SECONDARY METABOLITES COMPOSITION and GEOGRAPHICAL DISTRIBUTION of MARINE SPONGES of the GENUS Dysidea

COMPONENT 2C – PROJECT 2C4 Bioprospection & Active Marine Substances Training of Pacifi c students

June 2009

MASTER THESIS

SECONDARY METABOLITES COMPOSITION AND GEOGRAPHICAL DISTRIBUTION OF MARINE SPONGES OF THE GENUS Dysidea

Author: Mayuri Chandra Photo: ©IRD – Eric FOLCHER Photo: ©IRD The CRISP Coordinating Unit was integrated into the Secretariat of the Pacifi c Community in April 2008 to insure maximum coordination and synergy in work relating to coral reef management in the region.

The CRISP Programme is implemented as part of the policy developped by the Secretariat of the Pacifi c Regional Environment Programme to contribute to the conservation and sustainable development of coral reefs in the Pacifi c.

he Initiative for the Protection and Management of Coral Reefs in the Pacifi c (CRISP), T sponsored by France and prepared by the French Development Agency (AFD) as part of an inter-ministerial project from 2002 onwards, aims to develop a vision for the future of these unique eco-systems and the communities that depend on them and to introduce strategies and projects to conserve their biodiversity, while developing the economic and environmental services that they provide both locally and globally. Also, it is designed as a factor for integration between developed countries (Australia, New Zealand, Japan, USA), French overseas territories and Pacifi c Island developing countries.

The CRISP Programme comprises three major components, which are:

Component 1A: Integrated Coastal Management and watershed management - 1A1: Marine biodiversity conservation planning - 1A2: Marine Protected Areas CRISP Coordinating Unit (CCU) - 1A3: Institutional strengthening and networking Programme manager : Eric CLUA - 1A4: Integrated coastal reef zone and watershed management SPC - PoBox D5 Component 2: Development of Coral Ecosystems 98848 Noumea Cedex - 2A: Knowledge, monitoring and management of coral reef ecosytems New Caledonia - 2B: Reef rehabilitation Tel : (687) 26 54 71 - 2C: Bioprospection and marine active substances Email : [email protected] - 2D: Development of regional data base (ReefBase Pacifi c) www.crisponline.net Component 3: Programme Coordination and Development - 3A: Capitalisation, value-adding and extension of CRISP Programme activities - 3B: Coordination, promotion and development of CRISP Programme

COMPONENT 2C Bioprospection and Marine Active Substances

 PROJECT 2C-1 : Legal Output - Proposals for an improvement of legal framework in Pacifi c countries for insuring a better benefi t-sharing in bioprospection Component leader : Cécile DEBITUS  PROJECT 2C-2: IRD - UMR 152 Taxonomic Output - Improvement of scientifi c knowledge of reef benthic inverte- University of Paul Sabatier brates Toulouse II Faculty of Sciences 31062 Toulouse cedex 9  PROJECT 2C-3 : France Technological Output - Screening and identifi cation of Marine Active Subtances Tel : (33) 5 62 25 98 11 Fax : (33) 5 62 25 98 02  PROJECT 2C-4 : [email protected] Capacity building Output - Training of Pacifi c people in the fi eld of bioprospection

This training was eff ective thanks to the fi nancial support of the following agencies:

SECONDARY METABOLITES COMPOSITION AND GEOGRAPHICAL DISTRIBUTION OF MARINE SPONGES OF THE GENUS DYSIDEA

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

“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

ACKNOWLEDGEMENT

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,

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.

ABSTRACT

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.

iii

SOMMAIRE

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.

TABLE OF CONTENTS

ACKNOWLEDGEMENT ...... I

ABSTRACT ...... III

SOMMAIRE ...... IV

LIST OF FIGURES ...... VIII

LIST OF PLATES ...... IX

LIST OF TABLES ...... IX

LIST OF SCHEMES ...... IX

LIST OF ABBREVIATIONS ...... X

LIST OF STRUCTURES ...... XIII

1 INTRODUCTION ...... 1

1.1 INTRODUCTION ...... 1

1.2 BIOLOGY OF SPONGES AND THE GENUS DYSIDEA ...... 2 1.2.1 History and Background of Sponges ...... 2 1.2.2 Morphology & Classification ...... 3 1.2.3 The Genus Dysidea ...... 6

1.3 SPONGE – SYMBIONT ASSOCIATION ...... 13

1.4 SECONDARY METABOLITES AND CHEMOTAXONOMY ...... 15

1.5 NATURAL PRODUCTS AND DRUG DISCOVERY ...... 16

1.6 ANTIFUNGAL BIOASSAY ...... 18 1.6.1 Cryptococcus neoformans ...... 19 1.6.2 Candida albicans ...... 20 1.6.3 New Targets for Antimicrobial Drug Development ...... 20

1.7 RELEVANCE OF STUDY ...... 22

2 GENERAL METHODOLOGY...... 24

2.1 SAMPLE COLLECTION AND IDENTIFICATION ...... 24

2.2 EXTRACTION TECHNIQUES ...... 24 2.2.1 Technique 1 ...... 24 2.2.2 Technique 2 ...... 28

2.3 CHEMICAL ANALYSIS ...... 30 2.3.1 Thin Layer Chromatography (TLC) ...... 30 2.3.2 High Performance Liquid Chromatography (HPLC) ...... 30 2.3.3 Liquid Chromatography – Mass Spectroscopy (LC-MS) ...... 31

2.3.4 Nuclear Magnetic Resonance (NMR) Spectroscopy ...... 32

2.4 ISOLATION AND STRUCTURE DETERMINATION ...... 33 2.4.1 Dysidea Species R3141C ...... 33 2.4.2 Lamellodysidea herbacea ...... 34

2.5 ANTI-FUNGAL BIOASSAY ...... 35 2.5.1 Media (Sabouraud Dextrose Agar – SDA) Preparation ...... 35 2.5.2 Anti-fungigram Discs ...... 35 2.5.3 Fungal Inoculation ...... 36

3 RESULTS & DISCUSSION ...... 37

3.1 CHEMICAL SCREENING ...... 37 3.1.1 Lamellodysidea herbacea ...... 37 3.1.1.1 Thin Layer Chromatography ...... 37 3.1.1.2 High Performance Liquid Chromatography (HPLC) ...... 39 3.1.1.3 Liquid Chromatography – Mass Spectroscopy (LC-MS) ...... 41 3.1.1.4 Nuclear Magnetic Resonance Spectroscopy (NMR) ...... 43 3.1.1.4.1 Compound 1 ...... 44 3.1.1.4.2 Compound 2 ...... 46 3.1.1.4.3 Compound 3 ...... 49 3.1.2 Dysidea arenaria ...... 51 3.1.3 Dysidea avara ...... 52 3.1.4 CONCLUSION ...... 53

3.2 GEOGRAPHICAL DISTRIBUTION OF DYSIDEA SPECIES ...... 56 3.2.1 ECOLOGY OF THE GENUS DYSIDEA ...... 56 3.2.1.1 Lamellodysidea herbacea ...... 57 3.2.1.2 Dysidea arenaria and Dysidea avara ...... 57 3.2.1.3 Dysidea species (R3033, R3161, and R3141) ...... 58 3.2.2 SIMILARITIES AND DIFFERENCES IN CHEMICAL COMPOSITION AND GENETIC LINKAGE BETWEEN LAMELLODYSIDEA HERBACEA BASED ON GEOGRAPHICAL DISTRIBUTION ...... 59 3.2.2.1 Comparison of Lamellodysidea herbacea collected within New Caledonia...... 59 3.2.2.1.1 Comparison of the Chemical Profiles ...... 59 3.2.2.1.2 Comparison of the Chemical Profiles with Gene Mapping ...... 62 3.2.2.2 Comparison of Lamellodysidea herbacea in other Geographical Locations ...... 64 3.2.2.2.1 Comparison of the Chemical Profiles ...... 64 3.2.2.2.2 Comparison of the Chemical Profiles with Gene Mapping ...... 67 3.2.3 COMPARISON OF DYSIDEA ARENARIA AND D. AVARA FROM NEW CALEDONIA AND FIJI, AND 3 DYSIDEA SPECIES FROM THE SOLOMON ISLANDS ...... 68 3.2.4 CONCLUSION ...... 71

3.3 ISOLATION & STRUCTURE DETERMINATION OF COMPOUNDS FROM DYSIDEA SPECIES ...... 72 3.3.1 DYSIDEA SPECIES R3141C ...... 72

3.3.2 LAMELLODYSIDEA HERBACEA ...... 77 3.3.2.1 Sample R2252C (New Caledonia) ...... 77 3.3.2.2 Sample R3147C (Solomon Is.) ...... 78 3.3.2.3 Sample R3241C (Fiji Is.) ...... 80 3.3.2.4 Sample MAY-02-Dh2 (L. herbacea from Mayotte) ...... 81 3.3.3 CONCLUSION ...... 82

