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Novel metabolites from British Columbia nudibranchs Gustafson, Kirk 1984

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NOVEL BRITISH  M E T A B O L I T E S FROM COLUMBIA  NUDIBRANCHS  BY KIRK  GUSTAFSON  B.Sc,  The U n i v e r s i t y  THESIS  SUBMITTED IN PARTIAL  THE.  REQUIREMENTS  o f Delaware,  FOR  DOCTOR OF  1979  FULFILLMENT  T H E DEGREE  OF  PHILOSOPHY  in  THE  F A C U L T Y OF GRADUATE (Department  We  accept to  THE  this  of Chemistry)  thesis  the required  UNIVERSITY  OF  as  conforming  standard  BRITISH  September ©Kirk  STUDIES  COLUMBIA  1984  Gustafson,  1984  In  presenting  this  requirements of  B r i t i s h  i t  freely  agree for  for  an  available  that  I  by  understood  that  financial  his  or  reference  and  study.  I  extensive be  her or  shall  The U n i v e r s i t y o f B r i t i s h 1956 M a i n M a l l Vancouver, Canada V6T 1Y3  Date  (3/81)  ^a^3^  B  \  University shall  copying  granted  by  the  of  publication  not  be  allowed  ^  Columbia  of  this It  this  without  make  further  head  representatives.  C\r>i» rv\vS"V r  of  the  the  Library  permission.  Department  at  of  the  may  copying  gain  degree  fulfilment  that  for  purposes  or  p a r t i a l  agree  for  permission  scholarly  in  advanced  Columbia,  department for  thesis  thesis  of  my  is thesis my  written  ABSTRACT  Skin  extracts  nudibranchs  have  metabolites. of  The  from  structure  by  provided  glyceride  of  free  (1_5) .  the  of  novel  structural and  biosynthetic  3/7,  its  two  acid  These  and  includes  origin  compounds  diterpenoic was  farnesic  of  and  contain  2fJ  were  found  also acid  in  jL_5  1J3,  its  monoacetate  and by  the  the a  drimane  only  one  tentatively glyceride  The  2_7  21,  glyceryl  combination  interconversion  analysis.  of acid  and  2_2  was  Extracts  diterpenoic  established  chemical  and  nobilis  aldehyde  data.  and  Its  sesquiterpene  of  _18  been  methods  Anisodoris  the  glyceride  has  tricolor.  chemical  spectral  diffraction acid  Polycera  structure  glyceride acid  (7_5)  degraded  monoacetates  analysis,  montereyensis,  triophamine  synthesis.  The  mohtereyensis  c r y s t a l X-ray  isomeric  Columbia  source  compounds  classical  interpretation  spectral  single a  23^.  by  unambiguous  monocyclof arnesic  ether  their  odoriferous  sesquiterpenoic the  of  catalinae  deduced  the  Archidoris  rich  these  diacylguanidine  an  by  a  British  describes  of  studies  dihydroapof arnesal assigned  thesis  Triopha  was  confirmed  be  of  role.  unique  isolated  number  to  several  preliminary  physiological  a  proven This  investigation some  from  and/or  structure  of  c o l l e c t i o n of  A.  identified. in  addition  The to  iii glycerides  1Q_, 2 0 ,  isolated  from  glyceride  20. ^  activity labeled  the  is  Cadlina  n  glycerides  Preliminary  of  in  ether  Biosynthetic  acid  acid  ether  odhneri.  glyceryl  e  fish.  mevalonic  synthesized  skeleton  t  against  farnesic  odhneri.  Archidoris  an<  p o r t i o n of  22_ and  is  portion results  albicanyl  luteomarginata.  by  The  of  studies  T.  acetate  drimenoic  acid  antifeedant  showed t h a t the  that  is  24  by  and  C  into  Archidoris  triophamine and  l t +  terpenoid  montereyensis  catalinae (3_6)  been  into  glyceride  suggest  also  23^ e x h i b i t  incorporated  1_7 and 20_ by A .  situ  22_ h a v e  the  (75)  drimane  biosynthesized  by  iv TABLE OF  CONTENTS  Abstract  i i  Table o f Contents  iv  List  of Figures  vi  List  o f Schemes  viii  of Tables  viii  Lists List  of Plates  viii  Acknowledgments  ix  Abbreviations  x  INTRODUCTION I.  Nudibranch  Defenses  1  II.  Chemistry o f Other Opisthobranchs  5  III.  Nudibranch  9  Chemistry  CHEMICAL STUDIES OF TRIOPHA CATALINAE I.  Isolation  and S t r u c t u r a l  Triophamine  Elucidation of  (7^5)  II.  Biological  III.  S y n t h e s i s o f (± ) - T r i o p h a m i n e  35  Considerations  53 (75a)  55  CHEMICAL STUDIES OF ANISODORIS NOBILIS I.  D i h y d r o a p o f a r n e s a l (1_5)  65  CHEMICAL  STUDIES  OF A R C H I D O R I S  I.  Archidoris  montereyensis  II.  Archidoris  odhneri  III.  Antifeedant  IV.  Biosynthesis  V.  Additional  MONTEREYENSIS  116 118  Archidoris  Biosynthetic  Metabolites  Studies  SUMMARY AND D I S C U S S I O N EXPERIMENTAL BIBLIOGRAPHY  ODHNERI 72  Bioassay of  AND A .  120 123  124 i  132 152  vi LIST  OF F I G U R E S  1.  Phylogenetic  classification  2.  Typical  3.  Acylimine  4.  Interpretation  o f t h e HRMS o f 75.  4  5.  4 0 0 MHz  spectrum  46  6.  1 0 0 MHz  7.  1 0 0 MHz SFORD  8.  IR s p e c t r u m  9.  4 0 0 MHz  'H NMR  difference  10.  2 7 0 MHz  'H NMR  spectrum  o f 78  51  11.  4 0 0 MHz  'H NMR  spectrum  o f 83  52  12.  Comparison  dorid  nudibranch  tautomers  'H NMR 1  3  C NMR  triophamine  o f nudibranchs  1  3  '  o f triophamine  spectrum C NMR  2 4  (7j>)  42  o f 75 o f 75  spectrum  5  47 o f 75  48  o f 75  49  o f 4 0 0 MHz  NOE s p e c t r u m  'H NMR  resonances  (7j5) a n d s y n t h e t i c  o f 75  50  of natural  diastereomers  75b .  63  13.  Interpretation  o f t h e HRMS o f 15  69  14.  4 0 0 MHz  spectrum  70  15.  Computer of  *H NMR  generated  o f 15  stereochemical  representation  r7  76  16.  Interpretation  o f t h e HRMS o f 17  77  17.  4 0 0 MHz  spectrum  78  18.  1 0 0 MHz  19.  1 0 0 MHz SFORD  20.  IR s p e c t r u m  21.  4 0 0 MHz  22.  1 0 0 MHz  'H NMR 1  3  C NMR 1  3  3  C NMR  o f 17  spectrum  o f _17  'H NMR 1  spectrum  o f 17  C NMR  spectrum spectrum  79 o f 17  80 81  o f 97 o f 9_7  82 83  vii  23.  IR s p e c t r u m  o f 97  84  24.  400 MHz 'H NMR s p e c t r u m o f 1J3  87  25.  IR s p e c t r u m  88  26.  400 MHz  27.  IR s p e c t r u m  28.  Interpretation  29.  400 MHz H NMR s p e c t r u m o f 20  95  30.  100 MHz  96  31.  IR s p e c t r u m  32.  400 MHz 'H NMR d i f f e r e n c e NOE s p e c t r u m  33.  270 MHz 'H NMR s p e c t r u m o f 98  99  34.  400 MHz 'H NMR s p e c t r u m o f 21  100  35.  IR s p e c t r u m o f _21  101  36.  Interpretation  104  37.  400 MHz 'H NMR s p e c t r u m  38.  IR s p e c t r u m o f 22_  106  39.  Possible  mass s p e c t r a l f r a g m e n t a t i o n o f 100  108  40.  Possible  mass s p e c t r a l f r a g m e n t a t i o n o f 101  109  41.  400 MHz 'H NMR s p e c t r u m o f t h e d i t e r p e n o i c  o f 18  'H NMR s p e c t r u m o f 19  89  o f 19  90  o f t h e HRMS o f 20  94  1  acid(s)  1 3  C NMR s p e c t r u m o f 20  97 o f 20  o f t h e HRMS o f 22  100 a n d / o r  o f .22  101  42.  IR s p e c t r u m  43.  270 MHz 'H NMR s p e c t r u m  44.  IR s p e c t r u m o f _23 .  45.  270 MHz 'H NMR s p e c t r u m  46.  C, - h y d r o c a r b o n p o r t i o n n  o f 20  98  105  I l l  o f 100 a n d / o r  101  o f 23_  112 114 115  o f 21_ o f t r i o p h a m i n e (7_5)  117 125  viii  LIST  1.  Synthetic of  2.  sequence  the isomeric  SCHEMES  for production  acids  94  and 83a  Final  steps  i n the synthesis  (75a)  and t h e d i a s t e r e o m e r s 75b  LIST  1.  OF  Nudibranch  2.  'H a n d  3.  'H NMR  4.  Fish  5.  Results  1 3  C  OF  of  56 (±)-triophamine 59  TABLES  Metabolites NMR  Data  10  f o r Triophamine  Decoupling Studies  Antifeedant of  1 4  C  (25)  of Triophamine  38 (7j>) . . . .  Results  39 119  Mevalonic Acid  Incorporation  Experiments  122  LIST  OF  1.  Triopha  catalinae  2.  Anisodoris  3.  A r c h i d o r i s montereyensis  nobilis  PLATES  34 64 71  ix ACKNOWLEGEMENTS  I with  am  indebted  assistance  work.  t o numerous  and e n c o u r a g e m e n t  In p a r t i c u l a r ,  Andersen  people  I would  who h a v e  during the course  like  t o thank  f o r i n t r o d u c i n g me t o t h e f i e l d  chemistry.  His unfailing  provided  has  of this  D r . Raymond  of natural  enthusiasm  me  products  indeed  been  inspirational. The Steven  Ayer  extended the I  reliable is  and f r i e n d l y greatly  support  o f Mike  appreciated.  Warm  L e B l a n c and thanks  are  t o a l l o f t h e d i v e r s who a i d e d i n t h e c o l l e c t i o n o f  marine  organisms  am g r a t e f u l  that  t o Rosemary  were  essential  Armstrong,  who k i n d l y  sample o f a u t h e n t i c d r i m e n o l .  The p a t i e n t  staff  and MS  o f the departmental  acknowledged.  NMR  to this  research. supplied  a  and u n d e r s t a n d i n g  laboratories  are also  X LIST  OF A B B R E V I A T I O N S  DCC  =  Dicyclohexylcarbodiimide  DIBAL  =  Diisobutyaluminum  DMSO  =  Dimethyl  DNPH  =  E  =  Electronic  EtOH  =  Ethanol  GC  =  Gas c h r o m a t o g r a p h y  HPLC  =  High  pressure  HRMS  =  High  resolution  IR  =  LC  =  Liquid  MeOH  =  Methanol  NEN  =  New E n g l a n d  'H NMR  =  Proton  =  Carbon-13  NOE  =  Nuclear  RT  =  Room  S  =  Solvent  SFORD  =  Single  THF  =  Tetrahydrofuran  TLC  =  Thin-layer  UV  =  W  =  1 3  C  NMR  hydride  sulfoxide  2,4-Dinitrophenylhydrazine noise  liquid mass  chromatography spectroscopy  Infrared chromatography  Nuclear  nuclear  nuclear  resonance  magnetic  Overhauser  resonance  enhancement  temperature signal frequency  off-resonance  chromatography  Ultraviolet Water  magnetic  signal  decoupled  1. INTRODUCTION  I.  NUDIBRANCH  DEFENSES  M o l l u s c s Opisthobranchia n a t u r a l  of  t h e  have  been  p r o d u c t  opisthobranchs include are  sea  shell-less,  shells have  that  these  and  by  the  complete they  sponges,  preferred  from  colors  presence  the  of  of  animals,  fishes,  are  that  size  prey  a  seem  on  orders  are  and,  from  and  as  in  their  the  name  appendages. mm  and  their  c o e l e n t e r a t e s schemes  species  to  members  crustaceans, nudibranchs ,  that  very  blatantly  3  that  characterized  3-300  color  to  relative  defense.  respiratory  Selected  opisthobranchs, to  shell  colored,  This  seven  They  bryozoans  organism.  known  a  they  s p e c u l a t i o n  of  1).  reduced  though  brightly  to  largest  of  Even  chemical  camouflaged  substrate  conspicuous  of  in  greatly  1  form  synonyms  opisthobranchs  predators .  external  range  or  often  led  of  most  by  A d u l t  whose  protection.  (Figure  possess  ranges  While  scrutiny  y e a r s .  animals  has  s u b c l a s s  intense  r e c e n t  are  the  absence  include  Coloration the  some  comprise  nudibranchs  d i e t s  known  Opisthobranchia  implies, Adult  few  of  internal  l i t t l e and  u t i l i z e  Nudibranchs subclass  have  predation  opisthobranchs  i n  tissue  have  from  subject  snail.  forms very  G a s t r o p o d a ,  moving  naked  some  soft  animals  impunity  slow  provide  exposed  the  c h e m i s t s  are  slug  c l a s s  bright,  four  the  the  groups  asteroids but  .  mimic  advertise of  2  level  and of  MOLLUSCA  PHYLUM  GASTROPODA  CLASS  OPISTHOBRANCHIA  1  BULLOMORPHA  1  APLYSIAMORPHA  SUBCLASS  1  1  PLEUOBRANCHOMORPHA  PTEROPODA ORDER  SACOGLASSA  AEOLIDACEA  N.B.  NUDIBRANCHIA  ARMINACEA  Organisms c l a s s i f i e d a c c o r d i n g to  Figure  1.  Phylogenetic  PYRAMIDELLA  DENDRONOTACEA  Behrens .  DORIDACEA  SUBORDER  2  c l a s s i f i c a t i o n of nudibranchs  to  3. predation form  is  and  very  limited  particularly case.  low.  Therefore,  mobility  might  vulnerable to  It  is  sophisticated  believed  suggest  attack, that  defensive  while that  this  they  their  soft  nudibranchs  is  clearly  employ  strategies  to  in  with  body  a  are  not  the  variety  of  deter  potential  predators. Cryptic habitat  coloration  selection  nudibranchs. acquired they  Several  from  produce  their  were where  1  Such  not  color  cryptic  prey *, to  but  directly  while  others  match  the  offered  nudibranchs  to  no  fish  use  found when  rely  pigments  placed  of  their  a  by  nudibranchs in  Once  on  which  harassment that  concealment.  apparently  certain pigments  scheme  reduce  1  of  utilize  color  may  Thompson  acceptable  complementary  conspicuousness  coloration  predators,  still  the  species  themselves  surroundings. potential  reduces  concert  a  setting  discovered,  second  level  of  defense. The or  dorsum  exposed the  covering of  and  tissue  i t  while  sensory the  usually i t s  are  the  and  the  plume found  plume  near  substrate  nudibranch  is a anus.  is  disturbed,  i s termed  bulk  Other  branchial  branchial  nudibranchs  2).  apparatus  located  a  comprises  (Figure  rhinophores,  paired  to  outer  of  external and  foot.  around  a  involved most  foot in  mantle  organism's  features  include  Rhinophores  nudibranch's  respiratory The  the  the  structure  anchors  are  head, (gill)  the  animal  locomotion.  When  species  retract  these  external be  structures  attackers.  leaving  The  presence  dorid  (suborder  Doridacea)  as  p o s s i b l e  deterent  a  calcareous the is  spines  relative believed  be  quite  the  of  dorsum  spicules  nudibranchs to  acquired  importance to  only  of  dietary  spicules  in  has  p r e d a t i o n  from  exposed  in  5  the  would  mantle  been  .  to  of  postulated  Spicules  are  sponges.  However,  thwarting  predators  low . 3  Branchial Plume  Figure  2.  Typical  Nematocysts, coelenterates, c e r t a i n  dorid which  provide  a e o l i d , nematocysts  through  the  a  These  digestive  functional storage  nudibranchs  a  stinging  deterent  to  from  gland  the  are  surface.  in  organelles potential  Aeolidacea),  obtained  state  cells  skin  are  (suborder  Unfired  in  nudibranch  of  dietary  localized Thus  in  predators  organisms and  are  cynidocyst  projections  situated,  by for  n u d i b r a n c h s  nudibranch  specialized  produced  they  are  1  .  travel stored c e l l s  from  3  .  the  readily  5. encountered  during  predator. release  When  the  utilize  from  of  arsenal.  abundance  of  speculated  nature has  protection  = 1)  of  the  illustrates sequester organelle  part to  along of  species,  possible  an are can  their strong  the  The  7  by  their  nudibranchs  i s non-acidic .  interest  of  functions  solutions  some  number  possess  the release  s e c r e t i o n s and t h e i r much  defensive  a large  whose  Several  f o r  nudibranchs  that  a r e known  While  6  cells  phylum.  glands  pH  2  molested .  o f most  prompted  skin  a  cell  s e c r e t i o n s as  species  (H SO^,  as  intact  apparent  1  when  of these  an  defensive .  acidic  provides  secretion  Most  be  highly  surface  acid  defense  chemical  non-mucous  to  inquisitive  to selectively  of a different  employ  an  by nudibranchs  o f an animal  h a s become i n c r e a s i n g l y  defensive  skin  nematocysts  utilization  animal  by  i s disturbed, the cynidocyst  f o r i t s own  nudibranchs  secrete  inspection  stinging  ability  a dietary It  of  Nematocyst  remarkable  and  an a e o l i d  clouds  response.  the i n i t i a l  skin  chemical  defensive  natural  role  product  chemists.  II.  CHEMISTRY P r i o r  to  nudibranchs,  Rather,  the  OTHER  OPISTHOBRANCHS  discussing  an  opisthobranchs opisthobranch  OF  i s  overview in  chemistry highlights  the of  order.  the An  i s beyond and  chemical  chemistry exhaustive  t h e scope  general  defense  trends  of  other  review  of this are  of  of  thesis.  presented  6. along  with  findings  that  have  been  mirrored  in  nudibranch  work. Initial species  opisthobranch  because  hundred  mm)  they  and  compounds.  one  or  Many  compounds the  species'  and  .  from  highest of  extracts  that  the  were  differences products  b e e n  f o u n d  i n  a  '  1  0  '  c  demonstrated  digestive  the  alga  diet  are  products aplysin  1  2  .  that  its  In  these  A.  gland  laurinterol and  in  the  the  1  novel  include and  0  C-15  contain  o r  californica halogenated var.  (JL) a n d  pacifidiene  due  in  composition  of  to  is  and  a _ l .  8  of  of  dietary  have  also  s p e c i e s  o f  c l e a r l y  b  concentrates monoterpenes  and  stores  obtained  in  from  pacificum. concentrated  following ingestion. pacifenol (4)  time  geographical  Aplysia  m o r e  algae.  found  location  e_t  Aplysia  primarily  The  from  metabolites  modified  that  availability  isolated  one  instances,  indicates  collected,  coccineum  chemically  (3)  of  spp.  concentration  with  S t a l l a r d  Plocamium some  several  constituents  herbivors.  vary  Numerous  0  Aplysia  d i e t a r y sources,  these  algae.  1  length  diterpenes  metabolite  can  animals  seasonal  a 1 g a e  of  Aplysia  atoms.  d i g e s t i v e gland  year  from  c i r c u m s t a n t i a l evidence  Invariably  a  1 1  on  concentrations  9  more h a l o g e n  accumulates  the  high  isolated  focused  (average  sesquiterpenes ,  derivatives  Strong  large  contain  Products 8  acetate  are  often  monoterpenes ,  studies  (2)  are  respectively,  from The  the algal  converted within  to the  7. digestive  gland  of  of  the  known  of  Brasilenyne  (5_)  and  found  in  the  brasiliana,  Aplysia  a c t i v i t y  principles  both  c a l i f o r n i c a  1  .  3  Br 1  b i o l o g i c a l  active  A.  Br  2 Many  of  have  the  9  a  '  9  compounds b  |  1 1 1  animals'  possess  and  wall  defensive  and  demonstrated  type  a p p a r e n t l y  cis-dihydrorhodophytin body  some  secretions. which  (6) , 11  digestive  antifeedant  are  gland  activity  are  of  A.  toward  fish. Soft-bodied employ The  marine  chemical  defensive  opisthobranch  exudate  containing  polypropionate  molluscs  implicated  as  siphonarid  Siphonaria  to  than  Aplysia  thwart  produces  a  sesquiterpene  onchidal  (2)  deterrent  pectinatone in  pectinata.  