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6th European Conference on Marine Natural Products PROGRAMME & ABSTRACTS BOOK Edited by Luís Vieira, Carlos Gil Martins and Sara Cravo © 2009 CIMAR Associate Laboratory & CEQUIMED.UP Cover photograph (Porto Sunset) from Câmara Municipal do Porto website (www.cm-porto.pt) Conference website www.cimar.org/6ECMNP 6th ECMNP Secretariat Emília Afonso CIMAR | Rua dos Bragas, 289 | 4050-123 Porto | Portugal Tel (+351) 22 340 18 00 | Fax (+351) 22 339 06 08 E-Mail: : [email protected]

6th ECMNP

6th European Conference on Marine Natural Products

1 9 - 2 3 J u l y 2 0 0 9 , P o r t o , P o r t u g a l

Programme & Abstracts Book

This Conference is dedicated to the Great Masters Prof. Otto R. Gottlieb and Prof. Werner Herz

6TH EUROPEAN CONFERENCE ON MARINE NATURAL PRODUCTS

4

Table of Contents Welcome ........................................................................................................................... 5

Background ....................................................................................................................... 6

Organisation ...................................................................................................................... 7

Venue ................................................................................................................................ 8

Social Programme............................................................................................................. 8

Conference Themes........................................................................................................... 9

Conference Sponsors......................................................................................................... 9

Programme...................................................................................................................... 11

Opening Lecture.................................................................................................... 18

Plenary Lectures ................................................................................................... 20

APIVITA – PSE Award Lecture ............................................................................ 32

Invited Lectures..................................................................................................... 34

Oral Communications ........................................................................................... 46

Poster Communications ........................................................................................ 72

List of Participants ........................................................................................................ 173

Authors Index................................................................................................................ 182


5

Welcome Dear participants,

It is a great honour that the International Advisory Board of the 4th European Conference (4thECMNP) on Marine Natural Products, which took place in Paris during 12-16 September 2005, has decided to award us with the organisation of the sixth edition of this Conference (6th ECMNP). This Conference is jointly organized by CIMAR Associate Laboratory and Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP) and will take place at Hotel TUELA which is located at the heart of the city of Porto.

We are delighted with the great support of the scientific community which can be seen by the participation of many distinguished scientists working in the field of Marine Natural Products not only from Europe but also from all over the world. Besides the traditional topics of isolation & structure elucidation, synthesis and biological activity of marine natural products, we have also included the topics of marine toxins and biomaterials from the marine sources in this Conference. One of our main objectives, as established by the founders of this Euro-conference, is to stimulate young scientists to engage in research in this area. It is our great satisfaction to have a massive participation of PhD’s students in this Conference. In total, 97 posters, 25 oral communications, 12 invited lectures and 10 plenary lectures will be presented. Therefore, we expect an interesting debate and discussion on these topics as well as an exchange of ideas and perspectives during the Conference.

Porto will be an ideal platform to welcome this initiative since it was from here that ships set off in the discovery of new lands, thus connecting Portugal and the World. Taking into account that it was the Portuguese who were the first Europeans to reach Thailand in 1511, this event will be part of the celebrations to commemorate the five hundred years of that arrival. Hence, we feel very much honoured that HRH Princess Chulabhorn Mahidol from Thailand has graciously accepted to attend and deliver an opening lecture in this Conference.

I wish to thank many of my colleagues who have contributed in the preparation of this conference, especially the local organizing committee, the secretariat and assistants to the secretariat. I would like to thank the Scientific Committee and the International Advisory Board for their valuable advices and suggestions. I appreciate Phytochemical Society of Europe (PSE) for travelling bursaries for young scientists to attend this Conference. Furthermore, I would like to express my appreciation to the University of Porto for its support through the International Relations Office. Finally, I would like to thank Science and Technology Foundation (FCT), Ministry of Science, Technology and Higher Education of Portugal and all sponsors, especially Pharmamar (Spain) and Enzo Science (Switzerland) for their generous contributions to this Conference.

I very much hope that all participants can have a fruitful experience as well as a pleasant stay in Porto.

Anake Kijjoa

Chairman of the 6th ECMNP


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Background Taking off from Athens in 1997, and continuing with Santiago de Compostela in 1999, Elmau in 2002, Paris in 2005, Ischia in 2007, we are now arriving at the 6th Euroconference on Marine Natural Products taking place in Porto. Almost 200 participants have set the basis for another successful meeting of this widely accepted series of European Conferences.

The idea for the establishment of a European Series was born in 1997 after the realization that, mainly for financial reasons, not many European young scientists were able to participate in the major international conferences on Marine Natural Products. Europe used to be in the forefront of sciences and it seemed absolutely necessary to stimulate the interest of the young scientists in the fascinating areas of marine organisms’ research and simultaneously promote interactions with colleagues around the world.

The 5th European Framework Program and the Training and Mobility of Researchers activity supported the organization of the 1st event in Athens in November 1997 with over 50 fellowships for young scientists. This generous financial support continued till the 4th Conference. The 4th Conference in Paris and the 5th Conference in Ischia were self-financed but equally successful with the preceding events. In the Euroconferences, outstanding plenary lecturers are sharing the floor with young scientists presenting their work and getting the experience of the international scientific atmosphere. It is most encouraging to see that after 12 years and 5 events many of the young, in the initial events, scientists are now established and active cells in the European body.

Since the initial event it was decided that this series of Euroconferences should be organized in alternating years with the Gordon Conferences on Marine Natural Products. Of course, every three years the MaNaPro Symposia remain the most important appointment for all scientists interested in Marine Chemistry.

It is a great satisfaction to welcome in Porto more than 100 participants that are below 35 years old, a fact that makes everybody confident that Europe will continue for a long time to furnish prestigious contributions in Marine Chemistry.

Guido Cimino

Vassilios Roussis


7

Organisation

The 6thECMNP is co-organised by Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR) and Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP). CIIMAR is part of CIMAR Associate Laboratory.

Scientific Committee João Coimbra Madalena Pinto Maria São José Nascimento Artur Manuel Soares da Silva Madalena Humanes

International Advisory Board Angelo Fontana Marie-Lise Bourguet-Kondracki Guido Cimino Thomas Lindel Ricardo Riguera Vassilios Roussis

Local Organising Committee Anake Kijjoa (Chairman) Lars Bohlin Carlos Gil Martins Alexandre Lobo da Cunha Luís Mira Vieira Carlos Magalhães Afonso Emília Sousa Sara Cravo Honorina Cidade Ana Paula Almeida

Organising Secretariat Emília Afonso ([email protected])

CIIMAR, Rua dos Bragas, 289 4050-123 Porto, Portugal Tel (+351) 22 340 18 00 Fax (+351) 22 339 06 08

Assistants to the Secretariat Ana Sara Caetano Cordeiro Ana Sara Gomes Carla Sofia Fernandes Carlos Miguel Gonçalves de Azevedo Elisângela Costa Gisela dos Santos Adriano Júlia Manuela Marques dos Santos Bessa Marta Ramos Pinto Correia da Silva Raquel Alexandra Pinto Castanheiro Sónia Pereira dos Santos


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Venue

The 6th ECMNP will take place at HF Hotel Tuela, Porto, Portugal. The Tuela Porto is a 3 star hotel located in the Boavista area, just aside various shopping malls, traditional market (Bom Sucesso), Porto's Music Hall (Casa da Música) and at a 5 minutes walk to the Metro station (Casa da Música). It offers 197 elegantly decorated rooms completely equipped. The Tuela Porto was completely renovated at the end of 2004 and it now reflects the transformation of Porto city.

HF Hotel Tuela Rua Arq. Marques da Silva, 200 4150-483 Porto, Portugal Tel: + 351 226 194 100 Fax: + 351 226 195 160 www.hoteisfenix.com

Social Programme

Sunday 19th, 19h00 Welcome Party (Hotel Fénix)

all participants & registered accompanying persons

Tuesday 21st, 20h30 Dinner & Music at Praia da Luz beach

all participants & registered accompanying persons

Wednesday 22nd, 20h30 Gala Dinner and visit to Ferreirinha Port wine cellars

advanced registration (60 €) required

Thursday 23rd, full day

Douro Cruise (Porto-Régua-Porto)

advanced registration (60 €) required

Through the Douro river valley, the region where the Porto wine is produced, classified as World Heritage Patrimony of the Humanity. Lunch on board and return by train included in the price.


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Conference Themes

• Isolation, Structure Elucidation and Synthesis of Marine Natural Products

• Biological and Pharmacological Activities & Biotechnology of Marine Natural Products

• Biosynthesis of Marine Natural Products

• Ecology, Toxins & Biomaterials from the Marine Sources

Conference Sponsors Main Sponsors

PHARMA MAR, S.A. Unipersonal Society Avda. De los Reyes, 1 Pol. Ind. La Mina-Norte 28770-Colmenar Viejo Madrid, Spain Tel: + 34 91 846 6000 Fax: + 34 91 846 6001 Email: [email protected] http://www.pharmamar.com/

ENZO LIFE SCIENCES AG Industriestrasse 17, Postfach CH-4415 Lausen / Switzerland Telephone +41 61 926 89 89 Telefax +41 61 926 89 79 Email: [email protected] http://www.enzolifesciences.com/


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Sponsors

Additional Support


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Programme 19 July 2009 (Sunday)

10:00-16:30 Registration

17:00-17:30 Opening Ceremony

17:30-18:30 Opening Lecture: Prof. Dr. HRH Princess Chulabhorn Mahidol Marine Natural Products as an inspiration for Drug Discovery Chair: Anake Kijjoa

19.00- Get together party (Hotel Fénix)

20 July 2009 (Monday)

Morning Session

Isolation, Structure Elucidation and Synthesis of Marine Natural Products Chair: Vassilios Roussis

08:30-09:10 Plenary Lecture 1: Peter Proksch Bioactive Metabolites from Tropical Marine Invertebrates and Endophytic Fungi

09:10-09:40 Invited Lecture 1: Gabriel König Marine Microbial Metabolites - Focussing on Structural Diversity

09:40-10:00 Oral Communication 1: Stéphane La Barre NMR and MS Techniques for Rapid Characterization of Prokaryote - Eukaryote Associations: Spectroscopic Fingerprints of Marine Algae and of their Associated Microflora

10:40-10:20 Oral Communication 2: Yue Wei Guo Searching for New Bioactive Substances from South China Sea Marine Organisms

10:20-10:40 Oral Communication 3: Matthias Köck Dimeric Pyrrole-Imidazole Alkaloids – New Massadine Stereoisomers and their Configurational Assignment

10:40-11:00 Coffee Break

continuation

Chair: Angelo Fontana

11:00-11:40 Plenary Lecture 2: Chris Ireland Marine Natural Products Antitumor Agents

11:40-12:10 Invited Lecture 2: Jose Fernando Reyes New Bioactive Metabolites from Marine Invertebrates


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12:10-12:30 Oral Communication 4: RuAngelie Edrada-Ebel Metabolomic Profiling of Some Marine Sponges from the Irish and Celtic Seas by High Resolution FTMS and NMR with the aid of SIEVE Analysis

12:30-12:50 Oral Communication 5: Vatcharin Rukachaisirikul Metabolites from Marine-derived Fungi

12:50-13:10 Oral Communication 6: Yannick Viano Algal Diterpenoids as Antifouling Substances against a Marine Bacterial Biofilm

13:10-14:30 Lunch

Afternoon Session

Isolation, Structure Elucidation and Synthesis of Marine Natural Products (cont.)

Chair: Artur Silva

14:30-15:00 Invited Lecture 3: Paul B. Jones Photochemistry in the Metabolites of Placobranchus Ocellatus

15:00-15:20 Oral Communication 7: Pierangelo Luporini NMR Structures of a Psychrophilic Family of Water-Borne Signal Polypeptides Isolated from the Polar Protozoan Ciliate, Euplotes nobilii

15:20-15:40 Oral Communication 8: Mohamed Mehiri Njaoaminiums A, B, and C: Cyclic 3-Alkylpyridinium Salts from the Marine Sponge Reniera sp.

15:40-16:10 Invited Lecture 4: Robert Capon Australian Marine Biodiscovery


continuation

Chair: Madalena Pinto

16:30-17:10 Plenary Lecture 3: Eric Jim Thomas Approaches to the Total Synthesis of some Biologically Active Natural Products

17:10-17:40 Invited Lecture 5: Ali Al-Mourabit Progress in Biomimetic Total Synthesis of the Marine Palau'amine and Congeners

17:40-18:00 Oral Communication 9: Emiliano Manzo Synthesis of an Isocyanide Glyceryl-like Lipid Isolated from Actinocyclus papillatus

18:00-19:30 Poster Session [even numbers]


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21 July 2009 (Tuesday)

Morning Session

Biological and Pharmacological Activities & Biotechnology of Marine Natural Products

Chair: Marie-Lise Bourguet-Kondracki

08:30-09:10 Plenary Lecture 4: Laurent Meijer Protein Kinases Relevant to Human Diseases: Pharmacological Inhibitors Derived from Marine Organisms

09:10-09:40 Invited Lecture 6: Marc Diedrich Marine Compounds as Promising Anti-Cancer Agents

09:40-10:00 Oral Communication 10: Valeria Costantino Potent Anti-Tumor and Anti-Inflammatory Lead Structures from Caribbean Sponges

10:40-10:20 Oral Communication 11: Heonjoong Kang Novel Drug Leads for Hepatic Steatosis and Nonalcoholic Steatohepatitis (NASH)

10:20-10:40 Oral Communication 12: Carine Le Ker Search for Water-soluble Bioactive Marine Fungal Metabolites: Perfecting of an Extraction and Partial-Purification Method


continuation

Chair: Lars Bohlin

11:00-11:30 Invited Lecture 7: Orazio Taglialatela-Scafati Antimalarials from the Sea. Studies on the Mechanism of Action of Endoperoxide Derivatives from Marine Sponges

11:30-11:50 Oral Communication 13: Agostinho Casapullo Chemical Proteomics as a Tool in Target Discovery of Bioactive Small Molecules

11:50-12:10 Oral Communication 14: Heinz Schröder Marine Nanobiotechnology: Enzymes/Proteins from Sponges Acting at the Interface of Inorganic Chemistry and Biology

12:10-12:30 Oral Communication 15: Gäel Le Pennec Molecular Communication Actors within the Porifera/Bacteria Symbiotic Model. Insights into an Intimate Dialogue

12:30-12:50 Oral Communication 16: Yoel Kashman Salarins, Tulearins and Taumycins, Novel Marine Natural Products; Chemistry, Stereochemistry and Activity

12:50-14:30 Lunch


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Afternoon Session

Biosynthesis of Marine Natural Products Chair: Heinz Schröder

14:30-15:10 Plenary Lecture 5: Christian Hertweck Exploring Cryptic Pathways in Bacteria and Fungi

15:10-15:30 Oral Communication 17: Delphine Bry Biosynthesis of Pyridoacridines in C. dellechiajei Cell-Free Extracts

15:30-15:50 Oral Communication 18: Adele Cutignano The Bizarre Biogenetic Variability of Terpenes in the Antarctic Mollusc Austrodoris kerguelenensis

15:50-16:10 Oral Communication 19: Helena Gaspar Chemoecological Studies in Two Nudibranchs from the Portuguese Coast


continuation

Chair: Emilio Quiñoá

16:30-17:10 Plenary Lecture 6: William Gerwick Structures, Biological Activities and Biosynthesis of Intriguing Marine Cyanobacterial Metabolites

17:10-17:20 Ceremony for Apivita-PSE award

17:20-17:50 Apivita-PSE award Lecture: Angelo Fontana Chemistry of Signaling in Marine Diatoms

17:50-19:30 Poster Session [odd numbers]

20.30- Dinner & Music at Praia da Luz beach [all participants & registered accompanying persons] Announcement of the PSE Travelling Grants by Prof. Lars Bohlin, Chairman of PSE


15

22 July 2009 (Wednesday)

Morning Session

Ecology, Toxins & Biomaterials from the Marine Sources

Chair: Graziano Guella

08:30-09:10 Plenary Lecture 7: Adrianna Ianora Chemical Ecology of Secondary Metabolites and their Role in Driving Ecosystem Functionality in the Plankton

09:10-09:40 Invited Lecture 8: Joseph Pawlik The Chemical Ecology of Sponges on Caribbean Coral Reefs: How Natural Products Shape Natural Systems

09:40-10:00 Oral Communication 20: Charline Abed Chemotaxonomy as Valuable Approach to Study Sponges of the Family Irciniidae (Porifera, Dictyoceratida)

10:40-10:20 Oral Communication 21: Mikel A. Becero Relevant Scale of Chemical Variation in Aplysina aerophoba

10:20-10:40 Oral Communication 22: Tiago Silva Chitosan Derived from Squid Pens on the Development of Biomedical Membranes


Chair: Diaa Youssef

11:10-11:20 Oral Communication 23: Oriol Sacristán-Soriano Intraspecimen Variability of Natural Products in the Sponge Aplysina aerophoba

11:20-11:50 Invited Lecture 9: Paulo Vale Recent Developments in PSP Toxin Chemistry, Detection and Biotransformation

11:50-12:10 Oral Communication 24: Martino Forino 42-Hydroxy Palytoxin: a New Palytoxin Analog from Hawaiian Palythoa spp. Is this the Real Poison of the Legendary Hawaiian limu-make-o-Hana?

12:10-12:30 Oral Communication 25: Antonio Hernandez Daranas Bioactive Polyether Metabolites from Dinoflagellates: Structure Determination And Bioactivity

12:30-13:00 Invited Lecture 10: Ricardo Riguera Marine Biopolymers in Nanomedicine: Advances in Drug Delivery

13:00-14:30 Lunch


16

Afternoon Session

New Perspectives in Marine Natural Products

Chair: John Blunt

14:30-15:00 Invited Lecture 11: Tadeusz Molinski Nanomole-Scale Marine Natural Products

15:00-15:40 Plenary Lecture 8: Werner Müller The Power of Marine Genomics

15:40-16:20 Plenary Lecture 9: Murray Munro Marine Natural Products: From There to Here. What's next?

16:20-16:50 Ceremony of Professional Recognition to Guido Cimino Speech by Prof. Ernesto Fattorusso

16:50-17:30 Plenary Lecture 10: Guido Cimino From Sepiamelanin to Chemical Ecology of Opisthobranchs and Diatoms: an Exciting 40 Years Game in the Field of Marine Natural Products

17:30-18:00 Closing Remarks - Announcement of VII ECMNP by Lars Bohlin

20.30- Gala dinner and visit to Ferreirinha Port wine cellars [advanced registration (60 €) required]

23 July 2009 (Thursday)

full day Douro Cruise (Porto-Régua-Porto)

[advanced registration (60 €) required]

Through the Douro river valley, the region where the Porto wine is produced, classified as World Heritage Patrimony of the Humanity. Lunch on board and return by train included in the price.

Opening Lecture


18

OL

MARINE NATURAL PRODUCTS AS AN INSPIRATION FOR DRUG DISCOVERY Chulabhorn Mahidol

Chulabhorn Research Institute, Vipavadee Rangsit Highway, Bangkok 10210, Thailand

Recently the researches on marine natural products have intensified and marine organisms have been found to be a rich source for new drugs and leads for drug development. Even though only a small fraction of all marine species has been investigated, a plethora of new and diverse structures and structural features with interesting biological activities were isolated. We also have launched study of the natural products from marine organisms. This is, in part, due to the fact that most marine sources in Thailand are relatively unexplored, Thailand situates on the Indo-China Peninsula along which the coastal lines from the Gulf of Thailand to the Andaman Sea accounts for a total distance of 2,600 kilometers. As the biodiversity and variety of the marine ecosystems of these coastlines are expected to be unique. Many species of tunicates or ascidians, sponges, and soft corals have been collected from the East Coast of the Gulf of Thailand, and we have investigated the chemistry and

biological activities of various natural products isolated from these species.

The lamellarins are marine-derived natural products, which were first isolated in 1985 from mollusks, and subsequently found in ascidians, as well as marine sponges. These compounds have received much attention from various research groups worldwide due to their diverse biological activities1, especially cytotoxic activity and multidrug resistance (MDR) reversal in a number of cancer cell lines. Our study on the synthesis2 as well as cytotoxic property of the lamellarin alkaloids will also be presented.

1) Bailly, C. Curr. Med. Chem. Anti-Cancer

Agents 2004, 4, 363-378; b) Fan, H.; Peng, J.; Hamann, M.T.; Hu, J. F. Chem. Rev. 2008, 108, 264-287.

2) Ploypradith, P.; Mahidol, C.; Sahakitpichan, P.; Wongbundit, S.;Ruchirawat, S. Angew. Chem., Int. Ed. 2004, 43, 866–868.

Plenary Lectures


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PL 01

BIOACTIVE METABOLITES FROM TROPICAL MARINE INVERTEBRATES AND ENDOPHYTIC FUNGI

Peter Proksch

Heinrich-Heine-University Duesseldorf Institut fuer Pharmazeutische Biologie und Biotechnologie Universitaetsstr. 1 Geb. 26.23D-40225 DuesseldorfGermany

[email protected]

The oceans contain a vast biological diversity of species that have so far been utilized by mankind mainly as a source of protein. In the last decades, however, natural product chemists have started to discover the wealth of secondary metabolites that are produced by marine invertebrates such as sponges, tunicates, molluscs and others. Some of these natural products have advanced into clinical trials and the first compounds of marine origin have already entered the drug market.

Among marine invertebrates sponges (Porifera) have yielded the largest number of natural products discovered so far. This holds especially true for sponges from tropical habitats such as coral reefs that are characterized by a teeming biodiversity of both invertebrates and vertebrates.

The focus of our research activities is on the discovery of bioactive constituents of

sponges and other soft bodied invertebrates mainly from Indonesia and the South China Sea that are both hot spots of species diversity and still hold unspoiled coral habitats. Most of our research activities are related to drug discovery using cellular and/or target based screens such as inhibition of cancer relevant protein kinases or induction of apotosis. More recently, we have started to study anti fouling properties of sponge-derived natural products using barnacle larvae as model organism. A second focus is on new bioactive compounds from endophytic fungi isolated from Mangrove plants which line sea shores at undisturbed tropical sea shores.

In this presentation some of our latest findings related to the discovery of bioactive constituents from marine sponges and endophytic fungi will be presented.


21

PL 02

MARINE NATURAL PRODUCT ANTITUMOR AGENTS Chris M. Ireland

Department of Medicinal Chemistry 30 S. 2000 E. Room 308 University of Utah, Salt Lake City, UT 84112

[email protected] / http://www.pharmacy.utah.edu/medChem/faculty/ireland.html

The search for antitumor agents from the marine environment began in earnest about 25 years ago with didemnin B entering clinical trials. There are currently 14 compounds in clinical trials, and Yondelis® recently became the first marine natural product to be approved as a drug for treating cancer. The majority of agents to enter in clinical trials from marine organisms have been cytotoxic agents. As a consequence, they tend to suffer from lack of selectivity for tumor cells, and can cause collateral damage to

normal tissue. Our program has focused on targeting cellular components or pathways commonly over-expressed or selectively expressed in tumor cell lines to overcome these problems. The particular pathways we have focused on are kinase signaling pathways, the S26 proteasome, and Wnt signaling. Results from these projects will be presented along with studies towards creating marine natural products based HPLC peak libraries for HTS screening.


22

PL 03

APPROACHES TO THE TOTAL SYNTHESIS OF BIOLOGICALLY ACTIVE NATURAL PRODUCTS

Eric J. Thomas

The School of Chemistry, The University of Manchester, Manchester, M13 9PL, UK

[email protected]

The bryostatins, for example bryostatin 1 1, are marine natural products with important anti-cancer activity in particular when used in connection with other chemotherapy.1 Four total syntheses of bryostatins have been reported to date2 and interesting biologically active analogues with a cyclic acetal in place of the B ring have been prepared.3 Nevertheless there remains a need for improved synthetic access to bryostatins for further studies of structure activity relationships.

The classical Julia reaction has been used to form the C(16)-C(17) bond of intermediates for bryostatin synthesis but the yields obtained can be variable and the vigorous conditions used require several functional group modifications to be carried out after the Julia assembly step.2,4 Studies have been carried out on alternative strategies for assembly of bryostatins including the evaluation of metathesis and the modified one-step Julia. Metathesis has proved useful for the synthesis of analogues of bryostatins which lack the geminal dimethyl substituents at C(18).5,6 The modified Julia has given rise to the synthesis of several advanced intermediates for a

convergent synthesis of bryostatin 11 2.7 Aspects of this work will be described.

1. Hale, K. J.; Hummersone, M. G.; Manaviazar, S.; Frigerio, M. Nat. Prod. Reports 2002, 19, 413.

2. (a) Masamune, S. Pure Appl. Chem. 1988, 60, 1587; (b) Kageyama, M.; Tamura, T.; Nantz, M. H.; Roberts, J. C.; Somfrai, P.; Whitenour, D. C.; Masamune, S. J. Am. Chem. Soc. 1990, 112, 7407; (c) Evans, D. A.; Carter, P. H.; Carreira, E. M.; Prunet, J. A.; Charette, A. B.; Lautens, M. Angew. Chem. Int. Edn. 1998, 37, 2354; (d) Evans, D. A.; Carter, P. H.; Carreira, E. M.; Prunet, J. A.; Charette, A. B.; Lautens, M. J. Am. Chem. Soc. 1999, 121, 7540; (e) Ohmori, K.; Ogawa, Y.; Obitsu, T.; Ishikawa, Y.; Nishiyama, S.; Yamamura, S. Angew. Chem., Int. Ed. 2000, 39, 2290; (f) Trost, B. M.; Dong, G. Nature, 2008, 456, 485.

3. Wender, P. A.; Baryza, J. L.; Bennett, C. E.; Bi, F. C.; Brenner, S. E.; Clarke, M. O.; Horan, J. C.; Kan, C.; Lacote, E.; Lippa, B.; P. Nell, P. G.; Turner, T. M. J. Am. Chem. Soc. 2002, 124, 13648; (b) Wender, P. A.; DeChristopher, B. A.; Schrier, A. J. J. Am. Chem. Soc. 2008, 130, 6658; (c) Wender, P. A.; Horan, J. C.; Verma, V. A. Organic Lett. 2006, 8, 5299; (d) Wender, P. A.; Horan, J. C. Organic Lett. 2006, 8, 4581.

4. Manaviazar, S.; Frigerio, M.; Bhatia, G. S.; Hummerstone, M. G.; Aliev, A. E.; Hale, K. Org. Lett. 2006, 8, 4477.

5. Ball, M.; Bradshaw, B. J.; Dumeunier, R.; Gregson, T. J.; MacCormick, S.; Omori, H.; Thomas, E. J. Tetrahedron Lett. 2006, 47, 2223;

6. Trost, B. M.; Yang, H.; Thiel, O. R.; Frontier, A. J.; Brindle, C. S. J. Am. Chem. Soc. 2007, 129, 2206.

7. Allen, J. V.; Green, A. P.; Hardy, S.; Herron, N. M.; Lee, A. T. L.; Thomas, E. J. Tetrahedron Lett. 2008, 49, 6352.

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23

PL 04

PROTEIN KINASES RELEVANT TO HUMAN DISEASES: PHARMACOLOGICAL INHIBITORS DERIVED FROM MARINE ORGANISMS

Laurent Meijer

C.N.R.S., Protein Phosphorylation & Human Disease Group, Station Biologique, 29682 Roscoff, FRANCE

[email protected]

Phosphorylation of serine, threonine and tyrosine residues represents one of the most common post-translational mechanisms used by cells to regulate their enzymatic and structural proteins. Alterations in the phosphorylation of proteins represent a frequent feature associated with human disease. This is the reason for an exponentially growing investment in the discovery, optimization and therapeutic evaluation of small molecular weight, pharmacological inhibitors of protein kinases. It is estimated that 30-35% of drug discovery programs in the pharmaceutical industry currently target a protein kinase! Presently, over 130 kinase inhibitors are undergoing clinical evaluation against diseases such as cancers, inflammation, diabetes, and neurodegeneration.

Among the 518 human kinases, our laboratory has focused its efforts on four families of kinases: cyclin-dependent kinases (CDKs), glycogen synthase kinase -3 (GSK-3 and its Plasmodium ortholog PfGSK-3), casein kinases 1 (CK1) and dual-specificity tyrosine phosphorylation regulated kinases (DYRKs). These

kinases have attracted considerable interest because of their numerous key physiological functions such as regulation of cell division cycle, apoptosis, multiple neuronal activities, pain signaling, insulin release, transcription, RNA splicing, etc... Their involvement in human diseases such as cancers & leukemias, chronic & acute neurodegenerative disease (Alzheimer’s and Parkinson’s diseases, stroke), kidney diseases (glomerulonephritis, polycystic kidney disease), inflammation, type 2 diabetes, viral infections, unicellular parasites will be briefly reviewed.

Marine organisms constitute a particularly rich and relatively untapped source of new kinase inhibitory scaffolds. To illustrate this, we will describe a selection of molecules derived from marine organisms (meriolins, indirubins, hymenialdisine, lamellarins, etc…). The selectivity and intracellular mechanism of action of these compounds, their chemical synthesis and their pharmacological properties have been extensively studied and will be presented as representative examples of the multiple effects of kinase inhibitors in cells, tissues and organisms.


24

PL 05

EXPLOITING CRYPTIC BIOSYNTHETIC PATHWAYS IN MICROORGANISMS Christian Hertweck

Dept. Biomolecular Chemistry Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany

[email protected]

In the post-genomic era it has become increasingly apparent that the vast number of predicted polyketide biosynthesis genes of microorganisms is not reflected by the metabolic profile observed under standard fermentation conditions. In the absence of a particular, in most cases unknown trigger these gene loci remain silent. Since cryptic gene clusters could code for the biosynthesis of important virulence factors, toxins, or even drug candidates, new strategies for their activation are urgently needed to make use of this largely untapped reservoir of potentially bioactive compounds. A proof of principle is the successful induction of a silent fungal metabolic pathway, which led to the discovery of novel PKS-NRPS hybrid metabolites. Biosynthetic investigations may also provide surprising insights into complex biological systems. By studying the molecular basis of the polyketide macrolide rhizoxin from the blight fungus Rhizopus microsporus we found that the toxin is not produced by the fungus itself, but by bacteria that reside within the fungal cytosol. The endosymbionts could be isolated in pure culture to produce and engineer antitumoral rhizoxin derivatives.

Cell-based assays as well as tubulin binding experiments indicated that some new compounds are up to 10,000 times more active than rhizoxin and rank among the most potent antiproliferative agents known to date. Our progress in exploiting the hidden biosynthetic potential of fungi and bacteria is presented.

Further reading:

L.P. Partida-Martinez & C. Hertweck, Pathogenic Fungus Harbours Endosymbiotic Bacteria for Toxin Production, Nature 2005, 437, 884-888.

K. Scherlach, L. P. Partida-Martinez, H.-M. Dahse & C. Hertweck, Antimitotic Rhizoxin Derivatives from a Cultured Bacterial Endosymbiont of the Rice Pathogenic Fungus Rhizopus microsporus, J. Am. Chem. Soc. 2006, 128, 11529-11536.

S. Bergmann, J. Schümann, K. Scherlach, C. Lange, A.A. Brakhage & C. Hertweck, Genomics-Driven Discovery of PKS-NRPS Hybrid Metabolites from Aspergillus nidulans, Nature Chem. Biol. 2007, 3, 213-217.

T. Nguyen, K. Ishida, H. Jenke-Kodama, E. Dittmann, C. Gurgui, T. Hochmuth, S. Taudien, M. Platzer, C. Hertweck & J. Piel, Mosaic Structure of trans-AT Polyketide Synthases Reveals New Strategies for Natural Product Discovery and Pathway Dissection, Nature Biotechnol. 2008, 26, 225-233.


25

PL 06

STRUCTURES, BIOLOGICAL ACTIVITIES AND BIOSYNTHESES OF INTRIGUING MARINE CYANOBACTERIAL METABOLITES

Eduardo Esquenazi1, Rashel Grindberg1, Adam C. Jones1, lban Pereira1, Kevin Tidgewell1, Lena Gerwick1, Zhengyu Cao2, Thomas F. Murray2,

Pieter C. Dorrestein1,* and William H. Gerwick1,* 1 Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, the

Skaggs School of Pharmacy and Pharmaceutical Sciences, and the Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive MC 0212,

La Jolla, California 92093, USA and 2 Department of Pharmacology, School of Medicine, Creighton University, Omaha, NE 68178

[email protected], [email protected]

Marine cyanobacteria are among the richest groups of marine organisms for their wealth of structurally-diverse and biologically-active natural products. They often utilize an integrated polyketide synthase-nonribosomal peptide synthetase strategy to produce this broad array of nitrogen-rich frameworks, and then decorate these through oxidation, methylation or halogenation, thereby creating an enormous molecular diversity. Despite nearly 700 compounds now having been described from these organisms, our laboratory continues to encounter fundamentally novel molecular structures from new samples of these organisms.

Recently, we have been exploring innovative strategies by which to discover as well as study the biosynthesis of natural products from these organisms. One approach has been to modulate the

expression of natural product biosynthetic pathways that are normally ‘silent’. This approach is based on our findings of regulatory proteins and transcriptional promoter regions upstream of cyanobacterial natural product pathways. Another approach involves innovative applications of MALDI MS as well as other mass spectrometric methods. Coupled together, we are finding that marine cyanobacteria have an even greater capacity for natural products biosynthesis than we previously believed, and a number of these newly discovered natural products have potent and mechanistically interesting neuropharmacological properties.

1. Kevin Tidgewell, Benjamin R. Clark and William H. Gerwick, “The Natural Products Chemistry of Cyanobacteria” Comprehensive Natural Products Chemistry, Pergamon Press, Volume 8, 2009 (in press).


26

PL 07

CHEMICAL ECOLOGY OF DIATOM SECONDARY METABOLITES AND THEIR ROLE IN SHAPING PLANKTONIC INTERACTIONS

Adrianna Ianora1, Angelo Fontana2, Giovanna Romano1, Giuliana d’Ippolito2, Raffaella Casotti1, Adele Cutignano2, Guido Cimino1 and Antonio Miralto2

1 Stazione Zoologica Anton Dohrn, Villa Comunale 80121 Naples, Italy 2 CNR, Istituto di Chimica Biomolecolare, Via Campi Flegrei 34, 80078, Pozzuoli, Napoli, Italy

[email protected]

Diatoms are small eukaryotic plants with over 1600 species, constituting one of the major components of the phytoplankton in freshwater and marine environments. Diatom blooms are believed to initiate and support the cycle of secondary production and growth of fish larvae that depend predominantly on the eggs and larval stages of planktonic copepods, the dominant constituent of the zooplankton in most aquatic habitats. However, evidence has accumulated over the last decade that has progressively challenged the view that diatoms are good and harmless food items for copepod growth and survival. Numerous laboratory studies have shown that when copepods are fed certain diatom diets, the eggs produced either fail to develop to hatching or hatch into malformed nauplii. The compounds responsible for these effects are short-chain polyunsaturated aldehydes (PUAs) that arrest embryonic development in copepods and sea urchins, and have antiproliferative and apoptotic effects on human carcinoma cells. PUAs are cleaved from fatty acid precursors by enzymes activated within seconds after crushing of cells. The production of these compounds can also be accompanied or complemented by the synthesis of other products derived from the enzymatic oxidation of membrane lipids such as hydroxy acids, epoxyalcohols, and ω-oxo acids mainly derived from eicosapentaenoic acid (EPA) and chloroplastic C16-fatty acids. Such

compounds are activated chemical defences against grazers, potentially sabotaging future generations of copepods by inducing poor recruitment. This insidious mechanism, which does not deter the herbivore from feeding but impairs its recruitment, will restrain the cohort size of the next generation. Hence, certain diatom diets can negatively impact both copepod egg hatching success (up to 100%) and larval development and survival, with biomass build-up of blooms on the ocean floor that may have significant consequences for ocean ecology and biogeochemistry. Teratogens were unknown for the marine environment until their discovery in diatoms, whereas they are well known for higher terrestrial plants. This property may partially explain why diatoms cause problems in hatcheries where they are still widely used in aquaculture due to their ease of mass cultivation. Although the effects of such toxins are less catastrophic than those inducing poisoning and death, they are none-the-less insidious occurring through abortions, birth defects and reduced larval survivorship. My presentation will focus on some of the newest findings regarding this unique predator-prey relationship and discuss how chemical defence molecules, such as reactive PUAs and other recently described oxylipins from diatoms, can help shape plant-animal interactions and drive bloom dynamics in the plankton.


27

PL 08

THE POWER OF MARINE GENOMICS Werner E.G. Müller and Heinz‐C. Schröder

Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz; GERMANY

[email protected]

In the last decade the phylogenetically oldest metazoan phylum, the Porifera (sponges) gained special interest. Mainly due to the introduction of molecular biological techniques solid evidence was elaborated which indicated that this phylum provides a cornucopia of new information which allows a grasping for the understanding of the dynamics of evolutionary processes occurring during the Earth period of Ediacara until today. Furthermore, the species of this phylum are rich and valuable sources for bioprospecting, the translation of life-science discoveries into practical products or processes for the benefit of the society.

BIOPROSPECTING: The field of bioprospecting of Porifera may be of tremendous potential benefit for humans from the applied point of view. Taking into account that the chemical diversity of natural bioactive compounds is much higher than that of compounds synthesized in standard combinatorial chemistry approaches, and that natural compounds display an impressive selectivity, the high value of the secondary metabolites from natural resources in general and from sponges in particular can only be roughly imagined. Until now only in one case a bioactive compound from sponges is applied in clinics, arabinofuranosyladenine (ara-A) as antiviral drug; ara-A is a derivative of a lead structure isolated from a sponge.

THE FUTURE – EVOCHEMISTRY: Thanks to the progress initiated by the pressure of the society for a sustainable use of natural resources for human benefit, the

exploitation of natural biodiversity became possible through the application of the techniques of molecular biology and modern cell biology.

NOVEL DIRECTIONS: BIOMATERIALS. There is an increasing need for novel materials to be used as scaffolds in biomaterials in general and in tissue engineering (bone and cartilage) in particular. Siliceous sponges are unique in their ability to synthesize their silica skeleton enzymatically. The responsible enzymes, the silicateins which have been isolated from demosponges, polymerize alkoxide substrates to silica. Silica is an important component of materials such as bioactive glasses and composites based on glasses, ceramics and (organic) polymers. New strategies for the structure-directed synthesis of amorphous silica (biosilica) can now be envisaged.

CONCLUSION: It is fortunate that, according to the fossil records, the phylogenetic oldest metazoan phylum, the Porifera did not become extinct during the last 800 million years. Considerable impact in biotechnology cab be excepted from studies on the recombinant preparation of bioactive, low-molecular weight compounds and of the development of new biomaterials [biosilica] from marine sources. Müller WEG, Brümmer F, Batel R, Müller IM, Schröder HC (2003) Molecular biodiversity. Case study: Porifera (sponges). Naturwissenschaften 90: 103-120, • Schröder HC, Wang XH, Tremel W, Ushijima H, Müller WEG (2008) Biofabrication of biosilica-glass by living organisms. Nat. Prod. Rep. 25:455-474


28

PL 09

MARINE NATURAL PRODUCTS: FROM THERE TO HERE. WHAT’S NEXT?

Sunita Chumyuang1,2, Lin Sun1, John Blunt1, Tony Cole2, Murray Munro1, Siti Alwani Ariffin1,3,4, Hamidah Bakar3, Kalavathy Ramasamy3,

Jean‐Frédéric Weber3 and Paul Davis4

1 Department of Chemistry, University of Canterbury, Christchurch, New Zealand. 2 School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.

3 Faculty of Pharmacy, Universiti Teknologi MARA, Shah Alam, Malaysia. 4 School of Medicine and Health Sciences Building, University of Otago,

Wellington, New Zealand.

[email protected]

Over a 60-year period, marine natural products has advanced from infancy to maturity. Some 19,000 compounds have now been characterized, the rate of discovery continues to increase, the first marine-origin drugs are on the market, the emphasis is changing from macro- to microorganisms and such enormous advances have been made in separation and spectroscopic technologies that rapid structure determination is possible at the nanomolar level. But, to find new bioactive compounds it is increasingly

necessary to implement strategies that enhance the probability of discoveries and reduce time and costs.

In this presentation two projects will be discussed:

a. phenol pasteurization and OSMAC studies;

b. the role of NMR databases and CapProbe NMR spectroscopy in the study of New Zealand and Malaysian organisms.


29

PL 10

FROM SEPIAMELANIN TO CHEMICAL ECOLOGY OF OPISTHOBRANCHS AND DIATOMS: AN EXCITING 40 YEARS GAME IN THE FIELD OF MARINE

NATURAL PRODUCTS Guido Cimino

Istituto di Chimica Biomolecolare, C.N.R., Via Campi Flegrei, 34, 80078, Pozzuoli, Italy

[email protected]

The activity in the field of Marine Natural Products started the 8th of November 1962. In fact, exactly in that day, the author’s degree thesis began and the topic was “Structure of melanin from Sepia officinalis“. The work was performed at the University of Naples in the prestigious group of Alessandro Nicolaus under the supervision of Ernesto Fattorusso. Really, the main interest of the group was in studying structure and function of natural black (melanin) and red-brown (phaeomelanins) pigments. The marine source was only a lucky coincidence that anticipated the extensive work on marine organisms that started in 1969 and continued without any interruption until today.

In this communication, the most recent results obtained studying at ICB opisthobranchs and diatoms will be presented after a synthetic overview of the most significant moves played during this exciting 40 years game in the field of Marine Natural Products. After a “romantic” opening dedicated to the study of melanins (1) and phaeomelanins,(2) the study of marine organisms rapidly led to characterize an impressive number of new compounds exhibiting very unusual structures, prevalently terpenes and alkaloids. Substantially, the “structure hunt” ended in Kyoto (1988), 16th International Symposium on the

Chemistry of Marine Natural Products, presenting a series of alkaloids with absolutely unprecedented structural features: the saraines.(3)

Slowly, the scientific interest shifted “from the structure to the function”. Opisthobranchs were selected as model to investigate. These apparently unprotected molluscs are rarely victims of predators. In fact, they have elaborated a series of alternative defensive strategies which include the use of chemicals. The majority of the protective allomones were sequestered through the food chain from algae and other invertebrates. However, opisthobranchs were also able to biosynthesize many compounds structurally related to typical molecules possessed by their preys.(4) These aspects “from function, to ecology, to biosynthesis, to evolution” will be discussed with more details.

1. Fattorusso E.; Cimino G. Rend. Acc. Sc. Fis.

Mat. 1968, 35, 616-622.

2. Minale l.; Fattorusso E.; De Stefano S.; Magno S.; Cimino G.; Nicolaus R.A. Gazz. Chim. It., 1970, 100, 870-879.

3. Cimino G.; Puliti R.; Scognamiglio G.; Spinella A.; Trivellone E.; Mattia C.A.; Mazzarella L. Pure Appl. Chem. 1989, 61, 535-538.

4. Cimino G.; Fontana A.; Gavagnin M. Phythochem. Rev. 2004, 3, 285-307

APIVITA - PSE Award Lecture


32

AL

CHEMISTRY OF SIGNALING IN MARINE DIATOMS Angelo Fontana

CNR – Istituto di Chimica Biomolecolare, Via Campi Flegrei 34, 80078 Pozzuoli – Napoli (Italy)

[email protected]

Diatoms, which emerged as an independent lineage recently in the evolution of photosynthetic eukaryotes (Fehling et al, 2007; Kooistra et al., 2007), are responsible for almost 20% of the global primary production and play a key role in the carbon and silica biogeochemical cycles (Smetacek, 1999). The mechanism regulating growth and defence of these microalgae in seas and oceans is currently debated in consideration of conflicting pieces of evidence provided by independent studies in the last years. Recently, we have shown that marine diatoms produce a number of lipoxygenase products mostly derived from eicosapentaenoic acid (EPA) and chloroplastic C16-fatty acids (Cutignano et al., 2006; Fontana et al., 2007a). These molecules, generically named phycooxylipins, include hydroxy acids, epoxyalcohols, ω-oxo acids and polyunsaturated aldehydes, which have a recognized role as chemical mediators in many other organisms, including terrestrial plants and animals. Furthermore, a critical reading of the chemical structures of the molecules hitherto characterized suggests that phycooxylipins are the products of species-specific metabolic pathways in both centric (d’Ippolito et al., 2002; Cutignano et al., 2006; Fontana et al, 2007b) and pennate (Wendel & Jüttner, 1996; Pohnert, 2000) diatoms, despite the marked differences in the genomic structure of the two lineages.

After long dealing with the synthesis and ecological role of these compounds in laboratory cultures, we have matured the idea that the varied products of the lipoxygenase pathways may play a physiological function in regulating growth and death of plankton communities. The present communication deals with the chemistry and biochemistry of these signalling system, paying attention to the eco-physiological role and modulation of enzymes and/or molecules. 1. Cutignano A, d’Ippolito G, Romano G, Cimino

G, Febbraio F, Nucci R, Fontana A. 2006. ChemBioChem 7: 450-456.

2. d’Ippolito G, Romano G, Iadicicco O, Miralto A, Ianora A, Cimino G, Fontana A. 2002. Tetrahedron Lett. 43: 6133-6136.

3. Fehling J, Stoecker DK, Baladauf SL. 2007. In: P. G. Falkwoski and A.H Knoll eds. Evolution of Primary Producers in the Sea. Elsevier Academic Press, Burlington – MA, pp. 75-107.

4. Fontana A, d’Ippolito G, Cutignano A, Romano G, Ianora A, Miralto A, Cimino G. 2007a. Pur. Appl. Chem., 79: 481-490.

5. Fontana A, d’Ippolito G, Cutignano A, Romano G, Lamari N, Massa Gallucci A, Cimino G, Miralto A, Ianora A, 2007b. ChemBioChem 8: 1810-1818.

6. Kooistra W.H C F, Gersonde R., Medlin LK, Mann DG. 2007. In: P. G. Falkwoski and A.H Knoll eds. Evolution of Primary Producers in the Sea. Elsevier Academic Press, Burlington – MA, pp. 207-250.

7. Pohnert G. 2000. Angew. Chem. – Internat. Ed. 39: 4352-4354.

8. Smetacek V. 1999. Protist 150:25-32.

9. Wendel T, Jüttner F. 1996. Phytochemistry, 6: 1445-1449.

Invited Lectures


34

IL 01

MARINE MICROBIAL METABOLITES - FOCUSSING ON STRUCTURAL DIVERSITY

Gabriele M. König, S. Kehraus, A. Pontius, H. Greve, K. Neumann, Mahmoud F. Elsebai, Celso Almeida

Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, D-53115 Bonn, Germany, [email protected]

Nature has turned out to be most imaginative in the generation of biologically active metabolites, and natural products have often opened up completely new therapeutic approaches. Structural diversity of secondary metabolites is regarded as one of the major advantages of natural products in the drug discovery process (Molinski et al., 2009). Several examples from our research focussing on the secondary metabolites of marine microorganisms, predominantly fungal endophytes of marine algae and sponges, shall illustrate the unusual biosynthetic capabilities of these organisms.

Investigation of the algicolous fungus Phaeosphaeria spartinae and Microdiplodia sp. (Neumann et al., 2009) provided new hydroxylated and unsaturated polyketides, which were recognized as inhibitors of Human Leukocyte Elastase (HLE). Noduliprevenone, an inhibitor of P450 (CYP) 1A, which concomitantly induces NAD(P)H:quinone reductase (QR) is a unique marine fungal polyketide from

Nodulisporium sp. It is the first dimeric compound incorporating two unusual chromanone sub-units, substituted with a butanolide and a hydroxy-butanoic methylester moiety, respectively (Pontius et al., 2008). The sponge-derived fungus Stachylidium sp. yielded a cytotoxic extract, which contained new phthalides with unusual structural motives, a novel cytotoxic phthalimidine derivative, and new cyclic peptides. The phthalimidine respresents a new carbon skeleton, which poses intriguing biosynthetic questions. The cyclic peptides contain the amino acid residue N-methyl-3-(3-furyl)-alanine, which is a rare amino acid only reported once before in heptapeptides from the fungus Rhizopus microsporus. 1. T. F. Molinski, D. S. Dalisay, S. L. Lievens, J.

P. Saludes, Nature Reviews Drug Discovery 8, 69-85, 2009.

2. K. Neumann, S. Kehraus, M. Gütschow, G.M. König, Nat. Prod. Comm. 4, 347-354, 2009.

3. Pontius, A., Krick, A., Kehraus, S., Foegen, S.E., Müller, M., Klimo, K., Gerhäuser, C., König, G.M., Chem. Eur. J. 32, 9860-9863, 2008.


35

IL 02

NEW BIOACTIVE METABOLITES FROM MARINE INVERTEBRATES Fernando Reyes

Department of Medicinal Chemistry, PharmaMar S.A., Pol. Ind. La Mina Norte, Avda. de los Reyes 1, 28770-Colmenar Viejo (Madrid), Spain.

[email protected]

PharmaMar is a biopharmaceutical company founded in 1986 which is devoted to advancing in the treatment of cancer through the discovery, development and marketing of innovative drugs of marine origin. Yondelis®, developed by PharmaMar, was approved in 2007 by the European Medicines Agency (EMEA) for the treatment of patients with advanced soft tissue sarcoma, after failure of anthracyclines and ifosfamide, or who are unsuited to receive these agents. In late 2008 a registration dossier was submitted to the EMEA and Federal Drugs Administration (FDA) for Yondelis® when administered in combination with DOXIL®/Caelyx™ (pegylated liposomal doxorubicin) for the treatment of women with relapsed ovarian cancer (ROC). Aplidin®, Irvalec® and Zalypsis® are other PharmaMar compounds at different phases of clinical evaluation.