3.4 ANTI-FUNGAL BIOASSAY ...... 83

4 GENERAL CONCLUSION ...... 86

5 BIBLIOGRAPHY ...... 87

6 APPENDIX ...... 102

6.1 APPENDIX 1: DYSIDEA SPECIES STUDIED FOR CHEMICAL SCREENING ...... 102

6.2 APPENDIX 2: GUIDE TO DETERMINE HALOGENATION PATTERN AND NUMBER ...... 103 1 6.3 APPENDIX 3: HNMR DETAILS OF COMPOUNDS 1, 2, AND 3 FROM L. HERBACEA ...... 104 6.3.1 Appendix 3a: 1HNMR profiles of L. herbacea for Compound 1 ...... 104 6.3.2 Appendix 3b: 1-D and 2-D NMR Profiles of Compound 2 ...... 106 6.3.3 Appendix 3c: 1HNMR Profile of Compound 3 ...... 109

6.4 APPENDIX 4: NMR SHIFTS FOR ALL COMPOUNDS IDENTIFIED & THE YIELDS OBTAINED FOR THE FOUR

COMPOUNDS ...... 111 6.4.1 Appendix 4a: NMR shifts for all compounds identified ...... 111 6.4.2 Yields obtained for the isolated compounds ...... 112

6.5 APPENDIX 5: SPECTRAL DATA – DYSIDEA ARENARIA...... 113

6.6 APPENDIX 6: SPECTRAL DATA: DYSIDEA AVARA ...... 114

6.7 APPENDIX 7: COMPARISON OF L. HERBACEA, D. ARENARIA AND D. AVARA...... 115

6.8 APPENDIX 8: SPECIMENS STUDIED FOR ECOLOGICAL AND GEOGRAPHICAL COMPARISON ...... 117

6.9 APPENDIX 9: DETAILED MAP OF THE 6 SAMPLING SITES AND THE APPROXIMATE DISTANCE BETWEEN THEM .. 125

6.10 APPENDIX 10: SPECTRAL DETAILS OF R3147C AND R3241C FRACTIONS...... 127 6.10.1 : Appendix 10a: R3147C ...... 127 6.10.2 : Appendix 10b: R3241C ...... 130

6.11 APPENDIX 11: MAYOTTE SAMPLE (MAY-02-DH1) – STRUCTURE DETERMINATION ...... 132

6.12 APPENDIX 12: COMPARISON OF CHEMICAL PROFILES OF D. ARENARIA AND D. AVARA FROM NEW CALEDONIA

AND FIJI ...... 138 6.12.1 Appendix 12a: Dysidea arenaria (ELS scan and 1HNMR) ...... 138 6.12.2 Appendix 12b: Dysidea avara (ELS scan and 1HNMR) ...... 139

6.13 APPENDIX 13: DATA USED FOR STRUCTURE DETERMINATION OF THE BROMINATED COMPOUNDS IN DYSIDEA 4177 …………………………………………………………………………………………………………………………………………140

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LIST OF FIGURES

FIGURE 1 - ASCONOID SPONGE ...... 4

FIGURE 2 - SYCONOID SPONGE ...... 4

FIGURE 3 - LEUCONOID SPONGE ...... 5

FIGURE 4 - COMPOUNDS ISOLATED FROM DYSIDEA SPECIES...... 7

FIGURE 5 - CHLORINATED COMPOUNDS ISOLATED FROM DYSIDEA SPONGES ...... 8

FIGURE 6 – EXAMPLES OF CHLORINATED COMPOUNDS FOUND IN BOTH DYSIDEA SPONGES AND THE CYANOBACTERIA

LYNGBYA MAJUSCULA ...... 8

FIGURE 7 - DISTRIBUTION OF BROMINATED AND CHLORINATED COMPOUNDS FROM LAMELLODYSIDEA HERBACEA ...... 13

FIGURE 8 - POTENTIAL PHARMACEUTICALS FROM MARINE SPONGES ...... 17

FIGURE 9 - COMPOUNDS USED AS STANDARDS FOR TLC ANALYSIS ...... 38

FIGURE 10 - THREE TYPES OF CHEMICALLY DIFFERENT L. HERBACEA FOUND IN NEW CALEDONIA ...... 40

FIGURE 11 - PATTERN OF HALOGENATED CLUSTER SHOWING PRESENCE OF 4, 5, AND 6 BROMINE ATOMS ...... 41

FIGURE 12 – PATTERN OF REARRANGEMENT OF COMPOUND OBSERVED IN MASS ANALYSIS...... 43 1 FIGURE 13 - COMPOUND 1: MASS SPECTRUM, HNMR, AND STRUCTURE...... 44 1 FIGURE 14 - HNMR PROFILES OF ORGANIC EXTRACTS OF R2259C AND MAU-02-DH1...... 45

FIGURE 15 - COMPOUND 1 (3,5-DIBROMO-2-(3’,5’-DIBROMO-2’-METHOXYPHENOXY)PHENOL) ...... 46

FIGURE 16 - COMPOUND 2 (3,4,5,6-TETRABROMO-2-(3’,5’-DIBROMO-2’-HYDROXYPHENOXY)PHENOL)...... 48

FIGURE 17 - COMPOUND 3 (3,5,6-TRIBROMO-2-(3’,5’-DIBROMO-2’-HYDROXYPHENOXY)PHENOL) ...... 50

FIGURE 18 - COLLECTION SITES OF L. HERBACEA FOR GEOGRAPHICAL COMPARISON ...... 56

FIGURE 19 - MAP SHOWING COLLECTION SITES OF L. HERBACEA IN NEW CALEDONIA...... 60

FIGURE 20 - MAP ILLUSTRATING THE TERRAIN NEAR THE COLLECTION SITES OF R2259 AND R2279 ...... 61

FIGURE 21 - TAXONOMY TREE OF DYSIDEA SP. AFTER GENE MAPPING ...... 63

FIGURE 22 - GEOGRAPHICAL COMPARISON OF CHEMICAL CONSTITUENTS IN L. HERBACEA ...... 65

FIGURE 23 - BROMINATED DIPHENYL ETHERS FOUND IN DYSIDEA 4177 ...... 69 1 FIGURE 24 - HNMR AND HPLC (UV 254 NM) PROFILES OF R3141C ...... 72 1 FIGURE 25 - HNMR PROFILE OF R3141C-2B-FR.8 ...... 73 1 FIGURE 26: HNMR OF R3141C PEAK 5 (FRACTION OF R3141C) ...... 75

FIGURE 27: PUUPEHENONE DERIVATIVES LIKELY TO BE PRESENT IN DYSIDEA SPECIES R3141C ...... 76

FIGURE 28 - STRUCTURES FOR COMPOUNDS 2 AND 3 ...... 77

FIGURE 29 - PROPOSED STRUCTURE FOR COMPOUND 5 (3,4,5,6-TETRABROMO-2-(3’,5’-DIBROMO-2’-

METHOXYPHENOXY)PHENOL) ...... 78

FIGURE 30 - STRUCTURE OF COMPOUND 4 (3,5,6-TRIBROMO-2-(3’,5’-DIBROMO-2’-METHOXYPHENOXY)PHENOL) ... 79

FIGURE 31 - COMPOUNDS 6 AND 8 OBTAINED FROM SAMPLE R3241C ...... 80

FIGURE 32 - STRUCTURE OF COMPOUND 7 (4,6-DIBROMO-2-(4’,5’,6’-TRIBROMO-2’-HYDROXYPHENOXY)PHENOL .... 81

FIGURE 33 - INHIBITION OF CRYPTOCOCCUS NEOFORMANS AND WTCA BY EXTRACTS OF DYSIDEA SPONGES ...... 83

FIGURE 34 - ILLUSTRATION OF ACTIVITY BASED ON THE DIFFERENCE IN CHEMICAL CONSTITUENTS ...... 84

viii

LIST OF PLATES

PLATE 1 - DIFFERENCES IN PHENOLIC COMPOSITION WITHIN L. HERBACEA FROM NEW CALEDONIA...... 39

PLATE 2 - TLC PROFILE OF D. ARENARIA FROM NEW CALEDONIA ...... 51

PLATE 3 - D. AVARA (R2082C) COMPARED WITH REFERENCE...... 53

PLATE 4 - TLC OF R3141C-2B-FR.8-11 FOR THE DETERMINATION OF PEPTIDES AND PHENOLS...... 74

PLATE 5 - TLC OF R3141C-PURIF PEAK 7 FOR DETERMINATION OF PEPTIDES...... 75

PLATE 6 - INHIBITION OF CRYPTOCOCCUS NEOFORMANS BY SELECTED DYSIDEA SPECIES ...... 84

LIST OF TABLES

TABLE 1 - DYSIDEA SPECIES COLLECTED IN VARIOUS GEOGRAPHICAL LOCATIONS ...... 10 1 TABLE 2 - HNMR CHEMICAL SHIFTS OBTAINED FOR COMPOUND 1 ...... 45 1 13 TABLE 3 - H AND C NMR SHIFTS OBTAINED FOR COMPOUND 2 ...... 47 1 13 TABLE 4 - H AND C NMR SHIFTS OBTAINED FOR COMPOUND 3 ...... 50

