SECONDARY METABOLITES COMPOSITION AND GEOGRAPHICAL ...digilib.library.usp.ac.fj/.../index/assoc/HASH0112.dir/doc.pdf · SECONDARY METABOLITES COMPOSITION AND GEOGRAPHICAL DISTRIBUTION

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SECONDARY METABOLITES COMPOSITION AND GEOGRAPHICAL DISTRIBUTION OF MARINE

SPONGES OF THE GENUS DYSIDEA

By

Mayuri CHANDRA

A thesis submitted in partial fulfillment of the requirements for the degree of

Master of Science

School of Biological & Chemical Sciences,

Faculty of Science, Technology & Environment,

The University of the South Pacific

Suva, Fiji Islands

June, 2009

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“Man masters nature not by force but by understanding. This is why science has

succeeded where magic failed: because it has looked for no spell to cast on nature.”

- JACOB BRONOWSKI

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This project would not have been completed without the help and support of a

number of people. My heartfelt acknowledgement goes to the following people and

organisations:

This work is part of the C2 component of the Coral Reef Initiative in the South

Pacific (CRISP) project and funded by the Agence Française de Développement. The

research grant for the component completed in Fiji was obtained from the

Government of Taiwan. This project was also partly completed in New Caledonia

and France, and it was funded by the Government of France with the initiative of the

Embassy of France in Fiji and the Centre Régional des Œuvres Universitaires et

Scolaires (CROUS).

I am grateful to my supervisors, Professor Subramaniam Sotheeswaran and Associate

Professor Sadaquat Ali, for giving me an opportunity to pursue my interest in the

field of Natural Products research. This project was a good learning ground for me,

which provided great experiences along the way.

Furthermore, most of this project was completed in New Caledonia and France, and

my sincere gratitude goes to my external supervisors, Dr Sylvain Petek, Dr Isabelle

Bonnard and Dr Bernard Banaigs for their guidance.

Dr Sylvain Petek, my supervisor in New Caledonia, has been a great support and

mentor. The research team at the Institut des Recherche et Développement (IRD –

Noumea, New Caledonia) was really friendly and helped me greatly to adjust to the

new environment and language.

Dr Isabelle Bonnard at the Centre de Phytopharmacie, University of Perpignan via

Domitia (Perpignan, France), was always there for me whether I had any project-

related or personal problems. She is a great mentor and I was able to have a good

learning experience, which broadened my knowledge of research. Thanks to the staff

and students of Centre de Phytopharmacie, I was also able to explore France to some

extent, and the experience was amazing.

The sponge samples used in this study were obtained by diving; hence I would like to

thank the dive team of the IRD Centre of Nouméa, New Caledonia, for harvesting

the sponges. Furthermore, we highly appreciate the governments of New Caledonia,

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Vanuatu, Solomon and Fiji Islands for permitting us to collect our samples in their

countries, and the Fisheries Departments of these countries for their help and

assistance during the collections.

I would also like to thank Dr Cécile Debitus, Pierre Pério, and Camille Durand at the

Université Paul Sabatier, Toulouse, France, as well as Professor Valeria D’Auria at

the University of Naples, Italy, for providing us with technical support in our sample

analysis.

The anti-microbial testing was carried out partly at the IRD – Noumea, and partly at

the Institute of Applied Sciences (IAS), University of the South Pacific. I would like

to thank the microbiology team at the IAS (especially Pritesh, Kavita, Girish Lakhan,

and Mr. Klaus Feussner) for providing the necessary resources for completing this

part of the project.

I can not forget my family and friends for their never-ending support in the duration

of my project. For all the hours spent in the laboratory, there was always someone to

give company or talk to when things became tensed, confusing, or just tiresome. My

thanks goes to my friends Nirul, Ardarsh, Roselyn, Joslin, Sujlesh, Riteshma, and the

technical staff of the Faculty of Science and Technology, USP, Suva. Kirti and Shital

helped me quite a lot during my stay in France. We had a great time together.

Finally, the support, blessing and sacrifice of my parents, Mr. Vinod Chandra and

Mrs. Kala Chandra, have given me the strength to stay focused on my path to

complete this project.

Thank you, vinaka vakalevu, and merci beaucoup to everyone who has contributed to

this project in one way or another.

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Dysidea species – Lamellodysidea herbacea, Dysidea arenaria, and Dysidea avara –

from New Caledonia were chemically screened to identify the chemical constituents

of these sponges. Each species exhibited a specific profile, thus grouping the three

species chemically together in distinct taxa. The screening also identified the

presence of three different chemical types of L. herbacea present within the same

region, New Caledonia.

The pattern of occurrence of polybrominated diphenyl ethers (PBDE) in L. herbacea

was also mapped for distant geographical sites, which included New Caledonia,

Vanuatu, Fiji Islands, Solomon Islands, Moorea Island in French Polynesia, and

Mayotte. Comparison and analysis of the chemical fingerprints shows that all the

samples belong to the bromo-chemotype of compounds, and similarities and

differences exist within the same species in different geographical locations.

L. herbacea was further explored for compound isolations and characterization to

verify the chemical fingerprints of these sponges. Spectroscopic and chemical

analyses confirmed the presence of PBDEs in the L. herbacea analyzed. Eight

different PBDEs have been identified, where four of these (compounds 2, 6, 7, and 8)

have been successfully isolated and characterized.

Crude extracts of the Dysidea sponges were also tested for activity against the fungi

Cryptococcus neoformans and Candida albicans. Most extracts were active against

Cr. neoformans hence confirming the hypothesis that the action was due to the

inhibition of the proper functioning of the protein farnesyl transferase (PFT), which

is an essential enzyme in the metabolism of this fungus. Ca. albicans is able to

survive even without PFT. Since Plasmodium falciparum (the malarial parasite) is

also dependent on PFT for survival, this anti-fungal test is being developed to be

used as a pre-test for anti-plasmodial studies.

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L’étude et la comparaison des profils chimiques chez Lamellodysidea herbacea,

Dysidea arenaria, et Dysidea avara ont été menés afin d’identifier les métabolites

secondaires présents dans ces trois espèces d’éponges qui pourraient être utilisés

comme marqueurs taxonomiques. En comparant les profils chimiques, il est apparu

que chaque espèce Lamellodysidea herbacea, Dysidea arenaria, Dysidea avara

pouvaient être groupement, car chacune présente un profil chimique spécifique.

Les profils chimiques des différents échantillons de L. herbacea a ont été étudiés

pour différents sites géographiques. Les pays régions géographiques concernées sont:

la Nouvelle Calédonie, les Vanuatu, les îles Fidji, les îles Salomon, la Polynésie

Française (Moorea) et Mayotte. Les analyses chimiques et spectroscopiques

montrent que tous les échantillons de L. herbacea contiennent des diphénylethers

polybromés (PBDE, bromochémotype).

L. herbacea a été étudiée plus avant pour les composés isolement et la caractérisation

de manière plus approfondie au niveau de l’isolement et de la caractérisation de

composés pour vérifier les ‘empreintes digitales’ chimiques de ces éponges. Les

analysés spectroscopiques et chimiques ont a confirmé la présence de PBDE dans les

extraits de L. herbacea analysées. Huit différents PBDE ont été identifiés; trois types

chimiques de L. herbacea ont été caractérisés; et quatre composés (composés de 2, 6,

7 et 8) ont été isolés et caractérisés.

L’activité antifongique des extrait bruts de Dysidea a ont également été étudiée sur

deux souches de levure testées: pour l'activité contre les champignons Cryptococcus

neoformans et Candida albicans. La plupart des extraits ont été actifs contre sur Cr.

neoformans sans forcément l’être sur Ca. albicans, partant, confirmant l'hypothèse

selon laquelle l'action est le résultat de l'inhibition. Cette différence d’activité

suppose une inhibition du bon fonctionnement de la protéine farnesyl transférase

(PFTase), qui est un enzyme essentielle dans le métabolisme de Cr. neoformans alors

que ce champignon, Ca. albicans est capable de survivre même sans PFTase. Depuis

le Plasmodium falciparum (le parasite du paludisme) est également tributaire des

PFTase pour la survie, cette anti-fongiques test est développé pour être utilisé comme

un pré-test pour les études anti-plasmodiales.

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% – Percent

� – Wavelength

4Br-OH-OMe-PBDE – 3,5-dibromo-2-(3’,5’-dibromo-2’-methoxyphenoxy)phenol 79Br – Isomer of bromine79 81Br – Isomer of bromine81 35Cl – Isomer of chlorine35 37Cl – Isomer of chlorine37 13CNMR – Carbon Nuclear Magnetic Resonance Spectroscopy 1HNMR – Proton Nuclear Magnetic Resonance Spectroscopy

ACN – Acetonitrile

AFD – Agence Française pour le Développement (French Agency

for Development)

AIDS – Acquired Immuno-Deficiency Syndrome

APCI – Atmospheric Pressure Chemical Ionisation

Ca. albicans – Candida albicans

CARD-FISH – Catalyzed reporter deposition fluorescence in-situ

hybridization

CD3OCD3 – Deuterated Acetone

CDCl3 – Deuterated chloroform

CI – Conservation International

Cr. neoformans – Cryptococcus neoformans

CRISP – Coral Reef Initiative in the South Pacific

CROUS – Centre Régional des Œuvres Universitaires et Scolaires

D. arenaria – Dysidea arenaria

D. avara – Dysidea avara

D2O – Deuterated water

DCM – Dichloromethane

DMSO-d6 – Deuterated Dimethyl sulphoxide

DNA – Deoxyribonucleic acid

ELS – Evaporative Light Scattering (detector)

ESI – Electrospray Ionisation

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EtOAc – Ethyl acetate

EtOH – Ethanol

FACS – Fluorescent Activated Cell Sorter

FDA – Food and Drug Administration

g – Grams

GGTase-1 – Gerany geranyl transferase-1

H2O – Water

HCl – Hydrochloric acid

HIV – Human Immunodeficiency Virus

HMBC – Heteronuclear Multiple Bond Correlation

HPLC – High Performance Liquid Chromatography

HSQC – Heteronuclear Single Quantum Correlation

IRD – Institut de Recherche pour le Développement (Institute of

Research for Development)

IFRECOR – L’Initiative Française sur les Récifs Coralliens (The French

Coral Reef Initiative)

ITS – Internal Transcribed Spacers

J – Coupling constant

L. herbacea – Lamellodysidea (Dysidea) herbacea

LCBE – Laboratoire de Chimie, des Biomolécules et des

l’Environnement

LC-MS – Liquid Chromatography-Mass Spectroscopy

m/z – Mass to charge ratio

MeOD – Deuterated methanol

MeOH – Methanol

mg – Milligrams

MHz – Mega Hertz

mL – Millilitre

mu – Mass units

NMT – N-myristoyl Transferase

NaCl – Sodium chloride

Na2SO4 – Sodium sulphate

NI – Negative Ion

nm – Nanometers

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O. spongeliae – Oscillatoria spongeliae

PBDE – Polybrominated Diphenyl Ether

PDA – Photo Diode Array detector

PFT – Protein farnesyl transferase

PI – Positive ion

PLA2 – Phospholipase A2

ppm – Parts per million

RBF – Round bottom flask

Rf – Retention factor

Rt – Retention time

SDA – Sabouraud Dextrose Agar

SPE – Solid Phase Extraction

TLC – Thin Layer Chromatography

μL – Microlitre

UNF – United Nations Foundation

UV – Ultraviolet

VPA – Vanillin Phosphoric Acid

WTCA – Wild Type Candida albicans

WWF – World Wildlife Fund

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3,4,5,6-tetrabromo-2-(3’,5’-dibromo-2’-hydroxyphenoxy)phenol

Compound 3 R = H; R1 = Br; R2 = H

3,5,6-tribromo-2-(3’,5’-dibromo-2’-hydroxyphenoxy)phenol

Compound 4 R = Me; R1 = Br; R2 = H

3,5,6-tribromo-2-(3’,5’-dibromo-2’-methoxyphenoxy)phenol

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4,6-dibromo-2-(4’,5’,6’-tribromo-2’-hydroxyphenoxy)phenol

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Secondary Metabolites Composition and Geographical Distribution of Marine Sponges of the Genus Dysidea

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Oceans cover more than seventy percent of the surface of the Earth. It is an essential

element for life, and is also home to a vast number of organisms of all kinds, such as

phytoplanktons, fish, marine invertebrates and plants, to name a few. These

organisms have a very challenging environment for survival – it is constantly in

motion, nutrients are difficult to find, and there are risks of predation, competition

and fouling. It is even more challenging for sessile marine organisms to survive in

this environment. However, despite the difficulties of survival, the marine organisms

have developed remarkable means of protection and reproduction, and in many cases

this is achieved by utilizing their own biochemicals.

Biochemical defense mechanisms are subtle and widespread in the plant and animal

kingdom. Most of the marine invertebrates use their secondary metabolites as

chemical defence for protection from predators, to avoid competition and overgrowth

(Debitus, 1998), and to resist malignant microbial infection (Garson, 2001). These

metabolites are produced as a result of transformations and interconversions of the

vast number of organic compounds produced during the metabolic pathways of these

organisms (Voet & Voet, 2004). It is these secondary metabolites that have attracted

researchers from all over the world in the hope to find products that can be useful to

mankind in the form of drugs, as unique templates to serve as a starting point for

synthetic analogues, as interesting tools that can be used to better understand

biological processes (Farnswort, 1984; Sladi� & Gaši�, 2006), and for

chemotaxonomic analysis, which is the classification of organisms based on their

unique chemical components (Erpenbeck & Van Soest, 2006).

The biochemistry of marine invertebrate organisms has been gathering great interest

(Bergquist, 1978) for the past three decades. This has been encouraged mainly by the

recognition that the search for natural products with new molecular structures is

likely to prove rewarding in this little researched area (McConnell, Longley, &

Koehn, 1994).

Considering the identification of these marine organisms however, it is clear that

many specimens had been misclassified previously, and hence need to be re-


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classified correctly. In the past, classification of organisms was based generally on

morphological characteristics. Recent advancement in technology has led to the use

of electron microscopy to aid in the study of internal sections of organisms, and

genetic mapping for a much closer comparison. However, it is evident with the

various misclassifications of organisms that electron microscopy and genetic

mapping alone can not resolve all the problems of taxonomy. Hence, an additional,

rapid and simple method is required which can be used together with the current

methods to provide the necessary information needed for the proper taxonomy of the

numerous organisms.

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The first marine invertebrate group to be studied in any comprehensive way by those

searching for new compounds was the sponges (sedentary, filter-feeding metazoans

of the phylum Porifera) (Bergquist, 1978; Lévi, 1998). Up to now they have yielded

a great number of novel compounds over a very diverse chemical spectrum (Blunt, et

al., 2006), and there are still possibilities of more novel compounds to be discovered.

Metabolites have been discovered in simple and complex arrangements; many are

also halogenated or contain double bond conjugations and composite configurations,

which are quite unique and difficult to obtain synthetically (Debitus, 1998).

Sponges have exhibited a high incidence of biologically active compounds compared

to other organisms (Belarbi, Gómez, Christi, Camacho, & Grima, 2003; Debitus,

1998) as evidenced either by cytotoxic (Belarbi, Gómez, Christi, Camacho, & Grima,

2003; Haefner, 2003; Debitus, 1998), immunomodulatory (Belarbi, Gómez, Christi,

Camacho, & Grima, 2003; Debitus, 1998) or in some cases, antifouling effects

(Armstrong, McKenzie, & Goldworthy, 1999). It can thus be said that the secondary

metabolites from sponges offer a great potential for application in medicine and

industry (Debitus, 1998).


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Sponges inhabit a wide variety of marine systems and are found predominantly in

tropical and subtropical oceans but also in polar regions, deep sea and even in fresh

water systems (Hooper & Van Soest, 2002). Marine sponges (Phylum Porifera) are

the most ancient metazoan animal phylum, with a fossil record dating back nearly

600 million years and have existed relatively unchanged in their general structural

organization since the late Cambrian period.

They show a plausible evolutionary jump from unicellular to multicellular

organisms. Because they lack true tissues, these poriferans are also classed as

parazoans, and they exhibit a cellular-level organisation, where each type of cell

specialises to carry out a different task (Mather & Bennett, 1992). Similar cells are

not structured into tissues and bodies, thus forming loose aggregations of similar

types of cells. They also lack muscles, nerves, and internal organs (Brusca & Brusca,

2003).

The taxonomy of sponges depends on the composition of their skeletal materials

(Mather & Bennett, 1992) and their morphological characteristics (Hooper, 1998).

They can either have calcareous spicules (Class Calcarea), siliceous spicules (Class

Hexactinellidae) or spicules made of proteinaceous sponging fibers (Class

Demospongiae).

Furthermore, sponges do not have a definite body shape and are thus divided into

three body types, mainly asconoid, syconoid, and leuconoid (Brusca & Brusca,

2003).

Asconoid sponges are the simplest of all, with a central shaft (spongocoel) and

tubular body. It has several ostia (small pore-like openings) through which water

enters and a central osculum at the top of the sponge to expel water (Brusca &

Brusca, 2003).

Secondary Metabolites Co

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Figu

Syconoid sponges, on

but they contain a

spongocoel (Brusca &

Figu

The most complex or

chambers instead of

canals emptying into

omposition and Geographical Distribution of Marine Sponge

ure 1 - Asconoid sponge (Brusca & Brusca, 2003

n the other hand, also have a tubular body an

thicker body wall with radial canals whi

& Brusca, 2003).

ure 2 - Syconoid sponge (Brusca & Brusca, 2003

rganisation exists in leuconoid sponges consi

a spongocoel, and it also has several incur

a central osculum (Brusca & Brusca, 2003).

es of the Genus Dysidea

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3)

nd a single osculum,

ch empty into the

3)

isting of flagellated

rrent and excurrent


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Figu

All the water canals

which beat to create

incoming water, food

in and this sticks to t

and undergo intrace

incoming water is al

metabolic wastes diff

current through the os

Water flow is also a

generally hermaphrod

they do not undergo s

offspring has differen

a blastula forms whic

and floats around unt

into the sessile spo

reproduction is also

budding, whereby the

& Bennett, 1992).

The phylum Porifer

species, with new sp

2003). There are thre

Demospongiae, and


ure 3 - Leuconoid sponge (Brusca & Brusca, 200

s and spongocoel are lined with flagellated

water current to flow through the sponge.

d (mainly bacteria, debris and microscopic alg

the cells in the sponge which are later taken

llular digestion (Mather & Bennett, 1992;

lso rich in oxygen and this is diffused into

fuse out of the cells, flowing out of the spo

sculum (Lévi, 1998).

an important factor for reproduction in spo

dites (male and female gonads are present i

self-fertilization to avoid asexual reproductio

nt characteristics as that of either parent. Ofte

ch is released into the water. The blastula de

til it finds a suitable substrate, after which it s

onge form (Mather & Bennett, 1992; Lév

possible in sponges by the process of e

e parent and offspring have the same geneti

ra encompasses approximately 6000 taxon

pecies still being discovered (Lévi, 1998;

ee main classes of the phylum Porifera as see

d Hexactinellidae. A fourth class of


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d choanocyte cells

Together with the

gae) is also brought

into food vacuoles

; Lévi, 1998). The

the cells while the

onge with the water

onges. Sponges are

in one sponge), but

on, and the resulting

en after fertilization,

evelops into a larva

settles and develops

vi, 1998). Asexual

external or internal

c make-up (Mather

nomically validated

Brusca & Brusca,

en earlier: Calcarea,

f sponges (Class


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Sclerospongiae) has been mentioned in many other texts, but according to the World

Porifera Database, the sclerosponges are polyphyletic, hence accepted as

Demosponges (Van Soest, Boury-Esnault, Janussen, & Hooper, 2005).

