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Terpenoids from two British Columbia nudibranchs Hellou, Jocelyne 1981

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TERPENOIDS FROM TWO BRITISH COLUMBIA NUDIBRANCHS by JOCELYNE HELLOU BSc. Universite de Montreal, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1980 © Jocelyne Hellou, 1980 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that-the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date Rl DE-6 (2/79) I i A b s t r a c t The two B r i t i s h Columbia nudibranchs C a d i i n a luteomarginata and Acanthador i s nanaimoensis have sweet f r a g r a n c e s . The p o s s i b l e importance of odours i n the i n t e r a c t i o n s of marine organisms i n i t i a l l y aroused our chemical c u r i o s i t y . The r e s u l t s of our r e s e a r c h concerning the s t r u c t u r a l i n v e s t i g a t i o n of seven t e r p e n o i d s obtained from the organic e x t r a c t s of these two opisthobranch molluscs i s presented i n t h i s t h e s i s . F i v e of the molecules i s o l a t e d from luteomarginata have been i d e n t i f i e d as f u r o d y s i n (53), f u r o d y s i n i n (54), m i c r o c i o n i n - 2 (55), a l b i c a n y l a c e t a t e (51) and a l b i c a n o l (52). A s i x t h molecule, luteone (57) which g i v e s the sweet fragrance to C. luteomarginata has been p a r t i a l l y c h a r a c t e r i z e d . A c r y s t a l l i n e d e r i v a t i v e of t h i s methyl ketone has been submitted for X-ray d i f f r a c t i o n a n a l y s i s . The sweet fragrance of A_;_ nanaimoensis has been r e l a t e d to the presence of a s e s q u i t e r p e n o i d ( e x i s t i n g as two c o n s t i t u t i o n a l isomers, i n a 5:1 r a t i o ) . Three h y p o t h e t i c a l s t r u c t u r e s based on s p e c t r a l a n a l y s i s , chemical r e a c t i o n s and b i o s y n t h e t i c reasoning are proposed. The b i o l o g i c a l o r i g i n of the seven t e r p e n o i d s has a l s o been i n v e s t i g a t e d and i s d i s c u s s e d i n t h i s t h e s i s . Table of Contents T i t l e Page i A b s t r a c t i i Table of Contents i i i L i s t of F i g u r e s v i L i s t of Schemes • v i i L i s t of Tables x L i s t of Appendices x i Acknowledgments x i i Chapter 1: Phylogeny and Defense Mechanisms of Opisthobranch M o l l u s c s * 1.1. Phylogeny 1 1.2. Defense mechanisms 3 1.3. Chemical communication 5 1.4. Summary 10 Chapter 2: The Chemistry of the Opisthobranch M o l l u s c s 11.1. Review ^ 11 Chart I 12 11.2. O r i g i n of the molecules 11.2.1. D i e t r e l a t e d molecules 27 11.2.2. From a symbiotic a s s o c i a t i o n 29 11.2.3. P o s s i b l y of a molluscan o r i g i n 30 11.3. Summary 31 Chart II 32 Chapter 3: I s o l a t i o n of the Compounds III .1. C o l l e c t i o n 33 I I I . 2 . C a d l i n a luteomarginata I I I . 2 . 1 . E x t r a c t i o n procedure ' 39 i v 111.2.2. Crude s e p e r a t i o n 41 111.2.3. I s o l a t i o n of f u r a n o s e s q u i t e r p e n o i d s from f r a c t i o n (1) 43 111.2.4. I s o l a t i o n of a l b i c a n y l a c e t a t e from f r a c t i o n (2) 4 5 111.2.5. I s o l a t i o n of a l b i c a n o l and luteone from f r a c t i o n (3) 46 111.2.6. I s o l a t i o n of s t e r o i d a l peroxides from f r a c t i o n (4) 47 I I I . 3. Acanthadoris nanaimoensis 111.3.1. E x t r a c t i o n procedure 48 111.3.2. I s o l a t i o n of the compounds 48 Chapter 4: I d e n t i f i c a t i o n of the Compounds IV. 1. From C a d l i n a luteomarginata IV.1.1. A l b i c a n y l a c e t a t e 49 IV.1.2.a. A l b i c a n o l 60 IV.1.2.b. Drimanol 67 IV.3.a. Fu r o d y s i n and f u r o d y s i n i n 73 IV.3.b. M i c r o c i o n i n - 2 87 IV.1.5.a. Luteone 93 IV.1.5.b. Pht h a l a t e e s t e r 104 IV. 2. From Acanthadoris nanaimoensis IV.2.1. Nanaimoal 105 IV.2.2. D e r i v a t i v e s 113 IV. 2.3. Minor isomer 134 Chapter 5: D i s c u s s i o n V. l . C a d l i n a luteomarginata V. l . l . B i o l o g i c a l aspects 136 V.1.2. Chemical and biochem i c a l aspects 141 V.2. Acanthadoris nanaimoensis V.2.1. B i o l o g i c a l aspects 149 V.2.2. Chemical and biochemical aspects 150 Exper imental A. C a d l i n a luteomarginata A . l . E x t r a c t i o n and crude s e p a r a t i o n 154 A.2. Se p a r a t i o n of the f u r a n o s e s q u i t e r p e n o i d s 155 A.3. A l b i c a n y l a c e t a t e 160 A.4. A l b i c a n o l 161 A.5. Drimanol 162 A.6. Luteone 163 A.7. DNPH d e r i v a t i v e of luteone 163 A.8. S t e r o i d a l peroxides 164 A. 9. D i s s e c t i o n s 164 B. Acanthadoris nanaimoensis B. l . E x t r a c t i o n and crude s e p a r a t i o n 165 B.2. Se p a r a t i o n of nanaimoal 165 B.3. DNPH d e r i v a t i v e of nanaimoal 166 B.4. Nanaimool 166 B.5. p a r a - c h l o r o p h e n y l i s o c y a n a t e d e r i v a t i v e 167 B.6. Reaction with 1 , 3 - p r o p a n e d i t h i o l 168 B.7. meta-bromobenzoate d e r i v a t i v e 168 B.8. para-bromobenzoate d e r i v a t i v e 169 B.9. O z o n o l y s i s 170 B.10. para-bromophenylhydrazone d e r i v a t i v e 171 B i b l i o g r a p h y 172 v i L i s t of F i g u r e s 1- D o r s a l view of a d o r i d nudibranch 1 2- Underwater photograph of C a d l i n a luteomarginata 35 3- Map p r e s e n t i n g the d i v i n g s i t e s where C. luteomarginata was c o l l e c t e d 36 4- Underwater photograph of Acanthadoris  nanaimoensis 37 5- Map p r e s e n t i n g the d i v i n g s i t e s where A. nanaimoensis was c o l l e c t e d 38 6- MS of a l b i c a n y l a c e t a t e 55 7- IR spectrum of a l b i c a n y l a c e t a t e 56 8- 270 MHz JH NMR spectrum of a l b i c a n y l a c e t a t e 57 9- 100 MHz *H NMR spectrum of f a t ( s ) 58 10- MS of a l b i c a n o l 64 11- IR spectrum of a l b i c a n o l 65 12- 270 MHz 'H NMR spectrum of a l b i c a n o l 66 13- MS of drimanol 70 14- IR spectrum of drimanol 71 15- 270 HMz XH NMR spectrum of drimanol 72 16- MS of f u r o d y s i n i n 78 17- IR spectrum of f u r o d y s i n i n 80 18A- 80 MHz XH NMR spectrum of f u r o d y s i n i n 81 18B- 270 MHz XH NMR spectrum of f u r o d y s i n i n 82 19- MS of a mixture of f u r a n o s e s q u i t e r p e n o i d s 83 20- IR spectrum of a mixture of f u r a n o s e s q u i t e r p e n o i d s 84 21A- 80 MHz 1H NMR spectrum of a mixture of f u r a n o s e s q u i t e r p e n o i d s 85 v i i 21B- 270 MHz XH NMR spectrum of a mixture of f u r a n o s e s q u i t e r p e n o i d s 86 22- MS of m i c r o c i o n i n - 2 90 23- IR spectrum of m i c r o c i o n i n - 2 91 24- 270 MHz *H NMR spectrum of m i c r o c i o n i n - 2 92 25A- MS of luteone (probe without heating) 97 25B- MS of luteone (probe heated at 200*C) 98 26- 100 MHz  1H NMR spectrum of r e l a t i v e l y pure luteone 99 27- 270 MHz XH NMR spectrum of luteone plu s a p h t h a l a t e e s t e r 100 28- MS of luteone's DNPH d e r i v a t i v e 101 29- IR spectrum of luteone's DNPH d e r i v a t i v e 102 30- 270 MHz JH NMR spectrum of luteone's DNPH d e r i v a t i v e 103 31- MS of nanaimoal 110 32- IR spectum of nanaimoal 111 33- 270 MHz XH NMR spectrum of nanaimoal 112 34- MS of nanaimoal's DNPH d e r i v a t i v e 115 35- IR spectrum of nanaimoal's DNPH d e r i v a t i v e 116 36- 270 MHz XH NMR spectrum of nanaimoal's DNPH d e r i v a t i v e 117 37- MS of nanaimool 118 38- IR spectrum of nanaimool 119 39A- 270 MHz 1H NMR spectrum of nanaimool 120 39B- 80 MHz 1 3 C NMR spectrum of nanaimool 121 39C- 100 MHz  1H NMR spectrum of nanaimool obtained a t : (a) 0'C (b) -20° C (c) -40° C 122 v i i i 40- MS of nanaimool's p a r a - c h l o r o p h e n y l i s o c y a n a t e d e r i v a t i v e 123 41- IR spectrum of nanaimool's para-c h l o r o p h e n y l i s o c y a n a t e d e r i v a t i v e 124 42- 270 MHz *H NMR spectrum of nanaimool's para-c h l o r o p h e n y l i s o c y a n a t e d e r i v a t i v e 125 43- IR spectrum of nanaimool's meta-bromobenzoate d e r i v a t i v e 126 44- 270 MHz *H NMR spectrum of nanaimool's meta-bromobenzoate d e r i v a t i v e 127 45- 270 MHz XH NMR spectrum of nanaimool's para-bromobenzoate d e r i v a t i v e 128 46- MS of more p o l a r compound obtained from o z o n o l y s i s r e a c t i o n 129 47- MS of l e s s p o l a r compound obtained from o z o n o l y s i s r e a c t i o n 130 48- MS of nanaimoal's para-bromophenylhydrazone d e r i v a t i v e 131 49- IR of nanaimoal's para-bromophenylhydrazone d e r i v a t i v e 132 50- 270 MHz *H NMR spectrum of nanaimoal's para-bromophenylhydrazone d e r i v a t i v e 133 L i s t of Schemes 1- Fragmentation p a t t e r n of a l b i c a n y l a c e t a t e 54 2- Fragmentation p a t t e r n of a l b i c a n o l 63 3- Fragmentation p a t t e r n of drimanol 69 4- Fragmentation p a t t e r n of f u r o d y s i n i n and f u r o d y s i n 79 5- Fragmentation p a t t e r n of m i c r o c i o n i n - 2 89 6- P o s s i b l e Retro D i e l s - A l d e r fragmentation p a t t e r n of the three h y p o t h e t i c a l s t r u c t u r e s of nanaimoal 109 7- B i o s y n t h e s i s of f a r n e s y l pyrophosphate 145 8- P o s s i b l e b i o s y n t h e s i s of f u r o d y s i n i n and f u r o d y s i n 146 9- P o s s i b l e b i o s y n t h e s i s of m i c r o c i o n i n - 2 147 10- P o s s i b l e b i o s y n t h e s i s of a l b i c a n y l a c e t a t e and a l b i c a n o l 148 11- P o s s i b l e b i o s y n t h e s i s of the nanaimoane s k e l e t o n 152 X L i s t of Tables I- Review of the opistobranchs chemistry 13 I I - lH NMR data f o r a l b i c a n y l a c e t a t e and a l b i c a n o l 59 I I I - lH NMR s h i f t s f o r a l b i c a n o l obtained from C. luteomarginata , D_j_ a l b i c a n s , and a s y n t h e s i s 61 IV- XH NMR data f o r drimanol 68 V- 1H NMR s h i f t s f o r f u r o d y s i n i n obtained from C. luteomarginata and from a Dysidea s p e c i e s 74 VI- Comparison of IR s p e c t r a 75 V I I - lH NMR s h i f t s f o r f u r o d y s i n obtained from C. luteomarginata f u r a n o s e s q u i t e r p e n o i d mixture and from a Disydea s p e c i e s 86 VIII 1H- NMR data f o r m i c r o c i o n i n - 2 obtained from C. luteomarginata and from a M i c r o c i o n a t o x y s t i l l a . 88 IX- R e s u l t s of d i s s e c t i o n 137 X- R e s u l t s obtained with the HPLC using a s i l i c a column 157 XI- R e s u l t s obtained with the HPLC using a re v e r s e d -phase column 158 x i L i s t of Appendices 1- Phylogenetic t r e e 177 2- XH NMR spectrum of a l b i c a n o l o b t ained from Dr.N.H. Andersen 178 3- IR spectrum of a l b i c a n o l obtained from Dr. N.H. Andersen 179 4- 1H NMR spectrum of f u r o d y s i n i n obtained from Dr. Wells 180 5- IR spectrum of f u r o d y s i n i n obtained from Dr. Wells 181 6-  1H NMR spectrum of f u r o d y s i n obtained from Dr. Wells 182 7- IR spectrum of f u r o d y s i n obtained from Dr. Wells 183 Acknowledgments The r e s e a r c h work- presented i n t h i s t h e s i s would have never been p o s s i b l e without the h e l p of s e v e r a l people. I would l i k e to take t h i s o p p o r t u n i t y to thank: The d i v e r s who helped i n the c o l l e c t i o n of the marine organisms. Without the e v e r l a s t i n g enthousiasm of Mike Le Blanc and e s p e c i a l l y the p a r t i c i p a t i o n o f : Ray Andersen, K i r k Gustafson, A l Hay, Jane P e t r o v i t c h , Rick Stonard, t h i s work would have never s t a r t e d . The courteous s t a f f of the NMR l a b o r a t o r y and MS l a b o r a t o r y , who have been understanding i n s p i t e of our small samples and without whom data c o u l d not have been a c q u i r e d . Dr. Sandra M i l l e n from the Zoology Department at U.B.C. , for her taxonomic h e l p . She i d e n t i f i e d the nudibranchs and sponges and provided us with underwater s l i d e s of the nudibranchs. Ron Long from SFU who g r a c i o u s l y allowed us to i n s e r t the underwater photographs of C a d l i n a luteomarginata and Acanthadoris nanaimoensis i n the t h e s i s . Dr. Wells and Dr. Andersen who sent us photocopies of *H NMR and IR s p e c t r a of f u r o d y s i n and f u r o d y s i n i n , and of a l b i c a n o l , r e s p e c t i v e l y . My s u p e r v i s o r , Dr. Andersen and the s e n i o r graduate student i n our r e s e a r c h group, R.J. Stonard, f o r t h e i r continuous h e l p , a d v i c e , and e n r i c h i n g c o n v e r s a t i o n s . Nancy Stonard f o r t y p i n g Table I and the c a p t i o n s under the s p e c t r a . x i i i A.E. Hay who wrote the computer program f o r p l o t t i n g the MS and who p a t i e n t l y helped me get acquainted with the use of the computer while w r i t i n g t h i s t h e s i s . 1 Chapter 1 Phyloqeny and Defense Mechanisms of Opisthobranch M o l l u s c s  I.1. Phylogeny: The phyllum Mollusca c o n t a i n s the second l a r g e s t number of i n v e r t e b r a t e s p e c i e s . Gastropoda r e p r e s e n t s i t s major c l a s s (see Chart 1 ) , with specimens found i n a l a r g e v a r i e t y of marine and t e r r e s t r i a l h a b i t a t s . Members of t h i s c l a s s have an asymmetric body and only one s h e l l ( u n i v a l v e ) . One of the three s u b c l a s s e s of gastropods i s the O p i s t h o b r a n c h i a , with mainly marine members c h a r a c t e r i z e d by a s h e l l and mantle c a v i t y reduced i n s i z e or absent. The Opis t h o b r a n c h i a can be f u r t h e r s u b d i v i d e d i n t o e i g h t o r d e r s , i n c l u d i n g : Sacoglossa, Nudibranchia and Cephalospidea. In the order Nudibranchia (nudibranchs are a l s o known as sea slugs or naked s h e l l f i s h ) the animals have no s h e l l or mantle c a v i t y and as i n d i c a t e d by t h e i r name, do not have i n t e r n a l g i l l s . T h e i r body assumes an elongated form with a secondary b i l a t e r a l symmetry. Among the Nudibranchia, members of the suborder Doridaceae are c h a r a c t e r i z e d by the presence of a secondary g i l l r i n g e n c i r c l i n g the d o r s a l a n u s 1 . tubercules or p a p i l l a e " "^ ""fV^ r rhinophores F i g u r e 1 D o r s a l View of a D o r i d Nudibranch 2 F i g u r e 1 shows a d o r s a l view of a d o r i d nudibranch. Members of t h i s suborder vary from 1 to 12 cm i n length and from 0.5 to 3 cm i n t h i c k n e s s . T h e i r c o l o r ranges from white to numerous shades of yellow and orange, o f t e n with some p a r t i c u l a r c o l o r p a t t e r n ("spots") on t h e i r mantle. They are found on rocky s u r f a c e s at depths of 2m to more than 18 m. A s p e c i a l morphological f e a t u r e worth mentioning i s the two rhinophores, which represent m o d i f i e d t e n t a c l e s used as sense organs. Experiments have shown that rhinophores are the most l i k e l y s i t e f o r chemoreception i n n u d i b r a n c h s 2 . 3 1 .2 . Defense mechanisms: Nudibranchs are slow-moving organisms without an e x t e r i o r s h e l l and with no other obvious p r o t e c t i v e f e a t u r e . Four groups of animals have been suggested as p o s s i b l e p r e d a t o r s 3 : other opisthobranchs, c r u s t a c e a n s , sea s t a r s and f i s h . The f a c t that nudibranchs a v o i d d i r e c t p r e d a t i o n has r a i s e d i n t e r e s t among b i o l o g i s t s . L.G. H a r r i s 3 has reviewed the r e s u l t s of s e v e r a l s t u d i e s concerning nudibranch d e f e n s i v e mechanisms. He groups them i n t o f i v e c a t e g o r i e s , as f o l l o w s : 1) B e h a v i o r a l response 2) S p i c u l e s 3) Nematocysts 4) C o l o r camouflage 5) Chemical s e c r e t i o n s Experiments have shown that upon d i s t u r b a n c e , the behavior of nudibranchs i s a l t e r e d . Immediate r e t r a c t i o n of rhinophores and g i l l s and sometimes swimming can be o b s e r v e d 4 . Although t h i s i s a response to being a t t a c k e d , i t does not e x p l a i n the a b i l i t y of nudibranchs to s u r v i v e s i n c e t h e i r escape i s u s u a l l y slow i n comparison to the p r e d a t o r ' s speed. S p i c u l e s of a c a l c a r e o u s nature are present i n the mantle of some d o r i d s p e c i e s . P a i n e 5 p o s t u l a t e d that these give the nudibranchs a rather r i g i d shape and make them d i f f i c u l t f o r other opisthobranchs to swallow. 4 Nematocysts are c e l l s c o n t a i n i n g s t i n g i n g s t r u c t u r e s o r i g i n a t i n g from ingested c o e l e n t e r a t e s . Stored by some nudibranchs, they can be r e l e a s e d when the animal i s d i s t u r b e d 8 . D i f f e r e n t types of nematocyst e x i s t and some nudibranchs appear to s e l e c t the type they s t o r e . I t i s b e l i e v e d that some types of nematocyst are more e f f e c t i v e a g a i n s t c e r t a i n p r e d a t o r s . Of the four nudibranch suborders, only three are known to a s s o c i a t e with c o e l e n t e r a t e s ; the E o l i d a c e a , Dendronotacea and Arminacea. D o r i d nudibranchs, which are predominantly a s s o c i a t e d with sponges, bryozoans and c o l o n i a l t u n i c a t e s 3 , are not a s s o c i a t e d with c o e l e n t e r a t e s and c o u l d not t h e r e f o r e u t i l i z e nematocysts f o r defense. C o l o r a t i o n may be i n t e r p r e t e d as a camouflage i n some cases (white nudibranchs on white c o r a l ) , or as a warning i n the case of b r i g h t l y c o l o r e d nudibranchs. Often, the pigmentation of the nudibranch v a r i e s with i t s d i e t . The a b i l i t y of f i s h to p e r c e i v e c o l o r i s s t i l l u n c e r t a i n 6 and i n f o r m a t i o n on other p r e d a t o r s i s l a c k i n g . Hence, aspects of t h i s d e f e n s i v e mechanism remain to be i n v e s t i g a t e d . The f i n a l and most i n t e r e s t i n g means of p r o t e c t i o n from a chemist's p o i n t of view are the s e c r e t i o n s emanating from epidermal glands. These s e c r e t i o n s are designated as d e f e n s i v e because they are u s u a l l y emitted when the animal i s d i s t u r b e d . It was re p o r t e d i n the e a r l y l i t e r a t u r e that the s e c r e t i o n s from e o l i d nudibranchs might be strong a c i d s such as HC1 and H ?S0 4 1 . Recently, i t has been shown that organic molecules with i n t e r e s t i n g s t r u c t u r e s are s e c r e t e d by some s p e c i e s 8 '. In some cases the d e f e n s i v e molecules are r e l a t e d to a d i e t a r y s o u r c e 8 . 