a  mucous  (8_)  1 6  also  predators.  binneyi  derivative, the  substances  Onchidella the  other  repugnant arid  15  ,  secretion  has .from  a  been the  8.  The  ability  dietary  organisms  Several  acquire  is  opisthobranchs  assimilate algal  to  not of  Ireland  et  metabolites  such  Placobranchus  ocellatus,  mollusc-chloroplast that  an  unique the  photosynthetically  diet.  as  pair.  opisthobranch  biosynthesis  of  secondary  functional  a l .  order active 1  7  to  organelles  aeolid  nudibranchs.  Sacoglossa  are  chloroplasts  showed  that  biosynthesized  This  was  actively  the  first  to  their  polypropionate (.9)/ situ  from by  the  demonstration  participates  metabolites.  known  from  9,1O-deoxytridachione are  from  in  the  9. It many  is  that  sophisticated  variety resist  of  from  the  chemically  systems  of  of  although  transformed  several  species  of  metabolites  are  via  a  these some  they  Using  of  is  often  constituents  i n g e s t i o n . situ  in  a  successfully  dietary  capable  developed  compounds  f o l l o w i n g  symbiotic  have  defense.  chemicals  source  diet,  molluscs  chemical  noxious  The  addition, unique  opisthobranch  potentially  predation.  directly are  clear  association  In  production with  algal  chloroplasts.  I l l .  NUDIBRANCH  CHEMISTRY  Nudibranchs investigations a_l.  and  2 5  1982.  in  Schulte  To  over  have  date,  f o r t y  an  functionality  and  source  and  the  be  are  largest  recurring provided  seen  nudibranchs.  of  our  numerous  have  d i v e r s i t y type.  that in  the  I.  majority  of  class  features. insight  into  the  a l l their  nudibranch  exhibit  In  of  about  Sesquiterpenes  and  from  s i z e ,  compilation  Table  a  addition, chemical  to  compounds  m o l e c u l a r a  et  up  reported  comments  nature.  work  These  For  chemical  Thompson  the  been  of  with see  by  summarize  nudibranchs.  activity  structural  of  Reviews  products  of  terpenoid  focus  nicely  9  metabolites  structural much  2  structural  biological  can  a_l.  amazing  nudibranch  chemicals  et  the years.  natural  known  It  recent  species  encompass  been  skin  comprise  number they ecology  of have of  TABLE I:  NUDIBRANCH METABOLITES  SPECIES Acanthodoris nanaimoensis (Ref. 18)  METABOLITES  COMMENTS These i s o m e r i c aldehydes r e p r e s e n t three new carbon s k e l e t o n s . A b i o g e n e t i c p r o p o s a l suggests t h a t acanthodoral (11) i s the p r e cursor of nanaimoal (10) and i s o a c a n t h o d o r a l  ^-CHO  cPC  \  02).  nanaimoal  CHO  acanthodoral  ^CHO  <J2)  Adalaria sp. (Ref. 19)  isoacanthodoral (12)  Compound (13) i s the major component of a mixture of s t e r o i d a l p e r o x i d e s .  Jl  TABLE I:  continued  SPECIES  METABOLITES  COMMENTS  Aldisa sanguinea cooperi (Ref. 20, 21)  In a d d i t i o n t o s t e r o i d a l ketone J^4, i t s analog with an a8-unsaturated a c i d was a l s o p r e s e n t . A c i d _1_4, the major s t e r o i d , has a n t i f e e d a n t p r o p e r t i e s toward g o l d f i s h . I t i s suggested t h a t cholestenone o b t a i n e d from the sponge A n t h r o a r c u a t a graceae may be converted by the nudibranch t o f i s h a n t i f e e d a n t J4^. G l y c e r y l ether 23>, r e p o r t e d f o r A r c h i d o r i s montereyensis i s a l s o found i n A. sanguinea c o o p e r i .  Anisodoris nobilis (Ref. 2 1 , 22)  The degraded s e s q u i t e r p e n e aldehyde dihydroapof a r n e s a l (JJ^) i s the p r i n c i p l e o d o r i f e r o u s c o n s t i t u e n t of the nudibranch. Doridosine (^6) which was i s o l a t e d from the nudibranch's d i g e s t i v e gland, i s water soluble and e x h i b i t s a v a r i e t y o f pharmacological a c t i v i t i e s . I t has a l s o been i s o l a t e d from the A u s t r a l i a n sponge Tedania d i g i t a t a .  d i h y d r o a p o f a r n e s a l (15) NH  iV>  cHr  H H0CH  2  ft  0 0 H  H  d o r i d o s i n e (16)  TABLE I:  continued  SPECIES  METABOLITES  Archidoris montereyensis (Ref. 21, 23)  OR 2  ( ^ r ^  /\  COMMENTS  V 7 R  H  J 8  R  J9  R  = H R  1  x  = Ac  x  = H  OH  [  I  J)  20  R = H  21  R =  AC  OH  22  OH 23  = H  2  R R  2  2  = H = Ac  Compound 1 7 and i t s 1 ° and 2° monoacetates are the major m e t a b o l i t e s . G l y c e r i d e 2£ and g l y c e r y l ether 23 a c t as f e e d i n g d e t e r e n t s to the t i d e pool s c u l p i n O l i g o c o t t u s maculosus while 1 7 i s i n a c t i v e . Compound 23 has s t r o n g a n t i b i o t i c a c t i v i t y and i s a l s o found i n the d i e t a r y sponge H a l i c h o n d r i a panicea. C - l a b e l e d mevalonic a c i d f e d t o A. montereyensis i s i n c o r p o r a t e d i n t o the t e r p e n o i d p o r t i o n s o f compounds V7 and 20. Thus the nudibranch seems t o a c q u i r e g l y c e r o l d e r i v a t i v e s from both d i e t a r y sources and de novo b i o s y n t h e s i s . Compounds 1 7 , 1 8 , 20, 22 and 23^ have a l s o been i s o l a t e d from the nudibranch A r c h i d o r i s odhneri while compound 23 has been found i n A l d i s a sanguinea c o o p e r i . ll+  TABLE I:  continued  COMMENTS  METABOLITES  SPECIES Archidoris odhneri (Ref. 24)  0  0R  24  R  x  = H  25 . R  1  = Ac  26  1  = H  R  R  R R  = H  2  2  2  = H = Ac  o  27  H  0  HO  2  F a r n e s i c a c i d g l y c e r i d e 2A and i t s 1° and 2° monoacetates are the p r i n c i p l e m e t a b o l i t e s . The i s o m e r i c g l y c e r i d e 27 i s found at a much lower abundance. Compounds \T_, ^8, 2:0, 22^ and 23, r e p o r t e d f o r A. montereyensis, are a l s o present i n t r a c e amounts. labeled mevalonic a c i d f e d t o the nudibranch i s i n c o r p o r a t e d i n t o the f a r n e s i c a c i d p o r t i o n of 24. G l y c e r i d e 2A shows a n t i b i o t i c a c t i v i t y a g a i n s t gram p o s i t i v e b a c t e r i a but no a n t i f e e d a n t a c t i v i t y towards f i s h .  TABLE I:  continued  SPECIES  METABOLITES  COMMENTS  Cadlina luteomarginata (Ref. 25, 26, 27) d e n d r o l a s i n (28)  pleraplysillin-1  (29)  3_0  microcionin-2 (31)  The compounds present i n C. luteomarginata e x t r a c t s show wide v a r i a b i l i t y depending on the l o c a t i o n and time o f year o f c o l l e c t i o n . Many of these m e t a b o l i t e s can be t r a c e d t o d i e t a r y sponge sources. F u r o d y s i n i n (33), p a l l e s c e n s i n - A (34) and p o s s i b l y the other furanosesquiterpenes are o b t a i n e d from Dysidea amblia, i d i a d i o n e (37) comes from L e i o s e l l a i d i a and compounds 39 and 40 have been t r a c e d t o A x i n e l l a sp. A l b i c a n o l (35) i s known from the l i v e r w o r t Diplophyllum a b l i c a n s and i s p r e s e n t i n the nudibranch p r i m a r i l y as a l b i c a n y l a c e t a t e (_36) • The source o f the degraded t e r p e n o i d luteone (38) i s not c l e a r . While s e v e r a l of these m e t a b o l i t e s have been d e t e c t e d i n the gut contents of C. luteomarginata, they are a l l s t o r e d i n the dorsum of the nudibranch. F u r o d y s i n i n , p a l l e s c e n s i n - A , i d i a d i o n e and compounds 39 and 40^ are t o x i c t o g o l d f i s h . They a l s o show v a r y i n g degrees of a n t i f e e d a n t a c t i v i t y toward g o l d f i s h and the sculpin Clinocottus analis. Albicanyl acetate i s a l s o an a n t i f e e d a n t toward g o l d fish. The nudibranch H y p s e l o d o r i s g h i s e l i n i i s known t o c o n t a i n d e n d r o l a s i n while H. p o r t e r a e c o n t a i n s f u r o d y s i n i n and H. c a l i f o r n i e n s i s contains dendrolasin, f u r o d y s i n i n and p a l l e s c e n s i n - A .  TABLE I:  continued  SPECIES  METABOLITES  COMMENTS  Cadlina luteomarginata (continued)  f u r o d y s i n (32)  f u r o d y s i n i n (33)  p a l l e s c e n s i n - A (34)  35  R = H  36  R = Ac  •  TABLE I:  continued  SPECIES  METABOLITES  Cadlina luteomarginata (continued)  0  0 i d i a d i o n e (37)  OHC  COMMENTS  jT X  A H luteone (38)  39  40  TABLE I:  continued  COMMENTS A t o t a l of f o u r a c e t y l a t e d d e r i v a t i v e s of 4 1 and 4 2 were a l s o r e p o r t e d . These f u r a n o d i t e r p e n e s are i d e n t i c a l t o , or are s t r u c t u r a l l y r e l a t e d t o , compounds p r e v i o u s l y i s o l a t e d from an A u s t r a l i a n Spongia sp.  Pu'ulenal (43) i s r e a d i l y h y d r o l y z e d to p o l y g o d i a l (55) which i s known to deter p r e d a t i o n by f i s h .  TABLE I:  continued  METABOLITES  SPECIES Chromodoris maridadilus (Ref. 30)  nakafuran-8 (44)  nakafuran-9 (45)  Chromodoris marislae (Ref. 31) m a r i s l i n (46)  aR=  ^0  b  R  COMMENTS Nakafuran-8 (44) and nakafuran-9 (45) were i s o l a t e d from the nudibranchs C. m a r i d a d i l u s and Hypselodoris godeffroyana and t r a c e d to the d i e t a r y sponge Dysidea f r a g i l i s . Both compounds possess a n t i f e e d a n t p r o p e r t i e s a g a i n s t the common r e e f f i s h e s Chaetodon spp. The nudibranch H y p s e l o d o r i s g h i s e l i n i a l s o c o n t a i n s nakafuran-9 and a r e l a t e d methoxy butenolide 67 while H y p s e l o d o r i s c a l i f o r n i e n s i s c o n t a i n s nakafuran-8.  M a r i s l i n (4f5) i s the p r i n c i p a l c o n s t i t u e n t and i t r e a d i l y rearranges to the sponge metabolite p l e r a p l y s i l l i n - 2 ( f o r m a l l y a [3,3] sigmatrophic rearrangement). This close s t r u c t u r a l r e l a t i o n s h i p i n d i c a t e s t h a t there may be chemical a l t e r a t i o n by the nudibranch of a m e t a b o l i t e t h a t i t o b t a i n s from i t s diet.  TABLE I:  continued  COMMENTS The rearranged d i t e r p e n e n o r r i s o l i d e (49) was a l s o found i n the south P a c i f i c sponge D e n d r i l l a sp. A b i o s y n t h e t i c p r o p o s a l f o r the formation of the n o r r i s a n e s k e l e t o n from the spongian s k e l e t o n was p r e s e n t e d .  A t o t a l of f i v e s c a l a r i n type tetracyclic terpenes were i s o l a t e d . Sednolxde (50) i n h i b i t s the growth of the marine bacterium V i b r i o anguillarum. An A u s t r a l i a n d o r i d nudibranch, Chromodoris s p l e n d i d a was a l s o found to c o n t a i n a mixture of 24methylscalarins. S i m i l a r Cgg s c a l a r i n d e r i v a t i v e s have been i s o l a t e d from sponges of the genus P h y l l o s p o n g i a .  TABLE I:  continued  SPECIES Chromodoris splendida (Ref. 33)  COMMENTS  METABOLITES see Chromodoris  sedna  OH  Coryphella lineata (Ref. 34, 35)  A number of p o l y h y d r o x y l a t e d s t e r i o d s were i s o l a t e d from C. l i n e a t a and from the h y d r o i d Eudendrium sp. upon which they prey. The major s t e r o i d i s compound J53. A similar mixture of p o l y h y d r o x y l a t e d s t e r o i d s was a l s o found i n the nudibranchs F l a b e l l i n a a f f i n i s and Hervia p e r e g r i n a .  Dendrodoris grandiflora (Ref. 35)  Fasciculatin i s o l a t e d from grandiflora. of the sponge  Dendrodoris krebsii (Ref. 36)  The nudibranchs D. k r e b s i i , I), n i g r a and D. t u b e r c u l o s a were found to c o n t a i n p o l y g o d i a l (55) and olepupuane (56). Polygodial i s a known f i s h and i n s e c t a n t i f e e d a n t while olepupuane was found to i n h i b i t f e e d i n g of the P a c i f i c damsel f i s h D a s c y l l u s aruanus. P o l y g o d i a l has a l s o been i s o l a t e d from the nudibranch Dendrodoris limbata while olepupuane has been found i n D o r i o p s i l l a a l b o p u n c t a t a and D o r i o p s i l l a j a n a i n a .  polygodial  (55)  olepupuane  (56)  (54) i s a f u r a n o s e s t e r t e r p e n e the d i g e s t i v e gland of D. I t i s known to be a m e t a b o l i t e Ircinia fasciculata.  TABLE I:  continued  SPECIES  METABOLITES  Dendrodoris limbata (Ref. 37, 38)  0  II  R-CH-CO  /\ H 57.  Dendrodoris nigra (Ref. 36)  Dendrodoris tuberculosa (Ref. 36)  see Dendrodoris  see Dendrodoris  krebsii  krebsii  COMMENTS A sesquiterpene with the drimane s k e l e t o n was found e s t e r i f i e d to a s e r i e s of f a t t y a c i d s with v a r y i n g degrees of u n s a t u r a t i o n . These e s t e r s were l o c a l i z e d i n the d i g e s t i v e gland while p o l y g o d i a l (55) was found i n e x t r a c t s of the mantle. P o l y g o d i a l i s a potent a n t i f e e d a n t a g a i n s t f i s h but the sesquiterpene e s t e r s a r e i n a c t i v e . l ^ C - l a b e l e d mevalonic a c i d f e d t o D. limbata i s i n c o r p o r a t e d i n t o p o l y g o d i a l and the sesquiterpene p o r t i o n of the e s t e r s . I t i s suggested t h a t the s e s q u i t e r p e n e e s t e r s a r i s e from the d e t o x i f i c a t i o n of p o l y g o d i a l . S i m i l a r sesquiterpene e s t e r s have a l s o been i s o l a t e d from the nudibranchs D o r i o p s i l l a albopunctata and D o r i o p s i l l a j a n a i n a .  TABLE I:  continued  METABOLITES  SPECIES Diaulula sandiegensis (Ref. 39, 40)  NH  2  H H0CH  isoguanosine (58) 2  ft  0 0 H H  COMMENTS Isoguanosine (58) was i s o l a t e d from a water e x t r a c t of the nudibranch's d i g e s t i v e g l a n d . I t i s a purine r i b o s i d e and e x h i b i t s a v a r i e t y of p h a r m a c o l o g i c a l a c t i v i t i e s . A s e r i e s of nine s t r a i g h t chain c h l o r i n a t e d a c e t y l e n e s of g e n e r a l s t r u c t u r e 59 were a l s o i s o l a t e d from s k i n e x t r a c t s . Compound 59 shows a n t i m i c r o b i a l a c t i v i t y a g a i n s t s e v e r a l b a c t e r i a and a y e a s t .  59 Doriopsilla albopunctata (Ref. 36)  see Dendrodoris limbata Dendrodoris k r e b s i i  Doriopsilla janaina (Ref. 36)  see Dendrodoris limbata Dendrodoris k r e b s i i  Flabellina affinia (Ref. 34, 35)  see Coryphella  Glossodoris gracilis (Ref. 35)  lineata  L o n g i f o l i n (60) i s a known metabolite from the sponge P l e r a p l y s i l l a s p i n i f e r a . I t i s a d e t e r e n t to f e e d i n g by the marine f i s h Chromis chromis and has a l s o been i s o l a t e d from the mantle and d i g e s t i v e gland of the nudibranch G l o s s o d o r i s valenciennesi.  TABLE I:  continued  SPECIES  METABOLITES  AcO  Glossodoris tricolor (Ref. 41)  AH  0-A  COMMENTS F u r o s c a l a r o l (61) and d e o x o s c a l a r i n (62) were a l s o i s o l a t e d from the d i e t a r y sponge Cacospongia m o l l i o r . Both compounds show a n t i f e e d a n t p r o p e r t i e s toward the marine f i s h Chromis chromis.  f u r o s c a l a r o l (61)  HO Aco  "y-o  TJ AH Glossodoris valenciennesi (Ref. 41)  see G l o s s o d o r i s  Hervia peregrina (Ref. 34, 35)  see C o r y p h e l l a  d e o x o s c a l a r i n (62) gracilis  lineata  Hypselodoris agassizi (Ref. 42)  A g a s s i z i n (63) i s s t r u c t u r a l l y r e l a t e d t o the sponge metabolite p a l l e s c e n s i n - G .  a g a s s i z i n (63)  TABLE I:  continued METABOLITES  SPECIES Hypselodoris californiensis (Ref. 42)  Cu euryfuran (64)  Hypselodoris danielae (Ref. 29)  COMMENTS Euryfuran (64) i s a metabolite of the sponge Euryspongia sp. Four other sponge metabolites, dendrolasin (28), nakafuran-8 (44), furodysinin (33) and pallescensin-A (34) were also isolated from H. c a l i f o r n i e n s i s . Hypselodoris porterae i s also known to contain euryfuran. Spiniferin-2 (65) i s a known metabolite of the sponge P l e r a p l y s i l l a spinifera.  spiniferin-2 (65)  o  Hypselodoris ghiselini (Ref. 42)  f i Y /\ H  ghiselinin (66)  X H > ° '  ^ M e O  67  In addition to ghiselinin (66), H. g h i s e l i n i contains dendrolasin (28), nakafuran-9 (45) and a methoxy butenolide 6>7 related to nakafuran-9.  TABLE I:  continued  SPECIES  METABOLITES  Hypselodoris godeffroyana (Ref. 42)  see Chromodoris m a r i d a d i l u s  Hypselodoris porterae (Ref. 42)  see Hypselodoris c a l i f o r n i e n s i s and C a d l i n a luteomarginata  Melibe leonina (Ref. 43)  ea  Petodoris atromaculata (Ref. 44)  Phyllidia pulitzeri (Ref. 41)  0  H C i C - C H - C H = CH(CH.) CH = CH"C = C - C H - C 5 C ( C H . ) -CH=CH-CH-C = CH I I OH OH OH  69  wT  uI ,  NC  I  a x i s o n i t r i l e - 1 (70)  COMMENTS  Compound 68 i s the p r i n c i p a l o d o r i f e r o u s c o n s t i t u e n t of M. l e o n i n a . T h i s degraded monoterpene i s a l s o p r e s e n t as a c a r b o x y l i c acid.  A s e r i e s of high molecular weight s t r a i g h t chain p o l y a c e t y l e n e s (C^g, C^g, C 5g) of general s t r u c t u r e 69 were i s o l a t e d from the nudibranch's d i g e s t i v e gland and from the d i e t a r y sponge P e t r o s i a f i c i f o r m i s . a  n  d  C  5 2  A x i s o n i t r i l e - 1 (70) o r i g i n a t e s i n the d i e t a r y sponge A x i n e l l a cannabina. I t i s i n a c t i v e as a f i s h a n t i f e e d a n t but i s t o x i c to f i s h a t c o n c e n t r a t i o n s as low as 8 ppm.  TABLE I:  continued  SPECIES  METABOLITES  Phyllidia varicosai (Ref. 45, 46)  COMMENTS The i s o c y a n o s e s q u i t e r p e n e s _71 and 72 are sequestered from the d i e t a r y sponge Hymeniacidon sp. They are the a c t i v e c o n s t i t u e n t s of the nudibranch's mucous s k i n s e c r e t i o n that i s l e t h a l to f i s h and crustaceans.  isocyanopupukeanane  socyanopupukeanane ( Phyllodesmium longicirra (Ref. 47)  < t r o c h e l i o p h o r o l (73)  T r o c h e l i o p h o r o l (7_3) i s obtained from the d i e t a r y s o f t c o r a l Sarcophyton t r o c h e l i o p h o r i u m and i s concentrated i n the c e r a t a of the nudibranch. Two other cembranoid d i t e r p e n e s r e l a t e d t o t r o c h e l i o p h o r a l were a l s o found i n both the nudibranch and the s o f t c o r a l .  