Demospongiae are by far the most diverse and ecologically significant group,

whereby 90 % of all species of sponges belong to this class (Lévi, 1998). It includes

approximately 5500 species in more than 10 orders and generally possesses the

leuconoid body type (Brusca & Brusca, 2003). Sponges of this class are asymmetric

and brightly coloured (such as orange, green, yellow, purple, or red) due to the

presence of pigment granules in their amoebocytes.

�� , �)'��'�$��

Sponges of the genus Dysidea belong to the Class Demospongiae, Order

Dictyoceratida, and Family Dysideidae. These sponges lack mineral spicules, and the

main skeleton consists of reticulation of sponging fibers (Bergquist, 1998). By the

year 2001, the genus Dysidea was reported to consist of 17 species (Munro & Blunt,

2001).

Lamellodysidea appears as a new genus in the most recent classification of sponges,

split off from Dysidea, because of the consistent presence of an encrusting basal

plate and the lack of orientation of the skeleton with respect to the surface (Sauleau

& Bourget-Kondracki, 2005). Currently, the genus Lamellodysidea includes the two

species L. herbacea and L. chlorea.

The marine sponge Dysidea is a prolific producer of structurally diverse secondary

metabolites including sesquiterpenes (Sera, Adachi, Nishida, & Shizuri, 1999),

sesterpenes, meroterpenes, and sterols (Cameron, Stapleton, Simonsen, Brecknell, &

Garson, 2000).

The following structures are a few examples of the diversity of compounds found in

sponges of the genus Dysidea:


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O

OMe OH

Br

Br

Br

Br

4,6-dibromo-2-(3',4',6'-tribromo-2'-methoxyphenoxy)phenolN

S

NHO

N

CCl3

O CCl3

Dysidenin

O

OH

OCH3

O

Br

O

OH

H

OHH

OH

OH

H

OH

Herbasterol

H

H

6-hydroxyfurodysinin-O-methyl-lactone

Figure 4 - Compounds isolated from Dysidea species.

Some sesquiterpene derivatives (such as dysidotronic acid; bolaquinone; two

sesquiterpene cyclopentenones [Dysidenones A and B]; and a sesquiterpene

aminoquinone, dysidine) isolated from the Dysidea sp. have shown selective

inhibitor profile against human phospholipase A2 (PLA2) and other leukocyte

functions, such as degranulation process and superoxide production (Giannini, et al.,

2001). PLA2 has also been recognized to be involved in a wide number of

pathophysiological situations, ranging from systemic and acute inflammatory

conditions to cancer (Giannini, et al., 2001; Giannini, et al., 2000).

In addition, certain Dysidea species, exemplified by Lamellodysidea herbacea,

Keller (1889) and L. chlorea, de Laubenfiels (1954), have given rise to

bromophenols (Hanif, et al., 2007) and polychlorinated peptides (Flatt, Gautshci,

Thacker, Musafija-Girt, Crews, & Gerwick, 2005). It is striking that these two

families of halogenated compounds have never been found together in the same

sponge. Hence, marine sponge Lamellodysidea (together with some Dysidea species)

occurs in two chemotypes, one containing sesquiterpenes and polychlorinated

peptides and the other polybrominated diphenyl ethers.

The polychlorinated peptides are an original family of molecules including dysidenin

(Ardá, Jiménez, & Rodríguez, 2004), dysidamides, dysideathiazoles, and chlorinated

diketopiperazines (Goetz, Harrigan, & Likos, 2001). Together with a furodysin

lactone, a well-known Dysidea-derived sesquiterpene, the discovery of the novel


�

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polychlorinated dysideaprolines and barbaleucamides was reported by Goetz,

Harrigan, and Likos (2001).

X2HC N

O

R2 O

N

CHY2

R1

S N

1

11 3

5

6

9

12

16

13

19 18

DysideaprolineA - R1 = H, R2 = CH3, X = Y = ClB - R1 = R2 = CH3, X = Y = ClC - R1 = R2 = H, X = Y = ClD - R1 = H, R2 = CH3, X = H, Y = ClE - R1 = H, R2 = CH3, X = Cl, Y = HF - R1 = H, R2 = CH3, X = Cl, Y2 = ClH

Cl3C

O

CH3

N

O

CCl3

R

S N

5

12

14

11

7

1

6

15

10

BarbaleucamideA - R = HB - R = CH3

Figure 5 - Chlorinated compounds isolated from Dysidea sponges

All polychlorinated peptides contain a trichloromethyl functionality on leucine-

derived precursors, and are very similar to pseudodysidenin and barbamide, which

have been isolated from the cyanobacteria Lyngbya majuscula (Jimenez & Scheuer,

2001).

Cl3C

O

CH3

N

OS N

Barbamide

Cl3C N CCl3

O

H

NO CH3

N

SPseudodysidenin

Figure 6 – Examples of chlorinated compounds found in both Dysidea sponges and the

cyanobacteria Lyngbya majuscula


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Some of these compounds exhibit interesting pharmacological activities such as

inhibition of iodide transport in thyroid cells, neurotoxicity and inhibition of protein

phosphatase (Sauleau, Retailleau, Vacelet, & Bourget-Kondracki, 2005).

About forty different polybrominated diphenyl ethers (PBDEs) have been isolated

from L. herbacea, L. chlorea, D. granulosa, D. dendyi, D. fragilis, and

Phyllospongia foliascens (Table 1). These molecules are reported to exhibit a variety

of bioactivities: antibacterial and antifungal properties, brine shrimp toxicity,

antimicroalgal activity, anti-inflammatory activity, and inhibition of a range of

enzymes implicated in tumor development such as inosine monophosphate

dehydrogenase, guanosine monophosphate synthetase, and 15-lipoxygenase. More

recently, PBDEs have been found to inhibit the assembly of microtubule protein, the

maturation of starfish oocytes, and also Tie2 kinase (Hanif, et al., 2007). This class

of halogenated compounds has been found in unrelated organisms such as the green

alga Cladospora fascicularis (Kuniyoshi, Yamada, & Higa, 1985), red alga

Ceramium tenuicorne, blue mussel Mytilis edilis (Malmvarn, Marsh, Kautsky,

Athanasiadou, Bergman, & Asplund, 2005), and unidentified sponge of the family

Callispongia (Capon, Ghisaberti, Jeffries, Skelton, & White, 1981), gastropterides

Sagaminopteron nigropunctatum and S. psychedelium often found on sponges

(whether they are true sponge feeders or not, is still debated) (Becerro & Paul, 2004),

Baltic salmon and marine mammals (Vetters, Stoll, Garson, Fahey, Gaus, & Muller,

2002). The fact that they are present in a wide variety of marine organisms makes us

think that they are more likely produced by a symbiont than the marine invertebrates

themselves.

Hence, it was suggested that the halogenated compounds from the sponges of this

genus are produced by cyanobacteria living in a symbiotic relationship with the host

sponge (Flowers, Garson, Webb, Dumbei, & Charan, 1998; Ridley, Bergquist,

Harper, Faulkner, Hooper, & Haygood, 2005).

The following table illustrates the different Dysidea species collected at various

geographical locations.


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Table 1 - Dysidea species collected in various geographical locations

Dysidea species

Localisation Compound Reference

Lamellodysidea herbacea

Indian Ocean; Zanzibar; Great Barrier Reef, Australia; Indonesia

Polybrominated diphenyl ether

Anjaneyulu, Rao, Radhika, Muralikrishna, & Connolly, 1996; Sionov, et al., 2005; Agrawal & Bowden, 2005; Handayani, et al., 1997; Hanif, et al., 2007

L. herbacea (with symbiotic cyanobacteria Oscillatoria spongeliae)

Papua New Guinea

Polychlorinated peptides; Polychlorinated ketide amino acid (Herbamide A); Polychlorinated tetrapeptide (dysidenin)

Flatt, Gautshci, Thacker, Musafija-Girt, Crews, & Gerwick, 2005; Clark & Crews, 1995

L. herbacea Great Barrier Reef, Australia; Pohnpei, FSM; Red Sea

Chlorinated metabolites; 5,5,5-trichloroleucine metabolite (Herbacic acid); Polychlorinated amino acid derivative; Polychlorinated pyrrolidinones

Dumdei, Simpson, Garson, Byriel, & Kennard, 1997; MacMillan & Molinski, 2000; Unson, Rose, Faulkner, Brinen, Steiner, & Clardy, 1993; Sauleau, Retailleau, Vacelet, & Bourget-Kondracki, 2005

L. herbacea Red Sea; Australia; Ethiopia

Polyhydroxysterols; Ichthyotoxic 9,11-secosterol (Herbasterol)

Sauleau & Bourget-Kondracki, 2005; Capon & Faulkner, 1985; Isaacs, Berman, Kashman, Gebreyesus, & Yosief, 1991

L. herbacea Yap State, Federated States of Micronesia

Betaines Sakai, Suzuki, Shimamoto, & Kamiya, 2004

�,�-disubstituted �-amino acid derivative (Dysibetaine)

Sakai, Oiwa, Takaishi, Kamiya, & Tagawa, 1999

Amino acid (neodysiherbaine A)

Sakai, et al., 2001

L. herbacea Palau; India; Fiji Sesquiterpene; Dysinin-type sesquiterpene

Sera, Adachi, Nishida, & Shizuri, 1999; Venkateswarlu, Biabani, Reddy, Chavakula, & Rao, 1994; Horton, Inman, & Crews, 1990

L. herbacea Ethiopia Dideoxyhexose Isaacs, Berman, Kashman, Gebreyesus, & Yosief, 1991

D. arenaria New Caledonia; FSM; Thailand

Sesquiterpene (9-hydroxyfurodysinin-O-ethyl Lactone); Sesquiterpenoids (arenarol & arenarone); Sesquiterpene ethers (arenarans)

Piggott & Karuso, 2005; Schmitz, Lakshmi, Powell, & van der Helm, 1984; Horton & Crews, 1995

D. arenaria Whitsundays Reef, Australia

Sterol ECTA Jacob, et al., 2003

D. avara Italy; Solomon Is.; Australia

Sesquiterpene hydroquinone (Avarol derivatives); Furanosesquiterpene (Thiofurodysinin)

Crispino, de Giulio, de Rosa, & Strazzullo, 1989; Alvi, Diaz, Crews, Slate, Lee, & Moretti, 1992; Capon & MacLeod, 1987


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D. fragilis South China Sea Chlorinated diketopiperizine (Dysidamide D)

Fu, Zeng, Su, & Pais, 1997; Su, et al., 1993

D. fragilis Mozambique Brominated diphenyl ether Utkina, Kazantseva, & Denisenko, 1987

D. fragilis Fiji; FSM Azacyclopropene & Derivatives; 2H-Azirines

Molinski & Ireland, 1988; Salomon, Williams, & Faulkner, 1995; Skepper & Molinski, 2008

D. fragilis Spain; Venice; Black Sea; Oahu

Furanosesquiterpenoid; Sesquiterpenes; Steroids; Polyhydroxylated sterols; Sesquiterpenoid bicyclo [3.3.1] nonane aldehyde lactone

Marin, Lopez, Esteban, Meseguer, Munoz, & Fontana, 1998; Aiello, Fattorusso, Menna, & Pansini, 1996; Milkova, Mikhova, Nikolov, Popov, & Andreev, 1992; Schulte, Scheuer, & McConnell, 1980

D. etheria Bermuda �,�-Epoxy �-Lactone Schram & Cardellina, 1985

Polyhydroxylated sterols West & Cardellina, 1988

Indoles Cardellina, Nigh, & VanWagenen, 1986

Sesquiterpene lactone (furodysinin lactone)

Grode & Cardellina, 1984

Furanosesquiterpenes (Nakafurans)

Cardellina & Barnekow, 1988

D. etheria Caribbean Sesterterpene (Dysidiolide) Takahashi, Dodo, Hashimoto, & Shirai, 2000 Gunasekera, McCarthy, Kelly-Borges, Lobkovsky, & Clardy, 1996

D. fusca New Caledonia (East Coast)

Drimane sesquiterpene Montagnac, Martin, Debitus, & Pais, 1996

D. chlorea Yap State, FSM Polychlorinate diketopiperazines (dysidamides I-T)

Fu, Ferreira, Schmitz, & Kelly-Borges, 1998

Sterol

D. chlorea Okinawa Sesquiterpenes (Heterumadysins A-D)

Ueda, Kadekaru, Siwu, Kita, & Uemura, 2006

D. cinerea Dahlak archipelago, Eritrea (Red Sea)

Sesterpenes (Bilosespens A & B)

Rudi, Yosief, Schleyer, & Kashman, 1999

D. frondosa Pohnpei Sesquiterpene hydroquinone deriv.

Patil, et al., 1997

D. cristagalli Spirits Bay, Northland, New Zealand

Sesquiterpene quinones McNamara, et al., 2005

D. amblia CA, USA Diterpenes Walker & Faulkner, 1981; Walker, Rosser, & Faulkner, 1984

D. dendyi Scott reef, North-west Australia

Brominated dibenzo-p-dioxins (spongiadioxin A & B)

Utkina, et al., 2001


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D. incrustans Coast of Tunisia, Mediterranean Sea

Polyoxygenated steroids (incrustasterol A & B)

Casapullo, Minale, Zollo, Roussakis, & Verbist, 1995

D. reformensis Gulf of California, Pacific Ocean, Mexico

Sesquiterpene hydroquinone

Carballo, Zubía, & Ortega, 2006 D. cachui

D. uriae Furanosesquiterpene (dendrolasin)

Dysidea sp. FSM; Philippines; Fiji

Polybrominated diphenyl ether

Fu & Schmitz, 1996; Zhang, Skildum, Stromquist, Rose-Hellekant, & Chang, 2007; Salva & Faulkner, 1990; Fu, Schmitz, Govindan, & Abbas, 1995

Dysidea sp. Philippines; Indonesia

Polychlorinated compounds (Dysideaprolines A-F & Barbaleucamides A-B); Polychlorinated dipeptide

Harrigan, Goetz, Luesch, Yang, & Likos, 2001; Ardá, et al., 2005

Dysidea sp. Palau; Red Sea; Australia; Philippines; Guam; Vanuatu; USA; PNG; South China Sea

Diterpenes of the dolabellane class; 14-membered macrolide; Diterpenes; Sesquiterpene ester (7-deacetoxy-olepupuane); Sesquiterpenes (Furodysin lactone & Pyrodysinoic acid); Drimane sesquiterpenoid; Sesquiterpene cyclopentenones and aminoquinones; Merosesquiterpenes; Meroterpenoid (Avinosol); Sesquiterpene hydroquinone (Bolinaquinone); Tetracyclic sesquiterpene - substituted quinone (Puupehenone)

Lu & Faulkner, 1998; Carmely, Cojocaru, Loya, & Kashman, 1988; Garson, Dexter, Lambert, & Liokas, 1992; Goetz, Harrigan, & Likos, 2001; Paul, Seo, Cho, Rho, Shin, & Bergquist, 1997; Giannini, et al., 2001; Pérez-García, Zubía, Ortega, & Carballo, 2005; Diaz-Marrero, et al., 2006; de Guzman, et al., 1998; Ciavatta, et al., 2007

Dysidea sp. Okinawa Benzothiazole-2-one analog

Suzuki, et al., 1999

Phyllospongia dendyi

Palau Polybrominated diphenyl ethers

Liu, Namikoshi, Meguro, Nagai, Kobayashi, & Yao, 2004

It was also noticed that most of the specimen collected and studied were obtained

from the tropical regions between the tropics of Cancer and Capricorn, as seen in the

next figure. Furthermore, there were no other distinguishable patterns of distribution

of Dysidea species with regards to the halogenated compounds isolated from them.

The figure below also defines the distribution of brominated and chlorinated

compounds obtained from the Dysidea species.


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Figure 7 - Distribution of brominated (green) and chlorinated (pink) compounds from

Lamellodysidea herbacea

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Sponges have been the focus of much recent interest due to the following two main

(and often interrelated) factors: they are a rich source of biologically active

secondary metabolites, and they form close association with a wide variety of

microorganisms.

Certain Demosponges are known to be associated with enormous amounts of

microorganisms contributing up to 40 – 60 % of the animal biomass, with density in

excess of 109 microbial cells per mL of sponge tissue, several orders of magnitude

higher than those typical for sea water (Hoffmann, Rapp, & Reitner, 2005). The vast

majority of these microorganisms are located extracellularly within the mesohyl

matrix (Becerro & Paul, 2004).

Investigation of these microorganisms through phylogenetic analysis and microscopy

has shown that there are members of at least eight different bacterial phyla

(Proteobacteria, Acidobacteria, Actinobacteria, Bacteroides, Chloroflexi,

Cyanobacteria, Gemmatimonadetes, Nitrospira), one bacterial candidate phylum

(Poribacteria) and one archaeal phylum (Crenarchaeota) (Grozdanov & Hentschel,

2007).

Although unrelated sponges can harbour similar microbial groups, it is more typical

for the symbiont communities to be specific for a given sponge species, irrespective


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of the collection site in a geographic region (Thacker & Starnes, 2003). It has been

shown that the microbial communities are passed on to the next sponge generation by

vertical transmission through the reproductive stages such as embryos and larvae

(Grozdanov & Hentschel, 2007).

The symbiont community can serve as a source of nutrition by transferring products

of metabolite processes to the host. For example, symbiotic cyanobacteria have been

shown to transfer organic carbon obtained through photosynthesis to the host

(Ridley, Faulkner, & Haygood, 2005). Other suspected benefits of the symbionts

include contribution to the structural rigidity of sponge and production of

halogenated natural products (Hildebrandt, Waggoner, Lim, Sharp, Ridley, &

Haygood, 2004).

Experimental support for the hypothesis of halogenated metabolites produced by

symbionts was gained when Unson and Faulkner (1993) investigated a specimen of

L. herbacea which contained the chlorinated compound 13-demethylisodysidenin

and were able to separate the auto-fluorescent cyanobacterium from the sponge cells

using a fluorescent activated cell sorter (FACS). Chemical examination revealed that

only cyanobacteria Oscillatoria spongeliae, the major symbiont of the sponge,

contained the chlorinated metabolite. Further experiments on a L. herbacea

containing chlorinated diketopiperazines (Flowers, Garson, Webb, Dumbei, &

Charan, 1998) and on a L. herbacea containing polybrominated diphenyl ethers

(Unson, Holland, & Faulkner, 1991) confirmed this hypothesis.

Additionally, a putative halogenase gene cluster involved in the chlorination of

barbamide has been identified and used to amplify a homolog sequence from L.

herbacea samples. Catalyzed reporter deposition fluorescence in-situ hybridization

(CARD-FISH) analysis showed that halogenase gene homologues are situated in the

cyanobacteria symbiont (Flatt, Gautshci, Thacker, Musafija-Girt, Crews, & Gerwick,

2005). Therefore O. spongeliae is likely the biosynthetic source of the chlorinated

metabolites. But there is still no evidence that this cyanobacterium is also responsible

for the production of brominated metabolites since a marine bacterium (not the

cyanobacterium O. spongeliae) isolated from the tropical Dysidea sp. has been

cultured on medium broth and has biosynthesized polybrominated diphenyl ethers

(Elyakov, Kuznetsova, Mikhailov, Maltsev, Voinov, & Fedoreyev, 1991; Voinov,

Elkin, Kuznetsova, Maltsev, Mikhailov, & Sasunkevich, 1991). There is still no


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report of O. spongeliae cultured independently from the host sponge producing these

metabolites. Furthermore, the Caribbean sponge Oligoceras violacea (syn. O.

hemorrhages) has been found to be heavily associated with O. spongeliae, but it

contains metabolites consisting of simple terpenes and dendrolasin as a major

constituent (Flatt, Gautshci, Thacker, Musafija-Girt, Crews, & Gerwick, 2005).

