Abstracts Book

Transcrição

Abstracts Book

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 European Conference on
Marine Natural Products
6th ECMNP
19-23 July 2009, Porto, Portugal
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
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
4
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
5
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
6
Organisation
th
The 6 ECMNP 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
7
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
Tuesday 21st, 20h30
Wednesday 22nd, 20h30
Thursday 23rd, full day
Welcome Party (Hotel Fénix)
all participants & registered accompanying persons
Dinner & Music at Praia da Luz beach
all participants & registered accompanying persons
Gala Dinner and visit to Ferreirinha Port wine cellars
advanced registration (60 €) required
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.
8
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/
9
Sponsors
Additional Support
10
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
09:40-10:00
Oral Communication 1:
10:40-10:20
10:20-10:40
10:40-11:00
Coffee Break
König
Marine Microbial Metabolites - Focussing on Structural Diversity
Stéphane La Barre
NMR and MS Techniques for Rapid Characterization of Prokaryote
- Eukaryote Associations: Spectroscopic Fingerprints of Marine
Algae and of their Associated Microflora
Yue Wei Guo
Searching for New Bioactive Substances from South China Sea
Marine Organisms
Matthias Köck
Dimeric Pyrrole-Imidazole Alkaloids – New Massadine
Stereoisomers and their Configurational Assignment
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
11
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
12:10-12:30
12:30-12:50
12:50-13:10
13:10-14:30
Lunch
Vatcharin Rukachaisirikul
Metabolites from Marine-derived Fungi
Yannick Viano
Algal Diterpenoids as Antifouling Substances against a Marine
Bacterial Biofilm
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
15:20-15:40
15:40-16:10
Invited Lecture 4: Robert
16:10-16:30
Coffee Break
Pierangelo Luporini
NMR Structures of a Psychrophilic Family of Water-Borne Signal
Polypeptides Isolated from the Polar Protozoan Ciliate, Euplotes
nobilii
Mohamed Mehiri
Njaoaminiums A, B, and C: Cyclic 3-Alkylpyridinium Salts from the
Marine Sponge Reniera sp.
Capon
Australian Marine Biodiscovery
continuation
Chair: Madalena Pinto
16:30-17:10
Plenary Lecture 3: Eric
17:10-17:40
Invited Lecture 5: Ali
17:40-18:00
18:00-19:30
Poster Session [even numbers]
12
Jim Thomas
Approaches to the Total Synthesis of some Biologically Active
Natural Products
Al-Mourabit
Progress in Biomimetic Total Synthesis of the Marine Palau'amine
and Congeners
Emiliano Manzo
Synthesis of an Isocyanide Glyceryl-like Lipid Isolated from
Actinocyclus papillatus
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
09:40-10:00
10:40-10:20
10:20-10:40
10:40-11:00
Coffee Break
Diedrich
Marine Compounds as Promising Anti-Cancer Agents
Valeria Costantino
Potent Anti-Tumor and Anti-Inflammatory Lead Structures from
Caribbean Sponges
Heonjoong Kang
Novel Drug Leads for Hepatic Steatosis and Nonalcoholic
Steatohepatitis (NASH)
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
11:50-12:10
12:10-12:30
12:30-12:50
12:50-14:30
Lunch
Agostinho Casapullo
Chemical Proteomics as a Tool in Target Discovery of Bioactive
Small Molecules
Heinz Schröder
Marine Nanobiotechnology: Enzymes/Proteins from Sponges Acting
at the Interface of Inorganic Chemistry and Biology
Gäel Le Pennec
Molecular Communication Actors within the Porifera/Bacteria
Symbiotic Model. Insights into an Intimate Dialogue
Yoel Kashman
Salarins, Tulearins and Taumycins, Novel Marine Natural Products;
Chemistry, Stereochemistry and Activity
13
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
15:30-15:50
15:50-16:10
16:10-16:30
Coffee Break
Delphine Bry
Biosynthesis of Pyridoacridines in C. dellechiajei Cell-Free
Extracts
Adele Cutignano
The Bizarre Biogenetic Variability of Terpenes in the Antarctic
Mollusc Austrodoris kerguelenensis
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
14
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
09:40-10:00
10:40-10:20
10:20-10:40
10:40-11:00
Coffee Break
Pawlik
The Chemical Ecology of Sponges on Caribbean Coral Reefs: How
Natural Products Shape Natural Systems
Charline Abed
Chemotaxonomy as Valuable Approach to Study Sponges of the
Family Irciniidae (Porifera, Dictyoceratida)
Mikel A. Becero
Relevant Scale of Chemical Variation in Aplysina aerophoba
Tiago Silva
Chitosan Derived from Squid Pens on the Development of
Biomedical Membranes
Chair: Diaa Youssef
Oriol Sacristán-Soriano
Intraspecimen Variability of Natural Products in the Sponge
Aplysina aerophoba
11:10-11:20
11:20-11:50
Invited Lecture 9: Paulo
11:50-12:10
12:10-12:30
12:30-13:00
Invited Lecture 10: Ricardo
13:00-14:30
Lunch
Vale
Recent Developments in PSP Toxin Chemistry, Detection and
Biotransformation
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?
Antonio Hernandez Daranas
Bioactive Polyether Metabolites from Dinoflagellates: Structure
Determination And Bioactivity
Riguera
Marine Biopolymers in Nanomedicine: Advances in Drug Delivery
15
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.
16
Opening Lecture
MARINE NATURAL PRODUCTS AS AN INSPIRATION FOR DRUG DISCOVERY
Chulabhorn Mahidol OL
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
18
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
1
activities , 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
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]
PL 01
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
20
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 spongederived 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.
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
PL 02
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.
21
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]
PL 03 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.
Me Me
H HO
MeO2C
BO
H
O
Me
Me
X
OH H
O
20
Me
O
OH
16
17
O
H
1 O
O
27
Me
OH
CO2Me
1 X = n-PrCH=CH-CH=CH-CO2
2X=H
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
22
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.
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.
23
PL 04
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]
PL 05 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.
24
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, GenomicsDriven 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.
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).
25
PL 06
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
2
Stazione Zoologica Anton Dohrn, Villa Comunale 80121 Naples, Italy
CNR, Istituto di Chimica Biomolecolare, Via Campi Flegrei 34, 80078, Pozzuoli, Napoli, Italy
[email protected]
PL 07
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 shortchain 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
26
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.
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 lifescience 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 PL 08
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
27
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
2
PL 09
Department of Chemistry, University of Canterbury, Christchurch, New Zealand.
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
28
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.
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]
PL 10
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, 535538.
4. Cimino G.; Fontana A.; Gavagnin
Phythochem. Rev. 2004, 3, 285-307
M.
29
APIVITA - PSE
Award Lecture
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
AL 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.
32
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
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,
IL 01 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
34
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.
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 IL 02
for cytotoxic activity, pre-fractionation of
bioactive extracts by RP-18 column
chromatography
and
selection
of
candidates for fractionation by dereplication based on HPLC-DAD-MSbioactivity 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.
35
PHOTOCHEMISTRY IN THE METABOLITES OF PLACOBRANCHUS OCELLATUS
Paul B. Jones Wake Forest University Winston-Salem, NC 27106
[email protected]
The photochemical conversion of 9,10deoxytridachione
(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
IL 03
certainty.
Our work has focused on trying to
understand the mechanism of this
photoreaction, how it relates to other 1,3cyclohexadiene 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 1,3,5hexatrienes.
Photochemistry of 3 -alkyl6,6,9,9-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
hν
O
OMe
O
1
36
2
OMe
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.
IL 04
37
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]
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.
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
2
IL 05 corrected very recently.
HN
N
O
NH
N
H
N
H
H
H N
7
N
NH2
O
Cl
HN
H OH
NH
N
NH
NH
H
N
H
N
H OH
NH
N
NH
N H
Cl
HN
O
NH
N
H H
O
O
H
NH
O
NH
N
O
NH
NH
NH
N H
HN
HN
HN
O
O
NH H
NH H
HN
NH
38
H OH
NH
N
palau'amine A
NH
NH
NH
H
H
N
kombu'acidine A
NH
H
O
H2N
O
2x clathrodine
H2N
H
N
NH
Cl
HN
NH
N
H
N
7
NH
NH
Cl
HN
O
NH
1. Scheuer and coll., J.Am.Chem.Soc., 1993, 115,
3376-3377.
2. Köch and coll., Angew. Chem., 2007, 46, 23202324.
3. Al-Mourabit, A.; Potier, P. Eur. J. Chem. 2001,
237-243.
HN
NH
O
O
NH
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, anticancer 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 IL 06
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.
