t s were r a p i d l y consumed. The r e s u l t s o f t h e b i o a s s a y a r e shown i n T a b l e I V . T h e m a j o r g l y c e r i d e s f r o m A. m o n t e r e y e n s i s a n d A. o d h n e r i p r o v e d i n a c t i v e a s f i s h a n t i f e e d a n t s . Two o f t h e t r a c e c o n s t i t u e n t s , d r i m a n e d e r i v a t i v e 2_0 a n d g l y c e r y l e t h e r 2_3 e x h i b i t e d p o t e n t a n t i f e e d a n t a c t i v i t y . W h i l e t h e r o l e o f t h e more a b u n d a n t s k i n c h e m i c a l s h a s y e t t o be d e f i n e d , t h i s p r e l i m i n a r y s t u d y showed t h a t a t l e a s t two n u d i b r a n c h m e t a b o l i t e s do i n h i b i t t h e f e e d i n g b e h a v i o r o f f i s h . T a b l e IV. F i s h A n t i f e e d a n t B i o a s s a y R e s u l t s Compound ' C o n c e n t r a t i o n ug/mg A c t i v i t y 3 C o n c e n t r a t i o n (J-g/mg A c t i v i t y D i t e r p e n o i c a c i d g l y c e r i d e 17 5 _a 160 -1 " - a c e t a t e 18 5 - 110 -S e s q u i t e r -p e n o i c a c i d g l y c e r i d e 20 18 + NT NT F a r n e s i c a c i d g l y c e r i d e 24 26 - 111 -G l y c e r y l e t h e r 23 18 + NT NT a) + p e l l e t n o t e a t e n - p e l l e t e a t e n NT) no t e s t 1 2 0 . I V . B i o s y n t h e s i s o f A r c h i d o r i s M e t a b o l i t e s We w e r e i n t e r e s t e d i n d e t e r m i n i n g t h e s o u r c e o f m e t a b o l i t e s i n A . m o n t e r e y e n s i s a n d A . o d h n e r i . C o m p o u n d s f o u n d i n n u d i b r a n c h s c a n o f t e n b e t r a c e d t o a n o r g a n i s m i n t h e i r d i e t . T h e s p o n g e H a l i c h o n d r i a p a n i c e a i s a d i e t a r y c o m p o n e n t o f b o t h A r c h i d o r i s s p e c i e s , a n d A . m o n t e r e y e n s i s w a s f r e q u e n t l y f o u n d e a t i n g t h e s p o n g e a t c o l l e c t i o n s i t e s . S p o n g e e x t r a c t s p r o v i d e d g l y c e r y l e t h e r 2 3 , b u t n o n e o f t h e t e r p e n o i c a c i d g l y c e r i d e s . A s m e n t i o n e d i n C h a p t e r 1 , d e  n o v o b i o s y n t h e s i s o f t h e s e s q u i t e r p e n e p o l y g o d i a l ( 5 _ 5 ) , b y t h e d o r i d n u d i b r a n c h D e n d r o d o r i s l i m b a t a h a s p r e v i o u s l y b e e n d e m o n s t r a t e d 3 8 . S i n c e a p r e d a t o r - p r e y r e l a t i o n s h i p w a s e s t a b l i s h e d f o r o n l y o n e o f t h e g l y c e r o l d e r i v a t i v e s , t h e p o s s i b i l i t y t h a t t h e o t h e r s w e r e s y n t h e s i z e d b y t h e n u d i b r a n c h s w a s i n v e s t i g a t e d . 1 1 + C a r b o n l a b e l e d m e v a l o n i c a c i d - d i b e n z y l e t h y l e n e d i a m i n e s a l t w a s i n j e c t e d i n t o t h e d i g e s t i v e g l a n d o f l i v e A . m o n t e r e y e n s i s a n d A . o d h n e r i s p e c i m e n s . T h e n u d i b r a n c h s w e r e i n c u b a t e d f o r 2 4 h i n a n a q u a r i u m a n d t h e n e x t r a c t e d w i t h m e t h a n o l . T h e e x t r a c t s w e r e f r a c t i o n a t e d i n t h e u s u a l f a s h i o n a n d c o m p o u n d s 20_ a n d 2 4 w e r e t a k e n t o p u r i t y b y H P L C . T o r e m o v e a n y r a d i o a c t i v i t y a s s o c i a t e d w i t h t h e g l y c e r o l m o i e t y o r w i t h t r a c e i m p u r i t i e s , t h e g l y c e r i d e s w e r e r e d u c e d w i t h D I B A L t o a l c o h o l s . P u r i f i c a t i o n b y H P L C p r o v i d e d a l c o h o l s 97_ a n d 9 8 w h i c h c r y s t a l i z e d f r o m s o l u t i o n a n d f a r n e s o l ( 1 0 2 ) , w h i c h w a s a n o i l . R e a c t i o n o f f a r n e s o l 121. w i t h d i n i t r o b e n z o y 1 c h l o r i d e i n t h e p r e s e n c e o f 4 - d i m e t h y l a m i n o p y r i d i n e g a v e t h e c r y s t a l i n e 3 , 5 -d i n i t r o b e n z o a t e d e r i v a t i v e 103. A l l d e r i v a t i v e s were c r y s t a l i z e d p r i o r to c o u n t i n g , the r e s u l t s of which are shown i n Tab l e V. S i g n i f i c a n t l e v e l s of 1 J *C meva lon ic a c i d i n c o r p o r a t i o n were observed f o r the te rpene p o r t i o n o f t e r p e n o i c a c i d g l y c e r i d e s 17_, 20. an<^ 24. T a b l e V . R e s u l t s o f l H C M e v a l o n i c A c i d I n c o r p o r a t i o n E x p e r i m e n t s N u d i b r a n c h A r c h i d o r i s  m o n t e r e y e n s i s A r c h i d o r i s o d h n e r i M e t a b o l i t e 24 D e r i v a t i v e u s e d i n c o u n t i n g 103 A c t i v i t y 3 D P M / m g 550 3260 1810 a ) d i s i n t e g r a t i o n s p e r m i n u t e / m i l l i g r a m 123 . V. A d d i t i o n a l B i o s y n t h e t i c S tud i e s P r e l i m i n a r y b i o s y n t h e t i c i n v e s t i g a t i o n s were a l s o c a r r i e d o u t w i t h T r i o p h a c a t a l i n a e a n d C a d 1 i n a  l u t e o m a r g i n a t a . Base h y d r o l y s i s o f t r i ophamine (7_5), i s o l a t e d from T . c a t a l i n a e t h a t had been fed 1 1 + C l a b e l e d a c e t a t e , gave the monoacyl d e r i v a t i v e 8_1 which showed s i g n i f i c a n t (1201 dpm/mg) r a d i o a c t i v i t y . In a s i m i l a r exper iment , a l b i c a n y l a ce t a t e {36) was r e cove red from C. l u t eomarg ina ta t h a t were i n j e c t e d w i th 1 1 + C l a b e l e d meva lon ic a c i d . The a ce t a t e moiety was c l eaved w i th base t o g i ve a l b i c a n o l (3j>) w i th an a c t i v i t y o f 3815 dpm/mg. Whi le these r e s u l t s i n d i c a t e i n s i t u b i o s y n t h e s i s by the nud ib r anchs , they shou ld be i n t e r p r e t e d w i th c a u t i o n . The n a t u r a l p r o d u c t s were t a k e n t o HPLC p u r i t y and c o n v e r t e d t o d e r i v a t i v e s p r i o r t o c o u n t i n g , bu t c r y s t a l s o f t h e d e r i v a t i v e s were not o b t a i n e d . The p o s s i b i l i t y t ha t the observed r a d i o a c t i v i t y . was a c t u a l l y a s s o c i a t e d w i th t r a c e i m p u r i t i e s can not be r u l e d o u t . I t was not p o s s i b l e to r e p e a t t h e e x p e r i m e n t s bu t q u e s t i o n s r a i s e d by t h e s e p r e l i m i n a r y r e s u l t s c e r t a i n l y warrent f u r t h e r s tudy . 35 R = H 36 R = Ac SUMMARY A N D D I S C U S S I O N C h e m i c a l i n v e s t i g a t i o n o f n u d i b r a n c h s r e q u i r e s t h e i s o l a t i o n a n d s t r u c t u r a l i d e n t i f i c a t i o n o f c o n s t i t u e n t s t h a t a r e o f t e n p r e s e n t i n t r a c e q u a n t i t i e s . R e c e n t s u c c e s s o f t h e s e s t u d i e s i s d u e i n l a r g e p a r t t o a d v a n c e s i n s p e c t r o s c o p i c a n d s e p a r a t i o n t e c h n i q u e s . N a t u r a l p r o d u c t c h e m i s t s a r e a l s o i n t e r e s t e d i n t h e s o u r c e , b i o s y n t h e s i s a n d p h y s i o l o g i c a l f u n c t i o n o f n o v e l c o m p o u n d s . A d d r e s s i n g t h e s e l a t t e r q u e s t i o n s r e m a i n s a f o r m i d a b l e c h a l l e n g e . I t i s d i f f i c u l t t o d e s i g n e x p e r i m e n t s t h a t c a n c l e a r l y d e f i n e t h e o r i g i n o r p r e c i s e r o l e o f s e c o n d a r y m e t a b o l i t e s . W i t h n a t u r a l p r o d u c t s f r o m m a r i n e o r g a n i s m s , s u c h s t u d i e s a r e o f t e n m a d e m o r e d i f f i c u l t b y l o g i s t i c a l l i m i t a t i o n s i m p o s e d b y t h e m a r i n e e n v i r o n m e n t . T h e w o r k p r e s e n t e d i n t h i s t h e s i s c o n c e n t r a t e s o n s t r u c t u r a l a s s i g n m e n t s o f n u d i b r a n c h p r o d u c t s . S e v e r a l i n v e s t i g a t i o n s b e y o n d t h e i n t i a l s t r u c t u r a l s t u d i e s w e r e a l s o a t t e m p t e d . T h e s t r u c t u r e o f t r i o p h a m i n e (7_5) , i s o l a t e d f r o m T r i o p h a c a t a l i n a e , w a s s o l v e d b y c l a s s i c a l m e t h o d s o f s p e c t r a l a n a l y s i s a n d d e g r a d a t i o n . T h i s a s s i g n m e n t w a s t h e n c o n f i r m e d b y a n u n a m b i g u o u s s y n t h e s i s o f t h e n a t u r a l p r o d u c t . T h e p r e s e n c e o f g u a n i d i n e i s n o t u n c o m m o n i n c o m p o u n d s f r o m m a r i n e s o u r c e s , h o w e v e r t h e d i a c y l g u a n i d i n e f u n c t i o n a l i t y r e p r e s e n t s a n e w c l a s s o f n a t u r a l c o m p o u n d . T h e a c y l r e s i d u e o f t r i o p h a m i n e h a s a n u n u s u a l c a r b o n 125. s k e l e t o n and i t s b i o s y n t h e t i c o r i g i n i s not o b v i o u s . I t i s d i f f i c u l t to r a t i o n a l i z e the b i o g e n e s i s of the C 1 Q - u n i t from an i s o p r e n o i d p r e c u r s o r , a l though t h i s p o s s i b i l i t y can not be r u l e d ou t . A l t e r n a t i v e l y , the hydroca rbon moiety cou ld r e s u l t from condensa t ion o f a ce t y l -CoA and two b u t y r i c a c i d e q u i v a l e n t s ( F i g u r e 4 6 ) . Such a pathway i s p u r e l y s p e c u l a t i v e as a p p r o p r i a t e b i o s y n t h e t i c s t u d i e s o f t r i ophamine were not a t tempted . 