��. ��(��(��/��(��

Comparative biochemistry has been applied to sponge classification at frequent

intervals (Bergquist, 1978), but serious attempts to combine comprehensive

taxonomy with detailed chemistry have been rare. It is known that certain structural

types of sponge metabolites occur in certain sponge families or orders and targeting

of particular sponge groups can be useful in the search for new chemical variants

(Debitus, 1998). Therefore, it is important that taxonomic and chemical programs

proceed together.

Chemotaxonomy is not a new branch of science, and it has been explored previously.

However, interest in the subject with regards to sponges has declined over the past

few years due to certain drawbacks such as ambiguity in the exclusive presence of

chemical markers, availability of specimen, homology of the compounds, their

variability in occurrence, and whether the compound is produced by the organism

being studied or by its symbiont (Erpenbeck & Van Soest, 2006).

In the past, there have been a lot of misclassifications of many organisms, with

sponges being one of them due to their primitive features (Erpenbeck & Van Soest,

2006), and over the accumulated years, many stored specimen would have lost their

very basic morphological characteristics which were initially used to classify them.

By the effective use of chemotaxonomy, these specimens can be reclassified

correctly, providing better knowledge of the organisms and their actual taxonomic

position. Furthermore, the presence or absence of particular chemical markers from

similar groups of organisms can provide a link to the phylogenetic relationships

(Erpenbeck & Van Soest, 2006).

Moreover, it has been seen that only a few stable and suitable chemical markers (for

example sterols, protein banding, steroids, scalarane sesterterpenes, alkylpiperidines,

fatty acids, brominated derivatives, pyrrole-2-carboxylic acid derivatives and other

compounds) have been identified for the higher taxa of sponges (Erpenbeck & Van


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Soest, 2006), but it is still not very clear on the lower species level. Hence, it was

interesting to carry out this study on the sponges of the genus Dysidea, since

considerable information is available on the types of compounds present in these

sponges, but the classification of the genus Dysidea is very difficult to carry out due

to the plasticity of their morphological characteristics. Added to this, any interesting

compounds coming across during this study were subject to isolation and

characterization to further aid in the chemotaxonomical analysis.

��0 ��1��

Drug discovery, production and sales are a world-wide multi-billion dollar industry

that impacts all levels of modern life. The quality of life is greatly dependent on the

continued access to safe and effective drugs, whether it is in the form of antibiotics

for the treatment of infections; analgesics for pain relief; specific drugs for complex

ailments such as cancer, malaria, or blood pressure, and formulations for pest control

in the homes, agricultural products, and livestock. Most of these drugs mainly have

the active ingredient that is either a bioactive natural product or synthetic derivative

of these natural products.

Although terrestrial plants and organisms have provided great medicines in the past

and still manage to produce effective natural products for pharmacology, researchers

are now slowly moving their focus on marine derived natural products for research

and drug discovery. It has been reported by Walsh (2004) that biotechnology experts

believe that the prospect of finding a useful bioactive natural product from marine

sources is 500 times higher than from terrestrial sources.

Over the past two decades, marine invertebrates have been a rich source of

biologically active and structurally unique secondary metabolites (Faulkner, 2000)

having high potential to be developed for disease treatment (Debitus, 1998). Among

these potential pharmaceuticals include bengamides A and B (isolated from Jaspidae

marine sponges) first reported in 1986 by Quinoa, et al., having antihelminthic

activity; jaspamide (from the genus Jaspis) having herbicidal and fungicidal activities

(Peng, et al., 2003); and cytotoxic activities of arenastatin A, a cyclodepsipeptide

isolated from Dysidea arenaria (Newman & Cragg, 2004).


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HN

OH OH

OH O

O

N

OR

HH3C(H2C)12CO2Bengamide A: R = HBengamide B: R = CH3

OH

HN O

O

NH

O

N O

O

NH

BrH3C

Jaspamide

O

O

H

OO

O

H

NH

HNO

H HO

H

O

Arenastatin A

Figure 8 - Potential pharmaceuticals from marine sponges

Despite the large number of compounds and the high variety of structurally different

natural products isolated, before the year 2004, no marine derived natural product

had reached the commercial sector (Sladi� & Gaši�, 2006), although quite a number

of interesting marine-derived compounds were in the process of approaching phase II

and phase III clinical trials for cancer, analgesia, allergy and cognitive diseases

(Newman & Cragg, 2004; Haefner, 2003).

However, there were exceptions. After years of research, a medical break-through

was seen when scientists identified the cure for acute myeloid leukemia from a

natural product derived from the Caribbean sponge, Cryptotetheya crypta (Thakur &

Müller, 2004). By the year 2004, 1-beta-D-arabinofuranosylcytosine (also known as

ARA-C or Arabinosyl Cytosine) was a marine derived natural product to provide a

product for clinical use. This drug is marketed under the brand name Cytosar-UR by

Pharmacia & Upjohn Company (Thakur & Müller, 2004; Newman & Cragg, 2004).

There was also another product, adenine arabinoside (Ara-A), which was

commercialized by Glaxo Smith-Kline as an antiviral agent (Newman & Cragg,

2004; Haefner, 2003).

Ziconotide is another natural product of marine origin for clinical use granted

recently by the United States Food and Drug Administration (FDA) (Skov, Beck, de

Kater, & Shopp, 2007). It is the synthetic form of the natural product �-conotoxin

M-VII-A, a peptide derived from the cone snail Conus magus. This compound is an

N-type calcium channel blocker, and is used as an analgesic to improve chronic pain.


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Ziconotide is marketed under the brand name Prialt. Trabectedin, also known as

ecteinascidin 743 or ET-743, has been derived from the sea squirt Ecteinascidia

turbinata (Fayette, Coquard, Alberti, Ranchere, Boyle, & Blay, 2005). It is in the late

stages of clinical trials as a chemotherapeutic and anti-neoplastic agent, and is

developed under the brand name Yondelis by Zeltia and Johnson and Johnson as an

anti-tumor drug. ET-743 acts by binding in the minor groove of DNA, blocking

transcription factors activity, and trapping protein from the nucleotide excision repair

system. Thus, it blocks cells in the G2-M phase and causes cell cycle arrest.

��2 ��

Fungal infections are increasingly becoming important medical problems as more

strains are now found to be resistant to the classical medications. There are a limited

number of effective antifungal agents in use, even though a lot of progress is being

made in the development of different drugs for the treatment of systemic mycoses

(Sionov, et al., 2005). Human fungal infections are most frequently caused by

Candida and Aspergillus species (Sionov, et al., 2005), and also Cryptococcus

species (Wang & Casadevall, 1996).

Fungi of the genus Cryptococcus and Candida belong to the same family

Cryptococcaceae (Order Cryptococcales) (Mehrotra & Aneja, 1990). They are the

imperfect forms of basidiomycetous and ascomycetous yeasts. Candida albicans and

Cryptococcus neoformans are opportunistic fungal pathogens of this order, and are

greatly found in patients with compromised immune systems (Jacob, et al., 2003;

Wang & Casadevall, 1996; Rhome, et al., 2007). Such patients include human

immunodeficiency virus / acquired immuno deficiency syndrome (HIV/AIDS)

patients, cancer patients undergoing intensive chemotherapy, recipients of bone

marrow and solid organ transplants with acute neutropenia, and also individuals

undergoing prolonged corticosteroid and antibiotic treatments (Sionov, et al., 2005).

Invasive mycoses can be life-threatening for such patients even though some may

have low virulence.


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The Cryptococcus species are encapsulated basidiomycetous yeasts (Rhome, et al.,

2007). They are widely distributed in soil and on decaying plant materials (Mehrotra

& Aneja, 1990). Cryptococcus neoformans and Cryptococcus gattii are two species

pathogenic to humans, and cause life-threatening meningoencephalitis. It is readily

air-borne, and the lung is the primary site of infection, with consequent metastasis to

other parts of the body (Phaff & Macmillan, 1978). It affects the body by the

overgrowth of yeast-like cells that crowd the normal cells so that they do not

function properly. Cr. neoformans, the asexual state of Filobasidiella neoformans

(Mehrotra & Aneja, 1990), causes a vast majority of cryptococcoses and life

threatening infections in 6 – 8 % of AIDS patients (Wang & Casadevall, 1996;

Martinez, Garcia-Rivera, & Casadevall, 2001).

Some of the virulence factors of Cr. neoformans identified in recent studies include

the presence of a capsule, its ability to grow at 37 oC, and the production of melanin

(Wang & Casadevall, 1996). Cr. neoformans can catalyze the formation of melanin

from a variety of phenolic precursors due to the presence of the phenoloxidase

enzyme system. Melanin is said to contribute to virulence after in vitro studies

suggest that it protects fungal cells against oxygen- and nitrogen- derived reactive

intermediates produced by the host effector cells. Melanized cells are hence able to

survive longer since they cannot be efficiently killed in vitro by microglia and

macrophages, and also because they are less susceptible to Amphotericin B, which is

the first line of defence against fungal infections. This suggests that cryptococcal

infections are persistent even with fungicidal therapy.

Another virulence factor under current research is the biosynthetic pathways of the

cryptococcal sphingolipids (Rhome, et al., 2007). It has been identified in this study

that the pathways provide an extremely rich reservoir of sphingolipid molecules and

fungus-specific metabolising enzymes. These enzymes regulate many cellular

functions essential for the viability, virulence and pathogenicity of the fungus.

Further studies are still being carried out on this factor, but it can be said that the

sphingolipid metabolism can provide new and better pharmacological targets.


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Fungi of the genus Candida are yeast-like organisms with a strong mycelial or

pseudomycelial growth and multilateral budding (Mehrotra & Aneja, 1990). They

have been isolated from soil, plant and animal material, animal faeces, and water.

Fungal infections caused by Candida species are more common compared to

Cryptococcal infections. Candida species belong to the ascomycetes group of fungi

(Vallim, Fernandes, & Alspaugh, 2004), and Candida albicans is the most common

fungi found in this genus. They are also opportunistic fungi and target mainly

immunocompromised individuals (Sionov, et al., 2005), similar to Cryptococcus

neoformans. Ca. albicans grows as yeast in man (Taber & Taber, 1978), and may

also attack the skin on damp folds on the body, such as the vagina (vaginal thrush) or

the mucous membranes in the mouth (oral thrush) (Stevenson, 1967; Mehrotra &

Aneja, 1990; Moran, Sullivan, & Coleman, 2002), as well as cause cutaneous

candidiasis, bronchocandidiasis and pulmonary candidiasis (Mehrotra & Aneja,

1990; Hunter, Barnett, & Buckelew, 1978, Macmillan & Phaff, 1978).

Over the years, Candida albicans has developed resistance against antifungal agents

such as imidazoles and triazoles (collectively known as “azoles”) because HIV

patients had been kept on low dose prophylactic fluconazole therapy to prevent

opportunistic fungal infections (Jacob, et al., 2003; Moran, Sullivan, & Coleman,

2002). This developing resistance calls for the search of new bioactive compounds

that can help in the cure of these infections.

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The first anti-fungal agent to be identified was Nystatin, by Hazen and Brown in

1950 (Calderone, 2002). It is a polyene in nature and has since encouraged the

discovery of other polyene-type antifungal agents, including amphotericine B. The

increase in fungal diseases later called for the development of azoles (Calderone,

2002). Azole-based antifungals consist of a range of major clinically important and

useful drugs, such as miconazole, fluconazole, ketoconazole, and itraconazole.

Many of the drugs currently available have undesirable side-effects and may be toxic

to some extent (Sionov, et al., 2005). The steady rise in the occurrence of Candida

infections over the years (Calderone, 2002), and the difficulty in treating


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Cryptococcus and Candida infections calls for the discovery and design of new drug

targets which can be more efficient and effective in treating such life-threatening

fungal infections (Sionov, et al., 2005). The fact that the limited number of antifungal

drugs that are available have restricted sources, and that they have similar modes of

activity makes it important to search for new agents that exhibit antifungal activity

with different modes of activity or that affect different sites of the fungal cells.

Taking this into account, recent studies (Gelb, et al., 2003; Vallim, Fernandes, &

Alspaugh, 2004) have been carried out to explore gene sequences and enzymes of

microbes as new targets for the development of therapeutics. Genetic studies have

shown that the gene coding for protein N-myristoyl transferase (NMT), is essential

for the viability of a range of species including Candida albicans, Cryptococcus

neoformans, Sacharromyces cerevisiae, and Drosophila melanogaster (Gelb, et al.,

2003). Inhibitors of NMT exhibit both selectivity and specificity in the treatment of

such fungal infections. This protein is found to be indispensible for most eukaryotes,

hence making it a candidate as drug target for human pathogens on the condition that

specific reagents can be developed, which are selective against the pathogen rather

than the human enzyme.

Another enzyme, the protein farnesyl transferase (PFT), is well established in anti-

tumor studies. Inhibitors of PFT are able to arrest the growth of transformed cells and

cause tumor xenographs (tissue transplant from one species to an unrelated species)

to shrink (Gelb, et al., 2003). They exhibit known pharmacokinetic properties, and

low cytotoxicity to humans when the therapeutic doses are exceeded. It has been

seen in a study (Vallim, Fernandes, & Alspaugh, 2004) that pharmacological

inhibition of this enzyme can cause dose-dependant cytostasis and prevention of

hyphal differentiation of Cr. neoformans. Hence, the RAM1 gene which codes for

the �-subunit of PFT is essential for the viability of Cryptococcus neoformans.

It has also been seen that in ascomycetous yeasts, such as Candida albicans and

Saccharomyces cerevisiae, PFT works with another enzyme geranylgeranyl

transferase 1 (GGTase-1) to partially compensate the absence of each other, thus

preventing any fatal effects to the fungi due to their absence (Vallim, Fernandes, &

Alspaugh, 2004). On the other hand, in the distantly related basidiomycetous fungus

Cryptococcus neoformans, PFT is not readily complemented by any other enzyme,

therefore its activity is vital for the survival of the fungi. This result suggests that


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protein prenylation is an interesting target for the further development of anti-

microbial drugs.

In this study, we investigated the activity of crude sponge extracts against Cr.

neoformans and Ca. albicans to see if the growth of both the species of fungi is

inhibited or not. Based on the results obtained, suitable conclusions and hypotheses

about the mode of activity of the products have been suggested for future

investigations.

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This study is part of a major project of the Coral Reef Initiative in the South Pacific

(CRISP) program. The CRISP program is funded by major world organizations

including Agence Française pour le Développement (French Agency for

Development - AFD), French Global Environment Facility, United Nations

Foundation (UNF), French Ministry of Foreign Affairs, Conservation International

(CI), World Wild Life (WWF), Institut de Recherche pour le Développement

(Institute of Research for Development - IRD) and L’Initiative Française sur les

Récifs Coralliens (The French Coral Reef Initiative - IFRECOR). Apart from these

organizations, there are several other technical and institutional partners.

The main aim of this program as a whole is to provide initiative for the protection

and sustainable development of coral reefs in the South Pacific, and involves a wide

range of scientific disciplines of study. The CRISP program is divided into three

main components: Marine Protected Areas and Integrated Coastal Management;

Knowledge, Management, Rehabilitation and Development of Coral Ecosystems;

and Institutional and Technical Support, Communication, Coordination and

Extension. This project is a contributing part of the second component of the CRISP

program (Knowledge, Management, Rehabilitation and Development of Coral

Ecosystems).

One of the main objectives of this project is to analyze and compare the chemical

characteristics and constituents of sponges of the Genus Dysidea, which can be

related to the chemical taxonomy of these organisms; and to highlight the external

factors affecting secondary metabolites production in these sponges. The

intraspecific variability often observed in metabolite production may be related to

environmental factors, the physiological state of the organism or its genotype.


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Endosymbiotic microorganisms might also influence such a production, particularly

in sponges that contain large amounts of endobiotic bacteria. In the present project,

three sponges of the genus Dysidea, collected in Fiji, New Caledonia, Solomon Is.,

Vanuatu, Moorea and Mayotte are used as biological models.

Other objectives of this study were directed to isolate any interesting compound that

may or may not appear in literature and characterize it to obtain a fair idea of the

class of compound it belongs to. This data would then further aid in the

chemotaxonomic analysis of the Dysidea sponges.

The aim of the study is:

• To obtain baseline levels of the production of secondary metabolites (HPLC);

• To compare the chemical fingerprints and constituents of the three sponges

Lamellodysidea herbacea, Dysidea arenaria, and Dysidea avara (1H-NMR,

HPLC, LC-MS, TLC);

• To characterize the major compounds of the Dysidea sponges (NMR, MS,

UV);

• And to study the geographical variability (HPLC) at different scales within a

single species, Lamellodysidea herbacea.


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Most of the sponge specimens (Dysidea sp.) for this study were collected in New

Caledonia, with geographically different samples also from Vanuatu, Solomon

Islands and Fiji Islands, during a scientific expedition between 2007 and 2008, by

researchers of Institut de Recherche pour le Développement (IRD), Noumea, New

Caledonia.

The collected sponges were placed in sea water during transportation from the

collection sites to the laboratory. It was then freeze-dried before it could be used. The

identification of the specimens was primarily carried out on-site, and voucher

specimens were sent to the Queensland Museum (Australia) for confirmation by Dr

John Hooper.

Another set of Lamellodysidea herbacea samples was provided at the Laboratoire de

Chimie, des Biomolecules, et des l’Environnement (LCBE), University of Perpignan,

France. These samples were collected from French Polynesia (Moorea Island) and

the Departmental Collectivity of Mayotte, in the year 2002.

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Two extraction techniques were used in this project, as outlined by the research

teams worked with. Furthermore, the first technique (Technique 1) was more suitable

for sponge samples of large quantities, while the second technique (Technique 2) was

more convenient for the small quantity of sponge material available. The two

extraction techniques employed are detailed below. In both the techniques employed,

the organic extract obtained was used for this study.

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For the first set of sponge material (Dysidea sp.), collected by researchers of IRD,

Noumea, New Caledonia, approximately 20 g of the freeze-dried sponge material

was weighed out and left to mechanically stir overnight in 100 mL of a mixture of

ethanol and water (80:20). After the initial extraction, the mixture was filtered

(Whatman 103 – 9 cm) using vacuum and the residue was again left to stir in 100 mL

ethanol for 45 minutes. This was then filtered again and the step was repeated a final


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time. By this step all the ethanol-soluble compounds were expected to have leached

out of the sponge material.

The residue was then freeze-dried and stored separately, while the filtrate was dried

under vacuum using a rotavapor to obtain the organic residue of interest.

The following figure outlines the process of hydro-alcoholic extraction:

Scheme 1 - Hydroalcoholic extraction of freeze-dried sponge material

The residue obtained after drying the filtrate was then dissolved in 100 mL

dichloromethane (DCM), filled in separating funnel 1, and 100 mL of distilled water

was added to it. The content of the separating funnel was mixed carefully and the

layers were let to separate, after which, the organic layer (bottom layer) was drained

out and filled in a clean separating funnel 2. To minimize losses from imperfect layer

separation, 100 mL of water was added to funnel 2 and the two layers were mixed

properly again before obtaining the washed organic layer. Meanwhile, to the first

separating funnel 1, containing the aqueous layer, fresh 100 mL DCM was added,

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mixed and organic layer collected in funnel 2. The above steps were repeated twice.

Finally, the aqueous layers from funnels 1 and 2 were combined (ca. 200 mL) and

freeze-dried to remove the water, while the organic layer (ca. 300 mL) was combined

and desiccated by passing it through anhydrous sodium sulphate salt, before filtering

the extract. The filterate obtained thereafter was dried under vacuum using a

rotavapor until a viscous, thick gum texture was reached. It was then transferred to

labeled vials and air dried to completion, after which the vials were stored between 4 oC to –20 oC, until analysed.