39
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, 300500 million people become ill with
malaria and 1-3 millions die.
Plakortin (1), a polyketide metabolite
in 1978 from Plakortis
halichondroides, was demonstrated to
possess a nanomolar antimalarial activity
IL 07 isolated
O
O
O
COOCH3
1
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.
40
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 1,2dioxanes through: i) isolation of
analogues;2 ii) preparation of semisynthetic 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.
O
COOCH2CH3
2
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.
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 spongeeating 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 IL 08
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: 13961399.
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: 352358.
7. Pawlik, Steindler, Henkel, Beer, Ilan, Limnol
Oceanogr 2007, 52: 907-911.
41
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
IL 09 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
42
the 11-hydroxysulfated GC1/2, while the
third sub-group was constituted by
analogues of GC3, lacking the 11hydroxysulfate. 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 semipurified GC toxins. Decarbamoyl
analogues are already detected by
common HPLC methods used for food
protection.
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 IL 10
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.
43
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
IL 11
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
The
complete
phorbasides
A-E.9
stereostructure of muironolide A (total
44
yield, 90 µg) was solved from NMR data
obtained using a 1.7 mm cryomicroprobe
(600 MHz), including strategic use of Jcoupled 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
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, MALDITOF
(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
OC 01
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
46
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.
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, 390392.
47
OC 02
DIMERIC PYRROLE-IMIDAZOLE ALKALOIDS – NEW MASSADINE
STEREOISOMERS AND THEIR CONFIGURATIONAL ASSIGNMENT
Matthias Köck1, Christine Cychon1, Gesine Schmidt1, Ian Seiple2 and Phil S. Baran2 1
2
Alfred-Wegener-Institut für Polar- und Meeresforschung in der Helmholtz-Gemeinschaft, Am
Handelshafen 12, D-27570 Bremerhaven, Germany.
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La
Jolla, CA 92037, USA
To date, about 30 dimeric pyrroleimidazole
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
OC 03 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
48
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.
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 labelfree 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, 15051508.
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, 20512057
49
OC 04
.
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
OC 05
50
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.
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 macroorganisms 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.
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.
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.
Finally, the anti-adhesion activity of
metabolites isolated in sufficient amount
was evaluated against a marine bacterial OC 06
biofilm of Pseudoalteromonas sp.
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,
O
HO
O
1. Costerton, J. W.; Lewandowski, Z.; Caldwell,
D. E.; Korber, D. R.; Lappin-Scott, H. M.
Annu. Rev. Microbiol. 1995, 49, 711-745.
H
O
H
H
OH
HO
Dictyotadimer A (1)
2
51
NMR STRUCTURES OF A PSYCHROPHILIC FAMILY OF WATER-BORNE SIGNAL
POLYPEPTIDES ISOLATED FROM THE POLAR PROTOZOAN CILIATE, EUPLOTES
NOBILII
1
1
Claudio Alimenti , Adriana Vallesi , 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 50150 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 familyspecific traits can be distinguished. The
OC 07
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
52
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 solventexposed 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.
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.
R1
8'
3
1
9'
1'
N
3'
7
13
R2
14
7'
N
,2X
16
OC 08
1 Njaoaminium A R1 = R2 = H
2 Njaoaminium B R1 = R2 = Me
3 Njaoaminium C R1 = H R2 = Me
53
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
4
papillatus, 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
OC 09 secondary chiral carbon of 2-isocyano1,3-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
54
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.
AcO
OC16H33
NC
1
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.
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
antiinflammatory 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.
55
OC 10
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
OC 11 will lead to develop a novel drug for the
treatment of the disease.
The nuclear receptors are ligandactivated transcription factors which
regulate many aspects of metabolism and
inflammation in liver. The receptors thus
become promising drug targets for
treatment of these diseases.
56
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.
SEARCH FOR WATER-SOLUBLE BIOACTIVE MARINE FUNGAL
METABOLITES: PERFECTING OF AN EXTRACTION AND PARTIALPURIFICATION METHOD
1*
Carine Le Ker , 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 watersoluble 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 watersoluble 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
57
OC 12
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]
OC 13
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
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
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
2. R. Aebersold, et al. Nature 2003, 422:198-208
58
through a spacer arm, the isolation of the
potential target(s) in a crude cell extract,
and its (their) identification by HR-ESIMSMS.
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
3. M.C. Monti, et al. Chem. Eur. J. 2009, 15:1155
– 1163
4. I. Posadas, et al. Biochem. Pharm. 2003, 165:
887-895
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 structuredirecting 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
lighttransmitting biomimetic optical fibres OC 14
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”).
59
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
OC 15
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 bacteriosponges are far from biofilm conditions
in their host do and how sponges mimic
60
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 bacteriosponges in order to detect new molecules
which can further be involved in this crosstalk. 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
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]
H3C (CH2)5
O
1'
8'
O
16
O
1
H3C
5
O
13
O
7
O
26
27
CH3
CH3
Ile-2
O
N
19
9
Tulearin B
18
13
17
30
22
CH3
29
1
HO
H3C
27
O
H2NOC
10
O
8
4
O
CH3
31
NH2
28
NH HN O
1
O
O
3
7
Taumycin A
(Selective compounds)
5
O
18
26
O
Ile-1
11
20
H3C O O
O
13
H3C (CH2)4
9
11
O
N
H
N
O
15
23
25
Salarin A
O
OO
19
21
15
16
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 OC 16
blood monocytes (PBM) in a dosedependent 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
61
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[2,3,4kl]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
OC 17 alkaloids.
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. BontempsSubielos, 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).
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
4. B. Steffan, K. Brix, and W. Putz, "Biosynthesis
of shermilamine B," Tetrahedron 49(28), 62236228 (1993).
Pyridoacridines with sulphur heterocycle Pyridoacridines with pyridine type heterocycle
Purple chromotype
2
1
11
10
9
D
A
B
C
8
N
H
6
Green chromotype
4
deAcshermilamine B
N
N
O
5
N
Figure 1:
pyrido[2,3,4-kl]acridine
skeleton
H
N
N
H
N
O
O
NH
shermilamine B
NH2
N
S
N
H
O
O
N
OH
N
hydroxyascididemine
N
N
S
N
S
N
H
S
NH2
N
H
N
N
H
N
Blue chromotype
N
deAckuanoniamine D
N
H
N
ascididemine
N
NH
kuanoniamine D
O
NH O
cystodimine A
Table 1: Pyridoacridines composition of purple, green and blue morphs of Mediterranean C. dellechiajei
62
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 coldadapted
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.
63
OC 18
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
1-3
Berlengas Islands and Setúbal Coast.
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 thresholdOC 19 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,
64
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
1-2
nudibranch. Feeding experiments with
[1-13C]-glucose demonstrated the ability
of D. pelseneeri to produce 15-entacetoxy-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
Castelluccio F, Mollo E, Cimino
Tetrahedron 2005, 61, 11032–11037;
G,
G,
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).
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 OC 20
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.
65
RELEVANT SCALE OF CHEMICAL VARIATION IN APLYSINA AEROPHOBA
Mikel A. Becerro1, Oriol Sacristan‐Soriano1, Nabil Majdi2 and Bernard Banaigs2 1
2
Center for Advanced Studies of Blanes (CEAB, CSIC), Acc Cala St. Francesc 14,
17300 Blanes (Girona), Spain.
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
OC 21 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
.
66
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
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, βchitin 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 OC 22
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,
Carbohydrate Polymers 35 (1998) 223-231
K.,
67
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
OC 23 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
68
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.
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, 42hydroxy 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.
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 42hydroxy 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
Toxicological studies on this new
palytoxin-like compound suggested that
the new palytoxin analog and palytoxin
itself share almost the same lethality.
2. Malo, D. Hawaiian Antiquites (B. P. Bishop
Museum Special Publication 2, ed. 2, Honolulu
1951), p.201-226
O
OH
OH
O
OH
O
OH
HO
H2N
OH
OH
O
OH
OH
Me
HO
OH
OH
OH
HO
OH
OH
HO
O
O
Me
OH
Me
OH
HO
O
OH
OH
HO
N
H
HN
OH
O
OH
OH
Me
OH
HO
HO
OH
Me
O
OH
O
O
OH
Me
OH
HO
OH
Me
OH
O
42
OH
HO
OH
HO
OH
OH
OH
Figure 1. Stereostructure of 42-hydroxy Palytoxin.
69
OC 24
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
OC 25
on the molecular details of important
cellular events. In this way, for example,
the remarkable selectivity of okadaic acid
and derivatives to inhibit some serinethreonine 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
.
70
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.
2.
3.
