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Novel secondary metabolites from endophytic marine-derived fungi [Elektronische Ressource] / vorgelegt von Celso Almeida

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224 pages
 Novel Secondary Metabolites from Endophytic Marine-derived Fungi Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Celso Almeida aus Lissabon, Portugal Bonn 2011 2   Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachterin : Prof. Dr. G. M. König 2. Gutachterin : Prof. Dr. E. Kostenis Tag der Promotion : 15. Februar 2011 Erscheinungsjahr : 2011 3   Vorveröffentlichungen der Dissertation/In Advance Publications of the Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät, vertreten durch die Mentorin/Betreuerin der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Parts of this study have been published in advance by permission of the Mathematisch-Naturwissenschaftlichen Fakultät, represented by the supervisor of this study: Publikationen / Research Papers Almeida C., Eguereva E., Kehraus S., Siering C. and König G.M., Hydroxylated Sclerosporin Derivatives from the Marine-derived Fungus Cadophora malorum, J. Nat. Prod., 2010, 73 (3), pp 476–478. Almeida C., Elsaedi S., Kehraus S. and König G. M.
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Novel Secondary Metabolites from Endophytic Marine-derived
Fungi







Dissertation
zur
Erlangung des Doktorgrades (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms-Universität Bonn









vorgelegt von
Celso Almeida
aus
Lissabon, Portugal

Bonn 2011
2
  
Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät
der Rheinischen Friedrich-Wilhelms-Universität Bonn























1. Gutachterin : Prof. Dr. G. M. König
2. Gutachterin : Prof. Dr. E. Kostenis


Tag der Promotion : 15. Februar 2011
Erscheinungsjahr : 2011


3
  
Vorveröffentlichungen der Dissertation/In Advance Publications of the Dissertation

Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Mathematisch-
Naturwissenschaftlichen Fakultät, vertreten durch die Mentorin/Betreuerin der Arbeit, in
folgenden Beiträgen vorab veröffentlicht:

Parts of this study have been published in advance by permission of the Mathematisch-
Naturwissenschaftlichen Fakultät, represented by the supervisor of this study:

Publikationen / Research Papers

Almeida C., Eguereva E., Kehraus S., Siering C. and König G.M., Hydroxylated Sclerosporin
Derivatives from the Marine-derived Fungus Cadophora malorum, J. Nat.
Prod., 2010, 73 (3), pp 476–478.

Almeida C., Elsaedi S., Kehraus S. and König G. M., Novel Bisabolane Sesquiterpenes from the
Marine-derived Fungus Verticillium tenerum, Nat. Prod. Commun. 2010, 5(4), 507-
10.

Almeida C., Part N., Kehraus S. and König G. M., Stachylines A – D from the Sponge-derived
Fungus Stachylidium sp. (accepted by Journal of Natural Products).

Almeida C., Kehraus S., Prudêncio M. and König G. M., Marilones A – J, Unusual Phthalides
from the Sponge-derived Fungus Stachylidium sp. (submitted to European Journal
of Organic Chemistry).

Almeida C., Kehraus S., Dimas K., Gütschow M. and König G. M., Marilines A – D, Novel
Phthalimidines from the Sponge-derived Fungus Stachylidium sp. (In preparation)

Almeida C., Kehraus S. and König G. M., Novel Furyl Derivative from the Sponge-derived
Fungus Stachylidium sp. (In preparation).

Almeida C., Kehraus S. and König G. M. Novel bioactive peptides from the Sponge-derived
Fungus Stachylidium sp. (In preparation).
4
  
Tagungsbeiträge/Research Presentations

Almeida C., Kehraus S., König G. M., Hydroxylated derivatives of a rare cadinane-type
sesquiterpene isolated from a cytotoxic Wardomyces inflatus extract. Poster presented at the
th7 Natural Products Joint Meeting of AFERP, ASP, GA, PSE & SIF, August 3-8, 2008,
Athens, Greece.

Almeida C., Kehraus S., König G. M., Hydroxylated derivatives of a rare cadinane-type
sesquiterpene isolated from a cytotoxic Wardomyces inflatus extract. Poster presented at the
Joint Meeting of the Deutsche Pharmazeutische Gesellschaft (DPhG), October 8-11, 2008,
Bonn, Germany.

Almeida C., König G. M., Novel secondary metabolites from the sponge-derived fungus
thStachylidium sp.; tracking bacterial endosymbionts. Oral presentation at the 50 Annual
Meeting of the American Society of Pharmacognosy, June 27-July 1, 2009, Honolulu,
Hawaii.