TABLE 5 - CHEMICAL SHIFTS OBTAINED FOR COMPOUND 4 ...... 79

TABLE 6 - CHEMICAL SHIFTS OBTAINED FOR COMPOUNDS 6 AND 8 ...... 80

TABLE 7 - CHEMICAL SHIFTS OBTAINED FOR COMPOUND 7 ...... 81

LIST OF SCHEMES

SCHEME 1 - HYDROALCOHOLIC EXTRACTION OF FREEZE-DRIED SPONGE MATERIAL ...... 25

SCHEME 2 - LIQUID-LIQUID PARTITIONING OF THE HYDROALCOHOLIC EXTRACT ...... 27

SCHEME 3 - COLD-EXTRACTION PROCEDURE FOR SMALL QUANTITIES OF SAMPLE...... 29

LIST OF ABBREVIATIONS

δ – Chemical Shift % – Percent λ – 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

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 xi

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

xii

LIST OF STRUCTURES

OR OH

Br O R1 2' 1 3' 1' 2 6

4' 6' 3 5 5' 4 Br Br

Br R2

Compound 1 R = Me; R1 = R2 = H 3,5-dibromo-2-(3’,5’-dibromo-2’-methoxyphenoxy)phenol

Compound 2 R = H; R1 = R2 = Br 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

Compound 5 R = Me; R1 = R2 = Br 3,4,5,6-tetrabromo-2-(3’,5’-dibromo-2’-methoxyphenoxy)phenol

OR OH

O Br 2' 1 3' 1' 2 6

4' 6' 3 5 5' 4 Br Br

R3 Br

Compound 6 R = Me; R3 = H 4,6-dibromo-2-(4’,6’-dibromo-2’-methoxyphenoxy)phenol

Compound 7 R = H; R3 = Br 4,6-dibromo-2-(4’,5’,6’-tribromo-2’-hydroxyphenoxy)phenol

Compound 8 R = Me; R3 = Br 4,6-dibromo-2-(4’,5’,6’-tribromo-2’-methoxyphenoxy)phenol

xiii

Br OH

O 2' 1 3' 1' 2 6

4' 6' 3 5 5' 4 Br Br Br

Compound 9 R4 = H

3,5-dibromo-2-(2’,4’-dibromophenoxy)phenol

Compound 10 R4 = Br

3,4,5-tribromo-2-(2’,4’-dibromophenoxy)phenol

xiv

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

1 INTRODUCTION

1.1 INTRODUCTION

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; Sladić & Gašić, 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-

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

1.2 BIOLOGY OF SPONGES AND THE GENUS DYSIDEA

1.2.1 History and Background of Sponges

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).

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

1.2.2 Morphology & Classification

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 Composition and Geographical Distribution of Marine Sponges of the Genus Dysidea

Figure 1 - Asconoid sponge (Brusca & Brusca, 2003)

Syconoid sponges, on the other hand, also have a tubular body and a single osculum, but they contain a thicker body wall with radial canals which empty into the spongocoel (Brusca & Brusca, 2003).

Figure 2 - Syconoid sponge (Brusca & Brusca, 2003)

The most complex organisation exists in leuconoid sponges consisting of flagellated chambers instead of a spongocoel, and it also has several incurrent and excurrent canals emptying into a central osculum (Brusca & Brusca, 2003).

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

Figure 3 - Leuconoid sponge (Brusca & Brusca, 2003)

All the water canals and spongocoel are lined with flagellated choanocyte cells which beat to create water current to flow through the sponge. Together with the incoming water, food (mainly bacteria, debris and microscopic algae) is also brought in and this sticks to the cells in the sponge which are later taken into food vacuoles and undergo intracellular digestion (Mather & Bennett, 1992; Lévi, 1998). The incoming water is also rich in oxygen and this is diffused into the cells while the metabolic wastes diffuse out of the cells, flowing out of the sponge with the water current through the osculum (Lévi, 1998).

Water flow is also an important factor for reproduction in sponges. Sponges are generally hermaphrodites (male and female gonads are present in one sponge), but they do not undergo self-fertilization to avoid asexual reproduction, and the resulting offspring has different characteristics as that of either parent. Often after fertilization, a blastula forms which is released into the water. The blastula develops into a larva and floats around until it finds a suitable substrate, after which it settles and develops into the sessile sponge form (Mather & Bennett, 1992; Lévi, 1998). Asexual reproduction is also possible in sponges by the process of external or internal budding, whereby the parent and offspring have the same genetic make-up (Mather & Bennett, 1992).

The phylum Porifera encompasses approximately 6000 taxonomically validated species, with new species still being discovered (Lévi, 1998; Brusca & Brusca, 2003). There are three main classes of the phylum Porifera as seen earlier: Calcarea, Demospongiae, and Hexactinellidae. A fourth class of sponges (Class 5

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

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.

1.2.3 The Genus Dysidea

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:

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

O CCl3 OMe OH N Br O Br

CCl3 O NH Br Br

Br S 4,6-dibromo-2-(3',4',6'-tribromo-2'-methoxyphenoxy)phenol Dysidenin N

OH H OCH3 O OH O H O OH OH H H OH H Herbasterol 6-hydroxyfurodysinin-O-methyl-lactone OH

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

Secondary Metabolites Composition and Geographical Distribution of Marine Sponges of the Genus Dysidea polychlorinated dysideaprolines and barbaleucamides was reported by Goetz, Harrigan, and Likos (2001).

9 CHY2 Dysideaproline R1 1 O 6 16 A - R1 = H, R2 = CH3, X = Y = Cl B - R1 = R2 = CH3, X = Y = Cl N C - R1 = R2 = H, X = Y = Cl 5 12 D - R = H, R = CH , X = H, Y = Cl X2HC N 1 2 3 11 3 13 E - R1 = H, R2 = CH3, X = Cl, Y = H F - R = H, R = CH , X = Cl, Y = ClH R2 O 1 2 3 2 SN

19 18

CH3

1 O R

Barbaleucamide 11 5 N CCl3 A - R = H 6 7 Cl3C B - R = CH3 12

O 10 SN

14 15

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).

H CH3 O Cl3C N CCl3 N Cl C 3 O O O N CH3 SN N

S Barbamide Pseudodysidenin

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

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

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.

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

Table 1 - Dysidea species collected in various geographical locations

Dysidea Localisation Compound Reference species Lamellodysidea Indian Ocean; Polybrominated diphenyl Anjaneyulu, Rao, Radhika, herbacea Zanzibar; Great ether Muralikrishna, & Connolly, Barrier Reef, 1996; Sionov, et al., 2005; Australia; Agrawal & Bowden, 2005; Indonesia Handayani, et al., 1997; Hanif, et al., 2007 L. herbacea Papua New Polychlorinated peptides; Flatt, Gautshci, Thacker, (with symbiotic Guinea Polychlorinated ketide Musafija-Girt, Crews, & cyanobacteria amino acid (Herbamide Gerwick, 2005; Clark & Oscillatoria A); Polychlorinated Crews, 1995 spongeliae) tetrapeptide (dysidenin) L. herbacea Great Barrier Chlorinated metabolites; Dumdei, Simpson, Garson, Reef, Australia; 5,5,5-trichloroleucine Byriel, & Kennard, 1997; Pohnpei, FSM; metabolite (Herbacic acid); MacMillan & Molinski, Red Sea Polychlorinated amino 2000; Unson, Rose, acid derivative; Faulkner, Brinen, Steiner, & Polychlorinated Clardy, 1993; Sauleau, pyrrolidinones Retailleau, Vacelet, & Bourget-Kondracki, 2005 L. herbacea Red Sea; Polyhydroxysterols; Sauleau & Bourget- Australia; Ichthyotoxic 9,11- Kondracki, 2005; Capon & Ethiopia secosterol (Herbasterol) Faulkner, 1985; Isaacs, Berman, Kashman, Gebreyesus, & Yosief, 1991 L. herbacea Yap State, Betaines Sakai, Suzuki, Shimamoto, Federated States & Kamiya, 2004 of Micronesia α,α-disubstituted α-amino Sakai, Oiwa, Takaishi, acid derivative Kamiya, & Tagawa, 1999 (Dysibetaine) Amino acid Sakai, et al., 2001 (neodysiherbaine A) L. herbacea Palau; India; Fiji Sesquiterpene; Dysinin- Sera, Adachi, Nishida, & type sesquiterpene 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; Sesquiterpene (9- Piggott & Karuso, 2005; FSM; Thailand hydroxyfurodysinin-O- Schmitz, Lakshmi, Powell, ethyl Lactone); & van der Helm, 1984; Sesquiterpenoids (arenarol Horton & Crews, 1995 & arenarone); Sesquiterpene ethers (arenarans) D. arenaria Whitsundays Sterol ECTA Jacob, et al., 2003 Reef, Australia D. avara Italy; Solomon Is.; Sesquiterpene Crispino, de Giulio, de Rosa, Australia hydroquinone (Avarol & Strazzullo, 1989; Alvi, derivatives); Diaz, Crews, Slate, Lee, & Furanosesquiterpene Moretti, 1992; Capon & (Thiofurodysinin) MacLeod, 1987 10