5 A comparison of the e f f e c t i v e n e s s of the senses of smell and t a s t e i n f i s h e s makes o d o r i f e r o u s s e c r e t i o n s appear more v i a b l e as a means of chemical defense than g u s t a t o r y mechanisms. J.E. Bardash 6 r e p o r t s t h a t : "smell responses at t h e i r acute l e v e l s can be t r i g g e r e d by a few molecules i n a f i s h ' s n a s a l chamber ... while t a s t e t h r e s h o l d s of f i s h e s , f a r lower than those of man ... r e q u i r e m i l l i o n s of molecules/cm 3 of a stimulant to bathe the animals t a s t e sensors on i t s body or in i t s mouth". A c r i t i c a l study of the r e l a t i v e importance of the d i f f e r e n t d e f e n s i v e mechanisms i n nudibranchs i s d i f f i c u l t due to the l i m i t e d amount of i n f o r m a t i o n a v a i l a b l e concerning predator-prey i n t e r a c t i o n s . However, i t seems very l i k e l y that chemical s e c r e t i o n s may play an important d e f e n s i v e r o l e . I t must be s t a t e d that such d e f e n s i v e substances have not been u n e q u i v o c a l l y proven to e x i s t i n nudibranchs. However, on the b a s i s of s t u d i e s i n v o l v i n g other organisms such as a r t h r o p o d s 1 0 , i t would be l o g i c a l to hypothesize that d e f e n s i v e s e c r e t i o n s are a l s o o p e r a t i v e i n nudibranchs. 6 1.3. Chemical communication : The p o s s i b l e e x i s t e n c e of a defense mechanism r e l y i n g on the sensory p e r c e p t i o n and d i s c r i m i n a t i o n of s p e c i f i c chemicals by animals i s a r e l a t i v e l y new f a c t o r to be c o n s i d e r e d i n e c o l o g i c a l d y n a m i c s 1 1 . T h i s process r e p r e s e n t s one aspect of chemical communication, a f i e l d of r e s e a r c h at the i n t e r f a c e of chemistry and b i o l o g y 1 2 . When the chemistry of the nudibranchs i s examined more c l o s e l y , two kinds of chemical communication may be encountered: 1) I n t e r s p e c i e s chemical communication. 2) I n t r a s p e c i e s chemical communication. An example of the f i r s t would r e f e r to defense substances used by the nudibranchs a g a i n s t p r e d a t o r s , while an example of i n t r a s p e c i e s chemical communication would be sexual a t t r a c t a n t s . For f u r t h e r d e t a i l s on the chemistry and b i o l o g y of these substances Whittaker and Feeny's paper 1 0 should be c o n s u l t e d . L i s t e d below are examples of molecules e x t r a c t e d from opisthobranch molluscs which may be i n v o l v e d i n chemical communicat i o n . 1) The nudibranch P h y l l i d i a v a r i c o s a i s known to produce a smelly substance l e t h a l to small f i s h and crustaceans 8 . I n v e s t i g a t i o n of the o d o r i f e r o u s compound s t a r t e d on the e x t r a c t s of the opisthobranch m o l l u s c . L a t e r , v a r i c o s a was found to feed on a sponge of the genus Hymeniacidon. Higher y i e l d s of the allomone, 9-isocyanopupukeanane (1) were obtained from the s p o n g e 1 3 and t h i s f a c i l i t a t e d the s t r u c t u r a l e luc i d a t i o n . 7 (1) Experiments 8 showed that the mucus s e c r e t e d by the nudibranch has no i l l e f f e c t s on a crab ( Metapograpsus messor ) or a nudibranch ( Placobranchus ianthobapsus) ; but that i t was l e t h a l to small crustaceans ( Lembos intermedius , Tisbe  r e t i c u l a t a ), f i s h ( M o l l i n e s i a l a t i p i n n a ) and a l o b s t e r ( S c y l l a r i d e s squammosus ) i n d i c a t i n g a c e r t a i n s p e c i f i c i t y i n the allomone's e f f e c t . 2) When the opisthobranch mollusc O n c h i d e l l a b i n n e y i i s m o l l e s t e d i t emits a d e f e n s i v e s e c r e t i o n . Arey and C o z i e r 5 r e p o r t e d that the s e c r e t i o n s from a r e l a t e d specimen, O n c h i d e l l a  floridanumn ward o f f the a t t a c k s of c e r t a i n i n t e r t i d a l f i s h e s . The major component of the e x t r a c t of the mucus s e c r e t e d by 0. b i n n e y i 1 4 has been i d e n t i f i e d as the monocyclic s e s q u i t e r p e n o i d o n c h i d a l ( 2 ) . 8 OAc (2) Although s e s q u i t e r p e n o i d ^ 2 was not t e s t e d i n the f i e l d f o r i t s chemical d e t e r r e n t p r o p e r t i e s , i t was shown to have a n t i b a c t e r i a l a c t i v i t y . R e c ently, t h i s same molecule has been i s o l a t e d from O n c h i d e l l a b o r e a l i s c o l l e c t e d in Barkley Sound ( R.J.Andersen, unpublished r e s u l t s ) . 3) Navanax inermis i s known to be a v o r a c i o u s predator on other opisthobranch m o l l u s c s 1 5 . I t l o c a t e s i t s prey by c o n t a c t -chemoreception. inermis recognizes the mucus t r a i l of an acce p t a b l e prey and f o l l o w s i t to i t s source. inermis a l s o produces a " t r a i l - b r e a k i n g pheromone". When s e c r e t e d by one i n d i v i d u a l t h i s pheromone causes an i n d i v i d u a l of the same sp e c i e s to turn o f f the course (at an angle g r e a t e r than 90) of the mucus t r a i l i t i s f o l l o w i n g . Navenones A, B and C (with s t r u c t u r e s 3, 4, and 5 r e s p e c t i v e l y ) have been e x t r a c t e d from the c o l o r e d s e c r e t i o n emited when the animals are i r r i t a t e d 1 ' . When these are present i n a 4:2:1 mixture, ( r a t i o found i n the e x t r a c t ) they e l i c i t the t r a i l - b r e a k i n g response. o 10 Summary: In t h i s chapter, the general d e f e n s i v e " s t r a t e g i e s " of nudibranchs i n the marine environment have been d e s c r i b e d and i t has been shown that chemical s e c r e t i o n s might p l a y an important r o l e i n the l i v e s of d o r i d nudibranchs. 11 Chapter 2 The Chemistry of Opisthobranch M o l l u s c s  11 .1. Review: Research i n marine n a t u r a l products chemistry has made s i g n i f i c a n t advances i n the l a s t ten years. The f i r s t book to appear i n t h i s f i e l d was e d i t e d by P.J. S c h e u e r 1 9 i n 1973, while one of the f i r s t review papers was w r i t t e n i n 1974 by D.J. Faulkner and R.J. A n d e r s e n 2 0 . Since then, many other review papers, symposia proceedings and s t r u c t u r a l papers have been p u b l i s h e d . Among these, two reviews deal with the chemistry of the molluscs i n g e n e r a l 2 1 , and with the opisthobranch molluscs in p a r t i c u l a r 2 2 . Th i s chapter presents a general review of the opisthobranch molluscs chemistry i n Table I. P r i m a r i l y molecules of the t e r p e n o i d c l a s s are re p o r t e d , along with t h e i r more i n t e r e s t i n g p r o p e r t i e s . Pigments, l i p i d s , s t e r o i d s and amino a c i d s are omitted, except i n a few cases. In Table I, the s p e c i e s are l i s t e d a c c o r d i n g to a ph y l o g e n e t i c scheme. The general s u b d i v i s i o n s i n the phyllum Mollusca are presented i n Chart I a c c o r d i n g to K o z l o f f ' s c l a s s i f i c a t i o n . F o l l o w i n g Table I, the o r i g i n of these molecules i s d i s c u s s e d . Phyllum Class Subclass Order (Genus) Suborder (Genus) Chart I Mollusca Amphineura B i v a l v i a Gastropoda Cephalopoda Scaphopoda Opisthobranchia Prosobranchia Pulmonata Sacoglossa ( Tridachia T r i d a c h i e l l a ) Nudibranchia Cephalospidea ( Aplysia Dolabella Navanax Basomatophora Stylocheilus ) Arminacea Dendronotacea Doridacea Eolidacea ( Archidoris ( Hervia Adalaria F l a b e l l i n a P h y l l i d i a Coryphella ) Chromodoris ) Stylomatophora ( Onchidella ) N.B. Organisms are c l a s s i f i e d according to Kozloff 17 C l a s s i f i c a t i o n Subclass: Opistobranchia Order: Sacoglossa T r i d a c h i e l l a diomedia 24 Tridachia c r i s p a t a 25 Table I Molecules (6) tridachione (7) 9,10-deoxytridachione (8) crispatone Remarks 14 In vivo C uptake experiments have shown that the secondary metabolites 6^  and ^ 7 are synthesized by a' mollusc-algal chloroplast symbiotic p a i r . This new carbon skeleton could o r i g i n a t e from a polyketide condensation of seven propionate u n i t s . These are the major compounds extracted from the saccoglossan T. c r i s p a t a . Metabolites O M e 5* a n c * ' a r e a^-so believed to be synthesized by a mollusc-algal c h l o r o p l a s t symbiotic p a i r . (9) crispatene Subclass: Opistobranchia Order: Cephalospidea Aplysia  angassl 26 (10) a p l y s i s t a t i n The sea hare's extract i n h i b i t s growth of fymphocetic leukemia c e l l s . This a c t i v i t y has been rel a t e d to a p l y s i s t a t i n ( i o l An isomeric substance has been found i n the extract of a Japanese marine algae, Laurencia nipponica, and a r e l a t e d molecule has been i s o l a t e d from the marine algae, Chondria o p p o s i t i c l a d a . Re-cen t l y , a p l y s i s t a t i n was obtained as a minor I g metabolite of the red marine algae, L. palisada. Aplysia (11) b r a s i l e n o l R=H Three rearranged sesquiterpeneids, 11, U 4 1 - 2 7 > 2 8 b r a s i l i a n a (12) b r a s i l e n o l acetate R=AC 12, and 13 have been i s o l a t e d from the d i g e s t -(13) e p i b r a s l l e n o l ive glands of A. b r a s i l i a n a c o l l e c t e d from the H - \^ OR O H • Gulf of Mexico. Subsequently, they were found i n the red algae Laurencia obtusa. When a small amount of a di g e s t i v e gland extract i s a p p l i e d to the s h e l l of a crab C a l l i n e c t e s A i i sapidus i t becomes re p u l s i v e to i t s usual A (13) predator: Octopus v u l g a r i s . (14) brasudol RX=H R2=OH Sesquiterpenoids 14 and 15 have been (15) isobrasudol R^OH R2=H extracted from t h i s same species and shown B r to be feeding deterrents to f i s h and sharks. II H 1 U l Aplysia (16) tribromotrichlororaonoterpenoid The highly polyhalogenated raonoterpenoid 29 c a l i f o r n i c a ' 16 i s an example of the novel compounds i s o -30,31,32,33 B r C ' \ ^ P lated from the d i g e s t i v e glands of A. c a l i f o r n -i c a . Monoterpenoid 16 was subsequently found / ^ ^ ^ / \ B r i n the red algae Placomium coccunum. I t i s a B r CI H member of a unique s e r i e s of monoterpenoids containing a v i n y l bromide function. (17) trichlorodibromomonoterpenoid Compound 17 was also i s o l a t e d from the midgut of the sea hares, but was not found i n P. coccinum. This might indi c a t e that some C H CI C l i / C l chemical transformation took place i n the | ^ B r d i g e s t i v e gland of the animals or that the CI molecule o r i g i n a t e s from a d i f f e r e n t a l g a l source. (18) prepacifenol epoxide When A. c a l i f o r n i c a was c o l l e c t e d at La J o l l a and C a r d i f f , C a l i f o r n i a the amount of 18, 19, 20, 21, 22, and 23 varied with d i v i n g s i t e and i n d i v i d u a l . These metabolites o r i g i n -ate p a r t l y from the seaweeds Laurencia p a c i f i c a and Placomium coccinum, and p a r t l y from i n vivo chemical transformations. Johnstonol(23)was extracted from a i r dried algae, while prepacifenol epoxide 18 was extracted from f r e s h material. Hence, john-stonol (23) i s believed to be an a r t i f a c t formed from prepacifenol epoxide(l8). The authors believe that the transformation may have occurred during the i s o l a t i o n procedure or in the midgut of the animals. (23) johnstonol HO Aplysia 34 dactylomela ' 35,36,37,38,39 (24) isodactylyne R^H R 2 = H x Q\ (25) dactylyne ^"V* R 2 = H B r\ .•CI B n \ \ (26) d a c t y l o l The sea hare A. dactylomela has been the source of a large v a r i e t y of compounds. The isomeric non-terpenoid C 15 ethers 24 and 25 have been shown to prolong pento-b a r b i t o l hypnosis i n mice. The non-halogenated b i c y c l i c sesquiter-pene i d 26 which represents a new carbon skele-ton has also been i s o l a t e d from t h i s extract. (27) dactyloxene A (28) isomer B,C (29) dactylenol R=H (30) dactylenol acetate R=Ac The related compounds 27 to 30 have also been obtained from the d i g e s t i v e glands of A. dactylotnela. The carbon skeleton of 28 • * ; — might a r i s e b i o g e n e t i c a l l y v i a a methyl mi-gration i n the skeleton of a known sesquiter-penoid such as trans ft-raonocyclofarnesol. Dactylenol converts to dactyloxene A and B by acid treatment. (31) 14-bromo obtus-lene-3,11-diol Aplysia  depilans (33) d i c t y o l A R=H (34) d i c t y o l B R=OH The cytotoxic halogenated diterpenoid d i o l , 31 with a new carbon skeleton i s r e l a t e d r*— to compounds obtained from the seaweed Laurencia  obtusa. The sesquiterpeneid ether 32, which was also extracted from the d i g e s t i v e glands of the sea hares, shows moderate a n t i - n e o p l a s t i c a c t i v i t y and cytotoxic a c t i v i t y . Compounds 24 to 32^  have a d i e t a r y o r i g i n . These novel guaiane-diterpenoids extract-ed from A. depilans were also found i n the brown algae Pachidicton corriaceum. o Aplysia k u r o d a i ^ Aplysia 1imac ina 42 (35) pachidictyol H. R R R R Me H Br Me OH (36) aplysin (37) debromoaplysin H Me 11 Me (38) a p l y s i n o l H Me Br CH ^ l l (39) a p l y s i a v i o l i n COOCH, COOCH, This Japanese sea hare was one of the f i r s t opistobranch molluscs to be studied. The new aromatic sesquiterpenoid toxins existed in d i f f e r e n t amounts depending on where and when the sea hares were c o l l e c t e d . The Aplysia sea hares are known to emit coloured defensive secretions. The "defensive pigment" 39^ i s excreted from the glandular c e l l s . D o l a t r i o l A and B are the f i r s t members of a new c l a s s of diterpenoids with an angular 6-7-5 r i n g system. These compounds have a n t i -neoplastic a c t i v i t y . This molecule i s one of fourteen d i t e r -penoids extracted from the d i g e s t i v e glands of the sea hare. A l l these molecules are related to 42 and e x i s t in d i f f e r e n t proportions de-pending on the i n d i v i d u a l . This novel 5,11-b i c y c l o carbon skeleton could be formed by the c y c l i z a t i o n of geranlygeraniol pyrophosphate. These three conjugated ketones are described along with t h e i r properties i n Chapter 1. O A c These toxins found i n the d i g e s t i v e glands of S. longicauda have an unprecedented struc-ture, with a phenylpentadecanoic acid bearing f i v e methyl substituents as a moiety. Moore* 3 has i s o l a t e d these two toxins from the blue-green algae Lyngbia g r a c i l i s . This h i g h l y f u n c t i o n a l i z e d amine o f a methoxy monoolefinic m y r i s t i c acid (C 14) i s non-toxic. Subclass: Opistobranchia Order: Nudibranchia Arch i d o r i s (46) farnesic acid glyceride J U .48 odhneri 0 OH Adalaria (47) s t e r o i d a l peroxide * 49 species H O ' The major metabolite of the nudibranch's extract might have a defensive r o l e . A s e r i e s of s t e r o i d a l peroxides have been extracted from t h i s nudibranch. The C 2 6 s t e r o l 47 was obtained as the major component. The major metabolite obtained from the extract of the nudibranch C. marislae has been i d e n t i f i e d as 48. The structure of m a r i s l i n (48) i s very c l o s e l y related to a metabolite obtained from the Mediterranean sponge P l e r a p l y s i l l a  s p i n i f e r a . The major component of the extract of the three nudibranchs, H. peregrina, F. a f f i n i s and C. l i n e a t a and of the hydroid Eudendrium sp. upon which they feed has been i d e n t i f i e d as the ster o i d 49. P h y l l i d i a (1) varicosa Subclass: Pulmonata Order: Stylomatophora Onchidella (2) binneyl OAc The allemone obtained from t h i s species has been described in Chapter I. The sesquiterpenoid ochidal (2) has been described in Chapter I. 27 11.2. O r i g i n of the molecules: A c e r t a i n p a t t e r n emerges from Table I that enables us to d i v i d e the molecules i n t o three c l a s s e s depending on t h e i r o r i g i n : 1) Those of a d i e t a r y o r i g i n 2) Those from of a symbiotic a s s o c i a t i o n 3) Those p o s s i b l y of a molluscan o r i g i n II.2.1. D i e t R elated Molecules : Sea hares are s o f t - b o d i e d and slow-moving organisms ( l i k e the nudibranchs) which have been known s i n c e a n c i e n t times to have poisonous p r o p e r t i e s . "Indeed such t o x i c m a t e r i a l s are b e l i e v e d to have been used by Agrippa, mother of Nero, to d i s p a t c h r e l a t i v e s b l o c k i n g h i s ascent to Roman emperor" ( G . R . P e t t i t 3 1 ) Sea hares are a l s o of a somewhat l a r g e r s i z e ( u s u a l l y 10 to 12 cm) than the nudibranchs ( u s u a l l y 1 to 5 cm). The above two reasons have made the sea hares i n t e r e s t i n g to study, e s p e c i a l l y members of the more common A p l y s i d e a f a m i l y . The chemistry of sea hares c o n s t i t u t e s the sub j e c t of over t h r e e - f o u r t h s of the l i t e r a t u r e r e p o r t e d i n t h i s chapter. The m a j o r i t y of the compounds i s o l a t e d from sea hares are present i n the d i g e s t i v e glands (midgut). Close examination of the e a t i n g h a b i t s of these animals or a n a l y s i s of the midgut content has demonstrated that the primary source of the molecules i n s e v e r a l cases was algae (brown, blue-green and 28 r e d ) . The sea hares concentrate the m e t a b o l i t e s obtained from t h e i r prey, and have, t h e r e f o r e , served ,as a u s e f u l source of a l g a l m e t a b o l i t e s , many of which have novel s t r u c t u r e s . D i s c o v e r i n g these molecules by studying the algae d i r e c t l y would have been more d i f f i c u l t because of the comparatively lower c o n c e n t r a t i o n s . In some cases the molecules e x t r a c t e d from the sea hares were m o d i f i e d i n comparison to the a l g a l m e t a b o l i t e s . A t y p i c a l example i s the sea hare A p l y s i a c a l i f o r n i c a 3 2 , from which molecules 19, 20, 21, and 22 were e x t r a c t e d (among o t h e r s ) , while only 19 and 21 were obtained from the algae L a u r e n c i a  pac i f i c a . T h i s o b s e r v a t i o n l e d to a l a b e l l i n g experiment which showed that 20 and 22 r e s u l t e d from chemical t r a n s f o r m a t i o n s t a k i n g pl a c e i n the d i g e s t i v e glands of A^ c a l i f o r n i c a . On some oc c a s i o n s , sponges have been i d e n t i f i e d as a d i e t a r y source of secondary m e t a b o l i t e s obtained from Opisthobranch m o l l u s c s . T h i s was shown to be the case with P h y l l i d i a v a r i c o s a ', which may r e t a i n the sponge compound f o r a d e f e n s i v e purpose. In a recent s t u d y 5 1 of the chemistry of nudibranch p r e d a t o r s and t h e i r prey, i t was found that H e r v i a p e r e g r i n a , F l a b e l l i n a a f f i n i s , and C o r y p h e l l a 1ineate c o n t a i n the same s t e r o i d as that found i n the h y d r o i d upon which they feed. 29 11.2.2. Molecules Derived from a Symbiotic A s s o c i a t i o n : The order Sacoglossa i s c h a r a c t e r i z e d by organisms a s s i m i l a t i n g p h o t o s y n t h e t i c a l l y v i a b l e c h l o r o p l a s t s from the algae upon which they feed. A l a b e l l i n g e x p e r i m e n t 5 2 done on three s p e c i e s of t h i s order; T r i d a c h i e l l a diomedea , T r i d a c h i a  c r i s p a t a , and Placobranchus o c e l l a t u s , showed that organic carbon f i x e d d u r i n g p h o t o s y n t h e s i s i s t r a n s f e r r e d to c h l o r o p l a s t - f r e e t i s s u e . When the chemistry of these three s p e c i e s was subsequently s t u d i e d , t r i d a c h i o n e (6) and 9,10-d e o x y t r i d a c h i o n e (7) were e x t r a c t e d from T. diomedea , c r i s p a t o n e (8) and c r i s p a t e n e (9) were e x t r a c t e d from T. c r i s p a t a , while 9,10-deoxytridachione {!) and photodeoxytridachione (50) were obtained from o c e l l a t u s . These r e s u l t s i n i t i a t e d a s e r i e s of carbon-14 l a b e l l i n g experiments on Placobranchus o c e l l a t u s , and i t was shown t h a t : 1) M e t a b o l i t e s 2 a n <3 50 are s y n t h e s i z e d by the m o l l u s c - c h l o r o p l a s t symbiotic p a i r . 