Cruz, P.G.; Norte, M.; J.M.; Fernández, J.J.;
Hernández Daranas, A. Chem. Eur. J. 2008,
14, 6948.
Napolitano, J.G.; Daranas, A.H.; Fernández,
J.J.; Norte, M. Anti-Cancer Agents Med.
Chem. 2009, 9, 122.
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 (AGL2005-07294-C04-01/ALI and CTQ2008-06754V04-01/PPQ). J.G.N. acknowledges MICINN for a
Ph.D. scholarship (FPU Program)
Poster Communications
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.
diketopiperazines and piperidiniums were
isolated from Axinella polypoides1-2
collected at Marseille. The chemistry of
these sponges and new pyrrole-2aminoimidazole
dimmers
will
be
described.
The
chemical
studies
of
both
dichloromethane/methanol
extracts
showed the presence of pyrrole-2aminoimidazole alkaloids in Axinella
polypoides from Lebanon while only
2. Gabant, M., 2008, PhD, Paris XI University.
PC 01
72
1. Known chemical studies: Crist, V.; Djerassi, C.
Steroids. 1983, 42, 331-343.
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 irregularlyintroduced C1 branches and oxygensubstituents, 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. Iriomoteolide8a exhibited cytotoxic activity against
human B lymphoma DG-75 and EpsteinBarr virus (EBV)-infected human B
lymphocyte Raji cells in vitro with IC50
values of 0.5 and 0.7 µg/mL, respectively.
PC 02
73
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.
74
HO
HO
H
H
H
H
PC 03 1
OH
OH
OH
O
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.
Br
H
Br
2
3
Br
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-diketoderivative 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-diketocembranoid 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 24 suggested that the 1,4-diketocembranoid 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.
PC 04
75
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
PC 05 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
NHAc
OAc
76
1
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, 57515755; 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.
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]
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.
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.
NH2
N
HO
O
N
OH OH
-
N
+
N
N
O
N
OH O
O P O
O
O
NH2
N
NH
O
O
O P O
O
NH2
O
P O
O
NH2
OH OH
1
O
N
OH O
O P O
O
O
N
N
N
N
NH2
N
O N
N
N
OH OH
PC 06
2
77
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.
PC 07
78
The structural elucidation, established by
NMR
spectroscopic
and
mass
spectrometric
analysis,
and
antiinflammatory 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.
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]
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.
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
OH
HO
Acknowledgement: Financial support came from
the Egyptian Government and from BMBF (project
No. 03F0415A)
OH
HO
H
OH
HO
HO
HO
H
OH
OH
O
CH 3
OH
CH 3
HO
OH
OCH
CH 3
COOH
H
H
H
R
H
H
OH
O
CH 3
H
R
3
H
R
H
O
OH
R
H
CH 3
H
HO
H 3C
CH 3
R=
O
CH 3
CH 3
CH 3
PC 08
CH 3
H
HO
H
COOH
79
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
PC 09
80
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.
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 27hydroxy-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
H
O
O
H
O
O
HO
O
OH
H
81
PC 10
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
2
Instituto de Química – Unesp, Araraquara, SP, Brazil
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,
PC 11
82
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
CHEMILUMINESCENT OXIDATIVE DEGRADATION OF DIKETOPIPERAZINES
AND EARLY PRECURSORS OF MARINE PYRROLE-2-AMINOIMIDAZOLE
METABOLITES
1
Ludmila Ermolenko , 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-surYvette, 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. AlMourabit, Org. Lett. 2008, 10, 493-496
2. N. Travert and A Al-Mourabit, J. Chem. Am.
Soc. 2004, 10, 10252-10253
Histidine 2h30 (lisse 30)
Hist / Tyr / Tryp / Leu / Pro
Tyrosine 0h45 (lisse 30)
Leucine 0h40 (s2) (lisse 30)
O
Proline 1h20 (s2) (lisse 30)
O
N
Tryptophane 0h30 (s2) (lisse 30)
12000
N
PC 12
10000
O2 (air)
8000
6000
chemiluminescence
HN
NH2
NH2
metabolite
formation
4000
2000
0
450
550
650
750
850
Wavelength (nm)
83
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
Amphidinium
strain
screening
of
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 masscultivation
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.
PC 13
84
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, 44694474.
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, 15671570.
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 1thiomethyldiscorhabdin G*/I (1), both
enantiomers
of
16,17dehydrodiscorhabdin W (2) and 3dihydrodiscorhabdin A (3).
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.
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
O
HN
O
H
N
H
N
HN
H3CS
O
H
N
S
NH
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.
S
NH
O
(+)-(6S,8S)-1
S
N
O
H
N
HN
S
NH
Br
O
O
(-)-(6S,6'aS)-2
Br
NH
Br
PC 14
OH
(+)-(3R,5R,6S,8S)-3
85
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
2
Instituto Universitario de Bio-Orgánica “Antonio González”, Universidad de La Laguna,
Francisco Sánchez 2, 38206 La Laguna, Tenerife, Spain.
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
PC 15
86
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 Jbased NMR configurational analysis in
combination with ROESY experiments.4
1.
2.
3.
4.
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.
Napolitano, J.G.; Daranas, A.H.; Fernández,
J.J.; Norte, M. Anti-Cancer Agents Med.
Chem. 2009, 9, 122.
Napolitano, J.G.; Norte, M.; Padrón, J.M.;
Fernández, J.J.; Hernández Daranas, A.
Angew. Chem. Int. Ed. 2009, 48, 796.
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).
SECONDARY METABOLITES FROM A MARINE SPONGE
AXINELLA POLYPOIDES
Belma Konuklugil1, Y. Y. Küçükecir1, Peter Proksch2, Bulent Gözcelioğlu3 1
2
University of Ankara, Faculty of Pharmacy,06100- Tandoğan, Ankara, Turkey.
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, LCMS). Futher studies are in progress.
Acknowledgements: this research has been
sponsored by Tübitak-Jülich ( SBAG-JULICH)-3
(104S409)
PC 16
87
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.
R2
PC 17
Br
O
Br
R1
88
Cl
1 R1 = OH,
2 R1 = OAc,
3 R1 = OAc,
R2 = OAc
R2 = OH
R2 = OAc
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 trapand API-CID-MS/MS examinations.
The MS fragmentation of these
compounds
is
similar
to
the
2
but
strongly
cyclostellettamines
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.
PC 18
89
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.
CO2H
PC 19
CH3
HO
HO
N
O
N
O
O
O
OH
3
2
1
HO
HO
HO
CH3
O
O
HO
O
4
90
HO
O
HO
O
5
O
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
2
Departamento de Química Fundamental, Facultade de Ciencias, Universidade da Coruña,
15071 A Coruña, Spain
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 2propanol 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
O
X
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 βoxidation. 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
H
N
X
O
O
H
N
O
HO
Parathiosteroids A-C: X = S
Parathiosteroid analogues: X = O or N
91
PC 20
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
PC 21
92
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.
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
Istituto di Chimica Biomolecolare, C.N.R., Via Campi Flegrei, 34, 80078, Pozzuoli, Italy
2
[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.
the sea-grasses Halophila stipulacea and
Posidonia
oceanica,
collected
in
Mediterranean Sea, that revealed to
contain novel secondary metabolites
including rare malonylated glycosylflavones (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; BinGui 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,
Phytochemistry, 1984, 23, 2609-2611.
In this communication we report the
results of our recent chemical studies on
A.
8. Agostani, S.; Desjobert, J.M.; Pergent, G.
Phytochemistry, 1998, 48, 611-617
PC 22
OH
O
O
H3CO
OH
HO
O
O
O
OCH3
HO
HO
O
OH
O
O
O
OH
O
1
OCH3
2
OH
93
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.
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
3-5
molecule.
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,
τ-[(2-methoxyBenzenepropanol,
ethoxy)methoxy]-β-methyl-,(R*,S*)-(.+PC 23 .)- and Tridecanol were the major
components. The EO and extracts were
tested against their antimicrobial activity,
however, no significant inhibition was
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.
94
1. Saunders, G.W.; Lehmkuhl, K.V., 2005. Eur. J.
Phy. 40, 293-312.
2. Pereira, R.C., et al., 2004 Bot. Mar. 47, 202208.
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.
NEW DOLABELLANES WITH ANTIBACTERIAL ACTIVITY FROM
THE BROWN ALGA DILOPHUS SPIRALIS
1
Efstathia Ioannou , 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, 2939 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
Dilophus
spiralis
constituents
of
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.
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.
PC 24
dolabellane skeleton
95
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-TOFMS 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
PC 25
96
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, 595662.
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.