The organic extract was labeled as the C extract, while the aqueous extract was

labeled as the B extract. For most of the analyses, the organic extract was used.

These crude extracts obtained were of varying quantities since different sponge

material enabled extraction at varying degrees. Generally, the L. herbacea extracts

were obtained between 3 – 6 % of the freeze-dried sponge material; while the D.

arenaria extracts were obtained between 0.8 – 1.5 %. See Appendix 1 for a list of the

yields and descriptions of the extracts.

The following scheme gives a general outline of the process of liquid-liquid

partitioning:


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Scheme 2 - Liquid-liquid partitioning of the Hydroalcoholic extract

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The second procedure used was for the Lamellodysidea herbacea samples from

Moorea Island and Mayotte due to the small quantities of freeze-dried sponge

material available.

Two grams of the freeze-dried sponge powder was weighed out and extracted using

DCM/Methanol (1:1) three times with 20 mL of solvent. Each time, the solvent was

added to the sponge material, ultrasonicated for ten minutes, then filtered on cotton

and dried using a rotary evaporator (rotavapor). Before the last drying however, one

gram of reverse-phase (C18) silica was added to the extract to adsorb the sample on

it.

After adsorption on the C18 silica and drying of the solvent, solid phase extraction

(SPE) was carried out on SPE Phenomenex Strata C18 cartridge (vol. = 18 mL; 2 g

C18 silica). The cartridge was first washed and activated using 10 mL of

DCM/Methanol (1:2), followed by conditioning it using 10 mL of Methanol (MeOH)

and then 10 mL of water.

The adsorbed C18 silica was then loaded on the column and washed with 10 mL of

MilliQ water to eliminated salt from the sample. The cartridge was left under

vacuum for 20 to 30 minutes to remove water from the column. Once dry, elution of

the extract was carried out using 9 mL of DCM/Methanol (1:1) under vacuum. The

eluted extracts were collected in 10 mL volumetric flasks and made to the 10 mL

volume with DCM/Methanol (1:1). This was labeled “Extract A”.

From Extract A, 1 mL of the sample was filtered for HPLC analysis (Extract A1) and

another 1 mL for LC-MS analysis (Extract A2). The remaining 8 mL of Extract A,

labeled as Extract A3 was then dried, weighed and stored for later use at -20 oC.

The following scheme briefly outlines this procedure:


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Scheme 3 - Cold-extraction procedure for small quantities of sample


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The initial TLC of the crude extracts was carried out on normal phase silica gel

(F254) plates with elution using 70 % heptane and 30 % ethyl acetate. Changes were

made to the eluent to 1:1 ratio for fractions obtained during the course of the study in

order to obtain better separation of the products.

The visualization reagents used included ultraviolet Gibb’s reagent for phenols;

vanillin phosphoric acid for steroids; sulfuric acid for organic compounds;

hydrochloric acid/ninhydrin solution for amines, amino acids, and amino sugars; and

dragendorff reagent for alkaloids and other nitrogen-containing compounds. The

procedures for preparation, staining and development of the visualization reagents

was followed closely as stated in “Dyeing reagents for thin-layer and paper

chromatography” (Merck, 1978). The compounds with double bond conjugation

were visualized by ultraviolet (UV) light at 254 nm and 366 nm.

However, in the case of hydrochloric acid/ninhydrin solution, the developed plate

was first sprayed with concentrated hydrochloric acid (HCl) and dried in air for 5

minutes after which it was placed between two glass plates and heated for 10 minutes

at 110 oC, the heating was then continued for another 10 minutes after removing the

top glass plate. This allowed the process for any peptide bond cleavage to take place

and to remove the excess HCl from the plate. After the 20 minutes of heating, the

TLC plate was then sprayed with ninhydrin solution and heated again for 10 minutes

at 110 oC, and this fixed any free nitrogen obtained from the peptide bond cleavage.

After the preliminary results obtained using TLC, the crude samples were analysed

using reverse-phase HPLC.

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The HPLC system used in this study was the Waters 2695 Separations module with

autosampler, coupled with Waters 996 Photodiode array detector, and the Polymer

Laboratories evaporative light scattering (PL-ELS) detector. The software used to

acquire data was the Waters Corporation Empower Pro (2002).


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All the crude extracts were put through a primary screening to see the elution of the

compounds of interest and for comparison with other samples. The crude extracts

were prepared at a concentration of 10 mg/mL.

The following conditions (Condition 1) were used for the primary screening:

Column: Phenomenex Gemini 5μ C6-Phenyl 110A (250 x 3.0 mm; 5 micron)

Solvent: Acidified Water (H2O) and Acetonitrile (ACN) (0.1% Formic Acid)

Gradient: 5 minutes at 90 % H2O; 90 to 0 % H2O in 30 minutes; 0 % H2O for 10

minutes

Flow Rate: 0.5 mL/min

Detection: Photo-Diode Array (PDA) detector (254 nm and 280 nm);

Evaporative Light Scattering (ELS) detector

After the primary screening, the HPLC conditions were adjusted to obtain better

separation of the peaks. The following conditions (Condition 2) were adapted for

further analysis:

Column: Phenomenex Gemini 5μ C6-Phenyl 110A (150 x 3.0 mm; 5 micron)

Solvent: Water (H2O) and Acetonitrile (ACN) - pH adjusted to 2.2 with Formic

Acid

Gradient: 50 to 0 % H2O in 15 minutes; hold at 0 % H2O for 15 minutes

Flow Rate: 0.4 mL/min

Detection: PDA detector (240 nm and 280 nm); ELS detector

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Mass analysis was carried out at the Laboratoire de Pharmacochimie des Substances

Naturelles et Pharmacophores Redox, UMR-152-IRD-Université Paul Sabatier,

Toulouse (France).

The liquid chromatography – mass spectroscopy (LC-MS) system used was the

Finnigan LCQ DECA XP Max with positive and negative electrospray (ES) and

atmospheric pressure chemical (APC) ionization techniques, coupled to an ion trap

analyser, with dual pump system.


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For this study, the ionization technique employed was the atmospheric pressure

chemical ionization (APCI), and data was obtained using both positive and negative

modes of analysis. The APCI technique was used because initial analysis with

electrospray ionization (ESI) did not provide appropriate mass profile. As for the

liquid chromatography part, the same column and conditions were used for the

elution as that for the HPLC condition 2. However, instead of the ELS, the column

was connected in series with PDA detector and the Finnigan APCI mass analyser.

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1-D and 2-D NMR analyses were carried out at the Laboratoire de Chimie, des

Biomolécules et de l’Environnement (University of Perpignan, France) and

Laboratoire de Pharmacochimie des Substances Naturelles et Pharmacophores Redox

(UMR-152-IRD-Université Paul Sabatier, Toulouse, France).

The NMR system used in Perpignan was the 400 MHz Delta NMR System, while the

system used in Toulouse was the 300 MHz Bruker NMR spectrometer system.

Preliminary proton NMR spectra of the crude extracts were obtained using the 300

MHz system and the isolated compounds were analysed for 1-D and 2-D NMR with

the 400 MHz system.

Based on the solubility of the samples, suitable NMR solvent was chosen for the

analyses. Generally, for most of the samples analyzed in the 300 MHz spectrometer,

deuterated chloroform (CDCl3) was used, but in some cases, deuterated methanol

(MeOD) or deuterated dimethyl sufoxide (DMSO-d6) was required. The analyses of

the isolated compounds on the 400 MHz system were carried out with deuterated

acetone (CD3COCD3 or acetone-d6).


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Fractionation Set 1:

Normal phase flash chromatography of 220 mg of Dysidea species R3141C was

carried out using the Ana-Logix Bottomless Solvent Reservoir (solvent pump 0962-

1). The column used was the SuperFlash SF10-4g Sepra Si-50 (1369600) – 5μm,

60Å, 9.4 x 1.5 cm. It had a mass of packing of 4 grams and a column volume of 4.8

mL, and the flow used during fractionation was 12 mL/min.

A solid deposition of the sample was made by adsorbing the extract onto minimum

amount of normal phase silica gel. The sample was eluted using 100 % heptane,

followed by 90/10 – heptane/ethyl acetate, 50/50 – heptane/ethyl acetate, 100 %

ethyl acetate (EtOAc), 50/50 – EtOAc/methanol, and 100 % methanol (MeOH). The

final washing was carried out using an 84/16 mixture of dichloromethane/MeOH. A

total of 8 fractions were collected in pre-weighed round bottom flasks (RBF), and

dried under vacuum rotavapor at 35 oC.

TLC analysis on the fractions was done using normal phase (NP) silica gel TLC

plates backed with aluminium. Elution was carried out using 70/30 – heptane/EtOAc,

and visualized with 50% sulfuric acid. HPLC analysis was also completed with the

Condition 2 used in the chemical screening.

Following the preliminary analyses, the fraction eluted with 50/50 – heptane/EtOAc

(R3141C-fr2b) was chosen for further fractionation.

Fractionation Set 2:

80 mg of R3141C-fr2b was further fractionated using the same method and column

as in fractionation set 1. However, the solvent composition this time started with

75/25 – heptane/EtOAc, and gradually increased the percentage of EtOAc to 40 %,

50 %, 60 %, 75 %, and 100 %. The final washing was done using 100 % MeOH.

A total of 31 fractions were collected which were reduced to 13 after comparison

using TLC. The 13 fractions were then analysed using HPLC (Condition 2).

Selected fractions from the two sets were sent for proton NMR and LC-MS analysis

in Toulouse. Following data analyses, four fractions (fr. 8, 9, 10, and 11) from the


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second set of fractionation were selected for compound purification using reverse-

phase semi-preparative HPLC.

Purification of Compound:

The HPLC system used for semi-preparative purification of the sample R3141C-2-

fr8-11 was the Waters 1525 Binary HPLC pump with manual injector, coupled with

Waters 2487 dual wavelength (�) absorbance detector. The software used to acquire

data was the Waters Corporation Breeze version 3.20 (2000).

The following conditions were used for the fractionation:

Column: Phenomenex Gemini C6-Phenyl 5μ

Dimensions: 250 x 10 mm

Flow Rate: 5 mL/min

Solvents: Water and Acetonitrile

Gradient: 40 % water for 30 minutes; 40 – 0 % water in 2 minutes; Hold at 0 %

water for 10 minutes.

The combined fractions (R3141C-2b-fr. 8, 9, 10, and 11) was dissolved in minimum

amount of MeOH and injected. After elution, a total of 7 fractions was collected and

analysed using analytical HPLC (Condition 2) and TLC.

The appropriate products were selected and sent to the University of Naples in Italy

for further analysis.

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Flash chromatography fractionation similar to the fractionation set 1 of sample

R3141C was carried out with selected L. herbacea samples. Elution solvent was

adjusted according to the samples using heptane and EtOAc. Fractions were air-dried

overnight under ventilator. Analyses were carried out using TLC and HPLC

(Condition 2).

Recrystallization:

For separating the crystals from the mixture, small amounts of hot acetonitrile (ACN)

was added to dissolve the fraction, and let to cool in an ice bath with the vial


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covered. Over time, the crystals formed and settled to the bottom, after which the

supernatant was decanted and the procedure was repeated.

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The fungal strains were cultured on Sabouraud dextrose agar prepared at a

concentration of 45 g/L. The medium was prepared as follows:

• Medium powder was suspended in de-ionized water, mixed carefully, and

brought to boil with constant mixing, until the solution turned translucent.

• It was then dispensed into bottles and slants as required while it was still hot

and in liquid state; and autoclaved for 15 minutes at 118 oC.

• After taking the medium out of the autoclave, it was left at room temperature

for 15 seconds and then transferred into a thermostatically controlled water

bath set at approximately 45 – 50 oC. This condition was maintained until the

medium was used.

• For use at a later date, the medium was refrigerated and turned into liquid

state in a thermostatically controlled water bath just before use.

• The tubes containing the media were left to cool in a slanted position.

The sterile petri-dishes to be used were first passed under ultra-violet (UV) light for

15 minutes to ensure that there was no contamination, and then the medium was

poured into the dishes. It was left to cool and settle for about 1 hour, after which the

medium was again passed under UV light for 15 minutes. The petri-dishes were then

closed, inverted, and stored in the refrigerator until used.

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Anti-fungigram discs saturated with the sponge extracts were prepared for the

analysis. Each disc had the capacity to hold 10 μL of the sample at a time and the

dosage to be tested was 1 mg of sample per disc. Hence, the samples were prepared

at a concentration of 10 mg/mL, and each sample was loaded 10 times on the disc,

giving enough time for the solvent to evaporate in between loads. The fractionated


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and purified compounds were loaded at a lower dose (0.1 mg per disc) compared to

the crude extracts. All the tests were done in duplicates.

Anti-fungigram discs of the solvents used for the sample preparation were also

prepared, to act as control for the assay. Furthermore, the antifungal agent Nystatin

was also tested with the samples for reference purposes.

Anti-fungigram discs were prepared in sterile conditions to avoid contamination.

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The culture plates were marked at the bottom with evenly spaced spots each

containing reference numbers to be used for identifying the sample disc used on a

particular plate. Each plate contained seven spots; hence it could be used to test

seven extracts at a time. After marking the plates appropriately, the culture

suspension was prepared.

It was important that all the steps undertaken during the bioassay was done in a

sterile condition so that chances of contamination could be eliminated. The

inoculation loop was first heated until red-hot and introduced into the culture slant

touching at the medium void of fungal growth. This way, the excess heat that could

kill the microorganism, was transferred onto the medium and the loop was left cool

enough to pick out the culture unharmed. A loop full of the fungi from the culture

slant was taken and suspended in test-tube containing sterilized physiological water

(1 % sodium chloride (NaCl) solution). This was then vortexed to give an even

suspension.

The culture plates were inoculated using the spread plate technique. A sterilized

Pasteur pipette was used to transfer approximately 1 mL of the suspension onto the

medium and the plate was turned around gently to evenly spread the suspension. The

excess suspension was drained out using the pastuer pipette and discarded into a

beaker of disinfectant.

The prepared anti-fungigram discs were then placed at the allocated positions on the

culture plates. Fungal cultures incorporated with the anti-fungigram discs were then

incubated for 48 hours, with monitoring at 24 hours.


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Chemical analysis is the first approach to identify chemicals/compounds of interest

in any branch of chemistry. It involves a wide range of techniques including use of

chemical identifiers and reactions, and instrumental analysis. It also resolves issues

such as identification, isolation, structure determination, and quantification of

molecules. Chemical screening can be used to identify known compounds and isolate

new ones.

A total of 14 Lamellodysidea herbacea, 9 D. arenaria, and 1 D. avara from New

Caledonia and one L. herbacea from Moorea Island (for comparison) were screened.

Full details of the samples studied are provided in Appendix 1. The techniques used

for chemical screening included thin layer chromatography (TLC), high performance

liquid chromatography (HPLC), 1-D nuclear magnetic resonance (NMR)

spectroscopy, and liquid chromatography - mass spectroscopy (LC-MS).

The aim of this screening was to acquire the chemical fingerprints (TLC and HPLC

chromatograms) of the sponge samples; to set up the chemical identity cards of the

different samples by identifying the compounds that are common within a particular

species; and to see whether the same compound(s) can be found in other species of

the Genus Dysidea. This could indirectly suggest the species that are more closely

related to each other and those that are far away in the phylogeny in terms of

chemical constituents of the sponges.

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Lamellodysidea herbacea has been one of the most studied species of the genus

Dysidea. It has been found in various locations around the world. The chemical

composition of this species was investigated to identify compounds that can be used

as chemical markers for chemotaxonomy of Dysidea sponges.

3.1.1.1 Thin Layer Chromatography

Thin layer chromatography (TLC) is the primary step in identifying the main groups

of compounds present in the sample of interest, and to obtain a general profile in

order to view the similarities and differences of the chemical composition of the


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samples. This is achieved by use of different visualization reagents which are

specific for particular functional groups, such as phenols, amines, double bond

conjugations, etc.

The standard compounds used as reference spots for TLC were cholesterol and a

tetrabromo-diphenyl-ether (4Br-OH-OMe-PBDE). The 4Br-OH-OMe-PBDE (3,5-

dibromo-2-(3’,5’-dibromo-2’-methoxyphenoxy)phenol) was chosen as standard

compound for the analysis due to its availability, and also because it was isolated

from L. herbacea collected previously from one of our sampling sites (Moorea

Island).

O

OHO

Br

Br

Br Br

3,5-dibromo-2-(3',5'-dibromo-2'-methoxyphenoxy)phenol(4Br-OH-OMe-PBDE)

HO Cholesterol

12

34

561'

2'3'

4'5'

6'

Figure 9 - Compounds used as standards for TLC analysis

L. herbacea indicated the presence of phenolic compounds due to positive

visualization with Gibb’s reagent (seen as a blue or purple spot, between Rf values of

0.27 and 0.57).

Furthermore, within the L. herbacea extracts from New Caledonia, two out of 14

samples showed differences in TLC profiles and indicated presence of the standard

compound (4Br-OH-OMe-PBDE) through direct correlation (blue spot with Gibb’s

reagent at Rf = 0.55). This visualization was interesting because the other L.

herbacea specimen collected in the same location (South of New Caledonia) did not

contain this compound and they all had similar TLC profiles. The L. herbacea

specimen from Moorea Island contained the 4Br-OH-OMe-PBDE as expected. The

figure below illustrates this point:


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Plate 1 - Differences in phenolic composition within L. herbacea from New Caledonia

(TLC eluent: 7/3 - Heptane/EtOAc; Reagent: Gibb's Reagent)

With vanillin phosphoric acid (VPA), all the L. herbacea from New Caledonia had

the same profile, and difference was noted only with the Moorea sample.

Dragendorff reagent and Hydrochloric acid/Ninhydrin solution failed to give

comparable profiles of the samples. It was evident from the TLC analysis of the

samples that two New Caledonian samples (R2259C and R2279C) contained the

standard compound due to the elution of the compound at Rf = 0.55. For

confirmation, the HPLC, NMR and mass profiles of the extracts were obtained.

3.1.1.2 High Performance Liquid Chromatography (HPLC)

The HPLC system was coupled with the photo-diode array (PDA) detector and the

evaporative light scattering (ELS) detector. The PDA detector can scan a wide range

of light absorption from 200 to 800 nm, hence making it suitable for detecting

compounds with chromophores and provide characteristic UV spectra. On the other

hand, the ELS detector is not specific; therefore it can be used to detect all types of

compounds including compounds lacking chromophores. Thus, coupling the HPLC

system with the PDA and ELS detectors enabled efficient total analysis of the

extracts.

The HPLC profiles of the Lamellodysidea herbacea samples were quite simple with

generally two or three main peaks detected by the PDA and ELS detectors. Added to

this, the retention time and UV absorption of the standard 4Br-OH-OMe-PBDE was

comparable to the samples suspected of having this compound. As seen in the TLC


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profiles, sample R2259C (L. herbacea from New Caledonia) had one extra peak for

4Br-OH-OMe-PBDE in its profile compared to the other samples of the same set,

while R2279C had only one peak corresponding to the standard PBDE.

Figure 10 - Three types of chemically different L. herbacea found in New Caledonia

(Above: Chemical difference seen in TLC visualization using Gibb's reagent. Below:

Chemical difference seen in RP-HPLC and the mass and UV absorption obtained for each

peak [HPLC Condition 2])

Each peak gave a distinctive UV absorption which could be used to identify the

compounds of interest. The standard 4Br-OH-OMe-PBDE absorbed UV light at

232.2 nm and 286.7 nm, and this was also seen in the peaks of compound 1 present

in L. herbacea samples R2259C and R2279C. Thus, LC-MS and NMR analyses were

carried out to determine the mass and orientation of the chemical constituents present


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in the analytes. The peaks corresponding to compounds 2 and 3 had slightly different

UV spectra with a 2nd absorption maximum at 291.5 nm.

