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Universit\1Micrc5films

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8408973

Okuda, Roy Kenichi

CHEMICAL ECOLOGY OF SOME OPISTHOBRANCH MOLLUSKS

University of Hawaii

UniversityMicrofilms

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PH.D. 1983

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UniversityMicrofilms

International

CHEMICAL ECOLOGY OF SO}ffi

OPISTHOBRANCH MOLLUSKS

A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THEUNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN CHEHISTRY

DECE1!BER 1983

By

Roy Kenichi Okuda

Dissertation Committee:

Paul J. Scheuer, ChairmanRobert S.H. LiuRichard E. Moore

Harcus A. TiusE. Alison Kay

iii

ACKNOWLEDGEMENTS

One of the most important aspects of a graduate student's tenure,

I feel, is the chance to meet and interact with many other people

in one's chosen field, and I am very grateful for this opportunity.

A large number of these individuals have contributed directly to the

completion of this dissertation, however, a need for brevity prevents

me from mentioning all but an important few.

I am very grateful to those who have assisted in the collection of

organisms, expecially Drs. Gary Schulte, Robin Kinnel, and Chris Ire

land, and Debbie Roll, Robert Armstrong, and Scott Johnson. It is

with especial pleasure that I acknowledge the involvement and interest

of Mr. Scott Johnson, whose expertise on the biology of opisthobranch

mollusks was very important in these studies.

I thank the various instrument specialists who have helped in the

procurement of spectral data. Dr. Walt Niemczura provided much assis

tance with routine and non-routine NMR experiments. Mrs. Lars Berg

knut, Normal Liu, and Carl Yanagihara provided mass spectral data. Dr.

Sheldon Crane maintained other important instruments.

Many useful ideas resulted from discussions with Drs. Gary Schulte,

Robin Kinnel, and Dilip de Silva, and Mr. Patrick Yu; their interest

in my work is greatly appreciated.

Personal communications with Professors Yoel Kashman and John ColI

have provided useful information and interesting insight into one of

the projects studied.

iv

I am very fortunate to have, at this stage in my scientific career,

two mentors whose efforts and interest in my development have been com

plementary, and are directly responsible to the completion of this dis

sertation.

As an undergraduate, Dr. William Fenical employed me for four years

and provided me with a stimulating environment in which to learn the

skills of scientific research. His enthusiasm and interest prompted me

to enter the field of marine natural products chemistry, in which I

have continued to work as a graduate student. His long-standing and

continued interest in me is deeply appreciated.

lowe an equivalent debt of gratitude to Professor Paul Scheuer,

who, as my graduate research adviser, has allowed me to pursue my inter

ests in marine natural products, and who has provided me with much

assistance and opportunities during my time as a graduate student. I

most certainly could not have learned or done so much had I been in any

other research group.

Finally, I thank my parents and family, whose ceaseless encourage

ment and support have been an important factor in the completion of

this goal.

v

ABSTRACT

Many opisthobranch mollusks (Class Gastropoda, Subclass Opistho-

branchia) are shell-le~s, slow moving invertebrates, and are ostensibly

unprotected and vulnerable to predation. Observations have shown, how-

ever, that these shell-less moliusks are rarely, if ever, eaten. The

phenomenon is at least in some cases a result of defense secretions by

opisthobranchs.

Five nudibranch species were investigated chemically. Addition-

ally, three of the principal food sources for three of the nudibranch

specie~were subjected to parallel investigations. Different degrees

of correlation were found between the metabolites of the predator-prey

pairs, and all nudibranchs were found to contain high concentrations

of specific metabolites. Structure elucidation was performed by spec-

tral analysis and chemical transformation.

Indoles ~ to Zand hokupurine ~) we~e isolated from extracts of

the dendrophyllid coral Tubastrea coccinea. Indoles ~ to ~ and hokuw

purine (8) were identified from the aeolid nudibranch Phestilla melano~

brachia, which lives and feeds exclusively on T. coccinea. P. melano-

brachia also contained large amounts of an unstable heteroaromatic

base (9), which was not found in the coral, and may be a defensive~

secretion.

Specimens of the dorid nudibranch Dendrodoris nigra and Dendro-

doris tuber~ulosa contained a mixture of polygodial (~) and olepupu

ane (11) in their skin extracts.~

The dorid nudibranch Chromodoris elisabethina contains the potent

vi

fish toxin latrunculin-A (~) in its skin extracts. Latrunculin-A is

a sponge metabolite, but was not isolated from the chief food source

of this dorid, the sponge Heteronema sp. Puupehenone (~) was isola-

ted as the major constituent of Heteronema sp.

Hypselodoris infucata is known to feed on the sponge Dysidea

fragilis. Surface extracts of ~. infucata yielded a mixture of naka-

furan-8 (~) and -9 (~), previously isolated from Dysidea and other

dorid nudibranchs.

10'\IV

CRO

CHO

AcO" ••.

OH

vii

o

viii

TABLE OF CONTENTS---

ACKNOWLEDGEMENTS •

ABSTRACT ..•

LIST OF TABLES • •

LIST OF ILLUSTRATIONS

iii

v

xii

xiii

I. INTRODUCTION

A. GENERAL . . . . . . . . . . . . . . . . . . . . . . . . 1

B. OPISTHOBRANCH MOLLUSKS

1. GENERAL BIOLOGY 3

2. CHEMICAL ECOLOGY OF THE ORDER ANASPIDEA - BACKGROUND 5

3. CHEMICAL ECOLOGY OF THE ORDER NUDIBRANCHIA -

BACKGROUND . . •

4. CHEMICAL ECOLOGY OF OTHER ORDERS OF OPISTHOBRANCHIA -

BACKGROUND • • . .

C. RESEARCH OBJECTIVES

II. RESULTS AND DISCUSSION

A. SECONDARY METABOLITES OF TUBASTREA COCCINEA

1. BACKGROUND AND COLLECTION

6

16

18

20

2.

3.

ISOLATION

CHARACTERIZATION OF 8,9-DIHYDROAPLYSINOPSIN

20

21

4. CHARACTERIZATION OF 6-BROMO-8,9-DIHYDROAPLYSINOPSIN. 25

5. CHARACTERIZATION OF 6-BROMOAPLYSINOPSIN

6. CHARACTERIZATION OF 6-BROMO-2'-DE-!-METHYL-

APLYSINOPS IN . . . . . • . . . . . • . . . . .

7. CHARACTERIZATION OF 2'-DE-N-~mTHYLAPLYSINOPSIN .

30

36

40

ix

TABLE OF CONTENTS (CONTINUED)

8. CHARACTERIZATION OF INDOLE-3-CARBOXALDEHYDE 45

9. CHARACTERIZATION OF 6-BROMOINDOLE-3-CARBOXALDEHYDE 48

10. CHARACTERIZATION OF HOKUPURINE. • . . . . • . . . 52

11. BIOLOGICAL ACTIVITY OF SECONDARY METABOLITES FROM

TUBASTREA COCCINEA. . . . . . . . . . . . . . . 59

B. SECONDARY ~1ETABOLITES FROM PHESTILLA MELANOBRACHIA

1. BACKGROUND AND COLLECTION 60

2. ISOLATION . • . . . 60

3. CHARACTERIZATION OF INDOLES AND HOKUPURINE 61

4. STUDIES ON A HETEROAROMTAIC BASE 61

C. DISCUSSION OF SECONDARY METABOLITES FROM TUBASTREA

COCCINEA AND PHESTILLA MELANOBRACHIA

D. SECONDARY METABOLITES FROM DENDRODORIS NIGRA AND

DENDRODORIS TUBERCULOSA

1. BACKGROUND AND COLLECTION .

2. ISOLATION .......•

3. CHARACTERIZATION OF POLYGODIAL

4. CHARACTERIZATION OF OLEPUPUANE

5. BIOLOGICAL ACTIVITY OF POLYGODIAL AND OLEPUPUANE

E. DISCUSSION OF SECONDARY METABOLITES FROM DENDRODORIS

NIGRA AND DENDRODORIS TUBERCULOSA .

F. SECONDARY METABOLITES OF CHROMODORIS ELISABETHINA

1. BACKGROUND .M~D COLLECTION

2. ISOLATION. . • . . . . .

65

67

67

68

71

77

77

80

80

III.

IV.


x

81

87

89

89

89

93

94

94

94

95

96

97

98

98

102

103

xi


3. 6-BROMOAPLYSINOPSIN. . • • • . . . • .

4. 6-BROMO-2'-DE-N-METHYLAPLYSINOPSIN

5. 2'-DE-N-METHYLAPLYSINOPSIN

6. INDOLE-3-CARBOXALDEHYDE ..•

104

105

105

106

7. 6-BROMOINDOLE-3-CARBOXALDEHYDE 106

8. HOKUPURINE. . . • • • . • • • . . 107

9. ACETYLATION OF HOKUPURINE -- HOKUPURINE ACETATE. 107

10. UNIDENTIFIED BASE FROM P. MELANOBRACHIA • . 108

E. COLLECTION - DENDRODORIS NIGRA AND DENDRODORIS

TUBERCULOSA . • . . . • . .

F. EXTRACTION AND ISOLATION

G. SPECTRAL DATA AND CHEMICAL TRANSFORMATION

1. POLYGODIAL

2. OLEPUPUANE

109

109

110

HI

3. ACID HYDROLYSIS OF OLEPUPUANE -- POLYGODIAL • . 111

H. COLLECTION OF CHROMODORIS ELISABETHINA AND HETERONEMA SP. • 112

I . EXTRACTION AND ISOLATION

J. SPECTRAL DATA

1. LATRUNCULIN-A

2. PUUPEHENONE.

K. COLLECTION - HYPSELODORIS INFUCATA

L. EXTRACTION AND ISOLATION

M. SPECT~~ DATA

1. NAKAFUR}u~-8 AND -9•..

V. REFERENCES

112

114

116

117

117

118

119

LIST OF TABLES----

Table

L PARTIAL SYSTEMATIC CLASSIFICATION OF PHYLUM MOLLUSCA • •

2. SECONDARY METABOLITES REPORTED FROM ORDER NUDIBRANCHIA •

3. 5- AND 6-BROMOINDOLES WITH SP3

CARBON ON C-3 • • • • • •

4. 5- AND 6-BROMOINDOLES WITH SP2

CARBON ON C-3 •

5. SPECTRAL DATA FOR COMPOUNDS RELATED TO STRUCTURE ~ • • • •

xii

Page

4

10

29

34

75

xiii

LIST OF ILLUSTRATIONS

Figure

1

2

3

4

Kupchan partition scheme • • • • • • • •

Mass spectrum of 8,9-dihydroaplysinopsin (~)

UV spectrum of 8,9-dihydroaplysinopsin (~) in methanol.

1H NMR spectrum (300 MHz) of 8,9-dihydroaplysinopsin (~)

in acetone-d6 • • • • • • • • • • • • • • • •

Page

21

22

23

23

5 UV spectrum of 6-bromo-8,9-dihydroaplysinopsin (~)

in methanol . . . . . . . . . . . . . . . . . 26

6

7

8

9

10

1H NMR spectrum (300 MHz) of 6-bromo-8,9-dihydro-

aplysinopsin (~) in acetone-d6 •••• • • • • •

Mass spectrum of 6-bromo-8,9-dihydroaplysinopsin (~)

UV spectrum of 6-bromoaplysinopsin (~) in methanol

Mass spectrum of 6-bromoaplysinopsin (~)

1H NMR spectrum (100 MHz) of 6-bromoaplysinopsin (41)

'\IV

27

2.7

3')

31

in Me ZSO- d6 . . . . • • • . • • . . . • . . • . • • •• 31

11 UV spectrum of 6-bromo-2'-de-~-methylaplysinopsin(~)

in methanol

12 IR spectrum of 6-bromo-2'-de-~-methylaplysinopsin(~)

in KBr.

37

37

13

14

15

Mass spectrum of 6-bromo-2'-de-N-methylaplysinopsin (~)

1H NMR spectrum (300 MHz) of 6-bromo-2'-de-N-methyl-

aplysinopsin (~) in Me2SO-d 6 • • • • • • . • •

lH _ lH NOE difference spectrum of 6-bromo-2'-de-N-methyl-

aplysinopsin (~) in Me2SO-d 6, 68.5 to 6.5.

38

38

40

xiv

LIST OF ILLUSTRATIONS (CONTINUED)

Figure Page

16 UV spectrum of 2'-de-!-methylaplysinopsin ~)

17

18

19

20

21

in methanol

IR spectrum of 2'-de-!-methylaplysinopsin ~) in KBr ••

Mass spectrum of 2'-de-!-methylaplysinopsin ~)

lH NlfR spectrum (300 MHz) of 2 '-de-~-methylaplysinopsin

(~~) in Me2SO-d 6 • • • . • . • . . • • • • • . . • • . •

lH _ lH NOE difference spectra of 2'-de-N-methyl-

aplysinopsin (~) in Me 2SO-d6• 09.35 to 5.85

UV spectrum of indole-3-carboxaldehyde ~) in methanol .

41

42

42

43

44

45

22 IR spectrum of indole-3-carboxaldehyde (~) in

chloroform 46

23

24

25

1H NMR spectrum (300 MHz) of indole-3-carboxaldehyde

(44) in chloroform-dIV\,

Mass spectrum of indole-3-carboxaldehyde (~)

IH NMR spectrum (300 MHz) of indole-3-carboxaldehyde

46

47

(f:k) in Me 2SO-d6 ... . • • • • . • • • • .

26 UV spectrum of 6-bromoindole-3-carboxaldehyde (~)

in methanol

27 IR spectrum of 6-bromoindole-3-carboxa1dehyde (~)

in chloroform •

28 lH NMR spectrum (300 MHz) of 6-bromoindole-3-carbox-

aldehyde (45) in ch1oroform-d . . . . • • • . . . . .'v\,

47

49

49

50

Figure

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43


Mass spectrum of 6-bromoindole-3-carboxaldehyde (~).

~ NMR spectrum (300 MHz) of 6-bromoindo1e-3-carbox-

aldehyde (~) in acetone-d6 • • • • •

Mass spectrum of hokupurine (~) ••••

UV spectrum of hokupurine (~) in methanol

IR spectrum of hokupurine (~) in methanol

~ NMR spectrum (300 MHz) of hokupurine (~) in

methano1-d4 • • • • • • . • • • . . • . • •

Mass spectrum of hokupurine acetate (~)

1 -H NMR spectrum (300 MHz) of hokupurine acetate (49)'\IV

in ch1oroform-d

IR spectrum of hokupurine acetate (~) in chloroform

UV spectrum of hokupurine acetate (~) in methanol

UV spectrum of heteroaromatic base (~) in methanol

Mass spectrum of heteroaromatic base (~) . • • • . .

IH ~ffi spectrum (300 ~lliz) of heteroaromatic base (~)

in methano1-d4 • • • . • . • . . . • • • . . . . . . .

1H NMR spectrum (300 MHz) of heteroaromatic base (~)

in Me 2SO-d6 •••.•• • • • • • . . • • . •

13C NMR spectrum (126.1 ~rnz) of heteroaromatic base (~)

in methanol-d4, broadband and noise decoupled

xv

Page

50

52

53

53

54

54

56

57

S7

58

62

62

63

63

64

xvi


Figure Page

44 Distribution of metabolites in T. coccinea and

P. melanobrachia

45 ~ N}ffi spectrum (300 MHz) of 5 min extract of

65

46

Dendrodoris nigra in benzene-d6 . . . . . . . ....

1H NMR spectrum (300 ~ffiz) of polygodial (~) in

68

chloroform-d . . . . . . . . . . . . . . . . 69

47 IR spectrum of polygodial (,a) in chloroform 70

48 UV spectrum of polygodial (~V in methanol 70

50 IR spectrum of olepupuane (~) in methylene chloride 72

51 1 (300 MHz) of olepupuane (52) inH NMR spectrum'\IV

52

53

54

55

56

57

58

benzene-d6

Mass spectrum of olepupuane (52)'\IV

1H NMR spectrum (300 MHz) of 5 min extract of

Chromodoris e1isabethina in ch1oroform-d

Mass spectrum of latruncu1in-A (~) • • •

UV spectrum of 1atruncu1in-A (~Z) in methanol

IR spectrum of latruncu1in-A (~) in methylene chloride •

1H NMR spectrum (300 MHz) of 1atrunculin-A (57) in'\IV

benzene-d6 . . . . . ..... . . . .