STRUCTURAL ELUCIDATION OF NEW COMMUNESINS FROM A MARINEDERIVED 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
13
C-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 LCHRMS 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. Marinederived 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.
97
PC 26
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]+
PC 27
98
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.
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 Nmethyl-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 (PartidaMartinez, 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.
PC 28
99
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
2hydroxycyclopentenone D-ring with a 14β
configuration. Some structural differences
appear on the side chain of the aglycon
PC 29
100
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.
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/AGRAAM/64150/2006). D. Gomes (BI) and A. P.
Oliveira (SFRH/BD/47620/2008) are indebted to
FCT for their grants.
101
PC 30
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 farnesylpyrophosphate (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
PC 31 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.
102
[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, 55265534
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
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–
H
R1
R2
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.
1 R1 = H, R2 = Br
2 R1 = Br, R2 = H
OH
PC 32
103
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
PC 33
104
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.
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, cytotoxicity, 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.
PC 34
105
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
PC 35 population, since fisheries have possibly
reached their maximum yield due to
overexploitation4.
106
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 4-22005, 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
ANTIBACTERIAL AND ANTIBIOFILM ACTIVITIES OF BRAZILIAN MARINEDERIVED 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
107
PC 36
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.
PC 37
108
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
proliferatoractivated
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.
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 ratelimiting 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. Bioassayguided 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, 119.
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.
109
PC 38
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,
PC 39 Coscinium fenestratum were studied. The
result of investigation indicated that the
natural polymer chitosan and natural
alkaloid showed the same inhibiting the
110
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.
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
PC 40
111
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 hydroalcoholic extract of Thalassia testudinum
(BM21) (once a day during 7 days)
following UVB exposure results in a doseeffect 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
PC 41
strong skin regenerating activity.
112
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.
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% G2M 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. PC 42
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.
113
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,
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,
sp., Nodulisporium sp.,
PC 43 Nigrospora
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 sponge114
associated fungi and the plant pathogenic
fungi on PDA for 14 days at 28 oC. The
results showed that all of the spongeassociated fungi could inhibit the mycelial
Alternaria
alternata,
growth
of
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.
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.
PC 44
115
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
PC 45 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:
116
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.
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
2
Immunopathology Laboratory, Butantan Institute, Sao Paulo, Brazil;
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.
PC 46
Financial support: FAPESP (03/13207-0)
117
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
PC 47 (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.
118
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.
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,
2
Division of Environmental Science, Faculty of Science, Ramkhamhaeng University,
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
Rhizoctonia
solani
and
alternata,
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.
119
PC 48
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.
PC 49
120
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
Ethnopharmacology 2005, 100, 80
of
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,
4
Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science,
[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.
PC 50
121
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
PC 51 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
122
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.
GRISEORHODIN A: BIOSYNTHETIC STUDIES AND COMBINATORIAL
BIOSYNTHESIS
1
Kathrin Reinhardt , 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/ETmediated 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.
123
PC 52
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.
PC 53
124
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).
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]
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.
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 AC isolated from a marine-derived
Trichoderma saturnisporum2 (Figure 1)
give a good model system to gain a deeper
understanding of fungal polyketide
biosynthesis.
O
O
OH
OH
7
HO
7
R
H
HO
O
HO
O
H
O
H
Vertinolide
OH
D (3R, 5S, 6R; at C-7, C-8 only relative
stereochemistry implied)
O
O
HO
O OH
O
O
Trichodermanone
A R = α-OH, R1 = CH3
B R = β-OH, R1 = CH3
C R = β-OH, R1 = H
OH
O
OH
O
R1
OH
PC 54
O
O
Trichodimerol
O
O
OH
Rezishanone C
O
O
OH
OH
HO
O
Bislingiquinolide
OH
O
HO
O
HO
O
OH O
O
O
H
H
O
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
O
HO
1. Cox, R. J., Org Biomol Chem 2007, 5, (13),
2010-26.
HO
OH
Bisvertinol
OH
Sorbicillinol
Figure 1 Sorbicillinoids found in Trichoderma saturnisporum and sorbicillinol, a proposed
biosynthetic intermediate
125
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.
PC 55
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
126
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): 667673.
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): 111136.
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.
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 BIOTECHPHARMA
PC 56
127
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.
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.
From the crude extract of the
Mediterranean sponge Clathrina clathrus,
two major compounds, the known
clathridine1 and a new analogue, have
been isolated.
Results will be discussed with regard to
the origin of C. clathrus metabolites.
PC 57
Moreover, the phylogenetic diversity of
the bacterial biota has been analyzed
through DGGE experiments.
1. Ciminiello et al., 1989, Tetrahedron, 45: 3873
O
CH3
N
O
N CH3
NH
O
N
N
O
Clathridine
128
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-DomagkStr. 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.
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, 709722.
2. Nguyen, TA. et al. (2008) Nat. Biotechnol., 26,
225-233.
3. Fisch, K. M., Gurgui, C. et al. Nat. Chem.
Biol. Accepted
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.
PC 58
129
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
13
C-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: 233253
2. Mukhopadhyay, J. et al., (2008). Cell 135: 295307
Acknowledgement: Financial support came from
the DFG research project 854
O
OH
O
O
PC 59
OH
H
N
O
O
Figure 1. Structure of corallopyronin:
130
SAM derived;
acetate derived
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 interlagoon 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).
PC 60
Fig. 1 Principal component analysis of LCMS data from Theonella n.sp. and
Spheciospongia vagabunda
131
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.
biosynthetic genes, a genomic library was
constructed. The cosmid library was
screened with pks- and nrps-primers
(cyclization-,
adenylationdomain
primers) for the corresponding gene
cluster.
Three positive cosmid clones could be
identified containing nrps- and pksfragments. Data concerning the sequences
of subclones of these cosmids will be
presented.
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
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
.
PC 61
132
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.
PC 62
133
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
2
University of Bonn, Germany
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
PC 63 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
134
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.
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,
(2005) J. Nat. Prod. 68: 459-461
2. Cadel-Six, S., Dauga, C., Castets,
Rippka, R. (2008) Mol. Biol. Evol.
2031-2041
3. Li, T.L., Huang, F., Haydock, S.F.
Chem. Biol. 11(1): 107-119
G. M.
A.M.,
25(9):
(2004)
PC 64
135
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 nonspecific CAS AD and specific (Atkin,
PC 65
Arnow, Csaky) chemical tests. Biological
136
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 / αketohydroxy 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 αhydroxyacids.
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)
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 ironregulated 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.
137
PC 66
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
PC 67 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
138
express the pathway heterologously to
produce plakortin by fermentation.
O
O
COOCH3
plakortin
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.
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.
PC 68
1. Villanueva, R.D., Sousa, A.M.M., Gonçalves
M.P., Nilsson, M., Hilliou, L. (2009), Journal
of Applied Phycology, in press.
139
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
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
[email protected]
PC 69
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.
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).
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.
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.
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.
140
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.
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 manmade 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 biosilicaglass by living organisms. Nat. Prod. Rep. 25:455474
PC 70
141
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
2
BIOALVO S.A., Edificio ICAT, Campus da FCUL, 1749-016 Lisboa, Portugal
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
PC 71 extreme and unique environments, one
can expect to isolate new microorganisms
able to produce metabolites and chemicals
with unique molecular structures and
142
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.
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 nearNernstian 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.
MAA+PclTPB
AAMPSO
MAA
NIP AAMPSO
NIP MAA
AAMPSO washed
MAA washed
AAMPSO + pClTPB
50 mV
50 mV
-6.0
-5.5
-5.0
-4.5
-4.0
log [LMG], M
-3.5
-3.0
-2.5
-6.0
-5.5
-5.0
PC 72
-4.5
-4.0
-3.5
-3.0
-2.5
log [LMG], M
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(4chlorophenyl)borate.
143
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
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.
2
[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
PC 73
144
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 SG4I38 with swollen chitin gave better effect on survival
plant than other mixed strains with 73.3 %.
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.
PC 74
145
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
PC 75
146
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.
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.
147
PC 76
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
PC 77 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
148
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.
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 reuse 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
Spurilla
particular,
individuals
of
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, 23012303.
5. He J., Magarvey N., Piraee M., Vining L.C.
Microbiology 2001, 147, 2817–2829.
149
PC 78
A NEW PEPTIDIC ALLELOCHEMICAL FROM THE FILAMENTOUS
CYANOBACTERIUM OSCILLATORIA SP.
ISOLATION AND STRUCTURAL STUDIES
1,2
Pedro N. Leão , 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
PC 79
150
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).
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. L4n2 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.
151
PC 80
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
PC 81 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.