Apart from these compounds, the company possesses a unique collection of more than 70.000 marine samples which

is being screened for the discovery of new chemical entities with antitumour properties. Our drug discovery research program includes not only the study of samples of macroorganisms but also culture broths produced by marine microorganisms as potential sources of new anticancer compounds. As a result of this work, three new molecules are currently in advanced pre-clinical development.

Our drug discovery activities include the preparation of aqueous and organic extracts of each sample, in vitro screening for cytotoxic activity, pre-fractionation of bioactive extracts by RP-18 column chromatography and selection of candidates for fractionation by de-replication based on HPLC-DAD-MS-bioactivity profile. This presentation focuses on some of the most recent bioactive molecules discovered at PharmaMar, including metabolites from sponges, tunicates and soft corals collected in different areas of the world.


36

IL 03

PHOTOCHEMISTRY IN THE METABOLITES OF PLACOBRANCHUS OCELLATUS Paul B. Jones

Wake Forest University Winston-Salem, NC 27106

[email protected]

The photochemical conversion of 9,10-deoxytridachione (1) to photodeoxytridachione (2) in the Sacoglossan mollusc, Placobranchus ocellatus, has been known since the late 1970s.1 This transformation involves the rearrangement of a 1,3-cyclohexadiene to a bicyclo[3.1.0]hexene.

Such transformations are well-known in 1,3-cyclohexadiecur via a variety of mechanisms.2 Neither the mechanism by which 1 is converted to 2 in the mollusc nor the role of these two metabolites in the life of the mollusc are known with certainty.

Our work has focused on trying to understand the mechanism of this photoreaction, how it relates to other 1,3-cyclohexadiene rearrangements and how, or if, this chemistry plays any role in the life of the molluscs that host it. We have approached this problem by studying the photochemistry of α- and γ-pyrones, the best chromophore in this class of polypropionates, investigating directly the photochemistry of 1 and by testing 1, 2, and related molecules as feeding deterrents.

The presentation will discuss our results to date, the implications for the role of photochemistry in Sacoglossa and on where the work will go from here. The results of photolysis of 1 and related molecules under a variety of conditions and the implications of these results for the biosynthesis of 2 and other Sacoglossan polypropionates will be discussed as will the differences between α- and γ-pyrones in acting as triplet sensitizers.

1. Ireland, C.; Scheuer, P.J. “Photosynthetic marine mollusks: in vivo carbon-14 incorporation into metabolites of the sacoglossan Placobranchus ocellatus.” Science 1979, 205, 922-923.

2. a) Dauben, W.G.; Rabinowitz, J.; Vietmeyer, N.D.; Wendschuh, P.H. "Photoequilibriums between 1,3-cyclohexadienes and 1,3,5-hexatrienes. Photochemistry of 3 -alkyl-6,6,9,9-tetramethyl-∆3,5(10)-hexalins. J. Am. Chem. Soc. 1972, 94, 4285-4292. b) Padwa, A.; Brodsky, L.; Clough, S. "The role of steric factors in the photochemistry of substituted cyclohexa-1,3-dienes." Chem. Comm. 1971, 417-418.

O

O

OMeO

O

OMe

hν

1 2


37

IL 04

AUSTRALIAN MARINE BIODISCOVERY Robert J. Capon

Division of Chemical and Structural Biology, Institute for Molecular Bioscience, University of Queensland, Carmody Road, St Lucia, Queensland, 4072, Australia

[email protected]

This presentation will cover selected recent progress from our laboratory into Australian marine invertebrates, algae and microbes, as a source of new cytotoxic alkaloids (mirabilins, discorhabdins), and sesterterpene tetronic acid modulators of ligand gated ion channels targeting chronic inflammatory pain pathways. Preliminary data on new natural product

antibiotics and possible neuroprotectants will also be presented, along with a brief introduction to a novel bioinfomatics approach to visualizing multidimensional chemical diversity, capable of rapid correlation of chemical and biological space, drug-like properties, geography, taxonomy and many other variables.


38

IL 05

PROGRESS IN BIOMIMETIC TOTAL SYNTHESIS OF THE MARINE PALAU'AMINE AND CONGENERS

Ali Al‐Mourabit

Institut de Chimie des Substances Naturelles du CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France

[email protected]

Since its discovery in 1993 by Kinnel and Scheuer1 in the marine sponge Stylissa Massa, the immunosuppressive palau’amine is considered as one of the major targets for organic total synthesis. Due to its original and complex structure (six heterocycles and eight contiguous stereocenters) palau’amine becomes a challenging target for several groups. Despite numerous approaches, the total synthesis of palau’amine has not been yet reported. Its relative configuration was corrected very recently.2

Our biogenetic hypothesis3 is based on the formation of the C7-C7’ bond through a dimerization process involving two tautomers of oroidin. Synthetic progress following our biomimetic strategy and the state of the art will be discussed.

1. Scheuer and coll., J.Am.Chem.Soc., 1993, 115,

3376-3377. 2. Köch and coll., Angew. Chem., 2007, 46, 2320-

2324. 3. Al-Mourabit, A.; Potier, P. Eur. J. Chem. 2001,

237-243.

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NH

OH

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NO

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NH

H

HH

Cl

NH

HNNH

OH

N

NO

HN

NH

NH

H

H

NH

O

NH

kombu'acidine A palau'amine A

NN

N

NNH

O

NHNH2

HN

H

NH

O

NH

H

7

7

2x clathrodine

NHO

HNNH

HN

HN

O

HNNH

O

HN

NHO

NH

H

HH2N

Cl

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NH

OH

N

NO

HN

NH

NH

H

HH

NH

O

NH

Cl

NHHN

NH

O

NHN

NH

NH

HH

OO

NH


39

IL 06

MARINE COMPOUNDS AS PROMISING ANTI-CANCER AGENTS Marc Schumacher 1,2, Florence Folmer1,2, Marcel Jaspers1, Mario Dicato2

and Marc Diederich2

1 Marine Natural Products Laboratory, Chemistry Department, University of Aberdeen, Old Aberdeen, Scotland

2 Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Luxembourg, Luxembourg

[email protected]

Over the past twenty years, numerous drug discovery programmes based on a continuously growing knowledge about the signal-transduction network that drives neoplastic transformation, and using rationally designed cancer therapeutics that target specific molecular events, have been launched. Most importantly, anti-cancer drug development has shifted dramatically from conventional cytotoxic drugs that affect DNA synthesis in both cancerous and healthy cells to drugs that modulate the activity of proteins which are specifically associated with cancer. For several years, the National Cancer Institute (NCI) in the United States of America has included marine natural products in its screening for novel anticancer drugs. To date, numerous terrestrial natural products and several marine natural products have entered clinical trials as anti-tumour agents, and several natural anticancer compounds are currently being used in the clinic.

The transcription factor κB (NF-κB) orchestrates the activation of a large number of genes that are involved in the regulation of various physiological and

pathological processes, including immune and inflammatory responses. The deregulated activation of NF-κB is associated with numerous inflammatory diseases, with diabetes, and with cancer development. NF-κB has hence become globally recognized as a promising target in drug discovery. With the aim to find new NF-κB inhibitors, we screened over 200 extracts from Fijian marine invertebrates and algae, as well as extracts from cultured Scottish and Costa Rican marine bacteria and from cultured cyanobacteria and microalgae. We observed a 17% hit rate (>50% inhibition of TNF-α induced NF-κB activity at a single test concentration of 100 µg/mL). Bioactive extracts from the Fijian sponge Rhabdastrella globostellata, from the crinoid Comanthus parvicirrus, from the soft corals Lobophytum sp. and Sinularia sp., and from the gorgonian Subergorgia sp. were purified in order to investigate the chemistry and the molecular mechanism of action of the compounds responsible for the NF-κB inhibitory activity observed in the crude extracts.


40

IL 07

ANTIMALARIALS FROM THE SEA. STUDIES ON THE MECHANISM OF ACTION OF ENDOPEROXIDE DERIVATIVES FROM MARINE SPONGES

Orazio Taglialatela‐Scafati

Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli “Federico II”, via D. Montesano, 49, 80131, Naples, Italy.

[email protected]

Malaria is an infectious disease caused by several protozoan species belonging to the genus Plasmodium coming in contact with humans through the vector contribution of mosquitoes of the genus Anopheles. Unfortunately, malaria still continues to be a major cause of morbidity and mortality in poorest tropical countries and it has been estimated that, each year, 300-500 million people become ill with malaria and 1-3 millions die.

Plakortin (1), a polyketide metabolite isolated in 1978 from Plakortis halichondroides, was demonstrated to possess a nanomolar antimalarial activity

against chloroquine-resistant strains of Plasmodium falciparum, devoid of cytotoxicity.1 During the last years our research group has been actively engaged in the investigation of the antimalarial activity of plakortin and related 1,2-dioxanes through: i) isolation of analogues;2 ii) preparation of semi-synthetic derivatives;3 iii) computational (molecular modelling and ab initio calculations) investigation of the mechanism of action; iv) reaction with Fe(II) inorganic salts to experimentally verify our hypothesis on the mechanism of action.

OO

COOCH3

1

OO

COOCH2CH3

2

As expected, these experiments highlighted the crucial role of the endoperoxide group, but also evidenced the key role of the alkyl side chain. Basing on these data, we have designed the total synthesis of simplified endoperoxide derivatives based on the plakortin skeleton and bearing its essential pharmacophoric portions (e.g. 2). In this lecture, details of our investigation on the mechanism of the antimalarial activity of plakortin derivatives and efforts aimed at their optimization will be presented.

1. Fattorusso, E.; Parapini, S.; Campagnuolo, C.; Basilico, N.; Taglialatela-Scafati, O.; Taramelli, D.; J. Antimicrob. Chemother., 2002, 50, 883-888.

2. Campagnuolo, C.; Fattorusso, E.; Romano, A.; Taglialatela-Scafati, O.; Basilico, N.; Parapini, S.; Taramelli, D. Eur. J. Org. Chem., 2005, 23, 5077-5083.

3. Fattorusso, C.; Campiani, G.; Catalanotti, B.; Persico, M.; Basilico, N.; Parapini, S.; Taramelli, D.; Campagnuolo, C.; Fattorusso, E.; Romano, A.; Taglialatela-Scafati, O. J. Med. Chem., 2006, 49, 7088-7094.


41

IL 08

THE CHEMICAL ECOLOGY OF SPONGES ON CARIBBEAN CORAL REEFS: HOW NATURAL PRODUCTS SHAPE NATURAL SYSTEMS

Joseph R. Pawlik

Department of Biology and Marine Biology Center for Marine Science UNC Wilmington, NC 28409 USA

[email protected]

Natural products chemistry has played an important role in altering the course of medical and agricultural science, but also of ecosystem function. Our ability to study the chemical ecology of marine environments developed most recently, with the advent of SCUBA diving and submersibles. After several decades of research, we are finding that insights from marine chemical ecology are profoundly altering our understanding of some marine communities.

Sponges are now the dominant organisms on Caribbean coral reefs. Until recently, it was believed that consumers had little effect on reef sponges, because sponge-eating fishes were thought to spread their predatory activities over all available species to the detriment of none in particular. But research on the chemical ecology of this system has transformed our understanding of it1. Laboratory and field experiments have revealed three distinct categories of sponges within the community: (1) defended species that are unpalatable to consumers because they contain secondary metabolites, (2) palatable species that sustain grazing by consumers yet are equally common as defended species on the reef, and (3) preferred species that are rapidly consumed when transplanted to the reef, and are found only in refuge habitats2,3. The secondary metabolites responsible for the chemical defenses of several species

have been isolated and identified using bioassay-guided fractionation and field experiments with natural populations of reef consumers4,5. To counter the effects of grazing by fishes, palatable species appear to heal, grow or reproduce faster than defended species6. Some sponge species compete with corals for space by producing metabolites that cause coral bleaching or that interfere with photosynthesis of coral symbionts7. The predictive value of the foregoing is becoming evident: over-fishing on coral reefs may result in a release from predation of sponge species that are competitively superior to corals, reinforcing the current state of low coral cover on Caribbean reefs.

1. Pawlik, Henkel, McMurray, López-Legentil, Loh, Rohde, Mar Ecol Prog Ser, 2008, 368: 137-143.

2. Pawlik, Limnol Oceanogr, 1998, 43: 1396-1399.

3. Dunlap, Pawlik, P.S.Z.N.I: Mar Ecol, 1998, 19: 325-337.

4. Grube, Assmann, Lichte, Sasse, Pawlik, Köck, J Nat Prod, 2007, 70: 504-509.

5. Morinaka, Pawlik, Molinski, J Nat Prod, 2009, online early.

6. Walters, Pawlik, Int Comp Biol, 2005,45: 352-358.

7. Pawlik, Steindler, Henkel, Beer, Ilan, Limnol Oceanogr 2007, 52: 907-911.


42

IL 09

RECENT DEVELOPMENTS IN PSP TOXIN CHEMISTRY, DETECTION AND BIOTRANSFORMATION

Paulo Vale

Instituto Nacional dos Recursos Biológicos / L-IPIMAR (INRB/L-IPIMAR), Av. Brasília, s/n, 1449-006, Lisboa, Portugal

[email protected]

The presence of hydrophobic analogues of paralytic shellfish poisoning toxins (PSTs) was studied in a Portuguese strain of the dinoflagellate Gymnodinium catenatum by pre-column oxidation HPLC coupled with fluorescence detection. Separation of hydrophobic PSTs analogues from hydrophilic analogues was done by water/methanol SPE partitioning on a C18 cartridge. Several unknown oxidation products (OxP), with emission spectra similar to known PSTs, appeared after periodate or hydrogen peroxide oxidation. The compounds producing these OxP were grouped into three major sub-groups according to SPE partitioning. The first one eluting up to 20% MeOH, produced the first set of OxP observed after the saxitoxin OxP. The second one eluting between 30% and 100% MeOH produced the second set of OxP. The third one eluted only with acidified 90% MeOH produced the third and last set of OxP. Additionally, the OxP corresponding to decarbamoyl gonyautoxins and decarbamoyl saxitoxins were also abundant, resulting from ester cleavage of the benzoate side chain of these compounds during the oxidation. LC-MS analysis demonstrated the second sub-group was constituted by analogues of

the 11-hydroxysulfated GC1/2, while the third sub-group was constituted by analogues of GC3, lacking the 11-hydroxysulfate. In addition to GC1/2 and GC3, novel analogues differing by 16 a.m.u. could be related, respectively, to the N1-hydroxyl analogues of GC1 through GC3, designated GC4 through GC6. A novel family of GC analogues, differing, by 16 a.m.u. from GC1 through GC6, were hypothesized to possess an extra hydroxyl in the benzoate side chain, and designated GC1a through GC6a. The first sub-group were hypothesized to constitute an additional novel family of GC analogues with a hydroxysulfate group instead of the hydroxyl group in the benzoate side chain, designated GC1b through GC6b. Despite the high proportion of benzoate analogues produced by G. catenatum, in bivalves these analogues were found only in trace levels. A widespread carbamoylase activity in bivalves, converting benzoate into decarbamoyl analogues, was confirmed by in vitro incubations of digestive glands with semi-purified GC toxins. Decarbamoyl analogues are already detected by common HPLC methods used for food protection.


43

IL 10

MARINE BIOPOLYMERS IN NANOMEDICINE: ADVANCES IN DRUG DELIVERY Ricardo Riguera

Departamento de Química Orgánica, Facultad de Química, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain

[email protected]

Chitosan (CS) is a biopolymer of marine origin particularly promising in the drug delivery area due to its ability to form nanoparticles and its biocompatibility, low toxicity and mucoadhesiveness. Its properties are highly dependent on the acetylation degree and MW, difficult to be accurately determined by NMR or other methods, due to its polymeric nature (1-2). In this communication we will show examples of drug delivery based on the use of nanoparticles derived from chemically modified CS. These nanoparticles carry inside the drug to be delivered while their surface is decorated with a variety of functions designed to improve their stability, their visibility in the cell and their selective accumulation on the target cells/organs.

The preparation, characterization and isolation of CS-modified polymers carrying different proportions of PEG

chains (3), fluorescent labels, molecules to be recognized by the target, and antibodies, as well as their transformation into nanoparticles (4) are to be illustrated with examples of medical significance.

In a different approach, the nanoparticles are formed first from the biopolymer and then functionalized on their surface using Click chemistry. Procedures to avoid the depolymerization by Cu+2 in the click reaction are described and exemplified by the decoration of CS-g-PEG-azide nanoparticles with cyclooctyne-IgG antibody, its recognition by the specific protein and its visual detection through a fluorescent tag. 1. Novoa, R.; Correa, J.; Fernández, E.; Riguera,

R. J. Am. Chem. Soc. 2007, 129, 15164. 2. Fernández, E.; Novoa, R.; Quiñoá, E.; Riguera,

R. Carbohydr. Polym. 2005, 61, 155. 3. Prego, C.; Torres, D.; Fernandez, E.; Novoa,

R.; Quiñoá, E.; Alonso, M.J. J. Control. Release 2006, 111, 299.

4. Fernández, E.; Novoa, R.; Quiñoá, E.; Riguera, R. Biomacromolecules, 2007, 8, 833. (b) Aktas, Y. et al. Bioconjugate Chem., 2005, 16, 1503.

5. Lallana, E.; Fernández, E.; Riguera, R. J. Am. Chem. Soc. 2009, 131, 5748.


44

IL 11

NANOMOLE-SCALE MARINE NATURAL PRODUCTS Tadeusz F. Molinski, Doralyn S. Dalisay, Brandon I. Morinaka and Colin Skepper

Department of Chemistry and Biochemistry, and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California,

San Diego 9500 Gilman Drive, MC-0358, La Jolla, CA 92093, U.S.A.

[email protected]

Drug discovery from marine natural products has a relatively short history, yet in 2004 and 2007 the search for 'drugs from the sea' finally brought two compounds to the market; the analgesic peptide Prialt®, from Conus magus, and the antitumor agent, Yondelis®, from the tunicate Ecteinascidia turbinata.1 Two contemporary themes in marine natural products are discovery of novel compounds from novel microbial sources, and genomic mining of natural products from sequences of uncultured sources including metagenomes,2,3 and predictive natural product structure elucidation from 'gene-gazing',4 However, yields of 'orphan natural products' from non-optimized heterologous expressions systems, or slow-growing microbes associated with invertebrates, are often limited; sometimes to only a few nanomole/liter, which is sufficient for mass spectrometry, but presents a challenge for full characterization by NMR. Revolutionary cryo-microprobe NMR spectroscopy5 now permits elucidation of structure from tiny amounts (~µg's),6 a feat that would have been impossible a few years ago.

This talk will describe new natural products at the nanomole-scale from marine sponges, cyanobacteria and tunicates, including muironolide A,7 a new chemical entity with a novel carbon skeleton, from the sponge Phorbas sp. which also gave phorboxazoles A, B,8 and phorbasides A-E.9 The complete stereostructure of muironolide A (total

yield, 90 µg) was solved from NMR data obtained using a 1.7 mm cryomicroprobe (600 MHz), including strategic use of J-coupled HSQC, FTMS, circular dichroism (CD) and synthesis. 1. Molinski, T. F.; Dalisay, D. S.; Lievens, S. L.;

Saludes, J. P. Nat. Rev. Drug. Discov. 2009, 8, 69-85.

2. Piel, J. Curr. Med. Chem. 2006, 13, 39.

3. (a) McAlpine, J. B. J. Nat. Prod.2009, 72, 566. (b) Gross, H. Curr. Opin. Drug. Discov. Devel. 2009, 12, 207.

4. (a) McGlinchey, R. P.; Nett, M.; Eustaquio, A. S.; Asolkar, R. N.; Fenical, W.; Moore, B. S. J. Am. Chem. Soc. 2008, 130, 7822. (b) Udwary, D. W.; Zeigler, L.; Asolkar, R. N.; Singan, V.; Lapidus, A.; Fenical, W.; Jensen, P. R.; Moore, B. S. Proc. Nat. Acad. Sci. USA 2007, 104, 10376.

5. Molinski, T. F. Curr. Opin. Drug. Discov. 2009, 12, 197.

6. (a) Dalisay, D. S.; Molinski, T. F. J. Nat. Prod. 2009, 72, 739. (b) Dalisay, D. S.; Rogers, E. W.; Edison, A. S. J. Nat. Prod. 2009, 72, 732. (c). Dalisay, D. S.; Molinski, T. F. Org. Lett. 2009, 11, 1967.

7. Dalisay, D. S.; Morinaka, B. I.; Skepper, C. K.; Molinski, T. F. J. Am. Chem. Soc. 2009, accepted.

8. (a) Searle, P. A.; Molinski, T. F. J. Am. Chem. Soc. 1995, 117, 8126.(b) Molinski, T. F. Tetrahedron Lett. 1996, 37, 7879. (c) Searle, P. A.; Molinski, T. F.; Brzezinski, L. J.; Leahy, J. W. J. Am. Chem. Soc. 1996, 118, 9422.

9. (a). Skepper, C. K.; MacMillan, J. B.; Zhou, G. X.; Masuno, M. N.; Molinski, T. F. J. Am. Chem. Soc. 2007, 129, 4150. (b) MacMillan, J. B.; Xiong-Zhou, G.; Skepper, C. K.; Molinski, T. F. J. Org. Chem. 2008, 73, 3699.

Oral Communications


46

OC 01

NMR AND MS TECHNIQUES FOR RAPID CHARACTERIZATION OF PROKARYOTE - EUKARYOTE ASSOCIATIONS: SPECTROSCOPIC FINGERPRINTS OF MARINE ALGAE AND OF THEIR ASSOCIATED

MICROFLORA Stéphane La Barre1,2, Stéphanie Salaün1,2, Nelly Kervarec3, Simon Dittami1,2, Aude Le

Bail1,2, Thierry Tonon1,2, Bénédicte Charrier1,2 and Philippe Potin1,2 1 Université Pierre et Marie Curie-Paris 6, UMR 7139 Végétaux Marins et Biomolécules, Station Biologique F-29682, Roscoff, France. 2 CNRS, UMR 7139 Végétaux Marins et Biomolécules, Station Biologique F-29682, Roscoff, France. 3 Service Commun de Résonance Magnétique Nucléaire, Université de Bretagne Occidentale, UFR Sciences et Techniques, 6 avenue Le

Gorgeu, BP 809 29285, Brest, France.

[email protected]

Mass spectrometry (MALDI-TOF) and nuclear magnetic resonance spectroscopy (HR-MAS) with, respectively, MALDI-TOF (matrix assisted laser desorption/ionization – time of flight) and HR-MAS (high resolution – magic angle spin) technologies, have been recently developed for the fingerprinting of microbial pathogens of medical importance. As complementary tools to detailed spectroscopic analyses of individual metabolites, these fingerprinting techniques can be used in routine taxonomic checks of environmental microbial strains, and in metabolomic studies of both prokaryote cells and eukaryote tissues.

At Station Biologique de Roscoff, we have created a database of HR-MAS NMR proton spectra of bacterial microcolonies developed from isolates of

the brown kelp Laminaria digitata, together with a MALDI-TOF MS database of the same strains. Both databases were completed with fingerprints from referenced exogenous isolates. Visual and in silico grouping of spectra of both databases closely followed molecular 16S rDNA taxonomy. In addition, some HR-MAS spectral variability was noted with culture age and when changing from rich to minimal culture medium, indicating that reliable databases must include spectra from strains cultivated under strictly identical conditions. Metabolomic applications of proton HR-MAS NMR spectroscopy allowed us to distinguish between wild type and mutant strains of the brown algal model Ectocarpus siliculosus the genome of which was fully sequenced and partly annotated in a project led at Roscoff.


47

OC 02

SEARCHING FOR NEW BIOACTIVE SUBSTANCES FROM SOUTH CHINA SEA MARINE ORGANISMS

Yue‐Wei Guo

State Key Laboratory of Drug Research, Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China

[email protected]

The oceans contain a vast biological diversity of species that have so far been utilized by mankind mainly as a source of protein. In the last few decades, however, natural products chemists have started to explore the wealth of the marine fauna and flora such as sponges, soft corals, mangroves and others for discovering bioactive secondary metabolites with the pharmaceutical application potential.1

South China Sea is in the tropical zone where marine biodiversity is abundant. Searching of marine natural products produced by South China Sea marine organisms has been carried out by our group since 2000. These years our continuous exploration of South China Sea marine biodiversity with the purpose to find new leads for drug discovery has led to isolation and characterization of numerous marine natural products with diverse chemical structures, extraordinary biological and pharmaceutical activities.2-5

In this communication the structures and biological activities of these marine metabolites, which might be useful as biomedical agents, will be described.

1. J. W. Blunt, B. R. Copp, W.-P. Hu, M. H. G. Munro, P. T. Northcote and M. R. Prinsep, Nat. Prod. Rep., 2009, 26, 170-244, and previous reports in this series.

2. a). S.-C. Mao, E. Manzo, Y.-W. Guo, M. Gavagnin, E. Mollo, M. L. Ciavatta, R. van Soest and G. Cimino, Tetrahedron, 2007, 63, 11108-11113; b) Z,-Y. Li, Z.-G. Yu, Y.-W. Guo, Helv. Chim. Acta., 2008, 91, 1553-1558.

3. W. Zhang, K. Krohn, J. Ding, Z.-H. Miao, X.-H Zhou, S.-H. Chen, G. Pescitelli, P. Salvadori, T. Kurtán, Y.-W. Guo J. Nat. Peod., 2008, 71, 961-966.

4. X.-C. Huang, J. Li, Z.-Y. Li, L. Shi, Y.-W. Guo, J. Nat. Prod., 2008, 71, 1399-1403.

5. Y. Li, Y. Zhang, X. Shen, Y.-W. Guo, Bioorg.& Med. Chem. Lett.,, 2009, 19, 390-392.


48

OC 03

DIMERIC PYRROLE-IMIDAZOLE ALKALOIDS – NEW MASSADINE STEREOISOMERS AND THEIR CONFIGURATIONAL ASSIGNMENT

Matthias Köck1, Christine Cychon1, Gesine Schmidt1, Ian Seiple2 and Phil S. Baran2 1 Alfred-Wegener-Institut für Polar- und Meeresforschung in der Helmholtz-Gemeinschaft, Am

Handelshafen 12, D-27570 Bremerhaven, Germany. 2 Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La

Jolla, CA 92037, USA


To date, about 30 dimeric pyrrole-imidazole alkaloids (PIA´s), like palau’amine1, massadine2, axinellamine3, are known from the marine sponge families Agelasidae, Axinellidae, Dictyonellidae, and Hymeniacidonidae.4 The recent total synthesis of massadine and massadine chloride generated a new stable stereoisomer, the 3,7-epi-form.5 This stereoisomerism correlates with axinellamines A and its 5,9-epi form B which are both found in nature whereas the corresponding epi-isomer was not isolated for the massadines. Therefore, several sponge samples of the genus Stylissa were investigated to answer the question if 3,7-epi-massadine is a natural product. Furthermore, the search was expanded to 2-epi-massadine which represents the second half (first half massadine) of the tetrameric PIA stylissadine B6 which is also unknown from natural sources so far. The relative configuration of these compounds was assigned using the floating chirality

rDG/DDD method which was successfully applied in the revision of the relative configuration of the PIA palau’amine.7

1. a) R. B. Kinnel, H.–P. Gehrken, P. J. Scheuer J. Am. Chem. Soc. 1993, 115, 3376–3377; b) R. B. Kinnel, R. Swali, G. Skoropowski, H.-P. Gehrken, P. J. Scheuer, J. Org. Chem. 1998, 63, 3281-3286.

2. a) S. Nishimura, S. Matsunaga, N. Fusetani, M. Shibazaki, K. Suzuki, K. Furihata, R. W. M. van Soest, Org. Lett. 2003, 5, 2255-2257; b) A. Grube, S. Immel, P. S. Baran, M. Köck, Angew. Chem. Int. Ed. 2007, 46, 6721-6724.

3. S. Urban, P. de Almeida Leone, A. R. Carroll, G. A. Fechner, J. Smith, R. J. Quinn, J. N. A. Hooper, J. Org. Chem. 1999, 64, 731-735.

4. M. Köck, A. Grube, I. B. Seiple, P. S. Baran, Angew. Chem. Int. Ed. 2007, 46, 6586-6590.

5. S. Su, I. B. Seiple, I. S. Young, P. S. Baran, J. Am. Chem. Soc. 2008, 130,16490-16491.

6. a) A. Grube, M. Köck, Org. Lett. 2006, 8, 4675-4678; b) M. S. Buchanan, A. R. Carroll, R. Addepalli, V. M. Avery, J. N. A. Hooper, R. J. Quinn, J. Org. Chem. 2007, 72, 2309-2317.

7. A. Grube, M. Köck, Angew. Chem. Int. Ed. 2007, 46, 2320-2324.


49

OC 04

METABOLOMIC PROFILING OF SOME MARINE SPONGES FROM THE IRISH AND CELTIC SEAS BY HIGH RESOLUTION FTMS AND NMR WITH THE

AID OF SIEVE ANALYSIS RuAngelie Edrada‐Ebel

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, The John Arbuthnott Building, Glasgow G4 0NR, Scotland

[email protected]

Not much literature has been published on natural products from sponges of the Irish and Celtic Sea. Metabolomic profiling was done on organic extracts of 12 sponge samples collected from the Irish and Celtic Sea using high resolution LCFTMS and NMR. The secondary metabolite profiles were then compared to those of sponges from the Pacific and the Mediterranean Region through the database MARINLIT with the aid of SIEVE, an automated label-free differential expression software. High resolution NMR was utilised to identify resonances that quantifies and confirms the presence of the secondary metabolite of interest. Preliminary chromatographic

separation of the active secondary metabolites was achieved on sponge extracts exhibiting an interesting chemical profile. One of the sponges studied is Haliclona simulans and the presence of Xestospongin derivatives1,2,3 with the MF of C28H50O3N2 as the major secondary metabolite was detected. Xestospongin is a selective and membrane-permeable inhibitor of IP3 receptor.4 Related MF (molecular formula), RDE (double bond equivalence) and MSMS fragmentation obtained from the HRFTMS data suggested the presence of two plausible new derivatives in the extract.

1. Nakagawa M Endo M Tanaka N Gen-Pei L Tetrahedron Lett. 1984, 25, 3227-3230.

2. Quirion JC, Sevenet T, Husson HP, Weniger B, Debitus C. J. Nat. Prod.,1992, 55, 1505-1508.

3. Moon S, MacMillan JB, Olmstead MM, Ta TA, Pessah IN, Molinski TF. J. Nat. Prod., 2002, 65, 249-254

4. Jaimovich E, Mattei C, Liberona J, Cardenas C, Estrada M, Barbier J, Debitus C, Laurent D, Molgó J. FEBS Letters, 2005, 579, 2051-2057


50

OC 05

.

METABOLITES FROM MARINE-DERIVED FUNGI Vatcharin Rukachaisirikul1, Kongkiat Trisuwan1, Nanthaphong Khamthong1,

Yaowapa Sukpondma1, Jariya Sakayaroj2 and Souwalak Phongoaichit3 1 Department of Chemistry and Center for Innovation in Chemistry (PERCH-CIC),

Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand; 2 National Center for Genetic Engineering and Biotechnology, Thailand Science Park,

Klong Luang, Pathumthani, 12120, Thailand; 3 Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai,

Songkhla 90112, Thailand

[email protected]

Marine-derived fungi are a rich source of structurally unique and biologically active secondary metabolites and a promising source of pharmaceutical leads. During our ongoing search for bioactive natural products from marine-derived fungi, many fungal isolates have been isolated from a gorgonian sea fan (Annella sp.), collected near the Similan Islands, Southern Thailand. They were screened for their potential to produce antimicrobial and

antioxidation substances. The fungi were selected on the basis of their biological data and 1H NMR profiles for further study on bioactive metabolites and new compounds. Structural elucidation of secondary metabolites, isolated from the marine-derived fungi in the genera Nigrospora, Penicillium, Xylaria and Fusarium, and their biological activities will be presented.


51

OC 06

ALGAL DITERPENOIDS AS ANTIFOULING SUBSTANCES AGAINST A MARINE BACTERIAL BIOFILM

Yannick Viano, Dominique Bonhomme, Mercedes Camps, Jean‐François Briand, Annick Ortalo‐Magné, Yves Blache and Gérald Culioli

Laboratoire MAPIEM (EA 4323), Université du Sud Toulon-Var, Av. de l’Université, BP 20132, 83957 La Garde Cedex, France.


All surfaces immersed in the marine environment are subject to the colonization by micro- and then macro-organisms that results in the formation of a complex biological layer named biofouling. As first colonizers of free surfaces, bacteria are of special importance. Moreover, their organization in biofilms leads to a significant effort to search natural antifoulants that will inhibit the formation of these particularly resistant three-dimensional structured communities1.

In this context, phytochemical analysis of two brown mediterranean algae (Dictyota sp. and Dictyota dichotoma) collected off the french and algerian coasts were realized. From their lipophilic extracts, six new diterpenoids and one new diterpenic dimer (1) were isolated together with eight known compounds.

The planar structure of each metabolite was characterized on the basis of spectroscopic data (RMN 1D and 2D, HRESIMS) and by comparison with literature. To our knowledge,

Dictyotadimer A (1) is the first diterpenic dimer of algal origin. A C-C-linkage between two different xenicane units is the main structural feature of this unusual asymmetric bis-diterpene.

The relative stereochemistry of these new compounds was determined through extensive 1H-1H NOESY analysis and molecular modeling calculations. In particular, for one of these compounds (2), the most favoured conformation of each stereoisomer was searched. This process afforded only one stereoisomer for which the most stable conformer was in good agreement with NOE data, experimental coupling constants and biosynthetic considerations.

Finally, the anti-adhesion activity of metabolites isolated in sufficient amount was evaluated against a marine bacterial biofilm of Pseudoalteromonas sp.

1. Costerton, J. W.; Lewandowski, Z.; Caldwell,

D. E.; Korber, D. R.; Lappin-Scott, H. M. Annu. Rev. Microbiol. 1995, 49, 711-745.

HO

HO

OH

OO

HO

H

H

Dictyotadimer A (1) 2


52

OC 07

NMR STRUCTURES OF A PSYCHROPHILIC FAMILY OF WATER-BORNE SIGNAL POLYPEPTIDES ISOLATED FROM THE POLAR PROTOZOAN CILIATE, EUPLOTES

NOBILII Claudio Alimenti1, Adriana Vallesi1, Bill Pedrini2, Kurt Wüthrich2, Pierangelo Luporini1

1 Dipartimento di Biologia molecolare cellulare animale, Università di Camerino, 62032 Camerino (MC), Italy.

2 Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA; Institut für Molekularbiologie und Biophysik, ETH, CH-8093 Zurich, Switzerland.

[email protected]

A variety of strains of Euplotes nobilii collected from Antarctic and Arctic waters have been found capable to constitutively secrete cell type-specific signal polypeptides of 50 to 63 amino acids (usually referred to as pheromones) in concentrations high enough (approx 50-150 micrograms of protein/liter of cell culture) to carry out NMR determinations of the relative molecular structures. The four determined pheromone structures all show in common a tight conservation of a three-helix bundle core, that is stabilized by four disulfide bonds and ensures a long-lasting integrity of these molecules in the natural environment. On this conserved scaffold, molecule- and family-specific traits can be distinguished. The individual traits appear to be primarily committed to confer specificity to the autocrine (mitogenic) and paracrine (sexual) signaling activity of each pheromone, and are mainly due to variations in the length and regularity of

the three helices, as well as in the shape and orientation of the carboxy-terminal tail. On the other hand, the family-specific traits appear to be evolved in functional correlation with cold-adaptation. Most relevant are: (i) the extension of polypeptide segments devoid of regular secondary structures, (ii) a unique distribution of polar and hydrophobic amino acids, (iii) the presence of solvent-exposed clusters of negatively charged amino acid side chains, and (iv) a central role of aromatic residues in anchoring particular regions of the molecular architecture. Overall these cold-adaptive modifications make the psychrophilic pheromone family of E. nobilii an elegant example of how a high level of global stability of the three-dimensional structures may be combined with sufficient levels of local structural plasticity for efficient functioning of environmental signaling molecules at physiologically low temperatures.


53

OC 08

NJAOAMINIUMS A, B, AND C: CYCLIC 3-ALKYLPYRIDINIUM SALTS FROM THE MARINE SPONGE RENIERA SP.

Mohamed Mehiri1, Grégory Genta‐Jouve1, Rogelio Fernández2, Olivier P. Thomas1, Fernando Reyes2, Philippe Amade1

1 LCMBA - UMR 6001 - University of Nice Sophia Antipolis, Parc Valrose, 06108 Nice, France. 2 Medicinal Chemistry Department, PharmaMar S.A., Pol. Ind. La Mina Norte,

Avenida de los Reyes 1, 28770 Colmenar Viejo, Madrid, Spain.

[email protected]

Three novel cyclic 3-alkylpyridinium salts, named njaoaminiums A, B, and C (1-3), were isolated from the marine sponge Reniera sp., collected off the coasts of Pemba Island, Tanzania. The structural determination of the compounds was based on 1D and 2D NMR studies and mass spectral determinations. Njaoaminiums B (2) and C (3) are the first examples of cyclic 3-alkylpyridinium salts bearing a methyl substituent on the alkyl

chains. These compounds are assumed to be biosynthetic precursors of the njaoamines, previously isolated in the same sponge. The absolute configurations of the methyls of 2 and 3 were assigned by comparison between experimental and TDDFT calculated circular dichroism spectra on the most stable conformer. Compound 2 showed weak cytotoxicity against the three human tumour cell lines MDA-MB-231, A549, and HT29.

NN

1 Njaoaminium A R1 = R2 = H2 Njaoaminium B R1 = R2 = Me3 Njaoaminium C R1 = H R2 = Me

, 2 X1

3

713 14 16

1'

3'

7'8'

9'

R1

R2


54

OC 09

SYNTHESIS OF AN ISOCYANIDE GLYCERYL-LIKE LIPID ISOLATED FROM ACTINOCYCLUS PAPILLATUS

Emiliano Manzo, Marianna Carbone, Yan Li, Maria Letizia Ciavatta, and Margherita Gavagnin

Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, I 80078-Pozzuoli (Naples), Italy

[email protected]

Marine natural products are often characterized by the presence of stereogenic centers and their synthesis is a challenge for the structural elucidation as well as the ‘in vitro’ production. Among marine substances, isocyanide compounds are attracting molecules due to their interesting biological activities.2,3 An unusual isocyanide lipid, compound 1, was recently isolated in our laboratory from the lipophilic extract of the mantle of the opisthobranch Actinocyclus papillatus,4 collected along Chinese coasts.

This compound is an ether lipid with a glyceryl-like moiety in which the secondary hydroxyl function is replaced by an isocyanide group. Its structure was established by spectroscopic methods whereas the stereochemistry of the secondary chiral carbon of 2-isocyano-1,3-propandiol moiety remained undetermined.

We report here a stereospecific synthesis of both (+)- and (-)-1, planned with the aim at establishing the stereochemistry of natural 1 by comparing its optical

properties with those of the synthetic products. In addition, the potential biological activity of 1, that was not evaluated due to the small amount of the natural product, could be now investigated.

1

AcO OC16H33

NC

1. On leave from Institute of Materia Medica, CAS, Shanghai, PR China.

2. Blunt, J.W.; Copp, B.R.; Hu, W.-P.; Munro, M.H.G.; Northcote, P.T.; Prinsep, M.R. Nat.Prod.Rep. 2009, 170-244, and previous reports in this series.

3. Garson, M.J.; Simpson, J.S. Nat.Prod.Rep., 2004, 164-179.

4. Carbone, M.; Gavagnin, M.; Mollo, E.; Tsoukatou, M.; Castelluccio, F.; Guo, Y-W.; Cimino, G. Poster communication, 5th ECMNP-2007, P-63.


55

OC 10

POTENT ANTI-TUMOR AND ANTI-INFLAMMATORY LEAD STRUCTURES FROM CARIBBEAN SPONGES

Valeria Costantino, Ernesto Fattorusso, Alfonso Mangoni, and C. Perinu

Dipartimento di Chimica delle Sostanze Naturali, via D. Montesano, 49 - 80131 Napoli - Italy

[email protected]

Marine natural products are playing an increasingly important role in biomedical research and drug discovery, either directly as drugs or as lead structures.

Caribbean marine sponges are well-known to produce a large array of new chemical structures with promising anti-cancer, anti-inflammatory, immunomodulating and anti-bacterial properties. Over the last 15 years our research group’s interest has been focused on the chemical study of these colorful animals living in the tropical oceans as a source of new structures to be used as leads in the search of new drugs(1,2).

Two topics of our ongoing research work will be discussed in the communication:

a. the antinflammatory activity of tedanol, a new brominated and sulphated diterpene alcohol having a pimarane skeleton, which was isolated from the Caribbean sponge T. ignis. Tedanol showed a significant anti-inflammatory in vivo activity at 1 mg/kg3, coupled with the inhibition of the COX-2 expression. This activity makes tedanol a novel potent COX-2 selective inhibitor, and could represent an excellent water-soluble anti-inflammatory molecule with minimal gastrointestinal toxicity.

b. The anti-tumor activity of three diterpene isonitriles isolated from the Caribbean sponge Pseudaxinella flava. The compounds have been first screened for their in vitro growth inhibitory activity (MTT assay) using the four human cell lines. Then, the gross mechanism of action of the isonitriles have been determined by means of computer-assisted phase contrast videomicroscopy analysis (see Figure 1)4. The digital movies shoved that the cancer cells treated with isonitriles are pro-autophagic agents and make the cells unable to undergo mitotic processes, resulting in a cytostatic effect.

1. V. Costantino, E. Fattorusso, A. Mangoni, M. Di Rosa, A. Ianaro, Bioorg. & Med. Chem. Lett., 271 (1999).

2. L. Barbieri, V. Costantino, E. Fattorusso, A. Mangoni, E. Aru, S. Parapini, and D. Taramelli, Eur. J. Org. Chem., 3279-3285 (2005).

3. MG Henriques, PM Silva, MA Martins, CA Flores, FQ Cunha, J Assreuy-Filho, RS Cordeiro. Braz J Med Biol Res.; 20(2):243-9 (1987).

4. C. Decaestecker, O. Debeir, P. Van Ham, R. Kiss Med Res Rev Vol. 27, No. 2, 149-176 (2007).

Figure 1 PC3 human prostate cancer cell line with Caterp-6 at µM after 8 minutes, 24 minutes and 3 days.


56

OC 11

NOVEL DRUG LEADS FOR HEPATIC STEATOSIS AND NONALCOHOLIC STEATOHEPATITIS (NASH)

Junyoung Hong1, Euno Kim1, Jung‐A Kim1, Sun Shin1, Hyukjae Choi1, Dongyup Hahn1, Hoosang Hwang1, Byungsoo Hwang2, Jungrae Rho2 and Heonjoong Kang1*

1 Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, Seoul National University, NS-80, 599 Gwanangno,

Gwanak-gu, Seoul 151-747, Korea; 2 Department of Oceanography, Kunsan National University,

Korea 82-2-880-5730 (O), 883-9289 (F)

[email protected]

Fatty liver disease is a serious problem in modern society and prevailed in up to 30% of the adult population, characterized from mild hepatic fatty liver to serious cirrhosis, eventually leading to hepatic carcinoma. Yet there is no obvious treatment for the disease at the moment. At initial stage hepatic steatosis viz fatty liver is caused possibly by one of the following mechanisms in the liver: 1) increased uptake of lipids; 2) elevated de novo synthesis of fatty acids; 3) impaired lipoprotein synthesis or secretion; 4) reduced β-oxidation. Later it develops into NASH along with severe inflammation. Thus precise blocking of any of the processes or in combination will lead to develop a novel drug for the treatment of the disease.

The nuclear receptors are ligand-activated transcription factors which regulate many aspects of metabolism and inflammation in liver. The receptors thus become promising drug targets for treatment of these diseases.

A combination of marine natural product isolation and automated bioassays led to identification of various compounds as agonists and antagonists for the nuclear receptors. The marine natural products we isolated had for example IC50 value as low as 20 nM against nuclear receptors. The ligands had good selectivity over the other types of receptors. Pharmacological treatment of mice with one of the ligands totally reversed drug-induced hepatic steatosis in vivo. In addition the above mentioned antagonist gave great improvement in the condition of NASH in a mouse model with modulation of lipid biosynthesis, lipid uptake and inflammation genes. The other ligands gave good antiobesity and anti-diabetic effects in mice models. These results clearly demonstrate ligands of nuclear receptors as drug leads to treat steatohepatitis and NASH as well as metabolic diseases such as obesity, diabetes.


57

OC 12

SEARCH FOR WATER-SOLUBLE BIOACTIVE MARINE FUNGAL METABOLITES: PERFECTING OF AN EXTRACTION AND PARTIAL-

PURIFICATION METHOD Carine Le Ker1*, Karina‐Ethel Petit1, Yves‐François Pouchus1, Jean‐François Biard1,

Joël Fleurence2 1 Université de Nantes, Pôle Mer et Littoral, Laboratoire MMS-EA2160, Faculté de Pharmacie, BP

53508, Nantes, F-44035 France; 2 Université de Nantes, Pôle Mer et Littoral, Laboratoire MMS-EA2160, Faculté des Sciences et

Techniques, BP 92208, Nantes, F-44322, France

* [email protected]

In the drug discovery research, marine fungi have proven to be a rich source of new molecules with pharmaceutical interest (Blunt et al., 2009). Majority of fungal compounds described in chemistry literature are lipid-soluble or medium polar compounds. However water-soluble products seem to be a promising group of original and bioactive molecules. For example gigantin and sarcin, two ribosome inactivating proteins, and an antifungal polypeptide have been isolated from the culture filtrate of an Aspergillus gigantus (Ng, 2004). So we have perfecting a technic to extract water-soluble compounds from fungal cultures.

Six marine-derived fungal strains from the Atlantic French coast have been cultured on solid and liquid media. A simple soaking methanolic extraction has been compared with aqueous extraction methods to catch the targeted products. But difficulties in water extraction lie in the fact that such compounds are stored in mycelia and their media. To do that, enzymatic digestion with agarase have been processed on agar-agar from solid culture to accede to products trapped in. Mycelia from both liquid and solid cultures have been cryo-crushed, breaking in this way their rigid cell walls. Then, aqueous extracts have been precipited [(NH4)2SO4, and TCA]. Proteins and

peptides have been measured and analysed by electrophoresis. Moreover, fractions were also investigated for their cytotoxicity on KB cells (Mossman, 1983) and neuroactivity on blowfly larvae (Zlotkin, 1971).

This study leads to a standardized method for water-soluble fungal metabolites extraction from mycelia and agar-agar. Some significant results are obtained in cytotoxicity and neuroactivity assays and are discusse. Bioactivity-guided fractionation of aqueous extracts is in progress according to classic methods in purification of water-soluble compounds (Shimizu and Li, 2005). 1. Blunt JW, Copp BR Hu W-P, Munro MHG,

Northcote PT, Prinsep MR (2004) Marine natural products. Nat Prod Rep 26:170-244

2. Mossman T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Microbiol Meth 65:55-63

3. Ng TB (2004) Peptides and proteins from fungi. Peptides 25:1055-1073

4. Shimizu Y, Li B (2005) Purification of water-soluble natural products. From Methods in Biotechnology Vol 20 Natural Products Isolation 2n4 ed: 415-438

5. Zlotkin E, Fraenkel G, Miranda F and Lissitzky S (1971) The effect of scorpion venom on blowfly larvae - A new method for the evaluation of scorpion venoms potency. Toxicon 9:1-8


58

OC 13

CHEMICAL PROTEOMICS AS A TOOL IN TARGET DISCOVERY OF BIOACTIVE SMALL MOLECULES

Maria Chiara Monti, Luigi Margarucci, Alessandra Tosco, Raffaele Riccio and Agostino Casapullo

Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy

[email protected]

One of the main questions affecting the new era of chemical biology is the comprehension of the interactions between small molecules and their macromolecular targets. Although the therapeutic potential of the most promising lead compounds is being evaluated in preclinical and clinical trials, often their intracellular partners remains unknown1. Nowadays, the progress in the field of mass spectrometry and bioinformatics has a significant impact on the recognition of biological interactors of small molecules. Indeed, the versatile combination of affinity purification and mass spectrometry, often called chemical proteomics, has been recently applied for the detection of macromolecular partners which specifically bind immobilized small drugs.2

Here, we report the chemical proteomic analysis of Petrosaspongiolide M (PM), an anti-inflammatory marine metabolite, inhibiting secretory PLA2s3 and also interfering with NF-κB pathway through

an unknown mechanism mediated by unclear target(s).4 Our experimental procedure consists of three steps: the immobilization of the natural compound on a solid support

through a spacer arm, the isolation of the potential target(s) in a crude cell extract, and its (their) identification by HR-ESI-MSMS.

On the basis of the first evidences, the multi-component proteasome machinery has been identified as the major specific partner of PM. Since the proteasome activity strictly affects NF-κB pathway, these results validate our chemical proteomics procedure.