1H NMR spectrum (300 MHz) of 1atrunculin-A (57) in'v\o

chloroform-d

72

73

81

82

82

83

83

84

59 COSY spectrum of latruncu1in-A (~) in ch1oroform-d,

contour plot 86

Figure

60

61

62

63

64


13C NMR spectrum (75.6 MHz) of latruncu1in-A (~)

in ch1oroform-d • • . . •• • • • • • • • •

u~ spectrum of puupehenone (~~) in cyc10hexane

IR spectrum of puupehenone (~) in carbon tetrachloride •

Mass spectrum of puupehenone (~) • • • • • • • •

1H NMR spectrum (300 MHz) of puupehenone (~~) in

ch1orofor~d • • • • • • . . • • • • . • • .

xvii

Page

88

91

91

92

92

1

I. INTRODUCTION

A. GENERAL

The use of secondary metabo~ites by plants and animals as part of

a strategy of survival is now recognized as a general phenomenon in

nature. Biological and chemical studies have revealed many of the in-

tricate and often ingenious mechanisms by which chemicals and chemical

signals are used by living organisms.

evolved out of these investigations.

1A new field, chemical ecology,

Chemical ecology is the study of the manner in which chemicals

mediate the behavioral and physiological interactions of plant and ani-

mal species with their environment, whether living or inanimate. Much

effort in chemical ecology is devoted to the chemical interactions

among living systems, often called chemical communication. Two broad

classes of chemical communication are defined. Intraspecific inter-

actions are mediated by pheromones: these may be used as trail markers,

alarm devices, or in various capacities related to mating. 2 Interspe

cific interactions involve signals called allelochemics,3 which may be

divided into two types. Kairomones are substances released by one or-

ganism, and provide an ultimate survival advantage to the receiving

organism; for example, a predator may find its prey by orienting to a

kairomone. Allomones provide an adaptive advantage to the releasing

organism, and are most often used as defensive agents against preda-

4tors. Allomones may .act as "repellents" (e.g. predator does not ingest)

" " (b b d Lnges t ) 5or as antifeedants predator ites or tastes, ut oes not ~ngest •

Allomones, or defensive chemicals, have been extensively investi-

2

gated, and a large body of literature exists which deals with allo-

6 7mones in plants and arthropods. Many allomones are synthesized de

~ by the organisms, though in some cases a predator may use a meta-

bolite obtained from its prey as an allomone.

In the marine environment, chemical defense appears to play an

important role in the ability of many plant and animal species to

withstand predation. The ecological success of otherwise seemingly

vulnerable sessile (non-motile, permanently attached) organisms, such

as sponges, soft corals, and gorgonians, is often attributed to the

8unusual secondary metabolites found in many of these species. The

survival ability of other benthic organisms, such as gastropod mol-

lusks of the Subclass Opisthobranchia, has also been attributed to

chemical defense. 9, 10

B. OPISTHOBRANCH MOLLUSKS

1. GENERAL BIOLOGY

3

Members of the Phylum Mollusca constitute one of the largest

11animal phyla known, second in numbers only to the Arthropoda. Table

I depicts a partial systematic classification of the Phylum Mollusca.

The largest subgroup within the Phylum Mollusca is the Class Gastropo-

da, which contains three subclasses: Prosobranchia (marine and fresh-

water snails), Pulmonata (terrestrial, air-breathing snails) and Opis-

thobranchia (almost exclusively marine). Some opisthobranch mollusks

are characterized, in part, by the lack of an external shell. Hence,

these animals are soft-bodied, as well as slow-moving. However, des-

pite an obvious lack of physical protection, observations show that

these opisthobranchs without shell are rarely taken as food by reef

. 1 h i fl.·sh and crabs. 12-l 4 I i 11anl.ma s, suc as carn vorous t s now genera y

recognized that these opisthobranchs use one of two main strategies

for defense. A biological mechanism, used by members of the Super-

family Aeolidacea, involves the use of stinging cells (nematocysts),

which the nudibranch obtains from its coelenterate food. A more

general strategy of defense involves the use of specific chemical sub-

stances to deter predation. Defensive chemicals are reported from

most of the orders of the Subclass Opisthobranchia; however, most of

the observations have involved the orders Anaspidea and Nudibranchia.

The results of some of these investigations will be briefly reviewed.

TABLE 1. PARTIAL SYSTEMATIC CLASSIFICATION OF PHYLUM MOLLUSCAl 1

Prosobranchia Pulmonata

Mollusca..~_.------// '--"-:'~.~--'--'---

-..-.--- / ..--------~~~----_.----._--------Cephalopoda Gastropoda Monoplacophora Scaphopoda

----/''''''. "'-.......' ~

OPi_~;~hO~~~~Chias-» •••~~.:,~ • .:' .... -.- • - -'

. -

,

.-

--Bivalvia

Phylum

Class Amphineura

Subclass

Order Thecosomata-,

Cephalaspidea Anaspidea Notaspidea'" --.-...

Sacoglossa Nudibranchia

Superfamily Acteonacea

Bu11acea

Philinacea

\Aplysiacea

Pleurobranchaea

Oxynoacea

Doridacea

Dendronotacea

Arminacea

Juliacea Aeolidacea

Plakobranchacea Onchidiacea

.po.

5

2. CHEMICAL ECOLOGY OF THE ORDER ANASPIDEA - BACKGROUND

Opisthobranchs of the Order Anaspidea are commonly known as "sea

hares." The principal family os this order is the Aplysiidae, which

includes the genera Aplysia, Stylocheilus, and Dolabella. All aply-

siids lack an external shell, hence they are soft-bodied. Sea hares

feed exclusively on algae. l l Some species are notable for their ex

treme proliferation and are used widely inbi010gica1 studies.15

In the first century A.D., the naturalist Pliny reported that

certain maladies, including death, would befall a person unfortunate

16enough to touch or look at a sea hare! Though extracts from some

aplysiids have been demonstrated to be toxic when injected into

"17 Id f h "" i kID1ce, no ev~ ence or uman tox~c~ty s nown.

At least part of the negative reputation of sea hares may be

related to the habit of some species of emitting a purple dye when

disturbed. Chemical studies have shown that the major constituent of

this dye is a linear tetrapyrrole,18 apparently derived from the

erithrobilin pigments of the red algae upon which the animal feeds.19

The ink itself is harmless~20 and in one case was observed to have no

effect on the palatability of food. 2l It has been suggested that the21

ink may serve as a warning signal for potential predators.

Chemical scrutiny of numerous species of aplysiids has resulted

in the isolation and identification of a wide variety of chemical

22structural types. Most studies have involved constituent of the

digestive, or midgut, gland, due to its large size (5-10% of the whole

animal weight) and to an unusually high concentration of secondary

metabolites within this organ. 22 The metabolites isolated from the

6

digestive glands are typical algal compounds, and reflect the diet of

the particular aplysiid at the time of collection.

The secondary metabolites found in sea hares are believed to

serve the animal in a defensive capacity. Though the highest concen-

tration of metabolites occurs within the digestive gland, some levels

are also maintained in the epidermins, which is the point of first

. h d 21contact W1t any pre ator. In nature, aplysiids have no known pre-

23 13 21 24dators, and feeding studies with fish, gulls, and sharks

have demonstrated that sea hares are not considered as palatable by

these animals.

3. CHEMICAL ECOLOGY OF THE ORDER NUDIBRANCHIA - BACKGROUND

Nudibranchs are typically carnivorous mollusks. Many species feed

on sponges, while others feed on coelenterates, worms, or other small

animals. Most nudibranchs are benthic creatures, though some species

have developed the ability to swim. l l

There are numerous accounts of nudibranchs which, when disturbed,

release secretions, some of these are odorous. It can now be reason-

ably stated that these secretions represent examples of the chemical

defense used by these animals. In an early account, for example,

Crozier described "repugnatory glands" from the mantle of Chromodoris

25zebra. When the animal is disturbed, a white, oily discharge is

released from these glands. When applied to food particles, this

25exudate caused predators to reject the food as unpalatable. In a

survey of 24 species of opisthobranchs, Thompson found that two re-

leased acidic skin secretions, but that all species were rejected as

7

13food by fish. In a complementary survey, Edmunds found that five of

25 opisthobranchs studied released acidic defensive secretions.14 A

26later study suggested that the acid was sulfuric acid. None of

these studies were concerned with the mechanism by which non-aicd

secreting nudibranchs avoided predation.

In a 1928 treatise on opisthobranchs, Risbec noted that specimens

27of Phyllidia exude a strong-smelling mucus. This observation was

corroborated in 1963 by Johannes, who discovered that Phyllidia vari-

~ produced an unpleasant-smelling mucus that was toxic to small

28fish and crustaceans. Subsequent collection of P. varicosa allowed

Burreson and Scheuer to isolate and characterize this allomone from

the nudibranch. A chance discovery of P. varicosa actively feeding

29on the sponge Hymeniacidon sp. revealed the ultimate source of this

metabolite, an unusual sesquiterpene isonitrile given the name 9-iso

30 31cyanopupukeanane (t). '

The isolation of 9-isocyanopupukeanane from the food sponge of ~.

varicosa clearly establishes a dietary source of this metabolite for

the nudibranch, which subsequently uses it for its own defense.

8

Indeed, many dorid nudibranchs are sponge feeders and have specific

diets. It also seems to be a general phenomenon that many dorid spe-

cies sequester specific secondary metabolites from their prey, and use

these metabolites as allomones. Table 2 lists nudibranchs which have

been investigated chemically, along with their geographical distribu-

tion and the type of metabolite isolated. Some metabolites are known

to be derived from a specific sponge source, and a direct correlation

has been made: other compounds are known metabolites. Most of these

substances are believed to act as defensive chemicals.

The Hawaiian dorid nudibranchs Chromodoris maridadilus and Hypse-

lodoris godeffroyana were found to release a pungent secretion when

handled. Chemical and spectral analysis of the two isomeric compo-

nents of the skin extracts of both animals resulted in the structures

of nakafuran-8 (~) and -9 (~). Both metabolites were also found in

the sponge Dysidea fragilis, the main food of these nudibranchs.

Nakafuran-8 and -9 demonstrated antifeedant properties in feeding stu

32dies involving the butterfly fish, Chaetodon sp.

Chromodoris albonotata, a relatively rare Hawaiian dorid, was

9

observed to discharge a white secretion when handled. Initial spectral

data of the major constituent of the secretion revealed that it was a

sesquiterpene bearing an enol acetate and an unsaturated aldehyde.

Further studies revealed that this metabolite, pu'ulenal (~), contained

the drimane skeleton. 9

CHO

A Gulf of California dorid, Chromodoris marislae, contained a

sesquiterpene ester, marislin (~). Though the sponge food of £. maris-

lae was not identified, a close structural similarity between marislin

(~) and the sponge metabolite pleraplysillin-Z (~) was noted.33

R= :0OR 0

0

06 R='"

0

A series of CZ6 tetracyclic terpenes was isolated from Chromo

doris sedna. These were found to be of the "scalarin" type of ring

system. represented by ZO-hydroxy-ZO-methyl-deoxoscalarin (Z). The

TABLE 2. SECONDARY METABOLITES REPORTED FROM ORDER NUDIBRANCHIA

Family Phyllidiidae

Phyllidia pulitzeriP. varicosa

sesquiterpene isonitrilessesquiterpene isonitriles

MediterraneanHawaii

Refs.

4230, 31

Family Dorididae

Subfamily Chromodorinae

Chromodoris albonotataC. maridadilusC. marislaeC. sednaHypseIOdoris agassiziH. californiensis!!. ghiselini!!. godeffroyanaH. porterae

Subfamily Diaululinae

Diaulula sandiegensis

Peltodoris atromaculata

drimane esequiterpenefuran sesquiterpenesfuran sesquiterpenesC-26 terpenefuran sesquiterpenesfuran sesquiterpenesfuran sesquiterpenesfuran sesquiterpenesfuran sesquiterpenes

chlorinated acetylenesisoguanosinesterol, polyacetylenes

Hawaii 9Hawaii 32Gulf of Calif. 33Baja Calif. 34W. coast of Mexico 36Gulf of Calif., San Diego 36Gulf of Calif. 36Hawaii 32San Diego 36

San Diego 37Monterey Bay, Channel Is. 38Mediterranean 39, 40

Subfamily Archidoridinae

Archidoris odhneri

Subfamily Dendrodorinae

Dendrodoris limbataD. grandiflora

farnesic acid glycerides

drimane sesquiterpenesfuran sesterterpene

British Columbia

MediterraneanMediterranean

41

42, 4344

....o

TABLE 2. SECONDARY METABOLITES REPORTED FROM ORDER NUDIBRANCHIA (CONTINUED)

Fanuly Dorididae Ref.

Other Subfamilies

British ColumbiaSan Diego

Aldisa sanguineaCadlina luteomarginata

Casella atromarginataGlossodoris tricolorG. valenciennesi

C-25 sterolfuran sesquiterpenesC-23 terpene, furan sesqui-

terpene, drimane sesquiterpenes British Columbiafuran diterpenes Sri Lankasesterterpenes Mediterraneanfuran sesquiterpenes Mediterranean

4510

46474242

..........

12

34structure of sednolide (~) was determined by x-ray analysis. Scala-

35rins are known sponge metabolites, but the food of £. sedna was not

found. 34

Studies of four species of Hypselodoris from California and Mex-

ico revealed that all contained mixtures of some of ten sesquiterpenes

of different types. Representative structures include nakafuran-8 (~)

and -9 (~), agassizin (~), ghiselinin (~), and euryfuran (~).36

9v

10vv

11vv

13

Specimens of Diaulula sandiegensis from San Diego contained mix-

tures of nine chlorinated acetylenes, represented by structures 12 and'VU

13. Sponges upon which Q. sandiegensis were observed to feed did not'VU

contain these metabolites, which are believed to act as antifeedants. 37

12 (lZ, 3E, 9Z)'VU

13 (lZ, 3Z, 9Z)'VU

Interestingly, specimens of Q. sandiegensis from Monterey were found

38to contain the hypotensive agent isoguanine (14) in their extracts.'VU

It is not clear if the chemistry of the two California collections is

different ot if this finding is a result of different isolation tech-

nique.

14vv

39A unique cyclopropane-containing sterol, petrosterol (15), and'VU

40high molecular weight polyacetylenes (~) were isolated from the

dorid Peltodoris atromaculata, and from its sponge food, Petrosia

ficiformis. These resnlts confirm the strong predator-prey relation-

14

39ship observed be~een these two species.

A British Columbia dorid, Archidoris odhneri, was found to con

41tain farnesic acid derivatives (~ - ~) in its extracts.

Rl=R2=H

Rl =H, R2=CH3

Rl =CH3

, R2=H

Investigations into the skin extracts of Dendrodoris 1imbata

revealed that it contains the previously known plant metabolite poly

godial {~).42 The digestive gland, however, contained a series of

CRO

CRO

fatty acid esters based on the drimane skeleton (~, R= C16 to Cl 8

alkyl chains), but not polygodial. 43 D. grandiflora contained onlyoII

RO-Cro/J

15

fasciculatin (~), a known sponge metabolite, in its extracts. 44

Aldisa sanguinea from British Columbia has yielded a C25

steroid

(~), active as an antifeedant in the goldfish bioassay.45

H 0

o

Cadlina luteomarginata has been investigated by two groups, and

numerous metabolites have been identified from its extracts. Speci-

mens from San Diego were found to contain various combinations of

twelve metabolites, most of which were sesquiterpene furans. A close

correlation between the chemical constituents of nudibranch and

10sponge food was observed. Studies of f. luteomarginata from British

Columbia resulted in the isolation of an unusual C23

degraded terpene,

luteone (~~), with a fruity odor reminiscent of that of the nudibranch

16

46itself.

A dorid collected from Sri Lanka, Casella atromarginata, contained

47six furanosesquiterpenes (~ - ~). Furans ~ and ~ had previously

48been identified from an Australian Spongia sp., thus strongly sug-

gesting a dietary source of these metabo1ites. 47

0

" 0"\'

R10

OAc OR2

~ R2 Rl R2

kk Ac OAc ~ H Ac

25 Ac H ~ H H'VU

26 H OAc'VU

a H H

Glossodoris tricolor from the Mediterranean has been found to

contain the known sponge metabolite scalaridia1 (~), along with two

42related sesterterpenes. Another sponge metabolite, longifo1in (~),

d . f' d f G l' i 42 All b Ii' ~ d twas i entJ. ae rom • va encJ.ennes • meta 0 tes were r oun 0

17

inhibit feeding in fish. 42

AcO CHO

CHO

31'\IV

When disturbed, Onchide1la binneyi secretes copious amounts of a

white exudate from pores in glands along the dorsal surface of its

body. The structure of the major constituent of the secretion was

found to be a sesquiterpene which was given the name onchida1 (~).49

~HOOAc

32'\IV

4. CHEMICAL ECOLOGY OF OTHER ORDERS OF OPISTHOBRANCHIA -

BACKGROUND

Opisthobranchs of the Order Sacoglossa are unique in that many

species maintain intact, living chloroplasts within their bodies. The

chloroplasts originate from the algae upon which the sacog1ossan feeds,

and are transported into the tissues of the digestive diverticulum by

18

50an unknown mechanism.