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

D. fragilis South China Sea Chlorinated Fu, Zeng, Su, & Pais, 1997; diketopiperizine Su, et al., 1993 (Dysidamide D) D. fragilis Mozambique Brominated diphenyl ether Utkina, Kazantseva, & Denisenko, 1987 D. fragilis Fiji; FSM Azacyclopropene & Molinski & Ireland, 1988; Derivatives; 2H-Azirines Salomon, Williams, & Faulkner, 1995; Skepper & Molinski, 2008 D. fragilis Spain; Venice; Furanosesquiterpenoid; Marin, Lopez, Esteban, Black Sea; Oahu Sesquiterpenes; Steroids; Meseguer, Munoz, & Polyhydroxylated sterols; Fontana, 1998; Aiello, Sesquiterpenoid bicyclo Fattorusso, Menna, & [3.3.1] nonane aldehyde Pansini, 1996; Milkova, lactone Mikhova, Nikolov, Popov, & Andreev, 1992; Schulte, Scheuer, & McConnell, 1980 D. etheria Bermuda β,γ-Epoxy γ-Lactone Schram & Cardellina, 1985 Polyhydroxylated sterols West & Cardellina, 1988 Indoles Cardellina, Nigh, & VanWagenen, 1986 Sesquiterpene lactone Grode & Cardellina, 1984 (furodysinin lactone) Furanosesquiterpenes Cardellina & Barnekow, (Nakafurans) 1988 D. etheria Caribbean Sesterterpene (Dysidiolide) Takahashi, Dodo, Hashimoto, & Shirai, 2000 Gunasekera, McCarthy, Kelly-Borges, Lobkovsky, & Clardy, 1996 D. fusca New Caledonia Drimane sesquiterpene Montagnac, Martin, Debitus, (East Coast) & Pais, 1996 D. chlorea Yap State, FSM Polychlorinate Fu, Ferreira, Schmitz, & diketopiperazines Kelly-Borges, 1998 (dysidamides I-T) Sterol D. chlorea Okinawa Sesquiterpenes Ueda, Kadekaru, Siwu, Kita, (Heterumadysins A-D) & Uemura, 2006 D. cinerea Dahlak Sesterpenes (Bilosespens Rudi, Yosief, Schleyer, & archipelago, A & B) Kashman, 1999 Eritrea (Red Sea) D. frondosa Pohnpei Sesquiterpene Patil, et al., 1997 hydroquinone deriv. D. cristagalli Spirits Bay, Sesquiterpene quinones McNamara, et al., 2005 Northland, New Zealand D. amblia CA, USA Diterpenes Walker & Faulkner, 1981; Walker, Rosser, & Faulkner, 1984 D. dendyi Scott reef, North- Brominated dibenzo-p- Utkina, et al., 2001 west Australia dioxins (spongiadioxin A & B)

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

D. incrustans Coast of Tunisia, Polyoxygenated steroids Casapullo, Minale, Zollo, Mediterranean Sea (incrustasterol A & B) Roussakis, & Verbist, 1995 D. reformensis Gulf of California, Sesquiterpene Carballo, Zubía, & Ortega, D. cachui Pacific Ocean, hydroquinone 2006 Mexico D. uriae Furanosesquiterpene (dendrolasin) Dysidea sp. FSM; Philippines; Polybrominated diphenyl Fu & Schmitz, 1996; Zhang, Fiji ether Skildum, Stromquist, Rose- Hellekant, & Chang, 2007; Salva & Faulkner, 1990; Fu, Schmitz, Govindan, & Abbas, 1995 Dysidea sp. Philippines; Polychlorinated Harrigan, Goetz, Luesch, Indonesia compounds Yang, & Likos, 2001; Ardá, (Dysideaprolines A-F & et al., 2005 Barbaleucamides A-B); Polychlorinated dipeptide Dysidea sp. Palau; Red Sea; Diterpenes of the Lu & Faulkner, 1998; Australia; dolabellane class; 14- Carmely, Cojocaru, Loya, & Philippines; membered macrolide; Kashman, 1988; Garson, Guam; Vanuatu; Diterpenes; Sesquiterpene Dexter, Lambert, & Liokas, USA; PNG; South ester (7-deacetoxy- 1992; Goetz, Harrigan, & China Sea olepupuane); Likos, 2001; Paul, Seo, Cho, Sesquiterpenes (Furodysin Rho, Shin, & Bergquist, lactone & Pyrodysinoic 1997; Giannini, et al., 2001; acid); Drimane Pérez-García, Zubía, Ortega, sesquiterpenoid; & Carballo, 2005; Diaz- Sesquiterpene Marrero, et al., 2006; de cyclopentenones and Guzman, et al., 1998; aminoquinones; Ciavatta, et al., 2007 Merosesquiterpenes; Meroterpenoid (Avinosol); Sesquiterpene hydroquinone (Bolinaquinone); Tetracyclic sesquiterpene - substituted quinone (Puupehenone) Dysidea sp. Okinawa Benzothiazole-2-one Suzuki, et al., 1999 analog Phyllospongia Palau Polybrominated diphenyl Liu, Namikoshi, Meguro, dendyi ethers 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.

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

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

1.3 SPONGE – SYMBIONT ASSOCIATION

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

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

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

1.4 SECONDARY METABOLITES AND CHEMOTAXONOMY

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 15

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

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.

1.5 NATURAL PRODUCTS AND DRUG DISCOVERY

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).

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

OH O O H R N N OH

OH OH O

Bengamide A: R = H H O H3C(H2C)12CO2 Bengamide B: R = CH3 HN O

H O O N Br N O O H O O HN H C O 3 H H NH O N O O H H H Jaspamide 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 (Sladić & Gašić, 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 ω-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. 17

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

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.

1.6 ANTIFUNGAL BIOASSAY

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.

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

1.6.1 Cryptococcus neoformans

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.

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

1.6.2 Candida albicans

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.

1.6.3 New Targets for Antimicrobial Drug Development

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

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

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 β-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

Secondary Metabolites Composition and Geographical Distribution of Marine Sponges of the Genus Dysidea protein prenylation is an interesting target for the further development of antimicrobial 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.

1.7 RELEVANCE OF STUDY

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. 22

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

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.

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

2 GENERAL METHODOLOGY

2.1 SAMPLE COLLECTION AND IDENTIFICATION

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.

2.2 EXTRACTION TECHNIQUES

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.

2.2.1 Technique 1

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 24

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

20 g Freeze dried powder

100 mL (more if necessary) of a

solution of EtOH/H2O (80/20) Stirring 1 night

Filtration

Filtrate Solid Residue 100 mL (more if necessary) of EtOH - Stirring 45 minutes

Filtration

Evaporation Filtrate Solid Residue 100 mL (more if necessary) of Residue EtOH - Stirring 45 minutes 100 mL DCM 100 mL H2O Filtration

Liquid-liquid partitioning Filtrate Solid Residue

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,

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

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

Residue of filtrate from Hydroalcoholic extraction

+ 100 mL DCM

+ 100 mL H2O

+ 100 mL H2O STEP 1 Separating Funnel No. 1 Separating Funnel No. 2

Liquid-liquid partition Liquid-liquid partition

Organic Layer 1 Organic Layer 1’

STEP 2 Separating Funnel No. 1 With aqueous layer 1 Separating Funnel No. 2 With aqueous layer 1’ + 100 mL DCM Liquid-liquid partition Liquid-liquid partition

Organic Layer 2 Organic Layer 1’ & 2’

Separating Funnel No. 1 STEP 3 With aqueous layer 1 Separating Funnel No. 2 With aqueous layer 1’ + 100 mL DCM

Liquid-liquid partition Liquid-liquid partition

Aqueous Layer Organic Layer 3 Aqueous Layer Organic Layer 1 1’ 1’, 2’ & 3’

Freeze drying Drying on Na2SO4 Filtration on paper Evaporation

Extract B Extract C

Scheme 2 - Liquid-liquid partitioning of the Hydroalcoholic extract

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

2.2.2 Technique 2

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:

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

2g Freeze-dried

SPE Phenomenex 3 x 20mL DCM/MeOH (1:1) with Strata C18 Cartridge 10 min Ultrasonication each time Washing

Filter on cotton & dry 10mL DCM/MeOH (rotavapor) after each (1:2) Conditioning Add 1g RP-C silica 18 10mL MeOH; before last drying 10mL Water

Load adsorbed C18 silica on column

10mL MilliQ water to wash out salt from sample

Dry under vacuum for approx. 30 min to remove water Elution

9mL DCM/MeOH (1:1) under vacuum; collected in 10mL volumetric flask

Make up to mark with DCM/MeOH (1:1) = Extract A

1mL 1mL 1mL

Extract A1 Extract A2 Extract A3 Filtered for HPLC Filtered for LC-MS Dried, weighed & stored at -20 oC for TLC & Bioassays

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

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

2.3 CHEMICAL ANALYSIS

2.3.1 Thin Layer Chromatography (TLC)

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.