Almeida C., König G. M., Novel Secondary Metabolites from the Sponge-Derived Fungus
th Stachylidium sp.; Tracking Bacterial Endosymbionts. Poster presented at the 6 European
Conference on Marine Natural Products, July 19 - 23, 2009, Porto, Portugal.

Almeida C., Kehraus S., Dimas K., and König G. M., Marilines A – D, Novel Phthalimidines
from the Sponge-derived Fungus Stachylidium sp. Poster presented at the International
Conference New Biotrends in Green Chemistry, December 1-2, 2010, Dortmund, Germany.









5
  





























To my mother, Marilia, and father, Benjamim



6
  
Acknowledgements
I wish to express my sincere gratitude and admiration to my supervisor Prof. Dr. G. M. König for
the expert guidance, encouragement and kind support during the course of this project. I would
like to thank her for providing excellent scientific advice, working facilities, for letting me express
my creativity, and especially for understanding and guidance in unexpected personal difficulties
that arose during the research. I have nothing else than to be proud and consider to be a truly
privilege to have performed a PhD under the orientation of Prof. Dr. G. M. König.

Special thanks go to Prof. Dr. E. Kostenis for officiating as second referee.

Many specific tasks involved in this study were performed in cooperation with other members of
the Institute for Pharmaceutical Biology, Uni Bonn. For this work cordial thanks go to:
Dr. Stefan Kehraus for indispensable help concerning the implementation and interpretation of
special NMR experiments, for proofreading manuscripts and thesis, and for providing friendly,
professional laboratory support during all phases of this study. I would also like to express my
extreme admiration for the NMR interpretation skills of Dr. Kehraus.
Ekaterina Eguereva for introducing me to the work with marine-derived fungi. Also thanks for
recording all LC-MS spectra and friendly talks during the research.
Edith Neu for conducting agar diffusion assays and for being such a friendly person.
Natalja Part, for her outstanding lab work and for being a good friend.
E. Gassen for friendly assistance and for resolving all administrative questions.
I would like to thank all members of the Institute for Pharmaceutical Biology, University of Bonn,
present or past for cooperation and friendship, with special regards to Hendrik Greve and Ana
Kralj for the great help in the first year in the lab.

Extra-special thanks go to Sanem Insu Tezkan, words cannot describe her existance.
As for Rita Roque, Rui Branco and Paulo Figueiredo, thank you for making the days in Germany
so special, I met you here but the friendship will be for life.
Also for Maria Laura Vinuela which will be in my heart for the rest of my life.
Another thanks goes to Ricardo Baptista Lopes, long time friend that is allways there when i
return to Portugal.
A final thankfull word for “pre-PhD“ personalities, namely Dr. Rob Verpoorte (Leiden
University, The Netherlands) for all the knowledge given during my Masters, and Dr. Artur Silva
7
  
(Aveiro University, Portugal) for teaching exquisidly the basics of NMR spectroscopy to a
biologist, which was essencial for this PhD work.

Financial support for this project was provided by FCT (Fundação para a Ciencia e Tecnologia,
Portugal) and is gratefully acknowledged.

International scientific cooperation acknowledgements. I thank the efforts of Dr. K. Dimas
(Biomedical Research Foundation of Academy of Athens, Greece) for the cytotoxicity assays,
Prof. Dr. M. Gütschow (Institute for Pharmaceutical Chemistry, University of Bonn, Germany)
for performing the panel of proteases inhibition assays, Dr. L. Meijer (Protein Phosphorylation &
Disease, CNRS, Roscoff, France) for performing the protein kinases assays and Dr. C.
Pannecouque (Rega Institute for Medical Research, Leuven, Belgium) for performing the HIV-1
and HIV-2 antiviral assays; I also kindly thank Dr. M. Prudêncio (Malaria Unit, Institute for
Molecular Medicine, University of Lisbon, Portugal) for performing the antiplasmodial activity
assays, Indra Bergval (KIT Biomedical Research, Royal Tropical Institute, Amsterdam,
Netherlands) for performing the M. tuberculosis activity assays, Dr. Marc Diederich (LBMCC,
Luxembourg) for performing the NF-kB activity assays, Dr. Steinar Paulsen (University of
Tromsø, MabCent, Tromsø, Norway) for performing the anti-diabetic activity assays and Dr. K.
Shimokawa (Department of Chemistry, Nagoya University, Japan) for performing the 3T3-L1
murine adipocytes assay; the Ki determinations and antagonist functional data that was generously
provided by the National Institute of Mental Health's Psychoactive Drug Screening Program,
Contract # HHSN-271-2008-00025-C (NIMH PDSP). The NIMH PDSP is directed by Bryan L.
Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie
Driscol at NIMH, Bethesda MD, USA; I kindly thank also the remaining antiviral tests performed
by the U.S. National Institute of Health which were supported by contracts NO1-A1-30048
(Institute for Antiviral Research, IAR) and NO1-AI-15435 (IAR) from Virology Branch, National
Institute of Allergic and Infectious Diseases, NIAID; also a thankful word for Dr. M. Engeser and
Dr. B. Sondag (Kekulé-Institute for Organic Chemistry and Biochemistry, University of Bonn) for
the High Resolution Mass Spectrometry efforts. Synthetic reference compounds for CD spectra
comparison with verticonols A/B were kindly provided by Dr. Kenji Mori, Emeritus Professor
from the University of Tokyo, Japan; special thanks to Carsten Siering (Kekulé Institute for
Organic and Inorganic Chemistry, University of Bonn) for CD spectral measurements and
invaluable discussions.
8
  