1. L. Sleno, et al. Current Opinion in Chemical Biology 2008, 12:46–54

2. R. Aebersold, et al. Nature 2003, 422:198-208

3. M.C. Monti, et al. Chem. Eur. J. 2009, 15:1155 – 1163

4. I. Posadas, et al. Biochem. Pharm. 2003, 165: 887-895


59

OC 14

MARINE NANOBIOTECHNOLOGY: ENZYMES/PROTEINS FROM SPONGES ACTING AT THE INTERFACE OF INORGANIC CHEMISTRY AND BIOLOGY

Heinz C. Schröder, Matthias Wiens, Filipe Natalio and Werner E.G. Müller

Institut für Physiologische Chemie, Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz, Germany

[email protected] / [email protected]

Biosilica formation of siliceous sponges has attracted much attention in the development of novel bionic approaches. Silica is an important material in nanotechnology. The chemical production of silica typically requires harsh conditions. Siliceous sponges, by contrast, form their silica skeleton at low temperature and near-neutral pH using specific enzymes: silicateins. Recombinant silicateins are of high interest for a variety of medical and technical applications (patents: EP 1320624; US 7,169,589B2). Silicateins immobilized to surfaces (metals, metal oxides, silicon wafers etc) can be used for biocatalytic formation of silica nanolayers/particles for various applications in medicine (e.g., bone regeneration), dentistry (e.g., protective coatings), and microelectronics (soft lithography). Biocatalytic encapsulation of transformed bacteria in a silica shell does not affect the growth of the bacteria and enables new applications in industrial biotechnology and biosensorics. Silicatein does not only catalyze polycondensation of silica but also the synthesis of other metal oxides like titania and zirconia

showing semiconductor, piezoelectric, dielectric and/or electrooptic properties. Immobilization of His-tagged silicatein onto nanoparticles allows the fabrication of core-shell materials of alternating metal oxide layers with complex architectures and properties. Biosilica coatings of surfaces (e.g., of medical implants) are bioactive and promote new bone formation. The recent discovery of silintaphin-1 which forms the “core” of the silicatein filaments of sponge spicules markedly extends the application range of silicateins. Silintaphin-1 has structure-directing activity: it directs the assembly of silicatein molecules or silicatein immobilized on functionalized metal oxide particles to filamentous or rigid nanostructures. The combined action of silintaphin-1 and silicatein makes biocatalytic formation of light-transmitting biomimetic optical fibres feasible. Acknowledgements. This work was supported by grants from the European Commission (STREP “BIO-LITHO”; Marie-Curie ITN “BIOMINTEC”), Eurostars (“SILIBACTS”) and BMBF (Center of Excellence “BIOTECmarin”).


60

OC 15

MOLECULAR COMMUNICATION ACTORS WITHIN THE PORIFERA/BACTERIA SYMBIOTIC MODEL. INSIGHTS INTO AN INTIMATE DIALOGUE

Johan Garderes, Jasnizat Bin Saidin, Gaël Le Pennec

Laboratoire de Biotechnologie et Chimie marines. Université de Bretagne Sud-UEB. BP 92116 – 56326 Lorient. France.

[email protected]

For over 500 million years, it is assumed that Porifera live in close association with numerous bacterial partners (1), even in certain cases exceeding its own biomass. As filter feeders, sponges naturally prey on bacteria. But, curiously, part of them, the so-called bacterio-sponges, support peculiar relationships with their host: they are accepted within cells of eukaryote. Such association cannot be governed unless a clear recognition of the different actors of this symbiosis. Besides an immunological context already existing in porifera (2), sponge must have developed molecular communication means to converse with its own bacterial populations. However, bacteria must also communicate with sponge in order, at least, to be recognized as a symbiont and not as food supply. Bacteria produce communication molecules belonging to the quorum sensing mechanisms (QS) in particular conditions, those of biofilm. QS regulates numerous parameters such as cell motility, expression of virulence factors, etc. (3), and probably symbiosis. Thus, we searched for such molecules and we identified AcylHomoserine Lactone (AHL) in crude sponge extracts: the C6-, C7-, and 3-oxo-C12-HSL. As bacterio-sponges are far from biofilm conditions in their host do and how sponges mimic

those conditions propitious to bacterial AHL production? Do sponges are able to recruit and select bacteria for mutual symbiosis and how do they proceed? Communication molecules may be a good clue. Looking for potential molecules of communication in our model of study, the Demospongiae Suberites domuncula, we found elements requisite for cathecolamines production, well-known hormones in higher eukaryotes as elements of dialogue with prokaryotes (4). In the mean time, extracts were prepared from S. domuncula stimulated with bacterial exoproducts and were tested on bacterio-sponges in order to detect new molecules which can further be involved in this cross-talk. Comprehensive studies of their mechanisms of action may lead to the discovery of new drugs with potential applications in medicine or in biotechnologies in order to replace or enhance antibiotic action or to deceive eukaryotic cells in order to induce them in a way of phagocytosis of therapeutic bacteria.

1. Li CW, Chen JY, Hua TE. 1998. Science. 279:879-882

2. Wiens M, Korzhev M, Perovic-Ottstadt S, Luthringer B, Brandt D, Klein S, Müller WE. 2007. Mol Biol Evol. 24:792-804.

3. de Kievit TR, Iglewski BH. 2000. Infect. Immun. 68/4839-4849

4. Pacheco, AR & Sperandio, V. 2009. Curr. Opin. Microbiol. 12:192-198


61

OC 16

SALARINS, TULEARINS AND TAUMYCINS, NOVEL MARINE NATURAL PRODUCTS; CHEMISTRY, STEREOCHEMISTRY AND ACTIVITY Drorit Neumann1, Nathalie Ben‐Califa1, Ashgan Bashira2, Amira Rudi2

and Yoel Kashman2 1 Department of Cell and Developmental Biology, Sackler Faculty of Medicine and

2 School of Chemistry, Tel-Aviv University, Israel

[email protected]

O

OCH3

O

O

O(CH2)5H3C

OO

NH

H3C O

O

1 5

911

15

19

1'

22

2325

OON

CH3O 27

Salarin A

21

16

26

713

8' O

(CH2)4H3C

CH3

HO

OCH3

H3CO

H3C

NH2

O

1

4

13

10

8

31

28

27

29

18

17

30O

20

26

H2NOCTulearin B

NH HN

O

O

NO

Ile-2

Ile-1

3

57913 11

1915

1618

O

OO

1

Taumycin A(Selective compounds)

Bio-guided (brine shrimp test) separation of the extract of the Madagascar Fascaplysinopsis sp. sponge collected in Salary Bay afforded three groups of novel compounds designated salarins, tulearins and taumycins.

The salarins and tulearins possess unprecedented macrolides carrying unique functional groups, making them interesting subjects for chemical studies. Salarin A includes ten functional sites, each being a potential precursor for many derivatives. The tulearins possess one or more carbamoyl groups which are rare in nature and appear in bioactive compounds like geldanamycin and erythromycin. The taumycins embody the rare 12-membered oxodepsipeptide ring carrying an unsaturted heptyloxazole appendix – again a complex molecule with many functionalities that can be changed.

Preliminary studies showed that Salarin C selectively inhibits proliferation of human and murine tumor cell lines, and mitogen (phytohemaglutinin)-activated peripheral blood monocytes (PBM) in a dose-dependent manner, without substantial influence on viability of normal PBM. 1. Bishara A, A. Rudi, M. Aknin, D. Neumann,

N. Ben- Califa, Y. Kashman. Salarins A and B and tulearin a: new cytotoxic sponge-derived macrolides. Org Lett. 10(2):153-6 (2008).

2. Bishara, A., A. Rudi, M. Aknin, D. Neumann, N. Ben-Califa, and Y. Kashman. Taumycins A and B, two bioactive lipodepsipeptides from the Madagascar sponge Fascaplysinopsis sp. Org Lett 10:4307-10 (2008).

3. Bishara A., A. Rudi, M. Aknin, D. Neumann, N. Ben-Califa and Y. Kashman Salarin C, a new cytotoxic sponge-derived nitrogenous macrolide Tetrahedron Lett. 49: 4355-4358 (2008).

4. Ashgan Bishara, Amira Rudi, Israel Goldberg, Maurice Aknin, Yoel Kashman. Tulearins A, B and C; structures and absolute configurations. Tetrahedron Lett. In press


62

OC 17

BIOSYNTHESIS OF PYRIDOACRIDINES IN C. DELLECHIAJEI CELL-FREE EXTRACTS

Delphine Bry, Nataly Bontemps, and Bernard Banaigs

Laboratoire de Chimie des Biomolécules et de l’Environnement, Université de Perpignan via Domitia, 66860 Perpignan Cedex, France

[email protected]

Pyridoacridines are polyaromatic alkaloids based on a common heteroatomic tetracycle, the pyrido[2,3,4-kl]acridineskeleton (figure 1). Pyridoacridines vary in structure by fusion of new rings type pyridine, thiazole or thiazinone heterocycles. Tetracyclic and pentacyclic pyridoacridines with sulphur heterocycle generally have a hanging or cyclised ethylamine type chain (table 1). From the ascidian C. dellechiajei found in western Mediterranean we have described chemical pyridoacridines variability of various observed chromotypes (purple, green, blue)1 (table 1), shown the use of pyridoacridines for defence mechanism against predation2, shown a genetic variability, associated to the morphological differences and chemical variability3 and characterised news alkaloids.

Pyridoacridines were only isolated from various phyla of marine invertebrates (sponges, sea anemones, ascidians…). Whereas the importance of this chemical family their biosynthesis is unknown. In 1993, Steffan et al. have shown that both

tryptophan and tyrosine are biosynthetic precursors to Shermilamine B4. In order to determine if tryptophan and tyrosin are biosynthetic precursors to the tetracyclic skeleton of the pyridoacridines in both chemotypes and to know if the fifth cycle (thiazinone, thiazole or pyridine) could be elaborated from a third amino acid we developed a cell-free extract method on two chromotypes (purple and blue). 1. S. Lopez-Legentil, R. Dieckmann, N. Bontemps-

Subielos, X. Turon, and B. Banaigs, "Qualitative variation of alkaloids in color morphs of Cystodytes (Ascidiacea)," Biochemical Systematics and Ecology 33(11), 1107-1119 (2005).

2. S. Lopez-Legentil, X. Turon, and P. Schupp, "Chemical and physical defences against predators in Cystodytes (Ascidiacea)," Journal of Experimental Marine Biology and Ecology 332, (1), 27-36 (2006).

3. S. López-Legentil and X. Turon, "How do morphoytpes and chemotypes relate to genotypes? The colonial ascidian Cystodytes (Ascidiacea: Polycitoridae)." Zoologica Scripta 34(1), 3-14 (2005).

4. B. Steffan, K. Brix, and W. Putz, "Biosynthesis of shermilamine B," Tetrahedron 49(28), 6223-6228 (1993).

NH

N

A B C

D1

2

4

568

1011

9

Figure 1: pyrido[2,3,4-kl]acridine

skeleton

Pyridoacridines with sulphur heterocycle Pyridoacridines with pyridine type heterocyclePurple chromotype Green chromotype Blue chromotype

NH

N

S

HN O

NHO

NH

N

S

N

NHO

NH

N

S

HN O

NH2

NH

N

S

N

NH2

shermilamine B kuanoniamine D

deAcshermilamine BdeAckuanoniamine D

N

N

O

N

OH

N

N HN

ONH

hydroxyascididemine

cystodimine A

N

N

O

N

ascididemine

Table 1: Pyridoacridines composition of purple, green and blue morphs of Mediterranean C. dellechiajei


63

OC 18

THE BIZARRE BIOGENETIC VARIABILITY OF TERPENES IN THE ANTARCTIC MOLLUSC AUSTRODORIS KERGUELENENSIS

Adele Cutignano,1 Conxita Avila,2 Wen Zhang,1 Manuel Ballesteros,2

Guido Cimino1 and Angelo Fontana1

1 Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy. 2 Dept. Animal Biology (Invertebrates), Faculty of Biology, University of Barcelona,

Av. Diagonal 645, 08028, Barcelona, Catalonia, Spain

[email protected]

World-wide occurring marine molluscs of the family Dorididae (Doris, Archidoris, Austrodoris) contain glycerol derivatives in which at least one hydroxy group of the polyol is esterifed with a terpenoic acid. These compounds are generally named terpene glycerides and are suggested to play as chemical deterrents. The cold-adapted nudibranch Austrodoris kerguelenensis from Antarctica, as well as Archidoris odhneri and Archidoris montereyensis from Arctic and sub-Arctic regions, do not diverge from this role and contain terpene glycerides providing chemical defense against sympatric predators.1 Nevertheless, the chemical arsenal of Archidoris and Austrodoris (≡ Doris) shows deep differences that apparently reflect a profound diversity of the biosynthetic pathways underlying the synthesis of these secondary metabolites. In fact, whereas Archidoris molluscs collected along the Pacific coasts of Canada possess the unvarying presence of two major terpene moieties, the glycerides isolated from A. kerguelenensis are featured by a surprisingly various and erratic family of terpenes.

In the last years, we have carried out a deep survey on these molluscs by addressing both the biosynthesis of the deterrent molecules and the chemical

variability that seems to characterize the animals from the South Pole. Here we discuss the main results of these studies, paying attention to the recent hypothesis of an allotropic speciation as key factor leading to the observed chemical variability in A. kerguelenensis.2 In particular, we report the distribution of these terpene glycerides within two distinct populations of A. kerguelenensis from Weddell Sea and Ross Sea. Data are obtained by one-to-one analysis of 33 individuals and reveal an extraordinary specimen-dependent variability of the terpene moiety that, as far as we know, does not have precedent in marine invertebrates.

1. Davies-Coleman MT, Faulkner DJ, Tetrahedron (1991) 47: 9743–9750; Gavagnin M, Trivellone E, Castelluccio F, Cimino G, Cattaneo-Vietti R, Tetrahedron Lett (1995) 36: 7319–7322; Gavagnin M, De Napoli A, Castelluccio F, Cimino G, Tetrahedron Lett (1999) 40: 8471–8475; Gavagnin M, De Napoli A, Cimino G, Iken K, Avila C, García FJ, Tetrahedron Asymm (1999) 10: 2647–2650; Gustafson K, Andersen RJ, Chen MHM, Clardy J, Hochlowski JE, Tetrahedron Lett. (1984), 25: 11-14; Graziani E.I, Andersen RJ, Krug P.J., Faulkner DJ, Tetrahderon (1996) 52: 6869-6878

2. Wilson NG, Schrodl M, Halanuch KM, Molecular Ecology (2009) 18:965-984.


64

OC 19

CHEMOECOLOGICAL STUDIES IN TWO NUDIBRANCHS FROM THE PORTUGUESE COAST

Helena Gaspar

Instituto Nacional de Engenharia, Tecnologia e Inovação (INETI), Unidade de Produtos Naturais (PN), Estrada do Paço do Lumiar, Edifício F, 1649-038 Lisboa, Portugal,

[email protected]

Nudibranchs are shell-less gastropods that are often protected by deterrent compounds. These defensive compounds are usually obtained by bioaccumulation (or biotransformation) of dietary metabolites and, less often, by de novo biosynthesis. Despite extensive research on these animals, information on chemical ecology of nudibranchs from the Portuguese coasts is scarce. In this report, we discuss the secondary chemistry of two nudibranch species collected off Berlengas Islands and Setúbal Coast.1-3

Hypselodoris fontandraui had tavacpallescensin, a sesquiterpene that was also present in the Dysidea sponge on which the nudibranch feeds on. Tavacpallescensin is highly concentrated in the mantle border of the nudibranch, exceeding considerably the threshold-value of 3.5 mg/mL. At this concentration tavacpallescensin significantly deters sympatric predators. The association between the colour pattern and deterrence of H. fontandraui demonstrates that H. fontandraui is a true aposematic member of a Müllerian circle of blue-coloured nudibranch species.3 Further investigation on another putative sponge-prey of H. fontandraui, the sponge Fasciospongia sp., led to the isolation of three new isomeric furanosesquiterpenes, isomicrocionin-3, (-)-microcionin-1, (-)-isomicrocionin-1,

along with the known (-)-ent-pallescensin A and (-)-pallescensin-1.4

Doriopsilla pelseneeri, was characterized by a mixture of several sesquiterpenes including four new pelseneeriols compounds. All these compounds are biosynthesized de novo by the nudibranch.1-2 Feeding experiments with [1-13C]-glucose demonstrated the ability of D. pelseneeri to produce 15-ent-acetoxy-pallecensin-A and drimane esters through the mevalonate pathway. These experiments, together with the occurrence of monocyclofarnesyl metabolites, brought further details on the biogenetic pathway operating in these marine invertebrates.2 1. Gaspar H, Gavagnin M, Calado G,

Castelluccio F, Mollo E, Cimino G, Tetrahedron 2005, 61, 11032–11037;

2. Gaspar H, Ferreira T, Cutignano A, Calado G, Cimino G, Fontana A, J. Nat. Prod. 2008, 71(12), 2053-2056;

3. Haber M, Cerfeda S, Carbone M, Calado G, Gaspar H, Neves R, Maharajan V, Cimino G, Gavagnin M, Ghiselin MT, Mollo E, Evol. Ecol. 2009 (submitted);

4. Gaspar H, Santos S, Carbone M, Rodrigues AS, Rodrigues AI, Uriz MJ, Feio SMS, Humanes M, Gavagnin M, J. Nat. Prod. 2008, 71 (12), 2049-2052.

Acknowledgments - This research was partially funded by an Italian-Portuguese bilateral project (FCT/CNR) and by the FCT project (PTDC/MAR/65854/2006).


65

OC 20

CHEMOTAXONOMY AS VALUABLE APPROACH TO STUDY SPONGES OF THE FAMILY IRCINIIDAE (PORIFERA, DICTYOCERATIDA) Charline Abed1, Gaby Khalaf3, Ghazi Bitar4, Olivier P. Thomas2,

Mohamed Mehiri2, Thierry Perez1 1 DIMAR - UMR 6540, Station Marine d’Endoume,

rue de la Batterie des Lions, 13007 Marseille, France. 2 LCMBA - UMR 6001 - University of Nice Sophia Antipolis, Parc Valrose, 06108 Nice, France.

3 National Centre of Marine Sciences, Batroun, Lebanon, 4 Lebanese University, Hadeth, Lebanon.

[email protected]

Marine sponges of the Dictyoceratida order represent a rich source of unusual secondary metabolites of the terpene family. Among these sponges, the family IRCINIIDAE yield many bioactive terpenes exhibiting antibacterial, antiviral, cytotoxic and anti-inflammatory activities, but also reported as valuable chemotaxonomic markers.1, 2

The taxonomic classification of the IRCINIIDAE family is still unclear, notably the status of Sarcotragus is viewed as uncertain and the distinction of some Ircinia species remains a polemic subject because of the plastic nature of these species.3

To clarify this biological classification disorder, we used a chemotaxonomical approach.

The HPLC chemical fingerprints of 33 sponges of the IRCINIIDAE family collected from six different areas over the Mediterranean Sea: Gibraltar Straits (Ceuta - Spain), North Western basin

(Marseilles and Corsica island - France, and Monaco) and Eastern basin (Crete - Greece and Lebanon) were obtained. The data were processed and analyzed by multivariate analyses (HCA) and, according to their synapomorphic chemical markers, we identified seven different chemical groups. In the present study, we showed that Ircinia and Sarcotragus genera are chemically characterized by chemotaxonomic markers belonging to furanoterpenes and prenylated hydroquinones families respectively.

The bioactivity of the isolated secondary metabolites was also studied and some of the linear prenylated hydroquinones displayed moderate antioxidant properties compared to Vitamin E. 1. Perry, N. B.; Battershill, C. N.; Blunt, J. W.;

Fenwick, G. D.; Munro, M. H. G.; Bergquist, P. R. Biochem. Syst. Ecol. 1987, 15: 373-376.

2. Bergquist, P. R. Sponges; Hutchinson of London: London, 1978; pp 202-216.

3. Cook, S. C. & Bergquist, P. R. 2002. Systema Porifera: A Guide to the classification of sponges. Hooper, J. N. A. & Van Soest, R. W. M. eds. Kluwer Academic/Plenum Publ., New York: 1022 - 1027.


66

OC 21

RELEVANT SCALE OF CHEMICAL VARIATION IN APLYSINA AEROPHOBA Mikel A. Becerro1, Oriol Sacristan‐Soriano1, Nabil Majdi2 and Bernard Banaigs2

1 Center for Advanced Studies of Blanes (CEAB, CSIC), Acc Cala St. Francesc 14, 17300 Blanes (Girona), Spain.

2 Laboratoire de Chimie des Biomolécules et de l’Environnement, Université de Perpignan Via Domitia, 52 Ave Paul Alduy, 66860 Perpignan Cedex, France.

[email protected]

Understanding the production of natural products is an area of major interest in ecology with strong biotechnological implications. We now know that the concentration of secondary metabolites is subject to strong variations but we know far less on the factors that regulate their concentration. Understanding the scale at which natural products vary the most is critical because it sheds light on the type of factors that regulate their production. The sponge Aplysina aerophoba is a common Mediterranean sponge inhabiting shallow waters in the Mediterranean and its area of influence in Atlantic Ocean. This species contains large concentrations of brominated alkaloids (BA) that play a number of ecological roles in nature. Our research investigates the ecological variation in BAs of Aplysina aerophoba from a scale of hundred of meters to thousand kilometers. We used a nested design to sample sponges from two geographically distinct regions (Canary Islands and Mediterranean, over 1000 km), with two zones within each region (less than100 km), two locations within each zone (less than 5 km), and two

sites within each location (less than 500m). In each of the 16 sites, we sampled 10 specimens of Aplsyina aerophoba that were taken to the lab for the quantification of natural products and chlorophyll a. We used HPLC to quantify multiple BAs and a spectrophotometer to quantify chlorophyll a. Our results show a striking degree of variation in both natural products and chlorophyll content. Significant variation in chlorophyll content occurred exclusively at the largest geographic scale between Mediterranean and Canarian samples (p=0.008). Variation in natural products occurred at the smallest local scale (p=0.001), preventing any larger scale to be significant. The factors behind the ecological variation in natural products and cholophyll seem to act at contrasting scales, but a further analysis of the major BAs investigated revealed that the concentration of isofistularin-3 also varied the most between geographic regions. Concentrations of chlorophyll a and isofistularin-3 are in fact negatively correlated (r=-0.432, p<0.001). Our results underline the complex control of the production of secondary metabolites, with factors acting at both small and large geographic scales affecting multiple secondary metabolites

.


67

OC 22

CHITOSAN DERIVED FROM SQUID PENS ON THE DEVELOPMENT OF BIOMEDICAL MEMBRANES

Tiago H. Silva, Simone S. Silva, Joaquim M. Oliveira, João F. Mano, Rui A. Sousa and Rui L. Reis

3B´s Research Group – Biomaterials, Biodegradables and Biomimetics, Dept. of Polymer Engineering, University of Minho,

Campus de Gualtar, 4710-057 Braga, Portugal [email protected]

Since 1990, research on chitin and chitosan has received an increasing attention due to the exhibited biological properties, such as the biodegradation in the human body and immunological, antibacterial and wound healing activity. Furthermore, chitosan has been found to be good candidate as a support material for drug delivery and tissue engineering.

Chitin is present in nature in two crystalline forms: α and β. Due to its relative abundance and easy accessibility, α-chitin has been by far more studied and for the same reason, chitosan has been prepared almost exclusively from α-chitin. However, β-chitin may be a very interesting alternative source of chitosan, since their weak intermolecular forces make it more reactive and with higher affinity for solvents, which is expected to be reflected in the properties of derived chitosan. In this perspective, chitosan derived from β-chitin may constitute a biomaterial with great potential in the biomedical field.

In this work, the conditions for the isolation of β-chitin from the

endoskeletons of squids and for the consecutive deacetylation for the production of chitosan have been studied in order to achieve a chitosan with adequate properties for its use on the development of biomedical applications, taking into consideration in particular the deacetylation degree, molecular weight and crystallinity. The obtained materials were characterized by Fourier Transform Infrared (FTIR) spectroscopy, Nuclear Magnetic Resonance (RMN) spectroscopy and X-Ray Diffraction (XRD) analysis.

The obtained chitosan was further used on the development of membranes by solvent casting and their chemical and mechanical properties have been studied by FTIR spectroscopy and Dynamic Mechanical Analysis (DMA), respectively, and compared with the ones exhibited by membranes produced with commercial chitosan derived from α-chitin.

1. Muzzarelli, R.A.A., Peter, M.G. (Eds.), Chitin

Handbook, European Chitin Society, 1997. ISBN 88-86889-01-1.

2. Shimojoh, M., Fukushima, K., Kurita, K., Carbohydrate Polymers 35 (1998) 223-231


68

OC 23

INTRASPECIMEN VARIABILITY OF NATURAL PRODUCTS IN THE SPONGE APLYSINA AEROPHOBA

Oriol Sacristán‐Soriano1, Bernard Banaigs2 and Mikel A. Becerro1 1 Centro de Estudios Avanzados de Blanes (CEAB, CSIC). Acc. a la Cala St. Francesc 14,

17300 Blanes (Girona), Spain. 2 Laboratoire de Chimie des Biomolécules et de l’Environnement, Université de Perpignan Via

Domitia, 52 Ave Paul Alduy, 66860 Perpignan Cedex, France.

[email protected]

Sponges have long been known as a rich source for bioactive natural products. These secondary metabolites have a major role against predators, pathogens or foulers and show useful pharmaceutical and biotechnological applications. In order to increase the required amount of those compounds to set up the preclinical and clinical trials, it is essential to know more about some biological and ecological traits of sponge chemistry. Whether the production of compounds varies within a species or whether their biosynthesis is environmentally regulated has received little attention by the scientific community. We investigated the intraspecimen variability in the production of natural products in the sponge Aplysina aerophoba, which harbours large amounts of brominated alkaloids (BA). We also tested the effect of light on secondary metabolite abundance in A. aerophoba by a four-month experiment. We used high

performance liquid chromatography (HPLC) to quantify four major BAs. Our results show a striking variation in BA abundance within and between Aplysina specimens. The bottom zone of the chimney-like structure of the sponge and the choanosome tissue were more enriched in BAs than the top zone and the ectosome. The abundance of secondary metabolites does not seem to be regulated by light. However, the alkaloid content increased over the time course of the experiment probably due to seasonal changes. Our findings are a further step in the search for drugs from marine organisms with the purpose of obtaining the required amount of natural products without threatening the marine biodiversity. Biological and ecological studies are extremely useful to settle the basis of applied and biotechnological research.


69

OC 24

42-HYDROXY PALYTOXIN: A NEW PALYTOXIN ANALOG FROM HAWAIIAN PALYTHOA SPP. IS THIS THE REAL POISON OF THE LEGENDARY HAWAIIAN

LIMU-MAKE-O-HANA? Martino Forino,1 Patrizia Ciminiello,1 Carmela Dell’Aversano,1 Emma Dello Iacovo,1

Ernesto Fattorusso,1 Laura Grauso,1 Luciana Tartaglione,1 Aurelia Tubaro,2 Marco De Bortoli, 2 Mark Poli,3 and Gary Bignami4

1 Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli “Federico II”, via D. Montesano, 49, 80131 Napoli, Italy. 2 DEMREP, Universita` degli Studi di Trieste, Via Valerio 6,

34127 Trieste, Italy. 3 U.S. Army Medical Research Institute of Infectious Diseases, Toxicology and Aerobiology Division, Fort Detrick, Maryland

4 Hawaii Biotechnology Group, Inc., Aiea, Hawaii.

[email protected]

Our work based on in-depth HRLC-MS analysis along with extensive NMR study led us to structurally characterize a never reported palytoxin-like compound, 42-hydroxy palytoxin, from P. tubercolosa and P. toxica samples collected off the Hawaiian coasts. This new toxin and palytoxin itself appeared to be the major components of the toxic extract from P. tubercolosa sample; whilst 42-hydroxy palytoxin was proven by far the main palytoxin derivative in P. toxica.

Toxicological studies on this new palytoxin-like compound suggested that the new palytoxin analog and palytoxin itself share almost the same lethality.

As P. toxica sample we analyzed was from the legendary tide pool located in Hana district1, where for centuries native Hawaiians have collected the deadly seaweed used for making their spears fatal2, we were tempted to venture an answer to the ancient mystery involving the famous limu-make-o-Hana. Might 42-hydroxy palytoxin be accounting for the lethal effect of the “deadly seaweed of Hana”? 1. Moore, R. E.; Scheuer, P. J. Science

(Washington, D.C.) 1971, 172, 495-498. Palytoxin: a new marine toxin from a Coelenterate

2. Malo, D. Hawaiian Antiquites (B. P. Bishop Museum Special Publication 2, ed. 2, Honolulu 1951), p.201-226

O

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OH

OHMe

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OH

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OH

OH

OH

OH

OH

OHOHHO

OH

OH

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MeOH

OHHO

OHOH

OH Me HO

OH

OH

OH

Me

Me

OH

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OH

HO

OH

OH

42

Figure 1. Stereostructure of 42-hydroxy Palytoxin.


70

OC 25

BIOACTIVE POLYETHER METABOLITES FROM DINOFLAGELLATES: STRUCTURE DETERMINATION AND BIOACTIVITY

Antonio H. Daranas, José G. Napolitano, Tamara Vilches, José Javier Fernandez and Manuel Norte

Instituto Universitario de Bioorgánica “Antonio González”. Departamento de Química Orgánica. Universidad de La Laguna. 38206. La Laguna. Tenerife (Canary Islands).

[email protected]

Over the last years our research group has been focused on the study of microalgal metabolites as a source of bioactive compounds. DSP (Diarrhetic Shellfish Poisoning) toxins have unique chemical features and after their isolation focused natural product chemist attention, not only because of their public health repercussion and economical impact on the shellfish industry, but also to establish the real origin of the poisoning. Moreover the isolation and the structure elucidation of minor new toxins is imperative for designing proper countermeasures such as their detection in contaminated samples and for the determination of their biosynthetic pathway. In addition, marine toxins are more than just tools of biological chemistry; they are also powerful molecular probes that shed light on the molecular details of important cellular events. In this way, for example, the remarkable selectivity of okadaic acid and derivatives to inhibit some serine-threonine protein phosphatases (PP1 and PP2A) lead directly to the discovery and characterization of some members of that family of soluble proteins. However, despite serious advances in structure determination techniques relatively little is

known about the biosynthetic pathways or structural/functional relationships of these secondary metabolites. The structures of some compounds obtained from artificial cultures of the dinoflagellate Prorocentrum belizeanum will be discussed on the basis of their spectroscopical data, essentially obtained by NMR techniques, in combination with molecular modelling studies. In addition the utility of these new compounds to understand the structural requirements necessary to inhibit protein phosphatases will be commented on the basis of molecular modelling simulation results.

1. Cruz, P.G.; Norte, M.; J.M.; Fernández, J.J.;

Hernández Daranas, A. Chem. Eur. J. 2008, 14, 6948.

2. Napolitano, J.G.; Daranas, A.H.; Fernández, J.J.; Norte, M. Anti-Cancer Agents Med. Chem. 2009, 9, 122.

3. Napolitano, J.G.; Norte, M.; Padrón, J.M.; Fernández, J.J.; Hernández Daranas, A. Angew. Chem. Int. Ed. 2009, 48, 796.

Acknowledgements: The authors acknowledge financial support from the Spanish MEC (AGL-2005-07294-C04-01/ALI and CTQ2008-06754-V04-01/PPQ). J.G.N. acknowledges MICINN for a Ph.D. scholarship (FPU Program)

.

Poster Communications


72

PC 01

PYRROLE-2-AMINOIMIDAZOLES ALKALOIDS FROM AXINELLA CF POLYPOIDES SPONGE

Clarisse Lejeune1, Marion Gabant1, Marie‐Thérèse Martin1, Odile Thoison1, Thierry Perez2, Ghazi Bitar3 and Ali Al‐Mourabit1

1 Institut de Chimie des Substances Naturelles du CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France

2 CNRS, station marine d’Endoume, rue de la batterie des lions, 13007 Marseille, France 3 Lebanese University, Faculty of Sciences (Section I), Department of Biology,

Hadath/Lebanon, Liban

[email protected]

In the frame of ECIMAR research program, marine sponges samples, identified as Axinella polypoides and collected off various Mediterranean sites, were studied. LC-MS chromatograms comparison clearly showed that the samples of Axinella polypoides collected from Marseille coast and Lebanon shores are different.

The chemical studies of both dichloromethane/methanol extracts showed the presence of pyrrole-2-aminoimidazole alkaloids in Axinella polypoides from Lebanon while only

diketopiperazines and piperidiniums were isolated from Axinella polypoides1-2

collected at Marseille. The chemistry of these sponges and new pyrrole-2-aminoimidazole dimmers will be described.

1. Known chemical studies: Crist, V.; Djerassi, C. Steroids. 1983, 42, 331-343.

2. Gabant, M., 2008, PhD, Paris XI University.


73

PC 02

IRIOMOTEOLIDE-8A, A NOVEL 25-MEMBERED MACROLIDE FROM DINOFLAGELLATE AMPHIDINIUM SPECIES

Masashi Tsuda and Keiko Kumagai

Center for Advanced Marine Core Research, Kochi University, Kochi 783-8502, Japan

[email protected] Marine dinoflagellates Amphidinium species are known as producers of unique polyketide-like metabolites, macrolides and long-chain polyhydroxyl compounds. Amphidinium macrolides have various carbon chains as well as irregularly-introduced C1 branches and oxygen-substituents, and some of them exhibit potent cytotoxicity and antitumor activity. We have developed the methodology for screening of novel cytotoxic macrolides using genomics and metabonomics analyses, and discovered the new macrolide-producing Amphidinium HYA024 strain collected off Iriomote Island, Japan. We have isolated several cytotoxic macrolides, designated as iriomoteolides, from the Amphidinium strain . Further investigation of extracts of HYA024 led to the isolation of a new cytotoxic macrolide, iriomoteolide-8a. In this symposium, we describe the isolation and structural elucidation of iriomoteolide-8a.

The Amphidinium strain HYA024 was cultivated in 400 L seawater medium, and then the harvested algal cells were extracted with MeOH/toluene. The toluene-soluble materials of the extract were subjected to several column chromatographies, and one of cytotoxic fractions were separated by C18 HPLC to afford iriomoteolide-8a. Structure elucidation of iriomoteolide-8a was carried out using detailed analyses of 2D NMR in CD3OD.

Iriomoteolide-8a is a novel 25-membered macrolide with four ether rings, and had the largest molecular weight in the Amphidinium macrolides. Iriomoteolide-8a exhibited cytotoxic activity against human B lymphoma DG-75 and Epstein-Barr virus (EBV)-infected human B lymphocyte Raji cells in vitro with IC50 values of 0.5 and 0.7 µg/mL, respectively.


74

PC 03

BROMINATED DITERPENES FROM THE RED ALGA SPHAEROCOCCUS CORONOPIFOLIUS

Vangelis Smyrniotopoulos, Constantinos Vagias, Vassilios Roussis

Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece

[email protected]

The occurrence of bromoditerpenes in marine algae seems to be quite limited, since they have been found only in some red seaweeds (Rhodophyta) of the genus Laurencia (order Ceramiales) and in Sphaerococcus coronopifolius (order Gigartinales).

During the course of our investigations for the isolation of biologically active compounds from marine organisms of the Greek seas, we studied the secondary metabolite content of the red alga Sphaerococcus coronopifolius, collected from the north coasts of Corfu Island.

Chromatographic separations of the organic extract of S. coronopifolius led to the isolation of a number of known metabolites, along with a new diterpene methyl ketone featuring a novel norsphaerol-like skeleton (1), a guanacastane diterpene alcohol (2) and a metabolite possessing bromocorodienol skeleton (3). The structures of the new natural products, as well as their relative stereochemistry, were established by means of spectral data analyses, including 1D and 2D NMR experiments and MS spectrometry.

BrH

H

OH

O

Br

OHHO

H

H

OHHO

HBr

1 2 3


75

PC 04

LEPTOGORGOLIDE, A 1,4-DIKETO-CEMBRANOID FROM LEPTOGORGIA SPP. Mercedes Cueto 1, Ana R. Díaz‐Marrero 1, Gina Porras1, Luís D’Croz2,3,

Manuel Lorenzo4, Aurelio San‐Martín5 and J. Darias1 1 Instituto de Productos Naturales y Agrobiología del CSIC.

Avda Astrofísico F. Sánchez, 3, 38206 La Laguna, Tenerife, Spain, 2 Smithsonian Tropical Research Institute, P. O. Box 2072, Balboa, Panama,

3 Departamento de Biología Marina y Limnología. Estafeta Universitaria, Universidad de Panamá, Panama,

4 Universidad de Magallanes, Avenida Bulnes 01855, Punta Arenas, Chile, 5 Universidad de Chile, Departamento de Química, Santiago de Chile, Chile

[email protected]

Octocorals of the genera Pseudopterogorgia, Alcyonium, Gersemia, Lophogorgia, Leptogorgia, and Sinularia have the ability to biosynthesize highly oxygenated diterpenoids based on a 14-membered carbocyclic cembrane skeleton1 into which a substituted furan ring and a γ-lactone subunit are embedded. The oxidative cleavage of the furan ring may lead to a 1,4-diketo-derivative and naturally occurring metabolites with this feature are frequently found, mainly in species of genus Pseudopterogorgia, Alcyonium, Gersemia and Sinularia.

Herein we report on the structures of four new cembranoids 1-4 along with the known compound pukalide,2 isolated from this species. In connection with our recently introduced concept of genus specific oxidation, by which the oxidation profile of C-18 of furanocembranolides

provides a criterion as chemotaxonomical marker for octocorals,3 it is worth to note that for the first time a 1,4-diketo-cembranoid 1 with an oxidized C-18 as a methyl ester has been discovered in Leptogorgia. Thus, the ocurrence in Leptogorgia of compound 1 and the related furanocembranolide equivalents 2-4 suggested that the 1,4-diketo-cembranoid congeners may follow a parallel genus-dependent C-18 specific oxidation.

1. (a) Rodríguez, A. D. Tetrahedron, 1995, 51,

4571-4618. (b) Roethle, P. A.; Trauner, D. Nat. Prod. Rep. 2008, 25, 298-317.

2. Missakian, M. G.; Burreson, B. J.; Scheuer, P. J. Tetrahedron, 1975, 31, 2513-2515.

3. Dorta, E.; Díaz-Marrero, A-R.; Brito, I.; Cueto, M.; D'Croz, L.; Darias, J. Tetrahedron, 2007, 63, 9057-9062.


76

PC 05

NEW ANTIMICROBIAL 2-AMINO-ALKEN-3-OLS FROM THE MEDITERRANEAN COLONIAL ASCIDIAN PSEUDODISTOMA CRUCIGASTER

Maria Letizia Ciavatta1, Genoveffa Nuzzo1, Emiliano Manzo1, Guido Villani1, Anna Zanfardino2, Mario Varcamonti2 and Margherita Gavagnin1

1 Istituto di Chimica Biomolecolare, C.N.R., Via Campi Flegrei, 34, 80078, Pozzuoli, Italy, 2 Dip. di Biologia Strutturale e Funzionale, Università Federico II di Napoli,

Via Cinthia, 80126 Napoli, Italy

[email protected]

Among marine invertebrates, ascidians represent a prolific source of structurally novel compounds, especially rich in nitrogen.1 Aminoalcohols from the marine environment display structures related to sphinganines, with a carbon chain different in length and unsaturation; like sphingolipids they have shown various bioactivities. Species belonging to Pseudodistoma and Clavelina genera2 are the main producers of aminoalcohols, but these kind of molecules were also isolated from some sponges3 and a mollusc.4 Previous studies on the genus Pseudodistoma have led to the characterization of several aminoalcohols possessing cytotoxic and antimicrobial activities.5

In this communication we describe six new antimicrobial 2-amino-alken-3-ols (i.e. 1) from the Mediterranean colonial ascidian Pseudodistoma crucigaster, collected off the Northern coasts of Sardinia. The new compounds have been isolated from both lipophilic and polar extracts of the ascidian and characterized as acetyl derivatives. The structure elucidation was carried out by means of NMR and mass techniques. The relative

stereochemistry was suggested by NOE difference experiments on their oxazolidinone derivatives obtained by chemical transformation, whereas the absolute stereochemistry was established by applying the modified Mosher’s method to their N-acetyl derivatives. The results of antibacterial and antifungal tests performed on natural and acetylated compounds will be also presented.

1. Blunt, J. W.; Copp, B. R.; Hu, W-P.; Munro, M. H. G.; Northcote, P. T.; Prisep, M. R. Nat. Prod. Rep., 2007, 24, 31-86

2. Aiello, A.; Fattorusso, E.; Giordano, A.; Menna, M.; Navarrete, C.; Munoz, E. Bioorg. Med. Chem., 2007, 15, 2920-2926

3. Gulavita, N.K.; Scheuer, P.J. J. Org. Chem., 1989, 54, 366-369; Jimenez, C.; Crews, P. J. Nat. Prod., 1990, 53, 978-982.

4. Rinehart, K.L.; Fregeau, N.L.; Warwick, R.A. (1998) US Patent, 6107520c

5. Hooper, G.J., Davies-Coleman, M.T.; Coetzee, P.S. Nat. Prod. Lett., 1995, 6, 31-35; Rashid, M.A.; Gustafson, K.R.; Cartner, L.K.; Pannell, L.K.; Boyd, M.R. Tetrahedron, 2001, 57, 5751-5755; Garrido, L.; Zubia, E.; Ortega, M.J.; Naranjo, S.; Salvà, J. Tetrahedron, 2001, 57, 4579-4588; Jares-Erijman, E.A.; Bapat, C.P.; Lithgow-Bertelloni, A.; Rinehart, K.L.; Sakai, R. J. Org. Chem., 1993, 58, 5732-5737.

NHAc

OAc 1


77

PC 06

NATURAL 2',5'-LINKED OLIGORIBONUCLEOTIDES FROM MARINE SPONGES Annika Lopp1, Tõnu Reintamm1, Hans Tore Rapp2,

Friederike Hoffmann3 and Merike Kelve1

1 Tallinn University of Technology, Department of Gene Technology, Akadeemia tee 15, 12618 Tallinn, Estonia

2 University of Bergen, Centre of Geobiology and Department of Biology, Box 7800, N-5020 Bergen, Norway

3 Sars International Centre for Marine Molecular Biology, Thormohlensgate, 555008 Bergen, Norway

[email protected]

2',5'-oligoadenylate synthetases (OAS) form a family of enzymes that belong to the interferon (IFN)-induced antiviral proteins in higher animals and catalyse the formation of 2',5'-linked oligoadenylates (2-5A). In vitro, OAS can catalyse the transfer of the 5'-nucleotidyl moiety of a donor nucleoside triphosphate to a 2'-OH end of an acceptor molecule, such as ATP, NAD+ or tRNA. However, the in vivo existence of 2',5'-oligonucleotides other than 2-5A has not been proved yet. Sponges, the evolutionarily most basal multicellular animals, also possess OAS.

Here, we demonstrate that besides 2-5A, other 2',5'-linked hetero-oligomers are present in the sponge Thenea muricata (Demospongiae). HPLC analysis of the perchloric acid extract of the sponge revealed the presence of co-oligomers formed from ATP and GTP. After dephosphorylation, the amount of total 2',5'-ApG (1) in the extract formed up to 20 per cent from that of the total 2',5'-ApA oligomers. Besides, the existence of a 2',5'-linked hetero-oligomer (2) formed from NAD+ and ATP was shown.

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1 2


78

PC 07

A NEW GERANYLHYDROQUINONE FROM THE NEW ZEALAND ASCIDIAN APLIDIUM SCABELLUM

Januario A. H., Pearce A. N., Chan S. T. S., Webb V. A., Harper J. and Copp B. R.*

Núcleo de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, Franca, Brazil Department of Chemistry, University of Auckland, Auckland, New Zealand

National Institute of Water & Atmospheric Research (NIWA) Ltd., Private Bag 14-901, Kilbirnie, Wellington, New Zealand

Malaghan Institute of Medical Research, P.O. Box 7060, Wellington South, New Zealand.

*[email protected]

Ascidians of the genus Aplidium (Polyclinidae) are noted for their ability to biosynthesise prenylated quinones and hydroquinones with interesting biological activities.1

Our screening of New Zealand marine organisms for new anti-inflammatory lead compounds identified an extract of the ascidian Aplidium scabellum as exhibiting potent ability to inhibit the respiratory burst of stimulated human neutrophils.2

Bioassay-directed fractionation of a methanolic extract of A. scabellum using combinations of reversed–phase C18 flash CC and size exclusion Sephadex LH20 yielded the new geranylhydroquinone analogue 1.

The structural elucidation, established by NMR spectroscopic and mass spectrometric analysis, and anti-inflammatory properties of 1 will be presented.

1. Zubía, E.; Ortega, M. J.; Salvá, J., Mini-Reviews

in Organic Chemistry, 2005, 2, 389 – 399.

2. Pearce, A. N.; Chia, E. W.; Berridge, M. V.; Maas, E. W.; Page, M. J.; Harper, J. L.; Webb, V. L.; Copp, B. R. Tetrahedron, 2008, 64, 5748 – 5755. Pearce, A. N.; Chia, E. W.; Berridge, M. V.; Clark, G. R.; Harper, J. L.; Larsen, L.; Maas, E. W.; Page, M. J.; Perry, N. B.; Webb, V. L.; Copp, B. R. J. Nat. Prod., 2007, 70, 936-940.


79

PC 08

NEW SECONDARY METABOLITES FROM THE MARINE-DERIVED FUNGUS PHAEOSPHAERIA SPARTINAE

Mahmoud F. Elsebai, S. Kehraus, Gabriele M. König

University of Bonn, Institute for Pharmaceutical Biology, Nussallee 6, 53115 Bonn, Germany.

[email protected]

Marine derived-fungi are an important source of pharmacologically active natural products. The algicolous fungus Phaeosphaeria spartinae originated from the alga Ceramium sp. which was collected at the German coast (Büsum, North Sea). Its ethyl acetate extract showed high inhibitory activity toward acetylcholinesterase and papain. Further investigation of the crude extract provided new hydroxylated and

unsaturated polyketides, an unusual steroidal compound and an acyclic sesquiterpene. The structures of the compounds were established on the basis of spectroscopic studies. Their biological activities are under investigation. Acknowledgement: Financial support came from the Egyptian Government and from BMBF (project No. 03F0415A)

HO

H R

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OH

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R=


80

PC 09

BIOACTIVE BENZODIAZEPINE ALKALOIDS FROM A SPONGE-ASSOCIATED FUNGUS EXOPHIALA

Viviane N. Nenkep, Xavier N. Siwe, Alain S. Leutou, Guohua Yang, Zhile Feng, Keumja Yun, and Byeng W. Son*

Department of Chemistry, Pukyong National University, Nam-gu, Busan 608-737, South Korea

*[email protected]

Marine-derived microorganisms such as bacteria and fungi have proven to be a rich source of new biologically active secondary metabolites. As a part of our search for bioactive substances from the marine-derived microorganism, the marine sponge-associated fungus was studied because the mycelium extract showed potent ultraviolet (UV)-A protecting activity. An assay-guided purification resulted in the isolation of a new benzodiazepine alkaloid, circumdatin I, and two known circumdatins C and G from the marine isolate of fungus Exophiala sp. We report here on the isolation and structural elucidation of these compounds. The structure of the circumdatins was elucidated by spectral

data analysis and comparisons of their data with those of previously reported. The absolute stereochemistry of circumdatin I was determined by comparison of optical rotation and CD experiments with circumdatins C and G. The circumdatins I, C, and G were evaluated for UV-A protecting activity, and they exhibited an UV-A protecting activity with ED50 values of 98, 101, and 105 µM, respectively, which are more potent than the positive control, oxybenzone (ED50, 350 µM) used as sunscreen agent currently.1 1. Son, B. W. et al., J. Antibiot. 2008, 61(1), 40-42.


81

PC 10

FULL RELATIVE STEREOCHEMISTRY ASSIGNMENT AND CONFORMATIONAL ANALYSIS OF 13,19-DIDESMETHYL SPIROLIDE C VIA NMR- AND

MOLECULAR MODELING-BASED TECHNIQUES. A STEP TOWARDS THE COMPREHENSION OF SPIROLIDE MECHANISM OF ACTION

Laura Grauso, Patrizia Ciminiello, Bruno Catalanotti, Carmela Dell’Aversano, Emma Dello Iacovo, Caterina Fattorusso, Ernesto Fattorusso, Martino Forino, Angela Leo, Luciana

Tartaglione.

Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli Federico II, via D. Montesano, 49, 80131 Napoli, Italy.

[email protected]

The spirolide content of massive cultures of Alexandrium ostenfeldii1 collected along the North-western Adriatic coasts of Italy in November 2003 was determined by Liquid Chromatography- Mass Spectrometry and 1D- and 2D-NMR techniques. Among the detected spirolides, three resulted major compounds and were unambiguously identified as 13-desmethyl spirolide C2, 13,19-didesmethyl spirolide C3 and 27-hydroxy-13,19-didesmethyl spirolide C4. During our ongoing studies on Adriatic A. ostenfeldii, we have recently succeeded in elucidating the full relative stereochemistry of 13,19-didesmethyl spirolide C through NMR- and Molecular Modeling-based techniques. Besides this, our studies have also contributed to shed some light upon 13,19-didesmethyl spirolide C conformational behavior in solution. This could pave the way towards a more in-depth comprehension of spirolide mechanism of action. In fact, so far pharmacological studies have identified spirolides as fast-acting toxins5, as in the mouse bioassay they induce rapid onset of symptoms akin to those reported

for the acute toxicity of paralytic shellfish poisoning (PSP) toxins, followed by death within minutes from the intraperitoneal injection. It has been shown that spirolides affect Ca2+ channels and hypothesized that their pharmacophore is represented by the uncommon cyclic imine moiety. Beyond this information, though, spirolide toxicity is far from being totally and unambiguously defined.

1. Ciminiello, P., Dell'Aversano, C., Fattorusso, E., Magno, S., Tartaglione, L., Cangini, M., Pompei, M., Guerrini, F., Boni, L., Pistocchi, R. (2006). Toxicon 47, 597-604.

2. T. Hu, I. W. Burton, A. D. Cembella, J. M. Curtis, M. A. Quilliam, J. A. Walter and J. L. C. Wright, J. Nat. Prod., 2001, 64, 308.

3. S. L. MacKinnon, J. A. Walter, M. A. Quilliam, A. D. Cembella, P. LeBlanc, I. W. Burton, W. R. Hardstaff and N. I. Lewis, J. Nat. Prod., 2006, 69, 983.