152
Higher eukaryotes communicate with
bacteria using hormones, notably by
catecholamines (Hugues et al., 2008).
Suberites
Futhermore,
the
sponge
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, PerovicOttstadt 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.
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 algaebacteria 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
PC 82
153
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
6
invertebrates so far . Specimens of a well
represented tunicate genus from the
Antarctic Ocean have been studied here.
The
genus
Aplidium
(Chordata:
PC 83
Ascidiacea) includes a large number of
species distributed all over the world, with
around 40 Antarctic species described7.
This group of colonial ascidians is
154
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: 19111922
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.
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.
2.
3.
4.
5.
Ludmila Ph. Paradina, Natalie N. Kulikova,
Alexander N. Suturin Yelena V. Saibatalova.
Geostandards and Geoanalytical Research.
2007, 28: 225-232.
Maria Fátima Araújo, Ana Conceição, Teresa
Barbosa, Maria Teresa Lopes , Madalena
Humanes . X-Ray Spectrometry. 2003,32:428433
Uthaiwan, K., Pakkong, P., Noparatnaraporn,
N., Vilarinho, L. , Machado, J. Invertebrate
Reproduction and development .2003.44(1) 5361.
Araujo, Maria Fatima, Alexandra Cruz,
Madalena Humanes, Maria Teresa Lopes,Jose
Armando.
Chemical Speciation and
Bioavailability 1999: 25-36
Oxford Instruments Industrial Analysis Group.
ED2000 Operator’s Manual. 1995 (Handbook).
Oxford Instruments Industrial Analysis Group.
PC 84
155
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,
Nsulfocarbamoyl,
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
PC 85 species strongly contaminated during G.
catenatum blooms occurring at the
156
Portuguese coast. The presence of M1 was
tentatively identified in a variety of
bivalve species, ranging from estuarine
habitat:
blue
mussels
(Mytilus
galloprovinciallis),
cockles
edule)
and
clams
(Cerastoderma
(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.
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 (GCMS) analyses of their methyl esters and Nacyl 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, 1118:1, 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, biologicallyactive 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
PC 86
157
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 (DTX1), YTXs do not cause diarrhoea when
orally administered.3-5
However, study of these toxins does not
deal solely with the repercussions on
PC 87
158
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).
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.
PC 88
159
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 2vinyl
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 nearPC 89 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
160
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
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.
161
PC 90
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
2
CIMAR University of Porto
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,
PC 91
162
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.
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 spraydrying coagulation,4 suspension crosslinking,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 10100 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 (10100 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 1100 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
163
PC 92
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
PC 93
164
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.
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 PC 94
antioxidant and radical scavenging
properties they can be used in other areas,
like medicine.
165
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 selfassembly 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
(DA) of CS was
PC 95 acetylation degree
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
166
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.
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 Nhydroxysuccinimide
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.
167
PC 96
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
PC 97
168
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 sevenmember 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.
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
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
A new peptide isolated recently by
PharmaMar from a sample of Spongia sp.
contains in its structure the cyclic
tripeptide A possessing the mentioned
Newhouse, T.; Lewis, C. A.; Baran, P., J. Am.
Chem. Soc., 2009, 131, 6360
S
O
N
N
O
Trp-1
N
S
S
N
N
O
N
S
O
OH
HN
N
O
Trp-2
O
O
R
PC 98
HN
N
N
R
R
O
Ile-1
O
Kapakahine B
Chaetocochins A
Tripeptide A
169
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,8,8ahexahydropyrrolo[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 bromoderivative
3a-bromo-1,2,3,3a,8,8ahexahydropyrrolo[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.
PC 99
170
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
EXPLORATIVE SOLID-PHASE EXTRACTION FOR ACCELERATED NATURAL
PRODUCTS DISCOVERY AND PURIFICATION
Maria Johansen1, Kristian Fog Nielsen2, Lone Gram3, Thomas Ostenfeld Larsen4 1
[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 (ESPE) 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 itself 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.
171
PC 100
PRO-APOPTOTIC SECONDARY METABOLITES ISOLATED FROM THE
CYANOBACTERIA LYNGBYA MAJUSCULA
Annabel Simon‐Levert1, Anne‐Marie Genevière2, Bernard Banaigs1 and Isabelle Bonnard‐Cussac1 1
Laboratoire de Chimie des Biomolécules et de l'Environnement. UP. Perpignan. France
2
Laboratoire 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.
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
NMeVal 2
O
Pla 3
PC 99
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.
N
Val 1
O
N
O
O
O
HN
O
O
Pro 4
N
O
Hmoya 6
Hmoea 6
Hmoaa 6
NMeIle 5
172
R
Tiahuramide A : R =
Tiahuramide B : R =
Tiahuramide C : R =
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]
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]
ROCHA, FABIOLA DUTRA
Universidade Federal de Minas Gerais
[email protected]
173
AMADE, PHILIPPE
Université de Nice
[email protected]
LA BARRE, STEPHANE
Station Biologique de Roscoff
[email protected]
BANAIGS, BERNARD
Laboratoire de Chimie des Biomolécules et de
l'Environnement
Université de Perpignan.
[email protected]
LE JEUNE, CLARISSE
ICSN - CNRS
[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
[email protected]
DUFOUR-SCHROIF, COSIMA
COVALMAR
[email protected]
DUPLAT, DENIS
COVALMAR
[email protected]
GAEDERES, JOHAN
Université de Bretagne Sud
[email protected]
GROVEL, OLIVIER
[email protected]
KERZAON, ISABELLE
[email protected]
174
LE KER, CARINE
[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
[email protected]
VIANO, YANNICK
MAPIEM
[email protected]
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
[email protected]
KÖNIG, GABRIELE
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
[email protected]
SCHRÖDER, HEINZ C.
Institut für Physiologische Chemie
Angewandte Molekularbiologie
Universität Mainz
[email protected]
SCHÄBERLE, TILL
University of Bonn
[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]
175
ROUSSIS, VASSILIOS
School of Pharmacy
Natural Products
[email protected]
VAGIAS, CONSTANTINOS
School of Pharmacy
Natural Products
[email protected]
IRELAND
PEDDIREDDI, SUDHAKAR
Centre for Applied Marine Biotechnology
[email protected]
QUINN, GERARD
Letterkenny Institute of Technology
[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
[email protected]
GAVAGNIN, MARGHERITA
Istituto di Chimica Biomolecolare - CNR
[email protected]
ISRAEL
FAIBISH, HANNY
Ben Gurion University
[email protected]
GRAUSO, LAURA
[email protected]
KASHMAN, YOEL
Tel Aviv University School of Chemistry
[email protected]
GUELLA, GRAZIANO
University of Trento
[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]
176
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
University of Napoli Federico II
[email protected]
MANZO, EMILIANO
Institute of Biomolecular Chemistry (CNR)
[email protected]
CHAMYUANG, SUNITA
The University of Canterbury
[email protected]
SPOLITI, MAURIZIO
"La Sapienza"
University of Rome
[email protected]
COPP, BRENT R.
University of Auckland
[email protected]
TAGLIALATELA-SCAFATI, ORAZIO
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]
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]
JAPAN
KUMAGAI, KEIKO
Kochi University
[email protected]
PERANDER, MARIA
University of Tromsø
MabCent-SFI
[email protected]
TSUDA, MASASHI
Kochi University
[email protected]
SANDSDALEN, ERLING
Northern Research Institute
[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]
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]
177
ADRIANO, GISELA DOS SANTOS
Faculty of Pharmacy
University of Porto
CEQUIMED-UP
[email protected]
CIDADE, HONORINA
Faculty of Pharmacy
University of Porto
CEQUIMED-UP
[email protected]
AFONSO, CARLOS MANUEL MAGALHÃES
Faculty of Pharmacy
University of Porto
CEQUIMED-UP
[email protected]
COIMBRA, JOÃO
Institute of Biomedical Sicences Abel Salazar
CIIMAR
[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]
178
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
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]
GONÇALVES, MARIA DO PILAR FIGUEROA
Faculdade de Engenharia - UP
[email protected]
SCHULZE, CORINNA
CIMAR, Universidade do Porto
[email protected]
GUEDES, ANA CATARINA AFONSO
ESB/CBQF - UCP
[email protected]
SILVA, ARTUR MANUEL SOARES
Department of Chemistry
University of Aveiro
[email protected]
GUERREIRO, JOANA RAFAELA LARA
ICETA - Instituto de Ciências e Tecnologias
Agrárias e Agro Alimentares
[email protected]
KIJJOA, ANAKE
CIIMAR
[email protected]
LEÃO, PEDRO
CIIMAR-University of Porto
SIO - University of California, San Diego
[email protected]
MARTINS, CARLOS GIL
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
CIIMAR
[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
[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]
179
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
[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]
QUIÑOA, EMILIO
Universidad de Santiago de Compostela
[email protected]
REYES, JOSE FERNANDO
[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]
MARTIN, VICTOR S.