Chemical studies of Tridachiella diomedea from the Gulf of Calif-

ornia yielded the propionate-derived metabolite tridachione (~), and

its 9, lO-deoxy isomer. Tridachia crispata from the Caribbean elabo

rated crispatone (~) and crispatene (~).5l

0 .... 0

OMe 33

·····;v~e

'\IV

0

0

0 ....:~1:X-~

I I0

Studies of Navanax inermis (Order Bullomorpha) have shown that

these animals, when molested, release a bright yellow secretion which

is reported to act as a "trail-breaking alarm pheromone." The struc-

tures of three of the components of this secretion have been reported

52

~as navenones A - C (~ - ,u).

~ R=N

0 ~ R= V-~ OH~ ~ ~ R )'0

37 R= ~I'\IV

19

C. RESEARCH OBJECTIVES

The biological and chemical literature contains ample examples

which suggest that in many nudibranch species chemical defense appears

to be a primary means of protection from predation. It was of interest

to continue these studies with nudibranch species which have not been

previously investigated. The principal objective of this research was

the isolation and structural elucidation of secondary metabolites from

nudibranch species, with parallel investigations of the metabolites of

the appropriate prey (if known) in order to ascertain the biological

origin of the constituents of the nudibranch extracts. It was also

intended to study the activity of the nudibranch metabolites as medi

ators of chemical defense. In this manner, a general understanding

of the role of secondary metabolites in the ecology of the nudibranch

would be gained.

20

II. RESULTS AND DISCUSSION

A. SECONDARY METABOLITES OF TUBASTREA COCCINEA

1. Background and Collection

Tubastrea coccinea is an ahermatypic, or non-reef-building, coral

which is common in Hawaiian waters. The normal habitat of this colo-

nial coral is in dimly lit caves, ledges, or tidepools, where it is

easily distinguished by its bright orange coloration and large polyp

size. I. coccinea does not contain symbiotic zooxanthe11ae within its

53tissues.

Colonies of T. coccinea were collected by SCUBA at -13m from

Pupukea, Oahu, and by snorkeling at -1m in Kaneohe Bay, Oahu. Pick

axes were used to break off the colonies. After the animals were re-

turned to the laboratory, as much detritus as possible was removed

before extraction.

2. ISOLATION

Colonies of T. coccinea were extracted three times with 95%

ethanol over a period of several days. The combined alcohol extracts

were evaporated and lyophilized to yield a dark brown residue. This

residue was partitioned according to the Kupchan scheme (Figure 1),54

which resulted in four discrete fractions.

Preliminary investigations of the hexanes fraction revealed that

it consisted mainly of steroids, carotenoids, and fatty acid deriva

tives, while the carbon tetrachloride fraction contained mostly tri

glycerides and chlorophylls. ~ NMR spectroscopy revealed that the

chloroform and aqueous methanol partition fractions were rich in

21

Lyophilized Ethanol

Extract

Hexanes/aq. MeOH, 9:1

IHexanes fraction

ICC14 fraction

CC14/aq. MeOH, 8:2

CHC1 3/aq. MeOH, 6:4

ICHC13 fraction

(~, ~, ~, ~,

ft!t, ~, /tk)

Figure 1. Kupchan partition scheme. 54

Iaq. MeOH fraction

(~, ~, !tR.)

aromatic metabolites. Each of these fractions was subjected to Seph-

adex LH-20 column chromatography, followed by reversed-phase HPLC to

obtain pure metabolites.

2. CHARACTERIZATION OF 8,9-DIHYDROAPLYSINOPSIN

8,9-Dihydroaplysinopsin (~) was isolated as a white solid. Mass

spectroscopy (Figure 2) contained a molecular ion at ~/~ 256, which

was shown by high-resolution peak matching to correspond to a molecu-

lar formula of Cl4Hl6N4o. The UV spectrum (Figure 3) contained absorp

tion maxima at 223 (g=22350), 242 (g=5450), and 282nm (g=3850), which

are indicative of a simple indole chromophore. Presence of indole was

1further implicated by the presence in the H NMR spectrum (Figure 4) of

four mutually coupled aromatic proton signals between 07.05 and 7.63,

22

~

t

!~E

! II5~

Figure 1. Mass spectrum of 8,9-dihydroaplysinopsin (~).

corresponding to protons H-4 to H-7 of indole, and a one proton signal

at 07.14 (H-2), which sharpened when a broad signal at 010.00 (H-l) was

irradiated or exchanged with D20. Finally, a strong fragment ion in

the mass spectrum at~/~ 130 was assigned to fragment~, which is typi

3cal of indoles which bear an sp carbon at C-3, as in the amino acid

tryptophan.

Fragment ~ indicates that the immediate substituent on C-3 of1

indole is a methylene. Inspection of the H NMR spectrum reveals.that two non-equivalent protons of a methylene do indeed exist, at

23

2230I:1 (€=22350).242mn (e..5450)282nm (€=3850)291nm (€=3350)

I200

I250

A (nm)

I300

I350

Figure 1. UV spectrum of 8,9-dihydroaplysinopsin (~) in methanol.

L

I1()

I8

I6

Io PPM

Figure~. ~ N}ffi spectrum (300 MHz) of 8,9-dihydroaplysinopsin (~)

in acetone-d6•

24

03.39 and 3.09, each a doublet of doublets with ~=5.2, l4.lHz. Irra-

diation of either proton signal caused the other to collapse to a

doublet with J=5.2Hz. Simultaneously, an apparent triplet at 03.93

(~=5.lHz) was simplified to a doublet, J=5.lHz. Irradiation of the

triplet induced both patterns at 03.39 and 3.09 to collapse to a doub-

let, each with a coupling consant of l4.lHz, thus establishing that

these are geminal protons. These results also show that a methine

bearing carbon is attached to the methylene, and a partial structure

~ may be advanced.

Partial structure ~ accounts for ClOHgN and six degrees of unsat

uration, leaving C4H7N30

and three sites of unsaturation to be assigned.

Further analysis of the lH NMR spectrum revealed the presence of two

N-methyls at 02.61 (s) and 2.76 (broad s), and a broad, D20 exchange

able proton signal at 07.34, which is in the proper location for an

imine proton. Thus, all protons are assigned. Irradiation of the

imine proton at 07.34 caused the broad methyl at 02.78 to sharpen mar-

kedly. This can only be explained if the imine carbon is directly

attached to the nitrogen bearing the methyl. An !-methyl, a carbon.

and an oxygen remain to be assigned. In order to satisfy two addi-

tional degrees of unsaturation, a carbonyl and a ring must be involved.

Based on biogenetic and stability arguments, the most plausible ar-

rangement for these structural fragments was as shown in structure ~.

25

Attachment of the carbonyl and a nitrogen to C-9 is based on an

assumed origin and analogy with tryptophan. Assignment of the final

proton was limited by spectral data.

Indole ~ is the 8,9-dihydro derivative of a known sponge meta-

bolite, aplysinopsin (~), which had been isolated from sponges of the

55 56genera Aplysinopsis, Thorecta, and Dercitus. '

4. CHARACTERIZATION OF 6-BROMO-8,9-DIHYDROAPLYSINOPSIN

6-Bromo-8,9-dihydroaplysinopsin (~) was isolated as a white

powder. UV spectroscopy (Figure 5) revealed absorption maxima typical

of an indole nucleus with peaks at 228 (£=20050), 279 (£=2100), 288

(£=2700), and 296nm (£=2700).1

The H NMR spectrum (Figure 6) of k~

differed from that of 8,9-dihydroaplysinopsin (~) only in the region

of the aromatic signals. These signals were less complexly coupled in

~, as one less aromatic proton is present in this region. The mass

spectrum (Figure 7) of ~ exhibited molecular ions at ~/~ 334 and 336

26

of equal intensity, indicating the presence of bromine in this mole-

cule. This was confirmed by high-resolution measurements, which

79yielded a molecular formula of C14H15 BrN40 for the ion at ~/~ 334.

The bromine was placed on the indole nucleus as a result of ions in the

mass spectrum at ~/~ 208/210, which correspond to fragment c. Since-v

irradiation of the N-H proton at 010.39 caused a sharpening of a one

Br

proton singlet at 07.15 (H-2), the bromine was assigned to the benze-

noid ring of the indole. Analysis of the three remaining benzenoid

228nm (€ =20050)235-240nm (shoulder)278nm (€ =2100)288nm (€ =2700)296nr;l (e =2700)

·r200

l250

A (nm)

l300

I350

Figure 1. UV spectrum of 6-bromo~8,9-dihydroaplysinopsin(iQ) in

methanol.

27

iii i

10IB

I6

I I. i i

2 0 PPM

Figure 2,.

II..~

i~-!~

O! z..i

1H NMR spectrum (300 MHz) of 6-bromo-8,9-dihydroaplysinopsin

(~) in acetone-d6•

r:

II0.-...

~ ! 0

~ ! ~ ~ ~ S S 8 2 ~ S ~ 81- 0 Po ~ S 0 :.. .. .. .. .. .. PO .. ...

Figure 7. Mass spectrum of 6-bromo-8,9-dihydroaplysinopsin (iR).

28

protons at 07.59 (d, ~=8.5), 7.09 (dd, ~=1.7, 8.5), and 7.55 (d, ~=1.7)

by double resonance experiments clearly showed that these protons were

arranged either on t4e 4, 5, 7 or on the 4, 6, 7 positions of indole:

bromine is thus on C-5 or C-6.

Placement of the bromine on indole was accomplished by comparing

the chemical shifts of the benzenoid protons of ~ with those of known

5- and 6-bromoindoles, which also bear an alkyl substituent on C-3.

Table 3 lists these values for several bromoindoles, and it is seen

that the resonances of the benzenoid protons of ~ agree best with

placement of bromine at C-6.

The molecular formula of ~ indicates that the only difference

from 8,9-dihydroaplysinopsin (~) is substitution of a bromine for a1

proton. Additionally, the remainder of the H NMR spectrum of ~ was

nearly the same as for~. The structure of ~ could thus be completed

by direct analogy as 6-bromo-8,9-dihydroaplysinopsin.

B

TABLE 3. 5- AND 6-BROMOINDOLES WITH SP3 CARBON ON C-3

Solvent H-2 H-4 H-5 H-6 H-7 Ref.

yH3oN

>= NH acetone-d607.15 7.59 7.09 Br 7.55 this

fVl workN

I

Br u CH 3

NHZ I II CD3CN/CD3OD 07.15 7.53 7.17 Br 7.57 57

N r .............." <;»: vHBi ~ - N - u·

H

+NMe 3

CF3COOD

07.20 7.33 Br 7.57 58

Br~"""'N/ lJUU

HNMe

B~~'-../ 2acetone-d6 06.97 7.70 Br 7.13, 7.25 56

Br e3CF3COOD 07.20 7.46 Br 7.31 58

N\0

30

5. CHARACTERIZATION OF 6-BROMOAPLYSINOPSIN

6-Bromoaplysinopsin (~) was isolated as a yellow solid. UV

spectroscopy (Figure 8) revealed absorption maxima at 233 (g=l5000),

260-290 (g=5700), and 380nm (g=l0700). The mass spectrum (Figure 9)

contained parent ions at ~/~ 332 and 334 of equal intensity, indicating

that this molecule contained one bromine. High resolution peak match

ing established a molecular formula of Cl4G1379BrN4o for the ion at

~/~ 332, requiring ten sites of unsaturation.

1The basic features of the H NMR spectrum (Figure 10) closely

resembled those of bromoindole 40: signals for four indole ring pro'v,",

tons, one indole !-H, and two !-methyls were clearly seen. However,

signals for a -CH-CH2- moiety were not observed in the spectrum of kk.

Instead, a sharp one proton olefinic signal appeared at 06.56. This

I200

I250

I300

380nm (e:=10700)260-290 (e:=5700)233nm (e:=l5000)

I350

A (nm)

I400

Figure~. UV spectrum of 6-bromoaplysinopsin (kk) in methanol.

& !

31

Figure 1. Mass spectrum of 6-bromoap1ysinopsin <~).

: ... ii, ! i. i ..... : ..•. i I, •. "' ••. ' I' .' •. ,'.•. ,',. i

, ,•. __ ••.. _. _. L... .. _. •.__ •••.••

fII1:'Iil:1

j\.1I:J.iIiI!

II .u° II

. I j I- oE I ti!:1 I

-ll JJJLJn]_h_-O_O

~ __0

0_-

- jUuL _'0'.. . . . ! . . . J • • • • • I t • ! • t I • ! 1 : • • • '.' ! • • " , , • ! : • • • • I' • r • " _~ " • • : • • t • • • I • • '.' • • •

7 6 5 4 3 z PPM

Figure 10.1H NMR spectrum (100 MHz) of 6-bromoap1ysinopsin <tVin Me

ZSD-d 6•

32

signal could best be accomodated by placing a double bond on the C-8 -

C-9 bridge between the indole and the heterocyclic ring, resulting in

structure~, which is a brominated analog of aplysinopsin (~). This

Br

4la~

assignment seemed pertinent , since other members of the aplysinopsin

series were identified from the same extracts.

Supporting evidence for this structure was provided by an analy-

sis of the mass spectrum of~. Aplysinopsin shows fragment ions in

its mass spectrum at ~/~ 155 and 169, which are believed to correspond

to fragments ~ and ~, respectively.55 The mass spectrum of ~ contains

pairs of ions at ~/~ 233 and 235, and at 247 and 249, which correspond

to the expected fragment ions t and ~, respectively. Thus, the rela-

tionship of indole ~ with aplysinopsin is established.

X R ~~

~ H H 155

~ H CH3

169

f Br H 233/235~

~Br CH3 247/249

The multiplicities and coupling constants of the three benzenoid

protons at 07.90 (d, ~=8.0), 7.25 (dd, ~=1.5, 8.0), and 7.62 (d, ~=1.5)

indicated that they were on the 4, 5, 7, or 4, 6, 7 positions of in-

dole: this relative arrangement was confirmed by double resonance

33

experiments. Bromine was finally assigned to C-6 of indole by compari-

son of the chemical shifts of the benzenoid protons of 5- and 6-bromo

2indoles bearing an sp carbon at C-3 (Table 4). The structure of

indole it is thus 6-bromoaplysinopsin.

B

TABLE 4. 5- AND 6-BROMOINDOLES WITH Sp2 CARBON ON C-3

Solvent H-2 H-4 H-5 H-6 H-7 Refs.

R

D-:YX:>=NH I this~ R=CH3 DMSO-d6 08.73 7.90 7.25 Br 7.62 work

~ NoNfa R=H DMSO-d6 08.09 7.88 7.13 Br 7.54 this

Br H CH work, 593

H

QJ~N)=O DMSO-d6 08.16 7.77 7.26 Br 7.63 60~ NONBr H

H

627.57Br7.327.8867.43CDCl3

HOacetone-d6 08.26 8.18 7.41 Br 7.79 this44

,-~ 'N

, '\IVCDC13 07.81 8.16 7.40 7.59

work, 61Br Br

OOMe

Br

w.po

TABLE 4. 5- AND 6-BROMOINDOLES '~ITH SP2 CARBON ON C-3 (CONTINUED)

Solvent 11-2 H-4 H-5 H-6 H-7 Refs.

BrQ1CHOacetone-d6 -- o 8.23 Br 7.37, 7.51 61

~ NH

Be .e-; ~HN

)==NHDMSO-d

6 s 8.31, 8.09, Br 7.27 7.43 59NI * *eH3

8.36 8.13

*isolated as mixture of E and Z isomers- -wU1

sin

36

6. CHARACTERIZATION OF 6-BROMO-2 '-DE-B.-METHYLAPLYSINOPSIN

6-Bromo-2'-de-B.-methylaplysinopsin (~) was isolated as a yellow

solid. UV spectroscopy (Figure 11) revealed absorption maxima at 221

(E=23000), 287 (E=5200), and 385nm (E=9800). IR spectroscopy (Figure

12) showed a carbonyl band at l685cm-l• The mass spectrum (Figure 13)

contained parent ions of equal intesity at ~/~ 320 and 318, consis

tent with the presence of one bromine. Salient features of the lH NMR

spectrum (Figure 14) include four aromatic protons, one olefinic pro-

ton, and one B.-methyl signal.