0 F i g u r e 46 a) C^ Q -hydrocarbon p o r t i o n of t r i ophamine (75) b) p o s s i b l e p o s i t i o n of a ce t a t e u n i t s The p resence o f t r i ophamine i n the mucous s e c r e t i o n o f the nud ib ranch suggests i t p l a y s a de f ens i v e r o l e , but t h i s was not s u b s t a n t i a t e d i n a f i s h a n t i f e e d a n t b i o a s s a y . A more thorough i n v e s t i g a t i o n o f the n a t u r a l p r o d u c t ' s e f f e c t on p o t e n t i a l p r e d a t o r s i s i n o r d e r . T r iophamine was a l s o found i n the d o r i d P o l y c e r a t r i c o l o r . S ince a d i e t a r y 1 2 6 . s o u r c e f o r t r i o p h a m i n e c o u l d n o t b e l o c a t e d , t h e p o s s i b i l i t y o f d e n o v o b i o s y n t h e s i s w a s i n v e s t i g a t g e d . T e n t a t i v e r e s u l t s i n d i c a t e t h a t t r i o p h a m i n e i s i n d e e d p r o d u c e d b y t h e n u d i b r a n c h . T h e o d o r i f e r o u s c o n s t i t u e n t o f A n i s o d o r i s n o b i l i s , d i h y d r o a p o f a r n e s a l ( 1 _ 5 ) , w a s i s o l a t e d i n o n l y t r a c e a m o u n t s . I t s s t r u c t u r e w a s p r o p o s e d o n t h e b a s i s o f s p e c t r a l i n t e r p r e t a t i o n a n d c o r r e l a t i o n w i t h r e l a t e d a l d e h y d e 6 8 . D i h y d r o a p o f a r n e s a l c o n t a i n s o n l y 1 4 c a r b o n s , y e t i s c l e a r l y d e r i v e d f r o m a r e g u l a r i s o p r e n o i d p r e c u r s o r . A s s u c h , i t r e p r e s e n t s t h e f i r s t n o r - s e s q u i t e r p e n e i s o l a t e d f r o m a n u d i b r a n c h . C h e m i c a l s t u d i e s o f A r c h i d o r i s m o n t e r e y e n s i s r e v e a l e d a s e r i e s o f t e r p e n o i c a c i d g l y c e r i d e s . T h e s t r u c t u r e o f t h e p r i n c i p a l m e t a b o l i t e 1T_ w a s d e d u c e d f r o m s p e c t r a l d a t a b u t f i n a l l y p r o v e n b y X - r a y a n a l y s i s . I t s a b s o l u t e c o n f i g u r a t i o n w a s e s t a b l i s h e d v i a r e d u c t i o n t o a k n o w n c o m p o u n d . T h e d i t e r p e n e c a r b o n s k e l e t o n o f g l y c e r i d e 1_7 h a s p r e v i o u s l y b e e n o b s e r v e d i n f u r a n o d i t e r p e n e s f r o m t h e n u d i b r a n c h C a s e l l a a t r o m a r g i n a t a 2 8 a n d a s e r i e s o f s p o n g e m e t a b o l i t e s r e l a t e d t o i s o a g a t h a l a c t o n e ( 1 0 4 ) 7 5 a n d a p l y s i l l i n ( 1 0 5 ) 7 6 . W h i l e t h i s s k e l e t o n i s r a t h e r r a r e i n n a t u r e , i t c a n a r i s e f r o m a s t r a i g h t f o r w a r d c y c l i z a t i o n o f t h e a c y c l i c p r e c u r s o r a l l - t r a n s - g e r a n y l g e r a n i o l p y r o p h o s p h a t e . OAc OAc 104 105 D i t e rpenes o f mixed b i o g e n e s i s are known from a v a r i e t y of marine o r g a n i s m s 7 7 . In most c a s e s , the d i t e r p e n o i d c o m p o n e n t i s a c y c l i c a n d b o u n d t o an a r o m a t i c o r c a r b o h y d r a t e m o i e t y . Compound _17 r e p r e s e n t s a r a r e p o l y c y c l i c d i t e r p e n e o f mixed b i o g e n e s i s . The o n l y o the r d i t e r p e n e g l y c e r o l d e r i v a t i v e p r e v i o u s l y r e p o r t e d from a marine source i s the e the r O - g e r a n y l g e r a n y l g l y c e r o l 106, i s o l a t e d from the brown seaweed D i l aphus f a s c i o l a 7 8 . OH 106 The s t r u c t u r e and abso lu t e s t e r e o c h e m i s t r y o f the s e squ i t e rpene p o r t i o n o f g l y c e r i d e was e s t a b l i s h e d by chemica l i n t e r c o n v e r s i o n t o d r imeno l j)8. The drimane carbon s k e l e t o n , found i n s e squ i t e rpenes from t e r r e s t r i a l and 1 2 8 . m a r i n e o r g a n i s m s , i s a c o m m o n s t r u c t u r a l f e a t u r e o f s e v e r a l n u d i b r a n c h m e t a b o l i t e s . C o m p o u n d 2 0 , l i k e m a n y d r i m a n e s e s q u i t e r p e n e s , e x h i b i t s a n t i f e e d a n t a c t i v i t y . T h e d i v e r s e a r r a y o f c h e m i c a l f u n c t i o n a l i t i e s o b s e r v e d i n t h e s e s e s q u i t e r p e n e s s u g g e s t s t h a t t h e b i o c h e m i c a l b a s i s f o r t h e i r a c t i v i t y i s i n t i m a t e l y a s s o c i a t e d w i t h t h e d r i m a n e c a r b o n s k e l e t o n . T h e s e s q u i t e r p e n o i c a c i d g l y c e r i d e 22. w a s i s o l a t e d i n e x t r e m e l y l o w y i e l d a n d i t s s t r u c t u r e d e d u c e d f r o m s p e c t r a l i n t e r p r e t a t i o n . R e l a t i v e l y f e w s e s q u i t e r p e n e s p o s s e s s i n g t h e s i m p l e m o n o c y c l o f a r n e s y l s k e l e t o n h a v e b e e n f o u n d i n n a t u r e , h o w e v e r s u c h c o m p o u n d s h a v e b e e n r e p o r t e d f r o m a l g a e 7 9 a n d s p o n g e s 8 0 . T h e m e t h y l e s t e r o f t r a n s -m o n o c y c l o f a r n e s i c a c i d 1 0 7 h a s b e e n i s o l a t e d f r o m t h e d i e t a r y s p o n g e H a l i c h o n d r i a p a n i c e a 8 1 . T h i s s p o n g e h a s a l s o b e e n t h e s o u r c e o f m i x e d s e s q u i t e r p e n e - h y d r o q u i n o n e d e r i v a t i v e s 8 2 . H . p a n i c e a w a s t h e s u s p e c t e d s o u r c e o f A . m o n t e r e y e n s i s m e t a b o l i t e s , b u t a n a l y s i s o f s p o n g e s a m p l e s s h o w e d n o s i g n o f a n y t e r p e n o i d c o m p o u n d s . A s i n g l e c o n s t i t u e n t , g l y c e r y l e t h e r 23 . ' w a s f o u n d i n b o t h t h e n u d i b r a n c h a n d s p o n g e . T h i s s u g g e s t s t h a t e t h e r 22_ i s o b t a i n e d f r o m t h e s p o n g e d i e t . 129. 107 The nud ibranch A. odhne r i was known to c o n t a i n a f a m i l y of f a r n e s i c a c i d g l y c e r i d e s 2 4 . C l o se examina t ion r e vea l ed t h a t i t a l s o con ta ined a number of t e r p e n o i c a c i d g l y c e r i d e s i d e n t i f i e d from A. mon te reyens i s . We were unable t o f i n d a d i e t a r y source f o r any o f these subs t ances . C o m b i n i n g s p e c t r a l a n a l y s i s a n d b i o g e n e t i c c o n s i d e r a t i o n s , the s t r u c t u r e o f a f r e e d i t e r p e n o i c a c i d i s o l a t e d from A. montereyens is cou ld be t e n t a t i v e l y a s s i gned as 100 or 101. There i s some ev idence t h a t the i s o l a t e d p roduc t was a c t u a l l y a mixture o f the two o l e f i n i c i somers . The proposed s t r u c t u r e s are c l e a r l y r e l a t e d to o the r n a t u r a l compounds such as r e t i n o l (108) and the a l g a l m e t a b o l i t e c a u l e r p o l ( 1 0 9 ) 8 3 . Whi le the f r e e a c i d o n l y occured i n e x t r a c t s from a s i n g l e c o l l e c t i o n o f A. mon te reyens i s , the compos i t i on o f the t e r p e n o i c a c i d g l y c e r i d e s was q u i t e c o n s i s t e n t i n a l l c o l l e c t i o n s . The cause of t h i s apparent d i s c r e p a n c y i s not known. 130. 108 OH 109 An incorporation study with 1 4C labeled mevalonic acid indicated that the two Archidoris species are capable of terpene biosynthesis. Care was taken throughout t h i s experiment to e l i m i n a t e t r a c e contaminants and t h e i r a s s o c i a t e d r a d i o a c t i v i t y . We are c o n f i d e n t that the measured r a d i o a c t i v i t y resulted from de novo incorporation of the labeled precursor into the terpene residues. This i s only the second instance • where d i r e c t biosynthesis of secondary metabolites has been demonstrated i n nudibranchs. The p h y s i o l o g i c a l function of nudibranch skin chemicals i s o f t e n presumed to be defensive, but e v a l u a t i n g a substance's defensive p o t e n t i a l remains a problem. Antifeedant bioassays require a negative response from the assay organism, making i t d i f f i c u l t to avoid subjective interpretation of the test subjects behavior. The bioassay of Archidoris constituents u t i l i z e d f i s h that were active and voracious feeders. Rejection of food p e l l e t s treated 131. with the sesquiterpenoic ac id g lycer ide ^0 and g l y c e r y l e ther 23 was i n such dramat ic c o n t r a s t to the r a p i d consumption of the other treated p e l l e t s that we f e e l these metabolites can t rue ly i n h i b i t feeding by f i s h . EXPERIMENTAL G e n e r a l The 1H and 1 3 C