4. P. Ciminiello, C. Dell'Aversano, E. Fattorusso, M. Forino, L. Grauso, L. Tartaglione, F. Guerrini, and R. Pistocchi, J. Nat. Prod., 2007, 70, 1878.

5. Richard, D., Arsenault, E., Cembella, A., Quilliam, M. A. (2000) In Intergovernmental Oceanographic Commission of UNESCO: Harmful Algal Blooms 2000. (Hallegraeff, G. M., Blackburn, S. I., Bolch, C. J., Lewis, R. J., Eds.) pp. 383-386.

N

OO

HH

HO

O

H

O

O

OH


82

PC 11

AROMATICS POLYKETIDES PRODUCED BY AN ENDOPHYTIC FUNGUS OF BOSTRYCHIA RADICANS

Marcia Nasser Lopes1, Abe RO1, Luca AN1, Araújo AR1, Bolzani VS1, Erbert C2, Lopes JLC2 and Debonsi HM2

1 Instituto de Química – Unesp, Araraquara, SP, Brazil 2 Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Ribeirão Preto, SP, Brazil

[email protected]

Brazil is blessed due to its great biodiversity, which constitutes one of the most important sources of biologically active compounds. Considering that marine organisms have been shown to be one of the most promising sources of new bioactive compounds for the treatment of different human diseases, and Brazilian coastline, with 8000 km largely unexplored, represents a great potential for finding new pharmacologically active secondary metabolites. In the search for novel secondary metabolites from microbes, we have focused on fungi associated with marine algae. The algae, Bostrychia radicans, were collected in a rocky shore located in Ubatuba, São Paulo State, Brazil. The strain of endophytic fungi was obtained from sterilized,

sectioned portions sampled from thallus. The endophytic fungal strains were grown in solid rice medium. The mycelia masses were submitted to extraction with methanol and subsequently fractioned using hexane and ethyl acetate, giving the crude extracts after evaporation of the solvent. After analysis by the 1H NMR and HPLC-DAD, the crude extracts were fractionated by HPLC prep, affording the isolation of several aromatics polyketides. The structures of these compounds were elucidated through a series of 1D and 2D NMR experiments. The crude extracts and pure compounds were sent for antifungal, antioxidant and antimalaric bioassays. Acknowledgements: BIOPROSPECTA-FAPESP


83

PC 12N

N

O

O

HNNH2

NH2

O2 (air)

metaboliteformation

chemiluminescence

CHEMILUMINESCENT OXIDATIVE DEGRADATION OF DIKETOPIPERAZINES AND EARLY PRECURSORS OF MARINE PYRROLE-2-AMINOIMIDAZOLE

METABOLITES Ludmila Ermolenko1, Céline Ratinaud1, Serge Mazères2, Marion Gabant1,

Céline Moriou1 and Ali Al‐Mourabit1,2 1 Institut de Chimie des Substances Naturelles, CNRS, 1 Avenue de la Terrasse 91198 Gif-sur-

Yvette, France 2 Institut de Pharmacologie et de Biologie Structurale, CNRS, 205 route de Narbonne 31077

Toulouse Cedex 4, France

[email protected]

Marine sponges beloning the Agelasidae and Axinellidae families are known to produce pyrrole-2-aminoimidazole alkaloides. We presume an important role of pyrrole-amino acid diketopiperazines as a key precursor in the biosynthesis of this class of C11N5 compounds.1 Our biosynthetic hypothesis is based on the observation that the diketopiperazine cyclo(Pyr-Pro) underwent skeletal spontaneous oxidative rearrangement when exposed to atmospheric oxygen and guanidine to provide dispacamide, a natural product.2 Along with the formation of this pyrrole-2-aminoimidazole metabolite, a secondary reaction corresponding to a chemiluminescent decarboxylation of a dioxetanone intermediate was discovered.

The mechanism of this decarboxylation is close to the one described for bioluminescent luciferins. The reaction produces photons during the dioxetanone decarboxylation. We present here mechanistic studies of the spontaneous oxidation of selected diketopiperazines and their new chemiluminescent properties. The correlation between the diketopiperazines oxidation, the chemiluminescence and the biomimetic synthesis of pyrrole-2-aminoimidazole metabolites will be discussed. 1. C. Vergne, J. Appenzeller, C. Ratinaud, M.-T.

Martin, C. Debitus, A. Zaparucha and A. Al-Mourabit, Org. Lett. 2008, 10, 493-496

2. N. Travert and A Al-Mourabit, J. Chem. Am. Soc. 2004, 10, 10252-10253

Hist / Tyr / Tryp / Leu / Pro

0

2000

4000

6000

8000

10000

12000

450 550 650 750 850Wavelength (nm)

Histidine 2h30 (lisse 30)

Tyrosine 0h45 (lisse 30)

Tryptophane 0h30 (s2) (lisse 30)

Leucine 0h40 (s2) (lisse 30)

Proline 1h20 (s2) (lisse 30)


84

PC 13

AMPHIRIONIN-1, A NOVEL CYTOTOXIC POLYKETIDE FROM DINOFLAGELLATE AMPHIDINIUM SPECIES

Keiko Kumagai and Masashi Tsuda

Center for Advanced Marine Core Research, Kochi University, Kochi 783-8502, Japan

[email protected]

Marine dinoflagellates Amphidinium species are known as producers of unique secondary metabolites, such as cytotoxic macrolides and long-chain polyketides. 1,2 We have developed the methodology for screening of Amphidinium strain producing cytotoxic macrolides using genomics and metabonomics analyses,3 and discovered the Amphidinium HYA024 strain collected off Iriomote Island, Japan. We have isolated several cytotoxic macrolides, designated as iriomoteolides, from the Amphidinium strain.4,5 Further investigation of extracts of HYA024 led to the isolation of a new cytotoxic polyketide, amphirionin-1, possessing a oxetane ring. In this symposium, we describe the isolation and structural elucidation of amphirionin-1.

The Amphidinium strain HYA024 were cultivated in 400 L of medium, and then the harvested algal cells obtained by mass-cultivation were extracted with MeOH/toluene. The toluene-soluble materials of the extract were subjected to several column chromatographies and one of cytotoxic fractions were separated by C18 HPLC to afford amphirionin-1.

Structure elucidation of amphirionin-1 was carried out using detailed analyses of 2D NMR and MS-MS spectra.

Amphirionin-1 is a novel a C24-linear chain carboxylic acid with oxetane ring, five methyls and four hydroxyl groups. This is the first secoacid-like polyketide from the dinoflagellate Amphidinium sp., although many macrolides had been isolated so far.

1. Kobayashi, J., Tsuda, M., Nat. Prod. Rep.,

2004, 21, 77-93.

2. Kobayashi, J., Kubota, T., J. Nat. Prod., 2007, 70, 451-460.

3. Iwamoto, R., Kobayashi, J., Horiguchi, T., Tsuda, M., Phycologia, 2005, 44, S104.

4. Tsuda, M., Oguchi, K., Iwamoto, R., Okamoto, Y., Kobayashi, J., Fukushi, E., Kawabata, J., Ozawa, T., Masuda, A., Kitaya, Y., Omasa, K., J. Org. Chem., 2007, 72, 4469-4474.

5. Oguchi, K., Tsuda, M., Iwamoto, R., Okamoto, Y,; Kobayashi, J., Fukushi, E., Kawabata, J., Ozawa, T., Masuda, A., Kitaya, Y., Omasa, K., J. Org. Chem., 2008, 73, 1567-1570.


85

PC 14

DECONSTRUCTING THE DISCORHABDINS: ABSOLUTE CONFIGURATION AND BIOLOGICAL ACTIVITIES

T. Grkovic, C. Lam and Brent R. Copp

Department of Chemistry, University of Auckland, Auckland, New Zealand

[email protected]

As part of a survey of New Zealand Latrunculia spp. sponges, we recently reported the first examples of enantiomeric pairs of the discorhabdin alkaloids.1 Absolute configuration was assigned by comparison of observed experimental data with the results of time dependent density functional theory (TDDFT) calculations of electronic circular dichroism (ECD) spectra. In addition to a diverse array of known discorhabdin alkaloids, we have isolated a number of new analogues including 1-thiomethyldiscorhabdin G*/I (1), both enantiomers of 16,17-dehydrodiscorhabdin W (2) and 3-dihydrodiscorhabdin A (3).

Application of our ECD dataset was used to assign the absolute configuration of these thioether-containing discorhabdins. In addition to these new natural products, a library of semi-synthetic analogues has also been screened for antitumour and

anti-infective properties yielding a number of new lead compounds worthy of closer attention.

The structural elucidation and assignment of absolute configuration to new and known members of the discorhabdin family of alkaloids will be presented as will our more recent results directed towards optimising the pharmaceutical potential and understanding the mechanism of action of this intriguing class of biologically active marine natural products.

1. Grkovic, T.; Ding, Y.; Li, X.-C.; Webb, V. L.; Ferreira, D.; Copp, B. R. Enantiomeric Discorhabdin Alkaloids and Establishment of their Absolute Configurations using Theoretical Calculations of Electronic Circular Dichroism Spectra. Journal of Organic Chemistry, 2008, 73, 9133-9136.

HN

NH

HN

O

O

S

(+)-(6S,8S)-1

H3CS

HN

NH

HN

O

O

S

Br

HN

N

NH

O

O

S

Br

(-)-(6S,6'aS)-2

HN

NH

HN

O

OH

S

Br

(+)-(3R,5R,6S,8S)-3


86

PC 15

NEW INSIGHTS INTO THE STEREOCHEMISTRY OF BELIZEANOLIDE: J-BASED CONFIGURATION ANALYSIS USING 2D HOMO- AND

HETERONUCLEAR CORRELATION SPECTROSCOPY José G. Napolitano1, Manuel Norte1, José J. Fernández1

and Antonio Hernández Daranas.1,2 1 Instituto Universitario de Bio-Orgánica “Antonio González”, Universidad de La Laguna,

Francisco Sánchez 2, 38206 La Laguna, Tenerife, Spain. 2 Departamento de Ingeniería Química y Tecnología Farmacéutica, Universidad de La Laguna,

Francisco Sánchez 2, 38071 La Laguna, Tenerife, Spain.

[email protected]

The immense biodiversity of the marine biosphere has proved to be an extraordinary source of new bioactive natural products.1 One of the most interesting groups of marine metabolites is formed by macrolides, macrocyclic lactones that not only possess unique molecular architectures, but also exhibit an unparalleled range of biological activities.2 Due to their molecular complexity, structural elucidation of marine macrolides frequently involves a challenging assignment, and the determination of the stereochemistry of each individual domain of these natural products could represent a difficult task sometimes.

Recently, we reported the isolation and structural determination of belizeanolide, a new 52-membered macrolide obtained form artificial cultures of the marine dinoflagellate Prorocentrum belizeanum, together with its open form belizeanolic

acid.3 Herein, we present preliminary results on the stereochemistry of the three five-membered ether rings and some highly flexible carbon domains of belizeanolide through the application of J-based NMR configurational analysis in combination with ROESY experiments.4

1. Blunt, J.W.; Copp, B.R.; Hu, W.-P.; Munro,

M.H.G.; Northcote, P.T.; Prinsep, M.R. Nat. Prod. Rep. 2009, 26, 170.

2. Napolitano, J.G.; Daranas, A.H.; Fernández, J.J.; Norte, M. Anti-Cancer Agents Med. Chem. 2009, 9, 122.

3. Napolitano, J.G.; Norte, M.; Padrón, J.M.; Fernández, J.J.; Hernández Daranas, A. Angew. Chem. Int. Ed. 2009, 48, 796.

4. Matsumori, N.; Kameno, D.; Murata, M.; Nakamura, H.; Tachibana, K. J. Org. Chem. 1999, 64, 866.

Acknowledgements: The authors acknowledge financial support from the Spanish MEC (CTQ2008-06754-V04-01/PPQ). J.G.N. acknowledges MICINN for a Ph.D. scholarship (FPU Program).


87

PC 16

SECONDARY METABOLITES FROM A MARINE SPONGE AXINELLA POLYPOIDES

Belma Konuklugil1, Y. Y. Küçükecir1, Peter Proksch2, Bulent Gözcelioğlu3

1 University of Ankara, Faculty of Pharmacy,06100- Tandoğan, Ankara, Turkey. 2 Heinrich-Heine-Universität, Institute of Pharmaceutical Biology, Universitätsstr. 1, 40225

Düsseldorf, Germany 3 Department of Biology,Faculty of Sciences, 06100- Tandoğan, Ankara, Turkey.

[email protected]

In the last 25 years, research has been focused on the sea as source of substances with potential biological activity due to the discovery of novel compounds, which display biological properties such as antibacterial, antihelmentic and antitumoral. In the course of our continuing investigations towards the isolation of biological active compounds from marine sponges of the Turkish seas, we have recently examined the specimens of Axinella polypoides growing at Bodrum in the Turkish Aegean coast. Chemical investigation of methanolic extract of the sponges led to the identification of spongiacidin F, hymenialdisin, hymenidin, stevensin, oroidin.

All of them were identified using different spectroscopic techniques (HPLC, LC-MS). Futher studies are in progress.

Acknowledgements: this research has been sponsored by Tübitak-Jülich ( SBAG-JULICH)-3 (104S409)


88

PC 17

NEW HALOGENATED SESQUITERPENES FROM THE BRAZILIAN RED ALGA LAURENCIA CATARINENSIS

Miriam Falkenberg1, Cintia Lhullier1, Efstathia Ioannou2, Paulo Antunes Horta3, Eloir Paulo Schenkel1, Constantinos Vagias2, Vassilios Roussis2

1 Programa de Pós-graduação em Farmácia, Universidade Federal de Santa Catarina, Campus Trindade, 88040-970 Florianópolis, Brazil,

2 Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece,

3 Programa de Pós-graduação em Biologia Vegetal, Universidade Federal de Santa Catarina, Campus Trindade, 88040-970 Florianópolis, Brazil

[email protected]

Red algae belonging to the genus Laurencia are found throughout the world and have been the subject of intensive research. Secondary metabolites from these algae are predominantly sesquiterpenes, diterpenes and C15 acetogenins that are usually characterized by the presence of halogen atoms in their structures.1

In our ongoing investigations of marine organisms, we studied the secondary metabolites of Laurencia catarinensis Cordeiro-Marino & Fujii, collected from Ilha do Arvoredo, Santa Catarina, Brazil. Extraction of the dried alga with CH2Cl2/MeOH, followed by a series of chromatographic separations led to the isolation of five known compounds, along

with three new caespitol derivatives (1-3). To the best of our knowledge, this is the first report on the chemistry of L. catarinensis.

The structure elucidation of the isolated metabolites and the assignment of their relative stereochemistry were based on analyses of their spectroscopic data, including 1D and 2D NMR and MS experiments.

The evaluation of the biological activity of the isolated compounds is currently in progress. 1. Blunt, J.W., Copp, B.R., Hu, W., Munro,

M.H.G., Northcote, P.T., Prinsep, M. Marine Natural Products. Nat. Prod. Rep., 2008, 25, 35-94.

O

R2

ClBr

R1

Br1 R1 = OH,2 R1 = OAc,3 R1 = OAc,

R2 = OAcR2 = OHR2 = OAc


89

PC 18

STUDY OF THE MS FRAGMENTATION OF THE HALICLAMINES Gesine Schmidt and Matthias Köck

Alfred-Wegener-Institut für Polar- und Meeresforschung in der Helmholtz-Gemeinschaft, Am Handelshafen 12, D-27570 Bremerhaven, Germany

[email protected]

Two new 3-alkyl-pyridinium alkaloids, haliclamines E and F, were identified in the crude extract of the Arctic sponge Haliclona viscosa using LC-MS methods. Due to the scarcity of sponge material available, the compounds were not isolated and the structure elucidation relied on the chromatographic comparison with synthetic compounds.1

The MS fragmentation pattern of the haliclamines in general was examined on the basis of the synthetic compounds. Naturally occurring as well as to-date unreported haliclamines of (a) equal alkyl chain length and (b) chain length differences of one, two or three methylene

groups were subject to extensive ion trap- and API-CID-MS/MS examinations.

The MS fragmentation of these compounds is similar to the cyclostellettamines2 but strongly influenced by the tetrahydropyridine moiety and the ionization method applied in mass spectrometric analyses.3

1. G. Schmidt, C. Timm, M. Köck, Org. Biomol.

Chem. 2009, accepted for publication. 2. A. Grube, C. Timm, M. Köck, Eur. J. Org.

Chem. 2006, 1285-1295. 3. G. Schmidt, C. Timm, E. Lichte, M. Köck,

2009, in preparation.


90

PC 19

CONSTITUENTS OF PETROSIA (PETROSIA) HOEKSEMA, COLLECTED FROM THE GULF OF THAILAND

Julia Bessa1, Anake Kijjoa1, Rawiwan Wattanadilok2, Sumaitt Puchakarn2 and Pichai Sonchaeng3

1 ICBAS - Instituto de Ciências Biomédicas de Abel Salazar and CIIMAR, Universidade do Porto, 4099-003-Porto, Portugal.

2 Bangsaen Institute of Marine Science, Burapha University, Bangsaen, Chonburi 20131, Thailand.

3 National Science Museum, Technopolis, Klong 5, Pathumthani 12120, Thailand [email protected]

The marine sponges of the genus Petrosia are found to be a source of interesting bioactive secondary metabolites including cytotoxic polyacetylenes, contignasterol, sulfated sterols, cytotoxic polycyclic alkaloids and purine derivatives.

In the course of our investigation on anticancer compounds from the marine sponges from the Gulf of Thailand, we have isolated p-hydroxybenzoic acid (1), 3-methylmaleimide-5-oxime (2), maleimide-5-oxime (3), tetillapyrone (4) and nortetillapyrone (5) from the ethyl acetate extract of Petrosia (Petrosia) hoeksema, collected in the Gulf of Thailand at Chantaburi Province.

Very recently tetillapyrone (4) and nortetillapyrone (5), originally isolated from the sponge Tetilla japonica (family Tetillidae, order Spirophorida)1, have also been found in Haliclona cymbiformis and Haliclona baeri (family Haliclonidae,

order Haplosclerida)2 as well as from Cliona patera (family Clionidae, order Hadromerida)3, in shallow waters in the Gulf of Thailand. These isolations of tetillapyrone (4) and nortetillapyrone (5), from widely different taxa may suggest an extraneous source of these compounds as they have so far not been found in other representatives of these genera. 1. Wattanadilok, R., Sonchaeng, P., Kijjoa, A.,

Damas, A.M., Gales, L., Silva, A.M.S., Herz, W. (2001). Journal of Natural Products 64, 1056.

2. Wattanadilok, R., Sawangwong, P., Rodrigues, C., Cidade, H., Pinto, M., Pinto, E., Silva, A., Kijjoa, A. (2007). Marine Drugs 5, 40.

3. Sawangwong, P., Wattanadilok, R., Kijjoa, A., Silva, A.M.S., Eaton, G., Herz, W. (2008). Biochemical Systematics and Ecology 36, 493.

We thank FTC e Fundação para a Ciência e Tecnologia (Project POCI/MAR/58114/2004), FEDER and Ciência - Inovação 2010, for support.

OH

CO2H

O ONOH

O ONOH

CH3

O

O OOH

OH

OH CH3O

O OOH

OH

OH

1 32

4 5


91

PC 20

CYTOTOXIC STEROIDS FROM MARINE ORGANISMS: ISOLATION, SYNTHESIS AND STRUCTURE/ACTIVITY STUDIES OF NEW

THIOESTER STEROIDS FROM PARAGORGIA SP. Javier Jesús Poza1, Rogelio Fernández2, Fernando Reyes1, Jaime Rodríguez1

and Carlos Jiménez1

1 Departamento de Química Fundamental, Facultade de Ciencias, Universidade da Coruña, 15071 A Coruña, Spain

2 Medicinal Chemistry Department, PharmaMar S.A.U., Pol. Ind. La Mina Norte, Avda. de los Reyes 1, 28770 Colmenar Viejo, Madrid, Spain.

[email protected]

Steroids isolated from marine organisms have unusual and intriguing structures with different biological activities mainly cytotoxic.1 Our research group is involved in a research program focused on the isolation of marine steroids with cytotoxic activity from different phyla and their chemical synthesis.2

Bioassay-guided fractionation of the 2-propanol extract of the soft coral Paragorgia sp. collected in Madagascar yielded three novel unusual cytotoxic thioester steroids derivatives named parathiosteroids A–C. Their structures were established by spectroscopic methods (mainly 1D and 2D NMR) and confirmed by synthesis. They represent the first isolation of natural steroids bearing a C22 thioester in their side chain. Due to the cytotoxic activities displayed by the natural steroids against a panel of 3 human tumor cell lines at µM level, several analogues were prepared in order to deduce some structure-activity relationships such as that the XCH2CH2NHCOCH3 moiety (X= S, O, NH) in the side chain is essential for the antiproliferative activity and a low degree

of oxidation on A-ring results in higher bioactivity.

The isolation of these compounds is remarkable not only because they are the first natural steroids isolated that bear a C22 thioester but also because they could be biosynthetic intermediates for the degradation pathway of the steroid side chain through activation with CoA and β-oxidation. In such a case, this is the first report of the isolation of a degradation intermediate bearing a fragment of a steroid-CoA thioester. Their structures, synthesis, biological activity data, biological significance, and the deduced structure-activity relationships will be presented.3

1. D’Auria, M.V.; Minale, L.; Riccio, R. Chem. Rev. 1993, 93, 1839–1895.

2. a) González, N.; Barral, M. A.; Rodríguez, J.; Jiménez, C. Tetrahedron 2001, 57, 3487–3497. b) Deive, N., Rodríguez, J., Jiménez, C. J. Med. Chem. 2001, 44, 2612–2618

3. Poza, J. J.; Fernández, R.; Reyes, F.; Rodríguez, J.; Jiménez, C. J. Org. Chem. 2008, 73, 7978–7984.

O

XHN

O

O

HO

XHN

O

O

Parathiosteroids A-C: X = S

Parathiosteroid analogues: X = O or N


92

PC 21

BROMINATED METABOLITES FROM RED SEA SPONGE SUBEREA MOLLIS Diaa T.A. Youssef1,* and Lamiaa A. Shaala2

1 Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt

2 Department of Pharmacy, Hospital of Suez Canal University, Suez Canal University, Ismailia 41522, Egypt

[email protected]

Marine sponges of the order Verongida are of much current biological and chemical interest. They are characterized by elaboration of typical brominated metabolites which are biogenetically related to tyrosine. These metabolites are considered as distinct markers for Verongid sponges. Diverse biological activities for these compounds have been reported including antifungal, antibacterial, cytotoxic and enzyme inhibitory effects. Previous study in our laboratory on Suberea mollis resulted in the isolation and identification of a

number of cytotoxic, antioxidants and antimicrobial compounds, which has prompted us to further explore this sponge.

Reinvestigation of the sponge resulted into isolation and identification of two new amides, subereamide A and B together with several known compounds. The structural determinations of the compounds were based on extensive interpretation of high-field NMR spectra and HRFABMS data. The biological study of these compounds will be discussed.


93

PC 22

NATURAL PRODUCTS FROM MARINE PHANEROGAMS Fatma Bitam1, Maria Letizia Ciavatta2, Marianna Carbone2, Emiliano Manzo2,

Ernesto Mollo2, Guido Villani2 and Margherita Gavagnin2 1 Faculté des Sciences, Département de Chimie, Université de Batna, Batna 05000, Algeria

2 Istituto di Chimica Biomolecolare, C.N.R., Via Campi Flegrei, 34, 80078, Pozzuoli, Italy

[email protected]

In recent decades, marine phanerogams have been regarded with great interest in estimation of the ecological and economic values of various marine ecosystems. On the other hand, only recently an increasing number of chemical studies have been carried out on these organisms, with respect to the terrestrial counterpart, leading to the isolation and characterization of secondary metabolites.1 Different bioactivities (antifouling, antibacterial, antialgal, antiviral, antifungal, anti-inflammatory, cytotoxicity) have been also evidenced in sea-grass metabolites.2 Previous chemical investigation of genus Halophila reported flavones and flavone glycosides,3,4 sulphated phenolic compounds5 and a macrocyclic glucoterpenoid6 whereas genus Posidonia has been extensively studied from an ecological point of view. Sterols5 and phenolic products6 have been described from genus Posidonia, in particular with regards to the potential applications of phenolic compounds as antioxidants.

In this communication we report the results of our recent chemical studies on

the sea-grasses Halophila stipulacea and Posidonia oceanica, collected in Mediterranean Sea, that revealed to contain novel secondary metabolites including rare malonylated glycosyl-flavones (i.e. 1) and lignans (i.e. 2), respectively.

1. Blunt, J. W.; Copp, B. R.; Hu, W-P.; Munro, M. H. G.; Northcote, P. T.; Prisep, M. R. Nat. Prod. Rep., 2009, 26, 170-244.

2. Shu-Hua Qi1; Si Zhang; Pei-Yuan Qian; Bin-Gui Wang, Bot. Mar., 2008, 51, 441-447.

3. Meng, Y.; Krzysiak, A.J.; Durako, M.J.; Kunzelman, J.I.; Wright, J.L.C. Phytochemistry, 2008, 69, 2603-2608.

4. Mollo, E.; Gavagnin, M.; Carbone, M.; Castelluccio, F.; Pozone, F.; Roussis, V.; Templado, J.; Ghiselin, M.T., Cimino, G. Proc. Nat. Acad. Sci., 2008, 105, 4585-4586.

5. McMillan, C. Aquat. Bot., 1986, 25, 63-72.

6. Gavagnin, M.; Carbone, M.; Amodeo, P.; Mollo, E.; Vitale, M.R.; Roussis, V.; Cimino, G. J. Org. Chem., 2007, 72, 5625-5630.

7. Sica, D.; Piccialli, V.; Fasullo, A. Phytochemistry, 1984, 23, 2609-2611.

8. Agostani, S.; Desjobert, J.M.; Pergent, G. Phytochemistry, 1998, 48, 611-617

O

1 2

HO

OO

O

HO

OCH3

OH

OCH3

H3CO O

OOH

OOH

HO OH

OH

O O

O


94

PC 23

GC-MS ANALYSES AND IN VITRO ANTIMICROBIAL AND ANTIOXIDANT ACTIVITY OF THE ESSENTIAL OIL AND NON-VOLATILE EXTRACT

FRACTIONS OF THE MARINE RED ALGA PLOCAMIUM BRASILIENSE Vanessa Gressler1, Nair S. Yokoya2, Mutue T. Fujii2, Patrícia P.M. da Silva1,

Fabiana C. Missau1, Pio Colepicolo1, Ernani Pinto1 1 Universidade de São Paulo, Av. Prof. Lineu Prestes, 580, 13B, São Paulo, Brazil.

2 Instituto de Botânica, Av. Miguel Estéfano, 3687, São Paulo, Brazil.

[email protected]

Marine algae comprise a few thousands of species and are widely distributed throughout the world’s oceans, however, Plocamium brasiliense (Rhodophyta) is only distributed in the Brazilian coastal waters.1,2 In this paper, we report the identification of volatile organic compounds (VOC) and the investigation of the antimicrobial and antioxidant activities of the essential oil (EO) and non-volatile fractions from P. brasiliense.

VOC were extracted with Clevenger apparatus and analyzed by GC-MS. Freeze-dried P. brasiliense samples were extracted using hexane, dichloromethane, ethyl acetate and methanol. All extracts and the EO were tested to verify their antimicrobial (by MIC method) and antioxidant activities (by DPPH and chemiluminescence method). The GC-MS data and Kovat’s indices were used to identify 15 oil components. Butylated hydroxytoluene (BHT), Triphenylphosphine oxide, Phytol, Benzenepropanol, τ-[(2-methoxy-ethoxy)methoxy]-β-methyl-,(R*,S*)-(.+-.)- and Tridecanol were the major components. The EO and extracts were tested against their antimicrobial activity, however, no significant inhibition was

observed with potency up to 500 µg.mL-1. The EO, the hexane and ethyl acetate extracts showed excellent antioxidant activity (92.3; 97.4; 87.7% respectively) at 500 µg/mL by performing the chemiluminescence assay. For the EO, the high antioxidant activity observed is probably because of the presence of more than 27% of BHT which is known as a potent synthetic/natural antioxidant molecule.3-5

The genus Plocamium is poorly studied concerning the composition of the EO, as well as their biological activities. The investigation of active substances in algae can be an alternative tool to find new molecules that can be tested and used as pharmaceuticals.

1. Saunders, G.W.; Lehmkuhl, K.V., 2005. Eur. J. Phy. 40, 293-312.

2. Pereira, R.C., et al., 2004 Bot. Mar. 47, 202-208.

3. Gomez, E., et al., 1993. J. Agric. Food Chem., 41(10), 1669-1676.

4. Ito, N., et al., 1986. Food and Chem. Tox. 24(10/11), 1071-1082.

5. Huang, H-L., Wang, B.G., 2004. J. Agric. Food Chem. 52, 4993-4997.


95

PC 24

NEW DOLABELLANES WITH ANTIBACTERIAL ACTIVITY FROM THE BROWN ALGA DILOPHUS SPIRALIS

Efstathia Ioannou1, M. Mukhlesur Rahman2, Simon Gibbons2, Constantinos Vagias1, Vassilios Roussis1

1 Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece,

2 Centre for Pharmacognosy and Phytotherapy, School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK

[email protected]

Brown algae of the family Dictyotaceae are known to produce a wide range of diterpenes featuring different carbon frameworks, many of which possess cytotoxic, antibacterial, algicidal, ichthyotoxic and antifeedant activities.

In the framework of our research program towards the isolation of bioactive natural products from marine organisms of the Greek seas, a chemical study on the constituents of Dilophus spiralis specimens, collected from Elafonissos Island, south of Peloponnese, was undertaken.

Extraction of the freeze-dried alga with CH2Cl2 and subsequently MeOH, followed by a series of chromatographic separations led to the isolation of thirty dolabellane diterpenes, twenty two of which are new natural products.

dolabellaneskeleton

The structure elucidation and the assignment of the relative configuration of the isolated compounds were based on analyses of their spectroscopic data (NMR, MS, IR). When possible, the proposed structures were confirmed by single crystal X-ray diffraction analysis, whereas their absolute stereochemistry was determined using the modified Mosher’s method and chemical interconversions.

The antibacterial activity of the isolated metabolites was evaluated against multidrug-resistant (MDR) and methicillin-resistant (MRSA) strains of Staphylococcus aureus. A number of the tested dolabellanes exhibited noteworthy levels of activity.


96

PC 25

MALDI-TOF-MS, A SUITABLE METHOD FOR METABOLITE SCREENING?

Christine Cychon, Gesine Schmidt, and Matthias Köck

Alfred-Wegener-Institut für Polar- und Meeresforschung in der Helmholtz-Gemeinschaft, Am Handelshafen 12, D-27570 Bremerhaven, Germany

[email protected]

Next to standard LC-MS applications, MALDI1-TOF mass spectrometry is a powerful method to analyze and characterize macromolecules like proteins2, carbohydrates3, oligonucleotides4 and synthetic polymers5. Research on small molecules did not, for traditional and technical reasons, focus on MALDI, but applications in the investigation of bacteria regarding their chemotaxonomy6 and secondary metabolism7 are becoming more frequent. During our search for new natural products, we are applying MALDI-TOF-MS for a direct screening of secondary metabolites in sponge tissue.8 Due to the simple sample preparation, this method allows a broad insight into the compound spectrum at an early stage of investigation. Besides this, challenging questions regarding the qualitative and quantitative sample preparation, compound distribution and stability, etc. have emerged. Here, we want to discuss

our experiences with this procedure using the sponge Stylissa caribica as an example. What are the possibilities and perspectives of MALDI-TOF-MS and where are the limits in natural products research?

1. Matrix assisted laser desorption/ionization 2. Chaurand, D. Cornett, R. M. Caprioli, J.

Proteome Res. 2006, 5, 2889-2900. 3. D. Harvey, Mass. Spectom. Rev. 2006, 25, 595-

662. 4. E. Nordhoff, M. Schürenberg, G. Thiele, C.

Lübbert, K. Kloeppel, D. Theiss, H. Lehrach, J. Gobom, Int. J. Mass. Spectrom. 2003, 226, 163-180.

5. G. Montaudo, F. Samperi, M. Montaudo, Prog. Polym. Sci. 2006, 31, 277-357.

6. D. Dickinson, M. La Duc, M. Satomi, J. Winefordner, D. Powell, K. Venkateswaran, J. Microbiol. Meth. 2004, 58, 1-12.

7. M. Erhard, H. Von Doehren, P. Jungblut, Nature Biotech. 1997, 15, 906-909.

8. A. Grube, T. Maier, M. Kostrzewa, M. Köck, Z. Naturf. 2007, 62, 600-604.


97

PC 26

STRUCTURAL ELUCIDATION OF NEW COMMUNESINS FROM A MARINE-DERIVED PENICILLIUM EXPANSUM LINK BY LC-ESI-HRMS/MS Isabelle Kerzaon1*, Olivier Grovel1, Nicolas Ruiz1, Thibault Robiou du Pont1,

Jean‐François Biard1, Yves François Pouchus1 1 Université de Nantes, Pôle Mer et Littoral, Laboratoire MMS-EA2160, Faculté de Pharmacie, BP

53508, Nantes, F-44000 France

[email protected]

In the fields of natural products, investigation of marine-derived fungi is relatively recent but the discovery of structurally original substances has demonstrated their potential as a promising source of new compounds (Bugni and Ireland, 2004). In our search for new bioactive fungal substances, an uncommon marine-derived Penicillium expansum strain was studied on various culture media for compounds exhibiting a neuroactivity on a blowfly larvae assay (Zlotkin et al., 1971). Communesin B was identified as the main active compound of the neuroactive extract using bio-guided fractionation and analyses by spectroscopic methods including 1H and 13C-NMR. Communesin B is one of the eight members of an indole alkaloids series with an unusual carbon skeleton and a dimethyl epoxide moiety, except for communesin F. CID-MS² of communesins produces fragmentation patterns characteristic of the dimethyl epoxide (Jadulco et al., 2004). Culture extracts were dereplicated using LC-ESI-MS/MS and LC-HRMS to search for the presence of other communesin-related compounds. Analyses of MS/MS data, retention time index (RI) and accurate masses led to the identification of the four known communesins A, D, E and F. The exhaustive investigation of their MS/MS data allowed us to develop a predictive model for the identification of the variable

substituents. The search for other compounds corresponding to this model allowed to identify seven new derivatives among the minor molecules of the crude extracts. Structural hypotheses were made for the seven new derivatives by analyses of their fragmentation data, and LC-HRMS allowed to confirm the structural elucidation of new communesins I-O.

The use of fragmentation patterns in dereplication appears to be a powerful way for the identification and first-step structural elucidation of new derivatives belonging to a chemical series. Furthermore the detection of new molecules in a marine-derived strain, belonging to a widely studied terrestrial species, seems to exemplify the chemical diversity of marine-derived fungi.

1. Bugni, T. S., Ireland, C. M., 2004. Marine-

derived fungi: a chemically and biologically diverse group of microorganisms. Nat. Prod. Rep. 21, 143-163.

2. Jadulco, R., Edrada, R. A., Ebel, R., Berg, A., Schaumann, K., Wray, V., Steube, K., Proksch, P., 2004. New Communesin Derivatives from the Fungus Penicillium sp. Derived from the Mediterranean Sponge Axinella verrucosa. J. Nat. Prod. 67, 78-81.

3. Zlotkin, E., Fraenkel, G., Miranda, F., Lissitzky, S., 1971. The effect of scorpion venom on blowfly larvae--A new method for the evaluation of scorpion venoms potency. Toxicon 9, 1-2.


98

PC 27

NEW STRUCTURAL EVIDENCES FOR SARAINES A-C, MACROCYCLIC ALKALOIDS FROM THE MEDITERRANEAN SPONGE

RENIERA (HALICLONA) SARAI Ines Mancini1, Andrea Defant1, Lucija Raspor2, Graziano Guella2,

Tom Turk2 and Kristina Sepčić2 1 Laboratorio di Chimica Bioorganica, Dipartimento di Fisica, Università di Trento,

via Sommarive 14, I-38100 Povo Trento, Italy. 2 Department of Biology, Biotechnical Faculty, University of Ljubljana,

Večna pot 111, 1111 Ljubljana, Slovenia.

[email protected]

The structure of saraines A-C (1-3), reported from the sponge Reniera sarai (order Haploslerida) collected in the bay of Naples (Italy),1 includes a unique diazatricycloundecane core inducing a peculiar behaviour responsible of spectroscopic anomalies and intricacy in chromatographic purification.2 It was

explained by a strong "proximity effect" between the tertiary ammine and aldehyde groups, involved in an equilibrium with the cyclic zwitterionic species.

On a sample of saraines A-C recently isolated from the same sponge species collected in the Northern Adriatic Sea (Cres Island, Croatia), we report here the results from a structural study based on: a) electrospray ionization (ESI)-MS spectra recorded in positive ion mode showing signals for mixed [2M+H]+ clusters in neutral conditions and [M+H]+

pseudomolecular ions in acidic media, b) the conversion to a stable O-methyl ammonium salt, c) density functional theory (DFT) calculations on the amine/aldehyde form, resulting in a perfect agreement with the definition by the Bürgi–Dunitz angle, the latter one able to forecast the preferred spatial approach in the nucleophilic addition to a carbonyl group.

In addition, biological activities including antibacterial assays were investigated for saraines A-C, their protonated forms and related compounds saraines 1-3 and isosaraines 1-3 isolated from the same sponge. 1. G.Cimino, C.A. Mattia, L. Mazzarella, R.

Puliti, G. Scognamiglio, A. Spinella, E.Trivellone, Tetrahedron, 1989, 45, 3863.

2. G. Cimino, G. Scognamiglio, A. Spinella, E. Trivellone, J. Nat. Prod. 1990, 53, 1519.


99

PC 28

NOVEL SECONDARY METABOLITES FROM THE SPONGE-DERIVED FUNGUS STACHYLIDIUM SP.; TRACKING BACTERIAL ENDOSYMBIONTS

Celso Almeida, Gabriele M. König

Institute for Pharmaceutical Biology, Bonn University, Nussallee 6, D 53115 Bonn, Germany

[email protected]

The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia cf. C. flammea. Cultivation on a biomalt medium with added sea salt yielded an extract which was cytotoxic toward several cancer cell lines. Chemical investigation of this extract led to the isolation of several new compounds including new phthalides with unusual structural motives, a novel phthalimidine derivative, and new cyclic peptides. The latter contained the amino acid residue N-methyl-3-(3-furyl)-alanine, which is a rare amino acid only reported once before in heptapeptides from the fungus Rhizopus

microsporus (Steyn et al., 1983). Interestingly these peptides were later found to be produced by a bacterial endosymbiont in the fungus (Partida-Martinez, et al., 2007). Based on the biosynthetic molecular structures hereby reported, preliminary molecular biology and other biological assays we consider a bacterial association as likely.

1. Partida-Martinez, et al., (2007) App. Env. Microb., 73, 3, 793-797.

2. Steyn et al., (1983) J. Chem. Soc. Chem. Commun., 47-49.


100

PC 29

STEROIDAL GLYCOSIDES FROM THE MARINE SPONGE PANDAROS ACANTHIFOLIUM

Olivier P. Thomas1, Nadja Cachet1, Erik L. Regalado2, Grégory Genta‐Jouve1, Mohamed Mehiri1 and Philippe Amade1

1 University of Nice Sophia Antipolis, LCMBA - UMR 6001 CNRS Institut de Chimie de Nice Parc Valrose, 06108 Nice, Cedex 2, France.

2 Department of Chemistry, Center of Marine Bioproducts, Loma y 37 Alturas del Vedado, C.P. 10400 Havana, Cuba

[email protected]

In the course of our study on bioactive secondary metabolites produced by Caribbean marine sponges we report herein our first results on the very little studied Pandaros acanthifolium1 collected off the French Martinique Island coast.

The chemical composition of this Caribbean sponge was investigated and led to the isolation of a large number of steroidal glycosides named pandarosides on the basis of detailed spectroscopic analyses, including 2D NMR and HRESIMS studies. All pandarosides are characterized by a rare 2-hydroxycyclopentenone D-ring with a 14β configuration. Some structural differences appear on the side chain of the aglycon

part of the molecule where an additional saturated or unsaturated methyl or ethyl group is present at C-24. Furthermore an unsaturation was evidenced at the C-8/C-9 position in some cases. For the sugar portion, one or two sugar units were present linked to the first sugar residue at C-2 or C-3. The methyl esters were also isolated during the purification process. The absolute configuration of the aglycon part of the molecules was assigned by comparison between experimental and TDDFT calculated circular dichroism spectra on the more stable conformer.

1. Schmitz et al J. Am. Chem. Soc. 1981, 103,

2467-2469.


101

PC 30

CODIUM TOMENTOSUM AND PLOCAMIUM CARTILAGINEUM : CHEMICAL APPROACH AND ANTIOXIDANT POTENTIAL

Daniela Gomes1, Andreia P. Oliveira1, Pedro Trindade1, Patrícia Valentão1, Paula Guedes de Pinho1, Teresa Mouga2 and Paula B. Andrade1

1 REQUIMTE/Department of Pharmacognosy, Faculty of Pharmacy, Porto University, R. Aníbal Cunha, 164, 4050-047 Porto, Portugal.

2 Escola Superior de Turismo e Tecnologia do Mar, Instituto Politécnico de Leiria, Santuário N.ª Sra. Dos Remédios, Apartado 126, 2524-909 Peniche, Portugal.

[email protected]

The use of seaweed species as alternative materials to extract natural antioxidant compounds has attracted the attention of biomedical scientists. The green algae Codium tomentosum Stackhouse and the red algae Plocamium cartilagineum (Linnaeus) P. S. Dixon are intertidal marine seaweeds which live in harsh environment, having, therefore, a protective antioxidant defense system. Our study aimed to determinate the chemical composition (organic acids, phenolics and volatile compounds), as well as to evaluate the antioxidant potential of C. tomentosum and P. cartilagineum from the Atlantic Ocean surrounding Portugal.

For the first time, the profile of organic acids was analysed in these matrices. The analysis of aqueous extracts by HPLC/UV revealed seven and four organic acids in green and red species, respectively. In P. cartilagineum these compounds were present in vestigial amounts, while C. tomentosum exhibited a higher content, being oxalic acid the main compound. No phenolics, UV-absorving compounds, were identified by reversed-phase

HPLC/DAD. The volatiles composition was determined by GC/MS. For the first time, a total of forty-one compounds were identified, arising from several chemical classes: alcohols, aldehydes, esters, halogenated compounds, ketones, monoterpenes, norisoprenoid derivatives, among others. Norisoprenoid derivatives and aldehydes were predominant. The main volatiles in green and red seaweeds were limonene and benzophenone, respectively.

Both species revealed considerable antioxidant activity against reactive oxygen (superoxide radical) and reactive nitrogen (nitric oxide) species, in a concentration dependent manner. The identified compounds may partially contribute to the noticed anti-radical effects.

Acknowledgement: The authors are grateful to Fundação para a Ciência e Tecnologia (FCT) for financial support (PTDC/AGR-AAM/64150/2006). D. Gomes (BI) and A. P. Oliveira (SFRH/BD/47620/2008) are indebted to FCT for their grants.


102

PC 31

NEW DITERPENOIDS FROM DIFFERENT STRAINS OF THE MARINE CILIATE EUPLOTES RARISETA

Graziano Guella1,2, Emanuela Callone1, Rita Frassanito1, Ines Mancini1, Graziano Di Giuseppe3, Francesco Frontini3 and Fernando Dini3

1 Laboratory of Bioorganic Chemistry, Department of Physics, University of. Trento, Via Sommarive 14 38050 Povo (TN), Italy;

2 CNR, Institute of Biophysics, Unit at Trento, Via alla Cascata 56/C, 38050 Povo (TN), Italy; 3 Department of Biology, University of Pisa, via Volta 6, 56126 Pisa, Italy

[email protected]

Recently, marine interstitial ciliates belonging to the genus Euplotes have emerged as a rich source of new skeleton sesqui- and diterpenes bearing various functionalities [1] and displaying activities against other ciliates, competing for space and resources, and even inducing apoptotic action towards mouse (AtT-20) and rat (PC12) tumour cell lines. [2] From a structural point of view, all the secondary metabolites so far isolated are regular, often polycyclic, terpenoids, which are supposed to originate from the usual biogenetic precursors farnesyl-pyrophosphate (FPP) for sequiterpenoids and geranyl-geraniol pyrophosphate (GGP) for diterpenoids through the involvement of different cyclase and/or oxidase enzymes in the overall biosynthetic pathway.

A homogeneous, though multifaceted, secondary metabolic character emerges from a detailed investigation of secondary metabolites produced by the morphospecies Euplotes rariseta In particular, from the NZ2 strain collected at Omaha Bay in New Zealand we have isolated two irregular diterpenoids which are the C5 homologues of rarisetenolide.

[3] From the strain UBt22 collected in Brazil (Ubatuba), on the other hand, other diterpenoids with a new skeleton have been found, while LC-ESI-MS analysis of the raw organic extracts of both the strain OMAN1 (Oman), formerly assigned to the morphospecies E. quinquecarenatus, and the strain SM1 (Venezuela, S. Margarita,) before classified as E. parkei, shows the presence of secondary metabolites built on the same prenyl-rarisetane skeleton. Our findings on chemodiversity from secondary metabolites strongly suggest a wide polymorphism in E.rariseta populations. [4]

1. a) G. Guella, F. Dini, A. Tomei, F. Pietra, J.

Chem. Soc., Perkin Trans. I 1994, 2, 161-166; b) G.Guella, F. Dini, F. Pietra, Angew. Chem. Int. Ed. 1999, 38, 1134-1136;. c) Guella G., Callone E., Di Giuseppe G., Frassanito R., Frontini F., Mancini I., Dini F., European Journal of Organic Chemistry, 2007, 5526-5534

2. D. Cervia, M. Garcia-Gil, E. Simonetti, G. Di Giuseppe, G. Guella, P. Bagnoli, F. Dini, Apoptosis, 2007, 12, 1349-1363.

3. G. Guella, F. Dini, F. Pietra, Helv. Chim. Acta 1996, 79, 2180-2189.

4. G. Guella et al., to be published


103

PC 32

BROMINATED SESQUITERPENES WITH A NOVEL CARBOCYCLE FROM THE MOLLUSC APLYSIA PUNCTATA

Anastasia Petraki, Efstathia Ioannou, Constantinos Vagias, Vassilios Roussis

Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece

[email protected]

Sea hares are shell-less, slow-moving marine molluscs that have been proven a rich source of bioactive natural products, which are generally contained in minute amounts and are often considered to be of dietary origin. A possible role of the secondary metabolites acquired through their diet in the defense system of the sea hares has been suggested.

Species of the genus Aplysia (Mollusca, Aplysiidae), like other sea hares, thrive on algae, usually on Laurencia species, acquiring and accumulating many algal metabolites in their digestive glands.

Specimens of the mollusc Aplysia punctata were collected from Achladi Bay in Maliakos Gulf, Greece, at a depth of 2–

4 m in April of 2008. Extraction of the freeze-dried animals with a mixture of CH2Cl2/MeOH, followed by a series of chromatographic separations, led to the isolation of a number of known compounds, already isolated from species of the red alga Laurencia, along with two new brominated sesquiterpenes (1 and 2). The new natural products feature an unprecedented bicyclic skeleton, possibly derived by cyclization of the common humulene skeleton.

The structures and relative configurations of the isolated natural products were established on the basis of detailed NMR and MS spectroscopic analysis.

OH

HR2

R1

1 R1 = H,2 R1 = Br,

R2 = BrR2 = H


104

PC 33

BROMINATED CUPARENE SESQUITERPENES AND C15 ACETOGENINS FROM THE SOUTH AFRICAN MARINE ALGA LAURENCIA FLEXUOSA

Maryssa G. Mann1, John J. Bolton2 and Denzil R. Beukes1

1 Division of Pharmaceutical Chemistry, Faculty of Pharmacy, Rhodes University, Grahamstown, 6140;

2 Department of Botany, University of Cape Town, Private Bag, Rondebosch, 7701, South Africa

[email protected]

In our continuing search for new biologically active natural products from South African marine algae we have investigated the chemistry of the endemic red alga Laurencia flexuosa.

Fractionation of the crude organic extract of Laurencia flexuosa resulted in the isolation of a new brominated cuparene

sesquiterpene, cupalaurendiol, and two known C15 acetogenins. The structures of the compounds were determined by standard spectroscopic methods and derivatization. Interestingly, cupalaurendiol dehydrates in CDCl3, to form a new cyclopenta[b]benzofuran derivative.


105

PC 34

STRUCTURAL DIVERSITY AND BIOLOGICAL ACTIVITIES OF HALOGENATED METABOLITES IN RED ALGAE GENUS LAURENCIA FROM

NORTH BORNEO ISLAND Charles S. Vairappan and Suzuki Minoru

Laboratory of Natural Products Chemistry, Institute for Tropical Biology and Conservation,

Universiti Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia.

[email protected]

Malaysia, one of the 12 mega-diversity countries of the world is rich with tropical marine life such as seaweed, soft corals, sponges and invertebrates, and most of these marine heritage are in North Borneo Island of Sabah. Seaweed in particular is abundant and could be found growing in lagoons, estuaries, rocky shores and coral reefs. At present close to 339 specific and infra-specific taxa of marine algae are known to exist in Sabah and from this 147 Rhodophyceae, 112 Chlorophyceae and 80 Phaeophyceae have been identified. Red algae genus Laurencia in particular, is found growing as dense mats on rocky shores, deteriorating reefs and as biofoulers on commercial seaweed farms.