2) In v i v o photochemical conve r s i o n takes plac e from pyrone 7 to 50. These o b s e r v a t i o n s l e a d I r e l a n d and Scheuer to propose that the pyrones c o u l d be s e r v i n g as sunscreens to the sacoglossans. 30 11.2. 3 . P o s s i b l y of a Molluscan O r i g i n : Molecules which have not been shown to o r i g i n a t e from the d i e t or from a symbiotic a s s o c i a t i o n are i n c l u d e d i n t h i s category. O n c h i d a l 1 4 (2) r e p r e s e n t s a good example. T h i s s e s q u i t e r p e n o i d has been obtained from the e x t r a c t s of two r e l a t e d s p e c i e s ; O n c h i d e l l a b i n n e y i and O n c h i d e l l a b o r e a l i s , c o l l e c t e d at two d i f f e r e n t g e o g r a p h i c a l l o c a t i o n s ; C a l i f o r n i a and B r i t i s h Columbia. The f a c t that t h i s molecule has been obtained from organisms l i v i n g i n two d i f f e r e n t environments would tend to e l i m i n a t e the d i e t as the source of o n c h i d a l . U n f o r t u n a t e l y i n s u f f i c i e n t i n f o r m a t i o n i s a v a i l a b l e to be able to prove that the m e t a b o l i t e i s molluscan. 31 11.3. Summary: T h i s chapter has reviewed the compounds i s o l a t e d from opisthobranch m o l l u s c s , and has d e s c r i b e d t h e i r p r o p e r t i e s . I t has shown that the secondary m e t a b o l i t e s of opisthobranchs can have three d i f f e r e n t o r i g i n s : 1) The d i e t 2) A symbiotic a s s o c i a t i o n 3) The mollusc i t s e l f The v a r i e t y of molecular s t r u c t u r e s that can be encountered when studying the chemistry of the opisthobranchs has a l s o been i l l u s t r a t e d . Chart II Compounds is o l a t e d from Cadiina luteomarginata 55 Microcionin-2 56 Luteone 33 Chapter 3 I s o l a t i o n of the Compounds III .1. C o l l e c t i o n : As p a r t of a program aimed at studying the chemistry of B r i t i s h Columbia nudibranchs, we have examined the e x t r a c t s of C a d i i n a luteomarginata and Acanthadoris nanaimoensis. I n t e r e s t in these two organisms arose when we n o t i c e d t h e i r sweet fragrance while s o r t i n g d i f f e r e n t specimens a f t e r a d i v i n g e x p e d i t i o n . We are aware of very few b i o l o g i c a l s t u d i e s i n v o l v i n g these two s p e c i e s . The e a t i n g h a b i t s of d o r i d nudibranchs from C a l i f o r n i a , i n c l u d i n g C^ luteomarginata and A_;_ nanaimoensis, i s the sub j e c t of two p a p e r s 5 3 5 4 . The d i f f e r e n t stages of the development of C^ l a e v i s specimens from the Un i t e d Kingdom i s the s u b j e c t of another p a p e r 5 5 , and the sweet fragrance of C. luteomarginata has been r e p o r t e d 5 6 . C a d i i n a luteomarginata and Acanthadoris nanaimoensis are c l a s s i f i e d as f o l l o w s 1 7 : Phyllum: M o l l u s c a , C l a s s : Gastropoda, S u b c l a s s : O p i s t o b r a n c h i a , Order: Nudibranchia, Suborder: Doridaceae. C a d l i n a luteomarginata i s white, except f o r a yellow band bo r d e r i n g the edge of the f o o t , the edge of the mantle, and yellow on the t i p s of the g i l l s and d o r s a l t u b e r c u l e s ( F i g . 2 ) . Specimens were c o l l e c t e d by SCUBA from Howe Sound and Barkley Sound at depths of 5 to 18 m (see F i g . 3 f o r c o l l e c t i o n s i t e s ) . 34 C. luteomarginata v a r i e d i n s i z e from 2 to 6 cm. Acanthadoris nanaimoensis v a r i e s i n c o l o r from white to mauve-grey, with red on the edge of the g i l l s and rhinophores. The t i p s of the p a p i l l a e are yellow ( F i g . 3). Specimens of 2 to 4 cm i n l e n g t h were c o l l e c t e d at depths of 3 to 12 m i n the Howe Sound area, near G a l i a n o I s l a n d , and i n Barkley Sound (see F i g . 5 f o r c o l l e c t i o n s i t e s ) . These two nudibranch s p e c i e s are predominantly found on rock s u r f a c e s , and they tend to e x i s t i n areas exposed to strong c u r r e n t s , such as p o i n t s and submarine c l i f f s . We have a l s o noted that A_j_ nanaimoensis i s more abundant from the end of J u l y u n t i l at l e a s t the end of October. During t h i s p e r i o d specimens are found at a shallower depth ( i n t e r t i d a l ) than d u r i n g the r e s t of the year. Diving site 1- BIink Bonny (6-10m) 2- Boyer Island (6-10m) 3- Copper Cove (5-10m) 4- Sunset Beach (10-18m) 5- Earl's Cove (12-18m) 6- Nanat Island (6-12m) 7- Galiano Island (6-10m) 8- Ross Island (3-10m) 9- Execution Rock (5-12m) F i g u r e Map p r e s e n t i n g t h e d i v i n g s i t e s w h e r e C j a J l n a l u t e o m a r g i n a t a was c o l l e c t e d cn 39 111.2. C a d l i n a luteomarginata 111,2.1 E x t r a c t i o n Procedure: Immediately a f t e r c o l l e c t i o n , the specimens were immersed i n MeOH and s t o r e d at room temperature f o r three days. The supernatant was then decanted and evaporated i n vacuo. The r e s u l t i n g c o n c e n t r a t e d e x t r a c t was p a r t i t i o n e d between b r i n e and CHC1 3(or CH 2 C l 2 ). The organic phase was then d r i e d and evaporated to give a sweet s m e l l i n g o i l y r e s i d u e . To a v o i d contamination and l o s s of the s e s q u i t e r p e n o i d s , s e v e r a l p r e c a u t i o n a r y measures were taken throughout the manipulation of, the samples: 1) A l l s o l v e n t s used at d i f f e r e n t stages i n the l a b o r a t o r y , were p u r i f i e d by d i s t i l l a t i o n ( i . e . CH 2 C l 2 , EtOAc) or were HPLC (high performance l i q u i d chromatography) grades ( i . e . CHC1 3, hexane). 2) The i n v e r t e b r a t e s were soaked only i n g l a s s j a r s . 3) Aluminum f o i l was p l a c e d under a l l l i d s to prevent contamination from p l a s t i c i z e r s . 4) D i r e c t use of t e c h n i c a l grade acetone (or any other s o l v e n t ) s t o r e d i n p l a s t i c c o n t a i n e r s was avoided when d r y i n g glassware. 5) The samples were never heated when ev a p o r a t i n g s o l v e n t s . 6) Samples were never d r i e d under the high vacuum pump. Prec a u t i o n s (1) to (4) (above) enabled the e l i m i n a t i o n of s e v e r a l UV ( u l t r a v i o l e t ) absorbing spots on TLC ( t h i n l a y e r 40 chromatography) that were generated by the work up procedures. These are b e l i e v e d to be due to p l a s t i c i z e r s ( r e f e r to chapter 4). Measures (5) and (6) i n c r e a s e d the y i e l d s s l i g h t l y by p r e v e n t i n g l o s s e s due to the v o l a t i l i t y of the compounds. 41 111.2.2. I n i t i a l s e p a r a t i o n : Two methods f o r the f i r s t s tep of the p u r i f i c a t i o n of the e x t r a c t were t r i e d : 1) A s i l i c a column u s i n g a s o l v e n t g r a d i e n t i n c r e a s i n g i n p o l a r i t y from hexane to c h l o r o f o r m to e t h y l a c e t a t e (with 10% stepwise increments). 2) A f a s t s i l i c a column using only one s o l v e n t followed by s i l i c a g e l PTLC ( p r e p a r a t i v e t h i c k l a y e r chromatography) using the same s o l v e n t . The f i r s t method was used only twice. D i f f i c u l t i e s a s s o c i a t e d with t h i s procedure were: 1) The presence of a l a r g e number of compounds with s i m i l a r r e t e n t i o n times ( e s p e c i a l l y i n the more p o l a r f r a c t i o n s ) which are not r e s o l v e d on the column. 2) Large volumes of s o l v e n t s used , which n e c e s s i t a t e evaporation of the d i f f e r e n t t e s t tube f r a c t i o n s p r i o r to TLC. 3) Losses due to t r a n s f e r s from one c o n t a i n e r to another. 4) Above a l l , l o s s e s due to the v o l a t i l i t y of the molecules, e s p e c i a l l y the o d o r i f e r o u s compound, luteone (56) (which a l s o seems to decompose r a p i d l y ) . The second method was a p p l i e d i n the m a j o r i t y of cases, because i t i s f a s t e r and minimizes l o s s e s due to v o l a t i l i t y and t r a n s f e r s . 42 The second method i n v o l v e s chromatographing the e x t r a c t on a s i l i c a column (using CH 2C1 ? or CHC1 3 ) f i r s t . T h i s quick chromatographic step separates the very p o l a r compounds, which do not migrate i n the s o l v e n t systems employed (carbohydrates, amino a c i d s , . . . ) , from the remainder of the e x t r a c t . Then, a f t e r e v a p o r a t i o n of the s o l v e n t , PTLC on s i l i c a g e l using the same eluent p r o v i d e s the best crude s e p a r a t i o n . T h i s step enables the s u b d i v i s i o n of the e x t r a c t i n t o four f r a c t i o n s c o n t a i n i n g the f o l l o w i n g compounds: 1) Fu r a n o s e s q u i t e r p e n o i d s 2) A l b i c a n y l a c e t a t e 3) A l b i c a n o l and luteone 4) S t e r o i d a l peroxides where f r a c t i o n (1) i s the l e a s t p o l a r . L a t e r , each f r a c t i o n i s r e p u r i f i e d s e p a r a t e l y . The m a j o r i t y of the molecules i n v e s t i g a t e d d i d not have an u l t r a - v i o l e t chromophore at 254 or 280 nm, but a l l charred a f t e r s p r a y i n g with H 2 S0 4 and h e a t i n g . The c h a r r i n g procedure was a p p l i e d i n order to f o l l o w the m i g r a t i o n of the d i f f e r e n t substances on the s i l i c a g e l p l a t e s . The steps that allowed the i s o l a t i o n of s i x pure m e t a b o l i t e s are d e s c r i b e d s t a r t i n g with the l e a s t p o l a r f r a c t i o n . For p r e c i s e q u a n t i t a t i v e d e t a i l s ( s o l v e n t systems, Rf , . . .) r e f e r to the experimental s e c t i o n . 43 111.2.3. I s o l a t i o n of F u r a n o s e s q u i t e r p e n o i d s from  F r a c t i o n ( 1 ) ; T h i s f r a c t i o n of the e x t r a c t was obtained only i n the case of nudibranchs c o l l e c t e d i n Barkley Sound. The l o c a t i o n of the d i v i n g s i t e s are i n d i c a t e d i n F i g . 3. The composition of t h i s f r a c t i o n of the e x t r a c t v a r i e d with each c o l l e c t i o n . F u r o d y s i n i n (54) was i s o l a t e d from nudibranchs c o l l e c t e d d u r i n g the b e g i n i n g of March, at E l l i s I s l a n d , and near Execution Rock. In a separate c o l l e c t i o n performed at the beginning of May, nudibranchs were c o l l e c t e d mainly around E l l i s I s l a n d . A mixture of f u r a n o s e s q u i t e r p e n o i d s c o n t a i n i n g f u r o d y s i n (53) and f u r o d y s i n i n (54) was obtained from f r a c t i o n (1) of the e x t r a c t of these organisms. A t h i r d c o l l e c t i o n took p l a c e i n mid June, near Nanat I s l a n d , E l l i s I s l a n d and Execution Rock. F u r o d y s i n i n (54) was again the only compound present in t h i s f r a c t i o n . On the l a s t d i v i n g e x p e d i t i o n which occured at the end of J u l y , specimens were c o l l e c t e d c l o s e to Execution Rock. M i c r o c i o n i n - 2 (55) was the only f u r a n o s e s q u i t e r p e n o i d present i n the e x t r a c t of these nudibranchs. Even though the composition of f r a c t i o n (1) v a r i e d with each c o l l e c t i o n , the p u r i f i c a t i o n of the furan c o n t a i n i n g compounds was c a r r i e d out i n the same general manner. F r a c t i o n (1) was rechromatographed on s i l i c a g e l p l a t e s developed with a non-polar s o l v e n t (hexane). In the three cases where only one major molecule was found, t h i s one-step p u r i f i c a t i o n was suf f i c i e n t . The s e p a r a t i o n was more d i f f i c u l t when isomeric mixtures were present i n the e x t r a c t . The presence of two isomers was 44 i n d i c a t e d by c a r e f u l o b s e r v a t i o n of the c h a r r i n g of the substances. A f t e r s p r a y i n g the s i l i c a p l a t e with H2SO^ and h e a t i n g , the f u r a n o s e s q u i t e r p e n o i d band ( a l s o v i s i b l e i n short UV, at A=254 nm, as one band) becomes "pi n k - r e d " . Upon prolonged c h a r r i n g , the upper part of the, band becomes "dark-blue", while the lower p a r t becomes " b l u e - r e d " . The t h i n upper part of the band was c o l l e c t e d s e p a r a t e l y from the lower p a r t . The lower p a r t was then rechromatographed s e v e r a l times i n the same s o l v e n t . T h i s repeated procedure f a c i l i t a t e d the i s o l a t i o n of the m e t a b o l i t e present i n the upper part of the band as a pure compound which was subsequently i d e n t i f i e d as f u r o d y s i n i n (54). The lower part remained a mixture. S e v e r a l d i f f e r e n t chromatographic methods were t r i e d i n order to p u r i f y t h i s f r a c t i o n . These i n c l u d e d : 1) reversed-phase p l a t e s 2) TLC on alumina 3) HPLC using a s i l i c a column 4) HP1C using a reversed-phase column 5) GC U n f o r t u n a t e l y a l l were u n s u c c e s s f u l ( r e f e r to the experimental s e c t i o n ) . 45 111.2.4 . I s o l a t i o n of A l b i c a n y l Acetate from  F r a c t i o n ( 2 ) : T h i s f r a c t i o n of the e x t r a c t was obtained from nudibranchs c o l l e c t e d at a l l s i t e s . I t rep r e s e n t s the major c o n s t i t u e n t of the e x t r a c t (yield=0.5% of crude e x t r a c t ) . The main d i f f i c u l t y i n the p u r i f i c a t i o n of t h i s molecule was i t s s e p a r a t i o n from a f a t band ( y i e l d =3.0% of crude e x t r a c t , F i g . 9) which had a very s i m i l a r Rf and e x i s t e d i n a much l a r g e r amount than the s e s q u i t e r p e n o i d . I s o l a t i o n of pure a l b i c a n y l a c e t a t e (51) was made p o s s i b l e by developing f r a c t i o n (1) i n two s u c c e s s i v e s o l v e n t systems (CH. C l 2 rhexane and CHC1 3: EtOAc). 46 111.2.5 I s o l a t ion of A l b i c a n o l and Luteone from  F r a c t i o n (3): These two molecules e x i s t i n approximately one te n t h of the c o n c e n t r a t i o n of a l b i c a n y l a c e t a t e (52j • A l b i c a n o l (52) and luteone (56) were present i n the nudibranchs c o l l e c t e d at a l l d i v i n g s i t e s i n d i c a t e d on the map presented in F i g u r e 3. The p u r i f i c a t i o n of these two compounds was d i f f i c u l t because they were present i n such minor amounts, they are extremely v o l a t i l e , and they were always contaminated with f a t s , p h t h a l a t e e s t e r s and "decomposition products". When f r a c t i o n (3) was chromatographed i n a s o l v e n t system that enabled the s e p a r a t i o n of the two molecules, only " p a r t i a l l y pure" substances were obtained, as i n d i c a t e d by the *H NMR of luteone ( F i g . 26). Attempts to p u r i f y the samples f u r t h e r , r e s u l t e d i n lowering the y i e l d s and i n c r e a s i n g the r e l a t i v e amount of the i m p u r i t i e s ( F i g . 27). I s o l a t i o n of the f r a g r a n t compound, luteone was most s u c c e s s f u l as a d i n i t r o p h e n y l h y d r a z o n e (DNPH) d e r i v a t i v e . In the case of a l b i c a n o l (5^2), a l b i c a n y l a c e t a t e (51) was hy d r o l y z e d and the product obtained compared to the n a t u r a l substance by TLC, *H NMR and MS. 47 I I I . 2 . 6 . S t e r o i d a l Peroxides from F r a c t i o n (4): S t e r o i d a l peroxides are present i n the most p o l a r f r a c t i o n . They have a lower Rf than c h o l e s t e r o l ( Rf=0.1 i n CHC1 3) and char with H 2S0 4 i n a very c h a r a c t e r i s t i c manner (green). They were compared by TLC to a mixture of s t e r o i d a l peroxides e x t r a c t e d from two other organisms s t u d i e d i n our l a b o r a t o r y 4 ' . I d e n t i f i c a t i o n of the i n d i v i d u a l components was not c a r r i e d f u r t h e r . 48 111.3 Acanthador i s nanaimoensis I I . 3.1. E x t r a c t ion Procedure: A. nanaimoensis was c o l l e c t e d p r i m a r i l y i n Barkley Sound, in the v i c i n i t y of G a l i a n o I s l a n d , and near Sunset Beach ( F i g . 5). T h e i r e x t r a c t i o n was c a r r i e d i n the same manner as that d e s c r i b e d f o r Cj_ luteomarginata. I I I . 3 .2. I s o l a t ion of the Compounds: S i l i c a t h i n l a y e r chromatographic a n a l y s i s of the e x t r a c t of these nudibranchs i n d i c a t e d the presence, of one major non-p o l a r m e t a b o l i t e , d e s i g n a t e d as nanaimoal, and of s t e r o i d a l p eroxides recognized by t h e i r c h a r a c t e r i s t i c c h a r r i n g (green) and Rf (0.1 i n CHC1 3). The s e s q u i t e r p e n o i d was r e a d i l y p u r i f i e d from the crude e x t r a c t by chromatography on s i l i c a g e l p l a t e s using CH 2 Cl2 as the e l u e n t . T h i s procedure a l s o a f f o r d e d p a r t i a l l y p u r i f i e d s t e r o i d a l p e r o x i d e s . Addendum: On one o c c a s i o n when Acanthadoris hudsoni was c o l l e c t e d at Sunset Beach, nanaimoal was obtained i n the nudibranch's e x t r a c t . 