NMR s p e c t r a were r e c o r d e d on B r u k e r WH-400, B r u k e r WP-80, N i c o l e t - O x f o r d 270, V a r i a n XL-100 and V a r i a n CFT-20 s p e c t r o m e t e r s . T e t r a m e t h y l s i l a n e was employed a s a n i n t e r n a l s t a n d a r d a n d C D C 1 3 a s s o l v e n t u n l e s s o t h e r w i s e i n d i c a t e d . L o w - r e s o l u t i o n mass s p e c t r a were r e c o r d e d on a n A . E . I . MS-902 s p e c t r o m e t e r a n d h i g h -r e s o l u t i o n mass s p e c t r a w e r e r e c o r d e d o n a n MS-50 s p e c t r o m e t e r . I n f r a r e d s p e c t a w e r e r e c o r d e d o n a P e r k i n - E l m e r model 710B s p e c t r o m e t e r w i t h CHC1 3 as s o l v e n t and u l t r a v i o l e t a b s o r b a n c e s were measured w i t h a C a r y - 1 4 o r B a u s c h and Lomb S p e c t r o n i c 2000 s p e c t r o p h o t o m e t e r . O p t i c a l r o t a t i o n s w e r e m e a s u r e d o n a P e r k i n - E l m e r m o d e l 141 p o l a r i m e t e r u s i n g a 10 cm c e l l and CHC1 3 o r MeOH as s o l v e n t . A F i s h e r - J o h n s a p p a r a t u s was u s e d t o d e t e r m i n e m e l t i n g p o i n t s and t h e s e v a l u e s a r e u n c o r r e c t e d . Merck S i l i c a G e l 60 PF-254 was u s e d f o r p r e p a r a t i v e TLC, Merck S i l i c a G e l 230-400 Mesh was u s e d f o r f l a s h c h r o m a t o g r a p h y a n d M e r c k S i l i c a G e l 60 P F - 2 5 4 w i t h C a S 0 1 + , l / 2 H 2 0 was employed i n r a d i a l TLC. H i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y was p e r f o r m e d on a P e r k i n - E l m e r S e r i e s 2 i n s t r u m e n t w i t h a P e r k i n - E l m e r LC-25 r e f r a c t i v e i n d e x d e t e c t o r . A Whatman Magnum-9 P a r t i s i l 10 column was u s e d f o r p r e p a r a t i v e HPLC. A l l c h r o m a t o g r a p h y s o l v e n t s were HPLC 1 3 3 . o r r e a g e n t g r a d e . G a s c h r o m a t o g r a p h y w a s p e r f o r m e d o n a H e w l e t t - P a c k a r d 5 8 3 0 A i n s t r u m e n t u t i l i z i n g a t h e r m a l c o n d u c t i v i t y o r f l a m e i o n i z a t i o n d e t e c t o r . C o l l e c t i o n D a t a A l l s p e c i m e n s w e r e c o l l e c t e d b y h a n d u s i n g S C U B A . C o l l e c t i o n s w e r e m a d e a t a d e p t h o f 1 - 2 0 m i n B a r k l e y S o u n d , B r i t i s h C o l u m b i a . S a m p l e s w e r e i m m e d i a t e l y i m m e r s e d i n m e t h a n o l o r c h l o r o f o r m a n d s t o r e d a t l o w t e m p e r a t u r e . E x t r a c t i o n a n d C h r o m a t o g r a p h y o f T r i o p h a m i n e ( 7 5 ) A v a r i e t y o f p r o c e d u r e s w e r e e m p l o y e d t o e x t r a c t a n d p u r i f y t r i o p h a m i n e . T h e f o l l o w i n g s e q u e n c e w a s f o u n d t o b e t h e m o s t e f f i c i e n t . S e v e n t y - f i v e f r e s h l y c o l l e c t e d s p e c i m e n s o f T r i o p h a c a t a l i n a e w e r e i m m e d i a t e l y i m m e r s e d i n m e t h a n o l . T h e s o l v e n t w a s d e c a n t e d a n d t h e n u d i b r a n c h s w a s h e d 3 t i m e s w i t h 5 0 0 m i m e t h a n o l . E v a p o r a t i o n o f t h e c o m b i n e d e x t r a c t s g a v e a n a q u e o u s s u s p e n s i o n t h a t w a s p a r t i t i o n e d b e t w e e n w a t e r a n d c h l o r o f o r m . T h e c h l o r o f o r m f r a c t i o n w a s d r i e d o v e r s o d i u m s u l f a t e a n d e v a p o r a t e d t o y i e l d a d a r k v i s c o u s o i l ( 3 . 1 g , 7 . 8 % d r y w e i g h t ) . T h e o i l w a s f r a c t i o n a t e d b y f l a s h c h r o m a t o g r a p h y o n a c o l u m n o f s i l i c a g e l ( 1 6 0 g ) u s i n g 1 5 % e t h y l a c e t a t e i n h e x a n e a s e l u a n t . T r i o p h a m i n e w a s v i s u a l i z e d a s a s t r o n g UV ( 2 5 4 nm) a b s o r b i n g s p o t o n T L C . F r a c t i o n s f r o m t h e c o l u m n w h i c h c o n t a i n e d t r i o p h a m i n e w e r e f u r t h e r p u r i f i e d b y p r e p a r a t i v e 1 3 4 . t h i n - l a y e r c h r o m a t o g r a p h y ( 1 : 1 h e x a n e / d i e t h y l e t h e r , R f = 0 . 5 ) t o y i e l d 75 ( 5 1 mg) a s a l i g h t y e l l o w o i l . I n s o m e i n s t a n c e s , f i n a l p u r i f i c a t i o n b y H P L C o n P a r t i s i l - 1 0 w i t h c h l o r o f o r m w a s r e q u i r e d t o r e m o v e a p e r s i s t e n t l i p i d i m p u r i t y . 7_5 o i l ; [ a ] D = - 7 ° ( C = 1 . 7 , M e O H ) ; U V ^ m a x = 2 5 1 n m ( e 1 2 0 0 0 , M e O H ) ; H R M S o b s . 3 6 3 . 2 8 8 5 , C 2 1 H 3 7 N 3 ° 2 c a l * 3 6 3 . 2 8 8 5 ; I R ( C H C 1 3 ) 3 3 3 0 , 1 7 0 0 , 1 6 3 5 , 1 1 6 5 c m - 1 ; ' H NMR ( C D C l g ) 8 0 . 9 1 ( t , J = 7 H z , 3H ) , 0 . 9 5 ( t , J = 7 H z , 3 H ) , 1 . 5 6 ( d , J = 7 H z , 3 H ) , 1 . 4 6 - 1 . 6 7 ( m , 2 H ) , 2 . 0 1 ( q , J = 7 H z , 2 H ) , 2 . 1 2 ( d d , J = 6 , 1 3 H z , I H ) , 2 . 3 0 - 2 . 4 8 ( m , 2 H ) , 5 . 2 1 ( q , J = 7 H z , I H ) p p m ; 1 3 C NMR ( 1 0 0 M H z , a c e t o n e - d 6 ) 6 1 2 . 1 ( q ) , 1 2 . 9 ( q ) , 1 3 . 1 ( q ) , 2 3 . 2 ( t ) , 2 6 . 1 ( t ) , 4 0 . 1 ( t ) , 5 0 . 3 ( d ) , 1 2 0 . 5 ( d ) , 1 4 0 . 4 ( s ) , 1 5 8 . 9 ( s ) , 1 8 5 . 6 ( s ) p p m ; M S , m / z 3 6 3 ( 6 0 ) , 3 4 8 ( 1 0 ) , 3 3 4 ( 2 5 ) , 2 8 1 ( 2 5 ) , 2 3 8 ( 6 0 ) , 2 2 3 ( 5 0 ) , 2 0 5 ( 4 5 ) , 8 6 ( 1 0 0 ) . T h e m e t h a n o l e x t r a c t s o f 3 0 s p e c i m e n s o f P o l y c e r a t r i c o l o r w e r e f r a c t i o n a t e d i n a n i d e n t i c a l m a n n e r t o p r o v i d e 5 mg o f t r i o p h a m i n e (7_5) . H y d r o g e n a t i o n o f T r i o p h a m i n e ( 7 5 ) A s o l u t i o n o f 7_5 ( 8 mg) i n e t h a n o l w i t h 5% P d o n c h a r c o a l a s c a t a l y s t w a s s t i r r e d u n d e r H 2 f o r 4 8 h . T h e c h a r c o a l w a s f i l t e r e d o f f a n d t h e s o l u t i o n e v a p o r a t e d t o y i e l d a y e l l o w o i l . P r e p a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y ( 1 : 1 h e x a n e / e t h e r , R f = 0 . 4 ) p r o v i d e d 4 mg ( 4 9 % y i e l d ) o f t e t r a h y d r o t r i o p h a m i n e ( 7 8 ) . H R M S , o b s 3 6 7 . 3 1 9 8 , c 2 i H i + i N 3 0 2 c a l . 3 6 7 . 3 1 9 8 ; ' H NMR ( 2 7 0 M H z , C D C 1 3 ) 6 0 . 8 3 ( t , J = 7 H z , 1 3 5 . 3 H ) , 0 . 8 4 ( t , J = 7 H z , 3 H ) , 0 . 9 3 ( t , J = 7 H z , 3 H ) , 1 . 1 5 - 1 . 4 0 ( m , 6 H ) , 1 . 5 5 - 1 . 6 5 ( m , 3 H ) , 2 . 7 9 ( m , I H ) p p m ; M S , m / z 3 6 7 ( 1 0 ) , 3 3 8 ( 1 5 ) , 2 9 6 ( 3 0 ) , 2 8 3 ( 7 5 ) 2 6 8 ( 3 0 ) , 2 4 0 ( 7 0 ) . M o n o a c y l t r i o p h a m i n e 8 1 A m e t h a n o l i c s o l u t i o n o f t r i o p h a m i n e ( 1 0 mg) w a s t r e a t e d w i t h s e v e r a l d r o p s o f 1 N N a O H a n d s t i r r e d a t r o o m t e m p e r a t u r e f o r 2 h . T h e r e a c t i o n m i x t u r e w a s e v a p o r a t e d i n  v a c u o a n d p a r t i t i o n e d b e t w e e n w a t e r a n d e t h y l a c e t a t e . T h e e t h y l a c e t a t e s o l u b l e m a t e r i a l w a s p u r i f i e d b y p r e p a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y ( 9 : 1 C H C l 3 / M e O H R f = 0 . 1 ) t o y i e l d 2 mg ( 3 4 % y i e l d ) o f 8 1 . UV \ m a x = 2 3 2 ( e 1 0 0 0 0 , M e O H ) ; M S , m / z 2 1 1 ( M + ) , 1 9 6 ( 1 0 ) , 1 8 2 ( 1 5 ) , 1 2 9 ( 1 5 ) , 8 6 ( 1 0 0 ) . D i m e t h y l p y r i m i d i n e D e r i v a t i v e o f G u a n i d i n e 8 2 T o 1 5 mg o f t r i o p h a m i n e (7_5) i n 1 . 0 mjl MeOH w a s a d d e d 5 d r o p s o f 5 N N a O H . T h e r e a c t i o n w a s s t i r r e d a t r o o m t e m p e r a t u r e f o r 2 4 h a t w h i c h t i m e g u a n i d i n e c o u l d b e d e t e c t e d b y T L C ( 6 0 : 3 5 : 5 C H C 1 3 / M e O H / a c e t i c a c i d , R f = 0 . 3 ) u s i n g t h e F C N P s p r a y r e a g e n t 5 6 . T h e s o l u t i o n w a s a c i d i f i e d w i t h H C 1 a n d p a r t i t i o n e d b e t w e e n H 2 0 a n d e t h e r . T h e a q u e o u s l a y e r w a s e v a p o r a t e d t o d r y n e s s y i e l d i n g 5 mg o f F C N P p o s i t i v e m a t e r i a l . T h i s w a s s t i r r e d w i t h 1 2 mg N a H C 0 3 a n d 1 . 0 mA 2 , 4 - p e n t a n e d i o n e a t 1 3 0 ° f o r 3 0 m i n . T h e r e a c t i o n m i x t u r e w a s e v a p o r a t e d i n v a c u o a n d t r i t u r a t e d w i t h C H C 1 3 . P r e p a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y ( 1 : 4 a c e t o n i t r i l e / e t h y l a c e t a t e , R f = 0 . 1 5 ) o f t h e C H C 1 3 s o l u b l e m a t e r i a l p r o d u c e d 1 . 0 m g ( 2 0 % y i e l d o v e r a l l ) o f t h e 4 , 6 -d i m e t h y l p y r i m i d i n e d e r i v a t i v e 8 2 . UV \ m a x = 2 2 8 a n d 2 8 9 nm ( M e O H ) ; M S , m / z 1 2 3 ( M + ) ; ' H NMR ( 8 0 M H z , C D C l g ) 6 2 . 3 1 ( s , 6 H ) , 5 . 0 5 ( b s , 2 H ) , 6 . 