TABLE I:  continued  SPECIES Polycera tricolor (Ref. 21)  see Triopha c a t a l i n a e  Roboastra tigris (Ref. 48)  see Tambje abdere  Tamb j e abdere (Ref. 48)  OMe  tambjamine-A  Tambj e eliora (Ref. 48) Triopha catalinae (Ref. 49)  COMMENTS  METABOLITES  (74)  Tambjamine-A (74) and three r e l a t e d b i p y r r o l e s were t r a c e d to the d i e t a r y bryozoan S e s s i b u g u l a t r a n s l u c e n s . Tambjamines B, C and D d i f f e r from Tambjamine A by the presence of bromine and/or an isobutylamine f u n c t i o n a l i t y . These compounds shown a n t i b i o t i c a c t i v i t y and i n h i b i t c e l l d i v i s i o n of f e r t i l i z e d sea u r c h i n eggs. They have a l s o been found i n the nudibranchs Tambje e l i o r a and Roboastra t i g r i s .  see Tambje abdere  0  NHL 0  1  2  H  triophamine  (75)  Triophamine (75) i s p r e s e n t p r i m a r i l y i n the mucous s e c r e t i o n of T. c a t a l i n a e but does not d e t e r f e e d i n g by g o l d f i s h . I t has a l s o been i s o l a t e d from the nudibranch P o l y c e r a tricolor.  28. The were  f i r s t  the  (7^i)45  a n (  secretion  j  i  the  dietary  storing  that  Biologically also  by  active  2  and  These  formamide  analog  derivaties  not  40  been  the  furan  nudibranch s  or  are  Glossodoris species  2  9  ' * . 1  2  .  These  or also  has  b e e n  constituents  and  several  their  of  A l l  of  3  5  from and  these  a  mucous  both  found  clearly  sponge  1  origin  and  Cadlina  isoprenoid  marine  a l o n g  and  allomones.  regular  sponges,  w i t h Except  t h e i r for  luteomarginata,  the such  nudibranchs. i n  series  of  n u d i b r a n c h seven  i s o l a t e d are them  have  f r o m  of  furan-containing  the  demonstrated  possess  Chromodoris  C.  in  t o w a r d  which  number  related  localized  a c t i v i t y  derivatives,  known  g r a c i l i s  of  In  from  A  a n t i f e e d a n t  Sesquiterpenes, residue  5  mantle  1  t o x i c i t y  2  defensive  derivatives.  ring.  toxic  two  sequestering  f u n c t i o n a l i t y  f u r a n o s e s q u i t e r p e n e s luteomarginata  of  have  C.  These  This  1  o c c u r  from  nudibranch  were  pulitzeri *  fish.  reported  common is  to  o f t e n  isothiocyanate  sequiterpenes  as  compounds  toxic  and  A n o t h e r  use  a  sp.  capable  Phyllidia  compounds  have  of and  Hymeniacidon  for  •  6  isocyanosesquiterpenes  are  isothiocyanate  72^  varicosa  are  a  9-isocyanopupukeanane  isomer  Phyllidia  from .  5  from  constituents  metabolites  luteomarginata  i s o c y a n o  active  nudibranchs  known  skeletons  sesquiterpene  sponge  sponge  reported  2-isocyano  s  are  established  are  t  produced  the  products  rearranged  i s o n i t r i l e s  in  natural  f i s h . a  furan  m a r i s l a e  3  1  ,  Hypselodoris compounds  can  be  traced  known  to  a  sponge  that  C.  furan  sigmatropic  are  can  the  s t i l l  retained  reduced  activity (77)  has  3  2  .  These  against no  repellent  from  of  a  related  handling  variance.  (4_5)  from  to  are  obtain  the  Dysidea  properties  they  and  is  by  a  of  46  [3,3] to  has  show  sponge  metabolites absent  or  benefit  in and  nakafuran-8 Dysidea  antifeedant  sesquiterpene, not  J76  been  its  sponge  apparently  derive  (46) .  maridadilus  furanosesquiterpenes Another  sponge  .  Some  Chromodoris known  1  and  others  are  the  relationship  nudibranch  while  3  to  suggests  marislin  transformation  routine  nudibranchs  which  inactive.  the  nudibranch  fish.  The  formally  substantial  two  into  50  related  evidence  transform  predator-prey  nakafuran-9  metabolites are  during  godeffrayana  nudibranchs.  that  and  concentration.  and  f r a g i l i s  the  (7[6) are  extracts  show  by  Hypselodoris (44)  compounds  conclusive  established,  structurally  chemically  rearrangement  a  are  Circumstantial  can  spontaneously  When  prey  or  pleraplysillin-2  two  occurs  source  products.  marislae  metabolite These  sponge  upial  present  in  r e t a i n  sponge  and  reject  the  those  30.  77  Carbon s k e l e t o n s sesquiterpenes previously C.  from  discussed  3 6  ,  interesting antifeedant The sources Cimino limbata  marine  constituents  of  the  to or  (£3)  et is  carbon  3  of  established  l l  of  d_e  *C-labeled  notable in  including olepupuane  (_35) is  2  6  .  While  not  clear,  derivatives  the it  is  exhibit  activities.  secondary  recently  8  (12)  occurs  euryfuran  drimane  and  aldehydes  products,  skeleton  biological  godeffroyana  skeleton  albicanol  other  capable  sesquiterpene.  and  t h a t , many  firmly al.  9  note  acquisition is  2  Compounds  represent  carbon  s u c h as  sources.  isoacanthodoral  ,  natural  nudibranch  isomeric  and 1 8  of  H.  the  (1_1)  drimane  and  pu'ulenal  to  nanaimoensis  The  significance  majority  varicosa,  addition  Acanthodoris  terrestrial  P_.  acanthodoral  exceptions.  (56)  from  in  (_10) ,  nudibranch  i n the  a r e w e l l known f r o m o t h e r  maridadilus,  nanaimoal  observed  metabolites for  nudibranchs,  demonstrated novo  from  that  synthesis  mevalonic  acid  dietary however  Dendrodoris  of  a  fed  drimane to  the  nudibranch (5_5) . a  potent  from  insect  acids,  a  number 5 1  sesquiterpene also  suggested polygodial  ,  2 8  Polygodial Dendrodoris species  which  polygodial esters  3 6  .  mixed  did  not  ghiselini * . 1  another  example  found  2  2  8  ,  of  the  not  of  of  fatty It  was  esters  from  of  and  two  but  no  to  the  related  nudibranch  of  species  esters  directly  a  ambiguous.  other  the  as  which  label.  mixed  glycerides  Archidoris  sesquiterpenes  a  cembranoids  of a  2  ,  is  from  derivatives  metabolites  (4^), also  from  dietary soft  Sesterterpenes  n u d i b r a n c h s o r i g i n  luteomarginata  Norrisolide 3  i n sponge  Cadlina  norrisi  These  are  in  remains  three  contain  acid  Chromodoris  scalarin  contain  i t serves  series  C  is  been  d e t o x i f i c a t i o n  relationship from  mantle,  biogenesis.  atromarginata  to  the  s  5_7,  a l i +  polygodial  previously  esters  the  1  where  to  contain from  they  f u r a n o d i t e r p e n e s  traced  to  has  plants  esterified  farnesic  D i t e r p e n e s  number  It  mixed  isolated  While  are  2  is  D o r i ops i11a  57_,  odhneri *»  The  this  been  of  ;  arise  but  has  higher  dialdehyde  nudibranch  fish.  of  found  they  the  i n the  against  antifeedant  were  into  localized  antifeedant  drimane  of  incorporated  Polygodial,  isolated an  was  and  a  2 5  from and  rearranged  obtained  from  Phyllodesmium  i n c l u d e C a s e l l a  Hypselodoris diterpene  a  sponge  from while  longicirra**  7  were  coral. nudibranchs  fall  straight-chain  apparently  originate  into furano  two  groups;  compounds.  i n d i e t a r y sponges  or  32.  result C  2 6  from  transformation  methylated  scalarins,  sednolide  (5_0),  Biological  activity  was  ichthyotoxicity deterrence  of  from  luteomarginata  dietary  from  converted  by  A  the  arise  from  It  suggested  recently  number  been  polyacetylenes Peltodoris  bipyrroles  occur  dietary  bryozoan.  novel purine  Dialula  sandiegensis array  nudibranchs  Tambjamine-A  is  of  Other  feeding  tricolor * . 1  the  C  (3_8)  from  from  Cadlina of  sanguinea  modification  of  graceae, bile  may  of  Diaulula  have  chlorinated sandiegensis * , 1  another 8  be  acids.  constituents  series  a  A4-cholesta-3-one,  observed  (7_4)  source  and  series  three  s p e c i e s and  nitrogenous  0  of  related originate  metabolites  r i b o s i d e s from A n i s o d o r i s n o b i l i s 3 9  1  aldehyde  9  Aldisa  that  i n s e v e r a l nudibranch  include  The  from  the  polyhydroxylated  1  alcohols.  a  obtained  .  and  non-terpenoid A  the  3  the  a t r o m a c u l a t a * ** p r o v i d e d  acetylenic  in  of  by  3  also  chemical  to the  reported. were  by  Anthroarcuata  nudibranch  growing  1 9  derivatives  sponge  sedna  Glossodoris  are  peroxides  is  and  2 5  of  derivative  evidenced  luteone  2 3  series  novel  is  represented C  A  Chromodor i s  from  and  3  acid  the  the  (3_7)  Nudibranchs  compound.  obtained  and  .  possibly  2 0  are 1  Bile  3 1  cooperi  scalarins  steroidal  steroids *.  in  idiadione  leonina * 2 7  including  sesterterpenes  terpenes  Melibe  interesting  of  sponge p r o d u c t s .  found  of  two  Degraded 68  of  2 2  and  . natural  truely  products  remarkable.  identified Like  the  from other  33. opisthobranchs, obtain  complex, m o l e c u l e s  chemically are  nudibranchs  in  store.  In  for  biosynthesis  activity their a  some  the  associated  ascribed  chemical  allowed  dietary  cases  developed  from t h e i r  m o d i f y them f o l l o w i n g  selective  the  have  compounds t h e y  secondary  with  are  These  directly  defense  has  nudibranchs to  apparenty  eliminate  deterred  the m e t a b o l i c  t o p r o d u c e and m a i n t a i n a p r o t e c t i v e  shell.  to  and  responsible Antifeedant  constituents  f u n c t i o n as d e f e n s e a l l o m o n e s .  to  organisms  concentrate  metabolites.  nudibranch  ability  and may be a b l e  ingestion.  nudibranchs  of  diet  the  supports  Adaptation predation load  of and  required  Plate  1.  Triopha  catalinae.  Simon F r a s e r  Photographer:  University.  Ron  Long,  CHEMICAL  I.  ISOLATION  STUDIES  AND  OF  TRIOPHA  STRUCTURAL  CATALINAE  ELUCIDATION  OF  TRIOPHAMINE  (75) Triopha nudibranch decorated  catalinae of  with  (finger  like  from  white  a  the  (recently  suborder  orange  tipped  processes body  body  conspicuous  striking the  Due  1980.  Columbia to  most  support  feeds  San  from  west  in  assortment exposed  coast of  the  of  . coloration of  i n i t i a t e d  in  were  collected  in  Vancouver T.  Island. catalinae  outcroppings  This  the  was  habitats,  rocky  along Island,  investigation  study  communities.  throughout  2  This  found  conspicuous  an  this  is  Vancouver  catalinae  the  on  and  from  of  orange-spotted  environment.  C a l i f o r n i a  along an  length  1  in  papillae  average  bryozoans *  used  and  an  marine  abundance,  beautifully  projecting  white,  America  a  dorsum)  on  Triopha  is  is  2  the  the  predation,  bryozoan  collected  from With  Diego,  of  abundant  large  g i l l s  in  North  specimens  found  rhinophores,  catalinae's  relative  lack  Sound,  Although  was  its  T.  of  to  products The  Barkley  were  coast  apparent  natural  mm,  nudibranch  Pacific  British  and  quite  It  surface.  70  carpenteri)  doridacea.  extending  approximately is  Triopha  dorid  year  from  of  Triopha  which  nudibranch  depths  of  1-13  catalinae  were  meters. Freshly rapidly  collected  immersed  in  specimens  methanol  and  stored  at  room  temperature.  36. The  solvent  additional  was  decanted  methanol.  extracts  produced  between  chloroform  and  primarily  of  interest,  triophamine  fats,  steroid  'H  NMR  1  step  Triophamine  by  were  and  combined  partitioned  o i l  the  soluble composed  compound  silica of  ethyl  of  several  further  purified  0.5). (T5)  in  5%  ethyl  bright  orange  acetate/hexane while the  with  more  from to  a  chromatography  is  flash  acetate  with  polar  drawn t o t r i o p h a m i n e by  fraction  ether,  gel  ethyl  eluted  (7j>) and  crude  thin-layer  Triophamine  brownish  steroids  eluted  was  preparative Rf =  the  chloroform  the presence of i n t e r e s t i n g  the was  a  gradients  lipids  was  Attention and  The  fractionated  Triophamine  fraction  of  of  with  9  were e l u t e d w i t h 15%  absorbance  washed  (75) * .  Non-polar  mixtures. UV  to  pigments,  using  acetate/hexane. pigments  water.  concentrated  o i l was  hexane.  nudibranchs  an aqueous s u s p e n s i o n t h a t was  was  chromatography  the  E v a p o r a t i o n _in v a c u o  material  The  and  optically  the light (1:1  active  solvent  i t s strong  signals flash  i n the column.  yellow  o i l by  hexane/diethyl  (COCDD  -7.0°  (C  37. 1.7,  MeOH))  ^max  =  2  5  0  and n  i t contains  (  m  causes  a  14500,  MeOH/HCl),  causes  triophamine  \  m  a  hypsochromic  = 232 nm  x  The  ( T  1  as C  2 1  mass  spectrometry  Major  fragment  2  H  were  also  the 17  m/z  +  of  -*-  base  new  acid  4  n  m  (  £  (MeOH/NaOH)  substance  with  calc'd  from  of  loss  (m/z 2 8 1 ) a n d C H  1 Q  {75^)  triophamine  363.2885,  resulting  g  of  the  were  that  must  were  spectrum  'H  and  proton  exhibit  observed  showed  1  at  integrated  an  i n the  well  protons  obtained with  exchangeable  was  2  by electron-impact high  2  (M ,  nonexchangeable  signal  3  molecule  signals  'H NMR  spectra  with  to a  ^o  m  1  3  a  CH  was  resolution 363.2885). (m/z 3 4 8 ) ,  3  (m/z 2 3 8 ) r e s i d u e s  7  C  (7_5) l e d t o t h e c o n c l u s i o n  the 11  X ax  with  a  observed.  triophamine  Only  N 0  ions  Examination  scale  treatment  formula  3 7  6  the  with  = 30 m i n , e 1 0 0 0 0 ) .  2  (m/z 3 3 4 ) , C H  5  chromophore  Treatment  i n  t o decompose /  UV  MeOH).  shift  while  molecular  established  C H  12000,  £  a  that  DMSO-d  i n t h e NMR  1 3  C  a  f o r less  than  very  spectrum  When  broad  time  and  f o r only  the  one  of  symmetry.  resonances  I I ) .  6 9 . 4 and  of  NMR  as s o l v e n t  g  spectra  element  resolved  (Table  NMR  'H  NMR  additional  exchangeable  one p r o t o n  at  11  ppm  observed. It  double Thus,  was  apparent  the  number  triophamine  that of  each  protons  (7_5)  'H r e s o n a n c e indicated  contains  3  must by  account f o r integration.  exchangeable  and  34  38. TABLE  II.  *H  and  1 3  C  NMR  data  f o r Triophamine  *H NMR, 6  Carbon  a  1  (75)  3  1,1* 2,2'  2.30-2.48  3,3'  2.12  (m,  IH)  (dd, J =  2.30-2.42  6,  (m,  13 H z ,  IH)  185.6  (s)  50.3  (d)  40.1  (t)  140.4  (s)  5.21  (q, J =  7  Hz,  IH)  120.5  (d)  6,6'  1.56  (d, J =  7 Hz,  3H)  12.1  (q)  7,7'  2.01  (q, J  =  7 Hz,  2H)  23.2  (t)e  8,8'  0.95  (t, J  =  7 Hz,  3H)  12.9  (q)  9,9'  1.46-1.67  (m,  2H)  26.1  (t)e  =  7 Hz,  13.1  (q)  158.9  (s)  0.91  (t, J  3H)  11 NH^ NH  C  9.4  ( b r , IH)  11.0  ( b r , 0.5H)  4 0 0 MHz, C D C 1 1 0 0 MHz, D M S 0 - d May b e r e v e r s e d .  D  3  6  e  b  5,5'  10,10'  c  NMR, 6  IH)  4,4'  a  C  d  100 May  MHz, acetone-d be r e v e r s e d .  6  d  d  d  39. nonexchangeable resonances account 7)  observed  for  clearly  2  a  in  In  the  carbons.  showed  carbons, These  protons.  A  1  signals  methine  observations  3  1  C  and  spectrum  demonstrated  identical  9-carbon  a  C  3  NMR  SFORD  for  carbon  a d d i t i o n ,  3  a  facile that  NMR  the  must  experiment groups,  3  loss  of  C  g  H  each  with  17  each  methylene olefin. the  mass  contains  two  1  in  11  (Figure  trisubstituted  triophamine  segments,  of  spectrum  methyl  and  10  7  nonexchangeable  protons. The  structure  a n a l y s i s  of  decoupling  TABLE  'H  NMR  the  'H  Resonances  400  NMR  a  Decoupling  Irradiated  1.46-1.67  MHz,  The the  CDC1  1  3  C  presence  methyl  g  H  1  residue  7  spectra  and  a  was  assigned  series  signal  Studies  Observed  of  of  by  proton  Triophamine  Spectral  60.91 (t) 5.21 (q) 0.95 (t) 2.30-2.48 2.12 (d,d) 2.12 (d,d)  2.01 2.12 2.30-2.48 2.43  a  C  experiments.  III.  6  of  (75)  Changes singlet singlet singlet s h a r p e n up singlet ( d , J = 13 H z )  -> ->  -y -»•  ->  3  SFORD of  a at  NMR  experiment  trisubstituted 61.56  (3H,  d,  had  already  double  bond.  J  =  7Hz)  established An  o l e f i n i c  coupled  to  a  40. one-proton group  quartet  and  a  olefin.  A  coupled  to  revealed group. was to  proton  the  of  78  13Hz,  10)  IH)  and an  283,  deshielded  was  the  A  with  natural  coupled  prominant  2.30-2.42 ethyl  ppm  group.  contains  two  IH)  from  a  ppm  via a  chain methine  This  the hydrocarbon  chain  9-carbon  (J  = 7Hz)  coupled  ppm. of  The  ppm  'H  triophamine NMR  62.12 i n  spectrum  for a  pair  (dd,  J  =  triophamine  established  that  substitued  i n t h e mass  methylene  the  residues  Decoupling  protons  conclusion of  of  a  o f _78  indicated of  experiments  at the  that  general  had  multiplet  the methine  protons  6,  rearrangement,  one-proton  that  of  carbon  spectrum  McLafferty  moiety.  methylene  led to  ppm  attached t o the remainder  showed  the a l l y l i c the  was  7Hz)  7Hz)  was  i n tetrahydrotriophamine  triophamine  and  =  =  ethyl  at  A  of the  an  to the ethyl peak  (J  0.95  EtOH)  This  assignment.  62.37  to both  (m,  q,  resonances  observed  attached  the hydrocarbon molecule  that  methyl  J  (2H,  a t 60.91  (Pd/C,  shift.  resulting  this to  protons  upfield  group  supported  that  on  a  carbon  substituent  a t 1.46-1.67  2.30-2.42  the o l e f i n . m/z  triplet  revealed  methylene  methylene  at  at  t e t r a h y d r o t r i o p h a m i n e (7JB) .  undergone  of  62.01  residue  hydrogenation  (Figure  geminal  at  triplet  ethyl  indicated  t o t h e same  olefinic  multiplet  Catalytic gave  third  by a methyl  a two-proton  (75)  resonance  aliphatic  evidenced  = 7 H z ) ppm  attached  three-proton  that An  were  methylene a  (J  a t 5.21  proton  was  62.12  and  aliphatic  triophamine  structure  79,  41. which  was  consistent  fragmentation  pattern  0  with  the  observed  mass  (see Figure 4 ) .  