The data show a strong similarity with that of 6-bromoaplysinop

1(~). However, the H NMR and mass spectra indicate that kt lacks

one of the B.-methyls of the five-membered heterocyclic ring. Such a

compound was already known, and a comparison of the data for ~ with

literature values showed that these metabolites are identical. Indole

~ has the structure of 6-bromo-2'-de-~-methylaplysinopsin,which was

59previously isolated from the sponge Dercitus Spa

Br o

Placement of the methyl at B.-4' was justified by the absence in

the mass spectrum of peaks at ~/~ 247 and 249 which correspond to

fragment g. This peak is considered to be characterixtic of aplysi'V

nopsin derivatives bearing a methyl at N_2,.50, 59 The structure of

f d hes I 596-bromo-2'-de-!-methylaplysinopsin had been con irme by synt es~s.

221nm (€=23000)278nm (€=5200)3£5nm (€=9800)

37

I200

I250

I300

A (nm)

I350

I400

Figure 11. UV spectrum of 6-bromo-2'-de-N-methy1ap1ysinopsin (42) in- 'VV

methanol.

Figure 12. IR spectrum of 6-bromo-2'-de-!-methy1ap1ysinopsin (~)

in KBr.

38

§,IIII

II!

.'1 !~-;

II

II

a,II

~ ~ I'"

":'t""'!

2 III

·1i~ ..j,

o . I 1'1

IIII,I

IIII

IIII!:'iI,

""dII

IIii'I'

"!1 ;I

"I il,tol jl11 :1"i"" !:

. I I , I 'I i', o' , , " iii$ S e

~ ?a 0 '" :;.~." ::: ..... I. 0' ':. .\

Figure 13. Mass spectrum of 6-bromo-2'-de-N-methy1ap1ysinopsin (~).

Figure 14. of 6-bromo-2'-de-!-methy1ap1ysi-

a PPMI2

I'i

: o. IIG a 6

1H NMR spectrum (300 MHz)

nopsin (~) in M~2S0-d6.

39

Placement of the bromine at C-6 of indole was confirmed by compa-

rison of chemical shift values for the benzenoid protons with known

bromoindoles (Table 4).

1Close inspection of the H NMR spectrum of 6-bromo-2'-de-N-methyl-

aplysinopsin (~) reveals the presence of approximately ten percent of

a minor isomer. The isomers could not be separated by chromatography,

and probably represent isomers about the 8,9 olefin. A similar situa

tion has been reported for aplysinopsin (~).55

An attempt was made to determine the stereochemical arrangement

of 42 by NOE difference spectroscopy (Figure 15). Irradiation of the~

H-8 singlet at 06.77 resulted in enhancements of the signals at 08.27

(H-2) and 8.02 (H-4). Irradiation of the H-4 doublet at 08.02 resulted

in enhancements at 06.77 (H-8) and 7.25 (H-5). An NOE enhancement was

seen at 06.77 (H-8) when the signal at 8.27 was irradiated. These

results indicate that, in the time frame of the experiment, the olefi-

nic proton H-8 is in close proximity to both H-2 and H-8. Consequent-

ly, rotation must be occuring about the C-3 - C-8 bond. Thus, the

stereochemistry of 42 could not be unambiguously assigned as E or Z by~

this method.

40

irr___......... A.A'__ .--..,-_ __.-

irr

irr

I8.5

IB.C

I6.5" PPM

Figure 15. lH -~ NOE difference spectrum of 6-bromo-2'-de-N-methyl

aplysinopsin (~) in Me2SO-d6, 08.5 to 6.5.

7. CHARACTERIZATION OF 2'-DE-N-METHYLAPLYSINOPSIN

2'-De-~-methylaplysinopsin (~) occurs as a yellow solid. The UV

spectrum (Figure 16) contained maxima at 223 (g=18000), 280 (€=3900),

aDd 390nm (€=8700), while the IR spectrum (Figure 17) showed a carbo~

-1nyl stretch at l690cm , Mass spectroscopy (Figure 18) revealed a

41

molecular ion at ~/~ 240, which corresponded to a molecular formula of

C13H12N40, requiring ten sites of unsaturation.1

A close similarity between the H NMR spectrum of 43 (Figure 19),

and that of 6-bromo-2'-de-N-methylaplysinopsin (42) was noted. The- '\IV

only difference was the presence of one additional benzenoid proton and

an increase in the complexity in the benzenoid protons of 43. Since. '\IV

the mass spectrum supported the replacement of the bromine in 42 with'\IV

a proton, a structure for indole 43 could be drawn by analogy.'\IV

H

)=NHNICH3

223nm (e:=18000)280nm (e:=3900)390nm (e:=8700)

200I

250I

300I

350

A enm)

r450

Figure 16. UV spectrum of 2'de-~-methylaplysinopsin(~) in methanol.

42

---

Figure g.

co~l

Iii

IR spectrum of 2'-de-N-methylaplysinopsin (43) in KBr.'V\,

",. '".,. ".;:; '" " ~~:.. "

>-!:::nz::=~

j I.

e..

I~

~~'l

~.Iilli'll

I I' & i I I ,', I. ~ • I . I I' I

,:;:I

Figure 18. Mass spectrum of 2'-de-N-methylaplysinopsin (~).

\ 'I1~:-=;:=;:=r=1 =;==*":!:::;':::!!u......~I·-,~" i--,-=t- , i I Ju

'i2 10 0 f:j l.j

~-Iii I .

o PPM

43

Figure 19. 1H NMR spectrum (300 MHz) of 2'-de-!-methylaplysinopsin

(~) in Me2SO-d6•

As with bromoindole ~' assignment of the one !-methyl to !-4 was

based on the absence of a fragment ion at m/~ 169 in the mass spectrum

59of ~' corresponding to fragment ~. Indole ~ is 2'-de-!-methyl-

aplysinopsin, in accordance with previous nomenclature. It had also

been isolated from the sponge Dercitus sp., along with its 6-bromo

analog (~).59 The structure of ~ had been confirmed by synthesis. 59

The stereochemistry about the 8,9 olefin of 43 was investigated

by NOE difference spectroscopy (Figure 20). Irradiation at 06.81 (H-8)

produced enhancements at 07.97 (H-4) and 8.27 (H-2), while irradiation

at 07.97 (H-4) caused NOE enhancements at 06.81 (H-8) and 7.15 (presu-

mably H-5). Irradiation at 08.27 (H-2) caused an NOE effect to be seen

at 06.81 (H-8). These results are identical to those obtained with

44

llll'l

A1 I

9.0I I I

8.5iii8.0

iii7.5

iii

7.0iii

6.5iii

6.0 PPM

Figure ZO. ~ - lH NOE difference spectra of Z'-de-!-methylaplysinopsin

(~) in MeZSO-d6, 09.35 to 5.85.

the experiment performed on the analogous protons of 6-bromo-Z'-de-N-

methylaplysinopsin (42). Thus, rotation of the molecule must be occur'V\,

ing which allows H-8 to be in close proximity to both H-Z and H-4 of

indole. Consequently, the stereochemistry about the 8,9 olefin could

not be defined on the basis of these experiments.

45

8. CHARACTERIZATION OF INDOLE-3-CARBOXALDEHYDE

Indole-3-carboxaldehyde (44) was isolated as a tan crystallineIV\,

solid. UV spectroscopy (Figure '21) contained maxima at 207 (€=lOOOO)t

240 (€=5l00)t 258 (€=3900)t and 294nm (€=4200). The IR spectrum (Fig

ure 22) contained a carbonyl stretching band at l685cm-lt which was

shown by a lH NMR singlet at 010.05 (Figure 23) to be due to an alde-

hyde. Mass spectroscopy (Figure 24) showed a molecular ion at m/z 145,

which corresponded to a molecular formula of C9H7NO.

The lH NMR spectrum of ~ in chlorofor~d (Figure 23) or Me2SO-d6

(Figure 25) also contained a one proton DZO exchangeable signal and

signals for five aromatic protons. Irradiation of the !-H signal

caused one aromatic singlet (at 08.27 in Me 2SO-d6) to sharpen. The

other four protons could be seen to be mutually coupled by double

207nm240nm258nm294nm

(€=lOOOO)(€=5l00)(€=3900)(€=4200)

200I

250I

300

it (nm)350

Figure 21. UV spectrum of indole-3-aldehyde (~) in methanol.

46

Figure 22. IR spectrum of indole-3-carboxaldehyde (44) in chloroform."""

I4

I8

uI7

a ·PPM

Figure 23.1H NMR spectrum (300 MHz) of indole-3-carboxaldehyde (~)

in chloroform-d.

47

I.I

i

IIITT

8 ~M M

o:

I

! !." I I I I I ".

~ .~ ~ ~ ~

Figure 24. Mass spectrum of indole-3-carboxaldehyde (~) •

Io PPi"i

I2

I46

I8

.-lUll U Li I I I . I I I I

B 7

~ - .. l~i , I I i , I i I I I I ( i

Figure 25. ~ NMR spectrum (300 MHz) of indole-3-carboxaldehyde (~)

in MeZ

SD- d 6 •

48

resonance experiments.

Subtraction of the elements of aldehyde (CHO) from the molecular

formula results in C8H6N, which corresponds to a monosubstituted indole.

Based on the series of indoles already isolated from 1. coccinea, a

logical structure for ~ was indole-3-carboxaldehyde. Comparison of

spectral data of authentic indole-3-carboxaldehyde (Aldrich Chemical

Company) with that of ~ proved them to be identical in all respects.

~CHO

~N)JH

44"'"

9. CHARACTERIZATION OF 6-BROMOINDOLE-3-CARBOXALDEHYDE

6-Bromoindole-3-carboxaldehyde (~) was isolated as a light yellow

solid. The UV spectrum (Figure 26) showed maxima at 210 (e=11900), 217

(e=12l00), 244 (e=6900), 263 (e=6500), and 290 (e=4000): this is in

close agreement with the UV spectrum of indole-3-carboxaldehyde (kk).

-1The IR spectrum (Figure 27) showed a carbonyl band at l660cm , which

was assigned to an aldehyde carbonyl by virtue of a one proton singlet

1at 010.03 in the H NMR spectrum (Figure 28).

The mass spectrum of ~ (Figure 29) contained a four-line pattern

at ~/~ 225/224/223/222 of relative intensity 88/72/100/67. High-reso-

lution peak matching revealed that the peak at ~/~ 223 corresponded to

79a molecular formula of C9H6 BrNO, requiring seven sites of unsatura-

tion. Thus, the ions at ~/~ 225 and 223 are the parent ions, while the

mass peaks at ~/~ 224 and 222 are fragment ions of unusual intensity.

I200

I250

A (nm)

210nm217nm244nm263nm290nm

I300

(e=11900)(e=12100)

(e=6900)(e=6500)(e=4000)

I350

49

Figure 26. UV spectrum of 6-bromoindo1e-3-carboxa1dehyde (~) in

methanol •

....I ...IJnMII.cr - - .. 1M Me ... _

Figure 27. IR spectrum of 6-bromoindo1e-3-carboxa1dehyde (~) in

chloroform.

~1,rl.'JIIii I iii

S 8

50

I10

I(3

I6 o PPM

Figure 28. ~ NMR spectrum (300 MHz) of 6-bromoindole-3-carbox

aldehyde (~) in chloroform-d.

I-

~:: I-i

R.

0L..... ~

Ie I I T I • I I • I I I • I I I I I I I I I I

2 0 0 0 ! ! 0 ! ! g 2 0 S 0 §0 ow .. • :: • ~to to to ,.

Figure 29. Mass spectrum of 6-bromoindole-3-carboxaldehyde (~).

51

The data show that ~ is a brominated derivative of indole-3

carboxaldehyde (~). The ~ NMR spectrum (Figure 28) contained only

five other proton signals beside the aldehyde signal: each of these

protons could be assigned to positions on an indole ring by direct

analogy to previously isolated bromoindoles (Table 4). The structure

of ~ is thus confirmed as 6-bromoindole-3-carboxaldehdye.

Br

CHO

The position of the bromine on C-6 could be independantly deter-

I 63mined by the "solvent shift" method in H NMR spectra. It has been

observed that a marked downfield shift occurs in the signals of

protons H-2 and H-7 when the spectrum is measured in polar solvents.

The largest shift is observed for H-2, H-7 shifts moderately, and

little or no change is observed for H-4, H-5, and H-6. The chemical

shifts of the ring protons of 6-bromoindo1e-3-carboxaldehyde in

chloroform-d (Figure 28) versus the shifts of the same protons in

acetone-d6 Figure 30), are summarized in Table 4. As anticipated,

the largest shift (~O.42ppm) is associated with H-2. Only one other

signal in chloroform-d, at 07.59 (d, ~=1.6), shows a significant shift

(60.16ppm). This proton was thus assigned to H-7. The relationship

between this proton and H-4 and H-5 had previously been determined by

decoup1ing experiments.

6-Bromoindo1e-3-carboxaldehyde (~) had previously been reported

52

o PPM,2

ILj

I6

I

!,B

-1:=~~~~~:::;~~~~=;::~~~L.;:~:;:::~10

Figure 30. 1H NMR spectrum (300 MHz) of 6-bromoindole-3-carbox-

aldehyde (~) in acetone-d6•

61from a marine pseudomonad.

10. CHARACTERIZATION OF HOKUPURINE

Hokupurine (~) was isolated as a crystalline solid. Mass spec

troscopy (Figure 31) provided a molecular ion at ~/~ 179, which corres-

ponded to a molecular formula ofC7H9N50: this molecule thus incorpo

rated six sites of unsaturation and is clearly heteroaromatic. The UV

spectrum (Figure 32) in water exhibited absorption maxima at 214

(E=lllOO) and 290nm (E=10400). In base the maxima shifted to 218

(E=6400) and 278nm (E=lOlOO), while no change was seen in acid. The

-1IR spectrum (Figure 33) contained a broad absorption at 3600-2900cm ,

-1 1and bands at 1717 and l670cm • The H NMR spectrum of ~ in methanol-

53

i..o:e..

-

II :,I

, 1'1'I I • I I I I I I

~

!..-ZM.. ~

Figure 31. Mass spectrum of hokupurine (46).'\IV

,200

228nm (e: =7200)288nm (e: =11500)

I250 300 350

A (nm)

Figure 32. UV spectrum of hokupurine (46) in methanol.'\IV

...0:.,

I --

Figure 33.

.-

IR spectrum of hokupurine (46) in KBr.'\IV

.... 1M ~ ....

54

T

i •

141 I i

12i I

10I . I I

!3 6

I

I IJ~JJiii ! t

·4 2 o PPM

Figure 34. IH NMR spectrum (300 MHz) of hokupurine (~) in methanol-d4•

55

d4 (Figure 34) contained signals only for two methyls at 03.50 and 3.55,

and for a one proton singlet at 07.60. No other proton signals were

1observed, even when the H NMR spectrum was measured in Me 2SO-d6•

Determination of the carbon skeleton of kk with the available data

proved difficult. Initially, it was though that 46 was related to the'V\,

heterocyclic ring of the aplysinopsin series of metabolites already

isolated from the same extract. Though this seemed a good possibility

because of the mutual presence of two !-methyls and a high nitrogen

content, it was eventually ruled out. By subtracting the elements of

two methyls from the molecular formula, CSH 3N50 remains for the basic

skeletal framework. This formula corresponds to the purine base gua-

nine (47) or isoguanine (48), and suggested that the natural product'\IV 'V\,

46 was a di-N-methyl derivative of one of these purines. A search of

the literature, however, revealed that none of the known di-!-methyl

isomers of guanine or isoguanine had properties consistent with those

of 46. In particular, the UV spectrum of 46, and its behavior in acid'\IV '\IV

or base showed little similarity with the UV spectra of any of the

known bases.

Acetylation of ~ with acetic anhydride and pyridine resulted in

a single crystalline product (~). A molecular ion in the mass spec

trum of ~ at ~/~ 221 (Figure 35) was shown to correspond to a molecu-

56

O;-''T''''T''''''I"'''''I~Iw-T~I'''fif''''~,......r'&r-J~'''''''~~~T''l'''''+'r-rlL,-p.'I''T''I"'I''''l'''T'''l''''''''~1''''I''"I

Ie 0 ~ ~ sa!

Figure 35. Mass spectrum of hokupurine acetate (49).'\IV

lar formula of C9HllN502 by high resolution peak matching. A major

fragment ion at ~/~ 179 (M+ - 42) showed that an ~-acety1 was genera-

ted by this reaction. Further support for the ~-acety1 moiety was

provided by a three proton singlet at 02.31 in the ~ NMR spectrum

-1(Figure 36), and by a carbonyl band at 1720cm in the IR spectrum

(Figure 37). Other signals in the 1H NMR spectrum of 49 were two N-'\IV -

methyl singlets at 03.65 and 3.60, and a one proton singlet at 07.69.