3 8 ( s , I H ) p p m . C_i Q - C a r b o x y l i c A c i d 8 3 A f t e r r e p e a t e d a t t e m p t s , t h e C ^ 0 - c a r b o x y l i c a c i d 8_3 w a s i s o l a t e d i n l o w y i e l d f r o m b a s e - c a t a l y z e d h y d r o l y s i s o f t r i o p h a m i n e (7_5) . T o 1 5 mg o f 7_5 i n 3 m i MeOH w a s a d d e d 1 m i o f 1 N N a O H . A f t e r r e f l u x i n g f o r l h , 5 m i H 2 0 w a s a d d e d a n d t h e m i x t u r e 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 . T h e a q u e o u s l a y e r w a s a c i d i f i e d w i t h H C 1 a n d r a p i d l y 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 (3 x 1 5 m i ) . T h e e t h y l a c e t a t e f r a c t i o n s w e r e d r i e d o v e r N a 2 S 0 ( t a n d p u r i f i e d b y p r e p a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y ( 1 0 0 : 1 : 1 C H C 1 3 / M e O H / a c e t i c a c i d , R f = 0 . 3 ) t o p r o v i d e 1 . 5 mg ( 1 1 % y i e l d ) o f 8 3 . H R M S , o b s 1 7 0 . 1 2 9 8 , C 1 Q H 1 8 0 2 c a l . 1 7 0 . 1 3 0 7 ; I R ( C H C 1 3 ) 3 4 0 0 - 2 4 0 0 , 1 7 0 0 c m " 1 ; ' H NMR ( 4 0 0 M H z , C D C l g ) 6 0 . 9 5 ( t , J = 7 H z , 3 H ) , 0 . 9 7 ( t , J = 7 H z , 3 H ) , 1 . 5 7 ( d , J = 7 H z , 3 H ) , 1 . 5 2 - 1 . 6 2 ( m , 2 H ) , 2 . 0 4 ( q , J = 7 H z , 2 H ) , 2 . 1 4 ( d d , J = 7 , 1 5 H z , I H ) , 2 . 3 3 ( d d , J = 9 , 1 5 H z , I H ) , 2 . 4 7 (m , I H ) , 5 . 2 3 ( q , J = 7 H z , I H ) p p m ; M S , m/z 1 7 0 ( 2 0 ) , 1 4 1 ( 1 5 ) , 8 3 ( 8 5 ) , 7 3 ( 7 0 ) , 5 5 ( 1 0 0 ) . I f e x t r a c t i o n o f t h e a c i d i f i e d h y d r o l y s a t e i s n o t c a r r i e d o u t p r o m p t l y , t h e C . 0 - a c i d 8_3 l a c t o n i z e s t o t h e Y ~ l a c t o n e 9 5 . 137. 95, IR 1750 c m - 1 ; 'H NMR (100 MHz, CDClg) 60.93 ( t , J = 7Hz, 6H), 0.99 ( t , J = 7Hz, 3H), 1.55-1.80 (m, 8H), 2.62 (m, IH) ppm. D i a c e t y l g u a n i d i n e 80 To 390 mg o f g u a n i d i n e c a r b o n a t e was added an e x c e s s o f f r e s h l y d i s t i l l e d a c e t i c a n h y d r i d e . The m i x t u r e was k e p t u n d e r N 2 and h e a t e d t o 100°C f o r 90 min. E x c e s s a c e t i c a n h y d r i d e was e v a p o r a t e d i n v a c u o and t h e r e s u l t i n g s o l i d t r i t u r a t e d i n a c e t o n e . The a c e t o n e s o l u b l e m a t e r i a l was p u r i f i e d by p r e p a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y (1:4 a c e t o n e / C H C l g , R f = 0.3) t o g i v e 87 mg (28% y i e l d ) o f 80. UV \ m a x = 248nm ( e 16800, MeOH), \ m a x = 212nm (e 19900, MeOH/HCl); IR 3350, 1710, 1645 c m - 1 ; MS m/z 143 ( M + ) ; 1 3 C NMR (20 MHz, a c e t o n e - d g ) 626.0, 159.0, 180.1 ppm. B a s e - c a t a l y z e d h y d r o l y s i s o f 8 £ w i t h m e t h a n o l i c NaOH a t room t e m p e r a t u r e r a p i d l y p r o d u c e d m o n o a c e t y l g u a n i d i n e [UV X m a x = 230 nm ( e 14300, MeOH)]. p - N i t r o p h e n y l ( E ) - 2 , 4 - D i e t h y l - 4 - H e x e n o a t e (96) To a s t i r r e d s o l u t i o n o f t h e c a r b o x y l i c a c i d 83a (120 mg, 0.71 mmol) and p - n i t r o p h e n o l (150 mg, 1.08 mmol) i n 3 ml o f d r y a c e t o n i t r i l e was added 300 mg (1.47 mmol) o f N, N 1 - d i c y c l o h e x y l c a r b o d i i m i d e , and t h e r e s u l t a n t m i x t u r e was s t i r r e d a t room t e m p e r a t u r e f o r 12h. Removal o f t h e s o l v e n t u n d e r r e d u c e d p r e s s u r e p r o v i d e d a s o l i d w h i c h was t r i t u r a t e d 138. w i t h c h l o r o f o r m . F i l t r a t i o n o f t h e m i x t u r e , f o l l o w e d by r e m o v a l o f t h e s o l v e n t f r o m t h e f i l t r a t e , gave a s o l i d w h i c h was p u r i f i e d by f l a s h c h r o m a t o g r a p h y on 28 g o f s i l i c a g e l . E l u t i o n o f t h e column w i t h 1:3 h e x a n e - c h l o r o f o r m a f f o r d e d 180 mg ( 8 7 % y i e l d ) o f t h e d e s i r e d e s t e r £ 6 . mp 4 5 - 4 6 ° ; HRMS, o b s . 291.1496, C ^ H ^ N O ^ c a l . 291.1471; IR ( C H C l g ) , 1755 c m " l ; *H NMR (100 MHz, C D C l j ) 61.01 ( t , J = 7 Hz, 3H) , 1.04 ( t , J = 7 Hz, 3H), 1.42-1.86 (m, 2H), 1.62 (d, J = 7Hz, 3H), 2.13 (q, J = 7Hz, 2H) , 2.28-2.85 (m, 3H) , 5.33 (q, J = 7Hz, I H ) , 7.23 (d, J = 9Hz, 2H), 8.31 (d, J = 9Hz, 2H) ppm. (±)- T r i o p h a m i n e (75a) S u b j e c t i o n o f 14.3 g o f g u a n i d i n e h y d r o c h l o r i d e t o i o n - e x c h a n g e c h r o m a t o g r a p h y on a column o f 64.5 g o f Dowex 1-X8 (0H~ form) p r o v i d e d f r e e g u a n i d i n e as a h y g r o s c o p i c s o l i d . S m a l l amounts o f t h e l a t t e r m a t e r i a l were added a t l h i n t e r v a l s t o a s t i r r e d s o l u t i o n o f t h e p - n i t r o p h e n y l e s t e r 96 (34 mg, 0.12 mmol) i n 2 mi o f c h l o r o f o r m . The p r o g r e s s o f t h e r e a c t i o n was m o n i t o r e d by TLC ( s i l i c a g e l , 1:1 h e x a n e / d i e t h y l e t h e r ) . I t was f o u n d t h a t i f t h e r e a c t i o n was a l l o w e d t o p r o c e e d t o c o m p l e t i o n ( d i s a p p e a r a n c e o f 9 6 ) , a p p r e c i a b l e q u a n t i t i e s o f s i d e p r o d u c t ( s ) were f o r m e d a n d t h e y i e l d o f d e s i r e d m a t e r i a l was l o w . T h e r e f o r e , a f t e r a p p r o x i m a t e l y 50-75% o f t h e s t a r t i n g m a t e r i a l 96 h a d r e a c t e d , t h e r e a c t i o n m i x t u r e was f i l t e r e d and t h e c o l l e c t e d s o l i d was washed w i t h 4 x 5 m i o f 139. c h l o r o f o r m . Removal of the s o l v e n t from the combined f i l t r a t e gave a y e l l o w o i l which was s u b j e c t e d t o p r e p a r a t i v e t h i n - l a y e r chromatography. There was thus obtained 9.1 mg of the e s t e r 96 and 7.4 mg (48% y i e l d based on unrecovered 96) of a mixture of (±)-triophamine 75a and i t s diastereomers 75b. T h i s mixture was separated by r e p e a t e d ( t h r e e d e velopments) p r e p a r a t i v e t h i n - l a y e r c h r o m o t o g r a p h y on s i l i c a g e l 60 P F - 2 5 4 ( 1 9 : 1 h e x a n e / i s o p r o p y l a l c o h o l ) to g i v e 4.0 mg (26% y i e l d ) of (± )-triophamine which e x h i b i t e d i n f r a r e d , mass and 'H NMR s p e c t r a i d e n t i c a l with those of the n a t u r a l product. The diastereomers (2.5 mg; 16% y i e l d ) p r o v i d e d s p e c t r a which were s i m i l a r t o , b u t c l e a r l y d i f f e r e n t from n a t u r a l triophamine. The s p e c t r a l f e a t u r e s of the diastereomers are as f o l l o w s : HRMS obs. 363.2883, C 2 1 H 3 7 N 3 0 2 c a l . 363.2885; IR (CHC1 3 ) 3323, 1700, 1630, 1160 cm - 1; 'H NMR (400 MHz, CDC1 3) 60.91 ( t , J = 7Hz, 3H), 0.95 ( t , J = 7Hz, 3H), 1.46-1.67 (m, 2H), 1.56 (d, J = 7Hz, 2H) , 2.03 (q, J = 7Hz, 2H), 2.11 (dd, J = 6, 13Hz, IH), 2.28-2.37 (m, IH), 2.37-2.47 (m, IH), 5.20 (q, J = Hz, IH) ppm. E x t r a c t i o n and Chromatography of D i h y d r o a p o f a r n e s a l (15 ) A t o t a l of 22 specimens of A n i s o d o r i s n o b i l i s were e x t r a c t e d w i t h c h l o r o f o r m (2 x 800 mi). The c h l o r o f o r m was d r i e d over N a 2S0 4 and evaporated to o b t a i n a p l e a s a n t s m e l l i n g l i g h t brown o i l ( 820 mg) . The o i l was 1 4 0 . s e p a r a t e d b y f l a s h c h r o m o t o g r a p h y o n s i l i c a g e l ( h e x a n e -c h l o r o f o r m s t e p g r a d i e n t ) . M a t e r i a l e l u t i n g w i t h 1 5 % c h l o r o f o r m i n h e x a n e w a s f u r t h e r p u r i f i e d b y r a d i a l t h i n - l a y e r c h r o m a t o g r a p h y u s i n g 1 0 0 % h e x a n e a s t h e e l u a n t . F i n a l p u r i f i c a t i o n w a s a c h i e v e d b y p r e p a r a t i v e GC ( 3 % O V - 1 7 o n C h r o m o s o r b , i n i t i a l t e m p e r a t u r e 1 0 0 ° , r a t e 4 ° / m i n , RT 1 4 m i n . ) p r o v i d i n g 2 . 8 mg o f 1_5 a s a s w e e t s m e l l i n g , c o l o r l e s s o i l . H R M S , o b s . 2 0 8 . 1 8 2 5 , C 1 1 + H 2 1 + 0 c a l . 2 0 8 . 1 8 2 3 ; ' H NMR ( 4 0 0 M H z , C D C 1 3 ) 6 1 . 1 0 ( d , J = 7 H z , 3 H ) , 1 . 4 2 ( m , I H ) , 1 . 6 0 ( s , 3 H ) , 1 . 6 1 ( s , 3 H ) 1 . 6 9 ( s , 3 H ) , 1 . 7 8 ( m , I H ) , 1 . 9 6 - 2 . 1 3 ( m , 6 H ) , 2 . 3 6 ( m , I H ) , 5 . 1 2 ( m , 2 H ) , 9 . 5 8 ( d , J = 1 H z , I H ) p p m ; M S , m/z 2 0 8 ( 5 ) , 1 9 0 ( 5 ) , 1 7 5 ( 3 ) , 1 6 5 ( 5 0 ) , 1 5 0 ( 2 0 ) , 9 5 ( 2 5 ) , 81 ( 7 0 ) , 6 9 ( 1 0 0 ) . E x t r a c t i o n a n d C h r o m a t o g r a p h y o f A r c h i d o r i s M e t a b o l i t e s A t o t a l o f 8 0 s p e c i m e n s o f A r c h i d o r i s m o n t e r e y e n s i s w e r e e x t r a c t e d w i t h m e t h a n o l ( 3 x 1 j).) f o r 3 d a y s . T h e c o m b i n e d m e t h a n o l e x t r a c t s w e r e e v a p o r a t e d t o a n a q u e o u s s u s p e n s i o n a n d e x t r a c t e d w i t h c h l o r o f o r m (3 x 5 0 0 mj.) . T h e c o m b i n e d e x t r a c t s w e r e d r i e d o v e r s o d i u m s u l f a t e a n d e v a p o r a t e d t o y i e l d a b r o w n g u m ( 2 . 