2.3.2 High Performance Liquid Chromatography (HPLC)

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).

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

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

2.3.3 Liquid Chromatography – Mass Spectroscopy (LC-MS)

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.

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

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.

2.3.4 Nuclear Magnetic Resonance (NMR) Spectroscopy

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).

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

2.4 ISOLATION AND STRUCTURE DETERMINATION

2.4.1 Dysidea Species R3141C

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

Secondary Metabolites Composition and Geographical Distribution of Marine Sponges of the Genus Dysidea 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 (λ) 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.

2.4.2 Lamellodysidea herbacea

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

Secondary Metabolites Composition and Geographical Distribution of Marine Sponges of the Genus Dysidea covered. Over time, the crystals formed and settled to the bottom, after which the supernatant was decanted and the procedure was repeated.

2.5 ANTI-FUNGAL BIOASSAY

2.5.1 Media (Sabouraud Dextrose Agar – SDA) Preparation

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.

2.5.2 Anti-fungigram Discs

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

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

2.5.3 Fungal Inoculation

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.

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

3 RESULTS & DISCUSSION

3.1 CHEMICAL SCREENING

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.

3.1.1 Lamellodysidea herbacea

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

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

Br O 2' 1 3' 1' 2 6 4' 6' 3 5 5' 4 Br Br HO Cholesterol Br 3,5-dibromo-2-(3',5'-dibromo-2'-methoxyphenoxy)phenol (4Br-OH-OMe-PBDE)

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:

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

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

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

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

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

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.

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

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.

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

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.

3.1.1.4.1 Compound 1

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 1 analyser], HNMR [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

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

(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 δ J for δ J for δ MAU- J for δ R2259C R2259C R2279C R2279C 02-Dh1 MAU- Literature (NC) (Hz) (NC) (Hz) (Moorea) 02-Dh1 (ppm)* (ppm) (ppm) (ppm) (Hz) 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)

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

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 OH

Br O 2' 1 3' 2 1' 6 4' 6' 3 5 5' 4 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) 1 HNMR 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)

Figure 15 - Compound 1 (3,5-dibromo-2-(3’,5’-dibromo-2’-methoxyphenoxy)phenol)

3.1.1.4.2 Compound 2

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.

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

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.)

1 Following fractionation, the new HNMR 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.

13 The molecule was also analysed for C 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

300 MHz 400 MHz δ Lit. in (CDCl ) (Acetone-d ) H C # 3 6 CDCl 13C 1H 3 1H (ppm) HMBC (ppm)* (ppm) (ppm) 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)

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

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 1 analyser], HNMR [300 MHz; in CDCl3], and structure

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

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).

3.1.1.4.3 Compound 3

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:

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

Table 4 - 1H and 13C NMR shifts obtained for Compound 3

C # 300 MHz (CDCl3) 400 MHz (Acetone-d6) δH Lit. in 1 13 1 H (ppm) C (ppm) H (ppm) CDCl3 (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

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

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.

3.1.2 Dysidea arenaria

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

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

3.1.3 Dysidea avara

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)

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

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.

3.1.4 CONCLUSION

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, 53

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

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

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

3.2 GEOGRAPHICAL DISTRIBUTION OF DYSIDEA SPECIES

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:

Figure 18 - Collection sites of L. herbacea for geographical comparison (Source: Google Maps)

3.2.1 ECOLOGY OF THE GENUS DYSIDEA

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

Secondary Metabolites Composition and Geographical Distribution of Marine Sponges of the Genus Dysidea 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 57

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

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

3.2.2 SIMILARITIES AND DIFFERENCES IN CHEMICAL COMPOSITION AND GENETIC LINKAGE BETWEEN LAMELLODYSIDEA HERBACEA BASED ON GEOGRAPHICAL DISTRIBUTION

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

3.2.2.1.1 Comparison of the Chemical Profiles

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:

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

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).

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

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

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

3.2.2.1.2 Comparison of the Chemical Profiles with Gene Mapping

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:

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

R2278 L. herbacea NC R2274 L. herbacea NC R2256 L. herbacea NC R2259 L. herbacea NC R2261 L. herbacea NC R2280 L. herbacea NC R2262 L. herbacea NC R2252 L. herbacea NC R2271bis L. herbacea Vanuatu R3241 L. herbacea Fiji R2258 L. herbacea NC R2279 L. herbacea NC R2254 D. arenaria NC R2257 D. arenaria NC R2260 D. arenaria NC R2263 D. arenaria NC R2261 D. arenaria NC R3314 D. arenaria Fiji R3033 Dysidea 4177 Solomons R3161 Dysidea 4177 Solomons R3141 Dysidea sp. Solomons R3147 L. herbacea Solomons R3310 D. pallescens Fiji

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.

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

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

3.2.2.2.1 Comparison of the Chemical Profiles

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.

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

The following figure illustrates the comparative HPLC profiles of the geographically different L. herbacea with their finalized structures:

1 O OH 2 OH OH Br O Br O Br 1 6' 1' 2' 2 6 5' 3' 3 5 4' Br 4 Br Br Br Br Br Br

3 OH OH 4 O OH Br O Br Br O Br

Br Br Br Br Br Br

5 O OH 6 O OH Br O Br O Br

Br Br Br Br Br Br Br

OH OH 7 8 O OH O Br O Br

Br Br Br Br Br Br Br Br

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.

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

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.

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

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.

3.2.2.2.2 Comparison of the Chemical Profiles with Gene Mapping

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

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

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.

3.2.3 COMPARISON OF DYSIDEA ARENARIA AND D. AVARA FROM NEW CALEDONIA AND FIJI, AND 3 DYSIDEA SPECIES FROM THE SOLOMON ISLANDS

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

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

Br OH Br OH

O O 2' 1 3' 1' 2 6

4' 6' 3 5 5' 4 Br Br Br Br Br Br

Compound 9 Br 3,5-dibromo-2-(2',4'-dibromophenoxy)phenol Compound 10 3,4,5-tribromo-2-(2',4'-dibromophenoxy)phenol

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,

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

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.

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

3.2.4 CONCLUSION

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.

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

3.3 ISOLATION & STRUCTURE DETERMINATION OF COMPOUNDS FROM DYSIDEA SPECIES

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.

3.3.1 DYSIDEA SPECIES R3141C

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.

1 Figure 24 - HNMR [300 MHz; in CDCl3] and HPLC [Condition 2; UV 254 nm] profiles of R3141C

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

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 (Cα) were also visible in the region between 4 and 5 ppm.

1 Figure 25 - HNMR 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

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

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

Peak of degradation A Peak of degradation B

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.

1 Figure 26 - HNMR of R3141C peak 5 (fraction of R3141C) in CDCl3 75

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

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 OH HO O O O 20

O 18 15 H C24H32O5 14 Exact Mass: 400.22 H Mol. Wt.: 400.51 O

H 13 OH O

H O 12 11 O Puupehenone H C21H28O4 H Exact Mass: 344.20 C21H28O3 Mol. Wt.: 344.44 Exact Mass: 328.20 Mol. Wt.: 328.45

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; Sladić & Gašić, 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 (Sladić & Gašić, 2006). 76

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

3.3.2 LAMELLODYSIDEA HERBACEA

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:

OH OH OH OH

Br O Br Br O Br 2' 1 2' 1 3' 2 6 3' 2 1' 1' 6 6' 5 4' 3 4' 6' 3 5 5' 4 5' 4 Br Br Br Br

Br Br Br Compound 2 Compound 3 3,4,5,6-tetrabromo-2-(3',5'-dibromo-2'-hydroxyphenoxy)phenol 3,5,6-tribromo-2-(3',5'-dibromo-2'-hydroxyphenoxy)phenol

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.