There are four major species of Laurencia; L. snackeyi, L. majuscula, L. nanggii and L. similis; and each produces halogenated metabolites belonging to a particular chemical skeleton such as syndreans, chamigranes, acetogenin/bromoallene, and bromoindoles, respectively. To date a total of 35 halogenated metabolites have been identified from this genus and 13 were novel metabolites. These metabolites showed potent antimicrobial, cyto-toxicity, and antifeedent activities. Isolated chamigranes, C15-acetogenins and bromoindoles halogenated metabolites showed potent antibacterial activity against seaweed pathogens and human clinical microbes. Meanwhile, compounds from L. snackeyii showed strong toxic activity against marine fish fries.


106

PC 35

POLYCYCLIC AROMATIC HYDROCARBONS IN WILD AND CULTURED FISHES M. J. Ramalhosa1,2, P. Paíga1, S. Morais1, M. B. P.P. Oliveira2 and C. Delerue‐Matos1

1 REQUIMTE, Instituto Superior de Engenharia do Porto, Rua Dr. António Bernardino de Almeida 431, 4200-072 Porto, Portugal.

2 REQUIMTE, Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha 164, 4099-030 Porto, Portugal.

[email protected]

For the general population, the major routes of exposure to Polycyclic Aromatic Hydrocarbons (PAH) are from food and inhaled air. PAH enter the environment via the atmosphere from a variety of combustion processes and pyrolysis sources. PAH have been detected in a variety of foods as a result of the deposition of airborne PAH, and in fish and mussels from contaminated sediments and waterborne compounds1. The heavy PAH have been found to bioaccumulate in the fatty tissues of some marine organisms, and show clear evidence of mutagenicity/genotoxicity in experimental animals. Commission Regulation EC Nº 208/2005 of 4 February 2005 established a maximum level for benzo(a)pyrene (BaP) of 2 µg kg-1 (w/w) in muscle meat of fish2. The carcinogenic potential of total PAH concentrations contained in a food product is, in some cases, about 10 times higher than the content of BaP alone3.

Aquaculture has been developed in the past decades as a consequence of the increase of fish consumption by the world population, since fisheries have possibly reached their maximum yield due to overexploitation4.

The aim of the present work was to evaluate the PAH contamination in the wild fishes more consumed by Portuguese population such as horse mackerel (Trachurus trachurus), chub macherel (Scomber japonicus) and sardine (Sardina pilchardus) and in a cultured sea bass (Dicentrarchus labrax) to assess the dietary intake of these pollutants. 1. European Commission: Scientific Committee

on Food SCF/CS/CNTM/PAH/29Final 4 December 2002: Opinion of the Scientific Committee on Food on the risk to human health to Polycyclic Aromatic Hydrocarbons in food.

2. Commission Regulation (EC) 208/2005 4-2-2005, amending Regulation (EC) 466/2001 as regard polycyclic aromatic hydrocarbons, Official Journal of the European Union, Nº L34/3 8-2-2005.

3. Visciano, P., Perugini, M., Conte, F. and Amorena, M. (2008), Polycyclic aromatic hydrocarbons in farmed rainbow trout (Oncorhynchus mykiss) processed by traditional flue gás smoking and by liquid smoke flavourings, Food and Chemical Toxicology, 46, 1409-1413.

4. Fernandes, D., Zanuy, S., Bebianno, M.J. and Porte, C. (2008), Chemical and biochemical tools to assess pollution exposure in cultured fish, Environmental Pollution, 152, 138-146


107

PC 36

ANTIBACTERIAL AND ANTIBIOFILM ACTIVITIES OF BRAZILIAN MARINE-DERIVED FUNGI AGAINST STAPHYLOCOCCUS EPIDERMIDIS

Marina Scopel1, Alexandre J. Macedo1,2, Wolf‐Rainer Abraham3, Beatriz Mothes4, Cléa Lerner4 and Amélia T. Henriques1.

1 Faculdade de Farmácia and 2 Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Brazil.

3 Helmholtz-Zentrum für Infektionsforschung, Braunschweig, Germany. 4 Fundação Zoobotanica, Museu de Ciências Naturais, Porto Alegre, Brazil.

[email protected]

The increase observed on researches employing marine microorganisms has been evident during the last two decades. The chemical diversity of their secondary metabolites reported to date, has been showing an important potential to the discovery of new molecules with relevant biological activities1,2. Lately, studies concerning antimicrobial activities have been one of the greater targets for scientists due to the increase on bacteria resistance in hospital environment. Whitin this context, the biofilm formation can be observed through endogenous (f. e. cystic fibrosis3 and osteomyelitis4) as well as exogenous (f. e. catheters5 and implants5) origin, both as an aspect supporting the most important generalized bacterial injuries. Therefore, this study aims at the investigation of the antimicrobial and antibiofilm potentials from fungi associated to marine organisms. Forty-two fungi associated to different marine organisms from South Brazil coast were isolated employing distinct culture media. The initial screening was carried out with microorganisms cultivated during seven, fourteen and twenty one days in Sabouraud broth on static mode. The liquid medium and mycelia were separated by vacuum filtration and kept frozen; the mycelia extracts were then obtained using ethyl acetate as solvent extractor by turbolysis. The antimicrobial activity was evaluated employing liquid

medium samples and mycelia extracts against Staphylococcus epidermidis ATCC 35984. The potential activity towards biofilm formation and degradation of the biofilm previously formed was tested only with the liquid medium samples. The agar diffusion test and the cristal violet methods were used for the screening tests. Results showed antibacterial activity for two liquid medium and for at least eight mycelia extracts, mainly on fourteen and twenty one fermentation days. As to the activity on the biofilm formation, twelve liquid medium presented positive effects, and five promoted the degradation of the previously formed biofilm, both with less than forty per cent of inhibition of the formation and degradation. In summary, these results encourage us to doing further research into the chemical composition characterization for these compounds.

1. König, G. M. et al ChemBioChem. 7:229-238, 2006.

2. Blunt, J. W. et al. Nat Prod Rep, 25:35-94, 2008.

3. Moreau-Marquis, S. et al. Pulm Pharmacol Ther. 21:595–599, 2008.

4. Brady, A.R. FEMS Immunol Med Microbiol. 52:13–22, 2008.

5. Donlan, R.M. Emerg. Infect. Dis.7(2):277-281, 2001.

Financial support: CNPq


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BIOASSAY GUIDED DISCOVERY OF ANTI-DIABETIC AGENTS FROM MARINE INVERTEBRATES

Steinar M Paulsen, Marte Albrigtsen, Jeanette Hammer Andersen, Trond Jørgensen

MabCent-SFI, Tromsø Science Park, University of Tromsø, NO-9037 Tromso, Norway

[email protected]

Eating is essential to life, and its episodic nature requires physiological adaptations to avoid excess or insufficiency in circulating fuels, especially glucose and lipids. Our modern lifestyle with an increasing imbalance between energy intake and energy expenditure, often resulting in obesity, is a challenge to this fine tuned energy adaptation. Chronic disruption of the energy balance causes plasma glucose imbalance, hypertrophy and hyperplasia of adipocytes causing metabolic disorders such as type 2 diabetes mellitus (T2DM). A number of potential drug targets have been identified and investigated with respect to treatment of metabolic syndromes and T2DM.

Developed and released drugs have revealed moderate efficiency and many have shown low specificity with adverse effects. The drug screening campaign at MabCent is focused mainly on three targets: 1) Potentiators of glucose uptake, the enzyme protein tyrosine phosphatase PTB-1B, peroxisome proliferator-activated receptors regulating the expression of genes involved in the control of lipid metabolism, glucose homeostasis and inflammatory processes. Our screening campaigns rely on both cell based assays and isolated target. Screening strategy and initial results will be presented.


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STUDY ON BIOACTIVITY OF EXTRACTS FROM PORTUGUESE MARINE SPONGE OF ERYLUS GENUS

A. Guedes1, V. Cachatra1, M. Humanes1, J. Xavier4, J. M. Nogueira1, BIOALVO2, H. Gaspar3, S. Santos1

1 Centro de Química e Bioquímica, DQB, FCUL, Lisboa, Portugal 2 BioAlvo, Edifício ICAT, Campus da FCUL, Lisboa, Portugal

3 Instituto Nacional de Engenharia, Tecnologia e Inovação, Lisboa, Portugal 4 Institute for Biodiversity and Ecosystem Dynamics and Zoological Museum of Amsterdam, University of Amsterdam, Netherlands and CIBIO - Pólo Açores, Centro de Investigação em

Biodiversidade e Recursos Genéticos, and Departamento de Biologia, Universidade dos Açores, Ponta Delgada, Portugal.

[email protected]

The enzyme indoleamine 2,3-dioxygenase (IDO), which catalyzes the first and rate-limiting step of Kynurenine pathway (KP), the major route of tryptophan catabolism, has attracted special attention from scientific community due to its contribution to the escape of tumors from the host’s immune response, and its role in amyotrophic lateral sclerosis, AIDS, Alzheimer’s disease, cerebral malaria, etc.1 The search for IDO inhibitors, definitively appears as a research niche worth focusing on.

Marine sponges of the genus Erylus are a source of various classes of metabolites, namely saponins belonging to the steroidal or triterpenoid series and glycollpids, some of which have antileukemic activity or activity against Ehrlich carcinoma cells2. In view of these facts we decided to access the bioactivity of the extracts from two Portuguese sponges from Erylus genus: Erylus sp, collected in the Gorringe Bank and Erylus

discophorus collected of Berlengas islands. Organic crude extracts of Erylus sp proved to be active as IDO inhibitor in concentrations below 0.1mg/mL, using the Global Platform Screening for Drug Discovery (GPS D2) technology developed by BIOALVO. Bioassay-guided fractionation of these crude extracts afforded three fractions which kept the same level of activity. The less polar fraction proved to be a mixture of steroids commonly found in marine sponges: 24-methylene-cholesterol, cholesterol, β-sitosterol, brassicasterol. The same chemical profile was also found in the organic extracts of Erylus discophorus.

1. Y. Chen, G. J. Guillemin, International

Journal of Tryptophan Research, 2009, 2, 1-19.

2. A. S. Antonov, A. I. Kalinovsky, V. A. Stonik, S. S. Afiyatullov, D. L. Aminin, P. S. Dmitrenok, E. Mollo, G. Cimino, Journal of Natural Products, 2007, 70, 169-178.


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FUNGICIDAL ACTIVITY OF OLIGOMER CHITOSAN TO INHIBIT PLANT PATHOGENIC FUNGI

Narong Singburaudom

Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.

[email protected]

The efficiency of chitosans to inhibit plant pathogenic fungi, Sphaceloma ampelinum, were studied in vitro by using three difference molecular weight (MW) chitosans. 1% of chitosan solution was prepared by using 2% acetic acid as solvent and then it was diluted to different concentrations and tested for antimicrobial activity in vitro. The result of investigation on antifungal activity of acetic acid revealed that 2% acetic acid at concentration 100,000 ppm inhibited completely the growth of S. ampelinum, indicated by 0 value of optical density (OD) or 100% transparancy of culture medium, as compared to the lower concentration at 10,000 ppm it did not inhibit the growth of fungus that indicated by the same OD value as control treatment,1.2 and 1.1, respectively. The higher MW chitosan showed the better fungus inhibiting than lower MW chitosan, at concentration of 10000 ppm that gaved the lowest number of conidia 0.5x104 spores per ml, as compared to the lowest MW chitosan gave the number of conidia 52x105 spores per ml. The antifungal activity of commercial oligomer and natural polymer chitosan and natural alkaloid extracted from plant, Coscinium fenestratum were studied. The result of investigation indicated that the natural polymer chitosan and natural alkaloid showed the same inhibiting the

fungus at concentration 10,000 ppm and the higher MW chitosan showed the better inhibiting than lower MW chitosan. Natural alkaloid mixed chitosans did not increase inhibiting efficiency of the fungi as compared to alkaloid alone treatment. The higher MW chitosan mixed alkaloid exhibited the higher inhibiting at concentration of 10,000 ppm. The efficiency of oligomer chitosans were tested for inhibiting plant pathogenic fungi in vitro and in vivo. The result indicated that chitosans at concentration 10,000 ppm inhibited the mycelial growth 26.7% by average from all tested fungi in this experiment. Lower MW chitosans showed the better inhibiting than the higher MW chitosans. Natural polymer chitosan could not inhibit the anthracnose fungus, Colletotichum gloeosporioides, when infected mango fruits were dipped into chitosan solution at concentration 10,000 ppm. Sraying with oligomer chitosans onto maize plants to protect the fungus, Helminthosporium turcicum, the causal agent of northern corn leaf blight disease indicated that the lower MW chitosans exhibited the better inhibiting than higher MW chitosans. The investigation suggested that high MW chitosans have the higher potential to inhibit the plant pathogenic fungi in vitro where the lower MW chitosans exhibited more potential to inhibit the fungi in vivo.


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BIOLOGICAL ACTIVITY AND COMPOUNDS OF MARINE-DERIVED FUNGI ISOLATED FROM TROPICAL OR SUBTROPICAL SEAS

Kustiariyah Tarman1, Ulrike Lindequist1, Kristian Wende1, Michael Lalk1, Martina Wurster1, Beate Cuypers2, Gudrun Mernitz2

1 Department of Pharmaceutical Biology, Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Germany

2 RessourcenZentrum Marine Organismen GmbH, Greifswald, Germany

[email protected]

Twelve marine-derived fungi have been isolated from tropical or subtropical seas around Indonesia and Chile and screened for their biological activities. Extracts of culture broth of the marine-derived fungi exhibited considerable cytotoxic activity against cultivated human 5637 cells, a urinary bladder carcinoma cell line, as well as antimicrobial activity against human and fish pathogenic bacteria. In order to optimize the production of bioactive compounds the fungi were cultivated in media of different salinity. Nine strains exhibited more bioactive compounds if cultivated under low salinity. Predominantly, ethyl acetate extracts were the most active against the test organisms. Bacillus subtilis and Vibrio anguillarum were the most sensible test organisms representing human- and fish pathogenic bacteria, respectively.

The most active strain was KT27 with MIC 125 µg/ml against B. subtilis and 15.6 µg/ml against V. anguillarum. The lethal cytotoxic concentration (LD50) of the ethyl acetate extracts from strain KT27 and KT30 was 4 µg/ml for both strains, KT13 and KT19s (cultivated with marine salt) showed medium activity (approximately 40 µg/ml) and the rest was weak toxic or did not show activity. A chromane derivative has been isolated from KT13 and its structure has been elucidated. Keywords: antimicrobial, cytotoxic, marine fungi, marine natural product


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PHYTOCHEMICAL AND PHARMACOLOGICAL STUDY OF THE BIOACTIVE SEAGRASS THALASSIA TESTUDINUM

Eric L. Regalado1, M. Rodríguez1, A.R. Concepción2, J.A. Pino3, C. Nogueiras4, L. Laguna1, Olivier P. Thomas5, O. Valdés2 and V. Bueno2

1 Centro de Bioproductos Marinos, Loma y 37, Alturas del Vedado, La Habana, Cuba. 2 Centro Nacional de Genética Médica, La Habana, Cuba.

3 Instituto de Investigaciones para la Industria Alimenticia, La Habana, Cuba 4 Centro de Productos Naturales (CPN ), Facultad de Química, Universidad de la Habana, Cuba

5 University of Nice Sophia Antipolis, LCMBA - UMR 6001 CNRS, Nice, France

[email protected]

The incidence of various disorders related to exposure to solar ultraviolet radiation has increased in the last years. Among several effects, UV exposure to the skin results in generation of reactive oxygen species and inflammatory responses which lead to skin damage. Thus, one approach to protect human skin against the harmful effects of UV irradiation is the use of naturally occurring herbal compounds with antioxidant and/or anti-inflammatory effects. Topical application of the hydro-alcoholic extract of Thalassia testudinum (BM21) (once a day during 7 days) following UVB exposure results in a dose-effect inhibition of UVB-induced skin erythema, scabs, roughness and wrinkly of the skin which was confirmed by histological studies. A bioassay-guided separation of BM21 by means of the antioxidant and skin regenerating assays combined with extensive separation techniques allowed us to isolate and identify thalassiolin B1, which was found to scavenge DPPH radical and showed strong skin regenerating activity.

On the other hand, the chemical profile of the volatile constituents of this plant obtained by GC and GC/MS analyses allowed us to identify and quantify 143 compounds (more than 95% of the composition) and revealed the high content of ethyl (Z)-1-propenyl disulfide (31%), methyl (Z)-1-propenyl disulfide (2.8%) and methyl (E)-1-propenyl disulfide (2.6%). The sulfur organic compounds constitute a diverse and important subdivision of organic substances, among them, a large number of sulfur compounds have been widely used as sulfa drugs and dermatological agents. Thus, taking into account the biological results combined with the chemical analyses on the volatile and non volatile constituents of this plant, BM21 could be a great candidate as bioactive principle for the development of cosmetic product formulations.

1. Rowley DC, Hansen MS, Rhodes D, Sotrifer

CA, Ni H, Mccammon JA, Bushman FD, Fenical W (2002). Thalassiolins A–C: new marinederived inhibitors of HIV cDNA integrase. Biorgan Med Chem 10: 3619–3625.


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BIOPROSPECTION OF CYTOTOXIC COMPOUNDS IN THE NORTHEASTERN COAST OF BRAZIL: CELL CYCLE ARREST AND APOPTOSIS INDUCTION OF

LEUKEMIC CELLS TREATED WITH A NOVEL STAUROSPORINE ANALOG FOUND IN THE ENDEMIC TUNICATE EUDISTOMA VANNAMEI

Paula C. Jimenez1, D.V. Wilke1, E.G. Ferreira1, C.O. Pessoa1, M.O.Moraes1, E.R. Silveira3, N.P. Lopes2 and L.V. Costa‐Lotufo1

1 Depto. de Fisiologia e Farmacologia – UFC, Fortaleza, Ceará, Brasil; 2 Depto. de Física e Química – FCFRP, USP, São Paulo, Brasil;

3 Depto. de Química Orgânica e Inorgânica – UFC, Brasil.

[email protected].

Introduction: Eudistoma vannamei Millar, 1977 is an endemic tunicate from the northeastern Brazilian coast, widely distributed over the rocky beaches of Ceará State. Previously, the crude extract showed an interesting bioactivity profile. Bioassay-guided fractionation yielded a highly cytotoxic alkaloid (STPD’), which was identified as a novel close analog to staurosporine (STP). This study reveals a kinetic analysis of the novel alkaloid on cell cycle progression and apoptosis induction of HL-60 cells (leukemia), in comparison to STP.

Methods: IC50 for STPD’ and STP was obtained after 72h incubation on HL-60, HCT-8, MDA MB-435 and SF-295 cell lines using the MTT assay and in normal human leucocytes, by the Alamar Blue assay. Morphological analysis of treated or untreated cells was carried out by H/E staining. Cell count, viability, cell cycle studies and evaluation of apoptosis features were accessed by flow cytometry and western blotting. Cells were treated and analyzed after 24, 48 and 72h incubation. Reversibility of the effect was evaluated after 24h drug treatment followed by 24h drug-free incubation.

Results and Discussion: Tumor cell lines were 5 to 20 times more susceptible to STPD’ than STP, while normal cells showed less, however equal, susceptibility to both compounds. Cell cycle studies indicate that STPD’ induces a G2-M arrest (at 40ng/mL, 45, 63 and 94% of arrested cells after 24, 48 and 72h treatment, respectively, against 9, 10 and 13% for the non-treated culture). Moreover, G2-M arrest effect is irreversible following removal of STPD’. STP induces 83% G2-M arrest at 200ng/mL after 24h incubation, while longer incubation periods will provoke a substantial increase in polyploidy. Expression-rate of cell cycle related proteins (Cdc2p34, Cdk2, cyclin A and cyclin B1) paired with morphological analysis of STPD’-treated cells placed on glass slides suggest that arrest is actually occurring in G2 phase. A slight decrease in viability and cell count and an increase in sub-G1 population of 40ng/mL STPD’-treated cells will occur significantly only after 72h incubation. Anexin-7AAD double-stained cells show somewhat higher, though significant, apoptotic features only after 72h treatment with 40ng/mL STPD’. Support: FINEP, CAPES, CNPq and InCB.


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THE IN VITRO ANTAGONISTIC EFFECT OF MARINE SPONGE-ASSOCIATED FUNGI AGAINST PLANT PATHOGENIC FUNGI Tida Dethoup1, Leka Manoch1, Jamrearn Buaruang2,

Siangjeaw Piriyaprin3 and Anake Kijjoa4

1 Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand

2 Division of Environmental Science, Faculty of Science Ramkhamhaeng University Huamark, Bangkok 10240, Thailand

3 Land Development Department, Ministry of Agriculture and Cooperatives, Bangkok10900, Thailand

4 Instituto de Ciências Biomédicas de Abel Salazar and CIIMAR, Universidade do Porto, 4099-003 Porto, Portugal

[email protected]

Fungi from the marine environment have shown great potential as an important source of pharmacologically active metabolites and biological activities of their metabolites are mainly focused in the area of antibiotic and anticancer properties, and in lesser extent in other selective activities such as cell cycle inhibition, antagonism of platelet activating factors, antiviral activity and radical scavenging activity1. However, the antagonistic activity of the marine-derived fungi on other microorganisms has not been extensively explored. Consequently, we have investigated the antagonistic activity of the marine sponge-associated fungi against plant pathogenic fungi. Now, we report the antagonistic activity of the marine-derived fungi Chaetomiun globosum, C. minutum, Curvularia lunata, Emericella variecolor, Eupenicillium parvum, Menmoniella echinata, Nigrospora sp., Nodulisporium sp., Penicillium sp. and Speggazzinia tessarthra, isolated from the marine sponges Mycale armata, Haliclona sp. and Chalinula sp., collected in the Gulf of Thailand, on ten plant pathogenic fungi (Alternaria alternata, Colletotrichum capcisi, C. gloeosporioides, Fusarium oxysporum, Helminthosporium oryzae, Lasiodiplodia theobromae, Phytophthora palmivora, Pythium aphanidermatum, Rhizoctonia solani and Sclerotium rolfsii), using a dual culture of the sponge-

associated fungi and the plant pathogenic fungi on PDA for 14 days at 28 oC. The results showed that all of the sponge-associated fungi could inhibit the mycelial growth of Alternaria alternata, Colletotrichum capcisi, Fusarium oxysporum, Helminthosporium oryzae and Phytophthora palmivora. While Emericella variecolor, Nodulisporium sp., Chaetomiun globosum and Penicillium sp. effectively inhibited the mycelial growth of Colletotrichum gloeosporioides, the rest of fungi produced only a moderate inhibition of the radial growth of this plant pathogen. Interestingly, none of the fungi tested were able to control Pythium aphanidermatum, Lasiodiplodia theobromae, Rhizoctonia solani and Sclerotium rolfsii. Acknowledgements: The authors wish to thank Kasetsart University Research Development Institute (KURDI) and Thailand Research Fund (TRF) for the support this project.

1. Bugni, T.S., Ireland, C. (2004). Marine-derived fungi: a chemically and biologically diverse group of Microorganisms. Nat. Prod. Rep.21, 143-163.


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SCREENING FOR COMPOUNDS FROM ARCTIC MARINE INVERTEBRATES WITH ANTI-CANCER ACTIVITIES AT MABCENT-SFI

Maria Perander1, Trine Stiberg2, Jonas Eriksson3, Ole Morten Seternes1, Jeanette Hammer Andersen2, Trond Ø. Jørgensen1.

1 MabCent-SFI and 2 Marbio, Tromsø Science Park, University of Tromsø, 9037 Tromsø; 3 Lytix BioPharma AS,Tromsø Science Park, 9294 Tromsø, Norway

[email protected]

MabCent-SFI is a research-based innovation centre on marine bioactives and drug discovery, hosted by the University of Tromsø, Norway. The main objective is to identify and purify biological active compounds from Arctic and sub-Arctic marine organisms.

The goal of the anti-cancer screening project at MabCent-SFI is to identify compounds that kill cancer cells or inhibit the progression of cancer. Two strategies to identify drugs with potential anticancer activities are used. The first strategy is a classical approach where marine extracts are screened for cytotoxic or cytostatic activities towards a panel of cancer cell lines.

The second strategy is to target signaling pathways or proteins with important functions in cancer initiation and progression. Biochemical and cell-based assays are currently being employed or developed to identify inhibitors of cancer cell growth, the NF-κB signaling pathway, and members of the protein kinase super family.


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TOXICITY AND ANTIBACTERIAL ACTIVITIES OF ANTARCTIC MARINE NATURAL PRODUCTS:

PRELIMINARY RESULTS FROM THE ACTIQUIM PROJECT Jennifer Vázquez1*, Blanca Figuerola1, Sergi Taboada1, Francisco Javier Cristobo2,

Laura Núñez‐Pons1, Conxita Avila1 1 Departament of Animal Biology (Invertebrates), Faculty of Biology, Univesity of Barcelona, Av.

Diagonal 645, 08028 Barcelona, Spain. 2 Ministerio de Ciencia e Innovación, Instituto Español de Oceanografía. Centro Oceanográfico de

Gijón, C/ Príncipe de Asturias 70 bis, 33212 Gijón, Asturias, Spain.

* [email protected]

The aim of the ACTIQUIM project is to determine the ecological activity of the marine natural products obtained from Antarctic benthic organisms by doing in situ chemical ecology assays. The experimental work took place in the Spanish Antarctic Base “Gabriel de Castilla” at Deception Island (South Shetland Islands, Antarctica) during the Austral Summer of 2008-2009. The experiments were performed using ethereal extracts from different benthic invertebrates collected in a previous campaign (ECOQUIM project, 2003-04), from the Eastern Weddell Sea and the vicinities of Bouvet Island. These invertebrates belong to different phyla: Porifera, Cnidaria, Bryozoa, Echinodermata, Chordata and Hemichordata. When possible, invertebrate samples were divided into parts to evaluate the location of active compounds within the organisms. Extracts were tested to prove their potential activity against different organisms collected by scuba diving at different points of Deception Island. The objective of the assays was to determine 1) their cytotoxicity in different moments of the life cycle of a common Antarctic sea urchin (Sterechinus neumayeri); 2) their toxicity against a common copepod; and 3) their antifouling activity against a bacteria from the marine sediment.

The preliminary results of the three experiments we are presenting are:

1) Cytotoxicity in different moments of the development of Sterechinus neumayeri: in 10 out of the 14 species assayed the blastula stage of the life cycle of the sea urchin did not develop.

2) Toxicity against copepods: 24 different species from 6 different phyla were tested. More than 30% of the extracts displayed significant toxic activity against the copepods.

3) Antibacterial activity: using a paper disc diffusion method in marine agar, 30 different species were screened against a bacterial strain isolated from the marine environment. Of these, 8 species exhibited some type of antibacterial activity.

More experiments with other species extracts and also with isolated compounds will be performed next season (2009-10) to further develop the studies presented here.


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MARINE SPONGE DEPSIPEPTIDE INCREASES GAP JUNCTIONS LENGTH IN HTC CELLS TRANSFECTED WITH CX-43-GFP

Marisa Rangel1, S.C. Pfister2, M. Ionta2, R.F. Piva2, R.A.S. Ferreira2 and G.M. Machado‐Santelli2

1 Immunopathology Laboratory, Butantan Institute, Sao Paulo, Brazil; 2 Department of Cell Biology and Development, Biomedical Sciences Institute, University of Sao

Paulo, Sao Paulo, Brazil. [email protected]

Connexins are membrane proteins that form gap junction channels between adjacent cells. Connexin 43 (Cx43), the most widely expressed member of the connexin family, has a rapid turnover rate and its degradation involves both the lysosomal and ubiquitin-proteasome pathway.

The goal of this work is to study the effects of Geodiamolides, natural peptides from marine sponge that disrupt microfilaments (Rangel et al., 2006), in connexin assembly or degradation in plasma membrane.

Hepatocarcinoma cell line (HTC) expressing Cx43-GFP were submitted to treatment with Geodiamolides A, B, H and I solutions at 100nM concentration (2 h). Microfilaments and nuclei were also stained, and all analyses were performed under a confocal laser scanning microscope. The gap junctions length was measured in control and treated cells, and ANOVA followed by a Newman-Keuls multiple comparison test was performed.

Amongst the four peptides tested, only Geodiamolide H statistically enhanced the gap junctions channels length. Geodiamolide A seemed to have a less pronounced effect, but it was not significant.

According to our results, the treatment with Geodiamolide H could interfere with the delivery of connexins to the degradation structures, similar to proteasomal pathways, keeping the connexins assembled and accumulating gap junction plaques. Further experiments with the cells treated with Geodiamolide H, using the fungal antibiotic Brefeldin A (BFA), will be performed in order to uncouple events leading to gap junction assembly from those related to gap junction removal (Laird et al., 1995), since BFA is known to block protein trafficking within a fused ER/Golgi compartment.

1. Rangel M, Prado MP, Konno K, Naoki H, Freitas J C, Machado-Santelli GM. Cytoskeleton alterations induced by Geodia corticostylifera depsipeptides in breast cancer cells. Peptides, v. 27, p. 2047-2057, 2006.

2. Laird DW, Castillo M, Kasprzak L. Gap junction turnover, intracellular trafficking, and phosphorylation of Connexin-43 in Brefeldin A-treated rat mammary tumor cells. J. Cell. Biol., v. 131, n. 5, p. 1193-1203, 1995.

Financial support: FAPESP (03/13207-0)


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ATTEMPTS TO IDENTIFY NATURAL ANTIOXIDANTS BEARING DNA PROTECTION FEATURES, PRODUCED BY SCENEDESMUS OBLIQUUS

A. Catarina Guedes1, Helena M. Amaro1, Ricardo D. Pereira1, Rui Seabra2, Paula Tamagnini2,4, Pedro Moradas‐Ferreira2,3 and F. Xavier Malcata

1 Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal;

2 IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, P-4150-180 Porto, Portugal;

3 Faculdade de Ciências, Universidade do Porto, Departamento de Botânica, Edifício FC4, Rua do Campo Alegre, s/nº P-4169-007 Porto, Portugal;

4 ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Largo Abel Salazar 2, P-4099-003 Porto, Portugal

[email protected]

The latest decade has witnessed a growing interest for compounds possessing antioxidant properties, and which can be obtained from natural sources – as such natural compounds can play relevant roles upon health, via ingestion as part of one’s diet. Evidence gathered through a large number of studies has indeed supported the hypothesis that antioxidants help prevent and control growth of certain tumors, as well as incidence and severity of cardiovascular and degenerative diseases. Supplementation of normal foods with natural antioxidants (e.g. like carotenoids) will thus be beneficial to the consumer, in terms of active contribution toward his health condition (Goldberg, 1996; Guerin, 2003).Microalgae and cyanobacteria are potential sources of the aforementioned (high-added value) antioxidant ingredients – and they hold the further advantage that they are autotrophic (and so not energy-demanding) and can be cultivated in large-scale bioreactors (and so economically feasible).The major aim of this research effort was to find (novel) antioxidant features in intracellular extracts of a microalga, Scenedesmus obliquus (M2-1) – which had revealed the highest intracellular antioxidant capacity in previous comprehensive screenings, particularly upon DNA integrity.

Hence, the antioxidant and pro-oxidant capacity of several amounts of said microalgal extract were analyzed. No pro-oxidant effect was observed, and all extracts exhibited antioxidant activity; the strongest DNA protection was provided by 200 µL of extract. Our experimental results, supplemented by available bibliography, raised the possibility that carotenoids were the main responsible for the in vitro DNA protection effect. To check whether that situation held, carotenoids were extracted and analysed by HPLC; the major compounds identified were lutein (2.69 ± 0.09 mglutein /g microalga), neoxanthin (0.56 ± 0.02 mglutein eqivalent / gmicroalga), β-carotene (0.40 ± 0.03 mglutein

equivalent /g microalga),, and violaxanthin (0.14 ± 0.01 mglutein equivalent /g microalga). Our microalga has therefore a rich content of lutein and neoxanthin, as well as relevant amounts of violaxanthin and β-carotene – which may account for, at least in part, its antioxidant capacity.

1. Goldberg, I. 1996. Functional Foods: designer foods, pharmafood, nutraceuticals. Chapman and Hall: London, UK: p. 3.

2. Guerin, M., Huntley, M. E., Olaizola, M. 2003; Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol. 21: 210-215.


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ANTIFUNGAL ACTIVITIES OF THE CRUDE EXTRACTS OF MARINE SPONGE-ASSOCIATED FUNGI AGAINST PLANT PATHOGENIC FUNGI

Leka Manoch1, Tida Dethoup1, Jamrearn Buaruang2, Siangjeaw Piriyaprin3, and Anake Kijjoa4

1 Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand

2 Division of Environmental Science, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand

3 Land Development Department, Ministry of Agriculture and Cooperatives, Bangkok10900, Thailand

4 Instituto de Ciências Biomédicas de Abel Salazar and CIIMAR, Universidade do Porto, 4099-003 Porto, Portugal

[email protected]

Recently much of attention has been paid on marine sponge-associated fungi as an important source of biologically active secondary metabolites. In this context, our group has started to investigate the compounds produced by these organisms as a potential arsenal for the plant pathogen. Consequently, we have isolated the marine fungi associated with the marine sponges Clathria reinwardti and Xestospongia testudinaria, collected from the Gulf of Thailand near Ko Samaesan, Chonburi province. Among the isolates, we have identified five marine fungi, namely Emericella variecolor, Eurotium cristatum, Curvularia lunata, Cladobotyum varium and Acremonium sp. These fungi were cultured on the malt extract agar media with 70% of sea water. In order to evaluate the biological activity of the fungal metabolites, the ethyl acetate crude extracts of these fungi were obtained and tested against 10 plant pathogenic fungi: Phytophthora palmivora, Pythium aphanidermatum, Fusarium oxysporum, Helminthosporium oryzae, Alternaria alternata, Colletotrichum capcisi, C. gloeosporioides, Lasiodiplodia theobromae, Rhizoctonia solani and Sclerotium rolfsii.

Preliminary results showed that the crude extracts of these marine-associated fungi were selective against the plant pathogenic fungi tested. Thus, at 10,000 ppm concentration, the crude extract of Curvularia lunata effectively inhibited (70-74%) mycelium growth of Alternaria alternata, Rhizoctonia solani and Colletotrichum gloeosporioides whereas the crude extract of Eurotium cristatum could inhibit mycelium growth (42-45%) of Phytophthora palmivora, Pythium aphanidermatum, Colletotrichum capcisi and Sclerotium rolfsii. However, while the crude extracts of Emericella variecolor and Acremonium sp. were found to inhibit the mycelium growth (38% and 40%) of Lasiodiplodia theobromae and Fusarium oxysporum respectively, the crude extract of Cladobotyum varium was found to be inactive against all the fungi tested.

Acknowledgements: The authors wish to thank Kasetsart University Research Development Institute (KURDI) and Thailand Research Fund (TRF) for the support this project.


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ANTIBACTERIAL ACTIVITY OF COMPOUNDS FROM MARINE ALGAL-DERIVED ENDOPHYTIC FUNGI

Cíntia Erbert1, A.L.L. Oliveira1, R. Felício1, N.A.J.C. Furtado1, R. Conti1, M.T. Pupo1, J.L.C. Lopes1 and H.M. Debonsi1

1 Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Ribeirão Preto, SP, Brazil

[email protected]

Marine organisms constitute a source of potential bioactive substances that can be converted into new drugs, with an interesting array of metabolites and diverse biological activities (Hill 2007). Given this context, strains of endophytic fungi isolated from two species of red seaweed genus Bostrychia, B. radicans and B. tenella, were studied to verify their chemical and biological properties. The algae were collected in a rocky shore (B. radicans and B. tenella) and mangrove (B. radicans) located in Ubatuba vicinity, São Paulo State, Brazil. Several strains of endophytic fungi were obtained from sterilized, sectioned portions of thallus from the samples. Nine of these strains were selected according to morphological aspects and cultivated in rice solid medium. The mycelia masses were submitted to extraction with methanol and subsequently fractioned using hexane and acetate. The fractions were submitted to antimicrobial activity against Staphylococcus saprophyticus (ATCC6538) and S. aureus (ATCC15305), two strains of bacteria that cause serious hospital infections.

As a result, four strains were active against S. aureus (MIC values of 350, 300, 50 and 30 µg/mL) and two were active against S. saprophyticus (MIC values of 70 and 40 µg/mL). The presence of activity is very interesting in the case of concentrations below 100 µg/mL (Ríos & Recio 2005). These active fractions were analyzed by GC-MS (DB-5 column), and the class of compounds were identified by comparison with mass spectra library. The major chemical constituents from more active samples are mainly steroidal and aromatic derivatives, beside halogenated and nitrogenated compounds. These data can be seen as a first step that can lead to the discovery of promising metabolites against bacterial infections.

Acknowledgements: BIOPROSPECTA-FAPESP, CNPq, CAPES

1. HILL, R.A. Annu. Rep. Prog. Chem., Sect. B 2007, 103, 125

2. RÍOS, JL & Recio, MC Journal of Ethnopharmacology 2005, 100, 80


121

PC 50

ANTIMICROBIAL ACTIVITY OF SEA FAN-DERIVED FUNGI Sita Preedanon1, Jariya Sakayaroj2, Sakanan Plathong3, Vatcharin Rukachaisirikul4 and Souwalak Phongpaichit1*

1 Department of Microbiology and Natural Products Research Center,, Faculty of Science, Prince of Songkla University, Songkhla, Thailand.

2 Phylogenetics Laboratory, National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathum Thani, Thailand.

3 Centre for Biodiversity of Peninsular Thailand, Department of Biology, Faculty of Science, Prince of Songkla University, Songkhla, Thailand.

4 Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Songkhla, Thailand.

[email protected]

A total of 163 fungi isolated from gorgonian sea fans from the south of Thailand were investigated. Crude ethyl acetate extracts of the culture broth and crude hexane and ethyl acetate extracts of the fungal mycelium were tested for their antimicrobial activity by microdilution method. One hundred and eight out of 337 (32%) extracts from 77 fungal isolates (47%) exhibited antimicrobial activity against at least one test microorganism. Crude ethyl acetate extract of the culture broth of Nigrospora sp. F13 showed strongest antifungal activity against M. gypseum (MIC 1 µg/ml) comparable to standard drug miconazole.

Two new compounds, derivatives of dechlorogriseofulvin and chlorogriseofulvin, were isolated from Nigrospora sp. F13. Griseofulvin and dechlorogriseofulvin had antifungal activity against M. gypseum with MIC values of 2 and 32 µg/ml, respectively. The results indicated that sea fan-derived fungi are a potential source of new compounds and antimicrobial agents.


122

PC 51

EVALUATION OF SAMPLES FROM 4 ALGAE FROM THE BRAZILIAN COASTFOR ANTIOXIDATIVE ACTIVITY

Fabíola D. Rocha, Angélica R. Soares, Peter J. Houghton, Renato C. Pereira, Valéria L. Teixeira and Maria Auxiliadora C. Kaplan.

Universidade Federal de Minas Gerais, Brasil

[email protected]

Antioxidant activities of samples from 4 seaweeds, Stypopodium zonale, Caulerpa racemosa, Lobophora variegata and Spatoglossum schroederii, collected from the Brazilian Coastline, were evaluated for their antioxidant activity (AA), using several in vitro assay systems. The samples were found to have different levels of AA properties in the models tested. The radical scavenging activity against 2,2-diphenyl-1-picrythydrazyl (DPPH) was higher to S. zonale (EDC STY BU and EDC STY NO) extracts and Aqueous phase from L. variegata (Aq Phase LOB). While the other samples, in front of the quantitative DPPH test showed no significant activity, but the autobiography showed yellow spots on the TLC plates, revealed with DPPH solution (0.2% w/v in MeOH), representative of the presence of antioxidant substances. Most of tested samples displayed a profile similar to the kinetics of reaction of Trolox in the TEAC (Trolox equivalent antioxidant capacity) assay, which means, the major effect on the inhibition of absorbance at 734 nm appears rapidly in the first 30 seconds of reaction, continuing with a lower speed and then becomes constant. Only the Aq Phase LOB showed no such profile. For this sample, the decrease in absorbance was gradual throughout the time of reaction and does not finish the final 5 minutes of reaction. This fact is important to be considered, this may indicate an antioxidant

mechanism of action different from that of the Trolox and the other samples. The best results in terms of value of TEAC were to EDC STY BU (0.54), EDC STY NO (0.45), and Fase Aq LOB (0.42). The following hierarchy of AA on the inhibition of 2-thiobarbituric acid-reactive substances (TBARS) formed during the lipid peroxidation induced by the Fe (III) / ascorbate system in lipossomes of bovine brain, was found: STY (BU) (EC50 = 0.0035 mg/ml), EDC STY (NO) (EC50 = 0.023 mg/ml), Aq Phase LOB (EC50 = 1.0 mg/ml), extract in acetone from C. racemosa, EMeCO CR, (EC50 = 1.6 mg/ml) and extract in acetone from L. variegata, EMeCO LOB, (EC50 = 2.1 mg/ml). The test for evaluating the protective effect of samples on the normal cellular function of fibroblasts, exposed to oxidative stress induced by H2O2, the best results were observed for the EMeCO LOB and the Aq Phase LOB, and the maximum protective effect was observed for the lower concentrations tested, 3.12 and 6.25 g/ml, while in higher concentrations there was no protective effect against the deleterious effects of H2O2. The results indicated that Stypopodium zonale, Caulerpa racemosa and Lobophora variegata possess antioxidant activity to various degrees and appear useful in leading to the development of therapeutic products to protect against certain diseases.


123

PC 52

GRISEORHODIN A: BIOSYNTHETIC STUDIES AND COMBINATORIAL BIOSYNTHESIS

Kathrin Reinhardt1, Zeynep Yunt1, Minna Eklund1, Zhongli Xu2, Christian Hertweck2, Torsten Bruhn3, Gerhard Bringmann3 and Jörn Piel1

1 Kekulé-Institute of Organic Chemistry and Biochemistry, University of Bonn, Germany. 2

Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology-Hans-Knoell-Institute (HKI), Jena, Germany,

3 Institute of Organic Chemistry, University of Würzburg, Germany

[email protected]

Griseorhodin A belongs to the rubromycin family of aromatic polyketides. The unique feature of rubromycins is a spiroketal moiety that causes it to adopt a nonplanar shape. Due to this structure the rubromycins are strong inhibitors of human telomerase and retroviral reverse transcriptase.1, 2 Griseorhodin A belongs to the most heavily oxygenated aromatic polyketides and is characterised by its epoxyspiroketal moiety, which is crucial for telomerase inhibition. We have previously cloned, sequenced and heterologously expressed the entire griseorhodin A gene cluster in Streptomyces albus.3

In order to obtain new rubromycins with improved pharmacological activity and better application attributes such as a higher solubility, a deeper insight into the formation of the spiroketal is needed. Hence the functions of various tailoring enzymes were investigated by Red/ET-mediated deletion of single genes and subsequent heterologous expression in Streptomyces albus.4 Via this technique we were able to generate and explore 20 modified gene clusters. Metabolic analysis allowed for the assignment of 14 genes to various stages of griseorhodin A tailoring and pharmacophore generation.

Characterisation of the produced polyketides provided direct evidence that the pharmacophore is formed by cleavage of four carbon-carbon bonds. In the final step an epoxidation takes place catalyzed by an unprecedented oxidoreductase pair that utilizes a saturated substrate.5 The poster discusses current insights into the enzymology of this unusual pathway. In addition, the novel chimeric compound benarhodin A was obtained by combinatorial biosynthesis, using genes of the griseorhodin A and benastatin clusters. This work provides future perspectives for improving the pharmacological profile of pentangular polyketides by utilizing diverse oxidoreductases from the griseorhodin A gene cluster by engineered biosynthesis.

1. Bringmann, G.; Kraus, J.; et al. Eur J of Org Chem 2000, 2729-2734.

2. Ueno, T.; Takahashi, H.;et al. Biochemistry 2000, 39, 5995-6002.

3. Li, A.; Piel, J. Chem. Biol. 2002, 9, 1017-1026.

4. Lackner, G.; Schenk, A.; et al. J. Am. Chem. Soc. 2007, 129, 9306-9312.

5. Yunt, Z.; Reinhardt, K.; et al. J. Am. Chem. Soc. 2009, 131, 2297–2305.


124

PC 53

APPROXIMATION TO THE BIOSYNTHESIS OF BELIZEANOLIC ACID Tamara S. Vilches1, José G. Napolitano1, Antonio H. Daranas1,,

José J. Fernández1 and Manuel Norte1

1 Instituto Universitario de Bioorgánica “Antonio González”, Departamento de Química Orgánica, Avda Astrofísico Francisco Sánchez 2, 38206, La Laguna, Tenerife, España.

2 Departamento de Ingeniería Química y Tecnología Farmacéutica, Universidad de La Laguna, La Laguna, Tenerife, España.

[email protected]

Over the last 30 years, marine dinoflagellates have become a prolific source of new bioactive compounds. One of the most interesting groups of marine natural products isolated from dinoflagellates is formed by macrolides, macrocyclic lactones which typically possess a pattern of oxygenation, alkylation and dehydration along the primary aliphatic chain that is indicative of a polyketide biosynthetic origin.1

Recently, the identification of Belizeanolide, a new cytotoxic macrolide and its opened form (belizeanolic acid) from the culture media of Prorocentrum belizeanum has been published.2 Our research group have been interested in the study of the biosynthetic origin of marine natural products over the last two decades. Therefore belizeanolic acid is an excelent candidate to undertake such studies.

Herein, preliminary results on the biosynthesis of Belizeanolic acid through the incorporation of acetate units in Prorocentrum belizeanum will be discussed. The biosynthetic origin of this compound is being studied by means of the analysis of 13C-NMR quantitative spectroscopy.

1. Napolitano J. G., Hernández A., Norte M.,

Fernández J. J., Anti-Cancer Agents Med Chem 2009, 9,122

2. Napolitano J. G., Norte M., Padrón J. M., Fernández J. J., Hernández A., Angew Chem Int Ed. 2009, 48, 796

The authors thank the financiation of the MEC (2008CTQ-06754-C04-01/PPQ); T.V. to the ULL by the scholarship SEGAI-CajaCanarias and J. G. N. to the MEC by the Scholarship F. P. U. The strain of P. belizeanum was facilitated by S. Fraga, of the CCVIEO (Vigo, Spain).


125

PC 54

CHEMOGENETIC TOOLS FOR EXPLORING AND EXPLOITING FUNGAL CHEMISTRY

Katja M. Fisch, Rozida Mohd Khalid, Russell J. Cox

School of Chemistry, University of Bristol, UK

[email protected]

Current knowledge concerning the function of fungal polyketide synthases (PKSs) is very limited. This is due to the limited number of proven links between PKS gene clusters and natural products (less than 20 to date 1). Furthermore, the iterative use of PKS domains in the enzymes, makes predicting fungal PKS biosynthetic pathways severely challenging. Thus, up to now it has not been possible to predict the chemical structure of a natural product produced by a certain fungal PKS gene cluster from its sequence information alone. The diverse and chemically highly complex structures of sorbicillinoids, e.g. trichodermanone A-C isolated from a marine-derived Trichoderma saturnisporum2 (Figure 1) give a good model system to gain a deeper understanding of fungal polyketide biosynthesis.

Our proposed biosynthesis of the trichodermanones A-C involves up to three PKSs and a crucial oxygenase. A 50,000 clone gene library from T. saturnisporum was screened for non reducing PKS genes based on phylogeny and a gene cluster harbouring two PKS genes and several regulatory genes was isolated. A discussion of ongoing work on knockout and RNAi approaches to confirm the biosynthesis of the sorbicillinoids will highlight opportunities and technical challenges of modern fungal genome / chemistry interplay.

1. Cox, R. J., Org Biomol Chem 2007, 5, (13), 2010-26.

2. Neumann, K.; Abdel-Lateff, A.; Wright, A. D.; Kehraus, S.; Krick, A.; König, G. M., European Journal of Organic Chemistry 2007, 2007, (14), 2268-2275.

O

OHO

O

O

HO

O

R

R1

HO

H

H

A R = α-OH, R1 = CH3B R = β-OH, R1 = CH3C R = β-OH, R1 = H

O

OHO

HO

O

HO

O

D (3R, 5S, 6R; at C-7, C-8 only relative stereochemistry implied)

7 H7H

H

O

OO

OH

O

HO

O

O

HOOH

OH

OH

OOH

OOH

O

O OOH

OH

OO

OH

HO O

OO

OH

O

O

HO

OH

O OH

HO

TrichodermanoneVertinolide

Trichodimerol

Bislingiquinolide Bisvertinol Sorbicillinol

Rezishanone C

Figure 1 Sorbicillinoids found in Trichoderma saturnisporum and sorbicillinol, a proposed

biosynthetic intermediate


126

PC 55

MARINE-DERIVED FUNGI METABOLOMICS: CHEMICAL INVESTIGATION OF A PENICILLIUM WAKSMANII ZALESKI STRAIN

Marieke Vansteelandt, Olivier Grovel, Olivia Fossi Tankoua, Karina Petit, Thibaut Robiou Du Pont, Jean‐François Biard, Yves‐François Pouchus

Université de Nantes, Pôle Mer et Littoral, Laboratoire MMS-EA2160, Faculté de Pharmacie, 1 rue G. Veil-BP 53508, Nantes, F-44035 France

[email protected]

Penicillium is one of the most common genus of Ascomycetes, either in terrestrial or marine environment. Penicillium waksmanii Zaleski is an ubiquitous fungal species. Despite of this, there have been very limited investigations on its metabolome, with only 13 secondary metabolites described: the alkaloids brevianamide A and fellutanine A (Kozlovskii et al., 1997), the four pyrones pyrenocines A, B, D and E, three silvatine-derived dioxopiperazines (Amagata et al., 1998), griseofulvin (Petit et al., 2004) and the three pentacyclic spiroindolinones PF1270A-C (Kushida et al., 2007).