7 g , 8 . 3 % d r y w e i g h t , 3 3 . 7 m g / a n i m a l ) . T h e g u m ( 1 . 5 g ) w a s s u b j e c t e d t o f l a s h c h r o m a t o g r a p h y o n a c o l u m n o f s i l i c a g e l ( 1 3 0 g ) w i t h a s t e p g r a d i e n t o f h e x a n e - e t h y l a c e t a t e m i x t u r e s . E l u t i o n w i t h 2 0 % e t h y l a c e t a t e i n h e x a n e r e m o v e d n o n - p o l a r f a t s a n d p i g m e n t s . T h e 141. a c e t y l a t e d g l y c e r i d e s were e l u t e d w i t h a 1:1 s o l u t i o n o f h e x a n e - e t h y l a c e t a t e w h i l e t h e n o n - a c e t y l a t e d g l y c e r i d e s were e l u t e d w i t h 100% e t h y l a c e t a t e . M a t e r i a l e l u t e d w i t h (1:1) h e x a n e - e t h y l a c e t a t e was f r a c t i o n a t e d by r a d i a l t h i n - l a y e r c h r o m a t o g r a p h y ( s i l i c a g e l ) , e l u t i n g s o l v e n t c h l o r o f o r m - m e t h a n o l ( 9 9 : 1 ) . F i n a l s e p a r a t i o n was a c h e i v e d b y LC o n P a r t i s i l - 1 0 w i t h h e x a n e - e t h y l a c e t a t e - m e t h a n o l (85:15:1) e l u a n t t o y i e l d i n t h e o r d e r o f t h e i r e l u t i o n , compounds 18_ (24 mg; 1.6% o f c r u d e e x t r a c t ) , 21. ( 2 m 9 " 0*13% °f c r u d e e x t r a c t ) and IS) (3 mg; 0.2% o f c r u d e e x t r a c t ) . 18 mp 117-119° ( H e x a n e / E t 2 0 ) ; [ a ] D = -53.7° (C = 0.13, C H C 1 3 ) ; HRMS o b s . 420.2884, C 2 5 H 4 Q 0 5 c a l . 420.2876; IR (CHC1 3) 3600-3350, 2930, 1730, 1230 c m - 1 ; 'H NMR (400 MHz, CDC1 3) 6 0.82 ( s , 3H), 0.87 ( s , 3H), 0.92 ( s , 3H), 0.96 ( s , 3H), 1.61 ( s , 3H), 2.11 ( s , 3H), 2.41 (bs , IH, e x c h a n g a b l e ) , 2.97 ( b s , I H ) , 4.08-4.25 (m, 5H), 5.53 (bs, I H ) ; MS, m/z 420 ( 2 ) , 402 ( 5 ) , 347 ( 5 ) , 286 ( 1 0 0 ) , 192 ( 6 5 ) , 177 ( 7 0 ) , 117 (60) . 21 o i l ; [<x] D = +15.3° (C = 1.2, C H C 1 3 ) ; HRMS o b s . 352.2221, C 2 0 H 3 2 O 5 c a l . 352.2250, IR (CHClg) 3400, 2930, 1720, 1230 c m - 1 ; 'H NMR (400 MHz, CDC1 3) 6 0.88 ( s , 3H), 0.92 ( s , 3H), 0.98 ( s , 3H), 1.61 ( s , 3H), 2.11 ( s , 3H), 2.48 (b s , IH, e x c h a n g a b l e ) , 2.95 ( b s , I H ) , 4.05-4.24 (m, 5H), 5.55 ( b s , I H ) . MS, m/z 352 ( 3 ) , 334 (4) 279 ( 5 ) , 218 ( 5 5 ) , 190 ( 4 5 ) , 124 ( 8 0 ) , 117 ( 8 0 ) , 109 ( 1 0 0 ) , 95 ( 8 0 ) . 1 4 2 . 1 9 o i l ; [ a ] D = - 3 3 . 0 ° ( C = 0 . 8 3 , C H C 1 3 ) ; H R M S o b s . 4 2 0 . 2 8 7 6 , C 2 5 H l + Q 0 5 c a l . 4 2 0 . 2 8 7 6 ; I R ( C H C l g ) 3 6 0 0 - 3 2 1 0 , 2 9 2 5 , 1 7 2 5 , 1 2 3 0 , 1 0 5 5 c m ~ l ; ' H NMR ( 4 0 0 M H z , C D C l g ) 6 0 . 8 2 ( s , 3 H ) , 0 . 8 7 ( s , 3 H ) , 0 . 9 1 ( s , 3H ) , 0 . 9 4 ( s , 3 H ) , 1 . 6 0 ( s , 3 H ) , 2 . 0 9 ( s , 3 H ) , 2 . 9 4 ( b s , I H ) , 3 . 7 6 ( d , J = 6 H z , 2 H ) , 4 . 2 6 ( d d , J = 7 a n d 1 2 H z , I H ) , 4 . 3 2 ( d d , J = 6 a n d 1 3 H z , I H ) , 5 . 0 6 ( m , I H ) , 5 . 5 2 ( b s , I H ) ; M S , m/z 4 2 0 ( 5 ) , 4 0 2 ( 3 ) , 2 8 6 ( 9 0 ) , 2 5 8 ( 2 0 ) , 1 9 2 ( 5 5 ) , 1 9 1 ( 5 0 ) , 1 7 7 ( 7 5 ) , 1 1 7 ( 1 0 0 ) , 95 ( 8 0 ) , 81 ( 8 0 ) . T h e 1 0 0 % e t h y l a c e t a t e f r a c t i o n f r o m t h e f l a s h c o l u m n w a s s u b j e c t e d t o r a d i a l t h i n - l a y e r c h r o m a t o g r a p h y e l u t i n g w i t h c h l o r o f o r m - m e t h a n o l ( 9 5 : 5 ) . F i n a l s e p a r a t i o n w a s a c h i e v e d b y L C o n P a r t i s i l - 1 0 w i t h h e x a n e - e t h y l a c e t a t e - m e t h a n o l ( 5 0 : 4 0 : 2 ) e l u a n t t o y i e l d i n t h e o r d e r o f t h e i r e l u t i o n , c o m p o u n d s 1J7 ( 3 8 m g ; 2 . 5 % o f c r u d e e x t r a c t ) , 23 (2 m g ; 0 . 1 3 % o f c r u d e e x t r a c t ) , 2 0 (3 m g ; 0 . 2 % o f c r u d e e x t r a c t ) a n d 22^ (2 m g ; 0 . 1 3 % o f c r u d e e x t r a c t ) . 1 7 , mp = 1 2 5 - 1 2 6 ° ( H e x a n e / E t 2 0 ) , [ a ] D = - 1 2 . 5 ° ( c = 0 . 4 , C H C 1 3 ) ; H R M S , o b s . 3 7 8 . 2 7 7 2 , C ^ H ^ C ^ c a l . 3 7 8 . 2 7 7 0 ; I R ( C H C 1 3 ) 3 6 0 0 - 3 3 0 0 , 2 9 0 0 , 2 8 5 0 , 1 7 3 0 , 1 4 6 0 , 1 1 7 0 c m - l ~ ; *H NMR ( 4 0 0 M H z , C D C 1 3 ) 6 0 . 8 2 ( s , 3 H ) , 0 . 8 7 ( s , 3 H ) , 0 . 9 2 ( s , 3 H ) , 0 . 9 6 ( s , 3 H ) , 1 . 6 1 ( b s , 3 H ) , 1 . 9 2 - 1 . 9 9 ( m , 2 H ) , 2 . 4 4 ( b s , 2 H , e x c h a n g a b l e ) , 2 . 9 6 ( b s , I H ) , 3 . 6 3 ( d d , J = 6 a n d 1 2 H z , I H ) , 3 . 7 0 ( d d , J = 4 a n d 1 2 H z , I H ) , 3 . 9 5 ( m , I H ) , 4 . 1 5 ( d d , J = 7 a n d 12 H z , I H ) , 4 . 2 2 ( d d , J = 5 a n d 1 2 H z , I H ) , 5 . 5 4 ( b s , I H ) p p m ; 1 3 C NMR ( 1 0 0 M H z , C D C 1 3 ) 1 5 . 6 3 ( q ) , 1 5 . 7 3 ( q ) , 143. 18.52 ( q ) , 18.73 ( q ) , 21.15 ( t ) , 21.68 ( t ) , 22.74 ( t ) , 33.20 ( s ) , 33.43 ( q ) , 36.70 ( s ) , 37.53 ( s ) , 40.01 ( t ) , 41.95 ( t ) , 54.45 ( d ) , 56.58 ( d ) , 62.76 ( d ) , 63.64 ( t ) , 65.17 ( t ) , 70.47 ( d ) , 124.32 ( d ) , 128.60 ( s ) , 173.35 ( s ) ppm; MS, m/z 378 ( 3 0 ) , 363 ( 5 ) , 347 ( 1 0 ) , 286 ( 6 5 ) , 258 ( 3 5 ) , 192 ( 1 0 0 ) , 177 ( 8 0 ) , 95 ( 7 5 ) . 23 HRMS, o b s . 316.2973, C 1 9 H 1 + O 0 3 c a l . 316.2977; IR (CHC1 3) 3600-3250, 2900, 1110 c m - 1 ; 'H NMR (400 MHz, CDC1 3) 6 0.86 ( t , J = 7 Hz, 3H), 1.26 (bs , 28H), 1.59 (m, 2H), 3.44 ( t , J = 6 Hz, 2H), 3.50 (m, 2H), 3.64 (dd, J = 6 and 12 Hz, I H ) , 3.67 (dd, J = 4 and 12 Hz, I H ) , 3.82 (m, IH) ppm; MS, m/z 316 ( 2 ) , 285 ( 5 ) , 255 ( 1 5 ) , 225 ( 1 5 ) , 71 ( 8 0 ) , 57 ( 1 0 0 ) . 20 mp = 9 4 - 9 5 ° ; [ a ] D = +23.1 (c = 0.93, C H C 1 3 ) ; HRMS, o b s . 310.2142, c 1 8 H 3 o ° ( + c a l ' 310.2144; IR (CHClg) 3600-3300, 2900, 1730, 1165 c m _ l ; 'H NMR (400 MHz,CDCl 3) 6 0.89 ( s , 3H), 0.92 ( s , 3H), 0.98 ( s , 3H), 1.62 ( b s , 3H) , 2.48 ( b s , 2H, e x c h a n g a b l e ) , 2.96 ( b s , I H ) , 3.63 (dd, J = 6 and 12 Hz, I H ) , 3.70 (dd, J = 4 and 12 Hz, I H ) , 3.95 (m, I H ) , 4.15 (dd, J = 7 and 12 Hz, I H ) , 4.22 (dd, J = 5 and 12 Hz, I H ) , 5.57 (bs, IH) ppm; 1 3 C NMR (100 MHz, CDC1 3) 614.9, 18.7, 21.3, 21.9, 23.7, 33.1, 33.3, 40.5, 42.2, 42.3, 49.5, 62.3, 63.6, 65.2, 70.5, 124.6, 128.8, 173.4 ppm; MS, m/z 310 ( 1 5 ) , 295 ( 5 ) , 279 ( 1 0 ) , 218 ( 7 5 ) , 190 ( 4 5 ) , 187 ( 4 0 ) , 124 ( 7 0 ) , 109 (1 0 0 ) , 95 ( 7 5 ) . 12 o i l ; [ a ] D = +9.7° (C = 0.3, C H C 1 3 ) ; UV \ m a x = 228 nm ( e 1800, MeOH); HRMS o b s . 310.2146, C ^ H ^ O ^ c a l . 1 4 4 . 3 1 0 . 2 1 4 4 ; I R ( C H C l g ) 3 4 3 0 , 2 9 4 5 , 1 7 0 0 , 1 6 4 5 , 1 1 5 5 c m " 1 ; ' H NMR ( 4 0 0 M H z , C D C 1 3 ) 6 0 . 8 5 ( s , 3 H ) , 0 . 9 3 ( s , 3 H ) , 2 . 1 6 ( s , 3 H ) , 3 . 6 2 ( d d , J = 1 2 a n d 7 H z , I H ) , 3 . 7 0 ( d d , J = 12 a n d 5 H z , I H ) , 3 . 9 5 ( m , I H ) , 4 . 1 8 ( d d , J = 1 2 a n d 7 H z , I H ) , 4 . 2 4 ( d d , J = 1 2 a n d 5 H z , I H ) , 4 . 5 5 ( s , I H ) , 4 . 7 9 ( s , I H ) , 5 . 7 0 ( s , I H ) ; M S : m / z 3 1 0 ( 2 ) , 2 9 5 ( 5 ) , 2 7 9 ( 5 ) , 2 3 7 ( 1 0 ) , 2 1 9 ( 3 0 ) , 2 0 3 ( 3 5 ) , 1 7 6 ( 8 5 ) , 1 2 4 ( 3 0 ) , 1 0 9 ( 8 5 ) , 9 5 ( 9 0 ) , 8 2 ( 1 0 0 ) , 6 9 ( 9 0 ) . T h e d i t e r p e n o i c a c i d ( s ) t e n t a t i v e l y i d e n t i f i e d a s 1 0 0 a n d / o r 1 0 1 w a s i s o l a t e d f r o m o n l y o n e o f t h e m a n y c o l l e c t i o n s o f A . m o n t e r e y e n s i s m a d e d u r i n g t h e c o u r s e o f t h i s s t u d y . F r a c t i o n a t i o n o f t h e c h l o r o f o r m s o l u b l e m a t e r i a l b y f l a s h c h r o m a t o g r a p h y u s i n g 3 0 % e t h y l a c e t a t e i n h e x a n e a s e l u a n t p r o v i d e d a c r u d e m i x t u r e w h i c h d i d n o t c o n t a i n a n y o f t h e t e r p e n o i c g l y c e r i d e s . F i n a l p u r i f i c a t i o n b y p r e p a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y ( 1 % MeOH i n C H C 1 3 , R f = 0 . 4 ) g a v e 2 . 3 mg o f t h e a c i d ( s ) a s a l i g h t y e l l o w o i l . HRMS 3 0 4 . 2 3 5 6 o b s . C 2 0 H 3 2 ° 2 c a l * 3 0 4 - 2 4 0 2 ' * I R ( C H C 1 3 ) 3 2 2 5 - 2 5 0 0 , 1 6 9 5 , 1 6 5 0 c m " 1 ; ' H NMR ( 4 0 0 M H z , C D C 1 3 ) 6 0 . 8 3 ( s , 3 H ) , 0 . 9 1 ( s , 3 H ) , 1 . 6 0 ( s , 3 H ) , 2 . 2 0 ( b s , 3 H ) , 4 . 5 3 ( b s , I H ) , 4 . 7 6 ( b s , I H ) , 5 . 0 7 ( b s , I H ) , 5 . 7 0 ( s , I H ) p p m ; MS m / z 3 0 4 ( 1 5 ) , 2 8 9 ( 4 0 ) , 1 7 7 ( 5 0 ) , 1 2 4 ( 5 0 ) , 1 0 9 ( 8 5 ) , 81 ( 1 0 0 ) . T e n s p e c i m e n s o f A r c h i d o r i s o d h n e r i w e r e e x t r a c t e d w i t h m e t h a n o l a n d s u b j e c t e d t o a s e p a r a t i o n a n d p u r i f i c a t i o n s c h e m e i d e n t i c a l t o t h a t u s e d w i t h A . m o n t e r e y e n s i s . I n 1 4 5 . a d d i t i o n t o 4 3 mg o f f a r n e s i c a c i d g l y c e r i d e 2 4 ' 1 *5 mg o f t h e p o s i t i o n a l i s o m e r 27_ w e r e r e c o v e r e d . 