Instituto Universitario de Bio-Organica Antonio
Gonzalez
[email protected]
SWEDEN
BOHLIN, LARS
Division of Pharmacognosy
Department of Medicinal Chemistry
Biomedical Center
Uppsala University
[email protected]
NAPOLITANO, JOSE GABRIEL
IUBO-AG
[email protected]
THAILAND
HRH PRINCESS CHULABHORN MAHIDOL
Chulabhorn Research Institute
NOYER, CHARLOTTE
Centro de Estudios Avanzados de Blanes
(CEAB, CSIC)
[email protected]
PONS, LAURA NUÑEZ
Universitat de Barcelona
[email protected]
180
DETHOUP, TIDA
Department of Plant Pathology
Faculty of Agriculture
Kasetsart University
[email protected]
MANOCH, LEKA
[email protected]
PAKKONG, PANNEE
[email protected]
PASANPHAN, WANVIMOL
[email protected];[email protected]
PHONGPAICHIT, SOUWALAK
Department of Microbiology
Faculty of Science
Prince of Songkla University
[email protected]
PONGSAMART, SUNANTA
Chulalongkorn University
[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]
PRASATSRISUPAB, JARIYA
Department Agriculture
[email protected];[email protected]
FISCH, KATJA MARIA
School of Chemistry
University of Bristol
[email protected]
RUKACHAISIRIKUL, VATCHARIN
Prince of Songkla University
[email protected]
TABUDRAVU, JIOJI
Aquapharm Biodiscovery Ltd
[email protected]
SINGBURAUDOM, NARONG
[email protected]
THOMAS, ERIC JIM
School of Chemistry
The University of Manchester
[email protected]
SONCHAENG, PICHAI
National Science Museum
Thailand
[email protected]
UNITED STATES OF AMERICA
GERWICK, WILLIAM
Scripps Institution of Oceanography
Univ. California San Diego
[email protected]
SUWANBORIRUX, KHANIT
Faculty of Pharmaceutical Sciences
Chulalongkorn University
[email protected]
TUNKIJJANUKIJ, SURIYAN
Faculty of Fisheries
[email protected]
VICHIEN, KITPREECHAVANICH
Dept of Microbiology
Faculty of Science
[email protected]
TUNIS
HAMROUNI BUONOMO, SOUHIR
National Institute of Agronomy
[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]
181
Authors Index
-AAbe, R. O.
Abed, Charline
Abraham, Wolf-Rainer
Abreu, Maria Helena
Acosta, Gerardo A.
Albericio, Fernando
Albrigtsen, Marte
Alimenti, Claudio
Allard, Marc
Almeida, Celso
Al-Mourabit, Ali
Álvarez, Mercedes
Alves, M. R.
Alves, V. D.
Amade, Philippe
Amaro, Helena M.
Andersen, Jeanette Hammer
Anderson, Sally
Andrade, C. T.
Andrade, Paula B.
Araújo, A. R.
Araújo, M.
Ariffin, Siti Alwani
Avila, Conxita
PC-11
OC-20
PC-36
PC-91
PC-98, PC-99
PC-98, PC-99
PC-37
OC-07
PC-74
IL-01, PC-28
IL-05, PC-01, PC-12
PC-98, PC-99
PC-76
PC-68
OC-08, PC-29
PC-47
PC-37, PC-44
PC-58
PC-95
PC-30
PC-11
PC-94
PL-09
OC-18, PC-45, PC-83
-BBakar, Hamidah
Ballesteros, Manuel
Banaigs, Bernard
Bandarra, Narcisa M.
Baran, Phil S.
Barnathan, Gilles
Barreto, B. N.
Bashira, Ashgan
Becerro, Mikel A.
Ben Rejeb Jenhani, Amel
Ben Saidin, J.
Ben-Califa, Nathalie
Bessa, Julia
Beukes, Denzil R.
Biard, Jean-François
Bignami, Gary
BIOALVO
Bitam, Fatma
Bitar, Ghazi
Blache, Yves
PL-09
OC-18
OC-17, OC-21, OC-23, PC-101
PC-75
OC-03
PC-86
PC-95
OC-16
OC-21, OC-23, PC-57, PC-77, PC-88
PC-77
PC-81
OC-16
PC-19
PC-33
OC-12, PC-26, PC-55, PC-86
OC-24
PC-38
PC-22
OC-20, PC-01
OC-06
182
Blunt, John
Bolton, John J.
Bolzani, V. S.
Bonnard-Cussac, Isabelle
Bonhomme, Dominique
Bontemps, Nataly
Bouhired, Sarah
Boumghar, Yacine
Bourguet-Kondracki, Marie-Lise
Briand, Jean-François
Bringmann, Gerhard
Bruhn, Torsten
Bry, Delphine
Buaruang, Jamrearn
Bueno, V.
Bugni, Tim S.
PL-09
PC-33
PC-11
PC-101
OC-06
OC-17
PC-56
PC-74
PC-57, PC-69, PC-80
OC-06
PC-52
PC-52
OC-17
PC-43, PC-48
PC-41
PC-60
-CCachatra, V.
Cachet, Nadja
Calado, P.
Calejo, M. T.
Callone, Emanuela
Camps, Mercedes
Cao, Zhengyu
Capon, Robert J
Carbone, Marianna
Casapullo, Agostino
Casotti, Raffaella
Castelluccio, Francesco
Catalanotti, Bruno
Chan, S. T. S.
Charrier, Bénédicte
Chirachanchai, Suwabun
Choi, Hyukjae
Choofong, Surakarn
Chumyuang, Sunita
Ciavatta, Maria Letizia
Ciminiello, Patrizia
Cimino, Guido
Cole, Tony
Colepicolo, Pio
Concepción, A. R.
Conti, R.
Copp, B. R.
Costa-Lotufo, L. V.
Costantino, Valeria
Cox, James E.
Cox, Russell J.
PC-38
PC-29
PC-71
PC-96
PC-31
OC-06
PL-06
IL-04, PC-97
OC-09, PC-22, PC-78, PC-83
OC-13
PL-07
PC-83
PC-10
PC-07
OC-01
PC-92
OC-11
PC-92
PL-09
OC-09, PC-05, PC-22, PC-78
OC-24, PC-10
PL-07, PL-10, OC-18, PC-78
PL-09
PC-23
PC-41
PC-49
PC-07, PC-14
PC-42
OC-10, PC-67
PC-60
PC-54
183
Crews, Phillip
Cristobo, Francisco Javier
Cueto, Mercedes
Cuevas, Carmen
Culioli, Gérald
Cutignano, Adele
Cuypers, Beate
Cychon, Christine
PC-63
PC-45
PC-04
PC-99
OC-06
PL-07, OC-18, PC-90
PC-40
OC-03, PC-25
-DD’Croz, Luís
d’Ippolito, Giuliana
da Silva, Patrícia P.M.
Dagorn, Flore
Dalisay, Doralyn S.
Daranas, Antonio Hernández
Darias, J.
Davis, Paul
De Bortoli, Marco
de Pinho, Paula Guedes
de Souza, H. K. S.
Debonsi, H. M.
Defant, Andrea
Delerue-Matos, C.
Dell’Aversano, Carmela
Dello Iacovo, Emma
Dethoup, Tida
Di Giuseppe, Graziano
Díaz-Marrero, Ana R.
Dicato, Mario
Diederich, Marc
Dilokkunanant, Uraiwan
Dini, Fernando
Dittami, Simon
Domart-Coulon, Isabelle
Domínguez, Humberto J.
Dorrestein, Pieter C.
Du Pont, Thibaut Robiou
Dufour-Schroif, Cosima
Dumay, Justine
Duplat, Denis
PC-04
PL-07, PC-90
PC-23
PC-86
IL-11
OC-25, PC-15, PC-53, PC-87
PC-04
PL-09
OC-24
PC-30
PC-95
PC-11, PC-49
PC-27
PC-35, PC-68
OC-24, PC-10
OC-24, PC-10
PC-43, PC-48
PC-31
PC-04
IL-06
IL-06
PC-73
PC-31
OC-01
PC-57, PC-80
PC-87
PL-06
PC-26, PC-55
PC-69
PC-86
PC-69
-EEdrada-Ebel, RuAngelie
Eklund, Minna
El Mehdi, Naima
El Omari, M.
Elsebai, M. F.