Study of marine strains of Penicillium is relatively recent, but the discovery of many novel substances such as penicillones A and B (Liu et al., 2005) demonstrated that they represent a promising source of original natural products.

The aim of this work was to investigate the metabolome of a marine-derived strain of P. waksmanii isolated from seawater sampled on the Atlantic coast of France. Cultures on different media were extracted with ethyl acetate and crude extracts were directly dereplicated by LC/DAD/MSn. Metabolic profiles analyses led to the detection of only two compounds already known to be produced by this species: griseofulvin and pyrenocine E. Metabolites such as penicillic acid, orsellinic acid and fumagillin were identified for the first time in P. waksmanii. All the other

compounds observed seem to be new for this species, as their molecular weights, MS2 fragmentations and UV spectra were correlated neither with P. waksmanii known metabolites nor with the most common fungal compounds (Nielsen and Smedsgaard, 2003), 2003). Some of them are currently under structural investigation.

1. Amagata T, Minoura K and Numata A (1998) Cytotoxic metabolites produced by a fungal strain from a Sargassum alga. J Antibiot 51(4): 432-434.

2. Kozlovskii A G, Vinokurova N G, Zhelifonova V P, et al. (1997) Alkaloid formation by penicillia of the series Fellutana and Canescentia. Microbiology (Moscow)(Transl. of Mikrobiologiya) 66(4): 429-433.

3. Kushida N, Watanabe N, Okuda T, et al. (2007) PF1270A, B and C, novel histamine H3 receptor ligands produced by Penicillium waksmanii PF1270. J Antibiot 60(11): 667-673.

4. Liu W, Gu Q, Zhu W, et al. (2005) Penicillones A and B, two novel polyketides with tricyclo [5.3.1.03,8] undecane skeleton, from a marine-derived fungus Penicillium terrestre. Tetrahedron Lett 46(30): 4993-4996.

5. Nielsen K F and Smedsgaard J (2003) Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology. J Chromatogr A 1002(1-2): 111-136.

6. Petit K E, Mondeguer F, Roquebert M F, et al. (2004) Detection of griseofulvin in a marine strain of Penicillium waksmanii by ion trap mass spectrometry. J Microbiol Methods 58(1): 59-65.


127

PC 56

PHENYLNANNOLONE A: THE BIOSYNTHESIS OF AN MDR REVERSAL AGENT Sarah Bouhired, Gabriele M. König

Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany

[email protected]

Myxobacteria are gliding bacteria that belong to the δ-Proteobacteria and are known for their unique biosynthetic capabilities. Our research is aimed towards discovering unique structures from bacterial sources that have not been investigated to date, and at deciphering novel biosynthetic pathways.

Among myxobacteria, Nannocystis spp. are most closely related to marine myxobacteria and their secondary metabolism has hardly been investigated. Nannocystis exedens is a myxobacterial strain that produces compounds of a new group of natural products named phenylnannolones. Phenylnannolone A was obtained from a culture of N. exedens that was isolated from the intertidal region of Crete. Phenylnannolone A had inhibitory activity towards the ABCB1 gene product p-glycoprotein and reversed daunorubicin resistance in cultured cancer cells.

Phenylnannolone A has an unusual structural architecture. It is composed of an ethyl-substituted polyene chain linked to a pyrone moiety on one side and to a phenyl ring on the other. The investigation of the biosynthesis with labelled precursors revealed acetate, butyrate and phenylalanine as building blocks for phenylnannolone A. The labelling pattern suggested novel biochemical reactions for the biosynthesis of the starter unit.

To determinate the biosynthetic gene cluster for phenylnannolone A and to analyse in detail the unique biochemistry leading to the C6-C3 starter unit, a genomic library of N. exedens is currently constructed and screened with suitable primers.

1. B. Ohlendorf et al. (2008) Phenylnannolones

A–C: Biosynthesis of New Secondary Metabolites from the Myxobacterium Nannocystis exedens, ChemBioChem., 9(18), 2997-3003

Acknowledgement: Financial support is provided by the NRW graduate school BIOTECH-PHARMA


128

PC 57

STUDY OF THE BACTERIA ASSOCIATED WITH CLATHRINA CLATHRUS AND EVALUATION OF THEIR CONTRIBUTION TO SECONDARY METABOLISM

Mélanie Roué1, Isabelle Domart‐Coulon2, Mikel Becerro3, Thierry Perez4 and Marie‐Lise Bourguet‐Kondracki1

1 Molécules de Communication et Adaptation des Micro-organismes, FRE 3206 CNRS-MNHN, 57 rue Cuvier (C.P. 54), 75005 Paris, France;

2 Biologie des Organismes Marins et Ecosystèmes, UMR 5178 CNRS-MNHN-UPMC, 57 rue Cuvier (C.P. 51), 75005 Paris, France;

3 Centre d'Estudis Avançats de Blanes (CEAB-CSIC), C/ Accés a la Cala St. Francesc, 14, 17300 Blanes, Spain;

4 Diversité, Evolution et Ecologie Fonctionnelle Marine, UMR 6540 DIMAR CNRS-Université de la Méditerranée, Station Marine d’Endoume, Rue de la Batterie des Lions, 13007 Marseille, France.

[email protected]

Sponges represent a highly diverse source of bioactive natural compounds. They are also known to host a large community of micro-organisms and these associations raise the question about origin of the metabolites isolated from marine sponges. Calcareous sponges provide a good source of investigation because they have been little studied, both in terms of chemistry and microbiology, unlike siliceous sponges. Despite their low biomass, the study of their bacteria may allow to elucidate the origin of some secondary metabolites isolated from marine sponges.

From the crude extract of the Mediterranean sponge Clathrina clathrus, two major compounds, the known clathridine1 and a new analogue, have been isolated.

Furthermore, different populations of bacteria have been observed in the mesohyl of C. clathrus through microscopic studies. In order to evaluate the contribution of the associated bacteria to the production of the clathridine and its analogue, we investigated the cellular localization of these compounds by separating the different cell populations found in C. clathrus using differential centrifugation.

Moreover, the phylogenetic diversity of the bacterial biota has been analyzed through DGGE experiments.

Results will be discussed with regard to the origin of C. clathrus metabolites.

1. Ciminiello et al., 1989, Tetrahedron, 45: 3873

O

O N

NNH

N

N CH3

CH3 O

O

Clathridine


129

PC 58

TARGETING SECONDARY METABOLITE BIOSYNTHETIC GENES FROM THE METAGENOME OF THE SPONGE, MYCALE SP.

Sonia A. van der Sar1, Katja M. Fisch1, Cristian Gurgui1, Tu Anh Nguyen1,

Sally Anderson2, Vicky Webb2 and Jörn Piel1

1 Kekulé Institute for Organic Chemistry and Biochemistry, University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany

2 National Institute of Water and Atmospheric Research, Marine Biotechnology Section, Greta Point, Wellington, New Zealand

[email protected]

Many natural products from marine sponges are suspected to be produced by symbiotic bacteria. Their cultivation usually fails, thus making their direct study and exploitation for sustainable drug production difficult. One approach in overcoming this problem is to identify and express biosynthetic gene clusters from the metagenomic DNA of these sponges.

Mycalamide A, a member of the pederin family, peloruside A, a macrolide related to the bryostatins and pateamine A, a macrolide recently shown to have potential as an anticancer drug,1 are natural products that have been isolated from the New Zealand marine sponge, Mycale sp., but are suspected to be produced by symbiotic bacteria living within the sponge.

The vast genomic and metabolic complexity of bacteriosponges requires new laboratory protocols to be developed. Specifically, the poor quality of total DNA isolated from sponges according to standard procedures, the difficulty to identify genes of interest among numerous homologs, and the time-consuming screening procedures to identify and isolate the rare positive clones, have all been addressed.

In addition to the improvement of strategies for the construction of metagenomic libraries, used for the study of sponge polyketide biosynthesis, a new approach to screening2 has allowed a program to be developed that allows us to probe the Mycale sp. metagenome for the desired PKS gene clusters. Using these methods, several DNA fragments have been obtained that are good candidates for the mycalamide A and pateamine A gene clusters.3

1. Low, W-K. et al. (2005) Mol. Cell, 20, 709-722.

2. Nguyen, TA. et al. (2008) Nat. Biotechnol., 26, 225-233.

3. Fisch, K. M., Gurgui, C. et al. Nat. Chem. Biol. Accepted


130

PC 59

BIOSYNTHESIS OF THE MYXOBACTERIAL ANTIBIOTIC CORALLOPYRONIN Till Schäberle, Öezlem Erol‐Hollmann, A. Schmitz, Gabriele M. König

Institute for Pharamaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany [email protected]

Myxobacteria are potent producers of interesting natural products with various biological activities1. One example is corallopyronin, an antibiotically active compound discovered in the 1980s but not developed for clinical use (Reichenbach et al., 1985). It is an inhibitor of the bacterial DNA-dependent RNA polymerase (RNAP), showing no activity against eucaryotic RNA polymerase. The compund binds to the so called “switch region” of the RNAP and thus prevents the interaction of RNAP with promoter DNA2.

We obtained corallopyronin (Figure 1) from a Myxococcus coralloides strain, which was isolated from a soil sample collected in Belgium. The structural characteristics of corallopyronin suggest that its biosynthesis involves a gene cluster composed of polyketidesynthetase (PKS) and non ribosomal peptide synthetase (NRPS) modules. By using 13C-enriched precursors, insights into the biosynthesis of corallopyronin were

obtained. Feeding experiments with labeled S-adenosyl-methionine (SAM) as well with [1-13C]- and [2-13C]-acetate revealed the origin of the methyl groups. Two out of five methyl groups are derived from acetate by the action of HMG-CoA synthases. The remaining three are derived from SAM through the catalytic activity of methyltransferases. Feeding with labeled glycine allowed the conclusion that the nitrogen originated from an NRPS incorporated glycine molecule.

To verify these results and to get some more insights into the biosynthesis of this unusual antibiotic, we are currently elucidating the corresponding gene cluster.

1. Müller, R. et al. (2003). J. Biotech. 106: 233-253

2. Mukhopadhyay, J. et al., (2008). Cell 135: 295-307

Acknowledgement: Financial support came from the DFG research project 854

Figure 1. Structure of corallopyronin: SAM derived; acetate derived

O

OH

ONH

O

O

O

OH


131

PC 60

SPONGE METABOLOMICS: LCMS PROFILING Mary Kay Harper1, Tim S. Bugni1, James E. Cox2, Jason Reppart1, Patricia R. Sutcliffe3,

Monika A. Schlacher‐Hoenlinger3, John N. A. Hooper3, Chris M. Ireland1 1 Department of Medicinal Chemistry, 2 Health Sciences Metabolomics Core Research Facility,

University of Utah, Salt Lake City, Utah, USA 3 Biodiversity Program, Queensland Museum, South Brisbane, Queensland, Australia

[email protected]

Marine sponges are a rich source of diverse natural products, some of which have demonstrated utility in chemosystematics. We recently initiated a pilot study to evaluate LCMS metabolite profiling for assessing intraspecific chemical variability of marine sponges from the Great Barrier Reef. We hypothesize that comparative analysis of overall chemical profiles will reveal characteristic patterns of metabolite distribution that may assist in taxonomic classification or distinction of chemotypes within species.

The samples analyzed in this study were collected by dredge or trawl from inter-lagoon seabed areas during The Great

Barrier Reef Seabed Biodiversity Project. This preliminary study focused on five of the sponges most frequently encountered during this survey: Theonella n.sp., Spheciospongia vagabunda, Coscinoderma nardorus, Xenospongia patelliformis and Paracornulum n.sp.

Although LC/ESI-MS is one of the most sensitive methods for metabolite profiling, comparative analyses of this data present several challenges. Tools to analyze and detect statistical differences in these profiles will be presented. For example, principle component analysis was performed on LCMS profiles using SIMCA-P+, ver. 12.0 (Fig. 1).

Fig. 1 Principal component analysis of LCMS data from Theonella n.sp. and

Spheciospongia vagabunda


132

PC 61

SIPHONAZOLE Öezlem Erol‐Hollmann, T. Höver, M. Nett and Gabriele M. König

Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany

[email protected]

In a screening approach for new compounds from novel bacterial taxa siphonazole was isolated from a Herpetosiphon strain1. Apart from our recent investigations no secondary metabolites are known from this genus. Herpetosiphon species are gram-negative filamentous heterotrophs with the ability to glide on solid surfaces. Bacteria of the genus Herpetosiphon have been found in soil, freshwater and sewage treatment plants.

The structure of siphonazole represents a polyketide with incorporated amino acids (threonine, glycine) and an unusual diene containing side chain. For the characterization of the siphonazole

biosynthetic genes, a genomic library was constructed. The cosmid library was screened with pks- and nrps-primers (cyclization-, adenylation- domain primers) for the corresponding gene cluster.

Three positive cosmid clones could be identified containing nrps- and pks-fragments. Data concerning the sequences of subclones of these cosmids will be presented.

1. Nett, M., Erol, O., Kehraus, S., Köck, M., Krick, A., Eguereva, E., Neu, E., König, G.M. (2006). Siphonazole, an unusual metabolite from Herpetosiphon sp. Angew. Chem. Int. Ed. 45, 3863-3867

.


133

PC 62

MICROBIAL TRANSFORMATION OF CYCLONERODIOL BY STREPTOMYCES SP.

Zhile Feng, Viviane N. Nenkep, Xavier N. Siwe, Alain S. Leutou, Guohua Yang, Keumja Yun and Byeng W. Son*

Department of Chemistry, Pukyong National University, Nam-gu, Busan 608-737, South Korea

* [email protected]

Selectivity is an essential requirement in synthetic organic chemistry. The regioselectivity of enzymes is a fundamental strength of biocatalysis, and enzymes can modify complex or symmetric molecules without any need for protecting groups. As part of a program to explore the biological transformation of bioactive metabolites produced by fungi isolated from marine habitats, microbial transformation of the bioactive sesquiterpene, cyclonerodiol, isolated from marine-derived fungus Botrytis sp., was studied. A two-stage fermentation protocol1 was used to obtain metabolites of cyclonerodiol on a preparative scale. Fermentation of cyclonerodiol for two

weeks with a marine isolate of the actinomycete bacteria Streptomyces sp. afforded the cyclonerotriols. The stereostructure of the metabolites obtained was assigned on the basis of detailed spectroscopic data analyses. Cyclonerodiol and cyclonerotriol showed a moderate cytotoxic activity with IC50 values of 172.1 and 145.7 µM against human cervical carcinoma cells (HeLa cells).2

1. Smith, R. V. and J. P. Rosazza. 1975J. Pharm. Sci. 1975, 64, 1737-1759.

2. Son, B. W. et al., Enz. Microbial Technol. 2007, 40, 1188-1192.


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BIOSYNTHESIS OF PSYMBERIN BY AN UNCULTIVATED BACTERIAL SYMBIONT OF THE SPONGE PSAMMOCINIA AFF. BULBOSA

Cristian Gurgui1, Katja M. Fisch1, Nina Heycke1, Stefan Taudien2, Matthias Platzer2, Brent K. Rubio3, Sarah J. Robinson3, Phillip Crews3 and Jörn Piel1

1 University of Bonn, Germany 2 Leibniz Institute for Age Research, Jena, Germany

3 University of California at Santa Cruz, USA

[email protected]

The highly potent and selective antitumor compound psymberin (= irciniastatin A) belongs to the pederin family of polyketides reported from diverse marine sponges and terrestrial beetles1,2. Psymberin is an extremely rare natural product that could only be structurally characterized from the marine sponge Psammocinia aff. bulbosa after combining 600 extracts collected over an 11-year period. Since the sponge harbors large numbers of as-yet uncultivated symbiotic bacteria, we aimed to obtain insights into the true biosynthetic origin of psymberin by isolating the biosynthetic genes. Initial trials to detect the genes in the symbiotic association failed due to the presence of numerous polyketide biosynthesis pathways. This problem was addressed by developing a novel phylogeny-based strategy that can specifically detect polyketide synthase (PKS) gene clusters of interest in a single nested PCR step4,5. The method allowed us to rapidly isolate the candidate genes from a 410,000 clone metagenomic fosmid library3. Sequencing of three isolated fosmids revealed a gene cluster with an architecture that precisely matches the pederin-isocoumarine hybrid structure of psymberin. The isolated genomic region exhibits a typical bacterial organization and a high sequence

similarity to bacterial genes, which clearly suggests a symbiont origin of psymberin. An analysis of different chemotypes of P. aff. bulbosa showed that they harbor closely related bacteria, but that the symbiont in the psymberin-negative chemotype lacks the PKS genes. Comparison with the pederin and onnamide genes revealed that psymberin is an ancient natural product, from which the other members of the pederin family have evolved5. This is the first example of a complete biosynthetic pathway for a sponge-derived natural product, and our results suggest new perspectives for the sustainable production of marine drug candidates using bacterial expression systems. 1. Piel, J. Proc. Natl. Acad. Sci. U. S. A. 99,

14002-14007 (2002). 2. Piel, J., Hui, D., Wen, G., Butzke, D., Platzer,

M., Fusetani, N. and Matsunaga, S. Proc. Natl. Acad. Sci. U. S. A. 101, 16222-16227 (2004).

3. Hrvatin, S. and Piel, J. J. Microbiol. Methods 68, 434-436 (2007).

4. Nguyen, T., Ishida, K., Jenke-Kodama, H., Dittmann, E., Gurgui, C., Hochmuth, T., Taudien, S., Platzer, M., Hertweck, C. and Piel, J. Nat. Biotechnol. 26, 225-233 (2008).

5. Fisch, K., Gurgui, C., Heycke, N., van der Sar, S. A., Anderson, S. A., Webb, V. L., Taudien, S., Platzer, M., Rubio, B. K., Robinson, S. J., Crews, P. and Piel, J. Nat. Chem. Biol., accepted.


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HALOGENASES IN THE CYANOBACTERIUM FISCHERELLA AMBIGUA

Mustafa El Omari, Gabriele M. König

Institute for Pharmaceutical Biology, University of Bonn, Nußallee 6, 53115 Bonn, Germany

[email protected]

Cyanobacteria are enormously diverse microorganisms, i.e. in terms of their morphology, physiology and secondary metabolism. From our strain of the filamentous Fischerella ambigua, the biologically active natural products ambigol A, B, C were isolated1.

Currently, the biosynthesis of these chlorinated natural products is investigated. From PCR strategies a nucleotide sequence was obtained, containing two conserved motifs in FADH2-depending halogenases, GxGxxG and WxWxIP. Furthermore this sequence shows highest homology to AerJ 2 from Microcystis aeruginosa NIES-98, the halogenase involved in the aeruginosin biosynthesis. This enzyme was identified to chlorinate a phenolic moiety and thus, the putative halogenase detected within the F. ambigua genome is presumably

involved in ambigol formation. In order to obtain the complete genes, a fosmid library was constructed and screened with homologous primers. One clone, containing the phenolic halogenase sequence was subcloned. Partial sequence analysis lead to the assumption that the aromatic moieties are generated by the shikimate pathway and are chlorinated in ortho- and para- position, followed by phenolic oxidative coupling. This biosynthetic pathway is compareable to that of teicoplanin biosynthesis3

1. Wright, A. D., Papendorf, O., König, G. M. (2005) J. Nat. Prod. 68: 459-461

2. Cadel-Six, S., Dauga, C., Castets, A.M., Rippka, R. (2008) Mol. Biol. Evol. 25(9): 2031-2041

3. Li, T.L., Huang, F., Haydock, S.F. (2004) Chem. Biol. 11(1): 107-119


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SIDEROPHORE PRODUCTION IN PHOTO- AND HETEROTROPHIC BALTIC BACTERIA DURING GROWTH ON AN IRON-DEFICIENT MEDIUM

Alicja Kosakowska, Agnieszka Lewandowska, Lidia Żeglińska

Institute of Oceanology, Polish Academy of Sciences, Powstańców Warszawy 55, 81 – 712 Sopot, Poland

[email protected]

Recently, the interest and knowledge about the role of iron in aquatic ecosystems have increased. This trace nutrient is required by cells and organisms for optimal growth and suitable metabolism. Under conditions of iron stress, the many bacteria and some cyanobacteria and algae secrete organic compounds, called siderophores that specifically bind and solubilize Fe.

This contribution deals with the isolation and identification of siderophore-like substances in culture of cyanobacteria Anabaena variabilis and Nodularia spumigena as well as heterotrophic bacteria under low iron conditions. The three bacteria for experiments were isolated from the coastal surface water from the Gulf of Gdańsk. After 16S rDNA analysis the bacteria were determined as Micrococcus luteus, Bacillus silvestris and Erythrobacter flavus.

The iron-binding ligands were isolated by extraction and liquid column chromatography. The siderophore-like substances were analyzed in the examined samples by the chemical assays: by non-specific CAS AD and specific (Atkin, Arnow, Csaky) chemical tests. Biological

activity of extracts was determined using specific bioassays with the apply of mutated bacterial strains (Microbacterium flavescens JG-9 and Morganella morganii SBK-3).

The extracts examined showed strongly positive responses. Detectable levels of hydroxamate and α-hydroxy / α-ketohydroxy acids type siderophores were produced by Anabaena variabilis and Nodularia spumigena. During our work we proved that concentration of iron has influence on the growth of all examined bacteria: < 10-7 mol Fe / dm3 was insufficient for optimum growth. However tested microorganisms grew under iron-limited conditions, because – how we revealed – they can produce hydroxamate-type siderophores. Additionally, M. luteus and B. silvestris can produce α-ketoacids and α-hydroxyacids.

Acknowledgement: The study was partially supported by the Polish State Committee for Scientific Research (grant No. 2 PO4E 026 30, 2006-2008) and by the statutory programme of the Institute of Oceanology, PAS (grant No.II.3)


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NEW SIDEROPHORES FROM PHOTOBACTERIUM DAMSELAE SUBSP. PISCICIDA Alba Souto1, Amable J. Rivas2, Carlos R. Osorio2, Jaime Rodríguez1,

Manuel L. Lemos2 and Carlos Jiménez1

1 Departamento de Química Fundamental, Facultade de Ciencias, Universidade da Coruña, 15071 A Coruña, Spain.

2 Department of Microbiology and Parasitology, Institute of Aquaculture and Faculty of Biology, University of Santiago de Compostela, Campus Sur, Santiago de Compostela 15782, Spain

[email protected]

Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida) is the causative agent of fish pasteurellosis, a disease which causes large economical losses in marine aquaculture worldwide. This is, since 1990, the major pathological problem in the culture of sea bream and sea bass in the mediterranean countries, including Spain.1

This pathogen was known to have a high affinity iron uptake system, through the synthesis of siderophores and iron-regulated membrane proteins which act as receptors for the siderophore-iron complex.2,3 Siderophores are excreted to the culture medium, where they chelate iron in a specific manner, then transport this element into the cell, using specific receptors in the membrane, and release the iron in the cytoplasm. However, the chemical structure of the siderophore produced by this bacterium is totally unknown.

In our continuing investigations on the study of siderophores from fish pathogenic bacteria4 and taking into account that its production is an important virulence factor, our specific goal will be to chemically characterize the siderophore(s) produced by P. damselae. These iron-acquisition pathways could be

exploited in the development of new antimicrobials against pasteurellosis.

In the first step of this project, we were able to optimize the bacterial growth under iron deficient conditions in order to maximize the production of siderophores. The following step was the bioguided extraction and fractionation of the culture supernatant in order to locate the fraction containing siderophores. So, the cell-free supernatants (the cells were removed by centrifugation and filtration) were passed through a XAD-7 lipophilic resin and subjected to liquid-liquid extraction using AcOEt at different pHs. The first method allowed us to concentrate the siderophore activity in one of the obtained fractions, which will be subjected to further fractionation in order to isolate the siderophore.

1. Magariños, B; Toranzo, A.E.; Romalde, J.L. Ann. Rev. Fish Dis. 1997, 6, 41.

2. Magariños, B; Romalde, J.L.; Lemos, M.L. Barja, J.L.; Toranzo, A.E. Appl. Environ. Microbiol. 1994, 60, 2990.

3. Osorio; C.R,; Juiz-Río, S.; Lemos, M.L. Microbiology 2006, 152, 3327.

4. Soengas, R.G., Anta, C. Espada, A.; Paz, V.; Ares, I.R.; Balado, M.; Rodríguez, J. ; Lemos, M.L.; Jiménez, C. Tetrahedron Lett. 2006, 47, 7113.


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A BIOTECHNOLOGICAL APPROACH FOR PLAKORTIN PRODUCTION: NEW PERSPECTIVES FOR BIOACTIVE COMPOUNDS FROM MARINE SOURCE Valeria Costantino1, Ernesto Fattorusso1, Lena Gerwick2, William H. Gerwick2,

Alfonso Mangoni1, Jörn Piel3, Roberta Teta 1 1 Dipartimento di Chimica delle Sostanze Naturali, University of Napoli Federico II, via

Domenico Montesano, 49 80131 Napoli, Italy; 2 Scripps Institution of Oceanography, 8602 La Jolla Shores Drive La Jolla, CA 92037, USA;

3 Kekulè Institute of Organic Chemistry and Biochemistry, Gerhard-Domagk-Strasse 1D-53121 Bonn, Germany

[email protected]

A variety of limiting factors currently affect the full exploitation of bioactive natural products from marine invertebrates, the most important being the supply problem. Large-scale total synthesis is usually prevented by the complex structure of most natural products, while a massive collection of the organisms producing compounds of industrial interest appears unrealistic. New perspectives were opened by the discovery that several (and probably a significant portion) of these bioactive compounds are produced by bacterial symbionts, but even so, production by fermentation is often impossible because very few symbiotic species can be cultured with the present techniques.

We are currently exploring a biotechnological approach for the inexpensive and reproducible production of marine natural compounds. Our research is focused on the study of the biosynthetic pathway of plakortin (and related compounds), a polyketide peroxide with interesting antimalarial properties which is present in the marine sponge Plakortis simplex and is biosynthesized by a uncultivable bacterial symbiont of the sponge.1 We intend to identify, isolate and sequence the biosynthetic gene cluster for plakortin (presumably coding for a type-I polyketide synthase), and subsequently to

express the pathway heterologously to produce plakortin by fermentation.

plakortin

OO

COOCH3

Metagenomic DNA from Plakortis simplex was cloned to generate a 50,000 clone library which was PCR-screened in search of the PKS gene cluster involved in the biosynthesis of plakortin. One positive clone was isolated and fully shotgun sequenced. The PKS gene is located at one end of the insert, so that only a small part of the PKS gene cluster is present in the insert (9 kbp). Following these encouraging results, we are currently searching for the remaining part of the cluster.

The latest results of this project will be presented. The success of this research will demonstrate the feasibility of this strategy for the large-scale production of natural products, which could be applied to many other bioactive compounds from marine invertebrate. 1. M. Laroche, C. Imperatore, L. Grozdanov, V.

Costantino, A. Mangoni, U. Hentschel, E. Fattorusso, Marine Biology 2007, 151, 1365–1373.


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DEVELOPMENT AND OPTIMIZATION OF AN ECO-FRIENDLY AGAR EXTRACTION PROCESS FROM THE RED SEAWEED

GRACILARIA VERMICULOPHYLLA A.M.M. Sousa1, V.D. Alves1, S. Morais2, C. Delerue‐Matos2, Maria P. Gonçalves1*

1 REQUIMTE, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal.

2 REQUIMTE, Instituto Superior de Engenharia do Porto, Rua Dr. António Bernardino de Almeida 431, 4200-072 Porto, Portugal.

* [email protected]

Gracilaria vermiculophylla is an invasive species, newly established at Ria de Aveiro, north-western Portugal. This type of marine macroalgae constitutes a threat to the ecological balance of coastal ecosystems. The use of plants like seaweeds in aquaculture can produce sustainable and cost-effective operations that reduce the environmental impacts of effluents resultant of aquaculture mechanism. The intensive use of these photoautotrophic organisms as biofilters leads to large amounts of seaweed waste. Furthermore, Gracilaria is a red algal genus that biosynthesizes agar, a polymer that is extensively used in food and pharmaceutical industry as gelling and stabilizing agent. Traditionally, agar is hot-extracted with water for several hours. When Gracilaria genus is used, an additional alkali-treatment prior to the extraction is necessary to enhance the extract gelling properties1. The amount of time required, solvent and energy spent during this kind of procedure, justify the development of an alternative agar extraction process. The popularity of microwave-assisted extraction (MAE) has risen rapidly over the last decade and it

has proven to be effective for extracting several components. The major benefits of MAE are the decreased extraction times, reduced solvent consumption and increased sample throughput. The objectives of this work included the study of a potential application of the invasive species G.vermiculophylla, after being used in aquaculture systems, and the development and optimization of a new eco-friendly agar MAE process. The effect of time, solvent volume, temperature and stirring speed of the step were investigated in order to achieve appropriate agar yields, gel strengths, gelling and melting temperatures, as well as sulphate and 3,6-anhydro-L-galactose contents. The Response Surface Methodology was the statistical tool used in the optimization process. Due to the novelty of the extraction process, the optimization started with a 25 (2 levels and 5 factors) design to which more levels were added to achieve an optimum plateau regarding the selected parameters.

1. Villanueva, R.D., Sousa, A.M.M., Gonçalves M.P., Nilsson, M., Hilliou, L. (2009), Journal of Applied Phycology, in press.


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INDUSTRIAL DEVELOPMENT OF A NATURAL ANTIOXIDANT INGREDIENT WITH ANTIMICROBIAL PROPERTIES PRODUCED BY A MARINE BACTERIA

Cosima Dufour‐Schroif1, Delphine Pichon1, Elodie Quévrain2, Marie‐Lise Bourguet‐Kondracki2, Denis Duplat1

1 COVALMAR, 191 avenue Daumesnil, 75012 Paris, France 2 Molécules de Communication et Adaptation des Micro-Organismes, Muséum National

d’Histoire Naturelle, FRE 3206 CNRS/MNHN, 57 rue Cuvier (C.P. 54), 75005 Paris, France

[email protected]

Providing the cosmetic industry with ingredients from natural origin is today a real challenge to suppliers of raw materials. COVALMAR is a research and development company specialized in the discovery of natural active molecules produced by marine microorganisms and their production at the industrial scale for the cosmetic industry.

COVALMAR is developing, in collaboration with the Muséum National d’Histoire Naturelle and the CNRS, a natural antioxidant extract with antimicrobial properties as a novel ingredient for cosmetic products. The bioactive extract, rich in natural parabens, is produced by a marine bacteria belonging to the genus Microbulbifer (Alteromonodaceae), strain L4-n2, isolated from the marine sponge Leuconia nivea in temperate seawater (Concarneau, France). Nine antimicrobial molecules were already isolated, characterized and patented 1, 2; other antimicrobial and antioxidant molecules are being identified.

The optimization of culture parameters in flask and bioreactor like pH, temperature, salinity, nutrients, stirring and oxygenation increased the production of bioactive molecules by a factor three.

Moreover, the antioxidant activity of the bioactive extract, measured with the ORAC assay (Oxygen Radical Absorbance Capacity), was enhanced to 50% in comparison to the activity of ascorbic acid. The same extract showed antimicrobial activities against Staphylococcus aureus (ATCC 6538) and Candida albicans (ATCC 10231).

To obtain the commercially natural bioactive cosmetic ingredient, several extraction methods were considered for technical and economical constraints with respect to the cosmetic industry. The chosen industrial extraction method compared to the laboratory method, using dichloromethane, provided comparable yield and the bioactive molecules were extracted selectively.

1. Quévrain E, Domart-Coulon I, Pernice M, Bourguet-Kondracki ML. (2009) Novel natural parabens produced by a Microbulbifer bacterium in its calcareous sponge host Leuconia nivea. Environ Microbiol. Ahead of print.

2. Bourguet-Kondracki ML, Domart-Coulon I, Quévrain E (2008) Paraben compounds. Muséum National d’Histoire Naturelle; Centre National de la Recherche Scientifique; Université Paris Curie. EP1980612.


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BIOFABRICATION OF INORGANIC-ORGANIC COMPOSITES AND NEW BIOMATERIALS USING SPONGE SPICULES AS BLUEPRINTS

Filipe Natalio1, Matthias Wiens1, Heinz‐C. Schröder1, Xiahong Wang2 and Werner E.G. Müller1*

1 Institute for Physiological Chemistry, Department of Applied Molecular Biology, Johannes Gutenberg-Universität Mainz, Duesbergweg 6, D-55099 Mainz, Germany.

2 National Research Center for Geoanalysis, 26 Baiwanzhuang Dajie, CHN-100037 Beijing, China.

[email protected]

While most forms of multicellular life have developed a calcium-based skeleton, a few specialized organisms form their Bauplan using silica (SiO2). Among all animals only sponges (phylum Porifera) are able to polymerize silica (biosilica) enzymatically, at ambient temperature and pressure, in order to form their siliceous skeletal elements (spicules). Sponge biomineralization (spiculogenesis) in model organism S.domuncula was recently elucidated using 3D primary sponge cells – primmorphs. The first steps of spicule growth occurs inside highly specialized sponge cells containing biopolymers (galectin and collagen), silicatein and silintaphin-1. The final shape of the spicule is originally determined by presence of a templating crystalline nanostructure. The possibility to induce controlled formation of hybrid nanomaterials (SiO2/TiO2) with unique properties within sponge cells (Bionanofactories) using a synergetic effect of cell metabolism and silicatein

catalytic properties opens a new door to new biologically-formed composite materials. Siliceous structures can grow from some micrometers up to several meters demonstrating an incredible property to transmit light similar to man-made optical fibers. With biological exploitation of spiculogenesis (silicatein and silintaphin-1), a molecular toolbox was generated opening the possibility for fabrication of the first bioinspired light waveguides structures. Sililicatein, have attracted increasing attention because of their potential applications in the field of nanobiotechnology, and biomedicine catalyzing a wide variety of metal oxides. Moreover, the application of silica polymerizing enzyme in biomedicine is highlighted on basis of biosilica-mediated regeneration of tooth and bone defects.

Schröder HC, Wang XH, Tremel W, Ushijima H, Müller WEG (2008) Biofabrication of biosilica-glass by living organisms. Nat. Prod. Rep. 25:455-474


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BUGS AND BRAIN – THE KEY TO SUCCESS? TREATING NEURODEGENERATIVE DISEASES WITH A UNIQUE EXTRACT

COLLECTION FROM MICROORGANISMS COLLECTED IN AZORES - PHARMABUG LIBRARY

Christophe Roca1, T. Tenreiro2, R. Tenreiro2, A. Santos1, P. Calado1, Helena Vieira1 1 BIOALVO S.A., Edificio ICAT, Campus da FCUL, 1749-016 Lisboa, Portugal

2 BioFIG, Center for Biodiversity, Functional & Integrative Genomics, Edificio ICAT, Campus da FCUL, 1749-016 Lisboa, Portugal

[email protected]

Marine natural products are becoming an unlimited source of unique molecular structures leading to drugs in all major disease areas. PharmaBUG collection was created to take advantage of these untapped resources, and consists of 208 aqueous and organic extracts produced from microorganisms collected at the hydrothermal vents near Azores islands along the Middle-Atlantic Ridge. The collection gathers novel microbial samples obtained from water samples, small animals, sediments and chimneys in four hydrothermal areas: Menez Gwen, Monte Saldanha, Lucky Strike and Rainbow. Menez Gwen, Rainbow and Lucky Strike have intense hydrothermal activity and are characterized by the presence of chimneys where superheated water (ca. 300ºC) and other compounds are expelled. Monte Saldanha is characterized by a hydrothermal field yet in formation presenting small orifices scattered throughout the seafloor, where water temperature is around 3-4ºC. From these extreme and unique environments, one can expect to isolate new microorganisms able to produce metabolites and chemicals with unique molecular structures and

potential biological activity. Development of these molecular entities into drug-like compounds for unmet therapeutic needs can prove valuable to the fuelling of pharmaceutical industry pipelines.

Organic and aqueous extracts were produced from the biomass obtained from pure microbial culture. Biological assays using the Global Platform Screening for Drug Discovery (GPS D2) technology, developed by BIOALVO, were performed to identify extracts with therapeutic potential. A first screening allowed the identification of 10 different extracts with strong activity against a specific cellular stress involved in many neurodegenerative diseases. Two organic extracts produced from different isolates gave very high potency. Interestingly, the two isolates were both found in Rimicaris sp., suggesting similarities between isolates. Further characterization of the active extracts is currently ongoing with the purpose of isolating the active compound responsible for the observed activity. The final goal is to develop the active compound into a drug for the treatment of a wide range of neurodegenerative diseases.


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BIOMIMETIC SENSORS FOR LEUCOMALACHITE GREEN Felismina T. C. Moreira, M. Goreti F. Sales

REQUIMTE, Instituto Superior de Engenharia do Porto, R. Dr. António Bernardino de Almeida, 431, 4200-072 Porto, Portugal.

[email protected]

Concerns about chemical pollution from marine fish farming center around medications or other treatments used to keep farmed fish disease and parasite free. This is the case of malachite green (MG), a popular substance and, in some cases, the only choice to treat and prevent fungal and parasitic infections1. The use of this drug is not allowed under current European Union (EU) regulations. Being a potential carcinogenic, mutagenic, and teratogenic compound [2], it has been banned in many nations outside the EU as well.

The use of MG is linked to the presence of leucomalachite green (LMG) in farmed or wild fish and water. LMG is the reduction product and major metabolite of MG. To

detect it, new biomimetic sensors based on molecularly-imprinted polymers (MIP) are proposed. The sensors exhibit a near-Nernstian response (Fig. 1), with slopes and detection limits ranging 45.8 – 81.2 mV decade-1 and 0.28 – 1.01 µg mL-1, respectively.

The potentiometric sensors are independent from the pH of test solutions within 3 –5, and are successfully applied to monitor LMG in environmental water samples. 1. S.J. Culp, F.A. Beland, R.H. Heflich, R.W.

Benson, L.R. Blankenship, P.J. Webb, P.W. Mellick, R.W. Trotter, S.D. Shelton, K.J. Greenlees, M.G. Manjanatha, Mutation Research 506 (2002) 55–63;

2. D. Alderman. J. Fish. Dis. 8 (1985) 289-298.

-6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5

log [LMG], M

MAA+PclTPB MAA NIP MAA MAA washed

50 mV

-6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5

log [LMG], M

AAMPSO

NIP AAMPSO

AAMPSO washed

AAMPSO + pClTPB

50 mV

Fig. 1 Potentiometric response of LMG selective electrodes of different MIP materials. Non-imprinted polymers (NIP) are used as control. MAA: methacrylic acid; AAMPSO: acrylamido-2-methyl-1-propanesulfonic acid; pClTPB: tetrakis(4-chlorophenyl)borate.


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BIOLOGICAL CONTROL OF TOMATO DISEASE BY CHITIN DEGRADING ACTINOMYCETE STRAINS WITH SWOLLEN CHITIN

Vichien Kitpreechavanich1, Prapassorn Rugthaworn1,2, Uraiwan Dilokkunanant 2, Somsiri Sangchote3, and Nattayana Piadang4

1 Department of Microbiology, Faculty of Science 2 Kasetsart Agricultural and Agro-Industrial Product Improvement Institute,

Kasetsart Department of Plant Pathology 3 Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand

4 Radiation Ecology Group, The Irradiation for Agriculture Research Program, Office of Atoms for Peace, Bangkok 10900, Thailand.

[email protected]

Two wild type strains; SG4 and SJ9 and three isolates of gamma ray induced mutant actinomycete strains; SG4I-17, SG4I-38 and SJ9I-15 were tested for their ability on the production of chitinase and substance that inhibit the growth of Sclerotium rolfsii which was the tomato disease causing agent. The wild type strain of SG4 gave the highest yield of crude extract 0.032 % (w/v) after growing on liquid chitin medium for 10 days, and its MIC against the growth of S. rolfsii was 1.0 µg/µl,. The strain SJ9 produced the highest chitinase activity, which was 24.9 mU/ml after 5 days cultivation. Application of each actinomycete strain with swollen chitin decreased 6-13% the tomato stem and root rot disease which was higher than applying actinomycete alone at 14 days in pot scale. The strains SG4I-38 and SG4 showed higher survival of tomato plant, 73.3 and 66.6%, respectively. The two treatments gave chitinase activity, 4.3 and 5.5 mU/g

dried soil, and the crude extract, 0.037 and 0.034 %(g/g dried soil), respectively with their MIC to inhibit the growth of S. rolfsii was the same at 4.1 µg/µl. Application of mixed strains between SG4 and SG4I-38 with swollen chitin resulted in 73.3 % survival plant which was higher than other mixed strains with significant difference at confident interval of 95%. Chitinase activity, 5.4 mU/g dried soil and the highest yield of crude extract, 0.039 %(g/g dried soil) with its MIC to the growth of S. rolfsii of 4.1 µg/µl were detected.

Keyword: Chitin degrading Actinomycetes, Biological Control, Antifungal substance, Tomato Stem and Root Rot Disease, Sclerotium rolfsii Application of mixed strains between SG4 and SG4I-38 with swollen chitin gave better effect on survival plant than other mixed strains with 73.3 %.


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SUPERCRITICAL CARBON DIOXIDE AND SOLVENT EXTRACTION OF UNGAVA’S BROWN SEAWEED OIL

Yacine Boumghar1 , Naima El Mehdi1, Fatna Maifi1, Guy Rochefort2 and Marc Allard2 1 Centre d’études des procédés chimiques du Québec (CÉPROCQ, 6220, Sherbrooke East, Montréal,

Québec, H1N 1C1 CanadaFax: +1 (514) 251-3696 2 Nunavik Biosciences Inc., 1111 boulevard Dr Frederik-Philips, Saint-Laurent, Québec, H4M

2X6, Canada

[email protected]

Nunavik Biosciences Inc. developed a procedure to prepare Dry Algal Powder (DAP) from fucoids (Fucus distichus, Fucus vesiculosus) marine macroalgae. DAP was obtained using a sequential procedure consisting in harvesting the algae, washing it in fresh water, removing the extraneous materials, drying to constant weight, and then grinding the dry material to a standardized powder. During a program seeking to identify biologically active fractions from DAP, it was revealed that an oil rich in fatty acids could be extracted.

Conventional extraction was optimized by means of different organic solvents: hexane isopropanol, ether petroleum, ethyl acetate in two modes: maceration and Soxhlet, with different extraction times. Hexane was the most promising solvent and Soxhlet gives the better yields i.e. 3%.

Concurrently, a series of extraction experiments using supercritical carbon

dioxide were performed either alone or combined with ethanol as cosolvent. The experiments have been constructed on a factorial design to examine simultaneously the effects of multiple independent variables and their degree of interaction, including temperature and pressure of extraction reactor, cosolvent concentration, and extraction time. Further to obtain an oil refinement, a bleaching method was developed to remove coloured materials from the oil, such as chlorophyll and chlorophyll breakdown products, brown-coloured compounds. Importantly, GCMS analyses of bleached versus unbleached established clearly that bleaching does not affect the fatty acid profile of the algal oil.

The paper emphasizes the analysis of the extraction performance of each extraction technique as well as the results of a benchmarking study driven by yield and extract composition.


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PRELIMINARY AQUACULTURE TRIAL WITH CATOSTYLUS TAGI Zilda B. Morais1, Narcisa M. Bandarra2 and Teresa G. Pereira2

1 Cooperativa Egas Moniz, Centro de Polímeros Biomédicos, 2829-511 Caparica, Portugal 2

INRB/IPIMAR, Instituto de Investigação das Pescas e do Mar; Inovação Tecnológica e Valorização dos Produtos da Pesca, Av. de Brasília, 1449-006 Lisboa, Portugal

[email protected]

Along the Portuguese continental coast occurs Catostylus tagi, a Rhizostomeae jellyfish, which is particularly abundant in the summer at the Sado estuary. Although C. tagi belongs to a family of edible medusae, as far as we know, it is not consumed by humans. An alternative use for it could be as aquafeed1, given that the screening for toxic heavy metals in C. tagi revealed only aluminum, and in the same range of other local seafood2. Due to its chemical composition, C. tagi could be regarded as a mineral source, with the extra profit of a LCPUFAs fatty acid profile2.

In the present study, gilthead seabream (Sparus aurata) juveniles were fed with experimental diets where C. tagi was the only marine component, being soya meal the protein source and vegetable oil the oil source3. The growth trial involved 9 groups of 15 fish each, with an average body weight of 18 g, and consisted of two stages. In the first, during 9 weeks, three isonitrogenous diets were formulated in order to include the jellyfish ground mineral as a replacement for the commercial mineral premix. In the second

stage, which lasted for 1 month, fish were redistributed in the tanks in order to verify changes in the growth profile.

At the end of the first stage, the fatty acid profile of the seabream samples did not reflect marked differences between the mineral premix diet and the C. tagi’s diets. However, the contents for EPA and DHA were below the normal values for seabream feed on fish meal and fish oil.

On the other hand, the ICP results for mineral content, on whole body and muscle, showed no significant differences between seabream feed on the mineral premix diet or on C. tagi’s diets.

1. Pauly D., Graham W., Libralato S., Morissette L., Palomares M.L.D. (2009) Hydrobiologia, 616, 67–85.

2. Morais Z.B., Pintão A.M., Costa I.M., Calejo M.T., Bandarra N. and Abreu P. (2009) Journal of Aquatic Food Product Technology, 18, 90-107.

3. Pereira, T.G. and Oliva-Teles, A. (2002) Aquaculture Research, 33, 1183-1189.


147

PC 76

FRESH TURBOT FILLETS PACKAGED UNDER MODIFIED ATMOSPHERE: CONVENIENCE, QUALITY AND SAFETY

Joana Santos1, F. Lisboa1,2, N. Pestana1,2, M.R. Alves1,3, M.B.P.P. Oliveira1* 1 ,2 REQUIMTE, Department of Bromatology and Microbiology, Faculty of Pharmacy,

University of Porto, Portugal 3 ESTG - Instituto Politécnico de Viana do Castelo

* [email protected]

Convenience is nowadays one of the key that consumers look for in their foodstuffs. But, they also want safety and quality. The consumption of fresh fish in Southern Europe countries is common, although this product is generally perceived as a relatively inconvenient type of food1. Fresh fish is usually sold as whole in the traditional markets, but preparation and cooking of this product doesn’t suit the actual lifestyle of new consumers. It has been claimed that packaging of fish fillets in a modified atmosphere (MA) allows this product to meet the new consumer’s convenience preferences2.

Fish is a highly perishable product that begins to lose quality immediately after death. Packaging in a MA may extend product quality for a longer period, allowing at the same time a better convenience for transport and fast cooking2. Turbot (Psetta maxima) is a marine flatfish, highly appreciated for their lean, white and firm flesh and has a high economic value.

To study the best atmosphere to preserve the quality of fresh turbot fillets, they were packed under 3 modified atmospheres (AIR: control; B: 10 % O2/ 40 % CO2; C: 10 % O2/ 60% CO2; D: 10 % O2/ 80 % CO2) and stored at 2 + 1ºC for 30 days. Assessment of fillets’ quality

and safety from the four test groups was carried out within a 5 day interval, through the evaluation of microbiological, physical and chemical aspects, that change with fish degradation.

Fillets packed under MA showed a different quality evolution when compared with the control fillets. The signs of degradation reached rejection threshold values in the AIR packages firstly in all evaluate parameters. In the packages with a MA there were no visible signs of degradation during 30 days, although the package B exceeds the microbiological limit after 15 days. These findings indicates the protective effect of the different atmospheres especially those with a higher percentage of CO2 (C e D). Also the need for an accurate safety study to establish the conditions that guarantee that this technology doesn’t mask the degradation signs, when the product is no longer safe, is pointed out.

1. Olsen, S.V., Scholderer, J., Brunsø, K., Verbeke, W. 2007. Exploring relationship between convenience and fish consumption: A cross-cultural study. Appetite, 49:84-91.

2. Sivertsvik, M., Jeksrud, W.K., Rosnes, J.T. (2002). A review of modified atmosphere packaging of fish and fishery products – significance of microbial growth, activities and safety. International Journal of Food Science and Technology, 37, 107-127.


148

PC 77

PROTECTING THE BABIES: CHEMICAL DEFENSES IN EGGS AND RECRUITS OF APLYSIA DEPILANS

Souhir Hamrouni Buonomo1, Mohamed Salah Romdhane1, Amel Ben Rejeb Jenhani1

and Mikel A. Becerro2

1 National Institute of Agronomy, 43, Rue Charles Nicole, Cité Mahrajène, 1080 Tunis, Tunisia 2 Center for Advanced Studies of Blanes (CEAB, CSIC), Acc Cala St. Francesc 14,

17300 Blanes (Girona), Spain

[email protected]

Chemical defenses against predators are common in many benthic marine organisms. Sea hares are particularly well defended chemically since they have opaline, ink, and secondary metabolites sequestered from their food sources as an impressive arsenal of unrelated chemical weapons that effectively deter multiple predators. We know fairly well how these systems operate in a number of sea hare species, although most information available refers to adult specimens. Far less known is how these chemical defenses operate throughout the lifespan of a species. Here, we provide evidence of chemically mediated feeding deterrence of multiple stages of egg masses and recruits of the sea hare Aplysia depilans. We collected freshly laid eggs, well developed eggs prior to hatching, and an intermediate stage of development (eggs 1, eggs 3, and eggs 2 respectively). We also collected small recruits (<5 mm in length) and juveniles (<2 cm). All samples were taken to the lab and we ran a series of experiments to test for their deterrence against the natural predator Anemonia sulcata. The first experiments tested whether egg masses, recruits, and juveniles where defended against the anemone and used unmanipulated samples and small mussels as controls. Another set

of experiments tested whether egg masses, recruits, and juveniles were chemically defended. Samples were extracted with dichlormethane/methanol and offered to anemone to test for deterrency. Extracts were added at natural concentrations to an artificial diet and offered to the anemone with appropriate controls. Our results show that all three egg stages were deterrent against the anemone (eggs 1: p<0.001, eggs 2: p=0.001, eggs 3: p<0.001; n=20 for all experiments). The deterrent activity was chemically mediated as extracted egg masses were readily eaten by the anemone while the extracts were deterrent when incorporated in the artificial diets (p<0.001 for all stages). Small recruits showed no protection against the anemone since all replicates were readily eaten and failed to release opaline. Juveniles were deterrent (n=17, p=0.023) and all of them released opaline secretion. Extracted juveniles were all eaten while the extracts were deterrent when incorporated in the artificial diet (n=18, p<0.05). Our results show that sea hares rely heavily on secondary chemistry to protect their eggmasses, but recruits are not chemically defended. The chemical defense appears with the capacity to secrete opaline at a juvenile stage.