An additional proton, whose existence was shown by the molecular for-

mula, was not observed. The UV spectrum of ~ (Figure 38) showed

absorption maxima at 223 (E=6800), 250 (E=3300), 298 (E=7000), and a

broad shoulder at 320-330nm.

Data from the monoacetate did not provide any new information on

IJi)

I(0;

Ie

I'I o rrn r

57

Figure 36.1 .

H NMR spectrum (300 MHz) of hokupurine acetate (49) in'VU

chloroform-d.

............ .. . - - - - . . . .,~

: i. I

'.' ,, I~ !.

"

.:. L .;

:-t-. &.

...

Figure]I. IR spectrum of hokupurine acetate (~) in chloroform.

223nm (e:=6800)250nm (e:=3300)298mr.. (e:=7000)

58

I200

I250

I300

A (nm)

I350

I400

Figure 38. UV spectrum of hokupurine acetate (49) in methanol.'\/\, .

the molecular framework of the natural product. Because of the diffi-

culty in isolating further amounts of 46, attempts were made to solve"'"

the structures of 46 and 49 by x-ray crystallography. Crystals of'\/\, -vv

monoacetate 49 were first prepared and analyzed by Ms. Gayle Matsu'lI"

moto and Dr. Jon Clardy of Cornell University. An !i-acetyl dimethyl

ated isoguanine structure for 49 resulted from their studies. 64

"'"

49"'"

Structure ~, however, is not necessarily the most stable tauto-

59

mer of the natural product. Crystals of the natural base were even-

tually prepared and subjected to x-ray analysis by the same crystal

lographers. Structure 46 was determined for the natural base. 64

46'v\,

Nitrogen-8, therefore is in the Lmf.no tform in the natural base 46. The'\/\,

position of the amino proton could not be fixed to N-5 or ~-7, probably

due to rapid equilibrium between these positions. Structure 46 also'VI"

explains the anomalous UV spectrum observed for this purine base.

Since the carbonyl is isolated by the two ~-methyls, it cannot enter

into conjugation with the rest of the purine ring system.

Purine 46 is formally 1,3-dimethylisoguanine, and is a hithero'VI"

- 65unreported compound. It has been given the trivial name hokupurine:

derivative 49 thus is known as hokupurine acetate.'\/\,

11. BIOLOGICAL ACTIVITY OF SECONDARY METABOLITES FROM TUBASTREA

COCCINEA

No in vitro activity was found by any of the indoles 38, 40, 41,'\IV '\/\, -vv

,1, 43, 44, or 45, or by hokupurine (46) against Candida albicans," v u '\/\, '\IV 'VI" '\I\,

Bacillus subtilis. Pseudomonas sp. Escherevia coli, or Staphylococcus

aureus •

60

B. SECONDARY METABOLITES FROH PHESTILLA MELANOBRACHIA


The aeolid nudibranch Phestilla melanobrachia (Superfamily Aeoli-

dacea, Family Aeolidiidae) lives in intimate association with, and

feeds exclusively upon, dendrophyllid corals such as T. coccinea. The

pigmentation of the nudibranch is apparently derived from the particu-

lar type of coral it is eating, as evidenced by feeding studies with

dendrophyllid corals of different colors. In Hawaii, ~. melanobrachia

feeding on!. coccinea maintains the same orange coloration as its

66 67prey. '

Six specimens of P. melanobrachia were collected from beds of T.

coccinea at Pupukea, Oahu by Mr. Scott Johnson. The collection was

made at night, when the animals are actively feeding on the coral

polyps.

2. ISOLATION OF METABOLITES

The live nudibranchs were extracted three times with methanol.

The combined extracts were then evaporated in vacuo to a brown residue.

This residue was partitioned according to the Kupchan scheme (Figure

1) •

Preliminary investigations indicated that the compositions of the

hexanes and carbon tetrachloride fractions were similar to those of T.

coccinea, and so were not investigated further. The chloroform and

methanol fractions were found to be rich in aromatic metabolites: the

1chloroform fraction appeared identical by H NMR spectroscopy to the

corresponding fractions from T. coccinea, but the methanol fraction

61

was dominated by aromatic signals not seen in the coral extracts. Pu-

rification of metabolites was accomplished by Sephadex LH-20 chroma to-

graphy, followed by reversed-phase HPLC, when necessary.

3. CHARACTERIZATION OF INDOLES AND HOKUPURINE

Five indoles identified from the extracts of T. coccinea were also

found in P. melanobrachia. These are: 6-bromoaplysinopsin (41), 6~

bromo-2'-de-~-methylaplysinopsin(~), 2'-de-~-methylaplysinopsin(~),

indole-3-carboxaldehyde (~), and 6-bromoindole-3-carboxaldehyde l~).

In addition, hokupurine (46) was also isolated from the nudibranch~

extract. All spectral data for each of these metabolites correlate

exactly with the data from the corresponding coral metabolites.

4. STUDIES ON A HETEROAROMATIC BASE

The methanol partition fraction of !. me1anobrachia contained one

metabolite which was not found in the extracts of T. coccinea. This

metabolite was isolated as a yellow oil and was found to be extremely

base labile, but stable in acidic solutions. The UV spectrum (Figure

39) exhibited maxima at 225, 278, 286, and 298nm. Electron-impact

mass spectroscopy (Figure 40) contained peaks at ~/~ 239 and 237 of

equal intesity, suggesting presence of bromine. The identity of these

peaks as the parent ions, however, is confused by the initial fragment

ions at ~/~ 220 and 222, which correspond to loss of 17amu. The 1H

NMR spectrum of ~ in methano1-d4 (Figure 41) reveals only five pro

tons, while the spectrum in Me 2SD-d6 (Figure 42) contains additional

broad signals at 08.94 (lH) and 7.65 (2H). In either solvent, two of

the protons are coupled by a 14Hz coupling constant, thus indicating

62

225nm286nm293nm

200I

250I

300I

350I

400r

450

A (nm).

Figure 39. UV spectrum of heteroaromatic base (50) in methanol'\IV

;,:.r :

!;.,.

I·

~. 0 ~. 1"\r: '1' .,g C.-

0'

~l

I., I"l

i II

I

II

~

"1 II II I I II t1!1 I~! 11 11

:i ,,;j '" u

I II 1.;11

. 1 ~ /' ~l! " IIo I 1,1 J I l~ I I!~ :j j'

tn _ -I" i i 1 t· ; . I It· J I ! . ~ i 4 t ••• ( I . I ( I I' I I I . I . I . I . i .1 I"' I . I • I . ! 1 ! . I

~:: '=' :~ :; ~ ~ :;(.I rj "

Figure 40. Mass spectrum of heteroaromatic base (~).

63

I

I

~II l I. ,I

UUL__,,)VU.. P' .;

I I i I I I'!O 8 6 4' 2 0 F'?iVi

Figure 41.1H NMR spectrum (300 MHz) of heteroaromatic base (50) in

'\IV

methano1-d4·

o Ffn

Figure 42. ~ NMR spectrum (300 MHz) of heteroaromatic base (~) in

Me2SO-d6·

64

that they are located on a trans-disubstituted double bond. The other

protons are singlets and show no coupling. The l3C NMR spectrum (Fig-

ure 43) contains five doublet carbons at 0110.5, 109.7, 10S.2, 107.1,

and 104.4; 1these correspond to the five observed protons in the H

NMR spectrum. Four other singlet carbons at 0146.6, 137.1, 136.7, and

119.2 show that this molecule is fully heteroaromatic, and that it

contains nine carbons. Assuming that all protons are observed in the

1H NMR spectra, a formula of C9HSN3

may be derived.

Comparison of the spectral data with those of known heteroaroma-

tic systems failed to reveal a ring structure with similar physical

data. In particular, the l3C NMR chemical shift values could not be

related to any known system. No relationship was seen with indole or

purine structures.

" IIIiI

I

III"I,I

II;;'~I.I , I,I Ii----...--:_-"-__-i.--...Jl.l!,....I=-- --JI '"---,----

IZl lit IlL W..... 'J I•

Figure 43. l3c NMR spectrum (126.1 MHz) of heteroaromatic base (50) in'\IV

methanol-d4, broadband (full) and noise (inset) decoupled.

65

Some chemical degradations of hetp.roaromatic base 50 such as cata'\IV

lytic hydrogenation, ozonolysis, and acetylation, have been attempted,

but have yielded little information on the ring structure. Slow decom-

position and finite sample availability preclude further degradative

studies; an attempt at producing a crystalline derivative will be

made.

C. DISCUSSION OF SECONDARY METABOLITES FROM TUBASTREA COCCINEA AND

PHESTILLA MELANOBRACHIA

Figure 44 lists the distribution of the metabolites found in the

nudibranch Phestilla melanobrachia and its prey, the coral Tubastrea

coccinea. Five indoles found in the coral were also isolated from

extracts of the nudibranch. 6-bromo-8,9-dihydroaplysinopsin (40) and'\IV

8,9-dihydroaplysinopsin (38) were not detected in P. melanobrachia,'\IV

8,9-dihydroaplysinopsin (38)'\IV

6-bromo-8,9-dihydroaplysinopsin (40)'\IV

6-bromoaplysinopsin (~)

6-bromo-2'-de-!-methylaplysinopsin (~)

2'-de-N-methylaplysinopsin (43)- '\IV

indole-3-carboxaldehyde (44)'\IV

6-bromoindole-3-carboxaldehyde (45)'\IV

hokupurine (46)'\IV

heteroaromatic base (~)

Metabolite Source

T. coccinea P. melanobrachia

X

X

X X

X X

X X

X X

X X

X X

X

Figure 44. Distribution of metabolites in T. coccinea and P. melano

brachia.

66

but this might be a function of limited sample availability. since

these indoles were minor constituents of the coral extracts. Hoku-

purine (46) was isolated from both animal species, but the unidenti'\IV

fied heteroaromatic base (50) was found only in extracts of the aeolid.'\1\0

The origins of indoles 41. 42, 43, 44, and 45, and of hokupurine'\IV '\IV '\IV '\IV '\IV

(~) in P. melanobrachia are clearly dietary. It is not ·known. however.

if these metabolites are maintained within the body tissues of the

nudibranch, or if they were extracted as constituents of the digestive

gland of the nudibranch.

The isolation of large amounts of one metabolite, heteroaromatic

base 50. from only K. melanobrachia leads to speculation that this meta'\IV

bolite may have a special purpose, such as a defense secretion, for

this animal. Harris has reported that ~. melanobrachia, unlike other

aeolid nudibranchs, does not bear stinging nematocysts in its cerata;

thus, the animal does not have an obvious means of defense. Histologi-

cal studies of the cerata revealed that the tips contain large secre-

tory cells, which "exude droplets of a clear, viscous material which

dissolve slowly in sea water" when the cerata are disturbed. 66 Unfor-

tunately. the method of extraction used for the chemical studies of P.

melanobrachia does not allow an identification of anyone metabolite

from the cerata.

Biogenetically. it is interesting to find similar or identical

metabolites, such as the aplysinopsins, in both a coral and sponges,

68though indoles are well known from marine sources.

Part of the results of this research have been published.69

67

D. SECONDARY METABOLITES FROM DENDRODORIS NIGRA AND DENDRODORIS

TUBERCULOSA


The dorid nudibranch Dendrodoris nigra (Superfamily Dorididae,

Family Dendrodoridiae) is usually dark brown or black with white spots,

and may grow to 30mm in length. ~. nigra is extremely soft in body

texture, and is one of the more common dorids found in Hawaii. D.

tuberculosa is a much larger species, reaching l50mm in length. The

body texture of ~. tuberculosa is rather firm, and its mantle (dorsal

f) · . . 70 B h .sur ace conta1ns numerous warty proJect10ns. ot spec1es are

sponge feeders, and are "porostomes," meaning that they lack a rasping

71tongue (radula) and feed by sucking up pieces of sponge.

Collections of Q. nigra were made at Ala Moana reef, Fort Kameha-

meha reef, and Pupukea, Oahu. D. tuberculosa were found at the Ala

Moana and Pupukea sites. Both D. nigra and~. tuberculosa are noctur-

nal species, and generally hide under rocks or in dark crevices during

the day. These dorids were never observed during the day at Pupukea;

all animals from this site were collected at night. Specimens of Den-

drodoris were found at Ala Moana reef and Fort Kamehameha only by

turning rocks during the day.

2 • ISOLATION

It has been observed that some dorid nudibranchs maintain mantle

glands which release secretions when the animal is disturbed. 25 Since

it is physically impossible to separate the mantle from the remainder

of the nudibranch body without causing extensive discharge from these

68

glands, the following extraction procedure was utilized: live animals

were extracted with distilled hexanes or benzene for 5-l0min, then

transferred to fresh solvent for longer term extraction.

In all cases, the short term extract of Q. nigra or D. tuberculosa

1consisted of a specific mixture, as shown by its H NMR spectrum (Fig-

ure 45). Separation of this extract was achieved by silica gel HPLC,

which afforded only two components, 51 and 52, in a 1:1 ratio. The'V\I 'V\I

long term extracts were complex mixtures of metabolites, including

steroids, pigments, and fatty acid derivatives, and contained only

minor amounts of 51 and 52.'V\I 'V\I

3. CHARACTERIZATION OF POLYGODIAL

Polygodial (~) was isolated as a white solid. Salient features

1of the H NMR spectrum (Figure 46) included signals for aldehydic

I10

IB

Iti

Il'1

Figure 45. lH NMR spectrum (300 MHz) of 5 min extract of Dendrodoris

nigra in benzene-d6•

69

protons at 09.54 (d, ~=4.4) and 9.46 (s), and a vinylic proton at 07.13

-1(m). IR bands (Eigure 47) at 1720 and l677cm established that the

singlet aldehyde was a,S-unsaturated and that the doublet aldehyde was

aliphatic. The UV spectrum (Figure 48) contained an absorption maximum

at 230nm (€=7000), which was assigned to the vinylic aldehyde.

The mass spectrum of ~ (Figure 49) contained a small parent ion

at ~/~ 234 (5% relative intensity) which could not be accurately mea-

sured by peak-matching. However, the base peak at ~/~ 206 was found to

correspond to a formula of C14H220. Since aldehydes are known to lose

CO readily, the molecular formula of ~ may be established as C15H2202'

requiring five sites of unsaturation. The aldehydes and vinyl group

fulfill three sites of unsaturation. Since no evidence for other

functionalities was seen, 51 must be bicyclic. The presence of three~

IB

I6

I~

Figure 46. ~ NMR spectrum (300 MHz) of po1ygodia1 (51) in chloroform-d.~

70

Figure ire IR spectrum of polygodial (~) in chloroform.

230nm (g=7000)

I I200 250 300 350

A (rrm)

Figure 48. UV spectrum of polygodial (~) in methanol.

71

1aliphatic methyls in the R NMR spectrum strongly implicates that 51 is

'\1\0

a sesquiterpene.

In the course of studies of the allomone pu'ulenal (4) from the'"

dorid Chromodoris albonotata, Dr. Gary Schulte hydrolyzed 4 to obtain'"

polygodial (51), as well as its epimer at C-9 (53).9, 72 The similari-'\1\0 '\1\0

ty of the spectral data of polygodial with the Dendrodoris spp. dialde-

hyde was immediately observed. Comparison of data for 51 with litera'\1\0

ture data confirmed that dialdehyde 51 was polygodial. 73, 74'\1\0

51'\1\0

CRO

CRO

53'\1\0

Polygodial (51) was distinguished from its 9a epimer, or 9-epi'\IV

polygodial (~), by comparison of the chemical shifts and of the coup

ling constants for the aldehydic protons in chloroform-d. Polygodial

and the Dendrodoris spp. dialdehyde show aldehydic signals at 09.54

(d, ~=4.4) and 9.46 (s), while in 9-epi-polygodial (~) these signals

appear at 09.94 (d, ~=2.0) and 9.43 (s).

4. CHARACTERIZATION OF OLEPUPUANE

Olepupuane (~) was isolated as a white solid. The IR spectrum

-1(Figure 50) contained a strong, broad carbonyl band at l752cm ,and

~ NMR (Figure 51) signals at 01.64 (s, 3H) and 1.56 (s, 3H) support

the presence of two acetates. Other features of the ~ NMR sp~ctrum

- - .......... cr - - - ...... 1M ... 1M

•

72

Figure 50. IR spectrum of olepupuane (~) in methylene chloride.

~ lA ~ ~~A~~ V

I --- ""'-I I I I I I8 6 4 2 0 PPM

Figure 51- lH NMR spectrum (300 MHz) of olepupuane (~V in benzene-d6•

73

include protons at 06.68 (d, ~=2.3) and 6.12 (d, ~=1.8), each of which

are coupled to a proton at 02.72 (m), and a multiplet at 05.64. Sig-

nals for three aliphatic methyls appear at 00.76, 0.61, and 0.54.