3.1.1.3 Liquid Chromatography – Mass Spectroscopy (LC-MS)

Apart from the mass of a compound, mass analysis can give a fair idea of the type of

halogenation present, if any. The two main types of halogenation patterns seen

generally in L. herbacea are brominated and chlorinated isotopes. These two types of

atoms have a characteristic pattern of isotopic peaks. The chlorine atom has an

abundance ratio of 35Cl : 37Cl = 3:1, while the bromine atom has a ratio of 79Br : 81Br

= 1:1. This can be used as an easy way of identifying the presence of these halogens.

Furthermore, the pattern of the peak obtained can also indicate the number of

halogen atoms in the particular compound. (See Appendix 2 for the guide to identify

the pattern of halogenation and the number of halogen atoms in a compound.)

The LC-MS analysis was carried out using both negative ion (NI) and positive ion

(PI) mode APCI, and it was observed that the NI mode of ionization was more

informative than the PI mode. Therefore, based on the NI-APCI spectra of the

Dysidea samples, L. herbacea exhibited presence of Bromine isotopes corresponding

to the main peaks in the PDA scan. The pattern of spectra obtained for each peak

indicated the presence of 4, 5, or 6 bromo- compound.

Figure 11 - Pattern of halogenated cluster showing presence of 4, 5, and 6 bromine

atoms in the L. herbacea studied [HPLC Condition 2 + Finnigan APCI Mass analyser].


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This confirmed that the phenolic compounds indicated by the Gibb’s reagent in TLC

analysis of the L. herbacea samples were actually brominated phenols, and apart

from the standard 4Br-OH-OMe-PBDE, the samples also contain other types of

polybrominated diphenyl ethers (PBDE).

However, one complication faced with these results was that for each peak in the

PDA scan, there were two corresponding signals observed in the mass spectra.

Usually, the difference in mass between the two signals matched with the mass of

one bromine atom, and in few cases, it corresponded to a mass of 45 mass units.

Hence, either there were some rearrangements or fragmentations taking place in the

system during mass analysis; or there were two compounds present in the same peak

which could not be separated properly.

The second hypothesis does not quite hold true since this pattern was seen in all the

L. herbacea samples analysed. Furthermore, for two compounds having different

mass and structure, their retention times would differ to some extent, and they cannot

possibly be present in the same peak and retention time.

Therefore, the first hypothesis of rearrangements or fragmentations during the course

of the analysis seemed possible. This pattern has been previously observed in a study

by De Rijke, et al. (2003), where a comparative study was done on flavonoids using

LC-APCI-MS and LC-ESI-MS to determine their analytical performances. In this

study, it was seen that the APCI method, generally produced less fragments

compared to ESI method, thus making it suitable for crude extract analysis. Added to

this observation, it was noticed that with the use of formic acid in the eluent, there

tends to be a formic acid adduct [M+45]- forming with the negative ion mode of

ionization. The adduct formation is highly dependent on the acidity of the compound,

and so this pattern is not seen with all the polybrominated diphenyl ethers (PBDE) in

this analysis. According to their acidity, the formation of the formate ion with neutral

PBDE (monohydroxylated PBDE) successfully competes with proton abstraction

from the weakly acidic PBDE (dihydroxylated PBDE) to form an [M-H]- anion. This

would explain why a mass gain of 45 mass units was observed in some of the

Lamellodysidea herbacea samples only.


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Additionally, in another study by De la Fuente, et al. (2003), it was seen that some

PBDEs did lose a bromine atom to form [M-Br]- ion in mass analysis using NI-

APCI. Therefore, this also supports our first hypothesis.

Figure 12 – Pattern of rearrangement of compound observed in mass analysis [HPLC

Condition 2 + Finnigan APCI Mass analyser].

Thus, the L. herbacea samples studied generally contain penta- and hexa-PBDE,

with the exception of a few samples (2 New Caledonian L. herbacea [R2259C and

R2279C], and Moorea samples).

3.1.1.4 Nuclear Magnetic Resonance Spectroscopy (NMR)

NMR analysis of all the samples aided in confirming the observations made with the

other chemical analyses (TLC, HPLC and LC-MS). All the crude extracts of the

Dysidea samples were organic extracts; hence analysis was carried out in deuterated

chloroform (CDCl3). Isolated compounds were studied using acetone-d6.

From the proton NMR profiles of the extracts, it was seen that the L. herbacea

samples had interesting chemical shifts around 7 - 8 ppm. This shift corresponds to

the aromatic protons, and the profile matched with the PBDE possibility.


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The final conclusion was made using the proton NMR data (and in some cases, with

the 13CNMR and 2-D NMR spectra), whereby compounds were assigned to the

different chemical shifts to arrive to a specific structure.

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Since compound 1 (ref. Figure 10) gave the major peak in the HPLC profile of

R2259C - type 1 - (and the major spot in the TLC profile), it should show major

peaks in the proton NMR profile. This was confirmed due to the presence of 4 major

chemical shifts in the aromatic region of the profile, which corresponds well to the

presence of 4 protons in the structure with 4 bromine atoms, one hydroxy group and

one methoxy group. The chemical shifts of the hydroxy and methoxy groups were

observed at 5.40 ppm and 4.04 ppm, respectively.

Figure 13 - Compound 1: Mass spectrum [HPLC Condition 2 + Finnigan APCI Mass

analyser], 1HNMR [300 MHz; in CDCl3], and structure

The four protons gave doublets with coupling constant close to 2 Hz, which indicates

that the protons are meta-coupled.

Furthermore, for comparison purposes, the NMR profile of R2259C was compared to

the profile of another L. herbacea from Moorea (MAU-02-Dh1) and New Caledonia


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(R2279C), which contained only the standard compound, hence having clearer NMR

profiles. It was seen that the three profiles matched well in terms of chemical shift

pattern.

The figure below illustrates the NMR profiles of R2259C and the L. herbacea from

Moorea Island (Full NMR profiles of R2259C, R2279C and L. herbacea from

Moorea are given in Appendix 3a):

Figure 14 - 1HNMR profiles of organic extracts of R2259C and MAU-02-Dh1 (L.

herbacea from New Caledonia and Moorean Is., respectively), in CDCl3, 300 MHz

spectrometer.

The following table (Table 2) shows the comparison between the chemical shifts

obtained in this analysis for R2259C, R2279C, and the L. herbacea from Moorea

Island, with literature values:

Table 2 - 1HNMR chemical shifts obtained for Compound 1 in L. herbacea from

New Caledonia (R2259C and R2279C) and Moorea Is. (in CDCl3, 300 MHz)

Proton � R2259C

(NC) (ppm)

J for R2259C

(Hz)

� R2279C

(NC) (ppm)

J for R2279C

(Hz)

� MAU-02-Dh1

(Moorea) (ppm)

J for MAU-02-Dh1

(Hz)

� Literature

(ppm)*

H4 7.35 1.2 7.36 2.0 7.38 2.2 7.33

H4’ 7.45 1.9 7.46 2.2 7.48 2.2 7.40

H6 7.19 - 7.20 2.0 7.21 2.3 7.09

H6’ 6.78 2.1 6.80 2.2 6.83 2.2 6.54

-OH 5.40 5.08 5.34

-OMe 4.04 3.81 4.06 3.78

* (Fu, Schmitz, Govindan, & Abbas, 1995)


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A clear correlation could be seen for the chemical shifts obtained for the three

sponge specimens and the reference values available for the compound. Therefore, it

was finalized that Compound 1 in the New Caledonian sponges R2259C and

R2279C is 4Br-OH-OMe-PBDE, and it has the following structure and details:

O

OHO

Br

Br

Br Br

3,5-dibromo-2-(3',5'-dibromo-2'-methoxyphenoxy)phenol (4Br-OH-OMe-PBDE)

Mol. Wt. = 531.82 mu

M/z (APCI-NI mode) = 531.07 mu

TLC Rf = 0.55 - in 7:3 - heptane:EtOAc, Gibb's reagent

HPLC Rt = 31.429 min & 10.804 min (for HPLC condition 1 & 2, respectively)1HNMR shifts (CDCl3, 300 MHz): 7.36 ppm (H4, d, 2.01 Hz); 7.46 ppm (H4', d, 2.19 Hz); 7.20

ppm (H6, d, 2.04 Hz); 6.80 ppm (H6', d, 2.16 Hz); 3.81 ppm (-OMe); 5.08 ppm (-OH)

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56

2'1'

6'5'

4'

3'

Figure 15 - Compound 1 (3,5-dibromo-2-(3’,5’-dibromo-2’-methoxyphenoxy)phenol)

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Compound 2 was seen to be the most abundant product in all the type 2 L. herbacea

from New Caledonia, and it was the second most abundant compound in the type 3

sample as seen in the HPLC profile in Figure 10. The TLC profile could only be

used as an estimation of the location of the product since it was almost fused with

Compound 3. Therefore, only HPLC, mass spectra and NMR could be used for the

structure determination.

In the HPLC profile, compound 2 eluted at a retention time (Rt) of 32.631 and 10.519

minutes for HPLC conditions 1 and 2, respectively. As for the mass analysis, the

mass data obtained with NI-APCI showed two masses, 593.28 and 674.62 mu, with a

difference of 81.34 mu. This correlates to the loss of a bromine atom, in which case

the mass of 674.62 mass units is the pseudomolecular ion [M-H]- of the product, and

593.28 mu corresponds to [M-Br]- ion, which would have formed due to the

conditions involved in mass analysis.


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Furthermore, the mass spectra of compound 2 suggested the presence of 6 bromine

atoms. In the literature cited, only one PBDE could be found which had a molecular

weight of 675.58 mu, and 6 bromine atoms.

However, since the 1HNMR profile of the crude samples did not come out too well,

the L. herbacea sample R2252C was fractionated to see if a better profile could be

obtained. (Details of the fractionation and product isolation are provided in Section

3.3.2.1.)

Following fractionation, the new 1HNMR profile (at 300 MHz in CDCl3) contained 4

proton signals: two were doublets with coupling constants of 3 Hz, while the two

singlets at 6.01 and 6.04 ppm would be due to the hydroxy groups present on the

compound. This has been confirmed by the disappearance of the two peaks when

deuterated water (D2O) was added.

The molecule was also analysed for 13C NMR (at 100 MHz in acetone-d6) and 2-D

NMR (at 400 MHz in acetone-d6).

12 carbon signals and 2 proton signals were visible in the 13C NMR, 1H NMR and

the 2D NMR (HSQC and HMBC). Table 2 indicates the chemical shifts obtained

with the NMR analyses of compound 2.

Table 3 - 1H and 13C NMR shifts obtained for Compound 2

C #

300 MHz (CDCl3)

400 MHz (Acetone-d6)

�H Lit. in CDCl3 (ppm)* 1H (ppm)

13C (ppm)

1H (ppm)

HMBC

1 150.49 2 140.62 3 119.31 4 121.60 5 127.22 6 116.48 1’ 146.89 2’ 145.34 3’ 112.19 4’ 7.45 130.40 7.41 C2’; C3’; C5’; C6’ 7.21 5’ 111.74 6’ 6.68 117.12 6.85 C2’; C4’; C5’ 6.36

-OH 6.01 -OH 6.04

* (Norton, Croft, & Wells, 1981)


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The positioning of the protons can be justified:

• First, based on the C and H chemical shifts according to the substitution on

the aromatic ring. The low chemical shifts of the C bearing bromines allow us

to conclude that they are on positions 5’ and 3’. Furthermore, the hypothesis

of C bearing bromines on positions 4’ and 6’ is not consistent with the

chemical shifts of the C bearing H according to literature.

• Second, based on the long range H-C coupling data obtained. On the HMBC

spectrum, H6’ proton is coupled with C2’, C4’ and C5’, and the H4’ proton is

coupled with C2’, C3’, C5’ and C6’. The HSQC spectrum allows to correlate

H4’ (7.41 ppm) to the C appearing at 130.4 ppm, and the H6’ (6.85 ppm) to

the C at 117.1 ppm.

This arrangement is obtained for compound 2 illustrated in Figure 16.

Figure 16 illustrates the mass spectra, 1HNMR profile and the structure obtained for

Compound 2 (Full NMR spectra [1HNMR, 13CNMR, HSQC and HMBC] of

compound 2 is attached in Appendix 3b):

Figure 16 - Compound 2 (3,4,5,6-tetrabromo-2-(3’,5’-dibromo-2’-

hydroxyphenoxy)phenol): Mass spectrum [HPLC Condition 2 + Finnigan APCI Mass

analyser], 1HNMR [300 MHz; in CDCl3], and structure


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Hence, with the improved NMR profiles, compound 2 was assigned the structure of

the 3,4,5,6-tetrabromo-2-(3’,5’-dibromo-2’-hydroxyphenoxy)phenol found in

literature (Norton, Croft, & Wells, 1981).

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Compound 3 was the second abundant compound found in type 2 samples. It was

isolated together with compound 2 but could not be purified properly; hence it still

contained traces of compound 2. Thus, the HPLC and NMR profiles of compound 3

contained a mixture of compounds 2 and 3.

From the NI-APCI mass spectra, compound 3 had a pseudomolecular ion mass [M-

H]- of 594.77 mu, and five bromine atoms present in the product.

In the proton NMR profile of the product, there were only three signals observed:

two meta-coupled doublets, and one singlet. Eight possible structures exist for a 5Br-

PBDE with a mass of around 595 mu, and two meta-coupled protons.

These eight possibilities were isomers of each other and three have been isolated

previously in independent research by Hanif, et al. (2007) and Fu, Schmitz,

Govindan, & Abbas (1995) from Dysidea sponges.

Proton and carbon chemical shift data of compound 3 were very similar to compound

2 except for the carbon bearing the 3rd proton at 128.54 ppm and the corresponding

singlet at 7.61 ppm. After correlation of the chemical shifts and comparing them with

the literature, compound 3 was assigned to be 3,5,6-tribromo-2-(3’,5’-dibromo-2’-

hydroxyphenoxy)phenol. This compound has been previously found in literature

(Norton, Croft, & Wells, 1981).

The chemical shifts obtained for compound 3 are provided in the following table:


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Table 4 - 1H and 13C NMR shifts obtained for Compound 3

C # 300 MHz (CDCl3) 400 MHz (Acetone-d6) �H Lit. in CDCl3 (ppm)* 1H (ppm) 13C (ppm) 1H (ppm)

1 151.54 2 139.88 3 117.54 4 7.61 128.54 7.64 7.51 5 124.01 6 115.47 1’ 147.12 2’ 145.28 3’ 112.15 4’ 7.47 130.26 7.41 7.41 5’ 111.70 6’ 6.68 117.02 6.74 6.50

*Norton, Croft, & Wells, 1981

The following figure illustrates the mass spectra, NMR profile and structure obtained

for compound 3 (Full NMR profile of compound is provided in Appendix 3c):

Figure 17 - Compound 3 (3,5,6-tribromo-2-(3’,5’-dibromo-2’-hydroxyphenoxy)phenol):

Mass spectrum [HPLC Condition 2 + Finnigan APCI Mass analyser], 1HNMR [300

MHz; in CDCl3], & Structure


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Overall, the three compounds identified in L. herbacea were closely related diphenyl

ethers with varying numbers of bromine, hydroxyl, and methoxy substituents.

Appendix 4 illustrates the 1-D and 2-D NMR shifts and the yields obtained for all the

compounds found in this study.

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Dysidea arenaria has been noted to produce sterols (Jacob, et al., 2003),

sesquiterpene lactones (Piggott & Karuso, 2005), and sesquiterpene ethers (Horton &

Crews, 1995). In this analysis, however, it was difficult to identify particular

compounds due to the complex analytical profiles obtained.

In the TLC profile of the D. arenaria samples, two to three main groups of

compounds could be identified in the extracts. This was visualized with the help of

vanillin-phosphoric acid reagent and seen as pink or black spots. Furthermore, with

long-wave UV light (366 nm), it was possible to conclude that there were fluorescent

compounds also present in the extract.

Plate 2 - TLC profile of D. arenaria from New Caledonia after visualization using

vanillin phosphoric acid and UV light (360 nm). (Eluent: 7/3 - Heptane/EtOAc)

However, this failed to register clearly in the HPLC profile of these extracts. The

PDA scan gave a lot of peaks hence one peak could not be compared to another, and

in the ELS scan, three clusters of peaks could be observed but there was a lot of

noise registered as well. Overall, the HPLC profiles of all the D. arenaria samples

from New Caledonia were quite similar with differences in their peak intensities. In


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the mass spectra of these extracts, the masses obtained did not match with the masses

of D. arenaria products reported in literature.

Furthermore, looking at the proton NMR profile of D. arenaria, most of the peaks

were found to have chemical shifts between 0.5 to 3.5 ppm, and there were no peaks

observed in the aromatic region. It can thus be supposed that the main compound(s)

present in the D. arenaria are sterols, aglycones and other hydrocarbons lacking an

aromatic moiety. (See Appendix 5 for PDA + ELS + LC-MS + NMR)

Additional conclusions about the Dysidea arenaria species can not be made without

further study of the samples.

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Dysidea avara is a rich source of pharmacologically active ingredients such as

avarol, avarone, and their derivative compounds, etc., (Munro & Blunt, 2001; Marin,

et al., 1998; Shen, Lu, Chakraborty, & Kuo, 2003). According to Minale, Riccio, &

Sodano (1974), avarol has been found to be present at about 6 % of the sponge dry

weight, which would in turn make it the most abundant compound present in the

crude extract. However, the different chemical analyses failed to identify this product

in the sample R2082, which has been identified as Dysidea avara.

The TLC profile of the D. avara from New Caledonia had a complex profile similar

to the D. arenaria collected in the same area. This is also true for the HPLC profile

to some extent, because of the presence of similar clusters of peaks observed in the

two sample sets. For mass analysis, the only peaks detected were in the lipidic region

of the chromatogram. This confirmed the proton NMR profile since the main

chemical shifts were observed between 0.5 to 3.0 ppm. (See Appendix 6 for PDA +

ELS + LC-MS + NMR)


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Plate 3 - D. avara (R2082C) compared with reference [Avarol (Left) and D. arenaria

(Right)].

Thus, for D. avara, like the D. arenaria, confirmed conclusions could not be made

due to the complexity of the data obtained, but it is possible that the main compounds

present in this sample are sterols and other hydrocarbons.

When comparing all the species studied (L. herbacea, D. arenaria, and D. avara),

each profile had a distinct characteristic (See Appendix 7). Reverse-phase HPLC

analysis of the crude extracts confirmed the initial conclusions of the TLC results

that the separate species of Dysidea are chemically different also, and that members

of the same species do have similar chemical composition, with a few exceptions. In

comparison to L. herbacea, D. arenaria and D. avara did not exhibit presence of

phenolic or halogenated compounds, and the profiles obtained were not simple also.

A lot of compounds were seen to be UV active and were moderately hydrophobic,

but their quantities were too small to be detected properly.

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Chemical analysis helps to answer quite a lot of questions about the nature of a

compound or a complex mixture of compounds. Chemical identity cards of the

Dysidea sponges have permitted to see that the different species have specific

chemical profiles and that it is possible within a small location (New Caledonia) to

differentiate the sponges at the species level, according to their chemical fingerprints.