27 HRMS 3 1 0 . 2 1 4 3 o b s . C 1 8 H 3 Q 0 ^ c a l . 3 1 0 . 2 1 4 4 ; ' H NMR ( 2 7 0 M H z , C D C 1 3 ) 6 1 . 5 6 ( s , 6 H ) , 1 . 6 0 ( s , 3 H ) , 2 . 1 6 ( s , 3 H ) , 3 . 8 1 ( t , J = 6 H z , 4 H ) , 4 . 9 0 ( p e n t e t , J = 7 H z , I H ) , 5 . 0 3 ( b s , 2 H ) , 5 . 6 8 ( s , I H ) p p m ; M S , m / z 3 1 0 ( 1 5 ) , 2 1 9 ( 2 0 ) , 2 1 8 ( 2 0 ) , 8 2 ( 9 0 ) , 6 9 ( 1 0 0 ) . R e p e t a t i v e H P L C s e p a r a t i o n o f A . o d h n e r i e x t r a c t s p r o v i d e d p u r e s a m p l e s o f g l y c e r i d e s 17. a n d 1 8 . T r a c e q u a n t i t i e s o f 2 0 , 22^ a n d 23. w e r e a l s o e v i d e n c e d b y c h a r a c t e r i s t i c r e t e n t i o n t i m e s i n t h e H P L C t r a c e a n d d i a g n o s t i c s i g n a l s i n t h e ' H NMR s p e c t r u m o f a p a r t i a l l y p u r i f i e d m i x t u r e . R e d u c t i o n o f 17 A s o l u t i o n o f 17. ( 2 0 mg) i n 0 . 5 m l o f t o l u e n e w a s s t i r r e d a t RT f o r 8 h i n t h e p r e s e n c e o f e x c e s s D I B A L . A d d i t i o n o f 5 m i o f E t O H , f o l l o w e d b y f i l t r a t i o n o f t h e m i x t u r e a n d e v a p o r a t i o n o f t h e s o l v e n t s , r e s u l t e d i n o n e m a j o r p r o d u c t . P r e p a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y ( 1 : 4 , e t h y l a c e t a t e / h e x a n e R f = 0 . 3 ) p r o v i d e d 1 3 mg ( 8 5 % y i e l d ) o f w h i t e c r y s t a l l i n e 9 7 . mp 1 2 6 - 1 2 7 ° ( h e x a n e / E t 2 0 ) ( l i t . 1 2 5 - 1 2 6 " ) 6 9 ; [ a ] D = - 9 ° (C = 0 . 1 , C H C 1 3 ) , ( l i t . - 9 ° ) 6 9 ; H R M S , o b s . 2 9 0 . 2 6 2 3 , C 2 0 H 3 i ( O c a l . 2 9 0 . 2 6 0 9 ; I R ( C H C 1 3 ) 3 6 0 0 - 3 3 0 0 , 2 9 2 0 , 2 8 5 0 , 1 4 5 0 , 1 3 9 0 c m - ! ; ' H NMR ( 4 0 0 M H z , C D C 1 3 ) 6 0 . 8 2 ( s , 3 H ) , 0 . 8 4 ( s , 3 H ) , 0 . 8 7 ( s , 3 H ) , 0 . 9 0 ( s , 3 H ) , 1 . 7 8 ( s , 3 H ) , 2 . 0 6 ( d t , J = 4 a n d 1 3 H z , I H ) , 3 . 7 2 ( d d , 1 4 6 . J = 6 a n d 1 2 H z , I H ) , 3 . 8 5 ( d d , J = 4 a n d 1 2 H z , I H ) , 5 . 5 0 ( b s , I H ) ; 1 3 C NMR ( 1 0 0 M H z , C D C l g ) 6 1 5 . 8 , 1 8 . 6 , 1 8 . 9 , 2 1 . 7 , 2 2 . 7 , 2 3 . 7 , 2 9 . 7 , 3 3 . 2 , 3 3 . 4 , 3 6 . 4 , 3 7 . 4 , 4 0 . 1 , 4 1 . 7 , 4 2 . 1 , 5 5 . 0 , 5 6 . 4 , 5 8 . 1 6 0 . 9 , 1 2 3 . 9 , 1 3 2 . 7 ; M S , m / z 2 9 0 ( 4 0 ) , 2 7 5 ( 5 ) , 2 6 0 ( 5 ) , 2 4 5 ( 5 ) , 1 9 2 ( 1 0 0 ) , 1 7 7 ( 9 5 ) , 9 5 ( 4 0 ) , 8 1 ( 6 0 ) , 6 9 ( 8 5 ) . R e d u c t i o n o f 1 8 a n d 1 9 S e p a r a t e s o l u t i o n s o f lfi ( 1 5 mg) a n d 19_ ( 6 mg) w e r e t r e a t e d w i t h D I B A L a n d s u b s e q u e n t l y p u r i f i e d i n a m a n n e r i d e n t i c a l t o t h e r e d u c t i o n o f 1_7. T h i s p r o d u c e d 8 mg ( 7 7 % y i e l d ) a n d 3 mg ( 7 2 % y i e l d ) r e s p e c t i v e l y o f w h i t e c r y s t a l l i n e w h i c h w a s 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 e r e d u c t i o n p r o d u c t o f 1 7 . R e d u c t i o n o f 2 0 A s o l u t i o n o f 20_ ( 6 mg) w a s t r e a t e d w i t h D I B A L a n d s u b s e q u e n t l y p u r i f i e d i n a m a n n e r i d e n t i c a l t o t h e r e d u c t i o n o f 1 7 . T h i s p r o d u c e d 2 . 6 mg ( 5 0 % y i e l d ) o f w h i t e c r y s t a l i n e 9 8 . mp 9 4 - 9 5 ° ( l i t . 9 5 - 9 6 ° ) 7 1 . [ a ] D = - 2 0 ° ( c = 0 . 0 8 , C H C 1 3 ) ( l i t . - 2 0 ° ) 7 1 ; H R M S , o b s 2 2 2 . 1 9 8 4 , C 1 5 H 2 6 0 c a l . 2 2 2 . 1 9 8 4 ; ' H NMR ( 4 0 0 M H z , C D C l g ) 6 0 . 8 3 7 ( s , 3 H ) , 0 . 8 4 4 ( s , 3 H ) , 0 . 8 6 7 ( s , 3 H ) , 1 . 7 6 ( s , 3 H ) , 3 . 7 3 ( d d , J = 1 3 a n d 7 . 2 H z , I H ) , 3 . 8 4 ( d d , J = 1 3 a n d 5 H z , I H ) p p m ; M S , m / z 2 2 2 ( 1 0 ) , 1 9 1 ( 1 0 ) , 1 2 4 ( 4 5 ) , 1 0 9 ( 7 5 ) , 8 5 ( 7 5 ) , 8 3 ( 1 0 0 ) , 6 9 ( 6 0 ) . R e d u c t i o n p r o d u c t 9 8 w a s i d e n t i c a l t o a u t h e n t i c 1 4 7 . d r i m e n o l b y ' H NMR , M S , T L C ( e t h y l a c e t a t e / h e x a n e ( 1 : 4 ) , R f = 0 . 3 o n P o l y g r a m 0 . 2 5 mm s i l i c a g e l p l a t e s ) a n d GC ( c o - i n j e c t i o n o f 9 8 a n d a u t h e n t i c d r i m e n o l g a v e a s i n g l e p e a k : i n i t i a l t e m p e r a t u r e 1 4 0 ° , r a t e 1 0 ° / m i n , RT = 6 . 7 5 m i n o n 3% 0 V - 1 7 ) . i '+C I n c o r p o r a t i o n S t u d i e s o f A r c h i d o r i s M e t a b o l i t e s A t o t a l o f 2 2 s p e c i m e n s o f A . m o n t e r e y e n s i s a n d 1 3 s p e c i m e n s o f A . o d h n e r i w e r e e m p l o y e d i n t h e i n c o r p o r a t i o n s t u d i e s . T w o u C i o f [ 2 - l l * C ] R S - m e v a l o n i c a c i d -d i b e n z y l e t h y l e n e d i a m i n e s a l t ( N e w E n g l a n d N u c l e a r ; 4 5 m C i / m m o l e ) i n ( 1 : 1 ) e t h a n o l / s t e r i l e s e a w a t e r ( 0 . 0 5 m i ) w a s d i r e c t l y i n j e c t e d i n t o t h e d i g e s t i v e g l a n d o f e a c h a n i m a l b y m e a n s o f a s y r i n g e . A f t e r i n j e c t i o n , t h e n u d i b r a n c h s w e r e p l a c e d i n a r u n n i n g s e a w a t e r a q u a r i u m f o r 2 4 h a n d t h e n e x t r a c t e d w i t h m e t h a n o l i n t h e n o r m a l m a n n e r . T h e e x t r a c t s w e r e f r a c t i o n a t e d a n d t h e g l y c e r i d e m e t a b o l i t i e s p u r i f i e d b y H P L C a s p r e v i o u s l y d e s c r i b e d f o r b o t h s p e c i e s . C o m p o u n d s _17 a n d ^ 0 o b t a i n e d f r o m A . m o n t e r e y e n s i s , w e r e r e d u c e d t o a l c o h o l s 97_ a n d 9 8 r e s p e c t i v e l y b y D I B A L i n t o l u e n e . T h e a l c o h o l s w e r e p u r i f i e d b y H P L C ( P a r t i s i l - 1 0 , 8% e t h y l a c e t a t e / h e x a n e ) a n d c r y s t a l i z e d f r o m e t h e r / h e x a n e p r i o r t o 1 J t C c o u n t i n g . C o m p o u n d _24, o b t a i n e d f r o m A . o d h n e r i w a s r e d u c e d t o f a r n e s o l ( 1 0 2 ) a n d t h e n c o n v e r t e d t o i t s 3 , 5 - d i n i t r o b e n z o a t e d e r i v a t i v e 1 0 3 . C o m p o u n d 1 0 3 w a s c r y s t a l i z e d f r o m e t h e r / h e x a n e p r i o r t o c o u n t i n g . T h e 1 4 8 . s a m p l e s ( 0 . 5 - 4 mg) w e r e d i s s o l v e d i n 1 0 m i A q u a s o l ( N E N ) s c i n t i l l a t i o n c o u n t i n g f l u i d . R a d i o a c t i v e c o u n t s w e r e d e t e r m i n e d i n a U n i l u x I I I l i q u i d s c i n t i l l a t i o n s y s t e m . Q u e n c h i n g w a s c o r r e c t e d b y e x t e r n a l s t a n d a r d i z a t i o n . R e d u c t i o n o f 2 4 A s o l u t i o n o f 2 4 ( 5 2 mg) w a s t r e a t e d w i t h D I B A L a n d s u b s e q u e n t l y p u r i f i e d i n a m a n n e r i d e n t i c a l t o t h e r e d u c t i o n o f 17_. F i n a l p u r i f i c a t i o n b y H P L C ( P a r t i s i l - 1 0 , 8% e t h y l a c e t a t e i n h e x a n e ) p r o v i d e d 2 6 mg ( 6 5 % y i e l d ) o f f a r n e s o l ( 1 0 2 ) a s a c o l o r l e s s o i l . 3 , 5 - d i n i t r o b e n z o a t e D e r i v a t i v e o f F a r n e s o l ( 1 0 2 ) T o a s o l u t i o n o f 1 0 2 ( 2 6 m g ; 0 . 1 1 7 m m o l e ) i n 1 . 0 m i p y r i d i n e w a s a d d e d 4 - d i m e t h y l a m i n o p y r i d i n e ( 2 0 m g , 0 . 1 6 4 m m o l e ) a n d 3 , 5 - d i n i t r o b e n z o y l c h l o r i d e ( 3 1 m g , 0 . 1 3 5 m m o l e ) . A f t e r s t i r r i n g a t RT f o r 1 2 h , 5 m i m e t h a n o l w a s a d d e d a n d t h e m i x t u r e w a s s t i r r e d f o r a n a d d i t i o n a l 2 h . T h e r e a c t i o n m i x t u r e w a s f i l t e r e d t h r o u g h a p l u g (1 x 1 cm d i a . ) o f s i l i c a g e l u s i n g c h l o r o f o r m a s e l u a n t . R e m o v a l o f t h e s o l v e n t u n d e r r e d u c e d p r e s s u r e f o l l o w e d b y p r e p a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y ( 2 5 % e t h y l a c e t a t e i n h e x a n e ) p r o v i d e d 3 9 mg ( 8 6 % y i e l d ) o f 1 0 3 mp 7 7 - 7 9 ° ; ' H NMR ( 8 0 M H z , C D C 1 3 ) ; 1 . 5 8 ( s , 6 H ) , 1 . 6 4 ( s , 3 H ) , 1 . 7 9 ( s , 3 H ) , 1 . 9 0 - 2 . 1 9 ( m , 8 H ) , 4 . 9 3 ( d , J = 8 H z , 2 H ) , 5 . 0 4 ( m , 2 H ) , 5 . 4 5 ( t , J = 8 H z , I H ) , 9 . 2 1 ( m , 3 H ) ; MS m / z 4 1 6 ( 2 0 ) , 4 0 1 ( 5 ) , 1 9 5 ( 5 0 ) , 1 3 6 ( 6 0 ) , 9 3 ( 5 0 ) , 81 ( 1 7 0 ) , 6 9 ( 1 0 0 ) . 1 4 9 . A d d i t i o n a l 1 I n c o r p o r a t i o n S t u d i e s A t o t a l o f 1 0 s p e c i m e n s o f C a d l i n a l u t e o m a r g i n a t a w e r e e a c h i n j e c t e d w i t h f i v e u C i o f [ 2 - 1 4 C ] R S - m e v a l o n i c a c i d - d i b e n z y l e t h y l e n e d i a m i n e s a l t ( 4 5 m C i / m m o l ) i n 0 . 