NH  0  2  79  78  The means  geometry  experiment 65.21  (Figure  caused  methylene proton proton  at  molecule  2.12  * .  and No  3  resonances.  effect  and  a  of  enhancement This  group  spins  was  NOE s 1  that  t o the relaxation  5 3  must  ,  Since  at  of the  NOE s  of  1  Certain  to  i n  direct  the  i n the  same  olefinic  the olefin  proton  o f the methylene the magnitude of  to the sixth  the olefin  be c i s .  lead  5 2  allylic  molecules.  observed  proportional  the  enhancement  can  negative  substituent.  distance  of  by  (NOE)  proton  homonuclear  i n small  demonstrated  i s inversely  methylene  elucidated  Enhancement  negative  Negative  indirect  significantly  internuclear  chain  Overhauser  was  enhancement  ppm.  t h e main h y d r o c a r b o n  this the  positive at  bond  Irradiation of the o l e f i n  arrangements 1  contributes  Nuclear  a r e n o t uncommon  NOE s 5 2  the double  9).  2.30-2.42  signals  positive  in  a  proton  geometrical  ethyl  about  of a difference  spectral  proton  The o l e f i n  power  of  and t h e main therefore  has  42. an  (E)  configuration.  The C  3  H  3  N  0  3  remaining and  2  i t  unsaturation. was  highly  i n f r a r e d  two  1  C  3  C  NMR  clear moiety.  a  at  at  was  Low  s i g n a l  observed  as  in  their  the  NMR  From  acyl  R  3.  NH  2  imine  spectra its  the  presence  a  acyl  tautomers  of  l o s t  observations diacylated to  imine  NH  N  2  which  observed  was  on  the  i t  was  guanidine almost symmetry  results tautomers.  0  N H  triophamine  R  (75).  the  as  exist The  the  studies  -40°C  triophamine  R  Acylimine  NMR  at  known  0  H  'H  5  two  An  suggested  tautoraer "*.  of  ppm  and  these  are  6158.9  8)  symmetry  of  of  (Figure  1  temperature  consisted  e q u i l i b r i u m between  0  of  of  units  substituent.  broadening  Monoacylguanidines  exclusively  -  at  temperature  element  lowered.  triophamine  c m  consisted  three  observed  6 1 8 5 . 6 ppm  room  (7_5)  guanidine  1700  carbons.  scale  that  resonance  at  The  triophamine incorporate  of  resonance  -60°C.  temperature  Figure  3  of  to  suggestive  at  time  rapid  1  s u b s t a n t i a l  increased NMR  The  carbonyl  showed  had  absorption  remaining of  portion  from  43. To  test  prepared  by  anhydride. =  248  t h i s  reacting The  nm  conclusion  UV  MeOH/HCl)]  guanidine  MeOH)  were (7_5) .  h y d r o l y s i s  (MeOH/NaOH,  It  monoacety lguanidine Under  converted C^max  to  =  2  3  2  n  (  agreement  with  c a r b o n y l  those  room  diacetylguanidine difference (6172.7)  is  between  reported  a  0  NH  2  X  m  a  =  x  derivative  81  MeOH)]  i n almost  Levy  et  1  3  C  and  NMR 180.1  5  5  i n  .  2  2  0  give 14300,  (E  UV  ppm  well  are  with  i n  magnitude  o f good  of  r e l a t i v e  and  was  absorbance  r e s o n a n c e s  (6177.2)  NH  0  t o  The d e s h i e l d i n g  equal  f o r  triophamine  whose  t r i o p h a m i n e  a l .  nm  correlates  H N^N H  230  conditions  propionamide  by  19900,  U  t e m p e r a t u r e )  triophamine.  c a r b o n s  nm  m  base-catalyzed  6159.0 of  212  [\ ax  rapid  The  at  =  x  80  underwent  m o n o a c e t y l g u a n i d i n e . diacetylguanidine  a  compound  a c e t i c  o b s e r v e d  10000,  £  m  with  was  those  reaction  monoacyl  m  \  of  8_0  t o  with  similar i t s  and  comparable  triophamine  MeOH).  carbonate  characteristics  16800,  (e  diacety lguanidine  to  the t o the  acetamide  44. Final catalyzed Using  proof  hydrolysis  the  FCNP  nitroprusside), mixture 0.3]. of  was  by  also  (MeOH/NaOH,  guanidine  could  was  48h) 5 6  be  acetyl  base-  of triophamine  (75) .  the  i n low y i e l d  -  i n the  acid  (50:35:5),  acetone  allowed  i t s 4,6-dimethylpyrimidine  attempts,  isolated  by  detected  3  with  obtained  (ferricyanate  [CHC1 /MeOH/acetic  as  82  structure  reagent  TLC  repeated  the  spray  Treatment  guanidine  After  of  10-carbon  reaction R  of  =  f  isolation  derivative  carboxylic  as a p r o d u c t  83  the  sodium  acid  82. 83_  hydrolysis.  -CH-  Figure  4.  Interpretation  - C2H1  o f t h e HRMS o f  75.  PPM 16)  Figure  6.  100 75  MHz b r o a d b a n d in acetone-d  6 *  decoupled  1  3  C  NMR  spectrum  of  Figure  7.  100  MHz  SFORD  1  3  C  NMR  spectrum  of  75. i n  CDC1 . 3  MICROMETERS ( m) M  4000  3600  3200  2800  2400  2000  1800  1600  1400  1200  1000  800  600  400  FREQUENCY ( C M ) 1  vo  Figure  8.  IR  spectrum o f  75 i n  CHClg.  Figure  9.  4 0 0 MHz ' H NMR d i f f e r e n c e NOE spectrum of 75 when the o l e f i n i c proton at 65.21 ppm was irradiated: a) enhanced signals of the main chain methylene p r o t o n s ; b) enhanced signal of t h e v i n y l m e t h y l group c) a l i p h a t i c methyls.  Figure  10.  270 MHz 'H NMR s p e c t r u m o f 78 i n  CDC1 . 3  53. II.  BIOLOGICAL A  number  compounds ough 1981. marine the  of  these  have  also  our  impetus  implicit  system.  showed  that  produced  a  s e c r e t i o n .  in  triophamine  a  biological be  should  not  chemical  i t  '  5  thor5  7  in  derivatives  of  However,  to  .  9  is  the  can  be  to  in  inhibit  On  the  f i r s t  the  a did  these  fish  the  basis that  of  extraction  gram  seems  in  of a  tests these  for  possess  number powerful  triophamine  has  goldfish  no  In  bacteria  large  negative  a  bioassay  positive  unit  the  feeding.  a  to  mucous  s u i t e d  While  in  specimens  surface  deter  triophamine  Freshwater  chemical  antifeedant  not  the  exclusively  i d e a l l y  growth.  two  to  mobilized  against  a  l i v e  outer  are  was  Triophamine  guanidine  in  concluded  prior  7_5.  rapidly  assay  containg  employed  localized  in  l o g i s t i c s but  catalinae  repeatedly,  triophamine  deterent.  8  A  Chevolot  (7_5)  organism was  near  activity,  be  (7_5)  stored  standard  inactive.  5  Triopha  the  rich  failed  compounds  by  guanidine  nudibranch  prodded  substance,  goldfish,  addition,  the  exudate  Such  defensive using  that  When  where  provided  d e s c r i b e d  studying  triophamine  strategically  nudibranch  to  for  mucous  organisms.  triophamine  Dissecting  mantle.  containing  diacylaguanidine.  assumption  defense  was  been  guanidine  marine  other  knowledge,  occurring  unique  from  compounds  origin of  and  reported  several  The  of  been  then  naturally  be  interesting  Since  best  the  of  have  review  CONSIDERATIONS  proved  results, value  might  as  i t a  respond  54. differently  than  triophamine  deters  The of  egg  rarely  food  several  vividly  sessile  marine  previously to  diet,  search  encountered  was  of  preliminary within  0  .  the  triophamine.  4  C  i n an  incorporation  the nudibranch  found  i n extracts  of  Polycera  tricolor.  P.  (see Chapter  a  group  compounds  nudibranchs  a of  i t i s  suggests 4, V ) .  their  species  reveal  possible  dietary  have  source f o r  bryozoan to  of  have t h e  through  dietary  failed  undetected study  unusual  puzzle.  to find  sites  and  by  obtained  While  lipids  other  bryozoans,  analysis  egg  enjoyed  many  metabolites  collection  produced  nitrogenous  Since  t o be  predation i s  to predation  on  which  undertaken  originates 1  6  Chemical at  triophamine  from  isolated  sequester  triophamine.  presence  exclusively  appear  on  antifeedants.  (T5) o r  nudibranch  These  o f T. c a t a l i n a e  egg masses  subsequently  dorid  but  chemical  an unanswered  ( T 5 ) was  animals  been  ability a  remains  feeds  nature  immunity  consist  deposited  eggs would  triophamine  apparent  colored  also  no  species  and  animals  f o r  that  material.  colored  f o r many  be  fish.  spongy  Nudibranch  search  but  The  egg masses  tricolor  soft,  of Triopha  steroids  Triophamine the  a  than  o r i t may  nudibranch  The c o n s p i c u o u s  extracts  metabolites.  other  brightly  source  prompted  Methanol  these  often  observed.  masses  of  substrates.  suitable  counterparts  o f numerous  rings  are  unobstructed  marine  predators  masses  concentric  structures  a  their  the that  source,  i t i s  a  produced  55. III.  SYNTHESIS A  synthesis  provide  coupling  of  scheme two  synthesized the  these  the  Chong  organocopper  C  with  -acids  Cy -acids  are  Q  afforded  with  of  f o l l o w e d  to  the  of  In 90  to  a  of  to  the  (see  by  88  addition  91.  The  fashion  Chong  who  6 1  At  about  the the  established. method  6 2  produce  syntheses  of  the  1).  ester  84  J35  84 was  in  with THF  was  to  of  provided 9_3 w a s  reacted  copper  lithium at  methyl  reagent  groups  y i e l d e d the  the  -48°C  THF 87_  in  Q9_  via  residues  the  8_6  and  and  9_0  species  d e r i v a t i v e  trimethyl  at  Complete  vinylithium  produced  trimethylhydrazide  in  produced.  N , N , N ' - t r i m e t h y l h y d r a z i d e  similar  and  project  developed  Scheme  when  moieties  9_1  This  been  Their  t r a n s m e t a l a t i o n  acid  83_  (Z)-3-(tri-n-butylstannyl)-  isomer  Conjugate  -acid  geometry  cuprate  ethyl  ester  Q  sterospecifically  8-acetylenic  geometric  1  to The  molecule.  not  olefins.  Alternately,  C  and  the  the  (tri-n-butylstannyl)  2-ethylpropenoic 92.  a,  workup,  the  respectively. 89  the  86_.  corresponding  88  of  upon  reduction  (Z^)  the  recently  (tri-n-butylstannyl)  2-pentenoate - 7 8 °C  and  structure.  Piers  had  reagents  summarized below  Reaction (phenylthio)  (E)  with  portion  a  of  undertaken  guanidine.  initiated  applied  was  proposed  to  triophamine  employing 1 Q  the  moieties  was  in  (7_5)  production  hydrocarbon  bonds  and  of  conjunction  synthesis  olefinic Piers  proof  in  (75a)  triophamine  involved  of  performed  time  (±)-TRIOPHAMINE  definative  synthetic  was  OF  of  hydrazide  1,4-addition were  removed  CH,CH C=C-C0 Et o  Scheme  1.  9  S y n t h e t i c sequence f o r p r o d u c t i o n of the i s o m e r i c a c i d s 94 and 8 3 a . Note: the " a " d e s i g n a t e s a racemic synthetic product that structurally c o r r e s p o n d s w i t h m a t e r i a l d e r i v e d from a n a t u r a l source.  57. via  reductive  products  of  cleavage.  9_2 a n d  Synthetic identical  in  93_ p r o d u c e d  product a l l  from  natural  NMR)  the  isomeric  d i s t i n c t l y This  different  confirmed  the  double  difference by  94  the  unambiguous should  using  HC1,  acetic  produced  product.  p a r t i c i p a t e d  a  in  from now  facile the  mind,  the  the  reaction  mixture  that in  the the  mixture The  3°  carbonium  but acid. of  deduced  p r e d i c t e d  the  by of  from  the  established  hydrolysis  of  oxalic  as  the  major bond  process.  With  t h i s  of  triophamine  75. i n  the  acid.  (7_5)  presence  low  yield to  portion  of  of  of  75_ i s  which  base 8J3  It  is  prone  protonation is  was  acid  explain.  A c i d i f i c a t i o n  Markovnikov ion  or  double  d i f f i c u l t  of  95  acid  of  carbon-carbon  persistent  to  to,  83a.  hydrocarbon  leads  'H  portion  definitively  hydrolysis  presence  and  configuration  p-toluenesulfonic  was  £33  natural  been (E)  acid  (IR  hydrocarbon  h y d r o l y s i s  the  spectra  similar  acid-catalyzed  hydrolysis  NMR  Spectra  of  The  now  of  that  the  process,  lactonization  o l e f i n .  acid,  the  While  apparent  hydrolysis  was  that  indicated in  re-examined. is  of  'H  very  spectra  reduction  o l e f i n i c  2,4,4-triethylbutyrolactone  This  observation  the  triophamine,  noted  and  (7_5) .  data.  synthesis  be  of  crude  respectively.  p r e v i o u s l y  spectral in  83a  were  the  the  IR  those  94  experiment,  It  acid  acid  had  bond NOE  to  structure  of  and  triophamine  which  interpretation  94  of  provided  from,  the  t r i o p h a m i n e  83a  respects  isolated of  Oxidation  to  of  the  of  the  generated  can  58. then  be  acid.  attacked The  Y~  l  a  by c  t  o  n  the  carbonyl  95 was  e  a c i d i f i e d hydrolysate  of  oxygen  of  subsequently  the  carboxylic  isolated  from  the  75.  95  The s t r a t e g y converting  acid  of  the  83a  into  a  suitable  coupling  it  requires  nucleophilic  attack  carbonyl,  a  of  hydroxyl  residue  Treatment  of  the  to  remainder of the  derivative with  the  presence  of  p-nitrophenyl the  guanidine.  Coupling  the  more  the  96  in  guanidine made  on  which  unit  to  activated  ester  ratio  would  studies  have  acylation  of  9_6 9_6  the  a  the  group. in  afforded  negative  the  acid  replaced  the of  change  nucleophiles.  guanidine  complete  reaction  The a b i l i t y  toward to  then  p-nitrophenol  yield.  stabilize of  and  leaving  83a w i t h  excellent  involved  coupling  favorable  acid  reactivity  stoichiometric Scheme  a  derivative  N,N'-dicyclohexylcarbodiimide  ester  the  of  83a was  carboxylic  p-nitrophenyl  increased  Since  synthesis  in  a  synthesis  2:1 (see  2).  Previous obtained conditions  on  used,  N,  shown  that  guanidine  N'-diacyl  while  are  derivatives  the  dependent  products on  c a n be p r o d u c e d  the in  Final steps i n the synthesis of the (S,S) enantiomers of (±)-triophamine the (R,S) and (S,R) diastereomers 75b.  (R,R) (75a)  and and  60. good  y i e l d  6  3  .  treatment  of  p r o d u c e d  a  From  own  guanidine m i x t u r e  derivatives.  studies,  carbonate of  the  in  p-nitrophenyl  for  ester  predominated,  acetic  be  t r i a c e t y l  hydrolyzed  We  set  out  guanidine  or  t r i a c y l  i f  selective  the  hydrolysis  that  anhydide  and  could  ethanol.  known  diacylating  96^  achieve  was  d i a c e t y l  refluxing  conditions  i t  with  Triacetylguanidine  diacetylguanidine suitable  our  of  to  to  find  with  the  d e r i v a t i v e a  single  acyl  group. I n i t i a l carbonate to  react  attempts  salts  of  with  described treated  by  the  acylate  guanidine  were  guanidine  salts.  Banker  with  to  et  al.  6  1  *,  a c y l a t i n g  the  ester  the  reacted  monoacyl  derivative,  guanidine reaction product  total  was  75a  is  could  to  an  be  guanidine  portions  were  added  reaction  monitored of  at by  96).  was  of  was lh TLC If  i n  of the  Under  found by  96  that  adding  in  the  an  was  presence  of  to  reaction  was  its free the  diacyl of  free  Guanidine  free  weight  of  Rather,  the  appearance  with  amounts  determined.  (the  yield  varying  accurate  and  conditions  desired  small  intervals  failed  guanidine  96  chloroform.  so  not  the  of  or  procedure  these  systematically  hygroscopic added  base  reaction  By  formed  excess  extremely  disappearance  no  observed. i t  free  96  a  tetramethylguanidine  but  conditions,  guanidine base  with  as  Employing  9_6  guanidine.  h y d r o c h l o r i d e  unsuccessful  agent  N, N, N ' , N ' - t e t r a m e t h y 1 96  the  small  course of  75a  allowed  the  of  the  and  the  to  go  to  61. completion,  as  appreciable yield  of  50-75% was  of  quantities  75a  was  the  starting  stopped  material. (based (75a)  on  diastereomers  contain  carbon].  a  As  acid  In 83a  residues  single  about  the  is  The  mixture  are  represent  a  mixture  diastereomers thin-layer  the  while  of  diastereomers  were  product. showed  triophamine.  and  the  approximately reaction  chloroform  soluble  provided of  moieties  a  48%  yield  (±)-triophamine  center  optically must  (±)-triophamine,  racemic  (R,R), and the  (R,S)  and  'H  in  a l l  spectra but  ('H  Resonances  IR  respects NMR  distinct between  two  acid of  (S,R).  products  75b.  repeated and  or  constitute  (75a)  NMR  the  o l e f i n i c  (S,R)  diastereomers  by  (R,R)  and  products  (R,S)  The  be  combinations  (S,S), (S,S)  active,  When  four  (75)  methine  the  mixture.  guanidine  identical  subtle  C-2(2')  centers  achieved  The  triophamine  [the  is  chiral  of  ( ±) - t r i o p h a m i n e  was  96,  the  mixture  unwanted  chromatography.  natural  authentic  a  (R,R)  of  75b  (±)-triophamine  a  of  to  (75a)  S e p a r a t i o n  of  when  of  75b.  formed,  the  (±)-triophamine  s i l i c a  two  as  coupled  of  on  formed  reacted,  the  triophamine  synthesis  configurations  o f f  chiral  the  produced  are  96^ h a d  hydrocarbon  natural  configuration  product(s)  material  96)  disappearance  Therefore,  f i l t e r i n g  identical  the  side  low.  unrecovered its  The  (S,S).  of  very  by  by  Chromatography  and  each  determined  and  from  preparative spectra to IR)  those of  differences 61.5  i t s  and  2.5  of of the from ppm  62. i n the  'H NMR s p e c t r a were most u s e f u l  desired  product  displayed  a  (see  slightly  two-proton m u l t i p l e t 2.12  (dd,  difference and  J  =  was  2.37-2.47  two-proton triophamine.  7, the  in  at  figure  the  12).  altered  coupling  IH)  ppm.  diastereomers  pattern  The  clearest  one-proton m u l t i p l e t s  diastereomers  multiplet  The  the  in  the  6 1 . 4 9 - 1 . 6 7 and a s h a r p e n e d s i g n a l  14Hz, two  in distinguishing  at  at  spectral 62.28-2.37  t h a t were c o a l e s c e d t o  2.30-2.48  ppm  in  at  a  authentic  -I  1  2.5  1 2.0  Figure  12.  1  1  1.5  —I  PPM  (6)  2.5  1  1  2.0  1  1  1.5  C o m p a r i s o n o f ' H NMR ( 4 0 0 M H z , C D C 1 ) r e s o n a n c e s b e t w e e n 6 1 . 5 - 2 . 5 ppm o f : a) natural triophamine (75) and b) m i x t u r e o f s y n t h e t i c d i a s t e r e o m e r s 7 5 b . 3  PPM  (6) CT> f  Plate  2.  Anisodoris Simon F r a s e r  nobilis.  