149

PC 78

CHEMICAL STUDY OF AEOLID NUDIBRANCHS OF THE GENUS SPURILLA FROM DIFFERENT GEOGRAPHICAL AREAS

Stella García Matucheski1, Marianna Carbone2, Claudia Muniain1, Ernesto Mollo2, Maria Letizia Ciavatta2, Guido Villani2, Guido Cimino2, Margherita Gavagnin2

1 Lab. Ecología Química y Biodiversidad Acuática. Ins. Inv. e Ing. Ambiental. UNSAM. Peatonal Belgrano 3563 1º (1650) San Martín, Buenos Aires, Argentina.

2 Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, 80078 Pozzuoli, Naples, Italy

[email protected]

Aeolid nudibranchs are known to sequester nematocysts from cnidarian preys, storing such defensive structures in dorsal appendages, named cerata, for a re-use in their own defense.1 Despite of this, a possible defensive role of non-dietary aeolid secondary metabolites has been also proposed, while an origin of such compounds from symbionts has been hypothesized.2

In this report, we present the results of a recent study carried out on population on aeolids belonging to the genus Spurilla, from different geographical areas. In particular, individuals of Spurilla neapolitana, collected off Lake Lucrino (Naples) in Italy, have been investigated in comparison with specimens of Spurilla sp. from Larralde (Chubut) in Argentina. The compound (-)-bursatellin (1), previously characterized from herbivorous sea hares of the genus Bursatella,3,4 has been isolated from both Spurilla species, while differences between the secondary metabolite patterns of two aeolids, related to minor terpenoidic components, have been also detected. Remarkably, the structure of 1 is closely related to

chloramphenicol (2), the bacteriostatic antimicrobial produced by the filamentous Gram-positive soil bacterium Streptomyces venezuelae and certain other actinomycetes,5 and this might suggest a possible origin of bursatellin from symbiontic bacteria. In addition, chemical comparisons between the nudibranchs and their preys indicated that the compound is not food-derived in Spurilla nudibranchs. Finally, the possible role of bursatellin in chemical defense against predators and/or its probable influence on the microbial community composition of the nudibranch gut or skin will be discussed for further developments of the present study. 1. Greenwood P.G., Mariscal R.N. Tissue & Cell

1984, 16, 719-730. 2. Ciavatta M.L., Trivellone E., Villani G.,

Cimino G. Gazzetta Chimica italiana 1996, 126, 707-710.

3. Gopichand Y., Schmitz F.J. Journal of Organic Chemistry 1980, 45, 5383-5385.

4. Cimino G., Gavagnin M., Sodano G., Spinella A., Strazzullo G., Schmitz F.J., Gopichand Y. Journal of Organic Chemistry 1987, 52, 2301-2303.

5. He J., Magarvey N., Piraee M., Vining L.C. Microbiology 2001, 147, 2817–2829.


150

PC 79

A NEW PEPTIDIC ALLELOCHEMICAL FROM THE FILAMENTOUS CYANOBACTERIUM OSCILLATORIA SP. ISOLATION AND STRUCTURAL STUDIES

Pedro N. Leão1,2, Alban Pereira2, Vítor M. Vasconcelos1,3 and William H. Gerwick2,4 1 CIIMAR/CIMAR-LA – Centre for Marine and Environmental Research,

University of Porto, Portugal 2 Scripps Institution of Oceanography, University of California San Diego,

La Jolla, CA, United States 3 Department of Zoology and Anthropology, Faculty of Sciences, University of Porto, Portugal

4 Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, United States

[email protected]

Allelopathy refers to the chemically mediated effects (positive or negative) of one plant on another plant (including microalgae and cyanobacteria). This phenomenon is thought to play an important role in cyanobacterial ecology. We had previously reported that the spent medium from one strain of the filamentous cyanobacterium Oscillatoria sp. inhibited the growth of the eukaryotic microalga Chlorella vulgaris1. This motivated the bioassay guided isolation of the compound responsible for the allelopathic activity. An active pure compound was obtained after HPLC purification of a methanolic fraction of the extracted cyanobacterial biomass. A combination of NMR and MS was used to study the structure of the compound. The

molecule is of peptidic nature, has a molecular weight of 1531 Da and is structurally related to the cyanobacterial metabolites tychonamides and pahayokolides. The spent medium did not appear to contain the isolated compound; however, a smaller (1312 Da) and structurally related compound was present. We hypothesize that the intracellular 1531 Da compound is converted to a 1312 Da extracellular form that mediates the allelopathic interaction.

1. Leão PN, Vasconcelos MTSD, Vasconcelos VM (2009) Allelopathic activity of low cell densities of cyanobacteria on microalgae. Eur J Phycol (in press).


151

PC 80

PARABEN COMPOUNDS AS CHEMICAL MEDIATORS FROM THE BACTERIAL MARINE STRAIN MICROBULBIFER SP. L4-N2

Elodie Quévrain1, Isabelle Domart‐Coulon2,and Marie‐Lise Bourguet‐Kondracki1

1 Molécules de Communication et Adaptation des Micro-Organismes, Muséum National d’Histoire Naturelle, FRE 3206 CNRS/MNHN, 57 rue Cuvier (C.P. 54), 75005 Paris, France

2 Laboratoire de Biologie des Organismes Marins et Ecosystèmes, Muséum National d’Histoire Naturelle, UMR 5178 MNHN-CNRS-UPMC, 57 rue Cuvier (C.P. 51), 75005 Paris, France

[email protected]

Over the last decade, sponge-associated micro-organisms have been increasingly studied because they provide a sustainable source of biologically active natural products, previously thought to be produced by marine sponges. 1

The cultivable bacterial community of the calcareous sponge Leuconia nivea (class Calcarea, order Calcaronea, family Baeriidae) collected off Concarneau (Northeast Atlantic, France) was characterized. The antimicrobial activity of heterotrophic bacteria isolated from this sponge has been examined and their impact on the sponge bacterial associates has been studied.

One of the most active strains isolated from L. nivea was phylogenetically affiliated with the genus Microbulbifer (sub-class gamma-Proteobacteria, family Alteromonadaceae) with 99.8% sequence homology with the 16S RNA of Microbulbifer arenaceous.2,3 Bacterial metabolites from this Microbulbifer strain L4-n2 were purified and structurally elucidated. 3 We found a broad variety of natural parabens, including four novel structures and the known ethyl and butyl paraben.4 Parabens are compounds widely used as preservatives in cosmetics, food

and pharmaceuticals which are the core of an industrial R&D project in collaboration with CoValMar.

Their natural role in the marine environment has not yet been explored. The major natural paraben compound was detected by LC/MS in the whole sponge’s crude extract during all seasons, showing its persistent in situ production within the sponge. Moreover, Microbulbifer sp. L4-n2 was localized in the sponge body wall using fluorescence in situ hybridization with a probe specific to L4-n2 phylotype and M. arenaceous.3 Co-detection for the first time in the sponge host of both paraben metabolites and Microbulbifer sp. L4-n2 indicate production of natural parabens in a sponge, which have an ecological role as chemical mediators.

1. Piel J. Curr. Med. Chem. 2006, 13, 39-50.

2. Tanaka T., Yan L., Burgess JG., Curr. Microbiol., 2003, 47, 412-416.

3. Quévrain E., Domart-Coulon I., Pernice M., Bourguet-Kondracki ML. Environ. Microbiol., 2009, DOI: 10.1111/j.1462-2920.2009.01880.x

4. Peng X., Adachi K., Chen C., Kasai H., Kanoh K., Shizuri Y., Misawa N., Appl. Environ. Microbiol., 2006, 72, 5556-5561.


152

PC 81

INTER-KINGDOM MOLECULAR DIALOGUE IN A SYMBIOSIS EUKARYOTE/PROKARYOTE MODEL BETWEEN THE DEMOSPONGE

SUBERITES DOMUNCULA AND BACTERIA Johan Gardères1, J. Ben Saidin1, L. Taupin1, M. Wiens2 and Gäel Le Pennec1

1 Laboratoire de Biotechnologies et Chimie Marines. Lorient. France. 2 Institut für Physiologische Chemie. Mainz. Deutschland.

[email protected]

Sponges live in close association with many bacteria for over 600 millions years. 40 to 60% of the sponge biomass consist of prokaryotes. Thus, numerous relationships must co-exist in a symbiotic point of view: commensal, opportunist, etc. to govern the homeostasis of this community. Thus, molecular communications must have been developed between partners, defining role(s) and function(s) of each protagonist. Accordingly, each partner, in physiological conditions, must be able to produce communication molecules.

In particular depending Quorum Sensing conditions, bacteria produce communication molecules like homoserine lactone (HSL). Out of the bacterial biomass conditions present in biofilm, we were able to detect the C6-HSL, the C7-HSL and the 3oxoC12-HSL using LC-ESI-MS/MS in sponge extracts. This is the first report of HSL detection in crude extract of marine invertebrate organisms reflecting an in vivo HSL production within a marine eukaryote. Then, we investigated effects of these molecules on sponge cells. In vitro stimulation with HSL modified the electrophoresis pattern of sponge proteins. Future characterization of the de novo protein synthesis will allow to better understanding molecular perception of bacterial dialogue molecules in sponges.

Higher eukaryotes communicate with bacteria using hormones, notably by catecholamines (Hugues et al., 2008). Futhermore, the sponge Suberites domuncula is a useful model to study metazoan physiology since it possesses most metabolic pathways that are also found in human and other vertebrates (Wiens et al., 2001; Müller et al., 2004; Schröder et al., 2005; Wiens et al., 2007). Thus, we searched for catecholamine production in this sponge. Genes for enzymes regulating metabolism of catecholamines have been identified in S. domuncula. Catecholamines represent an interesting target of research in sponge communication for bacteria.

Hugues DT, Sperandio V. (2008) Nature Reviews Microbiology. 6: 111-120.

Müller WE, Thakur NL, Ushijima H, Thakur AN, Krasko A, Le Pennec G, Indap MM, Perovic-Ottstadt S, Schröder HC, Lang G, Bringmann G. (2004) J Cell Sci. 117: 2579-90.

Schröder HC, Perović-Ottstadt S, Grebenjuk VA, Engel S, Müller IM, Müller WEG. (2005) Genomics 85: 666-78.

Wiens M, Diehl-Seifert B, Müller WEG. (2001) Cell Death Differ. 8: 887-98.

Wiens M, Korzhev M, Perović-Ottstadt S, Luthringer B, Brandt D, Klein S, Müller WEG. 2007.. Mol Biol Evol 24: 792-804.


153

PC 82

ECOLOGICAL IMPACT OF BIOLOGICALLY ACTIVE METABOLITES PRODUCED BY LAMINARIA SACCHARINA ASSOCIATED

PSEUDOMONAS SP. STRAINS Kerstin Nagel, I. Schneemann, J. Wiese, I. Kajahn, A. Labes, J.F. Imhoff

Kieler Wirkstoff-Zentrum and IFM – GEOMAR

[email protected]

Laminaria saccharina belongs to the brown macroalgae and is distributed in temperate to polar rocky coastal ecosystems1. Recently, we described a specific association of bacterial communities with different parts of Laminaria saccharina. The alga was shown to harbour a quite diverse community of antimicrobially active bacteria, including Pseudomonas strains2. Antimicrobial activity seems to be widespread among algae-associated bacteria: From the epiphytical bacterial community of Laminaria, 210 isolates were obtained. 130 strains inhibited the

growth of at least one microorganism from a standard test panel. In this study, biologically active secondary metabolites produced by L. saccharina associated Pseudomonas sp. strains were extracted, purified and identified. It was shown that the Pseudomonas sp. strains produce a number of compounds displaying a strong antimicrobial activity. The ecological function of these compounds in the algae-bacteria interaction will be discussed.

1. Bartsch, I. et al. 2007. Europ J Phycol 43:1-86. 2. Wiese, J. et al. 2009. Mar Biotechnol (NY)

11:287-300


154

PC 83

CHEMICAL ECOLOGY OF TUNICATES OF THE GENUS APLIDIUM (CHORDATA: ASCIDIACEA) FROM ANTARCTIC WATERS

Laura Núñez Pons1* , Roberto Forestieri2, Michela Nappo1, Rosa Mª Nieto3, Jaime Rodríguez González3, Carlos Jiménez3, Mercedes Varela4, Alfonso Ramos5, Francesco

Castelluccio2, Marianna Carbone2, Margherita Gavagnin2 and Conxita Ávila1

1 Dept Biologia Animal (Invertebrats), Fac. Biologia, Univ. Barcelona, Catalunya, Espanya. 2 Istituto di Chimica Biomolecolare - CNR, Pozzuoli, Napoli, Italia.

3 Dept Química Fundamental, Fac. Ciencias, Univ. A Coruña, España. 4 Dept Ecoloxia e Bioloxia Animal, Fac. Ciencias do Mar, Univ. Vigo, España.

5 Dept Ciencias del Mar y Biología Aplicada. Univ. Alicante, España.

[email protected]

Sessile marine invertebrates are especially vulnerable to mobile predators and competitors fighting intensely for space and food. The ability of these organisms to chemically defend themselves can play a significant role in their survival, regulating prey-predator interactions and structuring marine communities1,2,3. Classically predation pressure as well as production of marine chemical defenses was hypothesized to decrease with latitude4. However, an intense predation caused by macroinvertebrates, such as sea stars, has been observed in Antarctic communities, commeasurable to that reported in temperate and tropical waters5. Even if chemistry in polar seas is still understudied compared to other areas, a good number of natural products has been isolated from Antarctic marine invertebrates so far6. Specimens of a well represented tunicate genus from the Antarctic Ocean have been studied here. The genus Aplidium (Chordata: Ascidiacea) includes a large number of species distributed all over the world, with around 40 Antarctic species described7. This group of colonial ascidians is

considered a very rich source of secondary metabolites8. But even though a number of natural products have been discovered in Aplidium ascidians, the knowledge of the putative ecological functions of these compounds remains mostly unknown. We report here some examples of chemical ecology studies conducted with species of the genus Aplidium from Antarctica, as well as the results obtained in bioassays employing sympatric predators, both with crude extracts and isolated compounds.

1. Paul, V. J. (1992). New York, Comstock Publications Association, Ithaca

2. Pawlik, J. R. (1993) Chem. Rev. 93: 1911-1922

3. Hay, M. E. (1996). J. Exp. Mar. Biol. Ecol. 200: 103-134

4. Bakus, G. J. and G. Green (1974). Science 185:951-953

5. Amsler, C. D. et al. (2000). In: Davidson, W., C. Howard-Williams & P. Broady (eds.), Proc. 7th SCAR Int. Biol. Sym. N.Z. Nat. Scie. Christchurch, New Zealand: 158-164

6. Avila, C. et al. (2008). Mar. Ecol. 29: 1-70 7. Varela, M. (2007). PhD. Dept. Univ. Alicante 8. Zubía, E. et al. (2005). Mini-Rev. Org. Chem.

2: 389-399.


155

PC 84

ELEMENTAL COMPOSITION OF MARINE SPONGE FROM COASTAL OF THAILAND

Pannee Pakkong1, Pichan Sawangwong2, Wanvisa Phongphern1 1 Department of Applied radiation and Isotopes, Faculty of Science,

Kasetsart University ,Thailand 2 Environmental Program, Faculty of Science, Burapa University, Thailand

[email protected]

Analytical studies of sponges have recently become a matter of interest, since these organisms have been reported to accumulate high levels of some elements, including heavy metals, which may have allowing their application as environmental pollution indicators. In the present work, 10 marine sponges collected at the upper Gulf of Thailand were classified and analyzed by secondary target energy-dispersive x-ray fluorescence spectrometry (EDXRF), OXFORD ED2000. Some major, minor and trace elements could be determined: Al, P, S, K, Ca, V, Mn, Fe, Ni, Cu, Zn, As, Br, Rb, Sr, Ba, I and Pb. Besides these elements, sponges are constituted by fairly high percentages of elements that do not emit characteristic x-ray lines. Calibration was performed using NIST-8414 Bovine muscle standard reference materials. Precision, accuracy and detection limits for the range of elements determined by software Isis 300 for chemical analysis of these organisms. Results for the chemical composition indicated the absence of any trace metal contamination in the region.

Certain elemental contents determined in some species indicated a clear selective bioaccumulation of particular trace elements, such as Ni, Zn and As, which is not dependent on local influences.

Keywords: marine sponge; EDXRF; elemental composition.

1. Ludmila Ph. Paradina, Natalie N. Kulikova, Alexander N. Suturin Yelena V. Saibatalova. Geostandards and Geoanalytical Research. 2007, 28: 225-232.

2. Maria Fátima Araújo, Ana Conceição, Teresa Barbosa, Maria Teresa Lopes , Madalena Humanes . X-Ray Spectrometry. 2003,32:428-433

3. Uthaiwan, K., Pakkong, P., Noparatnaraporn, N., Vilarinho, L. , Machado, J. Invertebrate Reproduction and development .2003.44(1) 53-61.

4. Araujo, Maria Fatima, Alexandra Cruz, Madalena Humanes, Maria Teresa Lopes,Jose Armando. Chemical Speciation and Bioavailability 1999: 25-36

5. Oxford Instruments Industrial Analysis Group. ED2000 Operator’s Manual. 1995 (Handbook). Oxford Instruments Industrial Analysis Group.


156

PC 85

UNUSUAL SAXITOXIN METABOLITES IN BIVALVES CONTAMINATED BY GYMNODINIUM CATENATUM

Paulo Vale

Instituto Nacional dos Recursos Biológicos / L-IPIMAR (INRB/L-IPIMAR), Av. Brasília, s/n, 1449-006, Lisboa, Portugal

[email protected]

Toxin profiles of bivalve molluscs contaminated after ingestion of dinoflagellates that produce saxitoxin (STX) analogues have been grouped in three families: carbamoyl, N-sulfocarbamoyl, and decarbamoyl. Recently, hydroxybenzoate analogues were characterised as an important fraction of the STX analogues produced by Gymnodinium catenatum. However, their metabolisation in bivalves increases the percentage of the decarbamoyl group, while reducing the contribution of hydroxybenzoate groups to the total toxin profile. New and unusual bivalve metabolites were recently described as an important toxin fraction in mussels from southeast Nova Scotia (Canada) contaminated by Alexandrium tamarense, originated by single or double hydroxylation at C11 position of STX and B1. These possess very low fluorescence, and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS) is the best technique to study its presence.

HILIC-MS was implemented to look for these new metabolites in several bivalve species strongly contaminated during G. catenatum blooms occurring at the

Portuguese coast. The presence of M1 was tentatively identified in a variety of bivalve species, ranging from estuarine habitat: blue mussels (Mytilus galloprovinciallis), cockles (Cerastoderma edule) and clams (Ruditapes decussatus), to oceanic habitat: clams (Donax trunculus) and razor clams (Ensis spp.). Despite absence of standards, it was hypothesized it could contribute to an important fraction of the profile of STX analogues. Regarding the three estuarine species, curiously M1 was more abundant in bivalve species that usually retain longer PSP toxins, in the following order: mussels > cockles > clams.

Hydroxylation of B1 results in M1 and M3. The high abundance of B1 and B2 in G. catenatum raised the question if B2 can also be hydroxylated. However, the single quadrupole MS instrument available was not adequate to screen for these analogues due to its poor selectivity and response. The presence of traces of M3 and hydroxilated B2, presenting the same molecular mass, could not be confirmed with mass spectra, but were suspected from positive selected ion monitoring ratios.


157

PC 86

PHOSPHOLIPID FATTY ACIDS OF THE TUNICATES EUDISTOMA SP. AND LEPTOCLINIDES SP. FROM THE GULF OF TADJOURA (DJIBOUTI) Flore Dagorn, Justine Dumay, Gaëtane Wielgosz‐Collin, Vony Rabesaotra,

Jean‐François Biard and Gilles Barnathan

Université de Nantes, Pôle Mer et Littoral, MMS EA 2160, Equipe Lipides marins à activité biologique, Faculté de Pharmacie, 1 rue G. Veil, BP 53508, Nantes F-44035, France

[email protected]

Tunicates and ascidians (class Ascidiacea) are marine invertebrates now well-known to be a rich source of unique and biologically active metabolites, lipids and lipophilic compounds (Kornprobst, 2005; Bergé and Barnathan, 2005), in particular from the Eudistoma genus. In spite of this interesting ascidian chemistry, very few studies have been directed towards the ascidian lipids.

As part of our ongoing comparative studies, this work aims at extending knowledge on ascidian lipids, two tropical tunicates, Eudistoma sp. and Leptoclinides sp., from the Gulf of Tadjoura (Djibouti) and to compare with data previously published on other species living at different places (Viracaoundin et al., 2003). Eudistoma sp. and Leptoclinides sp. were composed of respectively 0.86 % and 0.82 % of lipids (related to dry matter) including phospholipids (38.2 and 30.2 % respectively). Gas chromatography-mass spectrometry (GC-MS) analyses of their methyl esters and N-acyl pyrrolidide derivatives revealed

twenty-nine fatty acids (FA) in Eudistoma sp. and twenty-one in Leptoclinides sp., ranged from C12 to C24 chain lengths. In both organisms, the most abundant FA (>10%) were the 14:0, 9-16:1, 9-18:1, 11-18:1, 20:5n-3 acids. Leptoclinides sp. contained a considerable amount (24.7%) of the 20:5n-3 acid (8.7% in Eudistoma sp.). Fatty acid compositions of Eudistoma species were found quite different, and this is the first investigation on lipids from Leptoclinides species.

Kornprobst JM (2005) Substances Naturelles d’Origine Marine. Tec&Doc-Lavoisier, Paris, France, Tome 2, pp. 1589-1729

Bergé JP, Barnathan G (2005) Recent advances in FA from lipids of marine organisms : molecular biodiversity, roles as biomarkers, biologically-active compounds and economical aspects. In Marine Biotechnology, Y. Le Gal, R. Uber (Eds), Series Adv. Biochem. Eng. Biotechnol., Springer, Heidelberg, Germany, 96, 49-125

Viracaoundin I, Barnathan G, Gaydou EM, Aknin M (2003) Phospholipid FA from Indian Ocean Tunicates Eudistoma bituminis and Cystodytes violatinctus. Lipids 38:85-88


158

PC 87

STUDIES OF MARINE TOXINS FROM DINOFLAGELLATES CULTURES Humberto J. Domínguez1, José J. Fernández1, M. Norte1,

Maria L. Souto1 and Antonio H. Daranas1,2 1 Instituto Universitario de Bioorgánica “Antonio González” Departamento de Química Orgánica.

Universidad de La Laguna. La Laguna 38206, Canary Islands. Spain. 2 Departamento de Ingeniería Química y Tecnología Farmacéutica; Universidad de La Laguna;

Av. Astrofísico Francisco Sánchez 1, La Laguna, 38071, Canary Islands, Spain.

[email protected]

Some species of dinoflagellates have interest because they are producers of potent biotoxins, responsible for the "Harmful Algal Blooms" (HABs) also known as red tides. In recent years they have been produced an increase of the HABs, which has a great impact on environmental as well as on the public health. 1

Protoceratium reticulatum have been identified a producer of yessotoxin (YTX).2 YTXs have been associated with diarrhetic shellfish poisoning events (DSP) because they are often simultaneously extracted with DSP toxins, and give positive results when tested in the conventional mouse bioassay for DSP toxins. However, recent evidence suggests that YTXs should be excluded from the DSP toxins group, because unlike okadaic acid (OA) and dinophyisistoxin-1 (DTX-1), YTXs do not cause diarrhoea when orally administered.3-5

However, study of these toxins does not deal solely with the repercussions on

public health, as these substances have various and complex structures and activities, making them valuable tools for studying cellular processes. In this communication we report the isolation and structure determination of new derivatives of DSP toxins.

1. Paz, B.; Daranas, A.H.; Norte, M.; Riobo, P.; Franco, J.M.; Fernandez, J.J. Mar. Drugs 2008, 6, 73-102

2. Murata, M.; Masanori, K.; Lee, J.-S.; Yasumoto, T. Tetrahedron Lett. 1987, 28, 5869-5872

3. Rhodes, L.; McNabb, P.; de Salas, M.; Briggs, L.; Beuzenberg, V.; Gladstone, M. Harmful Algae 2006, 5, 148-155

4. Paz, B.; Riobó, P.; Fernández, M. L.; Fraga, S.; Franco, J. M. Toxicon 2004, 44, 251-258

5. P. Riobó, B. Paz, J.M. Franco Soler J. Phycology. 2004, 41(1), 212-225.

The authors thank the financiation of the MEC (2008CTQ-06754-C04-01/PPQ); H.D. to the MEC by the Scholarship F. P. U. The strain of P. reticulatum was facilitated by S. Fraga, of the CCVIEO (Vigo, Spain).


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PC 88

CHEMICAL FINGERPRINTING OF SPONGIA AGARICINA ACROSS THE NORTHWESTERN MEDITERRANEAN

Charlotte Noyer1, Olivier P. Thomas2, Mikel A. Becerro1 1 Department of Marine Ecology, Centro de Estudios Avanzados de Blanes (CEAB, CSIC),

Acceso Cala St. Francesc 14, Blanes 17300, Girona, Spain. 2 Laboratoire de Chimie des Molécules Bioactives et des Arômes (LCMBA), UMR CNRS-UNSA

6001, Université de Nice - Sophia Antipolis, Faculté´ des Sciences, Parc Valrose, 06108 Nice Cedex 02, France.

[email protected]

Spongia agaricina is a well known sponge traditionally collected as a bath sponge. Various compounds, such as furanoterpenes, have been isolated from this species but little is known about the spatial and temporal variability of these secondary metabolites. Here, we report the patterns observed in space and time for the metabolites of Spongia agaricina. We sampled seven populations of S agaricina covering a distance of 1200 km of the Northwestern Mediterranean. We obtained crude extracts that were compared between sponge populations, geographic area, and sampling season using HPLC chemical fingerprints. We purified and used the major compound nitenin as an internal reference to analyze the chemical fingerprints, and performed quantitative analyses using calibration curves. Concentrations of nitenin and dihydronitenin varied unsignificantly regardless location or season. However,

we found significant differences in chemical fingerprints between sponge populations based on the ELSD chromatograms. 12-epi-scalarin contributed the most to the overall chemical dissimilarities between the geographic areas of Catalonia and Marseille. In fact, we found a significant correlation between geographic distance and dissimilarities values of chemical fingerprints. At regional geographic scales, we found significant differences between the 3 populations sampled in Catalonia (spread over 100 km). At local geographic scales, we found no differences between the 3 populations sampled in Marseille (spread over 20 km). Our results suggest that variation in secondary metabolites in S. agaricina might be driven by factors associated with medium and large scale geographic distance.


160

PC 89

NEW ENROFLOXACIN SENSORS FOR AQUACULTURE ENVIRONMENT Ayman H. Kamel, Felismina T. C. Moreira, J. Rafaela L. Guerreiro and M. Goreti F. Sales

REQUIMTE, Instituto Superior de Engenharia do Porto, R. Dr. António Bernardino de Almeida, 431, 4200-072 Porto, Portugal.

[email protected]

Aquaculture is the fastest-growing form of global food production and cultures both saltwater of freshwater species. This significant growth has been observed over the past decade, after the introduction of veterinary medicines, such as enrofloxacin (ENR), one of the several antimicrobials administered to fish in aquaculture environment1. Its wide use has led to environmental and food spread of medicines, and may result in the emergence of antibiotic-resistant bacteria in water environments2,3. ENR residues are also potentially persistent4 and may be found in fish5 and water, posing environmental and food-safety problems. To avoid this, aquaculture facilities should handle small ENR doses that are strictly controlled.

Hence, this work proposes a new small biomimetic sensor for ENR capable of host-guest interactions and potentiometric transduction. The artificial host is imprinted in methacrylic acid and/or 2-vinyl pyridine based polymers. Molecularly imprinted particles are dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. The potentiometric sensors exhibit a near-Nernstian response in steady state evaluations. Slopes and detection limits range 48 – 63 mV decade-1 and 0.28 – 1.01 µg mL-1, respectively. The sensors are independent from the pH of test solutions within 4–7. Good selectivity is

observed towards potassium, calcium, barium, magnesium, glycine, ascorbic acid, creatinine, norfloxacin, ciprofloxacin and tetracycline. In flowing media, the sensors display good reproducibility (RSD of ± 0.7%), fast response, good sensitivity (47 mV decade-1), wide linear range (1.0x10-5 – 1.0x10-3 M), low detection limit (0.9 µg mL-1), and a stable baseline for a 5x10-2 M acetate buffer (pH 4.7) carrier. The sensors are successfully applied to field monitoring of ENR in fish samples. The method offers the advantages of simplicity, accuracy, and automation feasibility.

1. Council Regulation (EEC) 2377/90 of 26 June 1990. Consolidated with previous amendments and published by 19 November 2005

2. F.C. Cabello, Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment, Environment. Microb. 8 (2006) 1137–1144

3. T. Maki, I. Hirono, H. Kondo, T. Aoki, Drug resistance mechanism of the fish-pathogenic bacterium Lactococcus garvieae, J. Fish Diseases 31 (2008) 461–468

4. S. Gräslund, B.E. Bengtsson, Chemicals and biological products used in south-east Asian shrimp farming, and their potential impact on the environment - a review, The Sc. Tot. Environ. 280 (2001) 93–131

5. W.H. Xu, X.B. Zhu, X.T. Wang, L.P. Deng, G. Zhang, Residues of enrofloxacin, furazolidone and their metabolites in Nile tilapia (Oreochromis niloticus), Aquaculture 254 (2006) 1–8


161

PC 90

PHYCO-OXYLIPINS AS SPECIES-SPECIFIC CHEMICAL MARKERS Nadia Lamari1, Marina Montresor2, Carmen Minucci2, Adele Cutignano1,

Giuliana d’Ippolito1 and Angelo Fontana1 1 CNR-Istituto di Chimica Biomolecolare, Via Campi Flegrei 34, 80078 Pozzuoli - Napoli, Italy

2 Phytoplankton Ecology and Evolution, Stazione Zoologica Anton Dohrn, Villa Comunale 80121 Napoli, Italy

[email protected]

Diatoms are a group of unicellular microalgae responsible for extensive blooms and thus play a crucial role in the global carbon cycle. Species of the pennate marine genus Pseudo-nitzschia are significant contributors to phytoplankton blooms in coastal and oceanic waters and some of these species can produce domoic acid, a neurotoxin causing Amnesic Shellfish Poisoning. The taxonomy of diatoms is mainly based on morphological characters such as ultrastructural features of their siliceous frustule. Genetic analyses carried out with molecular markers (LSU rDNA and ITS regions) highlighted the presence of an unsuspected level of cryptic diversity: different and reproductively distinct genotypes exist behind what was considered a single morpho-species.1 Phyco-oxylipins (lipoxygenase-derived fatty acid derivatives in marine diatoms) have been reported from many different genera of centric marine diatoms.2 Recently, lipoxygenase products have been described in the marine pennate diatom P. delicatissima.3 Here we report

liquid chromatography-mass spectrometry profiles of five different Pseudo-nitzschia species isolated from the Gulf of Naples, including three P. delicatissima-like pseudo-cryptic species (see ref. 1 for further details), to evaluate the spectrum of phyco-oxylipins diversity. Up-to-date analysis shows that a large fraction of diatoms’ secondary metabolites arise from lipoxygenase pathways. These molecules, that likely play as functional mediators, may have also work as chemical markers to assist the taxonomy of this lineage of microalgae.

1. Alberto Amato, Wiebe H.C.F. Kooistra, Jung Hee Levialdi Ghiron, David G. Mann, Thomas Pröschold and Marina Montresor, 2007, Protist, 158 (2), 193- 207.

2. Giuliana d’Ippolito, Adele Cutignano, Raffaella Briante, Ferdinando Febbraio, Guido Cimino and Angelo Fontana, 2005, Org. Biomol. Chem., 3, 4065 – 4070.

3. Giuliana d’Ippolito, Nadia Lamari, Marina Montresor, Giovanna Romano, Adele Cutignano, Andrea Gerecht, Guido Cimino and Angelo Fontana, 2009, New Phytologist, in press.


162

PC 91

MYCOSPORINE-LIKE AMINO ACIDS (MAAS) FROM SEAWEEDS: RELATION WITH NITROGEN AVAILABILITY AND POTENTIAL APPLICATION AS

PHOTOPROTECTOR AND ANTIOXIDANT COMPOUNDS Maria Helena Abreu1, Nathalie Korbee2, Rui Pereira1, Isabel Sousa Pinto1,

Célia Gil2 and Félix L. Figueroa2 1 CIMAR University of Porto

2 Department of Ecology. University of Málaga. 29071- Málaga SPAIN

[email protected]

Recent trends in drug research from natural sources have shown that algae are promising organisms to furnish novel biochemically active compounds. Mycosporine-like amino acids (MAAs) are a family of intracellular water-soluble N-compounds involved in photoprotection against ultraviolet radiation (UV). These substances are already being commercially explored as suncare products for protection of skin. The potential of MAAs in photoprotection can be considered high due to a double function: (1) UV chemical screening with high efficiency for UVB and UVA regions of the solar spectrum, and (2) their antioxidant capacity. MAAs have been reported in several aquatic organisms around the world. Among macroalgae,

Rhodophytes accumulate MAAs in different types and concentrations in function of the species. Besides solar radiation, other environmental factors such as salinity, temperature and nutrient availability may affect MAAs accumulation. Seaweed based Integrated Multi- Trophic Aquaculture (IMTA) systems are gaining importance as they help to mitigate the environmental problems caused by several forms of fed aquaculture. The seaweed biomass here produced will be N-enriched and thus might contain high levels of MAAs. Preliminary results obtained from experimental work done in these systems with the red macroalgae, Gracilaria vermiculophylla, Gracilaria cornea and Hypnea spinella are presented.


163

PC 92

CHITOSAN NANOPARTICLE FORMATION: SYSTEMATIC CONDITION BASED ON GAMMA IRRADIATION AND CHEMICAL MODIFICATION STUDIES

Wanvimol Pasanphan1, Pakjira Rimdusit1, Surakarn Choofong1 and Suwabun Chirachanchai2

1 The Department of Applied Radiaiton and Isotopes, Faculty of Science, Kasetsart University 2 The Petroleum and Petrochemical College, Chulalongkorn University

[email protected]

Chitosan has been paid more interest as a biopolymer proper for a drug delivery system based on its biocompatibility,1 bioactivity,2 and biodegradability3 due to its bio-copolysaccharide structure. Over the past few years, “nano” scaled material is presently defined as a little word with big potential. Therefore, the development of chitosan to material for the drug delivery system is also been developed in term of nanoparticle. In the past, chitosan particle has been prepared via spray-drying coagulation,4 suspension cross-linking,5 chemical modification,6 etc. The size previously observed was mostly higher than 100 nm. Here, the work focuses on an approach to prepare the chitosan nanoparticle in the potential range of 1-100 nm, which is defined as a nanoscale.7 The systematic preparation is carried out by using gamma irradiation as a simple and effective method together with common chemical modification one. In this way, gamma radiation doses are 5, 10, 20, 40, and 100 kGy and chemical modification is performed by conjugating with deoxycholic acid molecule. The effect of irradiated chitosan forms, i.e. (i) solid state, (ii) colloidal, and (iii) acidic solution, to molecular weight by viscometer, to chemical structure by FTIR, and to particle size by TEM are studied. The molecular weight of chitosan is reduced by radiation degradation to be 10, 25, and >1000 times for chitosan irradiating in the form of solid state, colloidal, and acidic solution respectively, when gamma radiation dose is increased to 100 kGy. Additionally, the chemical structure still remains. The particle size decreases when the radiation dose is

increased. For solid chitosan, the size is reduced to 150-100 nm and 100-30 nm, when the radiation doses of 5 kGy and 10-100 kGy, respectively, while the size of the original one is 350-150 nm. In the case of colloidal chitosan, the sizes observed are 80-60 nm (5 kGy) and 70-30 nm (10-100 kGy). The relationship between the radiation dosage and the particle size in the case of irradiating chitosan in acidic solution can not be observed. After conjugating with deoxycholic acid, the size of chitosan particle irradiated in solid form decreases to 125, 100-50, and 70-50 nm and it greater reduces to 125, 90, and 50-30 nm in colloidal form when the chitosan is irradiated with the doses of 0, 5, 10-40 kGy, respectively. In conclusion, gamma radiation can easily reduce the particle size of chitosan in the range of 1-100 nm. The chemical modification with deoxycholic acid not only provides hydrophobic core for nanoparticle but also effectively reduces the particle size of chitosan. Irradiation of chitosan in colloidal form is even more systematically effective in preparation of chitosan nanoparticle than that of solid and acidic solution form. 1. Richardson, S. C., Kolbe, H. V., Duncan, R. Int. J.

Pharm., 1999, 178: 231-243. 2. Dumitriu, S., Popa, D M. I., Cringu, A., Stratome,

A. Colloid. Polym. Sci. 1989, 267: 595-599. 3. Yamamoto, H. Amaike, M. Macromolecules. 1997,

30: 3936-3937. 4. Mi., F. Wong, T., Shyu, S., Chang, S. J. Appl.

Polym. Sci. 1999, 71: 747-759. 5. Denkbas, E., Odabasi, M. J. Appl. Polym. Sci. 1999,

71: 1637-1643. 6. Yoksan, R., Akashi, M., Hiwatari, K.I.,

Chirachanchai, S. Biopolymers 2003, 69: 386-390 7. Ratner, M., Ratner, D. 2007. Nanotechnology: a gentle

introduction to the next big idea. Printice Hall PTR: New Jersey. p.7


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PC 93

EFFECTS OF IRRADIATED CHITOSAN ON THE THE GROWTH OF SOYBEAN Jariya Prasatsrisupab 1, Vitaya Thananuson1, Pannee Pakkong2

1 Senior Expert Office, Department of Agriculture, Chatujak, Bangkok 10900, Thailand 2 Faculty of Science, Kasetsart University, Chatujak, Bangkok 10900, Thailand

[email protected]

A 18 kilogram sand pot experiment was conducted to determine the effects of chitosan which were irradiated with gamma ray at doses of 25, 50, 75, 100 Krad on the growth of soybean comparing with control (no rhyzobium, no chitosan), rhyzobium and rhyzobium plus chitosan. Nitrogen-free nutrient solution (Broughton and Dillworth, 1970) was applied 1 litre in the pot at the beginning of the experiment and applied 50 ml once a week. Water was applied to keep the sand moisture in field capacity level. Phosphorus, as Triple Superphosphate (TSP), at 5 g/pot and potassium as muriate of potash (KCl) at 3 g/pot were incorporated into the pot. The treatments with rhyzobium, soybean seeds (SJ. 5) were inoculated with rhyzobium before growing. The 25 ml solution of irradiated chitosan (100 ppm) was sprayed into the soybean plant every two week up to harvesting (50 days after growing). Control plants were also sprayed with

distilled water without chitosan. Agronomic data on plant height, number of nodes, number of branches, number of flowers were recorded upon the harvest of experiment. The oven dried weight of soybean plants, roots and nodules were recorded.

The results showed that soybean plants in control and chitosan without rhyzobium became yellow. Agronomic data of soybean which were treated with rhyzobium plus chitosan and only rhyzobium gave the non significant difference result but gave the significantly better result than in control and only chitosan. The effects were observed in the different doses of irradiated chitosan plus rhyzobium on plant height, number of nodes, number of branches, number of flowers and oven dried weight of soybean plant, root and nodule. Chitosan irradiated at 50 and 75 Krad gave the best result.


165

PC 94

SPONGES AS A NEW SOURCE OF MELANIN Marco Araújo1, J. Xavier2, Madalena Humanes1

1 Centro de Química e Bioquímica – Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Edifício C8, Campo Grande, 1749-016 Lisboa, Portugal

2 Institute for Biodiversity and Ecosystem Dynamics (IBED) and Zoological Museum of Amsterdam (ZMA), University of Amsterdam, Mauritskade 57, 1092 AD Amsterdam, The Netherlands and CIBIO - Pólo Açores, Centro de Investigação em Biodiversidade e Recursos

Genéticos, e Departamento de Biologia, Universidade dos Açores, Rua Mãe de Deus, 9501-855 Ponta Delgada, Portugal.

[email protected]

The aim of this work is to isolate and characterize a black pigment present in some marine sponges facing the possibility of being melanin. Melanins are hydrophobic, negatively charged and paramagnetic polymers, which can cover a wide range of values of molecular mass, depending on their structure and their origin.

These organic and amorphous polymers absorb UV radiation, transforming it in chemical energy, but they have also antioxidant and semiconductor properties.

The extraction and isolation method was adapted from the method for the extraction of the black pigment of the fungus Aspergillus nidulans. The isolation of the black pigment from sponges was achieved, although in small amounts, but free of silica. The next step consisted in the characterization of the isolated pigment using techniques such as UV-Vis, DRX, IV and SEM-EDS.

The obtained results suggest that we were in presence of melanin polymer with a high degree of purification.

The images obtained by SEM showed different structures and arrangements for

all the analysed melanins, suggesting that we were in presence of a polymer with a very disordered and variable structure.

The higher content in carbon and oxygen observed in EDS analysis compared to the proportion of the same elements in commercial melanin, suggests a more complex structure of these natural melanins. Oxygen content can also constitute an evidence of the radical scavenging and antioxidant capacity, characteristic of the majority of melanins. Furthermore, the small content in silicon observed through the EDS analysis of the black pigments, supports the affirmation above about its degree of purification.

Technological applications for these polymers arise great expectations, for instance photovoltaic panels, due to their capacity of converting the absorbed UV radiation in chemical energy, The ability of quenching the water molecule, in a reaction where resultant products are hydrogen, oxygen and electrons, can be used to power generation; due to their antioxidant and radical scavenging properties they can be used in other areas, like medicine.


166

PC 95

CHITOSAN-PROTEIN COMPLEX COACERVATES: EFFECT OF PROCESS VARIABLES

Bianca N. Barreto1,2, H. K. S. de Souza2, P. Sampaio3, C. T. Andrade1 and M. P. Gonçalves2

1 Instituto de Macromoléculas Professora Eloisa Mano, IMA- Universidade Federal do Rio de Janeiro, P.O. Box 68525, 21945-970 Rio de Janeiro, Brazil.

2 REQUIMTE, Chemical Engineering Department, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal. 3 - IBMC – Instituto de Biologia Molecular e

Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.

[email protected]

Complex coacervation is a particular case of associative phase separation, which is induced by electrostatic interactions between oppositely charged polymers. Great interest was shown in complex coacervation because of its implication in many biological processes like self-assembly of biological macromolecules and its use in many industrial applications such as microencapsulation1. In this context, chitosan is one of the biopolymers that can be used to form the matrix of coacervates. Chitosan [poly(b-(1-4)-2-amino-2-deoxy-D-glucose)] is a natural cationic polysaccharide with widespread applications in food processing, as well as in agriculture and biomedicine.

In the present work, we characterized coacervates of chitosan-whey protein isolate complexes. A chitosan sample (CS) was obtained by a two-stage heterogenous deacetylation of chitin from shrimp (Penaus Schmitti) wastes. The acetylation degree (DA) of CS was determined by 1H NMR spectrometry and its intrinsic viscosity evaluated in 0.250 M acetate buffer, at 25 ºC, by extrapolation to zero concentration of Kraemer and Huggins’ equations. The viscosity-average molecular mass was calculated by the

Mark – Houwink – Sakurada equation. The interaction of CS with whey protein isolate (WPI) was studied, in 0.100 M acetate buffer, at pH 3 - 5.5 and 25 ºC, using turbidity measurements, rheology and confocal microscopy.

Turbidity measurements were used to provide information about the formation of insoluble complexes. At pH 3, when both CS and WPI are positively charged, no measurable aggregation was detected. When the pH was raised to 5.5, where the protein charge is expected to be negative, the turbidity increased sharply, suggesting that a much stronger interaction takes place. At this pH, the maximum turbidity was obtained for a CS:WPS mixing ratio of 1:9.5 (w/w). CS/WPI coacervates were prepared in the above conditions and their rheological behaviour studied, using dynamic and steady shear experiments. The diffusivity of CS and WPI in the coacervate phase was studied by fluorescence recovery after photobleaching (FRAP) using a confocal laser scanning microscope. The results will be presented and discussed in terms of the structure of the coacervates.

1. Gouin, S., Trends in Food Science and Technology, 15, (2004), 330.


167

PC 96

CROSSLINKING STUDIES ON A NOVEL MARINE COLLAGEN Maria T. Calejo, L.L. Gonçalves, Zilda B. Morais and Ana I. Fernandes

Centro de Polímeros Biomédicos, Instituto Superior de Ciências da Saúde Egas Moniz, Campus Universitário, Quinta da Granja, Monte de Caparica, 2829-511 Caparica, Portugal.

[email protected]

Collagen is regarded as one of the most useful biomaterials for drug delivery1. This structural protein has traditionally been isolated from animals but the need for alternative collagen sources (e.g. marine) has been highlighted. Sustained release of bioactive molecules from collagen matrices is beset with difficulties since they exhibit an effective pore size too large to control drug release by hindered diffusion, a fast degradation rate and low mechanical strength2 and, therefore, the introduction of exogenous crosslinking (CL) is often considered mandatory1.

The present work evaluates the structural properties of collagen extracted from Catostylus tagi3, a medusa from the Portuguese coast, before and after CL. Two CL agents [carbodiimide (EDC) and genipin (GP)] were used and microstructure (before and after microparticle production) of native collagen compared, with that of chemically crosslinked collagen, by SEM.

Both GP and EDC revealed to be effective CL agents for the reticulation of C. tagi collagen, though distinct structures may have been produced. The optimal concentration of the CL agent and time of contact with the biomaterial were primarily addressed. Maximum CL was attained within the first hour and the

degree of CL increased with rising concentrations of EDC or GP. Since GP and EDC react with the protein amino groups by different mechanisms, the use of both chemicals lead to improved CL, which was further accentuated by N-hydroxysuccinimide addition. The resistance of the matrices towards biodegradability was evaluated by enzymatic digestion and, as expected, CL resulted in a slower degradation of collagen. EDC-crosslinked microparticles particles were spherical, and presented reduced size and a smoother surface than the non-crosslinked ones. Conversely, disk-shaped particles were obtained for GP-treated samples, which presented an even smoother surface and lower porosity. Interestingly, the matrix formed with GP is flexible and seems to be susceptible to shape modification.

1. Lee, C.H., Singla, A., Lee, Y. (2001) Int. J. Pharm., 221, 1-22.

2. Wallace, D. G., Rosenblatt, J. (2003) Adv. Drug. Deliv. Rev., 55, 1631-49.

3. Calejo, M.T., Morais, Z.B., Fernandes, A.I. (2009) J. Biomater. Sci. Polym. Ed. (in press).

This work was financially supported by FCT (PPCDT/SAU-FCF/57911/2004). The collaboration of N. Desai and E. Cardoso is also gratefully acknowledged.


168

PC 97

NOVEL CYTOTOXIC DITERPENOIDS FROM A SOUTHERN AUSTRALIAN MARINE SPONGE PHORBAS SP. Hua Zhang and Robert J. Capon*

Institute for Molecular Bioscience University of Queensland, Australia

[email protected]

In the search of novel and bioactive molecules from natural sources, marine organisms continue to be a highlight due to its wide biodiversity. During our investigation into anticancer agents from Australian marine invertebrates, one sponge extract of the Phorbas genus came to our interest for its significant cytotoxicity against our selected human cancer cell lines. Bioassay guided fractionation and purification yielded the known phorbasins B (1) and C (2)1 together with eight additional analogues phorbasins D−K (3−10).2,3 Their partial absolute structures were assigned on the interpretation of detailed spectroscopic

data. Phorbasins D−F (3−5) are the first reported examples of terpenyl-taurines linked via an amine moiety, meanwhile phorbasins E and F (4 and 5) are dimers incorporating an unprecedented seven-member heterocycle. All the compounds except phorbasins D (3) and K (10) showed LC50 values of 5−30 µM against our cancer cell lines. 1. McNally, M.; Capon, R. J. J. Nat. Prod. 2001,

64, 645-647.

2. Zhang H.; Capon R. J. Org. Lett. 2008, 10, 1959−1962.

3. Zhang H.; Major, J. M.; Lewis, R. J.; Capon R. J. Org. Biomol. Chem. 2008, 6, 3811−3815.


169

PC 98

A CYCLIC TRIPEPTIDE WITH NOT ONLY AMIDE BONDS

Svetlana Savina1, Pau Ruiz‐Sanchis1, Gerardo A. Acosta1, Alberto Rodríguez2, Rogelio Fernández2, Fernando Albericio3 and Mercedes Álvarez1,4

1 Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028-Barcelona, Spain.

b Pharma Mar S. A., Avda. de los Reyes 1, E-28770 Colmenar Viejo, Madrid, Spain. 3 Department of Organic Chemistry,University of Barcelona, E-08028 Barcelona, Spain.

4 Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, 08028-Barcelona, Spain.