49 Chapter 4 I d e n t i f i c a t i o n of the Compounds  IV.1. From C a d l i n a luteomarginata  I V . 1 . 1 . A l b i c a n y l a c e t a t e : The new compound a l b i c a n y l a c e t a t e (51) has been i s o l a t e d as the major c o n s t i t u e n t of the organic e x t r a c t of C a d l i n a  luteomarginata . (51) The s t r u c t u r a l e l u c i d a t i o n was achieved through i n t e r p r e t a t i o n of s p e c t r a l data, b i o s y n t h e t i c arguments and chemical c o r r e l a t i o n . The HRMS (high r e s o l u t i o n mass spectrum) of a l b i c a n y l a c e t a t e (51) give s a parent ion of weak i n t e n s i t y at m/e= 264.2088 ( c a l c u l a t e d 264.2082) corresponding to the molecular formula C 1 7 H 2 8 0 2 . The molecule undergoes a f a c i l e rearrangement e x p e l l i n g a c e t i c a c i d to gi v e the base peak at m/e=204 ( C 1 5 H ^ 4 ) (Scheme 1 ) . T h i s rearrangement c o u l d i n v o l v e the oxygen of the ca r b o n y l f u n c t i o n and H-9 which would give r i s e through a M c L a f f e r t y rearrangement to fragment m/e=204 (A); or i t c o u l d i n v o l v e the second oxygen of the e s t e r and a proton on C-15 which would give fragment m/e = 204 ( B ) 5 7 . A major peak at m/e = 137 ( C ^ H ^ ) c o u l d a l s o r e s u l t from two d i f f e r e n t pathways. F i r s t , the cleavage of the a l l y l i c bond 50 C(9)-C(10) and then cleavage of the C(6)-C(7) bond v i a a rearrangement, would give a fragment (m/e=137) with a double bond between C ( 5 ) - C ( 6 ) . Fragment 137 cou l d a l s o r e s u l t from the a l l y l i c cleavage of the C(5)-C(6) bond of fragment 204(B). In t h i s case, the fragment obtained has a double bond between C(10)-C(15) . The m/e=137 peak i s g e n e r a l l y of a s i g n i f i c a n t i n t e n s i t y when a C(8)-C(20) double bond i s present i n the d i t e r p e n o i d s of the labdane f a m i l y . For example, i n the mass spectrum of man o o l 5 8 (57), a fragment ion of m/e=137 re p r e s e n t s the base peak. (57) Other ions that can be seen and which are i d e n t i f i e d i n the mass spectrum ( F i g . 6) r e s u l t from a l o s s of CH 3(m/e=249), C^H^O (m/e=222) and CH 3CO (m/e=221) from the parent i o n . A l s o , from the base peak at m/e=204(A or B), a l o s s of C H 3 i s i n d i c a t e d by the peak at m/e=189 (A or B). From the intense peak at m/e=204(B), an a l l y l i c cleavage of the C(5)-C(6) bond would give r i s e to fragment m/e=123. E x o c y c l i c double bonds are known to r a p i d l y isomerize to an e n d o c y c l i c p o s i t i o n 5 ' . T h i s c o u l d happen to the molecular fragments of m/e=137 and 123. These c o u l d then give r i s e through a r e t r o D i e l s - A l d e r rearrangement to fragments m/e=109 and 95 51 r e s p e c t i v e l y . The IR of a l b i c a n y l a c e t a t e shows a strong band corresponding to the ca r b o n y l f u n c t i o n at 1740 cm" 1, and a weaker one at 900 cm"1 a p p r o p r i a t e f o r a v i n y l i d i n e double bond ( F i g . 7). The 1H NMR spectrum ( f i g u r e 8) r e v e a l e d s e v e r a l f e a t u r e s of the molecule. Four s i n g l e t s i n t e g r a t i n g f o r three protons each c o u l d be de t e c t e d at 0.76, 0.81, 0.88 and 2.01 ppm. The f i r s t three s i g n a l s , c orresponding to methyl groups on quaternary carbons, were assigned to a geminal dimethyl f u n c t i o n a l i t y and a bridgehead methyl. The d e s h i e l d e d s i g n a l i n d i c a t e d the presence of a methyl next to a ca r b o n y l . Two doublets (J=lHz) at 4.51 and 4.85 ppm suggested the presence of an e x o c y c l i c double bond. From s e v e r a l proton-proton d e c o u p l i n g experiments, the chemical s h i f t s and c o u p l i n g c o n s t a n t s corresponding to the fi-r i n g protons were assig n e d as l i s t e d i n Table I I . The p a r t i a l s t r u c t u r e ^ CH —CH^-OAc was deduced from the chemical s h i f t s and the i r r a d i a t i o n of each of the C - l l protons. When the dd (doublet of doublets) at 4.34 ppm (H-12) was i r r a d i a t e d , the e f f e c t on the dd at 4.18 ppm (H-12') was not d e t e c t a b l e due to the p r o x i m i t y of t h i s l a t t e r s i g n a l to the i r r a d i a t e d one ( s a t u r a t i o n ) , however the complex m u l t i p l e t at 2.02 ppm (H-9a, H-7a) was a f f e c t e d . When the dd at 4.18 ppm (H-12) was i n turn i r r a d i a t e d , the e f f e c t on the dd at 4.34 ppm (H-12) was l i k e w i s e not d i s c e r n i b l e , but the m u l t i p l e t at 2.02 ppm (H-9a) was pert u r b e d . I r r a d i a t i o n of the ddd (J=13,4.5,3 Hz) at 2.41 ppm (H-7e) c o l l a p s e d the s i g n a l at 1.33 ppm (H-6a) to a ddd by e l i m i n a t i n g 52 a 4.5 Hz c o u p l i n g , c h a r a c t e r i s t i c of an "ae" ( a x i a l e q u a t o r i a l ) c o u p l i n g c o n s t a n t . I t a l s o c o l l a p s e d the dddd at 1.73 (H-6e) i n t o a ddd, e l i m i n a t i n g a 3 Hz c o u p l i n g , c h a r a c t e r i s t i c of an "ee" c o u p l i n g constant and changed the m u l t i p l e t at 2.02 (H-7a, H-9a) ( probably e l i m i n a t i n g a c o u p l i n g of 13 Hz, c h a r a c t e r i s t i c of a geminal c o u p l i n g c o n s t a n t ) . These r e s u l t s i n d i c a t e d that the s i g n a l at 2.41 ppm was a p p r o p r i a t e f o r a d e s h i e l d e d proton i n an e q u a t o r i a l p o s i t i o n (H-7e), next to a double bond. I t a l s o enabled the i d e n t i f i c a t i o n of the chemical s h i f t s corresponding to the three protons to which H-7e was coupled. I r r a d i a t i o n of the complex m u l t i p l e t at 2.02 ppm, a f f e c t e d the s i g n a l s at 4.34 (H-12), 4.18 (H-12), 2.41 (H-7e), 1.73 (H-6e) and 1.33 ppm (H-6a). A l l of these s i g n a l s showed the disappearance of one c o u p l i n g , . i n d i c a t i n g that they were coupled to only one of the two protons present i n the m u l t i p l e t at 2.02 ppm. The value of the c o u p l i n g constants enabled the assignment of the s i g n a l s at 1.73 and 1.33 ppm as being H-6e and H-6a, r e s p e c t i v e l y . F i n a l l y , i r r a d i a t i o n of the dddd at 1.73 (H-6e) c o l l a p s e d the ddd at 2.41 (H-7e) i n t o a dd and s i m p l i f i e d the three other s i g n a l s at 2.02 (H-7a, H-9a), 1.33 (H-6a), and 1.13 (H-5a)ppm. T h i s l a s t experiment r e v e a l e d the chemical s h i f t of the C-5 proton and the value of the c o u p l i n g constants confirmed the s t e r e o c h e m i s t r y at t h i s p o s i t i o n . The s p e c t r a l data a v a i l a b l e , t h e r e f o r e , i n d i c a t e d that the present molecule c o n t a i n e d : 1) Four s i t e s of u n s a t u r a t i o n : a c a r b o n y l , an 53 e x o c y c l i c double bond and two r i n g s . 2) A s e s q u i t e r p e n o i d framework plus an a c e t a t e f u n c t i o n a l i t y . 3) Fragment NCH-CH_-OAc 4) Fragment - (Q)-CH-CH 2 -CH2 iC-) ( w i t h i n a r i n g ) . 5) Three methyl groups (attached to quaternary c a r b o n s ) . Furthermore, when a p p l y i n g b i o s y n t h e t i c reasoning (Scheme 10), the s p e c t r a l data d e s c r i b e d above agreed with the proposed s t r u c t u r e of a l b i c a n y l a c e t a t e (51). Confi r m a t i o n of the proposed s t r u c t u r e was obtained by the h y d r o l y s i s of a l b i c a n y l a c e t a t e (51) to a l b i c a n o l (52). The a l b i c a n o l obtained from t h i s r e a c t i o n was i d e n t i c a l (by *H NMR and IR) to the a l b i c a n o l i s o l a t e d by A n d e r s e n 6 0 from the l i v e r w o r t D i p l o p h y l l u m a l b i c a n s FIGURE 6 M S O F ALBICANYL ACETATE 57 50 59 Table II *H NMR data f o r a l b i c a n y l a c e t a t e and a l b i c a n o l (The c o u p l i n g constants f o r 51 were deduced from proton-proton d e c o u p l i n g experiments. The c o u p l i n g c o n s t a n t s f o r 52 were deduced by analogy with 51) H on C# A l b i c a n y l a c e t a t e (51) 2 - A l b i c a n o l (52) (27 0MHz, CDC1 3) (27 0MHz, CDC1 3) 3 H 15 0.76 (s) 0.72 (s) 3 H 13 0.81 (s) 0.81 (s) 3 H 14 0.88 (s) 0.88 (s) H 5a 1.13 (dd, 3,12) 1.12 (dd, 3,13) H 6a 1.33 (dddd, 13,12,12,4.5) 1.34 (dddd, 13,13,13,4) H 6e 1.73 (dddd, 13,4.5,3,3) 1.75 (dddd, 13,4.5,3,2) H 7a 2.01 (ddd, 13,12,4.5) 2.02 (ddd, 13,13,4) H 7e 2.41 (ddd, 13,4.5,3) 2.41 (ddd, 13,4,2) H 9a 2.01 (dd, 9,4) 2.02 (dd, 11,4) CU2OAc 2.01 (s) — H 11 4.18 (dd, 9,11) 3.76 (dd, 11,11) H 11' 4.34 (dd, 4,11) 3.84 (dd, 11,4) H 12 4.51 (d, 1) 4.65 (d, 1.5) H 12' 4.85 (d, 1) 4.93 (d, 1.5) The f i r s t number i n each of the second and t h i r d columns repr e s e n t s the chemical s h i f t (S) values in ppm, r e l a t i v e to TMS £=0, while the numbers i n the parentheses represent the c o u p l i n g c o n s t a n t s in Hz ( H e r t z ) . 60 IV.1.2. A l b i c a n o l ; A l b i c a n o l (52) was i s o l a t e d from a more p o l a r f r a c t i o n of the e x t r a c t . Comparison of t h i s compound by TLC , XW NMR and MS has been made with the product obtained from the h y d r o l y s i s of a l b i c a n y l a c e t a t e . (52) The HRMS of a l b i c a n o l shows a parent ion at m/e=222.1990 ( c a l c u l a t e d 222.1977) corresponding to C 1 5 H 2 6 0 . The base peak at m/e=137 r e s u l t s from the same type of fragmentation as i n the case of a l b i c a n y l a c e t a t e (Scheme 2). Other fragments which are marked on the spectrum ( F i g . 10) r e s u l t from a l o s s of CH 3 from m/e=207, from a rearrangement i n v o l v i n g the OH group (m/e=204 B) or from a l o s s of H 20 (m/e=204 A). A l o s s of CH 3 from fragment m/e=204 (A or B) i s i n d i c a t e d by the peak at m/e=189 (A or B). As i n the case of a l b i c a n y l a c e t a t e peaks at m/e=123, 109 and 95 are present i n the spectrum of 52. The IR spectrum of the molecule shows bands that correspond to the OH group at 3350 and 1030 cm"1 and to the v i n y l i d i n e group at 890 cm"1 (Figure 11). The 1H NMR r e s u l t s enabled the assignment of a l l the protons on the B r i n g . The chemical s h i f t s and c o u p l i n g c o n s t a n t s are l i s t e d i n Table II to f a c i l i t a t e comparison with a l b i c a n y l a c e t a t e . T h i s spectrum i s presented i n F i g u r e 12. 61 The v a l u e s of the chemical s h i f t s obtained by A n d e r s e n 6 0 f o r the l i v e r w o r t d e r i v e d a l b i c a n o l and by P e l l e t i e r 6 1 who i s o l a t e d a l b i c a n o l from a s y n t h e t i c scheme are l i s t e d i n Table I I I . Table III j_H NMR s h i f t s f o r a l b i c a n o l obtained from  D. a l b i c a n s (*), from C. luteomarginata (**) and  s y n t h e t i c a l l y (***) H on C# 1- (*) J.,2 2- (**) .2,3 3- (***) .1,3 60MHz,CDC13 270MHz.CDC^ 60MHz,CDC13 3 H 15 0.75 .03 0.72 .03 0.75 .00 3 H 13 0.84 .03 0.81 .04 0.85 .01 3 H 14 0.89 .01 0.88 .02 0.90 .01 H 9a 3.79 .02 3.81 .08 3.89 .10 H 12 4.64 .01 4.63 .21 4.84 .20 H 12' 4.92 .01 4.93 .23 5.16 .24 We next turned our a t t e n t i o n to the absolute c o n f i g u r a t i o n of a l b i c a n o l and a l b i c a n y l a c e t a t e i s o l a t e d from C. luteomarginata . The r e l a t i v e s t e r e o c h e m i s t r y at the r i n g j u n c t i o n and at C-9 was determined by A n d e r s e n 6 0 u t i l i z i n g l a n t h a n i d e s h i f t reagents. *H NMR spectroscopy i n d i c a t e d that the a l b i c a n o l i s o l a t e d from C^ luteomarginata possessed the same r e l a t i v e c o n f i g u r a t i o n as Andersen's molecule. Andersen et a l 6 0 f a i l e d to re p o r t an o p t i c a l r o t a t i o n f o r a l b i c a n o l but d i d 62 determine i t s absolute c o n f i g u r a t i o n by r e d u c t i o n t o, and c o r r e l a t i o n with, (+) drimanol. We reduced our sample of a l b i c a n o l f o l l o w i n g Andersen's procedure to generate drimanol. The major product of the hydrogenation r e a c t i o n d i s p l a y e d an M D = + 1 5 ° (c 0.4, CHC1 3 , M.P.=100'C). The p o s i t i v e value of the s p e c i f i c r o t a t i o n i n d i c a t e s that we have the same enantiomer as drimanol (M.P. = 109-111° C, W =+15) obtained from the re d u c t i o n of a l b i c a n o l i s o l a t e d from the l i v e r w o r t . The ab s o l u t e c o n f i g u r a t i o n i s thus (5S),(9R),(10R). 123 o a. 0.0 Z0 .0 40.0 - r -60. ~~T 80 95 109 T I00.0 2 2 2 120.0 M/E r J40.0 109 i r 160.0 2 0 4 1207 leo.o T " " I ZOO.O - r — r 2Z0 .0 I 1 240.0 FIGURE 10 MS OF ALBICANOL 65 67 IV.1.3. Drimanol: (58) The parent peak at m/e=224.2142 ( c a l c u l a t e d 224.2133) i n the HRMS of drimanol (58) corresponds to a molecular formula of C 1 5 H 2 g O , i n d i c a t i n g an a d d i t i o n of two hydrogen atoms to a l b i c a n o l ( F i g . 13). The base peak at m/e=123 ( C g H 1 5 ) may r e s u l t from a two step fragmentation (Scheme 3). Drimanol f i r s t rearranges with l o s s of water, g i v i n g the weak peak at m/e=206 ( C 1 5 H 2 6 ) , then an a l l y l i c cleavage of the C(5)-C(6) bond generates the base peak at m/e=123. Daughter ions corresponding to a l o s s of CH 3 from the parent peak (209) and a l o s s of CH 3 + H 20 (191) are a l s o observed. A rearrangement of the e x o c y c l i c double bond of fragment m/e=123, fo l l o w e d by a r e t r o D i e l s - A l d e r would e x p l a i n the peak at m/e=109. The IR spectrum of the molecule shows bands at 3610, 3470 and 1030 cm"1 ( F i g . 14) i n d i c a t i n g the presence of an a l c o h o l . As a r e s u l t of the XH NMR spectrum and one decoup l i n g experiment ( F i g . 15) we were able to a s s i g n the chemical s h i f t s as l i s t e d i n Table IV. The proton-proton decoupling experiment was performed by i r r a d i a t i n g the doublet at 0.96 ppm (C-12 methyl), which c o l l a p s e d the m u l t i p l e t at 2.15 ppm i n t o a ddd with c o u p l i n g constants of 5, 5 and 2 Hz. T h i s would mean that the s i g n a l at 2.15 ppm corresponds to proton 8e. By e l i m i n a t i n g 68 the c o u p l i n g of H-8 with the three methyl protons of C-12, two "ae" c o u p l i n g s and an "ee" c o u p l i n g were d e t e c t e d . Table IV NMR data f o r drimanol H on C# Chemical S h i f t s (ppm) 270MHz C D C l 3 3 H 13 0.82 (s) 3 H 14 I 0.85 (s) 3 H 15 J 0.85 (s) 3 H 12 0.96 (d, 7.5) H 8e 2.15 (m, 7.5,7.5,7.5,5,5,2 deduced) H 11 3.59 (dd, 10.5, 10.5) H 11 3.86 (dd, 10.5, 4.5) T h i s r e s u l t i n d i c a t e s that the r e d u c t i o n of the double bond at C-8 took p l a c e from the l e s s h i n d e r e d <* s i d e of the molecule g i v i n g a major product with the CH 3 group in an a x i a l p o s i t i o n . The XH NMR spectrum shows that a minor amount of the molecule with a methyl in the e q u a t o r i a l p o s i t i o n was probably a l s o formed, i n a r a t i o of approximately 1:10. To the best of our knowledge t h i s i s the f i r s t r e p o r t of 1U NMR data for aVimano]. S cheme 3 F r a g m e n t a t i o n p a t t e r n o f d r i m a n o l FIGURE 13 MS OF DRIMANOL 71 73 IV.1.4.a. F u r o d y s i n i n and Furodysin ± The l e a s t p o l a r c o n s t i t u e n t s of the organic e x t r a c t of C a d i i n a luteomarginata have been i d e n t i f i e d as f u r o d y s i n i n (54), f u r o d y s i n (53) and m i c r o c i o n i n - 2 (55). Furod y s i n (53) and f u r o d y s i n i n (54) along with other r e l a t e d m e t a b o l i t e s have been p r e v i o u s l y i s o l a t e d from a Dysidea s p e c i e s of sponge c o l l e c t e d i n A u s t r a l i a 6 2 . • ( 5 3 ) ( 5 4 ) The HRMS of f u r o d y s i n (MS shown i n F i g . 16) or of the mixture ( F i g . 19) give s a parent ion at m/e=216.1512 ( c a l c u l a t e d 216.1509) corresponding to a molecular formula of C 1 5 H 2 0 0 . The base peak present at m/e = 122 (CgH l 00) i s due to an extremely f a c i l e Retro D i e l s - A l d e r rearrangement (Scheme 4 ). The remainder of the fragment ions have much weaker i n t e n s i t i e s (<10%). The only other peak which c o u l d be r a t i o n a l i z e d i s at m/e=201. T h i s a p p e a r e n t l y r e s u l t s from a l o s s of a methyl group from the parent molecule. The XH NMR spectrum of f u r o d y s i n i n ( F i g . 18A) i n d i c a t e s the presence of two protons on a furan r i n g (6.25, 7.23 ppm), an o l e f i n i c proton (5.63 ppm), a v i n y l i c methyl (1.66 ppm) and two s l i g h t l y d e s h i e l d e d methyls (1.19 ppm) bonded to a quaternary carbon. Comparison of t h i s s p e c t r a l i n f o r m a t i o n to that of known molecules of marine o r i g i n f a c i l i t a t e d assignment of the 74 s t r u c t u r e . Table V compares the chemical s h i f t s of C. luteomarginata f u r o d y s i n i n (54) a c q u i r e d i n two d i f f e r e n t s o l v e n t s and that a c q u i r e d by Wells et a l 6 2 f o r f u r o d y s i n i n (54) e x t r a c t e d from Dysidea . Table V 1H NMR s h i f t s of f u r o d y s i n i n obtained from  C. luteomarginata (*) and from Dysidea sp. (*•*) 1- (*) 2- (*) 3- (**) & 2,3 ( 80MHz,CDCy (270MHz ,CC1) 4 (270MHz,CCD 4 1.16 1.17,1. 18 1.15 .02 1.66 1.64 1.60 .04 2.25 2.24 2.19 .05 2.73 2.69 2.64 .05 5.63 5.57 5.50 .07 6.25 6.08 6.01 .07 7.23 7.08 6.98 .10 From d i r e c t v i s u a l comparison of the *H. NMR s p e c t r a (nudibranch versus sponge), (Fig.. 18A versus appendix 4), one can see that the C_^  luteomarginata m e t a b o l i t e i s i d e n t i c a l to the molecule obtained by Wells et a l . ' 2 Owing to the s m a l l amount of sample, an IR spectrum of C. luteomarginata d e r i v e d f u r o d y s i n i n (54) was obtained i n s o l u t i o n ( F i g . 