Photographer:  University.  Ron  Long,  65. CHEMICAL  I.  DIHYDROAPOFARNESAL The  d o r i d  (average of  size  British  variety  nudibranch  80-100mm)  the  emits  commonly  referred  Fuhrman  and  and  a  aJL.  2 2  also  prompted  specimens  6  a  by  the  emulsion  combined  a of  extracted the  and  from  sea  chemical  isolated  the  from  the  observed  in of  coast in  depths  Because A.  a of the  of  its  nobilis A.  is  nobilis  investigation.  In  N-methylpurine  extracts  is  for  the  water,  lemon.  aqueous It  large  found  to  unique  responsible  nudibranch's  with  is  zone  aroma.  speckled  investigation  smell  were  the  and  coloration,  constituent  produces  the  fruity  along  is  of  the  pharmacologically  the  t o x i c i t y  these the  to  mice  e x t r a c t s  Australian  6  5  .  sponge  .  present  nobilis  retains  6  removed  gland.  o r i g i n a l l y is  sponges  yellow  (JL6)  abundant  on  previous  probably  d i g i t a t a  The  were  et  is  Doridosine Tedonia  of  as  digestive  crabs  A.  to  doridosine  nudibranch's active  When  bright  n o b i l i s  intertidal  pleasant,  and  subject  riboside  m. a  smell  1980,  f a i r l y  from  pleasant  the  and  habitats 100  NOBILIS  A n i s o d o r i s  feeds  than  ANISODORIS  (15)  It  nudibranch  was  OF  Columbia.  of  greater  an  STUDIES  of  pleasant  voluminous the with  mucous.  Anisodoris odor. mucous  organism.  concentrated  to  a  When  secretion  as  which  collected  methanol  chloroform fragrant  was  perturbed,  Freshly  chloroform, Successive  nobilis  formed extracts  brown  o i l .  66. Fractionation lipids, DNPH  of  the  steroids  and  p o s i t i v e .  preparative aldehyde of  1_5  extract  single  i n  0.1  s i l i c a  a  to  nature  was  y i e l d  and  t o t a l  gel  compound  p u r i f i c a t i o n  volatile  r e s u l t e d  on  odoriferous  chromatography  The  (approximately  a  F i n a l  gas  15^.  crude  low  y i e l d  provided that  was  achieved  by  the  o l e f i n i c  natural  abundance  of  o n l y  2.8  mg  e s t a b l i s h e d  by  mg/animal).  0  15  The  molecular  formula  electron  impact  units  unsaturation.  14)  of  displayed  indicative groups 1.60,  1.61  each.  Two  65.12  ppm.  three  a  of  were  HRMS  and  and  1.69  o l e f i n i c It  units  aldehyde  and  spectrum  also  The  was of two  H  2  400  that  protons evident  unsaturation olefin  0  was  that 'H at  L5 NMR  spectrum =  broadened  compound  for as  was  at  (Figure Hz)  ppm, methyl  singlets three a  at  6  protons  multiplet  acyclic,  accounted  doublet  1  three  o l e f i n i c  functionalities.  methyl  (J  Three  observed  were  possessed  69.58  integrated  were the  4  MHz  s l i g h t l y  ppm  a  t  residue.  by  revealed  l  doublet  aldehyde  evidenced  l  revealed  one-proton  an  C  as  at the  for  by  one  The  'H  NMR  61.10  (J  =  7  Hz)  and  six  a l l y l i c  methylene  ppm.  One-proton  1.96-2.13 were and  appropriate aldehyde The  that  NMR  a  multiplets  methylene  spectrum  compound  principle  a  as  broad  at  that  multiplet  61.42  is  8 to  and  both  1.78 an  at ppm  olefin  substituent.  'H  of  for  protons  of  the  68,  of  lj>  recently  nudibranch  showed  great  isolated  Melibe  as  s i m i l a r i t y  the  to  odoriferous  leonina * . 1  3  0  68  The  fragrant  evidently  compound  the  from  ^-analog,  150  by  a  (M -CH CHCH0 +  doubly  (Figure  degraded The  contains the  and  a l l y l i c  a r b i t r a r i l y a  +  3  rearrangement  unit  prominent  the  mass  H)  cleavage 13).  been  The  The  spectral  peak  which  at  as  m/z  of  trans.  69  the the  (2,6,10-  ion  a  at  is m/z  McLafferty  resulting terminal  central  Aldehyde  was  structure  fragment from  expels  geometry  assigned  proposed  r e s u l t i n g  base  nobilis  dihydroapofarnesal  trimethyl-5,9-undecadien-l-al). supported  Anisodoris  from  isoprene  olefin  _15_ i s  a  has  clearly  sesquiterpene. mucous  s e c r e t i o n  dihydroapofarnesal  predatory  interesting  to  sea note  star that  of JU5  A n i s o d o r i s is  known  Pycnopodia the  to  n o b i l i s be  repugnant  helianhoides *.  exudate  1  from  which  Melibe  It  to is  leonina  68.  elicits  a  similar  and  contains  60^ as  and  volatile  nature  antifeedant  escape its of  bioassay.  reported previously  response  principal  P.  component.  JL5 p r e v e n t e d  its  product but  industry  6 8  .  helianthoides The  testing  Dihydroapofarnesal  as a n a t u r a l  f o u n d use i n t h e p e r f u m e  from  low in  7  yield a  has . not appears  6  to  fish been have  m/z 1 6 5 (50%)  m/z 1 9 0 (5%)  15 ~~  / A  m/z (5%)  208  m/z 1 5 0 (20%)  N.  7=^-  7=\  m/z 81 (70%)  m/z 69 (100%)  Figure  13.  Interpretation  X  of  the  HRMS  of  15.  Plate  3.  Archidoris Long,  montereyensis.  Simon F r a s e r  University.  Photographer:  Ron  72. CHEMICAL  I.  STUDIES  Archidoris The  OF  ARCHIDORIS  dorid  nudibranch  resembles  nobilis.  montereyensis  A. but  is  dominated methanol gram  positive  Barkley  pressure  gel  mixtures.  was using  The  subjected  to  step  material  radial  chloroform-methanol hexane-ethyl samples  of  The  (Calrjj  =  eluted  thin-layer (95:5).  major -12.5°,  1_7,  20_,  metabolite CHC1 ), 3  were  with  17_,  was  crystaline  in  between  extract  Bioassay  which guided  chromatography  100%  ethyl  separation  and  .  2 1  methanol.  hexane-ethyl  (50:40:2) 2_2  Bacillus  collected in  chromatography  Final  the  Partitioning organic  of  crude  reduced  flash  gradients  and  m  under  activity. by  i t s against  immersed  an  25  sponge  investigation  concentrated  provided  acetate-methanol  compounds  aureus  suspension.  performed  that  activity  montereyensis  and  to  shallow,  observation  chemical  Anisodoris  subtidal  relatively  antibiotic  antibiotic  lemon  from  immediately  decanted  water  the  fractionation silica  ODHNERI  montereyensi s  sea  Staphylococcus  and  aqueous  and  retained  The  present  B.C.  an  in  Archidoris  was  to  chloroform  the  of  methanol  ranges  common  bacteria  Sound,  speckled  exhibited  prompted  A r c h i d o r i s  the  communities.  Specimens  The  most  extracts  subtilis  A.  montereyensis  superficially  depth,  M O N T E R E Y E N S I S AND  acetate  acetate eluting  by  on  was with  HPLC w i t h  eluant provided  a  pure  23. an solid.  optically Electron  active impact  73. HRMS  e s t a b l i s h e d  Absorbances  in  h y d r o x y l  i t s  the  IR  spectrum  (3600-3300  functionalities. (100  molecular  MHz,  A  CDC1 )  c m  -  and  resonance spectrum  3  revealed  )  1  formula  at  as  the  e s t e r  6173.4  supported  C^HggO^.  (1730  ppm  the  presence  in  the  of  c m 1  3  C  presence  of  -  1  )  NMR an  ester.  The  'H  suggestive carbinol doubled Hz)  NMR of  that at  alcohol.  A  exchanged  an  IH)  the  contained  3  ppm  primary  resonances  derivative  one  and  singlet  IH)  24,  at  of  to  seen  two 12  a  secondary ppm  two  that  hydroxyl (J  = 7,  appropriate  12 for  resonances  glycerol  previously  = 4,  one-proton  distinctive the  (J  as  A  64.15  ppm w e r e  of  The  62.44  presence  These  those  of  highly  moiety.  3.70  proton.  deshielded  Hz,  to  Hz)  were  indicative  the  alcohol.  identical acid  was  12  12  for  doublets = 5,  alcohol  = 6,  supported  2  (J  (J  two-proton  D 0  4.22  primary  integrated  doubled  v i r t u a l l y  CDC1 )  63.63  broad,  Two  farnesic  at  a  63.95  esterified  were  of  each  with  and  MHz,  l-acyloxy-2,3-dihydroxypropane  doublets  multiplet  Hz,  a  protons  ppm  groups.  (400  unit  isolated  in from  74. Archidoris at  m/z  the  odhneri **.  Prominent  2  286  (M -C H 0 +  alcohol  3  8  )  and  258  of  the  ester  3  component  peaks (M -C +  in 1 +  was  the  H O 8  a  l 4  mass  )  spectrum  confirmed  glycerol  that  residue.  0  OH 24  The the  molecular  ester  suggested  esterified  to  unsaturation by  the  and  The  by a  at  1.61  a  for  and an  The  1  3  C  3  C  twelve  the  that  the  six  signal  a  a  is  be  in  a  (d)  and 65.54  a  an  that  three  spectral isoprene  128.6  (s) ppm.  methyl  methyl  ester  carbonyl.  accounted acyl the  for  residue. remaining and  suggested  structure  at  appropriate  methine  data  three  four  ppm  for was  (bs)  vinyl  diterpenoid  established  of  that  contained  substituents the  acid  units  olefin  2.93 to  of  accounted  revealed  at  also  portion  1  total  at  0.96,  singlet  Y  c  diterpenoic  c l e a r l y  methylene,  regular  e  could  6124.3  and  methyl  This  i  five  spectrum  0.92  experiment of  at  I7_  NMR  carbons  carbons.  17_ h a d  'H  l  trisubstituted  of  one-proton  and  the  proton  0.87,  ester  NMR  a  t  17_ w a s  of  resonances  The  °^  O  glyceride  and  methine  consisted  quarternary  Two  p o r t i o n  twenty  H  compound  o l e f i n i c  broad,  SFORD  compound  NMR  60.82,  a l l y l i c  of  C  parent  carbonyl  rings.  olefin,  eight 1  the  terpenoid  singlets  A  in  single  carbocyclic  that  ( 2 0 3l ^  glycerol.  ester  evidenced ppm  formula  shown.  three that The  75. base  peak  retro  in  the  Diels-Alder  hypothesis. resulted to  be  a  .to  2 3  defined  with  C H 5  a  spectral  product, X-ray  of  trace  the  at  the  loss  m/z C  m/z  resulting  ring,  310  parent  from  ambiguity  ^7 and  by  not  was  rational  envisioned.  quantity  verified  conducted  20  presumed  A  be  limited  analysis  was  a  this  compound  which  could  from  supported  glyceride.  a  the • s t r u c t u r e  diffraction  192,  contamination  peak  from  g  such  X-ray only  DIBAL  the  absolute  experiment  confirmed  relative  stereochemistry  the  absolute  specific with  of  at  of  by  by  the  single  Clardy  and  .  The  The  cleavage  persistent  for  this  crystal Chen  a  loss  natural  spectrum  However,  in  mechanism Due  mass  configuration to  the  rotation  known (CCXDD  literature configuration  was  -  value as  97  9°)  6 9  shown  9J_.  of  =  (5S,  8R,  the  by  reducing  reduction - 9  °)  9R  of  JL7 the  product  confirmed 10S,  but  molecule.  Comparison  the  (Calrj,  structure of  determined  alcohol =  the  14R).  the  Figure  15.  Computer g e n e r a t e d s t e r e o c h e m i c a l r e p r e s e n t a t i o n of obtained by single crystal X-ray diffraction analysis.  OH  Figure  16.  I n t e r p r e t a t i o n of  the  HRMS o f  17.  17  160  I  Figure  120  18.  I  XX)  T" 80  100 MHz b r o a d band d e c o u p l e d 17 i n C D C 1 . 3  -r60  1 3  C  NMR  40  spectrum  20  of  PPM (6)  vo  MICROMETERS 6  3.5  4000  3600  3200  2800  I  2400  2000  1800  ium) !  i  1600  10  7  1400  1200  1000  1 1 1 2 13 14  800  16 IB 20  25  600  400  F R E Q U E N C Y (CM"')  CO  Figure  20.  IR  s p e c t r u m o f _17 i n  CHClg.  T  1  1—:  r  1  '  1  ——i  1  1  r-  PPM  Figure  22.  1 0 0 MHz b r o a d 97 i n C D C 1 , .  band  decoupled  1  3  C  NMR  spectrum  of  (6)  MICROMETERS (/im)  4000  3600  3200  2800  2400  2000  1800  1600  1400  1200  1000  800  600  400  FREQUENCY (CM"') CO Figure  23.  IR  spectrum  of  91_  i n  CHC1 3  85. Material i n i t i a l two of  with  fractionation  minor JL8,  peaks  suggested  i t  glycerol  at  m/z  and  a  deshielded  in  well  +  'H  protons  i n  with  alcohol  97_  to  the  a  sharp  reduction  the  JL8.  product  of  the  1 Q  to  0 ) 5  the  singlet  at  ppm.  A  ppm,  which  and  coupling  glyceryl  acetate  1-acy  17.  6  diterpenoid  2.48  shifts  was  mass  contained  18  64.08-4.25  the  formula  +  methyl  Reduction  which  contained  acetate  at  the  (M -C H  of  of  proton  chemical  established in  respects  those  for  in  intense  d  of  spectrum  at  n  a n d 258  h  to  reported  residue  0 )  x Q  a  addition  to  the  3-acetoxypropane gave  H  NMR  multiplet  extracts  2 5 m}°5'  5  identical  with  previously  DIBAL  C  exchangeable  patterns  2_5,  as  (1:1)  The m o l e c u l a r  1T_.  f r o m _17 b y  five-proton  corresponded  to  (M -C H  addition  single  acetate  montereyensis  HRMS  The  virtually 1_7,  A.  286  differed  of  hexane-ethyl  related  by  fragment.  resonances portion  of  metabolites  established  spectral  62.11  eluted  loxy-2-hydroxyof  acetate  identical  in  1J3 a l l  86. A  second  monoacetyl  positional of and  C  2 5  H  1 + Q  isomer 0  M -C H +  6  and  5  1 Q  at  residue  at  IH),  4.32  identical the  The  5  of  62.09 63.76 (dd, to  glycerol  Reduction of  J  as  18  showed  0 .  characteristic singlet  of  glyceride  1  the  J  = 6,  those moiety  also  losses  NMR  spectrum  contained  portion  Resonances = 6 Hz, 14 H z ,  of  2H), IH)  19_ was  19 w i t h DIBAL a l s o  to  had a m o l e c u l a r  spectral  previously of  appeared  mass  terpenoid  ppm. (d,  H  it  19,  of  the  4.26  and  observed  JL7  (dd,  M -C H O +  5  for  (m,  1 0  1 +  resonances  and  J  a  formula  a  glyceryl  5.06  acetylated  of  be  methyl acetate  = 7,  12 H z ,  IH)  ppm were  26_ and  confirmed  at  the  gave a l c o h o l  97.  2-position.  90.  u X  u c 5 o > •J z u D  o  O  E D  u  -p O d) ft w  OS M  CN <D U  3  •H  NOISSIWSNVbi  !N30d3d  A  third  minor  purification active C  solid  18 30°'+*  cm  H  - 1  )  I  t  and  glyceride  ([<X]D  i- f  s  n  ester  Fragments 190  of  in t  8  l f  +23.1°)  =  r a r e  d  mass  was  with  spectrum  that  a  _2°_  w  a  - 1  )  formula  bands.  (M -C Hg0 ) +  3  glyceride  a  of  (3600-3300  absorbance 218  HPLC  optically  hydroxyl  m/z s  an  molecular  cm  at  during  20_ w a s  showed  (1730  spectrum  revealed  isolated  Compound  carbonyl  the  (M -C[ H O ) +  metabolite  and  3  ester.  OH  Resonances 62.48 (dd, Hz,  (2H, J  =  IH)  that  and  alcohol. methyl  12  suggested  =  H  21  R  =  Ac  in  the  Hz,  4.22  'H  1.62, that  IH),  (dd,  glycerol Methyl  at  R  exchangeable), 4,  the  20  the  (400  MHz,  3.63  (dd,  J  3.95 J  =  5,  fragment  singlets and  NMR  at  a  single  C, _  acyl  (m, 12  Hz,  60.89,  12  4.15 IH)  Hz,  0.92  portion  IH),  (dd,  ppm,  acylated  olefinic  spectrum  3  = 6,  IH),  was  CDC1 )  J  =  3.70 7,  on  a  primary  0.98,  a  proton  at  5.57  the  12  established  and  of  at  ester  vinyl  was  ppm a  92. b i c y c l i c  sesquiterpenoic  singlet  deshielded  a l l y l i c  methine  e x p e r i m e n t  in  32).  orientation in  the  an  obvious  with  DIBAL  (NMR,  HRMS)  identical  the  p r o v e d  of  the  sesquiterpenoid  t h e  GC  a b s o l u t e of  20_ i s  98  diaxial  other  protons  and  to  m/z  109,  supplied  by  drimenol  =  shown  of  an  reduced 9J3.  The  alcohol  -20°)  ([OC]D  (5S,  be  from  Armstrong)  c o n f i g u r a t i o n as  the  obtained  R.  ([a]rj  of  9S,  =  s  retro  loss  time  a  could  124,  drimenol  w  of  was.  those  20.  a l l  hypothesis at  (see  1-3  derivative  retention  rotation of  a  structure,  respects  value  protons  two  fragments  the  and  optical  portion  this  an  difference  satisfied  ring,  (kindly  literature t h a t  that  B  and  of  p a r t i c i p a t e s  alkyl  sesquiterpene  a l l  (±)-drimenol  Comparison with  in  two  drimane  for  confirm  known  98  authentic  To  the  the  data  The  spectral  of  NOE  indicated  proposal  mass  methyl.  of  proton  support  A  p r o t o n  NOE  system.  cleavage  to  t h i s  one-proton  characteristic  carbonyl.  methine  Further  spectral were  the  broadened  was  relaxation  structural  prominent  ester  t h a t  ring  found  A  ppm  observed  between  data.  additional  an  the  spectral  Diels-Alder  to  The  b i c y c l i c  in  62.96  showed  significantly Figure  a  to  acid.  of -20°) o f 10S).  7  .  0  98 7  1  the  93. A  related  montereyensis diagnostic M -C H +  6  NMR  1 0  O  5  A  20  were  gave  extracts.  The  spectral  and a  resonance  revealed  that  also  at  62.11 be  a  formula at  fragment  5  observed  d r i m e n o l 98.  i n 2_1.  -20  1 Q  0  H  A.  3 2 ° 5 ' and  £ t  i n t h e 'H glyceryl  64.05-4.24  ppm  was 1 , 3 - d i a c y l a t e d .  characteristic of the sesquiterpenoid  also  from  +  monoacetylated at  <  M -C H  ( s , 3H) ppm  m u l t i p l e t  the glycerol  recovered  molecular  fragments  i t to  five-proton  demonstrated Resonances  2_1, w a s  mass  spectrum  ester.  monoacetate  Reduction  of  portion  2_1 w i t h  of  DIBAL  CH=0H  m/z 279 (10%)  m/z 295 (5%)  -C H Q 3  20 —  e  3  m/z 310 (15%)  Retro  m/z 218 (75%)  Diels-Alder  -co  Cleavage  •CH, m/z 190 (45%)  m/z 187 (40%)  m/z 109 (100%)  m/z 124 (70%)  Figure  28.  Interpretation  of  the  HRMS  of  20.  VO  Figure  30.  100 MHz b r o a d band d e c o u p l e d 20 i n CDC1 . 3  1  NMR  spectrum  of  2.0 Figure  32.  1.5  1.0  0.5  1  400 MHz 'H NMR d i f f e r e n c e NOE s p e c t r u m o f 20 when t h e a l l y l i c m e t h i n e p r o t o n a t 6 2 . 9 6 ppm was irradiated: a) enhanced s i g n a l s o f two alkyl protons; b) incompletely subtracted methyl signals.  r  PPM (6)  102. Another yield  trace  after  extensive  optically  active  formula  C,  of  metabolite  o  H  o n  could  HPLC  be  isolated  purification.  (CaDp  o i l  only  +9.7°)  =  It  with  a  in  low  was  an  molecular  0,.  22  Compound cm  - 1  ),  a  mass  resonances 12  Hz,  J  showed  spectral  [63.62  IH),  (dd,  2_2  infrared fragment  (dd,  J  =  3.95  (m,  1H),  5,  12  Hz,  =  7,  IH)  unsaturated  the  UV  (1700  absorbance cm )  methyl  Methyl  ppm.  accounted in  22,  for  (X  m  ppm)  were  methylene The  moiety a  aB  three  indicating  and  IH) , J  =  3  3.70  7,  12  The  228  nm,  MeOH),  an  NMR  olefinic at  evidenced  and  ester  and  of  the  total  units  the  C.  acyl  IR a  portion  of  a  of  an  was  from  spectrum deshielded  0.93 at  ppm).  