Table of contents Page
1. Introduction …………………………….…………………………………….….1
2. Scope of the present study …………………………………….………...……..14
3. General methodology ……………………………………………….…………15
4. Marilines A – D, novel phthalimidines from the sponge-derived
fungus Stachylidium sp. ......................................................................................18
5. Marilones A – J, unusual phthalides from the sponge-derived
fungus Stachylidium sp. ......................................................................................36
6. Stachylines A – D from the sponge-derived fungus Stachylidium sp. ................56
7. Novel furyl derivative from the sponge-derived fungussp. .......70
8. Endolides A – J, N-methylated peptides from the sponge-derived
fungus Stachylidium sp. ......................................................................................75
9. Hydroxylated sclerosporin derivatives from the marine-derived
fungus Cadophora malorum …………………………………….……….…….76
10. Novel bisabolane sesquiterpenes from the marine-derived Fungus
Verticillium tenerum ...........................................................................................84
11. Discussion ……………………………………………………………..….……92
12. Summary …………………………………………………………..…..……….96
13. Appendix
13.1. Bioactivity results ……………………………………..…..……………109
13.2. Protocols and media ………………………………...…………………..121
13.3. Complete 2D NMR data ………………………….………...…………..127
13.4. Spectroscopic data supporting information ………………....………….147
1 1313.5. H and C NMR spectra
1 13 13.5.1. H and C NMR spectra of new molecules ……………….....160
1 1313.5.2. H and C NMR spectra of known molecules …………...…..185
9
  
Abbreviations

°C degrees Celsius
1D one dimensional
2D two dime
T[ α] specific rotary power, sodium D-line (589 nm); T: temperature D
δ NMR chemical shift [ppm]
λ wavelength [nm]
-6μ micro (10 )
-6μg 10 gram
-6μl 10 liter
-6 -6μM 10 molar, micromolar (= 10 mol/L)
-1ν wave number [cm ]
ASW artificial seawater
ATR attenuated total reflection
AU absorbance units
BMS biomalt salt medium
br broad (in connection with NMR data)
c concentration
C 18 C-18 modified silica gel
calcdcalculated
CD circular dichroism
CDCl chloroform-d 3
CDCN acetonitrile-d 3 3
CDOD methanol-d3 4
CH Cl dichloromethane (DCM) 2 2
CHCN acetonitrile 3
conc. concentration
COSY correlated spectroscopy
-2cm 10 meter
CZ Czapek medium
d doublet (in connection with NMR data)
DAD diode array detector
10
  
DCM dichloromethane
DEPT distortionless enhancement by polarization transfer
-1dmol 10 mol
DNA deoxyribonucleic acid
EC half maximal effective concentration (drug concentration causing 50
50% of maximal effect)
e.g. example given (for example)
EI electron ionization
ESI electron spray ionization
et al. et alii [Lat.]: and others
EtOAc ethyl acetate
EtOH ethanol
g gram
GI growth inhibition
h hour
H BO boric acid 3 3
HMBC heteronuclear multiple-bond correlation
HMGCoA hydroxymethylglutaryl-CoA
HPLC high performance liquid chromatography
HR high resolution
HSQC heteronuclear single quantum correlation
Hz Hertz
HO water 2
IC half maximal inhibitory concentration (drug concentration 50
causing 50% inhibition)
i.e. that is
IR infrared
J spin-spin coupling constant [Hz]
3kDa kilo Dalton (= 10 Dalton)
L liter
LC liquid chromatography
leu leucine
m meter
11
 

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