0 5 m l s t e r i l e s e a w a t e r . A f t e r i n c u b a t i n g f o r 2 4 h t h e n u d i b r a n c h s w e r e e x t r a c t e d w i t h m e t h a n o l . T h e c h l o r o f o r m s o l u b l e p o r t i o n o f t h e e x t r a c t w a s f r a c t i o n a t e d b y f l a s h c h r o m a t o g r a p h y o n a c o l u m n o f s i l i c a w i t h c h l o r o f o r m a s e l u a n t . F r a c t i o n s c o n t a i n i n g a l b i c a n y l a c e t a t e (3_6) w e r e c o m b i n e d a n d f u r t h e r p u r i f i e d b y H P L C ( P a r t i s i l - 1 0 , 5% e t h y l a c e t a t e / h e x a n e ) t o g i v e 3 . 2 mg o f p u r e 3_6. T h i s m a t e r i a l w a s a d d e d t o 6 . 8 mg o f " c o l d " a l b i c a n y l a c e t a t e a n d t h e n h y d r o l y z e d b y s t i r r i n g a t RT i n a m e t h a n o l i c p o t a s s i u m c a r b o n a t e s o l u t i o n . A f t e r 1 2 h t h e r e a c t i o n m i x t u r e w a s f i l t e r e d a n d p a r t i t i o n e d b e t w e e n w a t e r a n d e t h e r . T h e e t h e r s o l u b l e m a t e r i a l w a s p u r i f i e d b y p r e p a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y t o y i e l d 3 . 5 mg o f a l b i c a n o l (3_5) w i t h a n a c t i v i t y o f 1 2 2 1 d p m / m g . A f t e r c o r r e c t i n g f o r t h e 6 8 % d i l u t i o n w i t h " c o l d " m a t e r i a l , t h e a l b i c a n o l h a d a l l t C a c t i v i t y o f 3 8 1 5 d p m / m g . _35 ' H NMR 6 0 . 7 3 ( s , 3 H ) , 0 . 8 1 ( s , 3 H ) , 0 . 8 8 ( s , 3 H ) , 3 . 7 6 ( d d , J = 1 1 , 1 1 H z , I H ) , 3 . 8 6 ( d d , J = 1 1 , 4 H z , I H ) , 4 . 6 5 ( b s , I H ) , 4 . 9 3 ( b s , I H ) p p m . A t o t a l o f 1 0 s p e c i m e n s o f T r i o p h a c a t a l i n a e w e r e e a c h i n j e c t e d w i t h 5 u C i o f [ 2 - 1 ' * C ] a c e t i c a c i d s o d i u m s a l t ( 1 - 3 1 5 0 . m C i / m m o l ) i n 0 . 0 5 m l s t e r i l e s e a w a t e r . A f t e r i n c u b a t i n g f o r 2 4 h t h e n u d i b r a n c h s w e r e e x t r a c t e d a n d w o r k e d u p a s p r e v i o u s l y d e s c r i b e d t o y i e l d 3 . 5 mg o f t r i o p h a m i n e ( 7 5 ) . T h i s m a t e r i a l w a s c o m b i n e d w i t h 6 . 5 m g o f " c o l d " t r i o p h a m i n e , d i s s o l v e d i n 1 m i m e t h a n o l a n d s t i r r e d a t RT i n t h e p r e s e n c e o f 0 . 5 m i 1 N s o d i u m h y d r o x i d e . T h e r e a c t i o n m i x t u r e w a s 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 n d p u r i f i e d b y p r e p a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y t o g i v e 2 . 2 mg o f t h e m o n o a c y l d e r i v a t i v e 8_1 w i t h a n a c t i v i t y o f 4 2 0 . 4 d p m / m g . W h e n t h e 6 5 % d i l u t i o n w i t h " c o l d " m a t e r i a l w a s a c c o u n t e d f o r , c o m p o u n d 8_1 s h o w e d a n a c t i v i t y o f 1 2 0 1 d p m / m g . F i s h A n t i f e e d a n t B i o a s s a y A n t i f e e d a n t a c t i v i t y w a s a s s e s s e d b y o b s e r v i n g t h e f e e d i n g r e s p o n s e o f t h e t i d e p o o l s c u l p i n O l i g o c o t t u s  m a c u l o s u s t o w a r d f o o d p e l l e t s ( W a r d e l y S h r i m p P e l l e t s ) t r e a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f d o r i d m e t a b o l i t i e s . T h e s c u l p i n s w e r e c o l l e c t e d i n t e r t i d a l l y a t B a r k l e y S o u n d , B r i t i s h C o l u m b i a a n d w e r e s t a r v e d f o r 2 4 h p r i o r t o t e s t i n g . T e s t c o m p o u n d s w e r e a p p l i e d t o f o o d p e l l e t s w i t h a c e t o n e w h i c h w a s e v a p o r a t e d a t r o o m t e m p e r a t u r e . C o n t r o l p e l l e t s w e r e t r e a t e d w i t h s o l v e n t o n l y . S i n g l e p e l l e t s w e r e r a n d o m l y a d d e d t o a g r o u p o f 4 0 f i s h ( 5 - 8 cm i n l e n g t h ) i n a 1 0 g a l l o n a q u a r i u m a n d t h e f e e d i n g r e s p o n s e w a s o b s e r v e d f o r u p t o 1 h . T h e f i s h e x h i b i t i e d v o r a c i o u s f e e d i n g b e h a v i o u r t o w a r d c o n t r o l p e l l e t s a n d p e l l e t s t r e a t e d w i t h i n a c t i v e 1 5 1 . c o m p o u n d s . T h e s e p e l l e t s w e r e a g g r e s s i v e l y a t t a c k e d b y g r o u p s o f f i s h a n d c o n s u m e d w i t h i n 5 - 1 0 m i n . I n d i g e s t i b l e h a r d p a r t s i n t h e p e l l e t s w e r e i g n o r e d . T e s t c o m p o u n d s t h a t w e r e a c t i v e c a u s e d a m a r k e d c e s s a t i o n o f f e e d i n g . A f t e r i n i t i a l m o u t h i n g a n d r e j e c t i o n , t h e f i s h a v o i d e d t h e a c t i v e p e l l e t s w h i c h r e m a i n e d u n e a t e n a f t e r 1 h . T h e f e e d i n g a b i l i t y o f t h e f i s h w a s v e r i f i e d b y f e e d i n g t h e m u n t r e a t e d p e l l e t s b o t h b e f o r e a n d a f t e r e a c h t e s t . I n a l l c a s e s t h e u n t r e a t e d p e l l e t s w e r e r a p i d l y c o n s u m e d . 152. BIBLIOGRAPHY 1. Thompson, T.E. J . Mar. B i o l . A s s o c . U.K. i 9 6 0 , 39, 123. 2. B e h r e n s , D.W. " P a c i f i c C o a s t N u d i b r a n c h s " ; S e a C h a l l e n g e r s : L o s Osos, C a l i f o r n i a , 1980. 3. H a r r i s , L.G. C u r r e n t T o p i c s Comp. P a t h o b i o l . 1973, 2, 213. 4. McDonald, G. ; Nybakken, J.W. "Guide t o N u d i b r a n c h s o f C a l i f o r n i a " ; A m e r i c a n M a l a c o l o g i s t s I n c . : M e l b o u r n e , F l o r i d a , 1980. 5. P a i n e , R . J . V e l i g e r 1963, 6, 1. 6. Thompson, T.E. J . Mar. B i o l . A s s o c . U.K. 1960, 39, 115. 7. Edmunds, M. P r o c . M a l a c . S o c . Lo n d . 1968, 38, 121. 8. F o r example s e e : a) Imp e r a t o , F. ; M i n a l e , L. and R i c c i o , R. E x p e r i e n t i a 1977, 33, 1273. b) S t a l l a r d , M.O. and F a u l k n e r , D.J. Comp. Bio c h e m . P h y s i o l . 1974, 49B, 25. 9. F o r example s e e : a) P e t t i t , G.R.; H e r a l d , C L . ; A l l e n , M.S.; Von D r e e l e , R.B.; V a n e l l , L.D.; Kao, J.P.Y. and B l a k e , W. J . Am. Chem. S o c . 1977, 99, 262. b) H o l l e n b e a k , K.H.; S c h m i t z , F . J . ; H o s s a i n , M.B. and Van Der Helm, D. T e t r a h e d r o n 1979, 35, 541. c) Yamamura, S. and H i r a t a , Y. T e t r a h e d r o n 1963, 19, 1485. 1 5 3 . 1 0 . F o r e x a m p l e s e e : a ) M i n a l e , L . a n d R i c c i o , R . T e t r a h e d r o n L e t t . 1 9 7 6 , 2 7 1 1 . b ) M a t s u d a , H . ; T o m i i e , Y . ; Y a m a m u r a , S . a n d H i r a t a , Y . C h e m . C o m m . 1 9 6 7 , 8 9 8 . c ) P e t t i t , G . R . ; H e r a l d , C L . ; E i n c k , J . J . ; V a n e l l , L . D . ; B r o w n , P . a n d G u s t , D . J . O r g . C h e m . 1 9 7 8 , 4 3 , 4 6 8 5 . 1 1 . K i n n e l , R . B . ; D i e t e r , R . K . ; M e i n w a l d , J . ; V a n E n g e n , D . ; C l a r d y , J . ; E i s n e r , T . ; S t a l l a r d , M . O . a n d F e n i c a l , W. P r o c . N a t l . A c a d . S c i . U . S . A . 1 9 7 9 , 7 6 , 3 5 7 6 . 1 2 . I m p e r a t o , F . ; M i n a l e , L . a n d R i c c i o , R. E x p e r i e n t i a  1 9 7 7 , 3 3 , 1 2 7 3 . 1 3 . S t a l l a r d , M . O . a n d F a u l k n e r , D . J . C o m p . B i o c h e m .  P h y s i o l . 1 9 7 4 , 4 9 B , 3 7 . 1 4 . S e e f o r e x a m p l e : S c h m i t z , F . J . ; M i c h a u d , D . P . a n d S c h m i d t , P . G . J . A m . C h e m . S o c . 1 9 8 2 , 1 0 4 , 6 4 1 5 . 1 5 . I r e l a n d , C . a n d F a u l k n e r , D . J . B i o r g . C h e m . 1 9 7 8 , 1_, 1 2 5 . 1 6 . B i s k u p i a k , J . E . a n d I r e l a n d , C M . T e t r a h e d r o n L e t t .  1 9 8 3 , 3 0 5 5 . 1 7 . I r e l a n d , C . a n d S c h e u e r , P . J . S c i e n c e 1 9 7 9 , 2 0 5 , 9 2 2 . 1 8 . a ) A y e r , S . W . H e l l o u , J . ; T i s c h l e r , M . a n d A n d e r s e n , R . J . T e t r a h e d r o n L e t t . 1 9 8 4 , 1 4 1 . b ) A y e r , S . W . ; A n d e r s e n , R . J . ; C u n - h e n g , H . a n d C l a r d y , J . J . O r g .  C h e m . 1 9 8 4 , i n p r e s s . 1 9 . S t o n a r d , R . J . ; P e t r o v i c h , J . C . a n d A n d e r s e n , R . J . S t e r o i d s 1 9 8 0 , 3 6 , 8 1 . 1 5 4 . 2 0 . A y e r , S . W . a n d A n d e r s e n , R . J . T e t r a h e d r o n L e t t . 1 9 8 2 , 1 0 3 9 . 2 1 . G u s t a f s o n , K . a n d A n d e r s e n , R . J . T e t r a h e d r o n 1 9 8 4 , s u b m i t t e d f o r p u b l i c a t i o n . 2 2 . F u h r m a n , F . A . ; F u h r m a n , G . J . r K i m , Y . H . ; P a v e l k a , L . A . a n d M o s h e r , H . S . S c i e n c e 1 9 8 0 , 2 0 7 , 1 9 3 . 2 3 . G u s t a f s o n , K . ; A n d e r s e n , R . J . ; C h e n , M . H . M . ; C l a r d y , J . a n d H o c h l o w s k i , J . E . T e t r a h e d r o n L e t t . 1 9 8 4 , 2 5 , 1 1 . 2 4 . A n d e r s e n , R . J . a n d S u m , F . W . T e t r a h e d r o n L e t t . 1 9 8 0 , 7 9 7 . 