and 4.55  exocyclic of  5, 4.24  (65.70  singlets  unsaturated four  IH),  deduced  proton  60.85 by  for  NMR =  presence  readily  'H  J  d i a g n o s t i c  be  the  1700 'H  (dd, Hz,  could  observed  was  3  ppm]  in  and  +  residue.  =  x  resonances  (62.16  singlets  exocyclic 4.79  and  - 1  vinyl  ester  (3425,  (M -C Hg0 )  Hz,  (dd,  l-acyloxy-2,3-dihydroxypropane aB  ion  12  4.18  absorbances  an and  olefin  unsaturation a  monocyclic  103 . sesquiterpenoid. identical  to  Resonances  those  reported  trans-y-monocyclofarnesic acid the  minor  metabolite  22^ was  spectral  fragmentation  peaks  m/z  at  with this  176  proposal  (C  of  1 3  H  (see  for (99)  )  a  n  the  7 2  ,  as  the  included w  a  were  ester  of  structure  shown.  ^8^13^' 36).  residue  methyl  thus  which  d  Figure  99  acyl  assigned  pattern, 2 0  the  s  Its  of  mass  prominent consistent  m/z 176 (85%) Figure  36.  Interpretation  of the  HRMS o f  22.  107. A  very  collection of  C  minor  o f A.  2 0 3 2°2  constituent montereyensis.  indicating  H  Infrared  absorbances  presence  o f an a c i d  spectrum  of  methyl  an  2.20  unsaturated carbon. one-proton 1.60 and and  monocyclic An  m/z at  g  difficult  a  9  to  isomeric  m/z  177  should  The  result  peak  compound  i n a  at  NMR  vinyl an  to  aB  the  B  by  a  singlet  60.83  and  4.53  at  0.91  (bs,  metabolite  i s  consistent  with  the  1 6  ),  109  this  IH)  was  a  reasonable  on  strong  bond  1 3  )  and  hypothesis  be 40),  t h e mass  the  (CgH  at  m/z  177  signal  of  expected this  spectrum  between a t m/z  NMR  intense  fragmentation  could  (Figure  with  showed  observed from  101  ion  of  at  the  a  f o r  methyl  methylene  'H  evidenced  that  support  rationalize  cleavage  was  singlets  spectrum 9  consistent  and  attached  proposal  124 , ( C H  fragment  i n the  the  acid.  mass  which  formula  revealed  - 1  ( b s , IH)  vinyl  suggested  and  prominent  the  entirely  ppm  cm  appropriate  a  single  of unsaturation.  1695  group  and  a  a molecular  olefin  methyl  only  Resonances  methyl  structural  3  (C H )  and  were  65.07  i n  units  65.70  exocyclic  IH)  +  However,  allylic  an  (M -CH ),  81  at  grounds  100.  289  ppm the  diterpenoic  biosynthetic  5  trisubstituted  of  obvious  compound  3H)  with  (bs,  of  proton  aliphatic  resonances 4.76  total  functionality.  multiplet Two  found  I t had  a t 3225-2500  (bs,  second  ppm.  a  olefinic  acid  A  was  a  data  i s  signals  at  base  peak  (Figure  39).  (C  1 3  )  and  the  i s not A  C-10  (C-H .). 1 C  i s  While  t o produce  either.  123  2 1  100.  structure  C-9  H  doubly of  101  While  108.  z  304  15%  Figure  39.  Possible  mass s p e c t r a l  fragmentation of  100.  109.  m/z 304 15%  m/z 289 40%  .  m/z 177 50%  Figure  40.  P o s s i b l e mass s p e c t r a l f r a g m e n t a t i o n o f p r o d u c e a peak a t m/z 1 7 7 .  101  to  110. a  peak  at  m/z  c o n s i d e r a b l y  123 more  is  observed,  i n t e n s e .  rearrangement  in  compound  124  an  analogous  ion,  but  compound  101  It  is  isomers ions  generally olefin  yield of  a l l  both  100  quite  of but  proof the one  and  and  proton  natural  a  at  a l l y l i c readily  transfer  mixture  the  mass  101.  65.07 of  could  peak  124  is  M c L a f f e r t y  provide and  the  cleavage  m/z in  plausible.  sharp, at  An  100  that  isolated,  proton  Definative  less  possible  was  from  seems  the  the  collection  the  ppm  may  and A.  in  support  its  was  two  o l e f i n i c  shows  fragment  the  asymmetric  structure(s)  of  the  spectrum  Resonances  but  product  of  'H  NMR  are  of  the  nature this  prevented  absence  montereyensis.  from  argument. by  a  low  extracts  Figure  41.  400 MHz 'H NMR spectrum of the diterpenoic acid(s) tentatively identified as 100 and/or 101. Peak C i s an u n i d e n t i f i e d contaminant.  113 . The  final  montereyensis spectral +  7  had  a  3  ions,  resonances  Hz,  I H ) , 3.68  (dd, J  i n good  agreement  glycerol  at  ethers  7 3  .  =  5,  A  C  the  m e t a b o l i t e as t h e known Ether  against alone  discernable  activity  to  observed  be  i g  2  ether  0  5  fatty  The  (dd, J  = 6,  acid  (m,  IH)  225 1  H  12 ppm  values f o r  alkyl  spectrum  residue  establishing  1-0-hexadecyl  glycerol  in-vitro .antibiotic  activity  and B a c i l l u s  n C  mass  and  2  IH) and 3.82  compound  and  3  A.  moiety.  3.62  for  subtilis, the  f o r c r u d e A. m o n t e r e y e n s i s  23  4 Q  +  4H),  responsible  OH  H  from  (M -C H 0 )  i n t h e 'H NMR  aureus  C  reported l i t e r a t u r e  23. shows p o t e n t  Staphylcoccus appears  (m,  reduced  J 6  clearly  71  255  12 Hz,  with  was  (2_3) *.  m/z  63.41-3.53  isolated  formula  ) suggestive of a glyceryl  NMR  were  constituent  molecular  fragment  (M -C H 0 3  trace  l6 33 H  and i t  antibacterial extracts.  n C  HO OH  23  l6 33 H  116. II.  Archidoris The  principal  odhneri acid  were  were  rigorous was  HPLC  of  farnesic  purification  a  low  acyl  63.81  Hz,  demonstrated  spectrum acid  The was  extracts  r e s p e c t s  to  montereyensis. present  in  purified  =  and  _17 in the  r e t e n t i o n  signals  mixture  trace  of  in the  the  ^0  of  4.90  They  and  molecular  were  They  'H  spectrum  =  6  secondary  in  the  mass  diterpenoic A.  identical  in  a l l  of  A.  2^3 w e r e evidenced  HPLC  glycerides.  HO  J  from  were  the  a  isolated  ether  in  of  glycerol  the  The  7J_.  times  terpenoic  a  (pentet,  pattern  and  amounts.  NMR  glyceride  m e t a b o l i t e s  _22  After  the  at  A.  of  21_ h a d  subsequently  c o r r e s p o n d i n g  Glycerides  of  characteristic  and  structure  yield.  extracts  isomer  esterified  were  JL8  farnesic  constituents.  fragmentation with  low  4H)  Archidoris  three  Methanol  .  signals  Hz,  of  the  Resonances  was  observed  be  Compound  NMR  6  i t  extremely  c h a r a c t e r i s t i c diagnostic  J  consistent  glycerides  odhneri  (t,  to  positional  residue.  at  hydroxyl.  'H  2 4  extracts  trace  yield.  and  fragment IH)  J26 for  C ^ H ^ O ^  acid  and  re-examined  in  in  reported  2_5  2A,  isolated  formula  metabolites  previously  glycerides  odhneri  24  odhneri  of  trace a  also by and  p a r t i a l l y  Figure  45.  270  MHz  'H  NMR  spectrum  of  27  in  CDC1 . 3  118. I l l .  Antifeedant The  function  presumed test  to  this  and  A.  theory,  the  available isolated sculpin These  of  nudibranch  involve  odhneri  Since  Bioassay  glycerol  were  assays from  in  chemical  the  Oligocottus fish  are  regularly  p o t e n t i a l  p r e d a t o r . and  Commercial  of  Control  were  offering  pellets  and  monitoring  period  of  time.  verified the  testing  assessed by  by  as  a  the  were  the  active  q u i c k l y  i n h i b i t i o n  to,  was  or  pellets  of  avoidance  pellets  r e j e c t e d . observed,  were In  both  feeding  c o l l e c t e d to  t e s t i n g . varying  i n  acetone. assay  and  treated  during the  a  fixed  fish and  was after  i n h i b i t i o n  treated  cases  subsequently  The  behavior  by  the  legitimate  before  the  mouthed a l l  where  a  control  of  pool fish.  with  Feeding  of,  test  acetone.  a b i l i t y  was  tide  were  response  pellets.  cessation  a  p r i o r  with  sculpins  feeding  treated  The  metabolites  feeding  untreated  marked  indifference  Although  The  offering of  their  h  than  areas  coated  only  activity.  constituents  as  in  To  montereyensis  represent  were  treated  alternately  chosen  24  nudibranch  A.  those  s c u l p i n s  for  p e l l e t s  concentrations  involved  The  organism.  screened.  thus  generally  compound  only  was  and  starved  shrimp  pellets  more  were  is  antifeedant  encountered  collected  the  from  fish  required  maculosus  were  i n t e r t i d a l l y  for  quantity  nudibranchs  of  derivatives  nudibranchs,  sufficient  chemicals  defense  evaluated often  skin  the  was  followed pellet.  fish,  they  where  feeding  offered  c o n t r o l  119. pellets are  were  shown  rapidly i n  montereyens i s antifeedants.  consumed.  Table and  IV. A.  Two 2_0  antifeedant  activity.  skin  chemicals  showed the  that  Table  IV.  has  at  feeding  and  While  y e t t o be  least  two of  Antifeedant  Diterpenoic acid g l y c e r i d e 17  5  1 "-acetate  5  Farnesic acid g l y c e r i d e 24  major  the  glycerides  proved  trace  inactive  constituents,  ether the  of  2_3  role  defined,  nudibranch  of this  bioassay from as  potent abundant  preliminary  metabolites  do  fish  drimane  exhibited t h e more  A.  study inhibit  Bioassay  Activity  Results  3  Concentration (J-g/mg  Activity  160  -  -  110  -  18  +  NT  NT  26  -  111  -  18  +  NT  NT  18  Sesquiterpenoic acid g l y c e r i d e 20  results  fish.  'Concentration ug/mg  Compound  Glyceryl e t h e r 23  the  glyceryl  behavior  Fish  The  odhneri  of  derivative  The  a) NT)  _a  + p e l l e t not eaten - pellet eaten no t e s t  120. IV.  Biosynthesis We  were  metabolites in  their  diet. of  terpenoic novo  demonstrated  3  established  .  8  nudibranchs 1 1 +  salt  the  that was  Carbon  was  the  with  methanol.  A.  for  24  The  HPLC.  To  remove  provided and  moiety  h  is  a  dietary  montereyensis  but  in  sites.  none  of  Chapter  polygodial has  in  the  1,  de  (5_5),  by  previously  been  relationship  was  derivatives,  the  synthesized  by  the  or  an  aquarium  were  and which  trace to 98  24  an  The and  were  then  A.  extracted  in  the  to  purity  associated  with  the  from  Reaction  usual by the  glycerides  Purification  crystalized o i l .  live  nudibranchs  taken  impurities,  which  of  fractionated  alcohols.  was  gland  specimens.  radioactivity  DIBAL  (102),  digestive  in  with  97_  acid-dibenzylethylenediamine  20_ a n d  any  with  alcohols  farnesol  organism  collection  23,  of  Compounds  an  A.  glycerol were  the  extracts  compounds  reduced  the  odhneri  and  were  ether  limbata  mevalonic  fashion  glycerol  at  mentioned  others  into  and  incubated  and  predator-prey of  source  investigated.  injected  were  a  to  panicea  sesquiterpene  one  labeled  montereyensis  As  Dendrodoris  only  traced  sponge  glyceryl  Since  for  p o s s i b i l i t y  the  the  odhneri.  species,  glycerides.  nudibranch  be  A.  Halichondria  eating  of  and  often  provided  biosynthesis dorid  determining  Archidoris  found  acid  in  can  sponge  both  extracts  Metabolites  montereyensis  The  frequently  Sponge  the  A.  nudibranchs  component was  Archidoris  i n t e r e s t e d  in  found  of  of  by  HPLC  solution farnesol  121. with  dinitrobenzoy1  chloride  4-dimethylaminopyridine dinitrobenzoate derivative  All results of  1J  *C  terpene 24.  derivatives of  which  mevalonic portion  the  the  shown  acid  presence  crystaline  of 3,5-  103.  were c r y s t a l i z e d p r i o r  are  of  gave  in  in  Table  incorporation  terpenoic  acid  V. were  to  counting,  Significant observed  glycerides  17_,  the  levels for 20.  the an<  ^  Table  V.  Results  of  l H  C  Mevalonic  Acid  Incorporation  Metabolite  Nudibranch  Experiments D e r i v a t i v e used in counting  A c t i v i t y DPM/mg  550  A r c h i d o r i s montereyensis  3260  1810  A r c h i d o r i s odhneri  24  a)  d i s i n t e g r a t i o n s  per  minute/milligram  103  3  123 . V.  Additional Biosynthetic Preliminary  carried  out  biosynthetic with  luteomarginata. isolated  from  acetate,  gave  significant  T.  luteomarginata acid.  The  albicanol results they  should  derivatives observed  taken  prior were  the  preliminary  can  not  not  to  to  be  fed 8_1  of  HPLC  1 1 +  . was  but  certainly  out.  35  R = H  36  R = Ac  from  to  The  these  natural  converted  not  to  of  the  that  the  with  trace  possible  raised  further  give  nudibranchs,  possibility  was  C.  mevalonic  crystals  questions warrent  similar  While  associated It  showed  a  base  the  and  but  actually  ruled  with  caution.  The  labeled  which In  by  (7_5),  C  C labeled  purity  obtained.  1 1 +  3815 dpm/mg.  with  Cad1ina  recovered  cleaved  counting,  experiments results  with  biosynthesis  interpreted  radioactivity  impurities repeat  be  were  derivatives  was  w i t h an a c t i v i t y situ  been  was  {36)  also  triophamine  derivative  acetate  were  and  radioactivity.  moiety  in  of  had  were i n j e c t e d  acetate  indicate  products  dpm/mg)  that  (3j>)  that  monoacyl  albicanyl  catalinae  hydrolysis  catalinae  the  investigations  Triopha  Base  (1201  experiment,  Studies  by  study.  to  these  SUMMARY AND  Chemical isolation are  these  investigation  and  often  structural  present  studies  spectroscopic chemists  are  also  origin  or  natural  by  to  the  thesis  marine  from  s t r u c t u r e catalinae,  spectral  analysis by  product.  The from  functionality acyl  an  of  that  and  of  a  It  is  define  the  of  are  imposed  presented  beyond  With  studies  limitations  assignments  (7_5)  by  new  ,  in  this  nudibranch the  i n t i a l  This  however class has  of  from  methods  assignment  synthesis  triophamine  i s o l a t e d  classical  guanidine  sources,  represents  such  and  these  metabolites.  work  degradation.  unambiguous  marine  biosynthesis  clearly  in  product  Addressing  can  of  attempted.  solved  of  advances  challenge.  l o g i s t i c a l The  success  Natural  compounds.  triophamine  was  to  organisms,  by  also  that  source,  i n v e s t i g a t i o n s  presence  residue  the  structural  were  Triopha  compounds  on  constituents  part  secondary  environment.  studies  confirmed  of  d i f f i c u l t  the  Recent  formidable  marine  Several  The  in  a  role  requires  techniques.  experiments  concentrates  structural  large  novel  remains  design  more  i n  of  of  quantities.  interested  precise  products.  The  due  function  nudibranchs  identification  separation  products  made  of  trace  is  questions  d i f f i c u l t  often  in  and  physiological latter  DISCUSSION  was  the  of then  n a t u r a l  is  not  the  diacyl  guanidine  natural  compound.  of  an  uncommon  unusual  in  carbon  125. skeleton  and  difficult  to  its  rationalize  an  isoprenoid  be  ruled  result  biosynthetic  although  Alternatively,  from c o n d e n s a t i o n o f  equivalents  (Figure  speculative  as  not of  this  the  obvious.  the  C  1 Q  -unit  possibility  hydrocarbon  It  46).  Such  a  can  moiety  pathway  biosynthetic  is from  is  not  could  a c e t y l - C o A and two b u t y r i c  appropriate  t r i o p h a m i n e were n o t  is  the b i o g e n e s i s  precursor,  out.  origin  acid  purely  studies  of  attempted.  0  Figure  46  a)  C^ -hydrocarbon p o r t i o n of  b)  p o s s i b l e p o s i t i o n of  Q  The p r e s e n c e the  nudibranch  was  not  more on  thorough  in  triophamine  suggests  substantiated  potential  found  of  the  it in  investigation predators dorid  is  plays a  in  Polycera  acetate  in  the  the  mucous s e c r e t i o n  antifeedant natural  order.  role,  but  bioassay.  product's  Triophamine  tricolor.  (75)  units  a defensive  fish of  triophamine  Since  this A  effect  was a  of  also  dietary  126. source of  de  for  triophamine  novo  could  biosynthesis  i n d i c a t e  that  not  was  be  located,  investigatged.  triophamine  is  indeed  the  p o s s i b i l i t y  Tentative  results  produced  by  the  nudibranch. The  odoriferous  dihydroapof arnesal Its  structure  (1_5),  was  interpretation  from  represents  a  was  the  on  correlation contains  regular  of  isolated  proposed  and  Dihydroapofarnesal derived  constituent  14  isoprenoid  f i r s t  in  the  with  only  Anisodoris  nobilis,  only  trace  amounts.  basis  of  s p e c t r a l  related carbons,  aldehyde yet  precursor.  nor-sesquiterpene  is As  68.  clearly such,  isolated  i t  from  a  nudibranch. Chemical series  of  studies  terpenoic  of acid  principal  metabolite  f i n a l l y  proven  by  compound.  diterpene  previously nudibranch  Casella  (105)  nature,  can  t h e  i t  a c y c l i c  pyrophosphate.  7  6  in  via  .  arise  to While from  p r e c u r s o r  spectral Its  reduction of  8  and  a  a  skeleton  the  data  but  a b s o l u t e to  furanoditerpenes 2  series  is  straightforward  a  of  a  known  glyceride  isoagathalactone this  revealed  structure  from  skeleton  atromarginata  related  a p l y s i l l i n  carbon  observed  The  a n a l y s i s .  established  been  metabolites  deduced  X-ray  was  montereyensis  glycerides.  1T_ w a s  configuration The  Archidoris  from of  (104) rather  1_7  has the  sponge 7  5  rare  cyclization  and in of  a l l - t r a n s - g e r a n y l g e r a n i o l  OAc OAc  104  Diterpenes of  marine  component  diterpene marine  is  7 7  .  In  acyclic  glycerol is  most  and  moiety.  diterpene  source  isolated  o f mixed b i o g e n e s i s  organisms  carbohydrate polycyclic  105  mixed  ether  the  to  _17  an  previously  variety  diterpenoid aromatic  represents  biogenesis.  derivative the  cases,  bound  Compound of  a r e known f r o m a  The  a  rare  only  reported  other from  O-geranylgeranylglycerol  f r o m t h e brown seaweed D i l a p h u s  fasciola  7  8  or  a  106,  .  OH  106  The  structure  sesquiterpene chemical skeleton,  and  portion  of  interconversion found  in  absolute  stereochemistry  glyceride to  was  d r i m e n o l j)8.  