Few natural products like kapakahines1 isolated from the sponge Cribrochalina olemda and epipolythiodioxopiperazines2 isolated from the fungus Chaetomium cochliodes possess a new structural feature: two tryptophan residues (Trp-1 and Trp-2) linked by a non-amide bond. An unusual N-C bond between the indole nitrogen of Trp-1 and the C3a of Trp-2 characterizes these compounds. Synthetic approximations to these Trp-Trp systems have been studied by few groups.3

A new peptide isolated recently by PharmaMar from a sample of Spongia sp. contains in its structure the cyclic tripeptide A possessing the mentioned

Trp-Trp unit. The synthetic results in the preparation of tripeptide A will be presented. 1. Nakao, Y.; Yeung, B. K. S.; Yoshida, W. Y.;

Scheuer, P. J., J. Am. Chem. Soc., 1995, 117, 8271

2. Li, G. Y.; Li, B. G.; Yang, T.; Yan, J. F.; Liu, G. Y.; Zhang, G. L., J. Nat. Prod., 2006, 69, 1374

3. Matsuda, Y.; Kitajima, M.; Takayama, H., Org. Lett., 2008, 10, 125 Espejo, V. R.; Rainier, J. D., J. Am. Chem. Soc., 2008, 130, 12894 Newhouse, T.; Lewis, C. A.; Baran, P., J. Am. Chem. Soc., 2009, 131, 6360

N NN

OS

OH

OS

NN

N

O

O

S

S

O

NHN

O

OR

O

HN

N NO

RR

Trp-1

Trp-2

Ile-1

Kapakahine B Chaetocochins A Tripeptide A


170

PC 99

ORTHOGONAL PROTECTING GROUPS OF CYCLIC TRYPTOPHAN HEXAHYDROPYRROLO[2,3-B]INDOLE

Pau Ruiz‐Sanchis1, Svetlana Savina1, Gerardo A. Acosta1, Carmen Cuevas2,

Fernando Albericio1,3 and Mercedes Álvarez1,4

1 Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028-Barcelona, Spain.

2 Pharma Mar S. A., Avda. de los Reyes 1, E-28770 Colmenar Viejo, Madrid, Spain. 3 Department of Organic Chemistry,University of Barcelona, E-08028 Barcelona, Spain.

4 Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, 08028-Barcelona, Spain.

A tricyclic unit of 1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (HPI) has been found in several natural products with important biological activity. The biosynthetic precursor of this heterocycle is a tryptophan (Trp) unit. An intramolecular cyclization of the α-amino group over the indole position two could be the natural origin of HPI.

The results of two alternative procedures for the synthesis of the HPI and its bromo-derivative 3a-bromo-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (Br-HPI) following the biosynthetic way will be presented.

The use of orthogonal protecting groups to achieve such scaffold has been mandatory for further reactions and will be discussed. Several combinations of orthogonal protecting groups have been explored for the three functional groups of the tricyclic compound HPI.

Moreover, it is possible to obtain the two Br-HPI diastereomers depending on the synthetic pathway used.

The proper chemical methodology allows stereoselective control of the three stereocenters. The proper orthogonal protecting groups will allow to control their elimination and further reactivity of the functional groups in order to build the skeleton of the natural product. 1. Hochlowski, J. E.; Mullaly, M. M.; Spanton, S.

G.; Whittern, D. N.; Hill, P.; McAlpine, J. B., J. Antibiot. 1993, 46, 380

2. Roe, J. M.; Webster, A. B.; Ganesan, A., Org. Lett. 2003, 5, 2825

3. Schkeryantz, J.; Woo, J.; Siliphaivanh, P.; Depew, K.; Danishefsky, S., J. Am. Chem. Soc. 1999, 121, 11964


171

PC 100

EXPLORATIVE SOLID-PHASE EXTRACTION FOR ACCELERATED NATURAL PRODUCTS DISCOVERY AND PURIFICATION

Maria Johansen1, Kristian Fog Nielsen2, Lone Gram3, Thomas Ostenfeld Larsen4

[email protected]; [email protected]; [email protected]; [email protected]

When purifying a natural product, the more specific information available on the target compound prior to purification, the more effective the purification (1,2). If no prior knowledge about the target compound(s) is available (e.g. from dereplication), a purification strategy is normally developed on the go or by standard fractionation methods. However, for extracts containing mainly unknown compounds, or when targeting bioactive compounds this lack of strategy beforehand can result in poor recoveries and, at worst, a permanent loss of activity in the extract.

Therefore, we at Centre for Microbial Biotechnology have developed a so called Explorative Solid-Phase Extraction (E-SPE) kit consisting of a set of different SPE columns with orthogonal selectivities, which in a fast and easy way will indicate the optimum purification strategy on a small scale in the exploratory stage of the discovery process before moving on to a semi-preparative or preparative scale. This will allow a more rational approach to the purification process. When linked to a bioassay system, E-SPE can potentially reveal information about the active compound that can help the ensuing purification, for example by suggesting a purification step that removes the main part of inactive extract components or a step that selectively captures the active compound. By using different types of stationary

phases, the different functionalities of the molecules can be exploited to obtain pure compounds in the fewest possible chromatographic steps. When using an elution matrix (3) for visualisation of the bioactivity, the extract can be easily evaluated.

The method has been validated (in triplica) on 25 different marine bacteria with antibacterial effects, such as growth inhibition of Vibrio anguillarum and Staphylococcus aureus or quorum sensing inhibition. Further 8 filamentous fungi with anticancer effects have been included in the study. The E-SPE kit has proven it-self to be fast, easy and reproducible in use and has therefore been implemented as a standard screening procedure at CMB when dealing with new extracts.

1. Houghton, P. J.; Raman, A. Laboratory Handbook for the Fractionation of Natural Extracts; 1 ed.; Chapman & Hall: London, 1998.

2. Cannell, R. J. P.; Dufresne, C.; Gailliot, F. P.; Venkat, E.; Kothandaraman, S.; Salituro, G. M.; Stead, P.; Gibbons, S.; Gray, A. I.; McAlpine, J.; Shankland, N.; Florence, A. J.; VanMiddlesworth, F.; Shimizu, Y.; Silva, G. L.; Lee, I.-S.; Kinghorn, A. D.; Wright, A.; Verrall, M. S.; Warr, S. R. C. Natural Products Isolation; 1st ed.; Humana Press Inc.: Totowa, 1998; Vol. 4th.

3. Cardellina, J. H.; Munro, M. H. G.; Fuller, R. W.; Manfredi, K. P.; Mckee, T. C.; Tischler, M.; Bokesch, H. R.; Gustafson, K. R.; Beutler, J. A.; Boyd, M. R. Journal of Natural Products 1993, 56(7), 1123-1129.


172

PC 99

PRO-APOPTOTIC SECONDARY METABOLITES ISOLATED FROM THE CYANOBACTERIA LYNGBYA MAJUSCULA

Annabel Simon‐Levert1, Anne‐Marie Genevière2, Bernard Banaigs1

and Isabelle Bonnard‐Cussac1 1Laboratoire de Chimie des Biomolécules et de l'Environnement. UP. Perpignan. France

2Laboratoire Arago, UMR CNRS 7628 UPMC, Banyuls-sur-mer. France

[email protected]

In marine environment, cyanobacteria used to be studied due to their toxicity during algal bloom. High majority of compounds isolated from these marine organisms are nitrogen derivatives (lipopeptides, despipeptides, amides, alkaloids) with pharmacological potential: enzymatic inhibitors, antibiotics, immunosuppressive and antiproliferative compounds (Gerwick et al, 2001)1. More than 30 % of secondary metabolites isolated from cyanobacteria were characterized from Lyngbya majuscula (Burja et al, 2001)2 and many of them are described for their toxicity, antiproliferative or antimicrobial effects. The major class of compounds isolated from Lyngbya majuscula are lipopeptides, such as majusculamides or laxaphycines. From specimens collected in Moorea, we isolated by bio-guided purification three cyclic peptides, tiahuramides A-C.

As we could observe during bio-guided purification, these compounds show antiproliferative activities on bacteria, cancer cells and sea urchin eggs. The more active peptide, tiahuramide B, was used to characterize the way of action of these compounds. In vitro experiments allowed us to conclude that tiahuramide B cause apoptosis of sea urchin embryos and assays are still running to observe their effect on cancer cells.

As we could observe during bio-guided purification, these compounds show antiproliferative activities on bacteria, cancer cells and sea urchin eggs. The more active peptide, tiahuramide B, was used to characterize the way of action of these compounds. In vitro experiments allowed us to conclude that tiahuramide B cause apoptosis of sea urchin embryos and assays are still running to observe their effect on cancer cells. 1. Gerwick WH., Tan LT. and Sitachi N. 2001.

Nitrogen-containing metabolites from marine cyanobacteria. Z. Naturforsch. 56: 75-184

2. Burja AM., Banaigs B., Abou-Mansour E., Burgess GJ and Wright PC. 2001. Marine cyanobacteria- a prolific source of natural products. Tetrahedron. 57:9347-937

Tiahuramide A : R =Tiahuramide B : R =Tiahuramide C : R =

N

ON

HN

NO

OOO

OO O

R

Pla 3

NMeVal 2

Val 1

Pro 4

NMeIle 5

Hmoya 6Hmoea 6Hmoaa 6


173

List of Participants ARGENTINA MATUCHESKI, STELLA GARCIA Instituto de Investigación e Ingeniería Ambiental Universidad Nacional de San Martin [email protected] AUSTRALIA CAPON, ROB Institute for Molecular Bioscience [email protected] ZHANG, HUA Institute for Molecular Bioscience [email protected] AUSTRIA PFLUGFELDER, BETTINA Marinomed Biotechnologie GmbH [email protected] BRAZIL ERBERT, CÍNTIA Universidade de São Paulo - USP [email protected] FALKENBERG, MIRIAM Federal University of Santa Catarina [email protected] GRESSLER, VANESSA Universidade de São Paulo [email protected] JANUÁRIO, ANA HELENA Universidade de Franca [email protected] JIMENEZ, PAULA Universidade Federal do Ceará [email protected] LOPES, MARCIA NASSER Instituto de Química UNESP [email protected] RANGEL, MARISA Laboratório de Imunopatologia Instituto Butantan [email protected] ROCHA, FABIOLA DUTRA Universidade Federal de Minas Gerais [email protected]

SCOPEL, MARINA Universidade Federal do Rio Grande do Sul [email protected] CANADA BOUMGHAR, YACINE CÉPROCQ [email protected] ROCHEFORT, J.GUY Nunavic Biosciences Inc [email protected] CUBA REGALADO, ERIK LUIS Center of Marine Bioproducts (CEBIMAR) [email protected] DENMARK JOHANSEN, MARIA Technical University of Denmark [email protected] EGYPT EL-HEMIELY, AHMED MOUSTAFA Faculty of Science Cairo University [email protected] YOUSSEF, DIAA Suez Canal University [email protected] ESTONIA REINTAMM, TÕNU Tallinn University of Technology Department of Gene Technology [email protected] FRANCE ABED, CHARLINE University of Nice Sophia-Antipoli [email protected] AL-MOURABIT, ALI Institut de Chemie des Substances Naturelles du CNRS [email protected]


174

AMADE, PHILIPPE Université de Nice [email protected] BANAIGS, BERNARD Laboratoire de Chimie des Biomolécules et de l'Environnement Université de Perpignan. [email protected] BARNATHAN, GILLES University of Nantes Faculty of Pharmacy - MMS [email protected] BONNARD-CUSSAC, ISABELLE LCBE - University of Perpignan [email protected] BONTEMPS, NATALIE University of Perpignan [email protected] BOURGET-KONDRACKI, MARIE-LISE Muséum National d'Histoire Naturelle [email protected] BRY, DELPHINE Laboratoire de Chimie des biomolécules et de l'Environnement Université de Perpignan [email protected] DAGORN, FLORE University of Nantes Faculty of Pharmacy - MMS [email protected] DUFOUR-SCHROIF, COSIMA COVALMAR [email protected] DUPLAT, DENIS COVALMAR [email protected] GAEDERES, JOHAN Université de Bretagne Sud [email protected] GROVEL, OLIVIER University of Nantes Faculty of Pharmacy - MMS [email protected] KERZAON, ISABELLE University of Nantes Faculty of Pharmacy - MMS [email protected]

LA BARRE, STEPHANE Station Biologique de Roscoff [email protected] LE JEUNE, CLARISSE ICSN - CNRS [email protected] LE KER, CARINE University of Nantes Faculty of Pharmacy - MMS [email protected] LE PENNEC, GAËL Université de Bretagne Sud [email protected] LIOUDMILA, ERMOLENKO ISCN - CNRS [email protected] MEHIRI, MOHAMED Laboratoire de Chimie des Molécules Bioactives et des Arômes (LCMBA) Institut de Chimie de Nice (ICN), UFR [email protected] MEIJER, LAURENT CNRS – Station Biologique de Roscoff [email protected] PICHON, DELPHINE COVALMAR [email protected] PICOT, LAURENT Laboratoire LIENSs CNRS UMR 6250 [email protected] ROUE, MELANIE Muséum National d'Histoire Naturelle [email protected] THOMAS, OLIVIER P. University of Nice Sophia Antipolis [email protected] VANSTEELANDT, MARIEKE University of Nantes Faculty of Pharmacy - MMS [email protected] VIANO, YANNICK MAPIEM [email protected]


175

GERMANY ALMEIDA, CELSO HENRIQUE GUERREIRO Institute for Pharmaceutical Biology [email protected] BOUHIRED, SARAH Insitute for Pharmaceutical Biology University Bonn [email protected] CYCHON, CHRISTINE Alfred-Wegener-Institut [email protected] EKLUND, MINNA University of Bonn [email protected] EL OMARI, MUSTAFA Institute for pharmaceutical biology University of Bonn [email protected] EROL-HOLLMANN, OEZLEM Pharmaceutical Biology University Bonn [email protected] GURGUI, CRISTIAN Kekulé-Institute of Organic Chemistry and Biochemistry [email protected] HERTWECK, CHRISTIAN Leibniz Institute for Natural Product Research [email protected] KÖCK, MATTHIAS Alfred-Wegener-Institut [email protected] KÖNIG, GABRIELE Institute for Pharmaceutical Biology University of Bonn [email protected] MOUSTAFA, MAHMOUD FAHMI ELSEBAI Pharmaceutical Biology [email protected] MÜLLER, WERNER E. G. Institut für Physiologische Chemie Abteilung Angewandte Molekularbiologie Universität Mainz [email protected] NAGEL, KERSTIN Kieler Wirkstoff-Zentrum and IFM GEOMAR [email protected]

NATALIO, FILIPE ANDRÉ DA SILVA RAMINHOS Institute of Physiological Chemestry and Pathobiochemistry University Mainz [email protected] PIEL, JOERN Kekule Institute of Organic Chemestry and Biochemestry University of Bonn [email protected] PROKSCH, PETER Institute for Phaemaceutical Biology University Duesseldorf [email protected] REINHARDT, KATHRIN Kekulé-Institute for Organic Chemistry and Biochemistry [email protected] SCHMIDT, GESINE Alfred-Wegener-Institut [email protected] SCHRÖDER, HEINZ C. Institut für Physiologische Chemie Angewandte Molekularbiologie Universität Mainz [email protected] SCHÄBERLE, TILL University of Bonn Institute for Pharmaceutical Biology [email protected] TARMAN, KUSTIARIYAH Institute of Pharmacy Greifswald University [email protected] YUNT, ZEYNEP Universität Bonn [email protected] VAN DER SAR, SONIA Kekulé Institut für Organische Chemie und Biochemie Universität Bonn [email protected] GREECE IOANNOU, EFSTATHIA University of Athens School of Pharmacy Department of Pharmacognosy and Chemistry of Natural Products [email protected]


176

ROUSSIS, VASSILIOS University of Athens School of Pharmacy Department of Pharmacognosy and Chemistry of Natural Products [email protected] VAGIAS, CONSTANTINOS University of Athens School of Pharmacy Department of Pharmacognosy and Chemistry of Natural Products [email protected] IRELAND PEDDIREDDI, SUDHAKAR Centre for Applied Marine Biotechnology [email protected] QUINN, GERARD Letterkenny Institute of Technology [email protected] ISRAEL FAIBISH, HANNY Ben Gurion University [email protected] KASHMAN, YOEL Tel Aviv University School of Chemistry [email protected] ITALY BONADIES, FRANCESCO "La Sapienza" University of Rome [email protected] CASAPULLO, AGOSTINO University of Salerno [email protected] CIAVATTA, MARIA LETIZIA Istituto di Chimica Biomolecolare [email protected] CIMINIELLO, PATRIZIA Universitá di Napoli Dipartimento di Chimica Delle Sostanze Naturali [email protected] CIMINO, GUIDO Institute of Biomecular Chemistry [email protected]

COSTANTINO, VALERIA Dipartimento di Chimica delle Sostanze Naturali Universià "Federico II" di Napoli [email protected] CUTIGNANO, ADELE CNR-Istituto di chimica Biomolecolare [email protected] FATTORUSSO, ERNESTO Universitá di Napoli Dipartimento di Chimica Delle Sostanze Naturali [email protected] FONTANA, ANGELO CNR - Istituto Chimica Biomolecolare [email protected] FORINO, MARTINO Dipartimento di Chimica delle Sostanze Naturali [email protected] GAVAGNIN, MARGHERITA Istituto di Chimica Biomolecolare - CNR [email protected] GRAUSO, LAURA Dipartimento di Chimica delle Sostanze Naturali [email protected] GUELLA, GRAZIANO University of Trento [email protected] IANORA, ADRIANNA Stazione Zoologica Anton Dohrn [email protected] LAMARI, NADIA CNR - Institute of Biomolecular Chemistry [email protected] LEONE, ANTONELLA CNR, National Research Council Institute of Science of Food Production Lecce [email protected] LUPORINI, PIERANGELO Dipartimento di Biologia [email protected] MANCINI, INES University of Trento [email protected] MANGONI, ALFONSO Dipartimento di Chimica delle Sostanze Naturali University of Napoli Federico II [email protected]


177

MANZO, EMILIANO Institute of Biomolecular Chemistry (CNR) [email protected] SPOLITI, MAURIZIO "La Sapienza" University of Rome [email protected] TAGLIALATELA-SCAFATI, ORAZIO Dipartimento di Chimica delle Sostanze Naturali Università di Napoli Federico II [email protected] VALLESI, ADRIANA Università di Camerino [email protected] JAMAICA GALLIMORE, WINKLET University of the West Indies Mona Campus [email protected] JAPAN KUMAGAI, KEIKO Kochi University [email protected] TSUDA, MASASHI Kochi University [email protected] KOREA KANG, HEONJOONG Seoul National University [email protected] LUXEMBOURG DIEDERICH, MARC Laboratoire de Biologie Moleculaire et Cellulaire du Cancer [email protected] MALAYSIA VAIRAPPAN, CHARLES S. Institute for Tropical Biology and Conservation [email protected] NEW ZEALAND BLUNT, JOHN University of Canterbury [email protected]

CHAMYUANG, SUNITA The University of Canterbury [email protected] COPP, BRENT R. University of Auckland [email protected] MUNRO, MURRAY University of Canterbury [email protected] NORWAY GUDIMOVA, ELENA UoT (PINRO) [email protected] JØRGENSEN, TROND Ø. University of Tromsø [email protected] PAULSEN, STEINAR University of Tromso [email protected] PERANDER, MARIA University of Tromsø MabCent-SFI [email protected] SANDSDALEN, ERLING Northern Research Institute [email protected] SØRUM, UNN MABIT programme Norinnova AS [email protected] PEOPLE’S REPUBLIC OF CHINA GUO, YUE-WEI Shanghai Institute of Materia Medica-CAS [email protected] POLAND KOSAKOWSKA, ALICJA Institute of Oceanology Polish Academy of Sciences [email protected] PORTUGAL ABREU, MARIA HELENA CIIMAR Faculdade de Ciências da Universidade do Porto [email protected]


178

ADRIANO, GISELA DOS SANTOS Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected] AFONSO, CARLOS MANUEL MAGALHÃES Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected] AFONSO, EMÍLIA CIIMAR [email protected] ALMEIDA, ANA PAULA Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected] ARAÚJO, MARCO FILIPE CERQUEIRA Faculdade de Ciências da Universidade de Lisboa [email protected] AZEVEDO, CARLOS MIGUEL Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected] BARRETO, BIANCA NATIVIDADE Universidade Federal do Rio de Janeiro FEUP [email protected] BESSA, JÚLIA MANUELA MARQUES DOS SANTOS Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto [email protected] CACHATRA, VASCO MIGUEL CANDEIAS Departamento de Química e Bioquímica Faculdade de Ciências da Universidade de Lisboa [email protected] CALEJO, MARIA TERESA REBELO Centro de Polímeros Biomédicos Instituto Superior de Ciências da Saúde Egas Moniz [email protected] CASTANHEIRO, RAQUEL ALEXANDRA PINTO Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected]

CIDADE, HONORINA Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected] COIMBRA, JOÃO Institute of Biomedical Sicences Abel Salazar CIIMAR [email protected] CORDEIRO, ANA SARA CAETANO Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected] COSTA, ELISANGELA Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected] CRAVO, SARA MANUELA MENDONÇA DA SILVA Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected] CUNHA, ALEXANDRE LOBO Institute of Biomedical Sicences Abel Salazar CIIMAR [email protected] FERNANDES, CARLA SOFIA GARCIA Faculty of Pharmacy, University of Porto CEQUIMED-UP [email protected] FERREIRA, MARIA JOÃO DANTAS RAMALHOSA FFUP ISEP [email protected] FONSECA, MARIA SÃO JOSÉ NASCIMENTO Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected] GASPAR, HELENA Instituto Nacional de Engenharia, Tecnologia e Inovação [email protected] GOMES, ANA SARA Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected]


179

GONÇALVES, MARIA DO PILAR FIGUEROA Faculdade de Engenharia - UP [email protected] GUEDES, ANA CATARINA AFONSO ESB/CBQF - UCP [email protected] GUERREIRO, JOANA RAFAELA LARA ICETA - Instituto de Ciências e Tecnologias Agrárias e Agro Alimentares [email protected] KIJJOA, ANAKE Institute of Biomedical Sicences Abel Salazar CIIMAR [email protected] LEÃO, PEDRO CIIMAR-University of Porto SIO - University of California, San Diego [email protected] MARTINS, CARLOS GIL Institute of Biomedical Sicences Abel Salazar CIIMAR [email protected] MORAIS, ZILDA BRAGA Cooperativa Egas Moniz de Ensino Superior [email protected] MOREIRA, FELISMINA TEIXEIRA COELHO ICETA - Instituto de Ciências e Tecnologias Agrárias e Agro Alimentares [email protected] OLIVEIRA, ANDREIA PATRÍCIA DA SILVA ICETA/REQUIMTE Department of Pharmacognosy Faculty of Pharmacy Porto University [email protected] PINTO, MADALENA MARIA MAGALHÃES Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected] SANTOS, JOANA MANUELA DA SILVA Serviço Bromatologia Faculdade Farmácia da Universidade Porto [email protected] SANTOS, SÓNIA PEREIRA Institute of Biomedical Sicences Abel Salazar CIIMAR [email protected]

SCHULZE, CORINNA CIMAR, Universidade do Porto [email protected] SILVA, ARTUR MANUEL SOARES Department of Chemistry University of Aveiro [email protected] SILVA, MARTA CORREIA Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected] SILVA, TIAGO HENRIQUES 3B's Research Group University of Minho [email protected] SOUSA, MARIA EMÍLIA Faculty of Pharmacy University of Porto CEQUIMED-UP [email protected] VALE, PAULO INRB, I.P./L. IPIMAR [email protected] VIEIRA, LUIS MANUEL DE MIRA Institute of Biomedical Sicences Abel Salazar [email protected] VIEIRA, HELENA MARGARIDA MOREIRA DE OLIVEIRA Bioalvo, S.A. [email protected] SOUTH AFRICA BEUKES, DENZIL R. Rhodes University [email protected] SOUTH KOREA FENG, ZHILE Pukyong National University [email protected] SON, BYENG W. Pukyong National University [email protected] SPAIN BECERO, MIKEL A. Center for Advanced Studies of Blanes [email protected]


180

CUETO, MERCEDES Instituto de Productos Naturales y Agrobiología del CSIC [email protected] CUEVAS, CARMEN Pharma Mar, S.A. Sociedad Unipersonal [email protected] DARANAS, ANTONIO HERNANDEZ Instituto Universitario de Bio-Organica [email protected] DÁRIAS, JOSÉ CSIC [email protected] FERNÁNDEZ, JOSÉ MARÍA Pharma Mar, S.A. Sociedad Unipersonal [email protected] GONZÁLEZ, JENNIFER VÁZQUEZ Universidad de Barcelona Facultad de Biología [email protected] HERNÁNDEZ, LIBRADA Mª CAÑEDO Instituto Biomar, León [email protected] JIMENEZ, CARLOS Universidad de A Coruña [email protected] MARTIN, MANUEL NORTE University of La Laguna [email protected] MARTIN, VICTOR S. Universidad de La Laguna Instituto Universitario de Bio-Organica Antonio Gonzalez [email protected] NAPOLITANO, JOSE GABRIEL Universidad de La Laguna IUBO-AG [email protected] NOYER, CHARLOTTE Centro de Estudios Avanzados de Blanes (CEAB, CSIC) [email protected] PONS, LAURA NUÑEZ Universitat de Barcelona [email protected]

QUIÑOA, EMILIO Universidad de Santiago de Compostela [email protected] REYES, JOSE FERNANDO Pharma Mar, S.A. Sociedad Unipersonal [email protected] RIGUERA, RICARDO Universidad de Santiago de Compostela [email protected] RODRIGUEZ, ALBA SOUTO Universidade da Coruña Facultade de Ciencias [email protected] RODRÍGUEZ, HUMBERTO JOSÉ DOMÍNGUEZ Universidad de La Laguna IUBO [email protected] SACRISTÁN-SORIANO, ORIOL Centre d'Estudis Avançats de Blanes (CEAB-CSIC) [email protected] SANCHIS, PAU RUIZ Parc Cientific de Barcelona (PCB) [email protected] SAVINA, SVETLANA Parc Cientific de Barcelona (PCB) [email protected] SWEDEN BOHLIN, LARS Division of Pharmacognosy Department of Medicinal Chemistry Biomedical Center Uppsala University [email protected] THAILAND HRH PRINCESS CHULABHORN MAHIDOL Chulabhorn Research Institute DETHOUP, TIDA Department of Plant Pathology Faculty of Agriculture Kasetsart University [email protected] MANOCH, LEKA Department of Plant Pathology Faculty of Agriculture Kasetsart University [email protected]


181

PAKKONG, PANNEE Kasetsart University [email protected] PASANPHAN, WANVIMOL Kasetsart University [email protected];[email protected] PHONGPAICHIT, SOUWALAK Department of Microbiology Faculty of Science Prince of Songkla University [email protected] PONGSAMART, SUNANTA Chulalongkorn University [email protected] PRASATSRISUPAB, JARIYA Department Agriculture [email protected];[email protected] RUKACHAISIRIKUL, VATCHARIN Prince of Songkla University [email protected] SINGBURAUDOM, NARONG Department of Plant Pathology Faculty of Agriculture Kasetsart University [email protected] SONCHAENG, PICHAI National Science Museum Thailand [email protected] SUWANBORIRUX, KHANIT Faculty of Pharmaceutical Sciences Chulalongkorn University [email protected] TUNKIJJANUKIJ, SURIYAN Faculty of Fisheries Kasetsart University [email protected] VICHIEN, KITPREECHAVANICH Dept of Microbiology Faculty of Science Kasetsart University [email protected] TUNIS HAMROUNI BUONOMO, SOUHIR National Institute of Agronomy [email protected]

TURKEY GOZCELIOGLU, BULENT Ankara University [email protected] KONUKLUGIL, BELMA University of Ankara Faculty of Pharmacy [email protected] UNITED KINGDOM EDRADA-EBEL, RUANGELIE Strathclyde Institute of Pharmacy and Biomedical Sciences [email protected] FISCH, KATJA MARIA School of Chemistry University of Bristol [email protected] TABUDRAVU, JIOJI Aquapharm Biodiscovery Ltd [email protected] THOMAS, ERIC JIM School of Chemistry The University of Manchester [email protected] UNITED STATES OF AMERICA GERWICK, WILLIAM Scripps Institution of Oceanography Univ. California San Diego [email protected] HARPER, MARY KAY University of Utah [email protected] IRELAND, CHRIS M. University of Utah [email protected] JONES, PAUL B. Wake Forest University [email protected] MOLINSKI, TADEUSZ University of California [email protected] PAWLIK, JOSEPH R. University of North Carolina Wilmington [email protected]


182

Authors Index - A - Abe, R. O. PC-11Abed, Charline OC-20 Abraham, Wolf-Rainer PC-36 Abreu, Maria Helena PC-91 Acosta, Gerardo A. PC-98, PC-99 Albericio, Fernando PC-98, PC-99 Albrigtsen, Marte PC-37 Alimenti, Claudio OC-07 Allard, Marc PC-74 Almeida, Celso IL-01, PC-28 Al-Mourabit, Ali IL-05, PC-01, PC-12 Álvarez, Mercedes PC-98, PC-99 Alves, M. R. PC-76 Alves, V. D. PC-68 Amade, Philippe OC-08, PC-29 Amaro, Helena M. PC-47 Andersen, Jeanette Hammer PC-37, PC-44 Anderson, Sally PC-58 Andrade, C. T. PC-95 Andrade, Paula B. PC-30 Araújo, A. R. PC-11 Araújo, M. PC-94 Ariffin, Siti Alwani PL-09 Avila, Conxita OC-18, PC-45, PC-83 - B - Bakar, Hamidah PL-09Ballesteros, Manuel OC-18 Banaigs, Bernard OC-17, OC-21, OC-23, PC-101 Bandarra, Narcisa M. PC-75 Baran, Phil S. OC-03 Barnathan, Gilles PC-86 Barreto, B. N. PC-95 Bashira, Ashgan OC-16 Becerro, Mikel A. OC-21, OC-23, PC-57, PC-77, PC-88 Ben Rejeb Jenhani, Amel PC-77 Ben Saidin, J. PC-81 Ben-Califa, Nathalie OC-16 Bessa, Julia PC-19 Beukes, Denzil R. PC-33 Biard, Jean-François OC-12, PC-26, PC-55, PC-86 Bignami, Gary OC-24 BIOALVO PC-38 Bitam, Fatma PC-22 Bitar, Ghazi OC-20, PC-01 Blache, Yves OC-06


183

Blunt, John PL-09 Bolton, John J. PC-33 Bolzani, V. S. PC-11 Bonnard-Cussac, Isabelle PC-101 Bonhomme, Dominique OC-06 Bontemps, Nataly OC-17 Bouhired, Sarah PC-56 Boumghar, Yacine PC-74 Bourguet-Kondracki, Marie-Lise PC-57, PC-69, PC-80 Briand, Jean-François OC-06 Bringmann, Gerhard PC-52 Bruhn, Torsten PC-52 Bry, Delphine OC-17 Buaruang, Jamrearn PC-43, PC-48 Bueno, V. PC-41 Bugni, Tim S. PC-60 - C - Cachatra, V. PC-38Cachet, Nadja PC-29 Calado, P. PC-71 Calejo, M. T. PC-96 Callone, Emanuela PC-31 Camps, Mercedes OC-06 Cao, Zhengyu PL-06 Capon, Robert J IL-04, PC-97 Carbone, Marianna OC-09, PC-22, PC-78, PC-83 Casapullo, Agostino OC-13 Casotti, Raffaella PL-07 Castelluccio, Francesco PC-83 Catalanotti, Bruno PC-10 Chan, S. T. S. PC-07 Charrier, Bénédicte OC-01 Chirachanchai, Suwabun PC-92 Choi, Hyukjae OC-11 Choofong, Surakarn PC-92 Chumyuang, Sunita PL-09 Ciavatta, Maria Letizia OC-09, PC-05, PC-22, PC-78 Ciminiello, Patrizia OC-24, PC-10 Cimino, Guido PL-07, PL-10, OC-18, PC-78 Cole, Tony PL-09 Colepicolo, Pio PC-23 Concepción, A. R. PC-41 Conti, R. PC-49 Copp, B. R. PC-07, PC-14 Costa-Lotufo, L. V. PC-42 Costantino, Valeria OC-10, PC-67 Cox, James E. PC-60 Cox, Russell J. PC-54


184

Crews, Phillip PC-63 Cristobo, Francisco Javier PC-45 Cueto, Mercedes PC-04 Cuevas, Carmen PC-99 Culioli, Gérald OC-06 Cutignano, Adele PL-07, OC-18, PC-90 Cuypers, Beate PC-40 Cychon, Christine OC-03, PC-25 - D - D’Croz, Luís PC-04d’Ippolito, Giuliana PL-07, PC-90 da Silva, Patrícia P.M. PC-23 Dagorn, Flore PC-86 Dalisay, Doralyn S. IL-11 Daranas, Antonio Hernández OC-25, PC-15, PC-53, PC-87 Darias, J. PC-04 Davis, Paul PL-09 De Bortoli, Marco OC-24 de Pinho, Paula Guedes PC-30 de Souza, H. K. S. PC-95 Debonsi, H. M. PC-11, PC-49 Defant, Andrea PC-27 Delerue-Matos, C. PC-35, PC-68 Dell’Aversano, Carmela OC-24, PC-10 Dello Iacovo, Emma OC-24, PC-10 Dethoup, Tida PC-43, PC-48 Di Giuseppe, Graziano PC-31 Díaz-Marrero, Ana R. PC-04 Dicato, Mario IL-06 Diederich, Marc IL-06 Dilokkunanant, Uraiwan PC-73 Dini, Fernando PC-31 Dittami, Simon OC-01 Domart-Coulon, Isabelle PC-57, PC-80 Domínguez, Humberto J. PC-87 Dorrestein, Pieter C. PL-06 Du Pont, Thibaut Robiou PC-26, PC-55 Dufour-Schroif, Cosima PC-69 Dumay, Justine PC-86 Duplat, Denis PC-69 - E - Edrada-Ebel, RuAngelie OC-04Eklund, Minna PC-52 El Mehdi, Naima PC-74 El Omari, M. PC-64 Elsebai, M. F. IL-01, PC-08


185

Erbert, C. PC-11, PC-49 Eriksson, Jonas PC-44 Ermolenko, Ludmila PC-12 Erol-Hollmann Ö. PC-59, PC-61 Esquenazi, Eduardo PL-06 - F - Falkenberg, Miriam PC-17Fattorusso, Caterina PC-10 Fattorusso, Ernesto OC-10, OC-24, PC-10, PC-67 Felício, R. PC-49 Feng, Zhile PC-09, PC-62 Fernandes, A. I. PC-96 Fernandez, José Javier OC-25, PC-15, PC-53, PC-87 Fernández, Rogelio OC-08, PC-20, PC-98 Ferreira, E. G. PC-42 Ferreira, R. A. S. PC-46 Figuerola, Blanca PC-45 Fisch, Katja M. PC-54, PC-58, PC-63 Fleurence, Joël OC-12 Folmer, Florence IL-06 Fontana, Angelo Apivita PSE Award Lecture, PL-07, OC-18, PC-90 Forestieri, Roberto PC-83 Forino, Martino OC-24, PC-10 Frassanito, Rita PC-31 Frontini, Francesco PC-31 Fujii, Mutue T. PC-23 Furtado, N. A. J. C. PC-49 - G - Gabant, Marion PC-01, PC-12Gardères, J. PC-81 Garderes, Johan OC-15 Gaspar, Helena OC-19, PC-38 Gavagnin, Margherita OC-09, PC-05, PC-22, PC-78, PC-83 Genevière, Anne-Marie PC-101 Genta-Jouve, Grégory OC-08, PC-29 Gerwick, Lena PL-06, PC-67 Gerwick, William H. PL-06, PC-67, PC-79 Gibbons, Simon PC-24 Gomes, Daniela PC-30 Gonçalves, L. L. PC-96 Gonçalves, M. P. PC-68, PC-95 González, Jaime Rodríguez PC-83 Gözcelioğlu, B. PC-16 Gram, Lone PC-100 Grauso, Laura OC-24, PC-10 Gressler, Vanessa PC-23 Greve, H. IL-01


186

Grindberg, Rashel PL-06 Grkovic, T. PC-14 Grovel, Olivier PC-26, PC-55 Guedes, A. Catarina PC-47 Guedes, A. PC-38 Guella, Graziano PC-27, PC-31 Guerreiro, J. Rafaela L. PC-89 Guo, Yue-Wei OC-02 Gurgui, Cristian PC-58, PC-63 - H - Hahn, Dongyup OC-11Hamrouni Buonomo, Souhir PC-77 Harper, J. PC-07 Harper, Mary Kay PC-60 Henriques, Amélia T. PC-36 Hertweck, Christian PL-05, PC-52 Heycke, Nina PC-63 Hoffmann, Friederike PC-06 Hong, Junyoung OC-11 Hooper, John N. A. PC-60 Horta, Paulo Antunes PC-17 Houghton, Peter J. PC-51 Höver, T. PC-61 Humanes, M. PC-38, PC-94 Hwang, Byungsoo OC-11 Hwang, Hoosang OC-11 - I - Ireland, Chris M. PL-02, PC-60Ianora, Adrianna PL-07 Ioannou, Efstathia PC-17, PC-24, PC-32 Ionta, M. PC-46 Imhoff, J. F. PC-82 - J - Januario, A. H. PC-07Jaspers, Marcel IL-06 Jiménez, Carlos PC-20, PC-66, PC-83 Jimenez, P. C. PC-42 Johansen, Maria PC-100 Jones, Adam C. PL-06 Jones, Paul B. IL-03 Jørgensen Trond Ø. PC-37, PC-44 - K - Kajahn, I. PC-82Kamel, Ayman H. PC-89 Kang, Heonjoong OC-11


187

Kaplan, Maria Auxiliadora C. PC-51 Kashman, Yoel OC-16 Kehraus, S. IL-01, PC-08 Kelve, Merike PC-06 Kervarec, Nelly OC-01 Kerzaon, Isabelle PC-26 Khalaf, Gaby OC-20 Khamthong, Nanthaphong OC-05 Kijjoa, Anake PC-19, PC-43, PC-48 Kim, Euno OC-11 Kim, Jung-A OC-11 Kitpreechavanich, Vichien PC-73 Köck, Matthias OC-03, PC-18, PC-25 König, Gabriele M. IL-01, PC-08, PC-28, PC-56, PC-59, PC-61, PC-64 Konuklugil, B. PC-16 Korbee, Nathalie PC-91 Kosakowska, Alicja PC-65 Küçükecir, Y. Y. PC-16 Kumagai, Keiko PC-02, PC-13 - L - La Barre, Stéphane OC-01Labes, A. PC-82 Laguna, L. PC-41 Lalk, Michael PC-40 Lam, C. PC-14 Lamari, Nadia PC-90 Larsen, Thomas O. PC-100 Le Bail, Aude OC-01 Le Ker, Carine OC-12 Le Pennec, Gaël OC-15, PC-81 Leão, Pedro N. PC-79 Lejeune, Clarisse PC-01 Lemos, Manuel L. PC-66 Leo, Angela PC-10 Lerner, Cléa PC-36 Leutou, Alain S. PC-09, PC-62 Lewandowska, Agnieszka PC-65 Lhullier, Cintia PC-17 Li, Yan OC-09 Lindequist, Ulrike PC-40 Lisboa, F. PC-76 Lopes, J. L. C. PC-11, PC-49 Lopes, M. N. PC-11 Lopes, N. P. PC-42 Lopp, Annika PC-06 Lorenzo, Manuel PC-04 Luca, A. N. PC-11 Luporini, Pierangelo OC-07


188

- M - Macedo, Alexandre J. PC-36Machado-Santelli, G. M. PC-46 Mahidol, HRH Princess Opening lecture Maifi, Fatna PC-74 Majdi, Nabil OC-21 Malcata, F. Xavier PC-47 Mancini, Ines PC-27, PC-31 Mangoni, A. OC-10 Mangoni, Alfonso PC-67 Mann, Maryssa G. PC-33 Mano, João F. OC-22 Manoch, Leka PC-43, PC-48 Manzo, Emiliano OC-09, PC-05, PC-22 Margarucci, Luigi OC-13 Martin, Marie-Thérèse PC-01 Matucheski, Stella García PC-78 Mazères, Serge PC-12 Mehiri, Mohamed OC-08, OC-20, PC-29 Meijer, Laurent PL-04 Mernitz, Gudrun PC-40 Minoru, Suzuki PC-34 Minucci, Carmen PC-90 Miralto, Antonio PL-07 Missau, Fabiana C. PC-23 Mohd Khalid, Rozida PC-54 Molinski, Tadeusz F. IL-11 Mollo, Ernesto PC-22, PC-78 Monti, Maria Chiara OC-13 Montresor, Marina PC-90 Moradas-Ferreira, Pedro PC-47 Moraes, M. O. PC-42 Morais, S. PC-35, PC-68 Morais, Zilda B. PC-75, PC-96 Moreira, Felismina T. C. PC-72, PC-89 Morinaka, Brandon I. IL-11 Moriou, Céline PC-12 Mothes, Beatriz PC-36 Mouga, Teresa PC-30 Müller, Werner E.G. PL-08, OC-14, PC-70 Muniain, Claudia PC-78 Munro, Murray PL-09 Murray, Thomas F. PL-06 - N - Nagel, K. PC-82Napolitano, José G. OC-25, PC-15, PC-53 Nappo, Michela PC-83


189

Natalio, Filipe OC-14, PC-70 Nenkep, Viviane N. PC-09, PC-62 Nett, M. PC-61 Neumann, Drorit OC-16 Neumann, K. IL-01 Nguyen, Tu Anh PC-58 Nielsen, Kristian F. PC-100 Nieto, Rosa M. PC-83 Nogueira, J. M. PC-38 Nogueiras, C. PC-41 Norte, Manuel OC-25, PC-15, PC-53, PC-87 Noyer, Charlotte PC-88 Núñez-Pons, Laura PC-45 Nuzzo, Genoveffa PC-05 - O - Oliveira, A. L. L. PC-49Oliveira, Andreia P. PC-30 Oliveira, Joaquim M. OC-22 Oliveira, M. B. P. P. PC-35, PC-76 Ortalo-Magné, Annick OC-06 Osorio, Carlos R. PC-66 - P - Paíga, P. PC-35Pakkong, Pannee PC-84, PC-93 Pasanphan, Wanvimol PC-92 Paulsen, Steinar M. PC-37 Pawlik, Joseph R. IL-08 Pearce, A. N. PC-07 Pedrini, Bill OC-07 Perander, Maria PC-44 Pereira, Alban PL-06, PC-79 Pereira, Renato C. PC-51 Pereira, Ricardo D. PC-47 Pereira, Rui PC-91 Pereira, Teresa G. PC-75 Perez, Thierry OC-20, PC-01, PC-57 Perinu, C. OC-10 Pessoa, C. O. PC-42 Pestana, N. PC-76 Petit, Karina-Ethel OC-12, PC-55 Petraki, Anastasia PC-32 Pfister, S. C. PC-46 Phongoaichit, Souwalak OC-05, PC-50 Phongphern, Wanvisa PC-84 Piadang, Nattayana PC-73 Pichon, Delphine PC-69 Piel, Jörn PC-52, PC-58, PC-63, PC-67


190

Pino, J. A. PC-41 Pinto, Ernani PC-23 Pinto, Isabel Sousa PC-91 Piriyaprin, Siangjeaw PC-43, PC-48 Piva, R. F. PC-46 Plathong, Sakanan PC-50 Platzer, Matthias PC-63 Poli, Mark OC-24 Pons, Laura Núñez PC-83 Pontius, A. IL-01 Porras, Gina PC-04 Potin, Philippe OC-01 Pouchus, Yves François OC-12, PC-26, PC-55 Poza, Javier Jesús PC-20 Prasatsrisupab, Jariya PC-93 Preedanon, Sita PC-50 Proksch, Peter PL-01, PC-16 Puchakarn, Sumaitt PC-19 Pupo, M. T. PC-49 - Q - Quévrain, Elodie PC-69, PC-80 - R - Rabesaotra, Vony PC-86Rahman, M. Mukhlesur PC-24 Ramalhosa, M. J. PC-35 Ramasamy, Kalavathy PL-09 Ramos, Alfonso PC-83 Rangel, M. PC-46 Rapp, Hans Tore PC-06 Raspor, Lucija PC-27 Ratinaud, Céline PC-12 Regalado, Erik L. PC-29, PC-41 Reinhardt, Kathrin PC-52 Reintamm, Tõnu PC-06 Reis, Rui L. OC-22 Reppart, Jason PC-60 Reyes, Fernando IL-02, OC-08, PC-20 Rho, Jungrae OC-11 Riccio, Raffaele OC-13 Riguera, Ricardo IL-10 Rimdusit, Pakjira PC-92 Rivas, Amable J. PC-66 Robinson, Sarah J. PC-63 Roca, C. PC-71 Rocha, Fabiola D. PC-51 Rochefort, Guy PC-74 Rodríguez, Alberto PC-98


191

Rodríguez, Jaime PC-20, PC-66 Rodríguez, M. PC-41 Romano, Giovanna PL-07 Roué, Mélanie PC-57 Roussis, Vassilios PC-03, PC-17, PC-24, PC-32 Rubio, Brent K. PC-63 Rudi, Amira OC-16 Rugthaworn, Prapassorn PC-73 Ruiz, Nicolas PC-26 Ruiz-Sanchis, Pau PC-98, PC-99 Rukachaisirikul, Vatcharin OC-05, PC-50 - S - Sacristan-Soriano, Oriol OC-21, OC-23Saidin, Jasnizat Bin OC-15 Sakayaroj, Jariya OC-05, PC-50 Salah Romdhane, Mohamed PC-77 Salaün, Stéphanie OC-01 Sales, M. Goreti F. PC-72, PC-89 Sampaio, P. PC-95 Sangchote, Somsiri PC-73 San-Martín, Aurelio PC-04 Santos, A. PC-71 Santos, J. PC-76 Santos, S. PC-38 Savina, Svetlana PC-98, PC-99 Sawangwong, Pichan PC-84 Schäberle, T. PC-59 Schenkel, Eloir Paulo PC-17 Schlacher-Hoenlinger, Monika A. PC-60 Schmidt, Gesine OC-03, PC-18, PC-25 Schmitz, A. PC-59 Schneemann, I. PC-82 Schröder, Heinz C. PL-08, OC-14, PC-70 Schumacher, Marc IL-06 Scopel, Marina PC-36 Seabra, Rui PC-47 Seiple, Ian OC-03 Sepčić, Kristina PC-27 Seternes, Ole Morten PC-44 Shaala, Lamiaa A. PC-21 Shin, Sun OC-11 Silva, Simone S. OC-22 Silva, Tiago H. OC-22 Silveira, E. R. PC-42 Simon-Levert, Annabel PC-101 Singburaudom, Narong PC-39 Siwe, Xavier N. PC-09, PC-62 Skepper, Colin IL-11


192

Smyrniotopoulos, Vangelis PC-03 Soares, Angélica R. PC-51 Son, Byeng W. PC-09, PC-62 Sonchaeng, Pichai PC-19 Sousa, A. M. M. PC-68 Sousa, Rui A. OC-22 Souto, Alba PC-66 Souto, Maria L. PC-87 Stiberg, Trine PC-44 Sukpondma, Yaowapa OC-05 Sun, Lin PL-09 Sutcliffe, Patricia R. PC-60 - T - Taboada, Sergi PC-45Taglialatela-Scafati, Orazio IL-07 Tamagnini, Paula PC-47 Tankoua, Olivia Fossi PC-55 Tarman, Kustiariyah PC-40 Tartaglione, Luciana OC-24, PC-10 Taudien, Stefan PC-63 Taupin, L. PC-81 Teixeira, Valéria L. PC-51 Tenreiro, R. PC-71 Tenreiro, T. PC-71 Teta, Roberta PC-67 Thananuson, Vitaya PC-93 Thoison, Odile PC-01 Thomas, Eric J. PL-03 Thomas, Olivier P. OC-08, OC-20, PC-29, PC-41, PC-88 Tidgewell, Kevin PL-06 Tonon, Thierry OC-01 Tosco, Alessandra OC-13 Trindade, Pedro PC-30 Trisuwan, Kongkiat OC-05 Tsuda, Masashi PC-02, PC-13 Tubaro, Aurelia OC-24 Turk, Tom PC-27 - V - Vagias, Constantinos PC-03, PC-17, PC-24, PC-32Vairappan, Charles S. PC-34 Valdés, O. PC-41 Vale, Paulo IL-09, PC-85 Valentão, Patrícia PC-30 Vallesi, Adriana OC-07 van der Sar, Sonia A. PC-58 Vansteelandt, Marieke PC-55 Varcamonti, Mario PC-05


193

Varela, Mercedes PC-83 Vasconcelos, Vítor M. PC-79 Vázquez, Jennifer PC-45 Viano, Yannick OC-06 Vieira, H. PC-71 Vilches, Tamara S. OC-25, PC-53 Villani, Guido PC-05, PC-22, PC-78 - W - Wang, Xiahong PC-70Wattanadilok, Rawiwan PC-19 Webb, Vicky A. PC-07, PC-58 Weber, Jean-Frédéric PL-09 Wende, Kristian PC-40 Wielgosz-Collin, Gaëtane PC-86 Wiens, Matthias OC-14, PC-70, PC-81 Wiese, J. PC-82 Wilke, D. V. PC-42 Wurster, Martina PC-40 Wüthrich, Kurt OC-07 - X - Xavier, J. PC-38, PC-94Xu, Zhongli PC-52 - Y - Yang, Guohua PC-09, PC-62Yokoya, Nair S. PC-23 Youssef, Diaa T.A. PC-21 Yun, Keumja PC-09, PC-62 Yunt, Zeynep PC-52 - Z - Zanfardino, Anna PC-05Żeglińska, Lidia PC-65 Zhang, Hua PC-97 Zhang, Wen OC-18

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