OC-04
PC-52
PC-74
PC-64
IL-01, PC-08
184
Erbert, C.
Eriksson, Jonas
Ermolenko, Ludmila
Erol-Hollmann Ö.
Esquenazi, Eduardo
PC-11, PC-49
PC-44
PC-12
PC-59, PC-61
PL-06
-FFalkenberg, Miriam
Fattorusso, Caterina
Fattorusso, Ernesto
Felício, R.
Feng, Zhile
Fernandes, A. I.
Fernandez, José Javier
Fernández, Rogelio
Ferreira, E. G.
Ferreira, R. A. S.
Figuerola, Blanca
Fisch, Katja M.
Fleurence, Joël
Folmer, Florence
Fontana, Angelo
Forestieri, Roberto
Forino, Martino
Frassanito, Rita
Frontini, Francesco
Fujii, Mutue T.
Furtado, N. A. J. C.
PC-17
PC-10
OC-10, OC-24, PC-10, PC-67
PC-49
PC-09, PC-62
PC-96
OC-25, PC-15, PC-53, PC-87
OC-08, PC-20, PC-98
PC-42
PC-46
PC-45
PC-54, PC-58, PC-63
OC-12
IL-06
Apivita PSE Award Lecture, PL-07, OC-18, PC-90
PC-83
OC-24, PC-10
PC-31
PC-31
PC-23
PC-49
-GGabant, Marion
Gardères, J.
Garderes, Johan
Gaspar, Helena
Gavagnin, Margherita
Genevière, Anne-Marie Genta-Jouve, Grégory
Gerwick, Lena
Gerwick, William H.
Gibbons, Simon
Gomes, Daniela
Gonçalves, L. L.
Gonçalves, M. P.
González, Jaime Rodríguez
Gözcelioğlu, B.
Gram, Lone
Grauso, Laura
Gressler, Vanessa
Greve, H.
PC-01, PC-12
PC-81
OC-15
OC-19, PC-38
OC-09, PC-05, PC-22, PC-78, PC-83
PC-101
OC-08, PC-29
PL-06, PC-67
PL-06, PC-67, PC-79
PC-24
PC-30
PC-96
PC-68, PC-95
PC-83
PC-16
PC-100
OC-24, PC-10
PC-23
IL-01
185
Grindberg, Rashel
Grkovic, T.
Grovel, Olivier
Guedes, A. Catarina
Guedes, A.
Guella, Graziano
Guerreiro, J. Rafaela L.
Guo, Yue-Wei
Gurgui, Cristian
PL-06
PC-14
PC-26, PC-55
PC-47
PC-38
PC-27, PC-31
PC-89
OC-02
PC-58, PC-63
-HHahn, Dongyup
Hamrouni Buonomo, Souhir
Harper, J.
Harper, Mary Kay
Henriques, Amélia T.
Hertweck, Christian
Heycke, Nina
Hoffmann, Friederike
Hong, Junyoung
Hooper, John N. A.
Horta, Paulo Antunes
Houghton, Peter J.
Höver, T.
Humanes, M.
Hwang, Byungsoo
Hwang, Hoosang
OC-11
PC-77
PC-07
PC-60
PC-36
PL-05, PC-52
PC-63
PC-06
OC-11
PC-60
PC-17
PC-51
PC-61
PC-38, PC-94
OC-11
OC-11
-IIreland, Chris M.
Ianora, Adrianna
Ioannou, Efstathia
Ionta, M.
Imhoff, J. F.
PL-02, PC-60
PL-07
PC-17, PC-24, PC-32
PC-46
PC-82
-JJanuario, A. H.
Jaspers, Marcel
Jiménez, Carlos
Jimenez, P. C.
Johansen, Maria
Jones, Adam C.
Jones, Paul B.
Jørgensen Trond Ø.
PC-07
IL-06
PC-20, PC-66, PC-83
PC-42
PC-100
PL-06
IL-03
PC-37, PC-44
-KKajahn, I.
Kamel, Ayman H.
Kang, Heonjoong
PC-82
PC-89
OC-11
186
Kaplan, Maria Auxiliadora C.
Kashman, Yoel
Kehraus, S.
Kelve, Merike
Kervarec, Nelly
Kerzaon, Isabelle
Khalaf, Gaby
Khamthong, Nanthaphong
Kijjoa, Anake
Kim, Euno
Kim, Jung-A
Kitpreechavanich, Vichien
Köck, Matthias
König, Gabriele M.
Konuklugil, B.
Korbee, Nathalie
Kosakowska, Alicja
Küçükecir, Y. Y.
Kumagai, Keiko
PC-51
OC-16
IL-01, PC-08
PC-06
OC-01
PC-26
OC-20
OC-05
PC-19, PC-43, PC-48
OC-11
OC-11
PC-73
OC-03, PC-18, PC-25
IL-01, PC-08, PC-28, PC-56, PC-59, PC-61, PC-64
PC-16
PC-91
PC-65
PC-16
PC-02, PC-13
-LLa Barre, Stéphane
Labes, A.
Laguna, L.
Lalk, Michael
Lam, C.
Lamari, Nadia
Larsen, Thomas O.
Le Bail, Aude
Le Ker, Carine
Le Pennec, Gaël
Leão, Pedro N.
Lejeune, Clarisse
Lemos, Manuel L.
Leo, Angela
Lerner, Cléa
Leutou, Alain S.
Lewandowska, Agnieszka
Lhullier, Cintia
Li, Yan
Lindequist, Ulrike
Lisboa, F.
Lopes, J. L. C.
Lopes, M. N.
Lopes, N. P.
Lopp, Annika
Lorenzo, Manuel
Luca, A. N.
Luporini, Pierangelo
OC-01
PC-82
PC-41
PC-40
PC-14
PC-90
PC-100
OC-01
OC-12
OC-15, PC-81
PC-79
PC-01
PC-66
PC-10
PC-36
PC-09, PC-62
PC-65
PC-17
OC-09
PC-40
PC-76
PC-11, PC-49
PC-11
PC-42
PC-06
PC-04
PC-11
OC-07
187
-MMacedo, Alexandre J.
Machado-Santelli, G. M.
Mahidol, HRH Princess
Maifi, Fatna
Majdi, Nabil
Malcata, F. Xavier
Mancini, Ines
Mangoni, A.
Mangoni, Alfonso
Mann, Maryssa G.
Mano, João F.
Manoch, Leka
Manzo, Emiliano
Margarucci, Luigi
Martin, Marie-Thérèse
Matucheski, Stella García
Mazères, Serge
Mehiri, Mohamed
Meijer, Laurent
Mernitz, Gudrun
Minoru, Suzuki
Minucci, Carmen
Miralto, Antonio
Missau, Fabiana C.
Mohd Khalid, Rozida
Molinski, Tadeusz F.
Mollo, Ernesto
Monti, Maria Chiara
Montresor, Marina
Moradas-Ferreira, Pedro
Moraes, M. O.
Morais, S.
Morais, Zilda B.
Moreira, Felismina T. C.
Morinaka, Brandon I.
Moriou, Céline
Mothes, Beatriz
Mouga, Teresa
Müller, Werner E.G.
Muniain, Claudia
Munro, Murray
Murray, Thomas F.
PC-36
PC-46
Opening lecture
PC-74
OC-21
PC-47
PC-27, PC-31
OC-10
PC-67
PC-33
OC-22
PC-43, PC-48
OC-09, PC-05, PC-22
OC-13
PC-01
PC-78
PC-12
OC-08, OC-20, PC-29
PL-04
PC-40
PC-34
PC-90
PL-07
PC-23
PC-54
IL-11
PC-22, PC-78
OC-13
PC-90
PC-47
PC-42
PC-35, PC-68
PC-75, PC-96
PC-72, PC-89
IL-11
PC-12
PC-36
PC-30
PL-08, OC-14, PC-70
PC-78
PL-09
PL-06
-NNagel, K.
Napolitano, José G.
Nappo, Michela
PC-82
OC-25, PC-15, PC-53
PC-83
188
Natalio, Filipe
Nenkep, Viviane N.
Nett, M.
Neumann, Drorit
Neumann, K.
Nguyen, Tu Anh
Nielsen, Kristian F.
Nieto, Rosa M.
Nogueira, J. M.
Nogueiras, C.
Norte, Manuel
Noyer, Charlotte
Núñez-Pons, Laura
Nuzzo, Genoveffa
OC-14, PC-70
PC-09, PC-62
PC-61
OC-16
IL-01
PC-58
PC-100
PC-83
PC-38
PC-41
OC-25, PC-15, PC-53, PC-87
PC-88
PC-45
PC-05
-OOliveira, A. L. L.