sesquiterpenes  from  of  established  The d r i m a n e  the by  carbon  terrestrial  and  128. marine  organisms,  nudibranch  a  of  sesquiterpenes is  structural  Compound  exhibits  chemical  activity  common  metabolites.  sesquiterpenes, array  is  20,  antifeedant  that  intimately  the  like  with  the  glyceride  22.  several  many  drimane  The  observed  biochemical  associated  of  activity.  f u n c t i o n a l i t i e s  suggests  feature  diverse  i n  basis  these  for  their  drimane  carbon  skeleton. The  sesquiterpenoic  extremely  low  yield  interpretation. the  simple  nature, a l g a e  7  been  and  such  the  no  2  .  obtained  metabolites, of  any  glyceryl and  from  sponge.  the  sponge  have  has  1  .  been  been  reported  This  found  of  in from  trans-  from  the  has  also  sponge  sesquiterpene-hydroquinone  but  analysis  suspected  terpenoid  diet.  spectral  have  isolated  8  in  possessing  been  the  This  from  ester  was  ether  isolated  s  methyl  panicea  panicea  a  sesquiterpenes  The  mixed  w  deduced  skeleton  107  of  H.  sign  constituent, nudibranch  few  .  0  acid  source 8  8  Halichondria  montereyensis showed  structure  compounds  s p o n g e s  sponge  derivatives  its  monocyclofarnesyl  monocyclofarnesic dietary  and  Relatively  however 9  acid  23.'  of  source sponge  compounds. w  a  s  suggests  found that  of  samples A  in  A.  single  both  ether  22_  the is  129.  107 The n u d i b r a n c h A . of  farnesic  that  it  also  identified dietary  acid  o d h n e r i was known t o  glycerides  2 4  from A.  source  montereyensis.  for  any o f  these  spectral the  isolated  montereyensis  product  from A. or  101.  was  actually  such  caulerpol  (109)  extracts  as  from a  composition consistent discrepancy  of in is  8 3  retinol  .  While  single the  all  are  a  and  free  to  find  a  biogenetic  diterpenoic  evidence of  the  (108)  that  two  related and  free  collection  of  terpenoic  acid  collections.  The  n o t known.  glycerides  c o u l d be t e n t a t i v e l y  clearly  the  acid  revealed  We were u n a b l e  of  some  a mixture  The p r o p o s e d s t r u c t u r e s compounds  is  terpenoic  family  substances.  structure  There  a  examination  analysis  considerations,  100  Close  c o n t a i n e d a number o f  Combining  as  .  contain  assigned  the  isolated  olefinic  isomers.  to other  the  algal  acid  only  A.  natural  metabolite occured  montereyensis,  glycerides cause  acid  of  was  this  in the  quite  apparent  130.  108  OH  109 An  incorporation  indicated terpene  that  the  study with  two  Care  experiment  to  associated  radioactivity.  of the only  eliminate  the  second  is  has  are  capable  throughout  contaminants  We  are  from de  novo  their  that  the  incorporation  i n t o the terpene r e s i d u e s . direct  this  and  confident  of  This i s  biosynthesis  of  been demonstrated i n nudibranchs.  p h y s i o l o g i c a l f u n c t i o n of nudibranch s k i n chemicals  often  presumed  substance's Antifeedant assay  taken  i n s t a n c e • where  secondary m e t a b o l i t e s The  trace  l a b e l e d mevalonic a c i d species  was  r a d i o a c t i v i t y resulted  labeled precursor  C  Archidoris  biosynthesis.  measured  1 4  to  defensive bioassays  organism,  be  potential  require  making  it  interpretation  of the  of  Archidoris  constituents  and  voracious  feeders.  defensive,  but  remains  a negative  difficult  t e s t subjects  to  fish of  a  avoid  food  that  a  problem.  response  behavior.  utilized  Rejection  evaluating  from  the  subjective The were  pellets  bioassay active treated  131. with ether  the 23  sesquiterpenoic was  consumption of metabolites  in  such  acid  glyceride  dramatic  contrast  the other t r e a t e d p e l l e t s  can t r u e l y i n h i b i t  ^0  and to  glyceryl  the  t h a t we f e e l  f e e d i n g by f i s h .  rapid these  EXPERIMENTAL  General The  1  WH-400, Varian as  an  H  and  Bruker CFT-20  1 3  C  WP-80,  on  standard  resolution  an  Perkin-Elmer  model  ultraviolet  Bausch  and  rotations  A  were  measured  using  and  , 1 +  liquid 2  l/2  2  instrument A  were  and was  with  60  on cell  p r e p a r a t i v e HPLC.  mass  were  employed unless  spectra and  high-  an  MS-50  on  CHC1 with  3  a  Perkin-Elmer  and  CHC1  used  or  3  to  were  recorded  with  and  on  as  a  solvent  Cary-14  or  Optical  model  141  MeOH a s  solvent.  determine  melting  uncorrected. PF-254  employed  Whatman  a  was  Merck  a  XL-100  spectrophotometer.  230-400  was  Bruker  solvent  measured  2000  are  Gel  chromatography  detector. for  H 0  cm  Gel  Silica  chromatography CaS0  10  values  Silica  Merck  specta  apparatus  these  Merck TLC,  a  as  3  recorded  spectrometer  Spectronic  Varian  spectrometer  were  absorbances  Fisher-Johns  points  710B  Lomb  polarimeter  CDC1  MS-902  Infrared  on  T e t r a m e t h y l s i l a n e was and  spectra  spectrometer.  recorded  Low-resolution  A.E.I.  mass  were  N i c o l e t - O x f o r d 270,  indicated.  recorded  and  spectra  spectrometers.  internal  otherwise  NMR  was  used  for  Mesh  was  used  Silica in  radial  performed  Perkin-Elmer Magnum-9  Gel  on  60  TLC. a  A l l chromatography  for  flash  PF-254  with  High  pressure  Perkin-Elmer  LC-25  Partisil  preparative  Series  refractive  10  column  was  s o l v e n t s were  index used HPLC  133. or  reagent  grade.  Hewlett-Packard conductivity  Collection  5830  or  were  Columbia.  methanol  or  Extraction  and  of  methanol. 3  The  Triopha  a  dark  gel  with  500 gave  over  viscous  o i l  g)  on  a  thermal  absorbing  spot  on  contained  triophamine  SCUBA.  m in  Barkley  Sound,  was  and  sulfate  TLC. were  the  7.8%  dry  ethyl  Fractions further  as  a  from  was  chloroform  The  to o i l  a  column  of  in  hexane  as  strong the  purified  in  the  evaporated  on  acetate  of  that  weight).  chromatography  be  nudibranchs  The  and  to  immersed  suspension  sodium  and  c o l l e c t e d  Evaporation  chloroform.  visualized  found  f r e s h l y  and  15%  extract  immediately  aqueous  g,  (75)  sequence  methanol.  in  temperature.  to  were  (3.1  immersed  employed  mi  flash  was  using  Triophamine  were  an  hand  low  decanted  using  Triophamine  at  was  water  by  1-20  Seventy-five  dried  (160  of  catalinae  between  was  stored  by  immediately  following  solvent  extracts  eluant.  and  of  were  procedures  fractionated  s i l i c a  performed  detector.  depth  Samples  of  times  partitioned  a  a  Chromatography  The  combined  fraction  was  u t i l i z i n g  collected  e f f i c i e n t .  specimens  was  at  triophamine.  most  yield  were  chloroform  variety  washed  instrument  ionization  made  British  the  A  flame  specimens  Collections  purify  chromatography  Data  A l l  A  Gas  UV  (254  column by  nm) which  preparative  134. thin-layer 0.5)  to  chromatography  yield  instances,  f i n a l  chloroform  C  =  H  cm-1; Hz, J  o i l ;  251nm  N  c  a  l  *  [  a  ]  363.2885;  J  612.1 50.3  =  (q), (d),  223  (50),  specimens identical  7 H z , IH)  A charcoal  as  charcoal  was  yield (1:1  a  of  ether,  367.3198;  'H  363 . 2885,  0.95 (t,  (m,  1165  J  = 7  2H), 2.01 (q,  2.30-2.48  (m, 2 H ) ,  acetone-d ) 6  26.1(t),  40.1 (t),  1 8 5 . 6 (s) ppm; (25),  238 (60),  extracts  fractionated  o f 30 i n  an  Pd  on  (7_5) .  (75) i n  stirred and  = 0.4)  (78).  UV  1700, 1635,  The methanol were  l i p i d  MeOH) ;  o b s.  334 ( 2 5 ) , 281  ethanol under  the  Preparative Rf  1.7,  158.9 (s),  mg)  o f f  o i l .  with  5 mg o f t r i o p h a m i n e  (8  was  f i l t e r e d  tetrahydrotriophamine cal.  7_5  Partisil-10  NMR ( 1 0 0 M H z ,  t r i c o l o r  =  some  = 7 H z , 3H) ,  140.4 (s),  to provide  o i l .  Rf  In  =  3330,  23.2 (t),  (100).  ether,  p e r s i s t e n t  13Hz,IH),  13.1 (q),  catalyst  yellow  hexane/  = 6,  of Triophamine  solution  J  a  HRMS 3  3  Polycera  Hydrogenation  on  (C  (CHC1 )  ppm; 1 C  205 ( 4 5 ) , 86  manner  -7°  MeOH);  (60), 348 (10),  of  HPLC  = 7Hz, 3H), 1.46-1.67  120.5 (d),  m/z 363  yellow  remove  80.91 (t,  12.9 (q),  MS,  by  =  D  IR  = 7Hz, 2H), 2.12 (dd, J J  a  t o  e  3H), 1.56 (d,  (q,  light  ( 1 2 0 0 0 ,  ' H NMR ( C D C l g )  5.21  as  required  7_5  21 37 3°2  mg)  hexane/diethyl  p u r i f i c a t i o n  was  impurity. ^max  (51  75  (1:1  2  5%  f o r  48h.  solution  thin-layer  provided  HRMS,  H  with  evaporated  3  to  chromatography  4 mg ( 4 9 % y i e l d )  obs 367.3198,  NMR ( 2 7 0 M H z , C D C 1  The  )  60.83  (t,  c  i i+i 3 2 H  2  of  J  N  0  =7Hz,  135. 3H), (m,  0.84  (t,  6H),  (10),  J  = 7Hz,  1. 5 5 - 1 . 6 5  338  (15),  (m,  296  with  temperature vacuo  and  ethyl  acetate  yield  2  MeOH);  283  drops  2h.  The  partitioned  thin-layer  MS,  m/z  = 7Hz, IH)  268  (30),  3H),  ppm;  1.15-1.40  MS,  240  triophamine  1  N  NaOH  reaction  of  (M ),  m/z  367  (70).  (10  was  ethyl  3  (10),  \  m  a  at  room  acetate.  in The  by  preparative  R  =  =  x  182  was  evaporated  purified  UV  mg)  stirred  CHCl /MeOH  81.  196  +  and  was  (9:1  and  mixture  water  material  yield) 211  of  between  soluble  (34%  J  (m,  (75)  of  chromatography  mg  2.79  solution  several for  (t,  81  methanolic  treated  0.93  3H),  (30),  Monoacyltriophamine A  3H),  f  232  (15),  0.1) (  10000,  e  129  to  (15),  86  (100).  Dimethylpyrimidine To drops  1 5 mg o f of  5  temperature detected using with  the HC1  layer  mA  mixture  TLC  and  NaOH. 24h  at  material.  This  2,4-pentanedione evaporated  1.0  reaction  82  mjl M e O H w a s  was  time  guanidine  6  .  between  to  The H 0  was at in  and  2  dryness  solution  yielding  stirred 130°  for  vacuo  and  with 30  12  R  was  ether.  at  =  f  mg  mg  min.  triturated  be 0.3)  a c i d i f i e d  The  5  5  room  could  acid,  3  5  added  stirred  CHC1 /MeOH/acetic  reagent  partitioned  in  which  (60:35:5 spray  Guanidine  (7_5)  The  evaporated  was  of  triophamine  for  FCNP  was  positive 1.0  by  N  Derivative  aqueous of  FCNP  NaHC0  The with  3  and  reaction CHC1  3  .  Preparative ethyl  thin-layer  acetate,  p r o d u c e d  Rf  1.0  =  mg  nm  62.31  (MeOH);  (s,  MS, 5.05  C_i - C a r b o x y l i c  Acid  After  in  triophamine mi  of  and  To  mixture  was  ethyl dried  over  x  Na S0 2  Q  H  1  NMR 7Hz, J  =  15Hz, 170  8  0  2  (400  15  7Hz,  2.47  (20),  141  extraction  mg  (m,  the  the  C.  0  7_5  with  NMR  a  =  x  (80  IH)  in  (dd,  4 , 6 -  228  MHz,  and  CDClg)  ppm.  mi  l h ,  ethyl  5  IR  J  83  acidified 8_3  J  3H),  5.23  =  The  J  =  73  7Hz,  IH)  lactonizes  to  were  cm"1; (t,  (dd,  J  ppm;  MS,  not  carried l  a  c  t  o  n  = (q,  =  (100).  Y ~  'H J  2.04  55  the  0.3)  170.1298,  2H),  2.33  is  with  =  f  0.97  (m,  (70),  R  1700  IH),  hydrolysate  aqueous  thin-layer  obs  3H) ,  1  added  fractions  HRMS,  7Hz,  added  was  2  acid,  1.52-1.62  (q,  was  H 0  3400-2400,  15Hz,  (85),  of  preparative  83.  3  7,  hydrolysis  extracted  acetate  by  (CHC1 )  =  was  mi  rapidly  of  (t,  8_3  acetate.  3  yield)  acid  MeOH  CHC1 /MeOH/acetic  IH),  -acid  3  for  purified  = 7Hz,  (15),  of  of  60.95  J  2.14  IH),  promptly,  (11%  CDClg )  2H),  m  base-catalyzed  ethyl  MHz,  (d,  \  material the  0  The  170.1307;  1.57  of  C ^ -carboxylic  mi).  and  ( t  the  and  cal.  3H),  (s,  HC1  mg  1  6.38  'H  with  to C  soluble  3  UV  +  refluxing  (100:1:1  1.5  2H),  15  chromatography provide  (M );  extracted  (3  acetonitrile/  o v e r a l l )  123  from  After  acidified  acetate  CHC1  82.  attempts,  (7_5) .  the  (1:4  83  yield  N NaOH.  layer  (bs,  low  1  the  y i e l d  m/z  repeated  isolated  of  derivative  6H),  Q  0.15) (20%  dimethylpyrimidine 289  chromatography  9, m/z If  e  out 9  5  .  137. 95,  IR 1750 c m  6H),  0.99  - 1  ;  (t,J  'H NMR  =  7Hz,  ( 1 0 0 MHz,  CDClg)  3H), 1.55-1.80  60.93  (m,  ( t , J = 7Hz,  8 H ) , 2.62  (m,  IH)  ppm.  Diacetylguanidine To  3 9 0 mg  freshly under  of guanidine  distilled  N  and  2  anhydride  purified  19900,  (M ); +  1  3  C  m  a  R  X  m  a  f  =  x  NMR  = 2 3 0 nm  p-Nitrophenyl To  a  The  248nm  0.71 mmol)  of  dry  of  reduced  1645  (28%  was  cm  \  m  a  ;  - 1  =  x  MS  159.0,  (1:4  yield)  of  212nm  m/z  180.1  methanolic  143 ppm.  NaOH  monoacetylguanidine  solution  at [UV  was  temperature  (96)  of the carboxylic  a n d p - n i t r o p h e n o l ( 1 5 0 mg,  a t room  solid  material  MeOH),  (E)-2,4-Diethyl-4-Hexenoate  acetonitrile  acetic  MeOH)].  1  under  mg  of  kept  chromatography  with  produced  added  N -dicyclohexylcarbodiimide, stirred  8£  Excess  soluble  626.0,  g  was  the resulting  87  1710,  acetone-d )  14300,  e  give  16800,  e  3350,  rapidly  stirred  mg,  (  IR  and  an e x c e s s  mixture  min.  acetone  to  added  The  thin-layer  hydrolysis  (  was  f o r 90  i n vacuo  0.3)  ( 2 0 MHz,  temperature  x  =  MeOH/HCl);  Base-catalyzed room  100°C  preparative  acetone/CHClg,  (e  to  i n acetone.  \  carbonate  anhydride.  evaporated  by  UV  acetic  heated  was  triturated  80.  80  and  300 the  f o r 12h.  mg  acid  1.08 mmol) ( 1 . 4 7 mmol)  resultant Removal  pressure provided a solid  83a (120  which  i n 3 ml of  mixture  N, was  of the solvent was  triturated  138. with  chloroform.  removal was  Filtration  of the solvent  purified  by f l a s h  o f t h e column  180  (87% yield)  HRMS,  the mixture,  the filtrate,  chromatography  Elution mg  from  of  with  of  o b s . 291.1496,  1:3  followed  gave  a solid  the desired  ester  cal.  ( 1 0 0 MHz, C D C l j )  which  o n 28 g o f s i l i c a g e l .  hexane-chloroform  C^H^NO^  by  £6.  afforded  mp  291.1471;  IR  61.01 ( t , J =  45-46°; (CHClg),  1755  c m " l ; *H NMR  7 H z , 3H) ,  1.04  ( t , J = 7 Hz, 3 H ) , 1.42-1.86  3H),  2.13 ( q , J = 7 H z , 2H) , 2 . 2 8 - 2 . 8 5  7Hz,  I H ) , 7.23 ( d , J = 9 H z , 2 H ) , 8.31 ( d , J = 9 H z , 2H) ppm.  (m, 2 H ) , 1.62 ( d , J = 7 H z , (m, 3H) ,  5.33 ( q , J =  (±)-Triophamine (75a) Subjection ion-exchange 1-X8  Small  intervals  ester  96  1:1  to  a  appreciable  and  Therefore, material the  of  the after  on a free  column  hydrochloride o f 64.5 g  guanidine  i n 2  as  of  mi  t o proceed  t o completion  of  approximately  solid  was  desired  was  found  side  washed  that  The  i fthe  (disappearance  product(s)  material  50-75%  the reaction  added a t  by TLC ( s i l i c a g e l ,  It  yield  hygroscopic  the p-nitrophenyl  ether).  of  to  o f Dowex  of chloroform.  was m o n i t o r e d  quantities  a  m a t e r i a l were  solution  mmol)  96 h a d r e a c t e d , collected  guanidine  of the latter  0.12  was a l l o w e d  96),  g  stirred  hexane/diethyl  formed  and  amounts  of the reaction  reaction of  provided  ( 3 4 mg,  progress  14.3  chromatography  (0H~ form)  solid. lh  of  of  the  mixture with  was  4  was x  were low.  starting filtered 5  mi  of  139. chloroform.  Removal  filtrate  gave  a  of  yellow  preparative  thin-layer  obtained  mg  on  9.1  its  96)  of  diastereomers  repeated  spectra  were  a  with  (2.5  as  follows:  HRMS o b s .  IR  (CHC1 )  3323,  CDC1 ) 3  60.91  1.46-1.67 2H),  2.11  2.37-2.47  Extraction A  2H),  (dd, (m,  J  IH),  and  total  =  =  the  mg  yield  based  75a  and  separated  by  C  2 1  mass  natural  1160 3H),  H  3 7  3  cm ;  13Hz,  (q, J = Hz,  and  NMR  product.  The  NMR  22  specimens  of  J  =  7Hz,  (q, J =  2.28-2.37  (2  was  over  Na S0  evaporated  light  2  4  and  brown  o i l  x  800  ( 820  are  (m,  MHz, 3H), 7Hz, IH),  ppm.  Anisodoris  chloroform  natural  (400  Chromatography of D i h y d r o a p o f a r n e s a l of  which  c a l . 363.2885;  2H) , 2.03  IH)  of  'H  from  2  (t,  IH),  yield)  spectra  'H  -1  7Hz,  (19:1  diastereomers  N 0  0.95  thin-layer  (26%  different  with  smelling  thus  PF-254  provided  extracted dried  60  f e a t u r e s of the  (d, J =  5.20  was  4.0  yield)  7Hz,  6,  (48%  infrared,  of  1630,  1.56 J  gel  363.2883,  1700,  mg  to  was  preparative  clearly  spectral  (t,  (m,  16%  but  The  subjected  (±)-triophamine  give  those  mg;  to,  to  combined  There  mixture  exhibited  triophamine.  3  This  the  was  7.4  of  s i l i c a  which  similar  and  mixture  on  identical  diastereomers  e s t e r 96  alcohol)  (± ) - t r i o p h a m i n e  from  which  developments)  chromotography hexane/isopropyl  o i l  75b.  (three  solvent  chromatography.  of the  unrecovered  the  mi). to mg)  nobilis  The obtain  .  (15 )  The  were  chloroform a  pleasant o i l  was  140. separated  by  flash  chloroform  step  chloroform  in  thin-layer Final on  HRMS,  (400  MHz,  1.60  (s,  CDC1 )  (m,  1  ppm;  IH)  (20),  Extraction A  3H)  2.36 m/z 81  with  and  208  to  yield  a  r a d i a l  the  eluant.  GC  (3%  (s,  OV-17  4°/min,  3H) ,  RT  (m,  1.78  2H),  (5),  175  14  colorless  1.42  3H) ,  15%  by  208.1823;  Archidoris of  'H  (3),  NMR  (m,  IH),  (m,  IH),  9.58  (d, 165  J  =  (50),  Metabolites  Archidoris  (3  x  1  j).)  for  evaporated  chloroform  d r i e d  brown  with  (100).  were  with  Hz,  5.12  of  methanol  were  7  (hexane-  smelling,  cal.  190  specimens  extracted  0  1.69  (5),  as  rate  sweet  =  69  hexane  100°,  2 1 +  IH),  extracts  e x t r a c t s  evaporated  (m,  (70),  80  methanol  combined  over  gum  (2.7  montereyensis 3 to  (3  x  an  500  sodium g,  days.  The  aqueous  mj.) .  The  s u l f a t e  and  8.3%  dry  weight,  mg/animal). The  a  (s,  of  extracted  suspension  on  1.61  H  1 1 +  eluting  preparative  a  J  Chromatography  total  combined  33.7  (d,  (25),  by  gel  p u r i f i e d  100%  as C  1.10  MS,  and  1_5  208.1825,  6H),  95  of  6  3  3H),  using  temperature  mg  obs.  1.96-2.13 Hz,  2.8  s i l i c a  f u r t h e r  achieved  i n i t i a l  providing  o i l .  