01epupuane is transparent in the UV region.

Electron-impact mass spectroscopy (Figure 52) revealed a very

weak (1% relative intensity) molecular ion at ~/~ 336, with more in-

tense fragments at ~/~ 276 and 234. The fragment peak at ~/~ 276 was

measured by high resolution peak matching and was found to have the

molecular formula C17H2403. Since this fragment corresponds to a loss

of AcOH from the parent ion, the molecular formula of 52 was calculated'VU

as C19H2805. Presence of three alipatic methyls in the 1H NMR spectrum

suggested that 52 was terpenoid. Definite proof was provided by a hy'VU

drolysis of 52, which was converted by mild acid to polygodia1 (51),'VU 'VU

I

•

Figure~. Mass spectrum of olepupuane (~).

74

thus establishing the ring skeleton and stereochemistry at C-9 of di-

acetate ~).

The molecular formula of ~ requires six sites of unsaturation.

Four of these are explained by the acetates and two rings of the dri-

mane skeleton. Only a ring and a double bond could be accomodated for

the other two degrees of unsaturation. Two structures, ~ and ~'

could be formulated to explain the basic spectral features and the

facile acid-catalyzed conversion to polygodial (~).

52a~

OAc

~c

Structure ~ contains an unusual and highly strained 2,5-diace

toxytetrahydrofuran ring fused to C-8 and C-9 of the drimane ring skel

eton. This structure was ruled out because the lH NMR chemical shifts

and coupling constants which would necessarily be assigned to H-IO

(06.68, d, ~=2.3) and H-ll (06.12, d, ~=1.8) are inconsistent with

74-77reported values for structurally related compounds (Table 5).

The correct structure for olepupuane (52a) incorporates a 2-ace~

toxydihydrofuran with the drimane skeleton. This arrangement of func-

tional groups is closely related to that seen in the sponge metabolite

• 78 79heteronem1n (~).' Like heteronemin, the acetal proton (H-lO)

and the vinylic proton (H-ll) of olepupuane (~) are each doublets of

about 2Hz coupling each, and both are coupled to the same allylic

proton (H-9, 02.71, m). A Dreiding model of olepupuane reveals that

75TABLE 5. SPECTRAL DATA FOR COMPOUNDS RELATED TO STRUCTURE 53

'\IV

Solvent H-7 H-9

2.70

(rn)

H-IO

5.10

(d , ~=4.5)

H-ll Ref.

74

AcO,•.

not

reported 5.50

(bs)

5.26

(d, ~=4,0)

4.15

(d , ~=11),

4.50

(d, ~=11)

75

H

CDC13

5.53 3.12 6.13

(m) (bd) (d , ~=4)

4.26

(m)

76

4.03

(d , J=12),CC1

45.36 5.12

4.39 77(m) (d, ~=4) (d , ~=12)

76

Ac~~.

HO

OAc

in order to observe the 2Hz coupling constant between H-ll and H-9, the

acetate on C-ll must be in the a-orientation, as the corresponding ace-

tate in heteronemin (~). If the acetate on C-ll were a-oriented, the

dihedral angle between H-9 and H-ll would be approximately 300, requir

ing a coupling constant of about 7Hz between them. aO The proton chemi-

cal shift and coupling constant of H-7, however, bears a difference

from the corresponding signal in heteronemin. Proton-7 occurs as a

doublet of doublets (~=3.7, 3.7) which was shown by an NOE experiment

to be in close proximity to H-6 axial at 01.70. Further analysis of a

Dreiding model of ~ showed that this could only be explained if the

C-7 acetate were a-oriented, resulting in structure ~.

AcO~

~OAC

52~

77

In an independant and simultaneous study, researchers at the

Scripps Institution of Oceanography isolated olepupuane from several

species of porostome nudibranchs. Sufficient material was obtained by

them for a l3C NMR spectrum, whic contained signals for only one acetal

carbon (699.7) and one acetate-bearing carbon (665.8), thus confirming

81~ as the correct structure of olepupuane.

5. BIOLOGICAL ACTIVITY OF POLYGODIAL AND OLEPUPUANE

Olepupuane showed mild in vitro activity against Bacillus subti-

lis, but no activity against Candida albicans, Escherechia coli, Pseu-

domonas sp., or Staphylococcus aureus. Polygodial was inactive against

all of these microorganisms.

E. DISCUSSION OF SECONDARY METABOLITES FROM DENDRODORIS NIGRA AND

DENDRODORIS TUBERCULOSA

The short term method of extracting ~. nigra and D. tuberculosa

was intended to isolate secondary metabolites which may be localized

at or near the mantle surface of these nudibranchs quickly and speci-

fically. The isolation of polygodial (~) and olepupuane (~) as the

only constituents of the short term extracts indicated that these me-

tabolites were indeed from the body surface. Additional evidence is

shown by the complexity of the long term extracts, the constituents

of which probably arise from slower extraction of internal portions of

the nudibranchs.

The composition of the short term extracts proved to be consis-

tent; all collections of D. nigra and~. tuberculosa showed an approx-

imately 1 to 1 ratio of polygodial and olepupuane. None of the animals

78

were found in as·sociation with, or feeding upon, a particular sponge.

Common sponges found in the habitats of the Dendrodoris spp. were

screened, but none contained either polygodial or olepupuane.

Polygodial (~) was first isolated as a constituent of the plant

Polygonum hydropiPer,82 and has been found in other plants. 73, 74

Polygodial has also been found in the skin extracts of the Mediterra-

nean Dendrodoris limbata and is reported to be a defensive secretion

f h " "1 42o t ~s an~ma • The digestive gland of ~. limbata also contains a

series of mixed fatty esters (20) based on the drimae skeleton, and~

43showed a structural similarity to olepupuane. Feeding studies using

(2-l 4C)-mevalonic acid have shown that polygodial is synthesized de

novo by ~. limbata. 83 This is a surprising result, since all other

metabolites thus far isolated from dorid nudibranchs have been implica-

ted or directly shown to be derived from a dietary source. The rela-

tionship between~. limbata, and~. nigra and~. tuberculosa is ob-

vious, and it is suggested that both polygodial and olepupuane may be

biosynthesized directly by the nudibranchs.

Polygodial (~) has been reported to act as an insect73 and fish4 2

antifeedant. The activity of polygodial is believed to be due to the

ability of the dialdehyde portion to react with the -SH or -NHZ resi-

dues of amino acids, which are reported to be associated with chemo

receptors of insects. 84, 85 D'Ischia et al. have studied the reac-

tivity of polygodial with primary amines, and have found that polygo-

d " 1 d hI" H 9 . unstable adduct ~~.85~a an met y a~ne react at p to g1ve an ~

79

Kubo and Ganjigan have investigated the kinetics of the reaction of

polygodial with cysteine, and have shown that polygodial reacts three

times faster than 9-epi-polygodial (~), which is formed from polygo

dial by base treatment.84

The difference in reactivity is probably

due to the difference in the proximity of the aldehyde groups, which

are much closer in polygodial. Interestingly, Kubo has noted a elose

correlation between the spatial orientation of dialdehyde groups in

other natural products with a hot taste of these metabolites in humans.

In all cases that have been studied, the hot-tasting substances con

tained dialdehydes with the same steric orientation as polygodial.84

Olepupuane (~) was tested by the researchers at the Scripps

Institution of Oceanography, and was found to inhibit feeding in the

Pacific damselfish (Dascyllus aruanus) when applied to food pellets at

concentrations of 5_50~g/mg.8l One possible explanation for the acti-

vity of olepupuane may involve its facile hydrolysis to yield polygo-

dial and acetic acid, both of which are unpalatable.

An account of this research has been published.81

80

F. METABOLITES OF CHROMODORIS ELISABETHINA


Chromodoris elisabethina is a variably colored, striped dorid

nudibranch found throughout tropical waters. At Enewetak Atoll, Mr.

Scott Johnson has observed C. elisabethina to feed principally upon

the sponge Heteronema sp.

Specimens of f. elisabethina were collected from Enewetak Atoll

by Mr. Scott Johnson between June 1982 and May 1983; specimens of f.

elisabethina were collected from Guam Island in July 1981 by Drs. Gary

Schulte and Chris Ireland. The Guam specimens were kept frozen until

extracted, while most of the Enewetak dorids were stored in isopropyl

alcohol until processed. One collection at Enewetak was extracted

for Smin in isopropyl alcohol, then transferred to fresh solvent for

storage.

2 • ISOLATION

The isopropyl alcohol extracts were evaporated in vacuo and the

yellow residue partitioned according to the Kupchan scheme (Figure 1).

The major constituent of the crude extract partitioned into carbon

tetrachloride; HPLC on silica gel afforded latrunculin-A (~), which

crystallized from petroleum ether/ethyl acetate mixtures. The 5 min

extract of C. elisabethina contained virtually pure latrunculin-A, as

shown by lH ~1R (Figure 53) and HPLC of this extract.

i' , , • i '10 9

, I 'a

ii'7

81

Figure 53. ~ NMR spectrum (300 MHz) of 5 min extract of Chromodorls

elisabethina in chloroform-d.

3. CHARACTERIZATION OF LATRUNCULIN-A

High resolution mass spectroscopy (Figure 54) established the

formula C22H31N05S for latrunculin-A (~). from the molecular ion at

m/~ 421. The UV spectrum (Figure 55) showed a single chromophore at

2l8rim (E=18700). The IR spectrum (Figure 56) was highlighted by bands

-1at 3670. 3560. and 3400cm • for NH or OH stretching frequencies. and

-1 13by a broad carbonyl band at l690cm • Initial C NMR experiments

were inconclusive, but showed that ~ contained at least three double

bonds.

The ~ NMR spectrum of ~ was richly detailed. Complementary in-

formation was obtained through decoupling experiments in both.

benzene-d6 (Figure 57) and chloroform-d (Figure 58). Valuable coup-

!

I!

~

!!3~ II

II~

In I

IIj';~

·+r"T"'l-r""r'To~~~~...;::r;:;:;r;:fWll...p.,~""""~r-rJr~"""'~,....,..~~"""~l""T""T"'T"""''T'''''1

i!. • I sa! ! ! ! ! • a ! ! I I a I .. I • I ! ! J I.

Figure 54. Mass spectrum of latrunculin-A (~).

82

Z19nm (e=18700)

200I

250

A (nm)

r300 350

Figure 55. UV spectrum of latrunculin-A (~) in methanol.

r.

"

"t",·

•... ~.. ;,

-, . e ; t •

83

..'.'

--,' -....

===L..:::!~:l==i.U.i.~::-JI.,;..""-'''''''''I~'': 7.'; - -____________\V..VEII.'\'~·!l~~ .~::_'. _

Figure 56. IR spectrum of latrunculin-A (~) in methylene chloride.

Figure 57.1H NMR spec trum

benzene-d6•

(300MHz)of latrunculin-A (57)

"'"in

LCLCL

84

85

ling data were also obtained from a two-dimensional COSY (Homonuclear

J-resolvc;xl spectrsocopy) experiment (Figure 59). Analysis of the data

be drawn.»:

.- '-, , 2.87, 2.69H H

I

H HI

~ "6.3';<-- - 2.23

.b-

allowed a partial structure,,e. to

5 95 5.74• ". H H

4.99··, H

The trans and cis nature of the two double bonds were determined by

coupling constants of 15.0 and 10.5 Hz respectively, between the protons

of each olefin. This diene also accomodates the UV chromophore at

2l8nm.

Irradiation of the apparent triplet at 03.83 (~=7.3) resulted in

the collapse into two doublets with ~=12.0Hz each, centered at 03.46 and

3.38, from a complex multiplet in that region. Irradiation of the

multiplet, in turn, resulted in the collapse of the signal at 03.83

into a singlet as the only observed effect. Partial structure i wasv

drawn for this fragment, where X and Yare heteroatoms.

X - CH - CH2 - Y

03.83 (r , ~=7.3») t 03.46, 3.38 (each dd, ~=7.3, 12)

i

'"At this point, it became apparent that 57 showed physical features

\IV

similar to those of latrunculin-A, a macrocyc1e previously isolated

h R d S L u1 . f· 78 All d f 57from tee ea sponge atrunc ia magn~ ~ca. ata or """ were

"~o

COSY-16 o f Latrunculin Ij"

Contour Plot t300 MHZ

d ~/L ....

."'" LCDC13 ,'"" [

11 lJ' [!iff Co· IL

a r..."r!a ", L Na a .8 » I-e ~; C. [.

~9Jf1 ~ e.cJ a ft '- IT)

LL

00 [J LI

.. 0 ..".l-

()A C:l [; :rIr

,/ t;3 '-II.

L~. D

~- in.. l.L

p UI(:.a~ ""IL

~! }(l I\,) "I.I

r,: .., U1.- l~1.- I.!.

CJ (!! ~ II•.

l.i . r--..I,

f' !

I :I I I I I , : I I I i I I I I I 1 I I I . ,

I I I I I I I I I I I I

" I' ' ......

Figure 59. COSY spectrum of latrunculin~A (~) in chloroform-d,

contour plot.

86

found to correlate closely with literature values for latrunculin-A.

0yyo~

o°

57'V\,

1Additionally, an H NMR spectrum of latrunculin-A, kindly provided by

Professor Y. Kashman, matched exactly that of ~.

With this knowledge, careful analysis of decoupling and COSY ex-

87

periments allowed a complete assignment of all protons in latrunculin-

A.

13A C NMR spectrum (Figure 60) of latrunculin-A from~. elisa-

bethina was later obtained, and all chemical shifts were:identical to

78those of the authentic compound.

4. BIOLOGICAL ACTIVITY OF LATRUNCULIN-A

Latrunculin-A was found to show strong antibacterial activity

against Candida albicans, but no activity against Staphylococcus

aureus, Bacillus subtilis, Escherchia coli, and Pseudomonas sp.

Fish toxicity studies of latrunculin-A were performed using

commercially available goldfish. Fish were tested at concentrations

of lmg/L water, wtih simultaneous controls. Within 5min, the fish

88

Figure 60. 13C NMR sp~ctrum (75.6 MHz) of latrunculin-A (~) in

chloroform-d.

exposed to latrunculin-A were more active and agitated than the

controls; two of the test fish repeatedly attempted to jump out of

the container. By 15 to 35min, the test fish appeared to be partially

paralyzed, and could swim only in short bursts. As paralysis pro-

gressed, a test fish would lie on its side and be incapable of proper

orientation. By 50min, all test fish had died. All controls showed

normal behavior during the test period.

G. SECONDARY METABOLITES OF HETERONEMA SP.


The brown encrusting sponge Heteronema sp. grows as one of the

most common sponges at Enewetak Atoll, Marshall Islands. Mr. Scott

Johnson has identified this sponge as the main food of the dorid C.

elisabethina at Enewetak.

Pieces of Heteronema sp. were collected between June 1982 and

December 1982 and stored in isopropyl alcohol until processed.

2. ISOLATION

The isopropyl alcohol extract of Heteronema sp. was decanted and

filtered. After evaporation of solvent in vacuo, the resulting brown

residue was subjected to the Kupchan scheme (Figure 1). Each of the

resulting fractions was analyzed by TLC and lH NMR for possible con-

tent of latrunculin-A, but no evidence for its presence was seen.

The single most abundant metabolite from the extract of Hetero-

nema sp. occurred in the hexanes fraction of the partition. This

fraction was chromatographed on Sephadex LH-20. The chief fraction

was purified by HPLC on silica gel, affording puupehenone (~).

3. CHARACTERIZATION OF PUUPEHENONE

Puupehenone (~) was isolated as a yellow amorphous solid. UV

spectroscopy (Figure 60) contained maxima at 307 (£=7300), 317

(£=8600), and 329nm (£=5500), while the IR spectrum (Figure 61) con

tained a band at 3400cm-l for an alcohol moiety, and bands at 1630 and-1

l6l5cm The mass spectrum (Figure 62) showed a molecular ion at

89

90

~/~.328. Salient features of the lH NMR spectrum (Figure 63) included

a D20 exchangeable singlet at 06.83, confirming the presence of an al

cohol, and olefinic or aromatic signals at 06.64 (d. ~=7.0), 6.18 (s),

and 5.85 (s). Four methyls were also observed, at 01.22, 0.90, 0.83,

and 0.81, suggesting a terpenoid structure for~. The data also sug

gested that ~ contained a quinoid moiety.

Relatively few yellow terpenoid metabolites are known from marine

sources, and a search of the literature revealed that one compound,

puupehenone, contained identical physical data with that of the Hetero

nema compound, thus establishing the structure of~. Indeed, puupehe

none was initially isolated from a sponge from Enewetak which was not

described taxonomically, but which bears a morphological resemblance to

87Heteronema sp.