Oliveira, Andreia P.
Oliveira, Joaquim M.
Oliveira, M. B. P. P.
Ortalo-Magné, Annick
Osorio, Carlos R.
PC-49
PC-30
OC-22
PC-35, PC-76
OC-06
PC-66
-PPaíga, P.
Pakkong, Pannee
Pasanphan, Wanvimol
Paulsen, Steinar M.
Pawlik, Joseph R.
Pearce, A. N.
Pedrini, Bill
Perander, Maria
Pereira, Alban
Pereira, Renato C.
Pereira, Ricardo D.
Pereira, Rui
Pereira, Teresa G.
Perez, Thierry
Perinu, C.
Pessoa, C. O.
Pestana, N.
Petit, Karina-Ethel
Petraki, Anastasia
Pfister, S. C.
Phongoaichit, Souwalak
Phongphern, Wanvisa
Piadang, Nattayana
Pichon, Delphine
Piel, Jörn
PC-35
PC-84, PC-93
PC-92
PC-37
IL-08
PC-07
OC-07
PC-44
PL-06, PC-79
PC-51
PC-47
PC-91
PC-75
OC-20, PC-01, PC-57
OC-10
PC-42
PC-76
OC-12, PC-55
PC-32
PC-46
OC-05, PC-50
PC-84
PC-73
PC-69
PC-52, PC-58, PC-63, PC-67
189
Pino, J. A.
Pinto, Ernani
Pinto, Isabel Sousa
Piriyaprin, Siangjeaw
Piva, R. F.
Plathong, Sakanan
Platzer, Matthias
Poli, Mark
Pons, Laura Núñez
Pontius, A.
Porras, Gina
Potin, Philippe
Pouchus, Yves François
Poza, Javier Jesús
Prasatsrisupab, Jariya
Preedanon, Sita
Proksch, Peter
Puchakarn, Sumaitt
Pupo, M. T.
PC-41
PC-23
PC-91
PC-43, PC-48
PC-46
PC-50
PC-63
OC-24
PC-83
IL-01
PC-04
OC-01
OC-12, PC-26, PC-55
PC-20
PC-93
PC-50
PL-01, PC-16
PC-19
PC-49
-QQuévrain, Elodie
PC-69, PC-80
-RRabesaotra, Vony
Rahman, M. Mukhlesur
Ramalhosa, M. J.
Ramasamy, Kalavathy
Ramos, Alfonso
Rangel, M.
Rapp, Hans Tore
Raspor, Lucija
Ratinaud, Céline
Regalado, Erik L.
Reinhardt, Kathrin
Reintamm, Tõnu
Reis, Rui L.
Reppart, Jason
Reyes, Fernando
Rho, Jungrae
Riccio, Raffaele
Riguera, Ricardo
Rimdusit, Pakjira
Rivas, Amable J.
Robinson, Sarah J.
Roca, C.
Rocha, Fabiola D.
Rochefort, Guy
Rodríguez, Alberto
PC-86
PC-24
PC-35
PL-09
PC-83
PC-46
PC-06
PC-27
PC-12
PC-29, PC-41
PC-52
PC-06
OC-22
PC-60
IL-02, OC-08, PC-20
OC-11
OC-13
IL-10
PC-92
PC-66
PC-63
PC-71
PC-51
PC-74
PC-98
190
Rodríguez, Jaime
Rodríguez, M.
Romano, Giovanna
Roué, Mélanie
Roussis, Vassilios
Rubio, Brent K.
Rudi, Amira
Rugthaworn, Prapassorn
Ruiz, Nicolas
Ruiz-Sanchis, Pau
Rukachaisirikul, Vatcharin
PC-20, PC-66
PC-41
PL-07
PC-57
PC-03, PC-17, PC-24, PC-32
PC-63
OC-16
PC-73
PC-26
PC-98, PC-99
OC-05, PC-50
-SSacristan-Soriano, Oriol
Saidin, Jasnizat Bin
Sakayaroj, Jariya
Salah Romdhane, Mohamed
Salaün, Stéphanie
Sales, M. Goreti F.
Sampaio, P.
Sangchote, Somsiri
San-Martín, Aurelio
Santos, A.
Santos, J.
Santos, S.
Savina, Svetlana
Sawangwong, Pichan
Schäberle, T.
Schenkel, Eloir Paulo
Schlacher-Hoenlinger, Monika A.
Schmidt, Gesine
Schmitz, A.
Schneemann, I.
Schröder, Heinz C.
Schumacher, Marc
Scopel, Marina
Seabra, Rui
Seiple, Ian
Sepčić, Kristina
Seternes, Ole Morten
Shaala, Lamiaa A.
Shin, Sun
Silva, Simone S.
Silva, Tiago H.
Silveira, E. R.
Simon-Levert, Annabel Singburaudom, Narong
Siwe, Xavier N.
Skepper, Colin
OC-21, OC-23
OC-15
OC-05, PC-50
PC-77
OC-01
PC-72, PC-89
PC-95
PC-73
PC-04
PC-71
PC-76
PC-38
PC-98, PC-99
PC-84
PC-59
PC-17
PC-60
OC-03, PC-18, PC-25
PC-59
PC-82
PL-08, OC-14, PC-70
IL-06
PC-36
PC-47
OC-03
PC-27
PC-44
PC-21
OC-11
OC-22
OC-22
PC-42
PC-101
PC-39
PC-09, PC-62
IL-11
191
Smyrniotopoulos, Vangelis
Soares, Angélica R.
Son, Byeng W.
Sonchaeng, Pichai
Sousa, A. M. M.
Sousa, Rui A.
Souto, Alba
Souto, Maria L.
Stiberg, Trine
Sukpondma, Yaowapa
Sun, Lin
Sutcliffe, Patricia R.
PC-03
PC-51
PC-09, PC-62
PC-19
PC-68
OC-22
PC-66
PC-87
PC-44
OC-05
PL-09
PC-60
-TTaboada, Sergi
Taglialatela-Scafati, Orazio
Tamagnini, Paula
Tankoua, Olivia Fossi
Tarman, Kustiariyah
Tartaglione, Luciana
Taudien, Stefan
Taupin, L.
Teixeira, Valéria L.
Tenreiro, R.
Tenreiro, T.
Teta, Roberta
Thananuson, Vitaya
Thoison, Odile
Thomas, Eric J.
Thomas, Olivier P.
Tidgewell, Kevin
Tonon, Thierry
Tosco, Alessandra
Trindade, Pedro
Trisuwan, Kongkiat
Tsuda, Masashi
Tubaro, Aurelia
Turk, Tom
PC-45
IL-07
PC-47
PC-55
PC-40
OC-24, PC-10
PC-63
PC-81
PC-51
PC-71
PC-71
PC-67
PC-93
PC-01
PL-03
OC-08, OC-20, PC-29, PC-41, PC-88
PL-06
OC-01
OC-13
PC-30
OC-05
PC-02, PC-13
OC-24
PC-27
-VVagias, Constantinos
Vairappan, Charles S.
Valdés, O.
Vale, Paulo
Valentão, Patrícia
Vallesi, Adriana
van der Sar, Sonia A.
Vansteelandt, Marieke
Varcamonti, Mario
PC-03, PC-17, PC-24, PC-32
PC-34
PC-41
IL-09, PC-85
PC-30
OC-07
PC-58
PC-55
PC-05
192
Varela, Mercedes
Vasconcelos, Vítor M.
Vázquez, Jennifer
Viano, Yannick
Vieira, H.
Vilches, Tamara S.
Villani, Guido
PC-83
PC-79
PC-45
OC-06
PC-71
OC-25, PC-53
PC-05, PC-22, PC-78
-WWang, Xiahong
Wattanadilok, Rawiwan
Webb, Vicky A.
Weber, Jean-Frédéric
Wende, Kristian
Wielgosz-Collin, Gaëtane
Wiens, Matthias
Wiese, J.
Wilke, D. V.
Wurster, Martina
Wüthrich, Kurt
PC-70
PC-19
PC-07, PC-58
PL-09
PC-40
PC-86
OC-14, PC-70, PC-81
PC-82
PC-42
PC-40
OC-07
-XXavier, J.
Xu, Zhongli
PC-38, PC-94
PC-52
-YYang, Guohua
Yokoya, Nair S.
Youssef, Diaa T.A.
Yun, Keumja
Yunt, Zeynep
PC-09, PC-62
PC-23
PC-21
PC-09, PC-62
PC-52
-ZZanfardino, Anna
Żeglińska, Lidia
Zhang, Hua
Zhang, Wen
PC-05
PC-65
PC-97
OC-18
193
June 2009
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