Meanwhile, it is not always possible, depending on the complexity of the chemical

fingerprints, to compare and analyze the sponge molecular content. In this study,


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only the L. herbacea showed simple fingerprints that could be easily analyzed: the

main compounds identified in the L. herbacea samples were polybrominated

diphenyl ethers (PBDE).

The halogenated compounds present in L. herbacea can generally be divided into

two chemotypes: the chloro-chemotype and the bromo-chemotype. This is because,

after observing the several L. herbacea compounds from literature, it was noticed

that when one type of halogenation exists in a sponge metabolite, the other type is

not present, and vice versa. All the New Caledonian L. herbacea samples analyzed in

this study contained polybrominated diphenyl ethers and not chlorinated peptides, so

they belong to the bromo-chemotype of compounds.

The production of the halogenated compounds is suspected to be the result of

symbiotic cyanobacteria living in the sponge tissues, and not the sponge itself. The

difference in the halogenations pattern is correlated to the presence of particular

strains of morphologically similar cyanobacteria, Oscillatoria spongeliae. The

different strains of cyanobacteria have not been found to co-exist on the same

sponge. Therefore, the PBDE’s found in this study can not be used as biomarkers for

sponge chemotaxonomy until it is proven that the compound is actually produced

only in the presence of Lamellodysidea herbacea. More specific compounds will

need to be searched for in order to successfully find a link between the classical

taxonomic technique and chemotaxonomy. Thus, for further study on

chemotaxonomy of this group of sponges, the chemical constituents of both the

sponge and the symbiont will need to be analysed; together and separately. Added to

this, more focus will need to be placed on obtaining sponge-specific compounds

from the sponges, such as sterols, since most are produced by the sponges

themselves.

Overall, the extracts from New Caledonia exhibited three types of profiles: type 1 L.

herbacea contained mainly compound 1 - 4Br-OH-OMe-PBDE (which corresponded

to the standard compound used in the analysis); type 2 was noted to contain two

major compounds, compounds 2 and 3 - 6Br-2OH-PBDE and 5Br-2OH-PBDE,

respectively; while type 3 included the two compounds found in type 2 L. herbacea

as well as the major compound in type 1 sample. Whether these differences in the

sponge molecular content are due to biotic or abiotic factors is an important question

that will be discussed in the next section, including results from a larger geographical


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location. However, the chemical analysis of different species of the Genus Dysidea

shows that variations in chemical composition of sponges exist which make them

exclusive to their species. This is a very important step towards chemotaxonomic

analyses.


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The second aspect of this project was to compare the different ecological niche and

geographical locations the sponges were collected in, and if there were similarities or

differences in the pattern of the constituents present in the samples. Comparisons

were made based on the availability of the samples. The three Dysidea species

focused on were Lamellodysidea herbacea, Dysidea arenaria and Dysidea avara,

and the geographical locations studied were New Caledonia, Vanuatu, Solomon

Islands, Fiji Islands, Moorea Island in French Polynesia, and Mayotte. For

comparison of chemical constituents by geographical location, only Lamellodysidea

herbacea was focused on generally due to its availability, simplicity of composition

for comparison purposes, and due to time constraints.

The following figure outlines the six sampling regions for this study:

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Figure 18 - Collection sites of L. herbacea for geographical comparison (Source: Google

Maps)

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Sponges of the genus Dysidea can be found in many colors, shapes and sizes. They

can either be encrusting on hard coral or stone surface, or exist as free-forming

colonies in sediment gaining their stability using a hold-fast. They can also be found

at various depths depending on the substrate, food availability and chances of

survival.

The Dysidea sponges in this study were collected at various depths (from 1 meter in

the lagoon to 20 meters near the reef). Being filter-feeders, they were commonly


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found in clear waters, but some specimens were also found surrounded by sediment.

Full details of the specimens studied are provided in Appendix 8.

3.2.1.1 Lamellodysidea herbacea

Almost all of the L. herbacea collected in New Caledonia were green or pale green

in color with the base encrusting and spreading horizontally on hard coral surface.

Small leaf-like (lamellate) projections were visible coming out from the base, and

were generally vertical. This was reported by Austin, (2003), to be a form of

protection from sedimentation, since less surface area of the sponge is exposed to the

sediments, while the surface area for water intake would be maximum and free of

clogging.

Some specimens were found to be in competition for space with other sessile marine

organisms while others had dominated their habitats. In certain specimens, where

competition was not too much of a problem, vertical growth could be observed;

hence some samples had larger colonies than others.

Out of the L. herbacea collected in New Caledonia, two specimens were slightly

different in general appearance and environment than the others: samples R2259 and

R2279. R2259 was collected in an area which had some sedimentation, while R2279

contained a slightly different pigmentation and was surrounded by quite a few green

and red algae.

Similarly, small differences could be observed in the appearance of L. herbacea

collected in Fiji and Solomon Islands, especially in the pigmentation of these

sponges. Added to this, the Solomon Island sponge was collected in waters that were

slightly darker than other sites, and as for the Fijian L. herbacea, it was found

growing among other sea-weeds and algae.

For the samples from Moorea and Mayotte, specimens were collected in the lagoon

between the depths of 1 to 1.5 meters. They were found in clear waters close to hard

corals, and were not surrounded by algal or sea-weed growths.

3.2.1.2 Dysidea arenaria and Dysidea avara

The D. arenaria specimens collected in New Caledonia were grey in color and had

lobate finger-like projections coming out from the main stem. The colonies were

quite closely clustered, and they were found growing on sandy substrate, surrounded


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by sea-weed and algae. Some samples also had sedimentation in the water around

them.

The only D. arenaria obtained from Fiji (R3314), did not quite look like the samples

collected in New Caledonia. This species was pink in color, and had the appearance

of a perforated ball instead of digitate projections from the main stem. Furthermore,

this particular specimen grew out from the middle of a cluster of sea-weed.

For Dysidea avara samples, there was only one specimen each from New Caledonia

(R2082) and Fiji (R3203).

The Fijian sample was red to maroon in color when outside the water, and it had

rigid stems like that of hard corals. The ecological comparison of the two specimens

could not be completed due to lack of data about the ecology of these sponges.

3.2.1.3 Dysidea species (R3033, R3161, and R3141)

Three Dysidea sponges received from the Solomon Islands were unclassified species,

where two of these specimens (R3033 and R3161) were identified to be closer to

each other than the third sponge (R3141). Hence, the two similar species were

classed as Dysidea 4177 while R3141 was labeled Dysidea sp.

Sponge R3033 was purple in color with digitate projections as seen in hard corals.

However, they were not clustered together into one colony. The sponge was actually

scattered on a rocky surface, but still linked to each other. This species was present in

close proximity of algae and sea-ferns.

Sponge R3161 was also purple in color, with digitate projections branching out from

the main stem which was rooted to a rocky surface. This specimen was more colonial

compared to R3033. It was found in an area with less light, and the color of the

sponge out of the water was dark purple.

The last Dysidea sp. (R3141) was found growing on a rocky surface also inhabited

by other algae, sea-weeds and marine invertebrates, and it was dark maroon in color.

It had more of a leaf-like appearance, as if it were a marine plant.


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Geographical distribution of the Dysidea sponges was chosen as a part of this study

due to the fact that the sponges were collected in different geographical locations,

with each specimen providing a distinctive profile. Hence it was interesting to note

the chemical and genetic similarities and differences of these sponges within the

same region, between different regions, and if possible between the South Pacific

Ocean and the Indian Ocean.

Since L. herbacea was the only Dysidea species obtained from all the regions

studied, this analysis was focused only on this particular species.

3.2.2.1 Comparison of Lamellodysidea herbacea collected within New Caledonia

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Most of the L. herbacea sponges were obtained from the South of New Caledonia. It

was seen in the chemical screening (Section 3.1) of these samples, that there were

three types of L. herbacea: Type 1 containing mainly the 4Br-OH-OMe-PBDE; Type

2 having 5Br- and 6Br-2OH-PBDE; while the third kind (Type 3) having the two

compounds from type 1 and 4Br-OH-OMe-PBDE.

The following map shows the exact locations where samplings were done and where

the chemically different specimens were collected:


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Figure 19 - Map showing collection sites of L. herbacea in New Caledonia. (White: Type

1 sponges; Blue: Type 2 sponges; Yellow: Type 3 sponges)

It can be seen that the Type 3 and Type 1 samples were collected very near to the

mainland, and this could have been a factor in the different chemical patterns seen. A

closer look at the landscape close to the collection sites reveal that these samples

were collected near mountainous terrain. Hence, mineral and sediment wash-off from

the land could have enhanced or suppressed the production of particular compounds

in the two samples. This theory could be feasible since the other collection sites were

either far from land and on the reef, or close to land (not as close as R2259 and

R2279) but near flat terrain (see Figure 20).


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Figure 20 - Map illustrating the terrain near the collection sites of R2259 and R2279

(Source: Google Maps)

The geographical locations of particular sites are therefore also an important factor to

note when sampling in the same region because the slightest difference in conditions

can lead to differences in their chemical constituents (Mather & Bennett, 1992).

These invertebrates are quite sensitive to changing conditions around them, and

constant supplies of minerals and sediment can eventually cause them to change in

abundance and genetic make-up in the long term.

This point was also proven in a study by Maldonado, Giraud, and Carmona (2008),

where survival of asexually produced recruits of the demosponge Scopalina

lophyropoda was observed by exposing them to different levels of sedimentation. It

was seen during this study that the initial rate of mortality of the sponges was due to

sedimentation, but after a certain point, the sponges exhibited reversal of mortality

irrespective of sedimentation and had revived. This pattern was more evident in the

sponges placed in the harbor where the amount of silt was the highest. It was

concluded in this study that an unidentified genetic component was responsible for

the adaptation to sedimentation and thus, in the long term, with constant

sedimentation into their habitat, can eventually alter their genetic composition.

Hence, as perspectives for future work, new sampling of the L. herbacea can be done

in the region of the positions of R2259 and R2279 to see if samples around the


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vicinity have the same chemical profile or not. Studies can also be done on L.

herbacea exposed to different levels of sedimentation, to see if it is able to survive

the stress or not. If so, then the changes in chemical composition due to stress can

also be studied, thus proving our statement further. This study can be extended by

incorporating changes in genetic composition, if any, due to the exposure to stress.

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Selected samples used for obtaining the chemical profiles of the sponges were also

sent to a collaborative laboratory for phylogenetic mapping studies.

The analysis was carried out by sequencing the internal transcribed spacer (ITS)

region, since it is easy to amplify even from small quantities of DNA, and it has a

high degree of variation even between closely related species. The taxon set was

extended with Dysideidae from the collection of the Queensland Museum and

additional sequences from Genbank identified as Dysideidae (Erpenbeck & Van

Soest, 2006; Erpenbeck, Breeuwer, Parra, & Van Soest, 2006). A total of 74 taxa

with 644 characters were used to reconstruct the phylogeny. Character positions

which could not be aligned unambiguously were excluded from the alignment.

Phylogenetic reconstructions were performed with Bayesian Inference and

Maximum Likelihood Methods. The trees were rooted with Carteriospongia

foliascens of the family Thorectidae (Dictyoceratida).

The following taxonomy tree was obtained after analysis:


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Figure 21 - Taxonomy tree of Dysidea sp. after gene mapping (Courtesy of Dr. Dirk

Erpenbeck)

It was observed that most of the L. herbacea samples collected from New Caledonia

are genetically very closely related to each other. However, two L. herbacea

specimens from New Caledonia (R2258 and R2279) were seen to be slightly

distantly related to the first set of L. herbacea. Out of these two specimens, the

chemical profile and differences of R2279 correlates well with the phylogenetic tree

obtained of the samples. This however is not true for the samples R2258 and R2259

from New Caledonia. The chemical profiles of these two specimens suggest that

R2279 is more closely related to R2259 rather than R2258. This is quite opposite to

what is observed in the phylogenetic tree.

Since the genetic analysis was carried out using material from sponge tissue

complete with symbionts, phylogenetic study of the symbiotic cyanobacteria present

in these sponge samples is yet to be completed. It will be very interesting to see if the

chemical variations could be related to the genetic variations of the symbiont.

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However, the hypothesis of the environmental factors influencing the chemical

contents of the sponges is also plausible and needs to be studied.

3.2.2.2 Comparison of Lamellodysidea herbacea in other Geographical

Locations

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The total number of locations studied for this comparison was six, and the sites

included New Caledonia (as discussed earlier), Vanuatu, Fiji, Solomon, Moorea and

Mayotte Islands. Appendix 9 illustrates the six sampling sites, and the approximate

distances between each sampling site.

As observed earlier with the ecological comparison of the L. herbacea samples from

all the sites, it was evident that there were minor differences in the samples in terms

of appearance and the area of collection. The extracts were thus put through similar

chemical analyses as seen in Section 3.1, to be able to compare the samples in terms

of their chemical composition.


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The following figure illustrates the comparative HPLC profiles of the geographically

different L. herbacea with their finalized structures:

O

OHOO

OHOH

O

OHOH

O

OHO

O

OHO

O

OHO

O

OHOH

O

OHO

Br

Br

BrBr

Br

Br

Br

Br

Br

Br

Br

Br

Br Br

Br Br

Br

Br

BrBr

Br

Br

Br

Br

Br

Br Br Br

Br

Br

Br

Br

Br

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Br

Br

Br

Br

Br

Br

1 2

3 4

5 6

7 8

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Figure 22 - Geographical comparison of chemical constituents present in L. herbacea

Following the required chemical analyses, it was clear that the New Caledonian Type

2 L. herbacea had exactly the same chemical profile as that of the Vanuatu sample.

The similarity between these two samples can not be compared directly in terms of

geographical distance, since the two sites are separated by quite a large distance

(approximately 830 km). But, similar phylogenetic profiles of both sponge and

symbiont, or an adaptation to a very similar environment could explain such

chemical similarities. Unfortunately, the ecological environment could not be

compared in this case, since ecological data of the Vanuatu sample was absent.


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On the other hand, looking at the samples from Solomon Islands (R3147), and Fiji

(R3241), variations could be observed when both were compared to the New

Caledonian samples. From the TLC and HPLC analysis, it was concluded that all the

samples contained compound 2 (6Br-2OH-PBDE). The difference in the Solomon

Islands L. herbacea was that it lacked compound 3 (5Br-2OH-PBDE), and instead

showed the presence of compound 4 (5Br-OH-OMe-PBDE) and compound 1 (4Br-

OH-OMe-PBDE); the latter was also seen in the Type 1 and Type 3 L. herbacea

from New Caledonia. The details of structure determination are provided in Section

3.3. These results were confirmed using the LC-MS and 1-D and 2-D NMR spectra

obtained, and the extract was also fractionated to obtain clearer NMR profiles.

After fractionation of the sponge R3147C, the above mentioned compounds 1, 2 and

3 (4Br-OH-OMe-PBDE, 6Br-2OH-PBDE and 5Br-OH-OMe-PBDE) were confirmed

to be present in the sample. Together with these compounds, a fourth brominated

compound was also found in the fractions. The m/z of the compound was 688.89 mu

and it contained 6 bromine atoms. It resembled a 6Br-OH-OMe-PBDE. The 1HNMR

chemical shifts of this 6Br-OH-OMe-PBDE indicate protons that are meta-directed

(discussed in Section 3.3.2.2). However, due to low mass of product and impurities,

further analyses could not be carried forward.

The sponge sample from Fiji (R3241) had been initially classed as Lamellodysidea

herbacea but later it was reclassified to the species Dysidea lizardensis. After

thorough literature search, no further detail could be obtained for this new species. It

was seen that this sample also contained the brominated compounds found in L.

herbacea. Sample R3241 contains the two compounds found in the Type 2 L.

herbacea from New Caledonia – compounds 2 and 3 (6Br-2OH-PBDE and 5Br-

2OH-PBDE, respectively). However, it also contained another peak in the HPLC

profile which did not give a mass and the 1HNMR data at hand was not sufficient to

make any conclusions. Hence, the sample R3241C was also fractionated to see if

better profiles could be obtained for comparison and structure determination.

Presence of compounds 2 and 3 was confirmed in the sample after fractionation. A

third product, compound 8 (5Br-OH-OMe-PBDE) was also found to be present in the

sample and its structure was determined using spectral analyses (LC-MS and 1H and 13C NMR). It can be seen from this result that the D. lizardensis (R3241) studied is

quite closely related to L. herbacea.


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The results of the two fractionations are further discussed in Section 3.3 and spectral

details are provided in Appendix 10.

The five L. herbacea from Moorea Island had exactly the same chemical profiles and

contained mainly compound 1 (4Br-OH-OMe-PBDE), as seen with the Type 1

sample from New Caledonia. This compound was actually predicted since the

standard 4Br-OH-OMe-PBDE used in this study was also isolated from a Moorean L.

herbacea. The similarity between the Type 1 sample and the sponges from Moorea

can not be compared directly in terms of geographical distance since the two sites are

separated by quite a large distance.

The samples from Mayotte Island were exactly the same in their own profiles, but

when compared to all other samples, there was a clear difference in the pattern of

chemical profiles of these Indian Ocean sponges to that of the Pacific Ocean sponges

(from New Caledonia, Solomon Is., Fiji, Vanuatu, and French Polynesia). The L.

herbacea from Mayotte Island consisted of three main compounds, and upon further

investigation using LC-MS and NMR, it was found to consist of compound 6 – an

isomer of Compound 1 (4Br-OH-OMe-PBDE), compound 7 – an isomer of

Compound 3 (5Br-2OH-PBDE), and compound 8 – an isomer of compound 4 (5Br-

OH-OMe-PBDE). The details for structure determination of the compounds in the L.

herbacea from Mayotte are provided in Appendix 11.

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Several contradictory observations can be made from the phylogenetic tree:

• The L. herbacea from Vanuatu (R2271bis) is closely related to most of the L.

herbacea from New Caledonia when comparing both the chemical

composition of the sponge as well as its phylogenic characteristics. So the

results of the phylogenetic tree and the chemical analysis match perfectly.

• The L. herbacea from Fiji (R3241), which has been later identified as

Dysidea lizardensis, is also genetically and chemically not very different

from the other L. herbacea from New Caledonia.

• On the other hand, the L. herbacea from Solomon Is. (R3147), where the

same type of PBDEs have been found compared to the other L. herbacea,

seems genetically very different from the other L. herbacea. This feature does


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not correlate with the previous observations. Considering that PBDE are

suspected to be produced by the cyanobacterial symbionts, a close

relationship between PBDE and genetic mapping of the sponges can not be

confirmed without further analyses.

Another very interesting aspect noticed with the phylogenic study is that Dysidea

sponges from different regions form different clades of the taxonomy tree. This

feature is especially visible with the samples collected from the Solomon Islands.

The four Dysidea species of sponges from the Solomon Islands is clustered together

and distinct from the L. herbacea and D. arenaria collected from New Caledonia,

Fiji and Vanuatu.

Although a direct correlation has not been observed between the chemical profile and

the phylogenetic analysis of the Dysidea sponges in this study, chemotaxonomic

studies do not finish here. Further investigation will need to be carried out with focus

on compounds that are specific to certain groups of sponges, and to phylogenetic

analysis of the cyanobacterial symbionts associated with each sponge.

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The HPLC analyses of Dysidea arenaria samples from New Caledonia and Fiji

showed considerable differences in their profiles. The New Caledonian specimens

seemed to contain various compounds with slight UV absorption, none of them being

the dominant constituent; and with the ELS detector, three groups of compounds

were detected to be dominant, whereby none of the compounds were UV active.

However, in case of the Fijian specimen, three main groups of compounds were

detected by the ELS detector, and all the signals had corresponding UV absorption as

well.