OH

Small amounts of metabolites clearly related to puupehenone were

isolated, but were not investigated in these studies.

91

307nm (€=7300)317nm (€"'S600)329nm (€=5500)

200 250 300

A (nm)

350 400

Figure 61. UV spectrum of puupehenone ~~) in cyc1ohexane •

...... 1M .....--................._-- -......-----~~..;.--ri-+H+H++4J

-I!

Figure 62. IR spectrum of puupehenone (~) in carbon tetrachloride.

92

J!

~

t!~

! s~..:f

Figure 63. Mass spectrum of puupehenone (~).

Ie

i.

LJI6

I4 PPM

Figure 64. 1H NMR spectrum (300 MHz) of puupehenone (58) in chloroform'\IV

d.

93

H. DISCUSSION OF SECONDARY METABOLITES FROM CHROMODORIS ELISABETHINA

AND HETERONEMA SP.

Latrunculin-A (~) was first isolated from the Red Sea sponge

Latrunculia magnifica, and its structure was determined by single crys-

1 1 . 86 1·ta x-ray ana ys~s. Latruncu ~n-A possesses strong icthyotoxicity

against marine and fresh water fish,88 and in vitro studies have dem-

onstrated that it disrupts microfilament organization in cultured mouse

neuroblastoma and fibroblast cells. 89

The short term extract suggests that f. elisabethina sequesters

latrunculin-A near its mantle surface, and discharges it when disturbed.

In the whole animal extract, latrunculin-A is clearly the most impor-

tant consituent, thereby strongly implicating latrunculin-A as a defen-

sive secretion. Additionally, its potent biological activity makes it

an effective substance for this purpose.

Latrunculin-A has also been isolated from C. elisabethina from

Townsville, Australia (Professor John ColI, personal communication),

and now appears to be specifically associated with f. elisabethina.

However, the main food of f. elisabethina at Enewetak, the sponge

Heteronema sp., does not produce latrunculin-A, but instead contains

puupehenone and congeners. Apparently, C. elisabethina obtains lat-

runculin-A from a minor dietary source, which is most probably a

sponge. Though the possibility cannot be ruled out, it is unlikely

that the dorid biosynthesizes latrunculin-A.

94

I. SECONDARY METABOLITES OF HYPSELODORIS INFUCATA


Hypselodoris infucata is a wide-ranging dorid nudibranch, reported

from both tropical and subtropical waters. 90 The animal is colorful,

with a background of cream, and liberal amounts of blue-black and

orange-yellow spots; the rhinophores ore red, and the gills are white

with red tips.90 In Hawaii, H. infucata is known to feed on the sponge

Dysidea fragilis. 9l

Specimens of ~. infucata were collected from Fort Kamehameha reef

and from the pilings of the University of Hawaii pier in Kaneohe Bay,

Oahu. Collections were made during July 1982 by snorkeling at -1m.

2. ISOLATION OF NAl(AFURAN-B AND -9

Live H. infucata were immersed in distilled methylene chloride for

5min, then transferred to fresh solvent for longer term extraction.

Analysis of the short term extract by TLC revealed that it contained

only two constituents, which exhibited the same Rf values as authentic

nakafuran-8 and -9. HPLC of the short term extract on silica gel affor-

ded nakafuran-8 (~) and -9 (~).

3. CHARACTERIZATION OF NAKAFURAN-B and -9

The identity of the metabolites from H. infucata as nakafuran-8

and -9 was facilitated by the knowledge of the association of this nudi

branch with Dysidea fragilis. 90 When molested, H. infucata released

a pungent odor which was the same as that of Dysidea collected from the

same sites. Additionally, the lH NMR spectrum of the 5min extract of

H. infucata (Figure 64) exactly matched that of a mixture of nakafuran-8

95

..!I

!:• III1/I:II, I!: I:1 ,I: ,:; Ii'i:; :, I

Figure 64. IH NMR spectrum (300 MHz) of 5 min extract of Hypselodoris

infucata in benzene-d6•

and -9 from the sponge.

All physical data for the metabolites 2 and 3 from H. infucata'" '" -32matched that for nakafuran-8 and -9, respectively.

J. DISCUSSION OF SECONDARY METABOLITES FROM HYPSELODORIS INFUCATA

In light of the close predator-prey relationship between R. infu-

cata and Dysidea fragilis, this discovery of nakafuran-8 and -9 in the

skin extracts of the nudibranch is not surprising. Since undisturbed

M. infucata are odorless, while molested animals release the aromatic-

smelling nakafurans, it can be presumed that these sesquiterpenes re-

present defensive secretions. H. infucata represents the third Hawaii-

an dorid in which nakafuran-8 and -9 is an apparent defensive secre-

tion; these metabolites were first reported from Chromodoris marida-

96

dilus and Hypselodoris godeffroyana. 32

III. CONCLUSIONS

Five species of nudibranch mollusks were investigated and were

found to contain high concentrations and specific types of secondary

metabolites. In four species, the metabolites were found to be loca-

lized at or near the body surface of the animal. Many of the isolated

metabolites had previously been shown to possess toxic or antifeedant

properties, and are believed to serve as defensive secretions for the

nudibranchs.

The principal foods of three of the nudibranch species were also

studied. Comparison of the metabolites of nudibranch and prey ranged

from no correlation to a high level of correlation. In at least on~

case, it is believed that the nudibranch synthesizes its defense meta-

bolites de novo.---

97

IV. EXPERIMENTAL

A. GENERAL

Optical rorations were recorded either on a Bendix-Ericsson ETL

NPL or a Rudolph Research Autopol II polarimeter. NMR spectra were

recorded on Varian model XL-lOO, Nicolet NT-300, or Brucker WM-500

spectrometers. Infrared spectra were determined on a Perkin Elmer

467 or Beckman IR-IO spectrophotometers. Ultraviolet spectra were

measured from a Cary-14 or Beckman ACTA CIII spectrophotometers.

Mass spectra were obtained using a Varian MAT 311 or a Finnigan 1015

instrument. Mass spectral data are reported as ~/~ (percent relative

intensity). NMR data are reported as chemical shift, 0 (multiplicity,

number of protons, coupling constant in Hz). BioSil A (ASTM 200-400

mesh, BioRad Laboratories) was used for all column chromatography.

involving silica gel. Sephadex LH-20 (Pharmacia) or BioBeads SX-8

(BioRad Laboratories) were employed for gel filtration column chroma

tography. All solvents used in all phases were distilled in glass

prior to use.

B. COLLECTION - TUBASTREA COCCINEA AND PHESTILLA MELANOBRACHIA

98

Colonies of Tubastrea coccinea were collected in May 1980 from

Kaneohe Bay, Oahu by snorkeling at -1m, and from Pupukea, Oahu by

SCUBA at -13m. Colonies were broken off with pick-axes. As collec

ted, the colonies contained much inert substrate, as well as attached

sponges and other organisms. As much excess debris as conveniently

possible was removed before extraction.

Six specimens of Phestilla melanobrachia were collected in

August 1980 by Mr. Scott Johnson. The animals were collected at

night from the beds of T. coccinea at Pupukea (-13m). The nudi

branchs were kept alive until extracted.

C. EXTRACTION AND ISOLATION - TUBASTREA COCCINEA AND PHESTILLA

MELANOBRACHIA

About 200g of ~. coccinea was collected from Kaneohe Bay, and

2.5kg from Pupukea. The extracts from both sites were found to be

identical in composition, and no distinction is made between them.

Owing to the bulkiness of the coral, extractions were performed

batchwise as needed. In a typical extraction, coral was extracted

three times with 95% ethanol. The extracts were combined and eva

porated in vacuo by rotary evaporation and lyophilization to yield a

dark brown solid. This residue was subjected to the Kupchan scheme

of partitioning (Figure 1), which results in four fractions. In a

representative extraction of 200g of coral, the following yields

were obtained:

crude ethanol extract

hexanes partition

2.5g

450mg

CC14

partition

CHC13 partition

MeOH partition

74mg

94mg

l830mg

99

Preliminary separations of the hexanes and carbon tetrachloride

partition fractions were made on BioBeads SX-8 in benzene (4.5x172cm).

The resulting fractions showed little of interest and further studies

were not pursued. The most efficient separations for the chloroform

and methanol partition fractions were achieved by chromarography on

Sephadex LH-20 (1.5xllOcm, 1:1 methanol:chloroform). Fractions from

this chromatography were most conveniently collected by visualizing

colored bands, all of them yellow. Chromatography of 94mg of chloro-

form fraction yielded the following results:

A

B

C

D

E

F

30m!

20m!

20ml

30ml

30m!

60ml

l8mg

20mg

2lmg

7mg

8mg

3mg

no aromatics, fatty metabolites

no aromatics

hokupurine, 8,9-dihydroaplysinopsin,

6-Bromo-8,9-dihydroaplysinopsin

indole-3-carboxaldehyde.

6-bromoindole-3-carboxaldehyde

2'-de-!-methylaplysinopsin. 6-bromo

2'-de-!-methylaplysinopsin

complex aromatics

Fractions ~. ~. and ~ were further separated by reversed-phase HPLC

(Knauer-Unimetrics, O.5x25cm, Lichrosorb RP-18, 7:3 methanol:water).

HPLC of fraction C under these HPLC conditions afforded first 6-bromo-

8,9-dihydroaplysinopsin, then, 8,9dihydroaplysinopsin, followed by

hokupurine. HPLC of fraction D yielded first indole-3-carboxaldehyde

then 6-bromoindole-3-carboxaldehyde. HPLC of fraction! provided

2'-de-!-methylaplysinopsin, followed by its 6-bromo isomer.

100

Sephadex LH-20 chromatography of the MeOH partition fraction (83mg) pro-

vided three yellow bands, each of which was collected as a fraction:

A

B

C.

SOml

30ml

SOml

47mg

20mg

l3mg

no aromatics, complex mixture

hokupurine, complex aromatics

2'-de-~-methylaplysinopsin,6-bromo-

2'-de-~-methylaplysinopsin

Purification of individual metabolites was accomplished by reversed-

phase HPLC under the same conditions as for the chloroform fractions.

~. melanobrachia was extracted in exactly the same manner as T.

coccinea, and the extract was processed by the Kupchan scheme (Figure

1). The recovered yields from 4.Sg of nudibranchs were:

crude ethanol extract

hexanes partition

CC14 partition

CHC13 parti tion

MeOH partition

480mg

27mg

19m9

39mg

400mg

Sephadex LH-20 chromatography (O.SxllOcm, 1:1 methanol:chloroform)

of the chloroform partition yielded a series of fractions nearly identi-

cal to those obtained from the coral chloroform fraction. However, a

significant difference was observed in the methanol partition fraction

of the nudibranch; Sephadex LH-20 chromatography (O.SxllOcm, 1:1 meth-

anol:chloroform) afforded three bands:

A 80ml 2Smg no aromatics; complex fats

B 40ml l4mg hokupurine, 2'-de-N-methylaplysi-

nopsin, 6-bromo-2'-de-~-methyl-

aplysinopsin

C 30ml 17mg heteroaromatic base ,mThe cons tituents of fraction B could be separated by reversed-phase

101

HPLC, under conditions previously utilized for the coral metabolites.

Heteroaromatic base ~ could be purified only by repeated chromatography

on Sephadex LH-20; this metabolite comprised approximately 0.005% of

the organic extract of P. melanobrachia and was not found in the extract

of T. coccinea. Heteroaromatic base ~ was insoluble in all solvents

except alcohols or 1% acetic acid (aqueous).

102

D. SPECTRAL DATA

1. 8,9-Dihydroaplysinopsin (~): [~]~5 -39.370 (~0.15, MeOH);

UV (MeOH) 223 (E=22350), 242 (E=5450), 282 (E=3850), 291nm (E=3350);

1a NMR (acetone-d6) 010.00 (broad s, IH, D20 exchangeable), 7.63 (d,

IH, ~=7.6), 7.34 (m, IH), 7.14 (5, IH), 7.05 (m, 2H), 3.93 (t, IH,

~=5.2), 3.34 (dd, IH, ~=5.2, 14.2), 3.09 (dd, IH, ~=5.2, 14.2), 2.£1

(s, 3H), 2.78 (5, 3H); electron-impact mass spectrum m/z 256 (M+,

11), k30 (100), 127 (90),83 (14), 77 (20), 57 (24); HRMS of ~/~

103

Br

25o2. 6-Bromo-8,9-dihydroap1ysinopsin (kR): [a]n -14.28 (£ 1.12, MeOH);

UV (MeOH) 228nm (E=20050), 235-240nm (shoulder), 278nm (E=2100), 288nm

1(E=2700), 296nm (E=2700); H NMR (acetone-d6) 010.38 (broad s, 1H,

D20 exchangeable), 7.59 (d, 1H, 1=8.5), 7.55 (d, 1H, 1=1.7), 7.15

(s, 1H), 7.09 (dd, 1H, 1=1.7, 8.5), 6.59 (broad d, 1H, 1=4.3), 3.96

(t, 1H, J=4.5), 3.31 (dd, 1H, 1=4.5,15.1), 3.13 (dd, 1H, 1=4.5, 15.1),

2.85 (s, 3H), 2.75 (s, 3H); electron-impact mass spectrum ~/~ 336 (16),

334 (18),210 (61), 208 (61), 197 (14),195 (15), 130 (21), 129 (53),

128 (51),127 (100), 102 (20), 83 (26), 69 (53),57 (25); HRMS of

m/~ 334.0408 (C14H1579BrN40, requires 334.0429).

Br

104

3. 6-Bromoap1ysinopsin (~): UV (MeOH) 380 (£=10700). 260-290

(£=5700), 233nm (£=15000); 1H NMR (Me 2SO-d6) 08.73 (s, 1H), 7.90

(d, 1H, ~=8.0), 7.62 (d, 1H, ~=1.5), 7.25 (dd, 1H, ~=1.5, 8.0), 6.56

(s, 1H), 3.30 (s, 3H), 3.08 (s, 3H); electron-impact mass spectrum

~/~ 334 (88), 333 (33), 332 (100), 331 (15), 254 (12), 249 (12),247

(10), 235 (35), 233 (32), 210 (3),208 (4),169 (8),154 (21),117

(19), 82 (9), 57 (18); HRMS of 332.0285 (C14H1379BrN40, requires

332.0273).

105

Br

4. 6-Bromo-2'-de-!-methy1ap1ysinopsin (~): UV (MeOH) 221 (£=23000),

-1 1278 (£=5200), 385 (£=9800); IR (KBr) 1685cm ; H NMR (Me 2SO-d6)

08.16 (5, 1H), 7.93 (d, IH, ~=8.3), 7.58 (d, 1H, ~=1.4), 7.16 (dd, 1H,

~=1.4, 8.3), 6.68 (5, 1H), 3.02 (5, 3H); electron-impact mass spec-

trum ~/~ 320 (100), 318 (100), 236 (19), 235 (17), 234 (29), 233 (13),

155 (33).

5. 2'-De-!-methy1aplysinopsin (~): UV (MeOH) 223 (£=18000), 280

-1 1(£=3900), 390nm (£=8700); IR (KBr) 1680cm ; JH NMR (Me~SO-d6)

08.20 (5, 1H), 7.90 (m, IH), 7.41 (m, IH), 7.10 (m, 2H), 6.75 (5. 1H),

3.03 (5, 3H); electron-impact mass spectrum ~/~ 240 (100), 155 (50),

154 (41), 130 (23), 129 (16), 128 (17), 127 (10); HRMS of m/~

1060-(CHO~N)J

H

6. Indo1e-3-carboxa1dehyde (~): UV (MeOH) 207 (E=10000), 240

(E=5100), 258 (E=3900), 294nm (E=4200); IR (CHC1 3) 3460, 3000, 2850,

1665, 1210, 1020cm-1; I H NMR (Me2SO-d6)

012.20 (s, 1H), 9.91 (s, 1H),

8.27 (s, 1H), 8.08 (d, 1H, ~=7.0), 7.51 (d, 1H, ~=7.4), 7.23 (m, 2H);

I H NMR (CDC1 3) 010.06 (s, IH), 8.72 (bs, 1H), 8.30 (d, 1H, ~=7.0),

7.84 (d, 1H, ~=7.4), 7.43 (m, 1H), 7.32 (m, 1H); electron-impact

mass spectrum ~/~ 145 (85), 144 (100),116 (40), 90 (11), 89 (45),

69 (16), 63 (26), 59 (13), 39 (16); HRMS of ~/~ 145.0520 (C9H7NC,

requires 145.0528).