17) and not as a KBr p e l l e t (Appendix 5). T h i s weak i n t e n s i t y spectrum does show c e r t a i n peaks c h a r a c t e r i s t i c of the f u n c t i o n a l i t y i n the molecule (Table V I ) . U n f o r t u n a t e l y 75 the s p e c t r a were obtained i n d i f f e r e n t phases and t h e r e f o r e can not be e x a c t l y compared. Table VI Comparison of the IR s p e c t r a of f u r o d y s i n ,  f u r o d y s i n i n , and of the f u r a n o s e s q u i t e r p e n o i d mixture f u r o d y s i n i n f u r o d y s i n i n f u r o d y s i n mixture KBr p e l l e t CC1 4 s o l u t i o n KBr p e l l e t CCl^ s o l u t i o n 3000-2850 3000-2850 3000-2850 3000-2850 1500 1515 1500 1515 1460-1440 1460 1480-1460-1440 1460 1380 1390 1390 1360 1370 1370 1360 1298 1310 1310 1300 1280 1290 1290 1195 1210 1210-1180-1170 1140 1150 1150 1150 1125 1140 1140 1065 1080 1080 1052 1070 1070 1030 900-892 915 915-905-895 888 840 860 860 840 76 In the case of the mixture of isomers, a v i s u a l i n s p e c t i o n of the 1H NMR spectrum of the mixture ( F i g . 21B) and of the f u r o d y s i n i n .(54) lH NMR spectrum ( F i g . 18B), enables the i d e n t i f i c a t i o n of " e x t r a " s i g n a l s . When comparing these " e x t r a " s i g n a l s to the spectrum of f u r o d y s i n (53) obtained from Wells ( s p e c t r a : Appendix 6, chemical s h i f t s : Table VII) a very good c o r r e l a t i o n i s observed. T h i s comparison plus the f a c t that the MS g i v e s a molecular formula of C 1 5 H 2 Q 0 and that f u r o d y s i n i n (54) and t h i s molecule have i d e n t i c a l Rf values on TLC i n d i c a t e s that f u r o d y s i n (53) e x i s t s i n the mixture. Table VII 1H NMR s h i f t s of f u r o d y s i n obtained from  C. luteomarginata (*) f u r a n o s e s q u i t e r p e n o i d  mixture and from a Dysidea s p e c i e s (**) 1- (*) 2- (*) 3- (**) A 2,3 (80MHz ,CDCy (270MHz ,CCD 4 (60MHz, CCD 1.26 1.23 1.23 0.00 1.70 1.66 1.61 .05 2.42 2.48 .05 5.63 5.55 5.50 .05 6.09 5.95 5.90 .05 7.14 6.98 7.03 .05 77 I t i s a l s o important to note that our mixture of f u r a n o s e s q u i t e r p e n o i d s appears to c o n t a i n a t h i r d molecule. T h i s i s i n d i c a t e d by the presence of weak s i g n a l s i n the lH NMR spectrum ( F i g u r e 21B) a t : 0.90 (3H, d, J=7Hz), 1.05 (6H, s ) , 3.42 (IH, m) ppm, and i n the downfield region around 5.91 ppm. T h i s l a t t e r s i g n a l p o i n t s to the presence of a proton on a furan r i n g . T h i s incomplete i n f o r m a t i o n , p l u s the f a c t that no s i g n a l s of higher mass can be dete c t e d i n the mass spectrum, ( F i g . 19) tends to suggest that we are d e a l i n g with a t h i r d isomer. U n f o r t u n a t e l y , not enough i n f o r m a t i o n i s a v a i l a b l e to allow us to propose a s t r u c t u r e . The IR spectrum of t h i s mixture, a l s o taken i n a CC14 s o l u t i o n i s shown i n F i g u r e 20. Agreement with the s p e c t r a of f u r o d y s i n (53) and f u r o d y s i n i n (54) obtained as KBr p e l l e t s (Appendix 7) i s only p a r t i a l (Table V I ) . MS OF FURODYSININ 79 Scheme 4 Fragmentation pattern of fu rodys in in and furodysin 201 20! 84 CO cn FIGURE 21B 270 MHz 1H NMR SPECTRUM OF A MIXTURE OF FURANOSESQUITERPENOIDS co cn 87 IV.1.4.b. M i c r o c i o n i n - 2 As mentioned in Chapter 3, the l a s t c o l l e c t i o n of C. luteomarginata i n Barkley Sound r e v e a l e d the presence of a d i f f e r e n t s e s q u i t e r p e n o i d . M i c r o c i o n i n - 2 (55) , a known me t a b o l i t e of the sponge M i c r o c i o n a t o x y s t i l i a , which has been p r e v i o u s l y r e p o r t e d by Minale and a l . 6 3 , was obtained from f r a c t i o n (1) of the nudibranch e x t r a c t . The molecular ion i n the HRMS (MS i s shown in Fig.22) of m i c r o c i o n i n - 2 (55) appears at m/e=218.1612 ( c a l c u l a t e d 218.1665) and corresponds to C 1 5 H 1 g O . Other fragments which are i d e n t i f i e d in Scheme 5 and on the p l o t t e d spectrum ( F i g . 22), correspond t o : a l o s s of methyl i n d i c a t e d by the peak at m/e=203; an a l l y l i c cleavage of C7-C8, which g i v e s fragments at m/e=137 and 81; and another a l l y l i c cleavage of C5-C7, which g i v e s fragments at m/e=123 and 95. The IR spectrum of the molecule shows bands at 1460 and 1380 cm - 1 i n d i c a t i n g the presence of m e t h y l g r o u p s >, and bands at 880 and 730 cm"1 i n d i c a t i n g a s u b s t i t u t e d furan r i n g ( F i g . 23). The 1U NMR spectrum of t h i s s e s q u i t e r p e n o i d i n d i c a t e s the presence of three protons c h a r a c t e r i s t i c of a fl-substituted furan r i n g a t : 7.21 (IH, d, J=lHz), 7.08 (lH,bs) and 6.13 (IH, 88 bs) ppm ( F i g . 2 4 ) . A s i n g l e o l e f i n i c f u n c t i o n a l i t y i s i n d i c a t e d by a broad s i n g l e t at 5.36 and a v i n y l i c methyl at 1.62 ppm. There i s a l s o a methyl s i n g l e t at 1.08 and a methyl doublet at 0.99 (d, 7Hz) ppm. The " e x t r a " s i g n a l s at 0.88 ppm may be caused by the presence of some f a t . T h i s i n f o r m a t i o n suggested s e v e r a l chemical s t r u c t u r e s and a l i t e r a t u r e survey showed that the data a v a i l a b l e 6 3 f o r m i c r o c i o n i n - 2 (55) c o r r o l a t e d w e l l with our molecule. The 1U NMR data of m i c r o c i o n i n - 2 obtained from luteomarginata and from M. T o x y s t i l l a are l i s t e d i n Table V I I I . Table VIII *H NMR data f o r m i c r o c i o n i n - 2 obtained from  C.luteomarginata (*) and from M. t o x y s t i l l a (**) 1- (**) 2- (*) a 1,2 ( c c i 4 ) (27 0MHz ,CCl 4 ) 0.99 0.98 .01 1.08 1.07 .01 1.64 1.62 .02 5.40 5.36 .04 6.14 6.13 .01 7.12 7 .08 .04 7.24 7.21 .03 93 IV.1.5.a Luteone: A f t e r the crude PTLC s e p a r a t i o n , f r a c t i o n number three r e t a i n e d the sweet fragrance of the nudibranchs. A n a l y t i c a l TLC i n d i c a t e d t hat two major molecules e x i s t e d i n t h i s f r a c t i o n . The l e a s t p o l a r c o n s t i t u e n t of the f r a c t i o n was d i f f i c u l t to o b t a i n in a pure form due to the d i f f i c u l t i e s o u t l i n e d i n Chapter 3 ( v o l a t i l i t y , small amount, decomposition). T h i s l e a s t p o l a r o d o r i f e r o u s c o n s t i t u e n t was named luteone. In order to decrease the v o l a t i l i t y of luteone, i n c r e a s e the amount of sample, and o b t a i n a c r y s t a l l i n e d e r i v a t i v e the crude f r a c t i o n three was react e d with 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e (DNPH). We were able to i s o l a t e luteone as a c r y s t a l l i n e DNPH d e r i v a t i v e from t h i s r e a c t i o n mixture. The HRMS of the luteone DNPH d e r i v a t i v e (MS i s shown i n F i g . 25) d i s p l a y e d a molecular ion at m/e=524.3000 ( c a l c u l a t e d 524.2988) which corresponded to C 2 9 H 4 Q N 4 0 5 suggesting a parent mass of m/e=344' f o r luteone. Examination of a HRMS of crude f r a c t i o n three showed a' peak at m/e=344.2735 ( c a l c u l a t e d 344.2706) corresponding to C, H., 0 . R 3 23 36 2 The fragmentation p a t t e r n observed i n t h i s spectrum appeared to i n d i c a t e a l o s s of H 20 (326), a l o s s of CH 3 + H 20 (299), and a l o s s of CH 3CO + H 20 + CH 3 (268). The ion at m/e=191 may r e s u l t from the formation of the fragment 59 ( C M H 5 , ) . 94 (59) The c h a r a c t e r i s t i c fragment at m/e=137 seen i n the MS of a l b i c a n y l a c e t a t e and a l b i c a n o l i s a l s o present i n t h i s spectrum, but i s of lower i n t e n s i t y . Fragment ions m/e=123, 109 and 95 a l s o appear. The fragmentation p a t t e r n i s thus very s i m i l a r to that d i s p l a y e d by a l b i c a n y l a c e t a t e and a l b i c a n o l and i t supports the p a r t i a l s t r u c t u r e 59 d e p i c t e d f o r the ion of m/e=191. The XH NMR spectrum ( F i g . 26) a l s o p o i n t e d to the presence of 59 i n luteone. Three methyl s i n g l e t s appeared at 0.60, 0.78 and 0.95 ppm and two one proton doublets (J=lHz) appeared at 4.46 and 4.86 ppm i n d i c a t i n g that the e x o c y c l i c double bond was a l s o p r e s e n t . The presence of a methyl ketone was i n d i c a t e d by the occurrence of a s i n g l e t at 2.11 ppm, i n t e g r a t i n g f o r three p rotons. A l l of the above mentioned s i g n a l s were more c l e a r l y d e f i n e d i n the XH NMR spectrum of the luteone DNPH d e r i v a t i v e ( F i g . 3 0 ) . The data a v a i l a b l e account f o r only part of the s t r u c t u r e of the f r a g r a n t compound. I t s molecular formula ( C 2 3 H 3 e 0 2 ) corresponds t o s i x s i t e s of u n s a t u r a t i o n , four of which are known: r i n g s A and B, an e x o c y c l i c double bond, and a c a r b o n y l . However, more d e t a i l e d i n f o r m a t i o n c o u l d not be a b s t r a c t e d from 95 the 1H NMR of the d e r i v a t i v e , except that two isomers were present i n an approximate r a t i o of 4.5:1. The major isomer has the methyl and n i t r o a n i l i n o group present i n a syn c o n f i g u r a t i o n . T h i s assignment i s made on the b a s i s of the lH NMR ( F i g . 30) of the d e r i v a t i v e . The hydrazone NH s i g n a l f o r the major isomer can be seen at 10.12 ppm ( s ) , while a weaker s i n g l e t appears at 10.08 ppm ( 20% as i n t e n s e ) . The methyl ketone s i n g l e t of the syn isomer appears at 2.07 ppm ,while the corresponding s i g n a l f o r the a n t i isomer appears at 2.15 ppm, and i t again shows 20% of the i n t e g r a t e d i n t e n s i t y . Other s i g n a l s a l s o seem to be doubled i n the 1H NMR s p e c t r a . S i g n a l s corresponding to H-6, H-5 and H-3 on the benzene r i n g appear at 7.96 (d, J=10 Hz), 8.32 (dd,J=2.5, 10 Hz) and 9.15 (d, J=2.5 Hz) ppm, r e s p e c t i v e l y . Other i d e n t i f i a b l e f e a t u r e s a r e : the protons on the e x o c y c l i c double bond at 4.89 (s) and 4.56 (s) ppm and the three methyl s i n g l e t s at 0.64, 0.76 and 0.78 ppm. a n t i - - _ s y n DNPH d e r i v a t i v e s are expected to e x i s t i n the thermodynamically more s t a b l e a n t i - c o n f i g u r a t i o n 6 * " 6 7 . A s e r i e s of *H NMR s t u d i e s on DNPH d e r i v a t i v e s by Karabatsos et a l 6 4 - 6 7 showed t h a t : 96 1) ^-hydrogens syn to the a n i s o t r o p i c group are s h i e l d e d . 2) /3and ^-hydrogens syn to the a n i s o t r o p i c group are d e s h i e l d e d . If we assume that the major isomer of the luteone DNPH d e r i v a t i v e has the thermodynamically most s t a b l e c o n f i g u r a t i o n , then our r e s u l t s agree with these o b s e r v a t i o n s . The s i n g l e t c orresponding to the methyl ketone of the major isomer, which i s expected to be syn to the d i n i t r o a n i l i n o group , i s at higher f i e l d than the corresponding s i g n a l i n the a n t i - i s o m e r . The IR spectrum of t h i s d e r i v a t i v e i s d i s p l a y e d i n F i g . 29. The s t r o n g e s t bands appearing at 1540 and 1340 cm"1 are due to the NH bond and to the n i t r o f u n c t i o n s , r e s p e c t i v e l y . A weaker band at 1700 cm - 1 i s a p p r o p r i a t e f o r the imine (C=N). As s t a t e d p r e v i o u s l y , luteone c o u l d be i s o l a t e d i n only very small q u a n t i t i e s . T h i s c o n d i t i o n made i t impossible to e l u c i d a t e the s t r u c t u r e of the compound through chemical degr a d a t i o n s . To circumvent t h i s problem, a c r y s t a l l i n e d e r i v a t i v e (DNPH) has been submitted f o r X-ray d i f f r a c t i o n 11 7 fl 3 47 11 I I . I I. 147 I I " • r — 1 1 I "1 I — T — T — I 1" " "! 0.0 ZOO 40.0 60.0 00.0 100.0 120.0 140.0 M/E FIGURE 25A 229 i — r 160.0 ~r r ieo.o ~i —r 200.0 I "T 2Z0.0 —1 r Z40.0 • III.. I 1 1 260.0 280.0 MS OF LUTEONE (PROBE WITHOUT HEATING) ID -Nl FIGURE 25B MS OF LUTEONE (probe heated at 200 C) 100 FIGURE 28 MS OF LUTEONE'S DNPH DERIVATIVE 10 2 o 104 IV.1.5.D Phth a l a t e e s t e r : The presence of p h t h a l a t e e s t e r s (see Chapter 3) made i t d i f f i c u l t t o o b t a i n s p e c t r a l data on luteone. The p h t h a l a t e e s t e r (60) was i d e n t i f i e d as the major c o n s t i t u e n t of the i m p u r i t i e s a s s o c i a t e d with the f r a g r a n t molecule. O O (60) I t s s t r u c t u r e was deduced from rH NMR ( F i g . 27B) and MS ( F i g . 25B) data which were i d e n t i c a l with the known s p e c t r a of t h i s molecule 6 8 6 '. 105 IV.2. From Acanthadoris nanaimoensis:  IV.2.1. Nanaimoal: The major c o n s t i t u e n t of the e x t r a c t of Acanthadoris  nanaimoensis has a sweet f r a g r a n c e . Although the p u r i f i c a t i o n of t h i s compound was s t r a i g h t f o r w a r d , as i n d i c a t e d i n the previous chapter, the s t r u c t u r a l e l u c i d a t i o n has presented a great many d i f f i c u l t i e s . The lH NMR s p e c t r a ( F i g . 33) of the i s o l a t e d compound i n d i c a t e d the presence of two c l o s e l y r e l a t e d s e s q u i t e r p e n o i d aldehydes. The al d e h y d i c proton of the major component appears as a t r i p l e t (J=3Hz) at 9.83 ppm while the corresponding s i g n a l f o r the minor component appears at 9.71 ppm ( t , J=3Hz). T h i s second s i g n a l has about 20% of the i n t e n s i t y of the major s i g n a l . The c o u p l i n g p a t t e r n of the ald e h y d i c s i g n a l i n d i c a t e s the presence of a methylene group next to the c a r b o n y l . T h i s methylene appears as a dd at 2.25 ppm (J= 3, 14 Hz) i n d i c a t i v e of the presence of a quaternary carbon next to the methylene. Two methyl s i n g l e t s appear at 0.98 ppm, while a t h i r d one i s found at 1.05 ppm. S i g n a l s i n the  1H NMR spectrum at 2.70, 2.93, 3.77, 5.22 and 5.37 ppm are a t t r i b u t a b l e to i m p u r i t i e s , s i n c e they are not present i n s p e c t r a of l a t e r d e r i v a t i v e s . The MS ( F i g . 31) of t h i s mixture of compounds, i t s hydrogenation product ( F i g . 37) and DNPH d e r i v a t i v e ( F i g . 34) i n d i c a t e a molecular formula f o r nanaimoal of C 1 5 H 2 Q 0 , (parent peak i n the HRMS at 220.1836, c a l c u l a t e d 220.1821). The MS of nanaimoal a l s o i n d i c a t e s that t h i s molecule i s e a s i l y o x i d i z e d to the corresponding a c i d which i s observed at m/e=236. The fragmentation p a t t e r n of nanaimoal shows ions due to the l o s s of 106 CHO (m/e=191), a l o s s of CH 3 (m/e = 205) and l o s s e s of CpHjO and C ?H 40 (m/e=177 and 176). Loss of a methyl group from the fragment at m/e=176 generates the base peak at m/e=161. The molecular formula i n d i c a t e s the presence of four s i t e s of u n s a t u r a t i o n i n the molecule. One s i t e of u n s a t u r a t i o n i s accounted f o r by the aldehyde. A t e t r a s u b s t i t u t e d double bond i s apparent from the presence of a m u l t i p l e t at 2.00 ppm i n the  1H NMR and the absence of o l e f i n i c protons. The 1 3 C NMR of nanaimool ( F i g . 39C") shows two s i g n a l s at 133.4 and 125.5 ppm co n f i r m i n g the e x i s t e n c e of the o l e f i n i c f u n c t i o n a l i t y . The two remaining s i t e s of u n s a t u r a t i o n must t h e r e f o r e be assign e d to two r i n g s . From the s p e c t r a l data a v a i l a b l e , s e v e r a l f e a t u r e s of t h i s molecule can be d i s c e r n e d : 1) Four s i t e s of u n s a t u r a t i o n : a c a r b o n y l , a t e t r a s u b s t i t u t e d double bond and two r i n g s . 2) A s e s q u i t e r p e n o i d framework. 3) Fragment -C-CH2"CHO 4) Three methyl groups (attached to quaternary c a r b o n s ) . A p p l y i n g b i o s y n t h e t i c reasoning a l l o w s three h y p o t h e t i c a l s t r u c t u r e s , i , i i , and i i i to be drawn. ( i ) ( i i ) ( " i ) 107 There are only two small d i f f e r e n c e s between these three s t r u c t u r e s suggested f o r nanaimoal. F i r s t , these molecules have a d i f f e r e n t number of methylene groups next to the double bond. Second, they would give r i s e to d i f f e r e n t fragments i n the mass spectrum, i f a r e t r o D i e l s - A l d e r fragmentation o c c u r s . There are two methylene groups next to the double bond i n s t r u c t u r e i . In molecules i i and i i i three CH groups r e s i d e next to the double bond, one of which should be a doublet of d o u b l e t s . A c a r e f u l examination of the *H NMR s p e c t r a enables one to d i s c e r n a two proton m u l t i p l e t at 2.00 ppm and a s i g n a l which i s e i t h e r a doublet o v e r l a p p i n g a m u l t i p l e t , or two m u l t i p l e t s , i n t e g r a t i n g f o r 2 or 3 H, around 1.73 ppm. T h i s i s observed i n the s p e c t r a of nanaimoal ( F i g . 33), nanaimool ( F i g . 39) and some of t h e i r d e r i v a t i v e s ( F i g . 36 and 42). The XH NMR data are not s u f f i c i e n t l y c l e a r to enable the e l i m i n a t i o n of any of the proposed s t r u c t u r e s . Mass s p e c t r a l a n a l y s i s i s a l s o incapable of unambiguously d i f f e r e n t i a t i n g between these three molecules. For each of the h y p o t h e t i c a l s t r u c t u r e s i , i i , and i i i (Scheme 6) i t i s p o s s i b l e to imagine a l o s s of a methyl group fo l l o w e d by a Retro D i e l s -A l d e r t a k i n g p l a c e i n r i n g A or B. I f t h i s fragmentation p a t t e r n i s a p p l i e d to each s t r u c t u r e , i t would give r i s e (along with other fragments) t o : i 177 i i 177-121 i i i 177-149 I t i s p o s s i b l e to r a t i o n a l i z e the important fragment ion at m/e=121 from s t r u c t u r e i i o n l y . 108 The o r i g i n of MS fragmentation ions cannot always be e a s i l y e x p l a i n e d and the smaller (m/e) the fragments become the g r e a t e r i s the d i f f i c u l t y i n r a t i o n a l i z i n g t h e i r e x i s t e n c e . T h e r e f o r e , i t does not seem very p o s s i b l e to choose a s t r u c t u r e f o r nanaimoal on t h i s b a s i s . Since no c o n v i n c i n g s p e c t r a l argument c o u l d be found to prove the molecular s t r u c t u r e of nanaimoal, d e r i v a t i z a t i o n and subsequent X-ray d i f f r a c t i o n a n a l y s i s seemed l i k e the l o g i c a l path to f o l l o w . Scheme 6 Possible Retro Diels-Alder fragmentation pattern of the three hypothetical structures of nanaimoal 1 09 149 no o o L C D o ID o I I I I 1 I I — 0 001 0 08 0 09 O'Ot? m ' i N r i 3 a O U J 1 RE O CD o U . o < o < < o o 10 0 0? 0 0 FIGURE 32 IR SPECTRUM OF NANAIMOAL --o 112 \ 113 IV. 2.2 D e r i v a t i v e s : The f o l l o w i n g e i g h t d e r i v a t i v e s 7 0 have been prepared on small amounts of nanaimoal (see experimental f o r r e a c t i o n d e t a i l s ) . 