2 5 . T h o m p s o n , J . E . ; W a l k e r , R . P . ; W r a t t e n , S . J . a n d F a u l k n e r , D . J . T e t r a h e d r o n 1 9 8 2 , 3 8 , 1 8 6 5 . 2 6 . H e l l o u , J . ; A n d e r s e n , R . J . a n d T h o m p s o n , J . E . T e t r a h e d r o n 1 9 8 2 , 3 8 , 1 8 7 5 . 2 7 . H e l l o u , J . ; A n d e r s e n , R . J . ; R a f i i , S . ; A r n o l d , E . a n d C l a r d y , J . T e t r a h e d r o n L e t t . 1 9 8 1 , 4 1 7 3 . 2 8 . d e S i l v a , E . D . a n d S c h e u e r , P . J . H e t e r o c y c l e s 1 9 8 2 , 1 7 , 1 6 7 . 2 9 . S c h u l t e , G . R . a n d S c h e u e r , P . J . T e t r a h e d r o n 1 9 8 2 , 3 8 , 1 8 5 7 . 3 0 . S c h u l t e , F . ; S c h e u e r , P . J . a n d M c C o n n e l l , O . J . H e l v . C h i m . A c t a 1 9 8 0 , 6 3 , 2 1 5 9 . 3 1 . H o c h l o w s k i , J . E . a n d F a u l k n e r , D . J . T e t r a h e d r o n L e t t . 1 9 8 1 , 2 7 1 . 3 2 . H o c h l o w s k i , J . E . ; F a u l k n e r , D . J . ; M a t s u m o t o , G . K . a n d C l a r d y , J . J . O r g . C h e m . 1 9 8 3 , 4 8 , 1 1 4 2 . 1 5 5 . 3 3 . H o c h l o w s k i , J . E . ; F a u l k n e r , D . J . ; B a s s , L . S . a n d C l a r d y , J . J . O r g . C h e m . 1 9 8 3 , 4 8 , 1 7 3 8 . 3 4 . C i m i n o , G . ? De R o s a , S . ; De S t e f a n o , S . a n d S o d a n o , G . T e t r a h e d r o n L e t t . 1 9 8 0 , 3 3 0 3 . 3 5 . C i m i n o , G . ; De S t e f a n o , S . ; De R o s a , S . ; S o d a n o , G . a n d V i l l a n i , G . B u l l . S o c . C h i m . B e l g . 1 9 8 0 , 8 9 , 1 0 6 9 . 3 6 . O k u d a , R . K . ; S c h e u e r , P . J . ; H o c h l o w s k i , J . E . ; W a l k e r , R . P . a n d F a u l k n e r , D . J . J . O r g . C h e m . 1 9 8 3 , 4 8 , 1 8 6 6 . 3 7 . C i m i n o , G . ; De R o s a , S . ; De S t e f a n o , S . a n d S o d a n o , G . T e t r a h e d r o n L e t t . 1 9 8 1 , 1 2 7 1 . 3 8 . C i m i n o , G . ; De R o s a , S . ; De S t e f a n o , S . ; S o d a n o , G . a n d V i l l a n i , G . S c i e n c e 1 9 8 3 , 2 1 9 , 1 2 3 7 . 3 9 . F u h r m a n , F . A . ? F u h r m a n , G . J . ; N a c h m a n , R . J . a n d M o s h e r , H . S . S c i e n c e 1 9 8 1 , 2 1 2 , 5 5 7 . 4 0 . W a l k e r , R . P . a n d F a u l k n e r , D . J . J . O r g . C h e m . 1 9 8 1 , 4 6 , 1 4 7 5 . 4 1 . C i m i n o , G . ; De R o s a , S . ; De S t e f a n o , S . a n d S o d a n o , G . C o m p . B i o c h e m . P h y s i o l . B 1 9 8 2 , 7 3 , 4 7 1 . 4 2 . H o c h l o w s k i , J . E . ; W a l k e r , R . P . ? I r e l a n d , C . a n d F a u l k n e r , D . J . J . O r g . C h e m . 1 9 8 2 , 4 7 , 8 8 . 4 3 . A y e r , S . W . a n d A n d e r s e n , R . J . E x p e r i e n t i a 1 9 8 3 , 3 9 , 2 5 5 . 4 4 . C a s t i e l l o , D . ; C i m i n o , G . ; De R o s a , S . ; De S t e f a n o , S . a n d S o d a n o , G . T e t r a h e d r o n L e t t . 1 9 8 0 , 5 0 4 7 . 4 5 . B u r r e s o n , B . J . ; S c h e u e r , P . J . ; F i n e r , J . a n d C l a r d y , J . J . A m . C h e m . S o c . 1 9 7 5 , 9 7 , 4 7 6 3 . 1 5 6 , 4 6 . H a g a d o n e , M . R . ; B u r r e s o n , B . J . ; S c h e u e r , P . J . ; F i n e r , J . a n d C l a r d y , J . H e l v . C h i m . A c t a 1 9 7 9 , 6 2 , 2 4 8 4 . 4 7 . C o l l , J . C . ; B o w d e n , B . F . ; T a p i o l a s , D . M . ; W i l l i s , R . H . ; D j u r a , P . ; S t r e a m e r , M . ; T r o t t , L . a n d W e b b , L . T e t r a h e d r o n 1 9 8 4 , s u b m i t t e d f o r p u b l i c a t i o n . 4 8 . C a r t e l B . a n d F a u l k n e r , D . J . J . O r g . C h e m . 1 9 8 3 , 4 8 , 2 3 1 4 . 4 9 . G u s t a f s o n , K . a n d A n d e r s e n , R . J . J . O r g . C h e m . 1 9 8 2 , 4 7 , 2 1 6 7 . 5 0 . C i m i n o , G . ; D e S t e f a n o , S . a n d M i n a l e , L . E x p e r i e n t i a  1 9 7 4 , 3 0 , 8 4 6 . 5 1 . N a k a n i s h i , K . a n d K u b o , I. I s r . J . C h e m . 1 9 7 7 , 1 6 , 2 8 . 5 2 . a ) H a l l , L . D . a n d S a n d e r s , J . K . M . J . A m . C h e m . S o c . 1 9 8 0 , 1 0 2 , 5 7 0 . b ) S a n d e r s , J . K . M . a n d M e r s h , J . D . O r g . M a g . R e s . 1 9 8 2 , 1 8 , 1 2 2 . 5 3 . N o g g l e , J . H . a n d S c h i r m e r , R . E . " T h e N u c l e a r O v e r h a u s e r E f f e c t : C h e m i c a l A p p l i c a t i o n s " ; A c a d e m i c P r e s s : N e w Y o r k , 1 9 7 1 . 5 4 . M a s u m o t o , K . a n d R a p o p o r t , H . J . O r g . C h e m . 1 9 6 8 , 3 3 , 5 5 2 . 5 5 . L e v y G . C . a n d N e l s o n , G . L . " C a r b o n - 1 3 N u c l e a r M a g n e t i c R e s o n a n c e f o r O r g a n i c C h e m i s t s " ; W i l e y - I n t e r s c i e n c e : N e w Y o r k , 1 9 7 2 . 5 6 . F i s h b e i n , L . a n d C a v a n a u g h , M . A . J . C h r o m a t o g . 1 9 6 5 , 2 0 , 2 8 3 . 1 5 7 . 5 7 . C h e v o l o t , L . i n " M a r i n e N a t u r a l P r o d u c t s , C h e m i c a l a n d B i o l o g i c a l P e r s p e c t i v e s V o l . I V " , P . J . S c h e u e r E d . ; A c a d e m i c P r e s s , 1 9 8 1 . 5 8 . N a k a m u r a , H . ; W u , H . ; K o b a y a s h i , J . ; O h i z u m i , Y . ; H i r a t a , Y . ; H i g a s h i j i m a , T . a n d M i y a z a w a , T . T e t r a h e d r o n L e t t . 1 9 8 3 , 4 1 0 5 . 5 9 . C i m i n o , G . ; De R o s a , S . ; De S t e f a n o , S . ; S e l f , R. a n d S o d a n o , G . T e t r a h e d r o n L e t t . 1 9 8 3 , 3 0 2 9 . 6 0 . F o r e x a m p l e s e e : C a r l S J . S . a n d C h r i s t o p h e r s e n , C . J .  O r g . C h e m . 1 9 8 1 , 4 6 , 3 4 4 0 . 6 1 . P i e r s , E . ; C h o n g , J . M . ; G u s t a f s o n , K . a n d A n d e r s e n , R . J . C a n . J . C h e m . 1 9 8 4 , 6 2 , 1 . 6 2 . P i e r s , E . ; C h o n g , J . M . ; M o r t o n , H . E . T e t r a h e d r o n L e t t .  1 9 8 1 , 4 9 0 5 . 6 3 . G r e e n h a l g h , R. a n d B a n n a r d , R . A . B . C a n . J . C h e m . 1 9 6 1 , 3 9 , 1 0 1 7 . 6 4 . B a r k e r , P . L . ; B e n d l e r , P . L . a n d R a p o p o r t , H . J . O r g .  C h e m . 1 9 8 1 , 4 6 , 2 4 5 5 . 6 5 . F u h r m a n , F . A . ; F u h r m a n , G . J . a n d De R i e m e r , K . B i o l .  B u l l . 1 9 7 9 , 1 5 6 , 2 8 9 . 6 6 . Q u i n n , R . J . ; G r e g s o n , R . P . ; C o o k , A . F . a n d B a r t l e t t , R . T . T e t r a h e d r o n L e t t . 1 9 8 0 , 5 6 7 . 6 7 . N y b a k k e n , J . a n d A j e s k a , R . A . V e l i g e r 1 9 6 7 , 1 9 , 1 9 . 6 8 . G o r a , J . a n d A n t c z a k , U . P e r f u m e r F l a v o r i s t 1 9 8 0 , 5_, 3 1 . 1 5 8 . 6 9 . C i m i n o , G . ; De R o s a , S . ; De S t e f a n o , S . a n d M i n a l e , L . T e t r a h e d r o n 1 9 7 4 , 3 0 , 6 4 5 . 7 0 . A r m s t r o n g , R . J . P h . D . t h e s i s 1 9 8 3 , C h e m i s t r y D e p t . , U n i v . o f B r i t i s h C o l u m b i a . 7 1 . P e l l e t i e r , S . W . ; L a j s i a , S . ; O h t s u k a , Y . a n d D j a r m a t i , Z . J . O r g . C h e m . 1 9 7 5 , 4 0 , 1 6 0 7 . 7 2 . S c h u l t e - E l t e , K . H . ; M u l l e r , B . L . a n d O h l o f f , G . N o u v e a u J . C h i m . 1 9 7 8 , 2 , 4 2 7 . 7 3 . D o , M . N . a n d E r i c k s o n , K . L . T e t r a h e d r o n L e t t . 1 9 8 3 , 5 6 9 9 . 7 4 . P e t t i t , G . R . a n d F u j i i , Y . J . N a t u r a l P r o d s . 1 9 8 2 , 4 5 , 6 4 0 . 7 5 . C i m i n o , G . ; De R o s a , S . ; De S t e f a n o , S . a n d M i n a l e , L . T e t r a h e d r o n 1 9 7 4 , 3 0 , 6 4 5 . 7 6 . K a z l a u s k a s , R . ; M u r p h y , P . T . a n d W e l l s , R . J . T e t r a h e d r o n L e t t . 1 9 7 9 , 9 0 3 . 7 7 . F e n i c a l , W. i n " M a r i n e N a t u r a l P r o d u c t s , C h e m i c a l a n d B i o l o g i c a l P e r s p e c t i v e s , V o l . I I " , P . J . S c h e u e r E d . ; A c a d e m i c P r e s s : New Y o r k , 1 9 7 8 , p . 1 7 4 . 7 8 . A m i c o , V . ; O r i e n t e , G . ; P i a t t e l l i , M . ; T r i n g a l i , C ; F a t t o r u s s o , E . ; M a g n o , S . a n d M a y o l , L . E x p e r i e n t i a  1 9 7 7 , 3 3 , 1 2 7 4 . 7 9 . H o w a r d , B . M . a n d F e n i c a l , W. T e t r a h e d r o n L e t t . 1 9 7 6 , 4 1 . 1 5 9 . 8 0 . a ) C i m i n o , G . ; De S t e f a n o , S . ; G u e r r i e r o , A . a n d M i n a l e , L . T e t r a h e d r o n L e t t . 1 9 7 5 , 1 4 1 7 . b ) C i m i n o , G . ; De S t e f a n o , S . ; G u e r r i e r o , A . a n d M i n a l e , L . T e t r a h e d r o n L e t t . 1 9 7 5 , 3 7 2 3 . 8 1 . C i m i n o , G . ; De S t e f a n o , S . a n d M i n a l e , L . E x p e r i e n t i a  1 9 7 3 , 2 9 , 1 0 6 3 . 8 2 . C i m i n o , G . i n " M a r i n e N a t u r a l P r o d u c t s C h e m i s t r y " , D . J . F a u l k n e r a n d W . H . F e n i c a l , E d s . ; P l e n u m P r e s s : New Y o r k 1 9 7 7 , p . 6 1 . 8 3 . B l a c k m a n , A . J . a n d W e l l s , R . J . T e t r a h e d r o n L e t t . 1 9 7 6 , 2 7 2 9 . 

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