7. 6-Bromoindole-3-carboxaldehyde (~): UV (MeOH) 210 (E=11900),

217 (E=12100), 244 (E=6900), 263 (E=6500), 290nm (E=4000); IR (CHC13)-1 L

3420, 1660cm ; ~ NMR (CDCl) 010.03 (s, 1H), 8.79 (s, IH), 8.163

(d, IH, ~=8.4), 7.81 (5, IH), 7.59 (d, 1H, ~=1.6), 7.40 (dd, IH, ~=1.6,

8.4); 1H NMR (acetone-d6) 010.01 (s, IH), 8.23 (s, 1H), 8.13 (d, 1H"

~=8.4), 7.75 (d, 1H, ~=1.6), 7.37 (dd, 1H, ~=1.6, 8.4); e1ectron

impact mass spectrum ~/~ 225 (88),224 (72), 223 (100), 222 (67),196

(15),194 (21), 143 (19),115 (30),109 (23),97 (29), 95 (46), 84

(41), 83 (35), 69 (50); HR¥~ of ~/~ 222,9616 (C9H679BrNO,

requires

222.9633).

107

NH

CH31~:~o N

ICH3

8. Hokupurine (~): UV (MeOH) 228 (£=7200), 288nm (£=11500); UV

(H20) 214 (£=11100), 290nm (£=10400); IR (KBr) 3600-2900, 1710,

1670, l610cm-1; I H NMR (CD30D)

03.50 (s, 3H), 3.55 (s, 3H), 7.60

(s, IH); electron-impact mass spectrum ~/~ 179 (100), 150 (55), 149

(69), 121 (45).

9. Acetylation of hokupurine Hokupurine Acetate (~): To i.5mg of

hokupurine (kk) was added lmL of pyridine and lmL of acetic anhydride.

The solution was allowed to stir at room temperature for l2h. Solvents

were removed in vacuo; traces of pyridine were removed by toluene azeo-

trope. The resulting white residue (1.6mg) crystallized from CHCl3 as

hokupurine acetate (~): UV (MeOH) 223 (£=6800), 250 (£=3300), 298

(£=7000), 320-330nm (shoulder); IR (CHC13) 3005, 1720,1650, 1570,

-1 11540, 1365, 1020cm; H NMR (CDC13) 02.31 (s, 3H), 3.65 (s, 3H),

3.60 (s, 3H), 7.69 (s, 1H); electron-impact mass spectrum ~/~ 221

(35), 206 (100), 179 (17), 150 (26), 144 (90); HRMS of ~/~ 221.0887

108

10. Unidentified base from P. me1anobrachia (49): UV (MeOH) 225,~

1286. 293nm; H NMR (CD OD) 06.90 (d. 1H. J=14). 6.85 (5. 1H). 6.663 -

1(5. 2H). 6.13 (d. IH. 1=14); H NMR (He2SO-d6) 08.94 (broad s. IH).

7.65 (broad s. 2H), 7.10 (d, 1H, 1=14), 6.90 (5. IH). 6.70 (5. 2H),

136.00 (d. IH. ~=14); C NMR (CD30D) 0146.6 (5). 137.1 (5). 136.7 (5).

119.2 (5).110.5 (d). 109.7 (d). 109.2 (d). 107.1 (d). 104.4 Cd);

electron-impact mass spectrum ~/~ 210/209/208/207 (1:1:1:1 relative

intensity). 222/220 (1:1). 237/239 (1:1).

109

E. COLLECTION - DENDRODORIS NIGRA AND DENDRODORIS TUBERCULOSA

Specimens of Dendrodoris nigra were collected from Fort Kameha-

meha reef, Ala Moana reef, and Pupukea. Oahu; Dendrodoris tuberculosa

were found at Ala Moana and Pupukea. Collections at Pupukea were made

from the large tidepool adjacent to Shark's Cove during summer months

(May to August) when wave action is low. Dendrodoris spp. were found

at the other two sites at all times of the year. All collections were

made by snorkeling at -0.5 to -2m. At Pupukea. Q. nigra and Q. tuber-

culosa were found only at night. when they could be observed while

crawling about exposed. At Ala Moana and Fort Kamehameha, these

dorids could be found only by turning rocks.

F. EXTRACTION AND ISOLATION

Whole live.!!. nigra and D. tuberculosa were extracted with dis·-

tilled hexanes or benzene for 5-10min. The animals were then trans-

ferred to fresh solvent for longer term extraction. The short and

long term extracts were separately evaporated in vacuo and analyzed by

1TLC and H NMR. The short term extract, a pale yellow oil, was found

to consist of only two components, while the long term extract con-

tained only minor amounts of these two metabolites, and consisted

mainly of sterols, pigments and fatty acid derivatives. The short

term extract was chromatographed by HPLC using silica gel (Knauer-

Unimetrics, Lichrosorb Si-60, 0.5x25cm, 7:3 hexanes:ethyl acetate)

yielded olepupuane '~) followed by polygodial (~): these metabo-

lites were isolated in a 1:1 ratio.

Yields of the polygodial/olepupuane fraction in the extracts of

110

D. nigra and D. tuberculosa remained remarkably constant, both between

the two species and collecting sites. Typically, the yield of polygo-

dial + olepupuane was 0.03% to 0.04% of the wet weight of the animal.

For example, two~. nigra from Pupukea weighing 1.5g contained 0.5mg

(0.03%) of the drimanes. A 23g specimen of ~. tuberculosa from the

same site yielded 10.Omg (0.04%) of these metabolites.

G. SPECTRAL DATA AND CHEMICAL TRANSFORMATIONS

CHO

CHO

25 01. Polygodial (~): [a]D -97 (~0.35, CHC13); UV (MeOH) 230nm

-1 1(e=7000); IR (CHC13) 2923, 1720, 1677, 895cm; H NMR (CDC13)

09.54 (d, lH, I=4.4) , 9.46 (s. lH), 7.13 (m, lH), 2.84 (m, lH), 2.55

2.30 (m, 2H), 1.95-1.15 (m, 7H), 0.95 (s, 3H), 0.93 (s, 3H), 0.90 (s,

3H); electron-impact mass spectrum~/~ 234 (5), 206 (100),191 (73),

121 (61), 109 (52); HRMS of m/~ 206.1675 (C14H220, requires

206.1671).

111

AcO"'"

""OAc

25 02. Olepupuane (~): [a]D -83.3 (c 0.54, CHC13); IR (CH

2C12)3024,

-1 12992, 1732, 1423, l262cm; H NMR (C6D6) 06.70 (d, lH, ~=2.3), 6.13

(d, lH, ~=1.8), 5.63 (dd, lH, ~=3.5, 3.8), 2.72 (m, lH), 1.75 (ddd, lH,

J=2.2, 3.6, 14.4), 1.64 (s. 3H), 1.56 (s, 3H), 1.48 (m, lH), 1.4-1.1

(m, 5H), 0.05-0.90 (m, 2H), 0.76 (s, 3H), 0.61 (s, 3H), 0.54 (s, 3H);

electron-impact mass spectrum ~/~ 336 (1), 276 (53), 234 (100), 219

(15), 162 (13), 124 (23), 109 (48), 91 (16), 81 (21), 69 (23); H~:S

of ~/~ 276.1702 (C17H2403' requires 276.1705), 234.1601 (C15H2202'

requires 234.1620).

3. Acid Hydrolysis of Olepupuane -- Polygodial: One milligram of

olepupuane (~) was dissolved in 1.OmL methylene chloride. A small

crystal of £-toluenesulfonic acid was added and the solution was

stirred at room temperature for lh. TLC (silica gel, 7:3 hexanes:

ethyl acetate) revealed that no starting material remained, and that

a single UV absorbing spot with an Rf value identical to that of poly

godial appeared. The reaction mixture was subjected to HPLC (Knauer-

Unimetrics, Lichrosorb Si-60, 7:3 hexanes:ethyl acetate) and yielded

0.6mg of a dialdehyde, which showed identical UV, IH NMR, and mass

spectra to that of polygodial (~).

112

H. COLLECTION OF CHROMODORIS ELISABETHINA AND HETERONEMA SP.

Chromodoris e1isabethina was collected from Enewetak Atoll, Mar-

shall Islands by Mr. Scott Johnson between June 1982 and May 1983;

Collections from Guam Island were made in Ju1u 1981 by Drs Gary

Schulte and Chris Ireland. Heteronema sp. was collected at Enewetak

in June and November 1982.

I. EXTRACTION AND ISOLATION

Specimens of C. e1isabethina from Enewetak were stored in iso-

propyl alcohol as they were collected. The isopropyl alcohol extract

was concentrated in vacuo to a brown solid residue. This residue was

subjected to the Kupchan scheme (Figure 1). In a typical extraction,

8 specimens of dorids weighing 1.lg (extracted weight) yielded:

crude isopropyl alcohol extract

petroleum ether fraction

CC14 fraction

CHC13 fraction

MeOH fraction

250mh

49mg

3lmg

7mg

170mg

By TLC (silica, 1:1 petroleum ethyl acetate), the crude extract con-

tained one major constituent, which partitioned specifically into the

carbon tetrachloride fraction. HPLC of this fraction on silica gel

(Knauer-Unimetrics, Lichrosorb Si-60, O.5x25cm, 1:1 petroleum ether:

ethyl acetate) afforded pure latrunculin-A (~). Latrunculin-A

crystallized from mixtures of petroleum ether and ethyl acetate.

Specimens of C. e1isabethina from Guam were kept frozen until

workup. These animals were extracted with isopropyl alcohol, then

processed exactly as the Enewetak specimens. Latrunculin-A was the

storage.

113

major metabolite of the Guam specimens.

Yields of latrunculin-A from Chromodoris elisabethina were ex-

ceptionally high. The Guam samples contained 1% latrunculin-A, while

the Enewetak specimens ranged from 0.27 to 0.50% latrunculin-A content,

based on extracted weight of the animals.

One collection of C. elisabethina from Enewetak was extracted

for 5min in isopropyl alcohol, then transferred to fresh solvent for

lH NMR and TLC analysis of the short term extract revealed

that it consisted of nearly pure latrunculin-A.

Specimens of Heteronema sp. were stored in isopropyl alcohol

after collection. Removal of the solvent in ~~ yielded a dark

brown residue, which was partitioned by the Kupchan scheme (Figure 1).

Yields from a 11.5g piece of sponge are:

crude isopropyl alcohol extract

petroleum ether partition

CC14 partition

CHC13 partition

MeOH partition

250mg

64mg

42mg

57mg

103mg

TLC and lH NMR analysis of all fractions revealed that latrunculin-A

was not a constituent of the sponge extract. The major component

appeared in the petroleum ether fraction and was isolated by chromato-

graphy of this fraction on Sephadex LH-20 (l.5xllOcm, 1:1 CHCl3:MeOH).

Puupehenone (~) was isolated at approximately 0.2% of the wet weight

of the sponge.

114

J. SPECTRAL DATA

HN~),-So

2l9nm (e:=18700);

lH NMR (CDC13)

1. Latrunculin-A (~): [~]~5

IR (CH2C12)

06.38 (dd, lH,

o+145 (£ 0.12, CHC13); UV (MeOH). -1

3670, 3560, 3400, 2920, 2860, l690cm ;

~=10.3, 15.0), 5.95 (t, 1H, ~=10.5).

5.74 edt, lH, J=5.0, 15.0), 5.67 {s, 1H),5.65 (s. 1H, D20 exchange

able), 5.41 (t, lH, ~=2.l), 4.99 (t, 1H, ~=10.3), 4.24 em, lH), 3.87

(5, 1H, D20 exchangeable), 3.83 (dd, 1H, ~=7.5, 7.5), 3.42 (m, 3H),

2.87 em, 1H), 2.69 em, 2H), 2.24 em, 2H), 2.04 em, lH), 1.94 em, 1H),

1.91 (s , 3H), 1.77 em, 1H), 1.70 em, lH), 1.42 (m, 3H), 1.07 em, lH),

0.96 (d, 3H, ~=6.4); ~ NMR (C6D6) 06.48 Cd, 1H, ~=10.3, 15.0), 6.01

(t, 1H, ~=10.5), 5.52 (dt, lH, ~=5.0, 15.0), 5.61 (s , 1H), 5.44 (s,

IH, D20 exchangeable), 5.01 (t, lH, J=2.l), 4.96 (t, lH, ~=10.3), 4.20

(m, lH) , 4.25 (s, lH, D20 exchangeable), 3.23 (dd, lH, ~=7.3, 7.4) ,

2.78 (m, 3H), 2.49 em, 1H), 1.91 (m, 2H) , 1.68 (m, 2H), 1.43 (m, lH),

1.36 (5, 3H), 1.33 em, 1H), 1.28 (m, 3H), 1.01 (d, 3H, J=6.4), 0.85

115

(m, 2H); 13C NMR (CDC13) 0174.7, 165.4, 158.4, 136.5, 131.7, 127.1,

126.0, 117.3, 97.2, 68.1, 62.3, 61.3, 34.9, 32.6, 31.7, 31.5, 31.0,

30.4, 29.2, 28.7, 24.5, 21.6; electron-impact mass spectrum ~/~ 421

(M+, 5), 403 (M+ - H20, 21), 385 (M+ - 2H20, 15), 301 (24), 149 (24),

135 (23), 123 (24), 121 (35), 110 (48), 107 (50), 102 (41), 93 (53);

HRMS of ~/~ 421.1949 (C22H31N05S, requires 421.1923), 301.1823

(C19H2503' requires 301.1804).

116

OH

a

2. Puupehenone (~): UV (cyc1ohexane) 307 (£=7300), 317 (£=86(0),

329nm (£=5500); IR (CCI4)

3400, 2950, 2876, 1630, 1616cm-1; I H NMR

(CDCI3) 06.83 (s, IH, D20 exchangeable), 6.64 (d, IH, ~=7.0), 6.19

(s, IH), 5.85 (s, IH), 2.16 (m, 1H), 2.03 (d, IH, ~=7.0), 1.22 (s,

3H), 0.90 (s, 3H), 0.83 (s, 3H), 0.81 (s, 3H); electron-impact mass

spectrum~/~ 328 (12), 313 (8),213 (11), 211 (33), 204 (8), 178 (16),

177 (100), 176 (16),109 (9),95 (13), 81 (17), 69 (18),56 (17);

HRMS of ~/~ 328.2026 (C2IH2803' requires 328.2039).

117

K. COLLECTION - HYPSELODORIS INFUCATA

Hypselodoris infucata were collected from Fort Kamehameha reef

(1 specimen) and from the pilings of the University of Hawaii pier in

Kaneohe Bay (2 specimens). All were collected by snorkeling at -1m.

Collections were made in July 1982.

L. EXTRACTION AND ISOLATION - HYPSELODORIS INFUCATA

Live H. infucata were immersed in distilled methylene chloride

for 5min, then transferred to fresh solvent for long term extraction.

TLC of the short term extract (silica gel, 5:95 CH2C12:hexanes) re

vealed only two spots, which appeared at identical Rf values as naka

furan-8 and -9, which were spotted on the same plate. HPLC of the

short term extract (Knauer-Unimetrics, Lichrosorb Si-60, 0.5x25cm,

5:95 CH2C12:hexanes) yielded nakafuran-8 (t) and -9 (~) as the only

components of this extract.

TWo specimens of H. infucata weighing 2.5g yielded 3mg of a

yellow oil from the short term extract. From this oil was isolated

1.8mg of nakafuran-8 (~' 0.0007% wet animal weight) and 0.9mg of

nakafuran-9 (~' 0.0004% wet animal weight).

M. SPECTRAL DATA

U8

11. Nakafuran-8 (~): UV (hexane) 321nm (£=4300); H NMR (CDC1 3)

07.11 (d. 1H, ~=1.6). 6.05 (dd, 1H. ~=1.6. 1.0), 5.95 (dd, LH, ~=7.1.

1.0), 3.44 (m, 1H), 2.46 (m, 1H), 2.23 (m. 1H), 2.05 (m, IH), 1.)0

1.80 (m. 2H), 1.76 (d, 3H, ~=1.0), 1.25 (m, 2H), 1.05 (s, 3H). 0.85

(d, 3H, ~=7.1); electron-impact mass spectrum ~/~ 216 (100), 201

(55), 147 (15), 121 (32), 120 (47), 109 (15),91 (39).

1Nakafuran-9 (~): UV (hexane) 218nm (£=3300); H NMR (CDC1 3)

07.10 (d, IH, ~=1.7), 6.04 (dd, 1H, ~=1.7, 1.0), 3.16 (m, 1H), 2.40

2.30 (m, 2H), 1.91 (ro, IH), 1.80-1.65 (m, 3H), 1.56 (d, 3H, ~=1.0),

1.54 (s, 3H), 1.35 (m, 1H), 1.05 (s, 3H); electron-impact mass

spectrum ~/~ 216 (100), 201 (36), 161 (32), 126 (20), 109 (24),

107 (31), 91 (52).

119

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