1) A d i n i t r o p h e n y l h y d r a z o n e d e r i v a t i v e : T h i s r e a c t i o n gave one main product i n high y i e l d . I t a l s o r e p r e s e n t s the best " s o l i d " d e r i v a t i v e obtained. However, the formation of h i g h l y c r y s t a l l i n e m a t e r i a l c o u l d not be induced. 2) A bromophenylhydrazone d e r i v a t i v e : The product of t h i s r e a c t i o n was not as s t a b l e as the DNPH d e r i v a t i v e . H y d r o l y s i s seemed to take p l a c e when the compound was l e f t f o r long p e r i o d s in MeOH. AlsOj an i n s o l u b l e s o l i d was formed on st a n d i n g . 3) Nanaimool: The a l c o h o l d i d not c r y s t a l l i z e when any of the f o l l o w i n g s o l v e n t s were used e i t h e r alone or i n combination: MeOH, CH^CN, CHC1 , hexane or CC1,. 3 3 4 4) and 5) A para-bromobenzooate d e r i v a t i v e and a meta- bromobenzoate d e r i v a t i v e of nanaimool: The products obtained from these two r e a c t i o n s h y d r o l y z e d r e a d i l y to the a l c o h o l . Part of the m a t e r i a l obtained decomposed to give an i n s o l u b l e s o l i d . 6) A p a r a - c h l o r o p h e n y l i s o c y a n a t e d e r i v a t i v e of nanaimool: The d e r i v a t i v e obtained i n t h i s case was a s t a b l e s o l i d , but d i d not give any h i g h l y c r y s t a l l i n e m a t e r i a l . 7) A p r o p a n e d i t h i o l d e r i v a t i v e : T h i s r e a c t i o n d i d not proceed without h e a t i n g . Upon r e f l u x i n g , nanaimoal was l o s t from the r e a c t i o n mixture owing to i t s extreme v o l a t i l i t y . 8) An o z o n o l y s i s react i o n : More than two products were obtained i n t h i s case. The MS of the two major r e a c t i o n products appeared to i n d i c a t e a rearrangement. 114 Constant f a i l u r e to o b t a i n c r y s t a l l i n e d e r i v a t i v e s has at l e a s t two main causes. F i r s t , because of the small amount of sample used ( t y p i c a l l y 4 mg of aldehyde or a l c o h o l =2x10" 5 moles), t r a c e amounts of water (1 m i c r o l i t e r 5x10" 5 moles) and a c i d ( o r i g i n a t i n g from the s i l i c a p l a t e s or s o l v e n t s ) c r e a t e major problems of decomposition. Second, the minor component, as i t can be seen i n the *H NMR s p e c t r a of the d e r i v a t i v e s , c o u l d not always be separated from the major component. Mixtures of compounds can not be expected to produce h i g h l y c r y s t a l l i n e s o l i d s . o a. a. coto a a CM a o 0.0 T 176 2 80 40.0 60.0 320.0 1 r"—i 1 r — i 1 1 ]60.0 200.0 24).0 280.0 320.0 M / E 400 1 1 1 1 1 360.0 400.0 440.0 FIGURE 34 MS OF NANAINOAL'S DNPH DERIVATIVE LP 207 o a . CO a . -a 1 7 7 a a . 0.0 20.0 10.0 60.0 80 .0 i r 100.0 16 } ~ V 120 .0 M/E 140.0 "T T T—r 160.0 IK 9 179 T 7 160.0 222 ^ r 200.0 JL I 220.0 ~ l 1 210.0 FIGURE 37 MS OF NANAIMOOL 113 122 FIGURE 39C 2 0,1 MHZ ^ 3C NMR SPECTRUM OF NANAIMOOL In O CM I 1 1 1 1 1 1 r 0 001 0 08 0 09 0 0t? {/)'INI'13h CO . o V o ro o co o o . CD .9 CO o oUJ O o CO o o 003 C O o w o > > M w o w H u o co M z w se fa o fa o s u p-l hJ o o 2 fa o 00 s 126 127 "I 1 1 1 1 1 —i 1 r 8 7 b 5 + 3 2 1 FIGURE 45 0 MHZ XH NMR SPECTRUM OF NANAIMOOL PARA BROMOBENZOATE DERIVATIVE oo a a - a . "co . a tr. e 3 a I r o.o —i r 20.0 100.0 n — r 140.0 n— 160.0 2 3 6 r — r ieo.0 - I r 200.0 1 — r — r 220.0 ~i 1 240.0 40.0 60.0 eo. 120.0 M/F. FIGURE 46 MS OF MORE POLAR PRODUCT OBTAINED FROM OZONOLYSIS REACTION 1 7 » a . IS —to cn i — i — r — i — i — i — " i — " i i i 0.0 ZO.O 10.0 60.0 eo.o 100.0 T — r T 2 3 6 21 8 2 21 II .I . U I20.0 )<W.O M/E J60.0 1B0.0 T r 200.0 r r 2Z0.0 -I 1 Z40.0 FIGURE 47 MS OF LESS POLAR PRODUCT OBTAINED FROM OZONOLYSIS REACTION 8 w 00 ir* o r 0 001 008 0 09 m INI ~l "Of 13d o o o ID ro o [NJ m o co 9 CM obJ C N J ^ : o ID O rsj o CO o o 003 0"0 00 <f W Pi > M H < > M P i W a w z o O J H EC Z w P S p-l i o Pi PQ cu <: o s fa o 132 134 IV.2.3. Minor Component: I t i s important to b r i e f l y d i s c u s s the nature of the minor component present i n nanaimoal samples. I t i s apparent from the JH NMR of nanaimoal ( F i g . 33), nanaimool ( F i g . 39) and some of the d e r i v a t i v e s ( F i g . 36 and 42), that i t i s the s i g n a l c orresponding to the protons on the carbon bearing the oxygen f u n c t i o n a l i t y which are most f r e q u e n t l y doubled. Since, the MS does not show any " e x t r a " peaks, the isomerism c o u l d c o n c e i v a b l y be of a st e r e o c h e m i c a l nature (conformational isomerism). However, when the *H NMR spectrum ( F i g . 39) of nanaimool was obtained i n acetone at reduced temperatures of 0s, -20° and -40° C, a new s i g n a l developed on the downfield s i d e (^3.7 ppm) of the c a r b i n o l protons (^3.6 ppm) at -40°C. The appearance of t h i s s i g n a l tends to i n d i c a t e the " f r e e z i n g out" of two con f o r m a t i o n a l isomers of nanaimool. Since there was no change in the s i g n a l at A J 3 . 5 ppm over the temperature range of a c q u i r e d s p e c t r a , i t would appear that the isomer i n d i c a t e d by the u p f i e l d s i g n a l i s c o n s t i t u t i o n a l or c o n f i g u r a t i o n a l . The proposed s t r u c t u r e s f o r nanaimoal have only one c h i r a l c e n t r e and thus the only c o n f i g u r a t i o n a l isomer p o s s i b l e would be an enantiomer which would have i d e n t i c a l *H NMR s p e c t r a . Thus the minor component appears to be a c o n s t i t u t i o n a l isomer. When the meta-bromobenzoate d e r i v a t i v e of nanaimool was p u r i f i e d by performing e i g h t r e p e t i t i v e developments of the PTLC ( i n 10 % CHC1 3/C 6H 1 4 ) , the 1H NMR (Fig.^44) showed the presence of only one set of protons around 4.4 ppm suppo r t i n g the e x i s t e n c e of a c o n s t i t u t i o n a l isomer. C l e a r l y t h i s sequence of op e r a t i o n s would not allow the s e p a r a t i o n of conformers or 135 enantiomers. It should be mentioned that the s e p a r a t i o n of the two isomers was t r i e d with a l l of the d e r i v a t i v e s , i n an attempt to in c r e a s e our chances of c r y s t a l l i z i n g the compounds. The methods t r i e d were: 1) Reversed-phase p l a t e s (with d e r i v a t i v e s 2-4-5). 2) HPLC using a reversed-phase column (with d e r i v a t i v e s 4-6). 3) Repetetive developments of a s i l i c a p l a t e i n a non po l a r s o l v e n t (with d e r i v a t i v e s 2-4-5-6). 4) C r y s t a l l i z a t i o n s i n a l a r g e number of s o l v e n t s (with a l l the d e r i v a t i v e s ) . 5) GC (with 3). These d i d not always succeed i n s e p a r a t i n g the d e r i v a t i v e s of the two isomers, or when they d i d , no h i g h l y c r y s t a l l i n e substances were obtained. In the case of the para-bromobenzoate and para-bromophenylhydrazone d e r i v a t i v e s , the p u r i f i c a t i o n methods produced a r e l a t i v e l y higher amount of the p r e v i o u s l y minor isomer. 136 Chapter 5 D i s c u s s i o n  V.1. C a d i i n a luteomarginata : V. 1.1. B i o l o g i c a l Aspects : A s e r i e s of s i x compounds has been e x t r a c t e d from the d o r i d nudibranch C a d i i n a luteomarginata. The m e t a b o l i t e s have been d i v i d e d i n t o two groups on the b a s i s of the frequency of t h e i r appearance. A- Compounds c o n s t a n t l y p r e s e n t : a l b i c a n y l a c e t a t e (51), a l b i c a n o l (52) and luteone (56). B- Compounds v a r y i n g with d i v i n g s i t e : f u r o d y s i n (53), f u r o d y s i n i n (54) and m i c r o c i o n i n - 2 (55). Molluscan m e t a b o l i t e s are o f t e n d i e t a r y i n o r i g i n . A s e r i e s of simple d i s s e c t i o n experiments were performed i n an attempt to prove a d i e t a r y o r i g i n f o r the C_^  luteomarginata s e s q u i t e r p e n o i d s . Six to twelve f r e s h l y c o l l e c t e d nudibranchs were d i s s e c t e d , and t h e i r mantle and stomach e x t r a c t s worked up s e p a r a t e l y . A n a l y t i c a l TLC i n d i c a t e d that the group A molecules were generaly present only i n the mantle. The group B molecules, i f present at a l l , were in the stomach. On one o c c a s i o n , both the group A and the group B molecules were found to e x i s t i n both e x t r a c t s . These r e s u l t s are summarized in Table IX. 137 Table IX R e s u l t s of d i s s e c t i o n s Date C o l l e c t i o n Mantle Stomach march 8 E l l i s I s . Group A may . 6 Nanat I s . Group A Group B June 9 E a r l ' s Cove Group A and B Group A and B June 27 Sunset B. Group A o c t . 4 E l l i s I s . Group A The r e s u l t s of the d i s s e c t i o n performed on June 9 showed that the group A and B molecules e x i s t e d i n a higher c o n c e n t r a t i o n i n the mantle than i n the stomach. T h i s r e s u l t e l i m i n a t e s the p o s s i b l e contamination of the mantle e x t r a c t by the group B molecules (from the stomach), but does not e l i m i n a t e the p o s s i b l e contamination of the stomach e x t r a c t by the group A molecules (from the mantle). The only safe statement that can be drawn from the above mentioned r e s u l t s , i s : the group B s e s q u i t e r p e n o i d s are probably of a d i e t a r y o r i g i n and the d i e t source v a r i e s with d i v i n g s i t e s . There can be two i n t e r p r e t a t i o n s of the r e s u l t s concerning the group A s e s q u i t e r p e n o i d s : 1) I f the group A molecules are d i e t a r y i n o r i g i n , t h i s d i e t a r y source i s present at a l l d i v e s i t e s . 138 F i n d i n g the group A molecules i n the mantle e x t r a c t more o f t e n than i n the stomach e x t r a c t , can be p a r t i a l l y e x p l a i n e d by the f a c t that the nudibranchs are known to go s e v e r a l days without e a t i n g . I f the molecules of group A are d i e t a r y i n o r i g i n the nudibranchs must t r a n s p o r t them to the mantle more s e l e c t i v e l y or e f f i c i e n t l y than the molecules of group B. 2) I f the group A molecules are always found only i n the mantle, they are p o s s i b l y of a molluscan b i o s y n t h e t i c o r i g i n . The d i s s e c t i o n o b s e r v a t i o n s r a i s e d two a d d i t i o n a l q u e s t i o n s : 1) What i s the d i e t a r y source of these te r p e n o i d s ? 2) Why do the nudibranchs r e t a i n c e r t a i n chemicals i n t h e i r mantle? To answer the f i r s t q u e s t i o n , the l i t e r a t u r e was searched f o r p o s s i b l e d i e t a r y sources and throughout our d i v e s , c a r e f u l o b s e r v a t i o n of the organisms a s s o c i a t e d with luteomarginata was one of our primary concerns. The b i o l o g i c a l l i t e r a t u r e 5 3 5 4 r e v e a l e d that the three i n t e r t i d a l sponges: H a l i c h o n d r i a panicea , M y x i l l a i n c r u s t a n s and a H i g g i n s i a sp., c o n s t i t u t e d the known d i e t of the nudibranch. Furthermore, a l i t e r a t u r e survey on the re p o r t e d chemistry of these sponges r e v e a l e d that a p o s s i b l e precursor of the group A molecules, namely methyl trans-monocyclofarnesate had been rep o r t e d from H a l i c h o n d r i a panicea 7 1 . A p o s s i b l e o r i g i n to the group A molecules c o u l d t h e r e f o r e be assigned to chemical t r a n s f o r m a t i o n s of 61 t a k i n g place i n the stomach or i n the mantle of the nudibranchs. 139 C O O M e (61) H a l i c h o n d r i a panicea was not observed i n t e r t i d a l l y while d i v i n g . T h e r e f o r e a sample was c o l l e c t e d at the docks of the Royal Vancouver Yacht Club, i n S t a n l e y Park. E x t r a c t i o n and p a r t i a l c h a r a c t e r i z a t i o n by *H NMR spectroscopy of the molecules obtained from t h i s s p e c i e s r e v e a l e d only the presence of f a t . The monocyclofarnesol d e r i v a t i v e 61 r e p o r t e d i n the l i t e r a t u r e 6 ' was not found. We have been unable to f i n d the other sponges r e p o r t e d to be p a r t of C^ luteomarginata's d i e t . F u r o d y s i n and f u r o d y s i n i n have been r e p o r t e d 6 2 to be m e t a b o l i t e s from a Dysidea s p e c i e s of sponge, while m i c r o c i o n i n -2 i s a m e t a b o l i t e 6 3 from the sponge M i c r o c i o n a t o x y s t i l l a . These two sponges have not p r e v i o u s l y been o b s e r v e d 5 3 5 4 to be p a r t of C. luteomarginata 's d i e t . I t i s a l s o i n t e r e s t i n g to note that these m e t a b o l i t e s 53, 54, and 5_5 were e x t r a c t e d from sponges c o l l e c t e d i n A u s t r a l i a and I t a l y r e s p e c t i v e l y . From our underwater o b s e r v a t i o n s , no sponge, bryozoans or t u n i c a t e s were found a s s o c i a t e d with C\ luteomarginata. T h i s leaves the d i e t a r y o r i g i n of a l b i c a n y l a c e t a t e , a l b i c a n o l and luteone unknown. Specimens of C_^_ Luteomarginata have been given to J . Thompson at S c r i p p s I n s t i t u t e of Oceanography for gut content a n a l y s i s , which might shed new l i g h t on the subject of C. luteomarginata's d i e t . 140 As f a r as the r o l e of the m e t a b o l i t e s i s concerned, i t i s p o s s i b l e that a l b i c a n y l a c e t a t e , a l b i c a n o l and luteone, one at a time or i n combination are r e t a i n e d f o r chemical communication. The reasons f o r t h i s a r e : 1) The compounds are r e t a i n e d i n the nudibranchs mantle. 2) The compounds e x i s t a l l year round and at a l l the d i v i n g s i t e s r e p o r t e d . 3) Luteone i s a f r a g r a n t molecule. Experiments aimed at determining the b i o s y n t h e t i c o r i g i n of the group A molecules and t h e i r p o s s i b l e r o l e i n chemical communication would be q u i t e c h a l l e n g i n g . 141 V.1.2 . Chemical and Biochemical Aspects j_ The s i x molecules obtained from the e x t r a c t of C a d l i n a  luteomarginata belong to the t e r p e n o i d f a m i l y . B i o s y n t h e t i c a l l y , s e s q u i t e r p e n o i d s are d e r i v e d from the r e p e t i t i v e condensation of three u n i t s of i s o p e n t e n y l pyrophosphate l e a d i n g , as o u t l i n e d i n Scheme 7, to the formation of f a r n e s y l pyrophosphate. C y c l i z a t i o n of f a r n e s y l pyrophosphate and secondary t r a n s f o r m a t i o n s generate the s e s q u i t e r p e n o i d s . No l a b e l i n g s t u d i e s i n v o l v i n g any of the s i x substances t r e a t e d i n t h i s s e c t i o n have been undertaken. However, a p o s s i b l e b i o s y n t h e t i c pathway can be proposed f o r each molecule. M e t a b o l i t e 62 obtained from the sponge Dysidea herbeceae has been proposed by Wells et a l . 6 0 as a p o s s i b l e immediate p r e c u r s o r f o r f u r o d y s i n (53) and f u r o d y s i n i n (54). The probable route l e a d i n g to the b i o s y n t h e s i s of these two isomers i s presented i n Scheme 8. (62) 142 The t h i r d f u r a n o s e s q u i t e r p e n o i d , m i c r o c i o n i n - 2 (55) has a rearranged carbon s k e l e t o n , where m i g r a t i o n of a methyl from C-4 to C-5 has occured. The p o s s i b l e b i o s y n t h e t i c pathway l e a d i n g to m i c r o c i o n i n - 2 i s presented i n Scheme 10. A l b i c a n y l acetate (51) and a l b i c a n o l (52) have drimane s k e l e t o n s . T h e i r s t r u c t u r e s c o u l d a r i s e from the b i o s y n t h e t i c pathway o u t l i n e d i n Scheme 1 1 , which i n v o l v e s t r a n s t r a n s f a r n e s o l as a p r e c u r s o r . The drimane s e s q u i t e r p e n o i d s have the t r a n s (5o<:, 10^) s t e r e o c h e m i s t r y at the A-B r i n g j u n c t i o n . I t i s i n t e r e s t i n g to note that the i r e s a n e s , another f a m i l y of s e s q u i t e r p e n o i d s with the same carbon s k e l e t o n , but the opposite a b s o l u t e c o n f i g u r a t i o n (5ft, 10<), are a l s o known. S e v e r a l hypothesis concerning the b i o s y n t h e t i c pathways l e a d i n g to the formation of these two d i f f e r e n t f a m i l i e s have been put forward. ° In 1 9 5 9 , D j e r a s s i 7 2 n o t i c e d that a l l the molecules known at the time, which had the 5 ft, 10 ac "wrong" c o n f i g u r a t i o n ( i n r e f e r e n c e to most d i t e r p e n e s and t r i t e r p e n e s ) seemed to a l s o have an oxygen attached at C-3 ( i r e s a n e , dehydroiresane, f a r n e s i f e r o l A , . . . ) . The author suggested: "that the s t e r e o c h e m i s t r y requirements of the enzyme system promoting r i n g c l o s u r e of the open ch a i n s e s q u i - and d i t e r p e n o i d p r e c u r s o r by OH ... are such to y i e l d the 5^, 1 0 * a b s o l u t e c o n f i g u r a t i o n " L a t e r , e p e r u i c a c i d 7 3 a d i t e r p e n o i d without an oxygen a t t a c h e d to C-3, but with the "wrong" absolute c o n f i g u r a t i o n was d i s c o v e r e d , i n v a l i d a t i n g D j e r a s s i ' s h y p o t h e s i s . Other a u t h o r s 7 4 143 have suggested that s e s q u i t e r p e n o i d s which had the " r i g h t " a b s o l u t e c o n f i g u r a t i o n were degraded d i - or t r i t e r p e n o i d s . In a s e r i e s of biomimetic experiments, van Tamelen 7 5 t r e a t e d the t e r m i n a l monoepoxide of t r a n s t r a n s f a r n e s y l a c e t a t e with boron t r i f l u o r i d e e t h e r a t e and obtained a product having the drimane s t e r e o c h e m i s t r y . He performed the same r e a c t i o n on the monoepoxide of t r a n s c i s f a r n e s y l a c e t a t e and o b t a i n e d a s e s q u i t e r p e n o i d from the i r e s a n e f a m i l y . His experiments thus showed that i t i s the geometry of the p r e c u r s o r which determines the s t e r e o c h e m i s t r y of the products. Andersen i n a r e v i e w 7 6 concerning s e s q u i t e r p e n o i d b i o g e n e s i s , o f f e r e d another i n t e r p r e t a t i o n to the e x i s t e n c e of two d i f f e r e n t a b s o l u t e c o n f i g u r a t i o n s f o r one carbon s k e l e t o n . He observed that i n the case where fungi and marine organisms have been shown to make the same s e s q u i t e r p e n o i d s as v a s c