Similarly, looking at the chemical analyses of the D. avara samples from New

Caledonia and Fiji, it was evident that, like in D. arenaria, the two samples had quite

different profiles in HPLC. The New Caledonian sponge gave a lot of small peaks for

PDA scan, and one main cluster of compounds was observed with many other peaks

equivalent to noise. However, with the Fijian D. avara, two main compounds exist as


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seen by the correlation between the PDA and the ELS scans of the extracts. (Details

of these correlations can be found in Appendix 12)

With TLC analysis, the Fijian samples of D. arenaria and D. avara also contained

phenolic compounds as visualized by Gibb’s reagent by a brownish-blue spot. These

were seen at Rf 0.11 and 0.21 for D. arenaria, and Rf 0.11 and 0.39 for D. avara.

However, due to the small quantities of the Fiji samples, and also because of time

constraints, further analyses could not be carried forward in order to determine the

exact point of chemical difference between the New Caledonian and Fijian D.

arenaria and D. avara sponges. As observed in the phylogenetic tree (Figure 21),

most of the D. arenaria samples collected from New Caledonia are more closely

related to each other than the L. herbacea obtained from the same region. The

chemically different D. arenaria from Fiji is also genetically different.

Chemical analyses (TLC, HPLC, LC-MS, and 1HNMR) of Dysidea 4177 samples

(R3033C and R3161C) indicated presence of polybrominated diphenyl ethers

(PBDE), basically 4Br-OH-PBDE (3,5-dibromo-2-(2’,4’-dibromophenoxy)phenol -

in both R3033C and R3161C) and 5Br-OH-PBDE (3,4,5-tribromo-2-(2’,4’-

dibromophenoxy)phenol – only in R3161C).

O

Br

Br

OH

Br Br

O

Br

Br

OH

Br

Br

Br

Compound 93,5-dibromo-2-(2',4'-dibromophenoxy)phenol Compound 10

3,4,5-tribromo-2-(2',4'-dibromophenoxy)phenol

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Figure 23 - Brominated diphenyl ethers found in Dysidea 4177

The complete chemical screening details and structure determination is provided in

Appendix 13.

Three species of the genus Dysidea investigated previously have shown the presence

of brominated phenols, such as those described in Figure 23. These include L.

herbacea from Zanzibar (Sionov, et al., 2005), D. fragillis from Mozambique

(Utkina, Kazantseva, & Denisenko, 1987), and D. dendyi from Australia (Utkina,


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Denisenko, Virovaya, Scholokova, & Prokof'eva, 2002). It can be thus hypothesized

that due to the presence of brominated phenols in these Dysidea 4177 samples, they

may be linked more closely to L. herbacea, Dysidea fragillis, and Dysidea dendyi

than the other species. However, this hypothesis can not be proven until

classifications of these specimens are completed using morphological comparison

and genetic mapping.

As far as we know, the hypothesis of close links between Dysidea 4177 and L.

herbacea is not consistent with the genetic mapping of our extracts. As seen in the

phylogenetic tree (Figure 21), Dysidea 4177 from Solomon Is. are genetically

similar, but a little different from other Dysidea sp. from the Solomons, particularly

L. herbacea (R3147). On the other hand, they are very different from the L. herbacea

from New Caledonia. A large range study on the samples of L. herbacea, D. fragilis,

and D. dendyi from the Solomon Is. would be necessary to evaluate the chemical and

genetic proximity of these sponges.

From the different brominated diphenyl ether compounds observed in Dysidea

species, it was seen that there was a variety of the hydroxylated (-OH) or

methoxylated (-OMe) groups present in the molecules; apart from the number of

bromine atoms. Some compounds contained either 1 or 2 –OH’s or –OMe’s, while

others contained both –OH and –OMe. This pattern was not very particular of the

species of Dysidea or the location it was collected in. However, it was observed that

when molecules with 1-OH group were found, compounds with 2-OH groups were

not seen to be present (Handayani, et al., 1997; Zhang, Skildum, Stromquist, Rose-

Hellekant, & Chang, 2007; Fu & Schmitz, 1996). Nonetheless, this observation was

rejected in one study (Fu, Schmitz, Govindan, & Abbas, 1995), where these

compounds have been isolated together.

The chemical screening of Dysidea sp. specimen R3141C, indicated profiles that

were totally different from the other Dysidea species analysed in this study, and it did

not reveal any phenolic compounds by TLC either. This observation is surprising

since this sample is genetically close to the other Dysidea species from Solomon Is.

that contain PBDEs. Due to the lack of information about this species, it was chosen

for fractionation and product isolation, details of which are provided in Section 3.3.


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The ecological and geographical environments are major influences on the type and

amount of chemical compounds produced by a particular sponge. Due to their sessile

nature, these invertebrates tend to adapt to their constantly changing environments,

and eventually exhibit properties that are different when compared to the members of

the same species from other regions of the world. If these changes are not constant

throughout one particular region, variations seem to arise within the same region, as

seen with the New Caledonian sponges. This is another reason why chemotaxonomy

is difficult to apply to these invertebrates.

In order to determine the exact conditions due to which variations between the same

species occur in different geographical locations, more extensive studies need to be

carried out, where the sponge biology, ecology, and symbiosis can be analysed in

detail. Added to this, the geography, weather pattern and currents of the surrounding

environment should also be taken into account. This will allow better comparisons to

be made between organisms.


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Apart from the chemical analysis of the Dysidea sponges from New Caledonia, other

sponges of the genus Dysidea were also analyzed and certain constituents were

successfully isolated and characterized.

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The shape and appearance of the Dysidea species R3141 was intriguing since it was

not at all like the other sponges. Thus, it was interesting to determine the chemical

constituents of this particular sample. It was also chosen for fractionation since it

contained totally different chromatographic and spectral profiles compared to L.

herbacea, D. arenaria, D. avara and the two Dysidea 4177 species analysed in this

investigation. The general 1HNMR was interesting since most peaks were seen

scattered throughout the profile with major peaks seen in the region of 0.5 to 2.0

ppm. The other peaks were quite small but it could be seen that they were basically

of the same height, which would indicate presence of peptides or related compounds.

Furthermore, no other details were known about this sample from the chemical

analyses since it did not even register a mass in the LC-MS profile of the crude

extract.

Figure 24 - 1HNMR [300 MHz; in CDCl3] and HPLC [Condition 2; UV 254 nm]

profiles of R3141C


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A series of normal phase column chromatography and reverse phase semi-

preparative HPLC were required for the isolation of three compounds from this

species. (Details of the general isolation procedure are provided under

Methodology.)

The fraction (R3141C-fr2b) eluted using a 50/50 mixture of heptane and ethyl

acetate (EtOAc), was seen to contain most of the compounds found in the crude

extract. Hence it was fractionated a second time.

The interesting fractions in this set were fractions 8 to 11 (R3141C-2b-fr. 8, 9, 10,

and 11). These samples had similar HPLC profiles, and contained a compound which

was detectable by the ELS detector at 8.920 minutes, and which weakly absorbed

UV light at 214 nm. It also gave a mass-to-charge ratio (m/z) of 395.28 mass units.

The NMR profile looked very much like that of a peptide since signals were seen all

over the spectrum and most of the signals were present in the 0.5 to 3.5 ppm region,

with a few signals in the aromatic and -NH region also. Signals which could

correspond to the alpha carbon (C�) were also visible in the region between 4 and 5

ppm.

Figure 25 - 1HNMR profile of R3141C-2b-fr.8 [300 MHz; in CDCl3]

TLC profiling was carried out on the sample, and visualization was done using

Gibb’s reagent, HCl/ninhydrin and ninhydrin only. The Gibb’s visualization

confirmed that the compound did not have a phenolic group. Furthermore, an orange


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spot appeared when using HCl/ninhydrin reagent and it was absent when only

ninhydrin was used. Plate 4 illustrates the TLC profiles obtained.

From this visualization, it was proposed that there was a peptide without free

nitrogen (free nitrogen can be detected by ninhydrin reagent only). Since the mass

obtained from LC-MS was quite low (395.28 mass units), there was a possibility that

the compound was a small cyclic peptide.

Plate 4 - TLC of R3141C-2b-fr.8-11 for the determination of peptides and phenols.

Eluent – 1/1 - heptane/EtOAc; Reagent - HCl/Ninhydrin (Left); Ninhydrin (Middle); and

Gibb’s reagent (Right)

This fraction was then purified using reverse phase semi-preparative HPLC. Out of

the 7 fractions collected, fraction 5 (R3141C-purif peak 5) was identified as the peak

of interest. However, the mass of the product obtained was very low. After verifying

all the fractions obtained, fraction 7 (R3141C-purif peak 7) was seen to contain 2

new products: which was not detectable in all the HPLC analyses carried out before

the purification using semi-preparative HPLC, and which could be two products of

degradation.

One hypothesis of such a behavior was that degradation of the compound of interest

occurred in the column during semi-preparative fractionation.

Hence, TLC of peak 7 (the supposed degradation products of peak 5) was carried out

with visualization using HCl/Ninhydrin and Ninhydrin only: 2 spots were seen with

different Rf than the original spot of peak 5, one spot being only visible with

HCl/Ninhydrin.


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Plate 5 - TLC of R3141C-purif peak 7 for determination of peptides. Eluent – 1/1 -

heptane/EtOAc; Reagent - HCl/Ninhydrin (Left) and Ninhydrin (Right)

The original compound (peak 5) and the products of degradation were sent to the

University of Naples in Italy to be analyzed further.

Conclusions of the products obtained, as determined at the University of Naples,

suggest that the fractions contained a mixture of several compounds related to

Puupehenone. The 1HNMR (Figure 26) was composed of clusters of peaks generally

in the 0.6 – 2.6 ppm region. It is possible to say that these Puupehenone derivatives

are not methanol or ethyl acetate adducts (-OMe or O-CO-Me), since there are no

major peaks seen around 3.5 ppm in the 1HNMR spectrum.

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Figure 26 - 1HNMR of R3141C peak 5 (fraction of R3141C) in CDCl3

Peak of degradation B

Peak of degradation A


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These compounds are not peptides as predicted from the HCl/Ninhydrin reagent on

TLC. However, it is possible that when the TLC was sprayed with HCl, it formed a

hemiacetal which reacted with the ninhydrin giving an orange spot. So far, all

reactions with ninhydrin have been correlated to nitrogen-containing compounds.

Due to the low mass of the compounds obtained, it was not possible to confirm the

above hypothesis and the exact structure of the derivatives.

The following figure illustrates the possible Puupehenone derivatives present in the

sample R3141C:

O

O

OO

H

H

H

H

HOO

OOH

C21H28O3Exact Mass: 328.20Mol. Wt.: 328.45

C24H32O5Exact Mass: 400.22

Mol. Wt.: 400.51

Puupehenone

1112

13

14

1518

20

O

H

H

OH

O

O

C21H28O4Exact Mass: 344.20Mol. Wt.: 344.44

Figure 27: Puupehenone derivatives likely to be present in Dysidea species R3141C

Puupehenone and its derivatives belong to the sesquiterpene-substituted quinone

group of compounds (Ciavatta, et al., 2007; Bourguet-Kondracki, Lacombe, &

Guyot, 1999), constituting an important group of marine natural products in terms of

their bioactivities. They have been found to occur in sponges of the order Verongida,

genus Hyortis (Hamann, Scheuer, & Kelly-Borges, 1993; Sladi� & Gaši�, 2006;

Piña, Sanders, & Crews, 2003) and the order Dictyoceratida, genus Dysidea

(Ciavatta, et al., 2007). Puupehenone-related metabolites have been found to exhibit

biological activities such as cytotoxicity, anti-viral, anti-fungal, immunomodulatory

(Hamann, Scheuer, & Kelly-Borges, 1993; Ciavatta, et al., 2007), antibacterial, anti-

plasmodial (Bourguet-Kondracki, Lacombe, & Guyot, 1999), as well as anti-

tuberculosis and antitumor activities (Sladi� & Gaši�, 2006).


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The L. herbacea samples from New Caledonia (R2252C), Solomon Islands

(R3147C) and Fiji Islands (R3241C) were fractionated in order to get a confirmation

of the exact structure of the PBDE present in them. All three samples were only put

through the first set of fractionation.

3.3.2.1 Sample R2252C (New Caledonia)

It was seen in the sample R2252C after drying of the fractions that crystals had

formed in one of the fractions. After recrystallization using acetonitrile, the product

was obtained as an almost pure product with slight traces of the other compound.

From the HPLC, LC-MS, and 1H and 13C NMR analyses, it was evident that the

crystals obtained was compound 2 (3,4,5,6-tetrabromo-2-(3’,5’-dibromo-2’-

hydroxyphenoxy)phenol), while the supernatant product contained a mixture of

compound 2 and compound 3 (3,5,6-tribromo-2-(3’,5’-dibromo-2’-

hydroxyphenoxy)phenol). The chemical shifts obtained have been presented in

Tables 3 and 4 in Section 3.1.1.4.2 and 3.1.1.4.3, respectively.

The NMR signals of compounds 2 and 3 matched very closely to that found in

literature (Norton, Croft, & Wells, 1981); hence the structure of the compounds were

assigned as follows:

O

OH OH

Br

Br

Br

Br

Br

Br

Compound 23,4,5,6-tetrabromo-2-(3',5'-dibromo-2'-hydroxyphenoxy)phenol

6

5

1

43

22'

1'6'

5'4'

3'

O

OH OH

Br

Br

Br Br

Br

Compound 33,5,6-tribromo-2-(3',5'-dibromo-2'-hydroxyphenoxy)phenol

6

5

1

43

22'

1'6'

5'4'

3'

Figure 28 - Structures for Compounds 2 and 3

Furthermore, the melting point of the main product (Compound 2 – 3,4,5,6-

tetrabromo-2-(3’,5’-dibromo-2’-hydroxyphenoxy)phenol) was also determined and

was found to be in the range of 191.0 oC to 191.6 oC. This is very close to the value

obtained in literature, which is between 194 oC to 195 oC (Norton, Croft, & Wells,

1981). Thus, the conclusions of compounds 2 and 3 made in Section 3.1.1 have been

confirmed.


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3.3.2.2 Sample R3147C (Solomon Is.)

In case of the fractions of sample R3147C, crystals were also observed in the

samples, but recrystallization using hot acetonitrile failed, and crystals of the

products could not be obtained.

From the fractions of R3147C, the first fraction yielded two compounds as observed

in the HPLC chromatogram, where the first compound did not have UV absorption,

but exhibited a mass-to-charge ratio (m/z) of 609.06 mu. This compound did not

show presence of halogenation in the mass spectrum, and was more in the non-polar

region of the spectra. However, there was an aromatic moiety in this compound due

to the chemical shifts observed in the 1HNMR profile. The second (major) compound

(compound 5) in the first fraction had an m/z of 688.89 mu and the isotopic spectra

confirmed the presence of 6 bromine atoms. The closest possible structure with 6

bromine and mass of 689 mu found in literature was a 6Br-OH-OMe-PBDE.

O

O OH

Br

Br

Br

Br

Br

Br

Compound 53,4,5,6-tetrabromo-2-(3',5'-dibromo-2'methoxyphenoxy)phenol

12

34

5

61'2'

3'

4'5'

6'

Figure 29 - Proposed structure for compound 5 (3,4,5,6-tetrabromo-2-(3’,5’-dibromo-

2’-methoxyphenoxy)phenol)

NMR data could not be obtained to confirm this hypothesis due to the small quantity

of product available.

In the second fraction, a mixture of 3 compounds was obtained, out of which the

major compound was determined to be compound 4 (5Br-OH-OMe-PBDE). The m/z

of this compound was obtained to be 608.98 mu with the isotopic spectra indicating 5

bromine atoms. A fragment peak of the compound was also obtained at m/z of

346.47 mu, which corresponds to the loss of the ring containing the methoxy group

and 2 bromine atoms, hence leaving the second phenolic ring with three bromine

atoms. The 13C and 1H-NMR (Table 5) correlate well with the following structure:


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O

OHO

BrBr

Br

BrBr

165

43

23'

5'

1'

6'4'

2'

Compound 43,5,6-tribromo-2-(3',5'-dibromo-2'-methoxyphenoxy)phenol

Figure 30 - Structure of Compound 4 (3,5,6-tribromo-2-(3’,5’-dibromo-2’-

methoxyphenoxy)phenol)

Table 5 - Chemical shifts obtained for Compound 4 (at 400 MHz, in acetone-d6)

C # 13C (ppm)

1H (ppm)

�H Lit. in Acetone-d6 (ppm)*

13C 1H 1 151.50 150.8 2 139.78 139.0 3 117.65 117.0 4 128.71 7.65 (s) 127.9 7.65 (s) 5 124.05 123.3 6 115.80 115.1 1’ 152.64 151.9 2’ 147.20 146.5 3’ 120.13 119.5 4’ 130.38 7.48 (d, 2.2 Hz) 129.7 7.48 (d, 2.2 Hz) 5’ 117.62 116.9 6’ 118.18 6.82 (d, 2.2 Hz) 117.5 6.81 (d, 2.5 Hz)

-OMe 61.76 4.00 61.1 4.01 *Hanif, et al., 2007

The third fraction yielded compound 2, and thus confirmed the presence of this

compound in the sample. The 1HNMR chemical shifts obtained were also

comparable to the chemical shifts of this compound isolated from the other L.

herbacea from New Caledonia. (Spectral data of the fractions are present in

Appendix 10)


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3.3.2.3 Sample R3241C (Fiji Is.)

Just like in sample R3147, the fractions obtained for R3241C also showed presence

of crystals, but isolation of the pure compound was not successful using acetonitrile.

From the spectral data of the fractions, two of the compounds were easily identified

to be compound 2 (6Br-2OH-PBDE) and compound 3 (5Br-2OH-PBDE). Two other

compounds present in this specimen were compound 6 (4Br-OH-OMe-PBDE -

isomer of Compound 1) and compound 8 (5Br-OH-OMe-PBDE - isomer of the

Compound 4 obtained in R3147 extracts - Figure 30).

O

Br

R

Br

O

OH

Br

Br

R = H --> Compound 6 : 4,6-dibromo-2-(4',6'-dibromo-2'-methoxyphenoxy)phenolR = Br --> Compound 8 : 4,6-dibromo-2-(4',5',6'-tribromo-2'-methoxyphenoxy)phenol

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62'

1'

6'5'

4'

3'

Figure 31 - Compounds 6 and 8 obtained from sample R3241C

Table 6 - Chemical shifts obtained for Compounds 6 and 8 (in Acetone-d6, at

400 MHz)

C # Compound 6 Compound 8 Literature 13C

(ppm)

1H (ppm)

13C (ppm)

1H (ppm)

*Compd. 6 (CDCl3)

**Compd. 8 (CDCl3)

1 145.17 145.13 142.8 142.7 2 147.55 147.24 145.2 144.7 3 117.15 6.62 117.19 6.71 115.7 (6.53) 116.3 (6.51) 4 111.52 111.62 110.0 111.5 5 129.91 7.377 130.11 7.39 129.2 (7.33) 129.3 (7.34) 6 111.90 112.00 111.5 110.1 1’ 141.14 142.16 139.9 140.8 2’ 155.29 154.00 153.4 152.0 3’ 117.76 7.425 119.02 7.64 116.5 (7.09) 116.6 (7.28) 4’ 120.71 119.52 119.8 119.1 5’ 128.31 7.49 123.52 127.5 (7.40) 122.8 6’ 123.02 118.6 122.4

-OMe 3.87 3.87 56.6 (3.78) 56.7 (3.78) *Fu, Schmitz, Govindan, & Abbas, 1995 **Liu, Namikoshi, Meguro, Nagai, Kobayashi, & Yao, 2004

(Spectral data of the fractions are present in Appendix 10)


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3.3.2.4 Sample MAY-02-Dh2 (L. herbacea from Mayotte)

L. herbacea from Mayotte contained similar brominated compounds as that obtained

from L. herbacea from the other sampling sites. Three main compounds could be

isolated from this sample: compound 6 (4Br-OH-OMe-PBDE - isomer of Compound

1), compound 7 (5Br-2OH-PBDE - isomer of Compound 3), and compound 8 (5Br-

OH-OMe-PBDE - isomer of Compound 4).