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

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 OH

Br O Br 2' 1 2 3' 1' 6

4' 6' 3 5 5' 4 Br Br

Br Br Compound 5 3,4,5,6-tetrabromo-2-(3',5'-dibromo-2'methoxyphenoxy)phenol

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:

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

O OH

Br O Br 1 3' 2' 1' 2 6 4' 6' 3 5 5' 4 Br Br

Br Compound 4 3,5,6-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)

13 1 C # C H δH Lit. in Acetone-d6 (ppm) (ppm) (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)

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

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 OH

O Br 2' 1 3' 2 1' 6 4' 6' 3 5 5' 4 Br Br

R Br R = H --> Compound 6 : 4,6-dibromo-2-(4',6'-dibromo-2'-methoxyphenoxy)phenol R = Br --> Compound 8 : 4,6-dibromo-2-(4',5',6'-tribromo-2'-methoxyphenoxy)phenol

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 1H 13C 1H *Compd. 6 **Compd. 8 (ppm) (ppm) (ppm) (ppm) (CDCl3) (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)

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

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 OH

O Br 2' 1 2 3' 1' 6

4' 6' 3 5 5' 4 Br Br

Br Br

Compound 7 - 4,6-dibromo-2-(4',5',6'-tribromo-2'-hydroxyphenoxy)phenol

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* 13 1 # C (ppm) H (ppm) HMBC in CDCl3 (ppm) 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

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

3.3.3 CONCLUSION

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.

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

3.4 ANTI-FUNGAL BIOASSAY

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.

Inhibition of Cryptococcus & WTCA by Organic extracts Dysidea sponges 20 18 16 14 12 10 8 6 4 2 0 fr.4 fr.1 fr.4 fr.3 fr.4 fr.5 fr.1 fr.3 fr.4 ------PBDE PBDE - - R2252C R2256C R2259C R2261C R2262C R2265C R2267C R2269C R2270C R2272C R3147C R3241C R2274C R2278C R2279C R2280C R2254C R2257C R2260C R2263C R2264C R2268C R2271C R3203C R3314C R2082C R3033C R3161C R3141C R3310C R2271bisC R2247bisC R2261bisC 2OH OMe - purifpeak purifpeak 6 - R3141C R3241C R3241C R3147C R3147C R3147C R2252C R2252C R2252C MooreaDh1 - DCM control DCM MayotteDh1 Nystatin Ref. Nystatin 6Br OH - 4Br Crypto WTCA R3141C

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

Secondary Metabolites Composition and Geographical Distribution of Marine Sponges of the Genus Dysidea 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. 84

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

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.

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

4 GENERAL CONCLUSION

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.

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

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Venkateswarlu, Y., Biabani, M. A., Reddy, M. V., Chavakula, R., & Rao, J. V. (1994). A new sesquiterpene from the Andaman sponge, Dysidea herbacea. J. Nat. Prod. , 57 (6), 827-828.

Vetters, W., Stoll, E., Garson, M. J., Fahey, S. J., Gaus, C., & Muller, J. F. (2002). Sponge halogenated natural products found at parts-per-million levels in marine animals. Environmental Toxicology and Chemistry , 10 (21), 2014-2019.

Voet, D., & Voet, J. G. (2004). Biochemistry (3rd ed.). USA: John Wiley and Sons, Inc.

Voinov, V. G., Elkin, Y. N., Kuznetsova, T. A., Maltsev, I. I., Mikhailov, V. V., & Sasunkevich, V. A. (1991). Use of mass spectrometry for the detection and identification of bromine-containing diphenyl ethers. J. Chromatogr. , 586, 360- 362.

Walker, R. P., & Faulkner, D. J. (1981). Diterpenes from the sponge Dysidea amblia. J. Org. Chem. , 46 (6), 1098-1102.

Walker, R. P., Rosser, R. M., & Faulkner, D. J. (1984). Two new metabolites of the sponge Dysidea amblia and revision of the structure of Ambliol B. J. Org. Chem. , 49 (26), 5160-5163.

Walsh, D. (2004). Take two clams and call me in the morning. Proceedings of the United States Naval Institute, 130(11), p. 90.

Wang, Y., & Casadevall, A. (1996). Susceptibility of melanized and non-melanized Cryptococcus neoformans to the melanin-binding compounds Trifluoperazine and Chloroquine. Antimicrobial Agents and Chemotherapy , 40 (3), 541-545.

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West, R. R., & Cardellina, J. H. (1988). Three new polyhydroxylated sterols with the 5β-configuration from the sponge Dysidea etheria. J. Org. Chem. , 54, 3234-3236.

Xu, Y.-M., Johnson, R. K., & Hecht, S. M. (2005). Polybrominated diphenyl ethers from a sponge of the Dysidea genus that inhibit Tie2 kinase. Bioorganic & Medicinal Chemistry , 13, 657-659.

Zhang, H., Skildum, A., Stromquist, E., Rose-Hellekant, T., & Chang, L. C. (2007). Bioactive polybrominated diphenyl ethers from the marine sponge Dysidea sp. J. Nat. Prod. , (Article in Press).

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6 APPENDIX

6.1 Appendix 1: Dysidea species studied for Chemical Screening

Freeze- Mass % Mass Description dried of Yield of of Ext. C # Ext. Code Species Location Sponge Ext. C of Ext. B (g) (g) Ext. C (g) New Green; 1 R2252C L. herbacea Caledonia 16.174 0.6989 4.32 1.4202 Gummy; (NC) Semi-solid; 2 R2256C L. herbacea NC 20.943 0.9612 4.59 1.9702 Sticky substance 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 L. 20.320 0.8606 4.23 1.1297 5 R2262C NC herbacea? 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 11* R2274C L. herbacea NC 12* R2278C L. herbacea NC 13* R2279C L. herbacea NC 14* R2280C L. herbacea NC MAU-02- 2.0260 0.1237 6.11 - 15 L. herbacea Moorea Dh1 16* R2247bisC D. arenaria NC Yellow- brown; 20.824 0.1850 0.89 0.9106 17 R2254C D. arenaria NC Gummy; 18 R2257C D. arenaria NC 21.664 0.2633 1.22 1.1141 Semi-solid; Sticky 19* R2260C D. arenaria NC substance. 20 R2261bisC D. arenaria NC 21.844 0.3226 1.48 2.0333 21* R2263C D. arenaria NC 22* R2264C D. arenaria NC 23 R2268C D. arenaria NC 21.321 0.2012 0.94 1.2007 24* R2271C D. arenaria NC Gummy; Yellow- 25 R2082C D. avara NC 1.0006 0.0316 3.16 brown; sticky; semi- solid extract. *Extracts obtained from IRD Nouméa.

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6.2 Appendix 2: Guide to determine halogenation pattern and number

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6.3 Appendix 3: 1HNMR details of Compounds 1, 2, and 3 from L. herbacea

6.3.1 Appendix 3a: 1HNMR profiles of L. herbacea for Compound 1

[300 MHz; in CDCl3]

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6.3.2 Appendix 3b: 1-D and 2-D NMR Profiles of Compound 2

Compound 2 - 1H-NMR at 400 MHz (in acetone-d6)

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Compound 2 - 13C-NMR at 400 MHz (in acetone-d6)

Compound 2 – HSQC at 400

MHz (in acetone-d6)

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Compound 2 – HMBC at 400

MHz (in acetone-d6)

OH OH

Br O Br 2' 1 3' 1' 2 6

4' 6' 3 5 5' 4 H Br H Br

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

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6.3.3 Appendix 3c: 1HNMR Profile of Compound 3

Compound 3 – 1H-NMR at 400

MHz (in acetone-d6)

Compound 3 (7.64 ppm) Compound 2 (7.41 ppm) Compound 2 (6.85 ppm) Compound 3 Compound 3 (6.74 ppm) (7.40 ppm)

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Compound 3 – 13C-NMR at 400

MHz (in acetone-d6)

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6.4 Appendix 4: NMR shifts for all compounds identified and the Yields obtained for the four compounds

6.4.1 Appendix 4a: 1H & 13C NMR shifts for all compounds identified

Compound 1 2 3 4 6 7 8 C# H C H C H C H C H C H C H C 1 150.49 151.54 151.50 145.17 144.88 145.13 2 140.62 139.88 139.78 147.55 146.75 147.24 3 - - 119.31 117.54 117.65 6.62 117.15 6.73 116.71 6.71 117.19 4 7.36 - 121.60 7.64 128.54 7.65 128.71 111.52 111.34 111.62 5 - - 127.22 124.01 124.05 7.377 129.91 7.38 129.76 7.39 130.11 6 7.20 - 116.48 115.47 115.80 111.90 111.69 112.00 1’ 145.34 145.28 147.20 155.29 151.63 154.00 2’ 146.89 147.12 152.64 141.14 140.70 142.16 3’ 6.80 6.85 117.12 6.74 117.02 6.82 118.18 118.23 123.02 4’ - - 111.74 111.70 117.62 7.49 128.31 123.05 123.52 5’ 7.46 7.41 130.40 7.40 130.26 7.48 130.38 120.71 122.64 119.52 6’ - - 112.19 112.15 120.13 7.425 117.76 7.49 122.35 7.64 119.02 -OH 5.08 6.01 -OH - 6.04 -OMe 3.81 - 4.00 61.76 3.87 3.87

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6.4.2 Yields obtained for the isolated compounds