u l a r p l a n t s , the molecules were found to be enantiomeric. T h i s l e a d him to propose that there has been a " d r a m a t i c a l r e v e r s a l i n o p t i c a l s p e c i f i c i t y that a p p a r e n t l y o c c u r r e d between the most p r i m i t i v e l a n d p l a n t s and the more evolved ones." L a t e r , Andersen et a l 6 0 obtained a l b i c a n o l from a l i v e r w o r t (lower p l a n t ) and showed that i t s a b s o l u t e s t e r e o c h e m i s t r y i s the same as that of drimanol a n a t u r a l product i s o l a t e d from a t r e e (higher p l a n t ) 7 7 , l e a d i n g him to suggest that the drimane fa m i l y c o n s t i t u t e d an e x c e p t i o n . Recently, two f e e d i n g d e t e r r e n t s obtained from sea h a r e s 2 8 , brasudol' and i s o b r a s u d o l ( r e p o r t e d i n chapter 2) were shown to have the same a b s o l u t e c o n f i g u r a t i o n as -eudesmol a compound obtained from eucalyptus 144 o i l . T h i s i n d i c a t e d that although there were some examples f a v o u r i n g the p r o p o s a l , t h i s r u l e d i d not always apply. The drimane s k e l e t o n ( (+) a l b i c a n o l or (+) drimanol) have been shown to e x i s t i n three d i f f e r e n t p h y l l a 7 6 (or branches, see Appendix 1): a higher p l a n t Drimys w i n t e r i , a lower p l a n t D i p l o p h y l l u m a l b i c a n s , and a marine i n v e r t e b r a t e C a d l i n a  luteomarginata , f u r t h e r demonstrating that marine organisms and f u n g i need not produce m e t a b o l i t e s enantiomeric with those produced by v a s c u l a r p l a n t s . Scheme 7 145 Biosynthesis of f a r n e s y l pyrophosphate 146 Scheme 8 Possible biosynthesis of furodysin and furodysinin furodysinin furodysin Scheme 9 Possible biosynthesis of microcionin-2 147 1 4 8 Scheme 10 Possible biosynthesis of a l b i c a n y l acetate and a l b i c a n o l 149 V.2 Acanthadoris nanaimoensis:  V.2.1 B i o l o g i c a l Aspects: The o r i g i n of the s e s q u i t e r p e n o i d ( s ) obtained from Acanthadoris nanaimoensis has a l s o been i n v e s t i g a t e d . In order to f i n d out i f nanaimoal had a d i e t a r y o r i g i n or a molluscan o r i g i n , three to s i x nudibranchs were d i s s e c t e d and the stomachs and mantles e x t r a c t e d and worked up s e p a r a t e l y . The experiment was performed on two o c c a s i o n s and showed both times that nanaimoal e x i s t e d i n both stomachs and mantles. T h i s i n d i c a t e s that although the nudibranchs a c q u i r e nanaimoal from t h e i r d i e t , i t i s r e t a i n e d i n t h e i r s k i n . The d i e t a r y source of nanaimoal has not been found while d i v i n g . The only c l u e to i t s p o s s i b l e o r i g i n can be found i n the l i t e r a t u r e 5 4 , where i t i s r e p o r t e d that A_;_ nanaimoensis feeds on compound a s c i d i a n s . The e x t r a c t s of a p i n k i s h specimen and a p u r p l i s h specimen of a common l o c a l compound a s c i d i a n t e n t i t a t i v e l y i d e n t i f i e d as D i s t a p l i a o c c i d e n t a l i s have been examined, but no t r a c e of s e s q u i t e r p e n o i d s has been found. 150 V.2.2. Chemical and Biochemical Aspects: Although the f i n a l s t r u c t u r a l proof i s not a v a i l a b l e y e t, the three p o t e n t i a l s t r u c t u r e s f o r nanaimoal a l l have new carbon s k e l e t o n s . The only l i t e r a t u r e r e f e r e n c e 7 5 to a r e l a t e d molecule i s a s y n t h e t i c r e p o r t of compound 63. U n f o r t u n a t e l y , no s p e c t r a l data are a v a i l a b l e . I l l N H O (63) P o s s i b l e b i o s y n t h e t i c pathways l e a d i n g to the formation of h y p o t h e t i c a l molecules i , i i , and i i i are o u t l i n e d i n Scheme 11. These p o s s i b l e b i o s y n t h e t i c routes would tend to favour s t r u c t u r e i i which i n v o l v e s the usual isomer of f a r n e s y l pyrophosphate, as a pr e c u r s o r and which i n v o l v e s a w e l l known r i n g A c y c l i z a t i o n step. On the other hand, s i n c e i^ and iii would i n v o l v e the same p r e c u r s o r , i t c o u l d e x p l a i n the e x i s t e n c e of two s t r u c t u r a l isomers. Each of these two molecules would a r i s e from a d i f f e r e n t second c y c l i z a t i o n step. T h i s second p r e c u r s o r , which i n v o l v e s the c y c l i z a t i o n of a methyl group has been observed i n the b i o s y n t h e s i s of at l e a s t one other marine s e s q u i t e r p e n o i d . The marine . sponge P l e r a p l y s i l l a s p i n i f e r a 7 " has been repo r t e d to c o n t a i n p l e r a p l y s i l l i n - 1 (64). 151 (64) which may i n v o l v e the same type of c y c l i z a t i o n between C-12 and C-13. These o b s e r v a t i o n s , p l u s the data r e p o r t e d i n the pre v i o u s chapter, where: the XH NMR data would tend to support s t r u c t u r e i the MS fragments would favour s t r u c t u r e s i i or i i i show that the s t r u c t u r a l e l u c i d a t i o n of the Acanthadoris  nanaimoensis compound(s) i s a very d i f f i c u l t and i n t e r e s t i n g problem. 1 5 2 Scheme 11 Possible biosynthesis of the nanaimoane C skeleton OX i i i i i i 153 Experimental 1H NMR s p e c t r a were recorded on N i c o l e t - O x f o r d H-270 and V a r i a n XL-100 spectrometers, while 1 3 C NMR s p e c t r a were obtained on a Bruker WP-80 spectrometer. TMS was used as an i n t e r n a l standard and CDC1 as a s o l v e n t , except when mentioned otherwise. Low r e s o l u t i o n MS were obtained on an A.E.I. MS-902 spectrometer. The HRMS were recorded on A.E.I. MS 50 spectrometer and the i n t e n s i t i e s f o r the p l o t t e d mass s p e c t r a were c a l c u l a t e d from the low r e s o l u t i o n s p e c t r a . P l o t t i n g s t a r t s with the peak at m/e=32 and only peaks of a 3% i n t e n s i t y and higher are i n d i c a t e d . IR s p e c t r a (CHC1 3 s o l u t i o n s ) were recorded on a Perkin-Elmer Model~70B spectrometer and o p t i c a l r o t a t i o n s were measured on a Perkin-Elmer Model-141 p o l a r i m e t e r , u s i n g a 1 dm c e l l . M e l t i n g p o i n t s were obtained on a Fis h e r - J o h n s apparatus and values are u n c o r r e c t e d . UV absorbance s p e c t r a were recorded on Cary-14 spectrophotometer, using 1 cm c e l l s - a n d MeOH as a s o l v e n t . Values of e x t i n c t i o n c o e f f i c i e n t s are only approximate due to the d i f f i c u l t y i n o b t a i n i n g a ccurate weights of v o l a t i l e m a t e r i a l s . Merk s i l i c a g e l 60 PF-254 was used f o r PTLC and Merk s i l i c a g e l 60, p a r t i c l e s i z e 0.063-0.200 mm (70-230 mesh ASTM) was used f o r columns. So l v e n t s were HPLC grade or d i s t i l l e d reagent grades. A P e r k i n Elmer s e r i e s 2 LC equipped with a P e r k i n Elmer LC-55-s UV d i g i t a l scanner, and a Bondapak C18 or a L i c h r o s o r b SI-60 column was used f o r HPLC. GC a n a l y s i s were performed on Hewlett Packard 5830A GC equipped with an OV-1 or OV-17 column and e i t h e r a thermal c o n d u c t i v i t y or a flame i o n i z a t i o n d e t e c t o r . 154 A) C a d i i n a luteomarginata: A.1. E x t r a c t i o n and Crude S e p a r a t i o n ; Immediately a f t e r c o l l e c t i o n , the nudibranchs were put i n a g l a s s j a r c o n t a i n i n g MeOH (125 ml of MeOH/100 nudibranchs). About one hundred and seventy f i v e to two hundred and f i f t y specimens f i l l a 15 cm long by 6 cm i n diameter j a r . A f t e r soaking the animals f o r three days, the supernatant was f i l t e r e d through Whatman #1 f i l t e r paper and evaporated to about one t h i r d of the o r i g i n a l volume. The c o n c e n t r a t e d e x t r a c t was then p a r t i t i o n e d between b r i n e and C H C l 3 o r b r i n e and CH 2 C l 2 . The organic phase was d r i e d over Na 2 S0 4 , f i l t e r e d and evaporated to g i v e a sweet s m e l l i n g o i l y r e s i d u e . The e x t r a c t i o n procedure was repeated four times to give about one gram (1.03) of crude m a t e r i a l from one j a r of nudibranchs. A l l the weights given i n t h i s s e c t i o n were taken from one p a r t i c u l a r c o l l e c t i o n and represent t y p i c a l v a l u e s . The weights of a l l v o l a t i l e compounds are i n e r r o r s i n c e i t was impossible to remove a l l t r a c e s of s o l v e n t under high vacuum without s u b s t a n t i a l l o s s e s of the v o l a t i l e s e s q u i t e r p e n o i d s . The crude e x t r a c t was chromatographed on a s i l i c a column (45x3 cm, CH 2C1 2 or CHCl^ e l u e n t ) . E l u t i o n was continued u n t i l the s t e r o i d a l peroxides stopped e l u t i n g o f f the column, 300 mg of m a t e r i a l was obtained ( y i e l d 30%). The crude m a t e r i a l from the column was subsequently chromatographed on 1mm s i l i c a g e l p l a t e s developed i n CH 2C1 2 or CHC1 3. Four f r a c t i o n s were c o l l e c t e d from the PTLC p l a t e s : 155 1) f u r a n o s e s q u i t e r p e n o i d s Rf= 0.6 to 0.8 2) a l b i c a n y l a c e t a t e Rf = 0.4 to 0.6 3) a l b i c a n o l and luteone Rf = 0.2 to 0.4 4) s t e r o i d a l peroxides Rf = 0 to 0.15 A.2 S e p a r a t i o n of the F u r a n o s e s q u i t e r p e n o i d s :  A. 2.a. F u r o d y s i n i n : Three r e p e t i t i v e developments of f r a c t i o n (1) on 50 cm long s i l i c a p l a t e s (hexane) give the best p u r i f i c a t i o n of f u r o d y s i n i n (Rf=0.15). 2 mg (yield=0.1% of crude e x t r a c t ) of r e c r y s t a l l i z e d f u r o d y s i n i n (hexane) were obtained, M.P.=75°C. IR (CHC1 3, F i g . 17) 2960, 2910, 2870, 1460, 1210, 1080, 1070 cm" 1. XH NMR (CC1 4 , F i g . 18B) 1.17 (s, 3H-14+3H-15), 1.64 (s, 3H-13), 2.25 (dd, 1H-9, J=17.5, 12.5 Hz), 2.69 (m, 1+ dd, 1H-9', J=17.5, 5.5 Hz), 5.57 (m, 1H-1), 6.08 (d, 1H-12, J=2Hz), 7.08 (bs, 1H-11). MS (m/e r e l a t i v e i n t e n s i t y , F i g . 16) 216(20), 201(8), 122(100). A.2.b. Mixture C o n t a i n i n g Furodysin and F u r o d y s i n i n : A May 1980 c o l l e c t i o n of C a d i i n a luteomarginata ,from E l l i s I s l a n d , produced a mixture of f u r o d y s i n and f u r o d y s i n i n . F r a c t i o n (1) obtained from the crude s e p a r a t i o n was developed over PTLC p l a t e s i n hexane. The upper part of the band which c h a r r e d blue a f t e r s p r a y i n g with H 2 S0 4 (Rf = 0.15), was e x t r a c t e d s e p a r a t e l y from the lower p a r t which c h a r r e d pink-blue (Rf = 0.14). The m a t e r i a l i n the upper part was i d e n t i f i e d as 156 f u r o d y s i n i n , the m a t e r i a l i n the lower p a r t as a mixture of f u r o d y s i n and f u r o d y s i n i n p l u s an unknown. D i f f e r e n t chromatography techniques were t r i e d to separate the mixture: A.2.b.1. Reversed-phase p l a t e s : The f u r o d y s i n , f u r o d y s i n i n mixture was rechromatographed on reversed-phase p l a t e s developed with three d i f f e r e n t s o l v e n t systems: MeOH and H 20, CH 3CN and H 20, t e t r a h y d r o f u r a n and H ?0, t r i e d in d i f f e r e n t percent compositions. In a l l i n s t a n c e s , 80% of the organic s o l v e n t i n water was needed to o b t a i n an Rf between 0.2 and 0.4. In a l l cases, upon development with I 2 , only one spot c o u l d be v i s u a l i z e d . A.2.b.2. TLC on alumina: A n a l y t i c a l TLC on n e u t r a l alumina using combinations of CHCl 3:EtOAc (0% to 100% of EtOAc), and c h a r r i n g with H 2S0 4 r e v e a l e d the presence of a green spot at the o r i g i n of the TLC s t r i p , i n d i c a t i n g that a p o s s i b l e decomposition of the molecules had occured. A.2.b.3. S i l i c a g e l HPLC: W e l l s 6 0 r e p o r t e d s e p a r a t i n g a mixture of four f u r a n o s e s q u i t e r p e n o i d s obtained from an A u s t r a l i a n sponge by using "HPLC on a s i l i c a column". We attempted to repeat t h i s s e p a r a t i o n on s i l i c a using d i f f e r e n t combinations of isopropanol:hexane with v a r i o u s flow r a t e s (UV d e t e c t o r 252 nm). Changes i n the s o l v e n t system a f f e c t e d only the r e t e n t i o n time, g i v i n g i n a l l cases, only one peak. R e s u l t s were as presented i n Table X. 157 Table X Re s u l t s obtained with the HPLC using a s i l i c a column % i sopropanol/hexane 5 2 1 0 flow rat e (ml/min) 1.2 1.2 .8 .6 Rt (min) 1.62 2.21 5.73 8.10 A.2.b.4 Reverse phase HPLC: Reversed-phase HPLC (column Bondapak C-18) using MeOH/ H 20 as a so l v e n t system was somewhat more s u c c e s f u l at s e p a r a t i n g the mixture. When the flow r a t e was maintained at 0.6 ml/min., r e s u l t s of the s e p a r a t i o n were as i n d i c a t e d i n Table XI: 158 Table XI_ R e s u l t s obtained with the HPLC using a r e v e r s e  phase column % of MeOH in H, 0 99 90 80 78 75 70 Rt (min) of A 4.56 5.08 6.05 6.69 7.18 9.31 & (A), ( B ) (min) .46 1.22 3.96 5.12 6.48 12.32 Rt (min) of B 5.02 6.31 10.01 12.30 13.17 21.63 A ( B ) , (C) (min) .46 .53 .88 .78 .74 — Rt (min) of C 5.48 6.84 10.89 13.08 13.91 A represents a minor component preceded by s e v e r a l i m p u r i t i e s B r e p r e s e n t s the isomer present i n a minor amount C represents the isomer present i n a major amount Although the reversed-phase column seemed more e f f e c t i v e at s e p a r a t i n g f u r o d y s i n and f u r o d y s i n i n than reversed-phase p l a t e s , we c o u l d only o b t a i n .two o v e r l a p p i n g peaks. 159 I ,. 1 ill Ju LL A f t e r c o l l e c t i o n of a, b, c, e x t r a c t i o n of each f r a c t i o n with C H C l 3 , evaporation of the organic s o l v e n t , TLC and c h a r r i n g with H^SO^, the three f r a c t i o n s developed the same c o l o r . T h i s r e v e a l e d that f r a c t i o n s a, b and c were s t i l l m ixtures. IR (CHC1 3, F i g . 20) 2960, 2920, 2870, 1460, 1080, 1070 cm* 1. Furodysin (*), f u r o d y s i n i n ('**), unknown (.***) XH NMR (CC1 , Fig.21B) 0.90***(d, J=7Hz), 1.05*** ( s ) , 1.17** ( s ) , 1.23* ( s ) , 1.64** ( s ) , 1.69* ( s ) , 5.57* + ** (m), 5.91*** ( s ) , 5.93* ( s ) , 6.08** ( s ) , 6.98* ( s ) , 7.08** ( s ) . MS (m/e r e l a t i v e i n t e n s i t y F i g . 19) 216(25), 201(14), 122(100). A.2.c . Microc ionin-2 When f r a c t i o n (1) obtained from the crude e x t r a c t of C.  luteomarginata c o l l e c t e d at the end of J u l y , was run on PTLC p l a t e s i n hexane, m i c r o c i o n i n - 2 was obtained i n a 0.3% y i e l d (Rf = 0.15) . IR (CHC1 3, F i g . 23) 2940, 2860, 1460, 1380, 1165, 1030, 880 cm" 1. *H NMR (CC1 4 , F i g . 24) 0.99 (d, 3H-13, J=7Hz), 1.06 (s, 3H-15), 1.62 (s, 3H-14), 1.96 (bs, 2), 2.32 (m, 2), 5.36 (bs, 1H-3), 6.13 (s, 1H-11), 7.08 (s, 1H-10), 7.21 (s , l H - 1 2 ) . MS (m/e r e l a t i v e i n t e n s i t y , F i g . 22) 218(30), 203(14), 137(4), 123(100), 95(38), 81(44). 160 M i c r o c i o n i n - 2 l i k e the other furan c o n t a i n g compounds i s very unstable and has a high tendency to give p o l a r degradation p r o d u c t ( s ) . A.3. A l b i c a n y l a c e t a t e : F r a c t i o n (2) obtained from the crude s e p a r a t i o n was run on s i l i c a g e l PTLC p l a t e s developed i n CH ? C l ^ :hexane (2:1). A band which had an Rf of 0.30 was c o l l e c t e d and rechromatographed on s i l i c a g e l PTLC using CHCl 3:EtOAc (20:1). The major band having an Rf of 0.48 was c o l l e c t e d . A n a l y t i c a l TLC spots corresponding to a l b i c a n y l a c e t a t e i n i t i a l l y char pink when sprayed with U? S04 and heated, however, a f t e r s t anding at room temperature f o r s e v e r a l hours they become blue. 5 mg of a l b i c a n y l a c e t a t e (yield=0.5% of crude e x t r a c t ) show W 0= + 24* (c.5, CHC1 3)., IR (CHC1 3, F i g . 6) 2920, 2860, 1740, 1460, 1440, 1400, 1380, 1240, 1040, and 900 cm " l . lH NMR (CDC1 3, Fig.8) 0.76 (s, 3H-15), 0.81 (s, 3H-13), 0.88 (s, 3H-.14), 1.13 (dd, lH-5a, J = 13, 12 Hz), 1.33 (dddd, lH-6a, J=13, 12, 12, 4.5 Hz), 1.73 (dddd, lH-6e, J= 13, 4.5, 3, 3 Hz), 2.01 (ddd, lH-7a, J=13, 12, 4.5 Hz +dd, lH9-a, J=9, 4 Hz + s, 3), 2.41 (ddd, lH-7e, J= 13, 4.5, 3 Hz), 4.18 (dd, 1H-11, J=9, 11 Hz) 4.34 (dd, 1H-11', J=4, 11 Hz), 4.51 (d, 1H-12, J=lHz), 4.85 (d, 1H-12', J=l Hz). Values of c o u p l i n g constants l i s t e d were e i t h e r d i r e c t l y c a l c u l a t e d or deduced from proton-proton decoupling experiments. MS (m/e r e l a t i v e i n t e n s i t y , F i g . 6) 264(3), 249(3), 222(3), 221(3), 204(100), 189(33), 137(78), 123(33), 109(22), 95(30). 161 A.4. A l b i c a n o l ; A.4.a. Obtained from the H y d r o l y s i s of A l b i c a n y l a c e t a t e : 12 mg ( 4 . 5 x l 0 - 2 mmoles) of a l b i c a n y l a c e t a t e were d i s s o l v e d i n 1 ml of MeOH and about 20 mg of K 2C0 3 were added to the s o l u t i o n . The r e a c t i o n was l e f t s t i r r i n g o v e r n i g h t . The mixture was f i l t e r e d through g l a s s wool, and p a r t i t i o n e d between water and e t h e r . A f t e r three e x t r a c t i o n s , the organic s o l v e n t was evaporated. The residue was then p u r i f i e d by running on p t l c p l a t e s i n CH^Cl^, to gi v e a l b i c a n o l , with an Rf = 0.2. 9 mg (4x10" 2 moles) of a l b i c a n o l were obtained (yield=88%) from t h i s r e a c t i o n . Leaving a l b i c a n y l a c e t a t e i n MeOH for three days d i d not give a l b i c a n o l ( d i d not a f f e c t the compound at a l l ) , p r o v i n g that the compound obtained as a minor c o n s t i t u e n t of the e x t r a c t of C_^  luteomarginata i s not an a r t i f a c t . A.4.b. Se p a r a t i o n of A l b i c a n o l from F r a c t i o n (2) of the Crude  E x t r a c t : A l b i c a n o l was a l s o obtained d i r e c t l y from the crude e x t r a c t , by developing f r a c t i o n (3) on s i l i c a g e l PTLC p l a t e s f i r s t i n CHC1 3:EtOAc (8:1, Rf = 0.46) and then i n CHCl 3:EtOAc (20:1, Rf = 0.31). A n a l i t y c a l TLC spots corresponding to a l b i c a n o l char blue when sprayed with H 2S0 4 and heated. 1 or 2 mg ( 5 x l 0 - 3 mmoles, yield=0.05 to 0.1% of crude e x t r a c t ) were obtained + 13' (c .6, CHC1 3) and M. P. = 68-69°C ( r e c r y s t a l l i z a t i o n from carbon t e t r a c h l o r i d e or hexane). IR (CHC1 3, F i g . 