The chemical shifts obtained for compounds 6 and 8 were almost the same as that

obtained for these compounds in sample R3241C (the structures were confirmed by

HSQC and HMBC spectra). The structure of compound 7 (isomer of compound 3)

was confirmed as follows using 1-D and 2-D NMR:

OH

Br

Br

Br

O

OH

Br

Br

Compound 7 - 4,6-dibromo-2-(4',5',6'-tribromo-2'-hydroxyphenoxy)phenol

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5

62'

1'

6'5'

4'

3'

Figure 32 - Structure of Compound 7 (4,6-dibromo-2-(4’,5’,6’-tribromo-2’-

hydroxyphenoxy)phenol)

Table 7 - Chemical shifts obtained for Compound 7 (in acetone-d6, 400 MHz)

Carbon #

Compound 7 Literature* in CDCl3 (ppm) 13C (ppm) 1H (ppm) HMBC

1 144.88 2 146.75 3 116.71 6.73 144.88 / 129.76 / 111.34 6.46 4 111.34 5 129.76 7.38 144.88 / 116.71 / 111.69 7.23 6 111.69 1’ 151.63 2’ 140.70 3’ 122.35 7.49 140.70 / 123.05 / 118.23 7.26 4’ 118.23 5’ 123.05 6’ 122.64

*Fu, Schmitz, Govindan, & Abbas, 1995


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The compounds obtained from sample R3141C seems to be quite sensitive in terms

of stability, and the exact reason for the degradation behavior is not known yet.

The fractionations of L. herbacea sample R2252C from New Caledonia and the L.

herbacea from Mayotte were successful in isolating the major compounds of interest,

and to confirm their structures. Pure compounds of L. herbacea R3147C and D.

lizardensis R3241C could not be obtained but the fractions were clear enough to

provide some data to confirm the compounds present in the samples.

Thus, the compounds obtained from this analysis include tetra-, penta-, and hexa-

brominated diphenyl ethers, with either two hydroxy groups or one hydroxy and one

methoxy groups.


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Polybrominated diphenyl ethers are a class of phytopathogenic fungicides (Peng, et

al., 2003). The antifungal bioassay in this study was carried out using Cryptococcus

neoformans (ATCC 32045) and wild type Candida albicans (WTCA).

It was evident from the results obtained that although the Dysidea species showed

growth inhibition of Cryptococcus neoformans (and a few samples showed inhibition

of WTCA), the inhibition was not as good as the control (Nystatin) it was tested

with. Nystatin is one of the first commercial drugs used for the treatment of

opportunistic fungal infections. This however, was a preliminary test to see if the

crude samples were active or not, and if purified, it could show interesting activity at

the appropriate dosage.

Figure 33 - Inhibition of Cryptococcus neoformans and WTCA by crude and

fractionated extracts of Dysidea sponges (Dosage of crude extracts = 1 mg per disc;

Dosage of fractionated extracts = 0.1 mg per disc)

It was also interesting to see that the two compounds 1 and 2 had a vast difference in

their activity patterns. Compound 1 (4Br-OH-OMe-PBDE) was more active than

Compound 2 (6Br-2OH-PBDE), which was barely active. Similarly, when

comparing Compound 1 with the brominated metabolites of Dysidea sp. 4177

(Compounds 9 and 10 from samples R3033C and R3161C), it is clear that the penta-

bromo-diphenyl ether (Compound 10) is more active than Compound 1, followed by

the tetra-bromo-diphenyl ether (Compound 9). The following figure shows the

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inhibition of Cryptococcus neoformans by the crude extracts of R3033C, R3161C

and R3203C, and Compounds 1 (4Br-OH-OMe-PBDE) and 2 (6Br-2OH-PBDE).

Plate 6 - Inhibition of Cryptococcus neoformans by selected Dysidea species (R3033C and

R3161C – Dysidea 4177; R3310C – Dysidea pallescens; 4Br-OH-OMe-PBDE – Compound

1; 6Br-2OH-PBDE – Compound 2)

This result indicates a notable pattern of activity: as the number of bromine atoms

increase, the compounds exhibit higher activity (this is true in the case of Dysidea

4177 – R3033 and R3161). However, compounds with two hydroxy groups are less

active compared to compounds with either one hydroxy group, or a hydroxy-

methoxy group attached to it.

Figure 34 - Illustration of activity against Cr. neoformans and Ca. albicans based on the

difference in chemical constituents of the various sponges.


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Added to this, slight distinction in activity could also be seen within L. herbacea,

especially with the specimens containing Compound 1. These samples (R2259C and

R2279C) have been discussed previously regarding their unique chemical profiles

compared to other L. herbacea samples collected from New Caledonia (Section

3.2.2.1). Furthermore, even though brominated compounds have been found to be

responsible for the biological activity against the fungi, it is important to note that

there are other active compounds present in the Dysidea arenaria and Dysidea avara

specimens that have also showed slight activity.

It is also clear from the results that most of the Dysidea extracts are more active

against Cryptococcus neoformans than Candida albicans. This was the expected

result for the preliminary anti-malarial studies, and it can now be further investigated

for anti-plasmodium activities. As discussed before, one of the differences between

Cr. neoformans and Ca. albicans is that the former species requires the protein

farnesyl transferase (PFT) for viability, whereas the latter fungus can survive without

it. The result obtained in this assay proves that these compounds inhibit the PFT from

functioning normally. Consequently, it suggests a plausible mode of action for any

activity shown against the Plasmodium species, which is also dependent on the

proper functioning of the PFT for viability.

Although the specimens tested for anti-fungal activities did not prove to be very

challenging, it can still be a candidate for various other assays. It can also be tested

directly on Plasmodium species in order to determine another mode of action,

separate from the PFT inhibition.

A study carried out by Peng, et al., (2003) indicated that comparing activity of

sponge-derived PBDEs and manzamine alkaloids against weeds, fungi, and malarial

parasites, the latter group of metabolites showed more promising activity against

Plasmodium berghei. Therefore, this group of metabolites can also be explored in

order to find a drug candidate for Plasmodium infections.


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Sponge metabolites have been the targets of research for the past three decades. Due

to the large biodiversity of these organisms, there are still quite a lot of species that

have not been discovered and studied. Many analyses of sponge metabolites are

directed towards drug discovery. Apart from this, chemotaxonomy is also a very

important research field, whereby chemical constituents of these organisms can be

used together with biological taxonomy to classify the various organisms accurately.

The aspect of chemotaxonomy was partly studied in this research, and it was found

that one of the biggest problems is that the main extractable chemicals are produced

by symbionts and not by the host itself. The main group of compounds found in this

study was PBDEs. These compounds are suspected to be produced by the symbiont

cyanobacteria, Oscillatoria spongeliae, present in L. herbacea sponges. Due to this

reason, a sponge-specific metabolite could not be identified as yet, which could have

been a candidate for a chemical marker in the Dysidea sponges.

An intra- and inter-geographical comparison was also carried out with L. herbacea

from New Caledonia, Vanuatu, Fiji, Solomon Is., Moorea, and Mayotte islands. It

was observed that the sponges are quite sensitive to variations, and due to this,

certain specimen of the same species gave slightly different profiles, while the others

were quite the same. It would be interesting to know if all the L. herbacea studied

contain the same strain of cyanobacteria since all extracts contained brominated

diphenyl ethers (bromo-chemotype).

Also in this study, eight compounds have been identified with the help of spectral

analysis. Out of these eight compounds, four compounds (Compound 2, 6, 7 and 8)

have been isolated successfully, while the others were obtained as mixtures or

impure products. Finally, the anti-fungal assay against Cryptococcus neoformans and

Candida albicans mainly showed moderate activity against Cr. neoformans,

suggesting that the mode of action was via the inhibition of the enzyme protein

farnesyl transferase. This result indicates that the activity of these compounds against

the Plasmodium species is also possible through the same mode.


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Agrawal, M. S., & Bowden, B. F. (2005). Marine sponge Dysidea herbacea

revisited: Another brominated diphenyl ether. Marine Drugs , 3, 9-14.

Aiello, A., Fattorusso, E., Menna, M., & Pansini, M. (1996). The Chemistry of the

demosponge Dysidea fragilis from the Lagoon of Venice. Biochemical

Systematics and Ecology , 24 (1), 37-42.

Alvi, K. A., Diaz, M. C., Crews, P., Slate, D. L., Lee, R. H., & Moretti, R. (1992).

Evaluation of new sesquiterpene quinones from two Dysidea sponge species as

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Anjaneyulu, V., Rao, K. N., Radhika, P., Muralikrishna, M., & Connolly, J. D.

(1996). A new tetrabromodiphenyl etherfrom the sponge Dysidea herbacea of the

Indian Ocean. Indian Journal of Chemistry , 35B, 89-90.

Ardá, A., Jiménez, C., & Rodríguez, J. (2004). A study of polychlorinated leucine

derivatives: Synthesis of (2S,4S)-5,5-dichloroleucine. Tetrahedron Letters , 45,

3241-3243.

Ardá, A., Rodríguez, J., Nieto, R. M., Bassarello, C., Gomez-Paloma, L., Bifulco, G.,

et al. (2005). NMR J-based analysis of nitrogen-containing moieties and

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Tetrahedron , 61, 10093-10098.

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sponges on scallops for natural product research and antifouling. Journal of

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http://www.mareco.org/khoyatan/spongegardens

Becerro, M. A., & Paul, V. J. (2004). Effects of depth and light on the secondary

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West, R. R., & Cardellina, J. H. (1988). Three new polyhydroxylated sterols with the

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Bioactive polybrominated diphenyl ethers from the marine sponge Dysidea sp. J.

Nat. Prod. , (Article in Press).


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# Ext. Code Species Location

Freeze-dried

Sponge (g)

Mass of

Ext. C (g)

% Yield

of Ext. C

Mass of

Ext. B (g)

Description of Ext. C

1 R2252C L. herbacea New

Caledonia (NC)

16.174 0.6989 4.32 1.4202 Green;

Gummy; Semi-solid;

Sticky substance

2 R2256C L. herbacea NC 20.943 0.9612 4.59 1.9702

3 R2259C L. herbacea NC 20.326 1.1974 5.89 2.8710

4 R2261C L. herbacea NC 20.649 0.7715 3.74 1.7336

5 R2262C L.

herbacea? NC

20.320 0.8606 4.23 1.1297

6 R2265C L. herbacea NC 20.536 1.1865 5.78 1.7566

7 R2267C L. herbacea NC 21.186 0.6942 3.28 0.6137

8 R2269C L. herbacea NC 20.664 1.0117 4.90 1.7546

9* R2270C L. herbacea NC

10 R2272C L. herbacea NC 20.421 0.7392 3.62 0.9323





15 MAU-02-

Dh1 L. herbacea Moorea

2.0260 0.1237 6.11 -

16* R2247bisC D. arenaria NC Yellow-brown;

Gummy; Semi-solid;

Sticky substance.

17 R2254C D. arenaria NC 20.824 0.1850 0.89 0.9106

18 R2257C D. arenaria NC 21.664 0.2633 1.22 1.1141

19* R2260C D. arenaria NC

20 R2261bisC D. arenaria NC 21.844 0.3226 1.48 2.0333



23 R2268C D. arenaria NC 21.321 0.2012 0.94 1.2007


25 R2082C D. avara NC 1.0006 0.0316 3.16

Gummy; Yellow-brown;

sticky; semi-solid extract.

*Extracts obtained from IRD Nouméa.


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Station Sample Area of Collection

Photograph of Sample Description

ST720 R2252

L. herbacea

New Caledonia

(22°32.736S 166°26.588E)

Colour - pale

green; Encrusting on hard surface;

Finger-like projections in

some regions, no out-growth on the

other.

ST1048 R2256

L. herbacea

New Caledonia

(22°41.566S 166°38.704E)

Green, lamellate projections from

an encrusting base; in competition for space with other sessile marine

organisms.

ST1052 R2261

L. herbacea

New Caledonia

(22°21.249S 166°14.783E)

Green, lamellate to lobate projections from an encrusting

base; in competition for space with other sessile marine

organisms.

ST1053 R2262

L. herbacea

New Caledonia

(22°16.456S 166°14.311E)

Green, lamellate projections from

an encrusting base; little competition, therefore upward

growth of the leaf-like projections.


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ST1055 R2265

L. herbacea

New Caledonia

(22°01.865S 165°57.579E)

Green sponge with

encrusting base attached to rocky

surface, small projections; found in the middle of

many sessile invertebrates.

ST956 R2267

L. herbacea

New Caledonia

(21°51.695S 165°43.429E)

Green sponge with encrusting base

attached to rocky surface, small

projections; found in the middle of

many sessile invertebrates.

ST1059 R2269

L. herbacea

New Caledonia

(22°10.980S 166°06.066E)




many sessile invertebrates;

small colony seen.

ST254 R2272

L. herbacea

New Caledonia

(22°17.013S 166°11.035E)




many sessile invertebrates;

small colony seen.

ST1060 R2270

L. herbacea

New Caledonia

(22°23.201S 166°17.065E)



projections; found in the crevice between two rocks; small colony seen.


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ST1132 R2274

L. herbacea

New Caledonia

(22°19.350S 167°02.027E)

Green sponge with

encrusting base attached to flat

rocky surface, not so many

projections seen; small colony.

ST1135 R2278

L. herbacea

New Caledonia

(22°06.832S 167°00.321E)

Blue-green sponge

with encrusting base attached to

flat rocky surface, not so many

projections visible; small colony.

ST1127 R2280

L. herbacea

New Caledonia

(21°32.777S 165°12.996E)

No Photo Available

ST1050 R2259

L. herbacea

New Caledonia

(22°23.295S 166°47.301E)

Green sponge with lamellate/lobate

projections; surrounded by

sediment.

ST1136 R2279

L. herbacea

New Caledonia

(22°08.287S 166°56.955E)

Blue-green, encrusting sponge; large but generally flat colony, very

few lamellate projections visible;

occupying large surface area of a rock containing other organisms.

ST613 R2271bis

L. herbacea

Vanuatu (13°30.910S

167°21.100E)

167°21.100E)

No Photo Available


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ST840 R3147

L. herbacea

Solomon Is. (09°11.055S

160°11.982E)

Blue-grey sponge with lamellate

projections; small colony encrusted

on a rocky surface; habitat a little

darker than that for New Caledonian

samples.

FJ4 R3241

L. herbacea

Fiji Is. (16°18.847S

178°54.595E)

Blue-grey sponge with lamellate

projections (purple when out of the water); colony encrusted on a

rocky surface with coral and algae growing on it.

MAU-02-Dh1

L. herbacea

Moorea Is.

Found in lagoon

with clear water at a depth of 1 - 1.5 m; Encrusting on hard corals with a

big lamellate colony; in close proximity of two cyanobacteria,

Anabaena torulosa and an unidentified

species.

MAU-02-Dh2

L. herbacea

Moorea Is. No Photo Available

Found in lagoon with clear water at a depth of 1 - 1.5 m; Growing close to hard corals; No

algae seen growing around the samples.

MAU-02-Dh3

L. herbacea

Moorea Is.

Found in lagoon with clear water at a depth of 1 - 1.5 m; Encrusting on hard corals with a

big lamellate colony; in close proximity of two cyanobacteria,

Anabaena torulosa.


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MAY-02-Dh1

L. herbacea

Mayotte Is. No Photo Available

MAY-02-Dh2

L. herbacea

Mayotte Is. No Photo Available

ST124 R2247bis

D. arenaria

New Caledonia

(22°20.611S 166°25.970E)

No Photo Available

ST534 R2254

D. arenaria

New Caledonia

(22°20.700S 166°20.000E)

Grey lobate colony clustered together (only one colony seen); growing on sandy substrate;

surrounded by sea-weed and algae.

ST1049 R2257

D. arenaria

New Caledonia

(22°31.008S 166°36.212E)



ST1051 R2260

D. arenaria

New Caledonia

(22°19.699S 166°23.463E)




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ST1054 R2263

D. arenaria

New Caledonia

(22°18.936S 166°21.176E)


surrounded by sea-weed and algae; sedimentation

seen.

ST1053 R2261bis

D. arenaria

New Caledonia

(22°16.456S 166°14.311E)



seen.

ST1057 R2264

D. arenaria

New Caledonia

(21°51.971S 165°45.628E)



seen.

ST1058 R2268

D. arenaria

New Caledonia

(22°07.065S 166°06.556E)

No Photo Available

ST1061 R2271

D. arenaria

New Caledonia

(22°15.529S 166°20.155E)

Grey lobate colony clustered together (only one colony seen - small in

size); growing on sandy substrate;

surrounded by sea-weed and algae;

sedimentation seen slightly.


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FJ18 R3314

D. arenaria

Fiji Is. (16°06.762S

179°11.264W)

Pink in color; growing in the

middle of a group of sea-weeds; has

the appearance of a perforated ball (not

lobate like the other New Caledonian samples).

ST612 R2082

D. avara

New Caledonia

(20°32.930S 167°10.790E)

No Photo Available

FJ1 R3203 D. cf. avara

Fiji Is. (16°46.600S 178.24.344E)

Red in color when outside water; rigid-looking

stems like that of hard corals.

ST823 R3033

Dysidea 4177


158°14.228E)

Purple in color; growth pattern as

seen in hard corals but scattered;

found on a rocky outgrowth;

surrounged by algae and sea-

ferns.

ST846 R3161

Dysidea 4177


160°44.682E)

Purple in color; growth pattern as

seen in hard corals with finger-like

projections arising from a common

stem; dark purple in color when out

of the water.


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ST840 R3141

Dysidea sp.


160°11.982E)

Maroon in color, and leaf-like appearance;

growing with other sea-weeds and

algae.


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Site 1 Site 2 Distance

(km)

NC

Vanuatu 836.89 Solomon 1427.4 Fiji 1420.71 Moorea 4471.05 Mayotte 12115.62

Vanuatu

Solomon 871.29 Fiji 1226.96 Moorea 4488.15 Mayotte 12655.56

Solomon Fiji 2091.66 Moorea 5277.34 Mayotte 12164.15

Fiji Moorea 3237.39 Mayotte 13584.88

Moorea Mayotte 15787.65


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*Note: The fractions obtained contained a mixture of compounds; hence pure

compounds could not be isolated. However, fractionation did help to confirm the

major products present in the sample.


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1HNMR chemical shifts obtained for Compounds 9 and 10:

Proton � Compd. 9 (ppm)

J for Compd. 9 (Hz)

� Lit. (ppm)*

� Compd. 10 (ppm)

J for Compd. 10 (Hz)

� Lit. (ppm)**

H4 7.36 2.2 7.37 - - -

H6 7.23 2.1 7.12 7.46 - 7.43

H3’ 7.80 2.3 7.85 7.79 2.9 7.84

H5’ 7.32 2.4, 8.8 7.36 7.30 7.35

H6’ 6.47 8.8 6.42 6.44 8.3 6.51

* (Xu, Johnson, & Hecht, 2005)

** (Fu & Schmitz, 1996)

Documents

SECONDARY METABOLITES COMPOSITION AND GEOGRAPHICAL ...digilib.library.usp.ac.fj/.../index/assoc/HASH0112.dir/doc.pdf · SECONDARY METABOLITES COMPOSITION AND GEOGRAPHICAL DISTRIBUTION