Compound Name Amount Mass of Isolate Yield Description of of Extract (mg) Compound used (mg) 2 3,4,5,6-tetrabromo-2- 302.5 9.1 3.0 % White crystals (3’,5’-dibromo-2’- Mpt. = 191 – 191.6 oC hydroxyphenoxy)phenol 6 4,6-dibromo-2-(4’,6’- 5956.2 81.8 1.37 % Mixture of two these dibromo-2’- compounds; methoxyphenoxy)phenol Crystalline yellow- 8 4,6-dibromo-2-(4’,5’,6’- white compounds tribromo-2’- methoxyphenoxy)phenol 7 4,6-dibromo-2-(4’,5’,6’- 5956.2 248 4.16 % Crystalline yellow- tribromo-2’- white compounds hydroxyphenoxy)phenol

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6.5 Appendix 5: Spectral Data – Dysidea arenaria

1 [HPLC Condition 2 + Finnigan APCI-MS; HNMR at 300 MHz in CDCl3]

RT: 0.00 - 34.99 24.32 100 D. arenaria 90 Total PDA scan 80 2.45 2.68 70

60 1.48 15.90

50 9.93 12.05 15.47 20.83 5.07 18.88 24.70 40 6.30 8.57 17.95

Relative Absorbance Relative 30 25.85 29.35 20

0 13.18 100

90 D arenaria 80 Total Mass scan 70 . 60

50 13.52

40 20.52 21.74

Relative Abundance Relative 30 18.02 22.23 15.15 20 15.36 22.71 10 24.68 2.52 2.75 6.67 9.15 12.71 29.60 30.19 0 0 5 10 15 20 25 30 Time (min) D. arenaria ELS scan

D. arenaria - 1HNMR at 300 MHz

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6.6 Appendix 6: Spectral Data: Dysidea avara

1 [HPLC Condition 2 + Finnigan APCI-MS; HNMR at 300 MHz in CDCl3]

RT: 0.00 - 34.98 2.62 100 D. avara 90 Total PDA scan 80

70 3.42 60 15.85 50 18.82 24.32 40 19.92 23.05 14.43 17.57 24.72

Relative Absorbance Relative 30 10.18 13.38 30.23 20 9.48 7.35 10 5.83

0 21.75 100

90 D. avara 80 Total Mass scan 22.26 70

40 20.54 22.76 2.71 3.51 22.91

Relative Abundance Relative 30 19.89 18.00 23.35 20 14.92 13.48 24.90 5.95 11.36 26.14 10 2.07 29.10 30.94 0 0 5 10 15 20 25 30 Time (min) D. avara ELS scan

D. avara - 1HNMR at 300 MHz

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6.7 Appendix 7: Comparison of L. herbacea, D. arenaria and D. avara.

TLC profile of L. herbacea, D. arenaria and D. avara after visualization using Vanillin Phosphoric Acid:

HPLC profile comparison of the 3 species of Dysidea studied (HPLC Condition 2; UV absorption at λ = 254 nm):

1.00

0.80

0.60 AU

0.40

L. herbacea 0.20 D. avara

D. arenaria 0.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 Minutes

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1 HNMR of the 3 species investigated (at 300 MHz; in CDCl3):

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

6.8 Appendix 8: Specimens studied for Ecological and Geographical comparison

Area of Station Sample Photograph of Sample Description Collection

Colour - pale green; Encrusting New on hard surface; R2252 Caledonia Finger-like ST720 L. (22°32.736S projections in herbacea 166°26.588E) some regions, no out-growth on the other.

Green, lamellate projections from New R2256 an encrusting base; Caledonia ST1048 L. in competition for (22°41.566S herbacea space with other 166°38.704E) sessile marine organisms.

Green, lamellate to lobate projections New from an encrusting R2261 Caledonia base; in ST1052 L. (22°21.249S competition for herbacea 166°14.783E) space with other sessile marine organisms.

Green, lamellate projections from New R2262 an encrusting base; Caledonia ST1053 L. little competition, (22°16.456S herbacea therefore upward 166°14.311E) growth of the leaf- like projections.

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Green sponge with encrusting base New attached to rocky R2265 Caledonia surface, small ST1055 L. (22°01.865S projections; found herbacea 165°57.579E) in the middle of many sessile invertebrates.

Green sponge with encrusting base New attached to rocky R2267 Caledonia surface, small ST956 L. (21°51.695S projections; found herbacea 165°43.429E) in the middle of many sessile invertebrates.

Green sponge with encrusting base attached to rocky New R2269 surface, small Caledonia ST1059 L. projections; found (22°10.980S herbacea in the middle of 166°06.066E) many sessile invertebrates; small colony seen. Green sponge with encrusting base attached to rocky New R2272 surface, small Caledonia ST254 L. projections; found (22°17.013S herbacea in the middle of 166°11.035E) many sessile invertebrates; small colony seen. Green sponge with encrusting base attached to rocky New R2270 surface, small Caledonia ST1060 L. projections; found (22°23.201S herbacea in the crevice 166°17.065E) between two rocks; small colony seen.

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Green sponge with encrusting base New R2274 attached to flat Caledonia ST1132 L. rocky surface, not (22°19.350S herbacea so many 167°02.027E) projections seen; small colony.

Blue-green sponge with encrusting New R2278 base attached to Caledonia ST1135 L. flat rocky surface, (22°06.832S herbacea not so many 167°00.321E) projections visible; small colony.

New R2280 Caledonia ST1127 L. No Photo Available (21°32.777S herbacea 165°12.996E)

Green sponge with New R2259 lamellate/lobate Caledonia ST1050 L. projections; (22°23.295S herbacea surrounded by 166°47.301E) sediment.

Blue-green, encrusting sponge; large but generally New flat colony, very R2279 Caledonia few lamellate ST1136 L. (22°08.287S projections visible; herbacea 166°56.955E) occupying large surface area of a rock containing other organisms. Vanuatu (13°30.910S R2271bis 167°21.100E) ST613 L. No Photo Available

herbacea

167°21.100E)

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

Blue-grey sponge with lamellate projections; small R3147 Solomon Is. colony encrusted ST840 L. (09°11.055S on a rocky surface; herbacea 160°11.982E) habitat a little darker than that for New Caledonian samples. Blue-grey sponge with lamellate projections (purple R3241 Fiji Is. when out of the FJ4 L. (16°18.847S water); colony herbacea 178°54.595E) encrusted on a rocky surface with coral and algae growing on it. Found in lagoon with clear water at a depth of 1 - 1.5 m; Encrusting on MAU- hard corals with a 02-Dh1 big lamellate Moorea Is. L. colony; in close herbacea proximity of two cyanobacteria, Anabaena torulosa and an unidentified species. Found in lagoon with clear water at MAU- a depth of 1 - 1.5 02-Dh2 m; Growing close Moorea Is. No Photo Available L. to hard corals; No herbacea algae seen growing around the samples. Found in lagoon with clear water at a depth of 1 - 1.5 MAU- m; Encrusting on 02-Dh3 hard corals with a Moorea Is. L. big lamellate herbacea colony; in close proximity of two cyanobacteria, Anabaena torulosa.

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MAY- 02-Dh1 Mayotte Is. No Photo Available L. herbacea MAY- 02-Dh2 Mayotte Is. No Photo Available L. herbacea New R2247bis Caledonia ST124 D. No Photo Available (22°20.611S arenaria 166°25.970E)

Grey lobate colony clustered together New R2254 (only one colony Caledonia ST534 D. seen); growing on (22°20.700S arenaria sandy substrate; 166°20.000E) surrounded by sea- weed and algae.

Grey lobate colony clustered together New R2257 (only one colony Caledonia ST1049 D. seen); growing on (22°31.008S arenaria sandy substrate; 166°36.212E) surrounded by sea- weed and algae.

Grey lobate colony clustered together New R2260 (only one colony Caledonia ST1051 D. seen); growing on (22°19.699S arenaria sandy substrate; 166°23.463E) surrounded by sea- weed and algae.

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

Grey lobate colony clustered together (only one colony New R2263 seen); growing on Caledonia ST1054 D. sandy substrate; (22°18.936S arenaria surrounded by sea- 166°21.176E) weed and algae; sedimentation seen. Grey lobate colony clustered together (only one colony New R2261bis seen); growing on Caledonia ST1053 D. sandy substrate; (22°16.456S arenaria surrounded by sea- 166°14.311E) weed and algae; sedimentation seen. Grey lobate colony clustered together (only one colony New R2264 seen); growing on Caledonia ST1057 D. sandy substrate; (21°51.971S arenaria surrounded by sea- 165°45.628E) weed and algae; sedimentation seen.

New R2268 Caledonia ST1058 D. No Photo Available (22°07.065S arenaria 166°06.556E)

Grey lobate colony

clustered together (only one colony New seen - small in R2271 Caledonia size); growing on ST1061 D. (22°15.529S sandy substrate; arenaria 166°20.155E) surrounded by sea- weed and algae; sedimentation seen slightly.