11) 3350, 2920, 2850, 1730, 1640, 1460, 162 1390, 1370, 1030 and 890 cm" 1. *H NMR (CDC1 , F i g . 12) 0.72 (s, 3H-15), 0.81 (s, 3H-13), 0.88 (s, 3H-13, 1.12 (dd, lH-5a, J=3, 13 Hz), 1.34 (dddd, lH-6a, J=13, 13, 13, 4 Hz), 1.75 (dddd, 1H-6e, J=13, 4.5, 3, 2 Hz), 2.02 (ddd, lH-7a, J=13, 13, 4 Hz + dd, lH-9a, J = l l , 4 Hz), 2.41 (ddd, lH-7e, J=13, 4, 2 Hz), 3.76 (dd, 1H-11, J = l l , 11Hz), 3.84 (dd, 1H-11', J = l l , 4Hz), 4.65 (d, 1H-12, J=1.5Hz), 4.93 (d, 1H-12', J=1.5 Hz). MS (m/e r e l a t i v e i n t e n s i t y , F i g . 10) 222(38), 207(13), 204(17), 189(14), 137(100), 123(33), 109(28), 95(25). A.5. Drimanol Obtained from the Hydrogenation of A l b i c a n o l : 9 mg (4x10* 2 mmoles) of a l b i c a n o l were d i s s o l v e d i n 1 ml of con c e n t r a t e d a c e t i c a c i d and 5 mg of Pt0 2 were added to the s o l u t i o n . The mixture was s t i r r e d overnight under a hydrogen p r e s s u r e . I t was then f i l t e r e d through g l a s s wool and p a r t i t i o n e d between H 20 and CHC1 3 and the organic s o l v e n t was evaporated. 6 mg (2.7x10' 2 mmoles) of drimanol were obtained (yi e l d = 6 8 % ) , a f t e r r e c r y s t a l l i z a t i o n from hexane, M. P. = 100°C and M D = + 15° (c .5, CHC1 3 ) . IR (CHC1 3, F i g . 14) 3620, 3470, 2950, 2890, 2870, 1470, 1400, 1380, 1140, 1100, 1030, 998 and 980 cm - 1 . *H NMR (CDCl 3 , F i g . 15) 0.82 (s, 3), 0.85 (s, 6), 0.96 (d, 3H-12, J=7.5 Hz), 2.15 (m, lH-8e, c o u p l i n g deduced from proton-proton d e c o u p l i n g experiment J= 7.5, 7.5, 7.5, 5, 5, 2 Hz), 3.59 (dd, 1H-11, J= 10.5, 10.5 Hz), 3.86 (dd, 1H-11', J=10.5, 4.5 Hz). MS (m/e r e l a t i v e i n t e n s i t y , F i g . 11) 224(34), 209(24), 206(5), 191 (3), 123(100), 109(22), 95(32). 163 A.6.Luteone A f t e r crude s e p a r a t i o n , TLC of f r a c t i o n (3) showed the presence of two major compounds. The l e s s p o l a r one was i s o l a t e d by chromatographing crude f r a c t i o n (3) on s i l i c a , p l a t e s using f i r s t CHC13 :EtOAc (8:1, Rf = 0.58) and then CHCl 3:EtOAc, (20:1, Rf = 0.4) . At t h i s step of the i s o l a t i o n procedure, *H NMR re v e a l e d that luteone was s t i l l contaminated with i m p u r i t i e s ( p h t a l a t e e s t e r , see Chapter 4). Fu r t h e r attempts to p u r i f y luteone l e d to an i n c r e a s e i n the  1 U NMR s i g n a l s corresponding to the ph t h a l a t e ( F i g . 26, 27) Luteone(*) and p h t h a l a t e e s t e r (**) XH NMR (CDC1 3, F i g . 27) 0.60* ( s ) , 0.78* ( s ) , 0.95* + ** (s=m), 2.11* ( s ) , 4.22** ( t , J=7 Hz), 4.32** ( t , J= 7 Hz), 4.46* ( b s ) , 4.86* (bs) , 4.84** ( s ) . MS (m/e r e l a t i v e i n t e n s i t y , F i g . 25B) 344(3), 326(3), 299(4), 268(4), 191(15), 149(72), 137(18), 123(20), 109(50), 95(32), 43(100). A.7. DNPH D e r i v a t i v e of Luteone: From a subsequent c o l l e c t i o n , 12 mg of f r a c t i o n (3) were d i s s o l v e d i n 1 ml of MeOH. To t h i s s o l u t i o n , 0.5 ml of a mixture c o n t a i n i n g : 10 mg of 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e i n 1 ml of MeOH and 2 drops of HC1 was added and the r e a c t i o n was l e f t s t i r r i n g f o r one hour. A f t e r t h i s p e r i o d , c r y s t a l s formed i n the f l a s k . The supernatant was removed and the c r y s t a l s d i s s o l v e d i n CHC13 and chromatographed on s i l i c a g e l p l a t e s developed with CH 2C1 2. The DNPH c o n t a i n i n g band was e l u t e d from the s i l i c a g e l with 164 a c e t o n i t r i l e , the e x t r a c t was evaporated and the res i d u e r e c r y s t a l i z e d from MeOH (yield=3mg, M. P.= ). IR (CHC1 3, F i g . 29) 3310, 2920, 2850, 1700, 1670, 1600, 1510, 1340, 1320 cm" 1. XH NMR (CDC1 3, F i g . 30) 0.64 (s, 3), 0.76 (s, 3), 0.78 (s, 3), 2.07 (s, 3), 4.56 (s, 1), 4.89 (s, 1),7.96 (d, 1, J=10 Hz), 8.32 (dd, 1, J=2.5, 10Hz), 9.15 (d, 1, J=2.5 Hz), 10.12 (s, 1). MS (m/e r e l a t i v e i n t e n s i t y , F i g . 28) 524(9), 495(12), 449(15), 327(28), 326(32), 299(32), 161(80), 135(68), 123(52), 121(76), 109(95), 107(76), 95(100), 93(76), 81(95), 55(80). A.8. S t e r o i d a l p e r o x i d e s : A f t e r crude s e p a r a t i o n , a n a l y t i c a l TLC of f r a c t i o n (4) i n CHCI3 (Rf=0.1) showed the presence of a spot which c h a r r e d green with H 2S0 4. T h i s s t e r o i d a l peroxide mixture was co-chromatographed with two samples obtained i n our l a b o r a t o r y * ' (Rf=0.54 i n EtOAc). XH NMR (CDC13) of t h i s sample showed the presence of a doublet of doublets at 6.35 ppm, c h a r a c t e r i s t i c of t h i s c l a s s of compounds. A.9. D i s s e c t i o n s : 6 to 12 nudibranchs were d i s s e c t e d by performing a l o n g i t u d i n a l cut i n the mantle of the animals, and removing the " i n s i d e " (stomach) from the r e s t of the body. The stomachs and mantles were then soaked i n MeOH f o r three days and e x t r a c t i o n took plac e as d e s c r i b e d i n A . l . A n a l y t i c a l TLC were i n t e r p r e t e d q u a l i t a t i v e l y . (The crude e x t r a c t s were weighed and then d i s s o l v e d i n an amount of c h l o r o f o r m which gave the same 165 c o n c e n t r a t i o n i n the two s o l u t i o n s , and then rechromatographed on a n a l y t i c a l TLC s t i p s ) . B) From Acanthadoris nanaimoensis:  B.1. E x t r a c t ion and Crude Separat i o n : Immediately a f t e r c o l l e c t i o n , the nudibranchs are put to soak i n a g l a s s j a r c o n t a i n i n g MeOH. About 250 animals f i l l the three q u a r t e r s of a 14 cm long by 8 cm i n diameter j a r . A f t e r soaking f o r three days, the e x t r a c t i o n procedure takes p l a c e i n the same manner as f o r C^ luteomarginata. T y p i c a l l y , 1.3 g of crude r e s i d u e were obtained from the nudibranchs. The crude m a t e r i a l was chromatographed on a s i l i c a g e l column (35x3 cm, CH 2C1 2 e l u e n t ) . E l u t i o n stopped a f t e r c o l l e c t i o n of the s t e r o i d a l peroxides (200 mg, y i e l d = 16% of crude m a t e r i a l ) . B.2. S e p a r a t i o n of nanaimoal: 200 mg of p a r t i a l l y p u r i f i e d Acanthadoris nanaimoensis e x t r a c t was chromatographed on s i l i c a g e l p l a t e s using CH 2C1 ? as a developing s o l v e n t , r e s u l t i n g i n the s e p a r a t i o n of nanaimoal (Rf = 0.43) I*] = -7° (c 3.0, CHC1 3) and of the s t e r o i d a l p eroxides (Rf = 0.1) IR (CHCI3, F i g . 32) 2920, 2750, 1730, 1470, 1395, 1380, 930, 870, 800 and 750 cm - 1 . XH NMR (CDC1 3, F i g . 33) 0.98 (s, 6H), 1.05 (s, 3H), 2.0 (m, 2H), 2.25 (dd, 2H, J=14,3 Hz), 9.71 ( t , 3Hz, corresponding to .2H), 9.83 ( t , IH, J=3Hz). MS (m/e r e l a t i v e i n t e n s i t y , F i g . 31) 220(10), 205(3), 191(3), 176(70), 161(100), 121(30), 105(42). 166 B.3. DNPH D e r i v a t i v e of Nanaimoal: 3 mg ( 1 . 4 x l 0 - 2 mmoles) of nanaimoal was d i s s o l v e d i n 1 ml of MeOH and .5 ml of a mixture c o n t a i n i n g : 10 mg (5x10" 2 mmoles) of 2,4-phenylhydrazine i n 1 ml of MeOH and 2 drops of HCl, was added to t h i s s o l u t i o n . The r e a c t i o n was l e f t to s t i r f o r one hour, a f t e r t h i s p e r i o d , c r y s t a l s formed. The supernatant was removed and the c r y s t a l s d i s s o l v e d i n CHC1 3 and chromatographed on s i l i c a g e l p l a t e s developed with CH 2C1 2 (Rf=0.61). A f t e r e x t r a c t i o n with a c e t o n i t r i l e and eva p o r a t i o n of t h i s s o l v e n t , the s o l i d r e s idue was r e c r y s t a l l i z e d i n 5 drops of CHC1 3 p l u s 0.5 ml of MeOH to give 5 mg (1.3x10' 2mmoles) of a sample with a M. P. =126-128'C(yield= 90%). IR (CHC1 3 , F i g . 36) 3320, 2940, 2900, 1640, 1610, 1525, 1360, 1330, 1160, 860 and 750 cm" 1. lH NMR (CDC1 3, Fig.36) 11.03 (bs, IH), 9.12 (H-3, b s ) , 8.30 (H-5, dd, J=3,10 Hz), 7.93 (H-6, d, J=10 Hz), 7.55 ( t , IH, J=6Hz), 2.32 (dd, 2H, J=6,14 Hz), 2.04 (m, 2H), 1.00 (s, 3H), .99 (s, 3H), .98 (s, 3H). S i g n a l s f o r the minor s t e r e o c h e m i c a l isomer appear a t : 11.25 (b s ) , 7.06 ( t , J=6Hz), 2.79 (m). MS (m/e r e l a t i v e i n t e n s i t y , F i g . 34) 400(5), 280(40), 176(100), 161(60), 149(63). B.4. Nanaimool 15 mg ( 7x10" 2 mmoles) of nanaimoal were d i s s o l v e d i n 1 ml of MeOH, pl u s 5 drops of a c e t i c a c i d and about 5 mg of P t 0 2 were added to the s o l u t i o n . The r e a c t i o n was l e f t s t i r r i n g under a hydrogen atmosphere f o r 24 hours. I t was then f i l t e r e d through 167 g l a s s wool and p a r t i t i o n e d between H 20 and C H C l j . The organic s o l v e n t was evaporated and the residue obtained was chromatographed on PTLC p l a t e s , i n C H 2 C l 2 (Rf = 0.2). A f t e r e x t r a c t i o n and evaporation of the s o l v e n t , 9 mg (4x10* 2 mmoles) of nanaimool were obtained (60% y i e Id) , L ~ ] D =+8° (c 1.8, CHC1 ). IR ( C H C l 3 , . F i g . 38) 3600, 3400, 2920, 2850, 1460, 1380, 1360, 1020, 910 and 740 cm - 1 . lH NMR (CDC13 , F i g . 39A) 0.87 (s, 3H), 0.96 (s, 3H), 0.97 (s, 3H), 1.95 (m, 2H), 3.72 ( t , 2H, J=8 Hz, major isomer) and 3.63 ( t , minor isomer). MS (m/e r e l a t i v e i n t e n s i t y , F i g . 37) 222(40), 207(100), 189(30), 179(24), 177(33), 121(20). Proton-decoupled 1 3 C NMR ( C D C l 3 , F i g . 37) shows s i n g l e t s at 19.5, 21.5, 24.9, 27.9, 28, 30.8, 31.8,34.8, 39.9, 44.0, 59.7, 125.5 and 133.4 ppm. B.5. p a r a - c h l o r o p h e n y l i s o c y a n a t e D e r i v a t i v e : 4-5 mg (2x10" 2mmoles) of nanaimool were added to a CCl 4 s o l u t i o n c o n t a i n i n g 10 mg (6x10" 2mmoles) of p - c h l o r o p h e n y l i s o c y a n a t e . The r e a c t i o n mixture was corked and heated f o r 2-3 min. I t was then chromatographed on s i l i c a p l a t e s developed in CHC1 3. The new UV band appearing at Rf = 0.5 was c o l l e c t e d to o b t a i n 6-7 mg (1.5x10" 2mmoles) of p-chlorophenylurethane ( y i e l d = 80%). The r e c r y s t a l l i z a t i o n was most s u c c e s f u l i n MeOH/H2O (5:1, M.P.=194-198*C). IR (CHC1 3, F i g . 41) 3425, 2910, 2860, 1720, 1595, 1510, 1405, 1310, 1100, 840 cm ~ l . XH NMR (CDC1 3, F i g . 42) 0.90 (s, 3H), 0.96 (s, 3H), 0.97 (s, 3H), 1.74 (bd, 2H, J=16 Hz),1.97 (m, 2h, 8Hz), 4.14 ( t , J = l 0 Hz, minor isomer), 4.20 (2H, t , 10 Hz, 168 major isomer), 6.5 (bs, IH), 7.21 (d, 2H,J=9Hz), 7.26 (d, 2H, J=9HzO. MS (m/e r e l a t i v e i n t e n s i t y , F i g . 40) 377(8) 375(9), 204(40), 189(100), 161(28). B.6. Reaction with 1 , 3 - p r o p a n e d i t h i o l : 4mg (2x10" 2mmoles) of nanaimoal were added to 2 ml of benzene c o n t a i n i n g 0.2 ml of 1,3-propanedithiol(4x10" xmmoles). A c r y s t a l of p - t o l u e n e s u l f o n i c a c i d was added to t h i s s o l u t i o n . The r e a c t i o n mixture was l e f t s t i r r i n g o v ernight and then analyzed by TLC. The s t a r t i n g m a t e r i a l and the reagents c o u l d be seen by TLC (unreacted). Even so, the r e a c t i o n mixture was e x t r a c t e d s e v e r a l times with 10% NaOH and the organic l a y e r evaporated and co - chromatographed with nanaimoal to show the presence of the s t a r t i n g m a t e r i a l . The same r e a c t i o n mixture was prepared once more, and put to r e f l u x f o r 1 hr. A f t e r t h i s p e r i o d of time, the TLC showed that the aldehyde had completely disappeared. No new spot which had a UV and/or c h a r r e d c o u l d be d e t e c t e d by TLC. B.7. Meta-bromobenzoate D e r i v a t i v e : Another 4 mg (2x10" 2mmoles) of nanaimool were added to 0.5 ml (2x10" 2mmoles) of f r e s h l y d i s t i l l e d m-bromobenzoyl bromide. Four or f i v e drops of p y r i d i n e were added and the r e a c t i o n mixture heated for 4-5 min and then s t i r r e d f o r one hour at room temperature. A f t e r that p e r i o d of time, the s o l u t i o n was then chromatographed on a n a l y t i c a l TLC s t r i p s to show the appearance 169 of a new spot, with Rf = 0.65 i n CHC1 3, which had a UV and c h a r r e d when sprayed with S0 4 . The f l a s k c o n t a i n i n g the r e a c t i o n mixture was put on the high vacuum pump, f o r 2 hrs to e l i m i n a t e most of the p y r i d i n e p r e s e n t . I t was p u r i f i e d by s i l i c a PTLC (CHC1 3) and the band with Rf=0.65 was c o l l e c t e d . A f t e r rechromatographing on s i l i c a p l a t e s by developing e i g h t r e p e t e t i v e times i n hexane (Rf = 0.03), the p u r i f i e d e x t r a c t was then put to c r y s t a l l i z e i n d i f f e r e n t s o l v e n t s . JH NMR (CDC1 3, F i g . 44) 8.19 (H~2, b s ) , 7.99 (H-4, d, J=8 Hz), 7.70 (H-6, bd, J=8 Hz), 7.34 (H-5, t , J=8 Hz), 4.41 ( t , 2H, J=8 Hz), 2.00 (m, 2H, J=8 Hz), 0.97 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H). IR (CHC1 3,Fig. 43) 2940, 2870, 1720, 1470, 1300, 1275, 1140, 670 cm - 1. A f t e r f u r t h e r attempts to c r y s t a l l i z e the compound, TLC showed the presence of a spot corresponding to nanaimool and a p o l a r UV spot probably corresponding to m-bromobenzoic a c i d , while an unsoluble s o l i d appeared i n the f l a s k . No sample was submitted f o r MS. B.8. Para-bromobenzoate D e r i v a t i v e : 5 mg (2.2x10" 2mmoles) of nanaimool and 10 mg of p-bromobenzoyl c h l o r i d e (5x10" 2mmoles) were d i s s o l v e d i n 1 ml of CC1 4 and 4-5 drops of p y r i d i n e was added. This r e a c t i o n mixture was heated f o r 3-4 min and s t i r r e d f o r 1 hr at room temperature. A f t e r t h i s p e r i o d of time, i t was analyzed by TLC and the appearance of a new spot was observed (UV and H 2S0 4 p o s i t i v e , Rf 170 = 0.65 i n CHC1 3). The organic s o l v e n t was evaporated from the s o l u t i o n and the res i d u e put on the high vacuum pump f o r one hour. I t was then p u r i f i e d by chromatography ( r e p e t e t i v e developments of a s i l i c a p l a t e i n hexane). The UV band with Rf =0.03 was c o l l e c t e d , e x t r a c t e d and the sol v e n t evaporated. XH NMR (CDC1 3, F i g . 45) 7.92 (d, J=10 Hz, major isomer), 7.91 (d, J=10 Hz, minor isomer), 7.60 (d, J=10 Hz, two isomers), 4.43 ( t , J=8 Hz, minor isomer), 4.34 ( t , J=8 Hz, major isomer), 1.4 (m), 1.28 ( s ) , 1.18 ( s ) , 1.02 ( s ) , 0.93 ( s ) , 0.88 ( s ) , 0.86 (s) and 0.84 (s) ppm. The number of protons i n t h i s case i s u n c e r t a i n . Not enough m a t e r i a l was present f o r an IR. No sample was submitted f o r MS. B.9. O z o n o l y s i s : Ozone was passed through a s o l u t i o n c o n t a i n i n g 2-3 mg of nanaimool i n 1 ml of MeOH, durin g 5 min. at -78°C. Oxygen was then bubbled through to f l u s h the remaining ozone i n the s o l u t i o n , and dimethyl s u l f i d e was added i n excess. A f t e r 1 hr, the s o l v e n t was evaporated and the res i d u e p u r i f i e d by chromatography on s i l i c a g e l TLC s t r i p s developed i n CHC1 3. Two major bands were c o l l e c t e d Rf= 0.14 and 0.48, and due to the small amount of samples, only MS were obtained on these o i l s . MS (m/e r e l a t i v e i n t e n s i t y , F i g . 46 and 47 r e s p e c t i v e l y ) of product with Rf=0.14 236(8), 223(5), 83(100), and of product with Rf=0.48 236(16), 221(5), 179(100). 171 B. 10. P-bromophenylhydrazone D e r i v a t i v e : To 10 mg (4.5x10" 2mmoles) of nanaimoal present i n 1 ml of MeOH, 20 mg (.lmmoles) of p-bromophenylhydrazine and 4 drops of HC1 were added. T h i s s o l u t i o n was then heated s e v e r a l times f o r 2-3 min. p e r i o d s . A f t e r observing that a l l the nanaimoal (R = .51) was re a c t e d to g i v e a new UV and H 2S0 4 p o s i t i v e spot (on TLC), the r e a c t i o n mixture was p u r i f i e d by s i l i c a g e l chromatography p l a t e s u sing CHC1 3 as a developing s o l v e n t . A f t e r development of the p l a t e s and e x t r a c t i o n of the band with CHC1 3, TLC (Rf=0.53 i n CHC1 3) showed the presence of only one spot. IR (CHC1 3, F i g . 49) 3480, 2940, 1720, 1475, 1100, 740 cm" 1. 1H NMR (CDC1 3, F i g . 50) shows methyls f o r the major molecule 0.80 ( s , 3H), 0.84 (s, 6H), and methyls f o r the minor molecule 0.93 (s, 3H), 0.99 (s, 3H), 1.38 (s, 3H). MS (m/e r e l a t i v e i n t e n s i t y , F i g . 48) 388(1), 386(1), 373(3), 371(3), 234(37), 176(80), 105(100). A l l attempts to c r y s t a l l i z e t h i s product f a i l e d . 172 B i b l i o g r a p h y 1- Barnes, R.D. I n v e r t e b r a t e s Zoology. W.B. Saunders Company. 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JACS 1979, 101, 1275. 26- P e t t i t , G.R.; Hera l d , C.L.; A l l e n , M.S.; Van Dr e e l e , R.B.; V a n n e l l , L.D.; Kao, J.P.Y.; Blake, W. JACS 1977, 99, 262. 27- S t a l l a r d M.O.; F e n i c a l W. Tet. 1978, 34, 2077. 28- D i e t e r , R.K.; R i n n e l , R.; Meinwald J . ; E i s n e r , T. Tet. L e t t . 1979, 19,1645. 29- Faulkner, D.J.; S t a l l a r d , M.O. JACS 1973, 95, 3413. 30- Faulkner, D.J.; S t a l l a r d , M.O. Tet. L e t t . 1973, 14, 1171. 31- Faulkner, D.J.; S t a l l a r d , M.O.; I r e l a n d , C. Tet• L e t t . 1974, 40, 3571. 32- Faulkner, D.J.; S t a l l a r d , M.O.; Comp. Biochem. Phy. 1974, 49B, 25-35 and 37-41. 33- I r e l a n d , C ; Faulkner, D.J.; S t a l l a r d , M.O. JOC 1976, 41, 2461. 34- -Schmitz, F . J . ; Vanderah, D.J. JOC 1976, 34, 80. 35- Schmitz, F . J . ; Hollenbeak, K.H.; Vanderah, D.J. JOC 1978, 34, 80. 36- Schmitz, F . J . McDonald, F . J . Tet l e f t . 1974, 29, 2541. 37- Schmitz, F . J . ; Mc Donald, F.H.; Vanderah, D.J. 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Tet. 1965, 21, 1175. 74- Parker, W.; Roberts, J.S. Quart. Rev. Chem. Soc. 1967, 21, 331 ( r e f . W i t h i n ) . 75- van Tamelen E.E.; S t o r n i , A.; H e s s l e r , E . J . ; Schwartz, M. JACS 1963, 85, 3295. 176 76- Andersen, N.H.; Ohta, Y.; S y r d a l , D.D. B i o o r g a n i c Chemistry 1978, 2, 1. 77- Appel, H.H.; Brooks, C.J.W. JCS 1959, 3322. 78- Scheuer, P.J. Chemistry of Marine N a t u r a l Products Academic Press, N.Y., 1975. 79- Cimino, G.; De Stefano, S.; Minale, L.; T r i v e l l o n e , E. T e t . 1972, 28, 4761. APPENDIX 1 PHYLOGENETIC TREE (Ref. 78) The phylogenetic tree. 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 9 10 It 12 14 3000 2000 1B0O 1400 1400 1200 1000 6 0 0 APPENDIX 3 IR SPECTRUM OF ALBICANOL OBTAINED' FROM DR. N.H. ANDERSEN APPENDIX 4 H NMR SPECTRUM OF FURODYSININ OBTAINED FROM DR. WELLS **vt NUMULH CU ' APPENDIX 7 IR SPECTRUM OF FURODYSIN OBTAINED FROM DR. WELLS co 

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