was  on  Material  was  chromatography  p u r i f i c a t i o n  min.)  were  gradient). hexane  Chromosorb,  150  chromotography  gum  column  of  hexane-ethyl acetate  in  (1.5  g)  s i l i c a  acetate  hexane  was gel  subjected (130  g)  mixtures.  removed  non-polar  to  flash  with  a  Elution fats  chromatography  step with  and  gradient 20%  pigments.  of  ethyl The  141. acetylated  glycerides  hexane-ethyl were  eluted  acetate with  Material  eluting  separation  order  crude  solvent  mg; 0 . 2 % o f c r u d e 18  mp  117-119°  3600-3350,  3  by  LC  on  acetate  compounds  was  (silica  (99:1).  Final  Partisil-10  (85:15:1)  eluant  with  to yield  18_ ( 2 4 mg;  9 " 0*13% °f c r u d e  2  (CHC1 ) CDC1 )  m  of  glycerides  chromatography  chloroform-methanol  (Hexane/Et 0);  HRMS  3  2  solution  i n  1.6% o f  e x t r a c t ) a n d IS)  (3  extract).  CHC1 ); 3  thin-layer  elution,  21. (  1:1  (1:1) hexane-ethyl  acetate-methanol  extract),  a  acetate.  acheived  of their  with  the non-acetylated  with  radial  was  hexane-ethyl the  100% e t h y l  by  eluted  while  eluted  fractionated gel),  were  obs.  420.2884, 2930,  [ a ] C  2  5  H  4  =  D  0  Q  -53.7° c a l .  5  1730, 1230 c m  - 1  ;  (C  = 0.13,  420.2876;  'H NMR  IR  ( 4 0 0 MHz,  6 0.82 ( s , 3 H ) , 0.87 ( s , 3 H ) , 0.92 ( s , 3 H ) , 0 . 9 6  (s,  3H),  1.61 ( s , 3 H ) , 2.11 ( s , 3 H ) , 2.41 ( b s , I H , e x c h a n g a b l e ) ,  2.97  ( b s , I H ) , 4.08-4.25  (2),  402  ( 5 ) , 347  (m, 5 H ) , 5.53 ( b s , I H ) ; MS, m/z 4 2 0  ( 5 ) , 286  (100),  192  ( 6 5 ) , 177  ( 7 0 ) , 117  (60) . 21  o i l ; [<x]  352.2221, 1720,  C  2  0  H  1230 c m  D  3  - 1  = +15.3° 2  O  ;  5  (C = 1.2, C H C 1 ) ; 3  c a l . 352.2250, 'H NMR  ( 4 0 0 MHz,  IR  HRMS o b s .  (CHClg)  CDC1 ) 3  3400,  6 0.88  2930,  (s,  0.92  ( s , 3 H ) , 0.98 ( s , 3 H ) , 1.61 ( s ,  (bs,  IH, e x c h a n g a b l e ) ,  2.95  5.55  ( b s , I H ) . MS, m/z  352 ( 3 ) , 3 3 4 ( 4 ) 2 7 9 ( 5 ) , 2 1 8 ( 5 5 ) ,  190  3 H ) , 2.11 ( s , 3 H ) ,  3H),  ( b s , I H ) , 4.05-4.24  ( 4 5 ) , 124 ( 8 0 ) , 117 ( 8 0 ) , 109 ( 1 0 0 ) ,  95 ( 8 0 ) .  2.48  (m, 5 H ) ,  142. 19  o i l ;  [ ] a  420.2876, 2925, (s,  C  2  1725,  3H),  =  D  H  5  l  +  -33.0° 0  Q  0.87  (s,  3H),  2.09  4.26  (dd,  J  IH),  5.06  (m,  IH),  258  (20),  286 95  (s,  (90), (80), The  was  7  100%  to  by  elution,  23  mg;  (2  extract) mp  and  of  192  (55),  acetate  )  (400  MHz,  0.96  (s,  CDC1 )  7  3H),  and IH)  IH);  MS,  191  (50),  J J  m/z  = = 6  420  177  3600-3210,  3H),  (d,  obs.  CDClg)  (s,  (dd,  0.82  6  1.60  6  (s,  Hz,  and  (5),  2H),  13  402  (75),  J  12  Hz,  ppm;  13  2900,  (38  117  Hz, (3),  (100),  of  (s,  (bs,  2.96  NMR  3H) ,  (bs, 12  4.22 (100  in  2.5% 20  [ ] a  1730,  (dd,  =  D  of  extract),  0.2%  -12.5°  c a l .  (s,  3.63  of  crude  3H), (m,  (dd,  = 5  CDC1 )  and  (m, 12  15.63  =  cm l~;  *H  -  0.92 2H) ,  J  (c  378.2770;  1170  3.95  3  e t h y l  order  crude  mg;  1460,  IH),  the  was  extract).  0.87  J  of  (3  1.92-1.99  Hz,  MHz,  yield  to  mg;  separation hexane-  C ^ H ^ C ^  IH),  column eluting  w i t h  crude  3H),  flash  chromatography  extract),  2850,  = 4 and  C  1J7  the  Final  eluant  378.2772,  IH),  from  (95:5).  2  1.61  (dd,  fraction  0.13%  6 0.82  3  3.70  4.32  (Hexane/Et 0) ,  obs.  exchangable) ,  IH),  MHz,  3.76  P a r t i s i l - 1 0  mg;  125-126°  3600-3300,  3  (CHClg)  0.94  thin-layer  crude  22^ ( 2  (CHC1  (bs,  (bs,  compounds  HRMS,  3  IH),  HRMS  3  (400  3H) ,  IH),  5.52  on  CHC1 );  =  Hz,  CHC1 );  IR  NMR  (s,  (50:40:2)  0.13%  =  'H  (bs,  radial  LC  their  J  2.94  ethyl  acetate-methanol  2H,  0.91  chloroform-methanol  a c h i e v e d  17,  3H),  12  0.83,  (80).  subjected  with  cm~l;  and  =  420.2876;  1055  3H),  =  81  c a l .  5  1230,  (C  = 6 IH), Hz, (q),  0.4, IR NMR  (s,  3H),  2.44  (bs,  and  12  Hz,  4.15  (dd,  IH),  5.54  15.73  (q),  143. 18.52 (s),  ( q ) , 18.73 ( q ) , 21.15 ( t ) , 21.68 ( t ) , 22.74 33.43  54.45  ( q ) , 36.70  ( d ) , 56.58  (d),  124.32  ( s ) , 37.53  ( d ) , 62.76  ( d ) , 128.60  ( s ) , 40.01 ( t ) , 41.95  ( d ) , 63.64  ( s ) , 173.35  ( s ) ppm;  MS,  363 ( 5 ) , 3 4 7 ( 1 0 ) , 2 8 6 ( 6 5 ) , 2 5 8 ( 3 5 ) , 192  (80),  95 ( 7 5 ) .  HRMS,  o b s . 316.2973,  3600-3250, (t, =  1110 c m  6 H z , 2 H ) , 3.50 (dd, J  316 20  1 9  - 1  H  0  1 + O  ;  c a l . 316.2977;  3  'H NMR  ( 4 0 0 MHz,  m/z  378  (100),  177  IR  (CHC1 )  CDC1 ) 3  3  6 0.86  J = 7 H z , 3 H ) , 1.26 ( b s , 2 8 H ) , 1.59 (m, 2 H ) , 3.44 ( t , J  3.67  =  4  (m, 2 H ) , 3.64  a n d 12 H z ,  ( d d , J = 6 a n d 12 H z ,  I H ) , 3.82  mp  =  94-95°;  2900,  1 8  H  3o°(+  a  l  3.70 ( d d ,  '  310.2144; NMR  I H ) ppm;  IR  (CHClg)  HRMS, o b s .  3600-3300, 6  (400 MHz,CDCl ) 3  ( s , 3 H ) , 1.62  ( b s , 3H) ,  0.89 ( s , 2.48 ( b s ,  2.96 ( b s , I H ) , 3.63 ( d d , J = 6 a n d 12 H z ,  J = 4 a n d 12 H z , I H ) , 3.95 (m, I H ) , 4 . 1 5  1C 3  NMR  ( d d , J = 5 a n d 12 H z , I H ) ,  ( 1 0 0 MHz,  CDC1 )  614.9,  3  18.7,  21.9,  23.7, 33.1, 33.3, 40.5, 42.2, 42.3, 49.5, 62.3,  65.2,  70.5, 124.6,  (5),  279  (100),  m/z  (100).  3  7 a n d 12 H z , I H ) , 4.22  (bs,  ( 8 0 ) , 57  = +23.1 ( c = 0 . 9 3 , C H C 1 ) ;  ( s , 3 H ) , 0.98  exchangable),  =  c  D  _  0.92  IH),  ]  a  1 7 3 0 , 1 1 6 5 c m l ; 'H  3H), 2H,  c  [  IH),  (m, I H ) ppm; MS,  ( 2 ) , 2 8 5 ( 5 ) , 2 5 5 ( 1 5 ) , 2 2 5 ( 1 5 ) , 71  310.2142,  J  2900,  C  (t),  ( t ) , 65.17 ( t ) , 70.47  (30),  23  ( t ) , 33.20  128.8,  1 7 3 . 4 ppm; MS,  ( 1 0 ) , 218 ( 7 5 ) , 190  ( 4 5 ) , 187  m/z  (dd, 5.57  21.3, 63.6,  310  ( 1 5 ) , 295  ( 4 0 ) , 124  ( 7 0 ) , 109  95 ( 7 5 ) .  12  o i l ;  nm  (  e  [  a  ]  1800,  D  = +9.7°  MeOH);  (C = 0 . 3 , C H C 1 ) ; 3  HRMS  obs.  UV  310.2146,  \  m  a  x  = 228  C^H^O^ c a l .  144. 310.2144; NMR  IR  (400  3H),  MHz,  3.62  Hz,  = 12  IH);  (30),  and  MS:  69  (90).  The  101  material  contain  =  o i l .  (s, (bs,  IH),  3.70  (dd,  of  (s,  295  (5),  124  acid(s)  of  2.3  HRMS  304.2356 1695,  of  obs.  C  1650  2 0  H  cm" ; 1  'H  c  NMR  l  *  IH),  5.70  (5),  237  (10),  95  4  (400  -  4  I  1.60  (s,  3H) ,  2.20  (bs,  IH),  4.76  (bs,  IH),  5.07  (bs,  IH),  5.70  (s,  289  (40),  124  (50),  in  not  CHC1 , 3  yellow (CHC1 )  R  3  3  3H),  (50),  in  did  CDC1 )  (s,  177  s o l u b l e  light  MHz,  of  p u r i f i c a t i o n  ' *  2  many  acetate  MeOH  0  100  course  0.91  (15),  as  the  a 2  82  the  3H),  304  (90),  which  as  219  of  ethyl  (1%  0  5  (s,  Final  3  and  4.79  mixture  a  (s,  4.24  30%  acid(s)  32°2  = 12  chloroform  glycerides.  the  J  identified  chromatography  mg  2.16  IH),  during  using  crude  3H) ,  'H  Hz,  one  the  cm"1;  (85),  only made  1155  7  109  tentatively  terpenoic  gave  279  (30),  a  thin-layer  and  IH),  from  provided  the  = 12  4.55  (2),  (85),  J  chromatography  0.4)  3225-2500,  m/z  flash  preparative  Rf  Hz,  montereyensis  eluant  any  (s,  F r a c t i o n a t i o n  by as  0.93  (dd,  IH),  1645,  3H) ,  i s o l a t e d  A.  study.  7  1700,  (s,  4.18  310  176  of  and  Hz,  was  collections  by  5  2945,  0.85  6  diterpenoic  and/or  hexane  )  3  IH),  m/z  (35),  3430,  = 12  (m,  203  (100),  t h i s  J  3.95  J  (s,  CDC1  (dd,  IH),  (dd,  (CHClg)  60.83  3H), IH)  109  4.53  ppm; (85),  MS 81  (100) . Ten methanol scheme  specimens and  of  Archidoris  subjected  identical  to  that  to  a used  odhneri  were  separation with  A.  and  extracted  with  p u r i f i c a t i o n  montereyensis.  In  145. addition the 27  to  43  mg  positional HRMS  MHz, (t,  3  5.68 (90),  69  4H),  4.90  ppm;  quantities  of  at  mixture  of  n  3H),  = 7Hz,  (15),  d  'H  IH),  219  of  mg  of  NMR  (270  (s,  3H) ,  3.81  5.03  (bs,  2H),  (20),  218 of  w  e  r  i n  the  and  18.  evidenced  HPLC  spectrum  82  odhneri  17.  also  e  (20),  A.  glycerides  23.  NMR  'H  2.16  separation  times  the  RT  of  for  5  and  17.  mi  8  white  6 9  6  1.78  in  in  the  presence  evaporation  ;  a  D  2920,  (s,  the  =  (s, 3H),  =  0.3)  -9°  2850, 3H), 2.06  C  2 0  H  trace  by and  of  a  p a r t i a l l y  of  toluene  O  1450, 0.84 (dt,J  0.1,  1390  (s,  the  resulted  in  one  chromatography  = 4  13  yield)  (hexane/Et 0)  ( l i t .  2  ( l i t .  290.2609;  cm !; -  and  mg  (1:4,  (85%  3  3H) ,  DIBAL. of  CHC1 ),  c a l .  excess  0.87 13  was  f i l t r a t i o n  solvents,  126-127° =  3 i (  of  provided  mp (C  ml  by  thin-layer  97.  290.2623,  0.82  of  Rf  0.5  followed  Preparative  [ ]  obs.  3600-3300,  mg)  EtOH,  crystalline  125-126")  (20  h  of  acetate/hexane  3H),  J  HPLC  a  310.2144;  (s,  310  22^  1*5  17  ethyl  )  cal.  samples  in  product.  3  m/z  20,  major  CDC1  0^  1.60  r e t e n t i o n  solution  Addition  HRMS,  Q  24'  mixture.  stirred  of  3  (pentet,  pure  signals  Reduction A  H  8  6H) ,  of  c h a r a c t e r i s t i c  purified  1  glyceride  recovered.  Repetative  provided  diagnostic  C  MS,  (100).  extracts Trace  obs. (s,  IH)  acid  27_ w e r e  61.56  = 6Hz, (s,  farnesic  isomer  310.2143  CDC1 ) J  of  'H (s, Hz,  IR  NMR 3H) , IH),  -9°)  6  9  ;  (CHC1 ) 3  (400 0.90 3.72  MHz, (s, (dd,  146. J  =  6  and  (bs,  IH);  12 1  Hz,  C  3  IH),  NMR  22.7,  23.7,  29.7,  55.0,  56.4,  58.1  (5),  260  (5),  69  (85).  (60),  Reduction  of  18  y i e l d )  CDClg)  33.4,  60.9,  and  to  the  of  and  product  Reduction  of  of  of  of  produced  2.6  This  94-95°  ( l i t .  3  222.1984; 3H), Hz, (10), (60).  IH),  20°)  NMR  (s, 3.84  191  ( l i t .  -  'H  0.867  lfi  (15  mg)  40.1,  41.7,  42.1,  290  (95),  of  1_7.  and  (40),  275  95  (40),  (6  mg)  19_  purified  This  in  a  produced  81  in  a l l  were manner  8  r e s p e c t i v e l y  identical  (6  20_ in  CHC1 )  21.7,  mg  of  (77% white  respects  to  the  DIBAL  and  17.  purified  mp  5.50  18.9,  m/z  177  y i e l d )  was  subsequently  98.  (100),  IH),  18.6,  37.4, MS,  Hz,  20  solution  17.  12  615.8,  subsequently  (72%  which  reduction  and  19  mg  crystalline  = 4  132.7;  192  reduction  3  J  36.4,  123.9,  (5),  DIBAL  and  A  MHz,  solutions  with  identical  (dd,  33.2,  245  Separate treated  (100  3.85  (400  3H),  Reduction  a  J  124  was  manner mg  (50%  1  ;  HRMS,  MHz,  =  13  (45),  product  yield)  of  white  [ ]  =  -20°  a  D  the  o b s 2 2 2 . 1 9 8 4 , 6 0.837  3H), and  109 98  with to  CDClg) (s,  treated  identical  95-96°)71.  1.76  (dd,  (10),  7  mg)  5  3.73 Hz,  (75), was  (dd, IH)  85  identical  (c 1  5  J  =  to  2  6  0.08,  0  0.844 13  MS, 83  =  H  3H),  ppm; (75),  crystaline  C  (s,  reduction  c a l . (s,  and  7.2  m/z  222  (100),  69  authentic  147. drimenol Rf  =  by  0.3  'H  on  on  i'+C  of  i n i t i a l  3%  MS,  Polygram  (co-injection peak:  NMR,  98  TLC  0.25  and  total  Studies  of  of  22  A.  s t u d i e s .  140°,  gel  (1:4),  plates)  drimenol  rate  and  gave  10°/min,  mCi/mmole) directly means  (1:1)  a  of  extracted  a  RT  a  GC  single  = 6.75  min  fractionated  HPLC  as  alcohols alcohols  obtained 97_  were  acetate/hexane) counting.  reduced  to  98  and  England seawater  HPLC  derivative ether/hexane  DIBAL  and  h  in  and  then  extracts  _17  reduced  to  8%  ethyl  odhneri  Compound  The  prior  converted  to  were  toluene.  A.  by  Compounds  were  from  103. prior  animal  by  ether/hexane  then  was  purified  (Partisil-10,  from  mi)  The  species.  obtained  (102)  24  metabolities  by  45  nudibranchs  manner.  montereyensis,  _24,  each  normal  both  a c i d -  (0.05  of  the  13  Nuclear;  for  for  crystalized  farnesol  from  (New  gland  and  incorporation  aquarium  the  by  Compound  the  RS-mevalonic  respectively  purified  in  *C]  glyceride  A.  montereyensis  injection,  described  3,5-dinitrobenzoate crystalized  in  the  from  and  l  digestive  seawater  and  previously  l  s a l t  After  Metabolites  employed  [2-  the  methanol  were  ^0  into  running  with  A.  ethanol/sterile  syringe.  in  of  were  uCi  injected  of  placed  in  Archidoris  specimens  odhneri  Two  of  dibenzylethylenediamine  C  s i l i c a  authentic  temperature  Incorporation  specimens  1 J t  mm  acetate/hexane  0V-17).  A  and  (ethyl  to was  to  i t s  103  was  counting.  The  148. samples  (0.5-4  mg)  s c i n t i l l a t i o n determined  was  Reduction  of  A  in  as  of  colorless  a  mmole) After the  was  and  gel  at  was  was  solvent  in  a  of  RT  for  stirred  f i l t e r e d using  under  for  8Hz,  IH),  counts  were  s c i n t i l l a t i o n  system.  standardization.  39  26  1.58  reduced  4.93 9.21  (s,  (m,  J  mg  to  DIBAL  the  (65%  yield)  and  reduction  (Partisil-10,  (26  Farnesol  mg;  8%  of  ethyl  farnesol  (102)  0.117  chloride h,  an  5  mi  mmole)  a  1.64 =  3H) ;  8Hz, MS  (s,  m/z  mp  416  1  1.79 (m,  (20),  (s,  401  mmole). and  dia.)  of  of  the  preparative in  hexane)  NMR  (80  3H),  2H) ,  0.164  reaction  cm  acetate 'H  mi  added  The  by  77-79°;  5.04  was  Removal  followed  3H),  2H) ,  x  1.0  mg,  0.135  2 h.  eluant.  ethyl  103  mg,  (1  in  (20  methanol  plug  as  (25% of  (31  additional  pressure  yield)  6H),  (d,  with  identical  of  chloroform  (86%  treated  HPLC  through  mg  8H),  by  12  provided  (m,  Radioactive  4-dimethylaminopyridine  chromatography  3  (NEN)  was  manner  102  thin-layer  CDC1 );  mg)  3,5-dinitrobenzoyl  mixture  s i l i c a  (52  Derivative  added  s t i r r i n g  mixture  10  o i l .  solution  pyridine  Aquasol  external  provided  3,5-dinitrobenzoate To  mi  liquid  purification  hexane)  a  III by  24  purified  Final  acetate  Unilux  corrected  solution  17_.  (102)  a  fluid.  in  24  subsequently of  dissolved  counting  in  Quenching  were  1.90-2.19  5.45  (5),  MHz,  (t,  195  J  =  (50),  149. 136  (60),  93  Additional A  (50),  (170),  69  I n c o r p o r a t i o n  1  total  each  81  of  10  i n j e c t e d  Studies  specimens  with  five  of  were  seawater.  extracted  p o r t i o n  of  with  the  chromatography eluant.  on  and  added  to  hydrolyzed  6.8  soluble  material  activity  of  dilution  with  =  IH),  with  dpm/mg.  3.76  5  10 uCi  _35  3_6.  1  were  5%  ethyl  This  acetate  methanolic  and  material and  then  potassium  mixture  ether.  The  was ether  albicanol  (3_5)  with  correcting  for  the  the  thin-layer  albicanol 60.73  (s,  11Hz, (bs,  Triopha acetic  IH)  had  3H),  IH),  an 68%  a  l l t  0.81  3.86  C  (s,  (dd,  J  ppm.  catalinae  acid  as  (3_6)  of  4.93  [2- '*C]  chloroform  reaction  = 11,  of  f l a s h  preparative  NMR  J  IH),  specimens of  'H  by  by  After  (dd,  (bs,  the water  mg  material,  dpm/mg.  4.65  of  3.5  a  ml  soluble  (Partisil-10,  in  12h  0.05  nudibranchs  acetate  at  between  in  the  with  pure  were  RS-mevalonic  chloroform  albicanyl  RT  C ]  24h  "cold"  yield  3H),  of  purified  "cold"  3815  total  injected  was  to  (s,  4Hz, A  of  4  f r a c t i o n a t e d  HPLC  mg  After  1221  of  0.88  11,  mg  for  s i l i c a  by  3.2  partitioned  chromatography  activity  give  1  albicanyl  purified  solution. and  of  luteomarginata  mCi/mmol)  The  was  column  s t i r r i n g  f i l t e r e d  incubating  containing  to  by  carbonate  3H),  a  [ 2 -  (45  methanol.  further  acetate/hexane)  of  salt  e x t r a c t  Fractions  combined  was  After  Cadlina  uCi  acid-dibenzylethylenediamine sterile  (100).  sodium  were  each  salt  (1-3  150. mCi/mmol) 24  h  in  the  m a t e r i a l  was  When  0.5  mi  the  65%  N  8_1  compound  Fish  Antifeedant  Bioassay  Antifeedant  activity  maculosus treated The  was  were  treated  randomly gallon  up  to  toward  1 h.  at  with to  a  aquarium The  control  of  solvent group and  fish  of  the  to  food  40  feeding  and  was  at  metabolities.  pellets  voracious  pellets  Pellets)  Barkley  cm  to  Sound, testing.  with  acetone  Control  pellets  pellets in  was  length) observed  feeding  treated  the  Oligocottus  prior  response  the  dpm/mg.  Shrimp  dorid  (5-8  of  accounted  sculpin  Single  fish  mg  by  observing  temperature. only.  in  reaction  420.4  by  24 h  RT  dpm/mg.  (Wardely  for  at  purified 2.2  of  i n t e r t i d a l l y  exhibitied  pellets  give  of  " c o l d "  The  and  as (75).  of  stirred  1201  pool  starved  room  to  assessed  tide  applied  evaporated  added  10  were  and  material  activity  pellets  were  mg  acetate  "cold"  collected  6.5  for  up  triophamine  activity  was  worked  hydroxide.  concentrations  and  compounds  which  food  were  Columbia  an  the  varying  sculpins  Test  of  toward  with  B r i t i s h  showed  response  an  with  of  methanol  ethyl  with  incubating  and  w i t h  chromatography  for,  feeding  mg  sodium  with  dilution 8_1  3.5  1 mi  1  thin-layer  derivative  yield  in  After  extracted  combined  extracted  preparative monoacyl  to  was  of  seawater.  were  dissolved  presence  mixture  sterile  described  triophamine, the  ml  nudibranchs  previously T h i s  0.05  with  were in  a  for  behaviour inactive  151. compounds. groups hard were  of  These fish  parts  in  active  i n i t i a l  and the  pellets  which  a b i l i t y  of  pellets  both  untreated  were  consumed  within  pellets  caused  mouthing  pellets  and  a  marked  fish  before  pellets  cessation  and  were  the  uneaten  was  verified after  rapidly  5-10  ignored.  rejection,  remained  the  were  aggressively  by  each  min. 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