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

Pink in color; growing in the middle of a group of sea-weeds; has R3314 Fiji Is. the appearance of a FJ18 D. (16°06.762S perforated ball (not arenaria 179°11.264W) lobate like the other New Caledonian samples). New R2082 Caledonia ST612 No Photo Available D. avara (20°32.930S 167°10.790E)

Red in color when R3203 Fiji Is. outside water; FJ1 D. cf. (16°46.600S rigid-looking avara 178.24.344E) stems like that of hard corals.

Purple in color; growth pattern as seen in hard corals R3033 Solomon Is. but scattered; ST823 Dysidea (08°47.266S found on a rocky 4177 158°14.228E) outgrowth; surrounged by algae and sea- ferns. Purple in color; growth pattern as seen in hard corals R3161 Solomon Is. with finger-like ST846 Dysidea (08°58.067S projections arising 4177 160°44.682E) from a common stem; dark purple in color when out of the water.

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

Maroon in color, and leaf-like R3141 Solomon Is. appearance; ST840 Dysidea (09°11.055S growing with other sp. 160°11.982E) sea-weeds and algae.

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

6.9 Appendix 9: Detailed map of the 6 sampling sites and the approximate distance between them

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

Distance Site 1 Site 2 (km) Vanuatu 836.89 Solomon 1427.4 NC Fiji 1420.71 Moorea 4471.05 Mayotte 12115.62 Solomon 871.29 Fiji 1226.96 Vanuatu Moorea 4488.15 Mayotte 12655.56 Fiji 2091.66 Solomon Moorea 5277.34 Mayotte 12164.15 Moorea 3237.39 Fiji Mayotte 13584.88 Moorea Mayotte 15787.65

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

6.10 Appendix 10: Spectral details of R3147C and R3241C fractions

6.10.1 : Appendix 10a: R3147C [HPLC Condition 2 + PDA 254 nm + APCI-

MS; 1HNMR & 13CNMR at 400 MHz in Acetone-d6 and at 300 MHz in

CDCl3]

R3147C – 1H-NMR at 400 MHz (in acetone-d6)

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

R3147C – 13C-NMR at 400 MHz (in acetone-d6)

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

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

6.10.2 : Appendix 10b: R3241C [HPLC Condition 2 + PDA 254 nm + APCI-

MS; 1HNMR at 300 MHz in CDCl3]

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

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

6.11 Appendix 11: Mayotte sample (MAY-02-Dh1) – Structure determination

[HPLC Condition 2 + PDA 254 nm; 1-D & 2-D NMR at 400 MHz in Acetone- d6]

MAY2-3 – 1H-NMR for Compounds 6

and 8 at 400 MHz (in acetone-d6)

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

MAY2-3 – 13C-NMR for Compounds 6

and 8 at 400 MHz (in acetone-d6)

MAY2-3 –HSQC for Compounds 6 and

8 at 400 MHz (in acetone-d6)

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

MAY2-3 – HMBC for Compounds 6 and 8 at 400 MHz (in acetone-d6)

O OH

H O Br 2' 1 3' 1' 2 6

4' 6' 3 5 5' 4 Br H Br H H Br Compound 6: 4,6-dibromo-2-(4’,6’-dibromo-2’-methoxyphenoxy)phenol

O OH

H O Br 2' 1 3' 1' 2 6

4' 6' 3 5 5' 4 Br H Br H Br Br Compound 8: 4,6-dibromo-2-(4’,5’,6’-tribromo-2’-methoxyphenoxy)phenol

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

1 MAY2-5 – H-NMR for Compound 7 at 400 MHz (in acetone-d6)

13 MAY2-5 – C-NMR for Compound 7 at 400 MHz (in acetone-d6)

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

MAY2-5 – HSQC for Compound 7 at 400

MHz (in acetone-d6)

MAY2-5 – HMBC (1) for Compound 7 at 400 MHz (in

acetone-d6)

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

MAY2-5 – HMBC (2) for Compound 7 at 400 MHz (in

acetone-d6)

OH OH

H O Br 2' 1 3' 1' 2 6

4' 6' 3 5 5' 4 Br H Br H Br Br

Compound 7: 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

6.12 Appendix 12: Comparison of Chemical Profiles of D. arenaria and D. avara from New Caledonia and Fiji

6.12.1 Appendix 12a: Dysidea arenaria [HPLC Condition 2 + ELS scan;

1HNMR at 300 MHz in CDCl3]

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

6.12.2 Appendix 12b: Dysidea avara [HPLC Condition 2 + ELS scan;

1HNMR at 300 MHz in CDCl3]

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

6.13 Appendix 13: Data used for structure determination of the brominated compounds in Dysidea 4177

[HPLC Condition 2 + PDA 245 nm + APCI-MS; 1HNMR at 300 MHz in CDCl3]

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

1HNMR chemical shifts obtained for Compounds 9 and 10:

Proton δ J for δ Lit. δ J for δ Lit. Compd. Compd. (ppm)* Compd. Compd. (ppm)** 9 (ppm) 9 (Hz) 10 (ppm) 10 (Hz) 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)

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June 2009

SSecondaryecondary MMetabolitesetabolites CCompositionomposition Composante 2 aandnd GGeographicaleographical DDistributionistribution ooff MMa-a- sous-composante 2C rrineine SSpongesponges ooff tthehe GGenusenus DDysedeaysedea

RÉSUMÉ ABSTRACT L’étude et la comparaison des profi ls chimiques Chemical screening of Lamellodysidea herbacea, chez Lamellodysidea herbacea, Dysidea arenaria, Dysidea arenaria, and Dysidea avara was carried et Dysidea avara ont été menés afi n d’identifi er les out to identify the chemical constituents of these métabolites secondaires présents dans ces trois es- sponges, which can be used in the chemical taxo- pèces d’éponges qui pourraient être utilisés comme nomy of these organisms. A total of 37 samples marqueurs taxonomiques. 37 échantillons de Dysi- of Dysidea species have been worked on. Out of dea ont été collectés. Parmi ces 37 échantillons, the 37 samples obtained, there were 24 Lamello- 24 échantillons de Lamellodysidea herbacea, 9 de dysidea herbacea, 9 Dysidea arenaria, 1 Dysidea Dysidea arenaria, 1 Dysidea avara et 3 spécimens avara, and 3 Dysidea species which were not clas- non identifi és de Dysidea ont été étudiés. sifi ed to the species level at the time of study. En comparant les profi ls chimiques, il est apparu It was observed from the screening that each spe- que chaque espèce Lamellodysidea herbacea, Dy- cies Lamellodysidea herbacea, Dysidea arenaria, sidea arenaria, Dysidea avara pouvaient être diffé- and Dysidea avara had a specifi c profi le, thus dif- renciées, car chacune présente un profi l chimique ferentiating the three species chemically at a higher spécifi que. taxonomic level. Des études précédentes ont montrés que deux ché- Earlier studies of L. herbacea have shown the iso- motypes de L. herbacea ont été identifi és d’après lation of either polybrominated diphenyl ethers leur composition en métabolites secondaires, un (PBDEs), or chlorinated amino acid derivatives contenant des diphénylethers polybromés (PBDE) from these species. Spectroscopic and chemical et un contenant des dérivés d’acides aminés chlo- analyses show that the main group of compounds rés. Les analyses chimiques et spectroscopiques present in the samples of L. herbacea was the montrent que tous les échantillons de L. herbacea PBDEs. Nine different PBDEs have been identifi ed contiennent des diphénylethers polybromés. Sept and three different chemical types of L. herbacea différents PBDE ont été identifi és et trois types have been characterized. The pattern of occur- chimiques de L. herbacea ont été caractérisés. Le rence of PBDE in L. herbacea was also mapped profi l chimique de L. herbacea a été étudié pour for distant geographical sites. The different geogra- différents sites géographiques. Les régions géogra- phical locations involved in this study were New phiques concernées sont: Nouvelle-Calédonie, Va- Caledonia, Vanuatu, Fiji Islands, Solomon Islands, nuatu, îles Fidji, îles Salomon, Polynésie française Moorea Island in French Polynesia, and Mayotte. (Moorea) et Mayotte. L’analyse et comparaison des Comparison and analysis of the chemical fi nger- différents profi ls chimiques a montré que tous les prints shows that all the samples belong to the bro- échantillons font partie du bromochémotype. mo-chemotype of compounds. Un composé de type PBDE a été isolé avec Out of the nine PBDEs identifi ed, four compounds succès à partir d’un specimen de L. herbacea de have been successfully isolated and characterized Nouvelle Calédonie. Ce composé a été identifi é using spectroscopic analyses. par analyses spectroscopiques comme étant le 3,4,5,6-tétrabromo-2-(3’,5’-dibromo-2’-hydroxyphenoxy)phenol.